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Phylogenetic analysis of different genotypes of Newcastle disease virus using the F gene coding sequences suggests that these two strains belong to genotype VII.2, in class II of avian paramyxoviruses. The genomes of two newly emerged Newcastle disease virus strains, chicken/Indonesia/Mega/001WJ/2013 and chicken/Indonesia/Cimanglid/002WJ/2015, from disease outbreaks in chickens in Indonesia are reported. Phylogenetic analysis of different genotypes of Newcastle disease virus using the F gene coding sequences suggests that these two strains belong to genotype VII.2, in class II of avian paramyxoviruses. Orthoavulavirus (\u2013Newcastle disease (ND) is one of the most severe infectious diseases of chickens. The causative agent, ND virus (NDV), is a member of the avian genus viridae) . NDV is viridae) , 3. NDV viridae) \u20136. Receniridae) \u2013.de novo assembled using Unicycler v0.4.4 with default parameters (HQ697255), respectively. A gap in the sequence of Mega/001WJ was closed using reverse transcriptase PCR (Qiagen) and Sanger sequencing under BioProject number PRJNA613298.The genome sequences for Mega/001WJ and Cimanglid/002WJ were deposited in GenBank with accession numbers"} +{"text": "Scientific Reports 10.1038/s41598-019-55068-z, published online 10 December 2019Correction to: This Article contains a typographical error in the Acknowledgements section.http://ncn.gov.pl), grant DEC 2016/21/B/ST7/02241. The results published here are in part based upon data generated by TCGA managed by the NCI and NHGRI. Information about TCGA can be found at http://cancergenome.nih.gov.\u201d\u201cThis project has received funding from the European Union\u2019s Horizon 2020 Research and Innovation Programme under grant agreement No. 66740, the Academy of Finland, the Sigrid Jus\u00e9lius Foundation, Finnish Cancer Associations and by National Science Centre, Poland (should read:https://ncn.gov.pl), grant DEC 2016/21/B/ST7/02241. The results published here are in part based upon data generated by TCGA managed by the NCI and NHGRI. Information about TCGA can be found at https://cancergenome.nih.gov.\u201d\u201cThis project has received funding from the European Union\u2019s Horizon 2020 Research and Innovation Programme under grant agreement No. 667403 for HERCULES, the Academy of Finland, the Sigrid Jus\u00e9lius Foundation, Finnish Cancer Associations and by National Science Centre, Poland ("} +{"text": "Cell Death & DiseaseCorrection to: 10.1038/cddis.2017.193 published online 18 May 2017Since online publication of this article, the authors noticed that there was an error in Figs."} +{"text": "This article has been corrected: During the assembly of images for Figure 5, the image for Day 1 in the control (PBS) group, seen in panel A, was erroneously copied to Day 2 as well. The corrected Figure 5, obtained using the original data, is shown below. The authors declare that these corrections do not change the results or conclusions of this paper.109894-109914. https://doi.org/10.18632/oncotarget.22493Original article: Oncotarget. 2017; 8:109894\u2013109914."} +{"text": "Drosophila melanogaster (DGRP) allowed us to rediscover three known cases of adaptation at the loci Ace, Cyp6g1, and CHKov1 known to be driven by soft sweeps, and detected additional candidate loci for recent and strong sweeps. Surprisingly, all of the top 50 candidates showed patterns much more consistent with soft rather than hard sweeps. Recently, Harris et al. 2018 criticized this work, suggesting that all the candidate loci detected by our haplotype statistics, including the positive controls, are unlikely to be sweeps at all and that instead these haplotype patterns can be more easily explained by complex neutral demographic models. They also claim that these neutral non-sweeps are likely to be hard instead of soft sweeps. Here, we reanalyze the DGRP data using a range of complex admixture demographic models and reconfirm our original published results suggesting that the majority of recent and strong sweeps in D. melanogaster are first likely to be true sweeps, and second, that they do appear to be soft. Furthermore, we discuss ways to take this work forward given that most demographic models employed in such analyses are necessarily too simple to capture the full demographic complexity, while more realistic models are unlikely to be inferred correctly because they require a large number of free parameters.Whether hard sweeps or soft sweeps dominate adaptation has been a matter of much debate. Recently, we developed haplotype homozygosity statistics that (i) can detect both hard and soft sweeps with similar power and (ii) can classify the detected sweeps as hard or soft. The application of our method to population genomic data from a natural population of Cyp6g1, Ace, and CHKov1. Recently, Harris et al. 2018 have claimed that the selective sweeps we identified are false positives and are more likely to be explained by an admixture demographic history. Moreover, they claim that these neutral non-sweeps are more likely to be hard sweeps rather soft sweeps. Here we re-assess our and Harris et al\u2019s work and find that given a reasonably well-fitting demographic model, soft sweeps are a dominant mode of adaptation in North American Drosophila melanogaster.Whether hard versus soft sweeps dominate adaptation has long been a matter of great debate. Recently, we proposed novel statistics that can identify and differentiate hard and soft sweeps and found that soft sweeps are surprisingly common in North American Drosophila melanogaster. Among our top ranking candidates are three well-known soft sweeps at the loci Drosophila melanogaster. Recent studies suggest that (i) ~50% of amino acid changing and non-coding substitutions in D. melanogaster evolution were adaptive, and (ii) there are abundant signatures of adaptation in the population genomic data detectable as reductions of neutral diversity in the regions of higher functional divergence and as elevation in the frequencies of derived alleles above neutral expectations base pairs, where Ne is the population size and rho is the recombination rate. As an example, sweeps with s = 0.05% are likely to generate sweeps spanning 10kb windows when rho = 5*10^-7. As rho increases, only those selective sweeps with s>0.05% should be observed in 10kb windows.To circumvent the issue of defining a window size, another approach is to use H-scan or nSL to identWindows defined in terms of SNPs versus base pairs. Windows defined in terms of SNPs are all guaranteed to have the same number of SNPs, which then can be used to define the number and frequencies of haplotypes in a window. These SNP-based windows are fully capable of detecting complete hard sweeps . The selection coefficient and partial frequency of the sweeps were drawn from uniform priors ranging from 0 to 1.S1 FigAce, Cyp6g1, and CHKov1.Each point represents the mean Pi/bp value in a 10Kb window. Red vertical lines indicate the positions of the positive controls, (TIF)Click here for additional data file.S2 FigAce, Cyp6g1, and CHKov1.Each point represents the mean S/bp value in a 10Kb window. Red vertical lines indicate the positions of the positive controls, (TIF)Click here for additional data file.S3 Fig2015 [Ne = 106 model, (B) a constant Ne = 2.7x106 model, (C) a severe short bottleneck model, (D) a shallow long bottleneck model, (E) the implemented admixture model in Garud et al. 2015 [et al. 2015 [2015 . The disal. 2015 , and (F)al. 2015 . Short i(TIF)Click here for additional data file.S4 Fig, Harris et al. [, and Arguello et al [et al. 2013 [et al. 2013 [et al. 2013 [et al. 2018 [et al. 2019 [, Harris s et al. , and Arglo et al . The dislo et al , Harris lo et al , and Arglo et al . The modal. 2013 , simulatal. 2013 , simulatal. 2013 , simulatal. 2018 , simulatal. 2019 . Short i(TIF)Click here for additional data file.S5 FigFig 3J and 3K) (A) A variant of the Duchen et al. 2013 [et al. 2013 [The distribution of Pi/bp computed in short introns of length 10bps or longer in DGRP data is compared with Pi/bp values computed in two demographic models inferred in this paper to fit the DGRP A val. 2013 admixtural. 2013 . Short i(TIF)Click here for additional data file.S6 Fig2015 [Ne = 106 model, (B) a constant Ne = 2.7x106 model, (C) a severe short bottleneck model, (D) a shallow long bottleneck model, (E) the implemented admixture model in Garud et al. 2015 [et al. 2015 [2015 . These p2015 . The modal. 2015 , and (F)al. 2015 . The roo(TIF)Click here for additional data file.S7 Fig, Harris et al. [, and Arguello et al [et al. 2013 [et al. 2013 [et al. 2013 [et al. 2018 [et al. 2019 [, Harris s et al. , and Arglo et al . These plo et al , Harris lo et al , and Arglo et al . The modal. 2013 , simulatal. 2013 , simulatal. 2013 , simulatal. 2018 , simulatal. 2019 . The roo(TIF)Click here for additional data file.S8 FigFig 3J and 3K) (A) A variant of the Duchen et al. 2013 [et al. 2013 [These plots quantify the fit of the distributions plotted in al. 2013 admixtural. 2013 . (B) A val. 2013 admixtural. 2013 . The roo(TIF)Click here for additional data file.S9 Fig2015 [Ne = 106 model, (B) a constant Ne = 2.7x106 model, (C) a severe short bottleneck model, (D) a shallow long bottleneck model, (E) the implemented admixture model in Garud et al. 2015, and (F) the implemented admixture + bottleneck model in Garud et al. 2015. Short intron lengths matching those in data were used were used in simulations. Each simulation contains10x the number of short intron fragments as observed in the data. 2015 . The dis(TIF)Click here for additional data file.S10 Fig, Harris et al. [, and Arguello et al [et al. 2013 [et al. 2013 [et al. 2013 [et al. 2018 [et al. 2019 [, Harris s et al. , and Arglo et al . The dislo et al , Harris lo et al , and Arglo et al . The modal. 2013 , simulatal. 2013 , simulatal. 2013 , simulatal. 2018 , simulatal. 2019 . Short i(TIF)Click here for additional data file.S11 FigFig 3J and 3K) (A) A variant of the Duchen et al. 2013 [et al. 2013 [The distribution of S/bp computed in short introns of length 10bps or longer in DGRP data is compared with S/bp values computed in two demographic models inferred in this paper to fit the DGRP A val. 2013 admixtural. 2013 . Short i(TIF)Click here for additional data file.S12 Fig2015 [Ne = 106 model, (B) a constant Ne = 2.7x106 model, (C) a severe short bottleneck model, (D) a shallow long bottleneck model, (E) the implemented admixture model in Garud et al. 2015 [et al. 2015 [2015 . These p2015 . The modal. 2015 , and (F)al. 2015 . The roo(TIF)Click here for additional data file.S13 Fig, Harris et al. [, and Arguello et al [S10 Fig. The distribution of S/bp computed in short introns of length 10bps or longer in DGRP data is compared with S/bp values computed in a range of simulated neutral demographic models from Duchen et al. [et al. 2013 [et al. 2013 [et al. 2013 [et al. 2018 [et al. 2019 [, Harris s et al. , and Arglo et al . These pn et al. , Harris n et al. , and Argn et al. . The modal. 2013 , simulatal. 2013 , simulatal. 2013 , simulatal. 2018 , simulatal. 2019 . The roo(TIF)Click here for additional data file.S14 FigFig 3J and 3K) (A) A variant of the Duchen et al. 2013 [et al. 2013 [These plots quantify the fit of the distributions plotted in al. 2013 admixtural. 2013 . (B) A val. 2013 admixtural. 2013 . The roo(TIF)Click here for additional data file.S15 Fig2015 [Ne = 106 model, (B) a constant Ne = 2.7x106 model, (C) a severe short bottleneck model, (D) a shallow long bottleneck model, (E) the implemented admixture model in Garud et al. 2015 [et al. 2015. The number of analysis windows generated for the simulated models equals the number of analysis windows for the DGRP data, after excluding regions of low recombination rates. The red points indicate the H12 values for the top 50 peaks in the DGRP data.2015 . The DGRal. 2015 , and (F)(TIF)Click here for additional data file.S16 Fig, Harris et al. [, and Arguello et al [et al. 2013 [et al. 2013 [et al. 2013 [et al. 2018 [et al. 2019 [, Harris s et al. , and Arglo et al . The DGRlo et al , Harris lo et al , and Arglo et al . The modal. 2013 , simulatal. 2013 , simulatal. 2013 , simulatal. 2018 , simulatal. 2019 . The num(TIF)Click here for additional data file.S17 FigFig 3J and 3K and are (A) A variant of the Duchen et al. 2013 [et al. 2013 [The two models are depicted in al. 2013 admixtural. 2013 . (B) A val. 2013 admixtural. 2013 . The num(TIF)Click here for additional data file.S18 Fig2015 [Ne = 106 model, (B) a constant Ne = 2.7x106 model, (C) a severe short bottleneck model, (D) a shallow long bottleneck model, (E) the implemented admixture model in Garud et al. 2015 [et al. 2015 [2015 . These pal. 2015 , and (F)al. 2015 . The roo(TIF)Click here for additional data file.S19 Fig, Harris et al. [, and Arguello et al [et al. 2013 [et al. 2013 [et al. 2013 [et al. 2018 [et al. 2019 [, Harris s et al. , and Arglo et al . These pal. 2013 , simulatal. 2013 , simulatal. 2013 , simulatal. 2018 , simulatal. 2019 . The roo(TIF)Click here for additional data file.S20 FigFig 3J and 3K (A) A variant of the Duchen et al. 2013 [et al. 2013 [These plots quantify the fit of the distributions plotted in al. 2013 admixtural. 2013 . (B) A val. 2013 admixtural. 2013 . The roo(TIF)Click here for additional data file.S21 Fig(A) Quantile-quantile plot of H12 values within +/-1 SD of the median value in the DGRP data are compared with a random sample from the fitted Gaussian. The Gaussian was simulated with the mean equalling the median value of H12 in the DGRP data, and the standard deviation estimated from points within 1 standard deviation around the median (Methods). (B) Comparison of distribution of H12 values in DGRP data with that of a simulated Gaussian with a mean and standard deviation from (A). The vertical blue line indicates 11 standard deviations away from the mean of the simulated Gaussian distribution. The red points indicate the H12 values for the top 50 peaks in the DGRP data. (C) QQ-plot of H12 from the entire distribution of DGRP values compared with the same simulated Gaussian from (A). The distribution of H12 values in DGRP data has an extreme elevated tail compared to expectations under a Gaussian (D) QQ-plot comparing H12 values from a constant Ne = 2.7*10^6 model with a Gaussian fitted to its bulk (Methods). Neutral simulations lack the elevated tail present in the data.(TIF)Click here for additional data file.S22 FigWe computed Pi, S, and H12 in variants of the admixture model proposed by Duchen et al. 2013 . The adm(TIF)Click here for additional data file.S23 FigsSummary statistics S, Pi, H12, and LD were measured in admixture models with constant population sizes in Europe and North America . In S6 t(TIF)Click here for additional data file.S24 FigsSummary statistics S, Pi, H12, and LD were measured in admixture models with constant population sizes in Europe and North America . In S6 t(TIF)Click here for additional data file.S25 FigsSummary statistics S, Pi, H12, and LD were measured in admixture models with constant population sizes in Europe and North America . In S6 t(TIF)Click here for additional data file.S26 FigsSummary statistics S, Pi, H12, and LD were measured in admixture models with constant population sizes in Europe and North America . In S6 t(TIF)Click here for additional data file.S27 FigsSummary statistics S, Pi, H12, and LD were measured in admixture models with constant population sizes in Europe and North America . In S6 t(TIF)Click here for additional data file.S28 FigsSummary statistics S, Pi, H12, and LD were measured in admixture models with varying growth rates in Europe and North America . In S11\u2013(TIF)Click here for additional data file.S29 FigsSummary statistics S, Pi, H12, and LD were measured in admixture models with varying growth rates in Europe and North America . In S11\u2013(TIF)Click here for additional data file.S30 FigsSummary statistics S, Pi, H12, and LD were measured in admixture models with varying growth rates in Europe and North America . In S11\u2013(TIF)Click here for additional data file.S31 FigsSummary statistics S, Pi, H12, and LD were measured in admixture models with varying growth rates in Europe and North America . In S11\u2013(TIF)Click here for additional data file.S32 FigsSummary statistics S, Pi, H12, and LD were measured in admixture models with varying growth rates in Europe and North America . In S11\u2013(TIF)Click here for additional data file.S33 FigsSummary statistics S, Pi, H12, and LD were measured in admixture models with varying growth rates in Europe and North America . In S11\u2013(TIF)Click here for additional data file.S34 FigSummary statistics S, Pi, H12, and LD were measured in admixture models with varying admixture proportions between Europe and North America . Admixtu(TIF)Click here for additional data file.S35 FigSummary statistics S, Pi, H12, and LD were measured in admixture models with varying amounts of migration between Europe and North America . Migrati(TIF)Click here for additional data file.S36 Figth-30th and the (B) 31st\u201450th peaks in the DGRP scan.Same as (TIFF)Click here for additional data file."} +{"text": "Bone Research 10.1038/boneres.2017.44, published online 26 September 2017Correction to: During a reread of our article previously published in Bone Research, we regrettably found that the second lane of originally published Fig."} +{"text": "Experimental & Molecular MedicineCorrection to: 10.1038/emm.2016.168 published online 17 March 2017After online publication of this article, the authors noticed an error in Fig. In the version of this article originally published, Fig."} +{"text": "Cell Discovery (2020) 6:31Correction to: 10.1038/s41421-020-0168-9 Published online 04 May 20201, an error was identified in the Data availability section. The accession number of the dataset was updated. The corrected Data availability appears below:Following publication of the original articlehttps://bigd.big.ac.cn/gsa.The raw sequence data reported in this paper has been deposited in the Genome Sequence Archive in National Genomics Data Center (Nucleic Acids Res 2020), Beijing Institute of Genomics , Chinese Academy of Sciences, under Project Accession No. PRJCA002413 (GSA Accession No. CRA002497) that are publicly accessible at"} +{"text": "Differential globalization of industry\u2010 and non\u2010industry\u2010sponsored clinical trials. PLoS One. 10(12), 1\u201017. ,2https://doi.org/10.1136/bmjopen-2015-008932.Viergever, R.F., Li, K. (2015). Trends in global clinical trial registration: An analysis of numbers of registered clinical trials in different parts of the world from 2004 to 2013. BMJ Open. 5(9). Major reasons for this trend include the collective promotion of global health and the need to rapidly and effectively respond to threats to human health worldwide. The latest epidemic of Ebola Virus Disease (EVD) showed us that the systems put in place for the development of international clinical research were not ready to face the challenge of controlling that deadly disease.3https://doi.org/10.17226/24739.Keusch, G., McAdam, K., Cuff, P., Mancher, M., Busta, E.R. (2017). Integrating Clinical Research into Epidemic Response. Washington, D.C.: National Academies Press. ,4https://doi.org/10.1016/S0140-6736(17)31602-1.Keusch, G.T., McAdam, K.P.W.J. (2017). Clinical trials during epidemics. Lancet. 389(10088), 2455\u20102457. In addition to EVD, many diseases without new drugs and/or vaccines are listed in the reports published by the World Health Organization (WHO),5https://www.who.int/blueprint/en/.World Health Organization. (2018). A research and development Blueprint for action to prevent epidemics. Retrieved January 20, 2018, from the Global Health Security Agenda (GHSA),6https://www.ghsagenda.org/packages.Global Health Security Agenda.\u202f(2014). Action Packages. Retrieved April 26, 2019 from and the World Bank\u2010sponsored International Vaccine Task Force.7https://doi.org/10.1126/science.301.5637.1182b.Academy of Medical Sciences. (2018). Money and Microbes\u202f: Strengthening Clinical Research Capacity to Prevent Epidemics (English). Washington, D.C.: Publisher. The early 218https://crigh.org/://crigh.org/Clinical Research Initiative for Global Health. (2019). Overview. Retrieved July 24, 2019 from was launched in 2017 corresponding to the Organization for Economic Cooperation and Development (OECD) recommendations9OECD Global Science Forum. (2011). OECD Global Science Forum Facilitating International Cooperation in Non\u2010Commercial Clinical Trials Facilitating International Cooperation in Non\u2010Commercial Clinical Trials Organisation for Economic Co\u2010Operation and Development. Global Science Forum. for better global governance of international non\u2010commercial clinical research. CRIGH will encourage international cooperation to rapidly and efficiently respond to global health challenges which are mentioned above.The Clinical Research Initiative for Global Health (CRIGH)10https://doi.org/10.4172/2167-0870.1000219.Forjuoh, S.N. (2015). Challenges Associated with Multi\u2010institutional Multi\u2010site Clinical Trial Collaborations: Lessons from a Diabetes Self\u2010Management Interventions Study in Primary Care. Journal of Clinical Trials. 05(03). International clinical trials are even more complex than single\u2010country studies due to the diversity of legal and ethical frameworks. Researchers and sponsors who conduct an international and multi\u2010site trial face two problems with ethics reviews: the differences between the countries and duplicate reviews within one country.One of the major challenges is the need for multiple ethics reviews by institutional review boards (IRB) or research ethics committees (REC).11https://doi.org/10.1007/s00134-009-1544-yDruml, C., Wolzt, M., Pleiner, J., Singer, E.A. (2009). Research ethics committees in Europe: Trials and tribulations. Intensive Care Medicine. 35(9), 1636\u20101640. ,12https://doi.org/10.1136/medethics-2012-101282.Veerus, P., Lexchin, J., Hemminki, E. (2014). Legislative regulation and ethical governance of medical research in different European Union countries. Journal of Medical Ethics. 40(6), 409\u2010413. and USA13https://doi.org/10.1378/chest.15-0706.Grady, C. (2015). Institutional review boards purpose and challenges. Chest. 148(5), 1148\u20101155. are widely available, however, there is limited information on ethics review systems for countries in other areas. Therefore, the CRIGH Research Bioethics project, co\u2010chaired by Council on Health Research for Development (COHRED), National Cancer Center Japan, and National Institutes of Health, conducted a cross\u2010sectional survey to understand the differences of ethics review systems and how these could present obstacles for promoting international collaborative trials by non\u2010profit, academic organizations, and to formulate recommendations to overcome these obstacles to facilitate ethical international collaborative health research and clinical trials.Information on the current ethics review system in the EU14https://ec.europa.eu/health/sites/health/files/files/eudralex/vol-1/dir_2001_20/dir_2001_20_en.pdfEuropean Commission. (2019). Directive 2001/20/EC. Retrieved April 26, 2019 from the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) \u2013Guideline for Good Clinical Practice (GCP),15https://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Efficacy/E6/E6_R1_Guideline.pdf.ICH Harmonised Tripartite Guideline. (1996). Guideline For Good Clinical Practice. Retrieved April 26, 2019 from Standards and operational guidance for ethics review of health\u2010related research with human participants published by WHO,16https://www.ncbi.nlm.nih.gov/books/NBK310666/pdf/Bookshelf_NBK310666.pdf.World Health Organization. (2011). Standards and Operational Guidance for Ethics Review of Health\u2010Related Research with Human Participants.; 2011. Retrieved April 26, 2019 from and 45 CFR part 46.17https://www.govinfo.gov/content/pkg/CFR-2016-title45-vol1/pdf/CFR-2016-title45-vol1-part46.pdf.45 CFR 46. Retrieved April 26, 2019 from In addition, the information from COHRED\u2019s routine assessment of RECs using RHInnO Ethics18http://www.rhinno.net.RHInnO Ethics. Retrieved April 26, 2019 from was used for the development of the questionnaire. The questionnaire contained 17 items regarding national ethics review system.We developed the survey items referring to four international standards: the EU Clinical Trial Directive (Directive 2001/20/EC),We recruited experts of ethics review system in each country by snowball sampling in Latin America, Oceania, and South East Asia. In African countries, COHRED used its network of research ethics committees to recruit national experts. The experts included chairs of IRBs/RECs, committee members, and a clinical researcher. The survey questionnaire was sent via e\u2010mail or Google Forms to the experts in 41 countries. Data collection periods were from November 2017 to June 2018.We had 31 responses (response rate = 75.7%): 17 in Africa, 9 in Asia, 4 in Latin America, and one from Australia.The legal basis of IRB/REC and the composition of committee members were similarly regulated; 81% (n=25/31) had a national requirement to establish IRB/RECs. Regarding the composition of committee members, 90% (n=28/31) required more than five members including at least one non\u2010scientific member in a committee, and 97% (n=30/31) required Conflict Of Interest management of committee members.There was substantial variation in the number of IRB/RECs Figure , in the Figure Our survey showed the differences in characteristics of ethics review systems, some being potential hurdles for conducting international and multi\u2010site trials: multiple ethics approvals for multi\u2010site trials within a country, lack of trained committee members to properly review the study, or different timeframes for the review. We can make four recommendations for accelerating and improving ethics review of international, multi\u2010site trials based on our survey results.19European Commission, op. cit. note 14. The USA has also planned to mandate single IRB review for multi\u2010site trial within the U.S. National Institutes of Health (NIH) system.20https://doi.org/10.1111/cts.12447.Gordon, V., Culp, M., Wolinetz, C. (2017). Final NIH Policy on the Use of a Single Institutional Review Board for Multisite Research. Clinical and Translational Science. 10(3), 130\u2010132. However, only 13 countries mandate a \u201csingle opinion\u201d for a multi\u2010site trial within their national borders. One challenge is that a single IRB cannot review the local context of each institution included as a research site. Another challenge could be \u201cIRB shopping,\u201d because researchers can choose IRBs for the review, and they can submit protocols to multiple IRBs until one is found that will approve the protocol. Despite these challenges, \u201csingle opinion\u201d would be still beneficial for promoting international and multi\u2010site trials. Countries should at least provide an option for researchers to choose a \u201csingle opinion\u201d approach for a multi\u2010site trial within one country.First, countries should strive to adopt \u201csingle opinion\u201d for multi\u2010site clinical trials. A \u201csingle opinion\u201d approach to multi\u2010site trial review within a country will reduce duplicate reviews and delays caused by the need to re\u2010review. Duplicate reviews within a country are a huge hurdle to promote an international and multi\u2010site trial. The EU has already mandated \u201csingle opinion\u201d for multisite clinical trials carried out in more than one member state since 2001 based on its Clinical Trials Directive.Second, national requirements should include training and continuing education in research ethics for IRB/RECs members. The training of IRB/REC members could improve the protocol review, ensuring response in an adequate time and standardizing its quality. However, not all surveyed countries required training for IRB/REC members. A potential reason may be insufficient availability of educational tools and in multiple languages. Even if a country requires training for IRB/REC members, they cannot have effective education without resources. E\u2010learning approaches could be effective in countries with a large number of IRB/RECs and adequate internet infrastructure, and on\u2010site education could be implemented in countries with a small number of committees or low internet access. We need further discussion on the development of universal training tools in several languages and their availability through online platforms. Among its future activities, our group aims to assess the landscape of available tools.In addition to learning core competencies, IRB/REC members need to be up\u2010to\u2010date with changes in local regulation and their impact on ethics review . Moreover, we should consider streamlining the number of committees within a country to effectively and swiftly implement standardized education since countries with hundreds of IRB/REC represent a challenge to successful training.21https://www.who.int/ethics/review-committee/review_process/en/World Health Organization. (2018). Ethics Review Committee: review process. Retrieved December 25, 2018 from the IRB/REC human resources and their experience, and the internal processes within each country, setting a 60\u2010day maximum provides an estimate for investigators, who could than plan and prepare accordingly before the international trial starts. We need to set minimal timeframe of ethics review to harmonize the starting point of international trials.Third, national requirements should include an adequate timeframe for the ethics review process. It is essential to establish a reasonable timeframe for the trial review, because ethics review in a country which has no timeframe requirement would cause a bottleneck for promoting an international multi\u2010site trial. Although the timeframe may depend on the type of review,Ethics22RHInnO Ethics, op. cit. note 18. also can provide solutions for the issues above, especially in the countries with under\u2010resourced IRBs/RECs and research systems. Many countries in Africa have implemented the RHInnO Ethics platform with support from the European and Developing Countries Clinical Trial Partnership (EDCTP), research funders, and international non\u2010profits. By providing real\u2010time access to a virtual REC Administrator qualified to manage complex clinical research at the level required by, for example, the US Department of Health and Human Services, the RHInnO Ethics platform can provide the ability to conduct high level efficient review in time to virtually any REC Administrator23Kasule, M., Wassenaar, D.R., IJsselmuiden, C., Mokgatla, B. (2016). Silent Voices: Current and Future Roles of African Research Ethics Committee Administrators. IRB Ethics and Human Research. 38(1), 13\u201019. in Africa that has access to internet, and speed up clinical research substantially.24https://doi.org/10.4172/2155-6113.1000658.Mokgatla, B., Bahati, P., IJsselmuiden, C.I. (2017). Enhancing the Efficiency and Quality of African Research Ethics Review Processes \u2013 Through an Automated Review Platform. Journal of AIDS Clinical Research. 8(2), 2. Fourth, technological innovations such as web\u2010based ethics review management and expert decision support platforms including RHInnO While we realize that working globally entails working with and respecting national autonomy, especially in the ethics of research, we also want to emphasize that it is time to harmonize ethics review processes for international multi\u2010site trials as an effective and low\u2010cost manner to achieve global health."} +{"text": "Open Biol.10, 190273. (Published Online 26 February 2020) (doi:10.1098/rsob.190273)This correction refers to an error in the affiliation of author \u2018Tsung-Ming Chen\u2019. It should be:3Department and Graduate Institute of Aquaculture, National Kaohsiung University of Science and Technology, Kaohsiung, Taiwan.This has now been corrected."} +{"text": "Correction to: BMC Infect Dis 20, 300 (2020).10.1186/s12879-020-05033-3Incorrect description: The data of the TB cases in Guangxi from January 2012 to June 2019 was obtained from the Guangxi center for Disease Control and Prevention, China (please see Data source section.http://wsjkw.gxzf.gov.cn/zfxxgk_49572/ggws/fdcrbyqgb/t5518731.shtml).Corrected sentence: The data of the TB cases in Guangxi from January 2012 to June 2019 was obtained from the website of Guangxi Health Management Committee .The research did not involve any direct participation by human subjects. The TB data were extracted from monthly reports maintained on public website ("} +{"text": "The sixth author\u2019s name is spelled incorrectly. The correct name is: Nilson Ivo Tonin Zanchin.https://doi.org/10.1371/journal.pntd.0006871The correct citation is: Borja LS, Coelho LB, Jesus MSd, de Queiroz ATL, Celedon PAF, Zanchin NIT, et al. (2018) High accuracy of an ELISA test based in a flagella antigen of Leishmania in serodiagnosis of canine visceral leishmaniasis with potential to improve the control measures in Brazil\u2014A Phase II study. PLoS Negl Trop Dis 12(10): e0006871. The authors apologize for the errors in the published article."} +{"text": "Correction to: Cell Commun Signal (2016) 14:32https://doi.org/10.1186/s12964-016-0157-7Unfortunately, after publication of this article , it was AcknowledgementsWe are grateful for the technical support by Aruna Visavadiya, Ying Li, and Rhesa Dykes. Dr. Britta Engelhardt (Theodor Kocher institute) is thanked for providing the bEnd5 cells.FundingThis work was supported by NIH grant NS45734 and in part by NIH grant C06RR0306551 and the ETSU College of Medicine.Further to this, a duplicate image in Fig."} +{"text": "Toxoplasma gondii is a protozoan parasite that is the causative agent of toxoplasmosis, an infection with high prevalence worldwide. Most of the infected individuals are either asymptomatic or have mild symptoms, but T. gondii can cause severe neurologic damage and even death of the fetus when acquired during pregnancy. It is also a serious condition in immunodeficient patients. The life-cycle of T. gondii is complex, with more than one infective form and several transmission pathways. In two animated videos, we describe the main aspects of this cycle, raising questions about poorly or unknown issues of T. gondii biology. Original plates, based on electron microscope observations, are also available for teachers, students and researchers. The main goal of this review is to provide a source of learning on the fundamental aspects of T. gondii biology to students and teachers contributing for better knowledge and control on this important parasite, and unique cell model. In addition, drawings and videos point to still unclear aspects of T. gondii lytic cycle that may stimulate further studies. Toxoplasma gondii is the causative agent of toxoplasmosis that is a zoonosis of significant medical and veterinary importance and is transmitted by several pathways. Marked advances regarding the control of several infectious diseases caused by parasitic protozoa have taken place in the last decades, especially those that spend part of their life-cycle inside host cells. Nevertheless, the epidemiological control and development of new chemotherapeutic agents with low toxicity and high specificity continue to constitute great challenges. Some of these diseases are restricted to specific areas of the world, as in the case of Chagas disease. Others, like toxoplasmosis, are widely distributed throughout the world .Additional file 6: Figure S6. Tachyzoite. Abbreviations: c, conoid; R, rhoptry; A, acidocalcisome; m, microneme; DG, dense granule.Additional file 7: Figure S7. Four Membranes of apicoplast (arrows). Inset, relative position of the apicoplast (A) to the nucleus (N) and Golgi complex (GC). (Image courtesy Dr. Erica Martins Duarte).Additional file 8: Figure S8. Scheme of sporozoite.Additional file 9: Figure S9. Scheme of bradyzoite.Additional file 10: Figure S10. Bradyzoite. Amylopectin granules (arrow).Additional file 11: Figure S11. Sporocyst suture of curved plates (arrowheads).Additional file 12: Figure S12. 3D scheme of a sporulated oocyst containing two sporocysts with 4 sporozoites each.Additional file 13: Figure S13. Section view of a sporulated oocyst.Additional file 14: Figure S14. Tachyzoite (purple) adhered to a lymphocyte (beige) [17].Additional file 15: Figure S15. Tachyzoite (purple) invading a macrophage (beige).Additional file 16: Figure S16. Parasitophorous vacuole: rosette of tachyzoites (purple), filamentous network (pink). Host cell (beige)Additional file 17: Figure S17. Sequence of intracellular cycle. a Adhesion, secretion of ropthries. b Moving junction: T. gondii assumes an hourglass shape. c Secretion of dense granules inside the parasitophorous vacuole. d Division, formation of the intravacuolar network, accumulation of acidocalcisomes (green) in the residual body. e Rosette of parasites. f Individualization and egress of parasites.Additional file 18: Figure S18. Cystogenesis. a Invasion. b establishment of parasitophorous vacuole. c Division. d Bradyzoite secretion. e, f Cyst wall thickens, bradyzoites continue to divide. g The cyst inside the host cell.Additional file 19: Figure S19. Tissue cyst in the brain of a mouse. Bradyzoites (purple) surrounded by a thick cyst wall (yellow). Blood vessel (red).Additional file 20: Figure S20. Tissue cyst. A cystwall (arrowhead). Amylopectin granules (asterisk), granular matrix (black star).Additional file: Video S1. Part 1- Life cycle of T. gondii in the feline host.Additional file: Video S2. Part 2- Life cycle of T. gondii in the human intermediate host.Additional file 23. Slide show of T. gondii biological cycle, developmental stages and main organelles."} +{"text": "Bioinformatics (2019) doi: 10.1093/bioinformatics/btz828Correction text: The first version of this article placed the index of summation after, rather than below, the sum symbol in Equations 2, 3, and 4. This has now been corrected online. The publisher apologises for the error."} +{"text": "British Journal of Cancer (2014) 111, 1293\u20131304; 10.1038/bjc.2014.410, published online 22 July 2014Correction to: Since the publication of this paper, the authors have been alerted by a reader to a duplication of the NTS non-treated and treated bands appearing in Fig."} +{"text": "In the manuscript \u201cIn time: The value and global implications of newborn screening for severe combined immunodeficiency\u201d, DOI: 10.1590/1984-0462/;2018;36;4;00020, published in the Rev Paul Pediatr. 2018;36(4):388-397:Page 388:Where it reads:Cristina MeehanaJolan WalterIt should read:Cristina A. MeehanJolan E. WalterPage 389:Page 389: Where it reads:It should read:Page 390, first column:Where it reads:It should read:Page 390, second column:Where it reads:11,29,30However, since the implementation of SCID NBS depends on state legislatures, the implementation time is variable across the United States. Since the first pilot program began in Wisconsin in 2009, 47 of the 50 states, the District of Columbia and Puerto Rico have sequentially implemented or have committed to implement SCID NBS .It should read:11,29,30Since the first pilot program began in Wisconsin in 2008, all 50 states, the District of Columbia and Puerto Rico have sequentially implemented SCID NBS Where it reads:It should read:Page 394,Where it reads:[...] Analysis of screening of three million newborns for SCID after the initiation of SCID NBS confirmed a higher-than-expected prevalence of 1:58,000, increasing from 1:100,000 in 2009 prior to NBS. [...]It should read:25 [...][...] Analysis of screening of three million newborns for SCID after the initiation of SCID NBS confirmed a higher-than-expected prevalence of 1:58,000, increasing from 1:100,000 in 2008 prior to NBS.Where it reads:We thank and acknowledge Dr. Jane Carver from the University of South Florida, for their assistance in the editing of this document.It should read:We thank and acknowledge Dr. Jane Carver from theUniversity of South Florida for assistance in editing this document.Page 395,Where it reads:30. Jeffrey Modell Foundation. Newborn screening for SCID. Update on the implementation of newborn screening for SCID in the United States [Internet]. August 2018 . Available at: http://www.info4pi.org/ town-hall/newborn-screeningIt should read:30. Adapted from Jeffrey Modell Foundation. Newborn screening for SCID. Update on the implementation of newborn screening for SCID in the United States [Internet]. December 2018 . Available at: http://www.info4pi.org/ town-hall/newborn-screening."} +{"text": "Cyanoacrylate alone or in combination with other interventions, can be used to achieve variable rates of success in preventing rebleeding. Our study aims to assess the pooled risk of gastric and esophageal varices rebleeding after an initial treatment with cyanoacrylate alone and/or in combination with other treatments, by a systematic review of the literature and pooled analysis.STATA Version 15 which was also used to generate forest plots for pooled analysis. The random or fixed effect model was applied depending on the heterogeneity (I2).PubMed, EMBASE, SCOPUS, and the Cochrane library were searched for studies that reported the risk of rebleeding during the follow-up period after treatment of gastric or esophageal varices with either cyanoacrylate alone or in combination with other treatments. Standard error, upper and lower confidence intervals at 95% confidence interval for the risk were obtained using A total of 39 studies were found to report treatment of either gastric or esophageal varices with either cyanoacrylate alone or in combination with other treatments. When gastric varices are treated with cyanoacrylate alone, the risk of rebleeding during the follow-up period is 0.15. When combined with lipiodol; polidocanol or sclerotherapy the rebleeding risks are 0.13 (CI:0.03\u20130.22), 0.10(CI:0.02\u20130.19), and 0.10(CI:0.05\u20130.18), respectively. When combined with percutaneous transhepatic variceal embolization; percutaneous transhepatic variceal embolization; endoscopic ultrasound guided coils; or with ethanolamine, the rebleeding risk are 0.10(CI:0.03\u20130.17), 0.10(CI:0.03\u20130.17), 0.07(CI:0.03\u20130.11) and 0.08(CI:0.02\u20130.14), respectively.When esophageal varices are treated with cyanoacrylate alone, the risk of rebleeding is 0.29(CI:0.11\u20130.47). When combined with percutaneous transhepatic variceal embolization; sclerotherapy; or band ligation, the risks of rebleeding are 0.16(CI:0.10\u20130.22), 0.12(CI:0.04\u20130.20) and 0.10(CI:0.04\u20130.24), respectively. When combined with a transjugular intrahepatic portosystemic shunt; or ethanolamine, the risks of rebleeding are 0.06(CI: \u2212\u20090.01-0.12) and 0.02 (CI: \u2212\u20090.02-0.05), respectively.In treating both gastric and esophageal varices, cyanoacrylate produces better results in terms of lower risk of rebleeding when combined with other treatments than when used alone. The combination of cyanoacrylate with ethanolamine or with endoscopic ultrasound guided coils produces the lowest risk of rebleeding in esophageal and gastric varices, respectively. We call upon randomized trials to test these hypotheses. Liver cirrhosis is the leading cause of portal hypertension which in turn, leads to portal hypertension and gastrointestinal varices. Up to 17% of liver cirrhosis patients will develop esophageal varices, while 15% will develop gastric varices. Up to 30%, gastroesophageal varices will bleed within 2\u2009years .From older literature, half of the variceal hemorrhages would stop spontaneously however, the risk of rebleeding and mortality increases significantly . CurrentAlso known as \u201ctissue glue\u201d, tissue adhesives were approved by the United States of America\u2019s Food and Drug Authority in 1998, however, there have been previous studies reporting their use as back as the year 1981 , cyanoacCyanoacrylate can be used alone or in combination with other interventions, to achieve variable rates of successes in hemostasis, reducing mortality and prevention of rebleeding. Our study was aimed at assessing the overall risk of gastroesophageal rebleeding after an initial treatment with cyanoacrylate alone and/or in combination with other treatments, by a systematic review of literature and pooled analysis.The current study involved participants with bleeding gastroesophageal varices who underwent hemostasis by cyanoacrylate injection alone or in combination with other treatments. Observational and interventional studies reporting the risk of rebleeding after hemostasis treatment were included. Extending the external validity, eligible English published literature from across the world were included.Four online databases, namely PubMed, EMBASE, SCOPUS, and the Cochrane library were systematically searched with no time range specified. Secondary referencing of eligible studies extended the search scope. The last search was conducted on 4th March 2020.EndNote X9 which kept track of references.Advanced search tools employing MeSH and keywords, were utilized in all three online databases. Using PubMed, advanced search was done as; (cyanoacrylate [MeSH Terms]) AND endoscopic hemostasis [MeSH Terms]) AND esophageal varices [MeSH Terms]) OR gastric varices [MeSH Terms]) AND reble*. The search was repeated as; (adhes*) AND endosc*) AND varic*) AND reble*. The searches were independently performed by two authors; ZH and JS. Results were exported to Two authors screened titles and abstracts of all articles from online database searches to identify the most relevant articles in line with our study question. The relevant articles were sought for full texts and finally included studies were identified after thorough reading full text articles to assess inclusion and exclusion criteria. This process was done by two authors; ZH and JS with the third author, TL assisting to resolve discrepancies. The search, screening, and study identification process are summarized in Fig.\u00a0Before the data extraction process from full-text articles meeting eligibility criteria for inclusion, assessment for methodological biases was done by using the Joanna Briggs institute meta-analysis of statistics assessment and review instrument. PRISMA of the studies when analyzing the outcomes. The fixed effect model was used when I2 was less than 50% and the random effect model was used when I2 was more than 50% indicating significant heterogeneity.The risk of rebleeding was calculated dividing the number of patients rebleeding during the follow-up period after endoscopic hemostasis by the total number of patients that initially underwent the endoscopic hemostasis procedure. The denominator did not include patients lost during the follow up. Standard error, upper and lower confidence intervals for the risk, were obtained from the \u201cgenerate command\u201d in computer software Participants were considered to have been correctly diagnosed with upper gastrointestinal bleeding due to gastric or esophageal varices, and not due to other causes such as Mallory-Weiss tear or gastritis. Despite the country under which treatment was given, all patients were considered to have received standard care.Webb et al. (1981) did. (2003) and Smitet al. 20 [14] uti. (2004) and Zhan. (2007) used KorTable\u00a0A total of 39 studies reported 3630 who had either gastric or esophageal variceal and underwent hemostasis with cyanoacrylate alone or in combination with other treatments. A total of 497 had gastric or esophageal recurrent bleeding episodes during the follow-up period.Figure\u00a02 of 99.7%, p-Value<\u20090.05. This led us to conduct sensitivity analysis, eliminating peculiar studies from the analysis. Figure\u00a0Ramond et al. (1989) [Soga et al. (2010) [D\u2019Imperio et al. (1996) [Omar et al. (1998) [Noophun et al. (2005) [Rivet et al. (2009) [Cheng et al. (2010) [Binmoellar et al. (2011) [Tantau et al. (2013) [Kind et al. (2000) [Tan et al. (2006) [Procaccini et al. (2009) [Choudhuri et al. (2010) [Mishra et al. (2010) [Liao et al. (2013) [Singh et al. (2016) [Cheng et al. (2007) [Kuo et al. (2007) [Huo et al. (2009) [Kang et al. (2011) [Al-Baward et al. (2016) [Xiaoqing et al. (2019) [Evrad et al. (2003) [Hong et al. (2009) [Soga et al. (2010) [Liu et al. (2019) [Sigh et al. (2016), Procaccini et al. (2009), and Kind et al. (2000) were excluded for distinct follow-up times. Each of the 4 remaining studies had an estimate of 2\u2009years of follow-up. The resulting overall pooled risk was 0.15 with no significant heterogeneity .There was a significant heterogeneity observed with I. 1989) and Soga. 2010) were cas89 and S10 were . (2005) , Rivet e. (2009) , Cheng e. (2010) , Binmoel. (2011) and Tant. (2013) had less. (2000) , Tan et . (2006) , Procacc. (2009) , Choudhu. (2010) , Mishra . (2010) , Liao et. (2013) , Singh e. (2007) , Kuo et . (2007) , Huo et . (2009) , Kang et. (2011) , Al-Bawa. (2016) and Xiao. (2003) , Hong et. (2009) , Soga et. (2010) and Liu . (2019) were exc2 of 0.0%, p-Value\u2009=\u20090.53.7).Figure\u00a0Thakeeb et al. (1995) [Maruyama et al. (2010) [Thakeeb reported 3 (i.e. risk\u2009=\u20090.052) rebleeding events among gastric variceal patients; and one (risk\u2009=\u20090.017) rebleeding events among esophageal varices patients. Maruayama reported 10 (i.e. risk =0.5) rebleeding events among gastric varices patients. Figure\u00a0Two studies illustrated treatment with a combination of cyanoacrylate and ethanolamine; . 1995) and Maru. (2010) . Thakeeb and MarBhat et al. (2016) [Robles-Medranda et al. (2019) [Bhat et al. (2016) reported 10 rebleeding events out of 125 gastric varices patients who were followed-up. This corresponds to the risk of 0.08 . Robles-Medranda et al. (2019) reported 1 rebleeding event out of 27 gastric varices patients, which corresponds to the risk of 0.04 . Figure\u00a0Two studies illustrated treatment with a combination of cyanoacrylate and coils guided by endoscopic ultrasound; . 2016) and Robl. (2019) . Bhat et and RobZhang et al. (2007) [Zhang et al. (2008) [Tian et al. (2011) [Three studies illustrated treatment with a combination of cyanoacrylate and percutaneous transhepatic variceal embolization in gastroesophageal varices; . 2007) and Zhan. (2008) involved. (2011) involved and ZhaFeretis et al. (1995) [Dhiman et al.(2002) [Two studies assessed the efficacy of a combination of cyanoacrylate and sclerotherapy in the treatment of gastroesophageal varices. In one study, . (1995) comparedl.(2002) assessedShi et al. (2014) [p-Value of 0.04. In another study, Ma et al. (2018) [In their study, . (2014) compared. (2018) combinedDai et al. (2017) [Zeng et al. (2017) [. (2017) compared. (2017) comparedTable\u00a0Through decades-long progressive improvements in the treatment of gastroesophageal varices, cyanoacrylate has evolved to be one of the favored first lines of treatment. The current study was aimed at utilizing a systematic review of literature and pooled analysis to assess the overall risk of gastroesophageal rebleeding after an initial treatment with cyanoacrylate alone and/or in combination with other treatments.Hou et al. (2009) [Following the treatment of gastric varices with cyanoacrylate alone, 25 studies demonstrated different risks of rebleeding from the minimum of 0.04 to a maximum of 0.99 in another study, with the overall pooled risk of 0.30 . However, after getting rid of peculiar studies that increased heterogeneity, the resulting overall pooled risk was 0.15 . This risk of rebleeding coincides with that previously reported by . (2009) but diffRivet et al. (2009) [Evrad et al. (2003) [Esophageal varices treated with cyanoacrylate alone showed the risk of rebleeding ranging from 0.25 to 0.38 in different studies with the pooled overall risk of 0.29 . Following a fewer number of studies, a meta regression could not be conducted. However, authors believe that the reason for the differences between studies to be due to different methodological approaches between the studies as . (2009) followed. (2003) . The autThakeb et al. (1995) but differs from Maruyama who reported a higher risk of 0.5. The difference is accounted for by fewer sample size by Maruyama. On the other hand, when the combination is used to treat esophageal varices the risk of rebleeding is 0.017. From an otherwise weak basis, we hypothesis that esophageal varices in contrast to gastric varices, respond better to the combination of cyanoacrylate and ethanolamine, in terms of lower risk of rebleeding. We call upon clinical randomized clinical trials to test this hypothesis.When cyanoacrylate is combined with ethanolamine in the treatment of gastric varices the pooled risk of rebleeding after treatment is 0.08 . The result aligns with that reported by Bhat et al. (2016) [Robles-Medranda et al. (2019) [From our findings, when cyanoacrylate is combined with endoscopic ultrasound guided coils to treat gastric varices the pooled risk of rebleeding is 0.07. This finding is more or less similar to that reported by . (2016) but is h. (2019) . The reaZhang et al. (2007) [Tian et al. (2011) [When esophageal varices are treated with a combination of cyanoacrylate and percutaneous transhepatic variceal embolization the pooled risk of rebleeding is 0.16. This is coinciding with findings previously reported by . (2011) when theThe risk of rebleeding in gastric varices treated with cyanoacrylate with sclerotherapy was lower by 0.02 from that of esophageal varices treated with the same combination. The difference could partly be due to more or less the same number of sample sizes among the two studies descriptively analyzed. In combination with other treatments such as transjugular intrahepatic portosystemic shunt and balloon-occluded retrograde transvenous obliteration, it is evident that cyanoacrylate improves the efficacy of the treatment of gastroesophageal varices in terms of lowering rebleeding risk.Our study search was limited to English published literature; involved pooling of studies with different sample sizes, different study designs, and different follow-up durations. As demonstrated by Child-Pugh or the model for end-stage liver disease (MELD) classifications, different studies involved participants with different extents of liver damage/cirrhosis. Despite a few studies involving either emergent or electThese were thought to introduce heterogeneity in the pooled analysis. However, authors appraised eligible studies; performed sensitivity analyses, meta-regression, study exclusion, and used random effect models to deal with high heterogeneity among pooled studies. We also utilized PRISMA tools to minimize reporting biases.We call upon robust randomized studies taking into account biases encountered in our study and adequately matching participants by the extent of liver damage/cirrhosis; treatment urgency whether elective or emergency; lesion location and follow-up duration.In treating both gastric and esophageal varices, cyanoacrylate produces better results in terms of lower risk of rebleeding when combined with other treatments than when used alone. The combination of cyanoacrylate with ethanolamine or with endoscopic ultrasound guided coils produces the lowest risk of rebleeding in esophageal and gastric varices, respectively. We call upon randomized trials to test these hypotheses."} +{"text": "Nature Communications 10.1038/ncomms8769, published online 17 July 2015.Correction to: In this Article, there is an error in Fig."} +{"text": "Regenerative Biomaterials 2020doi: 10.1093/rb/rbaa022, Front. Bioeng. Biotechnol. 2017; 5:1-7.\u201d. This error has now been corrected, and the authors apologize for this oversight.In the originally published version of this article, the caption for Figure 8. did not credit the original source of the image: \u201cGrosso A, Burger MG, Lunger A et al. It takes two to tango: coupling of angiogenesis and osteogenesis for bone regeneration."} +{"text": "Chen should be included in the author byline. Joe W. Chen should be listed as the second author, and his affiliation is 1: UC Berkeley-UCSF Graduate Program in Bioengineering, University of California\u2013Berkeley, Berkeley, California, United States of America. The contributions of this author are as follows: Methodology. The correct citation is: Lu B, Chen JW, Maharbiz MM (2019) Ion concentration polarization (ICP) of proteins at silicon micropillar nanogaps. PLoS ONE 14(11): e0223732. https://osf.io/za9xc.The Data Availability statement is incorrect. The raw data underlying Figs 3 and 6 are not provided in the published paper and its Supporting Information files. The authors have provided the raw data via OSF:"} +{"text": "Correction to: Virol J 9, 163 (2012)http://www.virologyj.com/content/9/1/163Following publication of the original article , the autIn Fig. In Fig. In addition, we also repeated the experiments associated with Fig. We declare that the correction does not change the results or conclusions of this paper."} +{"text": "This article has been corrected: The authors requested to replace Figures 1, 2 and 5. The authors uploaded the wrong images by mistake during the preparation of the article. These corrections do not change any of the conclusions of the publication in any means. The corrected Figures 1, 2 and 5 are provided below. . https://doi.org/10.18632/aging.102552Original article: Aging. 2019; 11:11520\u201311540."} +{"text": "National Black HIV/AIDS Awareness Day (NBHAAD) is observed each year on February 7 to highlight the continuing disproportionate impact of human immunodeficiency virus (HIV) infection and acquired immunodeficiency syndrome (AIDS) on the U.S. black or African American (black) population. During 2018, blacks represented 13% of the U.S. population but accounted for 43% of all newly diagnosed HIV infections , was proposed. The plan calls for intensified efforts to diagnose, treat, prevent, and respond to HIV infections in the United States, with an overall goal of reducing new HIV infections by \u226590% by 2030 (MMWR issue presents data on CDC-funded HIV testing and outcomes among blacks who were tested in jurisdictions that are the initial focus of EHE. In these jurisdictions during 2017, blacks accounted for 43.2% of CDC-funded tests and 49.1% of newly diagnosed HIV infections (https://www.cdc.gov/hiv/group/racialethnic/africanamericans. Information about NBHAAD is available at https://www.cdc.gov/hiv/library/awareness/nbhaad.html. A study reported in this"} +{"text": "Int J Epidemiol 2017; 46:348\u201355. doi: https://doi.org/10.1093/ije/dyw098First published online: 8 June 2016, The originally published version of this article contained an algebraic definition of theregression model for interrupted time series (ITS) that could lead to erroneousinterpretations of the estimated parameters. This model was presented in the equation atpage 351, right column, and the following text. We provide here a more accuratedefinition.The correct equation should have been: The following text below the equation at page 351 (right column) and page 352 (leftcolumn), should read:\u201cwhere The R code in Finally, the dyaa118_Supplementary_DataClick here for additional data file."} +{"text": "The correct names are: Asghari Bano and M.D. Ali Babar. The correct citation is: Khan N, Bano A, Babar MDA (2020) Impacts of plant growth promoters and plant growth regulators on rainfed agriculture. PLoS ONE 15(4): e0231426. The word \u201cBiosciences\u201d is misspelled in the second affiliation. The correct affiliation is: Department of Biosciences, University of Wah, Wah Cantt, Pakistan.The publisher apologizes for the errors."} +{"text": "A third of Medicare beneficiaries are enrolled in Medicare Advantage (MA); however, little is known about MA beneficiaries diagnosed with Alzheimer\u2019s disease and related dementias (AD/ADRD). MA plans have incentives that may influence the type of beneficiaries who enroll/disenroll from plans and the documentation of diagnoses. We calculated the prevalence of diagnosed AD/ADRD in 2014 and 2016 in three MA plans representing ~30% of the MA market. We identified beneficiaries \u226565 years of age enrolled in the MA plans in 2014 and 2016. Among eligible beneficiaries, we identified individuals with AD/ADRD using ICD-9 (2014) and ICD-10 (2016) codes included in the Medicare Chronic Conditions Warehouse algorithms for AD/ADRD. We determined the age and sex of beneficiaries diagnosed with AD/ADRD, and whether they disenrolled from the MA plan for any reason within 364 days from the date they were first identified has having AD/ADRD . In 2014 and 2016 the prevalence of AD/ADRD diagnoses was 5.7% and 6.5%, respectively. In 2016, AD/ADRD beneficiaries were on average 82.4 (SD=7.3) years of age, 61.8% female, and had multiple comorbidities. By 364 days post-index, 32% of beneficiaries with diagnosed AD/ADRD had disenrolled from their plan. The characteristics of 2014 beneficiaries with diagnosed AD/ADRD were similar to their 2016 counterparts. In conclusion, MA beneficiaries with AD/ADRD are predominately female, have multimorbidity, and the age-stratified prevalence of AD/ADRD diagnoses is lower than rates reported in traditional Medicare."} +{"text": "Figure 1 was our self-made drawing modified and adapted from:In the original article, there was a mistake in Figure 1 as published. Netter's Surgical Anatomy and Approaches. Philadelphia, PA: Elsevier Saunders (2014).Delaney CP. Glob Surg. (2018) 4:3. doi: 10.15761/GOS.1000196Radjindrin A, Shanmugam V. Does lateral pelvic lymph node matters in rectal cancer. The mistake made was not to mention in the legend of Figure 1 and references that our self-made drawing was adapted from the sources mentioned previously. The authors apologize for any inconvenience, and under no circumstance was it the authors' intention to neglect to declare use of sources. The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.The references to Figure 1 have been updated in the original article.Netter's Surgical Anatomy and Approaches. Philadelphia, PA: Elsevier Saunders (2014).Delaney CP. Glob Surg. (2018) 4:3. doi: 10.15761/GOS.1000196Radjindrin A, Shanmugam V. Does lateral pelvic lymph node matters in rectal cancer."} +{"text": "The following information is missing from the Funding statement: Nicholas D. Juleff was funded by Wellcome Trust intermediate-level research fellowship 091726/Z/10/Z.In the Population structure section of the Materials and Methods, a reference is omitted from the first sentence of the first paragraph.The correct sentence is: SNV variants in the inoculum were called by SiNPle using an approximation of the Bayesian calling algorithm in Snape-pooled [41] suitable for high coverage.The reference is: Ferretti L, Tennakoon C, Silesian A, Freimanis G, Ribeca P. SiNPle: Fast and Sensitive Variant Calling for Deep Sequencing Data. Genes 2019, 10, 561."} +{"text": "JACMP has issued awards to several manuscripts deemed by the Board of Editors to be worthy of the designation \u201cBest Papers.\u201d These awards are selected by a committee of the Board of Editors and are supported by the American Association of Physicists in Medicine (AAPM). I offer my sincere congratulations to the authors of these outstanding contributions to the body of knowledge that comprises clinical medical physics.For the past several years, the http://www.jacmp.org/index.php/jacmp/article/view/4014/2902Yamak D, Pavlicek W, Boltz T, Panse P, Akay M. Coronary calcium quantification using contrast\u2010enhanced dual\u2010energy computed tomography scans. J Appl Clin Med Phys. 2013;14(3). http://www.jacmp.org/index.php/jacmp/article/view/4262/2885Shang Q, Sheplan Olsen L, Stephans K, Tendulkar R, Xia P. Prostate rotation detected from implanted markers can affect dose coverage and cannot be simply dismissed. J Appl Clin Med Phys. 2013;14(3). http://www.jacmp.org/index.php/jacmp/article/view/4062/2838Zhang A, Wen N, Nurushev T, Burmeister J, Chetty I. Comprehensive evaluation and clinical implementation of commercially available Monte Carlo dose calculation algorithm. J Appl Clin Med Phys. 2013;14(2). It is the intent to have one \u201cBest Paper\u201d award winner, the Editor\u2010in\u2010Chief Award, invited to the podium during the Awards and Honors Ceremony of the Annual Meeting of the AAPM to accept the award. http://www.jacmp.org/index.php/jacmp/article/view/4077/2780Beyer G. Commissioning measurements for photon beam data on three TrueBeam linear accelerators, and comparison with Trilogy and Clinac 2100 linear accelerators. J Appl Clin Med Phys. 2013;14(1). By listing the Best Papers of 2013, I would like to honor the award winners for their contributions to the clinical practice of medical physics, and thank the AAPM for its financial support.Michael D. Mills, PhDEditor\u2010in\u2010Chief"} +{"text": "Phys Ther. (2019) 83:237\u201352.\u201dIn the original article, the reference for Marsh et al. [referenParkinsons Dis. (2019) 2019:2478980. doi: 10.1155/2019/2478980.It should be Marsh R, Cole MH, Dissanayaka NNW, Au TR, Clewett S, O'Sullivan JD, et al. The CuePed trial: how does environmental complexity impact cue effectiveness? A comparison of tonic and phasic visual cueing in simple and complex environments in a Parkinson's disease population with freezing of gait. The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated."} +{"text": "The erratum adds funding information missing from the Acknowledgments section of the originally published version of the article. J. Med. Imag.7(4), 042804 (2020) doi: 10.1117/1.JMI.7.4.042804] was originally published in Vol.\u00a07, Issue 4 of the Journal of Medical Imaging (JMI) on 22 April 2020 with information missing from the Acknowledgments section. Specifically, the authors mistakenly omitted acknowledgment of financial support from FFG-FWO, Grant No.\u00a0861552. The article was republished with the correct, complete funding information on 21 December 2020.This article ["} +{"text": "The name should be indexed as Mbabazi PS. The correct citation is: Hotez PJ, Harrison W, Fenwick A, Bustinduy AL, Ducker C, Mbabazi PS, et al. (2019) Female genital schistosomiasis and HIV/AIDS: Reversing the neglect of girls and women. PLoS Negl Trop Dis 13(4): e0007025. There is an error in the article XML causing the eighth author\u2019s name to be indexed incorrectly. The name should be indexed as Kjetland EF."} +{"text": "Article title: The integration of sexual and reproductive health and rights into universal health coverage: a FIGO perspectiveAuthors: Faysal El KakJournal: Sexual and Reproductive Health MattersBibliometrics: Volume 28, Number 1, article 1829796DOI:https://doi.org/10.1080/26410397.2020.1829796This article was originally published in issue 28.1. The article belongs instead to issue 28.2, a themed issue on \u201cUniversal Health Coverage: Sexual and Reproductive Rights in Focus.\u201dThe article has now been republished in the correct issue, 28.2."} +{"text": "Correction to: Trials (2019) 20:725https://doi.org/10.1186/s13063-019-3770-0Originally published phraseAfter publication of our article the auth2018, with 120 patients randomized. Treatment with TMP finished in October 2019. We disposal the data at present. The current protocol version is 2.0, dated 28 September 2018.Correct phraseRecruitment started in September 2018 and is planned to end in October 2019, with 120 patients randomized. Treatment with TMP will be finished in March 2020. We disposal the data at present. The current protocol version is 2.0, dated 28 September 2018.Recruitment started in September 2018 and is planned to end in October"} +{"text": "Correction to: Experimental & Molecular Medicine10.3858/emm.2011.43.8.051, published online 16 June 2011After online publication of this article, the authors noticed an error in the Fig. The authors apologize for any inconvenience caused."} +{"text": "Keynote at the 20th European Conference on Eye Movement Research (ECEM) in Alicante, 22.8.2019Video stream:https://vimeo.com/361729502"} +{"text": "Keynote by Enkelejda Kasneci at the 20th European Conference on Eye Movement Research (ECEM) in Alicante, 18.8.2019,Video stream of presentation: https://vimeo.com/356314274"} +{"text": "The correct name is: Salim Benkhedda. The correct citation is: Alhabib KF, Gamra H, Almahmeed W, Hammoudeh A, Benkhedda S, Al Jarallah M, et al. (2020) Acute myocardial infarction and acute heart failure in the Middle East and North Africa: Study design and pilot phase study results from the PEACE MENA registry. PLoS ONE 15(7): e0236292. There is also an error in affiliation 5 for author Salim Benkhedda. The correct affiliation 5 is: Cardiology Oncology Collaborative Research Group, Faculty of Medicine, Benkhedda Benyoucef University, Algeria.A research assistant\u2019s name is also spelled incorrectly in the Acknowledgements section. The correct name is: Dahlia Djermane."} +{"text": "Correction: Journal of Neuroinfammation (2022) 19:38\u00a0https://doi.org/10.1186/s12974-022-02397-yThe part of the Data availability statement and Funding information section are missing in the original publication. The missing information are given below.Funding was provided by University of Rochester University Research Award: Targeting AXL in Alzheimer\u2019s disease, Grant Nos. F30AG061939, NIH R01 AG030149, NIH R56 AG066397\u00a0from the National Institutes of Health, and the National Institute on Drug Abuse Intramural Research Program.https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE171195.All data generated or analyzed during this study are available from the corresponding author on reasonable request. Viral vectors used in this work can be obtained from Addgene.org, plasmids 202,540 and 202,541. RNAseq datasets can be found at"} +{"text": "MGT110 4L ORF observed in the MAD/01/1998, MAU/01/2007 and TAN/01/2011 isolates. Furthermore, MOZ/01/2005 and Georgia 2007/1 only differ by a single synonymous SNP in the EP402R ORF, confirming that the closest link to Georgia 2007/1 is a virus that was circulating in Mozambique in 2005.Since the initial report of African swine fever (ASF) in Kenya in 1921, the disease has predominantly been confined to Africa. However, in 2007, an ASF genotype II virus of unknown provenance was introduced to Georgia. This was followed by its rampant spread to 73 countries, and the disease is now a global threat to pig production, with limited effective treatment and vaccine options. Here, we investigate the origin of Georgia 2007/1 through genome sequencing of three viruses from outbreaks that predated the genotype II introduction to the Caucasus, namely Madagascar (MAD/01/1998), Mozambique (MOZ/01/2005), and Mauritius (MAU/01/2007). In addition, genome sequences were generated for viruses from East African countries historically affected by genotype II and Tanzania (TAN/01/2011)) and newly invaded southern African countries (Zimbabwe (ZIM/2015) and South Africa (RSA/08/2019). Phylogenomic analyses revealed that MOZ/01/2005, MAL/04/2011, ZIM/2015 and RSA/08/2019 share a recent common ancestor with Georgia 2007/1 and that none contain the large (~550 bp) deletion in the Asfivirus within the Asfarviridae family, and it is currently the only known double-stranded deoxyribonucleic acid (dsDNA) arthropod-borne virus (arbovirus) [African swine fever (ASF), caused by African swine fever virus (ASFV), is a haemorrhagic fever of domestic pigs with mortality rates approaching 100% . This unbovirus) ,3. The Abovirus) ,5. The gbovirus) ,6.p72) gene encoding virus protein 72 (VP72) [p72 and central variable region (CVR)/B602L data from Mozambique and other East African countries, and for outbreak strains from Madagascar provided the first indication that the origin of Georgia 2007/1 was from the eastern seaboard of Africa [p72 and CVR, that are used for genotype assignment and intra-genotypic resolution, respectively [p72 region of genotype II viruses from diverse African countries confirmed that these ASFVs are homogeneous and share common ancestry [ASFVs group within 24 genotypes (I\u2013XXIV) based on the partial sequences of the C-terminal region of the B646L p2 gene en2 (VP72) ,9,10,11.2 (VP72) ,13. The f Africa . A subseancestry ,15.K177R gene sequence [The first report of ASFV genotype II in Georgia in 2007 marked the start of a major incursion into Eastern Europe, Russia, and later Asia, Western Europe and the Dominican Republic ,18,19,20sequence ,23. Diffhttps://www.sadc.int/member-states (accessed on 31 July 2023)) affected by genotype II. This was achieved by generating complete ASFV genome sequences for three viruses from Mozambique, Madagascar and Mauritius, which predate the 2007 excursion from Africa to Georgia, and four additional viruses (2011\u20132019) from the two SADC countries historically affected by genotype II and the two newly-affected countries .The aim of this study was to investigate the origin of Georgia 2007/1 and the evolutionary relationships of south-east African outbreak strains by sequencing ASFVs from seven southern African development community (SADC) countries Reference Laboratory for ASF at the Transboundary Animal Diseases Laboratory of the Agricultural Research Council\u2014Onderstepoort Veterinary Research (ARC-OVR) in South Africa. These isolates were selected based on temporal and geographical distribution to ensure the representation of seven of the eight SADC countries in which genotype II outbreaks have been reported/recorded. The viruses span a 21-year period using prescribed reaction conditions.The viruses were cultured in primary bone marrow cells (PBMCs), harvested, and purified using a sucrose gradient purification method, as previously described ,26. Virawww.clcbio.com (accessed on 31 July 2023)). Between 41,566,834 and 131,025,188 reads were obtained for each of the samples. Low quality reads and adaptor sequences were removed prior to mapping against the reference sequence Georgia 2007/1 (FR682468.2) and extracting the consensus sequence. De novo assembly was also performed with the reads, and the resulting consensus sequence was compared to the one generated from the mapping analysis. Deletions, insertions and unique point mutations were verified through PCR-Sanger sequencing, with the major deletion described in The viral DNA was used to construct a sequencing library using the Truseq Nano DNA library preparation kit according to manufacturers\u2019 instructions. The libraries were quality controlled using the Qubit fluorometer and PerkinElmer LabChip instruments. Sequencing was performed using an Illumina HiSeq 2500 instrument using the V4 SBS chemistry, generating 2\u00d7 125-bp paired-end reads for each of the samples. Data generated for all seven viruses were analysed using CLC Genomics workbench v9.5.2 and for newly advocated gene targets .Complete genome sequences from genotype II ASFs representing Africa, Georgia 2007/1 (FR682468.2) and representative genomes of the three geographical clades previously described in Europe, Russia and Asia were obtained from GenBank and used to create an alignment with the newly generated consensus sequences . The alihttps://inqababiotec.co.za/ accessed on 31 July 2023) for Sanger sequencing.An approximately 550 bp deletion was detected in MAD/01/1998, MAU/01/2007 and TAN/01/2011 in relation to Georgia 2007/1. In order to validate the deletion, primers were designed to bind to regions flanking the deletion. Primers ASFV6980-F: 5\u2032-CAT ACA GTG TTC CAT GGG ATA-3\u2032 and ASFV8820-R: 5\u2032-GGA CAA CTT CAT CCA ACG G- 3\u2032 were each used at 0.5 \u00b5M, with 2 \u00b5L DNA template in a 25 \u03bcL reaction containing 12.5 \u03bcL GoTaq green master mix . Amplification was performed using a T100 Thermal cycler at an annealing temperature of 55 \u00b0C. The PCR amplicons were run against the O\u2019GeneRuler DNA Ladder Mix and visualised on a 1% agarose gel stained with ethidium bromide prior to submission to Inqaba Biotech, Pretoria, South Africa . Amplification using primers flanking the deleted region, resulted in shorter amplicons for MAU/01/2007, MAD/01/1998, and TAN/01/2011, compared to the other four viruses, thus confirming the ~550 bp deletion .EP402R (I118I) was identified between MOZ/01/2005 and Georgia 2007/1 and the African isolates. A single synonymous SNP in the open reading frame (ORF) a 2007/1 . This SNa 2007/1 . Additioa 2007/1 , 40 synoa 2007/1 and 6 sia 2007/1 , were oba 2007/1 . In conta 2007/1 . Four sya 2007/1 .The polymorphisms were subsequently evaluated for their potential role as markers to delineate the isolates from Africa. Three of the SNPs (MGF360-13L: G177D), (A859L: A427E) and (O61R: I181I) were discriminated between a group consisting of Georgia 2007/1, MOZ/01/2005 and RSA/08/2019 compared to the remaining sequences from Africa . AdditioAn analysis of the intergenic regions (IGRs) indicated two SNPs and two indels in the sequence of TAN/01/2011, which cluster this isolate with previously determined sequences from Tanzania and Malawi . InteresSince the first introduction of genotype II ASFV to Georgia in 2007, the disease has spread across the Caucasus, Europe, Russia, Asia and the Dominican Republic . The comThe complete genome analyses performed in our study indicate that isolates from Mozambique (2005) and South Africa 2019) are more closely related to the Georgia 2007 virus than the latter is to the viruses currently circulating in Europe and Asia . The ful are moreThe viruses from South Africa (RSA/08/2019), Zimbabwe (ZIM/2015) and Malawi (MAL/04/2011), shared a recent common ancestor with Georgia 2007/1 and Mozambique (MOZ/01/2005); the percentage sequence identity ranged from 99.68% to 99.99% amongst the five ASFVs. In contrast to these closely related ASFVs, the sequences of isolates MAD/01/1998, MAU/01/2007 and TAN/01/2011 shared a recent common ancestor with the previously published viruses from Tanzania and Malawi (Tanzania/Rukwa/17/1 LR813622), MAL/19/Karonga (MW856068), TAN/17/Mbagala (ON409982), TAN/17/Kibaha (ON409979) and TAN/20/Mogorogo (ON409983). The divergence observed amongst the genotype II ASFVs from Africa further indicates that the Madagascar/2007 outbreak might have been related to the previous 1998 outbreak on the island, rather than a new introduction related to MOZ/01/2005 and subsequently Georgia 2007/1 ASFVs. A deletion of ~550 bp was detected in the genotype II genome sequences for Madagascar (MAD/01/1998), Mauritius (MAU/01/2007) and Tanzania (TAN/01/2011), which corresponds to a previously described deletion in the 5\u2032 region of Tanzania/Rukwa/17/1, MAL/19/Karonga, TAN/17/Mbagala, TAN/17/Kibaha and TAN/20/Mogorogo ,22,23., MAL/19/Additionally, a significant difference between MAL/04/2011 (this study) and MAL/19/Karonga was obseA comparison of the newly sequenced ASFVs to Georgia 2007/1, indicated 18 SNPs in the IGRs , and 51 Complete genome sequence analysis remains the gold standard in identifying novel SNPs and determining the evolution and molecular epidemiology of a virus population. Unfortunately, this is time-consuming and expensive, resulting in the study of individual markers to ascertain the spread and epidemiology of ASFVs during outbreaks. Various markers have been described to delineate the genotype II ASFVs circulating in Europe and Asia ,30. ThesEP402R between MOZ/01/2005 and Georgia 2007/1, it is suggested that a possible link exists between the founder ASFV in Georgia 2007/1 and viruses circulating concurrently in Mozambique. The study proved that the Georgia 2007/1 virus shared common similarities with RSA/08/2019 (South Africa), ZIM/2015 (Zimbabwe), MAL/04/2011 and MOZ/01/2005 (Mozambique). With regard to the evolutionary relationship between Georgia 2007/1 and ASFVs from this region, the study found a major divergence in viruses before and after Georgia 2007/1, highlighting the presence of ASFV genotype II clusters in Africa. These results underscore the need for sequencing additional genotype II strains from Africa with particular focus on earlier isolates in order to determine how diverse this genotype is.In conclusion, this study provides novel epidemiological findings on the origin of the virus introduced into Georgia in 2007. Due to a single synonymous SNP in the ORF"} +{"text": "J Clin Invest. 2022;132(18):e161400. https://doi.org/10.1172/JCI161400Original citation: J Clin Invest. 2023;133(11):e172059. https://doi.org/10.1172/JCI172059Citation for this corrigendum: After the publication of this paper, the authors became aware of errors in data analysis due to an issue with the barcoding of samples that caused some samples to be excluded. The authors have repeated all of the analysis and corrected Supplemental Figures 5, 6, and 9. The supplemental file has been updated online.The authors regret the errors."} +{"text": "Given two random variables X and Y, the IB finds the stochastic mapping M of X that encodes the most information about Y, subject to a constraint on the information that M is allowed to retain about X. Despite the popularity of the IB, an accessible implementation of the reference algorithm oriented towards ease of use on empirical data was missing. Embo is optimized for the common case of discrete, low-dimensional data. Embo is fast, provides a standard data-processing pipeline, offers a parallel implementation of key computational steps, and includes reasonable defaults for the method parameters. Embo is broadly applicable to different problem domains, as it can be employed with any dataset consisting in joint observations of two discrete variables. It is available from the Python Package Index (PyPI), Zenodo and GitLab.We present To solve this problem, we seek a third random variable M that solves the following optimization problem:I(\u00b7 : \u00b7) is Shannon\u2019s mutual information . MatpEugenio Piasini, University of Pennsylvania (developer)Alexandre Filipowicz, University of Pennsylvania (developer)Jonathan Levine, University of Pennsylvania (developer)Joshua Gold, University of Pennsylvania (consultant)Archive (1)Name: ZenodoPersistent identifier: 10.5281/zenodo.3625785Licence: GNU General Public License v3.0 or laterPublisher: Eugenio Piasini, Alexandre L. Filipowicz, Jonathan LevineVersion published: 1.1.0Date published: 22/02/2021Archive (2)Name: Python Package Index (PyPI)Persistent identifier:https://pypi.org/project/embo/Licence: GNU General Public License v3.0 or laterPublisher: Eugenio Piasini, Alexandre L. Filipowicz, Jonathan LevineVersion published: 1.1.0Date published: 22/02/2021Code repositoryName: GitlabPersistent identifier:https://gitlab.com/epiasini/emboLicence: GNU General Public License v3.0 or laterDate published: 22/02/2021LanguageEnglish(3)In , Embo haEmbo may be extended in several ways. Possible technical upgrades include improving the software\u2019s performance, for instance by rewriting the Blahut-Arimoto algorithm implementation in C, or by using performance-oriented Python libraries such as Numba or Cython. Features that may be added include the estimation of finite sample bounds for the IB . FinallyThe recommended support channel for Embo is via its GitLab projects, where issues can be reported, and patches and merge requests are welcome. Additionally, the maintainers can be contacted directly at their institutional email addresses."} +{"text": "Article title: Association of the endothelial nitric oxide synthase (eNOS) 4a/b polymorphism with the risk of incident diabetic retinopathy in patients with type 2 diabetes mellitus: a systematic review and updated meta-analysisAuthors: Shi Y., Fan, X., Zhang, K., & Ma, Y.Journal:Annals of MedicineBibliometrics: Volume 55, Number 01, 2226908DOI:http://dx.doi.org/10.1080/07853890.2023.2226908When the above article was first published, some of the overall case/control numbers in Table 3 and Table 4 were incorrect due to mistakes made when summing the case/control numbers from the original studies. This error only affected the case/control summary information presented in Table 3 and Table 4.The correct tables can be found below:"} +{"text": "South African Journal of Communication Disorders, 68(1), a832. https://doi.org/10.4102/sajcd.v68i1.832, there was a mistake in In the published article, Pillay, T., & Pillay, M. (2021). Contextualising clinical reasoning within the clinical swallow evaluation: A scoping review and expert consultation. The original incorrect:The revised and updated:The authors apologise for this error. The correction does not change the significance of study\u2019s findings or overall interpretation of the its results or the scientific conclusions of the article in any way."} +{"text": "This is a peer-review report submitted for the paper \u201cInfluence of Mass Media on Italian Web Users During the COVID-19 Pandemic: Infodemiological Analysis.\u201dThank you for providing me the opportunity of serving as a reviewer for this interesting manuscript . The stu1. The text presents some English errors. I recommend a grammar revision.2. Introduction: Present a broader view on the subject to justify the study objectives. Moreover, avoid introducing methodological issues in this section.3. Introduction: Why do people search for health-related information on the internet? How does the pandemic influence this behavior?4. Introduction: What is the impact of infodemiology studies on public health? Also, what is the importance of infodemiological studies to combat the infodemic?5. Introduction: What is Google Trends and what are its utility/advantages for infodemiological studies? The following studies may be helpful:JMIR Public Health Surveill 2020;6(2):e19702. doi: 10.2196/19702. PMID: 32401211Higgins TS, Wu AW, Sharma D, Illing EA, Rubel K, Ting JY, Snot Force Alliance. Correlations of online search engine trends with coronavirus disease (COVID-19) incidence: infodemiology study. JMIR Public Health Surveill 2020;6(2):e19374. doi: 10.2196/19374. PMID: 32338613Rovetta A, Bhagavathula AS. COVID-19-Related Web Search Behaviors and Infodemic Attitudes in Italy: Infodemiological Study. PLoS One 2017;12(10):e0186059. doi: 10.1371/journal.pone.0186059. PMID: 29049315Lotto M, Aguirre PEA, Rios D, Machado MAAM, Cruvinel AFP, Cruvinel T. Analysis of the interests of Google users on toothache information. JMIR Public Health Surveill 2019;5(2):e13439. doi:10.2196/13439. PMID:31144671Mavragani A, Ochoa G. Google Trends in infodemiology and infoveillance: methodology framework. PLoS One 2014;9(10):e109583. doi: 10.1371/journal.pone.0109583. PMID: 25337815Nuti SV, Wayda B, Ranasinghe I, Wang S, Dreyer RP, Chen SI, Murugiah K. The use of Google Trends in health care research: a systematic review. 6. Introduction: The third aspect proposed for investigation is not adequate. The authors go beyond the main scope by introducing a \u201cfake news\u201d analysis. The authors did not define what \u201cfake news\u201d is, and the analysis made is too superficial for the proposed outcome. I recommend that this step be better planned and analyzed, being described in a future manuscript.Discussion section.7. Introduction: Question R4 has already been widely discussed in the literature and can be removed. If necessary, this aspect can be presented in the 8. Methods: The data collection is confusing and needs to be better described. Why were these platforms chosen? What are the main media in Italy and which ones have been selected? Describe in detail how data were collected on each platform.9. Methods: Does analyzing search results on platforms really show the influence of the media? Why was a qualitative study not proposed?10. Methods: What is the infodemic scale? Present it in detail.11. Methods: Is the normalization of media data on the same scale as Google Trends correct? Since the absolute Google Trends data is blind, it doesn\u2019t seem like a valid comparison to me.12. Methods: What test was done to assess seasonality?13. Results: Avoid discussing the results in advance, as in \u201cEvidence supporting causation.\u201d14. Discussion: Only media influence is discussed about Google Trends data. What other factors might influence the findings? Why do people search for information related to COVID-19 during the pandemic?15. Discussion: Compare the findings with previous literature. Several studies have investigated users\u2019 COVID-19\u2013related interests on Google Trends, including in Italy.16. Discussion: Why are technical terms little used? Where does the Italian population learn about the pandemic? How do eHealth literacy and media literacy influence this process?Practical Implications subsection.17. Discussion: Include a Thank you for reviewing the manuscript based on my comments. In general, the suggestions were satisfactorily answered, and the quality of the paper has improved considerably."} +{"text": "The alaIn vitro and in vivo evaluation [the new therapeutic strategy is tested in vitro/in the laboratory and in vivo to assess its efficacy and safety] Cliunteers) ; (e) Regunteers) ; (f) Cliunteers) ; (g) Monunteers) . It is punteers) , 15. Thiunteers) . By the unteers) , 16. Theunteers) .To date, research on the use of stem cells for ASD is at the clinical trials stage \u201329 and tIn agreement with Price we belieAN: Conceptualization, Supervision, Writing\u2014original draft, Writing\u2014review and editing. AH: Supervision, Writing\u2014original draft, Writing\u2014review and editing. GM: Supervision, Writing\u2014review and editing. GN: Supervision, Writing\u2014review and editing. CL: Supervision, Writing\u2014review and editing."} +{"text": "Biol. Open (2019) 8, bio036244 (doi:10.1242/bio.036244).There was an error published in In The authors apologise to readers for this error, which does not impact the results or conclusions of this paper."} +{"text": "Scientific Reportshttps://doi.org/10.1038/s41598-023-37637-5, published online 25 July 2023Correction to: The Acknowledgements section in the original version of this Article was incomplete. It now reads:The authors extend their appreciation to the Deanship of Scientific Research at King Khalid University for funding this work through a large group Research Project under grant number (R.G.P.2/163/44). Princess Nourah bint Abdulrahman University Researchers Supporting Project number (PNURSP2023R192), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia.The original Article has been corrected."} +{"text": "Among gynecological cancers, cervical cancer is the most common cause of cancer\u2010related death in developing countries. This study analyzes the incidence, mortality, and burden of cervical cancer using the Global Burden of Disease (GBD) 2019 study.The GBD (2019) data on cervical cancer was extracted from the Global Health Data Exchange (GHDx) query tool. Age\u2010standardized rate (ASR) incidence, deaths, lost years of life (YLLs), years of life with disabilities (YLDs), and adjusted years of life with disabilities (DALYs) of cervical cancer in women were extracted. Data were extracted globally for 204 countries and groups based on a socio\u2010demographic index (SDI), World Health Organization (WHO) regions, continents, World Bank regions, and 22 GBD regions.The higher standardized age incidence of cervical cancer is in lower SDI countries, Africa, the African region (According to the WHO), and Sub\u2010Saharan Africa (According to GBD regions). The highest deaths of ASR is in countries with low SDI, low\u2010income group, Africa, the African region , and Sub\u2010Saharan Africa (According to GBD regions). According to SDI classification, the highest DALYs ASR is in low SDI countries, World Bank Low\u2010income countries, African and then American continents, African region, Sub\u2010Saharan Africa, and then Latin America & Caribbean\u2010WB (Based on GBD regions).In 2019, incidence, mortality, and DALYs of cervical cancer mostly affected countries with lower socioeconomic status. Given that cervical cancer is highly preventable, access to screening services and the presence of trained and knowledgeable health care staff can reduce illness, suffering, and death caused by this malignancy. It is recommended to use the national and international potentials to reduce the incidence of this malignancy. Also, early detection and treatment of precancerous lesions have prevented a significant proportion of morbidity and mortality associated with cervical cancer.At present, the development of effective prevention programs requires accurate statistics. Statistics, in addition to showing the current situation, make it necessary to take action to improve the situation, allocate the necessary budget and facilities, make purposeful planning, and finally select the best possible approach.2http://ghdx.healthdata.org/gbd-2019/data-input-sources).In summary, the GBD (2019) data on cervical cancer was extracted from the GHDx query tool. GBD (2019) has systematically and comprehensively estimated 286 causes of death, 369 causes of diseases and injuries, and 87 risk factors for 204 countries and regions. Geographically, the GBD divides the world into 7 main regions and 21 subregions. Detailed information on the data sources used in the present study can be found at GBD 2019 , years lived with disability (YLDs), disability\u2010adjusted life years (DALYs), age\u2010standardized incidence, and mortality rates per 100\u2009000 people in 2019 based on socio\u2010demographic Index (SDI) indicators. These variables also included World Bank income levels, continents, WHO regions, and GBD regions. The Socio\u2010demographic Index (SDI) is a composite indicator of a country's lag\u2010distributed income per capita, average years of schooling, and the fertility rate in females under the age of 25\u2009years.In this study, a description of the mentioned indicators was done separately for each group by crude and age\u2010standardized rates. Age\u2010standardized mortality rates can be used to compare national mortality rates without being affected by differences in age distribution across countries. Without such standardization, it would be difficult to determine if the different mortality rates are due to age or other factors.33.1In 2019, a total of 565\u2009541 new cases of cervical cancer with a confidence level (636435\u2013481\u2009524) were reported in women worldwide, with an incidence of ASR of 13.35 cases per 100\u2009000 people. Statistics show that the lower the SDI index is, the higher age\u2010standardized incidence rates of cervical cancer will be so that the highest standardized age incidence can be found in countries with low SDI and the lowest in countries with high SDI. This rate is equal to 8.91 in high SDI countries and 23.21 in low SDI countries. According to the World Bank classification, the incidence of ASR has the lowest value (9.21) in the high\u2010income countries and the highest value (30.29) in the low\u2010income countries. Among the continents, the highest incidence of ASR is in Africa (24.02) and the lowest is in Europe (10.79). According to the World Health Organization (WHO), the highest standard incidence is in the African region and the lowest is in the Eastern Mediterranean region.Also, in general and based on GBD regions, the highest incidence of ASR is in Sub\u2010Saharan African countries (WB) and then Latin America & Caribbean countries.The highest standardized incidence of cervical cancer has been reported to be in Kiribati (108.8), Palau (66.58), Solomon Islands (57), Guinea (53.61), Lesotho (52.77), Zimbabwe (48.95), Botswana (47.63), Eritrea (44.96), Guinea\u2010Bissau (44.77) and Haiti (44.12).However, the lowest standardized incidence of cervical cancer has been reported to be in Egypt (2.84), Syrian Arab Republic (3.25), Kuwait (3.62), Iran (3.99), Jordan (4.03), Iraq 4.61), Palestine (4.66), Turkey (4.67), Malta (4.94) and Saudi Arabia (4.95) and the highest value in the low\u2010income group (19.59). Among the continents, the highest ASR death is in Africa (15.49) and the lowest in Europe (4.02). According to the World Health Organization, the highest standard incidence is in the African region and the lowest in the European region.In 2019, a total of 280\u2009479 new deaths due to cervical cancer with the conference level (238864_313930) were reported among women in the world, with deaths ASR per 10Also in general and based on GBD regions, the highest death ASR is related to Sub\u2010Saharan Africa.The highest standardized deaths rate from cervical cancer has been reported in Kiribati (69.52), Guinea (36.16), Lesotho (35.96), Zimbabwe (31.39), Somalia (30.99), Eritrea (30.26), Palau (29.79), Solomon Islands (29.44), Central African Republic (29.31), and Guinea\u2010Bissau (29.28).Meanwhile, the lowest standardized death rate from cervical cancer has been reported in Kuwait (1.76), Egypt (1.77), Syrian Arab Republic (1.78), Finland (1.78), Malta (1.78), Iceland (1.9), Luxembourg (1.94), Jordan 2.04), Iran (2.06) and Australia (2.14) , from which 8\u2009712\u2009962 were related to YLLs cases and 242\u2009051 to YLDs cases. Also, the worldwide DALYs ASR was reported at 210.64 and this number for YLLs ASR and YLDs ASR was 204.89 and 5.75 respectively. According to SDI classification, the highest DALYs ASR is in low SDI countries and the lowest is in high SDI countries. The YLLs and YLDs ASR are also the highest in low SDI countries.According to the World Bank classification, the highest value of DALYs ASR is related to World Bank Low\u2010income countries and the lowest value is related to high\u2010income countries. Also in the case of YLLs ASR and YLDs ASR, the highest value is related to low\u2010income countries and the lowest value is related to high\u2010income countries.In different continents, the highest DALYs ASR belongs to the African and then American continents. For the YLLs ASR, the highest value belongs to the African continent and the lowest value belongs to the European continent, but for the YLDs ASR the highest value belongs to the African continent and the lowest value belongs to the Asian continent.According to the World Health Organization regions, the highest DALYs ASR is in the African region, followed by the American region, and the lowest is in the Eastern Mediterranean and European regions. But regarding YLDs ASR, the highest value is in Africa and the lowest is in the Eastern Mediterranean regions.Based on GBD regions, the highest standardized age of DALYs, YLLs ASR, and YLDs ASR are in Sub\u2010Saharan Africa and then Latin America & Caribbean\u2010WB.The highest DALYs ASR has been reported in Kiribati (2143.06), Guinea (1143.8), Lesotho (1087.77), Solomon Islands (1018.69), Somalia (1013.76), Eritrea (973.58), Zimbabwe (957.22), Central African Republic (955.25), Guinea\u2010Bissau (937.63) and Mozambique (915.04).And the lowest DALYs ASR has also been reported in Kuwait (44.34), Egypt (45.13), Syrian Arab Republic (46.56), Finland (47.49), Malta (50.94), Iran (54.11), Iceland (54.93), Jordan (55.25), Luxembourg (55.34) and Switzerland (58.09).The highest YLDs ASR has also been reported in Kiribati (39.28), Palau (29.01), Solomon Islands (23.15), Guinea (18.66), Botswana (18.44), Lesotho (17.93), Zimbabwe (17.11), Saint Vincent and the Grenadines (16.95), Sao Tome and Principe (16.65), and Nauru (16.64).Meanwhile, the lowest YLDs ASR has been observed in Egypt (1.14), Syrian Arab Republic (1.39), Kuwait (1.64), Iran (1.74), Jordan (1.78), Palestine (1.83), Turkey (1.97), Iraq (1.99), Sudan (2.21) and Saudi Arabia (2.23).The highest YLLs ASR has been reported in Kiribati (2103.78), Guinea (1125.13), Lesotho (1069.85), Somalia (999.57), Solomon Islands (995.55), Eritrea (957.88), Central African Republic (941.77), Guinea\u2010Bissau (921.96) and Mozambique (899.74).Meanwhile, the lowest YLLs ASR has been reported in Kuwait 42.7), Egypt (43.99), Finland (44.91), Syrian Arab Republic (45.18), Malta (48.55), Iceland (52.06), Iran (52.38), Jordan (53.47) and Switzerland (55.31) has collected data from various sources, data from some areas is limited, which can affect the results of this study. Second, GBD statistics were based on a set of data sources such as cancer registry data and cytological results, while access to these resources is limited in some low\u2010income countries, and estimating statistics may be erroneous. Third, due to several years delay in presentation of cancer data, up to date data not available.zohreh momenimovahed: Data curation ; investigation ; methodology ; project administration ; supervision ; validation ; writing \u2013 original draft ; writing \u2013 review and editing . Afrooz mazidi moradi: Conceptualization ; data curation ; formal analysis ; methodology ; writing \u2013 original draft ; writing \u2013 review and editing . Parang Maroofi: Data curation ; formal analysis ; methodology ; writing \u2013 original draft ; writing \u2013 review and editing . Leila Allahqoli: Conceptualization ; data curation ; methodology ; project administration ; supervision ; writing \u2013 original draft ; writing \u2013 review and editing . Ibrahim Alkatout: Conceptualization ; funding acquisition ; investigation ; project administration ; resources ; supervision ; validation ; writing \u2013 original draft ; writing \u2013 review and editing . Hamid Salehiniya: Conceptualization ; data curation ; formal analysis ; methodology ; project administration ; validation ; visualization ; writing \u2013 original draft ; writing \u2013 review and editing .The authors declare no conflict of interest.The study was approved by the ethics committee of the Birjand University of Medical Sciences . As we used routinely collected anonymized electronic data, patient consent was not required."} +{"text": "Food Science & Nutrition, https://doi.org/10.1002/fsn3.3158. The above article, published online on December 8, 2022 in Wiley Online Library (https://wileyonlinelibrary.com), has been retracted by agreement between the journal Editor in Chief Y. Martin Lo, and Wiley Periodicals LLC. The retraction has been agreed upon due to an error in which the incorrect version of the article was published.Preparation and optimization of soy (Katul cultivar) protein isolate cold\u2010set gels induced by CaCl"} +{"text": "BMC Vet Res\u00a019, 143 (2023)Correction: 10.1186/s12917-023-03700-6.Following publication of the original article , the autThe texts currently read:Publication costs financed by the Minister of Science and Higher Education in the rang\u0119 of the program entitled \u201cRegional lnitiative of Excellence\u201d for the years 2019\u20132023, Project No. 010/RID/2018/19, amount of funding 12.000.000 PLN.The texts should read:Publication costs financed by the Minister of Education and Science under the program entitled \u201cRegional Initiative of Excellence\u201d for the years 2019\u20132023, Project No. 010/RID/2018/19, amount of funding 12.000.000 PLN."} +{"text": "Study to compare the effect of casirivimab and imdevimab, remdesivir, and favipiravir on progression and multi-organ function of hospitalized COVID-19 patients Open Medicine 2023;18(1):20230768, DOI: Journal of Clinical Virology Plus: doi.org/10.1016/j.jcvp.2023.100151.Editorial Office decided to retract this article due to significant overlap with the other papers published by the same authors in"} +{"text": "Atypical parkinsonism and self\u2010mutilation: A new lens on the old concept. Clin Case Rep. 2021; 9:e04432. ( The retraction has been agreed upon due to an error at the publisher, which caused a duplicate of the article to be published. The correct version of the article is Salari, M, Etemadifar, M, Ghanbari, K. Atypical parkinsonism and self\u2010mutilation: A new lens on the old concept. Clin Case Rep. 2021; 11:e04958. (https://doi.org/10.1002/ccr3.4958).The above article, published online on 09 July 2021 in Wiley Online Library ("} +{"text": "Article title: Improving clinical outcomes of Barrett\u2019s esophagus with high dose proton pump inhibitors and cryoablationAuthors: Snady, H.Journal:Annals of MedicineBibliometrics: Volume 55, Number 01, pages 1256\u20131264DOI:https://dx.doi.org/10.1080/07853890.2023.2191002The article was processed and published with references 24 to 35 incorrectly ordered. The references have now been renumbered as per intended making sure that the numerical order of citations in the text is retained as such. The article also has one minor layout correction each in Tables 1, 2 and 4."} +{"text": "The majority of literature on cavitary pulmonary coccidioidomycosis is from four decades ago which was prior to the advent of triazoles and focused on surgical treatment. This observational study is a comprehensive retrospective study of pulmonary cavitary coccidioidomycosis from patients at Valley Fever Institute at Kern Medical over the last 12 years. This observational study aims to explore the spectrum of coccidioidal cavities and the evaluation and management of those cavities.IRB approved, retrospective review of electronic medical records of the Valley Fever Institute database was conducted. Demographics, comorbidities, types, and the number of cavities, complications, and medical and surgical treatment were gathered and compared to the literature. PubMed and Google Scholar were searched for cavitary pulmonary coccidioidomycosisOf the initially 276 identified patients, 137 met the inclusion criteria. This study found 52 (37.2%) patients with hemoptysis. One case (0.7%) required radiologic intervention to occlude the bleeding vessel, and one (0.7%) case of hemorrhage required right upper lobe lobectomy. Nine (6.6%) cases exhibited a ruptured cavity. Eight of those cases had initial chest tube placement, of which three did not require surgical intervention. Seven of 137 (5.1%) cases presented with a pleural effusion not associated with a cavity rupture. Five (3.7%) were due to primary coccidioidomycosis. Three of the coccidioidal effusions required therapeutic thoracentesis, and none required a chest tube or surgery. The mean duration of the initial antifungal treatment was found to be 563 days (n=80). In 35% (28/ 80) of them, a triazole was switched to another triazole for variable reasons, including treatment failure or side effects.Coccidioidal pulmonary cavitation remains a complex disease to evaluate and treat. This study's ethnic demographic differed from other cohorts. It also contradicts the notion that pulmonary coccidioidal cavitary disease and dissemination infrequently manifest in the same patient. In the present age of triazole therapy, indications and the need for surgery continue to decline. Further investigation needs to be conducted to evaluate medical therapy\u2019s efficacy and long-term outcomes.Rupam Sharma, PGY-1/MD, Astellas: Grant/Research Support George R. Thompson, III, MD, Astellas: Advisor/Consultant|Astellas: Grant/Research Support|Cidara: Advisor/Consultant|Cidara: Grant/Research Support|F2G: Advisor/Consultant|F2G: Grant/Research Support|Mayne: Advisor/Consultant|Mayne: Grant/Research Support|Melinta: Advisor/Consultant|Melinta: Grant/Research Support|Mundipharma: Advisor/Consultant|Mundipharma: Grant/Research Support|Pfizer: Advisor/Consultant|Pfizer: Grant/Research Support"} +{"text": "This erratum corrects the following:et\u00a0al. A consensus statement from the Japan Diabetes Society: A proposed algorithm for pharmacotherapy in people with type 2 diabetes. J Diabetes Investig. 2023; 14: 151\u2013164. https://doi.org/10.1111/jdi.13960Ryotaro Bouchi, Tatsuya Kondo, Yasuharu Ohta The below statement should have been included in the original version of this article:https://doi.org/10.11213/tonyobyo.65.419) released in Japanese on Volume 65 Issue 8, 2022, on the official website of the Japan Diabetes Society, and has been jointly published in Diabetology International and Journal of Diabetes Investigation .This article is the English version of the \u201cA consensus statement from the Japan Diabetes Society: A proposed algorithm for pharmacotherapy in people with type 2 diabetes\u201d (The online article has been corrected.The publisher apologizes for the error."} +{"text": "Article title: Phosphatidylinositide 3-kinase inhibition: A new potential target for the treatment of polycystic ovarian syndromeAuthors: Shah, K. N. & Patel, S. S.Journal: Pharmaceutical BiologyBibliometrics: Volume 54, Number 6, pages 975\u2013983DOI: https://doi.org/10.3109/13880209.2015.1091482In the version of this article initially published, there was a mistake in the images of normal control groups of Figures 2 and 3.Figures 2 and 3 are now corrected and the article has been republished online."} +{"text": "There was an error in the original publication . In the A correction has been made to the Results Section, Paragraph 1:In 2021, COVID-19 incidence in northwest Russia varied in waves, ranging from 258.8 (per 100 K/month) in April to 1185.8 in November. At the same time, increases in incidence with the achievement of local maxima were recorded in January (1113.5 per 100 K/month), June (822.3 per 100 K/month), and November (1185.8 per 100 K/month) .The authors state that the scientific conclusions are unaffected. This correction was approved by the Academic Editor. The original publication has also been updated."} +{"text": "Assessment of stress and anxiety in mice with colorectal cancer submitted to physical exercise, which DOI is: https://doi.org/10.1590/acb370508, published in the journal Acta Cir\u00fargica Brasileira, 2022;37(05)e370508, page 5, in the axis X of Figure 5:In the manuscript Instead of:Open ArmOpen ArmOpen ArmShould be:Open ArmClosed ArmCenter"} +{"text": "Page 7, Fig. 4: We found errors in the significance indicator lines of this figure. In particular, there is no significant difference of the percentage of BLV-infected cells between the *016:01/*009:02 group and *009:02/other allele group. Instead, there is a significant difference between the *016:01/*009:02 group and the other alleles group. We note that this does not affect the overall discussion and conclusions. The corrected Volume 8, No. 1, e00493-22, 2023,"} +{"text": "Correction: Journal of Neuroinflammation 2012, 9:251 http://www.jneuroinflammation.com/content/9/1/251Following publication of the original article , the co-Hence, her name has been updated in this correction article."} +{"text": "BMC Infectious Diseases (2023) 23:25310.1186/s12879-023-08197-wThe original publication of this article contained an incorrect funding number, the incorrect and correct information are available in this correction article. The original article has been updated.IncorrectThis work is a part of the research project financed by the National Science Center of Poland, grant number 2019/34/E/NZ6/00221.CorrectThis work is a part of the research project financed by the National Science Center of Poland, grant number 2019/35/B/NZ7/00942."} +{"text": "There are errors in the Funding section. The correct Funding statement is: The authors would like to acknowledge the Fundamental Research Grant Scheme (FRGS) from the Ministry of Higher Education, Grant no: FRGS/1/2018/TK04/UM/02/1 (FP091-2018A)."} +{"text": "In June 2021, IDSA updated its guideline to have fidaxomicin (FDX) as the preferred treatment over vancomycin (VAN) for patients with initial and recurrent CDI. The objective of this study was to examine changes in treatment pattern for CDI and variation in implementation of the guideline recommendation across hospitals.This was a retrospective, observational study using data from the PINC AI Healthcare Database, an electronic laboratory, pharmacy and billing data repository from about 25% of US hospitals. This study included patients aged 18 and older who received CDI treatment during the study period from 1/2020 to 6/2021 (pre-period) and from 10/2021 to 6/2022 (post-period). We first examined the treatment pattern in the pre- and post-periods by calculating the proportion of patients who received FDX on day one of treatment, only FDX, only VAN, only metronidazole (MTZ), switched from VAN or MTZ to FDX, and other treatment patterns. Then we examined proportion of patients receiving FDX on day one at the hospital level. We used a multilevel logistic regression model with hospital random effects to examine the association between hospital characteristics and FDX use. Patient and hospital characteristics were controlled.67,311 CDI patients from 798 hospitals met the inclusion criteria. Figure 1 shows the analysis of CDI treatment pattern at the patient level. The proportion of patients treated with FDX on day one increased from 2.4% in the pre-period to 6.9% in the post-period. Patients who received only FDX increased from 1.3% to 4.1%, and those with a switch from VAN/MTZ to FDX from 3.2% to 6.4%. At the hospital level, the mean use of FDX on day one increased from 2.6% (SD = 4.8%) to 7.9% (SD = 10.1%), and the median increased from 1.1% to 4.2% . We found that FDX use increased proportionately by all hospital characteristics except census region where greater increases were found in the South and Midwest. Hospitals had a statistically significant association with FDX use (p< 0.001) and 29.7% of the variance of FDX use on day one was explained by between-hospital variability.Adjusted Proportion of Treatment Starting with FidaxomicinUse of FDX for CDI treatment has increased since the publication of IDSA guideline in 2021, but it remains low with significant regional variation.Erik R. Dubberke, MD, MSPH, Abbott: Advisor/Consultant|AstraZeneca: Advisor/Consultant|Ferring Pharmaceuticals: Advisor/Consultant|Ferring Pharmaceuticals: Grant/Research Support|Merck and Co.: Advisor/Consultant|Pfizer: Advisor/Consultant|Pfizer: Grant/Research Support|Seres Therapeutics: Advisor/Consultant|Summit: Advisor/Consultant|Theriva Biologics: Grant/Research Support Qinghua Li, PhD, Merck & Co Inc: Employee Engels N. Obi, PhD, Merck & Co Inc: Employee Vladimir Turzhitsky, PhD, Merck & Co.: Full time employee of Merck & Co.|Merck & Co.: Stocks/Bonds Fakhar Siddiqui, MD, MBA, Merck & Co Inc.: Employee Brian H. Nathanson, Ph.D., Merck & Co., Inc: Advisor/Consultant"} +{"text": "The second author's name was spelled incorrectly. The correct name is: Jaap A. Wagenaar.The correct citation is: Bosman AB, Wagenaar JA, Stegeman A, Vernooij H, Mevius D (2012) Quantifying Antimicrobial Resistance at Veal Calf Farms. PLoS ONE 7(9): e44831. doi:10.1371/journal.pone.0044831There is also an error in the Materials and Methods section. In Statistical Analysis, the first sentence of the third paragraph of \"Logistic regression analysis\" should read: The estimated variance values of the random effects in the best fitting model were used to examine the variation in antimicrobial resistance on farm-level, pen-level and sample-level, with the variance of a standard logistic density fixed at \u03c02/3 [26]."} +{"text": "AbstractArachnida: Opiliones). Harvestmen accessible datasets in Iberian Peninsula are unknown, an only two other datasets available in GBIF are composed exclusively of harvestmen records. Moreover, only a few harvestmen data from Iberian Peninsula are available in GBIF network . This paper describes the data associated with the Opiliones kept in the BOS Arthropod Collection of the University of Oviedo, Spain (hosted in the Department of Biolog\u00eda de Organismos y Sistemas), filling some of those gaps. The specimens were mainly collected from the northern third of the Iberian Peninsula. The earliest specimen deposited in the collection, dating back to the early 20th century, belongs to the P. Franganillo Collection. The dataset documents the collection of 16,455 specimens, preserved in 3,772 vials. Approximately 38% of the specimens belong to the family Sclerosomatidae, and 26% to Phalangidae; six other families with fewer specimens are also included. Data quality control was incorporated at several steps of digitisation process to facilitate reuse and improve accuracy. The complete dataset is also provided in Darwin Core Archive format, allowing public retrieval, use and combination with other biological, biodiversity of geographical variables datasets.There are significant gaps in accessible knowledge about the distribution and phenology of Iberian harvestmen (PageBreak Purpose: Existing knowledge on the distribution of harvestmen in the Iberian Peninsula is still very fragmented of Ireland dataset of the National Biodiversity Data Centre (http://data.gbif.org/datasets/resource/10810), with 2,109 records (agmented . There aagmented . However groups) .Opiliones specimens deposited in the BOS Arthropod Collection (subcollection of Opiliones: BOS-Opi) of the University of Oviedo, Spain, comprising 16,455 specimens in 3,772 vials . As a result of this, the BOS-Opi dataset makes a significant contribution of primary data about Iberian harvestmen for ecological, faunistic and conservation studies. With the publication of this dataset, we aim to (1) providing a dataset with phenological and distribution data on harvestmen from the northern third of the Iberian Peninsula, and (2) describing the Opiliones subcollection of the BOS Arthropod Collection.The purpose of this paper is to document a dataset corresponding to Additional information: A list of publications citing harvestmen contained in this dataset (BOS-Opi) is provided in point 2 of the reference section.Project title: Informatizaci\u00f3n de la Colecci\u00f3n de Artr\u00f3podos BOS de la Universidad de Oviedo / Digitisation of the BOS Arthropod Collection of University of OviedoPersonnel digitisation: Torralba-Burrial APageBreakAdministrative contact: Anad\u00f3n ABOS-Opi determination specialist: Merino S\u00e1inz IBOS-Opi collectors: Collectors who have deposited more than 50 specimens include Merino S\u00e1inz I, Anad\u00f3n A, Fern\u00e1ndez-\u00c1lvarez F.A., Torralba-Burrial A, Ocharan Larrondo FJ, Melero Cimas VX, Monteser\u00edn Real S, Ocharan Ibarra R, Rosa Garc\u00eda R, and V\u00e1zquez Felechosa MTP. Franganillo Collection:Curator of Lastra CFunding: Digitisation of this biological collection was supported by the Spanish National R+D+i Plan and PCTI Asturias through a contract for ATB.PageBreakBiodiversity of Muniellos Biosphere Reserve\u201d was supported by the Asturias Regional Government .Almost 73% of the specimens were collected as part of the PhD Thesis by Study area description: Harvestmen specimens deposited in BOS Arthropod Collection are from the northern third of the Iberian Peninsula (eninsula . Most ofeninsula . The clieninsula . Oak andeninsula . Harvesteninsula .Design description: The digitisation process of this dataset (BOS-Opi) was carried out according to the workflow put in place for the Odonata subcollection (BOS-Odo) (http://www.gbif.es:8080/ipt). DarwinCore elements included in the dataset structure are listed in the dataset description section. Data quality controls of geographic, taxonomic and additional data associated with the harvestmen specimens were performed at several steps of digitisation process as an essential part of this Information Management Chain (BOS-Odo) . Prior tnt Chain , 2005b, PageBreakArthropod Collection, allowing free access of citizens, researches, environmental companies and government managements to biodiversity data kept in this Collection.Currently, dataset is being used to study phenological and life history differences of harvestmen species between areas in north Iberian Peninsula with different geographical/habitat features, species distribution and importance of opportunistic data in fill knowledge gaps when standardised sampling data are not available or are incomplete. Moreover, this dataset is considered as a dynamic catalogue of the harvestmen of BOS General taxonomic coverage description: All specimens were identified to species when preservation status, sex and life cycle phase permitted it. Sixty-two species were recorded from the northern third of the Iberian Peninsula and Phalangodidae (suborder Laniatores) are missing. As depicted in Sclerosomatidae , followed by Phalangiidae , Trogulidae , Nemastomatidae (14.0%: Nemastomella and Nemastoma). Other families represent less of 5% of the records descriptions and without figures. In four publications nomina dubia both in Araneae , current identifications more accurate and other specimens show a correct identification by Franganillo .No types are hosted among the ae e.g., and in Oes e.g., , are synes e.g., , or his es e.g., . In 1972es e.g., . The prees e.g., . A studyes e.g., . In Opilna dubia , and mosna dubia . IdentifAnimaliaKingdom: ArthropodaPhylum: ArachnidaClass: OpilionesOrder: Ischyropsalididae, Nemastomatidae, Phalangiidae, Sabaconidae, Sclerosomatidae, Sironidae, Travuniidae, Trogulidae.Family: Common names: Animals, Arthropods, Arachnids, Harvestmen.All specimens are from the northern part of the Iberian Peninsula . Most ofPageBreak40\u00b018'N and 43\u00b042'N Latitude; 8\u00b054'W and 0\u00b030'W Longitude.1900\u201320121977-presentParent collection identifier: Colecci\u00f3n de Artr\u00f3podos BOSCollection name: Colecci\u00f3n de Artr\u00f3podos BOS de la Universidad de Oviedo: Opiliones (BOS-Opi)Collection identifier:http://data.gbif.org/datasets/resource/15038Specimen preservation method: Ethanol 70\u00b0Curatorial unit: 3772 with an uncertainty of 0 )Curatorial unit: 16455 with an uncertainty of 0 (Specimens)Method description: The digitisation process of the Opiliones subcollection (BOS-Opi) was realised in accordance with the published workflow of the Odonata subcollection (BOS-Odo) (see Odo) see .Pre-digitisation phase: The preservation status of harvestmen specimens was reviewed prior to digitisation. Vials were changed when necessary and refilled with preservation liquid (ethanol 70\u00b0). Specimens were identified or identifications were reviewed when they were already noted. Identification labels were added when labels were lacking or otherwise incomplete. Specimens\u2019 vials were sorted alphabetically by family/genus/species names in trays, and hosted in metallic cabinets in a cold chamber.Digitisation phase: A database with DarwinCore v1.2 standard fields and other fields specific to different research projects was developed using MS EXCEL software. All biodiversity data available on the specimens\u2019 labels were included in the database.PageBreakhttp://www.ign.es/iberpix2/visor). Localities were sorted geographically for batch retrospective georeferencing, starting with larger batches from specimen labels or from associated publications were added to the database when available. If coordinates were not present on the specimen labels or in primary publications, retrospective georeferencing see was carr batches . CoordinThe database was converted and imported to, and managed with, ZOORBAR v2.1.1 software .Creation of the dataset: The dataset was exported as a file in DarwinCore v1.2 format and geographic coordinates were carried out with ZOORBAR v2.1.1 software. DarwinCore elements included in dataset structure are listed in the dataset description section. Data format, georeferenced coordinates and absence of ASCII anomalous characters were checked with DARWIN_TEST v.3.2 software (http://www.gbif.es/darwin_test/Darwin_test.php). Erroneous data were corrected and data cleaning was repeated to enhance the data quality .http://www.gbif.es:8080/ipt). Links to these data were also provided on the BOS Arthropod Collection website (http://www.unioviedo.es/BOS/Zoologia/artropodos). The offline version of the dataset includes the identification history of each specimen (4149 items), collection method, research project, and notes on materials derived from the specimens . This information is available on request.The dataset was transformed to a DarwinCore Archive format with metadata to ensure rapid discovery of this biodiversity resource and future publishing as a citable academic paper see . The datStudy extent description: Specimens are mainly from the northern third of the Iberian Peninsula (see geographic coverage section). The earliest specimens are from the 20th century (belonging to the P. Franganillo collection), but the general collection starts in 1977. However, only 9.73% of the items were collected prior to the year 2000, while 75.93% were collected between 2009 and 2012. The BOS-Opi dataset includes the record distributions by month specimens from the PhD dissertation by PageBreak2) specimens from the project \u201cCataloguing of the Biodiversity from the Biosphere Reserve of Muniellos\u201d (SW of Asturias province) (13.10%)3) specimens from other sources: collections from students in Biology and Forestry Engineering programs at the University of Oviedo, other research projects, practical courses, etc. (13.92%).PageBreakmuch lower number of specimens (1.2%) . Ethylene glycol was used as a fixation and preservation liquid in the pitfalls : OpilionesCharacter encoding: UTF-8Format name: Darwin Core Archive formatFormat version: 1.0Distribution:http://www.gbif.es:8080/ipt/archive.do?r=bos-opiPublication date of data: 2013-07-04Update police: Annually when necessary to transmit data of new specimens kept at BOS Collection.Language: SpanishLicenses of use: This dataset [BOS Arthropod Collection of University of Oviedo (Spain): Opiliones (BOS-Opi)] is made available under the Open Data Commons Attribution License: http://www.opendatacommons.org/licenses/by/1.0/.DarwinCore elements: The DarwinCore elements (http://purl.org/dc/terms/) included in the dataset published through the GBIF network describe the specimens\u2019 data to several levels. These elements are: Record data: type (basisofrecord), DateLastModified, InstitutionCode, CollectionCode, CatalogNumber, Collector, IndividualCount, Sex, YearCollected, MonthCollected, DayCollected, Notes (with info about habitat in most of cases); Geographic data: Country, StateProvince, Locality , MinimumElevation (meters), MaximunElevatium (meters), Latitude , Longitude , CoordinatePrecision (meters); Taxonomic data: Kingdom , Phylum , Class , Order , Family, Genus, Species (specificEpithet), ScientificNameAuthor (auPageBreakthorship of taxa name), ScientificName, Identified by, Yearidentified, Type status. Moreover, some DarwinCore elements were mapped to fixed values in the IPT as described in this data-paper: language, rights, rightsHolder, bibliographicCitation, references, datasetID, datasetName, ownerInstitutionCode.Object name: BOS Arthropod Collection of University of Oviedo (Spain): OpilionesCharacter encoding: iso-8859-1Format name: Darwin Core ArchiveFormat version: 1.0Distribution:http://data.gbif.org/datasets/resource/15038Metadata language: EnglishDate of metadata creation: 2013-06-12Hierarchy level: Dataset"} +{"text": "The name of the gene (BCMO1) was spelled incorrectly in the title.The title should be: Detection of a Cis eQTL Controlling BCMO1 GeneExpression Leads to the Identification of a QTG for Chicken Breast Meat ColorThe corrected citation is: Le Bihan-Duval E, Nadaf J, Berri C, Pitel F, Graulet B, et al. (2011) Detection of a Cis eQTL Controlling BCMO1 Gene Expression Leads to the Identification of a QTG for Chicken Breast Meat Color. PLoS ONE 6(7): e14825. doi:10.1371/journal.pone.0014825"} +{"text": "The name of the first author of the article is incorrect. The first author's correct name is: Chong Leong Gan. The correct Citation is: Chong Leong G, Uda H (2013) Comparative Reliability Studies and Analysis of Au, Pd-Coated Cu and Pd-Doped Cu Wire in Microelectronics Packaging. PLoS ONE 8(11): e78705. doi:10.1371/journal.pone.0078705. The correct abbreviation of the first author's name in the Author Contributions statement is: CLG."} +{"text": "Shigella Pathogenicity Puzzle: Spermidine Accumulation by Silencing of the speG Gene. The correct citation is: Barbagallo M, Di Martino ML, Marcocci L, Pietrangeli P, De Carolis E, et al. (2011) A New Piece of the Shigella Pathogenicity Puzzle: Spermidine Accumulation by Silencing of the speG Gene. PLoS ONE 6(11): e27226. There was a typographical error in the title. The correct title is: A New Piece of the"} +{"text": "The name of the first author is incorrectly represented in the Citation and Copyright. The correct Citation is: Abu Awad D, Gallina S, Bonamy C, Billiard S (2014) The Interaction between Selection, Demography and Selfing and How It Affects Population Viability. PLoS ONE 9(1):e86125. doi:10.1371/journal.pone.0086125.The Copyright statement should read: \"\u00a9 2014 Abu Awad et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.\""} +{"text": "Correction to:British Journal of Cancer (2011) 104, 1410\u20131417; doi:10.1038/bjc.2011.94When published originally, earlier in Volume 104, the authors noticed that they had omitted to include TRDRP grant (19XT-0051) in the acknowledgements, which funded a large portion of the work for this paper. The authors are now happy to correct this omission."} +{"text": "Acta Cryst. (2010), E66, o2633.Corrigendum to Acta Cryst. (2010), E66, o2633] is corrected.The author list in the paper by Ding [ In the paper by Ding (2010)"} +{"text": "The name of the third author was given incorrectly. The correct name is: Markus von Nickisch-Rosenegk. The correct citation is: Hoppe S, Bier FF, von Nickisch-Rosenegk M (2013) Rapid Identification of Novel Immunodominant Proteins and Characterization of a Specific Linear Epitope of Campylobacter jejuni. PLoS ONE 8(5): e65837. doi:10.1371/journal.pone.0065837. The abbreviation in Contributions correctly reflects the name as it is."} +{"text": "The word \"Systematic\" is misspelled in the title.The correct title is: The Scale of Faith Based Organization Participation in Health Service Delivery in Developing Countries: Systematic Review and Meta-Analysis.The correct citation is: Kagawa RC, Anglemyer A, Montagu D (2012) The Scale of Faith Based Organization Participation in Health Service Delivery in Developing Countries: Systematic Review and Meta-Analysis. PLoS ONE 7(11): e48457. doi:10.1371/journal.pone.0048457"} +{"text": "Porcine reproductive and respitatory syndrome virus (PRRSV) is a recently emerged pathogen and severely affects swine populations worldwide. The replication of PRRSV is tightly controlled by viral gene expression and the codon usage of translation initiation region within each gene could potentially regulate the translation rate. Therefore, a better understanding of the codon usage pattern of the initiation translation region would shed light on the regulation of PRRSV gene expression.In this study, the codon usage in the translation initiation region and in the whole coding sequence was compared in PRRSV ORF1a and ORFs2-7. To investigate the potential role of codon usage in affecting the translation initiation rate, we established a codon usage model for PRRSV translation initiation region. We observed that some non-preferential codons are preferentially used in the translation initiation region in particular ORFs. Although some positions vary with codons, they intend to use codons with negative CUB. Furthermore, our model of codon usage showed that the conserved pattern of CUB is not directly consensus with the conserved sequence, but shaped under the translation selection.The non-variation pattern with negative CUB in the PRRSV translation initiation region scanned by ribosomes is considered the rate-limiting step in the translation process. Nidovirales of family Arteriviridae [Porcine reproductive and respiratory syndrome virus (PRRSV) infection causes serious disease in swine populations with a series of clinical consequences, such as high mortality, reproductive failure, post-weaning pneumonia and growth reduction ,2. Basediviridae ,9. The Piviridae -13. Despiviridae ,14. Theriviridae -19. Moreiviridae .It is generally considered that the alternative synonymous codons are not used with equal frequencies among organisms, and the codon usage pattern plays a role in genes expressed at higher levels -30. Jacqhttp://www.ncbi.nlm.nih.gov/Genbank/ and the synonymous codon usage values (SCUV) for this virus were reported previously [st to the 50th residue) of ORF1a, ORF2, ORF3, ORF4, ORF5, ORF6 and ORF7 were used as targets for alignment analysis respectively.The 13 complete RNA sequences of PRRSV were downloaded from the National Center for Biotechnology Information (NCBI) eviously . Multipl0 = 1.00) for the development of serotype-specific codon usage [To calculate CUB, it is supposed that statistically equal and random usage of all available synonymous codons was the \"neutral point\" /(n/N), represents the relative abundance for a particular codon in the translation initiation region. ni represents the total number of a particular codon within the 1st to ith amino acids, Ni represents the total number of corresponding amino acid in the 1st to ith amino acid ones, n is the total number of a certain codon within the whole coding sequence, and N is the total number of corresponding amino acids within the whole coding sequence. When R value is equal to 1.00, it means that the frequency of this codon in the target region is equal to the frequency of this codon in the whole coding sequence; when R value is lower than zero, it implies that the frequency of this codon in the target region is lower than that of the whole coding sequence; when R value is higher than zero, it suggests that the frequency of this codon is higher than that of the whole coding sequence.We analyzed the codon usage characteristics of the translation initiation region depending on R values for codons of the target positions.To substantiate the characteristics of codon usage for positions with negative CUB in the target regions, we analyzed the target positions depending on the data, (i) the variations of codons and amino acids, (ii) The consensus amino acid sequence is based on the comparison of the strains in previous study . The posThe bars of all positions in the translation initiation region represented the CUB degree Figure . AlthougThe various extents of the conserved pattern of codon usage for their positions in PRRSV ORFs suggest that CUB associated with these positions might modulate the corresponding gene expression.R value for each codon was calculated and listed in Table R value indicated more preferential usage in the translation initiation site than that of the whole coding sequence. ij CUBvalue for each codon was listed in Table The R value for the codons with negative CUB vary compared with R = 1.00. However, some target positions contain the codons with negative CUB and R values > 1.00, suggesting that some new characteristics might influence the translation efficiency of the corresponding coding sequence. In translation initiation region of ORF1a, the non-preferential codons are preferentially used in the 4th (US and EU serotypes), 9th (US), 12th (EU), 19th (US), the 22nd (US and EU), 27th (US), 31st (US and EU) and 40th (US), while some non-preferential codons, which have R value < 1.00 or R value > 1.00, exist in the 16th (EU) and 30th (US and EU) positions. For ORF2, the non-preferential codons are more preferentially used in the 7th (EU), 8th (EU), 9th (EU), 11th (US), 20th (EU), 27th (EU), 30th (US), 33rd (US), 40th (EU), 43rd (EU), 44th (US) and 48th (EU) positions, while some non-preferential codons with R value > 1.00 or R value < 1.00 exist in the 12th (US) position. For ORF3, non-preferential codons exist in the 4th (US), 13th (US and EU), 17th (EU), 26th (US and EU), 31st (US and EU), 32nd (EU) and 37th (US) positions, while the non-preferential codons with R value > 1.00 or R value < 1.00 are used in the 5th (EU), 6th (US and EU), 7th (US), 11th (US and EU),16th (US) and 43rd (EU) positions. For ORF4, the non-preferential codons with R value > 1.00 are used in the 3rd (US and EU), 7th (US and EU), 20th (US and EU), 27th (US and EU), 28th (US and EU), 29th (US and EU), 38th (EU),40th (US and EU), 41st (US), 44th (EU) and 49th (US and EU), while some non-preferential nodons with R value > 1.00 or R value < 1.00 are used in the 31st (EU) position. For ORF5, the non-preferential nodons with R value > 1.00 are used in the 9th (EU), 12th (US), 14th (EU), 22nd (US) 23rd (US), 32nd (US), 36th (US), 39th (EU), 40th (EU), 44th (EU), 48th (EU) and 49th (EU), while non-preferential codon with R value > 1.00 or R value > 1.00 are used in the 8th (US), 24th (US), 46th (US) and 47th (US) positions. For ORF6, the non-preferential codons are used in the 3rd (US), 4th (EU), 7th (US), 13th (US), 14th (EU), 15th (EU), 19th (EU), 21st (US), 22nd (EU), 24th (EU), 26th (EU), 27th (US), 30th (EU), 31st (EU), 32nd (US), 37th (US), 40th (US), 45th (EU) and 48th (EU) positions, while some non-preferential codon are used in the 2nd (EU), 5th (US and EU), 46th (US) and 50th (US) positions. For ORF7, the non-preferential codons are chosen in the 11th (US), 32nd (US), 40th (US and EU), 41st (US), 43rd (EU), 44th (EU), 48th (US) and 50th (US) positions, while some non-preferential codon are used in the 3rd (US), 24th (EU), 25th (EU) and 35th (US) positions. The rest positions with negative CUB do not arise from the existence of non-preferential codons but contain some preferential codons (CUB > 0), implying that these positions do not affect the efficiency of gene translation. The degeneracy of the genetic code enables the same amino acid sequences to be encoded and translated in different ways. However, the synonymous codon usage is not purely random.The positions with negative CUB do not always use the codons with negative CUB, and the Drosophila species, suggesting that translation selection favors the conserved pattern of synonymous codon usage to enhance the accuracy of gene expression [CAT product [RNA virus possesses high mutation rates and therefore virus populations exist as dynamic and complex mutant distributions -41. Howepression . A lot opression . Non-unipression -52. Frompression . Howeverpression . Once a pression ,54-58. Kpression . Lavner pression . A relatpression ,61-65. Tpression ,67. Our pression ,63,64,68 product , the vie product . When th product ,71.In summary, the conserved non-preferential codons in the translation initiation region have a high relationship with the regulation of gene expression. And the conserved codons with negative CUB are preferentially used in the initial region, which may be explained by the minor codon modulator hypothesis and the translation selection. These codons within this critical region might play a negative role in regulation of gene expression.PRRSV: Porcine reproductive and respitatory syndrome virus; SCUV: synonymous codon usage values; CUB: codon usage bias; US: Northern American isolate; EU: European isolate.The authors declare that they have no competing interests.JHS and XXM carried out the molecular genetic studies, participated in the sequence alignment and drafted the manuscript. YLH, JDL and XSM participated in the sequence alignment. YXD and XNL participated in the design of the study and performed the statistical analysis. XPC conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript."} +{"text": "Correction to: British Journal of Cancer (2012) 106, 1453\u20131459; doi:10.1038/bjc.2012.98In revision of the above paper during the proofing and correction process, an earlier version of Figure 3 was resupplied for publication and, subsequently, published in error. The authors and publishers apologise for this mistake.The correct"} +{"text": "DNA RESEARCH 21, 53\u201368, (2014); doi:10.1093/dnares/dst040B. oleracea Repeat number: the 3 should be 12. The corrected part of the table is given below.There was an error in the first three rows of Table"} +{"text": "A reference was omitted from the reference list during the production process. There reference is: 53. Middleton FA, Strick PL (1994) Anatomical evidence for cerebellar and basal ganglia involvement in higher cognitive function. Science 266: 458-461.In place of reference 53, some erroneous formatting text was published in the PDF."} +{"text": "A funding organization for the second author, Claudio Eglueta, was incorrectly omitted from the Funding Statement. The Funding Statement should read: \"This work was supported by a DFG grant to NK and the VW-Foundation to CE. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\"In addition, affiliation number 2 for the second author is incorrect. The correct affiliation number 2 is: Institut f\u00fcr Physiologie I, Albert-Ludwigs-Universit\u00e4t, Freiburg, Germany.In addition, all Supplementary Figure files are missing in both hyperlink and thumbnail format. The Supplementary Figures can be viewed at the following links, as indicated:Figure S1: Click here for additional data file.Figure S2: Click here for additional data file.Figure S3: Click here for additional data file.Figure S4: Click here for additional data file.Figure S5: Click here for additional data file.Figure S6: Click here for additional data file.Figure S7: Click here for additional data file.Figure S8: Click here for additional data file."} +{"text": "Correction to: British Journal of Cancer (2012) 104, 1482\u20131486; doi:10.1038/bjc.2011.114After publication, it was noted that the dose category labels contained in The full, correct"} +{"text": "The classical symptoms of porcine epidemic diarrhea (PED) are acute diarrhea and dehydration. The isolated porcine epidemic diarrhea virus (PEDV) CH/GDGZ/2012 strain was obtained from the feces of diseased pigs in 2012 in southern China. We report the complete genome sequence of strain CH/GDGZ/2012, which might be useful for better understanding the molecular characteristics of this virus. Alphacoronavirus, the family Coronaviridae, and the order Nidovirales (Porcine epidemic diarrhea virus (PEDV), which is an enveloped, positive-sense, single-stranded RNA virus, is classified as a member of the genus ovirales , 2.The whole genome of the PEDV strain CH/GDGZ/2012 was amplified by 21 sets of primers and clonThe complete genome sequence of CH/GDGZ/2012 exhibits 96.6%, 97.4%, 97.9%, and 97.0% nucleotide homologies with the genomes of PEDV strains CV777, DR13, CH/FJND-3/2011, and CH/S, respectively . SimilarThese data may be useful for analyses of the epidemiology and evolutionary characteristics of PEDVs in southern China.KF384500.The complete genome sequence of PEDV CH/GDGZ/2012 strain has been deposited in GenBank under the accession no."} +{"text": "In Human Prion Disease and Relative Risk Associated with Chronic Wasting Disease by Samantha MaWhinney et al., an error occurred in the list of references. Missing from the list is reference no. 36: Belay ED, Maddox RA, Gambetti P, Schonberger LB. Monitoring the occurrence of emerging forms of Creutzfeldt-Jakob disease in the United States. Neurology. 2003;60:176\u201381.http://wwwnc.cdc.gov/eid/article/12/10/06-0019_article.htmThe corrected list of references appears in the online article at We regret any confusion this error may have caused."} +{"text": "There was an error in the citation in the online version of the article. The correct citation is \"Bouillard K, Nordez A, Hug F (2011) Estimation of Individual Muscle Force Using Elastography. PLoS ONE 6(12): e29261. doi:10.1371/journal.pone.0029261\""} +{"text": "There was an error in the name of the fourth author.The correct author name is: Nickolay V. BukoreshtlievThe correct citation is: Modulevsky DJ, Tremblay D, Gullekson C, Bukoreshtliev NV, Pelling AE (2012) The Physical Interaction of Myoblasts with the Microenvironment during Remodeling of the Cytoarchitecture. PLoS ONE 7(9): e45329. doi:10.1371/journal.pone.0045329"} +{"text": "The correct name of the second author is: Bashir AhmadThe correct citation is: Noreen S, Ahmad B, Hayat T (2013) Mixed Convection Flow of Nanofluid in Presence of an Inclined Magnetic Field. PLoS ONE 8(9): e73248. doi:10.1371/journal.pone.0073248The correct version of the grant number in the first sentence of the Funding statement should be: 25-130/1433 HiCi"} +{"text": "The name of the first author was listed incorrectly. The correct name is: Adzemovic MV. The correct citation is: Adzemovic MZ, Zeitelhofer M, Eriksson U, Olsson T, Nilsson I (2013) Imatinib Ameliorates Neuroinflammation in a Rat Model of Multiple Sclerosis by Enhancing Blood-Brain Barrier Integrity and by Modulating the Peripheral Immune Response. PLoS ONE 8(2): e56586. doi:10.1371/journal.pone.0056586. The correct abbreviation in Contributions is: MZA."} +{"text": "The name of the third author is incorrect. The correct name is: Bradley S. Peterson. The correct citation is: Hu S, Xu D, Peterson BS, Wang Q, He X, et al. (2013) Association of Cerebral Networks in Resting State with Sexual Preference of Homosexual Men: A Study of Regional Homogeneity and Functional Connectivity. PLoS ONE 8(3): e59426. The correct abbreviation in Contributions is: BSP."} +{"text": "The name of the first author was spelled incorrectly. The correct name is: Gesham Magombedze.In addition, the word \"Evaluation\" was spelled incorrectly in the article title. The correct title is: Evaluation of the \"Iceberg Phenomenon\" in Johne's Disease through Mathematical Modelling.The correct citation is: Magombedze G, Ngonghala CN, Lanzas C (2013) Evaluation of the \u201cIceberg Phenomenon\u201d in Johne's Disease through Mathematical Modelling. PLoS ONE 8(10): e76636. doi:10.1371/journal.pone.0076636.In addition, the number of the grant from the National Science Foundation is incorrect. The correct grant number is: NSF Award # EF-0832858."} +{"text": "Isolona and Monodora (Annonaceae). Both these genera, with 20 and 14 species, respectively, are widely distributed in African TRFs, with a few species occurring in slightly less humid regions such as in East Africa.The tropical rain forests (TRF) of Africa are the second largest block of this biome after the Amazon and exhibit high levels of plant endemism and diversity. Two main hypotheses have been advanced to explain speciation processes that have led to this high level of biodiversity: allopatric speciation linked to geographic isolation and ecological speciation linked to ecological gradients. Both these hypotheses rely on ecology: in the former conservation of ecological niches through time is implied, while in the latter adaptation via selection to alternative ecological niches would be a prerequisite. Here, we investigate the role of ecology in explaining present day species diversity in African TRF using a species level phylogeny and ecological niche modeling of two predominantly restricted TRF tree genera, A total of 11 sister species pairs were identified most of them occurring in allopatry or with little geographical overlap. Our results provide a mixed answer on the role of ecology in speciation. Although no sister species have identical niches, just under half of the tests suggest that sister species do have more similar niches than expected by chance. PCA analyses also support little ecological differences between sister species. Most speciation events within both genera predate the Pleistocene, occurring during the Late Miocene and Pliocene periods.Isolona and Monodora at the scale analyzed here. This is consistent with the geographical speciation model for TRF diversification. These results contrast to other studies for non-TRF plant species where ecological speciation was found to be an important factor of diversification. The Pliocene period appears to be a vital time in the generation of African TRF diversity, whereas Pleistocene climatic fluctuations have had a smaller role on speciation than previously thought.Ecology is almost always involved in speciation, however, it would seem to have had a little role in species generation within Isolona, Monodora, AnnonaceaeEcological niche modeling, species level phylogeny, ecological speciation, African tropics, The tropical rain forest (TRF) biome covers just ~7% of land but harbors over half of the planet's terrestrial biodiversity. The TRF of Africa represents the second largest extent of this biome after the Amazon basin, and contains high levels of species diversity and especially endemicity . Two maiUnderstanding the evolutionary processes responsible for high species richness of TRF has been a major focus of evolutionary biology , althougNew developments in ecological niche modeling (ENM) or species distribution modeling have provided important advances in the understanding of species distribution , as wellIsolona and Monodora (Annonaceae). Both genera contain small to large trees largely distributed across the African rain forests from West/Central Africa to East Africa. A recent monograph of both genera provides information about species delimitation as well as their distribution . In oner et al. ,29 proviMean divergence ages Table for all Isolona capuronii (1), I. pleurocarpa (7), Monodora carolinae (7), M. globiflora (6), M. hastipetala (4) and M. stenopetala (6), all being rare or localized species. Analysis of the variation of each bioclim variable between species underlines important ecological characteristics within and between species of each genus. For Isolona, differences between East and West/Central species were few , higher temperature seasonality (BC4) and lower annual precipitation (BC12) as well as other related variables such as BC13, BC14, BC16, BC17 and BC19 on all 19 bioclim variables and significance between principal components were tested under Mann-Whitney U test. Together, the first and second components explained between 59% and 87% of the variation among the 19 bioclim variables. The directionality of the loadings for components 1 and 2 were quite variable, Figures and 5 buMonodora laurentii within M. myristica; M. hastipetala within M. junodii; Isolona cooperi within I. campanulata; 2) species that overlapped for part of their variation ; 3) species with clearly separated ecological space, which was found in just one species pair: Monodora carolinae /M. stenopetala. For most comparisons one to two PC axes were not significantly different between species pairs Species with narrower distributions were included within the environmental variation of a more widely distributed species (for example Monodora species) no standard deviation (SD) was calculated, even though the respective AUCs were high, because the number of total samples was too low (for species with occurrence points less than 8 only one training point can be used from which the SD cannot be estimated). Although several species in this study exhibited low sample sizes, the resulting AUCs indicate that meaningful models have been produced is reported in Table S1. Response curves and variable importance were examined; however no general trends or consistent suite of variables were identified as the important factors for species' distributions.Monodora hastipetala /M. junodii, and all four tests for Isolona heinsenii /I. linearis.Potential distribution generated under ENM using Maxent between selected sister species are presented in Figures K of Blomberg et al. . Also, than 10 .Afromomum . A secondary calibration point was used, with the crown node of Isolona and Monodora set to 14.9 Ma (95% highest posterior density (HPD) 9.4-21). A similar age (14.4 (95% HPD 10.2-18.7) for this node was also found with a larger sampling of Annonaceae genera and with an updated fossil calibration hypothesis [For this analysis we used the chronogram of Couvreur et al. which inpothesis .Isolona and 737 for Monodora (Table S1).Locality data were compiled from Couvreur and reprAn estimate of the known geographic range for each species was produced in ArcGIS v 9.3 using a \"buffer\" approach. This method creates a buffer radius around each collection point for each species. Overlapping buffers for each species pair are then fused and the range overlap is calculated. Several other approaches can be used such as the \"quadrat\" [e.g. ]: the dihttp://www.worldclim.org at 30 arc seconds resolution [Using the 19 bioclim variables Table from httsolution , a set oEnvironmental niches were compared between sister species using Principal Component Analysis (PCA) on all 19 bioclim variables. Statistical tests between groups have generally relied on AMOVA or MANOVA methods either between the principal components of the PCA ,48 or onM. hastipetala (see below). Ecological niche models were generated using the maximum entropy method, Maxent version 3.3 [Ecological niche modeling was used in order to summarize the climatic tolerances of the sampled species, except for sion 3.3 . This ission 3.3 ,70,71. Msion 3.3 .Isolona and Monodora (below 12\u00b051'N and above 28\u00b07'S); latitudinal boundaries which roughly coincide with limits of the suitable land cover types for these species. The 19 bioclim layers and an elevation layer , niche models we unable to be generated in Maxent due to the low number of unique occurrence localities (4). Therefore, the known geographic range was used for niche similarity tests, rather than the Maxent output. The software quantifies niche similarity using two metrics: D [I, a measure derived from Hellinger distance. These metrics are calculated by comparing the estimated niche suitability values from individual pixels in Maxent model outputs, where those outputs have first been normalized such that all predicted suitability values in the geographic space sum to 1 [For all sister species pairs, we compared the Maxent outputs using the software ENMtools followintrics: D , and I, sum to 1 . Althougsum to 1 .D and I values were calculated for each of the pseudoreplicate models. The distribution of these similarity values was then compared to the D and I values calculated from the actual niche models for that species pair in the niche overlap test. This method tests the null hypothesis that the two species have equivalent ecological niches and is expected to be met only if both species tolerate exactly the same environmental conditions and have an equivalent set of environmental condition available to them [The niche identity test compares niche models generated with actual occurrence localities to pseudoreplicate models generated with points randomly selected from a pool of actual occurrence localities to determine if species pairs have equivalent niches. For the identity tests, 100 pseudoreplicates were created from the pooled localities for each pair of sister species and to them .D and I values were calculated for each pseudoreplicate model and the distribution of these values was compared to the niche overlap values calculated for the actual data. This method tests the null hypothesis that calculated niche overlap between two species is explained by differences in their environmental background. The null hypothesis is rejected if the calculated niche overlap falls outside the 95% confidence interval for the distribution of pseudoreplicate model values.The background similarity test compares differences in the environmental background of species pairs to determine if the two species are more or less similar than expected by chance. For each species pair, the niche model for the focal species is compared to a series of pseudoreplicate models generated by randomly sampling the 'background' (geographic range) of its sister species . In the K of Blomberg et al. [The phylogenetic signal for eacg et al. . The QVIg et al. . This apK is used to quantify the \"amount of phylogenetic signal relative to the amount expected for a character undergoing Brownian motion evolution along the specified topology and branch lengths\" [[K < 1 indicating low phylogenetic dependence of the variable and K > 1 indicating high phylogenetic signal of the variable. When K = 1, the variable exhibits the phylogenetic signal expected under the Brownian motion model (e.g. the null model). K was estimated for each of the 19 bioclim variables using the multiPhylosignal command in the picante (ver. 1.3) [engths\" [, page 73engths\" [. The staer. 1.3) R packagphyloclim ver. 0.8.1 [phyloclim. For each genus, a total of 1000 simulations were undertaken.We also tested for phylogenetic signal of niche differentiation by using an adaptation of the age-range correlation method of in whichr. 0.8.1 to generr. 0.8.1 as impleTLPC conceived and coordinated the study, participated in its design, undertook part of the statistical analyses and drafted the manuscript. HPM participated in the design of the study, performed part of the statistical and all GIS analyses and helped draft the manuscript. JJW participated significantly in the production of the data. LWC participated in the conception of the study. All authors read and approved the final manuscript.IsolonaVariation of bioclim variables BC1-6 for . Indicates the variation of bioclim variables BC1 to 6 for all sampled species in Isolona. West/Central African species: 1: Isolona congolana; 2: I. hexaloba; 3: I. pleurocarpa; 4: I. zenkeri; 5: I. campanulata; 6: I. cooperi; 7: I. dewevrei; 8: I. thonneri; 9: I. cauliflora. East African species: 10: I. heinsenii; 11: I. linearis. Malagasy species: 12: I. capuroni; 13: I. ghesquierei; 14: I. perrierii.Click here for fileIsolonaVariation of bioclim variables BC7-12 for . Indicates the variation of bioclim variables BC7 to 12 for all sampled species in Isolona. West/Central African species: 1: Isolona congolana; 2: I. hexaloba; 3: I. pleurocarpa; 4: I. zenkeri; 5: I. campanulata; 6: I. cooperi; 7: I. dewevrei; 8: I. thonneri; 9: I. cauliflora. East African species: 10: I. heinsenii; 11: I. linearis. Malagasy species: 12: I. capuroni; 13: I. ghesquierei; 14: I. perrierii.Click here for fileIsolonaVariation of bioclim variables BC13-18 for . Indicates the variation of bioclim variables BC7 to 12 for all sampled species in Isolona. West/Central African species: 1: Isolona congolana; 2: I. hexaloba; 3: I. pleurocarpa; 4: I. zenkeri; 5: I. campanulata; 6: I. cooperi; 7: I. dewevrei; 8: I. thonneri; 9: I. cauliflora. East African species: 10: I. heinsenii; 11: I. linearis. Malagasy species: 12: I. capuroni; 13: I. ghesquierei; 14: I. perrierii.Click here for fileIsolonaVariation of bioclim variable BC19 for . Indicates the variation of bioclim variable BC19 for all sampled species in Isolona. West/Central African species: 1: Isolona congolana; 2: I. hexaloba; 3: I. pleurocarpa; 4: I. zenkeri; 5: I. campanulata; 6: I. cooperi; 7: I. dewevrei; 8: I. thonneri; 9: I. cauliflora. East African species: 10: I. heinsenii; 11: I. linearis. Malagasy species: 12: I. capuroni; 13: I. ghesquierei; 14: I. perrierii.Click here for fileMonodoraVariation of bioclim variables BC1-6 for . Indicates the variation of bioclim variables BC1 to 6 for all sampled species in Monodora. West/Central African species 1: Monodora angolensis 2: M. crispata, 3: M. laurentii, 4: M. myristica, 5: M. tenuifolia, 6: M. undulata. East African species: 7: M. carolinae, 8: M. globiflora, 9: M. grandidieri, 10: M. hastipetala, 11: M. junodii, 12: M. minor, 13: M. stenopetala.Click here for fileMonodoraVariation of bioclim variables BC7-12 for . Indicates the variation of bioclim variables BC7 to 12 for all sampled species in Monodora. West/Central African species 1: Monodora angolensis 2: M. crispata, 3: M. laurentii, 4: M. myristica, 5: M. tenuifolia, 6: M. undulata. East African species: 7: M. carolinae, 8: M. globiflora, 9: M. grandidieri, 10: M. hastipetala, 11: M. junodii, 12: M. minor, 13: M. stenopetala.Click here for fileMonodoraVariation of bioclim variables BC13-18 for . Indicates the variation of bioclim variables BC7 to 12 for all sampled species in Monodora. West/Central African species 1: Monodora angolensis 2: M. crispata, 3: M. laurentii, 4: M. myristica, 5: M. tenuifolia, 6: M. undulata. East African species: 7: M. carolinae, 8: M. globiflora, 9: M. grandidieri, 10: M. hastipetala, 11: M. junodii, 12: M. minor, 13: M. stenopetala.Click here for fileMonodoraVariation of bioclim variable BC19 for . Indicates the variation of bioclim variable BC19 for all sampled species in Monodora. West/Central African species 1: Monodora angolensis 2: M. crispata, 3: M. laurentii, 4: M. myristica, 5: M. tenuifolia, 6: M. undulata. East African species: 7: M. carolinae, 8: M. globiflora, 9: M. grandidieri, 10: M. hastipetala, 11: M. junodii, 12: M. minor, 13: M. stenopetala.Click here for fileSpecies values. Indicates the number of unique data points as well as several ENM parameters for each species sampled in the molecular phylogeny used to generate the ENM. Bold values indicate species for which there were fewer than 8 unique data points.Click here for fileIsolona speciesPotential distribution of . Shows the rest of the models generated for Isolona species.Click here for fileMonodora speciesPotential distribution of . Shows the rest of the models generated for Monodora species.Click here for fileMonodora and Isolona speciesPotential distribution of the two last . Shows the rest of the models generated for Monodora and Isolona species.Click here for fileIsolonaDistribution of species in . Shows the geographical location of all data points for each species used in this study.Click here for fileIsolonaDistribution of species in . Shows the geographical location of all data points for each species used in this study.Click here for fileIsolona (continue from sup file 1) and MonodoraDistribution of species in . Shows the geographical location of all data points for each species used in this study.Click here for fileMonodora (continue from sup file 2)Distribution of species in . Shows the geographical location of all data points for each species used in this study.Click here for fileMonodora (continue from sup file 3)Distribution of species in . Shows the geographical location of all data points for each species used in this study.Click here for fileMonodora (continue from sup file 4)Distribution of species in . Shows the geographical location of all data points for each species used in this study.Click here for file"} +{"text": "Correction to:British Journal of Cancer (2011) 106, 133\u2013140; doi:10.1038/bjc.2011.504Upon publication of this paper earlier in Volume 106, the authors noticed an error in The authors would like to apologise for this error."} +{"text": "There was an error in the title of the article. The correct version of the title in the article is: Topographical Body Fat Distribution Links to Amino Acid and Lipid Metabolism in Healthy Obese WomenThe correct citation is: Martin F-PJ, Montoliu I, Collino S, Scherer M, Guy P, et al. (2013) Topographical Body Fat Distribution Links to Amino Acid and Lipid Metabolism in Healthy Obese Women. PLoS ONE 8(9): e73445. doi:10.1371/journal.pone.0073445"} +{"text": "Correction to: British Journal of Cancer (2010) 103, 1128\u20131135; doi:10.1038/sj.bjc.6605838During the final correction of the above paper prior to its publication in late 2010, an error was introduced in The publishers apologise for this mistake."} +{"text": "The middle initial is missing from the second author's name in the byline and citation. The correct spelling is: John S. DaviesThe correct citation is: Cleveland SB, Davies JS, McClure MA (2011) A Bioinformatics Approach to the Structure, Function, and Evolution of the Nucleoprotein of the Order Mononegavirales. PLoS ONE 6(5): e19275. doi:10.1371/journal.pone.0019275"} +{"text": "To the Editor:Sapovirus is the distinct genus within the family Caliciviridae; these viruses cause sporadic cases and outbreaks of gastroenteritis in humans worldwide . By using multiplex reverse transcription\u2013polymerase chain reaction (RT-PCR), 2 groups of diarrheal viruses were identified. The first group included astrovirus, norovirus, and sapovirus; the second group included rotavirus and adenovirus (http://sray.med.som.jhmi.edu/SCRoftware/simplot), the recombination site was identified at the polymerase-capsid junction. Before this junction, sequences of HU/5862/Osaka/JP and Sapporo/82 were highly homologous. However, homology between them was notably different after the junction, with a sudden drop in the identity for HU/5862/Osaka/JP. By using ClustalX, HU/5862/Osaka/JP shared a 96% identity in polymerase sequence and an 85% identity in capsid sequence with Sapporo/82. In contrast, homology was 99% in the capsid region between HU/5862/Osaka/JP and 8/DCC/Tokyo/JP/44. Since a polymerase sequence of 8/DCC/Tokyo/JP/44 was not available in GenBank because of the unsuccessful amplification, homology in the polymerase region between HU/5862/Osaka/JP and 8/DCC/Tokyo/JP/44 was unknown.The fecal specimen was positive for sapovirus. HU/5862/Osaka/JP clustered into the genogroup I genotype 8 (GI/8 the 8/DCC/Tokyo/JP/44 cluster) by usingAltogether, the findings underscored that HU/5862/Osaka/JP represented a novel, naturally occurring, recombinant sapovirus with GI/8 capsid and GI/1 polymerase. To determine whether the child was infected with this novel recombinant sapovirus or whether the novel recombinant sapovirus resulted from co-infection with 2 different viruses, Svppo (Sapporo/82-specific primer), Svdcc (8/DCC/Tokyo/JP/44-specific primer), and SLV5749 were used to amplify the capsid region (,Even though many molecular epidemiologic studies on sapovirus infection have been performed worldwide, reports documenting recombination in sapovirus are still limited. The first recombinant sapovirus identified was the Thai isolate Mc10 or the Japanese isolate C12 (In recent studies of sapovirus recombination, evidence for the location of the recombination event is lacking because of the distant geographic relationship of parent and progeny strains. HU/5862/Osaka/JP shared the closest sequences of polymerase and capsid with Sapporo/82 and 8/DCC/Tokyo/JP/44, respectively. Sapporo/82 was first isolated in 1982, and 8/DCC/Tokyo/JP/44 was isolated in 2000, both in Japan. Possibly, Sapporo/82 and 8/DCC/Tokyo/JP/44 were parental strains of HU/5862/Osaka/JP, and the event leading to the novel recombination might have occurred in Japan.The capsid region was used for genotype classification of sapovirus ("} +{"text": "The name of the second author was given incorrectly. The correct name is: Jean Michel Brunel. The correct citation is: Khelaifia S, Brunel JM, Drancourt M (2013) In-Vitro Archaeacidal Activity of Biocides against Human-Associated Archaea. PLoS ONE 8(5): e62738. doi:10.1371/journal.pone.0062738. The name is correctly reflected in the abbreviation in Contributions as it is."} +{"text": "The word \"Leishmaniasis\" is misspelled in the title. The correct title is: Monitoring Toxicity Associated with Parenteral Sodium Stibogluconate in the Day-Case Management of Returned Travellers with New World Cutaneous Leishmaniasis. The correct citation is: Wise ES, Armstrong MS, Watson J, Lockwood DN (2012) Monitoring Toxicity Associated with Parenteral Sodium Stibogluconate in the Day-Case Management of Returned Travellers with New World Cutaneous Leishmaniasis. PLoS Negl Trop Dis 6(6): e1688. doi:10.1371/journal.pntd.0001688"} +{"text": "The correct title and legend of Figure 2 presently appears with Figure 3. The correct, complete Figure 2 legend and title are:Time to resolution of viraemia.Kaplan \u2013 Meier survival analysis of time to resolution of plasma viraemia by treatment group (CQ or placebo) and population; A) Intention to treat population and B) Per Protocol population.doi:10.1371/journal.pntd.0000785.g003"} +{"text": "Acta Cryst. (2012), E68, m1025.Corrigendum to Acta Cryst. (2012), E68, m1025] is corrected.The author list in the paper by Mafud [ In the paper by Mafud (2012)"} +{"text": "Cryptococcus gattii, British Columbia, Canada, 1999\u20132007 . The article has been corrected online (www.cdc.gov/eid/content/16/2/251.htm).Some data were listed incorrectly in Table 1 and the text in the article Epidemiology of"} +{"text": "Correction to: British Journal of Cancer (2011) 104, 229\u2013234; doi:10.1038/sj.bjc.6606009Upon publication in early 2011, the authors noted the omission of an Acknowledgments section that they had intended to include. This is now included in full, below. The authors apologise for the original omission."} +{"text": "As a result of issues in the production process, there was an error in the name of the first author.The correct name of this author is: Leonardo Vieira NetoZAC1 and SSTR2 Are Downregulated in Non-Functioning Pituitary Adenomas but Not in somatotropinomas. PLoS ONE 8(10): e77406. doi:10.1371/journal.pone.0077406 The correct citation is: Vieira Neto L, Wildemberg LE, Colli LM, Kasuki L, Marques NV, et al. (2013)"} +{"text": "The word \"Protozoans\" is misspelled in the article title. The correct title is: On the Evolution of Hexose Transporters in Kinetoplastid Protozoans. The correct citation is: Pereira CA, Silber AM (2012) On the Evolution of Hexose Transporters in Kinetoplastid Protozoans. PLoS ONE 7(5): e36303. doi:10.1371/journal.pone.0036303"} +{"text": "There was a typographical error in the title and citation. The correct title is: Phenology of Scramble Polygyny in a Wild Population of Chrysomelid Beetles: The Opportunity for and the Strength of Sexual Selection. The correct citation is:Baena ML, Mac\u00edas-Ord\u00f3\u00f1ez R (2012) Phenology of Scramble Polygyny in a Wild Population of Chrysomelid Beetles: The Opportunity for and the Strength of Sexual Selection. PLoS ONE 7(6): e38315. doi:10.1371/journal.pone.0038315"} +{"text": "There was an error in the funding statement. The correct funding information is:http://www.cancerforskningsfond-umea.lions.se/), Ume\u00e5 University, Sweden (LP 09-1799). Funding for the Me-Can project was obtained from the World Cancer Research Fund (WCRF UK) (http://www.wcrf-uk.org/) for grant 2007/09 and from Wereld Kanker Onderzoek Fonds (WCRF NL) (http://www.wcrf.nl/) for grant R2010/247, as part of the WCRF International grant programme, and the Swedish Cancer Foundation (http://www.cancerfonden.se/sv/Information-in-English/)(2010/628). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.This study was supported by grants from Lion's Cancer Research Foundation ("} +{"text": "The word \"Level\" is misspelled in the article title. The correct title is: Associations between Perceived HIV Stigma and Quality of Life at the Dyadic Level: The Actor-Partner Interdependence Model. The correct citation is: Liu H, Xu Y, Lin X, Shi J, Chen S (2013) Associations between Perceived HIV Stigma and Quality of Life at the Dyadic Level: The Actor-Partner Interdependence Model. PLoS ONE 8(2): e55680. doi:10.1371/journal.pone.0055680"} +{"text": "Correction to: British Journal of Cancer (2011) 105, 682\u2013686; doi:10.1038/bjc.2011.276During the final editing of the above paper before its publication, errors were, unfortunately, introduced in equations 5 and 7. In both the equations, a minus sign had been deleted. The correct equations are now shown below.The authors and publishers apologise for any inconvenience this may have caused."} +{"text": "The name of the fourth author is incorrect. The correct name is: Masashi Kantake.The correct citation is: Jujo T, Sakao S, Tsukahara M, Kantake M, Maruoka M, et al. (2014) The Role of Matrix Metalloproteinase in the Intimal Sarcoma-Like Cells Derived from Endarterectomized Tissues from a Chronic Thromboembolic Pulmonary Hypertension Patient. PLoS ONE 9(1): e87489. doi:10.1371/journal.pone.0087489.The correct abbreviation of the fourth author's name in the Author Contributions statement is: MK."} +{"text": "There was an error in the article title. The correct title is: \"Testosterone Levels Are Negatively Associated with Fatherhood in Males, but Positively Related to Offspring Count in Fathers.\" The correct citation is: Pollet TV, Cobey KD, van der Meij L (2013) Testosterone Levels Are Negatively Associated with Fatherhood in Males, but Positively Related to Offspring Count in Fathers. PLoS ONE 8(4): e60018. doi:10.1371/journal.pone.0060018."} +{"text": "The name of the third author was incorrect. The correct name is: Thorhallur I Halldorsson.The correct citation is: Danielsen I, Granstr\u00f6m C, Halldorsson TI, Rytter D, Hammer Bech B, et al. (2013) Dietary Glycemic Index during Pregnancy Is Associated with Biomarkers of the Metabolic Syndrome in Offspring at Age 20 Years. PLoS ONE 8(5): e64887. doi:10.1371/journal.pone.0064887.The correct abbreviation in Contributions is: TIH."} +{"text": "Correction #1: Error in figure order. The images for Figures 7 and 8 were incorrectly switched. The image that appears as Figure 7 should be Figure 8, and the image that appears as Figure 8 should be Figure 7. The figure legends appear in the correct order.Correction #2: Error in Figure 1 legend.There is an error in the Figure 1 legend. The correct legend for Figure 1 is:1A and 1B: overlaid time courses of summarized microarray fluorescence for each yeast gene, as the of the mean-ratio , for the 0.7 h [11] and 5 h [10] period datasets, respectively. The bottom two panels show cluster averages for consensus and background clusters. The top panel shows the time courses of the dissolved O2 trace (DOT) in the culture medium in percent of the saturated concentration. Cluster colors and sizes (number of genes in each cluster) are given in the legend in Figure 1C. For clarity of visualization the time course data was normalized to a reference set that was selected for significant lack of oscillation . Individual time courses for each cluster are plotted in Figure S2. 1D: phase-phase plot comparing the phase-angles of all transcripts in the two experiments. The phase angles were shifted such that cluster A phase angles are just above 0\u00b0 in both datasets. Mapping back from frequency- to time-domain, we can locate the shifted phase angles of one cycle (0\u00b0 and 360\u00b0) in the time series plot , and use the same mapping in the top and right axes (in gray) of the phase-phase plot. The x- and y-extensions of each point scale with the transcript\u2019s scaled amplitude in the respective dataset, where the non-consensus clusters (lower case letters) have a smaller initial size. Dataset S1 provides raw summarized microarray intensities, and the clustering of all analyzed yeast genes.Correction #3: Errors in multiple URLs.There are several links in this article that are incorrect. The correct versions of these links are:The correct link found in the Genome Data Sources of the Discussion section is:http://www.tbi.univie.ac.at/~raim/data/2011/yeast/clusters/geneData.tar.gz [^] [^]The correct link found in the last paragraph of the Statistical DNA Profiles (SDP) section is:http://www.tbi.univie.ac.at/~raim/data/2011/yeast/clusters/geneData.tar.gz [^] [^]The correct links found in the Table S8 Supporting Information section are:http://www.tbi.univie.ac.at/~raim/data/2011/yeast/clusters/geneData.tar.gz [^] [^]andhttp://www.tbi.univie.ac.at/~raim/data/2011/yeast/clusters/ [^] [^]"} +{"text": "The third author's name is spelled incorrectly. The correct name is: Zainularifeen Abduljaleel. The correct citation is:Alanazi M, Pathan AAK, Abduljaleel Z, Shaik JP, Alabdulkarim HA, et al. (2013) Association between PARP-1 V762A Polymorphism and Breast Cancer Susceptibility in Saudi Population. PLoS ONE 8(12): e85541. doi:10.1371/journal.pone.0085541There is also an error in the second sentence of the Materials and Methods section. Patients included in the study had breast cancer, not cardiovascular disease. The correct sentence reads: These encompassed 99 patients with breast cancer and 96 healthy controls."} +{"text": "There was an error in the title of the article.Apostichopus japonicusThe correct title is: RNA-Seq Reveals Dynamic Changes of Gene Expression in Key Stages of Intestine Regeneration in the Sea Cucumber Apostichopus japonicus. PLoS ONE 8(8): e69441. doi:10.1371/journal.pone.0069441 The correct citation is: Sun L, Yang H, Chen M, Ma D, Lin C (2013) RNA-Seq Reveals Dynamic Changes of Gene Expression in Key Stages of Intestine Regeneration in the Sea Cucumber"} +{"text": "Corrigenda for five articles. Acta Cryst. (2011a), E67, m1496; Acta Cryst. (2011b), E67, m1497; Acta Cryst. (2011c), E67, m1498] and Chiririwa & Muller .The affiliation of one of the authors and a source of funding are both added in the following papers: Chiririwa & Meijboom ["} +{"text": "BN82451 is patented (P\u00e4tent portfolio made up of different family of patents: Novel of 2-(iminomethyl)amino-phenyl derivatives intermidiates; e.g. US6586454; 5-membered heterocycle derivatives: e.g. WO01/26656, WO2005/035510; Use of thiazole derivatives for preparing a medicament for protecting the mitochondria: e.g. WO 03/009843; Thiazoles derivatives for the treatment of dyskinesia: e.g. WO2007/006942 and Thiazoles derivatives for the treatment of restless legs syndrome: WO2007/006941) and is being developed by Ipsen. This does not alter our adherence to all the PLOS ONE policies on sharing data and materials.There is an error in the Competing Interests statement. The correct statement is: Co-authors Auguet M, Chabrier PE, Spinnewyn B are employed by IPSEN Innovation. The research reported in this manuscript has been funded in part by IPSEN Innovation ("} +{"text": "There was an error in the funding statement. The correct funding information is:This research is supported by UM High Impact Research Grant UM-MOHE UM.C/HIR/MOHE/08 from the Ministry of Higher Education Malaysia and High Impact Research Grant UM.C/625/1/HIR/003/1 from the University of Malaya. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."} +{"text": "MSM authors meant to be a check-list to adhere to in their submissions.This is a brief write-up for While writing, do look into the following:Abstract at the beginning [100-150 words].Add an Key Words after Abstract [4-6 key words from the text], separated by semi-colons.Add Author Affiliations, Correspondence address, and email id below Key Words.Add Introduction. Break the write-up into suitable paragraphs, with headings and subheadings as required.Start article with Flowchart of the paper, at the end of the write up but before Concluding Remarks, as a figure that includes salient features of the paper and its important findings, so readers can get a pictorial overview of the paper and the thought as it flows through the text. This helps both the reader understand the author and helps the author clarify his thoughts, both for the reader, and most importantly, for himself. For flowchart, see any MSM paper 2010 onwards.Add Concluding Remarks [100-150 words in 1-2 paragraphs which conclude the arguments and put things in a nutshell for the reader].Add Take Home message [3-4 lines] after concluding remarks. Take home should be the absolute gist of the paper and less than concluding remarks.Add Conflict of Interest [COI] statement, if any, after Take Home message .Add Declaration that it is your original unpublished work, not submitted for publication elsewhere.Add acknowledgements, if any, after DeclarationAdd Points 1-10 are mandatory requirements for any paper, including my own.Why Concluding remarks and Take Home?The reason why we insist on Concluding remarks and Take Home is to present the final summary and absolute gist of the paper toEncourage the reader to give the paper a second read.Re-understand the paper in a clearer manner after reading it the first time: often so many ideas are presented in the text of the paper that the reader may be drowned in attempting to make sense; Concluding remarks and Take home message help a reader get a clearer grasp.Start the first reading of the paper with some grounding in what the paper is trying to essentially say.Help clarify the author\u2019s thoughts to himself.All these methods make the author intelligible, the paper readable, and the thoughts digestible.Questions that the Paper Raises , after Acknowledgement/ Declaration.Add About the author with a recent high-resolution jpg photo to go along with your write-up, after Questions that the Paper RaisesAdd 100-150 words References in Modified Harvard style after About the author.Add Full length article: A paper may ordinarily not be more than 4500 words, complete with an abstract (150 words maximum), introduction and concluding remarks (150 words maximum). Each Article may contain not more than 30 references, 50% of which are 2000 AD or after. [This is so that recent work does not go unnoticed.]Editorial: An editorial may ordinarily not be more than 2500 words with max 30 references [50% of which are 2000 or later].Feature and Communications: Musings max 1500 words. Reflections, The Looking Mirror max 2500 words, with 20 references [50% 2000 or later]. Letters to editor word limit is 1000 words. MSM Book review word limit is 2000 words.Microsoft WORD, Times New Roman 12pt font size.All submissions in Review: Submissions are subject to editorial and peer review.Deadlines: Authors are advised to stick to deadlines of submissions strictly. As also carry out editorial corrections and peer review modifications as per time schedule given by the editorial office as follows: [The deadline for this submission is: \u2026]ReferencesAuthors must check authenticity of references and other facts quoted before submission. They must specially check that all references in text are included in reference list, and vice versa. No reference not in text should occur in the reference list. Do not prepare a separate Bibliography or Internet Citation list. They should be incorporated in the reference list itself, and only if quoted in the text.et al., 2011).In the text, references should occur with only surnames, and in the following form e.g. for one name ; for two names ; for more than 2 names and (b) maybe used after the year of publication. For example:Woodruff T., (2004a), Letters: The medical profession and the pharmaceutical industry: when will we open our eyes? eMJA, 181(8), p458-459.Woodruff T.G., (2004b), Pharmaceutical marketing, the PBS, and patient care, New Doctor, 81, p21-22.For Editorials:Angell M., (2000), Is Academic Medicine for Sale? , N Engl J Med, 342(20), p1516-1518.For Papers:iii.a. For a single authorSchafer A., (2004), Biomedical conflicts of interest: a defence of the sequestration thesis-learning from the cases of Nancy Olivieri and David Healy, J Med Ethics, 30, p8-24.et al may be used only after naming 6 authors]iii.b. For more than one author , The Encyclopedia of Philosophy, Vol 5 & 6, p343. New York: Macmillan.iv.e. Where a book in translatedHeidegger M., (1927/1962), Being and Time (Sein und Zeit). Translated by J. Macquarrie and E. Robinson. New York: Harper and Row.iv.f. Where an older book is republishedHeidegger M., (1927/1962), Being and Time (Sein und Zeit). Translated by J. Macquarrie and E. Robinson. New York: Harper and Row.iv.g. Where a book has been editedFlew A., (Ed.) (1983), A Dictionary of Philosophy. London: Pan Books in association with The Macmillan Press.iv.h Where a book has been edited by someone other than the authorHume D., (1739/1888), A Treatise of Human Nature. (Ed.) L Selby-Bigge. Oxford: Oxford University Press.iv.i. Where a book has been published by 2 publishersFlew A., , A Dictionary of Philosophy. London: Pan Books in association with The Macmillan Press.iv.j. Where a book is published by one but reprinted by anotherJames W., (1890/1999), The Principles of Psychology. Ch 9. (2 vols.). New York: Henry Holt .For Web References:National Institute of Mental Health, (2011), Schizophrenia. Available at http://www.nimh.nih.gov/health/topics/schizophrenia/index.shtml [Accessed 20 Dec 2010.].For News Paper/Magazine Articles:Harris G., (2004), As doctor writes prescription, drug company writes a check, New York Timesc, June 27, A1.Personal communicationsPersonal communication may not ordinarily be quoted as a reference. If at all done, it should be with the written permission of the communicator.Copyright21. Mens Sana Monographs. However, authors are permitted to disseminate their published work for non-commercial purposes freely by post or electronic means, and to put it up on their personal/institutional web sites for the information and knowledge of their viewers and fellow researchers, with due acknowledgement of the original source of publication (MSM). Authors should contact and obtain permission from MSM if they intend publication of their work in any other form later. Such requests are ordinarily granted on due acknowledgement of publication in MSM.Copyright of material published rests with the Typical Paper, Editorial, and other submissions22. For a typical paper, seehttp://www.msmonographs.org/article.asp?issn=0973-1229;year=2009;volume=7;issue=1;spage=10;epage=19;aulast=ChadwickAnd/orhttp://www.msmonographs.org/article.asp?issn=0973-1229;year=2010;volume=8;issue=1;spage=30;epage=51;aulast=VarmaFor a typical editorial, seehttp://www.msmonographs.org/article.asp?issn=0973-1229;year=2010;volume=8;issue=1;spage=6;epage=16;aulast=SchwartzFor a typical Book review, seehttp://www.msmonographs.org/article.asp?issn=0973-1229;year=2010;volume=8;issue=1;spage=146;epage=150;aulast=AndradeMSM Poem, seeFor typical http://www.msmonographs.org/article.asp?issn=0973-1229;year=2008;volume=6;issue=1;spage=277;epage=278;aulast=BrennerFor a typical Journalology article, seehttp://www.msmonographs.org/article.asp?issn=0973-1229;year=2008;volume=6;issue=1;spage=226;epage=236;aulast=HoeyFor a typical article in the Looking Glass, seehttp://www.msmonographs.org/article.asp?issn=0973-1229;year=2008;volume=6;issue=1;spage=41;epage=47;aulast=GilmanFor a typical article in Reflections, seehttp://www.msmonographs.org/article.asp?issn=0973-1229;year=2008;volume=6;issue=1;spage=135;epage=145;aulast=NarlikarFor a typical article in Musings, seehttp://www.msmonographs.org/article.asp?issn=0973-1229;year=2008;volume=6;issue=1;spage=131;epage=134;aulast=ArnoldFor typical obituary, seehttp://www.msmonographs.org/article.asp?issn=0973-1229;year=2008;volume=6;issue=1;spage=281;epage=284;aulast=RavehBest wishes."} +{"text": "The name of the third author was given incorrectly. The correct name is: Duolao Wang. The correct citation is: Gao W, Yan H, Wang D, Dang S, Pei L (2013) Severity of Anemia among Children under 36 Months Old in Rural Western China. PLoS ONE 8(4): e62883. doi:10.1371/journal.pone.0062883.In addition, the first sentence in Contributions was incorrectly separated into two sentences. The correct sentence is: \"Conceived and designed the experiments: WG SD LP.\""} +{"text": "LCH is the rare disease involving clonal proliferation of Langerhans cells, abnormal cells deriving from bone marrow and capable of migrating from skin to lymph nodes. The clinical presentation of LCH may occur in multiple organs: bone, skin, lymph nodes or pituitary gland, but clinical presentation of LCH rarely occurs in multiple endocrine systems.We presented a special case who was diagnosed with LCH and presentation of LCH occurred in multiple systems: pituitary gland, thyroid, adrenal gland.A 11 years old girl was hospitalized for lump on the neck. Past history: one year ago, she was diagnosed with autoimmune polyendocrine syndromes and treated with hormone replacement . Physical examination showed: she had a swollen lump on her neck, she had a temperature of 38\u00b0 C. She had no polyuria, no polydipsia. Her height was 141cm (-0.29 SD); her weight was 37 kg; her BMI was 18.5 (50th \u2013 70th) .She had normal growth velocity and normal pubertal development. Laboratory evaluation revealed : WBC : 10.3 \u00d7 109 /l ; CRP : 105.78 mg/l ; serum cortisol at 8 a.m : 16.3 nmol/l ; T3 : 1.8 nmol/l , T4 : 135.9 nmol/l , TSH : 0.002 mUI/ml ; blood osmotic pressure : 279 moms/kg, urinary osmotic pressure : 127 mosm/kg; plasma glucose level and electrolyte were normal. An MRI of brain showed: thickened pituitary stalk. A biopsy of the lump on her neck showed: features of Langerhans Cell; skeletal and long bone radiograph showed no osteolytic lesion.She was treated with hormone replacement and chemotherapy.Written informed consent was obtained from the patient's parent or guardian for publication of this Case report (and any accompanying images). A copy of the written consent is available for review by the Editor-in-Chief of this journal."} +{"text": "The correct citation is: Llibre JM, Raffi F, Moyle G, Behrens G, Bouee S, Reilly G, et al. (2016) An Indirect Comparison of Efficacy and Safety of Elvitegravir/Cobicistat/Emtricitabine/Tenofovir Disoproxil Fumarate and Abacavir/Lamivudine + Dolutegravir in Initial Therapy. PLoS ONE 11(5): e0155406. doi:The Abstract is missing a sentence at the end of the \u201cResults\u201d subheading. The additional sentence is: In an indirect comparison, we found no statistically significant differences in efficacy, serious adverse events, drug related adverse events, drug related serious adverse events, death or selection of viral resistance between E/C/F/TDF and ABC/3TC + DTG in initial therapy.There are numerical errors in the second sentence of the first paragraph in the Results section under the subheading \u201cVirological failure and resistance.\u201d The correct sentence is: In GS-US-236-0102, 7% of patients on E/C/F/TDF and 10% of subjects on EFV/FTC/TDF were considered virological failures at week 144, while in SINGLE, rates were 9% and 8% for ABC/3TC + DTG and EFV/FTC/TDF, respectively.There are errors in the first two sentences of the Conclusion. The correct sentences are: With the limitation that we did not perform a systematic review we can conclude that: The indirect efficacy comparisons do not show significant differences between E/C/F/TDF and ABC/3TC + DTG. For efficacy, the difference between both regimens at week 48, 96 and 144 were small and not statistically significant; Resistance and all safety results (except for discontinuation due adverse events) also do not show significant differences between the 2 regimens.The captions for Figs 3, 4 and 5 appear incorrectly in the published article. Please see Figs"} +{"text": "Nature Communications6: Article number: 10106 10.1038/ncomms10106 (2015); Published: 12032015; Updated: 03032016The original version of this Article contained an error in the spelling of the author Jennifer S. Stevens, which was incorrectly given as Jennifer J. Stevens. Furthermore, in Fig. 5, the titles for panels a and b were inadvertently switched. Both of these errors have now been corrected in the PDF and HTML versions of the Article."} +{"text": "Correction to:Cell Discovery (2015) 1, 15005; doi:10.1038/celldisc.2015.5; published online 19 May 2015During web production, there was an error in Supplementary information: Supplementary Tables S1 and S2 were omitted. The files are now installed in the online version of the paper.We apologize for any inconvenience that may have been caused by this error."} +{"text": "February 7 is National Black HIV/AIDS Awareness Day, an observance intended to raise awareness of human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS) and encourage action, such as HIV testing, to reduce the disproportionate impact of HIV/AIDS on blacks or African Americans in the United States. Two of the three goals of the National HIV/AIDS Strategy are to reduce new HIV infections and HIV disparities .Compared with other races and ethnicities, blacks had the highest HIV incidence in 2010, with an estimated rate of 68.9 per 100,000 population, which was nearly eight times the estimated rate of 8.7 among whites . Among bhttp://www.cdc.gov/features/blackhivaidsawareness. Information about blacks and HIV/AIDS is available at http://www.cdc.gov/hiv/risk/racialethnic/aa/index.html.Information about National Black HIV/AIDS Awareness Day is available at"} +{"text": "Regarding the paper titled: \u2018Empirical evidence of the effect of school gathering on the dynamics of dengue epidemics\u2019 by Carlos M Hern\u00e1ndez-Su\u00e1rez and Oliver Mendoza-CanoCitation: Glob Health Action 2016, 9: 28026 - http://dx.doi.org/10.3402/gha.v9.28026Published in Global Health Action 06 Jan 2016. In the Results and Discussion section, in Figure 5, the legend reads \u201cCleaning campaign in 2006 started at week 22.\u201d it should say \u201cCleaning campaign in 2007 started at week 22\u201d.The legend currently reads:Fig. 5. Reported accumulated dengue incidence in the state of Colima for the years 2006\u20132008. Source: National Center for Epidemic Surveillance. The beginning of the school year is at week 33. Cleaning campaign in 2006 started at week 22. Source: CENAVECE (12).The legend should read:Fig. 5. Reported accumulated dengue incidence in the state of Colima for the years 2006\u20132008. Source: National Center for Epidemic Surveillance. The beginning of the school year is at week 33. Cleaning campaign in 2007 started at week 22. Source: CENAVECE (12)."} +{"text": "Scientific Reports5: Article number: 13746; 10.1038/srep13746published online 09082015; updated: 01272016The Acknowledgements section in this Article is incomplete.\u201cThe study has been supported by EU (Marie Curie FP7 project #269140), Academy of Finland (grant #288151 and #265015), EPSRC (grant EP/M029042/1), and Russian Science Foundation (grant no. 14-22-00136). P.G.K. thanks Ministry of Education and Science of the Russian Federation (Grant No. 14.Z50.31.0009). Authors are grateful to Amin Abdolvand for valuable comments on the manuscript.\u201dshould read:http://dx.doi.org/10.5258/SOTON/384152).\u201d\u201cThe study has been supported by EU (Marie Curie FP7 project #269140), Academy of Finland (grant #288151 and #265015), EPSRC (grant EP/M029042/1), and Russian Science Foundation (grant no. 14-22-00136). P.G.K. thanks Ministry of Education and Science of the Russian Federation (Grant No. 14.Z50.31.0009). Authors are grateful to Amin Abdolvand for valuable comments on the manuscript. The data for this work is accessible through the University of Southampton Institutional Research Repository ("} +{"text": "The publisher apologizes for the error. The correct name is: Eva C. Arnspang. The correct citation for the original article is: Arnspang EC, Kulatunga P, Lagerholm BC (2012) A Single Molecule Investigation of the Photostability of Quantum Dots. PLoS ONE 7(8): e44355. doi: 10.1371/journal.pone.0044355.The first author\u2019s name in the text of the Correction notice published on April 9"} +{"text": "This article was republished on May 21, 2014, to correct an error in the title of the article.The title should read:Darevskia dahliClonal Diversity and Clone Formation in the Parthenogenetic Caucasian Rock Lizard The citation should read:Darevskia dahli. PLoS ONE 9(3): e91674. doi:10.1371/journal.pone.0091674Vergun AA, Martirosyan IA, Semyenova SK, Omelchenko AV, Petrosyan VG, et al. (2014) Clonal Diversity and Clone Formation in the Parthenogenetic Caucasian Rock Lizard Please download this article again to view the correct version. The originally published, uncorrected article and republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished corrected article.(PDF)Click here for additional data file."} +{"text": "Acta Cryst. (2014), E70, o133.Erratum to Acta Cryst. (2014), E70, o133], the conformation of the thiazine ring was reported incorrectly.In the paper by Yennawar & Silverberg [ The authors of Yennawar & Silverberg (2014)"} +{"text": "February 7 is National Black HIV/AIDS Awareness Day, an observance intended to raise awareness of human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS) and encourage action to reduce the disproportionate impact of HIV/AIDS on blacks in the United States. Compared with other races and ethnicities, blacks had the highest HIV incidence in 2010, with an estimated rate of 68.9 per 100,000 population, nearly eight times the estimated rate of 8.7 among whites of persons living with HIV, followed by whites (33.0%), and Hispanics (19.3%) (http://www.cdc.gov/features/blackhivaidsawareness. Information regarding blacks and HIV/AIDS is available at http://www.cdc.gov/hiv/risk/racialethnic/aa/index.html.Information regarding National Black HIV/AIDS Awareness Day is available at"} +{"text": "Dpy19l2 Knock-Out Mice Indicates That Sun5 Is Not Involved in Acrosome Attachment to the Nuclear Envelope. PLoS ONE 10(3): e0118698. doi:10.1371/journal.pone.0118698There is an error in the third author\u2019s name. The correct surname for the third author is: Abi Nahed. The correct given name is: Roland. The correct citation is: Yassine S, Escoffier J, Abi Nahed R, Pierre V, Karaouzene T, Ray PF, et al. (2015) Dynamics of Sun5 Localization during Spermatogenesis in Wild Type and The publisher apologizes for the error."} +{"text": "AbstractLimnatisLimnatisnilotica (LimnatisbacescuiLimnatispaluda (Limnatis leeches are well known species of endoparasitic leeches, Limnatisnilotica was recorded only once in Kazakhstan (nilotica , Limnatispaluda . The firspaluda . The secspaluda . Althougzakhstan .Limnatis from Almaty Province, Kazakhstan are identified as Limnatispaluda. This is the first record of Limnatispaluda from Kazakhstan. Mitochondrial COI and 12S data demonstrated that the present specimens are genetically close to an Israeli specimen identified as Limnatisnilotica. In addition, molecular data suggest that some Limnatis specimens whose DNA sequences have been reported were misidentified. According to the observed phylogenetic relationships, the taxonomic status of the known Limnatis species should be revisited.Specimens of the genus LimnatisLimnatis: L.nilotica and was stated to be a cattle leech there were determined for 2 specimens from Almaty Province. The extraction of genomic DNA and DNA sequencing methods followed Sequences of mitochondrial COI as well as 12S, tRNALimnatis, 10 previously published sequences were obtained from the INSDC for use in molecular phylogenetic analyses Type status:Other material. Occurrence: catalogNumber: Z700; recordedBy: Kanto Nishikawa; individualCount: 1; sex: hermaphrodite; Taxon: scientificName: Limnatispaluda ; Location: country: Kazakhstan; stateProvince: Almaty; verbatimLocality: Suygaty Valley, Almaty Province, Kazakhstan; decimalLatitude: 43.512778; decimalLongitude: 78.9708331; Identification: identifiedBy: Takafumi Nakano; Event: eventDate: 2013-06-21; Record Level: institutionCode: KUZType status:Other material. Occurrence: catalogNumber: Z701; recordedBy: Kanto Nishikawa; individualCount: 1; sex: hermaphrodite; Taxon: scientificName: Limnatispaluda ; Location: country: Kazakhstan; stateProvince: Almaty; verbatimLocality: Suygaty Valley, Almaty Province, Kazakhstan; decimalLatitude: 43.512778; decimalLongitude: 78.9708331; Identification: identifiedBy: Takafumi Nakano; Event: eventDate: 2013-06-21; Record Level: institutionCode: KUZType status:Other material. Occurrence: catalogNumber: Z702; recordedBy: Kanto Nishikawa; individualCount: 1; sex: hermaphrodite; Taxon: scientificName: Limnatispaluda ; Location: country: Kazakhstan; stateProvince: Almaty; verbatimLocality: Suygaty Valley, Almaty Province, Kazakhstan; decimalLatitude: 43.512778; decimalLongitude: 78.9708331; Identification: identifiedBy: Takafumi Nakano; Event: eventDate: 2013-06-21; Record Level: institutionCode: KUZType status:Other material. Occurrence: catalogNumber: Z703; recordedBy: Kanto Nishikawa; individualCount: 1; sex: hermaphrodite; Taxon: scientificName: Limnatispaluda ; Location: country: Kazakhstan; stateProvince: Almaty; verbatimLocality: Suygaty Valley, Almaty Province, Kazakhstan; decimalLatitude: 43.512778; decimalLongitude: 78.9708331; Identification: identifiedBy: Takafumi Nakano; Event: eventDate: 2013-06-21; Record Level: institutionCode: KUZBody firm, muscular, with constant width in caudal direction, dorsoventrally compressed, BL 22.64\u201336.73 mm, BW 4.47\u20139.82 mm Fig. 22.Somite I completely merged with prostomium Fig. a. somite333355555Male gonopore in XI b5/b6 Fig. . Female Eyes 5 pairs, in parabolic arc; 1st and 2nd pairs on II + III, 3rd pair on IV (a1 + a2), 4th pair on V (a1 + a2), and 5th pair on VI a2 . In the neighbour-joining tree based on COI sequences, this monophyletic lineage and one sequence obtained from the Afghan L.paluda formed a well-recovered clade (BS = 100%). In addition, the neighbour-joining trees revealed that sequences obtained from L.nilotica collected in Namibia and Croatia do not form a monophyletic group.Neighbour-joining trees generated based on both the COI Fig. and 12S L.paluda and the Israeli L.nilotica was 0.2% of teeth on each jaw (the latter species has numerous teeth on each jaw). He also mentioned that the numbers of teeth in L.paluda and L.nilotica never overlapped. L.paluda was at most 45, but that of L.nilotica ranged from 45 to 60. However, L.nilotica possessed 30\u201350 teeth on each jaw. Based on the specimens collected in Azerbaijam, Kazakhstan and Uzbekistan, L.nilotica bore 38\u201340 teeth on each jaw. In addition, L.bacescui possessed 50\u201354 teeth on each jaw . In addition, it is highly likely that L.cf.nilotica of Croatia and Bosnia and L.cf.nilotica of Namibia do not belong to the same species, because those specimens are paraphyletic consistently in the present phylogenetic trees, and the former is highly diverged from the latter (11.9% in COI and 3.9% in 12S). These uncorrected p-distance values are greater than those between L.paluda and the Namibian Limnatis species (7.3\u20137.4% in COI and 2.8% in 12S) as well as L.paluda and the Balkan Limnatis specimens (9.2\u20139.7% in COI and 2.8% in 12S).As mentioned above, the taxonomic identities of ioned in , becauseLimnatispaluda analysed in this study have low genetic divergences (0.2\u20130.5% in COI and 0% in 12S). The COI uncorrected p-distances between the present Kazakhstani specimens and the Israeli specimen are lower than that between the former and the Afghan specimen (0.5%) and that between the latter and the Afghan L.paluda (0.6%). The collection locality in Kazakhstan is ca. 4,000 km from Israel, and ca. 2,000 km from Afghanistan. In contrast, Israel is at most ca. 3,500 km from Afghanistan. Because few DNA sequences of L.paluda are available, it may be difficult to reveal its detailed genetic structure. However, the results of the mitochondrial genetic analyses at least shed light on the discordance between the COI genetic divergence between the Kazakhstani L.paluda and the Israeli specimen and the geographic distance between the collection localities. Limnatis species are endoparasitic leeches that attach to the nasopharyngeal cavities of mammals, they likely achieve long-distance dispersal. Except when they parasitise the mammalian nasopharyngeal cavities, Limnatis species generally inhabit a freshwater environment. One of the routes of the Silk Road passed through the Ili River Depression (Limnatispaluda have possibly dispersed by means of freshwater places along the trade route as stepping stones, and thus human activities may have influenced the distribution of L.paluda. In either case, further taxonomic studies of Limnatis species should be performed to clarify their taxonomic positions. In addition, future molecular studies should elucidate the biogeographical history of Limnatispaluda.It is noteworthy that the specimens of pression , near th"} +{"text": "Treatment of juvenile idiopathic arthritis (JIA) has changed dramatically since the introduction of biologics in 1999. Because of more insight in the cytokines involved in JIA the number of available biologic agents increased. Together with the introduction of these new drugs, new insights in the optimal treatment of JIA indicate that earlier and more aggressive therapy is associated with better outcomes. Whether these developments with regard to biologic treatment have resulted in better patients' outcomes in daily practice is not yet reported.To evaluate trends in prescription of biologics and influence on outcomes of Dutch JIA patients that started their first biologic between 1999 and 2010.The Arthritis and Biologicals in Children register aims to include all JIA patients in the Netherlands who use or used biologic agents since 1999. Patients were divided in time periods according to the year of introduction of first biologic agent. Trends in characteristics of patients before introduction of first biologic and effectiveness of the first biologic were analyzed over a 12 year period.343 non-systemic and 86 systemic JIA patients started at least 1 biologic agent between 1999 and 2010. Etanercept remained biologic of first choice for non-systemic JIA and anakinra has become first choice for systemic JIA. The use of systemic prednisone and synthetic disease-modifying anti-rheumatic drugs (besides methotrexate) prior to biologics decreased. During these 12 years of observation, biologics were prescribed after shorter disease durations; the proportion of patients with less than 1.5 years of disease duration before start of the first biologic agent increased from zero in the years 1999-2001 to 31% in 2008-2010. Disease activity and acquired sequelae at baseline decreased with regard to number of joints with arthritis (median of 18 in 1999-2001 decreased to 5 in 2008-2010), number of joints with limited motion (median of 12 in 1999-2001 decreased to 3 in 2008-2010) and functional disability (CHAQ) scores (median score of 1.8 in 1999-2001 decreased to 1.1 in 2008-2010). In systemic JIA biologics are now being introduced in patients with higher ESR values. These changes resulted in more patients with inactive disease and less joints with limited motion after 3 and 15 months of treatment in all JIA categories.Biologics are prescribed increasingly, are introduced earlier during the disease course and in JIA patients with lower disease activity. These changes are accompanied by better short-term disease outcomes. Etanercept remains biologic of first choice for non-systemic JIA and anakinra has become first choice for systemic JIA.M. Otten Grant/Research Support from: Abbott, Novartis, Roche, Pfizer, Consultant for: Roche, J. Anink: None declared., F. Prince: None declared., M. Twilt: None declared., S. Vastert Consultant for: Novartis, R. Ten Cate Grant/Research Support from: Pfizer, Consultant for: Pfizer, E. Hoppenreijs: None declared., W. Armbrust: None declared., S. Gorter: None declared., P. Van Pelt: None declared., S. Kamphuis Grant/Research Support from: Pfizer, glaxosmithkline, K. Dolman: None declared., J. Swart: None declared., J. Van den Berg: None declared., Y. Koopman-Keemink: None declared., M. Van Rossum: None declared., N. Wulffraat Grant/Research Support from: Pfizer, Novartis, Roche, abbvie, L. Van Suijlekom-Smit Grant/Research Support from: Dutch Board of Health Insurances, Dutch Arthritis Association, Pfizer, Abbott, Consultant for: Roche, Novartis."} +{"text": "Nucleic Acids Res. 2014 Feb;42(3):1831-44. doi: 10.1093/nar/gkt1032The authors would like to apologize for an error in the caption of Figure"} +{"text": "Reference 27 is omitted from the last sentence of the second last paragraph of the Methods section. The sentence should read: In addition, as a supplementary analysis, we made these comparisons for estimates from two models reported by Lazer et al., who developed regression models to improve upon GFT estimates .http://dx.doi.org/10.7910/DVN/24823 UNF:5:BJh9WzZQNEeSEpV3EWs+xg== IQSS Dataverse Network [Distributor] V1 [Version].The reference is: Lazer D, Kennedy R, King G, Vespignani A (2014) Replication data for: The parable of Google Flu: Traps in big data analysis. Available: In the \"Estimate\" column of S2 Table(DOCX)Click here for additional data file."} +{"text": "Nature Communications6: Article number: 7870 10.1038/ncomms8870 (2015); Published: 07282015; Updated: 06222016In Fig. 1b of this Article, the sex of patient 2.1 in family A is incorrect, and should be depicted as male. The correct version of this figure appears below.Figure 1"} +{"text": "Correction to:British Journal of Cancer (2016) 114, 88\u201395; doi:10.1038/bjc.2015.314; published online 10 December 2015British Journal of Cancer, one of the authors identified an error in the spelling of their name. Margreet L\u00fcchtenborg appears correctly in the author list above.Upon publication of the above paper in the"} +{"text": "Effect modification of FADS2 polymorphisms on the association between breastfeeding and intelligence: protocol for a collaborative meta-analysis. BMJ Open 2016;6:e010067. This paper has been resupplied with the correct license statement.Hartwig FP, Davies NM, Horta BL,"} +{"text": "International Journal of Environmental Research and Public Health [http://www.mdpi.com/1660-4601/11/11/11879.The authors wish to update the Acknowledgments in their paper published in c Health , doi:10."} +{"text": "In the online version of the article, a space was erroneously omitted from the second author's name. The correct name is Md. Imtaiyaz Hassan. The correct citation is:Shahbaaz M, Hassan MI, Ahmad F (2013) Functional Annotation of Conserved Hypothetical Proteins from Haemophilus influenzae Rd KW20. PLoS ONE 8(12): e84263. doi:10.1371/journal.pone.0084263The name and citation are correct on the PDF."} +{"text": "Nature Communications6: Article number: 10224 10.1038/ncomms10224 (2015); Published: 12212015; Updated: 03092016a contain black numerical labels that are incorrect, and light blue labels that are correct. A revised version of Fig. 5, with correct labels in black throughout, appears below.In Fig. 5 of this article, the three dot plots in the first row of panel Figure 5"} +{"text": "The following information is missing from the Funding section: This work was supported by Taylor's University through its Postgraduate Research Scholarship Programme to MSWP. The complete, correct funding information is as follows: This work was supported by Exploratory Research Grants Scheme ERGS/1/2012/STG08/TAYLOR/03/2) to YYC, and Fundamental Research Grant Scheme (FRGS/2/2010/SKK/TAYLOR/03/1) to YYC. This work was supported by Taylor's University through its Postgraduate Research Scholarship Programme to MSWP. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."} +{"text": "Database 2016 (2016), doi:10.1093/database/baw080The URL present in the abstract of the above article was included by mistake, and this has now been corrected online. The publisher apologizes for this error."} +{"text": "A 9-valent human papillomavirus (HPV) vaccine was licensed for use in females and males in the United States in December 2014 .http://www.cdc.gov/vaccines/who/teens/downloads/9vHPV-guidance.pdf).In February 2015, the Advisory Committee on Immunization Practices (ACIP) recommended 9-valent HPV vaccine as one of three HPV vaccines that can be used for routine vaccination of females and one of two HPV vaccines for routine vaccination of males. ACIP recommendations were published in a March 2015 report . Additio"} +{"text": "Additionally, this study was supported by the Grant Agency of the Czech Republic (Project GPP505/12/P647). Daniel Ricard was supported by project CZ.1.07/2.3.00/30.0032 , co-financed by the European Social Fund and the state budget of the Czech Republic.The following information is missing from the Funding section: This study received institutional support from the Czech Academy of Sciences and the CEKOPOT project (CZ.1.07/2.3.00/20.0204), co-financed by the European Social Fund and the state budget of the Czech Republic. Additionally, this study was supported by the Grant Agency of the Czech Republic (Project GPP505/12/P647). Daniel Ricard was supported by project CZ.1.07/2.3.00/30.0032 , co-financed by the European Social Fund and the state budget of the Czech Republic. Ecohydros S.L. provided support in the form of salaries for authors [AMH], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the \u2018author contributions\u2019 section.The complete, correct funding statement should read: This study received institutional support from the Czech Academy of Sciences (RVO:60077344,"} +{"text": "The publisher apologizes for the error.There is an error in the author name in the correction published on June 27, 2013. The correct name is Adzemovic MZ. The correct citation is: Adzemovic MZ, Zeitelhofer M, Eriksson U, Olsson T, Nilsson I (2013) Imatinib Ameliorates Neuroinflammation in a Rat Model of Multiple Sclerosis by Enhancing Blood-Brain Barrier Integrity and by Modulating the Peripheral Immune Response. PLoS ONE 8(2): e56586. doi:"} +{"text": "Nature Communications6: Article number: 10005 10.1038/ncomms10005 (2015); Published: 11242015; Updated: 06072016In the original version of this Article the chemical structures of 2-ketoisovaleric acid and isobutanal in Fig. 3a were incorrect. The correct version of Fig. 3 appears below.Figure 3"} +{"text": "The following information was missing from the Funding section: This work was supported in part by the NIH Epi4K Sequencing, Bioinformatics and Biostatistics Core grant number U01NS077303. There was an error in reference 15. The correct reference is: Nature. Advance online publication. doi:10.1038/nature12439 Epi4K Consortium & Epilepsy Phenome/Genome Project (2013). De novo mutations in epileptic encephalopathies."} +{"text": "Correction to:British Journal of Cancer (2016) 114, 723\u2013730; doi:10.1038/bjc.2016.41; published online 22 March 2016Updated online 26 May 2016: This article was originally published under a standard BJC licence, but has now been made available under a CC BY 4.0 licence. The PDF and HTML versions of the paper have been modified accordingly."} +{"text": "Streptococcus, Toronto, Ontario, Canada . The article has been corrected online (http://wwwnc.cdc.gov/eid/article/21/4/14-0759_article).An incorrect version of the Technical Appendix was provided online for the article Population Structure and Antimicrobial Resistance of Invasive Serotype IV Group B"} +{"text": "Nature Communications6: Article number: 6198 10.1038/ncomms7198 (2015); Published: 02052015; Updated: 02012016In Figure 2b of this article, the sets of arrows connecting 'I' and 'M' in the kinetic scheme are inverted. The correct version of Fig. 2 appears below.Figure 2 |"} +{"text": "AbstractMononychellus is represented by 28 herbivorous mites. Some of them are notorious pests of cassava (Manihot esculenta Crantz), a primary food crop in the tropics. With the exception of Mononychellus tanajoa (Bondar), their geographic distribution is not widely known. This article therefore reports observational and specimen-based occurrence data of Mononychellus species associated with cassava. The dataset consists of 1,513 distribution records documented by the International Center for Tropical Agriculture (CIAT) between 1975 and 2012. The specimens are held at CIAT\u2019s Arthropod Reference Collection (CIATARC). Most of the records are from the genus\u2019 native range in South America and were documented between 1980 and 2000. Approximately 61% of the records belong to M. tanajoa, 25% to M. caribbeanae (McGregor), 10% to M. mcgregori (Flechtmann and Baker) and 2% to M. planki (McGregor). The complete dataset is available in Darwin Core Archive format via the Global Biodiversity Information Facility (GBIF).The genus This genus includes several species of herbivorous mites that are major pests of cassava (Manihot esculenta Crantz), most notoriously Mononychellus tanajoa (Bondar). We report 1,513 distribution records of the genus, documented by the International Center for Tropical Agriculture (CIAT) between 1975 and 2012. Most of the records (53%) correspond to specimens preserved at CIAT\u2019s Arthropod Reference Collection (CIATARC). Prior to this contribution, only 30 distribution records of Mononychellus were accessible through the Global Biodiversity Information Facility (GBIF) data portal (accessed 1/13/2014). Accordingly, the CIATARC Mononychellus dataset should facilitate a much better understanding of the genus\u2019 geographic association with cassava.General taxonomic coverage description: Most records were identified to species level (98%) with the help of expert input . Only four species of the genus are reported. Approximately 61% of the records belong to Mononychellus tanajoa, 25% to Mononychellus caribbeanae (McGregor), 10% to Mononychellus mcgregori (Flechtmann and Baker) and 2% to Mononychellus planki (McGregor).PageBreakKingdom:Animalia.Phylum:Arthropoda.Class:Arachnida.Order:Trombidiformes.Family:Tetranychidae.Genus:Mononychellus.Species:Mononychellus caribbeanae, Mononychellus mcgregori, Mononychellus planki, Mononychellus tanajoa.Common name: Cassava Green Mite (for Mononychellus tanajoa), Cassava Green Mite Complex PageBreakGeneral spatial coverage: The Mononychellus specimens and observations of CIATARC are from South America (14 countries) and Central America , which represent the 99% of records, with Colombia and Venezuela are the best represented countries, followed by Brazil and Ecuador .PageBreakSpecimen preservation method: Specimens are preserved as microslide preparations in microscope slide boxes within cabinet drawers maintained at 21.0 \u00b1 0.4 C and 47.6 \u00b1 8.6 relative humidity. They are sorted numerically by species and country of origin.Curatorial unit: 3,510 with an uncertainty of 0 (microslide preparation).Method step description: The dataset integrates two data flows: observational records and specimen-based records, identified either to genus or to species. The former were digitized from field diagnostic forms completed by personnel extensively trained in mite identification. These identifications, however, were likely conducted on site without mounting and preserving samples. Alternatively, these observations may correspond to properly-mounted but lost specimens. In either case, our confidence in the identification of observational records is high to the genus level, but moderate to the species level. On the other hand, specimen-based records belong to verifiable samples properly-preserved at CIATARC following the guidelines of http://www.dane.gov.co/Divipola/ for Colombia, http://www.inec.gob.ec/estadisticas/?option=com_content&view=article&id=80 for Ecuador, etc. [accessed 2013/11/14]). Based on their locality names, we then geocoded the records using Google Maps (https://maps.google.com/), GeoNames (http://www.geonames.org/) or http://www.gbif.org/dataset/785cf038-7b79-4c2f-9e9e-eb940fcd4c0c).All biodiversity data available was digitized in a Microsoft Excel 2010 spreadsheet adopting the Darwin Core Archive format v1.2 . We updaSampling description: The records in the dataset have been documented in three ways:1) Records from CIAT\u2019s initial field explorations to document pests in cassava Records documented during the \u201cCassava Green Spider Mite Biological Control Project,\u201d led by CIAT, International Institute of Tropical Agriculture (IITA), Commonwealth Institute of Biological Control (CIBC) and Empresa Brasileira de Pesquisa Agropecu\u00e1ria (EMBRAPA) (n Africa .3) Records from other sources; including field inspections and collections conducted during routine farm visits by CIAT personnel, and from specimens submitted to CIATARC by fellow institutions and researchers .Object name: Darwin Core Archive Mononychellus distribution: data of the CIAT Arthropod Reference Collection of International Center for Tropical Agriculture (CIAT).Character encoding: UTF-8.Format name: Darwin Core Archive format.Format version: 1.0.Distribution:http://www.gbif.org/dataset/785cf038-7b79-4c2f-9e9e-eb940fcd4c0cPublication date of data: 2014-03-14.Language: English.Licenses of use: This dataset [Mononychellus Collection of CIAT Arthropod Reference Collection (CIATARC)] is made available under the Creative Commons Zero (CC0) 1.0.PageBreak"} +{"text": "Nature Communications6:8136 doi: 10.1038/ncomms9136 (2015); Published 09182015; Updated 06152016The original version of this Article failed to fully credit the use of the Ocean Data View software in figure 1 and supplementary figure 12, which appears below:http://odv.awi.de, 2016.Schlitzer, R., Ocean Data View,"} +{"text": "In Vivo and Assessment of the Specificities of the Anti-Bovine CD1 Antibodies. PLoS ONE 10(3): e0121923. doi:10.1371/journal.pone.0121923This article was republished on May 13, 2015, to correct the third and seventh authors\u2019 names. The third author\u2019s given name is Chema and surname is El Messlaki. The seventh author\u2019s given name is Ildiko and surname is Van Rhijn. The correct citation is: Nguyen TKA, Reinink P, El Messlaki C, Im JS, Ercan A, Porcelli SA, et al. (2015) Expression Patterns of Bovine CD1 The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "There is an error in reference 24. The correct reference is: Obirikorang C, Selleh PK, Abledu JK and Fofie CO (2013) Predictors of Adherence to Antiretroviral Therapy among HIV/AIDS Patients in the Upper West Region of Ghana. ISRN AIDS 2013: 873939.In"} +{"text": "Two grant numbers are inadvertently omitted in the Funding section. The correct funding information is as follows:http://www.dfg.de/en/) (DFG BO3790/1-1), DFG (FI1781/1-1) and the following grants from the ESRC (http://www.esrc.ac.uk/) (ES/L010690/1) and (ES/M009203/1). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Authors are funded by the following grants from the DFG (The publisher apologizes for this error."} +{"text": "There is an error in the first author\u2019s name in the article published on September 13, 2013 and in the correction published on December 17, 2015. The correct name is: Uzma Bashir.10.1371/journal.pone.0074018.The correct citation for the article is: Bashir U, Alam MM, Sadia H, Zaidi SSZ, Kazi BM (2013) Molecular Characterization of Circulating Respiratory Syncytial Virus (RSV) Genotypes in Gilgit Baltistan Province of Pakistan during 2011\u20132012 Winter Season. PLoS ONE 8(9): e74018. doi:10.1371/journal.pone.0145599. The publisher apologizes for the error.The correct citation for the correction is: Bashir U, Alam MM, Sadia H, Zaidi SSZ, Kazi BM (2015) Correction: Molecular Characterization of Circulating Respiratory Syncytial Virus (RSV) Genotypes in Gilgit Baltistan Province of Pakistan during 2011\u20132012 Winter Season. PLoS ONE 10(12): e0145599. doi:"} +{"text": "Haemophilus ducreyi Infections . The online edition of the article is correct, and the online PDF has been corrected (http://wwwnc.cdc.gov/EID/article/22/1/14-0425_article).Some references were cited incorrectly in the print and initial PDF editions of Epidemiology of"} +{"text": "There is an error in the title of this paper: \u201cCombing\u201d should be \u201cCombining.\u201d The correct title is: Ecological Change, Sliding Baselines and the Importance of Historical Data: Lessons from Combining Observational and Quantitative Data on a Temperate Reef Over 70 Years.10.1371/journal.pone.0118581The correct citation is: Gatti G, Bianchi CN, Parravicini V, Rovere A, Peirano A, Montefalcone M, et al. (2015) Ecological Change, Sliding Baselines and the Importance of Historical Data: Lessons from Combining Observational and Quantitative Data on a Temperate Reef Over 70 Years. PLoS ONE 10(2): e0118581. doi:"} +{"text": "In the Funding section, the grant number from the funder National Science Centre is listed incorrectly. The correct grant number is: 2011/03/D/HS6/05951."} +{"text": "Nature Communications6:6961 doi: 10.1038/ncomms7961 (2015); Published 04242015; Updated 2016The original version of this Article failed to fully credit the use of the Ocean Data View software in figure 3, which appears below:http://odv.awi.de, 2016.Schlitzer, R., Ocean Data View,"} +{"text": "The publisher apologizes for the errors.The first and third authors\u2019 names appear incorrectly in the citation. The correct names are: Kanthack TFD and Altimari LR. The correct citation is: Kanthack TFD, Guillot A, Altimari LR, Nunez Nagy S, Collet C, Di Rienzo F (2016) Selective Efficacy of Static and Dynamic Imagery in Different States of Physical Fatigue. PLoS ONE 11(3): e0149654. doi:"} +{"text": "Western Journal of Emergency Medicine :786\u2013787. DOI: 10.5811/westjem.2015.6.27582), there were the following errors in the published article:In the Original Research article entitled \u201cHydrocele of the Canal of Nuck,\u201d published in the October 2015 issue of the 1. On page 786, the following authors should have been included: Phillip Aguiniga, Jason Blake. They are research assistants at the Department of Emergency Medicine, Kern Medical Center, Bakersfield, California.We apologize for this error."} +{"text": "There are errors in the Funding section. The correct funding information is as follows:http://abc.uva.nl/research/nav), the European Union 7th Framework Programme (FP7/2007-2013) under grant agreement no. 604102 , and the Deutsche Forschungsgemeinschaft (DFG), SFB 936/Z1 (http://www.sfb936.net). These funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.This work was funded by the Amsterdam Brain and Cognition priority program; grant number: ABC2014-01 ("} +{"text": "A reader has brought to our attention that there are errors in references 27, 28, and 29.The correct reference for 27 is: Froeschle JE, Feorino PM, Gelfand HM. A continuing surveillance of enterovirus infection in healthy children in six United States cities. II. Surveillance enterovirus isolates 1960\u20131963 and comparison with enterovirus isolates from cases of acute central nervous system disease. American journal of epidemiology. 1966;83:455\u201346910.1002/rmv.326The correct reference for 28 is the same as for reference 31: Kim KS, Hufnagel G, Chapman NM, Tracy S (2001) The group B coxsackieviruses and myocarditis. Rev Med Virol 11: 355\u2013368. doi: The correct reference for 29 is: Modlin JF, Rotbart HA. Group B coxsackie disease in children. Current topics in microbiology and immunology. 1997;223:53\u201380The authors confirm that all other references are listed correctly."} +{"text": "Adseverin knockdown inhibits osteoclastogenesis in RAW264.7 cellsWENTING QI, YAN GAO, JUN TIAN and HONGWEI JIANGInt J Mol Med 34: 1483\u20131491, 2014; DOI: 10.3892/ijmm.2014.1941All the authors agree to the withdrawal of this manuscript. The retraction has been agreed to as the authors have admitted that the manuscript contains a substantial amount of material from the following study: Hassanpour S, Jiang H, Wang Y, Kuiper JW and Glogauer M: The actin binding protein adseverin regulates osteoclastogenesis. PLoS One 9: e109078, 2014. Thus, this cannot be considered as original work."} +{"text": "CORRECTIONRambam Maimonides Med J 2010;1(2):e0011. doi:10.5041/RMMJ.10011Youdim MBH. Why do we need multifunctional neuroprotective and neurorestorative drugs for Parkinson\u2019s and Alzheimer\u2019s disorders? , it was noted that the Acknowledgement regarding the source of the text was not published. The following Acknowledgement has been added:In the following paper: Acknowledgement: This article is reprinted with slight modifications from Youdim MBH. Why Do We Need Multifunctional Neuroprotective and Neurorestorative Drugs for Parkinson\u2019s and Alzheimer\u2019s Diseases as Disease Modifying Agents. Exp Neurobiol. 2010;19(1):1\u201314, under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/3.0/).The HTML and PDF versions of the paper have been corrected in the RMMJ.org.il archives.Published: August 31, 2015"} +{"text": "Cebus\u201d). The correct citation is: Makedonska J, Wright BW, Strait DS (2012) The Effect of Dietary Adaptation on Cranial Morphological Integration in Capuchins . PLoS ONE 7(10): e40398. doi:10.1371/journal.pone.0040398The word \u201cAdaptation\u201d is misspelled in the article title. The correct title is: The Effect of Dietary Adaptation on Cranial Morphological Integration in Capuchins (Order Primates, Genus \u201c"} +{"text": "AbstractLasiusalienoflavus Bingham, 1903. This species has hitherto been reported only from the Himalayas, and the present data are also based on specimens collected in the north-western part of the mountain range. Likewise other Himalayan ants, this species also shows restricted distribution, which suggests a rather high degree of endemism (45%) of this group in the Himalayas.The present paper provides a description of the male caste and re-description of the worker and queen castes of the poorly known ant species Lasius Fabricius, 1804 is hithrto known to comprise 111 extant species and 3 subspecies , Lasiusnearcticus Wheeler, 1906 Lasiusalienoflavus Bingham, 1903, Lasiustalpa Wilson, 1955 and Lasiusfallax Wilson, 1955 forming the flavus-group in a distinct subgenus Cautolasius Wilson, 1955, and provided a key to the Palaearctic species. Lasiussensu stricto from the Palaearctic, describing 17 new species, and updated Wilson's key to the Palaearctic species. Other significant contributions form the Palaearctic region include: Lasius (Dendrolasius) species of East Palaearctic.The genus bspecies . In the bspecies . A revisLasiusalienoflavus Bingham, 1903 from the Himalayas (above 8000 feet) with reports of worker and queen castes. Later on purely morphological grounds Cautolasius and placed Lasiusalienoflavus in it. This subgenus comprises a group of species showing a mixture of characters that put them in a position intermediate between Lasiussensu stricto and Chthonolasius. Subsequently, Lasiusalienoflavus and provided a key to the Himalayan members of the genus. In the discussion part he mistakenly assigned the species to the subgenus Chthonolasius. Recently Lasius from the Himalayas.Lasiusalienoflavus based on fresh material and for the first time provide a description of the male caste.Here we re-describe the worker and queen castes of The specimens were collected by handpicking. The morphological analysis was carried on with the aid of a Nikon SMZ 1500 stereo zoom microscope. All digital images were taken with a MP evolution digital camera and subsequently processed with Auto-Montage software and Adobe Photoshop CS5. All measurements were recorded in millimeters using oculometer between 50\u00d7 and 125\u00d7 to the nearest 0.001 mm and have been rounded to the nearest 0.01 mm (as the average recording error was approximately 0.005 mm). Specimens have been deposited in PUPAC, Punjabi University Patiala Ant Collection, Patiala. \u2013 Length of the head measured in full face view in a straight line from the middle of the anterior clypeal margin to the middle of the occipital margin. The head has to be carefully tilted to the position with the real maximumHL. \u2013 Maximum width of the head in full face view, behind the eyesHW. \u2013 Maximum eye length with eye in full face viewEL. \u2013 Diameter of eye measured perpendicularly to transect in EL and across structurally defined ommatidiaEW. \u2013 Maximum straight-line length of the scape excluding the basal neck and the condyleSL. \u2013 Weber\u2019s length \u2013 the length of mesosoma in profile from the margin of neck shield to the posterior margin of propodeal lobesWL. \u2013 Maximum width of the petiole from above, in dorsal viewPW. \u2013 The length of the gaster in lateral view from the anterior most point of first gastral segment to the posterior most point (excluding sting if present)GL. \u2013 Head size \u2013 the total of head length and head width divided by hundredHS. \u2013 Number of standing hairs projecting > 0.02 mm from dorsal profile of scape i.e. the number of hairs visible when looking at the small diameter of scape under transmitted-light conditions. The always present hairs on distal apex are not counted and the number refers to one scapenHS. \u2013 Number of standing hairs projecting > 0.02 mm from extensor profile of one hind tibia. The always present hairs on distal apex are not counted and the number refers to one tibianHHT. \u2013 The total outstretched length in profile from anterior clypeal margin to the posterior most point of gaster excluding stingTL. \u2013 CI = HL/HW \u00d7 100Cephalic Index \u2013 EI = EL/HW \u00d7 100Eye Index1 = SL/HL \u00d7 100 \u2013 SIScape Index2 = SL/HW \u00d7 100 \u2013 SIScape IndexBingham, 1903Lasiusalienoflavus Bingham, 1903 \u2013 Lasius (Cautolasius) alienoflavus \u2013 Type status:Other material. Occurrence: recordedBy: Irfan Gul; sex: 23 workers; Location: country: India; stateProvince: North-West Himalaya; verbatimLocality: Himachal Pradesh: Khajjiar; maximumElevationInMeters: 2100; Event: eventDate: 2010-07-01; Record Level: collectionCode: Insect (Ants)Type status:Other material. Occurrence: recordedBy: Irfan Gul; sex: 63 workers; Location: country: India; stateProvince: North-West Himalaya; verbatimLocality: Himachal Pradesh: Kharapathar; maximumElevationInMeters: 2800; Event: eventDate: 2008-08-13; Record Level: collectionCode: Insect (Ants)Type status:Other material. Occurrence: recordedBy: Irfan Gul; sex: 20 workers, 2 queens, 1 male; Location: country: India; stateProvince: North-West Himalaya; verbatimLocality: Himachal Pradesh: Manali; maximumElevationInMeters: 1800; Event: eventDate: 2010-06-17; Record Level: collectionCode: Insect (Ants)Type status:Other material. Occurrence: recordedBy: Irfan Gul; sex: 3 workers; Location: country: India; stateProvince: North-West Himalaya; verbatimLocality: Himachal Pradesh: Sungri; maximumElevationInMeters: 2600; Event: eventDate: 2008-08-14; Record Level: collectionCode: Insect (Ants)Type status:Other material. Occurrence: recordedBy: Irfan Gul; sex: 62 worker; Location: country: India; stateProvince: North-West Himalaya; verbatimLocality: Jammu & Kashmir: Sarthal; maximumElevationInMeters: 3000; Event: eventDate: 2010-06-04; Record Level: collectionCode: Insect (Ants)Type status:Other material. Occurrence: recordedBy: Irfan Gul; sex: 105 workers, 3 queens; Location: country: India; stateProvince: North-West Himalaya; verbatimLocality: Uttarakhand: Gangotri; maximumElevationInMeters: 3000; Event: eventDate: 2010-06-04; Record Level: collectionCode: Insect (Ants)Fig. 1Measurements: HL 0.82\u20130.91; HW 0.75\u20130.86; EL 0.12\u20130.15; EW 0.09\u20130.12; SL 0.70\u20130.80; WL 0.88\u20131.04; PW 0.20\u20130.24; GL 0.61\u20131.05; HS 0.78\u20130.88; CI 101\u2013109; EI 15\u201318; SI1 80\u201388; SI2 85\u201395; nHS+nHHT < 8; TL 2.5\u20132.8. n = 32.Head: Head roughly rectangular in full face view (CI = 101\u2013109); posterior margin of head straight; posterolateral corners rounded; a few setae present closer to posterior margin of head but not reaching the hind margin of eyes and less denser than in Lasiuselevatus Bharti et Gul, 2013; lateral sides of head more-or-less parallel, somewhat narrowing anteriorly; anterior clypeal margin broadly convex, clypeal carina absent; lateral clypeal profile convex; eyes almost round, size larger as compared to other species of the same group (EI = 15\u201318); mandibles triangular, the masticatory margin with 7 to 8 teeth, rarely 9, including denticles; antennae 12 segmented, scape long, distinctly surpassing the posterior margin of head .Mesosoma and petiole: Mesosoma with weakly convex promesonotal dorsum slightly depressed at the suture; propodeal dome rather hemispheric, as high as mesonotum, posterior slope of propodeum somewhat straight; area between propodeal spiracle and metapleural gland without distinct setae; in frontal view petiole with weakly convex sides and rather straight dorsum, in profile with steep and slightly convex anterior face and straight posterior face; gaster more-or-less ovate.Sculpture and pilosity: Head and mesosoma with shallow micropunctures; in general body smooth and fairly shiny with scattered pilosity; body covered with suberect to erect setae, abundant and longest on gaster; cuticular surface covered with smooth and rather dense pubescence; genae without standing hairs or setae; scape with subdecumbent to decumbent pubescence, a few setae present at the proximal end; hind tibia pubescence smooth, setae are normally present at the proximal end.Colour: The species is light to dark yellow in colour; the masticatory margin dark brownish and the eyes black in colour; pubescence pale-yellow.Fig. 2Measurements: HL 1.12\u20131.15; HW 1.32\u20131.37; EL 0.34\u20130.36; EW 0.26\u20130.27; SL 1.0\u20131.02; WL 2.33\u20132.45; PW 0.24\u20130.26; GL 2.30\u20132.70; HS 1.24\u20131.26; CI 81\u201384; EI 25\u201326; SI1 87\u201389; SI2 73\u201374; TL 5.7\u20136.3. n = 2.Resembles the worker, with modifications expected for caste and the following differences: body massive, hairy; lateral sides of head subparallel, narrowing towards the anterior margin; eyes much larger; mesosoma enlarged, dorsally flat, scutum and scutellum at the same level, propodeal declivity very steep; in profile view petiole compressed, in frontal view dorsum emarginate; gaster long and gibbous; setae scattered all over and short; head, mesosoma and gaster dark brown, legs dark yellow in colour.Fig. 3Measurements: HL 0.6; HW 0.7; EL 0.27; EW 0.18; SL 0.48; WL 1.3; PW 0.25; GL 1.7; HS 0.65; CI 86; EI 38; SI1 80; SI2 68; TL 3.6. n = 1.Head: Head roughly squarein full face view (CI = 86); Head broader at the posterior margin, narrowingtowards the anterior margin; posterior margin of head slightly convex; posterolateral corners rounded; Hairsdenser towards the posterior margin of head; lateral sides of head distinctly narrowinganteriorily; eyes very large, bulging beyond head outline in full-frontal view (EI = 38); three prominent ocelli present at the centre; antennae 13 segmented, filiform; scape long, distinctly surpassing the posterior margin of head ; clypeus smooth, without any carina in the middle; anterior clypeal margin broadly convex; lateral clypeal profile convex; mandiblewith an apical tooth and 3 denticles at the centre of masticatory margin.Mesosoma and petiole: Mesosomaenlarged to accommodate flight muscles; pronotum small; scutum smooth; scutellum somewhat raised; declivity steep; in frontal view petiole dorsallyround; gaster lengthened, tapering towards the apex.Sculptureand pilosity: Ingeneral body smooth with scattered pilosity; body covered with suberect toerect setae, abundant and longest on gaster; cuticular surface covered withsmooth and rather dense pubescence abundant towards the apex of gaster, antennaeand legs; Hairs more dense on the posterior margin of head, sparselydistributed over rest of the head; standinghairs or setae on genae sparse; mandibles with a few setae at the apicalportion; a few setae present on the anterior margin of clypeus; small blunthairs thinly present on the eyes; scape with subdecumbent to decumbentpubescence, a few setae present at the proximal end; hind tibia pubescencesmooth, setae are normally present at the proximal end.Genitals: Paramereselongated, roughly triangular, covered with long setae; cuspi with short peg-like teeth and bent towards digiti; digiti straight and long, about 3 timesas long as cuspi with round dorsum; penis valve projecting.Colour: brown with a blackish tinge; eyes black; legs and wings creamy-yellowish; body smooth and somewhat shiny all over.The overall distribution of the species is from the Himalaya but the specimens used for this study have been collected from the North-west range of Himalaya. All the collection areas were forested mountains surrounded by other mountains. The nests were very close to the surface with a depth of 3\u20135 inches. The soil surrounding the nest was mainly moist and covered by herbs. 23 specimens were collected from Winkler extraction.Lasiusalienoflavus Bingham, 1903 is well marked off from other reports of this genus and is a distinct species. However, it resembles Lasiusflavus (Fabricius) but can be readily distinguished from it by having the apical segment of the maxillary palp longer than the preapical one ."} +{"text": "A reference is omitted from the fifth sentence of the last paragraph in the Discussion section.The sentence should read: Our data are entirely consistent with and complementary to a parallel study conducted independently in the Rothstein, Klein, Kreicji, and Lisby laboratories .The reference is: Burgess RC, Sebesta M, Sisakova A, Marini VP, Lisby M, et al. (2013) The PCNA Interaction Protein Box Sequence in Rad54 Is an Integral Part of Its ATPase Domain and Is Required for Efficient DNA Repair and Recombination. PLoS ONE 8(12): e82630. doi:10.1371/journal.pone.0082630"} +{"text": "Nature Communications6: Article number:798310.1038/ncomms8983 (2015); Published: 08062015; Updated: 02172016.In Fig. 3d in this Article, the graph depicting flow cytometry of the anaerobic culture was inadvertently duplicated from that depicting the KCN-treated culture during the production process. The correct version of Fig. 3 appears below.Figure 3"} +{"text": "Correction to:Cell Discovery (2016) 2, 16012; doi:10.1038/celldisc.2016.12; published online 17 May 2016During web production, there was an error in Supplementary information: Supplementary Material was omitted. The files are now installed in the online version of the paper. We apologize for any inconvenience that may have been caused by this error."} +{"text": "The percentage of young adults aged 18\u201324 years who had never smoked cigarettes increased by more than 10 percentage points from 1999\u20132001 (65%) to 2011\u20132012 (76%). The increase was noted for men and for women. For each period, women were more likely than men to have never smoked cigarettes.Sources: Schoenborn CA, Adams PF, Barnes PM, Vickerie JL, Schiller JS. Health Behaviors of Adults: United States, 1999\u20132001. Vital Health Stat 2004;10(219).Adams PF, Schoenborn CA. Health behaviors of adults: United States, 2002\u20132004. Vital Health Stat 2006;10(230).Schoenborn CA, Adams PF. Health behaviors of adults: United States, 2005\u20132007. Vital Health Stat 2010;10(245).Schoenborn CA, Adams PF, Peregoy JA. Health behavior of adults: United States, 2008\u20132010. Vital Health Stat 2013;10(257).http://www.cdc.gov/nchs/nhis/quest_data_related_1997_forward.htm.National Health Interview Survey. Data and documentation for 2011 and 2012. Available at"} +{"text": "Petunia: the importance of stress severityPhysiological and molecular responses to drought in Jongyun Kim, Anish Malladi, and Marc W. van IerselJournal of Experimental Botany (2012) 63 (18): 6335\u20136345. doi: 10.1093/jxb/ers28556, 46\u201351).In the above paper, Figure 1 and a subset of the data in Figure 2 were previously published in a proceedings paper of the Southern Nursery Association Research Conference (Kim J, Malladi A, van Iersel MW. 2011. Physiological responses of petunia to different levels of drought stress. Proceedings of Southern Nursery Association Research Conference"} +{"text": "There is an error in the title. The word Fishes has an incorrectly capitalized I. The correct title should be \u201cDNA Barcodes for the Fishes of the Narmada, One of India's Longest River.\u201dThe correct Citation should read: Khedkar GD, Jamdade R, Naik S, David L, Haymer D (2014) DNA Barcodes for the Fishes of the Narmada, One of India\u2019s Longest Rivers. PLoS ONE 9(7): e101460. doi:10.1371/journal.pone.0101460Additionally, there are errors in"} +{"text": "The third author\u2019s name is spelled incorrectly in the correction published on August 11, 2014. The correct name is: K. Suzanne Scherf. The correct citation is: Halliday DWR, MacDonald SWS, Scherf KS, Tanaka JW (2014) A Reciprocal Model of Face Recognition and Autistic Traits: Evidence from an Individual Differences Perspective. PLoS ONE 9(5): e94013. doi:10.1371/journal.pone.0094013. The publisher apologizes for the error."} +{"text": "There is an error in reference 25. The correct reference is: Andres B, Ji Q (2008) A new pterosaur from the Liaoning province of China, the phylogeny of the Pterodactyloidea, and convergence in their cervical vertebrae. Palaeontology 51: 453\u2013469."} +{"text": "The correct citation is: Fortini LB, Vorsino AE, Amidon FA, Paxton EH, Jacobi JD (2015) Large-Scale Range Collapse of Hawaiian Forest Birds under Climate Change and the Need for 21st Century Conservation Options. PLoS ONE 10(10): e0140389. 10.1371/journal.pone.0140389. The publisher apologizes for the error.The publisher erroneously omitted the word \u201cfor\u201d from the article title. The correct title is: Large-Scale Range Collapse of Hawaiian Forest Birds under Climate Change and the Need for 21"} +{"text": "Correction to:British Journal of Cancer (2015) 114, 146\u2013150; doi:10.1038/bjc.2015.421; published online 15 December 2015British Journal of Cancer, one of the authors identified an error in the spelling of their name. V Sivasubramaniam appears correctly in the author list above.Upon publication of the above paper in the"} +{"text": "The third author\u2019s name is abbreviated incorrectly in the citation. The correct citation is: Nemeth M, Millesi E, Wagner KH, Wallner B (2014) Effects of Diets High in Unsaturated Fatty Acids on Socially Induced Stress Responses in Guinea Pigs. PLoS ONE 9(12): e116292. doi:10.1371/journal.pone.0116292There is an error in affiliation 2 for Karl-Heinz Wagner. Affiliation 2 should be: Department of Nutritional Sciences, University of Vienna, Vienna, Austria"} +{"text": "Reason for Erratum:Due to a production error the article was erroneously published in Frontiers in Neuroengineering, instead of Frontiers in Neuroscience. This mistake does not change the scientific conclusions of the article in any way and the publisher apologizes for the error.Old citation:In vivo comparison of the charge densities required to evoke motor responses using novel annular penetrating microelectrodes by Brunton EK, Winther-Jensen B, Wang C, Yan EB, Hagh Gooie S, Lowery AJ, and Rajan R (2015). Front. Neuroeng. 8:5. doi: 10.3389/fneng.2015.00005The original article has been updated."} +{"text": "Consolidation processes, involving synaptic and systems level changes, are suggested to stabilize memories once they are formed. At the synaptic level, dendritic structural changes are associated with long-term memory storage. At the systems level, memory storage dynamics between the hippocampus and anterior cingulate cortex (ACC) may be influenced by the number of sequentially encoded memories. The present experiment utilized Golgi-Cox staining and neuron reconstruction to examine recent and remote structural changes in the hippocampus and ACC following training on three different behavioral procedures. Rats were trained on one hippocampal-dependent task only (a water maze task), two hippocampal-dependent tasks , or one hippocampal-dependent and one non-hippocampal-dependent task (a water maze task followed by an operant conditioning task). Rats were euthanized recently or remotely. Brains underwent Golgi-Cox processing and neurons were reconstructed using Neurolucida software . Rats trained on two hippocampal-dependent tasks displayed increased dendritic complexity compared to control rats, in neurons examined in both the ACC and hippocampus at recent and remote time points. Importantly, this behavioral group showed consistent, significant structural differences in the ACC compared to the control group at the recent time point. These findings suggest that taxing the demand placed upon the hippocampus, by training rats on two hippocampal-dependent tasks, engages synaptic and systems consolidation processes in the ACC at an accelerated rate for recent and remote storage of spatial memories. Consolidation is distinguished into two specific components, synaptic, and systems consolidation. Synaptic consolidation occurs shortly after acquisition and refers to changes at the cellular level McGaugh, . SystemsSynaptic consolidation processes involve the induction of signaling cascades and second messenger systems, protein synthesis and gene expression alterations, all of which may be important synaptic pathways in the initiation of long-term memory storage processes Kandel, . Once thThe hippocampus critically contributes to the initial encoding and storage of memory representations , two different hippocampal-dependent tasks or one hippocampal-dependent and one non-hippocampal-dependent task . Because impaired hippocampal function does not alter OP task performance . Rats received no nesting material and no direct enrichment of any kind in their home cage. Food was restricted until rats reached 90% of their free-feeding baseline, which was maintained throughout the experiment. Prior to behavioral training, rats were given 5 chocolate pellets (45 mg) in their home cage and handled for 5 min daily. Principles of laboratory animal care were followed and all procedures were conducted in accordance with the Canadian Council on Animal Care and protocols approved by the Carleton University Animal Care Committee.Forty-eight Long Evans rats (190\u2013250 g) from Charles River, Quebec were used. Rats were housed individually in clear plastic cages (26 \u00d7 20 \u00d7 45 cm) and given The WM was located in a room within the animal housing area. The opaque, white, polypropylene pool measured 155 cm in diameter and 60 cm in height. The pool was filled to a depth of 37.5 cm with water that remained at approximately 21\u00b0C. The \u201cescape\u201d platform was made from clear Plexiglas and submerged approximately 2 cm below the surface of the water. Visual cues such as posters and geometric shapes were located on the walls around the room. The experimenter remained in the same position throughout all trials.RAM testing was done in a room within the animal housing area, located across the hall from the WM testing room. The maze was positioned 98.5 cm off the floor. Each arm measured 59 cm long and 11 cm wide. The distance between the ends of arms, where food reward was located, was 32.5 cm. Plastic inserts were placed on the sides of the maze arms to prevent animals from jumping across arms. Visual cues such as posters and geometric shapes were located on the walls around the room. The experimenter remained in the same position throughout all trials.Rats were tested in groups of six using six operant chambers . Each chamber was housed in an insulated box to minimize external noise. Each chamber possessed a pellet dispensing system, two levers separated by a food hopper, a houselight, a grid floor and a three light-panel. OP training took place in a room outside of the animal housing area.n = 24) or remotely . Eight experimental groups resulted (n = 6 in each group): (1) Control:Recent (2) WM:Recent (3) WM/RAM:Recent (4) WM/OP:Recent (5) Control:Remote (6) WM:Remote (7) WM/RAM:Remote (8) WM/OP:Remote.An overview of the behavioral procedure is shown in Figure Rats received 5 training trials per day for 5 days, with a different starting location for every trial within a day and randomized starting locations across days. The hidden platform was located in a fixed location within and across days. Rats were placed in the pool, facing the perimeter, and given a maximum of 60 s to locate the hidden platform. Rats that did not find the hidden platform within 60 s were guided to the platform by the experimenter. All rats remained on the platform for 15 s. Rats then received a 15 s rest period in a holding cage before the next trial. All movement within the pool was tracked using HVS Image 2100 Tracking System . Following the final trial of each day, rats were dried with a towel and placed in a holding cage on a heating pad in the housing room for 10\u201315 min after which they were returned to the home cage.Rats received one day of pretraining and 4 days of testing on the RAM. On the first trial of pretraining, chocolate pellets were located in the starting area, at the entrance to arms, within the arms as well as in the food holes located at the end of each arm. On trial 2 of pretraining, pellets were located within the arms and in food holes only. Trials 3\u20135 of pretraining had pellets located only in food holes.Days 2\u20135 were testing days, where pellets were located in food holes at the ends of 3 of the 5 arms. Baited arms were always the same for an individual rat but differed between rats. Each rat was given 5 trials per day. Trials were a maximum of 5 min each or ended when all food reward had been collected. Rats were placed in a holding cage for 30 s between trials while arms were re-baited. Performance on the maze was manually scored. Sessions were timed, and correct and incorrect arm entries were recorded. An arm entry was defined as all four feet inside an arm.Rats in the WM/OP condition were trained to lever-press for chocolate pellets over 5 days, 30 min per day. Upon pressing the lever to the left of the hopper two times (FR2), the house light extinguished, the panel lights above the lever changed from red to green and the pellet dispenser released one 45-mg chocolate pellet into the hopper. Presses on the right lever had no programmed consequences. Presses on the left lever were considered correct and presses on the right lever, incorrect. The number of lever presses, the number of times a rat poked its nose into the hopper and locomotor activity were recorded automatically .The Golgi method, originally named the \u201cBlack Reaction\u201d was developed by Camillo Golgi in 1873. The invention of the Golgi method marked a huge advancement in the field of neuroscience, largely thanks to Santiago Ramon y Cajal's use of the method. Curiously, and fortunately, the Golgi method stains only a small percentage of cells . Next, brains underwent incubation in 10% (8 h), 20% (8 h), and 30% sucrose (minimum 4 days). 200 \u03bcm thick sections were sliced on a vibratome and mounted onto gelatinized slides. Slides were placed in a humidified, dark box for 24 h before staining.Rats were placed into a Decapicone (Braintree) and decapitated. Brains were rapidly removed and hemisected . One hemisphere was placed in Golgi-Cox fixative and stored at room temperature, away from light, for 14 days. Following incubation in Golgi-Cox fixative, brains underwent 3 washes in DH2O (1 min), 28% Ammonium Hydroxide (40 min), DH2O (1 min), Kodak film fix A (40 min), DH2O (2 \u00d7 1 min), 50, 70, and 95% ETOH (1 min each), desiccated 100% ETOH (3 \u00d7 5 min), desiccated ETOH/Clearene/Chloroform solution (10 min), and desiccated Clearene (2 \u00d7 15 min). Slides were coverslipped with generous amounts of Permount mounting medium (Sigma) and placed in a desiccated box to dry for a minimum of 4 days.Slides were immersed in the following solutions: DHThree pyramidal neurons per rat were analyzed from each brain region. Four rats from each behavioral group were examined. All neuron reconstruction was performed by one experimenter who was unaware of the experimental condition. Brain regions were defined according to Paxinos and Watson . The ACCNeurons to be traced were selected at random, but had to meet predetermined criteria for analysis. Neurons had to be entirely impregnated, staining had to be uniform and complete within processes, the neuron had to be relatively isolated from surrounding impregnated cells or obstructions, and the cell body had to be centrally located within the 200 \u03bcm section depth . Separate analyses were carried out in the ACC and CA1 of the hippocampus and apical and basal dendrites were analyzed separately. Two-Way ANOVAs in each region for apical and basal dendrites were run with Behavioral group and Time (Recent or Remote) as the fixed factors and the characteristic of interest as the dependent variables.Four rats from each behavioral group were examined; three neurons per rat per brain area (ACC and CA1) were analyzed. Thus, resulting in 12 neurons per behavioral condition per brain region . For statistical analysis, the measurements of the three analyzed neurons per rat per brain region were pooled to get one value for each rat = 112.88, p < 0.001] but no main effect of behavior or time.Figure F = 2.82, p = 0.05] but no main effect of time.Two groups received training on the WM followed by training on the RAM: one assigned to receive a recent WM probe test and one assigned to receive a remote WM probe Figure . The numF = 12.73, p < 0.001] but no main effect of time.Two groups received training on the WM followed by training on the operant task: one assigned to receive a recent WM probe test and one assigned to receive a remote WM probe Figure . The numCell body size in the ACC was analyzed using a Two-Way ANOVA with Behavior and time (recent or remote) as the independent variables and cell body size as the dependent variable. No main effect of behavior or time was found (data not shown).Cell body size in the CA1 of the hippocampus was analyzed using a Two-Way ANOVA with Behavior and time (recent or remote) as the independent variables and cell body size as the dependent variable. No main effect of behavior or time was found (data not shown).Figure Number of branches. Figure Apical. A Two-Way ANOVA with Behavioral group and time (Recent or Remote) as the fixed factors and number of branches as the dependent variable revealed a main effect of behavior with group WM/RAM displaying the greatest number of branches (16). Fisher's LSD post-hoc analyses revealed group WM/RAM:Recent had significantly greater number of branches compared to groups WM/OP:Remote, Control:Recent, and Control:Remote (p < 0.05). Group WM/RAM:Remote had significantly greater number of branches compared to groups WM Only:Recent, WM/OP:Recent, WM/OP:Remote, Control:Recent, and Control:Remote (p < 0.05). Group WM Only:Remote had significantly greater number of branches compared to group Control:Remote (p < 0.05).Basal. A Two-Way ANOVA with Behavioral group and time (Recent or Remote) as the fixed factors and number of branches as the dependent variable revealed a main effect of behavior , with group WM/RAM displaying the greatest number of branches (18). Fisher's LSD post-hoc analyses revealed group WM/RAM:Remote had significantly greater number of branches compared to groups from WM Only:Recent, WM Only:Remote, WM/OP:Remote, Control:Recent, and Control:Remote, (p = 0.05). Group WM/OP:Recent had significantly greater number of branches compared to group Control:Recent (p < 0.05).Dendritic Length. Figure Apical. A Two-Way ANOVA with Behavioral group and time (Recent or Remote) as the fixed factors and dendritic length as the dependent variable revealed a main effect of behavior , with group WM/RAM displaying the greatest dendritic length (1016 \u03bcm). Fisher's LSD post-hoc analyses revealed group WM/RAM:Recent had significantly greater dendritic length compared to groups WM/OP:Remote, Control:Recent, and Control:Remote (p < 0.05). Group WM/RAM:Remote had significantly greater dendritic length compared to groups WM/OP:Remote, Control:Recent, Control:Remote (p < 0.05). Group WM/OP:Recent had significantly greater dendritic length compared to group Control:Remote (p = 0.05).Basal. A Two-Way ANOVA with Behavioral group and time (Recent or Remote) as the fixed factors and dendritic length as the dependent variable revealed a main effect of behavior , with group WM/RAM displaying the greatest dendritic length (839 \u03bcm). Fisher's LSD post-hoc analyses revealed group WM/RAM:Recent had significantly greater dendritic length compared to groups WM Only:Recent, WM Only:Remote, WM/OP:Remote, Control:Recent, and Control:Remote (p < 0.05). Group WM/RAM:Remote had significantly greater dendritic length compared to groups WM Only:Recent, WM Only:Remote, WM/OP:Remote, Control:Recent and Control:Remote (p < 0.05).Number of spines. Figure Apical. A Two-Way ANOVA with Behavioral group and time (Recent or Remote) as the fixed factors and number of spines as the dependent variable revealed a main effect of behavior , with group WM/RAM displaying the greatest number of spines (511). Fisher's LSD post-hoc analyses revealed group WM/RAM:Recent had significantly greater number of spines compared to groups WM Only:Recent, WM Only:Remote, WM/OP:Recent, WM/OP:Remote, Control:Recent and Control:Remote (p < 0.05). Group WM/RAM:Remote had significantly greater number of spines compared to groups Control:Recent and Control:Remote (p < 0.05).Basal. A Two-Way ANOVA with Behavioral group and time (Recent or Remote) as the fixed factors and number of spines as the dependent variable revealed a main effect of behavior , with group WM/RAM displaying the greatest number of spines (385). Fisher's LSD post-hoc analyses revealed group WM/RAM:Recent had significantly greater number of spines compared to groups WM Only:Recent, WM Only:Remote, WM/OP:Recent, WM/OP:Remote, Control:Recent, Control:Remote (p < 0.05). Group WM/RAM:Remote had significantly greater number of spines compared to groups WM Only:Recent, WM Only:Remote, Control:Recent, and Control:Remote (p < 0.05). Group WM/OP:Recent had significantly greater number of spines compared to groups WM Only:Recent and Control:Recent (p < 0.05).Spine density. Figure Apical. A Two-Way ANOVA with Behavioral group and time (Recent or Remote) as the fixed factors and spine density as the dependent variable revealed no main effects. Planned Fisher's LSD post-hoc comparisons revealed group WM/RAM:Recent had significantly greater spine density compared to group Control:Recent (p < 0.05).Basal. A Two-Way ANOVA with Behavioral group and time (Recent or Remote) as the fixed factors and spine density as the dependent variable revealed no main effects. A significant interaction between behavior X time was found. Fisher's LSD post-hoc comparisons revealed group WM/RAM:Recent had significantly greater spine density compared to groups WM Only:Recent, WM Only:Remote, WM/RAM:Remote, and Control:Recent (p < 0.05). Group WM/OP:Recent had significantly greater spine density compared to group Control:Recent (p < 0.05). Group Control:Remote had significantly greater spine density compared to group Control:Recent (p < 0.05).Summary of ACC findings. Group WM/RAM displayed the greatest and most consistent dendritic complexity in the ACC. At the recent time point, group WM/RAM was the sole group to show significantly increased dendritic complexity in apical dendrites compared to the control group.Figure Number of branches. Figure Apical. A Two-Way ANOVA with Behavioral group and time (Recent or Remote) as the fixed factors and number of branches as the dependent variable revealed a main effect of behavior with group WM/RAM displaying the greatest number of branches (38). Fisher's LSD post-hoc analyses revealed group WM/RAM:Recent had significantly greater number of branches compared to every other behavioral group (p < 0.05). Group WM/RAM:Remote had significantly greater number of branches compared to groups WM Only:Recent, WM Only:Remote, WM/RAM:Recent, Control:Recent, and Control:Remote (p < 0.05). Group WM/OP:Recent had significantly greater number of branches compared to group WM/OP:Remote (p < 0.05). Group WM/OP:Remote had significantly greater number of branches compared to groups WM/RAM:Recent, Control:Recent, and Control:Remote (p < 0.05).Basal. A Two-Way ANOVA with Behavioral group and time (Recent or Remote) as the fixed factors and number of branches as the dependent variable revealed a main effect of behavior with group WM/RAM displaying the greatest number of branches (16). A significant interaction between behavior X time was found . Fisher's LSD post-hoc analyses revealed group WM/RAM:Remote had significantly greater number of branches compared to groups WM Only:Remote, WM/OP:Recent, Control:Recent, and Control:Remote (p < 0.05). Group WM/OP:Recent had significantly fewer number of branches compared to groups WM/RAM:Remote, WM/OP:Remote (p < 0.05). Group WM/OP:Remote had significantly greater number of branches compared to group WM Only:Remote, WM/OP:Recent, and Control:Recent (p < 0.05).Dendritic length. Figure Apical. A Two-Way ANOVA with Behavioral group and time (Recent or Remote) as the fixed factors and dendritic length as the dependent variable revealed a main effect of behavior , with group WM/RAM displaying the greatest dendritic length (2259 \u03bcm). A significant interaction between behavior X time was found . Fisher's LSD post-hoc analyses revealed group WM/RAM:Recent had significantly greater dendritic length compared to groups WM Only:Recent, WM Only:Remote, WM/OP:Recent, Control:Recent, and Control:Remote (p < 0.05). Group WM/RAM:Remote had significantly greater dendritic length compared to group Contol:Remote (p < 0.05). Group WM/OP:Remote had significantly greater dendritic length compared to groups WM Only:Recent, WM Only:Remote, WM/OP:Recent, Control:Recent, and Control:Remote (p < 0.05).Basal. A Two-Way ANOVA with Behavioral group and time (Recent or Remote) as the fixed factors and dendritic length as the dependent variable revealed no main effects. Planned Fisher's LSD post-hoc comparisons revealed group WM/OP:Remote had significantly greater dendritic length compared to groups WM Only:Remote and Control:Recent. Group Control:Recent had significantly less dendritic length compared to groups WM Only:Recent and WM/OP:Remote (p < 0.05).Number of spines. Figure Apical. A Two-Way ANOVA with Behavioral group and time (Recent or Remote) as the fixed factors and number of spines as the dependent variable revealed a main effect of behavior , with group WM/RAM displaying the greatest number of spines (1793). Fisher's LSD post-hoc analyses revealed group WM/RAM:Recent had significantly greater number of spines compared to groups WM Only:Recent, WM Only:Remote, WM/OP:Recent, Control:Recent, and Control:Remote (p < 0.05). Group WM/RAM:Remote had significantly greater number of spines compared to group WM Only:Recent, WM Only:Remote, WM/OP:Reent, Control:Recent, and Control:Remote (p < 0.05). Group WM/OP:Recent had significantly fewer number of spines compared to groups WM/RAM:Recent, WM/RAM:Remote and WM/OP:Remote (p < 0.05). Group WM/OP:Remote had significantly greater number of spines compared to groups WM Only:Recent, WM Only:Remote, WM/OP:Recent, Control:Recent, and Control:Remote (p < 0.05).Basal. A Two-Way ANOVA with Behavioral group and time (Recent or Remote) as the fixed factors and number of spines as the dependent variable revealed a main effect of behavior , with group WM/RAM displaying the greatest number of spines (647). Fisher's LSD post-hoc analyses revealed group WM/RAM:Recent had significantly greater number of spines compared to group Control:Recent (p < 0.05). Group WM/RAM:Remote had significantly greater number of spines compared to groups WM Only:Remote, WM/OP:Recent, Control:Recent, and Control:Remote (p < 0.05). Group WM/OP:Recent had significantly fewer number of spines compared to groups WM/RAM:Remote and WM/OP:Remote (p < 0.05). Group WM/OP:Remote had significantly greater number of spines compared to groups WM Only:Remote, WM/OP:Recent, Control:Recent, and Control:Remote (p < 0.05).Spine density. Figure Apical. A Two-Way ANOVA with Behavioral group and time (Recent or Remote) as the fixed factors and spine density as the dependent variable revealed a main effect of behavior , with group WM/RAM displaying the greatest spine density (0.812). Fisher's LSD post-hoc analyses revealed group WM/RAM:Remote had significantly greater spine density compared to groups WM Only:Recent, WM Only:Remote, WM/OP:Recent, Control:Recent, and Control:Remote (p < 0.05).Basal. A Two-Way ANOVA with Behavioral group and time (Recent or Remote) as the fixed factors and spine density as the dependent variable revealed a main effect of behavior , with group WM/RAM displaying the greatest spine density (0.812). Fisher's LSD post-hoc analyses revealed group WM/RAM:Recent had significantly greater spine density compared to group Control:Recent (p < 0.05). Group WM/RAM:Remote had significantly greater spine density compared to groups WM/OP:Recent and Control:Recent (p < 0.05).Summary of CA1 findings. Group WM/RAM displayed the greatest dendritic complexity in the CA1. Group WM/RAM was the only group to show increased dendritic complexity at the recent time point. Group WM/RAM also showed persistent increases in the dendritic complexity characteristics analyzed as this group was found to have these characteristics significantly increased from the Control group at the remote time point as well. Group WM/OP:Remote consistently showed increases in dendritic complexity at the remote time point.A great deal of human research supports the idea of a time-limited role for the hippocampus in memory storage . Future behavioral studies utilizing one-day training procedures are underway so that early structural changes in the ACC and CA1 can be examined. A one-day training procedure would also help to diminish delay differences between the end of training on a behavioral task and neuron analysis. In the present study, there was a difference in the delay between the end of behavioral training and neuron analysis in rats trained on one or two tasks. Training on the RAM or OP tasks entailed 5 days of training followed by a day of rest then sacrifice. Rats trained on only the WM task had 6 days of rest prior to sacrifice. This difference could influence dynamic dendritic characteristics such as spine density. This difference may become more substantial when examining recent vs. remote time points.Neuron reconstruction data showed robust increases in dendritic complexity in the ACC and CA1 at recent and remote time points in groups trained on two hippocampal-dependent tasks. These results suggest competing hippocampal-dependent memories may result in more pronounced, or accelerated, increases in dendritic complexity in the ACC. Without hippocampal-dependent memories competing for hippocampal processing space, structural changes may be less distinct, or take a longer amount of time to come about. The introduction of a multiple memory behavioral procedure presents an innovative method through which structural changes associated with memory storage can be examined. Introducing variations in the type and number of tasks used during training provides an opportunity to examine and contrast morphological differences throughout the consolidation process, shedding light on the processes underlying remote memory storage.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Aquilegia pubescens and A. canadensis. The correct citation is: Noutsos C, Perera AM, Nikolau BJ, Seaver SMD, Ware DH (2015) Metabolomic Profiling of the Nectars of Aquilegia pubescens and A. canadensis. PLoS ONE 10(5): e0124501. doi:10.1371/journal.pone.0124501There is an error in the title. The correct title is: Metabolomic Profiling of the Nectars of The images for Figs Please see the corrected"} +{"text": "TP53 in gastric cancer (GC) has been investigated world-widely, but a comparison of mutation spectrum among GCs from various regions in the world are still sparsely documented. In order to identify the difference of TP53 mutation spectrum in GCs in Eastern Europe and in East Asia, we sequenced TP53 in GCs from Eastern Europe, Lujiang (China), and Yokohama, Kanagawa (Japan) and identified the feature of TP53 mutations of GC in these regions.Mutation spectrum of TP53. Mutation patterns were categorized into nine groups: six base substitutions, insertion, deletion and deletion-insertion. Within G:C\u2009>\u2009A:T mutations the mutations in CpG and non-CpG sites were divided. The Cancer Genome Atlas data having somatic mutation list of GCs from Whites, Asians, and other ethnicities were used as a reference for our data.In total, 689 tissue samples of GC were analyzed: 288 samples from East European populations , 268 from Yokohama, Kanagawa, Japan and 133 from Lujiang, Anhui province, China. DNA was extracted from FFPE tissue of Chinese, East European cases; and from frozen tissue of Japanese GCs. PCR products were direct-sequenced by Sanger method, and in ambiguous cases, PCR product was cloned and up to 8 clones were sequenced. We used No. NC_000017.11(hg38) as the reference sequence of The most frequent base substitutions were G:C\u2009>\u2009A:T transition in all the areas investigated. The G:C\u2009>\u2009A:T transition in non-CpG sites were prominent in East European GCs, compared with Asian ones. Mutation pattern from TCGA data revealed the same trend between GCs from White (TCGA category) vs Asian countries. Chinese and Japanese GCs showed higher ratio of G:C\u2009>\u2009A:T transition in CpG sites and A:T\u2009>\u2009G:C mutation was more prevalent in Asian countries.The divergence in mutation spectrum of GC in different areas in the world may reflect various pathogeneses and etiologies of GC, region to region. Diversified mutation spectrum in GC in Eastern Europe may suggest GC in Europe has different carcinogenic pathway of those from Asia.The online version contains supplementary material available at 10.1186/s41021-022-00257-y. GC inciay/home) . In mostay/home) , are knoay/home) .The time trends and geographic variation reflect the difference in causative factors of GC, such as differences in environmental factors, lifestyle, infection, traditional foods, and salty diet, as well as the genetic structure of individual populations.TP53 mutations, the most prevalent gene mutations found in human tumors, has provided important clues for environmental carcinogenesis [TP53 mutations in various human cancers in specific settings, such as tobacco smoking, UV damage, and aflatoxin exposure [TP53 in aflatoxin B-related hepatocellular carcinoma. These specific mutation spectra are currently being recapitulated as mutation signatures based on next-generation sequencing data, including single base substitution (SBS) 4, SBS 7, and SBS24 corresponding to tobacco, UV, and aflatoxin B, respectively [The spectrum of somatic ogenesis . In the exposure . For exaectively \u20138.TP53 database was created from voluminous mutation spectrum data in various cancers collected from different populations around the world. The IARC TP53 database by the NCI and other published reports indicated that the TP53 mutation observed in GC mainly involves G:C\u2009>\u2009A:T transitions [The nsitions .TP53 mutations in GC have mainly been found in Asians, Whites, Blacks, American Indians, Alaskan Natives, and Hawaiian natives.Although there are anecdotal reports on GC mutations from Poland , 11, infTP53 mutations in GC samples from East European countries and compared these with mutation spectra observed in GC samples from East Asian countries (China and Japan).In this report, for the first time, we characterized considerable numbers of In total, 689 GC tissue samples from three populations worldwide were analyzed: 288 samples from the European population , 268 from Japan, and 133 from China. The clinical profiles of the patients are summarized in Table\u00a0The pathology archives of GC formalin-fixed paraffin-embedded tissues (FFPE) were collected from three countries in Eastern Europe and Lujiang County, Anhui Province, China. Fresh GC tissues were obtained from the Pathology Department of Kanagawa Cancer Center, Yokohama. DNA was extracted from the FFPEs using the QIAamp DNA FFPE Tissue Kit , 13, whiTP53 gene sequencing was performed by direct sequencing using a polymerase chain reaction (PCR) product amplified using respective primer sets for each exon. Fragments covering exons 2 to 11 and the boundary regions of the TP53 gene were amplified via PCR using the HotStarTaq DNA polymerase (Qiagen). The PCR products were purified with Exo-SAP-IT and sequenced via the Sanger method using the BigDye Terminator Cycle Sequencing Reaction Kit, ver.3.1 and ABI 3130xL Genetic Analyzer (Thermo Fisher Scientific). PCR products exhibiting multiple bands were sequenced after subcloning them into a pGEM-T Easy vector system . Up to eight clones were sequenced, particularly upon confirming the presence of insertion/deletion mutations. The primers used are listed in Table STP53 reference sequence (Accession No. NC_000017.11(hg38 and GRCh38)). For the DNA samples from FFPEs that were difficult to amplify, and when amplification was not successful, the primer designs were modified to amplify different segments of TP53. The resulting sequences were assigned to the reference sequences. DNA mutations were described according to the international guidelines for gene nomenclature [nclature . In thisTP53 mutation patterns were categorized as follows: G:C\u2009>\u2009A:T, A:T\u2009>\u2009G:C, G:C\u2009>\u2009C:G, G:C\u2009>\u2009T:A, A:T\u2009>\u2009C:G, A:T\u2009>\u2009T:A, deletion, deletion-insertion, insertion, and splice-site mutations in exons 4 to 8. G:C\u2009>\u2009A:T mutations were subclassified based on their localization in CpG or non-CpG sites. We also grouped TP53 mutations found here according to the following hotspots proposed by Hainaut P [ainaut P : c.524G\u2009TP53 mutations was drawn using cBioPortal MutationMapper [A distribution map of _mapper) to deterTP53 gene was downloaded for Caucasian (n\u2009=\u2009278) and Asian (n\u2009=\u200989) populations from the Stomach Adenocarcinoma data in cBioPortal. The downloaded dataset included 148 Caucasian and 47 Asian patients with intrasomatic mutations. The percentage of base substitution patterns obtained for each race was determined. Since the nucleotide sequence files were not available, we extracted the patterns of G\u2009>\u2009A or C\u2009>\u2009T gene mutations to determine if they were in the CpG region, and the sequences of the mutations were compared against the reference genome (GRCh38) by extracting the sequences before and after the base substitution. Base substitutions at the splice site or region were preferentially classified as splice mutations.Information on somatic mutations except for synonymous mutations in the TP53 mutations. Ninety-six substitution types and sequence contexts were counted for each population. The percentage of each of the 96 substitution types was calculated from the total number of substitutions in each population. The SBS for each population was estimated using Signal [Substitutions in the coding sequence were determined from the somatic res.com) .This study was a retrospective, anonymous, and non-intervention study, and informed consent from the patients was waived. The research plan was agreed upon by all researchers and approved by the IRB of the Hamamatsu University School of Medicine (G-260 and 20-110), Kanagawa Cancer Center, and the Ethical Committee of the University of Medicine and Pharmacy of Targu-Mures, Romania (Agreement no. 124/28.07.2016).Statistical analyses were performed using the chi-square test, t-test, and Fisher\u2019s exact test with JMP, ver.11.2 test, p\u2009=\u20092\u2009\u00d7\u200910\u2212\u20095). Japanese patients were older than Chinese patients . There were no significant differences in sex among the three populations had TP53 mutations, and 404 (59%) were wild-type. The ratios of mutated cases were 29.5% (85/288), 57.1% (76/133), and 46.3% (124/268) in Eastern Europe, China, and Japan, respectively among the three regions; when the prevalence in Japan and China were combined (as East Asia), the prevalence was greater in East Asia than in Eastern Europe .A total of 689 genomic samples were successfully analyzed for TP53 were relatively evenly distributed among the three groups. Those at CpG sites involved several mutation assemblies, such as R175H and R248W/Q in Eastern Europe and Japan, and R273C/H in China for the three areas are presented. Mutation-accumulated codons, such as R175H/G, R248W/Q, and R273C/H/P, were observed in each population in five to ten cases , China (17.8%), and Japan 15.6%) (\u03c7.6% (\u03c72 tp\u2009=\u20090.04) than in East Asian countries. On the other hand, A:T\u2009>\u2009G:C showed a significantly higher prevalence in China and Japan than in Eastern Europe, especially in diffuse-type GCs in the diffuse type of GC in Eastern Europe, in which the prevalence of G:C\u2009>\u2009A:T at non-CpG sites was significantly higher 30.3%) [TET genes and methylation erasers were downregulated in mice with gastric inflammation, causing aberrant methylation [TP53 in East Asia, specifically G:C\u2009>\u2009A:T at CpG sites in GC, reflects the common infectious status of the stomach there. The increased ratio of G:C\u2009>\u2009T:A in GC in East Asian countries may also reflect increased oxyradical DNA damage caused by continued inflammation in the stomach. Non-environmental mechanisms may also play a role in the process. A considerable number of G:C\u2009>\u2009A:T mutations were also found in Eastern European cases, and the issue of whether G:C\u2009>\u2009A:T mutations at CpG and non-CpG sites are related to the histological type of GC is still an enigma.The higher frequency of G:C\u2009>\u2009A:T mutations at CpG sites in GC in Asia probably reflects chronic inflammation, specifically, chronic gastritis caused by chronic nfection \u201327. Chronfection , 29. G:Cnfection \u201332. Infli (CagA) . Recentlhylation . Thus, tTP53 mutations at CpG and non-CpG sites reminds us of several aspects of environmental gastric carcinogenesis [TP53 mutations at non-CpG sites had a more traditional dietary history, including nitrite, protein, and fat, particularly from animal sources, than in those with mutations at CpG sites. In addition, nitric oxide induced by gastritis may be used to produce N-nitroso compounds [N-Nitroso compounds can also be taken up by the human body through water, drugs, cosmetics, and tobacco. Since a successful experimental model of GC using N-nitroso compounds has been established [N-nitroso compounds [N-Nitroso compounds can generate alkylated guanine adducts, which contribute to G:C\u2009>\u2009A:T transitions. In contrast, deamination after the nitrosation of guanine and adenine produces xanthine and hypoxanthine, respectively. Hypoxanthine induces A:T\u2009>\u2009G:C transitional mutations [N-nitroso compounds remains a challenge. The generation of DNA adducts has been attributed to alkylating agents hypothetically [The implication of the difference in ogenesis . The epiompounds . N-Nitroablished , many inompounds . N-Nitroutations . These autations ; howeveretically \u201342; howeTP53 in understudied populations may encourage us to pursue the etiological varieties of GC in populations worldwide. We were not able to explain the exact causes of A:T\u2009>\u2009G:C in GC samples from East Asia. Hongyo et al. have already shown this mutation spectrum in their summary tables and stated that A:T\u2009>\u2009G:C mutations are prevalent in \u201cOriental\u201d regions but did not expound much on this finding [HPRT locus in CHO-K1 cells [The mutation spectrum of finding . Lee DH K1 cells . Some enK1 cells , may alsK1 cells \u201342; howeTP53 mutations, and the sample size was smaller than that generated by the international consortium. As such, hundreds of tumors from Eastern European residents were not compared with those from East Asian residents. It is necessary to confirm our findings by analyzing a large-scale sample set. Second, the designated \u201cChinese\u201d samples originated from a single institution only; thus, generalizing our findings in this population for Chinese patients with GC would be inappropriate, considering the extensive variations in environmental exposures for these patients. Third, the FFPE quality may not be perfectly controlled. No central pathological diagnosis was made. Primer coverage was not the same; thus, the detectability of splice site mutations may have differed. Currently, data on somatic mutations in human cancers using next-generation sequencing are accumulating, and the implications of our results may need to be re-evaluated. The mutation spectrum of ARID1A, which is currently the most prevalent mutated gene in all cancers, is also of interest.Our study has several obvious limitations. First, we only studied p\u2009=\u20090.04 in the two groups, East Europe vs. Asian countries). This apparent difference in the mutation spectrum in different histological subtypes is interesting; however, we must be careful in accepting this finding because we did not use a centralized pathological diagnosis system in this study. In each region, the numbers of blocks that were pathologically investigated were very different, and the method of histological subtyping differed among pathologists from each region. A more comprehensive approach, such as Massive Parallel Sequencing accompanied by centralized pathological assessment, will yield greater information.Another problem is the subjective bias of histological typing of GC in these three regions. G:C\u2009>\u2009A:T mutation prevalence was high (69.6%), especially in the diffuse type of GC in Eastern Europe, in which the ratio of G:C\u2009>\u2009A:T at non-CpG sites was significantly high (30.3%) . Mutation-distribution maps were created using the cBioPortal mutation mapper . Black dots indicate truncating mutations (nonsense and frameshift mutations). The light purple dot indicates a silent mutation. P53_TAD, TP53 transcriptional activation domain; P53, TP53 DNA-binding domain; P53 tetramer, TP53 tetramer domain.Additional file 2: Supplementary Figure S2.TP53 mutation types in the intestinal and diffuse types of GC samples from Eastern Europe, China, and Japan (exon 4-8). The pie graphs show the percentages of the mutations, including missense (blue), nonsense (orange), and silent mutations (gray), deletions (del) (yellow), deletion-insertion (delins) (light blue), insertions (ins) (light green), and splice site mutations (dark blue) in TP53 in GC samples from Eastern Europe, China, and Japan. The prevalence of silent mutations (gray) in diffuse-type GCs was significantly different between Europe and Asia (P\u2009<\u20090.01). * Statistically significant difference (p\u2009<\u20090.05).Additional file 3: Supplementary Figure S3. Mutation spectra in intestinal and diffuse-type GCs in East Europe, China, and Japan. The TP53 mutations were classified into six types of single nucleotide substitutions, as well as deletions (del), insertions (ins), deletion-insertion (delins), and splice mutations. The G:C\u2009>\u2009A:T transition was subdivided into G:C\u2009>\u2009A:T at CpG and non-CpG sites. Each spectrum is shown in the pie graph as follows: G:C\u2009>\u2009A:T at CpG sites (blue), G:C\u2009>\u2009A:T at non-CpG sites (orange), A:T\u2009>\u2009G:C (gray), G:C\u2009>\u2009C:G (yellow), G:C\u2009>\u2009T:A (light blue), A:T\u2009>\u2009C:G (light green), A:T\u2009>\u2009T:A (dark blue), del (brown), delins (dark gray), ins (light brown), and splice mutations (light navy). The prevalence of G:C\u2009>\u2009A:T at non-CpG sites (orange) in diffuse-type GCs in Asia was significantly different from that in Eastern Europe (p\u2009<\u20090.05). * Statistically significant difference (p\u2009<\u20090.05).Additional file 4: Supplementary Figure S4. Designations of populations based on TCGA classification. The majority of the data were from the \u201cWhite\u201d population. The \u201cAsian\u201d population was not defined, especially whether they were only residing in Asia or not. NA, not available.Additional file 5: Supplementary Figure S5. Mutation spectrum of the TP53 gene in GC in each population. Six substitution types are depicted in the bar graph as follows: C\u2009>\u2009A (light blue), C\u2009>\u2009G (black), C\u2009>\u2009T (red), T\u2009>\u2009A (gray), T\u2009>\u2009C (yellow-green), and T\u2009>\u2009G .Additional file 6: Table S1. Primers for PCR amplification of TP53 gene. Table S2. Histological type and TP53 mutation status. Table S3. Mutation distribution in TP53 (exon 4\u20138). Table S4. Mutation functions of TP53 (exon 4-8) in Eastern Europe, China and Japan. Table S5. Mutation spectrum of TP53 (exon 4\u20138) in Eastern Europe, China and Japan. Table S6. Hotspot mutation in Eastern Europe, China and Japan."} +{"text": "Scientific Reportshttps://doi.org/10.1038/srep19401, published online 14 January 2016Correction to: This Article contains an error in Equation 15, where the should read:An extra note of caution is warranted regarding the variances"} +{"text": "Funding statement. Several fundings were not included. The correct Funding statement appears below.In the published article, there was an error in the \u201cThis work was supported by U.K. Research and Innovation Biotechnology and Biological Sciences Research Council Grants BBS/E/I/00001825, BBS/E/I/00007030, BBS/E/I/00007031, BB/S01506X/1, BBS/E/I/00002529, BBS/E/I/00007039, BBS/E/I/00007032, BB/N002598/1 and BB/V019031/1. The authors would also like to acknowledge the Pirbright Flow Cytometry facility and support through the Core capability grant (BBS/E/I/00007039).\u201dThe authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."} +{"text": "Biol. Open (2020) 9, bio050435 (doi:10.1242/bio.050435)There was an error published in In The authors apologise to readers for the error, which does not impact the results or conclusions of this paper."} +{"text": "Interface Focus12, 20220020. (Published online 12 August 2022). (https://doi.org/10.1098/rsfs.2022.0020)The Funding section of the article \u2018Mathematical modelling of oxygen transport in a muscle-on-chip device\u2019, should read as follows:This project has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement no. 801423. This research was funded in whole, or in part, by the Engineering and Physical Sciences Research Council (EPSRC) (grant nos. EP/R029598/1 and EP/T008806/1). For the purpose of open access, the author has applied a creative commons attribution (CC BY) licence to any author accepted manuscript version arising."} +{"text": "Shallow groundwater (GW), defined as the water table of unconfined or perched aquifers that is near enough to the land surface to influence the vadose zone and the surface soil moisture, impacts land surface water, energy, and carbon cycles by providing additional moisture to the root zone via capillary fluxes. Although the interactions of shallow GW and the terrestrial land surface are widely recognized, incorporating shallow GW into the land surface, climate, and agroecosystem models is not yet possible due to the lack of groundwater data. Groundwater systems are affected by various factors, including climate, land use/land cover, ecosystems, GW extractions, and lithology. Although GW wells are the most direct and accurate way of monitoring water table depths at point scales, upscaling GW levels from point scale to areal or regional scale poses significant challenges.Here, we provide high spatiotemporal resolution global maps of the terrestrial land surface areas influenced by shallow GW from mid-2015 to 2021 (a separate NetCDF file for each year) in a 9 km spatial and daily temporal resolution. We derived this data from NASA's Soil Moisture Active Passive (SMAP) mission spaceborne soil moisture observations with a temporal resolution of 3 days and approximately 9 km grid resolution. This spatial scale corresponds to SMAP's \"Equal Area Scalable Earth\" (EASE) grids. The central assumption is that the monthly moving average of soil moisture observations and their coefficient of variation are sensitive to shallow GW regardless of the prevailing climate. We process the Level-2 enhanced passive soil moisture SMAP (SPL2SMP_E) product to detect shallow GW signals. The presence of shallow GW data is calculated by an ensemble machine learning model, which is trained using simulations from a variably saturated soil moisture flow model (Hydrus-1D). The simulations span various climates, soil textures, and lower boundary conditions.The spatiotemporal distribution of shallow GW data based on SMAP soil moisture observations is provided for the first time with this dataset. The data are of value in a wide variety of applications. The most direct use is in climate and land surface models as lower boundary conditions or as a diagnostic tool to verify model results. Some other applications may include flood risk analyses and regulation, identifying geotechnical issues such as shallow GW-triggered liquefaction, global food security, ecosystem services, watershed management, crop yield, vegetation health, water storage trends, and tracking mosquito-borne diseases by identifying wetlands, among other applications. Specifications Table\u2022An observation-based database representing shallow groundwater presence globally is provided. Given the high spatiotemporal resolution, the shallow GW dataset may contribute to the efforts of integrating GW into surface flux models as a calibration or diagnostic tool to assess the validity of such models. Moreover, the dataset helps reduce the model biases originating from the additional water added to the root zone due to shallow GW presence.\u2022The dataset presented here can potentially be helpful in a wide variety of research areas, including flood risk analyses and regulation, identifying geotechnical issues such as liquefaction, global food security, ecosystem services, watershed management, crop yield, vegetation health, water storage trends, and global distributions of wetlands.\u2022Another, more direct, use of the dataset may be to use it as LBC to the surface flux models. This option may improve the soil moisture flux simulation accuracies while reducing the computational cost of such models. Adopting this shallow GW database based on observations, as a driver to control the model LBC allows modelers to solve the Richards equation in 1-D instead of 3-D without losing the accuracy of results.1Shallow GW provides additional water to ecosystems and the land surface. The additional water resulting from the coupling between GW and the surface not only affects evapotranspiration but also has the potential to modify ecosystems and components of water, energy, and carbon cycles. The lack of global-scale shallow GW data with high spatial and temporal resolution hinders our understanding of terrestrial water, energy, carbon, and water cycle components.The theoretical background on generating the shallow GW dataset was explained in detail in 2Shallow GW data is provided in multidimensional NetCDF file contents. A separate NetCDF file is produced for each year from mid-2015 to 2021. Each data file covers the entire temporal range from January 1st to December 31st, except the file for the year 2015, which covers only between June 1st to December 31st due to SMAP data availability.The dataset has a \u201cShallow Groundwater Presence\u201d variable, indicating through a binary variable whether the region is under shallow groundwater influence or not. If this variable equals 1, the given pixels are under shallow GW influence. A pixel value of -9999 value indicates that there is no shallow GW influence or that there is not sufficient soil moisture data available to detect shallow GW presence.File name: ShallowGW2015.ncFiletype: NetCDF fileSpatial extent: Latitude: -180:180 Degrees North; Longitude: -85:85 Degrees EastSpatial resolution: 9 kmTemporal coverage: Start date:06/01/2015; End date:12/31/2015Temporal resolution: 1 dayFile name: ShallowGW2016.ncFiletype: NetCDF fileSpatial extent: Latitude: -180:180 Degrees North; Longitude: -85:85 Degrees EastSpatial resolution: 9 kmTemporal coverage: Start date:01/01/2016; End date:12/31/2016Temporal resolution: 1 dayFile name: ShallowGW2017.ncFiletype: NetCDF fileSpatial extent: Latitude: -180:180 Degrees North; Longitude: -85:85 Degrees EastSpatial resolution: 9 kmTemporal coverage: Start date:01/01/2017; End date:12/31/2017Temporal resolution: 1 dayFile name: ShallowGW2018.ncFiletype: NetCDF fileSpatial extent: Latitude: -180:180 Degrees North; Longitude: -85:85 Degrees EastSpatial resolution: 9 kmTemporal coverage: Start date:01/01/2018; End date:12/31/2018Temporal resolution: 1 dayFile name: ShallowGW2019.ncFiletype: NetCDF fileSpatial extent: Latitude: -180:180 Degrees North; Longitude: -85:85 Degrees EastSpatial resolution: 9 kmTemporal coverage: Start date:01/01/2019; End date:12/31/2019Temporal resolution: 1 dayFile name: ShallowGW2020.ncFiletype: NetCDF fileSpatial extent: Latitude: -180:180 Degrees North; Longitude: -85:85 Degrees EastSpatial resolution: 9 kmTemporal coverage: Start date:01/01/2020; End date:12/31/2020Temporal resolution: 1 dayFile name: ShallowGW2021.ncFiletype: NetCDF fileSpatial extent: Latitude: -180:180 Degrees North; Longitude: -85:85 Degrees EastSpatial resolution: 9 kmTemporal coverage: Start date:01/01/2021; End date:12/31/2021Temporal resolution: 1 day3To produce the shallow GW dataset, we applied a supervised EML model on SMAP L2 soil moisture observations. Surface soil moisture simulations representing SMAP observations are used to train the EML model . Below, 3.1The SMAP sensors capture L-band microwave emissions globally with approximately 3 days temporal and 36 km spatial resolution. We use the radiometer-based level 2 enhanced passive soil moisture (SPL2SMP_E Version 5) product, which has a 9 km grid resolution derived from a 36 km spatial resolution 3.2The Hydrus-1D model, which solves the Richards equation for variably saturated soil water flow, is used to simulate the surface soil moisture, mimicking SMAP observations. The model is driven by the Global Precipitation Measurement (GPM) mission precipitation data boundary conditions at five different depths, representing constant water table depths from 0.5 m to 2.5 m, in increments of 50 cm, are used. In addition, a simulation with free drainage lower boundary conditions is done. The total soil profile domain thickness is taken as 3 meters, but only the top 5 cm of the simulations are considered to represent SMAP soil moisture observations. The model simulations cover five years period from 2015 to 2019. We use the first year as the model spin-up period. The results of that year are not used in the data analyses.3.3The gentle adaptive boosting (GentleBoost), a variant of the adaptive boosting ensemble machine learning (EML) technique, is used to classify whether the surface soil moisture data is affected by shallow groundwater. To develop the EML method, about 1.2 million soil moisture data points are used in model training, and about 0.8 million data points are used in model testing. The daily coefficient of variation, minimum, maximum, and average surface soil moisture values are used as model predictors. A 30-day moving window average is used to calculate each predictor. We find that using a monthly time window width provides a sufficient number of sample points allowing us to analyze the soil moisture signals in a statistically meaningful way. At the same time, a 30-day window width does not smooth the shallow groundwater signals too much.Once the EML model is trained, we apply it to the SMAP observations covering the period from mid-2015 to the end of 2021. We adopt a data correction scheme as the last step before the final dataset, as recommended by The shallow GW dataset is evaluated against three datasets. First, we compare to baseflow estimations in the southeast US. Second, we check consistency with global scale wetland distributions estimated based on a global scale GW model outputs https://www.elsevier.com/authors/journal-authors/policies-and-ethics.The current work does not involve human subjects, animal experiments, or any data collected from social media platforms and meets the ethical requirements for publication in Data in Brief as stated in Mehmet Evren Soylu: Conceptualization, Investigation, Formal analysis, Methodology, Validation, Visualization, Writing \u2013 original draft; Writing \u2013 review & editing; Rafael L. Bras: Supervision, conceptualization, Writing \u2013 review & editing.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper."} +{"text": "In \u201cImproving Pelvic Floor Muscle Training Adherence Among Pregnant Women: Validation Study\u201d :e30989), the following errors were noted.1. Abstract:In the originally published paper, the first sentence in the abstract was stated asMobile health apps, for example, the T\u00e4t, have been shown to be potentially effective in improving pelvic floor muscle training (PFMT) among women, but their effectiveness in pregnant women was limited.This has been corrected to:Mobile health apps, for example, the T\u00e4t, have been shown to be potentially effective in improving pelvic floor muscle training (PFMT) among women, but they have not yet been studied among pregnant women.2. Methods, Intervention Mapping:The originally published paper was missing two references for this statement: The outcomes of the intention are self-efficacy (17 questions) and adherence (6 questions).This has been corrected to:The outcomes of the intention are self-efficacy (17 questions) (41) and adherence (6 questions) (42). 3. Methods, Cross-Sectional Study:The originally published paper was missing two references for this statement:The findings from this study provided input for the content of their educational videos and short notes on PFMT, which were captured as frequently asked questions (FAQ).This has been corrected to:The findings from this study provided input for the content of their educational videos and short notes on PFMT which were captured as frequently asked questions (FAQ).4. Results:The originally published paper stated the following in row 1, column 2 of Table 5:1.System credibility-expertise and authority.2. Primary support-Virtual rehearsal principleThis has been corrected to:System credibility-expertise and authority5. Results:The originally published paper stated the following as the title for the first column of Table 5:COM-B model and behavioral change techniques incorporated in the mHealth app.This has been corrected to:COM-B model and features of the mHealth app.6. Discussion:The originally published paper was missing one reference for this statement:The PSD component of the system\u2019s credibility and trustworthiness, with the expertise involved in the development, may add to the user\u2019s sense of safety and reliability regarding the KEPT app.This has been corrected to:The PSD component of the system\u2019s credibility and trustworthiness (55), with the expertise involved in the development, may add to the user\u2019s sense of safety and reliability regarding the KEPT app.6. References:In the corrected paper, the following citations have been newly added to the Reference List. As these new references have been numbered per the order of their in-text citations, the remaining citations in the reference list have been renumbered accordingly. 41. Sacomori C, Cardoso FL, Porto IP, Negri NB. The development and psychometric evaluation of a self-efficacy scale for practicing pelvic floor exercises. Brazilian Journal of Physical Therapy; 2013. [doi: 10.1590/S1413-35552012005000104] 42. Newman-Beinart NA, Norton S, Dowling D, Gavriloff D, Vari C, Weinman JA, Godfrey EL. The development and initial psychometric evaluation of a measure assessing adherence to prescribed exercise: the Exercise Adherence Rating Scale (EARS). Physiotherapy, 103(2); 2017, 180\u2013185. [doi: 10.1016/j.physio.2016.11.001]45. Alagirisamy P, Mohd Sidik S. Pelvic Floor Muscle Exercises During and After Pregnancy. Universiti Putra Malaysia Press Serdang 2020.46. Bo K, Berghmans B, Morkved S, Van Kampen M. Evidence-Based Physical Therapy for the Pelvic Floor-E-Book: Bridging Science and Clinical Practice, 2nd ed. London, UK: Elsevier Health Sciences; 2014. 3.55. Asklund I, Nystr\u00f6m E, Sj\u00f6str\u00f6m M, Umefjord G, Stenlund H, Samuelsson E. Mobile app for treatment of stress urinary incontinence: A randomized controlled trial. Neurourol Urodyn 2017 Jun;36(5):1369-1376. [doi: 10.1002/nau.23116] [Medline: 27611958]The correction will appear in the online version of the paper on the JMIR Publications website on April 11, 2022, together with the publication of this correction notice. Because this was made after submission to PubMed, PubMed Central, and other full-text repositories, the corrected article has also been resubmitted to those repositories."} +{"text": "Cell Death and Disease 10.1038/cddis.2016.292, published online 13 October 2016Correction to: The original version of this article unfortunately contained an error in fig. 3e. The authors apologize for the error. The correct figure can be found below."} +{"text": "To our knowledge, there is no Parkinson\u2019s disease (PD) gait biomechanics data sets available to the public.This study aimed to create a public data set of 26 idiopathic individuals with PD who walked overground on ON and OFF medication.Their upper extremity, trunk, lower extremity, and pelvis kinematics were measured using a three-dimensional motion-capture system . The external forces were collected using force plates. The results include raw and processed kinematic and kinetic data in c3d and ASCII files in different file formats. In addition, a metadata file containing demographic, anthropometric, and clinical data is provided. The following clinical scales were employed: Unified Parkinson\u2019s disease rating scale motor aspects of experiences of daily living and motor score, Hoehn & Yahr, New Freezing of Gait Questionnaire, Montreal Cognitive Assessment, Mini Balance Evaluation Systems Tests, Fall Efficacy Scale-International\u2013FES-I, Stroop test, and Trail Making Test A and B.https://figshare.com/articles/dataset/A_dataset_of_overground_walking_full-body_kinematics_and_kinetics_in_individuals_with_Parkinson_s_disease/14896881).All data are available at Figshare (This is the first public data set containing a three-dimensional full-body gait analysis of individuals with PD under the ON and OFF medication. It is expected to contribute so that different research groups worldwide have access to reference data and a better understanding of the effects of medication on gait. Several studies seek to understand the gait kinematics of Parkinson\u2019s disease (PD) patients to identify which parameters are altered with the disease. Compared to healthy individuals in general, it is known that these patients tend to present changes in the spatiotemporal parameters of gait, such as a decrease in step length, reduction in gait speed, and difficulty in the start and stop movement . HoweverA few gait datasets in PD are available in the literature . AlthougThe data collection was performed in the Laboratory of Biomechanics and Motor Control at the Federal University of ABC, Brazil. The local Ethics Committee approved this study (protocol number 21948619.6.0000.5594), and all patients signed a consent form before collecting data. The patients were on a stable dose of l-DOPA for at least 1 month. The idiopathic individuals with PD participated in two experimental sessions for 1 week, one in the ON condition of the medication and the other in the OFF condition. To be considered ON condition, participants had taken dopaminergic medication 1 h before starting the session to ensure dose stabilization. In the OFF condition, the participants spent at least 12 h without medication for Parkinson\u2019s disease . The ord2, Hoehn & Yahr (H&Y) scale between 1 and 4, and 13 with freezing of gait (FoG). Inclusion criteria were the absence of neurological or physical dysfunctions other than those associated with PD and no diagnosed vestibular, visual, or somatosensory dysfunctions as self-declared.A convenience sample of 26 idiopathic individuals with PD was recruited to participate in this study. The patients were recruited from local communities and included local neighborhoods and ambulatory movement disorders. The patients were interviewed to collect information about their demographic characteristics, socio-cultural characteristics, and overall health condition. Their ages varied from 44 to 81 years, body masses from 53.3 to 94.6 kg, heights from 151.5 to 179.0 cm, body-mass indexes (BMI) from 19.2 to 34.3 kg/mAll gait trials were performed barefoot, and the participants wore comfortable shorts (women wore sports bras). Participants were asked to perform overground walking trials at a self-selected comfortable speed. The marker-set protocol adopted for this study comprised: (a) in the lower limb, 26 anatomical reflective markers ; and (b)1.The researcher explained to each patient the process of data collection. The patient was informed that he or she would be monitored during the data collection. There should not be any verbal communication during the trials, but he or she could interrupt the data collection if desired. Furthermore, that assistance would be given if necessary.2.After these explanations, the patient signed the informed consent form.3.The researcher interviewed the subject to collect information about his or her clinical data, medication, and disease diagnosis.4.At the beginning of each session, two experienced physiotherapists in movement disorders applied the following scales: Unified Parkinson\u2019s Disease Rating Scale motor aspects of experiences of daily living (UPDRS-II) and motor aspects (UPDRS-III) , H&Y Ho, New Fre5.Participants rested for 10 min.6.Markers were placed directly onto the skin of the full body .7.X-axis of the laboratory coordinate system (LCS) . A templ8.After the calibration trial and a familiarization period, participants were instructed to walk at a comfortable self-selected speed along a 20-m walkway. Participants performed 20 trials. The participants did not use any aid during the trials.Standard gait analysis was collected using a motion-capture system that had 12 cameras , five force platforms embedded in the floor. The kinematic data were acquired at 150 Hz, and the data on ground-reaction forces were acquired at 300 Hz using a motion-capture system .The data processing was performed using Cortex 6.0. Visual 3D software version 6.00.33 performed all kinematics and kinetics calculations. To enable users to process the data in the Visual 3D software, a Visual 3D pipeline file is available at Figshare. The analysis of the overground trials considered only those files that contained at least one full gait cycle (stance and swing phase) detected by kinematics. Heel strike and toe-off were calculated bilaterally using the horizontal velocity of the heel marker method . The pub1 in both c3d and ASCII file formats. The data set comprises a file with metadata, and separate text files were generated for the markers, angular kinematics, and force signals at 150 Hz. The clinical characteristics of the patients are presented in The data is available at FigshareThe metadata file named PDGinfo.txt contains 61 information from each patient\u2019s anamnesis and clinical scales. Here is the coding for the metadata:1.ID: the file name of the stabilography trial .2.Gender: gender (F or M).3.Age: patient\u2019s age in years.4.Height (cm): height in meters .5.Weight (kg): weight in kilograms .6.2): body mass index in kg/m2.BMI (kg/m7.Ortho-Prosthesis: name of the orthosis or prosthesis the subject wears (\u201cNo\u201d if the patient did not wear any orthosis or prosthesis).8.Years of formal study: years of formal education.9.Disease duration (years): year from diagnosis.10.\u20131): total daily levodopa equivalent dose in mg\u22c5day\u20131 according to L-Dopa equivalent units (mg\u22c5day11.FoG group: presence (freezers) or not (non-freezers) of freezing of gait.12.NFoG-Q (score): score of New Freezing of Gait Questionnaire.13.Initial symptoms: self-reported initial symptoms.14.Is there a family history of PD? Who?15.Do you feel improvement after using the antiparkinsonian medicine?: Yes or No.16.Have you ever had any surgery? Which?17.Any rehabilitation or physical activity?: name of the rehabilitation or physical activity performed by the patient .18.Other diseases : name of the disability of the patient (\u201cNo\u201d if the patient did not present any disability).19.Handedness: a self-reported manual preference.20.ON\u2013Hoehn & Yahr: Hoehn & Yahr score in the ON medication.21.ON\u2013MoCA: MoCA score in the ON medication.22.ON\u2013miniBESTest: miniBESTest score in the ON medication.23.ON\u2013FES-I: FES-I score in the ON medication.24.ON\u2013UPDRS-II: total score of the UPDRS-II in the ON medication.25.ON\u2013UPDRS-II\u2013walking: score of item 4\u2013walking of the UPDRS-II in the ON medication.26.ON\u2013UPDRS-III: total score of the UPDRS-III in the ON medication.27.ON\u2013UPDRS-III\u2013Rigidity: score of item 5\u2013rigidity of UPDRS-III in the ON medication.28.ON\u2013UPDRS-III\u2013Gait: score of item 12\u2013rigidity of UPDRS-III in the ON medication.29.ON\u2013PIGD or TD: Postural Instability/Gait Difficulty (PIGD) or Tremor Dominant (TD) phenotypes in the ON medication, according to 30.ON\u2013UPDRS-III asymmetry: clinical asymmetry was defined as the difference between the summed UPDRS scores of the left and right body sides (items 3.3\u20133.8 and 3.15\u20133.17). The most affected body side was the side with the highest UPDRS score in the ON medication.31.ON\u2013Stroop-I time (s): time to complete part I of the Stroop test in the ON medication.32.ON\u2013Stroop-I error: number of errors presented in part I of the Stroop test in the ON medication.33.ON\u2013Stroop-II time (s): time to complete part II of the Stroop test in the ON medication.34.ON\u2013Stroop-II error: number of errors presented in part II of the Stroop test in the ON medication.35.ON\u2013Stroop-III time (s): time to complete part III of the Stroop test in the ON medication.36.ON\u2013Stroop-III error: number of errors presented in part III of the Stroop test in the ON medication.37.ON\u2013TMTA time (s): time to complete part A of the TMT in the ON medication.38.ON\u2013TMTA error: number of errors presented in part A of the TMT in the ON medication.39.ON\u2013TMTB time (s): time to complete part B of the TMT in the ON medication.40.ON\u2013TMTB error: number of errors presented in part B of the TMT in the ON medication.41.OFF\u2013Hoehn & Yahr: Hoehn & Yahr score in the OFF medication.42.OFF\u2013MoCA: MoCA score in the OFF medication.43.OFF\u2013miniBESTest: miniBESTest score in the OFF medication.44.OFF\u2013FES-I: FES-I score in the OFF medication.45.OFF\u2013UPDRS-II: total score of the UPDRS-II in the OFF medication.46.OFF\u2013UPDRS-II\u2013walking: score of item 4\u2013walking of the UPDRS-II in the OFF medication.47.OFF\u2013UPDRS-III: total score of the UPDRS-III in the OFF medication.48.OFF\u2013UPDRS-III\u2013Rigidity: score of item 5\u2013rigidity of UPDRS-III in the OFF medication.49.OFF\u2013UPDRS-III\u2013Gait: score of item 12\u2013rigidity of UPDRS-III in the OFF medication.50.OFF\u2013PIGD or TD: Postural Instability/Gait Difficulty (PIGD) or Tremor Dominant (TD) phenotypes in the OFF medication, according to 51.OFF\u2013UPDRS-III asymmetry: clinical asymmetry was defined as the difference between the summed UPDRS scores of the left and right body sides (items 3.3\u20133.8 and 3.15\u20133.17). The most affected body side was the side with the highest UPDRS score in the OFF medication.52.OFF\u2013Stroop-I time (s): time to complete part I of the Stroop test in the OFF medication.53.OFF\u2013Stroop-I error: number of errors presented in part I of the Stroop test in the OFF medication.54.OFF\u2013Stroop-II time (s): time to complete part II of the Stroop test in the OFF medication.55.OFF\u2013Stroop-II error: number of errors presented in part II of the Stroop test in the OFF medication.56.OFF\u2013Stroop-III time (s): time to complete part III of the Stroop test in the OFF medication.57.OFF\u2013Stroop-III error: number of errors presented in part III of the Stroop test in the OFF medication.58.OFF\u2013TMTA time (s): time to complete part A of the TMT in the OFF medication.59.OFF\u2013TMTA error: number of errors presented in part A of the TMT in the OFF medication.60.OFF\u2013TMTB time (s): time to complete part B of the TMT in the OFF medication.61.OFF\u2013TMTB error: number of errors presented in part B of the TMT in the OFF medication.The C3Dfiles folder contains a folder for each participant and medication condition. For each participant, the following files are available for each trial separately:1.c3d files : The c3d files can store both the 3D coordinates of the markers and the force signals in the same file. In addition, the static trial , which contains only marker trajectories, is available;2.Visual 3D pipeline file (Parkinson.v3s) to enable users to process the data in the Visual 3D software;3.Model templates file (Parkinson.mdh) that contains the definitions of all landmarks, segments, and segment properties;4.Angular kinematics of all frames of the joints of the full body on the right and left side;5.Linear Kinematics of all frames of the coordinates of the markers and center of mass (CoM);6.GRF of all frames on the right and left side in the anteroposterior, vertical and mediolateral directions;7.For each gait cycle, spatiotemporal parameters, such as stride and step length and duration, and cadence.The gait cycles folder provides the average ensemble data for each participant and medication throughout the full gait cycle of the following data:1.CoM: center of mass of the body;2.Files named with ending Ang: angular kinematics of the joints of the full body on the right and left side. Each gait cycle is organized into three columns:a.Elbow: flexion/extension, add/abduction, and pron/supination;b.Shoulder: flexion/extension, add/abduction, and int/external rotation;c.Trunk: lateral flexion, rotation, and flexion/extension;d.Pelvis: tilt, obliquity, and rotation;e.Hip: flexion/extension, add/abduction, and int/external rotation;f.Knee: flexion/extension, add/abduction, and int/external rotation;g.Ankle: dorsi/plantarflexion, inv/eversion, and add/abduction;h.Foot: dorsi/plantarflexion, inv/eversion, and int/external rotation.3.Files named with ending grf contain three tabs (1) mean and (2) standard deviation of all gait cycles of the GRF on the right and left side in the anteroposterior, vertical, and mediolateral directions;4.Files named with ending kinematics contain three tabs (1) mean and standard deviation of the spatiotemporal parameters; (2) mean of all gait cycles of the angular and linear kinematics on the right and left side; and (3) standard deviation of all gait cycles of the angular and linear kinematics on the right and left side.The following is a partial exploratory analysis of the data. The curves in this section represent the ensemble average across all participants, only the right leg and the pelvis curves. DOI:10.6084/m9.figshare.14896881). The study contains raw data comprising marker trajectories and GRFs and processed data comprising joint angles that characterize the gait pattern of each participant. A limitation is that there was no foot contact with the force plate in some trials. This circumstance limits the usability of the force plate data. Our sample is small and heterogeneous. Future collections may complement our data set.This study presents a public data set of overground walking kinematics and kinetics in ON and OFF medication for 26 individuals with PD , which may help with PD diagnosis, symptom monitoring, therapy management, rehabilitation, and fall risk assessment and prevention. In addition, improving Parkinson\u2019s disease individuals\u2019 conditions is a major challenge that can be addressed with new emerging technologies such as collaborative robots to assist them during rehabilitation treatments or wearable sensors and devices to monitor and alert them to fall-risk situations; machine learning techniques can increase the effectiveness of these assistive and signaling devices by giving them some awareness of the patients\u2019 situations.10.6084/m9.figshare.14896881.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: The studies involving human participants were reviewed and approved by Federal University of ABC. The patients/participants provided their written informed consent to participate in this study.TS: methodology, data curation, and writing\u2014original draft. TC, CO, RC, SH, EL, CB, and LS: methodology and data curation. MJ: writing\u2014review and editing and supervision. DC: conceptualization, methodology, formal analysis, writing\u2014review and editing, supervision, and project administration. All authors contributed to the article and approved the submitted version."} +{"text": "ACLF has a high risk of short-term mortality. ADAMTS13:AC and VWF:Ag are associated with ACLF development. We investigated the relationship between VWF:Ag/ADAMTS13:AC and prognosis of ACLF. In total, 101 patients with cirrhosis were enrolled in this study. The VWF:Ag/ADAMTS13:AC was associated with prognosis in the patients with ACLF in multivariate analysis. The cumulative survival of the patients with ACLF was significantly lower for patients with high VWF:Ag/ADAMTS13:AC compared with those with low VWF:Ag/ADAMTS13:AC. The VWF:Ag/ADAMTS13:AC predicted prognosis in patients with cirrhosis with ACLF.p < 0.05). The VWF:Ag/ADAMTS13:AC increased according to the progression of ACLF in patients with cirrhosis and predicted prognosis in patients with cirrhosis with ACLF.Acute-on-chronic liver failure (ACLF) has a high risk of short-term mortality. A disintegrin-like and metalloproteinase with thrombospondin type-1 motifs 13 (ADAMTS13) is a metalloproteinase that specifically cleaves multimeric von Willebrand factor (VWF). Imbalance between ADAMTS13 and VWF is associated with portal hypertension, which induces ACLF development. A previous study reported that ADAMTS13 activity (ADAMTS13:AC) and VWF antigen (VWF:Ag) are predictive biomarkers of ACLF development in patients with cirrhosis. This study investigated the changes in ADAMTS13:AC and VWF:Ag levels from before to after the development of ACLF to determine their usefulness as a prognostic biomarker in patients with ACLF. In total, 101 patients with cirrhosis were enrolled in this study. The level of ADAMTS13:AC and VWF:Ag was determined by an enzyme-linked immunosorbent assay. Cox proportional hazard regression analysis was conducted to determine independent prognostic factors for patients with liver cirrhosis in the post-ACLF group. ADAMTS13:AC levels gradually decreased in the order of non-ACLF group, pre-ACLF group, and finally post-ACLF group. VWF:Ag and the ratio of VWF:Ag to ADAMTS13:AC (VWF:Ag/ADAMTS13:AC) levels gradually increased in the order of non-ACLF group, pre-ACLF group, followed by post-ACLF group. VWF:Ag/ADAMTS13:AC and CLIF-C ACLF scores were associated with prognosis in the post-ACLF group in multivariate analysis. The cumulative survival of the post-ACLF group was significantly lower for patients with high VWF:Ag/ADAMTS13:AC (>9) compared with those with low VWF:Ag/ADAMTS13:AC (\u22649) (HR: 10.72, 95% confidence interval: 1.39\u201382.78, A disintegrin-like and metalloproteinase with thrombospondin type-1 motifs 13 (ADAMTS13) is a metalloproteinase that specifically cleaves multimeric von Willebrand factor (VWF) between the Tyr1605 and Met1606 residues in the A2 domain . ADAMTS1Acute-on-chronic liver failure (ACLF) develops in patients with LC after a bacterial infection, gastrointestinal bleeding, alcohol intake, or worsening of the underlying liver disease ,17. ACLFThis study investigated the changes in the imbalance of the ADAMTS13 enzyme\u2013VWF substrate from before to after the development of ACLF, to determine whether ADAMTS13:AC and VWF:Ag can be used as a prognostic biomarker in patients with ACLF.This retrospective observational study included a series of 101 patients with LC whose ADAMTS13:AC and VWF:Ag levels were assayed in our hospital between August 2012 and October 2021 . Among tg at 4 \u00b0C for 15 min and stored in aliquots at \u221280 \u00b0C until analysis. Plasma ADAMTS13:AC was determined via the sensitive chromogenic enzyme-linked immunosorbent assay (ELISA) [The samples were stored in plastic tubes containing 0.38% sodium citrate. Platelet-poor plasma was prepared by centrifuging at 3000\u00d7 , Japan) . The nor, Japan) .https://www.r-project.org, last accessed on 10 January 2023). EZR is a modified version of the R commander version 2.7\u20131 that includes statistical functions frequently used in biostatistics [p-value of <0.05 was considered significant.The statistical analysis was performed using EZR , which is a graphical user interface for R (p < 0.05) (p < 0.05) a. The VW < 0.05) b. Finall < 0.05) c.r = 0.492, p < 0.05), PT , and platelet count and Cre and PT and Cre (ADAMTS13:AC was directly correlated with Alb ( < 0.05) a\u2013c. In t < 0.05) d,e. VWF: < 0.05) f,g. Conv < 0.05) h,i.p < 0.05). The PT and ADAMTS13:AC levels in the survival subgroup of the post-ACLF group were higher than those of the mortality subgroup (p < 0.05 for both). The chronic liver failure consortium (CLIF-C) ACLF score, MELD score, VWF:Ag level, and VWF:Ag/ADAMTS13:AC level in the survival subgroup of the post-ACLF group were lower than those of the mortality subgroup . We performed univariate analysis using the factors that were reported previously for the association of prognosis of ACLF. To determine prognostic factors for patients with liver cirrhosis in the post-ACLF group, we performed multivariate analysis using VWF:Ag/ADAMTS13:AC, CLIF-C ACLF score, Cre, and MELD score, which had p-values <0.1 in the univariate analysis. The VWF:Ag/ADAMTS13:AC and CLIF-C ACLF scores were associated with prognosis in the post-ACLF group (p < 0.05 for both) (p < 0.05).The characteristics of the outcomes in patients with LC in the post-ACLF group are shown in or both) . The timor both) a. The AUor both) b\u2013d. The or both) e\u2013g. The In this study, ADAMTS13:AC, VWF:Ag, and VWF:Ag/ADAMTS13:AC levels gradually decreased, increased, and increased, respectively, in the order of the non-ACLF group, pre-ACLF group, and post-ACLF group. Our previous study reported that the VWF:Ag/ADAMTS13:AC became a predictive biomarker for ACLF development ,30, esopThe VWF:Ag/ADAMTS13:AC was demonstrated to be a prognostic biomarker for patients with LC with ACLF in the present study. A previous study reported that ACLF patients with high VWF:Ag levels had a lower survival rate than those with low levels . In addiPatients with LC have gut dysbiosis , which iThis study had several limitations, including enrolment at a single study center and a small sample size. Moreover, the occasional occurrence of VTE in patients with LC may affect the VWF:Ag/ADAMTS13:AC ,40. A prIn summary, the VWF:Ag/ADAMTS13:AC was increased according to progression of ACLF and was independently associated with prognosis in patients with ACLF."} +{"text": "Corrigendum to: Cranial Musculoskeletal Description of Black-Throated Finch (Aves: Passeriformes: Estrildidae) with DiceCTIntegrative Organismal Biology, Volume 3, Issue 1, 2021, obab007, https://doi.org/10.1093/iob/obab007Materials and methods, this should read: \u201cfledgling P. cincta ISIS No. 18031).\u201d instead of: \u201cfledgling P. cincta ISIS No. 18032).\u201d This error has now been corrected in the article online.In the originally published version of this manuscript, there was an error in a finch specimen number. In section In addition, the display of the Vietnamese abstract has been corrected in the PDF version of the manuscript."} +{"text": "PC provision is a moral obligation for all health professionals to protect public safety and wellbeing amid serious illness diagnosis and the heightened risk of suicide. Indeed, it is a matter of life and death.WER: Conceptualization, Writing\u2014Original Draft, Writing\u2014Review & Editing; MM: Writing\u2014Review & Editing; HMC: Writing\u2014Review & Editing.10.13039/100015348Cambia Health Foundation, 10.13039/100000867Robert Wood Johnson Foundation, and 10.13039/100017234The Rita and Alex Hillman Foundation. MM is funded by 10.13039/100000054NCI/10.13039/100000002NIH award number T32CA00946. WER and MM acknowledge 10.13039/100000054NCI/10.13039/100000002NIH award number P30CA008748. Unrelated to this manuscript, HMC has received grant funding from 10.13039/100008794Research Manitoba, 10.13039/100009395CancerCare Manitoba Foundation, 10.13039/501100000024Canadian Institutes of Health Research; receives book royalties from Oxford University Press; has received payment or honoraria from the Societa Italian\u00e1 Di Cure Palliative National and Saskatoon Hospice and Palliative Care Association; and serves on the ReSet Pharma/NYU advisory board for psilocybin protocol development in advanced cancer.WER has received funding unrelated to this manuscript from"} +{"text": "Bioscience Reports at the request of the authors due to concerns over the reliability of the results. The Editorial Office and the authors were alerted by a reader to apparent similarities between images in this manuscript with those from other manuscripts (from unrelated authors), which include the following:Figure 5C GAPDH/A549 bands and the GAPDH bands of both Fig. 5F from Liu et al. 2020 (doi: 10.1042/BSR20191330) and Fig. 4L from Fu et al. 2019 (doi: 10.18632/aging.102325),The Figure 3 A549 migration/si-NC, H1299 migration/si-circVANGL1 and the H1299 invasion/si-circVANGL1 panels with those from Figs. 6D and 7F from Yang et al. 2020 (doi.org/10.3389/fonc.2020.01104),Figure 2A (A549 and H1299/Bcl-2 and GAPDH bands) with bands from Fig. 3D from Sun et al. 2017 (doi: 10.18632/oncotarget.15846),The Figure 6A A549/GAPDH band with bands in both Fig. 3F from Chen et al. 2019 (doi: 10.1042/BSR20191050) and Fig. 4C from Li et al. 2019 (doi: 10.1042/BSR20181882).This article is being retracted from The authors have been unable to find the original data and are therefore unable to correct the article. They wish to retract the article. The Editor-in-Chief and Editorial Board agree with the retraction."} +{"text": "Proc. R. Soc. B288, 20210525. (Published online 23 June 2021). (https://doi.org/10.1098/rspb.2021.0525)Tetraselmis suecica growth rate. We hereby make the following three corrections in the Results, figure 1a and electronic supplementary material, table S1.We apologize for any inconvenience regarding the presentation of results on the Correction 1:Tetraselmis suecica (a). t-test pairwise comparisons show a significant difference of the red ALAN treatment from the dark .The first paragraph of the Results should read: The ALAN treatment did not have an effect on the growth rate of = 0.119) a. t-testCorrection 2:a) in Correction of panel (Correction 3: Correction of growth rate in electronic supplementary material, table S1."} +{"text": "Bioscience Reports at the request of the authors due to concerns over the reliability of the results. The Editorial Office and the authors were alerted by a reader to apparent similarities between images in this manuscript with those from other manuscripts (from unrelated authors), which include the following:Figure 5C GAPDH/A549 bands and the GAPDH bands of both Fig. 5F from Liu et al. 2020 (doi: 10.1042/BSR20191330) and Fig. 4L from Fu et al. 2019 (doi: 10.18632/aging.102325),The Figure 3 A549 migration/si-NC, H1299 migration/si-circVANGL1 and the H1299 invasion/si-circVANGL1 panels with those from Figs. 6D and 7F from Yang et al. 2020 (doi.org/10.3389/fonc.2020.01104),Figure 2A (A549 and H1299/Bcl-2 and GAPDH bands) with bands from Fig. 3D from Sun et al. 2017 (doi: 10.18632/oncotarget.15846),The Figure 6A A549/GAPDH band with bands in both Fig. 3F from Chen et al. 2019 (doi: 10.1042/BSR20191050) and Fig. 4C from Li et al. 2019 (doi: 10.1042/BSR20181882).This article is being retracted from The authors have been unable to find the original data and are therefore unable to correct the article. They wish to retract the article. The Editor-in-Chief and Editorial Board agree with the retraction."} +{"text": "The fourteenth author\u2019s name is spelled incorrectly. The correct name is: Iyad A. Yousef. There is also an error in affiliation 10 for author Iyad A. Yousef. The correct affiliation 10 is: Department of Physical Education, College of Education, Birzeit University, Ramallah, Palestine."} +{"text": "In \u201cThe Need for Standards Unification in Forensic Laboratory Practices: Protocol for Setting Up the Arab Forensic Laboratories Accreditation Center\u201d :e36778) the authors noted one error.In the originally published article, Reference 6 was incorrectly published as follows:Naglaa F. The Prevalence of Illicit Drugs and Alcohol in Road Traffic Accident Fatalities in the Eastern Region of Saudi Arabia. IJFMT 2020 Oct 7 [doi: 10.37506/ijfmt.v14i4.12122]This reference is now corrected as follows:Mahmoud NF, Al-Mazroua MK, Afify MM. The Prevalence of Illicit Drugs and Alcohol in Road Traffic Accident Fatalities in the Eastern Region of Saudi Arabia. Indian Journal of Forensic Medicine & Toxicology 2020;14(4): 3219-3225. [doi: 10.37506/ijfmt.v14i4.12122]The correction will appear in the online version of the paper on the JMIR Publications website on July 14, 2022, together with the publication of this correction notice. Because this was made after submission to PubMed, PubMed Central, and other full-text repositories, the corrected article has also been resubmitted to those repositories."} +{"text": "Nature Communications 10.1038/s41467-022-31529-4, published online 01 July 2022Correction to: Nat. Commun.8, 1\u20136 (2017). The correct form of Ref. 18 is: Nat. Commun.8, 14540 (2017).The original version of this Article contained an error in Ref. 18, which was incorrectly given with the wrong page number as: Nat. Commun.7, 1\u20136 (2016). The correct form of Ref. 22 is: Nat. Commun.7, 10983 (2016).The original version of this Article contained an error in Ref. 22, which was incorrectly given with the wrong page number as: Light Sci. Appl.7, 1\u20136 (2018). The correct form of Ref. 33 is: Light Sci. Appl.7, 74 (2018).The original version of this Article contained an error in Ref. 33, which was incorrectly given with the wrong page number as: Nat. Commun.11, 1\u20137 (2020). The correct form of Ref. 40 is: Nat. Commun.11, 897 (2020).The original version of this Article contained an error in Ref. 40, which was incorrectly given with the wrong page number as: This has been corrected in the PDF version of the Article."} +{"text": "Laparoscopic vs. Open Pancreaticoduodenectomy After Learning Curve: A Systematic Review and Meta-Analysis of Single-Center StudiesBy Feng Q, Xin Z, Qiu J and Xu M (2021). Front. Surg. 8:715083. doi: 10.3389/fsurg.2021.715083The journal and Chief Editors retract the 10 September 2021 article cited above.Following publication, the cited article was found to be substantially similar to a previously published study by Feng et al. in Gland Surgery :1655\u20131668; doi: 10.21037/gs-20-916). Our investigation, conducted in accordance with Frontiers\u2019 policies, concluded that this similarity was unacceptably high, and the Publisher is retracting the article.\u201cThis retraction was approved by the Chief Editors of Frontiers in Surgery and the Chief Executive Editor of Frontiers. The authors have agreed to the retraction.\u201d"} +{"text": "Erratum to: Hack B, Timalsina U, Tefera E, et al. Oral Prescription Opioids as a High-RiskIndicator for Hepatitis C Infection: Another Step Toward HCV Elimination. Journal of PrimaryCare & Community Health. January 2021. doi:10.1177/21501327211034379Incorrect versions of Figures 1 and 2 for this article were uploaded at the time ofpublication. The correct Figures are now published."} +{"text": "Int J Epidemiol 2020; doi: https://doi.org/10.1093/ije/dyaa226First published online: 8 December 2020, In the text of the results, several negative values in confidence intervals were published as positive values, and a hyphen was missing from a range of years indicating 2011 to 2016.These errors have now been corrected."} +{"text": "MYC/BCL2/BCL6 triple hit and TP53 deletion in a case of high-grade B cell lymphoma receiving CAR T cell immunotherapy. J Immunother Cancer 2021;9:e002029. doi:10.1136/jitc-2020-002029Wang J, Shang Z, Wang J, In the original published article, the funding statement was incomplete. The \u201c(No. 8170211 to MX)\u201d lost a number.The correct funding statement is: This work was supported by the National Natural Science Foundation of China (No. 81\u2009770\u2009211 to MX) and the National Natural Science Foundation of China (No.81830008 to JZ)."} +{"text": "CLINICS (Sao Paulo). 2021;76:e2914errhttps://doi.org/10.6061/clinics/2021/e2914, published in 2021.Erratum for: doi: Expression of Amphiregulin in Enchondromas and Central Chondrosarcomas, RESULTS section in the Abstract, remove the following sentences:In the article \u201cEnchondromas or chondrosarcomas? Please clarify. This will help understand the next sentence on enchondromas localized in short bones and long bones.\u201d"} +{"text": "Nymphalidae is the largest group of butterflies with high species richeness. Rhinopalpapolynice , a forest species, was discovered in the mid-stream of the Yuanjiang-Red River Valley of Yunnan Province for the first time, which represents the first record of the genus Rhinopalpa in China.The family R. polynice is the first record of the genus Rhinopalpa from China. The specimen was collected in the mid-stream of the Yuanjiang-Red River Valley of Yunnan Province. The female genitalia are described for the first time.The species Nymphalidae is a cosmopolitan family of Papilionoidea with high species richness, which includes about 6,100 species in 12 subfamilies and 350 genera and Dendrocnide (Urticaceae) in the wild , which i [1779]) . Rhinopa birmana . Rhinopathe wild . Three sthe wild . HoweverR. polynice was collected from Yuanyang County, southeast Yunnan, China, which sits in the mid-stream of the Yuanjiang-Red River Valley and is isolated from the sites in North Vietnam where R. polynice was previously recorded. The female genitalia are described for the first time. The specimen, collected in this study, is the first record of the genus Rhinopalpa in China. Both specimen and dissected genitalia are deposited in the insect collection of Southwest Forestry University (SFU), Kunming, China.In this study, a female Spread specimens were photographed by Canon 5DS with medium grey background and the photos were adjusted using Adobe Photoshop CS .To observe the female genitalia, the abdomen was treated with 1 ml 10% sodium hydroxide solution to digest soft tissue at 70\u2103 for 1 h and then dissected in a water-filled Petri dish under a stereoscope. The genitalia were then transferred to 80% glycerol for 12 h to render them transparent. A solutiion of 2% chlorazol black was used to dye the membranous parts for 10 min in order to obtain better photographic results. Photographs were taken with a Nikon SMZ1500 stereoscope and automatically stacked using Helicon Focus 7.5.8 . After observation and photography, a piece of card was cut, the genitalia were fixed to the card by white emulsion and pinned with the specimen to avoid confusion. The photographs were adjusted and arranged using Adobe Photoshop CS . Terminology of the female genitalia follows E0B18FEA-C7E3-5D41-B44C-DA0C61E125FDType status:Other material. Occurrence: recordedBy: Wen Shi; individualCount: 1; sex: female; lifeStage: adult; disposition: in collection; Taxon: scientificName: Rhinopalpapolynice ; kingdom: Animalia; phylum: Arthropoda; class: Insecta; order: Lepidoptera; family: Nymphalidae; genus: Rhinopalpa; specificEpithet: polynice; taxonRank: species; scientificNameAuthorship: ; vernacularName: The Wizard; taxonomicStatus: accepted; Location: country: China; stateProvince: Yunnan Province; county: Yuanyang County; locality: Shalatuo Village; verbatimElevation: 928m; verbatimCoordinates: 23\u00b06.047'N, 102\u00b034.43'E; Identification: identifiedBy: Huihong Zhang; dateIdentified: 2020; Event: samplingProtocol: sweep net; year: 2020; month: 9; day: 29; habitat: Evergreen broad-leaved forest; Record Level: basisOfRecord: PreservedSpecimenFemale daiyuanae Hu, Zhang & Cotton, 2018, Graphium wenlingae Hu, Cotton & Monastyrskii, 2019 and Losariadoubledayi , all of them being forest species in South Thailand to Malay Peninsula and ssp. birmana in Assam, Myanmar and Indochina (Rhinopalpap.birmana is different from R. p.eudoxia by paler colour on the upperside, plus better-defined hindwing subterminal black spot in space M1 and narrower forewing subterminal band (R. p.birmana. However, R. p.eudoxia, due to insufficient morphological differences. In this study, as we only examined one specimen, it is impossible to further analyse its subspecies status with such limited material. Hence, the subspecies identity of R. polynice in Yunnan still requires future study.In recent years, three cryptic species of butterflies were discovered from Indochina, species . Hence i forests , two subndochina . Rhinopanal band . Monasty"} +{"text": "International Journal of ObesityCorrection to: 10.1038/ijo.2016.206The original version of this article unfortunately contained a mistake in Table"} +{"text": "European Journal of Public Health, 2021, https://doi.org/10.1093/eurpub/ckab182In the originally version of the article, Figure 1 was published:but is now revised as follows:"} +{"text": "Vol 45 (08) 2015 | DOI: 10.1002/eji.201545451The presence of prolines in the flanking region of an immunodominant HIV\u20102 gag epitope influences the quality and quantity of the epitope generatedSabelle Jallow, Aleksandra Leligdowicz, Holger B. Kramer, Clayton Onyango, Matthew Cotten, Cynthia Wright, Hilton C. Whittle, Andrew McMichael, Tao Dong, Benedikt M. Kessler and Sarah L. Rowland\u2010JonesThe copyright line of the above article has been changed since first published on 24 June 2015 from the standard copyright to CC\u2010BY.The correct copyright of this article is:European Journal of Immunology published by Wiley\u2010VCH Verlag GmbH & Co. KGaA, Weinheim.\u00a9 2015 The Authors. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited."} +{"text": "R. Soc. Open Sci.7, 192151 (Published 5 August 2020) (doi:10.1098/rsos.192151)This correction refers to an error in the numeric values reported in This error has no implications on the results and conclusions drawn in the publication. The paper has now been updated."} +{"text": "There were errors in the extraction of numbers used to calculate the prevalence of limited health literacy, resulting in the incorrect extracted values for Souza, J. G., et al (2014), Kim, S. H. (2009), Chen, G. D., et al (2014), van der Heide, I., et al (2014), Aikens JE, Piette JD. (2009), Mancuso, J. M. (2010) and Wallace, A. S., et al (2010) in The Results section has also been affected by the errors in the extracted values. In the Included Studies subsection of the Results, there are errors in the second paragraph. The corrected paragraph should read: The study with the highest reported prevalence of limited health literacy (76.3%) was conducted to determine the mechanism through with health literacy exerted its influence on health outcomes related to diabetes care. It was a cross-sectional study involving 232 patients with T2DM attending regional hospital in Northern Taiwan. Health literacy was assessed using NVS. The mean age of the participants was 58.02 years (SD 9.49), 44.8% of participants were female, and 38.4% had received primary education or below. [55]The Pooled prevalence of limited HL: A meta-analysis subsection of the Results has also been affected and have been updated as a result. The corrected section should read: The pooled global prevalence of limited health literacy was 32.5% (95% CI: 24.9\u201340.1). Meta-analysis of all included studies yielded high heterogeneity ; which could primarily be explained by the country in which the study was conducted (p<0.001), the health literacy tool used (p = 0.002), participants\u2019 education levels (p<0.001), and the setting where the study was conducted (p<0.001). Most of the included studies (n = 18) were conducted in the USA. Thirteen of these studies measured functional HL specifically, these studies were included in a separate meta-analysis and presented in a forest plot in Figs"} +{"text": "Correction to: BMC Public Health 20, 1150 (2020)https://doi.org/10.1186/s12889-020-09258-4The authors of this study would li1). Reference 19 and 20 is reversed. The following description is correct.https://www.mhlw.go.jp/english/database/db-hw/vs01.html19. Ministry of Health, Labour and Welfare, Vital statistics in Japan 20. von Elm E, Altman DG, Egger M, Pocock SJ, Gtzsche PC. Vandenbroucke JP; STROBE initiative. Strengthening the reporting of observational studies in epidemiology (STROBE)statement: guidelines for reporting observational studies. BMJ. 2007;335:8068.2). In abstract, the descriptions do not match the results in the table 3 and 5. The following description is correct.Results: Numbers of target facilities changed yearly: 1857 in 2011, 2544 in 2013, and 2376 in 2016. The mean number of screening-test participants increased per facility, but the median increased or decreased. The mean number of positive individuals identified decreased. Multivariate analysis results revealed the number of screenings was reduced in Kanto and Chubu/Tokaiareas, although some areas (Hokkaido/Tohoku and Chugoku/Shikoku) and high volume in facility types increased. Regarding the positive rates, some areas exhibited decreases or increases by facility type. The number of western blotting (WB) implementations decreased in 2016, positive rates identified by WB decreased in 2016 andin all areas, and the number of facility types increased. The number of PCR participants increased in 2016 andin Kinki, but a decrease in facility type was observed. Positive rates were decreased in all areas (except Chubu/Tokaiarea) but facility types were increased.3) (P3) Results: the descriptions do not match the results in the table 3 and 5. The following description is correct.By multivariate analysis, some areas (Hokkaido/Tohoku and Chugoku/Shikoku) and facility types showed increased screening coverage although screening coverage in other areas (Kanto and Chubu) decreased. Positive rates were decreased in some areas and positive identification increased by facility type. Numbers of WB performed were decreased in 2016 and the positive identification rate was lower in 2016 and inall areas; however, facility types were increased. The number of PCR participants was markedly increased in 2016 and inKinki;"} +{"text": "Vol .14 (2021) No. 2 pp. 202\u20132302018 JAPAN Critical Limb Ischemia Database (JCLIMB) Annual ReportThe Japanese Society for Vascular Surgery JCLIMB CommitteeIn the above secondary publication article, numerical errors in tables were found after the publication. The erratum of the original Japanese article was published in Japanese Journal of Vascular Surgery Vol. 30 (2021) No. 4. The corrected version of table is given on the next page.Table 4-4p. 221, Incorrect:Correct:"} +{"text": "BMJ 2021;374:n2106, doi:10.1136/bmj.n2106, published 29 September 2021), two research team members were inadvertently missed in the article\u2019s submission. The article and PDF will be updated in due course.In this paper by Gray and colleagues ("} +{"text": "Correction to:Clin Trans Immunol 2019; 8: e1050. doi: https://doi.org/10.1002/cti2.1050. Published online 20 May 2019.The authors inadvertently excluded a funding body in the Acknowledgments section. The corrected version appears below:ACKNOWLEDGMENTSWe acknowledge support from the Cancer Council SA Beat Cancer Project Hospital Research Support Package, a Cancer Council Early Career Research Fellowship, the Health Services Charitable Gifts Board (Adelaide) and The Hospital Research Foundation.The authors apologise for this error."} +{"text": "Vol. 218, No. 4 | 10.1084/jem.20202187 | January 19, 2021The authors regret that the amino acid sequence of one of the T epitopes in The error appears in print and in PDFs downloaded before September 30, 2021."} +{"text": "J Am Heart Assoc. 2021;10:e019605. DOI: 10.1161/JAHA.120.019605), corrections were needed.In the article by Tarp et al, \u201cFitness, Fatness, and Mortality in Men and Women From the UK Biobank: Prospective Cohort Study,\u201d which published on March 13, 2021 and appeared in the March 16, 2021 issue of the Journal \u201d. No change has been made to the data in those rows.The publisher regrets the errors.https://www.ahajournals.org/doi/10.1161/JAHA.120.019605.The footnotes have been added to the current online version of the article, which is available here:"} +{"text": "In the original article published, the references are cited incorrectly in Tables 1, 2 and 3. The correct information is given below:The correct citation number for the study Plato et al. is reference number 39: Plato CC, Norris AH. Osteoarthritis of the hand: longitudinal studies. Am J Epidemiol. 1979;110(6):740\u2013746.The correct citation number for the study Kallman et al. is reference number 37: Kallman DA, et al. The longitudinal course of hand osteoarthritis in a male population. Arthritis Rheum. 1990;33(9):1323\u20131332.The correct citation number for the study Busby et al. is reference number 38: Busby J, et al. A longitudinal study of osteoarthritis of the hand: the effect of age. Ann Hum Biol. 1991;18(5):417\u2013424.The correct citation number for the study Kalichman et al. is reference number 43: Kalichman L, et al. Repeated measurement study of hand osteoarthritis in an apparently healthy Caucasian population. Am J Hum Biol. 2005;17(5):611\u2013621.The correct citation number for the study Kalichman et al. is reference number 44: Kalichman L, et al. Epiphyseal expansion in hand bones: association with age, sex, and hand osteoarthritis. Osteoarthr Cartil. 2008;16(5):560\u2013565.The correct citation number for the study Hoeven et al. is reference number 40: Hoeven TA, et al. Association of atherosclerosis with presence and progression of osteoarthritis: the Rotterdam Study. Ann Rheum Dis. 2013;72(5):646\u2013651.The correct citation number for the study Haugen et al. is reference number 41: Haugen IK, et al. The prevalence, incidence, and progression of hand osteoarthritis in relation to body mass index, smoking, and alcohol consumption. J Rheumatol. 2017;44(9):1402\u20131409.The correct citation number for the study Marshall et al. is reference number 42: Marshall M, et al. Metabolic risk factors and the incidence and progression of radiographic hand osteoarthritis: a population-based cohort study. Scand J Rheumatol. 2019;48(1):52\u201363."} +{"text": "The above article from Food Science & Nutrition, published online on 05 June 2020 in Wiley Online Library (http://onlinelibrary.wiley.com/), has been retracted by agreement between the authors, the Editor\u2010in\u2010Chief, Martin Lo, and Wiley Periodicals LLC. The retraction has been agreed due to an error in Formula 2 which invalidates the results of the study.Retraction: Zhang X, Zhang J, Chen T. An ANP\u2010fuzzy evaluation model of food quality safety supervision based on China's data. Food Sci Nutr. 2020;8: 3157\u20133163."} +{"text": "Correction to: BMC Cancer 22, 203 (2022)https://doi.org/10.1186/s12885-022-09299-5Following publication of the original article , an erroDr. Yehoda Martei\u2019s research work is supported by an NIH K01 Award\u2014K01TW011481. Bege Dauda\u2019s research is supported by the Penn Center for Global Genomics and Health Equity (PennGGHE) with funding from Genentech, Inc. and the Penn Medicine Office of Inclusion, Diversity, and Equity.The original article has been"} +{"text": "These corrigenda serve to correct figure errors in the article by Ju et al:, the 40X image for the nontarget control (NTC) condition was a duplicate of the RhoB\u20102 micrograph.In Figure Figure 4 is, as follows:The correct version of Additionally, in Figure https://kmplot.com/) for RhoB using: Affymetrix id 212099_at, median cutoff, JetSet best probe set, and 120\u2009months as follow up threshold.All plots were made from data using the online data portal (The correct version of Figure Neither error alters the results or conclusions of the study. The authors regret any inconvenience this may have caused.Reference:Ju JA, Godet I, DiGiacomo JW, GilkesDM. RhoB is regulated by hypoxia and modulates metastasis inbreast cancer.Cancer Reports. 2020;3:e1164.https://doi.org/10.1002/cnr2.1164"} +{"text": "Correction on:Pregnant women\u2019s knowledge of non-pharmacological techniques for pain relief during childbirthBy Maria A. Heim, Maria Y. MakuchEuropean Journal of Midwifery, Volume 6, Issue February, Pages 1-6Publish date: 7 February 2022https://doi.org/10.18332/ejm/145235DOI: https://doi.org/10.18332/ejm/145235. The pdf file has been replaced.In the original article, in the pdf file of the manuscript, the article DOI number did not correspond to the assigned DOI number. The correct manuscript DOI is"} +{"text": "Journal of Experimental Botany, Advance Access publication: 18 June 2020, doi: doi:10.1093/jxb/eraa285The version of this article that first published contained an error in the unit scale in Fig. 1E. The correct value of 0.025 nm has now been included. The Publisher apologizes for this error."} +{"text": "International Journal of Oral Science 10.1038/s41368-020-00101-5, published online 14 December 2020Correction to: 1 the authors reported the below errors.Following publication of this article,1. Reason for modification: ambiguous descriptionThird section of RESULTS: Effects of autophagy on the degradation of FN1Lines 17\u201320, paragraph 4: The data showed that CQ strongly inhibited autophagic activity and blocked the degradation of FN1 in SCC-25 cells and SCC-15 cells until 18\u2009h.Baf A1 strongly blocked the degradation of FN1 in SCC-15 cells until 18\u2009h.Corrected to: The data showed that CQ strongly inhibited autophagic activity and blocked the degradation of FN1 in SCC-25 cells and DISCUSSION:The first sentence of paragraph 3: Vimentin has been excluded as a marker of metastasis in a variety of cancers, such as breast cancer and non-small cell lung carcinoma.regarded as a marker of metastasis in a variety of cancers, such as breast cancer and non-small cell lung carcinoma.Corrected to: Vimentin has been 2. Reason for modification: Misaligned with the content of FigureSecond section of RESULTS: Autophagic activity in different OSCC cell linesLine 14: .Corrected to: .Third section of RESULTS: Effects of autophagy on the degradation of FN1Line 1, paragraph 4: Fig. 4kCorrected to: Fig. 4aThe last section of RESULTS: Roles of the PB1 and UBA domains of p62 in the degradation of FN1The fourth line from the bottom: .Corrected to: .3. Reason for modification: Mistakes of figure legend serial number sequenceLegend of Fig. 2:c Western blot data. d Migration assay data. e Western blot data.Line 2: c Migration assay data. d Western blot data. e AO staining data. f Western blot data.Corrected to: Legend of Fig. 4:h Quantification of the average fluorescence values of single cells in Fig. 3g.Line 5: h Quantification of the average fluorescence values of single cells in (g).Corrected to: Fig. 5:b, c serial number error, b label content is incorrect.Original Figure 5:Corrected to: Switch the serial numbers b and c. IB: BN1 changed to IB: FN1.Revised Figure 5:"} +{"text": "Correction to: Trop Med Health 49, 48 (2021)https://doi.org/10.1186/s41182-021-00340-0Following publication of the original article , a fundibold typeface.The updated Funding section is given below and the changes have been highlighted in FundingThis study is partially funded by Japan Agency for Medical Research and Development, Grant/Award Numbers: JP19fk0108104h0401, JP20fk0108104h0402.This work is in part funded by Nagasaki University . The original article has been"} +{"text": "The divThe content from Equations (1) and (2) in Section 2 needs to be corrected. The updated information is included below:The cited reference [27] needs to be corrected. The updated information is included below:divIt should be noted that, in Equation (2), we take into account the incompressibility of the nanofluid: The content from Equations (3) and (4) in Section 2 needs to be corrected. The updated information is included below:The References section needs to be corrected. The book title in Ref. [26] was incorrect. The updated information is included below:Fluid Mechanics, 2nd ed.; Pergamon: London, UK, 1987.26. Landau, L.D.; Lifshitz, E.M. The changes do not affect the scientific results or conclusions in the original published paper.The authors would like to apologize for any inconvenience caused to the readers by these changes."} +{"text": "PLoS Computational Biology, volume 3, issue 2: doi:10.1371/journal.pcbi.0030025In The set of four equations in the Materials and Methods section had three extra minus signs, which were incorrect. The following are the correct equations."} +{"text": "PLoS Clinical Trials, volume 1, issue 2: DOI: 10.1371/journal.pctr.0010012In The numbering of the reference citations in Table 2 is incorrect. The correct numbering is shown in the table below."} +{"text": "PLoS Pathogens, volume 2, issue 6: DOI: DOI: 10.1371/journal.ppat.0020066In The PDF version of the above article states that a previous version was published as an Early Online Release on June 23, 2006. This information appeared in error. No previous version of the article has been published."} +{"text": "PLoS Medicine, volume 3, issue 8: doi:10.1371/journal.pmed.0030295In Equation 1 appeared incorrectly. It should read as:The equations in the legend of Table 1 were also incorrect. The full model should be:The null model should be:"} +{"text": "Thousand Oaks (CA): Sage Publications; 2003) was omitted from the list of references; and several of the references were not numbered consecutively. Corrections were made on our Web site on June 15, 2007, and appear online at www.cdc.gov/pcd/issues/2007/apr/06_0076.htm. We regret any confusion or inconvenience this error may have caused.Because of an editing error, the references in the article \"From Heart Health Promotion to Chronic Disease Prevention: Contributions of the Canadian Heart Health Initiative\" were incorrectly presented. References (4) and (14) were omitted from the text of the article; one source (Creswell JW. Research design: qualitative, quantitative, and mixed methods approaches. 2"} +{"text": "PLoS Computational Biology, vol 2, issue 12: doi:10.1371/journal.pcbi.0020156In In the Acknowledgments section of this Perspective, the spelling of the name of Pavel Pevzner was incorrect."} +{"text": "PLoS Medicine, volume 4, issue 4, doi:10.1371/journal.pmed.0040074:In PLoS Medicine article, the article in The Lancet was still in press):The author inadvertently omitted the following citation that addresses the same topic Global burden of childhood hearing impairment and disease control priorities for developing countries. Lancet 369: 1314\u20131317.Lancet paper, as reference [10], in an earlier Correspondence article published in PLoS Medicine: Olusanya BO (2007) \u201cThe Right Stuff\u201d: The Global Burden of Disease. PLoS Med 4(2): e84. doi:10.1371/journal.pmed.0040084The author did, however, cite this"} +{"text": "Correction to:Molecular Psychiatry advance online publication, 8 December 2015; doi:10.1038/mp.2015.178Following publication of this paper, the authors noticed that"} +{"text": "Ianiropsis serricaudis (first record for the Iberian Peninsula and Lusitanian marine province), Paracerceis sculpta (first record for the Alboran Sea ecoregion), Paradella dianae, Paranthura japonica (earliest record for the Iberian Peninsula) and Sphaeroma walkeri. Photographs with morphological details for identification for non-taxonomic experts are provided, their worldwide distribution is updated and patterns of invasion are discussed. We report an expansion in the distribution range of all species, especially at the Strait of Gibraltar and nearby areas. Ianiropsis serricaudis and Paranthura japonica are polyvectic, with shellfish trade and recreational boating being most probable vectors for their introduction and secondary spread. The subsequent finding of the studied species in additional marinas over the years points at recreational boating as a vector and indicates a future spread. We call for attention to reduce lags in the detection and reporting of small-size exotics, which usually remain overlooked or underestimated until the invasion process is at an advanced stage.Effective management of marine bioinvasions starts with prevention, communication among the scientific community and comprehensive updated data on the distribution ranges of exotic species. Despite being a hotspot for introduction due to numerous shipping routes converging at the Strait of Gibraltar, knowledge of marine exotics in the Iberian Peninsula is scarce, especially of abundant but small-sized and taxonomically challenging taxa such as the Order Isopoda. To fill this gap, we conducted several sampling surveys in 44 marinas and provide the first comprehensive study of marine exotic isopods from the Iberian Peninsula, the southern side of the Strait of Gibraltar (northern Africa) and the Balearic Islands. Exotic species included Carcinus maenas and the Chinese mitten crab Eriocheir sinensis , both being aggressive competitors for native species, affecting aquaculture facilities and harvests and causing structural damage to river banks , despite having a much smaller size and being less notorious, also achieved a globally widespread distribution in a relatively short timeframe, as well as causing malfunctioning to pumps and fouling biomass to cages in aquaculture facilities and from the IPTM (Instituto Portu\u00e1rio e dos Transportes Mar\u00edtimos: http://www.atlanticstrategy.eu/en/partners/iptm-instituto-portu%C3%A1rio-e-dos-transportes-mar%C3%ADtimos-ip). Census data for the locality to which each marina belongs was obtained from the National Statistical Systems of Spain (http://www.ine.es), Portugal (http://www.ine.pt) and Morocco (http://www.hcp.ma) . Voucher material of each species was deposited in the Museo Nacional de Ciencias Naturales , Madrid, Spain. The rest of the material was kept in the Laboratorio de Biolog\u00eda Marina, University of Seville, Spain.Examined material was collected during several sampling surveys carried out from 2011 to 2017, in order to study the fouling epifauna in 44 marinas around the Iberian Peninsula, the Southern side of the Strait of Gibraltar (northern Africa) and Baleares. Marina choice was based on its vessel traffic and popularity as tourist locality (see .hcp.ma) . In 2011ibraltar . AdditioIaniropsis serricaudis, Paracerceis sculpta, Paradella dianae, Paranthura japonica and Sphaeroma walkeri had increased the number of exotic species, sometimes by 200% or more record of Paranthura japonica from the Iberian Peninsula. We report an extension in the distribution range for all species along the coasts of the Iberian Peninsula and adjacent waters.Five exotic marine isopods were found on fouling communities associated to marinas: walkeri . From thmore see . We provJaniropsis serricaudisIaniropsis notoensisIaniropsis serricaudisIaniropsis sp. Ianiropsis sp. St3: 2 males (MNCN 20.04/11439), 18 males and 119 females clinging on bryozoan Bugula neritina, floating pontoons, 07/05/2011.Material examined : Ianiropsis is similar to Janira and Carpias: three claws on walking legs, coxae visible in dorsal view and usually can only be definitely identified from the males. Ianiropsis can be distinguished from the other two by bearing an elongated carpus of male pereopod I (Carpias) or not elongated propodus and carpus at all (Janira) (I. serricaudis: (i) antennal peduncle segments 6 and 7 particularly elongated relative to the overall length of the antennae . Our speantennae ; (ii) heantennae , 1C daantennae .Laminaria, in water temperatures from 1.8\u00a0\u00b0C to 24\u00a0\u00b0C , 01/07/2017. St13: Six males, 13 females and 24 juveniles from Corallinaceae algae and green algae, 13/05/2017. St14.1: Four females and one juvenile on B. neritina, one male and one juvenile on Eudendrium sp., and one male and two female on Coralline algae, floating pontoons, 14/05/2017; four males, 12 females and 33 juveniles from fouling community on floating pontoons, 14/05/2017; one male and two females (MNCN 20.04/11443), three males six females and 16 juveniles collected from fouling community on floating structures, 02/07/2017. St14.2 One female and one juvenile from fouling substrates, floating structures, 02/07/2017. St34: One juvenile on A. verticillata, floating pontoons, 27/06/2011. St37: One female and one juvenile on A. verticillata, floating pontoons, 26/06/2011. St43: One female on Eudendrium sp., floating pontoons, 09/2012.Material examined : Paranthura species. These are: eyes well developed composed of less than 17 dark ommatidia; anterolateral angles of cephalon exceeding rostral projection; antenna 1 with 8 distinct articles , among algae (Sargassum) and in mussel beds and oyster reefs , 14 males, 224 females and 192 juveniles on fouling substrates, floating structures , 26/06/2017. St10: one female and four juveniles on fouling substrates, floating structures, 26/06/2017. St12: one female on fouling substrates, floating structures, 01/07/2017. St13: Three juveniles on B. neritina, one female and 10 juveniles on A. verticillata, floating pontoons, 17/05/2011; six juveniles on Coralline algae and green algae, floating pontoons, 13/05/2017. St14.1: One male, nine females, 19 juveniles on B. neritina, one male, 29 females, 23 juveniles on A. verticillata, floating pontoons, 17/05/2011; one female and six juveniles on A. verticillata, 12/2011; one juvenile on A. verticillata, one male and one female on hydrozoan Eudendrium sp., 05/2012; one juvenile on A. verticillata, 06/2012; one juvenile on A. verticillata, 07/2012; one female and 23 juveniles on A. verticillata, 08/2012; 15 females and 39 juveniles on A. verticillata, 09/2012; one female and five juveniles on A. verticillata, 10/2012; two females and nine juveniles on A. verticillata 11/2012; 8 females and 155 juveniles on fouling community, floating pontoons, 14/05/2017. St14.2: One male, six females and six juveniles on fouling substrates, floating structures, 01/07/2017. St16: One juvenile on B. neritina, floating pontoons, 17/05/2011. 18 females and 139 juveniles on fouling substrates, floating pontoons, /06/2017. St17: One male, 18 females and nine juveniles on fouling substrates, floating structures, 01\u221507\u22152017. St18: One juvenile on B. neritina, floating pontoons, 15/05/2011. St19: Two males, 18 females and 26 juveniles on fouling substrates, floating structures, 29/06/2017. St28: three females and seven juveniles on B. neritina, floating pontoons, 29/06/2011. St29: 8 females and 10 juveniles on B. neritina, floating pontoons, 29/06/2011. St30: Two juveniles on A. verticillata, floating pontoons, 28/06/2011. St31: One female and three juveniles on B. neritina, three females and seven juveniles on A. verticillata, floating pontoons, 28/06/2011. St34: five juveniles on B. neritina, six females and 54 juveniles on A. verticillata, floating pontoons, 27/06/2011. St40: Two juveniles on B. neritina, floating pontoons, 29/95/2011. St41: Seven juveniles on B. neritina, floating pontoons, 30/05/2011. St42: Two females and four juveniles on B. neritina, one juvenile on A. verticillata, floating pontoons, 30/05/2011.Material examined : Paracerceis, together with other Cerceis-like genera, can be distinguished by bearing pronounced marginal teeth on exopods of pleopods 1\u20133, especially obvious on pleopod 2 , five females and 36 juveniles collected from Corallinaceae algae and green algae, floating pontoons, 13/05/2017. St14.1: Two juveniles collected from fouling community, floating pontoons, 14/05/2017. St17: One male collected from fouling community of floating structures 01/07/2017. St23: One female collected from fouling substrates, floating structures, 28/06/2017. St22: One female from fouling substrates, floating structures, 28\u221506\u22152017 and one female on Bugula neritina, floating pontoons, 03\u221507\u221511.Material examined : Dynamenella dianae), Paradella can best be identified by males having a distinct dorsally-directed, Y-shaped and posteriorly closed pleotelson foramen; long, tapering and basally fused penial processes, and a long and basally narrow appendix masculina that usually extends beyond the distal margin of the endopod collected from fouling community, floating structures , 02/07/2017.Material examined : Sphaeroma can be distinguished from related genera like Exosphaeroma and Lekanesphaera by bearing a robust maxilliped, particularly the palp, articles II\u2013IV without lobes and a fringe of robust, plumose setae on internal border of endite see in an esand) see . Also inl, 1923) or in ascilities .The first evidence of its occurrence in the Mediterranean Sea took place in 2012, when it was found to be abundant in the Lagoon of Venice . The Lagversus the reported from native regions (up to seven). Moreover, our specimens were considerably large in comparison to those reported from Russia from thefemales) and fromfemales) . Whetherve range .I. serricaudis is probably linked to accidental introduction with shellfish transfers. This is a likely associated vector (see Crassostrea gigas) and the Japanese clam (Ruditapes philippinarum), and export to other countries of Europe (ia (USA) and its ia (USA) , San Quiia (USA) ; Puerto ia (USA) and nortia (USA) . It has ia (USA) ; and at ia (USA) . It was ia (USA) . From thia (USA) , Hong Koia (USA) , Taiwan ia (USA) and Japaia (USA) , to Austia (USA) and nortia (USA) . It is aia (USA) . In the ia (USA) , and decia (USA) and westia (USA) . In the ia (USA) . In the ia (USA) , collectia (USA) , collectParacerceis sculpta , being sP. dianae arrived to the Iberian Peninsula and the Mediterranean Sea from the Indian Ocean, from the Atlantic Ocean, or from both through multiple introductions. It was reported from the Italian coast in 1980 or the Madeira island system itself since the early 1980s . Its native range only includes localities from Japanaese coasts (Crassostrea gigas (Thunberg 1793) from the Senday Bay (Japan) was massively introduced (Crassostrea angulata (Lamarck 1819). Paranthura japonica probably remained unnoticed or misidentified since then (see Ruditapes philippinarum (Adams and Reeve 1850) during the 1970s; and secondary spread to further Mediterranean marinas at least since 2011. P. japonica was found in Barcelona, Benicarl\u00f3 and Mallorca , which are popular destinations for vessels cruising the western Mediterranean in between Barcelona to the West and northwestern Italy to the East . In 2014, two individuals of P. japonica were found within the Strait of Gibraltar\u2019s vicinity, in Chipiona rocky shores (C\u00e1diz) ; and three years later, it was abundant in marinas located in C\u00e1diz Bay. C\u00e1diz is a great hotspot for both international commercial shipping and pleasure craft, as well as a center for aquaculture production, including the Japanese clam Ruditapes philippinarum , and later on to C\u00e1diz marinas (present in 2017). It is to be noticed that P. japonica was not present in the bryozoan B. neritina in Puerto Am\u00e9rica marina in 2011; but it was found associated to the same host in 2017. This fact supports this record as a new arrival of NIS into a particular region, and thus represents a Marine Strategy Framework Directive indicator to establish C\u00e1diz Bay as a hotspot for marine introductions, following It was reported only recently from the Iberian Peninsula, from samples collected from fouling assemblages in marinas of the eastern coast in 2016 . Neverthppinarum . Just asi.e. role as prey-predator in the trophic chain, habitat selection, role in their ecosystem functioning) are of great need in here (see We have reported a distribution range extension for all exotic isopod species present in the studied areas, some of them proving to be polyvectic and well established in marinas. The next step is to evaluate their potential biological, social and economical impact, however, there are gaps of knowledge that hamper this task. Baseline studies delving into the ecology of all these species (here see . AlthougThere is a critical problem that keeps recurring and needs to be reduced: the lags in detection of a new arrival. In many occasions, much time lapse between the initial introduction and the report of it, with a bias for noticing invaders only after they become an abundant nuisance, due to inadequate monitoring or lack of taxonomic expertise see . This haIn order to be ready for decision making and implementation of invasion control, as well as assessment of future arrivals, prevention is the key; and all this starts with building comprehensive data on the presence and distribution range of exotic species, especially on new arrivals see . We cons10.7717/peerj.4408/supp-1Table S1List of introduced isopod species in European waters, updated with the findings of the present study. Name of the species, parasite/free-living status, origin, distribution in European waters, introduction status remarks and likely vectors of introduction are provided. MED, Mediterranean Sea; WMED, Western Mediterranean; CMED, Central Mediterranean; EMED, Eastern Mediterranean; ATL, Atlantic; NOR, North Sea; C, casual; E, established; NE, non-established; nd, no data available. Species with asterisk are those found to be present in the Iberian Peninsula.Click here for additional data file."} +{"text": "Correction to:Translational Psychiatry (2016) 6, e909; doi:10.1038/tp.2016.179; published online 4 October 2016In the online version of this paper, the second chart that appears in the Materials and Methods section, 'Real-time PCR' subsection, is incorrect. The correct chart appears below:The publisher regrets this error."} +{"text": "Correction to:Molecular Psychiatry (2017) 22: 1502-1508; advance online publication, 12 July 2017; doi: 10.1038/mp.2016.97In the first paragraph of the Results section and The corrected figure appears in previous page."} +{"text": "Correction to:Human Genome Variation (2016), 3, doi: 10.1038/hgv.2016.20; advance onlinepublication 7 July 2016After the online publication of this article, the authors noticed an error in the figureand legend of The correct figure should show that leftmost SNP is rs7939918.The correct The authors apologize for any inconvenience caused."} +{"text": "Current collection continues the series of BioMed Central special post-conference issues presenting the highlights from the set of meetings on bioinformatics and systems biology held in Novosibirsk and Moscow, Russia in 2017.http://conf.bionet.nsc.ru/belyaev100/en). This Memorial Conference included special session on medical aspects of the genomics. In 2017, \u201cVavilov Journal of Selection and Breeding\u201d published a series of memoirs publications about Prof. Belyaev (http://vavilov.elpub.ru/jour/issue/view/32/showToc).Year 2017 marks the 100-th anniversary since birth of Professor Dmitry K. Belyaev (1917\u20131985), Full Member of the USSR Academy of Sciences, world-famous visionary in evolution and genetics. In view of this memorable date, the Institute of Cytology and Genetics of the Siberian Branch of the Russian Academy of Sciences (ICG SB RAS) held international Belyaev Conference on Genetics and \u201cHigh throughput sequencing in genomics\u201d (NGS-2017) conferences in Novosibirsk (http://conf.nsc.ru/HSG2017/ru/hsg2017_hsg_thesis). Modern technologies in medicine more and more become interconnected with advances in sequencing in fundamental evolutionary studies. Previously published special issues of BMC Evolutionary Biology and BMC Genomics covered the proceedings of BGRS\\SB-2016 conference and SBB-2015 School in Novosibirsk [https://bmcgenomics.biomedcentral.com/articles/supplements/volume-15-supplement-12). The materials on evolutionary biology and genetics were recently published in BMC Evol Biol and BMC Genetics Supplements , correspondingly [Thematic issue of osibirsk as well This issue collected works on sequencing, genotyping, computational analysis and gene network reconstruction in human diseases.Anastasiya Snezhkina et al. describeThe work by Maxim Ivanov and colleagues discusseYu-Feng Huang et al. continueUlyana Boyarskikh and colleagues present Olga Saik et al. showed tGalatenko and colleagues identifiAbeer Fadda et al. discuss ASPM gene. The work represents an additional support for the clinical continuum between Seckel Syndrome and primary microcephaly.Andrey Marakhonov et al. describeBMC Evolutionary Biology , BMC Plant Biology , BMC Genetics \u00a0(published at the end of 2017), BMC Genomics , BMC Structural Biology and BMC Neuroscience . The Proceedings of the conference are available at http://conf.bionet.nsc.ru/belyaev100/enhttp://conf.bionet.nsc.ru/belyaev100/wp-content/uploads/sites/14/2017/01/BELYAEV_conf_2_08_2017.pdf.Follow-on series of related works in the areas of genomics, genetics, and plant biology discussed at \u201cBelyaev conference \u2013 2017\u201d and other related meetings in Novosibirsk are published in the Special Issues of http://conf.bionet.nsc.ru/bgrssb2018/en).The readers are welcome to visit Novosibirsk at the time of next XI-th BGRS\\SB-2018 conference on August 20-28th in 2018 ("} +{"text": "In the Funding section, a grant number from the funder National Science Centre, NCN Poland, UMO is listed incorrectly. The correct grant number is: 2015/17/D/NZ8/00782. The correct Funding statement is: This work was supported by the National Science Centre, NCN Poland, UMO-2016/21/B/NZ8/01542 to EG and 2015/17/D/NZ8/00782. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."} +{"text": "International Journal of Environmental Research and Public Health [http://www.mdpi.com/1660-4601/11/9/9897.The authors wish to update the Acknowledgments Section in their paper published in the c Health , doi:10."} +{"text": "Rahman RA. It should be Abdel Rahman R. The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way.In the original article, the name of one of the authors was incorrectly spelled in the citation section as Citation: Aristei S, Zwitserlood P and Rahman RA (2012) Picture-induced semantic interference reflects lexical competition during object naming. Front. Psychology3:28. doi: 10.3389/fpsyg.2012.00028This article was submitted to Frontiers in Language Sciences, a specialty of Frontiers in Psychology.Copyright\u00a9 2012 Aristei, Zwitserlood and Rahman.Citation: Aristei S, Zwitserlood P and Abdel Rahman R (2012) Picture-induced semantic interference reflects lexical competition during object naming. Front. Psychology3:28. doi: 10.3389/fpsyg.2012.00028This article was submitted to Frontiers in Language Sciences, a specialty of Frontiers in Psychology.Copyright \u00a9 2012 Aristei, Zwitserlood and Abdel Rahman.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "In the article titled \u201cPrenatal Diagnosis of Cardiac Diverticulum with Pericardial Effusion in the First Trimester of Pregnancy with Resolution after Early Pericardiocentesis\u201d , there wThe errors in the in-text citations of references in the Discussion should be corrected as follows:The original text: Ultrasonographic findings associated with diverticula include pericardial effusion, cardiomegaly, septal defects and arrhythmia with fetal death before delivery, and hydrops .The corrected text: Ultrasonographic findings associated with diverticula include pericardial effusion, cardiomegaly, septal defects and arrhythmia with fetal death before delivery, and hydrops .The original text: Thus, the observation of pericardial effusion makes it necessary to examine the cardiac function .The corrected text: Thus, the observation of pericardial effusion makes it necessary to examine the cardiac function .The original text: Five of them showed spontaneous resolution (71%) and 2 resulted in intrauterine death (29%): one of them, which occurred on week 26, was associated with trisomy 18 and the other, which occurred on week 29, was associated with treated twin-to-twin transfusion syndrome and death of one of the twins after treatment .The corrected text: Five of them showed spontaneous resolution (71%) and 2 resulted in intrauterine death (29%): one of them, which occurred in week 26, was associated with trisomy 18 and the other, which occurred in week 29, was associated with treated twin-to-twin transfusion syndrome and death of one of the twins after treatment .The original text: The prognosis of this entity is generally good, although the outcome largely depends on the size and location of associated anomalies. Cases of rupture, both pre- and postnatal, arrhythmia, fetal death, heart failure, and coronary insufficiency have been described . In these patients, serial control examinations are necessary to detect possible complications. In general, postnatal progression is good and surgery is not necessary in asymptomatic cases [19].The corrected text: The prognosis of this entity is generally good, although the outcome largely depends on the size and location of associated anomalies. Cases of rupture, both pre- and postnatal, arrhythmia, fetal death, heart failure, and coronary insufficiency have been described . In these patients, serial control examinations are necessary to detect possible complications. In general, postnatal progression is good and surgery is not necessary in asymptomatic cases [18].\u2009 Row 25: Williams et al. (2009) [3] should be Abi-Nader et al. (2009) [2].\u2009 Rows 29, 30, and 31: Abi-Nader et al. (2009) [2] should be Williams et al. (2009) [3].\u2009 Row 32: Williams et al. (2009) [3] should be Paoletti et al. (2012) [20].\u2009 Row 33: Paoletti and Robertson (2012) [20] should be Nam et al. (2010) [21].\u2009 Row 34: Nam et al. (2010) [21] should be Olor\u00f3n et al. (2011) [22].Errors in \u2009 Rows 4 and 11: Cavall\u00e9-Garrido et al.: the reference in the bibliography is [6].\u2009 Row 7: McAuliffe et al. [27] should be Del R\u00edo et al. [18].\u2009 Row 8: Pradhan et al. [28] should be Davidson et al. [15].\u2009 Row 9: McAuliffe et al. [27] should be Koshiishi et al. [17].\u2009 Row 10: Perlitz et al. [30] should be Menahem [31].\u2009 Row 12: Carles et al. [24] should be Johnson et al. [16].\u2009 Row 13: Cesko et al. [25] should be Bernasconi et al. [26].\u2009 Rows 14 and 15: Brachlow et al. [23] should be McAuliffe et al. [27].\u2009 Row 19: Williams et al. [3] should be Abi-Nader et al. [2].\u2009 Row 21: Abi-Nader et al. [2] should be Williams et al. [3].Errors in The corrected tables are shown in Tables"} +{"text": "The sections in QFP include the following: Determining the Client\u2019s Need for Services; Contraceptive Services; Pregnancy Testing and Counseling; Clients Who Want to Become Pregnant; Basic Infertility Services; Preconception Health Services; Sexually Transmitted Disease Services; and Related Preventive Health Services. In addition, the QFP includes an appendix entitled Screening Services for Which Evidence Does Not Support Screening.In April 2014, CDC published \u201cProviding Quality Family Planning Services: Recommendations of CDC and the U.S. Office of Population Affairs\u201d (QFP), which describes the scope of services that should be offered in a family planning visit and how to provide those services , and professional medical associations such as the American College of Obstetricians and Gynecologists and the American Academy of Pediatrics.2), and covered guidelines published during April 2014\u2013December 2015. This report summarizes recommendations from guidelines published during January 2016\u2013April 2017. CDC and the Office of Population Affairs prepared these updates by searching for materials from CDC, USPSTF, and other professional medical organizations that had recommendations referenced in the original QFP. When updated recommendations were identified, they were evaluated for changes in implications for providing family planning care. CDC and the Office of Population Affairs determined that none of the newly published recommendations marked a substantial shift in how family planning care should be provided, and therefore did not seek additional review to consider the implications for the QFP for this update. Technical reviews from clinical experts representing a broad range of family planning providers might be appropriate for future updates.The scope of preventive services related to reproductive health is constantly evolving as new scientific findings are published and clinical recommendations are modified accordingly. Being knowledgeable about the most current recommendations is an important step toward providing the highest quality care to patients. To keep QFP current with the latest recommendations, CDC and the Office of Population Affairs publish occasional updates that summarize newly published clinical recommendations. The first of these updates was published in March 2016 for emergency contraception.Revisions to the recommendations for postpartum women; women who are breastfeeding; women with known dyslipidemias, migraine headaches, superficial venous disease, gestational trophoblastic disease, sexually transmitted diseases (STDs), and human immunodeficiency virus (HIV) infection; and women who are receiving antiretroviral therapy.https://www.cdc.gov/mmwr/volumes/65/rr/rr6503a1_appendix.htmFor all 2016 updated recommendations, see Tables A1 and A2: Source: Curtis KM, Tepper NK, Jatlaoui TC, et al. U.S. medical eligibility criteria for contraceptive use, 2016. MMWR Recomm Rep 2016;65(No. RR-3).The 2016 CDC recommendations update earlier 2013 recommendations that address a select group of common, yet sometimes complex, issues regarding initiation and use of specific contraceptive methods.Recommendations have been updated regarding when to start regular contraception after UPA emergency contraceptive pills:Advise the woman to start or resume hormonal contraception no sooner than 5 days after use of UPA, and provide or prescribe the regular contraceptive method as needed. For methods requiring a visit to a health care provider, such as depo-medroxyprogesterone acetate (DMPA), implants, and intrauterine devices (IUDs), starting the method at the time of UPA use may be considered; the risk that the regular contraceptive method might decrease the effectiveness of UPA must be weighed against the risk of not starting a regular hormonal contraceptive method.The woman needs to abstain from sexual intercourse or use barrier contraception for the next 7 days after starting or resuming regular contraception or until her next menses, whichever comes first.Any nonhormonal contraceptive method can be started immediately after the use of UPA.The woman should be advised to have a pregnancy test if she does not have a withdrawal bleed within 3 weeks.New recommendations have been made regarding medications used to ease IUD insertion:Misoprostol is not recommended for routine use before IUD insertion. Misoprostol might be helpful in select circumstances .Paracervical block with lidocaine might reduce patient pain during IUD insertion.Source: Curtis KM, Jatlaoui TC, Tepper NK, et al. U.S. selected practice recommendations for contraceptive use, 2016. MMWR Recomm Rep 2016;65(No. RR-4).DepressionThe 2016 USPSTF recommendation for adults reaffirms the 2009 recommendation to screen all adults when staff-assisted depression care supports are in place. This replaces the 2009 recommendation regarding selective screening of adults.The 2016 USPSTF recommendation for adolescents aged 12\u201318 years reaffirms the 2009 recommendation to screen for major depressive disorder when systems are in place to ensure accurate diagnosis, effective treatment, and follow-up. The 2016 statement removes the recommendation of specific psychotherapies in recognition of decreased concern over the harms of pharmacotherapy in adolescents as long as they are adequately monitored.Sources:US Preventive Services Task Force. Screening for depression in adults. Rockville, MD: US Department of Health and Human Services, Agency for Healthcare Research and Quality; 2016.US Preventive Services Task Force. Screening for depression in children and adolescents. Rockville, MD: US Department of Health and Human Services, Agency for Healthcare Research and Quality; 2016.American College of Obstetricians and Gynecologists\u2019 Committee on Health Care for Underserved Women. Committee opinion no. 654: reproductive life planning to reduce unintended pregnancy.Obstet Gynecol2016;127:e66\u20139. 10.1097/AOG.000000000000131426942389CurtisKM, TepperNK, JatlaouiTC, U.S. medical eligibility criteria for contraceptive use, 2016.MMWR Recomm Rep2016;65(No. RR-3):1\u2013103. 10.15585/mmwr.rr6503a127467196CurtisKM, JatlaouiTC, TepperNK, U.S. selected practice recommendations for contraceptive use, 2016.MMWR Recomm Rep2016;65(No. RR-4):1\u201366. 10.15585/mmwr.rr6504a127467319American College of Obstetricians and Gynecologists\u2019 Committee on Obstetric Practice. Committee opinion no. 670: immediate postpartum long-acting reversible contraception.Obstet Gynecol2016;128:e32\u20137. 10.1097/AOG.000000000000158727454734American College of Obstetricians and Gynecologists\u2019 Committee on Gynecologic Practice Long-Acting Reversible Contraceptive Expert Work Group. Committee opinion no. 672: clinical challenges of long-acting reversible contraceptive methods.Obstet Gynecol2016;128:e69\u201377. 10.1097/AOG.000000000000164427548557PfeiferS, ButtsS, FossumG, ; Practice Committee of the American Society for Reproductive Medicine in collaboration with the Society for Reproductive Endocrinology and Infertility. Optimizing natural fertility: a\u00a0committee opinion.Fertil Steril2017;107:52\u20138. 10.1016/j.fertnstert.2016.09.02928228319American College of Obstetricians and Gynecologists\u2019 Committee on Health Care for Underserved Women. Committee opinion no. 654: reproductive life planning to reduce unintended pregnancy.Obstet Gynecol2016;127:e66\u20139. 10.1097/AOG.000000000000131426942389KimDK, RileyLE, HarrimanKH, HunterP, BridgesCB. Advisory Committee on Immunization Practices recommended immunization schedule for adults aged 19 years or older\u2014United States, 2017.MMWR Morb Mortal Wkly Rep2017;66:136\u20138. 10.15585/mmwr.mm6605e228182599RobinsonCL, RomeroJR, KempeA, PellegriniC; Advisory Committee on Immunization Practices (ACIP) Child/Adolescent Immunization Work Group. Advisory Committee on Immunization Practices recommended immunization schedule for children and adolescents aged 18 years or younger\u2014United States, 2017.MMWR Morb Mortal Wkly Rep2017;66:134\u20135. 10.15585/mmwr.mm6605e128182607US Preventive Services Task Force. Folic acid supplementation for the prevention of neural tube defects: preventive medication. Rockville, MD: US Department of Health and Human Services, Agency for Healthcare Research and Quality; 2017. https://www.uspreventiveservicestaskforce.org/Page/Document/UpdateSummaryFinal/folic-acid-for-the-prevention-of-neural-tube-defects-preventive-medicationUS Preventive Services Task Force. Depression in adults: screening. Rockville, MD: US Department of Health and Human Services, Agency for Healthcare Research and Quality; 2016. https://www.uspreventiveservicestaskforce.org/Page/Document/UpdateSummaryFinal/depression-in-adults-screening1US Preventive Services Task Force. Depression in children and adolescents: screening. Rockville, MD: US Department of Health and Human Services, Agency for Healthcare Research and Quality; 2016. https://www.uspreventiveservicestaskforce.org/Page/Document/UpdateSummaryFinal/depression-in-children-and-adolescents-screening1US Preventive Services Task Force. Syphilis infection in nonpregnant adults and adolescents: screening. Rockville, MD: US Department of Health and Human Services, Agency for Healthcare Research and Quality; 2016. https://www.uspreventiveservicestaskforce.org/Page/Document/UpdateSummaryFinal/syphilis-infection-in-nonpregnant-adults-and-adolescentsUS Preventive Services Task Force. Breast cancer: screening. Rockville, MD: US Department of Health and Human Services, Agency for Healthcare Research and Quality; 2016. https://www.uspreventiveservicestaskforce.org/Page/Document/UpdateSummaryFinal/breast-cancer-screening1US Preventive Services Task Force. Gynecological conditions: periodic screening with the pelvic examination. Rockville, MD: US Department of Health and Human Services, Agency for Healthcare Research and Quality; 2017. https://www.uspreventiveservicestaskforce.org/Page/Document/UpdateSummaryFinal/gynecological-conditions-screening-with-the-pelvic-examinationUS Preventive Services Task Force. Genital herpes infection: serologic screening. Rockville, MD: US Department of Health and Human Services, Agency for Healthcare Research and Quality; 2016. https://www.uspreventiveservicestaskforce.org/Page/Document/UpdateSummaryFinal/genital-herpes-screening11. GavinL, MoskoskyS, CarterM, . Providing quality family planning services: recommendations of CDC and the U.S. Office of Population Affairs.MMWR Recomm Rep2014;63(No. RR-04).247596902. GavinL, PazolK. Update: providing quality family planning services\u2014recommendations from CDC and the U.S. Office of Population Affairs, 2015.MMWR Morb Mortal Wkly Rep2016;65:231\u20134. 10.15585/mmwr.mm6509a326963363"} +{"text": "Cellular & Molecular Biology Letters launched with BioMed Central in January, 2016. Previously, its publishers were Walter De Gruyter in 2015, and Springer from 2006\u20132014. Content from 2015 (Volume 20) is available in the archive."} +{"text": "Corrigendum to:https://doi.org/10.1002/ece3.2528Casties I, Seebens H, Briski E (2016) Importance of geographic origin for invasion success: a case study of the North and Baltic Seas versus the Great Lakes\u2010St. Lawrence River region. Ecology and Evolution, 6(22), 8318\u20138329. The authors of the paper Casties et\u00a0al. (2016) want to note that Figure 1 appeared incorrectly. Please find the correct figure below."} +{"text": "Correction to:npj Systems Biology and Applications (2016) 2, 16002; doi:10.1038/npjsba.2016.2; published online 3 March 2016After online publication of this article, the authors noticed an error in the input files used for running the Prize Collecting Steiner Forest algorithm for With publication of this corrigendum, rectified"} +{"text": "De Novo Sequencing and Assembly Analysis of the Pseudostellaria heterophylla Transcriptome. PLoS ONE 11(10): e0164235.The second author\u2019s name is incorrect. The correct name is: Wei Zheng. The correct citation is: Li J, Zheng W, Long D, Ding L, Gong A, Xiao C, et al. (2016) P. heterophylla cultivar \u2018Shitai 1\u2019 was selected and grown in a commercial planting base in Shibing County, Guizhou Province, China.There is an error in the first sentence of the Methods section. The correct sentence is:"} +{"text": "Scientific Reports 10.1038/s41598-017-16850-z, published online 29 November 2017Correction to: The Acknowledgements section in this Article is incomplete.\u201cAdvanced Research Foundation, grant #7/019/2014-2017, supported the laser inscription experiments. Ministry of Education of Science of Russian Federation, grant #14.Z50.31.0009, have funded measurement, math treatment and analysis of the obtained data. The authors are grateful to Dr. R. Drevinskas for discussions of methods for measuring the optical phase.\u201dshould read:\u201cAdvanced Research Foundation, grant #7/019/2014-2017, supported the laser inscription experiments. Ministry of Education of Science of Russian Federation, grant #14.Z50.31.0009, have funded measurement, math treatment and analysis of the obtained data. A part of the study related to the measurements of refractive index change was also supported by the Royal Society (UK) International Exchanges Program (Grant IE150203). The authors are grateful to Dr. R. Drevinskas for helpful discussions.\u201d"} +{"text": "Correction to:Cell Discovery (2016) 2, 16037; doi:10.1038/celldisc.2016.37; Published 18 October 2016x axis and y axis were left out. The corrected In the initial published version of this article, an error was made in"} +{"text": "Scientific Data 3:160115 doi: 10.1038/sdata.2016.115 (2016); Published 20 December 2016; Updated 14 March 2017The original version of this Data Descriptor contained a typographical error in the spelling of the author A. Lamontanara, which was incorrectly given as A. Lamontara. This has now been corrected in the PDF and HTML versions of the Data Descriptor."} +{"text": "Correction to:Cell Death and Disease (2016) 7, e2388; doi:10.1038/cddis.2016.260; published online 29 September 2016Cell Death and Disease in 2016, the authors noted an error contained in Since the publication of this article in The corrected article appears online together with this corrigendum.The authors would like to apologize for any inconvenience caused."} +{"text": "Correction to: Translational Psychiatry (2016) 6, e713; doi:10.1038/tp.2015.209; published online 19 January 2016The authors noticed that an omission was made in the Acknowledgements section. The omitted data appear below.This project was partially funded by a grant from the Pennsylvania Department of Health."} +{"text": "Correction to:Clinical & Translational Immunology (2017) 6, e126; doi:10.1038/cti.2016.87; published online 20 January 2017.The volume number in the PDF of the above paper was published incorrectly. It should be 6 instead of 5. This has now been corrected in the PDF.The publishers wish to apologize for any inconvenience caused."} +{"text": "Dear Editors,Acta Neuropathol Commun. 2017; 5: 13 [We read with interest the publication by Wei et al., Mitochondrial DNA Point Mutations and Relative Copy Number in 1363 Disease and Control Human Brains, 7; 5: 13 . We disaSincerely,David K. Simon, MD PhD"} +{"text": "CDC is assisting ministries of health and working with other organizations to end the ongoing epidemic of Ebola virus disease (Ebola) in West Africa . The updAccording to the latest World Health Organization update on November 14, 2014 , a totalThe 2,705 new Ebola cases reported during October 19\u2013November 8 were more widely distributed geographically among districts in Guinea and Liberia compared with the 2,809 new cases reported during September 28\u2013October 18 .As of November 8, the highest cumulative incidence rates were reported by two prefectures in Guinea (Gu\u00e9ck\u00e9dou and Macenta), four counties in Liberia , and five districts in Sierra Leone . Evidenchttp://www.cdc.gov/vhf/ebola/outbreaks/guinea/index.html. The most up-to-date infection control and clinical guidelines on the 2014 Ebola epidemic in West Africa are available at http://www.cdc.gov/vhf/ebola/hcp/index.html.The latest updates on the 2014 Ebola epidemic in West Africa, including case counts, are available at"} +{"text": "AbstractOphiuroidea stored at the Italian National Antarctic Museum are presented, corresponding to 1595 individuals that belong to 35 species and 17 genera. Specimens were collected in 106 different sampling stations at depths ranging from 21 to 1652 m in the framework of 14 Antarctic expeditions to the Ross Sea, one to the Antarctic Peninsula, and one to the Falkland Islands . Three species, Amphiurajoubini Koehler, 1912, Amphiura (Amphiura) angularis Lyman, 1879, and Ophiuraflexibilis , are reported as new records for the Terra Nova Bay area, whose check-list of species increases from 15 to 18 species. The determination of these three new records was based both on morphological identification and molecular analyses (COI barcoding). Some of the genetically characterised specimens were also documented through photogrammetry and micro-computed tomography and represent the first bulk of 3D models that will be available through the MNA and Sketchfab websites, both for research and educational purposes.The distributional records of Museum collections have always been indispensable resources for biodiversity studies by providing data over a vast time span . The recOphiuroidea collected in the framework of several recent scientific expedition performed in the Southern Ocean and which are now permanently stored and curated at the Italian National Antarctic Museum . The Ross Sea expeditions were the Italian National Antarctic Program (PNRA) expedition \u201cIII\u201d (1987/1988), \u201cV\u201d (1989/1990), \u201cIX\u201d (1993/1994), \u201cX\u201d (1994/1995), \u201cXIII\u201d (1997/1998), \u201cXIV\u201d (1998/1999), \u201cXV\u201d (1999/2000), \u201cXVII\u201d (2001/2002), \u201cXIX\u201d (2003/2004), \u201cXXV\u201d (2009/2010), \u201cXXVII\u201d (2011/2012), \u201cXXVIII\u201d (2012/2013), \u201cXXIX\u201d (2013/2014) and the New Zealand \u201cTAN0802 IPY-CAML Oceans Survey 20/20\u201d voyage (2008). Additional samples collected outside the Ross Sea were obtained from the German ANT-XXIX/3, PS81 expedition (2013) and the US ICEFISH 2004 voyage (2004).In this paper new distributional data is provided for MNA collections, ii) helping researchers in crosschecking morphological data of sequenced species from specific geographic areas (in this case species from the Ross Sea and Terra Nova Bay in particular), and iii) producing materials useful for educational purposes. Virtual collections guarantee rapid and simultaneous access to accurate virtual representations of important museum materials, such as type materials (e.g. MNA 2644 and MNA 7784) and on photogrammetry for larger ones (i.e. MNA 7368). The distributional dataset is the fourth MNA contribution to the Antarctic Biodiversity Portal, which is the thematic Antarctic node for both the Ocean Biogeographic Information System (AntOBIS) and the Global Biodiversity Information Facility (ANTABIF) (http://www.biodiversity.aq). Previous contributions were: In parallel to this data set a series of ophiuroid 3D models have also been developed based on \u2018molecular vouchers\u2019 that are released with the aim of: i) providing the widest accessibility to the als e.g. or, as ials e.g. for smalProject title: Antarctic Ophiuroidea in the collection of the Italian National Antarctic Museum (MNA)Curator and Promoter: Stefano SchiaparelliPersonnel: Matteo Cecchetto, Maria Chiara Alvaro, Claudio Ghiglione, Alice Guzzi, Claudio Mazzoli, Paola Piazza, Stefano SchiaparelliFunding sources: The Ophiuroidea were collected during different Italian, New Zealand, German and American research projects and expeditions to Antarctica funded by: i) the Italian National Antarctic Research Program (PNRA), ii) the Ministry for Primary Industries , iii) the Alfred Wegener Institute Helmholtz Centre for Polar and Marine Research and iv) the National Science Foundation grant to H. William Detrich (North Eastern University). The full list of sponsors is listed below:PNRA research projectsItalian :\u2022 2.1.4.6 (Necton e risorse da pesca) \u2022 3.2.1.2.5 (Benthos) and 3.2.1.4 (Oceanografia geologica) \u2022 2d.2 (Ecology and Biogeochemistry of the Southern Ocean) \u2022 2d.2 (Ecology and Biogeochemistry of the Southern Ocean) \u2022 2b.3.1 (Struttura e dinamica delle cenosi marine di Baia Terra Nova) PageBreak\u2022 2b.3.1 (Struttura e dinamica delle cenosi marine di Baia Terra Nova) \u2022 8.5 (L\u2019area marina protetta di Baia Terra Nova: struttura e variazioni a breve e lungo termine) \u2022 4.7 \u2022 8.5 (L\u2019area marina protetta di Baia Terra Nova: struttura e variazioni a breve e lungo termine) \u2022 1.3 \u2022 2002/8.6 \u2022 2006/08.01 \u2022 2010/A1.10 \u2022 2009/A1.09 \u2022 2010/A1.10 Pleuragrammaantarcticum) \u2022 2010/A1.11 \u2022 2013/AZ1.18 (RAISE: Ricerche integrate sulla ecologia dell\u2019Antarctic Silverfish nel MarE di Ross) New Zealand research projects:NIWA)\u2022 IPY-CAML voyage - Census of Antarctic Marine Life programme - funded by the Government of New Zealand and administered by the Ocean Survey 20/20 CAML Advisor Group \u2022 Project 3.1 (M.C. Alvaro PageBreakAmerican research project:http://www.icefish.neu.edu) (M. Vacchi legit)\u2022 ICEFISH 2004 Cruise expedition, the first comprehensive international survey of the fishes of the Sub-Antarctic marine habitat and southeast Falkland Islands . The bathymetric range of sampling stations was from 21 to 1652 m.Design description: Data were assembled by revising Ophiuroidea voucher specimens curated by the Italian National Antarctic Museum collection (MNA), Section of Genoa, Genoa . These samples were collected in the framework of the above reported research expeditions, which had different aims and geographical scopes.Method step description: See sampling description below and flowchart of Figure Study extent description: The Ophiuroidea distributional data here considered originated from 106 different sampling stations ranging between 21 and 1652 metres of depth , of the National Institute of Water and Atmospheric Research (NIWA), of the Alfred Wegener Institute (AWI) and of the USA Antarctic Program. Sampling was performed through the deployment of a variety of sampling gears. Benthic sampling under the Italian PNRA was mainly performed by using a heterogeneous set of dredges (i.e. Charcot dredge and Triangular dredges) and Van Veen grabs of different volumes, plus an unconventional set of gears for sampling benthic fauna such as gillnets, trammel nets, long lines, also comprising a mid-water trawls that opportunistically collected benthic specimens due to accidental contact with the bottom during gear deployment failures. Two samples were collected during the \u201cXVII\u201d and \u201cXXV\u201d PNRA expeditions by SCUBA divers along the rocky cliffs of Tethys Bay. The NIWA expedition \u201cTAN0802 IPY-CAML\u201d (2008) used an epibenthic sled and a rough-bottom trawl (ORH). The German expedition \u201cANT-XXIX/3\u201d (PS81) collected the material through the deployment of an Agassiz trawl while an Otter trawl was used to sample the single specimen collected during the ICEFISH 2004 cruise. After the material has been acquired by the MNA and included in the collections, all the specimens were classified to the lowest possible taxonomical resolution on a morphological base mainly by following PNRA projects 2010/A1.10 \u201cBAMBi\u201d (Barcoding of Antarctic Marine Biodiversity) and PNRA 2013/AZ1.15 \u201cISOBIOTOX\u201d . DNA extraction and sequencing of partial cytochrome c oxidase subunit 1 (CO1) was carried out at the Canadian Centre for DNA Barcoding and sequences were uploaded on the BOLD platform . The primers used for amplification were LCOech1aF1 and HCO2198 (http://iobis.org/data/schema-and-meta-data) and according to the SCAR-MarBIN Data Toolkit (available at http://www.scarmarbin.be/documents/SM-FATv1.zip). The dataset was uploaded into AntOBIS (the Antarctic thematic node of OBIS). Vouchers stored at the Italian National Antarctic Museum (MNA), Section of Genoa, Genoa are preserved, according to the different lots, in 75% ethanol, frozen (-20\u00b0C) or dried obtained by sequencing a total of 167 specimens representative of all the different morphospecies found here. The outcomes of the molecular study will be the subject of another publication and here we release only the COI sequences corresponding to the specimens that have been used to produce 3D models.PageBreakat various steps in order to produce quality data and make consistent cross-references between the database and samples\u2019 labels to manage its collections and link all the data to the physical samples. Geo-referencing on board of the different research vessels was based on the interpolation of GPS satellite receivers and a gyrocompass. Station coordinates and sampling events were recorded during sampling activities based on various GPS systems.During all the phases of sorting, classification, and storage of samples at the Italian National Antarctic Museum, quality control and data cleaning have been undertaken els Fig. . The MNAGeneral taxonomic coverage description: This dataset focused on the Class Ophiuroidea and includes a total of 1595 specimens belonging to 17 genera and 35 different species. In the Southern Ocean, the Class Ophiuroidea numbers 219 species , thus representing one of the most important benthic groups of the Antarctic and sub-Antarctic regions. Amphiurajoubini Koehler, 1912, one specimen of Amphiura (Amphiura) angularis Lyman, 1879, and four specimens of Ophiuraflexibilis . These records have thus to be added to the Terra Nova Bay checklist provided by Chiantore et al. (2006) and increase the check-list of Terra Nova Bay ophiuroid species from 15 to 18. The determination of these new records was also molecularly cross-checked (and confirmed) through COI barcodes. The outcomes of the barcoding effort on Terra Nova Bay ophiuroids and other echinoderm classes will be the subject of a separate contribution.PageBreakKingdom: AnimaliaPhylum: EchinodermataClass: OphiuroideaOrder: EuryalidaFamily: GorgonocephalidaeGenera: Astrotoma, Astrochlamys, GorgonocephalusSpecies: Astrochlamysbruneus; Astrochlamyssol; Astrotomaagassizii; Gorgonocephalus sp.Kingdom: AnimaliaPhylum: EchinodermataClass: OphiuroideaOrder: OphiuridaFamily: Amphiuridae, Ophiacantidae, Ophiolepididae, Ophiuridae, OphiuridaeGenera: Amphiura; Ophiacantha; Ophiocamax; Ophioceres; Ophiocten; Ophiogona; Ophioleuce; Ophiolimna; Ophionotus; Ophioperla; Ophioplinthus; Ophiosparte; Ophiosteira; OphiuraSpecies: Amphiuraalgida; Amphiura (Amphiura) angularis; Amphiurabelgicae; Amphiurajoubini; Amphiura sp.; Ophiacanthaantarctica; Ophiacanthapentactis; Ophiacantha sp.; Ophiacanthavivipara; Ophiocamaxgigas; Ophioceresincipiens; Ophioctendubium; Ophioctenmegaloplax; Ophiogonadoederleini; Ophioleuceregulare; Ophiolimnaantarctica; Ophionotusvictoriae; Ophioperlakoehleri; Ophioplinthusbrevirima; Ophioplinthusgelida; Ophioplinthusmartensi; Ophioplinthus sp.; Ophiospartegigas; Ophiosteiraantarctica; Ophiosteirabullivanti; Ophiosteiraechinulata; Ophiuraambigua; Ophiura (Ophiuroglypha) carinifera; Ophiuracrassa; Ophiuraflexibilis; Ophiura sp.General geographic description: The Ross Sea, the Bransfield Strait in the Antarctic Peninsula and southeast of the Falkland Islands Figure , 4 and 5PNRA III expedition: -74.7125 and -74.79167; 164.03000 and 164.13667PNRA V expedition: -74.68745 and -74.87100; 164.02433 and 164.26083PNRA IX expedition: -74.72000; 164.14000PNRA X expedition: -74.69320 and -74.71688; 164.04272 and 164.14700PNRA XIII expedition: -74.68750; 164.14550PNRA XIV expedition: -74.68762 and -74.89950; 163.93748 and 164.10728PageBreakPNRA XV expedition: -74.71343 and -74.72053; 164.17060 and 164.17783PNRA XVII expedition: -72.20967 and -77.65133; 164.03418 and -166.79183PNRA XIX expedition: -71.28833 and -74.82167; 164.19167 and 170.65333PNRA XXV expedition: -74.69027 and -74.69768; 164.10255 and 164.13108PNRA XXVII expedition: -74.68562 and -74.71337; 164.03502 and 164.14903PNRA XXVIII expedition: -74.68090 and -74.77737; 164.05285 and 164.23640PNRA XXIX expedition: -74.68677 and -74.72283; 164.12278 and 164.24206New Zealand TAN0802 IPY-CAML voyage: -66.93250 and -73.24817; 170.87700 and 178.72367German ANT-XXIX/3 (PS81) expedition: -62.43250 and -63.00883; -56.28767 and -58.68483American ICEFISH 2004 cruise: -52.48498; -54.86978PNRA III expedition (1987/1988): January 15, 1988 - February 2, 1988PNRA V expedition (1989/1990): December 25, 1989 - December 29, 1989PNRA IX expedition (1993/1994): December 17, 1993PNRA X expedition (1994/1995): January 25, 1995 - February 8, 1995PNRA XIII expedition (1997/1998): February 21, 1998PNRA XIV expedition (1998/1999): January 25, 1999 - February 10, 1999PNRA XV expedition (1999/2000): February 10, 2000PNRA XVII expedition (2001/2002): January 4, 2002 - February 10, 2002PNRA XIX expedition (2003/2004): February 4, 2004 - February 21, 2004PNRA XXV expedition (2009/2010): December 10, 2009 - January 11, 2010PNRA XXVII expedition (2011/2012): January 27, 2012 - February 3, 2012PNRA XXVIII expedition (2012/2013): January 9, 2013 - January 31, 2013PNRA XXIX expedition (2013/2014): January 16, 2014 - February 3, 2014New Zealand TAN0802 IPY-CAML voyage (2008): February 19, 2008 - March 14, 2008German ANT-XXIX/3 (PS81) expedition (2013): January 26, 2013 - March 5, 2013American ICEFISH 2004 cruise (2004): June 26, 1905Parent collection identifier: Italian National Antarctic Museum (MNA) Section of Genoa, ItalyCollection name: MNA (Section of Genoa) Invertebrate Collection \u2013 Antarctic and sub-Antarctic OphiuroideaSpecimen preservation method: Part of the material collected during the older expeditions (roughly from 1988 to 2004) was fixed in ethanol or formalin immediately after isolation and kept in tubes or vials for further studies, while the remaining samples PageBreakwere dried out to facilitate morphological identifications. The specimens corresponding to these older expeditions are therefore generally not usable for molecular analyses. Instead, the material collected during recent expeditions (i.e. roughly from 2005 to 2014) was fixed in 75% ethanol or frozen immediately after isolation and kept in the same condition in order to preserve the DNA quality and integrity .ity Fig. . MoleculVirtual collection of vouchers and 3D models: 3D models were obtained from three barcoded specimens of the species Ophiosteiraechinulata (MNA 2644) and 3.95 \u03bcm (sample MNA 2644), respectively. Three-dimensional rendering and animations were performed with CTVox software package (Bruker).44) Fig. , O.antaca MNA 774 Fig. . The firMNA 7368) was produced by using photogrammetry and assembling pictures taken with a Nikon D700 equipped with a Nikon AF-S Micro Nikkor 105 mm (1: 2.8G ED) lens (corresponding to a resolution of 4256 x 2832 and a pixel size of 8.46 x 8.46 \u03bcm) in Agisoft Photoscan Professional 1.3.1. The MNA 3D models based on materials curated at the museum will be available from the MNA web site (www.mna.it) and from the Sketchfab gallery dedicated to cultural heritage and museums (https://sketchfab.com/MNA).The third model . The Darwin Core elements included in the dataset are: occurrence ID , institution code , basis of record , preparations (i.e. the preservation method), catalogue number , individual PageBreakcount, sampling protocol (i.e. sampling gear), event date , event remarks (i.e. expedition), field number (i.e. sampling station), field notes , maximum depth in meters, decimal latitude (DD), decimal longitude (DD) and scientific name.PageBreakObject name: MNA (Section of Genoa) \u2013 Antarctic and sub-Antarctic OphiuroideaCharacter encoding: UTF-8Format name: Darwin Core Archive formatFormat version: 1.0PageBreakDistribution: http://ipt.biodiversity.aq/resource.do?r=mna_antarctic_ophiuroideaLanguage: EnglishMetadata language: EnglishLicense of use: This dataset [MNA (Section of Genoa) \u2013 Antarctic and sub-Antarctic Ophiuroidea] is made available under the Creative Commons Attribution License (CC-BY) 4.0: http://www.creativecommons.org/licenses/by/4.0/legalcodeDate of metadata creation: 2017-05-04Hierarchy level: Dataset"} +{"text": "There is an error in reference 27. The correct reference is: Maru\u0161i\u0107 A, Bo\u0161njak L, Jeron\u010di\u0107 A. A systematic review of research on the meaning, ethics and practices of authorship across scholarly disciplines. PLoS ONE. 2011;6: e23477. doi: 10.1371/journal.pone.0023477There is an error in reference 52. The correct reference is: Ha\u00fcssler C, Sauermann H. Credit where credit is due? The impact of project contributions and social factors on authorship and inventorship. Res Policy. 2013;42: 688\u2013703.The second sentence of the fourth paragraph in the Introduction section should have cited reference 52 instead of 27.The correct sentence should read: Researchers may award guest authorship to colleagues in hopes of receiving reciprocal authorship on that colleague\u2019s publications or to increase the likelihood of an article\u2019s publication due to that colleague\u2019s political or reputational influence [52].The fourth sentence of the last paragraph in the Introduction section should have cited references 36, 53 instead of 28, 37. In the same sentence, the references 38,39,40,41,42,43,44,45,46,47,48 should be replaced with the following omitted references:Drenth JPH. Multiple authorship: the contribution of senior authors. JAMA J Am Med Assoc. 1998;280: 219\u2013221.Regalado A. Multiauthor papers on the rise. Science. 1995;268: 25.Modi P, Hassan A, Teng CJ, Chitwood Jr WR. \u201cHow many cardiac surgeons does it take to write a research article?\u201d: seventy years of authorship proliferation and internalization in the cardiothoracic surgical literature. J Thorac Cardiovasc Surg. 2008;136: 4\u20136.Onwude JL, Staines A, Lilford RJ. Multiple author trend worst in medicine. BMJ. 1993;306: 1345.Borry P, Schotsmans P, Dierickx K. Author, contributor or just a signer? a quantitative analysis of authorship trends in the field of bioethics. Bioethics. 2006;20: 213\u2013220.Epstein RJ. Six authors in search of a citation: villains or victims of the Vancouver convention? BMJ. 1993;306: 765\u2013767.https://www.geosociety.org/gsatoday/archive/17/7/pdf/i1052-5173-17-7-44.pdf.Engelder T. The coupling between devaluation of writing in scientific authorship and inflation of citation indices. GSA Today. 2007;17: 44\u201345. Available from: McDonald RJ, Neff KL, Rethlefsen ML, Kallmes DF. Effects of author contribution disclosures and numeric limitations on authorship trends. Mayo Clin Proc. 2010;85: 920\u2013927.Shaban S. Multiple authorship trends in prestigious journals from 1950 to 2005. Saudi Med J. 2007;28: 927\u2013932.http://ip-science.thomsonreuters.com/m/pdfs/klnl/8428096/swmultiauthor.pdf.King C. Multiauthor paper redux: a new peak at new peaks. Science Watch Nov-Dec. 2007; Available from: Cozzarelli NR. Responsible authorship of papers in PNAS. Proc Natl Acad Sci. 2004;101: 10495.The references given in the sixth sentence of the last paragraph in the Introduction section are incorrect. In this sentence, the references 41, 45 should be replaced with the following omitted references:Epstein RJ. Six authors in search of a citation: villains or victims of the Vancouver convention? BMJ. 1993;306: 765\u2013767.McDonald RJ, Neff KL, Rethlefsen ML, Kallmes DF. Effects of author contribution disclosures and numeric limitations on authorship trends. Mayo Clin Proc. 2010;85: 920\u2013927.The tenth sentence of the first paragraph in the Discussion section should have cited reference 27 instead of 52.The correct sentence should read: Our cluster analysis revealed two general groups among middle authors, one characteristic of more senior level researchers and the other more typical of junior level researchers [27].The structure of all affected sentences in the original article remains the same."} +{"text": "Correction to:Molecular Psychiatry advance online publication, 18 October 2016; doi:10.1038/mp.2016.179Following publication of the above article, the authors noticed that an obsolete version of the Supplementary Information (DOCX 18 kb)"} +{"text": "Correction to:Heredity (2016) 118, 52\u201363; doi:10.1038/hdy.2016.99; published online 2 November 2016Updated online 7 December 2016: This article was originally published under NPG's License to Publish, but has now been made available under a CC BY 4.0 license. The PDF and HTML versions of the paper have been modified accordingly.The corrected article appears in this issue together with this corrigendum. The publisher wishes to apologize for any inconvenience caused."} +{"text": "Retraction: Data in this paper was plagiarized from \u201cClinical application of contrast-enhanced ultrasonography in diagnosis of superficial lymphadenopathy,\u201d in the Journal of Ultrasound in Medicine. 2010; 29:735-40.Footnotes: The online version of the original article can be found under doi.52416-52422. doi: 10.18632/oncotarget.9385Original article: Oncotarget. 2016; 7:"} +{"text": "CDC is assisting ministries of health and working with other organizations to end the ongoing epidemic of Ebola virus disease (Ebola) in West Africa . The updAccording to the latest World Health Organization update on December 10, 2014 , a totalThere were 4,281 new Ebola cases reported during the 4-week period of November 9\u2013December 6, compared with the 2,705 new cases reported during the 3-week period of October 19\u2013November 8 .As of December 6, the highest cumulative incidence rates were reported by two prefectures in Guinea (Gu\u00e9ck\u00e9dou and Macenta), six counties in Liberia , and six districts in Sierra Leone . Evidenchttp://www.cdc.gov/vhf/ebola/outbreaks/2014-west-africa/index.html. The most up-to-date infection control and clinical guidelines on the 2014 Ebola epidemic in West Africa are available at http://www.cdc.gov/vhf/ebola/hcp/index.html.The latest updates on the 2014 Ebola epidemic in West Africa, including case counts, are available at"} +{"text": "AbstractIotarphiarufobrunnea Lee & Ahn, sp. n. is described from Tasmania. The new species is compared with another species of the genus, Iotarphiaaustralis Cameron. A description, habitus photograph and illustrations of the diagnostic characters are provided. Iotarphia Cameron. After detailed examination of the specimens and comparison with Iotarphiaaustralis Cameron (type species of Iotarphia), we concluded that these specimens represent a new species of the genus.While working on aleocharine beetles collected by the second author from the eastern and southern seashores in Tasmania, Australia, we found specimens very similar to the athetine genus PageBreakIotarphia and its single described species have been recorded only in a \u201cmaritime habitat\u201d from New South Wales and from Tasmania, both in Australia , Coal Point, Bruny Island , collected 25.ix.2014, A.W. Osborn. Paratypes: 4, of which 3 (QVM:2016:12:1052 to 1054) share common collection data with holotype, and 1 (QVM:2014:12:0125) collected from Lighthouse Bay, Bruny Is., collected 24.ix.2014, A.W.Osborn.Queen Victoria Museum and Art Gallery, Launceston, Tasmania (QVMAG).All type specimens have been placed in the PageBreakyellow, elytra reddish brown except for basal darker region; surface slightly glossy, densely pubescent with fine microsculpture. Head. Slightly transverse, approximately 1.1\u20131.2 times as wide as long, widest across eyes, narrower than pronotum; eyes slightly large and prominent, about 1.2 times as long as temples; gular sutures moderately separated, slightly diverged basally. Antennae ; 40:43:46:48:78 (mesotarsus); 48:55:58:54:80 (metatarsus); one empodial seta present, shorter than claw. Abdomen. Subparallel-sided; surface glossy and densely pubescent, with transverse and imbricate microsculpture; male tergite VIII , Tasmania, Australia Fig. .Iotarphiaaustralis, but can be distinguished by the characters provided in Table 43.34211\u00b0S and 147.32178\u00b0E) and (ii) from a sandy substrate in which some small rocks were present within the littoral zone at Lighthouse Bay .This species is similar to Staphylinidae species in the Tasmanian fauna to five: Iotarphiaaustralis (= Psammoporadelittlei Pace), Iotarphiarufobrunnea Lee & Ahn, sp. n., Teropalpuspictipes (Lea), Cafiuspacificus (Erichson), and Remussericeus (Holme).The description of the new species within the present paper brings the total number of coastal"} +{"text": "Correction to:Journal of Human Hypertension (2017) 31, 305\u2013312; doi:10.1038/jhh.2016.78; published online 22 December 2016The text of the licence type for this paper was incorrect on the full text version. The following is the correct text:The publishers wish to apologise for their error."} +{"text": "AbstractOpadometa are difficult to associate with conspecific females, and sex-matching errors may persist in the taxonomic literature. Recommended best practices for definitive sex matching in this genus suggest finding a male in the web of a female, or better yet, mating pairs.Males of Opadometa was observed hanging on a frame line of the web of a female Opadometasarawakensis, a species for which the male was previously undescribed. This occurred during a tropical ecology field course held at the Danau Girang Field Centre in Sabah, Malaysia. A taxonomic description was completed as a course activity.A male Opadometa species. Opadometa Males\u201d, in which they detailed the complexities of associating the rarely collected males of Opadometa with conspecific females. They included photographs of a male Opadometa collected in the vicinity of the red and blue female, but warned that it would be premature to conclude that these are conspecific. Males of Opadometa are rare in collections and notoriously difficult to associate with conspecific females, which are more than twice their length and much heavier. Confirmation of male-female conspecificity, they state, should be accepted, \u201c\u2026only if the males and females are found in the same web, or better still, are seen copulating.\u201d (p. 260).The cover of Koh and Ming\u2019s 2014 field guide to the Spiders of Borneo was graced with a striking but at the time undescribed red and blue Opadometa species based on a female specimen from Sarawak, Malaysia. The male was not described, and Dzulhelime et al. echoed some of the sentiment expressed by Koh and Ming regarding the difficulties of associating male and female Opadometa.Opadometa. The female spider matched Dzulhelmi et al.\u2019s (2015) description of Opadometasarawakensis. A survey of orb web-building spiders near DGFC found no other Opadometa species. The students resolved, along with lecturers and field station scientific staff, to describe the male and provide additional data on the female, as well as data on the ecology and behavior of the species, and submit their results in the form of a manuscript before the end of the course. This is the second contribution to spider taxonomy and natural history to be produced in this way in Sabah, Malaysia, found a mature male at the margin of the web of a red and blue this way .Opadometa are currently cataloged , O.fastigata , O.kuchingensis Dzulhelmi & Suriyanti, 2015, and O.sarawakensis Dzulhelmi & Suriyanti, 2015 were established on the order of 100 years ago and have not been revisited (Four species (plus eight subspecies) of ataloged : O.gratti, 2015 . The typhis name ; Fig. 1.evisited ; their sO.grata have been collected together with females of O.fastigata . Examination of illustrations of the male pedipalp in the legacy taxonomic literature reveals a clue: the Cymbial Basal Process (CBP) of specimens from New Guinea and New Hebrides extend more or less retrolaterally from the cymbium before curving distally ; males from elsewhere have the CBP extend nearly posteriorly before curving Dzulhelmi & Suriyanti, in Type status:Other material. Occurrence: catalogNumber: DGFCW2018022300; recordedBy: Jeremy Miller and Christian Freund; individualCount: 2; sex: 1 male, 1 female; lifeStage: adult; Taxon: scientificName: Opadometasarawakensis Dzulhelmi & Suriyanti, 2015; Location: country: Malaysia; stateProvince: Sabah; locality: Danau Girang Field Centre trails; verbatimElevation: 23 m; decimalLatitude: 5.41619; decimalLongitude: 118.0426; Event: eventDate: 2018-02-23; Record Level: institutionID: Universiti Malaysia Sabah; collectionID: Institute for Tropical Biology and Conservation, Borneensis; institutionCode: UMS; collectionCode: BORN; basisOfRecord: PreservedSpecimenType status:Other material. Occurrence: catalogNumber: DGFCW2018022402; recordedBy: Jeremy Miller, Christian Freund, Liselotte Rambonnet, Lianne Koets, Natasha Zulaikha, and Jozsef Geml; individualCount: 1; sex: female; lifeStage: adult; Taxon: scientificName: Opadometasarawakensis Dzulhelmi & Suriyanti, 2015; Location: country: Malaysia; stateProvince: Sabah; locality: Danau Girang Field Centre trails; verbatimElevation: 23 m; decimalLatitude: 5.40999; decimalLongitude: 118.04204; Event: eventDate: 2018-02-24; Record Level: institutionID: Universiti Malaysia Sabah; collectionID: Institute for Tropical Biology and Conservation, Borneensis; institutionCode: UMS; collectionCode: BORN; basisOfRecord: PreservedSpecimenType status:Other material. Occurrence: catalogNumber: DGFCW2018022611; recordedBy: Jeremy Miller and Christian Freund; individualCount: 1; sex: female; lifeStage: adult; Taxon: scientificName: Opadometasarawakensis Dzulhelmi & Suriyanti, 2015; Location: country: Malaysia; stateProvince: Sabah; locality: Danau Girang Field Centre trails; verbatimElevation: 22 m; decimalLatitude: 5.41623; decimalLongitude: 118.04273; Event: eventDate: 2018-02-26; Record Level: institutionID: Universiti Malaysia Sabah; collectionID: Institute for Tropical Biology and Conservation, Borneensis; institutionCode: UMS; collectionCode: BORN; basisOfRecord: PreservedSpecimenMale: from Sabah, Malaysia (DGFCW2018022300). Prosoma uniform orange. Eight eyes in two rows, with the medians closer together than to the laterals; posterior median eyes oriented slightly toward the front; lateral eyes touching. Sternum dusky orange, darker posteriorly. Chelicerae orange, enlarged, diverging distally, armed in front and basolaterally with strong macrosetae; macrosetae absent from frontal-basal region Fig. a; femur 3Abdomen gray dorsally with silvery patches and an anteriodorsal dark spot, black posteriorly and ventrally with two posteriolateral and one ventral orange spot, with a small anteriolateral black spot and a larger posteriolateral black spot, which joins with the black ventral marking.Palpal trochanter, femur, and tibia very long Fig. c. Cymbiahere). Our observations of females from DGFC largely agree with the description in 44Female: For description and diagnosis of female, see Male (DGFCW2018022300): Total length 2.8; carapace length 1.4, width 0.9; abdomen length 1.4, width 0.9, height 0.9.Female (DGFCW2018022300): Total length 6.2; carapace length 3.6, width 2.4; abdomen length 5.0, width 2.8, height 2.7.Female (DGFCW2018022402): Total length 8.1; carapace length 3.4, width 2.7; abdomen length 7.6, width 4.2, height 4.2.Female (DGFCW2018022611): Total length 5.0; carapace length 3.5, width 2.1; abdomen length 4.4, width 2.6, height 2.6.O.grata; Fig. 1a, b, c, d, g) in which the CBP extends almost retrolaterally from the cymbium before curving distally; distinguished from males illustrated from further West in Southeast Asia (presumably true O.fastigata) by the length of the CBP, which is shorter and more gradually curved in O.sarawakensis of male palp projects initially posteriorly Fig. c, distinsis Fig. c than insis Fig. c, d, whisis Fig. d and O.yes Fig. c, which sis Fig. a, b, d a3.2-3.5; .Opadometasarawakensis is known from lowland dipterocarp forest in Bako National Park, Sarawak and Maliau Basin, Sabah, Malaysia ; no other Opadometa species were encountered. Opadometasarawakensis build open-hub webs with an inflection point so that the top half is more steeply inclined than the bottom half. The specific angles were quite different between the two webs measured have a female/male size ratio of 2.2, meaning the female is more than twice the total length of the male.Sexual size dimorphism in Opadometa, Opadometa they found in proximity to the red and blue Opadometa was conspecific. In light of the new data presented here, it appears that the male photographed in Koh and Ming (p. 260) is in fact O.sarawakensis.Given the troubled history of matching sexes in O.sarawakensis presented here is behavioral and faunistic (only one Opadometa species found in survey of orbweaving spiders). Another possible line of evidence would be DNA barcode sequences (Taxon Expeditions) is involved with field trials of the MinION, a portable DNA sequencer from Oxford Nanopore Technologies while O.fastigata is found further West, this should clear up some of the confusion regarding the distribution, sex matching, and anatomical features found in this genus.More work clearly needs to be done to sort out the distributions of the known"} +{"text": "Scientific Reports 10.1038/s41598-017-02596-1, published online 07 June 2017Correction to: In this Article, the author contributions section is incomplete:\u201cDr. Benito-Le\u00f3n (jbenitol67@gmail.com) collaborated in: (1) the conception, organization and execution of the research project; (2) the statistical analysis design, and; (3) and the writing of the manuscript first draft and the review and critique of the manuscript. Dr. Mato-Abad (virginia.mato@urjc.es) collaborated in: (1) the conception, organization of the research project; (2) the statistical analysis design; and (3) the review and critique of the manuscript. Dr. Louis collaborated in: (1) the conception, organization of the research project; and (2) the review and critique of the manuscript. Dr. Hern\u00e1ndez-Tamames (juanantonio.hernandez@ctb.upm.es) collaborated in: (1) the conception, organization of the research project; and (2) the review and critique of the manuscript. Dr. \u00c1lvarez-Linera collaborated in: (1) the conception, organization of the research project; and (2) the review and critique of the manuscript. Dr. Domingo-Santos (gela_yo@hotmail.com) collaborated in: (1) the conception, organization of the research project; and (2) the review and critique of the manuscript. Dr. Luis Collado (lcollado@med.ucm.es) in: (1) the review and critique of the manuscript. Dr. Romero (juanpa5@hotmail.com) collaborated in: (1) the conception, organization of the research project; and (2) the review and critique of the manuscript.\u201dshould read:\u201cDr. Benito-Le\u00f3n (jbenitol67@gmail.com) collaborated in: (1) the conception, organization and execution of the research project; (2) the statistical analysis design, and; (3) and the writing of the manuscript first draft and the review and critique of the manuscript. Dr. Mato-Abad (virginia.mato@urjc.es) collaborated in: (1) the conception, organization of the research project; (2) the statistical analysis design; and (3) the review and critique of the manuscript. Dr. Louis collaborated in: (1) the conception, organization of the research project; and (2) the review and critique of the manuscript. Dr. Hern\u00e1ndez-Tamames (juanantonio.hernandez@ctb.upm.es) collaborated in: (1) the conception, organization of the research project; and (2) the review and critique of the manuscript. Dr. \u00c1lvarez-Linera collaborated in: (1) the conception, organization of the research project; and (2) the review and critique of the manuscript. Dr. Bermejo-Pareja (fbermejop2013@yahoo.es) collaborated in: (1) the conception, organization of the research project; and (2) the review and critique of the manuscript. Dr. Domingo-Santos (gela_yo@hotmail.com) collaborated in: (1) the conception, organization of the research project; and (2) the review and critique of the manuscript. Dr. Luis Collado (lcollado@med.ucm.es) in: (1) the review and critique of the manuscript. Dr. Romero (juanpa5@hotmail.com) collaborated in: (1) the conception, organization of the research project; and (2) the review and critique of the manuscript.\u201d"} +{"text": "Correction to:Horticulture Research (2016) 3, 16017; doi:10.1038/hortres.2016.17; Published online 04 May 2016Since the publication of this article, the authors have noticed an error in In addition, the author Xiuxin Deng should be removed from the author list.The authors would like to apologize for this error."} +{"text": "Three references that were cited in fifth sentence of the fifth paragraph in the Discussion were omitted from the Reference section. The sentence is: The extensive literature on \u201cbargaining\u201d in economics was also more focused on the case in which players are in a symmetric position, and usually did not investigate proportional bargaining solutions.The references are:RAND Journal of Economics, 17(2), 176\u2013188. http://doi.org/10.2307/2555382Binmore, K., Rubinstein, A., & Wolinsky, A. (1986). The Nash bargaining solution in economic modelling. Rationality and Society, 23, 151\u2013173. Retrieved from http://rss.sagepub.com/content/10/3/275.shortBinmore, K. (1998). The evolution of fairness norms. Philosophy of Science, 67(3), 490\u2013516. http://doi.org/10.1086/392792Alexander, J. M. (2000). Evolutionary Explanations of Distributive Justice. A reference that was cited in the sixth sentence of the fifth paragraph in the Discussion was omitted from the References section. The sentence is: An exception is the work by Kalai (1977) , who shows that individuals will compromise in different bargaining situations so as to keep their proportions of utility gains fixed.Econometrica, 45(7), 1623\u20131630. http://doi.org/10.2307/1913954The reference is: Kalai, E. (1977). Proportional Solutions to Bargaining Situations: Interpersonal Utility Comparisons."} +{"text": "Scientific Data 3:160030 doi:10.1038/sdata.2016.30 (2016); Published 10 May 2016; Updated 31 Jan 2017The original version of this Data Descriptor contained a typographical error in the spelling of the author Douglas B. Rusch, which was incorrectly given as Douglas Rush. This has now been corrected in the PDF and HTML versions of the Data Descriptor."} +{"text": "A reference is omitted from the first sentence of the second paragraph under the heading \u201cPpr Localizes to Mitochondria and Its Loss Causes a Progressive Defect in ERGs\u201d in the Results section. The sentence should read:CG14786/Lrpprc2 (ppr) , which is required for the coordination of mitochondrial translation .The causative mutations of the five alleles of this complementation group were mapped to Drosophila melanogaster LRPPRC2 is involved in coordination of mitochondrial translation. Nucleic Acids Res. Dec 16;42(22):13920\u201338. doi: 10.1093/nar/gku1132. Epub 2014 Nov 26.The reference is: Baggio F, Bratic A, Mourier A, Kauppila TE, Tain LS, Kukat C, et al. (2014)"} +{"text": "Across all these years, the Conference was chaired by Prof. Nikolay A. Kolchanov and Prof. Ralf Hofest\u00e4dt . In 2016, the multi-conference held parallel events and symposia on systems biology and biomedicine (SBioMed-2016) (http://conf.bionet.nsc.ru/ishg2016/en/), cognitive sciences (http://physiol.ru/csgb2016/), and mathematical modeling in biology (MM-HPC-BBB-2016) (http://conf.bionet.nsc.ru/mm-hpc-bbb-2016/en/). Since 2014, the BGRS Program Committee has collaborated with BioMed Central on full-text thematic issues reflecting the main science achievements of the conference series in past years. Recently BioMed Central had published several special issues based on best materials presented at the conference in BMC Genetics [http://bmcgenet.biomedcentral.com/articles/supplements/volume-16-supplement-1), BMC Genomics , BMC Evolutionary Biology , and BMC Systems biology .This special issue continues the series of BioMed Central special post-conference journal issues . All these issues collate the papers presented at BGRS\\SB-2016, 10th International Conference \u201cBioinformatics of Genome Regulation and Structure\\Systems Biology\u201d which took place at August 29 - September 2, 2016 in Novosibirsk, Russia. The BGRS conference series started in 1998 in Novosibirsk Akademgorodok in the tumour suppressor APC gene. It was shown that both putative promoters of APC (1A and 1B) drive transcription in an in vitro reporter experiment, many SNPs are functionally relevant and allele G of rs79896135 may be associated with the predisposition to colorectal cancer [The article by Kudryavtseva et al. highlights molecular mechanism of hexokinases function in tumorigenesis of human colorectal cancer and melanoma. The authors studied the effect of silencing hexokinase genes in colorectal cancer and melanoma cells using short hairpin RNA (shRNA) lentiviral vectors suggesting the HK1 and HK2 genes as the key therapeutic targets for reducing aerobic glycolysis .Two papers by Korbolina et al. and RyazThe paper by Elena Korbolina and colleagues describeThe ISIAH rat strain was developed by selection for high systolic arterial blood pressure (SABP) induced by restraint stress. Earlier studies showed that the ISIAH rats may be considered as a model of the human stress sensitive hypertensive disease with predominant involvement of the neuroendocrine hypothalamic-pituitary-adrenal (HPA) and sympathoadrenal systems in the pathogenesis of the hypertensive state .The studDendrolimus (Lepidoptera: Lasiocampidae), which are among the major pests of coniferous forests worldwide. The study clarifies the taxonomy of the moths of this genus in Eurasia using mitochondrial markers.The paper by A. Kononov et al. highlighFinally, the work by P. Drozdova et al. discussehttp://www.bionet.nsc.ru/files/2016/conference/BGRS2016.pdf.All BGRS\\SB-2016 Proceedings including \u201cBioinformatics and Systems Biology of Plants\u201d section are available at the multi-conference web-site: http://www.worldscientific.com/toc/jbcb/13/01) [http://vavilov.elpub.ru/jour/issue/view/15/showToc) (in Russian).Additionally, special issues on bioinformatics were published at the Journal of Bioinformatics and Computational Biology (http://conf.bionet.nsc.ru/sbb2016/en/) and Open Russian-German workshop on bioinformatics network \u201cSystems computational biology\u201d.As usual, BGRS/SB-2016 was accompanied by a number of satellite events, including already traditional Young Scientists School \u201cSystems Biology and Bioinformatics\u201d (SBB-2016) ("} +{"text": "The publisher apologizes for the error. The correct citation should be as follows: Gradidge PJ, Norris SA, Jaff NG, Crowther NJ (2016) Metabolic and Body Composition Risk Factors Associated with Metabolic Syndrome in a Cohort of Women with a High Prevalence of Cardiometabolic Disease. PLoS ONE 11(9): e0162247. doi:"} +{"text": "In the Author Contributions section, it should be noted that Norma-Rashid Yusoff also contributed to Supervision.The Funding section is incomplete. The complete funding information is: This work was supported by Malaysian Forest Research and Development Board (Grant number: GPP-0609-FA-02) received by CK; Postgraduate Research Fund from University of Malaya (Grant number: PV131/2012A) received by NY; Malaysian Economic Planning Unit and Ministry of Natural Resources and Environment, Malaysia. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Trogonoptera brookiana albescens). PLoS ONE 12(12): e0189450. https://doi.org/10.1371/journal.pone.0189450The abbreviation for the third author is incorrect in the citation. The correct citation is: Phon C-K, Kirton LG, Norma-Rashid, Y (2017) Monitoring butterflies using counts of puddling males: A case study of the Rajah Brooke\u2019s Birdwing (10.5176/2251-3353_GEOS14.37.There is an error in the URL link for Reference 9. The correct reference is: Baioumy HM, Nawawi M, Wagner K, Arifin MH. Geological setting and origin of non-volcanic hot springs in West Malaysia. GEOS 2014: Proceedings of the 3rd International Conference on Geological and Earth Sciences; Singapore. 2014. pp. 14\u201318. doi:The publisher apologizes for these errors."} +{"text": "The supporting information tables incorrectly include tracked changes that make them illegible. Please view the correct S1 Tabler-values obtained with Polar Plot analysis in individual rats and in groups of intact, saline and Riluzole treated animals. The values of SEM ranged from 0.64 to 1.99%. Abbreviations: L/Co-left/control, R/SNC-right-muscle with SNC, Sol-soleus, EDL-extensor digitorum longus. Abbreviations for statistical significance vs intact rats: *\u2014p < 0.001.The table contains mean (\u00b1 circular SD) of phase shifts of intralimb (L/Co Sol\u2014L/Co EDL and R/SNC Sol\u2014R/SNC EDL) and interlimb (R/SNC Sol\u2014L/Co Sol and R/SNC EDL\u2014L/Co EDL) coordination and (DOC)Click here for additional data file.S2 TableThe table contains mean (\u00b1SD) of cycle durations established based on the left and right Sol and EDL muscles, on control muscles and muscles with SNC in individual rats and in groups of intact, saline and Riluzole treated animals. The values of SEM ranged from 1.78 to 3.20%. Abbreviations: L/Co-left/control, R/SNC-right/muscle with SNC.(DOC)Click here for additional data file.S3 Tablep for significance of intercepts and correlation coefficients r in individual intact, saline and Riluzole treated animals. The values of p or significance of slopes and correlation coefficients were < 0.001 in all instances. Abbreviations: L/Co-left/control, R/SNC-right/muscle with SNC.The table contains slopes and intercepts of regression with the values of (DOC)Click here for additional data file.S4 Tablep for their significance as well as correlation coefficients r in individual intact, saline and Riluzole treated animals. Abbreviations: L/Co-left/control, R/SNC-right/muscle with SNC.The table contains slopes and intercepts of regression with the values of (DOC)Click here for additional data file.S5 Tablep < 0.011.The table contains mean (\u00b1SD) cycle duration, burst duration and duty factor in individual rats and in groups of intact, saline and Riluzole treated animals. The values of SEM ranged from 0.71 to 1.67%. Abbreviations: L/Co-left/control, R/SNC-right/muscle with SNC. Abbreviations for statistical significance vs intact rats: *\u2014(DOC)Click here for additional data file.S6 Tablep < 0.001.The table contains mean (\u00b1SD) cycle duration, burst duration and duty factor in individual rats and in groups of intact, saline and Riluzole treated animals. The values of SEM ranged from 0.57 to 3.96%. Abbreviations: L/Co-left/control, R/SNC-right/muscle with SNC. Abbreviations for statistical significance vs intact rats: *\u2014(DOC)Click here for additional data file.S7 Tabler with corresponding values of p for significance, for the relationship between the duty factor of burst of EMG activity of right muscle/muscle with SNC and the duty factor of burst of EMG activity of left muscle/control muscle for the Sol and EDL muscles in individual intact rats, saline and Riluzole treated animals. In addition the table contains the values of common correlation coefficients rw with the values of pw obtained with a test for the heterogeneity of correlation coefficients in the respective groups. Abbreviations: DF-duty factor, Sol-soleus, EDL-extensor digitorum longus.The table contains correlation coefficients (DOC)Click here for additional data file.S8 Tabler with the values of p obtained in individual rats and in group of intact rats for the left muscles as well as in individual rats and in groups of saline and Riluzole treated animals for the control muscles. The values of SEM ranged from 1.19 to 3.83%. Abbreviations for statistical significance vs intact rats: *\u2014p < 0.001.The table contains mean (\u00b1SD) of interval durations, predicted durations, slopes and intercepts of regressions as well as correlation coefficients (DOC)Click here for additional data file.S9 Tabler with the values of p obtained in individual rats and in group of intact rats for the right muscles as well as in individual rats and in groups of saline and Riluzole treated animals for muscles with SNC. The values of SEM ranged from 1.19 to 3.83%. Abbreviations for statistical significance vs intact rats: *\u2014p < 0.001.The table contains mean (\u00b1SD) of interval durations, predicted durations, slopes and intercepts of regressions as well as correlation coefficients (DOC)Click here for additional data file."} +{"text": "Owing to errors made by SAGE, the following article contains errors.Christoph Witzel (September-October 2016). An Easy Way to Show Memory Color Effects.10.1177/2041669516663751).i-Perception, 7(5), 1\u201311. (DOI: SAGE apologises to the author and to the readers. The following corrections apply:These corrections will be included in all subsequent versions of the article online."} +{"text": "Scientific Data 4:170179 doi: 10.1038/sdata.2017.179 (2017); Published 19 December 2017; Updated: 23 January 2018.In both the HTML and PDF versions of this Data Descriptor, the author name Jason Flannick was incorrectly listed as Flannick Jason."} +{"text": "Abstractspecies distribution modelling (SDM) and for model testing in a poorly documented marine region.The present dataset provides a case study for Echinodermata: Echinoidea) distribution. Echinoids were collected during oceanographic campaigns led around the Kerguelen Plateau since 1872. In addition to the identification of collection specimens from historical cruises, original data from the recent campaigns POKER II (2010) and PROTEKER 2 to 4 (2013-2015) are also provided. In total, five families, ten genera, and 12 echinoid species are recorded in the region of the Kerguelen Plateau.The dataset includes spatially-explicit data for echinoid .Future projections are provided for several parameters: they were modified from the Bio-ORACLE database . They arPageBreak Project title: Temporal, spatial, and sampling heterogeneities in species distribution modelling. A case study for the data-poor area of the Kerguelen Plateau.Personnel: Charl\u00e8ne Guillaumot, Alexis Martin, Salom\u00e9 Fabri-Ruiz, Marc El\u00e9aume, Thomas Sauc\u00e8deFunding: This study is part of a project funded by CNRS laboratory UMR6282 Biogeosciences and by the vERSO program . This is contribution no.14 to the vERSO project (www.versoproject.be), funded by the Belgian Science Policy Office . This is a contribution to the POKER program and the (Institut polaire fran\u00e7ais Paul-Emile Victor)IPEV program 1044 PROTEKER.6 km2 CCAMLR and by the (French Southern and Antarctic Lands)TAAF in the French (Exclusive Economic Zone)EEZ with scientific support from the Mus\u00e9um national d\u2019Histoire naturelle of Paris since 1978 Marine Protected Area: one of the world\u2019s largest MPA with an area of 65,000 km2 led in nearshore areas of the Kerguelen Islands. The spatial extent of the dataset was based on the bathymetric range of echinoids for species distribution modelling to be performed with limited extrapolations.In addition to the study of collection specimens sampled during historical cruises and identified at species level, the present work also provides original data collected during the recent oceanographic campaign POKER II (2010) and during three field summer campaigns of the Our project aimed at improving the robustness of existing modelling approaches in the case of areas for which only poor and heterogeneous biodiversity data are available, a situation prevailing in the region of the Kerguelen Plateau, and generally in the Southern Ocean .Data compilation from various sources implies temporal heterogeneities that may constitute a critical point when building species distribution models . SpatialOccurrence data were compiled from many oceanographic campaigns led over a long time-period starting with the Challenger Expedition in 1872 and ending with the recent PROTEKER campaigns that took place between 2013 and 2015 Table a. SpecimPageBreakPageBreakOccurrences are presence-only data for which different sampling tools, protocols, and strategies were used. Moreover, the study area was unevenly investigated, sampling effort being stronger in the northern than in the southern part of the Plateau Figure . AccordiThe environmental descriptors provided in the dataset were compiled from different sources and raw depth values measured in the field and mean winter (July to September) surface and seafloor temperatures and salinities) are available for the following six decades: 1955 to 1964, 1965 to 1974, 1975 to 1984, 1985 to 1994, 1995 to 2004, and 2005 to 2014.th IPCC report (2007). The modelled data correspond to the extrapolated means for two decades: 2087-2096 (here referred to as 2100) and 2187-2196 (here referred to as 2200) .All the environmental descriptors and metadata sources are detailed in the data catalog Table and dataSpecimens sampled during POKER II and PROTEKER 2, 3 and 4 campaigns were all identified by T. Sauc\u00e8de at the species level. Identifications and taxonomic accuracies are based on The final compiled dataset was checked for consistency using the WoRMS database in orderEnvironmental data relies on different sources as reported in Table Echinoidea (Echinodermata) occurring on the Kerguelen Plateau.The present dataset focuses on all species of the class PageBreakPageBreakPageBreakPageBreakEchinoids are common species of benthic communities in the Southern Ocean and on the Kerguelen Plateau . They arCtenocidarisnutrix is considered a Vulnerable Marine Ecosystems (VME) indicator species by (Commission for the Conservation of Antarctic Marine Living Resources)CCAMLR and is widely distributed on the Kerguelen Plateau.Echinoid studies take part in conservation issues. Echinoidea includes five families, ten genera, and 12 species. Species distribution is shown in Figure On the Kerguelen Plateau, the Class Phylum: EchinodermataClass: EchinoideaOrder: Camarodonta, Cidaroida, Holasteroida, SpatangoidaFamily: Ctenocidarinae, Echinidae, Plexechinidae, Pourtalesiidae, SchizasteridaeGenus: Abatus, Aporocidaris, Brisaster, Ctenocidaris, Dermechinus, Plexechinus, Pourtalesia, Rhynchocidaris, Sterechinus, TripylusSpecies: Abatuscordatus, Aporocidarismilleri, Brisasterantarcticus, Ctenocidarisnutrix, Dermechinushorridus, Plexechinussulcatus, Pourtalesiahispida, Pourtalesiadebilis, Rhynchocidaristriplopora, Sterechinusdiadema, Sterechinusneumayeri, TripylusabatoidesGeneral spatial coverage: the Kerguelen Plateau, Southern OceanCoordinates: -46\u00b0S and -56\u00b0S; +63\u00b0E and +81\u00b0ETemporal coverage: 1872\u20132015Echinoid occurrences available on the Kerguelen Plateau. Data from 1872 to 2015 collected with different sampling strategies and objectives, during different campaigns.Object name: Echinoids_Kerguelen_Plateau_1872_2015PageBreakCharacter encoding: x-MacRomanFormat name: Darwin Core Archive FormatFormat version: 3.0Distribution: http://ipt.biodiversity.aq/resource.do?r=echinoids_kerguelen_plateau_18- 72_2015Publication date of data: 12/07/2016Language: EnglishMetadata language: EnglishDate of metadata creation: 12/07/2016Hierarchy level: DatasetPageBreakand seafloor temperature, salinity and their respective amplitude data are available on the time coverage 1955-2012 and over six decades: 1955 to 1964, 1965 to 1974, 1975 to 1984, 1985 to 1994 and 1995 to 2004, and 2005 to 2012.Environmental variables in the region of the Kerguelen Plateau compiled from different sources and provided in the ascii raster format . Mean suth report).Future projections are provided for several parameters: they were modified after the Bio-ORACLE database . They arObject name: Environmental_Kerguelen_Plateau_1955_2012Format name: RasterFormat version: 1.0Distribution: https://data.aad.gov.au/metadata/records/Environmental_Kerguelen_Plateau_1955_2012doi: 10.4225/15/578ED5A08050FPublication date of data: 16/07/2016Language: EnglishMetadata language: EnglishDate of metadata creation: 16/07/2016Hierarchy level: Dataset"} +{"text": "The name and citation appear correctly in the PDF version.The fifth author\u2019s name is spelled incorrectly in the online version. The correct name is J. Frank Wharam. The correct citation is: Lopez Bernal JA, Lu CY, Gasparrini A, Cummins S, Wharam JF, Soumerai SB (2017) Association between the 2012 Health and Social Care Act and specialist visits and hospitalisations in England: A controlled interrupted time series analysis. PLoS Med 14(11): e1002427."} +{"text": "There are multiple errors in the manuscript.There is an error in reference 65. The correct reference is: \u201cSpeir M, Lawlor KE, Glaser SP, Abraham G, Chow S, Vogrin A, et al. Nat Microbiol. 2016 Feb 24;1:15034. doi: 10.1038/nmicrobiol.2015.34.\u201dThe following information is missing from the Funding section: \u201cResearch reported in this publication was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103652.\u201d"} +{"text": "Correction to:European Journal of Clinical Nutrition (2017); 71 (1); 51-55; doi: 10.1038/ejcn.2016.166; published online 14th September 2016Since the publication of the article, the authors have noticed an error in The authors apologise for any inconvenience caused by this error."} +{"text": "Nature Communications7: Article number: 13045; DOI: 10.1038/ncomms13045 (2016); Published: 10102016; Updated: 06142017This Article contains an error in Fig. 8a, for which we apologize. In Fig. 8a, the control 3 image for p-ERK staining was inadvertently duplicated from the control 1 image of p-ERK staining. The correct version of this figure appears below as"} +{"text": "In the Funding section, the grant number from EPSRC MOTION is listed incorrectly. The correct grant number is: EP/N03211X/2.There is an error in affiliation 4 for author Thrishantha Nanayakkara. Affiliation 4 should be: Dyson School of Design Engineering, Imperial College London, South Kensington Campus, London, United Kingdom."} +{"text": "CDC is assisting ministries of health and working with other organizations to control and end the ongoing outbreak of Ebola virus disease (Ebola) in West Africa . The updAccording to the latest World Health Organization update as of October 22, 2014 . The higThe geographic distribution of the number of Ebola cases reported during September 28\u2013October 18 changed from the distribution of cases reported during August 31\u2013September 23 .The map of the cumulative incidence of Ebola, as of October 18, indicates that the highest incidence rate was reported by two districts in Guinea (Gu\u00e9ck\u00e9dou and Macenta), five districts in Liberia , and four districts in Sierra Leone .http://www.cdc.gov/vhf/ebola/outbreaks/guinea/index.html. The most up-to-date clinical guidelines on the 2014 Ebola outbreak in West Africa are available at http://www.cdc.gov/vhf/ebola/hcp/index.html.The latest updates on the 2014 Ebola outbreak in West Africa, including case counts, are available at"} +{"text": "The publisher apologizes for the error.The fourth author\u2019s name is spelled incorrectly. The correct name is: Leila Sissani. The sixth author\u2019s name is spelled incorrectly. The correct name is: Saverio Stranges. The correct citation is: Sauvageot N, Leite S, Alkerwi A, Sissani L, Zannad F, Stranges S, et al. (2016) Association of Empirically Derived Dietary Patterns with Cardiovascular Risk Factors: A Comparison of PCA and RRR Methods. PLoS ONE 11(8): e0161298. doi:"} +{"text": "Correction to: British Journal of Cancer (2009) 101, 916\u2013923; doi:10.1038/sj.bjc.6605262After publication of the above paper in Volume 101, Number 6, the authors realised that they had intended to include an Acknowledgements paragraph in the article. The paragraph should read as follows:"} +{"text": "After the publication of this work , we becaIn the method section, see following paragraph: \"In the present study all assessments of hip abduction, hip external rotation, popliteal angle, knee extension and dorsiflexion of the foot with knee flexion from the start 1994 until 1 January 2007 were included (Table 2).\"In Table 2. Goniometer positioning and standardization procedure for all five joint angles, Extremity position Foot dorsiflexion. Supine. Knee in flexion.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1741-7015/8/49/prepub"} +{"text": "Correction to: British Journal of Cancer (2002) 87, 630\u2013634. doi:10.1038/sj.bjc.6600511 Unfortunately due to a typesetting error, Figure 2The correct version is reprinted below:The publisher would like to apologise for any inconvenience this may have caused."} +{"text": "Correction to: British Journal of Cancer (2009) 100, 1926\u20131936; doi:10.1038/sj.bjc.6605072In reference to"} +{"text": "A focal H5N1 outbreak in poultry was reported from Manipur, a north-eastern state, of India, in 2007. The aim of this study was to genetically characterize the Manipur isolate to understand the relationship with other H5N1 isolates and to trace the possible source of introduction of the virus into the country.Characterization of the complete genome revealed that the virus belonged to clade 2.2. It was distinctly different from viruses of the three EMA sublineages of clade 2.2 but related to isolates from wild migratory waterfowl from Russia, China and Mongolia. The HA gene, had the cleavage site GERRRRKR, earlier reported in whooper swan isolates from Mongolia in 2005. A stop codon at position 29 in the PB1-F2 protein could have implications on the replication efficiency. The acquisition of polymorphisms as seen in recent isolates of 2005\u201307 from distinct geographical regions suggests the possibility of transportation of H5N1 viruses through migratory birds.Considering that all eight genes of the earlier Indian isolates belonged to the EMA3 sublineage and similar strains have not been reported from neighbouring countries of the subcontinent, it appears that the virus may have been introduced independently. Highly pathogenic avian influenza (HPAI) A \u2013 H5N1 viruses have now appeared in about 60 countries causing devastating outbreaks in poultry with continued capacity to impact humans . The HonThe first outbreak of the H5N1 virus in India was reported from Maharashtra in January 2006 . Seven eThe aim of this study was to genetically characterize the Manipur isolate of 2007 to understand the relationship with other H5N1 isolates and to trace the possible source of introduction of the virus into the country.The state of Manipur (latitude 23\u00b083'N \u2013 25\u00b068'N and longitude 93\u00b003'E \u2013 94\u00b078'E) is known for some animal sanctuaries that are home to many exotic flora and fauna Figure . A largeSix clinical samples from different organs of a sick bird were received from Manipur. Specimens were processed for virus isolation in specific-pathogen-free (SPF) embryonated chicken eggs and Madin Darby Canine Kidney (MDCK) cell lines as described earlier . InoculaHemagglutination (HA) and Hemagglutination inhibition (HAI) tests were performed as described by Kendal et al. . Horse RRNA was extracted using QIAamp Viral RNA Minikit following manufacturers instruction. One-Step reverse transcription-PCR (RT-PCR) was performed using the QIAGEN one-step RT-PCR kit and WHO recommended diagnostic primer sets specific for influenza A HA (H5) and NA (N1) genes . RNA isoRNA isolated from the specimens was reverse transcribed as mentioned earlier . cDNA waFor phylogenetic analysis, representative sequences of the H5N1 viruses belonging to the Z genotype were selected from the GenBank based on sequence identity and geographical representation. In this process of selection, the sequences whose whole genome was available were preferred. Phylogenetic analysis was performed using the Bayesian approach for tree construction as implemented in Mr Bayes 3.2 . The GTRFJ719831\u2013FJ719838. The percent nucleotide identity (PNI) and percent amino acid identity (PAI) values were calculated as pairwise p-distances, for a dataset of about 80 representative sequences in each gene. For clarity, limited representative sequences are shown in the phylogenetic trees virus in HA and HAI.Samples tested in one step RT-PCR showed amplification of influenza A, H5 and N1 gene specific bands. A 219 base pair band appeared showing presence of H5 and 668 base pair for the presence of N1. Real Time RT-PCR analysis showed the presence of Avian influenza (H5N1) in all the specimens (Data not shown).Phylogenetic analysis of 41 whole genomes, all the eight gene segments concatenated, showed that the Manipur isolate was unique in the clade 2.2, as it did not cluster with the majority of the isolates in the EMA sublineages 1 to 3 with the A/Gf/Shantou/1341/06 isolate, followed by the A/Dk/Novosibirsk/56/05 and the A/Ck/Omsk/14/05 isolates with 98.99 PNI and 9 amino acid differences in all the three cases. It showed closest amino acid identity of 98.95 (8 differences) with several isolates including A/Ck/Egypt/22531/06, A/Sw/Slovenia/760/06, A/Ck/Gaza/714/06, A/Pg/Denmark/6632/06, A/Tk/Turkey/1/05 and A/Co/Croatia/1/05. In case of the PB1 gene, the Manipur isolate was closest in PNI (98.95) to the A/Ck/Kurgan/3/05 isolate with also the highest amino acid identity . The PA gene of the Manipur isolate had closest PNI (98.83) with the A/BHGs/Qinghai/59/05 isolate, followed by Azerbaijan/002115/06 with 7 amino acid differences. The closest PAI (99.16) with 6 amino acid differences was with the A/Ck/India/NIV33487/06 isolate (PNI 98.65). The Novosibirsk/05, Liaoning/05 and Mongolia/06 and Iran/06 showed PAI of 98.89 (7 aa differences) but higher PNI (98.7).The NP gene showed closest PNI with the A/Gs/Hungary/3413/07 isolate (98.9) and 1 amino acid difference. The closest PAI (99.79) also amounting to a single amino acid difference was with several isolates including the A/Tk/England/250/07, A/Gs/Qinghai/F/06, A/Md/Bavaria/1/06, A/Turkey/15/06, A/Krasnoozerskoe/627/05, A/Omsk/14/05, A/Dk/Novosibirsk/02/05, A/Ck/Kurgan/3/05, and A/Ws/Mongolia/3/05. The M gene shared the highest PNI (98.81) with the A/Dk/Novosibirsk/56/05 isolate. The NS gene had highest PNI with A/Dk/Novosibirsk/05 and A/Suzdalka/10/05 (98.96) followed by the A/BHGs/Qinghai/F/06 isolate (98.9). Again, in all these genes the Manipur isolate was distinct from the others and also did not cluster with any of the EMA sublineages.AB233320\u2013AB233322). Subsequently, the same pattern was noted in several isolates of 2007 from Egypt and Nigeria (EU148396). Another non synonymous mutation, S155N, at the N154 glycosylation site, observed in the Manipur isolate was also noted in the 2005 isolates of Novosibirsk, Qinghai, Tambov, Crimea, Omsk, Suzdalka, and Liaoning and majority of the EMA1 isolates. D54N in the Manipur isolate was shared with the A/Ck/Egypt/1079NAMRU3/07 isolate and the A/Ws/Mongolia/4/05 isolate. One unique mutation L297F in the Manipur isolate was not noted in any of the clade 2.2 isolates but was observed in A/Dk/HongKong/ww381/2000. T513A mutation was shared with A/Ck/Krasnador/199/06. Among synonymous nucleotide substitutions in the Manipur isolate, G42A, G705A, T861C, 983G, A1632G were shared with the Egypt/07 and/or Nigeria/07 isolates; T78C with A/Tk/SaudiArabia/67326/07; T573C with A/Gf/Shantou/1341/06, A/Pm/Liaoning/7/05 and A/Tk/CzechRep/10309-3/07; C1335T with A/Dk/Novosibirsk/56/05 and A/Ck/Sudan/21159/06.In the HA gene, among amino acid mutations, the Manipur isolate had a substitution K328R . This corresponds to the novel HA cleavage site, GERRRRKR that was originally found in three whooper swan isolates of Mongolia in 2005 viruses isolated in Europe, the Middle-Eastern region and Africa beyond 2005 . A minorThe molecular characterization of the viral proteins of the Manipur isolate was carried out for pathogenicity markers and sensitivity to antivirals. The HA of the Manipur isolate had the novel cleavage site (GERRRRKR) that was first reported in three whooper swan isolates in Mongolia, 2005 and also in the recent Nigeria/07 and Egypt/07 isolates. Though variations in the cleavage site such as RERRRKKR, RERRKKR and RERRRR have been linked to human H5N1 cases , no suchThe phylogenetic characterization of the Manipur isolate demonstrated that the virus isolated in 2007 in India was distinctly different from the viruses of the three EMA sublineages. Considering that all the eight genes of the 2006 isolates of India belonged to the EMA3 sublineage, with several mutations observed between the two strains, the possibility of local evolution can be excluded. Genetic analysis of the Manipur 2007 isolate clearly indicates a second introduction of the H5N1 virus in India. Among the other isolates that have been placed outside the EMA groups, the Manipur isolate was distinct and unique. It was related to viruses isolated from wild migratory waterfowl from Russia , China and Mongolia. A recent report stated tOverall, our findings suggest that the Manipur 2007 virus isolate is a unique variant and not related to the 2006 Indian isolate. The introduction of such a virus, either directly or indirectly, into India calls for improved surveillance in the country and subcontinent. The appearance of such variants is also serious concern for the emergence of even more highly pathogenic strains and a pandemic threat.BHGs: Bar headed goose; Ck: Chicken; Co: Cygnus olor; Dk: Duck; Gf: Guinea fowl; Gs: Goose; Md: Mallard; Pg: Peregrine; PgFc: Peregrine falcon; Pm: Piedmagpie; Sw: swan; Tk: Turkey; Ws: Whooper swan.The authors declare that they have no competing interests.ACM, AKC and SDP conceived and designed the experiments. AKC, SDP, BP, SR, SK and SSK performed the experiments, SSC, SMJ and AKC planned and performed the bioinformatics studies, SSC, AKC, SMJ and ACM analyzed the data, SSC, AKC and ACM wrote the paper. All authors read and approved the final manuscript."} +{"text": "Correction to: British Journal of Cancer (2006) 94, 1079\u20131085. doi:10.1038/sj.bjc.6603031Owing to an author error, the data presented in"} +{"text": "This report documents the additions and revisions to the nomenclature of HLA specificities following the principles established in previous reports methodology, where both alleles of a heterozygous individual are sequenced together, is insufficient evidence for assignment of an official designation.Sequence derived solely from the primers used to amplify an allele must not be included in the submitted sequence.Where possible, a novel sequence should be confirmed by typing of genomic DNA using a method such as PCR-SSOP or PCR-SSP. Where a new sequence contains either a novel mutation or a previously unseen combination of nucleotides (sequence motif), this must be confirmed by a DNA typing technique. This may require the use of newly designed probes or primers to cover the new mutation; these reagents should also be described.An accession number in a databank should have been obtained. Sequences may be submitted to the databases online at the following addresses:www.ebi.ac.uk/Submissions/index.htmlEMBL: www.ncbi.nlm.nih.gov/Genbank/submit.htmlGenBank: www.ddbj.nig.ac.jp/sub-e.htmlDDBJ: Full-length sequences are preferable though not essential; the minimum requirements are complete exons 2 and 3 for an HLA class I sequence and complete exon 2 for an HLA class II sequence.Where a novel sequence differs only within an intron or other non-coding part of the gene, a full-length sequence must be obtained, which covers all coding and non-coding regions. In the absence of a full-length genomic sequence from the most closely related allele that is identical in its exon sequence, it may be required that this also be sequenced and submitted before a name can be assigned to the novel sequence.Where possible, a paper in which the new sequence is described should be submitted for publication. Copies of draft publications can be submitted to the database by email or FAX.Sequences derived solely from tumour material will not be considered for nomenclature.HLA-A, -B and -DRB1 genes should be submitted for the material in which a novel allele has been defined. In addition the sample should have been characterised for the second allele at the locus of interest in a heterozygous individual.The complete HLA type for the DNA or other material, preferably cell lines, should, wherever possible, be made available in a publicly accessible repository or alternatively, at least in the originating laboratory. The WHO Nomenclature Committee will maintain documentation on this material.www.ebi.ac.uk/imgt/hla/subs/submit.html. Researchers are expected to complete a questionnaire relating to the sequence and provide a comparison of their new sequence with known related alleles. If the sequence cannot be submitted using the online web tools, researchers should contact hla@alleles.org directly for details of alternative submission methods.Submission of a sequence to the WHO Nomenclature Committee should be performed using the online submission tool available at Although at present it is only a recommendation that full-length sequences of the coding region of novel alleles be submitted it was widely felt that in the future this should become a requirement for submission. Such requirement would remove many of the currently encountered ambiguities in the assignment of names to alleles for which partial sequences have been submitted and should not be burdensome as sequencing techniques have improved substantially since the submission conditions were first devised. In cases where novel mutations or polymorphisms are detected in non-coding regions of the gene, it will be a requirement that full-length sequences be submitted of both the novel allele and its most closely related allele.It should be noted with some caution that cells from which only partial sequences have been obtained may later be shown to have different or novel alleles when further sequencing is performed. This is of particular importance in cases where partial sequences of what appears to be the same allele have been obtained from several different cells. In such cases, all cells studied have been listed in this report.Current practice is that official designations will be promptly assigned to newly described alleles in periods between Nomenclature Committee meetings, provided that the submitted data and its accompanying description meet the criteria outlined above. A list of the newly reported alleles is published each month in nomenclature updates in the journals Tissue Antigens, Human Immunology and the International Journal of Immunogenetics. The listing of references to new sequences does not imply priority of publication. The use of numbers or names for alleles, genes or specificities which pre-empt assignment of official designations by the Nomenclature Committee is strongly discouraged.The list of those genes in the HLA region considered by the WHO Nomenclature Committee is given in b. New Allele SequencesHLA-A, 913 HLA-B, 446 HLA-C, four HLA-E, 19 HLA-F, 31 HLA-G, 12 HLA-H, nine HLA-J, six HLA-K, five HLA-L, four HLA-P and three HLA-V alleles were named, making a total of 3249 class I alleles with official names. For HLA class II, 368 HLA-DRB1, 12 HLA-DRB3, one HLA-DRB4, one HLA-DRB5, seven HLA-DQA1, 45 HLA-DQB1, six HLA-DPA1, 22 HLA-DPB1, one HLA-DMB and four HLA-DOA alleles were named, making a total of 1198 class II alleles with official names. Eleven MICA alleles were named bringing their total to 68 and 12 MICB alleles bringing their total to 30 alleles, see st December 2009 is given in A total of 2558 HLA alleles have been named since the last report A*30:14L was named. The allele has a mutation in codon 164 encoding a cysteine residue contributing to a structurally critical disulphide bond in the \u03b12 domain of the HLA molecule. Expression studies performed on cells with this allele showed its protein to have a much-reduced expression compared to normal, and the allele name was thus given the suffix \u2018L\u2019 to indicate this low expression. Since then several other alleles have been reported that have also lost one of the two cysteine residues (position 101 and 164) that form the \u03b12 domain disulphide bond. It has not, however, been possible to ascertain the expression status of these alleles, due to a lack of viable material. The Nomenclature Committee considered the naming of these alleles during the 14th HLA and Immunogenetics Workshop. As a result of these discussions, it was decided to introduce an additional suffix, Q, to indicate a \u2018Questionable\u2019 expression level. The first seven alleles to receive this suffix have been named and are included in this report, A*23:19Q, A*32:11Q, B*13:08Q, B*35:65Q, B*39:38Q, C*02:25Q and C*03:22Q. It is anticipated that when further examples of these alleles are described, their expression status will be determined and the suffix changed accordingly.In February 2005 the allele HLA-DRB1*03, *11, *13, *14 and *08 family of alleles, for which the description of new alleles has revealed a continuum of allelic diversity rather than five discrete sub-families. It should be stressed that, although a goal is to indicate the serological grouping into which an allele will fall, this is not always possible. Most importantly the allele name should be seen as no more than a unique designation.As the database of HLA allele sequences has expanded, it has become increasingly difficult to maintain consistent linkage between allele names assigned on the basis of nucleotide sequences and the serological profiles of the encoded proteins. These difficulties are in part technological and in part due to the inherent biological properties of the HLA system. In the first category there is the increasing emphasis on DNA technology and consequent lack of a serological description for many newly discovered HLA alleles. In the second category is the finding that a newly defined antigen does not comfortably fit within any known serological grouping. This is especially true of the th International HLA and Immunogenetics Workshop in 2005 having been clearly identified as a novel antigen in a number of UCLA cell exchanges.Where this information is known, lists of the serological specificities or antigens associated with the alleles, is given in The convention of using a four-digit code to distinguish HLA alleles that differ in the proteins they encode was introduced in the 1987 Nomenclature Report The first two digits describe the allele family, which often corresponds to the serological antigen carried by the allotype. The third and fourth digits are assigned in the order in which the sequences have been determined. Alleles whose numbers differ in the first four digits must differ by one or more nucleotide substitutions that change the amino-acid sequence of the encoded protein. Alleles that differ only by synonymous nucleotide substitutions within the coding sequence are distinguished by the use of the fifth and sixth digits. Alleles that only differ by sequence polymorphisms in introns or in the 5\u2032 and 3\u2032 untranslated regions that flank the exons and introns are distinguished by the use of the seventh and eight digits.A*02 and B*15 allele families having more than 100 alleles A*92 and B*95 respectively. For HLA-DPB1 alleles, it was decided to assign new alleles within the existing system, hence once DPB1*9901 had been assigned, the next allele would be assigned DPB1*0102, followed by DPB1*0203, DPB1*0302 etc.In 2002 we faced the issue of the When these conventions were adopted it was anticipated that the nomenclature system would accommodate all the HLA alleles likely to be sequenced. Unfortunately this is not the case, as the number of alleles for certain genes is fast approaching the maximum possible with the current naming convention.With the ever increasing number of HLA alleles described it has been decided to introduce colons (:) into the allele names to act as delimiters of the separate fields. To facilitate the transition from the old to the new nomenclature, a single leading zero must be added to all fields containing the values 1 to 9 but beyond that no leading zeros are allowed. This will help to lessen any confusion in the conversion to the new style of nomenclature.HenceA*01010101 becomes A*01:01:01:01A*02010102L becomes A*02:01:01:02LA*260101 becomes A*26:01:01A*3301 becomes A*33:01B*0808N becomes B*08:08NDRB1*01010101 becomes DRB1*01:01:01:01A*02 and B*15 groups it will be possible to encode these in a single series. Thus the A*92 and B*95 alleles have now been renamed in to the A*02 and B*15 allele series. For example:For allele families that have more than 100 alleles such as the A*9201 becomes A*02:101A*9202 becomes A*02:102A*9203 becomes A*02:103 etcB*9501 becomes B*15:101B*9502 becomes B*15:102B*9503 becomes B*15:103 etcA*02:100 and B*15:100 will not be assigned. In cases of other allele families where the number of alleles reaches 100 these will be numbered sequentially, for example A*24:99 will be followed by A*24:100.The names DPB1 allele names that have been previously assigned names within the existing system have also be renamed, for example:The DPB1*0102 becomes DPB1*100:01DPB1*0203 becomes DPB1*101:01DPB1*0302 becomes DPB1*102:01DPB1*0403 becomes DPB1*103:01DPB1*0502 becomes DPB1*104:01 etcHLA-C allele names, but will be retained in the HLA-C antigen names, to avoid confusion with the factors of the complement system and epitopes on the HLA-C molecule often termed C1 and C2 that act as ligands for the Killer-cell Immunoglobulin-like Receptors.The \u2018w\u2019 will be removed from the Cw*0103 becomes C*01:03Cw*020201 becomes C*02:02:01Cw*07020101 becomes C*07:02:01:01 etcwww.ebi.ac.uk/imgt/hla) , 28 and The level of resolution achieved by many of the HLA typing technologies employed today does not always allow for a single HLA allele to be unambiguously assigned. Often it is only possible to resolve the presence of a number of closely related alleles. This is referred to as an ambiguous \u2018string\u2019 of alleles. In addition, typing strategies are frequently aimed at resolving alleles that encode differences within the peptide binding domains, but fail to exclude those that differ elsewhere. For some purposes it is helpful to provide codes that aid the reporting of certain ambiguous alleles \u2018strings\u2019. The decision was taken to introduce codes to allow for the easy reporting of:a. HLA alleles that encode for identical peptide binding domainsHLA alleles having nucleotide sequences that encode the same protein sequence for the peptide binding domains will be designated by an upper case \u2018P\u2019 which follows the allele designation of the lowest numbered allele in the group.For example the string of allele names below share the same \u03b11 and \u03b12 domain protein sequence encoded by exons 2 and 3.A*02:01:01:01/A*02:01:01:02L/A*02:01:01:03/A*02:01:02/A*02:01:03/A*02:01:04/A*02:01:05/A*02:01:06/A*02:01:07/A*02:01:08/A*02:01:09/A*02:01:10/A*02:01:11/ A*02:01:12/A*02:01:13/A*02:01:14/A*02:01:15/A*02:01:17/A*02:01:18/A*02:01:19/A*02:01:21/A*02:01:22/A*02:01:23/A*02:01:24/A*02:01:25/A*02:01:26/A*02:01:27/ A*02:01:28/A*02:01:29/A*02:01:30/A*02:01:31/A*02:01:32/A*02:01:33/A*02:01:34/A*02:01:35/A*02:01:36/A*02:01:37/A*02:01:38/A*02:01:39/A*02:01:40/A*02:01:41/ A*02:01:42/A*02:09/A*02:66/A*02:75/A*02:89/A*02:97:01/ A*02:97:02/A*02:132/A*02:134/A*02:140A*02:01PThis string can be reduced to b. HLA alleles that share identical nucleotide sequences for the exons encoding the peptide binding domainsHLA alleles that have identical nucleotide sequences for the exons encoding the peptide binding domains will be designated by an upper case \u2018G\u2019 which follows the allele designation of the lowest numbered allele in the group.For example the string shown below consists of alleles that have identical nucleotide sequences in exons 2 and 3.A*02:01:01:01/A*02:01:01:02L/A*02:01:01:03/A*02:01:08/A*02:01:11/A*02:01:14/A*02:01:15/A*02:01:21/A*02:09/A*02:43N/A*02:66/A*02:75/A*02:83N/A*02:89/A*02:97:01/A*02:97:02/A*02:132/A*02:134/A*02:140A*02:01:01GThis string can be reduced to www.ebi.ac.uk/imgt/hla) (These reporting codes will be implemented in April 2010 and will be made available through the IMGT/HLA Database (mgt/hla) , 28 and HLA-A gene, for example A*03:01 and the expressed protein product of the same gene A*03:01.Discussions took place on the use of nomenclature for defining HLA allele sequences at the gene and protein level. The committee recommended the use of standard genetic nomenclature where gene symbols are in uppercase and italicised and protein symbols are the same as the gene symbols but are not italicised. Using this approach it is possible to discriminate between an allele of the Additionally it was recommended that when reporting an ambiguous string of HLA alleles, a forward slash (/) should be used as the separator to indicate \u2018or\u2019. When reporting genotypes it was recommended to use a comma to indicate \u2018and\u2019. Hence an HLA type may be reported as:A*02:01/02:09, 03:01; B*07:02, 15:02/15:73; C*03:03, 07:02www.ebi.ac.uk/imgt/hla.The IMGT/HLA Sequence Database continues to act as the official repository for HLA sequences named by the WHO Nomenclature Committee for Factors of the HLA System , 28. TheThe IMGT/HLA Database is currently supported by the following organisations: Histogenetics, Abbott, Biotest, Invitrogen, One Lambda, Olerup SSP, Gen-Probe, The Anthony Nolan Trust (ANT), The American Society for Histocompatibility and Immunogenetics (ASHI), The European Federation for Immunogenetics (EFI), Innogenetics, BAG Healthcare, Be the Match Foundation and the National Marrow Donor Program."} +{"text": "The first author's name appears incorrectly. It should read: Boris V. Schmid. The correct citation should read: Schmid BV, Ke\u015fmir C, de Boer RJ (2008) The Specificity and Polymorphism of the MHC Class I Prevents the Global Adaptation of HIV-1 to the Monomorphic Proteasome and TAP. PLoS ONE 3(10): e3525. doi:10.1371/journal.pone.0003525"} +{"text": "British Journal of Cancer (2002) 87, 689\u2013689. doi:10.1038/sj.bjc.6600504www.bjcancer.comCancer Research UK\u00a9 2002 Correction to:British Journal of Cancer (2002) 86, 1270. doi:10.1038/sj/bjc/6600232 The authors would like to thank InSight Ltd., Rehovot, Israel for providing them with the anti-heparanase antibody and heparanase cDNA used in the study.The mentioned reagents are proprietary of InSight."} +{"text": "There was an error in the title of the article. The correct title should read: Functional and Molecular Effects of Arginine Butyrate and Prednisone on Muscle and Heart in the Dystrophin Deficient mdx mice. The correct citation is: Guerron AD, Rawat R, Sali A, Spurney CF, Pistilli E, et al. (2010) Functional and Molecular Effects of Arginine Butyrate and Prednisone on Muscle and Heart in the Dystrophin Deficient mdx mice. PLoS ONE 5(6): e11220. doi:10.1371/journal.pone.0011220"} +{"text": "Ma'i Suka: Individual, Familial, Cultural, and Environmental Stress Among Patients With Type 2 Diabetes Mellitus and Their Caregivers in American Samoa,\" which appeared in the July 2008 issue, was amended to include a footnote that was missing in the original publication. The footnote indicates that the column headed \"Total Participants\" includes 3 caregivers with diabetes. The changes were made June 25, 2008, and appear online at www.cdc.gov/pcd/issues/2008/jul/07_0101.htm.Table 2 in the article \"Living With"} +{"text": "Correction to:British Journal of Cancer (2004) 91, 1000. doi:10.1038/sj.bjc.6602070Due to an error, the ISBN number of the book reviewed above was given incorrectly. The correct number is shown below:ISBN \u2013 1588292282."} +{"text": "Correction to: British Journal of Cancer (2010) 103, 209-216; doi:10.1038/sj.bjc.6605747Owing to an error during final correction of this paper, Figure 3 was incorrectly reproduced. The correct"} +{"text": "Correction to:British Journal of Cancer (2003) 88, 502\u2013509. doi:10.1038/sj.bjc.6600797Unfortunately because of a typesetting error, Tables 1The publisher would like to apologise for any inconvenience this may have caused."} +{"text": "Correction to:British Journal of Cancer (2007) 96, 1293\u20131301. doi:10.1038/6603696In the abstract of the above paper, a peptide with correct sequence (TLMKKDKYTL) is mentioned. This sequence should also appear in The corrected"} +{"text": "Correction to: British Journal of Cancer (2008) 98, 1586\u20131592. doi: 10.1038/sj.bjc.6604303During the correction process for this article, incorrect versions of the graphs contained in The corrected"} +{"text": "Correction to:British Journal of Cancer (2009) 101, 441\u2013451; doi:10.1038/sj.bjc.6605186Owing to an error during the final correction of this paper, panels E and F in The correct panels 4E and 4F are reproduced below:"} +{"text": "Acta Cryst. (2009), E65, o2008.Corrigendum to Acta Cryst. (2009), E65, o2008] is corrected and the acknowledgements are updated.The list of authors in the paper by Zhi, Long, Chen & Ren ["} +{"text": "Correction to: British Journal of Cancer (2006) 95, 363\u2013365. doi:10.1038/sj.bjc.6603250Owing to an author error, there was a mistake in the presentation of results in"} +{"text": "Correction to:British Journal of Cancer (2003) 88, 613\u2013623; doi:10.1038/sj.bjc.66000681 The enzyme was prepared by the overexpression of biosynthetic arginine decarboxylase , who kindly provided the enzyme for CDN Wheatley"} +{"text": "Correction to:British Journal of Cancer (2010) 102, 232\u2013233. doi: 10.1038/sj.bjc.6605446Upon publication of this Letter to the Editor, it was noted that the affiliation for Dr Mark Nuijten had been presented incorrectly. The correct affiliation for Dr Nuijten is \u2018Ars Accessus Medica in Amsterdam, Dorpsstraat 75 1546 LG, The Netherlands\u2019."} +{"text": "El Mouzan et al. Prevalence of malnutrition in Saudi children: a community-based study. Ann Saudi Med 2010;30(5):381-385. PMID: 20697172. Some data in some of the tables (and the corresponding data in the text) are corrected as indicated. Changes are highlighted."} +{"text": "Correction to: British Journal of Cancer (2008) 99,, 1185\u20131190, doi: 10.1038/sj.bjc.6604657The surname of Driss Ait Ouakrim was published incorrectly when this paper appeared in volume 99, issue 7. The author's name is given correctly, above."} +{"text": "Correction to:British Journal of Cancer 2003; 89 (S1), S9\u2013S11 doi:10.1038/sj.bjc.6601078 Unfortunately, due to an error, the tables were omitted from the above article.All the three tables (The publisher would like to apologise for any inconvenience this error may have caused."} +{"text": "Correction for:10.1371/journal.pbio.0060265Welch DW, Rechisky EL, Melnychuk MC, Porter AD, Walters CJ, et al. (2008) Survival of migrating salmon smolts in large rivers with and without dams. PLoS Biol 6(10): e265 doi:In the legend below Table 1, the reference given at the end of the second sentence is incorrect. The sentence should read:\u201cAnnual survival (S) to the lowest listening line in the Fraser River, whole-river survival in the Columbia River (2006), and associated detection efficiencies (p) were calculated using the CJS model and program MARK [28]\u201d"} +{"text": "PLoS Pathog 3(4): e43. doi: PLoS Pathogens, volume 3, issue 4:In The funding statement for the above paper was incomplete. The full funding statement follows.Funding. This work was supported by grants from the National Institutes of Health . JDB is supported by K01 AI 066917 ."} +{"text": "Correction to: British Journal of Cancer (2006) 95, 87\u201390. doi:10.1038/sj.bjc.6603175P-values. The final sentence of the Results section should read:Owing to an author error, the final paragraph within the results section of this paper quoted erroneous P-values for trend: 0.199 and 0.043, for males and females, respectively).\u2019\u2018Rates were somewhat higher in deprived areas, but this trend is only significant for females ("} +{"text": "Correction to: British Journal of Cancer (2006) 96, 336\u2013340. doi:10.1038/6603492British Journal of Cancer website (http://www.nature.com/bjc).Owing to a publishing error, part of the intended"} +{"text": "Correction to: British Journal of Cancer (2009) 101, 1909\u20131918; doi:10.1038/sj.bjc.6605405Upon publication of this paper in the last issue, an error was spotted in the clinical data in"} +{"text": "Correction to Lindsay H, Yap VB, Ying H, Huttley GA: Pitfalls of the most commonly used models of context dependent substitution. Biology Direct 2008, 3:52 The published version of this article includesScripts used in the study. Archive of stand-alone web site presenting the central scripts used in this study.Click here for file"} +{"text": "Correction to:British Journal of Cancer (2003) 89, 1285\u20131289. doi:10.1038/sj.bjc.6601208A couple of errors have been noted in Table 2The Publisher would like to apologise for any inconvenience this may have caused."} +{"text": "Environ Health Perspect 118:20\u201332 (2010)], Orton et al. (2006) was cited on page 26 but was not included in the reference list. The reference is as follows:In the review by Rohr and McCoy [Rana pipiens. Environ Toxicol Chem 25:65\u201371.Orton F, Carr JA, Handy RD. 2006. Effects of nitrate and atrazine on larval development and sexual differentiation in the northern leopard frog"} +{"text": "Correction to: British Journal of Cancer (2008) 97, 857\u2013861, doi:10.1038/sj.bjc.6603942The authors of the above paper, first published in October 2007, would like to add the following acknowledgement to their paper:We thank Programme Hospitalier de Recherche Clinique (PHRC)."} +{"text": "Correction to:British Journal of Cancer (2006) 94, 1853\u20131863. doi:10.1038/6603190Owing to a publishing error,"} +{"text": "Correction to: British Journal of Cancer (2010) 103: 668\u2013675; doi:10.1038/sj.bjc.6605736Upon publication of the above paper in Volume 103, Issue 5, the authors noticed an error in The correct"} +{"text": "Correction to:British Journal of Cancer (2004) 90, 1756\u20131759. doi:10.1038/sj.bjc.6601763Due to an error, On page 1756 of this article, the second sentence in the Results section should read as follows:The incidence of hysterectomies with cervix removed increased from 2500 in 1991 to 2017 per million women in 1999."} +{"text": "Correction to: British Journal of Cancer (2007) 97, 73\u201384. doi: 10.1038/sj.bjc.6603835The authors of the above paper would like to add two author names to the list originally published in Volume 97, Number 1; the full author list is given above, including E Taoufik and E Probert."} +{"text": "Correction to: British Journal of Cancer (2006) 95, 1314\u20131320. doi: 10.1038/6603457Owing to an error on the author's part, the last named author (JY Shin) was omitted from the author listing when the paper was originally published. The full, correct author listing is now shown above."} +{"text": "Environ Health Perspect 118:A422\u2013A423 (2010)], one reference cited in the text was inadvertently omitted from the reference list. The reference for Grulich et al. (2007) is as follows:In the editorial by Fontham and Trapido [Grulich AE, van Leeuwen MT, Falster MO, Vajdic CM. 2007. Incidence of cancers in people with HIV/AIDS compared with immunosuppressed transplant recipients: a meta-analysis. Lancet 370(9581):59\u201367.EHP apologizes for the error."} +{"text": "Acta Cryst. (2010), E66, o217.Corrigendum to Acta Cryst. (2010), E66, o217] is corrected.The absolute configuration in the title of the paper by Wang, Zhang, Xu & Zhang [ The absolute configuration was established by anomalous-dispersion effects in diffraction measurements on the crystal. The revised scheme is shown below. In the paper by Wang"} +{"text": "Correction to: British Journal of Cancer (2006) 94, 1326\u20131332. doi:10.1038/6603101Owing to an author error, the incorrect"} +{"text": "Correction to: British Journal of Cancer (2009) 101, 395\u2013402; doi:10.1038/sj.bjc.6605155Upon publication of this paper in Volume 101, the authors noticed an error in the legend of"} +{"text": "There are errors in the Funding section. The correct funding information is as follows: This research was supported by the World Health Organization through a grant received from the Japan International Cooperation Agency (JICA). The World Health Organization was actively involved in study design, data collection and analysis, decision to publish, and preparation of the manuscript. JICA had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.http://www.who.int/gho/en/.]There is an error in reference 1. The correct reference is: World Health Organization. Global health observatory data repository 2015: WHO; 2015 There is an error in reference 7. The correct reference is: World Health Organization. Maternal mortality 2014; Fact sheet No. 348: WHO; 2014 There is an error in reference 12. The correct reference is: World Health Organization. Practical guide for the design, use and promotion of home-based records in immunization programmes: WHO; 2015 There is an error in reference 17. The correct reference is: Magwood O, Kpade V, Thavorn K, Oliver S, Mayhew AD, Pottie K. Effectiveness and cost-effectiveness of home-based records on maternal, newborn and child health outcomes: a protocol for a WHO systematic review and meta-analysis. Ottawa: Cochrane Equity Methods; 2017 [Available from: http://www.who.int/reproductivehealth/publications/maternal_perinatal_health/anc-positive-pregnancy-experience/en/.]There is an error in reference 18. The correct reference is: World Health Organization. WHO recommendations on antenatal care for a positive pregnancy experience: WHO; 2016 [Available from: http://www.who.int/topics/infant_newborn/en/.]There is an error in reference 19. The correct reference is: World Health Organization. Infant, Newborn: WHO; 2017 [Available from: There is an error in reference 21. The correct reference is: Cochrane Effective Practice and Organisation of Care (EPOC). What study designs should be included in an EPOC review? EPOC resources for review authors 2017 [Available from: epoc.cochrane.org/epoc-resources-review-authors.]There is an error in reference 24. The correct reference is: The Cochrane Collaboration. Review Manager (RevMan) version 5.3. 5.3 ed: The Cochrane Collaboration; 2014.There is an error in reference 35. The correct reference is: John Snow Inc. (JSI). Home-Based Record Redesigns That Worked: Lessons from Madagascar & Ethiopia. USA: JSI; 2016.There is an error in reference 36. The correct reference is: Japan International Cooperation Agency (JICA). Study on the use of the Maternal and Child Health Handbook in the Maternal and Child Health Project\u2013Knowledge, Lessons and Challenges. Japan: JICA; 2012."} +{"text": "Randomised controlled pilot feasibility trial of an early intervention programme for young infants with neurodevelopmental impairment in Uganda: a study protocol. BMJ Open 2019;9:e032705. doi: 10.1136/bmjopen-2019-032705.Nampijja M, Webb E, Nanyunja C, This article was previously published with an error.The first author of reference 2 was omitted. The correct reference is:et al. Global Research on Developmental Disabilities Collaborators. Developmental disabilities among children younger than 5 years in 195 countries and territories, 1990\u20132016: a systematic analysis for the global burden of disease study 2016. Lancet Glob Health 2018;6:e1100\u201321.Olusanya BO, Davis AC, Wertlieb D,"} +{"text": "International Journal of Oral Science (2015) 7, 205\u2013212; 10.1038/ijos.2015.29; published 18 September 2015Correction to: In the original version of this Article, Fig."} +{"text": "Correction to: British Journal of Cancer (2016) 94, 1765-1769; 10.1038/sj.bjc.6603170; published online 23 May 2006.Since the publication of this paper, the authors have reported that there is an error in the chemical structure for imatinib presented in Fig."} +{"text": "Correction to: BMC Systems Biology 2018, 12(Suppl 8):128https://10.1186/s12918-018-0651-1It was highlighted that the original article containeFig. 6 legendP-value <\u20090.05 and supporting reads number\u2009>\u2009=2) contains two domains: Biological Process and Cellular Component.GO enrichment analysis of the mRNA in the co-expression network. The GO analysis . Z.Z was partially supported by National Institutes of Health grants (R21CA196508 and R01LM012806). The funder had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.Fig. 6 legendcircRNA-mRNA co-expression network. Red box represents mRNA; blue triangle represents miRNA, and green circle represents circRNA.FundingPublication of this article was funded by the National Natural Science Foundation of China (No.81270700 and No. 61571223). Z.Z was partially supported by National Institutes of Health grants (R21CA196508 and R01LM012806). The funder had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript."} +{"text": "Wind coming from the West, compared to wind coming from other directions, was the most favourable for performance of all groups of finishers. Increasing precipitations worsened performances of top 100 and top 10 finishers . Wind speed, when increasing by 1 km/h, was related to worsened performance for all finishers , but not for top 100 group, where performances were 00:00:09 h:min:sec faster, p < 0.001. Pressure and WBGT were examined in uni-variable models: overall, performances worsened as pressure and WBGT increased. Our findings contributed to the knowledge about the effect of weather conditions on performance level in male marathon runners.This study investigated the effects of weather conditions on male performance during the Boston Marathon from 1897 to 2018. A total of 383,982 observations from 244,642 different finishers were analysed using Generalized Additive Mixed Models. All runners, annual top 100 finishers and annual top ten finishers were considered. Weather conditions, on race day, were: average air temperature (\u00b0C), precipitations (mm), wet-bulb globe temperature (WBGT) (\u00b0C), wind speed (km/h), wind direction and pressure (hPa). These effects were examined in multi-variable models with spline smooth terms in function of calendar year. Temperature, when increasing by 1 \u00b0C, was related to worsened performance for all groups (i.e., by 00:01:53 h:min:sec for all finishers, Environmental conditions seem to have an important effect on marathon running performance. Temperature, humidity and wind could influence the thermoregulatory anticipation to the increased heat gained during a marathon race . An ambiThe Boston Marathon has the longest tradition in marathon running since its start in 1897 and several studies have investigated the influence of environmental conditions on race performance ,13,14,15Therefore, the aim of the present study was to examine the effect of weather conditions on race time of male runners in the Boston Marathon from 1897 to 2018. This marathon race provided a unique model to conduct research on the effect of weather conditions considering their variability among calendar years. We hypothesized to find difference regarding existing literature when investigating the whole period of time and including all male annual finishers as well as the annual top 10 and the annual top 100 male finishers.All procedures used in the study were approved by the Institutional Review Board of Kanton St. Gallen, Switzerland, with a waiver of the requirement for informed consent of the participants given the fact that the study involved the analysis of publicly available data (01/06/2010).www.baa.org/races/boston-marathon/boston-marathon-history.aspx). To compete in this race, athletes must meet time standards which correspond to age and sex . Data, collected annually from 1897 to 2018 for men were obtained from the official race website (www.baa.org/races/boston-marathon.aspx). Available information from the race records were name and surname of the runners, sex and runners\u2019 nationality, year of competition, and race times.The Boston Marathon is the world\u2019s oldest annual marathon, where the course is a point-to-point race with its start in Hopkinton and finishing on Boylston Street in Boston, MA. (We cleaned the dataset removing runners with missing or questionable (unreliable) information on race time. Unfortunately, we did not have the complete list of all runners belonging to push rim wheelchair division, started on 1975 for men. We excluded this category eliminating runners with race time shorter than the annual top record. To identify observations from a single runner, we defined an id variable with name, surname, nationality and period of competition, supposing that a single runner could participate at most for 25 years.Temperature, and speed and direction of wind, seemed to have an influence on race time in the Boston Marathon ,13,14,22www.wunderground.com/history/airport/KBOS/2013/1/15/MonthlyHistory.html for 1930 to 2018 and from https://w2.weather.gov/climate/local_data.php?wfo=box for 1897 to 1929. WBGT was calculated with https://www.kwangu.com/work/psychrometric.htm using the dry bulb temperature and relative humidity obtained from www.wunderground.com and an altitude of 43 m above sea level.Weather data were obtained from two different websites for this period from 1897 to 2018 from Descriptive statistics were presented as means \u00b1 standard deviations for continuous variables and as number N (%) for categorical variables. Performance, or race time, was recorded in the format \u201chours:minutes:seconds\u201d. Different analyses were performed for the following groups of finishers: all runners, annual top 100 and annual ten finishers. Average performances, by sex and weather conditions, were reported for the following groups: temperature of 0\u20137, 8\u201315, 16\u201323, 24\u201330 \u00b0C, precipitations equal 0 or >0 mm, WBGT of 0\u20136, 7\u201310, 11\u201315, 16\u201320 \u00b0C, wind speed of 0\u201315, 16\u201317, 18\u201338 km/h, and pressure < 1015 or \u22651015 hPa. Wind direction was defined as: East , West , North , South . T-tests or ANOVA tests, with post-hoc Dunn\u2019s test for pairwise multi-comparison of means, where appropriate, were performed, but p-values were not shown in tables.p < 0.05.Data visualization was used to identify the uni-variable relationship between performance and each weather variable. Moreover, association between weather conditions was preliminary investigated through the correlation matrix, which showed the pairwise correlation among all the variables. As a result, the effects of calendar year on race time, together with the effects of weather conditions, were examined through multi-variable statistical models. Results were presented as estimates (standard errors). The acceptable type I error was set at Race Time (Y) ~ [Fixed effects (X) = Weather Conditions + S+ [Random effects of intercept = Runners]where S denoted a spline, changing with calendar year and with k basis dimension. K was set equal to nine. Observations before 1944 were discarded because of incomplete information about weather conditions. As a first analysis, all weather variables: temperature, WBGT, precipitations, wind direction, wind speed, and pressure were included into the multi-variable model. Subsequently, WBGT and pressure were discarded, in all groups of finishers, due to multi-collinearity, assessed by computing the variance inflation factor (or VIF) score. All predictors with VIF not greater than 10 were retained in the model. According to this criterion, in all finishers group, precipitations variable was also dropped. Instead, in top ten finishers the wind speed variable was discarded. Partial effects, which are the isolated effects of one particular predictor or interaction, were shown graphically. Moreover, the effects of pressure and WBGT on performance, discarded in the multi-variable model, where also assessed, more rigorously, through uni-variable linear mixed models, which corrected for repeated measurements.Different models were performed, one for each group of finishers. Calendar year was considered as a discrete value of a continuous variable. Temperature, precipitation, WBGT, wind speed and pressure were considered as continuous variables. Spline regression models were used, with a spline smooth term in function of calendar year and a linear term in function of the other effects. Preliminary data visualization and previous research ,23 justihttps://www.R-project.org/). In particular, we used the following R packages: PMCMR for Pairwise post-hoc Test for Multiple Comparisons of Means; ggplot2 for preliminary data visualization; GGally for plotting the correlation matrix and the association between explanatory variables; gamm4 for multi-variable mixed models with random effects on intercept; mgcv for statistical models visualization; lmer for linear mixed models.All statistical analyses were carried out using statistical package R, R Core Team (2016). R: A language and environment for statistical computing. . In Between 1897 and 2018, a total of 383,982 observations from 244,642 different finishers were recorded in the race results. In Before carrying out multi-variable analysis, association between weather variables was examined and reported in After that, the effects of each weather variable, on performance of all groups of finishers, were shown in p < 0.001 in all finishers; 00:00:37 (00:00:02) h:min:sec, p < 0.001 in top 100 finishers and 00:00:38 (00:00:03) h:min:sec, p < 0.001 in top 10 finishers. When wind speed increased by 1 km/h, performances of all finishers worsened but performances of top 100 group improved. In fact, the wind speed estimates was positive in all finishers: 00:00:19 (00:00:01) h:min:sec, p < 0.001 and negative in top 100 group: \u221200:00:09 (00:00:01) h:min:sec, p < 0.001. When precipitations increased by 1 mm, performances significantly worsened: by 00:00:04 (00:00:01) h:min:sec, p < 0.001 in top 100 group and by 00:00:05 (00:00:01) h:min:sec, p < 0.001 in top 10 group. Performances with wind coming from the West, were significantly better compared to the other wind directions (p < 0.001). In fact, estimates were positive, which meant worse performance compared to the West, the reference wind direction category. The wind direction had the greatest effects, in terms of estimates, in all finishers, compared to the other groups of finishers, when the wind came from the East and from the South. In fact, when the wind direction was the East, performances, compared to performances when wind direction was the West, were slower by: 00:11:18 (00:00:11) h:min:sec, p < 0.001, in all finishers, against 00:03:09 (00:00:27) h:min:sec, p < 0.001, in top 100 group and 00:03:54 (00:00:38) h:min:sec, p < 0.001 in top 10 group. Analogously, when the wind direction was the South, performances, compared to performances when wind direction was the West, were slower by 00:09:41 (00:00:10) h:min:sec, p < 0.001 in all finishers, against 00:01:13 (00:00:33) h:min:sec, p < 0.05 in top 10 group. In top 100 group, the difference between performances, when wind direction was the South and performances when wind direction was the West, was not significant. When the wind direction was the North compared to the West, the greatest difference was observed in all finishers: 00:05:30 (00:00:11) h:min:sec, p < 0.001 against 00:01:51 (00:00:38) h:min:sec, p < 0.001 in top 10 group and 00:01:30 (00:00:25) h:min:sec, p < 0.001 in top 100 group.In p < 0.001 in all finishers, 00:00:09 (00:00:01) h:min:sec, p < 0.001 in top 100 groups and 00:00:04 (00:00:02) h:min:sec, p < 0.05 in top ten group. Instead, when WBGT increased by 1 \u00b0C, performances of all groups of finishers were significantly slower: by 00:01:55 (00:00:01), p < 0.001 in all finishers, by 00:00:22 (00:00:03) h:min:sec, p < 0.001 in top 100 group and by 00:00:26 (00:00:06) h:min:sec, p < 0.001 in top 10 group.Since the uni-variable relationships, plotted in p < 0.001 in all groups. The greatest smoothing terms, in absolute value, were observed in all finishers where the trend was overall increasing, even if in some years was decreasing. This meant that performances had overall worsened over time. In top 100 and top 10 groups, instead, the temporal trend of performance was overall decreasing, even if not monotonically, meaning that performance had overall improved over time.Performances changed significantly over calendar year. In fact, in To visualize the most important results of p < 0.001) worsened performance by 00:01:53 h:min:sec for all finishers, 00:00:37 h:min:sec for annual top 100 finishers and 00:00:38 h:min:sec for annual top 10 finishers; (ii) wind coming from the West, compared to wind from other directions, was the most favourable for performance of all groups of finishers. In particular, for all finishers, performances when wind came from the West were: 00:09:41 h:min:sec faster (p < 0.001), compared to performances when wind came from the South, 00:11:18 h:min:sec faster (p < 0.001), compared to performances when wind came from the East and 00:05:30 h:min:sec faster (p < 0.001), compared to performances when wind came from the North; (iii) precipitations, when increasing by 1 mm, worsened performances of top 100 and of top 10 groups by 00:00:04 h:min:sec and 00:00:05 h:min:sec (p < 0.001), respectively; (iv) wind speed, when increasing, also worsened performances for all finishers , but not for top 100 group, where performances were 00:00:09 h:min:sec faster, p < 0.001; (v) pressure, when increasing, had a negative effect on performance in all group of finishers; (vi) WBGT when increasing, had a negative effect on performance of all groups of finishers.The main findings of the present study were that: (i) average temperature, when increasing by 1 \u00b0C, significantly (The direct relationship of race times with temperature, i.e., the hotter the temperature, the slower the race time, was in agreement with previous observations in marathon races ,12. For The findings of the present study were limited by the specific characteristics of the race in terms of participation and weatIn summary, race times in the Boston Marathon are influenced by temperature, pressure, precipitations, WBGT, wind coming from the West and wind speed. The magnitude and the direction of the influence of the abovementioned weather conditions on race time varied by performance level. Our findings improved the knowledge about the association between weather conditions and their relationship with performance level in male marathon runners. Considering the popularity of the particular marathon and of marathon races in general , the res"} +{"text": "Elizabethkingia Acquisition and Clinical Characteristics of Patients, South Korea . The corrected figure is shown, and the article has been corrected online (https://wwwnc.cdc.gov/eid/article/25/1/17-1985_article).Two locations in Figure 1 were shown incorrectly in Risk Factors for"} +{"text": "Bioinformatics (2019) doi: 10.1093/bioinformatics/bty873, 35, 1783\u20131785.In the original article, there was an error in the formatting of Table 1.This has been corrected and the corrected table appears below."} +{"text": "Correction to:Cell Death and Disease (2016) 7, e2419; 10.1038/cddis.2016.268, Published online 13 October 2016.Since the publication of the article, the authors became aware that Fig.\u00a0The authors apologise for any inconvenience caused."} +{"text": "There is an error in the reference for the source of the mathematical model used in this manuscript. The authors cited Chow&Hall 2008 instead of Hall et al. 2011. Both articles have Hall in their authorship. While Chow&Hall (2008) explains the model conceptualization and the model behavior with non-real simulations, Hall et al. (2011) explains the model implementation to the adult population with real data. The correct citation for reference #27 should read:27. Hall KD, Sacks G, Chandramohan D, Chow CC, Wang YC, Gortmaker SL, et al. Quantification of the effect of energy imbalance on bodyweight. Lancet. 2011;378: 826\u2013837. doi:10.1016/S0140-6736(11)60812-XThe reference is made several times throughout the paper, and therefore the following should be corrected:http://ensanut.insp.mx/). The final dataset after running the Hall and colleagues\u2019 model is available at the open science framework, available at https://osf.io/vfcm8/ (DOI: 10.17605/OSF.IO/VFCM8). The mathematical model used to estimate the impact on body weight is freely available within an R package version 1.0.0 named \u2018bw\u2019 in https://cran.r-project.org/web/packages/bw/index.html.The Data Availability Statement should read: Baseline added sugar consumption from SSBs and weight were obtained from the 2012 Mexico National Health and Nutrition Survey .\u201dThe first three sentences of the third paragraph in the Discussion section should read: Body weight modeling is a complex task, as many components and interrelationships need to be considered. In this paper, we used the model proposed by Hall and colleagues, which has been widely used to estimate the impact of nutritional policies, including the potential impact of sugar reformulation in SSBs [41]. Hall and colleagues\u2019 model is a physiological model that has been validated against experimental data and can be implemented using individual-level or aggregated data; however, other models to estimate weight change are available.\u201d"} +{"text": "This article has been corrected: The authors requested to replace Figure 3 and Figure 6. The mistakes of these figures are described below:Figure 3: the Westernblot of SDHB in Figure3B of MCF-7 flipped horizontally.Figure 6: the Westernblot of Cytc in Figure6B of MDA-231 was identical to Uqcrfs1 due to the layout mistakes.These corrections do not change any of the conclusions of the publication. The corrected Figure 3 and Figure 6 are provided below.https://doi.org/10.18632/aging.102442.Original article: Aging. 2019; 11:10203\u201310219."} +{"text": "Increasing wind speed was also related to worsened performances in all finishers and near elite groups. Kenyans and Ethiopians were the fastest nationalities. The sex differences were the largest in near elite groups. Our findings contributed to the knowledge of the performance in Boston Marathon across calendar years, considering as main effects weather conditions, country of origin and sex.This study examined the relationship of weather conditions, together with sex and country of origin, with running performance in the Boston Marathon from 1972 to 2018. A total of 580,990 observations from 382,209 different finishers were analyzed using Generalized Additive Mixed Models. Different groups and subgroups were considered such as all runners, near elite 101:200 finishers, near elite 21:100, annual top ten finishers and annual winners. Weather conditions, over the hours of the event, were average air temperature (\u00b0C), total precipitations (mm), wet-bulb globe temperature (WBGT) (\u00b0C), wind speed (km/h), wind direction and barometric pressure (hPa). These effects were examined in a multi-variable model, together with: sex, country of origin, calendar year, an interaction term country:sex and a spline smooth term in function of calendar year and sex. The average temperature, when increasing by 1\u00b0C, was related to worsened performance . Also, the pressure and wet-bulb globe temperature, when increasing, were related to worsened performances. Tail wind improved performances of all groups. Increasing precipitation was significantly ( To date, the influence of different environmental conditions such as ambient air temperature wind, precipitations, barometric pressure, humidity, dew point, cloud cover, solar irradiation and atmospheric pollutants have been investigated in marathon running performance \u20137. It isThe Boston Marathon has the longest tradition in marathon running (it started in 1897 and the first woman participated in 1972) and several studies investigated the influence of environmental conditions on race performance ,11. Howei.e. head wind, side wind, tail wind) and barometric pressure (hPa). These effects were analyzed together with sex, country of origin and calendar year. Based upon previous research, we hypothesized that increasing air temperature impaired running performance in both elite and sub-elite runners.The aim of the present study was to investigate the role of weather conditions, together with sex and country, on female and male performance in the Boston Marathon from 1972 to 2018 since in 1972 the first women officially participated in a marathon. We considered the effects, over the hours of the event, of average air temperature (\u00b0C), total precipitations (mm), wet-bulb globe temperature (WBGT) (\u00b0C), wind speed (km/h), wind direction . To compete in this race, athletes must meet time standards which correspond to age and sex . Data are freely available from the Boston Athletic Association website (www.baa.org) and Marathon Guide website (www.marathonguide.com). Data involved in this study are race results from 1972 to 2018 for women and men. Available information from the race records were name and surname of the runners, sex and runners\u2019 nationality, year of competition, and race times. We cleaned the dataset correcting for double coding of the same level of categories as reported earlier [www.wunderground.com/history/daily/us/ma/boston/KBOS and all units were converted to the metric system. All weather data was stored in www.kwangu.com/work/psychrometric.htm using the dry bulb temperature and relative humidity obtained from www.wunderground.com/ and an altitude of 43 m above sea level. The total precipitation (mm) was the sum over the duration of the race of the hourly amount of precipitations. The wind direction was the most frequent determination over the hourly observations which were classified as: head wind , side wind and tail wind [www.baa.org/races/boston-marathon/course-map).The Boston Marathon is the world\u2019s oldest annual marathon due to ti.e. or race time) was recorded in the format \u201chours:minutes:seconds\u201d. Average performances, by sex and weather conditions, were reported for the following groups: temperature of 0\u20137 \u00b0C, 8\u201315 \u00b0C and 16\u201324 \u00b0C; wind direction head, side or tail; total precipitations equal 0 and >0 mm; WBGT of 0\u20136 \u00b0C, 7\u201310 \u00b0C, 11\u201315 \u00b0C and 16\u201320 \u00b0C; wind speed of 9\u201317 km/h, 18\u201324 km/h and 25\u201339 km/h; pressure <1015 hPa and \u22651015 hPa. The effects of calendar year on race time, together with the effects of sex, country of origin and weather conditions, were examined through multi-variable statistical models. Results were presented as estimates (standard errors). Different analyses and regression models were performed for the following subgroups: all runners, annual top 101:200, annual top 21:100, annual top ten finishers and annual winners. The calendar year of the race was considered as a discrete value of a continuous variable. The country groups included the 8 most prevalent geographical areas in terms of participation . For annual top finishers (winners) country groups were only 4: Kenya-Ethiopia, Europe, USA and other countries. Weather characteristics such as: temperature, precipitations, WBGT, wind speed and pressure were considered as continuous variables. Wind direction as a categorical variable. The acceptable type I error was set at p<0.05. Preliminary data visualization and previous research [https://www.R-project.org/. In particular, we used the following R packages: ggplot2 for preliminary data visualization; gamm4 for multivariable mixed models with random effects on intercept; mgcv for statistical models visualization. The R script, used to manage data and to run the analyses, was provided in Descriptive statistics were presented as means \u00b1 standard deviations for continuous variables and as number N (%) for categorical variables. Performance . Instead, women performed better when there was head wind in most cases. Performances were also better, most frequently, with absence of precipitations for women and for men, on the contrary, with presence of precipitations. When considering wet-bulb globe temperature, when the level was 0\u20136 \u00b0C, both men and women performed better in all cases except all male finishers. In most cases, when wind speed was 18\u201324 km/h, men and women performed better. Both sexes performed better, in most cases for women and in all cases for men, when pressure <1015 hPa. In In In p<0.001 and a smaller effect for annual winners 00:00:20 (00:00:05) h:min:sec, p<0.001. For the annual top ten, a temperature variation effect on performance was not significant. As pressure increased by one hPa, performances significantly worsened with a greater effect for all finishers: 00:00:06 (00:00:00) h:min:sec, p<0.001 and a smaller effect for the annual top 101:200 finishers: 00:00:03 (00:00:00) h:min:sec, p<0.001. For the annual winners, an effect of pressure variation on performance was not significant. As wind speed increased by 1 km/h, performances significantly worsened with a greater effect for all finishers: 00:00:13 (00:00:00) h:min:sec, p<0.001 and a smaller effect for the annual top 21:100: 00:00:06 (00:00:01) h:min:sec, p<0.001. For the elite groups the effect of wind speed variation on performance was not significant. As precipitations increased by 1 mm, performances significantly worsened, with a greater effect for all finishers: 00:00:44 (00:00:01) h:min:sec, p<0.001 and equal effect for the other groups: estimate 00:00:10 h:min:sec. For the annual winners, a precipitations effect was not significant. A wet-bulb globe temperature effect was significant in all groups except winners. Analogously to the other weather effects, as wet-bulb globe temperature increased by 1 \u00b0C, performances worsened with a greater effect for near elite groups: 00:00:31 (00:00:03) h:min:sec, p<0.001 in top 21:100 group and a smaller effect for all finishers: 00:00:10 (00:00:02) h:min:sec, p<0.001. Performances of all groups were significantly slower with head and side wind compared to tail wind. The effects were greater for all finishers: compared to tail wind, head wind slowed down performance by 00:11:51 (00:00:10) h:min:sec, p<0.001 and side wind by 00:08:04 (00:00:09) h:min:sec, p<0.001. For the near elite finishers, in particular the top 101:200, the effects of wind direction were smaller: head wind slowed down performance by 00:03:06 (00:00:09) h:min:sec, p<0.001 and side wind by 00:01:37 (00:00:08) h:min:sec, p<0.001.As temperature, WBGT, pressure, precipitations or wind speed increased, performances significantly worsened in most cases. In fact, when average temperature increased by 1 \u00b0C, performances were slower with a greater effect for all finishers: 00:01:47 (00:00:01) h:min:sec, p<0.001, which was the comparison between Kenya\u2014Ethiopia and Central-South America, to a maximum of 01:45:14 (00:04:44) h:min:sec, p<0.001, which was the comparison between Kenya-Ethiopia and Asia. In the top ten group, the significant differences ranged from a minimum of 00:02:20 (00:01:04) h:min:sec, p<0.01, which was the comparison between Kenya\u2014Ethiopia and Asia, to a maximum of 00:03:00 (00:00:45) h:min:sec, p<0.001, which was the comparison between Kenya-Ethiopia and USA. The interaction terms country:sex represented the sex differences for each country group. For instance, the term Africa:M estimated how much greater the effect of being men on race time was for a runner from Africa, compared to a runner from Kenya-Ethiopia. From p<0.001. Men were significantly faster than women in all groups, with a greater effect in the near elite groups, in particular the top 101:200 finishers: 00:41:51 (00:00:11) h:min:sec, p<0.001 and a smaller effect in annual winners: 00:17:25 (00:01:24) h:min:sec, p<0.001. In all finishers group, being men was a significant factor when interacted with country. Moreover, performances changed significantly over calendar year for both sexes in all groups except men winners, where variations were small. The greater smoothing terms were observed in top 101:200 group. In all finishers the trend was overall increasing, after decreasing in the first ten years. In Athletes from Kenya and Ethiopia were significantly the fastest compared to every country group. The differences varied between sexes and between groups of finishers. The greater effects were observed for all finishers and the smaller effects for annual top 10 finishers. For women in all finishers group, the differences ranged from a minimum of 01:22:04 (00:04:45) h:min:sec, i) an increase of average temperature by 1 \u00b0C was related to worsened performance with greater effect for all finishers, (ii) increasing barometric pressure was related to worsened performances, (iii) wet-bulb globe temperature, analogously, when increasing was related to worsened performances, (iv) tail wind was related to improved performances of all groups, (v) precipitations, when increasing, were related to worsened performances, (vi) increasing wind speed was also related to worsened performances for all finishers and near elite groups.The aim of the present study was to investigate the role of weather conditions, together with sex and country, on performance in the Boston Marathon from 1972 to 2018. Different groups and subgroups of performance level were considered such as all runners, near elite 101:200 finishers, near elite 21:100, annual top ten finishers and annual winners. The main findings were . It was well-known that there was a progressive slowing of marathon performance as the ambient temperature increased from 5 \u00b0C to 25 \u00b0C [A first important finding was that increasing temperature was related to impaired performance in almost all groups of runners (to 25 \u00b0C . For thito 25 \u00b0C . In conthttps://sciencing.com/temperature-affect-barometric-pressure-5013070.html).A second important finding was that increasing barometric pressure worsened running performance. Most likely the increased barometric pressure is linked to an increased ambient temperature which is known to impair marathon running performance. Warm air causes air pressure to rise and American marathon races from 2001 to 2010 through with 1,791,972 participants\u2019 performances were analyzed, atmospheric pressure at sea level showed no effect on running performance [However, when the results of six European (www.weather.gov/tsa/wbgt). Little is known for the effect of wet-bulb globe temperature on marathon running performance. Ely et al. [i.e. Boston, New York, Twin Cities, Grandma\u2019s, Richmond, Hartford, and Vancouver Marathons) for a different range of years (i.e. 6 to 36 years) and different quartiles based on wet-bulb globe temperature and female and male finishers of different performance levels were analysed. The most likely explanation for the different findings is the fact that we analyzed a point-to-point race where wind might have a different effect on wet-bulb globe temperature compared to race held in one or several laps. Based on these disparate findings, future studies need to investigate more deeply the relationships between wet-bulb globe temperature and marathon running performance in other marathon races held in one or more laps.A third important finding was that an increase in wet-bulb globe temperature was related to a worsened performance in all groups except annual winners.Wet-bulb globe temperature is nowadays the most widely used index of heat stress in direcy et al. reportedi.e. wind from W, WNW, and WSW) improved race performance of all groups and increasing wind speed was also related to worsened performances for all finishers and near elite groups. Boston is a \u2018point-to-point\u2019 marathon from west to east (www.baa.org/races/boston-marathon/course-map). When the runners have a tailwind, they get a \u2018push\u2019 for 42 km. It is well-known for this race that headwinds on the day of the race slow winning times [A further important finding was that tail wind to 00:41:51 h:min:sec (top 101:200 finishers). Differences between countries and sexes in all finishers, compared to the other selection groups, were relatively smaller. For instance, the interaction term USA:M reduced the comparison Kenya-Ethiopia and USA, for men, by 00:11:59 h:min:sec. This term was almost twice the respective term in annual winners group (00:06:07 h:min:sec). But in all finishers, the performance difference Kenya-Ethiopia and USA was of 01:36:43 h:min:sec, compared to 00:06:30 h:min:sec in the winners group. Therefore, the effect in the winners was greater.e.g. topography, temperature) and participation . Thus, they should be generalized with caution to other marathon races. Future studies would be needed to confirm these findings in other large city marathons. Moreover, no information about age was available and repeated measurements within runners could not be exactly identified, although it could be reasonably supposed that two observations belonged to the same runner if they had in common both name, surname, sex, country and participation, once a year, in the same period of time.The findings of the present study were limited by the specific characteristics of this race in terms of environmental conditions and the qualifying standards could favour one sex and/or age group over the other. It was generally regarded that it was easier for women to qualify for Boston Marathon than men, which might influence the results. Although qualification criteria exist, about 10,000 runners participated annually on sponsor loyalties. Most likely, the number of this latter group of runners has increased most over the year, whilst qualifiers were stable over the years. It could be expected that the qualified age-groupers would perform better than their free-entry counterparts; however, with this information not available this speculative hypothesis could not be tested critically.It should be also mentioned that Boston Marathon was the only large city marathon in the World with \u2018qualifying times\u2019 Click here for additional data file.S1 File(R)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S1 FigPoints were observed average of time race. Lines were fitted curves.(TIF)Click here for additional data file."} +{"text": "Correction to: Journal for ImmunoTherapy of Cancer (2019) 7:15https://doi.org/10.1186/s40425-018-0472-1Following publication of the original article , the autThe correct files Additional file 2:Code and data for the validation dataset analyses. The R code and data used in the colorectal and endometrial/neuroendocrine validation analyses are included in this zip file. Code executes in the directory in which it is placed. (ZIP 456\u2009kb)"} +{"text": "AbstractRuehssiawoodburyana (Apocynaceae) was recently discovered at a single location on Norman Island in the British Virgin Islands. Despite an increase in the extent of occurrence and area of occupancy, this species meta-population is very limited with a total of 37 individuals known in the wild. The largest subpopulation, on Mona Island, has only 26 individuals. The species suitable habitat is experiencing a continuing decline due to urban development, grazing by feral ungulates and human-induced forest fires. Conservation action is urgently needed and should be directed towards establishing genetically representative ex situ collections, such as seed for long term storage and live material for propagation. This species is evaluated as Critically Endangered (CR), based on Criteria C2a(i)+D, according to the IUCN Red List Categories and Criteria (version 3.1) and guidelines (Thought to be endemic to the Commonwealth of Puerto Rico, idelines .Ruehssiawoodburyana on Norman Island, in the British Virgin Islands and discuss the species conservation status. Marsdeniawoodburyana is transferred to the genus Ruehssia to reflect the resurrection of that genus for species of Marsdenia native to the New World.Extensive and regular surveys to the region enable the discovery of new plant records for different countries and islands. In this paper, we record a new island record for In this paper, we present a species conservation profile for an endemic species to the British Virgin Islands and to the Commonwealth of Puerto Rico.RuehssiawoodburyanaScientific name: Species authority: (Acev.-Rodr) GoyderMarsdeniawoodburyana Acev.-Rodr., 1999 \u2013 Synonyms: PlantaeKingdom: TracheophytaPhylum: MagnoliopsidaClass: GentianalesOrder: ApocynaceaeFamily: Marsdenia R.Br. (Apocynaceae: Asclepiadoideae) occur in a single clade, according to a recent study using two plastid and two nuclear gene regions and it is planned to transfer species from other parts of the Americas in subsequent papers.In order to expedite the range extension of M.woodburyana Acev.-Rodr. to the British Virgin Islands and to permit timely publication of its conservation status, we here propose the formal transfer of this species from Marsdenia to Ruehssia.Ruehssiawoodburyana (Acev.-Rodr.) Goyder, comb. nov.BASIONYM: Marsdeniawoodburyana Acev.-Rodr., 1999 \u2013 Taxonomic notes: All the native New World species of the broadly delimited pan-tropical genus regions . The genRegion for assessment: GlobalReviewers: Clubbe, C.; Urbaniak, J.Editor: Barrios, S.; Hamilton, M.A.Reviewers: Clubbe, C.; Urbaniak, J.Editor: Barrios, S.; Hamilton, M.A.Biogeographic realm: NeotropicalCountries: Virgin Islands, BritishPuerto RicoMap of records (image): Map of records (Google Earth): Basis of EOO and AOO: ObservedBasis (narrative): Observed occurrences.Min Elevation/Depth (m): 10Max Elevation/Depth (m): 150Ruehssiawoodburyana is a rare plant species restricted to the Commonwealth of Puerto Rico and the British Virgin Islands (BVI). This species was originally described occurring exclusively at Ca\u00f1a Gorda within the Gu\u00e1nica State Forest in Gu\u00e1nica municipality on the island of Puerto Rico (R.woodburyana (O. Monsegur pers. comm. 2017). This species extent of occurrence (EOO) was estimated to be 5,649 km2 and the area of occupancy to be 32 km2 based on a 2\u00d72 km cell size : 5649Trend: IncreaseCauses ceased?: UnknownCauses understood?: UnknownCauses reversible?: UnknownExtreme fluctuations?: NoTrend: IncreaseCauses ceased?: UnknownCauses understood?: UnknownCauses reversible?: UnknownExtreme fluctuations?: NoAOO (km2): 32Number of locations: 7-8Justification for number of locations: The number of locations was calculated to be seven to eight, considering threats posed by human disturbance and human-induced fires at the different sites where the species has been recorded.Trend: UnknownExtreme fluctuations?: NoNumber of individuals: 37Trend: UnknownCauses ceased?: UnknownCauses understood?: UnknownCauses reversible?: UnknownExtreme fluctuations?: UnknownPopulation Information (Narrative): Originally described from Gu\u00e1nica State Forest as extremely rare, Abundance largest subpopulation: 26Number of subpopulations: 4Trend: UnknownExtreme fluctuations?: NoSevere fragmentation?: NoSystem: TerrestrialHabitat specialist: UnknownHabitat (narrative): A woody vine which can grow to eight metres long in tropical dry forest Figs . This spTrend in extent, area or quality?: Decline (observed)Habitat importance: Major ImportanceHabitats: 1.5. Forest - Subtropical/Tropical DryHabitat importance: Major ImportanceHabitats: 1.5. Forest - Subtropical/Tropical DryGeneration length (yr): 0Dependency of single sp?: NoEcology and traits (narrative): The generation length of this vine is unknown. More field observations are required.Justification for threats: This species is subjected to a variety of threats. Most locations are threatened by human disturbance which is causing habitat degradation and fragmentation, particularly through urban development and fire. In Puerto Rico, human-induced fires are frequent in Gu\u00e1nica State Forest along road PR 333 near Ca\u00f1a Gorda, the type locality. These seriously affect the quality of this species suitable habitat and may preclude the species natural recruitment. The habitat on Norman Island in the BVI was degraded by feral animals in the past, but these have now been removed, promoting the recovery of native vegetation. Despite the presence of feral goats and pigs on Mona Island, these animals are not thought to be impacting the species as feral mammal populations are managed through sports hunting. However, it is noted that no recruitment has been observed at this location in the recent years (J. Sustache pers. comm. 2018). At Laguna Cartagena and Cabo Rojo National Wildlife Refuges, there is no direct evidence of impact due to human-induced fires or feral animals, despite the presence of these threats. Within the municipality of Pe\u00f1uelas, this species suitable habitat is threatened by the expansion of industrial landfills, service roads and utility lines (O. Monsegur pers. comm. 2018). Climate change might already be impacting this species through more severe droughts and stronger tropical storms.Threat type: OngoingThreats: 1.3. Residential & commercial development - Tourism & recreation areas4.1. Transportation & service corridors - Roads & railroads4.2. Transportation & service corridors - Utility & service lines7.1. Natural system modifications - Fire & fire suppression11.2. Climate change & severe weather - DroughtsThreat type: OngoingThreats: 1.3. Residential & commercial development - Tourism & recreation areas4.1. Transportation & service corridors - Roads & railroads4.2. Transportation & service corridors - Utility & service lines7.1. Natural system modifications - Fire & fire suppression11.2. Climate change & severe weather - Droughtsex situ collections for this species despite attempts in recent years to collect seed from southern municipalities on the island of Puerto Rico (J. Sustache pers. comm. 2018).Justification for conservation actions: This species is found within protected areas across its natural range. In the Commonwealth of Puerto Rico, the species is recorded as occurring within the Gu\u00e1nica State Forest, Laguna Cartagena National Wildlife Refuge, Cabo Rojo National Wildlife Refuge and Mona Island Nature Reserve. It is also thought to occur within the Culebra National Wildlife Reserve, but further surveys are needed. Norman Island in the BVI is not a protected area, as it is privately owned. This species is listed as a Critical Element by the Department of Natural and Environmental Resources . MonseguConservation action type: NeededConservation actions: 1.2. Land/water protection - Resource & habitat protection3.4. Species management - Ex-situ conservation4.3. Education & awareness - Awareness & communicationsConservation action type: NeededConservation actions: 1.2. Land/water protection - Resource & habitat protection3.4. Species management - Ex-situ conservation4.3. Education & awareness - Awareness & communicationsJustification for use and trade: There are no known uses for this species.Justification for ecosystem services : Insufficient Information availableUse type: InternationalEcosystem service type: Less importantResearch needed: 1.2. Research - Population size, distribution & trends1.3. Research - Life history & ecology3.4. Monitoring - Habitat trendsex situ conservation collections. Further surveys are needed to look for potential undetected individuals and subpopulations within the species range, particularly in the US and British Virgin Islands. The areas and habitats, where this species occurs, should be closely managed and monitored.Justification for research needed: Conservation action and research should be directed to develop a better understanding of this species' ecology and population trends and develop Use type: InternationalEcosystem service type: Less importantResearch needed: 1.2. Research - Population size, distribution & trends1.3. Research - Life history & ecology3.4. Monitoring - Habitat trendsex situ conservation collections. Further surveys are needed to look for potential undetected individuals and subpopulations within the species range, particularly in the US and British Virgin Islands. The areas and habitats, where this species occurs, should be closely managed and monitored.Justification for research needed: Conservation action and research should be directed to develop a better understanding of this species' ecology and population trends and develop 6355BA10-D648-52AF-8F97-9E52B988512B10.3897/BDJ.7.e47110.suppl1Supplementary material 1Ruehssiawoodburyana known recordsGoogle Earth map showing Data type: occurencesFile: oo_307399.kmlhttps://binary.pensoft.net/file/307399Barrios, S.; Hamilton, H.; Monsegur, O.; Sustache, J."} +{"text": "Medical Law Review, 2019, doi:10.1093/medlaw/fwz009In the original version of this article, the following funding acknowledgement was mistakenly omitted:http://www.i-sense.org.uk>).James Wilson's research was supported by the i\u2010sense EPSRC IRC in Early Warning Sensing Systems for Infectious Diseases (Grant reference number EP/K031953/1, rol-6 repair template oligo. Additionally, 100\u00a0ng/\u03bcl crRNA against the locus of interest as well as 100-200ng/\u03bcl dsDNA repair template encoding the desired modification with around 50bp flanking homology arms were added to the mix. Note that for efficient degradation of DRSH-1 we needed to insert AID degrons at the N- and C-termini simultaneously as either degron alone was insufficient for depletion. Guide RNAs used and sequences inserted (in bold) were as follows:drsh-1 \u2013 N-terminal AID-tag \u2013 guide sequence used\u00a0= 5\u2032- TTAGATTTTCATTTAGATGT-3\u2032 \u2013 locus post-edit:ATGCCTAAAGATCCAGCCAAACCTCCGGCCAAGGCACAAGTTGTGGGATGGCCACCGGTGAGATCATACCGGAAGAACGTGATGGTTTCCTGCCAAAAATCAAGCGGTGGCCCGGAGGCGGCGGCGTTCGTGAAGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGACTACAAGGATGACGATGACAAGGGAGGAAGCGGAGGAGGAAGCGGTGGAGGAAGCGGAGGAGGAAGCGGAATGGAACAGAAACTCATCTCTGAAGAGGATCTGATGTCGGACGAAAAGATTTCAATGACGCTTAACTTCCCGAAACACAAGCGTAGTTAACGTTTTCATCTGAAATTGTAGACAGATTTAGATTTTCATTTAGdrsh-1 \u2013 C-terminal AID-tag - guide sequence used\u00a0= 5\u2032-GATACCAGCGACTAATTACG-3\u2032 \u2013 locus post-edit:ATGCCTAAAGATCCAGCCAAACCTCCGGCCAAGGCACAAGTTGTGGGATGGCCACCGGTGAGATCATACCGGAAGAACGTGATGGTTTCCTGCCAAAAATCAAGCGGTGGCCCGGAGGCGGCGGCGTTCGTGAAGGAACAGAAACTCATCTCTGAAGAGGATCTGTAGTTACGGGGTTATAATTATACTATGTCTGTTTGAATGTGATTCGGTTCTCAAGTGGTTTCAGAACATGCGCCGTCGTCTTGAACAAGATACCAGCGACGGAGGCAGCGGTGGTGGAAGCGGCGGGGGAAGCGGCGGTGGAAGCGGTGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGACTACAAGGATGACGATGACAAGpash-1 \u2013 C-terminal AID-tag \u2013 guide sequence used\u00a0= 5\u2032-AGGTGAATATACTATTTGTG-3\u2032 \u2013 locus post-edit:ATGCCTAAAGATCCAGCCAAACCTCCGGCCAAGGCACAAGTTGTGGGATGGCCACCGGTGAGATCATACCGGAAGAACGTGATGGTTTCCTGCCAAAAATCAAGCGGTGGCCCGGAGGCGGCGGCGTTCGTGAAGGAACAGAAACTCATCTCTGAAGAGGATCTGTAGGGAGGAAGCGGAGGAGGAAGCGGAGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGACTACAAGGATGACGATGACAAGTATATTCACCTCATATGTTTGTTGTTTTGTTGGTAGTTTTAATTTTTGGGGAAGACATGACGATTCATCATCCCCATCACATCAGAAACCACACAAAInserted sequences contained the TIR-1 recognition peptide , a FLAG (GACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGACTACAAGGATGACGATGACAAG) and a MYC (GAACAGAAACTCATCTCTGAAGAGGATCTG) tag, as well as GGSG linkers in between.To generate AID-tagged copies of ribed by , with moribed by . Brieflypash-1(ts) allele mj100 were kept constantly at the permissive temperature of 16\u00b0C. pash-1(ts) animals expressing the mirtron-51 transgene were selected every few generations for high levels of the co-expression marker elt-2p::dsRed, as this transgene was prone to undergo silencing. For experiments, L4 stage larvae were transferred to a fresh plate and shifted to 25\u00b0C approximately 16h later. In order to achieve maximal depletion of maternal miRNAs, mothers were kept at the restrictive temperature an additional 7h before harvesting early embryos (1-4 cell stage). Extending the time at the restrictive temperature was prohibited by the rapidly deteriorating health of gravid pash-1(ts) adults expressing mirtrons at 25\u00b0C. For backshift-experiments with mirtron-rescued pash-1(ts) animals, hatched L1s that developed under restrictive conditions were transferred to a fresh plate within 1h after hatching, allowed to develop at 16\u00b0C, and scored for their ability to reach adulthood within 7\u00a0days.Animals bearing the E.\u00a0coli OP50 at 20\u00b0C. For combined AID and RNAi treatment, NGM plates were supplemented with 1mM Carbenicillin, 1mM IPTG and 4mM Auxin . RNAiD plates were seeded with E.\u00a0coli HT115 expressing dsRNA which elicits an RNAi response against pash-1 mRNA library, ORF-ID T22A3.5). To ensure efficient mRNA knockdown, RNAi bacteria were grown freshly for every experiment in liquid LB culture, the medium was brought to 1mM IPTG around 1h before pelleting bacteria, and plates were seeded with a 20X concentrate . For experiments, L4s were transferred to RNAiD plates, embryos were harvested 24h later, and assayed as described below. While the AID-system rapidly depletes proteins within minutes to hours [pash-1(ts) animals, eventually owed to the temperature difference and/or deleterious side-effects of PASH-1(TS) at 25\u00b0C. Efficiency of RNAi-mediated pash-1 mRNA knockdown was assessed via RT-qPCR. Briefly, 5-10 embryos in the 2-cell stage were transferred to about 1\u00a0\u03bcl of Lysis Buffer using a thin glass needle. These samples were subjected to 10\u00a0min digestion at 65\u00b0C before heat inactivation of proteinase K for 1\u00a0min at 85\u00b0C. Next, crude lysates were reverse transcribed before performing qPCR using the GoTAQ qPCR Mastermix (Promega) according to the manufacturer\u2019s instructions. Relative expression was calculated according to the \u0394\u0394Cq-method using cdc-42 as a reference gene, the following primer sequences were employed:pash-1-F: 5\u2032-GCCTTCGAGAAAACGGGGAA-3\u2032; pash-1-R: 5\u2032-TGGCTCCCATTTCGGAGATT-3\u2032;drsh-1-F: 5\u2032-TGAGCTGGCTTTGGCTAATCT-3\u2032; drsh-1-R: 5\u2032-ACCCCGTAATTAGTCGCTGG-3\u2032cdc-42-F: 5\u2032-TGGGTGCCTGAAATTTCGC-3\u2032; cdc-42-R: 5\u2032-CTTCTCCTGTTGTGGTGGG-3\u2032Animals were kept constantly on standard NGM plates seeded with To assess the extent of protein degradation of PASH-1:AID:MYC and DRSH-1:AID:MYC upon Auxin treatment, L4s were transferred to Auxin plates prepared as described above. 24h later, gravid adults were collected in L\u00e4mmli-buffer containing 2.5% \u03b2-Mercaptoethanol (v/v), and subjected to multiple cycles of snap-freezing followed by boiling until embryos were disrupted. Proteins were separated via SDS-PAGE and transferred onto a Nitrocellulose membrane. As primary antibodies either monoclonal mouse anti-myc or a polyclonal rabbit anti-gamma tubulin were used. As secondary antibody, anti-mouse IgG HRP-linked Antibody or anti-rabbit IgG HRP-linked Antibody was applied followed by visualization using ECL reagent (Thermo Scientific).Embryos were obtained from day 1 gravid adults reared as described above, by slicing mothers in a drop of M9 buffer on a microscopic slide. 2-cell embryos of normal size were transferred to standard NGM plates or RNAiD plates and allowed to develop at the respective temperatures. Hatched animals were collected for subsequent analysis from plates within 1h after hatching. The final hatching rate was assessed > 24h after embryo collection by scoring the fraction of animals that successfully escaped the eggshell. To obtain samples at specific developmental stages for small RNA sequencing, 2-cell embryos were harvested via slicing, collected or allowed to develop on NGM plates before being selected manually by stage at given time points .myo-2prom::mCherry. Mean and range are plotted for each genotype; unpaired t test was used for statistical comparison.For phenotypic analysis of embryonic arrest or hatched L1 larvae, animals were mounted on a thin agar-pad on a microscopic slide sealed with a coverslip. Images were recorded at 400x magnification using an AxioImager Z2 (Zeiss) equipped with DIC and fluorescence optics, and analyzed via ImageJ. Arrest phenotypes were scored as follows: cell mass\u00a0= embryo fails to initiate bean stage; morphogenesis\u00a0= embryo begins to elongate but fails to reach 2-fold stage; elongation\u00a0= embryo completes 2-fold stage but arrests before hatching. Body measurements are based on DIC micrographs. Body length was measured as the length of a segmented path running through the mid body axis from head to tail. Body width was assessed as the arithmetic mean of 3 independent width measurements in the anterior, middle, and posterior part of each animal. Pharynx length was determined by the length of a segmented path running through the pharynx middle axis in animals expressing To profile miRNA levels, a modified version of the small RNA sequencing protocol described in was perf18-30nt-NNNNNNXXXXXAGATCGGAAGAGCAC-ACGTCT/3ddC/-3\u2032. After sufficient PCR-amplification as observed by SYBR green derived qPCR signal (requiring around 16-20 cycles), libraries were size-selected on an agarose gel to remove adaptor dimers and sequenced on a HiSeqV4 platform (Illumina). Small RNA reads were mapped and processed as in [https://github.com/lengfei5/smallRNA_nf/tree/master/dev_sRBC. To accurately quantify sRNAs, both mapped sequences and associated random nucleotides (functioning as UMIs) within the raw reads were quantified for miRNAs and piRNAs as well as spike-ins. To alleviate PCR amplification bias, UMI counts were used and normalized to spike-in as described in [https://github.com/lengfei5/smallRNA_analysis_philipp/tree/master/scripts. Every round of library preparation included one sample of a comparable amount of Arabidopsis thaliana total RNA to assess the extent of potential contamination. The average counts for reads mapping to C.\u00a0elegans miRNAs in contamination control samples (\u223c1% of the wild-type miRNA content) across four independent library preparations were subtracted from all samples as a background correction. For background-corrected, spike-in normalized, miRNA counts in molecules per embryo, see (XXXXX\u00a0= barcode): 5\u2032-ACACUCUUUCCCUACACGACGCUCUUCCGAUCUNNNN-(sRNA)ed as in . Modificed as in . The modribed in , allowinPlots were created using Prism, statistical analysis was also performed in Prism as described in the figure legends. For sRNaseq data statistical analysis was performed using DESeq2 . Figures"} +{"text": "Multiple HBV-derived T cell epitopes have been reported, which can be useful in a therapeutic vaccination strategy. However, these epitopes are largely restricted to HLA-A*02, which is not dominantly expressed in populations with high HBV prevalence. Thus, current epitopes are falling short in the development of a global immunotherapeutic approach. Therefore, we aimed to identify novel epitopes for 6 HLA supertypes most prevalent in the infected population. Moreover, established epitopes might not all be equally effective as they can be subject to different levels of immune escape. It is therefore important to identify targets that are crucial in viral replication and conserved in the majority of the infected population. Here, we applied a stringent selection procedure to compose a combined overview of existing and novel HBV-derived T cell epitopes most promising for viral eradication. This set of T cell epitopes now lays the basis for the development of globally effective HBV antigen-specific immunotherapies. in vitro assay to validate their HLA-binding capacity. Using this method, a total of 13 HLA binders derived from HBx and 33 binders from Pol were identified across HLA types. Subsequently, we demonstrated interferon gamma (IFN-\u03b3) production in response to 5 of the novel HBx-derived binders and 17 of the novel Pol-derived binders. In addition, we validated several infrequently described epitopes. Collectively, these results specify a set of highly potent T cell epitopes that represent a valuable resource for future HBV immunotherapy design.Immunotherapy represents an attractive option for the treatment of chronic hepatitis B virus (HBV) infection. The HBV proteins polymerase (Pol) and HBx are of special interest for antigen-specific immunotherapy because they are essential for viral replication and have been associated with viral control (Pol) or are still expressed upon viral DNA integration (HBx). Here, we scored all currently described HBx- and Pol-derived epitope sequences for viral indispensability and conservation across all HBV genotypes. This yielded 7 HBx-derived and 26 Pol-derived reported epitopes with functional association and high conservation. We subsequently predicted novel HLA-binding peptides for 6 HLA supertypes prevalent in HBV-infected patients. Potential epitopes expected to be the least prone to immune escape were subjected to a state-of-the-art IMPORTANCE Multiple HBV-derived T cell epitopes have been reported, which can be useful in a therapeutic vaccination strategy. However, these epitopes are largely restricted to HLA-A*02, which is not dominantly expressed in populations with high HBV prevalence. Thus, current epitopes are falling short in the development of a global immunotherapeutic approach. Therefore, we aimed to identify novel epitopes for 6 HLA supertypes most prevalent in the infected population. Moreover, established epitopes might not all be equally effective as they can be subject to different levels of immune escape. It is therefore important to identify targets that are crucial in viral replication and conserved in the majority of the infected population. Here, we applied a stringent selection procedure to compose a combined overview of existing and novel HBV-derived T cell epitopes most promising for viral eradication. This set of T cell epitopes now lays the basis for the development of globally effective HBV antigen-specific immunotherapies. Chronic hepatitis B virus (CHB) infection affects roughly 250 million people worldwide and is aImmunotherapy had already emerged in the 1990s as a promising option to treat CHB. T cell responses are considered essential for viral clearance but are scarce or exhausted in CHB patients \u20137. Still\u20138\u2013in vivo. Others have explored strategies to predict HLA-I epitopes from Pol but focused exclusively on a single HLA type or assessed only a limited number of HBV sequences or core antigen (HBcAg). However, the proteins X (HBx) and polymerase (Pol) also pose interesting targets, as both are vital for viral persistence \u201327 and i\u201330\u2013equences \u201340. TakeIn addition to a limited epitope repertoire, there is another hurdle in the development of HBV-directed immunotherapy. Established epitopes might not all be equally effective, as they can be subjected to different levels of viral mutagenesis and subsequent immune escape. Indeed, previous reports clearly demonstrate that HBV is subject to immune pressure and that mutation of epitope sequences leads to immune evasion \u201343 or ev\u201346\u2013Here, we have taken an effort to tackle the above-mentioned issues by integrating viral indispensability, genomic variation, HLA binding, and immunogenicity to identify the best HBx- and Pol-derived T cell epitopes for immunotherapy across 6 of the most prevalent HLA supertypes within the HBV-infected population. The results of this study pave the way for the development of globally effective HBV antigen-specific immunotherapies.n\u2009=\u200914) and Pol (n\u2009=\u200950) , left, w , left, (n\u2009=\u200950) Fig. 1,,n\u2009=\u200914 a\u2009=\u200950) 4648, 52\u201369We argued that epitopes in which the least conserved amino acid was still present in 80% of all sequences tested would be targetable in the majority of the population. Combining this criterion with the preference for functional association, we found 7 HBx-derived and 26 Pol-derived sequences reported as epitopes across HLA types to be preferential targets for global immunotherapy . We first predicted binders spanning 8 to 14 amino acids for supertype-representative HLA types using the established in silico prediction tool NetMHCpan to make a frequency distribution of predicted binders (P\u2009=\u20090.57 by a Mann-Whitney test). Predicted binders spanning 9 to 11 amino acids were subsequently aligned to our maps outlining conservation and function .The prediction yielded totals of 251 potential novel HLA binders for HBx and 1,655 for Pol , right, ed assay . For praed assay . For HLAin vitro binding assay. Peptides were classified as HLA binders when their binding capacity was higher than 25% of that of a known high-affinity peptide (Table S4). HLA-A*11:01 and HLA-A*03:01 were both tested as members of the HLA-A*03 supertype since many HBV-infected patients are Asian and HLA-A*11:01 is more prevalent in this population than the supertype representative HLA-A*03:01, which is more prevalent in Caucasians from blood donors who had previously resolved an HBV infection were expanded in the presence of peptide pools, followed by single-peptide restimulation and an interferon gamma (IFN-\u03b3) enzyme-linked immunosorbent assay (ELISA). As expected, IFN-\u03b3 production was detected in response to the well-established epitopes c18-27 and p549-557 . FurtherThe aim of this study was to rationally address two major hurdles in developing a generic antigen-based immunotherapy for HBV: (i) the lack of prioritization of epitopes in further studies toward clinical implementation and (ii) the shortage of non-HLA*02-restricted epitopes.To address the first issue, we ranked all currently described HBx- and Pol-derived epitopes according to conservation and association with viral indispensability. Conservation patterns were similar to those previously reported, with the most conspicuous observation being that the spacer domain of Pol is extremely variable , 42, 73.Four Pol-derived epitopes were more frequently described in acute or resolved infection than in chronic infection. Of these, p756-764 and p573-581 scored the highest in conservation. p756-764 contains at least 1 amino acid important for viral persistence, but no functional relevance has been described for p573-581. Since the DNA sequence coding for p573-581 completely overlaps that of HBsAg, we interrogated the literature for the functional relevance of amino acids in this overlapping part of HBsAg. However, no functional association was reported 7375. Altho76\u2013Although we rationalized to prioritize conserved peptide sequences, we reckon that regions containing prevalent sequence variation could still be interesting if immunogenicity is preserved. Novel T cell responses may arise due to cross-reaction between the variant and the original sequence , 81. Thi+ epitopes, while HBx seemed more subject to immune-pressure-induced deletion of epitopes vaccines. SLPs can be designed to harbor both HLA-I and HLA-II epitopes and are processed more efficiently by dendritic cells than whole proteins types covering the vast majority of the HBV-infected population that target the virus where it is most vulnerable. Collectively, the results of this study provide a valuable resource to guide future development of HBV-specific immunotherapies.n\u2009=\u20098,127) and Pol . Positions where a gap (indicated by \u201c\u2212\u201d) was most frequent were deleted, after which the dominating amino acid at each position was determined. Percentages of sequences containing the dominant amino acid were calculated as the conservation score. Combining all dominant amino acids for Pol led to the consensus sequence MPLSYQHFRKLLLLDDEAGPLEEELPRLADEGLNRRVAEDLNLGNLNVSIPWTHKVGNFTGLYSSTVPVFNPEWQTPSFPDIHLQEDIINRCQQFVGPLTVNEKRRLKLIMPARFYPNVTKYLPLDKGIKPYYPEHVVNHYFQTRHYLHTLWKAGILYKRETTRSASFCGSPYSWEQELQHGRLVFQTSKRHGDESFCSQSSGILSRSPVGPCIQSQLKQSRLGLQPQQGSLARRQQGRSGSIRARVHPTTRRSFGVEPSGSGHIDNSASSSSSCLHQSAVRKAAYSHLSTSKRQSSSGHAVELHNIPPSSARSQSEGPVFSCWWLQFRNSKPCSDYCLSHIVNLLEDWGPCTEHGEHHIRIPRTPARVTGGVFLVDKNPHNTTESRLVVDFSQFSRGNTRVSWPKFAVPNLQSLTNLLSSNLSWLSLDVSAAFYHLPLHPAAMPHLLVGSSGLSRYVARLSSNSRIINNQHGTMQNLHDSCSRNLYVSLLLLYKTFGRKLHLYSHPIILGFRKIPMGVGLSPFLLAQFTSAICSVVRRAFPHCLAFSYMDDVVLGAKSVQHLESLYTAVTNFLLSLGIHLNPNKTKRWGYSLNFMGYVIGSWGTLPQEHIVQKIKQCFRKLPVNRPIDWKVCQRIVGLLGFAAPFTQCGYPALMPLYACIQAKQAFTFSPTYKAFLCKQYLNLYPVARQRPGLCQVFADATPTGWGLAIGHQRMRGTFVAPLPIHTAELLAACFARSRSGAKLIGTDNSVVLSRKYTSFPWLLGCAANWILRGTSFVYVPSALNPADDPSRGRLGLYRPLLRLPFRPTTGRTSLYAVSPSVPSHLPDRVHFASPLHVAWRPP.A frequency table was downloaded from HBVdb V42.0 , 51 baseThe resulting consensus sequence for HBx was determined to be MAARLCCQLDPARDVLCLRPVGAESRGRPLSGPLGTLPSPSPSAVPADHGAHLSLRGLPVCAFSSAGPCALRFTSARRMETTVNAHQVLPKVLHKRTLGLSAMSTTDLEAYFKDCVFKDWEELGEEIRLKVFVLGGCRHKLVCSPAPCNFFTSA.in vitro binding assay as described below.These sequences were loaded into NetMHCpan3.0 to prediin vitro binding assay as described previously (Synthetic peptides (Peptide 2.0 Inc.) of selected potential HLA binders were used in an eviously . In briein vitro HLA binding assay were assessed for immunogenicity. Briefly, PBMCs were isolated by Ficoll density centrifugation from buffy coats of 9 donors who had previously resolved HBV infection. Buffy coats were provided by the local blood bank with corresponding 2-digit HLA types. Four-digit HLA typing was performed for 7 out of 9 donors using the global screening array (GSA) (Peptides with >25% binding in the tterdam) . All donP value was <0.05. For the few epitopes with a response in at least 25% of HCC patients, patient groups could not be compared due to the lack of data for CHB and acute hepatitis patients.The Hepitopes initiative previously performed an extensive literature search of the Medline and Embase databases to collect all HBV-derived HLA-I epitopes from 112 papers , 49. All55\u2013"} +{"text": "Bungarus multicinctus that binds irreversibly to the acetylcholine receptor extracellular domain, to the pH sensitive GFP Super Ecliptic pHluorin, and efficiently expressed it in Pichia pastoris. This sensor allows synaptic changes in pH to be measured without the need of incorporating transgenes into animal cells. Here, we show that incubation of the mouse levator auris muscle with a solution containing this recombinant protein is enough to fluorescently label the endplate post synaptic membrane. Furthermore, we could physiologically alter and measure post synaptic pH by evaluating changes in the fluorescent signal of pHluorin molecules bound to acetylcholine receptors. In fact, using this tool we were able to detect a drop in 0.01 to 0.05 pH units in the vicinity of the acetylcholine receptors following vesicle exocytosis triggered by nerve electrical stimulation. Further experiments will allow to learn the precise changes in pH during and after synaptic activation.Synaptic transmission triggers transient acidification of the synaptic cleft. Recently, it has been shown that pH affects the opening of postsynaptic channels and therefore the production of tools that allow to study these behaviors should result of paramount value. We fused \u03b1-bungarotoxin, a neurotoxin derived from the snake During synaptic transmission, the synaptic vesicle fuses to the presynaptic membrane and its content is released into the synaptic cleft. During this event, vesicular pH rises and falls again after endocytosis1, which is consistent with proton release into the synaptic cleft. Taking into account the very small volume of a synaptic vesicle (2\u2009\u00d7\u200910 \u221220 L) and the estimated pH before exocytosis (around 5.5), it has been calculated that there is less than one free proton per vesicle3. However, upon vesicle opening a process which includes deprotonation of proteins and neurotransmitters takes place and over 50 protons are released per vesicle3. Therefore, the synaptic cleft, which is a space 50\u00a0nM wide, is exposed to changes in pH due to the release of vesicle content and also to transmembrane fluxes owing to Ca2+/H+ exchange by the plasma membrane Ca2+ ATPase4. A transient drop in extracellular pH has been reported during synaptic activity in hippocampal sections5. It has been proposed that in photoreceptor terminals the liberation of vesicular protons results in an acidification of the synaptic cleft from 7.5 to 6.9, depending on the feedback inhibition of Ca2+ channels6. This study also showed that pH oscillations depend on the concentration of protons in the extracellular solution and that acidification is insignificant in acutely dissociated terminals, demonstrating that an intact synaptic cleft is essential.The content of synaptic vesicles has been shown to be acid1, a pH-sensitive green fluorescent protein, was fused to the extracellular domain of a postsynaptic membrane protein and used in lateral amygdala pyramidal neurons to report changes in extracellular pH at dendrite synapses7. After stimulation, the transfected pyramidal neurons transiently acidified neighboring dendrites and spines, a phenomenon which was followed by a slower increase in pH. The extent of pH fluctuations depended on stimulus frequency although quantification of the changes in proton concentration was not reported. In contrast to the established dogma, Stawarski et al. recently used genetically encoded fluorescent pH indicators to demonstrate that glutamatergic synaptic clefts alkalinize rather than acidify during neurotransmission at both the mouse calyx of Held and Drosophila neuromuscular junction (NMJ)8. Therefore, the exact changes in pH during synaptic transmission are not fully resolved.Recently, Super Ecliptic pHluorin (SEP)Bungarus multicinctus that binds irreversibly to the acetylcholine receptor extracellular domain, to SEP1 and efficiently expressed this fusion protein in Pichia pastoris. Using this tool, we were able to detect a drop in pH in the vicinity of the acetylcholine receptors following vesicle exocytosis triggered by nerve electrical stimulation.Changes in pH at the synaptic cleft at the mammalian NMJ have not been documented. With this aim, we developed a probe by fusing \u03b1 bungarotoxin (BTX), a neurotoxin derived from the snake P. pastoris and purified, resulting in an active protein of the expected size for de fusion ) was fitted to the data, where y0 represents the offset, ymax the dynamic range, and pK the pH where fluorescence is half maximal, a value that corresponds to minus the logarithm of the equilibrium constant for protonation9. The pK from the fit was 7.36.The sensitivity to pH of the SEP-BTX fusion protein probe was investigated by measuring the fluorescence intensity of the NMJ incubated at different pHs in a high capacity buffer (HEPES buffer 10\u00a0mM). A fluorescence vs pH curve was constructed taken the value at pH 9 as the maximal fluorescent output Fig.\u00a0A,B and t+ channel blocker diaminopiridine , known to increase the amplitude and prolong the time course of transmitter release10. As a result of DAP addition, fluorescence changes were significatively stronger on neuromuscular transmission17. Activation of ASICs requires changes in pH of 0.5 units or more, a value not reached in our measurements. One possibility is that the sensor also binds to other acetylcholine receptors located outside the synaptic cleft. Since these regions would not be exposed to the release of synaptic vesicle content, this event could mask the changes in pH reported by our SEP-BTX sensor in the synaptic cleft. However, it has been established that extra synaptic acetylcholine receptors are not expressed in normal adult mouse muscle both, by iontophoretic application of acetylcholine19 and by using labelled bungarotoxin20. Moreover, we focused the detection of fluorescent changes at the synaptic spot where only synaptic acetylcholine receptors are located. Secondly, it is worth mentioning that our measurements are highly dependent on the distance of the probe in relation with the source of the protons, which in our case is the synaptic cleft width. The high mobility of protons21 and the presence of buffers in the media could result in a very fast transient non accumulative increase in proton concentration not resolved with our detection system. Furthermore, the rapid dissipating proton concentration during the intervals between evoked transmitter release (10\u00a0ms at 100\u00a0Hz and 3.3\u00a0ms at 300\u00a0Hz) will strongly down average the fluorescent signal.Our experimental results using SEP-BTX have shown that acidification takes place at the NMJ synaptic cleft, confirming previous reports in other synapses23, we were able to show for the first time an acidification process due to transmitter release at the mouse NMJ. Synaptic cleft acidification and alkalization are probably not mutually exclusive processes. While they could operate in different types of synapses, it is also feasible that they occur at the same synapsis but with different kinetics. For example, following a rapid drop in pH, an over compensating mechanism driven by the Ca2+/H+ antiporter activity of plasma membrane Ca2+ ATPase4 and aimed at maintaining proton homeostasis could result in transient alkalinization8. Further experimental development of sensors located at the presynaptic membrane close to the vesicle release site and a fast image recording system are needed to obtain more realistic measurements of changes in the pH at the synaptic cleft.In summary, using a novel approach, in contrast to other reports showing an alkalization at CNS synapses9 in frame with BTX and followed by a TEV protease site and a 6XHis tag was subjected to codon optimization for expression in P. pastoris and synthetized by Integrated DNA Technologies (IDT). The sequence of the cloned DNA fragment, including EcoRI sites, is as follows:The DNA fragment containing SEPEcoRI site of pPic9 (Invitrogen Life Technologies Inc.) in frame with Saccharomyces cerevisiae \u03b1-factor signal sequence.5\u2032GAATTCTCTAAGGGTGAAGAGCTTTTCACCGGCGTGGTTCCTATTCTTGTGGAATTGGATGGTGACGTCAATGGTCACAAATTTTCAGTGTCAGGTGAAGGGGAGGGTGACGCCACTTACGGTAAATTGACTTTGAAGTTTATATGCACTACCGGCAAATTGCCAGTTCCATGGCCTACCTTGGTTACTACCTTAACTTACGGTGTTCAATGCTTTTCTAGATATCCTGACCATATGAAGAGACATGATTTTTTCAAGTCAGCCATGCCAGAGGGATATGTACAAGAACGTACAATTTTTTTTAAGGATGACGGTAATTACAAGACTCGTGCAGAAGTTAAGTTCGAAGGCGACACGCTGGTCAATCGTATTGAACTGAAGGGTATTGACTTCAAGGAGGATGGGAACATACTAGGACACAAATTAGAATATAATTATAACGATCACCAGGTTTACATTATGGCTGACAAACAAAAGAACGGCATTAAAGCTAACTTTAAAATTCGTCATAATATTGAGGATGGGGGAGTCCAATTAGCAGATCATTACCAACAGAATACACCAATAGGAGATGGTCCAGTGCTGTTGCCTGATAATCACTATCTTTTCACTACTTCTACTTTATCTAAAGACCCAAACGAGAAGAGGGACCATATGGTTTTGCTGGAATTTGTGACTGCAGCCGGGATTACTCACGGTATGGACGAGCTTTATAAAGGGCACGTCGGGATAGTTTGCCATACGACAGCCACCTCCCCCATTTCTGCTGTTACCTGTCCTCCAGGCGAAAACCTGTGCTACAGAAAAATGTGGTGTGATGCCTTCTGTTCTTCACGTGGTAAAGTTGTCGAGCTGGGATGCGCTGCCACCTGTCCTAGTAAGAAACCTTATGAAGAAGTAACATGTTGCTCTACAGATAAATGTAATCCACATCCTAAACAACGTCCTGGTTCGCGAGAGAACCTGTACTTTCAAGGACCCGGGCATCATCATCACCATCATTAAATTTAAATGAATTC3\u2032. This fragment was cloned into the P. pastoris was previously described24. Basically, pPic9-SEP-BTX-TEV-6XHis vector was linearized with DraI and used for transformation of P. pastoris strain GS115 (Invitrogen Life Technologies) by electroporation, and recombinant clones reverting histidine auxotrophy were selected on minimal MD plates 2SO4, 2% dextrose and 2% agar). Selection of clones expressing and secreting SEP-BTX was performed by transferring colonies to a nitrocellulose membrane placed on plates of MM minimal medium 2SO4, and 2% agar) with methanol as sole carbon source, for induction of the AOX1 promoter . After 4\u00a0days of growth at 30\u00a0\u00b0C, the nitrocellulose membranes were washed with distilled water and the secreted protein was revealed by western blot using a polyclonal rabbit anti-HIS antibody . For SEP-BTX production, recombinant clones were grown in 50\u00a0mL of BMGY medium 2SO4, 400\u00a0mg/L biotin, 1% glycerol, 100\u00a0mM potassium phosphate buffer, pH 6.0) for 48\u00a0h at 30\u00a0\u00b0C and 220\u00a0rpm. Cells were harvested by centrifugation for 5\u00a0min at 1500\u00a0g and resuspended in BMMY medium 2SO4, 400\u00a0mg/L biotin, 200\u00a0\u00b5M CuSO4 and 3% sorbitol) to a final OD600 nm\u2009=\u200910 and cultivated in 1000\u00a0mL shake flasks at 28\u00a0\u00b0C and 220\u00a0rpm. Sterile methanol was added every 24\u00a0h to maintain induction conditions.The protocol for protein expression in P. pastoris was previously described24. Briefly, P. pastoris cultures were harvested after 4\u00a0days of induction in BMMY and centrifuged at 1500\u00a0g for 10\u00a0min. The extracellular supernatant was concentrated by ultrafiltration and buffer exchanged to 300\u00a0mM NaCl, 50\u00a0mM sodium phosphate buffer, pH 8, containing 1\u00a0mM PMSF. SEP-BTX was purified by gravity flow Ni\u2013NTA affinity chromatography using a \u201cHis select\u201d nickel affinity gel , eluted with 300\u00a0mM NaCl, 50\u00a0mM sodium phosphate buffer, pH 6.5, 250\u00a0mM imidazole, 1\u00a0mM PMSF. Fractions containing SEP-BTX were pooled and buffer exchanged to 1\u00d7 PBS by ultrafiltration . Recombinant SEP-BTX was revealed by western blot. Culture supernatants and purification fractions were separated by reducing 12% SDS-PAGE and transferred to 0.45\u00a0\u03bcm nitrocellulose membranes . Western blot was performed by probing the membranes with 0.1\u00a0\u03bcg/mL of polyclonal rabbit anti-HIS antibody or a 1:1000 dilution of mouse anti-GFP antibody (Abcam) followed by 1:15,000 dilution of alkaline phosphatase-linked goat anti-rabbit antibody or goat anti-mouse antibody . Phosphatase activity was revealed by a chromogenic reaction using 5 bromo-4 chloro-3 indolyl phosphate and nitroblue tetrazolium as substrates .The protocol for protein purification from Levator auris longus (LAL) muscle of male C57BL/6\u00a0J mice (https://www.jax.org/strain/000664) as previously described25. Briefly, animals were supplied by the animal house of the FCEN-UBA. Animals were cared for in accordance with national guidelines for the human treatment of laboratory animals, similar to those of the US National Institutes of Health. All experimental protocols were approved by the Institutional Commission for the Care and Use of Laboratory Animals (CICUAL), FCEN-UBA. Animals were anesthetized with an overdose of 2% tribromoethanol (0.15\u00a0mL/10\u00a0g body weight) injected in the peritoneal cavity and exsanguinated immediately.Experiments were carried out on the left 2, 1 MgSO4, 12 NaHCO3, 1 Na2HPO4 and 11 glucose; continuously bubbled with 95% O2/5% CO2; pH 7.3 as previously described25.The muscle with its nerve supply was excised and dissected on a Sylgard-coated Petri dish containing physiological saline solution of the following composition (in mM): 137 NaCl, 5 KCl, 2 CaClLAL muscles were incubated in Ringer\u00b4s saline solution with the SEP-BTX sensor for 2\u00a0h and washed for 30\u00a0min. For each batch of SEP-BTX produced, containing 30\u201350\u00a0ng/\u00b5L of purified recombinant protein, preliminary experiments were done to identify the dilution (1/5 to 1/20) necessary to obtain a low background strong labelled NMJ. The preparation was then transferred to a 1.5\u00a0mL recording chamber. Experiments were performed at room temperature (20\u201323\u00baC). NMJs were visualized with Nomarsky interference optics and illuminated with a 488\u00a0nM light source .\u20136\u00a0g/mL was used during the experiment.The nerve was stimulated via two platinum electrodes isolated with vacuum grease coupled to a pulse generator . Supra threshold high frequency nerve stimulation was applied during 250\u00a0ms. To avoid using high concentrations of the reporter and to prevent any remaining muscle contraction during stimulation, curare up to 5\u2009\u00d7\u200910For fluorescent image acquisition, we used a BX51WI upright microscope with a 60X objective lens (Olympus) and an electron multiplying CCD camera , together with cell^M System Coordinator/cell^R real time controller software.The only exception is the image shown in Fig.\u00a0t-test.Data analysis was done using Clampfit 10.6 , Sigma Plot 10.0, SigmaStat 3.5 and Excel 2007 (Microsoft). Average data are expressed and plotted as mean\u2009\u00b1\u2009sem. Statistical significance was determined using paired or unpaired Student\u2019s"} +{"text": "FOXC2 are predominately associated with lymphedema. Herein, we demonstrate a key role for related factor FOXC1, in addition to FOXC2, in regulating cytoskeletal activity in lymphatic valves. FOXC1 is induced by laminar, but not oscillatory, shear and inducible, endothelial-specific deletion impaired postnatal lymphatic valve maturation in mice. However, deletion of Foxc2 induced valve degeneration, which is exacerbated in Foxc1; Foxc2 mutants. FOXC1 knockdown (KD) in human lymphatic endothelial cells increased focal adhesions and actin stress fibers whereas FOXC2-KD increased focal adherens and disrupted cell junctions, mediated by increased ROCK activation. ROCK inhibition rescued cytoskeletal or junctional integrity changes induced by inactivation of FOXC1 and FOXC2 invitro and vivo respectively, but only ameliorated valve degeneration in Foxc2 mutants. These results identify both FOXC1 and FOXC2 as mediators of mechanotransduction in the postnatal lymphatic vasculature and posit cytoskeletal signaling as a therapeutic target in lymphatic pathologies.Mutations in the transcription factor The lymphatic vasculature has a critical role in maintaining tissue homeostasis by returning interstitial fluid to the venous circulation, absorbing lipids from the digestive tract, and providing a network for immune surveillance and response . MutatioFOXC1 have primarily been dominantly associated with eye anterior segment defects, cerebellar malformation, and cerebral small vessel disease. In contrast, mutations in FOXC2 have been dominantly associated with lymphedema-distichiasis syndrome characterized by failure of lymph drainage in limbs, venous valve failure, and the growth of an extra set of eyelashes (FOXC2 mutations (FOXC1 and FOXC2 are closely related members of the forkhead box (FOX) transcription factor family with nearly identical DNA binding domains, similar expression patterns in mesenchymal tissues during development, and essential roles in cardiovascular developmental processes . Mutatioyelashes . Work fryelashes . Additioyelashes . Furtherutations . HoweverFoxc1 postnatally impairs valve maturation, while Foxc2 deletion impairs maturation and induces valve degeneration, as previously described in vitro, which also improves LEC barrier integrity in vivo, while valve degeneration is partially rescued in only Foxc2 mutants. Finally, via generation of transgenic mice that express Foxc2 within the Foxc1 locus, we show Foxc2 is capable of functionally substituting for Foxc1 in lymphatic development and maturation. Together, our data show a complementary role for FOXC1 in addition to FOXC2 as key mediators of mechanotransduction in the postnatal lymphatic valves and implicate new mechanistic targets for therapeutics in the treatment of lymphatic-associated diseases.Here, we report an essential role for FOXC1 during lymphatic valve maturation and maintenance. Detailed comparison of FOXC1 and FOXC2 expression and roles in lymphatic valves suggests some overlap with a broader importance for FOXC2, but more subtle, key contribution for FOXC1. In mice, endothelial cell (EC)-specific deletion of escribed . HoweverOur group previously reported that FOXC1 and FOXC2 expression co-localizes with PROX1 in lymphatic valve-forming cells at E17 and later at P3 . HoweverFoxc1 during murine embryonic development impairs lymphatic valve maturation (flFoxc1 mice (ERT2Chd5-Cre mice (Foxc1 mutant (EC-Foxc1-KO) mice. Tamoxifen was administered from P1-P5 to induce Cre-mediated recombination and we confirmed deletion of Foxc1 via qPCR analysis of isolated CD31-positive cells from hearts of P6 individuals and by immunostaining of the mesenteric lymphatic vasculature with antibodies against FOXC1, FOXC2, and VEGFR-3 protein ligand fibronectin-EIIIA to regulate the formation of the ECM core of valve leaflets mice and administered tamoxifen postnatally. PROX1 immunostaining showed no significant differences in total valve number of LEC-Foxc1-KO mice mice and administered tamoxifen from P1-P5 with inactivation confirmed by qPCR in ECs isolated from P6 hearts and by immunostaining of the mesenteric lymphatic vasculature with antibodies against FOXC1, FOXC2, and VEGFR-3 mice and administered tamoxifen from P1 to P5 with inactivation in compound mutant individuals verified by qPCR and immunostaining of the mesenteric lymphatic vasculature with antibodies against FOXC1, FOXC2, and VEGFR-3 developed severe chylous ascites compared to controls , indicatFOXC1 knockdown in LECs impairs cell adhesion, we slightly modified our protocol: LECs were first seeded on fibronectin-coated surfaces, then transfected with scramble control, FOXC1, FOXC2, or combined FOXC1/FOXC2 (50%:50%) siRNAs and kept under static conditions for 2 days. Given that FOXC2 inactivation and exposure to OSS in LECs results in increased actomyosin contractility suite of tools (https://ecrbrowser.dcode.org) -phosphorylated myosin light chain (pMLC) signaling pathway has been well established as a regulator of cytoskeletal contractility mechanisms . FurtherDKO mice . Recent DKO mice . Our RNADKO mice revealed embryos . Further embryos were notpression . Becausepression and our of tools was usedof tools in activof tools . Then, wode.org) tool to PRICKLE1 , ARHGAP2ARHGAP23 loci. ToPRICKLE1 , ARHGAP2P23 loci . We founin ECR-1 . Additio ECR-1\u20132 . Finally23 locus .ICAM1 that was not predicted to bind FOX transcription factors by in silico analysis allele, in which the Foxc1 coding region has been replaced with the cDNA coding (from the start codon to the stop codon) for Foxc2 and FoxCrostomes . Althougnditions . TherefoPecam1 or Sdc4 and LEC-specific deletion of Cdh5 in vivo resulted in impaired valve morphogenesis phenotypes and poor orientation and elongation of LECs with the direction of flow in mesentery collecting vessels for Foxc2 by using restriction enzyme digestion and PCR cloning. To facilitate the elimination of the selectable marker gene rNeo in mice, we introduced the ACN cassette flanked by two flox sites mice , c2/+Foxc1 , and c2/c2Foxc1 individuals.SalI-linearized targeting vector (100 \u03bcg) was then electroporated into TL1 ES cells (129S6) as described . The cel/+) mice were theGenotyping of mice for use in analysis was performed by Transnetyx Inc using real-time PCR.Dissection, immunostaining, and imaging of the lymphatic vasculature in mesentery tissue from 4 week old C57Bl6 mice for whole-mounts was performed as previously described . BrieflyThe lymphatic collecting vessel vasculature of neonates was analyzed by whole mount immunostaining of mesentery tissue harvested from pups at the indicated time. Briefly, mesentery tissue was dissected from the intestinal tract, laid out in plastic dish and left until it was firmly attached. Following fixation with 2% PFA in PBS, tissues were washed with PBS, then permeabilized and blocked in PBS solution containing 0.5% BSA, 5% serum, 0.3% Triton X-100, and 0.1% Sodium Azide. Tissues permeabilized/incubated with blocking buffer were then incubated with primary antibodies listed in Imaging was performed using a Zeiss AxioVision fluorescence microscope and Zeiss Axiovision software, Zeiss LSM-510 Meta, LSM-800 and LSM 880 confocal microscopes and Zeiss Zen Blue acquisition software, or using a Nikon A1 Confocal Laser Microscope System NIS-Elements software. Images were processed with Imaris and Adobe Photoshop software. Imaris colocalization function was used to produce pictures showing p-MLC2 , vinculiPpia) or 18S was used as an internal standard for mRNA expression. Primer sequences are provided in Hearts from neonatal mice were digested in collagenase Type I solution (2 mg/mL) for 40 min at 37\u00b0C with gentle agitation. Cells were then filtered through a 70 \u03bcm cell strainer and the pellet was resuspended in Buffer 1 . The cell suspension was then incubated with magnetic Dynabeads (Invitrogen) pre-coated with CD31 antibody to isolate the endothelial cell population. After several washes with Buffer 1, RNA was extracted from endothelial cells using RNA STAT solution (Tel-Test) followed by phenol-chloroform treatment. Extracted RNA was subjected to DNAse I treatment and concentration was determined using a NanoDrop machine (Thermo Scientific). cDNA was synthesized using an iScript reverse transcriptase kit (Bio-Rad) according to the manufacturer\u2019s instructions. Triplicates of cDNA samples for qPCR analysis were performed using a Fast qPCR machine (Applied Biosystems), Fast SYBR reaction mix (Applied Biosystems), and gene specific primer sets. Peptidylprolyl isomerase A coated with 40 \u00b5g/ml human fibronectin, cultured for 24 hr and then subjected to LSS (4 dyn/cm2), OSS (4 dyn/cm2 and flow direction change every 4 s) using Ibidi Pump system, or kept in static conditions for an additional 24 hr prior to fixation, immunostaining, and mounting using Ibidi Mounting Medium (ibidi GmbH).Flow experiments with cultured LECs were performed as described previously . Brieflyhttps://genome.ucsc.edu/ENCODE/)(2012). Putative sites in the human genome were then searched against the mm10 mouse genome using the ECR Browser (https://ecrbrowser.dcode.org)\u00a0(Putative FOX-binding sites were determined first by using the Hypergeometric Optimization of Motif EnRichment (HOMER) suite ofode.org)\u00a0 and rVishttps://www.promocell.com/product/human-dermal-lymphatic-endothelial-cells-hdlec/) were cultured and used according to the manufacturer's protocol. The cells were cross-linked with 1% formaldehyde, followed by sonication. The sheared chromatin was immunoprecipitated with dynabeads conjugated with anti-FOXC2 antibody , anti-FOXC1 antibodies , or control IgG . DNA extraction and PCR were performed as previously described from juvenile foreskin (escribed with priPRICKLE1 ECR-1.>hg19 chr12:42876176\u201342876523TGTTGGCTCCTGAACAACACTCCTCTTTGTGAAACTTACAGCACCCATTTGAAACAAGTTTCCAGAGAAAACACTTCAAGAAAGTGTTTGAGAAGTCACAAGACTCGAGTTGTAAAAACAAATTCCACACATAGCCTGGCTTATAAGGACACAGACTAACACCACACAAGGCGGTGCTCTAATGAGCCCATTATTTTCCATAATGGGGGATGCAGATATTTTCTCAAAATCGTGTTCTCCTCAGTCTTCTATTGATTTTTTGGATTTCTATTTTCAACAGTGGCCCGAGGAAACGGCAGCCAGACTTGACTCCAATGTACACACAGACTCAGGTTTCGCCCCGTCACCPRICKLE1 ECR-2.>hg19 chr12:42878066\u201342878454GAGCACCTACCGCCGCCCGCCCGCTCCATTCTCCCGAGCCCAGTGAGTGAAGCCGCTAAGATGCAAATACCCTAGGACGCTTATGTAAACTTCCCCCTCCCGCAGGTGCACGCGCGGGCCACGAAACGCTGGGAGAATATGAAAGGCCACCTCTTAAAGAAATCATCTCCACTCTGCCCATAACAATGATGTCAGCAAATGGCACATTTAAGCAAGTTCTCACTTAGAAGGGCTCATTAGCATATGAATTCTCTTAGGACTTTCCCTGCATTTCGGAGTGATTCCTACTGCTTAGCGCAGGAGATTTATTTTTATCAGTAAATAACAGCAAAGAAAAGGAGCCAGGTCACGCGATGTACTAACTCAAGTACACTACTGAGAGTTTTTACPRICKLE1 ECR-3.>hg19 chr12:42879624\u201342879943AGTTACTTGTAAATACGTTTTGTTATATTTTCAACAGGTACTGTCATGGTTTATTACCCATTGTAAGCGTTTTTAGTGATGAAGCAGTGTGGAGACAGTTGCAAGCTTCCTACAAAGGTTCAATCTCAATGAAACAGACTTTAGTCTGGTCTGAAAGGGGTTGTTATTGTCAAGGGTGAACTTATGCAGAATGGAGAGCAAGGCCCCCAACCAGCATCCTTTTGTTTCAGCCAGGTGGAATATTCATGCTTGCAGATCACACTTTAGGGGCCAGTTGAGAAAGGAAGCCCAAAAATTCACAGGGCTTGGTTCCCTCGCTCPRICKLE1 ECR-4 and ECR-5.>hg19 chr12:42981201\u201342982292TACATTAGCCATGACTTATTAAACTTGGAGATTTTCAAGTTCATCAGCAAGTGTGCAAACCCTTAACTGTGGGTAACATCCATTTATTTGTAGCACCTTTCGGTTTTAATATGTAGAGCACATGCATTGTTAACCTCTAAATCCTTTGT(ATAAACA)TTTCTGGAAGAGCTGGTAAAATATCTCCTTCTGTGTTTCCTGACTCGCCAGTTGATGGCATTTAGAAACCCTCTGGTACCAGCAGGTGCTGTATTTTGCTTTCTTCAGGCTCCAGCTGGGCTACAATGACAGATTCCTGTCCCAGGCCAAGCCTAGCCACCAAGGCTAGGACCACATTGGAGGCAAACTGAACCAGGCTCCACCAGACAGCAAGATGAATGGCTGCTGTTTAAGTTTAAAATCCCCTGGTGGGA(GTAAATATTGTTCCAGAGAAAAGCCTTGACAAATA)CTGCGTCATCCTTACAGAACTGTCTTGATTAAAGCAGAATCTTTGGATTAAGTTGATGCTCAATTCAAAATGTATCTATCTTGCTGTCATGGGATTTTTTTTTGTTTTTTTTTTCCTTTCTAGAGTCTGAAAACAGACATAATGTTGGGGACGGTCAAACAAGGCTGCCGGCTCCCAAGGGGCTAGAGTCCACTCCTGATAATAGAAGGCGGCTGAACACTGACACTTCACTGAGGATAATGGAGACAGCAAAGGCTTAGTGGGAAAGGGCCAGTTGTCACCTAAGTGACAGGCAACAGCTGAGCTCACACATCTGGAGCCGGACTACGGCAAACATTAGCAACCCTCACCAGTCTACACCTTGGGCCTGTCTGAAAAGACAGATGGAAGTTCCCTCTACTCCTAAAGTACATTAAAAAATGTCTGATGGTGAACCACATCAATTATATAACATCAACTGCAGGCACAGCCTTCCAAAGTACTGATTAAGACGAGGCAGTAGACAACACTGTATGCATGAACAGATACAAGATACCATTTCAGTGATTTGTCATTCATAAAACTTATCCTAAAAGACACATATACATGCATCCATTTGATAGCACAAATGCATGTTAACTCTGCAGGAGAGGCAGATTTTTACATGTPRICKLE1 ECR-6.>hg19 chr12:42982328\u201342982743CCTTTAGGCTGGTTCCCGTGGTGTGTTTGCCTTGTTCATGGTCTCAGTTCTGCCGCTGATACCCTTTTAAAAATCAGCAACCAAACGCGTTCGGCTTGTGATCCTGAACCCCCTTAGGCAAGCTGGAACTAAGCGTGATGCAGCCGTCCTCCCTCTCTCCCAACCCCCAACCTCGTTCTTCAGCCTCCTGAAGACAATCTGTGAACAATTTTCCCAAAGTCCCAAGAATAACACAGCACTGCCAATAGTCACTGGCGATGCCGTTTGTTTTTCTTAGAGGGTAATGAAAATTTAACAGCTTTCTGCTGCATCCTGAGTCCCGCTCCTAATAACTATTAACATGCCTAGTTTCTTCAACTTTTTCTACCTCAAGAGAGGAAGACGCTCCCATTTTTTCCCTATATCTGTGCTACATARHGAP21 ECR-1 and ECR-2.>hg19 chr10:25009076\u201325009514CTGTCATTGTTAATAAAAGCCAAGTGTGCAACAAACTGGAAATACTGCTTGCTAGCAAGGACAAGATGTGTCTAAATTCTTGGTTTCAGGACATCTCTTAAATGACCAAAAAAAAAAAAAAAAATCAAATAACATTAGTTCTT(GCAAACAGAATGCAAATACAATGCTAATTAAAGTATTCACAAGACAATGACAAATA)AGGCTTCAGGACCACAATACATATTATTATGTAACTGCAATACACATTAAGCAATCACCAGGTTGCAGGTAGGCCTTCCAAAAGGAGTTATTATGGTTTACCGTGATCAGAGGATTGTGGTGTTCCACTTAATCATGCTTTTGCCTGCAAGCAGGTGTTTACAGATGTCAAAAAGTAAAACACTGATTCTAAATGTAAACCTACAGTTCTGCCTAATAAATTGTACAGTAATAGCACACARHGAP21 ECR-3.>hg19 chr10:25017295\u201325017667CTATATTAATAAAAACTACAAGAAAGCTTTATACACTAAATCTAGGCAAGACATTTATGAAGATGAGAACTGTATCCTTAAAAGGTAAGTGTTGGCTTTGCTAATGACATAATATTGTTTTGGTGAACCAATATCAAGGGAAAAAATGTCAAAGCCAAAAATAGAGGCAAAGTATCCCAGCCCCTGGTGTGGAAGGCCATTCTATGATAATCTATGAATGATTTCTACTCTGAATATGTTAACAGAAGCTGGCACATCTGAGAAGCACAAGTGTTTGCTAGTGAATCCACAAATGAAATTTGCAATTTGGGTTGAAGCCTTTGCAAAACTACGTTAAGACCAATAGCCCTCAGAAGAGTAAGGGGTTTTGTTTARHGAP23 ECR-1.>hg19 chr17:36585363\u201336585767TTCTAGACACAGGCCCAGGACCCCGGGCTCTGCCGGCGAGGCTGCCCTCCCCTCTGCCCTCTCCGACCGGCTGTGGGTGGGTCAGAGCGCGGGGTGCCAGGGGCATTACTCAGCGCTGGGCTGCTCTGCCTGGGTTCTTTCATCTGCCAGCTGCTGAGGCTGGGGAGGGGCCAGCAGGGGCCTCCCAGCCCCATCCCCCCATCAGGGCCATTCCCTTACCTCTGAGCCTGGCTGCCCGCCCTGCAGGAGCCCCCCAGCAGGCCTCCCTGCTCCTAAGTTGAAGGGTTGAACACTGTCAGGCCAACAGTTTCCCTGAGCTCGGAAAAGAAATTCCCCGGGGTCCAGGTTGAGGTCAAGGCCAGGGCTGAGGCCTGTTCCTCTTTAGACAGGGCTGAAAGACTTGGGTo identify lymphatic valves, mesentery tissue was stained with PROX1 antibody and areas of high expression were quantified as mature (visible leaflets) or immature (no visible leaflets) on four lymphatic collecting vessels per individual. Total number of valves were determined and percent of mature valves was normalized to the total counted per individual. For assessment of apoptosis in lymphatic collecting vessels, mesentery tissue was immunostained with PROX1 and active caspase-3 antibody and the percentage of PROX1/caspase 3-positive LECs was quantified from 20X high-power fields generated from confocal z-stacks using Fiji software to determine the total number of LECs per field using thresholded PROX1 immunostaining. Quantification was completed from three biological replicates.in vitro quantifications, relative nuclear intensity levels were measured using Fiji software. Nuclei were considered as regions of interest from thresholded Hoechst staining pictures. FOXC1 or FOXC2 intensity was\u00a0then measured in each nuclei using RawIntDen function. For quantification of F-actin and p-MLC2 area per cell, LECs were fist manually segmented and defined as individual regions of interest, then F-actin (or p-MLC2) staining was thresholded using similar parameters for all pictures and F-actin+ (or p-MLC2+) area was measured per each region of interest. Quantification was completed from three independent experiments.For in vivo experiments was performed using GraphPad Prism v5 or v8. P values were obtained by performing a 2-tailed Student\u2019s t test or one-way ANOVA and Tukey\u2019s test. Data are presented as mean\u00a0\u00b1\u00a0standard deviation of representative experiments from at least three biological replicates. For quantification of apoptosis in LECs of compound Foxc1; Foxc2 mutant mice, the ROUT method with Q set to 1% was used to identify outliers in the data set that were then excluded from statistical analysis using student\u2019s t-test. P values less than 0.05 were considered statistically significant. Statistical analysis for in vitro experiments was performed using GraphPad Prism v8 to perform mixed-effects analysis and calculation of correlation coefficients. P values less than 0.05 were considered statistically significant for mixed-effects analysis.Statistical analysis for All procedures and animal studies were approved by Northwestern University\u2019s IACUC or by the Animal Ethics Committee of Vaud, Switzerland. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:This study provides new insight into the mechanically regulated activation of, and distinct roles for, FOXC1 and FOXC2 in lymphatic vessel valve development. The identification of unique roles for these closely related transcription factors is intriguing and will pave the way to a deeper understanding of the transcriptional mechanisms regulating their distinct activities. The study also implicates FOXC1 as a gene that may potentially underlie primary lymphoedema.Decision letter after peer review:eLife. Your article has been reviewed by three peer reviewers, including Natasha L Harvey as the Reviewing Editor and Reviewer #2, and the evaluation has been overseen by Didier Stainier as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Mark Kahn (Reviewer #3).Thank you for submitting your article \"Shear stimulation of FOXC1 and FOXC2 differentially regulates cytoskeletal activity during lymphatic valve maturation\" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:Here, Norden, Sabine and colleagues dissect the roles of transcription factors FOXC1 and FOXC2 in lymphatic vessel valve development, revealing that FOXC1 primarily controls focal adhesions in lymphatic endothelial cells, while FOXC2 primarily controls adherens junction integrity. The work builds on previous studies by the authors that have demonstrated (1) the importance of FOXC2 and shear stress for lymphatic vessel valve development and , (2) important roles for both FOXC1 and FOXC2 in embryonic lymphangiogenesis via regulation of RAS/ERK signalling . Aspects of this current study overlap with some of the authors' prior work and this should be clearly articulated in the manuscript where relevant. Distinctions and extensions of the work should also be clearly articulated. There are key novel and interesting findings described in this manuscript that address important questions regarding the distinct mechanisms by which FOXC1 and FOXC2 activity are regulated, together with the mechanisms via which these transcription factors regulate cytoskeletal architecture.Essential revisions:Foxc1 seems modest, and is characterized by a larger proportion of immature valves in mesenteric lymphatic vessels. It thus becomes important to know how the authors define immature valves. It is not obvious why those shown in Figure 2E, F are defined as immature, and not representing valves that are imaged from a different (90\u00b0) angle compared to valves that show the typical v-shape. Staining of the valve leaflets would demonstrate this more clearly. The inclusion of staining for markers of valve maturation such as \u03b15-laminin and/or \u03b19-integrin would help to demonstrate this point. To more convincingly demonstrate that Foxc1 modulates the Foxc2 KO phenotype, side-by-side comparison of Foxc2 single and Foxc1;Foxc2 double mutants should be shown. Of note, deletion of Foxc2 seems to be incomplete . Considering that the authors show that Foxc2 can compensate for Foxc1 function, Foxc1 deletion in combination with the incomplete Foxc2 deletion may just decrease total Foxc signaling sufficiently but not necessarily indicate distinct functions of the two genes as proposed. The question is whether Foxc1 deletion would affect the phenotype of Foxc2 mutants if Foxc2 is efficiently deleted. Why is Foxc1 staining apparent on the cell membrane and present in the Foxc1 mutant in Figure 3\u2014figure supplement 1?1) The phenotype caused by early postnatal deletion of Foxc1; Foxc2 mutants resulting in increased apoptosis in lymphatic collecting vessels' requires more supporting evidence. We see one image aiming to support the claim that cell elongation and junctional integrity is impaired but it is not clear how representative this image is. It is surprising that such a high number of apoptotic cells is detected in control vessels, drawing into question the specificity of the caspase-3 staining, and whether the signal is indeed detected in the LECs. No evidence is provided for cell elongation defects leading to increased apoptosis. This statement should also be demonstrated: \"Of note, apoptotic bodies were more frequently observed at branched areas of the lymphatic collecting vessels potentially indicating areas of valve degeneration.\" The authors should expand on the cellular mechanisms underlying the presumed rupture of lymphatic vessels and accumulation of chylous ascites in EC-Foxc1;Foxc2-DKO pups. Ultrastructural analyses could be informative here.2) The conclusion 'Cell elongation and junctional integrity is markedly impaired in compound EC- specific Foxc1;Foxc2 double mutants. Figure 3\u2014figure supplement 3D shows instead extremely high PROX1 levels, along with 'discontinuous junctions', making it difficult to judge if this is a representative image. The junctional phenotype presented in Figure 3\u2014figure supplement 3D is indeed dramatic , but the phenotype is seemingly different in Figure 3\u2014figure supplement 2D where the phenotype is not obvious at all. 'Discontinuous junctions' in the Foxc1;Foxc2 double mutants should be demonstrated more convincingly.3) Inconsistent quality of staining between different experiments and even panels in the same figure (as if staining and imaging is done at different time points and not using littermate mice) make assessment of the phenotypes difficult, especially when a single high magnification image is provided as a supporting evidence. For example \u2013 Figure 3\u2014figure supplement 2 VE-cadherin staining, Figure 2E-F CD31 staining. Figure 3 and Figure 3\u2014figure supplement 3H show low PROX1 levels overall in Foxc1 and Foxc2 have distinct functions in regulating the actin cytoskeleton, it is not clear why Foxc2 is capable of functionally substituting for Foxc1 in vivo.4) The authors should provide further evidence to demonstrate how relevant the described in vivo phenotypes are to those described in vitro. If 5) Previous studies have demonstrated that FOXC2 is regulated by oscillatory shear forces in LECs, and that such forces likely play a key role in valve development. In Figure 4 the authors use in vitro flow studies to conclude that FOXC1 expression is regulated primarily by laminar shear while FOXC2 primarily by oscillatory shear. However, the endothelial cells shown in Figure 4A do not appear aligned with flow as would be expected for laminar shear and the authors fail to identify a clear mechanism that would account for such a distinct regulation. This theory appears to rely primarily on antibody detection of nuclear FOXC1 which is not as robustly detected as FOXC2. In vivo studies, especially Figure 10, suggest that these transcription factors are functionally redundant at the protein level as replacement of FOXC1 with FOXC2 does not impair valve maturation. Much of the data shown are carefully analyzed, but these findings and their connection to where FOXC1 is best detected in vivo in the valve tip seems like an over-reach. More specific mechanistic data identifying how such remarkable distinctions in shear regulation are achieved should be provided.Foxc2-KO compared to mRNA? Foxc2 mRNA appears to be reduced by approximately 50% compared to control EC Foxc2 levels. Does this level of reduction in EC-Foxc2-KO animals explain the partial penetrance of the phenotype of chylous ascites?6) Figure 1\u2014figure supplement 1: What is the level of FOXC2 protein present in LEC of EC-Foxc2-KO compared to EC-Foxc1;Foxc2-DKO mice.7) Figure 3\u2014figure supplement 2: A lower power image depicting the broader profile of \u03b19-integrin expression throughout multiple valves would enable the reader to determine how \u03b19-integrin deposition is affected more globally in EC-Foxc1;Foxc2-DKO LEC? It would be informative to provide more detail as to why these three genes were selected for further analysis. Were they the only genes involved in RhoA/ROCK signalling that were changed in expression? It would also be informative to know whether RAS/ERK signalling is affected in mutant LEC at this stage of development, given the authors' previous work which demonstrated regulation of this key pathway by FOXC1 and FOXC2 in embryonic LEC.8) Figure 6: How do the changes in Prickle1, Arhgap21 and Arhgap23 levels compare to other gene expression changes in LEC-9) Figure 6D: Additional controls are required in which ChIP of both FOXC1 antibodies at a negative control region of the genome (e.g. an enhancer region demonstrated or predicted not to bind FOXC1) is assessed. This should demonstrate the selectivity of FOXC1 antibodies in terms of their specific versus non-specific/background binding to DNA and may help to explain the distinct results observed with each antibody in different regions of the genome.Foxc1c2/c2 mice. What is the remaining signal in FOXC1 stained c2/c2Foxc1 tissue?10) Figure 10D-F: It is not clear from the images presented that a call can be made regarding FOXC2 levels being modestly increased in Essential revisions:1) The phenotype caused by early postnatal deletion of Foxc1 seems modest, and is characterized by a larger proportion of immature valves in mesenteric lymphatic vessels. It thus becomes important to know how the authors define immature valves. It is not obvious why those shown in Figure 2E, F are defined as immature, and not representing valves that are imaged from a different (90 degrees) angle compared to valves that show the typical v-shape. Staining of the valve leaflets would demonstrate this more clearly. The inclusion of staining for markers of valve maturation such as \u03b15-laminin and/or \u03b19-integrin would help to demonstrate this point. To more convincingly demonstrate that Foxc1 modulates the Foxc2 KO phenotype, side-by-side comparison of Foxc2 single and Foxc1;Foxc2 double mutants should be shown. Of note, deletion of Foxc2 seems to be incomplete . Considering that the authors show that Foxc2 can compensate for Foxc1 function, Foxc1 deletion in combination with the incomplete Foxc2 deletion may just decrease total Foxc signaling sufficiently but not necessarily indicate distinct functions of the two genes as proposed. The question is whether Foxc1 deletion would affect the phenotype of Foxc2 mutants if Foxc2 is efficiently deleted. Why is Foxc1 staining apparent on the cell membrane and present in the Foxc1 mutant in Figure 3\u2014figure supplement 1?Foxc1, Foxc2, and compound Foxc1; Foxc2 mutant mice. To address the first part of this comment, we have included new representative images of \u03b19-integrin/VE-Cadherin immunostaining of lymphatic valve regions side-by-side with PROX1/CD31 immunostained lymphatic valves in Figure 2. Inset images of only the \u03b19-integrin channel more clearly show the presence of mature, intraluminal bi-leaflet valve structures in Control and EC-Foxc1-KO mice as well as valve regions that are considered immature that are not characterized by a distinct intraluminal leaflet structure by \u03b19-integrin immunostaining. In EC-Foxc2-KO mice, we have observed that overall, \u03b19-integrin expression is reduced compared to littermate control mice. However, there are regions characterized by the presence of intraluminal leaflets, as depicted by PROX1, CD31, and VE-Cadherin immunostaining, that are shortened in length. We describe these observations in the subsection \u201cFOXC1 and FOXC2 are required for postnatal lymphatic valve maturation and maintenance\u201d.The authors thank the reviewers for this comment and careful assessment of our described phenotypes in our Foxc2-KO mice and EC-Foxc1; Foxc2-DKO mice. Figure 2H and N depict representative images of a mesentery collecting vessel immunostained with PROX1/CD31 and Figure 2R and T depict 10X power images of collecting vessels immunostained with PROX1 to show the difference in phenotypes more clearly.To address the second part of this comment, we have also updated Figure 2 to include representative images showing side-by-side comparison of EC-Foxc1-KO, EC-Foxc2-KO, EC-Foxc1; Foxc2-DKO mice and respective littermate controls and included representative images in Figure 2\u2014figure supplement 1. In Figure 2\u2014figure supplement 1G, we show that FOXC2 expression is markedly reduced in EC-Foxc2-KO mice and FOXC1 expression is reduced compared to littermate controls, likely as a result of perturbed flow contributing to the reduced induction of FOXC1 expression. In contrast, Figure 2\u2014figure supplement 1E shows strong reduction of FOXC1 expression but no discernible difference in FOXC2 expression. Thus, the maintenance of FOXC2 expression in EC-Foxc1-KO mice may in part explain the differences in phenotype severity observed in Foxc2 mutant mice compared to Foxc1 mutant mice. We have revised our Discussion section (second paragraph) to note this important difference. The difference between our qPCR data, depicted in Figure 2\u2014figure supplement 1B, and our immunostaining evidence is likely attributable to the fact that CD31-positive cells were isolated from cardiac tissue to use for qPCR analysis. It is likely that FOXC2 expression levels are different between lymphatic endothelium in mesentery collecting vessels and the cardiac vasculature. Thus, this may explain the differences in deletion efficiency. The repeated immunostaining of collecting vessels with FOXC1, FOXC2, and VEGFR-3 antibodies also now shows clear expression of FOXC1 in the nucleus of both lymphatic endothelial cells and smooth muscle cells of arterioles. As shown in Figure 2\u2014figure supplement 1E, G, and I, there is no membrane signal detected. We suspect that there may be some cross-reactivity with our secondary antibodies used for the immunostaining combination of FOXC1 and VE-Cadherin, thus warranting our use of VEGFR-3 antibody to address this issue and verify the reduction of FOXC1 expression in the endothelium of collecting vessels.To address the third part of this comment, we performed immunostaining of FOXC1, FOXC2, and VEGFR-3 in mesentery collecting vessels of EC-2) The conclusion 'Cell elongation and junctional integrity is markedly impaired in compound EC- specific Foxc1; Foxc2 mutants resulting in increased apoptosis in lymphatic collecting vessels' requires more supporting evidence. We see one image aiming to support the claim that cell elongation and junctional integrity is impaired but it is not clear how representative this image is. It is surprising that such a high number of apoptotic cells is detected in control vessels, drawing into question the specificity of the caspase-3 staining, and whether the signal is indeed detected in the LECs. No evidence is provided for cell elongation defects leading to increased apoptosis. This statement should also be demonstrated: \"Of note, apoptotic bodies were more frequently observed at branched areas of the lymphatic collecting vessels potentially indicating areas of valve degeneration.\" The authors should expand on the cellular mechanisms underlying the presumed rupture of lymphatic vessels and accumulation of chylous ascites in EC-Foxc1;Foxc2-DKO pups. Ultrastructural analyses could be informative here.Foxc1; Foxc2 mutants and is accompanied by increased apoptosis throughout lymphatic collecting vessels\u2019 as the previous statement was not accurate. Our group previously reported that valves of LEC-Foxc2-KO mutant mice were characterized by increased apoptosis and apoptotic cells were often arranged in doublets with symmetrically organized PROX1-high apoptotic bodies, suggesting that apoptosis may occur in dividing cells. Additional evidence identified that half of the dying cells in LEC-Foxc2-KO mice were PROX1/Ki67-positive, demonstrating that valves of LEC-Foxc2-KO mice were characterized by abnormal activation of cell proliferation and increased cell death. This phenotype was associated with improper activation of TAZ signaling in the absence of FOXC2 . We have included a summary of this finding in the subsection \u201cCell elongation and junctional integrity is markedly impaired in compound EC-specific Foxc1; Foxc2 mutants and is accompanied by increased apoptosis throughout lymphatic collecting vessels\u201d. We then sought to characterize junctional integrity and apoptosis in compound Foxc1; Foxc2 mutant mice to assess if there was a synergistic effect of inactivation of both Foxc1 and Foxc2 in the lymphatic endothelium.We thank the reviewers for this helpful comment and careful critique of our data. We have revised our conclusion to more accurately state, \u2018Cell elongation and junctional integrity is markedly impaired in compound EC-specific Foxc1; Foxc2 mutant littermates that show apoptotic bodies are in closer proximity to branched regions.To further address this reviewer comment, we carefully re-evaluated our caspase-3 immunostaining and repeated analysis to characterize the percent of PROX1/cleaved caspase-3 positive lymphatic endothelial cells in 20X high-power field images. Figure 2\u2014figure supplement 4I-K has been updated to include new representative images and quantitative analysis. In addition to representative images of maximum intensity projections of PROX1/cleaved caspase-3 immunostained collecting vessels, we also show panels of z-sections from selected regions in higher magnification where PROX1/cleaved caspase-3 positive cells were identified. We also now show images from two compound 3) Inconsistent quality of staining between different experiments and even panels in the same figure (as if staining and imaging is done at different time points and not using littermate mice) make assessment of the phenotypes difficult, especially when a single high magnification image is provided as a supporting evidence. For example \u2013 Figure 3\u2014figure supplement 2 VE-cadherin staining, Figure 2E-F CD31 staining. Figure 3 and Figure 3\u2014figure supplement 3H show low PROX1 levels overall in Foxc1;Foxc2 double mutants. Figure 3\u2014figure supplement 3D shows instead extremely high PROX1 levels, along with 'discontinuous junctions', making it difficult to judge if this is a representative image. The junctional phenotype presented in Figure 3\u2014figure supplement 3D is indeed dramatic , but the phenotype is seemingly different in Figure 3\u2014figure supplement 2D where the phenotype is not obvious at all. 'Discontinuous junctions' in the Foxc1;Foxc2 double mutants should be demonstrated more convincingly.Foxc1; Foxc2-DKO mice more clearly depict that collecting vessels in these mice are characterized not only by discontinuous junctions, but the increased presence of overlapping junctions as well. We have also generated videos of 3D reconstructions of the collecting vessels depicted in Figure 2\u2014figure supplement 4A-H (Videos 1 \u2013 4), characterizing this observation.We thank the reviewers for this helpful comment and careful critique of our representative images. To address the first part of the reviewer comment, we have repeated immunostaining of tissues and re-evaluated the post-processing of our representative images for several figures to address issues with inconsistencies. This includes new images for PROX1/CD31 immunostaining in Figure 2C, E, I, and K and new images for PROX1/VE-Cadherin immunostaining in Figure 2\u2014figure supplement 4A-H that now show consistent PROX1 expression levels with representative images in Figure 2. Additionally, our new representative images for Figure 2\u2014figure supplement 3E-H and Figure 2\u2014figure supplement 4A-H show consistent VE-Cadherin expression levels among different tissue samples. Furthermore, our new images of VE-Cadherin immunostaining in EC-4) The authors should provide further evidence to demonstrate how relevant the described in vivo phenotypes are to those described in vitro. If Foxc1 and Foxc2 have distinct functions in regulating the actin cytoskeleton, it is not clear why Foxc2 is capable of functionally substituting for Foxc1 in vivo.Foxc2-KO and EC-Foxc1; Foxc2-DKO mutants in Figure 2\u2014figure supplement 4A-H that shows differences in the presence of discontinuous junctions in collecting vessels of EC-Foxc2-KO mice, but both discontinuous and overlapping junctions present in collecting vessels of EC-Foxc1; Foxc2-DKO mice. This new data recapitulates our in vitro evidence depicting the same observations in FOXC2-KD and FOXC1; FOXC2-KD cultured lymphatic endothelial cells as represented in Figure 4\u2014figure supplement 1, thus providing additional support for the relevancy of our in vivo and in vitro observations. In regard to the second part of the reviewer comment, although FOXC1 and FOXC2 are functionally similar, as characterized by their similar DNA-binding capacity, their transcriptional targets are likely to be different under oscillatory or laminar shear stress. For example, our group previously reported that loss of FOXC2 leads to distinct phenotypes in lymphatic endothelial cells under oscillatory shear stress and laminar shear stress . Thus, as FOXC1 is induced only by laminar shear, but FOXC2 is induced by both laminar and oscillatory shear, their downstream targets are likely different within the collecting vessels of lymphatic endothelial cells. We have also updated the fourth paragraph of the Discussion section to elaborate on these differences.We have now included new representative images of VE-Cadherin immunostaining in EC-5) Previous studies have demonstrated that FOXC2 is regulated by oscillatory shear forces in LECs, and that such forces likely play a key role in valve development. In Figure 4 the authors use in vitro flow studies to conclude that FOXC1 expression is regulated primarily by laminar shear while FOXC2 primarily by oscillatory shear. However, the endothelial cells shown in Figure 4A do not appear aligned with flow as would be expected for laminar shear and the authors fail to identify a clear mechanism that would account for such a distinct regulation. This theory appears to rely primarily on antibody detection of nuclear FOXC1 which is not as robustly detected as FOXC2. In vivo studies, especially Figure 10, suggest that these transcription factors are functionally redundant at the protein level as replacement of FOXC1 with FOXC2 does not impair valve maturation. Much of the data shown are carefully analyzed, but these findings and their connection to where FOXC1 is best detected in vivo in the valve tip seems like an over-reach. More specific mechanistic data identifying how such remarkable distinctions in shear regulation are achieved should be provided.As indicated in the Materials and methods section, the flow experiment presented in Figure 3 was run for only 24 hours (subsection \u201cCell transfection and immunostaining\u201d). This shorter time \u2013 24-hour flow instead of 48-hour flow presented in our previous publications \u2013 is not sufficient to drive an important cell elongation in the direction of flow. In In addition, we also provide the reviewer with the quantification of length:width ratio for static, LSS and OSS conditions presented in Figure 3, which shows that there is a significant elongation of cells under LSS in average . To clarThe observation that FOXC1 and FOXC2 expression are already different in LECs after only 24 hours under laminar shear stress is interesting as it suggests that FOXC1 upregulation by laminar flow is an early response of LECs to directional flow. How expression levels of FOXC1 and FOXC2 are distinctly regulated by the directionality of flow is a very interesting question that addresses the more general one of how LECs sense flow direction and are able to respond differently to LSS and OSS. Answering such a question would require further investigation and, in our opinion, is beyond the scope of this paper in which the central message is that each factor is differentially regulated by flow and plays slightly distinct but complementary roles in regulating the cell cytoskeleton in response to shear stress.6) Figure 1\u2014figure supplement 1: What is the level of FOXC2 protein present in LEC of EC-Foxc2-KO compared to mRNA? Foxc2 mRNA appears to be reduced by approximately 50% compared to control EC Foxc2 levels. Does this level of reduction in EC-Foxc2-KO animals explain the partial penetrance of the phenotype of chylous ascites?Foxc2-KO mice compared to littermate controls. We also suggested that the differences observed in reduction of mRNA levels compared to what we observe in regard to protein expression may be attributable to the different vascular beds analyzed. The difference in the penetrance of the phenotype of chylous ascites in the current study may be attributable to both the timeline of experimental analysis as well as the differences in Cre-drivers used. In our group\u2019s previous study, tamoxifen was administered to ERT2; Foxc2fl/flProx1-Cre mice beginning at P4 with approximately 50% of mutants developing chylous ascites 4 days after tamoxifen administration and nearly all mutants presenting chylous ascites 6-8 days after tamoxifen administration . In the current study, tamoxifen administration started at P1 and we primarily investigated tissues at P6. We believe it is likely that our ERT2; Foxc2fl/flCdh5-Cre mice would present chylous ascites at later time points.As previously described in our response to reviewer comment #1, Figure 2\u2014figure supplement 1F and G show a marked reduction of FOXC2 protein within collecting vessels of EC-7) Figure 3\u2014figure supplement 2: A lower power image depicting the broader profile of \u03b19-integrin expression throughout multiple valves would enable the reader to determine how \u03b19-integrin deposition is affected more globally in EC-Foxc2-KO compared to EC-Foxc1;Foxc2-DKO mice.Foxc2-KO mice at P6, but the collecting vessels of EC-Foxc1; Foxc2-DKO mice are absent of \u03b19-integrin-positive valve regions.We thank the reviewers for this suggestion. Figure 2\u2014figure supplement 3A-D now depicts 10X low power images of \u03b19-integrin/VE-Cadherin immunostaining to show that a few, \u03b19-integrin-positive degenerating valve regions are detected in collecting vessels of EC-8) Figure 6: How do the changes in Prickle1, Arhgap21 and Arhgap23 levels compare to other gene expression changes in LEC-Foxc1;Foxc2-DKO LEC? It would be informative to provide more detail as to why these three genes were selected for further analysis. Were they the only genes involved in RhoA/ROCK signalling that were changed in expression? It would also be informative to know whether RAS/ERK signalling is affected in mutant LEC at this stage of development, given the authors' previous work which demonstrated regulation of this key pathway by FOXC1 and FOXC2 in embryonic LEC.Foxc1; Foxc2-DKO mice and littermate controls in panel K. Figure 5\u2014figure supplement 1K now shows the changes in expression for RhoA, Rock1, and Rock2, and several GTPase activating proteins associated with regulation of RhoA signaling and endothelial barrier function and lumen maintenance . Here, we see that there is a modest, yet significant increase in Arhgap18 expression and a significant reduction of Arghap20, although its expression was generally lower compared to other GAPs. Because of the previously reported evidence of the formation of a physical complex between Prickle1 and Arhgap21/23 and our observation that all three genes were significantly downregulated in our RNA-seq analysis, we focused our investigation on these putative downstream targets. In regard to changes in RAS/ERK signaling, we attempted to perform whole-mount immunostaining of pERK using the same antibody utilized in our group\u2019s previous study but we were not successful in identifying a positive signal in control or mutant lymphatic valves at P6, testing the antibody at various concentrations. While we are interested in characterizing whether RAS-ERK signaling may be affected in our mutants during postnatal development in other lymphatic vascular beds, the authors believe that this is not of focus for the mechanism described in this study and is beyond its scope.We agree with the reviewers that providing additional details regarding the choice to focus on Prickle1, Arhgap21, and Arhgap23 would be helpful. We have now updated Figure 5\u2014figure supplement 1 in the revised manuscript to include additional data from our RNA-seq analysis for LECs isolated from the dorsal skin of embryonic LEC-9) Figure 6D: Additional controls are required in which ChIP of both FOXC1 antibodies at a negative control region of the genome (e.g. an enhancer region demonstrated or predicted not to bind FOXC1) is assessed. This should demonstrate the selectivity of FOXC1 antibodies in terms of their specific versus non-specific/background binding to DNA and may help to explain the distinct results observed with each antibody in different regions of the genome.ICAM1 promoter that is not predicted to bind to FOX transcription factors. Panel J then shows data from three separate experiments showing no detection of band signals from ChIP with our FOXC1 or FOXC2 antibodies. Within the text of the manuscript, we have also included relevant literature that has identified variations in immunohistochemistry related work for antibodies recognizing the same immunogen peptide that are dependent on the source of the manufacturer. This information is included in the second paragraph of the subsection \u201cFOXC1 and FOXC2 regulate LEC expression of negative RhoA signaling regulators PRICKLE1, ARHGAP21, and ARHGAP23\u201d.We thank the reviewers for this important suggestion. We have now included Figure 5\u2014figure supplement 1I and J to directly address this comment. Panel I depicts the location of primers used to amplify a transcriptionally active region of the c2/c2 mice. What is the remaining signal in FOXC1 stained Foxc1c2/c2 tissue?10) Figure 10D-F: It is not clear from the images presented that a call can be made regarding FOXC2 levels being modestly increased in Foxc1c2/c2Foxc1 mice, we have also included new representative images of FOXC1/VEGFR-3 immunostaining in Figure 9D and E. As previously stated in our reply to reviewer comment #1, we suspect that the remaining signal in the FOXC1 channel of Figure 9H is non-specific and may be related to a cross-reactivity issue of secondary antibodies. However, we believe that Figures 9F-H clearly depict a reduction in nuclear FOXC1 expression as expected.We agree with the reviewers that it is likely not accurate to describe that FOXC2 levels are modestly increased. Thus, this statement has now been removed from the revised manuscript. To verify that FOXC1 protein expression is absent in collecting vessels of"} +{"text": "Serratia marcescens TKU011 with the highest yield of 4.62 mg/mL at the optimal conditions of liquid medium with initial pH of 5.65\u20136.15 containing 1% \u03b1-chitin, 0.6% casein, 0.05% K2HPO4, and 0.1% CaSO4. Fermentation was kept at 25 \u00b0C for 2 d. Notably, \u03b1-chitin was newly investigated as the major potential material for PG production via fermentation; the salt CaSO4 was also found to play the key role in the enhancement of PG yield of Serratia marcescens fermentation for the first time. PG was qualified and identified based on specific UV, MALDI-TOF MS analysis. In the biological activity tests, purified PG demonstrated potent anticancer activities against A549, Hep G2, MCF-7, and WiDr with the IC50 values of 0.06, 0.04, 0.04, and 0.2 \u00b5g/mL, respectively. Mytomycin C, a commercial anti-cancer compound was also tested for comparison purpose, showing weaker activity with the IC50 values of 0.11, 0.1, 0.14, and 0.15 \u00b5g/mL, respectively. As such, purified PG displayed higher 2.75-fold, 1.67-fold, and 3.25-fold efficacy than Mytomycin C against MCF-7, A549, and Hep G2, respectively. The results suggest that marine chitins are valuable sources for production of prodigiosin, a potential candidate for cancer drugs.Marine chitins (MC) have been utilized for the production of vast array of bioactive products, including chitooligomers, chitinase, chitosanase, antioxidants, anti-NO, and antidiabetic compounds. The aim of this study is the bioprocessing of MC into a potent anticancer compound, prodigiosin (PG), via microbial fermentation. This bioactive compound was produced by Chitin, an abundant material, has been widely produced from fishery processing byproducts. Of the natural chitin-containing materials, shrimp shells, squid pens, and crab shells have the highest chitin content , and as Serratia marcescens and some other Gram-negative bacterial strains [Prodigiosin (PG), a red pigment is a typical alkaloid constituent produced by several bacterial genus, strains . PGs hav strains ,20,21,22 strains activiti strains . S. marcescens. However, numerous scientific parameters were not investigated in our previous studies, such as the kind of marine chitin (\u03b1 or \u03b2), protein sources, chitin/protein ratio, and supplementary minerals for the best PG productivity production by S. marcescens. All those previously unknown items were newly investigated in this study, and the PG produce from the medium containing marine chitin was also evaluated for its effect on four cancerous cell lines\u2014A549, Hep G2, MCF-7, and WIDR\u2014in this report.Due to the wild range of unique applications of PG, the production studies on this bioactive compound have been received with great interest ,24, and Serratia marcescens TKU011, compared to other materials; SPP was reported to contain approximately 60% chitin and 40% protein [S. marcescens, the chitins obtained from SPP (\u03b2-chitin) and shrimp shells (\u03b1-chitin) by using the method reported by Wang et al., 2006 [w/w) and used as the sole carbon and nitrogen source for fermentation by S. marcescens TKU011; SPP was also used as the control for comparison purpose. The results in w/w) give higher PG yield production (2.73 mg/mL) than that of SPP (2.45 mg/mL) fermented by S. marcescens TKU011, while \u03b1-chitin mixed with free protein at the ratio of 5/3 (w/w) reach the greatest PG yield production of 3.23 mg/mL. In addition to the use of \u03b1-chitin providing higher PG yield, and \u03b1-chitin could be more abundantly obtained from vast resources than \u03b2-chitin (mainly obtained from squid pens); thus, \u03b1-chitin was chosen for our further investigation. Based on the recent literature review, PG has been produced by S. marcescens with various types of carbon/nitrogen sources [Carbon source has been proven to play an important role in PG production via microbial fermentation . In prev protein . Thus, cl., 2006 were mix sources ,31,32,33 sources as the cS. marcescens strains, a total four strains including S. marcescens TKU011, S. marcescens CC17, S. marcescens TNU01, and S. marcescens TNU02 were conducted for fermentation. As shown in the S. marcescens TKU01, S. marcescens TNU01, and S. marcescens TNU02 showed their same level in PG production with the PG yield of 325\u2013335 mg/100mL, and 236\u2013243 mg/100mL when the medium contained newly designed C/N source (0.94% \u03b1-chitin and 0.56% Casein), and 1.5% squid pens, respectively. S. marcescens CC17 demonstrated the lowest production of PG yield.For the comparison of the PG producing by different S. marcescens TKU011 strain reached 335 (mg/100mL). However, two previous studies reported that with 2.0% sesame seed [S. marcescens TKU011 in this study. In these above cited reports [To date, PG has been produced from many carbon sources with multiple designed media ,31,32,33ame seed and the reports ,33, the S. marcescens TKU011, some chitinous materials, including chitosan (a derivative of chitin), N-acetyl-glucosamine (monomer of chitin), glucosamine (mono of chitosan), and some other carbon sources, such as cellulose and starch, were used for fermentation. As shown in S. marcescens with the greatest yield of 3.21 mg/mL, followed by its monomer N-acetyl-glucosamine with the PG yield production of 1.81 mg/mL, and all other tested carbon source give low yield PG production (\u2264 0.96 mg/mL). Thus, \u03b1-chitin was chosen as an excellent substrate for further investigation. To further investigate the effect of the combination of \u03b1-chitin and free protein source, a total of five protein sources\u2014beef extract, casein, nutrient broth, yeast extract, and peptone\u2014were combined with \u03b1-chitin used as sole C/N source for fermentation by Serratia marcescens to produce PG. The experimental results in w/w). This designed medium also reached the high PGs yield of 3.21 mg/mL, and as such used for next investigation. To investigate the effect of C/N sources on PG production by 2HPO4 was found to be the most suitable phosphate salt for PG biosynthesis by S. marcescens. Further experiments investigated the optimal added K2HPO4 was 0.05% 2SO4, MgSO4, CaSO4, CuSO4, (NH4)2SO4 as the basal salt solution. CaSO4 demonstrated good effect on PG production with the highest yield of 4.32 mg/mL in liquid medium with initial pH of 5.65\u20136.15 containing 1% \u03b1-chitin, 0.6% casein, 0.05% K2HPO4, and 0.1% CaSO4, fermentation was kept at 25 \u00b0C for 2 d. To achieve maximum production of prodigiosin, some parameters, including cultivation temperature e, initiaw/w) and used as the sole C/N source at the concentration of 1.5% (w/v) for fermentation by S. marcescens TKU011. PG was primary extracted from the cultured broth by ethyl acetate. The PG from the cell pellet extracted with acetone was mixed with the ethyl acetate layer. After evaporation to dry crude PG, this compound was further purified via silica gel column, and then finally isolated by thin layer chromatography. The procedure is summarized in \u03b1-chitin was mixed with casein with the ratio of 5/3 , A549 (Human lung carcinoma), Hep G2 (Human hepatocellular carcinoma), and WiDr (Human colon adenocarcinoma). The means of inhibition (%) with the same letter are not significantly different based on Duncan\u2019s multiple range test . CV (%) = 1.979533.50 value. IC50 value is a concentration of sample that may reduce 50% of cancerous cells; therefore, the smallest this value of the sample, the strongest anticancer activity it displayed. As shown in 50 values of 0.04, 0.06, 0.04, and 0.20 against A549, Hep G2, MCF-7, and WiDr, respectively . Four cancerous cell lines\u2014MCF-7 (Human breast adenocarcinoma), A549 (Human lung carcinoma), Hep G2 (Human hepatocellular carcinoma), and WiDr (Human colon adenocarcinoma)\u2014were purchased from the Bioresources Collection and Research Centre . Mitomycin C and Silicagel were obtained from Sigma Chemical Co. and Mitsubishi Chemical Co. , respectively. Reagents, solvents and common chemicals were used at the highest grade available.us study . S. marcus study , S. marcSerratia marcescens TNU01: 16S gene sequence of TGGCTCAGATTGAACGCTGGCGGCAGGCTTAACACATGCAAGTCGAGCGGTAGCACAGGGGAGCTTGCTCCCTGGGTGACGAGCGGCGGACGGGTGAGTAATGTCTGGGAAACTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGAGGGGGACCTTCGGGCCTCTTGCCATCAGATGTGCCCAGATGGGATTAGCTAGTAGGTGGGGTAATGGCTCACCTAGGCGACGATCCCTAGCTGGTCTGAGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGCGAGGAGGAAGGTGGTGAACTTAATACGTTCATCAATTGACGTTACTCGCAGAAGAAGCACCGGCTAACTCCGTGC.Serratia marcescens TNU02: 16S gene sequence of CTGGCTCAGATTGAACGCTGGCGGCAGGCTTAACACATGCAAGTCGAGCGGTAGCACAGGGGAGCTTGCTCCCCTGGGTGACGAGCGGCGGACGGGTGAGTAATGTCTGGGAAACTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGAGGGGGACCTTCGGGCCTCTTGCCATCAGATGTGCCCAGATGGGATTAGCTAGTAGGTGGGGTAATGGCTCACCTAGGCGACGATCCCTAGCTGGTCTGAGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGCGAGGAGGAAGGTGGTGAACTTAATACGTTCATCAATTGACGTTACTCGCAGAAGAAGCACCGGCTAACTCCGTG C.w/w) and used as the sole carbon and nitrogen source with the concentration of 1.5% (w/v) for fermentation. The medium containing 1.5% carbon and nitrogen source, 0.1% K2HPO4 and 0.1% FeSO4(NH4)2SO4 was fermented by S. marcescens TKU011 at 30 \u00b0C in 1 d, and then 25 \u00b0C over the next 2 d, shaking speed of 150 rpm, and a ratio volume of medium:flask of 1:2.5 (v/v). \u03b1-chitin mixed with casein at the ratio of 5/3 (w/w) reached the greatest PG yield production; as such, \u03b1-chitin/casein was used for comparison in the following experiments evaluating other carbohydrate sources and proteinous sources. \u03b1-chitin/casein at the ratio of 5/3 (w/w) finally proved best and was used for fermentation in the subsequent investigation, including the effect of added salts and some parameters.\u03b1-chitin and \u03b2-chitin obtained from shrimp shells and squid pens were mixed with casein with 6 ratios of 1/7, 2/6, 4/4, 5/3, 6/2, and 7/1 2SO4, 0.1% phosphate salt, and 1.5% C/N source were fermented at 30 \u00b0C in 1 d, and then at 25 \u00b0C over the next 2 d, shaking speed of 150 rpm, and a ratio volume of medium:flask of 1:2.5 (v/v). KH2PO4 was found to be the most suitable phosphate salt; as such, it was used to investigate its optimal concentration added to the medium. 0.025, 0.05, 0.1, 0.125, 0.15, 0.175, and 0.2% K2HPO4 and combined with 0.1% FeSO4(NH4)2SO4; the fermentation procedure was conducted at 30 \u00b0C in 1 d, and then 25 \u00b0C over the next 2 d, shaking speed of 150 rpm, and a ratio volume of medium:flask of 1:2.5 (v/v).The effect of phosphate salts and its optimal concentration added to culture medium on PG production: four kinds of phosphate salts\u2014KH4(NH4)2SO4, MgSO4, CaSO4, CuSO4, and (NH4)2SO4 were used as sulfate salts. The medium containing 0.05% K2HPO4 and 0.1% sulfate salt, 1.5% C/N source were fermented at 30 \u00b0C in 1 d, and then 25 \u00b0C over the next 2 d, shaking speed of 150 rpm, and a ratio volume of medium:flask of 1:2.5 (v/v). CaSO4 was found to be the most suitable sulfate salt; as such, it was used to investigate its optimal concentration added to the medium. 0.025, 0.05, 0.1, 0.125, 0.15, 0.175, and 0.2% CaSO4 and combined with 0.1% K2HPO4; the fermentation was conducted at 30 \u00b0C in 1 d, and then 25 \u00b0C over the next 2 d, shaking speed of 150 rpm, and a ratio volume of medium:flask of 1:2.5 (v/v).The effect of sulfate salts and its optimal concentration added to culture medium on PG production: FeSOThe effect of some parameters on PG production: some parameters including temperature programs , initial pH and period of cultivation time . w/v) hydrated potassium aluminum sulfate was added into this mixture, mixed, and then centrifuged at 1400 g for 5 min. The harvested supernatant was then mixed with a solution of methanol/0.5 N HCl at the ratio of 1/9, v/v. The final solution optical density was measured at 535 nm. PG purified from the culture broth was used as the standard to convert OD535 nm measurement to mass concentration via an appropriate calibration. PG was purified by the method previously described [g for 15 min. The supernatant was collected and mixed with ethyl acetate with the ratio 1/1. The mixture was kept in a funnel for 3 h and immediately shaken every 30 min. The PG dissolved in ethyl acetate layer was collected. The PG from the cell pellet was extracted with acetone, and centrifugated at 10000\u00d7 g for 15 min. The ethyl acetate layer and acetone containing PG were mixed, concentrated by evaporation of the solvent and then dissolved in ethyl acetate for further air oven drying at 55 \u00b0C to get dry crude PG powder. The crude PG was further purified by loading onto a silica open column for column chromatography, size: 0.040\u20130.063 mm) and eluted with methanol in chloroform with a ratio of 0/10\u20132/8 (v/v). The PG was finally isolated by thin layer chromatography (TLC) with the mobile phase system using methanol in chloroform with a ratio of 2/8 (v/v). After TLC separation, the lane contained PG was cut into small pieces, and methanol was used to dissolve PG. Then PG was concentrated in a rotary evaporator at 60 \u00b0C under vacuum. Finally, all the residue solvent was removed by keeping the sample in the oil pump in 12 h at 60 \u00b0C. The isolated PG was used to detect UV, MALDI-TOF MS and biological activities. PG concentration was determined according to the method previously described by Wang et al., 2012 . A mixtuescribed with mod50 values, were analyzed with the use of Statistical Analysis Software (SAS-9.4) provided by the SAS Institute Taiwan Ltd. .Four cancerous cell lines: A549, Hep G2, MCF-7, and WIDR were conducted to evaluate the anticancer activities. The bioassay was done according to the methods described in detail in our previous report . The sig2HPO4, and 0.1% CaSO4 for efficient biosynthesis of bioactive prodigiosin. The fermentation was maintained at 25 \u00b0C for 2 d. The prodigiosin was purified, qualified via UV and Mass. The purified prodigiosin was also evaluated for its anticancer properties. Notably, the purified PG displayed high inhibition on four cancerous cell lines. The results in this study suggest that the purified prodigiosin newly biosynthesized may be a potential candidate for cancer drugs.The current study established the novel designed medium containing 1% \u03b1-chitin, 0.6% casein, 0.05% K"} +{"text": "We show that CD28 stimulation in the absence of TCR strongly up-regulates IL-22 gene expression and secretion. As recently observed for IL-17A, we also found that CD28-mediated regulation of IL-22 transcription requires the cooperative activities of both IL-6-activated STAT3 and RelA/NF-\u03baB transcription factors. CD28-mediated IL-22 production also promotes the barrier functions of epithelial cells by inducing mucin and metalloproteases expression. Finally, by using specific inhibitory drugs, we also identified CD28-associated class 1A phosphatidylinositol 3-kinase (PI3K) as a pivotal mediator of CD28-mediated IL-22 expression and IL-22\u2013dependent epithelial cell barrier functions.IL-22 is a member of the IL-10 cytokine family involved in host protection against extracellular pathogens, by promoting epithelial cell regeneration and barrier functions. Dysregulation of IL-22 production has also frequently been observed in acute respiratory distress syndrome (ARDS) and several chronic inflammatory and autoimmune diseases. We have previously described that human CD28, a crucial co-stimulatory receptor necessary for full T cell activation, is also able to act as a TCR independent signaling receptor and to induce the expression of IL-17A and inflammatory cytokines related to Th17 cells, which together with Th22 cells represent the main cellular source of IL-22. Here we characterized the role of CD28 autonomous signaling in regulating IL-22 expression in human CD4 We found that IL-22 gene expression and secretion was strongly up-regulated by CD28 in human CD4+ T cells were enriched from PBMC by negative selection using an EasySepTM isolation kit and cultured in RPMI 1640 supplemented with 5% human serum , L-glutamine, penicillin and streptomycin. The purity of the sorted population was 95% to 99%, as evidenced by staining with anti-CD3 plus anti-CD4 Abs. Human na\u00efve CD4+CD45RA+ and effector/memory CD4+CD45RO+ T cells were sorted using a high speed cell sorter . Purity of sorted cells was consistently > 98%, and was acquired on a cytometer . PBMCs were derived from buffy coats or anonymous healthy blood (HD) donors provided by the Policlinico Umberto I . Written informed consent was obtained from blood donors and both the informed consent form and procedure was approved by the Ethics Committee of Policlinico Umberto I . CACO-2 epithelial cell line from human colon was provided by ATCC and cultured in DMEM supplemented with 10% FBS , L-glutamine, penicillin and streptomycin.Human primary CD4\u22121), anti-human CD3 , goat anti-mouse , anti-human CD3-PE (#555333), anti-human CD45RA BV421 (#562885), mouse anti-human STAT3 (#610189), anti-human CD45RO PE (#555493) (BD Biosciences); rabbit anti-human Lck , rabbit anti-human RNA polymerase II , anti-human RelB , anti-human GAPDH (#sc-25778), anti-human mucin 1 (MUC1) (#sc-7313) , rabbit anti-human RelA (#8242S) , mouse anti-human IL-6 , mouse anti-human IL-22 , anti-human CD4 FITC . Human recombinant IL-6 (rIL-6) was from Miltenyi Biotec . The following inhibitory drugs were used: PS1145 , S31-201 , AS605240 .The following antibodies were used: anti-human CD28 in 24 trans-well plates as indicated at 37\u00b0C. At the end of incubation, total cell extracts were obtained by lysing cells for 30\u00a0min on ice in 1% Nonidet P-40 lysis buffer , 1 mM EGTA, 1 mM MgCl2, 50 mM NaF, 10 mM Na4P2O7) in the presence of inhibitors of proteases and phosphatases . Proteins were resolved by SDS-PAGE and blotted onto nitrocellulose membranes. Blots were incubated with the anti-MUC1 or anti-GAPDH (1:400 dilution), extensively washed and after incubation with horseradish peroxidase (HRP)-labeled goat anti-rabbit or HRP-labeled goat anti-mouse developed with the enhanced chemiluminescence\u2019s detection system . Protein levels were quantified by densitometric analysis using the ImageJ 1.50i program .CACO-2 cells were plated at 2.5 \u00d7 10+ T cells were plated at 2 \u00d7 106 ml\u22121 in 24-well plate or 24-well plate inserts in the experiments of co-culture with CACO-2 cells (2.5 \u00d7 105 ml\u22121) and stimulated for the indicated times with control isotype Abs (Ig) or crosslinked anti-CD28 (2 \u03bcg ml\u22121), or anti-CD3 (2 \u03bcg ml\u22121) or anti-CD3 plus anti-CD28 Abs (2 \u03bcg ml\u22121). Secretion of human IL-22, IL-6, and MMP9 was measured from the supernatants of CD4+ T cells cultured alone or with CACO-2 cells and stimulated with control isotype Abs or crosslinked anti-CD28.2 (2 \u03bcg ml\u22121), by using human IL-6 (#DY206), IL-22 (#DY782), and MMP9 (#DY911) ELISA kits (R&D Systems). Data were analyzed on a Bio-Plex . The assays were performed in duplicate. The sensitivity of the assay was 9.4 pg ml\u22121 for IL-6 and 31.2 pg ml\u22121 for IL-22 and MMP9.CD4+ T cells were plated at 2 \u00d7 106 ml\u22121 in 24-well plate or 24-well plate inserts in the experiments of co-culture with CACO-2 cells and stimulated for the indicated times with control isotype matched Abs (Ig) or crosslinked anti-CD28 (2 \u03bcg ml\u22121), or anti-CD3 (2 \u03bcg ml\u22121) or anti-CD3 plus anti-CD28 Abs (2 \u03bcg ml\u22121). Total RNA was extracted by either CD4+ T cells or CACO-2 cells using Trizol according to the manufacturer\u2019s instructions and was reverse-transcribed into cDNA by using Moloney murine leukemia virus reverse transcriptase . TaqMan Universal PCR Master Mix and human IL-6, IL-22, MMP9, MUC1, and GAPDH primer/probe sets were from Thermo Fisher Scientific. The relative quantification was performed using the comparative CT method. The results were expressed as arbitrary units (AU) by using the mean value of cells stimulated with control Ig as CT calibrator or fold induction (F.I.) over control Ig-stimulated cells after normalization to GAPDH.CD4\u2212644 of the human IL-22 promoter, the pIL-22 (-644)-GFP construct was obtained by subcloning the PCR fragment into AseI-Hind III sites within the CMV promoter of pEGFP-N1 vector . The sequence of human IL-22 promoter fragment (\u2212644/+156) was verified by DNA sequencing and was as follow: 5\u2032-ATTAATACAATTTTAAGATATATTTACTTCTGCCTTAATTGTTATGATCACTTAAAAATAGTTCCAAAAAGGGAAGAAAACAATAATTAGATTAGCCAAGACAGTTATTTTTGAAACATAAGTCTGGTTTAGAATTCAGCATGTTTAAAAATGAGATAAAATTATTTTAATAATGGAATGATCTGTTAGCTGTCATTACCATTTACTTTAAAGCAGAGGATATAGGACATGGGTCCTTTTTTTCTGATCACCTCCAATGAGATAAGAATCTATAAAGCTGGTAGGAAAATGAGTCCGTGACCAAAATGCTTACTCGGTCACTATAGGAGATCAAAACATTTTATACTAAATCTGAACTCTACTAAGACAAAACAATTGTGTTCTTTGAAAAATATGTAGGGTTTAGAAAATTTCTGGGATTTGTCTGTAAAATACCCTCCGGGCTCTAATAGTGACGTTTTAGGAAAACACTTGCATCTCAAGGTGGAAAGGATAGAGGTGGTGTTTTGTGGGCTCCTGTGGTGGTTAGGTCGTTCTCAGAAGACAGTACTGGAAATTAGATAATTGCTGATGTCATATTTTTCACAATTAAAAAAAAGTCAGTATCCTGGGGGCTATAAAAGCAGCAGCTTCTACCTTCCCCGTCACAAGCAGAATCTTCAGAACAGGTAGGCGTTTCGGCAAACTTGGTACAATTGGTTAGTTTGACGAAATACTTCTTGACTAATTTTGTTCCTTCACGTTGTCTTCGACCAGGTTCTCCTTCCCCAGTCACCAGTTGCTCAAGTTAGAATTGTCTGCA-3\u2032.The pIL-22 (-644)-GFP construct containing the GFP under the control of the \u2212644 bp region upstream of the transcriptional start site and the +156 region upstream the translational start site was generated as previously described . BrieflyPlasmid vectors expressing HA-tagged human RelA, IKK\u03b1, IKK\u03b2 and NIK were previously described , 38.3 GFP-positive events by gating for SSC and FSC.CD28-positive Jurkat T cells were electroporated in 0.45\u00a0ml RPMI-1640 supplemented with 10% FBS with 1 \u03bcg of pIL-22(-644)-GFP together with pcDNA3 control vector 10 \u03bcg HA-RelA, or HA-IKK\u03b1 or HA-IKK\u03b2 or 20 \u03bcg HA-NIK. Twenty-four hours after transfection, the cells were analyzed by using a BD FACScan flow cytometer . Mean fluorescence intensity was calculated on a total of 5 \u00d7 107 CD4+ T cells were stimulated as indicated and chromatin immunoprecipitation (ChIP) assays were performed as previously described was performed for the human IL-22 promoter. Specific enrichment was calculated as previously described ((Ct of control ChIP-Ct of control Input)/2(Ct of specific ChIP - Ct of specific Input). The human IL-22 promoter primers used for each specific ChIP were as follow: RelA I, RelB I and pSTAT3 I, 5\u2032-GCTTACTCAGCCACTATAGGAGATCA-3\u2032 and 5\u2032-CCGGAGGGTATTTTACAGACAAATCC-3\u2032; RelA II, 5\u2032-ACCCTCCGGGCTCTAATAGTGAC-3\u2032 and 5\u2032-AGAACGACCTAACCACCACAGGA-3\u2032; pSTAT3 II and Pol II 5\u2032-CCTGTGGTGGTTAGGTCGTTCT-3\u2032 and 5\u2032-GCTGCTTTTATAGCCCCCAGGAT-3\u2032.10escribed . Brieflyescribed by using\u22121). CACO-2 cells were plated at 2.5 \u00d7 105 cells ml\u22121 in 24-well plates and treated with 10 \u03bcM AS605240 or DMSO, as vehicle control, for 48\u00a0h. Cytotoxicity was analyzed by a BD Biosciences FACScalibur by quantifying the percentage of PI positive cells. Results were calculated from at least three independent experiments.The cytotoxicity of AS605240 on CACO-2 cells was evaluated by propidium iodide (PI) staining was performed to evaluate differences between continuous variables through Prism 5.0 using Student t test. For multiple group comparisons, significant differences were calculated using nonparametric Mann\u2013Whitney + T cells from HD, relapsing-remitting MS (RRMS) and T1D patients (+ T cells from stable RRMS patients (+ T cells from HD with an agonistic anti-CD28 Ab (CD28.2) binding the same epitope recognized by B7 molecules (+ T cells from one representative HD induced a significant increase (p < 0.01) of IL-22 gene expression within 6\u00a0h that further increased 24 to 48\u00a0h and decreased 72\u00a0h after stimulation (+ T cells from a larger sample size (n = 7) (+ T cells from HD. As we previously observed for IL-17A (+CD45RA+ (+CD45RO+ (We have recently found that CD28 stimulation induces the expression of Th17 related cytokines in CD4patients \u201334. Morepatients . In ordeolecules or anti- (n = 7) . Consist (n = 7) , CD28 str IL-17A and other IL-17A , the ana+ T cells in a IL-6\u2013dependent manner.These data evidence that CD28 intrinsic signaling regulates IL-22 gene expression and secretion in both na\u00efve and effector/memory CD4+ T cells stimulated with agonistic anti-CD28.2 Abs. To do that, three distinct oligonucleotide probes were used; probe I (\u2212338 to \u2212203) for both STAT3 I and NF-\u03baB I binding sites, probe II (\u2212213 to \u2212110) for NF-\u03baB II binding site and probe III (\u2212131 to \u221220) for both STAT3 II binding site and TATA box (+ T cells from a larger sample size (n = 9) and was associated to CD28-induced transcriptional activation of IL-22 promoter, as evidenced by RNA polymerase II (pol II) promoter occupancy (The human IL-22 gene promoter contains two putative STAT3 responsive elements (STAT3 I and II) and two NF-\u03baB binding sites (NF-\u03baB I and II) upstream of the transcription start site that have been involved in IL-22 promoter trans-activation , 43 Fi, 44 and,ccupancy , 46 and ccupancy , or anti-CD28, or anti-CD3 or anti-CD3 plus anti-CD28 Abs and co-cultured in trans-well plates with CACO-2 cells for different times. The gene expressions of mucin 1 (MUC1), MMP9 and S100A9 anti-microbial peptide were then analyzed. The kinetic analysis evidenced that both MMP9 . Moreover, the up-regulation and MUC1 gene expression induced by co-culturing CACO-2 cells with CD28-stimulated CD4+ T cells was also associated with a strong increase of MUC1 protein content required for epithelial barrier functions and protection against extracellular pathogens , 9. We nreported . CD4+ T + T cells , 58 and (ROR\u03b3t) , 4. Here (ROR\u03b3t) Figure 1 kinetic and no s T cells , stimulapression , 60 resupression \u201363.+ T cells. We also found that activated STAT3 was also recruited to the proximal human IL-17A promoter and induced its trans-activation in response to CD28 stimulation . The patients/participants provided their written informed consent to participate in this study.MK performed most of the experiments, analyzed the data, interpreted the results, and helped in writing the manuscript. CA, SF, SC, MS, and MB performed parts of the experiments and data analyses. SA contributed with human samples for the study. MK, CA, SF, MS, and LB were involved in the discussion about the data. LT designed the study, coordinated the work, and wrote the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by: the Italian Foundation for Multiple\u00a0Sclerosis (FISM 2016/R/29), \u201cProgetto Ateneo\u201d and Istituto Pasteur Italia-Fondazione Cenci Bolognetti to LT; the Italian Ministry of Health and the Italian Foundation for Multiple Sclerosis to LB; the Italian Ministry of Health (GR-2016-02363725 and GR-2018- 12365529) to MS.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Fluorogen-activating proteins (FAPs) are innovative fluorescent probes combining advantages of genetically-encoded proteins such as green fluorescent protein and externally added fluorogens that allow for highly tunable and on demand fluorescent signaling. Previously, a panel of green- and red-emitting FAPs has been created from bacterial lipocalin Blc (named DiBs). Here we present a rational design as well as functional and structural characterization of the first self-assembling FAP split system, DiB-splits. This new system decreases the size of the FAP label to ~8\u201312\u2009kDa while preserving DiBs\u2019 unique properties: strong increase in fluorescence intensity of the chromophore upon binding, binding affinities to the chromophore in nanomolar to low micromolar range, and high photostability of the protein-ligand complex. These properties allow for use of DiB-splits for wide-field, confocal, and super-resolution fluorescence microscopy. DiB-splits also represent an attractive starting point for further design of a protein-protein interaction detection system as well as novel FAP-based sensors. The central idea is that reconstitution is followed by regain of a specific function which is abolished in the separated parts. Theoretically, many proteins can be divided into such fragments. In practice, the identification of a functional split protein is still nontrivial, although some success in direct evolution-basedin vivo protein-protein interaction detection4. Successful cleavage of the reporter protein, dihydrofolate reductase, fused to the C-terminal fragment of ubiquitin was happening only when both the C-terminal and mutated N-terminal fragments of ubiquitin were expressed as fusions to a leucine zipper homodimerization domain but not when expressed individually.Split proteins were first employed when using ubiquitin for 5, \u00df-lactamase6, thymidine kinase7, or luciferase8. This allows for real time and quantitative analysis of protein interactions in vitro as well as in model organisms. The desire for more user-friendly methods for detecting protein-protein interactions in complex environments and for identification of their precise cellular localization in combination with enormous progress in fluorescent microscopy techniques prompted the creation of fluorescent split proteins. This included split versions of green fluorescent protein (GFP)10, its differently colored derivatives and homologs13, far-red emitting phytochrome-based fluorescent proteins14, or even dual split reporters15.Later, this concept was applied to a number of other proteins. Many of them were enzymes like dihydrofolate reductase12.When used for protein-protein interaction detection, spontaneous self-association of split proteins is highly undesirable. Such self-association events will contribute to the false positive signal and decrease the overall sensitivity of the method. However, spontaneously self-complementing fluorescent split pairs were found to be useful. Their usage allows for substantial decrease of the tag size that is required to be fused to the protein of interest. Therefore, it diminishes potential influence of the tag on the protein of interest behavior16, IFP1.417, iRFP18, and UnaG19 find their ligands readily available in mammalian cells. Other FAPs like various dye-binding antibodies21, FAST22, DiBs23, and de novo computationally designed mFAPs24 require an exogenous supply of the chromophore. The latter group of FAPs provides multiple benefits. First, available synthetic molecules show a wide range of chemical and photophysical properties allowing for the creation of fluorescent probes with a desired combination of characteristics. Second, external addition of the ligand gives control over the timing and intensity of the fluorescent signal. Third, the noncovalent nature of interaction provides, in some systems, millisecond-scale blinking of the fluorescent signal caused by ligand binding-dissociation events. In these cases, the optimal signal density for high-resolution image reconstruction can be achieved simply by using an appropriate dye concentration. This approach circumvents usage of damaging levels of illumination intensities, which is common for many single-molecule localization microscopy techniques25.Fluorogen-activating proteins (FAPs) are a group of unrelated proteins capable of binding to non-protein ligands (fluorogens) and increase the fluorescence quantum yield and/or change spectral properties of these ligands. Some of these FAPs like miniSOG14 and reversible26 split systems, photoactive yellow protein-based splitFAST27, a label for correlative light and electron microscopy split-miniSOG28, and a bilirubin-binding UnaG-based split reporter uPPI29. Two of these FAP-based splits, IFP PCA26 and splitFAST27, require exogenous supply of the chromophore for imaging in eukaryotic cells. It also has been shown that the full-length UnaG fluorescence recovery under photobleaching conditions needs excess bilirubin added into the solution30. While IFP chromophore, biliverdin, forms a covalent adduct with the protein, only splitFast and uPPI seem to be suitable for use with binding and dissociation events-detecting single-molecule localization microscopy techniques like protein-PAINT23. However, to the best of our knowledge, such an application has not been shown yet for either probe. The reported usage of split-miniSOG for imaging at subdiffraction resolution via electron microscopy is limited to fixed samples.Following the extension of the list of reported FAP systems, FAPs-based splits, also known as bimolecular fluorescence complementation (BiFC) systems, begin to appear. That includes multiple bacteriophytochromes-based irreversiblein vitro screenings we created a panel of FAPs from bacterial lipocalin Blc (named DiBs) capable of recovering the fluorescence of synthetic analogs of green and red fluorescent proteins\u2019 chromophores31. Here we report on DiB-splits, a self-assembling FAP split system which has been inspired by the domain-swapped structure of a full-length DiB protein. This new FAP system reduces the size of the label needed to be conjugated with a protein of interest to ~8\u201312\u2009kDa and is compatible with wide-field, confocal, and super-resolution fluorescence microscopy. DiB-splits also offer an attractive template for design of a protein-protein interaction detection split system as well as FAP-based sensors.In our previous work, using a combination of computational and 32, previously characterized wild type Blc (wtBlc) protein35, as well as another Blc mutant, DiB1, that has been co-crystallized with the M739 . Our attempts to structurally characterize other DiB proteins23 in apo and bound states resulted in obtaining protein crystals of DiB3 in the apo form at low pH conditions (pH 3.5) which diffracted to 1.6\u2009\u00c5. The asymmetric unit contains only one protein chain. However, it forms a biological assembly (dimer) with a crystallographic symmetry mate. The intertwined dimer is caused by domain swapping: each of the two Blc-like eight-stranded beta-barrel folds is created by the N-terminus of one of two polypeptide chains and the C-terminus of the other , except for the region that connects the exchanging parts of the protein .The lipocalin fold contains a single eight-stranded continuously hydrogen-bonded antiparallel \u03b2-barrel complemented by an \u03b1-helix. This common fold has been observed for other lipocalin protein family membersher Fig.\u00a0. As obsein cellulo protein labelling23, we proposed that the observed domain swapping was driven mainly by the very low pH conditions of the crystallization buffer rather than the private DiB3 mutations (V74F and L141Q). To further evaluate this assumption, we calculated the interaction energies between N- and C-termini fragments of the wtBlc, DiB1, and DiB3 proteins using Rosetta38. Despite the fact that wtBlc protein seems to be slightly more stable, DiB3 is not an outlier . Such property is crucial for the successful creation of a split system. Usually designing a split protein involves laborious screening of multiple protein sites in order to select an appropriate cutting point First, we tested whether N- and C-termini fragments, created by separation of a protein chain in the hinge region, are indeed capable to form the lipocalin-like structure when brought together. We fused each of two fragments of DiBs1\u20133 with one of two leucine zipper peptides as shown on Fig.\u00a035 which locks side chains in the preferable conformation for ligand binding. The opposite effect, weaker binding seen in the case of the red-shifted DiB3-split-Zip:M739 complex, supports the previously suggested hypothesis of the alternative binding mode of the ligand in that complex .Split-Zip proteins showed properties similar to the \u201cparental\u201d full-length variants upon addition of the M739 fluorogen including binding affinities, fluorescence spectra maxima, and extinction coefficients Table\u00a0 and S5. 43. On the other hand, in the case of spontaneous self-assembly, the split form of the labelling system allows for a reduction in the size of the labelling tag that needs to be added to a protein of interest. Hence, it minimizes tags\u2019 influence on that protein44. Therefore, as a next step we checked the ability of the DiB N- and C-termini fragments to self-assemble. For this we deleted the leucine zippers as shown on Fig.\u00a0The behavior of a split system in the absence of additional \u201cattracting force\u201d like leucine zippers or other interacting proteins determines the range of its possible applications. If the assembly of the split protein is conditional (fails to self-assemble spontaneously), it can be used for investigation of protein-protein interactionsSelf-assembling split proteins retain their ability to bind and increase fluorescence of the fluorogen M739 Table\u00a0 and S5, 35 as well as a functional dimer34. However, according to our knowledge, the possibility of a trimer formation was never investigated before. According to size-exclusion chromatography conducted during purification routine of split proteins there were no signs of oligomerization of any kind . The residues of the hinge loop and two adjacent \u03b2-strands are also responsible for the main difference between wtBlc and DiB2-split monomers . We assessed the behavior of these constructs in separate transfections. Cells transfected with the TagBFP-splitC110\u2013177 construct alone showed uniform distribution of the blue fluorescence signal throughout cytoplasm and nuclei of the cells produced uniformly distributed blue fluorescent signal TagBFP-splitN1\u2013125\u2009+\u2009TagBFP-splitC126\u2013177 and (2) TagBFP-splitN1\u2013125\u2009+\u2009TagBFP-splitC110\u2013177. Upon addition of the M739 chromophore, recovery of the DiB2-specific fluorescent signal in green channel was observed only in the second pair . We speculate that this can be caused by somehow compromised integrity of the new splitN1\u2013125 fragment. For example, because of presence of an alternative conformation of the new C-terminus. Combination of two protein fragments with partial sequence overlap seems to allow for more efficient assembly or/and longer half-time of the functional complex.Next, we tested the ability of the split system to function 1\u2013125 and splitC110\u2013177 fragments in fusion with either histone H2B or vimentin, and conventional fluorescent protein TagBFP. The assembled split would be visible in a distinct localization in green detection channel if self-assembly occurs successfully. The blue detection channel would show the overall distribution of one of the split halves within a cell and allow for detection of aggregation, non-assembled portion, or other undesirable behavior. We first tested two pairs of H2B-fused proteins: (1) splitN1\u2013125 fragment fused with H2B and splitC110\u2013177 fused with TagBFP (H2B-splitN1\u2013125\u2009+\u2009TagBFP-splitC110\u2013177), and (2) splitN1\u2013125 fragment fused with TagBFP and splitC110\u2013177 fused with H2B (H2B-splitC110\u2013177\u2009+\u2009TagBFP-splitN1\u2013125). Upon transient transfection of the first pair of fusion proteins (H2B-splitN1\u2013125\u2009+\u2009TagBFP-splitC110\u2013177), the blue fluorescent signal of the TagBFP confirmed absence of aggregation both in the nucleus and in the cytoplasm , DiB2-split can be used as a protein-PAINT tag: bursts of fluorescence from individual protein-ligand interactions are clearly detectable Fig.\u00a0 of labelin vivo and was found to give bright and specific fluorescent signal indistinguishable from the one of the full-length protein.In this study, inspired by the obtained domain-swapped crystal structure of the DiB3 protein in its apo state, we designed and characterized a novel DiB-split FAP system. The two fragments of the split proteins were able to spontaneously reassemble fully restoring fluorogen-activating and spectral properties of the \u201cparental\u201d full-length DiBs. Crystallization of one of these proteins, DiB2-split, further corroborate the preservation of the lipocalin fold by the system. The DiB2-split was tested 23 label of a smaller size for super-resolution imaging in living cells. The decrease of the tag size provided through the split can diminish influence of the tag on the protein of interest. In vitro data suggest near complete assembly of DiB2 from split fragments, making DiB-splits a feasible replacement for full-length DiBs or fluorescent proteins in cases where the size of the molecular tag matters. Moreover, while DiB-split fluorescence does not require post-assembly chromophore maturation unlike self-assembling fluorescent proteins12, it can be used for immediate detection of different biological processes such as protein expression and early trafficking events or as a faster reporter of protein solubility.This DiB-split system presents a proof-of-principle demonstration of the potential of the lipocalin scaffold to create a split system. It is immediately applicable as a protein-PAINT46. Both systems do not require oxygen for their function and might be used in oxygen-deficient systems as it was previously shown with FAST47.DiB-splits as well as the parental full-length DiBs have lower signal-to-noise ratio in comparison with FAST family probes. However, FAST localization density decreased rapidly during data acquisition time in single-molecule localization microscopy regime and successful super-resolution imaging using FAST required protocol modifications and usage of oxygen scavengers1\u2013109) as well as the elongated one (splitN1\u2013125) can be redesigned for better stability. Moreover, mutagenesis of the new N- and C-termini could increase binding affinities for the fluorogen. In addition, there is potential for the design of a variety of other fluorescent tools. For example, redesign of the intramolecular interface of the DiB-split proteins reported here to promote higher independence of the N- and C-termini fragments can result in a new FAP-based tool for protein-protein interactions detection. Such tool would complement the existing mEos3.248 and PAmCherry1-based49 super-resolution imaging compatible BiFC labels providing additional benefit of fast and oxygen level independent measurements. Spatial proximity of the natural N- and C-termini of the protein makes DiB proteins a promising starting point for the design of DiB-based circularly permuted proteins. While the discovered split point is close to the ligand binding site, such circularly permuted proteins represent a promising starting point for DiB-based biosensors design. Successful permutation might also allow for the creation of a new self-assembling split system with smaller parts analogous to self-complementing split fluorescent GFP1110 and sfCherry1112 tags.DiB-splits would benefit from further optimization of the location of the split point. The original N-terminus fragment of the system and C-fragments (residues 110 to 177) of the DiB mutants23 as well as leucine zipper peptides with adjacent upstream or downstream portions of the vector from the pMRBad-Z-CspGFP and pET11a-Z-NspGFP plasmids, correspondingly, and the upstream portion of the pET11a-Z-NspGFP plasmid including His-tag. Second, we used the overlap PCR to create DNA fragments containing leucine zipper peptides fused with N- or C-fragments of the DiB mutants flanked by upstream and downstream portions of the vector. These fragments were digested with BamHI and XbaI or NcoI and BsrGI restriction enzymes and ligated in the original vectors. These vectors were further used to create split fragments without leucine zipper peptides. For this we amplified N-fragments of the DiB mutants with adjacent upstream portion of the vector introducing stop codon and BamHI restriction site instead of leucine zipper peptide coding sequence and C-fragments of the DiB mutants introducing start codon and NcoI restriction site instead of leucine zipper peptide. The PCR products were again digested with BamHI and XbaI or NcoI and BsrGI restriction enzymes and ligated in the original vectors.Plasmids pMRBad-Z-CspGFP (Addgene plasmid #40730) and pET11a-Z-NspGFP (Addgene plasmid #40729)53. The resulted constructs\u2019 amino acid sequences are provided below. The linker sequences are underlined.DiB2-split fusions with H2B, vimentin, and TagBFP were generated by Golden Gate Assembly1\u2013125>H2B-splitNDPPVATMASSPTPPRGVTVVNNFDCKRYLGTWYEIARFDHRFERGLEKVTATYSLRDDGGLNVINKGYNPDRGMWQQSEGKAYFTGAPTRAALKVSFFGPFYGGYNVIALDREYSG*MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKVLKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLPGELAKHAVSEGTKAITKYTSAK110\u2013177>TagBFP-splitCDPPVATMGPFYGGYNVIALDREYRHALVCGPDRDYLWILSRTPTISDEVKQEMLAVATREGFDVSKFIWVQQPGSG*MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKLVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCDLPSKLGHKLN110\u2013177>H2B-splitCDPPVATMGPFYGGYNVIALDREYRHALVCGPDRDYLWILSRTPTISDEVKQEMLAVATREGFDVSKFIWVQQPGSG*MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKVLKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLPGELAKHAVSEGTKAITKYTSAK1\u2013125>TagBFP-splitNDPPVATMASSPTPPRGVTVVNNFDCKRYLGTWYEIARFDHRFERGLEKVTATYSLRDDGGLNVINKGYNPDRGMWQQSEGKAYFTGAPTRAALKVSFFGPFYGGYNVIALDREYSG*MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKLVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCDLPSKLGHKLN1\u2013125>vimentin-splitNDPPVATGMASSPTPPRGVTVVNNFDCKRYLGTWYEIARFDHRFERGLEKVTATYSLRDDGGLNVINKGYNPDRGMWQQSEGKAYFTGAPTRAALKVSFFGPFYGGYNVIALDREYSG*MSTRSVSSSSYRRMFGGPGTASRPSSSRSYVTTSTRTYSLGSALRPSTSRSLYASSPGGVYATRSSAVRLRSSVPGVRLLQDSVDFSLADAINTEFKNTRTNEKVELQELNDRFANYIDKVRFLEQQNKILLAELEQLKGQGKSRLGDLYEEEMRELRRQVDQLTNDKARVEVERDNLAEDIMRLREKLQEEMLQREEAENTLQSFRQDVDNASLARLDLERKVESLQEEIAFLKKLHEEEIQELQAQIQEQHVQIDVDVSKPDLTAALRDVRQQYESVAAKNLQEAEEWYKSKFADLSEAANRNNDALRQAKQESTEYRRQVQSLTCEVDALKGTNESLERQMREMEENFAVEAANYQDTIGRLQDEIQNMKEEMARHLREYQDLLNVKMALDIEIATYRKLLEGEESRISLPLPNFSSLNLRETNLDSLPLVDTHSKRTLLIKTVETRDGQVINETSQHHDDLEG110\u2013177-TagBFP>splitCDPPVATMSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKLVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCDLPSKLGHKLN*MGPFYGGYNVIALDREYRHALVCGPDRDYLWILSRTPTISDEVKQEMLAVATREGFDVSKFIWVQQPGSG1\u2013109>TagBFP-splitNDPPVATMASSPTPPRGVTVVNNFDCKRYLGTWYEIARFDHRFERGLEKVTATYSLRDDGGLNVINKGYNPDRGMWQQSEGKAYFTGAPTRAALKVSFFSG*MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKLVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCDLPSKLGHKLN126\u2013177>TagBFP-splitCDPPVATMRHALVCGPDRDYLWILSRTPTISDEVKQEMLAVATREGFDVSKFIWVQQPGSG*MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKLVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCDLPSKLGHKLN>vimentin-DiB2DPPVATMASSPTPPRGVTVVNNFDCKRYLGTWYEIARFDHRFERGLEKVTATYSLRDDGGLNVINKGYNPDRGMWQQSEGKAYFTGAPTRAALKVSFFGPFYGGYNVIALDREYRHALVCGPDRDYLWILSRTPTISDEVKQEMLAVATREGFDVSKFIWVQQPGSG*MSTRSVSSSSYRRMFGGPGTASRPSSSRSYVTTSTRTYSLGSALRPSTSRSLYASSPGGVYATRSSAVRLRSSVPGVRLLQDSVDFSLADAINTEFKNTRTNEKVELQELNDRFANYIDKVRFLEQQNKILLAELEQLKGQGKSRLGDLYEEEMRELRRQVDQLTNDKARVEVERDNLAEDIMRLREKLQEEMLQREEAENTLQSFRQDVDNASLARLDLERKVESLQEEIAFLKKLHEEEIQELQAQIQEQHVQIDVDVSKPDLTAALRDVRQQYESVAAKNLQEAEEWYKSKFADLSEAANRNNDALRQAKQESTEYRRQVQSLTCEVDALKGTNESLERQMREMEENFAVEAANYQDTIGRLQDEIQNMKEEMARHLREYQDLLNVKMALDIEIATYRKLLEGEESRISLPLPNFSSLNLRETNLDSLPLVDTHSKRTLLIKTVETRDGQVINETSQHHDDLEGCorrectness of all obtained constructs was confirmed by sequencing.E. coli strain. Cells were grown in LB media supplemented with 100\u2009\u00b5g/mL ampicillin (full-length DiB proteins) or 100\u2009\u00b5g/mL ampicillin and 50\u2009\u00b5g/mL kanamycin (split-Zip and split proteins) at 37\u2009\u00b0C. Expression was induced by addition 0.04% L-arabinose (full-length DiB proteins) or 0.2% L-arabinose and 10\u2009\u03bcM IPTG (split-Zip and split proteins) at 0.8 OD. Cells were harvested after 3\u2009hours of expression at 37\u2009\u00b0C and were resuspended in PBS buffer, pH 7.4. Suspensions were frozen at \u221280\u2009\u00b0C and thawed at room temperature three times. DNA was destroyed by short sonication and the lysates were centrifuged to obtain cell-free extracts. The proteins were first purified using gravity flow columns with TALON metal affinity resin (Clontech) and further purified by size-exclusion chromatography on a HiLoad 16/600 Superdex 75\u2009pg or Superdex 200\u2009pg 10/300 GL column pre-equilibrated with 50\u2009mM sodium phosphate buffer, pH 6.0.All proteins were expressed in XJb(DE3) Autolysis (Zymo Research) 54 colorimetric assay (Bio-Rad) and bovine serum albumin standard. Single point absorption measurements (595\u2009nm) were performed using FlexStation 3 microplate reader (Molecular Devices). All measurements were performed in triplicate.Protein concentrations were estimated using the Bradford dye-binding method-basedHoriba Jobin Yvon Fluoromax-3 fluorometer was used to detect full fluorescence excitation and fluorescence emission spectra for excitation/emission maxima evaluation.Fluorescence quantum yield was measured relative to a known standard keeping all instrumental conditions identical. Previously characterized DiB:M739 complexes as well as free M739 chromophore were used as standards. Absorbance spectra were detected using double-beam Shimadzu UV-1800 UV/Vis spectrophotometer. Fluorescence spectra were measured using Horiba Jobin Yvon Fluoromax-3 fluorometer.23 using FlexStation 3 microplate reader (Molecular Devices). In brief, constant amount of the chromophore solution was added to protein solutions of different concentrations. The full fluorescence emission spectra were collected using wavelength close to protein-chromophore complex excitation spectrum maximum wavelength. Fluorescence intensity at complex emission spectrum maximum wavelength was extracted and used to determine apparent dissociation constants (Kd).Titrations were performed and analyzed as previously describedDiB3 was crystalized at 21\u2009\u00b0C in 0.8\u2009M sodium citrate, 50\u2009mM sodium borate, 0.1 sodium acetate, pH 3.5 with protein to precipitant volume ratio of 1:1 using hanging drop vapor diffusion technique. Crystals grew within 1\u20133 days and were flash frozen in liquid nitrogen using Parabar 10312 oil as cryoprotectant.DiB2-split crystals were obtained at 21\u2009\u00b0C in 1.6\u2009M ammonium sulphate, 0.1\u2009M MES, pH 4.5 with protein to precipitant volume ratio of 1:1 or in 1.6\u2009M ammonium sulphate, 0.1\u2009M MES, pH 4.5 supplemented with 0.1\u2009M Iron(III) chloride hexahydrate or 5% w/v n-Dodecyl-b-D-maltoside according to Hampton Research Additive Screen protocol using sitting drop vapor diffusion technique. Crystals grew within 1 week and were flash frozen in liquid nitrogen using Parabar 10312 oil as cryoprotectant.55. The crystal structures were solved by molecular replacement with MOLREP56 using the wtBlc structure (PDB ID 1QWD) as a search model. Models building and iterative refinement were performed with Coot57 and REFMAC58, respectively. The final statistics of the structures are shown in Supplementary Table S1. The models have been deposited into the Protein Data Bank (PDB ID 6UKK and 6UKL). Structure figures were prepared using PyMol .Diffraction data were collected at the Life Sciences Collaborative Access Team beamline 21-ID-G at the Advanced Photon Source, Argonne National Laboratory. The diffraction data were processed using xia2 software suite2. For transient transfections FuGENE 6 reagent (Roche) was used. Immediately before imaging DMEM was replaced with HHBS media (Hanks Buffer supplemented with 20\u2009mM Hepes).HEK293 and HeLa Kyoto cells were grown in Dulbecco\u2019s modification of Eagle\u2019s medium (DMEM) (PanEco) supplied with 50 U/ml penicillin and 50\u2009\u00b5g/ml streptomycin (PanEco), 2 mM L-glutamine (PanEco) and 10% fetal bovine serum at 37\u2009\u00b0C and 5% CO\u22122 of 488\u2009nm laser light intensity. Typical acquisitions were 10 000 frames taken at a frequency of 30\u2009Hz.Widefield fluorescence microscopy was performed with the Leica DMI6000B inverted microscope, Zyla 5.5 sCMOS camera (Andor), CoolLED pE-300 light source, GFP and BFP filter sets. Single-molecule localization super-resolution imaging of living cells was performed with Nanoimager S (ONI). Imaging in HILO mode was performed with \u20091.1\u00a0kW\u2009cm59.Localizations during acquisition were detected using NimOS 1.6.1.9898 . Super-resolution image reconstruction was performed using default values of photon, precision and sigma filters in NimOS. Data analysis was performed using a custom-made Python script. Image resolution was determined by decorrelation analysis pluginTo prepare DiB3 structure for analysis we reconstructed the intertwined dimer using crystallographic symmetry and saved the N-terminus portion of one protein chain (residues 24 to 109) and the C-terminus portion of the other protein chain (residues 114 to 175) as a single pdb file. Both wtBlc (PDB ID 1QWD) and DiB1 crystal structures contain two protein chains in the asymmetric unit. We separated the chains into different pdb files and used both for analysis. Each \u201coriginal\u201d chain was further separated into two chains mimicking DiB3-split and only residues 24 to 109, and 114 to 175 were left to make the length of all structures equal. Each of the 5 prepared structures was then relaxed and the interface energy between two chains was calculated using the following protocol:\u2009\u2009\u2009\u2009\u2009\u2009\u2009\u2009\u2009\u2009\u2009The protocol was repeated independently 100 times for each of the starting structures. The data obtained for two separate chains in the asymmetric unit from wtBlc and DiB1 crystal structures were combined. The distribution of the obtained interface energies between N- and C-terminus fragments is shown on Supplementary Fig. S2.Supplementary information.Video1Video2"} +{"text": "N-glycans and high-mannose type N-glycans, suggests that high-mannose-specific seaweed lectins are particularly well adapted as glycan probes for coronaviruses. This review presents a detailed study of the carbohydrate-binding specificity of high-mannose-specific seaweed lectins, demonstrating their potential to be used as specific glycan probes for coronaviruses, as well as the biomedical interest for both the detection and immobilization of SARS-CoV-2 to avoid shedding of the virus into the environment. The use of these seaweed lectins as replication blockers for SARS-CoV-2 is also discussed.Seaweed lectins, especially high-mannose-specific lectins from red algae, have been identified as potential antiviral agents that are capable of blocking the replication of various enveloped viruses like influenza virus, herpes virus, and HIV-1 in vitro. Their antiviral activity depends on the recognition of glycoprotein receptors on the surface of sensitive host cells\u2014in particular, hemagglutinin for influenza virus or gp120 for HIV-1, which in turn triggers fusion events, allowing the entry of the viral genome into the cells and its subsequent replication. The diversity of glycans present on the S-glycoproteins forming the spikes covering the SARS-CoV-2 envelope, essentially complex type The occurrence of lectins, formerly designated as hemagglutinins due to their capacity to agglutinate red blood cells from humans and various animals, in marine seaweeds has been recognized for a long time, following the pioneering works of Boyd et al. and BlunGriffithsia sp., exhibited inhibiting properties towards HIV-1 [Kappaphycus alvarezii (KAA-2) [Eucheuma serra (ESA-2) [Halimeda renschii (HRL-40) [N-glycans (HM-glycans) occurring at the surface of the influenza virus [Boodlea coacta (BCA) [Oscillatoria agardhii [Nostoc ellipsosporum [Microcystis aeruginosa [Scytonema varium [N-glycans associated to different glycoprotein receptors, e.g., hemagglutinin from influenza virus [Griffithsin (GRFT) from the red alga ds HIV-1 . High-ma (KAA-2) ,13, Euch (ESA-2) , and Hal(HRL-40) also recza virus ,11,12,14za virus ,11,13. Tagardhii , cyanovisosporum ,19,20,21ruginosa ,23, and a varium . Antivira varium , influena varium ,19, hepaN-glycans decorating the spikes arrayed on the surface of the SARS-CoV-2 envelope, essentially comprised of complex N-glycans and high-mannose N-glycans [Taking into account the antiviral properties and the well known diversity of -glycans ,26, one SARS-CoV-2 spikes consist of homotrimers of S-glycoproteins and play a key role in both the recognition and the subsequent membrane fusion events, resulting in the infection of the host cells ,28. SpikN-glycans of the S-glycoproteins forming the SARS-CoV-2 spikes [Due to the high number of high-mannose 2 spikes ,26,35, hAccording to their carbohydrate-binding specificity towards simple sugars, (seaweed) lectins can been classified in five main groups of Man-specific lectins, GlcNAc-specific lectins, Gal/GalNAc-specific lectins, Fuc-specific lectins, and Sia-specific lectins. Until now, the research on seaweed lectins has focused especially on the Man-specific lectins, and these lectins have been characterized in more detail.To date, a large number of seaweed lectins have been screened, but only a few lectins have been studied in detail or have been characterized at the molecular/structural level. In spite of these limitations, the amino acid sequences and some structural information have become available for some Man-specific seaweed lectins from the red algae (Rhodophyta), the yellow-green algae (Ochrophyta), and the green algae (Chlorophyta) .-Galanthus nivalis agglutinin)-related family of lectins consists of protomers organized in a \u03b2-prism II or \u03b2-trefoil. The red alga Grateloupia chiangii lectin (GCL) and the green alga Boodlea coacta lectin (BCA) present this type of structural organization.The GNA , Nannochloropsis , Ostreococcus , and Porphyra .The legume lectin-related family is made of protomers organized in a \u03b2-sandwich or jelly roll fold (two \u03b2-sheets). This structural scaffold occurs in a few lectins from the genera Man-specific seaweed lectins belong to a few well defined protein families which have been previously identified and characterized for the molecular organization of their protomers, especially in plants :-The GNAN-acetyllactosaminic type glycans and high-mannose type glycans.Man-specific seaweed lectins readily accommodate Man, oligomannosides, and high-mannose type glycan chains. In addition, most of them recognize the tri-mannosyl core Man\u03b11,3-Man\u03b11,6-Man occurring in both Grateloupia chiangii lectin (GCL) offers a nice example of a Man-specific red alga lectin with a \u03b2-prism II structure. The \u03b2-prism II scaffold consists of three bundles of four antiparallel \u03b2-strands arranged into a flattened trefoil-shaped structure around a central pseudoaxis. The GCL lectin dimer consists of two covalently linked swapped protomers organized in a \u03b2-trefoil in such a way that both protomers become oriented almost orthogonally with a \u03b2-prism II structure [The ogonally A. Each pogonally B,C. The tructure .Porphyra umbilicalis lectin (PUL) illustrates the \u03b2-sandwich organization of the lectin protomer, resulting from the covalent superposition of two strands of \u03b2-sheets connected by more or less extended loops, forming the front and back faces of the \u03b2-sandwich, respectively (Nannochloropsis gaditana (BU14), and in the green alga Ostreococcus tauri (OtL) [The ectively A,B. Thisectively B, form tectively C. The CBectively D. Two otri (OtL) , and two additional stacking interactions with aromatic residues Y28 and Y110 C. The CBThe CBS of griffithsin also accommodates dimannosides, e.g., Man\u03b11,6Man, via a similar network of 8 hydrogen bonds and stacking interactions with the Y28 and Y110 residues, but the second Man unit located at the reducing end of the disaccharide does not participate in the interaction A,B. A fr8) to the lectin towards high-mannose glycans, has been studied in detail by Sato et al. [The carbohydrate-binding specificity of another Man-specific lectin with a \u03b2-prism I structure from the red alga o et al. . The hig5) to the modeled KAA-2 lectin, showed the existence of four CBS located at both ends of the \u03b2-barrel forming each protomer of the KAA-2 dimer , e.g., cyanovirin-N (CV-N) from the cyanobacterium sosporum , microviruginosa ,23, scyta varium , and theinin OAA . All of inin OAA .N-glycans of the N-acetyllactosaminic type and three O-glycosylation sites T323, S325 and T678 are actually glycosylated [The spike S-glycoprotein consists of a single polypeptide chain of 1273 amino acids (140 kDa), containing 22 potential osylated ,35.N-glycosylation sites NXT/NXS are highlighted in dark blue and the O-glycosylation sites are shown in bold letters highlighted in yellow:As shown below the complete amino acid sequence of the S glycoprotein of SARS-CoV-2 is made of two S1 and S2 subunits. The RBD of subunit S1 is highlighted in green and the S1/S2 cleavage site for cathepsin and serine protease TMPRSS2 is highlighted in red. All the VNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGRSLEVLFQGPGHHHHHHHHSAWSHPQFEKGGGSGGGGSGGSAWSHPQFEKN- and O-glycans attached to the potential N- and O-glycosylation sites decorating the amino acid sequence of the S-glycoprotein of SARS-CoV-2 , are almost exclusively occupied by often sialylated, bi-, tri- and tetra-antennary N-glycans of the complex type , and GalNAcGalNeuAc A large diversity was observed in the types of N-glycans and high-mannose N-glycans. Moreover, high-mannose glycans predominantly occur at the top of the S-glycoprotein whereas complex N-glycans are localized at the bottom of the glycoprotein, close to the viral envelope surface.Bi- and tri-antennary glycans are predominantly represented, across all categories of the complex N-glycosylation sites, 331NIT and 343NAT, predominantly occupied by high-mannose N-glycans that should be readily accessible to Man-specific seaweed and cyanobacterial lectins.An interesting note is that the RBD , only contains two O-glycosylation sites T323 and S325, are rather buried at the top of the S-glycoprotein, in such a way that the O-glycans are not identified as key targets for the binding of GalNAc/T-Tn-specific lectins to the S-glycoprotein trimer. However, O-glycosylation site T678 is pretty well exposed at the bottom of the S-glycoprotein and therefore, should be accessible to the GalNAc/T-Tn-specific lectins. Accordingly, seaweed lectins with the corresponding specificity should not be relevant glycan probes for SARS-CoV-2, except for the single exposed O-glycosylation site T678 and two rather buried sites for O-glycosylation (T323 and S325) . In addist cells ,35.The spikes S-glycoprotein trimers covering the SARS-CoV-2 virions, mediate the binding to the ACE2 receptor through their RBD (S1 subunit), and the subsequent fusion of the viral membrane with the cell membrane (S2 subunit). The spike S-glycoprotein exhibits some flexibility and conformational motions of the S-glycoprotein are pH-dependent . HoweverBoodlea coacta, were shown to possess antiviral properties against various enveloped virus including influenza, herpes, and hepatitis C viruses, and HIV-1 or gp120 (HIV-1) glycoproteins (n = 0) to 100% (n = 3) (A detailed study of the binding-activity towards pyridylaminated (PA-)-oligosaccharides measured for Man-specific lectins of red algae , green a coacta ) and theagardhii ), showedproteins . The bin (n = 3) .An interesting note is that some of the high-mannose glycans of both the hemagglutinin of the influenza virus and the gp120 of HIV-1 recognized by the lectins, also decorate the S-glycoprotein forming the spikes occurring at the surface of the SARS-CoV-2 envelope. Accordingly, the algal Man-specific lectins should similarly interact with the SARS-CoV-2 through the recognition of their spike S-glycoproteins. In this respect, griffithsin (GRFT) was shown to inhibit both the replication and cytopathy of the coronavirus SARS-CoV .Kappaphycus alvarezii), HRL-40 , BCA (Boodlea coacta) and OAA (Oscillatoria agadhii), shows they are the best exposed at the surface of the S-glycoprotein monomer ), N122, N234, N331, N343, N616 and N717 occupied by the high-mannose glycans recognized by the Man-specific lectins KAA-2 and seaweed lectins specific for terminal Neu5Ac residues, could interact with the S-glycoprotein of SARS-CoV-2 RS-CoV-2 . Looking at T678 [http://www.rcsb.org/pdb/) [Burkholderia oklahomensis (PDB code 4GU8 and 4GK9) [Oscillatoria agardhii (PDB code 3OBL) [The atomic coordinates of griffithsin GRFT from de 2GTY) , and lecde 2GTY) , 6\u03b1-mannde 2GTY) and highde 2GTY) , were targ/pdb/) . Similarnd 4GK9) and the de 3OBL) were alsGrateloupia chiangii [Kappaphycus alvarezii [Porphyra umbilicalis [Homology modeling of other lectins including the \u03b2-prism II folded GCL from chiangii , the \u03b2-blvarezii , and theilicalis , was perilicalis using vailicalis , ANOLEA ilicalis , and theilicalis ,97, wereDocking of simple sugars and oligosaccharides was performed with YASARA and SwissDock . HydrophGriffithsia sp., readily recognized the high mannose N-glycans located on the very similarly glycosylated SARS-CoV S-glyco- protein [The S-glycoprotein on the surface of the SARS-CoV-2 virus is a highly glycosylated protein. Due to the exposed localization of high-mannose glycans at the top of the S-glycoprotein trimers many of these glycans are readily accessible to carbohydrate-binding proteins. Seaweed lectins represent well adapted glycan probes for the specific recognition of this type of viruses. In this respect, the Man-specific lectin griffithsin (GRFT) of the red alga protein ,63. More protein , GRFT waMoreover, the binding of seaweed lectins to SARS-CoV-2 virus could be applied in biomedical research, e.g., using Man-specific seaweed lectins (1) for detection purposes of the virus on various contaminated surfaces such as doorknobs or furniture elements, (2) as an efficient barrier to avoid the shedding into the environment of contaminating virions and, (3) as control reagents for the occurrence of viral particles in biotic/abiotic samples. Depending on the case, whether properly labelled, e.g., fluorochrome-labelled, Man-specific seaweed lectins could be used directly as glycan probes or unlabelled lectins could be further detected using properly labelled, e.g., fluorochrome-labelled, specific anti-lectin antibodies.Galanthus nivalis (snowdrop), NPA from Narcissus pseudonarcissus (daffodil) and APA from Allium porrum (leek). In addition, two targets for these Man-specific lectins in the replication cycle of SARS-CoV have been identified, one in the early phase of the replication cycle during viral attachment, and a second target at the end of the infection cycle [Lablab purpureus), which specifically recognizes N-glycans of the complex type occurring on the surface of coronavirus envelope, was demonstrated to neutralize SARS-CoV-2 and prevent both viral protein production and cytopathic effects in host (mice) cells [The antiviral properties of Man-specific seaweed lectins and the application of these lectins as blocking agents for the replication of enveloped viruses still requires more investigation. So far, the antiviral properties of Man-specific seaweed lectins, have been demonstrated essentially in in vitro conditions . Indeed,on cycle . More ree) cells .Hippeastrum hybridum, at the end of the SARS-CoV infection cycle [At the molecular level, the mechanism of action for Man-specific lectins is primarily referred to a masking effect of the molecular surface of S-glycoprotein RBDs due to their interaction with the Man-containing glycans, thus hampering the proper attachment of the virions to the host cell receptors and preventing the viral replication. However, the identification of a second target for HHA, the Man-specific lectin from on cycle , suggestAlthough lectins remain attractive anti-coronavirus candidates, at present it remains difficult to correctly assess the actual role of these natural compounds in the therapeutic armamentarium, to fight against SARS-CoV-2, the coronavirus responsible for the highly transmissible infectious COVID-19 ,105,106."} +{"text": "Correction to: IMA Fungus (2019) 10:15https://doi.org/10.1186/s43008-019-0015-5Incorrect sequence:The published article (Zhang and Zhang \">NAD4L_NC_001715Correct sequence:ATGCTTTTAGAAATAATAACAGCTTATAAAATAGGAACAATCTTATTTTTAATTGGAATTTTAGGTTTCATTATCAATAGACAAAATATTCTTTTACTTATTATCTCTATTGAAATGACTTTATTAGCTATTAGTTTTATTATTATTTGTTCTGCTCTTTTCCTTGATGATTCTGCAGCAGCTTGTTTTTCACTTTATATTTTAGCTCTTGCTGGTTCAGAAGCTGCAATTGGTCTTTCACTTTTAGTTTTATTCCATAGATTTAGAGGATCAGTATTAATTTCAGCTTCTCGACAATAG\"\">nad4L_NC_036382ATGAGTTTAACTTTAGTACTTTTTTTAATAGGAATCTTAGGATTCGTATTTAATAGAAAAAATATAATATTAATGCTTATTTCTATAGAAATAATGCTATTATCTATAACATTTTTAATATTGGTAAGTTCTATTAATCTTGACGATATAATAGGACAAACATATGCTATATACATTATAGTAGTTGCTGGTGCAGAATCTGCTATCGGTTTAGCTATTTTAGTAGCTTTTTATAGACTAAGAGGAAGTATCGCAATAGAATATAAATAA\"To be in concordance with this change, \u201cP263\u201d in the nad4L column should be changed to \u201cP239\u201d in Table 3.The authors would like to apologise for any inconvenience caused."} +{"text": "Bacillus species, B. CMC1) and a regulator molecule by a vapor-phased encapsulation method with simple steps of water sublimation and poly-p-xylylene deposition in chemical vapor deposition (CVD) process. Mechanically, the capsule construct exhibited a controllable shape and dimensions, and was composed of highly biocompatible poly-p-xylylene as the matrix with homogeneously distributed bacteria and CMC molecules. Versatility of the encapsulation of the molecules at the desired concentrations was achieved in the vapor-phased sublimation and deposition fabrication process. The discovery of the fabricated capsule revealed that viable living B. CMC1 inhabited the capsule, and the capsule enhanced bacterial growth due to the materials and process used. Biologically, the encapsulated B. CMC1 demonstrated viable and functional enzyme activity for cellulase activation, and such activity was regulatable and proportional to the concentration of the decorated CMC molecules in the same capsule construct. Impressively, 13% of cellulase activity increase was realized by encapsulation of B. CMC1 by poly-p-xylylene, and a further 34% of cellulase activity increase was achieved by encapsulation of additional 2.5% CMC. Accordingly, this synergistic effectiveness of the capsule constructs was established by combining enzymatic B. CMC1 bacteria and its regulatory CMC by poly-p-xylylene encapsulation process. This reported encapsulation process exhibited other advantages, including the use of simple steps and a dry and clean process free of harmful chemicals; most importantly, the process is scalable for mass production. The present study represents a novel method to fabricate bacteria-encapsulated capsule for cellulose degradation in bioremediation that can be used in various applications, such as wastewater treatment and transforming of cellulose into glucose for biofuel production. Moreover, the concept of this vapor-phased encapsulation technology can be correspondingly used to encapsulate multiple bacteria and regulators to enhance the specific enzyme functions for degradation of various organic matters.A regulatable bioremediation capsule material was synthesized with isolated single-strain bacteria ( Bioremediation provides promising solutions for the removal of environmental pollutants, toxic elements, and poisoning management for clinical purposes ,2,3. Varp-xylylenes of United States Pharmacopeia (USP) class VI with high biocompatibility and chemical resistance to strong acids, bases, and solvents was used for encapsulation during the fabrication process. The fabrication is performed in one step with a dry and clean vapor phase, which is desirable for sensitive biological substances such as cells, enzymes, growth factors, and other functional peptides and proteins [p-xylylene polymer on a template substrate that is eventually sublimated, and by manipulating the mass transport during the processing conditions, diverse species with distinct thermodynamic properties were subjected to sublimation and deposition within the confined space of the templates. Finally, transformation of the template resulted in the construction of composite materials with defined physical properties in terms of porosity, bulk size and geometry, and chemical functionality by the compartmentalized functional entities and the devised interfacial chemistries [Bacillus species CMC1 (hereafter referred to as B. CMC1) with specific enzymatic function and the same precision to localize and distribute B. CMC1 due to the encapsulation technique, enabling well-controlled bacterial growth activities and functions, and (ii) a regulator molecule, carboxymethyl cellulose (CMC), with a customizable encapsulation concentration to provide the same customizable stimulation dosages to regulate the enzymatic functions of the neighboring B. CMC1 bacteria. The fabricated and encapsulated capsules were composed of inhabitant bacteria from (i) and the surrounding conditioning regulator molecules from (ii), and the synergistic activities exhibited by (i) and (ii) were able to deliver a combination of controlled enzymatic factors for bioremediation in the devised microenvironments , 0.2 g CaCl2 , 0.2 g KNO2 , 2 g NH4NO3 , 0.3 g Na2HPO4 , 5 g glucose , and 2.5 mL tween 80 were added and then pH with NaOH to 7.0. The genomic DNA of the isolated bacterial strain was extracted using a commercial genomic DNA extraction kit . Then, the sequence of its 16S rRNA was confirmed by polymerase chain reaction by using the primers F8 (5\u2032GAGAGTTTGATCCTGGCTCAG3\u2032) and R1492 (5\u2032GGTTACCTTGTTACGACTT3\u2032). The obtained 16S rRNA sequence from the isolated bacteria wasWastewater and activated sludge were collected from the sewage of a pig farm in Taoyuan, Taiwan. All tools and reservoirs were sterilized before sampling. Activated sludge was stored immediately at 4 \u00b0C after collection. The wastewater and activated sludge were used for further culturing of environmental bacteria in liquid nutrient medium. In one liter of culture medium, 3 g peptone , 1 g yeast extract , 5 g NaCl , 0.2 g MgSOTGCTATACATGCAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTTTGAACCGCATGGTTCAAACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTACCGTTCGAATAGGGCGGTACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTAGGAGCCAGC.https://www.ezbiocloud.net/. The isolated bacteria were the Bacillus species with the top ranking of B. subtilis and was named Bacillus species CMC1 (B. CMC1) in the study.The sequence was further confirmed according to the 16S rRNA database at B. CMC1 was cultured for 30 h at 30 \u00b0C and then washed twice with diH2O by centrifugation and resuspended into final solutions , 1.5% CMC, or 2.5% CMC) to prepare samples with the same concentration of bacteria (~1 \u00d7 108 bacteria/mL) on the same day of capsule fabrication. The prepared bacterial solution with/without CMC was solidified by liquid nitrogen for later encapsulation. A home-built vapor deposition system was used for the encapsulation and fabrication process in this study , 0.2 g KCl , and 1 g MgSO4\u00b77H2O were added, and then the pH was adjusted to 6.3 with NaOH. Then, 9 g agar and 10 mg trypan blue were added. A single bacterial capsule (100 \u00b5L in volume) was seeded in the center of one 9 cm plate and left at 30 \u00b0C for up to 140 h for subsequent enzyme functioning zone quantification. For the quantification of DNA and RNA, a single bacterial capsule (100 \u00b5L in volume) was seeded in a 50 mL centrifuge tube with 30 mL of growth culture medium inside, and the culture was left at 30 \u00b0C for 50 h on an orbital shaker with a 100 rpm shaking speed. The same volume of the culture (5 mL) from encapsulated bacteria was centrifuged for genomic DNA extraction using a commercial extraction kit for the following DNA quantification. The same number of bacterial cells (2 \u00d7 108 cells) was used for total RNA extraction by using a commercial extraction kit for total RNA quantification. The concentrations of DNA and RNA were measured by a NanoDrop Spectrophotometer . For this, 1 \u00b5L of purified DNA and RNA were used in the analysis. Note that only the DNA and RNA with the 260/280 ratio between 1.8 and 2.0 were used for concentration measurements, ensuring the sufficient purity of DNA and RNA for further quantification analysis.Cellulase activity was examined on 9 cm agar plates by quantifying the size of the enzyme functioning zones. To one liter of cellulase visualizing agar, 1 g yeast extract , 1 g NHB. CMC1 in the porous parylene structure. A VK-9510 3D profile microscope was used to analyze the external architecture of the bacterial capsules and the existence of the bacteria inside the structure. SEM images were recorded using a NovaTM NanoSEM operated at a primary energy of 10 keV and a pressure of 5 \u00d7 10\u22126 Torr to detailed the internal structure of the bacterial capsule. EDS elemental point analyses were captured for the quantification of the studied elements. 3D analysis of the interior structure was performed by using Bruker Skyscan 2211 at 2.0 \u03bcm/pixel resolution. The setting of the voltage was 40 kVp, whereas the current was 700 \u03bcA at 8 Watt output with microfocus mode. Image reconstruction, ring artifacts, and beam-hardening correction were performed using reconstruction software, Instarecon . FT-IR spectra of the fabricated capsules were recorded by a Spectrum 100 spectrometer equipped with an ATR detector . The recorded spectra ranged from 600 to 4500 cm\u22121 with 4 scan times at 4 cm\u22121 resolution.A Nikon ECLIPSE 80i fluorescence microscope with a visible light source was used to visualize the crystal violet-stained p-xylylene in one continuous step [p-xylylene molecules to replace the resulting space when the sublimated ice/water molecules evaporated from the ice templates. The resultant construct consisted of a three-dimensional porous poly-p-xylylene matrix with encapsulated B. CMC1 bacteria in the matrix , and confocal microscopy to verify the localization and distribution of the bacteria . The imaructures a. The anB. CMC1, we hypothesized that the bacteria were resistant to low temperatures during iced template preparation and low-pressure conditions (approximately 10\u22123 Torr) during the sublimation and deposition process. Cultured samples of encapsulated B. CMC1 were compared to samples of B. CMC1 without encapsulation (positive control), and their cell densities were monitored by OD600 measurement. The results showed comparable bacterial growth patterns and activities in liquid medium for both types of bacteria during the culture time frame of 10 h various concentrations of CMC . Subsequently, the same sublimation and deposition process was performed to resolve the capsule construct that comprised a poly-p-xylylene matrix with simultaneous encapsulation (i) and (ii) the same matrix structure. In addition to the already confirmed encapsulation of B. CMC1, the use of FT-IR analysis indicated that the intensity of the characteristic \u2013C=O and \u2013CH\u2013O\u2013CH2 peaks from CMC observed at 1558 and 1050 cm\u22121, respectively, gradually increased from a low concentration to higher concentrations of CMC content in the fabricated capsules from poly-p-xylylene, additionally showed consistency by stoichiometry of the encapsulated CMC concentrations exhibited approximately 10 h of bacterial growth in the lag phase and showed consistency with the samples with only B. CMC1 encapsulation were studied in parallel to bare samples with unregulated B. CMC1 (0% CMC), and their enzymatic cellulase activities were first examined on agar plate results for up to 140 h to a 0.25% regulation was found, and an anticipated enhancement of the expression with increasing CMC regulation compositions was accordingly discovered with a 13.1% increase for 1.5% CMC and 27.4% for 2.5% CMC of the studied capsule samples. On the other hand, based on the same bacteria number to extract their total RNA, the results in B. CMC1 was regulated for the synergistic capsules, and the synergy was found to be consistent with the same regulatable and enhancement of the bacterial population in the aforementioned study. Collectively, the results unambiguously verified the hypothesis that synergic effectiveness was achieved by vapor encapsulation of (i) B. CMC1 and (ii) the CMC regulator in the same fabricated capsule constructs, and the regulation was achievable through the versatile use of the encapsulated CMC compositions.More evidence showing the enhancement of the enzyme activities was further verified through the analysis of the genomic information, including the amount of genomic DNA and total RNA from the cultured samples of the capsules at a time point of 50 h, which was in the early stage of stationary phase in the bacterial growth curve. Calculation and quantification of the genomic DNA was performed based on the same volume of bacteria obtained from the capsule samples, and as shown in B. CMC1 was achieved in the current study. Because of the versatility of various types of remedial bacteria and regulator molecules, unlimited applications are expected beyond those shown in the report. In addition, the use of water/ice templates and a dry and clean vapor-phased process preserved the sensitive biomolecules and their biological functions, and the final fabricated capsule construct was composed of a USP Class VI compatible poly-p-xylylene matrix. Due to this specific production process, this novel fabrication process can not only encapsulate functional bacteria, regulating molecules, but also other potential absorbing material to concentrate the organic matters for degradation. It was reported that the encapsulation of phenol metabolic bacteria in microfiltration membrane capsules alone can create a confined environment to enhance the bioremediation of efficiency of encapsulated bacteria [p-xylylene fabrication technology can constitute a defined inner porous structure to capture different size of organic particles to further concentrate organic matters and a controllable size and shape of the products to fit to different situations. Until now, most of the present studies still focused on identifying the bacteria with specific enzyme functions to use in the bioremediation process [Synergistic and regulatable bioremediation of cellulose by encapsulated bacteria . The pol process ,25,26. W"} +{"text": "Scientific Reports 10.1038/s41598-020-68172-2, published online 09 July 2020Correction to: The Supplementary Information file that accompanies this Article contains errors in Supplemental Table 1.her9 F, \u201cCCTGACGGAGAACTGAACACAAGACACACA\u201dThe primer sequence reported for should read:\u201cCGCCACACACACGCTCGTGT\u201dher9 R, \u201cTTTCTCAATGGTACGGCGGGTGCTCTGGGC\u201dThe sequence reported for should read:\u201cCGGCTTGTGGAACGCCCGAA\u201dher9 CR F, \u201cAAGCTTCCTGACGGAGAACTGAACACAAGACACACA\u201dThe sequence reported for should read:\u201cAAGCTTTGGCTTCTACCGGACTCAACTTTGGTGTTT\u201dher9 CR R, \u201cGAATTCTTTCTCAATGGTACGGCGGGTGCTCTGGGC\u201dFinally, the sequence reported for should read:\u201cGAATTCCCATGAAAACTTTATAAGTTCATATGAGATGCGC\u201d"} +{"text": "Recent approval of chimeric antigen receptor (CAR) T cell therapy by the European Medicines Agency (EMA)/Federal and Drug Administration (FDA) and the remarkable results of CAR T clinical trials illustrate the curative potential of this therapy. While CARs against a multitude of different antigens are being developed and tested (pre)clinically, there is still a need for optimization. The use of single-chain variable fragments (scFvs) as targeting moieties hampers the quick generation of functional CARs and could potentially limit the efficacy. Instead, nanobodies may largely circumvent these difficulties. We used an available nanobody library generated after immunization of llamas against Cluster of Differentiation (CD) 20 through DNA vaccination or against the ectodomain of CD33 using soluble protein. The nanobody specific sequences were amplified by PCR and cloned by Gibson Assembly into a retroviral vector containing two different second-generation CAR constructs. After transduction in T cells, we observed high cell membrane nanoCAR expression in all cases. Following stimulation of nanoCAR-expressing T cells with antigen-positive cell lines, robust T cell activation, cytokine production and tumor cell lysis both in vitro and in vivo was observed. The use of nanobody technology in combination with PCR and Gibson Assembly allows for the rapid and effective generation of compact CARs. CARs are synthetic chimeric receptors consisting of an antibody based extracellular part to recognize specific antigens expressed on the surface of tumor cells and an intracellular part containing (co)stimulatory signals derived from CD3\u03b6, CD28 and/or 4_1BB ,10,11. CH) and variable light (VL) regions and the generation of a single-chain variable fragment (scFv) for target recognition [H and VL of the different scFvs [CARs that obtained market authorization and those that are clinically tested rely on the generation of a monoclonal antibody, the sequencing of the variable heavy found in Camelidae [H gene family III and their small size [Nanobodies can potentially be an alternative antigen-binding moiety. Nanobodies, first described by Hamers-Castermans, are isolated from the Vamelidae . The strall size ,29. No oall size ,31,32,33all size . Finallyall size . Therefoall size . Both siall size ,40,41,42all size .We previously described the use of a dual specific CAR based on nanobodies . To expa1 CH2CH3 (Fc) spacer, the CD28 transmembrane domain and the CD28 and CD3\u03b6 intracellular signaling domains. The 4_1BB:\u03b6 construct contains the CD8\u03b1 hinge and transmembrane region followed by the 4_1BB and CD3\u03b6 intracellular signaling moieties. Both CAR backbone constructs were cloned in a retroviral plasmid that contains an IRES-eGFP sequence to allow easy detection of CAR-expressing cells. At the 5\u2032 position we incorporated a BamHI restriction site . The nanobody specific sequences were amplified by PCR using primers with 15\u201320 nucleotide overhangs complementary to the 5\u2032 retroviral plasmid (forward primer) or the 3\u2032 CAR sequence (reverse primer). The forward primer also encoded a leader sequence derived from the L-kappa murine leader sequence. The PCR products were purified, and Gibson Assembly was used to introduce the amplified sequence into the CAR vector . The nanobody sequences specific for human CD33 were derived from a llama nanobody phage library. This library was constructed from peripheral blood lymphocytes obtained after immunization of a llama with the extracellular domain of human CD33 protein. Three different nanobody clones were randomly selected and amplified by PCR, purified and cloned into the CAR backbone by Gibson Assembly, resulting in three CD33-specific nanoCAR constructs. After sequence confirmation, we produced retroviral particles and subsequently transduced peripheral blood mononuclear cells (PBMC). NanoCAR expression was confirmed by flow cytometry using an antibody specific for the nanobody protein . Transdu\u2212 ovarian cancer cell line SKOV3 was used as negative control. Most acute myeloid leukemia cells express CD33. A panel of AML cell lines was analyzed for CD33 expression A. Flow c+ cell lines. The cytotoxicity was driven by CAR binding to cognate antigens, since CD33+ cell lines were neglected by non-transduced T cells and CD33\u2212 SKOV3 cells did not elicit target cell lysis. Next, we evaluated cytokine production of our CD33-specific nanoCAR T cells 5 h after co-incubation with our target cell panel by intracellular staining for interferon-\u03b3 (IFN-\u03b3) and interleukin-2 (IL-2). The nanoCAR-expressing cells were able to produce both IFN-\u03b3 and IL-2 after incubation with CD33+ MOLM13 cells. Incubation with the CD33\u2212 cell line SKOV3 resulted in no significant expression of IL-2 or IFN-\u03b3 mice were intravenously injected with Thp1 cells expressing firefly luciferase. The cells were allowed to engraft and expand. After checking engraftment 7 days after injection, a single tail vein injection of nanoCAR T cells was administered day 0 in A. Follow+ hematopoietic precursor cells (HPC) were isolated from different cord blood donors and analyzed for CD33 expression. Only CD34dimCD38dim HPC expressed CD33 although at a lower level compared with leukemic cell lines through their CH2 domain. Earlier studies of the IgG1 Fc spacer showed off-target CAR T cell activation by FcR\u03b3+ myeloid and lymphoid cells and speculated that potential activation-induced cell death (AICD) could occur [We also noticed no survival benefit nor disease control in NSG mice injected with CD33-1-CD28:\u03b6 nanoCAR T cells. Furthermore, we were not able to detect any eGFPld occur . Other gld occur ,55,56,57In summary, we have shown that our technique of using nanobodies, PCR and Gibson Assembly is a rapid and efficient way to generate nanoCAR T cells with a 100% success rate for the six randomly selected nanobody clones. We chose two completely different antigens: CD20, a tetraspanner and CD33, a single pass receptor, to test our technique. We strongly believe that the use of nanobodies is advantageous over the use of scFvs, since nanobodies are monomeric structures that (i) will probably not aggregate on the T cell surface and therefore not induce premature T cell activation and exhaustion ; (ii) wiProcedures for immunization of llamas, preparation of mRNA, construction of the library, and panning were performed as previously described .l-glutamine , 100 IU/mL penicillin and 100 IU/mL streptomycin . SKOV3 was cultured in DMEM supplemented with 10% fetal calf serum , 2 mM L-glutamine , 100 IU/mL penicillin and 100 IU/mL streptomycin . Thp1 was cultured in RPMI supplemented with 0.05 mM \u03b2-mercaptoethanol, 10% fetal calf serum , 2mM L-glutamine , 100 IU/mL penicillin and 100 IU/mL streptomycin .All the cell lines were cultured as per American Type Culture Collection recommendations. RL, Raji, MOLM13, U937, were cultured in RPMI , supplemented with 10% fetal calf serum , 2 mM L-glutamine , 100 IU/mL penicillin and 100 IU/mL streptomycin . Jurkat and HL60 were cultured in IMDM supplemented with 10 % fetal calf serum , 2 mM Escherichia coli (High Efficiency), NEB) and plated on agar. After overnight incubation, colonies were selected and grown in liquid lysogenic broth overnight. Plasmids were isolated and sequenced. Colonies containing the correct plasmid were further cultured and midipreps (Qiagen) were performed. The different constructs, as shown in 5 cells per 6 cm dish and placed overnight in a 7% CO2 incubator at 37 \u00b0C. Next day, the plasmids encoding the nanoCAR constructs were transfected using calcium phosphate as follows: per 6 cm dish, 10 \u00b5g plasmid DNA was diluted in 36 \u00b5L 2M CaCl2 (homemade) and subsequently nuclease free water was added to a total volume of 300 \u00b5L. The DNA-CaCl2 solution was pipetted dropwise into a 15 mL polystyrene tube containing 300 \u00b5L 2\u00d7 HEPES-buffered saline (HBS) solution (homemade) while blowing air bubbles in the 2\u00d7 HBS buffer. The mixture was incubated for 15 min at room temperature and then added dropwise to the cells. Ten minutes before the addition of the DNA mixture, 2 mL medium was added to the cells containing 1 \u00b5L of a 200 mM chloroquine solution. Cells were placed in a 7% CO2 incubator at 37 \u00b0C overnight. Medium was refreshed at day one. At day two, cells were analyzed for transfection efficiency and transgene expression and reseeded in a T75 culture flask in selection medium containing 2 \u00b5g/mL puromycin (Sigma). After an additional two days, selection medium was replaced by medium without puromycin. At day six and day 10, the selection cycle was repeated. Finally, at day fourteen, retroviral supernatant was collected and was frozen at \u221280 \u00b0C until use. Phoenix A cells were analyzed for nanoCAR and eGFP expression at days of reseeding and at day fourteen. Viral particles were produced using the Phoenix A packaging cell line. Phoenix A cells were seeded at 7.5 \u00d7 10+ cells was determined by flow cytometry and T cells were stimulated with Immunocult Human CD3/CD28/CD2 T cell activator per fabricator instructions in cIMDM, in the presence of 10 ng/mL IL-12 . Cells were harvested 72 h after stimulation and resuspended in retroviral supernatant and centrifuged for 90 min at 1000\u00d7 g at 32 \u00b0C on retronectin coated plates. Buffy coats from healthy donors were obtained from the Belgian Red Cross and used following the guidelines of the Medical Ethical Committee of Ghent University Hospital, after informed consent had been obtained, in accordance with the Declaration of Helsinki. PBMC were isolated by Lymphrop gradient centrifugation. The percentage of CD3Transduced cells were detected by eGFP expression and after staining with a nanbody specific antibody. Transduced cells were sorted and expanded on irradiated allogenic feeder cells, consisting of a mixture of 40 Gy irradiated peripheral blood mononuclear cells and 50 Gy irradiated JY cells. Cells were cultured in cIMDM, supplemented with 1 \u03bcg/mL phytohemagglutinin (Roche) was added on day five and day ten. Cells were restimulated every seven\u2013fourteen days.Staining of surface markers was performed as described earlier . The fol+, Raji, RL and SKOV3. Cytotoxicity assay was performed as previously described . The folDetection of cytokine producing cells was performed as previously described , with th4 Thp1 cells) in cIMDM. Cells were stained with CD3, CD4 and CD8\u03b1 at the start of the co-culture and at day three, seven, ten and fourteen. At day seven of co-culture, 2 \u00d7 104 Thp1 cells were added to the remaining wells. Cell numbers were determined by flow cytometry. T cells expressing nanoCARs were incubated with Thp1 cells at an effector/target ratio of 0.025:1 was performed after intraperitoneal injection of 150 mg/kg bodyweight d-luciferin (Perkin Elmer) and 5 \u00d7 106 nanoCAR T cells were intravenously injected. Tumor progression was followed up by IVIS imaging. Mice were checked for overall health status and scarified when humane endpoints were reached. NSG mice were injected intravenously with 2 \u00d7 106 RL cells were subcutaneously injected. The cells were allowed to form a solid mass and at day eighteen, 5 \u00d7 106 nanoCAR T cells were intravenously injected. Tumor progression was followed up by caliper. Mice were checked for overall health status and scarified when human endpoints were reached. For the RL model, 2 \u00d7 10+ HPC were purified using CD34 MicroBead Kit (MACS Miltenyi Biotech) per manufactures instructions. T cells expressing the nanoCARs were cultured with CD34+ HPC in cIMDM supplemented with stem cell factor , thrombopoietin and FMS-like tyrosine kinase 3 ligand , all three at 100 ng/mL with PBMC at a 1:1 ratio. At the start and at 24 h, 48 h and at 72 h of the co-culture, cells were harvest and stained for CD3, CD34, CD38 and CD33. Subsequently, cells were analyzed by flow cytometry and absolute cell counts were determined.Cord blood was acquired through the Belgian Red Cross and used following the guidelines of the Medical Ethical Committee of Ghent University Hospital (EC/UZG 2015/0768), after informed consent had been obtained, in accordance with the Declaration of Helsinki. PBMC were isolated using density gradient centrifugation. CD34Underlined sequences indicate overlap with the LZRS vector, cursive sequences indicate BamHI site, sequences in bold indicate leader sequence, sequences in upper case indicate overlap with nanobody sequences and sequences in upper case and bold indicate overlap with CAR backbone.Nanobody specific sequence amplification.Fw primer:ggatccgggtggaccatcctctagactgccgccatggattttcaggtgcagattttcagcttcctgctaatcagtgcctcagtcataatgtctagaCAGGTGCAGCTGCAGGAG3\u20325\u2032Rev primer 4_1BB:\u03b6 backbone:ggatccACTGAGGAGACGGTGACCTG3\u20325\u2032GGTCGCGGCGCTGGCGTCGTGGTCRev primer CD28:\u03b6 backbone:ggatccACTGAGGAGACGGTGACCTG3\u20325\u2032AGGAGATTTGGGCTCGGCGGGCDNA sequence of CD28:\u03b6 CAR backbone construct:gggtggaccatcctctagactgccggatccgccggatccCGCCGAGCCCAAATCTCCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGGGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCTTCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAAAAGATCCCAAATTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCCTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGATAAatggattttcaggtgcagattttcagcttcctgctaatcagtgcctcagtcataatgtctagaGTDNA sequence of 4_1BB:\u03b6 CAR backbone construct:gggtggaccatcctctagactgccggatccgccggatccGACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAAatggattttcaggtgcagattttcagcttcctgctaatcagtgcctcagtcataatgtctagaGTData were analyzed with GraphPad Prism Software . Statistical differences between groups or conditions were determined by two-way ANOVA, followed by Bonferroni post hoc test. Survival curves were compared using the log-rank Mantel\u2013Cox test."} +{"text": "Ppt1, in cancer cells reduced priming and cytotoxic capacity of primed T cells. Exposure of antigen-primed T cells to macrophage-conditioned medium derived from macrophages treated with PPT1 inhibitors enhanced melanoma-specific killing. Genetic or chemical Ppt1 inhibition resulted in M2 to M1 phenotype switching in macrophages. The combination was associated with a reduction in myeloid-derived suppressor cells in the tumor. Ppt1 inhibition by HCQ, or DC661, induced cyclic GMP-AMP synthase/stimulator of interferon genes/TANK binding kinase 1 pathway activation and the secretion of interferon-\u03b2 in macrophages, the latter being a key component for augmented T cell\u2013mediated cytotoxicity. Genetic Ppt1 inhibition produced similar findings. These data provide the rationale for this combination in melanoma clinical trials and further investigation in other cancers.New strategies are needed to enhance the efficacy of anti\u2013programmed cell death protein antibody (anti\u2013PD-1 Ab) in cancer. Here, we report that inhibiting palmitoyl-protein thioesterase 1 (PPT1), a target of chloroquine derivatives like hydroxychloroquine (HCQ), enhances the antitumor efficacy of anti\u2013PD-1 Ab in melanoma. The combination resulted in tumor growth impairment and improved survival in mouse models. Genetic suppression of core autophagy genes, but not Inhibiting palmitoyl-protein thioesterase 1 (PPT1), a target of CQ derivatives like hydroxychloroquine (HCQ), enhances the antitumor efficacy of anti-PD-1 Ab in murine melanoma models. While there have been extensive efforts to combine other T cell\u2013stimulating factors with anti\u2013programmed cell death protein antibody anti\u2013PD-1 Ab), there is an increasing interest in identifying T cell\u2013independent strategies that will augment the efficacy of anti\u2013PD-1 Ab. Tumor cell autophagy has been identified as a major resistance mechanism to targeted therapy and chemotherapy. Chloroquine and hydroxychloroquine (CQ and HCQ) are the only drugs that have been tested as autophagy inhibitors in clinical trials in patients with cancer. However, there are conflicting reports about whether as a single agent CQ derivatives augment, impair, or have no effects on antitumor immunity Ab, ther.Ppt1 (siPpt1) were all able to convert M2 to M1 tumor-associated macrophages (TAMs). Conditioned media from macrophages treated with Ppt1 inhibitors were able to enhance T cell\u2013mediated cancer cell killing. Interestingly, the combination of HCQ and anti\u2013PD-1 Ab resulted in a change in TAM polarization and a significant reduction in myeloid-derived suppressor cell (MDSC) infiltration in vivo while each single agent did not. The mechanism by which PPT1 inhibitors produced macrophage phenotype switching was dependent on mitochondrial calcium release and p38 activation. PPT1 inhibition also induced the cyclic GMP-AMP synthase/stimulator of interferon genes/TANK binding kinase 1 (cGAS/STING/TBK1) pathway to induce IFN-\u03b2 release from macrophages. PPt1 inhibition\u2013induced IFN-\u03b2 release was critical for the augmentation of antigen-primed T cell killing of melanoma cells. These data suggest that this combination, which can be immediately tested in the clinic, could provide an alternative rational combination approach for melanoma immunotherapy.Recently, we identified the major molecular target of CQ derivatives as the lysosomal protein palmitoyl-protein thioesterase 1 (PPT1) , 4. Meanhttps://doi.org/10.1172/jci.insight.133225DS1). Tumors harvested at the end of the experiment were significantly smaller with combination compared with monotherapy arms , and Atg7. KD of these 3 genes in B16 cells produced reduced autophagic flux as evidenced by increase in p62. While Lc3b-II levels did not change much with siUlk1 or siPik3c3 as is often observed to an antitumorigenic M1) phenotype or mouse macrophages in vitro and in vivo phenotyp. Morpholhenotype . KD of crization . Ppt1 KD+ T cells play a major role in the elimination of tumor cells and regression of tumors by their cytotoxic activity activates Cre recombinase, and melanocytes express mutant Braf and lose Pten. Spontaneous melanoma tumors arise on the skin of these mice with 100% penetrance. In concordance with the B16 tumor model, we found that HCQ significantly enhanced the antitumor response of anti\u2013PD-1 Ab (+CD11b+Ly6C+Ly6G\u2013CX3CR1+ monocytes and spleen eosinophils (CD45+CD11b+Siglec F+) and no significant change in DCs (CD45+CD11b+CD11c+) and PMN-MDSCs (CD45+CD11b+Ly6CloLy6Ghi) T cells compartment in the spleen and tumor . There woLy6Ghi) . There wnd tumor . Taken tPpt1 also resulted in increased levels of p-p38 compared with siNon-target control . HCQ, DC control . PPT1 in control . Inhibit control .+/Ca2+ exchanger, completely abrogated the increased p38 phosphorylation induced by lysosomal inhibitors , while a comparison of DC661 versus control showed 47 significantly changed proteins out of a total of 3166 proteins identified with high confidence . MCM was subjected to proteome analysis. Comparison of HCQ versus control showed 64 significantly changed proteins CAGCATCTTCTTGGCAGACATAAATCAAGAGAGGTGTGTCAATGAGTCCTACAAGAAGAACCTGATGGCCCTCAAGAAGTTTGTGATGGTGAAATTCTTTAATGATTCCATTGTGGACCCTGTCGACTCTGAGTGGTTTGGATTTTACAGAAGTGGCCAAGCTAAGGAAACCATTCCCCTCCAGGAGAGCACTCTATACACAGAGGACCGCCTGGGGCTAAAGAAAATGGACAAAGCAGGAAAGCTAGTGTTTCTGGCTAAGGAAGGGGACCATCTTCAAATATCTAAAGAATGGTTTACTGCCCACATCATACCTTTTCTTAAGTGATGCCCTGGCACTTTATAGCAGAGTTCATGAAACCACAGCTCTTCCAAGCCATGTACATAGTTCATGCTCAGCCTGAACTCTAATCTAGCCTGCAACCAGCCCTTCTCTCCTCTTATCATCTAACATACCCTACTTGGAAAGATCTAAGATCTCAATCTTATCCTTTGCCGCCT. PpT1 siRNA pool 2 (Santa Cruz Biotechnology sc-142398) AGGCGGCAAAGGATAAGATTGAGATCTTAGATCTTTCCAAGTAGGGTATGTTAGATGATAAGAGGAGAGAAGGGCTGGTTGCAGGCTAGATTAGAGTTCAGGCTGAGCATGAACTATGTACATGGCTTGGAAGAGCTGTGGTTTCATGAACTCTGCTATAAAGTGCCAGGGCATCACTTAAGAAAAGGTATGATGTGGGCAGTAAACCATTCTTTAGATATTTGAAGATGGTCCCCTTCCTTAGCCAGAAACACTAGCTTTCCTGCTTTGTCCATTTTCTTTAGCCCCAGGCGGTCCTCTGTGTATAGAGTGCTCTCCTGGAGGGGAATGGTTTCCTTAGCTTGGCCACTTCTGTAAAATCCAAACCACTCAGAGTCGACAGGGTCCACAATGGAATCATTAAAGAATTTCACCATCACAAACTTCTTGAGGGCCATCAGGTTCTTCTTGTAGGACTCATTGACACACCTCTCTTGATTTATGTCTGCCAAGAAGATGCTG. Ppt1 siRNA single duplex CTGGTTGCAGGCTAGATTAGAGTTCAGGCTGAGCATGAACTATGTACATGGCTTGGAAGAGCTGTGGTTTCATGAACTCTGCTATAAAGTGCCAGGGCATCACTTAAGAAAAGGTATGATGTGGGCAGTAAACCATTCTTTAGATATTTGAAGATGGTCCCCTTCCTTAGCCAGAAACACTAGCTTTCCTGCTTTGTCCATTTTCTTTAGCCCCAGGCGGTCCTCTGTGTATAGAGTGCTCTCCTGGAGGGGAATGGTTTCCTTAGCTTGGCCACTTCTGTAAAATCCAAACCACTCAGAGTCGACAGGGTCCACAATGGAATCATTAAAGAATTTCACCATCACAAACTT. Fluo-4, AM, was used to stain the cells for calcium as per the manufacturer\u2019s instructions .DC661 was provided in-house. Purity of the sample was determined by NMR spectroscopy and liquid chromatography-mass spectrometry. Mouse melanoma B16-F10 (CRL-6475) and RAW 264.7 (TIB-71) were purchased from ATCC. Cell lines were tested for mycoplasma biannually and authenticated using short-tandem repeat fingerprinting. Cells were cultured in Dulbecco\u2019s modified Eagle medium (DMEM) supplemented with 4.5 g/L glucose, sodium pyruvate, phenol red, and 10% fetal calf serum . BMDMs were isolated as described previously .Total RNA was isolated by RNA isolation kit according to the manufacturer\u2019s protocol. Complementary DNA (cDNA) was synthesized using iScript Reverse Transcriptase kit with 500 ng of purified RNA as per manufacturer\u2019s protocol . The qPCR reaction was set up using SYBR Green PCR Master Mix containing 1 \u03bcL of cDNA. All measurements were carried out in duplicate, and Hsp90 was used as internal standard for \u0394CT calculations. Gene expression analysis was done using the following primers: iNos forward (AGGAGGAGAGAGATCCGATTTAG), iNos reverse (TCAGACTTCCCTGTCTCAGTAG); Retnla/Fizz1 forward (TGCCAATCCAGCTAACTATCC), Retnla/Fizz1 reverse (GCAAAGCCACAAGCACAC); Hsp90 forward (GGGAGCTCATCTCCAATTCATC), Hsp90 reverse (GTCCTGTTTGCTGGGAATGA); Arg1 forward (TACCTGCTGGGAAGGAAGAA), and Arg1 reverse (CTGTAAGATAGGCCTCCCAGA).Atg7 gRNA AACTCCAACGTCAAGCGGGT sequences for targeting mouse cells were used as described previously (XhoI and NheI sites of pLX-sgRNA followed by restriction digest and ligation. Sanger sequencing was used to confirm that the Atg7 gRNAs and nontargeting control RNA were correctly subcloned in the pLX-sgRNA vector. B16 cells were first transfected with the pCW-Cas9 vector and selected with puromycin 4 \u03bcg/mL for 6 days and then transfected with the pLX-sgRNA containing the Atg7 gRNA and nontargeting gRNA using Lipofectamine 3000 (Thermo Fisher Scientific) based on the manufacturer\u2019s instructions. After 48 hours\u2019 incubation, growth medium was changed to selection medium containing blasticidin 6 \u03bcg/mL. After blasticidin selection for 12 days, the cells were treated with doxycycline 0.5\u20131 \u03bcg/mL to express Cas9 and induce Atg7 deletion. Doxycycline was replenished every 2 days for 2 weeks, after which the cells were harvested and analyzed by immunoblotting for Atg7 deletion (The nontargeting guide RNA (gRNA) TAGCGAACGTGTCCGGCGT and eviously . The 2 seviously . Subclondeletion .Whole-cell lysates and lysosomal extracts were immunoblotted as previously described , 6.5) with an equal volume of Matrigel in the right flank of C57BL6/J mice. Daily injection of HCQ (60 mg/kg) and every-other-day injection of anti\u2013PD-1 Ab (200 \u03bcg) commenced at the tumor size of 50 mm3. CA PtenloxP Tyr::CreERT2BRaf mice (The Jackson Laboratory) were treated topically on the back with 4-HT to elicit V600EBRaf and to silence Pten expression. Tumors were measured using electric calipers . Tumor volume was calculated as L \u00d7 W2 \u00d7 0.5. In all animal experiments 2-tailed t test or 2-tailed t test for unequal variance was used to test the hypothesis that the addition of HCQ to anti\u2013PD-1 Ab is significantly different compared with anti\u2013PD-1 Ab + Veh.Tumor generation, measurement, and harvesting were performed as previously described , 9. BrieTumor was excised and digested with a tumor digestion kit according to the manufacturer\u2019s protocol.6) from the digested tumor were stained with antibodies (please see Cells (1 \u00d7 105) were cultured and irradiated with 25 Gy x-rays. Splenocytes were then cultured with (primed) or without (unprimed) irradiated B16 cells in the presence of IL-2 (5 IU/mL) and cocultured for 48 hours. Splenocytes cultured with concanavalin A (10 \u03bcg/mL) were used as a nonspecific T cell priming control. Priming was confirmed by IFN-\u03b3 ELISA of the supernatant. Primed splenocytes were then cocultured with freshly cultured B16 cells with various target (B16) to effector (splenocytes) ratios. The B16 cell death\u2013associated LDH release and then percentage cytotoxicity were measured according to the manufacturer\u2019s protocol .B16 tumor cells to remove cellular debris. The supernatant was passed through a 0.2 \u03bcm filter, concentrated 50-fold using an Amicon Ultra 3K membrane, electrophoresed for 0.5 cm into an SDS-PAGE gel, and stained with colloidal Coomassie . The entire stained region was excised, digested with trypsin, and analyzed by liquid chromatography-tandem mass spectrometry on a Q Exactive HF mass spectrometer (Thermo Fisher Scientific) coupled with a Nano-ACQUITY UPLC system using a 245-minute gradient and acquisition parameters as previously described were computed for each mouse using linear regression of the natural logarithm of tumor volume on time; the estimated slopes for the HCQ and anti\u2013PD-1 Ab or anti\u2013PD-1 Ab + Veh groups were analyzed using a 2-sample t test. All t tests were 2 tailed. A P value less than 0.05 was considered significant. P values presented in the figures are for the test of the hypothesis that the expected mean when HCQ is added to anti\u2013PD-1 Ab is significantly different from the expected mean for anti\u2013PD-1 Ab + Veh. Adjusted P values are indicated as * when the adjusted P < 0.05 and as ^ when the adjusted P < 0.10. All analyses were done using SAS/STAT software, version 9.4 of the SAS system for Windows.For continuous variables, a 2-sample All animal experiments were performed in accordance with the protocols approved by the University of Pennsylvania Institutional Animal Care and Use Committee.GS, RO, ENO, EJW, DG, XX, DWS, PAG, and RKA conceived experiments. GS, RO, ENO, JM, JA, VWR, SL, SP, JLL, SH, AR, VJ, MCN, JDW designed the experiments, conducted the experiments, and collected the data. GS, XX, ENO, EJW, DG, JDW, DWS, PAG, and RKA supervised experiments and analyzed the data. GS, DWS, DG, PAG, and RKA wrote the manuscript with assistance from all the other authors."} +{"text": "A large number of GEPP with varied 1O2 quantum yields have appeared recently; therefore, in the present work, the efficacy of different GEPP to photodynamically activate CCK1R was examined, as monitored by Fura-2 calcium imaging. KillerRed, miniSOG, miniSOG2, singlet oxygen protein photosensitizer (SOPP), flavin-binding fluorescent protein from Methylobacterium radiotolerans with point mutation C71G (Mr4511C71G), and flavin-binding fluorescent protein from Dinoroseobacter shibae (DsFbFP) were expressed at the plasma membrane (PM) in AR4-2J cells, which express endogenous CCK1R. Light irradiation of GEPPPM-expressing AR4-2J was found to all trigger persistent calcium oscillations, a hallmark of permanent photodynamic CCK1R activation; DsFbFP was the least effective, due to poor expression. miniSOG was targeted to PM, mitochondria (MT) or lysosomes (LS) in AR4-2J in parallel experiments; LED light irradiation was found to all induce persistent calcium oscillations. In miniSOGPM-AR4-2J cells, light emitting diode (LED) light irradiation-induced calcium oscillations were readily inhibited by CCK1R antagonist devazepide 2 nM; miniSOGMT-AR4-2J cells were less susceptible, but miniSOGLS-AR4-2J cells were not inhibited. In conclusion, different GEPPPM could all photodynamically activate CCK1R. Intracellular GEPP photodynamic action may prove particularly suited to study intracellular GPCR. Cholecystokinin 1 receptor (CCK1R) is activated by singlet oxygen ( Cholecystokinin 1 receptor (CCK1R) is expressed prominently in highly restricted brain regions, such as the basal ganglia, hippocampus, thalamus, hypothalamus, medulla oblongata ,6,7,8,9,1O2 or 1O2), usually generated in type II photodynamic action with chemical photosensitizer sulphonated aluminum phthalocyanine (SALPC) after a brief cellular incubation [CCK1R is unique among A class G protein-coupled receptors (GPCR) in that it is permanently activated ligand-independently by the lowest lying excited state molecular oxygen, the delta singlet oxygen . Energy transfer to ground state molecular oxygen results in the production of 1O2 (type II) [1O2 quantum yield (\u03d51O2) in type II photodynamic action. In contrast to chemical photosensitizers such as porphyrins and phthalocyanines, genetic manipulations will ensure that GEPP could be targeted to specified cell types or subcellular organelles with high precision. A typical photodynamic action involves three elements: light, a light-absorbing molecule (photosensitizer), and molecular oxygen. After absorption of a photon of a certain wavelength by a photosensitizer, the excited state photosensitizer molecule eventually undergoes either electron transfer or the energy transfer process. Electron transfer leads to the production of oxygen radicals (type I), such as superoxide anion (Otype II) ,30,31. T2\u2212.) [1O2 also [1O2 quantum yield (\u03d5\u03941O2 0.008) of more than eight-fold higher than O2\u2212. [KillerRed is the first major GEPP to emerge, initially thought to generate solely superoxide anion (O2\u2212.) ,34,35 bu1O2 also ,36, and han O2\u2212. . The minhan O2\u2212. ,39 but hhan O2\u2212. ,41, moduhan O2\u2212. , and forhan O2\u2212. . 1O2 quantum yields [Q103V) [1O2 quantum yield [1O2-generating flavin-binding fluorescent protein (FbFP) photosensitizers originally from other source organisms, such as Pp2FbFP (from Pseudomonas putida), DsFbFp (from Dinoroseobacter shibae), EcFbFP (from Bacillus subtilis), CreiLOV (from Chlamydomonas reinhardtii), Mr4511C71G (from Methylobacterium radiotolerans), and AsLOV2 (from Aveva sativa), have also appeared, all with desirably sufficient 1O2 quantum yields [Different variants of KillerRed and miniSOG have also appeared, either to monomerize the KillerRed dimer (SuperNova and GreenSuperNova) ,45, to br SOPP3) ,50,51,52 [Q103V) . A red fum yield . 1O2-genm yields ,57,58,59In view of the above developments, it has become pertinent to examine whether the newly emerged GEPP could also be used for photodynamic CCK1R activation. Further, numerous works have reported that G protein-coupled receptors (GPCR) function not only from PM but, also, from intracellular membranes ,61. The C71G, and DsFbFP in AR4-2J cells after light irradiation all photodynamically activated endogenous CCK1R in the rat pancreatic acinar tumor cell AR4-2J, triggering persistent calcium oscillations, with DsFbFP being the least effective. Interestingly, light irradiation of AR4-2J cells with miniSOG expressed at the plasma membrane (PM), mitochondria (MT), or lysosomes (LS) was found to trigger similarly persistent calcium oscillations, which were inhibited by CCK1R antagonist devazepide 2 nM with a graded sensitivity of PM > MT > LS. Therefore, GEPP expressed either at PM or intracellularly are both effective to photodynamically activate CCK1R, suggesting that photodynamic action may be particularly suited for the study of intracellular GPCR without the need for extracellularly added agonists to overcome multiple diffusion barriers. In the present work, it was found that plasma membrane (PM)-expressed KillerRed, miniSOG, miniSOG2, SOPP, Mr4511PM vector was bought from Evrogen . Ampicillin and kanamycin were from CWBio . Endotoxin-free plasmid extraction kit and DH5\u00e0 competent cells were from TianGen Biochemicals . MitoTracker\u2122 Red FM was from Invitrogen . LysoTracker Red was from Beyotime . Sulfated cholecystokinin octapeptide (CCK) and CCK1R antagonist devazepide were from Tocris Cookson . Dulbecco\u2019s modified Eagle\u2019s medium (DMEM)/F12 medium was bought from Invitrogen . Fura-2 AM was from AAT Bioquest . JetPRIME transfection reagent was from PolyPlus-transfection SA . Fetal bovine serum (FBS) was from Thermo Scientific . pKillerRed2 incubator under humidified atmosphere (5% CO2/95% air) at 37 \u00b0C, as reported previously [AR4-2J was bought from The American Type Culture Collection and cultured in DMEM/F12 supplemented with 20% fetal bovine serum in a COeviously ,62,63,64E. coli medium LB/kana and LB/amp were sterilized and culture plates made. Liquid E. coli medium LB/kana and LB/amp had the same composition but without agar. Solid PMpKillerRed was bought from Evrogen , proliferated in and harvested from competent E. coli. A mammalian codon-optimized miniSOG gene (GenBank accession number JX999997) was synthesized de novo from nucleotides at Genscript with the following full sequence: ATGGAAAAGAGCTTTGTGATTACCGATCCGCGCCTGCCAGACAACCCGATCATTTTCGCGAGCGATGGCTTTCTGGAGTTAACCGAATATTCTCGTGAGGAAATTCTGGGTCGCAATGGCCGTTTCTTGCAGGGTCCGGAAACGGATCAAGCCACCGTGCAGAAAATCCGCGATGCGATTCGTGACCAACGCGAAATCACCGTTCAGCTGATTAACTATACGAAAAGCGGCAAGAAATTTTGGAACTTACTGCATCTGCAACCGATGCGCGATCAGAAAGGCGAATTGCAATATTTCATTGGTGTGCAGCTGGATGGCTAG. This synthesized full miniSOG gene sequence was inserted into plasmid PMpKillerRed to replace the KillerRed sequence. Competent E. coli were infected with the recombinant plasmid, cultured on solid LB/kana. Bacteria colonies were picked and further cultured in liquid LB/kana with shaking overnight. Proliferated plasmid was extracted with sequence verification. The plasmid so obtained was named PMpminiSOG due to the presence of the PM-localization sequence in the original Evrogen plasmid. After transfection with plasmid PMpKillerRed or PMpminiSOG, positive expressing AR4-2J cells were named KillerRedPM- or miniSOGPM-AR4-2J cells, as reported before [Plasmid d before ,32. PMpminiSOG2, the miniSOG2 [miniSOG sequence in plasmid PMpminiSOG was replaced with the synthesized miniSOG2 sequence to obtain plasmid PMpminiSOG2 . The miniSOG2 sequence was: ATGGAGAAGAGCTTCGTGATCACCGACCCCCGCCTGCCTGACAACCCAATCATCTTCGCCAGCGACTCCTTCCTGGAGCTGACCGAGTACTCCAGGGAGGAGATCCTGGGAAGGAACCCACGGTTCCTGAGAGGACCTGAGACCGACCAGGCAACCGTGCAGAAGATCCACGACGCCATCCGCGACCAGAGGGAGATCACCGTGCAGCTGATCAACTACACCAAGAGCGGCAAGAAGTTCTGGAACCTGTTCCGGCTGCAGCCAATCAGAGACCAGAAGGGCGAGCTGCAGTACTTCATCGGCGTGCAGCTGGACGGCTAA. AR4-2J cells transfected with plasmid PMpminiSOG2 were named miniSOG2PM-AR4-2J cells. For the construction of plasmid miniSOG2 gene wasPMpSOPP, the SOPP amino acid sequence [SOPP gene was synthesized de novo after rat codon optimization. The miniSOG sequence in plasmid PMpminiSOG was replaced to obtain plasmid PMpSOPP , where the SOPP gene sequence was: ATGGAGAAGAGCTTCGTGATCACCGACCCCAGGCTGCCTGACAACCCAATCATCTTCGCCAGCGACGGCTTCCTGGAGCTGACCGAGTACTCCAGGGAGGAGATCCTGGGAAGGAACGGCCGGTTCCTGCAGGGACCCGAGACCGACCAGGCCACCGTGCAGAAGATCAGAGACGCCATCAGAGACCAGCGCGAGATCACCGTGCAGCTGATCAACTACACCAAGTCCGGCAAGAAGTTCTGGAACCTGCTGCACCTGCAGCCCATGCGGGACCAGAAGGGCGAGCTGCAGTACTTCATCGGCGTGCTGCTGGACGGCTAA. AR4-2J cells transfected with plasmid PMpSOPP as verified were named SOPPPM-AR4-2J cells.For the construction of plasmid sequence was usedC71GPMpMr5411, the Mr5411C71G protein sequence from [C71GMr5411 gene was synthesized de novo with rat codon optimization, which was used to replace the miniSOG sequence in PMpminiSOG . The C71GMr5411 sequence was ATGGAGACCGGAGGAACCGCCACCAGCCACGTGCCAGACGAGCTGAAGGCAGAGTCCCACAGAGGCGACCCTTTCGCCGCAGCCGTGAGGGCAACCAGGATGCCCATGATCATCACCGACCCTGCCCAGCACGACAACCCAATCGTGTTCGTGAACGACGCCTTCCTGAAGCTGACCGGCTACACCAGGATGGAGGTGGTGGGAAGAAACGGCCGCTTCCTGCAGGGACCAGACACCGAGGCAGCAGCAGTGGACAGACTGAGGGCAGCCATCAGGCGGGAGGAGGACATCAGAGTGGACCTGCTGAACTACCGCAAGGACGGCAGCACCTTCCAGAACGCCCTGTACGTGGGACCCGTGAGGGACGAGGCAGGACGGGTGGTGTACTTCTTCGCCAGCCAGCTGGACGTGTCCGAGCACTACGCCCTGACCGCAGAGATCGAGAGGCTGAAGGCCGCCCTGGCCGAGGCCGAGGCCAAGCTGGCCGCCCGGTAG. AR4-2J cells transfected with plasmid C71GPMpMr5411 were named C71GPMMr5411-AR4-2J cells.For the construction of plasmid nce from was usedDsFbFP gene [miniSOG from plasmid PMpminiSOG . The DsFbFP gene sequence was ATGAGGCGGCACTACCGCGACCTGATCAGGAACACCCCCATGCCTGACACCCCACAGGACATCGCAGACCTGCGCGCCCTGCTGGACGAGGACGAGGCCGAGATGAGCGTGGTGTTCAGCGACCCATCCCAGCCCGACAACCCTATGATCTACGTGTCCGACGCCTTCCTGGTGCAGACCGGATACACCCTGGAGGAGGTGCTGGGAAGGAACGCAAGATTCCTGCAGGGACCAGACACCAACCCACACGCAGTGGAGGCAATCAGGCAGGGCCTGAAGGCAGAGACCAGATTCACCATCGACATCCTGAACTACAGGAAGGACGGCAGCGCCTTCGTGAACAGACTGCGCATCAGGCCTATCTACGACCCAGAGGGCAACCTGATGTTCTTCGCCGGCGCCCAGAACCCCGTGCTGGAGTAG. Positive AR4-2J cells after transfection with plasmid PMpDsFbFP were named DsFbFPPM-AR4-2J cells.The bFP gene was syntMTpminiSOG was prepared by replacing the PM-localizing sequence ATGCTGTGCTGTATGAGAAGAACCAAACAGGTTGAAAAGAATGATGAGGACCAAAAGATC in PMpminiSOG with the mitochondrial (MT)-targeting sequence (MTS: ATGTCCGTCCTGACGCCGCTGCTGCTGCGGGGCTTGACAGGCTCGGCCCGGCGGCTCCCAGTGCCGCGCGCCAAGATCCATTCGTTGGGGGATCCACCGGTCGCCACC) . LSpminiSOG was prepared by replacing the PM-localizing ATGCTGTGCTGTATGAGAAGAACCAAACAGGTTGAAAAGAATGATGAGGACCAAAAGATC sequence in PMpminiSOG with the lysosomal (LS) sequence (LTS: ATGAAGGGACAGGGAAGCATGGACGAGGGAACCGCCGACGAGAGGGCCCCCCTGATCCGGACC). Competent E. coli were infected with the plasmid, further cultured on solid LB/kana. Bacteria colonies were picked and cultured in liquid LB/kana with shaking overnight. Propagated plasmids were extracted for sequence verification. The plasmid constructs were designated MTpminiSOG and LSpminiSOG; transformed AR4-2J cells were named miniSOGMT-AR4-2J and miniSOGLS-AR4-2J cells, accordingly.The plasmid ex 543 nm for KillerRedPM, and \u03bbex 488 nm for miniSOGPM, miniSOG2PM, Mr4511C71GPM, and DsFbFPPM. Mitochondrial or lysosomal expressions of miniSOG (\u03bbex 488 nm) were verified by colocalization with MitoTracker Red or LysoTracker Red (\u03bbex 543 nm) in confocal imaging (Zeiss LSM510 META), objective \u00d7 60 oil. AR4-2J cells were cultured in 6-well plates with one round glass cover-slip in each well, and to be transfected, cells were allowed to grow to 50\u201370% confluence. Plasmid (2 \u03bcg/well) and JetPRIME transfection reagent (4 \u03bcL/well) in JetPRIME buffer (200 \u03bcL) were added; then, AR4-2J cells were cultured for a further 24 h. Positive cellular GEPP expression was verified by confocal imaging of GEPP fluorescence: \u03bbPM-AR4-2J cells. AR4-2J cells cultured in a Petri dish (35 mm) were transfected; twenty-four hours later, cells were washed in PBS before the extraction of RNA. RNA concentration was determined with a Nanodrop2000 nanospectrometer . mRNA was reverse-transcribed with a GoScript Reverse Transcription Kit A5001 to obtain cDNA. To a PCR tube was added Oligo (dT) 1 \u03bcL, RNA 1 \u03bcg, 70 \u00b0C denaturation for 5 min, cooled on ice for 5 min, before the addition of the reaction buffer (\u00d75) 5 \u03bcL, RNAase inhibitor 1 \u03bcL, M-MLV reverse transcriptase 1 \u03bcL, dNTP (10 mM) 1.25 \u03bcL, topped up with diethyl pyrocarbonate (DEPC)-treated water to 25 \u03bcL. Reverse transcription conditions: 40 \u00b0C, 60 min and 70 \u00b0C, 15 min to obtain cDNA. PCR reaction: 2-\u03bcL cDNA template, 15-\u03bcL 2\u00d7Taq Master Mix , primers 1 \u03bcL each, topped up to 30 \u03bcL. Initial de-naturation 95 \u00b0C, 5 min; PCR cycles: 94 \u00b0C, 30 s, 60 \u00b0C, 30 s, 72 \u00b0C, 1.5 min, 30 cycles, and final prolongation 72 \u00b0C, 5 min. The RT-PCR product was run on 1% agarose gel with 0.01% GoodView added, 120 V, 40 min before imaging. PCR primers for DsFbFP were: forward 5\u2032-GGCACTACCGCGACCTGATC-3\u2032 and reverse 5\u2032-CTACTCCAGCACGGGGTTCT-3\u2032. Primers for internal reference GAPDH were: forward 5\u2032-GTGGAGTCTACTGGCGTCTT-3\u2032 and reverse 5\u2032-CCAGGATGCCCTTTAGTG-3\u2032.RT-PCR (reverse transcription-polymerase chain reaction): HiPure Total RNA Plus Mini Kit was used as instructed in the manufacturer\u2019s manual for RNA extraction from AR4-2J and DsFbFPPM-AR4-2J cells were irradiated with white light from a halogen cold light source equipped with a condenser (HLL201). AR4-2J cells expressing PM-localizing miniSOG, miniSOG2, SOPP/miniSOGQ103L, Mr4511C71G, DsFbFP, MT- or LS-localizing miniSOG were irradiated with blue LED . Power density was measured at the level of attached cells in the Sykes-Moore perfusion chamber with a power meter . Light-responding transfected AR4-2J cells were identified as GEPP-positive cells. KillerRed340/F380 and plotted against time with SigmaPlot , as reported before [N identical experiments.Parental control or transfected AR4-2J cells grown on glass cover-slips in 6-well plates were loaded with Fura-2 AM for 1 h after assembly in the Sykes-Moore perfusion chamber. Cytosolic calcium was measured in an inverted fluorescent microscope (Nikon TE-2000U) coupled to a Photon Technology International calcium measurement system with alternating excitations at 340 nm/380 nm (DeltaRam X); emitted Fura-2 fluorescence was detected with a charge-coupled device (CCD) camera . Calcium concentration was expressed as Fura-2 fluorescence ratios Fd before ,63,64,66N (as indicated) independent experiments were presented in bar graphs as mean \u00b1 SEM, unless specifically stated otherwise. Student\u2019s t-test was used for statistical analysis against controls, and p < 0.05 was taken as significant and indicated with an asterisk (*).All calcium tracings and other graphs were plotted with SigmaPlot. For calculation and comparison of the strength of induced calcium responses, the calcium peak area above the baseline was integrated . Statistical data from PMpKillerRed, PMpminiSOG, PMpminiSOG2, PMpSOPP, C71GPMpMr4511, and pDsFbFP , DsFbFP demonstrated markedly dimmer fluorescence, suggesting poorer protein expression, as shown in the confocal images in parental AR4-2J cells . In parallel experiments, white light irradiation from the halogen light source was found to induce sustained calcium oscillations in KillerRedPM-AR4-2J cells elicited persistent calcium oscillations in miniSOGPM- like previously reported ,32, we niniSOGLS a, with aoTracker b,c. \u22122, 1.5 min) was found to have no effect on the baseline calcium level in parental AR4-2J cells, but in these cells, CCK 20 pM induced marked calcium responses induced long-lasting calcium oscillations in miniSOGMT-AR4-2J cells similarly induced long-lasting calcium oscillations in miniSOGLS-AR4-2J cells identical experiments was calculated and plotted as bar graphs (i). Note the sharp difference in the calcium response after LED light irradiation in parental AR4-2J cells, and dark or light responses in MT or LS miniSOG-transfected AR4-2J cells (i). Quantitative analysis of calcium responses in original tracings, as represented in PM-AR4-2J cells were inhibited nearly completely by the CCK1R antagonist devazepide 2 nM but no effect on miniSOGLS photodynamic action . For the representative experiment shown in MT photodynamic action was inhibited without significance by devazepide 2 nM (from 100% to 65%) but significantly by devazepide 10 nM (from 100% to 29%) d. miniSO to 29%) d. C71G, and DsFbFP after light irradiation all photodynamically activated CCK1R to induce persistent cytosolic calcium oscillations in AR4-2J cells, but the photodynamic effect of DsFbFP was much reduced in comparison, likely due to poor protein expression. Permanent photodynamic CCK1R activation was achieved in AR4-2J cells by miniSOG expression not only at the plasma membrane (PM) but, also, in mitochondria (MT) and lysosomes (LS). Calcium oscillations induced by miniSOG photodynamic action at intracellular sites showed reduced sensitivity to inhibition by CCK1R antagonist devazepide 2 nM with the order of PM > MT > LS. In the present work, it was found that PM-expressed KillerRed, miniSOG, miniSOG2, SOPP, Mr45111O2 generated in type II photodynamic action with SALPC, KillerRed, or miniSOG as the photosensitizer [\u22122, 4 min and miniSOG with blue LED 450 nm, 85 mW\u2027cm\u22122, 1.5 min) triggered long-lasting calcium oscillations in both KillerRedPM-AR4-2J and miniSOGPM-AR4-2J cells (Q103L), Mr4511C71G, and DsFbFP expressed at the plasma membrane [bl50% miniSOG: 2.85 min and DsFbFP: 0.35 min) [PM-AR4-2J cells after blue LED irradiation (Dinoroseobacter shibae) has been known to be expressed in CHO-K1 cells as a dimer, which might also affect its fluorescence, unlike the monomeric miniSOG, miniSOG2, or SOPP, all sourced from Arabidopsis thaliana [Although the DsFbFP protein level was low, as shown by the dim DsFbFP fluorescence, DsFbFP mRNA were expressed at sufficient levels in AR4-2J cells, as verified by RT-PCR experiments g. Althou = 0.35) is the h.35 min) . This waadiation g. DsFbFPthaliana ,70. The thaliana might fa1O2 after light irradiation in a type II photodynamic action [1O2 of 0.03 (as reviewed in [2\u2212.) [1O2 [1O2 than O2\u2212., with an \u03d51O2 of 0.008, whilst monomeric KillerRed SuperNova has an \u03d51O2 of 0.02 [SOPP or miniSOGQ103L) has a \u03d51O2 of 0.25 [C71G sourced from Methylobacterium radiotolerans has a \u03d51O2 of 0.19 [1O2 among the lot at 0.33 [1O2 from 0.008 to 0.33 . Killerin [2\u2212.) , but mor\u2212.) [1O2 ,36,43. P of 0.02 . miniSOG of 0.02 . The sin of 0.25 . Mr4511C of 0.19 . DsFbFP at 0.33 . Althoug0.33 see , no signefficacy . This in\u22122, 1 min) induced phototoxicity in a small percentage of cells two hours after light irradiation of miniSOG-HEK293, miniSOG2-HEK293, and SOPP-HEK293 cells, but no marked difference was found in HEK293 cells expressing SOPP (9%) with a \u03d51O2 of 0.25 or miniSOG (11%) with a \u03d51O2 of 0.03 [1O2 of only 0.03 to permanently activated photodynamically CCK1R in miniSOGPM-AR4-2J cells in the present work might be related to the progressive photochemical transformation of the fluorophore flavin mononucleotide (FMN) to lumichrome and the photo-oxidization of internal residues in miniSOG to significantly increase its \u03d51O2 up to 10-fold [1O2 is not determined; see Avena sativa phototropin 1, light irradiation was found to induce progressive photochemical dissociation or the release of FMN from the AsLOV2 protein moiety, leading to significantly increased \u03d51O2 [It has been found by others that blue LED irradiation of the visible spectrum (full-spectrum white light was used in the present work), whereas all others by blue light (450 nm) (for the(450 nm) . Althoug(450 nm) might waC71G, and DsFbFP. It would be ideal if KillerRed could be subjected to further annotations, to shift its maximal excitation peak toward even longer wavelengths (a red shift), possibly by genetic code expansion [C71G, DsFbFP -induced calcium oscillations were inhibited by CCK1R antagonist devazepide 2 nM significantly in miniSOGPM-CHO-K1 cells, slightly in miniSOGMT-AR4-2J cells, but not at all in miniSOGLS-AR4-2J cells or lysosomal (LS) miniSOG were found to photodynamically trigger persistent calcium oscillations similarly . LED irr2J cells , althoug2J cells . The cal2J cells . We beli present ,76,77. S present and are present ; therefonometers could eaLS photodynamic CCK1R activation and the little inhibition afforded by CCK1R antagonist devazepide in miniSOGLS-AR4-2J cells. The insensitivity of lysosomal CCK1R to the antagonist devazepide 2 nM might also be due to limited accessibility of lysosomal CCK1R to extracellularly added devazepide 2 nM. Such a reduced sensitivity of intracellular GPCR to ligands was noted before for the nuclear membrane GPCR in cardiomyocytes, for example [The lysosomal accumulation of endocytosized CCK1R and partial CCK1R degradation ,79,80,81 example . 1O2 generation, due to its limited lifetime of 1 \u00b5s [1O2 in the cellular milieu has been suggested to be in the tens of nanometers or more (20\u2013150 nm) [1O2 could diffuse from the plasma membrane (PM), mitochondrial (MT), or lysosomal (LS) membranes as the origin to within a circle with a radius of 20\u2013150 nm. Although the emphasis in the present work was the subcellular localization of miniSOG expression and therefore of subcellular of 1 \u00b5s ,84,85 th\u2013150 nm) ,86,87,881O2 could well help to further investigate GPCR functions at these intracellular sites. Photodynamic GPCR activation/modulation might offer distinct advantages over conventional receptor pharmacology in that no ligand is needed for photodynamic activation after GEPP expression at defined intracellular sites. Only light irradiation is required to permanently activate the intracellular GPCR. The limited diffusion distance of photodynamically generated 1O2 (20\u2013150 nm) may ensure spatial precision and specificity. It may be noted that there is abundant evidence for GPCR expression, localization, and function at nuclear , mitocho1O2 activation of CCK1R. In the present work, all GEPP examined were found to elicit persistent calcium oscillations photodynamically, either from the PM, MT, or LS. We have previously found that the SALPC photodynamic activation of CCK1R in rat pancreatic acini involved the near quantitative transformation of the CCK1R protein dimer to the monomer . In addiC71G, and DsFbFP) reported in the literature were found to photodynamically activate the endogenous CCK1R in AR4-2J cells after plasma membrane expression. The miniSOG expression at intracellular sites was also found to induce persistent calcium oscillations or CCK1R activation (In conclusion, representative GEPP (KillerRed, miniSOG, miniSOG2, SOPP, Mr4511tivation . The pre"} +{"text": "Caenorhabditis elegans, the closest BUBR1 orthologue lacks the B56-interaction domain and Shugoshin is not required for meiotic segregation. Therefore, the role of PP2A in C. elegans female meiosis is unknown. We report that PP2A is essential for meiotic spindle assembly and chromosome dynamics during C. elegans female meiosis. BUB-1 is the main chromosome-targeting factor for B56 subunits during prometaphase I. BUB-1 recruits PP2A:B56 to the chromosomes via a newly identified LxxIxE motif in a phosphorylation-dependent manner, and this recruitment is important for proper chromosome congression. Our results highlight a novel mechanism for B56 recruitment, essential for recruiting a pool of PP2A involved in chromosome congression during meiosis I.Protein Phosphatase 2A (PP2A) is a heterotrimer composed of scaffolding (A), catalytic (C), and regulatory (B) subunits. PP2A complexes with B56 subunits are targeted by Shugoshin and BUBR1 to protect centromeric cohesion and stabilise kinetochore\u2013microtubule attachments in yeast and mouse meiosis. In Formation of a diploid embryo requires that sperm and egg contribute exactly one copy of each chromosome. The cell division in charge of reducing ploidy of the genome is meiosis, which involves two chromosome segregation steps after a single round of DNA replication . Female Protein Phosphatase 2A (PP2A) is a heterotrimeric serine/threonine phosphatase composed of a catalytic subunit C (PPP2C), a scaffolding subunit A (PPP2R1), and a regulatory subunit B (PPP2R2\u2013PPP2R5) . While tPP2A:B56 can be targeted to distinct sites by different proteins, and two mechanisms have been characterised at the structural level. While an N-terminal coiled coil domain in Shugoshin/MEI-S332 binds PP2A:B56 , other sCaenorhabditis elegans, the meiotic role of PP2A remains unexplored. The core components of the PP2A holoenzyme in C. elegans are LET-92 and PAA-1 (scaffolding A subunit), and the regulatory B subunits are B55SUR-6 and paa-1(RNAi),\u00a0oocytes microtubules do not organise into a bipolar structure (let-92(RNAi) oocytes, displaying a cluster of small foci activity (i.e. anaphase progression). Cytoplasmic securinIFY-1 degradation proceeded at similar rates in wild-type and LET-92-depleted oocytes in vivo, we attempted to tag endogenous LET-92 with GFP but were unsuccessful, presumably due to disruption of its native structure, in agreement with a recent report . We geneeiosis I . The fuseiosis I , in agreeiosis I . During eiosis I .C. elegans B56 orthologues, PPTR-1 and PPTR-2, and the single B55 orthologue, SUR-6 during metaphase of meiosis I with RNAi-mediated depletion of B56\u03b1PPTR-1 (\u2018pptr-1(RNAi)\u2019), which we will refer to hereafter as \u2018PPTR-1/2 depletion\u2019. While there was no significant change in GFP::PAA-1 localisation upon depletion of B56\u03b1PPTR-1 or deletion of B56\u03b3PPTR-2, no PAA-1 signal is detected associated with chromosomes upon double PPTR-1/2 depletion does not inhibit B56 localisation involved in targeting PP2A:B56 to meiotic chromosomes. Two of the most studied proteins involved in B56 targeting are Shugoshin and BubR1. The BubR1 orthologue, Mad3 spindle and Mad3lisation . We thery CRISPR and obse1 levels and a sl2 levels . ParticuC.\u00a0elegans B56 subunits display a dynamic localisation pattern similar to that of the kinase BUB-1 throughout meiosis I . Sequenc 282\u2013287 . When co 282\u2013287 . In addi elegans . The put elegans , with seL282A,V285A was indistinguishable from that of wild-type BUB-1 during metaphase I , but segregation was achieved in 100% of the cases with no PBE\u00a0defects were detected. Interestingly, the newly identified LxxIxE motif specifically recruits PP2A:B56 to metaphase chromosomes and anaphase central spindle, but not anaphase chromosomes.Using a fluorescence polarisation-based assay, we confirmed that the LxxIxE motif of BUB-1 binds to purified recombinant PPTR-2, and this binding is abolished by the L282A,V285A mutations . While laphase I , localisA mutant . B56\u03b3PPTxE motif . In cont spindle without spindle . Some GFteractor , is not teractor . Mutatioteractor , indicatPPTR-2 with higher affinity than non-phosphorylated LxxIxE peptide had a similar effect to that of the BUB-1L282A,V285A mutant: it significantly reduced B56\u03b1PPTR-1 localisation destabilises the kinase domain and prevents its interaction with Mad1MDF-1 the two pools of B56\u03b3PPTR-2.These results show that the BUB-1 kinase domain is important for recruitment of B56\u03b3C. elegans. PP2A is essential for meiosis I: depletion of the catalytic or scaffold subunits leads to severe spindle assembly defects, lack of chromosome segregation, and failure to achieve PBE. These effects are likely brought about by a combination of different PP2A subcomplexes with varying regulatory B subunits. PP2A:B56 regulates chromosome dynamics prior to segregation since depletion of the two B56 orthologues, PPTR-1 and PPTR-2, leads to alignment defects. We have uncovered a new phospho-regulated B56 LxxIxE motif in C. elegans BUB-1 that recruits PP2A:B56 and is important for chromosome alignment during meiosis I.In the present article, we have uncovered new roles for PP2A during oocyte meiosis in Depletion of the sole catalytic or scaffold PP2A subunits leads to massive meiotic failure and embryonic lethality. The earliest effect we could see in our experimental set\u00a0up is a failure to assemble a bipolar spindle. This will of course have a direct impact on any process that should occur after spindle assembly, including chromosome alignment and segregation, followed by PBE. However, BUB-1 depletion leads to severe spindle assembly and alignment defects, yet in the majority of cases, chromosomes segregate (with visible errors \u2013 lagging chromosomes) and polar bodies do extrude. Therefore, lack of a proper bipolar metaphase spindle is not sufficient to result in lack of segregation or PBE. PP2A complexes harbouring the B56 subunits PPTR-1 and PPTR-2 are required to target the phosphatase to chromosomes to regulate chromosome alignment in metaphase I. Furthermore, this chromosomal B56 pool is recruited by BUB-1 through its LxxIxE SLiM motif. Mutation of this motif that prevents binding to B56 subunits leads to alignment defects. B56 subunits however are not involved in spindle pole targeting of PP2A, suggesting that this spindle pole pool is the one relevant for spindle assembly. Our attempts to address a possible role for other B subunits were focused on RSA-1 and SUR-6 given their reported centrosomal roles during mitosis . HoweverC. elegans oocytes is highly dynamic, and differences have been observed between metaphase and anaphase. For example, while kinetochore localisation of the CLASP orthologue CLS-2 during metaphase depends on BUB-1, a pool of CLS-2 is able to\u00a0localise in the anaphase central spindle in a BUB-1-independent manner resemble those reported in the absence of kinetochore proteins . InteresC. elegans, the Aurora B orthologue, AIR-2, concentrates in the interface between homologous chromosomes closely resembling the consensus for PP2A:B56 reported in During meiosis in bivalent . Consistbivalent . During tochores and it iC. elegans BUB-1 .Strains used in this study were maintained at 20 degrees unless indicated otherwise. For a complete list of strains, please refer to For RNAi experiments, we cloned the different sequences in the L4440 RNAi feeding vector .600 of 0.6\u20130.8. Isopropyl-\u03b2-d-thiogalactopyranoside (IPTG)\u00a0was added to a final concentration of 1 mM, and cultures were incubated overnight at 20\u00b0C. Bacteria were then seeded onto NGM plates made with agarose and allowed to dry. L4 worms were then plated on RNAi plates and grown to adulthood at 20\u00b0C for 24 hr in the case of let-92(RNAi), bub-1(RNAi), and paa-1(RNAi) and 48 hr in all other cases.All sequences were inserted into L4440 using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs) and transformed into DH5a bacteria. The purified plasmids were then transformed into HT115(DE3) bacteria . RNAi clFor the generation of in situ-tagged GFP::SUR-6, we used the self-excising cassette method . In brieK718R,D847N, BUB-1L282A,V285A, BUB-1S283A,\u00a0and BUB-1L282A,V285A, K718R,D847N were generated by Sunybiotech.The strains AID::GFP::GSP-2, GFP::PAA-1, BUB-1.CTCGACAATATCATCTCCAGATTATTGGAAGATGcctaaagatccagccaaacctccggccaaggcacaagttgtgggatggccaccggtgagatcataccggaagaacgtgatggtttcctgccaaaaatcaagcggtggcccggaggcggcggcgttcgtgaagAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGgtaagtttaaacatatatatactaactaaccctgattatttaaattttcagCCAACACTTGTCACTACTTTCTgTTATGGTGTTCAATGCTTcTCgAGATACCCAGATCATATGAAACgGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACgtaagtttaaacagttcggtactaactaaccatacatatttaaattttcagGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTgtaagtttaaacatgattttactaactaactaatctgatttaaattttcagAACTTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAAGACGTAGAAAAGCTTAAT..GCTACTGACGACGCGATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGgtaagtttaaacatatatatactaactaaccctgattatttaaattttcagCCAACACTTGTCACTACTTTCTgTTATGGTGTTCAATGCTTcTCgAGATACCCAGATCATATGAAACgGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACgtaagtttaaacagttcggtactaactaaccatacatatttaaattttcagGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTgtaagtttaaacatgattttactaactaactaatctgatttaaattttcagAACTTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAAGGAGGTGGATCCGGTGGTGGATCCTCGGTTGTCGAAGAA.The wild-type sequence:AAGTACGAGGTGCCATCATGTTCGTGGGAAGTGTACATTTGCGACCAAATGCGGAATCGCCTGAAAGATCGAGGTTTGGAGCTGATGGCCAAATGTTGCATTATGGAAGTGATGGATGCTTATGTTTATTCAACTGCTTCGCTTCTTGTTAATCAGTACCACGAATATGGAACGCTGCTTGAATATGCGAATAACATGAAGGATCCGAATTGGCACATAACCTGCTTCTTGATTACCCAAATGGCCCGAGTTGTGAAGGAAGTCCATGCCTCTAAAATTATTCATGGAGATATCAAACCGGATAATTTTATGATCACCAGAAAgtatgggaaaacatttgttaattttagacgttatcttttttcagGATCGATGATAAATGGGGCAAAGATGCTCTGATGAGTAACGACAGCTTTGTCATCAAGATTATCGACTGGGGACGTGCCATTGACATGATGCCACTGAAGAACCAGCGTGTAACCGATGATCAAAGGACAGTAGCTGTGwas mutated to:CGCTACGAGGTGCCATCATGTTCGTGGGAAGTGTACATTTGCGACCAAATGCGGAATCGCCTGAAAGATCGAGGTTTGGAGCTGATGGCCAAATGTTGCATTATGGAAGTGATGGATGCTTATGTTTATTCAACTGCTTCGCTTCTTGTTAATCAGTACCACGAATATGGAACGCTGCTTGAATATGCGAATAACATGAAGGATCCGAATTGGCACATAACCTGCTTCTTGATTACCCAAATGGCCCGAGTTGTGAAGGAAGTCCATGCCTCTAAAATTATTCATGGAGATATCAAACCGGATAATTTTATGATCACCAGAAAgtatgggaaaacatttgttaattttagacgttatcttttttcagGATCGATGATAAATGGGGCAAAGATGCTCTGATGAGTAACGACAGCTTTGTCATCAAGATTATCAATTGGGGACGTGCGATTGACATGATGCCACTGAAGAACCAGCGTGTAACCGATGATCAAAGGACAGTAGCTGTGThe introduced changes are shown in red, and synonymous mutations are shown in cyan.The wild-type sequence:CTTTCACCAGTCAGTGAGAAAACGGTTGATGATGAGGAGGAAAAGAACGCCAATCTAAATCCTAGAAGACGTCATwas mutated to:GCATCACCAGCTAGCGAGAAAACGGTTGATGATGAGGAGGAAAAGAACGCCAATCTAAATCCTAGAAGACGTCATThe wild-type sequence:CATCTTTCACCAGTCAGTGAGAAAACGGTT GATGATGAGTTCAACGCCAATCTAAATCCTAGAAGACGTwas mutated to:CACCTTGCGCCAGTCAGTGAGAAAACGGTTGATGATGAGTTCAACGCCAATCTAAATCCTAGAAGACGTThe introduced changes are shown in red, and synonymous mutations are shown in cyan.See The wild-type sequence:CTTTCACCAGTCAGTGAGAAAACGGTTGATGATGAGGAGGAAAAGCGAAGCCGGATTTATTCGCCGCTGGTTGCAACGAAGGATGCTCACAGACCTGCACTTCGGAGCAAAATTGAGAATCCTCCAGCGACAGTGACACTTTCGTCGGATACAAAGTCTGCTTCGGAGAAAGATGTTAGTGATTCCGATGATGCAGATGATGATGAAAGACTCAAGATTATGACTGCCGGCAGAAAAGATGGTAACCCTCCAGACCGTTCCACAAGCATATCTTCCAACTATTCAACTGCTTCTGCAAGAACATCAAAGAGTGGAGCTGGATTGGATTTGATGGCGGAAAATAAGTGTTTGGAGGCACATGCTATGTTTTCCGACACTGTACATCTTGCTAGCGAAAAGACAATGGTCCTTGGCGATGATTCTGTCTTCGTTCCAGAAAGATCTTTAGCTACTACGCAGATAGTTACTGACTTTTCCGTGCTCTGTGATCCTGATCCGACAATGACCATTACACAGGAGCGTCCGAAAAAAGTGTCGAATGGGTTGAATGTTGTTTACGATGAGGCAGCCGAACCGGAAGAATCTCAGAAAGTTGAGGAATCTGAAGTACAACCCGAAATTGTCCTAGTTTCTCCAGTGACGCAAACCTCACCAGCTACAATGTTTAATGATAGTGGGTTTATCGAAAAATATAACAACTTATGTTTTAATTTTTAGTTTATGACGATGAAATCGAGTTTGGCTTTTTCAAACCGTCTCGTGGTAATTTCGTCACATCGACCCCCGCACAAGGAGTTCATTTGGTCAACATTGATGAATATTTCGGAAATAAAGAGGAGGAAAGCACTCACGAACAGGAAGCTCCAGTATTTGTTGCTCCAACCAGCAGTACTTTCAGTAAATTAGTAAGTGCCAGACAAATTTTCGACATACTATTCAAACTTTTTCAGACACGTCGAAAGTCACTAGCAGCAAATCAAGCCGTTCAGCCCTCAGTCACAGAGTCATCAAAGCCTGAACGATCAGATCCTAAAGATTCATCTATCGATTGTTTGACAGCTAATCTAGGAAGACGTCTTTCAATTGGTGCTGATGAAATTCCAAATCTCACTGAAAACAACGAATCTGAAATCACTGGTTGCAAGATTCGTCGGCGCAGTGAAATTATCAAGCAAGGAGACATCAATCCATGGGACGAAACTCTTCGAAAAAAATTGATGTGTCTTGTGCGTCCTCCCCAGAATATGCACGAGTTCCAAGAACGAGCACCGAAGATTCAAGCTCTGAGAGACTGCGAGGTTAGCGGAGAAAAGCTCCACATTCAAACTCTTATTGGTCAAGGTGGATACGCTAAAGTATACCGGGCTGTAACCGATGATCAAAGGACAGTAGCTGTGAAGTACGAGGTGCCATCATGTTCGTGGGAAGTGTACATTTGCGACCAAATGCGGAATCGCCTGAAAGATCGAGGTTTGGAGCTGATGGCCAAATGTTGCATTATGGAAGTGATGGATGCTTATGTTTATTCAACTGCTTCGCTTCTTGTTAATCAGTACCACGAATATGGAACGCTGCTTGAATATGCGAATAACATGAAGGATCCGAATTGGCACATAACCTGCTTCTTGATTACCCAAATGGCCCGAGTTGTGAAGGAAGTCCATGCCTCTAAAATTATTCATGGAGATATCAAACCGGATAATTTTATGATCACCAGAAAGTATGGGAAAACATTTGTTAATTTTAGACGTTATCTTTTTTCAGGATCGATGATAAATGGGGCAAAGATGCTCTGATGAGTAACGACAGCTTTGTCATCAAGATTATCGACTGGGGACGTGCCATTGACATGATGCCACTGAAGAACCAGCGTAACGCCAATCTAAATCCTAGAAGACGTCATwas mutated to:GCATCACCAGCTAGCGAGAAAACGGTTGATGATGAGGAGGAAAAGCGAAGCCGGATTTATTCGCCGCTGGTTGCAACGAAGGATGCTCACAGACCTGCACTTCGGAGCAAAATTGAGAATCCTCCAGCGACAGTGACACTTTCGTCGGATACAAAGTCTGCTTCGGAGAAAGATGTTAGTGATTCCGATGATGCAGATGATGATGAAAGACTCAAGATTATGACTGCCGGCAGAAAAGATGGTAACCCTCCAGACCGTTCCACAAGCATATCTTCCAACTATTCAACTGCTTCTGCAAGAACATCAAAGAGTGGAGCTGGATTGGATTTGATGGCGGAAAATAAGTGTTTGGAGGCACATGCTATGTTTTCCGACACTGTACATCTTGCTAGCGAAAAGACAATGGTCCTTGGCGATGATTCTGTCTTCGTTCCAGAAAGATCTTTAGCTACTACGCAGATAGTTACTGACTTTTCCGTGCTCTGTGATCCTGATCCGACAATGACCATTACACAGGAGCGTCCGAAAAAAGTGTCGAATGGGTTGAATGTTGTTTACGATGAGGCAGCCGAACCGGAAGAATCTCAGAAAGTTGAGGAATCTGAAGTACAACCCGAAATTGTCCTAGTTTCTCCAGTGACGCAAACCTCACCAGCTACAATGTTTAATGATAGTGGGTTTATCGAAAAATATAACAACTTATGTTTTAATTTTTAGTTTATGACGATGAAATCGAGTTTGGCTTTTTCAAACCGTCTCGTGGTAATTTCGTCACATCGACCCCCGCACAAGGAGTTCATTTGGTCAACATTGATGAATATTTCGGAAATAAAGAGGAGGAAAGCACTCACGAACAGGAAGCTCCAGTATTTGTTGCTCCAACCAGCAGTACTTTCAGTAAATTAGTAAGTGCCAGACAAATTTTCGACATACTATTCAAACTTTTTCAGACACGTCGAAAGTCACTAGCAGCAAATCAAGCCGTTCAGCCCTCAGTCACAGAGTCATCAAAGCCTGAACGATCAGATCCTAAAGATTCATCTATCGATTGTTTGACAGCTAATCTAGGAAGACGTCTTTCAATTGGTGCTGATGAAATTCCAAATCTCACTGAAAACAACGAATCTGAAATCACTGGTTGCAAGATTCGTCGGCGCAGTGAAATTATCAAGCAAGGAGACATCAATCCATGGGACGAAACTCTTCGAAAAAAATTGATGTGTCTTGTGCGTCCTCCCCAGAATATGCACGAGTTCCAAGAACGAGCACCGAAGATTCAAGCTCTGAGAGACTGCGAGGTTAGCGGAGAAAAGCTCCACATTCAAACTCTTATTGGTCAAGGTGGATACGCTAAAGTATACCGGGCTGTAACCGATGATCAAAGAACAGTAGCTGTGCGCTACGAGGTGCCATCATGTTCGTGGGAAGTGTACATTTGCGACCAAATGCGGAATCGCCTGAAAGATCGAGGTTTGGAGCTGATGGCCAAATGTTGCATTATGGAAGTGATGGATGCTTATGTTTATTCAACTGCTTCGCTTCTTGTTAATCAGTACCACGAATATGGAACGCTGCTTGAATATGCGAATAACATGAAGGATCCGAATTGGCACATAACCTGCTTCTTGATTACCCAAATGGCCCGAGTTGTGAAGGAAGTCCATGCCTCTAAAATTATTCATGGAGATATCAAACCGGATAATTTTATGATCACCAGAAAGTATGGGAAAACATTTGTTAATTTTAGACGTTATCTTTTTTCAGGATCGATGATAAATGGGGCAAAGATGCTCTGATGAGTAACGACAGCTTTGTCATCAAGATTATCAATTGGGGACGTGCGATTGACATGATGCCACTGAAGAACCAGCGTAACGCCAATCTAAATCCTAGAAGACGTCATThe introduced changes are shown in red, and synonymous mutations are shown in cyan.See C. elegans oocytes was used with minor modifications PVSEKTC. Serum was adsorbed with a non-phosphorylated peptide (RRRHLSPVSEKTC) followed by affinity purification with the antigenic, phosphorylated peptide. Different fractions were tested in immunofluorescence by incubating different dilutions with 1 \u00b5M and 10 \u00b5M of either non-phosphorylated or phosphorylated peptide. Only the phosphorylated peptide compited out the signal. Additionally, we used the BUB-1d-lysine -coated slide, and a 24\u00a0\u00d7\u00a024 cm coverslip was gently laid on top. Once the worms extruded the embryos, slides were placed on a metal block on dry ice for\u00a0>10 min. The coverslip was then flicked off with a scalpel blade, and the samples were fixed in methanol at 20\u00b0C for 30 min. After blocking in phosphate-buffered saline\u00a0(PBS) buffer plus 3% bovine serum albumin\u00a0and 0.1% Triton X-100 (AbDil), samples were incubated overnight at 4\u00b0C with anti-BUB-1 . We used the strain HY604, which is a temperature-sensitive allele of the the APC component MAT-1, that arrests in meiosis I prior to spindle rotation when moved to the restrictive temperature.For GFP immunoprecipitations, we followed a published protocol with minIP samples were run on 4\u201312% Bis\u2013Tris sodium dodecyl sulfate gels with MOPS running buffer, and the gel was stained using Quick Coomassie Stain (Generon). Bands of interest were cut and washed with water:acetonitrile (50:50), followed by a wash with 100 mM ammonium bicarbonate. The gel pieces were then washed with 100 mM ammonium bicarbonate:acetonitrile (50:50), followed by a final wash with acetonitrile. Gel pieces were dried using a SpeedVac.Samples were reduced with 10 mM DTT in 20 mM ammonium bicarbonate and alkilated\u00a0with 50 mM IAA (iodoacetamide)\u00a0in 20 mM ammonium bicarbonate. Samples were then washed sequentially with 100 mM ammonium bicarbonate, 100 mM ammonium bicarbonate:acetonitrile (50:50), and acetonitrile. Gel pieces were dried using a SpeedVac.Trypsin solution (12.5 \u00b5g/ml stock in 20 mM ammonium bicarbonate) was added to cover the gel pieces and incubated for 30 min on a shaking platform and incubated sample overnight at 30\u00b0C on a shaker. Peptides were extracted by standard procedures and reconstituted in 10 \u00b5l of 5% formic acid/10% acetonitrile. After vortexing for 1 min, water was added to 50 \u00b5l.Samples were run on an Ultimate 3000 RSLCnano system (ThermoFisher Scientific) coupled with\u00a0a Q-Exactive Plus Mass Spectrometer (Thermo Fisher Scientific). Peptides initially trapped on an Acclaim PepMap 100 (Thermo Fisher Scientific) and then separated on an Easy-Spray PepMap RSLC C18 column (Thermo Fisher Scientific). Sample was transferred to mass spectrometer via an Easy-Spray source with temperature set at 50\u00b0C and a source voltage of 2.1 kV. The mass spectrometer was operated on in data-dependent acquisition mode (top 15 method). MS resolution was 70,000 with a mass range of 350\u20131600. MS/MS resolution was 17,500.C. elegans proteome and BUB-1 specifically, using the Mascot Search Engine (Mascot Daemon Version 2.3.2). Type of search used was MS/MS Ion Search using Trypsin/P. Carbamidomethyl (C) was set as a fixed modification, and variable modifications were as follows: acetyl (N-term), dioxidation (M), Gln to pyro-Glu (N-term Q), oxidation (M), deamidation (NQ), and phosphorylation (STY).RAW data files were extracted and converted to mascot generic files (.mgf) using MSC Convert. Extracted data then searched against Peptide mass tolerance was\u00a0\u00b110 ppm (# 13C\u00a0=\u00a02) with a fragment mass tolerance of\u00a0\u00b10.6 Da. Maximum number of missed cleavages was 2.The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner C. elegans PPTR-1 (UniProtKB O18178) and PPTR-2 (UniProtKB A9UJN4-1). A guide tree was calculated from the distance matrix generated from sequence pairwise scores.All sequence alignments were performed using Clustal Omega , versionC. elegans PPTR-1 (S420-V435) and PPTR-2 (S449-A464) were used for the alignment. The canonical sequences of each B56 isoform were retrieved from UniProt , \u03b4 (S454-A469), \u03b2 (S409-V424), \u03b1 (S403-V418), \u03b5 (S395-V410) and UniProt .C. elegans BUB-1 , human BubR1 , and RepoMan were aligned with Clustal Omega or H1. At t\u00a0=\u00a00, 15 ng/\u00b5l Cdk1/Cyclin B was added, before incubation at 30\u00b0C for 30 min. Aliquots were taken immediately after Cdk1/CyclinB addition at t\u00a0=\u00a00 and then again after the 30 min incubation. All samples were then incubated at 70\u00b0C for 15 min in a final concentration of 1\u00d7 LDS buffer. Sodium dodecyl sulfate\u2013polyacrylamide gel electrophoresis\u00a0(SDS-PAGE) was then conducted on a NuPage 4\u201312% Bis\u2013Tris gel (Thermo) with MES buffer before being stained with ProQ Diamond (Thermo) and imaged using Bio-Rad ChemiDoc. Once fluorescence was recorded, Coomassie staining was performed. For the western blot, SDS\u2013PAGE was conducted as above with 67 ng of substrate protein per well before the western was conducted using a nitrocellulose membrane and 1\u00d7 NuPage transfer buffer (Thermo). The membrane was blocked using Intercept PBS blocking buffer (LI-COR), the primary antibodies used were anti-GST at 1:1000 (made in sheep), and anti-phospho\u00a0Ser 283 1:20,000 (made in rabbit). Secondary antibodies were anti-sheep IRDye 680RD anti-rabbit 800CW (LI-COR),\u00a0both at 1:50,000. The membrane was then imaged using LI-COR Odyssey CLx.Forty microlitre\u00a0reactions were set up containing 40 mM Tris\u2013HCl pH 7.5, 100 \u00b5M ATP\u00a0(Adenosine triphosphate), 10 mM MgClEscherichia coli strain BL21 (DE3) and the bacterial culture was incubated overnight at 37\u00b0C with shaking at 220 rpm. Bacteria were grown in TB medium at 37\u00b0C with shaking at 220 rpm until OD600 reached\u00a0~0.6\u20130.8 and induced with 150 \u03bcM IPTG. Induction was performed at 18\u00b0C with shaking at 220 rpm for\u00a0~16 hr. Cells were pelleted and lysed by sonication in 50 mM NaP, 300 mM NaCl, 10 mM imidazole, 10%\u00a0glycerol, 0.5 mM TCEP,\u00a0(tris(2-carboxyethyl)phosphine) and protease inhibitors. After binding to a Ni-NTA column, protein was washed with 20 mM imidazole and then eluted with 350 mM imidazole. The tag was cleaved with 6xHis-tagged TEV (Tobacco Etch Virus Protease)\u00a0protease overnight at 4\u00b0C, and the tag and protease were removed from the sample by binding to Ni-NTA. PPTR-2 was concentrated and further purified using a HiLoad 16/600 Superdex 200 pg size exclusion column.PPTR-2 was expressed from pHISTEV30a vector as a 6xHis- and Strep-tagged protein (plasmid fgp_445) in The following peptides were synthesised by peptides and elephants GmbH: BUB-1 FITC-Ahx-NPRRRHLSPVSEKTVDDEEE, pBUB-1 FITC-Ahx-NPRRRHLphSPVSEKTVDDEEE, and LAVA FITC-Ahx-NPRRRHASPASEKTVDDEEE. Reactions (35 \u00b5l) were set up in FP buffer containing peptide concentrations 0.5\u20131 \u00b5M. These were then used to create a 1:2 serial dilution series using FP buffer containing the same peptide concentrations as above as well as the indicated concentration of PPTR-2. Reactions were left for 30 min before triplicates of each dilution were aliquoted into a black 384-well plate, centrifuged , and analysed using the PheraStar FS.For time-dependent analysis, metaphase I was taken as time\u00a0=\u00a00 s. This frame was chosen as the previous frame where the first indication of chromosome separation was visible. All image analysis was done in Fiji . For totCentral spindle to chromosome ratio for PPTR-2::GFP was obtaFor the alignment/congression analysis, we selected videos in which the spindles were contained in a single Z-plane at \u221280 s. There, we established the spindle axes with a line extending from pole to pole and the \u2018metaphase plate\u2019 with a perpendicular line in the middle of the spindle see . A line Contingency tables were analysed using the Fisher\u2019s exact test (two\u00a0tailed), and the p\u00a0values are presented in the figures and/or in the text.The 4D TIFF files were converted to video (.avi) files using a custom-made macro that uses the StackReg Fiji plugin for image registration. Videos were assembled using maximum-intensity projections; hence, the videos might not match a specific panel within the main figures, which are single slices or partial projections in order to highlight specific characteristics. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:Using a combination of biochemistry, genetics and live imaging, Borja et al. show that the kinase BUB-1 recruits, through two regulatory subunits, the phosphatase PP2A to meiotic spindle chromosomes during oocyte meiosis I to promote proper congression of the chromosomes prior to anaphase. This recruitment occurs independently of the conserved protein Shugoshin, which has been shown by others to promote PP2A recruitment to chromosomes during mouse oocyte meiosis I. Moreover, Borja et al. show that phosphorylation of a peptide motif in BUB-1 promotes this recruitment, and that this BUB-1motif is likely targeted by CDK-1 for phosphorylation to provide proper temporal regulation of these events.Decision letter after peer review:[Editors\u2019 note: the authors submitted for reconsideration following the decision after peer review. What follows is the decision letter after the first round of review.]C. elegans Oocytes\" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and a Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Jakob Nilsson (Reviewer #2); Patrick Meraldi (Reviewer #3).Thank you for submitting your work entitled \"PP2A:B56 Regulates Meiotic Chromosome Segregation in eLife.Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in eLife; in particular whether BUB1 directly binds to PP2A, through the LxxIxE motif, and whether Cdk phosphorylation of this is important. After discussion, they concluded that the amount of time and effort that will be required for these experiments is such that the fairest thing to do is to return the manuscript to you.You will see from the reviews that all the reviewers agree that your study will be of interest to the meiosis community in identifying that BUB1 rather than Sgo recruits PP2A, but that also all agree that more experiments are required to provide firm evidence for a number of your conclusions that would be necessary for publication in Reviewer #1:C. elegans oocyte meiotic cell division. They show that PP2A and two conserved B56-type regulatory subunits are localized to spindle poles, chromosomes and the central spindle (with some differences for the B56 subunits), and that all required for spindle assembly, chromosome congression to the metaphase plate, and for chromosome segregation (with some redundancy for the two B56 subunits). The further show that a conserved LxxIxE motif in BUB-1 recruits most of one and some of the other B56 subunit, and that the kinase domain of BUB-1 is required to recruit the other (presumably through an intermediary). Thus in C. elegans, a novel form of PP2A recruitment functions, with the other known recruiters, Shugoshin and Mad3 , appearing not be required. The authors use mutational analysis to nicely document the requirements for both parts of BUB-1 in PP2A recruitment, and also identify a phosphorylated residue in BUB-1 that may be involved in CDK-1 regulation of the recruitment. The authors propose that BUB-1 recruitment of PP2A to the central spindle is important for chromosome segregation during anaphase in C. elegans oocyte meiosis I.Borja et al. describe their analysis of the requirements for the phosphatase PP2A during C. elegans oocyte meiosis, with results that will be of substantial interest to investigators studyng oocyte meiotic cell division, the advances are in my opinion to incremental, and also lacking in conclusiveness as to the actual mechanism involved. Therefore as written the manuscript is not suitable for publication in eLife, as summarized in the major comments below.While the authors provide an extensive analysis of the requirements for PP2A during 1) The authors provide clear evidence that PP2A is required for spindle assembly, congression of chromosomes to the metaphase plate, and chromosome segregation (with some caveats as to chromosome segregation noted below). While the authors favor and propose that the chromosome segregation defects are due to central spindle defects , they in fact provide no evidence to support this conclusion. It is entirely possible that earlier spindle assembly defects are responsible for the subsequent defects in both congression and segregation. The authors fail to address this possibility and provide no evidence to rule it out. Without some idea as to which targets PP2A acts through, the advance is incremental relative to what is known in other systems, only showing a variation on how LxxIxE motifs can recruit PP2A to spindle structures through BUB-1 instead of other factors. The authors discuss the AuroraB kinase AIR-2 as a possible target but provide no analysis of such a role. Without more mechanistic insight, the manuscript is of substantial interest but seems more appropriate for a more specialized journal such as Molecular Biology of the Cell.C. elegans mutants that chromosomes segregation and polar extrusion are not tightly correlated. The authors should establish a more direct approach to quantifying the defects in chromosome segregation; as written only representative examples are shown.2) In figures throughout the manuscript, the authors use polar body extrusion as a proxy for chromosome segregation defects. However, it is known from work on other 3) The authors show that there are severe spindle assembly defects after knockdown of PP2A but only show microtubules and chromosomes to document the defects. Given that the spindle assembly defects might be primarily responsible for subsequent defects in chromosome alignment and segregation, the authors should better characterize the spindle assembly defects using pole marker(s). Do mutant oocytes even establish a bipolar spindle? From the images, it seems likely they do not.4) The authors generate point mutations in both the LxxIxE motif and the kinase domain of BUB-1 to nicely document requirements for recruiting PP2A. However, the authors do not include any mention of whether these mutations are essential for oocyte meiotic cell division, or if the mutations are associated with any embryonic lethality . In fact, from the supplemental tables it appears that both mutant strains are homozygous viable, although this is never mentioned in the text. If these motifs are indeed responsible for recruiting PP2A and its essential functions to the spindle, then the mutants should result in defects identical to the PP2A knockdowns. Thus to verify the importance of these mutations, the authors would need to construct a balanced strain in which both motifs are mutated and document defects much like those observed in the PP2A knockdowns. More generally, the authors should provide a genetic analysis of the viability of the single mutants .Reviewer #2:C. elegans. Although the shugoshin proteins have been shown to be important regulators of cohesin during meiosis by recruiting PP2A-B56 in other systems the authors show this is not the case in C. elegans. Instead they show that the Bub1 protein recruits PP2A-B56 through a LxxIxE motif that resembles the one found in human BubR1. Mutation of this motif prevents the recruitment of the PPTR1 (one of the B56 isoforms) to the midbivalent and the central spindle and reduces the recruitment of the PPTR2 to the central spindle. The Bub1 variant with a mutated LxxIxE motif displays misalignments suggesting an imbalance in phosphorylations. The phenotype of the Bub1 mutant is not as severe as Bub1 RNAi but this could be because the kinase domain of Bub1 helps in recruiting PPTR2.This manuscript explores female meiosis and the role of Bub1 in targeting PP2A-B56 to chromosomes and central spindle for proper meiosis in eLife.Overall the paper is easy to read and the data are consistent. However, the paper is fairly descriptive and lacks to some degree mechanistic insight. I think that some additional experiments would make the work more interesting for the readers of 1) Characterization of the double Bub1 mutant with a mutated LxxIxE motif and mutations in the kinase domain. This would clarify if there is some redundancy in recruitment mechanisms2) Direct evidence that the LxxIxE motif of Bub1 facilitates binding to PPTR1/2 \u2013 IP or purified components3) Characterization of the LAPI mutation of the LxxIxE motif to determine if Cdk1 phosphorylation of this site is important. Reading the manuscript as it is now this is purely speculative despite they go through the effort of showing phosphorylation.Reviewer #3:C. elegans anaphase. It finds that PP2A plays a crucial role in chromosome alignment and segregation. This role depends on the B56 regulatory subunits. Moreover, the author show that the recruitment of the B56 subunits to the spindle apparatus and kinetochores in anaphase depends on the Bub-1 kinase.The study by Bel Borja and colleagues studies to which extent the PP2A phosphatase contributes to chromosome segregation in C. elegans anaphase, and is generally of good technical quality (see below), yet at the same time feels a bit thin in terms of results. In particular several major claims of the discussion are not supported by the data. Moreover, it is not clear to which extent the reported experiments are reproducible.The study explores a novel role of PP2A in 1) The authors do not indicate the number of independent experiments or the number of embryos on which the conclusions are based on. This information is essential to evaluate the reproducibility of the results. The authors use good statistical tests, but the reader must know whether the experiments are based on 5, 10 or 20 embryos.C. elegans anaphase by RECRUITING PP2A:B56 to the spindle apparatus. The presented experiments only show that the localization of the B56 depends on Bub-1, which is not the same thing. The authors could test by IP and in vitro pull-down that the claimed interaction is direct (in vitro), or that the proteins are part of the same complex (in vivo). This should be feasible since the authors already have performed IP experiments.2) The authors claim the Bub-1 acts on 3) The authors claim that PP2A:B56 counteracts Aurora-B during anaphase, but have no evidence for this claim. Does a Aurora-B hypomorph mutant attenuate the B56 loss phenotype? While a negative result would not mean that the model is wrong, a positive result would certainly help to bolster that claim. [Editors\u2019 note: the authors resubmitted a revised version of the paper for consideration. What follows is the authors\u2019 response to the first round of review.]You will see from the reviews that all the reviewers agree that your study will be of interest to the meiosis community in identifying that BUB1 rather than Sgo recruits PP2A, but that also all agree that more experiments are required to provide firm evidence for a number of your conclusions that would be necessary for publication in eLife; in particular whether BUB1 directly binds to PP2A, through the LxxIxE motif, and whether Cdk phosphorylation of this is important. After discussion, they concluded that the amount of time and effort that will be required for these experiments is such that the fairest thing to do is to return the manuscript to you.The new version of the manuscript has numerous new experiments to address reviewers\u2019 comments/concerns. Relating to the particular two points highlighted by the reviewing editor, we provide data showing that the BUB-1 LxxIxE motif binds the B56 orthologue PPTR-2 in vitro and Ser 283 phosphorylation increases affinity. Furthermore, mutating Ser 283 to Ala drastically inhibits B56 recruitment in vivo resulting in a chromosome alignment defect similar to the L282A,V285A mutant.We also performed in vitro kinase assays and showed that Cdk1 can phosphorylate Ser 283 in BUB-1 and developed a phospho-specific antibody recognising phosphor-Ser 283 and show that phosphorylated BUB-1 is detected \u2026 in vivo.Thanks to the new quantitative phenotypic analysis we performed, it became clear that B56 subunits play a role during chromosome alignment/congression prior to anaphase. Furthermore, this relies on B56 recruitment by the newly identified BUB-1 LxxIxE motif.Reviewer #1:[\u2026]1) The authors provide clear evidence that PP2A is required for spindle assembly, congression of chromosomes to the metaphase plate, and chromosome segregation (with some caveats as to chromosome segregation noted below). While the authors favor and propose that the chromosome segregation defects are due to central spindle defects , they in fact provide no evidence to support this conclusion. It is entirely possible that earlier spindle assembly defects are responsible for the subsequent defects in both congression and segregation. The authors fail to address this possibility and provide no evidence to rule it out.We agree we might have oversimplified the phenotypes and the connection between them. Our biggest mistake was to base the alignment analysis on subjective observations its classification as either \u201caligned\u201d or \u201cmisaligned\u201d.Firstly, we performed a new analysis related to the chromosome alignment phenotype. This is now detailed in the Materials and methods section and it provides more rigorous and less subjective quantifications which lead us to a better characterisation of this phenotype. This had a tremendous impact on the manuscript, because it is precisely this process of chromosome alignment where BUB-1 targeted PP2A:B56 plays a role. To avoid any potential amplification of the phenotypes due to partial loss of function in PP2A subunits, we decided to measure the phenotypes on the GFP::tubulin expressing strain and not on the strains with tagged PP2A subunits.PP2A is essential for the assembly of a bipolar spindle and chromosome alignment and segregation. While we agree with the reviewer that in this case it is difficult to tease out whether the alignment and segregation are only a direct consequence of this, depletion of the B56 subunits PPTR-1 and PPTR-2 does not affect spindle assembly but perturbs chromosome alignment/congression . Since this phenotype is measurable and independent from any noticeable spindle defect, we focused on this \u201cclean\u201d phenotype and sought to understand the molecular mechanisms driving it.Without some idea as to which targets PP2A acts through, the advance is incremental relative to what is known in other systems, only showing a variation on how LxxIxE motifs can recruit PP2A to spindle structures through BUB-1 instead of other factors. The authors discuss the AuroraB kinase AIR-2 as a possible target but provide no analysis of such a role. Without more mechanistic insight, the manuscript is of substantial interest but seems more appropriate for a more specialized journal such as Molecular Biology of the Cell.C. elegans, it was not clear what role(s) BUB-1 play. Therefore, we think we are providing a significant advance on the mechanisms in place to regulate meiosis I. While we are actively working on identifying PP2A substrates and its potential role in antagonising Aurora B, we think this will require an in depth analysis that will be the focus of another paper.While we respect the reviewer\u2019s view on the advance provided by our manuscript, we do believe we provide mechanistic insight into the targeting of PP2A:B56 in a context in which the previously known/characterised regulators (BubR1 and Shugoshin) do not play a role. On the other hand, while BUB-1 is known to be important during female meiosis in C. elegans mutants that chromosomes segregation and polar extrusion are not tightly correlated. The authors should establish a more direct approach to quantifying the defects in chromosome segregation; as written only representative examples are shown.2) In figures throughout the manuscript, the authors use polar body extrusion as a proxy for chromosome segregation defects. However, it is known from work on other We apologise for the misunderstanding. We did not intend to use PB extrusion as a proxy for chromosome segregation defects, but rather analysed it as an independent process. We are now making this clear in the manuscript. We analyse chromosome alignment defects (see below), lagging chromosomes, and polar body extrusion. For these three phenomena, we provide the quantitative assessment for each condition and show a representative image (or sets of images).3) The authors show that there are severe spindle assembly defects after knockdown of PP2A but only show microtubules and chromosomes to document the defects. Given that the spindle assembly defects might be primarily responsible for subsequent defects in chromosome alignment and segregation, the authors should better characterize the spindle assembly defects using pole marker(s). Do mutant oocytes even establish a bipolar spindle? From the images, it seems likely they do not.As requested by the reviewer, we have performed new experiments using GFP-ASPM-1 as a pole marker. As expected, LET-92-depleted oocytes fail to assemble a bipolar spindle . As mentioned above, in order to differentiate subsequent phenotypes from the spindle defect, we focused on the alignment/congression defects which are observed even in the presence of a seemingly normal bipolar spindle.4) The authors generate point mutations in both the LxxIxE motif and the kinase domain of BUB-1 to nicely document requirements for recruiting PP2A. However, the authors do not include any mention of whether these mutations are essential for oocyte meiotic cell division, or if the mutations are associated with any embryonic lethality . In fact, from the supplemental tables it appears that both mutant strains are homozygous viable, although this is never mentioned in the text. If these motifs are indeed responsible for recruiting PP2A and its essential functions to the spindle, then the mutants should result in defects identical to the PP2A knockdowns. Thus to verify the importance of these mutations, the authors would need to construct a balanced strain in which both motifs are mutated and document defects much like those observed in the PP2A knockdowns. More generally, the authors should provide a genetic analysis of the viability of the single mutants .Full analysis of embryo viability and brood size analysis is now presented for all the mutants used in the study .Reviewer #2:C. elegans. Although the shugoshin proteins have been shown to be important regulators of cohesin during meiosis by recruiting PP2A-B56 in other systems the authors show this is not the case in C. elegans. Instead they show that the Bub1 protein recruits PP2A-B56 through a LxxIxE motif that resembles the one found in human BubR1. Mutation of this motif prevents the recruitment of the PPTR1 (one of the B56 isoforms) to the midbivalent and the central spindle and reduces the recruitment of the PPTR2 to the central spindle. The Bub1 variant with a mutated LxxIxE motif displays misalignments suggesting an imbalance in phosphorylations. The phenotype of the Bub1 mutant is not as severe as Bub1 RNAi but this could be because the kinase domain of Bub1 helps in recruiting PPTR2.This manuscript explores female meiosis and the role of Bub1 in targeting PP2A-B56 to chromosomes and central spindle for proper meiosis in Overall the paper is easy to read and the data are consistent. However, the paper is fairly descriptive and lacks to some degree mechanistic insight. I think that some additional experiments would make the work more interesting for the readers of eLife.1) Characterization of the double Bub1 mutant with a mutated LxxIxE motif and mutations in the kinase domain. This would clarify if there is some redundancy in recruitment mechanisms.We have performed the suggested experiment. The results indicate that LxxIxE motif-mediated recruitment of B56 subunits operates mostly in the midbivalent and central spindle, whereas the kinase domain mediates mostly chromosome chromosomal recruitment of the B56 subunits .2) Direct evidence that the LxxIxE motif of Bub1 facilitates binding to PPTR1/2 \u2013 IP or purified components.We have performed in vitro binding experiments and present evidence for the direct interaction between the LxxIxE motif of BUB-1 and B56. We expressed recombinant, full-length PPTR-2 in bacteria and performed fluorescence polarisation experiments using fluorescently labelled LxxIxE motif peptides. We could observe that PPTR-2 bound the wild - type sequence and that L282A,V285A mutations abolished binding . On the contrary, binding was enhanced when the peptide was phosphorylated at Serine 283 .3) Characterization of the LAPI mutation of the LxxIxE motif to determine if Cdk1 phosphorylation of this site is important. Reading the manuscript as it is now this is purely speculative despite they go through the effort of showing phosphorylation.We have now expanded on the LxxIxE motif phosphorylation and its role in vivo.1) in vitro assays demonstrate that Cdk1 phosphorylates Serine 283 in vitro. This is now part of the New Figure 5C-E.S283A, achieving a similar effect to that of BUB-1L282A,V285A. Previous data showed that substituting I for V in the LxxIxE motif decreases affinity and we believe in this case this renders the motif more dependent on phosphorylation. These results are included in the New Figure 6.2) To analyse the role of Serine 283 phosphorylation in vivo, we mutated it to alanine in endogenous BUB-1. PPTR-1 and PPTR-2 localisation to the midbivalent and central spindle was significantly affected in BUB-13) In addition to the previously reported mass spec data, we now show the localisation of the L(pS)PVSE motif by immunofluorescence with a newly generated phospho-specific antibody .Reviewer #3:C. elegans anaphase. It finds that PP2A plays a crucial role in chromosome alignment and segregation. This role depends on the B56 regulatory subunits. Moreover, the author show that the recruitment of the B56 subunits to the spindle apparatus and kinetochores in anaphase depends on the Bub-1 kinase.The study by Bel Borja and colleagues studies to which extent the PP2A phosphatase contributes to chromosome segregation in C. elegans anaphase, and is generally of good technical quality (see below), yet at the same time feels a bit thin in terms of results. In particular several major claims of the discussion are not supported by the data. Moreover, it is not clear to which extent the reported experiments are reproducible.The study explores a novel role of PP2A in 1) The authors do not indicate the number of independent experiments or the number of embryos on which the conclusions are based on. This information is essential to evaluate the reproducibility of the results. The authors use good statistical tests, but the reader must know whether the experiments are based on 5, 10 or 20 embryos.We have now included all the information in the graphs. For intensity measurements, \u201cN\u201d is the number of experiments, and \u201cn\u201d is the number of oocytes. For the congression and alignment analysis, \u201cN\u201d is the number of spindles (=number of oocytes) and \u201cn\u201d is the number of bivalents analysed. In the latter analysis, information on the number of experiments used to obtain the data is stated in the figure legend.C. elegans anaphase by RECRUITING PP2A:B56 to the spindle apparatus. The presented experiments only show that the localization of the B56 depends on Bub-1, which is not the same thing. The authors could test by IP and in vitro pull-down that the claimed interaction is direct (in vitro), or that the proteins are part of the same complex (in vivo). This should be feasible since the authors already have performed IP experiments.2) The authors claim the Bub-1 acts on We thank the reviewer for raising this concern and we have now changed the wording when referring to this. We have performed in vitro binding experiments and present evidence for the direct interaction between the LxxIxE motif of BUB-1 and PPTR-2 . We expressed recombinant, full-length PPTR-2 in bacteria and performed fluorescence polarisation experiments using fluorescently labelled LxxIxE motif peptides. PPTR-2 bound the wild type sequence and that binding was abolished by the L282A,V285A mutations and enhanced when the peptide was phosphorylated at Serine 283.3) The authors claim that PP2A:B56 counteracts Aurora-B during anaphase, but have no evidence for this claim. Does a Aurora-B hypomorph mutant attenuate the B56 loss phenotype? While a negative result would not mean that the model is wrong, a positive result would certainly help to bolster that claim.As stated above, we think we are providing a significant advance on the mechanisms in place to regulate meiosis I by specific recruitment of PP2A/B56. While we are actively working on identifying PP2A substrates and its potential role in antagonising Aurora B, we believe this will require an in-depth analysis that will be the focus of another paper. We have now focused our discussion on our actual data and left this as an interesting, yet unsupported, hypothesis."} +{"text": "To assess the role of a protein, protein loss phenotypic studies can be used, most commonly through mutagenesis RNAi or CRISPR knockout. Such studies have been critical for the understanding of protein function and the identification of putative therapeutic targets for numerous human disease states. However, these methodological approaches present challenges because they are not easily reversible, and if an essential gene is targeted, an associated loss of cell viability can potentially hinder further studies. Here we present a reversible and conditional live\u2010cell knockout strategy that is applicable to numerous proteins. This modular protein\u2010tagging approach regulates target loss at the protein, rather than the genomic, level through the use of HaloPROTAC3, which specifically degrades HaloTag fusion proteins via recruitment of the VHL E3 ligase component. To enable HaloTag\u2010mediated degradation of endogenous proteins, we provide protocols for HaloTag genomic insertion at the protein N or C terminus via CRISPR/Cas9 and use of HaloTag fluorescent ligands to enrich edited cells via Fluorescence\u2010Activated Cell Sorting (FACS). Using these approaches, endogenous HaloTag fusion proteins present in various subcellular locations can be degraded by HaloPROTAC3. As detecting the degradation of endogenous targets is challenging, the 11\u2010amino\u2010acid peptide tag HiBiT is added to the HaloTag fusion to allows the sensitive luminescence detection of HaloTag fusion levels without the use of antibodies. Lastly, we demonstrate, through comparison of HaloPROTAC3 degradation with that of another fusion tag PROTAC, dTAG\u201013, that HaloPROTAC3 has a faster degradation rate and similar extent of degradation. \u00a9 2020 The Authors.Basic Protocol 1: CRISPR/Cas9 insertion of HaloTag or HiBiT\u2010HaloTagBasic Protocol 2: HaloPROTAC3 degradation of endogenous HaloTag fusions Targeted protein degradation using proteolysis targeting chimeras (PROTACs) is a rapidly growing research area and an exciting new modality of therapeutic treatment donor vector. After CRISPR pools are generated, further details are provided for enrichment of HaloTag positive cells using the HaloTag fluorophore, Janelia Fluor 646 (JF646) HaloTag ligand be placed immediately following the endogenous target start codons or any type of signal sequence that could be cleaved. If introducing HaloTag or HaloTag\u2010HiBiT at the C terminus, place the tag(s) immediately upstream of the native stop codon. Generation of genomic maps that include the CRISPR insertions will aid in ensuring the tags are placed in the proper reading frame. As CRISPR insertion success can be highly dependent upon the choice of guide RNA, it is advisable to test a minimum of two different guide RNA sequences. Initial studies appending the tag(s) on the N\u2010 or C\u2010terminal end of the protein using expression vectors can be used to determine the tag(s) effects on the protein's expression, binding interactions, and folding abilities. However, overexpression is not recommended for degradation studies with HaloPROTAC3, as little degradation will be detected due to the high expression levels of the fusion protein.Alt\u2010R CRISPR\u2010Cas9 tracrRNA Alt\u2010R CRISPR\u2010Cas9 CRISPR RNA Nuclease Free Duplex Buffer Alt\u2010R S.p. Cas9 Nuclease Desired cell line for CRISPR modification that is amenable to nucleofectionDPBS 0.05% trypsin/EDTA or 0.25% trypsin/EDTA , depending on cell lineComplete growth medium for cell type of choiceMirus Ingenio Solution HaloTag or HiBiT\u2010HaloTag double\u2010stranded (ds) DNA donor plasmid Janelia Fluor 646 (JF646) HaloTag Ligand 100\u00d7 Antibiotic/Antimycotic solution FACS buffer see Optional, for use of HiBiT\u2010HaloTag or HaloTag\u2010HiBiT CRISPR insertions: Nano\u2010GloHiBiT Lytic Detection System Heat block 50\u2010ml conical tubes Mirus Ingenio Kit Bio\u2010Rad Gene Pulser Xcell Electroporation SystemAppropriate incubator for cell lineClear, tissue\u2010culture\u2010grade 96\u2010well plates 5\u2010ml round\u2010bottom tube with cell strainer cap BD FACS Melody or similarWhite 96\u2010well plates Optional, for use of HiBiT\u2010HaloTag or HaloTag\u2010HiBiT CRISPR insertions: Luminometer such as the GloMax Discover Microplate Reader or CLARIOstar Plus (BMG Labtech)Additional reagents and equipment for basic cell culture techniques, including cell counting complex by combining 75 pmol Cas9 and 120 pmol tracrRNA:crRNA duplex:Add the Cas9 very slowly to avoid precipitation, and swirl with pipet tip.5Incubate RNP complex 10\u201020 min at room temperature.66 cells/reaction), remove cells from plasticware using appropriate splitting techniques, centrifuge cells at 200 \u00d7 g for 5 min, rinse cells with DPBS, and centrifuge again. For example, if using HEK293 cells, wash with DPBS, detach cells with trypsin, inactivate trypsin with DMEM + 10% FBS, and transfer cells to a conical tube for centrifugation.Determine total number of cells needed based on number of CRISPR reactions for the cell type, and split cells if they become confluent. If performing HiBiT\u2010HaloTag CRISPR insertions, HiBiT lytic luminescence assays can be performed to determine insertion efficiency in triplicate to a white 96\u2010well assay plate.29Dilute the LgBiT Protein 1:100 and the Nano\u2010Glo HiBiT Lytic Substrate 1:50 into an appropriate volume of room\u2010temperature Nano\u2010Glo HiBiT Lytic Buffer in a new tube. Mix by inversion.30Add 100 \u00b5l Nano\u2010Glo HiBiT Lytic Reagent to each well.31Mix the samples by placing the plate on an orbital shaker (300\u2010600 rpm) for at least 10 min.32Read luminescence with GloMax Discover Microplate Reader using a 0.5\u2010s integration time Fig. .Basic Protocol 2ent\u2010HaloPROTAC3 is an alternate procedure for the live\u2010cell kinetic degradation analysis of HiBiT\u2010HaloTag insertions to better understand degradation rate, optimal time of treatments, and kinetic dose\u2010response curves. The use of HiBiT for protein level detection is highly quantitative, directly correlative to the endogenous target protein level, and does not require the use of antibodies 0.05% trypsin/EDTA or 0.25% trypsin/EDTA , depending on cell lineComplete growth medium for cell type of choiceHaloPROTAC3 ligand ent\u2010HaloPROTAC3 ligand Dimethyl sulfoxide Optional materials for live\u2010cell kinetic degradation of HiBiT\u2010HaloTag fusions:LgBiT Expression Vector Nano\u2010Glo Endurazine Live Cell Substrate 2\u2010independent medium COFBS Six\u2010well platesAppropriate incubator for cell lineDilution reservoirs White 96\u2010well plates Optional material for live\u2010cell kinetic degradation of HiBiT\u2010HaloTag fusions: Luminometer such as the GloMax Discover Microplate Reader or CLARIOstar Plus (BMG Labtech)1Culture CRISPR/Cas9\u2010edited cells carrying HaloTag, HiBiT\u2010HaloTag , or HaloTag\u2010HiBiT insertions appropriately in preparation for the assay.If performing live\u2010cell degradation assays with HiBiT\u2010HaloTag CRISPR insertions proceed to optional live\u2010cell HiBiT\u2010HaloTag degradation assay at the end of Basic Protocol 2For adherent cells, wash cells with DPBS and trypsinize.3ent\u2010HaloPRTOAC3, and one for DMSO control.Count cells and plate 800,000 cells per well in a six\u2010well plate. Plate one well for HaloPROTAC3, one for 4For adherent cells, incubate cells overnight (18\u201024 hr) in an appropriate incubator. For suspension cells, proceed to step 5.5ent\u2010HaloPROTAC3, and (c) a volume of DMSO equivalent to that added in the PROTAC treatments.Add ligands to growth medium in well to obtain final concentrations of (a) 300 nM HaloPROTAC3, (b) 300 nM For example, if there is 2 ml of growth medium in well, add 500 \u00b5l 1.5 \u00b5M HaloPROTAC3 stock to make a solution of 300 nM final concentration.6Incubate cells in an appropriate incubator overnight.These recommended HaloPROTAC3 concentrations and treatment times can be increased or decreased depending upon target and desired level of degradation.7Detect degradation of endogenous HaloTag target fusion. If using HiBiT\u2010HaloTag CRISPR insertions, HiBiT lytic luminescence assays can be carried out to quantitate degradation following Basic Protocol 8Transfect the LgBiT vector into HaloTag\u2010 or HiBiT\u2010HaloTag\u2010edited cells using standard transient transfection and following manufacturer's recommendations.9Incubate plates in an appropriate incubator overnight (18\u201024 hr).102\u2010independent medium with the appropriate percentage of FBS for the cell type, or proper assay medium for cells, by diluting the stock reagent of the substrate 1:100. If the luminometer provides CO2 injection or cells do not require CO2, regular assay medium can be used.Prepare a 1\u00d7 solution of Nano\u2010Glo Endurazine Live Cell Substrate in CO11Aspirate cell culture medium from plate, and add 90 \u00b5l of the Endurazine solution to each well.122 level (if possible or needed) of cell line in use.Incubate plate for at least 2.5 hr at in an incubator set to the appropriate growing conditions for the cells to equilibrate the luminescence. During this incubation period, it is advisable to pre\u2010equilibrate the luminometer to the growth temperature and CO13ent\u2010HaloPROTAC3 either in CO2\u2010independent medium containing the appropriate percentage of FBS for the cell type or in proper assay medium for the cells.Prepare 10 \u00b5M solutions of HaloPROTAC3 and 14ent\u2010HaloPROTAC3 in dilution reservoirs using assay medium that contains DMSO at the same concentration as the 10 \u00b5M stock. Reserve the last well of the dilution series to contain DMSO alone, as a negative control. This results in a 10\u00d7 dilution series, which will be further diluted upon addition to cells for a final treatment concentration of 1 \u00b5M at the highest point.Perform threefold dilutions of 10 \u00b5M HaloPROTAC3 or 15ent\u2010HaloPROTAC3 ligand to each well of the 96\u2010well plate.Add 10 \u00b5l of the diluted HaloPROTAC3 or 16Immediately place the plate in a luminometer plate reader pre\u2010equilibrated to the growth temperature of the cell line in use.17Read luminescence at desired intervals. A recommended starting point is every 15 min over a 24\u2010hr total time frame.18ent\u2010HaloPROTAC3\u2010treated wells by those from the DMSO control . Examples of kinetic degradation profiles of HiBiT\u2010HaloTag CRISPR insertions are shown in Figures Calculate the fractional RLUs at each time point by dividing the RLU value from HaloPROTAC3\u2010treated or 1\u00d7 Hank's Balanced Salt Solution 10 mM HEPES 0.2% BSA 1\u00d7 Antibiotic/Antimycotic Solution N\u2010terminal HaloTag sequence:500\u2010bp upstream homology arm\u2010GCAGAAATCGGTACTGGCTTTCCATTCGACCCCCATTATGTGGAAGTCCTGGGCGAGCGCATGCACTACGTCGATGTTGGTCCGCGCGATGGCACCCCTGTGCTGTTCCTGCACGGTAACCCGACCTCCTCCTACGTGTGGCGCAACATCATCCCGCATGTTGCACCGACCCATCGCTGCATTGCTCCAGACCTGATCGGTATGGGCAAATCCGACAAACCAGACCTGGGTTATTTCTTCGACGACCACGTCCGCTTCATGGATGCCTTCATCGAAGCCCTGGGTCTGGAAGAGGTCGTCCTGGTCATTCACGACTGGGGCTCCGCTCTGGGTTTCCACTGGGCCAAGCGCAATCCAGAGCGCGTCAAAGGTATTGCATTTATGGAGTTCATCCGCCCTATCCCGACCTGGGACGAATGGCCAGAATTTGCCCGCGAGACCTTCCAGGCCTTCCGCACCACCGACGTCGGCCGCAAGCTGATCATCGATCAGAACGTTTTTATCGAGGGTACGCTGCCGATGGGTGTCGTCCGCCCGCTGACTGAAGTCGAGATGGACCATTACCGCGAGCCGTTCCTGAATCCTGTTGACCGCGAGCCACTGTGGCGCTTCCCAAACGAGCTGCCAATCGCCGGTGAGCCAGCGAACATCGTCGCGCTGGTCGAAGAATACATGGACTGGCTGCACCAGTCCCCTGTCCCGAAGCTGCTGTTCTGGGGCACCCCAGGCGTTCTGATCCCACCGGCCGAAGCCGCTCGCCTGGCCAAAAGCCTGCCTAACTGCAAGGCTGTGGACATCGGCCCGGGTCTGAATCTGCTGCAAGAAGACAACCCGGACCTGATCGGCAGCGAGATCGCGCGCTGGCTGTCGACGCTCGAGATTTCCGGCGAGCCAACCACTGAGGATCTGTACTTTCAGAGCGATAAC\u2010500\u2010bp downstream homology armC\u2010terminal HaloTag sequence:500\u2010bp upstream homology arm\u2010GAGCCAACCACTGAGGATCTGTACTTTCAGAGCGATAACGATGGATCCGAAATCGGTACTGGCTTTCCATTCGACCCCCATTATGTGGAAGTCCTGGGCGAGCGCATGCACTACGTCGATGTTGGTCCGCGCGATGGCACCCCTGTGCTGTTCCTGCACGGTAACCCGACCTCCTCCTACGTGTGGCGCAACATCATCCCGCATGTTGCACCGACCCATCGCTGCATTGCTCCAGACCTGATCGGTATGGGCAAATCCGACAAACCAGACCTGGGTTATTTCTTCGACGACCACGTCCGCTTCATGGATGCCTTCATCGAAGCCCTGGGTCTGGAAGAGGTCGTCCTGGTCATTCACGACTGGGGCTCCGCTCTGGGTTTCCACTGGGCCAAGCGCAATCCAGAGCGCGTCAAAGGTATTGCATTTATGGAGTTCATCCGCCCTATCCCGACCTGGGACGAATGGCCAGAATTTGCCCGCGAGACCTTCCAGGCCTTCCGCACCACCGACGTCGGCCGCAAGCTGATCATCGATCAGAACGTTTTTATCGAGGGTACGCTGCCGATGGGTGTCGTCCGCCCGCTGACTGAAGTCGAGATGGACCATTACCGCGAGCCGTTCCTGAATCCTGTTGACCGCGAGCCACTGTGGCGCTTCCCAAACGAGCTGCCAATCGCCGGTGAGCCAGCGAACATCGTCGCGCTGGTCGAAGAATACATGGACTGGCTGCACCAGTCCCCTGTCCCGAAGCTGCTGTTCTGGGGCACCCCAGGCGTTCTGATCCCACCGGCCGAAGCCGCTCGCCTGGCCAAAAGCCTGCCTAACTGCAAGGCTGTGGACATCGGCCCGGGTCTGAATCTGCTGCAAGAAGACAACCCGGACCTGATCGGCAGCGAGATCGCGCGCTGGCTGTCTACTCTGGAGATTTCCGGT\u2010500\u2010bp downstream homology armN\u2010terminal HiBiT\u2010HaloTag sequence:500\u2010bp upstream homology arm\u2010GTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGCGCAGAAATCGGTACTGGCTTTCCATTCGACCCCCATTATGTGGAAGTCCTGGGCGAGCGCATGCACTACGTCGATGTTGGTCCGCGCGATGGCACCCCTGTGCTGTTCCTGCACGGTAACCCGACCTCCTCCTACGTGTGGCGCAACATCATCCCGCATGTTGCACCGACCCATCGCTGCATTGCTCCAGACCTGATCGGTATGGGCAAATCCGACAAACCAGACCTGGGTTATTTCTTCGACGACCACGTCCGCTTCATGGATGCCTTCATCGAAGCCCTGGGTCTGGAAGAGGTCGTCCTGGTCATTCACGACTGGGGCTCCGCTCTGGGTTTCCACTGGGCCAAGCGCAATCCAGAGCGCGTCAAAGGTATTGCATTTATGGAGTTCATCCGCCCTATCCCGACCTGGGACGAATGGCCAGAATTTGCCCGCGAGACCTTCCAGGCCTTCCGCACCACCGACGTCGGCCGCAAGCTGATCATCGATCAGAACGTTTTTATCGAGGGTACGCTGCCGATGGGTGTCGTCCGCCCGCTGACTGAAGTCGAGATGGACCATTACCGCGAGCCGTTCCTGAATCCTGTTGACCGCGAGCCACTGTGGCGCTTCCCAAACGAGCTGCCAATCGCCGGTGAGCCAGCGAACATCGTCGCGCTGGTCGAAGAATACATGGACTGGCTGCACCAGTCCCCTGTCCCGAAGCTGCTGTTCTGGGGCACCCCAGGCGTTCTGATCCCACCGGCCGAAGCCGCTCGCCTGGCCAAAAGCCTGCCTAACTGCAAGGCTGTGGACATCGGCCCGGGTCTGAATCTGCTGCAAGAAGACAACCCGGACCTGATCGGCAGCGAGATCGCGCGCTGGCTGTCGACGCTCGAGATTTCCGGCGAGCCAACCACTGAGGATCTGTACTTTCAGAGCGATAAC\u2010500\u2010bp downstream homology armC\u2010terminal HaloTag\u2010VS\u2010HiBiT sequence:500\u2010bp upstream homology arm\u2010 GAGCCAACCACTGAGGATCTGTACTTTCAGAGCGATAACGATGGATCCGAAATCGGTACTGGCTTTCCATTCGACCCCCATTATGTGGAAGTCCTGGGCGAGCGCATGCACTACGTCGATGTTGGTCCGCGCGATGGCACCCCTGTGCTGTTCCTGCACGGTAACCCGACCTCCTCCTACGTGTGGCGCAACATCATCCCGCATGTTGCACCGACCCATCGCTGCATTGCTCCAGACCTGATCGGTATGGGCAAATCCGACAAACCAGACCTGGGTTATTTCTTCGACGACCACGTCCGCTTCATGGATGCCTTCATCGAAGCCCTGGGTCTGGAAGAGGTCGTCCTGGTCATTCACGACTGGGGCTCCGCTCTGGGTTTCCACTGGGCCAAGCGCAATCCAGAGCGCGTCAAAGGTATTGCATTTATGGAGTTCATCCGCCCTATCCCGACCTGGGACGAATGGCCAGAATTTGCCCGCGAGACCTTCCAGGCCTTCCGCACCACCGACGTCGGCCGCAAGCTGATCATCGATCAGAACGTTTTTATCGAGGGTACGCTGCCGATGGGTGTCGTCCGCCCGCTGACTGAAGTCGAGATGGACCATTACCGCGAGCCGTTCCTGAATCCTGTTGACCGCGAGCCACTGTGGCGCTTCCCAAACGAGCTGCCAATCGCCGGTGAGCCAGCGAACATCGTCGCGCTGGTCGAAGAATACATGGACTGGCTGCACCAGTCCCCTGTCCCGAAGCTGCTGTTCTGGGGCACCCCAGGCGTTCTGATCCCACCGGCCGAAGCCGCTCGCCTGGCCAAAAGCCTGCCTAACTGCAAGGCTGTGGACATCGGCCCGGGTCTGAATCTGCTGCAAGAAGACAACCCGGACCTGATCGGCAGCGAGATCGCGCGCTGGCTGTCTACTCTGGAGATTTCCGGTGTCTCCGTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGC\u2010500\u2010bp downstream homology armKey: blue, HiBiT; red, HaloTag; green, linker.Targeted protein degradation has resulted in an explosion of new avenues of research, from therapeutic drug discovery and clinical trials to the expansion of E3 ligase studies and phenotypic studies as compared to smaller insertions, for which single\u2010stranded oligodeoxynucleotide (ssODN) synthesis is possible and homology arms (30\u201050bp each) are shorter. The use of dsDNA donor vectors in general results in low insertion efficiency, and expected pool percentage of edited cells with this approach could range between 0.1% and 15%. For insertion of HaloTag or HiBiT\u2010HaloTag, the use of donor vector without a promoter is important to promote specific, on\u2010target insertion and minimize random integration of vector, which could then result in expression of the tag alone. To identify CRISPR HaloTag target\u2010edited cells from random integration, it is important to assess that the proper\u2010sized fusion is made by amplifying the genomic region by PCR or visualizing protein size on a protein gel with the HaloTag TMR ligand on untagged alleles, resulting in a knockout of the untagged protein copies. As a result, the entire target protein pool in these clones is expressed as a fusion to HaloTag; therefore, these clones are sufficient for phenotypic experiments with HaloPROTAC3.HaloPROTAC3 will degrade HaloTag target fusion proteins that are recruited to the VHL E3 ligase component, incorporated into active E2/E3 ligase complexes for ubiquitination, and then trafficked to the proteasome. VHL is expressed throughout the cytoplasm and nucleus, as well as in numerous cell types (Buckley et\u00a0al., As endogenous target proteins have highly variable expression levels that will not be significantly altered by HaloTag insertion, varying concentrations of HaloPROTAC3 may be necessary for successful and maximal degradation. Because HaloPROTAC3 binds irreversibly to HaloTag (Buckley et\u00a0al., ent\u2010HaloPROTAC3 (Buckley et\u00a0al., ent\u2010HaloPROTAC3 has significantly reduced affinity for VHL engagement (Buckley et\u00a0al., With any PROTAC degradation study, it is important to be certain that observed target protein loss is due to the specific PROTAC mechanism. For endogenous HaloTag proteins, this can be achieved using For the alternate procedures utilizing HiBiT\u2010HaloTag fusion proteins, protein levels are easily measured with luminescence (Daniels et\u00a0al., In the schematic shown in Figure ent\u2010HaloPROTAC3 at either 3 or 24 hr (Fig. As an example of HaloPROTAC3 degradation and phenotype studies of a target with no available degraders or specific PROTACs, a homozygous CRISPR clone of \u03b2\u2010catenin\u2010HaloTag\u2010HiBiT was generated in HEK293 cells stably expressing LgBiT. In Figure hr Fig. , demonst hr Fig. . These r hr Fig. and degr hr Fig. . Because hr Fig. , an orth hr Fig. and E. U hr Fig. , includi hr Fig. and E. T hr Fig. and loss hr Fig. .ent\u2010HaloPROTAC3 (Fig. ent\u2010HaloPROTAC3 shows that protein loss of each of these targets is specific to HaloPROTAC3. Each of the HaloPROTAC3 degradation profiles was then used to calculate the degradation rate (Fig. 50, the concentration of HaloPROTAC3 that gave half the degradation maximum (Fig. 50 values for each of these targets indicates that it is primarily HaloTag which is driving the degradation, with minimal influence by the different targets. This is desirable for a fusion tag PROTAC system as it needs to be broadly applicable to numerous targets. The kinetic analysis also shows how the optimal dose of HaloPROTAC3 and time of treatment to achieve degradation can be clearly understood from the profiles, saving significant time and yielding more detailed information as compared to western blot analysis.To demonstrate the ability to degrade endogenous HaloTag proteins at a variety of cellular locations, as well as to understand the quantitative parameters of HaloPROTAC3 degradation, the HiBiT kinetic studies outlined in the optional live\u2010cell luminescence degradation detection of HiBiT\u2010HaloTag CRISPR insertion protocol steps were performed Fig. . For theAC3 Fig. \u2010F to proAC3 Fig. \u2010F. All tAC3 Fig. , includiAC3 Fig. . This isAC3 Fig. , suggestate Fig. , as wellmum Fig. , for allmum Fig. . The simF36V\u2010HiBiT at the C terminus in HEK293 cells (Fig. F36V has low efficiency of insertion and edited cells cannot easily be identified or fluorescently enriched from the CRISPR pool.To quantitatively compare HaloPROTAC3 degradation with the dTAG PROTAC system, CRISPR clones of the EPOP target protein were generated by insertion of either HaloTag\u2010HiBiT or FKBP12lls Fig. . These clls Fig. or dTAG\u2010lls Fig. were obtlls Fig. , and dTAlls Fig. , showed lls Fig. , which wlls Fig. . A hook lls Fig. . This rells Fig. . The Hallls Fig. . TogetheBasic Protocol Basic Protocol Optional HiBiT Lytic and kinetic protocols: The HiBiT lytic assay to determine insertion efficiency within CRISPR pools or measure degradation of HiBiT\u2010HaloTag target proteins takes 30\u201060 min. The kinetic degradation assay takes 3 days to perform including plating, Endurazine equilibration, HaloPROTAC3 degradation, and data analysis.Elizabeth A. Caine: Data curation; formal analysis; investigation; writing\u2010original draft; writing\u2010review & editing. Sarah D. Mahan: Data curation; methodology. Rebecca L. Johnson: Methodology. Amanda N. Nieman: Methodology. Ngan Lam: Methodology; supervision. Curtis R. Warren: Conceptualization; supervision. Kristin M. Riching: Conceptualization; formal analysis; investigation. Marjeta Urh: Conceptualization; supervision; writing\u2010original draft; writing\u2010review & editing. Danette L. Daniels: Conceptualization; data curation; formal analysis; investigation; project administration."} +{"text": "ETS-driven prostate cancer initiation and progression remain poorly understood due to a lack of model systems that recapitulate this phenotype. We generated a genetically engineered mouse with prostate-specific expression of the ETS factor, ETV4, at lower and higher protein dosage through mutation of its degron. Lower-level expression of ETV4 caused mild luminal cell expansion without histologic abnormalities, and higher-level expression of stabilized ETV4 caused prostatic intraepithelial neoplasia (mPIN) with 100% penetrance within 1 week. Tumor progression was limited by p53-mediated senescence and Trp53 deletion cooperated with stabilized ETV4. The neoplastic cells expressed differentiation markers such as Nkx3.1 recapitulating luminal gene expression features of untreated human prostate cancer. Single-cell and bulk RNA sequencing showed that stabilized ETV4 induced a previously unidentified luminal-derived expression cluster with signatures of cell cycle, senescence, and epithelial-to-mesenchymal transition. These data suggest that ETS overexpression alone, at sufficient dosage, can initiate prostate neoplasia.The mechanisms underlying Sufficient protein dosage of ETV4 can directly transform prostate epithelial cells, which recapitulates early human disease. ETS family transcription factors including ERG and three highly conserved polyomavirus enhancer activator 3 (PEA3) subfamily members\u2014ETV1, ETV4, and ETV5\u2014are prevalent and mutually exclusive in prostate cancer; they occur early in the natural history of the disease models that overexpress ERG or ETV1 showed either minimal or no phenotype early time points and stabilized ETV4 , harboring mutations at the COP1-binding sites and therefore resistant to COP1-mediated degradation. We demonstrate that the stabilized ETV4AAA significantly increased the protein dosage of ETV4. ETV4AAA expression alone initiates widespread mPIN throughout the prostate epithelium within 2 weeks of activation. The mPIN fails to further progress to invasive cancer due to ETV4-mediated simultaneous activation of the p53-dependent senescence program, and ETV4AAA cooperates with Trp53 loss to promote the development of focally invasive prostate cancer. The neoplastic cells retain expression of prostate lineage differentiation markers such as Nkx3.1 similar to most cases of localized human prostate cancer. In contrast, Pten loss\u2013driven murine prostate cancers generally lose Nkx3.1 and are de novo castration resistant and distinct in human primary adenocarcinoma . We expressed enhanced green fluorescent protein (EGFP) vector control, ETV4WT, and ETV4AAA in A375 melanoma cells and found that, at baseline, protein level of ETV4AAA was significantly higher than that of ETV4WT. Treatment with MG132 that inhibits proteosomal degradation increased ETV4WT protein levels and endogenous ETV4 in vector-infected control cells but only minimally affected ETV4AAA protein level, indicating that ETV4AAA is constitutively stable (fig. S1A). To evaluate the possibility that, beyond protein stability, the mutations introduced in ETV4AAA may alter its transcriptional function, we expressed EGFP, ETV4WT, and ETV4AAA in two prostate cancer cell lines, 22Rv1 and PC3, and performed RNA sequencing (RNA-seq) analysis. In 22Rv1 cells, the protein level of ETV4AAA was significantly higher than that of ETV4WT due to degradation by COP1. PC3 cells harbor a deletion in COP1 and have increased baseline protein levels of ETV4 (AAA and ETV4WT exhibited similar protein levels (fig. S1B). Principal components analysis (PCA) of RNA-seq showed that the first principal component (PC) was separated by cell line and that the second PC was separated by ETV4 expression. In 22RV1 cells, ETV4AAA caused a much greater expression change as ETV4WT in the same direction. In PC3 cells that have baseline higher levels of ETV4, the transcriptome perturbation was less in general, and ETV4WT caused a greater change in gene expression (fig. S1C). We compared the global transcriptome change induced by ETV4AAA and ETV4WT. In 22Rv1 cells, there was a high correlation [Pearson correlation coefficient (r) =\u00a00.72], and ETV4AAA induced a greater change (fig. S1D). In PC3 cells, the global transcriptome change was also strongly correlated (Pearson r\u00a0=\u00a00.76), and ETV4WT induced slightly higher gene expression changes. These data suggest that ETV4AAA regulates a similar transcriptome to ETV4WT.Previous studies showed that transgenic and conditional WTETV4 and AAAETV4 followed by IRES-EGFP driven by the CAG promoter knocked into the Rosa26 locus mice with enlarged nuclei, prominent nucleoli, and cribriform growth in the anterior and dorsal prostate (WTETV4-expressing mice were indistinguishable from those expressing EYFP. While GFP immunohistochemistry (IHC) showed prevalent EGFP- or EYFP-positive cells in murine prostates of all three genotypes, ETV4 IHC detected weak nuclear ETV4 protein in ETV4WT prostate but strong nuclear ETV4 expression in the AAAETV4-expressing prostate cells . This highlights a lobe-specific sensitivity to specific oncogenic transformation as observed in other genetic engineered models of prostate cancer .To determine whether high ETV4 protein expression alone is sufficient to drive progression to invasive cancer, we administered TAM in 6- to 8-week-old mice and aged them for 6, 9, and 12 months for phenotypic assessment. Unexpectedly, the mPIN of the anterior and dorsal prostate of ETV4AAA . The proAAA in the mouse anterior prostate is comparable to that in human prostate cancer, we performed ETV4 IHC using the same conditions on a patient-derived xenograft (PDX) model with TMPRSS2-ETV4 fusion (MDA PCa 175-6) and a control PDX (MDA PCa 173-2) from MD Anderson (MDA PCa program) deposited in biobank , the most commonly used Cre driver in the prostate epithelium, that mediates continued recombination in prostate epithelial cells from puberty . The areas of mPIN were marked by positive EGFP staining. We reasoned that the continuous induction of ETV4AAA though a nonconditional Cre driver results in relatively constant phenotype over time. Despite the continuous induction that maintained mPIN throughout the time studied, there was no evidence of disease progression over time. These data suggest that the expression of AAAETV4 can directly induce mPIN with little latency and that persistent mPIN phenotype requires maintenance of the AAAETV4 expression in prostate epithelial cells; however, AAAETV4 expression alone is insufficient to mediate progression to invasive cancer.We chose ETS translocation alters the chromatin enhancer landscape and the AR cistrome in prostate cancer and RNA-seq, respectively. We chose the 2-week time point to enrich for direct targets. To specifically analyze prostate epithelial cells with the recombined allele, we sorted EPCAM-positive and EYFP- or EGFP-positive prostate epithelial cells by fluorescence-activated cell sorting (FACS) from T2; EYFP, WTT2; ETV4, and AAAT2; ETV4 murine prostates (fig. S4A).We next examined the molecular impact of stabilized ETV4 in the unique phenotypes associated with prostate tumorigenesis. Recent data have shown that WTETV4- and EYFP-expressing cells clustered closely, while AAAETV4-expressing cells exhibited marked changes in the accessible chromatin landscape > 2], with approximately equal number of peaks with significantly increased and decreased ATAC signals . The FOX and AP1 motifs were significantly enriched at both ETV4AAA\u2013up-regulated and ETV4AAA\u2013down-regulated peaks, and the nuclear receptor motif was enriched at the down-regulated peaks . Unsupervised clustering of ATAC signal intensity showed that andscape . We foun signals . Among tagnitude , indicated peaks . Consistpression .eq sites . ATAC-seAAA-induced transcriptome changes. Compared to EYFP-expressing control, AAAETV4 expression induced a greater number of statistically up-regulated genes (537) than down-regulated genes (250) . Hierarc control . Integra control . These dAAA in prostate epithelial cells, we performed gene set enrichment analysis (GSEA) using ~3000 curated genes sets and custom prostate cancer gene sets. There were many more gene sets enriched among genes significantly up-regulated by ETV4AAA expression than among genes down-regulated by ETV4AAA expression, also suggesting that ETV4AAA is primarily a transcriptional activator (fig. S4D). Among enriched gene sets are those genes associated with cell cycle and proliferation (CHANG_CYCLING_GENES), prostate cancer tumorigenesis in human prostate cancer (TCGA_PrCa_UP), and ERG target genes in prostate cancer (VCAP_siERG_DN and VCAP_Lenti_ERG_UP) . We furtWT- and ETV4AAA-expressing prostate cells; canonical ETS transcriptional targets, e.g., Dusp6 and Plat, were mildly induced by ETV4WT and much more significantly induced by ETV4AAA (Trp53), its downstream target p21 (Cdkn1a), and senescence-associated genes Cdkn2b, Cxcl1, Spp1, and Tgfb1 were all significantly up-regulated in ETV4AAA-expressing prostate cells compared to that in EYFP and ETV4WT controls. ETV4AAA prostates stained positive for p53 by IHC and for senescence-associated \u03b2-galactosidase \u2013EGFP was similar between ETV4 ETV4AAA . p53 is significantly higher in ETS fusion\u2013positive samples (42.42%) than that in ETS fusion\u2013negative samples (15.83%), consistent with analysis of the Memorial Sloan Kettering Cancer Center cohort . These data indicate that aberrant high level of ETS expression activates p53 and downstream senescence programs and that p53 loss might be a cooperative event that overcomes the barrier for human prostate tumorigenesis and progression.We analyzed the TCGA dataset of primary prostate cancer to correlate r cohort (20). Wee cancer . To assee cancer : (i) TCGAAA-mediated senescence and tumor progression, we crossed in the LoxP/LoxPTrp53 mice into the AAAT2; ETV4 mice to generate LoxP/LoxP; ETV4AAAT2; Trp53 mice. Two weeks after TAM injection, Western blot of prostate lysates shows decreased p53 protein , which include 19,854 genes, with a median of 1973 genes per cell and a median of 2039 cells per mouse. To visualize single cells of the global atlas, we used uniform manifold approximation and projection (UMAP) for dimension reduction. We then performed Leiden clustering on FACS-sorted live cells from prostates of T2; EYFP wild-type mice, there was expansion of epithelial cells over time from 2 weeks to 4 months , L2, and L3 that had been found in previous studies (fig. S5A and table S6) that highly expresses lateral prostate marker genes Msmb and Trpv6 (Tacstd2 (Trop2), Ly6a (Sca1), Psca, and Krt4; and exhibit enhanced progenitor characteristics (Foxi1 (fig. S5A and table S6).We next analyzed the identities of luminal cell clusters by the marker gene expression. We then annotated the luminal clusters based on the highly expressed marker genes and quantified the cell numbers in each group (fig. S5A). We first focused on T2; EYFP wild-type prostates, WTT2;ETV4 prostates did not comprise any new luminal clusters, suggesting that ETV4WT cannot transform luminal cells. However, WTT2;ETV4 prostates exhibited expansion of Luminal_1 clusters compared with T2;EYFP that is more pronounced from 2 weeks to 4 months . Furthermore, within the WTT2;ETV4 prostates, the expansion of Luminal_1 clusters over time was mostly due to WTETV4-expressing cells (dashed green and dashed cyan). These data suggest that, while expression of WTETV4 did not cause detectable histologic phenotype or new gene expression cluster, its expression was able to cause luminal expansion over time at the single-cell level.Compared to AAA in AAAT2; ETV4 and AAA;Trp53LoxP/LoxPT2; ETV4 prostates generated two new luminal clusters that express AAAETV4 with distinct transcriptome features from previously described luminal clusters cluster using GSEA using GSEAPY package . Pathway analysis showed that extracellular matrix (ECM) organization and multiple growth factor pathways are up-regulated (fig. S6B). To verify the scRNA-seq results, we analyzed the gene expression of L2 marker gene Tacstd2/Trop2 using immunofluorescence staining (fig. S6C). While EGFP (indicating ETV4)\u2013positive cells are Tacstd2 negative, there is a marked expansion of Tacstd2-positive cells in AAAETV4 mice and LoxP/LoxP; ETV4AAATrp53 mice. These Tacstd2-positive cells were found in regions of EGFP-positive, ETV4AAA-expressing cells, suggesting that ETV4AAA-positive cells, through paracrine interactions, expand the normal L2 population.In addition to ETV4 ETV4AAA . This suT2; EYFP and WTT2; ETV4 mice, Basal_2 cells were found exclusively in ETV4AAA-expressing mice, and Basal_3 cells had an intermediate gene expression and were found in all groups of mice (fig. S7A). Basal_2 cells highly express genes Col17a1, Areg, Ly6d, and Cd44 (fig. S7B). We performed immunofluorescence staining of Col17a1 and found a high expression in Ck5-positive basal cells adjacent to EGFP-positive (ETV4AAA-expressing) luminal cells in ETV4AAA mice (fig. S6D), suggesting that ETV4AAA expression in the luminal cells induced paracrine gene expression changes in basal cells.We next identified and quantified subclusters in basal, stroma, and hemopoietic populations. We identified four basal expression clusters. Basal_1 cells were found predominantly in Lama2, Zeb1, Wnt2, Wnt6, Wnt10a, and Rorb; Mesenchymal_2 is characterized by expression of Sult1e1, Fgf10, and Rspo1; Myofibroblast is characterized by expression of Acta2, Myh11, and Notch3. These clusters represent previously described Mesenchymal_1, Mesenchymal_2, and myofibroblast subsets (table S6) . Mesenchymal_3 is characterized by expression of Sod3 and Osr1, which are mesenchymal stem cell markers .We identified five stroma clusters: Mesenchymal_1 to Mesenchymal_4 and myofibroblast (fig. S7C). Mesenchymal_1 is characterized by expression of The myeloid cells and lymphocytes are characterized using the expression of the marker genes (fig. S7F). Marker genes of M1 macrophages, M2 macrophages, and myeloid-derived suppressor cells (MDSCs) are adopted from other publications level and a high dosage (ETV4AAA) level of ETV4 protein through increased ETV4 protein stability by mutating the conserved ExxVPD motifs responsible for COP1 degradation is significantly higher in ETS fusion\u2013positive samples than that in ETS fusion\u2013negative samples . MecAAA-driven murine prostate cancer model is that it maintains the characteristics of early-stage human prostate cancers, including prostate differentiation markers, such as Nkx3.1 expression. Comparably, prostates of Pten deletion, the most well-studied GEM model of prostate cancer, exhibit increased basal marker expression, lose Nkx3.1 expression, and are castration resistant de novo and mutated ETV4AAA cDNA into the plasmid in the middle of the two cassettes. The targeting plasmid was electrophoresed into albino C57BL/6J embryonic stem cells, and G418-resistant clones were isolated by the standard procedures. The clones were screened by Southern blotting. Positive clones were injected into C57BL/6J blastocysts by the MSKCC Mouse Genetics Core Facility, and chimeras were mated with albino C57BL/6J females. Germline transmission was confirmed in albino offspring using Southern blotting. For subsequent generations, genotyping was performed by quantitative polymerase chain reaction (PCR) using the following primers: hETV4_E3_F: GCCGCCCCTCGACTCTGAA; hETV4_E4_R: GAGCCACGTCTCCTGGAAGTGACT.Rosa26 targeting was as described by Srinivas GTACCAGAC(GCAGCAGCC)AGTGATGAGCAGTTTGTTCCTGAT(GCTGCTGCT)TTCCATTCAGAAAACCTAGCTTTCCACAGCCCCACCACCAGGATCAAGAAGGAGCCCCAGAGTCCCCGCACAGACCCGGCCCTGTCCTGCAGCAGGAAGCCGCCACTCCCCTACCACCATGGCGAGCAGTGCCTTTACTCCAGTGCCTATGACCCCCCCAGACAAATCGCCATCAAGTCCCCTGCCCCTGGTGCCCTTGGACAGTCGCCCCTACAGCCCTTTCCCCGGGCAGAGCAACGGAATTTCCTGAGATCCTCTGGCACCTCCCAGCCCCACCCTGGCCATGGGTACCTCGGGGAACATAGCTCCGTCTTCCAGCAGCCCCTGGACATTTGCCACTCCTTCACATCTCAGGGAGGGGGCCGGGAACCCCTCCCAGCCCCCTACCAACACCAGCTGTCGGAGCCCTGCCCACCCTATCCCCAGCAGAGCTTTAAGCAAGAATACCATGATCCCCTGTATGAACAGGCGGGCCAGCCAGCCGTGGACCAGGGTGGGGTCAATGGGCACAGGTACCCAGGGGCGGGGGTGGTGATCAAACAGGAACAGACGGACTTCGCCTACGACTCAGATGTCACCGGGTGCGCATCAATGTACCTCCACACAGAGGGCTTCTCTGGGCCCTCTCCAGGTGACGGGGCCATGGGCTATGGCTATGAGAAACCTCTGCGACCATTCCCAGATGATGTCTGCGTTGTCCCTGAGAAATTTGAAGGAGACATCAAGCAGGAAGGGGTCGGTGCATTTCGAGAGGGGCCGCCCTACCAGCGCCGGGGTGCCCTGCAGCTGTGGCAATTTCTGGTGGCCTTGCTGGATGACCCAACAAATGCCCATTTCATTGCCTGGACGGGCCGGGGAATGGAGTTCAAGCTCATTGAGCCTGAGGAGGTCGCCAGGCTCTGGGGCATCCAGAAGAACCGGCCAGCCATGAATTACGACAAGCTGAGCCGCTCGCTCCGATACTATTATGAGAAAGGCATCATGCAGAAGGTGGCTGGTGAGCGTTACGTGTACAAGTTTGTGTGTGAGCCCGAGGCCCTCTTCTCTTTGGCCTTCCCGGACAATCAGCGTCCAGCTCTCAAGGCTGAGTTTGACCGGCCTGTCAGTGAGGAGGACACAGTCCCTTTGTCCCACTTGGATGAGAGCCCCGCCTACCTCCCAGAGCTGGCTGGCCCCGCCCAGCCATTTGGCCCCAAGGGTGGCTACTCTTACTAG.ATGGAGCGGAGGATGAAAGCCGGATACTTGGACCAGCAAGTGCCCTACACCTTCAGCAGCAAATCGCCCGGAAATGGGAGCTTGCGCGAAGCGCTGATCGGCCCGCTGGGGAAGCTCATGGACCCGGGCTCCCTGCCGCCCCTCGACTCTGAAGATCTCTTCCAGGATCTAAGTCACTTCCAGGAGACGTGGCTCGCTGAAGCTCAGVPD(AAA)SDEQFVPD(AAA)FHSENLAFHSPTTRIKKEPQSPRTDPALSCSRKPPLPYHHGEQCLYSSAYDPPRQIAIKSPAPGALGQSPLQPFPRAEQRNFLRSSGTSQPHPGHGYLGEHSSVFQQPLDICHSFTSQGGGREPLPAPYQHQLSEPCPPYPQQSFKQEYHDPLYEQAGQPAVDQGGVNGHRYPGAGVVIKQEQTDFAYDSDVTGCASMYLHTEGFSGPSPGDGAMGYGYEKPLRPFPDDVCVVPEKFEGDIKQEGVGAFREGPPYQRRGALQLWQFLVALLDDPTNAHFIAWTGRGMEFKLIEPEEVARLWGIQKNRPAMNYDKLSRSLRYYYEKGIMQKVAGERYVYKFVCEPEALFSLAFPDNQRPALKAEFDRPVSEEDTVPLSHLDESPAYLPELAGPAQPFGPKGGYSY.MERRMKAGYLDQQVPYTFSSKSPGNGSLREALIGPLGKLMDPGSLPPLDSEDLFQDLSHFQETWLAEAQPb-Cre4 Hze/JB6.Cg-Gt(ROSA)26Sor) mice of conditional CAG-driven EYFP expression were purchased from the Jackson Laboratory in which exons 4 and 5 are flanked by LoxP sites were used as previously described . For mouse prostates, all tissues were fixed at 4\u00b0C overnight in 4% paraformaldehyde. Tissue processing, embedding, sectioning, and IHC staining was performed. Antibodies for IHC and Western blotting are as follows: chicken anti-GFP , rabbit anti-AR , rabbit anti-ETV4 , rabbit anti-Ki67 , rabbit anti-SMA , rabbit anti-p53 , rabbit anti-Nkx3.1 , mouse anti\u2013\u03b2-actin , and mouse anti\u2013glyceraldehyde-3-phosphate dehydrogenase . Tissue paraffin embedding, sectioning, and hematoxylin and eosin (H&E) staining were performed by the MSKCC core facility. IHC was performed by the MSKCC molecular cytology core using a Ventana Discovery XT. To generate lysates for Western blotting, tissue was homologized in radioimmunoprecipitation assay buffer using the FastPrep-24 system with Lysing Matrix A .Immunofluorescence staining of Tacstd2/Trop2 , Col17a1 , and Ck5 was performed on frozen sections. Sections were permeabilized with 0.5% Triton X-100 for 10\u00a0min and blocked with 10% normal donkey serum for 30\u00a0min at room temperature. Primary antibodies were incubated overnight. Secondary antibodies with Alexa Fluor 647 and Alexa Fluor 555 (Thermo Fisher Scientific) conjugation were applied on the second day. Image were taken with Leica TCS SP5 upright confocal microscope.Intraperitoneal injection of TAM was administered in 8-week-old mouse. Two weeks after TAM treatment, mouse prostate was digested 1 hour with collagenase/hyaluronidase and then 30\u00a0min with TrypLE Express Enzyme at 37\u00b0C to isolate single prostate cells. The prostate cells were stained with phycoerythrin/cyanine 7\u2013conjugated anti-mouse CD326 (EPCAM) antibody , and then, CD326 and EYFP double-positive cells were sorted out by flow cytometry, which are luminal cells mainly from anterior prostate and dorsal prostate. The mRNA or genomic DNA was extracted from these double-positive cells and then was used for ATAC-seq and RNA-seq analysis.https://github.com/kundajelab/atac_dnase_pipelines). Briefly, paired-end reads were trimmed, filtered, and aligned against mm9 using Bowtie2 ranges between 28 and 33, suggesting that the libraries have a high quality and were able to capture the majority of regions of interests.ATAC-seq was performed as previously described (P < 0.01 and |log2(FC)|\u00a0>\u00a02. For visualization, coverage bigwig files were generated using bamCoverage command from deepTools2 and were normalized using the size factor generated by DESeq2. The differential ATAC-seq peak density plot was generated with deepTools2 ] \u00d7 [\u2212log] as input for GSEA analysis using \u201cRun GSEA Pre-ranked\u201d with 1000 permutations (The extracted RNA was processed for RNA-seq by the Integrated Genomics Core Facility at MSKCC. The libraries were sequenced on an Illumina HiSeq-2500 platform with 51\u2013base pair (bp) paired-end reads to obtain a minimum yield of 40 million reads per sample. The sequenced data were aligned using STAR v2.3 calculated by DESeq2. P values were estimated using Wilcoxon rank t test and Student\u2019s t test.To assign ATAC-seq peaks to genes, ChIPseeker_v1.12.1 . After euthanasia, the prostates were dissected out and minced with scalpel and then processed for 1\u00a0hour of digestion with collagenase/hyaluronidase and 30\u00a0min of digestion with TrypLE . Live single prostate cells were sorted out by flow cytometry as DAPI\u2212. For each mouse, 5000 cells were directly processed with 10x Genomics Chromium Single Cell 3\u2032 GEM, Library & Gel Bead Kit v3, according to the manufacturer\u2019s specifications. For each sample, 200 million reads were acquired on NovaSeq platform S4 flow cell.2(X\u00a0+\u00a01)\u2013transformed for analysis of the combined dataset. The top 1000 highly variable genes were found using SCANPY (version 1.6.1) (Reads obtained from the 10x Genomics scRNAseq platform were mapped to mouse genome (mm9) using the Cell Ranger package (10x Genomics). True cells are distinguished from empty droplets using scCB2 package (https://arxiv.org/abs/1802.03426). We then performed Leiden clustering package ."} +{"text": "Euphorbiatithymaloides is one such medicinal plant that hasgained importance but is often confused with other plants of the samespecies. In order to address this issue, this study aimed to conducta conventional and molecular pharmacognostic study for the identificationof the root of E. tithymaloides. Theroot of the plant was studied for the macroscopic observations, andthen, the root was ground into coarse powder for microscopic studiesand to determine the physiochemical properties. The powder was subjectedto extraction with solvents such as ethanol, ethanol/water (1:1),hexane, and ethyl acetate. The extracts were then used for qualitativeand quantitative phytochemicalanalysis. The molecular study was performed with the DNA barcodingtechnique. The DNA was extracted from the root of the plant, and itspurity was examined by gel electrophoresis (1% w/v). The DNA was thenamplified using an Applied Biosystems 2720 thermal cycler for therbcL, matK, and ITS primers. The amplified primers were sequencedwith a 3130 Genetic Analyzer, and the generated sequences were searchedfor similarity in the GenBank Database using the nucleotide BLASTanalysis. The micro- and macroscopic studies revealed the morphologicaland organoleptic characters as well as the presence of medullary rays,fiber, cork, sclereids, parenchymal cells, and scalariform vessels.The physiochemical properties were found within the limit. The phytochemicalanalysis revealed the presence of terpenoids, flavonoids, saponins,and alkaloids. In addition, the alkaloidal content was high in theethanol extract (63.04 \u00b1 3.08 mg At E/g), while the phenol contentwas high in the hexane extract (10.26667 \u00b1 1.77 mg At E/g), andthe flavonoid content was high in the ethyl acetate extract (41.458\u00b1 1.33 mg At E/g). After the BLAST analysis from the GenBankdatabase, the rbcL, ITS, and matK primers showed a similarity percentageof 99.83, 99.84, and 100. The phylogenetic tree for the species closestto each primer was generated using the MEGA 6 software. The matK locihad the highest percentage similar to the rbcL and ITS loci, indicatingthat the matK loci can be used to identify the root of E. tithymaloides as a standalone. The results fromthis study can be used to establish a quality standard for E. tithymaloides that will ensure its quality andpurity.Adulteration and substitution of medicinal plants havebecome amatter of great concern in recent years. This might rapidly increase the production ofmedicinal plant-based products such as teas, tinctures, ointments,and other forms of medicine, which will become more accessible toconsumers, leading to improved health outcomes for many individuals.This is important for a variety of reasons, such as being more cost-effectivethan other medicines, being organic and natural, and being readilyavailable in many parts of the world. In addition, these plants oftenhave fewer side effects than other treatments especially syntheticdrugs and heavy metal formulations.2The use of medicinal plantsfor treating various illnesses anddisorders has a long history, stretching back centuries. In recentyears, the use of medicinal plants in modern medicine has seen a rapidincrease due to their potential benefits in treating conditions likecancer, chronic pain, and other prevalent health problems.3 as well as in more recent sources like the British Pharmacopoeia.4 Authentication and identification of herbal ingredientsare necessary to address the problem of adulteration and substitution.With time, plants have become increasingly important to human medicaltreatments and advancements. The extinction of certain medicinal plantscan have a drastic effect on the continued growth of medical treatments,leading to a greater understanding of the importance of these plants.Without them, it is impossible to make progress in many areas of medicalresearch. The regulation of medicinal plants is a very important anddifficult task, which is why it is necessary to have stringent lawsto ensure safety and quality and to distinguish between the adulterationand substitution of medicinal plants.5 Despiteincreased regulations, it is hard to guarantee the safety and qualityof herbal products. This not only affects the people consuming theseplants but also adversely affects the environment as well. The majorpart of quality control (QC) is to check the product for any adulterationor contamination to determine its safety and efficacy. Generally,QC is done with analytical techniques, which involve the use of electrophoretic,6 chromatographic ,7 and hyphenated methods ,8 including specific and non-specific detectorsystems. Some other aspects that are included in QC are physical examination,microscopical examination, chemical examination, microbiological examination,stability testing, toxicological examination, and a combination ofthe aforementioned techniques.9 Despitethese advances, adulteration and substitutions persist in the currentperiod. These conventional pharmacognostic studies and techniques,even the use of modern hyphenated analytical systems, also failedto detect adulterants and substitutions.As the medicinal plant industry continues to expand with betterprofits, substitution and adulteration of herbal ingredients havebecome more and more of an issue. These practices are documented inancient medical texts, such as the Ebers Papyrus,11 There areseveral techniques available, including conventional sequencing, non-Sangersequencing, random amplifiable polymorphic DNA, DNA barcoding, amplifiablefragment length polymorphism, microsatellites, single nucleotide polymorphism,and others. The limitation with a few of these methods is that noideal marker exists for all the species, and sometimes, it may misleadin identifying the taxonomy but could be highly beneficial in populationgenetics.12 Because of its accuracy andreliability to identify variations, DNA barcoding has gained the mostpopular of all methods. Traditional methods of plant identification,such as morphological analysis, can be subjective and prone to error,particularly in cases where species are closely related or have similarphysical characteristics.14 On the other hand,DNA barcoding enables accurate identification of a plant species basedon its distinct genetic make-up. Since every plant has a differentDNA sequence, it includes using a short, standardized DNA sequenceas a \u201cbarcode\u201d to identify a certain species of plant.The chloroplast gene rbcL, matK, and trnH-psbA are the most frequentlyused barcode region, but the ITS markerhas been also used in recent studies.16 In a study,the ITS marker was used for DNA barcoding of Euphorbiaspecies, and the researchers found that this marker was able to effectivelydistinguish between Euphorbia subgenusand closely related species. It was reported that the ITS marker mightbe used to identify Euphorbia speciesand to verify the authenticity of the plant material used in conventionalmedicine and the pharmaceutical industry.17 Similarly, Akilabindu in 2019 used chloroplast rbcL and matK genefrom Flacourtia inermis Roxb for barcoding.It was reported that rbcL gene showed 100% similarity and matK geneshowed 99.20% similarity with Flacourtia jangomas.18 These studies strongly suggest thatmatK, ITS, and rbcL markers are intensively used in plant DNA barcoding.Despite being a promising technique, employing DNA barcoding for theauthentication and identification of medicinal plants comes with anumber of limitations such as limited reference libraries, inter-and intra-specific variation, and quality of plant material, cost,and complexity.19On the other hand,molecular biology has had a significant impacton plant science research, allowing us to uncover many previouslyunknown genetic and behavioral traits in plants. These genetic traitsare very helpful in identifying plants of particular species in casesof adulteration and substitution.Euphorbia tithymaloides, also knownas \u201cdevil\u2019s-backbone\u201d or \u201ccoast spurge,\u201dis a medicinal plant that belongs to the family Euphorbiaceae. Itis widely distributed in Central and South America, including theAfrican and Caribbean. The plant is found in many other subtropicaland tropical areas as an invasive species. The plant is traditionallyused in folk medicine to treat various ailments, such as inflammatoryconditions, fever, and tumors. Some studies have also shown that extractsof E. tithymaloides have pharmacologicalactivities such as anti-inflammatory, anti-tumor, anti-diabetic, anti-cancer,anti-leishmanial, anti-malarial, anti-helminthic, anti-microbial,anti-oxidant, anti-ulcerogenic, and cytotoxicity.21 Extensive and elaborative research works have been carried out inthe aerial parts of the plant, while the roots are yet to be explored.Hence, this study aimed to carry out the pharmacognostical study of E. tithymaloides root by both conventional and molecularmethods.22.1E. tithymaloides were collected from Jamia Hamdard Herbal Garden, New Delhi, India.Department of Botany, Jamia Hamdard, New Delhi, India, helped to identifyand authenticate the plant. For future reference, a sample of theplant material was deposited in the herbarium under the voucher specimennumber BOT/DAC/2022/01. The roots were cleaned with water to removemud, broken into small pieces, and completely air-dried. The powderwas then pulverized into a coarse powder using an analytical millingmachine and stored in an airtight glass jar for use in the currentwork.In March 2022, root parts of 2.2E. tithymaloides wereexamined using visual perception. The color, odor, and taste of theroot were observed and recorded as organoleptic properties. The macroscopiccharacteristics of the root, such as shape, size, fracture, and othersurface features, were observed using the protocol mentioned in Indianpharmacopeia.22Thefresh roots of 2.3500 mgof a moderately fine (44/85) grounded root powder was immersed in10 mL of water (1:20) and left to stand overnight for 24 h. Subsequently,the contents were put into a Petri-plate, and a slide was preparedby placing the contents with a brush on a clean and dry slide. Thecontents were then examined using a Motic microscope moticam 3.0 MP,AE 2000, and images were taken.2.422 and WHO.23To assess the purity and quality of the crude drug, analytical standardsand physicochemical constants of the root were determined. The ashvalue , foamingindex, foreign matter, swelling index, moisture content, and extractivevalue of root powderedwere determined using the standard protocol provided by the IndianPharmacopoeia2.5The extractswere prepared by using the Soxhlet apparatus by increasing the polarityof a solvent such as ethanol/water (1:1), ethyl acetate, hexane, andethanol. For each solvent, 2 h was given for extraction with optimaltemperature.2.62.6.124To investigate the existence of different secondarymetabolites in the crude extract, qualitative phytochemical assayswere carried out using established methods. Saponin test with frothtest; terpenoid/steroid test with Liebermann\u2013Burchard reagent;flavonoid test with Shinoda test; Tannin test with ferrous(III) chloride;and alkaloid test with Dragendorff reagents.2.6.2The quantitative phytochemical tests were doneusing standard procedures to detect the number of secondary metabolitessuch as alkaloid, flavonoid, and phenolic compounds in the crude extract.2.6.2.125 Standard quercetin solutions of 20, 40, 60,80, and 100 g/mL were produced in 96% ethanol. The standard extractsolution (1 mg/mL) was prepared. 12 \u03bcL of 10% aluminium chloridesolution, 60 \u03bcL of extract solution, 180 \u03bcL of 96% ethanol,and 12 \u03bcL of 1 M sodium acetate were added to the mixture ina 96-well plate. 96 percent ethanol was used as a reagent blank. Afterbeing mixed, each substance was incubated for 40 min at room temperaturein a dimly lit area. The absorbance at 415 nm was measured with amicroplate reader. Total flavonoid content was calculated as quercetinequivalents per gram of plant extract.Total flavonoid content was determined using a slightly modifiedversion of the Sembiring et al. aluminium chloride colorimetric test.2.6.2.225 Ina flat-bottom microplate, a standard solution of extracts (1 mg/mL)was mixed with the Folin\u2013Ciocalteu reagent (1:4) in a 40 and400 \u03bcL ratio and shaken for 2 min. After 5 min, sodium carbonatesolution (100 g/L) was added (125 \u03bcL) and shaken for 1 min ata medium speed. Absorbance was measured at 765 nm using a VersaMaxAbsorbance Microplate Reader after 3 h at room temperature. The absorbancewas corrected by subtracting the ethanol control reaction from thesample reaction. Gallic acid in dilutions of 10, 50, 100, 150, and200 \u03bcg/mL was used as calibration standards. The total phenoliccontent was expressed in milligrams of gallic acid equivalents (GAE)per gram of plant extract.The Folin\u2013Ciocalteu method for a 96-well microplate wasoptimized based on Sembiring et al.2.6.2.326 Bromocresol green solution wasmade by dissolving 69.8 mg of BCG in 3 mL of 2 N NaOH and 5 mL ofdistilled water and then diluted to 1000 mL with distilled water.Phosphate buffer (pH 4.7) was prepared by adjusting the pH of 2 Msodium phosphate solution to 4.7 with 0.2 M citric acid. Atropinestandard solution was made by dissolving 1 mg of pure atropine in10 mL of distilled water. The extract was dissolved in 2 N HCl, filtered,and washed with CHCl3 (three times) and neutralized with0.1 N NaOH. 5 mL of BCG and 5 mL of phosphate buffer were added andcompletely extracted and diluted with chloroform.Accurately measured aliquots of atropine standard were mixed with BCG and phosphate buffer, extractedwith chloroform, and diluted with chloroform. The complex absorbancein chloroform was measured at 470 nm using a UV-Spectrophotometer.The blank was prepared similarly but without Atropine.The total alkaloid content was determined using the method by Ajanalet al.2.72.7.1The NucleoSpin PlantII, Macherey-Nagel kit instructions were followed to extract DNA fromthe plant root sample (13817). The gel electrophoresis (1% w/v) methodwas used to ensure the purity of the extracted DNA and the same wasdocumented using the Bio-Rad.2.7.2Master Mix PhirePlant (Thermo Scientific) and Applied Biosystems 2720 thermal cyclerwas used for the amplification of the primers selected 1. PCR miExoSAP-IT PCR Product Cleanup Reagent (Thermo Fisher)was usedto clean the PCR product. Prior to sequencing, 2% agarose gel electrophoresiswas used to validate the PCR product\u2019s purity. As a molecularstandard, a 100 bp DNA ladder was employed. Usingthe BIO-RAD GelDoc-XR gel documentation system, gel images were captured.2.7.3The DNA sequencing wasperformed with the PCR products purified with the ExoSAP. ABI BigDyeTerminator v3.1 Cycle Sequencing reaction kit was used.2.7.4The 3130Genetic Analyzer Automated DNA Sequencing Machine were used to generateDNA sequences in.ab1 and FASTA formats, and Sequencing Analysis 5.1software was used to do further analysis. A contig of the truncatedsequence was created using forward and reverse strand sequences. Consequently,a single FASTA sequence was produced and further investigated. Usingthe nucleotide BLAST analysis tool, the sequencing similarity of thegenerated samples was examined with the sequences in the GenBank Database.The Clustal W alignment was utilized for multiple sequence alignmentand comparing different sequences, and the degree of similarity betweenthem was determined. For all three of the sample sequences, a phylogenetictree was constructed using the MEGA 6 software utilizing the closest-matchingsource sequence data from the database (NCBI GenBank nucleotide sequence).33.1The macroscopiccharacteristics of the roots as shown in 3.2E. tithymaloides (L.) Poit that are diagnostically important.3.3E. tithymaloides were determinedand are reported in Total ash, watercontent, alcohol soluble extractive, water-soluble extractive value,acid insoluble ash value, soluble ash, and moisture content of theroot of 3.4The extracts were studied further to discoverwhich phytochemical compounds were present. Flavonoid, alkaloid, terpenoids,steroid, tannin, and saponins are examples of common phytochemistrycomponents found in the root extract 4.3.53.5.1y = 0.043x + 0.122, R2 = 0.9947. Among the four crude extracts, ethyl acetate containedthe highest amount of total flavonoid content compounds followed byhexane, ethanol, and then hydro alcoholic 4. Among 0.9987) 5.3.5.3y = 0.0302x + 0.171 R2 = 0.996) 5. Of all= 0.996) 5.3.6Pedilanthus tithymaloides chloroplast rbcL gene (AB267959.1)showed a maximum similarity of 99.83 , the proportion of duplicate trees in which the linkedtaxa grouped together is displayed next to the branches.28 The tree is rendered to scale with branch lengthsin the same units as the evolutionary distances used to estimate thephylogenetic tree. The evolutionary distances, which are measuredin terms of the number of base substitutions per site, were calculatedusing the maximum composite likelihood technique.29 Six nucleotide sequences were subject to this investigation.Codon positions 1st + 2nd + 3rd + noncoding were included. For eachsequence pair, all uncertain positions were eliminated (pairwise deletionoption). The final dataset had 671 positions altogether. In MEGA X,evolutionary studies were carried out.30 The closest plant species was used to draw the phylogenetic treefor the matches in the BLAST data .The final dataset had 671 locations altogether. In MEGA X, evolutionarystudies were performed.30 From the BLASThits, the distance matrix showed the closest distance between thenearby species 8. The phtermined 9. The fi of 100% 10, follo plotted 11.3.6.1aDNA sequencingThe following sequences were generated for the sample(13817-R(rbcl)):>13817-R(rbCL-F) ATATTGGATCAAGCTGGTGTTAAGATTATAAATTGACTTATTATACTCCTGAATATGAAACCAAAGATACTGATATCTTGGCAGCATTCCGAGTAACTCCTCAACCTGGAGTTCCACCTGAGGAAGCAGGAGCTGCCGTAGCTGCTGAATCTTCTACTGGTACATGGACACTGTGTGGACCGATGGGCTTACCAGTCTTGATCGTTATAAAGGACGATGCTACCACATCGAGCCCGTTGCTGGAGAAGAAAATCAATATATTGCTTATGTAGCTTACCCCTTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACCTCCATTGTGGGTAATGTATTTGGGTTCAAAGCCCTACGCGCTCTACGTCTGGAGGATTTACGAATCCCTACTTCTTATACTAAAACTTTCCAAGGGCCACCTCATGGCATCCAAGTTGAGAGAGATAAATTGACAAATATGGTCGCCCTCTATTGGGTTGTACTATTAAACCAAAATTGGGGCTATCCGCTAGAATTACGGTAGAGCGGTTTATGAATGTCTTCGCGGTGGATTGAATTATTTCCAGA.>13817-R(rbCL-R) TAAGGCAACCCCAAAACAGAGACTAAAGCAAGTGTTGGATTCAAGGCTGGTGTTAAAGATTATAAATTGACTTATTATACTCCTGAATATGAAACCAAAGATACTGATATCTTGGCAGCATTCCGAGTAACTCCTCAACCTGGAGTTCCACCTGAGGAAGCAGGAGCTGCCGTAGCTGCTGAATCTTCTACTGGTACATGGACAACTGTGTGGACCGATGGGCTTACCAGTCTTGATCGTATAAAGGACGATGCTACCACATCGAGCCCGTTGCTGGAGAAGAAAATCAATATATTGCTTATGTAGCTTACCCCTTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACCTCCATTGTGGGTAATGTATTTGGGTTCAAAGCCCTACGCGCTCTACGTCTGGAGGATTTACGAATCCCTACTTCTTATACTAAAACTTTCCAAGGGCCACCTCATGGCATCCAAGTTGAGAGAGATAAATTGAACAAATATGGTCGCCCTCTATTGGGTTGTACTATTAAACCAAAATTGGGGCTATCCGCTAAGAATACGTAGAGCGTA.>13817-R(rbCL) (assembled contig) TAAGGCAACCCCAAAACAGAGACTAAAGCAAGTGTTGGATTCAAGGCTGGTGTTAAAGATTATAAATTGACTTATTATACTCCTGAATATGAAACCAAAGATACTGATATCTTGGCAGCATTCCGAGTAACTCCTCAACCTGGAGTTCCACCTGAGGAAGCAGGAGCTGCCGTAGCTGCTGAATCTTCTACTGGTACATGGACAACTGTGTGGACCGATGGGCTTACCAGTCTTGATCGTTATAAAGGACGATGCTACCACATCGAGCCCGTTGCTGGAGAAGAAAATCAATATATTGCTTATGTAGCTTACCCCTTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACCTCCATTGTGGGTAATGTATTTGGGTTCAAAGCCCTACGCGCTCTACGTCTGGAGGATTTACGAATCCCTACTTCTTATACTAAAACTTTCCAAGGGCCACCTCATGGCATCCAAGTTGAGAGAGATAAATTGAACAAATATGGTCGCCCTCTATTGGGTTGTACTATTAAACCAAAATTGGGGCTATCCGCTAAGAATTACGGTAGAGCGGTTTATGAATGTCTTCGCGGTGGATTGAATTATTTCCAGA.bBLAST analysisLibrary details: molecule type: DNA, query length: 607bases databasename: nr.cDistance matrixDescription: nucleotide collection (nt) *program:BLASTN 2.2.28+.3.6.2aDNA sequencing.The following sequences were generated for the sample(13817\u2014ITS).>13817(ITS1) GGGCCGTAGCTACTGCAACGACCCGTGACATGTTCATAAACATGGGTGCCGGTGCGGGATTCGTCCGGCAACGGCATCCCACATGGGCCGGGCAGCGGGGACGCGGGTGGTGCACCCCGTGTTCTCCTTGTCCGGTTCCTTCTAACCAAACACCGACGCCAAACGCGTCAAGGAACTGCGAAAAAAAGGCAGCTTAGGCCCCGGAAACGGCGGTAACCAATGCTGTTTTGGAATAAAAACGACTCTCGGCAACGGATATCTCGGCTCTCGCATCGATGAAGAACGCAGCGAAATGCGATACTTGGTGTGAATTGCAGGATCCCGCGAACCATCGAGTCTTTGAACGCAAGTTGCGCCCGAAGCCTTTCGGCCGAGGGCACGCCTGCCCTGGGTGTCACTCAACTGTCGCCCCGACCCCCTCCTGAAAGGAGGGACGTGAGGGGCGGATGATGGCTTCCCGTGAGCTTTGCAGCCCGCGGTTGGCCCAAAATTCTGGTCCCTGGAACAGAATCCACGGCAATCGGTGGGTTGAAAGACCCTCGTAAAATGTCGTGGTTTCACGGAAGCCAGAGGCGGCAATTAGACCCCAGAGCGCATCCCAAAGCAGCGCTCGAACGGCGACCCCAGGTCAGGCGGGATTACCCGCTGAGTTTAAGCTTCGAGGGGGGGGGGGAGAGAAAAAA.>13817(ITS4) CTTTTTTTCGGCAGTTCCCGACGCCTTGGGTCGGGTTGGTAGAGACCGACAAGAGGAACACGGTGCACACCGGTCCCGCGCCCGCCCTTGGAGCCGTGCCGGACGATCCCGACCGGAAGCCAGTTTATGAAATGTCCCGGGTCGTTTGCGGGCGGGCCTGACAATGATCCTTTCGTAAGGGGGGGGCTGCGAAGGATCATGTCGAGGCCTGCCTAGCAAAACGACCCGTGAACATGTTCATAAACATGGCTGCCGGTGCGGGATTCGTCCGGCAACGGCATCCCCACATTGGCCGGGCAGCGGGGACGCGGGTGGTGCACCCCCGTGTTCTCCTTGTCCGGTTCCTTCTAACCCAAACACCGACGCCAAACGCGTCAAGGAACTGCGAAAAAAAGGCAGCTTAGGCCCCGGAAACGGCGGTAACCAATGCTGTTTTGGAATAAAAACGACTCTCGGCAACGGATATCTCGGCTCTCGCATGATGAAGAACGCAGCGAAATGCGATACTTGGTGTGAATTGCAGGATCCCGCGAACCATCGAGTCTTTGAACGCAAGTTGCGCCCGAAGCCTTTCGGCCGAGGGCACGCCTGCCTGGGTGTCACTCAACTGTCGCCCCGACCCCCTCCTGAAAGGAGGGACGTGAGGGGCGGATGATGGCTTCCCGTGAGCTTTGCAGCCCGCGGTTGGCCCAAAATTCTGGTCCCTGGAACAGAATCCACGGCAATCGGTGGTTGAAAGACCCTCGTAAAATGTCGTGGTTTCACGGAAGCCAGAGGCGGCAATTAGACCCCAGAGCGCATCCAAAGCAGCGCTCGAACGGCGACCCCAGTCAGGCGGATAGACCA.>13817-ITS(assembled contig) GGGTGCCGGTGCGGGATTCGTCCGGCAACGGCATCCCACATGGGCCGGGCAGCGGGGACGCGGGTGGTGCACCCCGTGTTCTCCTTGTCCGGTTCCTTCTAACCAAACACCGACGCCAAACGCGTCAAGGAACTGCGAAAAAAAGGCAGCTTAGGCCCCGGAAACGGCGGTAACCAATGCTGTTTTGGAATAAAAACGACTCTCGGCAACGGATATCTCGGCTCTCGCATCGATGAAGAACGCAGCGAAATGCGATACTTGGTGTGAATTGCAGGATCCCGCGAACCATCGAGTCTTTGAACGCAAGTTGCGCCCGAAGCCTTTCGGCCGAGGGCACGCCTGCCCTGGGTGTCACTCAACTGTCGCCCCGACCCCCTCCTGAAAGGAGGGACGTGAGGGGCGGATGATGGCTTCCCGTGCGCTTTGCAGCCCGCGGTTGGCCCAAAATTCTGGTCCCTGGAACAGAATCCACGGCAATCGGTGGTTGAAAGACCCTCGTAAAATGTCGTGGTTTCACGGAAGCCAGAGGCGGCAATTAGACCCCAGAGCGCATCCAAAGCAGCGCTCGAACGGCGACCCCAGGTCAGGCGGGATTACCCGCTGAGTTTAAGC.bBLAST analysis.Library details: molecule type: DNA, query length: 612bases databasename: nr.cDistance matrix.Description: nucleotide collection (nt) *program:BLASTN 2.2.28+.3.6.3aDNA sequencing.The following sequences were generated for the sample(13817M(matK)):>13817-M(MatK-F) ATAAGAGAAATTTCCGCATTTAATTATGTATCAGATGTATTAATACCTTATCCCATCCATCTTGAAAAATTGGTCCAAACCCTTCGTTTTTGGGTGACAGACCCTTCTTCTTTGCATTTTTTACGATTCTTTCTTCATCAGTATTGGAATTGGAACAGTCTTATTATTCCAAAGAAATCAATTTCGATTTTTCGAAAAAATAATCCACGATTTTTCTTGTTCATATATAATATTCATATATATCAATATGAATCCATCTTCTTTTTTCTTCGTAATCAGTCCTTTCATTTACGATCAACATTTTCTCGAGTCTTTCTTGAACGAATTTTTTTCTATGGAAAACTAGAACATTTTGCAGAAGTTTTTGCTAATGATTTTCAGACCATCCTAGGGTTGTTCAAGGAGCCTTTCATGCATTATGTTAGATATCAAGGAAAATCAATTCTGGCTTTAAAAGATAAGCCCTTTCTGATGAAAAAATGGAAATATTACCTTGTCAATTTATGTCAATGTCATTTTTATGTGTGGTTTCAACCAGAAAAGATCTATATCAATTCATTATCCAAAAATTCTCTCTATTTTGGGGGATATCTTTCAAGTGTACAAATCAATCCTTTGGTAGTACGGAGTCAAATGCTAGAAAATTCATATCTATAGCTAACGATGATACTATGAAGAAACTCGATACAATAGTTCCAATTACTCCTTTAATTAGATTATTGGCAAAATGCAATTTTGTAATGCAGTAGACATCCTATTAGTAAACCGATCCGGGCTCATTCATCCGATTCAGATATTATCGAACAATTTTGTGCGTATATGCAGAAATCTTTCTCATTATCTCGGGGGGGGGACTCACCAAAAA.>13817-M(MatK-R) CATCATCAAATATTTCCTTTTTAGAGGACAAATTTCCGCATTAATTATGTATCAGATGTACTAATACTATCCCATCCATCTTGAAAAATGGTCCAAACCCTTCGTTTTTGGGTGACAGACCATCTTCTTGCATTTTTTACGATTCTTCTTCATCAGTATTGGAATTGGAACAGTCTTATTATTCCAAAGAAATCAAATTTCGATTTTTCGAAAAAATAATCCACGATTTTTCTTGTTCATATATAATATTCATATATATCAATATGAATCCATCTTCTTTTTCTTCGTAATCAGTCCTTTCATTTACGATCAACATTTTCTCGAGTCTTTCTTGAACGAATTTTTTTCTATGGAAAACTAGAACATTTTGCAGAAGTTTTTGCTAATGATTTTCAGACCATCCTAGGGTTGTTCAAGGAGCCTTTCATGCATTATGTTAGATATCAAGGAAAATCAATTCTGGCTTTAAAAGATAAGCCCTTTCTGATGAAAAAATGGAAATATTACCTTGTCAATTTATGTCAATGTCATTTTTATGTGTGGTTTCAACCAGAAAAGATCTATATCAATTCATTATCCAAAAATTCTCTCTATTTTGGGGGATATCTTTCAAGTGTACAAATCAATCCTTTGGTAGTACGGAGTCAAATGCTAGAAAATTCATATCTAATAGCTAACGATAATACTATGAAGAAACTCGATACAATAGTTCCAATTACTCCTTTAATTAGATTATTGGCAAAAATGCAATTTTGTAATGCAGTAGGACATCCTATTAGTAAACCGATCCGGGCTCATTCATCCGATCAGATATATCGACAAATTTTGCCTATAATTCC.>13817-M(MatK)(assembled contig) TTATGTATCAGATGTATTAATACCTTATCCCATCCATCTTGAAAAATTGGTCCAAACCCTTCGTTTTTGGGTGACAGACCCTTCTTCTTTGCATTTTTTACGATTCTTTCTTCATCAGTATTGGAATTGGAACAGTCTTATTATTCCAAAGAAATCAATTTCGATTTTTCGAAAAAATAATCCACGATTTTTCTTGTTCATATATAATATTCATATATATCAATATGAATCCATCTTCTTTTTTCTTCGTAATCAGTCCTTTCATTTACGATCAACATTTTCTCGAGTCTTTCTTGAACGAATTTTTTTCTATGGAAAACTAGAACATTTTGCAGAAGTTTTTGCTAATGATTTTCAGACCATCCTAGGGTTGTTCAAGGAGCCTTTCATGCATTATGTTAGATATCAAGGAAAATCAATTCTGGCTTTAAAAGATAAGCCCTTTCTGATGAAAAAATGGAAATATTACCTTGTCAATTTATGTCAATGTCATTTTTATGTGTGGTTTCAACCAGAAAAGATCTATATCAATTCATTATCCAAAAATTCTCTCTATTTTGGGGGATATCTTTCAAGTGTACAAATCAATCCTTTGGTAGTACGGAGTCAAATGCTAGAAAATTCATATCTAATAGCTAACGATAATACTATGAAGAAACTCGATACAATAGTTCCAATTACTCCTTTAATTAGATTATTGGCAAAAATGCAATTTTGTAATGCAGTAGGACATCCTATTAGTAAACCGATCCGGGCTCATTCATCCGATTCAGATATTATCG.bBLAST analysis.cDistance matrix.Library details: molecule type: DNA, query length: 728bases databasename: nr description: nucleotide collection (nt) *program: BLASTN2.2.28+.4E. tithymaloides, was selected for the pharmacognosticstudy. Primarily, the macroscopic analysis of plants aids in identifyingauthentic materials. The E. tithymaloides root is macroscopically seen to be light brown on the outside andbuff color on the inside, with a fibrous texture and dusty and mildlybitter flavors. Similar to this, one of the essential factors in pharmacopeiais the study of powder microscopy. The powdered roots of E. tithymaloides revealed the presence of scalariformvessels with a pitted bordered wall, radially cut medullary rays,a group of fragmented oval-shaped parenchyma cells with a thin wallcontaining starch, a thick wall with an oval to rectangular-shapedcork, group of sclereids with polygonal wall, elongated, blunt end,and thin wall fibers.A pharmacognostic studyof a plant involves the scientific examinationof the plant\u2019s physical and chemical characteristics, as wellas its traditional uses. The goal of such a study is to establisha set of quality standards for the plant material that can be usedto ensure its authenticity and purity. One such plant, E. tithymaloides. It was reported that the ethanol,ethyl acetate, and hexane extracts showed the presence of steroids,triterpenes, saponins, tannin, and coumarin. It was observed thatthe leaves lack alkaloids, which makes the root of E. tithymaloides more advantageous for therapeuticuse than the leaves.31Preliminary phytochemical investigationof medicinal plants isthe initial step in the process of identifying and characterizingthe phytoconstituents present in a plant. The root powder was subjectedto extraction with solvents such as ethanol, ethanol: water, hexane,and ethyl acetate. These extracts were then used for the physiochemicaland phytochemical analysis, which indicated the presence of alkaloids,tannins, flavonoids, steroids, terpenoids, and traces of saponins.A similar study was conducted by Matisui et al. in 2017, where thephytochemical analysis was performed with the leaves of E.tithymaloides was found to be 7.5%, indicating thetotal amount of inorganic matter present in the plant. The water-solubleash value of 2% revealed the presence of water-soluble compounds suchas inorganic compounds, acids, and sugars. The hexane, alcohol, andhydro-alcohol soluble extractive values show the presence of polar-solublesolvents such as tannins, flavonoids, and alkaloids. Similarly, thefoaming index and moisture content were both found to be <100 and3.33%, indicating that no foaming agents and less moisture were presentin the root samples. The presence of moisture in the plant materialcan have a significant impact on the quality and stability of thephytoconstituents present in the material. Moisture serves as an idealmedium for the growth of bacteria and fungi, which can lead to thedegradation of the plant material and the loss of its medicinal properties.33 Additionally, moisture can also cause the hydrolysis and oxidationof moisture-sensitive phytoconstituents, such as alkaloids, flavonoids,and terpenoids, which can result in a decrease in their concentrationand effectiveness. These findings agree with the phytochemical analysis,which shows the presence of polar-soluble solvents and traces of saponins.Extractivevalues are a measure of the amount of certain activeor inert ingredients present in a drug. They are used to determinethe purity and potency of a drug and can help detect if a drug isexhausted or adulterated. The United States Pharmacopeia (USP) andthe European Pharmacopeia (EP) provide guidelines for the acceptablerange of extractive values for various drugs. Drugs that fall outsideof these ranges may be considered exhausted or adulterated and shouldnot be used. The total ash content of the root of 34 This study examined the total amount of flavonoids,phenols, and alkaloids. The aluminum chloride colorimetric test wasused to evaluate the sample\u2019s total flavonoid content. Theassay revealed that the ethyl acetate extract contains the highestamount of flavonoids, while the hydro-alcoholic extract showed thelowest amount. The ethyl acetate extract\u2019s high concentrationof flavonoids is comparable to the research conducted by Ch\u00e1vezet al. in 2022. It was reported that, when compared to hexane, water,and dichloromethane, the ethyl acetate extract of E.tithymaloides leaves contained a high amount of flavonoids.35 This indicates that E. tithymaloides in general, as a whole plant, possesses a good amount of flavonoids.Likewise, using gallic acid as a reference, the Folin\u2013Ciocalteumethod was used to quantify the total phenol concentration of theroot extracts. It was clear from the assay that the hexane extractshad a higher phenolic content than other extracts. The total alkaloidalcontent assay showed that the ethanol extract had a high amount ofalkaloids.The primary goal of quantitative chemical analysis is to estimatethe amounts of the plant\u2019s major phytoconstituents classes.Flavonoids, phenols, and alkaloids are three important classes ofphytoconstituents that are commonly found in medicinal plants. Theyare known for their medicinal properties and have been used in traditionalmedicine for centuries. Their presence in medicinal plants is oftenused as a measure of the plant\u2019s quality and potency.36 In this study, all three markers were used forthe identification of E. tithymaloides. The sequence match with rbcl loci showed a match of 98.64 to 99.83%with the top five hits from the BLAST analysis. Similarly, the sequencematch with the MatK loci was from 96.25 to 100%. These results arein contrast with the phylogenetic study conducted by El-Banhawy, 2020in the genus Euphorbia.37 It was reportedthat the rbcl was the least successful and matK genes were not significantlydifferent in identifying the Egyptian Euphorbia. However, the resultsof this study reveal a good identity score of 99.84 and 100 for Euphorbia plant grown in India. The difference inthe identification of the genus using the rbcl and matk loci couldbe varied due to the geographical location and environment of theplant growth. These genetic and geography-based changes and theiridentification by rbcl and ITS were expressed by Shawkat and Ahmed,2022 in a comparative study. It was reported that the rbcl and ITS2were able to provide a good resolution among the Euphorbiatirucalli, Euphorbia hirta, and Euphorbia peplus but showedonly a minor change in the evolution taxa and phylogenetic tree.39 These data are comparable to the results observedin this study as the phylogenetic tree and the distance matrix showsa minor change and were able to identify the closest species witha good matching percentage. The ITS is considered to be one of themost informative markers for species identification in plants, asit has a high degree of variation among different plant species. TheITS region is located between the 18S and 28S rRNA coding regionsand is transcribed as part of the rRNA precursor molecule. The ITSregion is highly variable, containing both coding and non-coding regions,which makes it a valuable marker for species identification. Additionally,the ITS region is present in multiple copies in the genome, whichincreases the chances of detecting variations among different species.38 Likewise, the ITS primers ITS1 and ITS4 in thisstudy revealed a good match from 95.11 to 99.84% from the BLAST analysis.The ITS loci results are comparable with the study experimented onby Kim et al., in 2020 where DNA barcoding was performed for KoreanEuphorbiaceae. It was reported that among the rbcl, ITS, and MatKloci, the ITS was the most beneficial and can be used to identifyother Korean Euphorbiaceae plants using ITS as a single barcode.40 Likewise, the results from this study show thatall the three loci were able to efficiently identify the E. tithymaloides. In addition, the 100% true matchwith the MatK loci shows that it can be used as a standalone to identify E. tithymaloides from the subspecies and other Euphorbiaceaeplants as well.The nuclear ribosomal DNA ITS regions ITS1 and ITS2and the chloroplastgenes matK, rbcL, and trnH-psbA are the markers in plant barcodingthat are most often investigated. These markers are highly informativefor identifying and differentiating plant species. Recently, the useof DNA barcoding based on the markers rbcl, ITS, and MatK has gainedsignificant traction in the field of plant species authentication.E. tithymaloides is the high demand for the plantand its medicinal properties. This has led to the collection of theplant from wild populations, which can lead to over-harvesting anddepletion of wild populations. Another reason is that E. tithymaloides is often confused with closely relatedspecies, and this can lead to misidentification and substitution.For example, E. tithymaloides is oftenconfused with Euphorbia lathyris, whichis not used for medicinal purposes and is considered toxic. Adulterationand substitution can have serious consequences for both the consumersand the environment. Consumers may be unknowingly consuming harmfulsubstances or receiving ineffective treatment, while wild populationsof medicinal plants may become endangered due to over-harvesting.To address these issues, it is important to establish and use DNAbarcoding techniques for the identification and authentication ofmedicinal plants, including E. tithymaloides, to ensure that the plant material being sold is authentic and unadulterated.Additionally, conservation measures should be implemented to protectwild populations of medicinal plants from over-harvesting, and regulationsshould be put in place to control the trade of medicinal plants.One of the main reasons for the adulterationand substitution of 5E. tithymaloides has been carriedout in this study and reported for the first time. The results fromthe micro-and macroscopic studies, phytochemical analysis, and DNAbarcoding had shown significant results in identifying the E. tithymaloides. It is important to keep in mindthat DNA barcoding is one tool among many that can be used to identifyand authenticate medicinal plants and it should be used in combinationwith other methods, such as morphological and chemical analysis, toensure accurate identification. The results of this pharmacognosticalstudy can be used to establish a set of quality standards for the E. tithymaloides, which can be used to ensure itsauthenticity and purity. This is important for ensuring the safetyand efficacy of traditional medicine and medicinal products made from E. tithymaloides.The conventional and molecularpharmacognostic study on the rootof"} +{"text": "The current study aims to investigate the regulatory impact of leptin or melatonin on bone metabolism as well as the underlying mechanism in conjunction with Sema4D .Rats were used to create the osteoporosis model utilizing the OVX (OVariectomize) technique. Rat tibial specimens from each side were collected for three-dimensional reconstruction and Micro-CT scanning examination. The Hematoxylin-osinstaining (HE) staining technique was used to determine the pathological condition of bone tissues. The ELISA (Enzyme-Linked Immunosorbent Assay) assay was used to measure the amount of estradiol present in the serum. In the current study, there were six groups: control, OVX, OVX\u2009+\u2009NL (no load group), OVX\u2009+\u2009Sema4D, OVX\u2009+\u2009Sema4D\u2009+\u2009leptin, and OVX\u2009+\u2009Sema4D\u2009+\u2009MT (melatonin). Rats were given injections of the Sema4D or leptin overexpressing vectors via the tail vein in accordance with the aforementioned classification. By using a high-resolution micro-CT technology, 3D bone structure was discovered. The activity of tartrate-resistant acid phosphatase-5b (TRAP-5b) and bone-derived alkaline phosphatase (BALP) in serum was assessed using an ELISA. The number of osteoclasts in the metaphysis of the upper tibia was determined using TRAP (tartrate-resistant acid phosphatase) staining. Immunohistochemistry was used to find leptin and bone morphogenetic protein-2 (BMP-2) expressions in bone tissue.The BV/TV (Bone volume/Tissue volume), Tb.N (Trabecular number), BMD , and BMC levels were significantly higher in the OVX\u2009+\u2009Sema4D\u2009+\u2009leptin and OVX\u2009+\u2009Sema4D\u2009+\u2009MT groups compared to OVX\u2009+\u2009NL, while Tb.Sp (Trabecular separation) levels were significantly lower. In contrast to the OVX group, the bone trabeculae in the OVX\u2009+\u2009Sema4D\u2009+\u2009leptin and OVX\u2009+\u2009Sema4D\u2009+\u2009MT groups had a relatively complete structure and tended to be organized closely. The amount of bone trabeculae grew drastically, whereas the proportion of TRAP-positive osteoclasts declined dramatically. BMP-2 and leptin were also elevated, while BALP and TRAP-5b activity was reduced.Leptin or melatonin improved Sema4d's role in trabecular bone microstructure, bone production, and repairment of trabecular bone loss in osteoporosis rats. A prevalent age-related illness is osteoporosis. Osteoporosis has recently emerged as a major global public health concern due to the sharp rise in patient numbers . StatistSema4D, a protein involved in brain signaling, belongs to the semaphorin family. It has been established that semaphorins, also known as immunological semaphorins, and immune control have a close association. According to reports, Sema4D is involved in a wide range of signal transduction pathways and performs a wide range of biological functions, including the control of B cells, T cells, and other immune cells as well as the development of the nervous system, platelet function, epithelial cell function, and endothelial cell function . Sema4D Using an OVX rat model, the current work intends to investigate the effects and processes of Sema4D, Sema4D paired with leptin, and Sema4D combined with melatonin on bone metabolism in an effort to identify possible molecular targets for osteoporosis therapeutic treatment.SD (Sprague Dawley) rats, 32 females, 200\u2013220\u00a0g Zhejiang Weilihua Experimental Animal Technology Co., Ltd., License No. SCXK (Zhejiang) 2019-0001), Rearing Environment: Temperature 20\u201326\u00a0\u00b0C, Humidity 40%- 70%. All research was done in compliance with international standards for the treatment and use of laboratory animals, and needless harm to the animals was avoided at all costs .Medicine refrigerator , refrigerated freezer , slicer , electric blast drying oven , thermostatic incubator , electric thermostatic incubator ; Pressure cooker , induction cooker , fume hood; Microscope , microtome , multifunctional enzyme marker ; First antibody: BMP-2 ; leptin; Sema4d ; Second antibody: horseradish enzyme-labeled goat anti-rabbit IgG (H\u2009+\u2009L) ; Tartrate-resistant acid phosphatase staining solution ; Rat tartrate-resistant acid phosphatase 5b (TRACP-5b) ELISA reagent ; Rat bone alkaline phosphatase (BALP) kit ; Melatonin (S20287) was purchased from Shanghai Yuanye Biotechnology Co., Ltd., with a relative molecular weight of 232.28 and a purity of more than 99%.Construct the PCR primer for the target fragment based on the sequence of the target gene Sema4D and Leptin ; As the carrier for digestion, the suitable restriction enzyme was chosen, and the pure linear carrier was extracted from the agarose gel; Then, perform PCR on the target fragment and recover the agarose gel to acquire the right fragment size; Link the linearization vector to the target fragment utilizing homologous recombination or the T4 technique; Transformed responsive DH5a or stbl3, covered with bacterial fluid and cultured for 12 to 16\u00a0h; Choose the moving monoclonal colony for validation; Choose the most appropriate positive clone for colony verification prior to sequencing; The extraction of plasmids was carried out on cloned samples with the right sequence. Anhui General Biology Co., Ltd. is responsible for the adenovirus packaging of the vector after the sequencing verification. The specific information on the viral vector is as follows :ATGAAGATGTGTGCCCCCGTCAGGGGGCTGTTCTTGGCCCTGGTGGCTGTGTGGAGGACCGCGGTGGCATTCGCCCCTGTGCCTCGGATCACCTGGGAGCACGGAGAGGTAGGTCTGGTGAAGTTTCACGAGCCAGGCATCTTTAACTACTCTTCCTTGCTGATGAGTGAAGACAAAGACACTCTGTATGTGGGTGCCCGGGAAGCTGTCTTTGCAGTGAATGCGCTGGACATCTCTGAGAAGCAACATGAGGTATACTGGAAGGTCTCTGAAGACAAAAAATCCAAGTGCGCAGAGAAGGGGAAATCAAAGCAGACGGAGTGCCTTAACTACATCCGAGTGCTGCAACCGCTTAGCAGCACTTCCCTCTACGTGTGTGGGACCAATGCGTTCCAGCCCACCTGTGACCACCTGAACTTGACCTCTTTCAAGTTTCTGGGGAAAAGCGAAGATGGCAAAGGAAGATGCCCCTTCGACCCCGCCCATAGCTACACATCCGTCATGGTCGGGGGAGAGCTCTACTCTGGGACTTCATATAATTTCTTGGGCAGCGAACCCATCATCTCTCGAAACTCTTCCCACAGTCCCCTGAGGACAGAGTACGCCATCCCTTGGCTAAACGAGCCTAGCTTCGTCTTTGCTGACGTGATCCACAAGAGCCCAGATGGTACAGAGGCTGAGGATGACAAGGTCTACTTCTTCTTTACGGAGGTGTCCGTGGAGTACGAGTTCGTCTTCAAGTTGATGATCCCGCGAGTTGCCAGGGTGTGCAAGGGCGACCAGGGCGGCCTGCGGACTTTGCAAAAAAAGTGGACCTCCTTCCTAAAGGCCAGACTGATCTGCTCCAGGCCAGACAGTGGCCTGGTCTTCAACATTCTTCAAGATGTGTTTGTGCTGAGGGCCCCGGGCCTCAAGGAACCTGTGTTCTATGCGGTCTTCACCCCACAGCTGAACAACGTGGGTCTGTCAGCGGTCTGTGCCTACACGCTGTCCACGGTGGAGGCCGTCTTCTCCCGAGGAAAGTACATGCAGAGTGCCACAGTGGAGCAGTCTCACACCAAGTGGGTACGCTACAATGGCCCAGTGCCCACTCCCCGGCCTGGAGCGTGTATCGACAGTGAGGCCCGGGCAGCCAACTACACCAGCTCCTTGAATCTCCCAGACAAAACGCTGCAGTTTGTCAAAGACCACCCTTTGATGGACGACTCGGTGACGCCAATAGACAACAGGCCGAAACTGATCAAAAAAGATGTCAACTACACCCAGATAGTGGTAGACAGGACCCAGGCCCTGGATGGGACCTTCTACGACGTCATGTTCCTCAGCACAGACCGGGGCGCTCTGCATAAAGCTGTCATCCTTGCAAAAGAGGTACACGTGGTTGAGGAGACCCAACTCTTCCAGGACTTCGAACCGGTCCTGTCTCTGCTGCTATCATCAAAGAAGGGGAGGAAGTTTGTCTATGCTGGCTCCAACTCAGGAGTGGTCCAAGCTCCCCTGGCCTTCTGCGGAAAGCACAGTAGCTGTGAAGACTGTGTGCTAGCACGGGACCCCTACTGCGCCTGGAGCCCAGCCATCAAGGCCTGTGTTACCTTGCACCAGGCAGAGGGCTCTAGCAGGGGCTGGATTCAGGACATGAGTGGCGACACGTCCTCGTGCCTGGATAAGAGTAAAGAAAGTTTCCATCAGCATTTTTTCAAGCACGGCGGCACAGCAGAACTCAAGTGTTTCCAAAAGTCCAACCTGGCCCGGGTGGTGTGGAAGTTCCAGAACGGCGAGTTGAAGGCTGTGAGTCCCAAGTATGGCTTTGTGGGCAGGAAGCACCTGCTCATCTTTAACCTGTCAGACGGAGACAGCGGTGTGTACCAGTGCCTGTCAGAGGAAAGGGTCAGGAATAAAACGGTCTCCCAGCTGCTCGCCAAGCACATCCTGGAAGTGAAAATGGTAGCTCGGATCCCCCCATCACCTACCTCACAGACTGCTCAGACAGAAGGTAGTAGGATCACATCCAAAATGCCTGTGGCGTCTACCCAGGGGTCCTCTCCCCCTACCCCGGCTCTGTGGGCAACCTCCCCCAGGGCTGCCACCCTACCTCCCAAGTCCTCCTCCACCGGCACGTCCTGTGAACCAAAAATGGTCATCAACACGGTCCCACAGCTCCACTCGGAGAAGACAGTGTATCTCAAGTCCAGTGACAACCGCCTGCTCATGTCTCTCCTCCTCTTCCTCTTTGTCCTCTTCCTCTGCCTCTTTTCCTACAACTGCTACAAGGGCTACCTGCCCGGACAGTGCTTAAAGTTCCGCTCAGCCCTGCTGCTCGCAAAGAAAAAACCCAAGTCAGAGTTCTCTGACCTGGAGCAGAGTGTGAAGGAGACGCTGGTAGAACCTGGGAGCTTCTCGCAGCAGAACGGCGACCAGCCCAAGCCAGCCTTGGATACCGGCTATGAAACCGAGCAGGACACTATCACCAGCAAGGTCCCCACCGATCGAGAGGACTCGCAACGTATCGACGAGCTCTCCGCCAGGGACAAACCGTTTGATGTCAAGTGTGAACTCAAGTTTGCAGACTCGGATGCCGACGGGGACTGA.ATGTGCTGGAGACCCCTGTGCCGGTTCCTGTGGCTTTGGTCCTATCTGTCCTATGTTCAAGCTGTGCCTATCCACAAAGTCCAGGATGACACCAAAACCCTCATCAAGACCATTGTCACCAGGATCAATGACATTTCACACACGCAGTCGGTATCCGCCAGGCAGAGGGTCACCGGTTTGGACTTCATTCCCGGGCTTCACCCCATTCTGAGTTTGTCCAAGATGGACCAGACCCTGGCAGTCTATCAACAGATCCTCACCAGCTTGCCTTCCCAAAACGTGCTGCAGATAGCTCATGACCTGGAGAACCTGCGAGACCTCCTCCATCTGCTGGCCTTCTCCAAGAGCTGCTCCCTGCCGCAGACCCGTGGCCTGCAGAAGCCAGAGAGCCTGGATGGCGTCCTGGAAGCCTCGCTCTACTCCACAGAGGTGGTGGCTCTGAGCAGGCTGCAGGGCTCTCTGCAGGACATTCTTCAACAGTTGGACCTTAGCCCTGAATGCTGA.The following is the map of the Semad4 adenovirus overexpression vector:The vector map of Leptin adenovirus overexpression is as follows:The occurrence and progression of osteoporosis are significantly influenced by the estrogen released by the ovaries. Rats' decreased estrogen secretion following ovariectomy results in a weaker suppression of osteoclasts. Bone production is outpaced by bone absorption. Because the bone is in a high stage of transition, the mass of the bone cannot be balanced, which causes more bone to be lost. The best model to research postmenopausal osteoporosis is the ovariectomized osteoporosis model , 20; AccBai et al. describeObtain serum samples and separate the supernatant for testing using a centrifuge. After treatment, BALP, TRAP-5b, and estradiol were identified according to the processes outlined in the instructions for the respective ELISA kit \u201327. Use Following the collection of tibia tissues from each animal, the tissues were washed in water for two hours. After dehydration with various concentrations of ethanol solution, tissues were dehydrated with xylene until translucent, followed by embedding for one hour and slicing. The slides were then roasted, dewaxed, hydrated, bathed in distilled water, colored in a hematoxylin aqueous solution for three minutes, and then differentiated using a hydrochloric acid ethanol differentiation solution for fifteen seconds. After being cleaned briefly with water and blue-returning solution for 15\u00a0s, slides were rinsed with water and stained with eosin for three minutes. The inverted microscope was then utilized to capture images .Sections of paraffin were deparaffinized for 5\u00a0min, then progressively incubated in 100% ethanol for 5\u00a0min, 90% ethanol for 2\u00a0min, and 70% ethanol for 2\u00a0min. The TRAP staining solution was then added for fixation at 4\u00a0\u00b0C for 60\u00a0s. After being washed with water, slices were immersed in TRAP incubation solution at 37\u00a0\u00b0C for 45\u201360\u00a0min, then stained with methyl green for 2 to 3\u00a0min. Pictures were captured using a microscope .The slides were washed in PBS for one hour before being incubated overnight with 10% goat serum. The slides were then incubated with primary antibodies against BMP-2 or leptin at 4\u00a0\u00b0C for 24\u00a0h, after which they were washed with PBS. The secondary antibody was then added, followed by a 24-h incubation at 4\u00a0\u00b0C, rinsing, and DAB staining. Finally, photos were captured with an inverted microscope .For statistical analysis, SPSS 20.0 software was used. Each experiment was conducted three times. The mean standard deviation was used to express the quantitative outcomes (X As shown in Fig.\u00a0As demonstrated in Fig.\u00a0As shown in Fig.\u00a0TRAP staining was used to evaluate the number of osteoclasts in metaphysis at the upper end of the tibia. Compared to control, the number of TRAP positive osteoclasts was dramatically increased in the OVX, OVX\u2009+\u2009NL, and OVX\u2009+\u2009Sema4D groups, among which the highest number of osteoclasts was observed in the OVX\u2009+\u2009Sema4D group. Furthermore, compared to the OVX group, dramatically declined TRAP positive osteoclasts were observed in the OVX\u2009+\u2009Sema4D\u2009+\u2009leptin and OVX\u2009+\u2009Sema4D\u2009+\u2009MT groups (Fig.\u00a0As shown in Fig.\u00a0As shown in Fig.\u00a0Sema4D plays a crucial function in the regulation of osteoclast formation. Sema4D expression is increased during osteoclast proliferation ; NegishiIn the present study, OVX was used to construct an osteoporosis rat model. Pathological results showed that the number of tibial trabeculae in OVX rats was significantly reduced, and the bone trabeculae were thin and fractured with low density, accompanied by an increased serum E2 level. These results were consistent with the research reports of Zhao and WangLeptin is reported to promote the proliferation of osteoblasts and the transformation of bone marrow stromal cells into osteoblasts. In previous experiments, an extremely repressed long-bone growth, reduced bone surface area, and decreased mineral and bone density level were observed in leptin deficient mice which were dramatically rescued by the injection of leptin, indicating that leptin played an important role in the differentiation and maturation of osteoblasts . It is rThe effect of melatonin on primary osteoporosis has been widely reported. St Hilaire et found that melatonin levels were lower in premenopausal women and were accompanied by increased bone resorption . Maria rIn conclusion, leptin and melatonin reversed the activation of osteoclasts caused by overexpression of Sema4D, which intensified the pathological process of osteoporosis. The target molecules for the clinical therapeutic development of osteoporosis were offered by the current study as potential and trustworthy candidates."} +{"text": "Pythium flevoense was diagnosed as the cause of dermatitis in a young adult female harbour porpoise (Phocoena phocoena) that had been trapped in a pound net in a temperate saltwater environment. Disease from Pythium sp. infection\u2014pythiosis\u2014is infrequently diagnosed in humans, horses, dogs, cattle, and few other mammalian species. Pythiosis is typically associated with exposure to tropical or subtropical freshwater conditions, and typically caused by Pythium insidiosum. However, until now, pythiosis has been reported in neither marine mammals nor temperate saltwater conditions, and P. flevoense is not known as a cause of pythiosis in mammals. This porpoise developed generalised dermatitis despite treatment and euthanasia was necessary. Histopathological evaluation revealed a chronic active erosive dermatitis, with intralesional hyphae morphologically consistent with a Pythium sp. PCR analysis and sequencing of affected skin matched Pythium flevoense with a 100% similarity to the reference strain. Additional diagnostics excluded other pathogens. Based on this case report, P. flevoense needs to be considered as a mammalian pathogen. Furthermore, harbour porpoises and possibly other marine mammals may be at risk of infection with P. flevoense, and pythiosis should be included in the differential diagnosis of dermatitis in marine mammals.The oomycete The online version contains supplementary material available at 10.1186/s13567-023-01226-1. Pythium species are oomycetes, fungus-like filamentous eukaryotic organisms belonging to the kingdom Stramenopila, phylum Oomycota, class Oomycetes, order Pythiales, family Pythiaceae, which may reproduce both sexually and asexually [Pythium insidiosum in humans and other animals may cause disease, called pythiosis (insidiosi), typically characterized by necrotising and granulomatous inflammation [sexually \u20133. Most sexually . Howeverammation , 3. The ammation , 3, suppammation .P. insidiosum occurs typically in (sub)tropical stagnant freshwater containing motile zoospores that enter skin lesions and invade tissues. Zoospores show tropism for skin and hair [Infection with and hair . Pythiosand hair \u20138. Pythiand hair , 3.Depending on the site of infection and involved species, cutaneous, lymphonodular, osseous, vascular, ocular, visceral, and disseminated forms of pythiosis may occur . VasculaP. insidiosum is morphologically discernible as hyaline, pauci-septate, nonparallel thin-walled (5\u201310\u00a0\u03bcm) hyphae with infrequent and irregular branching. As routine H&E stains may fail to stain these hyphae, enhancement of discernibility requires silver stains like Grocott silver stain [Histologically, er stain , 15.Pythium flevoense was isolated and identified for the first time in soil samples from the province Flevoland, The Netherlands in 1968 [Parabroteas sarci) in Argentina [Plecoglossus altivelis) in Japan [P. flevoense, and simultaneously the first case of pythiosis in a marine mammal, a harbour porpoise (Phocoena phocoena). in 1968 . Sporadirgentina and ayu in Japan . Here wend of May 2018, trapped in a pound net in the coastal seawaters near the town Kors\u00f8r in Denmark. It was freed and brought to the Marine Biological Research Center, University of Southern Denmark, and intended to be part of the porpoise collection at the neighbouring sea aquarium Fjord & B\u00e6lt at Kerteminde Bay which is permitted by Danish law to house bycaught harbour porpoises for research and conservation purposes. It was named Idun and kept alone in a quarantined saltwater basin. A separated basin housed another normal healthy harbour porpoise. Both basins were circulated continuously with the same fresh seawater from the bay. Despite treatments for a progressive generalised erosive dermatitis, the animal did not recover and was euthanised after 3\u00bd months . The carcass was stored on wet ice directly after euthanasia until autopsy was performed, 24\u00a0h after death.A female harbour porpoise, estimated to be 14 months of age, was found alive on the 22During autopsy the porpoise\u2019s external and internal organs were examined. The skin lesions were photographed and sampled for histology, bacteriology and molecular analysis. Internal organs and tissues were similarly sampled. Samples for histology of skin, lung, trachea, heart, kidney, spleen, lymph nodes, urinary bladder, liver, pancreas, stomach, small and large intestines were fixed by immersion in 10% neutral-buffered formalin. These fixed samples were routinely processed and embedded in paraffin. Tissue sections of 4\u00a0\u03bcm thick were mounted on glass slides, deparaffinised with xylene, rehydrated using graded alcohols, and stained according to routine histochemical protocols with haeSamples from affected skin were biopsied from the live animal on day 34 after rescue and taken during autopsy on day 105 after rescue. Samples for molecular analyses were stored in lysis buffer until processing. Tissue samples for bacteriology including affected skin were taken during autopsy on day 105, stored and frozen at \u221220\u2009\u00b0C and subsequently thawed a week later for further analyses. Fluorescent microscopy using Blancophor was performed on the skin samples (taken on day 34), to visualize fungal(-like) organisms. Fungal cultures from the same samples were performed using SGA agar and YGC agar with incubation at 35\u201337\u00a0\u00b0C for 2 weeks. Species identification included classical techniques including macro- and micromorphology and thermotolerance testing. Molecular analysis included internal transcribed spacer (ITS)-PCR and sequencing of which the results were compared with online sequence databases in July 2018, NCBI (BLAST) and CBS* database. . The sequence analysis was repeated in August 2021 specifically for this study.For bacteriological examination, samples of skin, lung, kidney, liver, spleen, uterus, tracheobronchial lymph node and adrenal gland were frozen at \u221220\u2009\u00b0C\u00a0and after thawing, cultured according to an in-house established standard protocol, including classical methods and commercial API identification kits , for bacterial species from marine mammals, as described previously . For virnd of May 2018 into the Marine centre (day 0), blood samples were collected. Results from standard haematological and clinical biochemical analyses on these samples were all within normal ranges, without any indication of immunosuppression, anaemia, or inflammation , days 3 to 29: enrofloxacin , days 20 to 104: tramadol , days 25 to 31: itraconazole , days 27 to 37: miconazol , days 29 to 49: terbinafine , days 31 to 45: posaconazole , days 45 to 101: voriconazole , days 45 to 104: amoxycillin with clavulanic acid , days 100 to 102: posaconazole , days 100 to 103: terbinafine , days 101 to 103: enilconazole (14.4% w/w baths), days 103 to 104: enrofloxacin .Macroscopic examination showed a female non-pregnant harbour porpoise in moderate physical condition , with a generalised dermatitis. The dermatitis was characterised by multifocal to coalescing lesions with an irregular rough surface , threadlike structures were discernible. These structures did not resemble true hyphae. They lacked the usual characteristics of fungal hyphae since they were of significantly variable wideness, had no distinct regular septa, and the tips were cut rather than rounded hyphal ends and fluorescence was not distinct as is usual for fungal hyphae.Aspergillus niger on YGC agar only. As A. niger is found ubiquitously as cause of black mold on certain fruits and vegetables, this single culture growth is considered more likely as a contaminant rather than a primary pathogen.After 2\u2009weeks, fungal culture of one of four skin samples (taken on day 34) grew Mucorales sp. (zygomycetes) and for Aspergillus sp. were all negative (likely confirming contaminated growth of Aspergillus niger in culture), but all four samples were positive for Pythium sp. The results of ITS sequencing in 2018 matched Pythium pectinolyticum with a similarity of >\u200999.5% compared with the reference strain CBS* 122643 . Sequence analysis was rerun in August 2021 for the current study, and this time showed a 100% sequence similarity to CBS strain 234.72 of Pythium flevoense, which had been submitted to the database after 2018, and thus the aetiological species was reclassified as P. flevoense in this study. The fasta of the sequenced porpoise skin samples were:Specific fungal PCRs on four skin samples (taken on day 34) for >ITS1&2GCCCGTCGCACCTACCGATTGAATGACTCGGTGAGAAATCGGGACCGTGAATCCGTTTGCTTCATTGCGAGTGGACTTATGGGAACTTTTTCTAACCTCGCCATTTAGAGGAAGGTGAAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTACCACACCCTAAAACTTTCCACGTGAACCGTTGTAAATATGTTCTGTGCTCTCTCTCGGGAGAGCTGAACGAAGGTGGCCTGCTTAATTGTAGACTGCCGATGTACTTTTAAACCCATTAAACTAATACTGAACTATACTCCGAAAACGAAAGTCTTTGGTTTTAATCAATAACAACTTTCAGCAGTGGATGTCTAGGCTCGCACATCGATGAAGAACGCTGCGAACTGCGATACGTAATGCGAATTGCAGAATTCAGTGAGTCATCGAAATTTTGAACGCACATTGCACTTTCGGGTTATGCCTGGAAGTATGCCTGTATCAGTGTCCGTACATCAAACTTGCCTTTCTTTTTTTGTGTAGTCAAGATTAGAAACGGCAGACTGTGAGGTGTCTCGCTGACTCCCTCTTCGGAGGAGAAGACGCGAGTCCCTTTAAATGTACGTTCGCTCTTTCTTGTGTTTAAGTAGAAGTGTGACTTTCGAACGCAGTGATCTGTTTGGATCGCTTTGCTCGAGTAGGCGACTTCGGTTAGGACATTAAAGGAAGCAACCCTATTGGCGGTATGTTAGGCTTCGGCCCGACTTTGCAGCTGACGGTGTGTTGTTTTCTGTTCTTTCCTTGAGGTGTACCTGTCTTGTGTGAGGCAATGGTCTaGGCAAATGGTTATTGTGTAGTAGGAAGTTGCTGCTCTTGAACGCCCTGTttTCGGATAGGGTAAAGGAGGCAACACCAATTTGGGATAGTCTTTGATTTATCATTGGCGCTCTTTCTAATTGGACCTGATATCAGGCAAGACTACCCGCTGAACTTAAGCATATTAATAAGCGGAGGAAAAGAAACTAACAAGGATTCCCCTAGTAACGGCGAGTGAAGCGGGATGAGCTCAAGCTTAAAATCTCTGTGCCAGTTTGGCATGGCGAATTGTAGTCTATGGAGGCGCTATCAGTGCGATTGTTCGGGG.Vibrio parahaemolyticus, considered a bacterial commensal of harbour porpoise\u2019s skin flora. Bacteriological cultures (of samples taken during autopsy on day 105) were unremarkable, with no growth obtained after 14 days of culture in all sampled sites, except for a few Pseudomonas sp. from the tracheobronchial lymph nodes and also from skin, along with a few Acinetobacter sp.Bacteriological PCR analysis of the affected skin samples (taken on day 34) were positive for Virological PCR analysis on samples from brain, lung, kidney and urinary bladder collected during autopsy on day 105 tested negative for the presence of morbilliviral RNA.Pythium insidiosum as the causative pathogen [P. flevoense infection as the cause of dermatitis in a harbour porpoise. To our knowledge, it is the first time both for the diagnosis of pythiosis in a marine mammal and for P. flevoense as a cause of disease in a mammalian species. P. flevoense infections were reported previously in non-mammalian species only; ayu fish larvae [Pythium species as the causative pathogen was indicated by histopathological evaluation and confirmed by DNA sequencing that identified this particular, pathogenic species. Histological examination, especially in silver stains, revealed intralesional presence of fungus-like hyphae of approximately 7\u201310\u00a0\u03bcm diameter with rare septations and irregular branching were detected increasingly at warmer freshwater temperatures\u2009>20\u00a0\u00b0C in Argentina [Pythium species have been isolated from seaweeds and algae in temperate coastal saltwater environments [P. flevoense infection was reported in ayu fish larvae reared in artificial saltwater conditions at 12\u201315\u2009\u00b0C in a fishery in Japan [Another unusual aspect of this case was the temperate saltwater environment, which is in contrast with the subtropical stagnant freshwater conditions, such as swamps and rice fields, where infections with ntil now , 29, 30.rgentina . Nonetheronments , 32. Morin Japan . Even soP. flevoense in the water. Skin wounds in animals and humans are reported to serve as potential port d\u2019entr\u00e9e for the infectious motile zoospores [P. flevoense was isolated from soil samples previously, another possible explanation is that the seawater in the bay and basin may have been contaminated via freshwater runoff from land. However, another harbour porpoise that was kept in a separated adjacent basin circulated with the same seawater remained healthy without any signs of dermatitis or skin lesions.As pythiosis is not known to be contagious between mammals, infection in our case most likely occurred at sea or in the marine centre, presumably from infectious zoospores of oospores . In our oospores , 34. Furoospores , 32. TheImmunosuppression and hemopathies are risk factors for contracting pythiosis. For example, there are scientific reports regarding pythiosis with haemoglobinopathies, anaemia and leukaemia as risk factors for infection in humans \u201312, and Phoca vitulina) and several species of cetaceans [Rhizopus sp. [Cryptococcus gatti [Candida albicans [Initial clinical examination and diagnostics implicated a fungal organism as a cause of disease in this case, although a specific identification at that time was not made. Marine mammals are known to be susceptible to fungal diseases. In particular, respiratory aspergillosis has been reported in harbour porpoises , harbouretaceans , 39. Raropus sp. , Cryptocus gatti and Candalbicans as causaDespite various antifungal and antibiotic treatments in this case, the clinical course subsequently developed into a progressive severe dermatitis. Retrospectively, such a clinical course, with difficulties in initial diagnosis and resistance to antifungal therapy, is typical for pythiosis, which is not a true fungal disease , 43, 44.P. flevoense as cause of this dermatitis was evidenced by its intralesional presence in combination with histological absence and microbiological exclusion of other potential pathogens, such as bacteria, true fungi, protozoa, and viruses. Also, the possibility of this particular Pythium species being an opportunistic infection was considered and excluded because: (1) there was no evidence from haematology, histopathology, or (molecular) microbiology to suggest underlying immunosuppression, and (2) infection occurred in a non-pregnant initially healthy young female harbour porpoise in good physical condition without co-morbidities. Furthermore, our findings of this new case of pythiosis in a harbour porpoise are consistent with observations from previous investigators that the host range of pythiosis is increasing [Pythium species, we recommend including P. flevoense as a differential diagnosis for dermatitis in marine mammals, and more specifically, in harbour porpoises that have been entangled in fishing nets.In conclusion, the diagnosis of ion, and infectiocreasing , 5. So gAdditional file 1. Results of blood analyses for haematologic and biochemistry parameters from harbour porpoise Idun. Reference intervals from free-ranging harbour porpoises from Danish waters with a healthy clinical appearance according to Siebert et al. [th and 90th percentiles for each blood parameter with their 95% bootstrapped confidence intervals, respectively. NA:\u00a0not available.t et al. . The low"} +{"text": "Human cytomegalovirus (HCMV) infection can lead to either lytic or latent infection, which is dependent on the regulation of the viral major immediate early promoter (MIEP). Suppression of the MIEP is a pre-requisite for latency and is driven by repressive epigenetic modifications at the MIEP during latent infection. However, other viral genes are expressed during latency and this is correlated with activatory epigenetic modifications at latent gene promoters. Yet the molecular basis of the differential regulation of latent and lytic gene expression by epigenetics is unclear. LUNA, a latent viral transcript, has been suggested to be important for HCMV latency and has also been shown to be important for efficient reactivation likely through its known deSUMOylase activity. Intriguingly, we and others have also observed that LUNA enhances latency-associated expression of the viral UL138 gene. Here, we show that in the absence of LUNA, the expression of multiple latency-associated transcripts is reduced during latent infection, which is correlated with a lack of activatory marks at their promoters. Interestingly, we also show that LUNA interacts with the hematopoietic transcription factor GATA-2, which has previously been shown to bind to a number of latency-associated gene promoters, and that this interaction is dependent on the deSUMOylase domain of LUNA. Finally, we show that the deSUMOylase activity of LUNA is required for the establishment and/or maintenance of an open chromatin configuration around latency-associated gene promoters. As such, LUNA plays a key role in efficient latency-associated viral gene expression and carriage of viral genome during latent carriage. Between 60 and 99% of global populations carry HCMV, depending on demographics; in part, this prevalence is made possible by the ability of the virus to establish lifelong persistence in the infected host. After primary lytic infection, HCMV can establish a quiescent or latent infection, which helps it avoid immune clearance . The lytDuring latent infection of undifferentiated myeloid cells, the MIEP is known to be targeted for repression to suppress the expression of key lytic IE genes by an overall balance of MIEP repressors ,7,8. HowAnother viral gene that has been shown to be expressed during latency is LUNA. It was one of the first genes identified to be transcribed during natural latency and has In this manuscript, we further investigate this transcriptional phenotype and demonstrate that LUNA is important for the expression of multiple latency-associated transcripts, which correlates with increased levels of activatory epigenetic marks at latent promoters. In silico analyses reveal that multiple latent promoters encode binding sites for the hematopoietic transcription factor GATA-2, and interestingly, we show that LUNA interacts with transfected GATA-2 by co-immunoprecipitation (Co-IP). Finally, we link GATA-2 binding and the formation of an open chromatin conformation at latent viral promoters with the deSUMOylase motif of the LUNA protein. Together, these data argue that LUNA contributes to the regulation of expression of latency-associated HCMV gene expression in myeloid cells through an interaction with an important hematopoietic transcription factor, GATA-2.2 at 37 degrees. CD14+ cells were isolated from venous blood as described previously [THP1 cells were cultured in RPMI-1640 (Gibco) supplemented with 10% FCS and 5% penicillin/streptomycin in 5% COeviously ,26. TB40TB40E-SV40-GFP BAC was used to generate the LUNA mutant, LUNA knockout, and revertant viruses. For this, gBLOCKs from IDT were utilised with primers for recombineering using the GalK/2-DOG selection method as described previously , using tTACCGCTTCGACGTCTTTGTCCGGTCAGGATCAGTGCCCGGGACAGTCCGCCTGTTGACAATTAATCATCGGCA 3\u2032 GalK insert LUNA (primer) GGTCTCTTTCCACGGAGCAACGTCATGCGCGGCGCCGTCTCCGAGTTTCTTCAGCACTGTCCTGCTCCTT LUNA G81A stop (gBLOCK) TACCGCTTCGACGTCTTTGTCCGGTCAGGATCAGTGCCCGGGACAGTCCGGCTTGAGTGTCCGAGTCCTCGTCGCCGCTGGCCTCCTCGAAGCCGGCAAACATGGCTTCGGACAGGGGGGTCGGCGTCGGTGTGGATGAGAGGTCATCTTCGTCGTCCTCTTCCTCTTCTTCCTCCTCTTCCTCGGTGGGTGGTAATCCGGGGGACTGCGGGAGAAACTCGGAGACGGCGCCGCGCATGACGTTGCTCCGTGGAAAGAGACC LUNA G233C (gBLOCK)TACCGCTTCGACGTCTTTGTCCGGTCAGGATCAGTGCCCGGGACAGTCCGGCTTGGGTGTCCGAGTCCTCGTCGCCGCTGGCCTCCTCGAAGCCGGCAAACATGGCTTCGGACAGGGGGGTCGGCGTCGGTGTGGATGAGAGGTCATCTTCGTCGTCCTCTTCCTCTTCTTCCTCCTCTTCCTCGGTGGGTGGTAATCCGGGGGACTCCGGGAGAAACTCGGAGACGGCGCCGCGCATGACGTTGCTCCGTGGAAAGAGACC.LUNA C234G (gBLOCK) TACCGCTTCGACGTCTTTGTCCGGTCAGGATCAGTGCCCGGGACAGTCCGGCTTGGGTGTCCGAGTCCTCGTCGCCGCTGGCCTCCTCGAAGCCGGCAAACATGGCTTCGGACAGGGGGGTCGGCGTCGGTGTGGATGAGAGGTCATCTTCGTCGTCCTCTTCCTCTTCTTCCTCCTCTTCCTCGGTGGGTGGTAATCCGGGGGACTGGGGGAGAAACTCGGAGACGGCGCCGCGCATGACGTTGCTCCGTGGAAAGAGACC.5\u2032 LUNA seq (60.4) (5\u2032 primer for sequencing the mutants) GCG TGT TGC ACG CTC ACC 3\u2032 LUNA seq (59.8) (3\u2032 primer for sequencing the mutants) CCG CCG TGG GTT TTG GAC 5\u2032 amp LUNA gB (55) TACCGCTTCGACGTCTTTG.3\u2032 amp LUNA gB (55.5) GGTCTCTTTCCACGGAGC.\u00ae Green RT-qPCR kit according to the manufacturer\u2019s instructions and the samples were amplified and detected using an ABI 7500 Fast Real-Time PCR machine (95 \u00b0C for 15 s and 60 \u00b0C for 45 s), as described previously [Chromatin immunoprecipitations were carried out as described previously . After sImmunoprecipitation analysis was carried out as described previously using anIsopeptidase inhibitor, G5 (Sigma), was used as described previously . First, Quantification of viral and cellular mRNAs was carried out by SYBR green detection using primers described previously , except Genome copy number was determined using droplet digital PCR as described previously ,30. BrieTo investigate directly whether LUNA is important for latency-associated viral gene expression, we analysed the levels of UL138 and vIL-10 RNA during latent infection in the absence of LUNA. Recombinant viruses were generated in which a premature stop codon was engineered at the start of LUNA, resulting in viruses unable to express LUNA protein but which did not disrupt the UL82 gene on the complementary DNA strand (TB40E-LUNAmut), which was then controlled for through production of a revertant virus with LUNA repaired (TB40E-LUNArev). It is worth pointing out that this equivalent mutation of LUNA in the context of the Merlin clinical isolate of HCMV had little impact on the growth of Merlin in fibroblasts and the expression of viral gene products, including UL82 . The datTo further understand the mechanisms important for these differences in levels of UL138 and vIL-10 gene expression in the presence or absence of LUNA, we analysed the chromatin structure around the promoters of these latency-associated genes . ConsistWe previously identified that the hematopoietic transcription factor GATA-2, which plays an important role in host gene expression in myeloid cells, is also important for the expression of both LUNA and UL144 mRNAs during latency by GATA-2 binding to their promoters . IntriguThere is no evidence to suggest that LUNA can function as a general transcription factor; thus, we hypothesised that LUNA could regulate latent gene expression via interaction with GATA-2. To investigate whether LUNA might enhance expression from GATA-2-bearing promoters by a direct interaction between LUNA and GATA-2, we carried out interaction assays using co-immunoprecipitation in cells overexpressing GATA-2 and LUNA, which showed a clear interaction between GATA-2 and LUNA . FurtherWe observed that LUNA expression resulted in the formation of active chromatin on viral latency-associated promoters, that all these promoters contained GATA-2 transcription factor binding sites, and that LUNA interacted with GATA-2 in a deSUMOylase-dependent manner. Thus, we hypothesised that the chromatin signature observed with a LUNA deletion virus should be phenocopied with a LUNA deSUMOylase mutant. To test this, we generated a LUNA mutant virus in which we introduced a point mutation in the deSUMOylase domain of LUNA, rendering LUNA devoid of deSUMOylase activity (TB40E-LUNApoint), and tested whether the loss of the deSUMOylase activity from LUNA affected the transcription of viral genes. An analysis of infected cells shows that the expression of both vIL-10 and UL138 is reduced in cells latently infected with the TB40E LUNApoint compared to the control A,B. NextFinally, we wished to determine whether these LUNA-mediated changes could have any impact on the biology of latency. It is well established that HCMV infection extends the lifespan of classically short-lived CD14+ monocytes, which could be important for virus dissemination in vivo. Furthermore, we have observed that the expression of vIL-10 enhances the survival of infected CD34+ cells in long-term culture. Thus, we hypothesised that the defect in latency-associated gene expression observed in the absence of LUNA could have an impact on monocyte survival. To this end, CD14+ cells were infected with wild-type or a LUNA deletion virus and cultured for 10 days, and monocyte viability was assessed. The data show that viral infection improves cell survival compared to mock cells A, consisPrevious studies have demonstrated a clear role for the latency-associated gene product, LUNA, in latent carriage and reactivation from latency ,22. HereWhilst the absence of LUNA does not prevent the establishment of latency in CD14+ monocytes, we show here that it significantly enhances the expression of latency-associated genes, which we hypothesise is at least in part via the myeloid transcription factor GATA-2; this included the expression of vIL-10, which was significantly reduced in the absence of LUNA.We have previously shown that vIL-10 drives the expression of cellular IL-10 in monocytes ,31, and It is important to highlight that vIL-10 has pleiotropic roles in HCMV infection, with key roles in immune regulation. Thus, the enhancement of vIL-10 production by LUNA during latency could also be important for immune evasion during latency in vivo. For example, vIL-10 directly modulates the immune response and the Our previous work has shown that LUNA is a functional deSUMOylase , and thiPML is generally repressive for viral IE gene expression, which would be advantageous during latency to help repress lytic gene expression. However, it is at present unclear how the virus maintains a repressive chromatin structure around the MIEP whilst at the same time allowing expression of latency-associated genes. The dispersal of repressive PML bodies during latency would be predicted to be disadvantageous for maintaining the general repressive chromatinization of the viral genome observed during HCMV latency; PML disruption is known to lead to activation of HCMV IE gene expression and gene expression of other viruses ,41. For In contrast, reactivation is likely regulated by the increase in differentiation-dependent transcriptional activators of the MIEP, coupled with a decrease in MIEP repressive factors, which can be enhanced by inflammatory signals that then allow viral IE gene expression and progTaken together, we believe that the deSUMOylation function of LUNA not only ensures that reactivation from latency is primed to go, by removing repressive PML bodies in latently infected cells, but is also crucial for latent carriage by enhancing the transcription of latency-associated genes."} +{"text": "Nature Communications 10.1038/s41467-022-32395-w, published online 13 August 2022Correction to: The original version of this Article contained an error in Table 1. The correct version of the 3rd row (Circle part 2) of the 3rd column (Sequence (5\u2019 -> 3\u2019)) states \u2018CGAGGTGCTTTTAGCACCTCGAAGTAAAGCTATCCACTGTCACCAACTACTAGATAAACGTCACACTTTTCGTGTGACG\u2019instead of the original, incorrect \u2018CGAGGTGCTTTTAGCACCTCGAAGTAAAGCTATCCACTGTCACCAACTACTAGATAAACGTCACACTTTTCGTGTGAC\u2019\u00a0where the final \u2018G\u2019 was omitted.This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Plant probiotics bacteria are live microbes that promote soil health and plant growth and build the stress-tolerant capacity to the plants. They benefit the plants by increasing nutrient absorption and release of stress-related phytohormones. These plant probiotic bacteria serve a better purpose to the plant when compared to chemical fertilizers. Use of chemical fertilizers such as arsenic and cadmium can lead to soil acidification and even release of harmful gases such as methane which further pollutes the environment.Corynebacterium spp., Bacillus spp., Lactobacillus spp., and Cytobacillus spp. The results were also examined using various bioinformatics tools for accuracy in their phylogenetic pattern.Different bacterial species were isolated from the agricultural fields of Tattiannaram, Telangana, and identified as the efficient rhizosphere bacteria with the essential qualities of plant growth promotion by evaluating the nitrogen-fixing ability on a selective media and various other methods. Upon the molecular characterization of the isolates, they were identified as The recognized species of plant probiotics have established roles in promoting plant growth and strengthening plant immunity. This research introduces an innovative methodology for evaluating and investigating recently identified bacterial isolates, focusing on their distinctive plant probiotic attributes. Through harnessing the potential of advantageous microorganisms and comprehending their interaction with plants and soil, our objective is to formulate inventive approaches to elevate crop productivity, enhance soil richness, and foster environmentally sustainable and robust agricultural methodologies. These characteristics exhibit promising potential for future incorporation into plant systems, fortifying growth and development, and underscoring their distinctive significance within the realm of agriculture.The online version contains supplementary material available at 10.1186/s43141-023-00615-5. Soil, the organic geological skin of the earth, is essential for life and habitat to an incredible microbial community. Microbes have mostly been regarded as stewards of the biosphere . Soil baBacilli and Pseudomonas being the most common genera reported [Plants interact with a range of microbes in both aerial and subsoil tissues, establishing associations that can be harmful (pathogenic), neutral, or advantageous to the host plant. Several studies have shown evidence of the ability of microbes to boost plant development in various cultivated species. One such criterion is the development of plant growth-promoting bacteria. Plant growth-promoting bacteria (PGPB) are one of the most generalized findings of plant probiotic microbes (PPM) in soil, having reported \u201317. Planreported . Upon coreported . Severalreported .Fusarium oxysporum is treated by Bacillus subtilisssp [Probiotics are living microbial species utilized to improve the health and vitality of the host. Microorganisms employed for probiotic purposes can be found in nature, but they must be correctly identified, isolated, and studied for virulence . Plant ptilisssp .. Plant tilisssp , 27.Haas and Keel coined the term plant probiotic bacteria (PPB) to describe a group of microorganisms that benefit plants and meet three necessary attributes that when combined result in improved plant protection: (i) niche colonization effectiveness and competitiveness, (ii) ability to induce systemic resistance (ISR) in their hosts, and (iii) existence of specific antagonistic traits on pathogens .Agriculture and forestry are constantly being impacted by increasing population, soil degradation, environmental degradation, and global warming . This haIn long-term agricultural production, nitrogen fertilizers are frequently used to boost early crop output. Long-term administration of nitrogen fertilizers, on the other hand, can alter the plant soil microbe system by altering the composition of vegetation and soil microbial populations \u201336. NitrThe entire methodology followed during the work is represented in Fig.\u00a0Soil samples were gathered from the Tattiannaram fields in Telangana, India Fig.\u00a0. TattianThe rhizosphere is considered an important plant nutrient source where most of the microbes make up this as a resident club. The first step in developing a producer strain is the isolation of concerned microorganisms from their natural habitats. A set of highly selective procedures which allows the detection and isolation of microbes producing the desired metabolite constitute primary screening. However, this is possibly the most critical step since it eliminates the bulk of unwanted useless isolates, either nonproducers or producers of known compounds. As a part of the primary screening technique, the isolation of the bacteria was done using basic lab equipment and certain media. To analyze for the various microbes within the collected soils, the basic medium such as nutrient agar was used, believing that majority of microbes can be screened. Each soil sample was serially diluted and plated as per the standard protocols of microbial isolation techniques. After the growth observed on basic media, the isolates with different morphological features were subcultured and further tested for biochemical properties. The biochemical properties were analyzed by performing IMViC tests, catalase, coagulase, oxidase, starch hydrolysis, organic acid production tests and blood haemolysis test, etc. In detail, the IMViC test helps to narrow down the group of bacteria and provide information about their metabolic capabilities. Plant probiotic bacteria often produce organic acids through their metabolic processes. These organic acids can help in solubilizing minerals, making them more available to plants. The ability to produce organic acids like citric acid and malic acid can be a desirable trait in plant probiotic bacteria. The purpose of performing various biochemical tests was tabulated in Table 2PO4 0.1%, MgSO4.7H2O 0.05%, NaCl 0.05%, FeSO4 0.01%, Na2Mo 0.0005%, CaCO3 0.2%, agar 1.5%). First, the isolates were inoculated on solid medium. Later to estimate their growth levels, the isolates were re-examined on liquid medium. This growth was analyzed for 24\u00a0h and 48\u00a0h by colorimetric analysis at 600\u00a0nm. Bacteria that thrive on Jensen\u2019s media might have the ability to fix nitrogen, making them potentially beneficial for plants. The organisms that have shown positive with Jensen\u2019s media were further examined if they can also utilize and tolerate to different chemical exposures such as zinc, phosphorus, and iron using modified zinc solubilizing medium , modified phosphate-solubilizing medium 2 0.02%, MgSO4.7H2O 0.010%, FeSO4 0.00001%, pH 7), and modified iron-solubilizing media , respectively. The growth rate was analyzed by measuring the OD which is set to 540\u00a0nm (nanometers) and 580\u00a0nm using a colorimeter. Plants often face challenges in accessing essential nutrients like zinc, iron, and phosphorus due to their limited solubility in the soil. Bacteria capable of solubilizing these minerals can enhance nutrient availability for plants. The organisms which have shown good growth rate and minimum standard deviation upon all the triplicate values were selected as the best isolates and moved for further examination. The selected isolates were inoculated to Ashby\u2019s liquid medium to confirm the nitrogen-fixing property [Later, the studies were carried out to screen out the nitrogen-fixing property using certain specific media such as Jensen\u2019s medium \u201d), inputting the 16S rRNA sequence into the query box, adjusting parameters, and subsequently initiating the BLAST search . SubsequLater, the bootstrapping technique was employed as it is crucial for making well-informed evolutionary interpretations, as it addresses the inherent noise in genetic data and the complexities of evolutionary processes . Using tUpon screening the isolates by considering the basic traits, a total of 101 isolates were found initially. Colony morphological testing, gram staining, and biochemical tests were performed. The organisms with similar traits were re-examined and eliminated. For the second stage of screening, 36 isolates were selected. The selected isolates were highlighted in Table Later, these 36 isolates were subjected to test their nitrogen-fixing capacity. Jensen\u2019s media was used to detect the presence of nitrogen-fixing bacteria. This media was prepared in broth consistency and inoculated with all the selected 36 isolates. All the isolates were grown as triplicates, and the average of all the readings was tabulated as the results in Table The seven best isolates which proved their ability of nitrogen fixation was further moved to examination such as zinc solubilizers, phosphate solubilizers, and zinc solubilizers. The result was tabulated in Table The isolates that shown best within chemical analysis tests were further examined using Ashby\u2019s nitrogen liquid medium in order to compare their main purpose to be a plant probiotic. Surprisingly, the isolate nos. KL-015, KL-076, and KL-089 have shown positive results indicating their potentiality for nitrogen fixation. Along with these isolates, remaining screened out isolates were subjected to the exposure to liquid medium of Ashby\u2019s. The isolate which had shown growth in liquid medium technique. Based on the gel amplicon picture, the isolates have shown the bands at app. 1000\u00a0kb Fig.\u00a0. The posCTGCACTTCGGGATAAGCTTGGGAAACTGGGTCTAATACCGGATAGGAACCATCTTTAGTGTGATGGTTGGAAAGTTTTTTCGGTGTAGGATGAGCTCGCGGCCTATCAGCTTGTTGGTGGGGTAATGGCCTACCAAGGCGGCGACGGGTAGCCGGCCTGAGAGGGTGTACGGCCACATTGGGACTGAGATACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCGACGCCGCGTGGGGGATGAAGGCCTTCGGGTTGTAAACTCCTTTCGCTAGGGACGAAGCTTTTTGTGACGGTACCTAGATAAGAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTGTCCGGAATTACTGGGCGTAAAGGGCTCGTAGGTGGTTTGTCGCGTCGTCTGTGAAATTCTGGGGCTTAACTCCGGGCGTGCAGGCGATACGGGCATAACTTGAGTGCTGTAGGGGTAACTGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGATGGCGAAGGCAGGTTACTGGGCAGTTACTGACGCTGAGGAGCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGGTGGGCGCTAGGTGTGAGGGTCTTTCTACGACTTTCGTGCCGTAGCTAACGCATTAAGCGCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGG.AGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTAGGAGCCAGCCGCCGAAGGTGGGACAGATGATTGGGGTGAAGTCGTAACAAGGTAGCCGTATC.ACCCGCGGTGCATTAGCTAGTTGGTAGGGTAAAGGCCTACCAAGGCATTGATGCATAGCCGAGTTGAGAGACTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTGGTGAAGAAAGATAGAGAGAGTAACTGATCTTTATTTGACGGTAATCAACCAGAAAGTCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGAAAGATAAGTCTGATGTGAAAGCCCTCGGCTCAACCGAGGAACTGCATCGGAAACTGTCTTTCTTGAGTGCAGAAGAGGAGAGTGGAACTCCATGTGTAGCGGTGGAATGCGTAGATATATGGAAGAACACCAGTGGCGAAAGCGGCTCTCTGGTCTGCAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAGTGCTAAGTGTTGGGAGGTTTCCGCCTCTCAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCTAGCGCAATCCCAAGAGATTGGGAGTTCCCTTCGGGGACGCTAAGACAGG.GGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAACTCTGTTGTCAGGGAAGAACAAGTACCGGAGTAACTGCCGGTACCTTGACGGTACCTGACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGTTCCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGAAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTTTGGTCTGTAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGCAGCAAACGCATTAAGCACTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCTCCTGACAACCCTAGAGATAGGGCGTTCCCCTTCGGGGGACAGGATGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAAAGGGCTGCAA.The above sequences were used to run the BLAST separately, and later on, the top 10 sequences were used to construct the cladogram using Clustal Omega tool as shown in Fig.\u00a0To select a bacteria as a potent plant probiotic, it must be beneficiary to humans also as the end result of this concept is growth of a healthy plant. As we know, the greatest ecological and agronomical advantages derived by plants from their contact with microbes are biological nitrogen fixation by bacteria. It is believed that the bacterial isolates which were finally identified within this work are the one which can convert the atmospheric nitrogen to ammonia using its nitrogenase enzyme, thus making it available for plant absorption. The presence of the nitrogenase enzyme decides the plant\u2019s maturity and adds economic value too. The bacteria may also be capable to utilize various chemicals such as zinc which stimulates enzymes that are involved in the creation of some proteins. It aids in the production of chlorophyll and certain carbohydrates, the transformation of starches to sugars, and its availability in plant tissue aids in the plant\u2019s resistance to low temperatures . Zinc is\u201cPlant probiotics\u201d is an emerging and intriguing concept within the realm of agriculture and plant science. This concept revolves around the idea of using beneficial microorganisms to enhance plant health and growth. This innovative approach delves into the analysis of plant immunity as well, aiming to better understand how these microorganisms can interact with plants\u2019 natural defense mechanisms. Numerous studies have drawn attention to the significant role that plant probiotics can play. These studies have identified specific microorganism isolates that have demonstrated particularly promising results. These promising isolates are then subjected to a thorough screening process to assess their potential. This assessment involves employing techniques such as biopriming, which entails treating seeds with these beneficial microorganisms to facilitate their growth and establishment in the plant\u2019s environment. Moreover, we are simultaneously working on optimizing the growth conditions and parameters for these beneficial microorganisms. This involves creating an environment that allows these microorganisms to thrive and interact harmoniously with the plant. The ultimate goal is to develop a technique that can reliably enhance plant health and performance. One aspect of research in this area focuses on investigating the specific compositions of microbial communities in the soil. Different combinations of microorganisms can have varying effects on soil fertility and subsequently impact the development of plants that grow in that soil. Through careful analysis, researchers seek to uncover the intricate relationships between these microorganisms and the overall health of both the soil and the plants. Early findings suggest that these microbial communities possess an adaptive capability, allowing them to adjust and thrive under different chemical conditions present in the soil. This adaptability opens up avenues to cultivate these microorganisms into potent plant probiotics that can potentially withstand a range of environmental challenges. In summary, the emerging field of plant probiotics holds immense promise for revolutionizing agriculture and plant cultivation.Additional file 1. BLASTN Results."} +{"text": "Myosin-X (MYO10), a molecular motor localizing to filopodia, is thought to transport various cargo to filopodia tips, modulating filopodia function. However, only a few MYO10 cargoes have been described. Here, using GFP-Trap and BioID approaches combined with mass spectrometry, we identified lamellipodin (RAPH1) as a novel MYO10 cargo. We report that the FERM domain of MYO10 is required for RAPH1 localization and accumulation at filopodia tips. Previous studies have mapped the RAPH1 interaction domain for adhesome components to its talin-binding and Ras-association domains. Surprisingly, we find that the RAPH1 MYO10-binding site is not within these domains. Instead, it comprises a conserved helix located just after the RAPH1 pleckstrin homology domain with previously unknown functions. Functionally, RAPH1 supports MYO10 filopodia formation and stability but is not required to activate integrins at filopodia tips. Taken together, our data indicate a feed-forward mechanism whereby MYO10 filopodia are positively regulated by MYO10-mediated transport of RAPH1 to the filopodium tip. Summary: Myosin-X transports lamellipodin (RAPH1) to filopodia tips to regulate filopodia functions. Cell migration is essential during embryonic development, immune surveillance and wound healing. Misregulation of cell migration is implicated in multiple diseases, including inflammation and cancer. One hallmark of cell motility is a high degree of plasticity, allowing cells to adopt different morphologies and migration modes . A shareFilopodia are small and dynamic finger-like actin-rich protrusions (1\u20135\u2005\u00b5m in length and 50\u2013200\u2005nm in width) and are often the first point of contact between a cell and its immediate surroundings. Filopodia contain cell surface receptors, such as integrins, cadherins and growth factor receptors, interacting with and interpreting various extracellular cues. Filopodia assembly is primarily driven by the linear polymerization of actin filaments with their barbed ends facing the plasma membrane . These fHere, we set out to identify novel MYO10 cargo molecules. Using GFP-Trap and BioID approaches combined with mass spectrometry, we identified lamellipodin (RAPH1) as a novel MYO10-binding partner. Using structured illumination microscopy, we report that the FERM domain of MYO10 is required for RAPH1 localization and accumulation at filopodia tips; thus, RAPH1 is likely an MYO10 cargo. We map the RAPH1 MYO10-binding site to a previously uninvestigated RAPH1 sequence, and demonstrate that RAPH1 is a critical positive regulator of filopodia formation and stability in cells. Our results indicate that, in filopodia, RAPH1 is not required for integrin activation. Instead, RAPH1 regulates MYO10 filopodia formation and stability.FERM, or GFP\u2013TLN1FERM , followed by mass spectrometry analysis , the main cargo-binding site in MYO10 . We perfanalysis A,B. TLN1analysis . We idenanalysis . Interesanalysis .FERM and that RAPH1 is biotinylated in cells expressing GFP\u2013MYO10\u2013BioID .RAPH1 is a member of the Mig-10/RIAM/lamellipodin (MRL) protein family, with MIG-10 being the of RAPH1 . RAPH1 wof RAPH1 , but itsof RAPH1 . In addiof RAPH1 ; Movie\u00a01\u0394F) in cells within RAPH1. RAPH1 comprises several conserved domains, including a Ras-association (RA) and a pleckstrin homology (PH) domain. RAPH1 also contains known profilin-, VASP- and multiple putative SH3-binding sites A. Furthe\u0394F filopodia showed that this construct could accumulate at the tip of MYO10-containing filopodia MYO10 is required to target RAPH1 to filopodia tips, and (2) the MYO10\u2013RAPH1 interaction contributes to formation of filopodia containing MYO10. We propose that MYO10 transports RAPH1 to filopodia tips, contributing to filopodia stability via yet unknown mechanisms, possibly involving RAPH1 interactions with other proteins such as VASP. However, our data do not fully exclude the possibility that RAPH1 simply diffuses to filopodia and that MYO10 only contributes to RAPH1 accumulation at filopodia tips without direct transport. Testing this would require performing two-color single-molecule imaging of MYO10 and RAPH1 to see whether these proteins move toward filopodia tips together. However, we find that RAPH1 is not very abundant in filopodia when the MYO10 FERM domain is missing, suggesting that RAPH1 is likely to be actively transported by MYO10.RAPH1 is presumably in a complex with MYO10, VASP and actin at filopodia tips. In this scenario, MYO10 could tether RAPH1 to filopodia tips using its motor domain, providing resistance against the retrograde actin flow in filopodia . Once tein vivo.Interestingly, both MYO10 and RAPH1 have been implicated separately as positive regulators of cancer cell migration and invasion in similar contexts . In addi\u22121 final concentration), and sorted for green fluorescence using a fluorescence-assisted cell sorter (FACS). All cell lines tested negative for mycoplasma. Cells were not authenticated.U2-OS osteosarcoma cells and MDA-MB-231 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FCS) . U2-OS cells were purchased from DSMZ . MDA-MB-231 cells were provided by ATCC. The U2-OS MYO10-GFP lines were generated by transfecting U2-OS cells using Lipofectamine 3000 (Thermo Fisher Scientific), selected using geneticin , 1:1000], anti-His tag , and anti-tubulin . Rabbit polyclonal antibodies used in this study were anti-RAPH1 , anti-MYO10 , and anti-GFP . Biotinylated proteins were detected using Streptavidin conjugated with Alexa Fluor\u2122 555 or Alexa Fluor\u2122 647 , both provided by Thermo Fisher Scientific (S21381 and S21374). The bovine plasma fibronectin was provided by Merck (341631). DAPI was provided by Thermo Fisher Scientific (D1306).U2-OS and MDA-MB-231 cells were transfected using Lipofectamine 3000 and the P3000TM Enhancer Reagent (Thermo Fisher Scientific) according to the manufacturer's instructions.47608 . The mScarlet-I-MYO10\u0394F (MYO10\u0394F-RFP) construct was previously described (145139). The GFP-VASP plasmid was Addgene plasmid #54297 (deposited by Michael Davidson). The GFP-RIAM(1-666) construct was Addgene plasmid #80028 plasmid was Addgene plasmid #escribed and is aThe GFP\u2013MYO10\u2013BioID construct was generated as follows. Flanking XbaI sites were introduced into BioID by PCR and the resulting amplicon was then inserted into a unique XbaI site in the EGFPC1-hMyoX plasmid resulting in an EGFP-MYO10-(stop codon)-BioID fusion gene. The stop codon between MYO10 and BioID was then replaced with a codon encoding valine (GTA) using a quick-change mutagenesis kit from Agilent and following the manufacturer's instructions.\u0394TBS [RAPH1 with amino acids (aa) 2\u201392 deleted] construct was created by inserting a custom gene block (IDT) in the EGFP-Lpd plasmid using the XhoI/HindIII sites. The sequence of the gene block is provided below:The GFP\u2013RAPH15\u2032-ATTAGACTCGAGCCGCGATGTGCTCTATAGAGCAGGAGCTCAGCAGCATTGGTTCAGGAAACAGTAAGCGTCAAATCACAGAAACGAAAGCTACTCAGAAATTGCCTGTTAGCCGACATACATTGAAACATGGCACCTTGAAAGGATTATCTTCTTCATCTAATAGGATAGCTAAACCTTCCCATGCCAGCTACTCCTTGGACGACGTCACTGCACAGTTAGAACAGGCCTCTTTGAGTATGGATGAGGCTGCTCAGCAATCTGTACTAGAAGATACTAAACCCTTAGTAACTAATCAGCACAGAAGAACCGCGTCAGCAGGCACAGTGAGTGATGCTGAAGTACACTCTATTAGTAATTCCTCCCATTCCAGCATCACTTCCGCAGCCTCCAGCATGGACTCTTTGGATATTGATAAAGTAACACGCCCTCAAGAGCTGGATTTGACACATCAAGGGCAGCCAATTACTGAGGAAGAACAGGCAGCAAAATTGAAAGCTGAGAAGATCAGAGTTGCCCTAGAGAAAATTAAAGAGGCACAAGTGAAAAAGCTGGTGATCAGAGTCCACATGTCTGATGACAGTTCTAAAACAATGATGGTGGATGAGAGGCAGACAGTAAGACAAGTACTGGATAACCTGATGGACAAATCCCACTGCGGTTATAGTTTAGACTGGTCACTGGTAGAAACCGTTTCTGAATTACAAATGGAGAGAATCTTTGAAGACCATGAAAACTTGGTTGAAAATCTTCTTAATTGGACAAGAGATAGCCAAAACAAGCTTATTAGA-3\u2032.F2 construct (RAPH1 aa 535\u2013868) was purchased from GenScript. The gene fragment was synthesized using gene synthesis and cloned into pGEX-4T-1 using the BamHI/XhoI sites.The RAPH1 fragments F1 (RAPH1 aa 1\u2013535), F2 (RAPH1 aa 535\u2013868), F3 (RAPH1 aa 868\u20131062), F4 (RAPH1 aa 1062\u20131250) and F5 (RAPH1 aa 536\u2013587) constructs were purchased from GenScript. Briefly, the gene fragments were synthesized using gene synthesis and cloned into pcDNA3.1(+)-N-eGFP using the BamHI/XhoI sites. The GST\u2013RAPH1\u0394536-587 (RAPH1 aa 536-587 deleted) construct was created by inserting a custom gene block (IDT) in the EGFP-Lpd plasmid using the HindIII/KpnI sites. The sequence of the gene block is provided below:The GFP\u2013RAPH15\u2032-AAGCTTATATTTATGGAGCGTATAGAAAAATATGCACTTTTCAAAAACCCACAGAATTATCTTTTGGGGAAAAAGGAAACAGCTGAGATGGCAGATAGAAACAAAGAAGTCCTCTTGGAGGAATGTTTTTGTGGAAGTTCTGTAACTGTACCAGAAATTGAAGGAGTCCTTTGGTTGAAGGATGATGGCAAGAAGTCCTGGAAAAAGCGTTATTTTCTCTTGCGAGCATCTGGTATCTACTATGTTCCCAAAGGAAAAGCAAAGGTCTCTCGGGATCTGGTGTGCTTTCTCCAGCTGGATCATGTCAACGTTTATTATGGCCAGGACTATCGGAACAAATACAAAGCACCTACAGACTATTGTCTGGTGCTGAAGCATCCACAAATCCAGAAGAAATCTCAATATATCAAATACCTTTGTTGTGATGATGTGAGGACACTGCATCAGTGGGTCAATGGGATCCGCATTGCAAAGTATGGGAAGCAGCTCTATATGAACTACCAAGAAGCCTTGAAGAGGACAGAGTCAGCCTATGATTGGACTTCCTTATCCAGCTCCAGCATTAAATCGGAAGAGTCCAGCAAGGCCAGAATGGAGTCTATGAATCGGCCCTACACTTCACTTGTGCCCCCTTTATCCCCGCAACCTAAGATAGTCACCCCCTACACTGCTTCACAGCCTTCACCACCTCTACCTCCTCCGCCACCCCCACCTCCTCCTCCACCACCCCCTCCACCACCCCCTCCTCCCCCACTCCCCAGCCAGTCTGCACCTTCTGCAGGCTCAGCAGCCCCAATGTTCGTCAAGTACAGCACAATAACACGGCTACAGAATGCGTCTCAGCATTCAGGGGCCCTGTTTAAGCCGCCAACACCCCCAGTGATGCAGTCACAGTCAGTGAAGCCTCAGATCCTGGTACC-3\u2032.The expression of RAPH1 was suppressed using 83\u2005nM siRNA and Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufacturer's instructions. siRNAs used were AllStars Negative siRNA control (cat. no. 1027418), RAPH1 siRNA #2 and RAPH1 siRNA #5 provided by Qiagen.Protein extracts were separated under denaturing conditions by SDS-PAGE and transferred to nitrocellulose membranes using a Mini Blot Module . Membranes were blocked for 30\u2005min at room temperature using 1\u00d7 StartingBlock buffer . After blocking, membranes were incubated overnight with the appropriate primary antibody (1:1000 in blocking buffer), washed three times in PBS, and probed for 1\u2005h using a fluorophore-conjugated secondary antibody diluted 1:5000 in the blocking buffer. Membranes were washed three times using PBS over 30\u2005min and scanned using an iBright FL1500 imaging system (Invitrogen).g for 5\u2005min at 4\u00b0C. Clarified lysates were incubated with GFP-Trap magnetic or agarose beads for 1\u2005h at 4\u00b0C. Complexes bound to the beads were isolated by centrifugation, washed three times with ice-cold lysis buffer, and eluted in Laemmli reducing sample buffer for 10\u2005min at 95\u00b0C.Cells transiently expressing bait GFP-tagged proteins were lysed in a buffer containing 20\u2005mM HEPES pH 7.4, 75\u2005mM NaCl, 2\u2005mM EDTA, 1% NP-40, as well as a cOmplete\u2122 protease inhibitor tablet , and a phosphatase inhibitor mix (Roche cat. no. 04906837001). Lysates were then centrifuged at 15,000\u2005Escherichia coli strain was transformed with plasmids encoding the relevant His-tagged or GST-tagged proteins. Bacteria were grown at 37\u00b0C in LB medium supplemented with ampicillin . Protein expression was induced with IPTG at 20\u00b0C. After 5\u2005h, bacteria were harvested by centrifugation (20\u2005min at 6000\u2005g) and resuspended in resuspension buffer . Bacteria were then lysed by adding BugBuster and a small spoonful of lysozyme . The suspension was mixed at 4\u00b0C for 30\u2005min. Cell debris were then pelleted by ultracentrifugation at 4\u00b0C for 1\u2005h. His-tagged MYO10 FERM was purified using a Protino Ni-TED 2000 packed column according to the manufacturer's instructions. The protein was eluted in multiple 1\u2005ml fractions, supplemented with 1\u2005mM AEBSF , and kept at 4\u00b0C until needed (for up to 1 week). For GST-tagged proteins, 600\u2005\u00b5l of equilibrated glutathione\u2013Sepharose 4B beads was added to the supernatant and agitated for 1\u2005h at 4\u00b0C. Beads were collected and washed four times with TBS supplemented with PMSF (1\u2005mM). Protein-bound beads were stored at \u221280\u00b0C until needed.The BL-21(DE3) F2 Sepharose beads were incubated with 10\u2005mM His-tagged MYO10FERM, and the mixture was rotated overnight at 4\u00b0C. Beads were then washed four times with TBS supplemented with 1\u2005mM PMSF. Proteins bound to beads were then eluted in 2\u00d7 Laemmli sample buffer at 80\u00b0C. Results were then analyzed by western blotting.GST and GST\u2013RAPH1g, +4\u00b0C, 2\u2005min). Biotinylated proteins were then incubated with streptavidin beads for 1\u2005h with rotation at +4\u00b0C. Beads were washed twice with 500\u2005\u03bcl wash buffer 1 [10% (w/v) SDS], once with 500\u2005\u03bcl wash buffer 2 , and once with 500\u2005\u03bcl wash buffer 3 . Proteins were eluted in 40\u2005\u03bcl of 2\u00d7 reducing sample buffer for 10\u2005min at 90\u00b0C.U2-OS cells stably expressing GFP\u2013MYO10 or GFP\u2013MYO10\u2013BioID were plated on fibronectin-coated plates in a medium containing 50\u2005\u03bcM biotin for 24\u2005h. After washing cells with cold PBS, cells were lysed, and debris were removed by centrifugation , gel lanes were sliced into five 2-mm bands. The slices were washed using a solution of 50% 100\u2005mM ammonium bicarbonate and 50% acetonitrile until all blue color vanished. Gel slices were washed with 100% acetonitrile for 5\u201310\u2005min and then rehydrated in a reducing buffer containing 20\u2005mM dithiothreitol in 100\u2005mM ammonium bicarbonate for 30\u2005min at 56\u00b0C. Proteins in gel pieces were then alkylated by washing the slices with 100% acetonitrile for 5\u201310\u2005min and rehydrated using an alkylating buffer of 55\u2005mM iodoacetamide in 100\u2005mM ammonium bicarbonate solution . Finally, gel pieces were washed with 100% acetonitrile, followed by washes with 100\u2005\u03bcl 100\u2005mM ammonium bicarbonate, after which slices were dehydrated using 100% acetonitrile and fully dried using a vacuum centrifuge. Trypsin was used to digest the proteins (37\u00b0C overnight). After trypsinization, an equal amount of 100% acetonitrile was added, and gel pieces were further incubated at 37\u00b0C for 15\u2005min, followed by peptide extraction using a buffer of 50% acetonitrile and 5% formic acid. The buffer with peptides was collected, and the sample was dried using a vacuum centrifuge. Dried peptides were stored at \u221220\u00b0C. Before liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) analysis, dried peptides were dissolved in 0.1% formic acid. The LC-ESI-MS/MS analyses were performed on a nanoflow HPLC system coupled to the Orbitrap Fusion Lumos mass spectrometer equipped with a nano-ESI source. Peptides were first loaded on a trapping column and subsequently separated inline on a 15\u2005cm C18 column . The mobile phase consisted of water with 0.1% formic acid (solvent A) and acetonitrile/water [80:20 (v/v)] with 0.1% formic acid (solvent B). Peptides were eluted with 40\u2005min method: from 8% to 43% of solvent B in 30\u2005min, from 43% to 100% solvent B in 2\u2005min, followed by a wash for 8\u2005min at 100% of solvent B. MS data was acquired automatically by using Thermo Xcalibur 4.4 software (Thermo Fisher Scientific). A data-dependent acquisition method consisted of an Orbitrap MS survey scan of mass range 350\u20131750\u2005Raw data from the mass spectrometer were submitted to the Mascot search engine using Proteome Discoverer 1.5 (Thermo Fisher Scientific). The search was performed against the human database SwissProt_2021_02, assuming the digestion enzyme trypsin, a maximum of two missed cleavages, an initial mass tolerance of 10\u2005ppm (parts per million) for precursor ions, and a fragment ion mass tolerance of 0.020 Dalton. Cysteine carbamidomethylation was set as a fixed modification, and methionine oxidation was set as a variable modification.FERM and TLN1FERM datasets, two biological replicates were combined. Proteins enriched at least twofold in MYO10FERM over GFP and over TLN1FERM and detected with more than ten spectral counts (across both repeats) were considered putative MYO10-binding proteins. The fold-change enrichment and the significance of the association used to generate the volcano Plots and detected with over five spectral counts were considered putative MYO10-binding proteins. To generate the MYO10no Plots A,B were The spinning-disk confocal microscope used was a Marianas spinning-disk imaging system with a Yokogawa CSU-W1 scanning unit on an inverted Zeiss Axio Observer Z1 microscope controlled by SlideBook 6 . Images were acquired using either an Orca Flash 4 sCMOS camera or an Evolve 512 EMCCD camera . The objective used was a 100\u00d7 oil objective.The structured illumination microscope (SIM) used was DeltaVision OMX v4 fitted with a 60\u00d7 Plan-Apochromat objective lens, 1.42 NA (immersion oil RI of 1.516) used in SIM illumination mode (five phases\u00d7three rotations). Emitted light was collected on a front-illuminated pco.edge sCMOS controlled by SoftWorx.The confocal microscope used was a laser scanning confocal microscope LSM880 (Zeiss) equipped with an Airyscan detector (Carl Zeiss) and a 40\u00d7 water (NA 1.2) or 63\u00d7 oil (NA 1.4) objective. The microscope was controlled using Zen Black (2.3), and the Airyscan was used in standard super-resolution mode.For the filopodia formation assays, cells were plated on fibronectin-coated glass-bottom dishes (MatTek Corporation) for 2\u2005h. Samples were fixed for 10\u2005min using a solution of 4% PFA, then permeabilized using a solution of 0.25% (v/v) Triton X-100 for 3\u2005min. Cells were then washed with PBS and quenched using a solution of 1\u2005M glycine for 30\u2005min. Samples were then washed three times in PBS and stored in PBS containing SiR-actin at 4\u00b0C until imaging. Just before imaging, samples were washed three times in PBS. Images were acquired using a spinning-disk confocal microscope (100\u00d7 objective). The number of filopodia per cell was manually scored using Fiji .2. Filopodia lifetimes were then measured by identifying and tracking all MYO10 spots using the Fiji plugin TrackMate . All live-cell imaging experiments were performed in normal growth medium, supplemented with 50\u2005mM HEPES, at 37\u00b0C and in the presence of 5% COrackMate . In TracrackMate . The traU2-OS cells transiently expressing the constructs of interest were plated on high tolerance glass-bottom dishes pre-coated first with poly-L-lysine and then with bovine plasma fibronectin . After 2\u2005h, samples were fixed and permeabilized simultaneously using a solution of 4% (w/v) PFA and 0.25% (v/v) Triton X-100 for 10\u2005min. Cells were then washed with PBS, quenched using a solution of 1\u2005M glycine for 30\u2005min, and, when appropriate, incubated with the primary antibody for 1\u2005h (1:100). After three washes, cells were incubated with a secondary antibody for 1\u2005h (1:100). Samples were then washed three times and incubated with SiR-actin at 4\u00b0C until imaging . Just before imaging, samples were washed three times in PBS and mounted in Vectashield (Vector Laboratories).To map the localization of each protein within filopodia, images were first processed in Fiji , and datThe preferential recruitment of protein to filopodia tips or shafts was assessed by calculating an enrichment ratio where the averaged intensity of the signal at the filopodia tip (bin 1\u20136) was divided by the averaged intensity at the filopodia shaft (bin 7\u201340).https://huygens.science.uva.nl/PlotsOfDifferences/) (https://github.com/guijacquemet/FiloMAP).Randomization tests were performed using the online tool PlotsOfDifferences (rences/) preprintrences/) . Volcanorences/) . Superplrences/) . All numClick here for additional data file.10.1242/joces.260574_sup1Supplementary informationClick here for additional data file."} +{"text": "The treatment of chronic inflammation with systemically administered anti-inflammatory treatments is associated with moderate-to-severe side effects, and the efficacy of locally administered drugs is short-lived. Here we show that inflammation can be locally suppressed by a fusion protein of the immunosuppressive enzyme indoleamine 2,3-dioxygenase 1 (IDO) and galectin-3 . Gal3 anchors IDO to tissue, limiting the diffusion of IDO-Gal3 away from the injection site. In rodent models of endotoxin-induced inflammation, psoriasis, periodontal disease and osteoarthritis, the fusion protein remained in the inflamed tissues and joints for about 1 week after injection, and the amelioration of local inflammation, disease progression and inflammatory pain in the animals were concomitant with homoeostatic preservation of the tissues and with the absence of global immune suppression. IDO-Gal3 may serve as an immunomodulatory enzyme for the control of focal inflammation in other inflammatory conditions. An anti-inflammatory enzyme fused with a tissue-anchoring protein and injected into inflamed tissues ameliorates local inflammation without causing systemic immune suppression, as shown in multiple rodent models of inflammatory diseases. A major challenge in the treatment of chronic inflammatory diseases is the development of therapeutics to safely and specifically direct resolution of inflammation in a site-specific manner1. Anti-inflammatory drugs such as glucocorticoids are pleiotropic, non-specifically affecting numerous pathways1 and are consequently accompanied by issues of toxicity, resistance and a wide array of serious adverse effects such as infection and defective wound healing3. Additionally, systemic immune modulation leads to disease states such as hypertension, osteoporosis, obesity, cataracts and diabetes3. Biologic immunosuppressive drugs functioning through either cytokine blockade, cell depletion or cell surface receptor blockade provide improved specificity and can effectively modulate immune responses to halt disease progression in certain, but not all patients4. However, such treatments can also increase susceptibility to infections and disrupt tissue homoeostasis, leading to cancer, exacerbation of congestive heart failure and neurologic events, among other pathologies4. Critically, each of these therapeutics require life-long continual use and clinical options to resolve chronic inflammation and restore tissue homoeostasis remain to be developed5.Chronic inflammation, characterized by professional immune cell and resident tissue cell interactions, irreversibly damages tissues of the body and is an associated risk factor for a host of diseases such as cardiovascular disease, diabetes and cancer6. Catabolism of the essential amino acid tryptophan (Trp) by the cytosolic enzyme indoleamine 2,3-dioxygenase 1 (IDO) and the resultant production of kynurenine metabolites is a general regulator of inflammation in response to sterile and pathogenic inflammatory stimuli, acting on both innate and adaptive immune cells8. IDO catabolism of Trp is also a contributing factor to promoting fetal tolerance in pregnancy, warding off autoimmunity and avoiding immune elimination in some forms of cancer9. Trp insufficiency via IDO activates the metabolic stress sensor general control nonderepressible 2 (GCN2) to regulate immune cell cycle10, while kynurenine pathway metabolites activate anti-inflammatory programmes, such as kynurenine binding to the aryl hydrocarbon receptor (AHR)11. IDO expression in immune cells such as macrophages and dendritic cells has been demonstrated to suppress T cell activation and proliferation while activating and promoting phenotypic maintenance of regulatory T cells14. Additionally, the terminal product of the kynurenine pathway, nicotinamide adenine dinucleotide (NAD+), regulates innate immunity function in macrophages during aging and inflammation15. In total, actions of IDO serve as a key mechanism maintaining homoeostasis, suppressing autoimmunity and shutting down excess inflammation10. Informed by these data, we envisioned therapeutic delivery of exogenous IDO as a regulator of chronic inflammatory diseases.The capability to direct cellular metabolism to programme immune responses has recently emerged as a new avenue for therapeutic immunomodulation19, we recently demonstrated the utility of model enzymes fused to galectin-3 , a carbohydrate-binding protein, as a generalizable means to restrict enzyme diffusion via binding to tissue glycans20. Gal3 binds N-acetyllactosamine and other \u03b2-galactoside glycans, as well as glycosaminoglycans, which are highly abundant in mammalian tissues22, collectively representing a more universal target than a specific extracellular matrix protein. Thus, Gal3 fusion to enzymes represents a promising approach to retain local enzymatic function at an intended site of action. Building upon this success, we engineered the IDO-Gal3 fusion with the expectation of creating a tissue-anchored IDO administered as a localized anti-inflammatory therapeutic to serve as positive feedback to further elevate expression9, endogenous Ido1 transcripts were quantified after treatment with IDO-Gal3 in both the LPS and psoriasis models could also potentially contribute to the resolution of inflammation. In sum, s.c. administration of IDO-Gal3 ameliorated skin inflammation both prophylactically and therapeutically, using two different inflammatory insults acting through different specific receptors (CD14/TLR4 for LPS and TLR7 for imiquimod)26.Given the capacity for endogenous cellular IDO gene expression and hard (bone) tissue destructionl29 Fig. , submandl29 Fig. . In vivol29 Fig. , whereasl29 Fig. . Retentil29 Fig. . The pril29 Fig. . Specifil29 Fig. and thicl29 Fig. , were prl29 Fig. and IL-1l29 Fig. , and chel29 Fig. . In contl29 Fig. , wherebyl29 Fig. , where tl29 Fig. . IDO-Gall29 Fig. . Further31 was utilized to test for the ability of IDO-Gal3 to reduce load-induced inflammatory joint damage. Intra-articular injection of IDO-Gal3 was locally retained, suppressed inflammation and spared joint tissue destruction , a form of osteoarthritis (OA), commonly arises after a ligament or meniscus tear, or following repeated overloading of a joint. A cyclic mechanical overloading PTOA mouse modelion Fig. , with meion Fig. and day ion Fig. and quanion Fig. by ex viion Fig. . Weekly ion Fig. and draiion Fig. , whereasion Fig. . Criticaion Fig. and artiion Fig. . Additioion Fig. and 20. We note that multiple injections of human IDO-Gal3 in mice led to the emergence of some anti-IDO-Gal3 antibodies 34. Intra-articular injection of IDO-Gal3 was locally retained, suppressed joint inflammation, reduced OA-associated pain and improved rat gait vectors between NcoI and XhoI sites. IDO-Gal3 genetic and protein sequences are provided in E. coli (ThermoFisher), selected on Luria-Bertani (LB) ampicillin (50\u2009\u03bcg\u2009ml\u22121) agar and incubated overnight at 37\u2009\u00b0C. Isolated colonies from the plates were subcultured in 5\u2009ml LB ampicillin broth overnight at 37\u2009\u00b0C with orbital shaking. Successful transformants were confirmed by Sanger sequencing (Genewiz). Positive DNA sequences were then transformed into the kanamycin B resistant expression strain, Origami B (DE3) E. coli (Novagen) and selected on LB ampicillin with kanamycin B (15\u2009\u03bcg\u2009ml\u22121) agar. Positive clones were picked, banked in LB with 10% w/v glycerol and used to preculture 5\u2009ml of LB ampicillin and kanamycin. Overnight precultures were used to inoculate 1l 2\u00d7 TY media with ampicillin and kanamycin B at 37\u2009\u00b0C and 225\u2009r.p.m. in an orbital shaker until approximate exponential growth phase 600\u2009nm\u2009=\u20090.6\u20130.8). IDO-Gal3 expression cultures were supplemented with 500\u2009\u03bcM \u03b4-Aminolevulinic acid (Sigma) at the time of inoculation. Recombinant protein expression was triggered using 0.5\u2009mM isopropyl \u03b2-d-1-thiogalactopyranoside (ThermoFisher) and incubated for 18\u2009h in an orbital shaker at 18\u2009\u00b0C. Bacteria were washed and pelleted with PBS via centrifugation with a superspeed centrifuge (ThermoFisher). Afterwards, the pellet was weighed, resuspended in 4\u2009ml PBS per gram pellet with protease inhibitor (ThermoFisher) and disrupted by sonic dismembration . Following dismembration, lysates were treated with 20 units per gram pellet DNAse I (ThermoFisher) and 800\u2009\u03bcg\u2009g\u22121 pellet lysozyme (ThermoFisher). The lysate was centrifuged to remove the insoluble fraction and the supernatant was collected by decanting. Supernatant containing soluble recombinant protein was loaded into an \u03b1-lactose agarose (Sigma) affinity column pre-equilibrated with PBS. Columns were washed with 20 column volumes PBS and recombinant proteins were eluted with 100\u2009mM \u03b2-d-lactose (Sigma) prepared in PBS. A final polishing step was performed by 200\u2009kDa size exclusion chromatography on AKTA pure chromatography system (GE Life Sciences) to remove \u03b1-lactose and further isolate IDO-Gal3. Protein purity was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS\u2013PAGE) and Coomassie staining. Endotoxin contaminants were removed by endotoxin removal solution (Sigma) following manufacturer instructions. Endotoxin content was analysed using Chromo-LAL kinetic chromogenic endotoxin quantification assay (Associates of Cape Cod) and determined to be below 0.1\u2009EU\u2009ml\u22121 in all stocks.NanoLuc is the tradename of an engineered deep-sea shrimp luciferase variant developed by Promega CorporationE. coli was purchased from R&D Systems as measured by its ability to oxidize l-tryptophan to N-formyl-kynurenine (NFK)). The specific activity of both proteins, IDO and IDO-Gal3, was measured before experiments to ensure maximal effect at the beginning of the assay, following the IDO manufacturer\u2019s protocol. IDO-Gal3 was reacted in equimolar amounts to IDO in the standard protocol, and activities were compared using the unit pmol\u2009NFK\u2009min\u22121\u2009pmol\u22121 IDO to more accurately compare activity.Recombinant human IDO expressed in \u03b1-lactose agarose affinity resin (Sigma-Aldrich). Proteins were eluted with a linear gradient of \u03b2-lactose (Sigma-Aldrich) in phosphate buffer.Affinity of IDO-Gal3 for lactose was determined using affinity chromatography in an AKTA Pure chromatography system (GE Life Sciences) equipped with consumer-packable glass column (GE Life Sciences) packed with CCATGGCGCACGCGATGGAAAACAGCTGGACCATCAGCAAAGAGTACCACATTGACGAGGAAGTTGGTTTCGCGCTGCCGAACCCGCAGGAAAACCTGCCGGACTTCTATAACGATTGGATGTTTATCGCGAAGCACCTGCCGGATCTGATTGAGAGCGGCCAGCTGCGTGAGCGTGTGGAAAAACTGAACATGCTGAGCATCGACCACCTGACCGATCACAAGAGCCAACGTCTGGCGCGTCTGGTTCTGGGTTGCATTACGATGGCGTACGTGTGGGGCAAAGGTCACGGCGACGTGCGTAAGGTTCTGCCGCGTAACATCGCGGTTCCGTACTGCCAACTGAGCAAGAAACTGGAACTGCCGCCGATTCTGGTGTATGCGGACTGCGTTCTGGCGAACTGGAAGAAGAAGGACCCGAACAAACCGCTGACCTATGAGAACATGGATGTGCTGTTCAGCTTTCGTGACGGTGATTGCAGCAAGGGCTTCTTTCTGGTGAGCCTGCTGGTTGAAATCGCGGCGGCGAGCGCGATCAAAGTGATTCCGACCGTTTTCAAGGCGATGCAGATGCAAGAGCGTGACACCCTGCTGAAAGCGCTGCTGGAAATCGCGAGCTGCCTGGAGAAGGCGCTGCAGGTGTTTCACCAAATTCACGATCACGTTAACCCGAAAGCGTTCTTTAGCGTGCTGCGTATCTACCTGAGCGGTTGGAAGGGCAACCCGCAGCTGAGCGACGGTCTGGTTTATGAGGGCTTCTGGGAAGATCCGAAAGAGTTTGCGGGTGGCAGCGCGGGTCAGAGCAGCGTGTTCCAATGCTTTGACGTTCTGCTGGGCATTCAGCAAACCGCGGGTGGCGGTCATGCGGCGCAGTTCCTGCAAGATATGCGTCGTTACATGCCGCCAGCGCACCGTAACTTCCTGTGCAGCCTGGAAAGCAACCCGAGCGTGCGTGAGTTTGTTCTGAGCAAAGGTGACGCGGGCCTGCGTGAAGCGTATGATGCGTGCGTGAAGGCGCTGGTTAGCCTGCGTAGCTACCACCTGCAGATCGTTACCAAATATATCCTGATTCCGGCGAGCCAGCAACCGAAAGAAAACAAGACCAGCGAGGACCCGAGCAAACTGGAGGCGAAGGGTACCGGCGGTACCGATCTGATGAACTTTCTGAAGACCGTGCGTAGCACCACCGAGAAGAGCCTGCTGAAAGAGGGTGGATCCGGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCGAATTCGCGGACAACTTCAGCCTGCACGATGCGCTGAGCGGTAGCGGTAACCCGAACCCGCAGGGTTGGCCGGGTGCGTGGGGTAACCAACCGGCGGGTGCGGGTGGCTACCCGGGTGCGAGCTATCCGGGTGCGTATCCGGGTCAGGCTCCGCCGGGTGCGTACCCGGGCCAAGCTCCGCCGGGTGCTTATCCTGGTGCGCCGGGCGCGTACCCGGGTGCGCCGGCGCCGGGCGTGTACCCGGGTCCGCCGAGCGGTCCGGGCGCGTATCCGAGCAGCGGCCAGCCGAGCGCGCCGGGTGCGTATCCGGCGACCGGCCCGTATGGTGCGCCGGCGGGTCCGCTGATTGTTCCGTATAACCTGCCGCTGCCGGGTGGCGTGGTTCCGCGTATGCTGATCACCATTCTGGGCACCGTGAAGCCGAACGCGAACCGTATCGCGCTGGACTTCCAACGTGGTAACGATGTTGCGTTCCACTTTAACCCGCGTTTTAACGAGAACAACCGTCGTGTGATTGTTTGCAACACCAAACTGGACAACAACTGGGGCCGTGAGGAACGTCAGAGCGTGTTCCCGTTTGAGAGCGGCAAGCCGTTCAAAATTCAAGTGCTGGTTGAACCGGACCACTTTAAGGTGGCGGTTAACGATGCGCACCTGCTGCAGTACAACCACCGTGTTAAGAAACTGAACGAAATCAGCAAACTGGGCATCAGCGGTGACATTGATCTGACCAGCGCGAGCTATAACATGATTCTCGAG, with restriction sites underlined.The sequence used was GSGGGSGGSGGSGGEFADNFSLHDALSGSGNPNPQGWPGAWGNQPAGAGGYPGASYPGAYPGQAPPGAYPGQAPPGAYPGAPGAYPGAPAPGVYPGPPSGPGAYPSSGQPSAPGAYPATGPYGAPAGPLIVPYNLPLPGGVVPRMLITILGTVKPNANRIALDFQRGNDVAFHFNPRFNENNRRVIVCNTKLDNNWGREERQSVFPFESGKPFKIQVLVEPDHFKVAVNDAHLLQYNHRVKKLNEISKLGISGDIDLTSASYNMILEHHHHHHMAHAMENSWTISKEYHIDEEVGFALPNPQENLPDFYNDWMFIAKHLPDLIESGQLRERVEKLNMLSIDHLTDHKSQRLARLVLGCITMAYVWGKGHGDVRKVLPRNIAVPYCQLSKKLELPPILVYADCVLANWKKKDPNKPLTYENMDVLFSFRDGDCSKGFFLVSLLVEIAAASAIKVIPTVFKAMQMQERDTLLKALLEIASCLEKALQVFHQIHDHVNPKAFFSVLRIYLSGWKGNPQLSDGLVYEGFWEDPKEFAGGSAGQSSVFQCFDVLLGIQQTAGGGHAAQFLQDMRRYMPPAHRNFLCSLESNPSVREFVLSKGDAGLREAYDACVKALVSLRSYHLQIVTKYILIPASQQPKENKTSEDPSKLEAKGTGGTDLMNFLKTVRSTTEKSLLKEG\u22121 lipopolysaccharide (LPS) in 40\u2009\u03bcl, after 24\u2009h or 120\u2009h post IDO-Gal3 treatment, to assess local and distal modulation of inflammation. At 2\u2009h after LPS administration, animals were euthanized and the injection site collected.B6 mice were administered 2.1\u2009\u03bcg IDO-Gal3 in 40\u2009\u03bcl PBS subcutaneously at the region of the hock and challenged with 2\u2009ng\u2009g\u03b2, IFN-\u03b3 and IL-6) using Applied Biosystems QuantStudio 12K Flex Real-Time PCR System. Results are presented as the ratio of gene expression to glyceraldehyde 3\u2010phosphate dehydrogenase (GAPDH) expression determined by the relative quantification method. Treatment groups were normalized to PBS only group.Soft tissue was separated from bone and stored in RNAlater RNA stabilization reagent (Qiagen) in preparation for qPCR. Soft tissues were homogenized and RNA purified using RNeasy Protect mini kit (Qiagen). Complementary DNA was synthesized from RNA using the High-Capacity cDNA Reverse Transcriptase kit (ThermoFisher) for use in qPCR in accordance with manufacturer instructions. qPCR analysis was run with primers specific for pro-inflammatory cytokines overnight. After fixation, tissues were washed in deionized water and decalcified by storing in 10% ethylenediaminetetraacetic acid (EDTA) at 4\u2009\u00b0C for 3 weeks. Samples were assessed every 2\u20133\u2009d for stiffness and EDTA solution replenished. Tissues were submitted in 70% ethanol to the University of Florida Molecular Pathology Core for processing, paraffin embedding, sectioning, mounting and staining with haematoxylin and eosin (H&E). Tissues were imaged using a Zeiss Axiovert 200M with a \u00d720 objective lens through the multidimensional acquisition module. Eleven images were taken per tissue and scored by two blinded independent individuals on the basis of cellular infiltration and epidermis hypertrophy .n\u2009=\u20093). At 30\u2009min after injection, the mice were euthanized, the hock and tibia tissue regions excised and flash frozen using liquid nitrogen. These samples were then submitted to the Southeast Center for Integrated Metabolomics at the University of Florida for mass spectrometric analysis of kynurenine levels.IDO-Gal3 (2.2\u2009\u03bcg) in 40\u2009\u03bcl PBS or 40\u2009\u03bcl PBS alone was injected into the hock site of B6 mice was collected in microtainer serum separator tubes and centrifuged at 1,500\u2009r.p.m. for 5\u2009min at room temperature. The serum was collected and stored at \u221220\u2009\u00b0C. Cytokine analysis was performed using a Luminex 200 system running xPONENT 3.1 software (Luminex) following manufacturer instructions.37. Briefly, mice were anaesthetized and their backs were shaved, followed by application of depilatory cream to remove any remaining fur. Each day for 14\u2009d total, 5% IMQ cream was applied to the backs of the mice. On the 3rd day of IMQ application, mice were subcutaneously injected with five 10\u2009\u03bcg doses of IDO-Gal3 in sterile saline distributed evenly throughout the back, a molar equivalent of NL-Gal3 delivered in the same volume, or a sterile saline control (n\u2009=\u200912 per group). Disease severity was measured each day using a modified version of the Psoriasis Area and Severity Index (PASI) where area of effect is not taken into account. Erythema (redness), scaling and thickening were scored independently and assigned a score on a scale of 0 to 4: 0, none, 1: slight, 2: moderate, 3: marked, 4: very marked. The cumulative score was reported as a measure of the severity of inflammation .Pre-clinical modelling of psoriasis was carried out in 8\u201312-week-old female C57BL/6j mice (Jackson Laboratory) in accordance with the Institutional Animal Care and Use Committee (IACUC) at the University of Florida. The model used was a modified version of a previously reported modelIn vivo imaging of fluorescently tagged IDO-Gal3 was carried out in 8\u201312-week-old female C57BL/6j mice (Jackson Laboratory) in accordance with the IACUC at the University of Florida. Before injection, IDO-Gal3 was incubated with IRDye 680RD NHS Ester following manufacturer instructions. Mice received a 40\u2009\u03bcl subcutaneous injection of fluorescently labelled IDO-Gal3 to the hock. Immediately following injection and every subsequent 24\u2009h following injection, mice were imaged using an IVIS Spectrum in vivo imaging system (PerkinElmer). Fluorescent images were captured using the AF680 emission filter, subject size 1.5\u2009cm, 0.2\u2009s exposure time, field of view B (6.6\u2009cm), medium binning (factor of 8) resolution and a 1F/Stop aperture. Relative fluorescent intensities were represented by a pseudo colour scale ranging from red (least intense) to yellow (most intense).\u22121\u2009cm\u22122\u2009sr\u22121). The specific amount of protein in tissue was determined by comparison with a standard curve of NanoLuc-Gal3 activity.NanoLuc-Gal3 (164\u2009pmol) in 40\u2009\u03bcl PBS was injected subcutaneously into the hock of B6 mice. At the prescribed time points, animals were euthanized in accordance with approved protocols. Organs and tissues of interest were collected, weighed, processed, incubated with furimazine and bioluminescence quantified by a luminometer. Bioluminescence images were acquired using an IVIS Spectrum in vivo imaging system. Living Image software v4.3.1 (PerkinElmer) was used to acquire the data immediately after furimazine administration. Exposure time for the bioluminescence imaging was 1\u2009s. Regions of interest (ROIs) were quantified as the average radiance (photons\u2009sListeria monocytogenes InIAM (strain 10403s). Tissue bacteria burdens in the liver and spleen were used as a metric for infection where the liver is local for gut infection and the spleen represents systemic spread of infection. On the day before infection, 10-week-old na\u00efve C57BL/6 mice (Taconic Biosciences) received IDO-Gal3 injection subcutaneously (hock), while control mice received sterile saline injection. The following day, mice were orally infected with 2\u2009\u00d7\u2009109 colony-forming units (c.f.u.) of Listeria monocytogenes per mouse. To prepare for infection, bacteria were cultured overnight in BHI broth at 37\u2009\u00b0C, shaking at 220 r.p.m. Before infection, a subculture of 2\u2009ml of the bacteria and 18\u2009ml BHI broth was cultured under the same conditions until reaching an OD600\u2009=\u20090.8. Bacteria were pelleted, resuspended in 500\u2009\u03bcl of sterile PBS, and 50\u2009\u03bcl of bacteria was pipetted onto a small square of white bread and fed to each mouse individually. Once the entire piece of bread was eaten, mice were returned to their cage. At day 7 post infection, mice were euthanized, followed by collection of the spleen and liver. Tissue was homogenized, suspended in 1% saponin for 1\u2009h, then plated at serial dilutions from undiluted to 1:1,000. BHI Agar plates were treated with streptomycin to limit non-specific bacterial growth, as this strain of L. monocytogenes is streptomycin resistant. C.f.u.s were counted at 24\u2009h post-plating.Immune suppression from IDO-Gal3 was evaluated in response to oral infection with 9P. gingivalis strain 381 and 2.5\u2009\u00d7\u2009109Aggregatibacter actinomycetemocomitans strain 29522 (ATCC) resuspended in 2% low-viscosity carboxy-methyl-cellulose (Sigma-Aldrich). Oral lavage was repeated every week for 5 weeks. Each week, 1\u2009d before the first day of infection (prophylactic) or 1\u2009d after the last day of infection (therapeutic), 10\u2009\u03bcl of IDO-Gal3 was injected into the submandibular space using a 30-gauge insulin syringe (Becton Dickenson). Each week of infection, on the first day of infection, before infection, microbial sampling of the oral environment was performed with calcium alginate swabs (ThermoFisher). One week following the last infection, the mandibles were collected to evaluate soft tissue soluble mediator expression and bone morphometric analysis.All mice were lavaged with 25\u2009\u03bcl 0.12% chlorhexidine gluconate (3\u2009M) for 3\u2009d. On days 4, 5, 6 and 7, mice received a 25\u2009\u03bcl oral lavage with 2.5\u2009\u00d7\u200910\u03b2, IL-10 and MCP1 according to manufacturer protocols. Data were acquired on a Luminex 200 system running xPONENT 3.1 software (Luminex) and analysed using a 5-paramater logistic spline-curve fitting method using Milliplex Analyst v5.1 software (Vigene Tech). Data are presented as pg\u2009ml\u22121 normalized to total protein .Mandibles with both soft tissue and bone were subjected to bead beating at two 2\u2009min intervals with 2\u2009min of cooling in between using 1.0-mm-diameter zirconia silica beads (BioSpec) in cell extraction buffer (ThermoFisher). The buffer was prepared with a protease inhibitor cocktail and PMSF protease inhibitor (Abcam) to allow for dissociation and lysis of all soft tissue while leaving the hard tissues intact. MILLIPLEX Multiplex assays (EMD Millipore) were used to probe resulting lysates for IL-6, IL-13 ROI was set with standardized dimensions of 1.5 \u2009\u00d7\u20094.0 \u2009\u00d7\u20090.9\u2009mm . Anatomical landmarks were used for the standardized positioning of the ROI: frontal plane, the roof of the furcation area between mesial and distal roots of the upper first molar; sagittal plane, anterior limit was the distal aspect of the mesial root of the first molar. The thickness of the ROI on the transversal plane was set to 50 slices (900\u2009\u03bcm) and counted towards the palatal /medial direction beginning from the image that included the centre of the upper first molar in its transversal width. A standardized threshold was set to distinguish between non-mineralized and mineralized tissues where total volume and total thickness of the ROI were calculated. Mean trabecular bone volume (BV) and thickness (BT) analysis assessed the percentage of mineralized tissue within the total volume/thickness of the ROI and is presented as the BV/TV ratio (mean trabecular bone volume) or BT/TT ratio (mean trabecular thickness). Vertical bone loss is the distance from the cementoenamel junction (CEJ) to the alveolar bone and was calculated at 12 sites over three molars and averaged to calculate the average vertical bone loss in mm.Mandibles were fixed in 4% buffered formalin for 24\u2009h, stored in 70% alcohol and scanned at 18\u2009\u03bcm resolution using a micro-CT system (Skyscan). Three-dimensional images were reconstructed and the resulting images re-oriented spatially using anatomical landmarks with the NRecon and DataViewer software (Skyscan). A standardized 5.4\u2009mmP. gingivalis 16S, A. actinomycetemocomitans 16S and total 16S using real-time PCR. The percentage of A. actinomycetemocomitans 16S and P. gingivalis 16S within the total 16S compartment was calculated using the following formula: Ct value of total 16S/Ct value of A. actinomycetemocomitans or P. gingivalis 16S. 16s rRNA For: AGA GTT TGA TCC TGG CTC AG; Rev: ACG GCT ACC TTG TTA CGA CTT; Pg For: CTT GAC TTC AGT GGC GGC AG; Rev: AGG GAA GAC GGT TTT CAC CA; Aa For: GTT TAG CCC TGG CCG AAG; Rev: TGA CGG GCG GTG TGT ACA AGG.Genomic DNA was isolated from microbial sampling of the oral environment using a DNeasy kit (Qiagen) according to manufacturer instructions. The gDNA was then probed for 30 that applies cyclic mechanical loading to the knees of aged (6 months) mice, causing mechanical damage and consequent inflammation and cartilage degradation31. The mice were anaesthetized and placed in a fixture with the knee in flexion; loading (9\u2009N) was applied axially for 500 cycles and loading sessions were done on the mice 5 times per week during the experiment. IDO or IDO-Gal3 was prepared at 143\u2009\u03bcM, and 20\u2009\u03bcl of each treatment was injected intra-articularly at the start of each week of the study, with each knee receiving 4 total treatments. At the end of the 4-week study, mice were euthanized for analysis of gene expression and histopathology.IDO-Gal3 activity was assessed in a post-traumatic osteoarthritis mouse modelPharmacokinetics of IDO and IDO-Gal3 retention after local injection at the disease site was assessed by intravital imaging over the course of 7\u2009d. Both proteins were labelled with Li-Cor IRDye 680RD NHS ester (Li-Cor Biosciences) to visualize and measure protein knee retention. Mice were subjected to mechanical loading for 2 weeks before each treatment was administered via intra-articular injection. Intravital imaging was performed immediately following injection and every subsequent 24\u2009h. The fluorescence signal measured at the joint over time was normalized to the initial measurement for each knee and an exponential decay was individually fit for each specimen. The AUC/bioavailability for each joint was calculated from the best-fit line of exponential decay. After 7\u2009d, the mice were euthanized and an ex vivo image was taken of each joint with the surrounding skin removed to increase measurement sensitivity.TaqMan qPCR in the knee joint and the popliteal lymph node that drains the knee joint. Following euthanization, knees and popliteal lymph nodes were excised. Combined joint tissue from the synovial wall and articular surface and the popliteal lymph nodes were homogenized with bead pulverization in Qiazol. RNA was extracted and purified using the RNeasy Plus mini kit from Qiagen and quantified using NanoQuant plate from Tecan in a microplate reader . The RNA was converted to cDNA using the iScript Synthesis kit from Bio-Rad (Hercules). Gene expression was calculated by the \u0394\u0394Ct method, normalizing to GAPDH and beta-actin (ACTB). TaqMan reagents were purchased from ThermoFisher and used according to provided protocols, using appropriate primers .Gene expression was evaluated by 38. OARSI scoring was based on medial and lateral tibial plateaus 39, and a generic score was concurrently assigned on the basis of H&E features and the safranin O staining of the tibial plateaus according to the DJD methodology .Tissue samples were fixed in 10% formalin and decalcified in 20% EDTA for 7\u2009d. A standard 8\u2009h cycle of graded alcohols, xylenes and paraffin wax was used to process tissues before embedding and sectioning at 5\u2009\u03bcm thickness. Sections were mounted on positively charged glass slides and stained with H&E using the Gemini autostainer (ThermoFisher). Safranin O staining was performed using the StatLab staining kit. Sections were imaged using Leica SCN400 slide scanner. Each joint was evaluated by at least two mid-frontal sections for both H&E and safranin O stains. A board-certified veterinary pathologist conducted histopathologic interpretations under blinded conditionsSixteen male Lewis rats were acquired from Charles River Laboratories. Rats were acclimated to the University of Florida housing facilities for 1 week. After acclimation, rats underwent baseline gait and von Frey behavioural testing. Following baseline behavioural testing, all rats received medial collateral ligament plus medial meniscus transection (MCLT\u2009+\u2009MMT) surgery to their right hind limb. Gait data were collected on weeks 3, 5 and 7 post-surgery using a Phantom Miro Lab320 (Phantom Camera Control 3.0), while von Frey testing was conducted weekly after surgery. At 8 weeks post-surgery, rats received a unilateral saline or IDO-Gal3 injection. Rats underwent von Frey testing the day following injection and gait testing 2\u2009d after injection. Both gait and von Frey data were collected weekly until euthanasia.\u22121) (Patterson Veterinary) intra-operatively and every 12\u2009h for 48\u2009h. Rats were grouped into a saline injection cohort (n\u2009=\u20097) or IDO-Gal3 injection cohort (n\u2009=\u20098). At 8 weeks after OA induction, rats received 30\u2009\u00b5l unilateral injections of either sterile saline or IDO-Gal3 (33\u2009pg\u2009\u00b5l\u22121) in the operated knee using sterile allergy syringes (Becton Dickinson). First, rats were anaesthetized using 2.5% isoflurane (Patterson Veterinary) and the operated knee was aseptically prepped as described above. Then the needle was inserted through the patellar ligament following the patellar groove into the joint space. The knee was flexed and the injection site was cleaned with sterile gauze and 70% ethanol.MCLT\u2009+\u2009MMT surgery rats were anaesthetized in a 2.5% isoflurane (Patterson Veterinary) sleep box. Rats were then aseptically prepped with betadine surgical scrub (Purdue Products) and 70% ethanol in triplicate, and transferred to a sterile field with anaesthesia maintained via mask inhalation of 2.5% isoflurane. During MCLT\u2009+\u2009MMT surgery, a 1\u20132\u2009cm midline skin incision was made along the medial aspects of the rat knee and the skin was retracted to reveal the medial collateral ligament. The medial collateral ligament was transected and the knee was placed in the valgus orientation to stretch the medial compartment and expose the medial meniscus. The medial meniscus was cut radially, and absorbable 5\u20130 vicryl braided sutures (Ethicon) were used for muscle closure and 4\u20130 ethilon nylon monofilament sutures (Ethicon) were used for skin closure. Rats recovered post-operatively in a warming box until weight bearing on all limbs. For pain management, rats received a subcutaneous injection of buprenorphine was applied to the plantar region of each hind foot. First, the 4.0\u2009g von Frey filament was applied. A less-stiff filament was applied if a paw withdrawal occurred, and a stiffer filament was applied if a paw withdrawal did not occur. Using these data, the force at which rats were equally likely to withdraw or tolerate was calculated via Chaplan\u2019s approximation1.Tactile sensitivity was assessed by measuring the 50% paw withdrawal threshold determined using the Chaplan up-down method for von Frey filaments\u22121 anti-CTXII, 8.9\u2009ng\u2009\u00b5l\u22121 anti-IL-6 or 357\u2009ng\u2009\u00b5l\u22121 anti-MCP1 and then moved to static incubation at 4\u2009\u00b0C overnight. Particles were then washed three times in PBS containing 2% BSA and 2\u2009mM EDTA (capture buffer), with final antibody amounts per particle measured to be 0.33\u2009ng\u2009Ab\u2009\u00b5g\u22121 particle (anti-CTXII), 0.33\u2009ng\u2009Ab\u2009\u00b5g\u22121 particle (anti-IL-6) and 13.0\u2009ng\u2009Ab\u2009\u00b5g\u22121 particle (anti-MCP1).Commercially available streptavidin-functionalized particles were coated with biotinylated antibodies for CTXII , IL-6 and MCP1 . Here, particles were washed 3 times in PBS, incubated for 2\u2009h on a tube revolver at room temperature in antibody mixes that contained either 8.9\u2009ng\u2009\u00b5l40. Briefly, 300\u2009\u00b5g of antibody-conjugated magnetic particles were suspended in 10\u2009\u00b5l of saline, then injected in the operated and contralateral knee. After 2\u2009h incubation in the knee, particles were collected via 5 repeated 50\u2009\u03bcl PBS washes of the knee, with collected fluid combined and particles in the fluid isolated via a magnetic separator. Collected particles were washed twice with capture buffer, then incubated for 15\u2009min in the 100\u2009mM Glycine-Tris buffer (pH 3.1) containing 2% BSA and 2\u2009mM EDTA (release buffer). Following biomarker release, magnetic particles were isolated by magnetic separation and the pH of the supernatant was adjusted to 8.3 for enzyme-linked immunosorbent assays (ELISA). CTXII was then measured in the supernatant using the Cartilaps CTXII ELISA kit according to manufacturer instructions, and CCL2 was quantified using a rat CCL2 ELISA kit (KRC1012 Life Technologies) according to manufacturer instructions and previously described modifications41. IL-6 was quantified using ELISA developed in the laboratory using biotin anti-rat IL-6 antibody (517703) and purified (coating) anti-rat IL-6 antibody . Coating antibody was diluted in 100\u2009mM NaHCO3 and 34\u2009mM Na2CO3 (pH 9.5), placed in coated microwells , incubated for 30\u2009min on a plate shaker then overnight at 4\u2009\u00b0C, washed 5 times with PBS with 0.5% Tween-20 (wash buffer), blocked for 1\u2009h with PBS containing 2% BSA and 10% heat-treated bovine serum, and finally washed 5 times with wash buffer. Samples and standards were pre-incubated with anti-IL-6 detection antibody (30\u2009min at room temperature and then overnight at 4\u2009\u00b0C), then added to ELISA plate and incubated for 3\u2009h. The plate was then washed 5 times, and 100\u2009\u03bcl of avidin-HRP was added to the plate and incubated for 30\u2009min. The plate was again washed 5 times, with 100\u2009\u03bcl of tetramethylbenzidine substrate added for 15\u2009min, followed by 100\u2009\u03bcl of stop solution (diluted sulfuric acid). Absorbance was read at 450 and 650\u2009nm. Particles were quantified in 60\u2009\u03bcl of capture buffer, with particle suspensions read for absorbance at 450\u2009nm using Synergy 2 Multi-Mode microplate reader, as previously described40.Following euthanasia via exsanguination, magnetic capture was performed in both the operated and contralateral knee, as previously describedFollowing magnetic capture, operated and contralateral knees were dissected and placed in 10% neutral buffered formalin (ThermoFisher) for 48\u2009h at room temperature. Following fixation, knees were decalcified using Cal-Ex (ThermoFisher) for 5\u2009d at room temperature, dehydrated through an ethanol ladder and embedded in paraffin wax via vacuum infiltration. Then, 10\u2009\u00b5m frontal sections were acquired, with at least one section taken at every 100\u2009\u00b5m through the loading region of the medial meniscus. Slides were stained with toluidine blue.n\u2009=\u20098) or bilateral injections of NL-Gal3 (n\u2009=\u20098) in both operated and contralateral knees. (Surgeries and injections were conducted the same way as described above). Knees were flexed, then injected with 50\u2009\u00b5l furimazine . Immediately after injection, knees were flexed again and luminescence was measured with IVIS using a 1\u2009s (and 60\u2009s) exposure time in field of view D. Furimazine injections and IVIS imaging were repeated at 1, 2, 4, 8, 12, 16, 20, 24 and 28\u2009d post NL or NL-Gal3 injection. For analysis, a ROI was drawn around the largest luminescent signal and copied to create an identical sized ROI for all knees.Sixteen male Lewis rats were acquired from Charles River Laboratories. Rats were acclimated to the University of Florida housing facilities for 1 week. Then rats underwent MCLT\u2009+\u2009MMT surgery. After 8 weeks, rats received a 50\u2009\u00b5l injection of either Nano-Glo Luciferase (NL) or Nano-Glo Luciferase Galectin-3 . Rats were IVIS imaged immediately following injection, then at 1, 2, 4, 8, 12, 16, 20, 24 and 28\u2009d post-injection. After imaging on day 28, rats were euthanized, and both operated and contralateral knees were dissected for joint tissue distribution analysis via IVIS. In vivo joint retention was measured in the operated and contralateral knees of 16 male Lewis rats. At 8 weeks post MCLT\u2009+\u2009MMT surgery, knees were aseptically prepped and rats received bilateral injections of NL at room temperate for roughly 1\u2009min. After incubation, tissues were removed from the 24-well plates and luminescence was measured with IVIS using auto-exposure in field of view C. For assessment, individual ROIs were drawn around the patellar tissue, tibial tissue, femoral tissue and meniscus. The ROI for each tissue was copied to create an identical sized ROI for the respective tissue.U-tests on psoriasis data. Study-specific analyses are reported in figure captions.Statistical analyses were performed using GraphPad Prism 8 and 9, with the following exceptions. Surgically induced osteoarthritis data were analysed using R Analytics 4.0.4 with RStudio 2022, and MATLAB 2020b was used for Mann-Whitney Further information on research design is available in the Supplementary InformationSupplementary methods and figures.Reporting Summary"} +{"text": "Caenorhabditis elegans. We show that orthologs of GPA2 and GPB5, together with thyrotropin-releasing hormone (TRH) related neuropeptides, constitute a neuroendocrine pathway that promotes growth in C. elegans. GPA2/GPB5 signaling is required for normal body size and acts through activation of the glycoprotein hormone receptor ortholog FSHR-1. C. elegans GPA2 and GPB5 increase cAMP signaling by FSHR-1 in vitro. Both subunits are expressed in enteric neurons and promote growth by signaling to their receptor in glial cells and the intestine. Impaired GPA2/GPB5 signaling causes bloating of the intestinal lumen. In addition, mutants lacking thyrostimulin-like signaling show an increased defecation cycle period. Our study suggests that the thyrostimulin GPA2/GPB5 pathway is an ancient enteric neuroendocrine system that regulates intestinal function in ecdysozoans, and may ancestrally have been involved in the control of organismal growth.In vertebrates, thyrostimulin is a highly conserved glycoprotein hormone that, besides thyroid stimulating hormone (TSH), is a potent ligand of the TSH receptor. Thyrostimulin is considered the most ancestral glycoprotein hormone and orthologs of its subunits, GPA2 and GPB5, are widely conserved across vertebrate and invertebrate animals. Unlike TSH, however, the functions of the thyrostimulin neuroendocrine system remain largely unexplored. Here, we identify a functional thyrostimulin-like signaling system in Glycoprotein hormones are key neuroendocrine factors that control diverse physiological processes, such as development, reproduction, energy homeostasis and growth , 2. In vDrosophila melanogaster and the mosquito Aedes aegypti and thyrotropin-releasing hormone (TRH), have been identified in C. elegans and were found to have conserved physiological roles, such as in the control of reproduction and growth , respectively. GPLA-1 and GPLB-1 resemble vertebrate GPA2 and GPB5 and are widely conserved in nematodes and gplb-1 (ibt4) knockout mutants display a significantly shorter body length compared to wild-type animals mutant carries a substitution of the glycine residue between the second and third cysteine (G63E), which is highly conserved and important for proper cysteine knot formation (gpla-1 (ok2127) mutant contains a large deletion, removing the start codon and the complete first exon of the gene cells. We co-expressed FSHR-1 with a cAMP response element (CRE)-luciferase reporter in HEK cells and quantified its activation by assessing bioluminescence levels in the presence of luciferin substrate and the enteric muscles of the hindgut, which are involved in defecation or hmc and the enteric muscles (gplb-1).To gain further insight into the role of thyrostimulin-like signaling in growth regulation, we investigated the expression patterns of defects 2E, show defects , 49, 50, elegans . These i elegans \u201355. We afecation \u201357. Takefshr-1 under its endogenous promoter recapitulated the reported expression of the receptor in the intestine and in multiple neurons in the head and exp-1(ox276) mutants are also significantly smaller than wild-type animals have a carboxytail extension, referred to as the \u201cseatbelt\u201d, that wraps around the alpha subunit to stabilize heterodimer configuration , 79, 80.erodimer , 28, 82.erodimer , 38, 82.solution . Howeversolution . Based osolution . Severalsolution , in whicC. elegans FSHR-1, is abundantly expressed in the alimentary canal superfamily that is required for intestinal calcium and pH oscillations plates seeded with Escherichia coli OP50 bacteria. All experiments were performed using 1-day adult hermaphrodites, unless mentioned otherwise. Wild-type (N2-Bristol), IBE7 flr-2 (ut5), IBE24 flr-2 (ok2127), IBE1 fshr-1 (ok778) and EG1285 lin-15B&lin-15A (n765) oxIs12 [unc-47p::GFP + lin-15(+)] strains were obtained from the Caenorhabditis Genetics Center . CB156 unc-25 (e156) and EG276 exp-1 (ox276) strains were a kind gift from the lab of W.R. Schafer . Deletion alleles for gpla-1 (ibt1) and gplb-1 (ibt4) were obtained by CRISPR/Cas9 genome editing, as described below. A full list of strains used in this study can be found in All fshr-1 (a isoform), gpla-1, and gplb-1 (a isoform) were cloned in front of a SL2 trans-splicing site and fluorescent reporter gene.All fluorescent reporter and rescue constructs were made using the MultiSite Gateway Three-Fragment cloning system (Invitrogen). Genomic DNA or cDNA sequences of fshr-1 (3110 bp), gpla-1 (4058 bp), and gplb-1 (3589 bp) were cloned from genomic DNA of wild-type C. elegans. Tissue-specific rescue transgenes were generated using promoter regions of rab-3 (1208 bp) (rgef-1 (3660 bp) (mir-228 (2217 bp) , 66, rge3660 bp) , ges-1 \u201363, and 2217 bp) . All confshr-1 in HEK cells was obtained by directionally cloning the fshr-1 cDNA into the pcDNA3.1/V5-His-TOPO vector (Invitrogen). The cDNA sequence of the fshr-1a gene isoform was amplified by PCR using cDNA from mix-staged wild-type C. elegans as template. The forward primer included a \u2018CACC\u2019 sequence at the 5\u2019 end that introduced a partial Kozak sequence for increased translation efficiency in mammalian cells.The plasmid for heterologous expression of gpla-1(ibt1) and gplb-1(ibt4) knockout alleles were generated by CRISPR/Cas9-mediated deletion of the gpla-1 or gplb-1 open reading frames. For each gene, two crRNA sequences were designed by looking for PAM sites (NGG) that were in the vicinity of the double stranded break site. After analysis of putative off-target sites (http://crispor.tefor.net/), we selected the highest scoring sequences based on their predicted on-target activities. Repair templates were designed to be in-frame and were codon-optimized for C. elegans (https://www.genscript.com/tools/codon-frequency-table). To simplify selection of the successfully edited worms, we used a Co-CRISPR technique in which the dpy-10 gene was also knocked out by CRISPR editing (The editing . We used editing for the editing Table S2gpla-1 (ibt3 [His::GPLA-1]) knockin allele was made by CRISPR/Cas9 mediated insertion of a His-tag at the N-terminus of the gpla-1 open reading frame, using a similar strategy as described above and the crRNA and repair template listed in The Below, the sequences of the gene knockouts and knockins are shown with flanks on the (-) strand. Exons are in grey, with start and stop codons in red, the remaining gene sequences after CRISPR gene editing are underlined, and with the inserted gene sequence in blue.gpla-1(ibt1) deletion:ATGGGCTCCAAAGCACGAGCACGACGACGTTTAAGTTGTTTTTTAAGCGTTTTTGTTGTGACATGCTTATTACAGTACTGCACAGCAGGTGTTACTAAGAATAATAGTTGCAAAAAAGTTGgtacgtcacgaaatctactaaacttcgatcagtgtcctttaaatttttttttcagGAGTGGAGGAACTTATAGATGAAGAAGGCTGTGATTTGATGATAATTCGAATCAATCGATGCAGTGGGCATTGCTTCTCATTTACATTTCCTAATCCCTTAACGAAAAAATATTCAGTGCATGCGAAGTGCTGCCGGATGGTTGAATGGGAAATGgttagtattttttaactacagaaatgacttctgaaaatttataacaaagttattacggaagccaaaattctgggaatgtgttttgcgcaacatgtaaaaaaaatctcgtagcgaaagctacagtaattctctaaataactactgtagtgcttgcgtcgaattacggacttgatttgcgatatatccttcgtttctctgtattactttctcatttttgttttttttttaaacattctatcgaaaaattgatgattaattcatttcgaaagccgagcccgtaaatcgtcacaagcgctacagtagtcatgtaaagaattactgtatcgctacgagatgttttaatattgttttccaagaatcaatttttatttttcagCTTGAAACAGAATTAAAATGTTCCAAAGGAAACCGAAATCTTCGAATACCATCTGCAACACAATGTGAATGTTTTGATTGTCTTGTTCGATAGttgcagtttccatctctttcatttctcattcttgaatctcttgagttttacctgcataatagatttaatttattttctctcctccagtgccacattcatccacatttccataaaatctcgttcccttaattatttctataataatctcgttgatacgggcatctaaaaatgcaaaaaatcaaagcgaaagaaggatgactgacataaattgtctcaattctttaaattctatttttaacttttcagcctataccttctcaaatggplb-1(ibt4) deletion:tgttgacgttaagctcttttgaaaaaaATGCTTATATTTCCCGTGATCACTATACTTCATATATTTTTGgtaactttttgaaatatcttctaaaatgaatttaatatgtaacaatattctaacaaaatttttaatattgatcagatagtttttccaaatttaagtattaaccaggcgcgaaattttccgaattttaggccaaaaatacggtgcccggtctcgacacgaatttttttattaggtaaaaatgggtgtgtgcctttaaagagtactgtaactttaaactttcgttgctgcggaacttttgtcgacttttcatagctattagataaaaataaaaaaatattcaatattttcaacaaatctttagaaaaactatgtaaaatcgataaaaattctgcaacaaaaatttgaagttacagtactctttaaaggcacacactcgattgtatttaacaaaaaaagtcgtgtcgagaccggttaccgtaatttttgcgcaaatcggaataatttcgcgcttgggtaataagcatcacatctccaactaatttaaaagcaaaagtgtgatttttaaattcagATTTCGGTTGAATCTGGAAAAGAATGCGAGTTTGCAATGCGATTGGTCCCAGGATTCAATCCACTTCGTCAAGTTGATGCAAATGGAAAAGAATGCCGAGGAAACGTGGAATTGCCATTTTGCAAGGGTTACTGTAAGACTAGCGAGgtgaatttccttttttttccgattcaaaaataatccaattaaatttcagAGTGGCACCCATGGCTTTCCACCACGAGTTCAAAATAGTAAAGTGTGCACATTGGTCACCACTTCAACTCGAAAAGTAGTTCTTGATGATTGTGATGATGGAGCCGATGAGAGTGTCAAGTTTGTAATGGTTCCACATGGAACTGATTGTGAATGTTCTGCAGTTCCACTTGAACAACATCATTCATAAattatcatacattcattcaaaaattcatcgaataaataaaagttttgtggpla-1(ibt3 [His::GPLA-1]) insertion:ttctttaaattctatttttaacttttcagcctataccttctcaaatgATGCATCACCATCACCATCACGGCTCCAAAGCACGAGCACGACGACGTTTAAGTTGTTTTTTAAGCGTTTTTGTTGTGACATGCTTATTACAGTACTGCACAGCAGGTGTTACTAAGAATAATAGTTGCAAAAAAGTTGgtacgtcacgaaatctactaaacttcgatcagtgektcctttaaatttttttttcagGAGTGGAGGAACTTATAGATGAAGAAGGCTGTGATTTGATGATAATTCGAATCAATCGATGCAGTGGGCATTGCTTCTCATTTACATTTCCTAATCCCTTAACGAAAAAATATTCAGTGCATGCGAAGTGCTGCCGGATGGTTGAATGGGAAATGgttagtattttttaactacagaaatgacttctgaaaatttataacaaagttattacggaagccaaaattctgggaatgtgttttgcgcaacatgtaaaaaaaatctcgtagcgaaagctacagtaattctctaaataactactgtagtgcttgcgtcgaattacggacttgatttgcgatatatccttcgtttctctgtattactttctcatttttgttttttttttaaacattctatcgaaaaattgatgattaattcatttcgaagccgagcccgtaaatcgtcacaagcgctacagtagtcatgtaaagaattactgtatcgctacgagatgttttaatattgttttccaagaatcaatttttatttttcagCTTGAAACAGAATTAAAATGTTCCAAAGGAAACCGAAATCTTCGAATACCATCTGCAACACAATGTGAATGTTTTGATTGTCTTGTTCGATAGttgcagtttccatctctttcatttctcattcttgaatctcttgagttttacctgcataatagatttaatttattttctctcctccagtgccacattcatccacatttccataaaatctcgttcccttaattatttctataataatctcgttgatacgggcatctaaaaatgcaaaaaatcaaagcgaaagaaggatgactgacataaattgtctcaamyo-2p::mCherry, unc-122p::dsRed, or unc-122p::GFP) and 1-kb DNA ladder (Thermo Scientific) as carrier DNA.Germline transformations were carried out by injecting constructs into the syncytial gonad of young adult worms together with a co-injection marker and resulting Z-stack projections were created and analyzed with ImarisViewer (v9.7.2) software. Adult worms were immobilized by mounting in 50 mM Sodium Azide solution in M9 buffer on a 2% agarose pad and covered with a glass cover slip. Expression patterns were confirmed in at least two independent transgenic strains. DVB and RME expression was confirmed by crossing with marker strain EG1285, which marks GABAergic neurons . Other chttps://github.com/jwatteyne/WormSizer). Synchronized L1 larvae were placed onto freshly seeded NGM plates one day post-synchronization and kept at 20\u00b0C until assaying. After 65 hours on food, 20 to 30 well-fed day one adults were transferred from the plates and anesthetized in 20 \u00b5L of 10 mM tetramisole hydrochloride solution (Sigma-Aldrich) in Milli-Q water on a 2% agarose pad. Images were captured with a ZEISS Axio Observer.Z1 at 5x magnification. Body size was measured from images of anesthetized day 1 adult worms on at least two independent days. After skeletonization, delineating the worm\u2019s outline and midline, the worm\u2019s total length and middle width were calculated using the calibration factor (pixels/\u00b5m) of the pictures. For the volume estimation, the midline of the worm was separated in 30 segments of the same size. The volume for each segment was determined by using the formula for the frustum of a cone, taking the natural shape of a worm into consideration. The summation of all these volumes was described as the worm\u2019s total volume. To assess growth throughout adulthood, body size measurements were executed for 5 subsequent days (day 1 to day 5 adulthood) for at least two independent time periods.Body size parameters of day one adults were measured using the custom WormSizer MATLAB script , luminal width was determined by measuring the average width (in \u00b5m) of the intestine at two different points in the posterior lumen. Body size parameters and luminal widths were plotted, and significance levels were calculated with GraphPad Prism 8 software.C. elegans N-terminal 6xHis-tagged GPLA-1 and non-tagged GPLB-1. Recombinant proteins were purified from cell lysate by affinity chromatography in PBS buffer and analyzed by Tricine-SDS-PAGE on a 8 \u2013 20% gel in Tris-Glycine running buffer and by subsequent Western blot using anti-His monoclonal antibodies. A similar approach was used to synthesize individual GPLA-1 and GPLB-1 proteins. N-terminal 6xHis-tagged GPLA-1 was expressed in HEK293 cells and purified by affinity chromatography. Non-tagged GPLB-1 was expressed in HEK cells and purified using ion exchange chromatography. Both recombinant GPLA-1 and GPLB-1 protein samples were analyzed by Tricine-SDS-PAGE.Recombinant proteins were synthesized and purified by OriGene\u2122 Technologies . To generate recombinant GPLA-1/GPLB-1, HEK293 cells were transfected with expression vectors encoding 2, in Dulbecco\u2019s Modified Eagle\u2019s Medium (DMEM)/Nutrient F-12 Ham (Gibco) supplemented with 10% heat inactivated Fetal Bovine Serum and 1% Penicillin/Streptomycin (Gibco). Co-transfection with the cAMP indicator CRE(6x)-luciferase and pcDNA3.1/fshr-1a was done in a 1:1 ratio when cells reached 60-70% confluency. Transfection medium contained Opti-MEM , CRE(6x)-luciferase and receptor plasmid DNA, Plus Reagent (Invitrogen) and Lipofectamine LTX (Invitrogen). One day post-transfection, fresh culture medium was added to the transfected cells. Two days post transfection, each well of a Bio-One CELLSTAR\u2122 96-well, Flat Bottom Microplate (Greiner) was loaded with a dilution series of recombinant protein compounds in 200 \u00b5M 3-isobutyl-1-methylxanthine (IBMX), which inhibits cAMP hydrolysis. Compound buffer (PBS with 10% glycerol) without hormone was added to the IBMX medium as a ligand-free negative control. Cells were detached, counted, pelleted and resuspended to a concentration of 10E-06 cells/mL in IBMX medium. Each well of the compound plate was supplemented with 50 \u00b5L of the cell suspension (50 000 cells/well) and the plate was incubated at 37\u00b0C for 3.5 h. Cells were then loaded with 100 \u00b5L SteadyLitePlus substrate (PerkinElmer) and incubated on a shaking plate for 15 minutes under dark conditions at RT. Finally, luminescence was measured twice for 5s (at 0s and 5s) per well at 469 nm on a Mithras LB 940 luminometer (Berthold Technologies). Measurements were performed in triplicate in at least 4 independent experiments. Luminescence values were plotted and significance levels were calculated with GraphPad Prism 8 software.The cAMP bioluminescence assay quantifies ligand-induced bioluminescent responses by measuring changes in intracellular cAMP levels after receptor activation. HEK293T cells were cultured in monolayer at 37\u00b0C in a humidified atmosphere with 5% COhttps://github.com/NathanDeFruyt/DefecationAnalysisBeetsLab). The length of one DMP cycle was defined by the time elapsed between two subsequent contractions of the posterior body wall muscles (pBocs). The average cycle length was calculated over 10 defecation cycles for each worm. aBoc frequency was defined as the ratio of aBoc over pBoc. Defecation parameters were plotted and significance levels were calculated with GraphPad Prism 8 software.The defecation motor program (DMP) was analyzed as previously described . AnimalsC. elegans GPLA-1 and GPLB-1 sequences were used as queries to identify sequences with highest similarity in selected model systems representative for their phylum by the Basic Local Alignment Search Tool (BLAST) of the National Center for Biotechnology Information (NCBI) database. The multiple sequence alignment program Clustal Omega was used to align sequences, and conserved residues were identified using BoxShade .The H. Sapiens GPA2 and GPB5 and The original contributions presented in the study are included in the article/SK, MI, JW, LS and IB designed the research. SK, MI, SD and EV performed the experiments. SK, MI and IB analyzed data. IB and LS supervised the project and were responsible for funding acquisition. SK and IB wrote the paper. All authors contributed to the article and approved the submitted version."} +{"text": "The kinase BsfK specifically catalyzes the phosphorylation of the precursor peptide BsfA on the Ser3 residue. BsfB1 performs dual functions to accelerate the post-translational phosphorylation and assist BsfB2 in leader peptide removal. Most importantly, the penultimate residue of leader peptide is an isoleucine rather than the conserved threonine and this isoleucine has a marked impact on the phosphorylation of Ser3 as well as leader peptide removal, implying that BsfB1 and BsfB2 exhibit a new substrate selectivity for leader peptide binding and excision. This is the first experimentally validated penultimate isoleucine residue in a lasso peptide precursor to our knowledge. In silico analysis reveals that the leader peptide Ile/Val(-2) residue is rare but not uncommon in phosphorylated lasso peptides, as this residue is also discovered in Acidobacteriaceae and Sphingomonadales in addition to Bradymonadales.Lasso peptides are ribosomally synthesized peptides that undergo post-translational modifications including leader peptide removal by B (or the segregated B1 and B2) proteins and core peptide macrolactamization by C proteins to form a unique lariat topology. A conserved threonine residue at the penultimate position of leader peptide is hitherto found in lasso peptide precursors and shown to be a critical recognition element for effective enzymatic processing. We identified a lasso peptide biosynthetic gene cluster ( C-terminal tail threads through the macrolactam ring formed by the N-terminal amino group and the carboxylic acid side chain of an aspartate or a glutamate located at position 7\u20139 of the core peptide via an isopeptide bond (D), which is responsible for excretion and self-resistance. B genes from actinobacteria and firmicutes are usually split into two genes: B1 encodes the N-terminal RiPP precursor recognition element (RRE) domain with homology to PqqD family proteins, while B2 encodes the C-terminal protease domain that cleaves the leader peptide of the precursor, after which the remanent core peptide is macrolactamized to form the mature lasso peptide with a characteristic lariat topology, in which the ide bond . The numide bond . Generalide bond . Many la peptide .Beyond the class-defining leader peptide removal and core peptide macrolactamization, nine unique post-translational modifications (PTMs) are reported in total in the biosynthesis of lasso peptides up to now , among wBradymonas sediminis FA350 is a Gram-negative, rod-shaped, and facultatively prey-dependent bacterium isolated from coastal sediments in Weihai, China. Phylogenetic analysis based on 16S rRNA gene sequences showed that this strain belongs to a novel bacterial order Bradymonadales and a novel family Bradymonadaceae in the class Deltaproteobacteria (B. sediminis FA350 (accession No. NZ_CP030032.1) and subsequent search for natural products BGCs allowed the identification of a lasso peptide BGC, which we subsequently named bsf (bsfA), a discrete RRE (bsfB1), a leader peptidase (bsfB2), a lasso peptide cyclase (bsfC) and an ABC-transporter (bsfD), two additional genes encoding a putative kinase (bsfK) and a predicted nucleotidyltransferase (bsfN) have been found in this BGC as well. Herein, we characterized the activities of BsfK, BsfB1, and BsfB2.bacteria . Sequencamed bsf . The canApexHF HS DNA polymerase for high-fidelity amplification from Accurate Biology Co. Ltd. (China). Chemical compounds and reagents were purchased from Bide Pharmatech Ltd. (China), Macklin Biochemical Technology Co., Ltd. (China) and J&K Scientific Ltd. (China) unless stated otherwise. Gene synthesis and codon optimization were performed at GENEWIZ, Inc. (China). Primer synthesis and DNA sequencing were performed at Shanghai Sangon Biotech Co. Ltd. (China). Primers used in this study are summarized in Biochemicals and media were purchased from Sinopharm Chemical Reagent Co. Ltd. (China) or Oxoid Ltd. (United Kingdom) unless stated otherwise. Enzymes were purchased from New England Biolabs Ltd. (United Kingdom) except 2O containing 0.1% formic acid) and solvent B (CH3CN containing 0.1% formic acid) with a flow rate of 0.3\u2009mL/min over a 25\u2009min period as follows: T\u2009=\u20090\u2009min, 5% B; T\u2009=\u20093\u2009min, 5% B; T\u2009=\u200918\u2009min, 95% B; T\u2009=\u200922\u2009min, 95% B; T\u2009=\u200923\u2009min, 5% B; and T\u2009=\u200925\u2009min, 5% B (mAU at 220\u2009nm). ESI-MS was performed on a Bruker AmaZon SL Ion Trap LC/MS spectrometer , and the data were analyzed using Bruker Daltonics DataAnalysis. ESI-HRMS analysis was carried out on a Bruker High Resolution Q-TOF mass spectrometry and the data were analyzed using Bruker Daltonics DataAnalysis. Tandem MS analysis was performed by collision-induced dissociation (CID) using He as collision gas.HPLC analysis was carried out on a Thermo Fisher Dionex UltiMate 3,000 UHPLC system using an Acclaim TM RSLC 120 C18 column by gradient elution of solvent A and purified by ethanol precipitation. The digested genomic DNA, p15A vector with homologous arms, T4 polymerase, and buffer were mixed and reacted in a thermocycler with the following program: 25\u00b0C, 60\u2009min; 75\u00b0C, 20\u2009min; 50\u00b0C, 60\u2009min. The mixture was then electroporated into the l-arabinose induced E. coli GB05-dir competent cells and plated on appropriate Luria-Bertani (LB)-agar plates to incubate at 37\u00b0C overnight. The bsf region (approximate 22.67\u2009kb) was cloned into the corresponding p15A-cm vectors in E. coli, and several constitutive promotors such as PTn5-Km, Papra, or the inducible promoter Ptet were subsequently inserted upstream to bsfC, respectively, leading to the constructions of p15A-cm-Tn5-km-bsf, p15A-cm-Papra-bsf, and p15A-cm-Ptet-km-bsf. Then, the plasmids were electroporated into the heterologous hosts E. coli BL21(DE3) or E. coli GB2005. In order to heterologous express the BGC in Burkholderiales hosts, the chloramphenicol resistance gene was then replaced with phiC31-site-specific integrase by recombineering, resulting the construction of p15A-phiC31-amp-Papra-bsf. This plasmid was then electroporated into Schlegelella brevitalea DSM 7029 or introduced into the specific chromosomal site of Burkholderia gladioli ATCC10248 and Burkholderia thailandensis E264 with the help of donor strain E. coli WM3064, which is auxotrophic for diaminopimelic acid , inserted into the BamHI-HindIII site of the plasmid pRSFDuet-1ATGGAAATGAAAAAAGCTAAAAACAAAATCTCTCGTAAACTGATCTACAAAAAACCGCAGCTGACCTACCACTCTACCCTGGCTGCTATCATCCGTGGTACCTCTGGTAACGAAGTTGACGGTTCTACCCCGGCTGACCGTGGTAACAACCCGGGTGACTAAbsfB1 (accession No. WP_162687378.1), inserted into the NdeI-XhoI site of the plasmid pET28aATGCGTGAAACCCCGATGACCGAAAACCTGTTCCCGCGTGACGCTGTTTTCTCTATCCGTGAAGACCTGGTTGTTGAACAGGTTGACGACGAATTCCTGGTTCTGGACCTGCGTGGTAACGAATACTTCGGTCTGAACGCTGTTGCTCGTCACATCTGGGCTGCTATCGACGCTGGTGACTCTCTGGCTGCTATCGCTGACTCTGTTTGCGAACGTTTCGAAGTTGAACGTGAACGTGCTGCTACCGACGTTGCTGACTTCATCGCTAACCTGCTGGAACAGCGTCTGGTTTCTCGTGTTGACGCTTAAbsfB2 (accession No. WP_111331306.1), inserted into the NdeI-XhoI site of the plasmid pET28aATGAAACCGATCCACCCGATCGCTCAGGCTGAAATGCTGGTTGAAGTTGCTATCGCTCGTGTTCTGTCTGAATTCCTGTCTATCGAAGACCTGCTGCGTCTGGTTGGTGAAGCTGCTCGTATCGCTCAGCAGTGGCCGGCTCGTTCTTGCGTTGCTTCTCCGTCTGCTGAAGCTATCAACGAAACCGCTGCTCTGTCTGAACTGCTGGCTCACCGTGCTTCTCGTCTGGTTCCGGGTGCTCAGTGCCTGCAGCGTGCTCTGGCTGGTCGTGTTTGGCTGGCTCGTCGTGGTATCGCTTCTGAAATCGTTGTTGGTTTCCGTAAACGTGGTGTTCTGGAAGGTCACGCTTGGCTGGAAGTTATGTCTCCGGACGGTCTGATCGAACTGTTCAACAACGCTGACGACGGTTACCGTGAATCTTTCCGTGAAGTTGCTGCTTAAbsfK (accession No. WP_204355063.1), inserted into the NdeI-XhoI site of the plasmid pET28aATGGAAAACACCGACCGTCTGGTTCGTTACCGTGCTTTCGGTCGTGACATCGACGGTCCGGCTGGTCTGTCTCTGTCTCCGGCTCCGGCTGAAGCTGCTCCGGGTGAAGTTCCGGTTCTGCGTCTGCAGCCGGACGCTTCTCTGCGTGTTTTCATCGACGAAAACCTGCCGCCGCTGCTGCACAACGTTGAAGACTACCCGGGTGGTCCGAAATTCTACGTTTGGCAGGAAGGTGAAGCTGTTGGTGTTCAGTACGACCGTTGGCGTACCCGTCTGATCCCGGGTGAAGGTCGTATCGACTTCGCTGAACTGCCGCCGTCTGGTCCGAAACGTGTTGAAGACGCTGGTGACGAATACGGTCGTTTCCGTTTCTCTCTGGCTATGGAACGTGTTTTCCTGCCGCTGTACGCTCTGTTCTCTATGCCGGACGCTGTTGCTCTGCACGGTTCTGCTGTTGTTCTGAACGGTGAAGCTTTCCTGTTCATCGGTCGTTCTGGTGCTGGTAAATCTACCACCGCTTACGAATTCGTTCGTCGTGGTGCTACCCTGCTGGCTGACGACCTGATCGTTGCTGACGTTGCTCGTGGTATCGCTCTGGGTGGTGCTCCGACCCTGCGTCTGTGGAAAGGTGAAGGTGCTCTGCCGGAAGCTCAGGAAGACCGTTCTCTGTGGCGTCACGACGCTTCTAAACGTTGGTTCCGTATCCCGGCTGAACGTGGTGCTGCTTCTGCTGTTCCGATCGCTGCTATCGTTATGCTGGACCCGGACACCCTGGGTGGTCAGCGTGACGTTCTGCCGGGTCTGGAAACCTCTCCGCAGCGTAAAGCTCTGACCGACCTGCTGGGTCAGACCTTCGACCTGTCTCACGGTACCCCGGAATGGATGGTTGCTCGTTTCCGTAACACCGCTCGTCTGATCCGTGAATACCCGTTCTACCACTTCCGTTACGTTAAATCTGCTGACGGTAAACCGACCCACATGGACGCTCTGTACCAGGCTATCGTTGGTCTGGCTTCTAAATAAbsfK was amplified by PCR with pET28a\u2009+\u2009bsfK as the template. After purification by agarose gel electrophoresis and restrictive digestion, the bsfK fragment was inserted into the NdeI-XhoI site of the plasmid pRSFDuet-1\u2009+\u2009bsfA to yield the recombinant plasmid pRSFDuet-1\u2009+\u2009bsfA\u2009+\u2009bsfK.The DNA fragment containing bsfB1 or bsfB2 was amplified by PCR, respectively. The fragments were inserted into the NcoI-HindIII site of pACYCDuet-1 and the NdeI-XhoI site of pCold-TF to yield the recombinant plasmids pACYCDuet-1\u2009+ bsfB1 and pCold-TF\u2009+ bsfB2, respectively.The DNA fragment containing bsfA was transferred into E. coli BL21 (DE3) for expression. BsfA, fused to an N-terminal 6\u2009\u00d7\u2009His tag was expressed at 20\u00b0C for 24\u2009h with 100\u2009\u03bcM isopropyl-\u03b2-d-thiogalactopyranoside induction and shaking at 200\u2009rpm. Cells were harvested by centrifugation at 4\u00b0C and re-suspended in buffer A containing 6 M guanidine hydrochloride, 20\u2009mM NaH2PO4 (pH 7.5), 500\u2009mM NaCl, 0.5\u2009mM imidazole. After disruption by a low-temperature, ultra-high-pressure homogenizer, the insoluble material was removed by centrifugation at 20,000\u2009g and 4\u00b0C for 1\u2009h. The soluble fraction was subjected to purification using a Ni Bestarose FF column . Briefly, the column was equilibrated with at least 2 column volumes of binding buffer. Then, the pre-treated sample was loaded onto the Ni Bestarose FF column, and washed with elution buffer containing a low concentration of imidazole (25\u2009mM) to remove the impurities. The sample was further eluted with elution buffer containing a high concentration of imidazole (1\u2009M) to obtain the purified BsfA peptide. The eluted fractions were desalted by reversed phase (RP) HPLC on a Shimadzu LC-20AT system equipped with a ReproSil 300 C4 column . The solvent used for RP-HPLC separation was deionized water with 0.1% trifluoroacetic acid , and acetonitrile with 0.1% TFA (solvent B). A gradient was applied: 5% B for 15\u2009min, ramp up to 100% B over 50\u2009min, hold at 100% B for 7\u2009min, and ramp to 5% B over 5\u2009min. Eluted fractions were monitored by UV absorbance at 220\u2009nm. Desalted precursor peptides were lyophilized and stored at \u221220\u00b0C. The peptide was validated by HPLC-ESI-(HR)MS analysis on an Acclaim TM RSLC 120 C18 column by gradient elution of solvent A (H2O containing 0.1% formic acid) and solvent B (CH3CN containing 0.1% formic acid) with a flow rate of 0.3\u2009mL/min over a 25\u2009min period as follows: T\u2009=\u20090\u2009min, 5% B; T\u2009=\u20093\u2009min, 5% B; T\u2009=\u200918\u2009min, 95% B; T\u2009=\u200922\u2009min, 95% B; T\u2009=\u200923\u2009min, 5% B; and T\u2009=\u200925\u2009min, 5% B (mAU at 220\u2009nm).The plasmid pRSFDuet-1\u2009+\u2009bsfA\u2009+\u2009bsfK was electroporated solely or co-electroporated with pACYCDuet-1\u2009+\u2009bsfB1 into E. coli BL21 (DE3) for expression. The precursor peptides were purified, desalted, lyophilized, and validated according to the procedures described above for unmodified BsfA precursor peptide.The plasmid pRSFDuet-1\u2009+\u2009bsfK was transferred into E. coli BL21(DE3) and expressed according to the procedures described above for BsfA. Cells were harvested by centrifugation at 4\u00b0C and re-suspended in lysis buffer containing 50\u2009mM Tris\u2013HCl (pH 8.0), 300\u2009mM NaCl, 5\u2009mM imidazole and 10% (v/v) glycerol. After disruption by a low-temperature, ultra-high-pressure homogenizer, the insoluble material was removed by centrifugation at 20,000\u2009g and 4\u00b0C for 1\u2009h. The soluble fraction was subjected to purification using a Ni Bestarose FF column. The column was equilibrated with at least 2 column volumes of binding buffer. Then, the pre-treated sample was loaded onto the Ni Bestarose FF column, and washed with binding buffer. The sample was further eluted with elution buffer using a stepwise gradient containing different concentrations of imidazole (10\u2013500\u2009mM). The elution fractions were determined by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis, and the fractions containing the recombinant protein were desalted using a PD-10 Desalting Column into storage buffer containing 50\u2009mM Tris\u2013HCl (pH 8.0), 100\u2009mM NaCl, 10% (v/v) glycerol, and 1\u2009mM DTT. The resulting protein was concentrated and stored at \u221280\u00b0C. The purity of the protein was determined by SDS-PAGE analysis, and the concentration was determined by the Bradford assay using bovine serum albumin (BSA) as the standard.The plasmid pET28a\u2009+\u2009bsfB1 and pCold-TF\u2009+\u2009bsfB2 were transferred into E. coli BL21 (DE3) for expression, respectively. The expression temperature was 15\u00b0C for TF-BsfB2 instead of 20\u00b0C for BsfK and BsfB1. The proteins were purified to homogeneity, and concentrated according to the procedures described above for BsfK.The plasmids pET28a\u2009+\u2009bsfA or pRSFDuet-1\u2009+ bsfA\u2009+\u2009bsfK as the template. Then, 1\u2009\u03bcL of DpnI enzyme was added to the PCR system and incubated at 37\u00b0C for 3\u2009h to remove the template. And the PCR system was electroporated into E. coli BL21 (DE3). After sequencing to validate the fidelity, the resulting precursor peptide variants were expressed individually, or co-expressed with BsfK and BsfB1 in E. coli BL21 (DE3), and purified to homogeneity, desalted, and lyophilized according to the procedures described above for the native precursor peptides.Plasmids containing site-directed mutations were generated by PCR amplification with pRSFDuet-1\u2009+ bsfK as the template. Then, 1\u2009\u03bcL of DpnI enzyme was added to the PCR system and incubated at 37\u00b0C for 3\u2009h to remove the template. The PCR system was electroporated into E. coli BL21 (DE3). After sequencing to validate the fidelity, the resulting BsfK variants were expressed in E. coli BL21 (DE3), purified to homogeneity and desalted according to the procedures described above for the native protein.Plasmids containing site-directed mutations were generated by PCR amplification with pET28a\u2009+ 4HCO3 buffer (pH 7.8) to a concentration of 500\u2009\u03bcM. 20\u2009\u03bcg trypsin was dissolved in 20\u2009\u03bcL resuspension buffer (50\u2009mM acetic acid) and heated at 30\u00b0C for 15\u2009min before utilization for maximum activity. 10\u2009\u03bcL resuspended trypsin was added to a total volume of 50\u2009\u03bcL BsfA reaction system, followed by incubation at 37\u00b0C for 2\u2009h. The reaction was quenched by adding an equal volume of CH3OH. After removal of precipitate by centrifugation, the mixtures were then subjected to HPLC-ESI-HRMS analysis according to the procedure described above for BsfA precursor peptide.The (phosphorylated) precursor peptide BsfA (or the BsfA variants) was dissolved in 50\u2009mM NH2, 5\u2009mM ATP, 200\u2009\u03bcM BsfA in the presence of 20\u2009\u03bcM BsfB1 and 20\u2009\u03bcM BsfK (or the BsfK variants). ATP was added last to initiate the reaction. The assays were quenched by adding an equal volume of CH3CN. After removal of precipitate by centrifugation, the mixtures were then subjected to HPLC-ESI-(HR)MS analysis according to the procedure described above for BsfA precursor peptide.The assays for BsfK were performed at 30\u00b0C for 5\u2009h in 100\u2009mM Tris\u2013HCl buffer (pH 8.0) containing 5\u2009mM MgCl2HPO4 buffer (pH 7.5) containing 100\u2009mM NaCl, 2\u2009mM MgCl2, 2\u2009mM ATP, 1\u2009mM DTT, 100\u2009\u03bcM BsfA in the presence of 20\u2009\u03bcM BsfB1 and 20\u2009\u03bcM TF-BsfB2. ATP was added last to initiate the reaction. After similar termination and centrifugation, the mixtures were then subjected to HPLC-ESI-(HR)MS analysis according to the procedure described above for BsfA precursor peptide.The assays for TF-BsfB2 were performed at 30\u00b0C for 12\u2009h in 100\u2009mM Kbsf cluster is prone to be silent under laboratory conditions, and the Bradymonas sediminis FA350 strain is difficult to genetically manipulate. Thus, heterologous expression of the bsf cluster has been tried in other hosts. Briefly, the DNA fragment containing all the bsf genes and the flanking regions was removed from the genomic DNA by restriction digestion and further connected to the vector p15A via Red/ET recombination. The obtained plasmid (p15A-bsf) was then transformed into E. coli BL21(DE3), E. coli GB2005 and Schlegelella brevitalea DSM 7029, or conjugated into Burkholderia gladioli ATCC10248 and Burkholderia thailandensis E264, respectively, for heterologous expression in different culture media. However, no mass corresponding to the predicted lasso peptide has been observed. Moreover, several constitutive promotors such as PTn5-Km, Papra and the inducible promoter Ptet have been inserted upstream to bsfC, respectively, but the mature lasso peptide has never been detected. This results in difficulty to demarcate the boundary between the leader and the core peptide in BsfA. It was reported that the conserved Tyr(-17), Pro(-14), and Leu(-12) residues in leader peptides were required for the recognition by the B1 proteins , Microvenator marinus V1718 (mmv), Lujinxingia litoralis B210 (llb), L. sediminis SEH01 (lss) and L. vulgaris TMQ2 (lvt), et al. motif motif , indicatocessing . Neverthpeptides .N-terminally hexaHis-tagged BsfA (BamHI\u2009+\u2009HindIII) and untagged BsfK (NdeI\u2009+\u2009XhoI). After purification, high performance liquid chromatography-electrospray ionization-high resolution mass spectrometry (HPLC-ESI-HRMS) was used for the detection of PTMs in the N-terminally tagged BsfA. Apart from the dominant unmodified BsfA , a small component with a mass increase of 79.9640\u2009Da was also detected, which was barely visible when bsfK was omitted with other proteins in the National Center for Biotechnology Information (NCBI) database.A BLASTP search using the BsfK protein sequence as input revealed that it was a hypothetical protein with a very low identity (<35%) with histidine containing phospho carrier protein (HPr) kinases . To vali omitted . This inNext, untagged BsfB1was introduced into the co-expression system above. In comparison with the BsfA and BsfK co-expression experiment , the monC-terminal Ser residues of the precursors , indicative of a distinct phosphorylated position from the known lasso peptides. Trypsin digestion and subsequent tandem MS analysis of phosphorylated precursor peptide pointed to the Ser3 residue as the modification site by BsfK . It was observed that while BsfA S3T was phosphorylated efficiently, no phosphorylation was observed with BsfA S3Y, surmising that BsfK is somewhat substrate tolerant . This fiN-terminally hexaHis-tagged BsfK was characterized in vitro in the presence of the BsfA-precursor, ATP and magnesium chloride. In accordance with the in vivo characterization, BsfK phosphorylated the precursor peptide in a very low yield, whereas no conversion was observed in the absence of ATP, magnesium chloride, or with heat-treated BsfK tag , which was confirmed by MS/MS fragmentation analysis . A similar result was obtained when unphosphorylated precursor peptide was used instead of phosphorylated precursor. The mass of unphosphorylated BsfA core peptide was detected and its identify was further verified by MS/MS analysis (Since the (TF) tag . HPLC-ES(TF) tag . The foranalysis . The latanalysis . These ranalysis , which iAcidobacteriaceae bacterium TAA 166 residue is also observed in a list of Sphingomonadales derived lasso peptide precursors I substitutions were still accepted as substrates from the processing machinery, albeit with lower turnover , 2014. I TAA 166 , and a n TAA 166 . Besidesecursors , suggestecursors . These aC-terminal serine residues were reported for ThcoK and SyanK (We were curious to know whether the leader peptide Ile(-2) residue in BsfA also plays a role in phosphorylation and leader peptide removal. This residue was targeted for replacement with other amino acids. Ile(-2) was first exchanged with the congener leucine residue in BsfA, which was then co-expressed with BsfB1 and BsfK. HPLC-ESI-HRMS analysis of the BsfA variant after purification and desalting revealed that only part of the precursor peptide was phosphorylated , comparend SyanK , whereasBradymonadales derived B1 proteins (The BsfA Ile(-2) substitution variants were furthermore utilized for the characterization of TF-BsfB2 activity. In contrast to the wild type BsfA that was cleaved almost completely, I(-2)T as well as I(-2)A exchanges completely hindered the removal of the leader peptide even in the presence of BsfB1 , 8B. In proteins . The intAla-replacements of Tyr(-17), Pro(-14), and Leu(-12) were also performed and tested for their effects on phosphorylation and leader peptide excision. The Y(-17)A and L(-12)A exchanges did not have any effect on the phosphorylation of BsfA, as the masses of unphosphorylated precursors are barely detected in HRMS analyses. Nevertheless, the P(-14)A variant precipitates immediately after addition to the BsfK assay, and no mass of the precursor is observed. Thus, double (Y(-17)A&P(-14)A) and triple (Y(-17)A&P(-14)A&L(-12)A) exchange variants of BsfA were generated. Surprisingly, neither the double nor the triple Ala substitution affects precursor phosphorylation . MoreoveAcidobacteriaceae and Sphingomonadales.Our results demonstrate that the kinase BsfK specifically catalyzes the phosphorylation of the Ser3 residue in BsfA, while BsfB1 performs dual functions to accelerate the post-translational phosphorylation and to assist BsfB2 in leader peptide removal. Moreover, it is noteworthy that the penultimate residue in the leader peptide is isoleucine rather than the conserved threonine in all other so far investigated lasso peptide precursors. This Ile(-2) residue has a profound effect on the monophosphorylation of Ser3 and leader peptide removal, both of which are decreased in Ile(-2) substitution variants. The presence of a penultimate Ile/Val residue was also observed in putative lasso peptide precursors from Paenibacillus dendritiformis C454, ThcoB2 from Thermobacillus composti KWC4, BaceB2 from Bacillus cereus VD115, PapoB2 from Paenibacillus polymyxa CR1, SyanB2 from Sphingobium yanoikuyae ATCC 51230 and PsmB2 from Bacillus pseudomycoides DSM 12442 are the only B2 proteins reported to be involved in the biosynthesis of phosphorylated lasso peptides can be found in the article/in-silico analyses. D-SM and Z-JD assisted with data analysis and manuscript preparation. GZ and YD analyzed the data and wrote the manuscript. GZ, XB, and YZ designed the work. All authors contributed to the article and approved the submitted version.YD carried out the experiments. YD, WN, and LP performed the This work was supported by the National Natural Science Foundation of China (21907057 and 32170038), the Natural Science Foundation of Jiangsu Province, China (BK20190201), the Future Plan for Young Scholars, and the Fundamental Research Funds (2019GN032) of Shandong University.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."} +{"text": "Numerous circular RNAs (circRNAs) have been recently identified in porcine tissues and cell types. Nevertheless, their significance in porcine spleen development is yet unelucidated. Herein, we reported an extensive overlook of circRNA expression profile during spleen development in Meishan pigs.CD226, MBD2, SAMD3, SIT1, SRP14, SYTL3 gene expressions via acting as miRNA sponges. Moreover, the circRNA_21767/miR-202-3p axis regulated SIT1 expression in a ceRNA manner, which is critical for the immune-based regulation of spleen development in Meishan pigs.Overall, 39,641 circRNAs were identified from 6,914 host genes. Among them, many circRNAs are up- or down-regulated at different time points of pig spleen development. Using WGCNA analysis, we revealed two essential modules for protein-coding genes and circRNAs. Subsequent correlation analysis revealed 67 circRNAs/co-expressed genes that participated in immnue-associated networks. Furthermore, a competing endogenous RNA (ceRNA) network analysis of circRNAs revealed that 12 circRNAs modulated Overall, our results demonstrated that the circRNAs were differentially expressed during different stages of porcine spleen development, meanwhile the circRNAs interacted with immune-related genes in a ceRNA-based fashion. Moreover, we presented biomedical researchers with RNAseqTools, a user-friendly and powerful software for the visualization of transcriptome profile data.The online version contains supplementary material available at 10.1186/s12864-023-09612-x. Sus scrofa) are an incredible resource for agriculture and food production, as well as biomedical research for the examination of human development, congenital diseases, and pathogen response networks [Pigs is a newly discovered form of non-coding RNA (ncRNA) known for its enclosed configuration, generated via precursor mRNA back-splicing . In contSpleen is a secondary lymphoid organ involved in older erythrocyte elimination, iron recycle, capture and destruction of pathogens, and induction of adaptive immune responses , 18. BeiTo explore the presence of circRNAs during spleen development, we assessed circRNAs expression in the spleen tissues of Meishan pigs at various developmental stage. We prepared and sequenced ribo-depleted total RNA-seq libraries, as shown in the flow chart Fig.\u00a0. Table STo characterize the circRNAs in the Meishan pig spleen tissues, we evaluated their sequences. In all, 39,641 candidate circRNAs were identified from 6,914 host genes, using\u2009\u2265\u20091 unique back-spliced read Fig.\u00a0A. This iEmerging evidences support the presence of DE circRNAs during porcine skeletal muscle development , howeverp\u2009=\u20092e-08). The heatmap shows the expression pattern of red module, where the expression of genes in the red module tends to increase with time . As depicted in Fig. 05) Fig. C. Moreov05) Fig. D. TherefBased on the above results, we identified 36 circRNAs in the red module, and 508 mRNAs in the darkturquoise module. We performed correlation analysis on the circRNAs/mRNAs co-expression using the Shiny framework and immunomodulation of several genes were significantly enriched. Cell adhesion molecules, which express on the cell surface, facilitate activities of several biological processes, including, immune response, inflammation, and embryogenesis [SIT1 was strongly modulated by the circRNA_21767/miR-202-3p axis. Based on a prior investigation, signaling threshold regulating transmembrane adaptor 1 (SIT1), encoded by the SIT1 gene, is a disulfide-linked homodimeric glycoprotein that interacts with the lymphocyte-specific transmembrane adaptor protein family to modulate the immunologic process [CircRNAs are known to modulate numerous physiological processes using distinct signaling networks . Althougogenesis . CD226 iogenesis . Herein, process . It is iSIT1, which may modulate immune activities. Our findings provide candidate ncRNA-based targets for future in vivo animal study on the molecular regulation of immune function within the porcine spleen. Finally, we designed a user-friendly software named RNAseqTools that is available for use for the visualization of transcriptomic data using the Shiny app (https://github.com/ChaoXu1997/RNAseqTool).In conclusion, this study presented the first reported catalog of circRNAs expression profile in the spleen tissues from Meishan pigs. We characterized numerous DE circRNAs that were relevant to the porcine spleen development. We also generated a comprehensive expression profile of various RNAs, particularly mRNAs and circRNAs, which, together, modulate a myriad of physiological processes within the Meishan pig spleen. Moreover, we identified a ceRNA network involving circRNA_21767/miR-202-3p/Overall, 24 healthy Meishan pigs were acquired from the Kunshan Conservation Ltd. (Permission No. JS-C-05). Our examination involved 8 developmental stages, namely, 1d, 7d, 14d, 21d, 28d, 35d, 120d, and 180d after birth. All piglets were provided with the same standard diet, with housing at an environmentally regulated facility. Three piglets were sacrificed per treatment via an intravenous administration of pentobarbital sodium to minimize suffering. The spleen was immediately extracted and frozen in liquid nitrogen. Our animal care and treatment protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of Yangzhou University (No. SYXK(Su)2021-0026).Splenic total RNA extraction employed TRIZOL following kit directions. RNA quality assessment employed an Agilent 2100 Bioanalyzer . Ribosomal RNA elimination from extracted RNA utilized the Ribozero\u2122 rRNA Removal Kit . Subsequently, linear RNA was eliminated via an RNAse R kit . Lastly, sequencing libraries were generated with rRNA-free and linear RNA-free RNA using the NEBNext\u00ae Ultra\u2122 Directional RNA Library Prep Kit .Sscrofa11.1). We uploaded the resulting RNA-seq information in the Gene Expression Omnibus (accession codes GSE228936). To construct the SAM file, we employed BWA [Sscrofa11.1 genome.Sequencing of generated libraries was done via the Illumina HiSeq 2500 platform . TopHat2 (v2.1.1) mapped the clear data against the porcine reference genome website, and the mRNA 3\u2019UTR sequences from BioMart [We retrieved the miRNA sequences from the miRbase (er 2022) . To expler 2022) predicteer 2022) to examiWe performed the GO term and KEGG pathway \u201349 enricGAPDH (for mRNA and circRNA) and U6 (for miRNA) served as controls for normalization purpose, and all qRT-PCR reactions were performed three times, and the average adjusted value was used for analysis. Relative gene expressions were computed using the 2\u2212\u0394\u0394Ct formula. All data analysis employed the unpaired 2-tailed Student\u2019s t-tests; *p\u2009<\u20090.05, **p\u2009<\u20090.01, and data are provided as mean\u2009\u00b1\u2009SD (standard error of mean).qPCR employed an Applied Biosystems qPCR apparatus and SYBR green master mix , as per kit protocols. The reaction mixture was composed of 5\u00a0\u00b5l SYBR Green Mixture , 1\u00a0\u00b5l cDNA template, 0.2\u00a0\u00b5l primer (each), and 3.6\u00a0\u00b5l deionised water, and the conditions were set as follows: 95\u00a0\u00b0C for 5\u00a0min, 40 cycles of 95\u00a0\u00b0C for 10\u00a0s, and 60\u00a0\u00b0C for 30\u00a0s. All employed primer sequences were summarized in Table SIT1-3\u2019UTR or circRNA_21767 downstream of the pmirGLO Dual-Luciferase vector. Next, we plated cells into a 24-well plates, and co-transfected the cells with the WT (SIT1-WT or circRNA_21767-WT), or Mut , and miR-202-3p-mimics (NC-mimics). Finally, we detected both firefly and renilla bioluminescence using the DLR Gene Assay Kit . The firefly luciferase activity was then adjusted with the control renilla luciferase activity for a measurement of the constructed reporter luciferase activity.We inserted wild-type (WT) or mutant-type (Mut) SIT1-WT:TCTGTGCTGACGCCTCAGTGTCTCCTCAGCCACAGGAAGTAGGCAGTGGGGGAGGGGGTTAGAGCCTGAGAGGATATGTATGGGTATC.TCCTCCCAACCCCCAAACTCCCAGGTTTTCAGTCCTCCTTCCGGAGTTTAATCAGATGCTCCCCACTCCGGCTGCCTCATGGSIT1-Mut:CTCACATCGGTATTCTAGTGTCTCCTCAGCCACAGGAAGTAGGCAGTGGGGGAGGGGGTTAGAGCCTGAGAGGATATGTATGGGTATC.TCCTCCCAACCCCCAAACTCCCAGGTTTTCAGTCCTCCTTCCGGAGTTTAATCAGATGCTCCCCACTCCGGCTGCCTCATGGcircRNA_21767-WT:CCCAGAATCTTTTAGTCCTGCCCTACAGCTGCACCTCGTACATCAAGCTCCATATAATGTTCCTCCTTACCTCTCAAAGAACGAATCAAATCTTGGGGACCTCTTAT.TAGTCTTGTATTGATGGTTTTGCACTATTTACAGAccctACCTGAAcctatccttccatccatccaaaAAATGTAcircRNA_21767-Mut:TTTGGAATCTTTTAGTCCCATTCTACAGCCATGTTCTGTACATCAAGCTCCATATAATGTTCCTCCTTACCTCTCAAAGAACGAATCAAATCTTGGGGACCTCTTAT.TAGTCTTGTATTGATGGTTTTGCACTATTTACAGAccctACCTGAAcctatccttccatccatccaaaAAATGTABelow is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2"} +{"text": "C. elegansvulva is a polarized epithelial tube that has been studied extensively as a model for cell-cell signaling, cell fate specification, and tubulogenesis. Here we used endogenous fusions to show that the spectrin cytoskeleton is polarized in this organ, with conventional beta-spectrin (UNC-70) found only at basolateral membranes and beta heavy spectrin (SMA-1) found only at apical membranes. The sole alpha-spectrin (SPC-1) is present at both locations but requiresSMA-1for its apical localization. Thus, beta spectrins are excellent markers for vulva cell membranes and polarity.The There, spectrins serve as linkers to connect plasma membranes and transmembrane proteins to the actin cytoskeleton . They play numerous roles in membrane and cytoskeletal organization and stability, tissue mechanics, and morphogenesis The spectrin cytoskeleton lines the cytoplasmic sides of cell plasma membranes . Vertebrates have multiple \u03b1-spectrin and \u03b2-spectrin genes. Invertebrates such asDrosophila melanogasterandCaenorhabditis elegans have just one \u03b1-spectrin and two \u03b2-spectrin genes, a conventional \u03b2-spectrin and a larger or \u201cheavy\u201d \u03b2-spectrin (\u03b2H).C. elegans\u03b1-spectrin is encoded byspc-1, and \u03b2-spectrin and \u03b2H-spectrin are encoded byunc-70andsma-1, respectively .SPC-1functions with eitherUNC-70orSMA-1to control different processes. In general,UNC-70is widely expressed and plays key roles in neuron and muscle structure , whileSMA-1is expressed more specifically in epithelial cells and affects tissue morphogenesis . We also discovered a role forSMA-1in blastomere cytokinesis .Spectrins exist as tetramers composed of two dimers, each with an alpha (\u03b1) and beta (\u03b2) subunit . InC. elegans, immunolocalization and/or transgenic reporter studies in the embryo detectedUNC-70/\u03b2-spectrin at many sites of cell-cell contact butSMA-1/\u03b2H-spectrin only at apical membranes , suggesting that apical vs. basal partitioning of different \u03b2-spectrins is conserved; however, there has been limited analysis of spectrin polarity in larval or adult epitheliaInC. elegansvulva. The vulva is a polarized epithelial tube used for egg-laying. It has been studied extensively as a model for cell-cell signaling, cell fate specification, and tubulogenesis . Vulva anatomy is well characterized and apical vs. basal cell surfaces can be distinguished easily using simple light microscopy of live animals.Here we visualized the spectrin cytoskeleton in theSPC-1/\u03b1-spectrin andUNC-70/\u03b2-spectrin have been reported previously . We used CRISPR/Cas9 to tag the endogenousSMA-1/\u03b2H-spectrin protein with GFP (Methods) and then examined the localization patterns of all three fusions in the developing vulva. At the mid-L4 larval stage, vulva cells are organized into a series of 7 stacked rings (named vulA-vulF), surrounding a central lumen cavity .SPC-1::GFP outlined all 7 rings . Thus, .2020) '.SMA-1/SPC-1tetramer formation, we found thatSMA-1/\u03b2H-spectrin is needed to localizeSPC-1/\u03b1-spectrin to apical membranes. In allsma-1(ru18)null mutants examined,SPC-1::GFP failed to localize to apical membranes and was found only at basolateral membranes .The similarly polarized distributions of \u03b2-spectrin and \u03b2H-spectrin inet al.1999). These rings adopt specific shapes and undergo stereotypical movements during morphogenesis. The vulva also must connect appropriately with the uterus to allow the passage of eggs, and with the sex muscles and neurons that control egg-laying . Many of these cell behaviors depend on the actin cytoskeleton and/or on interactions with apical or basal extracellular matrices . We observed a variety of vulva shape abnormalities insma-1mutants and the panels assembled with Adobe Illustrator.SMA-1with GFP was performed by Suny Biotech. The tag is inserted at theSMA-1C-terminus, immediately preceding the stop codon, as shown below. This endogenous fusion is functional based on normal body morphology, brood size and embryonic viability of the homozygotes.CRISPR/Cas9-mediated genome editing to tag endogenous1) Wild type sequence:TAGatacctcaccacacgctgatcttcataTTATTCAAGCGTGGATCCAAACATTCAAAG*sma-1; asterisk is where the GFP was insertedBold TAG is the stop codon ofsma-1(syb4954[SMA-1::GFP])V2) Precise sequence knock-in,CTGTTCAAGCGTGGATCCAAACATTCAAAGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGgtaagtttaaacatatatatactaactaaccctgattatttaaattttcagCCAACACTTGTCACTACTTTCTgTTATGGTGTTCAATGCTTcTCgAGATACCCAGATCATATGAAACgGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACgtaagtttaaacagttcggtactaactaaccatacatatttaaattttcagGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTgtaagtttaaacatgattttactaactaactaatctgatttaaattttcagAACTTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAATAGatacctcaccacacgctgatcttcataBold italics indicate silent mutations; underline indicates the GFP sequence.Strains used:GOU2043vab-10(cas602[VAB-10a::GFP])IGOU2936spc-1(cas815[SPC-1::GFP])Xunc-70(cas962[UNC-70::GFP)]VGOU3103sma-1(syb4954[SMA-1::GFP])VPHX4954sma-1(ru18) V;spc-1(cas815[SPC-1::GFP])XUP4241vab-10(cas602[VAB-10a::GFP]) I;sma-1(ru18)VUP4252"} +{"text": "Insults to the developing cerebellum can cause motor, language, and social deficits. Here, we investigate whether developmental insults to different cerebellar neurons constrain the ability to acquire cerebellar-dependent behaviors. We perturb cerebellar cortical or nuclei neuron function by eliminating glutamatergic neurotransmission during development, and then we measure motor and social behaviors in early postnatal and adult mice. Altering cortical and nuclei neurons impacts postnatal motor control and social vocalizations. Normalizing neurotransmission in cortical neurons but not nuclei neurons restores social behaviors while the motor deficits remain impaired in adults. In contrast, manipulating only a subset of nuclei neurons leaves social behaviors intact but leads to early motor deficits that are restored by adulthood. Our data uncover that glutamatergic neurotransmission from cerebellar cortical and nuclei neurons differentially control the acquisition of motor and social behaviors, and that the brain can compensate for some but not all perturbations to the developing cerebellum. Cerebellar injury in early life can impair the development of motor and social behaviors. Here, the authors show that cerebellar cell types are differentially important for the acquisition of these behaviors in pups and adult mice. Preterm injuries affect the exponential phase of granule cell proliferation, although they often also alter the early development of all glutamatergic neurons in the cerebellum. The resulting defects lead to long-term changes in gray matter volume in the cerebellar cortex that are correlated to the severity of neural deficits in infants6. Cerebellar cortical injuries further impact the development and function of downstream cerebellar nuclei neurons, which serve as the predominant output from the cerebellum and link it to the rest of the motor network7. Accordingly, cerebellar defects can also impair the development and function of the cerebral cortex9. The combined injury to the cerebellum and neocortex may help explain the broad neural deficits observed in many infants that experience cerebellar injury during the perinatal period.Cerebellar injury in preterm infants is often associated with movement disorders, language impairments, and social deficits11. Intriguingly, over time, patients largely recover these impaired neural functions with only minor residual symptoms, most commonly persisting in their motor coordination13. Patients and apes with lesions localized to the cerebellar nuclei mainly demonstrate motor symptoms with limited recovery following the injury15. When the cerebellar cortex is the primary site of the lesion, deficits in social cognition, language, and motor performance arise, but they often persist following the initial injury with limited recovery, especially in the non-motor domains affecting cognition, sociability, language, and affect2. Similarly, studies in rodents have provided compelling evidence that disrupting cerebellar cortical function during development can lead to motor impairments, altered vocalizations, and social deficits21.Accumulating evidence suggests that the site of injury within the developing cerebellum may determine behavioral outcomes. When damage is confined to the cerebellar nuclei neuron axons that project and travel through the superior cerebellar peduncle, affected patients can develop posterior fossa syndrome, which is hallmarked by ataxia, mutism, and changes in social interactionsThese clinical outcomes illustrate that while damage to the cerebellar cortex or to the downstream cerebellar nuclei neurons during infancy is sufficient to impair motor function, language, and social behavior, there are instances when the cerebellum can remarkably overcome perturbations and restore functions. Importantly, the degree of compensation may be linked to the cerebellar neurons that are primarily affected by the lesion, suggesting a unique role for each cerebellar neural subtype in the regulation of cerebellar-associated behaviors. These studies have inspired the need for a deeper examination of how the relatively few neuron types in the cerebellum contribute to a wide range of motor and non-motor functions. However, it remains largely unknown where in the circuit cerebellar-associated behaviors originate, whether the same neuronal subtypes contribute equally to the acquisition of these diverse behaviors, and whether the perturbation of all neuronal subtypes can be equally compensated for during development. Additionally, it remains specifically unexplored how the cerebellar nuclei contribute to the acquisition of different behaviors during postnatal life.22. The unique identities of the lineages can be used to manipulate GABAergic or glutamatergic cerebellar neurons. The Ptf1a lineage that originates in the ventricular zone gives rise to GABAergic neurons, including GABAergic cerebellar nuclei neurons and Purkinje cells23. In contrast, the Atoh1 lineage is derived from the rhombic lip and gives rise to glutamatergic neurons, including glutamatergic granule cells and cerebellar nuclei neurons to eliminate the transport of glutamate into the synaptic vesicles of Atoh1 lineage neurons. This manipulation results in an effective silencing of fast neurotransmission in the genetically targeted neurons from the Atoh1 lineage, which resulted in a lack of VGluT2 protein in pre-synaptic vesicles of the Atoh1 lineage, Vglut2-expressing neurons33. As a result, when an action potential arrives at the synapse, synaptic vesicles fuse to the pre-synaptic membrane but do not functionally affect the postsynaptic cells because no neurotransmitter is released into the synaptic cleft , and therefore the effects of our genetic manipulation do not actively and directly continue through adulthood. Unlike Atoh1 null and cerebellum-specific Atoh1 conditional knockout mice32, Atoh1Cre/+;Vglut2fl/fl mice were viable and survived into adulthood, likely because Atoh1Cre/+;Vglut2fl/fl mice had normal respiratory rhythms , but no Vglut2 expression was found in the granule cell layer assays in adult animals showed that nearly all cerebellar nuclei neurons from the yer Fig.\u00a0. We vali01) Fig.\u00a0. Immunohtex Fig.\u00a0. We visuice Fig.\u00a0 since, aAtoh1Cre/+;Vglut2fl/fl conditional knockout mice have no major differences in zonal stripe patterning of VGluT2+ mossy fiber synapses in the anterior or posterior zones of the cerebellum, regions into which the spinocerebellar projections are targeted and regularity (CV2). In line with our hypothesis, we found that loss of Vglut2 from the Atoh1 lineage resulted in a reduction of simple spike firing rate to the prelimbic area in the medial prefrontal cortex36. Other studies have also suggested that these long-range projections from glutamatergic cerebellar nuclei neurons may mediate non-motor behaviors49. We therefore tested whether the Vglut2-expressing, Atoh1 lineage neurons make long-range projections to areas previously implicated in non-motor behavior. To investigate the complete population of neurons that were affected by our manipulation, we used an intersectional reporter allele (Rosafsf-lsl-tdTomato) that expresses tdTomato only after FlpO- (Atoh1FlpO/+) and Cre- (Vglut2IRES-Cre) mediated excision of two stop cassettes. In these mice, only the neurons with a history of Atoh1 and Vglut2 expression express tdTomato VPM Fig.\u00a0 and the VPM Fig.\u00a0. We alsoVPM Fig.\u00a0 and red VPM Fig.\u00a0. Furtherlei Fig.\u00a0. We alsoons Fig.\u00a0. We provAtoh1Cre/+;Vglut2fl/fl mice , nearly three-quarters of Vglut2+ neurons in the interposed nucleus were also YFP+ (73.7\u2009\u00b1\u20091.3%), and around a sixth of Vglut2+ neurons in the fastigial nucleus were also YFP+ (16.5\u2009\u00b1\u20091.6%). Overall, around half of all Vglut2+ cerebellar nuclei neurons were also YFP+ (52.5\u2009\u00b1\u20091.1%) Fig.\u00a0.Ntsr1Cre-expressing nuclei neurons send long range projections to areas associated with motor and non-motor behaviors. To this end, we crossed the Ntsr1Cre mice to Atoh1FlpO/+;Rosafsf-lsl-tdTomato mice to obtain mice that expressed tdTomato solely in the Ntsr1Cre expressing cerebellar nuclei neurons , may affect adult behaviors. We found that compared to control mice, adult Ntsr1Cre/+;Vglut2fl/fl mice had no deficits in ambulatory activity and specific (only the glutamatergic transporter Vglut2 is deleted). We have used the elimination of vesicular transporters to study the contribution of specific circuit components in several previous studies. In these studies, we did not detect changes in synaptic connectivity or developmental compensation that overcome the functional elimination of neurotransmission72. Therefore, we propose that our genetic methods allow for testing how synaptic VGluT2-dependent neurotransmission only in neurons within the intersectional domain influences circuit function and mouse behavior. Our study confirms that Atoh1 lineage cerebellar nuclei neurons are necessary for the refinement of motor behavior, and we also unveil that Atoh1 granule cells contribute to social vocalizations during postnatal development.Our genetic silencing approach provides a compelling in vivo demonstration of how the function of This study provides in vivo experimental evidence showing that glutamatergic neurons in the cerebellum are critical for the acquisition of motor function and social behaviors. Specifically, the glutamatergic neurons in the cerebellar cortex and nuclei are differentially required for the early postnatal development of motor behavior and social behaviors. These data also raise the possibility that parallel signaling from the GABAergic nuclei neurons may provide some compensation for functional lesions of the glutamatergic nuclei neurons during development, leading to improvements in early motor and social deficits. By leveraging the ability to restore cerebellar-dependent behaviors after normalizing cerebellar cortical function, tapping into cerebellar neurons that communicate social and motor signals to extra-cerebellar regions may provide an ideal therapeutic target to restore motor and non-motor functions after developmental\u00a0brain injury and disease.Ai14 ;73Ai32 ;74Ai65 ;74Atoh1Cre;75Atoh1FlpO (JAX: 036541);32Ntsr1Cre (MMRRC: 030648);57Vglut2IRES-Cre (JAX: 028863);76Vglut2fl (JAX: 012898)72. We used ear clippings from pre-weaned pups for genotyping and identification of transgenic alleles. Mice of both sexes were used in all experiments. We considered the day the pups were born as postnatal day 0 (P0). We did not observe gross differences in weight between control and Atoh1Cre;Vglut2fl/fl conditional knockout mice . The ages of the adult mice were between two and fourteen months old. All mice were kept under a 14\u2009h/10\u2009h light/dark cycle, a daily temperature between 68 and 72\u2009F, and humidity between 30 and 70%.All mice included in the experiments for this manuscript were housed in a Level 3, AALAS-certified facility. The Institutional Animal Care and Use Committee (IACUC) of Baylor College of Medicine (BCM) reviewed and approved all studies that involved mice. We used the following mice for our experiments: 29. We anesthetized mice with Avertin and tested them for effective anesthesia by toe or tail pinch. Analgesia was also provided. We then accessed the chest cavity and penetrated the heart with a small butterfly needle for perfusion. We first perfused with 1\u2009M phosphate-buffered saline (PBS pH 7.4) to flush out blood until the liver turned clear. Next, we perfused the mouse body with 4% paraformaldehyde (PFA) to fix the tissue. After the tail and hind paws were stiff from fixation, we decapitated the mouse and dissected the brain from the skull or spinal cord from the spinal column. We post-fixed brain and spinal cord tissue in 4% PFA overnight or until the tissue was used for cryoprotection. We cryoprotected the tissue by serial sucrose gradients (15% \u2192 30% sucrose in PBS) at 4\u2009\u00b0C, each step until the tissue sinks. Finally, we froze the tissue in an optimal cutting temperature (OCT) solution and stored it at \u221280\u2009\u00b0C. Brain sections were cut into 40\u2009\u00b5m free-floating tissue sections, and spinal cord sections were cut into 25\u2009\u00b5m sections on the slide. Cut sections were stored at 4\u2009\u00b0C until it was used for immunohistochemistry (tissue was stored for a maximum of two months). Tissues from mice expressing the tdTomato allele were stored in aluminum foil at all steps to prevent photobleaching.We collected brain and spinal cord tissue for analyses as previously described29. We blocked tissue sections in 500\u2009\u03bcL 10% normal goat or normal donkey serum in 0.1% Triton-X in PBS (PBS-T) for 2\u2009h. We then incubated tissue sections overnight at room temperature in 500\u2009\u03bcL blocking solution with primary antibodies at desired concentrations. After washing the tissue sections three times in PBS-T, we incubated the tissue for two hours in 500\u2009\u03bcL PBS-T with secondary antibodies at desired concentrations. Subsequently, we incubated tissue sections with DAPI in PBS for 10\u2009min. After a final two washes, we mounted the sections using VECTASHIELD Vibrance\u00ae mounting medium . All mounted slides were stored at 4\u2009\u00b0C until they were imaged. We used the following primary antibodies: guinea-pig (gp)-anti-VGluT2 , rabbit (rb)-anti-VGluT1 , rb-anti-Parvalbumin (PV) , rb-anti-Neurogranin (NG) , rb-anti-carbonic anhydrase 8 (Car8) , and sh-anti-tyrosine hydroxylase (TH) . We used the following secondary antibodies which were conjugated to an Alexa-488 fluorophore: goat-anti-gp , goat-anti-rb , or goat-anti-sh .We stained free-floating tissue sections as previously describedVglut2, Vglut1, YFP, and tdTomato expression in unfixed, fresh frozen tissue cut in 25\u2009\u03bcm-thick coronal brain sections. Digoxigenin (DIG)-labeled mRNA antisense probes against Vglut2, Vglut1, YFP, and tdTomato were generated using an RNA DIG-labeling kit from Roche. The specific sequences of the antisense probes that we used were as follows:ISH was performed by the RNA ISH Core at Baylor College of Medicine using an automated robotic platform. ISH was used to visualize Vglut2: GGTGCTGGAGAAGAAGCAGGACAACCGAGAGACCATCGAGCTGACAGAGGACGGTAAGCCCCTGGAGGTGCCTGAGAAGAAGGCTCCGCTATGCGACTGCACGTGCTTCGGCCTGCCGCGCCGCTACATCATAGCCATCATGAGCGGCCTCGGCTTCTGCATATCCTTCGGCATCCGCTGTAACCTGGGCGTGGCCATCGTGGACATGGTCAACAACAGCACTATCCACCGCGGAGGCAAAGTTATCAAGGAGVglut1: CAGAGCCGGAGGAGATGAGCGAGGAGAAGTGTGGCTTTGTTGGCCACGACCAGCTGGCTGGCAGTGACGAAAGTGAAATGGAGGACGAGGCTGAGCCCCCAGGGGCGCCCCCCGCGCCGCCTCCGTCCTACGGGGCCACACACAGCACAGTGCAGCCTCCGAGGCCCCCGCCCCCTGTCCGGGACTACTGACCACGGGCCTCCCACTGTGGGGCAGTTTCCAGGACTTCCACTCCATACACCTCTAGCCTGAGCGGCAGTGTCGAGGAACCCCACTCCTCCCCTGCCTCAGGCTTAAGATGCAAGTCCTCCCTTGTTCCCAGTGCTGTCCGACCAGCCCTCTTTCCCTCTCAACTGCCTCCTGCGGGGGGTGAAGCTGCACACTAGCAGTTTCAAGGATACCCAGACTCCCCTGAAAGTCGTTCTCCGCTTGTTTCTGCCTGTGTGGGCTCAAATCTCCCCTTTGAGGGCTTTATTTGGAGGGACAGTTCAACCTCTTCCTCTCTTGTGGTTTTGAGGTTTCACCCCTTCCCCCAAGACCCCAGGGATTCTCAGGCTACCCCGAGATTATTCAGGTGGTCCCCTACTCAGAAGACTTCATGGTCGTCCTCTATTAGTTTCAAGGCTCGCCTAACCAATTCTACATTTTTCCAAGCTGGTTTAACCTAACCACCAATGCCGCCGTTCCCAGGACTGATTCTCACCAGCGTTTCTGAGGGAYFP: AGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGtdTomato: ATCAAAGAGTTCATGCGCTTCAAGGTGCGCATGGAGGGCTCCATGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGCGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCCCAGTTCATGTACGGCTCCAAGGCGTACGTGAAGCACCCCGCCGACATCCCCGATTACAAGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGTCTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGGCACGCTGATCTACAAGGTGAAGATGCGCGGCACCAACTTCCCCCCCGACGGCCCCGTAATGCAGAAGAAGACCATGGGCTGGGAGGCCTCCACCGAGCGCCTGTACCCCCGCGACGGCGTGCTGAAGGGCGAGATCCACCAGGCCCTGAAGCTGAAGGACGGCGGCCACTACCTGGTGGAGTTCAAGACCATCTACATGGCCAAGAAGCCCGTGCAACTGCCCGGCTACTACTACGTGGACACCAAGCTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAANissl staining was performed with the tissue sections mounted and dried overnight on the slides. The next day, the mounted sections were immersed in 100% xylene two times for five minutes each time. Subsequently, they were placed through a rehydration series of 3 immersions in 100% ethanol, followed by 95% ethanol, 70% ethanol, and tap water, with each step lasting 2\u2009min. Afterward, the sections were immersed in cresyl violet solution for approximately two minutes. They were then dehydrated following a reversed order of the rehydration series and followed by a final immersion in xylene, with each step lasting up to one minute. Coverslips were mounted on the slides immediately after using Cytoseal XYL mounting media .We acquired photomicrographs of cerebellar sections using a Leica DM4000 B LED microscope with a DPX365FX camera or using a Zeiss Axio Imager.M2 microscope with an AxioCam MRc5 camera. We stitched the whole mount images for the intersectional lineage tracing together using the Adobe Photoshop Photomerge function. We adjusted color brightness and balance using ImageJ software, and we cropped the images to the desired size for figures using Adobe Illustrator.77. Prior to surgery, mice were anesthetized using a mixture of ketamine (80\u2009mg/kg) and dexmedetomidine (16\u2009mg/kg). During the surgery, the mouse was placed on a heated surgery pad and received additional isoflurane (1%) when necessary. The mouse\u2019s head was stabilized by ear bars in a stereotaxic surgery rig. When the mouse was fully anesthetized, we removed the hair from the head and made an incision in the skin over the anterior part of the skull. Next, we used a dental drill to make a large craniotomy over the cerebellar cortex. The craniotomy spanned from around bregma \u22125.60\u2009mm to \u22126.64\u2009mm, and from around 0.5\u20133\u2009mm lateral to bregma. Purkinje cells across several lobules were randomly sampled from this craniotomy, preventing any potential bias from local activity patterns to acquire breath waveforms and used custom-written MATLAB (Mathworks) code to derive Tidal Volume, Respiratory Frequency, and Minute Volume parameters.We tested respiratory parameters in room air as described in our previous publication47. Mice were placed head down on a negative incline (\u221235\u00b0) that was covered with a sterile Poly-Lined drape. We measured the time until mice turned 90\u00b0 in either direction. Mice were tested three times at each time point. We suspended the test if mice did not turn within 60\u2009s or fell down the slope. We recorded the falls that occurred within 60\u2009s.We tested the negative geotaxis reflex at P7, P9, and P11 as previously described and shown47. Mice were placed in the supine position in an empty, clean cage. We measured the time until the mice turned onto their four paws. Mice were tested three times at each time point. We suspended the test if mice did not turn within 1\u2009min (60\u2009s).We tested the surface righting reflex at P7, P9, and P11, as previously described and shownAtoh1Cre/+;Vglut2fl/fl mice and littermate controls, vocalizations were recorded using a CM16 microphone (Avisoft Bioacoustics). Sound was amplified and digitized using UltraSoundGate 416H at a 250\u2009kHz sampling rate, and bit depth of 16 while Avisoft RECORDER software was used to collect the recordings. For Ntsr1Cre;Vglut2fl/fl mice and littermate controls, we measured vocalizations using a Noldus microphone and UltraVox XT software. Due to minimal congruency between the number of vocalizations recorded between these two recording systems79, we only compared mutants to control littermates whose vocalizations were measured using the same system.We measured pup ultrasonic vocalizations in a social isolation task at P7, P9, and P11. The ultrasonic vocalizations of each animal were monitored for 2\u2009min in a sound-attenuating chamber (Med Associates Inc.). For We measured open-field activity using automated Fusion software. Mice were placed in the center of an open field (40x40x30 cm chamber) that has photo beams for detecting horizontal and vertical activity. The chamber was placed in a room with the light set to 200\u2009lux and ambient white noise to 60\u2009dB. Each mouse was allowed to explore the chamber for 30\u2009min.81 to assess the sociability of adult mice. The apparatus consisted of three chambers\u2014a center chamber with doorways to two side chambers, all of equal dimensions . For three days prior to testing, the age- and sex-matched mice to be used as partners for Atoh1Cre/+;Vglut2fl/fl, Ntsr1Cre;Vglut2fl/fl, and their control littermates during the social interaction test were placed under a wire cup for 1\u2009h each day. The assay consisted of a 10-min habituation session followed by a 10-min test phase performed in dim lighting conditions (15\u2009lux). During the habituation session, the test subject was placed in the center chamber and allowed to freely explore for 10\u2009min. Once the subject returned to the center chamber, the doorways to the side chambers were blocked with plexiglass walls. Before the test phase starts, a novel partner mouse (pre-habituated to the apparatus 3 days prior) is placed under a wire cup in one side chamber, while a novel object (Lego block of similar size and color) is placed under a wire cup in the other side chamber. The plexiglass walls covering the doorways are then lifted, and the test session begins as the test subject is allowed to freely investigate all three chambers for 10\u2009min. The amount of time spent interacting with either the novel object or novel mouse during the two phases was scored manually by an experimenter blinded to genotype during the assay using ANY-Maze. Additional activity data that was acquired from each chamber during the two sessions was calculated automatically using ANY-Maze. The placement of the novel mouse and novel object within the side chambers was randomized to prevent chamber bias.We used the three-chamber test53. As is standard for this task, mice were placed on an accelerating rod on which they locomote according to the motion of the rod (4\u201340\u2009rpm over 5\u2009min) . Time was stopped and noted when one of three events occurred; the mouse fell off, the mouse made two consecutive rotations while hanging on the rod without walking, or the mouse successfully stayed on the rod for the total duration of the trial (5\u2009min). We recorded the trial duration for four trials per day for three consecutive days.We assessed rotarod performance using a previously established protocol51. The tremor monitor consists of a lightweight plastic container that is suspended in the air by eight elastic cords, one on each corner. The container is large enough for mice to move around and explore freely, providing us with readings related to movement. An accelerometer is mounted onto the underside of the container, where it functions to convert the detected movements into an electrical signal that is digitized using a Brownlee amplifier and records using Spike2 software. We also used the Spike2 software to detect the tremor power using a Fourier transform analysis based on two full minutes of mouse movements. For the recordings, mice were placed in the tremor monitor and were allowed to acclimate for three minutes. After this period, the recordings for analysis were initiated once the animals started actively exploring and utilizing the space.We recorded tremors using our custom-built tremor monitord for effect size (and reported these in the figure legends) by dividing the absolute difference in the parameter average of each group by the combined standard deviation.All statistical analyses were performed using MATLAB (Mathworks). When performing a two-way (sex*genotype) ANOVA, we did not observe an effect of sex or an interaction effect between sex and genotype on pup behavior. We, therefore, combined mice from both sexes in the experiments and statistical analyses. We performed a two-tailed T-test to assess differences between control and conditional knockout mice when only a one-time point was involved. For analyses of pup behavior and rotarod performance, we performed a repeated measures ANOVA analysis with a Tukey post hoc analysis to test for differences at each time point. For the analyses of electrophysiological recordings in Purkinje cells, we used a linear mixed model analysis with genotype as a fixed variable and mouse number as a random variable. We calculated Cohen\u2019s Further information on research design is available in the\u00a0Supplementary InformationPeer Review FileReporting Summary"} +{"text": "RNA undergoes complex posttranscriptional processing including chemical modifications of the nucleotides. The resultant-modified nucleotides are an integral part of RNA sequences that must be considered in studying the biology of RNA and in the design of RNA therapeutics. However, the current \u201cRNA-sequencing\u201d methods primarily sequence complementary DNA rather than RNA itself, which means that the modifications present in RNA are not captured in the sequencing results. Emerging direct RNA-sequencing technologies, such as those offered by Oxford Nanopore, aim to address this limitation. In this study, we synthesized and used Nanopore technology to sequence RNA transcripts consisting of canonical nucleotides and 10 different modifications in various concentrations. The results show that direct RNA sequencing still has a baseline error rate of >10%, and although some modifications can be detected, many remain unidentified. Thus, there is a need to develop sequencing technologies and analysis methods that can comprehensively capture the total complexity of RNA. The RNA sequences obtained through this project are made available for benchmarking analysis methods. N6-methyladenosine (m6A), and others are complex multistep modifications, resulting in wybutosine and a sugar-phosphate backbone. The bases and sugar of RNA undergo chemical alteration, leading to >150 modified nucleotides methods do not sequence RNA, rather RNA is converted back to DNA and the resultant DNA is sequenced; in the reverse transcription, all modifications are lost and the >150 modifications, it is necessary to train base-calling algorithms to establish the correspondence between the nucleotides and their corresponding current patterns. Many groups have developed methods to convert the current signals to sequence was cloned from human fibroblast DNA into the pcDNA3.1-GFP(1\u201310) (Addgene cat# 70219) vector. The clone sequence was as follows:UUGAGCAUUUCAUCCGGAGUCUGGCCGCCCUGACCUUCCCCCAGCCGCCUGCAGGGGGCGCCAGAGGGCCGGAGCACGGAAAGCAGCGGAUCCUUGAUGCUGCCUUAAGUCCGGCUCAGAGGGGCGCAGCGUGGCCUGGGGUCGCUAUCUUCCCAUCCGGAACAUCUGCCCUGCUGGGGGACACUACGGGCCUUCCCUUGCCUGAGGGUAGGGUCUCAAGGUCACUUGCCCCCAGCUUGACCUGGCCGGAGUGGCUAUAGAGGACUUUGUCCCUGCAGACUGCAGCAGCAGAGAUGACACUGUCUCUGAGUGCAGAGAUGGGGGCAGGGAGCUGGGAGAGGGUUCAAGCUACUGGAACAGCUUCAGAACAACUAGGGUACUAGGAACUGCUGUGUCAGGGAGAAGGGGCUCAAGGACUCGCAGGCCUGGGAGGAGGGGCCUAGGCCAGCCAUGGGAGUUGGGUCACCUGUGUCUGAGGACUUGGUGCUGUCUGGAUUUUGCCAACCUAGGGCUGGGGUCAGCUGAUGCCCACCACGACUCCCGAGCCUCCAGGAACUGAAACCCUGUCUGCCCCCAGGGUCUGGGGAAGGAGGCUGCUGAGUAGAACCAACCCCAGGUUACCAACCCCACCUCAGCCACCCCUUGCCAGCCAAAGCAAACAGGCCCGGCCCGGCACUGGGGGUUCCUUCUCGAACCAGGAGUUCAGCCUCCCCUGACCCGCAGAAUCUUCUGAUCCCACCCGCUCCAGGAGCCAGGAAUGAGUCCCAGUCUCUCCCAGUUCUCACUGUGUGGUUUUGCCAUUCAUCUUGCUGCUGAACCACGGGUUUCUCCUCUGAAACAUCUGGGAUUUAUAACAGGGCUUAGGAAAGUGACAGCGUCUGAGCGUUCACUGUGGCCUGUCCAUUGCUAGCCCUAACAUAGGACCGCUGUGUGCCAGGGCUGUCCUCCAUGCUCAAUACACGUUAGCUUGUCACCAAACAUACCCGUGCCGCUGCUUUCCCAGUCUGAUGAGCAAAGGAACUUGAUGCUCAGAGAGGACAAGUCAUUUGCCCAAGGUCACACAGCUGGCAACUGGCAGAGCCAGGAUUCACGCCCUGGCAAUUUGACUCCAGAAUCCUAACCUUAACCCAGAAGCACGGCUUCAAGCCCCUGGAAACCACAAUACCUGUGGCAGCCAGGGGGAGGUGCUGGAAUCUCAUUUCACAUGUGGGGAGGGGGCUCCCCUGUGCUCAAGGUCACAACCAAAGAGGAAGCUGUGAUUAAAACCCAGGUCCCAUUUGCAAAGCCUCGACUUUUAGCAGGUGCAUCAUACUGUUCCCACCCCUCCCAUCCCACUUCUGUCCAGCCGCCUAGCCCCACUUUCUUUUUUUUCUUUUUUUGAGACAGUCUCCCUCUUGCUGAGGCUGGAGUGCAGUGGCGAGAUCUCGGCUCACUGUAACCUCCGCCUCCCGGGUUCAAGCGAUUCUCCUGCCUCAGCCUCCCAAGUAGCUAGGAUUACAGGCGCCCGCCACCACGCCUGGCUAACUUUUGUAUUUUUAGUAGAGAUGGGGUUUCACCAUGUUGGCCAGGCUGGUCUCAAACUCCUGACCUUAAGUGAUUCGCCCACUGUGGCCUCCCAAAGUGCUGGGAUUACAGGCGUGAGCUACCGCCCCCAGCCCCUCCCAUCCCACUUCUGUCCAGCCCCCUAGCCCUACUUUCUUUCUGGGAUCCAGGAGUCCAGAUCCCCAGCCCCCUCUCCAGAUUACAUUCAUCCAGGCACAGGAAAGGACAGGGUCAGGAAAGGAGGACUCUGGGCGGCAGCCUCCACAUUCCCCUUCCACGCUUGGCCCCCAGAAUGGAGGAGGGUGUCUGGAUUACUGGGCGAGGUGUCCUCCCUUCCUGGGGACUGUGGGGGGUGGUCAAAAGACC5)] of the 5-mers of the RNA synthesized with just the canonical nucleotides.The sequence includes 691 distinct 5-mers, about 70% 5-deoxyadenosine, 2.5 mM MgCl2, and 5 mM Tris-HCl buffer pH 8.0. The digestion mixture was incubated at 37\u00b0C for 6 h.RNA from each sample (2 \u00b5g) was hydrolyzed in a 50-\u00b5L digestion cocktail containing 8 U benzonase, 5 U calf intestinal alkaline phosphatase, 0.15 U phosphodiesterase I, 0.1 mM deferoxamine, 0.1 mM butylated hydroxytoluene, 5 ng coformycin, 50 nM internal standard [N1-methyladenosine (m1A), m/z 282 \u2192 150, 1.2 min; m6A, m/z 282 \u2192 150, 8.7 min; 5-hydroxymethylcytidine (hm5C), m/z 274.1 \u2192 142.1, 0.88 min; Y, m/z 245 \u2192 191, 0.88 min. Signal intensities for each ribonucleoside were normalized by dividing by the sum of the UV signal intensities of the 4 canonical ribonucleosides recorded with an in-line UV spectrophotometer at 260 nm.After digestion, all 9 samples were analyzed by chromatography-coupled triple-quadrupole mass spectrometry (LC-MS/MS). For each sample, 600 ng of hydrolysate was injected into each of 3 technical replicates. Using synthetic standards, HPLC retention times of RNA modifications were confirmed on a Waters Acuity BEH C18 column coupled to an Agilent 1290 HPLC system and an Agilent 6495 triple-quadrupole mass spectrometer. The HPLC system was operated at 25\u00b0C and a flow rate of 0.3 mL/min in a gradient [Escherichia coli poly(A) polymerase, followed by Oxford Nanopore's protocol for direct RNA sequencing (SQK-RNA002). RNA samples were sequenced using an Oxford Nanopore MinION Mk1B device or a 5-cell GridION sequencer, with FLO-MIN106D flow cells . Reads were aligned to the clone sequence using minimap2 v2.17 , using tIn each sample, at each nucleotide position, we compared the RNA sequence to the corresponding sequence of the DNA sense strand, and then, we calculated the error rate as a ratio of mismatches to the total sequence reads at position (matched + mismatched). Those ratios are presented as percentages in the figures.For each sample, we also break down the errors by the 4 nucleotides in the DNA template. For each of the 4 nucleotides in the DNA, we looked for the number of mismatches in the RNA. In each sample, for each nucleotide, we determined the ratio of the number of mismatches to the total number of reads for the nucleotides and reported the ratios in percentages as A-to-X, C-to-X, G-to-X, and U-to-X where X is A, C, G, and U from Nanopore. The error rates calculated as implemented in our Python scripts are the same as those from EpiNano.To compute dwell times, reads were first resquiggled using f5c version 0.8 and principal component 1 (PC2) scores are plotted.Principal component analysis (PCA) was performed by using 7 variables for the 45 samples . The data were normalized by using the Z-score. The PCA was calculated as the total variance explained.We also carried out a PCA with up to 5,000 randomly selected reads from each sample and the current and dwell times for the 5-mers that are most different among the modifications were used as variables. To select the variables for the PCA, for each 5-mer in our data, we performed ANOVA to identify the current signals that are most variable among the samples vs within, the top 5% of the most variable current signals were used as variables. The same analysis was used to identify the dwell times for the PCA. The variables were normalized by Z-score. The PCA was calculated as the total variance explained. For each sample, we then looked for the sites with the most modified nucleotides based on the PC1 and PC2 scores. We identified the outlier sites as those with PC scores furthest by Euclidean distance from the center of the plots; then at those locations, in each read, we looked for errors and longer dwell times to identify the most likely modified nucleotides. For the m6A, biotinylated-C, and Y samples, we only looked at the A, C, and U, respectively.Another PCA was performed for each sample with 7 variables for each nucleotide. The data were also normalized by We synthesized a set of RNA transcripts with only canonical nucleotides and also with 10 different modified nucleotides. We then performed direct RNA sequencing using the Oxford Nanopore technology on those 2 kb RNA transcripts.O-methyladenosine (Am), 2\u2032-O-methylcytidine (Cm), 2\u2032-O-methylguanosine (Gm), and 2\u2032-O-methyluridine (Um). Each of the modified nucleotides was added with its corresponding canonical nucleotide in 1:100, 1:10, 1:5, and 1:2 proportions .Some of the IVT reactions included canonical nucleotides only, others included different proportions of a modified base or sugar nucleotide and the corresponding canonical nucleotide. The modified bases consist of m1A, m6A, biotinylated cytidine, hm5C, 5-methylcytidine (m5C), Y, and the modified sugars were 2\u2032-To verify that the modified nucleotides were incorporated into the RNA transcripts, the samples were checked by chemiluminescent assay and LC-MS/MS. The samples with biotinylated cytidine were checked by chemiluminescence using streptavidin-horseradish peroxidase. Additionally, to obtain a more accurate assessment of the modifications, 9 samples were analyzed by mass spectrometry. These include 1 sample synthesized with just canonical nucleotides and samples with m1A, m6A, hm5C, and Y at 2 concentrations . All the synthesized RNA transcripts were polyadenylated, and then sequenced using the Oxford Nanopore MinION. The base-calling was carried out using Guppy, alignment was performed with Minimap2 , and seqWe started by analyzing the 10 independently synthesized and sequenced sets of RNA with just canonical nucleotides. The samples were sequenced deeply with an average coverage of 5,563 reads and is prone to slippage in particular when transcribing A/T homopolymers and those from EpiNano in the samples with and without modified bases. We found that all 6 nucleotides with modified bases have highly significantly different , the cytidines in the samples with modified cytidines and the uridines in the Y samples. By motif analysis, we found that neighboring adenosines contributed to errors in the m6A samples but not in the m1A samples, this may allow for the 2 modifications to be distinguished. There is no such combination of features that allows us to tell apart the 3 modified cytidines. While all 6 modified bases led to significantly different dwell times in the pores, dwell time alone does not tell apart the different modifications.O-methylated sugars at different proportions in the IVT reactions. The samples are deeply sequenced . We also carried out PCA with sequence reads to ask whether we can distinguish them. We used a similar concept in population genetic studies where individuals are grouped by populations based on their genotypes of genetic markers that have different allele frequencies across populations . We usedMaterials and Methods) and at those positions, we annotated the sequence reads with errors or longer dwell time as modified nucleotides DNA template for IVT with 10 different modified nucleotides in a range of concentrations to generate RNA transcripts with modified bases or sugars. We also included RNA transcripts with only canonical nucleotides. The incorporation of the modified nucleotides was confirmed by chemiluminescence assay and mass spectrometry analyses. The RNAs were then sequenced on a Nanopore MinION with deep coverage, most samples having >5,000 reads. These are available for benchmarking algorithms for base-calling.We generated independently 10 sets of RNA with only canonical nucleotides for comparison with their counterparts with modified ribonucleotides. Direct RNA sequencing of the samples with only canonical nucleotides revealed that the error rate of Nanopore RNA sequencing is still >10%.Our analyses show that, while some modifications can be detected with existing algorithms such as Tombo and analysis of error patterns, we are far from sequencing RNA with all modifications. To obtain the complete sequences with all modifications, several technological advancements are needed. These include reducing the error rates to <0.1%, improving the resolution from 5-nucleotide to single-nucleotide, and developing base-callers that identify the canonical bases, known modifications, and yet-to-be discovered modifications. Given the large number of ribonucleotides per cell, even if we assume 10 pg of RNA per cell, there are >10 billion ribonucleotides in each cell, and thus, even a low error rate of 0.1% has many errors. Improvement in resolution to the single-nucleotide level will facilitate base-calling. Most probably, many modifications are transient and occur at a low level, so they are difficult to detect, and therefore, technical barriers such as poor resolution need to be improved. The biggest challenge of direct RNA sequencing is base-calling. Even if the instrument can capture current signals of a single nucleotide or uses methods such as exo-seq (RNA-sequencing technologies that provide the complete sequences with all the modifications, at affordable cost, will transform RNA biology and clinical medicine. The complete sequences will elucidate the basic aspects of RNA from splicing to stability. The sequences are crucial for the development of effective RNA-based therapeutics. The exact sequences are necessary for targeting specific RNA, replacing dysfunctional ones and in manufacturing vaccines. Development of sequencing technologies, coupled with analysis methods, is a necessary breakthrough to advance RNA biology and therapeutics.jkad200_Supplementary_DataClick here for additional data file."} +{"text": "Peng D, Ando K, Hu\u00dfmann M, Gloger M, Skoczylas R, Mochizuki N, Betsholtz C, Fukuhara S, Schulte-Merker S, Lawson ND, Koltowska K. 2022. Proper migration of lymphatic endothelial cells requires survival and guidance cues from arterial mural cells. Published 22 March 2022This error occurred during the misaligned \u201ccopy and paste\u201d from the primer list while drafting the paper. It did not affect anyone qPCR experiments or the results. We regret any confusion or difficulties it may have caused.In the Materials and methods session - FACS and qPCR analysis, there is a mix-up for the dll4 primersCorrected sequence:GGACAAATGCACCAGTATGCGTTTGCGCAGTCGTTAATGTOriginal sequence:CGTTCCCAGGAGCCTTTTCTTTGGGATCAGGGATGGGGATThe article has been corrected accordingly."} +{"text": "Antibody discovery is bottlenecked by the individual expression and evaluation of antigen-specific hits. Here, we address this bottleneck by developing a workflow combining cell-free DNA template generation, cell-free protein synthesis, and binding measurements of antibody fragments in a process that takes hours rather than weeks. We apply this workflow to evaluate 135 previously published antibodies targeting the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), including all 8 antibodies previously granted emergency use authorization for coronavirus disease 2019 (COVID-19), and demonstrate identification of the most potent antibodies. We also evaluate 119 anti-SARS-CoV-2 antibodies from a mouse immunized with the SARS-CoV-2 spike protein and identify neutralizing antibody candidates, including the antibody SC2-3, which binds the SARS-CoV-2 spike protein of all tested variants of concern. We expect that our cell-free workflow will accelerate the discovery and characterization of antibodies for future pandemics and for research, diagnostic, and therapeutic applications more broadly. Antibody discovery is bottlenecked by the individual expression and evaluation of antigen specific hits. Here, the authors build an antibody screening workflow leveraging cell-free protein synthesis that enables expression and evaluation of hundreds of antibody fragments in less than 24\u2009h. Antibodies have also recently garnered attention as potential countermeasures for emerging pathogens, having been used as both prophylaxis and therapy against infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus and coronavirus disease 2019 (COVID-19)6.Antibodies are widely used in diagnostics and as drugs. They are the critical component in immunoassays enabling rapid diagnostics8 sequences to a pool of 103 to 104 candidates targeting the desired antigen. However, once this pool of candidates is generated, state-of-the-art workflows rely on labor-intensive procedures , etc.) to evaluate and identify the best antibody candidates8. These procedures take weeks to months and represent a major speed and throughput bottleneck in antibody discovery.Contemporary workflows for antibody discovery commonly utilize synthetic selections or the isolation of single B cell clones from convalescent patients or animals to go from >1010 or non-overlapping epitopes12 to resist viral escape and increase the ability to neutralize viral variants13; both of which require intensive screening campaigns. A further challenge is that existing antibody discovery processes frequently have low efficiency, with few of the screened candidates having potent neutralizing activity, as has been the case for SARS-CoV-2 (Supplementary Table\u00a0The effort to identify antibodies against emerging threats like SARS-CoV-2 during the coronavirus disease 2019 (COVID-19) pandemic has highlighted the importance of (i) rapid and high-throughput antibody discovery platforms and (ii) identifying high-affinity antibodies targeting conserved15, the manufacture of proteins without living cells using crude extracts or purified components, is an attractive tool to overcome these limitations. A variety of CFPS systems for antibody expression have been developed22; however, few of these studies have focused on the functional screening of antibodies, and most methods rely on techniques that are not suitable for high-throughput screening like the use of purified plasmids or labor-intensive ELISAs23.Cell-free protein synthesis (CFPS)24, and (iv) acoustic liquid handling that enables a highly parallel and miniaturized workflow. This integrated workflow enables a single researcher to express and profile the antigen-specific binding of hundreds of antibodies in less than 24\u2009h. As a model, we apply our workflow to profile a diverse set of 135 previously published antibodies targeting the SARS-CoV-2 spike glycoprotein, and show that our workflow identifies all 8 neutralizing antibodies that had been granted emergency use authorization (EUA) by the United States Food and Drug Administration for the treatment of COVID-19. In addition, we screen 119 antibodies derived from mice immunized against the SARS-CoV-2 spike glycoprotein and identify several candidate neutralizing antibodies.Here, we describe a CFPS-based integrated pipeline for antibody expression and evaluation to address screening limitations in current antibody discovery pipelines. The workflow leverages four key developments Fig.\u00a0: (i) DNA25. The method consists of a Gibson assembly step, followed by PCR amplification of the linear expression template (LET) using the unpurified Gibson assembly product as a template. The key idea was to create a versatile approach for construction of DNA templates without the requirement of cell culture, allowing for DNA assembly and amplification in less than 3\u2009h entirely in 384-well plates.We first implemented a method for cell-free DNA assembly and amplification by adapting and optimizing recently reported protocols for rapid construction of DNA templates for CFPS26. These sequences were subsequently amplified by PCR to generate LETs strain, which contains mutations in the E. coli reductase genes trxB and gor to enable the formation of disulfide bonds in the cytoplasm27. By pretreating the extract with the reductase-inhibitor iodoacetamide (IAM) to further stabilize the redox environment30 and supplementing the reaction with purified E. coli disulfide bond isomerase DsbC and prolyl isomerase FkpA32, we successfully expressed and assembled full-length trastuzumab 31. Consistent assembly under the same experimental conditions is important for screening since individual conditions cannot be optimized for hundreds or thousands of antibodies. We thus opted to use the synthetically dimerized antigen-binding fragment format and, like Ojima-Kato et al.18, found that the assembly of sdFabs were more uniform than their corresponding Fabs in CFPS for a panel of antibodies for binding of the SARS-CoV-2 Receptor Binding Domain (RBD). Our determined rank order of IC50s Fig.\u00a0.Furthermore, we utilized AlphaLISA to develop a sdFab assembly screen to monitor antibody fragment expression and assembly in CFPS, a step that traditionally requires SDS-PAGE. The measurement immobilizes the heavy and light chains of the sdFab to the AlphaLISA beads, resulting in signal when the two chains are assembled 50. We used these assays as a rapid screen for S6P binding and ACE2 competition at a single unknown concentration of CFPS-derived antibody.Using the developed workflow, we next evaluated a set of 115 SARS-CoV-2 targeted antibodies that were selected based on the availability of sequence, structural, binding, and neutralization data, with 84 being drawn from Brouwer et al.FPS Fig.\u00a0. MeasureTo determine the robustness of the workflow, antibody fragments were expressed and evaluated in triplicate. We observed that independent replicates were consistent with one another and exhibited average coefficients of variation (standard deviation divided by the mean) in the range of 0.15\u20130.22 , r\u2009=\u20090.41 , and r\u2009=\u20090.40 for Figs.\u00a0While there is only a weak correlation between our AlphaLISA data and the corresponding Brouwer et al. S6P binding ELISA data, the RBD binding ELISA data, and the pseudovirus neutralization data 67. For all 8 EUA antibody fragments, we observed S6P binding, RBD binding, ACE2 competition, and assembly by the US Food and Drug Administration for prophylaxis or treatment of COVID-19 67. Notably, we observed that S2H97 exhibited ACE2 competition, which like S309, is reported to bind an epitope adjacent to the receptor binding motif and not compete with ACE2 for binding10. The antibody 54042-4 exhibited weak binding to the RBD but showed low assembly signal, indicative of poor expression or assembly , we observed binding and competition for 10 of 11 antibody fragments tested, with the results largely consistent with published literatureata Fig.\u00a0. Based o68. For these antibodies, the normalized S6P binding AlphaLISA measurement correlates well with loss in neutralization potency ). Also consistent with literature, S309 exhibited binding to the SARS-CoV spike protein, whereas no other EUA Ab is reported to bind SARS-CoV59.We further profiled the binding of this set of high interest antibodies against SARS-CoV-2 variants of concern (VOC) including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), and Omicron as well as several other human coronaviruses including SARS-CoV, MERS-CoV, HCoV-HKU1, HCoV-OC43, HCoV-NL63, and HCoV-229E Fig.\u00a0. Our dat64. S2K146 did not exhibit binding to the SARS-CoV spike, despite being reported to neutralize SARS-CoV65. S2P6 additionally exhibited binding to MERS-CoV and HCoV-OC43 consistent with the literature on this antibody61. For S2P6, we observed heterogeneity in binding signal to different S6P variants, possibly a result of the target epitope of S2P6 being near the C-terminus of S6P and near to the C-terminal avi tag (a site-specific biotinylation), which may impede immobilization on the AlphaLISA bead. Neutralization data against all VOC for the tested bnAbs are not available, but the binding profiles for those characterized are generally consistent with literature. Against Omicron BA.1, S2K146 exhibits strong binding whereas S2X259, S2H97, and SARS2-38 all exhibit reduced binding70. Similarly, S2K146 exhibits strong binding to all Omicron variants except BA.4/5, whereas S2X259 and S2H97 exhibit reduced binding to the other Omicron sub-lineages72. Taken together, our results indicate that the CFPS-derived antibody fragment binding patterns are consistent with those reported in literature.For the set of bnAbs, we observed cross-reactivity with the SARS-CoV spike protein for S2P6, S2X259, DH1047, C118, and S2H97 all of which are reported to bind to this antigen73 and isolated spike-positive activated B cells 10 days later using fluorescence-activated cell sorting. The pooled sorted B cells were sequenced to identify paired heavy and light chains, which were codon optimized and ordered commercially as synthetic DNA .No statistical method was used to predetermine sample size. Previous experience with the measurement techniques and their dynamic ranges was used to determine sample sizes. Large-scale experiments . The Benjamini and Hochberg False Discovery Rate procedure50 was used to correct for multiple testing. Statistical analyses were performed in python. Two-sided t-tests were performed using the scipy package and the FDR procedure was performed using the statsmodels package with a family-wise error rate of 5%. For antibody screening, the following samples were used as a measurement of background, and the combined data were used in the t-test. Assembly: No DNA and beads only controls. S6P binding: No DNA and beads only controls. RBD binding: No DNA and beads only controls. ACE2 competition: No DNA and \u03b1HER2.Samples were considered different from background if they exhibited Pearson correlation coefficients were calculated using GraphPad Prism 9.Single-cell BCR sequences were analyzed with Cell Ranger v3.1.0. AlphaLISA data were analyzed with Prism 9.5.1. Images were processed using ImageJ2 v2.9.0/1.53t. Plate reader data were processed using Python 3.8.8. The Python code to analyze plate reader data was not central to the research and was not deposited.18. Example plasmid maps of the aHER2 heavy and light chain sdFabs can be found in the Source data. Antibody sequences were collected from literature and their light chains were classified as either kappa or lambda via the terminal residue of the J-segment in the VL domain. The VH and VL domains were subsequently fused to their corresponding human constant heavy (Uniprot P0DOX5) or human constant light (kappa CL Uniprot P01834 or lambda 1 CL Uniprot P0CG04) chains. At the N-terminus of the VH and VL domains, we chose to include a modified expression tag based on the first 5-residues of the E. coli chloramphenicol acetyltransferase gene followed by a Tobacco Etch Virus (TEV) protease cleavage site 79 as opposed to the previously published SKIK tag80. The heavy chain was fused to the LZA heterodimer subunit (AQLEKELQALEKENAQLEWELQALEKELAQK) and a strep II tag or super FLAG (sFLAG) tag81. The light chain was fused to the LZB heterodimer subunit (AQLKKKLQALKKKNAQLKWKLQALKKKLAQK). Antibodies in the screen in Fig.\u00a0sdFabs were assembled based on a modified version of previously published protocolssdFab heavy chain constant strepII tagged:[MEKKIENLYFQS][VH_Sequence][ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC]GGGGS[AQLEKELQALEKENAQLEWELQALEKELAQK]GSSA[WSHPQFEK].sdFab heavy chain constant super FLAG (sFLAG) tagged:[MEKKIENLYFQS][VH_Sequence][ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC]GGGGS[AQLEKELQALEKENAQLEWELQALEKELAQK]GSSA[DYKDEDLL].sdFab light chain kappa:[MEKKIENLYFQS][VL_Sequence][RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC]GGGGS[AQLKKKLQALKKKNAQLKWKLQALKKKLAQK].sdFab light chain lambda 1:[MEKKIENLYFQS][VL_Sequence][GQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS]GGGGS[AQLKKKLQALKKKNAQLKWKLQALKKKLAQK].Proteins to be manufactured via CFPS were codon optimized using the IDT codon optimization tool and ordered as double-stranded linear DNA containing the desired Gibson assembly overhangs from IDT or GenScript. sfGFP was ordered containing the two pJL1 Gibson assembly overhangs. Antibody VH DNA was ordered with the pJL1 5\u2032 and the human IgG1 heavy chain constant 5\u2032 Gibson overhangs. Antibody VL DNA was ordered with the pJL1 5\u2032 and the human Ig light chain kappa or lambda 1 Gibson assembly overhangs. DNA was resuspended at a concentration of 50\u2009ng/\u03bcL and used without amplification.Additional linear DNA components for Gibson assembly were ordered as gblocks from IDT. These components were amplified using PCR using Q5 Hot Start DNA polymerase following manufacturer instructions. Amplified DNA was purified using the DNA Clean and Concentrate Kit and diluted to a concentration of 50\u2009ng/\u03bcL. Sequences of the utilized components are listed below, with Gibson assembly sequences being denoted by underlined lowercase text and primers for a given amplicon being listed below the DNA sequence.Gibson assembly overhangs:tttgtttaactttaagaaggagatatacat.pJL1 5\u2032 Gibson: gtcgaccggctgctaacaaagcccgaaagg.pJL1 3\u2032 Gibson: gcgtcaacaaaaggtccttcagttttcccattagcccct.Human IgG1 heavy chain constant 5\u2032 Gibson: cgcacggtcgcggcgccgtctgtctttatttttcctcct.Human Ig light chain kappa 5\u2032 Gibson: ggccaacccaaagcaaacccaactgtcactttgttcccg.Human Ig light chain lambda 5\u2032 Gibson: Linear pJL1 plasmid backbone (Addgene plasmid # 69496):gtcgaccggctgctaacaaagcccgaaaggAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGCCAATTCTGATTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGCTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCTTCGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGATGATATATTTTTATCTTGTGCAATGTAACATCAGAGATTTTGAGACACAACGTGAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGATCCCGCGAAATTAATACGACTCACTATAGGGAGACCACAACGGTTTCCCTCTAGAAATAATtttgtttaactttaagaaggagatatacat.pJL1_F: gtcgaccggctgcta.pJL1_R: atgtatatctccttcttaaagttaaacaaaattatttcta.Linear sdFab heavy chain constant strepII tagged:gcgtcaacaaaaggtccttcagttttcccattagcccctTCTTCTAAGTCAACTAGTGGCGGTACTGCCGCTCTTGGGTGTTTGGTTAAAGATTACTTCCCAGAACCGGTTACGGTCTCGTGGAACTCTGGTGCACTGACATCGGGCGTACATACATTTCCCGCAGTTTTGCAGTCTTCGGGACTGTATTCTCTTTCATCGGTGGTTACAGTCCCTAGCTCTTCCCTGGGTACACAGACCTACATTTGTAATGTTAATCATAAGCCGAGTAATACTAAGGTGGATAAAAAGGTGGAACCGAAGTCTTGTGGTGGTGGCGGGTCAGCTCAACTGGAGAAGGAGTTACAGGCACTGGAAAAAGAGAATGCTCAACTTGAGTGGGAATTACAGGCGTTAGAAAAAGAACTGGCCCAGAAGGGTTCTAGCGCATGGTCACATCCCCAGTTCGAAAAATAAgtcgaccggctgctaacaaagcccgaaagg.Linear sdFab heavy chain constant super FLAG tagged:gcgtcaacaaaaggtccttcagttttcccattagcccctTCTTCTAAGTCAACTAGTGGCGGTACTGCCGCTCTTGGGTGTTTGGTTAAAGATTACTTCCCAGAACCGGTTACGGTCTCGTGGAACTCTGGTGCACTGACATCGGGCGTACATACATTTCCCGCAGTTTTGCAGTCTTCGGGACTGTATTCTCTTTCATCGGTGGTTACAGTCCCTAGCTCTTCCCTGGGTACACAGACCTACATTTGTAATGTTAATCATAAGCCGAGTAATACTAAGGTGGATAAAAAGGTGGAACCGAAGTCTTGTGGTGGTGGCGGGTCAGCTCAACTGGAGAAGGAGTTACAGGCACTGGAAAAAGAGAATGCTCAACTTGAGTGGGAATTACAGGCGTTAGAAAAAGAACTGGCCCAGAAGGGTGGAGCCAGTCCAGCAGCTCCTGCGCCTGGCGGGGACTACAAAGATGAAGACCTTCTTTAAgtcgaccggctgctaacaaagcccgaaagg.IgGC_F: GCGTCAACAAAAGGTCCTTCAGTTTTC.pJL1_3\u2032Gib_R: CCTTTCGGGCTTTGTTAGCAGC.Linear sdFab light chain kappa constant:cgcacggtcgcggcgccgtctgtctttatttttcctcctTCTGATGAACAGCTTAAATCTGGGACAGCTTCTGTTGTATGTTTATTAAACAACTTTTACCCGCGTGAGGCAAAAGTTCAATGGAAGGTAGACAACGCACTGCAAAGCGGAAATTCGCAGGAGTCAGTTACCGAACAGGATTCCAAGGATAGTACCTACTCCTTAAGTTCAACATTAACCCTGTCAAAGGCGGACTATGAAAAACATAAGGTATATGCCTGCGAAGTAACTCATCAGGGCTTATCATCCCCAGTTACAAAATCTTTCAACCGTGGAGAATGCGGCGGCGGAGGTAGCGCGCAGCTTAAGAAAAAATTGCAAGCCCTTAAAAAAAAAAATGCCCAACTTAAATGGAAGCTGCAAGCCTTAAAAAAGAAATTGGCGCAGAAGTAAgtcgaccggctgctaacaaagcccgaaagg.kLC_F: TCGCGGCGCCGTCTG.pJL1_3\u2032Gib_R: CCTTTCGGGCTTTGTTAGCAGC.Linear sdFab light chain lambda 1 constant:ggccaacccaaagcaaacccaactgtcactttgttcccgCCCTCAAGCGAGGAACTTCAGGCTAATAAGGCCACGCTTGTTTGCCTGATCTCAGACTTTTATCCCGGTGCCGTAACAGTGGCTTGGAAGGCAGATGGTTCGCCGGTCAAAGCGGGCGTGGAAACTACAAAGCCATCGAAACAGTCAAACAATAAATATGCGGCATCAAGTTACTTGAGCCTTACCCCAGAACAGTGGAAGTCACACCGCTCGTACAGTTGTCAAGTTACACACGAGGGAAGTACAGTTGAAAAGACCGTTGCCCCAACTGAATGTTCAGGCGGTGGTGGCTCAGCGCAGTTAAAGAAAAAACTGCAGGCTTTGAAGAAAAAGAATGCTCAATTAAAGTGGAAATTGCAGGCGTTGAAGAAGAAACTTGCGCAGAAGTAAgtcgaccggctgctaacaaagcccgaaagg.lLC_F: GGCCAACCCAAAGCAAACC.pJL1_3\u2032Gib_R: CCTTTCGGGCTTTGTTAGCAGC.82.Linear sfGFP (same DNA sequence as Addgene Plasmid #102634). Note that the sequence of sfGFP is heavily modified and contains mutations from Bundy et al.tttgtttaactttaagaaggagatatacatATGAGCAAAGGTGAAGAACTGTTTACCGGCGTTGTGCCGATTCTGGTGGAACTGGATGGCGATGTGAACGGTCACAAATTCAGCGTGCGTGGTGAAGGTGAAGGCGATGCCACGATTGGCAAACTGACGCTGAAATTTATCTGCACCACCGGCAAACTGCCGGTGCCGTGGCCGACGCTGGTGACCACCCTGACCTATGGCGTTCAGTGTTTTAGTCGCTATCCGGATCACATGAAACGTCACGATTTCTTTAAATCTGCAATGCCGGAAGGCTATGTGCAGGAACGTACGATTAGCTTTAAAGATGATGGCAAATATAAAACGCGCGCCGTTGTGAAATTTGAAGGCGATACCCTGGTGAACCGCATTGAACTGAAAGGCACGGATTTTAAAGAAGATGGCAATATCCTGGGCCATAAACTGGAATACAACTTTAATAGCCATAATGTTTATATTACGGCGGATAAACAGAAAAATGGCATCAAAGCGAATTTTACCGTTCGCCATAACGTTGAAGATGGCAGTGTGCAGCTGGCAGATCATTATCAGCAGAATACCCCGATTGGTGATGGTCCGGTGCTGCTGCCGGATAATCATTATCTGAGCACGCAGACCGTTCTGTCTAAAGATCCGAACGAAAAAGGCACGCGGGACCACATGGTTCTGCACGAATATGTGAATGCGGCAGGTATTACGTGGAGCCATCCGCAGTTCGAAAAATAAgtcgaccggctgctaacaaagcccgaaagg.84. 20\u2009ng of purified, linear pJL1 backbone, 20\u2009ng of purified, linear sdFab VH or VL constant DNA, and 20\u2009ng of the protein open reading frame insert were combined in 2\u2009\u03bcL Gibson assembly reactions and incubated at 50\u2009\u00b0C for 30\u2009min. The unpurified assembly reactions were diluted in 40\u2009\u03bcL of nuclease-free water and 1\u2009\u03bcL of the diluted reaction was used as the template for a PCR to generate linear expression templates (LETs) for CFPS. Linear expression templates were amplified via PCR using the pJL1_LET_F (ctgagatacctacagcgtgagc) and pJL1_LET_R (cgtcactcatggtgatttctcacttg) primers in a 50\u2009\u03bcL PCR reaction using the Q5 Hot Start DNA polymerase following manufacturer instructions.Gibson assembly was used to assemble protein open reading frame DNA with the pJL1 backbone following the published protocol with the addition of 3.125\u2009\u03bcg/mL of ET SSB P. pyralis luciferase containing a c-terminal strepII tag used as a negative control is below and was cloned into the pJL1 vector.The DNA sequence of the atggaagacgctaagaacattaagaagggacctgctccattctaccccctcgaagacggcactgcaggtgagcagcttcataaagcgatgaagcgttatgcgttagttcctggcacgatcgccttcactgacgcgcacatcgaagtcaatatcacctacgctgaatactttgagatgagtgtgcgtctggcggaagccatgaagcgttatggccttaacacgaaccaccgcatcgttgtttgtagcgagaattccttacaattcttcatgcccgtccttggcgcgctgtttattggtgtggccgttgcaccagccaatgacatctataatgagcgcgagttgttgaactccatgaacatttctcaaccaacagtggtgttcgtttcaaagaaaggcttacagaaaatcttaaacgttcaaaagaaactgccgattatccagaagatcatcattatggatagtaagactgactaccagggcttccagtcaatgtatacattcgtgacgagtcacctgcccccgggttttaacgagtacgactttgtcccagagagctttgatcgcgacaagaccatcgccctcattatgaatagcagtggttcgacgggtagcccaaagggagtggccctgccccatcgtaccgcgtgcgtccgtttctcccatgcccgcgacccaattttcggcaatcaaatcatccccgacacggcaatcttgtcggtcgtcccgtttcaccatggctttggaatgtttacgacactcggttacctcatctgcggtttccgcgtcgttctgatgtatcgcttcgaggaagagttgttcttacgttcgcttcaggactacaagattcaatccgcccttctggtccccactttgttcagtttctttgctaagagcaccttaattgataagtatgacctctccaacttacacgagattgcgagcggtggtgctcccctcagcaaagaggttggagaggcggttgctaagcgttttcatctgcccggtatccgtcaaggttacggcctcaccgaaaccacttctgccattcttatcactccggaaggtgacgataagcctggggcagtgggtaaagttgtacccttcttcgaggctaaggttgtggatttagatacggggaagaccttaggtgtgaaccagcgcggtgaactgtgcgttcgcggtccgatgattatgtcgggttatgttaatgaccccgaggctacgaacgcgcttatcgataaggacggttggcttcattccggcgacatcgcttactgggatgaggatgagcacttcttcatcgttgaccgtctgaagagtctcatcaagtataagggatgtcaagtcgctccggcagagttagagagcatcttactccagcaccctaatatcttcgatgctggggttgccgggctcccaggcgacgatgccggcgagctgccggcggcggtagttgttttagagcatggcaagaccatgaccgaaaaggagattgtagactacgtcgcgagtcaagtaaccacagcgaagaagctccgcggtggagtggtctttgttgacgaggtgcctaaaggcctgacgggcaaacttgacgcgcgtaagatccgtgagatcctcatcaaagcgaagaagggtgggaagagtaagctggggagttcaggttggtcccacccgcaatttgagaagtga.E. coli OrigamiTM B(DE3) extracts were prepared using a modified version of established protocols86. Briefly, a 150\u2009mL OrigamiTM B(DE3) starter culture was inoculated in LB from a glycerol stock and cultured in a 250\u2009mL baffled flask at 37\u2009\u00b0C for 16\u2009h. The 2xYTP was prepared without glucose in 75% of the final volume and sterilized using an autoclave. A 4x glucose solution was prepared and autoclaved separately, then added to the medium immediately before use. The starter cultures were used to inoculate 1\u2009L of 2xYTPG media in a 2.5\u2009L Full-Baffle Tunair shake flask at an initial OD600 of 0.08. Cells were cultured at 37\u2009\u00b0C at 220 RPM in a shaking incubator. Cultures were grown until OD600 0.4-0.6, at which point the expression of T7 RNA polymerase was induced by the addition of IPTG to a final concentration of 0.5\u2009mM. Cells were harvested at an OD600 of 2.5 via centrifugation at 12,000\u2009\u00d7\u2009g for 1\u2009min at 4\u2009\u00b0C. Cell pellets were washed three times with 25\u2009mL S30 buffer per 50\u2009mL culture . Pellets were resuspended in 1\u2009mL S30 buffer per gram of cell mass. Cell suspensions were lysed using a single pass on an Avestin EmulsiFlex-B15 Homogenizer at a lysis pressure of 24,000 PSI. Cell debris was separated via centrifugation at 18,000\u2009\u00d7\u2009g for 20\u2009min, and the clarified lysate was collected, flash-frozen in liquid nitrogen, and stored at \u221280\u2009\u00b0C.E. coli MRE600 tRNA (Roche 10109541001), 100\u2009mM NAD, 50\u2009mM CoA, 4\u2009mM oxalic acid, 1\u2009mM putrescine, 1\u2009mM spermidine, 57\u2009mM HEPES pH 7.2, 2\u2009mM of each amino acid, 33.3\u2009mM PEP, 20% v/v E. coli extract, varying concentrations of DNA template, and the remainder water. The preparation of these reagents has been described in detail elsewhere87. For DNA templates, plasmids were used at a concentration of 8\u2009nM, and unpurified linear PCR products were used at 6.66% (v/v). For the expression of antibodies, each template was added to a final concentration of 6.66% (v/v). For antibody and sdFab expression 4\u2009mM oxidized glutathione, 1\u2009mM reduced glutathione, 14\u2009\u03bcM of purified DsbC, and 50\u2009\u03bcM FkpA were also supplemented to the reactions. In addition, for oxidizing CFPS reactions, cell-extracts were treated with 500\u2009\u03bcM iodoacetamide (IAM) at room temperature for 30\u2009min before use in CFPS88. All reaction components were assembled on ice and were either run as 12\u2009\u03bcL reactions in 1.5\u2009mL microtubes or 2\u2009\u03bcL reactions in 384-well plates . For 2\u2009\u03bcL reactions, components were transferred to the plate using an Echo 525 acoustic liquid handler. A mix containing all the CFPS components except for the DNA was dispensed from 384PP Plus plates using the BP setting. The DNA (unpurified PCR products) was dispensed from a 384LDV Plus plate using the GP setting. Reactions were allowed to proceed at 30\u2009\u00b0C for 20\u2009h.CFPS reactions were composed of the following reagents: 8\u2009mM magnesium glutamate, 10\u2009mM ammonium glutamate, 130\u2009mM potassium glutamate, 1.2\u2009mM ATP, 0.5\u2009mM of each CTP, GTP, and UTP. 0.03\u2009mg/mL folinic acid, 0.17\u2009mg/mL 86. Radioactive leucine was added to CFPS at a final concentration of 10\u2009\u03bcM of L-[14C(U)]-leucine , followed by precipitation of the expressed proteins and scintillation counting89. To quantify sfGFP fluorescence, 2\u2009\u03bcL of a CFPS reaction was diluted in 48\u2009\u03bcL of water in a Black Costar 96 Well Half Area Plate. Fluorescence was measured using a BioTek SynergyTM H1 plate reader with excitation and emission wavelengths of 485 and 528, respectively. Scintillation counts and fluorescence were fit to determine a standard curve for use with non-radioactive samples.To quantify sfGFP fluorescence, a standard curve was prepared using previously reported methodsTM . FluoroTectTM was included in the CFPS reaction at 3.33%v/v. After protein synthesis, RNAseA was added to 0.1\u2009mg/mL and the sample was incubated at 37\u2009\u00b0C for 10\u2009min. 3\u2009\u03bcL of the CFPS and RNAseA mixture were combined with 4x loading buffer and the samples were subsequently denatured at 70\u2009\u00b0C for 3\u2009min, then separated via SDS-PAGE and imaged using a LI-COR Odyssey Fc imager on the 600 channel. Densitometry was performed using the ImageJ software.To visualize antibody assembly, proteins were labeled during CFPS with FluoroTect77. DsbC and FkpA were ordered as gBlocks from IDT containing a c-terminal, TEV cleavable his tag (GSENLYFQSGSHHHHHHHHHH) and cloned into pET28a. Plasmid maps of both DsbC and FkpA are available in the Source Data. Plasmids were transformed into BL21 StarTM DE3, plated on LB agar, and cultured overnight at 37\u2009\u00b0C. 1\u2009L of Overnight Express TB was inoculated by scraping all colonies on a transformation plate and cultured at 37\u2009\u00b0C in 2.5\u2009L tunair flasks at 220\u2009rpm overnight. Cells were harvested, resuspended at a ratio of 1\u2009g cell mass to 4\u2009mL resuspension buffer , 1\u2009mg/mL lysozyme, 62.5\u2009U/mL cell suspension of benzonase ) and lysed using an Avestin B15 homogenizer at 24,000 PSI. The lysate was spun down 14,000\u2009\u00d7\u2009g for 10\u2009min and the clarified supernatant was incubated with Ni-NTA Agarose for 60\u2009min on an end-over-end shaker. The resin was spun down 2500l\u2009\u00d7\u2009g for 2\u2009min, the supernatant removed, resuspended in wash buffer , loaded on a gravity flow column, and subsequently washed with 20X resin volumes of wash buffer. Protein was eluted using elution buffer and exchanged into 50\u2009mM HEPES pH 7.4, 150\u2009mM NaCl using PD-10 desalting columns according to manufacturer instructions.Protein expression, purification, and his tag removal were performed similarly to previously reportedHis tags were removed via cleavage by ProTEV Plus . Before cleavage, 10% v/v glycerol was added to the protein. ProTEV Plus was added to a concentration of 0.5 U/\u03bcg purified protein and DTT was added to a concentration of 1\u2009mM. Cleavage reactions were carried out at 30\u2009\u00b0C for 4\u2009h. Free His tag and ProTEV Plus were removed by incubating with Ni-NTA Agarose for 1\u2009h at 4\u2009\u00b0C and collecting the supernatant. Proteins were subsequently concentrated to >1\u2009mg/mL . His tag removal was validated via SDS-PAGE and the AlphaScreen Histidine (Nickel Chelate) Detection Kit .90.AlphaLISA reactions were carried out in 50\u2009mM HEPES pH 7.4, 150\u2009mM NaCl, 1\u2009mg/mL BSA, and 0.00015\u2009v/v TritonX-100 (hereafter referred to as Alpha buffer). All components were dispensed using an Echo 525 liquid handler from a 384-Well Polypropylene 2.0 Plus microplate using the 384PP_Plus_GPSA fluid type. All components of the AlphaLISA reactions were prepared as 4x stocks and added as 0.5\u2009\u03bcL to the final 2\u2009\u03bcL reaction to achieve the desired concentration. All AlphaLISA reactions were performed with CFPS reactions diluted to a final concentration of 0.025\u2009v/v. AlphaLISA beads were combined to prepare a 4X stock in Alpha buffer immediately before use and added to the proteins to yield a concentration of 0.08\u2009mg/mL donor beads and 0.02\u2009mg/mL acceptor beads in the final reaction. All reactions were incubated with AlphaLISA beads for at least 1\u2009h before measurement. AlphaLISA measurements were taken on a Tecan Infinite M1000 Pro plate reader using the AlphaLISA filter with an excitation time of 100\u2009ms, an integration time of 300\u2009ms, and a settling time of 20\u2009ms. Before measurement, plates were allowed to equilibrate inside the instrument for 10\u2009min. For measurements involving sdFabs, protein A AlphaLISA beads were avoided due to the ability of protein A to bind human subgroup VH3 FabsThe impact of CFPS reagents on AlphaLISA was determined by serially diluting the specified reagents in Alpha buffer and combining them with the specified AlphaLISA conditions. The TrueHits kit was used to assess the impact of the CFPS reagents on the Alpha detection chemistry. CFPS reagents were mixed with the donor and acceptor beads and incubated for 2\u2009h before measurement. His tagged RBD and human FC tagged human ACE2 were used to evaluate the impact of CFPS reagents on capture chemistries. RBD and ACE2 were diluted in Alpha buffer, mixed at a final reaction concentration of 10\u2009nM each, combined with the CFPS reagents, and allowed to incubate for 1\u2009h. Donor and acceptor beads were subsequently added and allowed to incubate for a further hour before measurement. Protein A Alpha donor beads , Ni-Chelate AlphaLISA acceptor beads , and anti-6xhis AlphaLISA acceptor beads were utilized for detection.50 values were recorded from the product page at the time of purchase and converted to \u03bcg/mL assuming a MW of 150,000\u2009Da if reported in M. Antibodies were serially diluted in Alpha buffer and mixed with SARS-CoV-2 RBD at a concentration of 10\u2009nM in the final reaction and incubated for 1\u2009h. Mouse FC tagged human ACE2 was subsequently added and incubated for 1\u2009h, followed by simultaneous addition of the acceptor and donor beads. AlphaLISA detection was performed using Anti-Mouse IgG Alpha Donor beads and Strep-Tactin AlphaLISA Acceptor beads . IC50 values were calculated using Prism 9 by fitting the normalized data to [Inhibitor] vs. response\u2013Variable slope (four parameters) fit with the max constrained to a value of 1.The commercial neutralizing antibody ACE2 competition experiment was performed with the following antibodies: nAb1 , nAb2 , nAb3 , nAb4 . ELISA ICFor all antibody screening experiments, the different reagents and AlphaLISA conditions used are described in Supplementary Table\u00a0Assembly AlphaLISA reactions consisted of sdFab expressing CFPS and either Rabbit Anti-Human kappa light chain antibody or Rabbit Anti-Human lambda light chain . CFPS reaction containing the expressed sdFab of interest was mixed with the appropriate anti-light chain antibody and allowed to equilibrate for two hours before the simultaneous addition of the acceptor and donor beads.SARS-CoV-2 S6P binding AlphaLISA reactions consisted of sdFab expressing CFPS and SARS-CoV-2 S6P CFPS reaction containing the expressed sdFab of interest was mixed with the S6P and allowed to equilibrate for two hours before the simultaneous addition of the acceptor and donor beads.SARS-CoV-2 RBD binding AlphaLISA reactions consisted of sdFab expressing CFPS and SARS-CoV-2 RBD. CFPS reaction containing the expressed sdFab of interest was mixed with the RBD and allowed to equilibrate for two hours before the simultaneous addition of the acceptor and donor beads.ACE2 and RBD competition AlphaLISA reactions consisted of sdFab expressing CFPS, human ACE2, and SARS-CoV-2 S6P. CFPS reaction containing the expressed sdFab of interest was first mixed with S6P and allowed to incubate for 1\u2009h. Subsequently, ACE2 was added and allowed to equilibrate for a further 1\u2009h before the simultaneous addition of the acceptor and donor beads.77), SARS-CoV-2 S6P Beta/B.1.351 , SARS-CoV-2 S6P Gamma/P.1 , SARS-CoV-2 S6P Delta/B.1.617.2 , SARS-CoV-2 S6P Omicron/BA.1 , SARS-CoV-2 S6P Omicron/BA.2 , SARS-CoV-2 S6P Omicron/BA.2.12.1 , SARS-CoV-2 S6P Omicron/BA.4/5 , SARS-CoV S2P , MERS-CoV S2P , HCoV-HKU1 S , HCoV-OC43 S , HCoV-NL63 S , and HCoV-229E S .For SARS-CoV-2 variant and other non-SARS-CoV-2 coronavirus binding experiments, AlphaLISA measurements were carried out in the same manner as described for SARS-CoV-2 S6P. The following Hisx6-tagged proteins were used. SARS-CoV-2 S6P , SARS-CoV-2 S6P Alpha/ B.1.1.7 of human ACE2. All three components were incubated for an additional hour prior to simultaneous addition of AlphaLISA beads. Reactions were incubated for 2\u2009h prior to measurement.For RBD and ACE2 bridging experiments SARS-CoV-2 RBD, human ACE2, and the specified dilution of CFPS were incubated for 1\u2009h prior to the simultaneous addition of the AlphaLISA beads. Reactions were incubated for 2\u2009h prior to measurement.10 viral particles (vp) of ChAd-SARS-CoV-2-S73 in 50\u2009\u00b5l of PBS via intramuscular injection in the hind leg. Draining inguinal lymph nodes were collected 10 days later and processed into a single-cell suspension. Cells were stained with biotinylated recombinant SARS-CoV-2 spike (S2P) for 30\u2009min at 4\u2009\u00b0C then washed twice with FACS buffer followed by staining with anti-CD19 BV421 (BioLegend # 115537), anti-CD4 FITC (BioLegend # 100405), anti-IgD-PE-Cy7 (BioLegend # 405719), Streptavidin APC (BioLegend # 405207), aqua cell viability dye (Invitrogen L34957), and anti-mouse CD16/CD32 Fc block (BioLegend # 156607). Spike-positive activated B cells (live singlet CD4- CD19+ IgDlo Streptavidin+) were bulk sorted on BD FACSAriaII sorter.Female C57BL/6 (Strain: 000664) were purchased from The Jackson Laboratory. Six-week-old animals were immunized with 10The following 10x Genomics kits were used for libraries preparation: Chromium Single Cell 5\u2032 Library and Gel Bead Kit v2 (PN-1000006), Chromium Single Cell A Chip Kit (PN-1000152), Chromium Single Cell V(D)J Enrichment Kit, Mouse B cell (96rxns) (PN-1000072), and Single Index Kit T (PN-1000213). The GEM generation and barcoding was followed by cDNA preparation then GEM RT reaction and bead cleanup steps. Purified cDNA was amplified for 10\u201314 cycles then cleaned up using SPRIselect beads. cDNA concentration was determined by running samples on a Bioanalyzer. BCR target enrichments were done on the full-length cDNA followed by BCR libraries preparation as recommended by 10x Genomics Chromium Single Cell V(D)J Reagent Kits (v1 Chemistry) user guide. The cDNA Libraries were sequenced on Novaseq S4 (Illumina), targeting a median sequencing depth of 5000 read pairs per cell.Cell Ranger v3.1.0 for alignment against the GRCm38 mouse reference v3.1.0 , generating 3760 assembled high-confidence BCR sequences for 4420 cells. Sequences for screening were selected randomly from the top clonal groups with >10 members in the clonal group. Cellranger vdj output was then parsed using Change-O v0.4.6 within the immcantation suite. Additional quality control included examining sequences to be productively rearranged and have valid V and J gene annotations. Furthermore, only cells with exactly one heavy chain sequence paired with at least one light chain sequence were kept.Demultiplexed pair-end FASTQ reads from 10x Genomics single-cell V(D)J profiling were preprocessed using the \u201ccellranger vdj\u201d command from Further information on research design is available in the\u00a0Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Reporting Summary"} +{"text": "Shigella flexneri to selectively internalize into glioblastoma (GBM) brain tumor cells as an initial step to generating a bacterial-based drug delivery system.The use of microorganisms as drug delivery systems to treat cancer has expanded recently, including FDA approval of certain viruses as oncolytics. Microorganisms have several unique benefits compared to traditional pharmacologic agents including dose independence, the ability to produce therapeutic proteins locally within the tumor, and simplicity of administration. However, current microbial delivery systems such as AAV9 and herpes virus have limited cassette sizes, minimal cancer cell selectivity, and low innate cytotoxicity. To address these issues, we sought to generate a strain of S. flexneri that selectively internalize into GBM cells using iterative co-cultured assays.We generated After 50 rounds of co-culture, the new strain infected 95 percent of GBM cells in 2 hours. GBM-infecting Shigella demonstrate a 124-fold preference for internalizing in nine different GBM cell lines compared to Normal Astrocytes (NA) controls. Additionally, we developed an in-cell western to identify GBM-infecting Shigella clones that preferentially internalize in patient samples without iterative co-culture. Finally, we demonstrate internalization into GBM cells is mediated via a factor modified by myristoylation.In conclusion, here we present a novel bacterial platform that preferentially internalizes in brain tumor cells. This system provides numerous potential benefits over current interventions and other microbial strategies for treating brain tumors. Microorganism-based drug delivery systems are emerging as a promising approach to treating solid tumors \u20134. Most Glioblastoma (GBM) is the most common malignant, primary brain tumor observed in adults, and patients diagnosed with GBM demonstrate a median survival of only 15 months . The staShigella flexneri, a gram-negative intracellular bacterium similar to the viruses discussed above. Importantly, Shigella has a type 3 secretion system capable of administering toxic proteins into the host cell cytosol (30 g/L) + agar (15 g/L), and Congo Red (100 mg/L) sterile plates and incubated overnight at 30\u00b0C or 37\u00b0C. Individual colonies were expanded in sterile Tryptic Soy Broth (30 g/L) using a shaking incubator (250 rpm) at 30\u00b0C.Shigella was streaked out from the frozen stock onto TSB (30 g/L) + Congo Red (0.01% w/v) plates and incubated overnight at 37\u00b0C. Colonies were picked and grown for approximately 2 hours (OD650\u00a0=\u00a00.6) (\u00ae EHEC (Cat#: 751630). Band intensity was measured using ImageJ.At 24 hours prior to testing, 0\u00a0=\u00a00.6) . SamplesShigella colony was picked using a pipet tip and resuspended in 20 \u03bcL nuclease-free water. A colony was incubated for 10\u00a0min at 95\u00b0C and then centrifuged at 1,000 rcf for 1\u00a0min to remove membrane components.A single Supernatant at a volume of 5 \u03bcL was used as the template for a PCR assay to quantify the presence of DNA encoding for Shiga toxin(s). Potential DNA regions were amplified with the Phusion taq polymerase kit using previously described primers that are specific for Shiga toxin 1 (Stx1 \u2013 primer pair: ATGTCAGAGGGATAGATCCA and TATAGCTACTGTCACCAGACAAT), Shiga toxin 2 (Stx2 \u2013 primer pair: AGTTCTGCGTTTTGTCACTGTC and CGGAAGCACATTGCTGATT), or positive control virulence factor F (virF \u2013 primer pair: AGCTCAGGCAATGAAACTTTGAC and TGGGCTTGATATTCCGATAAGTC) . Bands wS. flexneri was streaked onto TSB agar plates with 0.01% Congo Red and grown at 37\u00b0C overnight. The next day, 96 red colonies of Shigella were picked using a filtered p200 pipet tip. Each colony was added into 400 \u03bcL of TSB in a deep-well block plate and incubated at 37\u00b0C, 250 rpm, until an OD650 of 0.6 was reached (approximately 2.5 to 3 hours). Shigella suspensions were combined and pelleted into a 50-mL conical tube. After washing three times with 25 mL of 10% glycerol, Shigella was resuspended in 5 mL of 10% glycerol. Shigella was electroporated with 200 ng of pRedTKI plasmid DNA (Addgene Plasmid #51628) and recovered in TSB for 1 hour at 30\u00b0C, followed by plating on TSB + 50 \u03bcg/mL Kan agar plates with 0.01% Congo Red overnight. The pRedTKI plasmid introduces the Lambda red genes necessary for homologous recombination along with conferring Kan resistance.Genes were deleted as previously described using standard Lambda red recombineering methods with minor modifications , 27. S. Shigella + pRedTKI colonies were picked and incubated at 30\u00b0C, 250 rpm, until an OD650 of 0.6 was reached (approximately 3 hours); during the last 30\u00a0min of incubation, l-arabinose was added at 10 mmol/L to induce the Lambda red recombination genes along with 50 \u03bcg/mL kanamycin to maintain transmission of the pRedTKI plasmid. Bacteria were pelleted in a 50-mL conical tube by centrifugation at 4,000 \u00d7 g, transferred to a 1.5-mL Eppendorf tube, washed three times with 500 \u03bcL of 10% glycerol, and resuspended in 50 \u03bcL of 10% glycerol. Using an electroporation cuvette with a 2\u00a0mm gap, Shigella were transfected via electroporation with 200 ng of I-SceI-flanking resistance cassette with 70-bp homology adjacent to the gene to be deleted. The cassette for MsbB1 was as follows: TGGTGCGGGGCAAGTTGTGCCGCTACACTATCACCAGATTGATTTTTGCCTTATCCGAAACTGGAAAAGCTAGGGATAACAGGGTAATATGGAGAAAAAAATCACTGGATATACCACCGTTGATATATCCCAATGGCATCGTAAAGAACATTTTGAGGCATTTCAGTCAGTTGCTCAATGTACCTATAACCAGACCGTTCAGCTGGATATTACGGCCTTTTTAAAGACCGTAAAGAAAAATAAGCACAAGTTTTATCCGGCCTTTATTCACATTCTTGCCCGCCTGATGAATGCTCATCCGGAATTACGTATGGCAATGAAAGACGGTGAGCTGGTGATATGGGATAGTGTTCACCCTTGTTACACCGTTTTCCATGAGCAAACTGAAACGTTTTCATCGCTCTGGAGTGAATACCACGACGATTTCCGGCAGTTTCTACACATATATTCGCAAGATGTGGCGTGTTACGGTGAAAACCTGGCCTATTTCCCTAAAGGGTTTATTGAGAATATGTTTTTCGTCTCAGCCAATCCCTGGGTGAGTTTCACCAGTTTTGATTTAAACGTGGCCAATATGGACAACTTCTTCGCCCCCGTTTTCACCATGGGCAAATATTATACGCAAGGCGACAAGGTGCTGATGCCGCTGGCGATTCAGGTTCATCATGCCGTTTGTGATGGCTTCCATGTCGGCAGAATGCTTAATGAATTACAACAGTACTGCGATGAGTGGCAGGGCGGGGCGTAATAGGGATAACAGGGTATAAAAGCCTCTCGCGAGGAGAGGCCTTCGCCTGATGATAAGTTCAAGTTTGCTTCAGAATATTCGAAATCT.The cassette for MsbB2 was as follows: AATTAAGGTTAGATGTATTCTCTGAATAAAATATTAATGATGATTATGGTAGGGGCATTCGCACTAAATATAGGGATAACAGGGTAATATGGAGAAAAAAATCACTGGATATACCACCGTTGATATATCCCAATGGCATCGTAAAGAACATTTTGAGGCATTTCAGTCAGTTGCTCAATGTACCTATAACCAGACCGTTCAGCTGGATATTACGGCCTTTTTAAAGACCGTAAAGAAAAATAAGCACAAGTTTTATCCGGCCTTTATTCACATTCTTGCCCGCCTGATGAATGCTCATCCGGAATTACGTATGGCAATGAAAGACGGTGAGCTGGTGATATGGGATAGTGTTCACCCTTGTTACACCGTTTTCCATGAGCAAACTGAAACGTTTTCATCGCTCTGGAGTGAATACCACGACGATTTCCGGCAGTTTCTACACATATATTCGCAAGATGTGGCGTGTTACGGTGAAAACCTGGCCTATTTCCCTAAAGGGTTTATTGAGAATATGTTTTTCGTCTCAGCCAATCCCTGGGTGAGTTTCACCAGTTTTGATTTAAACGTGGCCAATATGGACAACTTCTTCGCCCCCGTTTTCACCATGGGCAAATATTATACGCAAGGCGACAAGGTGCTGATGCCGCTGGCGATTCAGGTTCATCATGCCGTTTGTGATGGCTTCCATGTCGGCAGAATGCTTAATGAATTACAACAGTACTGCGATGAGTGGCAGGGCGGGGCGTAATAGGGATAACAGGGTAATAATTATAAAGTACAGGTATTTCCACTAGTTGTTTCTTACAGGTTACCAATCGAAACACATCCCCCTTCCG.Shigella was allowed to recover in Super Optimal broth with Catabolite repression (SOC) for 3 hours at 30\u00b0C and plated on TSB + 50 \u03bcg/mL Kan + 12.5 \u03bcg/mL chloramphenicol (Cam) agar plates with 0.01% Congo Red overnight.The primer pair for identifying MsbB1 was TTGAACTTATCATCAGGCGAAGGCCTC and CGGCTTTTTTTATTTGGTGCGGGG, and that for MsbB2 was CTGCTATCCGCTCTTTGGATGCA and CTACACAGTCCTCCGTGCCAA. Electroporated Shigella + pRedTKI + Cam resistance cassette was incubated at 30\u00b0C, 250 rpm, until an OD650 of 0.6 (approximately 3 hours) in 3 mL TSB + 50 \u03bcg/mL Kan + 20 mmol/L isopropyl \u03b2-d-1-thiogalactopyranoside (IPTG) to induce I-SceI expression to excise the Cam resistance gene. A sample of liquid culture was seeded onto TSB + 50 \u03bcg/mL Kan + agar plates with 0.01% Congo Red and incubated overnight. Modifications were verified by PCR.To complete the gene deletion protocol, a single colony of l-cysteine, NaOH, CaCl2, DNAse I, EDTA, and papain for 35\u00a0min, and then inactivated in serum media. Cells then underwent trituration, were strained through a 100-\u03bcm cell strainer, and were centrifuged for 5\u00a0min at 200 \u00d7 g, 4\u00b0C. The supernatant was aspirated, and cells were counted and replated at 750,000 cells/mL. Neurobasal media at a volume of 2 mL with B-27 supplement, GlutaMAX, and 1.25% FBS was added to a culture plate. Thirty percent of the media was replaced twice per week. After 3 days of culture, 5-fluoro-2\u2032-deoxyuridine (FUDR) was added to remove dividing cells. All mammalian cells were cultured at 37\u00b0C, 5% CO2.GBM cell lines were obtained from Dr. John Kuo or ATCC . Normal astrocytes were purchased from Lonza (Cat#: CC-2565) and grown according to the manufacturer\u2019s protocol without antibiotics. Mammalian cells were grown in tissue culture-treated T25 or T75 flasks in low-glucose Dulbecco\u2019s modified Eagle\u2019s medium (DMEM) with 10% fetal bovine serum (FBS), GlutaMAX, and 1% sodium pyruvate (without pen/strep) \u201330. CellMammalian cells were maintained in culture as described above. At 24 hours prior to co-culture assays, the media was changed without removing cells from the tissue culture flask. Cells grown to 80% confluency in a T25 flask were used for each round of co-culture.S. flexneri was streaked on TSB agar plates with 0.01% Congo Red and grown at 37\u00b0C overnight. The next day, 96 red colonies of Shigella were picked using a filtered p200 pipet tip and added to individual wells of a deep-well block plate that contained 400 \u03bcL of TSB. The deep-well block plate was incubated at 37\u00b0C, 250 rpm, until an OD650 of 0.6 was reached (approximately 2.5 to 3 hours). A control well was used to estimate the Shigella growth rate. After reaching the mid-log phase (0.6 OD650), individual wells were pooled, and the Shigella was concentrated to 2 \u00d7 108 cfu/mL (OD650 of 1.0\u00a0=\u00a08.0 \u00d7 108). Next, 250 \u03bcL of Shigella concentrate was added to ~5 mL of GBM, normal astrocyte (NA), or rNeuron media. Shigella-containing media at a volume of 5 mL was added to the T25 cell culture flask containing mammalian cells. The T25 culture flask was centrifuged at 200 \u00d7 g for 10\u00a0min and then incubated at 37\u00b0C, 5% CO2, for 30\u00a0min. After 30\u00a0min, culture media was removed by aspiration. The T25 flask was washed four times using 5 mL of phosphate-buffered saline (PBS) for 1\u00a0min with gentle agitation. Next, 5 mL of mammalian culture media containing 20 \u03bcg/mL of gentamicin was added to the T25 flask. The T25 flask was incubated at 37\u00b0C, 5% CO2, for an additional 90\u00a0min. After 90\u00a0min, the media was aspirated, and cells were washed four times with PBS, 1\u00a0min per wash, with gentle agitation.2, until GBM, NA, or rNeuron cells detached from the plate. Mammalian cell membranes were mechanically disrupted by pulling the cell suspension through a 27Ga needle 10 times. The mammalian cell lysate was centrifuged at 1,000 \u00d7 g for 2\u00a0min to pellet any membrane-associated Shigella. Mammalian cell lysate at a volume of 20 \u03bcL was plated onto a TSB + 0.01% Congo Red agar plate and grown at 37\u00b0C overnight. Individual colonies were expanded and frozen in 25% glycerol or used directly for the next round of co-culture assays.After the final wash, 1 mL of Accutase was added to the T25 flask. The T25 flask was incubated at 37\u00b0C, 5% COd-Lysine, No. 1.5 glass-bottom dishes .At 24 hours prior to internalization, GBM cells were grown to ~80% confluence using the protocol described above in the mammalian cell culture section on Poly-Shigella colony was picked, grown to the mid-log phase, and co-cultured with mammalian cells in the microscopy dish using the protocol described above in the co-culture assay section. After the final wash, cells were fixed with 4% paraformaldehyde (PFA) for 10\u00a0min and then washed three times with PBS.An individual PBS staining solution at a volume of 1 mL containing wheat germ agglutinin-fluorescein and Hoechst 33342 was added to the fixed cells and incubated for 15\u00a0min. Cells were washed three times with PBS. After the final wash, 1 mL of PBS was added to the dish so cells did not dry out during imaging. Cells were imaged using a 60\u00d7 objective on a Nikon Eclipse Ti2 confocal microscope. Images were processed and analyzed using the publicly available ImageJ software package.Shigella antibody in 1 mL PBS + 30 mg/mL BSA + 0.1% Triton X-100 for 90\u00a0min at room temperature. After incubation, cells were washed three times with PBS. Next, cells were incubated with anti-rabbit Alexa Fluor 488 secondary in PBS + 30 mg/mL BSA + 5% donkey serum + 0.1% Triton X-100 for 60\u00a0min. Cells were washed three times with PBS. Next, cells were incubated with Phalloidin Dye 594 and Hoechst 33342 in PBS for 15\u00a0min. Cells were imaged using a 60\u00d7 objective on a Nikon Eclipse Ti2 confocal microscope. Images were processed and analyzed using the publicly available ImageJ software package.Cells were permeabilized by adding 1 mL of 0.1% Triton X-100 in PBS for 5\u00a0min. Blocking buffer at a volume of 1 mL + 5% donkey serum + 0.1% Triton X-100) was added to each dish, and the cells were incubated at 4\u00b0C overnight. The next day, cells were washed three times with PBS. Next, cells were incubated with an anti-At 24 hours prior to internalization, GBM or NA cells were grown to 80% confluence as described in the mammalian tissue culture section above on a tissue culture-treated, black well, clear bottom, 96-well tissue culture plate.Shigella colonies were picked and expanded as described above in a deep block 96-well plate. Shigella culture at a volume of 40 \u03bcL from a single well was added to 200 \u03bcL of GBM or NA media previously plated into each well of the clear-bottom, black, 96-well plate. The 96-well plate was centrifuged at 200 \u00d7 g for 10\u00a0min and then incubated at 37\u00b0C, 5% CO2, for 30\u00a0min. Culture media was removed after 30\u00a0min by dumping the supernatant into a sterile glass dish containing bleach. The plate was washed four times with PBS using a similar method to remove the supernatant. Next, 200 \u03bcL of mammalian cell media containing 20 \u03bcg/mL gentamicin was added to each well. The plate was incubated at 37\u00b0C, 5% CO2, for 90\u00a0min. After incubation, the plate was washed four times with PBS. Cells were fixed by adding 200 \u03bcL of 4% PFA to each well and incubating for 10\u00a0min. The plate was washed three times with PBS. Cells were permeabilized by adding 200 \u03bcL of PBS containing 0.1% Triton X-100 to each well for 5\u00a0min. Blocking buffer containing 1% BSA and 1% donkey serum was added to each well, and the plate was incubated at 4\u00b0C overnight. The next day, the plate was washed three times with PBS. The plate was incubated with anti-Shigella antibody in PBS + 30 mg/mL BSA + 0.1% Triton X-100 (100 \u03bcL per well) for 90\u00a0min. The plate was washed with PBS three times. Finally, each well was incubated with IR800CW anti-rabbit secondary in PBS + 30 mg/mL BSA + 5% donkey serum + 0.1% Triton X-100 for 60\u00a0min. The plate was washed three times with PBS and imaged on a LI-COR Fc scanner using a 2-min medium resolution scan. A standard curve of Shigella was used on each plate to convert the IR800 signal to the number of bacteria per well. It was important to ensure that the moles of anti-Shigella antibody greatly exceeded the moles of Shigella to ensure the saturated binding assumption was valid in order to quantify the number of bacteria per well.Individual Shigella internalization in cell lines were determined using ANOVA with a 1% false discovery rate used as the threshold for significance. For microscopy assays, ANOVA was used to determine differences between groups with a 5% false discovery rate used as the threshold for significance.Data are generally presented as means with standard deviation. All validation experiments were conducted with a minimum of two independent replicates. For microscopy experiments, a minimum of three fields were quantified from at least two independent experiments. Significant differences between Shigella clones that preferentially internalize into brain tumors using iterative co-culture assays. Briefly, S. flexneri, an intracellular bacterium, was streaked onto agar plates. Clones containing the virulence factor needed to survive inside mammalian cells were expanded and incubated with a brain tumor cell line, U-251 GBM. Bacteria that internalized into GBM cells were harvested and used for additional rounds of co-culture.As described in Shigella strain that is safe to use as a therapeutic platform, we identified a strain that did not contain Shiga toxins. We probed S. flexneri strain 2475 serotype 2a for DNA regions encoding Shiga toxins using previously described primers. As expected, we did not observe a band at the expected size for the PCR product when stained with a DNA intercalating dye Hoechst 33342 (rendered blue). The number of GBM cells demonstrating an internalized bacterium increased after each round of Shigella co-culture, starting with 5% \u00b1 2.5% in round 10 and enriching to 95% \u00b1 1% by round 50 of co-culture. The percentage of GBM cells with an internalized bacterium was calculated by taking three randomly selected fields and dividing the total number of infected cells by the total number of U-251 cells present , phalloidin-AF594, and Hoechst 33342 then imaged via confocal microscopy. The DNA rod-like structures observed inside GBM cells throughout this study co-localized with an anti-Shigella antibody lines were incubated with GBM-infecting Shigella. Internalized Shigella was harvested and plated to count the number of internalized bacteria per group. GSC 115 showed the highest internalization at 123,800 bacteria/5 \u00d7 105 GBM cells. To demonstrate the specificity of the platform, GBM-infecting Shigella were incubated with normal astrocytes and internalized bacteria counted. Approximately 383 bacteria internalized/~300 NA cells (two separate patient cell lines) were observed. Next, GBM-infecting Shigella and compared the data to those of the parental strain. We identified 177 mutations in 46 genes . Removal of both MsbB1 and MsbB2 completely abrogated internalization to an average of <1 bacterium recovered. Confocal imaging of GBM cells treated with either R50, MsbB1 KO, or MsbB1 and MsbB2 KO demonstrate a significant decrease in cells internalizing Shigella (Shigella-positive cells dropped from 95% to 25% (p < 0.05 via ANOVA). Quantifying the fluorescent Shigella signal indicated that MsbB1 removal reduced the mean signal to 0.425 rfu compared to 6.16 rfu for R50 . Removal of both MsbB1 and MsbB2 resulted in 0% of cells with a Shigella internalized and a fluorescent signal of 0.009 rfu (~684-fold decrease). GBM cells that did demonstrate Shigella internalization after removal of myristoylation factor MsbB1 had similar numbers of internalized Shigella compared to R50. The growth curves of R50, MsbB1 KO, and MsbB1 and MsbB2 KO Shigella are presented in We removed MsbB1 and MsbB2 myristoylation factors from the round 50 GBM-infecting ineering \u201337, remoShigella Figure\u00a05S. flexneri clones that selectively internalize into GBM cell lines. We first selected a strain of Shigella that does not express Shiga toxins to improve the safety profile of the platform , doxycycline, or polymyxins, can serve as master suicide switch(es) to immediately halt GBM-infecting Shigella activity in the case of adverse events such as fever or general infection , 41. A rblastoma . Additioe genome , 27. Thun system , 20. In n system , 24, 27.nfection . It is iShigella to internalize into GBM cells as a foundation for a drug delivery system. We envision this platform serving as a tool for neurosurgeons to clean the margins of a GBM post-resection. GBM-infecting Shigella could be administered into the surgical cavity before closing to infect and eradicate cancerous cells in the invasive margin that cause disease recurrence. This approach bypasses the need for systemic administration and reduces the potential for internalization into off-target cells in the body. However, many steps are required before GBM-infecting Shigella are suitable for a drug delivery platform including stabilizing genetic material, controlling the bacterial population, ensuring intracellular replication, modulating immunogenicity, and weaponizing the system with cytotoxic proteins such as ribosome toxins (gelonin), cytokines (IL-2 and TNF), and autophagy-inducing proteins (caspases) \u201346. AddiShigella into GBM cells. Removal of MsbB1 and MsbB2 myristoylation enzymes completely abrogates the internalization of GBM-infecting Shigella in GBM cells. Previous reports indicate that removing MsbB genes does not impair the internalization of Shigella in intestinal cells, the typical target of wild-type Shigella or Salmonella Typhimurium invasion into tumor cells is essential for GBM-infecting Shigella to internalize into GBM cells. However, our data do not indicate what that factor is. Whole genome sequencing did not identify obvious mutations in MsbB genes or a known myristoylated protein; however, future RNA-sequencing, epigenetic, and/or proteomic studies of Shigella membrane-bound proteins could identify factors with differential copy number, alterations in promoters, and/or de-novo localization to bacterial membranes that are driving the observed changes in Shigella internalization in GBM cells. These studies can be narrowed bioinformatically by eliminating proteins of interest that do not contain an N-terminal glycine that is required for modification with myristic acid can be found in the article/Ethical approval was not required for the studies on humans in accordance with the local legislation and institutional requirements because only commercially available established cell lines were used. Ethical approval was not required for the studies on animals in accordance with the local legislation and institutional requirements because only commercially available established cell lines were used.AS designed assays and wrote the manuscript, GF designed assays and edited the manuscript, BD conceptualized the study and edited the manuscript, and BU conceptualized the study, designed assays, and wrote the manuscript. All authors contributed to the article and approved the submitted version."} +{"text": "Chlamydomonas reinhardtii delta tubulin (uni3) and epsilon tubulin (bld2) genes through an unknown mechanism. Our studies revealed that intact PCR products for both these genes could be obtained upon PCR amplification from plasmid templates carrying these genes. However, interestingly, purification of these PCR products led to their cleavage through an unidentified mechanism. This cleavage persisted despite using different PCR purification kits. Deleting a synthetic intron within the delta tubulin gene also did not have any effect on this cleavage.In this study, we report an unusual phenomenon of the self-cleavage of purified PCR products of codon-optimized Centrosomes consist of orthogonally-paired cylindrical centrioles enveloped by the protein-dense pericentriolar material .Chlamydomonas reinhardtii delta and epsilon tubulin proteins that are coded by theuni3 andbld2 genes respectively, play a crucial role in maintaining the proper triplet microtubule architecture of centrioles . To obtain further insights into the functions of these genes in regulating centriole architecture, we wanted to generate purified PCR products ofChlamydomonas reinhardtiidelta and epsilon tubulin cDNAs carrying synthetic introns that were codon-optimized for expression inC. elegans. These purified PCR products would enable us to perform downstream functional assays to further characterize the functions of these genes. Although we were successful in obtaining full length PCR products of codon-optimizedChlamydomonas reinhardtiidelta and epsilon tubulin genes, these PCR products exhibited an unexpected cleavage upon their purification using either the Invitrogen Purelink\u2122 Quick PCR purification kit or the Qiaquick\u00ae PCR purification kit.As shown in, both the delta and epsilon tubulin PCR products but not a non-relevant control PCR product (dyn-1) exhibited cleavage after their purification using the Invitrogen Purelink\u2122 Quick PCR purification kit or the Qiaquick\u00ae PCR purification kit. Purification of the delta and epsilon tubulin PCR products using a non-kit-based isopropanol precipitation method as well as upon their gel extraction using a Qiaquick Gel Extraction kit , both yielded this similar pattern of cleavage. To confirm that the cleaved fragments indeed correspond to the PCR products of the delta and epsilon tubulin genes, DNA sequencing of these lower sized fragments was performed after excising these separated bands from the agarose gel. Our sequencing results confirmed that the lower sized fragments that were observed between 650 bp and 1000 bp upon agarose gel electrophoresis of the purified delta and epsilon tubulin PCR productscontained DNA sequences from these respective genes. Our sequencing analysis identified a synthetic intron close to the genomic region where the delta and epsilon tubulin PCR products were being cleaved. Since the sequence of this synthetic intron was identical between the delta and epsilon tubulin genes, we questioned whether the presence of this synthetic intron was responsible for the cleavage of the purified PCR products of these genes. To address this, site-directed mutagenesis was performed to remove this synthetic intron from the sequence of the delta tubulin gene and the PCR purification procedure was repeated. As shown indeletion of the synthetic intron close to the cleavage region within the delta tubulin gene did not eliminate cleavage of the purified PCR product. These data indicate that this synthetic intron does not contribute to this observed cleavage phenomenon. In the future, studies should be directed to further characterize the molecular mechanisms mediating the cleavage of theChlamydomonas reinhardtii delta and epsilon tubulin PCR products upon purification. Since we have invested a considerable amount of time and resources on this project, we would like to share these findings with the scientific community and with other researchers working on these genes to prevent a further loss of their valuable time and resources.Centrosomes are the major microtubule organizing centers of most animal cells. They contribute to a variety of cellular functions including spindle assembly, cell polarity, cell shape and cilia assembly (Reviewed in NiggI) PCR conditions, analysis, and purificationPCR was performed using a C1000\u2122 Thermal Cycler . 35 cycles of amplification were used for 50 \u00b5L PCR reaction volume consisting of:uni3,bld2,dyn-1), 1 \u00b5L - 10 mM dNTPs, 2 \u00b5L- 50 mM MgSO4, 5 \u00b5L - 10X Hi-Fi Buffer, 0.2 \u00b5L - Platinum\u00aeTaqDNA Polymerase Hi -Fi enzyme, and molecular biology water was used to bring the final volume to 50 \u00b5L.1 \u00b5L- 10 \u00b5M forward primer, 1 \u00b5L- 10 \u00b5M reverse primer, 35 ng- plasmid template was obtained from a synthesized plasmid construct that contains the coding sequence for blue fluorescent protein with homology arms to theC. elegans dyn-1 gene.The control PCR product (dyn-1) PCR:PCR conditions for the control Site-directed mutagenesisuni3 gene except that the Phusion\u00ae High-Fidelity DNA Polymerase was used for PCR amplification.For performing the site-directed mutagenesis, the PCR reaction mixture comprised of all the same components used in PCR amplification of thePCR conditions for delta tubulin intron deletion using site-directed mutagenesis:Initial denaturation: 98\u00b0C - 30 sDenaturation: 98\u00b0C - 10 sAnnealing: 68.6\u00b0C - 30 sExtension: 72\u00b0C - 6 minFinal Extension: 72\u00b0C - 10 minSite-directed mutagenesis was followed by digestion with 5 units of Dpn1 for 2 hours, followed by bacterial transformation and plasmid purification using the QIAprep\u00ae Spin Miniprep kit. The isolated plasmid samples were sent for whole plasmid sequencing . Upon analyzing the sequencing results, the plasmid with successful intron deletion was selected and used for further analysis.Primers and sequences used for studyI) Primers for PCR amplification of Epsilon tubulinForward primer: 5\u2019- ATT CGA ATA TAT ATT GTC AGT TG -3\u2019Reverse primer: 5\u2019- ATA CGA GGA TTA TGG TAC AAG -3\u2019II) Primers for PCR amplification of Delta tubulinForward primer: 5\u2019- TTG TTT CTT TCT TTT AAT GTT AAA TAT TTC CAG AAC TAT GCC ATG -3\u2019Reverse primer: 5\u2019- GAT AAA ATA ATT ATT CGG GCA GTA ATA AAA CAG GGA TCT ATC ACT TC -3\u2019III) Primers for site-directed mutagenesis for Delta tubulin with one intron deletedForward primer: 5\u2019- TCG AGG AGG CCG GAC TCA AGG GAC AAT CCT CCG GAC CAG G -3\u2019Reverse primer: 5\u2019- CCT GGT CCG GAG GAT TGT CCC TTG AGT CCG GCC TCC TCG A -3\u2019(dyn-1):IV) Primers for amplification of controlForward primer: 5\u2019- AAA ATC GAT TTT CAG GTA GTT CAG C -3\u2019Reverse primer: 5\u2019- TTG ATC ACA GGG ATC AAC GCC -3\u2019Chlamydomonas reinhardtii delta tubulin (uni3) cDNA codon optimized for expression inC. elegans (contains synthetic introns (lowercase + underlined) and homology arms for CRISPR genome editing (only lowercase)):V) Sequence ofgtaagtttaaacatatatatactaactaaccctgattatttaaattttcagGTCGTCGCCGGAGCCCGTTCCGCCGCCGCCGCCTCCGGATCCTGGTGGCGTTACCCATCCTCCGGATACCTCGTCATGCAATCCGGATCCGGAAACAACTGGGCCCAAGGATTCCACGGATACGGACCACAAGTCCACGAGGACGCCCTCGACCTCGTCCGTAAGgtaagtttaaacagttcggtactaactaaccatacatatttaaattttcagGAGGTCGAGCACGCCGACTCCCTCACCGGATTCCTCCTCCTCCAATCCATGGCCGGAGGAACCGGAGCCGGACTCGGAACCTACGTCGCCGAGGCCCTCCGTGACGAGTACCACTCCGCCTTCGTCGCCAACTGCTGCGTCTGGCCATACGAGTCCGGAGAGGTCATCGTCCAACCATACAACACCCTCCTCACCCTCTCCCACCTCGCCGACGTCTCCGACGGACTCGTCCTCCTCGAGAACGAGGCCCTCCACCGTACCGCCGCCAAGCTCTACGGAATCGCCCGTCCATCCTTCGGAGTCCGTGGACGTGTCCTCGGACGTGCCGGAGAGTCCCGTGTCGAGGAGGCCGGACTCAAGgtaagtttaaacatgattttactaactaactaatctgatttaaattttcagGGACAATCCTCCGGACCAGGAGGATGGGGAGTCTGCACCGCCCCACTCGCCGAGCTCGTCACCCGTCTCTGCGGACACCCAGCCTACCGTCTCCTCACCCTCCGTTCCGTCCCACAACTCCCACCAGCCAACATCGACTTCACCACCTTCACCTGGCCAGCCCTCACCAAGCGTCTCCGTCAAATGCTCGTCACCGGATCCGTCCTCGAGGAGGGACTCGACTGGTCCATCACCCCACAATCCCCAGGAGCCGCCGCCGCCCTCGGAGCCGGACTCGCCGGACCAACCGTCAACCGTGCCCTCGCCTCCTGGCTCATCCTCCGTGGACAAGGAGCCGCCGAGGCCGACGTCGGAGAGTTCGCCGACCCAGCCCTCTCCGCCGCCTGGTCCCCAGAGCCACTCTCCGTCTCCTACTCCACCGGACGTTTCGGACGTTGCGCCATGTCCGCCTGCCTCCTCTCCAACGACCGTCACTGCGTCGGACCAATCCAACGTATGCAAGAGCACGCCTACGGAATGCTCGAGTCCCGTGCCTTCGTCCACCAATACGAGAAGTACGGACTCTCCGTCGCCGAGTTCCAAGACTGCTTCGCCCGTATCGAGGACATCGCCCAACGTTACGCCCGTCTCGACTACAAAGACCATGACGGTGATTATAAAGATCATGATATCGATTACAAGGATGACGATGACAAGATGCCTAAAGATCCAGCCAAACCTCCGGCCAAGGCACAAGTTGTGGGATGGCCACCGGTGAGATCATACCGGAAGAACGTGATGGTTTCCTGCCAAAAATCAAGCGGTGGCCCGGAGGCGGCGGCGTTCGTGAAGtgatagatccctgttttattactgcccgaataattattttatc-3\u20195\u2019-ttgtttctttcttttaatgttaaatatttccagaactATGCCATGCATCACCCTCCAACTCGGACAATGCGGAAACCAACTCGGATGCTCCCTCTTCAACACCCTCGCCACCGAGTTCTCCTCCCACGACTACGGAACCGACGCCGTCCACGAGTACTTCCGTCCATCCGCCGACCCAAACCTCTACACCGCCCGTTCCGTCCTCATCGACATGGAGCCAAAGChlamydomonas reinhardtii epsilon tubulin (bld2) cDNA codon optimized for expression inC. elegans (contains introns (lowercase + underlined) and homology arms for CRISPR genome editing (only lower case)):VI) Sequence ofgtaagtttaaacatatatatactaactaaccctgattatttaaattttcagATCCGTCTCACCGCCGAGGACTGCGACTCCCTCCAATCCTTCATGGTCCTCCACTCCCTCGGAGGAGGAACCGGATCCGGAGTCGGAACCTACATCGTCCGTATGCTCGCCGACGAGTTCCCAGGAGTCTTCCGTTTCACCGGATCCGTCTTCCCATCCGAGGACGACGACGTCGTCACCTCCCCATACAACGCCATGCTCGCCCTCGGACAACTCGTCGAGCACGCCGACTGCGTCCTCCCAATCGAGAACCAAGCCCTCATCGACATCGTCAACCGTACCGAGGCCGCCCGTGACCGTGCCGCCGCCGCCGACGCCGCCGCCTCCGCCGTCTCCGGACTCAAGgtaagtttaaacagttcggtactaactaaccatacatatttaaattttcagGGATCCGGAGGAGGATCCAAGCCATTCGACTCCATGAACGGAGTCGCCGCCTCCCTCCTCCTCCACCTCACCGCCTCCGTCCGTTTCGAGGGACCACTCAACGTCGACCTCAACGACATCACCATGAACCTCGTCCCATACCCACGTATGCACTTCCTCCTCTCCTCCATGTCCCCACTCCAACCACCACCAAAGGACAAGGACCCACGTACCCTCGACCAAGTCCGTGTCTTCGGAGACGTCTTCTCCCGTGAGCACCAACTCATCCGTGCCGACCCACGTGCCGCCACCTACCTCGCCTGCGGACTCATCGCCCGTGGACCAACCGCCACCATGGCCGACATCAACCGTAACGTCGCCCGTCTCCGTCCACAACTCAAGgtaagtttaaacatgattttactaactaactaatctgatttaaattttcagATGGTCCACTGGAACTCCGAGGGATTCAAGCTCGGAATCTGCTCCACCCCACCAGTCGGATGCCCATTCGGACTCCTCTGCCTCGCCAACAACACCGCCATCGCCCACACCTTCACCACCATGCGTGAGCGTTTCGACAAGCTCTACAAGCGTCGTTTCTACACCCACCACTACGAGCAATACATGGACCCAGGAGGATTCACCTCCGCCATGGAGGTCGTCGGAGACCTCACCGCCCAATACCGTGCCCTCGAGGGAGCCACCCAAGCCCCACCACTCACCCGTCTCCGTCCACGTGGACTCTCCTTCCTCCCAGGAGGTTCCGGTGGTTCTGGTGGATCCGGTAAGCCTATCCCAAATCCTTTGTTGGGTCTGGACTCCACGATGCCTAAAGATCCAGCCAAACCTCCGGCCAAGGCACAAGTTGTGGGATGGCCACCGGTGAGATCATACCGGAAGAACGTGATGGTTTCCTGCCAAAAATCAAGCGGTGGCCCGGAGGCGGCGGCGTTCGTGAAGtgataatattcatttaatccaacttgtaccataatcctcgtat-3\u20195\u2019- attcgaatatatattgtcagttgttctgtttgtcgtcgtgATGCCACGTGAGCTCGTCACCATCCAAGTCGGACAATGCGGAAACCAAGTCGGATGCCGTTTCTGGGAGCTCGCCCTCCGTGAGCACGCCGCCTACAACACCAAGGGAGTCTACGACGAGGCCCTCTCCTCCTTCTTCCGTAACGTCGACACCCGTGTCGAGCCACCACGTAACCTCCCAGTCGGAGAGGGACGTGGAGCCATCCGTACCCTCAAGGCCCGTTCCGTCATCGTCGACATGGAGTGCGGAGTCATCAACGAGATGCTCAAGGGACCACTCGGAGAGGTCCTCGACACCCGTCAACTCGTCTCCGACGTCTCCGGAGCCGGAAACAACTGGGCCCACGGACACCACGAGTACGGACCACGTTACCACGACGCCATCCTCGACAAG(dyn-1):VI) Sequence of the positive control used in this study5\u2019- AAAATCGATTTTCAGGTAGTTCAGCGTATAACCACCAGGATCAGCGATGGTTTCGGAATTGATTAAAGAAAATATGCACATGAAGCTCTACATGGAGGGAACCGTCGACAACCACCACTTTAAATGTACCTCCGAGGGAGAGGGAAAGCCATACGAGGGAACCCAAACCATGCGTATCAAGGTCGTCGAGGGTGGTCCGCTCCCATTCGCCTTTGATATCCTCGCCACCTCCTTCCTCTATGGTTCCAAGGTAAGTTTAAACATATATATACTAACTAACCCTGATTATTTAAATTTTCAGACCTTCATCAACCACACCCAAGGAATCCCAGACTTTTTTAAACAATCCTTCCCAGAGGGATTCACCTGGGAGCGTGTCACCACCTACGAGGACGGAGGAGTCCTCACCGCCACCCAAGACACCTCCCTCCAAGACGGATGCCTCATCTACAACGTCAAGGTAAGTTTAAACAGTTCGGTACTAACTAACCATACATATTTAAATTTTCAGATCCGTGGAGTCAACTTCACCTCCAACGGACCAGTCATGCAAAAGAAGACCCTCGGATGGGAGGCCTTCACCGAGACCCTCTACCCAGCCGACGGAGGACTCGAGGGACGTAACGACATGGCCCTCAAGCTCGTCGGAGGATCCCACCTCATCGCCAACGCCAAGGTAAGTTTAAACATGATTTTACTAACTAACTAATCTGATTTAAATTTTCAGACCACCTACCGTTCCAAGAAGCCAGCCAAGAACCTCAAGATGCCAGGAGTCTACTACGTCGACTACCGTCTCGAGCGTATCAAGGAGGCCAACAACGAGACCTACGTCGAGCAACACGAGGTCGCCGTCGCCCGTTACTGCGACCTCCCATCCAAGCTCGGACACAAGCTCAACTACCCATATGATGTTCCAGATTACGCTGGAGGATCTGGAGGCGGTTCTGGCGGAGGTTCTGGTATGTCGTGGCAAAACCAGGGAATGCAGGCGTTGATCCCTGTGATCAA \u2013 3\u2019"} +{"text": "Blumeria graminis forma specialis tritici (B.g. tritici) is the airborne fungal pathogen that causes powdery mildew disease on hexaploid bread wheat. Calmodulin-binding transcription activators (CAMTAs) regulate plant responses to environments, but their potential functions in the regulation of wheat\u2013B.g. tritici interaction remain unknown. In this study, the wheat CAMTA transcription factors TaCAMTA2 and TaCAMTA3 were identified as suppressors of wheat post-penetration resistance against powdery mildew. Transient overexpression of TaCAMTA2 and TaCAMTA3 enhanced the post-penetration susceptibility of wheat to B.g. tritici, while knockdown of TaCAMTA2 and TaCAMTA3 expression using transient- or virus-induced gene silencing compromised wheat post-penetration susceptibility to B.g. tritici. In addition, TaSARD1 and TaEDS1 were characterized as positive regulators of wheat post-penetration resistance against powdery mildew. Overexpressing TaSARD1 and TaEDS1 confers wheat post-penetration resistance against B.g. tritici, while silencing TaSARD1 and TaEDS1 enhances wheat post-penetration susceptibility to B.g. tritici. Importantly, we showed that expressions of TaSARD1 and TaEDS1 were potentiated by silencing of TaCAMTA2 and TaCAMTA3. Collectively, these results implicated that the Susceptibility genes TaCAMTA2 and TaCAMTA3 contribute to the wheat\u2013B.g. tritici compatibility might via negative regulation of TaSARD1 and TaEDS1 expression. Triticum aestivum L.) has served as a major staple food for thousands of years and provided about 20% of the calories consumed by humans [Blumeria graminis forma specialis tritici (B.g. tritici), leading to 5\u201350% yield losses [B.g. tritici-resistant wheat cultivars [B.g. tritici and identify key regulators of wheat resistance against powdery mildew disease.As one of the most widely grown small-grain cereal crops, bread wheat transcription factors, calmodulin-binding transcription activators (CAMTAs) play important roles in regulating plant growth, development, and responses to environmental stresses [CAMTA genes differentially respond to environmental cues like drought, salinity, and extreme temperatures in the model plant Arabidopsis thaliana [Arabidopsis mutant camta1 exhibited hypersensitivity to cold and drought stress, and AtCAMTA1 was shown to regulate the expression of cold and drought-responsive genes like AtRD26, AtERD7, AtCBF2, and AtRAB18 [Arabidopsis mutant camta6 exhibited hypersensitivity to NaCl treatment, and AtCAMTA6 was demonstrated to regulate expression of salt resilience-related genes, including + TRANSPORTER1HIGH-AFFINITY K, SALT OVERLY SENSITIVE1, and +/H+ ANTIPORTERNa [Arabidopsis AtCAMTA3 was shown to function in concert with AtCAMTA1 and AtCAMTA2 in suppressing plant defense responses [B.g. tritici remains largely unknown.As Castresses ,20,21. Fthaliana ,24,25,26 AtRAB18 ,24,25,26TIPORTER . In addiesponses ,30,31,32TaCAMTA2 and TaCAMTA3, were characterized as Susceptibility (S) genes contributing to wheat\u2013B.g. tritici compatibility. Transient overexpression of TaCAMTA2 and TaCAMTA3 resulted in enhanced wheat post-penetration susceptibility to B.g. tritici, while transient silencing of TaCAMTA2 and TaCAMTA3 led to attenuated wheat post-penetration susceptibility to B.g. tritici. Furthermore, overexpressing TaSARD1 and TaEDS1 could confer wheat post-penetration resistance against powdery mildew, while silencing TaSARD1 and TaEDS1 enhanced wheat post-penetration susceptibility to B.g. tritici. Moreover, TaCAMTA2 and TaCAMTA3 were demonstrated to negatively regulate the expression of the defense genes TaSARD1 and TaEDS1. These results strongly support that S genes TaCAMTA2 and TaCAMTA3 partially redundantly suppress wheat post-penetration resistance against B.g. tritici presumably via the negative regulation of expressions of defense genes TaSARD1 and TaEDS1.In this research, two CAMTA transcription factor genes, Arabidopsis CAMTA transcription factor AtCAMTA3 plays a vital role in the regulation of plant immunity [B.g. tritici interaction. To this end, we first searched the reference genome of the hexaploid bread wheat by using the amino acid sequence of Arabidopsis AtCAMTA3 (At2g22300) as a query and obtained TaCAMAT2 and TaCAMTA3, the most closely related homologs of AtCAMTA3, in bread wheat. Three highly homologous sequences of TaCAMAT2 genes separately located on chromosomes 4A, 4B, and 4D were obtained from the genome sequence of the hexaploid wheat and designated as TaCAMTA2-4A (TraesCS4A02G407100), TaCAMTA2-4B (TraesCS4B02G306300), and TaCAMTA2-4D (TraesCS4D02G304500). Similarly, three highly homologous sequences of TaCAMAT3 genes separately located on chromosomes 2A, 2B, and 2D were obtained from the genome sequence of the hexaploid wheat and designated as TaCAMTA3-2A (TraesCS2A02G163000), TaCAMTA3-2B (TraesCS2B02G188800), and TaCAMTA3-2D (TraesCS2D02G169900).Previous studies revealed that the immunity ,30,31,32Arabidopsis AtCAMTA3. In addition, TaCAMTA2-4A, TaCAMTA2-4B, TaCAMTA2-4D, TaCAMTA3-2A, TaCAMTA3-2B, and TaCAMTA3-2D proteins all contain a conserved CG-1 DNA-binding domain at their N-terminal parts, a transcription factor immunoglobulin-like (TIG) DNA-binding domain, several ankyrin repeats (ANK) in the middle parts, as well as two IQ CaMB motifs (IQXXXRGXXXR) at their C-termini increased from 56% for the empty vector (OE-EV) control to above 70% on wheat cells overexpressing TaCAMTA2 or TaCAMTA3 genes. These results suggested that overexpression of TaCAMAT2 and TaCAMTA3 could significantly enhance wheat post-penetration susceptibility to B.g. tritici.To study the function of TaCAMAT2 and TaCAMTA3 in the regulation of wheat\u2013B.g. tritici interaction, we employed transiently induced gene silencing (TIGS) assays to silence all endogenous TaCAMAT2 or TaCAMTA3 genes in the epidermal cell of the B.g. tritici-susceptible wheat cultivar Yannong 999. After inoculation of conidia from the virulent B.g. tritici isolate E09, the frequency of fungal haustorium formation in the transformed plant cells was scored. As shown in TaCAMAT2 or TaCAMTA3 genes resulted in a marked HI% decrease to about 27%, compared to 33% for empty vector (EV) controls. Significantly, simultaneous silencing of TaCAMAT2 and TaCAMTA3 could lead to a further decrease in HI% to approximately 13%, suggesting that TaCAMTA2 and TaCAMTA3 might partially redundantly suppress post-penetration resistance of wheat to B.g. tritici.To further verify the function of TaCAMAT2 or TaCAMTA3 genes in the leaves of the B.g. tritici-susceptible wheat cultivar Yannong 999. qRT-PCR showed that the endogenous transcript level of TaCAMAT2 or TaCAMTA3 was substantially reduced in the indicated VIGS plants declined to approximate 40% on BSMV-TaCAMTA2as plants and 47% on BSMV-TaCAMTA3as plants, compared with 55% for the BSMV-\u03b3 plants -induced gene silencing (BSMV-VIGS) to silence all endogenous S plants C. Therea\u03b3 plants D. NotablSYSTEMIC ACQUIRED RESISTANCE DEFICIENT 1 (AtSARD1) and ENHANCED DISEASE SUSCEPTIBILITY 1 (AtEDS1) in A. thaliana [Arabidopsis AtSARD1 (At1g73805) and AtEDS1 (At3g48090) as a query and obtained TaSARD1 and TaEDS1, the most closely related homologs of AtSARD1 and AtEDS1, in bread wheat. Five highly homologous sequences of TaSARD1 genes separately located on chromosomes 6A, 6B, and 6D were obtained from the genome sequence of the hexaploid wheat and designated as TaSARD1.1-6A (TraesCS6A02G091700), TaSARD1.1-6B (TraesCS6B02G119900), TaSARD1.1-6D (TraesCS6D02G080500), TaSARD1.2-6A (TraesCS6A02G296600), and TaSARD1.2-6D (TraesCS6D02G276800). Similarly, three highly homologous sequences of TaEDS1 genes separately located on chromosomes 5A, 5B, and 5D were obtained from the genome sequence of the hexaploid wheat and designated as TaEDS1-5A, TaEDS1-5B, and TaEDS1-5D [Previous studies revealed that AtCAMTA3 could regulate the expression of defense genes thaliana ,30,31,32aEDS1-5D .Arabidopsis AtSARD1. In addition, TaSARD1.1-6A, TaSARD1.1-6B, TaSARD1.1-6D, TaSARD1.2-6A, and TaSARD1.2-6D proteins all contain a CBP60-conserved domain resistance genes and quantitative trait loci (QTL) contributed to wheat resistance to B.g. tritici and have been employed in wheat breeding for powdery mildew resistance [B.g. tritici underlies wheat\u2019s susceptibility to powdery mildew. A plethora of wheat S genes have been identified to facilitate compatibility by inducing B.g. tritici (pre)penetration, suppressing wheat immunity, and supporting the sustenance of B.g. tritici [S genes TaWIN1, TaKCS6, and TaECR were revealed to facilitate the conidial germination of B.g. tritici by promoting the biosynthesis of wheat cuticular wax, whereas wheat S gene TaSTP13 encodes a sugar transporter facilitating wheat hexose accumulation for B.g. tritici acquisition [TaMLO, TaEDR1, and TaPOD70 genes contribute to wheat susceptibility to powdery mildew by suppressing plant defense responses [B.g. tritici by suppressing defense-related transcriptional reprogramming in bread wheat [Powdery mildew, caused by the adapted fungal pathogen oduction ,4. To imeraction ,4. Powdesistance ,4. Compa tritici ,35. For uisition ,39,40,41esponses ,45,46,47ad wheat ,51,52,53TaCAMAT2 and TaCAMTA3 were identified as the most closely related homologs of AtCAMTA3, which is consistent with the reported phylogenetic analysis of the CAMTA homologs in different species [TaCAMAT2 and TaCAMTA3 are characterized as wheat S genes contributing to the wheat post-penetration susceptibility to B.g. tritici in this study. Overexpression of TaCAMTA2 and TaCAMTA3 in the leaf epidermal cell by transient gene expression assays led to enhanced wheat susceptibility to B.g. tritici, while knockdown of TaCAMTA2 and TaCAMTA3 expression using transient- or virus-induced gene silencing resulted in compromised wheat post-penetration susceptibility to B.g. tritici. Interestingly, a gain-of-function mutation in SIGNAL RESPONSIVE1 (SR1), which encodes the Arabidopsis homologs of wheat TaCAMTA2 and TaCAMTA3, could suppress the edr2-associated powdery mildew resistance [sr1-4D single mutant is more susceptible to Arabidopsis powdery mildew (Golovinomyces cichoracearum), whereas the sr1-1 null mutant plants displayed enhanced post-penetration resistance against G. cichoracearum [Arabidopsis AtCAMTA1 was revealed to function partially redundantly with AtCAMTA2 and AtCAMTA3 in suppressing plant immunity [TaCAMAT2 and TaCAMTA3 could lead to a further decrease in the HI% and MI% compared with single silencing of TaCAMAT2 or TaCAMTA3, supporting the fact that TaCAMTA3 functions partially redundantly with TaCAMAT2 in suppressing wheat post-penetration resistance against B.g. tritici. In Arabidopsis, CAMTA transcription factors AtCAMTA1, AtCAMTA2, and AtCAMTA3 partially redundantly suppress the biosynthesis of salicylic acid (SA) and N-hydroxypipecolic acid (NHP), a metabolite duo essential for systemic acquired resistance (SAR) [S genes TaCAMAT2 and TaCAMTA3 in the regulation of SA and NHP biosynthesis, as well as SAR establishment, in bread wheat in future research.Through homology-based searching, species . TaCAMATsistance . The sr1racearum . In addiimmunity ,31,32. Ice (SAR) ,31,32. TTaSARD1 and TaEDS1 are identified as positive regulators of wheat resistance against B.g. tritici in this study. Overexpression of TaSARD1 or TaEDS1 in the leaf epidermal cell by transient gene expression assays led to enhanced wheat post-penetration resistance to B.g. tritici, while knockdown of TaSARD1 or TaEDS1 expression using transient- or virus-induced gene silencing resulted in increased wheat post-penetration susceptibility to B.g. tritici. In Arabidopsis, transcription factor AtSARD1 functions in concert with AtCBP60g to activate the expression of SID2 (SA INDUCTION DEFICIENT 2), which encodes isochorismate synthase 1 (ICS1), essential for pathogen-induced SA biosynthesis [Arabidopsis AtEDS1 was shown to heterodimerize with its partners, phytoalexin deficient 4 (PAD4) or senescence-associated gene 101 (SAG101), to play signaling roles in ETI as well as SA-dependent and SA-independent PTI pathways [TaPR1, TaPR2, and TaPR5 induced by B.g. tritici infection were attenuated by silencing of TaSARD1 or TaEDS1, suggesting that the SARD1-EDS1-SA defense axis might be partially conserved between model plant Arabidopsis and crop plant bread wheat. Therefore, it is intriguing to examine the potential regulation of wheat SA biosynthesis and signaling by TaSARD1 and TaEDS1 in future research.ynthesis ,55,56. Apathways ,62,63,64TaSARD1 and TaEDS1 were significantly enhanced by silencing TaCAMTA2 and TaCAMTA3. Notably, simultaneous silencing TaCAMAT2 and TaCAMTA3 could lead to a further increase in the expression levels of TaSARD1 and TaEDS1 compared with single silencing TaCAMAT2 or TaCAMTA3, indicating that TaCAMTA2 and TaCAMTA3 partially redundantly suppress expressions of TaSARD1 and TaEDS1. In Arabidopsis, AtCAMTA3 could bind to the promoter region of AtEDS1 by recognizing the CGCG box, thereby directly repressing the expression of AtEDS1 [AtSARD1 was demonstrated to be negatively regulated by partially redundant AtCAMTA1, AtCAMTA2, and AtCAMTA3, presumably via an indirect effect [SARD1 and EDS1 by partially redundant CAMTA3 and its homologs might be partly conserved between the model plant Arabidopsis and the important crop bread wheat. Indeed, the expressions of SA defense marker genes TaPR1, TaPR2, and TaPR5 induced by B.g. tritici infection were found to be potentiated by silencing TaCAMAT2 or TaCAMTA3 in this study. However, binding sites for TaCAMAT2 and TaCAMTA3 in the promoter regions of TaSARD1 and TaEDS1 genes remain to be identified.In this study, expression levels of f AtEDS1 ,29,30,31t effect ,29,30,31TaCAMAT2 and TaCAMTA3 are identified as wheat S genes partially redundantly suppressing post-penetration resistance against powdery mildew, presumably via negative regulation of the expressions of defense genes TaSARD1 and TaEDS1. Genetic manipulation of S genes TaMLO and TaEDR1 via targeting induced local lesions in genomes (TILLING) and genome editing techniques like transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated) 9 systems compromised wheat compatibility with B.g. tritici and conferred wheat resistance against powdery mildew [TaCAMAT2 and TaCAMTA3 in wheat breeding for powdery mildew resistance in future research.Herein, y mildew ,71,72,73B.g. tritici strain E09 was maintained on the leaves of Jing411 plants. Conidia of B.g. tritici strain E09 were used for the inoculation of Jing411 leaves in the study of wheat\u2013powdery mildew interaction. Arabidopsis thaliana used in this study was grown in the greenhouse under a 16 h/8 h light period at 23 \u00b1 1 \u00b0C with 70% relative humidity.The seedlings of bread wheat cultivar Yannong999 used in this study were grown in a growth chamber under a 16-h/8-h, 20 \u00b0C/18 \u00b0C day/night cycle with 70% relative humidity. The GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE (TaGAPDH) was set as the internal control and expressions of TaGAPDH, TaCAMTA2, TaCAMTA3, TaSARD1, TaEDS1, TaPR1, TaPR2 and TaPR5 were analyzed using the primers 5\u2032-TTAGACTTGCGAAGCCAGCA-3\u2032/5\u2032-AAATGCCCTTGAGGTTTCCC-3\u2032, 5\u2032-TACAGAAGTTGCAACAG-3\u2032/5\u2032-ATCTCCGTCGACTCCTCA-3\u2032, 5\u2032-CCTGACAAACAACTTGA-3\u2032/5\u2032-CGCCAGCTGCA TCGCTT-3\u2032, 5\u2032-GCGAGTAATGAAAGCAT-3\u2032/5\u2032-TTAATCAACTTGATCCC-3\u2032, 5\u2032-TGAAAGACAGGGTGGGT-3\u2032/5\u2032-CGAAGGCACAAGTCTCG-3\u2032, 5\u2032-GAGAATGCAGACGCCCAAGC-3\u2032/5\u2032-CTGGAGCTTGCAGTCGTTGATC-3\u2032, 5\u2032-AGGATGTTGCTTCCATGTTTGCCG-3\u2032/5\u2032-AAGTAGATGCGCATGCCGTTGATG-3\u2032, and 5\u2032-CTTCTACATCAAGA ACAACTG-3\u2032/5\u2032-CAGTCGCCGGTCTGGCAG-3\u2032.Total RNA was extracted from the wheat leaves using the EasyPure Plant RNA kit and 2 \u03bcg of RNA was used to synthesize the cDNA template using the TransScript one-step gDNA removal and cDNA synthesis supermix according to the manufacturer\u2019s instructions. The real-time PCR assay was performed using the ABI real-time PCR system with the qPCR Master Mix . The expression of traditional housekeeping gene TaCAMTA2, TaCAMTA3, TaSARD1, and TaEDS1 was cloned into the pCa-\u03b3bLIC vector to create the BSMV-TaCAMTA2as, BSMV-TaCAMTA3as, BSMV-TaSARD1as, and BSMV-TaEDS1as constructs using the primer pair 5\u2032-AAGGAAGTTTATACCATCATTAGCACTTGG-3\u2032/5\u2032-AACCACCACCACCGTCACTTTTGGAATTACATTC-3\u2032, 5\u2032-AAGGAAGTTTACATTATGCACCTGCGAGGA-3\u2032/5\u2032-AACCACCACCACCGTTCAGTGCACTTTGGTGAGC-3\u2032, 5\u2032-AAGGAAGTTTATGGTTCTAGTATCTATAAG-3\u2032/5\u2032-AACCACCACCACCGTGTTTGGAACCAGTTATTCG-3\u2032, and 5\u2032-AAGGAAGTTTAAGCGAATTCCCAACAGGTG-3\u2032/5\u2032-AACCACCACCACCGTAGACGGGGAAGTGTCAATC-3\u2032. The BSMV-mediated gene silencing in wheat leaves was performed as described by Zhi et al. (2020) [B.g. tritici strain E09 conidia. About 72 h post-B.g. tritici inoculation, leaf segments were fixed with ethanol: acetic acid solution and kept in the destaining solution . Before mounting for microscopy, B.g. tritici-infected leaves were stained with 0.1% (w/v) Coomassie Brilliant Blue R250 to visualize the fungal epiphytic structure, as reported previously [The antisense fragment of . (2020) . About 1eviously .TaCAMTA2, TaCAMTA3, TaSARD1, and TaEDS1 were, respectively, amplified using the primers 5\u2032-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCTACCATCATTAGCACTTGG-3\u2032/5\u2032-GGGGACCACTTTGTACAAGAAAGCTGGGTCCACTTTTGGAATTACATTC-3\u2032, 5\u2032-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCCATTATGCACCTGCGAGGA-3\u2032/5\u2032-GGGGACCACTTTGTACAAGAAAGCTGGGTCTCAGTGCACTTTGGTGAGC-3\u2032, 5\u2032-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCTGGTTCTAGTATCTATAAG-3\u2032/5\u2032-GGGGACCACTTTGTACAAGAAAGCTGGGTCGTTTGGAACCAGTTATTCG-3\u2032, and 5\u2032-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCAGCGAATTCCCAACAGGTG-3\u2032/5\u2032-GGGGACCACTTTGTACAAGAAAGCTGGGTCAGACGGGGAAGTGTCAATC-3\u2032, and cloned into the pIPKb007 vector using a Gateway cloning system to create the TIGS-TaCAMTA2, TIGS-TaCAMTA3, TIGS-TaSARD1, and TIGS-TaEDS1 constructs. The coding regions of TaCAMTA2-4A, TaCAMTA2-4B, TaCAMTA2-4D, TaCAMTA3-2A, TaCAMTA3-2B, TaCAMTA3-2D, TaSARD1.1-6A, TaSARD1.1-6B, TaSARD1.1-6D, TaSARD1.2-6A, TaSARD1.2-6B, TaSARD1.2-6D, TaEDS1-5A, TaEDS1-5B, and TaEDS1-5D were, respectively, amplified using the primers 5\u2032-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGCCGAGGGCCGGCGCTAC-3\u2032/5\u2032-GGGGACCACTTTGTACAAGAAAGCTGGGTCCTAGAAATAGCCCGGCAACG-3\u2032, 5\u2032-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGCCGAGGGCCGGCGCTAC-3\u2032/5\u2032-GGGGACCACTTTGTACAAGAAAGCT GGGTCCTAGAAATAGCCAGGCAACG-3\u2032, 5\u2032-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGCCGAGGGCCGGCGCTAC-3\u2032/5\u2032-GGGGACCACTTTGTACAAGAAAGCTGGGTCCTAGAAATAGCCCGGCAACG-3\u2032, 5\u2032-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGCGGAGATGCACAAGTAC-3\u2032/5\u2032-GGGGACCACTTTGTACAAGAAAGCTGGGTCTCACAAAATATTGGACATCG-3\u2032, 5\u2032-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGCGGAGATGCACAAGTAC-3\u2032/5\u2032-GGGGACCACTTTGTACAAGAAAGCTGGGTCTCACAAAACAGTGGACATCG-3\u2032, 5\u2032-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGCGGAGATGCACAAGTAC-3\u2032/5\u2032-GGGGACCACTTTGTACAAGAAAGCTGGGTCTCACAAAATAGTGGACATCG-3\u2032, 5\u2032-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGTCTGTGCGAAGGCCGCG-3\u2032/5\u2032-GGGGACCACTTTGTACAAGAAAGCTGGGTCTTAATCAACTTGATCCCAAC-3\u2032, 5\u2032-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGTCTGTGCGAAGGCCGCG-3\u2032/5\u2032-GGGGACCACTTTGTACAAGAAAGCTGGGTCTTAATCAACTTGATCCCAAC-3\u2032, 5\u2032-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGTCTGTGCGAAGGCCGCG-3\u2032/5\u2032-GGGGACCACTTTGTACAAGAAAGCTGGGTCTTAATCAACTTGATCCCAAC-3\u2032, 5\u2032-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGTCGGTGCGAAGGCCCCG-3\u2032/5\u2032-GGGGACCACTTTGTACAAGAAAGCTGGGTCTTAATCAACTTGATCCCAAC-3\u2032, 5\u2032-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGTCGGTGCGAAGGCCACG-3\u2032/5\u2032-GGGGACCACTTTGTACAAGAAAGCTGGGTCTTAATCAACTTGATCCCAAC-3\u2032, 5\u2032-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGCCGATGGACACCCCGCC-3\u2032/5\u2032-GGGGACCACTTTGTACAAGAAAGCTGGGTCTTACGAAGGCACAAGTCTCGC-3\u2032, 5\u2032-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGCCGATGGACACCCCGCC-3\u2032/5\u2032-GGGGACCACTTTGTACAAGAAAGCTGGGTCTTACGAAGGCACAAGTCTCGC-3\u2032, and 5\u2032-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGCCGATGGACACCCCGCC-3\u2032/5\u2032-GGGGACCACTTTGTACAAGAAAGCTGGGTCTTACGAAGGCACAAGTCTCGC-3\u2032, and cloned into the pIPKb001 vector. The single-cell transient gene silencing and expression were conducted essentially as described [B.g. tritici strain E09 conidia, the leaf segments were stained for GUS activity 48 h post-B.g. tritici inoculation. Before mounting for microscopic analysis, the leaves were stained with 0.1% (w/v) Coomassie Brilliant Blue R250 to visualize the fungal epiphytic structure.Antisense fragments of ., 2020) . Briefly"} +{"text": "Tmarus Simon, 1875 is a relatively large spider genus, currently includes 227 species distributed worldwide. Fanjing Mountain Nature Reserve is one of China\u2019s most biodiverse regions. However, Tmarus can be regarded as being poorly represented in Fanjing Mountain, with only one species having been recorded so far: T.fanjing Yang & Yu, 2022.Tmarus species were brought to light by those expeditions: T.fanjing Yang & Yu, 2022 and T.circinalis Song & Chai, 1990. T.fanjing is redescribed, based on new material and the female is described and illustrated for the first time. The supplementary micrographs of T.circinalis are given for the first time. The DNA barcodes and a distribution map of both species are provided for future use.Recently, various expeditions to Fanjing Mountain Nature Reserve were carried out by the authors. In this paper, two Tmarus Simon, 1875 is the second most speciose genus of Thomisidae Sundevall, 1833, with 227 valid species distributed worldwide so far, after Xysticus C. L. Koch, 1835 (293 species), 27 species of which are recorded from China ; for manThomisidae represents a substantial fraction of southwest China foliage-dwelling spiders , Lysitelesfanjingensis Wang, Gan & Mi, 2020, L.inflatus Song & Chai, 1990, Phartatangi Wang, Mi & Peng, 2016, Phrynarachnemammillata Song, 1990, Strigoplusguizhouensis Song, 1990, Tmarusfanjing Yang & Yu, 2022, Thomisuslabefactus Karsch, 1881 and Xysticuskurilensis Strand, 1907 to redescribe the male and report the female of T.fanjing for the first time; 2) to re-illustrate T.circinalis, based on new material from Mt. Fanjing and give supplementary micrographs; 3) to provide the DNA barcodes and a distribution map of T.fanjing and T.circinalis for future use.Recently various short, but intensive field collections in Fanjing Mountain have been conducted by staff of the Guizhou Normal University and Guizhou Education University. This paper reports our findings on the study of recently-available samples from the area, which revealed a new record species of Fanjing Mountain, Specimens in this study were collected by beating vegetation. Spiders were fixed and preserved in 95% ethanol. Specimens were examined with an Olympus SZX7 stereomicroscope; details were studied with an Olympus CX41 compound microscope. Female epigynes and male palps were examined and illustrated after being dissected. Epigynes were removed and cleared in warm lactic acid before illustration. The vulva was also imaged after being embedded in Arabic gum. Photos were made with a Cannon EOS70D digital camera mounted on an Olympus CX41 compound microscope. The digital images were taken and assembled using Helifocus 3.10.3. software package .T.circinalis in Hubei Province and Chongqing City were originated from ArcGIS . Due to lack of locality coordinates in previous publications, locality coordinates for cGIS see .Tmarusfanjing: YHTHO014, \u2642, GenBank ON796486; YHTHO013, \u2640, GenBank ON796487. Tmaruscircinalis: YHTHO015, \u2642, GenBank OR075896; YHTHO016, \u2640, GenBank OR075897.A DNA barcode was also obtained for the species matching. A partial fragment of the mitochondrial cytochrome oxidase subunit I (CO1) gene was amplified and sequenced for one male and one female specimen, respectively, using the primers LCOI1490 (5\u2019-GGTCAACAAATCATAAAGATATTG-3\u2019) and HCOI2198 (5\u2019-TAAACTTCAGGGTGACCAAAAAAT-3\u2019) . For addAll measurements were obtained using an Olympus SZX7 stereomicroscope and given in millimetres. Eye diameters are taken at the widest point. The total body length does not include chelicerae or spinnerets length. Leg lengths are given as total length . Most of the terminologies used in text and figure legends follows All specimens are deposited Museum of Guizhou Normal University, Guiyang, Guizhou, China.Yang & Yu, 202242208F9D-B77B-5D32-BABA-3628DD1444C6Type status:Holotype. Occurrence: recordedBy: Da Wang; Jaiyuan Xin; individualID: YHTHO001; individualCount: 1; sex: 1 male; lifeStage: 1 adult; behavior: foraging; preparations: whole animal (ETOH); associatedSequences: ON392063GenBank: ; occurrenceID: 7FC42291-EC27-5A7D-9D64-6E9F98746510; Taxon: order: Araneae; family: Thomisidae; genus: Tmarus; specificEpithet: fanjing; taxonRank: species; scientificNameAuthorship: Yang & Yu; taxonomicStatus: accepted; Location: continent: Asia; country: China; countryCode: CHN; stateProvince: Guizhou; county: Jiangkou; locality: Fanjingshan Nature Reserve; verbatimElevation: 755 m; decimalLatitude: 27.87; decimalLongitude: 108.80; Identification: identifiedBy: Jianshuang Zhang; dateIdentified: 12-12-2022; identificationReferences: Yang et al. 2022; Event: samplingProtocol: Beating; samplingEffort: 10 km by foot; year: 2021; month: 4; day: 18; Record Level: basisOfRecord: PreservedSpecimenType status:Other material. Occurrence: recordedBy: Haonan Zhang; individualCount: 2; sex: 1 male, 1 female; lifeStage: 2 adults; behavior: foraging; preparations: whole animal (ETOH); occurrenceID: A38DE598-7685-5DA5-AB10-C63D1031394B; Taxon: order: Araneae; family: Thomisidae; genus: Tmarus; specificEpithet: fanjing; taxonRank: species; scientificNameAuthorship: Yang & Yu; taxonomicStatus: accepted; Location: continent: Asia; country: China; countryCode: CHN; stateProvince: Guizhou; county: Jiangkou; locality: Fanjingshan Nature Reserve; verbatimElevation: 1060 m; decimalLatitude: 27.90; decimalLongitude: 108.58; Identification: identifiedBy: Hao Yu; dateIdentified: 12-12-2022; identificationReferences: Yang et al. 2022; Event: samplingProtocol: Beating; samplingEffort: 10 km by foot; year: 2022; month: 7; day: 19; Record Level: basisOfRecord: PreservedSpecimenType status:Other material. Occurrence: recordedBy: Cheng Wang; Xiaoqi Mi; individualID: YHTHO013, YHTHO014; individualCount: 4; sex: 2 males, 2 females; lifeStage: 4 adults; behavior: foraging; preparations: whole animal (ETOH); associatedSequences: ON796487; ON796486; occurrenceID: EE48AA39-2206-5DA9-8F4B-82BB2DA9B1D3; Taxon: order: Araneae; family: Thomisidae; genus: Tmarus; specificEpithet: fanjing; taxonRank: species; scientificNameAuthorship: Yang & Yu; taxonomicStatus: accepted; Location: continent: Asia; country: China; countryCode: CHN; stateProvince: Guizhou; county: Shiqian; locality: Fodingshan Nature Reserve; verbatimElevation: 858 m; decimalLatitude: 27.36; decimalLongitude: 108.0; Identification: identifiedBy: Cheng Wang; dateIdentified: 12-05-2022; identificationReferences: Yang et al. 2022; Event: samplingProtocol: Beating; samplingEffort: 10 km by foot; year: 2017; month: 4; day: 28; Record Level: basisOfRecord: PreservedSpecimenFemale . Overall body colour is dull brown in ethanol. Total length 6.34; carapace 2.33 long, 2.25 wide; abdomen 4.01 long, 2.23 wide.Chelicerae coloured as ocular area, both margins without teeth. Labium and endites uniformly light brown, endites depressed posteriorly, slightly convergent anteriorly, with dense scopulae on anterior margin; labium nearly diamond-shaped, anterior margin with sparse setae. Sternum yellowish-white, more or less cordiform or shield-shaped, 1.20 long, 0.93 wide.Carapace Fig. A dull brAbdomen Fig. A\u2013C elongLegs uniformly yellowish-white Fig. A and B. Epigyne Fig. C\u2013F. EpigMale .5'TATTTGGAGCGTGATCGGCTATAGTAGGAACTGCTATAAGAGTATTGATTCGAATAGAATTAGGTAATTCAGGAAGACTTTTTGGAAATGATCATTTATATAATGTAATTGTGACTGCTCATGCTTTTGTGATAATTTTTTTTATAGTTATACCTATTTTAATTGGAGGATTTGGTAATTGATTAGTACCTTTGATATTAGGGGCTCCTGATATATCTTTTCCTCGAATAAATAATTTATCTTTTTGGTTATTACCTCCTTCTTTATTTTTATTATTTATATCTTCTATAGTAGAAATAGGAGTAGGAGCTGGATGAACTGTATATCCACCTTTGGCTTCTAGTTTAGGTCATATAGGGAGATCAATGGATTTTGCTATTTTTTCTCTTCATTTAGCTGGGGCTTCTTCAATTATAGGGGCTGTAAATTTTATTTCTACTATTATTAATATACGAAGAGTAGGAATGACTATAGAAAAGGTGCCTTTATTTGTCTGATCGGTGTTAATTACTGCTATTTTACTTTTATTATCATTACCTGTTTTAGCAGGAGCTATTACTATATTATTAACAGATCGAAATTTTAATACTTCGTTTTTTGACCCTGCTGGTGGAGGGGATCCAATTTTATTTCAACATTTATTTTGATTTTT3' .5'TATTTGGAGCGTGATCGGCTATAGTAGGAACTGCTATAAGAGTATTGATTCGAATAGAATTAGGTAATTCAGGAAGACTTTTTGGAAATGATCATTTATATAATGTAATTGTGACTGCTCATGCTTTTGTGATAATTTTTTTTATAGTTATACCTATTTTAATTGGAGGATTTGGTAATTGATTAGTACCTTTGATATTAGGGGCTCCTGATATATCTTTTCCTCGAATAAATAATTTATCTTTTTGGTTATTACCTCCTTCTTTATTTTTATTATTTATATCTTCTATAGTAGAAATAGGAGTAGGAGCTGGATGAACTGTATATCCACCTTTGGCTTCTAGTTTAGGTCATATAGGGAGATCAATGGATTTTGCTATTTTTTCTCTTCATTTAGCTGGGGCTTCTTCAATTATAGGGGCTGTAAATTTTATTTCTACTATTATTAATATACGAAGAGTAGGAATGACTATAGAAAAGGTGCCTTTATTTGTCTGATCGGTGTTAATTACTGCTATTTTACTTTTATTATCATTACCTGTTTTAGCAGGAGCTATTACTATATTATTAACAGATCGAAATTTTAATACTTCGTTTTTTGACCCTGCTGGTGGAGGGGATCCAATTTTATTTCAACATTTATTTTGATTTTT3' . However, T.fanjing can be distinguished from T.piger by the following characters: for the males, embolus apex blunt in T.fanjing ; associatedSequences: GenBank: prepare to upload; occurrenceID: 9E9A0A7F-27B9-5732-81EE-36AD91E7A4E2; Taxon: order: Araneae; family: Thomisidae; genus: Tmarus; specificEpithet: Tmaruscircinalis; scientificNameAuthorship: Song & Chai; taxonomicStatus: accepted; Location: continent: Asia; country: China; countryCode: CHN; stateProvince: Guizhou; municipality: Jiangkou; locality: Fanjingshan Nature Reserve; verbatimElevation: 1025 m; decimalLatitude: 27.98; decimalLongitude: 108.69; Identification: identifiedBy: Hao Yu; dateIdentified: 15-01-2023; identificationReferences: Song and Zhu 1997; Event: samplingProtocol: Beating; samplingEffort: 10 km by foot; year: 2021; month: 7; day: 20; Record Level: basisOfRecord: PreservedSpecimenSee OR075896).5'TATTTGGGGCGTGGTCAGCTATAGTAGGAACTGCTATAAGAGTATTAATTCGAATAGAATTGGGTAATTCAGGAAGACTTCTTGGTAATGATCATTTATATAATGTAATTGTGACTGCTCATGCTTTTGTAATAATTTTTTTTATAGTTATGCCTATTTTAATTGGAGGTTTTGGTAATTGATTAGTACCTTTGATATTAGGAGCTCCTGATATATCTTTTCCTCGAATAAATAATTTATCTTTTTGGTTATTACCTCCTTCTTTATTTTTATTATTTATATCTTCTATAGTGGAGATAGGAGTAGGGGCTGGGTGAACTGTATATCCACCTTTAGCTTCTAGTTTGGGTCATATAGGAAGATCAATGGATTTTGCTATTTTTTCTCTTCATTTAGCTGGGGCTTCTTCAATTATAGGGGCTGTAAATTTTATTACTACTATTATTAATATACGTAGAGTAGGAATAACTATAGAAAAAGTGCCTTTATTTGTTTGATCAGTGTTAATTACTGCTATTTTACTTTTACTATCATTACCTGTTTTAGCAGGAGCTATTACTATATTATTAACAGATCGAAATTTTAATACATCGTTTTTTGACCCTGCTGGAGGGGGGGATCCAATTTTATTTCAACATTTATTTTGATTTTT3' .5'TATTTGGGGCGTGGTCAGCTATAGTAGGAACTGCTATAAGAGTATTAATTCGAATAGAATTGGGTAATTCAGGAAGACTTCTTGGTAATGATCATTTATATAATGTAATTGTGACTGCTCATGCTTTTGTAATAATTTTTTTTATAGTTATGCCTATTTTAATTGGAGGTTTTGGTAATTGATTAGTACCTTTGATATTGGGGGCTCCTGATATATCTTTTCCTCGAATAAATAATTTATCTTTTTGGTTATTACCTCCTTCTTTATTTTTATTATTTATATCTTCTATAGTGGAGATAGGAGTAGGGGCTGGGTGAACTGTGTATCCACCTTTAGCTTCTAGTTTGGGTCATATAGGGAGATCAATGGATTTTGCTATTTTTTCTCTTCATTTGGCTGGGGCTTCTTCAATTATAGGGGCTGTAAATTTTATTACTACTATTATTAATATACGTAGAGTAGGAATAACTATAGAAAAAGTGCCTTTATTTGTTTGATCAGTGTTAATTACTGCTATTTTACTTTTACTATCATTACCTGTTTTAGCAGGAGCTATTACTATATTATTGACAGATCGAAATTTTAATACATCGTTTTTTGACCCTGCTGGAGGGGGGGATCCAATTTTATTTCAACATTTATTTTGATTTTT3' , Chongqing City (Xiushan County) and Guizhou Province (Mt. Fanjing), China Fig. ."} +{"text": "In contrast, mutation of theC. elegansBRCA1 ortholog,brc-1,or itsbinding partner,brd-1, lead to only mild embryonic lethality. We show that inC. elegans,brc-1andbrd-1embryonic lethality is enhanced when53bp1 ortholog,hsr-9,is also mutated. This is not a consequence of activatingpolq-1-dependent microhomology-mediated end joining, aspolq-1mutation does not suppress embryonic lethality ofhsr-9;brc-1mutants. Together, these results suggest thatBRC-1-BRD-1andHSR-9function in parallel pathways and do not act antagonistically as in mammals.In mice, mutation of In contrast to what has been reported in mice, we observed elevated embryonic lethality inbrc-1andbrd-1null alleles . We found that thehsr-9;brc-1polq-1triple mutant had levels of embryonic lethality similar tobrc-1polq-1but higher thanhsr-9;brc-1, suggesting that the elevated embryonic lethality ofhsr-9;brc-1is not a consequence of activation of MMEJ . On thethality . To detof MMEJ . TherefCRISPR-mediated genome editing:hsr-9(xoe17)andpolq-1(xoe51)alleles were engineered by incorporating the stop-in cassettehsr-9repair template (gattttgcctcttaaataaaatttcagCAAAAAACCGAGGGGAGACTTGCAATAGGGAAGTTTGTCCAGAGCAGAGGTGACTAAGTGATAAGCTAGCTCTCGGATCATCTTGCAAACATGCTTATTGCTGgtaggtattgcaacc) and guide RNA (AGGGGAGACTTGCAATATCT) were injection intoN2and the resulting progeny were analyzed by PCR using TGAAATTAAGGTGGTCACTCGAAG and GTTGTTGTGGGGAGGCTGAA. Thepolq-1repair template (AGAGAATTCTCTGAAGATCCATTAATATTGCTTACCGAAGGGGAAGTTTGTCCAGAGCAGAGGTGACTAAGTGATAAGCTAGCAGAGTTTTCGCCGCAATTCTCAGACTTTGGTAATGATTTC) and guide RNA (ATTGCGGCGAAAACTCTCTT) were injected intoN2and the resulting progeny were analyzed by PCR using ATAGGCAAATGGCTGGACGG and TCAAAGCAGTCTTCTCGGCA. Worms were outcrossed a minimum of three times.Embryonic lethality:L4 hermaphrodites of indicated genotypes were picked onto individual plates and transferred to new plates every 24hr for 3 days. Embryonic lethality was determined by counting eggs and hatched larvae 24hr after removing the hermaphrodite and calculating percent as eggs/(eggs + larvae).Strains:"} +{"text": "Ribosome footprint profiling has demonstrated that ribosomes can be slowed or stalled on select mRNAs, often due to the presence of rare codons, stalling motifs, or via a ribosome-binding protein . Stalled ribosomes can act as physical roadblocks for trailing ribosomes and ultimately can cause ribosome collisions that stimulate no-go mRNA decay. Detecting stalled or slowed ribosomes in cells by ribosome footprint profiling or classic polysome profiling is laborious, technically challenging, and low throughput. Here, we present a protocol to assay for stalled ribosomes on in vitro\u2013transcribed reporter mRNAs using a robust, commercially available mammalian in vitro translation lysate and an optimized low-speed sucrose cushion. In short, we take advantage of the ability of puromycin to incorporate into the nascent polypeptide and cause the ribosome to dissociate from the mRNA during active elongation, as well as the ability to selectively pellet ribosomes through a low-speed sucrose cushion due to their large molecular weight. Stalled ribosomes are not actively elongating and do not incorporate puromycin, allowing the ribosome-bound mRNA to pellet in the low-speed sucrose cushion. RT-qPCR is used to quantify the amount of ribosome-bound reporter mRNA in the pellet. This workflow allows for direct assessment of stalled ribosomes and is fully amendable to insertion of putative stalling motifs in the target mRNA, as well as supplementation with recombinant proteins or small molecule inhibitors that target translation elongation.Key featuresThis protocol is optimized for cap-dependent in vitro translation in the dynamic linear range.Details for generating capped reporter mRNA in one day are provided.Requires as little as one day to complete if starting with in vitro\u2013transcribed mRNA.This protocol requires access to an ultracentrifuge and a real-time PCR system. Graphical overviewCaenorhabditis elegans) (Caenorhabditis elegans) . Elongatelegans) , which uelegans) . In yeaselegans) . Subsequelegans) . Upon laelegans) .Ribosome footprint profiling and classic polysome profiling can be used to detect stalled ribosomes; however, these approaches can be technically challenging, laborious, and rather low throughput. Additionally, ribosome profiling is not cost-effective when testing multiple specific mutations within reporter mRNAs or effector proteins. Here, we present a validated protocol that can be used to assess ribosome stalling in vitro that is medium-to-high throughput and can be performed in as little as one day if starting with in vitro\u2013transcribed mRNA.We take advantage of the selective nature of puromycin, an amino-acyl transfer RNA analog to incorporate into nascent polypeptides of only actively elongating ribosomes . PuromycReagents2-Propanol (isopropanol) 3\u2032-O-Me-m7G(5\u2032)ppp(5\u2032)G RNA Cap Structure Analog Agarose LE, quick dissolve Bromophenol blue Chloroform, ethanol stabilized 10\u00d7 CutSmart buffer Cycloheximide (CHX) Dimethyl sulfoxide (DMSO) Dithiothreitol (DTT) DNA Clean & Concentrator-25 kit DNase I (Rnase-free) E. coli Poly(A) polymerase 500 mM EDTA, pH 8.0 ULTROL grade Ethanol, 200 proof 10 mg/mL ethidium bromide Flexi Rabbit Reticulocyte Lysate System 37% (w/v) formaldehyde Glycerol, biotechnology grade Glycogen, molecular biology grade Hi-Di Formamide HiScribe T7 High Yield RNA Synthesis kit iScript Reverse Transcription Supermix iTaq Universal SYBR Green Supermix, 5 mL 2 M KCl, pH 7.4 2 1 M MgClMillennium RNA size marker 10\u00d7 MOPS buffer Nuclease-free water Puromycin dihydrochloride 6\u00d7 purple gel loading dye Quick-Load Purple 1 kb Plus DNA ladder RNA Clean & Concentrator-25 kit Rnase inhibitor, murine Sucrose, Ultra-pure, Rnase- & Dnase-free 10\u00d7 TBE buffer 1 M Tris-HCl, pH 7.4 TRIzol reagent Xylene cyanol FF pcDNA3.1(+)/nLuc-3XFLAG Dual promoter plasmid pCR II pGL4.13 Solutions100 mg/mL cycloheximide (CHX) (see Recipes)10 mg/mL CHX (see Recipes)5 mg/mL CHX (see Recipes)1 M DTT, Cleland\u2019s Reagent (see Recipes)70% (v/v) ethanol (see Recipes)1 mg/mL ethidium bromide (see Recipes)10 mM GTP (see Recipes)1\u00d7 MOPS buffer (see Recipes)10 mg/mL (~18 mM) puromycin (see Recipes)0.6 mM puromycin (see Recipes)2\u00d7 ribosome dilution buffer (see Recipes)RNA loading dye (see Recipes)RNA sample buffer (see Recipes)60% (w/v) sucrose (see Recipes)35% (w/v) sucrose, buffered (see Recipes)1\u00d7 TBE buffer (see Recipes)Recipes100 mg/mL cycloheximide (CHX) (store at -20 \u00b0C)10 mg/mL CHX 5 mg/mL CHX 1 M DTT, Cleland\u2019s Reagent (store at -20 \u00b0C)70% (v/v) ethanol1 mg/mL ethidium bromide10 mM GTP (store at -20 \u00b0C)1\u00d7 MOPS buffer10 mg/mL (~18 mM) puromycin (store at -20 \u00b0C)0.6 mM puromycin (store at -20 \u00b0C)2\u00d7 ribosome dilution buffer (store at 4 \u00b0C)RNA loading dyeRNA sample buffer (make fresh day of use)60% (w/v) sucrose35% (w/v) sucrose, buffered (store at 4 \u00b0C)1\u00d7 TBE bufferLaboratory supplies0.2 mL, open-top thick wall polycarbonate tube, 7 mm \u00d7 20 mm 1.7 mL microcentrifuge tube, clear 500 mL Erlenmeyer flask8-strip PCR tubes Hard-shell PCR plates, 96-well, thin-well IceIce bucketsLight-dry tissue wipes Microseal \u2018B\u2019 seals Nitrile glovesP10, P20, P200, and P1000 calibrated pipettesP10, P20, P200, and P1000 pipette tips ParafilmPlastic wrapTube racks-80 \u00b0C freezer-20 \u00b0C freezer4 \u00b0C refrigeratorAspiratorCFX Connect Real-Time System Eppendorf centrifuge 5430 Fume hoodGelDoc Go Gel imaging system with Image Lab Touch software MicrowaveMilliporeSigma Synergy ultrapure water purification system NanoDrop One Microvolume UV-Vis spectrophotometer OWL EasyCast B1 Mini Gel electrophoresis system OWL EasyCast B1A Mini Gel electrophoresis system Paper towelsPlate centrifuge, PerfectSpin P Pointed tweezersS100-AT3 fixed angle rotor Sorvall Discovery M120 SE micro-ultracentrifuge (Hitachi)T100 thermal cycler Vortex mixer Bio-Rad CFX MaestroMicrosoft Excelhttps://www.graphpad.com/quickcalcs/molarityform/)Prism GraphPad Molarity Calculator Oligo Calc: Oligonucleotide Properties Calculator Transcription and Translation Tool subcloned from pGL4.13 into pCR II, which is linearized with HindIII. For the experimental reporter, we typically use nanoLuciferase (nLuc) subcloned from pNL1.1 into pcDNA3.1(+), which is linearized with either XbaI or PspOMI. Both plasmids are available upon request from the corresponding author; pcDNA3.1(+)/nLuc-3XFLAG is also available from Addgene.ii. Choose a restriction endonuclease that cleaves downstream of the reporter coding sequence and either produces blunt ends or 5 overhangs. Restriction endonucleases that produce 3\u2032 overhangs should be avoided as they can elicit aberrant and antisense transcription . Choose In a microcentrifuge tube, combine 100 \u03bcL of plasmid DNA (300 ng/\u03bcL), 20 \u03bcL of 10\u00d7 CutSmart buffer, 20 \u03bcL of restriction enzyme , and 60 \u03bcL of Milli-Q water. Gently mix by inversion 20 times and collect contents by a short centrifugation spin .Pause point: Store completed digest at -20 \u00b0C.Digest the plasmid for 4 h at 37 \u00b0C and then hold at 4 \u00b0C. Note: Despite the excess of restriction enzyme in the reaction, we suggest a 4 h incubation for complete linearization due to the relatively large massof plasmid DNA.During the restriction digest, cast a 0.8% (w/v) agarose gel in an OWL EasyCast B1A Mini Gel electrophoresis system with a 1.5 mm 10-well comb. Add 100 mL of 1\u00d7 TBE buffer with 0.8 g of agarose in a 500 mL Erlenmeyer flask and then loosely plug the flask with a folded-up paper towel. Heat in the microwave for 1.5 min or until the agarose is completely dissolved. Allow to cool for 10 min on the benchtop and then add 5 \u03bcL of 10 mg/mL ethidium bromide. Gently swirl to mix and let the flask cool on the benchtop until it can be held comfortably for 10 s (or place in a 65 \u00b0C water or bead bath for 30 min). Pour ~70 mL into the casting tray and let the gel solidify at room temperature for ~1 h. Remove combs by gently pulling them up vertically. Remove and rotate the casting tray so that the wells are near the cathode. Fill the gel tank and completely cover the agarose gel with 1\u00d7 TBE buffer .Purify linearized plasmids using the DNA Clean and Concentrator-25 kit and the supplied solutions.g for 30 s, unless specified. To the 200 \u03bcL digest, add 800 \u03bcL of DNA binding buffer and mix by gentle inversion 20 times. Transfer mixture to the Zymo-spin column in a collection tube. Centrifuge and discard flowthrough. Wash the column with 200 \u03bcL of DNA wash buffer by centrifugation. Discard flowthrough and repeat wash step. Transfer column into a fresh and pre-labeled 1.7 mL microcentrifuge tube. Add 30 \u03bcL of nuclease-free water directly to the column (the white resin at the bottom of the column) and incubate at room temperature for 1 min. Elute by centrifugation . Store on ice for immediate use or at -20 \u00b0C for long-term storage.Perform all steps at room temperature and centrifugation at 16,000\u00d7 Determine DNA concentration and purity by UV spectrophotometry .Note: The Zymo-spin IICR column has a reported capacity of 25 \u03bcg of DNA. This protocol calls for 30 \u03bcg of DNA, which allows us to max out the column, resulting in most reporter plasmids eluting at ~1,000 ng/\u03bcL.Confirm linear plasmid integrity by 0.8% (w/v) agarose gel electrophoresis. Add 5 \u03bcL of Quick-Load Purple 1 kb Plus DNA ladder to the first well. On a piece of parafilm or in a microcentrifuge tube, gently mix 1 \u03bcL of purified linear plasmid DNA (250\u2013500 ng/\u03bcL), 1 \u03bcL of purple gel loading dye (6\u00d7), and 4 \u03bcL of Milli-Q water, and then load the entire sample into a single well. Run the agarose gel at 120 V (constant) for 30\u201345 min or until the desired resolution. Image gel via UV transillumination to confirm a single band that is running at the expected molecular weight.Note: If using two combs per casting tray, use the bottom half of the agarose gel before using the top half, as ethidium bromide in the gel runs toward the cathode. Unused parts of the gel may be stored for up to one week in plastic wrap at 4 \u00b0C.Synthesize in vitro\u2013transcribed reporter mRNA using the HiScribe T7 High Yield RNA Synthesis kit.To resuspend the ARCA cap analog to 40 mM, first quickly collect the contents (1 \u03bcmol) by a short centrifugation and then add 25 \u03bcL of nuclease-free water to the pellet. Gently mix by flicking the tube and collect the contents by brief centrifugation. Repeat the gently mixing procedure for a total of five times.It is critical to use 1 \u03bcL of 10 mM GTP and not 1 \u03bcL of 100 mM GTP. The 8:1 ARCA cap analog to GTP ratio ensures 90% co-transcriptional capping efficiency , 0.5 \u03bcL of RNase inhibitor, 1 \u03bcL of 10\u00d7 T7 reaction buffer, 1 \u03bcL of 100 mM ATP, 1 \u03bcL of 100 mM UTP, 1 \u03bcL of 100 mM CTP, 1 \u03bcL of 10 mM GTP, 2 \u03bcL of 40 mM ARCA cap analog, 1 \u03bcL of T7 RNA polymerase mix, and 0.5 \u03bcL of nuclease-free water in a PCR tube or microcentrifuge tube. ficiency .Notes:i. If transcribing multiple mRNAs at one time, assemble a master mix of all components except linear plasmid templates. Use 9 \u03bcL of master mix with 1 \u03bcL of linear plasmid template (~500 ng/\u03bcL). Reactions can also be scaled up linearly to 20 \u03bcL but beware of exceeding the binding capacity of the RNA clean up columns (see below).ii. Most T7 RNA polymerase\u2013mediated mRNA synthesis kits with a cap analog pre-mixed with the NTPs use a 4:1 ARCA cap to GTP ratio, which only provides ~80% co-transcriptional capping efficiency. These cap analog pre-mixed kits also do not provide the ability to generate A-capped or non-methylated G-capped mRNAs to test cap-dependency.Perform in vitro transcription for 2 h at 30 \u00b0C using a thermal cycler (for PCR tube) or heat block (for microcentrifuge tube).Note: In our hands, transcription at 30 \u00b0C yielded purer mRNA than transcription at 37 \u00b0C.To remove the DNA template, add 1 \u03bcL of DNase I (RNase-free) to each 10 \u03bcL reaction and gently mix by inversion. Incubate at 37 \u00b0C for 15 min.E. coli Poly(A) polymerase, and 28 \u03bcL of nuclease-free water, followed by incubation at 37 \u00b0C for 1 h.If polyadenylation of the RNA is desired, combine and gently mix on ice the 11 \u03bcL DNase-treated capped and transcribed mRNA reaction with 5 \u03bcL of 10\u00d7 Poly(A) polymerase buffer, 5 \u03bcL of 10 mM ATP, 1 \u03bcL of Note: This protocol uses thenuclease-treatedFlexi rabbit reticulocyte lysate (RRL) system from Promega. It is not entirely necessary to polyadenylate in vitro\u2013transcribed mRNA for efficient translation usingnuclease-treatedRRL. Polyadenylation provides a less than two-fold enhancement of reporter mRNA translation innuclease-treatedRRL Soto . If polyIf using a PCR tube, transfer the entire contents to a microcentrifuge tube.Purify mRNA using the RNA Clean and Concentrator-25 kit and the supplied solutions.g for 1 min, unless specified. To each 50 \u03bcL of mRNA sample, add 100 \u03bcL of RNA binding buffer and gently mix by inversion 20 times. Add 150 \u03bcL of 100% ethanol to each reaction and gently mix by inversion 20 times. Transfer the entire sample to the Zymo-Spin IICR Column in a collection tube and centrifuge. Discard the flowthrough. Add 400 \u03bcL of RNA prep buffer to the column, centrifuge, discard flowthrough, and place column back into the collection tube. Add 700 \u03bcL of RNA wash buffer to the column, centrifuge, discard flowthrough, and place column back into the collection tube. Add 400 \u03bcL of RNA wash buffer to the column, centrifuge for 4 min, discard flowthrough, and place column into a new RNase-free 1.7 mL microcentrifuge tube. Add 75 \u03bcL of nuclease-free water directly to the column matrix and allow to incubate at room temperature for 1 min. Elute by centrifugation. Store mRNA on ice moving forward.Perform all steps at room temperature and centrifugation at 16,000\u00d7 Determine RNA concentration and purity by UV spectrophotometry .Pause point.Aliquot mRNA in 3 \u03bcL volumes in PCR strips and store at -80 \u00b0C. Confirm in vitro\u2013transcribed mRNA quality and purity by denaturing agarose gel electrophoresis.Cast a denaturing 0.8%\u20131% (w/v) agarose formaldehyde gel in OWL EasyCast B1 Mini Gel electrophoresis system with a 1.5 mm 10-well comb placed in the top comb slot. Add 0.8\u20131 g of agarose to 80 mL of Milli-Q water in a 500 mL Erlenmeyer flask loosely plugged with a folded-up paper towel. Microwave for 1.5 min or until the agarose is dissolved and gently swirl to mix. Cool on countertop for 2 min.In a fume hood, add 10 mL of 10\u00d7 MOPS buffer and 10 mL of 37% formaldehyde to the flask containing dissolved agarose. Gently swirl to mix and allow to cool for 5\u201310 min.Pour 100 mL into the casting tray and let the gel solidify at room temperature for ~1 h in the dark by loosely covering with aluminum foil.Prepare mRNA samples by mixing 500 ng of in vitro\u2013transcribed mRNA, 1 \u03bcL of 1 mg/mL ethidium bromide, 1 \u03bcL of RNA loading dye (see Recipes), 5 \u03bcL of freshly-prepared RNA sample buffer (see Recipes), and nuclease-free water to 15 \u03bcL total volume.Prepare the RNA ladder by mixing 1 \u03bcL of Millennium RNA size marker (1 \u03bcg/\u03bcL), 1 \u03bcL of 1 mg/mL ethidium bromide, 1 \u03bcL of RNA loading dye (see Recipes), 3 \u03bcL of RNA sample buffer (see Recipes), and 4 \u03bcL of nuclease-free water.Heat ladder and samples at 70 \u00b0C for 5 min; then, place the ladder and samples on ice for 2 min.Remove combs by gently pulling up vertically. Remove and rotate the casting tray so that the wells are near the cathode. Fill the gel tank and completely cover the agarose gel with 1\u00d7 MOPS buffer .Load ladder and samples on the gel.Run at 60 V (constant) (or 5\u201310 V/cm gel width) in the dark for 3\u20134 h (when the dye front reaches 2 cm from the bottom of the gel) or until the desired resolution.Image gel via UV transillumination to confirm a single band is running at the expected molecular weight (polyadenylating mRNA will add to the expected molecular weight).Note: Non-polyadenylated in vitro\u2013transcribed mRNA will run as a single, crisp band at the expected molecular weight on a denaturing agarose gel. The same in vitro\u2013transcribed mRNA that is polyadenylated will appear ~100\u2013200 nt heavier as a slightly broader band. An additional control reaction lacking the Poly(A) polymerase can be included to better define the poly(A) tail length. Large smears spanning far beyond the expected molecular weight indicate RNA degradation, rolling circle transcription due to the presence of non-linear template DNA, nondenatured RNA secondary structure, or errors during RNA clean up steps. We have also found that making fresh, day-of-use RNA sample buffer is critical.In vitro translationPre-cool the Sorvall Discovery M120 SE micro-ultracentrifuge and S100-AT3 rotor to 4 \u00b0C prior to setting up in vitro translation reactions. The S100-AT3 rotor can hold up to 20 samples.Note: For each experimental mRNA, at least six samples should be prepared and translated: three replicates that are translated and not puromycin-treated and three replicates that are translated and puromycin-treated (step B3). Each of the six samples is mixed with a translated normalizing control mRNA-containing reaction (step B2), then diluted and layered on top of individual sucrose cushions (step C1).In vitro translation of normalizing control mRNA:be sure to use the ssRNA setting; see Software). Using its molecular weight from above and the online Prism GraphPad Molarity Calculator (see Software), calculate the mass required for 3 nM in 10 \u03bcL. This is the mass of mRNA required in the complete 10 \u03bcL in vitro translation reaction for 3 nM mRNA . Dilute the purified mRNA in nuclease-free water such that 2 \u03bcL of RNA contains the mass calculated above, resulting in a final concentration of 15 fmol/\u03bcL. Other similar online tools are available and would suffice.Dilute normalizing mRNA to 15 fmol/\u03bcL in nuclease-free water and keep on ice. To do so, use the plasmid DNA sequence from the first transcribed nucleotide of the T7 RNA polymerase promoter to the restriction endonuclease cut site and determine the corresponding RNA sequence using the online Transcription and Translation Tool (see Software). Then, calculate the molecular weight of the control mRNA reporter with the online Oligo Calc: Oligonucleotide Properties Calculator , 0.1 \u03bcL of 1 mM amino acid mixture minus leucine, 0.1 \u03bcL of amino acid mixture minus methionine, 0.2 \u03bcL of 25 mM Mg(OAc), 0.4 \u03bcL of 2.5 M KCl, 0.2 \u03bcL of RNase inhibitor, and 4 \u03bcL of nuclease-free water. Perform in vitro translation for 15 min at 30 \u00b0C in a thermal cycler.Immediately place samples on ice.To each sample, add 4 \u03bcL of 5 mg/mL CHX (see Recipes) . Gently mix by inversion 20 times and collect contents by a short centrifugation spin. Samples should now be 14 \u03bcL. Keep samples on ice until step C1.Note: Cycloheximide is added in step B2 to robustly inhibit elongation and preserve ribosome-bound mRNAs when added to experimental samples that contain puromycin in step B3.In vitro translation of experimental mRNA:Dilute experimental mRNA(s) to 15 fmol/\u03bcL (as in step B2a) in nuclease-free water and keep on ice.On ice in a PCR tube, set up the following 10 \u03bcL translation reaction: mix 2 \u03bcL of 15 fmol/\u03bcL in vitro transcribed mRNA, 3 \u03bcL of Flexi RRL (nuclease-treated), 0.1 \u03bcL of 1 mM amino acid mixture minus leucine, 0.1 \u03bcL of amino acid mixture minus methionine, 0.2 \u03bcL of 25 mM Mg(OAc), 0.4 \u03bcL of 2.5 M KCl, 0.2 \u03bcL of RNase inhibitor, and 4 \u03bcL of nuclease-free water. If translating multiple experimental reporters or many biological replicates, prepare a master mix with all components except for the mRNA; add 8 \u03bcL of the master mix to 2 \u03bcL of 15 fmol/\u03bcL in vitro\u2013transcribed mRNA.Perform in vitro translation for 15 min at 30 \u00b0C in a thermal cycler.Immediately place samples on ice.To each sample, add 2 \u03bcL of nuclease-free water or 0.6 mM puromycin (see Recipes) . Gently mix by inversion 20 times and collect contents by a short centrifugation spin.Place puromycin-containing samples on a thermal cycler and incubate at 30 \u00b0C for 30 min. Keep samples without puromycin on ice.Place all samples on ice and immediately add 2 \u03bcL of 10 mg/mL CHX (see Recipes) . Gently mix by inversion 20 times and collect contents by a short centrifugation spin. Keep samples on ice.All translation reactions should now be 14 \u03bcL.Ribosomal pelleting through low-speed sucrose cushionPrepare samples for low-speed ribosomal pelleting through a sucrose cushion.Mix a 14 \u03bcL normalizing control mRNA-containing reaction tube and a 14 \u03bcL experimental mRNA-containing reaction tube for a total sample volume of 28 \u03bcL. Keep samples on ice.Add 28 \u03bcL of ice-cold 2\u00d7 ribosome dilution buffer . Each sample should now be 56 \u03bcL.Gently mix by inversion 20 times and collect contents by a short centrifugation spin. Keep samples on ice.Prepare sucrose cushions and overlay samples.Label 7 mm \u00d7 20 mm polycarbonate thick-walled tubes. Mark a single spot on the rim of each tube . Place tAdd 130 \u03bcL of ice-cold 35% buffered sucrose to the bottom of each tube.Very carefully, overlay all 56 \u03bcL of the diluted sample from step C1 on top of the sucrose cushion.Low-speed centrifugationWithout disturbing the sample\u2013sucrose interface, place the tubes into the pre-chilled S100-AT3 rotor. Be sure to position the marked spot facing outward and toward the back of the rotor. This spot will indicate the side of the tube where the ribosome pellet will be located after centrifugation . Using pCarefully place the rotor into the pre-cooled Sorvall Discovery M120 SE micro-ultracentrifuge.g for 1 h at 4 \u00b0C. Use an acceleration setting of 9 (fastest setting) and a deceleration setting of 5 (middle setting).Centrifuge samples at 50,000\u00d7 Carefully, remove the tubes from the rotor and place them on ice using the homemade ice bucket for the 7 mm \u00d7 20 mm tubes. Using pointed tweezers for this step is helpful.Using a pipettor, remove and discard the supernatant without disturbing the pellet. The glossy clear pellet should be at the bottom edge of the tube below the mark that was facing outward and toward the back of the rotor during centrifugation.Resuspend the pellet in TRIzol.i. First, add 200 \u03bcL of TRIzol to a new, labeled, nuclease-free microcentrifuge tube.ii. Add 100 \u03bcL of TRIzol to the ribosome pellet in the 7 mm \u00d7 20 mm tube and mix 20 times by gently pipetting up and down. The pellet will dissociate from the tube wall when TRIzol is first added and will float in solution until it ultimately dissolves.iii. Transfer this 100 \u03bcL to the microcentrifuge tube (now containing 300 \u03bcL).iv. Add another 200 \u03bcL of TRIzol to the 7 mm \u00d7 20 mm tube to wash off any remaining ribosomes by gently pipetting up and down 10 times.v. Transfer the 200 \u03bcL sample to the labeled microcentrifuge tube Pause point: Store samples at -80 \u00b0C.Mix samples end-over-end at room temperature for 15 min at 15 rpm. Collect contents by a short centrifugation spin. RNA extractions, cDNA synthesis, and RT-qPCRRNA extractionsThaw samples at room temperature if necessary. Add 100 \u03bcL of chloroform to each sample.Mix vigorously by hand for 1 min (do not vortex).g at 4 \u00b0C.Centrifuge for 15 min at 12,000\u00d7 Without disturbing or touching the protein interface, carefully remove the top 200 \u03bcL clear aqueous layer and transfer it to a new, labeled, nuclease-free microcentrifuge tube.Add 1.5 \u03bcL glycogen (20 mg/mL) to each sample. Upon dispensing, wash the tip in the aqueous phase by gently pipetting up and down three times.To each sample, add 500 \u03bcL of 100% isopropanol. Gently mix by inversion 20 times.g at 4 \u00b0C.Centrifuge for 15 min at 12,000\u00d7 Note: When pelleting RNA in microcentrifuge tubes, place the hinge upright facing the outside of the rotor. This will allow you to predict the location of the pellet at the bottom of the tube on the same side of the hinge.Aspirate off the isopropanol until ~100 \u03bcL remains in the tube, leaving the RNA pellet untouched. Remove the final ~100 \u03bcL with a P200 pipette. The pellet should be white but very small.Add 600 \u03bcL of ice-cold 70% ethanol. Vortex each sample for 1 s.g at 4 \u00b0C.Centrifuge for 15 min at 12,000\u00d7 Aspirate off the ethanol until ~100 \u03bcL remains in the tube, leaving the RNA pellet untouched. Remove the final ~100 \u03bcL with a P200 pipette and finally a P10 pipette. Aspirate any remaining ethanol off the tube walls. Allow to air dry with the top open for 2\u20133 min at room temperature on the bench.Place tubes on ice, add 30 \u03bcL of nuclease-free water to the pellet, and let stand on ice for 2 min. Gently resuspend the pellet by pipetting up and down 20 times. Be sure to wash down the side of the tube (same side as the hinge) to ensure complete resuspension of the RNA pellet.cDNA synthesisOn ice, combine 16 \u03bcL of RNA and 4 \u03bcL of 5\u00d7 iScript Reverse Transcription Supermix. Mix by gentle inversion 20 times and collect the contents by a short centrifugation spin. Store the remaining 14 \u03bcL of RNA at -80 \u00b0C.Using a thermal cycler, reverse transcribe using:i. 25 \u00b0C for 5 min (priming)ii. 46 \u00b0C for 20 min (reverse transcription)iii. 95 \u00b0C for 1 min (reverse transcriptase inactivation and RNA cleavage)iv. Hold at 4 \u00b0CPause point: Store samples at -20 \u00b0C.To each 20 \u03bcL cDNA sample, add 180 \u03bcL of nuclease-free water for a 1:10 dilution. Gently mix by inversion 20 times and collect contents by a short centrifugation spin. RT-qPCRDesign a 96-well plate layout scheme for all samples and negative controls. Each cDNA sample will be amplified with two primer sets: one targeting the normalizing control mRNA and the other targeting the experimental mRNA. A no-template negative control (where water is added instead of cDNA) should also be included for each primer set. Perform at least technical duplicates for each sample and primer set.Dilute qPCR primers by mixing 510 \u03bcL of nuclease-free water, 45 \u03bcL of 10 \u03bcM forward primer, and 45 \u03bcL of 10 \u03bcM reverse primer. See General notes below for the primer sequences we used for FFLuc and nLuc.Our typical RT-qPCR reaction is 15 \u03bcL. Each well will contain 7.5 \u03bcL of iTaq Universal SYBR Green Supermix (2\u00d7), 1.5 \u03bcL of the 1:10 diluted cDNA, and 6 \u03bcL of diluted primers. For both the normalizing control and experimental primer sets, create a master mix of iTaq Universal SYBR Green Supermix and diluted primers. Add 13.5 \u03bcL of this master mix to the appropriate wells. Then, carefully add 1.5 \u03bcL of the appropriate diluted cDNA samples. The no-template negative control contains 13.5 \u03bcL of appropriate master mix and 1.5 \u03bcL of nuclease-free water.Seal the plate with a microseal \u2018B\u2019 seal. Be sure not to touch the top of the plastic directly, but rather apply pressure to the seal by using a clean tissue wipe (or Kimwipe). Remove and discard the perforated edges.Centrifuge the plate in a plate centrifuge for ~30 s to pull the samples to the bottom of the wells.Place the plate into the Bio-Rad CFX Connect Real-Time System.Run the following RT-qPCR program:i. One cycle of 95 \u00b0C for 3 min.ii. Forty cycles of 95 \u00b0C for 10 s, 60 \u00b0C for 30 s, followed by a Plate Read.iii. Melt curve from 65 \u00b0C to 95 \u00b0C with an increment of 0.5 \u00b0C for 5 s and a Plate Read.Note: If using new primer sets, peel back the plastic film after a run and take out 10 \u03bcL to confirm the expected size amplicon on a 2% (w/v) agarose gel.For each experimental mRNA, at least six samples should be prepared and translated: three replicates that are translated and not puromycin-treated and three replicates that are translated and puromycin-treated (step B3). Each of the six samples is mixed with a translated normalizing control mRNA reaction (step B2), then diluted and layered on top of individual sucrose cushions (step C1). During RT-qPCR, each cDNA should be assayed in at least technical duplicates. Primer sets for both the normalizing control mRNA and experimental mRNA should be used in separate wells on the same plate.t-test with Welch\u2019s correction. For nLuc as the experimental mRNA reporter, we typically observe a relative ~60% reduction in signal with puromycin treatment to the signal of the normalizing control mRNA to account for any error during RNA extraction and/or cDNA synthesis. Once the gene expression has been calculated by the CFX Maestro software, export the data and perform the remaining analysis in Excel. For each experimental reporter, group the without puromycin-treatment replicates and set to 100%. Then, determine the relative signal for each replicate with puromycin-treatment. Statistical comparisons can be made using an unpaired reatment .Two additional controls should be incorporated in step B3:1) A no-template negative control, where water is added instead of either reporter mRNA, is critical to ensure specificity of RT-qPCR primers and determine background levels of detection.2) To confirm that the low-speed sucrose cushions are working as expected to selectively pellet ribosome-bound mRNA, set up translation reactions with the experimental mRNA without puromycin, incubate for 15 min on ice instead of 30 \u00b0C (step B3c), and proceed as directed above. The ice-incubated sample should have very few, if any, ribosomes loaded on the experimental mRNA and should be minorly detected by RT-qPCR when compared to the 30 \u00b0C\u2013incubated sample.Journal of Biological Chemistry, DOI: 10.1016/j.jbc.2022.102660. See Figure 6E and 6F.This protocol was validated in Scarpitti et al. (2022) These in vitro translation reaction conditions have been optimized to be in the dynamic linear range for time and mRNA input for a range of reporter mRNAs . These sg sucrose cushion in the Sorvall Discovery M120 SE micro-ultracentrifuge and S100-AT3 rotor. Increased centrifugal force resulted in loss of specificity for pelleting ribosome-bound mRNAs in a translation-dependent manner. Both variables may have to be re-optimized if using different translation extracts, if making substantial alterations to the translation reaction conditions, and/or if using a different rotor.We empirically optimized the 0.1 mM puromycin for use with the in vitro translation reaction conditions described in this protocol. Increasing puromycin beyond 0.1 mM did not further reduce the amount of mRNA co-pelleted in a translation-dependent manner. We also optimized the 50,000\u00d7 If testing ribosome stalling by an mRNA-binding protein , we recoLastly, while we have always used FFLuc as the normalizing control mRNA and nLuc as the experimental mRNA, we believe other reporters would function just as well for either the normalizing control or experimental mRNA. We have previously cloned FFLuc from pGL4.13 into the dual promoter plasmid pCR II. The use of pCR II is not required, but pCR II/FFLuc under control of the T7 RNA polymerase promoter is available upon request. pcDNA3.1(+)/nLuc-3XFLAG, which contains a T7 RNA polymerase promoter upstream of nLuc, is available upon request and from Addgene.See below for reporter coding sequences and qPCR primer sequences.FireFly Luciferase ATGGAAGATGCCAAAAACATTAAGAAGGGCCCAGCGCCATTCTACCCACTCGAAGACGGGACCGCCGGCGAGCAGCTGCACAAAGCCATGAAGCGCTACGCCCTGGTGCCCGGCACCATCGCCTTTACCGACGCACATATCGAGGTGGACATTACCTACGCCGAGTACTTCGAGATGAGCGTTCGGCTGGCAGAAGCTATGAAGCGCTATGGGCTGAATACAAACCATCGGATCGTGGTGTGCAGCGAGAATAGCTTGCAGTTCTTCATGCCCGTGTTGGGTGCCCTGTTCATCGGTGTGGCTGTGGCCCCAGCTAACGACATCTACAACGAGCGCGAGCTGCTGAACAGCATGGGCATCAGCCAGCCCACCGTCGTATTCGTGAGCAAGAAAGGGCTGCAAAAGATCCTCAACGTGCAAAAGAAGCTACCGATCATACAAAAGATCATCATCATGGATAGCAAGACCGACTACCAGGGCTTCCAAAGCATGTACACCTTCGTGACTTCCCATTTGCCACCCGGCTTCAACGAGTACGACTTCGTGCCCGAGAGCTTCGACCGGGACAAAACCATCGCCCTGATCATGAACAGTAGTGGCAGTACCGGATTGCCCAAGGGCGTAGCCCTACCGCACCGCACCGCTTGTGTCCGATTCAGTCATGCCCGCGACCCCATCTTCGGCAACCAGATCATCCCCGACACCGCTATCCTCAGCGTGGTGCCATTTCACCACGGCTTCGGCATGTTCACCACGCTGGGCTACTTGATCTGCGGCTTTCGGGTCGTGCTCATGTACCGCTTCGAGGAGGAGCTATTCTTGCGCAGCTTGCAAGACTATAAGATTCAATCTGCCCTGCTGGTGCCCACACTATTTAGCTTCTTCGCTAAGAGCACTCTCATCGACAAGTACGACCTAAGCAACTTGCACGAGATCGCCAGCGGCGGGGCGCCGCTCAGCAAGGAGGTAGGTGAGGCCGTGGCCAAACGCTTCCACCTACCAGGCATCCGCCAGGGCTACGGCCTGACAGAAACAACCAGCGCCATTCTGATCACCCCCGAAGGGGACGACAAGCCTGGCGCAGTAGGCAAGGTGGTGCCCTTCTTCGAGGCTAAGGTGGTGGACTTGGACACCGGTAAGACACTGGGTGTGAACCAGCGCGGCGAGCTGTGCGTCCGTGGCCCCATGATCATGAGCGGCTACGTTAACAACCCCGAGGCTACAAACGCTCTCATCGACAAGGACGGCTGGCTGCACAGCGGCGACATCGCCTACTGGGACGAGGACGAGCACTTCTTCATCGTGGACCGGCTGAAGAGCCTGATCAAATACAAGGGCTACCAGGTAGCCCCAGCCGAACTGGAGAGCATCCTGCTGCAACACCCCAACATCTTCGACGCCGGGGTCGCCGGCCTGCCCGACGACGATGCCGGCGAGCTGCCCGCCGCAGTCGTCGTGCTGGAACACGGTAAAACCATGACCGAGAAGGAGATCGTGGACTATGTGGCCAGCCAGGTTACAACCGCCAAGAAGCTGCGCGGTGGTGTTGTGTTCGTGGACGAGGTGCCTAAAGGACTGACCGGCAAGTTGGACGCCCGCAAGATCCGCGAGATTCTCATTAAGGCCAAGAAGGGCGGCAAGATCGCCGTGTAAnanoLuciferase ATGGTCTTCACACTCGAAGATTTCGTTGGGGACTGGCGACAGACAGCCGGCTACAACCTGGACCAAGTCCTTGAACAGGGAGGTGTGTCCAGTTTGTTTCAGAATCTCGGGGTGTCCGTAACTCCGATCCAAAGGATTGTCCTGAGCGGTGAAAATGGGCTGAAGATCGACATCCATGTCATCATCCCGTATGAAGGTCTGAGCGGCGACCAAATGGGCCAGATCGAAAAAATTTTTAAGGTGGTGTACCCTGTGGATGATCATCACTTTAAGGTGATCCTGCACTATGGCACACTGGTAATCGACGGGGTTACGCCGAACATGATCGACTATTTCGGACGGCCGTATGAAGGCATCGCCGTGTTCGACGGCAAAAAGATCACTGTAACAGGGACCCTGTGGAACGGCAACAAAATTATCGACGAGCGCCTGATCAACCCCGACGGCTCCCTGCTGTTCCGAGTAACCATCAACGGAGTGACCGGCTGGCGGCTGTGCGAACGCATTCTGGCGTAART-qPCR primersF_pGL4.13 RT-qPCR: GCAGTACCGGATTGCCCAAGR_pGL4.13 RT-qPCR: GTCGGGGATGATCTGGTTGCF_nLuc (pNL1.1) RT-qPCR: CAGCGGGCTACAACCTGGACR_nLuc (pNL1.1) RT-qPCR: AGCCCATTTTCACCGCTCAG"} +{"text": "Bacillus cereus strain PC2 (GenBank accession No. MZ314010) and Streptomyces praecox strain SP1 (GenBank accession No. MZ314009). In-site bacterial and fungal isolates defined in the current study were proficient in cleaning wastewater of chlorpyrifos pesticide residues.Pollutants cause a huge problem for humans, animals, plants, and various ecosystems, especially water resources. Agricultural, domestic, and industrial waste effluents change the water quality and affect living microorganisms. Therefore, the current study aimed to identify possible microorganisms in wastewater as potential bioremediation agents of pesticide residues. Wastewater samples were collected from El-Khairy agricultural drainage, which receives agricultural and domestic wastes. Bacteria and fungi species were isolated as clean cultures. Wastewater samples were analyzed for pesticide residues via gas chromatography-mass spectroscopy (GC\u2013MS) system. Results uncovered the presence of ten pesticides ranging from 0.0817 to 28.162\u00a0\u00b5g/l, and the predominant pesticide was chlorpyrifos. Along with that, about nine species were relatively efficient in the removal of chlorpyrifos residues up to 2000\u00a0\u00b5g/l with removal percentages ranging from 24.16 to 80.93% under laboratory conditions. Two bacterial isolates proficiently degraded significant amounts of chlorpyrifos: Therefore, the current study aimed to isolate indigenous bacteria and fungi and screen their potential as bioremediation agents of wastewater of pesticide residues.Acetonitrile HPLC-grade and culture media were purchased from local chemical providers. DNA purification kit (Germany) and PCR clean-up kit were from Maxim Biotech Inc. (USA). The internal standard (TPP) and extraction (Cat#5982\u20130650) and dispersive SPE clean-up (Cat#5982\u20135056) kits were purchased from Technoscient for Lab & Optical Product, Cairo, Egypt. Certified reference standard materials of pesticides were obtained from ULTRA Scientific Analytical Solutions Table .Table 1L3/s were collected by water sampler into cleaned and sterilized one-liter Pyrex borosilicate dark glass bottles and plate count agar for isolation of the bacteria colonies at 25\u00a0\u00b0C. Nine samples were collected; 3 from each collection point as replicates and 3 plates per replicate were planted from each sample under sterilized conditions and incubated at 37\u00a0\u00b0C. The complete growth of the microbe was reached after about 7\u00a0days of incubation. Each microorganism was transferred into a new Petri dish. The subculture of each microbe was repeated several times until a visually clean culture was obtained.4 (anhy) and NaCl, respectively, were thoroughly mixed for 1\u00a0min. Then the internal standard triphenyl phosphate (TPP) solution was added to tubes and shaken for 30\u00a0s. Then tubes were centrifuged at 1350\u2009\u00d7\u2009g for 10\u00a0min . About 1\u00a0ml of supernatant (acetonitrile) was mixed by hand for 5\u00a0min with 25\u00a0mg PSA sorbent and 150\u00a0mg MgSO4 (anhy) and centrifuged for 5\u00a0min at 1350\u2009\u00d7\u2009g. About 500\u00a0\u00b5l of each tube was filtered through 0.22-\u03bcm PTFE filters into HPLC vials for GC\u2013MS analysis.Pesticide residues in wastewater were extracted and cleaned up using a modified method of Anastassiades . Separation conditions were as reported by AOAC AOAC, , where tThe intra-day assay (repeatability) and inter-day assay (intermediate precision) of the used analytical technique were calculated according to Ermer . Also, tThe radial growth of separated and identified fungi and bacteria on media mixed with the prevalent insecticide in wastewater, chlorpyrifos (CPF), was examined. Five concentrations of CPF were prepared in the growing media. Five plates of each microorganism were used as replicates per each concentration. Microorganisms were incubated with the CPF at 37\u00a0\u00b0C for 1\u00a0week, and then their radial growth was photographed and recorded. Then competence of growth fungi and bacteria on such media with the insecticide was calculated compared to control plates. This experiment was repeated six times. Then the performance of the potential bioremediation activity of organisms was examined using 500, 1000, and 2000\u00a0\u00b5g/l. The residues of CPF in media after the incubation time were measured using the GC\u2013MS as described previously.The fungal isolates were cultured onto clean growth media until pure cultures were obtained and used for various evaluations and identification following the manufacturer\u2019s guidelines.E. coli, forward, 5\u02b9 AGG ACG TGC TCC AAC CGC A \u02b93, and reverse, 5\u02b9 AAC TGG AGG AAG GTG GGG AT \u02b93 was performed by Macrogen Company . The 16S rRNA gene nucleotide sequences of isolated bacterial strains were submitted to the GenBank database under accession numbers MZ314009 and MZ314010.http://www.ebi.ac.uk/clustalw) . The pesticide residues and microbial isolates\u2019 performance and growth were expressed as mean\u2009\u00b1\u2009SD. Significant means were contrasted using Tukey\u2019s honest significant difference test (HSD) (05) SAS, .The analytical method used in the analysis of pesticides was accurate and suitable based on obtained values of repeatability and intermediate precision Ermer, . ResultsAnalysis of pesticides residues revealed the presence of lenacil, chlorpyrifos, cypermethrin, bifenthrin, carbofuran, and permethrin at 0.721, 28.16, 4.14, 0.052, 7.881, and 0.208\u00a0\u00b5g/l, respectively, in samples collected from site A were presented in Table Verification of the performance of isolated bacteria and fungi was examined by challenging their growth on media with 500, 1000, and 2000\u00a0\u00b5g/l of CPF Fig.\u00a0. ResultsAspergillus terreus, Aspergillus foetidus var. pallidus, Aspergillus fumigatus var. ellipticus, and Aspergillus fumigates according to the database identification program of RCMB For Aspergilli and Bacillus cereus (I). For further differentiation between the two bacterial strains, similar morphology, physiological, and biochemical traits were reported, except for starch hydrolysis and lactose production that were negative in B. cereus and positive in S. praecox. Also, B. cereus produced acid from sucrose, but S. praecox did not.The characteristics of the most efficient bacterial isolates (F and I) in degrading CPF were listed in Table Bacillus cereus PC2 (GenBank Acc# MZ314010) and Streptomyces praecox SP1 (GenBank Acc# MZ314009) were constructed via molecular identification. Results of DNA sequences of the 16S gene of F and I bacterial isolates were as the following for F, identified as Streptomyces praecox, with a sequence of the following:GACGGCCTTCGGGTTGTAAACCTCGGGCAGCAGGGAAGAAGCGCAAGTGACGGTACCTGCAGAAGAAGCGCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGCCCCAAGCGTTGTCCGGAATTATTGGGCGTAAAGAGCTCGTAGGCGGCTTGTCACGTCGGATGTGAAAGCCCGGGGCTTAAGGGGGGGTCTGCATTCGATACGGGCTAGCTAGAGTGTGGTAGCCCAGATCGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGGTGGCGTTGGCGGATCTCTGGGCCATTACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGTTGGGAACTAGGTGTTGGCGACATTCCACGTCGTCGGTGCCGCAGCTAACGCATTAAGTTGGGGGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATThe 16S rRNA gene sequences of Bacillus cereus with a DNA sequence of the following:CAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTAGGGAAGAACAAGTGCTAGTTGAATAAGCTGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGCGCGCAGGTGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGAGACTTGAGTGCAGAAGAGGAAAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGAGATATGGAGGAACACCAGTGGCGAAGGCGACTTTCTGGTCTGTAACTGACACTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGAAGTTAACGCATTAAGCACTCCGCCTGGGGAGTAThe second bacteria (I) was identified as S. praecox strain SP1 was compared with the other 16 species of Streptomyces in GenBank. The neighbor-joining phylogenetic tree revealed high homology between S. praecox strain SP1 and S. praecox strain 7445 within 24\u00a0h as the main metabolite with greater water solubility compared to TCP. Streptomyces sp. AC5 and AC7 species, effectively biodegraded CPF insecticide. The AC5 removed over 90% of CPF (50\u00a0mg/l) after 72\u00a0h. of incubation, while the AC7 strain was less effective , I (Bacillus cereus), and A (Aspergillus terreus) removed about 80.82, 80.93, and 75.59% of the 2000\u00a0\u00b5g/l of CPF in 1\u00a0week. Following the molecular identification of these species, they were registered in the GenBank as Streptomyces praecox strain SP1 and Bacillus cereus strain PC2 with accession Nos. MZ314009 and MZ314010, respectively.Pollution of water resources with pesticides might cause serious problems. Indigenous microorganisms help lessen the adverse effects of pesticide residues through degradation. Agricultural wastewater samples were analyzed for pesticide residues, and results showed the detection of lenacil, chlorpyrifos, cypermethrin, bifenthrin, carbofuran, tolfenpyrad, oxamyl, dicloran, simetryn, sulfotep, ethofumesate, and permethrin. Chlorpyrifos insecticide was the dominant compound in wastewater samples. Indigenous bacterial and fungal species were isolated, and their ability to degrade chlorpyrifos insecticide was examined. After 1\u00a0week of incubation of isolated bacteria and fungi with CPF, results revealed efficiency % of removal ranging from 31.41 to 90.82%. Specifically, F ("} +{"text": "Salmonella entericaserovar Typhimurium. Eight unique sRNAs were selected for deletion primarily based on their genomic location and/or putative targets. Quantitative and qualitative analyses confirm one of these, sRNA1186573, is required for efficient biofilm formation inS. entericafurther highlighting the significance of sRNAs duringSalmonellastress response.Small RNAs (sRNAs) are short noncoding RNAs of ~50-200 nucleotides believed to primarily function in regulating crucial activities in bacteria during periods of cellular stress. This study examined the relevance of specific sRNAs on biofilm formation in nutrient starved Salmonella entericais rod-shaped, motile, non-spore-forming gram-negative bacilli responsible for causing gastrointestinal disease and thousands of deaths across the world every year. Salmonella, like many other bacteria, forms biofilm when subjected to stressAcinetobacter baumanii, which is a classic biofilm formerE.coliandS. enterica:bssRandcsgD Small RNA (sRNA) are non-coding RNA molecules that are less than 200 nucleotides in length and have been found to control gene expression in many regulatory circuits in bacteria, such asSalmonella entericaserovar Typhimurium, we selected eight unique sRNAs for deletion primarily based on their genomic location and/or putative targets then performed qualitative and quantitative assays of biofilm formation on each mutant and controls . After 24-h of incubation at room temperature in a nutrient deficit condition (as nutrient starvation is an inducer of biofilm formation). In agreement with Congo Red assay results, standard microplate biofilm UV absorbance assays confirmed that the ability of the sRNA1186573 deletion mutant to form biofilms during nutrient deprivation was almost entirely lost, whereas the ability of the other sRNA deletion mutants to form biofilms did not significantly differ from wild type .To examine the relevance of specific sRNAs on biofilm formation inS. enterica, further highlighting the significance of sRNAs during Salmonella stress response. Finally of note, although RT-qPCR analyses confirm that sRNA1186573 deletion does not disruptcsgDgene expression, it is tempting to speculate that this sRNA may be involved with the post-transcriptional regulation ofcsgD, as csgD protein directly contributes to curli expression inSalmonella entericaIn conclusion, our results confirm that sRNA1186573 is required for efficient biofilm formation inPre-selection of sRNAs and mutant generationSalmonella entericaserovar TyphimuriumA total of 8 sRNAs inThe primers used for generating mutants are mentioned below:sRNA924744 F: CACATTCACCGCTTACACAGGTCTGAACAAGGGGAGGCGAGTGTAGGCTGGAGCTGsRNA924744 R: AAGGCTCCAGTATATTTTTAAAGGATTTTTGGCATAATGAACATATGAATATCCTCCTsRNA1170414 F: GTAGTAATAGCGGTAGTTCCCCGGCAGTGATGGTCACTCAGTGTAGGCTGGAGCTGCsRNA1170414 R: AATGATGAGAGCTTTTAAGATGACAAGACCACCACCGGCGACATATGAATATCCTCCsRNA1186573 F: GTAATGGCTAGATTGAAAACAGTTAGTGTAGGCTGGAGCTGCTTCsRNA1186573 R: CCCCATAAAATAAAGGCACCAGAAGTACTGACAGATGTTGCATATGAATATCCTCCTTsRNA176086 F: TGAATTTGACACTGCGCACAGGGCGAsRNA176086 R: ACGACCTGCTTCTGAGGCTTTCTCTTTsRNA2594511 F: AAATAAGATCCCGGCCAGCCTGATACsRNA2594511 R: CGTGAACTGGGGAACTGGAAAGATTTsRNA3551252 F: TTTTAATATCATTAAAATCAAAAGTATAGACATTCATAGCGTGTAGGCTGGAGCTGCsRNA3551252 R: TGACTATACTTATTTGAGATACAAAAACAGCGCAAGAGTGCATATGAATATCCTCCTsRNA4130247 F: ATCTTGTGCTATTGGCAAAACCTATGGTAACTCTTTAGGTGTGTAGGCTGGAGCTGCsRNA4130247 R: TCGTCCAAGTGCAGCCCCGCACGGTGGGATAATAATCACCACATATGAATATCCTCCTsRNA4720054 F: CACAAAACTTATGGATTTATGCGTATAATCCGCGGCGCAAGTGTAGGCTGGAGCTGCsRNA4720054 R: CGTTATTGTGTCACTGTCTTACACACCGGTAAGACAGCAGACATATGAATATCCTCCThe primers used for confirming the mutants are here below:sRNA924744 F: GAATCCCCAGCAAACCAAG; sRNA924744 R: GCAGGCATAGTGATGATTTCCsRNA1170414 F: CTATGGAGATCGCGAATGGT; sRNA1170414 R: GAATGTCCGTACAGGGTGTTGsRNA1186573 F: AGGCACCAGAAGTACTGACAGA; sRNA1186573 R: ACGGCTATTTCAACCCACAGsRNA176086 F: GACATATCATATTTAAAACGCAACA; sRNA176086 R: CGCGATGTTCTGCCATAATsRNA2594511 F: TCTTCGTTGAGTCGCCTTT; sRNA2594511 R: CGTAAATAAATGCCTGGAAGGsRNA3551252 F: ACCATCCCGACAGACAA; sRNA3551252 R: TTGGAAGTGAAACCTCTGCATsRNA4130247 F: AGCCAAGATGCAAGAATAGACA; sRNA4130247 R: CCACGCTAATCACGACCAsRNA4720054 F: TTACTTACCGGAGGCGACAT; sRNA4720054 R: GAAAATTCTCCATCGCGGcsgDgene expression are as follows:The primers used to confirm that sRNA1186573 deletion does not disruptCsgD_F_qPCR: GGTCAGCGGATTACAGGGTA; CsgD_R_qPCR: TCGCGATGAGTGAGTAATGCBiofilm formation assaySalmonella Typhimurium: (i) rdar , (ii) pdar , (iii) bar , and (iv) saw et al.Both qualitative (Congo-Red agar test"} +{"text": "Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a +sense single-strand RNA virus. The virus has four major surface proteins: spike (S), envelope (E), membrane (M), and nucleocapsid (N), respectively. The constitutive proteins present a high grade of symmetry. Identifying a binding site is difficult. The virion is approximately 50\u2013200 nm in diameter. Angiotensin-converting enzyme 2 (ACE2) acts as the cell receptor for the virus. SARS-CoV-2 has an increased affinity to human ACE2 compared with the original SAR strain. Topological space, and its symmetry, is a critical component in molecular interactions. By exploring this space, a suitable ligand space can be characterized accordingly. A spike protein (S) computational model in a complex with ACE 2 was generated using silica methods. Topological spaces were probed using high computational throughput screening techniques to identify and characterize the topological space of both SARS and SARS-CoV-2 spike protein and its ligand space. In order to identify the symmetry clusters, computational analysis techniques, together with statistical analysis, were utilized. The computations are based on crystallographic protein data bank PDB-based models of constitutive proteins. Cartesian coordinates of component atoms and some cluster maps were generated and analyzed. Dihedral angles were used in order to compute a topological receptor space. This computational study uses a multimodal representation of spike protein interactions with some fragment proteins. The chemical space of the receptors suggests the relevance of the receptor as a drug target. The spike protein S of SARS and SARS-CoV-2 is analyzed and compared. The results suggest a mirror symmetry of SARS and SARS-CoV-2 spike proteins. The results show thatSARS-CoV-2 space is variable and has a distinct topology. In conclusion, surface proteins grant virion variability and symmetry in interactions with a potential complementary target . The mirror symmetry of dihedral angle clusters determines a high specificity of the receptor space. Designing an antibody against the SARS-CoV-2 spike protein S is challenging due to its nonspecific nature, mainly conferred by each spike protein monomer . The spiRecent research and protein sequences designed to fit spike protein S failed to bind. A lack of specificity is a result of high mobility and flexibility combined with additional maximum solvent exposure of the monomer domains . A mass effect should virtually resolve this lack of specificity; in other words, by increasing the mass of the desired paratope, that will interact with the epitope (S protein), an alternative unavailable from the receptor\u2013ligand interaction point of view . Judging from the antigen\u2013antibody point of view, epitope amino acid (Aa) sequences located on the antigen must fit complementarily with the Aa chain of the paratope (antigen variable (VL) and constant regions (CR)) .f) of one argument from a given domain is a polynomial function if a polynomial exists. Like any other functions, polynomial functions can be represented by a graph in H2n. Stated precisely, this is a bilinear form. This is a symmetric form for n even , in which case the signature of M is defined to be the form\u2019s signature, and an alternating form for n odd . These can be referred to uniformly as \u03b5-symmetric forms, where \u03b5 = (\u22121)n = \u00b11, respectively, for symmetric and skew-symmetric forms . These f\u03b8 is the determinant, and cos\u03b8 represents the bound angle value in \u00c5, and if cos\u03b811, cos\u03b822, cos\u03b833, and cos\u03b844 are considered zero, the determinant matrix can be written as follows \u00d7 [\u2212165 \u2212 (\u2212130)] = (\u22120.35) \u00d7 (\u221235) = 1225. The convergence of the two functions is determined via the following relationship:On the function interval \u22120.5 \u2264 x \u2264 \u22120.15, function f1 = \u221214x6) and (f1(x))/(\u2212x6) is computed: (f1(x))/\u3016\u2212x\u30176 =10 + 8/x + 6/x2 \u2212 (0.0002)/x3 \u2212 (0.0556)/x4 \u2212 (0.146)/x5 + (110.3)/x6 also (f2(x))/(\u2212x6) = 14 + 10/x \u2212 7/x2 +5/x3 + (0.0265)/x4 \u2212 (3.0432)/x5 + (165.5)/x6. Because A is a constant, the limits of the two functions are 10 and 14, respectively. While the limits have positive values , both of the functions have the following convergence domain: \u2212\u221e < x < +\u221e.The Riemann theorem shows that via permutation of a series of terms, the sum can be made equal to any number given before. In the case of functions f1(x) and f2(x), these numbers are 10 and 14. So, the functions are convergent.y-axis is described by = (x(t), \u03b8, y(t)). In Cartesian coordinates, these becomes (y(t)cos(\u03b8), y(t)sin(\u03b8), z(t) and (x(t)cos(\u03b8), x(t)sin (\u03b8), y(t)) [The surface of revolution in Euclidean space is a surface created by a rotating curve around an axis of rotation . Here, t), y(t)) ,46. ProtRamachandran plots are a tool for visualizing energetically allowed domains for backbone dihedral angles \u03c8 against \u03d5 of amino acid residues in proteins. Dihedral angle values are annular (0 degrees is the same as 360 degrees). Ramachandran plots warp from right to left and bottom to top . The \u03c9 aThe property space of both spike proteins demonstrates similar characteristics of both molecules. Solvent exposure of both molecules shares common characteristics. Spike protein SARS and SARS-CoV-2 solvent exposure are similar .Binding site identification of potential therapeutic targets is crucial in every drug design process as the expected spike protein has various binding sites. Also, many binding sites in various spike protein regions are described in the literature. An efficient vaccine can be designed by identifying the energetically and structurally favorable binding site. In the present study, the ligand\u2019s interaction with its receptor (spike protein) is a general process of applying various binding sites.Furthermore, the mirror symmetry of SARS-CoV-2 compared with SARS can potentially suggest potential ligands that can be used as vaccines. Symmetric molecules of SARS-proven ligands can be considered in such a way. Also, as seen in The design of an appropriate paratope that can lead to a COVID-19 antibody in in silico methods was used together with secondary data derived from the literature. In order to find a suitable paratope, a set of very low (VL) random sequences were used to assess the chemical space of epitope\u2013paratope interactions see also .Firstly, a target for a potential antibody was established. Spike protein S was chosen as a target due to its volume, shape, and frequency at the external membrane of COVID-19 compared to proteins E, M, and N, respectively. Computational studies and published data show that spike S protein is a valid target ,50,51,52Also, an Aa sequence was chosen as an epitope. The Aa sequence was chosen from the literature ,54.A set of 47 PDB VL chains was chosen from the literature to explore the paratope space by evaluating the epitope\u2013paratope interactions using distinct computational techniques.https://www-cohsoftware.ch.cam.ac.uk, accessed on 1 July 2023). For example, the following sequence was obtained for 1A8J using Paratome: SYEGSDF. After checking that the sequence was of the outer external region and could contact the antigen, the probabilistic contribution of each Aa was computed using Parapred, in this case yielding the following values: S 0.1649, Y 0.486472, E 0.713605, G 0.32135, S 0.469778, and F 0.0840889. In order to take into account the epitopes\u2019 first light chains, interactions with the light chains were studied. The PSOPIA online server was used [Firstly, a light chain of 47PDB structures was used. Paratopes were detected for each structure using the Paratome online engine . Resultswas used . PSOPIA was used .iFrag, a protein\u2013protein interaction prediction server, was used to compute and quantify the interaction between epitopes and paratopes . First, Spike protein monomer:AYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVNCTFEYVSFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKGNYNYLYRKPFERDIYFPLQSYGFQPTNVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTSQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSACEII monomerSTTEELAKTFLETFNYEAQELSYQSSVASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQTYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADPFGEVFNATKFPSVYAWERKKISNCVADYSVLYNSTFFSTFKCYGVSATKLNVYADSFVVKGDDVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGNYNYKYRYLRHGKLRPFERDISNVPFSPDGKPCTPPALNCYWPLNDYGFYTTTGIGYQPYRVVVLSFEIn order to characterize the VL sequences, a cluster analysis was performed using molecular descriptors. The properties, after the cluster was computed, are the following: sum of atomic polarizabilities, number of hydrogen bond acceptors, number of acidic atoms, number of aromatic atoms, number of H donor atoms, the sum of the number of H bond acceptors and donors, number of heavy atoms, information content, medium information content, number of carbon atoms, number of hydrogen atoms, number of nitrogen atoms, number of oxygen atoms, Balaban index, difference in bonded atom polarizabilities, number of rotable single bonds, number of aromatic bonds, number of bounds, number of heavy bounds, number of single bounds, atomic connectivity index, atomic valence connectivity index, number of chiral centes, number of unconstrained chiral centers, density, diameter (most considerable vertex eccentricity in a graph), SlogP, molar refractivity, topological polar surface area, vertex adjency information, volume surface area, van der Waals acceptor surface area, molecular weight, winner path, Weiner polarizability, Zagreb index, surface rugosity, total energy, angular energy, electronic energy, non-bonded energy, solvation energy, strain energy, van der Waals energy, and globularity. As seen in The following PDB models were used: 6VXXfor SARS-CoV-2spike protein, 6CVR for SARS spike protein, 5X29for SARS-CoV-2 envelope protein, and3I6Gfor SARS-CoV-2 spike protein. Furthermore, the PDB models were energetically minimized, charges corrected, and protonated at pH 7.4, a salt concentration of 0.9 nmol/L, and at 315 K. The model preparations were performed using Schrodinger2009 and MOE 2009software packages ,58. ShapIn order to reduce the amount of data and to simplify the results, monomers for each protein were prepared computationally, as stated before. The trimers and pentamer PDB structures were energetically minimized, and charges were corrected using the AMBER force field available in MOE 2009 software package. For each monomer structure, the energy was minimized, charges corrected, and structures protonated at a physiological pH. Dihedral (torsion angles) for each monomer were computed using the Chemoffice 2008 software package.In probing the chemical space, some structural information, from simple dihedral angles to complex tertiary and quaternary organizations, was obtained computationally using Schrodinger 2009 software packages. Dihedral angles were represented using a scatter plot for each monomer . While an abrupt cut-off was observed in data, and an abrupt variation in data was noted, a logarithmic scale was used. Also, as discussed in the introductory part, by normalizing the data, clusters are more easily observed . A logarhttps://www.wolframalpha.com/, accessed on 1 July 2023) interface was used to perform a cluster analysis of all dihedral angles. To compare their function domains, the function table dn/dxn (f(x)) for n = 1 \u2026 4 was calculated for all four monomers. Surface plots were computed using the radius of gyration of each logarithmic trend line. Here, r2, while exploring a trend and not a correlation between two phenomena, is not significant. A histogram was computed using dihedral angle values on 300 intervals for all three monomers. Using logarithmic trendline equations, a derivate equation was computed for all three protein monomers to represent the dynamic of dihedral angles as a function graphically.In order to expand data dimensionality, a Ramachandran plot diagram was computed for all four monomers in order to characterize the dihedral angles of the structures using the Schrodinger 2009 software package. The Wolfram Alpha software (n/dxn (\u22120.059 log(x) + 6.1779) for n = 1 \u2026 4; for M (COVID-19) protein monomer table: dn/dxn(\u22121.51011 log(x) + 6.1779) for n = 1 \u2026 4; for E (COVID-19), protein monomer table: dn/dxn(\u22120.842 log(x) + 62.544) for n = 1 \u2026 4; and for S (COVID-19) protein monomer table: dn/dxn(\u22120.443 log(x) + 9.7581) for n = 1 \u2026 4.Radar plots were used to emphasize the dihedral angles of protein monomers. Furthermore, the surface of revolution for protein S for SARS and for M, E, and S for SARS-COV-2 were computed to retrieve the 3D dimensionality of monomers\u2019 dihedral angles. Surfaces were generated using the following formulas: for S (SARS) protein monomer table: dThe dimensionality of the COVID-19 interaction space is defined by its interaction with its core receptor. The interaction space has a finite dimensionality defined quantitatively by the polynomial discriminant. Judging by its 2D dimensionality, the interaction space has a negative and positive domain through which epitope\u2013paratope interactions are defined.SARS and SARS-CoV-2 spike proteins have similar solvent exposure and mirror symmetry regarding their dihedral angle chemical space. Both proteins have a similar solvent exposure even if their dihedral angle conformational space has mirror symmetry. Mirror symmetry explains to some extent the chemical inaccessibility of the spike protein SARS-CoV-2 as a receptor and the evasive but permanent chemical bonding in which it is involved."} +{"text": "Foxc1, Foxc2, or both in mice worsen I/R\u2010induced intestinal damage by causing defects in vascular regrowth, expression of chemokine CXCL12 and Wnt activator R\u2010spondin 3 (RSPO3) in blood ECs (BECs) and LECs, respectively, and activation of Wnt signaling in ISCs. Both FOXC1 and FOXC2 directly bind to regulatory elements of the CXCL12 and RSPO3 loci in BECs and LECs, respectively. Treatment with CXCL12 and RSPO3 rescues the I/R\u2010induced intestinal damage in EC\u2010 and LEC\u2010Foxc mutant mice, respectively. This study provides evidence that FOXC1 and FOXC2 are required for intestinal regeneration by stimulating paracrine CXCL12 and Wnt signaling.Intestinal ischemia underlies several clinical conditions and can result in the loss of the intestinal mucosal barrier. Ischemia\u2010induced damage to the intestinal epithelium is repaired by stimulation of intestinal stem cells (ISCs), and paracrine signaling from the vascular niche regulates intestinal regeneration. Here, we identify FOXC1 and FOXC2 as essential regulators of paracrine signaling in intestinal regeneration after ischemia\u2013reperfusion (I/R) injury. Vascular endothelial cell (EC)\u2010 and lymphatic EC (LEC)\u2010specific deletions of The transcriptional activity of FOXC1 and FOXC2 in lymphatic and blood endothelial cells in the small intestine contributes to vascular repair and intestinal regeneration after ischemia\u2010reperfusion injury by regulating CXCL12 and R\u2010spondin3 signalling, respectively. Endothelial cells (ECs) present in the blood and lymphatic vessels are crucial participants in the vascular\u2010dependent processes that restore damaged tissue because they control the secretion of paracrine factors from both the vessels themselves and nearby cells and other nonepithelial stromal cells express Wnt ligands in the ISC niche , is a homeostatic chemokine expressed in many cell types such as stromal cells, ECs, and fibroblasts in various tissues defective repair of intestinal BECs and LECs, (ii) reduced expression of CXCL12 and RSPO3 in intestinal BECs and LECs, respectively, and (iii) decreased activation of the Wnt/\u03b2\u2010catenin pathway in ISCs. Importantly, treatment with either CXCL12 or RSPO3 partially rescues the defects in intestinal repair and regeneration associated with EC\u2010 and LEC\u2010Foxc1/c2 deficiency. Chromatin immunoprecipitation (ChIP) assays reveal that both FOXC1 and FOXC2 proteins bind to the regulatory elements of the CXCL12 and RSPO3 loci in BECs and LECs, respectively. Together, our data show a new role for FOXC1 and FOXC2 as key transcriptional regulators of paracrine signaling in the intestinal blood/lymphatic vessels during postischemic intestinal repair/regeneration, and our findings may have important implications for the treatment of ischemic bowel disease by modulating the vascular paracrine signaling pathways.In this study, we report that FOXC1 and FOXC2 in intestinal BECs and LECs contribute to vascular repair and intestinal regeneration after I/R injury by regulating the expression of paracrine signaling factors. Inducible, EC\u2010 and LEC\u2010specific, single and compound mutant mice for Foxc1 and Foxc2 mRNA expression in intestinal BECs and LECs was evaluated via quantitative PCR (qPCR) of BECs and LECs isolated from the small intestine in adult mice , which were used as a relative control , LYVE1 (LEC marker), and FOXC1 Fig\u00a0. In the Foxc2\u2010CreERT2 knock\u2010in mice by oral gavage for 5 consecutive days and subjected to the I/R injury 12\u2009day post tamoxifen treatment. FOXC2\u2010GFP+ cells were detected mainly in the LECs in sham\u2010 and I/R\u2010intestines , which is characterized by gut microbiota induced intestinal inflammation and injury and intestinal ischemia arising from derangements in the intestinal microcirculation angiogenesis have been generally unsuccessful because global single and compound Foxc1/Foxc2\u2010mutant mice die perinatally with severe cardiovascular abnormalities mice, which (after the mutation is induced) are referred to as EC\u2010Foxc\u2010DKO mice and lymphatic (CD31+LYVE1+) vessels to confirm Cre\u2010mediated recombination in intestinal BECs and LECs and their control littermates. Recovery from intestinal damage was impaired in both EC\u2010Foxc1\u2010KO and EC\u2010Foxc2\u2010KO mice 24\u2009h after I/R injury were measured under homeostasis and at an early time point (3\u2009h) after I/R injury via qPCR. No significant difference was found in the levels of these regulators between sham\u2010operated mice vs. the control mice but a significant increase vs. the sham\u2010operated EC\u2010Foxc\u2010DKO mice. Furthermore, EC\u2010specific loss of Foxc1/c2 significantly reduced the proliferative response of intestinal epithelial cells to I/R injury as assessed via immunostaining of EpCAM and BrdU by breeding conditional\u2010null Foxc1fl/fl and Foxc2fl/fl mice intestinal epithelial cells cells regrow earlier than lacteals (LECs) in the villous stroma analyses of distal jejuna from control and EC\u2010Foxc\u2010DKO mice 18.5\u2009h after I/R injury. Dimensionality reduction and clustering analysis identified 22 transcriptionally distinct cell clusters after intestinal I/R injury and human umbilical vein endothelial cells by retro\u2010orbital injection 30\u2009min before intestinal ischemia. Quantification of Chiu scores was then performed 24\u2009h after I/R injury. In control mice, the rescue of intestinal mucosal damage was not significant after RSPO3 treatment at I/R\u201024\u2009h. However, in EC\u2010Foxc\u2010DKO mice, RSPO3 treatment could partially rescue the mucosal damage and complement also contribute to intestinal I/R injury , as well as the lacteal length of EC\u2010Foxc\u2010DKO mice 24\u2009h after I/R injury and stimulating Wnt signaling in the crypts of the intestinal epithelium.jury Fig\u00a0. Given emice Fig\u00a0. Moreovement Fig\u00a0. The numISCs Fig\u00a0 and celljury Fig\u00a0. However.5\u2009h Fig\u00a0. TogetheFoxc1/Foxc2 expression is crucial for the repair of the intestinal mucosa, BECs, and LECs after I/R injury and that the EC\u2010 and LEC\u2010Foxc\u2010DKO mutations in mice impair canonical Wnt/\u03b2\u2010catenin signaling in ISCs at the crypt base. Furthermore, our scRNA\u2010seq data indicate that RSPO3 expression is attenuated in LECs and stromal cells of the EC\u2010Foxc\u2010DKO mice after intestinal I/R injury, which is at least partially attributable to impairments in intestinal regeneration because ISC activity appears to be crucially dependent on Wnt/\u03b2\u2010catenin signaling in the subepithelial cellular microenvironment in the ISC niche , the extent of Foxc downregulation in the EC\u2010 and LEC\u2010specific mutant mouse lines may not be equal. However, the degree of impairments in intestinal repair is consistently greater in the EC\u2010Foxc\u2010DKO mutant line than in the LEC\u2010Foxc\u2010DKO mutant line, suggesting that Foxc1 and Foxc2 are required in both BECs and LECs for intestinal tissue repair. Equivalent experiments were also performed with adult mice carrying EC\u2010 (and LEC\u2010) specific KO mutations of each individual Foxc gene to determine the similarities and differences between the phenotypes associated with each deletion. While our qPCR and scRNA\u2010seq analyses show that expression levels of Foxc1 in intestinal ECs are higher than those of Foxc2, the phenotypic differences between EC\u2010 (or LEC\u2010) Foxc1\u2010KO and EC\u2010 (or LEC\u2010) Foxc2\u2010KO mice are not particularly distinct. Although the reason(s) for the phenotypic similarities remains unclear, recent evidence indicates that Foxc2 is essential for the maintenance of intestinal LECs and that treatment with antibiotics to deplete gut microbiota rescues the phenotype of LEC\u2010specific Foxc2 mutants, including lymphatic dilation in the small intestine \u2010specific Foxc genes in mice impair the regulation of RSPO3 in LECs with Cdh5\u2010CreERT2;Foxc1fl/fl (EC\u2010Foxc1\u2010KO), Cdh5\u2010CreERT2;Foxc2fl/fl (EC\u2010Foxc2\u2010KO), Cdh5\u2010CreERT2;Foxc1fl/fl;Foxc2fl/fl (EC\u2010Foxc\u2010DKO), Vegfr3\u2010CreERT2;Foxc1fl/fl (LEC\u2010Foxc1\u2010KO), Vegfr3\u2010CreERT2;Foxc2fl/fl (LEC\u2010Foxc2\u2010KO), Vegfr3\u2010CreERT2;Foxc1fl/fl;Foxc2fl/fl (LEC\u2010Foxc\u2010DKO) males, respectively, as described previously , mTmG/+;Vegfr3\u2010CreERT2;Foxc1fl/fl;Foxc2fl/fl (mTmG/LEC\u2010Foxc\u2010DKO) and mTmG/+;Foxc2\u2010CreERT2 mice were generated by crossing mTmG females with EC\u2010Foxc\u2010DKO, LEC\u2010Foxc\u2010DKO and Foxc2\u2010CreERT2 males, respectively. Genotyping of mice was performed by Transnetyx Inc.et\u00a0al,\u00a0For adult mice, Tamoxifen was dissolved in corn oil (Sigma #C8267) at 40\u2009mg/ml by shaking at 37\u00b0C for 3\u20134\u2009h. 7\u20138\u2010week\u2010old male adult mice were treated with 150\u2009mg/kg Tm by oral gavage once daily for 5 consecutive days. For neonatal mice, each individual was treated with Tm by oral gavage once daily from postnatal day 1 (P1) to day 5 (P5) , followed by dehydration in 30% sucrose, and OCT embedding. Ten or 15\u2009\u03bcm cryosections were cut and immunostained with CD31 and/or LYVE1 antibody surgery as previously described by intraperitoneal (i.p.) injection 2 or 18.5\u2009h before tissue dissection (at I/R\u201024\u2009h and I/R\u201018.5\u2009h respectively).et\u00a0al,\u00a0Distal jejunum was selected for this study because this segment has the most severe mucosal damage after intestinal I/R surgery compared with other segments according to pilot experiments. Different time points were chosen for different analysis purposes. For histological analysis, transcardial perfusion was performed on the adult mice with cold PBS followed by 4% PFA after anesthesia. Distal jejunum was harvested and cut longitudinally to expose the lumen. After several washes with PBS, the intestine was postfixed in 4% PFA at 4\u00b0C for 4\u2009h (for frozen or whole\u2010mount samples) or for O/N (for paraffin\u2010embedded samples). For qPCR and western blot on whole tissue lysates of the small intestine, blood was removed by transcardial perfusion with cold PBS. Distal jejunum was harvested, opened longitudinally, and washed with cold PBS, then snap\u2010frozen in liquid nitrogen for RNA isolation and protein extraction. For the tissue collection for neonatal intestinal whole\u2010mount staining, the neonates were euthanized at P7 after Tm treatment from P1 to P5. The proximal jejuna were collected, washed with cold PBS, and fixed in 4% PFA at 4\u00b0C for 4\u2009h, then subjected to the whole\u2010mount staining protocol. Proximal jejunum was collected from neonatal mouse due to the ease of operation and similar lacteal length/blood capillary network length ratio between proximal and distal jejuna . Based on the H&E staining, Chiu Score (Chiu 3 in PBS) for 2\u2009h at 4\u00b0C, and then incubated with the indicated primary antibodies according to the manufacturer's instructions.For IHC\u2010P, 4 or 15\u2009\u03bcm paraffin sections were deparaffinized, rehydrated, subjected to antigen retrieval, permeabilized with PBST, blocked with blocking buffer containing 5% donkey serum in PBST for 30\u2009min at room temperature (RT), and incubated with indicated antibodies Table\u00a0 in blockH&E staining images were acquired using an Olympus Vanox AHBT3 Research Microscope and an Apple iPhone 12 Pro Max. Fluorescent images were acquired using a Zeiss AxioVision fluorescence microscope, a Nikon A1 Confocal Laser Microscope, or a Nikon AXR confocal microscope with the software of Zeiss AxioVision SE64 Rel. 4.9.1 or NIS\u2010Elements Viewer, respectively. Images were processed and analyzed with Adobe Photoshop, Imaris, and Fiji (ImageJ) software. Imaris imaging software was used to create videos Movies\u00a0 and EV2 et\u00a0al,\u00a0et\u00a0al,\u00a0The mouse lacteal permeability was assessed as previously described in 100\u2009\u03bcl PBS to get the CD45\u2212LYVE1+ LECs. The CD45\u2212LYVE1\u2212 cell suspension was incubated with Dynabeads labeled with CD31 antibody (BD #553369) to get the CD45\u2212LYVE1\u2212CD31+ BECs. Finally, the sorted Epis, LECs, and BECs were used for RNA isolation and qPCR analysis. For the detection of gene deletion in ECs in EC\u2010Foxc\u2010DKO mice, CD45\u2212CD31+ ECs (including BECs and LECs) were isolated by using Dynabeads labeled with CD45 antibody (depletion of CD45+ cells) followed by Dynabeads labeled with CD31 antibody (positive selection of CD45\u2212CD31+ cells).Epithelial cells (Epis), blood endothelial cells (BECs), and lymphatic endothelial cells (LECs) were isolated from the distal jejunum for further qPCR analysis as previously described (McCarthy A RNeasy Mini Kit (Qiagen #74104) was used for RNA extraction from cells. TRIzol\u2122 Reagent (Invitrogen #15596026) was used to isolate RNA from whole distal jejunum. The concentration of RNA was determined using NanoDrop\u2122 2000 Spectrophotometers (Thermo Scientific). cDNA was synthesized using an iScript reverse transcriptase kit (Bio\u2010Rad #170\u20108891). qPCR was performed on triplicates of cDNA samples by using QuantStudio\u00ae 3 Real\u2010Time PCR System (Applied Biosystems), Fast SYBR reaction mix (Applied Biosystems), and gene\u2010specific primer sets. 18S (for mouse samples) and PPIA (for human cells) were used as internal standards for mRNA expression in mouse samples. Primer sequences are provided in Table\u00a0The frozen intestinal tissue was grinned using mortar and pestle chilled with liquid nitrogen, followed by lysis in RIPA buffer containing protease inhibitors (Roche #4693116001). After centrifugation, the supernatant of the tissue lysates was collected and mixed with 5\u00d7 Protein Loading Buffer. Equal amount of total protein for each sample was loaded and run on an SDS\u2013PAGE gel. Samples were transferred to 0.45\u2009\u03bcm nitrocellulose (Invitrogen) and western blotted with the antibodies listed in Table\u00a0Foxc\u2010DKO I/R\u201018.5\u2009h. Briefly, mice were anesthetized by isoflurane. Blood was removed by cardiac perfusion with cold PBS. The distal jejunum was then dissected, washed with cold PBS, and cut into small pieces. The tissue was processed for scRNA\u2010seq upon dissociating into single\u2010cell suspension in a digestion buffer as mentioned above for 35\u2009min at 37\u00b0C, followed by filtration through a 70\u2009\u03bcm and a 40\u2009\u03bcm cell strainer. Cells were washed with washing buffer for three times and resuspended in PBS with 0.04% BSA at a concentration of 1,200\u2009cells/\u03bcl (according to 10\u00d7 Genomics Document #CG00053 Rev B) before being passed through a 30\u2009\u03bcm MACS SmartStrainer. The cell viability was tested by using the Cellometer Auto 2000 Cell Viability Counter . The cell sample was processed for scRNA\u2010seq only when the cell viability was more than 70%. Average cell viability for samples was determined to be 80.96%.For single\u2010cell RNA sequencing, mouse distal jejunums were collected 18.5\u2009h after intestinal I/R surgery. Two mice were used for each group: control I/R\u201018.5\u2009h and EC\u2010Single\u2010cell 3\u2032 gene expression libraries were constructed by using the Chromium Next GEM Single\u2010Cell 3' Reagent Kits v3.1 according to the manufacturer's manual CG000204 Rev D. The single\u2010cell libraries were assessed for quality and then run by using paired\u2010end 50\u2009bp sequencing on the Illumina HiSeq 4000 platform . Ten thousand cells were targeted for each sample with a sequencing depth of 20,000 read pairs per cell.Foxc\u2010DKO mice using CellRanger. The matrix files were then utilized for data processing and downstream analysis using the BIOMEX browser\u2010based software platform and its incorporated packages developed in R using the CellRanger toolkit . Gene expression matrices were then generated from both control and EC\u2010FindClusters function in Seurat . Marker set analysis was then performed in BIOMEX on highly variable genes to identify the top 10 gene markers expressed in each initial cluster using a similar methodology described previously and previously reported the data package were then auto\u2010scaled and summarized by principal component analysis (PCA), followed by visualization using Uniform Manifold Approximation and Projection ) to reduce the data into\u00a0a two\u2010dimensional space. Graph\u2010based clustering was then performed in BIOMEX to cluster cells according to their respective gene expression profile using a methodology similar to the Plotly software was used for UMAP and volcano plot visualization.BIOMEX implementation of the et\u00a0al,\u00a0https://www.ebi.ac.uk/gxa/sc/home) and was exported to check Lgr5 expression in mouse small intestinal ECs.The scRNA\u2010seq data for mouse small intestinal ECs from the publication entitled \u201cSingle\u2010Cell Transcriptome Atlas of Murine Endothelial Cells\u201d . Putative sites in the human genome were then searched against the Genome Reference Consortium Mouse Build 38 (mm10) genome using the Evolutionary Conserved Region (ECR) Browser (https://ecrbrowser.dcode.org) and rVista 2.0 tools to identify conserved and aligned putative binding sites between mouse and human sequences. Conserved and aligned putative FOX\u2010binding site sequences are underlined and bolded within the human RSPO3 and CXCL12 ECRs shown below:Putative FOX\u2010binding sites in the TATTTACAAAACGTCTTGGAATGAGAATGAGCTGCTTGTGGTTCCTGTGGCTGATTCAGGGATGGTTTCCTACAGGCAGAGGATGCTGGTCAACCGAATGACCTCTCTGTAACTAACCCGTGCACCCCTGTGGTAAGGCTGTTTGGTCTTATAGGTACCTCTTCTAACTAAGCTTGGAGGGATTTGTTTTTGTGGTAAAGAACTTAGTAATAACCAAACGTCACTGTAAAGACAGATTTAATAATGTTAAGGTCCATCAGAGCCTACTCCTTCTACTACCAACAAGAGAAGCCAGAAATACACTGGGATGCCTTTAGATTCCTGTGCATCAATCTTTCTTTCTCTAAGGATTATGGTCTTGAAATGGATAATGATAGATTCCAAACACATGGAAATCTCTTGCCCCTTTTACTTTTTAGGATCTTTGCAAGCTTACAATATGTACACGTTTTCTGTAAGTCACCAATGCTGAGTTACTGGCATGAAAAATGACCCTGTTACTTGGAAAGTAGTTTCACTTACAAGTCCCCCAGGCCCTGTAATGTCTAAACCTCCTGTGCCACTTTATGTGACTACCCCGCCCCCACAGAGGAGCATGCACAGGAAAAGCAGACTTCCCTTCCCCCACACATTTCCTTAGTTATAAATATAGATTTATGAGATACTTTAATGTTCTAAAACAAATGAAAACCACCCAAGAGGAGCCTCACCAAACCTGAGGTTGTCCAGATTGCATTGACTAAGATTAAGTAAAAGATCATTCATCTCCAGAGGTCATGCAATTAATCTCAGAGTGGGAGTTAAAGCAATGACTAAGCAGAAAAGGAAGCCAAATACAAGCTCGTAACAAAAGGTGCTGGGGCTCCAACATCAAGGAACTTGTTATTTCCTTTTTATTTATTTATTTTTTTTTAATAGACCTAAAACACTCATTCCTTACTACTGGTTTCTTTGGGTCCTAAAATTCCACTTGGTTAGGTCAGCTATTTTCCATGACTATTTTTGATACGGTCAAACAAATACAAAGAATAAGCTTTTAAAAAACAATACCAGTAAATGGGGACACATTATATTGAATAAGGGTATTGTTAGCCAAATTCTAAGATTCATCTTAAATTGTTTTCTTATAAGAATTGTGTATTTACCATTTTAAAAATCACTATTATTTTAAAACACTTAGAAAGTGAACATTTGAAAATGATGTGCCTTTGGATGCTCTGTAATGTTAAGCAGATCCAGACATAAAGACAAAAGTAAATTCCAGAGTATTTTTGTAGCCATGGAATCACCATAAAAAGGGGTTTTTGACCCCAATGTTACCGTAACATTGTCTTCAGCATTTCATATTTAATTACAGTAGATTACTCACCAATATATGTTTACTTCTCTGCATTTAATGAGCAGTTGTTAACATGACTGAAACCATCTGATGATTTTTACCAAATGGAAAAATCTGCCTACAGGGGCAATAAAATAAATATTCAGAATAGAGAGAGGCAGTCATAAAAGACATTACCCGGTTGTAAACGGAGGCGGGTGGTGGTGATCTATTACCCCTGCCTCGGCAGCTTTCAACAGAGTTCTGGAATTCCAGGAGGGGCCCTGACCCAAGGCAATATTTACTTCTGCGGCTTCTTCATCAGGTCAGCATGGGTATAATTCTGTCTACCAGTTGACTGGAGCTGAGGTTTCGAGCAGGAAGTGCAAACCCTGAGTGCTTATAACTCAGGAGGAGTGAGGCACCCCTTCCCAGAGTATGCCAAGAAAAGCACATTGTACTGTCCTGGCTGCAGGGGTGAGGCCCCTGCACACCCAGGCCATTATCAGCTTTGTGCCCTGGCCAACAGCGCCTCTGGTTGGTGCATTTGTCAGCTGTATTTTACCCTAGAGCTCTGGGAGGCTCATCCTTTTTTGGTATACCACCACGTGGAGAGAGCAGAGTTTTAATAGTGTGGCTGCATCAAAACCTTCACCTTTCTCTGCTGAAGGAATGGCCTTCTCTTATGGGCAGGGAGGGTTTCCTAGGGAAAGCCCACCCAGGCAGGAGATGAGGAGAGCAGCATCTGAGCACACTTCATCCCACAGTGCCCATCCCATGAGTATCCTCCATAAATTACAAAGAAAGAAAAAAAATAGGGAAAAAACAAACCTTTATTTCTCGTAAATAAATTCCCACATACAGTAGGACGTTTATACCATGAAACAATTAGCATTTTATTGCTAGTGCATATAATGTCACATTTGATACAATTTTAGTACAAGTGAAAAAATACACTGTGGCTAACATTGAAAAGCTGCAATCACATTTATATATCATATATATTTCTTTACAAATTGCCAGTAGTTTGAGATAATAGAGAAGTATAAACTACTGACATTCATATGGCTCCACTTCAAATATATGAATTGTTCGACTATAAATATATTTTGAAATACATTTGTTTTCTAAAGAAACGTAAAAAAAAATGTGCACAAAAATATATATAAAAAAATGCCTTGCAAAAAGTTACAAATACCACCAGGACCTTCTGTGGATCGCATTTATGCATGGAAATGTCACCTTGCCAACAGTTCTGATTGGAACCTGAAACCCTGCTGTGGCTTCAGGAGGGGGTAGTGGCAAGATGATGGTTTATTCACTGATTTTTTCGCTTCTGATTTCGGAAACCTCAGAGTTTGTTAGTGCCTC CATGGCATACATAGGCTGGGTTAAAAAAAAAAAAAAAAGATCCAAAAACTTGAGCTGCAGATCTAATCTGCTCGTGAGAAAAGCCCATACACTGTCACACATGGGCTGTGAGAAGGGGTCTCAGACACCTGACTGCAGGCAGGCTTAACTATATAAACCAGAAACGTCTATAAGCTCCATCACTAACAACTAATGAATTTTATTTCAGRspo3 and Cxcl12 ECRs corresponding to the human RSPO3 and CXCL12 ECRs are shown below:As comparison, in mouse, the conserved and aligned putative FOX\u2010binding sites (underlined and bolded) within the mouse sequences of ATAAATAAACTGAGGAAATGTGTGGGGGAAGGGAGGGCTGCTTTTCCTGTGCATGTTCTTCTGTAGGGTGGGGCAGTCCTATAAAGTGGGTGAGGAGGGTTTGCCATTATGGGACCTGAGGGATTTGTACCAGAAACTCCTTTCCAAGGAAGGTCATTTTTTATGACAGGGGACTAGGGGTGGGGGAGTGCTTTAGACATGGACACTATTAATCTGTATTTGCAATGATGTCTGATAATTAAGCTCTTTATTGCAGAAACAAAGCCCTGCAGTCTTACGATGCCACACCAAGAAGCAGAGAACAGGAGGAGAACCATGGTCTGGGAAATAAACTTCAAAAGTCCCATATTAATAATTTTTCTCCTCCAGCAAGGCCACACCTAAGGGCTCCATCTCTCCCCCAGATATTATCACCTACTAGCTAGGGATCAAGTATTCAAAACATGAGCCGGGAAGAATTATTTCATATTTCAAGTATAATATTAGTCTGTAATCACAGTGATAATTGGTTATTAAGCTCTTCATTGGAACAACAAAGCCCTCCAGGCACAGTCTGAAATGATACCCATAACAAAGATCCTTCCTGTTGGGGTATAGGAGTTAGCTCCATAGAGGTCATTCAGTTGACCCGCTTCCCTGGCCTGCAGGAAACCATCCCTGAATAAGCCACAGGAACCACAAGCAGCTCGCTCTCATTCCAAGATGTTTCTATTTATTATATTAGTGAATACTATACTACAATTAGATATGAAATGCTGAAGACAATGTTAAGGTGAGAGTGGGTTCACAAACCTCTATATTGGTAGCTTCAGAATTGCAAAAACATTTGTGGAATATTGGTTTTGTCTCTATGCCTGGATCTGTTTAACATAACATAGTATCCACAGCACACTCTTTCAGACAGGGACTTTGTGAATCCTCTAAAACAACCATCATTTCTAAATGCAACAATAAGGAGTATTTTAGGTTTATTCAGGAGGAGGAAAAAAAAAAGGAAATAGCTCCTTGATGTTGGAGGCCAGGCCCCTCTTGTTATGGGCTTGCATGCTGGCTTTCTTTTCTGCTTAATCATCGCCTGAACTCCCACTCTAAGATTGCTTACATAACCCTGGGAGATGAATGATCTTCTACTTAATTTCAGCCAGTGCAATCTGGACAACCTCAGGTTGGATTGGTGAGGCTCCTCTTGGGTGGTTTTCACTTGCTTTAAGAACATTAAAATATCTCATAAATCTAGTAAATATTGCCTTGGGTCAGATCCTCTCCTGGAATCCCAGAACTGTTGAAAGCTGCCAAGGCAGGGGTAATAGATCACCACGACCCGCCTCTGTTTACAACCGGGTAATGCCTTTTACGACCGCCTCTCTCTGTCCTGAATATTTATTTTATTGCCCCTGTAGGGAGATTTTTCCATTTGGTAAAAATCATCAGATGGTTTCAGTCATGTTAACAACTGTTCATTAAACGCAGAGGGTAAACAGAGATGGAAAGGTTTGTTTCTTTCCTTTTTCTCTATTTATTTTTAACTTATGGAGAAAACTCAGGGAGTGAACCCAGGGAGGGAGGCATGTTCAGATACCAGCCATCAGTACCGTCTGCCCAAGTACAAACAAGCTGTATATCCCACTGACAATGGCCTGGTTATGTGGGACCCTCACCTTGACAGCCAGGACGGTACCATGAGCTTTTCTTGGCACACCCTGGAAAGGGTGCCTTACTCTTCCTGAGTTATGAGTGCTTAGGGCTCTGGCTTCCTGCTCAAAACCTCAACTCCAGTCAACTGGTGGACAGAATTATACCCACAATGACCTGCCGAAAAAGCCACAGAATATTTGTAGTCAATTCATATATTTGAAGTGGAGCCATAGTAATGCCAGTAGATATCTCTATGATCTTGAGCTACTGGCAACTTGTAAAGAAATATATATGACATATAAATGTATTGTAGCTTTCCGGTGTCAGCCACGGTGTATTTTTCCACTTGGAATGAAATTGTATCAACTGTGACATTATATGCACTAGCAATAAAATGCTAATTGTTTCATGCTGTAAACCTCCTACCGTATGTGGGAATTTATTTACCTGAAATAAAATCTACTAGTTGTTAGATGGAGTGCACATACATTTCTGAAGATGGAGAAAAACAGGTGTGCCTGCTGATCAGGTGCTGTGGGCTGAAGCCACAGTGGGGATTCTGGGTTCCAATCAGAAATGGAGACAAGATAAAACTTGCATACATTCTTATGATCACAGACGGCCCTGGTGGTTTTTGGTAACTATTTACAAGGCATTTTTTTACATATATTTTTGTGCACTTTTTATGTTTCTTTGGAAGACAAATGTATTTCAGAATAet\u00a0al,\u00a0CXCL12 promoters according to the prediction of JASPAR, as well as the binding sites at the ECRs of CXCL12 and RSPO3 mentioned above. The ChIP primers are listed below:The ChIP experiment was performed as previously described and LPS (5\u2009mg/kg) to perturb the normal intestinal colonization process; (ii) gavage with formula every 3\u2009h ; and (iii) exposure to brief episodes of hypoxia (60\u2009s in 100% N2) followed immediately by cold stress (10\u2009min at 4\u00b0C) twice daily. This protocol induces intestinal injuries ranging from epithelial injury to transmural necrosis resembling human NEC, which typically develop after 36\u2009h and has been widely used to study NEC pathogenesis per sample were analyzed for the fluorescent intensity (FI) of \u03b2\u2010catenin within the ISC by ImageJ. For quantification of FI of FOXC1 and FOXC2 in intestinal BECs and LECs, about 40\u2009~\u200950 cells were quantified by ImageJ. For analysis of BEC and LEC proliferation and apoptosis, confocal Z\u2010stacks were acquired using a 20\u00d7 objective from about 4\u2009~\u20098 different fields of 15\u2009\u03bcm paraffin sections for each sample. Area of blood vessels (CD31+LYVE1\u2212) and lymphatic vessels (CD31+LYVE1+) were measured using ImageJ software. Then, the number of BrdU+ or TUNEL+ cells per 0.1\u2009mm2 vessel areas was calculated and compared between groups. Measurements for the length of blood capillaries and lacteals were performed as previously described package . All experimental protocols and procedures used in this study were approved by the Institutional Animal Care and Use Committee (IACUC) at Northwestern University.Can Tan: Conceptualization; data curation; formal analysis; validation; investigation; visualization; methodology; writing \u2013 original draft; writing \u2013 review and editing. Pieter R Norden: Data curation; formal analysis; visualization; writing \u2013 original draft. Wei Yu: Data curation; formal analysis; investigation; visualization; methodology. Ting Liu: Data curation; investigation; methodology. Naoto Ujiie: Data curation; formal analysis; investigation. Sun Kyong Lee: Investigation. Xiaocai Yan: Investigation. Yaryna Dyakiv: Investigation. Kazushi Aoto: Resources. Sagrario Ortega: Resources. Isabelle G De Plaen: Resources; methodology. Venkatesh Sampath: Resources; funding acquisition. Tsutomu Kume: Conceptualization; supervision; funding acquisition; investigation; writing \u2013 original draft; project administration; writing \u2013 review and editing.The authors declare that they have no conflict of interest.AppendixClick here for additional data file.Expanded View Figures PDFClick here for additional data file.Movie EV1Click here for additional data file.Movie EV2Click here for additional data file.Source Data for Expanded View and AppendixClick here for additional data file.PDF+Click here for additional data file.Source Data for Figure 1Click here for additional data file.Source Data for Figure 2Click here for additional data file.Source Data for Figure 3Click here for additional data file.Source Data for Figure 4Click here for additional data file.Source Data for Figure 5Click here for additional data file.Source Data for Figure 6Click here for additional data file.Source Data for Figure 7Click here for additional data file.Source Data for Figure 8Click here for additional data file.Source Data for Figure 9Click here for additional data file.Source Data for Figure 10Click here for additional data file."} +{"text": "A critical step in the removal of polyubiquitinated proteins from macromolecular complexes and membranes for subsequent proteasomal degradation is the unfolding of an ubiquitin moiety by the cofactor Ufd1/Npl4 (UN) and its insertion into the Cdc48 ATPase for mechanical translocation. Here, we present a stepwise protocol for the assembly and purification of Lys48-linked ubiquitin chains that are fluorophore labeled at specific ubiquitin moieties and allow monitoring polyubiquitin engagement by the Cdc48-UN complex in a FRET-based assay.For complete details on the use and execution of this protocol, please refer to Williams et\u00a0al. (2023). \u2022Linkage-specific enzymatic synthesis of ubiquitin chains\u2022Ubiquitin labeling with fluorescent dyes\u2022Positional attachment of fluorescent dyes to ubiquitin moieties in polyubiquitin chains\u2022FRET-based assay for ubiquitin unfolding by Ufd1/Npl4 and insertion into Cdc48 Publisher\u2019s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. A critical step in the removal of polyubiquitinated proteins from macromolecular complexes and membranes for subsequent proteasomal degradation is the unfolding of an ubiquitin moiety by the cofactor Ufd1/Npl4 (UN) and its insertion into the Cdc48 ATPase for mechanical translocation. Here, we present a stepwise protocol for the assembly and purification of Lys48-linked ubiquitin chains that are fluorophore labeled at specific ubiquitin moieties and allow monitoring polyubiquitin engagement by the Cdc48-UN complex in a FRET-based assay. We also describe how chain variants containing a substrate protein or a Met1-linked ubiquitin on the C-terminus of the most proximal ubiquitin are constructed. We used these specifically Cy3-labeled chains together with Cdc48-Ufd1/Npl4 (UN) complexes that had a Cy5 acceptor fluorophore conjugated at a position in the central processing channel of Cdc48 to monitor polyubiquitin-chain engagement in a FRET-based assay. Our studies revealed that Cdc48-UN unfolds and engages ubiquitins in a position- and linkage-specific manner.The protocol below describes the assembly of Lys-48-linked polyubiquitin chains containing a Cy3-donor fluorophore on the N-terminus of the most proximal ubiquitin competent cells with the expression plasmids for tag-less wild-type S. cerevisiae ubiquitin or ubiquitin with a Met-Cys extension N-terminal to the native Met1 (MCM-Ub) by heat shock at 42\u00b0C in a water bath for 45 s. Recover cells for 1\u00a0h at 37\u00b0C in SOC medium.2.Prepare 50\u00a0mL starter cultures in dYT with the appropriate antibiotic and grow 14\u00a0h at 37\u00b0C.3.Inoculate 1\u00a0L of Terrific Broth containing kanamycin with 8\u00a0mL of starter culture and grow at 37\u00b0C while shaking at 180\u00a0rpm until OD600\u00a0= 1.0.4.Induce protein production for \u223c18\u00a0h at 18\u00b0C with 1\u00a0mM IPTG .5.Harvest cells by centrifugation at 3,500 rcf for 30\u00a0min at 18\u00b0C\u201320\u00b0C and resuspend the resulting pellets in NiA buffer and clarify the lysate by centrifugation at 27,000 rcf for 30\u00a0min at 4\u00b0C.7.Add glacial acetic acid to the supernatant until a pH of 4.5 is reached, as monitored by pH paper.8.Stir the solution for 30\u00a0min at 18\u00b0C\u201320\u00b0C to precipitate proteins other than ubiquitin.9.Clarify the solution by centrifugation at 27,000 rcf for 30\u00a0min at 4\u00b0C.Note: Soluble ubiquitin will be found in the supernatant.10.Dialyze the supernatant against 4\u00a0L of cation buffer A that has been equilibrated with cation buffer A. Elute ubiquitin from the column with a 0\u00a0mM\u2013350\u00a0mM NaCl gradient over 20 column volumes using cation buffer A and cation buffer B on a Cytiva \u00c4KTA Pure FPLC or equivalent equilibrated in GF buffer and cysteine-ubiquitin (MCM-Ub-His) were expressed and cells lysed using the same steps 1\u20136 as above.C-terminally His14.Following lysis, supernatants were incubated with Ni-NTA beads and batch bound for 30\u00a0min.15.Beads were washed with NiA buffer, and proteins were eluted with NiA buffer plus 350\u00a0mM imidazole.16.Eluates were concentrated with a 3\u00a0kDa Amicon spin filter and subjected to size-exclusion chromatography using a Superdex 75 16/600 column equilibrated in GF buffer.CRITICAL: TCEP at 1\u00a0mM was included in all purification steps above for MCM-Ub-His to keep the cysteine reduced for subsequent labeling with Cy3-maleimide.Timing: 3\u00a0days17.Ube1, the E2-E3 chimera gp78RING-Ube2g2, and PreScission protease were expressed and cells lysed using the same steps 1\u20136 as above for Ub and MCM-Ub.18.Following lysis, supernatants were incubated with Ni-NTA beads and batch bound for 30\u00a0min.19.Beads were washed with NiA buffer, and proteins were eluted with NiA buffer plus 350\u00a0mM imidazole.20.6-tag. The high-imidazole buffer was exchanged for NiA buffer by repeated concentration and dilution, and a subtractive step with Ni-NTA beads was used to remove uncleaved gp78RING-Ube2g2 and His-tagged TEV protease.The eluate for gp78RING-Ube2g2 was treated with TEV protease (at a TEV-to-E2-E3 w/w ratio of 1:200) for 14\u201316\u00a0h at 4\u00b0C to cleave the N-terminal His21.The flow-through containing gp78RING-Ube2g2 as well as the Ube1 and PreScission elutions from Ni-NTA beads were concentrated with spin filters and subjected to size-exclusion chromatography using a Superdex 75 16/600 (gp78RING-Ube2g2 and PreScission) or a Superdex 200 16/600 (Ube1) column equilibrated in GF buffer.Timing: 2\u20133 h22.MCM-Ub and MCM-Ub-His at 50\u00a0\u03bcM in GF buffer were incubated with 1\u00a0mM TCEP for 5\u00a0min to ensure cysteines are reduced for labeling.23.Sulfo-Cy3-maleimide was added at a 10-fold molar excess (500\u00a0\u03bcM) and labeling occurred for 30\u00a0min at 18\u00b0C\u201320\u00b0C. See 24.A 1\u00a0M stock of dithiothreitol (DTT) was added at 5\u00a0mM to quench unreacted maleimide dye, and the Cy3-labeled proteins were subjected to size-exclusion chromatography with a Superdex 75 increase 10/300 column equilibrated in GF buffer. 25.Cy3Ub and Cy3Ub-His preparations was calculated by comparing the total protein concentration determined by a Bradford assay and the concentration of Cy3 dye determined by absorbance at the excitation wavelength maximum (555\u00a0nm). Labeling efficiency for CRITICAL: Ensure that purified protein stocks are free of thiol-containing contaminants that can react with maleimide. Quenching the labeling reaction with DTT is important, as unreacted Cy3-maleimide can damage chromatography columns.Ub: MQIFVKTLTGKTITLEVESSDTIDNVKSKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKSTLHLVLRLRGG.MCMUb:MCMQIFVKTLTGKTITLEVESSDTIDNVKSKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGG.MCMUbHis:MCMQIFVKTLTGKTITLEVESSDTIDNVKSKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGGLEVLFQGPHHHHHH.D602AzF:Cdc48MGSSHHHHHHSQDPLEVLFQGPMGEEHKPLLDASGVDPREEDKTATAILRRKKKDNMLLVDDAINDDNSVIAINSNTMDKLELFRGDTVLVKGKKRKDTVLIVLIDDELEDGACRINRVVRNNLRIRLGDLVTIHPCPDIKYATRISVLPIADTIEGITGNLFDVFLKPYFVEAYRPVRKGDHFVVRGGMRQVEFKVVDVEPEEYAVVAQDTIIHWEGEPINREDEENNMNEVGYDDIGGCRKQMAQIREMVELPLRHPQLFKAIGIKPPRGVLMYGPPGTGKTLMARAVANETGAFFFLINGPEVMSKMAGESESNLRKAFEEAEKNAPAIIFIDEIDSIAPKRDKTNGEVERRVVSQLLTLMDGMKARSNVVVIAATNRPNSIDPALRRFGRFDREVDIGIPDATGRLEVLRIHTKNMKLADDVDLEALAAETHGYVGADIASLCSEAAMQQIREKMDLIDLDEDEIDAEVLDSLGVTMDNFRFALGNSNPSALRETVVESVNVTWDDVGGLDEIKEELKETVEYPVLHPDQYTKFGLSPSKGVLFYGPPGTGKTLLAKAVATEVSANFISVKGPELLSMWYGESESNIRDIFDKARAAAPTVVFLDELDSIAKARGGSLG\u2217AGGASDRVVNQLLTEMDGMNAKKNVFVIGATNRPDQIDPAILRPGRLDQLIYVPLPDENARLSILNAQLRKTPLEPGLELTAIAKATQGFSGADLLYIVQRAAKYAIKDSIEAHRQHEAEKEVKVEGEDVEMTDEGAKAEQEPEVDPVPYITKEHFAEAMKTAKRSVSDAELRRYEAYSQQMKASRGQFSNFNFNDAPLGTTATDNANSNNSAPSGAGAAFGSNAEEDDDLYSGSGSGSGSGSGLNDIFEAQKIEWHE.6-TEV-gp78RING-Ube2g2:HisMGSSHHHHHHDYDIPTTENLYFQGHMEARFAVATPEELAVNNDDCAICWDSMQAARKLPCGHLFHNSCLRSWLEQDTSCPTCRMSLNIADNNRVREEGTGSHMAGTALKRLMAEYKQLTLNPPEGIVAGPMNEENFFEWEALIMGPEDTCFEFGVFPAILSFPLDYPLSPPKMRFTCEMFHPNIYPDGRVCISILHAPGDDPMGYESSAERWSPVQSVEKILLSVVSMLAEPNDESGANVDASKMWRDDREQFYKIAKQIVQKSLGL.Timing: 2\u20133 hCy3Ub-Ubn). The proximal ubiquitin is the ubiquitin closest to the substrate and in case of unanchored chains has a free C-terminal glycine, while the distal ubiquitin extends away from the substrate and represents the last moiety added to a ubiquitin chain . For studies with Cdc48-Ufd1/Npl4, we aimed to generate heterogenous chains centered around Ub5 in length. The C-terminal His-tag on unlabeled MCM-Ub-His serves the same purpose as in the major step above. Only unlabeled Ub will be present at the most proximal position in a chain. Formation of Ub-Cy3Ub-Ubn, however,\u00a0requires two steps. In this first step, limiting Cy3Ub is added to MCM-Ub-His to form Ub-Cy3Ub\u00a0and small amounts of Ub-Cy3Ub-Cy3Ub. After a Ni-NTA purification step, Ub-Cy3Ub is mixed with unlabeled Ub to form Ub-Cy3Ub-Ubn.8.Cy3Ub, 1\u00a0\u03bcM E1 and 20\u00a0\u03bcM of the E2-E3 chimera, gp78RING-Ube2g2, in ubiquitination buffer.Create a mixture of 50\u00a0\u03bcM Ub-His, 5\u00a0\u03bcM 9.Cy3Ub and unreacted Ub-His. Initiate elongation by adding ATP to 10\u00a0mM. Ideally, the major products will be Ub-CRITICAL: The goal of this part of the protocol is to add limiting amounts of Cy3Ub to the ubiquitination reaction such that the major product is elongation of Ub-His by a single addition of Cy3-labeled ubiquitin (to form Ub-Cy3Ub). Conditions that lead to elongation of Ub-His by multiple additions of Cy3-labeled ubiquitin Cy3Ub (forming Ub-Cy3Ubn) should be avoided. Small-scale test reactions can be performed using different molar ratios of Ub-His to Cy3Ub to determine optimal ubiquitination reaction conditions in assay buffer with and without 4\u00a0\u03bcM UN in assay buffer supplemented with 1\u00a0mg/mL BSA and 1\u00a0mM ATP\u03b3S. These are the 2X Cy5Cdc48 and Cy5Cdc48-UN samples.Create samples of 4\u00a0\u03bcM 17.Cy5Cdc48 or Cy5Cdc48-UN sample. Incubate at 30\u00b0C in a thermocycler. The final concentrations are 0.2\u00a0\u03bcM Cy3Ub-Ubn or Ub-Cy3Ub-Ubn, 2\u00a0\u03bcM Cy5Cdc48, 0\u00a0\u03bcM or 2\u00a0\u03bcM UN, and 0.5\u00a0mg/mL BSA.Create mixtures consisting of 6\u00a0\u03bcL of the 2X polyubiquitin sample and 6\u00a0\u03bcL of the 2X Note: Pore-insertion kinetics are rapid (\u223c3 s)ref.18.Transfer 10\u00a0\u03bcL of each mixture into wells of a 384-well black plate pre-heated at 30\u00b0C and load into a microplate reader (BMG Labtech CLARIOstar Plus) equipped to measure fluorescence emission spectra of Cy3 and Cy5 following excitation at the absorbance maximum for Cy3.19.Incubate at 30\u00b0C and scan wells for fluorescence emission between 525\u00a0nm and 725\u00a0nm using an excitation wavelength of 480\u00a0nm CRITICAL: Since both Cy3Ub-Ubn and Ub-Cy3Ub-Ubn contain contaminating unlabeled polyubiquitin chains, pore-insertion experiments are performed with a 10-fold excess of Cy5Cdc48-UN to avoid any competition between labeled and unlabeled chains. However, these conditions lead to technical challenges due to some extent of direct Cy5-acceptor excitation when exciting the Cy3 donor, which is further exacerbated by the fact that Cdc48 homohexamers can be modified with multiple Cy5 fluorophores depending on the labeling conditions and efficiency. If there is excessive direct excitation of Cy5, the Cy5 emission peak will dominate the spectrum and, due to spectral overlap, introduce some background fluorescence in the region of the Cy3 emission peak as well. To minimize Cy5 fluorescence emission resulting from direct excitation, experiments should be performed with an Cy3-donor excitation wavelength of 480\u00a0nm or lower.20.Cy5Cdc48 and UN are synthesized in a single step in which unlabeled ubiquitin is added progressively to Cy3-labeled ubiquitin with a C-terminal His-tag in the presence of ubiquitination machinery. The C-terminal His-tag on the fluorophore-labeled species is critical, because it restricts the labeled ubiquitin to the first position in the chain by preventing conjugation of this moiety to itself or to other ubiquitins during the course of the reaction. A tight distribution of polyubiquitin chain lengths is expected are synthesized in two steps. In the first step, a limiting amount of Cy3-labeled ubiquitin is added to unlabeled ubiquitin whose C-terminus is blocked with a His6-tag to yield a ubiquitin dimer with the labeled species in the second position (Ub-Cy3Ub). The molar ratio of these species should be optimized such that the single addition of Cy3-labeled ubiquitin to unlabeled ubiquitin is the major product that can react with maleimide conjugated fluorophores. See Perform a dialysis or desalting step with a 7K MWCO spin desalting column to exchange for buffer compatible with maleimide labeling.Following conjugation, my cysteine-ubiquitin sample did not elute from the size-exclusion chromatography column or eluted in the void peak. See Attachment of hydrophobic fluorophores to ubiquitin can greatly alter its properties and lead to aggregation or adsorption to filter membranes. We have achieved the best results with sulfonated fluorophores that are more hydrophilic and water soluble. The stability of the fluorophore-conjugated ubiquitin can also depend on the labeling position. Ubiquitin labeled at an N-terminal cysteine may be more well-behaved than ubiquitin labeled at an engineered internal cysteine.My fluorophore labeling efficiency is low. See Ensure that the cysteine-containing ubiquitin sample is in a buffer free of thiol-containing reagents. Oxidation of cysteine during the purification steps prior to conjugation will decrease the labeling efficiency. Include a reducing agent throughout the purification to keep cysteines reduced.Cy3Ub-His, a diffuse distribution of chains was formed. See During the first round of chain extension on Decrease the amount of unlabeled ubiquitin in the reaction. If the concentration of unlabeled ubiquitin is too high, the processive E2-E3 ligase gp78RING-Ube2g2 will form a broad length distribution and distinct bands will not be visible on the gel.Cy3Ub is a ubiquitin trimer (Ub-Cy3Ub-Cy3Ub) or longer chain (Ub-Cy3Ub-Cy3Ubn). See The major product of the elongation of Ub-His with Cy3Ub to Ub-His.As shown in the example gel A, the maThe Cy3 fluorescence emission peak is masked by the Cy5 fluorescence emission peak originating from direct excitation of excess Cy5 fluorophores. See Cy5Cdc48 could be used in the assay to decrease the size of the Cy5 emission peak, but doing so may compromise the extent of donor quenching if the motor is no longer at concentrations that are saturating for polyubiquitin-chain binding. Another potential solution is to decrease the number of Cy5 fluorophores conjugated to Cdc48 hexamers by performing labeling reactions with lower concentrations of Cy5-maleimide.A lower concentration of a.martin@berkeley.edu).Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Andreas Martin (This study did not generate new unique reagents."} +{"text": "AbstractPageBreakCatharylla Zeller, 1863 is redescribed. Catharylla contiguella Zeller, 1872, C. interrupta Zeller, 1866 and Myelois sericina Zeller, 1881, included by Munroe (1995) in Catharylla, are moved to Argyria H\u00fcbner. Catharylla paulella Schaus, 1922 and C. tenellus are redescribed. Six new species are described by L\u00e9ger and Landry: C. bijuga, C. chelicerata, C. coronata, C. gigantea, C. mayrabonillae and C. serrabonita. The phylogenetic relationships were investigated using morphological as well as molecular data . The median and subterminal transverse lines of the forewing as well as the short anterior and posterior apophyses of the female genitalia are characteristic of the genus. The monophyly of Catharylla was recovered in all phylogenetic analyses of the molecular and the combined datasets, with three morphological apomorphies highlighted. Phylogenetic analyses of the morphology of the two sexes recovered three separate species groups within Catharylla: the chelicerata, the mayrabonillae, and the tenellus species groups. The possible position of Micrelephas Schaus, 1922 as sister to Catharylla, based on both morphological and molecular data, and the status of tribe Argyriini are discussed. The biogeographical data indicate that the chelicerata species group is restricted to the Guyanas and the Amazonian regions whereas the tenellus group is restricted to the Atlantic Forest in the South-Eastern part of Brazil. The mayrabonillae group is widespread from Costa Rica to South Bolivia with an allopatric distribution of the two species. COI barcode sequences indicate relatively strong divergence within C. bijuga, C. mayrabonillae, C. serrabonita and C. tenellus.The Neotropical genus Pyraloidea is one of the largest superfamilies of the order Lepidoptera. The monophyly of the group and that of its two main lineages, the Pyraliformes and Crambiformes , are well supported by morphological characters , but these share no evident common characteristics other than the snow white color of the wings.Catharylla is revised using both morphological and molecular data. Phylogenetic relationships within Catharylla and with putatively related taxa as well as the distribution of each species along with biogeographical hypotheses are analysed.In this work, PageBreakCatharylla and outgroup taxa investigated were borrowed from museums and private collectors as listed in Several of the MHNG specimens were kindly given to this institution by collaborators mentioned in the acknowledgments section. The specimens were usually well preserved, the color being sometimes faded. Many specimens from the BMNH were dissected by S. Bleszynski. Unfortunately, his preparations were usually poorly made and badly mounted, sometimes hampering the investigation of genitalia characters.The dissection and slide mounting methods follow Catharylla species, four additional crambine species were included in the dataset for phylogenetic analyses. The material investigated to build the morphological matrix is reported below in Apart from the eight PageBreakPageBreakCatharylla contiguella Zeller, 1872, and Catharylla interrupta Zeller, 1866 could not be found. With the help of the descriptions and illustrations, they were excluded from Catharylla because of the forewing pattern, which is like that of Argyria H\u00fcbner, 1818, with only one median transverse line. For Catharylla sericina Zeller, 1881, based on the description and a photograph of the type in the BMNH, the species was rejected from Catharylla based on the elongated forewing shape and the silvery white pattern without transverse lines. For Catharylla paulella Schaus, 1922, a photograph of the habitus and the genitalia of the female type from the USNM allowed to find other specimens of the same species; the male and female were then associated based on wing pattern. For Catharylla tenellus Zeller, 1839, a photograph of the habitus and the genitalia of the female type were available and the male of the species was associated based on wing pattern. For the descriptions, we followed the nomenclature and terminology used by The types of the two species The following measurements were made with the use of an ocular micrometer: length of labial palpus (base of segment I to apex of segment III), diameter of eye , length of forewing (from base to apex), length of uncus (from tegumen-uncus junction to apex of uncus), length of tegumen connection , length of papillae anales , lengths of anterior and posterior apophyses (from base to apex).Regarding the holotype data, the information was copied exactly as found on the labels with vertical slashes to express changes of lines. Abbreviations are spelled out in square brackets. We assume that the labels are rectangular and white, and that the text is in black ink, otherwise differences are indicated in brackets. Paratype data are reported by country in alphabetical order and the information is recorded without indication of line change. Collecting localities are reported as written on labels, with a question-mark when the locality could not be recovered. Dates and collectors\u2019 information were standardized and the latter placed in parentheses. The specimen depositories are reported with the use of the corresponding acronyms.PageBreakhttp://earth-info.nga.mil/gns/html/). The localities were reported on a text file (*.txt) and loaded on a map using DIVA-GIS 7.4.0.1 . The localities that were not registered in Google Earth were localized more or less precisely with the help of internet search engines or with gazeeters from the GEOnet Names Server (GNS) of the National Geospatial Intelligence Agency and the two nuclear genes wingless (353 bp) and EF-1\u03b1 (679 bp). These genes show different rates of substitution through time, with COI >> wingless > EF-1\u03b1 . COI peridoptera .Specimens used for molecular investigations are listed in Catharylla specimens collected no more than twenty years ago, a leg was sent to the Canadian Centre for DNA Barcoding (CCDB) at the Biodiversity Institute of Ontario (BIO) in Guelph. The barcode sequences of Catharylla are reported in each species description. The protocol for DNA extraction is found in the supplementary material of http://dev.ccdb.ca/docs/CCDB_Amplification.pdfFor DNA extractions were performed following the method of The primers used are recorded in PageBreakPageBreakPageBreakPCRs were performed using peqGOLD Taq DNA polymerase (PeqLab). In cases of weak or absent PCR result, a re-examination PCR was done using BIO-X-ACT Short Taq polymerase (Bioline). PCR protocols are given in Success of gene amplification was evaluated by an electrophoresis with 1% agarose gel, subsequent gel dying with GelRed and analysis under ultraviolet light. PCR products were purified using ExoSAP-IT (USB Corporation).Sequence PCR was done with the BigDye Terminator-Kit of Applied Biosystems. The amount of each product is reported in Sequence alignment was done with BIOEDIT 7.1.3 and PhyDhttp://www.ebi.ac.uk/ena/data/view/HG793012-HG793020; http://www.ebi.ac.uk/ena/data/view/HG793012-HG793020PageBreakGenetic distances between barcoding sequences of COI are given in Crambus pascuella and Crambus uliginosellus set as outgroups. Maximum parsimony analyses were performed using the Branch-and-bound method as searching algorithm with parameters left unchanged. The bootstrap resampling method with 1000 replicates was used. The resulting tree was a 50% majority-rule consensus tree with bootstrap (BS) values assigned to each node. BS supports are reported on the tree of The 21 characters are listed in PageBreakCatharylla bijuga and Catharylla coronata, and the whole COI gene for Catharylla paulella. For Catharylla bijuga, we used the sequence BC MTD 1840 to build the datasets as this sequence performed better than BC MTD 1839 in phylogenetic analyses. The COI sequence of LEP 972 was combined with that of wingless for LEP 973 given that the two samples come from the same population and are genetically very similar as attest the barcode sequences. We generated four different datasets from the sequence data: a complete dataset with all three genes sequences available for the 12 taxa (mol_1), a 12-taxa dataset with the 3rd codon position of COI deleted (mol_2), a dataset restricted to the 7 taxa for which the sequence of the COI gene and at least of one nuclear gene were available (nucl_1), and the same data as nucl_1 but with the 3rd codon position of the COI deleted (nucl_2). We used the programme RAxML over 0.95 are considered well supported. PP values are reported on the tree the morpho-matrix , (2) COIthe tree .Zeller, 1863http://species-id.net/wiki/CatharyllaCatharyllaCrambus tenellus Zeller, 1839, by subsequent designation by PageBreakCatharylla species have snow white to creamy white wings and short labial palpi. They can be separated from other Argyriini by the presence on the forewing of median and subterminal thin transverse lines, slightly curved, convex on costal 1/3. The labial palpi are also shorter in comparison to those of Vaxi. The highly variable male genitalia do not show any synapomorphy or generic diagnostic character. In females, a possible synapomorphy is the strongly reduced anterior and posterior apophyses of abdominal segments VIII and IX, but this is shared with some Crambini and a few other Crambinae (see inae see .Catharylla chelicerata) : R1 presicerata) backgrouTympanal organs : TransveCatharylla tenellus group, strongly developed. Juxta medially curved downward, narrowing toward rounded apex, slightly directed downward apically, basally triangular, sometimes with additional lobes at base or ventro-lateral projections. Vinculum of medium width; saccus short, rounded, directed anterad and slightly upward. Phallus straight or curved, usually more strongly sclerotized at apex; vesica without cornuti, with one cornutus, or with crest of cornuti.Male genitalia \u201333: UncuPageBreakrior apophyses 0.25\u20130.45 \u00d7 length of papillae anales, straight, regularly thin. Tergite VIII narrow, with postero-dorsal line of setae. Anterior apophyses reduced, 0.01\u20130.1 \u00d7 length of papillae anales. Sternite VIII about twice length of tergite, not connecting ventrally in tenellus species group. Sterigma present, strongly sclerotized except in tenellus species group, usually forming pockets of variable shape; reduced to sclerotized lamella antevaginalis in Catharylla bijuga. Ductus seminalis connecting posteriorly at base of ductus bursae. Ductus bursae long, at least 2 \u00d7 length of corpus, wide, basally curved. Corpus bursae usually rounded, egg-shaped, often enlarging progressively from ductus bursae, usually with one signum, sometimes without; corpus and ductus bursae covered with minute spicules.Female genitalia : PapillaThe genus is restricted to the Neotropical Region, from Costa Rica to Santa Catarina, Brazil, from sea level to 1300 m \u201346.Catharylla serrabonita in its environment, i.e. forested hills up to about 950 m in elevation, surrounded by cacao or coffee plantations in the lowlands. The moths were coming to light, usually very late (after 23:00).The biology of the species remains unknown. In the Serra Bonita Reserve in march 2011, we observed Catharylla has been placed in the Argyriini , Amazonas, P[ar]q.[ue] Nac.[ional] do Ja\u00fa, Rio Ja\u00fa, bg. Miratuc\u00fa, 1\u00b057'S, / 61\u00b049'W, 26\u201327.vii.1995, U[ltra] V[iolet] light sheet (R. W. Hutchings) (USNM). FRENCH GUIANA: 2 \u2642 with same data as holotype (BMNH); 5 \u2642, 1 \u2640 with same locality as holotype except i.1918 (1 \u2642), ii.1918 (S. M. Klages) ; 1 \u2642, 2 \u2640, Parcelles CIRAD de Combi, plantations exp\u00e9rimentales pk 1.5, 5\u00b018'N, 52\u00b055'30 W, 4.iii.2011, pi\u00e8ge lumineux [light trap] (B. Hermier) (MHNG); 3 \u2642, 2 \u2640 , Saint-Jean-du-Maroni (Le Moult) (BMNH); PageBreak1 \u2642, Saint-Laurent-du-Maroni (USNM); 1 \u2642 Roura, 3.6km E[ast] Roura at r[oa]d to Crique Gabrielle, 50m, 20.iv.1994, at light (J. S. Miller & C. Snyder) (AMNH); 1 \u2640 , Cayenne, iii.1917 (CMNH). GUYANA: 1 \u2642, New River 1938 (C.A. Hudson) (BMNH); 1 \u2642, Mallali [sic] (USNM). SURINAME: 1 \u2640 , Geldersland, Surinam River (USNM); 1 \u2640 , Sipaliwini Distr[ict], Thibiti area, Kabo Creek, partly swampy, primary forest on hilly slopes, ca. 2km from river, 29.v.1989 (J. Beerlink) (Schouten Coll.).COI barcode sequence of paratype BC MTD 01839 (654 bp): ACATTATATTTTATCTTCGGAATTTGAGCAGGAATAGTTGGAACATCCCTAAGACTTTTAATTCGAGCAGAATTAGGTAATCCAGGTTCTCTTATTGGTGACGACCAAATTTATAATACTATTGTTACTGCTCATGCATTTATTATAATTTTTTTTATAGTTATGCCAATTATAATTGGAGGATTCGGTAATTGATTAGTTCCATTAATATTGGGAGCACCAGATATAGCATTCCCACGAATAAATAATATAAGATTTTGATTACTCCCCCCCTCTTTAATCCTATTAATTTCTAGAAGAGTTGTAGAAAATGGAGCTGGAACAGGATGAACAGTTTACCCCCCACTTTCATCAAATATTGCTCATAGTGGTAGATCTGTAGATTTAGCAATTTTTTCTCTACACTTAGCAGGAATTTCATCAATCTTAGGAGCTATTAATTTTATTACAACAATTCTTAATATACGAATTAATGGTTTATCTTTCGATCAAATACCTTTATTTGTTTGATCTGTAGGAATTACAGCTTTACTTCTTCTCTTATCCTTACCCGTATTAGCTGGTGCTATTACTATACTTTTAACTGATCGAAATTTAAATACATCTTTTTTTGATCCTGCTGGAGGAGGAGATCCTATCCTTTACCAACACTTAOn the forewing , the sevPageBreakunderside dull white to light ochreous along costal margin, with marginal dashes pronounced. Hindwing snow white, veins slightly ochreous, with shiny aspect; marginal PageBreakline thin, brown, pronounced up to CuA1, then shiny white; fringes white; underside white, with same margin as on recto.Male (n = 17) : AntennaTympanal organs (n = 8): Tympanic pockets extending slightly beyond transverse ridge, rounded. Tympanic drum elongate, more or less oval, postero-laterally extended beyond transverse ridge.Male genitalia (n = 8) , 12: UncFemale (n = 10): Labial palpi: 1.1\u20131.4 mm long. Forewing length: 11\u201315 mm; frenulum triple.Female genitalia (n = 6) : PapillaThe species occurs in lowlands in the three Guianas and Brazil .bijugus, a, um which means \u201cyoked together, double\u201d, in reference to the bifid costal arm of the male genitalia.Bijuga comes from the Latin PageBreakCatharylla, he gave the manuscript name Catharylla ramona to this species, but never published it. The comparison of the tip of the tubular costal arm of the male genitalia and the female lateral projections of sternite VIII shows rather nicely that the male hooks the female genitalia during the mating process. The specimen collected in Parque Nacional do Ja\u00fa, Brazil, shows a divergence in COI barcode sequence of 5.05% with that of Roura, French Guiana. In morphology we find no significant difference corroborating this divergence. The relationships of this species to the others remain uncertain in our phylogenetic analyses.In some paratypes from French Guiana the collecting data mention a \u201cpk\u201d (=\u201dpoint kilom\u00e9trique\u201d). This kilometric marker refers to the distance of the collecting spot on the forest road to the nearest main road. CIRAD refers to the name of the research institution leading agronomical research on the Combi site. the Combi site. When S. Bleszynski looked into Diagnosis. The synapomorphies of the group are the quadrangular valva with a truncated apex and the hook-shaped gnathos in the male genitalia. The chelicerata species group can be separated from the other Catharylla species based on additional diagnostic characters. Externally, the forewing has a clear, dark brown costal band, and its length is usually over 14 mm. In male genitalia, the apex of the uncus is regularly rounded with a short, narrow projection medially and the vesica shows one large, curved, pointed cornutus, preceded by a string of 13\u201314 smaller cornuti increasing in size toward apex. In female genitalia, the ductus bursae shows a pronounced, tongue-shaped projection postero-ventrally.Notes. This group includes two species. The phylogenetic analyses restricted to the nuclear genes and the combined Bayesian analysis place the group as sister-group of the mayrabonillae species group , Amazonas, Rio Negro, Mirapinima, 8.iv.1972 (CNC); 9 \u2642, 1 \u2640, Reserva Ducke, km 26 Manaus\u2013Itacoatiara Highway, 15.iv.1972 (1 \u2642), 18.iv.1972 , 21.iv.1972 , 16.v.1972 (2 \u2642), 17.v.1972 , 18.v.1972 (CNC). FRENCH GUIANA: 5 \u2642, 1 \u2640, 36km SE, Roura (Camp Patawa), 21.xi.2007 , 29\u201330.xi.2007 , 30.xi.2007 (1 \u2642) (MHNG); 1 \u2640 with same data as holotype; 1 \u2642, same data as holotype except 2.ix.2011 (Hermier n\u00b0 24755); 1 \u2640, same data as holotype except /604/ and 4.iii.2011 (Hermier n\u00b0 24344); 2 \u2642, Beaus\u00e9jour, N[ationale] 1 pk 28.5, 4\u00b042'30\"N, 52\u00b023'30\"W, 3.vi.2011, pi\u00e8ge lumineux (Hermier n\u00b0 24545 & 24546) (B. Hermier) (MHNG); 1 \u2642, Route d\u2019Apatou pk 25.5 spk 2+4.4, 1.x.2011, pi\u00e8ge lumineux (B. Hermier) (Hermier n\u00b0 24956) (MHNG); 1 \u2642 , R[ou]te foresti\u00e8re de Saut L\u00e9odate pk 4.5, 4\u00b055'N, 52\u00b033'W, 31.x.1995 pi\u00e8ge lumineux (B. Hermier) (Hermier n\u00b0 8457) (MHNG).PageBreakOther specimens. 1 \u2640 , Nova Olinda, Rio Purus, v.1922 (S. M. Klages) (CMNH); 1 \u2640 , Teff\u00e9 [sic], vi.1906 (W. Hoffmanns) (BMNH).COI barcode sequence of paratype BC MTD 01703 (654 bp): ACTTTATATTTTATCTTTGGAATTTGAGCAGGAATAATTGGAACATCCTTAAGACTACTAATTCGAGCAGAATTAGGTAATCCTGGATCTCTTATCGGGGATGACCAAATTTATAACACTATTGTTACTGCTCATGCATTTGTAATAATCTTTTTTATAGTTATACCAATTATAATTGGTGGATTTGGAAACTGATTAGTACCTTTAATGCTAGGGGCACCAGATATAGCATTCCCTCGTATAAATAATATAAGATTTTGACTTCTTCCCCCCTCTTTAACCCTATTAATTTCAAGTAGAATTGTAGAAAATGGGGCAGGAACAGGATGAACCGTTTATCCACCTTTATCATCTAATATTGCCCATGGAGGCAGATCAGTAGATCTGGCAATTTTTTCACTACATTTAGCTGGAATTTCATCAATTTTAGGGGCAATTAATTTTATTACAACAATTATTAATATACGAATTAATAATCTTTCATTTGATCAAATACCCCTATTTGTTTGATCAGTAGGTATTACAGCATTACTATTACTTCTATCTTTACCAGTATTGGCGGGAGCTATTACCATACTTCTAACTGACCGAAATCTCAATACTTCCTTTTTTGATCCAGCAGGGGGGGGAGACCCTATTTTATATCAACACCTACatharylla gigantea, Catharylla chelicerata differs in having the male costal arm hook shaped, longer, and thinner than in Catharylla gigantea, and the juxta is strongly downcurved, apically conical whereas it is long, almost straight, without apical conical projection downward in Catharylla gigantea. In female genitalia the sterigma forms a strongly sclerotized symmetrical structure made of two asymmetrical bell-shaped cavities, opened anterad in Catharylla chelicerata whereas it forms a pair of shallow pockets opened posterad in Catharylla gigantea.From Male (n = 21) , 9: HeadPageBreaksclerotized, dorsal base of praecinctorium sclerotized. Tympanic drums elongate, bean shaped, posteriorly reaching transverse ridge or slightly beyond.Tympanal organs (n = 9): Transverse ridge almost straight medially. Tympanic pockets conical, extending slightly beyond transverse ridge. Tympanic bridge lightly PageBreakshaped, forming angle of about 100\u00b0 with axis of basal arms, about 1/4 longer than uncus. Tegumen arms narrow at base, enlarging progressively toward dorsum to 2 \u00d7 basal width, projected dorsally with bump at connection, connecting at distal 1/6. Cucculus densely setose, slightly directed upward on distal 1/3, apically truncated; basal 2/3 of costa of valva dorso-ventrally and laterally widened; costal arm hook-shaped, PageBreakPageBreakstrongly sclerotized, directed upward at about 45\u00b0 from costal arm base. Juxta triangular, curved downward, tip rounded and sac-like, basal lateral lobes curved ventrally. Saccus short, curved upward medially. Phallus narrow, S-shaped; vesica covered with tiny spicules, with one large, curved, pointed cornutus apically, preceded by string of 13\u201314 smaller cornuti increasing in size toward apex.Male genitalia (n = 9) , 14: UncFemale (n = 4): Labial palpi: 1.8\u20132.2 mm long. Forewing: 15\u201319.5 mm. Frenulum quadruple.PageBreakPageBreakanterior margin; covered with minute punctuation. Ventro-basal section of ductus bursae tongue shaped, strongly sclerotized; ductus bursae long, ventrally sclerotized, widened and looped in basal half; enlarging progressively into corpus bursae. Corpus bursae egg-shaped with one signum.Female genitalia (n = 3) : PapillaThe species was found in French Guiana and Brazil (Amazonas) .Chelicerata\u201d refers to the shape of the costal arms of the male valva, which look like mygalomorph chelicerae.\u201cCatharylla robustella and Catharylla tenellina by S. Bleszynski, as indicated on labels, but these names were never published. These two specimens are probably Catharylla chelicerata, but the bad genitalia preparations do not allow to see details, and therefore they are not included as paratypes.Two females included here have been named T. L\u00e9ger & B. Landrysp. n.http://zoobank.org/06B7837B-A5DE-4347-824E-B74127F028E2http://species-id.net/wiki/Catharylla_giganteaHolotype. \u2642, with labels as follows: \u201cBrazil: Amazonas, Manaus, | Reserva Ducke, AM-010, k[ilo]m[eter]. 26 | 2\u00b055'S, 59\u00b059'W, Dec[ember].13, 1993 | J. Bolling Sullivan & | Roger W. Hutchings | U[ltra]V[iolet] Light (Plateau Hut)\u201d; \u201cHOLOTYPE | Catharylla gigantea | T. L\u00e9ger & B. Landry\u201d [red label]; \u201cBL 1747 \u2642\u201d [light green label]. Deposited in USNM.Paratypes. 5\u2642, 2 \u2640. BRAZIL: 1 \u2642, Amazonas, Reserva Ducke, km. 26, Manaus\u2013Itacoatiara Highway, 15.v.1972 (CNC). FRENCH GUIANA: 1 \u2642, 1 \u2640 , Saint-Jean-du-Maroni (E. Le Moult) (BMNH); 1 \u2642 , Oyapok [sic] River, Pied Saut, iii.1918 (S. M. Klages) (CMNH). GUYANA: 2 \u2642, 1 \u2640 , Potaro, ii.1908 (2 \u2642), v.1908 (1 \u2640) (S. M. Klages) (BMNH).Catharylla chelicerata, Catharylla gigantea differs in having the male costal arm shorter, basally wide and tooth shaped while it is long, narrow throughout and hook shaped in Catharylla chelicerata. The juxta is long, tongue shaped, almost straight, and apically rounded, whereas it is downcurved and apically conical in Catharylla chelicerata. In female genitalia, the sterigma forms a pair of shallow pockets opened posterad whereas in Catharylla chelicerata the sterigma forms a strongly sclerotized symmetrical structure made of two asymmetrical bell-shaped cavities opened anterad.From PageBreakbrown scales at half of length, white tipped. Thorax with some brown at collar. Foreleg coxa white, femur white, ashen brown dorsally; tibia and tarsomeres brown-ochreous, distally ringed with dark brown. Midleg white with tibia-femur joint and base of tibia ashen; tarsomeres ochreous to brown ochreous with upperside brown to dark brown, white tipped. Hindleg white with tarsomeres II\u2013V ochreous to brown ochreous, upperside brown, with white tips. Forewing length: 13.5\u201314.5 mm; snow white with wide brown to dark brown costal line from base to apex; median and subterminal transverse lines faded brown; dark brown spots on termen forming more or less continuous line; fringes brass colored; underside white, with costal margin brown ochreous, outer margin with subtriangular spots. Hindwing snow white; marginal spots dark brown between R5, M1, M2, M3, and CuA1; fringe white; underside snow white, with same spots as on upperside.Male (n = 6) : Head wiTympanal organs (n = 5): Transverse ridge medially convex. Tympanic pockets extending slightly beyond transverse ridge, rounded. Tympanic bridge lightly sclerotized, dorsal base of praecinctorium sclerotized. Tympanic drums elongate, bean shaped.Male genitalia (n = 5) , 16: UncFemale (n = 2): Labial palpi: 2.5\u20133.1 mm long. Forewing length: 17.5\u201322 mm; frenulum triple.Female genitalia (n = 2) : PapillaCatharylla gigantea has been found in French Guiana, Guyana, and Brazil (Amazonas) (mazonas) .giganteus, a, um meaning very large.The name comes from the Latin The name was given to the species on manuscript labels by S. Blezynski, probably in reference to the large size of the female.PageBreakDiagnosis. The synapomorphies of the group are the dorsal furrow on the uncus, the uncus apex slightly bifid, the presence of a transtilla in male genitalia, and the absence of a ventral connection of sternite VIII in female genitalia. The tenellus species group can also be separated from the other Catharylla species based on the following additional diagnostic characters: the hindwings are creamy-white, and in female genitalia, the papillae anales are not produced.Notes. This group includes three species, including two new ones. Catharylla serrabonita and Catharylla tenellus form a monophyletic group within the tenellus species group http://species-id.net/wiki/Catharylla_tenellusCrambus tenellus Zeller, 1839: 174\u2013175Catharylla tenella : Argyria tenella : Platytes tenella : Holotype. \u2640, \u201cType\u201d [red ringed]; \u201cCatharylla | tenella Z[eller]. | Mon[ograph]. p[age]. 50 Am. anftr.\u201d [not clearly readable]; \u201cZell[er]. Coll[ection]. | 1884\u201d; \u201c\u2640 | Pyralidae | Brit[ish]. Mus[eum]. | Slide N\u00b0 | 1094\u201d. Deposited in BMNH.Other specimens examined. 20 \u2642, 7 \u2640. BRAZIL: 3 \u2642 , S\u00e3o Paulo, Ubatuba, Picinguaba, 23\u00b022'S, 44\u00b050'W, 2\u201320m, 22\u201324.ix.2001 (V. O. Becker n\u00b0132820) (Becker Coll.); 2 \u2642 with same data except 10\u201312.xi.2001 (V. O. Becker n\u00b0133712) (Becker Coll.); 2 \u2642, 1 \u2640 , S\u00e3o Paulo, Bertioga, 5 m, 5.xi.1995 (V. O. Becker n\u00b099090) (USNM); 1 \u2642 with same data (Becker Coll.); 1 \u2642 with same data except 15\u201317.v.1996 (V. O. Becker n\u00b0 99386) (Becker Coll.); 1 \u2642 with same data except 7\u20139.x.1996 (V. O. Becker Coll. n\u00b099757) (Becker Coll.); 1 \u2642 , S\u00e3o Paulo, S\u00e3o Luiz do Paraitinga, 23\u00b020'S, 45\u00b006'W, 900 m, 13\u201320.iii.2001 (V. O. Becker n\u00b0132356) (Becker Coll.); 1 \u2642 , S\u00e3o Paulo, 700 m (E. D. Jones) (BMNH); 1 \u2642 , Minas Gerais, Cara\u00e7a, 1300 m, 1\u20132.iv.1992 (V. O. Becker n\u00b085081) (USNM); 1 \u2640 with same data (Becker Coll.); 1 \u2642 with same data except 25.x.1994 (Becker Coll.); 1 \u2642, 1 \u2640 , Bahia, Porto Seguro, A. d\u2019Ajuda, 16\u00b027'S, 39\u00b003'W, 20 m, 12.vii.2009 (V. O. Becker n\u00b0144140) (Becker Coll.); 2 \u2642 with same data except 15.viii.2008 (V. O. Becker n\u00b0140808) (Becker Coll.); 1 \u2642 with same data except 1\u20133.v.2009 (V. O. Becker n\u00b0142784) (Becker Coll.); 1 \u2642, Paran\u00e0, Castro (USNM); 1 \u2640 , Rio de Janeiro, xi[day and year data missing] (H. H. Smith) (CMNH); 1 \u2640 , Rio de Janeiro, Corcovado, 457 m, 26.xii.1958 (E. P. Wiltshire) (BMNH). No locality data: 1 \u2642, 1 \u2640 ; 1 \u2640 , 1869 (NMW).COI barcode sequence of specimen BC MTD 1710 (654 bp): ACTCTATATTTTATCTTTGGAATTTGATCAGGAATAATTGGAACATCTTTAAGATTATTAATTCGAGCAGAATTAGGGAATCCTGGATCTCTAATTGGAGATGATCAAATTTATAACACTATTGTAACAGCCCATGCATTTATTATAATTTTTTTTATGGTTATACCAATTATAATTGGTGGATTTGGAAATTGATTGGTTCCATTAATATTAGGAGCCCCAGATATAGCTTTCCCCCGAATAAATAACATAAGATTTTGGTTATTACCCCCTTCCTTAACTCTTTTAATTTCTAGAAGAATTGTAGAAAATGGAGCTGGAACAGGATGAACGGTCTACCCCCCCCTTTCATCTAATATTGCCCATAGTGGAAGATCTGTAGATTTAGCAATCTTTTCTCTTCATTTAGCTGGAATTTCATCAATTTTAGGAGCTATTAATTTTATTACAACAATTATTAATATACGAATTAGTAATTTATCTTTTGATCAAATACCTTTATTTGTTTGATCAGTCGGTATTACAGCTTTACTTCTTCTTCTATCTTTACCTGTATTAGCAGGAGCTATTACTATACTTTTAACTGATCGAAATTTAAATACATCTTTTTTTGATCCTGCAGGAGGAGGAGATCCTATCTTATATCAACATTTACatharylla serrabonita and Catharylla coronata, Catharylla tenellus can be separated by the median transverse line, which is faintly convex towards costa, whereas it is more strongly convex in Catharylla coronata and Catharylla serrabonita. The male genitalia provide the best diagnostic characters. The most obvious refers to the transtilla, which forms a pair of short, narrow sclerotized arms with pointed tips, projecting posterad, with, in between, a pair of brushes directed medio-ventrally, whereas it forms a pair of arms pointing posterad with a string of spines ventrally in Catharylla serrabonita and Catharylla coronata. In female genitalia, the anterior angle of sternite VIII is directed downward into a more or less rounded projection covered with short spinules of same length, whereas it is projected anterad in Catharylla serrabonita, and it is not projected in Catharylla coronata.From PageBreaktips. Thorax slightly ochreous at collar. Foreleg coxa white; femur ochreous, dorsally dark brown; tibia and tarsomeres ochreous, distally ringed with dark brown. Midleg light ochreous with tibia-femur joint brown; tarsomeres II\u2013V dark brown on upperside, with white ringed tips. Hindleg white; tarsomeres as midleg. Forewing length: 10.5\u201312 mm; snow white; costal line ochreous, lightly pronounced from base to apex; median and subterminal transverse lines ochreous, median transverse line faintly convex towards costa; outer margin ochreous with 7 dark brown spots often triangular, strongly pronounced; fringes brass colored; underside ochreous with costal margin pronounced in basal half and marginal spots pronounced. Hindwing cream-coloured; outer margin with small ochreous brown spots forming more or less continuous line between Sc+R1, Rs, M1, M2, M3, CuA1 and CuA2; underside light ochreous, with marginal spots pronounced; fringes white.Male (n = 20) : Head whTympanal organs (n = 13): Transverse ridge more or less regularly rounded, medially more straight. Tympanic pocket extending slightly beyond transverse ridge. Tympanic drum ovoid, posteriorly not extended beyond transverse ridge. Tympanic bridge faintly sclerotized.Male genitalia (n = 13) , 27\u201329: Female (n = 7): Labial palpi: 1.6\u20132 mm; forewing length 12\u201316 mm; frenulum triple.Female genitalia (n = 7) : PapillaPageBreakThe species is known from Brazil in the Atlantic Forest .Notes. The species was described from \u201cone female collected in Brazil, near Rio de Janeiro\u201d. Hence, the lectotype designated by a label by S. Bleszynski is not warranted. This designation is presumably based on the fact that Catharylla tenellus, or that there is indeed a deep divergence in the COI barcode between populations of this species.Specimens from Porto Seguro, Brazil show a divergence of 3.34% in COI barcode sequences with the specimen from Ubatuba, Brazil. In morphology, differences in male genitalia are also observed: in the specimens from Bertioga, Cara\u00e7a, S\u00e3o Paulo and Ubatuba the costal arm of the valva is wide and 1/3 of the length of the cucullus, almost reaching its tip, and the dorsal edge at base is slightly produced . In the T. L\u00e9ger & B. Landrysp. n.http://zoobank.org/7E0EB0BE-44C4-42EC-9F4D-2C923E9299E6http://species-id.net/wiki/Catharylla_coronataHolotype. \u2642, with labels as follows: \u201cCol. BECKER | 81552\u201d; \u201cBRASIL:ES | Linhares, 40m | 20-29.ii.1992 | V.O.Becker Col\u201d; \u201cHOLOTYPE | Catharylla | coronata | L\u00e9ger & Landry\u201d [red label]. Deposited in Becker Collection.Paratypes. 21 \u2642, 4 \u2640. BRAZIL: 5 \u2642 with same data as holotype ; 2 \u2642 with same data as holotype (1 used for DNA barcoding BC MTD 01891) except 05\u201309.iv.1992 (V. O. Becker n\u00b082486); 6 \u2642, 1 \u2640 , Paran\u00e0, Rio Negro, 900 m, 8.ii.1973 (2 \u2642), 10.ii.1973 (1 \u2642), 11.ii.1973 (3 \u2642), 13.ii.1973 (1 \u2640) (A. & J. Razowski) (ISZP); 2 \u2642, 2 \u2640, Paran\u00e0, Curitiba, 920 m, 17.ii.1975 (1 \u2642) (V. O. Becker n\u00b010167), 20.ii.1975 (V. O. Becker n\u00b010168), 12.iii.1975 (V. O. Becker n\u00b010166), 10.x.1975 (1 \u2642) (V. O. Becker n\u00b04010) (Becker Coll.); 1 \u2642 , Paran\u00e0, Castro, 950 m (E. D. Jones) (BMNH); 1 \u2642, Paran\u00e0, Quatro Barras, 850 m, 27.ii.1970 (Laroca & Becker) (V. O. Becker n\u00b015442) (Becker Coll.); 1 \u2642 Rio de Janeiro, Novo Friburgo (BMNH); 1 \u2642 Sao Paulo, 700 m (E. D. Jones) (BMNH); 1 \u2642, 1 \u2640 , Santa Catarina, Rio Vermelho, 968 m, 18.ii.1973 (\u2642), 28. ii. 1973 (\u2640) (A. & J. Razowski) (ISZP); 1 \u2642, no locality data (V. O. Becker) (Becker Coll.).PageBreakCOI barcode sequence of paratype BC MTD 01890 (654 bp): ACTTTATATTTTATTTTTGGAATTTGAGCAGGAATAGTAGGAACATCATTAAGATTATTAATTCGAGCTGAATTAGGTAATCCTGGATCTCTTATTGGAGATGATCAAATCTATAATACTATTGTAACCGCTCATGCATTTATTATAATTTTTTTTATAGTTATACCAATTATAATTGGTGGATTTGGAAATTGATTAGTTCCCTTAATATTAGGAGCACCAGATATAGCTTTTCCTCGAATAAATAACATAAGATTTTGATTATTACCCCCCTCTTTAACTCTTTTAATTTCAAGAAGAATTGTAGAAAATGGAGCTGGAACAGGATGAACAGTTTACCCCCCACTTTCATCTAATATTGCCCATAGTGGAAGATCCGTAGATTTAGCAATCTTTTCCCTTCATTTAGCTGGAATTTCTTCAATTTTAGGAGCAATTAATTTTATTACAACAATTATTAATATACGAATCAATAATCTTTCATTTGATCAAATACCTCTTTTTGTTTGATCAGTAGGAATTACAGCTTTACTTCTTCTTTTATCATTACCAGTATTAGCTGGAGCTATTACTATACTTTTAACTGATCGAAATTTAAATACATCTTTTTTTGATCCCGCAGGAGGAGGAGATCCTATTTTATATCAACATTTACatharylla serrabonita and Catharylla tenellus, Catharylla coronata can be separated with characters of the male genitalia: the uncus is apically bifid and grooved on distal 1/5 in Catharylla coronata whereas it is only indented medially at apex in Catharylla serrabonita and Catharylla tenellus; the costal arm of the valva is short and the apex is curved inward in Catharylla coronata whereas the costal arm is longer and points postero-dorsally in the other two species; the transtilla forms a pair of sclerotized arms slightly bent inward distally, ventrally with a row of short spines increasing in size from base to apex whereas it forms a pair of short, narrow sclerotized arms with pointed tips, projecting posterad, and with a pair of brushes directed medio-ventrally in Catharylla tenellus and a pair of sclerotized arms strongly bent inward on distal 1/4 and with a string of long spines of same length medially along it in Catharylla serrabonita; the juxta is shorter than in Catharylla tenellus, and regularly narrowing toward apex whereas it is strongly narrowing on distal 1/4 in Catharylla serrabonita; the ventral projections of the juxta form a pair of shallow pockets whereas they are bell-shaped in Catharylla serrabonita and thumb-like in Catharylla tenellus; the vesica has a row of 6\u20137 cornuti in Catharylla coronata whereas it does not show any cornuti in Catharylla serrabonita and Catharylla tenellus. In the female genitalia of Catharylla coronata, the anterior angle of sternite VIII is not projected whereas it is rounded, projected anterad and covered with short spinules in Catharylla serrabonita, and projected downward in Catharylla tenellus. The anterior apophyses are quadrangular, anvil shaped whereas they are spine like in the other two species.From PageBreakline thin, light ochreous, apically faded; median transverse line light ochreous, concave on costal half, more or less disrupted; subterminal transverse line ochreous, curving toward base on costal half; R5 vein faintly marked apically with ochreous; outer margin ochreous with 7 pronounced dark brown spots more or less triangular between veins, sometimes connecting; fringes brass colored; underside white ochreous to ochreous, costal margin basally brown; outer margin with pronounced spots. Hindwing white to creamy white, usually with marginal brown spots between Sc+R1, Rs, M1, M2, M3, CuA1 and CuA2, forming more or less continuous line; fringes white; underside light ochreous, with dark brown marginal spots pronounced.Male (n = 21) : Head whTympanal organs (n = 7): Transverse ridge more or less regularly rounded. Tympanic pocket extending faintly beyond transverse ridge, rounded. Tympanic drum glomerular, not reaching transverse ridge.Male genitalia (n = 7) , 20: UncFemale (n = 4): Labial palpi: 1.6\u20132.2 mm long. Forewing length 14\u201316 mm. Frenulum triple.Female genitalia (n = 4) : PapillaThe species occurs in Brazil in the following states: Bahia, Espirito Santo, Paran\u00e0, Rio de Janeiro, Santa Catarina, Sa\u00f5 Paulo .PageBreakcoronatus, a, um: crowned, referring to the longitudinal string of short spines of the transtilla in the male genitalia.The name comes from the latin Catharylla coronata is the sister species of the Catharylla tenellus + Catharylla serrabonita pair , Esp\u00edrito Santo, Linhares, 40m, 25\u201330.i.1998 (V. O. Becker n\u00b0113929) ; 2 \u2642, 1 \u2640 with same except 20\u201329.ii.1992 (V. O. Becker n\u00b081552) ; 2 \u2642 with same data as holotype except 05\u201309.iv.1992 (V. O. Becker n\u00b082486) (USNM); 10 \u2642 Bahia, Camacan, Serra Bonita Reserve, 15\u00b023'S, 39\u00b033'W, 800 m, B. Landry, V. O. Becker, 1.iv.2011 (1 \u2642), 2.iv.2011 (2 \u2642), 3.iv.2011 , 5.iv.2011 (3 \u2642), 6.iv.2011 (1 \u2642), 7.iv.2011 (1 \u2642) (MHNG); 1 \u2642 with same data except vii.2010 (V. O. Becker) (Becker Coll.); 1 \u2642 Bahia, Porto Seguro, A. d\u2019Ajuda, 16\u00b027'S, 39\u00b003'W, 20 m, 12.vii.2009 (V. O. Becker n\u00b0144140) (Becker Coll.).COI barcode sequence of holotype LEP 979 (516 bp): TAGTTGGAACATCATTAAGACTATTAATTCGAGSAGAGTTAGGGAATCCTGGATCTCTTATTGGAGATGATCAAATTTATAATACTATTGKAACAGCTCATGSATTTATTATAATTTTTTTTATAGTTATACCAATTATAATTGGTGGATTTGGAAACTGACTAGTTCCATTAATATTAGGAGCCCCAGACATAGCTTTCCCCCGAATAAATAATATAAGATTTTGATTACTCCCCCCCTCTTTAACCCTTTTAATTTCCAGAAGAATTGTAGAGAATGGAGCTGGAACAGGATGAACGGTTTACCCCCCCCTTTCATCTAATATTGCTCATAGKGGAAGATCTGTAGATTTAGCAATTTTTTCTCTTCATTTAGSAGGAATTTCATCAATTTTAGGAGCAATTAATTTTATTACAACAATTATTAATATACGAATTAATAATTTATCTTTTGATCAAATACCGTTATTTGTCTGATCAGTTGGTATTACAGCTTTACTCCTTCTTTTATCTTTACPageBreakCatharylla coronata and Catharylla tenellus, Catharylla serrabonita can be separated by the zigzagging median transverse line with the short triangular dent at CuA2 and the pronounced creamy color of the hindwing. The male genitalia provide the best discriminant characters: in Catharylla serrabonita, the transtilla forms a pair of sclerotized arms bent inward in distal 1/4 and with a string of long spines of same length medially along it, whereas it forms a pair of short, narrow sclerotized arms with pointed tips projecting posterad, and with a pair of brushes directed medio-ventrally in Catharylla tenellus, and two sclerotized arms slightly bent inward distally, with a row of short spines increasing in size from base to apex in Catharylla coronata, and the juxta is apically narrow and pointed whereas it is triangular and regularly narrowed in Catharylla coronata and Catharylla tenellus. In female genitalia, the anterior angle of sternite VIII is projected anterad into a rounded protrusion covered with short spinules in Catharylla serrabonita, whereas it is projected downward in Catharylla tenellus and it is not projected in Catharylla coronata.From Male (n = 21) : Head whTympanal organs (n = 4): Transverse ridge more or less rounded, medially slightly flattened. Tympanic pocket extending faintly beyond transverse ridge, rounded. Tympanic drum glomerular, not reaching transverse ridge.Male genitalia (n = 4) \u201326: UncuFemale (n = 1): Labial palpi: 1.9 mm long. Forewing length: 14 mm. Frenulum triple.PageBreakbetween segment VIII and IX covered with microspines. Sternite VIII laterally about 5/3 length of tergite VIII; posterior margin of tergite VIII with line of setae; sternite VIII forming 2 triangular lobes regularly narrowing downward, not connected, densely covered with short spinules of same length; anterior angle of sternite VIII slightly projected anterad, rounded, covered with short spinules of same length. Anterior apophyses 0.03 \u00d7 length of papillae anales. Sterigma membranous, covered with spinules. Ductus bursae about 3 \u00d7 length of corpus bursae, narrow, basally directed downward and then bent upward. Corpus bursae elongate, ovoid, with one tiny signum.Female genitalia (n = 1) : PapillaThe species occurs in Brazil & 46.The name comes from that of the Serra Bonita Reserve founded by Vitor O. Becker and Clemira de Souza. It is managed by Instituto Uira\u00e7u in the State of Bahia, Brazil.Serra Bonita Reserve is located in the Atlantic Forest, in a hilly region of cacao plantations and scattered forest. Adults came late to light, usually after 23:00. Our molecular analysis of the COI barcode sequences highlighted that specimens from Serra Bonita respectively show 3.24 and 2.21 % base differences with those of Porto Seguro and Linhares. This divergence is possibly associated with slight morphological differences in male genitalia as shown in Diagnosis. We have not recovered any obvious synapomorphy for this group. It can be separated from the other Catharylla species based on the shorter forewing length, usually between 7.5 and 9.0 mm (maximum 10.5 mm). In male genitalia, the tegumen connection is more than two times longer than the uncus, the uncus is beak-shaped, with the apex narrowing to a point, the gnathos is bent at an angle of about 90\u00b0. In female genitalia, the basal line along the anal papillae is ventrally expanded onto a triangle, and the sterigma forms a pair of sclerotized pockets on each side of the middle, covered with short spines or spicules. The sterigma does not bear tiny setae on the ventral membrane of segment VIII.Notes. This group includes two species. The phylogenetic analyses restricted to the nuclear genes and the combined Bayesian analysis place the mayrabonillae group as sister to the chelicerata group , [Acre] Rio Branco, 1924 (Dengler) (SMNS); 2 \u2640, Amazonas, Manaus, Reserva Ducke, AM-010, km 26, 2\u00b055'S, 59\u00b059'W, 15.xii.1993, U[ltra]V[iolet] Light (J. B. Sullivan & R. W. Hutchings) (USNM); 1 \u2642 , Amazonas, Fonte Boa, ix.1906 (S. M. Klages) (BMNH); 1 \u2640 , Federal District, Esta\u00e7ao Florestal, Cabeca do Vedao, 1100m, 18.x.1971 (CNC); 2 \u2642, Maranh\u00e3o, Feira Nova, Faz[enda]. Retiro, 480m, 07\u00b000'S, 46\u00b026'W, 1\u20133.xii.2011 (V. O. Becker n\u00b0148263) (Becker Coll.); 2 \u2640, Par\u00e1, Bel\u00e9m, 20m, i.1984 (V. O. Becker n\u00b046981) (Becker Coll.); 1 \u2640, Par\u00e1, Capitao Poco, 25\u201331.i.1984 (V. O. Becker n\u00b097880) (Becker Coll.); 1 \u2642, Rondonia, Cacaul\u00e3ndia, 140m, xi.1991 (V. O. Becker n\u00b079592) (Becker Coll.); 1 \u2640, Rondonia, 62km S[outh] Ariquemes, Fazenda Rancho Grande, 165m, 10\u00b032'S, 62\u00b048'W, 18\u201326.iv.1991 (R. Leuschner) (USNM). COLOMBIA: 1 \u2642, Valle, J[un]ct[ion]. Old B\u2019[uena]v[en]tura R[oa]d. and Rio Dagua, 50m, 8.ii.1989 (J. B. Sullivan) (USNM). COSTA RICA: 1 \u2640 , Alajuela, Area de Conservacion Guanacaste, Estacion Caribe, 12.xi.2007 (S. Rios & H. Cambronero) (INBio); 1 \u2640, Alajuela, Area de Conservacion Guanacaste, Rio Negro, 25.i.2009 (H. Cambronero & F. Quesada) (INBio); 2 \u2642 , Alajuela, San Carlos, Arenal National Park, Send[ero] Pil\u00f3n, Rio Celeste, 700m, light trap, 17\u201319.x.2001 (G. Rodriguez) (INBio); 1 \u2642, Prov[incia] Guanacaste, F[in]ca Pasmompa, Est[acion] Pitilla, 5km SO S[an]ta Cecilia, 400m, xii.1990 (P. Rios & C. Moraga) (INBio). ECUADOR: 4 \u2640 with same data and deposition as holotype; 4 \u2642, 8 \u2640 (1 \u2642 used for DNA barcoding BC MTD 01844) with same data (USNM); 1 \u2640 , Napo, 6km NW Tena, Lumu Caspi, 0\u00b054'37\"S, 77\u00b049'32\"W, 590m, 29.ix.2002 (Schouten Coll.); 1 \u2640 , Pastaza, 1km N Santa Clara, 1\u00b016'02\"S, 77\u00b052'57\"W, 630m, 28.ix.2002 (Schouten Coll.). FRENCH GUIANA: 1 \u2642, 4 \u2640 , Saint Jean de Maroni (E. Le Moult) (BMNH); 2 \u2640, Piste Nancibo, km 6, 4\u00b041'N,; 52\u00b025'W, in logged rain forest, at 125W[atts] mer[cury]-vapor light and 15W[atts] U[ltra]V[iolet], 11.i.1985 (J.[-]F. Landry) (USNM); 1 \u2642, Roura, Montagne des Chevaux, xii.2008 (S. Delmas) (MHNG); 1 \u2642 , Cayen[ne] (BMNH). GUYANA: 1 \u2640 , Omai, vi.1908 (S. M. Klages) (BMNH); 1 \u2640 , Potaro i.1908 (S. M. Klages) (BMNH). PANAMA: 1 \u2640, Rio Trinidad, 12.iii [no year data] (A. Busck) (USNM). PERU: 1 \u2642 , Agnaytia, Huallaga, 400m, ix.1961 (CNC); 1 \u2640 , Yurimaguas, PageBreakHuallaga 14.iv.[19]20 (CMNH). SURINAME: 2 \u2640 , Kabo, 5\u00b016'N, 55\u00b044'W, Saramaca, black light, respectively 15\u201316.iii.1983 and 13\u201314.i.1983 (K.E.Neerling) (Schouten Coll.); 1 \u2640 , Sipaliwini Distr[ict]., Tibiti area, Kabo Creek, partly swampy primary forest on hilly slopes, ca 2km from river, vi.1989 (J. Beerlink) (Schouten Coll.).Other specimen examined. 1\u2640 (used for DNA sequencing Lep 1126), Peru, Hu\u00e1nuco, Rio Llullapichis, Panguana, 74,945\u00b0W / 9,614\u00b0S, 23.9.\u201310.10.2011 (SMTD).COI barcode sequence of paratype 07-SRNP-113921 (654 bp): ACATTATATTTTATTTTCGGGATTTGAGCAGGTATAGTAGGAACTTCACTTAGATTATTAATTCGTGCTGAATTAGGTAACCCTGGCTCTCTTATTGGAGATGATCAAATTTATAATACTATTGTAACAGCCCATGCATTTATTATAATTTTTTTTATAGTTATACCTATTATAATCGGTGGATTTGGAAATTGATTAGTTCCTTTAATATTAGGGGCACCAGATATAGCTTTCCCTCGAATAAATAACATAAGATTTTGATTATTACCACCATCATTAACTCTTTTAATTTCTAGAAGAATTGTAGAAAATGGAGCTGGAACAGGATGAACAGTTTATCCACCTTTATCATCTAATATTGCCCATGGGGGTAGATCTGTAGATTTAACAATTTTTTCATTACATTTAGCTGGAATTTCATCAATTTTAGGAGCTATTAATTTTATTACAACAATTATTAATATACGAATTAATAATTTATCATTTGATCAATTATCATTATTTATTTGATCAGTAGGAATTACTGCTTTACTTTTATTATTATCATTACCAGTTTTAGCTGGGGCTATTACTATACTTTTAACTGATCGAAATCTTAATACATCATTTTTTGATCCAGCAGGAGGAGGAGATCCAATTTTATATCAACATTTAmayrabonillae group are the shape of the forewing outer margin, which is slightly produced apically in Catharylla mayrabonillae and not produced in Catharylla paulella, and the forewing median transverse line with two strongly pronounced spots at 1/3 and 2/3 in Catharylla paulella, whereas these spots are lacking in Catharylla mayrabonillae. The hindwing of Catharylla mayrabonillae has a faded subterminal transverse line on costal half whereas the hindwing of Catharylla paulella lacks this marking. In male genitalia, the heavily sclerotized sacculus bears a dorso-lateral sclerotized string of short spines on distal 1/4 whereas the two processes of the costa are S-shaped in Catharylla paulella, and the apex of the phallus is trifid, rounded medially, shortly triangular laterally, whereas it is simply rounded in Catharylla paulella. In female genitalia, the sterigma forms double rounded cavities with a mustachio-shape arrangement of short spines in ventral view, and the ductus bursae is wide, progressively widening toward corpus in Catharylla mayrabonillae, whereas the sterigma forms a pair of shallow rounded pockets on each side of middle and the ductus bursae is narrow, with the rounded corpus bursae clearly differentiated from it in Catharylla paulella.The best discriminant characters externally between the two species of the PageBreakdorsally ashen brown, tibia and tarsomeres ochreous, distally ringed with dark brown. Midleg femur white, tibia light ochreous, basally brown, tarsomeres II\u2013V ochreous with tips ringed white. Hindleg white, except tarsomeres, as in midleg. Abdomen dull white. Forewing length: 7.5\u20138.5 mm; with apex slightly produced; costal line thin, ochreous or white in basal half, white in apical half; median transverse line ochreous, slightly undulated; subterminal transverse line ochreous; transverse lines enlarging into brown spot on costal margin with ochreous bar on costa following subterminal transverse line; terminal sector with light ochreous between veins, margin with thin, dark brown line from apex to CuA1, with two dark brown spots in cubital sector, with spot between CuA1 and CuA2 slightly displaced toward base; fringes brass colored; underside light ochreous with some brownish scales, with thin brown margin. Hindwing white with thin transverse subterminal line faded ochreous, in continuity with forewing median transverse line; outer margin line pronounced, dark brown; underside dull white with thin faded brown margin; fringes white.Male (n = 17) : Head win=7): Transverse ridge regularly rounded, medially slightly flattened. Tympanic pockets broadly rounded, extended widely beyond transverse ridge, connected medially at base of praecinctorium. Tympanic drum bean-shaped, elongated, extended beyond tympanic pockets.Tympanal organs , 31: UncFemale (n = 37): Labial palpi length: 1.1\u20131.3 mm. Forewing length: 9.5\u201310.5 mm; frenulum triple.PageBreakside of enlargement and posterior section. Corpus bursae circular to elongate, about as long as tergite VII; single signum faintly pronounced.Female genitalia (n = 16) : PapillaCatharylla and the only one so far found in Central America and in Venezuela, Columbia, Ecuador and Peru.The species has been found so far in Panama, Costa Rica, Colombia, Venezuela, Guyana, Suriname, French Guiana, Ecuador, Peru and Brazil . It is tCatharylla mayrabonillae is named in honor of Ms. Mayra Bonilla of San Jose, Costa Rica, in recognition of her artistic portrayal of the biodiversity and ecosystems of Costa Rica and her many years of support for the existence of the rain forest in Area de Conservacion Guanacaste.The relatively strong COI barcode divergence of 4.34% between samples LEP 1126 from Peru and 07-SRNP-113921 from Costa Rica is notabSchaus, 1922http://species-id.net/wiki/Catharylla_paulellaCatharylla paulella Schaus, 1922: 131; Holotype. \u2640, with labels as follows: \u201cSao Paulo | S.E. Brazil.\u201d; \u201cCollection | W[illia]mSchaus\u201d; \u201cType No. | 25533 | U.S.N.M.\u201d [orange label]; \u201cSLIDE | SB \u2640 | No.4641\u201d [light blue label]; \u201cCatharylla | paulella | type Sch[au]s\u201d [hand written]; \u201cGenitalia Slide | By SB | USNM 111,535\u201d [green rectangular label with thin black line submarginally]. Deposited in USNM.Other specimens examined. 2 \u2642, 7 \u2640. BOLIVIA: 2 \u2642 , Prov.[incia] del Sara, 450 m, iv.1910 (J. Steinbach) . BRAZIL: 1 \u2640 , Federal District, Planaltina, 15\u00b035'S, 47\u00b042'W, 1000 m, 3.xi.1977 (V. O. Becker n\u00b022055) (Becker Coll.), 1 \u2640 with same locality, 16.x.1990 (V. O. Becker n\u00b096854) (Becker Coll.); 1 \u2640, Maranh\u00e3o, Feira Nova, Faz[enda]. Retiro, 480m, 07\u00b000'S, 46\u00b026'W, 1\u20133.xii.2011 (V. O. Becker n\u00b0148263) (Becker Coll.); 1 \u2640 , Mato Grosso, Urucum, 15 miles S[outh]. of Columb\u00e1, 650 f[ee]t, 19. iv. [19]27, at light (C. L. Collenette) (BMNH); 1 \u2640, Par\u00e0, Bel\u00e9m, 20m, i.1984 (V. O. Becker n\u00b046993) (Becker Coll.); 1 \u2640 , S\u00e3o Paulo (BMNH); 1 \u2640 , S\u00e3o Paulo, S\u00e3o Luiz do Paraitinga, 23\u00b020'S, 45\u00b006'W, 900 m, 13\u201320.iii.2001 (V. O. Becker n\u00b0132357) (Becker Coll.).PageBreakTATTAATTCGTGCTGAATTAGGTAATCCTGGATCTCTTATTGGTGATGATCAAATTTATAATACTATTGTAACAGCTCATGCATTTATTATAATTTTTTTTATAGTTATACCTATTATAATTGGTGGATTTGGAAATTGATTAGTTCCTTTAATATTAGGTGCACCAGATATAGCTTTCCCTCGAATAAATAATATGAGATTTTGATTATTACCCCCATCATTAACTCTTTTATTTT?TAGAAGAATTGTCGAAAATGGAACTGGAACAGGATGAACAGTTTACCCACCCTTATCATCCAATATTGCTCATAGAGGTAGATCAGTAGATCTAGCAATTTTTTCTTTACATTTGGCTGGAATTTCATCAATCTTAGGAGCTATTAATTTTATTACAACAATTATCAATATACGAATTAATAATTTATCTTTTGATCAATTATCATTATTTATTTGATCTGTAGGTATTACAGCTTTACTTTTATTATTATCATTACCAGTTCTAGCTGGAGCTATTACTATACTTTTAACTGATCGAAATCTTAATACATCATTTTTTGATCCTGCAGGAGGAGGTGATCCTATCTTGTATCAACATTTACOI barcode sequence of specimen LEP 965 (654 bp): ACATTATATTTTATTTTTGGAATTTGAGCAGGTATACTAGGAACTTCACTTAGATCatharylla species by the forewing median transverse line with two strongly pronounced spots at 1/3 and 2/3. The forewing is also sparkled with dark brown scales, which is unique in the genus. In male genitalia, the two S-like projections of the costal arm of the valva discriminate this species from the other species of Catharylla. In female genitalia, the sterigma forms a pair of shallow rounded pockets on each side of middle, and the ductus bursae is narrow, with the rounded corpus bursae clearly differentiated from it in Catharylla paulella, whereas it forms double rounded cavities with a mustachio-shape arrangement of short spines in ventral view, and the ductus bursae is wide, progressively widening toward corpus in Catharylla mayrabonillae.This species can be easily separated from the other Male (n = 2): Head with ochreous chaetosemata. Antenna ochreous with white scales, with patch of dark brown scales at base. Maxillary palpus light ochreous, white tipped. Labial palpus: 1.4\u20131.7 mm long, light ochreous, white tipped. Thorax light ochreous at collar. Foreleg coxa whitish ochreous, femur light ochreous, dorsally ochreous; tibia and tarsomeres greyish brown, distally ringed with dark brown. Midleg and hindleg whitish ochreous; midleg tibia basally brown, hindleg tibia white; midleg and hindleg tarsomeres with white tips. Forewing length: 7\u20138 mm; costal line thin, brown or dirty white; median transverse line ochreous, with two dark brown strongly pronounced spots at 1/3 and 2/3; subterminal transverse line thin, ochreous, with small triangular spot on costal margin; with ochreous bar on costal margin following subterminal transverse line; outer margin ochreous with short dark brown lunules or dashes; fringes brass colored; underside light ochreous with brownish suffusion; with pronounced marginal spots. Hindwing white; outer margin with thin ochreous line in apical half; fringes white; underside dull white with dark brown marginal spots more or less connected on apical half.PageBreaklus with apex more thickly sclerotized, with blunt apical margin, with short triangular ventral projection; vesica covered on basal 1/4 with tiny spicules, with barely visible microspicules all along, with one wide and curved cornutus at about 1/4 length of phallus.Male genitalia (n = 2) , 33: UncFemale (n = 7) , 10: LabTympanal organs (n = 5) : TransveFemale genitalia (n = 5) : PapillaThe species has been found in Brazil and in Bolivia .Catharylla hibisca to specimens that appear to be Catharylla paulella. The BMNH S\u00e3o Paulo specimen is associated with slide n\u00b0 17692, but the genitalia on this slide seem to be wrongly associated, given the inscription \u201cwrong abdomen?\u201d on the label, as well as the size of the abdomen, which is much bigger than those of Catharylla paulella. Therefore, this specimen cannot be identified with certainty. An error is possible in the association of the sexes of this species as there are no series of both sexes from the same locality or other means of associating them with 100% confidence.The original description doesn\u2019t mention the original number or sex of the specimens but it is assumed that there was only one. S. Bleszynski gave the new name of The number of bases obtained for each barcode sequence is given in Catharylla species. We observe a relatively high divergence between different barcode haplotypes in Catharylla bijuga (5.05%), Catharylla mayrabonillae (4.34%), Catharylla serrabonita (3.24%) and Catharylla tenellus (3.34%), sometimes possibly associated with differences in morphological characters and with geographical distances. The divergence between Catharylla chelicerata samples LEP 1703 and LEP 1704 is of 0.15% (1 base) because they are issued from the same population. Sample LEP 1290 differs from samples LEP 1703 and LEP 1704 respectively by 0.62 and 0.46%. We observe no variation between samples LEP 1708, LEP 1709, LEP 1710 and LEP 1888 because they are all issued from the same population. The inter-specific variation in COI barcode sequences (6.29\u201316.84%) is always higher than the intraspecific variation (0.15\u20135.05%).As most of the data were restricted to few sequence samples per species, and some of the sequences came from the same populations, we don\u2019t have the definitive picture of the intraspecific variation in the COI barcode sequences of The 21 analysed characters are listed in PageBreakPageBreakPageBreakCatharylla is supported by the analysis of mol_1 and the combined Bayesian analysis and by the analyses of nucl_1 and nucl_2 . Three synapomorphies and one non-unique apomorphy support the group. The mayrabonillae group is well supported in all analyses except in the morphology-based analysis (BS support = 60), because no clear synapomorphy was found for the group. However, two non-unique synapomorphies and one reversal (observed only once in Catharylla) are observed . The tenellus group is well supported by the morphology-based analysis (BS support = 97) with four clear synapomorphies and two reversals , but show no support in other analyses, probably because only the barcode sequence was available for Catharylla coronata. The closer relationship between Catharylla serrabonita and Catharylla tenellus within the tenellus group is supported by the combined Bayesian analysis and by one non-unique apomorphy (but unique in Catharylla). The tenellus group as represented in the analyses of nucl_1 and nucl_2 (Catharylla serrabonita + Catharylla tenellus) is well supported . The chelicerata group is well supported by the morphology-based analysis (BS support = 96) and the combined Bayesian analysis (PP = 1.00). The group shows two synapomorphies , one non-unique apomorphy (7:1) and one reversal (11:0). Unfortunately, no sequence was available for Catharylla gigantea, thus we cannot compare the morphology with the molecular-based analyses. The closer relationships between the chelicerata and the mayrabonillae groups is strongly supported by the nucl_1 analysis (BS support = 100) and the combined Bayesian analysis (PP = 1.00). Two reversals support the group. The position of Catharylla bijuga as sister group of the other Catharylla species is weakly supported by the combined Bayesian analysis (PP = 0.90), and show no support in other analyses. The position of Micrelephas pictellus as sister group of Catharylla is supported by the analysis of mol_2 and nucl_2 (respective BS supports of 91 and 100). This node is supported by one synapomorphy . Node 10 (Argyria lacteella+ Micrelephas pictellus+ Catharylla) is supported by the analysis of mol_1 (BS support = 100) and the combined Bayesian analysis (PP = 1.00). One synapomorphy (2:1) supports the group. The monophyly of the two Crambus species chosen as outgroups is well supported in the mol_1 analysis (BS support of 100). The settings of MrBayes do not allow to choose two outgroups, therefore the monophyly of the two Crambus species is not supported by the combined Bayesian analysis. Three non-unique synapomorphies support the genus based on these two species . The synapomorphies of the different groups highlighted by the phylogeny are reported here below:The monophyly of Chelicerata group (node 3)8:1. Apex of valva quadriangular, truncated9:2. Gnathos regularly curvedTenellus group (node 5)5:1. Presence of a dorsal furrow on the uncus7:3. Apex of uncus slightly bifid10:1. Transtilla present20:1. Postvaginal sterigma absentCatharylla (node 8)11:1. Lateroventral projections on juxta17:1. Posterior apophyses/papillae anales < 0.519:1. Ventral membrane of segment VIII with tiny setaeCatharylla + Micrelephas (node 9)6:1. Uncus dorsally bare, or with few setaeCatharylla + Micrelephas + Argyria (node 10)2:1. Length of labial palpi/eye diameter < 2/1PageBreakPageBreakPageBreakCatharylla occurs northward from Middle America with locality records in Costa Rica (Area de Conservacion Guanacaste) at a latitude of 10\u00b054 N, southward to Rio Vermelho at a latitude of 27\u00b030 S, from sea level up to 1300 m . Within Catharylla, species and species groups show distinct distribution patterns. The chelicerata group is widespread in the Northern Amazonian rainforest of Brazil, and in the three Guyanas , whereas Catharylla paulella is widespread from Feira Nova (07\u00b000 S) down to S\u00e3o Paulo (23\u00b035 S) , in the Chaco formation while haplotype BC MTD 1840 is from the Humid Guyana province . Catharylla mayrabonillae barcode haplotype 07-SRNP-113921 is found in the Carribean subregion, whereas haplotype LEP 1126 is found in the Amazonian subregion. The barcode haplotypes of the Catharylla serrabonita populations from the two coastal localities of Linhares and Porto Seguro are more closely related, whereas the haplotype from the population of the forested hills of the Serra Bonita Reserve clearly diverges from the other two . They arFederal) , 44, wheFederal) . CatharyBolivia) , 44, whiBolivia) . Catharydiagonal formed bdiagonal , constitther two .PageBreakCatharylla (nodes 7 & 8). The reduced number of taxa , as well as the quality of the datasets (complete sets of genes for nucl_1 and nucl_2 vs partly complete sets of genes in mol_1 and mol_2) explain the better results obtained in nucl_1 and nucl_2, because taxa with incomplete data tend to lower the resolution and the bootstrap supports of the tree because of the great divergence of these sequences from those of other Catharylla species. However, the clear support brought by the morphological analysis places Catharylla coronata together with Catharylla serrabonita and Catharylla tenellus. The neighbor-joining (NJ) analysis of barcode sequences of Catharylla species (not represented here) places Catharylla coronata together with Catharylla serrabonita (divergence of 7.59% with the sequence BC MTD 1843) and Catharylla tenellus (divergence of 7.3% with the sequence BC MTD 1842), and thus corroborates the findings of the morphology-based analysis. Moreover, the species is morphologically very similar to Catharylla serrabonita especially regarding the transtilla in the male genitalia. The basal position of Catharylla bijuga in Catharylla, as sister taxon of other Catharylla species, is doubtful (PP = 0.90) and may be an artefact due to the great divergence of the barcode sequence (the lowest divergence with other Catharylla species is of 9.7% with sample BC MTD 1843 of Catharylla serrabonita). The supports brought by the combined Bayesian analysis have to be carefully considered since the results are sensitive to small variations of the taxon and character sampling, and the posterior probabilities tend to overestimate the strongness of the nodes along with Argyria, Urola and Vaxi, and Micrelephas in Crambini , Catharylla mayrabonillae (4.34%), Catharylla serrabonita (3.24%) and Catharylla tenellus (3.34%) could therefore suggest that these different barcode haplotypes represent different species. Some morphological variation in male genitalia was found in Catharylla serrabonita and Catharylla tenellus , as well as Catharylla serrabonita and Catharylla tenellus (both collected in Porto Seguro), while Catharylla coronata and Catharylla serrabonita are vicariant, Catharylla serrabonita being distributed north of Linhares, whereas Catharylla coronata is found south of this locality. The patterns of distribution suggest that the diversification of the tenellus group might have occurred in the Atlantic Forest, or have occurred in a wider area that reduced afterwards. In Catharylla tenellus, two forms of male valva linked to different COI barcode sequences seem to be geographically separated, with the form from Ubatuba (associated to barcode sequence BC MTD 1842), Bertioga, S\u00e3o Paulo (S\u00e3o Paulo State), and Cara\u00e7a (Minas Gerais) occurring more to the south than the form from Porto Seguro . The third form, observed in Cara\u00e7a (Minas Gerais) suggests that this locality could represent a point of contact between the two other forms. Regarding Catharylla serrabonita, the locality of Serra Bonita is a moist forest of middle elevation (800 m) , while t"} +{"text": "Drosophila SCA3 models with Hsp104, a powerful protein disaggregase from yeast, which is bafflingly absent from metazoa. Hsp104 suppressed eye degeneration caused by a C-terminal ataxin-3 (MJD) fragment containing the pathogenic expanded PolyQ tract, but unexpectedly enhanced aggregation and toxicity of full-length pathogenic MJD. Hsp104 suppressed toxicity of MJD variants lacking a portion of the N-terminal deubiquitylase domain and full-length MJD variants unable to engage polyubiquitin, indicating that MJD-ubiquitin interactions hinder protective Hsp104 modalities. Importantly, in staging experiments, Hsp104 suppressed toxicity of a C-terminal MJD fragment when expressed after the onset of PolyQ-induced degeneration, whereas Hsp70 was ineffective. Thus, we establish the first disaggregase or chaperone treatment administered after the onset of pathogenic protein-induced degeneration that mitigates disease progression.There are no effective therapeutics that antagonize or reverse the protein-misfolding events underpinning polyglutamine (PolyQ) disorders, including Spinocerebellar Ataxia Type-3 (SCA3). Here, we augment the proteostasis network of Drosophila models of SCA3. Hsp104 has no homologue in animals, but has an unusual ability to dissolve PolyQ aggregates in vitro, an activity that could be harnessed therapeutically. Indeed, Hsp104 suppressed degeneration caused by a C-terminal ataxin-3 (MJD) fragment containing the pathogenic expanded PolyQ tract, which accumulates in disease. However, Hsp104 enhanced aggregation and toxicity of full-length pathogenic MJD. Hsp104 rescued forms of MJD unable to engage polyubiquitin or with a deletion in the deubiquitylase domain indicating that MJD-ubiquitin interactions hinder protective Hsp104 activities. Importantly, Hsp104 suppressed toxicity of a C-terminal MJD fragment when expressed after the onset of PolyQ-induced degeneration, whereas Hsp70 was ineffective. Thus, we establish the first disaggregase or chaperone treatment administered after the onset of pathogenic protein-induced degeneration that mitigates disease progression.There are no effective therapeutics for any of the neurodegenerative disorders caused by expanded polyglutamine (PolyQ) tracts including Spinocerebellar Ataxia Type-3 (SCA3). These disorders are connected with the misfolding and aggregation of proteins bearing expanded PolyQ tracts in the neurons of affected individuals. In SCA3, ataxin-3 (MJD) is the protein that bears the PolyQ expansion and forms insoluble aggregates. Here, as a therapeutic strategy we introduce Hsp104, a powerful protein disaggregase from yeast, into Many neurodegenerative diseases, such as Alzheimer's Disease, Parkinson's Disease (PD), prion disease, and the collection of polyglutamine (PolyQ) disorders, including Huntington's Disease (HD) and the Spinal Cerebellar Ataxias (SCAs), are characterized by the formation of protein inclusions in the nervous system Despite the extraordinary structural stability of amyloid, a protein disaggregase from yeast, Hsp104, can rapidly solubilize amyloid. Hsp104 is a hexameric AAA+ (ATPases Associated with diverse cellular Activities) protein that couples ATP hydrolysis to translocation of substrate through a central pore, thus prying individual monomers from the amyloid fiber Curiously, Hsp104 has no homologue in metazoa. Indeed, until recently it was unclear whether the metazoan proteostasis network possessed any coupled protein disaggregase and reactivation machinery. It is now clear that Hsp110, Hsp70, and Hsp40 collaborate to promote the dissolution and reactivation of disordered aggregates in vitro, with the Josephin domain forming SDS-soluble linear polymers that then convert into SDS-insoluble PolyQ-driven amyloid fibers Spinocerebellar Ataxia Type 3 or Machado-Joseph Disease (MJD/SCA3) is the most prevalent dominantly inherited ataxia C. elegans, Hsp104 prevented aggregation and toxicity of GFP-tagged PolyQ in vivo after aggregates have already formed and degeneration has begun; a situation likely to mimic an actual therapy. Therefore, we created novel Hsp104 Drosophila lines to exploit well-characterized models of disease in combination with powerful genetic tools to temporally control the expression of Hsp104 after disease-associated aggregation and degeneration has begun.Hsp104 has been introduced to combat protein-aggregation disease in metazoan systems with various levels of success in vivo.Our studies reveal surprisingly distinct interactions of Hsp104 with the full-length versus a truncated version of the MJD protein. Importantly, we establish that Hsp104 possesses the ability to suppress the progression of degeneration when activated subsequent to onset of expression of the disease protein. These data indicate that protein context is central in Hsp104 interactions, and that Hsp104 displays the ability to halt the progression of pre-established disease Drosophila to evaluate its ability to prevent and potentially reverse aggregation of disease-associated human proteins, readily available in various fly models of disease. To achieve strong expression of the Hsp104 protein in the fruit fly, we codon-optimized the transgene for Drosophila (see ACAAA) before the start codon Drosophila using the GAL4/UAS system glass gene element for driving GAL4 expression, we instead used a driver line with reduced expression and the truncated C-terminal region of the protein containing the expanded glutamine tract (MJDtrQ78) To probe the mechanism underlying the dichotomous results found for the Hsp104 interaction with MJDtrQ78 and MJDnQ78, an in-depth investigation of the protein aggregates was performed. To slow protein aggregation such that we could analyze underlying protein accumulations in detail, we expressed the transgenes in the eye with an adult-onset driver rhodopsin1(rh1)-GAL4. Analysis of the PolyQ protein accumulations showed that Hsp104 altered the kinetics of inclusion formation for both MJD protein isoforms. By cryosectioning and subsequent immunohistochemistry (IHC), MJDtrQ78 formed compact inclusions that increased in size over time , top rowTo examine protein accumulation by biochemical methods, we used SDD-AGE (Semi-Denaturing Detergent\u2013Agarose Gel Electrophoresis), a protein agarose gel technique that can resolve amyloid aggregates in vitroNext, we assessed MJDnQ78 misfolding. In contrast to the truncated MJDtrQ78 isoform, the pathogenic full-length MJDnQ78 initially formed amorphous inclusions that did not become more numerous after day 1 . Thus, tWhile it is known that in select conditions, Hsp104 promotes amyloid formation of specific yeast prions We also examined variants lacking DUB activity through mutation of the active site in the Josephin domain, MJD-Q88-C14A , which cTo uncover additional mechanistic insight into the interactions with Hsp104, we examined inclusion formation and kinetics with adult-onset rh1-GAL4 expression. By IHC, the MJD variants formed accumulations in a manner roughly consistent with severity of eye degeneration , precludA summary of the effect of Hsp104 on MJD variants is presented in DPLDWB), which is structurally identical to wild-type but functionally inactive DPLDWB were innocuous into Hsp104 to ensure that Hsp104 could not engage substrate or hydrolyze ATP, creating the mutant known as Double Pore Loop Double Walker B the toxic MJDtrQ78 protein driven directly by a gmr element such that the disease-associated protein was constitutively expressed in the eye; (2) a drug-inducible gmr-GAL4 driver known as \u201cGeneSwitch\u201d (gmr-GS) to activate GAL4 expression only in the presence of the drug RU486 (mifepristone) Hsp104 is unique in its capacity to reverse pre-existing amyloids in yeast and DPLDWB had no effect and progressed in severity to d7 , Fig. 10o effect . Hsp104 Next, we examined the underlying protein aggregates by SDD-AGE and Western immunoblot. We observed that gmr-MJDtrQ78 had high levels of amyloid, and this was lessened with time . When tuin vivo, which is its ability to mitigate the course of protein-aggregation disease even after it has already initiated. Indeed, our studies show that Hsp104 is able to mitigate disease progression once it has begun, unlike the classical metazoan chaperone Hsp70. These studies provide new insight into the in vivo effects of Hsp104 in the context of a therapeutic agent.Here, we reveal key novel insights into the efficacy and interactions of Hsp104 with the pathogenic PolyQ protein MJD. Our studies reveal the surprising finding that Hsp104 interacts differentially with different forms of the MJD protein. Hsp104 is a potent suppressor of toxicity of the truncated protein, but an enhancer of toxicity of the full-length protein. These differences are determined by specific domains of MJD that are not directly implicated in aggregation. Our findings have also uncovered a heretofore unrecognized and important application of Hsp104 Our detailed investigations of the effects of Hsp104 on the MJD protein led to the unexpected result that Hsp104 has opposite effects on the toxicity of different versions of the MJD protein (MJDtrQ78 and MJDnQ78), despite the fact that these proteins contain the same pathogenic PolyQ stretch. These disparate actions indicate that Hsp104 might be a useful probe to understand the nature of aggregates and the toxicity imposed by them. Hsp70 suppressed both MJDtrQ78 aggregation and toxicity , 3C. By What might these biochemical changes be? Hsp104 can disrupt toxic soluble oligomers of various proteins, including Sup35, which may help explain why Sup35 prion formation is not intrinsically toxic to yeast In other settings, it has been suggested that chaperone-initiated formation of large, insoluble amyloid aggregates can actually be protective by sequestering potentially toxic pre-fibrillar conformers Moreover, our findings demonstrate that an agent with a mitigating effect on the truncated version of the MJD protein may act in a different manner against the full-length MJD protein. Thus, what is good for one may not be beneficial for the other. In MJD/SCA3, as well as other neurodegenerative diseases, fragmentation of the disease protein may initiate aggregation and this process is critical for disease progression Although Hsp104 enhances MJDnQ78 amyloidogenesis and toxicity, we found that elimination of functional domains not implicated in PolyQ aggregation facilitated the ability of Hsp104 to suppress MJD-associated degeneration. Elimination of UIM functionality or removal of a component of the Josephin domain (exon 2) restored the remodeling capacity of Hsp104. This suggests that MJDnQ78 pathogenicity is not intrinsically intractable, but is capable of being suppressed by Hsp104 if other domains of the protein are inactivated . Alternatively, potentiated or MJDnQ78-optimized Hsp104 variants might be developed that are able to overcome these hindrances via increased unfolding power Our studies underscore the importance of protein context in studying protein-misfolding diseases. Within the protein itself, neighboring domains not thought to be involved in aggregation may be impacting accumulation kinetics and the biochemical properties of inclusions in vitro have characterized aggregation of the full-length, pathogenic MJD protein as a two-step process in which the protein assembles first into SDS-soluble fibrillar polymers associating via the Josephin domain, and then converts to SDS-insoluble amyloid fibers driven by the PolyQ domain in vivo as well. Indeed, it is consistent with our observation that full-length MJDnQ78 forms amorphous accumulations that appear visually by IHC before they can be observed as SDS-insoluble amyloid aggregates by SDD-AGE may increase efficiency of such interactions and enable Hsp104 to rescue disease phenotypes. Moreover, if UIM binding to poly-ub chains is impairing access of Hsp104 to MJD, this suggests that co-administering an agent to modulate function of a neighboring domain may affect the access of a treatment to the aggregation-prone domain. Indeed, increasing global DUB activity coupled with Hsp104 induction could overcome antagonism due to poly-ub chains.DrosophilaChaperone treatment, and examination of Hsp70 in particular, has been an exciting avenue of research in the battle to combat and contain neurodegenerative disease An inducible system is particularly well suited for Hsp104 because of its unique ability to rapidly dismantle pre-existing amyloid aggregates. Since metazoan chaperones can only very slowly depolymerize amyloid Our experimental paradigm offers the exciting possibility to address the efficacy of Hsp104 (or other molecules) in a more genuine therapeutic setting. Indeed, we found that turning on Hsp104 was able to significantly suppress disease-associated degeneration. Interestingly, however, Hsp104 did not disaggregate MJDtrQ78 amyloid in these experiments . Thus, Hin vitro and in the most appropriate animal models. Moreover, the fact that Hsp104 is well tolerated by mammalian systems is encouraging Naturally, several barriers must be surmounted to translate Hsp104 into a therapeutic agent for human neurodegenerative disease Clostridium botulinum, as a therapeutic agent. Despite being a deadly toxin, botulinum toxin variants have found key clinical applications due to their highly potent and selective ability to cleave SNARE proteins and prevent secretion Finally, the concept of using a yeast protein as the basis for a therapeutic agent might at first glance seem implausible. However, it must also have seemed equally implausible to use a lethal protein toxin from the bacterium, DPLDWB were generated by standard techniques using the pUAST vector Drosophila and a Kozak sequence (ACAAA) was added prior to the start codon Transgenic flies expressing UAS-Hsp104 and UAS-Hsp104The full sequence of codon-optimized Hsp104 is:ATGAACGATCAGACCCAGTTCACCGAGCGCGCCCTGACCATCCTGACCCTGGCCCAGAAGCTGGCCAGCGATCACCAGCACCCCCAGCTGCAGCCCATCCACATCCTGGCCGCCTTCATCGAGACCCCCGAGGATGGCAGCGTGCCCTACCTGCAGAACCTGATCGAGAAGGGCCGCTACGATTACGATCTGTTCAAGAAGGTGGTGAACCGCAACCTGGTGCGCATCCCCCAGCAGCAGCCAGCCCCAGCCGAGATCACCCCAAGCTACGCCCTGGGCAAGGTGCTGCAGGATGCCGCCAAGATCCAGAAGCAGCAGAAGGATAGCTTCATCGCCCAGGATCACATCCTGTTCGCCCTGTTCAACGATAGCAGCATCCAGCAAATCTTCAAGGAGGCCCAGGTGGATATCGAGGCCATCAAGCAGCAGGCCCTGGAGCTGCGCGGAAACACCCGCATCGATAGCCGCGGAGCCGATACCAACACCCCCCTGGAGTACCTGAGCAAGTACGCCATCGATATGACCGAGCAGGCCCGCCAGGGAAAGCTGGACCCAGTGATCGGACGCGAGGAGGAGATCCGCAGCACCATCCGCGTGCTGGCCCGCCGCATCAAGAGCAACCCATGCCTGATCGGAGAGCCAGGAATCGGCAAGACCGCCATCATCGAGGGAGTGGCCCAGCGCATCATCGATGATGATGTGCCAACCATCCTGCAGGGAGCCAAGCTGTTCAGCCTGGATCTGGCCGCCCTGACCGCCGGCGCCAAGTACAAGGGCGATTTCGAGGAGCGCTTCAAGGGCGTGCTGAAGGAGATCGAGGAGAGCAAGACCCTGATCGTGCTGTTCATCGATGAGATCCACATGCTGATGGGCAACGGCAAGGATGATGCCGCCAACATCCTGAAGCCAGCCCTGAGCCGCGGACAGCTGAAGGTCATCGGAGCCACCACCAACAACGAGTACCGCAGCATCGTGGAGAAGGATGGAGCCTTCGAGCGCCGCTTCCAGAAGATCGAGGTGGCCGAGCCAAGCGTGCGCCAGACCGTGGCCATCCTGCGCGGACTGCAGCCCAAGTACGAGATCCACCACGGCGTGCGCATCCTGGATAGCGCCCTGGTGACCGCCGCCCAGCTGGCCAAGCGCTACCTGCCATACCGCCGCCTGCCAGATAGCGCCCTGGATCTGGTGGATATCAGCTGCGCCGGAGTGGCCGTGGCCCGCGATAGCAAGCCAGAGGAGCTGGATAGCAAGGAGCGCCAGCTGCAGCTGATCCAGGTGGAGATCAAGGCCCTGGAGCGCGATGAGGATGCCGATAGCACCACCAAGGATCGCCTGAAGCTGGCCCGCCAGAAGGAGGCCAGCCTGCAGGAGGAGCTGGAGCCACTGCGCCAGCGCTACAACGAGGAGAAGCACGGCCACGAGGAGCTGACCCAGGCTAAGAAAAAGCTGGATGAGCTGGAGAACAAGGCCCTGGATGCCGAGCGCCGCTACGATACCGCCACCGCCGCCGATCTGCGCTACTTCGCCATCCCCGATATCAAGAAGCAGATCGAGAAGCTGGAGGATCAGGTGGCCGAGGAGGAGCGCCGCGCCGGCGCCAACAGCATGATCCAGAACGTGGTGGATAGCGATACCATCAGCGAGACCGCCGCCCGCCTGACCGGCATCCCCGTGAAGAAGCTGAGCGAGAGCGAGAACGAGAAGCTGATCCACATGGAGCGCGATCTGAGCAGCGAGGTGGTGGGCCAGATGGATGCCATCAAGGCCGTGAGCAACGCCGTGCGCCTGAGCCGCAGCGGACTGGCCAACCCACGCCAGCCAGCCAGCTTCCTGTTCCTGGGCCTGAGCGGCAGCGGCAAGACCGAGCTGGCCAAGAAGGTGGCCGGCTTCCTGTTCAACGATGAGGATATGATGATCCGCGTGGATTGCAGCGAGCTGAGCGAGAAGTACGCCGTGAGCAAGCTGCTGGGCACCACCGCCGGCTACGTGGGCTACGATGAGGGCGGCTTCCTGACCAACCAGCTGCAGTACAAGCCCTACAGCGTGCTGCTGTTCGATGAGGTGGAGAAGGCCCACCCCGATGTGCTGACCGTGATGCTGCAGATGCTGGATGATGGCCGCATCACCAGCGGCCAGGGCAAGACCATCGATTGCAGCAACTGCATCGTGATCATGACCAGCAACCTGGGCGCCGAGTTCATCAACAGCCAGCAGGGCAGCAAGATCCAGGAGAGCACCAAGAACCTGGTCATGGGCGCCGTGCGCCAGCACTTCCGCCCCGAGTTCCTGAACCGCATCAGCAGCATCGTGATCTTCAACAAGCTGAGCCGCAAGGCCATCCACAAGATCGTGGATATCCGCCTGAAGGAGATTGAGGAGCGCTTCGAGCAGAACGATAAGCACTACAAGCTGAACCTGACCCAGGAGGCCAAGGATTTCCTGGCCAAGTACGGCTACAGCGATGATATGGGCGCCCGCCCCCTGAACCGCCTGATCCAGAACGAGATCCTGAACAAGCTGGCCCTGCGCATCCTGAAGAACGAGATCAAGGATAAGGAGACCGTGAACGTGGTGCTGAAGAAGGGCAAGAGCCGCGATGAGAACGTGCCAGAGGAGGCCGAGGAGTGCCTGGAGGTGCTGCCAAACCACGAGGCCACCATCGGAGCCGATACCCTGGGCGATGATGATAACGAGGATAGCATGGAGATCGATGATGATCTGGATTAA5\u2032-TCGAACCCAGTGGAAACCCTTGAAATGCCTTTAACTCGAGACGG-3\u2032 and 5\u2032-GTACCCGTCTCGAGTTAAAGGCATTTCAAGGGTTTCCACTGGGT-3\u2032), with a single copy of the 31 bp glass-binding site from the Rh1 proximal enhancer, were duplexed and ligated into the vector, producing p1\u00d7GR (1 copy of glass reporter). This plasmid was then modified to introduce the GAL4 coding sequence, excised from pGaTN Multiple insertion lines were characterized for each transgene. To create the 1\u00d7gr-GAL4 driver line, the pGMR (glass multimer reporter) vector 2) area was selected; particle analysis was performed with ImageJ and statistics performed with one-way ANOVA and unpaired t-test.Heads were frozen in Tissue Freezing Medium (Electron Microscopy Sciences) and sectioned at 12 \u00b5m by cryotome, and the tissue sections were then fixed with 4% paraformaldehyde. Immunohistochemistry was performed according to standard procedures using primary antibodies anti-HA 5B1D10 or anti-myc 9E10 (both mouse) alongside either anti-Hsp104 or anti-Hsp70 (both rabbit). Hsp70 staining was confirmed with human-specific anti-Hsp70 (mouse) alongside anti-HA Y11 or anti-myc A14 (both rabbit). Rabbit primary antibodies were preadsorbed at 1\u223625 with fixed, dissected wild-type larvae. Secondary antibodies were Alexa Fluor 594 Goat-anti-Mouse IgG , Alexa Fluor 488 Goat-anti-Rabbit IgG , Alexa Fluor 594 Goat-anti-Rabbit IgG , and Alexa Fluor 488 Goat-anti-Mouse IgG . Sections were co-stained with Hoechst nuclear dye and viewed with a Leica fluorescence microscope. A 75 \u00b5m\u00d775 \u00b5m square and anti-actin with secondary antibody Goat-anti-Rabbit-HRP . For MJD aggregation analysis through SDD-AGE (Semi-Denaturing Detergent Agarose Gel Electrophoresis) and accompanying Western immunoblots, heads were ground in lysis buffer A 4.0 mg/ml stock solution of RU486 (Sigma M8046) was prepared in 100% ethanol, and then 50 \u00b5l (200 \u00b5g) was added to pre-prepared food vials containing \u223c12 ml of food and gently shaken overnight"} +{"text": "Although Iph1 cleaved hallmark IDE substrates including insulin efficiently, its role in the ER stress response was independent of its catalytic activity since expression of inactive Iph1 restored normal sensitivity. Importantly, wild type as well as inactive human IDE complemented gene-invalidated yeast cells when expressed at the genomic locus under the control of +iph1 promoter. These results suggest that IDE has a previously unknown function unrelated to substrate cleavage, which links sensitivity to ER stress to a pro-survival role of the TORC1 pathway.Insulin Degrading Enzyme (IDE) is a protease conserved through evolution with a role in diabetes and Alzheimer's disease. The reason underlying its ubiquitous expression including cells lacking identified IDE substrates remains unknown. Here we show that the fission yeast IDE homologue (Iph1) modulates cellular sensitivity to endoplasmic reticulum (ER) stress in a manner dependent on TORC1 (Target of Rapamycin Complex 1). Reduced sensitivity to tunicamycin was associated with a smaller number of cells undergoing apoptosis. Wild type levels of tunicamycin sensitivity were restored in Human Insulin Degrading Enzyme (hIDE) or insulinase belongs to the M16A family of peptidases, which comprises large zinc-dependent metalloproteases found in all prokaryotic and eukaryotic organisms examined The budding yeast orthologue of hIDE, Ste23p, displays similar substrate specificity as mammalian IDE and, together with Axl1, the second yeast M16A metalloprotease, cleaves the precursor of the mating pheromone a-factor Cellular proteins are subjected to continuous damage and maintenance of protein homeostasis is central to all biological processes. A cellular compartment particularly susceptible to protein damage is the endoplasmatic reticulum (ER). Accumulation of misfolded proteins in the ER induces the Unfolded Protein Response (UPR) that increases the level of chaperones, stimulates retro-translocation of misfolded proteins to the cytosolic proteolytic system and attenuates general translation and transcription. If this response cannot resolve the ER stress, apoptotic pathways are engaged Schizosaccharomyces pombe has two: the non-essential +tor1 gene encodes the kinase forming the TORC2 complex, while the essential +tor2 gene encodes the kinase present in the TORC1 complex tor2-S1837E mutant, which is predicted to prevent interaction with the FKBP12-rapamycin complex in vitro, similarly to other organisms The generation and resolution of cellular stress is intimately linked to the evolutionary conserved target of rapamycin (TOR) kinase, which regulates cell growth according to nutrient and energy availability Unbridled activation of mammalian TOR exacerbates cellular stress and is linked to diabetes, cancer and a shorter life span S. pombe homologue of mammalian IDE as model.Several facts concerning IDE \u2013 its preference for amyloidogenic substrates able to cause cellular stress, its high expression in beta cells subjected to permanent ER stress, its dead-end chaperone function, indirect evidence for unidentified functions that might underlie its ubiquitous expression \u2013 prompted us to speculate that IDE might be implicated in the response to proteotoxic stress. To address this hypothesis, we took advantage of its evolutionary conservation and used the Strains are listed in iph1 with KanR marker: (5\u2032TGGCCTCTAAACAGTAATGCCTACGTACTGTGTGTATGTAAACACATAATTCAACCTATTGCCATATTTCTTACATATTACGGATCCCCGGGTTAATTAA-3\u2032) and (5\u2032CTAGCAGAAGAGTAGGTCTCGTCACACTTGTTTGGATAGCGAGAAAAACCGCAGTGCCAGAATGCAAAACTGAAATTAAGGAATTCGAGCTCGTTTAAAC3\u2032).Gene disruption and replacement were performed by PCR-based gene targeting iph1 with ura4+ marker:Primers used to to disrupt (5\u2032TATTACCCTTTTTTTGGGTGTAATAGCAGTAGTCAGAATTCTGGGTTGTTTTATCTTTTCCTTTCATAAATAAAAACGCCAGGGTTTTCCCAGTCACGAC-3\u2032) and (5\u2032GTGCCAGAATGCAAAACTGAAATTAAGATGAGAATATAAAATCAGTAAATTTGAGAATCGGATTAGGGAAAAAAAAAGCGGATAACAATTTCACACAGGA).tor1 with ura4+ marker in strain iph1-d ura4-D18: (5\u2032TGGAAGAATTGAACACCGCGACTATTAGAAAGTCTATCGTTTCACTCGCTCTCTTTGATTCATGGAGTATTTTAGTCGCCAGGGTTTTCCCAGTCACGAC 3\u2032) and (5\u2032TAAATTAATAACAACACGAAAAAAATTATCATAATCTCAAAAAACAGAAAACATCATTACCAAAAACTACACCATCAGCGGATAACAATTTCACACAGGA3\u2032).Primers used to disrupt iph1 coding sequence between position 188 and position 280 by the ura4+ gene to obtain strain iph1-M:Primers used to replace the (5\u2032GAGAGATCCGGAAACAGATAATGCAAGTGCAGCTATTGACGTTCACATCGGCAGTCAAAGCAATCCACGAGAGTTGCGCCAGGGTTTTCCCAGTCACGAC 3\u2032) and (5\u2032ATAGAGCATCATGAGACACTTCGAAGTAATAATTTGTATTATTAGAGGCTGTATAGGCGTTTGAAATTCCATTATGAGCGGATAACAATTTCACACAGGA 3\u2032).iph1-E71D was obtained by replacing the ura4+ gene in strain iph1-M with a DNA fragment of iph1 gene mutated at codon 71 (GAA to GAT). This fragment was synthesized, cloned and sequenced by GeneArt .Mutant The cDNA coding the hIDE protein was cloned into pCRBlunt plasmid. Site direct mutagenesis was used to change nt A390 to C to obtain the hIDE-E111D mutant as described in (5\u2032ATTCAACCTATTGCCATATTTCTTACATATTACCCTTTTTTTGGGTGTAATAGCAGTAGTCAGAATTCTGGGTTGTTTTATCTTTTCCTTTCATAAATAAAAAATGCGGTACCGGCTAGCG 3\u2032) and (5\u2032TGTTTGGATAGCGAGAAAAACCGCAGTGCCAGAATGCAAAACTGAAATTAAGATGAGAATATAAAATCAGTAAATTTGAGAATCGGATTAGGGAAAAAAAACTAGAGTTTTGCAGCCATGAAGTTAATATG-3\u2032).iph1-d ura+ strain and transformants were selected on standard 5 fluoroorotic acid (5-FOA) medium. Stable transformants expressing hIDE and hIDE-E111D from the iph1 promoter were selected.PCR- fragments were used to transform the Alignment was performed with Clustalw software. The tridimensional structure of Iph1 was performed using the Modeller software A full length cDNA of 2911 bp encoding Iph1 was amplified using a high fidelity enzyme and primers encoding a C-terminal extension by six histidine residues, inserted into pCRBlunt , sequenced completely to confirm the absence of errors, and transferred as XbaI/PstI fragment into the baculovirus transfer vector pVL1393 (Invitrogen). To produce the E71D mutant, site-directed mutagenesis was performed directly on the pVL1393-Iph1 plasmid. The primers used for the mutagenesis PCR were (5\u2032GGATTGGCGCACTTTTGTGATCATCTGTTGTTTATGGGGAC3\u2032) and (5\u2032GTCCCCATAAACAACAGATGATCACAAAAGTGCGCCAATCC3\u2032). After the PCR, the reaction mixture was digested with Dpn I and this material was used for transformation. Successful mutagenesis was confirmed by sequencing. Recombinant baculoviruses encoding wt Iph1 and Iph1-E71D were produced by co-transfection of Sf9 insect cells with the resulting plasmids and BaculoGold\u2122 virus DNA, followed by a plaque assay and plaque selection by PCR. Recombinant Iph1 and Iph1-E71D were produced in Hi5 insect cells after infection with the recombinant baculoviruses. Cells were harvested 72 h post-infection and lysed on ice for 30 min in 25 mM Tris, 50 mM phosphate, 300 mM NaCl, 10 mM imidazole, 1% Triton X-100, pH 8.0 in the presence of protease inhibitors. The lysis supernatant was harvested by centrifugation for 10 min at 14000 rpm. Then it was transferred on Ni-NTA beads (Invitrogen) equilibrated in 50 mM phosphate, 300 mM NaCl, 10 mM imidazole, pH 8.0 and left to bind on a turning wheel at 4\u00b0C overnight. The resin was washed with the same buffer containing 10 mM imidazole, then with 25 mM imidazole and the protein was eluted with buffer containing 300 mM imidazole. The recombinant protein was used directly for enzymatic assays. Insect cell-expressed recombinant hIDE carrying an N-terminal extension by 7 His residues was purchased from R&D Systems .Enzymatic activity towards the fluorogenic substrate Mca-RPPGFSAFK(Dnp) was measured by following the time-dependent increase in the fluorescence signal at 405 nm after excitation at 340 nm on a Mithras LB 940 plate reader . 10 \u00b5M of substrate were incubated with 10 ng of hIDE or 20 ng of Iph1 in 50 mM Tris, 150 mM NaCl pH 7.4 at 25\u00b0C and fluorescence was recorded for 10 min. The resulting time slope was used to calculate the digestion rate of the fluorogenic substrate.10 \u00b5g of insulin were incubated with 2\u2013500 ng hIDE or 8\u2013800 ng Iph1 in 50 mM Tris, 150 mM NaCl pH 7.4 for 3 h 20 min or 16 h in 300 \u00b5l final volume. Reactions were stopped by the addition of 30 \u00b5l 10% formic acid. Analysis of insulin digestions was performed by reversed phase HPLC on a \u03bcRPC C2/C18 ST 4.6/100 column . Digestion products were eluted using a 20\u201350% acetonitrile gradient, while monitoring the absorbance at 215 nm. For analysis by SDS-PAGE on 15% Tris-Tricine gels, 185 ng insulin were digested for 16 h with Iph1 WT, hIDE or Iph1 E71D . Bands were visualized using SYPRO Orange Protein Gel Stain .2O and spotted onto the indicated medium. TU, dithiotreitol and rapamycin treatments were performed on mid-log cultures grown in YNB. For each time point or drug concentration two to three dilutions were plated on YNB in triplicates and plates incubated at the appropriated temperature for 3\u20135 days. Colonies formed were counted and percent of survival calculated against time 0. All experiments shown were performed at least three times. TU was suspended in dimethyl sulfoxide (DMSO) at 10 mg/mL and used at 10 \u00b5g/mL otherwise stated. RA was suspended in DMSO at 0.5 mg/mL, and used at the final concentration of 300 ng/mL. DTT was suspended in H2O at 1 M and used at the final concentration of 50 mM.In drop tests, cells from exponentially growing cultures in either EMM or YNB were treated or not with tunicamycin for 45 min, serially diluted in HProtein extracts were prepared as described in Cells were treated with 10 \u00b5g/ml of TU for 45 min, collected, suspended in fresh medium without TU and incubated at 30\u00b0C with agitation. At 0, 4 and 6 hours from release, cells were processed as described in S. pombe encodes five putative metallopeptidases belonging to the M16 peptidase family that are potential orthologues of budding yeast Ste23p. Among these, the SPACUNK4.12c ORF on chromosome 1 encodes a protein sharing the highest degree of identity (37%) with hIDE .In order to compare the protease activity of Iph1 with hIDE we expressed recombinant wt Iph1 and a mutant Iph1 protein with a substitution of residue Glu71 in the catalytic site by Asp, both tagged by six C-terminal His residues, using the baculovirus system. The equivalent mutation in hIDE (E111D) decreases enzyme activity to<1% without affecting substrate binding Next, we sought to determine whether Iph1 is able to cleave insulin, the hallmark natural substrate of hIDE cleaved with high efficiency by it. We performed insulin digestions with various amounts of enzyme and analyzed the results by column and gel chromatography. After 16 h of digestion with either 500 ng hIDE or 800 ng Iph1, the insulin peak was no longer detectable in reversed phase chromatography and various new product peaks appeared on the chromatogram . Analysiiph1 ORF was replaced by the selectable marker KanR. Cells disrupted for iph1 (iph1-d) did not show any obvious phenotype and, in contrast to budding yeast ste23 null mutant, cells were not sterile.To study Iph1 function we constructed a haploid strain where the iph1-d) to wild-type (wt) cells. TU is an ER stressor that blocks protein N-glycosylation in the ER, leading to accumulation of misfolded proteins.IDE is highly expressed in murine pancreatic beta cells (our unpublished observation), in which the UPR is constitutively activated to handle glucose-triggered bursts of insulin synthesis that challenge the ER protein folding capacity iph1-d cells were more resistant to induced ER stress than wt was constructed by replacing in the haploid strain the +iph1 sequence with iph1-E71D. As shown in iph1-d cells is due to lack of the protein but not to lack of its protease activity. Thus, Iph1 has a function in the ER stress response that is unrelated to substrate degradation.Given that proteins belonging to the insulinase family have protease activity, we asked if increased survival to ER stress of tor1 (tor1-d) were slightly more sensitive than wt cells to TU in all experiments, however differences in survival between wt and tor1-d were at the limit of statistical significance (p\u200a=\u200a0.05) , indicating that the observed phenotype might be specific to ER stress.Fission yeast Tor1 kinase is required for the response to a wide range of stresses including heat stress and DNA damaging conditions \u200a=\u200a0.05) . In contng agent . The hig-d cells , indicat-d cells . Thus, rwt and tor1-d cells by iph1 deletion might depend on the Tor2 kinase that participates in the formation of the RA sensitive TORC1 complex as shown in wt cells and of tor1-d cells .Next to activating the UPR, ER stress is an inducer of autophagy that plays a cytoprotective role by preventing accumulation of misfolded proteins in apoptosis proficient cells We have shown that hIDE shares the protease independent function of Iph1 at least in yeast cells. Our results suggest that knock-out of mammalian IDE might also lead to ER stress resistance at least under certain metabolic conditions. This might be particularly relevant for pancreatic beta cells, in which excessive ER stress is linked to functional failure and diabetes development. The known capacity of IDE to degrade insulin is commonly viewed as mechanistic underpinning of its genetic link to diabetes. Our results suggest considering an additional or alternative possibility, namely that an implication of IDE in the response to proteotoxic stress might affect the survival of pancreatic beta cells in this pathology."} +{"text": "This paper aims to present a new genetic approach that uses rank distance for solving two known NP-hard problems, and to compare rank distance with other distance measures for strings. The two NP-hard problems we are trying to solve are closest string and closest substring. For each problem we build a genetic algorithm and we describe the genetic operations involved. Both genetic algorithms use a fitness function based on rank distance. We compare our algorithms with other genetic algorithms that use different distance measures, such as Hamming distance or Levenshtein distance, on real DNA sequences. Our experiments show that the genetic algorithms based on rank distance have the best results. In many important problems in computational biology a common task is to compare a new DNA sequence with sequences that are already well studied and annotated. Sequences that are similar would probably have the same function, or, if two sequences from different organisms are similar, there may be a common ancestor sequence In computational biology the problem that deals with this task is known as the closest string problem (CSP): given a set The standard method used in computational biology for sequence comparison is by sequence alignment. Sequence alignment is the procedure of comparing two sequences or more sequences by searching for a series of individual characters or characters patterns that are in the same order in the sequences. Algorithmically, the standard pairwise alignment method is based on dynamic programming; the method compares every pair of characters of the two sequences and generates an alignment and a score, which is dependent on the scoring scheme used, i.e. a scoring matrix for the different base-pair combinations, match and mismatch scores, or a scheme for insertion or deletion (gap) penalties.Although dynamic programming for sequence alignment is mathematically optimal, it is far too slow for comparing a large number of bases, and too slow to be performed in a reasonable time.Also, since some of the search solutions are inaccurate from a biological point of view, alternative approaches periodically are explored in computational biology. This important problem, known also as DNA sequence comparison, is ranked in the top of two lists with major open problems in bioinformatics The standard distances with respect to the alignment principle are edit (Levenshtein) distance rank distance (RD)To measure the similarity between strings Dinu proposes a new distance measure, termed To measure the distance between two strings with RD we scan (from left to right) both strings and for each letter from the first string we count the number of elements between its position in the first string and the position of its first occurrence in the second string. Finally, we sum up all these scores and obtain the rank distance. In other words, the rank distance measures the \u201cgap\u201d between the positions of a letter in the two given strings, and then sums up these values. Intuitively, the rank distance gives us the total non-alignment score between two sequences.ad hoc extension to arbitrary strings, without affecting the low computational complexity. In contrast, the extensions of Hamming distance are mathematically optimal but computationally too heavy, and lead to the edit-distance, which is the base of the standard alignment principle. Thus, the rank distance sides with Hamming distance rather than Levenshtein distance as far as computational complexity is concerned: a significant indicator is the fact that in the Hamming and rank distance case the median string problem is tractable Clearly, the rank distance gives a score zero only to letters which are in the same position in both strings, as Hamming distance does . On the other hand, an important aspect is the reduced sensitivity of the rank distance with respect to deletions and insertions. Reduced sensitivity is of paramount importance, since it allows the RD is easy to implement, does not use the standard alignment principle, and has an extremely good computational behavior. Another advantage of RD is that it imposes minimal hardware demands: it runs in optimal conditions on modest computers, reducing the costs and increasing the number of possible users. For example, the time needed to compare a DNA string of Traditionally, the Closest String Problem (CSP) is related to Hamming distance and it tries to find a minimal integer In When CSP emerged in bioinformatics, the problem was investigated from many points of view. These investigations implied the use of different distances. The most intensive studied approach was the one based on edit distance. In In many practical situations the alphabet is of fixed constant size . For some applications, one needs to encode the DNA or protein sequences on a binary alphabet that expresses only a binary property of the molecule, e.g. hydrophoby via rank distance are NP-hard. In this paper we use an approach based on genetic algorithms to propose an approximation of CSP and CSSP via rank distance.In In this section we introduce notation and mathematical preliminaries. We first introduce the rank distance and then we define closest string and closest substring problems.A ranking is an ordered list and is the result of applying an ordering criterion to a set of objects. Formally,Definition 1. Let A ranking defines a partial function on full rankings, while the others are partial rankings. We define the order of an object The rankings that contain all the objects of an universe Definition 2. Given two partial rankings In Definition 3. If The rank distance is naturally extended to strings. The following observation is immediate: if a string does not contain identical symbols, it can be transformed directly into a ranking (the rank of each symbol is its position in the string). Conversely, each ranking can be viewed as a string, over an alphabet equal to the universe of the objects in the ranking. The next definition formalizes the transformation of strings that have identical symbols into rankings.Definition 4. Let Example 1. If Observe that given Definition 5. Given Example 2. Consider the following two strings The computation of the RD between two rankings can be done in linear time in the cardinality of the universe. Our universe has precisely Let Problem 1 (Closest string via rank distance). Let The CSSP is a generalization of CSP where the objective is to find a string similar to substrings of the input.Problem 2 (Closest substring via rank distance). Let Homo sapiens, V00662), common chimpanzee , gorilla , donkey , rat , mouse , fat dormouse , and cow . Mitochondrial DNA (mtDNA) is the DNA located in organelles called mitochondria. The DNA sequence of mtDNA has been determined from a large number of organisms and individuals, and the comparison of those DNA sequences represents a mainstay of phylogenetics, in that it allows biologists to elucidate the evolutionary relationships among species. In mammals, each double-stranded circular mtDNA molecule consists of We test the genetic algorithm using mitochondrial DNA sequences extracted from several mammals available in the EMBL database: human we design two similar experiments. We have another artificial experiment for CSSP, and another experiment for CSP with great interest for biologist.For the first experiment we use the human, chimpanzee and donkey genomes. We want to find the closest string (or substring) of nucleotides between the human and chimpanzee DNAs on one hand, and between the human and donkey DNAs on the other hand. The goal of this experiment is to compare the distances obtained for the two strings (or substrings). Note that the donkey belongs to the Perissodactylae branch, while the human and the chimpanzee belong to the Primates branch. Since the human and the chimpanzee are both primates, the human-chimpanzee distance should be smaller than the human-donkey distance. In other words, we expect the biological classification of mammals to be reflected in the DNA.For the second experiment we use the rat, house mouse, fat dormouse and cow genomes. As in the former case, we want to find the closest string (or substring) of nucleotides between the rat and house mouse DNAs, between the rat and fat dormouse DNAs, and between the rat and cow DNAs. The goal of this experiment is to compare the distances obtained for the three strings (or substrings). Note that the cow belongs to the Cetartiodactylae branch, while the rat, the house mouse, and the fat dormouse belong to the Rodentia branch. We expect the rat-house mouse distance and the rat-fat dormouse distance to be smaller than the rat-cow distance. We have chosen this experiment because in We also use an artificial test case for the closest substring problem to point out our optimization of the genetic algorithm presented in Note that another study that shows experiments using Hamming distance for CSP and CSSP is Each of our experiments are performed using three different metrics: rank distance, Hamming distance and Levenshtein distance. We want to compare the results for each distance measure. We show graphs of the best candidate evolution for each metric used.After we determine the metric that has the best results, we will perform another experiment (using only this metric) with great interest for biologists. At present, no definitive agreement on either the correct branching order or differential rates of evolution among the higher primates exists, despite the research in this area. Joining human with chimpanzee and the gorilla with the orangutan is currently favoured, but the alternatives that group humans with either gorillas or the orangutan rather than with chimpanzees also have support With two experiments and three distance measures for the closest string problem, we have six test cases with associated graphs. For the closest substring there is an extra artificial experiment, generating nine test cases and six graphs associated to the real DNA experiments. In our latest experiment we use the distance measure that has the best performance on the former test cases. We investigate only the closest strings for DNA sequences of variable lengths and we present three more graphs.For each experiment we give the input strings, then we present the results obtained by using rank distance, Hamming distance and Levenshtein distance, respectively. An input string is a DNA sequence. The algorithm designed for CSRD needs at least two DNAs (of same length) to produce an output DNA sequence. The output DNA is the closest string to the input strings computed with rank distance. Using Hamming or Levenshtein distance in the selection process of the genetic algorithm is analogous. The algorithm designed for CSSRD need two DNAs (not necessary of same length) to produce the output DNA that represents the closest substring.Let us describe the genetic algorithm parameters and the format of the input and output data. The population size represents the number of chromosomes in a single generation. The crossover probability represents the percent of chromosomes (from a single generation) that get involved in the crossover operation. The mutation probability is similar to the crossover probability only that the chromosomes are mutated. The number of strings (DNA sequences) gives the number of input strings. The size of each DNA sequence is the number of nucleotides in every DNA sequence. We use different input parameters for each problem that we are trying to solve. The input parameters used for one experiment are the same for every investigated metric. We want to compare only the metrics used, without changing the genetic algorithm parameters or the genetic operations involved.The average time represents the mean time for There are two different settings for this experiment corresponding to CSP and CSSP, respectively. We present the test cases and results separately for each setting.In this setting we use the first 1. RANK DISTANCE TEST CASE 1: Population size: 2500; number of generations: 300; crossover probability: 0.36; mutation probability: 0.002; size of each DNA sequence: 200.HUMAN-CHIMPANZEE RESULT: Average time: 22 seconds; Distance achieved: 3698; Closest string: G T A C T A C G C G T T T A C T C T A C C A A A C G C A T A C T G A C A A A T G T C T G T T A G A T G G A T C C A T C T C C G C G T G T A C T G T C T A A A A G C G T A G C G T C A C G T A C G T C A A G C A G T G T T T C A G T C C C A C A A T C C A T T G C A C A T T A C T G A G C T C T C C A T T C G T C T C A C T C T T T T T A C G A A C A A T A T T A T C A A T G C A A A C G T G G G C C C T C T T C.HUMAN-DONKEY RESULT: Average time: 22 seconds; Distance achieved: 5001; Closest string: T G A A G A G C A T T C C A T A T C T A A C T C C T G A A G T A C A C G A A C G G A T A T G C A C T T T G C T T C G T T A C A C T A G C G T G G A C G T A C A T T C T C G G C T G A C C T T G G G C A T A T A A T A T T A A A G T A A C G G A G T C T A C A T C T A A T A T C A T C G T A A C C C A T A G A A T G T T A T A C C C T C A T C G T C C T T C G C C C A A G T G C C C T G C T T A A C T T C T C A T.2. HAMMING DISTANCE TEST CASE 1: Population size: 2500; number of generations: 300; crossover probability: 0.36; mutation probability: 0.002; size of each DNA sequence: 200.HUMAN-CHIMPANZEE RESULT: Average time: 1 min 14 seconds; Distance achieved: 73; Closest string: G A T C A T G T G G C T A T C A C C C T C A A A G C C A C T C A C G G G A A C T G T T C A G A C A T T T T T A C A T T A C C C C A T G A A G A T A T G C G C G T G G T A C T A T T C T G T C A A G C A G C A G T C A G A A A A C T C A C T C T T G C A A T A A C T G T C T T T G C T T G C T T C A T C A T C T T A A T C T C T A T C A T C A C T A G G T A C A A T A A T A C A G G C C G A C G C A C T G C A G C.HUMAN-DONKEY RESULT: Average time: 1 min 13 seconds; Distance achieved: 77; Closest string: G A T C A C A G A G C T A A A A G A C A A C A A A C C A C G C A C C T G A A A A T G C C A A G A T T T T G G T T C C T A C G C C T T G G G C A T A T A C A C T C G A T C C C G T T C T G A T A C T C T G T A A C C G G T G C A A C C A C T C A T G C A A G A T T C G T C A T T C C T G C C T G A A T G A T C T C T T T A T T G A A C T C T C C G A T C T T A A G G A G C A G G T G C G A A C A A A A T T A C T A.3. LEVENSHTEIN DISTANCE TEST CASE 1: Population size: 2500; number of generations: 300; crossover probability: 0.36; mutation probability: 0.002; size of each DNA sequence: 200.HUMAN-CHIMPANZEE RESULT: Average time: 24 min 12 seconds; Distance achieved: 63; Closest string: G T A T A C A C A G C T C T A C C C C C T A A A G C A A T A C C A C G G A A G A T C T T C C A T G G A T T T A T A T C A T C C T C T A A G C A A C A T G C A T G G T A G C C T T G C G A T T C G A T T G A G C T C G T G A G A C C C T A T A T C G C A T A C T G A T C C C C G A T C C T G G T C A T C C T A T T A A T C A T C C A T G T A A A G T T A C A A G T A T T A C A G C G C G C A G C A A T T A C A A C.HUMAN-DONKEY RESULT: Average time: 24 min 11 seconds; Distance achieved: 59; Closest string: G T T C A A T G T A C T A T C A C G A T A T A A A T C A A G G A G C T G T C A A T G C A C T T G G T A G T T T C C T C T G C G C T A T G C A C A C A T A G G G C A T T G C G A C C T G G A G C C T T A T T A T T A C T A T G A A G C A G A T T A A C A T G C A T T G A T T C C T G C C T C C C C A T A T A A T C C T C T A A A T C G C A C T C T A G A T C A A A T T A C A G G C G A A C A A G A C T C T A C T A.In this setting we use the first 300 nucleotides extracted from the human, chimpanzee and donkey genomes. We want to determine the human-chimpanzee and human-donkey closest substrings of 24 nucleotides.Here the substring size input parameter represents the desired length of the best substring .1. RANK DISTANCE TEST CASE 2: Population size: 500; number of generations: 100; crossover probability: 0.36; mutation probability: 0.02; size of each DNA sequence: 300; substring size: 24.HUMAN-CHIMPANZEE RESULT: Average time: 39 seconds; Distance achieved: 26; Closest substring: C T T A G T A A C T A T A T C G A G A C A A G C.HUMAN-DONKEY RESULT: Average time: 40 seconds; Distance achieved: 34; Closest substring: A C A T G C C T A T C T A C C C G T A A T A C C.2. HAMMING DISTANCE TEST CASE 2: Population size: 500; number of generations: 100; crossover probability: 0.36; mutation probability: 0.02; size of each DNA sequence: 300; substring size: 24.HUMAN-CHIMPANZEE RESULT: Average time: 1 min 36 seconds; Distance achieved: 7; Closest substring: C T A C A C A C G C A A G C C T T C C C T G C A.HUMAN-DONKEY RESULT: Average time: 1 min 38 second; Distance achieved: 7; Closest substring: A C G T A C G A A C C A T A C T A C A A G C T A.3. LEVENSHTEIN DISTANCE TEST CASE 2: Population size: 500; number of generations: 100; crossover probability: 0.36; mutation probability: 0.02; size of each DNA sequence: 300; substring size: 24.HUMAN-CHIMPANZEE RESULT: Average time: 7 min 4 seconds; Distance achieved: 4; Closest substring: T T G A T T C C T G C C T A T C T A T T A G C T.HUMAN-DONKEY RESULT: Average time: 7 min 3 seconds; Distance achieved: 4; Closest substring: A T G C T A C T C T T A A T C G C A C C T A C G.First, we must point out that rank distance, Hamming distance and Levenshtein distance use different scales, i.e. a rank distance of 100 is not equivalent to a Hamming distance of 100, nor a Hamming distance of 100 to a Levenshtein distance of 100. We would also like to point out that rank distance has a finer scale, possibly being able to detect subtle differences between DNA strings.As one might expect, the results indicate that the human genome is closer to the chimpanzee genome, than it is to the donkey genome.In the CSP setting, rank distance shows a great difference between the human-chimpanzee closest string and the human-donkey closest string. Levenshtein distance indicates that humans are closer related to donkeys than to chimpanzees, while Hamming gives the expected result as rank distance does. Both Hamming and Levenshtein distances show small differences between the two analysed strings. The evolution of the best closest string candidate for each distance measure is given in In the CSSP setting, RD is the only distance that can catch the subtle difference between the human-chimpanzee closest substring and the human-donkey closest substring, even if we use only In both CSP and CSSP settings, rank distance clearly outperforms Hamming and Levenshtein distances see and 2.As for the Human-Chimpanzee-Donkey experiment, there are two different settings corresponding to CSP and CSSP. We present the test cases and results separately for each setting.In this setting we use the first 150 nucleotides extracted from each of the rat, house mouse, fat dormouse and cow DNA sequences. We want to determine the rat-house mouse, rat-fat dormouse and rat-cow closest strings which also have 150 nucleotides.1. RANK DISTANCE TEST CASE 3: Population size: 1800; number of generations: 300; crossover probability: 0.36; mutation probability: 0.005; size of each DNA sequence: 150.RAT-HOUSE MOUSE RESULT: Average time: 12 seconds; Distance achieved: 454; Closest string: G T T G A A T C G T T A A T A T A C A A A G C A A G T A C A T G A A T C A G A A G T G A T A T T C T A A A A G C T T A G C A A C C A T C A A A T A T G T G G C C G T G T T C T A C A T T T A A G T G A A G A T G T A A A T C A A A C C T A A G C A T C A T G A C A T G C G A A T C A A G C A T A C C T A T T.RAT-FAT DORMOUSE RESULT: Average time: 12 seconds; Distance achieved: 1209; Closest string: G T A T A C T G T A G T A T A A A A A A T C T G A G A C C A T G A T A A T G T A C A G T A G G A T A C A T A C C T A A C C G C A A C A A T T G A T G C C G T G T A C G C T T A A T T T C A A T G T C T C T A G C A G G A A G A A A A T T T G C A A A C T T C C A A C G A A A G T C G C T A A A T G T C C A T.RAT-COW RESULT: Average time: 12 seconds; Distance achieved: 3321; Closest string: G T A T A A C A T G T C A C T G A A C C G A A T A C T A G T A A T G A A A A T T C G G C T C T T A T G C A A G A C T T A T A C T T T C A G G A G G A T C G A T T T T A G A A C A T G A A A A T G C T A G G C T G T A G T G G C G T A G A T C A C T A G G C A G C T G C T T G T T C T T T T G T C A A C T G G.2. HAMMING DISTANCE TEST CASE 3: Population size: 1800; number of generations: 300; crossover probability: 0.36; mutation probability: 0.005; size of each DNA sequence: 150.RAT-HOUSE MOUSE RESULT: Average time: 42 seconds; Distance achieved: 45; Closest string: G T T A A T G T A G C T T A T T A A C A A G G A A A G G A A T T G A A A A T G T T T A G T G G G T T C A A T A T T C C C A A T A A C C C A A A G G G T T G G T C C C G G G C C T G T A A A T A A A T T A A G G G T A G A A T A A A C A T T C A A A A C C C C C A A A A A C C G G G T T A A A A C C C T T T A.RAT-FAT DORMOUSE RESULT: Average time: 41 seconds; Distance achieved: 43; Closest string: G T T A A T G T A G C T T A T A A T A A G C A A A A C C A T T A A A A A G C T T T G G A T G G A A T C T A A A A C C C C T A A A A C A A A A A G T T T G G G C C C A G G C T T T T T A A T T G T T T G T A G G A A A A A T A A A C A T T G C A A C A A T C A C G A C A C C G G T A T A A A A C C C T T T A C.RAT-COW RESULT: Average time: 41 seconds; Distance achieved: 56; Closest string: A T T A A T G G A T A A T C T G C T A A T G C A A A G A C A T G A C A A T G C T G T G A T A G A T T T A G A A A T T C T A T A A T C A G G A A G G T T T T G G C A T T C A G C T A T G G T T G A C T G A G G G T A T G A T T C G A C A C A T A A A C T T C A A T A G G C C T T A G C A G A A T C T T T A G A.3. LEVENSHTEIN DISTANCE TEST CASE 3: Population size: 1800; number of generations: 300; crossover probability: 0.36; mutation probability: 0.005; size of each DNA sequence: 150.RAT-HOUSE MOUSE RESULT: Average time: 9 min 28 seconds; Distance achieved: 14; Closest string: G T T A A T G T A G C T T A T A A T A A A G C A A A G C A C T G A A A A G C T T A G A T G G A T C A A A T G A T C C C A T A A A C A C A A A G G T T T G G T C C T G G C C T A A A T A A T T A G A G G T A A A G A T C T A C A C A T G C A A A C C T C C A T A G A C C G G T G T A A A C A T C C C G T T A A.RAT-FAT DORMOUSE RESULT: Average time: 9 min 29 seconds; Distance achieved: 28; Closest string: T T A A T G A G C T T A A A A G C A A A G C A A C T G A A A T G C T T A G A T G G T A G C A A A T A T C C C A T A A A C A C A A A G G T T C T G G T C C C A G C C T T C T A T T A A T T A G A T T G T A T A G C A A G A T T A C A C A T G C A A C A T C A T G A A C C T G G T G T A A G A A T C C C T T A A.RAT-COW RESULT: Average time: 9 min 29 seconds; Distance achieved: 46; Closest string: G A C T A A T G G C T A T C A G A A T G C A A A G C A C A T G A A C A T G C T G C T G A G A T A G A T T T G A A A A T C T T T A A T A C T G G A A G G G T T G C T C C T G G A C T C A T A G C T A T G G A C G T A A G G C T T G A C A C A G C A T A C A T T G T A C C G G A G T A A A A T G C A C T T A A G.In this setting we use the first 300 nucleotides extracted from the rat, house mouse, fat dormouse and cow genomes. We want to determine the rat-house mouse, rat-fat dormouse and rat-cow closest substrings of 24 nucleotides.The substring size parameter is the desired length of the best substring.1. RANK DISTANCE TEST CASE 4: Population size: 700; number of generations: 110; crossover probability: 0.36; mutation probability: 0.03; size of each DNA sequence: 300; substring size: 24.RAT-HOUSE MOUSE RESULT: Average time: 1 min 25 seconds; Distance achieved: 0; Closest substring: A A A G C A A A G C A C T G A A A A T G C T T A.RAT-FAT DORMOUSE RESULT: Average time: 1 min 24 seconds; Distance achieved: 4; Closest substring: A T A A G A C A A G C A C T G A A A A T G C T T.RAT-COW RESULT: Average time: 1 min 25 seconds; Distance achieved: 22; Closest substring: A G A T A C G T T C A G T A C A T G A G T A C C.2. HAMMING DISTANCE TEST CASE 4: Population size: 600; number of generations: 110; crossover probability: 0.36; mutation probability: 0.03; size of each DNA sequence: 300; substring size: 24.RAT-HOUSE MOUSE RESULT: Average time: 2 min 5 seconds; Distance achieved: 0; Closest substring: T C A G C A G T G A T A A A T A T T A A G C A A.RAT-FAT DORMOUSE RESULT: Average time: 2 min 4 seconds; Distance achieved: 1; Closest substring: C C C C A T A A A C A C A A A G G T T T G G T C.RAT-COW RESULT: Average time: 2 min 4 seconds; Distance achieved: 7; Closest substring: G T A A T T G G A C A T A A A T T T T C A C A T.3. LEVENSHTEIN DISTANCE TEST CASE 4: Population size: 700; number of generations: 110; crossover probability: 0.36; mutation probability: 0.03; size of each DNA sequence: 300; substring size: 24.RAT-HOUSE MOUSE RESULT: Average time: 13 min 18 seconds; Distance achieved: 1; Closest substring: T A A A A A A G C A A A G C A C T G A A A A T G.RAT-FAT DORMOUSE RESULT: Average time: 13 min 19 seconds; Distance achieved: 1; Closest substring: T A A A C G A A A G T T T G A C T A A G C T A G.RAT-COW RESULT: Average time: 13 min 19 seconds; Distance achieved: 6; Closest substring: C A A A C A T C T A C C A C C C G G T T A A A A.The expected result for this experiment should indicate that the rat is closer to the house mouse and fat dormouse, than the cow. We would also like to catch even a finer difference between the rat-house mouse distance and the rat-fat dormouse distance.In the CSP setting, rank distance shows again a great difference between the rat-house mouse closest string, the rat-fat dormouse and the rat-cow closest string. Hamming is able to distinguish the rat from the cow genome, but it doesn\u2019t catch the difference between the rat-house mouse closest string and the rat-fat dormouse closest string. The rat-fat dormouse Hamming distance appears to be smaller than the rat-house mouse Hamming distance, which is wrong. Levenshtein distance works as good as rank distance in this case, giving the expected result. Our observations are supported by the graphs shown in In the CSSP setting, all distances perform very good and are able to put the rat genome near the house mouse and fat dormouse genomes rather than the cow genome. However, the rat-house mouse Hamming distance is very close to the rat-fat dormouse Hamming distance (the closest substrings differ only by one letter). The Levenshtein distance is the same for rat-house mouse and rat-fat dormouse closest substrings. The associated graphs are given in In both CSP and CSSP settings, all distances are able to put the rat near the house mouse and fat dormouse rather than the cow, which is the expected result see and 4. OFor this experiment we use only the CSSP setting. The goal of this experiment is to show the time improvement obtained by optimizing the genetic algorithm introduced in 1. TEST CASE 5: Population size: 500; number of generations: 100; crossover probability: 0.36; mutation probability: 0.02; size of DNA sequence 1\u223690; size of DNA sequence 2\u223690; substring size: 30.DNA Sequence 1: A A A A A A A A A A A A T T T T T T T T T T T T T T T T T T T T T T T T T G G G G G A A A A A A A A A A A A A A A A A A A A A A A A A A A G G G G G G G G G G T T T T T A A A A A A A A A A A A A A A A.DNA Sequence 2: C C C C C C C C C C G G G G G G G G G G T T T T T C C C C C C C C C C C C C C C C C C T T T T T T T T T T T T T T T T T T T T T T T T T G G G G G C C C C C C C C C C C C C C C C C.RANK DISTANCE RESULT: Average time: 10 seconds; Distance achieved: 0; Closest substring: T T T T T T T T T T T T T T T T T T T T T T T T T G G G G G.HAMMING DISTANCE RESULT: Average time: 35 seconds; Distance achieved: 0; Closest substring: T T T T T T T T T T T T T T T T T T T T T T T T T G G G G G.LEVENSHTEIN DISTANCE RESULT: Average time: 3 min 22 seconds; Distance achieved: 0; Closest substring: T T T T T T T T T T T T T T T T T T T T T T T T T G G G G G.Using an algorithm to compute rank distance in linear time and a hash table to store precomputed distances between DNA sequences, we are able to report a great improvement in terms of speed. The algorithm that computes rank distance in linear time was introduced in At the selection step, the genetic algorithm needs to sort the chromosomes in each generation by distance. In order to sort the chromosomes we must compare distances that are computed (or recomputed) between chromosomes and input sequences. Instead of computing the distances each time, we store the precomputed distances in a hash table. It is much faster to access a distance value stored in a hash table instead of computing it in linear time. Note that we also used the hash table optimization for Hamming and Levenshtein distances. This optimization helps us reduce the number of distances to be computed from For this test case, in We obtained the same closest substring for each of the three metrics. This result shows that if an exact common substrings exists, the genetic algorithm can find it disregading the metric used. This shows that the genetic algorithm is robust and it can find the optimal solution if the input parameters are properly set.We designed simple and clear experiments that can show the differences of the compared distances. In order to keep things simple, we used the genetic algorithms to determine the closest string or substring for only two DNA sequences. Of course, the algorithms work as well with multiple sequences at once, since the CSP and CSSP problems are generally defined for sets of strings.We mention that the results obtained are not influenced by the fact that the DNA strings are part of coding or non-coding sequences or within genes or part of intergenic regions. The DNA strings used in our experiments were selected without taking into consideration these aspects so the strings may be part of any kind of region. However, it is important for DNA strings used in the same experiment to be extracted from the same position because the alignment matters. In other words, it doesn\u2019t have sense to compare DNA from different regions that have different significance.All our experiments show that RD can be computed 2 times faster than Hamming distance and 10 to 15 times faster than Levenshtein distance. As the closest string (or substring) size increases the Levenshtein distance takes more time to compute when compared to rank distance and Hamming distance.Although the Hamming distance computes almost as fast as rank distance, the downside is that is gives inaccurate results compared to RD. The Levenshtein distance can easily be dismissed because is takes longer to compute and it is also unable to detect the subtle differences that rank distance detects by having a finer scale.Neither Hamming distance or Levenshtein distance were able to give the right answer in all our experiments (Levenshtein distance is wrong in TEST CASE 1 and Hamming distance is wrong in TEST CASE 3). Only rank distance has the expected outcome in all the experiments. Overall, we believe that rank distance is best suited for finding closest strings or substrings on DNA sequences. Due to this observation we conducted the following experiment using only RD.The goal of this experiment is to see if the DNA information can lead to one of the three distinct unrooted phylogenetic trees of higher primates. For this experiment we use only the CSP setting: we want to find the human-chimp closest string and the human-gorilla closest string and compare the associated rank distances. We perform four tests using DNA sequences of variable length and different input parameters for the genetic algorithm. We show graphs for the last three test cases which are more relevant.In the first test case (TEST CASE 6) we use the first The difference between the human, chimpanzee and gorilla mtDNA is very small and We present only the distance achieved for each closest string, because the strings are too long to be presented here.1. RANK DISTANCE TEST CASE 6: Population size: 7000; number of generations: 500; crossover probability: 0.36; mutation probability: 0.001; size of each DNA sequence: 800.HUMAN-CHIMPANZEE RESULT: Average time: 7 min 3 seconds; Distance achieved: 43207.HUMAN-GORILLA RESULT: Average time: 7 min 6 seconds; Distance achieved: 45544.2. RANK DISTANCE TEST CASE 7: Population size: 33000; number of generations: 2000; crossover probability: 0.36; mutation probability: 0.0002; size of each DNA sequence: 5000.HUMAN-CHIMPANZEE RESULT: Average time: 13\u201314 hours; Distance achieved: 426232;HUMAN-GORILLA RESULT: Average time: 13\u201314 hours; Distance achieved: 358525.3. RANK DISTANCE TEST CASE 8: Population size: 40000; number of generations: 2400; crossover probability: 0.36; mutation probability: 0.0001; size of each DNA sequence: 7000.HUMAN-CHIMPANZEE RESULT: Average time: 27\u201328 hours; Distance achieved: 682664.HUMAN-GORILLA RESULT: Average time: 27\u201328 hours; Distance achieved: 656806;4. RANK DISTANCE TEST CASE 9: Population size: 55000; number of generations: 2800; crossover probability: 0.36; mutation probability: 0.00005; size of each DNA sequence: 16000.HUMAN-CHIMPANZEE RESULT: Average time: 5 days and 6\u20137 hours; Distance achieved: 2412780.HUMAN-GORILLA RESULT: Average time: 5 days and 6\u20137 hours; Distance achieved: 2089976.We adjusted the genetic algorithm parameters to obtain the best results disregarding the higher computational time needed to get these results for the first three test cases. The graphs show that the size of the population used in the genetic algorithm is much higher than necessary because the best chromosome evolves very fast during the first In TEST CASE 9 the parameters are rather ajusted for speed than accuracy. We can obtain better approximations of the closest strings by using a population larger than The results for TEST CASE 6 shows that according to rank distance the human is near the chimpanzee rather than the gorilla. The graphs corresponding to TEST CASE 7, 8 and 9 from It seems that Note that in our last test case we used almost all of the entire mtDNA which is approximately Overall, the DNA information that RD was able to extract during this experiment seems to support the theory favours the phylogenetic tree that joins the human with the gorilla In this paper we presented two genetic algorithms designed for solving the closest string problem and closest substring problem, respectively. The genetic operations for the closest string problem have a strong mathematical background and are only inspired from nature. The genetic algorithm designed for the closest substring problem uses standard genetic operations.We tested these two algorithms using several experiments that involve DNA sequences extracted from mammals genomes. Each of these experiments were performed using three different metrics: rank distance, Hamming distance and Levenshtein distance. In all our experiments rank distance clearly outperforms Hamming and Levenshtein distances. On top of this, rank distance is the only distance able to catch subtle differences between DNA strings.By comparing the results for each distance measure, we are able to conclude that RD is best suited for finding closest strings or substrings on DNA sequences.We used our genetic algorithm with rank distance to bring some light in a case disputed by biology scientists: which is the closest human relative, the chimpanzee or the gorilla? The DNA information extracted by rank distance supports the theory that says the human closest relative is the gorilla. We also showed the importance of using DNA sequences that are long enough to obtain conclusive results. Too short DNA sequences can lead to confusing results.In the near future we would like to compare our genetic algorithms based on RD with other approaches, such as dynamic programming techniques. We strongly believe that our approach is comparable, in terms of precision and speed, with other approaches.We also want to investigate a possible approach to obtain better results. This approach combines the results coming from several parallel executions of the genetic algorithm. The best candidates from these parallel executions may be taken to form the first generation of another genetic algorithm. The best candidates will evolve together until the final result is achieved. The final result is expected to be an optimal solution. This approach could work very good with very high-dimensional input data.Genetic algorithms are adaptive searching techniques based on the principles of genetics see . The firWe used the classic general form of the genetic algorithm. For each problem, we used a different set of operations. The set of operations used for the closest substring problem are classical. The crossover and the mutation operations are the same operations found in nature. For the closest string problem the operations are only inspired from biology, but they rely on a mathematical background. We will later describe the structure of the chromosomes and the operations applied on each generation.Algorithm 1General Form.Initialization: Generate a random population that represents the first generation.1: Loop: For a number of generations apply the next operations:2: 2.a 1em Apply the crossover according to the probability of having a crossover.2.b 1em Apply mutations according to the probability of having a mutation.2.c 1em Select the best candidates for the next generation using a density of probability.Termination: Choose the best individual from the last generation to be the optimal ranking.3: An individual chromosome is a permutation of ranks. Each chromosome is a possible candidate for the optimal ranking. We need to convert each input DNA to a permutation. Note that any string can be converted to a permutation. Each letter of the string can be annotated with an index that starts at There are three forms of crossover that are used by the algorithm. Each time the crossover must occur we apply all three forms of crossover.The first crossover operation keeps the first part (prefix) of the individuals and completes the rest of the permutation according to the order given by the complementary chromosome. The second operation uses the same principle, but applies it at the other end of the chromosomes. This crossover operation keeps the last part (suffix) of the individuals and completes the rest of the permutation according to the order given by the complementary chromosome.The third crossover is a natural combination of the previous two. This crossover keeps both the prefix and the suffix of the chromosomes but completes the middle part according to the order found in the complementary chromosome.In order to successfully apply the crossover operations a certain cutting point should be randomly generated. There are six new individuals after the recombination because each crossover operation generates two new individuals. The best two individuals are chosen to replace the parent chromosomes. The optimality condition is used as a criterion to choose the best individuals.We have chosen this model (with 3 types of crossover) because the use of a single crossover usually destroys certain parts of the two individuals involved in the operation. For example, the crossover that keeps the prefixes will have to reorder the components of the suffix. If this single type of crossover is used, we would be unable to evolve the suffix part of the chromosome. This will generate populations with similar individuals that tend to have a bad pattern. In this pattern a good part and a bad part always appear. With our model we ensure that individuals do not follow this pattern and get close to the optimal ranking, but in different ways.The mutation operation may be applied to any chromosome. The mutation only needs one chromosome. To apply a mutation on an individual two positions are randomly chosen. The values at the two positions are swaped.To select the individuals for the new generation from the current generation we use a density of probability function. The new generation is involved in the next iteration of the algorithm. The first step is to sort the individuals on the maximal distances from the input rankings criterion in descending order. Then we generate indexes from the top to the bottom of the list of candidates. The indexes close to the top of the list are more probable. Note that one index can be generated several times; this is the case with the best candidates. There are also indexes that may never be generated; this is the case of the candidates close to the bottom of the list. The density probability function used to select the candidates for the next generation is the normal distribution of mean The graph of this function is represented in Note that in the implementation of the algorithm the fitness function was statistically approximated.The motivation for using this fitness function is based on test results. This fitness functions reduces the number of generations that are required to obtain a close-to-optimal solution. Helped by the crossover and mutation operations, the fitness function has a good generalization capacity: it doesn\u2019t favour certain chromosomes which could narrow the solution space and lead to local minima solutions.Each chromosome is a sequence of DNA of fixed length that represents a possible candidate for the closest substring. Note that a sequence of DNA is simply a strand of nucleotides that appear randomly in a sequence.The crossover operation between two chromosomes for the closest substring problem is straightforward. First, we need to generate a random cutting point. The prefixes of the two chromosomes remain in place, while the suffixes of the two chromosomes interchange. This is the standard crossover operation inspired directly from nature.To apply a mutation to a certain chromosome, one position is randomly chosen. The nucleotide found at that position will be changed with a new one. Multiple mutations may appear at the same chromosome, although this is very unlikely. This is the classic mutation operation that can also be found in nature.The selection operation used here is similar to the selection used for closest string problem and is based on the normal distribution of mean"} +{"text": "We show that snippets from this sequence, at 100 base pairs or longer, drive gene expression in vitro in a number of mammalian cells, and are thus candidates for use in protein production. We further show that expression is driven by the general transcription factors TFIIB and TFIID, both being ubiquitously present across cell types, which results in less tissue- and species-specific regulation compared to the viral promoter SV40. We lastly found that the strength of a promoter can be tuned up and down by modulating the counts of GC and CpGs in localized regions. These results constitute a \u201cproof-of-concept\u201d for custom-designing promoters that are suitable for biotechnological and medical applications.The choice of promoter is a critical step in optimizing the efficiency and stability of recombinant protein production in mammalian cell lines. Artificial promoters that provide stable expression across cell lines and can be designed to the desired strength constitute an alternative to the use of viral promoters. Here, we show how the nucleotide characteristics of highly active human promoters can be modelled via the genome-wide frequency distribution of short motifs: by overlapping motifs that occur Artificially engineered promoter sequences have the potential for use in industrial and biotechnological applications, such as recombinant protein production of biopharmaceuticals. Some human proteins require mammalian cell lines for proper production, with e.g. the Chinese hamster ovary (CHO) cell line being a widely used system for EPO, Interferon-\u03b2, Factor VIII, IX, etc The promoter is the genomic region around the transcription start site (TSS) of a gene, and acts as an essential component in gene regulation and transcription, its role being to interface with transcription factors (TFs) through protein-DNA binding. The TFs anchor the pre-initiation complex (PIC), specifying the exact point of initiation, and recruit RNA polymerase (Pol) II to start transcription SSRCGCC\u2019 General transcription factors (GTFs), organized in complexes TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH and TFIIJ, form a special class of TFs, in that they are ubiquitously present and both necessary and sufficient to enable Pol II transcription at significant levels, making these proteins desirable candidates as drivers of expression of artificial promoters. Only TFIIB and TFIID have been shown to exhibit sequence preference: the TATA-Binding Protein is most well characterized and binds to the TATA-Box, thereby establishing the TSS 25\u201330 base pairs downstream of its location. However, only about 10% of human promoters rely on a TATA-Box nucleotide composition, rather than sequence motifs, where we define the term \u201cnucleotide composition\u201d as the frequency patterns of mono-nucleotides, di-nucleotides, tri-nucleotides etc. This composition varies over the human genome on a large scale, recognizable as isochores in vitro in mammalian cells, as well as how expression levels depend on highly localized features in these sequences.Designing artificial sequences that attract TFIIB and TFIID requires determining the features that capture the interactions between these proteins and the DNA. Generally, a complicating factor is the lack of a one-to-one relationship between TFs and exact instances of binding motifs, so that neither motif consensus of short sequences nor Position Weight Matrices (PWM) are ideal representations of binding sites in vitro expression. Here, we first recapitulate how highly active promoters (mostly associated with both housekeeping and strong tissue-specific genes) differ from the genome-wide distribution in GC and CpG, as well as in other di-nucleotides. We then exploit these \u201cun-genomic\u201d features by devising a measure that tracks the genome-wide frequency of each short motif: the less common genome-wide, the more likely it is to reflect properties of a promoter. We subsequently overlap a set of uncommon motifs to build \u201cpromoter-like\u201d contiguous sequence, which allows for editing existing promoters, as well as to construct entirely artificial ones that work in a number of mammalian cells. We last show that TFIIB and TFIID bind to these promoters, which is reflected in their stability of expression level across multiple cell lines.Highly active promoters exhibit nucleotide patterns that are different from the majority of the genome Trinity . The resulting assembly consists of 38 Mb of sequence, residing in 27,000 disjoint transcripts. We eliminated non-full-length transcript assemblies of less prominently expressed genes by requiring sequences to contain open reading frames of 500 bp or more, aligned the remaining sequences to the human genome, and selected only transcripts with the 5\u2032 end falling within 50 bp of an annotated TSS To capture a set of highly active promoters, we sequenced the mRNA of the most highly expressed genes in human cerebellum tissue. We constructed two cDNA libraries, one normalized and one un-normalized library (both filtered for poly-A tails), and sequenced both libraries on one lane of Illumina each, yielding a total of 2 billion base pairs in 71 bp long reads. We then assembled the reads from both libraries into contiguous transcripts using the transcriptome assembly program While only 127 (7.3%) of the 1,746 promoter sequences contain one or more instances of a TATA-Box in the correct orientation, the sequences are clearly distinct from the genome-wide average by their high G/C content (66% vs. 40% genome-wide) as well as the average frequency of CpGs (9% vs. 1% genome-wide). \u03b1 score\u201d, see \u03b1 yields a potential spatial resolution of tens of nucleotides. annotated TSSWe defined a measure that tracks with G/C and CpG content see , incorpo\u03b1 score for each possible 12-mer, including those not present in the human genome. We selected sequences from two quintiles: (a) the top 5% represent the most \u201cun-genomic\u201d (or promoter-like) 12-mers; and (b) the percentile between 45\u201350%, which contains 12-mers with di-, tri-nucleotide etc. frequencies close to the genomic median, representing more \u201cnormal\u201d (or non-promoter-like) sequences. The 12-mers from each set were then independently assembled into \u201cconcatomers\u201d and non-promoter-like (for down-regulation) artificial constructs. As test case, we chose the promoter upstream of the TSS of the X-linked gene cancer/testis antigen 1A (CTAG1A), which exhibited strong in-vitro activity in human cell line HEK293. The CTAG1A promoter region contains three distinct regions of elevated \u03b1 scores with size-matched, but of higher \u03b1 score, snippets from the promoter-like concatomer.We tested whether we could modulate in-vitro promoter activity in HEK293 cells relative to the original sequence, as does replacement with \u201cnon-promoter\u201d sequences with \u03b1 scores of approximately half the original (\u201chCTAG1A-replace\u201d). Replacement with \u201cpromoter-like\u201d sequences with an \u03b1 score roughly twice that of the original (\u201chCTAG1A-UP\u201d) increases activity beyond that of the original sequence. This indicates that highly localized changes in sequence composition can drive up- and down-regulation of in-vitro gene expression.in-vitro expression, we pulled sequences from the promoter-like concatomer using different criteria and of different lengths in four mammalian cell lines: CHO (hamster ovary); P19 (mouse embryo); Vero (monkey kidney); and HEK293 (human kidney). Promoter strength of most constructs was comparable to, or exceeded activity of the SV40 core promoter, which is a routinely used viral promoter for recombinant protein expression in mammalian cell lines (Spodoptera frugiperda) for any of the constructs, suggesting fundamental differences in promoter mechanisms between insects and mammals .To create entirely artificial promoter constructs for ll lines . NotablyAll constructs contain at least one instance of a TATA-Box. In addition to two TATA-Boxes, ArS232 contains one perfect, and 12 imperfect instances of BRE, and one Inr. To examine to what extent the TATA-box is needed to drive expression, we constructed three variations : (i) remD values, and that these readings are thus somewhat more difficult to interpret than the TFIIB binding results.The general transcription factors TFIIB and TBP bind to the artificial promoter constructs: we monitored real-time binding of TFIIB to constructs ArS110, ArS300, ArS201 and ArS232 through measurement of quantitative protein kinetics . Figure in vitro transcription depending of the abundance of the binding proteins that drive the promoter.Artificially engineered promoter sequences have potential for use in industrial, biotechnological and medical applications involving recombinant protein production and gene therapy, since they can be designed to have different activities and be adapted to the specific requirements (strong or weak expression). The method proposed here yields constructs that appear less variable and species specifically regulated than the viral promoter SV40, a feature that would increase stability across cell types and conditions. By extension, it should be possible to build artificial test beds to determine the behavior of known binding sites, and subsequently design promoters that are targeted and regulated by specific transcription factors. Preliminary results already show promise that this is, in fact, the case, and future experiments will help expand our \u201cvocabulary\u201d of promoter elements, and to predict their effect on in vivo expression, in presence of additional factors, such as methylation, degradation by miRNAs etc. While it might not be possible to accurately predict the behavior of artificially designed promoters in living organisms in the immediate short term, modifying short sub-sequences to adjust relative expression levels should be. In addition, emerging fields such as Synthetic Biology We do not address here how these results translate to This work might also be relevant for the study of expression regulation on a more general level: our findings suggest that different sequences can respond to transcription factors in very similar ways, even though they share no nucleotide sequence similarity; this would provide an explanation as to why promoters are generally not conserved across species over their entire length, but exhibit a pattern of conservation peaks and troughs \u03b1 score measures the \u201cun-genomicness\u201d of short (12 nucleotides long) sequences, taking into account the genome-wide frequencies of di-nucleotides, tri-nucleotides etc. within the sequence. Let N denote the number of k-mers in the genome, and ki\u03d5 the genome wide occurrence count of the k-mer starting at position i (using zero-based counting) in the 12 base pair (bp) long sequence, then the score k\u03b1 iskThe For existing promoter templates as well as the CTAG1A promoter, we computed the scores for all overlapping 12 bp long sequences within, and assigned the score to the base at position 6.\u03b1 score, and extended the boundaries of each until its composite \u03b1 score became negative, which yields a 2% false positive rate on the promoter template set were designed as described in the Constructs ArS 50, 110, 201, 232 and 300 were selected from the 160,000 nucleotide (nt) long promoter-like concatomer. For identification and spacing of TATA Boxes, we used a promoter prediction tool trained on fruit fly E. coli and purification using NucleoSpin Extrakt II all plasmids were sequenced to confirm the original sequence. In case of CHO dhfr-, HEK293 and P19, 4\u00d710\u22276 cells were transfected with 10 \u00b5g of the firefly luciferase plasmids and co-transfected with 1 \u00b5g of the Renilla luciferase reporter vector pRL-SV40 as an internal standard using Amaxa's Nucleofector Kit V according to the manufacturer's instructions. 3\u00d710\u22275 VERO and MDCK cells were transfected with 1 \u00b5g DNA of the firefly luciferase constructs and co-transfected with 0,25 \u00b5g pRL-SV40 using Dreamfect Gold and CombiMag . 3\u00d710\u22276 Sf9 cells were transfected with 1 \u00b5g of the firefly luciferase constructs using Cellfectin II according to the manufacturer's instructions. Instead of the SV40 promoter the baculovirus derived immediate early promoter OplE2, which is active in insect cells was used for the positive control. Luciferase expression was measured 48 h post transfection on a Synergy 2 microplate reader with the Gen5 software using the Dual-Glo luciferase assay system . To normalize transfection efficiency, promoter activities are expressed as the ratio of firefly and Renilla luciferase activity. The pGL3-Promoter plasmid , containing the SV40 promoter served as positive control. The promoter activity of this viral promoter was set to 100%. All other measurements refer to this value within the same cell-line. The promoterless pGL3-Basic Vector was used as a negative control.All inserts have been assembled either by oligo synthesis followed by annealing and PCR or by gene synthesis and cloning into the reporter vector pGL3 Basic upstream of a firefly luciferase gene. After propagation in on and koff) and affinities (KD) were calculated using the Octet Data Analysis Software Version 6.3.For the identification of sequence-specific DNA binding of the transcription factors TFIIB and TBP the binding kinetics were measured by biolayer interferometry on an Octet QK instrument (ForteBio Inc.), which provides continuous real-time display of biomolecular interactions. Streptavidin biosensors were loaded with biotinylated DNA fragments (25 \u00b5g/ml) of the promoter constructs ArS110, ArS201, ArS232 and ArS300, or with the promoter constructs ArS232, ArS232 dT1, ArS232 dT2 and ArS232 dT12, generated by PCR amplification using 5\u2032 biotinylated primer (Sigma-Aldrich). Binding was conducted in 1\u00d7 Kinetics Buffer (ForteBio Inc.) with a protein concentration of 285 nM for TBP and 270 nM for TFIIB . Kinetic parameters (k>ArS 50ACGCACGCGGTATAAACGCGCGACCTATTCGCGACCGTATAGCGACCGGA>ArS 110CTACGCCGCGTAAATATCGCGCGCTAACGGTGCGCGTTAAAACGCCGACGCGTCATAAAGCGCCGGCGTATAAGCGCGCCGTACGTCGTCGAACCACGTTAGTCCGGACC>ArS 201AACGGTGCGCGTTAAAACGGCCGACGCGTCATAACCGCGACTCGTCGACGCAGCGCCGGCGTATAAGCGCGCCGTACGTCAACCGTCGACGTTAGTCCGACGATCGCGGCGTCTATACGCCGCGTCAATCGCGCGCGGTTCAACGTCGCGCTACGGGCGCGTATAAGTCGCGCGTATGGACCGCGTACGTCCTACGAGCGT>ArS 232TCGACGCGCGTATAACACGCGAGCGGTTCGAACGTTGGCGCGCTAACGCGAGTCGTACGCCCGTCAACGCGGATCAATCGCGCGACTTGTGCGCGACGTTAGACCGCCGATCGTCAAGCGCCGATCGGTAATCGGACGATTCGGATACGCGAGTTCGGACGTACGAGCGTGATACGGCGCGTAACGGTGCGCGTTAAAACGCCGACGCGTCATAACCGCGACTCGTCGACGC>ArS 300AACGGTGCGCGTTAAAACGCCGACGCGTCATAACCGCGACTCGTCGACGCAGCGCCGGCGTATAAGCGCGCCGTACGTCAACCGTCGACGTTAGTCCGACGATCGCGGCGTCTATACGCCGCGTCAATCGCGCGCGGTTCAACGTCGCGCTACGGGCGCGTATAAGTCGCGCGGTTAATACGCGCGGTGTACGCGGATGCCGGGGTCGCGTATAATCGGCGCGTATACCTCGCGCGTATACGCGGCGTATTACGGCCGCGTATAATTCGCGCGTATGGACCGCGTACGTCCTACGAGCGT>CTAG1A_originalCGGAGCACGTGACCGGTTCTCACCAACCCCGCCCCTCCCCAAGAGAGCCCGGGCCGGAAGGTGGCCGCAATGCCAGCTTGGACCCCTCACCCCTGAGCAGCCGGCTGTCCGCCGGACCCCTGTCCCGGGAGCCCTGCAGGGAGTCAGGCACTGCGGGGCCCAGCCTGTCCCATCCCCCGGGTCTCCCTCACATCGAGGAGCAAGACGGGCCTGGGAACACGGGGCCGGGACTGTGCGGCCATCGTCCCGGACCCTGCCTGCCCTGTCCGTCCTTGGGGGAGCGCCCAGGACAGACfCCCGGGGGGCAGGCCTCTAfACTGGGCTCAGCAGCCTCCGTCCCTGTCCTGGTCGCCCAGCTGGTGGGGTAGCTGGAACTGCATGTCTGGTCTCAGAGAGAAGGTCAGGGCCCACGAGGATGCGGAGGCAGAGAGGCTGCAGGAAGTTCCGCCCCCTGGCGTGAGATGGGCAGCCCGGGATCCTCAGGGCGCCTGCGCACAGGGGCCCTACTTCCGGCCCTGGGAGACCCCGAGTGAGCCC>CTAG1A_replaceTTACCTAAAACAGCCCAAAAGAGCAACCCCGCCCCTCCCCAAGAGAGCCCGGGCCGGAAGGTGGCCGCAATGCCAGCTTGGACCCCTCACCCCTGAGCCCACCACCACCTCCACCACCACTGTCCCGGGAGCCCTGCAGGGAGTCAGGCACTGCGGGGCCCAGCCTGTCCCATCCCCCGGGTCTCCCTCACATCGAGGAGCAAGACGGGCCTGGGAACACGGGGCCGGCCAAAGAAGCCCAAAAAGGCCCAGGAAACCCAAACTTTCCGTCCTTGGGGGAGCGCCCAGGACAGACCCCGGGGGGCAGGCCTCTAACTGGGCTCAGCAGCCTCCGTCCCTGTCCTGGTCGCCCAGCTGGTGGGGTAGCTGGAACTGCATGTCTGGTCTCAGAGAGAAGGTCAGGGCCCACGAGGATGCGGAGGCAGAGAGGCTGCAGGAAGTTCCGCCCCCTGGCGTGAGATGGGCAGCCCGGGATCCTCAGGGCGCCTGCGCACAGGGGCCCTACTTCCGGCCCTGGGAGACCCCGAGTGAGCCC>CTAG1A_deltaTCTCAGAGAGAAGGTCAGGGCCCACGAGGATGCGGAGGCAGAGAGGCTGCAGGAAGTTCCGCCCCCTGGCGTGAGATGGGCAGCCCGGGATCCTCAGGGCGCCTGCGCACAGGGGCCCTACTTCCGGCCCTGGGAGACCCCGAGTGAGCCCCAACCCCGCCCCTCCCCAAGAGAGCCCGGGCCGGAAGGTGGCCGCAATGCCAGCTTGGACCCCTCACCCCTGAGCTCCCGGGAGCCCTGCAGGGAGTCAGGCACTGCGGGGCCCAGCCTGTCCCATCCCCCGGGTCTCCCTCACATCGAGGAGCAAGACGGGCCTGGGAACACGGGGCCGGTCCGTCCTTGGGGGAGCGCCCAGGACAGACCCCGGGGGGCAGGCCTCTAACTGGGCTCAGCAGCCTCCGTCCCTGTCCTGGTCGCCCAGCTGGTGGGGTAGCTGGAACTGCATGTCTGG>CTAG1A_upCGTTTGACGGACGCCGTTCGCAGTCAACCCCGCCCCTCCCCAAGAGAGCCCGGGCCGGAAGGTGGCCGCAATGCCAGCTTGGACCCCTCACCCCTGAGCCGGAGCACGTGACCGGTTCTCACTCCCGGGAGCCCTGCAGGGAGTCAGGCACTGCGGGGCCCAGCCTGTCCCATCCCCCGGGTCTCCCTCACATCGAGGAGCAAGACGGGCCTGGGAACACGGGGCCGGATCGCGCAGCGATCGACGCCGGATCAACGCGATACGGTCCGTCCTTGGGGGAGCGCCCAGGACAGACCCCGGGGGGCAGGCCTCTAACTGGGCTCAGCAGCCTCCGTCCCTGTCCTGGTCGCCCAGCTGGTGGGGTAGCTGGAACTGCATGTCTGGTCTCAGAGAGAAGGTCAGGGCCCACGAGGATGCGGAGGCAGAGAGGCTGCAGGAAGTTCCGCCCCCTGGCGTGAGATGGGCAGCCCGGGATCCTCAGGGCGCCTGCGCACAGGGGCCCTACTTCCGGCCCTGGGAGACCCCGAGTGAGCCCTATAACACGCGAGCGGTTCGAACGTTGGCGCGCTAACGCGAGTCGTACGCTCGACGCGCG232\u2003 60TCGACGCGCG-----CACGCGAGCGGTTCGAACGTTGGCGCGCTAACGCGAGTCGTACGC 55dT1\u2003TATAACACGCGAGCGGTTCGAACGTTGGCGCGCTAACGCGAGTCGTACGCTCGACGCGCGdT2\u2003 60TCGACGCGCG-----CACGCGAGCGGTTCGAACGTTGGCGCGCTAACGCGAGTCGTACGC 55dT12\u2003**********\u2003*********************************************CCGTCAACGCGGATCAATCGCGCGACTTGTGCGCGACGTTAGACCGCCGATCGTCAAGCG 120232\u2003CCGTCAACGCGGATCAATCGCGCGACTTGTGCGCGACGTTAGACCGCCGATCGTCAAGCG 115dT1\u2003CCGTCAACGCGGATCAATCGCGCGACTTGTGCGCGACGTTAGACCGCCGATCGTCAAGCG 120dT2\u2003CCGTCAACGCGGATCAATCGCGCGACTTGTGCGCGACGTTAGACCGCCGATCGTCAAGCG 115dT12\u2003************************************************************CCGATCGGTAATCGGACGATTCGGATACGCGAGTTCGGACGTACGAGCGTGATACGGCGC 180232\u2003CCGATCGGTAATCGGACGATTCGGATACGCGAGTTCGGACGTACGAGCGTGATACGGCGC 175dT1\u2003CCGATCGGTAATCGGACGATTCGGATACGCGAGTTCGGACGTACGAGCGTGATACGGCGC 180dT2\u2003CCGATCGGTAATCGGACGATTCGGATACGCGAGTTCGGACGTACGAGCGTGATACGGCGC 175dT12\u2003************************************************************TTAAAACGCCGACGCGTCATAACCGCGACTCGTCGACGCGTAACGGTGCGCG232\u2003 232TTAAAACGCCGACGCGTCATAACCGCGACTCGTCGACGCGTAACGGTGCGCGdT1\u2003 227GTAACGGTGCGCG------CGCCGACGCGTCATAACCGCGACTCGTCGACGC 226dT2\u2003GTAACGGTGCGCG------CGCCGACGCGTCATAACCGCGACTCGTCGACGC 221dT12\u2003*************\u2003*********************************"} +{"text": "Fibroblast growth factor 19 (FGF19) is a hormone-like protein that regulates carbohydrate, lipid and bile acid metabolism. At supra-physiological doses, FGF19 also increases hepatocyte proliferation and induces hepatocellular carcinogenesis in mice. Much of FGF19 activity is attributed to the activation of the liver enriched FGF Receptor 4 (FGFR4), although FGF19 can activate other FGFRs in vitro in the presence of the coreceptor \u03b2Klotho (KLB). In this report, we investigate the role of FGFR4 in mediating FGF19 activity by using Fgfr4 deficient mice as well as a variant of FGF19 protein (FGF19v) which is specifically impaired in activating FGFR4. Our results demonstrate that FGFR4 activation mediates the induction of hepatocyte proliferation and the suppression of bile acid biosynthesis by FGF19, but is not essential for FGF19 to improve glucose and lipid metabolism in high fat diet fed mice as well as in leptin-deficient ob/ob mice. Thus, FGF19 acts through multiple receptor pathways to elicit pleiotropic effects in regulating nutrient metabolism and cell proliferation. FGF19 (fibroblast growth factor 19) and its murine ortholog Fgf15 are the founding members of the endocrine FGF subfamily that also includes FGF21 and FGF23 In addition to the effects on lipid and glucose metabolism, FGF19/Fgf15 has also been implicated in the regulation of hepatic BA metabolism and hepatocyte proliferation. FGF19/Fgf15 expression in the intestine is transcriptionally regulated by the nuclear BA receptor Farnesoid X Receptor (FXR) FGF19/Fgf15 is believed to act by activating FGF receptor (FGFR) homodimers complexed with a membrane bound protein \u03b2Klotho (KLB) In order to determine which of the metabolic effects elicited by FGF19 are mediated by FGFR4, we treated HFD-fed WT or Fgfr4 KO mice with recombinant FGF19 or vehicle control and studied metabolic phenotypes and gene expression. To achieve sustained exposure to FGF19, mice were implanted with osmotic pumps to continuously infuse FGF19 at 1 ng/hr. This achieved an average FGF19 serum concentration of 26 ng/ml, as determined by ELISA, about 50- to 250-fold higher than circulating FGF19 concentrations in humans To evaluate changes in systemic BA regulation, serum BA composition was determined by liquid chromatography-mass spectrometry . AlthougBased upon the results described above, we hypothesized that if we could generate FGF19 variants with specifically reduced FGFR4 activity, such molecules would retain beneficial metabolic effects while losing FGFR4-dependent actions such as the induction of hepatocyte proliferation and altered BA homeostasis. In order to quantitatively evaluate specific activation of FGFRs by FGF19, an FGF-responsive GAL-Elk1 luciferase reporter assay was introduced into rat L6 cells One chimeric construct classified as a class II molecule, consisting of amino acids 1-20 of FGF21 and 25-194 of FGF19 , was selected for large scale synthesis in CHO cells and this variant is referred to as \u201cFGF19v\u201d. When compared with FGF19 using the luciferase reporter assay, FGF19v protein exhibited a similar dose-dependent activity to FGF19 in L6 cells cotransfected with KLB and FGFR1c . HoweverActivity of FGF19v was further tested in vivo in comparison with FGF19 and FGF21 by intravenously injection into overnight fasted FVB mice. Livers were harvested at 4 hours post injection and hepatic mRNA expression was determined by qPCR. Genes that were acutely induced by FGF19 but not by FGF21, such as Egr-1 and c-Fos, were not efficiently induced by FGF19v, consistent with the reduced FGFR4 activity of FGF19v . FGF19v It has been previously proposed that FGFR4 mediates the induction of hepatocyte proliferation by FGF19 The in vitro and in vivo results described above raised the question as to whether FGF19v, a variant of FGF19 with reduced FGFR4 activity and proliferative potential, could improve hyperglycemia in diabetic animals similar to FGF21. FGF21, FGF19v (1ng/hr) or vehicle control was continuously infused subcutaneously into ob/ob mice using osmotic mini-pumps. While infusion did not significantly affect body weight , both FGThe mechanism by which FGF21 and FGF19 ameliorate hyperglycemia in diabetic animals is not well understood. Since FGF21 and FGF19v show very similar anti-diabetic effects in ob/ob mice, we hypothesize that commonly regulated pathways may contribute to their anti-diabetic effects. We identified a number of genes exhibiting commonly altered expression in ob/ob mice treated with FGF21 and FGF19v. In the liver, both proteins induced IGFBP2 (a recently demonstrated anti-diabetic protein) Although FGF19 has been shown to activate multiple FGFRs in the presence of the coreceptor KLB in vitro, contribution of each FGFR to the in vivo activity of FGF19 has been poorly defined. Our findings in Fgfr4 KO mice as well as using a FGF19 variant protein with reduced FGFR4 activity have delineated pathways downstream of FGF19. We have shown that FGFR4 is required for regulation of BA biosynthesis and hepatocyte proliferation as previously proposed Intriguingly, we found that both FGF19 and FGF21 acutely reduce hepatic expression of Cyp7a1 even in Fgfr4 KO mice . This FgFgfr4 has also been implicated in the regulation of lipid metabolism and glucose tolerance and may indeed mediate regulation of fat metabolism by endogenously produced Fgf15 protein Previously, therapeutic potential for FGF19 in the treatment of obesity and diabetes has been proposed Each FGF family protein consists of the structurally conserved central globular domain, and the flanking N-terminal and C-terminal segments that are structurally flexible and are divergent in primary sequence. In X-ray crystal structures of multiple FGF/FGFR complexes, the N-terminal segment of the FGF molecule makes specific contact with the FGFR and is believed to play an important role determining the specificity of the FGF-FGFR interaction In conclusion, our study demonstrates that FGFR4 is not required for beneficial pharmacological activity of FGF19, and that an engineered FGF19 variant mimicking the specificity of FGF21 could successfully be generated. Given the pleiotropic activities of FGF19 and FGF21 on multiple receptors , further exploration into altering receptor specificity of FGF19 or FGF21 to achieve specific activation of a particular FGFR may provide a safer and more predictable approach to exploit endocrine FGF pathways and provide new therapeutic options for the epidemic of obesity associated-disorders such as type 2 diabetes, nonalcoholic fatty liver disease and other manifestations of insulin resistance and the metabolic syndrome.The study protocols for all animal experiments were approved by the Genentech Institutional Animal Care and Use Committee (IACUC). The approval IDs for this study are: #08-1943, #08-2004, #08-2004A, #08-2004B, #08-2004C, #08-2136, #08-2136F, #09-1001, #09-1066, #10-1818.Unless otherwise noted, recombinant human FGF21, FGF19 and variants produced in transiently transfected CHO cell and purified to homogeneity in PBS were used for experiments. For some experiments, E. coli derived FGF21 was used. All the purified proteins were tested for activity by cell based GAL-Elk1 assays prior to use in for other assays. For experiments in All the constructs also possessed a signal sequence at the N-terminal end (cleaved upon secretion) and the flag tag (DYKDDDDK) at the C-terminal end. The sequences derived from FGF21 are shown in bold.1 (hFGF19): RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEKLKPGTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK2: RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAHPIPDSSPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK3: HPIPDSSPLLQFGGQVRQRYLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK4 (hFGF19v): HPIPDSSPLLQFGGQVRQRYLYTDDPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK5: HPIPDSSPLLQFGGQVRQRYLYTDDAQLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK6: HPIPDSSPLLQFGGQVRQRYLYTDDAQQTSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK7: LPGLPPALPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS8: RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS9: RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS10: RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS11: RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS12: RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGEDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS13: RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS14: RPLAFSDAGPHVHYGWGDPIRLRHLYTLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS15: RPLAFSDAGPHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK16: HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS17 (hFGF21): \u20138 hours in serum free media containing 25 mg/L porcine heparin (Sigma) and FGF protein at a various concentration. The cells were then lysed with PLB reagent (Promega) and luciferase activity in each well was determined using Dual-Glo Luciferase Assay System (Promega) and EnVision Multilabel Reader (PerkinElmer). Firefly luciferase activity was normalized to the co-expressed Renilla luciferase activity, and is shown as an average and standard error of the mean of the three replicas.All the cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37\u00b0C under 5% CO2. Rat L6 myoblasts in a 96-well plate were transiently-transfected with expression vectors encoding Renilla luciferase , human KLB, appropriate human FGFR, GAL4-Elk-1 transcriptional activator , and firefly luciferase reporter driven GAL4 binding sites , using FuGENE HD Transfection Reagent (Roche Applied Science). On the next day, the transfected cells were cultured for an additional 6A 96-well-plate was filled with 50 \u00b5L/well of 0.5% molten agarose in growth media. After the base agarose had solidified, about 670 HepG2 cells suspended in 50 \u00b5L top molten agarose solution (0.35% agarose in growth media) were added to the base agar in each well, and allowed to solidify. Following solidification, 20 \u00b5L of growth medium containing an appropriate amount of FGF protein was added to each well on designated day 0. On each of the subsequent days 2, 4, 6 and 8, a further 20 \u00b5L of growth medium with an appropriate amount of FGF protein was added to each well. A subset of the sample wells was also treated with protein synthesis inhibitor Geneticin (Invitrogen) to provide a background fluorescence signal. On day 9, 10 \u00b5L AlamarBlue reagent (Invitrogen) was added to each sample well and the plate was further incubated for 5 hrs. The resulting fluorescent intensity was measured using EnVision Multilabel Reader (PerkinElmer) and used as an indication of the total metabolic activity in each well. Each condition was tested in quintuplicate.FGFR-binding activity of FGF19 and FGF19v were measured as described in Mice were maintained in a pathogen-free animal facility at 21\u00b0C under standard 12 hr light/12 hr dark cycle with access to chow or a high fat, high carbohydrate diet and water ad libitum. Male mice were used for all of the experiments described. FGFR4 KO mice in C57BL/6 background were previously described Total cholesterol, triglyceride, \u03b2-hydroxybutylate (BHB), lactate (Thermo DMA) and nonesterified fatty acid (Roche) were determined by using enzymatic reactions. Serum insulin levels were determined by ELISA . BA composition was determined by liquid chromatography-mass spectrometry analysis as previously described Tissue RNAs were isolated by using QIAzol reagent (Qiagen). cDNA was synthesize with the Quantitect Reverse Transcription Kit (Qiagen). For real time qPCR, samples were run in triplicate in the ABI Prism 7900HT (Applied Biosystems) by using SYBR green universal mix (Invitrogen) or by Taqman universal mix (Roche) and normalized by levels of 36B4. Pre-designed Quantitect primers for GK, SHP, Cyp8b1, IGFBP2, and AFP were obtained from Qiagen and all other primers were designed using primer express software (Applied Biosystems). Sequences of in-house designed primers will be provided upon request.p-value <0.05 was considered statistically significant. Values were presented as means+/\u2212 standard error of the mean.Unpaired student's t-test (two-tailed) was used for statistical analyses to compare treatment groups using Prism 5 software (Graphpad) or Excel (Microsoft). A Figure S1FGF21 and 19 activates FGFR2c and FGFR3c in the presence of KLB. GAL-Elk1 luciferase assay in L6 cells. L6 cells were cotransfected with expression vectors for KLB and the indicated FGFR together with GAL-Elk1, SV40-Renilla Luciferase, and Gal-responsive luciferase reporter. Transfected cells were incubated with media containing increasing concentrations of FGF19 (\u25cb) or FGF21(\u25b4) for 6 hours before luciferase assays. Transcriptional activation was assessed by the relative luciferase activity normalized by Renilla luciferase activity and expressed as relative luciferase unit (RLU).(TIF)Click here for additional data file.Figure S2In vitro activity of FGF21, FGF19 and chimeric constructs. GAL-Elk1 luciferase assay in rat L6 cells. L6 cells were cotransfected with expression vectors for KLB and the indicated FGFR together with GAL-Elk1, SV40-renilla Luciferase, and Gal-responsive firefly luciferase reporter. Transfected L6 cells were incubated for 6 hours before luciferase assays with conditioned medium from 293 cells transiently transfected with each FGF construct indicated at the bottom. The number below each group corresponds to the number of the construct as indicated in (TIF)Click here for additional data file."} +{"text": "Escherichia coli, yeast and CHO have been exploited for producing recombinant human insulin and a variety of different recombinant insulin are extensively used.Plants are among promising and suitable platform systems for production of recombinant biopharmaceutical proteins due to several features such as safety, no need for fermentation, inexpensive investment, and fast and easy scale-up. Human insulin is one of the most widely used medicines in the world. Up to now different expression systems including This study reports on the transformation and expression of proinsulin gene in tomato plants for the first time in Iran.Agrobacterium tumefaciens strain LBA4404, and used for Agrobacterium mediated stable transformation of tomato plants. Presence of the desired gene in transgenic lines was confirmed through colony PCR and sequencing. The expression of the protein in transgenic lines was confirmed by immunodot blot assay.This study reports the cloning, transformation and expression of proinsulin gene in tomato plants. Specific primers were designed and used for PCR amplification and cloning of the proinsulin gene in the plant expression vector pCAMBIA1304. The recombinant construct was transferred into The presence of the proinsulin gene in the genomic DNA of transgenic tomato was confirmed by PCR. Also total protein of transgenic tomato was extracted and the expression of proinsulin was detected using dotblot assay.This survey addresses the possibility of proinsulin gene transfer and expression in tomato transgenic lines. This study can be used as a basis for future researches to produce human proinsulin in tomato and other candidate plants. Escherichia coli as insoluble inclusion bodies have been demonstrated and are commercially used. The major advantage of these approaches is that proinsulin can be produced in large scale, but the intricate process of refinement and the formation of accurate disulfide bonds during folding are critical cost factors is one of the most important and favorable vegetable crops. It is an ideal candidate plant for the production and delivery of oral vaccines. Being a short-duration crop and having the ability to grow in greenhouses adds to its advantages for exploring the possibilities of using this crop for biopharmaceutical production harboring the human proinsulin gene. This expression vector having a resistance gene for kanamycin (NPT II) was used for selection of the transformed bacteria, and the hygromycin resistance gene was used to create hygromycin resistance in plants to screen the transgenic line. CaMV35S (Cauliflower Mosaic Virus promoter which induces high level of transcription) promoter, NOS terminator sequence, the reporter genes of GUS and GFP (as reporter genes) encoding beta-glucuronidase and green florescent protein was used in front of the 35S promoter, and the sequences for restriction enzymes of Bst EII and Nco I were used to replace the desired gene with the reporter genes (Staphylococcus aureus (pro A-Pins) were cloned instead of the reporter genes. Proinsulin gene was obtained from the Pasteur Institute, Iran. The coding sequence is:er genes . The humATGGCGGGATTNAACCAATTTAATAAGGAACAACAAAATGCTTTCTATGAAATCTTACATTTACCTAACTTAAATGAAGAACAACGCAATGGTTTCATCCAGAGCTTAAAAGATGACCCAAGCCAAAGCGCTAACCTTTTAGCAGAAGCTAAAAAGCTAAATGATGCACAAGCACCAAAAGCTGATAACAAAGGATCCCGTCGCTTTGTTAACCAACACCTGTGCGGTTCTCACCTGGTTGAAGCTCTGTACCTGGTTTGCGGTGAACGTGGTTTCTTCTACACCCCGAAGACCCGTCGTGAAGCTGAAGACCTGCAGGTTGGTCAGGTTGAACTGGGTGGTGGTCCGGGTGCTGGTAGCCTGCAACCGCTGGCTCTGGAAGGTTCTCTGCAGAAGCGTGGTATCGTTGAACAGTGCTGCACCTCTATCTGCTCTCTGTACTACCAACTGGAAAACTACTGCAACStaphylococcus aureus spa gene for immunoglobulin G binding protein A\", and black is proinsulin , which has been deleted because there was no need for targeting recombinant protein . Proinsulin is converted into the bioactive hormone insulin by removal of its connecting peptide (C-peptide). PROTEIN A can cause high expression of recombinant proinsulin gene and high stability of recombinant proinsulin protein produced in the Escherichia coli, and this protein causes easier purification of human proinsulin bound to it. Proinsulin is converted to bioactive hormone insulin by carboxypeptidase-H (CP-H) from Arg-Arg and Lys-Arg sites in secreting vesicle for 30 minutes at room temperature. The explants were transferred into sterile paper to remove excess Agrobacterium, and were placed into coculture medium top side down (CM) for 2 days in the dark at 28\u02daC. Finally, inoculated explants were transferred into regeneration medium (RM) were surface sterilized by 70% ethanol for 30 seconds, and then rinsed in distilled water for 1 minute. Then seed sterilization continued by immersing in 3% NaClO and rinsed three times with sterile distilled water. Sterile seeds were germinated in the dark at 25\u02daC on Murashige and Skoog (MS) medium fium (RM) . The traGenomic DNA of transgenic plants was extracted, using the CTAB method . A quantFor the extraction of total soluble protein (TSP), 200 mg of young tomato leaves were used. The tomato leaves were ground in liquid nitrogen to a fine powder. Proteins were extracted by using 1000 \u00b5L of extraction buffer and 0.04% (v/v) 2- Mercaptoethanol). Cell debris was removed by two rounds of centrifugation , and the supernatant was used for dot blot analysis. Twenty nanograms of protein samples were directly spotted onto a nitrocellulose membrane. BSA 1% solution was added after dying and incubated for 1 hour at 25\u02daC. Solution was poured out and washing with PBS-T for 5 minutes was performed; this step was repeated 3 times. Then the primary antibody was added and incubated for 1 hour. The solution was poured out; washing with PBS-T was performed for 5 minutes, and repeated 3 times. Next, nitrocellulose membrane was incubated in anti-rabbit IgG conjugated with horseradish peroxidase as a secondary antibody for an hour with 1:4000 dilutions. Color development solution was DAB and 0.01% hydrogen peroxide in 50 mM Tris (pH = 7.5).E. coli strain DH5\u03b1. The constructed vector was confirmed by colony PCR, PCR, digestion and sequencing and mammalian cell cultures (0.1% cost reduction) . The fir"} +{"text": "AbstractAmynthas Kinberg, 1867 species belonging in family Megascolecidae s. stricto are sketched, dissected and described. Amynthas daeari Blakemore sp. n. has spermathecae in 6/7/8 complying with an Amynthas tokioensis spp-group, whilst Amynthas jinburi Blakemore sp. n. has spermathecal pores in 5 & 6 strictly complying with Amynthas canaliculatus-group. A definitive COI gene barcode is provided for the holotype of Amynthas daeari but the age since collection or preservation of the Amynthas jinburi type in 2000 precluded its mtDNA extraction at this time.Two Korean endemic pheretimoid Pheretima auct.) group of Oriental origin that provides approximately 970 valid species from 1,200 nominal taxa .Specimens, now in 80% Ethanol, are lodged in the NIBR facility. Small tissue samples taken for mtDNA COI barcoding as proposed 10 yrs ago by Discussion is confined to remarks after each species\u2019 description. For brevity, not all taxonomic authorities are cited in References as these may be sought elsewhere.urn:lsid:zoobank.org:act:E6B103F4-2FFE-4A05-BCD8-356A90754AC5http://species-id.net/wiki/Amynthas_daeari35.9801N, 127.2981E); collected 27th July, 2012 by Dr Hong-Yul Seo. DNA tissue sample code \u2013 w53.IV0000261261, mature specimen complete but broken in two at clitellum after being figured and dissected. Collected from small valley at Jeollabuk-do, Wanju-gun, Dongsang-myeon, Daea-ri with GMs anterio-median and shallow clefts laterally .Female pores. Single on 14.Spermathecal pores. 6/7/8 ca 0.3 C apart at edge of puckered area and lateral to GMs.Genital markings. Paired discs just median to male and spermathecal pores as noted; composite glands on spermathecal pore GMs but none found for GMs near male pores although the body here is macerated and they may well have broken off and dissipated.Septa. Nephridial forests on septa 5 & 6; 7/8 thin, 8/9/10 aborted.Dorsal blood vessel (dbv). Single.Hearts. Last hearts in 13 (preceding vascularization unclear/damaged).Gizzard. Single in 8-9.Calciferous glands. Absent.Intestine. Indeterminate as specimen macerated; caeca ventrally incised from 27; typhlosole not noted.Nephridia. Meroic.Male organs. Holandric, seminal vesicles in 11 & 12.Ovaries. In 13 as usual.Prostates. Racemose glands in 17-19, duct short and muscular.Spermathecae. Two pairs in 7 and 8; that in 7lhs inflated, that in 8lhs deflated .Gut contents. Coarse organic debris, i.e., a litter diet suggesting superficial feeding.Amynthas daeari Holotype.>w53 PageBreakGCTCATGCATTTTTAATAATCTTCTTTCTTGTAATACCAGTATTTATTGGTGGGTTTGGAAATTGACTTCTACCTCTAATACTAGGTGCCCCAGATATAGCTTTCCCGCGACTTAACAATATAAGATTCTGATTACTGCCCCCATCACTAATTTTACTAGTATCGTCTGCAGCAGTAGAAAAAGGTGCCGGAACAGGATGGACAGTGTACCCCCCACTTGCGAGAAACATTGCACATGCCGGCCCTTCAGTAGATCTTGCAATTTTTTCTCTCCATCTAGCCGGAGCATCATCAATTCTCGGTGCCATCAACTTCATTACTACCGTAATTAATATACGATGATCTGGGCTACGCTTAGAACGAATTCCTCTATTTGTATGAGCAGTTGTAATTACTGTAATTCTTTTACTTCTATCTTTACCAGTCTTAGCCGGTGCTATTACAATATTACTAACAGACCGAAACCTAAATACATCATTTTTTGATCCAGCGGGAGGAGGTGATCCAATTCTATATCAACACTTATTTCTATATTTCATTTTAGGAATTTGAGCTGGAATAATTGGGGCAGGAATAAGACTGCTTATTCGAATTGAGCTAAGACAGCCGGGCTCTTTTCTAGGAAGGGATCAACTCTATAATACAATTGTAACAAmynthas tappensis\u201d (AB542547.1) from Japan max. identity <88% this then is a different and likely new taxon. The closest match from current Korean studies with BLASTn identity 565/653 (87%) is WO49, an immature Amynthas sp. from Jeju that itself comes closest to the Amynthas tokioensis/Metaphire hilgendorfi spp. complexes ; about twenty other species, many placed in this group after 1972, have simple intestinal caeca. Only four have simple incised caeca as here, but they all differ in characteristics of their GMs, at least, and none of these latter are known from Korea (Blakemore unpublished). The incised caeca is assumed to be a characteristic transitional or intermediate from simple to complex/manicate. The GMs in 7-8 obviously correspond to those in 18 during amphimixis but it is not known whether they interlock serially. The shape of the spermathecae and spermathecal pores are further distinguishing characteristics of Amynthas daeari that, along with its objective DNA barcode data, now serve to define this taxon.Of urn:lsid:zoobank.org:act:9BA5299B-0BA0-4E50-8EAC-C76C2D66B2EAhttp://species-id.net/wiki/Amynthas_jinburi38.2961N, 128.3546E) just north of Seoraksan Park on East coast; collected 1st \u2013 2nd June, 2000 by unknown person(s) and deposited in NIBR. DNA tissue sample w61b (unsuccessful at this time).IV0000213690, sub-mature specimen, figured and dissected. From Gangwon-do, Goseong-gun, Ganseong-eup, Jinbu-ri .Length. 210 mm.Width. ca. 10 mm at male pore level.Segments. 143 with some secondary annulation (from preservation?).Colour. Bleached pale yellow in aged alcohol, possibly darker in life.Prostomium. Open epilobous.First dorsal pore. 11/12.Setae. >100 per segment; e.g. 100+ on 11 and 112 counted on segment 12; approximately 16 setae intervene between male pore pads that are asetal on 18.Nephropores. Not found.Clitellum. Slightly darker at 14-16.Male pores: On 18 on small, rounded and flat porophores.Female pores. Single on 14.Spermathecal pores. At posterior of 5 and 6 approximately 0.3 C apart.Genital markings. None (sub-adult?).Septa. Nephridial forests on septa 5 & 6; 5/6/7/8 thick, 8/9 thin to base of gizzard, 9/10 aborted.Hearts. Seen in 11-13 (aborted in 10?).Gizzard. Single in 8-9.Calciferous glands. Absent.Intestine. From 15; caeca simple elongate from 27; typhlosole not noted.Nephridia. Meroic.Male organs. Holandric, testes small in 10 &11; seminal vesicles in 11 & 12.Ovaries. Compact in 13; ovisacs not found in 14.Prostates: Racemose glands not fully developed in 18 on short, muscular duct.Spermathecae. Two pairs in 6 & 7 exiting to anterior of 5/6 and 6/7 in 5 & 6 or denatured by pH.Amynthas serenus from Pahang, Malaysia that also lacks GMs, and Amynthas? breviclitellatus from Vietnam that differs, at least, in its GMs in 7, 18 and 19. From \u201cK\u00f4ry\u00f4\u201d Korea (about 30 Km from Seoul), Amynthas fibulus fibulus is superficially similar but has spermathecal pores anteriorly in 6 & 7 (rather than posteriorly in 5 & 6) plus its caeca are incised ventrally (rather than smooth); ditto for Amynthas fibulus ranunculus that further has slits lateral to male pores. Interestingly, Amynthas fibulus closely resembles the current specimen\u2019s gland . It appears that many of Spermathecal pores in 5/6 and 6/7...at or near leading edge of vi, vii\u201d and no useful figures are provided for the reader to decide.It should be here noted that morrisi-group\u2019s possible nearest relatives from Korea would likely be Amynthas koreanus that, however, has manicate caeca; or Amynthas kobayashii and Amynthas geojeinsulae that both have male fields from 17-19 but differ in simple or incised caeca, respectively; or Amynthas assimilis Hong & Kim, 2002 that, like many of its similar cited taxa, has seminal grooves on 18.If spermathecal pores were in 5/6/7 in any of the above taxa, then the The current species has simple, superficial male pores on large disc-like pads on 18. Although not fully mature, it appears unique in the Korea fauna on its combination of this aspect of its male field, spermathecal pores in 5 & 6 and its profusion of setae that number more than 100 per segment, combined with simple elongate intestinal caeca.Fresh topotypic material is required to confirm these conclusions and to provide definitive DNA data, unless refinement of techniques allows extraction from older types."} +{"text": "Escherichia coli as a frequently utilized host organism for recombinant protein production offers different cellular locations with distinct qualities. The periplasmic space is often favored for the production of complex proteins due to enhanced disulfide bond formation, increased target product stability and simplified downstream processing. To direct proteins to the periplasmic space rather small proteinaceus tags that can be used for affinity purification would be advantageous.Staphylococcus aureus protein A was sufficient for the secretion of various target proteins into the periplasmic space of E. coli. Our experiments indicated the Sec pathway as the mode of secretion, although N-terminal processing was not observed. Furthermore, the solubility of recombinant fusion proteins was improved for proteins prone to aggregation.We discovered that domain D of the The tag allowed a straightforward affinity purification of recombinant fusion protein via an IgG column, which was exemplified for the target protein human superoxide dismutase 1 (SOD).E. coli. Domain D of S. aureus protein A protects the protein of interest against N-terminal degradation, increases target protein solubility and enables a straight-forward purification of the recombinant protein using of IgG columns.In this work we present a new secretion tag that combines several advantages for the production of recombinant proteins in E. coli remains an attractive host for the production of recombinant proteins, even though more complex proteins with posttranslational modifications, such as glycosylation patterns require alternative host systems ; SOD [BT008028.1]; Staphylococcal Protein A[P38507]:Domain D:GFPmut3.1 [proEDDIE [ADAQQNKFNKDQQSAFYEILNMPNLNEEQRNGFIQSLKDDPSQSTNVLGEAKKLNESQAPK NproEDDIE DNA sequence sSpAD (codon optimized):GCAGACGCACAACAGAATAAGTTTAACAAAGACCAGCAGAGCGCATTCTACGAAATTCTGAACATGCCGAATCTGAATGAGGAACAACGTAATGGCTTTATTCAGTCTTTAAAAGACGACCCATCTCAGAGCACCAACGTTCTGGGCGAAGCAAAGAAACTGAACGAATCTCAGGCACCAAAA"} +{"text": "Exposure of neurons to 2 \u00b5M A\u03b21\u201342 resulted in significant viability loss and cell apoptosis. Accumulation of reactive oxygen species (ROS), decreased mitochondrial membrane potential, and activation of caspase-9 and caspase-3 were also observed after A\u03b21\u201342 exposure. All these effects induced by A\u03b21\u201342 were markedly reversed by DG treatment. In addition, DG could alleviate lipid peroxidation and partially restore the mitochondrial function in A\u03b21\u201342-induced AD mice. DG also significantly increased the PGC-1\u03b1 expression in vivo and in vitro, while knocking down PGC-1\u03b1 partially blocked the protective effects, which indicated that PGC-1\u03b1 contributed to the neuroprotective effects of DG. Furthermore, DG significantly decreased the escape latency and search distance and increased the target crossing times of A\u03b21\u201342-induced AD mice in the Morris water maze test. Therefore, these results demonstrated that DG could attenuate A\u03b21\u201342-induced neuronal injury by preventing mitochondrial dysfunction and oxidative stress and improved cognitive impairment in A\u03b21\u201342-induced AD mice, indicating that DG exerted potential beneficial effects on AD.Mitochondrial dysfunction is a hallmark of beta-amyloid (A\u03b2)-induced neurotoxicity in Alzheimer's disease (AD), and is considered an early event in AD pathology. Diammonium glycyrrhizinate (DG), the salt form of Glycyrrhizin, is known for its anti-inflammatory effects, resistance to biologic oxidation and membranous protection. In the present study, the neuroprotective effects of DG on A\u03b2 Alzheimer's disease (AD), with typical pathological abnormalities including amyloid plaques, neurofibrillary tangles and neuron death, is the most prevalent neurodegenerative disease in vivo and CREB-dependent gene expression played critical roles in the neuroplasticity associated with cognitive function The peroxisome proliferator-activated receptor gamma coactivator 1 (PGC-1) are a small family of transcriptional coactivators which play a critical role in the control of glucose, lipid, and energy metabolism 1\u201342-induced oxidative stress and mitochondrial dysfunction partially via induction of PGC-1\u03b1 and alleviated A\u03b21\u201342-induced cognitive impairment, suggesting DG might be developed into a promising drug for treatment of AD.Glycyrrhizin (GL), which is extracted in liquorice root, has a wide range of pharmacological actions including anti-virus, anti-allergenic and anti-immune-mediated cytotoxicity 1\u201342 was dissolved in 1% NH3\u00b7H2O at a concentration of 1 \u00b5g/\u00b5l and incubated at 37\u00b0C for 5 days to allow for fibril formation. DG was purchased from Jiangsu Chia-Tai Tianqing Pharmacy Company. The male ICR mice (weight range: 15\u201320 g) were anesthetized and A\u03b21\u201342 was injected to bilateral hippocampus by infusion cannulae. DG was co-injected intraperitoneally with A\u03b21\u201342. The mice were randomly assigned into four groups: the normal mice with saline or DG , and A\u03b21\u201342-induced AD mice with saline or DG . All animal experiments were approved by the Animal Care Committee in Nanjing University and performed according to institutional guidelines. We made every effort to minimize the number of mice used and their suffering.The A\u03b25 cells/ml on poly-D-lysine-coated plates. Cells were maintained in Neurobasal media supplemented with B27 and 25 nM glutamine at 37\u00b0C in a humidified 5% CO2 incubator. The purity of neurons was over 95%. The cells at day 8 were incubated with 2 \u00b5M A\u03b21\u201342 with DG or saline for 24 h.Primary cortical neurons were prepared from E15\u201317 mouse embryo. Cortexes were dissected and plated at 4\u00d7102 incubator.HEK293T, BV-2 and RAW264.7 cells were obtained from American Type Culture Collection (ATCC) and maintained in DMEM containing 10% of heat-inactivated fetal bovine serum (FBS), 2 mmol/L of L-glutamine, 100 U/ml of penicillin, and 100 \u00b5g/ml of streptomycin at 37\u00b0C in a humidified 5% COSmall hairpin RNAs (shRNAs) were synthesized and subsequently cloned into pCMV-U6 vector using Bbsl and BglII . Five PGC-1\u03b1 shRNAs sequences (shP1\u2013shP5) were designed to target mouse PGC-1\u03b1 gene. The plasmid expressing scrambled shRNA (sh-con) was used as a negative control. ShRNA sequences were as follows:5\u2032-TTTGGCCATTGTTAAGACCGAGAATCTCGAGATTCTCGGTCTTAACAATGGCTTTTTG-3\u2032, Reverse: 5\u2032-GATCCAAAAAGCCATTGTTAAGACCGAGAATCTCGAGATTCTCGGTCTTAACAATGGC-3\u2032;shP1: Forward: 5\u2032-TTTGCCCATTTGAGAACAAGACTATCTCGAGATAGTCTTGTTCTCAAATGGGTTTTTG-3\u2032, Reverse: 5\u2032-GATCCAAAAACCCATTTGAGAACAAGACTATCTCGAGATAGTCTTGTTCTCAAATGGG-3\u2032;shP2: Forward: 5\u2032-TTTGCGGAGACTATTGAGCGAACCTTAACTCGAGTTAAGGTTCGCTCAATAGTCTTTTTTTG-3\u2032, Reverse: 5\u2032-GATCCAAAAAAAGACTATTGAGCGAACCTTAACTCGAGTTAAGGTTCGCTCAATAGTCTCCG-3\u2032;shP3: Forward: 5\u2032-TTTGCGGTAACTATGCAGACCTAGATACCTCGAGGTATCTAGGTCTGCATAGTTATTTTTTG-3\u2032, Reverse: 5\u2032-GATCCAAAAAATAACTATGCAGACCTAGATACCTCGAGGTATCTAGGTCTGCATAGTTACCG-3\u2032;shP4: Forward: 5\u2032-TTTGTCCAGTAAGCACACGTTTATTCTCGAGAATAAACGTGTGCTTACTGGATTTTTG-3\u2032, Reverse: 5\u2032-GATCCAAAAATCCAGTAAGCACACGTTTATTCTCGAGAATAAACGTGTGCTTACTGGA-3\u2032.shP5: Forward: 5\u2032-TTTGGCATTGCTTCTGTGTAAATTACTCGAGTAATTTACACAGAAGCAATGCTTTTTG-3\u2032, Reverse: 5\u2032-GATCCAAAAAGCATTGCTTCTGTGTAAATTACTCGAGTAATTTACACAGAAGCAATGC-3\u2032.sh-con: Forward: The oligonucleotides were synthesized by Biocolor BioScience and Technology Company . shRNAs were transfected into neurons using Lipofectamine 2000 according to the manufacturer's instructions. Cells were harvested for RT-PCR and western blotting at 24 h after the transfection.Apoptosis was determined by Annexin V-FITC apoptosis detection kit . After treatment, the cells were rinsed with PBS twice, centrifuged at 600 g for 10 min and resuspended in 0.5 ml binding buffer containing 5 \u00b5l Annexin V and 5 \u00b5l propidium iodide (PI), and then incubated for 15 min at 37\u00b0C in the dark. The apoptotic rate was examined by flow cytometry.Cell viability was determined using the conventional MTT assay. After treatment, primary cortical neurons were treated with 0.5 mg/ml MTT for 4 h at 37\u00b0C. The formazan crystals were dissolved in 100 ml of DMSO and the absorbance was measured at 570 nm in a plate reader. Cell survival rates were expressed as percentages of the value of normal cells.LDH is the most widely used marker in cytotoxicity study. At the end of incubation, the supernatant was collected from plates and the LDH content was determined using an LDH assay kit according to the manufacturer's instructions . LDH cytotoxicity was calculated by the following formula: LDH cytotoxicity\u200a=\u200a(sample OD\u2212blank OD)/(standard solution OD\u2212blank standard solution OD )\u00d72000.Change of the mitochondrial transmembrane potential in neurons was quantified by JC-1 . Briefly, neuronal cells were centrifuged at 600 g for 10 min, and resuspended in 0.5 ml medium containing 5 \u00b5M JC-1. After 20 min of incubation at 37\u00b0C in the dark, the cells were washed with PBS twice and resuspended in 0.5 ml PBS. Samples were analyzed by flow cytometry.To monitor intracellular accumulation of ROS, flow cytometry was used with commercial kit according to the manufacturer's instructions. After treatment, the cells were harvested, rinsed with PBS twice, centrifuged at 600 g for 10 min, and then resuspended in 10 \u00b5M DCFH-DA solutions. After 20 min of incubation at 37\u00b0C, cells were washed with PBS twice and resuspended in 0.5 ml PBS. Samples were analyzed by flow cytometry.The levels of 4-HNE from hippocampus and serum were measured by the ELISA kits according to the manufacturer's instruction. Briefly, supernatant from hippocampus or serum were added into the 96-well plate coated with purified anti-4-HNE antibody, and then HRP-labeled 4-HNE antibody was added. The absorbance was measured at 450 nm and the concentration of 4-HNE was determined by comparing the O.D. of the sample to the standard curve.For measurement of cytochrome c release, the mitochondrial and cytosol fractions were prepared according to the manufacturer's instructions . Briefly, mice hippocampus were washed twice with cold PBS, resuspended in fresh cytosolic extract buffer and incubated for 30 min on ice with frequent tube tapping. Tissues were homogenized on ice, and then nuclei, unbroken cells, and cell debris were pelleted at 600 g for 10 min at 4\u00b0C. The supernatant was spun again at 13,000 g for 20 min at 4\u00b0C. The supernatant was carefully transferred and the final pellet was used as the mitochondrial fraction. The cytochrome c levels were determined using a monoclonal antibody to cytochrome c by western blotting as described below.Equal amounts of protein were separated by SDS-PAGE and electrophoretically transferred onto polyvinylidene fluoride membranes. Membranes were blocked with 5% non-fat dry milk for 1 h and incubated overnight at 4\u00b0C with rabbit anti-cleaved caspase-3 , rabbit anti-caspase-3 , rabbit anti-caspase-9 , rabbit anti-PGC-1\u03b1 , mouse anti-cytochrome c , or mouse anti-GAPDH antibody. GAPDH was used as a loading control. The proteins were detected with horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies and visualized with chemiluminescence reagents provided with the ECL kit and exposure to film. The intensity of the blots was quantified with densitometry.Caspase-3 and caspase-9 activities of primary cortical neurons were measured by means of colorimetric assay kits , according to the manufacturer's instructions. In brief, harvested cells were incubated with 50 \u00b5l lysis buffer on ice for 30 min, followed by centrifugation at 10,000 g for 1 min at 4\u00b0C. Then, cells were suspended in 50 \u00b5l 2\u00d7reaction buffer and 5 \u00b5l caspase-3 or caspase-9 substrate incubating for 4 h at 37\u00b0C. Later, the absorbance was read in a microplate reader at 400 nm.Real-time PCR was performed as described previously 5\u2032-TGACACAACGCGGACAGAA-3\u2032, Reverse: 5\u2032-GGTAGGTGATGAAACCATAG-3\u2032;PGC-1\u03b1: Forward: 5\u2032-GCCAAGGCTGTGGGCAAGGT-3\u2032, Reverse: 5\u2032-TCTCCAGGCGGCACGTCAGA-3\u2032.GAPDH: Forward: The PCR products were analyzed on 1.5% agarose gels and visualized by ethidium bromide. The gel was visualized with UV-transilluminator and photographed.5\u2032-ATAAACGCGTAATGTGTGGCCGAACACACTGT-3\u2032, Reverse: 5\u2032-CGCCGAGATCTAAAGCTATTAAAAAGTAGGCT-3\u2032) to facilitate directional cloning. The PCR products were cloned into the pGL3 basic in sense orientation (designated as p-PGC-3K). The truncated constructs were made using the following primers:The promoter regions of mouse PGC-1\u03b1 (\u22123000 to 0 bp) were amplified using PCR, DNAs of primary cortical neurons as templates, and specific primers with MluI and BglII restriction enzyme cut sites engineered on the ends : Forward: 5\u2032-ATAAACGCGTAATGTGTGGCCGAACAC-3\u2032, Reverse: 5\u2032-GTCGAGATCTTCTACTTTCCACACAGTC-3\u2032;\u22123000\u20132000 bp (named as p-PGC-1K): Forward: 5\u2032-ATAAACGCGTAATGTGTGGCCGAACAC-3\u2032, Reverse: 5\u2032-CCGCCGAGATCTTCTGACTTTATATAGTC-3\u2032;\u22123000\u20131500 bp (named as p-PGC-1.5K): Forward: 5\u2032-ATAAACGCGTAATGTGTGGCCGAACACACT-3\u2032, Reverse: 5\u2032-GCCGAGATCTTCCAACCCTAGTGCCTTG-3\u2032;\u22123000\u20131000 bp (named as p-PGC-2K): Forward: 5\u2032-ATAAACGCGTAATGTGTGGCCGAACACACT-3\u2032, Reverse: 5\u2032-GCCGAGATCTGATTTTCTTTCTCTCTCTCCT-3\u2032;\u22123000\u2013500 bp (named as p-PGC-2.5K): Forward: 5\u2032-AAATAAACGCGTGGGGGTGTTGCCTTCAAAC-3\u2032, Reverse: 5\u2032-GCCCCGAGATCTAAAGCTATTAAAAAGTAGG-3\u2032.\u2212500\u20130 bp (named as p-PGC-500 bp): Forward: The sequence of \u2212100\u20130 bp in the PGC-1\u03b1 promoter (named as p-PGC-100 bp), CREB binding site mutation sequence (named as p-PGC-100 bp mutate) and deletion sequence (named as p-PGC-100 bp delete) were synthesized with MluI and BglII restriction enzyme cut sites as followers:5\u2032-CGCGTGAGGGCTGCCTTGGAGTGACGTCAGGAGTTTGTGCAGCAAGCTTGCACAGGAGAAGGGAGGCTGGGTGAGTGACAGCCCAGCCTACTTTTTAATAGCTTTA-3\u2032, Reverse: 5\u2032GATCTAAAGCTATTAAAAAGTAGGCTGGGCTGTCACTCACCCAGCCTCCCTTCTCCTGTGCAAGCTTGCTGCACAAACTCCTGACGTCACTCCAAGGCAGCCCTCA-3\u2032;p-PGC-100 bp: Forward: 5\u2032-CGCGTGAGGGCTGCCTTGGAGTGTGGTCAGGAGTTTGTGCAGCAAGCTTGCACAGGAGAAGGGAGGCTGGGTGAGTGACAGCCCAGCCTACTTTTTAATAGCTTTA-3\u2032, Reverse: 5\u2032-GATCTAAAGCTATTAAAAAGTAGGCTGGGCTGTCACTCACCCAGCCTCCCTTCTCCTGTGCAAGCTTGCTGCACAAACTCCTGACCACACTCCAAGGCAGCCCTCA-3\u2032;p-PGC-100 bp mutate: Forward: 5\u2032-CGCGTGAGGGCTGCCTTGGAGGGAGTTTGTGCAGCAAGCTTGCACAGGAGAAGGGAGGCTGGGTGAGTGACAGCCCAGCCTACTTTTTAATAGCTTTA-3\u2032, Reverse: 5\u2032-GATCTAAAGCTATTAAAAAGTAGGCTGGGCTGTCACTCACCCAGCCTCCCTTCTCCTGTGCAAGCTTGCTGCACAAACTCCCTCCAAGGCAGCCCTCA-3\u2032.p-PGC-100 bp delete: Forward: Renilla were cotransfected to cells followed by DG treatment for 24 h. The Luciferase activity was assayed by using the Promega Bright-N-Glo system as previously described All transfection experiments in this study were performed with Lipofectamine 2000 (Invitrogen) following the manufacturer's instructions. pPGCs and phRL-CMV The Morris water maze test was conducted as previously described post hoc test with day and treatment as the sources of variation. Otherwise comparison between two groups was statistically evaluated by Student's t-test and multiple group comparisons were analyzed by one-way ANOVA followed by Tukey post hoc test. Values of P<0.05 were considered statistically significant.The data were expressed as means \u00b1 SEM and analyzed by SPSS12.0 statistical analytical software . Group differences in the escape latency, searching distance and swimming speed during the Morris water maze test were analyzed using two-way analysis of variance (ANOVA) with repeated measures followed by Bonferroni 1\u201342 (2 \u00b5M) and different concentrations of DG or saline. As expected, the viability of cortical neurons exposed to A\u03b21\u201342 was reduced by 34.1% in comparison with the control group . To further investigate DG's ability to inhibit A\u03b2-induced oxidative stress, a marker of lipid peroxidation, 4-HNE was examined. As shown in 1\u201342-induced AD mice were significantly increased by 40.4% and 67.3% compared to control mice respectively (P<0.05), while 4-HNE levels were reduced 24.2% and 33.2% in the serum and hippocampus after DG treatment respectively (P<0.05).Emerging evidence suggests that mitochondrial dysfunction and oxidative stress are involved in A\u03b21\u201342 (P<0.01). However, the decrease of \u0394\u03c8 induced by A\u03b21\u201342 was greatly alleviated after DG treatment (P<0.01), indicating that DG protected mitochondrial against A\u03b21\u201342-induced injury. Meanwhile, the activities of caspase -9 and caspase-3, were assessed. As shown in 1\u201342-treated neurons, while DG-treated neurons exhibited lower caspase-9 and caspase-3 activities compared to A\u03b21\u201342-treated neurons (P<0.05).Mitochondrial membrane potential (\u0394\u03c8) is widely recognized as an indicator of mitochondrial functionality, which is measured by JC-1, a cationic lipophilic fluorescent. The results showed that there was a significant loss of \u0394\u03c8 in neurons treated with A\u03b21\u201342 , P<0.01.reatment , P<0.01,in vivo, the release of cytochrome c from the mitochondrial membrane as well as the subsequent activation of caspase-9 and caspase-3 was investigated by western blotting. The levels of cytosolic cytochrome c expression in A\u03b21\u201342-induced AD mice were significantly increased, which were significantly reversed by the treatment with DG , 6 h (1.47\u00b10.08-fold), 12 h (1.70\u00b10.11-fold), 24 h (2.11\u00b10.48-fold) and 48 h (1.24\u00b10.38-fold) , with si38-fold) . PGC-1\u03b1 pression . To demopression and the neurons .P<0.01). Also DG treatment significantly up-regulated the transcriptional activity of PGC-1\u03b1 by 4.08-fold in HEK293T cells, 1.98-fold in BV-2 cells and 1.62-fold in RAW264.7 cells at a concentration of 0.001 \u00b5g/\u00b5l, indicating that induction of transcriptional activity of PGC-1\u03b1 by DG may not be cell type specific (P<0.01), which indicated that \u2212500\u20130 bp in the promoter of PGC-1\u03b1 might be involved in the protective effects of DG \u200a=\u200a3.620, P\u200a=\u200a0.016; group x day: F\u200a=\u200a0.915, P\u200a=\u200a0.516). In addition, the search distance was also significantly decreased by DG treatment compared to AD mice \u200a=\u200a41.688, P\u200a=\u200a0.014; days: F\u200a=\u200a11.536, P\u200a=\u200a0.01; group x day: F\u200a=\u200a1.942, P\u200a=\u200a0.067, P<0.05, 1\u201342-induced AD mice.To explore whether DG could improve cognitive impairment in A\u03b21\u201342-induced AD model in vitro and in vivo, this study for the first time shows: 1) DG exerts neuroprotective effects and improves cognitive impairment; 2) DG rescues mitochondrial dysfunction and inhibits oxidative stress; and 3) DG increases the expression of PGC-1\u03b1, which might contribute to the neuroprotection of DG.In the A\u03b21\u201342-induced neuronal toxicity, and is considered as an early event in AD pathology. Several evidences indicated that A\u03b2 triggered mitochondrial dysfunction through a number of pathways such as increase of ROS, interaction with ABAD, impaired mitochondrial biogenesis, and alteration of mitochondrial dynamics 1\u201342-treated neurons. In addition, DG decreased lipid peroxidation and release of cytochrome c from the mitochondria, and the activation of caspase-9 and caspase-3 in A\u03b21\u201342-induced AD mice. Furthermore, this anti-oxidation function of DG could refrain neurotoxicity mediated by A\u03b21\u201342, that is, increased cell viability, decreased apoptosis and LDH release in A\u03b21\u201342-treated neurons.Mitochondrial dysfunction is a hallmark of A\u03b21\u201342-treated neurons.Regardless of the possible mechanisms of DG restraining oxidative stress, it is clear that PGC-1\u03b1 is a major regulator of mitochondrial biogenesis and is protective against oxidative damage 1\u201342-treated neurons and CREB might play an important role in induction of the transcriptional activity of PGC-1\u03b1. PGC-1\u03b1 was a direct target of CREB induction of gluconeogenesis in vivoIt is intriguing that DG treatment increased the expression of PGC-1\u03b1 in A\u03b21\u201342-induced toxicity in vitro and in vivo. DG significantly increased the viability of A\u03b21\u201342-treated neurons by inhibiting oxidative stress and reversing mitochondrial dysfunction. Furthermore, PGC-1\u03b1 upregulated by DG treatment might play an important role against A\u03b21\u201342-induced neurotoxicity. Findings of current study revealed new function and mechanism of DG on neurotoxicity induced by A\u03b21\u201342, suggesting that DG may be developed into a new drug for treatment of AD.Taken together, DG exerted neuroprotective effects against A\u03b2"} +{"text": "Escherichia coli cells, it has been much more challenging to create tRNA and tRNA-Synthetase pairs that enable UAAs incorporation, for use in mammalian systems. By altering the orthogonality properties of existing unnatural pairs, previously evolved pairs for use in E. coli could be used in mammalian cells. This would bypass the cumbersome step of having to evolve mutant synthetases and would allow for the rapid development of new mammalian pairs. A major limitation to the amount of UAA-containing proteins that can be expressed in the cell is the availability of UAA-charged orthogonal suppressor tRNA. By using a natural mammalian tRNA promoter, the amount of functional suppressor tRNA can be greatly increased. Furthermore, increasing recognition of the suppressor tRNA by the mutant synthetase will ultimately lead to the appearance of more UAA-charged tRNA.The ability to site-specifically incorporate unnatural amino acids (UAAs) into proteins is a powerful tool in protein engineering. While dozens of UAAs have been successfully introduced into proteins expressed by Methanocaldococcus jannaschii (M. jannaschii) tyrosyl-tRNA synthetase (TyrRS) to charge an amber codon-suppressing tRNA with an UAA Transfer ribonucleic acids (tRNAs) serve as an adaptor molecule, bridging between genetic information and amino acid sequences during protein biosynthesis. Aminoacyl-tRNA synthetases (aaRSs) catalyze the covalent attachment of amino acids to a corresponding tRNA and have evolved active sites that are highly specific for a given substrate M. jannaschii TyrRS can only be used in prokaryotic cells; they are not orthogonal in eukaryotic systems M. jannaschii TyrRS can thus be used in a eukaryotic system if they are manipulated into ignoring endogenous tRNAs.All of the existing unnatural aaRSs derived from M. jannaschii TyrRS can be made orthogonal in mammalian cells by using peptide transplantation involving the connective polypeptide 1 (CP1) domain of E. coli TyrRS M. jannaschii that is not orthogonal in mammalian cells can be made orthogonal by switching C1:G72 to G1:C72 thus generating 1bp-tRNACUAM. jannaschii TyrRS (aaRS) specific to an unnatural amino acid in E. coli allowing it to charge 1bp-tRNACUA with the same unnatural amino acid in mammalian cells We have previously shown that M. jannaschii in mammalian cells, the tRNA was previously flanked with sequences taken from a human tyrosyl-tRNA gene. Six tandem repeats of the resulting genes were cloned into plasmid pZeoSV2 (+) to create 6x_wt-tRNACUA of green fluorescent protein (GFP) gene in vector p-EGFP-N1 to afford p-GFP_39TAG. Mammalian HEK 293T cells were co-transfected with p-GFP_39TAG and the plasmid expressing the amber-suppressor tRNA. Suppression of the 39th amber codon TAG led to the expression of a full-length GFP in vivo region in recognizing its corresponding tRNA acceptor stem M. jannaschii TyrRSs (E. coli (pdb.org 1X8X) and M. jannaschii TyrRS (pdb.org 1J1U) were used to design six E. coli CP1 swapped mutants: TyrRS_36CP1, TyrRS_39CP1, TyrRS_39CP1_2, TyrRS_42CP1, TyrRS_44CP1, and TyrRS_fullCP1. Amino acid sequence alignment of the two TyrRS was used to design another three CP1-transplanted mutants: TyrRS_29RED, TyrRS_34RED, and TyrRS_38RED. The N-terminal regions of these swaps are similar to the structural homology designs, although the C-terminal region is based on a RED sequence that is shared by both TyrRSs. The crystal structure of Thermus thermophilus (T. thermophilus) TyrRS is also available, and it is known to charge a G1:C72-containing tyrosyl-tRNA T. thermophilus TyrRS and the M. jannaschii TyrRS, two additional CP1-substituted mutants were designed: TyrRS_39tt and TyrRS_45tt. All CP1-swapped TyrRSs also contained an Arg286 mutation that has previously been shown to increase recognition of the anticodon region of a suppressor tRNA by M. jannaschii TyrRSs Our next focus was manipulating the i TyrRSs Figure 4CUA, and with a plasmid containing a gene for a CP1-swapped TyrRS, harvested 72 hours later, and analyzed by FACS analysis. Since the tRNA is not recognized by any endogenous aaRS, full-length GFP could only be detected when the 1bp-tRNACUA was charged by the CP1-swapped TyrRS. Wild type E. coli TyrRS was used as a positive control and offered a 13.5-fold increase in fluorescence over that seen when cells were transfected with a plasmid harboring 1bp-tRNACUA only orthogonality in the mammalian host system; b) stop codon-suppression efficiency; c) expression of a protein, reaching yields up to five-fold higher than realized with previously used systems that rely on the amber suppression approach in mammalian cells. These improvements pave the way for efficient and specific incorporation of UAA into recombinant mammalian proteins.E. coli TOP10 cells and isolated using a Qiagen Miniprep Kit. HEK293T cells (ATCC\u00ae CRL-11268TM) were cultured in Dulbecco's Modified Eagle Medium (D-MEM) supplemented with 10% (v/v) fetal bovine serum in a 37\u00b0C humidified incubator containing 5% CO2. Transfections were performed using FuGENE HD according to product manual . Full-length GFP was excited at 460-500 nm and detected using a Nikon Eclipse TE2000-S microscope equipped with a FITC HyQ filter (Chroma).All plasmids were amplified in The commercially available plasmid p-EGFP-N1 (Clontech) was used to express a gene for EGFP using a CMV promoter. The 39th codon in the EGFP gene was mutated from TAC to TAG by site-directed mutagenesis (SDM) using primers designed by the Stratagene online primer design program5\u2032-GCGAGGGCGAGGGCGATTAGACCTACGGCAAGC-3\u20325\u2032-GCTTGCCGTAGGTCTAATCGCCCTCGCCCTCGC-3\u2032A QuikChange II Site-Directed Mutagenesis Kit was purchased from Stratagene , and site-directed mutagenesis was completed according to the provided protocol. Plasmids were sequenced by the University of Texas at Austin ICMB DNA Sequencing Facility. The new plasmid created was termed plasmid GFP_39TAG.The MCS-1 region from a commercially available plasmid, pTRE-TIGHT-BI (Clontech) was removed by digesting the plasmid with the KpnI and EcoRI restriction enzymes . Oligonucleotides, containing a sequence for the T7 promoter:5\u2032-GGGGTACCCCTAATACGACTCACTATAGGGGGAATTCC-3\u20325\u2032-GGAATT-CCCCCTATAGTGAGTCGTATTAGGGGTACCCC-3\u2032were annealed and digested with KpnI and EcoRI. The digested insert was then ligated using DNA T4 Ligase into the pre-digested p-TRE-TIGHT-BI plasmid to form plasmid p-TRE-TIGHT-T7. Next, oligonucleotides containing the sequence for the human H1 promoter were annealed and extended using a Klenow enzyme. The oligonucleotide sequences were:5\u2032-CAACCCGCTCCAAGGAATCGCGGGCCCAGTGTCACTAGGCGGGAAC ACCCAGCGCGCGTGCGCCCTGGCAGGAAGATGGCTGTGAGGGACAGGGGAGTGGCGCCCTGCAA-3\u20325\u2032-GAACTTATAAGATTCCCAAATCCAAAGACATTTCACGTTTATGGTG ATTTCCCAGAACACATAGCGACATGCAAATATTGCAGGGCGCCACTCCCCTGTCCCTCACAGCC-3\u2032The sequence was then amplified by PCR using the primers:5\u2032-GGAATTCCAATTCGAACGCTGACGTCATCAACCCGCTCCAAGG AATC-3\u20325\u2032-GAAGATCTGTGGTCTCATACAGAACTTATAAGATTCCCA-3\u2032The resulting product was digested with the KpnI and BglII restriction enzymes and ligated into a pre-digested p-TRE-TIGHT-T7 plasmid to generate plasmid pT7-H1.M. jannaschii TyrRS (amino acids 110-148) with the CP1 region of E. coli TyrRS (amino acids 129-172). The 5\u2032 end of the gene (containing sequences for amino acids 1-109) was amplified using the following primers:TyrRS_44CP1 was created by replacing the CP1 region of 5\u2032-AAGGATCCACCATGGACGAATTTGAAATGAT-3\u20325\u2032-ACATTCATATTGCCGAACCACTGGAATTCACTTCCAT-3\u2032The 3\u2032 end (containing amino acids 149-306) was amplified by PCR using primers:5\u2032-AGGGGATTTCGTTCACTGAGGTTATCTATCCAATAATGCA-3\u20325\u2032-CCCGAATTCTAATCTCTTTCTAATTGGCT-3\u2032E. coli CP1 region (amino acids 129-172) was amplified from a wild type E. coli TyrRS gene by PCR using the following primers:Finally, the 5\u2032-ATGGAAGTGAATTCCAGTGGTTCGGCAATATGAATGT-3\u20325\u2032-TGCATTATTGGATAGATAACTTCAGTGAACGAAATCCCCT-3\u2032All three PCR fragments were combined, denatured for 15 minutes at 85\u00b0C and elongated with a Klenow enzyme for 30 minutes at room temperature. The Klenow product was again amplified by PCR, purified, and digested with the HindIII and EcoRI restriction enzymes. Finally, TyrRS_44CP1 was created by ligating the digested product into a pre-digested pEF6-V5-His6-TOPO plasmid using T4 DNA ligase (NEB). All other CP1-transplanted mutants were created by PCR in a process similar to that described in the previous section. The chimeric gene was divided into three segments, and each was amplified by PCR. Finally, a joint gene was also amplified by PCR, digested, and ligated into plasmid pEF6-V5-His6-TOPO. Primers are listed for each of the segments, based on the size and sequence of the CP1 swap.TyrRS_36CP1:5\u2032-AAGGATCCACCATGGACGAATTTGAAATGAT-3\u20325\u2032-CGCAGGAAGGTCAGCACATTCATCTGGAATTCACTTCCATAAACAT-3\u20325\u2032-ATGTTTATGGAAGTGAATTCCAGATGAATGTGCTGACCTTCCTGCG-3\u20325\u2032-TGGATAGATAACTTCAGCAACAATCCCCTGATCTTCACGGTTGAGA-3\u20325\u2032-TCTCAACCGTGAAGATCAGGGGATTGTTGCTGAAGTTATCTATCCA-3\u20325\u2032-TGCATTATTGGATAGATAACTTCAGTGAACGAAATCCCCT-3\u2032TyrRS_39CP1:5\u2032-AAGGATCCACCATGGACGAATTTGAAATGAT-3\u20325\u2032-ACATTCATATTGCCGAACCAATAAACATATTTTGCCTT-3\u20325\u2032-AGGCAAAATATGTTTATTGGTTCGGCAATATGAATGT-3\u20325\u2032-ATAACTTCAGCAACCTTTGGCCCCTGATCTTCACGGTTGA-3\u20325\u2032-TCAACCGTGAAGATCAGGGGCCAAAGGTTGCTGAAGTTAT-3\u20325\u2032-TGCATTATTGGATAGATAACTTCAGTGAACGAAATCCCCT-3\u2032TyrRS_39CP1_2:5\u2032-GCGGATCCGCCACCATGGACGAATTTGAAATGATAAAGAGAA-3\u20325\u2032-ACATTCATATTGCCGAACCAATAAACATATTTTGCCTT-3\u20325\u2032-AAGGCAAAATATGTTTATTGGTTCGGCAATATGAATGT-3\u20325\u2032-ATAACTTCAGCAACCTTTGGCCCCTGATCTTCACGGTTGA-3\u20325\u2032-TCAACCGTGAAGATCAGGGGCCAAAGGTTGCTGAAGTTAT-3\u20325\u2032-GCTCTAGAGCTTATAATCTCTTTCTAATTGGCTCTAAAATCTTTATA AGTTCTTCAGCTACAGCATTTTTTAACCTCATTGGATGCAATTCCTT-3\u2032TyrRS_42CP1:5\u2032-GCGGATCCGCCACCATGGACGAATTTGAAATGATAAAGAGAA-3\u20325\u2032-TCATAGTTGTTCGCCGCGATTGCCTTTAACCCCATTGCTT-3\u20325\u2032-AAGCAATGGGGTTAAAGGCAATCGCGGCGAACAACTATGA-3\u20325\u2032-TTCAGCAACCTTTGGATTACGGTTGAGACGCTGCTTAACC-3\u20325\u2032-GGTTAAGCAGCGTCTCAACCGTAATCCAAAGGTTGCTGAA-3\u20325\u2032-GCTCTAGAGCTTATAATCTCTTTCTAATTGGCTCTAAAATCTTTATA AGTTCTTCAGCTACAGCATTTTTTAACCTCATTGGATGCAATTCCTT-3\u2032TyrRS_44CP1:5\u2032-AAGGATCCACCATGGACGAATTTGAAATGAT-3\u20325\u2032-ACATTCATATTGCCGAACCACTGGAATTCACTTCCAT-3\u20325\u2032-AGGGGATTTCGTTCACTGAGGTTATCTATCCAATAATGCA-3\u20325\u2032-CCCGAATTCTAATCTCTTTCTAATTGGCT-3\u20325\u2032-ATGGAAGTGAATTCCAGTGGTTCGGCAATATGAATGT-3\u20325\u2032-TGCATTATTGGATAGATAACTTCAGTGAACGAAATCCCCT-3\u2032TyrRS_fullCP1:5\u2032-AAGGATCCACCATGGACGAATTTGAAATGAT-3\u20325\u2032-ACATTCATATTGCCGAACCAATAAACATATTTTGCCTT-3\u20325\u2032-AGGCAAAATATGTTTATTGGTTCGGCAATATGAATGT-3\u20325\u2032-CCAACTGCAACATCAACGCCACCGTACTGTTTGTTCAGAC-3\u20325\u2032-GTCTGAACAAACAGTACGGTGGCGTTGATGTTGCAGTTGG-3\u20325\u2032-TGCATTATTGGATAGATAACTTCAGTGAACGAAATCCCCT-3\u2032TyrRS_29RED:5\u2032-GCGGATCCGCCACCATGGACGAATTTGAAATGATAAAGAGAA-3\u20325\u2032-ACATTCATATTGCCGAACCAATAAACATATTTTGCCTT-3\u20325\u2032-AAGGCAAAATATGTTTATTGGTTCGGCAATATGAATGT-3\u20325\u2032-TTGGATTTTCATCCTCTCTGTTGAGACGCTGCTTAACCGC-3\u20325\u2032-GCGGTTAAGCAGCGTCTCAACAGAGAGGATGAAAATCCAA-3\u20325\u2032-GCTCTAGAGCTTATAATCTCTTTCTAATTGGCTCTAAAATCTTTATA AGTTCTTCAGCTACAGCATTTTTTAACCTCATTGGATGCAATTCCTT-3\u2032TyrRS_34RED:5\u2032-AAGGATCCACCATGGACGAATTTGAAATGAT-3\u20325\u2032-ACATTCATATTGCCGAACCAATAAACATATTTTGCCTT-3\u20325\u2032-AGGCAAAATATGTTTATTGGTTCGGCAATATGAATGT-3\u20325\u2032-TTGGATTTTCATCCTCTCTGTTGAGACGCTGCTTAACCGC-3\u20325\u2032-GCGGTTAAGCAGCGTCTCAACAGAGAGGATGAAAATCCAA-3\u20325\u2032-TGCATTATTGGATAGATAACTTCAGTGAACGAAATCCCCT-3\u2032TyrRS_38RED:5\u2032-GCGGATCCGCCACCATGGACGAATTTGAAATGATAAAGAGAA-3\u20325\u2032-TCATAGTTGTTCGCCGCGATTGCCTTTAACCCCATTGCTT-3\u20325\u2032-AAGCAATGGGGTTAAAGGCAATCGCGGCGAACAACTATGA-3\u20325\u2032-TTGGATTTTCATCCTCTCTGTTGAGACGCTGCTTAACCGC-3\u20325\u2032-GCGGTTAAGCAGCGTCTCAACAGAGAGGATGAAAATCCAA-3\u20325\u2032-GCTCTAGAGCTTATAATCTCTTTCTAATTGGCTCTAAAATCTTTATA AGTTCTTCAGCTACAGCATTTTTTAACCTCATTGGATGCAATTCCTT-3\u2032TyrRS_39tt:5\u2032-AAGGATCCACCATGGACGAATTTGAAATGAT-3\u20325\u2032-GTGAGGCCCTCCAGCCACTCGGAATAAACATATTTTGCCTTTAAC-3\u20325\u2032-GTTAAAGGCAAAATATGTTTATTCCGAGTGGCTGGAGGGCCTCAC-3\u20325\u2032-ATAACTTCAGCAACCTTTGGGGGAATCCCCGCCTCGTACCGCTTC-3\u20325\u2032-GAAGCGGTACGAGGCGGGGATTCCCCCAAAGGTTGCTGAAGTTAT-3\u20325\u2032-TGCATTATTGGATAGATAACTTCAGTGAACGAAATCCCCT-3\u2032TyrRS_45tt:5\u2032-AAGGATCCACCATGGACGAATTTGAAATGAT-3\u20325\u2032-CTCGGAGTTGTAGCGGAGCTCAAATAACCCCATTGCTTCAAAAAC-3\u20325\u2032-GTTTTTGAAGCAATGGGGTTATTTGAGCTCCGCTACAACTCCGAG-3\u20325\u2032-ATAACTTCAGCAACCTTTGGGGGAATCCCCGCCTCGTACCGCTTC-3\u20325\u2032-GAAGCGGTACGAGGCGGGGATTCCCCCAAAGGTTGCTGAAGTTAT-3\u20325\u2032-TGCATTATTGGATAGATAACTTCAGTGAACGAAATCCCCT-3\u2032.CUA gene were annealed, extended using a Klenow enzyme, and amplified by PCR. The sequences of oligonucleotides were:Two oligonucleotides for the 1bp-tRNA5\u2032-GAAGATCTGCGGCGGTAGTTCAGCCTGGTAGAACGGCGGACTCT AAATCCGCATGTCGCTGGTTCAAATCCGGCCCGCCGCAGACAAGTGCGGTTTTTTT-35\u2032-CCAATGCATTGGTTGCCCGCTCGAGTAGAAAAAAACCGCACTTGTC TGCGGCGGGCCGGATTTGAACCAGCGACATGCGGATTTAGAGTCCGCCGTTCTA-3\u2032CUA plasmid.The PCR product was then digested with the BglII and PstI restriction enzymes and ligated into a pre-digested pT7-H1 (described above) plasmid to create the 1bp-tRNACUA was created in a similar fashion using the following oligonucleotides to create the tRNA gene:H1_2bp-tRNA5\u2032-GAAGATCTGGGGCGGTAGTTCAGCCTGGTAGAACGGCGGACTCTA AATCCGCATGTCGCTGGTTCAAATCCGGCCCGCCCCAGACAAGTGCGGTTTTTTT-3\u20325\u2032-CCAATGCATTGGTTGCCCGCTCGAGTAGAAAAAAACCGCACTTGTC TGGGGCGGGCCGGATTTGAACCAGCGACATGCGGATTTAGAGTCCGCCGTTCTA-3CUA was generated using the following oligonucleotides:Finally, H1_3bp-tRNA5\u2032-GAAGATCTGGTGCGGTAGTTCAGCCTGGTAGAACGGCGGACTCTA AATCCGCATGTCGCTGGTTCAAATCCGGCCCGCACCAGACAAGTGCGGTTTTTTT-3\u20325\u2032-CCAATGCATTGGTTGCCCGCTCGAGTAGAAAAAAACCGCACTTG TCTGGTGCGGGCCGGATTTGAACCAGCGACATGCGGATTTAGAGTCCGCCGTTCTA-3\u2032.E. coli TyrRS was amplified from a plasmid by PCR using the following primers:A gene for 5\u2032-GCTCTAGATTATTTCCAGCAAATCAGACAGTA-3\u20325\u2032-CGGGATCCATGGCAAGCAGTAACTTGATTAAA-3\u2032E. coli TyrRS in HEK293T cells.The PCR product was digested with the XbaI and BamHI restriction enzymes and ligated using T4 ligase into a pre-digested pEF6-V5 plasmid to express All new sequences of TyrRSs have been deposited in GenBank with the following accession numbers:TyrRS_36CP1: KF050826TyrRS_39CP1: KF050827TyrRS_39CP1_2: KF050828TyrRS_42CP1: KF050829TyrRS_44CP1: KF050830TyrRS_fullCP1: KF050831TyrRS_29RED: KF050832TyrRS_34RED: KF050833TyrRS_38RED: KF050834TyrRS_39tt: KF050835TyrRS_45tt: KF050836A BD FACSCalibur flow cytometer was used to gate approximately 10,000 cells, based on forward and side scatter. Each cell was then excited at a wavelength of 488 nm and the resulting emission was detected with an FL-1 filter (515\u2013545 nm). Positive fluorescent populations were gated based on negative or positive controls and analyzed using Cyflogic v.1.2.1 computer software ."} +{"text": "The aim of this study was to investigate the presence of nerve growth factor (NGF) and its receptors tyrosine kinase A (TrkA) and p75 in the ovaries of the wild ground squirrels during the breeding and nonbreeding seasons. In the breeding period, NGF, TrkA and p75 were immunolocalized in granulosa cells, thecal cells, interstitial cells and luteal cells whereas in the nonbreeding period, both of them were detected only in granulosa cells, thecal cells and interstitial cells. Stronger immunostaining of NGF, TrkA and p75 were observed in granulosa cells, thecal cells and interstitial cells in the breeding season compared to the nonbreeding season. Corresponding for the immunohistochemical results, immunoreactivities of NGF and its two receptors were greater in the ovaries of the breeding season then decreased to a relatively low level in the nonbreeding season. The mean mRNA levels of NGF, TrkA and p75 were significantly higher in the breeding season than in the nonbreeding season. In addition, plasma gonadotropins, estradiol-17\u03b2 and progesterone concentrations were significantly higher in the breeding season than in the nonbreeding season, suggesting that the expression patterns of NGF, and TrkA and p75 were correlated with changes in plasma gonadotropins, estradiol-17\u03b2 and progesterone concentrations. These results indicated that NGF and its receptors, TrkA and p75 may be involved in the regulation of seasonal changes in the ovarian functions of the wild ground squirrel. The nerve growth factor (NGF) belongs to a family of related proteins required for the survival, maintenance, and development of discrete neuronal populations in the central and peripheral nervous systems -3. It isIt is now well known that NGF and its receptors are expressed in the mammalian ovary, including women -14, ratsCitellus dauricus Brandt) is a typical seasonal breeder which has a strict and extremely compressed breeding period from April to May and a long period of sexual dormancy from June to the following March including a 6-month hibernation period [The wild ground squirrel . Wild female ground squirrels that were regarded as adults according to their body weights (242\u2013412\u00a0g) were captured on April 13 (10.2 hours of daylight) after emergence from hibernation in the breeding period (n\u2009=\u200910) and on June 9 (12.6 hours of daylight) in the nonbreeding period (n\u2009=\u20098) of 2009 in Hebei Province, PR China.Animals were anesthetized with 4% isoflurane and blood samples were rapidly collected from leg vein. Plasma samples were frozen and stored at -20C, after the blood samples were added heparin sodium and centrifuged . An overdose of pentobarbital was applied afterwards for euthanasia. Ovary and brain were quickly removed and dissected. Length, width and weight of each ovary were measured. A part of the tissues were fixed in 0.05\u00a0M phosphate-buffered saline containing 4% paraformaldehyde for histological and immunohistochemical observation, while the rest were immediately frozen in liquid nitrogen and stored at -80C for RNA isolation and protein extraction.Ovarian samples were dehydrated in ethanol series and embedded in paraffin wax. Serial sections (4\u00a0\u03bcm) were mounted on slides coated with poly-L-lysine . Sections were stained with hematoxylin-eosin (HE) for observations of general histology. The sections were screened using an Olympus photomicroscope with a\u2009\u00d7\u200920 objective lens and imaged with software Image-Pro Plus 4.5 . Every one in ten serial sections, and altogether 50 and 30 sections for the breeding and nonbreeding season ovary respectively were selected for follicle identification [2O2. Finally, the sections were counterstained with hematoxylin and NGF, TrkA and p75 were detected, respectively. The immunostained slides were scanned using the software Image-Pro Plus 4.5 at 20\u00d7 magnification. The specificity of NGF and its receptors, TrkA and p75 antibodies have been described previously [Ovarian sections were blocked with 10% normal goat serum to prevent the non-specific binding of the second antibody. The sections were then incubated with polyclonal primary antibody against NGF , TrkA or p75 for 12\u00a0h at 4C, and incubated with the second antibody, goat anti-rabbit IgG conjugated with biotin and peroxidase with avidin for 1\u00a0h at room temperature. The sections were visualized using a rabbit ExtrAvidin\u2122 staining kit in 150\u00a0ml of 0.05\u00a0M Tris\u2013HCl buffer containing 30\u00a0mg 3,3-diaminobenzidine plus 30\u00a0\u03bcl Heviously . The imm2O2 and visualized with Odyssey infrared imaging system. Brain tissue of wild ground squirrel was used as a positive control and water, instead of primary antisera, was used as a negative control. \u03b2-actin was selected as the endogenous control. The intensities of the bands were quantified using Quantity One software and expression ratios were calculated.Ovarian tissues were weighed and dissected into small pieces using a clean razor blade. The tissues were homogenized in a tissue homogenizer containing 300\u00a0\u03bcl of 10\u00a0mg/ml PMSF and incubated for 30\u00a0min on ice. Homogenates were centrifuged at 12,000\u00a0g for 10\u00a0min at 4C. Protein extracts (25\u00a0\u03bcg) were mixed with equal volumes of 2\u2009\u00d7\u2009Laemmli sample buffer. Equal amounts of proteins from each sample were loaded onto a 12% SDS-PAGE gel and electrophoretically separated at 18\u00a0V/cm and transferred to nitrocellulose membrane using a wet transblotting apparatus . The membrane was blocked in 3% BSA for 1\u00a0h at room temperature. Primary incubation of the membrane was carried out using NGF, TrkA or p75 antibody (1:1000 dilution) for 1\u00a0h at room temperature. Secondary incubation of the membrane was then carried out using an IRDye for 1\u00a0h at room temperature. Finally, the membrane was washed in 25\u00a0ml Tris-Buffered Saline with Tween-20 plus 3\u00a0\u03bcl HTotal RNA from each sample was extracted using ISOGEN . Approximately 1\u00a0g of ovarian tissues were thawed and immediately homogenized in 10\u00a0ml of ISOGEN\u2122. The homogenate was incubated for 5\u00a0min at room temperature to allow the complete dissociation of nucleoprotein complexes. After the addition of 2\u00a0ml of chloroform, the mixture was vigorously shaken for 3\u00a0min at room temperature and centrifuged at 12,000\u00a0g for 10\u00a0min at 4C. The aqueous phase was then transferred to a fresh tube and washed with an equal volume of chloroform. An equal volume of isopropanol was added, and the sample was kept for 10\u00a0min at room temperature. RNA was precipitated by centrifugation at 12,000\u00a0g for 10\u00a0min at 4C. The RNA pellet was washed twice with 75% ethanol, briefly dried under air, and dissolved in 100\u00a0\u03bcl of diethylprocarbonate-treated water.12\u201318 according to the manufacturer\u2019s protocol. The 20\u00a0\u03bcl of reaction mixture contained 4\u00a0\u03bcg of total RNA, 0.5\u00a0\u03bcg of oligo (dT)12\u201318, 2.5\u00a0mM MgCl2, 0.5\u00a0mM dNTP, 10\u00a0mM dithiothreitol, 20\u00a0mM Tris\u2013HCl (pH\u00a08.4) and 200 U of Superscript II enzyme. The first-strand cDNA was used for PCR amplification with the appropriate primers previously proved and 2.5 U of Taq polymerase . The amplification was under the following condition: 94C for 5\u00a0min for the initial denaturation of the RNA/cDNA hybrid, 30 cycles of 94C for 1\u00a0min, 52C for 1\u00a0min, and 72C for 2\u00a0min for amplification. The PCR product was electrophoresed in the 2% agarose gel and individual bands were visualized by ethidium bromide staining. Brain tissue of wild ground squirrel was used as positive control and water, instead of cDNA, was used as negative control. The housekeeping gene, RpL7, was selected as the endogenous control as it is an estrogen-independent gene. The bands were quantified using Quantity One software and expression ratios were calculated.The first-strand cDNA from total RNA was synthesized using Superscript II Reverse Transcriptase and oligo (dT)E. coli using TOPO TA Cloning Kit . Plasmids were extracted from the bacteria and positive clones containing the proper insert were sequenced in both directions using Thermo Sequenase II Dye Terminator Cycle Sequencing Premix Kit with an automatic sequencing system . After obtaining the sequence of each PCR product, we blasted with the known mRNA sequences of mouse , rat , bovine and human , find the homologous sequence fragments in each species and compare for homology using DNAman.The purified PCR products were ligated into pCR 2.1-TOPO and the ligation products were used to transform the competent 125I-labeled radioligands as described previously [Plasma concentrations of estradiol-17\u03b2 and progesterone were determined by double-antibody RIA systems using eviously . Antisereviously and progeviously was kindMeans and standard deviations were calculated. Data were analyzed using a one-way ANOVA and the means were compared for significance using Duncan\u2019s Multiple Range Test (P\u2009=\u20090.05) using the SPSS computer software package.Ovaries of breeding and nonbreeding seasons were observed morphologically and histologically Figure\u00a0. In lineImmunohistochemistry was performed to detect the localization pattern of NGF and its receptors in the wild ground squirrel ovary and representative stainings were shown in Figure\u00a0We then moved on to detect the protein and mRNA expression levels of NGF, TrkA and p75 using Western blot and PCR, and representative bands were shown in Figure\u00a0To further confirm the nature of the PCR signals, cDNA fragments of NGF, TrkA and p75 in ovarian tissues were sequenced and compared to the corresponding fragments in mouse, rat, bovine, and human. The partial mRNA sequences in the wild ground squirrel are as below:NGF#GGGGGACTCAGTGTGTGTGCTGGTGTCAGTGTGTGGGTTGGAGATAAGACCACAGCCACAGACATCAAGGGCAAGGAGGTGACAGTGCTGGCCGAGGTGAACATTAACAACAGTGTATTCAGACAGTACTTTTTTGAGACCAAGTGCCGAGCTrkA#GGAAGTGACATCTCTACCGCAGTTCAGCACCGAGAGCGATGTGTGGAGCTTTGGGGTGGTGCTCTGGGAGATCTTCACCTATGGAAAGCAGCCCTGGTACCAGCTCTCTAACACTGAGGCGATCGAGTGTATCACGCAGGGCCGGGAGCTGGAGCGGCCGCGCGCCTGCCCTCCTGATGTCTACGCCATCATGCGAGGCTGCTGGCAGCGAGAACCGAATCAACGCCTP75#CCAGTAGGGCAGTGTGGCGGAGCCTTGCGGAGCCATCCAGACCGTGTGTGAACCCTGCCTGGACAGTGTTACGTTCTCTGACGTGGTGAGCGCCACCGAGCCGTGCAAGCCGTGCACCGAGTGCCTGGGCCTGCAGAGTATGTCCGCTCCCTGTGTGGAGGCAGACGATGCCGTGTGCCGATGCTCCTATGGCTACTACCAGGACGAGGAGACTGGCCGCTGCGAGGCTTGCAGCGTGTGCGGGGTGGGCTCAGGATCGGTGTCCTCCCRpL7#AAGGATCTGCTGCTGCTTCTGTTCCAGATCTCAATGGCACCTTTGTTAAGCTCAACAAGGCTTCAATTAACATGCTGCGGATTGTGGAGCCATACATTGCATGGGGGTACCCCAACCTGAAGTCAGTAAACGAGCTCATCTACAAGCGAGGCTACGGCAAAATCAACAAGAAGCGGATTGCCTTGACAGATAATTCCTTGAATGCACGGTCTCTTThe 176-bp NGF cDNA nucleotide sequence identity was 92.11%, 90.20%, 81.70% and 86.18%, respectively; the 261-bp TrkA cDNA nucleotide sequence identity was 94.32%, 90.83%, 84.72% and 83.41%, respectively; the 295-bp p75 cDNA nucleotide sequence identity was 94.42%, 88.10%, 84.01% and 83.64%, respectively; the 207-bp RpL7 cDNA nucleotide sequence identity was 94.55%, 84.65%, 74.23% and 76.87%, respectively that involves both sex steroids and growth factors.The authors declare that they have no competing interests.BL participated in performing the experiments, analyzing the data and drafting the manuscript. XS, LB, SH, QL and YL assisted with sample collection, all experiments and helped revising the manuscript. YH and QW designed, supervised the study, and revised manuscript. GW and KT provided some reagents and revised the manuscript. All authors read and approved the final manuscript."} +{"text": "There is growing evidence that TET proteins, which convert 5-methylcytosine (5mC) to 5hmC, play important biological roles. To further understand the function of 5hmC, an analysis of the genome-wide localization of this mark is required.Here, we have generated a genome-wide map of 5hmC in human embryonic stem cells by hmeDIP-seq, in which hydroxymethyl-DNA immunoprecipitation is followed by massively parallel sequencing. We found that 5hmC is enriched in enhancers as well as in gene bodies, suggesting a potential role for 5hmC in gene regulation. Consistent with localization of 5hmC at enhancers, 5hmC was significantly enriched in histone modifications associated with enhancers, such as H3K4me1 and H3K27ac. 5hmC was also enriched in other protein-DNA interaction sites, such as OCT4 and NANOG binding sites. Furthermore, we found that 5hmC regions tend to have an excess of G over C on one strand of DNA.Our findings suggest that 5hmC may be targeted to certain genomic regions based both on gene expression and sequence composition. Regions were defined by using SICER (v.1.03). Only regions that were called by using both input and 'no antibody' as a background control with Benjamini corrected false discovery rate < 0.05 were kept. Finally, only regions called in both antibody hmeDIP-seq experiments were kept and analyzed. Gene ontology analysis was performed using the Genomic Regions Enrichment of Annotations Tool (GREAT) . PublishFully hydroxymethylated DNA was produced by endpoint PCR using Phusion polymerase and hm-dCTP followed by PCR purification . Unmethylated and fully methylated control DNAs were produced in the same manner with dCTP and m-dCTP , respectively. Various amounts of DNA were denatured, snap cooled and dotted onto positively charged nylon membranes . Membranes were crosslinked, blocked with 5% milk, and incubated with Active Motif 5-hmC antibody for 1 hour. Membranes were washed and then incubated with anti-rabbit secondary horseradish peroxidase-linked antibody for 1 hour, washed, and developed with ECL reagent (CST) and Biomax MS film (Kodak). DNA sequences were (primer sequences in bold):TACTCTATACTCTACTCATCATTACACGCGCGATATCGTTAACGATAATTCGCGCGATTACGATCGATAACGCGTTAATATGAGATATGAGATGTGTATG; 6 CG-TACTCTATACTCTACTCATCATTACAATATATATATCGTTAACGATAATTCGCGCGATTACGATTTATAATTAATTAATATGAGATATGAGATGTGTATG; 3 CG-TACTCTATACTCTACTCATCATTACAATATATATATAATTAATTATAATTCGCGAAATTACGATTTATAATTAATTAATATGAGATATGAGATGTGTATG; 1 CG-TACTCTATACTCTACTCATCATTACAATATATATATAATTAATTATAATTAACGAAATTATAATTTATAATTAATTAATATGAGATATGAGATGTGTATG.12 CG-Human stem cell genomic DNA 5 to 10 \u03bcg) was treated with the EpiMark 5-hmC and 5-mC Analysis Kit as per the included protocol (NEB). Briefly, DNA was either glucosylated with beta-glucosyltransferase and UDP-Glc or mock treated with beta-glucosyltransferase and no UDP-Glc for 12 to 18 hours. These reactions were then split into three and mock digested, digested with MspI, or with HpaII for at least 4 hours. Samples were treated with proteinase K that was then heat inactivated. All DNA were diluted to a final concentration of 16 ng/\u03bcl to be used for PCR analysis. Quantitative PCR was completed with iQ SYBR Green Supermix using a CFX384 Real-Time PCR Detection System (Biorad). Primers used for quantitative PCR are listed in Table S1 in Additional file 0 \u03bcg was 5hmC: 5-hydroxymethylcytosine; 5mC: 5-methylcytosine; bp: base pair; ESC: embryonic stem cell; H3K4me1: histone H3 mono-methylated at lysine 4; H3K27ac: histone H3 acetylated at lysine 27; hESC: human embryonic stem cell; hmeDIP: hydroxymethyl-DNA immunoprecipitation; RPKM: reads per kilobase per million mapped reads; TFBS: transcription factor binding site.HS, SP and SEJ designed the study. SF and SMK performed the experiments. HS and SEJ analyzed the data. HS wrote the paper.Supplementary figures and table. Figure S1: 5hmC dot blots on oligos with varying amounts of 5hmC. Figure S2: characterization of defined 5hmC peaks. Figure S3: correlation of 5hmC and gene expression. Figure S4: 5hmC and different classes of putative enhancers [nhancers . Figure nhancers . Figure nhancers ,14,15. FClick here for file"} +{"text": "L26 gene encodes a virion protein that is important for high titer viral replication. To identify specific domains within the UL26 protein that contribute to viral infection, we created a panel of site-directed UL26 mutant viruses and assessed their impact on phenotypes attributed to UL26. We find that the C-terminal 38 amino acids of the UL26 protein are absolutely necessary for UL26 function. A stop-insertion mutant that produced a truncated UL26 protein lacking this region behaved identically to UL26-null viruses. This included reduced accumulation of IE1 protein at early time points, smaller plaque size, reduced virion stability, and growth with similarly attenuated kinetics. This C-terminal truncation decreased the amount of UL26 packaged into the virion resulting in reduced delivery of UL26 to newly infected cells. Further, this C-terminal truncated UL26 exhibited substantially reduced nuclear localization compared to wildtype UL26. Translation of UL26 mRNA is initiated from two separate in frame methionines that give rise to a long and a short isoform of UL26. We find that the N-terminal 34 amino acids, which are unique to the long isoform of UL26, are also important for the function of the UL26 protein. A viral mutant that produces only the short isoform of UL26 and lacks these N-terminal 34 amino acids exhibits delayed IE1 accumulation, and demonstrates intermediate defects in viral plaque size, virion stability and viral growth kinetics. Ablation of the short UL26 isoform in the presence of the long UL26 isoform did not impact any of the in vitro phenotypes tested. These experiments highlight important domains within the UL26 protein that contribute to HCMV infection.The human cytomegalovirus (HCMV) U Human cytomegalovirus (HCMV), a betaherpesvirus, is a widespread opportunistic pathogen. HCMV causes severe disease in various immunosuppressed populations including the elderly, cancer patients receiving immunosuppressive chemotherapy, transplant recipients, and AIDS patients L32 and UL99 HCMV is a relatively large virus, with a \u223c240-kb DNA genome that encodes >200 open reading frames. The viral particle is enveloped and its genome is encased within a protein capsid. Packaged in between the capsid and the viral envelope is a protein layer called the tegument, a structural feature unique to herpes viruses L26 gene, which has been found to be critical for high-titer viral replication L26 protein is expressed with early kinetics, and synthesis of the protein initiates at one of two start codons resulting in 21- or 27-kDa products L26 deletion grow to lower final titers, with slower growth kinetics, and exhibit a small plaque phenotype L26 has been implicated in transcriptional activation of the immediate early promoter L26 also impacts the structural characteristics of virions. These mutants are less stable than wildtype virions and contain hypophosphorylated tegument constituents L26 protein localizes to the nucleus at early times post infection, and to viral assembly compartments at late time points While many HCMV tegument proteins are known to be important for HCMV replication, the mechanisms through which many of these proteins contribute to the infectious cycle are unclear. One such tegument protein is encoded by the UL26 that contribute to UL26-dependent phenotypes through the creation of a panel of mutant UL26 viruses. Site-directed mutagenesis was employed to target both of UL26\u2019s initiation methionines and to introduce stop codons throughout the UL26 ORF. Analysis of these mutant viruses indicates that the UL26 short isoform is dispensable in vitro when in the presence of the UL26 long isoform. In contrast, the extra N-terminal 34 amino acids of the long UL26 isoform was found to be important for UL26-dependent phenotypes, exhibiting intermediate defects in plaque size and virion stability in comparison to wildtype and UL26-null viruses. Lastly, the carboxy terminal 38 amino acids were found to be critical for wildtype replication as deletion of this domain phenocopied UL26-deletion viruses. Deletion of these of 38 amino acids reduced the nuclear localization and tegumentation of the resulting UL26 protein product. These studies indicate that distinct domains of UL26 contribute to different UL26-dependent phenotypes and shed light on how these domains contribute to HCMV replication.Here, we analyzed specific domains of UBAdwt, a bacterial artificial chromosome (BAC) clone of Ad169 4 cells per cm2. Prior to infection, cells were serum starved for 24 hours. In all infections, viral innocula were added to cells for a 2 hr adsorption period and then aspirated. For experiments involving measurement of viral titers via plaque assay, unbound virus was inactivated through a sodium citrate wash followed by a DMEM wash immediately following viral adsorption.MRC5 fibroblasts (passages 23\u201329) were cultured in Dulbecco\u2019s modified Eagle medium supplemented with 10% fetal bovine serum. The wild type HCMV strain used in this study was L26 mutants were derived from the BAdwt clone of Ad169 (Genebank accession number: FJ527563) L26 mutants constructed are: BAdUL26 double methionine deletion (referred to as DBmet\u0394 in the text); BAdUL26 1st Methionine deletion (referred to as 1stmet\u0394 in the text); BAdUL26 2nd Methionine deletion (referred to as 2ndmet\u0394 in the text); BAdUL26 double methionine deletion rescue (referred to as DBrescue in the text); BAdUL26 #68 stop codon mutant (referred to as #68stop in the text); BAdUL26 #107 stop codon mutant (referred to as #107stop in the text); BAdUL26 #146 stop codon mutant (referred to as #146stop in the text); BAdUL26 #185 stop codon mutant (referred to as #185stop in the text). Wild type BAdwt is referred as WT in the text and BAdUL26 transposon insertion virus L26 flanking sequences was recombined into BADwt through electroporation into E. coli (strain SW105) containing BAdwt. Recombination was screened by growth in kanamycin. The Kan/Isce I cassette containing BAC was then electroporated into GS1783 cells, which contain an arabinose-inducible I-Sce 1 restriction site used for negative selection L26 gene. The UL26 DBmet\u0394 mutant was created by deletion of the 2nd Methionine from the UL26 1stmet\u0394 mutant. The primers for generating mutant viruses via two sequential PCR reactions (first reaction to introduce Kan/Isce I cassette and second reaction to insert the point mutation) were as follows (5\u2032 to 3\u2032): UL26 1stmet\u0394 insertion:F-GGCCCTCGGTGCGCTACCGGGCCCACATTCAAAAGTTTGAGCGTCAll UTTCATAGGATGACGACGATAAGTAGGG; R-GCGGCTTCATGTGGCGTGACCTCCGACCTCGTGAGGCCGAAAACGGCGTACAACCAATTAACCAATTCTGATTAG;stmet\u0394 Met to ATCUL26 1: F -GCGCTACCGGGCCCACATTCAAAAGTTTGAGCGTCTTCATCTACGCCGTTTTCGGCCTCACGAGGTCGGAGGTCACGCCA;R\u2013TGGCGTGACCTCCGACCTCGTGAGGCCGAAAACGGCGTAGATGAAGACGCTCAAACTTTTGAATGTGGGCCC GGTAGCGC; stmet to ATC negative controlUL26 1:F-GCGCTACCGGGCCCACATTCAAAAGTTTGAGCGTCTTCATGTACGCCGTTTTCGGCCTCACGAGGTCGGAGGTCACGCCA; R- TGGCGTGACCTCCGACCTCGTGAGGCCGAAAACGGCGTACATGAAGACGCTCAAACTTTTGAATGTGGGCCCGGTAGCGC;ndmet\u0394 insertionUL26 2:F-GCGGCGCGTTATAAGCACCGTGGGGTCATCGACCGACAAGGCGCGGCGATAGGATGACGACGATAAGTAGGG;R- CGCATAAAATCGTCTAAATTCAAACCGCCGTCGGGTGCGCGCCTACTCGTCAACCAATTAACCAATTCTGATTAG; ndmet\u0394 Met to ATCUL26 2: F-ATAAGCACCGTGGGGTCATCGACCGACAAGGCGCGGCGATCACGAGTAGGCGCGCACCCGACGGCGGTTTGAATTTAGAC;R-GTCTAAATTCAAACCGCCGTCGGGTGCGCGCCTACTCGTGATCGCCGCGCCTTGTCGGTCGATGACCCCACGGTGCTTAT; ndmet to ATC negative controlUL26 2:F-ATAAGCACCGTGGGGTCATCGACCGACAAGGCGCGGCGATGACGAGTAGGCGCGCACCCGACGGCGGTTTGAATTTAGAC;R-GTCTAAATTCAAACCGCCGTCGGGTGCGCGCCTACTCGTCATCGCCGCGCCTTGTCGGTCGATGACCCCACGGTGCTTAT; UL26 DBmet\u0394: F-CGCCGTTTTCGGCCTCACGAG; R-GGTGCCGATGACGCGCAACTG. The single step PCR process employs only a single set of primers that contain homology sufficient for both recombination events UL26#185stop:F-CACGGTGACGTAGCAGCACGCGGCTCACGTAGCAGGCCGATTAGCGGATGACCTGGCCGTCGGAGGATGACGACGATAAGTAGGG;R-CTCGGGCCTGCGACGCGACGCCGACGGCCAGGTCATCCGCTAATCGGCCTGCTACGTGAGCCGCAACCAATTAACCAATTCTGATTAG; UL26#146stop:F-GCTCCACGTCTTCAAAGTAGCTGTGTAGCAGGCCGCGCTCTTACAGCTGCGGCAGCGAGTCGGAGGATGACGACGATAAGTAGGG; R-GAACTTTGTAGTGCGCGCCGCCGACTCGCTGCCGCAGCTGTAAGAGCGCGGCCTGCTACACAGCAACCAATTAACCAATTCTGATTAG; UL26#107stop:F \u2013CGGCCGCCACGCCGGCCACGCTGCGGTCCCAACTGAAAAGTTAGGCGAGTCCGATGGTGCCGAAGGATGACGACGATAAGTAGGG; R-CCAGGGTCAGTTGCGCGTCATCGGCACCATCGGACTCGCCTAACTTTTCAGTTGGGACCGCAGCAACCAATTAACCAATTCTGATTAG; UL26#68stop:F-GCGGGGTGAGGATGGTCTCCTCCACGTCGCAGACAAACAATTAGTAGCCGCGCGGATAGGGCAAGGATGACGACGATAAGTAGGG; R-GCGCGGTCGCCACCTGGATCTGCCCTATCCGCGCGGCTACTAATTGTTTGTCTGCGACGTGGACAACCAATTAACCAATTCTGATTAGL26 [7H19] , PP28 [10B4\u201329], UL83-coded pp65 (8F5) and pUL44 and cellular protein tubulin [Epitomics]. A rabbit UL26 N-terminal specific antibody was generated by Biomatik (http://www.biomatik.com/) using the following underlined sequence: MTSRRAPDGGLNLDD. The methionine preceding this sequence is the 2nd UL26 initiation methionine. The secondary antibodies were rabbit polyclonal and mouse monoclonal [Abcam].Protein accumulation was assayed by Western blotting. Protein from cell lysates was solubilized in disruption buffer , separated by either 10% or 15% SDS-PAGE, and transferred to nitrocellulose in Tris-glycine transfer buffer. Blots were then stained with Ponceau S to visualize protein bands and ensure equal protein loading. The membranes were blocked in 5% milk in Tris-buffered saline-Tween 20 (TBST), followed by incubation in primary antibody. After subsequent washes, blots were treated with secondary antibody and protein bands were visualized using the enhanced chemiluminescence (ECL) system (Pierce). The primary antibodies were specific for viral proteins UL123-coded IE1 [1B12], UViral DNA accumulation was monitored by real-time PCR. At various times post infection, medium was aspirated from cells and viral DNA was harvested in lysis buffer . The extracted nucleic acid was quantified and checked for purity through 260\u2236280 absorbance by NanoDrop. Quantitative PCR (qPCR) was performed using Fast SYBR green master mix, a model 7500 Fast real-time PCR system, and Fast 7500 software (Applied Biosystems) according to manufacturer\u2019s instructions. Viral DNA was quantified with specific primer pairs targeting UL83 (pp65), 5\u2032-CAG-GAA-GAT-TTG-CTG-CCC-GTT-CAT-3\u2032 (forward) and (5\u2032-GGC-TTT-ACG-GTG-TTG-TGT-CCC-AAA-3\u2032 (reverse).For immunofluorescence, MRC5 fibroblasts were grown on glass coverslips. At various time points post infection, cells were washed once with PBS, fixed with 2% paraformaldehyde in PBS for 20 min, washed three times with PBS, and permeabilized with 0.1% Triton X-100 and 0.1% SDS for 15 min, then washed twice with PBS containing 0.05% Tween-20. Cells were subsequently blocked by overnight incubation in PBS containing 2% bovine serum albumin (BSA), 5% goat serum, 5% human serum, and 0.3% Triton X-100. Cells were incubated with anti-UL26 sera [7H19] that had been diluted 1\u22362 in PBS containing 0.05% Tween-20 for 1 hr. Slides were subsequently washed with PBS containing 0.01% Tween-20 three times, incubated with fluorochrome-conjugated anti-mouse secondary antibody for 1 hr, and washed three times in the same buffer lacking antibody. Coverslips were mounted in slow-fade Gold antifade reagent (Molecular Probes) and DAPI . Confocal images were captured with FV1000 Olympus laser scanning confocal microscope. All images were captured under identical confocal settings.L26 and anti-pp65-specific western to ensure the linearity of UL26 and pp65 detection. Quantitation of UL26 and pp65-specific protein bands was performed using ImageLab software tools from BioRad.To produce partially purified virions for the analysis of their constituent proteins, WT and #185stop virus stocks were first clarified by low speed centrifugation and then centrifuged through a sorbitol cushion at 26K rpm for 1 hr. The virion pellet was then resuspended in T.N. buffer containing 20 mM Tris-HCl, PH 7.4, 100 mM NaCl, and 1.5% BSA and purified by centrifugation through a glycerol tartrate gradient as previously described Replicate cultures of MRC5 fibroblasts were infected with 25 PFU of the indicated recombinant virus. Representative plaques at day 15 post infection for each virus are shown. Areas of representative plaques for each virus were quantified by Image J and normalized to the WT plaque size. To investigate the stability of virion infectivity, an equivalent number of plaque forming units from freshly thawed viral stocks were incubated at 37\u00b0C for 0, 4, 8, or 20 hours. After the indicated incubation period, confluent MRC5 fibroblasts were infected. The percentage of plaques remaining relative to the 0 h control was plotted.To investigate virion stability after trypsin exposure, infected MRC5 cells (MOI\u200a=\u200a3.0) were harvested when the CPE reached 80%. The media containing infectious virus was reserved, and cells were scraped in a small volume of media and sonicated. The sonicated cells and reserved culture media were combined and centrifuged at 6,000 rpm for 30 min. The supernatants were then sedimented at 38,000\u00d7g for 60 min. The pellets containing virus were resuspended in serum-free minimal essential medium. Two hundred \u00b5l of either 2.5% trypsin (Invitrogen) or media was mixed with 1.8 ml of the resuspended virus, and incubated at 37\u00b0C for 30 or 60 minutes for the trypsin treated, or 0 minutes for the media control. To inactivate the trypsin, at the end of the prescribed intervals, calf serum was added to a final concentration of 10%. The suspension was then tittered by plaque assay. The percentage of plaques remaining relative to the media control was plotted.t-test unless otherwise indicated. A probability of value (p) <0.05 was considered statistically significant. For comparison of the viral growth between wildtype and the #185stop mutant from 48\u2013120 hpi a homoscedastic paired two-tailed ttest of viral titers at each time point was performed. Averages are plotted with either standard deviation (SD), or standard error of the mean (SE) as indicated.Statistical significance was assessed by a non-paired two tailed homoscedastic student\u2019s L26 gene have been shown to be growth attenuated L26 protein is expressed from a spliced mRNA transcript that is also responsible for the expression of the UL29, UL28, UL27, and UL29/28 open reading frames L26-null viruses contain large deletions within the UL26 open reading frame which could impact the expression of the other open reading frames that are expressed from this mRNA transcript. Further, it is unclear how the two separate isoforms of the UL26 protein contribute to HCMV infection. To address these issues, and to map the domains of UL26 that impact viral replication, we employed BAC-mediated recombineering to create a panel of viruses containing site-directed UL26 mutations. This panel included viruses containing a mutation ablating one or both initiating methionines as well as viruses containing stop codon insertions throughout the UL26 open frame.Viruses containing deletions in the UL26 protein is illustrated in L26 reading frames that could be stably expressed, stop insertions were made at locations that approximated transitions between these predicted domains , nor did the previously described transposon-deleted UL26 virus (UL26TI) and were not analyzed in further detail. The specific mutations of the remaining recombinant viruses, and the UL26 ORFs they produce, are illustrated in A plot of manavalan hydrophobicity domains . To veri isoform . Further(UL26TI) . The repisoforms . The stoL26 protein impacts production of viral progeny, we wanted to elucidate how specific domains of the UL26 protein contribute to viral replication. We first wanted to assess whether the double methionine deletion mutant (DBmet\u0394), grew with similar kinetics as the transposon insertion mutant (UL26TI). As shown in L26 ORF does not substantially impact the in vitro viral growth over and above what is observed with less disruptive targeting of the UL26 initiation methionines.As it has previously been found that deletion of the UL26 protein contribute to viral growth, we analyzed the replication of the individual methionine mutants and the #185 stop insertion mutant. At high MOI, the 2ndmet\u0394 virus, which only expresses the long isoform of UL26 . The #18(DBmet\u0394) . Similarl titers . Interesl titers . The comL26 is important for wildtype levels of IE1 accumulation L26 domains on viral gene expression, we analyzed the accumulation of viral proteins during infection. Upon infection at an MOI\u200a=\u200a3.0, both UL26-null viruses, UL26TI and DBmet\u0394, accumulated less IE1 at 4 hpi, but recovered by 24 hpi , viral DNA accumulated similarly between the WT and 2ndmet\u0394 from our panel of mutants, overlaid with agarose and incubated at 37\u00b0C for 15 days. Images of the resulting plaques were captured with subsequent analysis of the area of each plaque. Images of representative plaques are shown in L26-null viruses. The plaques produced by the 1stmet\u0394 virus were intermediate in size between the UL26-null viruses and WT HCMV, whereas the 2ndmet\u0394 virus produced plaques of WT size. These results indicate that the C-terminal 38 amino acids of the UL26 protein, and to a lesser extent, the N-terminal 34 amino acids are important for WT plaque size.We and others have observed that deletion of the UL26 protein is important for virion stability, inasmuch as prolonged incubation at 20\u00b0C causes UL26-defective virions to lose their ability to initiate infection faster than WT virions do. L26 mutations impact virion stability, viral supernatants containing equivalent PFUs were incubated at 37\u00b0C for various times and then plated. The percentage of plaques remaining after incubation in comparison to control was plotted in L26-null viruses demonstrated a \u223c50% drop in infectivity, a statistically significant difference . This suggests that large differences in virion protein constituents are likely not responsible for the decreased stability of mutant UL26 viruses. Another major question is the function of UL26 in the nucleus at early times. It seems likely that this nuclear UL26 is responsible for impacting IE1 gene expression at early times, although the potential mechanism involved still needs to be elucidated. Our identification of the important UL26 N-terminal and C-terminal domains will facilitate addressing these questions. The C-terminal 38-amino acids of UL26 are important for proper UL26 tegumentation, nuclear localization, and viral replication. Our studies highlight the importance of these C-terminal 38-amino acids for future study. Further functional analysis will distinguish how the specific residues within this domain contribute to UL26 nuclear localization and proper tegumentation, and subsequently to HCMV replication. Given its importance to HCMV infection, elucidating the mechanisms through which UL26 domains contribute to high-titer replication may shed light on possibilities for therapeutic intervention.A number of questions remain about U"} +{"text": "Short interfering RNAs (siRNAs) are widely used as tool for gene inactivation in basic research and therapeutic applications. One of the major shortcomings of siRNA experiments are sequence-specific off-target effects. Such effects are largely unpredictable because siRNAs can affect partially complementary sequences and function like microRNAs (miRNAs), which inhibit gene expression on mRNA stability or translational levels. Here we demonstrate that novel, enzymatically generated siRNA pools\u2014referred to as siPools\u2014containing up to 60 accurately defined siRNAs eliminate off-target effects. This is achieved by the low concentration of each individual siRNA diluting sequence-specific off-target effects below detection limits. In fact, whole transcriptome analyses reveal that single siRNA transfections can severely affect global gene expression. However, when complex siRNA pools are transfected, almost no transcriptome alterations are observed. Taken together, we present enzymatically produced complex but accurately defined siRNA pools with potent on-target silencing but without detectable off-target effects. RNA interference (RNAi) is not only a potent cellular pathway to silence endogenous or exogenous genes but is also a widely used tool for sequence-specific gene knockdown . The triThe core protein component of RISC and direct interaction partner of siRNAs is a member of the Argonaute (Ago) protein family. Ago proteins are characterized by PAZ, MID and PIWI domains . The PAZAfter an initial hype and the hope that RNAi develops into a potent novel strategy for therapy, it became clear that several problems associated with RNAi experiments have not been solved. Besides delivery issues in RNAi-based therapeutic approaches, one of the major problems of siRNAs are off-target effects ,21. MostHere, we report the development of a new generation of siRNA tools without off-target effects. Using an enzymatic approach, we generate complex pools of accurately defined siRNAs. While synergistically silencing one single on-target gene, each individual siRNA is present at very low concentrations, effectively diluting off-target effects below detection limits. The novel enzymatic strategy allows for the free combination of individually selected siRNA sequences to obtain optimal siRNA pools. We refer to our novel siRNA pools as siPools and overcome off-target effects, the major shortcoming of classical siRNA reagents.in vitro transcribed using an integrated T7 promoter sequence followed by RNA precipitation with 2.5 M LiCl. Sense and antisense strands were annealed by incubation at 95\u00b0C for 5 min followed by slow cooling to room temperature. Annealed dsRNAs were subsequently digested with 10 U/\u03bcg RNase T1 to obtain ds siRNAs with 2 nt 3\u2032 overhangs. RNase T1 digested siRNAs were purified by native 20% polyacrylamide gel electrophoresis and eluted from the gel overnight at 4\u00b0C in elution buffer followed by ethanol precipitation. To obtain siPools with 30 siRNAs, siPool #1 was mixed with siPool #2, and to generate siPool 45 with 45 different siRNAs, siPool #1 was mixed with siPool #2 and #3. siPool 60 containing 60 different siRNAs was mixed by the use of all four different siPools #1, #2, #3 and #4. TNRC6A, TNRC6B, TNRC6C and unspecific control siPools (neg. pools) each comprised of 30 individual siRNA sequences. For all template and siRNA sequences, see Supplementary Material. Ready-to-use siPools against various genes were purchased from siTools Biotech, Munich, Germany.DNA templates were synthesized by GeneArt (Life Technologies). SiPool templates were Antisense strands of siRNA sequences were depicted: PolG siRNA off-T: 5\u2032-AAUAUCCAGCGCUUCACCC-3\u2032, Scyl1 siRNA off-T: 5\u2032-ACAUUGUUGUGGAUGAGGC-3\u2032; Mad2 siRNA: 5\u2032-CCAAUCUUUCAGUUGUUCC3\u2032; neg. ctrl. siRNA: 5\u2032-UUGUCUUGCAUUCGACUAA-3\u2032. esiRNAs (Sigma) were delivered as RNase III digested double-stranded products derived from the following sequences, PolG esiRNA: 5\u2032-GGAAGAAGTGGGAGGTGGTTGCTGAACGGGCATGGAAGGGGGGCACAGAGTCAGAAATGTTCAATAAGCTTGAGAGCATTGCTACGTCTGACATACCACGTACCCCGGTGCTGGGCTGCTGCATCAGCCGAGCCCTGGAGCCCTCGGCTGTCCAGGAAGAGTTTATGACCAGCCGTGTGAATTGGGTGGTACAGAGCTCTGCTGTTGACTACTTACACCTCATGCTTGTGGCCATGAAGTGGCTGTTTGAAGAGT-3\u2032, Scyl1 esiRNA: 5\u2032-CAGCCGAGAAGCAAAAATTCTTCCAGGAGCTGAGCAAGAGCCTGGACGCATTCCCTGAGGATTTCTGTCGGCACAAGGTGCTGCCCCAGCTGCTGACCGCCTTCGAGTTCGGCAATGCTGGGGCCGTTGTCCTCACGCCCCTCTTCAAGGTGGGCAAGTTCCTGAGCGCTGAGGAGTATCAGCAGAAGATCATCCCTGTGGTGGTCAAGATGTTCTCATCCACTGACCGGGCCATGCGCATCCGCCTCCTGCAGCAGATGGAGCAGTTCATCCAGTACCTTGACGAGCCAACAGTCAACACCCAGATCTTCCCCCACGTCGTACATGGCTTCCTGGACACCAACCCTGCCATCCGGGAGCAGACGGTCAAGTCCATGCTGCTCCTGGCCCCAAAGCTGAACGAGGCCAACCTCAATGTGGAGCTGA-3\u2032.To generate low-complexity pools, the following siRNAs were used: PolG siRNA 1: 5\u2032-UCAUCCGACAGCCGAUACC-3\u2032, PolG siRNA 2: 5\u2032-AAUUCUUGCAGGUCCCACU-3\u2032, PolG siRNA 3: 5\u2032-GCUAUUACCAUCCUUGUGA-3\u2032, PolG siRNA 4: 5\u2032-UUAAACUGCAUUAGUAAGC-3\u2032. Scyl1 siRNA 1: 5\u2032-UUUCUCAGGAUCUACAGUGAG-3\u2032, Scyl1 siRNA 2: 5\u2032-UUGAGGUAUAUUCCCAACGGG-3\u2032, Scyl1 siRNA 3: 5\u2032-UUGGUUUCUACAAAGCGGUUG-3\u2032, Scyl1 siRNA 4: 5\u2032-UUGUACAAUAAAUACAUCUGU-3\u2032. Three siRNAs were mixed in different combinations with the corresponding siRNA off-T to obtain four different siRNA-containing low-complexity pools with possible Mad2 off-target effects. For pool #1, siRNA 1, 2, 3; for pool #2, siRNA 1, 2, 4; and for pool #3, siRNA 1, 3 and 4 were mixed with off-T siRNAs. Low-complexity pools consisting of siRNA 1\u20134 were used as non-Mad2 off-target controls (pool #4).HeLa cells were cultivated in Dulbecco's modified Eagle's medium substituted with 10% FCS and penicillin/streptomycin. siRNA transfections were done using Lipofectamine RNAiMax (Life Technologies) according to the manufacturer's protocol. Cells were harvested 24 h or 48 h after transfection.RNA was isolated 24 h after transfection followed by cDNA synthesis and quantitative polymerase chain reaction (qPCR). Following primers were used: PolG for: 5\u2032-TTCCAGGACCTGATGCAGTA-3\u2032, PolG rev: 5\u2032- ACAGGCAGGTAGGAGACACC-3\u2032, Scyl1 for: 5\u2032-CTGGAGGAAGTGGAGAAGGA-3\u2032, Scyl1 rev: 5\u2032-TCAGCTTGGAGGTGAGTGAG-3\u2032, Mad2 for: 5\u2032-AGATGACAGTGCACCCAGAG-3\u2032, Mad2 rev: 5\u2032-TCCAACAGTGGCAGAAATGT-3\u2032 GAPDH for: 5\u2032-ATGGGTGTGAACCATGAGAA-3\u2032, GAPDH rev: 5\u2032-GTGCTAAGCAGTTGGTGGTG-3\u2032, TNRC6A for: 5\u2032-CCCTCAGGTTCCAGTTTCAT-3\u2032, TNRC6A rev: 5\u2032-GCTGGTTTAGCTGGGATAGC-3\u2032, TNRC6B for: 5\u2032-ATGGTTCTGGCTTCAGCTCT-3\u2032, TNRC6B rev: 5\u2032-CATATTGGCTTCCTGTGTGG-3\u2032, TNRC6C for: 5\u2032-TAGCAAGCATGGTGCTATCC-3\u2032, TNRC6C rev: 5\u2032-GTACCGGCCATAGGAGTCAT-3\u2032, OAS1 for: 5\u2032-TGCCATTGACATCATCTGTG-3\u2032, OAS1 rev: 5\u2032-GAGCCACCCTTTACCACCT-3\u2032, INFB1 for: 5\u2032-AGTAGGCGACACTGTTCGTG-3\u2032, IFNB1 rev: 5\u2032-GAAGCACAACAGGAGAGCAA-3\u2032, IL6 for: 5\u2032-AATGAGGAGACTTGCCTGGT-3\u2032, IL6 rev: 5\u2032-GCAGGAACTGGATCAGGACT-3\u2032, STAT1 for: 5\u2032-AATTGACCTCGAGACGACCT-3\u2032, STAT1 rev: 5\u2032-CACATGGTGGAGTCAGGAAG-3\u2032.For western blot analysis cells were harvested and lysed in NET buffer 48 h after transfection. Proteins were separated via sodium dodecyl sulphate-polyacrylamide gel electrophoresis followed by semi-dry electroblotting. Following antibodies were used: polyclonal anti-MAD2 (Bethyl Laboratories) at a dilution of 1:5000 and a monoclonal mouse anti beta actin antibody (clone AC15 from Abcam) at a dilution of 1:5000 in TBS-Tween with 5% milk powder. Fluorescently labeled IRDye 800 CW antibodies were used as secondary antibodies (Li-COR). Western blots were imaged with an Odyssey Fluorescence scanner (Li-COR).To generate an off-target reporter construct, a modified pMIR dual luciferase reporter plasmid was usedHeLa cells were transfected with 10 nM siPools and lysed in NET buffer 48 h after transfection. Lysates were used for Ago2-siRNA co-immunoprecipitation. Protein-G sepharose beads (GE) were pre-incubated with monoclonal anti-Ago2 (11A9) antibody . LysatesHeLa cells were transfected with 3 nM Scyl1 siRNA, Scyl1 siPool 15, Scyl1 siPool 60, or mock transfected in three biological replicates. RNA was isolated 48 h after transfection and further processed for Affymetrix Human Gene 1.0 ST array analysis. The Human Gene 1.0 ST array platform from Affymetrix was used to assess transcriptome-wide expression profiles. Normalization of raw intensity values from CEL files was performed using variance stabilization , and thelimma , or mock transfected in three biological replicates. RNA was isolated 48 h after transfection. Transcription profiles were analyzed using the Human Gene 2.0 ST array platform (Affymetrix). Raw intensity values (from CEL files) were normalized using RMA (oligo ). The nsanalyzeR . All logAnalyses were performed within the statistical programming environment R and using Bioconductor packagesP-value for the number of repressed transcripts with a seed match for the Scyl1 siRNA to belong to the same distribution. For the pool 15 and pool 60, three additional siRNA seed sequences were drawn from the pools and tested analogously.Human 3\u2032-UTR sequences were retrieved from Ensembl version 68 for the transcripts represented on the cartridge microarray. The reverse compliment of the siRNA seed sequences (7 nt) was searched for in the 3\u2032-UTRs of the transcripts and 2292 transcripts with a seed sequence match were found. To analyze whether there is significant enrichment of transcripts with a seed-binding site among the repressed transcripts after siRNA transfection, the number of repressed transcripts with a seed match was compared to the mean number of transcripts with a random seed match. Seven thousand five hundred random seed sequences were drawn and from these, 2000 with similar numbers of transcripts with a seed match in the 3\u2032-UTR (range 1692\u20132892 transcripts per sequence) were drawn and the number of repressed transcripts with a seed match were recorded for each random seed sequence. The distribution of these values was used to calculate the We hypothesized that siRNA pools containing up to 60 individual siRNAs against one target possess very low off-target effects because of the very low concentration of each individual siRNA. However, chemical synthesis of high numbers of siRNAs is cost-intensive and therefore not practicable. Therefore, we set out to generate complex siRNA pools enzymatically. For our experiments, we chose the human target genes PolG and Scyl1. siRNAs with strong off-target effects against the gene MAD2 had been reported for both genes . These sFor siRNA pool production, we selected siRNA sequences using standard siRNA prediction tools, which are connected by non-complementary linker sequences Figure . The DNAThe two strands were transcribed, annealed and analyzed on a native polyacrylamide (PAA) gel , but not in the siPool transfections or siRNASince siPools and esiRNAs derive from longer dsRNA precursors and such precursors might cause interferon response, we tested the expression of interferon response genes after siRNA transfection Figure . siPoolsOff-target effects are a severe but often ignored error source in RNAi experiments. This is particularly important for genome-wide screening approaches, in which only a number of hits can be validated individually . In factO-methylation of the guide strand leads to a reduction of miRNA-like off-target effects presumably due to less efficient binding to the seed sequence of the off-target mRNA. For siRNA on-target activity, this modification seems to be tolerated and theobserved highlighEscherichia coli RNase III generates more discrete size products thus improving RNase III generated siRNAs have been discovered in various organisms and tissues ,59. Theshttp://www.ncbi.nlm.nih.gov/geo/) under accession GSE57674.Raw intensities and normalized expression data of the microarrays are publicly available at the NCBI Gene Expression Omnibus (GEO, Supplementary Data are available at NAR Online."} +{"text": "CCND1) and a 3\u2032UTR consisting of sequences from both the CCND1 3\u2032UTR and myotonic dystrophy kinase-related Cdc42-binding kinase's (MRCK) intron one. The resulting CCND1/MRCK mRNA is resistant to CCND1-targeted miRNA regulation, and targeting the MRCK region of the chimeric 3\u2032UTR with siRNA results in decreased CCND1 levels.The t translocation resulting in constitutive cyclin D1 expression is an early event in mantle cell lymphoma (MCL) transformation. Patients with a highly proliferative phenotype produce cyclin D1 transcripts with truncated 3\u2032UTRs that evade miRNA regulation. Here, we report the recurrence of a novel gene fusion in MCL cell lines and MCL patient isolates that consists of the full protein coding region of cyclin D1 (The online version of this article (doi:10.1186/s13045-016-0260-7) contains supplementary material, which is available to authorized users. CCND1) transcript [CCND1 mRNA has a long 3\u2032UTR (~3.1\u00a0Kb) that contains numerous destabilizing elements [CCND1 transcripts with truncated 3\u2032UTRs correlating with reduced survival [Mantle cell lymphoma (MCL) is considered incurable upon relapse . The halanscript . The CCNelements , 5. MCL survival . In somesurvival . In othesurvival , has beesurvival . Aside fsurvival .CCND1 mRNA. We observed 3\u2032rapid amplification of cDNA end (3\u2032RACE) products in all the MCL lines that would indicate that 3\u2032UTR shortening has occurred gene from MRCK, which triggers the subsequent addition of the poly(A) tail, creating a chimeric 3\u2032UTR. The observed CCND1/MRCK fusion gene likely is formed by a second translocation event between chromosomes 11 and 14, which positions the full open reading frame of CCND1 and a truncated 3\u2032UTR within intron one of MRCK or the truncated CCND1 3\u2032UTR from Jeko-1 (Tr-CCND1) and co-transfected them with three miRNA mimics known to repress CCND1. The FL-CCND1 reporter was downregulated in response to each mimic tested and 5% penicillin/streptomycin. All three MCL cell lines: Jeko-1, Granta-519, and SP-53 contain the t11;14) translocation [;14 per the manufacturer\u2019s protocol. HF Phusion buffer was used together with MgCl2. Primers were designed to provide the largest DNA PCR product possible, and were validated to work with genomic DNA. The primer sequences used were CCND1 forward 5\u00b4TCCGGAGCATTTTGATACCAG and MRCK reverse 5\u00b4TCCAATTCTGCTAGACCTTTGTGATA.Total mRNA was extracted using TRIzol reagent (Life Technologies). After DNase (Promega) treatment, cDNA synthesis was carried out using M-MLV Reverse Transcriptase (Life Technologies). For 3\u00b4RACE, oligo(dT25)T7 primer was used in cDNA synthesis, and the first round of PCR was performed using the CCND1 primer 5\u00b4 TGGTGAACAAGCTCAAGTGG and oligo(dT25)T7. The second round of PCR was performed using a nested CCND1 primer 5\u00b4TGGCATTTTGGAGAGGAAGTG and a T7 primer. All PCR was performed using pfu polymerase. The resulting PCR product was cloned using Zero Blunt TOPO PCR Cloning Kit for Sequencing (Life Technologies) and sequenced . The qRT-PCR protocol used, as well as the amplicons used to measure 7SK and CCND1, were previously described . The priThree different CCND1 3\u00b4UTR constructs were cloned downstream of the Renilla luciferase gene of psicheck2 (Promega) between Xho1 and Not1. To clone the gene fusion, the CCND1-MRCK sequence: CTCGAGGGGCGCCAGGACGGCGGGCGCCACCGCCACCCGCAGCGAGGGCGGAGCCGGCCCCAGGTGCTCCCCTGACAGTCCCTCCTCTCCGGAGCATTTTGATACCAGAAGGGAAAGCTTCATTCTCCTTGTTGTTGGTTGTTTTTTCCTTTGCTCTTTCCCCTAAAAAAGCATAATAGGTTAGCTGACCAATGATTAAAACATTATAGATCCGGAACGAACTAGAGAAGGCATGCAAAATTCAGATGAGAAAGTTTCTAAGTGATAGCTCCAGCTACCCCTCCAAATATCACAAAGGTCTAGCAGAATTGGAATTAAAAAAAAAAAGTCCTCTTGCACGGTTTATTTAAAATAAAGCCTTAACCTTAGGTGGTGCACCAAGTTGAACCTGACAGTGGAACTGTGTGGGTTTCAAGATCGAGTGATCAGAAAGGAACGGTAAACAAGCTGGGTGCAGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCGAGGCAGGTGGATCACCCGAGGTCAGGAGTACAAGACCAGCCTGGCCAACACTGTGGCGGCCGC was synthesized by GenScript and ligated into pUC57-Kan. After Xho1 and Not1 Restriction enzyme digestion, the sequence was directly cloned into the psicheck2 plasmid. The Jeko-1 specific truncated 3\u00b4UTR was cloned from Jeko-1 cDNA using the following primers: forward 5\u00b4GGCCCTCGAGGGGCGCCAGGCAGGCGGGCGC and reverse 5\u00b4GGCCGCGGCCGCTGCCTAGAACCCCACTACAGCTGTGC. Full length CCND1 3\u00b4UTR was cloned from the CCND1-pLightSwitch 3\u00b4UTR plasmid (S813994) from SwitchGear Genomics. The primers used were: forward 5\u00b4 GGCCGTCGACGGGCGCCAGGCAGGCGGGCGC and reverse 5\u00b4 GGCCGCGGCCGC CGTCTTTTTGTCTTCTGCTGGA.Either control siRNA , CCND1-sCells were lysed using RIPA buffer and 20ug of each protein sample was resolved on a 12% SDS-PAGE gel. After transfer to a PVDF membrane, blocking was done for 1hr in 5% non-fat milk resuspended in PBS (+0.001 % Tween 20). The membranes were probed with the monoclonal cyclin D1 antibody and polyclonal alpha tubulin antibody (Abcam). After probing with HRP-conjugated secondary antibody, proteins were detected by luminol.We used predicted CCND1-miRNA interactions using starBase software, which consolidates TargetScan, PicTar, PITA, Miranda and RNA22 miRNA prediction softwares, and overlaps the data with CLIP-Sequencing (CLIP-Seq.) data. We used a high stringency cutoff, where only miRNAs supported by >/=3 CLIP-Seq. experiments were selected, to reduce false positives ."} +{"text": "We have reconstituted a eukaryotic leading/lagging strand replisome comprising 31 distinct polypeptides. This study identifies a process unprecedented in bacterial replisomes. While bacteria and phage simply recruit polymerases to the fork, we find that suppression mechanisms are used to position the distinct eukaryotic polymerases on their respective strands. Hence, Pol \u03b5 is active with CMG on the leading strand, but it is unable to function on the lagging strand, even when Pol \u03b4 is not present. Conversely, Pol \u03b4-PCNA is the only enzyme capable of extending Okazaki fragments in the presence of Pols \u03b5 and \u03b1. We have shown earlier that Pol \u03b4-PCNA is suppressed on the leading strand with CMG . We propDOI:http://dx.doi.org/10.7554/eLife.04988.001 Cells must replicate their DNA before they divide so that the newly formed cells can each receive a copy of the same genetic material. DNA replication requires complex molecular machinery called a replisome, which comprises multiple proteins, enzymes, and other molecules. First, an enzyme called a helicase starts to unwind the double-stranded DNA into two single strands. This process continues while other enzymes, called polymerases, use the exposed single strands as templates to make complementary new strands of DNA. One of these new strands is built continuously and called the \u2018leading strand\u2019. The other newly forming strand\u2014the \u2018lagging strand\u2019\u2014is made in the opposite direction, as a series of short fragments that are later joined together.The replisomes in bacterial cells have been well studied, but many researchers are investigating the composition of the replisome in animals, plants, and fungi . Now, Georgescu et al. have essentially rebuilt a eukaryotic replisome from 31 different proteins in a test tube and confirmed that it can make both leading and lagging DNA strands\u2014just like in a normal cell. Further experiments revealed that the polymerase enzyme that operates on the leading strand cannot work on the lagging strand and vice versa. This exclusivity is unique to eukaryotic DNA replication, as bacterial polymerases can use either DNA strand as a template.Georgescu et al. then found that the eukaryotic polymerases are actively prevented from copying the \u2018wrong\u2019 strand of DNA and suggest that the helicase enzyme that unwinds the DNA might be behind this activity. Important future studies must now address how the replisome deals with obstacles created by certain DNA-binding proteins and damaged DNA and how it interfaces with the molecules that control cell division and DNA repair.DOI:http://dx.doi.org/10.7554/eLife.04988.002 Composition of the eukaryotic replisome and the function of its various proteins is an area of active investigation. Cellular studies reveal that eukaryotes use two different DNA polymerases for the leading and lagging strands, Pols \u03b5 and \u03b4, respectively . PrimingWhile in vitro synthesis of the leading strand replisome has been accomplished with the purified CMG complex from budding yeast , the disStudy of the eukaryotic replisome identifies a new process that has no precedent in bacterial systems. Bacteria use simple recruitment processes to attract and hold polymerases to the fork. These are typically mediated by polymerase interactions with other proteins at the replication fork, such as the helicase and sliding clamps . However32P-dCTP (leading) or 32P-dGTP (lagging).We expressed and purified yeast CMG helicase in our previous studies and demonstrated its function in leading strand synthesis with Pol \u03b5 . The disEscherichia coli. A mixture of cells containing the 4 subunits were lysed, and Pol \u03b1 was purified as an intact 4-subunit holoenzyme . The analysis also confirms that Pol \u03b1 cannot perform priming and extension in the absence of CMG (lanes 1\u20133) and that \u03d529 Pol cannot perform lagging strand synthesis (lanes 13\u201315).To determine if Okazaki fragments are distributed over the length of the DNA template, we used tive gel . The anaPol \u03b5 is the leading strand enzyme and presumably takes over the leading strand from Pol \u03b1 after this distributive enzyme dissociates from DNA. To determine if the Pol \u03b1/\u03b5 switch occurs as expected, we preloaded CMG on the forked DNA, then added increasing amounts of Pol \u03b1 either with or without Pol \u03b5, and stopped the reactions after 20 min. If Pol \u03b5 takes over the leading strand from Pol \u03b1, full-length products will be observed sooner in reactions that contain Pol \u03b5 because in the presence of CMG, this enzyme synthesizes DNA faster than Pol \u03b1. The results show full-length product in all the lanes containing Pol \u03b5 with Pol \u03b1 .In order to compare total leading and lagging strand synthesis rates, we setup standard replication reactions by loading CMG onto the nucleotide-biased forked DNA, followed by the addition of all three DNA polymerases in the presence of RFC and PCNA; the reactions were divided, and replication was initiated by the addition of either 32P-dCTP (leading) or 32P-dGTP (lagging) in the three polymerase replisome system shows similar amounts of leading and lagging strand synthesis . Indeed, polymerase recruitment by binding clamps and the helicase underlies attraction of polymerases to replication forks of bacteria, its phages, and the SV40 virus. Recruitment is also partly responsible for polymerase placement in eukaryotes. For example, Pol \u03b5 binds directly to CMG helicase, stabilizing it on the leading strand . HoweverWe were surprised to find that Pol \u03b1 polymerase activity is highly functional with CMG on both leading and lagging strands in the absence of other polymerases. Pol \u03b1 lacks the high fidelity of Pols \u03b5 and \u03b4 and does not provide bulk leading or lagging strand synthesis in cells, and thus processes must exist that suppress the polymerase activity of Pol \u03b1. In fact, we find many ways that Pol \u03b1 polymerase is suppressed. One mechanism is by Pol \u03b5 positioning on the lagging strand. Interestingly, Pol \u03b5 is also suppressed on the lagging strand; perhaps, Pol \u03b5 is suppressed from function on the lagging strand by the relative orientation in which CMG holds Pol \u03b5. On the leading strand, Pol \u03b5 simply prevents Pol \u03b1 polymerase extension by switching with it and becoming processive with CMG. This can be likened to the switch of Pol \u03b4 with Pol \u03b1 that prevents leading strand synthesis by Pol \u03b1 in the SV40 system . Pol \u03b1 pThe exclusion processes that underlie eukaryotic fork leading/lagging strand function are summarized in E. coli Pol III replicase complex, directing the single Pol \u03b5 molecule to the leading strand. Suppression of Pol \u03b1 by Pol \u03b5 on the lagging strand could perhaps be explained by competition of these polymerases for CMG, where Pol \u03b5 is suppressed from extending the primer. In this connection, the Pol2 gene encoding the catalytic polymerase actually contains two polymerase structures: the active polymerase/exonuclease on the N-terminal half and B-family polymerase in the C-terminal half of Pol2 that is presumed to be inactive . One posNumerous proteins travel with the replication fork, and it is not possible to know a priori how many are needed for functional leading and lagging strand replication. This is the first study to reconstitute leading/lagging strand replication with three pure polymerases in a eukaryotic system. It reveals that many of the proteins that move with forks are not required to recapitulate leading/lagging strand synthesis in vitro. For example, Ctf4/AND-1 is essential in most cells (not in budding yeast), and yeast Ctf4 helps recruit Pol \u03b1 bind to RPCs , yet theThe current report also reveals that Pol \u03b1 can prime the leading strand directly. To our knowledge, before this report, all proposed models of leading strand initiation in bacteria and SV40 show priming on the lagging strand of a bidirectional origin, which becomes the leading strand of one fork, rather than directly priming the leading strands . We showCellular studies indicate that nucleosomes are involved in determining Okazaki fragment size , but datReconstitution of cellular replisomes in vitro should provide a framework to explore the effects of other proteins that move with forks and of post-translational modifications that control eukaryotic forks in response to the cell cycle and DNA checkpoint mechanisms. Reconstituted systems should also enable detailed study of factors that maintain the epigenetic state of a cell during replication.2, 5 mM imidazole, 20 mM KOAc, and 350 mM KCl. Buffer D is 25 mM Tris-OAc pH 7.6, 40 mM K-OAc, 40 mM K glutamate, 2 mM Mg-OAc2, 1 mM DTT, 20% glycerol, and 0.25 mM EDTA. Stop buffer is 1% SDS, 40 mm EDTA. Buffer H is 20 mM Hepes pH 7.5, 10% glycerol, 1 mM EDTA, 2 mM DTT, 350 mM KCl, 1 mM ATP, and 4 mM MgCl2.Radioactive nucleotides were from Perkin Elmer. Unlabeled nucleotides were from GE Healthcare. DNA modification enzymes and \u03d529 DNA polymerase were from New England Biolabs. DNA oligonucleotides were from Integrated DNA Technologies. Protein concentrations were determined using the Bio-Rad Bradford Protein stain and bovine serum albumin as a standard. Buffer A is 20 mM Tris-HCl, pH 7.5, 5 mM DTT, 0.1 mM EDTA, and 4% glycerol. Buffer B is the same as buffer A except 20 mM Tris-acetate, pH 7.5 was used in place of 20 nM Tris-HCl. Buffer C is 25 mM Tris-Cl pH 7.9, 10% glycerol, 1 mM DTT, 1 mM MgClE. coli SSB , a strain constructed from W303 . Pol12 was transformed into E. coli BL21(DE3)codon plus RIL , then induced with IPTG for 8 hr at 15\u00b0C. Pri1 and Pri2 were co-expressed in E. coli BL21(DE3) cells by IPTG induction for 8 hr at 15\u00b0C. A 12 l culture of induced yeast cells for Pol1 and 1 l of each induced E. coli cultures for Pol12 and Pri1 and Pri2 were co-crushed in a cryogenic mill as described for CMG (g for 1 hr at 4\u00b0C. Anti-Flag agarose (1.2 ml) was added to the supernatant (80 ml) and incubated with slow rotation for 1.5 hr at 12\u00b0C. Beads were collected by centrifugation at 1500\u00d7g, washed twice with 5 ml 50 mM Hepes pH 7.4, 250 mM potassium glutamate, 1 mM EDTA, then loaded into a gravity column. The column was washed with 15 ml 50 mM Hepes pH 7.4, 250 mM potassium glutamate, 1 mM EDTA, 10% glycerol, and Pol \u03b1 was eluted with 5 ml 50 mM Hepes pH 7.4, 250 mM potassium glutamate, 1 mM EDTA, 10% glycerol containing 20 \u03bcg/ml Flag peptide. The eluent was diluted to a conductivity equal to 150 mM NaCl using 25 mM Hepes pH 7.4, 1 mM EDTA, and 10% glycerol, then applied to a 1 ml Heparin agarose column. Pol \u03b1 was eluted with a gradient of 100 mM to 1 M NaCl in 25 mM Hepes pH 7.4, 1 mM EDTA, 10% glycerol. Peak fractions were pooled, aliquoted, and stored at \u221280\u00b0C. Typical yield of Pol \u03b1 was about 3 mg.Proteins were purified as described: RPA , E. colicoli SSB , PCNA . The Pol for CMG . Frozen To make the linear fork DNA substrate, a 3.2 kb sequence of DNA was designed such that one strand lacked dC residues and thus the other strand lacked dG. The G-rich strand was examined to eliminate runs of four G residues. The resulting 3260 bp sequence was synthesized by Biomatik . The first four and last two bases represent overhangs generated by BsaI and BtsC1, respectively. Both of these enzymes cut outside of their recognition sequences, and their recognition sequences are excluded from the template.CGGTATTCTAACCACATTAATCTACACCTCTCACACACTACTCATATCATCTTCCAAAACCCACCTTTAAAAAACCCTTTATCCACACTCATCACCATTTCACCAACCTTTTTCTTAATTCTACACAAATCCAATTAACCTATCTCCAATTTTAACTCCATCACCTCTTATATTAACCCACCTACTACTCCAACAATACCCTATCAAATCTACTTCTATCTCAAAACTATCACCTACTCCTTCCATCATAATCCACTCTTATCAATTAAACAATTATCCTTCTTTCCCACCATCACTCACCATCTTTTCTTAACACCCTTAACATTTCCTTTTATAAAACACTTCCAATCCTATTTTCTCACTATCCCACCCACCATAAAAACTATCTCACCCTAACTCAACCCTTTCCCTCTCACCAACACTCCTTTATCACACACACTTTACCACACAAATCCCTCCATCATACACCTTTACCCTCAAATCCTAAACCACCTAACTATTCCACACAATATATCTACAAAAATTTACTTTTCCACATCTCCAACCCTTCCAACACCTTAATCCCAAACCTTAACTAACCTCTCTTTAATACTTCCTCCCATTCCCACCACATACACCAAATTATCTTCAACTCAAAAACCTAAACTCTCCTTTTTATTCCCTATAAAAACTCTTAACCCTCCAATATACAACTCTAACTAACTTCATTATCAACCAATCTTCCTCTACTTCCTCATCTTATAATTTATCCATTCAAAATAACCTACCTACCACCTCTCCTCTTCACTTCCTACCCTAAAATCACCACCTTATCCCTAATTTACCTCTTTCAACTTTCCTTAACCCAACTTCTCAATCCTACTTCACTTACTTCTTATAAAACCATCATTATCACACTACACATTACTCTATCTATCCAATCATCACCTTCTACAATCCAAACTATCCCACTACCCTCTCATTCTACCTTTTCATCTATCTCAAACTATCCACCAATCCATACCTCAAACTTTAACCACCCACTCCAAATCTACAACATAAAATTAACCTATCACATTCCAAACTAATCACCCTAACCCTAACACCCTTTTATCCTCACCAAAATTACCATTTTCCTCTTTACTCATAAACAAACATTCTCACCCATTTATAAAACACTTAATACCCACTTAATTCACTTCCTTTTTCTACCTCACCATCATCAACTCCTAATATCAACAACCCAAAATCACCACCTATATCCTCATCTCCTATATAAAAAACTTCACATCTCAACCTCAAACCACCTATTCCACTTAATCCCAATCAACCTATCAACTCTACAACCTACTCTTCAAATACATCTCCTATCACTTTCCCACCTCCTTCAATCAATTATAACTTTATCACCTAAACATTCTAAAATCTCCTATCCACTACATCACATAAACCTAAACCTACTACCAACCTACCATTATCCTTATCAAACATCATCAATTCCATCTTTCTTTATACCCTCTCCATATCTCTCTCATTAAAAAAACCAAAATCTAACAACTTCTTATTTCTAATCAAAAAAACAATCAACCTAACTCATAAAATCTTCACCTTAAAATTCCTTTACATTTAACAAATCCACTCTCCCTATCTTTTTCATATCAAACTTATCTAAACCACTATCCTCATTTATCCTAATACTCCATATACAACACCAAATTTCTTATATCTCATCTAAAATCCTCCACCAATATAAACTCCTCTTTACCATTTCCACTCAACACACCAAATCTTATTATTCCATCAAATCTAATCCATTACCATCATCAACCCTAATAAACCTACTTCCCAACTTTTATCTCTCCACTACCACACCAAAAATTAACCTCCTCTAAAAACTATCATTCCCTTTACCTCTTCCACATTCCACCTATAACTCCTCATCTTAAAACCAATCAACACCAAACAACTATCTCACCATATTTCCTCTCCAAACCAACAAATTAACAATCCTACCCACTCCAACCTCCACATTACTAATAACTAAACTTACCTTACCTACCACACCCTATCAACCATATTTAAAAAATTACTTATTCACTAACTAAAACATCACCCACAAACTTAAATCATCACCTCCTCTTTTCCACCTTATTCACCAACCCAATCTATCTATCTCACCTATACCTTTCCCTAATATCTTTTACTAACCCTATAAATACCACAATTCTAAAAAACCCATACTTATCTCACACATCACTTTATACTTCACTCTTAAAATACCCTCCAATATATATTACAACCCAAAAATATCTCCCTTCTATCTCCTACACACAAATTATACCACTTTTAAACCACTCCTCATCTCTAACCCAACCCTCTACAATTCCATACATCTCTACTATCAACATCACTCCTTCTTTTCCACTCTTCTCTCCACATCTTTATTAAACATCTCCTCCTCATTTTCACATAACTATTTACTAAATAAATTTACCTAAACTACATTTATTAAAACCCTACAACATACTCCTTATTCTCCTACCTACCATTCTCTAATCTCTTTACATTCTACTACTTTCCTACCTACTATCTCAATAAATTCACTTTCCTTTCACCACACACCAACACACCTTCCTCCAAATTCTTTATATCTCCTTCTCCTAACCAAATTCCTCACTAATAACATCTTACCTCCCTACCTTTTTCCTTCTACCCTCCACCATTTCCCAACCTCATACTCAATAATCAATTTACCCTCCCACAACATAACTCTATTAACACCCATTTTCTATCCATCAACTTCCTATTACTTAAATTATCTTTTAAACCAATAAAACTCCACCTCAACCACCCACCTCTCTCTTTCAATCCAATTTCAATCTTTCCAACCATTCTATCTACCCTAAACTATTAACTATCTATCTCACCCACAATCCCTCCTACAATTCACAACAACATTCCACCACTTCACTTTATCTTCACCTCCAAACTTATTCCTTCCCATTATCACCTTCTCCAAAACCCACAAACATCTAACTCTCATCTCTCAACACTTTCTACCCATTCTCTCTAACAAAATTCCCTTACTCTTTATTCACAAAACCACTAAAATCACCACAACAACCCAACAAAAACAAAATCCTCACTTACCTATACTCAATAAATCCTTCAACTCATTATTCTATTTCTAACCCTAAATCAAAACTCCCATATCTACCATTCTTTCCACTCAATTAAATCCCACCAACCCTTATTTTCCTCCAATAACTTAACC.The synthetic 3.2 kb DNA was cut from the plasmid using BsaI-HF and BtsCI and purified from low-melting agarose gel. 35 pmol of the 3.2 kb linear DNA template was ligated to a fivefold excess of forked junction on the BsaI end and to a 10-fold molar excess of a short blocking duplex on the BtsCI end. To make the forked junction, 1050 pmol 1T oligonucleotide was annealed to 175 pmol 160mer oligonucleotide, as described . The 16032P-dCTP, while for lagging strand replications, we used 20 \u03bcM dGTP and 10 \u03bcCi 32P-dGTP. Exceptions to this protocol are noted in the figure legends. Timed aliquots were removed and quenched upon adding SDS and EDTA to final concentrations of 0.5% and 20 mM, respectively. Quenched reactions were analyzed in 0.7% or 2% alkaline agarose gels and imaged in a Typhoon 9400 PhosphorImager .Replication assays contained 1.5 nM linear forked DNA, 24 nM CMG, 400 nM RPA (unless noted otherwise), 20 nM PCNA (unless noted otherwise), 6 nM RFC (unless noted otherwise), and Pol \u03b1, Pol \u03b5, and Pol \u03b4 as indicated in the figure legends, in 25 mM Tris-acetate pH 7.5, 10 mM Mg-acetate, 50 mM potassium glutamate, 5 mM DTT, 0.1 mM EDTA, 40 \u03bcg/ml BSA, 0.1 mM AMP\u2013PNP, 5 mM ATP, 200 \u03bcM each rCTP, rUTP, rGTP, 60 \u03bcM of each unlabeled dNTP, and 20 \u03bcM of the labeled dNTP. Reactions were staged as follows. CMG was added first and pre-incubated with the DNA and 0.1 mM AMP-PNP for 10 min at 30\u00b0C, then the noted polymerases together with the RFC and PCNA (where indicated) were added, along with dATP, dCTP for an additional 2 min. Replication was then initiated upon the addition of a solution containing the RPA, ATP, dTTP, and dGTP. It is important to note that for leading strand replication reactions, we used 20 \u03bc\u039c dCTP and 10 \u03bcCi 32P-dCTP (leading) or 32P-dGTP (lagging) was added. Timed reactions were stopped with an equal volume of 2\u00d7 STOP solution (40 mM EDTA and 1% SDS) and spotted on DE81 filter papers, then analyzed using a Perkin Elmer Liquid Scintillation Analyzer . Separately, we performed control replication reactions using \u03d5X174 ssDNA coated with RPA, containing a known amount of Gs and Cs, confirm that 32P-dCTP and 32P-dGTP are equally incorporated by each of the DNA polymerases in our experimental conditions.In order to compare total DNA synthesis rates on leading and lagging strand, we performed standard replication reactions containing all three DNA polymerases at 10 nM, CMG (30 nM), RFC (10 nM), PCNA (20 nM), and RPA (400 nM) in presence of 1 mM ATP, rNTPs , and 30 \u03bcM dNTPs; reactions were divided, and either 32P-dGTP to label the lagging strand; the second reaction utilized 1.5 nM unprimed forked DNA and 10 nM each Pol \u03b5 and Pol \u03b1 along with 20 \u03bcM dGTP and 10 \u03bcCi 32P-dGTP to label the lagging strand, and the third reaction contained DNA-primed forked DNA and 1 U of \u03d529 DNA polymerase along with 20 \u03bcM dCTP and 10 \u03bcCi 32P-dCTP to label the leading strand. Each reaction was quenched upon heating to 65\u00b0C for 10 min to inactivate the CMG and polymerases. Then, reactions were divided into three tubes, one was untreated, the second was treated with EarI, and the third was treated with PsiI, adjusting the reaction buffer for each enzyme with the commercial provided buffer. Reactions were analyzed in a 2% native agarose gel followed by autoradiography in a Typhoon 9400 PhosphorImager (GE/Molecular Dynamics).Three replication reactions were performed as described above with the following differences. The first reaction used 1.5 nM unprimed forked DNA and 10 nM Pol \u03b1 with 20 \u03bcM dGTP as well as 10 \u03bcCi 32P) dTTP and incubated at 30\u00b0C. At the times indicated, 25-\u03bcl aliquots were removed and quenched by addition of an equal volume of 1% SDS/40 mm EDTA. Products were analyzed in 0.7% alkaline agarose gels. Gels were dried, exposed to PhosphorImager screens, and imaged using a Typhoon 9400 PhosphorImager (GE/Molecular Dynamics).Reactions contained 1.5 nM \u03d5X174 circular ssDNA (as circles) primed with a DNA 30-mer and pre-incubated for 10 min with 420 nM RPA (as heterotrimer) in 20 mM Tris-Cl (pH 7.5), 50 mM potassium glutamate, 5 mM DTT, 0.1 mM EDTA, 40 \u03bcg/ml BSA, 8 mM MgOAc, 0.5 mm ATP, 5% glycerol, and 60 \u03bcM, each of dGTP and dATP. Reactions also contained the indicated amounts of RFC, PCNA, and Pol \u03b1 and were pre-incubated for 5 min at 30\u00b0C. DNA synthesis was initiated by adding 15 \u03bcl of 60 \u03bcM dCTP, 20 \u03bcM dTTP, 15 \u03bcCi of . Proteins were brought to a final volume of 200 \u03bcl in 100 mM sodium phosphate, 150 mM NaCl pH 8.0, and incubated with 50 \u03bcl (as a 10% slurry) Strep-Tactin magnetic beads for 1 hr at 4\u00b0C with end-over-end mixing. Beads were collected in a magnetic separator and washed twice in 200 \u03bcl 100 mM sodium phosphate, 150 mM NaCl pH 8.0, then eluted in 75 \u03bcl 10 mM biotin in 100 mM sodium phosphate, 150 mM NaCl pH 8.0 for 20 min on ice. Samples were analyzed in 8% SDS-PAGE followed by staining with Coomassie Blue Denville stain."} +{"text": "AbstractSymmerista H\u00fcbner is reviewed for Costa Rica, based on 49 wild-caught specimens. Four species are newly described: Symmerista luisdiegogomezi Chac\u00f3n, Symmerista inbioi Chac\u00f3n, Symmerista minaei Chac\u00f3n and Symmerista aura Chac\u00f3n. All are from the cloud forests of the Talamanca moutain range, southern Costa Rica. Photographs of the adults, male and female genitalia, and barcodes are also provided. The species Symmerista tlotzin Schaus (1892) is removed from Symmerista and assigned to the genus Elymiotis Walker as a new combination.The genus Notodontidae includes about 3500 described species worldwide , from the cloud forests at altitudes between 1000 and 2600 meters.H\u00fcbner (1821) established the genus species . SpeciesSymmerista tlotzin male genitalia morphology and barcodes , Santo Domingo de Heredia, Costa Rica were examined, sexed and identified. Of those, 12 were DNA barcoded and dissected. All holotypes and paratypes are deposited in the collections at INBio. Fifty-six specimens of reared and wild-caught Elymiotis tlotzin, previously known as Symmerista tlotzin, were also examined.Forty-nine specimens of Instituto Nacional de Biodiversidad, Santo Domingo de Heredia, Costa RicaINBio National Museum of Natural History, Smithsonian Institution, Washington DC, USAUSNM Adterminal lineAD Corpus bursaeCB Ductus bursaeDB ForewingFW HindwingHW Medial lineM Postmedial linePM Sternum 8ST8 Tergum 8T8 Wing lengthWLPageBreakTaxon classificationAnimaliaLepidopteraNotodontidaeH\u00fcbnerSymmerista H\u00fcbner, 1821. Verz. bekannt. Schmett. : 248.Noctua albicosta H\u00fcbner, 1809, Samml. eur. Schmett. 4: pl. 93, fig. 440. By subsequent designation by, Kirby, 1892, Syn. Cat. Lepid. Het. Het. 1: 572.Noctua albicosta occurs in Canada and the US.Type(s), [North America]: Mistakenly included by H\u00fcbner as a European species; Symmerista was originally proposed in the Noctuidae.Nye (1975) stated: Noctua albicosta is a form of Phalaena albifrons Smith, 1797, in Smith & Abbot, Nat. Hist. rarer lepid. Insects Georgia 2: 159, pl. 80.Watson et al. (1980) stated: Adults \u2013 Medium-sized notodontid moth, FW = 16\u201322 mm, females larger than males; male antenna pectinate nearly to apex, terminal 10\u201312 annulations simple or antenna bipectinate, six terminal flagellomeres without rami; antennae of female simple; labial palpus porrect; haustellum reduced to two small lobes, completely hidden by labial palpi; ocelli absent; eye smooth, round; thorax generally dark brown with beige tegula; all scales of thorax long and forked; scales of patagium and prothorax beige and cream colored. FW with the costa straight, apex marked, the outer margin evenly rounded; accessory cell present, narrow; R2-5, stalked, R5 arising beyond R2, M1 from apex of accessory cell or nearly so. Male genitalia \u2013 ST8 with a wide, deep emargination; valva membranous, finely pubescent, costulae absent; tegumen narrowed dorsally; uncus concave, the socii subquadrate and lobed or long, wide, pubescent at bases, narrow and flattened at apices; vinculum slightly sclerotized; manica membranous, fused to juxta; ventral process of phallus long and forked, its tip well beyond the tip of the medial, dorsal process; vesica bulbous with a scobinate patch.Female genitalia \u2013 Papillae anales membranous, covered with short, scattered setae; posterior apophyses long and slender; DB sclerotized for approximately two-thirds of its length; CB rounded; anterior vaginal plate asymmetrical, slightly swollen and inflated in middle.Symmerista is characterized by: haustellum reduced to two small lobes, completely hidden by labial palpi; valva membranous; costulae absent; uncus concave; manica membranous, fused to juxta and ductus bursae sclerotized for approximately two-thirds of its length. Symmerista differs of the genera Elasmia and Elymiotis by the following characteristics: Elasmia has a well-developed haustellum, sacculus pleated and saccular scent organ present, manica sclerotized, deciduous cornuti, costulae present, costal margin sclerotized, ST8 with long and curvy lateral sclerotized projections like forceps, midplate with tiny pleats and membranous; uncus elongate, setose, with two dorsal protuberances and two tiny spines at the apex. Elymiotis has the haustellum well developed, sacculus pleated, deciduous cornuti, costal margin sclerotized, St8 posterior margin irregularly sclerotized and convex with tiny lateral proyections, uncus thin and long with apex acute and setose. All of these characterisctics are absent in Symmerista.The genus PageBreakTaxon classificationAnimaliaLepidopteraNotodontidaeChac\u00f3nsp. n.http://zoobank.org/81A8E740-7368-45A7-9BBE-6113BBC43D3E16 specimens Holotype male: INB0003536238 , Costa Rica, Prov. Cartago, P.N. Tapanti, Macizo de La Muerte, Est. La Esperanza 9.69129-83.87683, 2600 m, September 2002, R. Delgado (INBio).Paratypes: 13 males, 2 females. 2 males: INB0003756237, INB0003756364 Costa Rica, Prov. Limon, Parque Internacional La Amistad, Valle del Silencio, Alrededor del Refugio y el Sendero Circular, 9.110281-82.961934, 2450 m, 22\u201327 September 2003, D. Rubi, R. Gonzalez, R. Delgado (INBio). 4 males: INB0003316532, INB0003316533, INB0003316534, INB0003316535 Costa Rica, Prov. Cartago, El Guarco, Macizo de la Muerte, Sector La Esperanza, 9.686771-83.87775, 2600 m, June 2001, R. Delgado (INBio). Male: INB0003352709 Costa Rica, Prov. Cartago, Reserva Forestal Rio Macho. El Guarco, Macizo de la Muerte, Sector La Esperanza, 9.686771-83.87775, 2600 m, August 2001, R. Delgado (INBio). 2 males: INB0003387641 (dissected), INB0003387642, Costa Rica, Prov. Cartago, Reserva Forestal Rio Macho, El Guarco, Macizo de la Muerte, 9.686771-83.87775, 2600 m, October 2001, R. Delgado (INBio). 2 males: INB0003536234 (COI barcoded), INB0003536235 (COI Barcoded), INB0003536237 (COI barcoded), Costa Rica, Prov. Cartago, Parque Nacional Tapanti, Macizo de La Muerte, Est. La Esperanza, 9.69129-83.876832, 2600 m, September 2002, R. Delgado (INBio). 2 males: INB0003545221 , INB0003545222 (COI barcoded) Costa Rica, Prov. Cartago, Parque Nacional Tapanti, Macizo de La Muerte, Est. La Esperanza, 9.69129-83.876832, 2600 m, October 2002, R. Delgado (INBio). Female: INB0003339209 Costa Rica, Prov. Cartago, El Guarco, Macizo de la Muerte, Est. La Esperanza. 9.686771-83.87775, 2600 m. July 2001. R. Delgado (INBio). Female: INB0003756274 Costa Rica, Prov. Limon, Parque Internacional La Amistad, Valle del Silencio, Alrededor Refugio y Sendero Circular, 9.110281-82.961934, 2450 m, 22\u201327 September 2003, D. Rubi, R. Gonzalez, R. Delgado (INBio).This species is named in honor of the late Professor Luis Diego G\u00f3mez Pignataro of San Jose, Costa Rica, for his outstanding contribution to our knowledge of Costa Rican biodiversity, his support of the Museo Nacional de Costa Rica, and for inspiring me to become a naturalist and taxonomist.Male genitalia: St8 posterior margin concave with a pair of short postero-lateral projections, projections robust and heavily sclerotized with blunt apices; valva membranous, finely pubescent, sacculus smooth, its margin uniform, costa straight with a distal protuberance near apex; valva with two triangular spine-like processes, one at base, the other at juxta near anellus; uncus slightly concave, somewhat helmet shaped, dorsal surface PageBreakrough, pubescent, ventral surface smooth with sparse pubescence; socii long, wide, pubescent at bases, narrow and flattened at apices, shape as in the figure; length of the phallus 3.1 mm, proximal part of phallus tube wide at the base, narrow in the middle, distal part of phallus tube robust, sclerotized, with a tubular lateral projection and rounded apex; proximal part of the vesica with a ventral scobinate patch, distal part of the vesica bulbous. Female genitalia: posterior apophyses longer and more slender than anterior apophyses; CB rounded and membranous, lacking a signum; DB wide and sclerotized; posterior margin of postvaginal plate sclerotized and emarginate.Dorsal FW ground color dark brown with costal margin black; an irregular, long thin cream-colored mark from the reniform spot to the apex; fringe dark brown with beige scales where veins touch termen; dorsal HW dark brown. Male , southern Costa Rica .Holotype male: INB0003487050 , Costa Rica, Prov. Cartago, El Guarco, Reserva Forestal Rio Macho, Macizo de la Muerte, Sector La Esperanza 9.686771-83.87775, 2600 m, May 2002, R. Delgado (INBio).Paratypes: Male: INB0003153991 Costa Rica, Prov. Cartago, El Guarco, San Isidro, Est. La Esperanza 9.687685-83.884582, 2450 m, March 2001, R. Delgado (INBio). 2 males: INB0003316531, INB0003316537 Costa Rica, Prov. Cartago, El Guarco, Macizo de la Muerte, Sector La Esperanza, 9.686771-83.87775, 2600 m, June 2001, R. Delgado (INBio). Male: INB0003320716 Costa Rica, Prov. Cartago, Parque Nacional Tapanti, El Guarco, San Isidro, Est. La Esperanza, 9.683922-83.876688, 2600 m, May 2001, R. Delgado (INBio). Male: INB0003334468 Costa Rica, Prov. Cartago, Parque Nacional Tapanti, Macizo de la Muerte, Est. La Esperanza, 9.686771-83.87775, 2600-2700 m, April 2001, R. Delgado (INBio). 3 males: INB0003339195, INB0003339197, INB0003339198 Costa Rica, Prov. Cartago, El Guarco, Parque Nacional Tapanti, Macizo de la Muerte, Est. La Esperanza 9.686771-83.87775, 2600 m, July 2001, R. Delgado (INBio). Male: INB0003387640 Costa Rica, Prov. Cartago, Reserva Forestal Rio Macho, El Guarco, Macizo de la Muerte, 9.686771-83.87775, 2600 m, October 2001, R. Delgado (INBio). Male: INB0003478225 , Costa Rica, Prov. Cartago, Parque Nacional Tapanti, Macizo de La Muerte, Est. La Esperanza, 9.69397-83.854504, 2700 m, 13\u201314 May 2002, J. Montero (INBio). 2 males: INB0003536236 , INB0003536239 (COI Barcoded) Costa Rica, Prov. Cartago, Parque Nacional Tapanti, Macizo de La Muerte, Est. La Esperanza, 9.69129-83.876832, 2600 m, September 2002, R. Delgado (INBio). Male: INB0003545219 Costa Rica, Prov. Cartago, Parque Nacional Tapanti, Macizo de La Muerte, Est. La Esperanza, 9.69129-83.876832, 2600 m, October 2002, R. Delgado (INBio). 3 males: INB0003756229, INB0003756321, INB0003756418 Costa Rica, Prov. Limon, Parque Internacional La Amistad, Valle del Silencio, Alrededor del Refugio y Sendero Circular, 9.110281-82.961934, 2450 m, 22\u201327 September 2003, D. Rubi, R. Gonzalez, R. Delgado(INBio). Male: INBIOCRI001359567 Costa Rica, Prov. Cartago, Quebrada Segunda, Parque Nacional Tapanti, 9.762583-83.788328, 1250 m, February 1993, G. Mora. Male: INBIOCRI002210601 Costa Rica, Prov. Cartago, La Represa, Tapanti, 9.695643-83.768399, 1800 m, July 1995, R. Delgado (INBio). Male: INBIOCRI002253344 Costa Rica, Prov. Cartago, Rio Grande de Orosi, desde Puente Rio Dos Amigos hasta la Represa, 9.695643-83.768399, 1400-1800 m, March 1995, R. Delgado (INBio). Male: INBIOCRI002423774 Costa Rica, Prov. Cartago, Rio Grande de Orosi, desde Puente Rio Dos Amigos hasta la Represa, 9.695643-83.768399, 1800 m, February 1995, R. Delgado (INBio). Male, INBIOCRI002427627 Costa Rica, Prov. Cartago, Rio Grande de Orosi, desde Puente Rio Dos Amigos hasta la Represa, 9.695643-83.768399, 1400-1800 m, 22 August\u201315 September 1995, R. Delgado (INBio).PageBreakThis species is dedicated to the Instituto Nacional de Biodiversidad (INBio) in recognition of its 25 years of support for developing an understanding of the biodiversity of Costa Rica and exporting that understanding to the nation and the world.Symmerista inbioi differs from Symmerista luisdiegogomezi on: dorsal FW ground color light brown; from the reniform spot to the apex there is an uniform long, thin, white cream-colored band; fringe reddish brown; dorsal HW dirty beige. Male genitalia: St8 wide at base, anterior margin convex, posterior margin slightly irregular with a pair of very short projections, these sclerotized with blunt apices; valva membranous, saccular margin slightly jagged, costal margin smooth, with a distal protuberance near the apex; elongate finger-shaped process on the saccular margin at the internal base of the valve; uncus plate slightly concave, with papillae and setae on the dorsal and ventral edge; socii elongated, broad at base, narrow and flattened at the apex, with papillae and setae in the dorsal surface; length of the phallus 3.9 mm, proximal part of phallus curved at the base, slightly narrow in the middle, distal part of phallus tube sclerotized, narrow at the base, robust, irregular and wide at the end, with a tubular lateral projection with the distal nipple; proximal part of the vesica with a dorsal scobinate patch, distal part of the vesica bulbous. The genital armature of Symmerista inbioi is more robust and larger than Symmerista luisdiegogomezi.Male (n Range) .DNA barcode of holotype male INB0003487050Symmerista inbioi | COI-5P:MHMXP003-08 | INB0003487050 | AACATTATATTTTATTTTTGGGATTTGAGCAGGTATAGTAGGAACTTCTTTAAGTCTATTAATTCGAGCTGAATTAGGAAACCCCGGATCACTTATTGGGGATGATCAAATTTATAATACAATTGTTACAGCCCATGCCTTTATTATAATTTTTTTTATGGTAATACCTATTATAATTGGGGGATTTGGTAATTGATTAGTCCCTCTTATACTAGGAGCCCCAGATATAGCATTCCCCCGCATAAATAATATAAGTTTTTGACTTTTGCCCCCTTCTTTAACCCTTTTAATTTCAAGAAGAATCGTAGAAAATGGAGCAGGAACTGGATGGACAGTGTACCCCCCACTATCCGCCAACATTGCCCATAGTGGAAGTTCTGTAGATTTAGCTATTTTTTCCCTTCATTTAGCTGGAATTTCCTCAATTTTAGGAGCTATTAATTTTATTACAACAATTATTAATATACGCCTCAATAATATATCTTTTGATCAAATACCTTTATTTGTTTGAGCTGTTGGAATTACAGCATTTTTACTTTTACTTTCTTTACCTGTTTTAGCGGGAGCTATTACAATACTACTAACTGACCGTAATTTAAATACATCCTTTTTTGACCCTGCTGGGGGAGGAGATCCAATTTTATACCAACATTTATTTTaxon classificationAnimaliaLepidopteraNotodontidaeChac\u00f3nsp. n.http://zoobank.org/71A2F18E-A8EC-4BF6-9991-ACEC39D207994 specimens Holotype female: INB0003339208 , Costa Rica, Prov. Cartago, El Guarco, Macizo de la Muerte, Estacion La Esperanza, 9.68677-83.87775, 2600 m, July 2001, R. Delgado (INBio). Paratypes: Female: INB0003155283 (COI barcoded), Costa Rica, Prov. Limon, Bratsi, Valle del Silencio, 9.107197-82.961749, 2472 m, 11\u201312 October 2000, R. Delgado (INBio). Female: INB0003352700 (COI barcoded), Costa Rica, Prov. Cartago, Reserva Forestal Rio Macho, El Guarco, Macizo de la Muerte, Sector La Esperanza, 9.686771-83.87775, 2600 m, August 2001, R. Delgado (INBio).Other material examined: 1 Male, INB00033387642 (dissected) Costa Rica, Prov. Cartago, Reserva Forestal Rio Macho, El Guarco, Macizo de la Muerte, 9.68677-83.87775, 2600 m, October 2001. R. Delgado (INBio).This species is dedicated to the Ministerio del Ambiente y Energ\u00eda (MINAE) of the government of Costa Rica in recognition of its 28 years of continuous and widespread support for the survival and conservation of the wild biodiversity of Costa Rica.PageBreakSymmerista minaei differs from Symmerista luisdiegogomezi on: dorsal FW ground color beige and light brown, square mark creamy near the reniform spot; beige mark in the apex; fringe beige yellow; dorsal HW beige. Male genitalia: T8 anterior margin slightly concave, posterior margin finely serrated with a window in the center; St8 lateral margins wide at the base, narrow to posterior margin, anterior margin concave, slightly sclerotized with a short projection in the center, posterior margin with robust projections, highly sclerotized on each side, with blunt apices, a little dome in the middle of the posterior margin; length of the phallus 3.3 mm, proximal part of phallus tube wide at base, distal part of phallus tube robust, sclerotized, with a tubular lateral projection rounded apex with the distal nipple; proximal part of the vesica with a ventral scobinate patch, distal part of the vesica bulbous. Female genitalia: Anterior and posterior apophyses the same size, long an slender; DB sclerotized; CB rounded, membranous and pleated; posterior margin of postvaginal plate sclerotized, slightly irregular, inverted V-shape.Male (n Range) .DNA barcode paratype female INB0003155283.Symmerista minaei | COI-5P:MHMXP006-08 | INB0003155283 | PageBreakTATTATAATTTTTTTTATAGTAATACCTATTATAATTGGGGGATTTGGTAATTGATTAGTCCCCCTTATGCTAGGAGCCCCAGATATAGCATTCCCACGTATAAATAATATAAGTTTTTGACTTTTACCCCCCTCCTTAACCCTTTTAATTTCAAGAAGAATCGTCGAAAATGGGGCAGGAACCGGATGGACAGTGTACCCCCCACTATCCTCCAATATTGCCCACAGTGGAAGTTCTGTAGATTTAGCTATTTTTTCCCTACATTTAGCTGGAATTTCATCAATTTTAGGGGCCATTAATTTTATCACAACAATTATTAATATACGTCTCAATAACATATCTTTTGATCAAATACCCTTATTTGTTTGAGCTGTTGGAATTACAGCATTTTTACTTTTACTTTCTTTACCTGTTCTAGGGAGCTATTACAATACTACTAACGGATCGTAATTTAAATACATCTTTTTTTGATCCTGCAGGAGGAGGAGATCCAATTTTATATCAACATTTATTTAACATTATATTTCATTTTTGGAATTTGAGCAGGTATAGTTGGAACTTCATAAGCCTATTAATTCGAGCTGAATTAGGAAATCCCGGATCCCTTATTGGAGATGATCAAATTTATAACACAATTGTTACAGCCCATGCCTTTaxon classificationAnimaliaLepidopteraNotodontidaeChac\u00f3nsp. n.http://zoobank.org/31DCFCFA-B792-406E-A667-CAAA17B5111F4 specimens Holotype male: INB0003116415 , Costa Rica, Prov. Cartago, Paraiso, P.N. Tapanti, Macizo de La Muerte, Estacion Quebrada Segunda, 9.762583-83.788328, 1300 m, November 2000, R. Delgado (INBio).Paratypes: 1 males, 2 females. Male: INBIOCRI002442681 (COI barcoded), Costa Rica, Prov. Puntarenas, Buenos Aires, Potrero Grande, Estacion Altamira, 1 Km. S del Cerro Biolley, 9.032987-83.010887, 1450 m, 13\u201326 May 1996, R. Villalobos (INBio). Female: INBIOCRI002549508 (COI barcoded), Costa Rica, Prov. Puntarenas, Coto Brus, Sabalito, Send. El ripario a 3 Km NE. de Progreso, 8.917676-82.78469, 1300 m, 6\u20139 April 1997, A. Picado (INBio). Female: INBIOCRI002754030 Costa Rica, Prov. Cartago, Turrialba, Tayutic, Moravia de Chirripo Shipiri, 9.837781-83.453639, 1000 m, 10 May 1983, D. H. Janzen & W. Hallwachs (INBio).This species is dedicated to Isidro Chac\u00f3n\u2019s daughter, Aura Chac\u00f3n, for 25 years of understanding an absent father obsessed with his work.Male genitalia: St8 anterior margin with a sclerotized triangular projection in the middle, posterior margin sclerotized, irregular, with a rectangular projection at the center bearing two highly sclerotic and sharply serrated structures; proximal part of phallus tube wide and short at the base, with a blade-like lateral projection, sharp at the distal apex; a small rounded projection and curve off the previous, distal part of phallus tube robust, sclerotized, with a small bulge on the ventral side; proximal part of the vesica with a ventral scobinate patch, distal part of the vesica bulbous. Female genitalia: papillae anales ovoid-triangular, setose; posterior apophyses longer and more slender than anterior apophyses; DB sclerotized; CB elongated, membranous and pleated, lacking a signum; margin distal of antevaginalis plate very sclerotized and lightly depressed at the center, concave to the sides, proximal margin convex.Dorsal FW ground color light gray; a white cream mark from the discal cell to the apex. PageBreakSymmerista aura differs from female of Symmerista meridionalis Thiaucourt, 2007 in the shape of the antivaginalis plate; the CB elongated, membranous and pleated, lacking a signum; margin distal of antevaginalis plate very sclerotized and lightly depressed at the center, concave to the sides, proximal margin convex. This is the description of the female genitalia of Symmerista meridionalis published by Thiaucourt in 2007 which mentiones the differences, \u201cFemale terminalia: distal edge of lamella postvaginalis slightly incurved; lamella antevaginalis rectangular, almost square; its distal margin strongly sclerotized; mouth of the ostium bursae oval, densely sclerotized under the margin; ductus bursae beyond the constriction under the ostium, forming a short funnel; bursa membranous inserted on the dorsal surface of the extremity of the duct; signum distinct.\u201d The male of Symmerista meridionalis is unknown.The female of Male .DNA barcode paratype male INB0003116415.Symmerista aura | COI-5P:MHMXP017-08 | INB0003116415 | ACATTATATTTTATTTTTGGGGTTTGAGCTGGGATAGTTGGAACTTCCCTAAGTTTACTAATTCGAGCTGAATTGGGTAACCCTGGATCTTTAATTGGAGATGATCAAATTTATAATACAATTGTAACAGCCCATGCTTTTATTATAATTTTTTTTATAGTTATACCCACCATAATCGGGGGATTTGGTAATTGACTAGTTCCTCTTATATTAGGGGCACCGGATATAGCATTTCCACGTATAAATAACATAAGTTTTTGACTTCTACCCCCTTCTTTAACCCTTTTAATTTCAAGAAGAATTGTCGAAAATGGAGCTGGAACAGGATGAACAGTGTACCCCCCATTGTCATCTAATATTGCTCATGGTGGTAGTTCCGTAGATTTAGCTATTTTTTCACTTCATTTAGTGGAATTTCTTCAATTTTAGGGGCTATTAATTTTATTACAACAATCATTAATATACGTCTTAATAATATATCTTTTGACCAAATACCTTTATTTGTGTGAGCTGTAGGGATTACAGCATTTTTACTTTTACTTTCTTTACCTGTATTAGCTGGAGCTATTACAATATTATTAACTGATCGTAATCTAAACACATCTTTTTTTGATCCCGCTGGAGGAGGAGATCCTATTTTATACPageBreakTaxon classificationAnimaliaLepidopteraNotodontidaecomb. n.11 males 12 females.PageBreakINB0003319696 Costa Rica, Prov. Guanacaste, Nicoya, P.N. Barra Honda, Sector Barra Honda, 10.169826-85.379137, 50 m, 25\u201330 December 2000, H. Mendez (INBio). Female: INBIOCRI001184551 Costa Rica, Prov. Guanacaste, Bagaces, P. N. Palo Verde, Estacion Palo Verde, 10.349119-85.352345, 10 m, 20 June 1993, U. Chavarria (INBio). Female: INB0003300310 Costa Rica, Prov. Guanacaste, Hojancha, Z.P. Nosara, Hojancha, R.F. Monte Alto, 10.011248-85.402778, 400 a 500 m, 27 July \u2013 3 August 2000, H. Mendez (INBio). Female: INBIOCRI000674401 Costa Rica, Prov. Guanacaste, Liberia, P.N.Sta. Rosa, Playa Naranjo, 10.802713 \u2013 85.67479, 0\u201310 m, March 1991, E. Alcazar (INBio). Female: INB0003956448 Costa Rica, Prov. Guanacaste, Nicoya, San Antonio, Humedal Mata Redonda, 10.328094-85.42111, 8 m, 6 July 2005, B. Gamboa, J. Azofeifa, J. Gutierrez, M. Moraga, Y. Cardenas (INBio).2 Males: INBIOCRI000584965 Costa Rica, Prov. Guanacaste, Bagaces, Ref. Nac. Fauna Silv. R. L. Rodriguez, Estacion Palo Verde, 10.349119-85.352345, 10 m, May 1991, U. Chavarria (INBio). Male: INB0003072431 Costa Rica, Prov. Guanacaste, Bagaces, Pque. Nal. Palo Verde, Sector Palo Verde, 10.366668-85.383266, 0\u201350 m, 3 May 2000, H. Mendez (INBio). Male: INBIOCRI000386810 Costa Rica, Prov. Guanacaste, Liberia, P. N. Sta. Rosa, Playa Naranjo, 10.80275-85.666572, 0\u201310 m, May 1991, E. Alcazar (INBio). Male: INBIOCRI002426620 Costa Rica, Prov. Guanacaste, Liberia, Sector Las Pailas, 4.5 Km. SW del Volcan Rincon de la Vieja, 10.776784-85.351913, 800 m, 24 June 1995, K. Taylor (INBio). Male: INB0004065577 Costa Rica, Prov. Guanacaste, Liberia, Santa Rosa Nat. Pk., 10.83641-85.615491, 300 m, 4\u20136 December 1979, D. H. Janzen (INBio). Male: Male: 94-SRNP-2964 Costa Rica, Area Conservacion Guanacaste, Prov. Guanacaste, Sector Santa Rosa, Estero Naranjo, 10.80426-85.68285, 2 m, 9 June 1994, Gusaneros. Male: 98-SRNP-9137 (COI barcoded), Costa Rica, Area Conservacion Guanacaste, Prov. Guanacaste, Sector Santa Rosa, Area Administrativa, 10.83764-85.61871, 295 m, 14 August 1998, Manuel Pereira. Male: 01-SRNP-17295 (COI barcoded), Costa Rica, Area Conservacion Guanacaste, Prov. Guanacaste, Sector Santa Rosa, Sendero Carbonal, 10.77594-85.65799, 7 m, 2 November 2001, Gusaneros. Male: 06-SRNP-13290 (COI barcoded), Costa Rica, Area Conservacion Guanacaste, Prov. Guanacaste, Sector Santa Rosa, Estero Naranjo, 10.80426-85.68285, 2 m, 31 May 2006, Eilyn Camacho.PageBreakGuanacaste, Prov. Guanacaste, Sector Santa Rosa, Area Administrativa (adult at light), 10.83764-85.61871, 295 m, 1 June 2011, Daniel H. JanzenFemale: 92-SRNP-736 Costa Rica, Area Conservacion Guanacaste, Prov. Guanacaste, Sector Santa Rosa, Vado Nisperal, 10.80212-85.65372, 10 m, 20 May 1992, Gusaneros. Female: 96-SRNP-1331 (COI barcoded), Costa Rica, Area Conservacion Guanacaste, Prov. Guanacaste, Sector Santa Rosa, Sendero Palo Seco, 10.79342-85.6666, 5 m, 31 May 1996, Gusaneros. Female: 96-SRNP-1332 Costa Rica, Area Conservacion Guanacaste, Prov. Guanacaste, Sector Santa Rosa, Sendero Palo Seco, 10.79342-85.6666, 5 m, 2 June 1996, Gusaneros. Female: 98-SRNP-9134 (COI barcoded), Costa Rica, Area Conservacion Guanacaste, Prov. Guanacaste, Sector Santa Rosa, Area Administrativa (adult at light), 10.83764-85.61871, 295 m, 2 August 1998, Guillermo Pereira. Female: 98-SRNP-9137 Costa Rica, Area Conservacion Guanacaste, Prov. Guanacaste, Sector Santa Rosa, Area Administrativa (adult at light), 10.83764-85.61871, 295 m, 14 August 1998, Guillermo Pereira. Female: 01-SRNP-17336 (COI barcoded), Costa Rica, Area Conservacion Guanacaste, Prov. Guanacaste, Sector Santa Rosa, Sendero Carbonal, 10.77594-85.65799, 7 m, 28 October 2001, Gusaneros. Female: 01-SRNP-17336 Costa Rica, Area Conservacion Guanacaste, Prov. Guanacaste, Sector Santa Rosa, Sendero Carbonal, 10.77594-85.65799, 7 m, 28 October 2001, Gusaneros. Female: 07-SRNP-112736 (COI barcoded), Costa Rica, Area Conservacion Guanacaste, Prov. Guanacaste, Sector Santa Rosa, Sendero los Patos (adult at light), 10.82097-85.63323, 251 m, 8 December 2007, H. Cambronero & S. Rios. Female: 11-SRNP-12732 (COI Barcoded), Costa Rica, Area Conservacion PageBreakAdults \u2013 in this genus, the species should be now in another genus.\u201d KIRBY (1892) lists Edema as a junior synonym of Symmerista Hubner, [1821].Nystaleini, but it differs from that of Symmerista .Eulophidae:Euplectrus (n=1).Elymiotis tlotzin have been collected in the dry forest ecosystem of Peninsula de Nicoya, and in the dry forests of Sector Santa Rosa and Sector Pailas of ACG, at elevations of 0 to 800 m. (Adult o 800 m. .DNA barcode female 11-SRNP-12732.Elymiotis tlotzin | COI-5P:MHMYM2073-11 | 11-SRNP-12732 | PageBreakAACATTATATTTTATTTTTGGAATTTGAGCAGGAATAGTAGGAACTTCTTTAAGTTTATTAATTCGAGCTGAATTAGGAAATCCAGGATCTTTAATTGGTGATGATCAAATTTATAATACTATTGTAACAGCTCATGCTTTTATTATAATTTTTTTTATAGTAATGCCTATTATAATTGGAGGATTTGGAAATTGACTAGTTCCATTAATATTAGGAGCCCCAGATATAGCTTTCCCCCGAATAAATAATATAAGATTTTGACTACTTCCACCCTCACTAACTTTATTGATTTCAAGAAGTATTGTAGAAAATGGAGCAGGAACTGGATGAACAGTTTATCCCCCCCTTTCATCTAA0TATTGCACATAGAGGAAGATCTGTAGATTTAGCAATTTTTTCACTTCATTTAGCTGGTATTTCATCGATTTTAGGAGCTATTAATTTTATTACAACGATTATTAATATACGACTTAATAACATAACTTTTGATCAAATACCTTTATTTGTTTGAGCAGTAGGAATTACAGCTTTTTTATTATTATTATCTTTACCTGTTTTAGCCGGAGCGATTACTATATTATTAACAGACCGTAATTTAAATACTTCATTTTTCGACCCTGCTGGTGGAGGAGATCCAATTCTTTATCAACATTTATTT"} +{"text": "Similar results were obtained using cell size (forward/side scatter) to fractionate MCF7 cells. Larger stem-like cells also showed increased hTERT-GFP levels, as well as increased mitochondrial mass and function. Thus, this simple and rapid approach for the enrichment of immortal anabolic stem-like cancer cells will allow us and others to develop new prognostic biomarkers and novel anti-cancer therapies, by specifically and selectively targeting this metabolic sub-population of aggressive cancer cells. Based on our proteomics and functional analysis, FDA-approved inhibitors of protein synthesis and/or mitochondrial biogenesis, may represent novel treatment options for targeting these anabolic stem-like cancer cells.Tumor cell metabolic heterogeneity is thought to contribute to tumor recurrence, distant metastasis and chemo-resistance in cancer patients, driving poor clinical outcome. To better understand tumor metabolic heterogeneity, here we used the MCF7 breast cancer line as a model system to metabolically fractionate a cancer cell population. First, MCF7 cells were stably transfected with an hTERT-promoter construct driving GFP expression, as a surrogate marker of telomerase transcriptional activity. To enrich for immortal stem-like cancer cells, MCF7 cells expressing the highest levels of GFP (top 5%) were then isolated by FACS analysis. Notably, hTERT-GFP(+) MCF7 cells were significantly more efficient at forming mammospheres and showed increased mitochondrial mass and mitochondrial functional activity, all relative to hTERT-GFP(\u2212) cells. Unbiased proteomics analysis of hTERT-GFP(+) MCF7 cells directly demonstrated the over-expression of 33 key mitochondrial proteins, 17 glycolytic enzymes, 34 ribosome-related proteins and 17 EMT markers, consistent with an anabolic cancer stem-like phenotype. Interestingly, MT-CO2 expression was increased by >20-fold. As MT-CO2 is encoded by mt-DNA, this finding is indicative of increased mitochondrial biogenesis in hTERT-GFP(+) MCF7 cells. Importantly, most of these candidate biomarkers were transcriptionally over-expressed in human breast cancer epithelial cells Telomerase plays a central role both in the biology of normal aging, as well as in the development of human cancers , 2. HoweRecently, Clarke and colleagues have taken advantage of the properties of human telomerase (hTERT), to enrich for a population of osteosarcoma cells with stem-like properties . For thiHere, we have adapted this approach to the study of breast cancer stem-like cells, with a focus on proteomics analysis, biomarker discovery and cell metabolism. Importantly, we demonstrate that hTERT-high-activity breast cancer cells form mammospheres with higher efficiency, and show a proteomics profile consistent with an anabolic cancer stem cell phenotype. In support of this notion, we also show that hTERT-high-activity breast cancer cells have increased mitochondrial mass and activity, consistent with an increase in mitochondrial biogenesis.To enrich for a population of immortal CSCs, we exploited a sensitive eGFP reporter system for the fluorescent detection of high telomerase transcriptional activity. Briefly, MCF7 cells were transduced with a lentiviral vector driving eGFP protein expression, under the control of a 1.5-kB fragment of the hTERT promoter. This DNA construct also contains a puromycin-resistance cassette for antibiotic-resistance selection. Schematic diagrams illustrating this overall experimental strategy and the construction of the hTERT-promoter-vector are shown in Figures After selection with puromycin, MCF7-hTERT-eGFP cells were subjected to FACS analysis to visualize the broad distribution of eGFP expression, which serves as a surrogate marker of telomerase activity. Importantly, fewer than 1 in a 100 cells in MCF7-hTERT-eGFP cell monolayers visually showed high GFP fluorescence. In striking contrast, there was a dramatic enrichment of GFP(+) cells in MCF7 cell mammospheres (> 50 \u03bcm), each containing usually 1-to-2 GFP-high cells and GFP-low (negative) groups. Then, five thousands cells from each group were seeded per well in 6-well low-attachment plates. These two groups were compared to the total unfractionated cell population.Remarkably, Figure In order to dissect the mechanism(s) by which high telomerase activity drives the survival and clonal expansion of CSCs, we next employed an unbiased label-free proteomics approach. The proteome of fractionated MCF7-hTERT-eGFP cells was directly determined, after FACS separation into GFP-high and GFP-low populations. For simplicity, we focused on the proteins that were over-expressed in GFP-high cells by > 1.5-fold. Our results are summarized in Tables Importantly, >30 mitochondrial-related proteins were over-expressed in GFP-high cells Tables . Most ofCSCs undergo an EMT, which facilitates cell migration, invasion and metastatic dissemination . Thus, wElevated protein synthesis is another important feature of the anabolic CSC phenotype . As predTaken together, hTERT-eGFP-high cells over-express >100 proteins related to an anabolic CSC phenotype.in vivo. For this purpose, we exploited a clinical data set of tumor samples from N = 28 breast cancer patients. These tumor samples were subjected to laser-capture micro-dissection, to separate epithelial cancer cells from adjacent tumor stroma [in vivo. Tables To determine the clinical relevance of our findings, we next assessed whether the hTERT proteomic targets that we identified in GFP-high cells were transcriptionally over-expressed, in human breast cancer cells r stroma . OverallTo directly validate the mitochondrial phenotype of hTERT-eGFP-high cells top 5%), we used two well-established fluorescent probes to quantitate mitochondrial membrane potential and mass, by FACS analysis. More specifically, we used MitoTracker Orange (561-nm) as a reporter for mitochondrial membrane potential and MitoTracker Deep-Red 640-nm) as a marker of mitochondrial mass. Importantly, Figures 0-nm as a%, we usein vivo [Previous studies using mouse mammary epithelial cells have demonstrated that stem-like cells can be enriched solely based on cell size . For exain vivo .larger cells and ii) smaller cells ) Figure . Interes) Figure .As such, larger cell size in MCF7 cells directly correlates with telomerase activity and mitochondrial mass/activity, which would be consistent with an anabolic CSC phenotype. These results provide independent validation for the idea that high hTERT activity (\u201cstemness\u201d) is functionally associated with increased mitochondrial mass and activity in breast cancer cells, and co-segregates with large cell size. Importantly, large cell size is determined by increased PI3K/AKT/mTOR-signaling, which drives significant increases in overall protein synthesis \u201314. ThisHere, we have used an hTERT-promoter-eGFP-reporter system to identify and purify a sub-population of MCF7 cells, with high hTERT transcriptional activity, by FACS analysis. These hTERT-eGFP-high cells formed mammospheres with greater efficiency, as predicted, consistent with the idea that this sub-population of cells is enriched in cancer stem-like cells. Importantly, proteomics analysis of these hTERT-eGFP-high MCF7 cells revealed the upregulation of mitochondrial proteins, glycolytic enzymes and EMT markers, as well as components of the protein synthesis machinery, such as ribosome-related proteins and chaperones for protein folding. Interestingly, MT-CO2 expression was increased by >20-fold. As MT-CO2 is encoded by mt-DNA, this finding is indicative of increased mitochondrial biogenesis in hTERT-eGFP-high MCF7 cells. We then functionally validated that hTERT-eGFP-high MCF7 cells show increases in mitochondrial mass and activity, using two distinct MitoTracker probes. Complementary results were obtained using cell size to fractionate MCF7 cells. Larger stem-like cells showed increased hTERT-GFP levels, as well as increased mitochondrial mass and function. Thus, these two independent approaches for the enrichment of immortal anabolic CSCs should allow the development of new prognostic biomarkers and related novel anti-cancer therapies.Interestingly, recent studies in aging and cancer have both directly linked telomerase activity to mitochondrial function, via the hTERT-p53-PGC1 signaling axis \u201318. Moreprotein synthesis machinery and ii) are associated with large cell size , which share several properties with CSCs or TICs \u201326. For We recently directly compared the proteome of MCF7 cell monolayers to MCF7-derived mammospheres, using label-free unbiased proteomics analysis. Consistent with our current findings, this analysis demonstrated that MCF7-derived mammospheres show the over-expression of >60 mitochondrial-related proteins and >80 components of the protein synthesis machinery , 29. ComImportantly, functional validation studies revealed that mammosphere formation could be efficiently blocked with well-established inhibitors of mitochondrial function and/or inhibitors of protein synthesis, such as oligomycin A and puromycin, respectively , 29. Morin vivo. Thus, the new hTERT metabolic targets that we identified here may be important for improving human breast cancer diagnosis and therapy.In summary, we show here that high telomerase activity metabolically defines a sub-population of anabolic CSCs, with increased mitochondrial mass and large cell size. Overall, greater than seventy hTERT targets that we identified in hTERT-eGFP-high cells were also transcriptionally elevated in human breast cancer cells MCF7 cells were purchased from the ATCC. Gibco-brand cell culture media (DMEM and DMEM/F12) was purchased from Life Technologies. The lentiviral vector encoding the hTERT promoter linked to eGFP was custom-made by Genecopoeia (USA). MitoTracker probes (Deep Red and Orange) were purchased from Molecular Probes/Invitrogen, via Life Technologies. The telomerase inhibitor IX was obtained commercially from Santa Cruz Biotech (USA).R cassette, and cloned into a lentiviral vector, custom-made by Genecopoeia: 5\u2032-TAAAATTGTGTTTTCTATGTTGGCTTCTCTGCAGAGAACCAGTGTAAGCTACAACTTAACTTTTGTTGGAACAAATTTTCCAAACCGCCCCTTTGCCCTAGTGCAGAGACAATTCACAAACACAGCCCTTTAAAAAGGCTTAGGGATCACTAAGGGGATTTCTAGAAGAGCGACCTGTAATCCTAAGTATTTACAAGACGAGGCTAACCTCCAGGAGCGTGACAGCCCAGGGAGGGTGCGAGGCCTGTTCAAATGCTAGCTCCATAAATAAAGCAATTTCCTCC GGCAGTTTCTGAAAGTAGGAAAGGTTACATTTAAGGTTGCGTTTGTTAGCATTTCAGTGTTTGCCGACCTCAGCTACAGCATCCCTGCAAGGCCTCGGGAGACCCAGAAGTTTCTCGCCCCTTAGATCCAAACTTGAGC AACCCGGAGTCTGGATTCCTGGGAAGTCCTAGCTGTCCTGCGGTTGTGCCGGGGCCCCAGGTCTGGAGGGGACCAGTGGCCGTGTGGCTTCTACTGCTGGGCTGGAAGTCGGGCCTCCTAGCTCTGCAGTCCGAGGCTTGGAGCCAGGTGCCTGGACCCCGAGGTTGCCCTCCACCCTGTGCGGGCGGGATGTGACCAGATGTTGGCCTCATCTGCCAGACAGAGTGCCG GGGCCCAGGGTCAAGGCCGTTGTGGCTGGTGTGAGGCGCCCGGTGCGCGGCCAGCAGGAGCGCCTGGCTCCATTTCCCACCCTTTCTCGACGGGACCGCCCCGGTGGGTGATTAACAGATTTGGGGTGG TTTGCTCATGGTGGGGACCCCTCGCCGCCTGAGAACCTGCAAAGAGAAATGACGGGCCTGTGTCAAGGAGCCCAAGTCGCGGGGAAGTGTTGCAGGGAGGCACTCCGGGAGGTCCCGCGTGCCCGTCC AGGGAGCAATGCGTCCTCGGGTTCGTCCCCAGCCGCGTCTACGCGCCTCCGTCCTCCCCTTCACGTCCGGCATTCGTGGTGCCCGGAGCCCGACGCCCCGCGTCCGGACCTGGAGGCAGCCCTGGGTCTCCGGATCAGGCCAGCGGCCAAAGGGTCGCCGCACGCACCTGTTCCCAGGGCCTCCACATCATGGCCCCTCCCTCGGGTTACCCCACAGCCTAGGCCGATTCGACCTCTCTCCGCTGGGGCCCTCGCTGGCGTCCCTGCACCCTGGGAGCGCGAGCGGCGCGCGGGCGGGGAAGCGCGGCCCAGACCCCCGGGTCCGCCCGGAGCAGCTGCG CTGTCGGGGCCAGGCCGGGCTCCCAGTGGATTCGCGGGCACAGACGCCCAGGACCGCGCTTCCCACGTGGCGGAGGGACTGGGGACCCGGGCACCCGTCCTGCCCCTTCACCTTCCAGCTCCGCCTCCTCCGCGCGGACCCCGCCCCGTCCCGACCCCTCCCGGGTCCCCGGCCCAGCCCCCTCCGGGCCCTCCCAGCCCCTCCCCTTCCTTTCCGCGGCCCCGCCCTCTCCTCGCGGCGCGAGTT-3\u2032.The following 1.5 kB sequence was used as the hTERT-promoter to generate the hTERT-eGFP-PuroR cassette were prepared and used to stably transduce MCF7 cells, according to the manufacturer's protocol (in the presence of 5 \u03bcg/ml polybrene). Twenty-four hours post-infection, media containing the virus was removed and replaced with standard media. Cells were then selected with puromycin (2 \u03bcg/ml), for up to 10 days. Please note that for most of the experiments described in this paper, we compared the GFP-high (top 5%) fraction to the GFP-low/negative (bottom 5%) fraction, unless stated otherwise. However, for label-free proteomics analysis, we compared the GFP-high (top 10%) population to the GFP-low (bottom 10%) population, to insure that enough material was collected for sample processing. For these experiments, singlet FACS gating of live cells was utilized.Lentiviral particles harboring the hTERT-eGFP-Puro2 in mammosphere medium (DMEM-F12/B27/20-ng/ml EGF/PenStrep) in non-adherent conditions, in culture dishes coated with (2-hydroxyethylmethacrylate) . Cells were grown for 5 days and maintained in a humidified incubator at 37\u00b0C at an atmospheric pressure in 5% (v/v) carbon dioxide/air. After 5 days for culture, spheres >50 \u03bcm were counted using an eye piece graticule, and the percentage of cells plated which formed spheres was calculated and is referred to as percentage mammosphere formation, and was normalized to one (1 = 100% MSE). Mammosphere assays were performed in triplicate and repeated three times independently.A single cell suspension of MCF7-hTERT-eGFP cells was prepared using enzymatic , and manual disaggregation (25 gauge needle) . Cells wCells were treated on day 0 with MST-312 at a concentration of 10 \u03bcM and compared to vehicle alone controls, processed in parallel, after 5 days of mammosphere culture. A dose-response analysis of the effects of MST-312 on parental MCF7 cells was first performed to establish an effective concentration for the inhibition of mammosphere formation; our results showed that 1 \u03bcM had little or no effect, while 10 \u03bcM inhibited mammosphere formation by approximately 70% (data not shown). Therefore, experiments with eGFP fractionated MCF7 cells were carried out at 10 \u03bcM.Fluorescent imaging of MCF7 cells in adherent conditions and non-adherent spheroid culture was performed using a Leica SP8 multi-photon DM6000 microscope to detect GFP expression and bright field images.Cell lysates were prepared for trypsin digestion by sequential reduction of disulphide bonds with TCEP and alkylation with MMTS . Then, tN = 28 human breast cancer patients) [To firmly establish the clinical relevance of our results from the quantitative proteomics, we re-analyzed the transcriptional profiles of epithelial breast cancer cells and adjacent tumor stromal cells that were physically separated by laser-capture microdissection (from atients) .For live cell sorting experiments, hTERT-eGFP transfected MCF7 cells were resuspended in PBS and sorted according to eGFP expression using the BD influx. Untransfected MCF7 cells were used as a negative control to determine positive GFP expression. Cells were sorted into two groups, those with the highest GFP expression and those with the lowest GFP expression. For FACs analysis experiments, hTERT-eGFP cells were labeled with MitoTracker Deep Red and MitoTracker Orange . Cells were kept of ice until analysis using the FACS Calibur. Results were analyzed using FlowJo software version 10. Cells were gated into two populations, those with the highest 5% of GFP expression, and those with the lowest 5% and negative GFP expression. The median fluorescent intensity of MitoTracker Deep Red (640nm) and MitoTracker Orange (561nm) was then determined within the two distinct GFP cellular populations. Additionally, cells were discriminated by cell size into two populations, small and large using SSC (side scatter) and FSC (forward scatter). The median intensity of eGFP, MitoTracker Deep Red, and MitoTracker Orange, within these populations, was then determined.To measure mitochondrial activity, cells were stained with MitoTracker Orange , whose accumulation in mitochondria is dependent upon membrane potential. To measure mitochondrial mass, cells were stained with MitoTracker Deep Red , localizing to mitochondria regardless of mitochondrial membrane potential. Cells were incubated with pre-warmed MitoTracker staining solution for 30-60 min at 37 \u00b0C. All subsequent steps were performed in the dark. Cells were washed in PBS, harvested, and re-suspended in 300 \u03bcL of PBS. Cells were then analyzed by flow cytometry. Data analysis was performed using FlowJo software.hTERT-eGFP-MCF7 cells, co-labeled with MitoTracker dyes, were analyzed by cell size using FlowJo software. Cells were separated by gating of forward scatter (FSC) and side scatter (SSC) plots into two populations for analysis, large cells and small cells . The median fluorescent intensity of eGFP and MitoTracker dyes were determined in each population of cells. Very similar results were obtained, with either singlet FACS gating or all live cell FACS gating.Statistical significance was determined using the Student's t-test or ANOVA, where appropriate. Values of less than 0.05 were considered significant. Data are shown as the mean \u00b1 SEM, unless stated otherwise."} +{"text": "Drosophila Leucine-rich repeat-containing G protein-coupled receptor 3 (Lgr3), and find body asymmetries similar to those found in insulin-like peptide 8 (dilp8) mutants, which fail to coordinate growth with developmental timing. Indeed, mutation or RNA intereference (RNAi) against Lgr3 suppresses the delay in pupariation induced by imaginal disc growth perturbation or ectopic Dilp8 expression. By tagging endogenous Lgr3 and performing cell type-specific RNAi, we map this Lgr3 activity to a new subset of CNS neurons, four of which are a pair of bilateral pars intercerebralis Lgr3-positive (PIL) neurons that respond specifically to ectopic Dilp8 by increasing cAMP-dependent signalling. Our work sheds new light on the function and evolution of relaxin receptors and reveals a novel neuroendocrine circuit responsive to growth aberrations.How different organs in the body sense growth perturbations in distant tissues to coordinate their size during development is poorly understood. Here we mutate an invertebrate orphan relaxin receptor gene, the The orphan ligand Dilp8 has been shown to coordinate growth and developmental timing in Drosophila. Here, using Gal4 drivers and CRISPR/Cas9 approaches, Garelli et al. identify a role for relaxin-like receptor Lgr3 in regulating the Dilp8 developmental delay pathway. Drosophila, the insulin/relaxin-like peptide (Dilp8), which ensures organ and body size coordination3dilp8 uncouples the endocrine communication between imaginal discs and the prothoracic gland, making dilp8 mutants susceptible to uncoordinated disc growth when intrinsic errors of development or noxious environmental stimuli affect the growth status of one or more discs. This results in an increase in random deviations from bilateral symmetry (fluctuating asymmetry (FA)), measurable at the population levelDrosophila3456How different organs in the body sense growth perturbations in distant tissues to coordinate their size and differentiation status during development is poorly understood1bona fide relaxin peptide homologues89Drosophila genome encodes two orphan receptors, Lgr3 and Lgr4, with clear homologies to vertebrate relaxin receptors neurons.Type C1 Leucine-rich repeat-containing G protein-coupled receptors (Lgrs) are a conserved protein family in metazoans that act as receptors for insulin-like peptides of the relaxin subfamily in vertebrates, where they play diverse roles in tissue homeostasis and remodelling, behaviour and reproductionLgr3, we remobilized an MB Minos elementLgr3+/+), which served as genetic background controls, and one imprecise excision allele, Lgr3ag1, which consists of a 3.8-kb deletion that completely removes exon 8 and partially removes exon 9 (Lgr3ag1 produces a transcript with a premature termination codon (PTC) that truncates the Lgr3 protein one amino acid after D326 are increased by an order of \u223c3 when compared with their Lgr3+/+ controls under control of the wing-pouch Beadex-Gal4 (Bx>) driver3Lgr3+/+ animals ; To generate a mutant for s exon 9 . Lgr3ag1ter D326 . We concarea FAi ). This p animals . This deLgr3 should be necessary for the developmental delay produced by ectopic Dilp8 expression in the absence of tissue growth abnormalities3UASP-dilp8::3xFLAG (UAS-dilp8a) transgene under the control of armadillo-Gal4 (arm>) was suppressed in larvae homozygous for Lgr3ag1 or trans-heterozygous for Lgr3ag1 and a deficiency that completely uncovers the Lgr3 locus (Lgr3Df(3)BSC321) )tubulin-Gal4 (tub>), and reduced Lgr3 activity by concomitant RNA intereference (RNAi) knockdown using a short hairpin (TRiP VALIUM22 (Lgr3-IR-V22)) lineLgr3 mRNA levels by \u223c85% (Lgr3 completely suppressed the dilp8-dependent delay (Lgr3 (TRiP VALIUM10 (Lgr3-IR-V10))dilp8-dependent delay, albeit to a lesser extent than Lgr3-IR-V22 (Lgr3 mRNA levels (\u223c50%) than Lgr3-IR-V22 . Coherennt delay . A secon3-IR-V22 . This pa3-IR-V22 , suggestLgr3 at the protein level, we used CRISPR/Cas9-mediated homologous repair15Lgr3ag5, hereafter named sfGFP::Lgr3, which contained an intact Lgr3-coding sequence downstream of the sfGFP insertion indels in the Lgr3-coding region , which induces apoptosis, tissue damage/regeneration, an imaginal-disc-specific Dilp8 upregulation and consequentially a robust delay in the onset of metamorphosissfGFP::Lgr3 ag5 allele encodes a functional receptor. Together, these results strongly suggest that the Lgr3 protein acts in a subpopulation of CNS neurons.To gain insight into the tissue and cellular expression pattern of nsertion . sfGFP::nsertion . No otheof Dilp8 . The effg region or by usWe were unable to detect sfGFP::Lgr3 expression in neurons directly innervating the ring gland , suggestsfGFP::Lgr3 neurons in more detail. sfGFP::Lgr3 expression was not homogeneous in the \u223c180 CNS cell bodies. It was most strongly expressed in a single pair of neurons in the very tip of the VNC, in a single dorso-ventral pair of midline neurons located deep in the thoracic segment of the VNC ), and in a pair of bilateral neurons localized in the anterior part of the pars intercerebralis ) -Gal4 driving a UAS-myr::tdTomato reporter indicates that many Lgr3-positive neurons are cholinergic, including all three major neuronal populations -Gal4 and Vesicular glutamate transporter promoter (VGlut)-Gal4, which drive expression in GABAergic and glutamatergic neurons, respectively, label very faintly the PIL neurons and the pair of distal VNC neurons and show no detectable staining in MIL neurons ) . All thrL1 stage . Co-stai neurons ). Co-sta neurons . MIL neu neurons . PIL neu neurons . The lat neurons . PIL neu neurons . These rLgr3-IR-V22 RNAi line to the panneuronal driver elav-Gal4 (elav>) and performed EMS assays -Gal4 did not rescue the EMS-induced delay and GMR19B09-Gal4 (GMR19B09>) throughout embryonic and larval development was compatible with developmental progression and led to a significant suppression of the EMS-dependent delay , selecteMS-delay , the GMRhoc test . These rGMR19B09> driver labels \u223c270 neurons, \u223c30 of which populate each brain hemisphereGMR19B09> neurons with myr::tdTomato in a sfGFP::Lgr3 background, we detect overlapping expression in \u223c10 neurons per hemisphere, 2 of which are the bright PIL neurons line drives expression in similar type of neurons, named #5 neurons25. MZ699>myr::tdTomato expression analysis in a sfGFP::Lgr3 background demonstrates that PIL neurons are a subset of #5 neurons -(FRTmCherry) luciferase reporterCRE-luciferase and the sfGFP::Lgr3 reporters in heterologous studies in human cell lines ery cell . We concDrosophila SL2/DL2 cells. In this assay, Dilp8 is covalently attached to TriCEPS2727Drosophila insulin-like receptor (InR)Drosophila cells assays27la cells . An endoCTLH2) -like factor derailed (Drl), neuroglian (Nrg), the laminin receptor integrin \u03b1-PS3 (ITA3) and the choline transporter (CTL)-like protein 2 (CTLH2) . Any of CTLH2) . While tDrosophila. Our study opens many questions for further research, such as the determination of which of the eight bilateral Lgr3-positive interneuron populations , and relayed through one or more steps before reaching the Lgr3-positive cells , it is tempting to propose a direct ligand\u2013receptor interaction between them. This possibility is supported by the strong genetic interaction between in vivo, nevertheless it strongly indicates that Dilp8 can consistently interact with a likewise strong receptor candidate for an Ilp, such as the InR. The LRC technique we used can identify receptors of interest with affinities spanning 4 orders of magnitude at expression levels as low as 2,000 receptors per cell . However, it is not yet clear how quantitative it can be relative to affinity constants. Dilp8 has been previously shown to modulate growth in vivo often in opposite ways depending on the observed tissueThor (4E-BP) in the larval fat body, which is consistent with a local increase in insulin/IGF-like signallingThor levels are higher in imaginal discs in the same animalsSecond, we have failed to identify Lgr3 among candidate Dilp8-binding cell surface receptors/co-receptors. Clearly, the biochemical identification of alternative cell surface-binding proteins such as the InR, Nrg and the RYK-like Drl37Third, the fact that ectopic expression of Dilp8 only leads to a detectable increase in cAMP signalling in PIL neurons, and not in other Lgr3-positive neurons , indicatsfGFP::Lgr3 or GMR19B09>myr::tdTomato expression in the ring gland or in neurons innervating the ring gland , UAS-rpr , Lgr3-IR-V10 , Lgr3-IR-V22 , elav-Gal4 (P{w[+mW.hs]=GawB}elavC155), elav-Gal4 (P{w[+mC]=GAL4-elav.L}2/CyO), w1118;Mi{ET1}Lgr3MB06848, w1118; snaSco/SM6a, P{w[+mC]=hsILMiT}2.4, tub-Gal4 , w1118; Lgr3Df(3)BSC321/TM6C Sb1 cu1, Gad1-Gal4 (P{w[+mC]=Gad1-GAL4.3.098}2/CyO), Cha-Gal4 , GMR19B09-Gal4 , GMR17G11-Gal4 , y1 w* P{y[+t7.7] w[+mC]=10XUAS-IVS-mCD8::RFP}attP18 P{y[+t7.7] w[+mC]=13XLexAop2-mCD8::GFP}su(Hw)attP8, and w1118; P{y[+t7.7] w[+mC]=GMR19B09-lexA}attP40w were obtained from the Bloomington Drosophila Stock Center at Indiana University. The stock 1 M{w[+mC]=Act5C-Cas9.P}ZH-2A w*y (reference #16) was obtained from Bestgene. The stock arm-gal4 was a gift from P. Domingos. The stocks UAS-dilp8b::3XFLAG and UAS-dilp8c::3XFLAG (reference #3) were a gift from M. Dominguez. The stock w*; P{GawB}Mz699 and UAS-myr::tdTomato/CyO; TM2/TM6B were a gift from M.L. Vasconcelos. Balanced lines were generated by crosses to the stock w1118; If/CyO; MKRS/TM6B, which was a gift from A. Jacinto. The stock w; CRE-F-luc (II) was a gift from J.C. Yin. The stock y w; ptth-HA was a gift from M. O'Connor. Stocks are maintained at low densities at 18\u2009\u00b0C in a 12-h light/dark cycle.The Drosophila Lgr3 locus by using the MiET1 transposase to remobilize the MB Minos element Lgr3MB06848 (reference #11), which is inserted in the seventh Lgr3 intron, <100\u2009bp from exon 7 and transferred to new vials every dayCy-Minos/+; Lgr3MB06848/MKRS or TM6B male adults were selected and individually crossed to the balancer strain w1118; If/CyO; MKRS/TM6B. A single eGFP-negative (lacking the Mi{ET1} element) male was selected from each cross and mated with w1118; If/CyO; MKRS/TM6B females. The putative Lgr3 excisions were balanced over TM6B to obtain the following genotypes w1118; +/+; Lgr3MB06848excision/TM6B. We obtained one imprecise excision that generated the Lgr3ag1 mutant allele and three precise excisions named Lgr3ag2, Lgr3ag3 and Lgr3ag4 all of which behaved the same way. In this study, the Lgr3ag2 line was used as the genetic background control for the imprecise excision allele Lgr3ag1.We generated a mutation in the m exon 7 . Virgin Lgr3ag1 deletion, we performed a series of PCR assays with primer pairs located around the Lgr3MB06848 insertion . This suggested that while the Lgr3ag1 deletion was very large, its breakpoints were confined within the Lgr3 locus. We then tried to amplify a 5.3-kb PCR product with a primer upstream of the Lgr3MB06848 position are lacking in the Lgr3ag1 leucine-rich repeat domains, which are critical for relaxin ligand binding in vertebrate relaxin receptorsTo molecularly characterize the nsertion . Lgr3ag1r3 locus . This alLgr3ag1 and controls Lgr3+/+ and the original Lgr3MB06848 stock. Lgr3ag1 generated a smear with a major band that was \u223c200\u2009bp smaller than the other control genotypes \u2013PCR analyses with mRNA isolated from o exon 9 . The resn 9 (326 . We concterminus .Drosophila and Homo sapiens codon-optimized complementary DNA (cDNA) corresponding to full-length Lgr3 was synthetized de novo and cloned into pUASPpUASP-Lgr3. This plasmid was injected into w1118 and two independent insertions were tested, pUASP-Lgr3a and pUASP-Lgr3b . A similar protocol was used to place the dilp8::3xFLAG construct described in reference #3 into pUASP, making pUASP-dilp8a.A http://tools.flycrispr.molbio.wisc.edu/targetFinder/)We used a CRISPR/Cas9-mediated homologous repair strategy15gRNA 1Fw: 5\u2032-CTTCGGAGCACTCAATTCCCACTC (CGG)-3\u2032Rv: 5\u2032-AAACGAGTGGGAATTGAGTGCTCC-3\u2032gRNA 2Fw: 5\u2032-CTTCGCAAACTCAAGTAGAATATCA (CGG)-3\u2032Rv: 5\u2032-AAACTGATATTCTACTTGAGTTTGC-3\u2032The PAM regions (CGG) are located right after the forward primers of both gRNAs 1 and 2, as shown above.As a repair cassette we designed the following sequence, where each colour represents the following:Lgr3 ATG siteOrange: Homology region up to Lgr3 ATGRed: Green: sfGFPBlue: Spacer (GSGSGS)Lgr3 ATG siteViolet: Homology region after ATGCGTAAGGGCGAGGAGTTGTTCACGGGAGTTGTGCCCATATTGGTTGAGCTGGATGGAGATGTGAATGGCCACAAGTTCAGTGTGCGGGGTGAGGGAGAAGGAGACGCAACAAACGGTAAGCTGACACTGAAGTTCATTTGTACTACGGGCAAGCTCCCGGTGCCATGGCCCACATTGGTCACCACCCTGACCTATGGCGTGCAATGCTTCGCCCGATATCCAGATCATATGAAGCAGCATGATTTCTTTAAGTCGGCCATGCCCGAGGGTTACGTACAAGAGCGCACTATTAGCTTTAAGGACGACGGTACGTATAAAACCAGGGCTGAGGTGAAGTTTGAGGGTGATACCCTGGTGAACCGCATTGAATTGAAGGGCATCGATTTTAAGGAGGACGGCAACATCCTGGGCCACAAGCTCGAATATAATTTTAATAGCCATAATGTTTACATTACCGCGGACAAGCAGAAGAATGGAATTAAGGCTAATTTCAAGATCCGACATAATGTGGAGGACGGATCCGTTCAGTTGGCCGATCACTACCAGCAAAACACCCCCATCGGAGATGGCCCCGTCCTGCTGCCCGATAACCACTACCTGAGTACCCAGTCCGTCCTGTCGAAGGATCCTAATGAGAAGCGGGATCATATGGTGCTGCTGGAGTTTGTGACTGCCGCCGGCATAACGCATGGAATGGACGAGCTGTATAAAGGCTCCGGTAGTGGTTCCGTCTACGGCAGGAGCATCGCCGTAGGCTTCTGTCTGATGACCGTCGTCCTTCTGCTGGCCGCCGTGATATTCTACTTGAGTTTGGGTGAGTCCTTAGAGTGATGTCCTTTCAAAATTCCATCATTCGCAAACCTAAATAATTTCTGAATCAAGAATGTTCAAAATCTTAGCAATTATTATACGCATAATTTGTGAAACTACTTAAAGTTCTTTTAAAACTTGAGCTGCTGTAAATTTCTATATATACTTTCGTATCCTTAAAGGGTTCCTTCGCTTGAAGCAAAAACCAAAATCAAATTCCAAACTGCAAA-3\u20325\u2032-CACTTAAAACTCTTCTCCGCGAGCTGTGAACATTAGCCAAATGAAGTGACAAGAAATTAACGCAAAAATAAAACAAGAAGACGGAGCGGTATAAGAAATAATAATATAAAAACTCAATGAGTCAGCACCGCATCAGCTCCTGCTGCTGTTGTTCTTCTTATTGCTGTTGTTTGTGGGGGCGTGGCCGGAGTGGGAATTGAGTGCTCCTAATGATGAACTCGGTCAAGGAGCCAGTGCAGCCATGGTGGCCAAGTAATTAGATAAGCGAGCGTGCAAAACAGGAGCAAACCGATAAATCGCCde novo into a pUC57 plasmid and co-injected with the two gRNAs into the stock y[1] M{w[+mC]=Act5C-Cas9.P}ZH-2A w[*], which strongly and ubiquitously expresses a human codon-optimized cas9 . Ten negative hits were also sequenced and two were retained as background controls (ag10 and ag11). The y1M{w[+mC]=Act5C-Cas9.P}ZH-2A w* cassette was removed by selecting against eye colour and stocks were kept as homozygous stocks, except for allele ag8, which was kept balanced over TM6B.These primers were designed to indicate the correct insertion of the sfGFP repair cassette from both sides in the genome. Positive hits were sequenced . The next morning, the flies were transferred to a fresh plate to lay eggs for 3\u20136\u2009h, depending on the experiment. To control for overcrowding, between 10 and 30 larvae were transferred to vials containing normal Egg collections were performed on normal food plates and larvae were reared at controlled densities without additional yeast . Newly molted third instar larvae were collected every 2\u2009h as described previously48Larvae were collected as described above and transferred 72\u2009h after egg laying to fresh food with 10\u2009mM of EMS (Sigma) or PBS as control. Developmental time was measured as indicated above. EMS food was prepared as follows: food was melted and cooled to 55\u2009\u00b0C, an appropriate volume of freshly made EMS stock solution in PBS was added and thoroughly mixed and 3\u2009ml per tube were dispensed. EMS and PBS tubes were kept in the dark as much as possible throughout the experiments.Brains of wandering third instar larvae or first instar larvae were dissected in cold PBS, fixed for 30\u2009min in 4% paraformaldehyde, rinsed with PBS with Triton (0.3%) (PBST), incubated with primary antibody overnight and with fluorescently labelled secondary antibody for 2\u201324\u2009h in PBST with 1% bovine serum albumin. Nuclei were counterstained with DAPI (Sigma) and tissues were mounted in Fluoromount-G (Southern Biotech). Antibodies used were: mouse anti-GFP 1:200 , mouse anti-nc82 1:250 (DSHB), mouse anti-HA 1:50 , rabbit anti-GFP 1:200 , rabbit anti-Dilp7 1:5000 (gift from I. Miguel-AliagaAleft-Aright/[(Aleft+Aright)/2]. Results were compared statistically using the F-test for the significance of the difference between the FAi of the samples, an appropriate test for dispersion3\u03b1/n) for multiple comparisons was applied to \u03b1=0.05.Pairs of the left and right wings of male individuals reared at 29\u2009\u00b0C and rinsed with ethanol were dissected in a glycerol/ethanol solution and mounted in glycerol. Photos were obtained in a Zeiss Axiovert 40 CFL microscope. The wing areas and wing lengths were calculated as previously described3Lgr3+/+ by a factor of \u223c23 .To control for measurement errors, we measured the area of the same wing three times. Values obtained were 18310.2\u00b130.5\u2009a.u., which gives a coefficient of variation (CV) of 0.17%, which is smaller than the CV of wing areas of control \u22121 (Roche), and incubated the mixture at 37\u2009\u00b0C for 1\u2009h, followed by 95\u2009\u00b0C for 5\u2009min, to inactivate the protease.gDNA was extracted from a group of flies or single fliesg for 1\u2009min, to lower tissue debris. An extra DNAse treatment was performed to reduce gDNA contamination. cDNA synthesis was performed using the Maxima First Strand cDNA Synthesis Kit for RT\u2013quantitative PCR (Thermo Scientific), following manufacturer's instructions.RNA was extracted using the Direct-zol RNA MiniPrep kit (Zymo Research), following manufacturer's instructions. The material used for the RT\u2013PCR experiments described in For this study, PCR and RT\u2013PCR primers were designed and their specificity tested using Primer BLAST or Primer3. A T100 Thermal Cycler (Bio-Rad) was used for performing the PCR steps. The following primers were used for PCR analyses described in Lgr3_salto_fw (expected product size 866\u2009bp)Fw: 5\u2032-CCGACGCCTTGCTGCTAACT-3\u2032Rv: 5\u2032-TTTATGGAGCGGGCGTGGTC-3\u2032Lgr3_exonshort Lgr3 (expected product size 331\u2009bp)Fw: 5\u2032-CCGACGCCTTGCTGCTAACT-3\u2032Rv: 5\u2032-GTGCGTTATGAGGTTGTGCTG-3\u2032Lgr3_exon3p Lgr3 (expected product size 240\u2009bp)Fw: 5\u2032-CGCCTTGTCGGTAATCCCAT-3\u2032Rv: 5\u2032-GTGGCTCCATTAAACTGCTGC-3\u2032Lgr3_exons Fw: 5\u2032-CCGACGCCTTGCTGCTAACT-3\u2032Rv: 5\u2032-CAAAGACCACCAACCAGGCGTA-3\u2032rp49 (control)Fw: 5\u2032-TTGAGAACGCAGGCGACCGT-3\u2032Rv: 5\u2032-CGTCTCCTCCAAGAAGCGCAAG-3\u2032qRT\u2013PCR experiments were performed using Lightcycler 96 (Roche) using the FastStart Essential DNA Green Master dye and polymerase (Roche). The final volume for each reaction was 10\u2009\u03bcl, consisting of 5\u2009\u03bcl of dye and polymerase (master mix), 2\u2009\u03bcl of 10 \u00d7 diluted cDNA sample and 3\u2009\u03bcl of the specific primer pairs. The following primers were used:rp49 (control)Fw: 5\u2032-TTGAGAACGCAGGCGACCGT-3\u2032Rv: 5\u2032-CGTCTCCTCCAAGAAGCGCAAG-3\u2032Lgr3Fw: 5\u2032-GCTGGGTGCCCATCATCGTTAT-3\u2032Rv: 5\u2032-CAAAGACCACCAACCAGGCGTA-3\u2032InRFw: 5\u2032-TGTCAGCTGCACAATAATAGGC-3\u2032Rv: 5\u2032-TGCACTTTTCAGGGCATTT-3\u2032DrlFw: 5\u2032-CGGAGTTCCATACCCAGATTAC-3\u2032Rv: 5\u2032-GCCTCTTGTTATATTTACAGGTCTTGG-3\u2032rp49 according to the formula: %rp49=(2\u0302\u2212(\u0394CqLgr3\u2212\u0394Cqrp49)) \u00d7 100. The geometric mean\u00b1s.e.m. of three biological repeats was used and data were analysed by one-tailed unpaired Student's t-test using Bonferroni corrections (\u03b1/n) for multiple comparisons, using \u03b1=0.05. This test assumes the independent samples have equal variances.Primer efficiency for qRT\u2013PCR was tested by serial dilution. Data were expressed as %9Drosophila SL2/DL2 cells (ATCC) cultured in Schneider medium supplemented with 10% foetal bovine serum. The cells were gently oxidized with NaIO4 and TriCEPS\u2013ligand complexes were incubated with the cells for 1.5\u2009h to allow capture of oxidized glycomoieties. Following cell lysis, trypsinization and biotin-mediated affinity purification of ligand\u2013receptor peptides, the glycosylated peptides were selectively released using PNGase F, and samples were analysed by Dualsystems Biotech AG on an LTQ Orbitrap XL (Thermo Scientific) spectrometer fitted with an electrospray ion source. The samples were measured in data-dependent acquisition mode in a 40-min gradient using a 10-cm C18 column. Peptide identifications were filtered to a false-discovery rate (FDR) of \u22641% and qua014 ref. . For stav2) ref. . The adjIn all experiments reported in this work, no data point was excluded. All data points, including outliers, are represented in the figures and were used in the statistical analyses. No blinding was done and no particular randomization method was used to attribute individuals to experimental groups.Accession codes: DNA sequence data has been deposited in GenBank under accession codes KT321103-KT321113.How to cite this article: Garelli, A. et al. Dilp8 requires the neuronal relaxin receptor Lgr3 to couple growth to developmental timing. Nat. Commun. 6:8732 doi: 10.1038/ncomms9732 (2015).Supplementary Figures 1-15 and Supplementary Tables 1-2"} +{"text": "Mitochondrial division, essential for survival in mammals, is enhanced by an inter-organellar process involving ER tubules encircling and constricting mitochondria. The force for constriction is thought to involve actin polymerization by the ER-anchored isoform of the formin protein inverted formin 2 (INF2). Unknown is the mechanism triggering INF2-mediated actin polymerization at ER-mitochondria intersections. We show that a novel isoform of the formin-binding, actin-nucleating protein Spire, Spire1C, localizes to mitochondria and directly links mitochondria to the actin cytoskeleton and the ER. Spire1C binds INF2 and promotes actin assembly on mitochondrial surfaces. Disrupting either Spire1C actin- or formin-binding activities reduces mitochondrial constriction and division. We propose Spire1C cooperates with INF2 to regulate actin assembly at ER-mitochondrial contacts. Simulations support this model's feasibility and demonstrate polymerizing actin filaments can induce mitochondrial constriction. Thus, Spire1C is optimally positioned to serve as a molecular hub that links mitochondria to actin and the ER for regulation of mitochondrial division.DOI:http://dx.doi.org/10.7554/eLife.08828.001 Mitochondria are structures within cells that provide the energy to power many biological processes that are essential for complex life. These structures are also highly dynamic and go through cycles of fission (in which a single mitochondrion splits in two) and fusion (in which two mitochondria merge into one). These processes both maintain the correct number of mitochondria in a cell and remove damaged ones, and defects in either can result in many diseases.Previous research had shown that mitochondria are in close contact with another cellular structure called the endoplasmic reticulum. The points of contact mark the sites where mitochondria undergo fission, as small tubes of the endoplasmic reticulum wrap around, and then constrict, to split a mitochondrion.Other recent work revealed that a protein called INF2 is anchored on the endoplasmic reticulum where it promotes mitochondrial constriction. This protein builds actin subunits into long filaments that provide the force for constriction. However, it was not clear how INF2 became active, and whether there are proteins on mitochondria that interact with INF2 or actin.Manor, Bartholomew et al. have now used a combination of microscopy-based methods and biochemical analysis to discover that a mitochondrial protein called Spire1C performs all of these roles. Spire1C is found on the outer membrane of mitochondria; it interacts with INF2 to drive the formation of actin filaments that constrict mitochondria. These results suggest that Spire1C bridges the endoplasmic reticulum with the network of actin filaments. Further experiments then showed that increasing Spire1C levels in cells resulted in the mitochondria becoming fragmented due to increased constriction. On the other hand, depleting Spire1C had the opposite effect and caused mitochondria to become unusually elongated. Following on from this work, the next challenge is to see if Spire1C is used differently or similarly in the different processes that involve mitochondrial fission.DOI:http://dx.doi.org/10.7554/eLife.08828.002 Mitochondrial division is a complex process that is essential for survival in mammals and is fInverted formin 2 (INF2) is a formin family protein that promotes actin filament polymerization in a regulated fashion , 2014. ASpire proteins are membrane-binding actin-nucleators that interact with and regulate formin proteins . SynergiVertebrates have two known Spire genes, Spire1 and Spire2. Each Spire protein contains highly conserved domains with specific capabilities, including: four actin-monomer binding WH2 domains necessary for nucleating actin filaments; an mFYVE domain that binds to intracellular membranes and facilitates oligomerization ; and an We identified and characterized a novel alternate splice-isoform of Spire1 that contains KIND and WH2 domains common to all Spire proteins, as well as a previously uncharacterized unique 58 amino acid alternate exon sequence (ExonC) . After dWhen cells were transfected with a myc-tagged Spire1C construct, the protein showed extensive co-distribution with the mitochondrial marker mitoRFP also shoTo examine whether Spire1C distributes on the surface or interior of mitochondria we compared the distribution of GFP-ExonC (marking Spire1C) with mitoRFP using structured illumination microscopy (SIM), which gives a twofold resolution improvement over conventional confocal imaging . GFP-ExoTo confirm Spire1C's mitochondrial outer membrane localization, we employed a fluorescence protease protection (FPP) assay , which cIn support of this notion, transmembrane domain prediction software indicateWe next performed photobleaching experiments to investigate the dynamics of Spire1C's association with mitochondrial membranes. Photobleaching of a portion of a mitochondrial element that expressed GFP-Spire1C resulted in rapid recovery of fluorescence, with replenishment arising first in regions close to the bleach site and later at regions further away , similarGiven that Spire proteins can nucleate actin via their highly conserved WH2-repeat domain , we nextGiven that actin assembly has been shown to play an important role in regulating mitochondrial fission , the obsSince ER tubules have been implicated in mitochondrial constriction and division , we inveOne way the KIND domain of Spire1C could affect ER-mediated mitochondrial division is by binding to ER-anchored INF2. To test this possibility, we performed in vitro GST pull-down assays. We found that the C-terminal half of INF2 (INF2-CT), but not the N-terminal half (INF2-NT), associated with a GST-tagged Spire1C KIND domain, but not GST alone . AdditioWe next tested whether disrupting Spire1C's interaction with INF2 inhibits mitochondrial fission. To test this hypothesis, we first asked whether removing the KIND domain from Spire1C blocks the increase in mitochondrial division associated with overexpressing a constitutively active INF2 mutant, INF2 A149 , 2014. CMitochondrial fission would be co-dependent on Spire1C and INF2 if Spire1C's interaction with INF2 drives ER-mediated mitochondrial constriction. To test this hypothesis, we employed confocal fluorescence imaging of ER and mitochondria to examine mitochondrial constriction sites in cells co-expressing the ER marker Ii33-mCherry and different variants of Spire1C. Mitochondria constriction was visible at sites of ER-mitochondria crossover slightly more frequently in cells overexpressing Spire1C compared to cells not overexpressing the construct . U2OS cells stably expressing GFP-INF2 was described in Ii33-mCherry was a generous gift from P Satpute . MitoEmerald and mitoRFP were gifts from A Rambold . Spire1C was amplified from mouse brain cDNA using the sequence of NM_194355 as a reference. We expected to obtain a sequence yielding protein corresponding to GI 37595748, however, it contained an additional 58 residues (ExonC). Thorough examination of all available mammalian Spire1 isoforms revealed at least 3 alternatively-spliced exons, which we refer to as exons A (majority of KIND domain), B (protein sequence AVRPLSMSHSFDLS), and C (protein sequence VPRITGVWPRTPFRPLFSTIQTASLLSSHPFEAAMFGVAGAMYYLFERAFTSRWKPSK).To obtain a full-length Spire1C construct, we amplified the mouse Spire1 gene AK129296, which contains the full KIND domain through the first 3 WH2 domains, along with NM_194355, which contains a partial KIND domain and ExonC without Exon B. A series of amplifications of partial gene sequences was then performed to obtain versions of mouse Spire1 that were \u00b1 each of exons A, B, and C . Each vaGGCGCGCCATGGAACTGCATACATTTCTGACCAAAATTAAGAGPrimer 1 GCGCTTAATTAATCAGATCTCGTTGATAGTCCGTTCTGAAGPrimer 2 GCGCGGCGCGCCATGGCCAATACCGTGGAGGCTGPrimer 3 GAGCATTAATTAATCTAGTCTGCTCCGTCTAATTTCTTCPrimer 4 GGCGGGCGCGCCATGGCGCAGCCCTCCAGPrimer 5 GACTTAATTAATCTAGTCTGCTCCGTCTAATTTCTTCPrimer 6 GGCGPrimer 7 CCATGTGCTCCAGGAAGAAGCCPrimer 8 CTGCCTTCCAAGCCATACTCTACTCTACPrimers for spire1 gene amplification and plasmid constructionunderlined.All primers are 5\u2032 to 3\u2032. AscI or PacI restriction sites are CGAGGCTGCAGATGAAGGCCCGGAAGATGAAGACGGAGAGAAGAGAAGCATCTCAGCCATCCGGTCCTATCAGGACGTTATGAAGATCTGTGCTGCTCACCTCCCAACTGAGTCGGAGGCACCCAATCATTATCAGGCAGTATGTCGGGCCCTGTTCGCAGAAACCATGGAACTGCATACATTTCTGACCAAAATTAAGAGTGCAAAGGAGAACCTTAAGAAGATTCAAGAAATGGAAAAGGGTGATGAATCTAGCACAGATCTGGAGGACCTGAAAAATGCAGACTGGGCCCGGTTCTGGGTACAAGCGGCGAGGGATTTGCGAAATGGGGTAAAAGCTAAGAAAGTCCAGCAGCGGCAGTACAACCCTCTGCCCATTGAGTACCAACTGACCCCTTATGAGATGGCCGCGGACGACATTCGGTGCAAAAGATACACCGCGAGAAAAGTAATGGTAAATGGTGACGTCCCCCCTCGGTTGAAAAAGAGTGCTCATGAGGTCGCCGCTGACTTTATCAGATCAAGACCCCCTGCAAATCCAGTTTCAGCCAGAAAACTGAAACCAACCCCACCACGGCCACGGAGCCTCCATGAAAGAGCAGCAGAAGAAATTAAAGCAGAAAGAAAGGCTCGGCCTGTGTCACCAGAAGAAATTAGACGGAGCAGACTAGCAGTGCGGCCACTTAGCATGTCTCACAGTTTTGACTTGTCAGATGTCACTACGCCAGAATCTCCAAAGAATGTTGGAGAATCATCTATGGTGAATGGAGGCTTAACATCTCAAACAAAAGAAAATGGGCTGAGCGCTGCCCAGCAGGGGTCMAQPSSPGGEGPQLGAAGGPRDALSLEEILRLYNQPINEEQAWAVCFQCCGSLRAAAARRQPHRRVRSAAQIRVWRDGAVTLAPAAAAAAEGEPPPASGQLGYSHCTETEVIESLGIIIYKALDYGLKENEERELSPPLEQLIDQMANTVEADGSKDEGYEAADEGPEDEDGEKRSISAIRSYQDVMKICAAHLPTESEAPNHYQAVCRALFAETMELHTFLTKIKSAKENLKKIQEMEKGDESSTDLEDLKNADWARFWVQVMRDLRNGVKLKKVQQRQYNPLPIEYQLTPYEMLMDDIRCKRYTLRKVMVNGDVPPRLKKSAHEVILDFIRSRPPLNPVSARKLKPTPPRPRSLHERILEEIKAERKLRPVSPEEIRRSRLAVRPLSMSHSFDLSSPKNVGESSMVNGGLTSQTKENGLSAAQQGSAQRKRLLKAPTLAELDSSDSEEEKSLHKSTSSSSASPSLYEDPVLEAMCSRKKPPKFLPISSTPQPERRQPPQRRHSIEKETPTNVRQFLPPSRQSSRSLDVTTPEVPRITGVWPRTPFRPLFSTIQTASLLSSHPFEAAMFGVAGAMYYLFERAFTSRWKPSKEEFCYPVECLALTVEEVMHIRQVLVKAELEKYQQYKDVYTALKKGKLCFCCRTRRFSFFTWSYTCQFCKRPVCSQCCSDEELQFPKELMEDWSTMEVCVDCKKFISEIISSSRRSLVLANKRARLKRKTQSFYMSSAGPSEYCPSERTINEIKKMRLPSKPYSTLPIFSLGPSALQRGESCSRSEKPSTSHHRPLRSIARFSTKSRSVDKKIND domainWH2 domains Alternate exon BAlternate ExonCSpire boxmFYVE domainSpire1 shRNA constructs were generated by cloning the sequences into Clontech's pSingle-tTS-shRNA vectors using the HindIII/XhoI restriction sites. The sequences cloned into the vector to knockdown Spire1C were 5\u2032-TCGAGGGATTAGACGTAGCAGATTATTCAAGAG ATAATCTGCTACGTCTAATCTTTTTTACGCGTA-3\u2032 and 5\u2032-TCGAGGCGAATAATCTCCTGACTAATTCAAGAGATTAGTCAGGAGATTATTCGTTTTTTACGCGTA-3\u2032 . Oligonucleotides for human INF2 siRNA were previously described in spire1C gene. A mouse tissue cDNA panel was used as a template for amplification using Primers 7 and 8. Amplified DNA containing ExonC was \u223c400 bp, while DNA lacking this exon was \u223c200 bp. Multiple oligomer sets were utilized to eliminate non-specific amplification while capturing as many on-target amplifications as possible. PCR reactions were run on a 2% agaorse gel, and bands of the appropriate size were excised from the gel and purified using a QIAquick gel extraction kit . Purified DNA was cloned into the pCR II-TOPO vector using the TOPO-TA cloning kit (Invitrogen). Sequence analysis was used to confirm the sequence of the amplified and inserted DNA.Oligos flanking ExonC were designed to amplify the The Spire1C gene was amplified from mouse cDNA as described above. Vectors used for protein purification include a modified avidin-6x his-MBP-TEV-3x FLAG-Precision construct under the P1 promoter, as well as a modified pGEX vector for N-terminal GST fusion proteins. All vector backbones were gifts of Dr. Aaron Straight and are described in ExonC and the C-terminal 50 residues of mouse Spire1 were cloned into the avi-his-MBP-TEV-3xFLAG-precision vector described above or a modified pGEX vector using standard techniques . Avi-hisg 4\u00b0C. Supernatant was applied to hydrated glutathione resin (2 ml bed volume per liter culture), protein bound for 1 hr at 4\u00b0C, and resin was washed extensively with lysis buffer. Protein was eluted with elution buffer and loaded onto a HiTrapQ anion exchange column. A salt gradient of 150 mM to 1 M was used for protein elution.GST fusion proteins were used for affinity column construction for affinity purification of antibodies. These proteins were purified by using single colonies of transformed Rosetta (DE3) cells to inoculate 400 ml Terrific Broth cultures containing 100 \u03bcg/ml carbenicillin, 34 \u03bcg/ml chloramphenicol, which was grown overnight at 37\u00b0C. This culture was diluted into 2 l of fresh TB with antibiotics and grown to an O.D. of 0.8\u20130.9, at which time it was moved to 23\u00b0C. After 1 hr at 23\u00b0C, cultures were induced with 0.5 mM isopropyl \u03b2-D-1-thiogalactopyranoside for 3\u20134 hr and harvested as described for purification of avi-his-MBP-TEV-3xFLAG-Precision proteins above. Cells were thawed in lysis buffer , sonicated, and lysates were centrifuged for 30 min at 125,000\u00d7Spire1 affinity columns were made using GST-fusion proteins following the method of Two rabbit polyclonal antibodies described above and three commercially available antibodies were used for examining expression patterns of Spire1 protein in various cell types. The antibodies discussed are: (1) Rabbit polyclonal anti-Spire1 C-term (affinity-purified), (2) Rabbit polyclonal anti-Spire1 ExonC (whole antisera), (3) Sigma mouse monoclonal anti-Spire1, (4) Abcam mouse monoclonal anti Spire1, and (5) Abnova rabbit antisera to Spire1. Notably, all of the commercially-available antibodies were targeted to residues 482\u2013584 of the Spire1 isoform lacking ExonC (NP_064533), and thus could only detect non-ExonC containing isoforms.Western blots were performed with 5\u201350 \u03bcg cell lysate and antibodies/antisera was tested at various concentrations, temperatures, and lengths of time for best conditions. Optimized conditions for all antibodies used in this work are described below. HRP-conjugated goat anti-rabbit secondary antibody was used at 1:20,000 in all cases.2, 1 mM EGTA, 10 mM Hepes-HCl pH 7.4, 1 mM DTT, 0.02% thesit , 10 \u03bcg/ml aprotinin, 2 \u03bcg/ml leupeptin, 0.5 mM benzamidine). INF2-CT and INF2-NT were purified as described (Spire-KIND (amino acids 1\u2013234) was expressed as a GST fusion protein in bacteria, and purified on glutathione-sepharose followed by Superdex200 gel filtration (GE Biosciences) of the glutathione-eluted GST-fusion protein. GST-KIND or GST alone was re-bound to glutathione-sepharose in binding buffer at 23\u00b0C for 1 hr before reading fluorescence anisotropy in an M-1000 fluorescence plate reader (Tecan Inc) at 530 nm excitation and 585 nm emission.INF2 C-term was expressed in bacteria, purified and labeled on its N-terminal amine with tetramethylrhodamine succinimide as described . LabeledCells were washed in phosphate buffered saline then fixed with 4% paraformaldehyde for 30 min. Cells were then permeabilized with 0.1% Triton X-100 for 30 min before being blocked overnight with 4% BSA at 4\u00b0C. The next day, cells were incubated with primary antibody for 2 hr, rinsed three times with PBS for 10 min each, then incubated with secondary antibodies (Invitrogen) for 1 hr, rinsed three times with PBS for 10 min each, then counterstained with phalloidin (Invitrogen) for 30 min, then rinsed with PBS three times, then mounted using ProLong Gold antifade reagent.Confocal images were acquired with an Apochromat 63\u00d7 1.4 NA objective lens on a Marinas spinning disk confocal imaging system using an EM charge-coupled device camera , or a 100\u00d7 Apo TIRF 1.49 NA objective on a Yokogawa CSU-X1 spinning disk system using an EM charge-coupled device camera . Cells were imaged in HEPES-buffered growth media. Confocal z-stacks were taken using 200 nm steps. Images were deconvoluted using Slidebook 6. Individual 16-bit tiff image files were exported, then processed using ImageJ.GFP-Spire1C photobleaching experiments presented in SIM imaging of fixed cells was performed using an ELYRA SIM (Carl Zeiss) with an Apochromat 63\u00d7 1.4 NA oil objective lens. Five angles of the excitation grid with five phases each were acquired for each channel and each z-plane, which were spaced at 110 nm each. SIM processing was performed using the SIM module of the Zeiss Zen software package. 16-bit grayscale tiffs were subsequently exported to ImageJ for quantification and processing into rendered colored images. Channels in maximum projection images were aligned in the xy-plane using maximum projection images of fluorescent beads.http://rsb.info.nih.gov/ij/plugins/mbf/index.html). When calculating Pearson's values, the mitochondria channel was used as a mask for colocalization. ER-mitochondria intersection sites were visually identified as regions where ER tubules could be clearly visualized crossing mitochondria\u2014these regions were always in the periphery of the cell, significantly restricting the total number of intersections that could be reliably identified. Mitochondria constriction sites were visually identified as regions defined by relative narrowing of mitochondria diameter or reduced fluorescence. Magnifications of boxed regions were generated using ImageJ. Color images of merged 16-bit tiffs exported from the microscope were generated using the ImageJ \u2018merge channels\u2019 function. Statistical analysis was performed using Excel . p-values were determined using the unpaired Student's t-test or ANOVA, as appropriate.All image analysis and processing was performed using ImageJ. Mitochondria lengths were measured manually by first setting the scale according to pixel size, drawing a line along the length of the mitochondria, then using ImageJ's \u2018measure\u2019 function. Colocalization analysis and rendering was performed using the colocalization plugin included in the MacBiophotonics ImageJ plugin bundle value r = R, and (ii) the tubule profile remains parallel to the tubule axis. While the tubule length L = 680 nm was required to remain constant during the deformation, the tubule surface area was free to change. This means that the membrane lateral tension was taken to be zero, which guaranteed that the membrane bending energy was the sole contribution to the membrane elastic energy.The boundary conditions for the energy minimization consisted in the requirements that at the tubule left and right edges (i) the tubule cross-sectional radius, The energy minimization and the shape determination for each pressure value were performed using Brakke's \u2018Surface Evolver\u2019 program . review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent and three reviewers, one of whom, Pekka Lappalainen, is a member of our Board of Reviewing Editors. One of the three reviewers, Liza Pon, has also agreed to share her identity.Thank you for submitting your work entitled \u201cA mitochondria-anchored isoform of the actin-nucleating Spire protein regulates mitochondrial division\u201d for peer review at The reviewers have discussed the reviews with one another and the Reviewing editor has drafted this decision to help you prepare a revised submission.Previous studies revealed that constriction of mitochondria, which precedes Drp1-mediated mitochondrial fission, occurs at sites of close contact between ER and mitochondria, and is driven in part by INF2-mediated actin polymerization. Here Manor et al. show that a specific splice-variant of an actin nucleating protein Spire (named Spire1C) localizes to the mitochondria outer membrane through a specific a-helical motif encoded by its alternate exon C. Importantly, they demonstrate that Spire1C promotes actin filament assembly at the mitochondrial surfaces and modulates mitochondrial fission through its actin-binding WH2 domains and its KIND domain, which specifically binds to INF2 formin. Collectively, these studies provide evidence that Spire1C and INF2 cooperate to form mitochondria-ER intersections, and to promote efficient actin filament assembly specifically at these sites.The majority of the data presented in the manuscript are convincing and the study provides fundamentally important new insights into the mechanisms of mitochondrial fission. However, there are few important issues that should be addressed to confirm the conclusions presented and to further strengthen this study.Essentials revisions:1) Most of the studies presented are based on Spire1C over-expression. The authors should thus perform the mitochondrial fission and \u2018mitochondrial constriction\u2019 assays also on Spire1C knockdown cells. The effects of Spire RNAi should also be verified with an additional shRNAi construct, or the authors could try to rescue the phenotype of Spire RNAi cells. Together, these experiments would perhaps also reconcile some inconsistencies in the data. For example, while cell quantification suggests that Spire1C overexpression induces mitochondrial fragmentation , the images presented in the manuscript either show normal or even elongated mitochondria.2) The lipid specificity assay is not particularly convincing and should be omitted from the manuscript. Lipid specificity of a transmembrane protein (or a hydrophobic protein motif that penetrates into the acyl chain region of the bilayer) cannot be studied using lipid strips, where the individual lipid molecules are most likely not organized into a proper bilayer. Thus, if the authors wish to examine lipid specificity of ExonC, they should instead perform proper vesicle co-flotation or co-sedimentation assays.3) The authors should provide better controls for the protease sensitivity assay. This assay would benefit from analysis of a protease sensitive IMS protein to confirm that the protease treatment conditions used degrades OM protein without affecting the integrity of the OM. Furthermore, based on the sequence analysis ExonC is predicted to consist of two a-helices, which both are long enough (15-20 residues) to span the mitochondrial outer membrane. Would it be possible that these helices could either make a \u2018hairpin\u2019, which spans the outer membrane twice, or that this region would instead just \u2018horizontally\u2019 penetrate into the acyl-chain region of the mitochondrial outer membrane without spanning the entire bilayer? To provide stronger support for the conclusions presented, the authors should repeat the assay with a C-terminal GFP-fusion of full-length Spire1C (to confirm that the C-terminus of full-length Spire1C indeed does not face the cytosol as proposed in the current version of the manuscript). 1) Most of the studies presented are based on Spire1C over-expression. The authors should thus perform the mitochondrial fission and \u2018mitochondrial constriction\u2019 assays also on Spire1C knockdown cells. The effects of Spire RNAi should also be verified with an additional shRNAi construct, or the authors could try to rescue the phenotype of Spire RNAi cells. Together, these experiments would perhaps also reconcile some inconsistencies in the data. For example, while cell quantification suggests that Spire1C overexpression induces mitochondrial fragmentation , the images presented in the manuscript either show normal or even elongated mitochondria.We have performed additional experiments using Spire knockdown cells (using two different shRNA constructs), and added the fission and constriction data to the manuscript , all of which is consistent with our other data.With regards to apparent inconsistencies in mitochondrial phenotype in Spire1C overexpressing cells: We found that in all conditions there was a range of mitochondrial lengths within each cell, as well as between cells. While the average mitochondrial lengths significantly changed depending on Spire or INF2 activities, nearly every cell has some mitochondria that are very short, and at least a couple that are longer. For the sake of clarity in some of our experiments , we used cells with longer mitochondria in order to be able to make measurements as necessary . In order to clarify our data in this regard, we have added as a supplemental figure a histogram showing the distribution of mitochondrial lengths for each condition .2) The lipid specificity assay is not particularly convincing and should be omitted from the manuscript. Lipid specificity of a transmembrane protein (or a hydrophobic protein motif that penetrates into the acyl chain region of the bilayer) cannot be studied using lipid strips, where the individual lipid molecules are most likely not organized into a proper bilayer. Thus, if the authors wish to examine lipid specificity of ExonC, they should instead perform proper vesicle co-flotation or co-sedimentation assays.We agree that co-flotation and/or co-sedimentation assays would better establish the lipid specificity of ExonC. This will require many more experiments that go beyond the scope of this work. Therefore, we have removed the lipid dot blot and will attempt to establish ExonC\u2019s lipid specificity in a future study.3) The authors should provide better controls for the protease sensitivity assay. This assay would benefit from analysis of a protease sensitive IMS protein to confirm that the protease treatment conditions used degrades OM protein without affecting the integrity of the OM. Furthermore, based on the sequence analysis ExonC is predicted to consist of two a-helices, which both are long enough (15-20 residues) to span the mitochondrial outer membrane. Would it be possible that these helices could either make a \u2018hairpin\u2019, which spans the outer membrane twice, or that this region would instead just \u2018horizontally\u2019 penetrate into the acyl-chain region of the mitochondrial outer membrane without spanning the entire bilayer? To provide stronger support for the conclusions presented, the authors should repeat the assay with a C-terminal GFP-fusion of full-length Spire1C (to confirm that the C-terminus of full-length Spire1C indeed does not face the cytosol as proposed in the current version of the manuscript).We think the reviewers may have overlooked our control protein in the protease protection assay in making his/her statement \u201cthis assay would benefit from analysis of a protease sensitive IMS protein to confirm that the protease treatment conditions used degrades OM protein without affecting the integrity of the OM.\u201d By definition, if our treatment conditions are not affecting the integrity of the OM, then any IMS protein would not be affected. This is exactly why we used the OMI-mCherry construct as a control in all of our experiments. OMI-mCherry localizes to the IMS \u2013 if the OM was being degraded by our digitonin treatment, then OMI-mCherry would escape into the cytoplasm. Similarly, if the OM was degraded enough that trypsin was able to digest proteins in the IMS, then OMI-mCherry would be depleted upon addition of trypsin. In none of our experiments did we observe depletion of OMI-mCherry mitochondrial fluorescence, indicating that the OM was indeed intact in all of our experimental conditions.We agree that it is important to be careful not to make any claims about the C-terminus of Spire1C being in the IMS, since our FPP assay only informs us that the C-terminal region of ExonC (which is in the middle of the full-length Spire protein) is protected from trypsinization. While our FPP assay leaves us confident that the C-terminus of ExonC is protected from the cytoplasm, we were unable to draw any conclusions from these assays as to the localization of the Spire1C C-terminus. While the secondary protein structure prediction software PHYRE identifies two putative alpha-helices within ExonC, only one of those helices was predicted to be a transmembrane domain by predictive software, so we chose not to postulate that there are two transmembrane domains in ExonC. However, we agree that the hairpin model the reviewers have proposed is just as likely to be correct, and is furthermore appealing since this would allow for the C-terminal domain of Spire1C to interact with other cytoplasmic proteins.We agree that an additional FPP assay using a C-terminus GFP-tagged Spire1C construct would ideally clarify the topology of the Spire1C C-terminus. In order to address the reviewers\u2019 comments, we performed an additional FPP assay using a new Spire1C-GFP construct with GFP on the C-terminus. Unfortunately, when we expressed this construct, the protein no longer localized properly to mitochondria, instead displaying a mostly cytoplasmic localization pattern (with barely detectable accumulation on mitochondria). Due to this disrupted localization pattern, we were unable to draw any conclusions from an FPP assay; upon addition of digitonin, all of the GFP fluorescence was almost immediately depleted, even without subsequent addition of trypsin. While this result may give some clue as to how Spire1C localizes to the OM, many more experiments that go beyond the scope of this work would have to be performed in order to determine what this result is telling us. Thus, we have mentioned these latest findings in the text, and modified our interpretation of our FPP data to include the likely possibility that Spire1C\u2019s ExonC localizes in a hairpin or horizontal fashion on the outer leaflet of the outer mitochondrial membrane."} +{"text": "MicroRNAs (miRNAs) play an important role in targeted gene silencing by facilitating posttranscriptional and translational repression. However, the precise mechanism of mammalian miRNA-mediated gene silencing remains to be elucidated. Here, we used a stem-loop array reverse-transcription polymerase chain reaction assay to analyse miRNA-induced mRNA recognition, cleavage, posttranscriptional modification, and degradation. We detected endogenous let-7 miRNA-induced and Argonaute-catalysed endonucleolytic cleavage on target mRNAs at various sites within partially paired miRNA:mRNA sequences. Most of the cleaved mRNA 5\u2032-fragments were 3\u2032-oligouridylated by activities of terminal uridylyl transferases (TUTases) in miRNA-induced silencing complexes and temporarily accumulated in the cytosol for 5\u2032-3\u2032 degradation or other molecular fates. Some 3\u2032-5\u2032 decayed mRNA fragments could also be captured by the miRNA-induced silencing complex stationed at the specific miRNA:mRNA target site and oligouridylated by other TUTases at its proximity without involving Argonaute-mediated RNA cleavage. Our findings provide new insights into the molecular mechanics of mammalian miRNA-mediated gene silencing by coordinated target mRNA recognition, cleavage, uridylation and degradation. HOX B8 mRNA, directed by the perfectly base-matched miR-196, is one of only a few cases of miRNA-directed mRNA decay reported in mammalian cells1011MicroRNAs (miRNAs) are noncoding RNAs that have been shown to posttranscriptionally regulate gene expression and protein synthesis, particularly in mammalian cells, by partially base-pairing to complementary sequences in the 3\u2032-untranslated regions (3\u2032UTRs) of their target mRNAs124et al.It is very important to provide direct evidence for miRNA-mediated target mRNA cleavage and degradation to access the precise mechanism of action in the miRNA-mediated suppression of target gene expression in mammalian cells. The techniques currently used to study the individual or global effects of miRNA action on its target mRNA, such as 5\u2032RACE, ribosome profiling, and RNA-deep sequencing, are powerful and informative2810TUSC2, also known as FUS1), to demonstrate the mammalian miRNA-mediated target mRNA cleavage and regulatory activities in human cells using a novel SLA\u2013RT-PCR assay could be posttranscriptionally added to the 3\u2032 ends of miRNA-cleaved 5\u2032-mRNA fragments in various organismsragments . These 3ragments using toragments .TUSC2 target site that is effective in all let-7 miRNA members. The expression of let-7 miRNAs was depleted in the cells transfected with miR-98 LNAi 48\u2009h after treatment, as shown by qRT-PCR analysis using let-7\u2013specific SL-RT primers and the 3\u2032 supplementary pairing region, and they share a conserved short stretch of base-paired (7\u201310 nt) sequences at the seed region were used as a negative control. The cel-miR-67 was confirmed to have minimal sequence identity with miRNAs in human, mouse, and rat and shown to have no significant biological impact when ectopically expressed in those mammalian cells. Overexpressed miR-622 and let-7d mRNAs were detected by qRT-PCR in H1299 cells transfected with a miR-622 or a let-7 expression vector, respectively mRNA targeted by miR-142-3p activity in HeLa cells by a U-track\u2013specific SLA\u2013RT-PCR and Sanger sequencing , were detected by agarose gel electrophoresis promoter, immediately followed by a 3\u2032UTR with two identical copies of let-7 target sequences (T1 and T2) directly derived from TUSC2 mRNA sequences arranged in tandem and a BGH poly(A) signalling sequence reporter plasmid-based model system with a defined let-7 target and SLA\u2013RT-PCR primer binding sequences and a fully functional mammalian mRNA structure to monitor the precise action of miRNA on its target in a time- and space-dependent manner. The reporter plasmid (pLJ-T214) consisted of an sequence .TUSC2 let-7 target sites were produced: one copy in the 3\u2032UTR of the endogenous TUSC2 mRNA transcript and two copies in the 3\u2032UTR of the exogenously expressed pLJ-T214 EGFP reporter transcript. Endogenous let-7\u2013mediated mRNA cleavage of those target sites would produce 5\u2032-mRNA fragments with identical 3\u2032 termini, which could be detected competitively by the same SL-RT primer in RT. The origins of the cleaved mRNA fragments could be determined by subsequent PCR amplification with either the endogenous or the pLJ-T214 transcript\u2013specific PCR primers, which would generate SLA\u2013RT-PCR amplicons of different sizes. The predicted sizes of the SLA\u2013RT-PCR products derived from target sites T1 and T2 were 240-209\u2009bp and 532-500\u2009bp, respectively, and those derived from the endogenous target site were predicted to be 230-198\u2009bp.In pLJ-T214\u2013transfected H1299 cells, three identical copies of TUSC2 mRNA fragments and internal references for defining the spatial effect of target sites on endogenous let-7 activity by the SLA\u2013RT-PCR assay or SLA-qRT-PCR. Total RNAs from pLJ-T214\u2013transfected H1299 cells at different time points after transfection were examined with the same set of SLA-RT primers used for the endogenous TUSC2 mRNA fragment detection by SLA-qRT-PCR in the T1 site were consistently stronger than those in its T2 counterpart at any given time; however, intensity patterns at both sites displayed similar trends over the course of transfection. The accumulation patterns of the 3\u2032-uridylated T2 fragments at 16 h after transfection catalyse the transfer of UMP residues to the 3\u2032 hydroxyl group of RNA and are involved in bulk degradation of histone mRNA in human cells2228A\u2013RT-PCR . Total RA\u2013RT-PCR . The unmA\u2013RT-PCR . The siRe3 genes showed me3 genes . The 3\u2032-e3 genes . In conte3 genes . No appae3 genes . The oliTUSC2 mRNAs at various sites in partially paired miRNA:mRNA sequences, predominantly within the miRNA seed region or in the 3\u2032 supplementary pairing region. Most of the cleaved mRNA 5\u2032-fragments were oligouridylated at their 3\u2032-termini by TUTase2 in the miRISC, while some 3\u2032-5\u2032 decayed mRNA fragments could also be intercepted by miRISCs stationed on their targets and oligouridylated by other TUTases in the proximity of the miRISC. These 3\u2032-oligouridylated mRNA 5\u2032-fragments accumulated in cytosol and appeared destined for 5\u2032-3\u2032 decay. These findings provide direct evidence for mammalian miRNA-mediated gene silencing by target mRNA recognition, cleavage and 3\u2032-uridylation, leading to degradation of the protein coding sequence\u2013containing 5\u2032-mRNA fragments via a possible bulk 5\u2032-3\u2032 decay pathway.Multiple models have been proposed for the molecular mechanics of miRNA-mediated posttranscriptional repression of target gene expression either by inhibition of mRNA translation, with no apparent impact on mRNA stability, or by initiation of mRNA decay1278307G cap-binding motif has been identified on Argonaute MID domains but was shown to be weak and could not compete with the cap-binding eIF4E to sustain translational suppression33Our findings suggest that the mRNA cleavage and 3\u2032-oligouridylation activities mediated by mammalian miRNAs with partial base-pairing to their target mRNA sequences in Ago2-miRISCs may be a common molecular mechanism for miRNA-targeted gene silencing. We have shown that target gene silencing mediated by let-7 family miRNAs and other human miRNAs could be initiated by Ago2-catalysed endonucleolytic cleavage on base-paired miRNA:mRNA target sites, which is consistent with the fact that the Ago2 endonucleolytic RNase H domain prefers paired bases as substrates15Most of the cleaved 5\u2032 mRNA fragments were oligouridylated at their 3\u2032-termini in the miRISC. Those 5\u2032 mRNA fragments derived from the initial cleavage sites on the same transcript that escaped oligouridine modification in the miRISC could be subjected to the exonucleolytic RNA 3\u2032-5\u2032 decay pathway and would then be intercepted and oligouridylated by other TUTases in proximity to the miRISC. These oligouridylated mRNA fragments could be recognised by the cytoplasmic heptameric Lsm1-7 complex and accumulated in P-bodies2836373839414243Our results provide new insights into the molecular mechanics of mammalian mRNA-mediated gene silencing by showing a cooperative action of miRNA, Ago2/TUTases and other essential molecular components in miRISCs mediating mRNA degradation initiated by mRNA cleavage and concurrent 3\u2032-uridylation. Further studies are needed to understand the precise mechanism of miRNA action in association with specific co-factors in miRISC on posttranscriptional regulation of target gene expression in a complex mammalian cellular system.RNase inhibitor was acquired from New England Bio-Labs (M0314L). DNA primers and RNA oligos were synthesized by Sigma-Aldrich. High-capacity cDNA first-strand synthesis kit, TaqMan Gene Expression Master Mix and TaqMan probes for miRNA quantification were purchased from Applied Biosystems (Life Technologies). The miRCURY LNA inhibitor against hsa-miR-98 was obtained from Exiqon. ExoSAP-IT for PCR Product Cleanup was obtained from Affymetrix. TRIzol LS Reagent, SYBR Green I and 1\u2009Kb-Plus DNA Ladder were obtained from Invitrogen (Life Technologies). Rabbit anti-Ago2 monoclonal antibody was purchased from Cell Signaling Technology. SiGENOME SMARTpool-EIF2C2 anti-Ago2 and ON-TARGET plus non-targeting siRNA as nonspecific controls (siR-NSCs) were purchased from Thermo Scientific.Human H1299 large cell lung cancer and HeLa cervical cancer cells were grown in Roswell Park Both Memorial Institute 1640 medium and Dulbecco modified Eagle medium supplemented with 10% foetal bovine serum (FBS). For transfection, plasmid DNA was adjusted to 0.5\u2009\u03bcg/\u03bcL and siRNAs (25\u2009\u03bcM) were dissolved in sterile water. Plasmid DNA or siRNA was mixed with an equal volume of 8\u2009mM DOTAP:Cholesterol liposome to make DOTAP:Chol:DNA complexes. DOTAP:Cholesterol liposomes were prepared in-house as described previouslySchizosaccharomyces pombe Cid1 enzyme, which showed that TUTase1 and TUTase3 are involved in histone mRNA uridylationmiRNA let-7 knockdown was carried out in H1299 cells transfected with 10\u2009\u03bcM of miRCURY LNA anti-miR-98 inhibitor packaged in a DOTAP:Chol:siRNA complex. After 24\u2009h of initial transfection, the medium was refreshed and cultures were returned to incubation for an additional 24\u2009h. Cells were then subjected to direct lysis in TRIzol LS Reagent, and total RNAs were isolated according to the vendor\u2019s recommendation. The let-7 miRNA levels were measured by qRT-PCR assays. Several putative TUTases have been identified in the human genome on the basis of a homology comparison with the U6-terminal transferase and the TUTase mRNA levels were measured by end-point RT-PCR assays as reported by Mullen et al.To determine the effect of TUTase knockdown on miRNA-mediated fragment distribution, we synthesized all TUTase siRNAs and PCR primers according to the ref. RNAs (50\u2009\u03bcg) were treated with 10\u2009\u03bcL of RNase-free DNase I and 20\u2009\u03bcL of RNase inhibitor in 100\u2009\u03bcL of reaction solution for 5 min at 37\u2009\u00b0C, 10\u2009min at 80\u2009\u00b0C and 15\u2009sec at 95\u2009\u00b0C. RT master mixes were prepared using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems), and aliquots containing 0.25\u2009\u03bcg RNA were mixed with 2.5\u2009\u03bcL of the appropriate 100\u2009nM SL-RT primers. The reactions were incubated by the following program: 1\u2009min at 20\u2009\u00b0C and 10\u2009sec at 37\u2009\u00b0C for 60 cycles, 30\u2009min at 37\u2009\u00b0C, 20\u2009min at 42\u2009\u00b0C, 5\u2009min at 80\u2009\u00b0C and hold at 4\u2009\u00b0C. The RT products were then subjected to further PCR amplification for end-point PCR evaluation or to real-time PCR for quantitation. Samples not subjected to RT were used as negative controls to avoid any potential DNA contaminants in the RNA preparations.For end-point PCR analyses of cDNA products primed by SLA-RT primers, PCR was performed in TaqMan Gene Expression PCR Master Mix with universal primer and transcript-specific primers. The RT product (1\u2009\u03bcL) was mixed in 30\u2009\u03bcL of PCR mixtures containing 0.5\u2009\u03bcM PCR primers. The PCR program started at 95\u2009\u00b0C for 10\u2009min, followed by 30 cycles of 15\u2009sec each at 95\u2009\u00b0C, 1\u2009min at 68\u2009\u00b0C and 20\u2009sec at 72\u2009\u00b0C for an additional 5 cycles, chased with 15\u2009sec at 95\u2009\u00b0C, 2.5\u2009min at 68\u2009\u00b0C and 2.5\u2009min at 72\u2009\u00b0C. PCR products were analysed by electrophoresis on 3% agarose gels in 1\u00d7 Tris-Boric acid buffer\u2206Ct method, where normalised \u2206Ct\u2009=\u2009Ct of Sample\u2009\u2212\u2009Ct of NTC (non-template control). The qRT-PCR conditions for miRNA detection were modified to 10\u2009min at 95\u2009\u00b0C, 5 cycles of pre-amplification of 15\u2009sec each at 95\u2009\u00b0C, 2.5\u2009min at 61\u2009\u00b0C and 2.5\u2009min at 72\u2009\u00b0C, then 40 cycles of 15 sec at 95\u2009\u00b0C, 1\u2009min at 60\u2009\u00b0C and 1\u2009sec at 72\u2009\u00b0C. Let-7 family miRNA, hsa-miR-622, hsa-miR-30a and hsa-RNU44 in H1299 cells were determined by SLA\u2013RT-PCR methods. The data were first normalised against hsa-RNU44, and then the changes in miRNA expression level were calculated by the following formula: \u2206\u2206Ct\u2009=\u2009\u2206Ct of miRNA target \u2212\u2009\u2206Ct of miRNA negative control4546For profiling of relative quantitative mRNA fragment abundance, a real-time SLA-qRT-PCR with ether the TagMan probe-based or SYBR-Green-based method was performed with 1\u2009\u03bcL of RT product primed by each SLA-RT primer in triplicate in 40\u2009\u03bcL of TaqMan Gene Expression PCR Master Mix with or without containing SYBR Green I, 0.5\u2009\u03bcM of universal primer and 0.5\u2009\u03bcM of transcript-specific primer. PCR reactions started at 95\u2009\u00b0C for 10\u2009min, followed by 10 cycles of pre-amplification of 15\u2009sec at 95\u2009\u00b0C, 2.5\u2009min at 68\u2009\u00b0C and 2.5\u2009min at 72\u2009\u00b0C, then 40 cycles of 15\u2009sec at 95\u2009\u00b0C, 1\u2009min at 68\u2009\u00b0C and 20\u2009sec at 72\u2009\u00b0C on a CFX384 Real-Time PCR Detection System (Bio-Rad). Melting curve analysis was used to confirm a single PCR product in each reaction. RNU44 was measured as a sample control, and RNA fragment relative abundance was calculated by the 2TUSC2 let-7 target site was synthesized by GenScript. A DNA insert was released by BsrGI/BglII and cloned into an EGFP expression cassette as 3\u2032UTR under a CMV promoter. The transcript 3\u2032UTR region on the plasmid was verified by Sanger sequencing. The details of the pLJ-T214 transcript are illustrated in et al.47A DNA fragment with two copies of let-7 targets identical to the EGFP open reading frame \u2009\u2009\u2009\u2009\u2009\u2009\u2009stop codonTGTACAAGTACGGCATGGACGAGCCCATAACCTTCAGCTGTGAGCGAAGAAACTCCCAGGCTCAATCAAGGTGTGGCTTCCATTGAGGAGCCCAGGCTGCCACAACCCTGAATAAACTCTGTTGGCGGTCGGTCCACAGTATTGGTTGGTGTTGGTTTGTGTGTGGACAAGAATTAGCATTTCAGCCGTTTGCTACCTCGATTCCTCCCTAGTCAGGCTAGCTAAAGGACTGACCGGCAAGTTGGACGCCCGCAAGATCCCCATAACCTTCAGCTGTGAGCGAAGAAACTCCCAGGCTCAATCAAGGTGTGGCTTCCATTGAGGAGCCCAGGCTGCCACAACCCTGAATAAACTCTGTTGGCGGTCGGTCCACAGTATTGGTTGGTGTTGGTTTGTGTGTGGACAAGAGGTAGTCAGAGCCTGATTCTTCCGACCATTTGTTCCCGCCTTCAGATGCTCGAGAGCATTTCAGCCGTTTGCTACCTCGATTCCTCCAGATCTCTAGTCAGGATAAACCCGCTGATCAGCCTCGA-BGH poly(A) Signal.TUSC2 let-7 target site was synthesized by GenScript. A DNA insert was released by BsrGI/BglII and cloned into an eGFP expression cassette as 3\u2032UTR under a CMV promoter. The transcript 3\u2032UTR region on the plasmid was verified by Sanger sequencing. The details of the pLJ-T722 transcript are illustrated in A DNA fragment with two copies of let-7 targets identical to the EGFP open reading frame \u2009\u2009\u2009\u2009\u2009\u2009\u2009stop codonTGTACAAGTACGGCATGGACGAGCAAGCGGCCGCCATAACCTTCAGCTGTGAGCGAAGAAACTCCCAGGCTCAATCAAGGTGTGGCTTCCATTGAGGAGCCCAGGCTGCCACAACCCTGAATAAACTCTGTTGGCGGTCGGTCCACAGTATTGGTTGGTGTTGGTTTGTGTGTGGACAAGAGGTAGTCAGAGCATTTCAGCCGTTTGCTACCTCGGGACGAGGTGCCTAAAGGACTGACCGGCAAGTTGGACGCCCGCAAGATCCCCATAACCTTCAGCTGTGAGCGAAGAAACTCCCAGGCTCAATCAAGGTGTGGCTTCCATTGAGGAGCCCAGGCTGCCACAACCCTGAATAAACTCTGTTGGCGGTCGGTCCACAGTATTGGTTGGTGTTGGTTTGTGTGTGGACAAGAGGTAGTCAGAGCCTGATTCTTCCGACCTACCTGCCATTTGTTCCCGCCTTCAGATGCTCGAGAGCATTTCAGCCGTTTGCTACCTCGTAGATCTGATAAACCCGCTGATCAGCCTCGA-BGH poly(A) Signal.PCR was performed on genomic DNA extracted from normal human bronchial epithelial cells with the following primers flanking the miR-622 precursor:GGACTAGTGAATTCTTTAGAGAAGCTGGACAAGTACTACTTGTAACTCGAGAAAAGTGTCATCTCAGCAGCTCSpeI/EcoRI and cloned into a CMV promoter\u2013driven expression plasmid with BGH poly(A) signal. The plasmid expression cassette was verified by Sanger sequencing.PCR fragments were digested with CMV promoter\u2013driven expression plasmid with BGH poly(A) signal was digested by EcoRI/XhoI and purified as a cloning vector. The correct clones were screened and verified by Sanger sequencing as a let-7d expression vector.The following oligonucleotides were synthesized by Sigma-Aldrich and annealed as inserts. A AATTCCCTAGGAAGAGGTAGTAGGTTGCATAGTTTTAGGGCAGGGATTTTGCCCACAAGGAGGTAACTATACGACCTGCTGCCTTTCTTAGGCTCGAGCCTAAGAAAGGCAGCAGGTCGTATAGTTACCTCCTTGTGGGCAAAATCCCTGCCCTAAAACTATGCAACCTACTACCTCTTCCTAGGGet al.TUSC2 5\u2032-mRNA fragment detection and quantification and nU-track detection, and the TUTase-siRNAs for each experiment, are summarised in SLA-RT primer was composed of stem loop and probe as reported by Chen TUSC2 mRNA fragments with various uridine tails were detected by qRT-PCR assay. The A4 RNA fragments of pLJ-T214 transcripts were represented by 528-bp and 234-bp amplicons and detectable in both unmodified and oligouridylated forms in total RNA samples from H1299 cells transfected with plasmid pLJ-T214. The A4 RNA fragments were further tested with SL-RT primers with different probes to confirm the oligouridine detection methods. Total RNAs from pLJ-T214-transfected H1299 cells were reverse-transcribed with 5 SL-RT primers with different probe compositions to compare their priming capabilities, and the results are shown in 18RNA fragment abundance varies with oligouridine lengths see . Let-7\u2013mHow to cite this article: Xu, K. et al. MicroRNA-mediated target mRNA cleavage and 3\u2032-uridylation in human cells. Sci. Rep.6, 30242; doi: 10.1038/srep30242 (2016)."} +{"text": "Understanding the quantitative functional consequences of human disease mutations requires silencing of endogenous genes and expression of mutants at close to physiological levels. Changing protein levels above or below these levels is also important for system perturbation and modelling. Fast design optimization demands flexible interchangeable cassettes for endogenous gene silencing and tuneable expression. Here, we introduce \u2018TEMTAC\u2019, a multigene recombineering and delivery system for simultaneous siRNA-based knockdown and regulated mutant (or other variant) expression with different dynamic ranges. We show its applicability by confirming known phenotypic effects for selected mutations for BRAF, HRAS, and SHP2. Recent work provided evidence that quantitative rather than qualitative differences in the proteomic inventory between different cell types and tissues are the cause of functional differences123456Gene silencing based on short interfering RNAs (siRNA) has been demonstrated in several organisms, including in cultured mammalian cells11in vitro, optionally in high-throughput by robotics15Plasmid-based gene delivery systems have become essential for molecular and cell biology. However, the number of available selection markers and the physical space that needs to be available to allow entry of multiple plasmids into cells are technically limiting. These problems are exacerbated by imbalanced delivery if multiple plasmids are used, resulting in heterogeneous cell populations which interferes with read-out. These impediments can be circumvented by combining independent modules containing genes of interest, regulatory elements and other desired functionalities into a single multifunctional multigene delivery plasmid. This is a viable option if the combination of the modules is sufficiently straight-forward. Addressing this challenge, we previously developed the \u2018ACEMBL\u2019 system to enable fast and flexible generation of multigene delivery constructs by an automatable technique called tandem recombineering (TR) to combine \u2018Donor\u2019 and \u2018Acceptor\u2019 plasmid modules via reversible Cre-loxP fusion Tunable Endogenous Mammalian TArget Complementation by multiplexed plasmid-based recombineering. Retaining the original ACEMBL concept, we developed this novel system which affords the means to simultaneously knockdown endogenous targets via RNA interference89in vitro exploiting the Cre-Lox recombination reaction, which can be carried out optionally in high-throughput by robotics applying automated routines we have implementedHere, we introduce TEMTAC, a novel system for http://www.invivogen.com/sirna-wizard). Donor D1 containing the shRNAs was transiently transfected in HEK293 or HeLa cells. After 24 and 48\u2009hours, cells were lysed and endogenous protein levels detected by Western blotting, real time PCR, and deep sequencing. We observed the best silencing effect 48 h post transfection and no down-regulation using random (\u2018scrambled\u2019 scrb) shRNAs as a control contains the silencing cassette for knocking down the endogenous gene by RNA interference . In one 192021 control . IncomplE. coli) mutant and three VP16 (transcription activation domain of herpes simplex virus). In the presence of the inducer doxycycline (dox), the TETon3G-dox complex binds to the TRE minimal promoter (TRE-pMCVmin) and induces the expression of the gene of interest (GOI).Donor D2.1 (pMDS-GOI-TETon3G), constitutively expresses the TETon3G transactivator , a fusion protein of a Tet repressor (Donor D2.2 (pMDS-GOI-TEToff) is based on the TEToff advanced inducible expression system (Clontech). This vector expresses the TEToff advanced transactivator (TetR) and also contains a TRE-based module that expresses high levels of the GOI in the absence of dox.Donor D2.3 (pMDS-GOI-TETon-PGK), is a modification of Donor D2.1, where the expression of the rtTA is downstream of a minimal phosphoglycerate kinase (PGK) promoter .Donor D2.4 (pMDS-GOI-TETon-tTS) is a modification of TETon to reduce basal expression levels. This is achieved by adding an internal ribosome entry site (IRES) and a tTS , which is a fusion of the TetR and the KRAB-AB silencing domain of the Kid-1 protein, after the transactivator (rtTA). As tTS competes with rtTA by binding specifically to the TRE, it suppresses the transcription in the absence of dox.Achieving expression levels that are close to endogenous levels is a crucial prerequisite for analysing the physiological effect of mutations. This is particularly important when studying signalling proteins because of their frequent involvement in downstream activation and feedback loops, which often function at very precise protein levels that need to be maintained meticulously. A major problem in many TET regulated systems is basal expression (without the inducer being present), especially for proteins that are present at markedly low abundance in cells. Furthermore, depending on the protein of interest, different dynamic expression ranges of mutants would be highly desirable to properly recapitulate physiological events. To address this, we created as Donor D2 variants four different TET-related expression system versions with defined properties in HEK293 cells grown in serum-containing medium. Exogenous expression of this oncogenic mutation tends to be higher as compared to wildtype (WT) . To rectify this, we took advantage of our TET-regulated system to tune the expression levels of WT and V600E to be close to identical and Donor D2 (pMDS-GOI) were created based on the original plasmids of the ACEMBL system for protein complex expression in prokaryotic hostsde novo from scratch. All DNA elements . Sequences are provided in the shRNAs and genes of interest (GOI) were inserted into the TEMTAC system using standard restriction-ligation cloning methods or, alternatively, by Gibson cloningHEK293, HeLa and GH-HEK293 cells were cultured in Dulbecco\u2019s modified Eagle\u2019s medium supplemented with L-glutamine and 10% (v/v) heat-inactivated TET-free fetal calf serum (Clontech ref. 631106) .HEK293 or HeLa cells were seeded in 96-well-plates (Thermo Scientific 165306) and grown to 60% confluency in normal growth medium. Cells were transfected with 40\u2009ng of the four different Donor D2 plasmids containing luciferase as the GOI (Donor D2.1-D2.4) or with the fully Cre-assembled (A-D1-D2) pTEMTAC composite plasmid (containing siRNA scramble) using lipofectamine 2000 . 24\u2009hours after transfection, cells were supplemented with indicated amounts of doxycycline (Sigma-Aldrich). 24\u2009hour later the luciferase assay was performed according to the manufactures instructions .HEK293 cells were seeded on 6 well plates and grown to 60% confluency in normal growth medium. Cells were transfected with 2.5\u2009\u03bcg of the fully Cre-assembled A-D1-D2 composite plasmid containing mCherry as the GOI (containing siRNA scramble) using lipofectamine 2000 (Invitrogen). 24\u2009hours after transfection, cells were supplemented with indicated amounts of doxycycline . 24\u2009h later YFP and mCherry fluorescence was imaged on an inverted DMI-6000 Leica wide-field fluorescent microscope equipped with a Leica DFC 350FX camera with a 20\u00d7 objective.GH-HEK293 cells were seeded on 6-cm dishes in normal growth medium, grown to 60% confluency, and transfected with 2.5\u2009\u03bcg of pTEMTAC (A-D1-D2) plasmid or, alternatively, pMDC-RNAiDual (shRNA against SHP2 or scramble shRNA) plasmid, using lipofectamine 2000 (Invitrogen) according to the manufacturer\u2019s instructions. 24\u2009hours after transfection, cells were supplemented with indicated amounts of doxycycline (Sigma-Aldrich) and incubated in minimal medium (DMEM plus 2\u2009mM glutamine and 1% serum). 24\u2009hours after transfection, cells were stimulated with 125\u2009ng/ml growth hormone for 10\u2009minutes, washed with PBS and resuspended in 200\u2009\u03bcl of lysis buffer . Lysed cells were analysed by western blot.HEK293 were seeded on 6-cm dishes in normal growth medium, grown to 60% confluency, and transfected with 2.5\u2009\u03bcg of pTEMTAC (containing BRAF/HRAS WT or V600E/G12V as GOI) or pMDC-RNAiDual (shRNA against BRAF/HRAS or scramble shRNA) plasmid, using lipofectamine 2000 (Invitrogen) according to the manufacturer\u2019s instructions. 48\u2009hours after transfection, cells were supplemented with indicated amounts of doxycycline (Sigma-Aldrich) and incubated in normal growth medium. 24\u2009hours after transfection, cells were washed with PBS and resuspended in 200\u2009\u03bcl of lysis buffer . Lysed cells were analysed by western blot.Cell lysates were loaded on SDS gels and separated by electrophoresis; gels were then transferred onto nitrocellulose membrane using the iBlot system (Invitrogen), and the blots were incubated 1\u2009h at room temperature in TBS, Tween 0.1%+5% milk. The primary antibody was incubated at 4\u2009\u00b0C overnight (1:1000 dilution), and the HRP-coupled secondary antibody (1:10 000 dilution) was incubated 1\u2009h at room temperature, both in TBS Tween 0.1%\u2009+\u20090.5% milk. Blots were developed using high sensitivity ECL reagent (Thermo) and visualized using the Fujifilm LAS-3000 developer. Bands were analyzed using ImageJ. The following antibodies were used for Western blotting: SHP2 , BRAF , total RAS , growth hormone receptor , phospho-ERK Thr202/Tyr204 , phospho-MEK Ser217 and Ser221 and \u03b2-actin .\u00ae Green RNA-to-CTTM 1-Step Kit, Applied Biosystems), 0.15\u2009\u03bcM of forward and reverse primers and 60\u2009ng of RNA following the manufactures instructions. For PCR the cycling parameters were (i) reverse transcription , (ii) activation of polymerase and initial denaturation , (iii) 40 cycles of denaturation , annealing, extension and read fluorescence , (iv) final hold (4\u2009\u00b0C). The following primers were used. For endogenous BRAF 5\u2032ATCCCAGAGTGCTGTGCTGT3\u2032 (forward) and 5\u2032TCTCCAACACTTCCACATGC3\u2032 (reverse), for exogenous BRAF 5\u2032AATGTTGCGCCGTTTACAG3\u2032 (forward) and 5\u2032TCTCCAACACTTCCACATGC3\u2032 (reverse), for endogenous HRAS 5\u2032TGCCATCAACAACACCAAGT3\u2032(forward) and 5\u2032ACGTCATCCGAGTCCTTCAC3\u2032 (reverse), for exogenous HRAS 5\u2032GAGGGCTTCCTGTGTGTCTT3\u2032 (forward) and 5\u2032TCTTTGACGCGCTTAATTTG3\u2032 (reverse), for endogenous SHP2 5\u2032CTTGTACTCCAACGCCACCC3\u2032 (forward) and 5\u2032CTGTGCTGAAGTTTTGGCAGG3\u2032 (reverse) and for total SHP2 5\u2032GGTGTGGAGGCAGAAAACCT3\u2032 (forward) and 5\u2032TTGATGTGGGTGACAGCTCC3\u2032(reverse).Cells were grown and transfected as before. RNA was isolated using miRNeasy Mini Kit (Qiagen) following the manufactures protocol including the optional DNase digestion step. PCR reactions were prepared using qPCR mix (Power SYBRTM II RT (Invitrogen) with random oligonucleotides. In the first PCR all target genes were amplified for 5 cycles using Phusion DNA polymerase (New England Biolabs) with the following degenerated adaptor primers: for BRAF (region of V600E)RNA was extracted as described for real time PCR. cDNA synthesis was done using SuperScript5\u2032CCCTACACGACGCTCTTCCGATCTCTTCATGAAGACCTCACAG3\u20325\u2032CCCTACACGACGCTCTTCCGATCTaCTTCATGAAGACCTCACAG3\u20325\u2032CCCTACACGACGCTCTTCCGATCTgaCTTCATGAAGACCTCACAG3\u20325\u2032CCCTACACGACGCTCTTCCGATCTcgtCTTCATGAAGACCTCACAG3\u20325\u2032CCCTACACGACGCTCTTCCGATCTagatCTTCATGAAGACCTCACAG3\u2032(forward) and5\u2032TTCAGACGTGTGCTCTTCCGATCTCTGTTCAAACTGATGGGACC3\u20325\u2032TTCAGACGTGTGCTCTTCCGATCTaCTGTTCAAACTGATGGGACC3\u20325\u2032TTCAGACGTGTGCTCTTCCGATCTgaCTGTTCAAACTGATGGGACC3\u20325\u2032TTCAGACGTGTGCTCTTCCGATCTcgtCTGTTCAAACTGATGGGACC3\u20325\u2032TTCAGACGTGTGCTCTTCCGATCTagatCTGTTCAAACTGATGGGACC3\u2032(reverse), for HRAS (region around G12V)5\u2032CCCTACACGACGCTCTTCCGATCTGACGGAATATAAGCTGGTGG3\u20325\u2032CCCTACACGACGCTCTTCCGATCTaGACGGAATATAAGCTGGTGG3\u20325\u2032CCCTACACGACGCTCTTCCGATCTcaGACGGAATATAAGCTGGTGG3\u20325\u2032CCCTACACGACGCTCTTCCGATCTgctGACGGAATATAAGCTGGTGG3\u20325\u2032CCCTACACGACGCTCTTCCGATCTatcaGACGGAATATAAGCTGGTGG3\u2032(forward) and5\u2032TTCAGACGTGTGCTCTTCCGATCTGTCGTATTCGTCCACAAAGTG3\u20325\u2032TTCAGACGTGTGCTCTTCCGATCTaGTCGTATTCGTCCACAAAGTG3\u20325\u2032TTCAGACGTGTGCTCTTCCGATCTcaGTCGTATTCGTCCACAAAGTG3\u20325\u2032TTCAGACGTGTGCTCTTCCGATCTatcGTCGTATTCGTCCACAAAGTG3\u20325\u2032TTCAGACGTGTGCTCTTCCGATCTatcaGTCGTATTCGTCCACAAAGTG3\u2032(reverse), for SHP2 (region around D61G)5\u2032CCCTACACGACGCTCTTCCGATCTAGTAAAAGTAACCCTGGAGAC3\u20325\u2032CCCTACACGACGCTCTTCCGATCTaAGTAAAAGTAACCCTGGAGAC3\u20325\u2032CCCTACACGACGCTCTTCCGATCTgaAGTAAAAGTAACCCTGGAGAC3\u20325\u2032CCCTACACGACGCTCTTCCGATCTtgaAGTAAAAGTAACCCTGGAGAC3\u20325\u2032CCCTACACGACGCTCTTCCGATCTctgaAGTAAAAGTAACCCTGGAGAC3\u2032(forward) and5\u2032TTCAGACGTGTGCTCTTCCGATCTGACCAACTCAGCCAAAGTG3\u20325\u2032TTCAGACGTGTGCTCTTCCGATCTaGACCAACTCAGCCAAAGTG3\u20325\u2032TTCAGACGTGTGCTCTTCCGATCTgaGACCAACTCAGCCAAAGTG3\u20325\u2032TTCAGACGTGTGCTCTTCCGATCTtgaGACCAACTCAGCCAAAGTG3\u20325\u2032TTCAGACGTGTGCTCTTCCGATCTctgaGACCAACTCAGCCAAAGTG3\u2032(reverse), and for SHP2 (region around C459G)5\u2032CCCTACACGACGCTCTTCCGATCTGAGGTGCACCATAAGCAGGAG3\u20325\u2032CCCTACACGACGCTCTTCCGATCTaGAGGTGCACCATAAGCAGGAG3\u20325\u2032CCCTACACGACGCTCTTCCGATCTgaGAGGTGCACCATAAGCAGGAG3\u20325\u2032CCCTACACGACGCTCTTCCGATCTtgaGAGGTGCACCATAAGCAGGAG3\u20325\u2019CCCTACACGACGCTCTTCCGATCTctgaGAGGTGCACCATAAGCAGGAG3\u2032(forward) and5\u2032TTCAGACGTGTGCTCTTCCGATCTTCACAATGAACGTCCCTGTC3\u20325\u2032TTCAGACGTGTGCTCTTCCGATCTaTCACAATGAACGTCCCTGTC3\u20325\u2032TTCAGACGTGTGCTCTTCCGATCTgaTCACAATGAACGTCCCTGTC3\u20325\u2032TTCAGACGTGTGCTCTTCCGATCTtgaTCACAATGAACGTCCCTGTC3\u20325\u2032TTCAGACGTGTGCTCTTCCGATCTctgaTCACAATGAACGTCCCTGTC3\u2032(reverse).The resulting amplicons were used for determination of cycle numbers for the second PCR with the TruSeq dual barcoding primers. The primary PCR product was amplified using NEBNext 2x High Fidelity PCR Master Mix (New England Biolabs), with 200nM primer concentration, and 0.1x SYBR Green I monitoring the amplification in a Roche LightCycler 480. The cycle number for the final library PCR was chosen to get a sufficient amount of product without reaching the plateau phase of the PCR. Then 2.5\u2009\u03bcl of the primary PCR product was used for amplification with the NEBNext Master Mix and barcode primers but without SYBR Green, for the number of cycles determined from the qPCR. PCR products were purified using AMPure XP beads and concentration was determined on a Bioanalyzer DNA 1000 chip. Samples were sequenced using MiSeq v3 chemistry with 130 cycle single reads in a MiSeq sequencer (Illumina). Flanking reads were subtracted by mutant reads to calculate the reads corresponding to the endogenous expression level.How to cite this article: Beltran-Sastre, V. et al. Tuneable endogenous mammalian target complementation via multiplexed plasmid-based recombineering. Sci. Rep.5, 17432; doi: 10.1038/srep17432 (2015)."} +{"text": "MAC are AIDS-causing, zoonotic lentiviruses that jumped to humans and rhesus macaques, respectively, from SIVSM-bearing sooty mangabey monkeys. Cross-species transmission events such as these sometimes necessitate virus adaptation to species-specific, host restriction factors such as TRIM5. Here, a new human restriction activity is described that blocks viruses of the SIVSM/SIVMAC/HIV-2 lineage. Human T, B, and myeloid cell lines, peripheral blood mononuclear cells and dendritic cells were 4 to >100-fold less transducible by VSV G-pseudotyped SIVMAC, HIV-2, or SIVSM than by HIV-1. In contrast, transduction of six epithelial cell lines was equivalent to that by HIV-1. Substitution of HIV-1 CA with the SIVMAC or HIV-2 CA was sufficient to reduce HIV-1 transduction to the level of the respective vectors. Among such CA chimeras there was a general trend such that CAs from epidemic HIV-2 Group A and B isolates were the most infectious on human T cells, CA from a 1\u00b0 sooty mangabey isolate was the least infectious, and non-epidemic HIV-2 Group D, E, F, and G CAs were in the middle. The CA-specific decrease in infectivity was observed with either HIV-1, HIV-2, ecotropic MLV, or ALV Env pseudotypes, indicating that it was independent of the virus entry pathway. As2O3, a drug that suppresses TRIM5-mediated restriction, increased human blood cell transduction by SIVMAC but not by HIV-1. Nonetheless, elimination of TRIM5 restriction activity did not rescue SIVMAC transduction. Also, in contrast to TRIM5-mediated restriction, the SIVMAC CA-specific block occurred after completion of reverse transcription and the formation of 2-LTR circles, but before establishment of the provirus. Transduction efficiency in heterokaryons generated by fusing epithelial cells with T cells resembled that in the T cells, indicative of a dominant-acting SIVMAC restriction activity in the latter. These results suggest that the nucleus of human blood cells possesses a restriction factor specific for the CA of HIV-2/SIVMAC/SIVSM and that cross-species transmission of SIVSM to human T cells necessitated adaptation of HIV-2 to this putative restriction factor.HIV-2 and SIV MAC/SIVSM lineage, with restriction being greatest for SIVSM and the least for epidemic HIV-2. Here we show that this dominant-acting, antiviral activity is specific for the capsid and blocks the virus after it enters the nucleus. The evidence suggests that, in order to jump from sooty mangabey monkeys to humans, the capsid of these viruses changed in order to adapt to this antiviral activity. In keeping with the practice concerning anti-lentiviral activities we propose to call this new antiviral activity Lv4.HIV-1 and HIV-2, the two lentiviruses that cause AIDS in humans, are members of a family of such viruses that infect African primates. HIV-1 is a zoonosis that was transmitted to humans from chimpanzees. HIV-2 was transmitted to humans from sooty mangabey monkeys. In several documented cases of cross-species transmission of lentiviruses it has been shown that replication of the virus in the new host species necessitated that the virus adapt to species-specific antiviral factors in the host. Here we report that human blood cells possess an antiviral activity that exhibits specificity for viruses of the HIV-2/SIV CPZ), and perhaps from gorillas (SIVGOR) [CPZ itself is probably a recombinant virus that resulted from co-infection of a chimp with viruses transmitted from a red-capped mangabey (SIVRCM) and a greater spot-nosed monkey (SIVGSN) [CPZ did not cause disease in chimpanzees but extensive observation of feral animals has demonstrated that this is not the case [Human immunodeficiency virus type 1 (HIV-1) is the major cause of the acquired immune deficiency syndrome (AIDS) pandemic. Among the immunodeficiency viruses that infect at least 40 of the primate species in sub-Saharan Africa, the simian immunodeficiency viruses (SIVs) found in central African chimpanzees and gorillas are monophyletic with HIV-1 . Each of(SIVGOR) \u20136. SIVCP(SIVGSN) . Until rthe case .Cercocebus atys) on multiple occasions [SM, but cross-species transmission to another non-native host, rhesus macaques (SIVMAC), resulted in AIDS [HIV-2, a second AIDS-causing virus that has highest prevalence in West Africa, was transmitted to people from sooty mangabey monkeys family can disrupt retroviral replication in a species-dependent manner [Though transmission of primate lentiviruses to humans has occurred on multiple occasions and may still be occurring , these et manner \u201318. TRIMt manner . Moreovet manner ,21 and it manner . TRIM5\u03b1 t manner and has t manner . Inhibitt manner \u201327. Addit manner \u201330. Finat manner ,30\u201332.MAC and SIV from African green monkeys (SIVAGM) [HIV-1 is inhibited by TRIM5\u03b1 from a number of African and Asian monkey species, such as rhesus macaques, African green monkeys, and sooty mangabeys ,33. The (SIVAGM) ,36\u201339.MAC replication are not inhibited by TRIM5\u03b1 in human cells. These experiments, however, all used immortalized adherent cell lines such as TE671 (rhabdomyosarcoma) [MAC or HIV-1 in a range of mammalian cell lines [MAC vectors in several human cell lines, e.g. Raji (B lymphocyte) and in the T lymphocyte cell lines Jurkat, HuT78 and CEM. This raised the possibility that lentiviruses could be inhibited in a cell-type specific fashion in human cells. In the work presented here, we investigated restriction to SIVMAC replication in peripheral blood lymphocytes (PBLs) as well as in various cell lines. Our data reveal a TRIM5\u03b1-independent restriction activity targeting SIVMAC, and the related SIVSM and HIV-2, in human blood cells.Thus, available data suggest that the early post-entry stages of SIVsarcoma) ,40,41, Hsarcoma) or HeLa sarcoma) ,31,43. Hll lines . They foNL4-3 and SIVMAC239, as previously described [nef was replaced with GFP coding sequence, such that the fluorescent reporter was expressed from the respective LTR. The two vectors were produced in parallel by collecting supernatant from transfected 293T cells. The vector-containing supernatants were checked for reverse transcriptase activity [Human cell lines were challenged with VSV G-pseudotyped, single-cycle vectors derived from HIV-1escribed . In eachactivity , normaliactivity , and theMAC transduction efficiency was 4 to 20-times less than that of HIV-1NL4-3 when the two vectors were used to challenge any of a panel of T cell lines, including Jurkat, SupT1, and CEM-SS cells, the Burkitt lymphoma-derived B cell line Raji, or the myelomonocytic cell lines U937 and THP-1 . Based on these parameters, the decrease in apparent infectivity of SIVMAC did not appear to be explained by poor expression of the GFP reporter from the SIV LTR. The latter point was demonstrated more conclusively by using 3-part lentiviral vectors in which the GFP reporter was expressed from the HIV-1 and SIVMAC vectors using an identical spleen focus-forming virus (SFFV) promoter were prepared, stimulated with PHA for three days, and challenged with the single-cycle vectors. SIVMAC transduction was less efficient than for HIV-1NL4-3 , had transduction activity more similar to that of SIVMAC239 is the retroviral determinant that confers sensitivity to several restriction factors, including Fv1 [MAC239 or HIV-2ROD. Neither of the two chimeras had transduction activity on CRFK cells or on HeLa cells.The experiments described above suggest that SIVding Fv1 , TRIM5 [ding Fv1 ,45, and ding Fv1 \u201357, the ROD and SIVMAC239 CA back to amino acid 202, using HIV-1 CA sequences to encode amino acids 203 to 230 . As with the 2-part vectors, no infectivity was observed unless CA amino acids 203\u2013230 were provided by HIV-1. Among the chimeras generated, representatives from Groups AB, A, D, E, F, and H, and from a primary SIVSM, were sufficiently infectious to evaluate CRFK-normalized transduction efficiency on Jurkat T cells. As a general trend, chimeras generated with CA from the epidemic Groups (A and B) were the most infectious on Jurkat T cells, those from the non-epidemic Groups were less infectious, and that from SIVSM was the least infectious bearing restriction-sensitive cores [MAC-specific restriction activity in Jurkat T cells with SIV VLPs were also unsuccessful.Given the results described above, evidence was sought that the cell type-specific defect in SIVve cores . Flat, eve cores ,60. Atte2O3 rescues retroviruses from CA-specific restriction by TRIM5 but has no effect on retrovirus transduction efficiency in the absence of TRIM5-mediated restriction [2O3 blocks TRIM5-mediated restriction is not known, though the effect results in increased reverse transcription and correlates with disruption of mitochondrial membrane potential [Astriction ,47,61,62otential ,61.MAC transduction of human blood cells might be restricted by TRIM5, or by a cellular factor with similar properties, the effect of As2O3 on SIVMAC transduction was assessed. As2O3 had no effect on the transduction efficiency of VSV G-pseudotyped, 2-part vectors for either SIVMAC239 or HIV-1NL4-3 in TE671 , and the other which is not restricted (B-MLV) . The throbserved . Also, aved + T cellswo weeks .+ T cells from one of two representative blood donors are shown in + T cells, was not sufficient to assess the efficiency of TRIM5 knockdown. Instead, a lentiviral vector derived from the equine infectious anemia virus (EIAV-GFP) was utilized [NL4-3 infectivity , or the isogenic vector bearing the SIVMAC239 CA (sCA-GFP), that were shown schematically in Human blood cells such as Jurkat T cells might be less permissive for SIVA flow cytometry-based assay was established that discriminates infected heterokaryons from those cells that fail to form heterokaryons . PrimaryAs a control, HeLa-RFP cells were engineered to express TvA (HeLa-RFP-TvA); in these cells, transduction with SIV CA-GFP was 1.6-fold higher than with HIV-1 CA-GFP . ChallenMAC239 transduced human blood cells less efficiently than did HIV-1. Lower SIVMAC239 transduction efficiency relative to HIV-1 was observed with all human blood-derived cells tested here, including cell lines of lymphoid and myeloid lineage, human PBMCs and primary CD4+ T cells, and, as previously described [MAC239 as by HIV-1.The characteristics of a previously unreported retroviral restriction activity in human blood cells are described here. The first clue to the existence of this restriction activity was that SIVescribed ,50,74, mMAC239-specific, restriction factor in human blood cells\u2014as opposed to the lack of a cofactor for SIVMAC239 replication in these cells\u2014was supported by the finding of a block to SIVMAC239 replication in Jurkat/HeLa-heterokaryons of equal magnitude to the block in Jurkat T cells. Similar heterokaryon experiments demonstrated the presence of a dominant restriction activity prior to the cloning of the retroviral restriction factors APOBEC3G, TRIM5, and TETHERIN [The presence of a dominant-acting, SIVTETHERIN ,43,75,76TETHERIN , and theTETHERIN .MAC239-specific restriction activity described here will be called Lv4. Whether this activity is due to a single factor, or due to a multi-factor complex, remains to be determined. Knockdown experiments presented here showed that Lv4 is distinct from TRIM5 , 731744GCTCAGCAAGCAGCAGCTGACACAGGAAACAACAGCCAGGTCAGCCAAAATTACCCAGTGCAACAAGTAGCTGGCAATTATGTCCATGTGCCGTTAAGTCCCCGAACCTTAAATGCCTGGGTAAAATTAGTGGAGGAAAAGAAGTTCGGGGCAGAAATAGTACCAGGATTTCAGGCACTATCAGAGGGATGTACCCCTTATGATATCAATCAAATGCTAAATTGTGTGGGAGAACACCAGGCAGCCATGCAAGTCATTAGAGAAATAATCAATGAAGAGGCGGCAGACTGGGACCAGCAACACCCGATACCAGGTCCACTGCCAGCAGGACAACTTAGAGACCCCAGAGGATCAGATATAGCGGGAACCACCAGCACAGTAGAGGAACAAATACAGTGGATGTACAGGGGTCAAAATTCCGTCCCAGTGGGGAACATTTATAGAAGATGGATTCAATTAGGATTGCAGAAATGTGTCAGGATGTACAATCCTACTAATATACTAGATGTAAAACAAGGGCCAAAAGAACCCTTCCAAAGCTATGTAGATAGATTCTACAAAAGCCTACGGGCAGAACAAGCAGACACAGCCGTGAGAGCATGGATGACAGAAACACTACTGGTCCAGAATGCTAACCCAGATTGCAAGCTAGTACTC>HIV-2(A), GH123TGTACAACAGACAGGCGGTGGCAACTATATCCACGTGCCACTGAGCCCCCGAACTCTAAATGCTTGGGTAAAATTAGTAGAGGACAAGAAGTTCGGGGCAGAAGTAGTGCCAGGATTTCAAGCACTCTCAGAAGGCTGCACGCCCTATGATATCAACCAAATGCTTAATTGTGTGGGCGATCACCAAGCAGCTATGCAAATAATCAGAGAGATTATCAATGACGAAGCAGCAGATTGGGATGCACAGCACCCAATACCAGGCCCCTTACCAGCAGGGCAGCTTAGAGACCCAAGGGGGTCTGACATAGCAGGAACAACTAGCACAGTAGAAGAACAGATCCAGTGGATGTATAGGCCACAAAATCCCGTGCCGGTAGGGAACATCTACAGAAGATGGATCCAGATAGGGCTACAGAAGTGTGTCAGGATGTACAACCCAACTAACATCTTAGACGTAAAGCAGGGACCAAAGGAACCGTTCCAGAGCTATGTGGACAGGTTCTATAAAAGCTTGAGGGCAGAACAAACAGATCCGGCAGTAAAGAACTGGATGACCCAAACGCTGCTAATACAGAATGCCAACCCAGACTGCAAGTTAGTACTA>HIV-2(D), L33083AGTGCAGCAAGTCGGCGGAAATTATGTCCACCTACCGCTGAGTCCCAGAACATTAAATGCATGGGTTAAGTTAGTGGAGGACAAAAAATTCGGGGCAGAGGTAGTGCCAGGGTTTCAGGCACTATCGGAAGGCTGCACTCCGTATGACATCAATCAGATGCTAAATTGTGTAGGAGAACATCAGGCAGCCATGCAGATCATAAGGGAAATAATCAATGATGAGGCAGCAGATTGGGATCAGCAGCATCCACAACCAGGCCCACTACCAGCAGGACAGCTCAGAGATCCACGAGGATCTGATATAGCAGGAACCACTAGCACAGTGGAGGAACAAATACAGTGGATGTACAGGCAGCAGAATCCCATACCAGTTGGAAATATCTATAGGAGATGGATCCAGCTAGGGTTACAGAAATGTGTCAGAATGTACAACCCAACTAACATTCTGGATATAAAACAAGGGCCAAAAGAGACGTTCCAGAGCTATGTAGATAGATTCTACAAAAGCTTGAGGGCAGAACAAACAGACCCAGCAGTGAAAAATTGGATGACACAAACACTGCTGATTCAGAATGCTAACCCAGATTGCAAGTTAGTACTA>HIV-2(E), L33087AGTGCAACAGATAGGAAATAACTATGTGCACTCTCCACTGTCCCCAAGAACATTGAATGCATGGGTCAAATTAGTAGAAGAAAAGAAATTTGGAGCAGAGGTAGTGCCAGGCTTCCAGGCATTATCAGAAGGATGCACCCCGTATGACATCAACCAGATGCTTAATTGCGTGGGGGAACATCAGGCAGCCATGCAAATTATCAGAGAGATAATCAATGAAGAAGCAGCAGATTGGGACGTACAGCATCCAAGAGGGCAACCGCCAGCACAGGGCCTAAGAGACCCATCAGGATCAGACATAGCAGGGACAACCAGTACCCCCGCAGAACAAATAGAGTGGATGTACAGGAATCCAAATCCAATCCCTGTGGGAGACATCTATAGAAGATGGATCCAGCTAGGGCTCCAGAAATGTGTCAGAATGTATAATCCAACAAACATTCTGGACGTCAAACAGGGGCCCAAAGAATCTTTTCAGAGCTATGTAGATAGATTCTACAAAAGCTTGAGGGCAGAACAAACAGACCCAGCAGTGAAAAATTGGATGACACAAACACTGCTGATTCAGAATGCTAACCCAGATTGCAAGTTAGTACTA>HIV-2(F), U75441AGTGCAGCAGGTAGGAGGAAATTACACCCATATTCCTCTGAGTCCGAGGACATTAAATGCTTGGGTTAAATTAGTAGAGGAAAAGAAATTTGGGGCAGAAATAGTGCCAGGCTTCCAAGCATTGTCAGAAGGCTGCACCCCTTATGATATTAATCAAATGTTAAATTGTGTAGGGGAACATCAGGCAGCCATGCAAATAATCAGGGAAATAATCAATGAAGAAGCAGCCGACTGGGATCAGAATCATCCAAGGCAGCTGCCAGCGCCACCAGGGCTGCGTGATCCGTCAGGATCTGACATTGCAGGAACAACTAGTACAGTACAAGAACAGATAGAATGGATGTACAGACAGGGTAACTCAATCCCAGTAGGGGACATTTACAGAAGATGGATCCAAATAGGCCTTCAAAAATGTGTAAGAATGTACAATCCTACTAATATCCTAGATGTAAAACAGGGACCAAAAGAACCATTTCAAAGCTATGTAGATAGATTCTACAAAAGCTTGAGGGCAGAACAAACAGACCCAGCAGTGAAAAATTGGATGACACAAACACTGCTGATTCAGAATGCTAACCCAGATTGCAAGTTAGTACTA>HIV-2(H), AY5308GGTGCAGCAGATAGGTGGCAATTATGCCCACCTACCTCTAAGTCCTAGAACACTCAATGCCTGGGTAAAACTGGTAGAGGAGAAAAAATTTGGAGCAGAAGTAGTGCCAGGATTTCAGGCACTCTCAGAGGGCTGCACGCCCTATGATATTAATCAAATGTTAAATTGCGTGGGAGAACATCAAGCTGCTATGCAAATTATCAGGGAAATAATTAATGATGAAGCAGCAGATTGGGACACACAGCACCCAAACCAAGGCCCACCACCAGCAGGGCAACTTAGAGAGCCAAGAGGTTCTGATATTGCAGGAACAACTAGCACAGTGGAAGAGCAGATACAGTGGATGTACAGGCCGCAAAATCCAATACCGGTGGGTAACATCTATCGGAGATGGATCCAATTGGGCCTACAAAAATGTGTTAGAATGTACAATCCAACTAATATCTTAGATATAAAGCAAGGGCCAAAGGAGCCATTTCAAAGTTATGTAGATAGATTCTACAAAAGTTTGAGAGCAGAACAAACAGATCCAGCAGTGAAAAATTGGATGACTCAGACGCTGCTGATTCAGAATGCTAACCCAGACTGCAAACTCGTGTTA>SIVSME041, HM059825AGTGCAGCAAGTAGGTGGCAATTATACCCACCTACCCTTAAGTCCAAGAACATTAAATGCTTGGGTAAAATTGATAGAAGAGAAAAAATTTGGGGCAGAAGTAGTGCCAGGATTCCAAGCACTATCAGAAGGCTGCACTCCCTATGACATCAATCAGATGCTAAATTGTGTAGGGGAGCATCAATCAGCCATGCAAATTATTAGAGAAATTATAAATGAAGAAGCTGCTGATTGGGATTTACAACACCCACAGCCAGGTCCAATACCAGCAGGACAACTTAGAGACCCGAGAGGATCAGACATTGCAGGAACTACTAGCACAGTAGAAGAACAAATTCAATGGATGTATAGGCAGCAAAACCCTATACCAGTAGGTAACATTTACAGAAGGTGGATCCAATTAGGGCTGCAAAAATGTGTAAGGATGTATAATCCAACAAACATTTTAGATGTGAAACAAGGACCAAAAGAGCCATTTCAAAGCTATGTAGATAGATTCTACAAGAGTCTAAGAGCAGAACAAACAGACCCAGCAGTGAAAAATTGGATGACTCAAACACTGCTGATTCAAAATGCTAACCCAGATTGCAAATTGGTGCTCgag-pol plasmid and pONY8.0 is an EIAV GFP-packaging vector [pMD2.G encodes the vesicular stomatitis virus glycoprotein (VSV G) and psPAX2 encodes HIV-1 Gag and Gag-Pol . pCIG3N g vector .env glycoprotein for virion pseudotyping was expressed from pAB6 [pAPM is a lentiviral vector expressing puromycin-resistance and a miR30-based knockdown cassette from the spleen focus forming virus LTR ,63,64. Trom pAB6 . Far redrom pAB6 was clonCell lines were either grown in DMEM or RPMI , supplemented with 10% fetal calf serum as described before ,104,105.PBMC were separated by Ficoll density centrifugation, stimulated with PHA for 3 days, and cultured in RPMI supplemented with antibiotics, 10% fetal bovine serum, and 20 IU/ml hIL-2 ,106.+ T lymphocytes were enriched from PBMC by positive selection using magnetic beads (Miltenyi Biotec). Typically the resulting population was >99% CD4+. Cells were stimulated for 24 hrs on NUNC maxisorp plates that had been coated with 2 \u03bcg/ml anti-CD3 antibody and 2 \u03bcg/ml anti-CD28 antibody (BD Biosciences) in RPMI with 10% FBS, glutamax (Invitrogen), and 20 IU/ml hIL-2. Two wks after primary stimulation, cells were re-stimulated using plate-bound anti-CD3 and anti-CD28 antibodies.CD4MAC239, and the CA chimera vectors described above, were prepared by co-transfection of the indicated plasmids with pMD2.G in 293T cells, as described [6 cells were plated per 10-cm plate. The next day, cells were transfected using Lipofectamine 2000 (Invitrogen) and 20 \u03bcg of pAPM, 15 \u03bcg of psPAX2 and 5 \u03bcg of pMD2.G. Supernatant was collected and passed through a 0.45 \u03bcM filter at 48 hrs and at 72 hrs post-transfection, and used immediately to transduce target cells.VSV G-pseudotyped viral stocks of HIV-1, SIVescribed . Virion escribed and by tescribed . For pro4 of the indicated target cells, in 0.4 ml media per well, in 24-well plates. As2O3 (Sigma) was prepared as described [Reporter virus-containing supernatant was titrated onto 4 x 10escribed and, wheescribed .+ T cells were spinfected with shRNA-encoding APM vectors twice, at 24 hr and 48 hr after stimulation with plate-bound anti-CD3 and anti-CD28 antibodies. Spinfection was done at 1,130 rcf for 90 mins, using 2 ml of freshly produced virus supernatant for each well of a 6-well plate containing 5 x 105 stimulated lymphocytes. Cells were put in 5 \u03bcg/ml of puromycin for 72 hrs, 2 days after the first spinfection.Jurkat cells or primary CD44)2SO4, 20 mM KCl, 5 mM MgCl2, 0.1 mg/ml BSA, 1/20,000 SYBR Green I (Sigma), and 200 \u03bcM dNTPs. All reactions and quantitation of product were carried out with a Biorad CFX96 cycler. The RT step was 42\u00b0C for 20 min, and the PCR was programmed for 40 cycles of denaturation at 95\u00b0C for 5 s, annealing 55\u00b0C for 5 s, extension at 72\u00b0C for 20 s and acquisition at 80\u00b0C for 5 s. A standard curve was obtained using known concentrations of recombinant HIV-1 RT (Ambion).Virus-containing supernatant was harvested 48 hr post-transfection, clarified by low-speed centrifugation, and filtered through 0.45 \u03bcm pore filters (Sarstedt). Reverse transcriptase (RT) activity in the supernatant was quantified using a modified Sybr green I-based, real-time PCR, enhanced RT assay ,109. VirCell-free virions were normalized by RT-activity and incubated with CRFK, Hela or Jurkat cells in 6-well plates for 12 hrs, for full-length linear cDNA and 2-LTR circles, or 48 hrs, for Alu PCR. For each virus and cell type, infections were also performed in the presence of 40 \u03bcM AZT, to control for contamination of plasmid DNA in the PCR reaction. Cells were harvested and washed extensively with PBS. Total DNA was extracted , quantified, and subjected to real-time PCR with a Biorad CFX96 cycler.4)2SO4, 20 mM KCl, 5 mM MgCl2, 0.1 mg/ml BSA, 1/20,000 SYBR Green I (Sigma), and 200 \u03bcM dNTPs. The PCR was programmed for 40 cycles of denaturation at 95\u00b0C for 5 s, annealing 55\u00b0C for 5 s, extension at 72\u00b0C for 20 s and acquisition at 80\u00b0C for 5 s. Provirus was quantified by Taqman-based ALU-PCR according to the protocol described by Butler et al. [Full-length linear retroviral cDNA and 2-LTR circles were detected with SYBR-Green I based reactions using 100 ng template DNA and 320 nM of each primer pair in 20 mM Tris-Cl pH 8.3, 5 mM , at 37\u00b0C. Cells were incubated for another 2 mins and then 2 ml of serum-free DMEM was added slowly over a period of 4 minutes at 37\u00b0C with constant, gentle agitation. An additional 5 ml of serum-free DMEM was added and cells were incubated for 5 min at 37\u00b0C. Cells were then pelleted and resuspended in complete medium before seeding in 24-well plates. 6 hours later, cells were challenged with ALV-A Env-pseudotyped vectors. A negative fusion control sample was also produced with no PEG addition. Infected cell cultures were analyzed using a FACS-Canto (BD) 48 hrs after vector challenge. Fluorescence acquisition was performed using blue (488 nm) and red (633 nm) lasers. Dead cells were excluded from the analysis based on propidium iodide staining.2 x 10http://grants.nih.gov/grants/policy/hs/faqs_aps_definitions.htm.Human peripheral blood mononuclear cells (PBMC) were obtained from anonymous, untraceable blood donors. This research is therefore considered non-human subjects research by our Institutional Review Board, based on NIH guidelines (45 CFR 46.102(f)):"} +{"text": "TRHR) gene, rs1800795 in the interleukin-6 (IL6) gene and rs6552828 in the coenzyme A synthetase long-chain 1 (ACSL1) gene. To gain insight into their functionality (which is yet unknown), here we determined for the first time luciferase gene reporter activity at the muscle tissue level in rs7832552 and rs6552828. We then compared allele/genotype frequencies of the 3 abovementioned variants among centenarians and healthy controls of the same ethnic and geographic origin (Spain). We also studied healthy centenarians and controls from Italy, and centenarians and healthy controls from Japan. The THRH rs7832552 T-allele and ACSL1 rs6552828 A-allele up-regulated luciferase activity compared to the C and G-allele, respectively (P = 0.001). Yet we found no significant association of EL with rs7832552, rs1800795 or rs6552828 in any of the 3 cohorts. Further research is needed with larger cohorts of centenarians of different origin as well as with younger old people.There are several gene variants that are candidates to influence functional capacity in long-lived individuals. As such, their potential association with exceptional longevity deserves analysis. Among them are rs7832552 in the thyrotropin-releasing hormone receptor ( The oldest old population (\u226585 years) is rapidly expanding among westerners Waite, . HoweverMSTN) gene gene), might also influence the likelihood of reaching EL gene, rs16892496 and rs7832552 in the interleukin-6 (IL6) gene, where the G-allele is associated with higher transcription in vitro analysis of 379,319 SNPs in US Caucasians of both genders (aged 50 years on average) revealed an association of lean body mass with 2 SNPs in tight linkage disequilibrium within the thyrotropin-releasing hormone receptor (2peak)], a recent GWAS study in sedentary Caucasians found that, among 324,611 SNPs, the strongest association with the VO2peak response to exercise was found to acyl coenzyme A synthetase long-chain 1 (ACSL1) gene polymorphism rs6552828 \u2014among Spanish centenarians (cases) and healthy controls matched by ethnic and geographic origin and also in 2 other geographically and ethnically-independent replication cohorts .In order to analyze their functionality at the muscle level, we measured for the first time luciferase gene reporter activity in MluI/NheI in the 5\u2032 and XhoI in the 3\u2032 (see below -in bold) of the sequences obtained from the genomes of:A-allele and one individual homozygous for the rs16892496 C-allele (see below -underlined)one individual homozygous for the rs16892496 AArs16892496 AGTGGGATACAAGTCACTCTCAGGCTTGAAAAATGAGTAGGCATTCACTAGGCCAACATAAAATACAAGAAGACCCTCCAGTCTGCAGAAGTAGTCAATGACTCGAGGCTAGCTATGAAAGATCTACGTTAAAACATAAGGTTAAGCTGTGCAGTGTACAGAAGAGACAAGAAAGTGGTACTTACTGTGCATAAGGTTGAAGAGCAAGCCCCCCCrs16892496 CGTGGGATACAAGTCACTCTCAGGCTTGAAAAATGAGTAGGCATTCACTAGGCCAACATAAAATACAAGAAGACCCTCCAGTCTGCAGAAGTAGTCAATGACTCGAGGCTAGCTATGAAAGATCTACGTTAAAACATAAGGTTAAGCTGTGCAGTGTACAGAAGAGACAAGAAAGTGGTACTTACTGTGCATAAGGTTGAAGAGCAAGCCCCCC-allele and one homozygous for the rs7832552 T-allele (see below -underlined)one individual homozygous for the rs7832552 CCrs7832552-CGAATGCTCTTAATAAACAGGATCGATCAAGGGTGCTTGACTCTTGTTGTTCATGTGCAAGTATAGTGGCTTTTTTGTGCCTCAACAAAACCATCAAGAGTCTCGAGGAGCTCATTAGCCTTGTGACAAAAGCAACGCACTCCATTTTGCACACAGTACTTGACTTTATTTTGCTACTGCCTTGACCTCAAAGGAATGTGATAGTGTGAGGTATTrs7832552-TGAATGCTCTTAATAAACAGGATCGATCAAGGGTGCTTGACTCTTGTTGTTCATGTGCAAGTATAGTGGCTTTTTTGTGCCTCAACAAAACCATCAAGAGTCTCGAGGAGCTCATTAGCCTTGTGACAAAAGCAACGCACTCCATTTTGCACACAGTACTTGACTTTATTTTGCTACTGCCTTGACCTCAAAGGAATGTGATAGTGTGAGGTAA-allele and one individual homozygous for the rs6552828 G-allele (see below -underlined)one individual homozygous for the rs6552828 rs6552828-AAAAGAAAGTACAGAATAGTATTTGAGATCCTAGATGCAGCCGGACGCGGTGGCTCATGCCTGTAATCCCAGCACTTTGGGAAGCCGAGGCGGGTGGATCACCCTCGAGGAGCTCCAAGACATTATAGCCAAAAGAAACAAACAGATAAATTGGTGTGCATAAACTTTAAACCAACCACCAGATATCTAAAGAGGGAATACAGCACAGTGTTGGArs6552828-GGGAGAAAGTACAGAATAGTATTTGAGATCCTAGATGCAGCCGGACGCGGTGGCTCATGCCTGTAATCCCAGCACTTTGGGAAGCCGAGGCGGGTGGATCACCCTCGAGGAGCTCCAAGACATTATAGCCAAAAGAAACAAACAGATAAATTGGTGTGCATAAACTTTAAACCAACCACCAGATATCTAAAGAGGGAATACAGCACAGTGTTGGAThe fragment, including the allele, was directly inserted into the pGL3-promoter at the restriction recognition sites We used mice skeletal muscle C2C12 cell lines to study muscle-specific expression. We performed cell cultures, transfections and dual-luciferase reporter assays following the procedures previously reported by our group , University of Pavia , and National Institute of Health and Nutrition, (Japan) Medical Research Institute and Keio University (Japan)] and the study followed the tenets of the Declaration of Helsinki for Human Research. Written consent was also obtained from each participant.Two groups of Spanish subjects were assessed: (i) 138 cases ; and (ii) 334 healthy controls . All the subjects were of the same Caucasian (Spanish) descent for \u22653 generations. The major diseases among the centenarians were osteoarthritis (66%), hypertension (57%), dementia (51%) and cardiovascular disease . The DNA of a convenience sample of 355 younger disease-free controls with no reported family history of high longevity (>90 years) was collected during 2008-2012 in the European University of Madrid.Two groups of subjects from Northern Italy (mainly from Lombardy and Piedmont) were studied: (i) 79 cases ; and (ii) 316 healthy controls . All patients and controls were Caucasian whites of Italian descent for \u22653 generations The Italian centenarians were free of major age-related diseases, i.e., severe cognitive impairment, clinically evident cancer, CVD, renal insufficiency or severe physical impairment descent were assessed: (i) 742 cases ; and (ii) 499 healthy controls . The group of cases was gathered from 2 cohorts, which are described in detail elsewhere because the genotype distributions of both SNPs are completely linked according to available HapMap for both European and Asian populations (sorted as a Supplementary file 1) and previous research has shown that both SNPs are in strong linkage disequilibrium (r2 = 0.98). All genotyping was performed only for research purposes with the researchers who performed the genotyping being blinded to the participants' identities. For quality control, a random ~20% of the samples of each cohort were genotyped again, with no differences in the results compared with the initial genotyping.As mentioned above, only one DNA was extracted from the participants' buccal cells using a standard phenol chloroform protocol and the genotype analyses were performed in the Biomedicine laboratory at the European University, Madrid (Spain). The DNA samples were diluted with sterile water and stored at \u221220\u00b0C until analysis. Genotyping was performed by Real-Time PCR and using the TaqMan\u00ae rs7832552, rs6552828, and rs1800795 SNP genotyping assays with a Step One Real-Time PCR System .Genomic DNA was purified from blood leukocytes using the QiaAmp DNA Mini kit according to the manufacturer's protocol. Genotyping was performed at the Cellular Pathophysiology and Clinical Immunology Laboratory (University of Pavia) using the TaqMan\u00ae rs7832552, rs6552828, and rs1800795 SNP genotyping assays .Total genomic DNA was extracted from blood leukocytes with a QIAamp DNA Blood Mini or Maxi Kit . Genotyping of rs7832552, rs6552828, and rs1800795 was performed at the Institute of Health and Sports Science and Medicine (Juntendo University) with Real Time Thermocycler using TaqMan SNP genotyping assay method. PCR 384-well plates were read on LightCycler 480 using the end-point analysis mode. Allelic discrimination analysis was performed with a LightCycler 480 SW software version 1.5.1.62 .2-test. Genotype/allele frequencies of cases vs. controls within each cohort were compared using the \u03c72-test with Yates' correction and the association between genotypes/alleles and EL within each of the 3 cohorts was analyzed with logistic regression analysis after adjusting for sex. All statistical analyses were performed using the PASW and corrected for multiple comparisons using the Bonferroni's method -that is, the threshold P-value was obtained by dividing 0.05 by the number of studied polymorphisms (P = 0.05/3 = 0.017).One-way analysis of variance was used to compare the relative luciferase activity in the different plasmids of each SNP. Allele frequencies were calculated by gene-counting. We tested Hardy-Weinberg equilibrium (HWE) using \u03c7P \u2264 0.001); the THRH rs16892496 A-allele up-regulated luciferase activity compared to the C-allele (upper panel), the THRH rs7832552 T-allele up-regulated luciferase activity compared to the C-allele (middle panel), and the ACSL1 rs6552828 A-allele up-regulated luciferase activity compared to the G-allele (lower panel).The results of luciferase report analyses are presented in Figure THRH rs7832552, 97.2% in cases and 100% in controls; ACSL rs6552828, 97.2% in cases and 99.1% in controls; IL6 rs1800795, 100% in cases and 94.9% in controls. The distribution of all genotypes was consistent with the HWE in both groups (P > 0.05), except for IL6 rs1800795 in the control group (P < 0.01).Rate of genotyping success was as follows: 2 = 1.21, P = 0.27) or genotype frequency distributions of THRH rs7832552 did not differ between cases and controls . Using logistic regression analysis, no significant associations were found between EL and rs7832552, including when analyzing both sexes separately (data not shown). No differences were found for IL6 rs1800795 in allele or genotype distributions , with no significant association with EL after adjusting for sex or when analyzing both sexes separately -data not shown) or for ACSL1 rs6552828 .The results of genotype/allele frequency distributions as well as of binary logistic regression adjusted by sex are shown in Table P > 0.05), except for THRH rs7832552 in the control group (P = 0.04).Rate of genotyping success was 100% for all gene variants. The distribution of all genotypes was consistent with the HWE in both groups and genotype frequency distributions of THRH rs7832552 did not differ between groups and no significant association was found between this polymorphism and EL using logistic regression adjusted by sex, or when analyzing both sexes separately (data not shown). Similar results were found for IL6 rs1800795 and ACSL1 rs6552828 , with no significant association between these two polymorphisms and EL using logistic regression adjusted by sex, or when analyzing both sexes separately (data not shown).The results of genotype/allele frequency distributions as well as of binary logistic regression adjusted by sex are shown in Table THRH rs7832552, 97.0% in cases and 100% in controls; IL6 rs1800795, 98.7% in cases and 100% in controls; ACSL1 rs6552828, 95.9% in cases and 99.2% in controls. The distribution of all genotypes was consistent with the HWE in both groups (P > 0.05), except for rs6552828 in centenarians (P = 0.02).Rate of genotyping success was as follows: THRH rs7832552 , rs1800795 , or rs6552828 , with no significant association between any of the SNPs and EL using logistic regression adjusted by sex or when analyzing both sexes separately (data not shown).The results of genotype/allele frequency distributions as well as of binary logistic regression adjusted by sex are shown in Table in vitro approach) the potential functional consequences of the rs16892496, rs7832552, and rs6552828 SNPs, with the A-allele, T-allele and A-allele up-regulating luciferase activity compared to the other alleles, respectively. The THRH rs7832552 and ACSL1 rs6552828 SNPs are intronic genomic variants and, as such, could potentially alter the stability and/or alternative splicing of mRNA, as well as transcription factor binding might favor EL. Yet a GWAS study reported that the TRHR rs7832552 SNP was associated with lean body mass in US Caucasians genotype had 2.55 kg higher lean body mass compared to the other subjects. There is some scientific rationale in postulating that higher TRHR expression might help preservation of muscle mass in long-lived individuals: TRHR stimulates the hypothalamic-pituitary-thyroid axis, thereby leading to the release of thyroxin, a hormone that plays an important role in the development of skeletal muscle as well as in attenuating age-related changes in tissue function , we used convenience samples, which increases the risk of bias induced by population stratification. The SNPs rs1800795 and rs7832552 did not meet HWE in Spanish and Italian controls, respectively. In this regard, deviation from HWE does not necessarily reflect genotyping errors none of them was associated with EL. Similarly, no association was found for rs1800795. More research is needed in the field with other cohorts, using larger population samples, as well as younger elderly to assess the potential link between these genetic variants and the human aging process.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Protein synthesis is tightly regulated and alterations to translation are characteristic of many cancers. Translation regulation is largely exerted at initiation through the eukaryotic translation initiation factor 4\u00a0F (eIF4F). eIF4F is pivotal for oncogenic signaling as it integrates mitogenic signals to amplify production of pro-growth and pro-survival factors. Convergence of these signals on eIF4F positions this factor as a gatekeeper of malignant fate. While the oncogenic properties of eIF4F have been characterized, genome-wide evaluation of eIF4F translational output is incomplete yet critical for developing novel translation-targeted therapies.To understand the impact of eIF4F on malignancy, we utilized a genome-wide ribosome profiling approach to identify eIF4F-driven mRNAs in MDA-MB-231 breast cancer cells. Using Silvestrol, a selective eIF4A inhibitor, we identify 284 genes that rely on eIF4A for efficient translation. Our screen confirmed several known eIF4F-dependent genes and identified many unrecognized targets of translation regulation. We show that 5\u2032UTR complexity determines Silvestrol-sensitivity and altering 5\u2032UTR structure modifies translational output. We highlight physiological implications of eIF4A inhibition, providing mechanistic insight into eIF4F pro-oncogenic activity.Here we describe the transcriptome-wide consequence of eIF4A inhibition in malignant cells, define mRNA features that confer eIF4A dependence, and provide genetic support for Silvestrol\u2019s anti-oncogenic properties. Importantly, our results show that eIF4A inhibition alters translation of an mRNA subset distinct from those affected by mTOR-mediated eIF4E inhibition. These results have significant implications for therapeutically targeting translation and underscore a dynamic role for eIF4F in remodeling the proteome toward malignancy.The online version of this article (doi:10.1186/s13059-014-0476-1) contains supplementary material, which is available to authorized users. Energetically, protein synthesis is the most costly step on the path toward gene expression and is thus a rigidly controlled process. In eukaryotes, protein synthesis occurs in three phases: translation initiation, elongation and termination. Although translation is controlled at multiple stages, regulation is primarily exerted at initiation, the phase in which 80S ribosomes assemble onto mRNA transcripts. Regulation of initiation is mediated by multiple factors, many of which converge on the assembly of the eukaryotic initiation factor 4F (eIF4F). This heterotrimeric complex is composed of eIF4E, the rate-limiting protein which binds the 5\u2032-7-methylguanosine cap on cellular mRNA transcripts; eIF4A, a DEAD-box RNA helicase; and eIF4G, a scaffolding protein which bridges eIF4E and eIF4A, and recruits eIF3 and the 43S pre-initiation complex. Formation of eIF4F is tightly controlled by multiple mitogenic signaling pathways, namely mitogen-activated protein kinase (MAPK) and phosphoinositide-3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR), and has been shown to stimulate translation of mRNAs involved in cell proliferation, growth, survival, cell cycle progression, and DNA damage repair \u20133. Moreoin vitro [Significant progress has been made toward understanding the machinery that drives protein synthesis. However, the underlying mechanisms by which individual eIF4F components contribute to translation regulation in the cell remain ambiguous. Emerging methods that allow for global dissection of translation have bolstered the long standing knowledge that translation is subject to considerable regulation and thus plays a key role in regulating gene expression \u201313. Studin vitro . These din vitro and in vivo, thereby impairing translation initiation [eIF4A is an integral part of the heterotrimeric eIF4F complex and the only component with known enzymatic activity. While several eIF4A-regulated genes have been identified, in-depth studies have yet to provide a genome-wide description of the eIF4A target gene landscape. We sought to comprehensively define the cellular mRNAs regulated by eIF4A and investigate the mRNA features that confer dependence on this helicase by directly blocking its activity. To achieve this, we employed the potent and specific eIF4A inhibitor Silvestrol. This compound has been shown to selectively target the RNA helicase activity of eIF4A both itiation ,22, and itiation ,24. Moreitiation . Here we35S-methionine incorporation into newly synthesized proteins. To mitigate off-target or secondary effects of drug treatment, we chose to treat cells with a low concentration of Silvestrol for a short time period. We selected a concentration of 25 nM Silvestrol, which had negligible effects on global translation after 2\u00a0hours of exposure and a difference in distribution of \u2206G values from genes with increased TE was also notable compared with the insensitive pool (P =4.25\u2009\u00d7\u200910-7) while those from genes with increased TE were comparatively shorter and slightly higher in those from genes with increased TE (P =8.08\u2009\u00d7\u200910-6) and ROCK1 , were significantly more sensitive to Silvestrol than unstructured controls, CMV alone and PFN2 . The introduction of ARF6mut into our luc2CP reporter decreased the sensitivity of our reporter construct to Silvestrol analysis of Silvestrol-sensitive genes revealed that a large proportion of eIF4A-dependent genes were involved in cell cycle progression 4 motif is present in both the ARF6 and ARF6mut 5\u2032 UTR reporter constructs .Cells were seeded at a density of 1\u2009\u00d7\u2009105 cells/well in 6-well format and grown overnight. 24\u00a0hours after seeding, cells were washed in 1\u00d7 phosphate-buffered saline for 10\u00a0minutes prior to harvest. Cells were lysed in RIPA buffer containing complete EDTA-free protease inhibitors , and phosphatase inhibitors . Total protein was trichloroacetic acid and used to normalize radioactive counts in TCA precipitated protein.MDA-MB-231 cells were seeded at a density of 3\u2009\u00d7\u2009106 cells/plate in 15\u00a0cm plates and grown overnight. Cells were then treated with DMSO or Silvestrol (25 nM) for 2\u00a0hours prior to harvest. At time of harvest, 100\u00a0\u03bcg/ml cycloheximide was added to each plate and incubated for 1\u00a0minute. Cells were washed twice in ice-cold 1\u00d7 PBS +100\u00a0\u03bcg/ml cycloheximide and lysed in ice-cold polysome lysis buffer , 1% TritonX-100 ). Sucrose gradients (10 to 50%) were created by layering 10% and 50% sucrose solutions , 10 or 50% RNase-free sucrose) into Seton tubes followed by mixing with the BioComp Gradient maker set at an 81.5\u00b0 angle at 16\u00a0rpm . Lysates were layered on top of each gradient and subjected to ultracentrifugation in an SW41-Ti rotor at 35,000\u00a0rpm at 4\u00b0C for 3\u00a0hours. Polysome profiles were analyzed using a BioComp Gradient Station at constant speed with optical monitoring at a 260\u00a0nm wavelength. For polysome fractionation experiments, 1\u00a0ml fractions were collected and stored at -80\u00b0C for subsequent RNA isolation followed by quantitative RT-PCR.Cells were seeded at a density of 4\u2009\u00d7\u2009102, 1\u00a0mM DTT, 8% glycerol, 100\u00a0\u03bcg/ml cycloheximide (Sigma), 24 U/ml Turbo DNAse (Ambion)). Lysates were clarified, treated with RNAse I (Ambion) and overlaid on a 34% sucrose cushion. Monosomes were isolated by centrifugation at 69,000\u00a0rpm for 4\u00a0hours in a TLA110 rotor. Ribosome protected RNA fragments (RFs) were isolated from monosome fractions by acidic-phenol extraction and used to generate libraries for sequencing. The genome-aligned RF profiles in Additional file Ribosome profiling was performed as previously described ,41 with Total RNA was extracted from MDA-MB-231 cells using Trizol following the manufacturer\u2019s guidelines. Polyadenylated RNA was isolated from the total fraction using Oligotex mRNA kit . The resulting mRNA was partially fragmented by alkaline hydrolysis with sodium carbonate to generate approximately 150-nucleotide fragments on average. RNA fragments of 40 to 100 nucleotides were isolated by gel extraction and used to generate libraries for mRNA-Seq. The genome-aligned mRNA profiles in Additional file et al. [Strand-specific libraries were generated as described ,41 with et al. . SamplesSequence analysis was performed as described . Briefly5\u2032 UTR sequences were retrieved from Ensemble using RefSeq mRNA numbers as query. Folding energies of the resulting sequences were analyzed using CONTRAfold and McCaCyclinD1 and ROCK1 were reverse transcribed from total RNA isolated from MDA-MB-231 cells and the resulting cDNA was amplified by high-fidelity PCR using 5\u2032 UTR-specific oligonucleotides. The 5\u2032 UTRs for ARF6, ARF6mut and PFN2 were synthesized by Blue Heron Biotechnology . 5\u2032 UTRs were subcloned into the NcoI site of vector pMH2 to create vectors pCR300, pCR301, pCR302, pCR303, and pCR304 . Luciferase assays were performed by adding 100\u00a0\u03bcl/well Luciferase Assay Reagent (Promega E1501) and incubating for 10\u00a0minutes at room temperature before measuring luminescence. For luc2CP mRNA analysis, 4\u2009\u00d7\u2009105 cells/well were seeded in 6-well plates and grown overnight. Cells were then treated with the indicated amounts of Silvestrol for 40\u00a0minutes and lysed using Trizol reagent. RNA was isolated from Trizol lysates using standard methods. The abundance of luc2CP and \u03b2-actin mRNA was subsequently analyzed by quantitative RT-PCR.293\u00a0T cells were co-transfected with 5\u2032 UTR-bearing Prior to RNA extraction from polysome fractions, 5\u00a0ng of Luciferase Control RNA (Promega) was added to each fraction. Samples were treated with 200\u00a0\u03bcg/ml Proteinase K for 1\u00a0hour at 50\u00b0C and RNA was extracted using the standard hot acid phenol method. RNAs were precipitated using isopropanol and resuspended in 10\u00a0mM Tris pH7.luc2CP mRNA) levels, transcript abundance was normalized to \u03b2-actin mRNA measurements.Total RNA was converted to cDNA using SuperScript III reverse transcriptase (Invitrogen) with oligo dT primers according to the manufacturer\u2019s instructions. Transcript levels were measured by quantitative PCR using SYBR green FAST PCR mix and oligo pairs specific to each transcript. In polysome fractionation experiments, transcript levels were normalized to luciferase control RNA abundance. To measure luciferase reporter mRNA antibody at room temperature for 30\u00a0minutes, washed, resuspended in PI/RNase solution (BD Biosciences #550825) and analyzed by fluorescence-activated cell sorting (FACS).MDA-MB-231 cells were seeded in 6-well dishes at 3.5\u2009\u00d7\u200910MDA-MB-231 cells were treated with 25 nM Silvestrol and lysed in RIPA buffer containing complete EDTA-free protease inhibitors (Roche #11836170001), and phosphatase inhibitors . Lysates were quantified using BCA reagent (Thermo Scientific #23228). Lysates were subjected to SDS-PAGE (NuPAGE) using 15 to 25\u00a0\u03bcg of cell lysate per well and transferred to PVDF (Invitrogen).The following antibodies were purchased from commercial suppliers: CyclinD1 , CyclinB1 , CyclinE1 , Bcl2 (BD Pharmingen #551097), Parp(Asp214) , Arf6 , GAPDH , \u03b2-Tubulin .Cell lines used in this study were obtained from ATCC. Cells were tested for mycoplasma by the Novartis Institutes for Biomedical Research and were mycoplasma-free._CATCTCCATGGGCAGAACTGGGAGGAGGAGT_3\u2032; oARF6rev: 5\u2032_GACCTCCATGGCGCGTCGGAGGAGCCGGGGCCG_3\u2032; oCCND1fwd:_5\u2032_GACCTCCATGGGCTTAACAACAGTAACGTCACACGG_3\u2032; oCCND1rev:_5\u2032_CAGCTCCATGGCTGGGGCTCTTCCTGGGCAG_3\u2032; oROCK1fwd:_5\u2032_GACCTCCATGGGCGCUGGUUCCCCTTCCGAGCGT_3\u2032; oROCK1rev:_5\u2032_CATCTCCATGGGTGTTGCTGCTGCTGTGACAATGCCCT_3\u2032; oPFN2fwd:_5\u2032_GAATCTCCATGGGCCGCTGGTTTGTCAGCC_3\u2032; oPFN2rev:_5\u2032_CTCGAGCCATGGCTTCGAGCCCTTCGC_3\u2032; oARFmut_ampF:_5\u2032_CCATGGTTGGGACGTGCACTGGCAGCCGGC_3\u2032; oARFmut_ampR:_5\u2032_GTGGGAGTTGCCTCCTAAGCTAATATGTGC_3\u2032oARF6fwd: 5\u2032_ATGTTGCAGGTGAGATGTGGT_3\u2032,ARF6_qP_F8: 5\u2032_TACCTGCTCCAGTCACCAATG_3\u2032,ARF6_qP_R8: 5\u2032_AGCCTCGCCTTTGCCGA_3\u2032,ActB_qP_F1: 5\u2032_GCGCGGCGATATCATCATC_3\u2032,ActB_qP_R1: 5\u2032_TTCTGCCCCTGCCAAATCTT_3\u2032,BCL2_qP_F3: 5\u2032_CATCTGAGAACCTCCTCGGC_3\u2032,BCL2_qP_R3: 5\u2032_TGAGGGACGCTTTGTCTGTC_3\u2032,CCND1_qP_F2: 5\u2032_CTTCTGCTGGAAACATGCCG_3\u2032,CCND1_qP_R2: 5\u2032_ACCTGCCCCTTACTCTGACT_3\u2032,CDK6_qP_F2: 5\u2032_AGCACCCAGTAAGACATCCAG_3\u2032,CDK6_qP_R2: 5\u2032_TGAAAGCCGCACTGATGGAT_3\u2032,ROCK1_qP_F1: 5\u2032_GCCATGAGAAAACACATTGCAG_3\u2032,ROCK1_qP_R1: 5\u2032_ATCCGGAAGCGACCAACGCC_3\u2032,Luciferase_F: 5\u2032_GTCGGGAAGACCTGCCACGC_3\u2032,Luciferase_R: 5\u2032_ATCCACCTTAACAGCCACGG_3\u2032,Luc2CP_F: 5\u2032_CAGGGTGTCTATCCATGCCG_3\u2032.Luc2CP_R: 5\u2032ARF6mut sequence. The (CGG)4 motif is shown in bold.Mutated residues are indicated by lower case lettering in the ARF6wt:_5\u2032_AGAACUGGGAGGAGGAGUUGGAGGCCGGAGGGAGCCCGCGCUCGGGGCGGCGGCUGGAGGCAGCGCACCGAGUUCCCGCGAGGAUCCAUGACCUGACGGGGCCCCGGAGCCGCGCUGCCUCUCGGGUGUCCUGGGUCGGUGGGGAGCCCAGUGCUCGCAGGCCGGCGGGCGGGCCGGAGGGCUGCAGUCUCCCUCGCGGUGAGAGGAAGGCGGAGGAGCGGGAACCGCGGCGGCGCUCGCGCGGCGCCUGCGGGGGGAAGGGCAGUUCCGGGCCGGGCCGCGCCUCAGCAGGGCGGCGGCUCCCAGCGCAGUCUCAGGGCCCGGGUGGCGGCGGCGACUGGAGAAAUCAAGUUGUGCGGUCGGUGAUGCCCGAGUGAGCGGGGGGCCUGGGCCUCUGCCCUUAGGAGGCAACUCCCACGCAGGCCGCAAAGGCGCUCUCGCGGCCGAGAGGCUUCGUUUCGGUUUCGCGGCGGCGGCGGCGUUGUUGGCUGAGGGGACCCGGGACACCUGAAUGCCCCCGGCCCCGGCUCCUCCGACGCGCCAUG_3\u2032.ARF6mut:_5\u2032_CCAUGGuUGGGAcGuGcAcUgGcAGcCgGcAGaGAGCCCGCcCaCcGcuacGCGGCUuacccgucCcgAgCcAcUaggCcCGAGGAUCCAUGACCUGACGGGGCCCCGGAGCCGCGCUGCCUCUCGGGUGUCCUGGGUCGGUGGGGAGCCCAGUGCUCGCAGGCCGGCGGGCGGGCCGGAGGGCUGCAGUCUCCCUCGCGGUGAGAGGAAGGCGGAGGAGCGGGAACCGCGGCucuagaCGCGCGGCGCCUGCGGGGGGAAGGGCAGUUCCGGGCCGGGCCGCGCCUCAGCAGGGCGGCGGCUCCCAGCGCAGUCUCAGGGCCCGGGUGGCGGCGGCGACUGGAGAAAUCAAGUUGUGCGGUCGGUGAUGCCCGAGUGAGCGuauacagUcGaCaUaUuagCUUAGGAGGCAACUCCCACGCAGGCCGCAAAGGCGCUCUCGCGGCCGAGAGGCUUCGUUUCGGUUUCGCGGCGGCGGCGGCGUUGUUGGCUGAGGGGACCCGGGACACCUGAAUGCCCCCGGCCCCGGCUCCUCCGACGCGCCAUG_3\u2032.CyclinD1:_5\u2032_CCAUGGGCUUAACAACAGUAACGUCACACGGACUACAGGGGAGUUUUGUUGAAGUUGCAAAGUCCUGGAGCCUCCAGAGGGCUGUCGGCGCAGUAGCAGCGAGCAGCAGAGUCCGCACGCUCCGGCGAGGGGCAGAAGAGCGCGAGGGAGCGCGGGGCAGCAGAAGCGAGAGCCGAGCGCGGACCCAGCCAGGACCCACAGCCCUCCCCAGCUGCCCAGGAAGAGCCCCAGCCAUG_3\u2032.ROCK1:_5\u2032_GCUGGUUCCCCUUCCGAGCGUCCGCGCCCCGCAUGCGCAGUCUGCCCCGGCGGUCUCCGUUUGUUUGAACAGGAAGGCGGACAUAUUAGUCCCUCUCAGCCCCCCUCGCCCCACCCCCCAGGCAUUCGCCGCCGCGACUCGCCCUUUCCCCGGCUGGGACCGCAGCCCCUCCCAGAAGCUCCCCCAUCAGCAGCCGCCGGGACCCAACUAUCGUCUUCCUCUUCGCCCGCUCUCCAGCCUUUCCUCUGCUAAGUCUCCAUCGGGCAUCGACCUCGCCCUGCCCCACCGGACACCGUAGCAGCAGCCCCAGCAGCGACGGGACAAAAUGGGAGAGUGAGGCUGUCCUGCGUGGACCAGCUCGUGGCCGAGACUGAUCGGUGCGUCGGGCCGGGCCGAGUAGAGCCGGGGACGCGGGGCUAGACCGUCUACAGCGCCUCUGAGCGGAGCGGGCCCGGCCCGUGGCCCGAGCGGCGGCCGCAGCUGGCACAGCUCCUCACCCGCCCUUUGCUUUCGCCUUUCCUCUUCUCCCUCCCUUGUUGCCCGGAGGGAGUCUCCACCCUGCUUCUCUUUCUCUACCCGCUCCUGCCCAUCUCGGGACGGGGACCCCUCCAUGGCGACGGCGGCCGGGGCCCGCUAGACUGAAGCACCUCGCCGGAGCGACGAGGCUGGUGGCGACGGCGCUGUCGGCUGUCGUGAGGGGCUGCCGGGUGGGAUGCGACUUUGGGCGUCCGAGCGGCUGUGGGUCGCUGUUGCCCCCGGCCCGGGGUCUGGAGAGCGGAGGUCCCCUCAGUGAGGGGAAGACGGGGGAACCGGGCGCACCUGGUGACCCUGAGGUUCCGGCUCCUCCGCCCCGCGGCUGCGAACCCACCGCGGAGGAAGUUGGUUGAAAUUGCUUUCCGCUGCUGGUGCUGGUAAGAGGGCAUUGUCACAGCAGCAGCAACACCCAUG_3\u2032PFN2:_5\u2032_CGCUGCGGUAAGGAGCAGCCGCCACAGGCACAGCCGCUUCGCAGCCUCCCGCCGCUGGUUUGUCAGCCCCGCGGCUGCGGGCGGCCGGGCGGCCGAGCGCGCUCUGAGGUUCGUCCCUCAUCGCUGAACCCGCGUCCUCCCGCCGCAGCUCCUCGGGGAGGGGGGCGGUCGGUGCCUGCGCAGAGCCGCCUCCUCCCCGCCCCCGCCCCGCCUCCCCCCGCGCCGCCGCCGCCCGCUACCGCCGCCGCCGCCGCUGCGCCUGCUGCUCCUCGCCGUCCGCGCUGCAGUGCGAAGGGCUCGAGCCAUG_3\u2032.Monosome preparations pelleted in ultracentrifuge tubes were washed with 1\u00a0ml ice-cold ethanol by addition of the ethanol followed by a 5\u00a0minute incubation on ice and aspiration of the ethanol via a vacuum trap. The pellet was allowed to air dry. Monosome pellet was dissolved in 100\u00a0\u03bcl of a solution of 20\u00a0mM ammonium bicarbonate, 8\u00a0M urea and 2\u00a0mM Tris(2-carboxyethyl)phosphine hydrochloride. MS-Grade Lys C (Thermo) was dissolved in water to a concentration of 0.5\u00a0\u03bcg/\u03bcl and 2\u00a0\u03bcl was added to the dissolved pellet. The Lys C digestion was incubated for 4\u00a0hours at room temperature. The digestion was further diluted with an additional 98\u00a0\u03bcl of water. MS-Grade Trypsin (Thermo) was also dissolved in water to a concentration of 0.5\u00a0\u03bcg/\u03bcl and 2\u00a0\u03bcl was added to the diluted Lys C digestion. This trypsin digestion was incubated overnight at room temperature.Digestion samples were prepared for liquid chromatography-tandem mass spectrometry (LC-MS/MS) by acidification. The digestion sample (20\u00a0\u03bcl) was acidified by the addition of 2\u00a0\u03bcl of 10% formic acid; 20\u00a0\u03bcl of this sample was injected onto a 2.1\u2009\u00d7\u200950\u00a0mm Extend-C18 column at 400\u00a0\u03bcl/minute flow rate. Elution of peptides was over a 51\u00a0minute gradient from 5 to 40% acetonitrile with 0.1% formic acid as the modifier, and a flow rate of 200\u00a0\u03bcl/minute. Data acquisition was on an Agilent 6530 Q-ToF instrument equipped with a dual electrospray source, and a reference mass of 922.009798 enabled. MS/MS spectra were acquired in Auto MS/MS mode with triggering of precursor ions of +2 charge state and higher and individual ions were excluded for 9\u00a0seconds after triggering an MS/MS scan. Up to five precursors were selected per MS scan, and MS/MS scans were for 50,000 counts or 333 mS, whichever came first. LC-MS/MS data were exported to a Mascot Generic Format file using MassHunter Qual 5.0 (Agilent) and searched against SwissProt 51.6 with taxonomy limited to human, without modifications other than variable pyroglutamate formation at Q and E, MS tolerance of 10\u00a0ppm and MS/MS tolerance of 0.1\u00a0Da.Digestion samples were prepared for LC-MS/MS by acidification. The digestion sample (10\u00a0\u03bcl) was acidified by the addition of 10\u00a0\u03bcl of 1% formic acid; 4\u00a0\u03bcl of this sample was injected onto a 4\u00a0mm 40\u00a0nL trap column at a flow rate of 3\u00a0\u03bcl/minute and analyzed on a 75\u00a0\u03bcm\u2009\u00d7\u2009150\u00a0mm column containing Zorbax 300SB-C18 (5\u00a0\u03bcm beads) at a flow rate of 400\u00a0nl/minute. Elution of peptides was over a 100\u00a0minute gradient from 5 to 40% acetonitrile with 0.1% formic acid as the modifier. Data acquisition was on an Agilent 6550 Q-ToF instrument equipped with the ChipCube source, and a reference mass of 1221.990637 enabled. MS survey scans were acquired at a rate of 5\u00a0Hz, and MS/MS spectra were acquired in Auto MS/MS mode with triggering of precursor ions of +2 charge state and higher. Individual ions were excluded for 18\u00a0seconds after triggering an MS/MS scan. Up to five precursors were selected per MS scan, and MS/MS scans were for 25,000 counts or 200 mS, whichever came first. LC-MS/MS data were exported to a Mascot Generic Format file using MassHunter Qual 5.0 and searched against SwissProt 51.6 with taxonomy limited to human, without modifications other than variable pyroglutamate formation at Q and E, MS tolerance of 5\u00a0ppm and MS/MS tolerance of 0.1\u00a0Da.5\u2032 UTR sequences were retrieved from Ensemble using RefSeq mRNA names as query. Sequences were broken into 20-nucleotide fragments in a stepwise manner beginning from the first nucleotide (n) and proceeding in 1 nucleotide steps through the length of the UTR. The structure of each fragment was analyzed using the CONTRAfold algorithm and the resulting free energy values were plotted based on their position across the length of the UTR, as were the percentage GC values.5 cells in serum-free medium. Medium containing 5% fetal bovine serum was aliquoted to lower wells and cells were incubated for 22\u00a0hours. When measuring migration in the presence of Silvestrol, compound was included in the medium in both upper and lower chambers at the concentrations indicated. To assess cell migration after 22\u00a0hours, upper chambers were transferred to wells containing 1\u00d7 PBS +8\u00a0\u03bcM Calcenein AM (Life Technologies) and incubated for 40\u00a0minutes. Fluorescence was evaluated by excitation at 485 nM wavelength followed by measuring emission at 520 nM.Migration assays were performed using trans-well migration chambers (BD Biosciences). MDA-MB-231 cells were seeded into upper chambers at a density of 0.5\u2009\u00d7\u2009105 cells/well in RPMI supplemented with 10% fetal bovine serum and grown for 24\u00a0hours to generate a monolayer. The following day cells were scratched to produce a wound in the monolayer and wound closure was monitored by microscopy using the Incucyte Live Cell Imaging system .Cells were seeded into 24 well plates at a density of 6\u2009\u00d7\u200910The next generation sequencing data presented in this paper have been deposited in NCBI\u2019s Gene Expression Omnibus and are"} +{"text": "Here we use transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 technologies to interrogate the functional relevance of predicted miRNA response elements (MREs) to post-transcriptional silencing in zebrafish and Drosophila. We also demonstrate an effective strategy that uses CRISPR-mediated homology-directed repair with short oligonucleotide donors for the assessment of MRE activity in human cells. These methods facilitate analysis of the direct phenotypic consequences resulting from blocking specific miRNA\u2013MRE interactions at any point during development.MicroRNA (miRNA) target recognition is largely dictated by short \u2018seed\u2019 sequences, and single miRNAs therefore have the potential to regulate a large number of genes. Understanding the contribution of specific miRNA\u2013target interactions to the regulation of biological processes Identifying miRNA response elements (MREs) within target mRNAs can be done computationally but the functional validation of putative MREs remains challenging. Here, Bassett et al. describe applications of genome engineering to target and assess the functional significance of MREs in different organisms and stages of development. To date, the standard strategy used to validate miRNA targets has relied on artificial sensor assays whereby the 3\u2032-untranslated region (UTR) of the gene of interest (or a region flanking the predicted MRE) is coupled to a reporter gene. Although valuable, a critical limitation of this technology is its inability to infer the physiological relevance of a putative MRE in vivo, as these assays do not recapitulate the endogenous context and stoichiometry of miRNAs and their targets. An alternative strategy is provided by target protector (TP) oligonucleotides, which overlap with the seed region and a unique flanking sequence in the target 3\u2032UTR, and can therefore block miRNA access to the MRE8910in vivo, few studies have been performed to address potential toxicity or off-target effects of TP oligonucleotides. Furthermore, TP assays are in most cases limited to transient effects in systems where oligonucleotides can be supplied by injection or transfectionMicroRNA (miRNA)-mediated post-transcriptional silencing represents an essential regulatory layer underlying cellular function, development and disease12ry high ~0%67, andin vivo. To generate targeted insertion and deletion (indel) mutations within MREs, we use both transcription activator-like effector nucleases (TALENs) in zebrafish and the clustered regularly interspaced short palindromic repeats (CRISPR)-associated nuclease (Cas9) in Drosophila. In both systems, indels are created as a result of inefficient non-homologous end joining repair at the nuclease cleavage site. Using this approach, we have accurately defined bona fide MREs in these two species and have directly addressed their functional relevance in an endogenous context in vivo. Furthermore, we describe a novel approach for the identification of active MREs in cell culture systems, using CRISPR-mediated homology-directed repair (HDR) with short oligonucleotide donors. This now allows the effect of defined deletions or alterations within MREs to be rapidly assessed in heterogeneous cell populations. Finally, we present an online algorithm that computationally predicts CRISPR target sites neighbouring all predicted MREs in several species, and thereby show that these techniques may be generally applicable for investigation of the vast majority of MREs.To address these limitations, we have used genome engineering technologies to genetically ablate endogenous MREs within putative target genes lefty2 (lft2) 3\u2032UTR, causing a midline development phenotype in the embryo as a consequence of impaired Nodal signallinglft2 DNA of single experimental embryos showed that lft2 expression level at shield stage was significantly increased as compared with controls . miR-430 MRE disruption, as assayed by the resistance to Bsp1286I digest, was observed in DNA samples obtained from all tested embryos that exhibited mutant phenotypes , indicating highly efficient editing events at the targeted locus. These results establish the feasibility of this approach for analysis of miRNA\u2013target regulatory interactions during zebrafish embryonic development, and validate the functional relevance of this MRE in the lft2 3\u2032UTR. Notably, such mutants could be used to generate stable lines, allowing phenotypic analysis at all stages of development and during adult life.In zebrafish, morpholino TP oligonucleotides have been used to block a predicted miR-430 site in the lft2 DNA . Customilft2 DNA . The spaion site . In agreion site , with a bantam (ban) in the enabled (ena) gene in Drosophila. Previous work showed that overexpression of ban in the wing imaginal disc can reduce Ena levels in the context of a 3\u2032UTR reporter assay, and that this effect was dependent on a conserved ban target siteTo realize the potential of our strategy in elucidating the functional significance of MREs throughout development, we next investigated the effect of removing a well-studied target site for the miRNA ban target site in the ena 3\u2032UTR . The presence of the mutation in animals used for all subsequent steps was validated by PCR (ban using the decapentaplegic driver (dpp-GAL4) that expresses in a stripe of cells across the anterior/posterior boundary of the wing imaginal disc (ena-3\u2032UTR-sensor construct (ena-3\u2032UTR) and endogenous Ena protein levels (n>30). No change in Ena levels was observed in control discs expressing CD8-EGFP under the same GAL4 driver . This result indicates that overexpressed ban can directly target ena via this predicted MRE in its 3\u2032UTR.To create a genomic deletion in the predicted MRE, we designed a synthetic guide RNA (sgRNA) that would target the Cas9 nuclease to the predicted na 3\u2032UTR . The expquencing . We decid by PCR and sequd by PCR . We overnal disc . Confirmn levels , n>30. N4 driver . When th protein , n>30. Tban regulates Ena expression on either side of the dorsal/ventral (D/V) boundary of the wing imaginal discsban-MREmut animals revealed no apparent changes to endogenous Ena expression pattern in the wing imaginal discs (compare n=10), or any detectable phenotypes in the adult wings (n=10). This indicates that although ban overexpression is able to downregulate Ena through this MRE, the endogenous expression pattern of Ena in the wing disc is not dependent on this target site. Thus, although it remains possible that ban participates in establishing the identity of the D/V boundary in wing discs, it is unlikely this activity is mediated by tuning Ena expression through this predicted MRE. These results emphasize the importance of directly establishing the in vivo physiological relevance of a MRE, and suggest that caution should be exerted when inferring its function from indirect assays.It was previously suggested that endogenous compare . We also compare , n=10, olt wings , n=10. Tin vivo are critical for understanding the function of MREs in the context of the whole organism, cell culture systems offer a valuable tool for studying miRNA function in a well-defined context, and a means to directly investigate their role in human cells. We therefore developed a novel strategy to validate functional MREs in cultured cells, using the CRISPR/Cas9 system to enhance homologous recombination, and generate defined sequence alterations of a MRE of interest. The approach involves co-transfection of the Cas9/sgRNA targeting a MRE with two ~140\u2009nt single-stranded DNA (ssDNA) oligonucleotide templates for HDR. One of these HDR templates inserts a T3 \u2018barcode\u2019 downstream of the target MRE locus, but maintains the intact MRE . Interestingly, C9orf7 is targeted by miR-92a via a non-canonical motif through complementary base pairing with its 3\u2032 end, demonstrating that such interactions can be biologically relevant. The other putative MREs in PCMTD1 and MAPRE1 failed to show this effect, suggesting that they may not play a role in regulating the abundance of these transcripts, at least in this particular cell type. One consideration when designing this strategy is to determine whether integration of the barcodes creates or inadvertently removes endogenous MREs. To test this, the sequences generated upon integration of T7-MREmut and T3-MREWT barcodes were screened for miRNA target sites using the PITA algorithmmut barcode, as intended , and revealed that both of these MREs are functional in this cell type intended . All butell type .Based on these results, we propose that this strategy can be applied broadly to the identification of physiologically active MREs when the target mRNA is expressed at sufficient levels to allow detection by qPCR of homologous integration within the cDNA. In addition, our results suggest that care must be taken when extrapolating the functional relevance of a MRE from studies of miRNA binding to the target transcript or changes in gene expression resulting from miRNA inhibition.www.microrna.org, August 2010 release1921To establish the versatility of these technologies, and whether they can be generally applied to analysis of miRNA\u2013MRE interactions, we performed computational analysis to identify CRISPR target sites in the vicinity of all predicted MREs in several organisms . CRISPR http://miR-CRISPR.molbiol.ox.ac.uk/fulga/miR-CRISPR.cgi). Information is provided for five species: human, mouse, rat, Drosophila and Caenorhabditis elegans. Upon entering a gene symbol and/or miRNA identifier, CRISPR sgRNA target sites are generated for each predicted MRE, along with the distance between the PAM and MRE, the forward and reverse oligonucleotides required for sgRNA production, and the number and details of putative off-target sites.CRISPR target sites for all predicted MREs are available via the miR-CRISPR online web interface lft2 3\u2032UTR at the miR-430-binding site, separated by a 16-nt spacer. RVD arrays containing HD, NG, NI and NN monomers were assembled using the Golden Gate TALEN and TAL Effector Kit 2.0 as previously describedin vitro transcription of corresponding TALEN mRNAs. The RVD sequences of the left and right TALENs are:The TALE nuclease was designed using the TALEN Targeter algorithm , and capped TALEN mRNAs were in vitro transcribed using the mMESSAGEmMACHINE SP6 kit . RNAs were then polyadenylated with the polyA tailing kit and purified with the RNeasy mini kit .Plasmid pMTB2-Goldy A measure of 25\u2009pg of each TALEN mRNA was injected into single-cell stage embryos, using the Picospritzer II microinjector (Parker Instrumentation). Embryos were incubated in E3 mediumlft2 antisense probe was generated by PCR amplification of cDNA from 12\u2009hours post fertilization wild-type zebrafish, using the following primers: SP6-lft2-F 5\u2032-GATTTAGGTGACACTATAGgaccacagcgatctcactca-3\u2032 and T7-lft2-R 5\u2032-TAATACGACTCACTATAGGGgactggagggattttgtcc-3\u2032. The PCR product was gel extracted and purified by ethanol precipitation. 1.5\u2009\u03bcg of template DNA was transcribed using T7 RNA polymerase in the presence of digoxygenin (DIG)-labelled dNTPs . Probes were purified using G-50 micro-columns . Embryos were permeabilized with 1% H2O2, equilibrated with hybridization buffer dimethylammonio]-1-propanesulfonate (CHAPS), 100\u2009\u03bcg\u2009ml\u22121 Heparin) and incubated overnight with lft2 DIG-labelled probes. Embryos were blocked with 20% sheep serum+2% Boehringer Blocking Reagent . Probes were detected with an anti-DIG-alkaline phosphatase antibody and the signal was developed using 30\u2009\u03bcg\u2009ml\u22121 of each nitro-blue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl-phosphate .The template for Bsp1286I for 4\u2009h at 37\u2009\u00b0C. Digested products were then analysed by gel electrophoresis (1% agarose). To identify the nature of indel mutations, PCR products were cloned into pGEM-T easy vector and DNA was isolated from individual clones and sequenced using an SP6 primer.gDNA was extracted by homogenizing single zebrafish embryos in 50\u2009\u03bcl of 50\u2009mM NaOH, followed by incubation for 20\u2009min at 95\u2009\u00b0C, cooling to 4\u2009\u00b0C and addition of 5\u2009\u03bcl of 1\u2009mM Tris-HCl pH=8 to neutralize the solutionFor coupled transcriptional/genomic analysis of single injected zebrafish embryos, total RNA and DNA were extracted using the RNAqueous Micro Kit . cDNA was synthesized using the Superscript III Reverse transcriptase kit . For quantitative PCR, the following gene-specific primers were used: Lft2-F (5\u2032-CTGGCAGGAATACTCAGGGG-3\u2032), Lft2-R (5\u2032-TGGCCTCCATGTCGAACA-3\u2032), ActB-F (5\u2032-AATCCCAAAGCCAACAGAGA-3\u2032), ActB-R (5\u2032-ACATACATGGCAGGGGTGTT-3\u2032). The levels of Lft2 transcript in individual embryo analysis were calculated using the standard curve method.20NGG). Potential off target sites within the Drosophila genome were identified by BLAST and using the CRISPR design tool ( http://crispr.mit.edu). The closest off-target site contained four mismatches to the 20\u2009nt target sequence. Cas9 mRNA was in vitro transcribed from plasmid MLM3613 (ref. TAATACGACTCACTATAGGCGTATGAGATCGTGTGCTTGTTTTAGAGCTAGAAATAGC-3\u2032) and a reverse oligonucleotide encoding the remainder of the sgRNA sequence (sgRNA-R=5\u2032-AAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC-3\u2032). Reaction solutions (100\u2009\u03bcl) were cycled on a GStorm thermal cycler , 72\u2009\u00b0C 10\u2009min, 10\u2009\u00b0C \u221e) and purified with a PCR purification kit . In vitro transcription was performed with 300\u2009ng purified DNA template for 4\u2009h at 37\u2009\u00b0C using the Megascript T7 kit , and sgRNA purified by phenol chloroform extraction and isopropanol precipitation. sgRNAs were diluted to 1\u2009\u03bcg\u2009\u03bcl\u22121 in water and stored in aliquots at \u221280\u2009\u00b0C was used with an Injectman NI2 micromanipulator and Femtotip II needles . Embryos were incubated at 25\u2009\u00b0C for the remainder of the development. Putative mosaic flies were crossed individually to yw; Sco/CyO flies. These flies were removed after 7 days, and analysed for mutant tissue by HRMA ; ena-3\u2032UTR-sensor and UAS-bantam were a generous gift from Marco MilanDrosophila CRISPR injections are provided at the OxfCRISPR website ( http://www.oxfcrispr.org)A measure of 0.5\u2009\u03bcg sgRNA and 10\u2009\u03bcg Cas9 mRNA were precipitated with ethanol to purify and concentrate, and resuspended in water at 0.5\u20131\u2009\u03bcg\u2009\u03bcl by HRMA . Only cr by HRMA . During \u22121 proteinase K , and heating to 37\u2009\u00b0C for 30\u2009min or 60\u2009min, followed by inactivation at 95\u2009\u00b0C for 2\u2009min and blocked in 5% normal goat serum in PBS-T. Discs were then incubated overnight at room temperature with primary antibodies mouse anti-Enabled , rabbit anti-GFP and mouse anti-wingless , washed three times 20\u2009min each in PBS-T and incubated for 2\u2009h at room temperature with anti-mouse A568 , anti-rabbit A488 and DAPI . Samples were then mounted in Slowfade and imaged on a Zeiss 780 confocal microscope with a \u00d7 25 oil immersion objective and \u00d7 0.7 digital zoom. Images were acquired using similar laser power, pinhole size and gain parameters, and adjusted post-acquisition for background intensity and contrast correction. For assessing Ena expression in the dpp-GAL4>UAS-ban background, a minimum of 30 third instar larval wing imaginal discs were analysed. The results displayed in ban-MREmut lines. To confirm the nature of ban-MREmut indels in animals processed for immunofluorescence and imaging, half of each larvae was snap frozen immediately after dissection and subjected to gDNA extraction followed by PCR validation and sequencing.20NGG were chosen as close as possible to the predicted MREs in the 3\u2032UTRs of PCMTD1 (5\u2032-GGCAATATATGAGTGCAATA-3\u2032), MAPRE1 (5\u2032-CCTTGGGATCTGCCAGGCTG-3\u2032) and C9orf7 (5\u2032-GTGGCATCTGAGGCCGGGAG-3\u2032) in human cells, and Pck (5\u2032-TCCATAGTGCCTTTAACAAT-3\u2032) and CG13088 (5\u2032-CATTTCTACCTCAATCCGTC-3\u2032) in Drosophila cells. Target sequences were cloned into the pX330 vector Target sites of the form NPCMTD1 targetCCGGGCAATATATGA GTGCAATAACAATACAAGATATTGAATAATTTAGCTTTAAAAAATCCCACAAATTTTATG AAATTTT-3\u20325\u2032-TTACTTTTGTTGCAATCACTGTTGTTGGGTTGCTGTATATATATTPCMTD1 ssOligoT7TAATACGACTCACTATAGGGAACAATACAAGATATTGAATAATTTAGCTTTAAA AAATCCCACAAATTTTATGAAATTTT-3\u20325\u2032-TTACTTTTGTTGCAATCACTGTTGTTGGGTTGCTGTATATATATTGCGGGCAATATATGAPCMTD1 ssOligoT3GTGCAATAATTAACCCTCACTAAAGGGAAACAATACAAGATATTGAATAATTTAGCTTTAAAAAATCCCACAAATTTTATGAAATTTT-3\u20325\u2032-TTACTTTTGTTGCAATCACTGTTGTTGGGTTGCTGTATATATATTGCGGGCAATATATGA MAPRE1 targetGGCAGGCTGGTGTTTTCGGTATCTGCTGTTCACAGCTCTCCA CTGTAATCCGAATACTTTGCCAGTGCA-3\u20325\u2032-CTTTCTGGACCTCTGGCAAAGGGAGTGGTCAGTGAAGGCCATCGTTACCTTGGGATCTGCMAPRE1 ssOligoT7TAATACGACTCACTATAGGGGTGTTTTCGGTATCTGCTGTTCACAGCTCTCCACTGTAATCCGAA TACTTTGCCAGTGCA-3\u20325\u2032-CTTTCTGGACCTCTGGCAAAGGGAGTGGTCAGTGAAGGCCATCGTTACCTTGGGATCTGCMAPRE1 ssOligoT3CAGGCTGGGAATTAACCCTCACTAAAGGGAGTGTTTTCGGTATCTGCTGTTCACAGCTCTCCACTGTAATCCGAATACTTTGCCAGTGCA-3\u20325\u2032-CTTTCTGGACCTCTGGCAAAGGGAGTGGTCAGTGAAGGCCATCGTTACCTTGGGATCTGCC9orf7 targetAGGCCGGGAGTGGCATCTGAGGCCAGGAGTGGCAGGCTGGTGGGCTGGGCGTGGGGTT TTCTGGGCCCT-3\u20325\u2032-AACTGTTTCCCAGGAACACCTCTCGGGCCCATCTGCGTCTGAGGCTGGGAGTGGCATCTGC9orf7 ssOligoT7TAATACGACTCACTATAGGGGTGGCATCTGAGGCCAGGAGTGGCAGGCTGGTGGGCTGGGCGTG GGGTTTTCTGGGCCCT-3\u20325\u2032-AACTGTTTCCCAGGAACACCTCTCGGGCCCATCTGCGTCTGAGGCTGGGAGTGGCATCTGC9orf7 ssOligoT3AGGCCGGGAAATTAACCCTCACTAAAGGGAGTGGCATCTGAGGCCAG GAGTGGCAGGCTGGTGGGCTGGGCGTGGGGTTT TCTGGGCCCT-3\u20325\u2032-AACTGTTTCCCAGGAACACCTCTCGGGCCCATCTGCGTCTGAGGCTGGGAGTGGCATCTGPCK targetCCGTCCATAGTGCCTTTAACAATCGACCATATGTATCTATATACACGCCGACTCAGCCGAG ATCAG-3\u20325\u2032-ATTGGATCTTAGCTTAAGTTTTCAGACGCAATCTCGTGCCTCGAATCTGCGCAATCGACGPCK ssOligoT7TAATACGACTCACTATAGGGATAGTGCCTTTAACAATCGACCATATGTATCTATATACA CGCCGACTCAGCCGAGATCAG-3\u20325\u2032-ATTGGATCTTAGCTTAAGTTTTCAGACGCAATCTCGTGCCTCGAATCTGCGCAATCGACGPCK ssOligoT3CCGTCCAATTAACCCTCACTAAAGGGAATAGTGCCTTTAACAATCGACCATATGTATCTATATACACGCCGACTCAGCCGAGATCAG-3\u20325\u2032-ATTGGATCTTAGCTTAAGTTTTCAGACGCAATCTCGTGCCTCGAATCTGCGCAAT CGACGCG13088 targetC TCCGTCGGTATACGAATATATATGTAGATGGAGATCCAA ATGATATATCCTGAGTAAAATGTTGTA-3\u20325\u2032-GAATTCAGTCCAGACTCGTAGTAGTCATTTGAAAAGACTTAAATGACATTTCTACCTCAACG13088 ssOligoT7 TAATACGACTCACTATAGGGGGTATACGAATATATATGTAGATGGAGATCCAAATGATATATCCTGAG TAAAATGTTGTA-3\u20325\u2032-GAATTCAGTCCAGACTCGTAGTAGTCATTTGAAAAGACTTAAATGACATTTCTACCTCAACG13088 ssOligoT3TCCGTCCAATTAACCCTCACTAAAGGGAGGTATACGAATATATATGTAGATGGAGATCCAAATGATATATCCTGAGTAAAATGTTGTA-3\u20325\u2032-GAATTCAGTCCAGACTCGTAGTAGTCATTTGAAAAGACTTAAATGACATTTCTACCTCAA 2 in DMEM with 25\u2009mM glucose , 10% fetal bovine serum and 1% penicillin-streptomycin . S2R+ cells were cultured at 25\u2009\u00b0C in Schneider\u2019s Drosophila medium , 10% fetal bovine serum and 1% penicillin-streptomycin . Cas9/sgRNA expression vectors (1\u2009\u03bcg) and homology oligonucleotides (0.5\u2009\u03bcg each T3/T7 oligo) were co-transfected into HEK293T cells in a six-well dish using polyethylenimine (Sigma-Aldrich) as previously describedHEK293T cells were cultured at 37\u2009\u00b0C and 5% CO\u22121 of proteinase K , and incubation at 55\u2009\u00b0C for a minimum of 2\u2009h. Proteins were removed by phenol/chloroform extraction, and DNA precipitated with 2.5 volumes ethanol. DNA was quantified with a Nanodrop spectrophotometer and diluted to a concentration of 100\u2009ng\u2009\u03bcl\u22121 before analysis. RNA was extracted using a miRNeasy mini kit including the additional DNAse treatment step. cDNA was synthesized from 1\u2009\u03bcg RNA with the Quantitect Reverse Transcription kit and diluted twofold before analysis.gDNA was extracted from zebrafish embryos by addition of 300\u2009\u03bcl lysis buffer containing 200\u2009\u03bcg\u2009ml\u2212\u0394Ct.Quantitative PCR was performed with gene-specific forward primers , and either T3as (5\u2032-TCCCTTTAGTGAGGGTTAATT-3\u2032) or T7as (5\u2032-CCTATAGTGAGTCGTATTA-3\u2032) reverse primers. Amplification was performed with SybrGreen JumpStart Taq Ready Mix and detected on an ABI 7500 Fast thermal cycler. Results are shown as a ratio of the signal with the T7 primer to that with the T3 primer for gDNA and cDNA for three independent biological replicates, each analysed in technical triplicate. The final signal ratio was obtained by calculating the difference in Ct between T7 and T3 primers (\u0394Ct) during the exponential amplification phase, and subsequently transforming the resulting values using 2www.microrna.org, August 2010 Release) for four species, Drosophila, human, mouse and rat. A custom perl script was used to identify potential CRISPR target sites within a 200-bp window around each of the MREs from the appropriate genomic sequences . Potential off-targets for each of the CRISPR target sites were identified using the BatMis algorithmMREs for conserved miRNAs with a \u2018good mirSVR score\u2019 were predicted using the miRanda algorithm .Supplementary Figure 1 and Supplementary Table 1"} +{"text": "The zinc finger proteins ZNF644 and WIZ regulate the G9a/GLP complex for gene repression. Published 19 March 2015TTCGATGATTCCCAGATACTAGTACGGTCAG-3\u2032 (WIZ \u2018WT DNA target\u2019).There was an error in the WIZ binding sequence used for the EMSA experiments. The incorrect sequence was 5\u2032-GAGTCTCACTCACGCGCCATTCCATTCCATTCAGATACTAGTACGGTCAG-3\u2032 (WIZ \u2018WT DNA target\u2019).The correct sequence is 5\u2032-GAGTCTCACTCACGCGCThe article has been corrected accordingly."} +{"text": "TMPRSS2:ERG gene fusion is common in androgen receptor (AR) positive prostate cancers, yet its function remains poorly understood. From a screen for functionally relevant ERG interactors, we identify the arginine methyltransferase PRMT5. ERG recruits PRMT5 to AR-target genes, where PRMT5 methylates AR on arginine 761. This attenuates AR recruitment and transcription of genes expressed in differentiated prostate epithelium. The AR-inhibitory function of PRMT5 is restricted to TMPRSS2:ERG-positive prostate cancer cells. Mutation of this methylation site on AR results in a transcriptionally hyperactive AR, suggesting that the proliferative effects of ERG and PRMT5 are mediated through attenuating AR\u2019s ability to induce genes normally involved in lineage differentiation. This provides a rationale for targeting PRMT5 in TMPRSS2:ERG positive prostate cancers. Moreover, methylation of AR at arginine 761 highlights a mechanism for how the ERG oncogene may coax AR towards inducing proliferation versus differentiation.The DOI:http://dx.doi.org/10.7554/eLife.13964.001 Prostate cancers are among the most common types of cancer in men, which, like other cancers, are driven by genetic mutations. Roughly half of all prostate cancers contain a genetic change that incorrectly fuses two genes together, causing the cells to produce abnormally high levels of a protein called ERG.ERG is a transcription factor, a protein that binds to specific sequences of DNA to influence the activity of nearby genes. ERG represses genes that help to prevent prostate cancers from growing, and so promotes prostate cancer development. Like most other transcription factors, ERG is difficult to target with drugs and no therapies that directly prevent the activity of ERG currently exist.Mounir et al. wanted to find out whether ERG cooperates with other proteins to cause prostate cancer cells to grow, with the hope that these proteins could be more easily targeted with a drug. By using various biochemical techniques in human prostate cancer cell lines, Mounir et al. found that ERG interacts with an enzyme called PRMT5. This interaction enables PRMT5 to chemically modify other proteins to change their activity. In the case of prostate cancer cells, PRMT5 inappropriately modifies the androgen receptor, a protein that regulates the growth of normal prostate cells. This abnormal modification contributes to the excessive growth of the cancer cells.Although PRMT5 will be easier to target with drugs than ERG, it also has many other roles besides those described by Mounir et al. Much more work is therefore needed to investigate whether PRMT5 could be safely targeted to treat patients with prostate cancer.DOI:http://dx.doi.org/10.7554/eLife.13964.002 TMPRSS2:ERG fusion is highly prevalent and lethal . Drugs tG fusion . TMPRSS2 in mice . ERG is in mice , suggest in mice . We explTMPRSS2:ERG positive PC cells, we performed a pooled short hairpin RNA (shRNA) screen in TMPRSS2:ERG and AR-positive VCaP prostate cancer cells, using ERG-negative 22Rv1 cells as a control . The shRNA pool targets 648 genes involved in transcriptional and epigenetic regulation . Identified proteins includedTMPRSS2:ERG positive NCI-H660) and one PRMT5 hairpin likely to have minimal off-target effects. This shRNA shows a trend of sensitivity in ERG-positive lines, in agreement with our findings -inducible shRNA vectors targeting PRMT5 and a non-targeting control shRNA (NTC). PRMT5 knockdown was robust in all cell lines . Robust findings .PRMT5 is a protein arginine methyltransferase that regulates multiple signaling pathways through the mono- and symmetric di-methylation of arginines on its target proteins .To deterPSA, NKX3-1 and SLC45A3 to investigate ERG, AR, and PRMT5 recruitment to the AR targets cterized binding cterized . We alsocterized . In VCaPcterized . As expecterized , ERG expcterized , and ERGcterized . Like ERcterized .To extend these findings to a genome wide scale, we performed AR and ERG ChIP-seq experiments in androgen (DHT or R1881) stimulated VCaP cells upon PRMT5 knockdown . ERG and AR recruitment were robust in these experiments, as judged by recovery of the canonical DNA binding sites of these proteins . In agrePSA or NKX3-1 loci and symmetric di-methylation (SDMA) levels compared to wild-type controls , suggest3-1 loci . We ther3-1 loci . To confenotypes . Like VCcontrols . On the sus VCaP , suggestsus VCaP . In thessus VCaP , suggestTo further understand if the observed AR methylation was directly dependent on PRMT5, we performed biochemical assays using purified PRMT5 (complexed with its requisite binding partner MEP50/WDR77) and AR LBD. PRMT5 activity, as judged by production of SAH (the by-product of SAM-dependent substrate methylation), was observed in the presence of AR LBD as substrate, but not in the presence of ERG ETS DNA binding domain or pointed (PNT) domain. PRMT5 activity in the presence of AR LBD further increased with the addition of ERG ETS domain protein to the reaction, but not with PNT domain . Unlike To identify the arginine methylation site(s) on AR, we cloned AR cDNA and mutated all arginines in the LBD to lysine . We expressed each construct in AR-negative, ERG-negative RWPE-1 prostate cells. R1881 stimulates exogenous AR to induce target gene expression in these cells. ERG expression represses this effect and induPSA locus as a model AR-regulated gene. Relative to WT AR, the AR R761K mutant showed enhanced recruitment to PSA and increased PSA expression. Moreover, R761K mutation prevented ERG-dependent attenuation of AR recruitment to PSA and PSA expression and tested for mycoplasma contamination using the MycoAlert Mycoplasma Detection kit (Lonza).Redundant siRNA Activity or RSA metric was used as described were used for further analysis targeting transcriptional and epigenetic regulators similar to a previously reported library . shRNA lescribed . Brieflyhttp://tools.proteomecenter.org/software.php]) using a false positive threshold of <1% for protein identifications (See Nuclear extracts of VCaP cells (~600 million cells) were pre-cleared using agarose beads and used for pulldown with either an ERG antibody (Santa Cruz#353) or an anti-rabbit IgG antibody. All antibodies were pre-coupled to beads , washed then used for pulldown. Immunoprecipitations were eluted at pH2.5 followed by TCA protein precipitation, alkylated with iodoacetamide, and separated on a NuPage 4\u201312% Bis-Tris gradient gel (Invitrogen). Complete gel lanes were excised using a LEAP 2DiD robot and in-gel digested with trypsin (Tecan Freedom EVO 20). Peptide sequencing for the resulting resulting 16 digest samples was performed by liquid chromatography-tandem mass spectrometry using an Eksigent 1D+ high-pressure liquid chromatography system coupled to a LTQ-Orbitrap XL mass spectrometer (Thermo Scientific). Peptide mass and fragmentation data were searched against a combined forward-reverse IPI database (v3.55) using Mascot 2.2 (Matrix Science). Peptide and protein validation were done using Transproteomic pipeline v3.3sqall was previously described, as were the sequences of the nontargeting control and ERG shRNA inserts and stable cell line generation. PRMT5 sPRMT5 shRNA#1:AGGGACTGGAATACGCTAATTCTCGAGAATTAGCGTATTCCAGTCCCTPRMT5 shRNA#2:AGGGACTGGAATACGTTAATTGTTAATATTCATAGCAATTAGCGTATTCCAGTCCCTPRMT5 shRNA#3:GCGGATAAAGTTGTATGTTGTGTTAATATTCATAGCACAGCATACAGCTTTATCCGCAll shRNA were expressed from a puromycin resistant vector. Lentiviral production and cell transduction was as previously described .CGCGATTGGAACACGTTGATT .The Dox-inducible ERG constructs named ERG or ERG DNAx used for stable and inducible expression in 22Rv1 cells were generated as previously described . The cDNCAGAATCAAGCTCTACGCCGT .For shPRMT5-2: the GCGGATAAAGCTGTATGCTGT target sequence was converted to Full sequence of scPRMT5 below:scPRMT5 sequence: ATGTACCCCTATGACGTGCCAGATTACGCCATGGCGGCGATGGCGGTCGGGGGTGCTGGTGGGAGCCGCGTGTCCAGCGGGAGGGACCTGAATTGCGTCCCCGAAATAGCTGACACACTAGGGGCTGTGGCCAAGCAGGGGTTTGATTTCCTCTGCATGCCTGTCTTCCATCCCAGGTTCAAGCGCGAGTTTATTCAGGAACCTGCTAAGAATCGGCCCGGTCCCCAGACACGATCAGACCTACTGCTGTCAGGACGCGATTGGAACACGTTGATTGTGGGAAAGCTTTCTCCATGGATTCGTCCAGACTCAAAAGTGGAGAAGATTCGCAGGAACTCCGAGGCGGCCATGTTACAGGAGCTGAATTTTGGTGCATATTTGGGTCTTCCAGCTTTCCTGCTGCCCCTTAATCAGGAAGATAACACCAACCTGGCCAGAGTTTTGACCAACCACATCCACACTGGCCATCACTCTTCCATGTTCTGGATGCGGGTACCCTTGGTGGCACCAGAGGACCTGAGAGATGATATAATTGAGAATGCACCAACTACACACACAGAGGAGTACAGTGGGGAGGAGAAAACGTGGATGTGGTGGCACAACTTCCGGACTTTGTGTGACTATAGTAAGAGGATTGCAGTGGCTCTTGAAATTGGGGCTGATTTGCCCTCTAATCACGTCATTGATCGCTGGCTTGGGGAGCCCATCAAAGCAGCCATTCTCCCCACTAGCATTTTCCTGACCAATAAGAAGGGATTTCCTGTTCTTTCTAAGATGCACCAGAGGCTCATCTTCCGGCTCCTCAAGTTGGAGGTGCAGTTCATCATCACAGGCACCAACCACCACTCAGAGAAGGAGTTTTGTAGCTACCTGCAGTACCTGGAATACTTAAGCCAGAACCGTCCTCCACCTAATGCCTATGAACTCTTTGCCAAGGGCTATGAAGACTATCTGCAGTCCCCGCTTCAGCCACTGATGGACAATCTGGAATCTCAGACATATGAAGTGTTTGAAAAGGACCCCATCAAATACTCTCAGTACCAGCAGGCCATCTATAAATGTCTGCTAGACCGAGTACCAGAAGAGGAGAAGGATACCAATGTCCAGGTACTGATGGTGCTGGGAGCAGGACGGGGACCCCTGGTGAACGCTTCCCTGCGGGCAGCCAAGCAGGCCGACCGCAGAATCAAGCTCTACGCCGTGGAGAAAAACCCAAATGCCGTGGTGACGCTAGAGAACTGGCAGTTTGAAGAATGGGGATCCCAGGTCACGGTAGTCAGCTCAGACATGAGGGAATGGGTGGCTCCAGAGAAAGCAGACATCATTGTCAGTGAGCTTCTGGGCTCATTTGCTGACAATGAATTGTCGCCTGAGTGCCTGGATGGAGCCCAGCACTTCCTAAAAGATGATGGTGTGAGCATCCCCGGGGAGTACACTTCCTTTCTGGCTCCCATCTCTTCCTCCAAGCTGTACAATGAGGTCCGAGCCTGTAGGGAGAAGGACCGTGACCCTGAGGCCCAGTTTGAGATGCCTTATGTGGTACGGCTGCACAACTTCCACCAGCTCTCTGCACCCCAGCCCTGTTTCACCTTCAGTCACCCTAATCGCGACCCCATGATTGACAACAACCGCTATTGCACCTTGGAATTTCCTGTGGAGGTGAACACAGTACTACATGGCTTTGCCGGCTACTTTGAGACTGTGCTTTATCAGGACATCACTCTGAGTATCCGTCCAGAGACTCACTCTCCTGGGATGTTCTCATGGTTTCCTATTCTGTTTCCCATCAAGCAGCCCATAACGGTACGTGAAGGCCAAACCATCTGTGTGCGTTTCTGGCGATGCAGCAATTCCAAGAAGGTGTGGTATGAGTGGGCTGTGACAGCACCAGTCTGTTCTGCTATTCATAACCCCACAGGCCGCTCATATACCATTGGCCTCTGA.Generation of the PRMT5 catalytic dead mutant was performed by site-directed mutagenesis through mutation of G365 to A and R368 to A .2 incubator at 37\u00b0C. Doxycycline was used at 100 ng/ml. Cell proliferation was measured in 6-well plates (Corning) using automated confluence readings . R1881 (Sigma) and charcoal-stripped serum (Omega Scientific) were used where indicated.VCaP, 22Rv1, LNCaP and RWPE-1 cell lines (ATCC) were grown in vendor-recommended media and maintained in a humidified 5% CO2\u20135\u00a0ug of AR, ERG or IgG control antibody was coupled to 1mg of magnetic beads according to manufacturer\u2019s protocol (Invitrogen Dynabeads Antibody Coupling kit#143.11D). After coupling, 1\u00a0mg of the antibody/bead mixture was incubated with 1\u20135\u00a0mg of protein lysate overnight under rotation at 4\u00b0C. IP samples were then washed with RIPA buffer containing protease/phosphatase inhibitor cocktail for 3\u20134 washes and resuspended in non-reducing loading buffer, boiled and loaded on a gel for western blot analysis. Immunoprecipitations were performed using the following antibodies: anti-ERG antibody (Epitomics# 2805\u20131), anti-HA antibody (Roche#11815016001), anti-AR antibody (Thermo Scientific# MA5-13426) or anti-IgG antibody .Procedures were previously described and the Generation of labeled cDNA, hybridization to Affymetrix U133plus2 human arrays, and data normalization were performed as described . For theBlack line represents expressed probe set position and is ranked by average fold-change. Blue, green, and red lines indicate where the probe sets mapping to genes in the androgen receptor activation signatures appear in our data set and show the cumulative sum of the probe sets in the androgen receptor activation signatures that overlap with our gene list (only the highest expressing probe set was used per gene). The dashed line represents the hypothetical cumulative sum for a random list of genes that are unenriched.PSA, NKX3-1 and SLC45A3 and VIC-labeled probe for Beta-2-macroglobulin (B2M) as a normalization control. Samples were run on a 7900HT Real-Time PCR machine (Applied Biosystems) and data was analyzed and normalized according to manufacturer\u2019s instructions (2-\u0394Ct method).RNA isolation was performed as previously described . Taqman VCaP, 22Rv1 and RWPE-1 cells were treated as specified followed by cross-linking with 1% formaldehyde for 10\u00a0min. Cells were next lysed in 1% SDS and sonicated until DNA ladder is below 1\u00a0kb (Diagenode). Sheared chromatin was then used for IP with specific primary antibodies pre-complexed with Protein A/G Dynabeads and incubated overnight under rotation at 4\u00b0C. The next day, ChIP samples were washed with RIPA buffer and TE followed by reverse crosslinking using 1%SDS and 30\u00a0ug/ml proteinase K (Invitrogen) at 65\u00b0C for 6\u2009hr with beads. The eluates were then purified using the QIAquick PCR purification kit (Qiagen) and used for qPCR with the following primer sets:PSA -4100: acctgctcagcctttgtctc AND ttgtttactgtcaaggacaatcgPSA -3800: agaattgcctcccaacactg AND cagtcgatcgggacctagaaPSA -100: cttccacagctctgggtgt AND aaaccttcattccccaggacPSA +700: agccccagactcttcattca AND atgcagatttggggaatcagNKX3-1 -2800: gagagcagctgttcctccac AND acgagccttttccacctttcNKX3-1 -200: agggaggagagctggagaag AND tcctccctaggggattcctNKX3-1 +2150: accaggatgaggatgtcacc AND cagggacagagagagccttgNKX3-1 -+10800: tctctcgttggctcctgatt AND ccagcttttgttccttcctgNKX3-1 +62100: cggtttattgcccatgaaga AND aacagggctcacagtgctttVCaP cells harboring Dox inducible shRNAs targeting PRMT5 where grown to 80% confluency and re-seeded (day 0) into full media containing 100\u00a0ng/ml Doxycycline. On day 3 media was replaced with \u2018hormone reduced\u2019 media, containing 100\u00a0ng/ml Doxycycline. Cells were stimulated with adding indicated ligands (DHT (Sigma), R1881 [Sigma]) or vehicle on day 4 and harvesting of cells was performed on day 5. Cells were harvested by fixation using 1% methanol free formaldedyde in PBS at room temperature. Fixation was stopped after 8\u2009min by replacing fixation buffer with ice cold PBS containing 125\u00a0mM glycine and 5\u00a0mg/ml BSA. Cells were further washed once using ice-cold PBS and re-suspended into 500\u00a0ul of PBS containing Complete Protease Inhibitors (Roche). Cells where then pelleted and supernatant removed, and the resulting pellet either snap frozen in liquid Nitrogen or immediately re-suspended in lysis buffer for further processing.3 and 1% (w/v) SDS. Crosslink reversal was done at 65C\u00b0C for 6\u2009hr and ChIP DNA were isolated using DNA purification beads (MagBio). ChIP-seq libraries were generated using the KAPA HTP library preparation kit (Kapa biosciences). All handling of samples after sonication was done on using a Sciclone NGS Workstation . Sequencing was performed on an Illumina NextSeq500 instrument.For ChIP the resulting cell lysate was sonicated using a Covaris E210 instrument according to manufacturers recommendations. Each ChIP reaction was performed using soluble fraction chromatin corresponding to 7.5\u00a0ug purifed DNA and 4\u00a0ug of antibodies. Antibodies were allowed to bind overnight before capture on protein A magnetic beads . Bound beads where washed 4 times in RIPA buffer containing 500\u00a0mM LiCl, and 2 times with TE buffer before being re-suspended in containing 100\u00a0mM NaHCOReads passing Illumina standard QC were mapped to genome version Hg19 using BWA, and binding sites (\u2018peaks\u2019) were identified using MACS2, evolutionary conservation scores at peak locations was calculated using Phastcons and enriched DNA motifs using MDscan and Seqpos. These were performed using the ChiLin QC pipeline (liulab.dfci.harvard.edu/WEBSITE/software). Peaks from AR ChIP-sequencing samples with a MACS2 enrichment score higher than 10 were extended to a uniform 400\u00a0bp across all samples and overlapping peaks where collapsed to generate a union of all peaks. This resulted in a Cistrome of 25,593 peaks that were used in all genome wide ChIP analyses. Using the features of this Cistrome as GTF, read counts from BWA mapped Bam files were processed using the Qlucore 3.1.19 software. Heatmaps and statistical test (two-sided t-test using correction for multiple hypothesis testing) of differential binding scores on the 25,593 features were performed in Qlucore v3.1.19. The AR and ERG ChIPseq datasets are available at the NCBI Gene Expression Omnibus (Accession number GSE79128).RWPE-1 cells were treated as specified, fixed with 4% paraformaldehyde for 45 min at room temperature (Electron Microscopy Sciences), blocked with 5% goat serum, 0.5% Triton X-100 in PBS for 2\u2009hr and incubated with 1:50 dilutions of AR (LSBIO #LS-C87494) and symmetric di-methyl arginine antibodies (CST#13222) in 5% goat serum and 0.05% Triton X-100. Fixed samples were incubated overnight at 4\u00b0C in primary antibody before incubations with proximity ligation assay (PLA) secondary antibodies .The secondary antibody incubation, ligation, amplification and final wash steps were performed according to the manufacturer\u2019s specifications. Confocal microscopy was performed using an LSM 510 META with a 40x C-Apochromat objective, NA 1.2. Images were collected and processed using Zen software .Homo sapiens androgen receptor (AR) sequence was synthesized to include a 5\u2019 NotI and 3\u2019 BamHI sites and used as a template for the mutagenesis of each arginine in the ligand binding domain of AR into lysine . Following sequence verification to ensure mutation incorporation, each AR mutant sequence was cloned into the pLVX vector via the 5\u2019 NotI and 3\u2019 BamHI as previously described and incubated with 1:50 dilution of AR antibody (LSBIO #LS-C87494). Confocal microscopy was performed using an LSM 510 META with a 40x C-Apochromat objective, NA 1.2. Images were collected and processed using Zen software .The heterodimeric structure of PPAR\u03b3-RXR\u03b1\u00a0(PDB Code: 3DZY) was used as a template to overlay individual domain structures of AR including the DBD in complex with DNA (PDB Code: 1R4I) and the LBD in complex with coactivator peptide TIF2(iii) and ligand R1881 (PDB Code: 2AO6). A superposition was achieved using secondary structure matching in COOT . The AR \u00b0C. Cells were resuspended in buffer A containing 50\u2009mM Tris-HCl (pH 7.3), 150\u2009mM NaCl, 10% glycerol, 0.25\u2009mM TCEP, and 10 \u00b5M DHT, to which 50 \u00b5g/ml DNase I and protease inhibitor cocktail (Roche) were added. Cells were lysed using an M-110L Microfluidizer at 18,000 psi, followed by the addition of 0.5% CHAPS to the lysate prior to high speed centrifugation. For one-step batch purification, the soluble extract was incubated with 2\u2009ml of glutathione sepharose 4 fast flow medium for 1\u2009hr at 4\u00b0C with rotational mixing. The sepharose medium was washed in buffer A with the addition of 0.5% CHAPS. Elution was accomplished by resuspending and incubating the media for 10 min in the wash buffer plus 10\u201320\u2009mM reduced glutathione. The eluted fractions were then combined and concentrated to 0.3 mg/ml.The gene encoding human AR LBD (residues 663\u2013919), was inserted into a pGEX-6P-1 vector and expressed as a GST-tagged fusion protein in BL21 Star (DE3) cells. Cells were grown in TB2 medium containing 10 \u00b5M dihydrotestosterone (DHT) and induced with 1\u2009mM IPTG for 14 \u201316\u2009hr at 16S-adenosyl-L-homocysteine (SAH) product formation utilizing liquid chromatography-tandem mass spectrometry (LC-MS/MS). 0.5\u20131\u00a0uM of PRMT5/MEP50 recombinant enzyme was incubated with 50\u00a0uM SAM, 2\u00a0uM GST-AR LBD and/or 5\u00a0uM ETS or PNT ERG protein for 2\u2009hr at 37\u00b0C. Reactions were quenched to 0.1% HCOOH followed by addition of -SAH (SAH-D4) in 20% DMSO as an internal standard for MS quantification. Samples were sonicated with a Hendrix SM-100 sonicator (Microsonics Systems) and centrifuged. SAH was separated from the reaction mixture by reversed phase chromatography using polar endcapped C18 reversed phase columns and detected using a 4000 QTRAP Hybrid Triple Quadrupole/Linear Ion Trap LC-MS/MS system (AB Sciex).PRMT5 enzymatic activity was assessed by monitoring In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Kevin Struhl as the Senior Editor.Thank you for submitting your work entitled \"ERG signaling in prostate cancer is driven through PRMT5-dependent methylation of the Androgen Receptor\" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission. As you will see below, the reviewers have some concerns that will require quite an additional effort to address. Ordinarily, we allow just two months to return a revised version but we are concerned that this required experiments may take longer to complete. To help us assess the likelihood of a successful completion of the required additional work, please send us a note indicating how you plan to proceed and an estimate of the time it will take to complete the work.Summary:In this manuscript, the authors present two major findings: 1) PRMT5 methylates AR at R761 and these methylation results in decreased AR recruitment at target genes and decreased AR function and 2) ERG targets AR to be methylated. Overall, the findings address long-standing questions of ERG function in prostate tumorigenesis and possibly posit new therapies to ERG translocated prostate cancers. This would be certainly relevant to the prostate field. In addition, it is appropriate for a general audience for implications of a general enzyme that affects specific programs. The experiments are well done and logical. The manuscript is well written. However, at this point, there are several critical experiments that should be performed to solidify the conclusion.Essential revisions:1) Definition of the ERG and AR binding genome wide with and without PRMT5 and ERG knockdown and correlation with the gene expression is very important for the conclusion. Is AR binding specifically depleted in ERG co-bound sites? The changes genome wide may not reflect the selected sites, as highlighted by the effect of FOXA1 on AR. This does require time and effort. But AR and ERG ChIP-seq is well optimized and ChIP-seq today is cost effective so this is eminently doable.2) One weakness of the manuscript, intrinsic to prostate cancer research, is the use of a single ERG positive cell line. So any hit of the screen could be ERG specific, or even more likely, VCAP specific. Since there are no other available ERG dependent cell lines, the authors should note in the manuscript this major caveat. It would be very helpful if the authors could examine xenografts or perform some validation of the mechanisms in primary human tissue samples. For example, authors can IP AR from several ERG positive and ERG negative prostatectomy specimens to assess for methylation.3) Mass spec evidence of methylation at R761 would be helpful. Since you know what you're looking for, IP of AR in VCAP and another ERG negative line and looking for this should not be hard and would provide definitive evidence. The mutagenesis is highly suggestive, but could still change conformation in a way such that R761 is not the direct methylation site. Essential revisions:1) Definition of the ERG and AR binding genome wide with and without PRMT5 and ERG knockdown and correlation with the gene expression is very important for the conclusion. Is AR binding specifically depleted in ERG co-bound sites? The changes genome wide may not reflect the selected sites, as highlighted by the effect of FOXA1 on AR. This does require time and effort. But AR and ERG ChIP-seq is well optimized and ChIP-seq today is cost effective so this is eminently doable.KLK3 (aka PSA) and NKX3-1, the ChIPseq dataset shows a significant increase of AR recruitment upon PRMT5 knockdown at 6% of the total AR-bound cistrome. Genome-wide recruitment of ERG was not significantly affected upon PRMT5 knockdown, including to the sites where AR recruitment was increased . This data agrees with our model that ERG recruits PRMT5 to attenuate AR activity to a significant subset of its target genes.We thank the reviewers for raising this important point. To extend our findings beyond individual genes, we performed ChIPseq for both ERG and AR in the presence of the androgen molecules R1881 and DHT, and in the absence or presence of PRMT5 knockdown. This data is provided in 2) One weakness of the manuscript, intrinsic to prostate cancer research is the use of a single ERG positive cell line. So any hit of the screen could be ERG specific, or even more likely, VCAP specific. Since there are no other available ERG dependent cell lines, the authors should note in the manuscript this major caveat. It would be very helpful if the authors could examine xenografts or perform some validation of the mechanisms in primary human tissue samples. For example, authors can IP AR from several ERG positive and ERG negative prostatectomy specimens to assess for methylation.TMPRSS2:ERG positive models, we have explored the ERG/PRMT5 interaction using additional model systems. First, in TMPRSS2:ERG transgenic mice vs. control mice ; AR immunoprecipitates from the TMPRSS2:ERG mouse prostates show increased SDMA and MMA signals at the correct size for AR. Fifth, we show in We thank the reviewers for acknowledging the challenges of obtaining appropriate prostate models and patient specimens. To address the general lack of We further attempted to confirm our findings in human tissue samples. In the absence of an AR-R761me2s-specific antibody for immunoblotting or IHC, we planned to use a chromogenic in situ proximity ligation assay (PLA) with primary antibodies recognizing AR and symmetric di-methyl arginine (SDMA) to detect AR-R761me2s in human prostate cancer samples. First, we focused on optimizing PLA for formalin-fixed paraffin-embedded (FFPE) tissues, using FFPE RWPE-1 prostate cells, FFPE human prostate cancer samples, and two primary antibodies against Ki67 . While numerous individual spots representing positive signal were detected in the nuclei of RWPE-1 prostate cells, few, if any, spots were seen in the human tissues see , suggest3) Mass spec evidence of methylation at R761 would be helpful. Since you know what you're looking for, IP of AR in VCAP and another ERG negative line and looking for this should not be hard and would provide definitive evidence. The mutagenesis is highly suggestive, but could still change conformation in a way such that R761 is not the direct methylation site.We agree that mass spectrometry data would be an important support of our findings. We made several attempts at mass spec identification of the arginine site using AR immunoprecipitated from VCaP cells. Unfortunately, we could not identify this site by mass spectrometry. In our hands, arginine methylation has been generally difficult to detect, including on histones, which are 'classic' PRMT5 targets. We suspect that R761me2s does not occur on the entire population of AR within the cell lysate, which 'masks' the detection of methylated AR by mass spec. This may be in line with the observation that PRMT5 knockdown does not affect AR recruitment to all possible sites in our ChIPseq experiments.While not directly addressing the reviewers\u2019 question, we provide additional supporting data that PRMT5 can methylate purified AR ligand binding domain in vitro. These biochemical assays demonstrate PRMT5 activity on purified AR but not ERG, and that addition of purified ERG DNA binding domain can facilitate greater PRMT5 activity on AR. This data is provided in"} +{"text": "AbstractPhyllodonta latrata (Guen\u00e9e) has been applied to what is a complex of three undescribed species in Costa Rica. They are very similar in maculation, but can be differentiated by genitalic characters and barcodes. P. alajuela Sullivan, sp. n. occurs at lower altitudes in the northwestern part of Costa Rica whereas P. intermediata Sullivan, sp. n. and P. esperanza Sullivan, sp. n. are found at partially overlapping altitudes in the central mountain ranges.Historically, the name PageBreakPhyllodonta Warren currently consists of approximately 26 species of medium-sized geometrid moths that occur from the southern United States to Argentina (Phyllodonta latrata (Guen\u00e9e) was described from Brazil and Colombia; in the British Museum the specimen from Nova Friburgo is labeled as the type. It is unlikely that specimens from Colombia are conspecific. In Costa Rica, specimens that are very similar to Phyllodonta latrata occur from about 500\u20133000 m, but mtDNA barcode sequences of distinct clusters suggest a complex of species. Four additional species from Peru and Ecuador have been subject to barcode analysis but no haplotype clusters overlap those from Costa Rica. There are no specimens of Phyllodonta latrata from Brazil that have been barcoded nor have the types of Phyllodonta latrata nor Phyllodonta succedens been dissected. Currently, Phyllodonta succedens is placed as a synonym of Phyllodonta latrata in the British Museum collections, although Phyllodonta nolckeniata (Snellen) as a synonym.The genus rgentina . Currentrgentina . UnpubliPhotographic methods used herein are described in Natural History Museum, London, UKBMNH Instituto Nacional de Biodiversidad, Santo Domingo de Heredia, Costa RicaINBio J. Bolling Sullivan, Beaufort, North Carolina, USAJBS National Museum of Natural History, Washington, District of Columbia, USAUSNMPageBreakPhyllodonta latrata (Gn.), Phyllodonta succedens (Wlk.) and Phyllodonta nolckeniata (Snellen) are distinguished by their rich brown color and will be referred to as the Phyllodonta latrata complex. The antemedial and postmedial lines are usually distinct and are wavy or undulating. On the underside, these lines are distinct and the characteristic whitish blotch and Phyllodonta sarukhani Beutelspacher are similar in general color but the wing markings and genitalia are very different , Cartago Province, 1275 m, 7\u20139 July 2008, J. Bolling Sullivan (DNA voucher #11-CRBS-381) (INBio). Paratypes: 5\u2642, 4\u2640: 1\u2642, same data as holotype (DNA voucher #11-CRBS-288); 1\u2642, same data as holotype but 12\u201317 February 2005 (07-CRBS-1206), (dissection #JBS-3305); 1\u2642, Costa Rica, Parque National Volcan Poas , Alajuela Province, 2500 m, 7\u20138 August 2007, J. Bolling Sullivan (07-CRBS-359), (dissection #JBS-2006); 2\u2640, same data (07-CRBS-357), (JBS-2007); 3\u2642, Costa Rica, Villa Mills , Cartago Province, 2845 m, J. Bolling Sullivan , 2\u2640, same data . BMNH, INBio, JBS, USNM.Holotype male: Costa Rica, Tapanti Parque ; the vesica is unarmed with pouches emanating from the left side . In the female, the collar on the ductus bursae is narrow and the signum on the corpus bursae is crescent shaped, often with a medial kink and with the anterior and posterior sides narrowly separated. In the other two species the collar is wider and the signa are more oval shaped, never kinked.Maculation does not seem to be diagnostic for distinguishing this species. It is best characterized by barcode data and the genitalia. It has the highest altitude distribution but does overlap that of Male. . Wings\u2013forewings warm brown with prominent antemedial and postmedial lines that undulate, in both cases forming two outward bulges. Antemedial bulges separated by prominent cleft. Both lines black edged with lighter grayish scales proximally. Black scaling distal to reniform at costa and forming a diffuse line paralleling antemedial line. Reniform spot small, black and forming center of a gray circle. Forewings long, outer margin truncated at costa with a distinct notch medially, forewing length 23.0 mm . Hindwing with prominent postmedial line, margin with submedial notch. Underside of wings warm brown, forewing with postmedial line prominent, medial line visible, antemedial line absent. White blotch subterminal at notch, larger black blotch proximal to it. Small discal spot present. Hindwing underside with prominent postmedial line, prominent discal spot, small yellow line of scales distal to postmedial line on both wings. Cream streak from wing base widening to anal angle. Male Genitalia is:Twenty nine specimens have been barcoded and exhibit twelve haplotypes that differ from each other by a maximum of 0.8%. They differ from those of PageBreakGATGAACTGTTTATCCTCCTTTATCTTCTAATATTGCTCACGGTGGTAGTTCTGTTGACCTTGCTATTTTTTCATTACATTTAGCTGGTATTTCATCAATTTTAGGGGCTATTAATTTTATTACTACAATTATTAATATACGATTAAATAATTTATCTTTTGATCAAATACCTTTATTTGTATGAGCAGTAGGAATTACTGCATTTTTATTATTATTATCATTACCTGTTTTAGCTGGAGCTATTACTATATTATTAACAGATCGAAATTTAAATACATCTTTTTTTGATCCTGCTGGAGGAGGAGATCCAATTTTATACCAACATTTATTTAACATTATATTTTATTTTTGGGATTTGAGCTGGAATAGTAGGAACATCTTTAAGTTTATTAATTCGAGCTGAATTAGGAAATCCTGGATCTCTAATTGGAGATGATCAAATTTATAATACTATTGTAACTGCTCATGCTTTTATTATAATTTTCTTTATAGTAATACCTATTATAATCGGAGGATTTGGAAATTGATTAGTTCCTTTAATATTAGGAGCTCCTGATATGGCTTTCCCTCGAATAAATAATATAAGATTTTGATTACTTCCACCTTCTATTACATTATTAATTTCTAGAAGAATTGTGGAAAATGGAGCTGGGACAGKnown from above 1200 m in the Talamancas as well as the Central Volcanic and Tilaran ranges in Costa Rica. In flight throughout the year.Nothing is known about the biology of this species. Its range probably extends into the other mountain ranges in Costa Rican and perhaps in northern Panama.Taxon classificationAnimaliaLepidopteraGeometridaeSullivansp. n.http://zoobank.org/FEE6C9E5-A097-4088-983C-D0573F1F0E1C9.43\u00b0N, 83.46\u00b0W) Cartago Province, 1475 m, 7\u20138 July 2008, J. Bolling Sullivan (10-CRBS-283) (INBio), Paratypes. 5\u2642, 1\u2640: 2\u2642, 1\u2640, same data as holotype , 10-CRBS-282 (JBS-5409)); 2\u2642, Costa Rica, Tapanti Parque , Cartago Province, 1275 m, 7\u20139 July 2008, J. Bolling Sullivan, (11-CRBS-286 (JBS-5416); 1\u2642, Costa Rica, Vera Blanca, La Paz Waterfall Garden , Alajuela Province, J. Bolling Sullivan (07-CRBS-1202), (JBS-3306) .Costa Rica, tunnel road, Tapanti Parque , the vesica is unarmed and pouches emanate from the right side . In the female, the collar on the ductus bursae is narrow (broad in Phyllodonta esperanza)and the signum on the corpus bursae is peanut-shaped with moderate spacing between the anterior and posterior sides .Maculation is not diagnostic for identifying this species. It is best characterized by barcode data and the genitalia. In the male, the distal side of the socius is usually straight . Wings\u2013forewings warm brown with prominent antemedial and postmedial lines that undulate, in both cases forming two outward bulges. Antemedial bulges separated by cleft. Both lines black edged with lighter grayish scales proximally. Black scaling distal to reniform at costa and forming a diffuse line paralleling antemedial line. Reniform spot small, black and forming center of a gray circle. Wings long, outer margin truncated at costa with a distinct notch medially, forewing length 23.1 mm . Hindwing with prominent postmedial line, margin with submedial notch. Underside of wings warm brown, forewing with postmedial line prominent, medial line visible, antemedial line absent. White blotch subterminal at notch, larger black blotch proximal to it. Small discal spot present. Hindwing underside with prominent PM line, prominent discal spot, small yellow line of scales distal to PM line on both wings. Cream streak from wing base widening to anal angle. Male Genitalia is:Seven specimens have been barcoded and exhibit two haplotypes that differ from each other by a maximum of 0.5%. They differ from those of AACATTATATTTTATTTTTGGAATTTGAGCTAGAATAGTGGGAACGTCTTTAAGTTTATTAATTCGAGCAGAATTAGGGAATCCTGGGTCTTTAATTGGAGATGATCAAATTTATAATACTATTGTAACTGCACATGCTTTTATTATAATTTTCTTTATAGTAATACCTATTATAATTGGGGGATTTGGAAATTGATTAATTCCTTTAATACTAGGGGCTCCTGATATAGCTTTCCCTCGAATAAATAATATAAGATTTTGGTTACTTCCACCTTCCATTACATTATTAATTTTTAGAAGAATTGTAGAAAATGGAGCTGGAACAGGATGAACAGTTTACCCACCTTTATCTTCTAATATTGCTCATGGGGGTAGTTCTGTTGATCTTGCTATTTTTTCATTACATTTAGCTGGTATTTCATCAATTTTAGGAGCTATTAATTTCATCACCACAATTATTAATATACGATTAAATAATTTATCTTTTGATCAAATACCTTTATTTGTATGAGCGGTAGGAATTACTGCATTTTTATTATTATTATCATTACCTGTTTTAGCTGGAGCTATTACTATATTATTAACCGATCGAAATTTAAATACATCTTTTTTTGACCCTGCTGGTGGAGGAGATCCAATTTTATACCAACATTTATTTKnown from between 1275 to 2280 m in the Talamancas, the Central Volcanic range, and the Tilaran range in Costa Rica. The moths are probably in flight throughout the year.Nothing is known about the biology of this species. Its range may extend into the other mountain ranges in Costa Rican and perhaps into northern Panama.Taxon classificationAnimaliaLepidopteraGeometridaeSullivansp. n.http://zoobank.org/F193BB75-F3BF-42EC-9292-C10D65E1533110.22\u00b0N, 85.62\u00b0W), Alajuela Province, 850 m, 7\u201311 February PageBreak2005, J. Bolling Sullivan (INBio). Paratypes: 4\u2642, 1\u2640: 2\u2642, same data as holotype , 1\u2642, 1\u2640, Costa Rica, Upata. Estacion San Gerardo , Alajuela Province, 550 m, 17\u201321 July 2006, J. Bolling Sullivan , JBS-3314). 1\u2642, Costa Rica, Upata Bijagua, Alberque Heliconias , 800 m, Alajuela Province, J. Bolling Sullivan (JBS-3310).Holotype male: Costa Rica, San Ramon Reserva Biol. Alberto M. Brenes Estacion Biol. . The female has a broadened collar on the ductus bursae (narrow in Phyllodonta esperanza)and the signum on the corpus bursae is almost round with equal-sized spines around it .Maculation does not seem to distinguish this species. It is best characterized by barcode data and the genitalia. To date it has been found below 1200 m and with no other member of the Male. . Wings\u2013forewings warm brown with prominent antemedial and postmedial lines that undulate, in both cases forming two outward bulges. Antemedial bulges separated by cleft. Both lines black edged with lighter grayish scales proximally. Black scaling distal to reniform at costa and forming a diffuse line paralleling antemedial line. Reniform spot small, black, and forming center of a gray circle. Wings long, outer margin truncated at costa with a distinct notch medially, forewing length 24.1 mm . Hindwing with prominent postmedial line, margin with submedial notch. Underside of wings warm brown, forewing with postmedial line prominent, medial line visible, antemedial line absent. White blotch subterminal at notch, larger black blotch proximal to it. Small discal spot present. Hindwing underside with prominent PM line, prominent discal spot, small yellow line of scales distal to PM line on both wings. Cream streak from wing base widening to anal angle. Male Genitalia is:Twenty eight specimens have been barcoded and exhibit 9 haplotypes that differ from each other by a maximum of 1%. They differ from those of PageBreakGAGATGATCAAATTTATAATACTATTGTAACTGCTCATGCTTTTATTATAATTTTTTTTATGGTAATACCTATTATAATTGGGGGATTTGGGAATTGATTAGTTCCTTTAATATTGGGGGCCCCAGATATAGCTTTCCCACGAATAAATAATATAAGATTTTGATTACTTCCGCCTTCTATTACACTTTTAATTTCTAGAAGAATTGTAGAAAATGGAGCCGGAACTGGATGAACTGTCTACCCTCCTTTATCTTCTAATATTGCCCACGGTGGTAGTTCTGTTGATCTTGCTATTTTTTCATTACATTTAGCTGGTATTTCATCAATTTTAGGGGCTATTAATTTTATTACAACAATTATTAATATACGATTAAATAACTTATCTTTTGATCAAATACCTTTATTTGTTTGAGCTGTAGGAATCACTGCATTTTTATTATTATTATCATTACCTGTTTTAGCTGGAGCTATTACTATATTATTAACTGATCGAAATTTAAATACATCTTTTTTTGACCCTGCTGGAGGAGGAGACCCAATTTTATATCAACATTTATTCAACATTATATTTTATTTTTGGAATTTGAGCTGGAATAGTAGGTACATCTTTAAGTTTATTAATTCGAGCGGAATTAGGAAACCCTGGGTCTTTAATTGKnown from 500 to 1150 m in the provinces of Alajuela and Guanacaste. Moths are probably in flight throughout the year.Witheringia solanacea L. H\u00e9r. in the Solanaceae. One record may represent a second species on Brugmansia \u00d7 candida (Solanaceae). The present range of Phyllodonta alajuela seems to be limited to the Northwestern provinces of Costa Rica and may extend into Nicaragua. Southward and eastward its range is unknown. Specimens of the Phyllodonta latrata complex from Anchicaya and Calima Dam in Valle Province of Colombia (four dissections) have similar male genitalia, but the basal spines on the vesica are larger; no specimens of it have been barcoded. Species of non-migratory moths common to Colombia and Costa Rica are often distributed in the western lowlands of both countries.Phyllodonta latrata (Gn.) complex is represented in Costa Rica by three new species that can be differentiated by barcodes and genitalic features. Phyllodonta alajuela occurs at lower altitudes whereas Phyllodonta intermediata and Phyllodonta esperanza overlap at intermediate altitudes; but are often separate at the altitude extremes of their ranges. At the conclusion of this study, two male specimens in the Phyllodonta latrata complex from the states of Bahia and Rio De Janeiro in Brazil were borrowed from the USNM and dissected. Both dissections showed characters not seen in any other members of the complex and are supported by other less obvious characters and geographic separation.The complex nature of species grouped under the name otropics . ConstanPhyllodonta latrata complex in South America. Specimens from lowland western Colombia may be conspecific with Phyllodonta alajuela but proof is lacking. Two male specimens taken above 2500 m in the central Andean range (Alto Rio Quindio) were dissected and differed from each other and all species from Costa Rica. Two specimens from coastal Brazil also were unique. Additional specimens barcoded from Ecuador and Peru are distinct. Thus, it is likely that the type locality for Phyllodonta latrata (Brazil) and high altitude locations throughout the Andes will provide a surprising number of additional species in the complex.Barcodes and genitalic analyses also support the differentiation of other populations of the PageBreak"} +{"text": "AbstractDisphragis notabilis (Schaus) species-group are reviewed, including the types and their dissected genitalia. Disphragis hemicera (Schaus), stat. rev., is elevated to species rank, D. normula (Dognin) is retained as a synonym of D. notabilis, D. sobolis Miller is confirmed as distinct from D. hemicera, and D. bifurcatasp. n., is newly described. Both D. hemicera and D. bifurcata occur in Costa Rica. The known ranges of the other species are outlined. Defining characters of each species are presented and a key to species is provided. Unusual variation in the genitalia is noted.The four described taxa in the Disphragis notabilis (Schaus), described from French Guiana, has been applied to prominent moths throughout Central and South America. Miller described Disphragis sobolis from Ecuador and indicated that genitalic characters reveal yet another member of the complex in Ecuador from Costa Rica and Disphragis normula (Dognin) from Peru, so it is necessary to examine all named taxa in order to classify the species found in Costa Rica.The name Ecuador . CollectPhotographic methods used herein are described in Natural History Museum, London, UKBMNH Instituto Nacional de Biodiversidad, Santo Domingo de Heredia, Costa RicaINBio J. Bolling Sullivan collection, Beaufort, North Carolina, USAJBS National Museum of Natural History, Washington, District of Columbia, USAUSNMHeterocampa notabilis Schaus was named from French Guiana. Both Heterocampa hemicera Schaus from Costa Rica and Heterocampa normula Dognin from Peru are listed as synonyms of Disphragis notabilis by Disphragis sobolis from the mountains of eastern Ecuador and noted that it was easily separated from specimens of Disphragis notabilis by its darker color and more mottled appearance , Limon Province, 354\u2019, 1\u20134 July 2008, J. Bolling Sullivan. INBio. Paratypes: 11\u2642, 3\u2640: 4\u2642, same data as holotype ; 1\u2640, 22 March 2003, Monty Wood (JBS-3030); 1\u2642, Costa Rica, Est. Biol. La Selva , Heredia Province, 50\u2013150 m, 21\u201330 June 2003, Monty Volovsic (JBS-3040), 2\u2642, 29 Aug. \u20132 Sept. 2003, J. Bolling Sullivan (JBS-3038); 2\u2642, Costa Rica, Upata Estacion San Gerardo , Alajuela Province, 550 m, 17\u201321 July 2006, J. Bolling Sullivan, B. Espinosa (JBS-3035); 1\u2642, Costa Rica, Puriscal Chires, Mastatal , San Jose Province, 400 m, 16\u201318 Oct. 2011, J. Bolling Sullivan; 1\u2642, Costa Rica, Verugua Rainforest Campamento , Limon Province, 400\u2013500 m, 12\u201316 March 2010, J. Bolling Sullivan (11-CRBS-2066), (JBS-5427); 1\u2640, Costa Rica, Tuis, 2500\u2019, June, W. Schaus 1910-110. (BM-); 1\u2640, Costa Rica, Cashi, 8\u201310 1912 (Lankester), Rothschild Bequest, B. M. 1939-1. (BM-). Holotype male: Costa Rica, Reserva Hitoy Cerere . Longest rami 0.44 mm. Thorax a blend of fine brown and cream scales giving a tan appearance. Metathorax bearing a central white spot with row of darker brown scales anteriorly. Abdomen with appressed brown scaling. Forewing (17.5 mm N = 10) elongate, rounded apically and with broad tan subcostal streak from base of wing to apex. Streak encloses chocolate reniform spot and has several slightly darker brown lines crossing obliquely from costa. Basal dash below streak paralleling costa. White streak below basal dash; warm brown patch distal to white streak bordered by white; wavy antemedial (AM) and postmedial (PM) lines. Chocolate shading from middle of forewing below costal streak and forming a wedge to margin . Weak gray crescent on lower half of margin. Hind wing fuscous with darker margin and veins, weak darker brown anal markings almost a spot at anal angle. Underside of forewing fuscous, anal margin and cell yellowish. Basal 3/4 of hind wing yellowish, margin brown and well differentiated. Legs a mixture of brown and white scales, appearing almost yellowish, with white scales forming rings at distal end of tarsal segments. Tibial spines 0-2-4. Male genitalia and with fasciculate antennae. Female genitalia is:Five barcoded specimens exhibit two haplotypes that differ from each other by a maximum of 0.15%. They differ from AACCTTATATTTCATTTTTGGAATTTGAGCAGGAATAGTAGGAACCTCTTTAAGTCTTCTAATTCGTGCTGAATTAGGAACCCCCGGGACTTTAATTGGAGATGATCAAATTTATAATACTATTGTAACAGCTCATGCTTTCATTATAATTTTTTTTATAGTAATACCTATTATAATTGGAGGATTTGGAAATTGATTAGTACCTTTAATATTAGGAGCCCCAGACATAGCTTTCCCACGAATAAATAATATAAGTTTTTGATTATTACCTCCTTCTTTAATACTTTTAATTTCGAGAAGTATTGTAGAAAATGGAGCAGGAACAGGATGAACAGTTTACCCACCACTGTCATCTAATATTGCTCATAGAGGAAGCTCTGTTGATTTAGCCATTTTTTCCCTTCACTTAGCTGGTATTTCATCAATTTTAGGGGCTATTAATTTTATCACAACAATTATTAATATACGATTAAATAATATATCTTTTGATCAAATACCTTTATTTGTGTGAGCTGTAGGAATTACTGCTTTTTTACTTTTACTTTCTCTCCCAGTTCTAGCTGGAGCTATTACTATACTTTTAACTGATCGTAATTTAAATACATCTTTTTTTGACCCTGCAGGGGGAGGAGATCCTATTTTATACCAACATTTATTTPageBreakKnown from Guatemala to Colombia , and probably extending south into northern Ecuador.Disphragis hemicera.This species occurs at lower altitudes and moderate elevations (1000 m) where it occurs with Taxon classificationAnimaliaLepidopteraNotodontidaestat. rev.Heterocampa hemicera Schaus, 1910, Annals and Magazine of Natural History 6: 582.Costa Rica.Disphragis hemicera and Disphragis sobolis from Disphragis notabilis and Disphragis bifurcata. Their appearance is mottled, grayish brown with a distinct dark band next to the PM line. Males may be distinguished by the shape of the phallus, which in Disphragis sobolis has a distinct dorsal projection. Females can be separated by the shape of the genital plate, which in Disphragis sobolis is bifurcate at the distal tip and in Disphragis hemicera has a middle phalanx with lateral \u201cwings\u201d from the base. Geographic distribution also separates Disphragis hemicera from Disphragis sobolis, with Disphragis hemicera in Central America and western Colombia, and Disphragis sobolis along the western slopes of the Andes.Maculation will usually separate Male. . Rami noticeably longer than in Disphragis bifurcata, longest rami 0.53 mm. Thorax a blend of brown and cream scales giving a tan appearance. Metathorax with a central white spot with row of darker brown scales anteriorly. Abdomen with appressed brown scaling. Forewing elongate, rounded apically and with broad light brown subcostal streak from base of wing to apex. Streak encloses chocolate reniform spot and has several slightly darker brown lines crossing obliquely from costa. Basal dash below streak perpendicular to thorax. White streak below dash; warm brown patch distal to white streak bordered by white; AM and PM lines wavy. Distinct brown line bisecting warm brown patch. Chocolate shading from middle of forewing below costal streak and forming a wedge to margin . Gray crescent on lower half of margin with distinct brown band inward to PM line. Hind wing uniformly fuscous with brown anal markings forming something of a spot at anal margin. Light streak along anal edge. Underside of forewing fuscous with yellowish subapical crescent along costa. Basal half of hind wing yellowish, no well-differentiated margin. Legs a mixture of brown and white scales appearing somewhat yellowish with white scales forming rings at distal end of tarsal joints. Tibial spines 0-2-4. Male genitalia is:Fifty eight barcoded specimens exhibit seven haplotypes that differ from each other by a maximum of 0.30%. They differ from those of AACTTTATATTTTATTTTTGGAATTTGAGCAGGAATAGTAGGAACTTCTTTAAGTCTTTTAATTCGTGCTGAATTAGGAACCCCCGGGACTTTAATTGGAGATGATCAAATTTATAATACTATCGTAACAGCTCATGCTTTTATTATAATTTTTTTTATAGTTATACCTATTATAATTGGAGGATTTGGAAATTGATTAGTCCCTTTAATACTAGGAGCACCAGATATAGCTTTCCCACGAATAAATAATATAAGTTTTTGACTATTACCCCCTTCTTTAATACTTCTAATTTCAAGAAGTATTGTAGAAAATGGAGCTGGTACAGGATGAACAGTTTATCCCCCACTGTCATCAAATATTGCTCACGGAGGAAGCTCTGTTGATTTAGCTATTTTTTCCCTTCATTTAGCGGGTATTTCCTCAATTTTAGGGGCTATTAATTTTATTACAACAATTATTAATATACGATTAAATAATATATCTTTTGATCAAATACCTTTATTTGTATGAGCTGTAGGAATTACTGCTTTTCTACTTTTACTTTCACTCCCAGTATTAGCTGGAGCTATTACTATACTTTTAACCGATCGTAATTTAAATACATCTTTTTTCGACCCTGCTGGGGGAGGAGATCCTATTTTATACCAACATTTATTTPageBreakDisphragis hemicera occurs throughout Costa Rica at moderate altitudes. It is found south along the western coast of Colombia and may extend to the west coast of Ecuador. The northern limits are unknown but it probably occurs at least into Nicaragua.Disphragis hemicera is by far the most common member of the group in Costa Rica and appears to be absent below 500 m. At moderate altitudes both Disphragis hemicera and Disphragis bifurcata occur together.Taxon classificationAnimaliaLepidopteraNotodontidaeHeterocampa notabilis Schaus, 1906, Proceedings of the United States National Museum 29: 253.Heterocampa normula Dognin, 1909, Annales de la Soci\u00e9t\u00e9 entomologique de Belgique 53: 81. Disphragis notabilis: French Guiana; Disphragis normula: PeruDisphragis notabilis and Disphragis bifurcata from Disphragis hemicera and Disphragis sobolis. Disphragis notabilis and Disphragis bifurcata are warm brown, not mottled or brownish gray like Disphragis hemicera and Disphragis sobolis. The male antennal pectinations are shorter in Disphragis notabilis than in Disphragis hemicera and Disphragis sobolis. Males of Disphragis notabilis are easily distinguished by their moderately wide socii, which taper to a single point with many ventral spines. In males of Disphragis bifurcata the socii are much broader and are bifurcate at the upturned apex. Females must be sorted by maculation and geography. Disphragis notabilis is Amazonian in distribution whereas Disphragis bifurcata occurs from central and western Colombia north into Central America.Maculation characters can usually be used to separate Male. . Longest rami 0.34 mm, shortest of all species. Thorax a blend of brown and cream scales giving a tan appearance. Metathorax bearing a central white spot with row of darker brown scales anteriorly. Abdomen with appressed brown scaling. Forewing elongate, rounded apically and with broad tan subcostal streak from base of wing to apex. Streak encloses chocolate reniform spot and has several slightly darker brown lines crossing obliquely from costa. Basal dash below streak perpendicular to thorax, abbreviated relative to that of Disphragis bifurcata. White streak below dash; warm brown patch distal to white streak bordered by white; AM and PM lines wavy. Chocolate shading from middle of wing below costal streak and forming a wedge to margin . Weak gray crescent on lower half of margin. Warm brown from patch expanded almost to margin and reducing size of chocolate wedge seen in Disphragis bifurcata. Hind wing fuscous with darker margin, weak darker brown anal markings almost forming a spot. Underside of forewing fuscous, anal margin and cell yellowish. Basal 3/4 of hind wing yellowish, margin brown and moderately differentiated. Legs a mixture of brown and white scales appearing almost yellowish with white scales forming rings at distal end of tarsal joints. Tibial spines 0-2-4. Male genitalia is:Two barcoded specimens exhibit 2 haplotypes that differ from each other by 0.30%. They differ from those of AACTTTATATTTCATTTTTGGAATTTGAGCAGGAATAGTAGGAACCTCTTTAAGTCTTCTAATTCGTGCTGAATTAGGAACCCCCGGGACTTTAATTGGAGATGACCAAATTTATAATACTATCGTAACAGCTCATGCTTTCATTATAATTTTTTTTATAGTAATACCTATTATAATTGGAGGATTTGGAAATTGATTAGTACCTTTAATATTAGGAGCCCCAGACATAGCTTTCCCACGAATAAATAATATAAGTTTTTGATTATTACCTCCTTCTTTAATACTTTTAATTTCAAGAAGTATTGTAGAAAATGGAGCAGGAACAGGATGAACAGTTTACCCACCACTGTCATCTAATATTGCCCATAGAGGAAGCTCTGTTGATTTAGCCATTTTTTCCCTTCACTTAGCCGGTATTTCATCAATTTTAGGGGCTATTAATTTTATCACAACAATTATTAATATACGATTAAATAATATATCTTTTGATCAAATACCTTTATTTGTATGAGCTGTAGGAATTACTGCTTTTTTACTTTTACTTTCTCTTCCAGTTCTAGCTGGAGCTATTACTATACTTTTAACTGATCGTAATTTAAATACATCTTTTTTTGACCCTGCAGGGGGAGGAGATCCTATTTTATACCAACATTTATTTThis species occurs throughout the Amazon basin from western Venezuela eastward and southward to at least Bolivia.Disphragis notabilis is by far the most common member of the group in South America, however, earlier references to this species should be confirmed in light of the additional species described here.Taxon classificationAnimaliaLepidopteraNotodontidaeMiller, 2011Disphragis sobolis Miller, 2011. In Ecuador.Male. . Rami noticeably longer than in Disphragis hemicera, longest 0.59 mm. Thorax a blend of brown and cream scales giving a tan appearance. Metathorax bearing a central white spot with row of darker brown scales anteriorly. Abdomen with appressed brown scaling. Forewing elongate, rounded apically and with broad light brown subcostal streak from base of wing to apex. Streak encloses chocolate reniform spot and has several slightly darker brown lines crossing obliquely from costa. Brown scaling throughout as well as several black streaks. Basal dash below streak perpendicular to thorax and greatly reduced in length. White streak below dash; warm brown patch distal to white streak bordered by white; AM and PM lines wavy. Distinct brown line bisecting warm brown patch. Chocolate shading from middle of forewing below costal streak and forming a wedge to margin more extensive than in Disphragis hemicera. Prominent gray crescent on lower half of margin with distinct brown band inward to PM line. Hind wing uniformly fuscous with brown anal markings almost a spot. Light streak along anal edge. Underside of forewing fuscous with yellowish subapical crescent along costa. Basal half of hind wing yellowish, no well-differentiated margin. Legs a mixture of brown and white scales, appearing almost yellowish with white scales forming rings at distal end of tarsal joints. Tibial spines 0-2-4. Male genitalia is:One specimen has been barcoded and differs from that of AACTTTATATTTTATTTTTGGAATTTGAGCAGGAATAGTAGGAACCTCTTTAAGTCTCCTAATTCGTGCTGAATTAGGAACCCCCGGGACTTTAATTGGAGATGATCAAATTTATAATACTATTGTAACAGCTCATGCTTTTATTATAATTTTTTTTATAGTAATACCCATTATAATTGGAGGATTTGGTAATTGATTAGTTCCTCTAATATTAGGAGCTCCAGATATAGCTTTCCCACGAATAAATAATATAAGTTTTTGATTATTACCCCCCTCTCTAATACTTTTAATTTCAAGAAGTATTGTAGAAAATGGAGCAGGAACAGGATGAACAGTTTACCCCCCACTGTCATCAAACATTGCTCATAGAGGAAGATCTGTTGATTTAGCTATTTTTTCCCTTCACTTAGCAGGTATTTCATCAATTTTAGGAGCTATTAATTTTATTACAACAATTATTAATATACGATTAAATAACATATCTTTTGATCAAATACCTTTATTTGTTTGAGCTGTAGGAATTACTGCTTTTTTACTTTTACTCTCTCTTCCAGTATTAGCAGGAGCTATTACTATATTATTAACCGATCGTAATTTAAATACATCTTTTTTTGACCCCGCTGGGGGAGGAGATCCTATTTTATATCAACATTTATTTPageBreakThis species appears to be limited to the eastern slopes of the Andes from Bolivia to Villavicencio, Colombia.Disphragis sobolis was recently described from Ecuador; the species appears to have a much greater geographical range and occurs to almost 3000 m. The lower altitude limits of its range are undefined, as is the southern boundary.PageBreakDisphragis notabilis complex is typical of many neotropical species. When studied in detail, they frequently are found to consist of a number of very similar species that do have structural differences, can be separated by barcoding, and occupy different altitudes or geographic ranges. The correct generic placement of this complex is not in Disphragis and makes the delineation of species and their defining characters with female specimens extremely difficult. Fortunately, such extreme variation has not been observed in males.A second difficulty encountered in this study was the hyper-variation of the female genital plate in hemicera . This inDisphragis notabilis complex found in Colombia (and probably Ecuador) remains to be elucidated but should highlight the individual habitat requirements of each species. Neither larvae nor foodplants are known. The geographical area where Disphragis bifurcata and Disphragis notabilis come into contact should be particularly interesting because the two species differ by 1.4% in their barcodes, a magnitude lower than between most congeneric species. However, there are a number of characters that separate the two species and characters do not seem to intergrade in individuals from central Colombia and western Venezuela.The exact distribution of the four species of the"} +{"text": "Nicotiana tabaccum L.) and rustica tobacco (Nicotiana rustica L.) were analysed between the third intron and the fourth intron of the propylene alcohol dehydrogenase gene. The results showed that the alcohol dehydrogenase gene is a low-copy nuclear gene. The intron sequences have a combination of single nucleotide polymorphisms and length polymorphisms between common tobacco and rustica tobacco, which are suitable to identify the different germplasms. Furthermore, there are some single nucleotide polymorphism sites in the target sequence within common tobacco that can be used to distinguish intraspecific varieties.A pair of primers was designed to amplify the propylene alcohol dehydrogenase gene sequence based on the cDNA sequence of the tobacco allyl-alcohol dehydrogenase gene. All introns were sequenced using traditional polymerase chain reaction (PCR) methods and T-A cloning. The sequences from common tobacco ( Nicotiana sp.) is one of many plants currently used as models for molecular studies and is an economically important crop. The interspecific phylogenetic relationships within tobacco are complicated due to complex hybridization events within the tobacco lineage. DNA molecular markers have been used to identify germplasms and to accelerate the breeding cycle. The second generation of molecular markers that include random amplification of polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP) and simple sequence repeats (SSR) has been employed broadly to differentiate tobacco varieties,[Tobacco .The specific primers were designed to target the boundary between the third intron and the fourth intron of the tobacco propylene alcohol dehydrogenase gene, and the IP sequences were identified as follows, IP-S: GTGCTGGAAGCAAAGAAAAG, IP-A: CAATTCCATCAGGGAAGTAC. The PCR materials included 5\u00a0\u03bcL of 10 \u00d7 PCR buffer, 4\u00a0\u03bcL of dNTP (2.5\u00a0mmol/L each), 1\u00a0\u03bcL of each primer (10\u00a0\u03bcmol/L), 1\u00a0\u03bcL of the DNA template and 1.5\u00a0U of PCR was performed at 94\u00a0\u00b0C for 5\u00a0min, then 94\u00a0\u00b0C for 30\u00a0s, 58\u00a0\u00b0C for 30\u00a0s, 72\u00a0\u00b0C for 1\u00a0min for 30 cycles, with a hold at 72\u00a0\u00b0C for 20\u00a0min. The PCR products were ligated to a pGEM-T vector and sequenced on an ABI 3730 sequencer after electrophoretic detection in a 1.0% agarose/EB gel.The propylene alcohol dehydrogenase gene of sample C1 contained four introns and five exons that were 225, 178, 167, 83 and 379\u00a0bp long . The boundary sequence between the intron and exon segments were the sites of interest for which a primer was designed to target.Nicotiana rustica) exhibited two bands when both primers, IP-S and IP-A, were used, while those of common tobacco (sun-shined tobacco and flue-cured tobacco) exhibited only one band on the 1.0% agarose/EB gel .In the common tobacco, the shorter fragment was 559-bp long in the target region, and the two introns found were 223 and 253\u00a0bp. There were no length differences in this fragment among the 12 samples sequenced, and the consensus sequence was as follows:GTAAGTTCCATTGCGTTGGATCTTCATAGTGTTGAATCTATCTTCATTCTTCGTTATGTTAATCATTGTGCTCCGTCCACCATCAAACCCAAAGGTTGGGGTTGGGGTTGGGGTTAGGGTTGGGTGGGTTATGCCTTTGGGGGGAAAGAGAGGTTTTAGTTTAATAGACCATGATGTTTAAAACTCTTGTTATTTAATGTCTCTTTTTTATTCATTTCCAAAGGTTGATCTGTTGAAGAGCAAATTTGGTTTTGACGAGGCTTTTAACTATAAAGAGGAGCAGGATTTAAATGCAGCTTTGAAGAGGTGAGTAATCTTACTTCCTGCTACAAAACAATCTACCTATTGTACAAAAGCAGTTAAAGATGTTCTGTGAAAAGTATGATGATATTATGTAGATAGTGTGAAACTTATAGAAAGACTTTAAGATATGCCTACGAAAACCGAGGAACATGGATCATTTCAAATCTGGCTTTTCATGAATATGGAACCCCATTCACTAGATTTTAGTGCGAAATTCCTAGACTTGAAGTGACTGGAAATACTGGTTTGTTATTAGwww.ebi.ac.uk/Tools/clustalw2/). The results indicated that there were two transitions and one transversion in addition to three gaps within the third intron. The two transitions and one transversion were also present within the fourth exon, but no amino acid replacement had occurred. There were four transitions and one transversion within the fourth intron as well . These polymorphic sites within the third and fourth intron may function as references to distinguish the varieties of common tobacco studied here.In the common tobacco, the longer sequence obtained was between 588 and 591-bp long in the target region, and the two introns were either between 194 and 197\u00a0bp or 311\u00a0bp in length. The sequences from 13 samples were analysed with the software ClusterW2 (In the rustica tobacco, the shorter sequences were 442-bp long in the target region, and the two introns found were 195 and 164\u00a0bp in length. No length polymorphisms were found in this fragment among the 12 samples sequenced, and the consensus sequence obtained was as follows:GTAAGTTCCTTCATAGTGTTGAAGCTCTCTTCATTCTTCGTTATGTTAATTATTGTGCTCCGTCCTCCATCAAACCCAAAGGTTGGGGTTTTGTGGGTTATGAGGAGGAGAAAGGGGTATACCTTTGGGGGGGAAGAGATGTTTTAGTTTAATAGACCATGATGTTTAACTCTCTTTTTATATTCATTTCCAAAGGTTGATCTGTTGAGGAACAAATTTGGTTTTGACGAAGCTTTTAACTATAAAGAGGAGCAGGACTTAAATGCAGCTTTGAAGAGGTGAGTAATCTTACTACTTGGCCTGGTGTTAGAGAAGCAGTTAGCAACAGCTTTTCATCAATATTTCCATAGAATGATATTTATAATGGAAGTCCCAAAGTAAAGATGTTCAGTGAAAAGTATGCCTGTGAAAACTGAGGGAAATAATACTGGTTTCTTATTAGIn the rustica tobacco, the longer sequence was 583-bp long in the target region, and the two introns found were 220 and 280\u00a0bp in length. No polymorphisms were found in this fragment among the 12 samples sequenced as well, and the consensus sequence was as follows:GTAAGTTCCATTGCGTTGGATGTTCATAGTGTTGAAGCTATCTTCATTCTTCGTTATGTTAATCATTGTGTTCTGTCCACCGTCAAACCCAAAGGTTGGGGTTTTGTGGGTTATGAGGAGGAGAAAGGTATACCTTTGGGGGGAAGAGATGTTTTAGTTTAATAGACCATGATGTTTAACTATCTTTTTATTTAATGTCTCTTTTTATTCATTTCCAAAGGTTGATCTGTTGAAGAGCAAATTTGGTTTTGACGAAGCTTTTAACTATAAAGAGGAGCAGGACTTAAATTCAGCTTTGAAGAGGTGAGTAATCTTACTTCCTGTTAGAAAACAATCTACCTATTGTAACAAACTTGCCCTGGTGTTAGAGAAGCAGTTAGCAACAGCTTTTCCTTTTCATGTAAACGGAATCATCGATATTTCCATAGAATGATATTTATTCCTTGAACTGAAAAATAGAAGTCCCAAAGTAAAGATGTACAGTGAAAAGTATGAAGATTGTGTGAAAATTATAGAAAAGACTTTAGGATATGCGTATGAAAACTGAGGGAAATTCCTCCCTGGAATACTGGTTTCTTATTAGIn this study, the tobacco propylene alcohol dehydrogenase gene proved to be a low-copy nuclear gene. It could have formed as a result of divergent evolution after gene duplication, or by a parallel evolutionary process between the two sets of genomes, as the tobacco plants tested were all allotetraploid. Although the PCR product for the allyl-alcohol dehydrogenase gene of sample C1 was sequenced successfully, this does not mean that only one copy of the target gene is present in common tobacco. There were obvious intron length polymorphisms between common tobacco and rustica tobacco, which were mostly due to different modes of evolution of the two kinds of tobacco.Nicotiana sylvestris.[N. tomentosiformis,[N. otophora or from introgressive hybridization of N. otophora and N. tomentosiformis within the subgenus of common tobacco.[N. rustica originated from a natural chromosome duplication event after ancestral hybridization of N. paniculata and N. undulate.[N. rustica L. group and Nicotiana tabaccum L. group, after an analysis that used AFLP methods coupled with unweighted pair group method with arithmetic mean analysis, which was consistent with our results in this work. Furthermore, the two categories of tobacco can be distinguished using only one pair of primers according to the results of this work.It is thought that the maternal ancestor of common tobacco was lvestris. It is alsiformis, N. otoph tobacco. It has bundulate. Yang et\u00a0undulate. divided The two copies of the propylene alcohol dehydrogenase gene found in tobacco each contained four introns and five exons. The length polymorphisms of the intron existed intraspecifically as well as interspecifically. The primers produced here, IP-S and IP-A, were able to easily differentiate common tobacco from rustica tobacco in a 1% agarose gel. Some SNPs of the intron within common tobacco were found, and may be useful in the identification of varieties of this species.http://dx.doi.org/10.1080/13102818.2014.907651.Supplemental data and research materials for this article can be accessed at"} +{"text": "Mycobacterium avium and Mycobacterium abscessus for potential antiviral activity. Both of these IFITMs conferred a moderate level of resistance to influenza virus in human cells, identifying them as functional homologues of IFITM3. Analysis of sequence elements shared by bacterial IFITMs and IFITM3 identified two hydrophobic domains, putative S-palmitoylation sites, and conserved phenylalanine residues associated with IFITM3 interactions, which are all necessary for IFITM3 antiviral activity. We observed that, like IFITM3, bacterial IFITMs were S-palmitoylated, albeit to a lesser degree. We also demonstrated the ability of a bacterial IFITM to co-immunoprecipitate with IFITM3 suggesting formation of a complex, and also visualized strong co-localization of bacterial IFITMs with IFITM3. However, the mycobacterial IFITMs lack the endocytic-targeting motif conserved in vertebrate IFITM3. As such, these bacterial proteins, when expressed alone, had diminished colocalization with cathepsin B-positive endolysosomal compartments that are the primary site of IFITM3-dependent influenza virus restriction. Though the precise evolutionary origin of vertebrate IFITMs is not known, our results support a model whereby transfer of a bacterial IFITM gene to eukaryotic cells may have provided a selective advantage against viral infection that was refined through the course of vertebrate evolution to include more robust signals for S-palmitoylation and localization to sites of endocytic virus trafficking.Interferon induced transmembrane proteins (IFITMs) found in vertebrates restrict infections by specific viruses. IFITM3 is known to be essential for restriction of influenza virus infections in both mice and humans. Vertebrate IFITMs are hypothesized to have derived from a horizontal gene transfer from bacteria to a primitive unicellular eukaryote. Since bacterial IFITMs share minimal amino acid identity with human IFITM3, we hypothesized that examination of bacterial IFITMs in human cells would provide insight into the essential characteristics necessary for antiviral activity of IFITMs. We examined IFITMs from Interferon-induced transmembrane proteins (IFITMs) encoded by a variety of vertebrates, including pigs, chickens, bats, fish, mice, and humans, have been shown to restrict cellular infection by specific viruses ,3,4,5,6.The essentiality of mouse IFITM3 in restricting influenza virus infection was demonstrated by showing that IFITM3 knockout mice succumb to sublethal doses of virus and exhibit higher lung titers and more severe lung pathology than wild type mice ,15. SimiAn extensive evolutionary study of IFITMs posited that vertebrate IFITMs originated from a horizontal gene transfer from bacteria to a common ancestor of choanoflagellates and metazoans . WhetherExpression constructs in the pCMV-HA vector or pCMV-myc vectors encoding mouse and human HA-IFITM3, human myc-IFITM3, and mouse HA-IRGM1 have been previously described ,20,21. UMAV IFITMGAATTCCCATGACCCAACCACCTCCTCCCCCGCCACCACCAGGATATCCCCCACAACAGCCAGCCGCTCAGGCTCCTAATAACTATCTGGTGTGGTCTATCCTTGTCACTCTCTTCTGCTGCCTGCCGTTTGGCATTGTCGCTATCGTAAAGAGCTCTCAAGTGAACGGACTTTGGGCACAGGGTAGATATGCTGAGGCACAGGCCTCCGCAGACAGTGCCAAGAAATGGGTGATATGGAGCGCAGTTATAGGCGTCGTGGTGGGAATAATCTATGGAATCCTTATGGCCGTAGGCGCCCTCAACACAAATACAAACGCGGCCCTCGCCGCGATGTTTTAGTAAGTCGAC MAB IFITMGAATTCCCATGAGTGATGAAACCAAAAGCGACGAGCCTACAGGCGCTATCACCACACCGACCCCTCCTCCCCCACCGGCTCCTGCCTCTGTGACTGGCCCACCCAAACCCCCACCCACTAACGTGGGTTGGGCCGTCGCTAGCGTGATTTTTTTCTGGCCTCTGGCATTTAGCGCATTCACCAATGCACTGAATGTGACTCAGTTTTGGCTGACGGGGCAGTATGATCGGGCCCAGGAGTCTAGCGATCGGGCCAAGCTCCTGGGAAAGATTGCCCTCCTGACCGGGTTGGTACTGCTGTTCCTGTTCATCACCCTCCGCATTGCCTGCGCCATCTGGTGGCACTCACATGGTGGGGGATGGGGTCATCATGGCGGATGGCATAGGAGTTGGGACGACGGCGGGTGGGATGGCCCTGGCCCCATCGGGCCTATGGGTAGGCCGGGTCGCGACAACTAGTAAGTCGAC2 at 37 \u00b0C. For imaging experiments, cells were grown on sterilized glass coverslips in 12-well plates. For all other experiments, cells were grown in 12-well or 6-well plates. Cells were transfected overnight using LipoJet according to the manufacturer\u2019s instructions. For biochemical experiments, 2 ug/well of each plasmid was transfected into cells grown in 6-well plates. For infection experiments, 1 ug/well of each plasmid was transfected into cells grown in 12-well plates, with the exception of plasmid encoding HA-MAB IFITM for which 0.5 ug/well was transfected. For examination of palmitoylation, transfected cells were labeled with 50 uM alk-16 for 1 h as previously described in detail [HEK293T cells were obtained from the ATCC and were grown in DMEM supplemented with 10% FBS in a humidified incubator with 5% COn detail ,22,23. In detail ,22,23. In detail . Sendai n detail . Cells wFor palmitoylation experiments, cells were lysed with 1% Brij Buffer , pH 7.4) containing EDTA-free protease inhibitor cocktail. For co-immunoprecipitation experiments, cells were lysed with 1% digitonin in PBS containing EDTA-free protease inhibitor cocktail. Immunoprecipitations were performed with either anti-HA or anti-myc EzView Affinity Gel (Sigma). For palmitoylation experiments, immunoprecipitations were washed 3 times with standard RIPA buffer , and for co-immunoprecipitation experiments, the immunoprecipitations were washed 5 times with 0.1% digitonin in PBS. All detergents were purchased from Sigma. Western blotting was performed with anti-HA antibody or anti-myc antibody . For immunofluorescence and flow cytometry, cells were fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.1% Triton X100 in PBS for 10 min, and blocked with 2% FBS in PBS for 10 min. For immunofluorescence, cells were stained with the anti-HA or anti-myc antibodies described above that were directly labeled with Alexafluor 488 or 555 using 100 ug antibody kits from Life Technologies. Slides were mounted with Prolong Gold Antifade Reagent containing DAPI from Life Technologies and images were taken using an Olympus Fluoview FV10i confocal microscope. Quantification of Manders overlap coefficients was performed using the Just Another Colocalization Plugin (JACoP) for ImageJ software. For flow cytometry, cells were stained with anti-IFITM3 antibody and anti-rabbit-Alexafluor-488 secondary antibody (Life Technologies), or with anti-HA antibody directly conjugated to Alexafluor-488 and anti-influenza NP antibody directly conjugated to Alexafluor-647 as previously described [Mycobacterium abscessus (MAB) was significantly divergent from the majority of the remaining mycobacterial IFITMs (Mycobacterium avium (MAV) IFITM as a representative of the more typical mycobacterial IFITMs (Evolutionary studies of the IFITM gene family have identified numerous IFITM genes in different vertebrates ,26,27. Ol IFITMs A. For ful IFITMs A. Clustal IFITMs B, 65.6%,l IFITMs B. Howevel IFITMs D. One iml IFITMs B. We, anl IFITMs B.S-palmitoylated cysteines, including an S-palmitoylated di-cysteine motif, within the first hydrophobic domain [S-palmitoylation site on murine IFITM1 [Despite the major differences between the amino acid sequences of IFITM3 and mycobacterial IFITMs, a similar fundamental domain structure could be identified in each of these proteins. Two hydrophobic domains separated by approximately 30 amino acids are defining characteristics of IFITMs and were present in both MAV and MAB IFITMs B. Most vc domain ,31,32 (Fe IFITM1 (Figure e IFITM1 . InteresGiven the important differences and striking similarities between the mycobacterial IFITMs and IFITM3 B, we souWe synthesized codon optimized MAB and MAV IFITM genes for insertion into the pCMV-HA mammalian expression vector in-frame with the HA tag of the vector, and transfected these constructs into HEK293T cells. After transfection, we observed high expression of MAB IFITM, far greater than IFITM3 expression from the same vector, and a more similar expression level of MAV IFITM A. Thus, S-palmitoylation of IFITM3 is important for its proper membrane targeting and maximal antiviral activity [S-palmitoylation sites on IFITM3 (S-palmitoylation site present on murine IFITM1 [S-palmitoylation of these IFITMs (S-palmitoylation (S-palmitoylation of IFITMs is a driver of maximal antiviral activity [activity ,12,36. Uactivity ,23,45,46n IFITM3 B. MAB IFn IFITM3 B that ise IFITM1 . Using ce IFITMs B. We nexe IFITMs C. Similae IFITMs C. Consisoylation A,B, mutaoylation C. Overalactivity .IFITM3 was reported to homodimerize and also to heterodimerize with other IFITMs ,47. ThisN-terminus that interacts with endocytic adaptor complexes, resulting in the effective trafficking of IFITM3 from the plasma membrane to endosomes [S-palmitoylation that we observed for mycobacterial IFITMs compared to IFITM3 (IFITM3 has been reported to co-localize with early and late endosomal markers and with markers of lysosomes and multi-vesicular bodies ,29,36,48ndosomes ,29. Thisndosomes (Figure o IFITM3 A,B, thiso IFITM3 C.Monosiga brevicollis [Monosiga brevicollis. IFITMs were also identified in multiple ancient bacteria species, and given the identifiable sequence similarities between bacterial IFITMs and metazoan IFITMs (S-palmitoylation (Though the exact evolutionary origin of vertebrate IFITMs is unknown, an IFITM with a splicing pattern that is conserved in all metazoans was identified in vicollis , a unicen IFITMs B, it wasn IFITMs C,D. Confn IFITMs D, and thoylation , which iS-palmitoylation level and that positioning of this domain evolved toward optimal modification in vertebrates. Alternatively, other determinants may affect interaction with the as yet unidentified palmitoyltransferase enzyme that modifies IFITMs. Further interrogation of MAV IFITM may shed light on these important questions.Many aspects of IFITM3 cell biology that are not fully understood may possibly be clarified by study of the mycobacterial IFITMs. For example, IFITM3 is known to be optimally active when expressed at high levels in cells, and its turnover is promoted by post-translational ubiquitination . HoweverThe precise mechanism of antiviral action of IFITMs is also currently unknown. Two potentially complementary models have been proposed for the anti-influenza virus activity of IFITM3. One, IFITM3 may directly alter fluidity of endolysosomal membranes ,10,49 th"} +{"text": "AbstractHarpacticella Sars, 1908 is described from a tidal pool on Jeju Island, Korea. Harpacticellajejuensissp. n. is closely related to Harpacticellaitoi Chang & Kim, 1991, with regard to the structure of P1 exp-1 and enp-1, the length of P1 exp-1 and exp-2, and the setal number of the P5 exopod in males. However, the new species is clearly distinguishable from Harpacticellaitoi by the combined following characters: six setae on the P5 exopod in females, one naked seta on the inner margin of P1 exp-2, the short endopod of P1 compared to the exopod, and a naked long seta on the proximal inner margin of the P5 exopod of males. The mtCOI partial sequence is provided as a DNA barcode for the new species.A new species of the genus Harpacticella Sars, 1908 is a genus of harpacticoid copepods, family Harpacticidae Dana, 1846. The genus has been reported from various habitats , mostly in Asian waters , and Harpacticellaamurensis Borutzky, 1952 were described from freshwater, Harpacticellalacustris Sewell, 1924 and Harpacticellaitoi Chang & Kim, 1991 from brackish water, and Harpacticellaoceanica It\u00f4, 1977 from the marine environment.The first reported . Among tHarpacticella from a tidal pool. Here, we describe the new species and provide a key to species in the genus Harpacticella. Partial mtCOI sequence was also obtained as a DNA barcode for the new species.During a study of the harpacticoid community along the coast of Jeju Island of Korea, we collected a new species of Samples were collected by hand net (63 \u00b5m mesh-size) from a tidal pool on the coast of Jeju Island, Korea. Specimens were preserved in 99% ethanol. Specimens were dissected in lactic acid, and the dissected parts were mounted on slides with lactophenol mounting medium. Preparations were sealed with transparent nail varnish. All drawings were prepared using a drawing tube attached to an Olympus BX51 differential interference contrast microscope., antennuleA1; , antennaA2; , aesthetascae; , exopodexp; , endopodenp; , first to sixth thoracopodP1-P6; , proximal segment of a three-segment ramusexp (enp)-1; , caudal ramiCR. Specimens were deposited in the National Institute of Biological Resources, Incheon, Korea (NIBR). Scale bars in figures are indicated in \u00b5m.Descriptive terminology is adopted from Molecular analysis. For DNA extraction, fixative materials (99% Et-OH) were removed from specimens by washing with distilled water, and DNA was extracted using a tissue DNA purification kit . Amplifications were performed in 20 \u00b5l reactions volumes containing extracted tissue DNA, primers LCO-1490 (5\u2019-GGT CAA CAA ATC ATA AAG ATA TTG G-3\u2019) and HCO-2198 (5\u2019-TAA ACT TCA GGG TGA CCA AAA AAT CA-3\u2019) , and PCRPageBreakTaxon classificationAnimaliaHarpacticoidaHarpacticidaehttp://zoobank.org/77BE96F4-597C-44D0-A07E-03A4D993A5F233\u00b013.949'N; 126\u00b030.653'E) on Beophwan beach, Seoguipo-shi, Jeju Island, Korea.A tidal pool (Holotype: 1\u2640 (NIBRIV0000304111) in 70% ethanol from the type locality. Paratypes: 5\u2640\u2640 (NIBRIV0000304112) in 70% ethanol, 2\u2640\u2640 (NIBRIV0000304113 \u2013 NIBR0000304114) dissected on 11 and 10 slides, respectively, and 2\u2642\u2642 (NIBRIV0000304115 \u2013 NIBR0000304116) dissected on 11 and 2 slides, respectively. All from the type locality and collected by R. Jeong on 3 March 2013.KM272559, 619 bp).DNA-barcode (mtCOI) sequence and trace were submitted to GenBank . Maximum width at posterior margin of cephalosome . Urosome gradually tapering posteriorly. Body surface armed with some sensilla Figs .Prosome Fig. 4-segmenRostrum Fig. well devUrosome Figs , 2C 5-seGenital double somite Figs , 2C, 7A Anal somite Fig. without PageBreakPageBreakPageBreakPageBreakouter corner; seta III as long as lateral seta II; apical seta IV unipinnate, slightly longer than urosome; apical seta V bipinnate, as long as whole body; apical seta VI similar in length to seta III; dorsal seta VII bare and bi-articulate at its base.Caudal ramus Figs , 2D wideAntennule 7-segmented Fig. . AesthetAntenna Fig. 3-segmenMandible Fig. with larMaxillule Fig. with praMaxilla Fig. : syncoxaMaxilliped Fig. : syncoxaSwimming legs 1\u20134 Figs , 5A\u2013B biP1 Fig. : coxa wiPageBreakP2 Fig. : coxa wiP3 Fig. : coxa wiP4 Fig. : coxa wiArmature formulae as follows:PageBreakP5 Fig. : exopod Male. Total body length of examined samples 631\u2013650 \u00b5m . Greatest width at posterior margin of cephalosome. Cephalosome with sensilla along lateral margin. Other prosomites also with sensilla along lateral margin Fig. .Prosome Fig. , 4-segmeUrosome Figs , 7E 6-seAntennule Fig. 6-segmenP5 Figs , 7D, basPageBreakPageBreakPageBreakP6 Fig. : small fmitochondrial oxidase subunit I; partial cds; 619 bpPageBreakGATTTTTGATGCCCTCTCTTATATTAATAATTATTAGAAGAGTTGTTGAAGGCGGGGCAGGGACAGGGTGAACTGTTTACCCCCCTTTAAGAAGAAATTTAGCACATGCAGGAGGCTCGGTGGATTTAGTAATTTTTTCTTTACATTTAGCAGGAGTTTCTTCCTTATTAGGGGCTGTAAATTTTATTAGGACTTTAAGAAATCTTCGAGTATTCGGGATGTATTTTGACCAAGTGCCGTTATTTTGTTGATCTGTCTTGGTTACAGCTGTTCTATTACTTTTATCACTGCCTGTATTAGCGGGGGCAATTACTATATTGTTGACCGATCGAAACATTAATTCAAGCTTCTATGATGTTAACTTTATATCTTTTAAGGGGGATATGAGCGGGAGTTATGGGGGCGGCAATAAGAGTTATTATTCGGCTTGAATTAGGACAGCCTGGGACTTTAATTAAGGATGAGCAAATTTATAATGTTTTAGTGACTTCGCATGCTTTTATTATAATTTTCTTTATGGTTATACCAATTTTAATTGGGGGGTTTGGAAACTGGTTAGTTCCTTTAATATTAGGAGCTCCTGATATGGCCTTTCCTCGATTAAATAATTTGAGATTCTThe specific name refers to the type locality of Jeju Island, Korea.Harpacticella based on the combination of following character sets: a) 7-segmented antennule in the female, b) 2-segmented antennary exopod, c) 3-segmented P1 endopod and exopod, d) only one seta on the inner edge of P2 enp-2 and e) spiniform outer spine of exp-3 P3 and P4 six setae on the P5 exopod of females P1 see Fig. ; (4) naksee Fig. , but plusee Fig. ; (5) naksee Fig. , but it see Fig. .PageBreakPageBreakHarpacticella species have a wide distribution ranging from freshwater to true marine environments, and have been found in Asian waters, American waters, and the Aldabra Atoll in the Indian Ocean is the most ubiquitous species; it has been recorded in China, Japan, and the northwest coast of the USA for use in future studies; no sequences have been obtained from congeners to date, even though it would be interesting to determine the phylogenetic relationships among congeners based on analysis of mtCOI sequences.DNA barcoding is an efficient tool to identify species, especially morphologically similar species . This ba"} +{"text": "Previous studies have demonstrated that the trafficking defects of Nav1.1/Nav1.2 are involved in the dementia pathophysiology. However, the detailed mechanisms are not fully understood. Moreover, whether the impaired miRNAs regulation linked to dementia is a key player in sodium channel trafficking disturbance remains unclear. The cognitive impairment induced by chronic cerebral ischemia through chronic brain hypoperfusion (CBH) is likely reason to precede dementia. Therefore, our goal in the present study was to examine the role of microRNA-9 (miR-9) in regulating Nav1.1/Nav1.2 trafficking under CBH generated by bilateral common carotid artery occlusion (2VO).The impairment of Nav1.1/Nav1.2 trafficking and decreased expression of Nav\u03b22 were found in the hippocampi and cortices of rats following CBH generated by bilateral 2VO. MiR-9 was increased in both the hippocampi and cortices of rats following CBH by qRT-PCR. Intriguingly, miR-9 suppressed, while AMO-miR-9 enhanced, the trafficking of Nav1.1/Nav1.2 from cytoplasm to cell membrane. Further study showed that overexpression of miR-9 inhibited the Nav\u03b22 expression by targeting on its coding sequence (CDS) domain by dual luciferase assay. However, binding-site mutation or miR-masks failed to influence Nav\u03b22 expression as well as Nav1.1/Nav1.2 trafficking process, indicating that Nav\u03b22 is a potential target for miR-9. Lentivirus-mediated miR-9 overexpression also inhibited Nav\u03b22 expression and elicited translocation deficits to cell membrane of Nav1.1/Nav1.2 in rats, whereas injection of lentivirus-mediated miR-9 knockdown could reverse the impaired trafficking of Nav1.1/Nav1.2 triggered by 2VO.We conclude that miR-9 may play a key role in regulating the process of Nav1.1/Nav1.2 trafficking via targeting on Nav\u03b22 protein in 2VO rats at post-transcriptional level, and inhibition of miR-9 may be a potentially valuable approach to prevent Nav1.1/Nav1.2 trafficking disturbance induced by CBH. Since voltage-gated sodium channel (VGSC) is necessary in the initiation and propagation of action potentials in neurons, it is a valuable therapeutic target for neurological disorders, such as epilepsy and chronic neuropathic pain \u20133. Recen+ channels (VGSCs) are macromolecular protein complexes, which are composed of \u03b1-subunits (Nav1.1\u2013Nav1.9) and \u03b2-subunits , in which \u03b1-subunits are necessary for forming a functional ion-selective channel and \u03b2-subunits affect ion channel gating and trafficking to regulate the voltage-dependency and density of VGSC on the cell membrane. It has been reported that mutation of Nav1.1, a remarkable feature of Dravet syndrome, could induce higher seizure activity and cognitive dysfunction [BACE1-transgenic mice, accompanied with disturbed Nav\u03b22 [hAPP) transgenic mice [BACE1-null mice [BACE1) in BACE1-deficient or over-expressing mice [APP) mice [Voltage-gated Nafunction . Interesed Nav\u03b22 . And resnic mice . Furtherull mice . These sull mice \u201317, and ull mice . Interesing mice , and thePP) mice . HoweverChronic brain hypoperfusion (CBH)-mediated chronic cerebral ischemia and consequent cognitive impairment , 22 is mMicroRNAs are small non-coding RNA, which regulate protein synthesis. MicroRNA-9 (miR-9), enriched in central nerve system (CNS) , contribIn this study, our data provide strong evidence that miR-9 regulates Nav1.1/Nav1.2 trafficking by post-transcriptional regulating SCN2B gene under CBH status.P\u2009<\u20090.01 vs sham). In order to explore whether the decreased surface protein expression of Nav1.1/Nav1.2 was due to the reduction of total Nav1.1/Nav1.2 or the impairment of trafficking, the total protein levels of Nav1.1/Nav1.2\u00a0were evaluated. The data showed that the total protein levels of Nav1.1 and Nav1.2 were significantly increased in the hippocampi and cortices of 2VO rats rather than reduced. These results suggested that the decreased expression of Nav1.1/Nav1.2 on the cell membrane were due presumably to the impaired trafficking of Nav1.1 and Nav1.2 protein from cytoplasm to cell membrane in 2VO rats rather than the changes of their total protein levels.Previous studies have reported that Nav1.1/Nav1.2 trafficking was changed after dementia , 20, 40.P\u2009<\u20090.01). However, their mRNA expression of Nav\u03b22 (SCN2B) was not changed consistently with the protein expression of Nav\u03b22 .MicroRNAs have long been known to control post-transcriptional gene regulation and are essential for neuronal function . MiR-9 ihttp://www.targetscan.org/), we found that SCN2B has a poorly conservative \u2018seed\u2019 sequence of miR-9 in its 3\u2019UTR and the length of 3\u2019UTR of SCB2B in rat was very short based on the database of the University of California Santa Cruz (UCSC) and the National Center for Biotechnology Information (NCBI) Genome Browsers. Since recent studies have reported that microRNAs regulate protein expression by binding to the coding regions of target protein [http://bibiserv.techfak.uni-bielefeld.de/rnahybrid/) and found that the CDS domain of SCN2B is likely to serve as potential targets for miR-9. We next identified whether there are binding sites for miR-9 on the CDS domain of SCN2B gene at the position of 336\u2013358 and 575\u2013597 with highly conservative regions , however, scrambled negative control and inhibitor of miR-9 had no effect on luciferase activity compared with control group , and the difference of luciferase activity between inhibitor of miR-9 and scramble negative inhibitor control was also not detected . For each of the two binding sites, we generated and tested several variants by introducing silent mutations designed to disrupt the putative base pairings between the microRNAs and the corresponding predicted targets. Silent mutations introduced into the predicted targets at the position of 336\u2013358 of SCN2B CDS disrupted the ability of miR-9 to repress the translation of the SCN2B-CDS . However, simultaneous introduction of silent mutations at the position of 575\u2013597 of SCN2B CDS did not abolish the downregulation of SCN2B by miR-9 . Silent mutations introduced into both two predicted targets at the positions of 336\u2013358 and 575\u2013597 of SCN2B CDS, MutSCN2B-1 & 2 disrupted the ability of miR-9 to repress the translation of the SCN2B-CDS, strongly suggesting that miR-9 uniquely targets at the position of 336\u2013358, rather than 575\u2013597, of SCN2B CDS.Since the protein level of Nav\u03b22 was reduced in both hippocampi and cortices in 2VO rats, which is discrepancy with the unchanged SCN2B expression, the alternation of miR-9 is highly expected in this process. By searching the database of microRNA targets , implying that the SCN2B shows a great potential as the target for miR-9.To observe the influence of miR-9 on the protein translation of SCN2B, we analyzed the protein levels of Nav\u03b22 in primary cultured neonatal rat neurons (NRNs) co-transfected with miR-9 mimics. The successful transfection of miR-9 was identified Fig.\u00a0 by qRT-PP\u2009<\u20090.05) and Nav1.2 total protein. And they were prevented in the presence of AMO-9 was injected directly into CA1 region of bilateral hippocampus of each rat and significantly higher expression of miR-9 in both hippocampi and cortices was observed at 8\u00a0weeks after injection compared with the negative control oligonucleotide . The surface expressions of both Nav1.1 and Nav1.2 proteins were markedly reduced in both hippocampi and cortices of rats with lenti-pre-miR-9 treatment, which was reversed by lenti-pre-AMO-miR-9 , even though the total protein levels of Nav1.1 and Nav1.2 were increased in both hippocampi and cortices of rats with lenti-pre-miR-9 treatment, which were reversed by lenti-pre-AMO-miR-9 . These data suggested that miR-9 plays an important role in trafficking and cellular distribution of Nav1.1/ Nav1.2.To verify the functional role of miR-9 on Nav1.1/Nav1.2 trafficking NC, Fig.\u00a0. ImportaP\u2009<\u20090.01). And furthermore, the surface protein expression of Nav1.1 was increased and the total protein of Nav1.1 level was decreased by lenti-pre-AMO-miR-9 compared with that in 2VO control rats . Similarly, lenti-pre-AMO-miR-9 effectively improved the impaired trafficking of Nav1.2 from cytoplasm to cell membrane under the same experimental condition .Our data displayed that 2VO results in miR-9 increase, and over-expression of miR-9 then induces the trafficking defects of Nav1.1/Nav1.2 in vitro. In order to evaluate the protective effect of AMO-miR-9 on the onset of the trafficking defects of Nav1.1/Nav1.2 in 2VO rats, lenti-pre-AMO-miR-9 was injected into the hippocampus area. At 8\u00a0weeks after injection, miR-9 level was significantly decreased in both hippocampi and cortices compared with 2VO rats Fig.\u00a0. To our In this study, our observations have demonstrated, for the first time that CBH induces the trafficking defects of Nav1.1/Nav1.2 in hippocampi and cortices areas in 2VO rats, leading to the decrease in the expression of Nav\u03b22 protein. Further study has shown that the increased miR-9 negatively regulated the expression of Nav\u03b22 protein by binding to the target in CDS region of SCN2B gene. This observation provides a novel mechanism to modify the reduction in Nav1.1/Nav1.2 membrane trafficking, and careful monitoring the changes in miR-9 level and the expression for Nav1.1/Nav1.2 and targeted gene are considerably necessary during CBH.BACE1-trangenic mice although total Nav1.1 and Nav1.2 levels are elevated [BACE1-null mice [BACE1-trangenic mice [It has been known that Nav1.1, Nav1.2, and Nav1.6 are abundant in the central nervous system, whereas Nav1.3 is mostly present during embryonic stage . Nav1.6 elevated , 14, andull mice . In the nic mice .BACE1 affects Nav1.1 and Nav1.2 surface trafficking differentially [BACE1 activity leading to decrease in surface levels of Nav1.1 in neuronal cells [APP and BACE1 expression [APP and BACE1 is unknown and need to be elucidated further.Previous studies have demonstrated that Nav\u03b22 subunit, an auxiliary subunit of Nav channel, participates in channel trafficking, re-localization, and interaction of both Nav1.1 , 47 and entially . Furtheral cells . Interespression , 41. WheAs far as we know, microRNAs are newly discovered and commonly considered as modulators of protein expression at post-transcriptional level, which are associated with the pathogenesis in multiple kinds of diseases \u201353. In tOur study provides strong evidences that miR-9 increases in both hippocampi and cortices, and inhibited the expression of Nav\u03b22, which in turn blocked the trafficking of Nav1.1 and Nav1.2 from cytoplasm to plasma membrane Figs.\u00a0, 4 and 5In addition, our important finding here is that miR-9 regulates endogenous Nav\u03b22 expression by targeting its coding sequence (CDS) region rather than not 3\u2019UTR of SCN2B Fig. . Additioin vivo study supports the data collected from our in vitro observations that the upregulation of miR-9 induced by both CBH and lenti-pre-miR-9 could also disturb the trafficking of both Nav1.1/Nav1.2 by downregulation of Nav\u03b22 expression. On the contrary, lenti-pre-AMO-miR-9 injection into hippocampus markedly prevents the abnormal trafficking of both Nav1.1 and Nav1.2 following either CBH or lenti-pre-miR-9 treated normal rats accompanied by increased Nav\u03b22 expression. These results combination with our in vitro data suggested that the inhibition of miR-9 in hippocampi and cortices in CBH model rats would be a way to prevent sodium channel dysfunction after CBH. An understanding of miR-9-Nav\u03b22-Nav1.1/Nav1.2 trafficking pathway could yield to the potential therapeutic targets for the prevention of abnormal electrical activation induced by CBH.More importantly, our in vitro and in vivo, we did not provide evidence whether these changes could induce abnormal sodium channel currents and its dynamics characteristics in hippocampi and cortices of 2VO rats. These need to be studied further.In the present study, though we have demonstrated the regulation effect of miR-9 on the trafficking of Nav1.1/Nav1.2 by inhibiting the expression of Nav\u03b22 both MiR-9 plays a key role in regulating the process of Nav1.1/Nav1.2 trafficking via targeting on Nav\u03b22 protein in 2VO rats at post-transcriptional level, and inhibition of miR-9 may be a potentially-valuable approach to prevent the Nav1.1/Nav1.2 trafficking disturbance induced by CBH.ad libium. Rats used for operation of permanent, bilateral common carotid artery occlusion (2VO) and stereotaxic injection of the lentiviral vectors were anesthetized with chloral hydrate and maintained by administrating 0.5-1.0\u00a0% isoflurane. The depth of anesthesia was monitored by detecting reflexes, heart rate and respiratory rate. Samples for qRT-PCR, and Western blot assay were obtained from the hippocampi and cortices of rats after anesthetized with chloral hydrate following by confirmation of death by exsanguination. Tissues for primary neuron culturing were from neonatal SD rats after administration of 20\u00a0% isoflurane and confirmation of death by cervical dislocation. All animal procedures were approved by the Institutional Animal Care and Use Committee at Harbin Medical University (No.HMUIRB-2008-06) and the Institute of Laboratory Animal Science of China (A5655-01). All procedures were conformed to the Directive 2010/63/EU of the European Parliament.Male Sprague\u2013Dawley rats were housed at 23\u2009\u00b1\u20091\u00a0\u00b0C with 55\u2009\u00b1\u20095\u00a0% of humidity and maintained on 12\u00a0h dark\u2013light artificial cycle (lights on at 07:00 A.M.) with food and water available The method for preparation of 2VO rat was according to the previous study , 62. BriThe hippocampi and cortices regions were removed from the postnatal day 0 (P0) rat pups. After tissues were dissected and triturated, they were plated onto cell plates precoated with 10\u00a0\u03bcg/mL poly-D-lysine and cultured in the culture media containing neurobasal medium with 2\u00a0% B27 supplement and 10\u00a0% fetal bovine serum . After 3\u00a0days, the neurons were treated with 5\u00a0\u03bcM cytosine arabinoside to inhibit astrocyte proliferation. For all experiments, the neurons were used at 14\u00a0days after plating .MiR-9 mimics and AMO-miR-9 (5\u2019-UCAUACAGCUAGAUAACCAAAGA-3\u2019) were synthesized by Shanghai GenePharma Co., Ltd . AMO-9 contains 2\u2019-O-methyl modifications at every base and a 3\u2019 C3-containing amino linker. Additionally, a scrambled RNA was used as a negative control . The Nav\u03b22-masking antisense oligodeoxynucleotides (ODNs) were synthesized by Shanghai Sangon Biological Engineering Technology and Service Co., Ltd. Nav\u03b22 masking antisense-ODN-1 was 5\u2019-ATGCCTTCGTCTTCTAGCTGC-3\u2019, which masks the binding sites of miR-9, located in the position 336\u2013358 of SCN2B CDS (coding sequence) region; Nav\u03b22 masking antisense-ODN-2 was 5\u2019TCCTCTTCGGTCTTCAGGTCA-3\u2019\u2009, which masks the binding sites of miR-9, located in the position of 575\u2013597 of SCN2B CDS region. Five nucleotides or deoxynucleotides at both ends of the antisense molecules were locked by a methylene bridge connecting between the 2\u2019-O- and the 4\u2019-C atoms.Thirty pmol/mL miR-9 and/or AMO-9, ODNs or NC siRNAs were transfected into neonatal hippocampal and cortical neurons with X-treme GENE siRNA transfection reagent according to the manufacturer\u2019s instructions. Forty-eight hours after transfection, cells were collected for total RNA isolation or protein purification.miR-9 (\u201ctop strand\u201d oligo: TGCTGTcTTTggTTaTcTagcTgTaTgaGTTTTGGCCACTGACTGACTcaTacagagaTaaccaaaga) and its complementary chain (\u201cbottom strand\u201d oligo: CCTGTcTTTggTTaTcTcTgTaTgaGTCAGTCAGTGGCCAAAACTcaTacagcTagaTaaccaaagaC); (2) pre-AMO-miR-9 (\u201ctop strand\u201d oligo: TGCTGTcaTacagcTagaTaaccaaagaGTTTTGGCCACTGACTGACTcTTTggTTcTagcTgTaTga) and its complement (\u201cbottom strand\u201d oligo: CCTGTcaTacagcTagaaccaaagaGTCAGTCAGTGGCCAAAACTcTTTggTTaTcTagcTgTaTgaC); (3) negative control (\u201ctop strand\u201d oligo: tgctgAAATGTACTGCGCGTGGAGACGTTTTGGCCACTGACTGACGTCTCCACGCAGTACATTT) and its complement (\u201cbottom strand\u201d oligo: cctgAAATGTACTGCGTGGAGACGTCAGTCAGTGGCCAAAACGTCTCCACGCGCAGTACATTTc). We then cloned the double-stranded oligonucleotides (ds oligo) generated by annealling the top and bottom strand oligos into the pcDNA\u21226.2-GW/\u00b1 EmGFP-miR vector and transformed the ligated mixture into competent E. coli. After colony was purified and identified as the correct expression clone, the pre-microRNA expression cassette was transferred to other Gateway\u00ae adapted destination vectors utilizing PolII promoters and formed a new miRNA expression clone containing attR substrates. The vector was identified after analyzing the plasmid sequence . The titers of the vectors used for experiments were 9.25\u2009\u00d7\u2009108 transducing U/ml. Virus suspensions were stored at \u221280\u00a0\u00b0C until use and were briefly centrifuged and kept on ice immediately before injection.Using the BLOCK-iT polII miR-RNAi expression vector with the EmGFP kit from invitrogen, three single-stranded DNA oligonucleotides were designed as follows: (1) pre-After anaesthetized, rats were placed onto a stereotaxic frame described as previous study . InjectiBefore the luciferase activity assay, plasmid design and construction was shown as in the below figure: a 707\u00a0bp fragment from the coding region of SCN2B containing the putative binding sequences for miR-9 (position 336\u2013358 and position 575\u2013597 of SCN2B CDS) was amplified by PCR, cloned into the pSICHECK-2-control vector.GACGAAGGC)ATTTACAACTGCTAATCACCAACCCTCCAGACCGCCACCGTGGCCATGGCAAGATCTACCTGCAGGTCCTTCTAGAAGGCCCCCAGAGCGGGACTCCACGGTGGCAGTCATCGTGGGTGCCTCAGTGGGGGGTTTCCTGGCTGTGGTCATCTTGGTGCTGATGGTGGTCAAATGTGTGAGGAGGAAAAAAGAGCAGAAGCTGAGC(ACGGATGACCTGAAGACCGAAGA)GGAAGGCAAGACGGATGGCGAGGGCAACGCGGAAGATGGCGCCAAGTAACCGGAAGCTTGCCCTGAAGCCCCTTCCTGTGTCCTGTCTCCTCTCACTCTCTGCCCTGT; mutSCN2B CDS (bold nucleotide): ATGCACAGGGATGCCTGGCTACCTCGCCCTGCCTTCAGCCTCACGGGGCTCAGTCTGTTTTTCTCTTTGGTGCCCTCGGGGCGGAGCATGGAAGTCACAGTCCCCACCACTCTTAGTGTCCTCAACGGGTCTGATACCCGCCTGCCCTGTACCTTCAACTCCTGCTATACCGTGAACCACAAGCAGTTCTCTCTGAACTGGACTTACCAGGAGTGTAGCAATTGCTCAGAGGAGATGTTCCTCCAGTTCCGAATGAAGATCATCAACCTGAAGCTGGAGCGGTTTGGAGACCGCGTAGAGTTCTCGGGGAACCCCAGTAAGTACGACGTGTCAGTGACTCTAAAGAA(CGTGCAGCTAGAAATCGATCGC)ATTTACAACTGCTACATCACCAACCCTCCAGACCGCCACCGTGGCCATGGCAAGATCTACCTGCAGGTCCTTCTAGAAGTGCCCCCAGAGCGGGACTCCACGGTGGCAGTCATCGTGGGTGCCTCAGTGGGGGGTTTCCTGGCTGTGGTCATCTTGGTGCTGATGGTGGTCAAATGTGTGAGGAGGAAAAAAGAGCAGAAGCTGAGC(ACGGATGACCTGAAATCGATCGA)GGAAGGCAAGACGGATGGCGAGGGCAACGCGGAAGATGGCGCCAAGTAACCGGAAGCTTGCCCTGAAGCCCCTTCCTGTGTCCTGTCTCCTCTCACTCTCTGCCCTGTMutagenesis nucleotides were carried out using direct oligomer synthesis for the CDS region of Nav\u03b22-binding site 1 and Nav\u03b22-binding site 2. Point mutations were introduced into a possible miR-9 binding site located in the coding region of SCN2B (position 336\u2013358 and position 575\u2013597 of SCN2B CDS). MutSCN2B-1 represents that \u201cGACGAAGG\u201d was mutated \u201cATCGATCG\u201d in the position 336\u2013358 of SCN2B CDS, MutSCN2B-2 represents that \u201cGACCGAAG\u201d was mutated \u201cATCGATCG\u201d in the position 575\u2013597 of SCN2B CDS. MutSCN2B-1&2 represents that both sites were mutated. All constructs were sequence verified. Rat SCN2B CDS and mutSCN2B CDS sequences were shown as following: SCN2B CDS sequences (bold nucleotide showed the putative binding sequences for miR-9): ATGCACAGGGATGCCTGGCTACCTCGCCCTGCCTTCAGCCTCACGGGGCTCAGTCTGTTTTTCTCTTTGGTGCCCTCGGGGCGGAGCATGGAAGTCACAGTCCCCACCACTCTTAGTGTCCTCAACGGGTCTGATACCCGCCTGCCCTGTACCTTCAACTCCTGCTATACCGTGAACCACAAGCAGTTCTCTCTGAACTGGACTTACCAGGAGTGTAGCAATTGCTCAGAGGAGATGTTCCTCCAGTTCCGAATGAAGATCATCAACCTGAAGCTGGAGCGGTTTGGAGACCGCGTAGAGTTCTCGGGGAACCCCAGTAGTACGACGTGTCAGTGACTCTAAAGAA as well as 0.5\u00a0\u03bcg of psi-CHECKTM-2-target DNA (firefly luciferase vector) and 1\u00a0\u03bcl blank plasmid using lipofectamine 2000 transfection reagent according to the manufacturer\u2019s instructions. After 48\u00a0h of transfection, Firefly and renilla luciferase activities, as indicated by relative luminescence units (RLU) were determined using luciferase assay kits and luminometer according to the manufacturer's instructions.The sequence of miR-9 mimic is 5\u2019-UCUUUGGUUAUCUAGCUGUAUGA-3\u2019 (synthesized based on the sequence of rno miR-9 (miRBase Accession No. MIMAT0000781)); that of miR-NC is 5\u2019-UUCUCCGAACGUGUCACGUAA-3\u2019; the sequence of the antisense 2\u2019-O-methyl (2\u2019-O-Me) oligonucleotide for miR-9 is 5\u2019-UCAUACAGCUAGAUAACCAAAGA-3\u2019, that of inhibitor-NC is 5\u2019UUCUCCGAACGUGUCACGUTT-3\u2019; HEK293T cells (plated at 40\u00a0%\u2009~\u200950\u00a0% confluence) were transfected with 20\u00a0\u03bcmol/l miRMiR-9 level was quantified by the TaqMan\u00ae MicroRNA Reverse Transcription Kit and the TaqMan\u00ae Gene Expression Master Mix . The TaqMan qRT\u2013PCR probes and primers for miR-9, were designed by Applied Biosystems [SCN2B forward: CTCTCTGAACTGGACTTACC and SCN2B reverse: GGTTGGTGATGTAGCAGTTG; \u03b2-actin forward: GGAAATCGTGCGTGACATTA and \u03b2-actin reverse: AGGAAGGAAGGCTGGAAGAG. All reactions were performed in triplicate, and the expressions of microRNAs data were shown as Delta-Delta Ct method.Total RNA was purified with the Trizol Reagent , according to the manufacturer\u2019s instructions as described previously . MiR-9 losystems . U6 was Both the total protein and surface protein samples were extracted from hippocampi and cortices of rats or primary cultured neurons for immunoblotting analysis. For the total protein analysis, frozen tissue was homogenized with 1000\u00a0\u03bcl solution contained 40\u00a0% SDS, 60\u00a0% RIPA and 1\u00a0% protease inhibitor in each 200\u00a0mg brain tissue. The homogenate was then centrifuged at 13,500\u00a0rpm for 30\u00a0min and the supernatants (containing cytosolic and membrane fractions) were collected. The method of surface protein extraction was using Mem-PER Eukaryotic Membrane Protein Extraction Reagent Kit according to the manufacturer's instructions. Protein concentrations were measured spectrophotometrically using a BCA kit . Protein samples were fractionated by SDS-PAGE (10\u00a0% polyacrylamide gels for sodium channels) then transferred to PVDF membrane. The primary anti-Nav1.1, Nav1.2, Nav\u03b22 antibodies were used and \u03b2-actin \u03b2-actin was selected as an internal control of total proteins, mouse anti-human transferrin receptor (TfR) was selected as an internal control of surface proteins. Western blot bands were captured on the Odyssey Infrared Imaging System and quantified with Odyssey v1.2 software by measuring the band intensity (area\u2009\u00d7\u2009OD) in each group and normalizing to the internal control.The cultured neonatal rat neurons were transcardially perfused by 4\u00a0% buffered paraformaldehyde, pH7.4. After blocking, cultured neonatal rat neurons were incubated with the anti-\u03b2-Tubulin III antibody or anti- Nav1.1, Nav1.2, Nav\u03b22 antibodies overnight at 4\u00a0\u00b0C, and then the cultured neonatal rat neurons were washed and incubated with the secondary antibodies conjugated to Alexa Fluor 488 and Alexa Fluor 594 for 1\u00a0h at room temperature.t-test was applied for comparisons between the two groups. Multi-group\u2019s comparisions were performed by One-way ANOVA. SPSS19.0 software was used for all statistical analyses. P\u2009<\u20090.05 was considered significant.Data were described as mean\u2009\u00b1\u2009s.e.m for experimental data. The two-tailed Student\u2019s"} +{"text": "There is an error in the third sentence of the \u201c16S library preparation\u201d subsection of the Materials and Methods. The first primer listed is incorrect. The correct sentence is: The V6 region was amplified with custom barcoded primers that consist of a region that anneals to the V6 region of interest, followed by a unique five basepair barcode, and the Illumina sequencing adapter [44]:V6-L [5\u2019- ACACTCTTTCCCTACACGACGCTCTTCCGATCTnnnnnCWACGCGARGAACCTTACC-3\u2019]V6-R [5\u2019-CGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCTnnnnnACRACACGAGCTGACGAC-3\u2019]"} +{"text": "Traditional fermented cheese whey (TFCW), containing probiotics, has been used both as a dairy food with ethnic flavor and a medicine for cardiovascular disease, especially regulating blood lipid among Kazakh. We therefore investigated anti-atherosclerotic effects of TFCW in atherosclerotic rabbits and identified lactic acid bacteria (LAB) and yeasts in TFCW.Atherosclerotic rabbits were induced by administration of atherosclerotic diet for 12\u00a0weeks and divided randomly into three groups and treated for 4\u00a0weeks with Simvastatin (20\u00a0mg/kg) or TFCW (25\u00a0mg/kg) and (50\u00a0mg/kg). In addition, a normal control group and an atherosclerotic group were used for comparison. All drugs were intragastrical administered once daily 10\u00a0mL/kg for 4\u00a0weeks. Body weight (BW), lipid profiles, C-reactive protein (CRP), vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1) were tested and theromatous plaques and the number of foam cells and infiltrating fibroblast cells in the thoracic aorta endothelium was evaluated by hematoxylin and eosin stainin. LAB and yeasts were isolated and purified by conventional techniques and identified using morphological and biochemical properties as well as gene sequences analysis.P\u2009<\u20090.05) compared to atherosclerotic group, and increased HDL-C (P\u2009<\u20090.05) compared to normal controls. Histological analysis showed TFCW reduced VCAM-1 expression and formation of atheromatous plaques on the aortic endothelium of atherosclerotic rabbits.After 4\u00a0weeks of treatment, high and low dose TFCW decreased serum TC, TG, LDLC, CRP, VCAM-1 and ICAM-1 (Seven classes of LBA from two different genera including Lactobacillus brevis, Lactobacillus kefianofaciens, Lactobacillus helveticus, Lactobacillus Casei, Lactobacillus plantarum, Lactobacillus kefiri and Lactococcus lactic as well as 2 classes of yeasts from two different genera including Saccharomyces unisporus and Issatchenkia orientalis were isolated and identified from TFCW. In summary, TFCW, containing 7 classes of LBA and 2 classes of yeasts, has significant anti-atherosclerotic potential in atherosclerotic rabbits and may modulate lipid metabolism and protect aorta in the atherosclerotic condition, which might be related to various probiotics acting through reducing the CRP, VCAM-1 and ICAM-1 levels and protecting the aortic endothelium. The public health burden of cardiovascular disease (CVD) is substantial, as CVD remains the leading cause of mortality and morbidity worldwide and atherosclerosis is the major cause of CVD , 2.Traditional fermented cheese whey (TFCW), by-product of cheese-making, has been widely used as a traditional dairy medication for regulating blood lipid among Kazakh people . Indeed,Lactobacillus and Bifidobacterium and a few yeast species including Saccharomyces boulardi [S. boulardii, one of the probiotic yeasts, provides anti-inflammatory and host immunity stimulatory effects [Probiotics mainly include boulardi . Lactic boulardi and atheboulardi in anima effects and lowe effects .However, anti-atherosclerotic effects of TFCW have not been experimentally demonstrated and no LAB or yeast has been found in TFCW. The aims of this study were to investigate anti-atherosclerotic effects of TFCW in a rabbit model of atherosclerosis and to identify LBA and yeast in TFCW.Traditional fermented cow\u2019s milk is the source of the cheese whey. Experimental TFCW samples were manufactured by standard procedures in 10\u00a0L vats in Altay Kanas Dairy Co. Ltd., . Fresh cow\u2019s milk samples were obtained from Jimunai Saur farm and skimmed in centrifuging at 3000\u2009\u00d7\u2009g for 30\u00a0min, homogenized under the pressure of 1.5\u2009~\u20091.7 Mpa and pasteurized by high temperature short time (HTST) then cooled to about 30\u00a0\u00b0C and fermented by inoculation with traditional home made Kazak yogurt purchased from Jimunai Saur farm at 37\u00a0\u00b0C for 12\u00a0h. After ferment, the whey was filtered in sterile gauze and dialyzed in cellulose membrane under constant magnetic stirring at 8\u00a0\u00b0C, also performed lactose removal by periodic water exchange. The experimental TFCW was stored at \u221220\u00a0\u00b0C until further use.Sodium pentobarbital was purchased from Merck & Co., (Germany). Simvastatin was purchased from Merck Sharp & Dohme Pty Ltd., . VCAM-1, ICAM-1 and CRP ELISA kits were purchased from Shanghai Senxiong Technology Co. Ltd., . Man Rogosa Sharpe (MRS) was purchased from Merck Sharp & Dohme Pty Ltd., . All media for cultivation of zymocytes were purchased from Hangzhou Microbial Reagent Co. Ltd., .ad libitum access to food and water. After a week of adaptive feeding, all the rabbits were randomly divided into 5 groups with 12 in normal group and 12 in atherogenic group, the normal control groups were given regular die and the atherogenic models were developed using an atherogenic diet for 12\u00a0weeks. The atherogenic diet consisted of 3\u00a0% cholesterol, 0.5\u00a0% sodium taurocholate, 0.2\u00a0% propylthiouracil, 5\u00a0% sugar, 10\u00a0% lard, and 81.3\u00a0% standard laboratory rabbit chow, which were provided by Experimental Animal Center of Xinjiang Medical University, China. After developing atherogenic models, Group 1 was treated with saline in a matched volume; Group 2 (atherogenic group) had atherogenic rabbits treated with saline in a matched volume; Group 3 (positive control) had atherogenic rabbits administered with simvastatin 20\u00a0mg/kg; Group 4 and Group 5 were treated with TFCW 25\u00a0mg/kg and 50\u00a0mg/kg, respectively (low and high doses). Simvastatin and TFCW were intragastrical administered once daily 10\u00a0mL/kg for 4\u00a0weeks. All animals received care in compliance with the Chinese Convention on Animal Care, and the study was approved by the Institutional Ethics Committee of Xinjiang Medical University.Sixty male white New Zealand rabbits, weighing 1.95-2.05\u00a0kg, specific pathogen free (SPF), were provided by Experimental Animal Center of Xinjiang Medical University, China and placed in separate cages and maintained on a 12-h day/night cycle at an ambient temperature, with At the end of experiments, all rabbits were fasted for 12\u00a0h, weighed, anesthetized with sodium pentobarbital and continually monitored until total loss of consciousness as indicated by a total lack of response after a foot pinch. Blood samples were collected from abdominal aorta, allowed to clot on ice and subsequently subjected to centrifugation (3500\u00a0rpm at 4\u00a0\u00b0C for 10\u00a0min), where after serum aliquots were stored at \u221280\u00a0\u00b0C for further analysis. Serum TC, TG, LDL-C and HDL-C were examined via an automatic biochemical analyzer . CRP was determined by rate nephelometry . Serum ICAM-1 and VCAM-1 were determined using commercially-available ELISA kits according to manufacturer instruction.Aorta was harvested from rabbits, placed immediately in formaldehyde 10\u00a0%, embedded in paraffin 24\u00a0h later, cut at 5\u00a0\u03bcm, stained with hematoxylin and eosin (H&E), and then scanned to assess pathological changes. For immunohistochemical staining, sections were incubated with anti-VCAM-1 and anti-F4/80 at 37\u00a0\u00b0C for 1\u00a0h, color developed with 3,3\u2032-diaminobenzidine tetrahydrochloride and counterstained with hematoxylin. Samples in the absence of the primary antibodies were used as negative controls. Slides were observed under a light microscope, and images were subjected to statistical evaluation of positively stained cells in 10 random fields of view at a magnification of\u2009\u00d7\u2009400. The average numbers of positively stained cells were counted per high power field (HPF).2) production were analyzed. All isolates were presumptively identified as LAB strains based on their ability to grow on MRS agar plates, Gram-positive staining, and a catalase activity-negative phenotype [Agar plates with Man Rogosa Sharpe (MRS) broth suitable for lactobacillus growth were used for initial isolation of LAB single colonies. Single bacterial colonies were initially separated based on their morphological differences on agar plates. Cell morphology was observed under light microscopy after Gram staining. Catalase activity, carbohydrate fermentation, acidogenicity, aciduricity , and gas . Significance was defined as p\u2009<\u20090.05), whereas Simvastatin group and low and high dose TFCW group showed significantly lower serum TC, TG and LDL-C than did atherogenic group (p\u2009<\u20090.05). TFCW significantly increased HDL-C levels compared to normal control group (p\u2009<\u20090.05), but no significant difference was observed in treated groups with TFCW, compared with atherogenic group. Data indicated that TFCW affects lipid metabolic parameters and TFCW treatment could effectively improve lipid metabolism in atherogenic rabbits.Table\u00a0P\u2009<\u20090.05). Outstandingly, the simvastatin and low and high dose TFCW groups had significantly lower CRP (P\u2009<\u20090.05) at values 3.43\u2009\u00b1\u20090.80\u00a0mg/L, 3.33\u2009\u00b1\u20090.50\u00a0mg/L and 1.34\u2009\u00b1\u20090.90\u00a0mg/L, respectively, compared with the atherogenic group. CRP in low and high dose TFCW was significantly decreased, compared to atherogenic group (P\u2009<\u20090.05). Data indicate that TFCW affects CRP and TFCW treatment could effectively improve inflammatory status in atherogenic rabbits. No significant differences were recorded in treated groups with TFCW on body weight compared with atherogenic group.Serum CRP was measured to determine inflammatory status of experimental groups as shown in Table\u00a0p\u2009<\u20090.05), while Simvastatin and low and high dose TFCW group showed significantly decreased ICAM-1 and VCAM-1, compared to atherogenic group (p\u2009<\u20090.05). Data indicated that TFCW might affect ICAM-1 and VCAM-1 and TFCW treatment might effectively inhibit adhesion of circulating inflammatory cells to endothelial cell walls in atherogenic rabbits.Adhesion molecules ICAM-1 and VCAM-1 play important roles in perpetuation of inflammation. Table\u00a0Histopathological examination of aorta sections from experimental groups were shown in Fig.\u00a0Immunohistochemical evaluation of VCAM-1 expression in aorta section and histomorphometric analysis from experimental groups were shown in Fig.\u00a0Lactobacillus species was rod-shaped, slender short or long, and the majority of Lactobacillus exhibited chain-like arrangement. We successfully isolated and purified seven isolates of LBA and identified them as L. brevis, L. kefianofaciens, L. helveticus, L. casei, L. plantarum, L. kefiri and Lactococcus lactic based on morphological characteristics, physiological tests, biochemical tests, and 16S rDNA and 16S rRNA sequence homology with NCBI Reference Sequences NC 008497.1, NC 015602.1, NC 006814.3, NC 008526.1, NC 004567.2, NC 015428.1 and NC 017486. All the test results and sequence homology were shown in Table\u00a0L. brevis:GCTGACTCCCGAAGGTTATCTCACCGGCTTTGGGTGTTACAAACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCAACTTCATGTAGGCGAGTTGCAGCCTACAATCCGAACTGAGAACGGCTTTAAGAGATTAGCTTAGCCTCACGACTTCGCGACTCGTTGTACCGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCACCAGAGTGCCCAACTGAATGCTGGCAACTGATAATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCATTCTGTCCCCGAAGGGAACGTCTTATCTCTAAGATTGGCAGAAGATGTCAAGACCTGGTAAGGTTCTTCGCGTAGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTGAAGGGCGGAAACCCTCCAACACTTAGCACTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTACAGACTAGACAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTCCACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTCTCCCAGTTTCCGATGCACTTCTCCGGTTAAGCCGAAGGCTTTCACATCAGACTTAAAAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAAATACCGTCAACCCTTGAACAGTTACTCTCAAAGGTGTTCTTCTTTAACAACAGAGTTTTACGAGCCGAAACCCTTCTTCACTCACGCGGCATTGCTCCATCAGACTTTCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATTACCCTCTCAGGTCGGCTACGTATCATCGTCTTGGTGGGCCTTTACCTCACCAACTAACTAATACGCCGCGGGATCATCCAGAAGTGATAGCCGAANCCACCTTTCAAACAAAATCCATGCGGATTTTGTTGTTATACGGTATTAGCACCTGTTTCCAAGTGTTATCCCCTGCTTCTGGGCAGATTTCCCACGTGTTACTCACCAGTTCGCCACTCGCTTCATTGTTGAAATCAANGCAAGCACGTCATTCAACGGAAGCTCGTTCGACTL. kefianofaciens:CTGCTTAGACGGCTCCTTCCTTGCGGTTAGGCCACCGGCTTTGGGCATTGCAGACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCGTGCTGATCCGCGATTAATAGCTATTCCAGCTTCGTGCAGTCGAGTTGCAGACTGCAGTCCGAACTGAGAACAGCTTTCAGAGAATTGCTTGCCTTTGCAGGCTCGCTGCTCGTTGTGCTGCCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGACTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCATTAGAGTGCCCAACTTAATGCTGGCAACTAATAACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCACCACCTGTCTTAGCGTCCCCGAAGGGAACTTTGTATCTCTACAAATGGCACTAGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTGAGAGGCGGAAGCCTCCCAACACTTAGCACTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTGCAGACCAGAGAGCCGCCTTCGCCACTGGTATTCTTCCATATATCTACGCATTCCACCGCTACACATGGAGTTCTACTCTCCTCTTCTGCACTCAAGAAAAACAGTTTCCGATGCAATTCCTCGGTTAAGCCGAGGGCTTTCACATCAGACTTATTCTTCCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGACTTTCTGGTTGATTACCGTCAAATAAAGGCCAGTTACTACCTCTATCCTTCTTCACCAACAACAGAGCTTTACGGTCCGAAAACCTTCTTCACTCACGCGGCGTTGCTCCATCAGACTTGCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTANGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATCAGTCTCTCAACTCGGCTATGCATCACTGCCTTGGTAGGCCGTTACCTTACCAACTAGCTAATGCACCGCGGGTCCATCCTTTAGCGACAGCTTGCGCCGCCTTTTAAAAGCTGTTCATGCGAACTGCTTTCTTATCCGGTATTAGCACCTGTTTCCAAGTGGTATCCCAGACTTAAGGGCAGGTTCCCCACGTGTTACTCACCCATCCGCCGCTCGCTTTCCCAGCGTCCTCACCGAAGTGATTCTGCTGGTTCCGCTCGCTCGACTTGCATGTATTAGL. helveticus:TTAGACGGCTCCTTCCCGAAGGTTAGGCCACCGGCTTTGGGCATTGCAGACTTCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCGTTCTGATCCGCGATTACTAGCGATTCCAGCTTCGTGCAGTCGAGTTGCAGACTGCAGTCCGAACTGAGAACAGCTTTCAGAGATTCGCTTGCCTTCGCAGGCTCGCTTCTCGTTGTACTGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGACTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCATTAGAGTGCCCAACTTAATGCTGGCAACTAATAACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCACCACCTGTCTTAGCGTCCCCGAAGGGAACTCCTAATCTCTTAGGATGGCACTAGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTGAGAGGCGGAAACCTCCCAACACTTAGCACTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTGCAGACCAGAGAGTCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTCCACCGCTACACATGGAGTTCCACTCTCCTCTTCTGCACTCAAGAAAAACAGTTTCCGATGCAGTTCCTCGGTTAAGCCGAGGGCTTTCACATCAGACTTATTCTTCCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGACTTTCTGGTTGATTACCGTCAAATAAAGGCCAGTTACTACCTCTATCCTTCTTCACCAACAACAGAGCTTTACGATCCGAAAACCTTCTTCACTCACGCGGCGTTGCTCCATCAGACTTGCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGTTCAGTCTCTCAACTCGGCTATGCATCATTGCCTTGGTAAGCCGTTACCTTACCAACTAGCTAATGCACCGCGGGGCCATCCCATAGCGACAGCTTACGCCGCCTTTTATAAGCTGATCATGCGATCTGCTTTCTTATCCGGTATTAGCACCTGTTTCCAAGTGGTATCCCAGACTATGGGGCAGGTTCCCCACGTGTTACTCACCCATCCGCCGCTCGCGTCCCCAGCATCATTACCGAAGTAAATCTGCTGGTTCTGCTCGCTCGACTGCATGTATL. casei:TAGACGGCTCGCTCCCTAAAAGGGTTACGCCACCGGCTTCGGGTGTTACAAACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCGTGCTGATCCGCGATTACTAGCGATTCCGACTTCGTGTAGGCGAGTTGCAGCCTACAGTCCGAACTGAGAATGGCTTTAAGAGATTAGCTTGACCTCGCGGTCTCGCAACTCGTTGTACCATCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTTACTAGAGTGCCCAACTAAATGCTGGCAACTAGTCATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCATTTTGCCCCCGAAGGGGAAACCTGATCTCTCAGGTGATCAAAAGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAATGCTTAATGCGTTAGCTGCGGCACTGAAGGGCGGAAACCCTCCAACACCTAGCATTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTACAGACCAGACAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTTCACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTTTCCCAGTTTCCGATGCGCTTCCTCGGTTAAGCCGAGGGCTTTCACATCAGACTTAAAAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGATAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTGGATACCGTCACGCCGACAACAGTTACTCTGCCGACCATTCTTCTCCAACAACAGAGTTTTACGACCCGAAAGCCTTCTTCACTCACGCGGCGTTGCTCCATCAGACTTGCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTANNAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATCAACCTCTCAGTTCGGCTACGTATCATCGCCTTGGTGAGCCATTACCTCACCAACTAGCTAATACGCCGCGGGTCCATCCAAAAGCGATAGCTTACGCCATCTTTCAGCCAAGAACCATGCGGTTCTTGGATCTATGCGGTATTAGCATCTGTTTCCAAATGTTATCCCCCACTTAAGGGCAGGTTACCCACGTGTTACTCACCCGTCCGCCACTCGTTCCATGTTGAATCTCGGTGCAAGCACCGATCATCAACGAGAACTCGTTCGACTGCATGTATAGCL. plantarum:GGCGTGCCTAATACATGCAAGTCGAACGAACTCTGGTATTGATTGGTGCTTGCATCATGATTTACATTTGAGTGAGTGGCGAACTGGTGAGTAACACGTGGGAAACCTGCCCAGAAGCGGGGGATAACACCTGGAAACAGATGCTAATACCGCATAACAACTTGGACCGCATGGTCCGAGTTTGAAAGATGGCTTCGGCTATCACTTTTGGATGGTCCCGCGGCGTATTAGCTAGATGGTGGGGTAACGGCTCACCATGGCAATGATACGTAGCCGACCTGAGAGGGTAATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAACTCTGTTGTTAAAGAAGAACATATCTGAGAGTAACTGTTCAGGTATTGACGGTATTTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCTTCGGCTCAACCGAAGAAGTGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGTATGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCATACCGTAAACGATGAATGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATACTATGCAAATCTAAGAGATTAGACGTTCCCTTCGGGGACATGGATACAGGTGGTGCATGGTTGTCGTCAGCTGGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTATCAGTTGCCACCATTAAGTTGGGCACTCTGGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAAGGAGTTGCGAACTCGCGAGAGTAAGCTAATCTCTTAAAGCCATTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCAAAGTCGGTGGGGTAACCTTTTAGGAACCAGCCGCCTAAGGTGGL. kefiri:CTTAGACGGCTGGTCCCCGAAGGTTACCTCACCGGCTTTGGGTGTTACAAACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGTGGCATGCTGATCCACGATTACTAGCGATTCCAACTTCATGCAGGCGAGTTGCAGCCTGCAATCCGAACTGAGAACGGCTTTAAGAGATTAGCTTGACCTCGCGGTTTCGCGACTCGTTGTACCGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTTGCTAGAGTGCCCAACTGAATGCTGGCAACTAACAATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCATTCTGTCCCCGAAGGGAACGCCTAATCTCTTAGGTTGGCAGAAGATGTCAAGACCTGGTAAGGTTCTTCGCGTAGCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTGAAGGGCGGAAACCCTCCAACACTTAGCACTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTACAGACCAGACAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTTCACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTCTCCTGGTTTCCGATGCACTTCTCCGGTTAAGCCGAAGGCTTTCACATCAGACCTAAGAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTGGATACCGTCAAGATGTCAACAGTTACTCTGACACCTGTTCTTCTCCAACAACAGAGTTTTACGAGCCGAAACCCTTCATCACTCACGCGGCGTTGCTCCATCAGACTTTCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATTACCCTCTCAGGTCGGCTACGTATCATTGCCTTGGTAGGCCATTACCTTACCAACAAGCTAATACGCCGCGGGTCCATCCTAAAGTGATAGCCGAAGCCATCTTTTAAACCAAAACCATGTGGTTTTGGTTGTTATACGGTATTAGCACCTGTTTCCAAGTGTTATCCCCTACTTCAAGGGCAGGTTACCCACGTGTTACTCACCAGTTCGCCACTCGTTTCGTGTTAAATCATTTAAATGCAAGCATCTAAAATCAATAACGGAAACGCGTTCGACTTGCATGTATLactococcus lactic:GGTCTTACCTTAGGAAGCGCCCTCCTTGCGGTTAGGCAACCTACTTCGGGTACTCCCAACTCCCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCGTGCTGATCCGCGATTACTAGCGATTCCGACTTCATGTAGGCGAGTTGCAGCCTACAATCCGAACTGAGAATGGTTTTAAGAGATTAGCTAAACATCACTGTCTCGCGACTCGTTGTACCATCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTATCACCGGCAGTCTCGTTAGAGTGCCCAACTTAATGATGGCAACTAACAATAGGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTATCCCGTGTCCCGAAGGAACTTCCTATCTCTAGGAATAGCACGAGTATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTATTGCGTTAGCTGCGATACAGAGAACTTATAGCTCCCTACATCTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGAGCCTCAGTGTCAGTTACAGGCCAGAGAGCCGCTTTCGCCACCGGTGTTCCTCCATATATCTACGCATTTCACCGCTACACATGGAATTCCACTCTCCTCTCCTGCACTCAAGTCTACCAGTTTCCAATGCATACAATGGTTGAGCCACTGCCTTTTACACCAGACTTAATAAACCACCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTCGGGACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTCCCTTTCTGGGTAGTTACCGTCACTTGATGAGCTTTCCACTCTCACCAACGTTCTTCTCTACCAACAGAGTTTTACGATCCGAAAACCTTCTTCACTCACGCGGCGTTGCTCGGTCAGACTTTCGTCCATTGCCGAAGATTCCCTACTGCTGCCTCCCGTANGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATCACCCTCTCAGGTCGGCTATGTATCATCGCCTTGGTGAGCCTTTACCTCACCAACTAGCTAATACAACGCGGGATCATCTTTGAGTGATGCAATTGCATCTTTCAAACTTAAAACTTATGTTTAAAGTTGTTATGCGGTATTAGCATTCGTTTCCAAATGTTGTCCCCCGCTCAAAGGCAGATTCCCCACGCGTTACTCACCCGTTCGCTGCTCTTCAAATTGGTGCAAGCACCAATCTTCATCGCTCAACTTGCATGATTAGSeven gram-positive, catalase-negative bacilli were isolated from TFCW. Most of the bacilli had yellow or white colonies with uneven edges and a rough and dull surface. Some of the colonies in the center were translucent, with neat edges. The type colony size was approximately 0.5-3.0\u00a0mm. Morphology of the separated S. unisporus and I. orientalis based on morphological characteristics, physiological and biochemical tests and 26S rDNAD1/D2 sequence homology with sequences AY 707865 and EU 019220 in Genbank. All the test results and sequence homology were shown in Table\u00a0S. unisporus:TGCATATTCAATAAGCGGAGGAAAAGAAACCAACCGGGATTGCCTTAGTAACGGCGAGTGAAGCGGCAAAAGCTCAAATTTGAAATCTAGTACCTTCGGTGCTCGAGTTGTAATTTGTAGAGGGATACTTTGGGGCCGTTCCTTGTCTATGTTCCTTGGAACAGGACGTCATAGAGGGTGAGAATCCCGTGTGGCGAGGAGTGCGGTTCTATGTAAAGTGCCTTCGAAGAGTCGAGTTGTTTGGGAATGCAGCTCTAAGTGGGTGGTAAATTCCATCTAAAGCTAAATATTGGCGAGAGACCGATAGCGAACAAGTACAGTGATGGAAAGATGAAAAGAACTTTGAAAAGAGAGTGAAAAAGTACGTGAAATTGTTGAAAGGGAAGGGCATTTGATCAGACATGGTGTTTTGCGCCCTCTGCTCCTTGTGGGTGGGGGAATCTCGCAGCTCACTGGGCCAACATCAGTTTTGGTGGTCGGATAAATCCGTAGGAATGTGGCTTGCCTCGGCAAGTGTTATAGCCTGCGGGAATACGGCCAGCTGGGACTGAGGACTGCCACTTTTGTCAAGGATGTTGGCATAATGGTTATATGCCGCCCGTCTTGAAACACGGACCAAI. orientalis:GCATATCAATAAGCGGAGGAAAAGAAACCAACAGGGATTGCCTCAGTAGCGGCGAGTGAAGCGGCAAGAGCTCANATTTGAAATCGTGCTTTGCGGCACGAGTTGTAGATTGCAGGTTGGAGTCTGTGTGGAAGGCGGTGTCCAAGTCCCTTGGAACAGGGCGCCCAGGAGGGTGAGAGCCCCGTGGGATGCCGGCGGAAGCAGTGAGGCCCTTCTGACGAGTCGAGTTGTTTGGGAATGCAGCTCCAAGCGGGTGGTAAATTCCATCTAAGGCTAAATACTGGCGAGAGACCGATAGCGAACAAGTACTGTGAAGGAAAGATGAAAAGCACTTTGAAAAGAGAGTGAAACAGCACGTGAAATTGTTGAAAGGGAAGGGTATTGCGCCCGACATGGGGATTGCGCACCGCTGCCTCTCGTGGGCGGCGCTCTGGGCTTTCCCTGGGCCAGCATCGGTTCTTGCTGCAGGAGAAGGGGTTCTGGAACGTGGCTCTTCGGAGTGTTATAGCCAGGGCCAGATGCTGCGTGCGGGGACCGAGGACTGCGGCCGTGTAGGTCACGGATGCTGGCAGAACGGCGCAACACCGCCCGTCTTGAAACACGGACCAWe identified two yeast isolates to be Major finding of the current study is that treatment with TFCW significantly modified lipid profile and reduced CRP, ICAM-1 and VCAM-1 in atherosclerotic rabbit model. Preventive effects of TFCW in atherogenic rabbits were also demonstrated by reduction in VCAM-1 expression and formation of atheromatous plaques on aortic endothelium. In fact, accumulation of cholesterol and lipids leads to foam cell formation, which is regarded as a critical process in development of atherosclerosis . OverwheL. casei [L. helveticus [L. plantarum [L. lactis [In this study, 7 potential probiotic lactobacillus species, including L. casei , L. helvlveticus , L. planlantarum and L. l. lactis , which a. lactis and redu. lactis , 23 both. lactis , 25 and . lactis . LAB or . lactis .Lactocillus fermenterum ME-3 improves antioxidant activities in human blood, thus providing antiatherogenic activity [In addition, goat milk fermented with activity . Consumpactivity , 29.S. unisporus is ubiquitously present in fermented milk, cheese and kefir-based milk products and may produce vitamins and interact with LAB, which may enhance LAB growth [S. unisporus contains middle chain fatty acids up to C 14:0 to 18:1 and produces a high percentage of palmitoleate. Palmitoleic acid, an omega-7 monounsaturated fatty acid, is a major constituent of human adipose tissues and is considered antioxidant [I.orientalis exhibits a higher tolerance for pH, bile, and heat stress for survival in gastrointestinal environment as a probiotic [I. orientalis commonly exists in cheeses and other fermentation milk products and exhibits ability to scavenge 1,1 diphenyl-2-picrylhydrazyl and to inhibit lipid peroxidation, thus presenting antioxidant activity as a potential probiotic in fermented milk products [We also identified two probiotic yeasts in TFCW. B growth . S. unisioxidant . I.orienrobiotic . I. orieproducts .Notably, oxidized LDL in vascular wall seems to be a key factor in atherosclerosis, because oxidized LDLs might recruit monocytes and favor their transformation into foam cells through a receptor-mediated intake (scavenger pathway). Moreover, cytotoxic oxidized form of LDLs are likely responsible for endothelial cell damage and macrophage degeneration in atherosclerotic human plaque . PolyunsIn conclusion, current study indicates that 7 LAB and 2 yeasts identified from TFCW and TFCW have significant anti-atherosclerotic potential in atherosclerotic rabbits and may modulate lipid metabolism and protect aorta in the atherosclerotic condition, which might be related to various probiotics acting through reducing the CRP, VCAM-1 and ICAM-1 levels and protecting the aortic endothelium."} +{"text": "Mitochondrial dysfunction would ultimately lead to myocardial cell apoptosis and death during ischemia-reperfusion injuries. Autophagy could ameliorate mitochondrial dysfunction by autophagosome forming, which is a catabolic process to preserve the mitochondrial\u2019s structural and functional integrity. HO-1 induction and expression are important protective mechanisms. This study in order to investigate the role of HO-1 during mitochondrial damage and its mechanism.The H9c2 cardiomyocyte cell line were incubated by hypoxic and then reoxygenated for the indicated time . Cell viability was tested with CCK-8 kit. The expression of endogenous HO-1(RT-PCR and Western blot) increased with the duration of reoxygenation and reached maximum levels after 2 hours of H/R; thereafter, the expression gradually decreased to a stable level. Mitochondrial dysfunction (Flow cytometry quantified the ROS generation and JC-1 staining) and autophagy were induced after 6 hours of H/R. Then, genetic engineering technology was employed to construct an Lv-HO1-H9c2 cell line. When HO-1 was overexpressed, the LC3II levels were significantly increased after reoxygenation, p62 protein expression was significantly decreased, the level of autophagy was unchanged, the mitochondrial membrane potential was significantly increased, and the mitochondrial ROS level was significantly decreased. Furthermore, when the HO-1 inhibitor ZnPP was applied the level of autophagy after reoxygenation was significantly inhibited, and no significant improvement in mitochondrial dysfunction was observed.During myocardial hypoxia-reoxygenation injury, HO-1 overexpression induces autophagy to protect the stability of the mitochondrial membrane and reduce the amount of mitochondrial oxidation products, thereby exerting a protective effect. To date, mitochondria have been thought to play an important role in myocardial ischemia-reperfusion (I/R) injury . It is wIn contrast, heme oxygenase-1 (HO-1), which belongs to the low-molecular weight heat shock protein HSP family, induction and expression are important protective mechanisms during cell stress \u20136. In raTherefore, in this study, we chose to use genetic engineering techniques to construct an Lv-HO1-H9c2 cell line that enables a steady increase in the expression of exogenous HO-1. After myocardial hypoxia-reoxygenation injury, the RFP-GFP-LC3 double-labeled adenovirus was used to detect autophagy, and flow cytometry was used to measure mitochondrial membrane potential and mitochondrial ROS levels to investigate the mechanism by which HO-1 protects mitochondrial function following myocardial hypoxia-reoxygenation injury.The effect of different durations of hypoxia/reoxygenation on myocardial cytotoxicity was measured. As shown in Cell viability was compared between the four groups . SignifiCell viability was compared between the four groups . An Lv-HCell viability in the four groups was compared . After ZHO is the initial and rate-limiting enzyme that catalyzes the oxidative metabolism of heme. Three HO isozymes exist, and HO-1 is the only isoenzyme that exhibits inductive expression. During cardiac ischemia-reperfusion injury, ROS generation can increase the expression of HO-1 , consistThe autophagic dysfunction of myocardial cells has attracted attention as one of the mechanisms underlying ischemia-reperfusion injury ,24,25. IIn addition, in this study, flow cytometry detection showed that during autophagy, the mitochondrial membrane potential was significantly higher and the Therefore, clearing damaged mitochondria is extremely important. Autophagy, the major degradation pathway involved in mitochondrial quality control, has been reported as a cellular adaptive response to oxidative stresses . PreviouInterestingly, in our studies, rapamycin could up-regulate the expression of the HO-1 protein . It is aTo define the role of HO-1, most studies have used methods such as employing adeno-associated viruses to achieve instantaneous HO-1 over-expression ,14 or knWith HO-1 overexpression, LC3II levels were significantly increased after reoxygenation, and p62 protein expression was significantly decreased . MoreoveRecent studies have shown that an HO-1 activator can activate transcription factor EB (TFEB), a regulator that induces lysosome and autophagosome formation, to protect against LPS-induced oxidative stress in heart tissue . MeanwhiIn summary, we based our conclusions on evidence that showed that during myocardial hypoxia-reoxygenation injury, HO-1 overexpression induces autophagy to protect the stability of the mitochondrial membrane and reduce the amount of mitochondrial oxidation products, thereby exerting a protective effect against myocardial hypoxia-reoxygenation injury. By studying the molecular and regulatory mechanisms of mitochondrial function protection, we will obtain further knowledge regarding the mechanism underlying the development of ischemic heart disease, information that will aid in for the development of new disease prevention and treatment strategies.Dulbecco\u2019s Modified Eagle\u2019s Medium (DMEM) and other cell culture supplies, such as trypsinogen, were purchased from Invitrogen. Fetal calf serum was purchased from Gibco BRL . A Cell Counting Kit-8 was purchased from Dojindo . HO-1 and pAbR rabbit antibody were purchased from Enzo Life Sciences Inc. . p62 and LC-3 antibody were purchased from Abcam (USA), and GAPDH antibody was obtained from CST (USA). AntiRabbit was obtained from Sigma (USA). BSA was obtained from Invitrogen (USA). TritonX-100 was obtained from Sigma (USA). A JC-1 Mitochondrial Membrane Potential Detection Kit was purchased from Beyotime (China). Confocal microscopy and imaging systems (ZEISS-Axio) were used at the Wuhan University School of Basic Medical Sciences.34H32N4O4Zn and a molecular weight of 626.03 g/mol.Zinc protoporphyrin (ZnPP), a HO-1 inhibitor, was supplied by Sigma. The purity of ZnPP (\u226595%) was determined by HPLC; the inhibitor was soluble in water and has the molecular formula C2. The medium was replaced every 2\u20133 days, and the cells were subcultured or subjected to experimental procedures at 80\u201390% confluence. In all experiments, the cells were rendered quiescent by serum starvation for 24 h before treatment. For the H/R experiments, hypoxic conditions were created by incubating the cells in an anaerobic chamber equilibrated with 2.5% O2, 5% CO2 and 92.5% N2 at 37\u00b0C for 24 h. The cells were then reoxygenated under normoxic conditions in a humidified atmosphere (95% air/5% CO2) at 37\u00b0C for the indicated time . Normoxic control cells were incubated at 37\u00b0C under 95% air/5% CO2. In the Rapamycin experiments, the cells were pretreated for 1 hour with the autophagy inducer Rapamycin (100 nM) and then subjected to hypoxia and reoxygenation. In the ZnPP experiments, the cells were pretreated for 6 hours with the HO-1 inhibitor ZnPP (10 \u03bcM) and then subjected to hypoxia and reoxygenation.The H9c2 cardiomyocyte cell line (rat embryonic cardiomyoblasts) was maintained in our laboratory and obtained from the American Type Culture Collection . The H9c2 cells were maintained in high-glucose DMEM supplemented with 10% v/v fetal calf serum at 37\u00b0C in a humidified atmosphere containing 5% COA lentiviral vector was constructed, and a lentiviral shuttle plasmid and its secondary packaging of the original vector plasmid were prepared; the three plasmid vectors were subjected to endotoxin-free extraction to obtain materials of high purity. Next, the plasmid vectors were co-transfected into 293T cells. The medium was replaced with complete medium at 6 hours after transfection. The supernatant, which was rich in lentivirus particles, was collected after 48 and 72 hours of culture and filtered using a 0.45 \u03bcm filter (Millipore), and the virus was then concentrated by ultracentrifugation. Next, a high-titer lentivirus concentrate was obtained after concentration. A vector map of pHBLV-CMVIE-IRES-puro is shown in The pHBLV-CMVIE-IRES-puro vector contains a CMVIE promoter to initiate the expression of target genes and contains a puromycin resistance gene as a marker. The HO-1primer designs as ATGGAGCGTCCGCAACCCGACAGCATGCCCCAGGATTTGTCAGAGGCCCTGAAGGAGGCCACCAAGGAGGTGCACACCCAGGCAGAGAATGCTGAGTTCATGAGGAACTTTCAGAAGGGCCAGGTGACCCGAGACGGCTTCAAGCTGGTGATGGCCTCCCTGTACCACATCTATGTGGCCCTGGAGGAGGAGATTGAGCGCAACAAGGAGAGCCCAGTCTTCGCCCCTGTCTACTTCCCAGAAGAGCTGCACCGCAAGGCTGCCCTGGAGCAGGACCTGGCCTTCTGGTACGGGCCCCGCTGGCAGGAGGTCATCCCCTACACACCAGCCATGCAGCGCTATGTGAAGCGGCTCCACGAGGTGGGGCGCACAGAGCCCGAGCTGCTGGTGGCCCACGCCTACACCCGCTACCTGGGTGACCTGTCTGGGGGCCAGGTGCTCAAAAAGATTGCCCAGAAAGCCCTGGACCTGCCCAGCTCTGGCGAGGGCCTGGCCTTCTTCACCTTCCCCAACATTGCCAGTGCCACCAAGTTCAAGCAGCTCTACCGCTCCCGCATGAACTCCCTGGAGATGACTCCCGCAGTCAGGCAGAGGGTGATAGAAGAGGCCAAGACTGCGTTCCTGCTCAACATCCAGCTCTTTGAGGAGTTGCAGGAGCTGCTGACCCATGACACCAAGGACCAGAGCCCCTCACGGGCACCAGGGCTTCGCCAGCGGGCCAGCAACAAAGTGCAAGATTCTGCCCCCGTGGAGACTCCCAGAGGGAAGCCCCCACTCAACACCCGCTCCCAGGCTCCGCTTCTCCGATGGGTCCTTACACTCAGCTTTCTGGTGGCGACAGTTGCTGTAGGGCTTTATGCCATGTGAAfter H9c2 and Lv-HO1-H9c2 cells were cultured in 96-well plates and received the described treatments, 10 \u03bcl of CCK-8 solution was added to each well (1/10 dilution); the plates were then incubated for a further 3 h. The absorbance was measured at 450 nm using a microplate reader . The mean optical density (OD) of five wells in the indicated groups was used to calculate the percent cell viability according to the following formula: Percent cell viability = OD treatment group/OD control group\u00d7100%. The experiments were repeated 3 times.\u00aePremix Ex Taq\u2122 Perfect Real-Time Kit. To measure mouse gene expression, the following SYBR Green real-time PCR primers were used:Total RNA was isolated from cells using TRIzol Reagent (Invitrogen) and purified using the RNeasy Total RNA Isolation Kit (Invitrogen). Real-time quantitative PCR was performed using the ABI 7900 Real-Time PCR System (Applied Biosystems) and the SYBR5\u2032- TGCACATCCGTGCAGAGAAT -3\u2032HO-1: Forward 5\u2032-CTGGGTTCTGCTTGTTTCGC -3\u2032; product length = 147\u2003\u2003\u2003\u2003Reverse 5\u2032\u2014CACCATCTTCCAGGAGCGAG -3\u2032GAPDH: Forward 5\u2032\u2014AAATGAGCCCCAGCCTTCTC -3\u2032; product length = 114\u2003\u2003\u2003\u2003Reverse Proteins were isolated from the cells that had been subjected to hypoxia and reoxygenation using standard protocols, and the protein concentrations in the lysates were determined using the Bradford Protein Assay Kit . Equal quantities of proteins (30\u201350 lg/lane) were subjected to 8%\u201312% SDS-PAGE, depending on the target proteins, electrotransferred onto nitrocellulose membranes, and incubated with primary antibodies against HO-1 , p62 , LC-3-II , GAPDH , and antiRabbit . After incubating the membranes with the corresponding secondary antibodies, protein bands were detected using a chemiluminescence imaging analysis system (Fujifilm), and quantitation was performed using Quantity One 4.4.0 software (Bio-Rad).4 cells/mL) was added. Cells were transfected with Ad-mCherry-LC3-GFP adenovirus (MOI = 50); 36 hours later, the plates were pre-incubated for 24 hours in an incubator . Next, 4% PFA was used to fix the cells for more than 10 minutes; the cells were then washed in PBS (5 minutes x 3 times). Then, Antifade mounting medium was added, and the cells were imaged under a fluorescent microscope. RFP-GFP-LC3 double-labeled adenovirus was used for testing. GFP degrades in the acidic environment used. When red and green fluorescent images are merged, the yellow spots that appear indicate autophagosomes. Red spots indicate autophagic lysosomes. In normal phagosome-lysosome fusions, there will be more red fluorescence than yellow fluorescence. In impeded autophagy, where phagosome-lysosome fusion does not work, yellow fluorescence will be dominant.To 6-well plates , 2 mL of Lv-HO1-H9c2 cell suspension and reactive nitrogen species (RNS) rapid oxidation. After binding nucleic acid oxidation products, the probe can produce large amounts of fluorescence by flow cytometry, thus, the changes of mitochondrial reactive oxygen species content may be reflected.Using flow cytometry to detect the 5,5',6,6'-tetrachloro-1,1',3,3'-. tetraethylbenzi-midazolylcarbocyanine iodide (JC-1) staining. After incubation with the JC-1 staining solution at 37\u00b0C in the cell incubator for 10 min, cells were washed with JC-1 staining buffer two times and then analyzed using a fluorescence microplate reader. In the normal mitochondria, JC-1 aggregate to form a polymer in the mitochondrial matrix, the polymer sends a strong red fluorescence ; While in the unhealthy mitochondrial, due to the decline or loss of the mitochondrial membrane potential, JC-1 monomers just can be present in the cytoplasm, resulting in a green fluorescence . Therefore, using flow cytometry to observe the color changes reflects very directly the early change of mitochondrial membrane potential.5 cells were harvested and placed in EP tubes. The cells were rinsed with 0.1 mmol/L PBS (pH = 7.4) and collected brief centrifugation with trypsin EDTA-free. After that, the cells were resuspended in 500 \u03bcL of binding buffer, mixed with 5 \u03bcL of Annexin V-FITC, and then kept in darkness for 10 min at room temperature. Then, 5 \u03bcL of propidium iodide (PI) was added and the samples were kept in darkness for 10 min at room temperature. Approximately 300 \u03bcL of binding buffer was added, and the samples were analyzed using flow cytometry within 1 h. The blank control was the cells of the control group without Annexin V-FITC and PI. The Annexin V-FITC single-labeled control consisted of cells labeled with Annexin V-FITC. The PI single-labeled control consisted of cells labeled with PI.After treatment, 1~5\u00d710The data are presented as means \u00b1 SEM. Statistical analysis was performed using the Mann-Whitney test for two-group comparisons. Significant differences between multiple treatments were calculated using one-way analysis of variance followed by the Bonferroni post hoc test when appropriate. Western blot densities were analyzed using the Kruskal-Wallis test followed by Dunn's post hoc test. Probabilities of 0.05 or less were considered statistically significant.S1 Fig(A) Cell viability; (B) HO1 QPCR; (C)WB analysis.(RAR)Click here for additional data file.S2 Fig(A) Cell viability (CCK-8); (B) WB analysis; (C) Confocol-RFP-GFP-LC3; Flow cytometry detection of changes in JC-1 and MitoSox; (F) apoptosis rate (AnnexinV-PI).(RAR)Click here for additional data file.S3 Fig(A) Cell viability (CCK-8); (B) HO1 QPCR; (C) WB analysis; (D)Confocol-RFP-GFP-LC3; Flow cytometry detection of changes in JC-1 and MitoSox; (G) apoptosis rate (AnnexinV-PI).(RAR)Click here for additional data file.S4 Fig(A) Cell viability (CCK-8); (B) HO1 QPCR; (C) WB analysis; (D)Confocol-RFP-GFP-LC3; Flow cytometry detection of changes in JC-1 and MitoSox; (G) apoptosis rate (AnnexinV-PI).(RAR)Click here for additional data file."} +{"text": "Monoclonal antibodies (mAbs), because of their unique specificity, are irreplaceable tools for scientific research. Precise mapping of the antigenic determinants allows the development of epitope tagging approaches to be used with recombinant proteins for several purposes. Here we describe a new family of tags derived from the epitope recognized by a single highly specific mAb (anti-roTag mAb), which was obtained from a pool of mAbs reacting with the rotavirus nonstructural protein 5 (NSP5). The variable regions of the anti-roTag mAb were identified and their binding capacity verified upon expression as a single-chain/miniAb. The minimal epitope, termed roTag, was identified as a 10 amino acid sequence (SISSSIFKNE). The affinity of the anti-roTag/roTag interaction was found to be comparable to that of the anti-SV5/SV5 tag interaction. roTag was successfully used for detection of several recombinant cytosolic, secretory and membrane proteins. Two additional variants of roTag of 10 and 13 amino acids containing O-glycosylation susceptible sites (termed OG-tag and roTagO) were constructed and characterised. These tags were useful to detect proteins passing through the Golgi apparatus, the site of O-glycosylation. In biological sciences development of new specific monoclonal antibodies (mAbs) is a pressing requirement for several aspects in the field: from basic research on protein function, to medical diagnosis, prophylaxis and therapy of several pathogenic conditions As tags are often short (6\u201315 amino acids in length), they are generally presumed to have no effect on the biological functions of the tagged proteins. However, if located in inappropriate positions, they might interfere with protein structure, function and interactions. In addition, not all mAb are suitable for every immunodetection method, as in the case of mAb specific for non-linear epitopes. For those reasons, it is useful to develop mAbs and epitope tags of different sequence characteristics or that can be fused in different positions of the target protein to increase the chances of success in tagging applications.Here we describe and characterize a new 10 amino acids long epitope tag (roTag) derived from the sequence of the rotavirus (RV) non-structural protein 5 (NSP5). NSP5 has an essential role during the RV replication cycle, as it is essentially required for the assembly of viroplasms, the sites of viral genome replication and initial assembly of progeny virus ++-purified His-tagged NSP5 protein of the RV porcine OSU strain A panel of anti-NSP5 mAbs were generated from BALB/c mice immunized with a NiTo establish the epitope on NSP5 we took advantage of a series of NSP5 deletion mutants To further define the epitope, a series of progressive N-terminal deletion mutants, NSP5-\u0394N3, NSP5-\u0394N8, NSP5-\u0394N13, NSP5-\u0394N18, NSP5-\u0394N23 and NSP5-\u0394N28, were assayed with a polyclonal anti-NSP5 serum or with 1F2 by WB. As shown in PSISSSIFKNE) suggesting involvement of S12 and S14. In fact, a tag initiating in S12 (tag 12\u201324) resulted in a pattern of bands equally recognized by anti-SV5 and 1F2, both in the intracellular and in the secreted material and because of the relevant impact of P11 on O-glycosylation, we defined S12 as the N-terminal border of the epitope and termed roTag the peptide 12\u201321 and P-roTag the one starting in P11 (11\u201321). The C-terminal border of the anti-roTag/1F2 epitope was confirmed to be E21, as peptides 9\u201318, 9\u201319 and 9\u201320 were not detected by anti-roTag . All theper se sufficient to cause retention in the ER and to completely prevent O-glycosylation, according to three different criteria: unchanged mobility, lack of secretion and detection by mAb anti-roTag.O-glycosylation was confirmed for roTagO, P-roTag and OG-tag by treatment of supernatants containing the reporter protein (with either of the three tags) with a glycosidase cocktail. As shown in L-linker-VH) fused to the hinge-CH2-CH3 domains of the \u03b3H chain of murine IgG2b. The resulting recombinant antibodies containing the scFv the 10 amino acids long roTag (peptide 12\u201321) that can be efficiently used with different proteins, in N-terminal, middle and C-terminal positions and in different cellular compartments , approved by the ICGEB Trieste Ethics Committee for Animal Experimentation. Animals were anesthetized (isoflurane) before venipuncture and sacrifice (asphyxiation in carbon dioxide). All efforts were made to minimize suffering.++-purified His-tagged NSP5 protein Balb/c mice have been immunized with NiNSP5 deletion mutants were previously described 5\u2032-GATATTGTGATGACCCAGTCTCCA-3\u2032, CK-2 3\u2032: 5\u2032-TGGATACAGTTGGTGCAGC-3\u2032, VHS-2 5\u2032: 5\u2032-TGTGCACTCYSAGGTSMARCT-3\u2032, CH\u03b3 3\u2032: 5\u2032-GGCCAGTGGATAGAC-3\u2032. From pUC18 VL and VH regions were amplified with primers 1F2-VL-for: 5\u2032TATGGTGCACTCTGATGTTGTGATGACCCAGACTCCA-3\u2032, 1F2-VL-rev: 5\u2032-TATAACTAGTGCTGCCTTTCAGCTCCAGCTTGGT-3\u2032, VH-for 5\u2032: 5\u2032-TCTCTCGAGCAAAGGTCAGGTCCAACTGCAGCAGTC-3\u2032, 1F2-VH rev: 5\u2032-AGTTCCGGAGGAGACGGTGACTGA-3\u2032 and cloned ApaLI/BspEI into pUT vector downstream of a leader peptide H2 e CH3 of IgG2b, which were previously amplified from murine splenocytes and inserted in pcDNA3 by BspEI/XbaI (primers MHG2B-1 5\u2032: 5\u2032-CCCTCCGGACCCATTTCAACAATCA-3\u2032 and MHG2B-2 3\u2032: 5\u2032-TCTTCTAGAGCTCATTTACCCGGAGA-3\u2032).After cDNA synthesis VL and VH were amplified and cloned in pUC18 using primers VKB-2 5\u2032: 5\u2032-GATCCCTTCCCTCAATTTCTTCTAGTATCTTTAAAAATGAATCGFor mapping the anti-roTag epitope the following synthetic oligonucleotides encoding the different epitope tags were cloned as BamHI/EcoRI, downstream of SV5 in a plasmid containing scFv-SV5-BAP previously described 5\u2032-AATTCTTAAGAAGACGATTCATTTTTAAAGATACTAGAAGAAATCTTCTTAAG-3\u2032, 9-24-2 3\u2032: 5\u2032-GATCCCTTCCCTCAATTTCTTCTAGTATCTTTAAAAATGAAGGTTGAGGGAAGG-3\u2032, 9-24G-1 5\u2032: 5\u2032-AATTCTTAAGAAGAACCTTCATTTTTAAAGATACTAGAAGAAATTGAGGGAAGG-3\u2032, 9-21-1 5\u2032: 5\u2032-GATCCCTTCCCTCAATTTCTTCTAGTATCTTTAAAAATGAAGGTTAAG-3\u2032, 9-21-2 3\u2032: 5\u2032-AATTCTTAACCTTCATTTTTAAAGATACTAGAAGAAATTGAGGGAAGG-3\u2032, 12-24-1 5\u2032: 5\u2032-GATCCATTTCTTCTAGTATCTTTAAAAATGAATCGTCTTCTTAAG-3\u2032, 12-24-2 3\u2032: 5\u2032-AATTCTTAAGAAGACGATTCATTTTTAAAGATACTAGAAGAAATG-3\u2032, 9-16-1 5\u2032: 5\u2032-GATCCCTTCCCTCAATTTCTTCTAGTTAAG-3\u2032, 9-16-2 3\u2032: 5\u2032-AATTCTTAACTAGAAGAAATTGAGGGAAGG-3\u2032, 10-21-1 5\u2032: 5\u2032-GATCCGGTGGCCTTCCCTCAATTTCTTCTAGTATCTTTAAAAATGAAGGTTAAG-3\u2032, 10-21-2 3\u2032: 5\u2032-AATTCTTAACCTTCATTTTTAAAGATACTAGAAGAAATTGAGGGAAGGCCACCG-3\u2032, 11-21-1 5\u2032: 5\u2032-GATCCGGTGGCCCCTCAATTTCTTCTAGTATCTTTAAAAATGAAGGTTAAG-3\u2032, 11-21-2 3\u2032: 5\u2032-AATTCTTAACCTTCATTTTTAAAGATACTAGAAGAAATTGAGGGGCCACCG-3\u2032, 12-21-1 5\u2032: 5\u2032-GATCCATTTCTTCTAGTATCTTTAAAAATGAAGGTTAAG-3\u2032 12-21-2 3\u2032: 5\u2032-AATTCTTAACCTTCATTTTTAAAGATACTAGAAGAAATG-3\u2032, 9-20-1 5\u2032: 5\u2032-GATCCCTTCCCTCAATTTCTTCTAGTATCTTTAAAAATTAAG-3\u2032, 9-20-2 3\u2032: 5\u2032-AATTCTTAATTTTTAAAGATACTAGAAGAAATTGAGGGAAGG-3\u2032, 9-19-1 5\u2032: 5\u2032-GATCCCTTCCCTCAATTTCTTCTAGTATCTTTAAATAAG-3\u2032, 9-19-2 3\u2032: 5\u2032-AATTCTTATTTAAAGATACTAGAAGAAATTGAGGGAAGG-3\u2032, 9-18-1 5\u2032: 5\u2032-GATCCCTTCCCTCAATTTCTTCTAGTATCTTTTAAG-3\u2032, 9-18-2 3\u2032: 5\u2032-AATTCTTAAAAGATACTAGAAGAAATTGAGGGAAGG-3\u2032, roTagG KDEL-1: GATCCCTTCCCTCAATTTCTTCTAGTATCTTTAAAAATGAAGGTAAGGATGAGCTTTAAG, roTagG KDEL-2: AATTCTTAAAGCTCATCCTTACCTTCATTTTTAAAGATACTAGAAGAAATTGAGGGAAGG, OG-Tag KDEL-1: GATCCCTTCCCTCAATTTCTTCTAGTATCTTTGGAAAGGATGAGCTGTAAG, OG-Tag KDEL-2: AATTCTTACAGCTCATCCTTTCCAAAGATACTAGAAGAAATTGAGGGAAGG.TTCTTCTTAAG-3\u2032, 9-24G-2 3\u2032: 5 cells/well) by standard calcium phosphate technique Sp2/0 myeloma cells (ATCC CRL-1581) were cultured in RPMI 1640 supplemented with 10% fetal calf serum (FCS); hybridoma clones were grown in the same medium supplemented with 2% Hybridoma Media Supplement (Sigma-Aldrich) and 1 mM sodium pyruvate. MA104 and HEK 293T cells were grown in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% fetal calf serum (FCS). Cells were co-transfected in 6-well plates supplemented with protease inhibitors cocktail (Sigma-Aldrich). In the experiments shown in Immunofluorescence experiments were performed after cells were fixed in 3.7% paraformaldehyde in PBS for 10 min at room temperature. Coverslips were washed in PBS and blocked with 1% bovine serum albumin (BSA) in PBS for 30 min and incubated with mouse anti-NSP5 serum (1:500) in PBS\u20131% BSA, supernatants of anti-roTag hybridoma for 1 h at room temperature. After three washes in PBS, the slides were stained for 45 min with rhodamine isothiocyanate-conjugated secondary antibody (Sigma), washed, and mounted with ProLong mounting medium (Molecular Probes). Samples were analyzed by confocal microscopy (Zeiss LSM510).3-Na2CO3 50 mM, pH 9.5 (100 \u00b5l/well). After reaction with HRP-labeled goat anti-mouse gamma Fc (Jackson Immunoresearch) plates were developed with tetramethylbenzidine (TMB) reagent (Sigma) and blocked with H2SO4. O.D. at 450 nm was read on a BioRad microplate reader 550. ELISA comparison of relative affinity of recombinant anti-SV5 and anti-roTag was performed by capturing the double-tagged (SV5-roTag) reporter ELISA to determine relative antibody concentrations was performed by capturing supernatants containing Abs on polystyrene microplates (Nunc Maxisorp C96) coated with 0.1 \u00b5g/ml of goat anti-mouse gamma in buffer NaHCOFigure S1(A) Full gel, of non-infected and RV-infected MA104 cell extracts reacted with mAb 1F2. (B) Full gel of lanes shown in (TIF)Click here for additional data file."} +{"text": "G\u03b1s, which transduces mitogenic signals, in 30% of the analyzed JGCTs.Ovarian granulosa cell tumors are the most common sex-cord stromal tumors and have juvenile (JGCTs) and adult forms. In a previous study we reported the occurrence of activating somatic mutations of We have searched for alterations in other proteins involved in ovarian mitogenic signaling. We focused on the PI3K\u2013AKT axis. As we found mutations in AKT1, we analyzed the subcellular localization of the mutated proteins and performed functional explorations using Western-blot and luciferase assays.We detected in-frame duplications affecting the pleckstrin-homology domain of AKT1 in more than 60% of the tumors occurring in girls under 15 years of age. The somatic status of the mutations was confirmed when peritumoral DNA was available. The JGCTs without duplications carried point mutations affecting highly conserved residues. Several of these substitutions were somatic lesions. The mutated proteins carrying the duplications had a non-wild-type subcellular distribution, with a marked enrichment at the plasma membrane. This led to a striking degree of AKT1 activation demonstrated by a strong phosphorylation level and by reporter assays.AKT1 as a major event in the pathogenesis of JGCTs. The existence of AKT inhibitors currently tested in clinical trials opens new perspectives for targeted therapies for these tumors, which are currently treated with standard non-specific chemotherapy protocols.Our study incriminates somatic mutations of \u2022We detected duplications affecting the pleckstrin-homology domain of AKT1 in >\u00a060% of the Juvenile Granulosa cell tumors studied.\u2022The JGCTs without duplications carried point mutations affecting highly conserved residues.\u2022Mutated proteins with duplications displayed an enrichment at the plasma membrane and a striking phosphorylation level. Our study points to AKT1 as a major driver in the pathogenesis of JGCTs.Sex cord stromal tumors involve granulosa, theca or stromal cells, alone or combined. The most common ones are ovarian granulosa cell tumors (GCTs), which represent up to 5\u20138% of all ovarian tumors . Two disof AGCTs . This muof AGCTs . With reof AGCTs . In anote latter . FSHR sie latter that phoe latter . Becausee latter , we hypo22.1This study involves a cohort of 16 histologically proven JGCTs, occurring in girls under 15\u00a0years of age, collected between 1994 and 2014 . The available clinical data are displayed in 2.2Twelve samples were formalin-fixed paraffin-embedded (FFPE) (T1\u2013T12). Four tumors were obtained as frozen samples (T13\u2013T16). We isolated genomic DNA and RNA from FFPE tumors using the AllPrep DNA/RNA FFPE Kit (Qiagen) and the frozen ones were processed using standard procedures. To assess the somatic status of the mutations, we extracted DNA from manually isolated peritumoral tissue fragments (only available for 8 samples). We also analyzed the COV434 cell line, supposed to derive from a JGCT .G\u03b1s gene, potentially harboring the activating mutations R201C and R201H, was performed as described , supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin .2.4AKT1RED\u2013EcoR1-F: 5\u2032AGCTTCGAATTCGCCACCATGAGCGACGTGGCTATTGTGAAGG3\u2032AKT1-F2: GTGGACCACTGTCATCG3\u2032AKT1RED\u2013BamH1-R: 5\u2032ACCGGTGGATCCCG GGCCGTGCCGCTGGCCGAGTAGGAGAAC3\u2032InsertionT3R: 5\u2032CGATGACAGTGGTCCACTGCAGGCAGCGGATGATGAAGGTGTTGGGCCGGGGCCAGCGGATGATGAAGG3\u2032InsertionT8R: 5\u2032CGATGACAGTGGTCCACTGCAGGCAGCGGATGATGAAGGTGTTGGGCCGGGGCCGCAGGCAGCGGATGATGAAGG3\u2032InsertionT5R: 5\u2032CGATGACAGTGGTCCACTGCAGGCAGCGGATGATGAAGGTGTTGGGCCGGGGCCGCTCGCAGCGGATGATGAAGGTGTTGG3\u2032InsertT11R: 5\u2032CGATGACAGTGGTCCACTGCAGGCAGCGGATGATGAAGGTGTTGGGCCGGGGCCGCTCCAGGCAGCGGATGATGAAGG3\u2032InsertT12R: 5\u2032CGATGACAGTGGTCCACTGCAGGCAGCGGATGATGAAGGTGTTGGGCCGGGGCCGCTCCGTCTTCATCAGCTGGCGGATGATGAAGGTGTTGGG3\u2032InsertT14R: 5\u2032CGATGACAGTGGTCCACTGCAGGCAGCGGATGATGAAGGTGTTGGGCCGGGGCTGCAGGCAGCGGATGATGAAGG3\u2032InsertT15R: 5\u2032CGATGACAGTGGTCCACTGCAGGCAGCGGATGATGAAGGTGTTGGGCCGGATGATGAAGGTGTTGG3\u2032InsertT16R: 5\u2032CGATGACAGTGGTCCACTGCAGGCAGCGGATGATGAAGGTGTTGGGCCGGGGCCGCTGGCAGCGGATGATGAAGG3\u2032E17K F: 5\u2032GCCACCATGAGCGACGTGGCTATTGTGAAGGAGGGTTGGCTGCACAAACGAGGGAAGTACATCAAGACCTGG3\u2032Q79K-F: 5\u2032CATCATCCGCTGCCTGAAGTGGACCACTGTCATCG3\u2032Q79K-R: 5\u2032CGATGACAGTGGTCCACTTCAGGCAGCGGATGATG3\u2032W80R-F: 5\u2032CATCATCCGCTGCCTGCAGAGGACCACTGTCATCG3\u2032W80R-R: 5\u2032CGATGACAGTGGTCCTCTGCAGGCAGCGGATGATG3\u2032Q79K\u2013W80R-F: 5\u2032CATCATCCGCTGCCTGAAGAGGACCACTGTCATCG3\u2032Q79K\u2013W80R-R: 5\u2032CGATGACAGTGGTCCTCTTCAGGCAGCGGATGATG3\u2032.The plasmids driving the expression of wild-type and mutated AKT1 fused to the mCherry protein were constructed by fusion PCR. Briefly, for the insertion mutations, two PCRs were performed to generate the 5\u2032 and 3\u2032 portions of the AKT1 coding sequence using, respectively, AKT1RED\u2013EcoR1-F primer and the corresponding mutagenic R primer and AKT1-F2 primer along with AKT1RED\u2013BamH1-R. After purification of the PCR products, they were quantified, mixed in similar amounts and allowed to undergo eight cycles of PCR in the absence of primers, to generate the full-length mutated coding regions. Then, a final PCR reaction was performed using the EcoR1\u2013BamHI primers. For E17K, we used the primers E17K F and AKT1RED\u2013BamH1-R to generate the amplicon in a single PCR. The amplified EcoR1\u2013BamHIs were cloned (EcoR1\u2013BamHI) into digested pDsRed vector to produce fusion proteins in frame with the mCherry. All constructs were sequenced to exclude the presence of PCR-induced mutations. The sequences of the primers used are the following:2.5HeLa cells were transfected with constructs driving the expression of AKT1 fused to the mCherry protein. Cells were seeded, in 24-well plates containing sterile coverslips, 16\u00a0h before transfection to be confluent at the time of transfection. Cells were transfected using the calcium-phosphate method with 250\u00a0ng of plasmid per well and rinsed 24\u00a0h after transfection. At this point, cells were serum-starved or not for 24\u00a0h. Forty-eight hours after transfection, cells were washed with phosphate-buffered saline solution (PBS) and fixed for 15\u00a0min with paraformaldehyde (PFA 4%). They were washed three times in PBS and nuclei were stained with Hoechst 33342 . Coverslips were mounted on microscope slides using the fluorescence mounting medium DakoCytomaton . Cells were visualized with an epifluorescence microscope provided with an ApoTome module.For Western-blot studies, HeLa cells were transfected with the constructs driving the expression of wild-type or mutated AKT1 forms. One day after transfection cells were rinsed and serum-starved or not for 24\u00a0h before lysis. Electrophoresis and Western-blot were performed as previously described . The priDual-Luciferase Reporter Assays involved the reporter promoter 4XDBE-luc which contains 4 copies of the FOXO response element (DAF-16 family member-binding element or DBE) upstream of a minimal promoter driving the expression of the firefly luciferase gene . Each ex33.1G\u03b1s altering protein position 201 samples, the allelic combinations (i.e. haplotypes) of the other co-occurring mutations could not be worked out, thus preventing their experimental exploration. In total, 14 out of 16 JGCTs harbor putative driver alterations of AKT1.Given the high degree of identity between and AKT2 . These m3.21H10, 3O96 and 4EJN3O964EJN). The tandem duplications described here involve the 6th beta strand, according to the ribbon model displayed in The tandem duplications described here alter the PHD of AKT1 . The PHDAKT1 in our experimental setting. The pervasive presence at the plasmalemma of the elongated AKT1 proteins suggested the existence of a high level of activation. To test this hypothesis, we analyzed the phosphorylation status of seven AKT1 variants carrying the in-frame duplications, which is supposed to reflect protein activation or by the presence of a myristoylation signal, artificially introduced or provided by the viral Gag sequence fused to Akt in v-Akt . StructuAlthough JGCTs are of good prognosis, it would be interesting to study the properties of the filopodia-like processes induced by the mutated AKT1 carrying the duplications because of the known roles of filopodia/invadopodia in sensing, migration and intercellular interactions . These pAKT1 mutations and the involvement of the PI3K\u2013AKT pathway in regulating cellular proliferation and survival suggests that the molecular lesions reported here are driver events. Irrespective of the precise role of AKT1 in the origin and/or progression of the JGCTs, which is yet to be studied, the uncovering of the AKT1 insertions will facilitate the molecular diagnostics of JGCTs. Our findings open also targeted therapeutic perspectives because inhibitors of the PI3K\u2013AKT\u2013mTOR pathway(s) are being tested in clinical trials partners. Finally, we cannot exclude that granulosa cells may need a strong level of AKT1 activation to become transformed, which would only be achieved with the observed duplications or with several AKT1 mutations per cell, such as Q79K and W80R. The fact that most of the JGCTs harbor l trials .GEFLUC (Groupement des Entreprises Fran\u00e7aises dans la Lutte contre le Cancer), the Centre National de la Recherche Scientifique (CNRS), the Universit\u00e9 Paris Diderot and the Agence Nationale pour la Recherche (Projet ICEBERG).This work was supported by LB, ALT, AA and RAV conceived and designed the study. LB, ALT, AA, LG, BL, DF and RAV developed the methodology. LB, ALT, AA, LG, BL and RAV acquired data. LB, ALT, AA, SS, DF, CS, NK, LG and RAV analyzed and/or interpreted data. All authors wrote and provided final approval of the manuscript.The authors declare no competing interests.Tumoroth\u00e8que Necker-Enfants Malades and Tumoroth\u00e8que Montpellier.This project was approved by the ethics committees of the tumor repositories The funding sources had no role in study design, data collection, data analysis, data interpretation, or writing of the manuscript. The corresponding author had full access to all data and had the final responsibility for the decision to submit for publication."} +{"text": "AbstractHeteranassa Smith , native to the southwestern United States and Mexico, includes two recognized species, namely Heteranassamima (Harvey) and Heteranassafraterna Smith. These are separated mainly by subtle differences in wing color and pattern, leading to speculation about the validity of the described species. This study examines variation in external and internal morphology across the geographic range of the genus, aiming to clarify species limits, describe morphology, and provide a comprehensive assessment of variation within the genus. Results indicate that Heteranassafraternasyn. n., is a junior synonym of Heteranassamima. Heteranassa Smith, 1899, is a genus of moths native to warmer desert regions of southwestern United States southward to southern Mexico, currently containing two valid species. Heteranassamima , described from Texas, and Heteranassafraterna Smith, 1899, described from Death Valley, California. PageBreakan additional species, Heteranassaminor Smith, 1899, with Heteranassafraterna. Heteranassa feed on mesquite (Prosopis sp.) and Acacia (Acacia sp.) (both Fabaceae) and are multivoltine , White Sands National Monument, Otero Co., New Mexico, , Cuatrocienagas Protected Area, Cuatroci\u00e9nagas, Coahuila, Mexico, , Pima Co., Arizona, (July 2012), and Socorro Co., New Mexico (October 2012). Specimens were collected with a sheet trap using 15W UV fluorescent lamp, 175W Mercury Vapor lamp, or a 175W, 6500K metal halide lamp. Death Valley specimens were collected at incandescent or fluorescent outdoor lighting at the Furnace Creek Ranch Hotel.Specimen loans were generously provided by the following institutions: University of Alberta Strickland Entomology Museum, Edmonton, Alberta (F.A.H. Sperling)UASM Essig Entomology Museum, University of California, Berkeley (J. Powell)EMEC Natural History Museum of Los Angeles County, Los Angeles, California (B. Brown)LACM University of Arizona Insect Collection, Tucson (W. Moore)UAIC Arizona State University Entomology Collection, Tempe (T. Dowling)ASUT Cornell University Insect Collection, Ithaca, New York (J. Liebherr)CUIC Kansas State Entomological Museum, Manhattan (G. Zolnerowich)KSUCUnited States National Museum and the Lepidoptera and Biodiversity McGuire Center for . A complete list of specimens examined is included in Suppl. material Heteranassa. A list of dissected specimens is included in Suppl. material Specimens were also examined during visits to the PageBreakwatch glass with distilled water, and scales were removed with a fine brush. Once clear of scales, the integument of the abdomen was cut along the left pleural membrane, and the genital capsule removed. On male specimens setae were carefully removed from the membranous costal region of valves with a fine camel\u2019s hair brush. The aedeagus was separated from the valves by grasping the distal end with fine\u2013tipped forceps, and gently pulling to separate from the juxta. The ductus seminalis was then cut where it enters the side of the proximal part of the aedeagus. The vesica was then carefully teased out of the aedeagus with a #20 minuten with the tip bent to a right angle, held in a standard pin vice, and with water pressure from a syringe. A syringe with a modified 30 gauge needle . Line drawings were made in Adobe Illustrator with a Wacom Intuos 4 drawing tablet from photographs or sketches made with a drawing tube attached to a Wild M5 stereomicroscope.Photographs of dissected specimens, genitalia, and adults were made using a Visionary Digital imaging system (Prosopisglandulosa Torr.) foliage and bark. Larvae were reared on Prosopisglandulosa foliage.Eggs were obtained from gravid females collected in Box Canyon, Pima Co., AZ (18 July 2012). The females were placed in brown paper bags with Honey Mesquite , Toxonprucha species and Coxina species are frequently misidentified as Heteranassa. Of these, Acritogrammametaleuca is the most similar to Heteranassa Homopteramima Harvey, 1876: 155\u2013156.Eubolinamima ; Campometramima ; Elousamima ; Draudt and Gaede (in Seitz) 1923: 478.Heteranassamima ; Campometrafraternasyn. n.; Smith, 1899: 104, Heteranassafraterna ; Elousafraterna ; Draudt and Gaede (in Seitz) 1923: 478.Campometraminor Smith, 1899: 104\u2013105; Elousaminor : Draudt & Gaede (in Seitz) 1923: 478.Heteranassaminorsyn. n.; , PageBreakThis is the only species in the genus and can be diagnosed with the generic combination (see above).Heteranassamima . Holotype, labeled: \u201cHomoptera mima, type, Harvey, Holotype, 15/9, 73.\u201d The specimen and associated labels were examined through high-resolution photographs provided by the BMNH. Type locality: Texas [USA]pe, Fig. \u2640 in theHeteranassafraterna . Lectotype . Lectotype, barcodes from California, Arizona, New Mexico, Texas, and northern Mexico showed a maximum divergence of less than 0.8%. One haplotype* dominated the sample, representing more than half of the specimens; the other barcodes included 36 haplotypes that had no more than two base-pair differences from each other. One haplotype, restricted to central and southern Texas, departed from this pattern in being 0.8% different from those from farther west. This is most probably the haplotype that should be associated with the name Heteranassamima, it being described from this part of Texas. However, this \u201ceastern\u201d haplotype is found with \u201cwestern\u201d haplotypes in central Texas and there is no indication in genital structural characters, or wing color or pattern, that Heteranassa includes more than a single species. The barcodes of Heteranassa are so divergent that they give no indication of a close relationship to any other erebid genus, other than belonging in the subfamily Erebinae, tribe Omopterini. Heteranassa specimens from Texas and Mexico are frequently confused with some species associated with the genus Coxina Guen\u00e9e, which can have a similar superficial pattern, but the barcodes are more than 10% different and the two genera do not appear to be closely related. (D. Lafontaine pers. comm.).Barcode variation in *CNCNoctuoidea13382 PageBreakTATTATAATTTTCTTTATAGTTATACCAATTATAATTGGAGGATTTGGAAATTGATTAGTCCCCTTAATATTAGGAGCTCCTGATATAGCTTTCCCTCGAATAAATAATATAAGTTTCTGATTATTACCCCCATCTTTAACTCTTTTAATCTCAAGAAGAATCGTAGAAAATGGAGCAGGAACAGGATGAACAGTTTACCCCCCACTTTCATCTAACATTGCTCATAGAGGAAGATCAGTAGATTTAGCAATTTTCTCTCTTCATTTAGCTGGAATTTCATCAATTTTAGGAGCTATTAATTTTATTACTACTATTATCAATATACGATTAAATAGATTAATATTTGACCAAATACCTTTATTTGTTTGAGCTGTTGGTATTACTGCTTTTTTACTATTATTATCTTTACCTGTTTTAGCTGGAGCTATTACTATACTCTTAACAGATCGAAATTTAAATACTTCCTTTTTTGATCCTGCTGGAGGAGGAGATCCTATTCTTTACCAACATCTATTTAACTTTATATTTTATTTTTGGAATTTGAGCAGGAATAGTAGGAACCTCTTTAAGTTTATTAATTCGTGCTGAATTAGGAAACCCTGGTTCTTTAATTGGAGATGATCAAATTTATAATACTATTGTTACAGCTCATGCTTTLACM.Warm, arid habitats from California to Texas, northward to Oklahoma, and south as far as Oaxaca, Mexico Fig. . A singlHeteranassa wing pattern and coloration is continuous, with many specimens appearing intermediate to the phenotypes described by Heteranassa contains a single, highly variable species, Heteranassamima. Studies of another erebine genus, Catocala, have shown that pressure from avian predators may drive high levels of polymorphism in forewing pattern and coloration than they are in Elousaschausi and the other Coxina species we have dissected. We have examined 10 species in these genera from the Caribbean and South America. Specimens belonging to this group that we collected in the Nicaraguan highlands appear more similar to Elousaschausi specimens from Argentina and Coxina specimens from Mexico, south Texas, and Florida, than they do to Caribbean Elousa or North American Heteranassa. Future research could test the monophyly of Coxina and Elousa with respect to Heteranassa, and how these genera speciated in North and South America and the Caribbean.During the course of this research, we became aware of potential taxonomic affinities with the neotropical genera PageBreak"} +{"text": "Staphylococcus aureus and vancomycin-resistant Enterococcus, there is not much information describing its activity against gram-negative bacteria. In this study, we report the effect of methylglyoxal against multidrug-resistant Pseudomonas aeruginosa (MDRP) using 53 clinically isolated strains. We also assessed the effect of deleting the five multidrug efflux systems in P. aeruginosa, as well as the efflux systems in Escherichia coli and Salmonella enterica serovar Typhimurium, on MICs of methylglyoxal. Our results indicate that methylglyoxal inhibits the growth of MDRP at concentrations of 128\u2013512 \u03bcg/ml (1.7\u20137.1 mM) and is not recognized by drug efflux systems.Honey has a complex chemistry, and its broad-spectrum antimicrobial activity varies with floral source, climate, and harvesting conditions. Methylglyoxal was identified as the dominant antibacterial component of manuka honey. Although it has been known that methylglyoxal has antibacterial activity against gram-positive bacteria, including methicillin-resistant Pseudomonas aeruginosa is endemic among critically ill patients, and multidrug-resistant strains are increasingly being isolated in intensive care units has been defined as P. aeruginosa resistant to imipenem, amikacin, and ciprofloxacin foraging on a shrub known as manuka (Leptospermum scoparium) that is indigenous to New Zealand. Manuka honey is broad in spectrum, able to inhibit a diverse range of bacterial and yeast pathogens, and equally effective against multidrug-resistant bacteria from wounds reported to be <3% (v/v) and NKS233 (\u0394tolC) were constructed as described previously and NKE95 (\u0394tolC), we performed gene disruption using procedures described previously ; acrB-P2 (GAACAGTCCAAGTCTTAACTTAAACAGGAGCCGTTAAGACCATATGAATATCCTCCTTAG); acrD-P1 (TGAAAAAGGCGACACATTGGCATGTCGCCTTTTTTATTGCGTGTAGGCTGGAGCTGCTTC); acrD-P2 (AAGCCTACAACGATACGCAGAAACACGAGGTCCTCTTTTACATATGAATATCCTCCTTAG); mdtA-P1 (ATCATTCCGCGAAACGTTTCAGGAAGAGAAACTCTTAACGGTGTAGGCTGGAGCTGCTTC); mdtC-P2 (GAGATACACCACCGGCGTGGTATACAGCGTAAGGAGCTGGCATATGAATATCCTCCTTAG); mdtE-P1 (TTAAAGAACCGTTATTTCTCAAGAATTTTCAGGGACTAAAGTGTAGGCTGGAGCTGCTTC); mdtF-P2 (AGGCTGAACCTTCATGTTCAACCTTACTCTCATTTACACGCATATGAATATCCTCCTTAG); acrE-P1 (TTGGGTAAATAACGCGCTTTTGGTTTTTTGAGGAATAGTAGTGTAGGCTGGAGCTGCTTC); acrF-P2 (AAATAATAAAGGCACCCGAAAGCGCCTTTATGTTTCTGATCATATGAATATCCTCCTTAG); tolC-P1 (ACTGGTGCCGGGCTATCAGGCGCATAACCATCAGCAATAGGTGTAGGCTGGAGCTGCTTC); and tolC-P2 (TTACAGTTTGATCGCGCTAAATACTGCTTCACCACAAGGACATATGAATATCCTCCTTAG). The chloramphenicol resistance gene cat or the kanamycin resistance gene aph, flanked by Flp recognition sites, was amplified by PCR using the primers listed above. The resulting PCR products were used to transform E. coli MG1655, which harbors plasmid pKD46, expressing Red recombinase. The chromosomal structures of the mutated loci were verified by PCR; cat and aph were further eliminated using the plasmid pCP20, as described previously . The inoculated agar plates were examined after incubation at 37\u00b0C for 16 h. MIC was the lowest concentration of a compound that inhibited cell growth.Antibacterial activities were determined on Muller Hinton II agar plates containing methylglyoxal (32\u20132048 \u03bcg/ml), imipenem (0.0625\u20132048 \u03bcg/ml), amikacin (0.125\u20134096 \u03bcg/ml), or ciprofloxacin (0.0078\u20132048 \u03bcg/ml) . Agar plates were prepared using the two-fold agar dilution technique. Bacteria were grown at 37\u00b0C overnight and then tested at a final inoculum volume of 1 \u00d7 10E. coli and S. enterica strains were grown in Luria\u2013Bertani broth , and P. aeruginosa (PAO1 and PMX52) strains were grown in Muller Hinton II (MHII) broth . Bacterial cells were cultured overnight at 37\u00b0C, and then 100 \u03bcl of cell cultures were diluted in 5 ml of the same medium. The diluted bacterial cells were incubated at 37\u00b0C until OD600 reached 0.5. Then, the bacterial cells were diluted in the same medium to an OD600 of 0.05. This diluted bacterial cells were incubated in NUNC Edge 96-well plates with shaking at 37\u00b0C for 7 h. Bacterial growth was monitored using an Infinite M200 Pro plate reader .P. aeruginosa resistant to imipenem , amikacin (\u226532 \u03bcg/ml), and ciprofloxacin (\u22654 \u03bcg/ml) . Although PMX52 was more susceptible to amikacin and ciprofloxacin than PAO1, MIC of methylglyoxal for these strains was the same. This suggests that methylglyoxal is not recognized by drug efflux systems in P. aeruginosa. To confirm whether same phenomenon could be observed in other gram-negative bacteria, we determined MICs of methylglyoxal for the efflux-deficient mutants of E. coli and S. enterica serovar Typhimurium. There are five RND-type drug efflux systems in E. coli, and all of them require the TolC outer membrane channel for their function , NKE1329 (\u0394acrB acrD mdtABC mdtEF acrEF), and NKE95 (\u0394tolC) strains. The susceptibility of NKE1329 and NKE95 to methylglyoxal was same as that of the wild-type strain, although they were more susceptible to ciprofloxacin than the wild-type strain. S. enterica serovar Typhimurium harbors at least nine drug efflux systems belonging to RND, multidrug and toxic compound extrusion, and ATP-binding cassette (ABC) superfamilies , NKS196 (\u0394acrAB acrEF acrD mdtABC mdsABC emrAB mdfA mdtK macAB), and NKS233 (\u0394tolC) strains. Although NKS196 and NKS233 were more sensitive to ciprofloxacin than the wild-type strain ATCC14028s, MICs of methylglyoxal for ATCC14028s, NKS196, and NKS233 were the same. In addition to MIC determination using agar plates, we tested the effect of methylglyoxal on bacterial growth in liquid medium. The growth of E. coli and Salmonella strains was inhibited by methylglyoxal at a concentration of 256 \u03bcg/ml, and the growth of P. aeruginosa (PAO1 and PMX52) strains was inhibited at 512 \u03bcg/ml, which is consistent with MICs determined . Methylglyoxal is a key antimicrobial component of manuka honey, and manuka honey has previously been suggested as a topical treatment option for burn patients infected with P. aeruginosa (Mavric et al., P. aeruginosa, E. coli, and S. enterica.In this study, we showed that methylglyoxal equally inhibits drug-susceptible The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "The correct sentence is: Then an exchange of ADP with ATP in ATP domains of MutS takes place shown in the \u201cSynthetic DNA Fragments\u201d subsection of the Materials and Methods. Please see the correct structures here.GGGTGCC I (G/C)CAAGCCTATGCCCTCAGCACCCACCCACGGGTTCGGATACGGGAGTCGTGGGTGGGTGCC II (G/T)CAAGCCTATGCCCTCAGCACCCATCCACGGGTTCGGATACGGGAGTCGTGGGTG-GGTGCC III (G/Tg)CAAGCCTATGCCCTCAGCACCCATgCCACGGGTTCGGATACGGGAGTCGTGGGTA-GGTGCC IV (A/Tg)CAAGCCTATGCCCTCAGCACCCATgCCACGGGTTCGGATACGGGAGTCGTGGGT"} +{"text": "Correction: Genome Biol 19, 205 (2018)https://doi.org/10.1186/s13059-018-1581-3Following the publication of the original paper , the autATCTTGTGGAAAGGACGAAACAThe sequence for Primer 1: SAMlibrary-HiSeq_50bp-F1 has two extra bases inserted (in bold) ACACTCTTTCCCTACACGACGCTCTTCCGATCTThe correct sequence should be:ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTTGTGGAAAGGACGAAACA"} +{"text": "Retinopathies are multifactorial diseases with complex pathologies that eventually lead to vision loss. Animal models facilitate the understanding of the pathophysiology and identification of novel treatment options. However, each animal model reflects only specific disease aspects and understanding of the specific molecular changes in most disease models is limited. Here, we conducted transcriptome analysis of murine ocular tissue transduced with recombinant Adeno-associated viruses (AAVs) expressing either human VEGF-A, TNF-\u03b1, or IL-6. VEGF expression led to a distinct regulation of extracellular matrix (ECM)-associated genes. In contrast, both TNF-\u03b1 and IL-6 led to more comparable gene expression changes in interleukin signaling, and the complement cascade, with TNF-\u03b1-induced changes being more pronounced. Furthermore, integration of single cell RNA-Sequencing data suggested an increase of endothelial cell-specific marker genes by VEGF, while TNF-\u03b1 expression increased the expression T-cell markers. Both TNF-\u03b1 and IL-6 expression led to an increase in macrophage markers. Finally, transcriptomic changes in AAV-VEGF treated mice largely overlapped with gene expression changes observed in the oxygen-induced retinopathy model, especially regarding ECM components and endothelial cell-specific gene expression. Altogether, our study represents a valuable investigation of gene expression changes induced by VEGF, TNF-\u03b1, and IL-6 and will aid researchers in selecting appropriate animal models for retinopathies based on their agreement with the human pathophysiology. Accordingly, anti-VEGF treatment has emerged as standard-of-care for wet AMD5 and DME6. Another common hallmark of many retinopathies is inflammation: proinflammatory cytokines such as TNF-\u03b1 as well as IL-6 proteins are upregulated in the vitreous of DR8 and uveitis patients10.Retinopathies such as diabetic retinopathy (DR), age-related macular degeneration (AMD), uveitis or retinopathy of prematurity (ROP) display complex pathologies in patients including vasculopathies, inflammation, neurodegeneration, and fibrosis, ultimately leading to blindness. A number of molecular changes have been associated with these retinal pathologies, for example changes in expression of Vascular Endothelial Growth Factor (VEGF), Tumor Necrosis Factor alpha (TNF-\u03b1), or Interleukin-6 (IL-6). VEGF causes vascular leakage and pathological neovascularization, and VEGF protein has been shown to be upregulated in wet AMD12. Transgenic mice expressing VEGF15, intraocular injection of recombinant VEGF protein18 or recombinant adeno-associated viruses (AAVs) expressing VEGF23 further demonstrated that VEGF is not only necessary but also sufficient to cause vasculopathies. Accordingly, anti-VEGF treatment prevents neovascularization in the OIR model24 and these preclinical studies paved the way for modern anti-VEGF therapies. Furthermore, inflammatory processes observed in patients of retinopathies can also be modeled in rodents, for example by uveitis mouse models such as endotoxin- or antigen-induced uveitis27 or transgenic mice lacking the Aire gene29. Similarly, recombinant proteins or AAV-mediated expression of TNF-\u03b1 and IL-6 induces inflammation in the rodent eye, although the direct function of IL-6 is more controverse32. Again, anti-TNF-\u03b1 or anti-IL-6 treatment improves the induced pathologies in diverse uveitis models35.Various pre-clinical rodent models have directly or indirectly shed light on the function of VEGF, TNF-\u03b1, or IL-6 in retinopathies. One frequently used animal model displaying vascular pathologies, similar to those observed in proliferative retinopathies such as wet AMD or ROP, is the oxygen induced retinopathy (OIR) model19. We further demonstrated that AAV-mediated expression of human VEGF, TNF-\u03b1, and IL-6 in the murine eye leads to pathway-specific, human-relevant retinal pathologies. In brief, AAV-driven expression of VEGF induced vascular leakage and neovascularization. On the other hand, pro-inflammatory cytokines TNF-\u03b1 and IL-6 both activated immune cells. TNF-\u03b1 additionally led to vasculitis, fibrosis, and development of fibrotic epiretinal membranes.In the context of pre-clinical research, AAV mediated gene transfer has recently emerged as a powerful method to create novel animal models. As a main benefit, AAVs allow for long-term expression of a transgene in a tissue- and cell-type-specific manner. Previously, we have shown that AAV-mediated expression leads to constant and long-term expression of human transgenes at 1, 3 and 6\u00a0weeks after IVT-injection38. Similarly, the transcriptome of multiple rodent models of retinopathies has been sequenced and analyzed42. For the OIR model, several studies have identified time point-dependent gene expression changes related to hypoxia, angiogenesis, and inflammation47. However, comparisons with other mouse models of retinopathies are still missing. Additionally, modern single cell RNA-sequencing (scRNA-Seq) approaches gave unprecedented insights into the cellular organization of healthy and diseased mammalian ocular tissues54. In more detail, Heng et al. demonstrated by single cell sequencing of Aire-/- mice, a spontaneous uveoretinitis model, that a very diverse population of immune cells invades the retina of Aire-/- mice and that Th1 cells represent the main effector T cells in this model, highlighting the great potential of such single cell sequencing approaches. As the number of available bulk and single-cell datasets in the context of retinopathies increases, so does the potential to integrate data from various retinopathy models and compare them on a molecular level.In the recent years, next generation sequencing techniques allowed for a detailed investigation of the molecular changes in retinopathies and brought about a better understanding of disease progression in AMD and DR patientsIn this study, we provide transcriptomic analysis of intravitreally injected recombinant AAVs expressing human VEGF, TNF-\u03b1, or IL-6 in mice. We observed that AAV-mediated expression of human transgenes introduced distinct transcriptomic responses: While TNF-\u03b1 and IL6 displayed overlapping gene expression changes, VEGF overexpression led to a more distinct response compared to TNF-\u03b1 and IL-6. In more detail, investigating pathways affected by each experimental condition, VEGF led to a specific regulation of extracellular matrix (ECM)-related genes, while TNF-\u03b1 and IL-6-induced changes in interleukin signaling. We further identified specific gene expression changes associated with TNF-\u03b1 that included cellular adhesion molecules, such as Madcam1 and we further demonstrated a conserved regulation of MAdCAM-1 by TNF-\u03b1 in human retinal endothelial cells. By integration of single cell RNA-Sequencing data, we could show an increase of T-cell-specific genes following TNF-\u03b1 expression and, indeed, TNF-\u03b1 mediated T-cell invasion was validated by immunofluorescence. In addition to assessing transcriptome changes in the various transgenic mice models, we generated a detailed time-course of transcriptome changes in the established OIR model. Comparing transcriptome changes in AAV overexpression models and OIR mice, we observed the largest overlap between AAV-VEGF treated eyes and the late response (P16) in the OIR model, most prominently including changes in the ECM pathway and endothelial cell specific marker genes. Prominently, OIR-specific changes in gene regulation included neuronal signaling pathways which were not observed after AAV-VEGF transgene expression. In conclusion, our study suggests that each animal model produces distinct gene expression profiles and a careful selection of models according to the requirements of different research questions is necessary.8\u00a0VG/eye), while AAV-TNF-\u03b1 and AAV-IL-6 were injected at 1\u2009\u00d7\u2009109\u00a0VG/eye. Eyes of all animals were imaged in vivo directly before tissue dissection and RNA sequencing to validate expected pathologies and grade phenotype severity. In line with our previously published study19, AAV-stuffer injections did not induce obvious retinal pathologies at both concentrations based on Blue Autofluorescence (BAF) imaging . Since no mRNA staining was observed in AAV-stuffer control treated eyes , choroid, sclera and the ciliary body. Apart from two samples that were excluded due to a low RNA integrity number (RIN), six biological replicates per group were sequenced. Sequencing libraries were sequenced at an average depth of 31.3 million reads, with 80.1% mapping to mRNA transcripts and low ribosomal content (2.1%), suggesting overall good quality of the RNA sequencing data. As expected, human transgenes showed strong expression in their respective treatment groups indicated the largest variance in gene expression between retina and eye cup samples Fig.\u00a0d. The reVcam1 and Madcam1 and chemokines such as Ccl2 were among the strongest deregulated genes, supporting the well-known function of TNF-\u03b1 in regulating immune cell adhesion to endothelial cells56. To confirm the RNA sequencing results, Ccl2 expression was validated by qRT-PCR, which demonstrated similar expression patterns with the highest expression of Ccl2 found in AAV-TNF-\u03b1 treated eyes genes in the three treatment groups AAV-VEGF, TNF-\u03b1, and AAV-IL-6 in comparison to their respective AAV-stuffer control, as well as AAV-stuffer compared to non-injected controls by AAV-VEGF, AAV-TNF-\u03b1 or AAV-IL-6 treatment. Only 1 pathway was enriched in the retina of all three treatment groups Fig.\u00a0a, namelySince only very few genes were significantly differentially regulated in AAV-VEGF transduced eye cup tissue, only AAV-TNF-\u03b1 and AAV-IL-6 treatment was compared in the eye cup tissue of endothelial and perivascular cell-specific marker genes after AAV-VEGF treatment was observed were mostly positive , P13, P14, P15 and P16. PCA demonstrated a clear separation between the OIR model and controls model, presenting both neovascularization and neurodegeneration. Neonatal mice are exposed to 75% oxygen from postnatal day 7 to 12, and thereafter return to room air (21% oxygen) until day 17. At day 7, the retinal vasculature of mouse pups is still immature and vulnerable to high oxygen conditions. Loss of capillaries in the center of the retina leads to the formation of an avascular area. Upon return to room air at day 12 the area becomes hypoxic and Hif1\u03b1 dependent VEGF expression has been shown to trigger angiogenesis, peaking at P17ols Fig.\u00a0. At late54 demonstrated that, among others, also vascular cell-specific marker genes were enriched in the OIR data set in OIR retina compared to those identified in the AAV-VEGF model Fig.\u00a0c,d. The Finally, we aimed to understand which pathways were affected in the AAV-VEGF driven retinopathy models compared to the OIR model into mouse eyes with a biologically inactive sequence, produced very few gene expression changes. AAV-mediated expression of hTNF-\u03b1, hIL-6, or hVEGF, led to high expression of these human transgenes, which in turn produced strong and distinct transcriptome changes. Although measurement errors in RNA-Seq caused by the simultaneous presence of human and murine versions of the AAV-expressed transgene are theoretically possible, we expect no large mis-quantification, since human transgenes are codon optimized and possess a V5 tag, hence they are sufficiently dissimilar to their endogenous transcript sequence. In addition, we did not observe any reduction in the percentage of uniquely mapped reads in the presence of AAV expressed transgenes (data not shown).AAVs have emerged as a useful tool to quickly generate novel animal models that allow a high degree of flexibility regarding timing and tissue-specific expression. In our study, we chose the ShH10 capsid for expression of the three human transgenes. Even on a global scale, measured via a high-throughput method such as RNA-Seq, injection of the control capsid secondary to AMD72. In the past, transgenic animals modifying the expression of complement genes, or mice expressing human variants of complement-related genes, have proven extremely useful to understand their contribution to disease progression78. To further develop novel and effective therapies, animal models with robust activation of the complement pathway are needed. In LPS-induced uveitis model, complement-related genes including C3 are upregulated39, suggesting complement activation. Similarly, experimental autoimmune uveitis (EAU) leads to activation of the complement system and blockage of the complement system ameliorates disease pathology79. Activation of the complement system is also visible in the laser-induced choroidal neovascularization (CNV) model, and inhibition of various complement factors had beneficial effects82. However, both the experimental uveitis models and laser CNV model are transient and only allow for short-term investigations. In contrast, in the presented AAV-TNF-\u03b1 model, C3 showed strong and sustained upregulation at 3 and 6\u00a0weeks after IVT injection of AAV-TNF-\u03b1, thus, allowing long-term studies of the complement system. In addition, as a proof-of-mechanism we showed that treatment with the neutralizing TNF-\u03b1 antibody golimumab reduces C3 expression, demonstrating that complement activation is reversible in our model. In summary, the AAV-TNF-\u03b1 induced mouse model provides a valuable tool to study the role of the complement system in disease progression, featuring long-term TNF-\u03b1 and C3 expression, which is comparable to the human pathology that develop over decades.In our analysis, significantly changing genes present in the complement cascade were strongly and specifically upregulated in eyes injected with AAV-TNF-\u03b1. Among them, the complement pathway factor C3 was one of the strongest upregulated genes in AAV-TNF-\u03b1 injected eyes. C3 plays a central role in the activation of the classical and alternative complement pathway and mutations in C3 and other members of the complement cascade correlate with higher risk of AMDIcam1, Vcam1 and Madcam1 were highly upregulated in AAV-TNF-\u03b1 injected eyes, strengthening the previous observation of immune cell infiltration and vasculitis in AAV-TNF-\u03b1 injected eyes by histology19. MAdCAM-1 is induced by TNF-\u03b1 in diverse endothelial cells and recruits T-cells to inflamed tissues85 and, therefore, has recently been identified as an interesting target to treat inflammatory bowel diseases86. In addition to Madcam1 upregulation, we observed T-cell infiltration in our AAV-TNF-\u03b1 driven uveitis-like model, in line with well-described role for T-cells in uveitis patients and uveitis rodent models88. Although there is general interest in the role of TNF-\u03b1 and CAMs in the context of retinopathies, to our knowledge there exists only one recent study by Peng et al. demonstrating a direct function for murine Madcam1 in retinal degeneration83. In line with Peng et al., we demonstrated upregulation of Madcam1 in AAV-TNF-\u03b1 injected eyes correlating with an invasion of T-cells. Expanding on this observation, we demonstrated that MAdCAM-1 is also upregulated in TNF-\u03b1 stimulated HRMECs, suggesting that the TNF-\u03b1 induced regulation of MAdCAM-1 may also be relevant in human retinal cells.In addition to C3, cellular adhesion molecules (CAMs), such as 12. To capture transient expression dynamics present in the OIR model, we collected mouse eyes at 5 subsequent time points after hyperoxic treatment. Indeed, other authors have performed analysis of OIR gene expression, however, many studies relied on microarray data only assessing part of the transcriptome in contrast to the whole transcriptome analysis presented here. On the other hand, OIR RNA-Seq studies included less dense sampling of timepoints compared to our study, in which we provide more detailed insights into time-dependent gene expression changes. The study of Yang et al.46 focused on gene expression changes during and shortly after hyperoxic treatment, while our study was mostly interested in gene expression changes after the return to normoxic conditions and a detailed comparison to AAV induced gene expression changes. Nevertheless, we observed shared gene expression signatures of key genes between the two studies, for example VEGF and Edn2 gene were both upregulated at P13.In order to better understand the differences between various retinopathy disease models, we compared gene expression profiles of the generated AAV-driven retinopathy models to RNASeq data of the frequently used OIR model24. Thus, as expected, DE genes in the AAV-VEGF model showed the largest overlap with those identified in the OIR model. By integration of single-cell RNA-Seq expression data, we showed that within the time-course measured for the OIR model, endothelial cell-specific genes were first down-regulated and then up-regulated starting from P15. Also other studies have found downregulation of angiogenesis-related genes such as CD34 in the OIR model at P12, while endothelial cell-specific genes such as Esm1 or Edn2 were upregulated at P1789. Esm1 is a well-known gene induced by VEGF in tip cells with a crucial role in angiogenesis and its blockage inhibits neovascularization of diverse rodent models of neovascularization including the OIR, laser CNV and the rho/VEGF transgenic mouse91. Accordingly, we also observed upregulation of the Esm1 gene in both the OIR dataset but also the AAV-VEGF driven model, resembling very well the observed neovascularization phenotype. Since we provide a high time-resolution of gene expression changes in the OIR model, we were able to pinpoint the switch between down- and up-regulation of endothelial cell-specific genes to P14. These gene expression changes fit very well with the phenotypic changes present in the OIR model, where at P12 the development of the vasculature is impaired and avascular areas are present, and later on, neovascularization peaks at around P1793.It is well known that VEGF is a major driver of the OIR model and anti-VEGF treatment prevents neovascularization in the OIR model95. Furthermore, neuronal death and decreased retinal function has been observed in the OIR model97, probably due to ischemia and malnutrition induced by hypoxia. Accordingly, the OIR model has been discussed as the more appropriate model for retinopathy of prematurity (ROP)93, a disease affecting human preterm infants. While very young mouse pups are needed for the OIR model, old or diabetic mice may be combined with AAV overexpression of human transgenes, to mirror more closely age-related or diabetes-associated human retinopathies such as AMD or DR in the future.Although many pathways including ECM-related pathways and endothelial cell-specific responses were similar between the OIR model and AAV-VEGF treated mice, we also observed differences between these disease models: For example, in the OIR model, but not AAV-VEGF, neuronal signaling pathways and synapse-related genes were significantly enriched for DE genes. In line with this observation, we observed a significant overlap of neuronal cell-specific genes and DE genes identified in the OIR model. For the OIR model pups at age P12 to P17 are used, a timepoint which corresponds to a time-point where vascular and neuronal networks are still developing in the murine retinaDespite our efforts to ensure optimal experimental design, certain limitations apply, most prominently that AAV and OIR sequencing data sets were generated in independent experiments. Furthermore, also from a biological perspective, the models differ, since the OIR model uses very young mouse pups during development, in contrast to our AAV-induced models that were developed using adult mice. While in the OIR study 3 or 4 samples where included at each time-point, the AAV study was performed with 5 or 6 replicates per group. In addition, library preparation and sequencing was dependent on different protocols. To alleviate these limitations, our analysis is based on relative fold changes between treatment and respective controls, rather than on absolute expression values, thereby accounting for differences in library preparation.Altogether, here we characterized the gene expression profiles of AAV-VEGF, TNF-\u03b1, or IL-6 driven retinopathy mouse models, and combined these measurements with publicly available singe-cell RNA expression data. Our study gives unique insights into the molecular and cell-specific changes leading to retinal pathologies, hereby uncovering novel potential treatment options for retinal diseases, and at the same time offering the means to test them in a stable disease model comparable to the human pathology. By further measuring gene expression changes in the well-established OIR model, we compared new and established animal models for retinopathies and provide researchers with important information guiding the decision of which animal model best suits the need of a given pre-clinical research project.C57BL/6\u00a0J mice were purchased from Charles River . 6\u20138-week-old male mice were used for the AAV study. Pregnant females were purchased for the OIR study and male and female pups at P12 were used for the OIR experiments. Mice were housed in individually ventilated cages in groups of 2\u20135, constant temperature and a 12-h light/dark cycle. Mice had ad libitum access to standard rodent chow and water. Animal experiments were performed in accordance with the German animal welfare act, the guidelines of the Federation of the European Laboratory Animal Science Association (FELASA), the ARRIVE guidelines and were reviewed and approved by the governmental body responsible for animal welfare in the state of Baden W\u00fcrttemberg .19.The AAV-stuffer control construct includes a non-coding sequence from the 3\u2019 UTR of the UBE3A gene and has been described elsewhere (Strobel 2015). AAVs used in this study were packaged into the ShH10 capsid (Klimczak 2009). AAV production and quantification by quantitative PCR was done according to previously published protocols (Strobel 2019). The following human codon-optimized sequences were included in each of the recombinant AAVs:Plasmids with the ubiquitous CAG promoter were generated encoding for three human, codon optimized transgenes that have a V5-tag sequence at the 3\u2019 end of the gene were generated previouslyAAV-hIL6\u2014ATGAACAGCTTCAGCACCAGCGCCTTCGGACCTGTGGCTTTTTCTCTGGGACTGCTGCTGGTCCTGCCTGCCGCTTTTCCAGCTCCTGTTCCTCCTGGCGAGGACAGCAAAGATGTGGCCGCTCCTCATAGACAGCCTCTGACCAGCTCCGAGCGGATCGATAAGCAGATCCGGTACATCCTGGATGGCATCAGCGCCCTGCGGAAAGAGACATGCAACAAGAGCAACATGTGCGAGAGCAGCAAAGAGGCCCTGGCCGAGAACAACCTGAACCTGCCTAAGATGGCCGAAAAGGACGGCTGCTTCCAGAGCGGCTTCAACGAGGAAACCTGCCTGGTCAAGATCATCACCGGCCTGCTGGAATTCGAGGTGTACCTGGAATACCTGCAGAACAGATTCGAGTCCAGCGAAGAACAGGCCAGAGCCGTGCAGATGAGCACCAAGGTGCTGATCCAGTTCCTGCAGAAGAAGGCCAAGAACCTGGACGCCATCACCACACCTGATCCTACCACAAATGCCAGCCTGCTGACAAAGCTGCAGGCCCAGAATCAGTGGCTGCAGGACATGACAACCCACCTGATTCTGCGGAGCTTCAAAGAGTTTCTGCAGAGCAGCCTGCGGGCCCTGAGACAAATGGGAGGCGGAGGATCTGGCGGAGGCGGATCTGGAAAGCCCATTCCTAATCCTCTGCTGGGCCTCGACAGCACCTGATGATAA.AAV-hTNFa\u2014ATGAGCACCGAGAGCATGATCAGAGATGTGGAACTGGCCGAGGAAGCCCTGCCTAAGAAAACAGGCGGACCTCAGGGCAGCAGAAGATGCCTGTTTCTGAGCCTGTTCAGCTTCCTGATCGTGGCAGGCGCCACCACACTGTTCTGTCTGCTGCACTTTGGAGTGATCGGCCCTCAGAGAGAGGAATTCCCCAGAGATCTGTCCCTGATCTCTCCACTGGCTCAGGCTGTGCGGAGCAGCTCTAGAACACCTAGCGATAAGCCTGTGGCTCACGTGGTGGCCAATCCTCAGGCTGAAGGACAGCTGCAGTGGCTGAATAGAAGGGCCAACGCTCTGCTGGCCAACGGCGTGGAACTGAGAGATAATCAGCTGGTGGTGCCCAGCGAGGGCCTGTACCTGATCTATAGCCAGGTGCTGTTCAAAGGCCAGGGCTGCCCTTCTACACACGTGCTGCTGACCCACACCATCAGCAGAATCGCCGTGTCCTACCAGACCAAAGTGAACCTGCTGAGCGCCATCAAGAGCCCCTGTCAGAGAGAAACACCTGAGGGCGCCGAAGCCAAGCCTTGGTACGAACCTATCTATCTCGGCGGCGTGTTCCAGCTCGAGAAGGGCGATAGACTGAGCGCCGAGATCAACAGACCCGACTACCTGGATTTTGCCGAGAGCGGCCAGGTGTACTTCGGCATTATTGCTCTCGGAGGCGGAGGAAGTGGTGGCGGAGGATCTGGCAAGCCCATTCCTAATCCTCTGCTGGGCCTCGACTCCACCTGATGATAA.AAV-hVEGF\u2014ATGAACTTCCTGCTGAGCTGGGTGCACTGGTCACTGGCTCTGCTGCTGTATCTGCACCACGCCAAATGGTCACAGGCCGCTCCTATGGCTGAAGGCGGAGGACAGAATCACCACGAGGTGGTCAAGTTCATGGACGTGTACCAGCGGAGCTACTGTCACCCCATCGAGACACTGGTGGACATCTTCCAAGAGTACCCCGACGAGATCGAGTACATCTTCAAGCCTAGCTGCGTGCCCCTGATGAGATGCGGCGGCTGTTGTAACGATGAAGGCCTGGAATGCGTGCCCACCGAGGAATCCAACATCACCATGCAGATCATGCGGATCAAGCCCCACCAGGGCCAGCATATCGGCGAGATGTCTTTCCTGCAGCACAACAAGTGCGAGTGCAGACCCAAGAAGGACCGGGCCAGACAAGAGAATCCTTGCGGCCCTTGCAGCGAGCGGAGAAAGCACCTGTTTGTGCAGGACCCTCAGACCTGCAAGTGCTCCTGCAAGAACACCGACAGCAGATGCAAGGCCCGGCAGCTGGAACTGAACGAGAGAACCTGCAGATGCGACAAGCCTAGAAGAGGTGGCGGAGGATCTGGCGGAGGCGGATCTGGAAAGCCCATTCCTAATCCTCTGCTGGGCCTCGACAGCACCTGATGATAA.8\u00a0viral genomes (VG)/eye (low dose) or 1\u2009\u00d7\u2009109\u00a0VG/eye (high dose) in 1\u00a0\u00b5L AAV buffer. Note that AAV-VEGF was injected at a concentration of 1\u2009\u00d7\u2009108\u00a0VG/eye and AAV-TNF-\u03b1 and AAV-IL-6 at 1\u2009\u00d7\u2009109\u00a0VG/eye with matching AAV-stuffer controls. In an independent mouse cohort, 2\u00a0weeks after AAV-TNF-\u03b1 treatment, eyes were IVT injected with 1\u00a0\u00b5L of vehicle or neutralizing anti-TNF-\u03b1 Golimumab . If IVT injection failed (e.g. due to damage of a major blood vessel or the lens), the eye was excluded from analysis.Mice were intravitreally injected with the different AAVs under isoflurane anesthesia and after local anesthetic eye drops were applied . Both eyes of 6 mice per group were injected with either 1\u2009\u00d7\u20091019. In brief, mice were anesthetized by intraperitoneal injection with 60\u201390\u00a0mg/kg ketamine (Medistar) and 6\u20138\u00a0mg/kg xylazine . Pupils were dilated with 5\u00a0mg/mL tropicamide and phenylephrine . A Spectralis HRA/OCT device (Heidelberg Engineering) equipped with a 55\u00b0 widefield lens was used for recording of Optical Coherence Tomography (OCT) pictures and Autofluorescence (AF) images. For Fundus Fluorescein Angiography (FFA), 200\u00a0\u00b5L of a 0.2% fluorescein solution (Alcon) were injected subcutaneously and recorded 90\u00a0s after injection with the Spectralis HRA/OCT (Heidelberg Engineering). Mice were euthanized by cervical dislocation. The retina and the eye cup were dissected, and flash frozen in liquid nitrogen.In vivo imaging was done as described previously11). In brief, 7-days-old male and female pups with their mothers were transferred to a hyperoxic chamber (75% oxygen). At P12, oxygen concentration was slowly reduced back to normoxic conditions (21% oxygen) within approximately 3\u00a0h. Control mice remained at normoxic conditions for the whole experiment. Mice were euthanized at P12, P13, P14, P15 and P16 by cervical dislocation. Dissected retinae were stored in RNAlater (Invitrogen).The OIR experiments were performed according to previously published protocols were homogenized in 700\u00a0\u00b5L Qiazol (Qiagen) using a Precellys Evolution homogenizer (Bertin Technologies). Retina samples were homogenized by ceramic beads MPbio, 6913-500) and eye cup samples by metal beads and processed in 4 separate batches. Total RNA was extracted using the miRNeasy Micro Kit according to the manufacturer\u2019s tissue protocol including on-column DNase treatment . Total RNA samples were quantitatively and qualitatively assessed using the fluorescence-based Broad Range Quant-iT RNA Assay Kit (Thermo Fisher Scientific) and the Standard Sensitivity RNA Analysis DNF-471 Kit on a 96-channel Fragment Analyzer (Agilent), respectively. Concentrations averaged at 48.7\u00a0ng/\u00b5L while RIN ranged from 5.2 to 9.6, with a median at 9.2. Two samples with RIN below 6 were excluded from downstream processing: AAV-stuffer (high) eye cup replicate 4 and AAV-VEGF eye cup replicate 5. See Table 3-500 andRetinae were dissected from 12 to 16-day postnatal C57BL/6\u00a0J mice (n\u2009=\u200932). RNAlater-preserved tissues were disrupted and homogenized in CK14 tubes with RLT buffer using a Precellys Evolution homogenizer (Bertin Technologies). Total RNA was then extracted using the MagMAX-96 total RNA isolation kit including a DNase digestion prior to final elution . Total RNA samples were quantitatively and qualitatively assessed on a Synergy microplate reader with a Gen5 Take3 module (BioTek), and the Eukaryote total RNA 6000 Nano microfluidic chip on a 2100 Bioanalyzer system (Agilent), respectively. Concentrations averaged at 136.3\u00a0ng/\u00b5L while RIN of selected samples were above 8.4. All samples were further processed for library preparation. See Table 36.70 retina- and eye cup-derived RNA samples were normalized on the MicroLab STAR automated liquid platform (Hamilton). Total RNA input of 250\u00a0ng was used for library construction with the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina #E7760, together with the NEBNext Poly(A) mRNA Magnetic Isolation Module #E7490 upstream and the NEBNext Multiplex Oligos for Illumina #E7600 downstream . The only deviation from the manufacturer\u2019s protocol was the use of Ampure XP beads (Beckman Coulter) for double-stranded cDNA purification, instead of the recommended SPRIselect Beads. The index PCR was performed with 12 cycles, while the final library was eluted in 35\u00a0\u00b5L. mRNA libraries were then quantified by the High Sensitivity dsDNA Quanti-iT Assay Kit (ThermoFisher) on a Synergy HTX (BioTek). Library molarity averaged at 149\u00a0nM. mRNA libraries were also assessed for size distribution and adapter dimer presence (<\u20090.5%) by the High Sensitivity Small Fragment DNF-477 Kit on a 96-channel Fragment Analyzer (Agilent). All 70 sequencing libraries were then normalized on the MicroLab STAR (Hamilton), pooled and spiked in with PhiX Control v3 (Illumina). The library pool was subsequently clustered on an S4 Flow Cell and sequenced on a NovaSeq 6000 Sequencing System (Illumina) with dual index, paired-end reads at 2\u2009\u00d7\u2009100\u00a0bp length , reaching an average depth of 31.3 million Pass-Filter reads per sample (7.0% CV). The description of RNA library preparation and sequencing closely aligns with the one given in Becker et al.Total RNA input of 100\u00a0ng was used for library construction with the TruSeq Stranded mRNA LT kit\u2014Set A , together with the SuperScript II Reverse Transcriptase , according to the manufacturer\u2019s protocol. The quality of mRNA libraries was assessed for adapter and heterodimer presence using the DNA 1000 microfluidic chip , while library molarity was measured using the Quant-iT PicoGreen dsDNA Assay Kit . All 32 sequencing libraries were then normalized, pooled and spiked in with PhiX Control v3 (Illumina). The library pool was subsequently clustered using the TruSeq SR Cluster Kit v3 , and sequenced with TruSeq SBS Kit v3 reagents on a HiSeq 2000 Sequencing System (Illumina) with single index, single-read reads at 1\u2009\u00d7\u200951\u00a0bp length , reaching an average depth of 24.9 million Pass-Filter reads per sample (13.1% CV).2. The LUNA-FL cell counter (LogosBio) with Acridine Orange dye was used for cell quantification before seeding. HRMECs were stimulated with 10\u00a0ng/mL recombinant human TNF-\u03b1 for 24\u00a0h at 37\u00a0\u00b0C. Cells were washed with PBS and lysed in RLT buffer provided by Qiagen and RNA extracted using the RNeasy Mini Kit according to the manufacturer\u2019s manual.Primary human retinal microvascular endothelial cells were cultivated in Endothelial Cell Growth Medium on plates pre-coated with 0.1% gelatin . Cells were cultivated in a humidified chamber at 37\u00a0\u00b0C and 5% COcDNA synthesis was done using the high-capacity cDNA Kit and qRT-PCR was performed with the Taqman Universal PCR Master Mix on a QuantStudio 6 Real-Time PCR system (Applied Biosystems). Relative expression (fold induction) was calculated using the \u0394\u0394CT method and the 18S (for mouse tissue) and POLR2A (for HRMECs) genes were used as a normalization controls. The following Taqman probes were used in this study: 18S (Hs99999901_s1), POLR2A (Hs00172187_m1), Madcam1 (Hs00369968_m1), CCL2 (Mm00441242_m1) and a custom-made Taqman probe detecting human codon-optimized VEGF with a V5 tag .57. In brief, 2\u00a0weeks after IVT injection of AAV-TNF-\u03b1, 100\u00a0\u00b5g golimumab (Simponi) were injected intravitreally. 6\u00a0weeks after AAV injection, whole eyes were enucleated, flash frozen in liquid nitrogen, and homogenized in lysis buffer supplemented with proteinase inhibitor Pefabloc SC with metal beads in a Precellys Evolution tissue homogenizer (Bertin Technologies). C3 expression was measured with the Mouse C3 SimpleStep ELISA Kit according to the manufacturer\u2019s protocol and measured with a SpectraMax Plus 384 plate reader (Molecular Devices).Eyes for validation of C3 expression were generated from an independent mouse cohort that is published elsewhere19. Eyes were enucleated 3\u20136\u00a0weeks after IVT injection and directly fixed in 4% paraformaldehyde for 48\u00a0h at 4\u00a0\u00b0C. Eyes were dehydrated, incubated in xylol, and infiltrated with paraffin using a tissue processing machine . Immunohistochemical stainings were performed on 3\u00a0\u00b5m sections with the Opal Multiplex IHC Kit (Akoya Biosciences) and carried out on the automated Leica Bond platform . Antigen retrieval was done with the BOND Epitope Retrieval Solution 1 at 95\u00a0\u00b0C and pH 6.0 for 20\u00a0min. Polyclonal rabbit anti-CD3 antibody was diluted 1:200 and used as a pan-T-cell marker. OPAL polymer anti-rabbit-HRP secondary antibody and OPAL 570 reagent were used to develop the fluorescent signal. Nuclei were stained with spectral DAPI and slides were mounted with ProLong Antifade Mounting Medium . Immunofluorescence stainings were imaged using a laser-scanning microscope LSM700 . RNAscope in situ hybridizations for human VEGF, TNF-\u03b1 and IL-6 were performed externally at ACDBio/Biotechne using the RNAscope 2.5 LSx Red assay. Custom-made probes were designed based on the codon-optimized, V5-tagged human transgenes expressed by each AAV. As technical controls, ACD Positive Control and ACD Negative Control probes were used. Epitope retrieval was done for 15\u00a0min at 88\u00a0\u00b0C and proteolysis was performed with Protease III for 15\u00a0min at 40\u00a0\u00b0C at ACDBio. Slides were imaged using an AxioScan.Z1 slide scanner .Eyes for histological analysis were generated in an independent experiment that was published elsewhereIn case of the OIR dataset GRCm38.96 was used as the reference genome. For the AAV study, a custom genome was created by merging codon optimized sequences for hIL6, hTNFa, and hVEGF into the mouse genome. Generation of genomic indices and read alignment was carried out via STAR (version 2.5.2b). Quantification on gene level was performed using FeatureCounts (version 1.5.1) and RSEM (version 1.3.0), while discarding multimapping reads. Quality control metrics were obtained using MultiQC (version 1.0), with information assembled from STAR, picardmetrics (version 0.2.4), and fastQC (version 0.11.5), among other sources.Genes with a read count value of below 10 across all samples in either the AAV or OIR study were removed from our analysis. We normalized counts expression values using the voom function provided by limma (version 3.44.3). In the AAV study we corrected for an identified batch effect associated with the RNA extraction batch (variable: rna_extraction_batch) using the ComBat function (sva package version 3.40.0). Principal component analysis was performed using the pcaMethods R package (version 1.80.0) selecting for the top 1000 most variable genes.Differential gene expression was calculated using the lmFit function provided by the limma R package. Significantly changing genes were selected based on a BH-adjusted p-value\u2009<\u20090.05. Pathway information was retrieved using the misgdbr R package (version 7.1.1). Gene set enrichment analysis was performed using the over-representation analysis (ORA) function provided by Clusterprofiler version 4.0.2. Direct comparisons between gene sets derived from the AAV experiment and the OIR study were carried out using a hypergeometric test provided by the stats R package (version 4.1.0). Venn diagrams were plotted using ggvenn (version 0.1.9).http://feb2021.archive.ensembl.org/. Raw counts were further processed using the Seurat R package (version 4.0.3). Cell specific genes were identified for each individual dataset and cell type via the FindMarkers function. Genes were defined as cell specific if the BH-adjusted p-value\u2009<\u20090.01 and the gene was not identified as specific to any other cell type (unique genes only). As above, the overlap between cell specific marker genes and genesets derived from the AAV or OIR studies were quantified by the ORA function part of clusterProfiler.Single cell RNA-Seq datasets were obtained from GSE1352229, GSE130636, and GSE135922. If required, human ensembl IDs were mapped to mouse homolog using the biomaRt R package (2.48.2), with host parameter set to Supplementary Figure 1.Supplementary Table 2.Supplementary Table 3."} +{"text": "E. coli genome codes for four different POTs, known as Di- and tripeptide permeases A-D (DtpA-D). DtpC was shown previously to favor positively charged peptides as substrates. In this study, we describe, how we determined the structure of the 53\u00a0kDa DtpC by cryogenic electron microscopy (cryo-EM), and provide structural insights into the ligand specificity of this atypical POT. We collected and analyzed data on the transporter fused to split superfolder GFP (split sfGFP), in complex with a 52\u00a0kDa Pro-macrobody and with a 13\u00a0kDa nanobody. The latter sample was more stable, rigid and a significant fraction dimeric, allowing us to reconstruct a 3D volume of DtpC at a resolution of 2.7\u00a0\u00c5. This work provides a molecular explanation for the selectivity of DtpC, and highlights the value of small and rigid fiducial markers such as nanobodies for structure determination of low molecular weight integral membrane proteins lacking soluble domains.Proton-coupled Oligopeptide Transporters (POTs) of the Major Facilitator Superfamily (MFS) mediate the uptake of short di- and tripeptides in all phyla of life. POTs are thought to constitute the most promiscuous class of MFS transporters, with the potential to transport more than 8400 unique substrates. Over the past two\u00a0decades, transport assays and biophysical studies have shown that various orthologues and paralogues display differences in substrate selectivity. The Membranes of cells compartmentalize metabolic processes and present a selective barrier for permeation. To preserve the characteristic intracellular milieu, membrane transporters with specialized functions have evolved to maintain the nutrient homeostasis of cells . Many ofE. coli genome codes for four different POTs named Di- and tripeptide permease A-D (DtpA-D), also known as YdgR (=DtpA), YhiP (=DtpB), YjdL (=DtpC) and YbgH (=DtpD). They cluster in pairs, DtpA and B (sequence identity 51%), and DtpC and D (sequence identity 56%) with around 25% identity between them. DtpA and B exhibit a prototypical substrate preference similar to the human PepT1 transporter fused the transporter to split-sfGFP , ii) rai. This resulted in a 52\u00a0kDa Pro-macrobody (short Mb26), and we expected it to bind to the periplasmic side of the transporter as seen in other MFS transporter-Nb complexes , or ten residues (split sfGFP-DtpC1-470) of the transporter were deleted. We then assessed proper folding and complementation by monitoring the fluorescence of the chromophore on an HPLC system (FL) and lowest in the most truncated version (split sfGFP-DtpC1-470). In order to extend this observation to other MFS transporters, we repeated this experiment with the human POT homologue PepT1, and noticed a similar trend upon shortening of the termini. Yet, since the decrease of fluorescence was only minor in split sfGFP-DtpC1-475 in comparison to split sfGFP-DtpCFL, we proceeded to imaging with the shorter construct in the presence of Nb26.Since MFS transporters typically lack additional domains outside their transport unit, which is a major impediment for accurate particle alignment in single particle cryo-EM approaches, we assessed three fiducial marker strategies introducing additional density outside of detergent micelles containing DtpC, by analyzing the quality of 2D class averages . To obtaomplexes . In a thC system . All con(1\u2013475)-Nb26 sample allowed clear visualization of the transmembrane helices after clustering a small subset of particles, but the majority of particles clustered in classes with blurry density for the split sfGFP fiducial, or with the two complementary parts \u03b21-6 and \u03b27-11 not assembled . HoweverAs we obtained the best 2D class averages for DtpC with the fiducial marker Nb26, we proceeded to a large data collection and coul\u03b1-atoms). The peptide binding site of DtpC is exposed to the cytoplasmic side organized in two helical bundles and additional two\u00a0TMs specific for the POT family (known as HA and HB domains). It is highly similar compared to the previously determined DtpD structure with an mic side , 4. Almomic side .Shewanella oneidensis (PepTSo), where the inter-bundle periplasmic salt bridge is formed between TM5 and TM7, the IF state is in all other analyzed structures stabilized by a salt bridge on the tip of TM2 and TM7 and R294 and hydrogen bonds between H37 and D293 (TM7) as well as R28 (TM1) and N421 . We also and TM7 . Additio1XXE2R motif on TM1 involved in proton coupling and ligand binding, and ii) the ability to accommodate dipeptides, tripeptides, and peptidomimetics, which relies on a set of conserved residues located in the central binding cavity. In DtpC, the E1XXE2R motif, has evolved to Q1XXE2Y . In all high resolution X-ray structures of canonical POTs, R is in salt-bridge distance to E2 and the C-terminus of substrate peptides. Mutation of either E1 or E2 in the conventional E1XXE2R motif to glutamine residues abolishes uptake the presence of the Es uptake . A revereases it . In addiproposed . This bi1XXE2Y motif instead of E1XXE2R.In DtpC, we now observe that the side chain pocket has a different architecture and characteristic in comparison with the one of canonical POTs. It displays an overall more acidic groove caused by the presence of the aspartate residue 392, conserved among atypical POTs. This residue has been predicted to be involved in substrate coordination and mutation of this residues in DtpC and homologues DtpD (corresponding residue is D395) abolished transport activity . CanonicE. coli POTs structures. Lastly, it displays some of the challenges related to high resolution cryo-EM structure determination of MFS transporters devoid of soluble domains, and manifests once again, the benefit of fiducial markers in overcoming those.In summary, our work provides new insights into promiscuous versus selective substrate recognition in POTs and constitutes a step forward towards completing the family of Escherichia coli genome, and cloned into a pNIC-CTHF vector by ligation-independent cloning (LIC). This vector contains a C-terminal His-Tag and a Tobacco Etch virus (TEV) cleavage site and a kanamycin resistance gene as selectable marker. The first 6\u00a0N-terminal beta strands of sfGFP were fused to the N-terminus of DtpC, and the beta strands 7 to 11 fused to the C-terminus. We named this construct split sfGFP-DtpCFL. Two additional constructs were cloned with truncations of 5 (split sfGFP-DtpC1-475), and 10 residues (split sfGFP-DtpC1-470), on the C-terminal side of DtpC.The full-length cDNA of DtpC wild type (WT) was amplified from the HsPepT1 was previously cloned into a pXLG vector containing an expression cassette composed of an N-terminal Twin-Streptavidin tag followed by the HRV-3C protease recognition sequence a C-terminal truncation of 36 residues (split sfGFP-HsPepT11-672), and ii) a C-terminal truncation of 36 residues and a N-terminal truncation of 10 residues (split sfGFP-HsPepT110-672) were cloned.sequence . SimilarE. coli C41(DE3) cells grown in terrific broth (TB) media supplemented with 30\u00a0\u03bcg/ml kanamycin according to established procedures , and the pellet was stored at -20\u00b0C until further use. Cell pellets were resuspended in lysis buffer glycerol, 15\u00a0mM imidazole, with 3\u00a0ml of lysis buffer per Gram of wet weight pellet), supplemented with lysozyme, DNase and 0.5\u00a0mM tris(2-carboxyethyl)phosphine (TCEP). The cells were lysed by three cycles using an Avestin Emulsiflex homogenizer at 10,000\u201315,000 psi. Recovered material was centrifuged to remove non-lysed cells and the supernatant was subjected to ultracentrifugation to separate the membrane fraction . Membranes were resuspended in lysis buffer supplemented with cOmplete EDTA-free protease inhibitors (Roche), and solubilized by adding 1% n-Dodecyl-\u03b2-D-Maltoside (DDM) detergent (Anatrace). The sample was centrifuged for 50\u00a0min at 90,000 \u00d7 g, and the supernatant was applied to Ni-NTA beads for immobilized-metal affinity chromatography (IMAC) on a gravity column. The beads were pre-equilibrated in lysis buffer and incubated with the solubilized membrane proteins for one hour at 4\u00b0C on a rotating wheel. Loaded beads were washed with buffer with increasing imidazole concentrations . The proteins were eluted from the column with a buffer containing high imidazole concentration and combined with 1\u00a0mg of TEV protease to perform the His-tag cleavage during dialysis overnight at 4\u00b0C. The dialysis buffer contained 20\u00a0mM HEPES at pH 7.5, 150\u00a0mM NaCl, 5% glycerol, 0.5\u00a0mM TCEP, 0.03% DDM. The cleaved protein was recovered by negative IMAC, concentrated to 4\u00a0ml using a 50\u00a0kDa concentrator (Corning\u00ae Spin-X\u00ae UF concentrators) and run on an \u00c4KTA Pure system , using a HiLoad 16/ 600 Superdex 200 column for DtpC, and a Superdex 200 Increase 10/300 column for the split sfGFP-DtpC constructs. Fractions containing the protein were pooled, concentrated, flash frozen and stored at -80\u00b0C until further use.Recombinant DtpC, and the three split sfGFP-DtpC constructs were expressed in ocedures . CultureHsPepT1 constructs, expression was done in mammalian cells as described previously . After 20\u00a0min of incubation on a rotating wheel, the suspension was transferred to a gravity column. Following two wash steps with 300\u00a0mM NaCl, 20\u00a0mM HEPES (pH 7.5), 0.03% DDM, and 0.003% CHS, split sfGFP-HsPepT1comnstructs were eluted with 0.03% DDM, 0.003% CHS, 150\u00a0mM NaCl, 20\u00a0mM HEPES (pH 7.5), and 10\u00a0mM desthiobiotin (Sigma-Aldrich).For the split sfGFP-eviously . BrieflyTo generate DtpC specific nanobodies, two non-inbred llamas were injected six times at weekly intervals with a mixture of 94 different proteins including DtpC purified in the detergent DDM (50\u00a0\u00b5g of each antigen weekly). After 6\u00a0weeks of immunization, two separate phage display libraries were constructed, one from each animal, in the pMESy2 vector, which is a derivative of pMESy4 that contain a C-terminal EPEA-tag for affinity purification. After pooling both libraries, nanobodies were selected against individual antigens in two rounds of parallel panning in 96-well plates containing one immobilized antigen in each well. After two selection rounds on DtpC, 60 clones were picked for sequence analysis, 13 clones encoded antigen-specific nanobodies as tested in ELISA, grouping them in 5 different sequence families. A nanobody family is defined as a group of nanobodies with a high similarity in their CDR3 sequence . Nanobodies from the same family derive from the same B-cell lineage and likely bind to the same epitope on the target. Immunizations, library construction, selection by panning and nanobody characterization were performed according to standard procedures . Five naE. coli WK6 cells and purified following standard procedures. Specifically, the cell pellet was resuspended in TES buffer supplemented with one protease inhibitor tablet (Roche). Osmotic shock was performed by the addition of diluted TES buffer to release the periplasmic proteins. The solution was first centrifuged for 20\u00a0min at 10,000 \u00d7 g and additionally for 30\u00a0min at 100,000 \u00d7 g. The supernatant was applied to CaptureSelect beads (Thermo Fisher Scientific), which were equilibrated with wash buffer . After three column volumes of washing, the nanobody was eluted with 20\u00a0mM HEPES, pH 7.5, 1.5\u00a0M MgCl2. The nanobodies were further purified on a HiLoad 16/600 Superdex 75\u00a0pg column in 20\u00a0mM HEPES, pH 7.5, 150\u00a0mM NaCl, 5% glycerol, concentrated with a 5\u00a0kDa cut-off concentrator, flash-frozen and stored at -80\u00b0C until further use.The nanobodies were expressed in E. coli WK6 cells as above. The cell pellet was resuspended in TES buffer supplemented with one protease inhibitor tablet (Roche). Osmotic shock was performed by the addition of diluted TES buffer to release the periplasmic proteins. The solution was first centrifuged for 20\u00a0min at 10,000 \u00d7 g and additionally for 30\u00a0min at 142,000 \u00d7 g. The supernatant was further purified by immobilized-metal affinity chromatography (IMAC) on a gravity column. The beads were pre-equilibrated in 20\u00a0mM NaPi at pH 7.5, 300\u00a0mM NaCl, 5% glycerol, 15-30\u00a0mM imidazole, 0.5\u00a0mM TCEP and incubated. Loaded beads were washed with increasing imidazole concentrations . The proteins were eluted from the column with a buffer containing high imidazole concentration and combined with 1\u00a0mg of 3C protease to perform the His-tag cleavage. The cleaved protein was recovered by negative IMAC, concentrated to 0.5\u00a0ml using a 30\u00a0kDa concentrator (Corning\u00ae Spin-X\u00ae UF concentrators) and run on an \u00c4KTA Pure system , using a Superdex 75 Increase 10/300 column. Fractions containing the protein were pooled, concentrated, flash frozen and stored at -80\u00b0C until further use.The nanobody 26 (Nb26) was first inserted into a pBXNPH3 vector containing a C-terminal penta-histidine tag preceded of a HRV-3C protease recognition sequence. The maltose binding protein (MBP) was then inserted in frame with the 3\u2019 end of the nanobody, with two proline residues as a linker between the two genes similar as described in . The resulting Pro-macrobody 26 (Mb26) was expressed in 1-475-Nb26 with a Prometheus NT.48 device . The purified proteins were diluted to 16 \u00b5M, and the complexes were formed using a 1:1.5\u00a0M ratio of membrane protein: fiducial. The fluorescence at 330 and 350\u00a0nm was recorded over a temperature gradient scan from 15\u00b0 to 95\u00b0C and processed in GraphPad Prism 9.0 (GraphPad Software).The differential scanning fluorimetry method was used to follow the thermal unfolding event of Nb17,Structures with the following sequences were used as input for AlphaFold2 structure prediction , and AMBMSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNKTPSQPRAIYYIVAIQIWEYFSFYGMRALLILYLTHQLGFDDNHAISLFSAYASLVYVTPILGGWLADRLLGNRTAVIAGALLMTLGHVVLGIDTNSTFSLYLALAIIICGYGLFKSNISCLLGELYDENDHRRDGGFSLLYAAGNIGSIAAPIACGLAAQWYGWHVGFALAGGGMFIGLLIFLSGHRHFQSTRSMDKKALTSVKFALPVWSWLVVMLCLAPVFFTLLLENDWSGYLLAIVCLIAAQIIARMMIKFPEHRRALWQIVLLMFVGTLFWVLAQQGGSTISLFIDRFVNRQAFNIEVPTALFQSVNAIAVMLAGVVLAWLASPESRGNSTLRVWLKFAFGLLLMACGFMLLAFDARHAAADGQASMGVMISGLALMGFAELFIDPVAIAQITRLKMSGVLTGIYMLATGAVANWLAGVVAQQTTESQISGMAIAAYQRFFSQMGEWTLACVAIIVVLAFATRFLFSTPNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEFVTAAGITHGMDELYK.MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNKTPSQPRAIYYIVAIQIWEYFSFYGMRALLILYLTHQLGFDDNHAISLFSAYASLVYVTPILGGWLADRLLGNRTAVIAGALLMTLGHVVLGIDTNSTFSLYLALAIIICGYGLFKSNISCLLGELYDENDHRRDGGFSLLYAAGNIGSIAAPIACGLAAQWYGWHVGFALAGGGMFIGLLIFLSGHRHFQSTRSMDKKALTSVKFALPVWSWLVVMLCLAPVFFTLLLENDWSGYLLAIVCLIAAQIIARMMIKFPEHRRALWQIVLLMFVGTLFWVLAQQGGSTISLFIDRFVNRQAFNIEVPTALFQSVNAIAVMLAGVVLAWLASPESRGNSTLRVWLKFAFGLLLMACGFMLLAFDARHAAADGQASMGVMISGLALMGFAELFIDPVAIAQITRLKMSGVLTGIYMLATGAVANWLAGVVAQQTTESQISGMAIAAYQRFFSQMGEWTLACVAIIVVLAFATRFLFSTPTNMIQESNDNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEFVTAAGITHGMDELYK.MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNKTPSQPRAIYYIVAIQIWEYFSFYGMRALLILYLTHQLGFDDNHAISLFSAYASLVYVTPILGGWLADRLLGNRTAVIAGALLMTLGHVVLGIDTNSTFSLYLALAIIICGYGLFKSNISCLLGELYDENDHRRDGGFSLLYAAGNIGSIAAPIACGLAAQWYGWHVGFALAGGGMFIGLLIFLSGHRHFQSTRSMDKKALTSVKFALPVWSWLVVMLCLAPVFFTLLLENDWSGYLLAIVCLIAAQIIARMMIKFPEHRRALWQIVLLMFVGTLFWVLAQQGGSTISLFIDRFVNRQAFNIEVPTALFQSVNAIAVMLAGVVLAWLASPESRGNSTLRVWLKFAFGLLLMACGFMLLAFDARHAAADGQASMGVMISGLALMGFAELFIDPVAIAQITRLKMSGVLTGIYMLATGAVANWLAGVVAQQTTESQISGMAIAAYQRFFSQMGEWTLACVAIIVVLAFATRFLFSTPTNMIQESNDGGGGGNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEFVTAAGITHGMDELYK.One hour before vitrification, the purified protein complexes were thawed on ice and run on a Superdex Increase 200 5/150 column in 0.015% DDM, 100\u00a0mM NaCl, 10\u00a0mM HEPES (pH 7.5), 0.5\u00a0mM TCEP in order to remove the excess of empty detergent micelles earlier generated upon sample concentration. The top fraction reached a concentration ranging between 3 and 6\u00a0mg/ml, and for each sample, 3.6\u00a0\u03bcl were applied to glow-discharged gold holey carbon 2/1 300-mesh grids (Quantifoil). Grids were blotted for 4\u00a0s at 0 force and 1-s wait time before being vitrified in liquid propane using a Mark IV Vitrobot (Thermo Fisher Scientific). The blotting chamber was maintained at 4\u00b0C and 100% humidity during freezing.All movies were collected using a Titan Krios (Thermo Fisher Scientific) outfitted with a K3 camera and BioQuantum energy filter (Gatan) set to 10\u00a0eV. Automated data acquisitions were set using EPU (Thermo Fisher Scientific). The applied defocus ranged between -0.9\u00a0\u00b5m and -1.8\u00a0\u00b5m in all datasets.2 was used with 2.8\u00a0s exposure time, fractionated in 50 frames. For split sfGFP-DtpC1-475-Nb26, movies were collected at a nominal magnification of \u00d7,130,000 and a physical pixel size of 0.67\u00a0\u00c5, with a 50-\u03bcm C2 aperture and 100-\u03bcm objective aperture at a dose rate of 19.0 e\u2212/pixel per second. A total dose of 57 e\u2212/\u00c52 was used with 3\u00a0s exposure time fractionated in 40 frames.For DtpC-Nb26 and DtpC-Mb26, movies were collected at a nominal magnification of \u00d7105,000 and a physical pixel size of 0.85\u00a0\u00c5, with a 70-\u03bcm C2 aperture and 100-\u03bcm objective aperture at a dose rate of 19.5 e\u2212/pixel per second. A total dose of 75 e\u2212/\u00c5All movies were motion-corrected using Relion-3.1 own impl1-475-Nb26, 7602 movies were collected, 1,049,399 coordinates were picked and used for 2D averaging and clustering. For DtpC-Nb26, 24,333 movies were collected, 6,464,070 coordinates were picked and used for 2D averaging and clustering, and 878,428 particles were used in the final 3D reconstruction. Briefly, DtpC-Nb26 dimeric population was clustered using 3D class averaging in Relion3.1 were collected on the EMBL P12 beamlineGraphs were generated using GraphPad Prism 9.0 (GraphPad Software). Molecular graphics and analyses performed with UCSF ChimeraX-1.2.5 . Figures"} +{"text": "Since the ancestors of modern humans separated from those of Neanderthals, around 100 amino acid substitutions spread to essentially all modern humans. The biological significance of these changes is largely unknown. Here, we examine all six such amino acid substitutions in three proteins known to have key roles in kinetochore function and chromosome segregation and to be highly expressed in the stem cells of the developing neocortex. When we introduce these modern human-specific substitutions in mice, three substitutions in two of these proteins, KIF18a and KNL1, cause metaphase prolongation and fewer chromosome segregation errors in apical progenitors of the developing neocortex. Conversely, the ancestral substitutions cause shorter metaphase length and more chromosome segregation errors in human brain organoids, similar to what we find in chimpanzee organoids. These results imply that the fidelity of chromosome segregation during neocortex development improved in modern humans after their divergence from Neanderthals. Neural stem cells in modern humans have a longer metaphase and fewer chromosome errors compared with Neanderthals and chimpanzees. FOXP2 Kinesin 8], KNL1 (a.k.a. CASC5), and SPAG5 (a.k.a. astrin), which are highly expressed in the germinal zones of the developing neocortex and are associated with mitotic spindle, kinetochore, and chromosome segregation functions. The kinetochore is a complex, three-dimensional (3D), multiprotein structure mediating the attachment of chromosome centromeres with the ends of kinetochore microtubules that carry the ancestral variants of KIF18a and KNL1 are used to generate cerebral organoids, shorter metaphases, less SAC-positive kinetochores, and more chromosome segregation defects are observed in APs. Together, our data suggest that the three amino acid substitutions in KIF18a and KNL1 cause fewer chromosome inheritance errors to occur in APs of modern humans than in archaic humans and apes.P > 0.05; P > 0.05; fig. S1, A, B, and D). Organoids can therefore be used to study the number of SAC-positive kinetochores in cerebral tissue APs.An active SAC, triggered, e.g., by one or more kinetochores not being properly attached to kinetochore microtubules, delays anaphase onset and therefore prolongs metaphase in the fetal human neocortex and human organoid APs, respectively, than in chimpanzee organoid APs . Thus, human metaphase APs have more SAC-positive kinetochores than chimpanzee APs.To investigate whether the previously described metaphase prolongation in human relative to chimpanzee APs mice; P < 0.001; P < 0.001) that did not differ (P > 0.05) from the hKIF18a-hKNL1-hSPAG5 mice with the six amino acid substitutions , two in KNL1 (H159R and G1086S), and three in SPAG5 , albeit not significantly so versus 4.6 min (hKNL1 substitution 2) versus 4.6 min (wt), respectively; fig. S4, see also In contrast, in hSPAG5 mice, the length of the AP metaphase was 4.4 min and hence similar to wt mice , indicatNotably, the durations of prometaphase, which precedes metaphase, and of anaphase, which follows metaphase, did not show any significant changes in any of the mouse lines analyzed .We also explored whether the modern human-specific changes in KIF18a and KNL1 may influence general aspects of cortical development. However, we did not observe any effects on the overall morphology, thickness, or perimeter of the neocortex in adult mice humanized for KIF18a and KNL1 . Consistent with this, the adult mice did not show any change in the distribution or numbers of Ctip2KIF18a and the two codons in KNL1 back to the ancestral, Neanderthal-like state in the H9 ESC line. Individual cells subjected to the relevant RNA guides and donor DNAs were expanded to cell lines, among which we selected two independent ancestralized lines [aKif18a-aKNL1 lines 1 (L1) and 2 (L2)] and two lines where none of the three codons had been changed at the midpoint of the dark period . After positive plug detection in the morning, the cumulus complexes were isolated and zygotes were removed with a treatment of hyaluronidase [final concentration of 0.1% (801 U/ml)]. CRISPR-Cas9 solutions were injected into the male pronucleus of fertilized zygotes by using a motor-driven manipulator-based microinjection stage. About 2 hours after injections, the surviving embryos were transferred into Crl:CD1(ICR) pseudopregnant recipient female mice (ca. 20 embryos per recipient).The recipient mice were mated with vasectomized males Crl:CD1 (ICR). After detection of a copulation plug in the morning of the transfer day, pseudopregnant mice were used for unilateral surgical embryo transfer into the oviduct as described with 2 \u03bcl of genomic tail DNA, 0.25% dimethyl sulfoxide, and 0.5 \u03bcM forward and reverse primer. Primer sequences (target-seq-primer) and PCR conditions are in table S1. Products were Sanger sequenced either with the forward and reverse PCR primers or if required with internal sequencing primers. Primary candidates have been identified by sequence alignments of potentially modified PCR fragments against the wt and modified reference sequence. The products from putatively edited mice were cloned (TOPO) and verified by Sanger sequencing of 10 to 20 randomly selected clones. Founders carrying the respective genome modification either homo- or heterozygously were propagated and genotyped by Sanger sequencing of PCR fragments. From the second generation onward, allele-specific PCRs were performed on 2 \u03bcl of crude buccal DNA in a total of 10 \u03bcl using the REDExtract-N-Amp PCR ready Mix (Sigma-Aldrich) according to the manufacturer\u2019s protocol using 0.5 \u03bcM forward, reverse, and mutant primer. The readout is done on standard agarose gels. Primers and PCR conditions are in table S1.Kif18a, Knl1, and Spag5 in the wt and humanized embryonic mouse telencephalon was confirmed by PCR, using generated cDNA as templates, followed by Sanger sequencing of the PCR products. Wt and gene-edited sequences were mapped to the Kif18a, Knl1, and Spag5 genes, respectively, using Geneious Prime (version 2020.2.4). Data showed the expression of only the wt Kif18a, Knl1, and Spag5 in E11.5 wt embryonic mouse telencephalon, and the expression of only the gene-edited hKif18a, hKnl1, and hSpag5 in E11.5 humanized hKIF18a-hKNL1-hSPAG5 and LightCycler 96 Instrument (Roche). Gene expression analyses by qPCR showed that the relative mRNA levels of Kif18a, Knl1, and Spag5 were not significantly different between E11.5 wt and humanized embryonic mouse telencephalon .For Sanger sequencing and real-time qPCR, total RNA was isolated from E11.5 wt and humanized mouse dorsolateral telencephalon using the RNAeasy Micro Plus Kit (Qiagen) according to the manufacturer\u2019s instructions. cDNA was synthesized using the Maxima First-Strand cDNA Synthesis Kit (Thermo Fisher Scientific). The expression of PAG5 see embryoniThe following primers were used for PCR:Forward primer for Kif18a (5\u2032-AGAAAAGGCGGTGCAGTTCT-3\u2032),Reverse primer for Kif18a (5\u2032- GGAATCTTCCCGGACAGCAA-3\u2032);Forward primer for Knl1 H159R (5\u2032-CCCCAGACAAGTCAAGCAGAA-3\u2032),Reverse primer for Knl1 H159R (5\u2032-TCAACTCCATACACTCATTGCC-3\u2032);Forward primer for Knl1 G1086S (5\u2032-TGGATATCACCAAGAGTTGCAC-3\u2032),Reverse primer for Knl1 G1086S (5\u2032-CAAAACTGAAGCCCTTTCTGTC-3\u2032);Forward primer for Spag5 D410H and E162G (5\u2032-TGAATCTCGGTTTGTCGCCT-3\u2032),Reverse primer for Spag5 D410H and E162G (5\u2032-TTCCTCCCCTGGATCGACAT \u22123\u2032);Forward primer for Spag5 P43S (5\u2032-GTTCAAATAGAGGCGGCGGG-3\u2032),Reverse primer for Spag5 P43S (5\u2032-GTTCTTTCCCACCAGCTACAAG-3\u2032).The following primers were used for qPCR:Forward primer for Kif18a (5\u2032-CAAACTCAGGACCACTTGCTGT-3\u2032),Reverse primer for Kif18a (5\u2032-ATGGGAACGAGAAGACACTGC-3\u2032);Forward primer for Knl1 (5\u2032-CCTCTGGGGGAGATGGCTACAT-3\u2032),Reverse primer for Knl1 (5\u2032-GATGGACTTTGTTGGGCTGAGA-3\u2032);Forward primer for Spag5 (5\u2032-GTCTCACCCTCTTCTTACAGGC-3\u2032),Reverse primer for Spat5 (5\u2032-GCTGGTTCTGGCACTTCATCTA-3\u2032);Forward primer for Actb (5\u2032-CGGGACCTGACAGACTACCTC-3\u2032),Reverse primer for Actb (5\u2032-GGTGGTGAAGCTGTAGCCACG-3\u2032).AAVS1 locus from the H9 human ESC line that we generated as previously described :KIF18A_sg1: 5\u2032-TGTTATAAAGAAACAAAATAKIF18A_sg2: 5\u2032-GATTTGTAGTTTTCTTTCCAKNL1_H109R_sg1: 5\u2032-ATTTGCATGTTTCCTTTCACKNL1_G1086S_sg1: 5\u2032-TGAATGAACCTCTATCAAGCAssDNA KIF18A_K67R: 5\u2032-AGTTGACGTTTCATCAAAAACAGCATCAAATACAAATTTAAGATCCTTGTTTTGTCTCTTTATAACATTTTGATTTGTAGTTTTCTTTCCATGGAAAAAACTGACTssDNA KNL1_H159R: 5\u2032-TGTTGTAAAATGCCTTTTTAAAAGTTTGCTTTTGTTCTGATCTCTTATATTTTGCTTTCATTATAGTTTTCAATTATAGAACATACCCATGAAAGGAAACATGCAAATGACCAGACAGTCATTTTTTssDNA KNL1_G1086S: 5\u2032-TCTTGTCATTTTTTAGCTTAAGGCTTTTTCTTCTCTGACTTTTGCCTGATAGAGGTTCGTTCAGAAATCCAGGACTTTGTACATCTTTGATo derive colonies from single cells, cells were incubated with StemFlex with supplement and CloneR for 1 day and then sorted with a Cytena cell printer.Genomic DNA of each colony was isolated, a region of ~200 base pairs (bp) around the cut site was amplified and sequenced, and from sequences, the editing state was evaluated as described amplification included 1\u00d7 ddPCR Supermix for probes , 0.2 \u03bcM of each primer and 0.2 \u03bcM probe (both IDT) for target and reference, together with 1 \u03bcl genomic DNA in QuickExtract DNA Extraction Solution (Lucigen). After droplet generation with the QX200 Droplet generator (Bio-Rad), the PCR was for 5 min at 95\u00b0C, followed by 42 cycles of 35 s at 95\u00b0C (at a ramp rate of 1.5\u00b0C/s) and 65 s at 54\u00b0C for KIF18A and KNL1_G1086S or 65 s at 56\u00b0C for KNL1_H109R (at a ramp rate of 1.5\u00b0C/s), and 5 min at 98\u00b0C. Readout was in a QX200 Droplet reader (Bio-Rad), and allele copy numbers were determined relative to the FOXP2 reference and unedited controls.Deletions bigger than ~200 bp and therefore not detected by the amplicon sequencing KNL1_G10862_upSNP_F: 5\u2032-ACACTCTTTCCCTACACGACGCTCTTCCGATCTAAGCAATCCCACACCTGACTKNL1_G10862_upSNP_R: 5\u2032-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCTTTCGTCCTCTAACACATCCAKNL1_G10862_downSNP_F: 5\u2032-ACACTCTTTCCCTACACGACGCTCTTCCGATCTGCCCTGGAGGATAAAGAGGAKNL1_G10862_downSNP_R: 5\u2032-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGAATCGGTGACTTCCAGATCAKNL1_H159R KNL1_H159R_upSNP_F: 5\u2032-ACACTCTTTCCCTACACGACGCTCTTCCGATCTAAGACCCTTCAGAATCCTACCCKNL1_H159R_upSNP_R: 5\u2032-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTGTATCCTCTGTGTGGCTGAAKNL1_H159R_downSNP_F: 5\u2032-ACACTCTTTCCCTACACGACGCTCTTCCGATCTGCAGAGCCTGTCAAATCCTTKNL1_H159R_downSNP_R: 5\u2032-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCCAGGTGTCGGTTAAGCTGTKIF18A KIF18A_upSNP_F: 5\u2032-ACACTCTTTCCCTACACGACGCTCTTCCGATCTGTGGGTAAGGGAGTGGGAATKIF18A_upSNP_R: 5\u2032-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGCTTCAGAGCCTGCACTCTTKIF18A_downSNP_F: 5\u2032-ACACTCTTTCCCTACACGACGCTCTTCCGATCTGCAACCGTTTTAGCCAAGATKIF18A_downSNP_R: 5\u2032-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCTCCCCGAATGTCTTTCTCATo find large-scale chromosomal deletions, duplications, or karyotype abnormalities, we performed \u201cshallow\u201d whole genome sequencing to an average genome coverage 0.10 to 0.15 of edited and nonedited colonies as well as the cell line from which the colonies were derived, as described (KIF18a and KNL1 in edited and nonedited cell lines as well as their \u201cmother\u201d cell line was measured with CellsDirect One-Step qRT-PCR Kit (Invitrogen) using TaqMan probes for KIF18a (FAM Hs0105428_m1), KNL1 (FAM Hs00538241_m1), and a \u201chousekeeping gene\u201d PPIB (VIC Hs00168719_m1). The producer\u2019s protocol was followed to set up reactions. For a subset of reactions, reverse transcriptase was inactivated by 15-min incubation at 70\u00b0C. The cycler program for RT-qPCR was 50\u00b0C for 10 min, 95\u00b0C for 2 min, then 50 cycles of 95\u00b0C for 3 s, 53\u00b0C for 15 s, and 60\u00b0C for 30 s.Expression of Organoids were generated from the H9 ESCs and cultured as described and immunohistofluorescence (fixed). Mouse neocortex was fixed, embedded, cryosectioned, and prepared for immunohistofluorescence as described for multiphoton excitation, and using 63\u00d7 Plan-Apochromat 1.4 numerical aperture (NA) oil or 40\u00d7 C-Apochromat 1.2 NA W objectives .http://imagej.nih.gov/ij/). Brightness and contrast were recorded and adjusted linearly. Measurements of mitotic phases were performed as described . Because at least some datasets in most experiments did not pass normality tests (Shapiro-Wilk and Kolmogorov-Smirnov tests), nonparametric Mann-Whitney"} +{"text": "The structure of mammalian V-ATPase with mEAK-7 shows how a TLDc domain-containing protein can bind the proton pump to form an activity-sensitive interaction. V-ATPases are rotary proton pumps that serve as signaling hubs with numerous protein binding partners. CryoEM with exhaustive focused classification allowed detection of endogenous proteins associated with porcine kidney V-ATPase. An extra C subunit was found in \u223c3% of complexes, whereas \u223c1.6% of complexes bound mEAK-7, a protein with proposed roles in dauer formation in nematodes and mTOR signaling in mammals. High-resolution cryoEM of porcine kidney V-ATPase with recombinant mEAK-7 showed that mEAK-7\u2019s TLDc domain interacts with V-ATPase\u2019s stator, whereas its C-terminal \u03b1 helix binds V-ATPase\u2019s rotor. This crosslink would be expected to inhibit rotary catalysis. However, unlike the yeast TLDc protein Oxr1p, exogenous mEAK-7 does not inhibit V-ATPase and mEAK-7 overexpression in cells does not alter lysosomal or phagosomal pH. Instead, cryoEM suggests that the mEAK-7:V-ATPase interaction is disrupted by ATP-induced rotation of the rotor. Comparison of Oxr1p and mEAK-7 binding explains this difference. These results show that V-ATPase binding by TLDc domain proteins can lead to effects ranging from strong inhibition to formation of labile interactions that are sensitive to the enzyme\u2019s activity. ATP hydrolysis in the A3B3 subcomplex of the V1 region induces rotation of the rotor subcomplex, which contains subunits D, F, d, and a ring of membrane-embedded c subunits are large, membrane-embedded protein complexes that function as proton pumps in eukaryotic cells. V-ATPase activity is essential for acidification of numerous intracellular compartments including endosomes, lysosomes, and secretory vesicles . In somesubunits . In the the ring . Rotatiothe ring , station regions , with su1 region and subuociation .V-ATPase\u2013driven acidification is necessary for targeting and post-translational modification of proteins in the Golgi , degradaV-ATPases interact with other proteins in the cell . In partAnalysis of V-ATPase from kidney tissue identified ARHGEF7, DMXL1, EZR, NCOA7, OXR1, RPS6KA3, SNX27, and nine subunits of the CCT complex as V-ATPase associated proteins . Two of 1 and VO regions .To identify low abundance complexes between mammalian V-ATPase and its binding partners, we first determined the subunit composition and structure of V-ATPase prepared from porcine kidney. We then subjected images of this complex to an exhaustive 3D classification strategy designed to detect low-population structures. This procedure revealed a small population of V-ATPase complexes with super-stoichiometric occupancy of the C subunit, and a second small population of complexes with an additional density bound to the catalytic Aignaling and an mmEAK-7 has been proposed to activate the mTOR pathway but was not previously suggested to interact with V-ATPase , 2019. T1 region, and subunits a1, c, c\u2033, d1, e2, RNAseK, ATP6AP1/Ac45, and ATP6AP2/PRR in the VO region Fig 1A). O region . AlthougO region . DespiteO region , the speMass spectrometry analysis of trypsin digested V-ATPase sample.Table S1 Mass spectrometry quantification of V-ATPase subunits.Table S2 left and 1 regions at 3.6\u20134.0 \u00c5 resolution and the VO regions at 3.7\u20135.8 \u00c5 resolution . This density appears to correspond to an additional copy of subunit C . This density abuts subunit B, subunit A, and peripheral stalk 3. The shape of the density does not correspond to any of the known subunits of V-ATPase. It is not clear if the fraction of particle images with this additional density represents the true fraction of V-ATPase complexes in cells with this protein bound, or if the interaction is more common but is disrupted during purification of V-ATPase. The list of proteins identified by mass spectrometry of the preparation (Table S1) was inspected to identify candidate proteins that could explain the density. After excluding V-ATPase subunits, the most abundant \u223c50-kD proteins based on iBAQ scores (intensity-based absolute quantification) were the \u03b1 and \u03b2 subunits of mitochondrial ATP synthase followed by mEAK-7. Structures are known for both subunit \u03b1 and \u03b2 from ATP synthase , which regulates dauer formation in nematodes , followed by a region resembling an EF-hand domain (cyan), a TLDc domain , and a C-terminal \u03b1 helix that is separated from the rest of the protein by an extended linker region (red) . The N tleft) as well as a B1 subunit (right), facilitated mainly by hydrophobic interactions. This \u03b1 helix is formed from sequence in the protein that was previously proposed to bind mTOR . Binding involving subunit A occurs through apparent ionic interactions between Thr354 and Gln317 from mEAK-7 with backbone atoms from residues Arg553 and Thr557 from subunit A, respectively, and hydrophobic interactions between Pro316 from mEAK-7 and Ala559 from subunit A (middle). Residues Gly276 and His277 from the TLDc domain of mEAK-7 interact with backbone atoms from Phe481 and Pro482 of subunit B1 (right). Together the TLDc domain and C-terminal \u03b1 helix form a pincer-like grip around B1 confirmed the lysosomal localization of the protein, as shown previously (lower). Endogenous mEAK-7 expression in HEK293T cells is nearly undetectable . Whereas 3D classification identified a population comprising \u223c25% of particle images where the density for mEAK-7\u2019s N-terminal domain and TLDc domain are weak and poorly resolved, suggesting that they are loosely attached via the TLDc domain, density for mEAK-7\u2019s C-terminal \u03b1 helix remains strong and well resolved even in this 3D class . In contrast, when the C-terminal \u03b1 helix is truncated most V-ATPase complexes in rotational State 2 do not bind mEAK-7 (upper middle). Only \u223c22% of particle images showed any density for the N-terminal domain and TLDc domain of mEAK-7 when the C-terminal \u03b1 helix was truncated, and even this density was weak and poorly resolved (lower middle). These experiments indicate that mEAK-7\u2019s C-terminal \u03b1 helix is important for the tight binding of V-ATPase by mEAK-7.To understand the relative importance of mEAK-7\u2019s TLDc domain and C-terminal \u03b1 helix for V-ATPase binding, we prepared a construct of mEAK-7 with the C-terminal \u03b1 helix removed by truncation at residue Ser415. This construct was expressed heterologously in is state , left. Wupper left). In contrast, with ATP added a population of particles appeared with mEAK-7 entirely absent in rotational State 2 (upper right). In this state, even density for the C-terminal \u03b1 helix is missing . Thus, it appears that rotation of the rotor, driven by ATP hydrolysis can result in the displacement of mEAK-7 from V-ATPase and disruption of the cross-link between the enzyme\u2019s rotor and stator. Despite several biochemical experiments, we were unable to demonstrate that mEAK-7 can be fully dissociated from V-ATPase by the addition of ATP to the complex, including experiments where we washed immobilized V-ATPase:mEAK-7 extensively with a buffer containing ATP before elution of V-ATPase. These experiments suggest that mEAK-7 can remain associated to V-ATPase, either by its TLDc domain or its C-terminal \u03b1 helix, during ATP hydrolysis.The observation that mEAK-7, which crosslinks the rotor and stator of V-ATPase in the structure, does not affect the enzyme\u2019s activity in vitro or in cells suggests that the crosslink is broken during rotary catalysis. To determine the effect of ATP hydrolysis on mEAK-7 crosslinking of the rotor and the stator, V-ATPase was mixed with a \u223c20\u00d7 molar excess of mEAK-7, ATP was added to 10 mM and mixed, and cryoEM grids were frozen within 5 s. Given the concentration of V-ATPase (20 mg/ml) and the enzyme\u2019s specific ATPase activity (2.9 \u00b1 0.72 \u03bcmol ATP/min/mg), these conditions ensure that the grids were frozen before the supply of ATP was consumed. In the absence of ATP, it was impossible to find a 3D class in rotational State 2 that lacked density for mEAK-7 completely and density for mEAK-7\u2019s C-terminal \u03b1 helix was always clear , an article was published showing that the yeast TLDc protein Oxr1p binds to the yeast V1 complex, releasing subunit H and promoting dissociation of V1 from VO (1 complex (1:Oxr1p is inhibited even though subunit H is absent (left) and mEAK-7 (middle) shows notable differences in their interactions with V-ATPase: Oxr1p binds the enzyme in rotational State 1, whereas mEAK-7 binds it in rotational State 2. Furthermore, Oxr1p attaches to peripheral stalk 2, whereas mEAK-7 attaches to peripheral stalk 3. Most notably, Oxr1p binding forces the peripheral stalk 2 into a highly strained conformation (left) that is not seen in any of the peripheral stalks in V-ATPase with mEAK-7 (center). This strained peripheral stalk explains why Oxr1p binding promotes dissociation of V1 from VO, whereas mEAK-7 binding does not. Recent structures of yeast V1 and V1\u2206C complexes show a sharp bending of peripheral stalk 2 induced by interaction with subunit H (right). The interaction of subunit H with peripheral stalk 2 and the resulting bending of the peripheral stalk was proposed to be responsible for inhibiting ATP hydrolysis of V1, as disruption of the interaction prevents subunit H from inhibiting ATP hydrolysis was added to the membrane fraction at 8 ml/g and resuspended with a Dounce homogenizer. Approximately 6 g of membranes were used for each preparation. The detergent glycol diosgenin (GDN) was added to 1% (wt/vol) and incubated with gentle mixing overnight. After solubilization, V-ATPase purification and assay was performed with no added lipids at 37\u00b0C as described previously . All subsequent steps were performed at 4\u00b0C. Each kidney was dissected to separate the softer cortex and medulla tissue from the more rigid pelvis tissue. The cortex and medulla tissue were blended with homogenization buffer for 1 min in a total volume of \u223c500 ml for four kidneys at high speed followed by a 10-s pause and an additional 1 min of blending. Cell debris was removed by centrifugation at 800eviously , except eviously . BrieflySus scrofa (UniProt ID: A0A4X1T484) was synthesized in a pET28a(+) vector with an N-terminal 6\u00d7 His tag (GenScript). The codon optimized sequence for mEAK-7 was:Recombinant mEAK-7 from GGGAATTCAAAAAGTAGGTCTGGACAAGGTCTTTGCAGCCGTTTCCTGCCGGAGGAACAAGCGGAAGTGGACGGTCTGTTCGATGCACTCAGCTCTGAGAAGCTGTCCTCACGGACCAGCCCGCGTAGCTTTAGCCTGCAAGCGCTGAAATCTCACGTAGGCGAAGCGTTGCCACCGGAGATGGTTACGCGTCTGTTCGAGGGTATGCGTCGTGCGGATCCGACAGGCAAGGCGACCGGTCCGAGCGCACGCATCAGCCAGGAGCAATTTACCTTGAGTATGAGCCATTTGCTGCGCGGCAGCAGCGAGGAAAAATCCTTGGTTATTCTGGCAATGGCTGCCGCTACCGATGGCCCAGCGGAAGCCCGTGAGGTTCTGCGCTTCACGGAAGACCTGGTGGGCAGCGTCGTGCATGTCTTACACTACCGCCAAGAGCTGCGCGGTTGGACCCAGAAACAGGCCTCTGGTTCCCCGCCTCGTGTTCAGGCGTTGGCGGCACAATTATTCTCCGAGCTGAAGCTGCAGGACGGCGAGAAGCTGCCGGGTCCGCAGCGTCTGGACTGCGATTGTGATCGTGCAGTCGTGGAAGCGTGGCTGTTCCGCGCTCCGCATGTTGCAACCTTTCTGTCCGTGGTGATTCATCAGGGGTTTCGCTTGCTGCGCTCCAGCCTGGACTTGGCGACTCTGCTGCCAGAACGTCAAGTTGACCGAGGCCGTGAATTTGCGTCGCTGCTGGACGTGCTGAGCGTTGCCTATATCAACAGTCACCTGCCGCGTGATCTCCGTCATCGTTGGCGTCTCCTTTTCGCCACGGCTCTGCACGGTCACAGCTTTGCTCAATTGTGCGGTCGTATCACCCAGCGCGGCCCGTGCGTGGTGCTGTTGGAAGATCAGGATGGCCACGTTTTTGGCGGCTTCGCGTCTTGTAGCTGGGAAGTGAAACCGCAGTTTCAGGGTGACAGCAAGTGCTTTCTTTTCTCGATCTGCCCGGCTATGGCGGTTTACACCTGCACCGGGTATAACGATCATTACATGTATCTGAATCATGGTCAGCAGACCATTCCGAATGGTCTGGGTATGGGTGGTCAACACAACTACTTCGGTCTGTGGGTTGACGTTGATTTTGGTAAAGGTCACTCCAAAGCAAAACCGACCTGTACCACGTACAGCAGCCCACAACTGTCGGCTCAAGAGGACTTCCGCTTCGAAAAGATGGAAGTTTGGGCAGTGGGCGACCCGTCTGTCACTCAACCGGCGAAAAGCAGCAAGTCCATCCTGGACGGCGACCCGGAGGCGCAAATTCTGCTTGAGGCGAGCGGCAGAAGCCGTCACTCCGAAGGTTTGCGTGCGGTGCCGGAGGACGATTAA.\u22121 kanamycin (Sigma-Aldrich) and grown overnight at 37\u00b0C. A transformed colony was used to inoculate a starter culture containing 10 ml of 2xYT medium supplemented with 50 \u03bcg ml\u22121 kanamycin. The starter culture was grown overnight at 37\u00b0C with shaking at 240 rpm, and 10 ml of this culture was used to inoculate 1 liter of 2xTY medium supplemented with 50 \u03bcg ml\u22121 kanamycin. The culture was grown at 37\u00b0C with shaking at 240 rpm to an OD of \u223c0.8, with the temperature then reduced to 7\u00b0C and protein expression was induced overnight by addition of 1 mM isopropyl \u03b2-D-1-thiogalactopyranoside (IPTG) (Fisher). All further steps were performed at 4\u00b0C. Cells were harvested by centrifugation at 4,000g for 15 min and the cell pellet was resuspended in PBS and centrifuged again at 4,000g for 30 min. The cell pellet was then resuspended in lysis buffer at \u223c8 ml/g of wet cell pellet. The resuspended cells were lysed by sonication (Q Sonica Q500) with 2 s on, 2 s off at 30% amplitude for 10 min, and cell debris was removed by centrifugation at 130,000g for 45 min with a Type 70 Ti Rotor (Beckman Coulter). The supernatant was filtered through a 0.22 \u03bcm filter (Sigma-Aldrich) and loaded onto a HisTrap HP 5 ml column previously equilibrated with HisTrap buffer . The column was washed with 10 column volumes (CV) of HisTrap buffer followed by elution with five CV of HisTrap buffer with imidazole added to 300 mM. Eluted protein was concentrated with a 10-kD MWCO concentrator (Amicon) to \u223c500 \u03bcl before two rounds of gel filtration, first with a Superose 6 Increase 10/300 column and then a Superdex 75 10/300 column. The purified protein was pooled, flash frozen, and stored at \u221280\u00b0C for subsequent use.ArcticExpress competent cells (Agilent) were transformed with the plasmid and plated onto Luria broth (LB) agar supplemented with 50 \u03bcg ml2 at 50 mM (0.4 \u03bcl) was applied on the grid, and immediately before plunge freezing, 1.6 \u03bcl of V-ATPase:mEAK-7 mixture was added and mixed by pipetting for 2 s. This procedure results in a final concentration of 10 mM ATP and requires a total of 5 s from mixing to plunge freezing.Purified V-ATPase was concentrated to \u223c20 mg/ml and 1.2 \u03bcl was applied onto the copper side of nanofabricated gold grids in the e\u2212/\u00c52, were collected. For V-ATPase with recombinant mEAK-7, mEAK-7\u0394Cterm, mEAK-7 plus ATP, and mEAK-7 plus EDTA with EGTA, cryoEM data were acquired with the same electron microscope. However, for these datasets, a lower magnification was used with a calibrated pixel size of 1.3225 \u00c5/pixel, and 5,142, 2,013, 5,498, and 2,468 movies consisting of 29 exposure fractions each with total exposures of 36, 37, 44, and 41 e\u2212/\u00c52, respectively, were collected. For the V-ATPase with mEAK-7 and calcium, a Thermo Fisher Scientific Glacios electron microscope and Falcon 4 camera with Selectris X energy filter was used. The slit width was 10 eV, and aberration-free image shift was used during data collection. The calibrated pixel size was 0.895 \u00c5/pixel and 3,209 movies with 1,288 frames in EER mode .For V-ATPase alone, movies were aligned with UCSF MotionCor2 through elion v3 and proceference . 2D claseference . This coolishing and tranRelion signal subtraction and focused classification strategy and was applied to the map. Map-to-model FSC was calculated with proc3d in EMAN (All raw (WIFF and WIFF.SCAN) files were saved in ProHits . mzXML fiz.org/) .5 cells/ml and were transfected with FuGENE 6 (Promega) transfection reagent 24 h later. RAW264.7 cells were seeded on 18 mm glass coverslips at a density of 5 \u00d7 105 cells/ml and transfected 24 h later with FuGENE HD. Cells were transfected with 3 \u03bcl of FuGENE per 1 \u03bcg of total plasmid. Both cell types were imaged 18\u201324 h post-transfection.The sequence for mEAK-7 was amplified from the pET28a(+) vector from GenScript for Gibson assembly (New England Biolabs), cloning into the pmCherry-N1 (Clonetech) expression vector for mammalian cells with the forward primer GTCGCCACCATGgggaattcaaaaagtaggtctggacaaggtctttgcagccgtttc and the reverse primer CTCGCCCTTGCTCACatcgtcctccggcaccgcacgcaaaccttcggagtgacggcttct, which maintains the C-terminal mCherry tag. The reverse primer CTCGCCCTTGCTCACttaatcgtcctccggcaccgcacgcaaaccttcggagtgacggct was used to create a plasmid with a stop codon before the mCherry tag for expression of mEAK-7 without the tag. HeLa and HEK293T cells were seeded on 18 mm glass coverslips at a density of 3 \u00d7 10To visualize lysosomal compartments, HeLa cells transiently expressing mEAK-7-mCherry were incubated with 250 \u03bcg/ml fluorescein isothiocyanate (FITC)-conjugated 10 kD-dextran at the time of transfection and chased with complete medium 1 h before imaging. HEK293T cells were transiently transfected with mEAK7-mCherry and LAMP1-GFP to label the lysosomes. Coverslips with cells were mounted in a Chamlide magnetic chamber, incubated at 37\u00b0C in HBSS medium, and visualized with a Zeiss Axiovert 200 M confocal microscope operated by Volocity v6.3 software. Images were acquired using a 63\u00d7/1.4 NA oil objective (Zeiss) with an additional 1.5\u00d7 magnifying lens and processed using Volocity v6.3 and Adobe Illustrator.+ solutions at different pH values , containing 10 \u03bcM nigericin and 5 \u03bcM monensin. Cells were imaged 5 min after the addition of each solution to obtain the 490 nm/440 nm fluorescence ratio corresponding to each pH standard. A calibration curve was prepared from a least-squares fit of mean background-subtracted fluorescence ratios as a function of pH. The pH measurements were compared with an unpaired two-tailed t test.To measure pH of lysosomes, HEK293T cells transiently transfected with unlabelled mEAK-7 and PLC\u03b4-PH-RFP were incubated with 250 \u03bcg/ml FITC-conjugated 10-kD dextran at the time of transfection and chased with complete medium 1 h before imaging to visualize lysosomal compartments. RAW264.7 cells co-transfected with unlabelled mEAK-7 and PLC\u03b4-PH-RFP were incubated with FITC-conjugated zymosan particles, which were taken up into phagosomes, 1 h before imaging and were chased with complete medium 30 min before imaging to wash away unbound particles. Steady-state lysosomal and phagosomal pH were determined by exciting FITC labelled lysosome/phagosomes at 481 \u00b1 15 nm and 436 \u00b1 20 nm, respectively, and collecting emitted light at 520 \u00b1 35 nm. The pH-dependent fluorescence intensity of FITC when excited at \u223c490 nm was used as described below to determine the steady-state pH of lysosomes. The relatively pH-insensitive fluorescence intensity of FITC when excited at \u223c440 nm was used to control for photobleaching during image acquisition. Multiple fields of cells were imaged for each condition, and the data were processed with Volocity v6.3. To convert fluorescence ratios to pH values, cells were sequentially subjected to isotonic KEMD-26385, EMD-26386, EMD-26387, and EMD-26388. Models have been deposited into the PDB with accession codes 7U8O, 7U8P, 7U8Q, and 7U8R. Mass spectrometry data for proteins that bind mEAK-7 were deposited into the ProteomeXchange with accession code PXD034953, through partner MassIVE with accession code MSV000089750.CryoEM maps have been deposited into the EMDB with accession codes"} +{"text": "The second near-infrared (NIR-II) window is a fundamental modality for deep-tissue in vivo imaging. However, it is challenging to synthesize NIR-II probes with high quantum yields (QYs), good biocompatibility, satisfactory pharmacokinetics, and tunable biological properties. Conventional long-wavelength probes, such as inorganic probes and organic dyes (which contain large \u03c0-conjugated groups), exhibit poor biosafety, low QYs, and/or uncontrollable pharmacokinetic properties. Herein, we present a bioengineering strategy that can replace the conventional chemical synthesis methods for generating NIR-II contrast agents. We use a genetic engineering technique to obtain a series of albumin fragments and recombinant proteins containing one or multiple domains that form covalent bonds with chloro-containing cyanine dyes. These albumin variants protect the inserted dyes and remarkably enhance their brightness. The albumin variants can also be genetically edited to develop size-tunable complexes with precisely tailored pharmacokinetics. The proteins can also be conjugated to biofunctional molecules without impacting the complexed dyes. This combination of albumin mutants and clinically-used cyanine dyes can help widen the clinical application prospects of NIR-II fluorophores. It is currently difficult to synthesise NIR-II probes with good quantum yields, biocompatibility and pharmacokinetics. Here the authors report a strategy to alter these properties by modifying the protein coatings with biofunctional molecules, and generate long-wavelength fluorophores for in vivo imaging. The development of high-quality NIR-II fluorophores is essential to realizing deep bioimaging11 and facilitating clinical translation. For example, clinically used cyanine dyes14 were discovered to possess a tail peak in the NIR-II window when viewed using an InGaAs camera20. However, the majority of organic NIR-II fluorophores exhibit very low fluorescence quantum yields (QYs)19 and/or poor pharmacokinetic properties. Moreover, multiple and complex synthetic steps are followed to synthesize these fluorophores22. The photoluminescence of small molecules is controlled by their energy gap, which depends on their chemical structure through \u03c0-conjugated groups23. Hence, long-wavelength fluorophores bear large \u03c0-conjugated groups 26. However, fluorophores with large \u03c0-conjugated groups usually have inherently low QYs and poor pharmacokinetics18.Near-infrared II (NIR-II) fluorescence imaging is preferrable to near-infrared I (NIR-I) imaging as this window provides reduced tissue scattering and autofluorescence, thus achieving deeper tissue penetration with superior contrast and resolution3. Additionally, large conjugated fluorophores are usually hydrophobic and do not exhibit satisfying pharmacokinetic properties22. In recent years, efforts have been made to synthesize rigid structures bearing electron-withdrawing groups at the center of the scaffold and/or to introduce shielding groups to reduce collisional quenching29. For example, rigid cyclohexanol structures have been introduced at the center of cyanine/polymethine dyes to produce improved dye variants. Researchers introduced a rigid alkyl thiophene moiety to increase the dihedral angle in a donor-acceptor-donor (D-A-D) dye to improve QYs19. Shielding units containing long polyethylene glycol (PEG) groups have also been used to develop good-performance D-A-D dyes and reduce collisional quenching. Some researchers have introduced dialkoxy-substituted benzene- and/or fluorene-based groups as the shielding units to develop S-D-A-D-S fluorophores that exhibit relatively high QYs. By modifying the PEG and S groups, the dispersion of these molecules in solution can be improved and nonradiative decay caused by collisional quenching/\u03c0-\u03c0 stacking can be reduced31.Large \u03c0-bridges can potentially induce nonradiative decay through vibrational relaxation, collisional quenching, and/or \u03c0-\u03c0 stacking-induced quenching. Molecular simulation studies revealed that the low QYs exhibited by fluorophores containing large \u03c0-bridges could be attributed to the properties of the excited states of the \u03c0-bridges. The excited states can be directly attacked and subsequently quenched by water molecules and they are also prone to oxidation7 that can potentially influence the QY of the dyes. Moreover, some cyanine/polymethine dyes, such as the clinically used indocyanine green (ICG), do not possess modifiable units in their scaffolds and so cannot be easily conjugated to other moieties. Most importantly, achieving a good-performance fluorophore with high QY, biosafety, clinical translation potential, and satisfying pharmacokinetic properties as well as terrific biofunction is extremely challenging. We have previously reported that cyanine dyes can \u201chitchhike\u201d with serum albumin in vivo to produce ultra-bright NIR-II fluorophores with improved pharmacokinetics16. The cyanine dyes inserted into a hydrophobic pocket of albumin, which stabilized the dyes and so reduced nonradiative decay and synergistically increased the extent of \u03c0-conjugation. The protein shell also protected the dyes from collisional quenching and provided biocompatibility. Inspired by this work, here we screen high-affinity recombinant subunits of albumin that could be used to generate a series of editable protein shells to chaperone dyes. Using yeast to express fragments of albumin, we successfully identify the exact binding domain for cyanine dyes. This allows us to re-engineer a series of minimal subunit-incorporating albumin variants. We use this genetic engineering technique to tune the size and brightness of cyanine/polymethine dye@protein complexes to identify the exact binding domain for cyanine dyes. HSA consists of three independent subdomains that contain highly homologous sequences: domain I (DI), domain II (DII), and domain III (DIII)Kd\u2009=\u20090.57\u2009nM) than HSA (Kd\u2009=\u20091.7\u2009nM) and slower dissociation (Koff\u2009=\u20090.1\u2009s\u22121) with DIII compared to HSA Fig.\u00a0. IR-783 PBS Fig.\u00a0. We furtPBS Fig.\u00a0. The resThe binding behavior and loading capacity of cyanine dyes and albumin were further studied by ultra-high performance liquid chromatography\u2013mass spectrometry (UHPLC-MS). Each DIII molecule bound only one IR-783 molecule . The results indicated that the dye-labeled peptide sequence was C(dye)C(carbamidomethylation)TESLVNR. The b and y ion series were further analyzed to identify the exact binding residue. The full mass (MS1) of the peptide was calculated to be 1770.749\u2009m/z (measurement accuracy\u2009<\u20095 ppm). The measurement accuracy for the fragments was\u2009<\u200910 ppm . The results revealed that the Cys476 residue was the potential binding site and Cl\u2013C groups and to understand the reaction mechanism , prostate-specific membrane antigen-617 (PSMA-617) and cyclic Arg-Gly-Asp (cRGD), which specifically bind to somatostatin receptor (SSTR), PSMA and integrin \u03b1V\u03b23, respectively. SH-modified TATE, PSMA-617 and cRGD were first reacted with maleimide-NHS ester. Following this, the purified products were conjugated to DIII Fig.\u00a0. The reaPichia pastoris with their 3D structures retained in DIII that binds cyanine dyes, resulting in increased brightness. Using this information, the independent DIIIa and DIIIb subunits were expressed by recombinant ned Fig.\u00a041\u201343. Thnts Fig.\u00a0. To idennts Fig.\u00a0. The brints Fig.\u00a0. Thus, tSince DIIIa is a fragment of DIII with approximately half its molecule weight, we hypothesized that the rate of renal excretion of IR-783@DIIIa would be higher than that of IR-783@DIII. The pharmacokinetic properties of IR-783@DIIIa were evaluated in healthy mice Fig.\u00a0. As expe32. The three domains of albumin are homologous of endogenous albumin if the DI, DII, or DIII units are substituted with each other. Hence, we re-engineered a recombinant HSA plasmid with 3 copies of the DIII sequence (triple DIII or TDIII) and (glycine-glycine-glycine-glycine-serine)2 (-[Gly-Gly-Gly-Gly-Ser]2-). After the recombinant Pichia pastoris clones were identified by polymerase chain reaction (PCR), TDIII was expressed in Pichia pastoris and verified by SDS-PAGE without altering the inserted cyanine dyes. However, the ideal pharmacokinetics of a fluorophore depend on the imaging application. For example, renally cleared probes are preferrable for molecular imaging due to their safety, while long-circulating probes are useful for vascular or lymphatic imaging. Long-circulating probes are also preferred for imaging-guided surgery. As dye@DIII/DIIIa are rapidly cleared through the renal system, they cannot function as long-circulating probes. IR-783@albumin was shown to be a long-circulating probe with a circulation time of 19 days, comparable to that of endogenous albuminII) Fig.\u00a0. To retaAGE Fig.\u00a0. And alsAGE Fig.\u00a0.Fig. 6Dyn\u2009=\u20095). The dye-to-protein molar ratios investigated were 0.25:1, 0.5:1, 0.75:1, 1:1, 1.5:1, 2:1, 3:1, 4:1, 5:1, 6:1, 8:1 and 16:1. The samples were then exposed to a xenon lamp (780\u2009nm) and the fluorescence intensity of each sample was detected by NIR-I (700\u2013900\u2009nm) and NIR-II (1100\u20131300\u2009nm) detectors were mixed with various molar concentrations of IR-783 NIR-II imaging was performed in mice with shaved heads, and vessels underneath the skull and skin were imaged with high resolution and imaged lymphatic vessels. The ultra-bright IR-783@TDIII was able to illuminate the tiny lymphatic vessels with a resolution of 87.8 \u03bcm per pixel Fig.\u00a0. NIR-II 16. Thus, dye@TDIII could be excellent candidates for imaging-guided surgery with translational potential.Intraoperative navigation technique is widely used in the field of medicine. Currently, various cyanine dyes are used in the clinic, but these exhibit rapid photobleaching and clearance. We hypothesized that dye@TDIII could address the existing problems in the field. We first compared IR-783@TDIII to the clinically used ICG to study the optical properties of the probes for binding cyanine dyes. To our surprise, we observed that Cl-containing cyanine dyes formed covalent bonds with endogenous or mutant albumin, while Cl-free dyes formed non-covalent bonds. Some researchers have reported that Cys34 on albumin could covalently bind with IR-78351. However, the binding site was speculated on Cys34 since it is the only free cystine on albumin. In this study, we systematically studied and discovered a binding site (Cys476) on subdomain IIIa which could also covalently bond with Cl-containing cyanine dyes, and this finding has never been reported before. To confirm our finding, we used domain III or subdomain IIIa (without Cys34) instead of full albumin to react with Cl-containing dyes, and systematically studied the nucleophilic substitution of Cys476 and Cl-containing dyes. Based on this finding, we designed a series of recombinant albumin variants to chaperone Cl-containing dyes for in vivo NIR-II bioimaging. Furthermore, we determined that the interaction between cyanine dyes and albumin occurs in two independent stages and studied the mechanism of brightness enhancement. In the first stage, dyes bind to albumin or albumin mutants through non-covalent bonds, resulting in increased brightness though strengthened TICT. In the second stage, a \u201cclasp\u201d formed by the SH\u2013 group of Cys476 and the Cl\u2013C group of Cl-containing dyes stabilizes the dyes through nucleophilic substitution. One group has observed some absorption spectra shifts but with no explanation52, while, according to this phenomenon, another group provided with a possible explanation that a noncovalent adduct would be slowly transformed covalent51. However, they did not provide with more evidence for the speculation.The discovery of an albumin chaperone that functions as a brightness amplifierThis discovery provides a platform for the development of dyes that can attach themselves to serum albumin for improved pharmacokinetics. Cyanine dyes protected with a shell of albumin mutant or variant were shown to have enhanced optical properties. We also showed that the shells can be modified by fusion with a biofunctional peptide or conjugation with a small molecule. Additionally, the sizes of the bioengineered shells can be tuned to tailor the pharmacokinetic properties and clearance pathway. These excellent properties make the molecules suitable candidates for use in the fields of molecular imaging and imaging navigated surgery. In this study, we presented several applications in biofunctional imaging and imaging-guided surgery using complexes composed of bioengineered albumin fragments of various sizes with tailored pharmacokinetics and clearance pathways. We also designed an albumin mimic composed of three tandem DIII, which showed the highest brightness enhancement and generated dye complexes with albumin-like in vivo behavior.In conclusion, we demonstrated an alternative strategy to conventional chemical modification techniques to develop targeted NIR-II probes. We developed a facile strategy to design bright and editable NIR-II fluorophores that exhibit tailored pharmacokinetic properties to address the problems of existing NIR-II probes. Our innovation uses existing cyanine dyes with peak emission at ~800\u2009nm, which could achieve full color imaging in both the NIR-I and NIR-II windows to provide accurate diagnosis and guide surgery. We believe that development of cyanine dyes with peak emission at 1000\u20131500\u2009nm will further promote our genetic engineering strategy. More importantly, this genetic engineering strategy would provide us with a platform for modifying NIR-II dyes and exhibit an additional perspective to design dyes. Our strategy can help the design and development of NIR-II probes with clinical translation potential in the near future.The investigators adhered fully to the \u201cGuide for the Care and Use of Animals\u201d by the NIH Clinical Center Animal Care and Use Committee (NIHACUC), and all animal work was conducted in compliance with protocols approved by the NIHACUC.The aimed sequences of DIIIa and DIIIb were designed as follows. To make sure the connection part of DIIIa and DIIIb including, we designed an overlay segment with several amino acid residues (highlighted by blue color) on each of them.The amino acid sequence of DIIIa (381\u2013494):LEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSThe amino acid sequence of DIIIb (490\u2013585):KTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKATo make sure the subdomains were successfully expressed and purified , we used two strategies to achieve the small proteins: His tag and GST tag. Typically, a thrombin cleavage site was insert between tag and the targeting protein, respectively.GST tag sequence:MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLEVLFQGPLGSPEFPGRLERPHRDHence, the GST-LVPRGS-DIIIa/DIIIb:MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLEVLFQGPLGSPEFPGRLERPHRDLVPRGSLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVS*MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLEVLFQGPLGSPEFPGRLERPHRDLVPRGSKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKA*After achieved the above corresponding pPIC9K recombinant plasmid, we transformed them into GS115 strain. Specifically, the pPIC9K-DIIIa/DIIIb plasmid was linearized with XhoI and EcoR1, respectively. Then, GS115 strain was transformed by electroporation. Recombinant Pichia clones were isolated on RD plate. After the clones appeared in the plate, washed the clones with sterile water, and put them onto YPD\u2009+\u2009GS115 (1000\u2009\u00b5g/ml) plate. Recombinant Pichia clones were identified by PCR.PCR primes, suggest by Invitrogen yeast protocol, are documented as below:5\u2032AOX: 5\u2032 ctgctgatagcctaacgttc 3\u20323\u2032AOX: 5\u2032 gctgatcaggagcaagctcg 3\u2032\u22121 in 25\u2009mM sodium phosphate, 100\u2009mM sodium sulphate, 0.05 % (w / v) sodium azide, pH 7.0. The column was TSK G3000SWXL (Tosoh Bioscience), and the eluted proteins were detected by UV at 280\u2009nm compared to an HSA standard. Finally, proteins were analyzed using SDS-PAGE in PhastGelTM electrophoresis system and appropriate media.Then, yeast (1\u2009mL) was inoculated into 10\u2009ml BMMD medium (2 % w / v glucose), and grown for 48\u2009h on a rotary shaker of 250 r.p.m. at 30\u2009\u00b0C. After that, 4\u2009ml of each culture was inoculated in 2\u2009\u00d7\u2009200\u2009ml BMMD media to grow for 120\u2009h on a rotary shaker of 250\u2009r.p.m at 30\u2009\u00b0C. Proteins produced by recombinant Pichia were purified using the AlbuPure matrix (ProMetic BioSciences). Briefly, the supernatant was collected after centrifugation and filtered with 0.2\u2009\u00b5m vacuum filter membranes (Millipore). Subsequently, a Pall Filtron LV system equipped with an Omega 10\u2009kDa filter was employed to further concentrate the filtered supernatant. The column was equilibrated with 50\u2009mM sodium acetate pH 5.3 and loaded with the concentrated supernatant. After that, the column was washed with 10 column volume (CV) of equilibration buffer and 50\u2009mM ammonium acetate pH 8.0 (10 CV) to elute the unbound fraction. The bound protein was eluted with a series of buffers, 50\u2009mM ammonium acetate, 10\u2009mM octanoate pH 8.0, 50\u2009mM ammonium acetate, 30\u2009mM sodium octanoate pH 8.0 or 200\u2009mM potassium thiocyanate, respectively. Then, the eluted protein was concentrated and filtered against 10 CV of 50\u2009mM NaCl by Vivaspin20 10\u2009kDa PES (Sartorius). GP- HPLC was performed to quantify the HSA variants. Samples were chromatographed at a flow rate of 1\u2009ml\u2009minThe DNA sequence of TDIII:ATGAAGTGTTGTGCTGCTGCTGACCCACACGAATGTTACGCTAAGGTTTTCGACGAGTTCAAGCCATTGGTTGAGGAACCACAGAACCTGATCAAGCAGAACTGTGAGTTGTTCGAGCAGCTGGGTGAGTACAAGTTCCAGAACGCTTTGTTGGTCAGATACACCAAGAAGGTCCCACAGGTTTCCACTCCAACCTTGGTTGAAGTCTCCAGAAACCTTGGTAAGGTCGGTTCCAAGTGTTGTAAGCACCCTGAGGCTAAGAGAATGCCATGTGCTGAAGATTACTTGTCCGTCGTCTTGAACCAGTTGTGCGTCTTGCACGAAAAGACTCCAGTTTCCGACAGAGTTACCAAGTGCTGTACTGAGTCCTTGGTCAACAGACGTCCATGTTTCTCTGCTTTGGAGGTCGACGAAACCTACGTGCCAAAAGAGTTCAACGCTGAGACTTTCACTTTCCACGCTGACATCTGTACCCTGTCCGAAAAAGAGAGACAGATCAAGAAGCAGACTGCCTTGGTCGAGTTGGTTAAGCACAAGCCAAAGGCTACCAAAGAGCAGTTGAAGGCTGTTATGGATGACTTCGCTGCCTTCGTTGAGAAGTGTTGCAAGGCTTTGGAAAAGTGTTGCGCAGCTGCAGATCCTCATGAGTGTTACGCCAAAGTCTTTGATGAGTTTAAGCCCCTGGTCGAAGAACCCCAAAACTTGATTAAGCAAAACTGCGAACTGTTTGAGCAATTGGGCGAGTACAAATTTCAAAACGCCCTGCTGGTTAGGTACACTAAGAAAGTTCCTCAGGTGTCTACCCCAACTTTGGTCGAGGTTTCTAGGAACTTGGGTAAAGTGGGTTCTAAGTGCTGCAAACATCCAGAGGCCAAAAGAATGCCTTGCGCAGAGGACTACTTGTCTGTTGTTCTGAACCAGCTTTGTGTGCTGCACGAGAAAACCCCAGTCTCTGATAGAGTCACCAAATGTTGCACCGAGTCTCTGGTTAACCGTAGACCATGTTTTTCCGCCTTGGAAGTGGATGAGACTTACGTCCCTAAAGAGTTTAACGCCGAAACCTTTACCTTTCACGCCGATATCTGCACTTTGTCTGAGAAAGAGCGTCAGATTAAGAAACAAACCGCTCTGGTCGAACTTGTCAAGCACAAACCTAAAGCCACAAAAGAACAACTGAAGGCCGTCATGGACGATTTTGCCGCTTTTGTTGAGAAATGCTGTAAGGCCCTTGAAAAGTGCTGTGCCGCAGCCGATCCACATGAATGCTATGCTAAAGTGTTCGATGAGTTCAAACCACTTGTGGAAGAACCTCAGAATCTTATCAAACAAAATTGCGAGCTTTTCGAACAGTTGGGAGAGTATAAGTTTCAAAATGCCTTGTTGGTGCGTTACACAAAAAAGGTGCCTCAAGTCTCCACTCCTACTCTGGTTGAGGTTTCCCGTAACCTGGGAAAAGTTGGTAGCAAATGCTGCAAGCACCCCGAAGCTAAACGTATGCCTTGTGCCGAGGATTATCTGAGCGTTGTCTTGAATCAGCTGTGTGTCCTTCATGAGAAAACTCCCGTTTCTGACCGTGTCACTAAGTGTTGTACCGAAAGCTTGGTGAACAGAAGGCCTTGCTTTTCTGCTCTGGAAGTTGACGAGACATATGTTCCCAAAGAGTTCAACGCAGAAACATTCACATTTCATGCAGACATCTGCACACTTAGCGAGAAAGAAAGGCAAATCAAAAAGCAAACAGCCCTGGTTGAGCTGGTCAAACATAAGCCCAAGGCCACAAAAGAGCAGCTTAAAGCAGTAATGGACGATTTCGCTGCATTTGTCGAAAAGTGTTGTAAAGCCTAAGCGGCCGCThe amino acid sequence of TDIII MKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKALEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKALEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKA*The His tag was used in this sequence design since the TDIII is a big protein with molecule weight similarly equal to wild type albumin.The amino acid sequence fused with cleavage site for thrombin and His tag (TDIII-LVPRGS-His tag).MKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKALEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKALEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKALVPRGSHHHHHH.Most of the cyanine dyes were purchased from Sigma-Aldrich. IR-12N3 was purchased from Nirmidas Biotech Co. Indocyanine Green for human injection was purchased from Dandong Yichuang Pharmaceutical Co., LTD. PBS was purchased from HyClone. Human Serum Albumin (HSA) and Bovine Serum Albumin (BSA) were purchased from Sigma-Aldrich. Fetal Bovine Serum (FBS) was purchased from Neuromics. Sucrose was purchased from Sigma-Aldrich.Binding affinity of cyanine dyes with albumin variants was determined by biolayer interferometry (BLI) by interaction using an Octet Red96 system (fort\u00e9Bio), respectively. Because of the same protocol, binding affinity of IR-783 with HSA was taken as an example. IR-783 powder was freshly dissolved in anhydrous DMSO (26.7\u2009mM). Then, IR-783 is serially diluted into different concentrations by PBS. The assay protocol was briefly described as follows. After washing with 1\u2009\u00d7\u2009PBS for 60\u2009s, biotinylated HSA (1\u2009\u03bcg/mL) was loaded to the biosensor for 600\u2009s, and after another 60\u2009s washing, quenching with biocytin was performed for 180\u2009s. Followed by another 60\u2009s washing, association for 600\u2009s and dissociation for another 600\u2009s were performed in turn. Data was calculated and analyzed using Octet Analysis software v 7.0.Synthesis of dye@HSA and dye@HSA-domains/variants complex shared the same protocol and IR-783-DIII complex was taken as an example. DIII powder was dissolved in 1XPBS with a concentration of 20\u2009mg/mL (855\u2009\u00b5M); IR-783 powder was freshly dissolved in anhydrous DMSO (26.7\u2009mM). 500\u2009\u00b5L DIII PBS solution was first added in 500\u2009\u00b5L PBS under slight vortexing. Then, 8, 16, 32, 48, 96 \u03bcL\u00a0of\u00a0the IR-783 (26.7\u2009mM) was added into the DIII solutions, respectively . Finally, the suspension was vortexed for 30\u2009s and heated at 60\u2009\u00b0C for 10\u2009min.Since we do not know if the conjugation of TATE/PSMA-617 would impact the binding of IR-783, we attempted to add IR-783 before or after the conjugation of TATE/PSMA. Results indicated that both routes did not lead to obvious fluorescence diminishing; hence, we conclude that tailoring DIII does not impact the in vitro and in vivo behavior of the fluorophores. IR-783@DIII or DIIIa (TDIII) was reacted with maleimide-PEG-NHS ester (Sigma) at RT. for 4\u2009h under shaker. Then products were filtered with 10 k filter against PBS for 5 times to move unreacted maleimide-PEG-NHS ester. The maleimide-PEG-NHS labeled compounds were incubated with PSMA-SH and TATE-SH peptides (molar ratio of protein and peptide was 1:5), as described in our previous paper. After that, the conjugates were washed with 10\u2009K filter against PBS for 5 times.UV-Vis-NIR spectrophotometer (Cary 6000i) with background correction was employed to measure the optical absorption spectrum. Fluorescence spectrophotometry was carried out on a Hitachi F-7000 fluorescence spectrophotometer. Fluorescence microscopy was performed on an Olympus fluorescence microscope.DIII-cRGD was mixed with HCCA (\u03b1-cyano-4-hydroxycinnamic acid) in a 1:1 ratio, 2\u2009\u03bcL of sample and 2\u2009\u03bcL of HCCA. Sample matrix was mixed and loaded onto a MALDI target plate (MTP) 384 by direct droplet method. It was allowed to dry for a few minutes. MALDI-TOF mass spectrometry was performed on Autoflex maX (Bruker) instrument. Finally, the data was plotted using Graphpad 8.0.Mice were shaved using Nair hair removal cream and anesthetized using isoflurane before placing them for injection of imaging agents. For each imaging experiment, at least 3 mice were used as a parallel group cohort. All NIR-II images were collected on a Princeton InGaAs array. The excitation laser was an 808\u2009nm laser set-up. Emission was typically collected with different long pass filters. A lens set was used for obtaining tunable magnifications, ranging from 1\u00d7 (whole body) to 2.5\u00d7 (high magnification) by changing the relative position of two NIR achromats . NIR-II microscopy was built based on the previous report, and the excitation laser was a 785\u2009nm laser set-up.2, 37\u2009\u00b0C). All cells were tested to be free of mycoplasma.AR42J (from ATCC) were cultured in F-12k medium with 20% heat-inactivated FBS and 1% penicillin and streptomycin. PC3 (from ATCC), PC3-PIP (from NIBIB at NIH) and 4T1-fluc cell lines (from ATCC) were cultured in RPMI-1640 medium with 10% heat-inactivated FBS and 1% penicillin and streptomycin. Cells were grown in a humidified atmosphere (5% CO6) were inoculated to nude mice to grow subcutaneous tumor, respectively. 4T1-fluc cells (1.0\u2009\u00d7\u2009105) were inoculated in the mammary fat pad to establish the orthotopic mammary tumor model/metastatic tumor model.Nude mice , C57BL/6j mice and balb/c mice were purchased from Jackson\u2019s Laboratory . Bedding, nesting material, food, and water were provided ad libitum. Ambient temperature was controlled at 20 to 22\u2009\u00b0C with 12\u2009h light/12\u2009h dark cycles. AR42J or PC3 cells were intravenously injected with PBS, DIII, IR-783@DIII, IR-783@HSA, IR-783@DIIIa, and IR-783@TDIII, respectively. Peripheral blood was collected to analyze subtypes of lymphatic cells. Briefly, blood was collected from the treated mice on day 3, day 7 and day 14. Blood cells were enriched by centrifugation. Red blood cells were lysed using ACK lysis buffer for 10\u2009min at room temperature. Then, cells were washed twice in PBS and stained with the according antibodies for 15\u2009min. After that, cells were washed with FCS buffer (PBS buffer with 0.1% FBS), and resuspended for flow cytometric analysis. Flow cytometry was conducted on a Beckman CytoFlex S flow cytometer. Data were analyzed using FlowJo V10 version.The ground state (S0) geometries of IR-783 and ICG molecules were optimized using the B3LYP-GD3GJ/6-31\u2009G(d) method dispersion correction. The excited state (S1) properties were calculated using the time-dependent (TD) LC-BLYP*/6-31\u2009G(d) method with the polarizable continuum model (PCM). The HOMO and LUMO distributions were displayed using VMD code. All the (TD)DFT calculations were performed using Gaussian 16 software.Further information on research design is available in the\u00a0Supplementary InformationReporting SummaryDescription of Additional Supplementary FilesSuppl Movie 1Suppl Movie 2Suppl Movie 3"} +{"text": "Caenorhabditis elegans. We observed that both the catalytic (KIN-3::V5) and regulatory (KIN-10::2xMyc) subunits of the Casein Kinase II (CK2) holoenzyme complex are associated with meiotic DNA, enriched in the midvalent rings during meiotic divisions in fertilizedC. elegans oocytes.By using CRISPR/Cas9 genome-editing, we have generated epitope-tagged KIN-3 and KIN-10 expressing strains at the endogenous C-terminal loci in C. eleganscatalytic and regulatory subunits are encoded bykin-3andkin-10, respectively . We used CRISPR/Cas9 genome-editing to generate epitope-tagged KIN-3::V5 and KIN-10::2xMyc expressing strains at the endogenous C-terminal loci. By staining early embryos with commercially available epitope antibodies, we observed the localization of KIN-3::V5 and KIN-10::2xMyc in fertilizedC. elegansoocytes. During meiosis in fertilizedC. elegansoocytes, both the catalytic (KIN-3) and regulatory (KIN-10) subunits of the CK2 holoenzyme are associated with meiotic DNA, enriched around the center of the bivalent, referred to as the ring complex . It has been shown that AIR-2/Aurora B and KLP-19 localize to the ring complexes associated with meiosis I bivalent and meiosis II chromosomes. Both AIR-2 and KLP-19 are required for proper chromosome segregation duringC. elegansmeiosis . The close association of KIN-3 and KIN-10 with meiotic DNA suggests that CK2 kinase activity might influence chromosome organization and segregation during meiotic divisions in theC. elegans oocyte. In support of this, previous work has reported that depletion of CK2 results in polar body extrusion failure and extra DNA, likely due to meiotic errors in fertilizedC. elegansoocytes . A study in porcine oocytes has also shown that CK2 localizes to meiotic chromosomes and that CK2 activity is required for normal meiotic progression . Thus, CK2 function during meiotic division appears to be evolutionarily conserved.The kinase Casein Kinase II (CK2), a tetrameric holoenzyme, consists of two catalytic (CK2\u03b1) and two regulatory (CK2\u03b2) subunits . TheC. elegansCulture:All strains were derived from the wild-type Bristol N2 strain and maintained on MYOB plates seeded withEscherichia coliOP50 at 20\u00b0C.Immunostaining and Confocal Microscopy: Immunofluorescence and confocal microscopy were performed as described . For immunostaining, the following primary and secondary antibodies were used at 1:3000 dilutions: \u03b1-Myc , \u03b1-V5 , and Alexa Fluor 488 and 568 secondary antibodies . Confocal microscopy was performed using a Nikon Eclipse Ti-U microscope equipped with a Plan Apo 60\u00d71.4 NA lens, a Spinning Disk Confocal (CSU X1), and a Photometrics Evolve 512 camera. MetaMorph software was used for image acquisition and Adobe Photoshop/Illustrator 2022 for image processing.CRISPR/Cas9 Genome Editing: For genome editing, we used the co-CRISPR technique described previously . To design crRNA, we used the CRISPOR webserver . Animals were microinjected with a mixture of commercially available SpCas9 and custom-designed oligonucleotides including crRNAs at 0.4\u20130.8 \u00b5g/ml tracrRNA at 12 \u00b5g/ml, and single-stranded DNA oligonucleotides at 25\u2013100 ng/ml. After injection, we screened fordpy-10(cn64) II/+rollers in F1 progeny and genotyped F2 for the epitope-tag insertion. The genome editing was verified by Sanger Sequencing . All theC. elegansstrains generated in this study produce nearly 100% viable progeny.Single-stranded DNA oligonucleotides homologous repair templates for genome editing were as follows.GGTAAGCCTATCCCAAATCCTTTGTTGGGTCTGGACTCCACGTAAAATTTCTTTCTATTTTTTTTTTAATTTTCCTGKIN-3::V5 tag at the C-terminus (5'-3'): CATCGAATTCCGCTTCTTCTCAATCCTCCGATGCTAAAATTGACGGCGCTGGAGGTTCCGGTGGTTCTGGTGGATCCGAACAAAAACTGATATCTGAAGAAGACCTTGAGCAGAAGTTGATTAGTGAGGAGGATCTTTGAGCCACTTTCTTCCTTATTTTTGTTTTGATTTCKIN-10::2xMyc tag at the C-terminus (5'-3'): CAAAACAACACGACTCCAGCCGGGCAACAATCTGGCGGCCAGTTCAACAACTATGGTCTCGGTGGCTCTGGTGGAAGTGGAGGCTCAN2: wild-type (CGC), MTU137:kin-3(mhs464[KIN-3::V5]) I (This study), MTU598:kin-10(mhs688[KIN-10::2xMyc]) I (This study)"} +{"text": "S-adenosyl-L-methionine (SAM) L-tyrosine lyase and catalyzes the L-tyrosine C\u03b1\u2013C\u03b2 bond break to produce dehydroglycine and p-cresol while the radical SAM L-tryptophan lyase NosL cleaves the L-tryptophan C\u03b1\u2013C bond to produce 3-methylindole-2-carboxylic acid. It has been difficult to understand the features that condition one C\u2013C bond break over the other one because the two enzymes display significant primary structure similarities and presumably similar substrate-binding modes. Here, we report the crystal structure of L-tyrosine bound ThiH from Thermosinus carboxydivorans revealing an unusual protonation state of L-tyrosine upon binding. Structural comparison of ThiH with NosL and computational studies of the respective reactions they catalyze show that substrate activation is eased by tunneling effect and that subtle structural changes between the two enzymes affect, in particular, the hydrogen-atom abstraction by the 5\u00b4-deoxyadenosyl radical species, driving the difference in reaction specificity.2-iminoacetate synthase ThiH is a radical ThiH is a radical SAM L-tyrosine lyase involved in the biosynthesis of the thiazole ring of vitamin B1. Here, the authors report the crystal structure of ThiH in complex with its L-tyrosine substrate, revealing an unexpected protonation state and tunneling effect that lowers the reaction energy barrier. It enables, for example, the formation of acetyl coenzyme A by pyruvate dehydrogenase3 and pyruvate: ferredoxin oxidoreductase4 as well as the transfer of a glycoaldehyde from a ketosugar to an aldosugar by transketolase5. Dehydroglycine (DHG), also termed 2-iminoacetate, is a key precursor in the bacterial biosynthesis of the thiazole ring that constitutes the core of TPP. In aerobic prokaryotes, such as the extensively studied Bacillus subtilis, DHG is produced by the oxygen-dependent glycine oxidase7. Such glycine oxidation is not possible under anaerobic conditions and anaerobes, including the facultative bacterium Escherichia coli, use instead the 2-iminoacetate synthase ThiH that catalyzes DHG formation from L-tyrosine processing , derived from vitamin B1, is a key ubiquitous cofactorS-adenosyl-L-methionine (SAM) superfamily13 and contains one [Fe4S4] cluster that reductively cleaves SAM to form both methionine and a highly reactive 5\u00b4-deoxyadenosyl radical (5\u00b4-dA\u2022) species. Subsequently, 5\u00b4-dA\u2022 abstracts from L-tyrosine, a hydrogen atom, long proposed to be the phenolic hydrogen atom10. By analogy, the same reaction step would be catalyzed by the radical SAM tyrosine lyase HydG involved in the assembly of the [FeFe]-hydrogenase active site15. ThiH and HydG are closely related members of the radical SAM superfamily sharing 27% sequence identity16. Both enzymes catalyze the cleavage of L-tyrosine C\u03b1\u2013C\u03b2 bond to generate p-cresol16 and DHG17. The latter is used by HydG at a second active site of the enzyme to synthesize the CO and CN\u2013 diatomic ligands20 that are part of the so-called [FeFe]-hydrogenase H-cluster22. Conversely, ThiH transfers DHG to ThiG, where it is further processed to produce the thiazole ring of thiamine11.ThiH is a member of the radical 23, involved in the synthesis of the thiopeptide antibiotic nosiheptide24. NosL shares about 23% primary structure identity with them25. But, whereas ThiH and HydG cleave the L-tyrosine C\u03b1\u2013C\u03b2 bond17, NosL cleaves the L-tryptophan C\u03b1-C bond instead and produces a 3-methylindole-2-carboxylic acid (MIA)27 , an observation supported by a subsequent biochemical study28. In addition, structure-based sequence comparisons of NosL with tyrosine lyases ThiH and HydG show that, in the latter enzymes, the corresponding [L-Tyr-NH\u2022] would be formed25. Using electron paramagnetic resonance spectroscopy (EPR), a methylene-centered p-cresyl radical intermediate (p-cresyl\u2022) was trapped during L-tyrosine scission by HydG15 and, more recently, the 5\u00b4-dA\u2022 was trapped using p-coumaric acid instead of L-tyrosine29.These two tyrosine lyases are often compared to the radical SAM tryptophan lyase NosLThermosinus carboxydivorans (Tc) in complex with its L-tyrosine substrate. In this study, an unexpected substrate protonation state, supported by its interactions in our X-ray model, has been confirmed by molecular dynamics (MD) simulations. More specifically, deprotonation of the L-tyrosine hydroxyl moiety is in agreement with the very recent report that, in HydG, the ensuing p-cresyl\u2022 intermediate is most probably deprotonated as well, and corresponds to a 4-oxidobenzyl radical instead30. The description of the mechanism of direct hydrogen atom abstraction by 5\u00b4-dA\u2022 at the amino nitrogen position of L-tyrosine, obtained by using hybrid quantum mechanical / molecular mechanical (QM/MM) methods, supports a highly controlled and tight local environment, inducing a tunneling effect during hydrogen transfer that we have estimated analytically to be about 50% of the reaction barrier. Additional calculations performed on both ThiH and NosL show that this step corresponding to the radical formation [L-Tyr-NH\u2022] ([L-Trp-NH\u2022] in NosL) is crucial for the orientation of the subsequent bond cleavage step . In addition, comparison of ThiH with HydG and NosL has revealed unique structural motions, most likely responsible for specific substrate access and product release sites, showing that these enzymes work as assembly lines. Thus, by combining X-ray crystallography and computational chemistry, we provide a rarely achieved mechanistic insight into the hydrogen-atom abstraction step catalyzed by radical SAM enzymes and underscore how subtle changes at the active site afford both substrate selectivity and chemical regiospecificity.Here, we report the crystal structure of ThiH from TcThiH was incubated with its substrate L-tyrosine, 5\u00b4-deoxyadenosine (5\u00b4-dA) and methionine as mimics of the SAM cleavage products prior to crystallization. Brownish prism-shaped crystals that diffracted to 1.27\u2009\u00c5 resolution were obtained. In this crystal form, two independent ThiH molecules termed chains A and B were present in the asymmetric unit. Surface interaction calculations using the PDBePISA server31 supported the fact that the protein does not form quaternary structures in the crystals and would likely correspond to monomers in solution, in agreement with our previous observation when performing size exclusion chromatography. The refined model . We have also performed a 250-ns molecular dynamics (MD) simulation of L-Tyr bound TcThiH (COO\u2212) Fig.\u00a0.TcThiH numbering), all the key residues involved in L-tyrosine binding are strictly conserved in both proteins and 0.25\u2009\u00c5 (molecule B), in the same range derived from comparing molecules A and B of the same crystal (see above). The Fo-Fc residual electron density map at the substrate-binding site indicates features that do not correspond to L-tyrosine, best modeled as two water molecules and a sulfate ion from the crystallization condition 25, in order to identify what makes such difference in substrate specificity. TcThiH and SaNosL share about 23% identity mainly located at key positions either to stabilize the tridimensional architecture or because they are important for their function. Yet, the two structures are very similar and display a RMSD of 2.1\u2009\u00c5 for 281 C\u03b1-atoms superimposed. The main structural differences lay in the N-terminal \u03b1-domain. As can be seen in Fig.\u00a0TcThiH and SaNosL, respectively). Their respective dihedral angles are also the same. Strikingly, the main significant difference corresponds to a one-residue insertion at the C-terminal end of strand \u03b28 and the L-tryptophan bound SaNosL structure we had previously solved25, both crystallized with 5\u00b4-dA, we first focused on the 5\u00b4-dA\u2022 attack on their respective substrates. Since the attack occurs for both models at the substrates\u2019 amino group for [5\u00b4-dA\u2022\u2009+\u2009L-Tyr]-TcThiH and [5\u00b4-dA\u2022 + L-Trp]-SaNosL, respectively at the start of the scan. By contrast, for NosL, the C5\u00b4 and adenine parts are energetically crossing, i.e. the characters of LUMO and LUMO\u2009+\u20091 are exchanged and their corresponding energies remain close within ~0.2\u2009eV range. This means that 5\u00b4-dA\u2022 is influenced by subtle changes in its environment. This results in an inflection of the energy increase in addition to the first lowest point (\u03a6\u2009=\u2009\u221247.9\u00b0) mirroring that of ThiH. As it turns out, this energy scan inflection for NosL opens an alternative path leading ultimately to the C\u03b1\u2013C bond scission whereas the absolute minimum common to both ThiH and NosL would lead for NosL to the non-productive C\u03b1\u2013C\u03b2 bond scission . Hence, we decided to further investigate the impact of a hydrogen tunneling effect for the reaction to proceed and 25\u2009\u00b0C (SaNosL), corresponding to the optimal growth of T. carboxidivorans and S. actuosus, respectively.The transition state geometries for these reactions were validated by frequency calculations. Strikingly, the unique imaginary frequency exhibits a large absolute value greater than 2000 cmect Fig.\u00a038. In adp-cresyl\u2022 and DHG to extract a sampling of active site conformations corresponding to normal structural \u201cbreathing\u201d in order to determine whether some of them could separate the products further. As a reference, we first scanned the C\u03b1\u2013C\u03b2 bond break using a reduced quantum model consisting of about 300 atoms extracted from our QM/MM model of [L-Tyr-NH\u2022], and observed that the energy reaches a minimum when C\u03b1 and C\u03b2 are about 2.7\u2009\u00c5 apart but goes back up at greater scanned distances are necessary for product release.However, a product escape route is observed through a gate defined by residues Y76 and Y181 Fig.\u00a0, left anydG Fig.\u00a0, center;osL Fig.\u00a0, right. TcThiH has shown that the substrate acquires an unusual protonation state upon its binding at the active site. Indeed, the phenol functional group of L-tyrosine is deprotonated and the resulting phenolate stabilized by four anion-dipole interactions with strictly conserved residues in ThiH but also in the tyrosine lyase HydG between the NcoI and BamHI restriction sites, leading to a protein containing an N-ter Strep-tag and corresponding to the pTcThiH construct. The corresponding DNA sequence is as follow:The synthetic gene of ThiH from CCATGGGACATCATCATCATCATCACTCCGGCACGTTCTATGATGTGATTGAAGATTACCGCCACTTTGATTTCGCAGCGTATTTCGCGAAAGTTACCGATTCTGACGTCCGTCGCATCCTGCGTCAGGATCGCCTGTCAGCCCTGGACTTTCTGACGCTGCTGTCGCCGCAAGCGGAAGCCTATCTGGAAGAAATGGCACAGAAAGCTCATCGTCTGACCGTTCAACACTTTGGCCGCACCATGCTGCTGTATACGCCGCTGTACCTGGCGAACTATTGCGTGAATCAGTGCGTTTACTGTGGTTTCCAACTGAAAAACAAACTGGAACGTAAAAAACTGACCCTGGCGGAAGTGGAACAGGAAGCCCAACTGATTGCGGCCACCGGCCTGAAACATATTCTGATCCTGACGGGTGAAAGTCGCCAGCACTCACCGGTCTCGTATATCAAAGATTGTGTGAACATCCTGAAAAAATACTTTAGCTCTATCAGCATCGAAATTTATCCGCTGACCCAGGAAGAATACGCAGAACTGATCGGCGCTGGTGTTGACGGCCTGACGATTTACCAGGAAGTGTATAACGAAGAAGTTTATGCGGAAATGCATCCGGCCGGCCCGAAACGTAATTACCGTTTCCGCCTGGAAGCACCGGAACGTGCATGCCAGGCCGGTATGCGTACCGTGAACATCGGCGCTCTGCTGGGTCTGAATGATTGGCGCCAGGAAGCATTTTTCACGGGTCTGCATGCTGATTATCTGCAACGTCGCTTTCCGGACGTGGAAGTTAGTATTTCCCCGCCGCGTATGCGTCCGCACCTGGGCGGTTTCCCGCCGCGTGTGGTTGTCAGCGATCAGAACCTGGTCCAATATGTGCTGGCATTTCGTCTGTTCATGCCGCGTAGTGGTATCACCCTGTCCACGCGTGAAAATGGTCGTCTGCGTGACGCGATGGTCCGTCTGGGCGTGACCAAAATGAGCGCAGGTTCTTGTACGGCTGTTGGCGGTCGTAGCGATCAGGAAGCCGTCGGCCAGTTCCAAATTTCTGACGAACGCACCGTTGCGGAAGTCGCAGCTATGCTGTACGCCCAGGGTTATCAACCGGTGTACAAAGATTGGCAGGCACTGAGGATCCwhich leads to the following protein sequence:MWSHPQFEKASGTFYDVIEDYRHFDFAAYFAKVTDSDVRRILRQDRLSALDFLTLLSPQAEAYLEEMAQKAHRLTVQHFGRTMLLYTPLYLANYCVNQCVYCGFQLKNKLERKKLTLAEVEQEAQLIAATGLKHILILTGESRQHSPVSYIKDCVNILKKYFSSISIEIYPLTQEEYAELIGAGVDGLTIYQEVYNEEVYAEMHPAGPKRNYRFRLEAPERACQAGMRTVNIGALLGLNDWRQEAFFTGLHADYLQRRFPDVEVSISPPRMRPHLGGFPPRVVVSDQNLVQYVLAFRLFMPRSGITLSTRENGRLRDAMVRLGVTKMSAGSCTAVGGRSDQEAVGQFQISDERTVAEVAAMLYAQGYQPVYKDWQAL.thiH gene was coexpressed with the isc operon and the SAM synthase gene coding metK from E. coli using an E. coli BL21(DE3) strain containing both pTcThiH and our home-made pRSF-ISC-MetK plasmids25. Cells were grown aerobically at 37\u2009\u00b0C under agitation in 4\u2009L of TB medium supplemented with 50\u2009mM MOPS/KOH, pH 7.1, 0.5% D-glucose, ampicillin (100\u2009\u00b5g.ml\u22121) and kanamycine (50\u2009\u00b5g.ml\u22121) up to an OD600 of about 0.6. They were subsequently transferred in 2\u2009L sealed bottles into a glove box with an anaerobic atmosphere containing less than 5\u2009ppm O2 under constant stirring at 20\u2009\u00b0C. After about 15\u2009min, the bottles were opened to equilibrate the medium with the atmosphere of the glove box and the medium was supplemented with 10\u2009mL MEM Vitamin solution 100X (SIGMA M6895); 4\u2009g fumarate; 500\u2009\u00b5L 0.5\u2009M cysteine and 750\u2009\u00b5L 0.2 M L-methionine per L of culture. Fifteen more minutes later, the medium was enriched with 250\u2009\u00b5L 1\u2009M ammonium iron(III) citrate. Protein expression was induced when the OD600 reached 1 with addition of 1\u2009mM final concentration isopropyl-\u03b2-D-1-thiogalactopyranoside and cells were further grown overnight before being harvested and frozen in liquid nitrogen. All the purification steps were performed under anaerobic conditions. Cell pellets were resuspended in the glove box in buffer A supplemented with EDTA-free protease inhibitor cocktail. They were subsequently disrupted by sonication and the crude-extract was cleared by centrifugation in sealed tubes at 15,000\u2009\u00d7\u2009rpm during 30\u2009min at 4\u2009\u00b0C. The clarified supernatant was filtered, and loaded onto a streptavidin-agarose column (Iba\u00ae) equilibrated with anaerobic cleared lysate was injected into a Strep-tactin column equilibrated with buffer A. After an extensive wash step, the protein was eluted with a 1\u2009mM desthiobiotin solution in buffer A. The iron-sulfur cluster was subsequently reconstituted. The protein was incubated 15\u2009min with 5\u2009mM DTT prior to the addition of 200\u2009nM NifS from Azotobacter vinelandii. 5 excess FeCl3 and L-cysteine were then added and the solution was kept under mild stirring overnight at 20\u2009\u00b0C. The protein was further purified to homogeneity by gel filtration using a GE-healthcare highload 16/600 S200 prep-grade column equilibrated with buffer B (50\u2009mM Tris pH 8 and 100\u2009mM NaCl). The protein eluted as two peaks corresponding to a dimer and a monomer, respectively. Only the fractions corresponding to the monomer (major peak) were pooled, concentrated to 10\u2009mg/mL and stored in liquid nitrogen. Iron content was determined using the method of Fish48 and indicated over 3.9 iron equivalents per ThiH molecule.The recombinant 4)2SO4, 100\u2009mM MES buffer pH 6.5, 15% dioxane and 10\u2009mg/mL TcThiH. The protein was preincubated with 1\u2009mM 5\u00b4-dA and 1 mM L-methionine. When required, 5-excess L-tyrosine was added to the crystallization drop. Crystals were cryoprotected using 25% glycerol in addition to the crystallization condition and were subsequently mounted in cryoloops before flash cooling in liquid propane in the glove box49. Data were collected at SOLEIL at beamlines PROXIMA-1 and PROXIMA-2A and were processed using the XDS package50 as a search model. The initial model was manually corrected using COOT52 and refined using PHENIX53 to an Rwork\u2009=\u20090.153 and Rfree\u2009=\u20090.174 with good geometry and over 98% residues in the most allowed region of the Ramachandran plot54. This model was subsequently used to solve the ligand-free TcThiH crystal structure following the same procedure, leading to a model with good geometry. The corresponding statistics are reported in Supplementary Table\u00a0Crystals were obtained using the vapor diffusion method. Initial conditions were determined by screening 1248 conditions using a Gryphon robot setup in a glove box and were subsequently manually optimized. Crystals suitable for X-ray diffraction experiments were obtained with 1.4\u20131.6\u2009M (NH55.Details of all calculations are given in the Supplementary Information. All MD simulations, QM and QM/MM calculations were performed within the Schr\u00f6dinger suiteFurther information on research design is available in the\u00a0Supplementary information filePeer Review FileReporting Summary"} +{"text": "We found that LTBR and CREB1 exhibited a significant upregulation in lungs of mouse model of BPD. LTBR and CREB1 expression were also increased by hyperoxia in A549 and ATII cells. According to results of cell counting kit-8 assay and flow cytometry analysis, silencing of LTBR rescued the suppressive effect of hyperoxia on cell viability and its promotive effect on cell apoptosis of A549 and ATII cells. Bioinformatics revealed CREB1 as a transcriptional factor for LTBR, and the luciferase reporter assay and ChIP assay subsequently confirmed it. The NF-\u03baB pathway was regulated by LTBR. CREB1 induced LTBR expression at the transcriptional level to regulate NF-\u03baB pathway and further modulate A549 and ATII cells viability and apoptosis. In conclusion, this study revealed the CREB1/LTBR/NF-\u03baB pathway in BPD and supported the beneficial role of LTBR silence in BPD by promoting viability and decreasing apoptosis of lung epithelial cells.Bronchopulmonary dysplasia (BPD) is a prevalent chronic pediatric lung disease. Aberrant proliferation and apoptosis of lung epithelial cells are important in the pathogenesis of BPD. Lymphotoxin beta receptor (LTBR) is expressed in lung epithelial cells. Blocking LTBR induces regeneration of lung tissue and reverts airway fibrosis in young and aged mice. This study is aimed at revealing the role of LTBR in BPD. A mouse model of BPD and two A review research concludes that BPD may be caused by various factors like premature birth, fetal growth restriction, mechanical ventilation, oxygen toxicity, inflammation, and genetic susceptibility [Bronchopulmonary dysplasia (BPD), initially described by Northway et al., is caused by oxygen supply and mechanical ventilation in premature infants with severe respiratory distress syndrome . Accorditibility . Despitetibility , pulmonatibility were devtibility . Thus, iBPD is a chronic disease featured by disrupting the alveolar together with microvascular development of the peripheral lung. The regeneration or induced growth of type II alveolar epithelial (ATII) cells has been the focus feature of lung regeneration research . Thus, e\u03b2 receptor (LTBR) signaling in ATII cells induces kinase NIK through nonclassical nuclear factor-kappaB (NF-\u03baB) signaling. Blocking LTBR signaling limits broncho-associated lymphoid tissue formation and reduces alveolar epithelial cell apoptosis, thereby driving alveolar regeneration [Activation of the LTneration . LTBR sineration . LTBR mRneration . LTBR exneration , a contiTranscription factors respond to stimuli of the external environment or signals at different stages of development, activate or inhibit gene transcription, and thus control the expression of different genes. It is very important to identify key transcription factors in BPD. cAMP responsive element binding protein 1 (CREB1) regulates late-stage lung development in mammals . CREB1 i\u03baB signaling plays a crucial role in cell survival, differentiation, and innate immunity [\u03baB dimers are not active in the cytoplasm of cells related to a suppressive protein, I\u03baB. Under the conditions of cytokines, growth factors, or bacterial products, I\u03baB kinases IKK-\u03b1 and -\u03b2 activate NF-\u03baB, leading to I\u03baB phosphorylation and degradation. Clearance of I\u03baB allows translocation of NF-\u03baB complexes into the nucleus and modulates a variety of downstream targets that affect inflammation, cell adhesion, and cell survival [\u03baB-mediated gene modulation is implicated in the pathogenesis of BPD [The NF-immunity . NF-\u03baB dsurvival . Many sts of BPD .in vivo and its role in the apoptosis of lung epithelial cells in vitro. Hyperoxia-stimulated A549 and ATII cells were used as the in vitro model of BPD [\u03baB pathway was assessed, providing new ideas for understanding the pathogenesis of BPD.This study is aimed at evaluating the expression of LTBR in the BPD lung injury mouse model l of BPD , 19. Then = 8) or the control group (n = 8). To establish the BPD model, mice were exposed to 100% O2 in a medium-sized airtight polypropylene chamber on postnatal days 1-4, followed by recovering for 10 days [Neonatal C57BL/6 mice of both sexes were used for establishing the model of BPD. Animal studies were conducted in compliance with the ARRIVE guidelines under the approval of the animal care and ethic committee of First Affiliated Hospital of Nanjing Medical University. Mice were housed under controlled standard conditions . Neonatal mice in the normoxia group were exposed to room air and fed by lactating mice. Mice were randomly divided into the BPD model group and dissolved in RNase-free water. Reverse transcription of total RNA into cDNA was performed using a High-Capacity cDNA Reverse Transcription Kit . Next, the QuantiFast SYBR\u00ae Green PCR Kit (QIAGEN) was used for a rapid and specific quantitative detection of target cDNA. PCR analysis was performed on a LightCycer\u00ae480II Instrument, 96-well block . LTBR or CREB1 expression was calculated using the 2t method and was Mouse LTBR, forward, 5\u2032-TGAACACTGGAACCATCTC-3\u2032; reverse, 5\u2032-ACACTCATTGTCCAGATACAC-3\u2032.Human LTBR, forward, 5\u2032-GAAGGGTAACAACCACTGC-3\u2032; reverse, 5\u2032-CTTGGTTCTCACACCTGGT-3\u2032.Mouse CREB1, forward, 5\u2032-GAAGAGGAGACTTCAGCCC-3\u2032; reverse, 5\u2032-TAATGGCAATGTACTGCCCA-3\u2032.Human CREB1, forward, 5\u2032-ATGCAGCTGTAACAGAAGC-3\u2032; reverse, 5\u2032-CATAGATACCTGGGCTAATGTG-3\u2032.Mouse GAPDH, forward, 5\u2032-ACTCTTCCACCTTCGATGC-3\u2032; reverse, 5\u2032-CCGTATTCATTGTCATACCAGG-3\u2032.Human GAPDH, forward, 5\u2032-TCAAGATCATCAGCAATGCC-3\u2032; reverse, 5\u2032-CGATACCAAAGTTGTCATGGA-3\u2032.The radial alveolar count (RAC) determines alveolar septation and is a test of alveologenesis. Through images at 100\u00d7 magnification, a perpendicular line was made from a respiratory bronchiole to the nearest pleural edge or fibrovascular septum. Airspaces or saccules through this line were counted . Scion I2. A549 and ATII cells were exposed to different concentrations of O2 in a standard modular chamber at 37\u00b0C to assess the effects of hyperoxia on LTBR and CREB1 expression. The normoxia condition contains 21% O2. Based on the results of the preliminary assays, A549 and ATII cells were exposed to 85% O2 for 48\u2009h to mimic the in vitro model of BPD. Si-NC, si-LTBR, si-CREB1, empty pcDNA 3.1, pcDNA 3.1-LTBR, and pcDNA 3.1-CREB1 were commercially provided by GenePharma . These oligonucleotides or vectors were transfected into A549 and ATII cells using Lipofectamine 3000 for 24\u2009h at 37\u00b0C. The transfection sequences used were as follows: si-NC: 5\u2032-AACCATCACTTACAAGAAACC-3\u2032; si-CREB1: 5\u2032-GCTCGATAAATCTAACAGTTA-3\u2032; si-LTBR: 5\u2032-CCATCCATACTTCCCTGACTT-3\u2032; pcDNA3.1: 5\u2032-CATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGCGTTTAAACTTAAGCTTGGTACCGAGCTCGGATCCACTAGTCCAGTGTGGTGGAATTCTGCAGATATCCAGCACAGTGGCGGCCGCTCGAGTCTAGAGGGCCCGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCAT-3\u2032; pcDNA 3.1-CREB1: 5\u2032- ATGACCATGGAATCTGGAGCCGAGAACCAGCAGAGTGGAGATGCAGCTGTAACAGAAGCTGAAAACCAACAAATGACAGTTCAAGCCCAGCCACAGATTGCCACATTAGCCCAGGTATCTATGCCAGCAGCTCATGCAACATCATCTGCTCCCACCGTAACTCTAGTACAGCTGCCCAATGGGCAGACAGTTCAAGTCCATGGAGTCATTCAGGCGGCCCAGCCATCAGTTATTCAGTCTCCACAAGTCCAAACAGTTCAGATTTCAACTATTGCAGAAAGTGAAGATTCACAGGAGTCAGTGGATAGTGTAACTGATTCCCAAAAGCGAAGGGAAATTCTTTCAAGGAGGCCTTCCTACAGGAAAATTTTGAATGACTTATCTTCTGATGCACCAGGAGTGCCAAGGATTGAAGAAGAGAAGTCTGAAGAGGAGACTTCAGCACCTGCCATCACCACTGTAACGGTGCCAACTCCAATTTACCAAACTAGCAGTGGACAGTATATTGCCATTACCCAGGGAGGAGCAATACAGCTGGCTAACAATGGTACCGATGGGGTACAGGGCCTGCAAACATTAACCATGACCAATGCAGCAGCCACTCAGCCGGGTACTACCATTCTACAGTATGCACAGACCACTGATGGACAGCAGATCTTAGTGCCCAGCAACCAAGTTGTTGTTCAAGCTGCCTCTGGAGACGTACAAACATACCAGATTCGCACAGCACCCACTAGCACTATTGCCCCTGGAGTTGTTATGGCATCCTCCCCAGCACTTCCTACACAGCCTGCTGAAGAAGCAGCACGAAAGAGAGAGGTCCGTCTAATGAAGAACAGGGAAGCAGCTCGAGAGTGTCGTAGAAAGAAGAAAGAATATGTGAAATGTTTAGAAAACAGAGTGGCAGTGCTTGAAAATCAAAACAAGACATTGATTGAGGAGCTAAAAGCACTTAAGGACCTTTACTGCCACAAATCAGAT-3\u2032 and pcDNA 3.1-LTBR: 5\u2032-ATGGAAGCGACAGGAATCTCATTAGCATCTCAATTAAAGGTGCCTCCATATGCGTCGGAGAACCAGACCTGCAGGGACCAGGAAAAGGAATACTATGAGCCCCAGCACCGCATCTGCTGCTCCCGCTGCCCGCCAGGCACCTATGTCTCAGCTAAATGTAGCCGCATCCGGGACACAGTTTGTGCCACATGTGCCGAGAATTCCTACAACGAGCACTGGAACTACCTGACCATCTGCCAGCTGTGCCGCCCCTGTGACCCAGTGATGGGCCTCGAGGAGATTGCCCCCTGCACAAGCAAACGGAAGACCCAGTGCCGCTGCCAGCCGGGAATGTTCTGTGCTGCCTGGGCCCTCGAGTGTACACACTGCGAGCTACTTTCTGACTGCCCGCCTGGCACTGAAGCCGAGCTCAAAGATGAAGTTGGGAAGGGTAACAACCACTGCGTCCCCTGCAAGGCCGGGCACTTCCAGAATACCTCCTCCCCCAGCGCCCGCTGCCAGCCCCACACCAGGTGTGAGAACCAAGGTCTGGTGGAGGCAGCTCCAGGCACTGCCCAGTCCGACACAACCTGCAAAAATCCATTAGAGCCACTGCCCCCAGAGATGTCAGGAACCATGCTGATGCTGGCCGTTCTGCTGCCACTGGCCTTCTTTCTGCTCCTTGCCACCGTCTTCTCCTGCATCTGGAAGAGCCACCCTTCTCTCTGCAGGAAACTGGGATCGCTGCTCAAGAGGCGTCCGCAGGGAGAGGGACCCAATCCTGTAGCTGGAAGCTGGGAGCCTCCGAAGGCCCATCCATACTTCCCTGACTTGGTACAGCCACTGCTACCCATTTCTGGAGATGTTTCCCCAGTATCCACTGGGCTCCCCGCAGCCCCAGTTTTGGAGGCAGGGGTGCCGCAACAGCAGAGTCCTCTGGACCTGACCAGGGAGCCGCAGTTGGAACCCGGGGAGCAGAGCCAGGTGGCCCACGGTACCAATGGCATTCATGTCACCGGCGGGTCTATGACTATCACTGGCAACATCTACATCTACAATGGACCAGTACTGGGGGGACCACCGGGTCCTGGAGACCTCCCAGCTACCCCCGAACCTCCATACCCCATTCCCGAAGAGGGGGACCCTGGCCCTCCCGGGCTCTCTACACCCCACCAGGAAGATGGCAAGGCTTGGCACCTAGCGGAGACAGAGCACTGTGGTGCCACACCCTCTAACAGGGGCCCAAGGAACCAATTTATCACCCATGACTGA-3\u2032.Lung epithelial A549 cell line was commercially provided by ATCC and was cultured in F-12\u2009K Medium supplemented with 10% FBS. Human ATII cells and HEK-293\u2009T cells were purchased from Procell and cultured in human ATII cell complete culture medium and DMEM, respectively. Cells were maintained in a 37\u00b0C incubator in the atmosphere of 95% and 5% CO2. Next, cells were added with 10\u2009\u03bcL of silencing or overexpressing plasmids. Ten \u03bcL of CCK-8 solution was added, and then cells were cultured in the incubator for 2\u2009h. OD value at 450\u2009nm was read using a SpectraMax iD5 microplate reader .A CCK-8 kit was applied to detect A549 and ATII cell viability. In brief, transfected cells were plated in 96-well plate and precultured in an incubator at 37\u00b0C and 5% CO\u03bcg/lane) was separated by 11% SDS-PAGE at 110\u2009V for 2\u2009h and transferred to 0.22\u2009\u03bcm PVDF membranes . The membranes were incubated with LTBR antibody , CREB1 antibody , IKK\u03b1 (phospho T23) antibody , IKK\u03b1 antibody , p52 antibody , RELB antibody , or GAPDH antibody at 4\u00b0C overnight. After washing with PBS, membranes were incubated with the secondary antibody IgG for 2\u2009h at room temperature. Proteins were detected by enhanced chemiluminescence based on the manufacturer's instructions. Protein band grey intensity was quantified using the Quantity One software .Total protein was extracted from lung tissues and A549 and ATII cells using RIPA lysis buffer . Protein concentration was measured with a BCA protein assay kit . Total protein for 15\u2009min at room temperature in dark. Finally, the cells were resuspended in 400\u2009\u03bcL 1\u2009\u00d7\u2009Binding Buffer, mixed on ice, and analyzed by FACS using flow cytometry within one hour. Cells were gated into four groups: Annexin V\u2212/PI\u2212, Annexin V+/PI+, Annexin V+/PI\u2212, and Annexin V\u2212/PI+. In this study, cell apoptosis rate was defined as the percentage of Annexin V+/PI+ cells (in late apoptosis stage).A549 and ATII cells were harvested, washed with PBS, and fixed with 1% paraformaldehyde overnight at 4\u00b0C. Next, cells were washed again, resuspended in 100\u2009\u03bcg, Abcam) or anti-IgG for one night. After washing and elution, samples were treated with 5\u2009M NaCl for heating, and then incubated with proteinase K for 1\u2009h. The bound DNA fragments were purified via DNA Extraction Kit and analyzed by real-time PCR.This assay was performed with a ChIP assay kit following the manufacturer's instructions. Briefly, A549 and ATII cells were cross-linked with 1% formaldehyde and sonicated to shear DNA to lengths between 200~1000 base pairs. Cell lysates were incubated at 4\u00b0C with protein A/G beads coated with the anti-CREB1 antibody .Wild type (WT) LTBR gene promoter at different sites and the mutated (wt) sites were subcloned into the pGL3 luciferase reporter vector (GeneCopoeia). Mutation was performed using a QuickMutation\u2122 Site-Directed Mutagenesis Kit . HEK-293\u2009T tool cells at the concentration of 5 \u00d7 10t-test and one-way ANOVA with Dunnett's or Tukey's post hoc test. A p < 0.05 was deemed as statistically significant. For in vitro studies, three\u2009biological\u2009replicates \u00d7 three\u2009technical\u2009replicates were applied.Data are presented as mean \u00b1 SD. For expression and luciferase activity values, data are represented as fold changes to mean control. For cell viability values, data are represented as percentage (%) of mean control. GraphPad Prism 8 was used to perform statistical tests including unpaired two-tailed Student's 2 in A549 cells expression data of LTBR in clinical lung tissues of BPD are lacked; (2) there is a close association of LTBR and WNT/ignaling , while t\u03baB pathway and further modulated lung epithelial cell viability and apoptosis. This study may aid in understating the pathogenesis and treatment of BPD.In conclusion, this work innovatively demonstrated the upregulation of LTBR and CREB1 in BPD. LTBR and CREB1 are positive regulators of cell apoptosis and negative regulators of cell viability of A549 and ATII cells under hyperoxia. CREB1 induced LTBR expression at the transcriptional level to regulate NF-"} +{"text": "Synema Simon, 1864 is a relatively large genus of family Thomisidae Sundevall, 1833 and currently includes 124 species distributed worldwide, except for the Polar Regions. However, Synema can be regarded as being poorly represented in China, with only seven species, three of which are endemic.Synema from Guiyang City in China, is described under the name of S.guiyang J. Zhang, Q. Lu & H. Yu, sp. nov. Detailed descriptions and photographs of the new species are provided. DNA barcodes of the species were obtained to confirm matching of the sexes and for future use in molecular studies.A new spider species of the genus Thomisidae is one of the largest spider families, with 171 genera and 2159 valid species distributed worldwide, 51 genera and 306 species of which are recorded from China , Tmarus Simon, 1875 (226 species) and Thomisus Walckenaer, 1805 (143 species) . Currently, only seven Synema species are known from China, amongst them, three are endemic . Despite being common, the genus remains inadequately studied because: more than half of the species are known from a single sex or juveniles ; original descriptions are rather brief and lack illustrations or illustrations are inadequate . CurrentSynema specimens of crab spiders in the same location, which are with similar habitus, markings, leg spination and other characters .A DNA barcode was also obtained for the species matching. A partial fragment of the mitochondrial cytochrome oxidase subunit I (CO1) gene was amplified and sequenced for 21 specimens, using the primers LCOI1490 (5\u2019-GGTCAACAAATCATAAAGATATTG-3\u2019) and HCOI2198 (5\u2019-TAAACTTCAGGGTGACCAAAAAAT-3\u2019). For additional information on extraction, amplification and sequencing procedures, see All measurements were obtained using an Olympus SZX7 stereomicroscope and given in millimetres. Eye diameters were taken at the widest point. The total body length does not include the length of the chelicerae or spinnerets. Leg lengths are given as total length . Most of the terminologies used in text and figure legends follow All specimens are deposited at the Museum of Guizhou Education University, Guiyang, Guizhou, China .J. Zhang, Q. Lu & H. Yusp. n.BB6AFCEC-3FC5-5F68-A0A0-CFB80E085396C659D2A5-4612-4DCA-9EA3-1A43DBB0A5DCType status:Holotype. Occurrence: recordedBy: Hao Yu; individualID: YHTHO002; individualCount: 1; sex: male; lifeStage: adult; behavior: foraging; preparations: whole animal (ETOH); associatedSequences: GenBank: ON435709; Taxon: order: Araneae; family: Thomisidae; genus: Synema; specificEpithet: guiyang; scientificNameAuthorship: J. Zhang, Q. Lu & H. Yu,; Location: continent: Asia; country: China; countryCode: CHN; stateProvince: Guizhou; county: Guiyang City; locality: Guiyang Forest Park; decimalLatitude: 26.55540319; decimalLongitude: 106.75898910; Identification: identifiedBy: Hao Yu; dateIdentified: 2021-11; Event: samplingProtocol: by hand; samplingEffort: 10 km by foot; year: 2021; month: 8; day: 10; Record Level: institutionID: MGEU; basisOfRecord: PreservedSpecimenType status:Paratype. Occurrence: recordedBy: Hao Yu; individualID: YHTHO003; individualCount: 1; sex: female; lifeStage: adult; behavior: foraging; preparations: whole animal (ETOH); associatedSequences: GenBank: ON435708; Taxon: order: Araneae; family: Thomisidae; genus: Synema; specificEpithet: guiyang; scientificNameAuthorship: J. Zhang, Q. Lu & H. Yu,; Location: continent: Asia; country: China; countryCode: CHN; stateProvince: Guizhou; county: Guiyang City; locality: Guiyang Forest Park; decimalLatitude: 26.55540319; decimalLongitude: 106.75898910; Identification: identifiedBy: Hao Yu; dateIdentified: 2021-11; Event: samplingProtocol: by hand; samplingEffort: 10 km by foot; year: 2021; month: 8; day: 10; Record Level: institutionID: MGEU; basisOfRecord: PreservedSpecimenMale (holotype) Fig. A\u2013C. TotaCarapace Fig. A, C yellAbdomen Fig. A\u2013C elongLegs basically yellowish-brown Fig. E, all lePalp Fig. A\u2013D. TibiFemale .DNAbarcode: 5'TATTTGGAGCTTGATCTGCTATAGTAGGGACGGCTATAAGAGTGTTAATTCGTATGGAATTAGGA AGATCTGGAAGATTATTAGGAAATGATCATCTTTATAATGTAATTGTTACCGCTCATGCTTTTGTCATGATTTTTTTTATA GTAATACCTATTTTAATTGGGGGTTTTGGAAATTGATTAGTACCTTTAATGTTAGGGGCTCCTGATATATCTTTCCCTCG GATAAATAATTTATCTTTTTGATTATTACCCCCTTCATTATTTTTACTATTTATATCTTCTATAGTAGAGGTAGGTGTGGGG GCAGGATGAACTGTTTATCCTCCTCTAGCTTCTAGAGTTGGGCATATAGGAGGATCTATAGATTTTGCTATTTTTTCTTT ACATTTAGCTGGGGCTTCTTCTATTATAGGGGCGGTTAATTTTATTTCTACTATTATTAATATACGAACTAGAGGTATAAG AATAGAAAAGGTTCCTTTGTTTGTATGATCTGTATTAATTACAGCTATTTTACTTCTTTTGTCTTTACCTGTATTAGCAGG TGCTATTACTATATTATTAACTGATCGTAATTTTAACACTTCTTTTTTTGATCCTGCAGGGGGAGGGGATCCAATTTTATT TCAACATTTGTTTTGATTTTT3' .S.albomaculatum and S.chikunii in having the similar habitus ; anterior metatarsi and tibiae with dense hairs (vs. few hairs in Spilosynema); PER slightly recurved (vs. distinctly recurved in Spilosynema). Strictly based on this diagnosis, it seems that the new species should be assigned to the genus Spilosynema . Therefore, it is very strange (or interesting) that S.guiyang sp. nov. exhibits typical somatic features of Spilosynema and genitalic features of Synema. In view of the fact that somatic characters are either poorly marked or variable, they are thus not sufficient for distinguishing the Spilosynema and Synema. Consequently, S.guiyang sp. nov. is assigned tentatively to the genus Synema in the present paper for the lack of a better solution.However, some genitalic characteristics are not mentioned in the diagnosis of ned Fig. B, D. In"} +{"text": "Caulobacter crescentus cells that display and secrete a self-interacting protein. This protein formed a de novo matrix and assembled cells into centimeter-scale ELMs. Discovery of design and assembly principles allowed us to tune the composition, mechanical properties, and catalytic function of these ELMs. This work provides genetic tools, design and assembly rules, and a platform for growing ELMs with control over both matrix and cellular structure and function.Engineered living materials (ELMs) embed living cells in a biopolymer matrix to create materials with tailored functions. While bottom-up assembly of macroscopic ELMs with a de novo matrix would offer the greatest control over material properties, we lack the ability to genetically encode a protein matrix that leads to collective self-organization. Here we report growth of ELMs from Engineered living materials (ELMs) embed living cells in a biopolymer matrix to create novel materials with tailored functions.\u00a0In this work, the authors engineered bacteria to grow novel macroscopic materials that can be reshaped, functionalized, and used to filter contaminated water while also showing that the stiffness of these materials can be tuned through genetic changes. Engineered living materials (ELMs)4 are inspired by naturally occurring living materials, but use synthetic biology to introduce tailored, non-natural functions. By incorporating engineered cells into a biopolymer matrix, these materials can function as living sensors5, therapeutics6, biomanufacturing platforms7, electronics8, energy converters9, and structural materials10. While cells confer functionality to ELMs, the matrix assembles the material and controls the bulk material composition, structure, and function11.Naturally occurring living biomaterials, such as bones or wood, grow bottom-up from a small number of progenitor cells into macroscale structures11 because secreting recombinant biopolymers at concentrations that gelate is challenging12 and because the assembly of micrometer-sized cells into centimeter-scale materials requires self-organization across length scales spanning four orders of magnitude. Engineering principles to achieve this assembly are unknown12. Therefore, most macroscopic ELMs have been produced by adopting a top-down approach (such as 3D printing) to incorporate living cells into an exogenous matrix14 or by processing microscopic ELMs that grow a synthetic biomolecular matrix into macroscopic materials19. The few autonomously produced, macroscopic ELMs have been created by genetically modifying existing nanocellulose matrices20 or genetically manipulating the mineralization of silica matrices. However, these two approaches to autonomously produced, macroscopic ELMs have afforded little genetic control over the mechanical properties, e.g., ~1.2\u20131.4-fold change in the storage modulus21. This tunability is much more limited than the tunability of naturally occurring materials, chemically synthesized materials, or macroscopic ELMs produced by processing23.Since the matrix plays such a key role in generating material properties, one primary goal of the field is to create ELMs that both have a synthetic biomolecular matrix\u2014that can control these properties\u2014and grow autonomously into macroscopic structures. However, such bottom-up, de novo ELMs are considered well beyond the current state-of- artEscherichia coli has been engineered to display interacting proteins, such as leucine zippers24 or antigen-nanobody pairs25, via outer membrane proteins. Engineered strains that display interacting pairs will self-assemble into cell\u2013cell aggregates that flocculate25; however, these aggregates are microscopic and must be processed to form larger materials18. In contrast, micron-sized colloidal particles that display DNA have been programmed to self-assemble into both microscopic21 and macroscopic crystalline solids26. Over two decades of work on these systems has established central principles that underlie their self-assembly27. One of these central principles is that the interactions between particles must be mediated by high-density surface modifications, e.g., 1 DNA molecule per 27\u2009nm26. Since the outer membrane proteins used for bacterial adhesins are displayed at ~5% of this density, i.e., 1 nanobody per 640\u2009nm28, we hypothesized that a matrix composed of self-interacting proteins displayed on bacteria at high density could lead to the formation of macroscopic solid materials.We posit that new strategies for developing synthetic biomolecular matrices to self-assemble bacteria into macroscopic ELMs can be informed by prior work on surface-engineered bacteria and surface-modified colloidal particles. The surface of Caulobacter crescentus for high-density peptide display29 and biopolymer secretion23. The S-layer forms a 2D crystalline layer on the extracellular surface of C. crescentus, opening the possibility of displaying proteins at a density of up to 1 protein per 70\u2009nm30. Leveraging this prior work, here we describe the autonomous formation of macroscopic living material from C. crescentus engineered to display a synthetic, self-interacting, protein matrix based on the S-layer scaffold. We demonstrate that the mechanical properties\u00a0of this material can be genetically controlled over a factor of ~25x. We also describe unexpected findings indicating that the protein matrix plays a multifaceted role in the material formation and that material assembly occurs through a multi-step process mediated by the air\u2013water interface.We have previously engineered the surface layer (S-layer) of the oligotrophic bacterium C. crescentus31, we sought to create bottom-up ELMs composed of cells that interact at high density through a surface-bound, de novo matrix. To minimize native cell\u2013cell interactions, we started with a C. crescentus background that lacks the adhesive holdfast and therefore cannot form a biofilm33; we refer to this as the wild-type strain. Next, we designed a displayed bottom-up de novo (BUD) protein by replacing the native copy of the surface layer (S-layer) RsaA30 based on human tropoelastin that contains 60 repeats of the Val-Pro-Gly-X-Gly motif 37 (ELP60). ELPs form elastic materials, are flexible, and self-associate in a concentration-dependent fashion. Thus, this region is designed to influence material properties, promote solution accessibility, and add non-covalent self-interactions. SpyTag38 was used as a functionalization tag, as it covalently binds to fusion proteins containing SpyCatcher. We also introduced a FLAG tag as an epitope marker. Lastly, the C-terminal domain of the BUD protein, consisting of the last 336 residues of RsaA, was chosen to mediate protein secretion29 and to self-associate39. We refer to this BUD protein-expressing strain of C. crescentus as the BUD-ELM strain. In this way, we designed a C. crescentus strain to display a high-density, surface-bound, elastin-based matrix across its entire surface on all sides , and the BUD protein shows this same apparent difference in molecular weight (observed 102 vs. expected 86\u2009kDa). Interestingly, we found secreted BUD protein in the medium under both static and shaking conditions limit\u201390\u2009\u00b1\u20095% of cadmium was removed to obtain the holo forms of the enzyme. After confirming the activity of holo GDH in both cases , which couples oxidation of glucose to the reduction of a soluble electron carrierier Fig.\u00a0. This deIn summary, we developed macroscopic living materials that autonomously grow from engineered bacteria and that can be genetically encoded to have a wide range of mechanical properties. Specifically, we show that the expression of a self-interacting protein\u2014the BUD protein\u2014enables macroscopic material formation Fig.\u00a0. When diE. coli with self-interacting proteins displayed at ~10% the density of our engineered C. crescentus strains lead to small cell\u2013cell aggregates25. However, additional studies that systematically vary the surface density are needed to test this hypothesis. Another design rule that will be critical to understand and explore in future work is the nature and strength of the self-interactions in the BUD protein. We selected the RsaA690-1026 and ELP60 domains because prior reports demonstrate they can self-aggregate44. However, additional studies are needed to identify the nature of self-interactions and their strengths in the existing BUD protein and the range of self-interactions that permit the assembly of macroscopic materials.Our work identifies design rules that lead to the autonomous formation of BUD-ELMs and suggests other design rules to be tested. We identify a secreted matrix as a design constraint for this class of centimeter-scale, autonomously forming BUD-ELMs. Our work also indicates that a surface-anchored protein matrix is necessary for these materials to be cell-rich. Our data also suggests that this surface-anchored protein matrix may need to be present at high-density for cell-rich materials. This suggestion is supported by previous literature that shows that 18, we suggest that the use of the air\u2013water interface to locally concentrate and order hydrophobic biomolecules into a matrix may represent a general assembly principle for macroscopic ELMs. The genetic tools and C. crescentus platform developed here will permit systematic exploration of design and assembly rules for programming the growth of centimeter-scale structures using living cells as building blocks.This work also identified assembly principles for the autonomous formation of macroscopic materials. We have demonstrated that nucleation of a pellicle at the liquid-air interface and hydrodynamically-driven coalescence and collapse of the pellicle are required to form macroscopic ELMs. Since pellicle formation is also a key step in nanocellulose-based living materialsC. crescentus BUD-ELM platform developed herein is the highly reproducible, autonomous formation of engineered living materials. Growing BUD-ELMs from an engineered strain of C. crescentus requires only control of the temperature, media composition, flask and culture volume, shaking speed, and shaking orbit. We envision this simplicity will enable the ready adoption of this platform by other researchers. The second advantage of this platform is that the modularity of the BUD protein and the ease of engineering protein biopolymers offer much greater opportunities for introducing desirable properties into the matrix11. The BUD-ELM variants described herein have storage moduli that ranges between 13\u2009kPa, comparable to nanocellulose-based materials, and 0.5\u2009kPa, comparable to printed curli fiber-based materials. Introducing sites for chemical crosslinking into the ELP domain could allow the BUD-ELMs to be developed into elastomers45. More broadly, this work enables leveraging known polypeptides and proteins that exhibit desirable optical, electrical, mechanical, thermal, transport, and catalytic properties46. We envision specific matrix properties that can be combined synergistically with existing cellular functions such as sensing, biomolecule production, and information processing. Thus, this work multiplies the opportunities to program ELMs tailored for applications in human health, energy, and the environment.By creating BUD-ELMs with a de novo, modular protein matrix, this work greatly expands the ability to tailor macroscopic ELMs for specific applications. One of the key advantages of the \u2206ELP60\u2014RCC004, and \u2206SpyTag\u2014RCC005) from the wild-type47 (MFm126), we cloned integration plasmids designed to facilitate the incorporation of synthetic DNA sequences into the rsaA locus using homologous recombination. The integration plasmid used to generate the original BUD-ELM strain (pSMCAF008) was cloned by inserting a target sequence into the multicloning site of the backbone plasmid pNPTS138 (GenBank: MK533795.1) using restriction enzymes (ApaI upstream and NheI downstream). The target sequence encoded the ELP60-SpyTag flanked by 800\u2009bp of homology regions up- and downstream of the native rsaA central domain (rsaA750\u20132073). The integration plasmids used to generate the \u2206ELP60 and \u2206SpyTag BUD-ELM strains were cloned from the plasmid pSMCAF008 using Golden Gate assembly (PCR primers listed below).All strains and plasmids used in this work are listed in Supplementary Table\u00a029: plasmid pSMCAF008, pSMCAF017 or pSMCAF018 was electroporated into E. coli WM3064 cells and subsequently conjugated overnight into C. crescentus NA1000 \u0394sapA::Pxyl-mkate2 (MFm126) on a PYE agar plate containing 300n\u2009\u03bcM DAP. The culture was then plated on PYE with 25\u2009\u03bcg/ml kanamycin to select for integration of the plasmid and removal of E. coli cells. Successful integrants were incubated in liquid PYE media overnight and plated on PYE supplemented with 3% w/v sucrose to select for excision of the plasmid and sacB gene, leaving the target sequence in the genome. Integration of the sequences was confirmed by colony PCR (with primers SMCAF070 and SMCAF065) using a touchdown thermocycling protocol with an annealing temperature ranging from 72\u201362\u2009\u00b0C, decreasing 1\u2009\u00b0C per cycle. The PCR amplicons have been fully sequenced.The BUD-ELM strains were generated using the two-step recombination techniqueSMCAF142: TGGGTCTCAAGGGCGGTTCGGGAGGAGGCSMCAF143: TGGGTCTCAGCAATCCAAACGAGAGTCTAATAGAATGAGGTCSMCAF144: TGGGTCTCCTTGCAACTGGTCTATTTTCCTCTTTTGSMCAF145: TAGGTCTCCCCCTTGTCATCGTCGTCCTTG.SMCAF168: CAGGTCTCTTGTCGCACCTGATTGCCCGASMCAF169: CAGGTCTCTGCTGGGACACCACCGCCAGGSMCAF170: CTGGTCTCCCAGCTGACCCGGCCTTCGGCSMCAF171:\u2009CTGGTCTCTGACAATCTATCGATTGTATGGGAAGCCCG.SMCAF070: TATGACGTTTGCTCTATAGCCATCSMCAF065: GAGGATCAGACGTTCGCTTAG.CCAATGATCGTAATACGACTCACTAGTGGGGCCCGCGCCACTCGGTCGCAGGGGGTGTGGGATTTTTTTTGGGAGACAATCCTCATGGCCTATACGACGGCCCAGTTGGTGACTGCGTACACCAACGCCAACCTCGGCAAGGCGCCTGACGCCGCCACCACGCTGACGCTCGACGCGTACGCGACTCAAACCCAGACGGGCGGCCTCTCGGACGCCGCTGCGCTGACCAACACCCTGAAGCTGGTCAACAGCACGACGGCTGTTGCCATCCAGACCTACCAGTTCTTCACCGGCGTTGCCCCGTCGGCCGCTGGTCTGGACTTCCTGGTCGACTCGACCACCAACACCAACGACCTGAACGACGCGTACTACTCGAAGTTCGCTCAGGAAAACCGCTTCATCAACTTCTCGATCAACCTGGCCACGGGCGCCGGCGCCGGCGCGACGGCTTTCGCCGCCGCCTACACGGGCGTTTCGTACGCCCAGACGGTCGCCACCGCCTATGACAAGATCATCGGCAACGCCGTCGCGACCGCCGCTGGCGTCGACGTCGCGGCCGCCGTGGCTTTCCTGAGCCGCCAGGCCAACATCGACTACCTGACCGCCTTCGTGCGCGCCAACACGCCGTTCACGGCCGCTGCCGACATCGATCTGGCCGTCAAGGCCGCCCTGATCGGCACCATCCTGAACGCCGCCACGGTGTCGGGCATCGGTGGTTACGCGACCGCCACGGCCGCGATGATCAACGACCTGTCGGACGGCGCCCTGTCGACCGACAACGCGGCTGGCGTGAACCTGTTCACCGCCTATCCGTCGTCGGGCGTGTCGGGTTCGGGCGGTTCGGGAGGAGGCTCGGGTGACTACAAGGACGACGATGACAAGGGAGTTGGCGTCCCAGGAGTTGGAGTCCCAGGAGGGGGCGTTCCGGGCGCAGGAGTTCCTGGAGTAGGAGTTCCAGGAGTGGGCGTGCCAGGGGTGGGCGTCCCAGGTGGGGGAGTTCCCGGAGCAGGTGTGCCTGGGGGCGGCGTGCCTGGAGTCGGAGTTCCGGGGGTGGGTGTACCGGGTGGAGGCGTACCAGGCGCGGGAGTGCCGGGCGTGGGCGTGCCAGGCGTCGGTGTACCCGGCGTTGGTGTTCCGGGCGGAGGTGTCCCCGGAGCTGGGGTTCCCGGTGGGGGTGTACCGGGCGTCGGGGTTCCCGGTGTGGGTGTCCCAGGTGGCGGCGTTCCCGGGGCGGGCGTACCTGGAGTGGGTGTGCCAGGAGTCGGCGTCCCAGGAGTCGGCGTACCAGGAGGTGGTGTTCCCGGGGCCGGAGTTCCCGGCGGAGGAGTTCCCGGCGTCGGCGTCCCTGGGGTCGGCGTCCCGGGAGGTGGAGTACCCGGAGCAGGAGTGCCGGGAGTCGGTGTACCTGGTGTCGGTGTCCCTGGTGTAGGTGTCCCGGGTGGTGGGGTGCCAGGTGCTGGCGTACCTGGGGGGGGGGTTCCTGGCGTAGGCGTTCCGGGGGTGGGCGTTCCGGGCGGCGGGGTGCCGGGAGCAGGTGTCCCCGGCGTTGGTGTACCGGGGGTTGGTGTCCCAGGCGTAGGTGTGCCCGGTGGAGGGGTGCCGGGAGCTGGAGTGCCTGGAGGGGGTGTACCAGGGGTCGGTGTTCCCGGTGTAGGAGTACCGGGGGGCGGAGTCCCAGGAGCCGGCGTGCCGGGTGTTGGAGTCCCGGGAGTCGGAGTCCCTGGGGTAGGCGTTCCAGGGGGAGGGGTCCCCGGTGCAGGGGTTCCTGGCGGTGGTGTCCCAGGCGGTTCGGGAGGAGGCTCGGGTGCGCATATCGTAATGGTCGATGCATACAAGCCCACGAAAGGAGGTTCAGGCGGCGGAAGCGGTGGTGGAAGCGGAGGTGGGTCAGGCGGAGGCTCAGGGGGAGGTTCGGGTGGCGGTTCGGGAGGAGGCTCGGGTGCTGACCCGGCCTTCGGCGGCTTCGAAACCCTCCGCGTCGCTGGCGCGGCGGCTCAAGGCTCGCACAACGCCAACGGCTTCACGGCTCTGCAACTGGGCGCGACGGCGGGTGCGACGACCTTCACCAACGTTGCGGTGAATGTCGGCCTGACCGTTCTGGCGGCTCCGACCGGTACGACGACCGTGACCCTGGCCAACGCCACGGGCACCTCGGACGTGTTCAACCTGACCCTGTCGTCCTCGGCCGCTCTGGCCGCTGGTACGGTTGCGCTGGCTGGCGTCGAGACGGTGAACATCGCCGCCACCGACACCAACACGACCGCTCACGTCGACACGCTGACGCTGCAAGCCACCTCGGCCAAGTCGATCGTGGTGACGGGCAACGCCGGTCTGAACCTGACCAACACCGGCAACACGGCTGTCACCAGCTTCGACGCCAGCGCCGTCACCGGCACGGGCTCGGCTGTGACCTTCGTGTCGGCCAACACCACGGTGGGTGAAGTCGTCACGATCCGCGGCGGCGCTGGCGCCGACTCGCTGACCGGTTCGGCCACCGCCAATGACACCATCATCGGTGGCGCTGGCGCTGACACCCTGGTCTACACCGGCGGTACGGACACCTTCACGGGTGGCACGGGCGCGGATATCTTCGATATCAACGCTATCGGCACCTCGACCGCTTTCGTGACGATCACCGACGCCGCTGTCGGCGACAAGCTCGACCTCGTCGGCATCTCGACGAACGGCGCTATCGCTGACGGCGCCTTCGGCGCTGCGGTCACCCTGGGCGCTGCTGCGACGCTAGCTGACTGGGAAAACCCTGGCGTTAATCGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTC.C. crescentus strains into 80\u2009mL of PYE in a 250\u2009mL glass flask. All cultures were grown in an Innova 44 incubator shaker with a 2-inch orbit. These cultures were grown at 30\u2009\u00b0C at 250 rpm, and BUD-ELMs typically formed within ~24\u201330\u2009h. To explore the effect of growth parameters on BUD-ELM size, the flask volume, shaking speed, and culture volume were varied from 125 to 500\u2009mL, 0 to 250 rpm, and 25 to 160\u2009mL, respectively. The complete list of conditions tested can be found in Supplementary Tables\u00a0Unless indicated otherwise, BUD-ELMs were grown by inoculating a single colony of 2 piece was broken off from the desiccated material and inoculated into 80\u2009mL of fresh PYE and grown under standard conditions. We detected BUD-ELM formation in 48\u2009h for material dried over 7 or 14 days, and in 72\u2009h for material dried over 21 days.To test the ability of BUD-ELMs to re-seed their own growth, BUD-ELMs grown under standard conditions were collected, transferred in a petri dish for 7, 14, or 21 days and left on the bench at room temperature. The material dried out in 24\u201348\u2009h. To re-seed material growth, a ~0.3 to 0.5\u2009cmE. coli were assembled from existing constructs by substituting the mRFP sequence with the GFP sequence (below). Similarly, plasmids pSMCAF032 and pSMCAF029 used for the expression of GDH and SpyCatcher-GSH from E. coli were assembled by introducing the GDH sequence in the same position. These plasmids were transformed into chemically competent BL21(DE3) cells (New England Biolabs \u2013 C2527H); single transformants were selected using ampicillin resistance.Plasmids pSMCAF015 and pSMCAF016 used for the expression of GFP and SpyCatcher-GFP from ATGCGTAAAGGCGAAGAGCTGTTCACTGGTGTCGTCCCTATTCTGGTGGAACTGGATGGTGATGTCAACGGTCATAAGTTTTCCGTGCGTGGCGAGGGTGAAGGTGACGCAACTAATGGTAAACTGACGCTGAAGTTCATCTGTACTACTGGTAAACTGCCGGTTCCTTGGCCGACTCTGGTAACGACGCTGACTTATGGTGTTCAGTGCTTTGCTCGTTATCCGGACCATATGAAGCAGCATGACTTCTTCAAGTCCGCCATGCCGGAAGGCTATGTGCAGGAACGCACGATTTCCTTTAAGGATGACGGCACGTACAAAACGCGTGCGGAAGTGAAATTTGAAGGCGATACCCTGGTAAACCGCATTGAGCTGAAAGGCATTGACTTTAAAGAAGACGGCAATATCCTGGGCCATAAGCTGGAATACAATTTTAACAGCCACAATGTTTACATCACCGCCGATAAACAAAAAAATGGCATTAAAGCGAATTTTAAAATTCGCCACAACGTGGAGGATGGCAGCGTGCAGCTGGCTGATCACTACCAGCAAAACACTCCAATCGGTGATGGTCCTGTTCTGCTGCCAGACAATCACTATCTGAGCACGCAAAGCGTTCTGTCTAAAGATCCGAACGAGAAACGCGATCATATGGTTCTGCTGGAGTTCGTAACCGCAGCGGGCATCACGCATGGTATGGATGAACTGTACAAATAA.GACGTTCCGCTGACCCCGAGCCAGTTTGCGAAAGCGAAAAGCGAGAACTTCGACAAAAAAGTCATCCTGAGCAACCTGAATAAACCGCACGCTCTGCTGTGGGGTCCGGATAATCAGATTTGGCTGACCGAACGCGCAACCGGTAAAATTCTGCGCGTTAACCCGGAAAGCGGCAGCGTTAAAACCGTCTTTCAGGTTCCGGAAATCGTTAACGACGCAGACGGTCAAAACGGTCTGCTGGGTTTTGCGTTTCATCCGGACTTCAAAAACAACCCGTACATCTACATCAGCGGCACCTTCAAAAACCCGAAAAGTACCGACAAAGAGCTGCCGAATCAGACCATCATCCGTCGCTATACCTACAACAAAAGCACCGACACCCTGGAAAAACCGGTTGATCTGCTGGCAGGTCTGCCGAGTAGTAAAGATCATCAGAGCGGTCGTCTGGTAATTGGTCCGGACCAGAAAATCTACTATACCATTGGCGATCAGGGCCGTAACCAACTGGCATACCTGTTTCTGCCGAACCAAGCACAACATACCCCGACCCAACAAGAACTGAACGGCAAAGACTACCACACCTACATGGGCAAAGTTCTGCGTCTGAATCTGGACGGTAGCATTCCGAAAGACAACCCGAGCTTCAACGGCGTTGTTAGCCATATCTATACCCTGGGTCACCGTAATCCGCAAGGTCTGGCATTTACCCCGAACGGTAAACTGCTGCAGTCTGAACAGGGTCCGAATTCTGACGACGAAATCAACCTGATCGTTAAAGGCGGCAATTACGGTTGGCCGAACGTTGCAGGCTATAAAGACGATAGCGGCTATGCATACGCGAATTATAGCGCAGCGGCAAACAAAAGCATCAAAGACCTGGCCCAGAACGGTGTTAAAGTTGCAGCAGGCGTTCCGGTTACCAAAGAAAGCGAGTGGACCGGCAAAAACTTTGTTCCGCCGCTGAAAACCCTGTATACCGTCCAGGACACCTACAACTATAACGATCCGACCTGCGGCGAAATGACCTATATTTGCTGGCCGACCGTTGCACCGAGTTCTGCATACGTTTACAAAGGCGGCAAAAAAGCGATCACCGGTTGGGAAAATACCCTGCTGGTTCCGAGTCTGAAACGCGGCGTTATCTTCCGCATCAAACTGGATCCGACCTATAGTACCACCTACGACGATGCCGTTCCGATGTTCAAAAGCAACAACCGTTATCGCGACGTTATTGCAAGTCCGGACGGTAACGTTCTGTACGTTCTGACCGATACCGCAGGTAACGTTCAGAAAGACGACGGTAGCGTTACCAATACCCTGGAAAATCCGGGTAGCCTGATCAAATTCACCTACAAAGCGAAATGA.E. coli BL21(DE3) harboring plasmids pSMCAF015 and pSMCAF016, for expression of GFP and SpyCatcher-GFP, respectively, were inoculated in 25\u2009mL of RM minimal media with 0.2% w/v glucose and 100\u2009\u00b5g/mL ampicillin. After ~16\u2009h of growth at 37\u2009\u00b0C and 250 rpm, cells were used to inoculate 0.5\u2009L of RM minimal media with 0.2% v/v glycerol, 100\u2009\u00b5g/mL ampicillin and 0.0004% antifoam (Antifoam 204) to a final OD600 ~0.05. The cultures were allowed to grow at 37\u2009\u00b0C until mid-log phase. Protein production was induced with 0.2% w/v l-arabinose with incubation at 30\u2009\u00b0C for ~17\u2009h.Single colonies of g for 30\u2009min, resuspended in lysis buffer and lysed using Avestin Emulsiflex C3 Homogenizer. The lysate was centrifuged at 12,000 \u00d7 g for 1\u2009h and the supernatant was collected for protein purification. The proteins were purified using Immobilized Metal Affinity Chromatography (IMAC) with a HisTrap FF column and buffers containing 50\u2009mM Tris pH 8.0, 300\u2009mM NaCl, 5% v/v glycerol, and 10\u2013250\u2009mM Imidazole. After protein purity was confirmed by SDS-PAGE, the protein was dialyzed into TEV-cleavage buffer and the 6 x His-tag was cleaved using TEV protease by agitation at 4\u2009\u00b0C for 4\u2009h. The cleaved protein was stored at \u221280\u2009\u00b0C in 50\u2009mM NaPO4 pH 8.0, 300\u2009mM NaCl, and 5% v/v glycerol.Cells were harvested by centrifugation at 8000 \u00d7 2O and lyophilized for 5\u2009h in a Labconco Freezone 4.5 freeze dryer. Tubes were then weighted.Eight BUD-ELMs were grown under standard conditions. Samples were harvested from liquid cultures, placed in Eppendorf tubes, washed once in ddHl-lysine coated silicon substrates were immersed in Falcon\u2122 round-bottom polypropylene culturing tubes containing 3\u2009mL fresh C. crescentus cell culture at an OD600 of 0.3\u20130.5. Culture tubes were then centrifuged at 3000 \u00d7 g for 10\u2009min to immobilize the cells onto the silicon substrate. The silicon substrate was washed with 2\u2009mL of sterile PYE to remove loosely-bound cells before being mounted to a metal puck and transferred to the AFM sample stage. In situ AFM was performed on an Asylum Cypher AFM using soft tapping mode. A fluid cell and two syringe pumps were assembled to control liquid flow and PYE medium was supplied to maintain cell viability during imaging. The AFM probe consisted of a sharp silicon tip on a silicon nitride cantilever with a spring constant of 0.09\u2009N/m. Cells were imaged in native state without fixation. A 100\u2013200\u2009mV amplitude setpoint was used to apply minimum forces (~0.2\u2009nN) to cells during imaging.Poly-2 precleaned silicon substrate was dipped into the pellicle forming cell culture with an entry angle of ~60\u00b0 perpendicular to the water surface. The silicon substrate was then retrieved, and the pellicle structure was dried under an N2 atmosphere for 2\u2009h. The dried pellicle structure on the substrate was mounted to a metal puck and transferred to the AFM sample stage. AFM imaging was performed on an Asylum Cypher AFM using soft tapping mode in air. A Tap-150 tip (BudgetSensors) with a 5\u2009N/m force constant was used to image the pellicle structure.To bind the BUD-ELM pellicle to a silicon substrate, a 2\u2009cmSmall pieces of BUD-ELMs grown under standard conditions were placed between a slab of PYE agarose (1.5% w/v) and a glass coverslip-bottomed 50-mm Petri dish with a glass diameter of 30\u2009mm (MatTek Corporation) and imaged with an optical inverted microscope. Optical microscopy data were acquired using the software NIS-Elements AR (version 4.51.01). All microscopy pictures presented were generated using ImageJ software (version 2.0.0-rc-69/1.52p).d-xylose\u2014to induce the expression of mKate2, in a 125\u2009mL flask and grown for 24\u2009h at 30\u2009\u00b0C at a shaking speed of 250\u2009rpm. BUD-ELMs of similar dimensions were collected and washed twice with 1\u2009mL of 0.01\u2009M Phosphate-buffered saline (PBS), in a centrifuge tube. They were then incubated in 1\u2009mL of 0.01\u2009M PBS, at 30\u2009\u00b0C, with the following staining agent: 80\u2009\u00b5g of SpyCatcher-GFP or GFP for 1\u2009h, 1% Congo Red (Thermo Fisher Scientific\u2014D275) or 100\u2009\u00b5g DiO (DiOC18(3) \u2212 3,3\u2032-Dioctadecyloxacarbocyanine Perchlorate) for 20\u2009min. Samples were washed three times with 1\u2009mL 0.01\u2009M PBS and then a small amount was placed between a slab of PYE agarose (1.5% w/v) and a glass coverslip-bottomed 50-mm Petri dish with a glass diameter of 30\u2009mm (MatTek Corporation). To acquire the low-magnification images to distinguish the matrix from the cells.Single colonies of BUD-ELM strain RCC002) were inoculated in 30\u2009mL PYE with 0.15% were inoC. crescentus BUD-ELM strain (RCC002) were cultured in standard (shaking) or static (not shaking) conditions until they reached stationary phase . The supernatant of each culture was extracted and loaded onto a TGX Stain-Free\u2122 gel (Biorad). After running, the gel was transferred to a 0.2\u2009\u03bcm nitrocellulose membrane and blocked for 1\u2009h at room temperature with SuperBlock\u2122 blocking buffer (Thermo Scientific). Membranes were then washed four times in TBST buffer before incubation in a 1:5000 dilution of Monoclonal ANTI-FLAG\u00ae antibody antibody from Sigma Aldrich \u2013 A8592-.2MG) produced in mouse, clone M2, purified immunoglobulin, buffered aqueous glycerol solution) solution for 1\u2009h at room temperature. Membranes were washed an additional four times in TBST buffer before Clarity Max Western ECL Substrate (Biorad) was applied and the membrane was imaged for chemiluminescence. Each sample lane represents an independent sample, grown from separate individual colonies.For immunoblot analysis of culture supernatant, cultures of \u0394ELP60 BUD-ELM strains were grown under standard conditions. Planktonic cells from each colony were collected and washed 3 times in fresh PYE media to remove any free BUD protein not attached to the cell surface. After washes, all samples were normalized to an OD600 value of 0.225 in 0.5 x PYE, 1 x Laemmli buffer. Each sample was then boiled for 5\u2009min, and 10\u2009uL was loaded onto a TGX Stain-Free\u2122 gel (Biorad). All subsequent steps follow the protocol for immunoblotting described above. Protein molecular weight has been determined by image analysis with ImageJ.For whole-cell immunoblot analysis, the original and L) values.To determine the apparent BUD-ELM size, the bottom of glass flasks where BUD-ELM grew was imaged using a Canon EOS 77D camera. Flasks were positioned within a reflective photobox on a clear plastic surface such that the bottom of the flask stood approximately 11.5\u2009cm above the camera lens. These images were separated into RGB channels using MATLAB R2020b. A subset of the blue channel images was then input into the image classification software ilastik (version 1.3.3), as a training set for the autocontext workflow. The first stage of training separated images into three different classifications: background, scattered material, and bundled material. Scattered material was defined as overlapping regions of small aggregates not associated with each other, whereas bundled material referred to larger, connected pieces of material. The second stage of training distinguished bundled material from the rest of the image. Both stages of training utilized all 37 features provided within the ilastik workflow . From the results of the training set, the second stage segmentation masks for all blue channel images were calculated, and loaded into MATLAB R202b. From these masks, the flat area of each piece of material was calculated, and the top five percentile of size from each image was averaged to yield a representative size measurement. For each image, a conversion rate between pixels and squared centimeters was determined using the standard flask diameter as a reference point. Size measurements were averaged between samples and plotted with respect to their calculated (d5)(kLa); Ne\u2019 \u2013 modified Newton number (dimensionless); P \u2013 power input (W); Re \u2013 Reynold\u2019s number (dimensionless); VL \u2013 culture volume (m3); \u03b7app \u2013 dynamic apparent viscosity (Pa\u2219s); \u03c1 \u2013 liquid density (kg/m3); d0 \u2013 orbital shaking diameter (m); D \u2013 diffusion coefficient (m2/s); v \u2013 kinematic viscosity (m2/s); g \u2013 acceleration of gravity (m/s2).ELP60, and \u2206rsaA1\u2013250 strains, respectively) were evaluated on a strain-controlled rheometer (ARIES G2) equipped with an 0.1\u2009rad 8-mm diameter cone plate. BUD-ELMs were grown in standard conditions. An approximate volume of 100\u2013200\u2009\u00b5L of BUD-ELMs were collected into a 1.5\u2009mL centrifuge tube and spun for 10\u2009s at 3200 rcf with a mini centrifuge (VWR\u00ae \u2013 C0803). This allowed for the material to collect at the bottom as a homogeneous paste. The supernatant was removed and 150uL of fresh PYE were added on top of BUD-ELMs to prevent desiccation. Strain sweep experiments from 0.1 to 100% strain amplitudes were performed at a fixed frequency of 3.14\u2009rad/s. Frequency sweep experiments from 100 to 0.1\u2009rad/s were performed at a 0.35% strain amplitude. Data were acquired using TRIOS software (version 4.2.1.36612).The rheological properties of BUD-ELMs produced from strains RCC002, RCC004 and MFm152 and incubated with 7\u2009mL of 6 ppm CdCl2 (Sigma Aldrich \u2013 202908) in ddH2O for 90\u2009min on an orbital shaker. After incubation, the Cd2+ concentration of the supernatant was measured by ICP-MS. Specifically, 5\u2009\u00b5L of the supernatant was diluted in 4.995\u2009mL 1% HNO3 with 5\u2009\u00b5g/mL Indium (In), as standard for data analysis (Perkin Elmer N9303741). This diluted solution was run on a Perkin Elmer Nexion 300 ICP-MS with two isotopic measurements (Cd2+ 111 and Cd2+ 112) and In 115 as the internal standard. Data was acquired using Syngistix software.To measure the ability of BUD-ELMs to bind CdE. coli BL21(DE3) harboring pSMCAF032 and pSMCAF029, for expression of GDH and SpyCatcher-GDH, respectively, were inoculated in 25\u2009mL of Terrific broth (TB) with 100\u2009\u00b5g/mL ampicillin. After ~16\u2009h of growth at 37\u2009\u00b0C and 250\u2009rpm, cells were used to inoculate 0.5\u2009L of TB with 0.02% antifoam (Antifoam 204) and 100\u2009\u00b5g/mL ampicillin to a final OD600 ~0.05. The cultures were allowed to grow at 37\u2009\u00b0C until the mid-log phase. Protein production was induced with 0.2% w/v L-arabinose with incubation at 30\u2009\u00b0C for ~17\u2009h.Single colonies of g for 5\u2009min, resuspended in lysis buffer (0.01\u2009M MOPS) and lysed using sonication. After centrifugation at 12,000 \u00d7 g for 1\u2009h, the GDH (or SpyCatcher-GDH) in the supernatant was reconstituted by adding a final concentration of 3\u2009mM Ca2+ and 0.06\u2009mM PQQ and incubated for 15\u2009min at 4\u2009\u00b0C. BUD-ELMs, washed once with 0.01\u2009M PBS, were incubated with reconstituted and non-reconstituted cell lysates for 2\u2009h at 4\u2009\u00b0C and then washed three times with 0.01\u2009M PBS. A small piece of functionalized BUD-ELMs , 1\u2009mL DCPIP (20\u2009mg dissolved in 5\u2009mL of DI water), and 1\u2009mL phenazine methosulfate (PMS) (45\u2009mg dissolved in 5\u2009mL of DI water) were prepared. Analytical samples (~3\u2009uL) were mixed with the reagent to 190\u2009\u00b5L. The reaction was initialized by adding 10\u2009\u00b5L glucose (2\u2009M). Glucose consumption was correlated to the consumption of DCPIP (2:1 ratio), which was quantified colorimetrically by absorption at 600\u2009nm. A representative curve is shown in Supplementary Fig.\u00a0The activity of GDH functionalized material was quantified with a modified colorimetric 2,6-dichlorophenol (DCPIP) assayFigure\u00a0Figures\u00a0Figures\u00a0The blots shown in Fig.\u00a0Further information on research design is available in the\u00a0Supplementary InformationReporting SummaryPeer Review File"} +{"text": "Liquid\u2013liquid phase separation (LLPS) governs a variety of mesoscale cellular processes. However, less is known about how cells utilize LLPS to drive cellular function. Here, we examined the destruction complex (DC), an organelle which controls Wnt signaling and whose components phase separate. Through a combination of advanced microscopy, CRISPR, computational modeling, and optogenetics, we find that the DC is nucleated by the centrosome and that this nucleation drives efficient signal transduction. Our work not only uncovers a biological function for LLPS but also highlights nucleation as a general method for controlling the function of intracellular condensates. Finally, our findings suggest a thermodynamic coupling between Wnt signal transduction and the cell cycle which could lead to insights into Wnt-driven cancers. Wnt signal transduction is controlled by the destruction complex (DC), a condensate comprising scaffold proteins and kinases that regulate \u03b2-catenin stability. Overexpressed DC scaffolds undergo liquid\u2013liquid phase separation (LLPS), but DC mesoscale organization at endogenous expression levels and its role in \u03b2-catenin processing were previously unknown. Here, we find that DC LLPS is nucleated by the centrosome. Through a combination of CRISPR-engineered custom fluorescent tags, finite element simulations, and optogenetic tools that allow for manipulation of DC concentration and multivalency, we find that centrosomal nucleation drives processing of \u03b2-catenin by colocalizing DC components to a single reaction crucible. Enriching GSK3\u03b2 partitioning on the centrosome controls \u03b2-catenin processing and prevents Wnt-driven embryonic stem cell differentiation to mesoderm. Our findings demonstrate the role of nucleators in controlling biomolecular condensates and suggest tight integration between Wnt signal transduction and the cell cycle. The canonical Wnt signaling pathway is a conserved , morphog2Extracellular Wnt ligands inhibit DC function through a mechanism that is still unclear, but likely involves selective recruitment of DC components to the signalosome, a biomolecular condensate on the plasma membrane that includes Wnt/Frizzled/LRP5/6 clusters and, whe8The \u201cmolecular crucible\u201d model posits that DC LLPS promotes \u03b2-cat degradation in Wnt OFF conditions via concA biophysical mechanism for regulating condensates is control over their nucleation. This principle was recently explored with a synthetic optogenetic system , but theSlimb and localization of \u03b2-cat at the cell membrane, consistent with previous work in fixed specimens (Movies S2 and S3). \u03b2-cat puncta became more difficult to distinguish at higher cytoplasmic concentrations produced by activated Opto-LRP6, but dissolution nearly always preceded appreciable dilute-phase \u03b2-cat accumulation, indicating that they were not simply obscured by higher background levels. Further, of light-stimulated cells, those that were resistant to optogenetic activation maintained their \u03b2-cat puncta . Together, these results indicate that activation of the Wnt pathway causes perinuclear puncta to dissolve, and the presence of these puncta is inversely related to Wnt pathway activation at the population and single-cell levels.To establish whether directly activating the Wnt receptor controls the existence of the puncta, we transduced to-LRP6) K and L. to-LRP6) F. We fout puncta I and J. CSNK1A1, GSK3B, and AXIN1, genes encoding the kinases CK1\u03b1 and GSK3\u03b2 that sequentially phosphorylate \u03b2-cat in the DC, and the primary DC scaffold.We next sought to determine 1) what, if any, cellular structure was organizing these puncta, 2) whether all DC components were colocalized with puncta, and 3) whether these were solid or liquid-like condensates. Because of the sensitivity of LLPS systems to protein concentration , we deciSI Appendix, Fig. S2A): We observed single condensates in G1, two condensates in G2/S, and a \u201cfinger-like\u201d pattern\u2014suggesting association with the mitotic spindle\u2014during late mitosis. These observations, combined with previous reports of perinuclear enrichment of CK1\u03b1, GSK3\u03b2, and Axin1 in fixed cells confirmed that tdmRuby3-CK1\u03b1, tdmRuby3-GSK3\u03b2, and tdmRuby3-\u03b2-cat puncta were indeed colocalized to the centrosome.We found that all tagged proteins were localized into one or two perinuclear puncta A. Timelaed cells \u201331, led ed cells B and GM1Left) that colocalized with centrosomal markers and replicated following cell cycle progression . As protein concentration increased, Axin1, but not APC, caused formation of extracentrosomal puncta throughout the cytoplasm . Interestingly, extracentrosomal Axin1 condensates did not reliably induce formation of extracentrosomal GSK3\u03b2 condensates in these experiments, but often resulted in deenrichment of centrosomal puncta . We reason that this was due to extracentrosomal Axin1 condensates competing for relatively scarce GSK3\u03b2, thereby diluting across all condensates in the cytoplasm.When overexpressed, Axin and APC cross the phase boundary and form liquid condensates in the cytoplasm that are hypothesized to concentrate DC kinases and \u03b2-cat . The facytoplasm C\u2013E. To dSI Appendix, Fig. S2F). These findings establish that all DC components necessary for phosphorylating \u03b2-cat, prior to its ubiquitination, are localized at the centrosome throughout the cell cycle and suggest that DC centrosomal nucleation is generalizable to multiple cell types.The 293Ts are commonly used in experiments probing DC mesoscale structure in\u00a0vivo , 10, butNext, we sought to determine the material state of the centrosomal DC using FRAP on CRISPR-tagged CK1\u03b1, GSK3\u03b2, and \u03b2-cat, as well as of Axin1 and APC at low levels of induction. All centrosomal DC components exhibited mean half-maximal recovery times (\u03c41/2) between 10 s and 60 s E\u2014like inSI Appendix, Fig. S3 A\u2013D). Indeed, synthetic DC scaffolds with these simple attributes have been shown to rescue aberrant Wnt signaling . We found that the nucleated system processed \u03b2-cat and its intermediates more quickly . See SI Appendix, Fig. S3F). This efficiency gain was maintained over a large range of reaction rates . As expected, in systems with high reaction rates, the effect of nucleated phase separation is no longer observed.To test the effects of nucleation on \u03b2-cat processing, we compared simulations in the presence and absence of a nucleation region C. We fou quickly D over a SI Appendix, Fig. S3 H and I, Movie S6). Together, these results demonstrate that nucleation of DC components has the potential to increase \u03b2-cat processing and that a tunable control parameter of this process is the free energy of mixing.Given our findings that nucleation drives efficient processing of \u03b2-cat, we hypothesized that In silico analysis of the DC indicates that processing efficiency in the presence of a nucleator is dependent on client condensation. Imaging of GSK3\u03b2 showed relatively weak enrichment in centrosomal puncta compared to CK1\u03b1, suggesting that increasing nucleation of GSK3\u03b2 would increase the degradation rate of \u03b2-cat in\u00a0vivo. Changing concentration alters both propensity to undergo LLPS and reaction rate and therMovie S7). Notably, activation of Opto-GSK3 strictly resulted in the formation of one or two perinuclear puncta and did not form extracentrosomal condensates, contrasting with results from studies using Cry2 alone and eGFP to human GSK3\u03b2 (\u201cOpto-GSK3\u201d hereafter) and stably transduced it into 293Ts A. Upon ly2 alone . Thus, wSI Appendix, Fig. S4 A and B). We observed a similar effect when analyzing total \u03b2-cat by Western blotting and immunofluorescence staining . Given the modest accumulation of \u03b2-cat in response to Wnt-3a in 293Ts, we tested to see whether Opto-GSK3 clustering was sufficient to blunt \u03b2-cat accumulation induced by either CHIR or a Dox-inducible \u03b2-cat overexpression construct. Indeed, activation of Opto-GSK3 also inhibited both methods for driving \u03b2-cat accumulation in a light-dependent manner . These results demonstrate that increasing DC client nucleation at the centrosome dictates Wnt signal transmission across a wide range of activation regimes.To determine whether increased centrosomal condensation of GSK3\u03b2 controlled Wnt signal transmission, we activated Opto-GSK3 in cell lines with three distinct methods for increasing the cellular concentration of \u03b2-cat: ligand-induced, kinase inhibition, and dox-induced gene up-regulation. We found that Opto-GSK activation abolished both Wnt-3a-induced \u03b2-cat accumulation and transcriptional activation as measured by TOPFlash fluorescence D and E. Changes in \u03b2-cat concentration differentiate a variety of stem cell populations, including human embryonic stem cells (hESCs) , 40. HavBuilding on recent discoveries suggesting that LLPS plays a role in DC structure, we sought to understand how the biophysics of DC proteins regulate DC function in live cells. Through a combination of superresolution microscopy, in silico modeling, and optogenetic methods to isolate and probe the phase diagram, we discovered that the mesoscale structure of the DC is a liquid condensate nucleated by the centrosome. The complementarity of these methods allowed us to identify a function for nucleation: acceleration of the catalytic action of DC proteins, thereby promoting efficient processing of \u03b2-cat.The presence of many cytoplasmic Axin1 and APC droplets in mildly overexpressed cellular conditions has beenOur results support a \u201cmolecular crucible\u201d model of \u03b2-cat degradation, in which multivalent DC scaffolds concentrate DC clients in nucleated droplets to increase \u03b2-cat phosphorylation rate. Assembly line models for \u03b2-cat degradation have been proposed , 42, andWe found that centrosomal DCs cease to concentrate \u03b2-cat under Wnt ON and GSK3\u03b2 chemical inhibition, but the mechanism for this change remains unclear. Multiple DC components that bind \u03b2-cat\u2014including Axin1, GSK3\u03b2, and CK1\u03b1\u2014are also binding partners of the Frizzled-LRP6 signalosome , 45, a kDrosophila embryos leads to only minor tissue-level defects in canonical Wnt signaling and overall morphology coupling the DC to the centrosome? Axin1 is known to associate with \u03b3-tubulin and is arphology , 48, indFinally, centrosomal nucleation of the DC suggests a potential function in coordinating cell cycle progression with Wnt signaling. We found two DC droplets in cells with duplicated centrosomes, suggesting that the DC is split along with centrosomes during mitosis. Nonnucleated droplets would be randomly partitioned into daughter cells, leading to potentially detrimental asymmetry in Wnt signaling capacity of the growing tissue. Cell cycle synchronization could be a method of reducing heterogeneity in Wnt-induced stem cell differentiations.Overall, our studies suggest an integral role for LLPS nucleation in regulating the activity of membraneless organelles in\u00a0vivo. The power of observing proteins in their endogenous contexts, coupled with the ability to precisely tune interaction strength without altering protein function or concentration, enables the functional dissection of membraneless organelles.2 Dulbecco\u2019s Modified Eagle Medium, high glucose GlutaMAX medium supplemented with 10% fetal bovine serum , and 1% penicillin-streptomycin. The hiPSC WTC was gifted by the B.P. laboratory (purchased from Coriell). The hiPSCs were propagated on Matrigel-coated tissue culture plates using serum-free essential 8 (Gibco) culture conditions in standard environments consisting of 5% carbon dioxide at 37\u2009\u00b0C. Experiments in hESC lines were performed using the H9 hESC cell line purchased from the William K. Bowes Center for Stem Cell Biology and Engineering at University of California, Santa Barbara (UCSB). Cells were grown in mTeSR Plus medium (Stem Cell Technologies) on Matrigel (Corning)-coated tissue culture dishes and tested for mycoplasma in 2-mo intervals.Human 293T cells were cultured at 37\u2009\u00b0C and 5% COPiggyBac (CMV) Backbone fwd: tgacgcccgccccac rev: ggtaagctttttgcaaaagcctaggcc. H2B + 18AA linker fwd: cctaggcttttgcaaaaagcttaccatgccagagccagcgaagtc rev: GCATATTTTCCTTGATGAGTTCACTCATccCagTatGtcCgcCggAg. mTagBFP2 fwd: ATGAGTGAACTCATCAAGGAAAATATGCACATG rev: CGTCCCCGCAGGTCAACAAACTTCCGCGACCTTCTCCGCTCCCATTGAGCTTATGGCCGAGTTTGCTG.-3\u2032X-Flag-NLS-Cas9-HA-Avidin fwd: GGAAGTTTGTTGACCTGCGGGGACGTGGAAGAAAACCCGGGTCCAgactataaggaccacgacggagactac rev: gctgcgggtcgtggggcgggcgtcaggatccagacgccgcagThe pPig_H2B-mTagBFP2::t2A::Cas9-Avidin was constructed via subcloning human H2B, mTagBFP2, and Cas9-Avidin provided by M.Z.W. into an expression vector bearing a cytomegalovirus CMV promoter and flanking PiggyBac transposase-compatible inverted terminal repeats using Gibson Assembly according to supplier instructions. Each of the PCR fragments used was amplified using the following primers:rd gen tet ON-responsive promoter, and EF1\u03b1-driven Blasticidin selection cassette. The following primers were used: XLone Backbone fwd: taaactagtagaccacctcccctgcg, rev: ggtacctttacgagggtaggaagtgg, human Axin1 fwd: cacttcctaccctcgtaaaggtaccatgaatatccaagagcagggtttcccc, rev: CCATgctTCCgCCgCCACTACCgCCgtccaccttctccactttgccgatgatc, 7AA link- tdmRuby3 fwd: GGcGGTAGTGGcGGcGGAagcATGGTTAGCAAAGGGGAGGAGC, rev: gcaggggaggtggtctactagtttaCTTGTACAGCTCGTCCATGCCGXLone-Axin-tdmRuby3 was constructed via PCR and Gibson Assembly, subcloning from the following constructs: Flag-Axin1 purchased from Addgene (#109370), tdmRuby3 from M.Z.W. into XLone-GFP purchased from Addgene (#96930) containing-3\u2032XLone-bCat-tdmRuby3 was constructed via PCR and Gibson Assembly, subcloning from the following constructs: XLone-Axin-tdmRuby3 (above) and Human Beta-catenin GFP purchased from Addgene (#71367). The following primers were used: XLone Backbone fwd: GGcGGTAGTGGcGGcGGAagcATGGTTAGCAAAGGGGAGGAGC, rev: ggtacctttacgagggtaggaagtgg, human bcat fwd: cacttcctaccctcgtaaaggtaccatggctactcaagctgatttgatggagttg, rev: CCATgctTCCgCCgCCACTACCgCCcaggtcagtatcaaaccaggccagcpPig-Hygro Backbone fwd: GGACGTGGAAGAAAACCCGGGTCCAatgggtaaaaagcctgaactcaccgc, rev: cattccacagggtcgacagtacaagc,Cuo + CMV fwd: cttgtactgtcgaccctgtggaatgcgttacataacttacggtaaatggcccgc,rev: actgatcatatgaagctgcagccatgaattcggtaccggatccagtcgactag,APC fwd: atggctgcagcttcatatgatcagttgttaaagcaag,rev: CCATgctTCCgCCgCCACTACCgCCaacagatgtcacaaggtaagacccagaatg,7AAlinker-tdmirfp670nano fwd: GGcGGTAGTGGcGGc,rev: ggcgccaaaacccggcgcggaggccttaGGACTGCTGTATTGCAATGCCAACTAC,UbC-CymR-V5-T2A fwd: ggcctccgcgccggg,rev: TGGACCCGGGTTTTCTTCCACGTCCCCGCAGGTCAACAAACTTCCGCGACCTTCTCCGCTCCCcgtagaatcgagaccgaggagaggThe pPig_CuO-APC-tdmIRFP670::CymR was constructed via PCR and Gibson Assembly from the following constructs: pCuo CA Rac1 CMV + cumate operon purchased from Addgene (#84643), human APC open reading frame (ORF) purchased from Addgene (#16507), tdmirfp670nano from M.Z.W., and human ubiquitin C-driven CymR Cuo repressor purchased from Addgene (#119907) into pPig-Hygro transposase backbone from M.Z.W. PCR fragments were amplified using the following primers:Arabadopsis thaliana, tdmIRFP from M.Z.W. and human GSK3\u03b2 purchased from Addgene (#16260) ORFs were supplied to VectorBuilder for cloning and EF1\u03b1-driven expression into third-generation lentiviral backbone. Vectorbuilder provided the desired final, sequenced plasmid.The pLV_Cry2-tdeGFP-GSK3b was obtained via synthesis and cloning services provided by Vector Builder Inc. Full details are available upon request, but, briefly: primary plasmids containing ATGGCTGAAGGCAGCGTGGCCCGACAGCCAGACCTTTTGACTTGTGACGATGAACCAATCCACATACCGGGGGCAATACAACCTCATGGTCTCCTTCTGGCGCTTGCTGCCGACATGACTATAGTGGCCGGCTCTGACAACTTGCCGGAATTGACCGGACTTGCTATTGGGGCGTTGATTGGGCGCTCTGCCGCTGATGTATTTGATTCCGAGACACATAATAGGCTTACTATAGCCCTCGCCGAACCAGGGGCTGCCGTCGGCGCTCCTATAACAGTTGGGTTCACGATGCGAAAAGATGCTGGGTTCATTGGTAGCTGGCATCGCCACGATCAACTTATCTTCCTTGAGCTTGAACCCCCTCAACGGGACGTTGCGGAACCCCAAGCTTTCTTTAGAAGGACCAATTCAGCCATAAGGCGCCTTCAGGCCGCAGAGACATTGGAGTCCGCGTGTGCGGCAGCAGCGCAGGAAGTACGAAAGATCACGGGATTTGACCGGGTTATGATTTACAGATTCGCATCTGATTTCTCCGGGGAAGTCATCGCGGAGGATCGGTGTGCAGAAGTGGAAAGCAAGCTTGGTTTGCATTACCCCGCATCTACGGTTCCGGCCCAAGCGAGGAGACTGTATACGATAAACCCAGTGAGGATCATACCTGACATAAATTATAGACCGGTTCCCGTTACGCCAGACCTGAACCCCGTCACAGGCAGGCCAATAGACTTGTCTTTTGCAATCCTGCGGTCAGTCTCACCTGTTCACCTCGAGTTTATGAGGAACATAGGGATGCATGGGACGATGAGCATCTCAATCCTGAGAGGTGAACGGCTCTGGGGACTTATTGTTTGTCATCATCGCACACCGTATTACGTTGACCTTGATGGTCGCCAGGCCTGCGAACTCGTAGCTCAAGTATTGGCCTGGCAGATCGGTGTTATGGAGGAAAGCGGTCATGGGACTGGGAGTACAGGTAGCGGCAGCTCTAGTGGCACCTCCandTAGCGGCAGCTCTAGTGGCACCTCCATGGCAGAAGGGTCCGTAGCAAGGCAACCTGACTTGTTGACCTGTGATGATGAACCGATTCACATTCCTGGAGCAATTCAACCGCATGGGCTGCTCCTTGCTTTGGCAGCGGACATGACGATCGTCGCCGGCTCCGATAACCTGCCCGAGTTGACGGGCTTGGCGATAGGAGCCCTGATAGGCCGCTCAGCCGCTGACGTATTCGATAGCGAAACGCATAACCGGCTTACAATCGCCTTGGCTGAACCGGGCGCGGCCGTGGGAGCACCGATTACTGTAGGCTTTACAATGAGAAAAGACGCCGGCTTTATCGGGTCATGGCACCGACATGACCAGCTGATTTTCCTGGAATTGGAGCCCCCGCAGCGGGATGTAGCCGAACCACAGGCCTTCTTCCGGCGCACTAACTCCGCAATTAGGAGACTGCAGGCAGCTGAGACTTTGGAATCAGCATGCGCGGCAGCTGCACAAGAAGTCCGGAAAATCACGGGTTTTGACCGAGTCATGATCTATAGATTCGCGAGCGATTTCTCAGGAGAAGTTATTGCGGAAGACCGATGCGCGGAGGTAGAATCTAAGCTTGGGTTGCACTACCCCGCCTCCACCGTTCCGGCGCAAGCCAGACGGCTCTATACCATTAATCCGGTGCGGATCATTCCAGATATAAATTACCGGCCTGTACCTGTGACACCGGATTTGAACCCTGTCACGGGCCGACCGATAGACCTCAGCTTCGCTATATTGCGATCTGTGTCACCGGTCCACCTCGAGTTTATGAGGAATATAGGCATGCATGGTACAATGTCCATTTCCATTCTCCGGGGTGAACGGCTTTGGGGCCTCATCGTTTGTCACCATCGAACACCGTATTACGTCGATCTCGACGGCAGACAGGCATGTGAGTTGGTCGCTCAGGTACTCGCTTGGCAGATAGGGGTAATGGAGGAGThe pPig_8XTOP_tdIRFP_Puro was constructed via PCR and Gibson Assembly from the following constructs: pPig_H2B-mTagBFP2::t2A::Cas9-Avidin (above), M50 Super 8\u00d7 TOPFlash purchased from Addgene (#12456), and codon-optimized tandem (td) IRFP ordered from Twist Biosciences as overlapping gene fragments with the sequences:PiggyBacPuro backbone fwd: ACCTGCGGGGACGTGGAAGAAAACCCGGGTCCAatgaccgagtacaagcccacggtg,rev: cattccacagggtcgacagtacaagcaaaaag.8\u00d7 TOPFlash fwd: cttgtactgtcgaccctgtggaatgaagtgcaggtgccagaacatttctc,rev: GTCGGGCCACGCTGCCTTCAGCCATggtggctttaccaacagtaccgg.tdIRFP1 fwd: ATGGCTGAAGGCAGCGTGGC,rev: GGAGGTGCCACTAGAGCTGC. tdIRFP2fwd: TAGCGGCAGCTCTAGTGGCAC,rev: GGTTTTCTTCCACGTCCCCGCAGGTCAACAAACTTCCGCGACCTTCTCCGCTCCCCTCCTCCATTACCCCTATCTGCCAAGCGPCR fragments were amplified using the following primers:E. coli Transformation Kit and Buffer Set (Zymo Research #T3002), cultured on lysogeny broth agar plates to select for antibiotic resistance using standard workflows for molecular cloning and DNA production and pMD 2.G at a 1:0.88:0.11 mass ratio using standard PEI-based transfection procedures (50). Cells were incubated for 24 h before replacing with fresh media and allowing for lentiviral production for an additional 48 h. Supernatant was harvested, filtered through a 0.22-\u03bcm filter and added to plated cells for transduction. Note that all steps for lentiviral production, transduction, and subsequent maintenance of cell lines were carried out in the presence of far-red light or the complete absence of light in an attempt to eliminate the possibility of Cry-2 opto-GSK3 clustering interference with cell growth or virus production.Genomic edits in 293Ts were carried out in cells constitutively expressing Cas9 to maximize editing efficiency.fwd: acgcgccctgtagcgrev: cttaatgcgccgctacagggcgcgtggtacctctagagccatttgtctgc,assembled, cloned, purified, and verified as described in the previous section. The baseline pCab_minimal construct was then subsequently used for production of gRNAs targeting exon1 of the human genomic loci of CTNNB1, CSNK1a1, and GSK3B. Primers creating one to three (depending on protospacer adjacent motif site availability/predicted on/off-target editing scores) unique protospacers targeting the 50-bp window surrounding the first codon of each gene were annealed and cloned into the pCAB_minimal via BbsI digestion and ligation using standard protocols (CTNNB1_1 fwd: caccgTGAGTAGCCATTGTCCACGC rev: aaacGCGTGGACAATGGCTACTCACTNNB1_2 fwd: caccgTGAAAATCCAGCGTGGACAA rev: aaacTTGTCCACGCTGGATTTTCAcCTNNB1_3 fwd: caccGCGTGGACAATGGCTACTCA rev: aaacTGAGTAGCCATTGTCCACGCCSNK1a1_1 fwd: caccGGCCAAGCCCCGACACCTCT rev: aaacAGAGGTGTCGGGGCTTGGCCCSNK1a1_2 fwd: caccgAGGCTGAATTCATTGTCGGA rev: aaacTCCGACAATGAATTCAGCCTGSK3B fwd: caccCGAAGAGAGTGATCATGTCA rev: aaacTGACATGATCACTCTCTTCGA vector expressing guide RNA (gRNA) and Cas9 obtained from M.Z.W. was subcloned to remove the unnecessary Cas9 ORF via PCR using the following primers:rotocols . The folAXIN1 gRNAs were ordered complete from IDT. Four different protospacer sequences were used (in separate reactions) with the same homology-directed repair (HDR) template to maximize chance of target locus cutting. Cells from each reaction were then pooled 7 d after transfection and subsequently enriched together.AXIN1_1: GGCCGTCCTGCCCGTCTTTGAXIN1_2: GTCTTTGAGGAGAAGATCATAXIN1_3: gTCTTTGAGGAGAAGATCATCAXIN1_4: GGAGAAGATCATCGGCAAAG.Protospacer sequences:tdmRuby3: GGcGGTAGTGGcGGcGGAagcATGGTTAGCAAAGGGGAGGAGCTTATAAAGGAAAATATGAGAATGAAAGTTGTCATGGAAGGTTCAGTGAATGGCCATCAGTTTAAATGTACAGGTGAAGGCGAGGGACGCCCTTATGAAGGAGTCCAAACTATGAGGATCAAAGTCATAGAGGGAGGTCCTCTCCCCTTCGCCTTCGATATCCTCGCCACCTCTTTCATGTATGGTTCAAGAACATTTATCAAGTATCCTGCCGATATACCAGACTTCTTTAAGCAGTCATTTCCAGAAGGTTTCACTTGGGAACGAGTCACTAGGTATGAGGACGGCGGGGTTGTGACAGTAACTCAAGACACCTCTTTGGAAGATGGTGAGTTGGTCTACAACGTGAAGGTACGCGGGGTTAATTTCCCTTCTAACGGGCCTGTTATGCAAAAGAAGACAAAGGGTTGGGAGCCAAATACCGAGATGATGTATCCTGCAGATGGTGGCCTGCGGGGCTATACCGACATCGCTCTGAAGGTAGACGGCGGGGGCCACCTCCATTGTAATTTTGTAACCACTTACAGGTCTAAGAAGACCGTGGGTAACATTAAGATGCCAGGGGTTCATGCTGTCGACCATAGATTGGAGCGGATAGAAGAAAGCGACAACGAGACCTACGTCGTGCAACGCGAAGTCGCAGTAGCCAAGTATTCCAATCTCGGGGGAGGTATGGATGAACTCTATAAAGGCGGATCCGGTGGTGTGTCCAAGGGAGAAGAACTGATCAAAGAGAACATGAGGATGAAGGTCGTGATGGAGGGCAGCGTCAACGGACACCAATTCAAGTGCACCGGAGAGGGAGAAGGCAGACCATACGAGGGCGTGCAGACAATGAGAATTAAGGTGATCGAAGGCGGACCACTGCCTTTTGCTTTCGACATTCTGGCTACAAGCTTCATGTACGGCAGCAGGACCTTCATTAAATACCCCGCTGACATCCCTGATTTTTTCAAACAAAGCTTCCCTGAGGGCTTTACCTGGGAGAGAGTGACAAGATACGAAGACGGAGGCGTCGTCACCGTCACACAGGATACAAGCCTGGAGGACGGAGAACTGGTGTATAACGTCAAAGTCAGAGGAGTGAACTTTCCCAGCAATGGCCCCGTGATGCAGAAAAAGACCAAAGGCTGGGAACCTAACACAGAAATGATGTACCCAGCCGACGGAGGACTGAGAGGATACACAGACATTGCCCTCAAAGTGGATGGAGGAGGACATCTGCACTGCAACTTCGTCACAACCTACAGATCCAAGAAAACAGTCGGAAATATCAAGATGCCTGGCGTGCACGCCGTGGATCACAGGCTGGAAAGGATTGAGGAGTCCGATAATGAAACATATGTGGTCCAGAGGGAGGTGGCCGTCGCTAAATACAGCAACCTGGGCGGCGGCATGGACGAGCTGTACAAGGGGGGATCAGGAGGaGGctctCTNNB1 fwd: ATAAAAAGACATTTTTGGTAAGGAGGAGTTTTCACTGAAGTTCAGCAGTGATGGAGCTGTGGTTGAGGTGTCTGGAGGAGACCATGAGGTCTGCGTTTCA CTAACCTGGTAAAAGAGGATATGGGTTTTTTTTGTGGGTGTAATAGTGACATTTAACAGGTATCCCAGTGACTTAGGAGTATTAATCAAGCTAAATTTAAATCCTAATGACTTTTGATTAACTTTTTTTAGGGTATTTGAAGTATACCATACAACTGTTTTGAAAATCCAGCGTGGACAGGcGGTAGTGGcGGcGGAagcrev: TAGGGAACCACCTAACAGTTACTCACTGAATCAGTGGAAGAATGGTACTGCATCCAGGCTCCAGAAGCAGTCATCCAGACTAGATTCCTGCTGGTGGCTT GTTTGCTATTTCACCAAGCCATTAGGAGGAGTGAGCAGAAAATGGAGCAAAAGGTAGCCTGACAAGTAAGCAGGGAGAGAGGAAAGCAGGGGGATCTCAGCCAGACTGGCTTAATGGCAACGAAGCAGAGCCCCAATTCAGTAACTAAAGATTTAATGACACAAACCTTGAGTAGCCATagagCCtCCTCCTGATCCCCCBlunt-end PCR products were used in conjunction with gRNAs to template genomic edits containing desired knock-ins. Blunt-end, double-stranded HDR templates were created from templates obtained via DNeasy Blood and Tissue genomic prep of the 293T cell line to be edited (see next section). PCR was conducted using primers targeting amplicons of a 500- to 1,000-bp window centered on the intended cut site. The following primers were used to amplify genomic loci homology regions:CSNK1a1fwd: CCAGCCCGCGACGTC rev: CTTGACCCTTTTAGGGAGACAGCGGSK3B fwd: GATTTGCCCTCTCTTTTCTCTCCTCC rev: CCAAATAAATATCATATTATCTCAATTCAAGGTTAATGAGACCGNote that CTNNB1 homology arms were synthesized (requiring no genomic amplification step) and provided as a generous gift from Integrated DNA Technologies.Generic tdmRuby3 insert fwd: GGcGGTAGTGGcGGcGGAagcATGGTTAGCAAAGGGGAGGAGC, rev: agagCCtCCTCCTGATCCCCCCTTGTACAGCTCGTCCATGCCCSNK1a1 upstream homology arm rev: gctTCCgCCgCCACTACCgCCCCTGAGAGACGAAGATGGAGGCCSNK1a1 downstream homology arm fwd: GGGGGATCAGGAGGaGGctctATGGCGAGTAGCAGCGGCGSK3B upstream homology arm rev: gctTCCgCCgCCACTACCgCCGATCACTCTCTTCGCGAATCACCGSK3B downstream homology arm fwd: GGGGGATCAGGAGGaGGctctATGTCAGGGCGGCCCAXIN1 upstream homology arm fwd: CTTCACCCACATGTGGTCATTGCACAXIN1 upstream homology arm rev: CGGCAAAGTGGAGAAGGTGGACGGcGGTAGTGGcGGcGGAagcAXIN1 downstream homology arm fwd: GGGGGATCAGGAGGaGGctctTGATAGGCTGGTGGGCTGGCCAXIN1 downstream homology arm rev: CACCTGAAGCTGGCAGCAGGThe above amplicons were then used in a second round of PCR to obtain separate upstream and downstream homology arms that flanked desired knock-ins, and overlap extension was used to construct the final desired amplicons bearing tdmRuby3 and 7AA GS linker. The following primers were used:Note that original upstream fwd and downstream rev primers listed above for isolating genomic loci were reused in the present step and thus not repeated here.Bare 293T cells were first cotransfected using polyethyleniminePEI with theCRISPR chassis cells were then cotransfected with one of the constructed gRNA plasmids and respective HDR templates at a 2:1 HDR template:gRNA plasmid molar ratio and allowed 72 h to reach steady-state expression. Similar to the process described above, cells were subject to two rounds of FACS to obtain a clonal population. Knock-in validation was accomplished via a combination of fluorescence microscopy, genomic PCR, and sequencing . In the case of all intended knock-ins, spatiotemporal fluorescence expression of cell populations was binary (either fluorescent or not) and uniform , suggesting that selected clones were broadly representative of overall edited populations.The 293Ts were cotransfected as described in the previous section with PiggyBac and compatible XLone-Axin-tdmRuby3 and pPig_CuO-APC-tdmIRFP670::CymR expression cassettes. Seventy-two hours after transfection, cells were selected in 1 \u03bcM Blasticidin and 100 \u03bcg/mL Hygromycin B Gold . Blast+/Hygro+ cells were then clonally sorted via FACS as described in the previous section to obtain a uniform population for experiments. For iPSCs, Both Piggyback and Donor plasmids were chemically transfected when cells reached 30% confluency using Lipofectamine Stem Transfection Reagent (manufacturer\u2019s protocol). Following transfection, Blasticidin selection (1 \u03bcM) was initiated 5 d later. At the end of Blasticidin selection, 12 clones were manually picked under a dissection microscope and continuously cultured in Blasticidin (1 \u03bcM) for an additional week. Upon fluorescence signal confirming successful integration, Blasticidin (1 \u03bcM) treatment ceased, and one clone was chosen for the remaining experiments.CHIR 99021 (STEMCELL Technologies #72052) was resuspended in DMSO according to supplied manufacturer recommendations and diluted to 5\u00d7 concentrated stocks in culture medium immediately prior to use on cells. In all cases, CHIR was used at 10 \u03bcM. Doxycycline hyclate (Sigma Aldrich #D9891-1G) was resuspended in phosphate-buffered saline (PBS) and diluted to 5\u00d7 desired concentration in culture medium prior to use. Stock cumate solution (System Biosciences #QM100A-1) was diluted to 5\u00d7 in culture medium prior to use.SI Appendix, Fig. S4 I and J was 100 ng/mL.The \u201cLow\u201d dose of Dox referred to in Recombinant Human Wnt-3a (R&D Systems 5036-WN-010) was resuspended in in PBS containing 0.1% bovine serum albumin according to supplied manufacturer recommendations and diluted to 5\u00d7 concentration in culture medium immediately prior to use. In all cases, Wnt-3a was used at a final concentration of 1 \u03bcg/mL.Primary antibodies used for immunofluorescent markers of the centrosome were \u03b1-GM130 and \u03b1-\u03b3-tubulin . The secondary used for both stains was \u03b1-Ms-Alexa-488 . Tissue fixation and staining was carried out using standard protocols using cold methanol . Immunof2. Glass-bottom culture plates (Cellvis #P96-1.5H-N) were pretreated with bovine fibronectin (Sigma #F1141) in the case of 293Ts or Matrigel in the case of H9 and iPSCs, and cells were allowed to adhere to the plate before subsequent treatment or imaging. FRAP was performed via custom Nikon NIS Elements JOBs function and 488-nm FRAP laser .All live and fixed cell imaging experiments were carried out using a Nikon W2 SoRa spinning-disk confocal microscope equipped with incubation chamber maintaining cells at 37\u2009\u00b0C and 5% COSpatial patterning of light during timelapse fluorescent imaging sessions was accomplished via purpose-built microscope-mounted LED-coupled digital micromirror devices (DMDs) triggered via Nikon NIS Elements software. Stimulation parameters were optimized to minimize phototoxicity while maintaining continuous activation of Cry-2. For DMD-based stimulation on the microscope, the final settings for \u201cLight ON\u201d were 25% LED power (\u03bb = 455 nm), 2-s duration pulses every 30 s. For experiments that did not require frequent confocal imaging, cells were stimulated via a benchtop LED array purpose built for light delivery to cells in standard tissue culture plates (\u201cOptoPlate\u201d) adapted from previously established designs . The samAll quantification of raw microscopy images was carried out using the same general workflow: background subtraction > classification > measurement > normalization > statistical comparison. Subcellular segmentation of nuclear fluorescence was performed via custom Matlab scripts using H2B-mTagBFP2 brightness, size, and circularity to mask objects. When experimental conditions did not permit segmentation via H2B-mTagBFP2 nuclear fluorescence (such as with live-cell optogenetic stimulation), cells were selected at random using custom ImageJ macro that generates random regions of interest (ROIs) (available upon request). Unless otherwise noted, mean fluorescent intensity of ROIs were measured and subsequently processed. Raw measurements were compiled, processed, and plotted via custom Matlab scripts, available upon request.Statistical parameters are provided in https://fenicsproject.org/) to solve the modified Cahn\u2013Hilliard partial differential equations using the finite element method. In our simulation, we represent the volume fraction of each DC protein, SI Appendix, Fig. S3B), and Ri is the added reaction term such thatWe used the Python-based FEniCS computing environment . We generate a grid mesh with closed boundary conditions to mimic the closed system within a cell. A layer is generated for each simulated component, and \u00b15% noise of the initial value is added to induce inhomogeneities. The FEniCS package partial differential solver is called to generate the chemical potential with respect to each component. The final step is to define the output file path and then use the built-in Newton solver to generate the simulation. The simulations are then rendered using Paraview software. A detailed Python notebook of the simulations is available on https://github.com/MZWLab/Lach2022.First, all parameters are defined . This allowed us to test the sensitivity of a single metric to alterations in our model\u2019s parameters. In Movie S6) and also increased the nucleation efficiency.We defined the nucleation efficiency of \u03b2-cat processing for a given simulation by comparing the ratio of the integrated P4-\u03b2-cat to \u03b2-cat between identical simulations with and without a nucleator (Supplementary FileSupplementary FileSupplementary FileSupplementary FileSupplementary FileSupplementary FileSupplementary FileSupplementary File"} +{"text": "Ctenidae Keyserling, 1877 has a worldwide distribution with 584 species belonging to 49 genera. Amongst these, 141 species are from Asia, including 130 species assigned to Cteninae Keyserling, 1877.The spider family Cteninae are reported from Asia: Amauropelmakrabi sp. n. , Am.phangnga sp. n. , Am.saraburi sp. n. ; Anahitamedog sp. n. ; Bowieninhbinh sp. n. and B.vinhphuc sp. n. from the robustus-species group; B.borneo sp. n. from the chinagirl-species group; B.engkilili sp. n. ; B.sabah sp. n. from the scarymonsters-species group. The male of An.popa J\u00e4ger & Minn, 2015 and the female of B.fascination J\u00e4ger, 2022 (robustus-species group) are described for the first time. B.fascination J\u00e4ger, 2022 is reported from China for the first time. In addition, the DNA barcodes of all the species in this study were obtained, except for B.vinhphuc sp. n.Nine new species belonging to three genera of Ctenidae Keyserling, 1877 has a worldwide distribution, but mainly occurs in the tropical and subtropical regions . Both Amrom Asia .Amauropelma, Anahita and Bowie are described from Asia. This brings the total number of Ctenidae to 150 species in Asia, of which 139 species belong to Cteninae . Photos were stacked with Helicon Focus\u00ae (Version 7.6.1) or Zerene Stacker\u00ae (Version 1.04) and processed in Adobe Photoshop CC2019\u00ae. All measurements are in millimetres (mm) and were obtained with an Olympus SZX16 stereomicroscope with a Zongyuan CCD industrial camera. Total length does not include the chelicerae. Palp and leg measurements are shown as: total length . Leg segments were measured on their dorsal side. The distribution map was generated with ArcGIS 10.2 (ESRI Incorporated Company). References to figures in the cited papers are listed in lowercase (fig. or figs); figures from this paper are noted with a capital letter . The type material is deposited in the Institute of Zoology, Chinese Academy of Sciences (IZCAS) in Beijing, China.Size classes are used according to Terminology and taxonomic descriptions follow B.vinhphuc sp. n. A partial fragment of the mitochondrial cytochrome oxidase subunit I (COI) gene was amplified and sequenced, using the following primers: forward: LCO1490 (5\u2019-CWACAAAYCATARRGATATTGG-3\u2019) and reverse: HCO2198 (5'-TAAACTTCAGGGTGACCAAAAAATCA-3') and reverse: HCO2198 (5'-TAAACTTCAGGGTGACCAAAAAATCA-3') , except ATCA-3') . COI p-dATCA-3') . For addS. Li & Yaosp. n.C5E9E60C-A668-5AE7-A9FF-89D05F8A802757EA8144-E44C-4F59-8B3A-0D16719CB5D5Type status:Holotype. Occurrence: recordedBy: Z. Chen; individualCount: 1; sex: female; lifeStage: adult; Taxon: order: Araneae; family: Ctenidae; genus: Amauropelma; Location: country: Thailand; stateProvince: Krabi; verbatimLocality: Ao Luk District, Klang Cave; verbatimElevation: 36 m a.s.l.; verbatimLatitude: 8\u00b020.268'N; verbatimLongitude: 98\u00b044.707'E; Event: year: 2015; month: 10; day: 12; Record Level: institutionCode: IZCAS-Ar 43530MaleUnknown.Female (IZCAS-Ar 43530): PL 3.3, PW 2.4, AW 1.6, OL 3.1, OW 1.6. Eye diameters and interdistances: AME 0.10, ALE 0.13, PME 0.11, PLE 0.11, AME\u2013AME 0.04, AME\u2013ALE 0.11, PME\u2013PME 0.06, PME\u2013PLE 0.24, AME\u2013PME 0.06, ALE\u2013PLE 0.08, clypeus AME 0.12, clypeus ALE 0.17. Palp and leg measurements: palp 3.8 , I 10.3 , II 9.2 , III 9.0 , IV 12.2 . Leg formula 4123. Spination of palp and legs: palp 130, 100, 1111, 1212; femora I p002, d111, r010, II p010, d111, r010, III p111, d111, r012, IV p002, d111, r102; patellae I\u2013IV 001; tibiae I\u2013II v22222, III p11, d111, r11, v222, IV p111, d11, r11, v222; metatarsi I\u2013II v222, III p112, d010, r112, v222, IV p112, r112, v222. Chelicerae with 3 promarginal, 4 + 1 retromarginal teeth, without denticles. Retromargin of chelicerae close to fang base without bristle. Tarsi and metatarsi without scopula. Claw tufts arising separately, but intermingle with each other distally. Palpal claw with 3 secondary teeth, leg claws I\u2013II with 3, III with 2 and IV with 3 secondary teeth. Position of tarsal organ: I 0.76, II 0.72, III 0.68.Copulatory organ Fig. a and b. Colour Fig. c and d. Ctenidae . The new species can be distinguished from all known congeners by the median plate roughly heart-shaped and with a mating plug :OP561682).TGTTTGGAGCTTGAGCTGCTATAGCAGGAACTGGAATAAGAGTGTTGATTCGAATAGAGTTAGGTCATCCTGGTAGATTGTTAGGAGATGATCATTTATATAATGTTATTGTAACTGCTCATGCTTTTGTAATGATTTTTTTTATAGTAATACCAATTTTGATTGGTGGATTTGGAAATTGATTAGTTCCGTTGAGATTGGAGCACCTGATATATCATTTCCTCGAATAAATAATTTGTCGTTTTGATTACTACCTCCTTCTTTATTTTTATTAATAATATCATCAATAGTAGAAATAGGTGTTGGAGCGGGATGAACTGTTTATCCTCCTTTAGCATCTAGTATTGGGCATATAGGAAGATCTATAGATTTTGCTATTTTTTCTCTTCATTTGGCTGGAGCTTCTTCTATTATAGGAGCAGTAAATTTTATTTCTACTATTATTAATATACGGTTGTATGGAATGAGTATAGAAAAGGTTCCTTTGTTTGTGTGGTCTGTTTTTATTACTGCTATTTTGTTATTATTGTCGTTACCTGTGTTAGCAGGTGCTATTACTATATTATTGACTGATCGAAATTTTAATACTTCTTTTTTTGACCCTGCGGGAGGGGGAGATCCTATTTTGTTTCAACATTTATTTTGATTTTTTG : PL 3.3, PW 2.8, AW 1.2, OL 3.0, OW 1.9. Eye diameters and interdistances: AME 0.09, ALE 0.12, PME 0.10, PLE 0.10, AME\u2013AME 0.04, AME\u2013ALE 0.13, PME\u2013PME 0.08, PME\u2013PLE 0.21, AME\u2013PME 0.05, ALE\u2013PLE 0.08, clypeus AME 0.17, clypeus ALE 0.22. Palp and leg measurements: palp 3.8 , I 13.5 , II 11.6 , III 10.9 , IV 14.8 . Leg formula 4123. Spination of palp and legs: palp 131, 100, 1101; femora I p021, d211, r112, II\u2013III p012, d111, r012, IV p102, d111, r012; patellae I\u2013IV 001; tibiae I p010, v22222, II p100, r100, v22222, III p11, d111, r11, v222, IV p11, d11, r11, v222; metatarsi I v222, II p112, r010, v222, III p112, d010, r112, v222, IV p112, r112, v2222. Chelicerae with 3 promarginal, 4 retromarginal teeth, without denticles. Retromargin of chelicerae close to fang base without bristle. Tarsi and metatarsi without scopula. Claw tufts arising separately, but intermingle with each other distally. Leg claws I with 7 and II with 6 secondary teeth. Position of tarsal organ: I 1.37, II 0.92, III 0.85.Palp Fig. a\u2013c. Pate9Colour Fig. a and b. FemaleUnknown.Variation: Paratype male (IZCAS-Ar 43532): PL 3.4, OL 2.4.Ctenidae . The new species can be distinguished from all known congeners by the embolus tip with an extension :OP718556).GGTGGGTTCGGAAATTGATTGGTTCCTTTGATGTTAGGAGCTCCTGATATATCATTTCCTCGTATAAATAATTTGTCTTTTTGGTTACTTCCTCCTTCTTTATTTTTGTTATTAATATCTTCTATGGTGGAAATAGGAGTGGGAGCAGGATGAACTGTCTATCCTCCTTTAGCTTCTAGAATAGGGCATGTGGGAAGATCAATAGATTTTGCGATTTTTTCTCTTCATTTAGCTGGAGTTTCTTCTATTATGGGAGCGGTTAATTTTATTTCTACTATTATTAATATGCGATTATATGGAATAACTATAGAAAAGGTTCCTTTATTCGTTTGATCAGTTTTTATTACTGCAGTTTTGTTGTTGTTATCATTACCTGTGTTAGCAGGTGCTATTACTATATTATTGACAGATCGAAATTTTAATACTTCTTTTTTTGATCCTGCAGGGGGTGGAGATCCAATTTTATTTCAACATTTATTCTGATTTTTTGGTCACCCTGGAAAGTTTAA : PL 4.5, PW 3.7, AW 1.6, OL 3.2, OW 2.1. Eye diameters and interdistances: AME 0.12, ALE 0.14, PME 0.14, PLE 0.14, AME\u2013AME 0.07, AME\u2013ALE 0.16, PME\u2013PME 0.08, PME\u2013PLE 0.26, AME\u2013PME 0.07, ALE\u2013PLE 0.13, clypeus AME 0.16, clypeus ALE 0.28. Palp and leg measurements: palp 5.2 , I - , II 15.2 , III 14.3 , IV 19.4 . Leg formula 4123. Spination of palp and legs: palp 131, 100, 210; femora I p112, d111, r111, II p211, d111, r211, III p112, d111, r112, IV p112, d111, r002; patellae I 001, II\u2013IV 101; tibiae I p010, r110, v22222, II p100, r100, v22222, III\u2013IV p11, d111, r11, v222; metatarsi I v222, II p110, r110, v222, III p112, d010, r112, v222, IV p112, d010, r112, v2222. Chelicerae with 3 promarginal, 4 retromarginal teeth, without denticles. Retromargin of chelicerae close to fang base without bristle. Claw tufts arising separately, but intermingle with each other distally. Leg claws II with 1 and III\u2013IV with 2 secondary teeth. Position of tarsal organ: IV 1.58.Palp Fig. a\u2013c. Pate9Colour Fig. c and d. Female (IZCAS-Ar 43534): PL 5.6, PW 4.4, AW 2.8, OL 4.9, OW 3.1. Eye diameters and interdistances: AME 0.14, ALE 0.20, PME 0.15, PLE 0.16, AME\u2013AME 0.12, AME\u2013ALE 0.32, PME\u2013PME 0.16, PME\u2013PLE 0.53, AME\u2013PME 0.08, ALE\u2013PLE 0.20, clypeus AME 0.13, clypeus ALE 0.21. Palp and leg measurements: palp 6.0 , I 19.3 , II 18.3 , III 17.0 , IV 23.0 . Leg formula 4123. Spination of palp and legs: palp 131, 100, 1111, 2112; femora I p021, d111, r111, II\u2013III p112, d111, r112, IV p112, d111, r002; patellae I\u2013II 000, III\u2013IV 101; tibiae I \u2013II v22222, III\u2013IV p11, d111, r11, v222; metatarsi I\u2013II v222, III\u2013IV p112, d010, r112, v222. Chelicerae with 3 promarginal, 4 retromarginal teeth, without denticles. Retromargin of chelicerae close to fang base without bristle. Sparse scopula restricted almost entirely to tarsi, only metatarsi I\u2013II with sparse scopula hairs. Claw tufts arising separately, but intermingle with each other distally. Palpal claw with 3 secondary teeth, leg claws I with 3, II\u2013III with 2 secondary teeth.Copulatory organ Fig. a and b. Colour Fig. e and f. Variation: Second paratype female (IZCAS-Ar 43535): PL 4.6, OL 4.7.Ctenidae . The new species can be distinguished from all known congeners by the embolus slender .Male (IZCAS-Ar 43533):TGTTTGGAGCTAGATCTGCTATAGCGGGAACGGCAATAAGAGTTTTAATTCGTATGGAATTAGGAAATTCTGGAAGATTATTAGGGGATGATCATTTATATAATGTAATTGTGACAGCTCATGCTTTTATTATGATTTTTTTTATAGTAATACCGATTTTGATTGGTGGTTTTGGAAATTGATTAGTGCCTTTAATGTTAGGAGCTCCTGATATATCTTTTCCTCGGATGAATAATTTGTCTTTTTGATTACTTCCACCTTCTTTGTTTTTATTATTCATATCTTCTATGGTGGAAATGGGTGTAGGAGCTGGATGAACTGTTTATCCACCTTTGGCTTCTAGAATTGGTCATGCTGGAAGATCTATGGATTTTGCTATTTTTTCTTTACATTTAGCTGGGGCTTCTTCAATTATAGGAGCGGTGAATTTTATTTCTACTATTATTAATATACGATTATCTGGAATAAGAATGGAGAAGGTTCCATTATTTGTTTGATCTGTTCTTATTACTGCAATTTTATTATTATTATCTTTGCCGGTATTAGCTGGTGCTATTACTATATTGTTGACTGATCGAAATTTTAATACTTCTTTTTTTGATCCGGCTGGGGGAGGGGATCCTATTTTATTTCAACATTTATTTTGATTTTTTG (GenBank accession number OP572099).Female (IZCAS-Ar 43534):TGTTTGGAGCTTGATCTGCTATAGCGGGAACGGCAATAAGAGTTTTAATTCGTATGGAATTAGGAAATTCTGGAAGATTATTAGGGGATGATCATTTATATAATGTAATTGTGACAGCTCATGCTTTTATTATGATTTTTTTTATAGTAATACCGATTTTGATTGGTGGTTTTGGAAATTGATTAGTGCCTTTAATGTTAGGAGCTCCTGATATATCTTTTCCTCGGATGAATAATTTGTCTTTTTGATTACTTCCACCTTCTTTGTTTTTATTATTCATATCTTCTATGGTGGAAATGGGTGTAGGAGCTGGATGAACTGTTTATCCACCTTTGGCTTCTAGAATTGGTCATGCTGGAAGATCTATGGATTTTGCTATTTTTTCTTTACATTTAGCTGGGGCTTCTTCAATTATAGGAGCGGTGAATTTTATTTCTACTATTATTAATATACGATTATCTGGAATAAGAATGGAGAAGGTTCCATTATTTGTTTGATCTGTTCTTATTACTGCAATTTTATTATTATTATCTTTGCCGGTATTAGCTGGTGCTATTACTATATTGTTGACTGATCGAAATTTTAATACTTCTTTTTTTGATCCGGCTGGGGGAGGGGATCCTATTTTATTTCAACATTTATTTTGATTTTTTG : PL 2.7, PW 2.3, AW 0.9, OL 2.4, OW 1.4. Eye diameters and interdistances: AME 0.13, ALE 0.10, PME 0.22, PLE 0.19, AME\u2013AME 0.10, AME\u2013ALE 0.22, PME\u2013PME 0.16, PME\u2013PLE 0.23, AME\u2013PME 0.11, ALE\u2013PLE 0.14, clypeus AME 0.10, clypeus ALE 0.37. Palp and leg measurements: palp 4.1 , I missing, II 13.0 , III 10.9 , IV 16.0 . Leg formula 4123. Spination of palp and legs: palp 023, 000, 0211; femora II p112, d111, r012, III p112, d111, r112, IV p112, d111, r012; patellae II\u2013IV 101; tibiae II p110, d101, r100, v22222, III p11, d111, r11, v222, IV p11, d111, r11, v22; metatarsi II p111, r111, v222, III p112, d010, r112, v222, IV p112, d010, r112, v2222. Chelicerae with 3 promarginal, 4 + 1 retromarginal teeth and with elongated patch of 6 tiny denticles along entire cheliceral furrow. Leg claws II with 9, III with 5 and IV with 7 secondary teeth. Position of tarsal organ: II 1.06, III 0.85, IV 1.14.Palp Fig. a\u2013c. Palp14Colour Fig. c and d. Female (IZCAS-Ar 43537): PL 2.9, PW 2.4, AW 1.2, OL 3.5, OW 2.0. Eye diameters and interdistances: AME 0.13, ALE 0.11, PME 0.19, PLE 0.18, AME\u2013AME 0.13, AME\u2013ALE 0.26, PME\u2013PME 0.21, PME\u2013PLE 0.24, AME\u2013PME 0.15, ALE\u2013PLE 0.17, clypeus AME 0.10, clypeus ALE 0.38. Palp and leg measurements: palp 3.2 , I 10.4 , II 9.3 , III 8.3 , IV 12.4 . Leg formula 4123. Spination of palp and legs: palp 020, 010, 010, 2012; femora I p011, d111, r021, II p011, d111, r011, III p012, d111, r112, IV p002, d111, r012; patellae I\u2013II 000, III\u2013IV 101; tibiae I v22212, II v22222, III\u2013IV p11, d111, r11, v222; metatarsi I\u2013II v222, III p112, d010, r112, v222, IV p112, r112, v2222. Chelicerae with 3 promarginal, 4 retromarginal teeth and with elongated patch of 5 tiny denticles along entire cheliceral furrow. Palpal claw with 5 secondary teeth, leg claws I\u2013II with 5, III with 4 and IV with 7 secondary teeth. Position of tarsal organ: I 0.79, II 0.72, III 0.70, IV 1.00.Copulatory organ Fig. a and b. Colour Fig. e and f. Ctenidae . The species resembles A.maolan Zhu, Chen & Song, 1999 (see 14A.maolan), by the median plate with a large n-shaped sclerite , by the lateral teeth pointing postero-medially and by the spermathecae nearly cylindrical .Small 1999 see a\u2013c and f1999 see b, but caThe specific name refers to the type locality and is a noun in apposition.China :.OP572101).TGTTTGGAGCTTGAGCTGCTATAGCTGGAACAGCAATAAGAGTTTTAATTCGAATGGAATTAGGACATTCTGGTAGATTGTTAGGAGATGATCATTTATATAATGTAATTGTAACGGCTCATGCTTTTGTTATAATTTTTTTTATAGTAATACCTATTTTGATTGGGGGCTTTGGTAATTGGTTGGTTCCTTTAATGTTAGGGGCTCCGGATATATCTTTTCCTCGAATAAATAATTTATCCTTTTGATTATTACCGCCTTCTTTATTTTTGTTGTTTATATCTTCTATAGTTGAGATAGGGGTTGGAGCAGGTTGAACGGTTTATCCTCCTTTAGCTTCTAGAATTGGGCATATGGGAAGTTCAATGGATTTTGCTATTTTTTCTTTACATTTAGCAGGTGCTTCTTCTATTATAGGTGCTGTGAATTTTATTTCTACTATTATTAATATACGATTAATAGGAATAACAATGGAGAAGATCCCTTTATTTGTATGATCGGTTTTTATTACTGCAATTTTATTATTATTATCTTTACCTGTTTTAGCAGGAGCTATTACTATATTATTGACTGATCGAAATTTTAATACTTCTTTTTTTGACCCTGCTGGAGGTGGAGATCCTATTTTATTTCAACATTTATTTTGATTTTTTG :OP572102).TGTTTGGAGCTTGAGCTGCTATAGCTGGAACAGCAATAAGAGTTTTAATTCGAATGGAATTAGGACATTCTGGTAGATTGTTAGGAGATGATCATTTATATAATGTAATTGTAACGGCTCATGCTTTTGTTATAATTTTTTTTATAGTAATACCTATTTTGATTGGGGGCTTTGGTAATTGGTTGGTTCCTTTAATGTTAGGGGCTCCGGATATATCTTTTCCTCGAATAAATAATTTATCCTTTTGATTATTACCGCCTTCTTTATTTTTGTTGTTTATATCTTCTATAGTTGAGATAGGGGTTGGAGCAGGTTGAACGGTTTATCCTCCTTTAGCTTCTAGAATTGGGCATATGGGAAGTTCAATGGATTTTGCTATTTTTTCTTTACATTTAGCAGGTGCTTCTTCTATTATAGGTGCTGTGAATTTTATTTCTACTATTATTAATATACGATTAATAGGAATAACAATGGAGAAGATCCCTTTATTTGTATGATCGGTTTTTATTACTGCAATTTTATTATTATTATCTTTACCTGTTTTAGCAGGAGCTATTACTATATTATTGACTGATCGAAATTTTAATACTTCTTTTTTTGACCCTGCTGGAGGTGGAGATCCTATTTTATTTCAACATTTATTTTGATTTTTTG : PL 3.3, PW 2.7, AW 0.9, OL 3.3, OW 1.8. Eye diameters and interdistances: AME 0.14, ALE 0.12, PME 0.22, PLE 0.17, AME\u2013AME 0.08, AME\u2013ALE 0.23, PME\u2013PME 0.16, PME\u2013PLE 0.21, AME\u2013PME 0.18, ALE\u2013PLE 0.13, clypeus AME 0.10, clypeus ALE 0.55. Palp and leg measurements: palp 3.9 , I 15.4 , II 12.7 , III 11.0 , IV 17.3 . Leg formula 4123. Spination of palp and legs: palp 151, 000, 122; femora I p021, d111, r112, II p112, d111, r112, III p111, d111, r111, IV p012, d111, r112; patellae I\u2013IV 101; tibiae I p010, d101, r110, v22222, II p10, d101, r110, v22222, III\u2013IV p11, d111, r11, v222; metatarsi I p111, d001, r111, v222, II p111, d111, r111, v222, III p111, d012, r111, v222, IV p112, d011, r112, v222. Chelicerae with 3 promarginal, 4 + 2 retromarginal teeth and with elongated patch of 3 tiny denticles along entire cheliceral furrow. Retromargin of chelicerae close to fang base with 2 bristles. Sparse scopula restricted almost entirely to tarsi. Leg claws I with 6, II\u2013III with 5 and IV with 4secondary teeth. Position of tarsal organ: I 1.89, II 1.03, III 0.75, IV 1.56.Palp Fig. a\u2013c. Palp14Colour Fig. c and d. Female (IZCAS-Ar 43539): See Fig. Ctenidae . The species can be distinguished from all known congeners by the embolus arising at 12 o\u2019clock position, long and laminar, running around tegulum, its tip situated distally :OP572105).TATTTGGGGCTTGAGCTGCTATAGCGGGTACTGCAATAAGAGTTTTGATTCGAATGGAATTAGGACATCCTGGAAGATTATTAGGTGATGATCATTTATATAATGTTATTGTAACAGCTCATGCTTTTGTTATGATTTTTTTTATAGTTATACCTATTTTAATTGGTGGTTTTGGAAATTGGTTAGTTCCTTTAATATTAGGAGCTCCGGATATATCATTTCCTCGAATAAATAATTTATCTTTTTGGTTATTACCTCCTTCTTTGTTTTTATTGTTTATATCTTCTATAGTTGAAATAGGTGTAGGAGCAGGGTGAACAGTTTATCCTCCTTTAGCTTCTAGAATTGGGCATGCAGGGAGATCTATGGATTTTGCTATTTTTTCTTTACATTTAGCGGGTGCTTCTTCTATTATAGGGGCTGTAAATTTTATTTCTACTATTATTAATATACGATTAATAGGAATGACTATAGAGAAGGTTCCTTTGTTTGTTTGATCTGTTTTTATTACTGCAATTTTATTATTGTTATCTTTACCAGTGTTAGCTGGTGCTATTACAATATTATTAACTGATCGTAATTTTAATACTTCTTTTTTTGATCCTGCTGGAGGAGGAGATCCAGTTTTATTTCAGCATTTGTTTTGATTTTTTG :OP572104).TTTTTGGAGCTTGAGCCGCTATAGCGGGTACTGCAATAAGAGTTTTAATTCGAATAGAATTAGGGCATCCTGGGAGATTATTAGGTGATGATCATTTATATAATGTTATTGTAACAGCTCATGCTTTTGTTATAATTTTTTTTATAGTTATACCTATTTTAATTGGTGGTTTTGGAAATTGGTTAGTTCCTTTAATGTTAGGAGCTCCGGATATATCATTTCCTCGAATAAATAATTTATCTTTTTGATTATTACCTCCTTCTTTGTTTTTATTGTTTATATCTTCCATGGTTGAAATAGGTGTGGGAGCAGGATGGACAGTTTATCCTCCTTTAGCTTCTAGAATTGGGCATGCGGGAAGATCTATGGATTTTGCTATTTTTTCTTTACATTTAGCGGGTGCTTCTTCTATTATAGGAGCTGTAAATTTTATTTCGACTATTATTAATATACGATTAATAGGAATGACTATAGAGAAGGTTCCCTTATTTGTTTGATCTGTTTTTATTACTGCAATTTTATTGTTATTATCTTTACCAGTATTAGCTGGTGCTATTACGATGTTGTTAACTGATCGTAATTTTAATACTTCTTTTTTTGACCCTGCTGGGGGAGGGGATCCGGTTTTATTTCAACATTTATTTTGATTTTTTG : See Fig. 28Female (IZCAS-Ar 43541): PL 9.9, PW 7.8, AW 4.1, OL 11.0, OW 7.3. Eye diameters and interdistances: AME 0.29, ALE 0.27, PME 0.32, PLE 0.39, AME\u2013AME 0.31, AME\u2013ALE 0.71, PME\u2013PME 0.43, PME\u2013PLE 1.32, AME\u2013PME 0.32, ALE\u2013PLE 0.38, clypeus AME 0.42, clypeus ALE 0.95. Palp and leg measurements: palp 10.0 , I 23.0 , II 21.4 , III 18.6 , IV 26.1 . Leg formula 4123. Spination of palp and legs: palp 131, 100, 131, 3020; femora I p021, d111, r111, II p112, d111, r111, III\u2013IV p111, d111, r112; patellae I\u2013II 000, III\u2013IV 101; tibiae I\u2013II v22222, III\u2013IV p11, d111, r11, v222; metatarsi I\u2013II v222, III p111, d012, r111, v222, IV p121, d012, r111, v2122. Chelicerae with 3 promarginal, 4 + 1 retromarginal teeth and with elongated patch of 27 tiny denticles along entire cheliceral furrow. Retromargin of chelicerae close to fang base with 11 bristles. Ventral tarsi and metatarsi I\u2013II with sparse scopula. Palpal claw with 5 secondary teeth, leg claws I\u2013II with 1, III with 2 and IV with 3 secondary teeth. Position of tarsal organ: I 1.41, II 1.34, III 1.25, IV 1.44.Copulatory organ Fig. a. EpigynColour Fig. e and f. Ctenidae . The species is assigned to the robustus-species group with the characteristics of stout tegular apophysis, simple stout embolus with broad base, presence of retro-proximal cymbial outgrowth, RTA arising medially to distally from palpal tibia, female possesses a transversally median plate with lateral teeth situated mainly laterally and not reaching the epigastric furrow. It resembles B.candidate J\u00e4ger, 2022 (see B.candidate), by the spermathecae separated by less than their diameter and smaller chamber with distinct external rim and by the fertilisation ducts pointing laterally . For the diagnosis of male, see Large-sized 2022 see a, but caChina :OP572108).TATTTGGATCTTGGGCTGCTATAGCTGGGACAGCTATAAGAGTATTAATTCGTATAGAGCTAGGTCATTCTGGTAGATTATTTGGTGATGATCATTTATATAATGTAATTGTTACAGCTCATGCTTTTGTAATAATTTTTTTTATGGTTATGCCTATTTTAATTGGTGGTTTTGGAAACTGATTAGTTCCTTTGATATTAGGGGCTCCTGATATATCTTTTCCTCGTATAAATAATTTATCTTTTTGATTACTCCCTCCTTCATTATTTTTGTTATTTATATCTTCTATGGTTGAGATAGGGGTGGGAGCTGGTTGGACAGTGTATCCTCCTTTAGCTTCTAGTATTGGCCATATAGGAAGATCAATAGATTTTGCTATTTTTTCTTTACATTTAGCGGGAGCTTCTTCTATTATAGGGGCTGTTAATTTTATTTCTACAATTATTAATATACGTTTGTATGGAGTAAGAATAGAAAAGGTGCCTTTATTTGTATGATCTGTTCTAATTACTGCAGTATTATTGCTTTTATCTTTACCTGTATTAGCAGGTGCTATTACTATATTATTAACTGATCGTAATTTTAATACTTCTTTTTTTGACCCGGCTGGAGGAGGGGATCCAGTTTTATTTCAACATTTATTTTGATTTTTTG :OP572107).TATTTGGATCTTGGGCTGCTATAGCTGGGACAGCTATAAGAGTATTAATTCGTATAGAGCTAGGTCATTCTGGTAGATTATTTGGTGATGATCATTTATATAATGTAATTGTTACAGCTCATGCTTTTGTAATAATTTTTTTTATGGTTATGCCTATTTTAATTGGTGGTTTTGGAAACTGATTAGTTCCTTTGATATTAGGGGCTCCTGATATATCTTTTCCTCGTATAAATAATTTATCTTTTTGATTACTCCCTCCTTCATTATTTTTGTTATTTATATCTTCTATGGTTGAGATAGGGGTGGGAGCTGGTTGGACAGTGTATCCTCCTTTAGCTTCTAGTATTGGCCATATAGGAAGATCAATAGATTTTGCTATTTTTTCTTTACATTTAGCGGGAGCTTCTTCTATTATAGGGGCTGTTAATTTTATTTCTACAATTATTAATATACGTTTGTATGGAGTAAGAATAGAAAAGGTGCCTTTATTTGTATGATCTGTTCTAATTACTGCAGTATTATTGCTTTTATCTTTACCTGTATTAGCAGGTGCTATTACTATATTATTAACTGATCGTAATTTTAATACTTCTTTTTTTGACCCGGCTGGAGGAGGGGATCCAGTTTTATTTCAACATTTATTTTGATTTTTTG : PL 7.6, PW 5.9, AW 3.0, OL 5.8, OW 3.9. Eye diameters and interdistances: AME 0.26, ALE 0.19, PME 0.38, PLE 0.30, AME\u2013AME 0.19, AME\u2013ALE 0.29, PME\u2013PME 0.22, PME\u2013PLE 0.43, AME\u2013PME 0.18, ALE\u2013PLE 0.23, clypeus AME 0.11, clypeus ALE 0.55. Palp and leg measurements: palp 7.8 , I 21.3 , II 19.6 , III 15.9 , IV 23.1 . Leg formula 4123. Spination of palp and legs: palp 151, 100, 101; femora I p021, d111, r112, II p112, d111, r112, III p212, d111, r112, IV p112, d111, r012; patellae I\u2013IV 101; tibiae I p110, d111, r210, v22222, II p110, d111, r110, v22222, III p11, d200, r11, v222, IV p11, d111, r11, v222; metatarsi I\u2013III p112, d010, r112, v222, IV p112, d010, r112, v2222. Chelicerae with 3 promarginal, 4 retromarginal teeth and with elongated patch of 19 tiny denticles along entire cheliceral furrow. Retromargin of chelicerae close to fang base with 6 bristles. Ventral tarsi and metatarsi I\u2013II with sparse scopula. Right leg claws I\u2013III with 2 and IV with 3 secondary teeth. Position of tarsal organ: I 1.27, II 1.28, III 1.06, IV 1.36.Palp Fig. a\u2013c. RTA 28Colour Fig. a and b. FemaleUnknown.Variation: Paratype male (IZCAS-Ar 43543): PL 7.4, OL 5.7.Ctenidae . The new species is assigned to the robustus-species group with the characteristics of stout tegular apophysis, simple stout embolus with broad base, presence of retro-proximal cymbial outgrowth and RTA arising subdistally from palpal tibia. It resembles B.dodo J\u00e4ger, 2022 (see B.dodo) and by the RTA distally bifurcated .Medium-sized 2022 see b, but caThe specific name refers to the type locality and is a noun in apposition.Vietnam :OP572110).TACTTGGATCTTGGGCTGCTATGGCAGGGACAGCTATAAGAGTATTAATTCGGATGGAATTAGGCCATTCTGGGAGATTGTTAGGTGATGATCATTTATACAATGTAATTGTTACTGCACATGCTTTTGTAATGATTTTTTTTATAGTAATGCCTATTTTAATTGGGGGTTTTGGAAATTGGTTAGTACCTTTGATATTAGGGGCTCCTGATATATCTTTTCCTCGAATAAATAATTTGTCTTTTTGGTTACTTCCTCCTTCGTTATTTTTATTATTTATATCTTCAATAGTTGAGATAGGAGTTGGAGCTGGATGAACGGTATATCCTCCTTTAGCTTCTAGTATTGGTCATATAGGGAGATCTATAGATTTTGCTATTTTTTCTTTACATTTAGCGGGGGCTTCTTCTATTATAGGAGCGGTAAATTTTATTTCTACGATTATTAATATGCGTTTGTATGGGATGACTATAGAGAAAGTACCTTTATTTGTGTGATCTGTTTTAATTACTGCGGTATTGTTATTATTGTCTTTACCTGTTTTAGCAGGTGCTATTACTATATTGTTAACTGATCGAAATTTTAATACTTCTTTTTTTGATCCGGCTGGGGGTGGTGATCCTGTTTTGTTTCAACATTTATTTTGATTTTTTG : PL 6.4, PW 5.0, AW 2.4, OL 3.1, OW 3.5. Eye diameters and interdistances: AME 0.23, ALE 0.24, PME 0.28, PLE 0.26, AME\u2013AME 0.22, AME\u2013ALE 0.42, PME\u2013PME 0.25, PME\u2013PLE 0.41, AME\u2013PME 0.20, ALE\u2013PLE 0.21, clypeus AME 0.21, clypeus ALE 0.53. Palp and leg measurements: palp 7.4 , I 17.7 , II 15.9 , III 13.4 , IV 19.5 . Leg formula 4123. Spination of palp and legs: palp 141, 100, 1010; femora I p021, d111, r112, II p112, d111, r1111, III p1111, d111, r1111, IV p002, d111, r111; patellae I\u2013IV 101; tibiae I p110, d111, r21, v22222, II p20, d111, r110, v22222, III\u2013IV p11, d111, r11, v222; metatarsi I p11, d002, r111, v222, II p111, r111, v222, III p111, d012, r211, v222, IV p122, d012, r111, v2122. Chelicerae with 3 promarginal, 4 retromarginal teeth and with elongated patch of 12 tiny denticles along entire cheliceral furrow. Retromargin of chelicerae close to fang base with 4 bristles. Sparse scopula restricted almost entirely to tarsi, only metatarsi I\u2013II with sparse scopula hairs. Leg claws I\u2013III with 3 and IV with 4 secondary teeth. Position of tarsal organ: I with 1.17, II 1.07, III 0.90, IV 1.09.Palp Fig. a\u2013c. RTA 28Colour Fig. c and d. Female (IZCAS-Ar 43728): PL 6.7, PW 5.2, AW 3.1, OL 8.5, OW 7.0. Eye diameters and interdistances: AME 0.25, ALE 0.21, PME 0.28, PLE 0.26, AME\u2013AME 0.22, AME\u2013ALE 0.48, PME\u2013PME 0.29, PME\u2013PLE 0.57, AME\u2013PME 0.23, ALE\u2013PLE 0.23, clypeus AME 0.19, clypeus ALE 0.58. Palp and leg measurements: palp 6.9 , I 16.3 , II 15.0 , III 13.1 , IV 18.8 . Leg formula 4123. Spination of palp and legs: palp 131, 000, 1111, 2101; femora I p021, d111, r021, II p112, d111, r021, III p112, d111, r112, IV p111, d111, r001; patellae I\u2013II 000, III\u2013IV 101, tibiae I\u2013II v22222, III\u2013IV p11, d111, r11, v222; metatarsi I\u2013II v222, III p112, d010, r112, v222, IV p112, r112, v2222. Chelicerae with 3 promarginal, 4 retromarginal teeth and with elongated patch of 10 tiny denticles along entire cheliceral furrow. Retromargin of chelicerae close to fang base with 5 bristles. Sparse scopula restricted almost entirely to tarsi, only metatarsi I\u2013II with sparse scopula hairs. Palpal claw with 6 secondary teeth, leg claws I with 1, II\u2013III with 2 and IV with 3 secondary teeth. Position of tarsal organ: I 1.03, II 0.97, III 0.95, IV 1.42.Copulatory organ Fig. b. EpigynColour Fig. e and f. Variation: Paratype male (IZCAS-Ar 43545): PL 7.4, OL 6.5. Second paratype female (IZCAS-Ar 43729): PL 7.6, OL 7.8.Ctenidae . The new species is assigned to the robustus-species group with the characteristics of stout tegular apophysis, simple stout embolus with broad base and short apical part, presence of retro-proximal cymbial outgrowth, RTA arising subdistally from palpal tibia, female possesses a transversally oval median plate with lateral teeth situated laterally and not reaching the epigastric furrow. It resembles B.yassassin J\u00e4ger, 2022 (see 28B.yassassin), by the median plate with distinct humped areas best seen in posterior view , by the spermathecae separated by less than their diameter, larger chamber round and apically swollen and by the fertilisation ducts pointing medially .Small to medium-sized 2022 see a\u2013c and l2022 see b, but caThe specific name refers to the type locality and is a noun in apposition.Vietnam : PL 3.8, PW 2.9, AW 1.2, OL 3.2, OW 2.2. Eye diameters and interdistances: AME 0.17, ALE 0.15, PME 0.22, PLE 0.21, AME\u2013AME 0.14, AME\u2013ALE 0.11, PME\u2013PME 0.24, PME\u2013PLE 0.27, AME\u2013PME 0.11, ALE\u2013PLE 0.13, clypeus AME 0.09, clypeus ALE 0.37. Palp and leg measurements: palp 5.1 , I 12.1 , II 10.9 , III 9.6 , IV 15.1 . Leg formula 4123. Spination of palp and legs: palp 151, 100, 1110; femora I p012, d111, r012, II\u2013III p112, d111, r112, IV p112, d111, r002; patellae I\u2013II 100, III\u2013IV 101; tibiae I p010, r010, v212222, II p100, d001, r110, v22222, III\u2013IV p11, d111, r11, v222; metatarsi I p111, r011, v222, II p111, d002, r111, v222, III p111, d012, r111, v222, IV p111, d012, r111, v2212. Chelicerae with 3 promarginal, 4 retromarginal teeth and with elongated patch of 8 tiny denticles along entire cheliceral furrow. Retromargin of chelicerae close to fang base with 4 bristles. Sparse scopula on all tarsi. Leg claws I\u2013II with 5 and III\u2013IV with 4 secondary teeth. Position of tarsal organ: I 1.07, II 0.89, III 0.72, IV 1.06.Palp Fig. a\u2013c. RTA 28Colour Fig. a and b. FemaleUnknown.Ctenidae . The new species is assigned to the chinagirl-species group with the characteristics of compact embolus, thick-walled, with rounded edges and with an entire margin, the tegular apophysis is longitudinally elongated, not covering the embolus. It resembles B.abdulmajid J\u00e4ger, 2022 (see B.abdulmajid), by the tibia having no distinct broad longitudinal ridge ventrally and by the conductor nearly quadrilateral .Small 2022 see d, but caThe specific name refers to the type locality and is a noun in apposition.Malaysia :OP572103).GGTTTGGTGCTTGGGCTTCTATAGCAGGTACGTCTATAAGAGTTTTGATTCGAATGGAATTAGGACATTCTGGAAGATTATTAGGGGATGATCATTTATATAATGTTGTTGTTACTGCTCATGCTTTTGTTATAATTTTTTTTATAGTGATACCTATTTTAATTGGTGGTTTTGGAAATTGGTTGGTTCCTTTAATATTAGGAGCTCCTGATATATCTTTTCCTCGTATAAATAATTTGTCGTTTTGATTACTTCCTCCTTCTTTATTTTTATTGTTTATATCTTCTATGACTGAGATAGGGGTGGGAGCTGGTTGGACGGTTTATCCACCTTTGGCTTCTGGAATTGGTCATGCAGGAAGATCGATAGATTTTGCTATCTTTTCTCTCCATTTAGCAGGTGCTTCTTCTATTATAGGAGCTATTAATTTTATTTCTACGATTATTAATATACGATTATTAGGAATGAGAATGGAAAAGGTTCCTTTGTTTGTATGGTCTGTTTTTATTACTGCAGTTTTATTATTATTATCTTTACCTGTTTTAGCGGGTGCTATTACTATATTATTAACGGATCGTAATTTTAATACTTCTTTTTTCGATCCTGCTGGAGGAGGGGATCCAATTTTATTTCAACATTTATTTTGATTTTTTG : PL 4.5, PW 3.6, AW 2.3, OL 3.9, OW 2.3. Eye diameters and interdistances: AME 0.21, ALE 0.11, PME 0.26, PLE 0.23, AME\u2013AME 0.15, AME\u2013ALE 0.31, PME\u2013PME 0.27, PME\u2013PLE 0.35, AME\u2013PME 0.13, ALE\u2013PLE 0.17, clypeus AME 0.14, clypeus ALE 0.41. Palp and leg measurements: palp 4.7 , I missing, II 10.8 , III 10.2 , IV 14.4 . Leg formula 4123. Spination of palp and legs: palp 131, 001, 112, 2012; femora II p110, d111, r112, III p012, d111, r112, IV p002, d111, r112; patellae II 000, III\u2013IV 101; tibiae II v22222, III\u2013IV p11, d111, r11, v222; metatarsi II v222, III p112, d010, r112, v222, IV p112, d010, r112, v2222. Chelicerae with 3 promarginal, 4 retromarginal teeth and with elongated patch of 5 tiny denticles along entire cheliceral furrow. Retromargin of chelicerae close to fang base with 5 bristles. Sparse scopula restricted almost entirely to tarsi. Palpal claw with 7 secondary teeth, leg claws II\u2013IV with 3 secondary teeth. Position of tarsal organ: II 0.75, IV 0.85.Copulatory organ Fig. . EpigynaColour Fig. c and d. Ctenidae . The new species resembles B.withinyou J\u00e4ger, 2022 (see B.withinyou), by the median plate with two separate sclerotised patches laterally and a longer median bulge in anterior half and by the spermathecae separated by more than twice their diameter .Small 2022 see b, but caThe specific name refers to the type locality and is a noun in apposition.Malaysia :OP572106).TATTTGGGGCTTGAGCTTCTATAGCTGGTACATCTATAAGTGTTTTGATTCGTATGGAGTTGGGACATTCGGGGAGAATATTGGGAGATGATCATCTCTATAATGTTATTGTTACTGCTCATGCTTTTGTTATAATTTTTTTTATAGTTATACCTATTTTAATTGGAGGTTTTGGTAATTGGTTGGTTCCTTTAATGTTAGGGGCTCCTGATATGTCGTTTCCTCGAATAAATAATTTGTCTTTTTGGTTACTTCCTCCTTCTTTATTTTTATTGTTTATGTCTTCTATAACTGAAATAGGGGTAGGAGCTGGTTGAACGGTGTATCCTCCTTTGGCTTCAAGAATGGGTCATGCTGGTAGATCTATGGATTTTGCTATTTTTTCTCTTCATTTAGCTGGGGCGTCTTCTATTATAGGTGCTATTAATTTTATTTCTACTATTATTAATATGCGTTTATTAGGAATGAGAATAGAGAAAGTTCCTTTGTTTGTATGGTCTGTTTTTATTACTGCGGTATTGTTATTATTGTCTCTTCCTGTTTTGGCAGGAGCTATTACTATATTGTTAACTGATCGTAATTTTAATACTTCTTTTTTTGATCCGGCTGGAGGGGGAGATCCGATTTTATTTCAACATTTATTTTGATTTTTT : PL 6.4, PW 5.1, AW 2.3, OL 4.8, OW 3.6. Eye diameters and interdistances: AME 0.22, ALE 0.17, PME 0.27, PLE 0.23, AME\u2013AME 0.25, AME\u2013ALE 0.40, PME\u2013PME 0.34, PME\u2013PLE 0.49, AME\u2013PME 0.17, ALE\u2013PLE 0.29, clypeus AME 0.11, clypeus ALE 0.52. Palp and leg measurements: palp 8.2 , I 20.5 , II 17.4 , III 14.5 , IV 22.4 . Leg formula 4123. Spination of palp and legs: palp 161, 001, 111; femora I p021, d111, r1111, II p112, d111, r112, III p001, d222, r112, IV p012, d111, r112; patellae I\u2013IV 101; tibiae I p110, d111, r11, v22222, II p110, d11, r11, v22222, III\u2013IV p11, d111, r11, v222; metatarsi I\u2013II p111, d012, r111, v222, III p111, d002, r111, v222, IV p111, d012, r111, v222. Chelicerae with 3 promarginal, 4 retromarginal teeth and with elongated patch of 23 tiny denticles along entire cheliceral furrow. Retromargin of chelicerae close to fang base with 6 bristles. Sparse scopula on all tarsi and metatarsi I\u2013III. Leg claws I with 5, II with 4, III with 5 and IV with 6 secondary teeth. Position of tarsal organ: I with 1.64, II 1.15, III 0.96, IV 1.28.Palp Fig. a\u2013c. RTA 28Colour Fig. c and d. Female (IZCAS-Ar 43733): PL 5.9, PW 4.4, AW 3.0, OL 5.2, OW 3.5. Eye diameters and interdistances: AME 0.23, ALE 0.19, PME 0.27, PLE 0.25, AME\u2013AME 0.20, AME\u2013ALE 0.44, PME\u2013PME 0.33, PME\u2013PLE 0.52, AME\u2013PME 0.17, ALE\u2013PLE 0.28, clypeus AME 0.13, clypeus ALE 0.51. Palp and leg measurements: palp 5.8 , I 13.9 , II 12.8 , III 11.3 , IV 16.7 . Leg formula 4123. Spination of palp and legs: palp 131, 001, 112, 203; femora I p021, d111, r011, II p012, d111, r111, III p112, d111, r112, IV p002, d111, r112; patellae I\u2013II 000, III\u2013IV 101; tibiae I\u2013II v22222, III\u2013IV p11, d111, r11, v222; metatarsi I\u2013II v222, III\u2013IV p111, d012, r111, v222. Chelicerae with 3 promarginal, 4 retromarginal teeth and with elongated patch of 12 tiny denticles along entire cheliceral furrow. Retromargin of chelicerae close to fang base with 7 bristles. Sparse scopula restricted almost entirely to tarsi, only metatarsi I\u2013II with sparse scopula hairs. Palpal claw with 5 secondary teeth, leg claws I with 2, II with 3, III with 4 and IV with 3 secondary teeth. Position of tarsal organ: I 0.97, II 0.84, III 0.75, IV 1.06.Copulatory organ Fig. a and b. Colour Fig. e and f. Ctenidae . The new species is assigned to the scarymonsters-species group with the characteristics of embolus with a basal, ventral bulge , tegulum bulging proximally at tegular apophysis base, the subdistally arising, apically pointed RTA in males, transversally oval median plate and lateral teeth situated at posterior margin of epigyne and spermathecae bottle gourd-shaped. It resembles B.neukoeln J\u00e4ger, 2022 (see 28B.neukoeln), by the tegular apophysis longitudinally orientated , by the conductor nearly quadrilateral and by the constrictive anterior width/widest width: 1/2 .Medium-sized 2022 see a\u2013c and l2022 see a, but caThe specific name refers to the type locality and is a noun in apposition.Malaysia :OP572111).GGTTTGGAGCTTGAGCTTCTATAGTAGGAACATCTATAAGAGTATTAATTCGTATAGAATTAGGACATTCTGGAAGATTATTAGGAGATGATCATTTATATAATGTAGTTGTTACTGCTCATGCTTTTGTTATGATTTTTTTTATAGTAATGCCTATTTTAATTGGAGGTTTTGGAAATTGATTAGTTCCTTTAATATTAGGGGCTCCTGATATATCTTTTCCTCGAATAAATAATTTATCATTTTGATTACTTCCTCCTTCTTTATTCTTATTATTTATGTCTTCTATAACTGAGATAGGAGTAGGAGCTGGTTGAACAGTATATCCTCCCTTAGCTTCTAGAATAGGACATATGGGAAGATCAATGGATTTTGCTATTTTTTCTCTTCATTTAGCTGGGGCTTCCTCTATTATAGGAGCTATTAATTTTATTTCTACAATTATTAATATACGATTATTGGGAATAAGAATAGAGAAAGTTCCATTATTTGTGTGGTCTGTTTTTATTACTGCGGTATTGTTGTTATTGTCTTTACCTGTTTTAGCAGGTGCTATTACTATATTATTAACGGATCGTAATTTTAATACTTCTTTTTTTGACCCAGCTGGGGGGGGGGATCCCATTTTATTTCAACATTTATTTTGATTTTTGC :OP572109).GGTTTGGAGCTTGAGCTTCTATAGTAGGAACATCTATAAGAGTATTAATTCGTATAGAATTAGGACATTCTGGAAGATTATTAGGAGATGATCATTTATATAATGTAGTTGTTACTGCTCATGCTTTTGTTATGATTTTTTTTATAGTAATGCCTATTTTAATTGGAGGTTTTGGAAATTGATTAGTTCCTCTAATATTAGGGGCTCCTGATATATCTTTTCCTCGAATAAATAATTTATCATTTTGATTACTTCCTCCTTCTTTATTCTTATTATTTATGTCTTCTATAACTGAGATAGGAGTAGGAGCTGGTTGAACAGTATATCCTCCCTTAGCTTCTAGAATAGGACATATGGGAAGATCAATGGATTTTGCTATTTTTTCTCTTCATTTAGCTGGGGCTTCCTCTATTATAGGAGCTATTAATTTTATTTCTACAATTATTAATATACGATTATTGGGAATAAGAATAGAGAAAGTTCCATTATTTGTGTGGTCTGTTTTTATTACTGCGGTATTGTTGTTATTGTCTTTACCTGTTTTAGCAGGTGCTATTACTATATTATTAACGGATCGTAATTTTAATACTTCTTTTTTTGACCCAGCTGGGGGGGGGGATCCCATTTTATTTCAACATTTATTTTGATTTTTGC , Am.krabi sp. n. , Am.matakecil Miller & Rahmadi, 2012 , Am.phangnga sp. n. and Am.saraburi sp. n. , the PME and AME are almost the same size also is a cave-dwelling species and its eyes are strongly reduced in size and pigments are absent are almost the same size (The posterior median eyes of all Li, 2022 . For theame size . These re absent . It is wame size , so thes"} +{"text": "Drosophila melanogaster, insulin/insulin-like growth factor (IIS) and mTOR-mediated signaling within adipocytes regulates oogenesis. To facilitate comparative study of nutrient-sensing pathway activity in the fat body, we developed antibodies to assess IIS (anti-FOXO) and mTOR signaling (anti-TOR) across three nymphalid species (Lepidoptera). By optimizing whole-mount fat body immunostaining, we find FOXO nuclear enrichment in adult adipocytes, like that observed inDrosophila. Additionally, we show a previously uncharacterized TOR localization pattern in the fat body.Nutritional stress impacts many insect species that have differing reproductive strategies and life histories, yet it is unclear how nutrient-sensing signaling pathways mediate tissue-specific responses to changes in dietary input. In Drosophila melanogaster. For example, nutrient sensing pathways, like IIS, mTOR, and the amino acid response (AAR) pathway, function within adultDrosophilaadipose tissue to control distinct stages of oogenesis . However, insects as a group that potentially includes 5.5 million species have diverse life histories. For example, female reproductive strategies are influenced by differences in ovarian dynamics , egg carbon:nitrogen ratios , and ovariole numbers . Insects also vary in distribution of the adipose tissue within the body, which could potentially affect inter-organ communication.Drosophila is therefore an excellent model for one such suite of traits but is not representative of all insects. We must develop additional diverse insect models in order to understand the variety of mechanisms controlling life history responses to dietary stress across insects.Resource allocation is inextricably linked to tradeoffs between life-history traits, allowing organisms to adapt to a dynamic environment . Nutritional control of the tradeoff between survival and reproduction is one of the best-characterized examples of this phenomenon and has been described for a variety of organisms, including insects. In several insect species, nutrients are reallocated from the energy intensive process of oogenesis to support somatic tissue maintenance during nutrient deprivation . This ensures that organisms survive beyond suboptimal nutritional conditions and do not waste resources on reproduction when the likelihood of offspring survival is low. While it is likely that multiple tissues sense and respond to nutrient input, we are just beginning to understand how inter-organ communication mediates whole organism responses in the heavily studied insect model,Drosophila melanogasterand various butterfly species within the Nymphalidae (Lepidoptera). These species differ in fat body distribution , ovarian dynamics , egg C:N ratio and completeness of the adult diet .Given that nutrient sensing signaling pathways control the response of insect fat body to changes in dietary input, which in turn affects allocation to egg production and other life history functions , we are interested in comparing the operation of these pathways and their outcomes amongDrosophila.Upon insulin receptor activation, Akt, the main effector kinase, acts on several targets to regulate cellular metabolism, survival, proliferation, and growth as well as reproduction . When phosphorylated by Akt, the transcription factor FOXO is restricted from the nucleus, where it controls expression of genes that mediate the negative effects of reduced IIS . In addition, Akt indirectly activates the mTOR kinase by inhibiting the negative regulatory complex of mTOR, Tsc1/Tsc2 . The mTOR kinase can also be activated by the Ragulator complex which senses amino acid status . In both cases, mTOR activation happens at the lysosome.The response to dietary stress of the IIS pathway has been well characterized inDrosophila, the transcriptomes for our non-model lepidopteran species have very limited similarity to those ofDrosophila melanogaster. Therefore, the likelihood of successful use ofD. melanogasterantibodies in the lepidopteran species is low. In contrast, our identified FOXO and TOR transcriptomes for the three lepidopteran species had high percent similarity (> 95%), indicating likely success in developing an antibody for each protein that would work in all three butterfly species.To gain a better understanding of how IIS functions within lepidopteran fat tissue, we want to visualize changes in localization of FOXO and mTOR. One approach to tracking localization of these components is to use species-specific antibodies for immunocytochemistry. While the required antibodies for FOXO and mTOR are available forAgraulis vanillae andHeliconius charithonia, we first determined the protein sequences for those genes for each species. We downloaded amino acid sequences forBombyx mori(Lepidoptera: Bombycidae) andHeliconius melpomene melpomene for FOXO and TOR from uniprot.org. We then used GeneiousPrime to generate transcriptome sequences for these proteins. We blasted these sequences againstA. vanillae andH. charithoniatranscriptomes to locate the sequences for each gene for these species. Transcriptome data forH. charithoniacame from Catalan et al (2018), and forA. vanillaefrom Hanly et al (2019). Additionally, we used transcriptome and amino acid sequences forSpeyeria mormonia, because we are interested in experiments with that species in the future. These RNA sequences were converted to amino acid sequences for determination of epitopes joint among the species. Epitope identification and antibody production were done by Genscript. Rabbits were used for polyclonal antibody production. We also used GeneiousPrime to compare the similarity of FOXO and TOR RNA sequences among our three species, as well as withDrosophila melanogaster(Diptera: Drosophiladae).In this report, we describe the development of Lepidoptera-specific antibodies for FOXO and mTOR to track changes in localization of FOXO and mTOR. To generate antibodies for FOXO and TOR inDrosophilasince adipocytes in fruit flies from nutrient-rich conditions show low-nearly undetectable levels of FOXO nuclear localization . The majority of mTOR immunoreactivity shows diffuse cytoplasmic localization with slight enrichment at cell-cell junctions . Under nutrient replete conditions, mTOR localizes to lysosomes where it can be activated by amino acids . Thus, future studies will assess if the mTOR staining pattern in butterfly adipocytes colocalizes with lysosomal markers. For both FOXO and mTOR immunolabeling, we are confident that the observed staining patterns are specific since there is no fluorescence in samples incubated without primary antibody . Moreover, the nuclear localization of FOXO is quite different from the cytoplasmic localization of mTOR . There is stronger fluorescence intensity at the lowest antibody concentration for tissue treated with Tween 20 than that treated with Triton X-100 . At higher antibody concentrations, detergent type is not a major factor in immunostaining quality. Taken together, we achieve the best signal-to-noise ratio for both antibodies when using Tween 20 as the detergent and 2.5 \u03bcg/mL of anti-FOXO and 5 \u03bcg/mL of anti-mTOR .We used a standard whole-mount immunostaining protocol to test immunoreactivity of these antibodies in lepidopteran fat body. We varied antibody concentration and permeabilization solution detergent (Triton X-100 versus Tween 20). We find that both antibodies show immunoreactivity in lepidopteran fat body at all three concentrations with varying robustness in all three butterfly species tested. For both antibodies, higher concentrations produced stronger signal . FOXO immunoreactivity showed enrichment in the nucleus with diffuse labeling in the cytoplasm . Since FOXO translocation to the nucleus results from reduced IIS pathway activity, this data suggests that the butterflies from which the fat body originated may have been under nutrient stress. Alternatively, lepidopterans may have a higher baseline of FOXO-dependent gene expression thanDrosophilato other insects that differ in their fat body distribution, mode of ovarian function (e.g. stem cell-supported versus not), and adult diet macronutrient composition?In the future, we will further optimize the immunostaining protocol by modifying additional steps in the protocol, such as fixative type and fixation time. This work demonstrates the feasibility of using these newly developed lepidopteran antibodies to answer questions like how generalizable are nutrient sensing mechanisms inButterfly collectionAgraulis vanillae females were collected in Sunrise, Florida in January 2022.Heliconius charithonia females came from Davie, Florida and south Texas in January 2022 and fall 2021 respectively.AntigensFOXO: MSLPRGGSYQSPWSSQTALSELEGAMGEFEPVGELAEVGFEPQTRARSNTWPQPRPENYVDAEDPGSKKNSNQNLSGAPPLPTSATTKKNSSRRNAWGNLSYADLITQAITSSQEKRLTLSQIYEWMVQNVPYFKDKGDSNSSAGWKNSIRHNLSLHNRFMRVQNEGTGKSSWWMINPDAKPGKSVRRRALSMETSKTEKRRGRVKKKPDALRNGVTADATPSPSSSISESLDIFPDSPIHSSFQLSPDFRQRASSNASSCSRLSPIPSLIPTEPEWSTDYTPSGDFASTSDFSPADYTQDQDLAGSLADSMKLHGADPFLNTYVPTTSSSSSGGSYRFTYGACPRHPHGGCACAPLYPAHPAHPTHSHQHSLDHFVRPPPPADPADIMQTENSQTQMVATSDATLMNGGIMVQTGPMGPTTVMGQIMGALNTGLSEDLNIEALEHSFDCNVDEVIRHELSMDGTLDFNFPQQNTAMAAEAESQFRAPSAPVPTTLSGGGASQTPYTVAPSWVH.TOR: MYMFLLKGHEDLRQDERVMQLFGLVNTLLQADPDTFRRDLVIQRYAVIPLSTNSGLIGWVPHCDTLHSLIKDYREKRKILLNIEHRIMQRMASDLEKLMLMQKVEVFEHALEHTAGDDLAKLLWLKSPSSEVWFERRTNYTRSLAVMSMVGYILGLGDRHPSNIMLDRVTGKFLHIDFGDCFEVAVTRDKFPEKIPFRLTRMLINAMEVTGIEGTYRRTCESVMEVLHRHRDSVMAVLEAFVYDPLLNWRLIDAGRRAGAPDDDGDASPQRPDDAPAETNLNKRALAIVNRVRDKLTGRDFTHIEENSVSVQRQVDLLIQQATSNENLCQCYVGWCPFW.Whole-mount adipose tissue immunostaining\u03bfC until mounted on slides.Females were sacrificed by crushing the ventral nerve and their abdomens were immediately dissected in cold phosphate buffered saline (PBS) to obtain fat body. Fat bodies were placed in 1.5ml microfuge tubes that were previously coated with 3% bovine serum albumin (BSA). Fat bodies were fixed in 1ml of 4% paraformaldehyde diluted in cold PBS (PFA) for 30 minutes at room temperature, rocking on a nutator. The PFA was then removed, and samples were rinsed twice with 1ml of PBS containing either 0.1% Triton X-100 (PBTX) or Tween 20 (PBTW) as a detergent. After rinsing, samples were washed three times with 1ml of 0.1% PBTX or PBTW, rocking each time for 15min on a nutator at room temperature. After the last wash, samples were incubated in 1ml of a blocking solution (0.1% PBTX or PBTW in 5% BSA) overnight at room temperature on a nutator. Samples were incubated in the indicated primary antibody concentration, diluted in blocking solution, overnight at room temperature. After washing three times for 15 minutes with PBTX or PBTW, samples were incubated in goat anti-rabbit AlexaFluor 488 secondary antibody diluted 1:250 in blocking solution and protected from light by covering with foil. Samples were then washed three times for 15 minutes with 0.1% PBTX or PBTW, protected from light. Following removal of wash solution, two drops of VectaShield containing DAPI were added. Samples were stored at 41X PBS \u2013 made from 10X PBS 3% BSA \u2013 made from 30% BSA 4% PFA \u2013 made from 16% PFA Triton X-100 - VWR, 0694-1LTween 20 \u2013 VWR, 0777-1LBlocking solution \u2013 0.1% PBTX or PBTW and 5% BSA in 1XPBSRabbit anti-FOXO \u2013 GenScriptRabbit anti-TOR \u2013 GenScriptGoat anti-rabbit IgG AlexaFluor 488 \u2013 Invitrogen, A27034"} +{"text": "Caenorhabditis elegans synthetic lethality screening. We identified the bromodomain protein, BET\u20101, as a key regulator of actin function and longevity. Overexpression of bet\u20101 preserves actin function at late age and promotes life span and healthspan in C. elegans. These beneficial effects are mediated through actin preservation by the transcriptional regulator function of BET\u20101. Together, our discovery assigns a key role for BET\u20101 in cytoskeletal health, highlighting regulatory cellular networks promoting cytoskeletal homeostasis.The actin cytoskeleton is a three\u2010dimensional scaffold of proteins that is a regulatory, energyconsuming network with dynamic properties to shape the structure and function of the cell. Proper actin function is required for many cellular pathways, including cell division, autophagy, chaperone function, endocytosis, and exocytosis. Deterioration of these processes manifests during aging and exposure to stress, which is in part due to the breakdown of the actin cytoskeleton. However, the regulatory mechanisms involved in preservation of cytoskeletal form and function are not well\u2010understood. Here, we performed a multipronged, cross\u2010organismal screen combining a whole\u2010genome CRISPR\u2010Cas9 screen in human fibroblasts with in vivo Caenorhabditis elegans (2), we identified BET\u20101/BRD4 as a novel regulator of the actin cytoskeleton (3). BET\u20101/BRD4 promotes actin filament stability in C.\u00a0elegans and senescent cells, which has direct impact on organismal health, survival, and life span.Using a cross\u2010species screening approach combining genome\u2010wide CRISPR\u2010Cas9 screening in human fibroblasts (1) and synthetic lethality screening in ACSRactin cytoskeletal stress responseEVempty vectorGOgene ontologyRNAiribonucleic acid interference1Caenorhabditis elegans , mediated by the heat\u2010shock transcription factor, HSF\u20101. HSF\u20101 is activated under thermal stress and promotes protein homeostasis through the upregulation of chaperones and other genes related to protein quality control , which promotes actin function, longevity, and healthspan in a multicellular eukaryote.To identify previously unidentified regulators of actin function, we adopted a multipronged, cross\u2010organismal screening approach. Since actin is one of the most highly conserved proteins, both in terms of sequence and functional homology, we held the rationale that an evolutionary\u2010conserved perspective could provide a powerful method to identify key regulators of actin function. We first utilized a CRISPR/Cas9\u2010driven growth\u2010based genetic screen in human cells to identify genes required for survival under actin stress. Actin stress was applied by exposure to cytochalasin D, a drug that inhibits actin polymerization by binding to F\u2010actin filaments and preventing the addition of actin monomers and exhibits visible perturbations of actin function without affecting whole organismal physiology. Animals treated with 10% act\u20101 RNAi develop normally to adulthood but display notable perturbations of actin quality in muscle cells Figure\u00a0. Importas Figure\u00a0. Therefobet\u20101, egrh\u20101 (Early Growth factor Response factor Homolog), F53B6.5, and ikb\u20101 (I Kappa B homolog) resulted in delayed development when combined with 10% act\u20101 RNAi but exhibit no physiological phenotypes when knocked down alone. RNAi knockdown of ZK512.4 did not exhibit a developmental defect but showed sterility (no eggs were visible on the plate at day 1 of adulthood) when combined with 10% act\u20101 RNAi, despite showing visible progeny formation when knocked down alone. ZK512.4 is a predicted ortholog of human SRP9 , a critical part of protein targeting to the endoplasmic reticulum , a major transcription factor that has been implicated in multiple diseases including cancer , which removes methionine residues from nascent polypeptide chains and are also implicated in cancer for 24\u2009h to induce senescence , the heat\u2010shock response (HSR), or oxidative stress response. We did observe a mild activation of genes involved in the endoplasmic reticulum UPR (UPRER). These data were further verified by direct comparison with a dataset identifying targets of the UPRER transcription factor, XBP\u20101 Figure\u00a0. Consist6 Figure\u00a0, which e1 Figure\u00a0, suggest2.4bet\u20101B overexpressing worms was act\u20103 , which has been previously linked to aging , another gene whose reduction has been linked to longevity , we opted for a secondary screening platform in bet\u20101, the C. elegans ortholog of human BRD2 and BRD4. bet\u20101 encodes a double bromodomain protein that has been originally characterized for its role in cell fate decisions dosage has been previously linked to longevity in C57B6/J mice, although the underlying molecular basis was not identified , but may be detrimental where actin dynamics are required .What was intriguing was that BET\u20101's impact on actin function is seemingly context dependent. We found that bet\u20101 overexpressing animals, with act\u20103 specifically being highly induced. We also observed changes in other cytoskeletal genes, including different regulatory factors. These genes showed opposing effects when compared to bet\u20101 loss of function animals and were largely dependent on the HAT, mys\u20101. Importantly, the transcriptional changes induced by BET\u20101B were largely distinct from the UPRMT, HSR, and the oxidative stress response.Since we see that all visible BET\u20101B protein is found within the nucleus and localizes to puncta which in no way resembles actin filaments, we believe that BET\u20101B does not impact actin through direct interactions. Moreover, the beneficial effects of BET\u20101B on actin and life span are dependent on MYS HATs, which acetylate histones at specific lysine residues, allowing BET\u20101B binding to alter gene expression , which can impact organismal health. An exciting area of future work is to investigate whether a BET\u20101B driven ACSR \u2013 possibly in coordination of other cytoskeletal regulators including DAF\u201016 and HSF\u20101 \u2013 can drive overall stress resilience and longevity.However, we did observe a mild overlap with UPRbet\u20101B overexpression was dependent on the insulin/IGF\u20101 transcription factor, DAF\u201016. While DAF\u201016 itself has not been directly tied to cytoskeletal function, FOXO transcription factors have direct impacts on actin function in mammalian systems expressing hTERT and Cas9 were used for the CRISPR\u2010Cas9 based screen as previously described and live/dead Aqua staining was performed as per manufacturer's protocol. Briefly, cells were stained with both dyes for 20\u2009min at room temperature in Annexin V binding buffer . Dyes were washed once with Annexin V binding buffer, resuspended with Annexin V binding buffer, and flow cytometry was used to quantify staining. For statistical analysis, two\u2010way ANOVA with Dunnett's test was used to compare the ratio of live cells between the 0\u00a0\u03bcM control against each drug\u2010treated group. 3 technical replicates of 2 biological replicates were performed.For Annexin V staining, BJ fibroblasts were treated with cytochalasin D at 0\u201310\u00a0\u03bcM concentrations for 24\u2009h and 3\u2010h treatment with 30\u2009mM H4.2Human primary IMR\u201090 fibroblasts were grown in DMEM, supplemented with 10% FBS, 100\u2009U/ml penicillin, 100\u2009\u03bcg/ml streptomycin, and 2\u2009mM glutamine at 37\u00b0C in a humidified atmosphere with 5% CO2 and 3.5% oxygen per 100\u2009\u03bcl culture medium. The old medium from senescent IMR\u201090 cells was aspirated and 120\u2009\u03bcl MTS assay solution per well was added. After 2\u00a0h incubation, 100\u2009\u03bcl MTS assay solution aspirated from the plate in a fresh 96\u2010well clear plate and the absorbance at 495\u2009nm was taken immediately using Microplate Reader.To examine the adhesion capacity of senescent cells after drug treatments, VybrantTM Cell Adhesion Assay Kit was used according to the manufacturer's protocol with modification. Briefly, 30\u201340% confluent IMR\u201090 cells were treated with etoposide and BET inhibitor/degraders drugs in 6\u2010wells plates as previously mentioned. After 8\u2009days, cells from each well were trypsinized and pelleted down after neutralizing the trypsin with complete media. The cells were washed once with PBS and resuspended in 1\u00a0ml serum free DMEM containing 5\u00a0\u03bcM Calcein AM solution for 30\u2009min. Then cells were washed twice with complete media to remove the excess Calcein AM. Finally, the Calcein AM labeled cells were suspended in 700\u2009\u03bcl complete media and 100\u2009\u03bcl of suspension was seeded per well in a PhenoPlate\u201096 (PerkinElmer). Cells from each treatment were seeded in total 6 wells of 96 wells. After 4\u00a0h incubation, 3 wells from each treatment were washed 3 times with PBS to assay the adhering cells and 3 wells were maintained without washing to assay the total cells. Fluorescence was measured using Clariostar Multi\u2010Mode Microplate Reader using FITC channel. The percentage of adhered cells were calculated from the fluorescence of adhered cells against total cells seeded after background subtraction.To detect the level of actin polymerization, we used Alexa Fluor 488 Phalloidin , because this bicyclic peptide specifically binds to polymerized F\u2010actin. Approximately 12,000 IMR90 cells were seeded in a PhenoPlate\u201096 (PerkinElmer) and treated with etoposide and drugs, as previously mentioned. After 8\u2009days of etoposide treatment, cells were fixed with 4% paraformaldehyde. After washing the fixed cells with PBS three times, cells were permeabilized with 0.2% Triton\u2010X and blocked with 1% BSA solution in PBS for 30\u2009min to reduce the background staining. After blocking, F\u2010Actin of the cells was stained with 165\u2009nM Alexa Fluor 488 Phalloidin for 1\u2009h at room temperature. Cells were washed with PBS three times and nuclei were stained with DAPI. The fluorescence of the wells was measured by using Clariostar Multi\u2010Mode Microplate Reader.4.3E. coli B strain and switched to growth at 20\u00b0C on HT115 E. coli K strain for all experimentation. HT115 bacteria carrying a pL4440 empty vector control or expressing double\u2010stranded RNA containing the sequence against a specific target gene were used for all experimentation. Experiments are performed on age\u2010matched animals synchronized using a standard bleaching protocol and bleached in 1.8% sodium hypochlorite and 0.375\u2009M KOH diluted in M9 until all carcasses were digested. Intact eggs were then washed 4\u00d7 with M9 solution followed by L1 synchronization by floating eggs in M9 overnight in a 20\u00b0C incubator on a rotator for a maximum of 16\u2009h. Synchronized animals are always grown on standard RNAi plates .All strains used in this study are derivatives of the N2 wild\u2010type worm from the Caenorhabditis Genetics Center (CGC) and are listed below. Worms are maintained at 15\u00b0C on OP50 bet\u20101 overexpression, isoforms A, B, and C were defined as per as a co\u2010injection marker, and 100\u2009ng/\u03bcl of pD64 vehicle as filler DNA. Worms positive for the fluorescent pharynx were selected to identify stable arrays. Integration was performed by gamma irradiation where L4 worms were irradiated with 4000\u20134400 rems of radiation and integration events were selected by finding animals that maintained 100% frequency of co\u2010injection marker in the F3 generation. Lines were then backcrossed into N2 a minimum of 8x to eliminate mutations. For overexpression of 3xHA::GFP::bet\u20101B, a GFP sequence containing introns and a 3xHA cassette was cloned upstream of the bet\u20101B coding sequence. Injection and integrations were performed by SUNY Biotech.For bet\u20101(uth41) mutant line, we used a Cas9\u2010RNA protocol as published on the IDT website via the Dernberg lab. Briefly, a mixture of 15\u2009\u03bcM trRNA, and 22\u2009\u03bcM crRNA (accatgggcaagtccgcgac) were incubated for 5\u00a0min at 95\u00b0C, cooled, then mixed with 24\u2009\u03bcM Cas9 protein and left at room temperature for 5\u00b0C. 2.5\u2009ng/\u03bcl of pEK2 (myo\u20102p::tdtomato) was added as a co\u2010injection marker and this mixture was injected into C. elegans gonads. All progeny positive for the co\u2010injection marker were selected, then sequenced for INDELs that incorporated a premature stop codon. The bet\u20101(uth41) mutant has a premature stop codon at amino acid 17.To synthesize the 4.4ATGTCTGAGGGCAGCGGAGACCAATCACAACAACGACCATGGGCAAGTCCGCGACAGCAACCAATCAAAGGAATCGTACAGCCACGAGTACTTCCACCATTCGGAAAGCCAACACGACACACAAACAAACTGGACTACATTATGACAACAGTACTCAAAGAGGCTGGAAAACATAAACATGTCTGGCCGTTTCAGAAGCCCGTCGATGCGGTTGCTTTATGTATTCCTCTATATCACGAGAGAGTCGCCCGACCAATGGACTTGAAAACAATCGAGAATAGACTGAAAAGTACTTATTACACATGTGCTCAAGAATGCATTGATGATATCGAAACAGTTTTCCAAAACTGCTACACATTCAATGGGAAAGAGGACGACGTGACAATTATGGCCCAAAATGTGCACGAAGTGATAAAAAAGTCACTGGAACAAGCACCTCGCGAAGAGCATGATATGGATGTTTATTGGGGAAAAAATAAGAAAAAACC GGCAAAAAGTGACGGTGGATCGAAATCTTCGTCGAGCAAGAAGAATGATGCTCGTGGACCATCTGAAGCACCGTCAGAGGCTGGAAGTGAAGTTTCGTCTGTAACAACAGCATCAGCAGC AGCCCCGACGGTTTCTGAGTCTGCGAGTGTTGCCGCGAAGCCAGAACGAAAAGTGGCCGGAAAGAAGACGGGAAAACGAAAAGCCGAATCAGAAGATGACGAGAAGCCGGAACCTTTGA GAGCAAAACGAGAGGTGGCTGTTGTCAAAAAAGAAGTTCATCAGCCATTGCTCCCAAGTATGAAGCCCTGTCTGAAGCTGCTCAATGATTTTTCTACAAAAAAATATCAGGAATTTGCTTGGC CATTCAACGAACCAGTAGACGCTGAACAACTGGGACTCCATGATTATCATAAAATTATCAAAGAACCAATGGATCTGAAATCAATGAAAGCAAAAATGGAAAGTGGAGCATACAAGGAACCTTCAGATTTCGAGCATGATGTTCGTTTAATGCTCAGGAATTGTTTTCTTTATAATCCAGTCGGTGATCCGGTTCACAGTTTTGGTCTTAGGTTTCAAGAAGTTTTTGATAGACGATGGGCTGAACTAGGTGATTCGAGTTCTCGTGCTTCATCAGTTGCACCTCAATCAGCTCCGATTGCTCCAACTCCGAAAGTAGCAAAATCAAGTGCTCCAAAAGAACCGAAAGAGTCTCGAAAAGAGCATAAAAAGGAGACGACTTTTGAAGCAAGCGGTGCAAAATCGGAGGATTTAATGCAGATAAACAACGCGTTGAGCATGATTCGAGAACGTGAGGAAAAGCTTAAAGCAGAGCTCGCCGCTGCACAAGC GATAAAGGATAAACTGACGAGTGTGAAGAATCGACGAGAAGATAATCCGAATGAGCCATTTCCGGAGAAGCTTATCAATGAGACAAGAGCCTTGTGCACGACGCAAGTTGGACAAAATGCTTCAAGTTCTTCAGCTTCTTCTGCTGCTTTGAGGAACGGACGAAGCAAAAAAGCAGCATCCGCACGTCTCTATGGTTACGAATTTGATTCGGATGATGAGGATAATAAGATGGCACTGACTTATGAGGAAAAACGAAACTTGAGCAATCTGATTAATAATTTACCCAACAATCAACTCAACACCATAATTTCGATTATTCAACGGAGAGAACGAAGCGCTCTGATGCAACAACAACTCGATGACAGTGAGGTTGAACTGGATTTCGAATCACTTGGAGATATGTGCCTGAGAGAAATGGGTGCATTTATCAAAACAATTCCAACATTAAACGGAAATGGCGATGATGAGAAGCCGAAAACGTCTTCGAATCCGACATCTTCTGGAGCAACAGGATCAAAGGGTTCGTCGTCGTTGGAGAGCAAAAATGGA AAGAAAAAGAAAAACTTCAATATGTCCGAATCCTCGGATGATGAGACGTCGAATAGTCGAAAACGTCGAAAGAGAGAGAGCAGTGAATCACAGAGCTCTTCGTCCAGTGATGATGATTCAGATGATGAGGATAGGCCGAGTATTCCCCGTAAATCAGGTCAACCACCATCAACATCACGTGAATGGAATCAATCATCAGCTCCTCCACCACGAATGGGAGGAATGGGAGGACAACCACCAATGTCACGAGTACCTGCATCATCATCCACATCTGTATCAGCAATCGGAAAGAACAACGCAGCCGCCTCGTCGAATTCATATCAAGCTCCAAAACCTGCACCAGTACCAGCACCAACATCATCAAGACCTCCGGCAGCACCGAGACCACCGTCAAAACCAAAGAAAACGGGTGGAGCGAGTATTCTTGATACTCTACTTCCAGATACATTTGGAGCATCACCTCCCCAGTTTTTCCAGTCGCAACCAACAACGTCGGCTACGATTAGATCACCAACGGAAAGCCAACCCGGGAATGGTGAAGACGAGCAGACCAGGATTCAGAGGATGCGGATGGAGGCAAAGCGAGCCCGCCAAAAAGAAGACGAAGGCAGTGTCTCGTTGTCAAACCAAATGGAAATGATGGCTGCATTTGAATTTGATAATACATATTAA4.5ATGTCTGAGGGCAGCGGAGACCAATCACAACAACGACCATGGGCAAGTCCGCGACAGCAACCAATCAAAGGAATCGTACAGCCACGAGTACTTCCACCATTCGGAAAGCCAACACGACACACAAACAAACTGGACTACATTATGACAACAGTACTCAAAGAGGCTGGAAAACATAAACATGTCTGGCCGTTTCAGAAGCCCGTCGATGCGGTTGCTTTATGTATTCCTCTATATCACGAGAGAGTCGCCCGACCAATGGACTTGAAAACAATCGAGAATAGACTGAAAAGTACTTATTACACATGTGCTCAAGAATGCATTGATGATATCGAAACAGTTTTCCAAAACTGCTACACATTCAATGGGAAAGAGGACGACGTGACAATTATGGCCCAAAATGTGCACGAAGTGATAAAAAAGTCACTGGAACAAGCACCTCGCGAAGAGCATGATATGGATGTTTATTGGGGAAAAAATAAGAAAAAACC GGCAAAAAGTGACGGTGGATCAAATCTTCGTCGAGCAAGAAGAATGATGCTCGTGGACCATCTGAAGCACCGTCAGAGGCTGGAAGTGAAGTTTCGTCTGTAACAACAGCATCAGCAGC AGCCCCGACGGTTTCTGAGTCTGCGAGTGTTGCCGCGAAGCCAGAACGAAAAGTGGCCGGAAAGAAGACGGGAAAACGAAAAGCCGAATCAGAAGATGACGAGAAGCCGGAACCTTTGA GAGCAAAACGAGAGGTGGCTGTTGTCAAAAAAGAAGTTCATCAGCCATTGCTCCCAAGTATGAAGCCCTGTCTGAAGCTGCTCAATGATTTTTCTACAAAAAAATATCAGGAATTTGCTTGGCCATTCAACGAACCAGTAGACGCTGAACAACTGGGACTCCATGATTATCATAAAATTATCAAAGAACCAATGGATCTGAAATCAATGAAAGCAAAAATGGAAAGTGGAGCATACAAGGAACCTTCAGATTTCGAGCATGATGTTCGTTTAATGCTCAGGAATTGTTTTCTTTATAATCCAGTCGGTGATCCGGTTCACAGTTTTGGTCTTAGGTTTCAAGAAGTTTTTGATAGACGATGGGCTGAACTAGGTGATTCGAGTTCTCGTGCTTCATCAGTTGCACCTCAATCAGCTCCGATTGCTCCAACTCCGAAAGTAGCAAAATCAAGTGCTCCAAAAGAACCGAAAGAGTCTCGAAAAGAGCATAAAAAGGAGACGACTTTTGAAGCAAGCGGTGCAAAATCGGAGGATTTAATGCAGATAAACAACGCGTTGAGCATGATTCGAGAACGTGAGGAAAAGCTTAAAGCAGAGCTCGCCGCTGCACAAGCGATAAAGGATAAACTGACGAGTGTGAAGAATCGACGAGAAGATAATCCGAATGAGCCATTTCCGGAGAAGCTTATCAATGAGACAAGAGCCTTGTGCACGACGCAAGTTGGACAAAATGCTT CAAGTTCTTCAGCTTCTTCTGCTGCTTTGAGGAACGGACGAAGCAAAAAAGCAGCATCCGCACGTCTCTATGGTTACGAATTTGATTCGGATGATGAGGATAATAAGATGGCACTGACTTATGAGGAAAAACGAAACTTGAGCAATCTGATTAATAATTTACCCAACAATCAACTCAACACCATAATTTCGATTATTCAACGGAGAGAACGAAGCGCTCTGATGCAACAACAACTCGATGACAGTGAGGTTGAACTGGATTTCGAATCACTTGGAGATATGTGCCTGAGAGAAATGGGTGCATTTATCAAAACAATTCCAACATTAAACGGAAATGGCGATGATGAGAAGCCGAAAACGTCTTCGAAT CCGACATCTTCTGGAGCAACAGGATCAAAGGGTTCGTCGTCGTTGGAGAGCAAAAATGGA AAGAAAAAGAAAAACTTCAATATGTCCGAATCCTCGGATGATGAGACGTCGAATAGTCGAAAACGTCGAAAGAGAGAGAGCAGTGAATCACAGAGCTCTTCGTCCAGTGATGATGATTCAGATGATGAGGATAGGCCGAGTATTCCCCGTAAATCAGGTCAACCACCATCAACATCACGTGAATGGAATCAATCATCAGCTCCTCCACCACGAATGGGAGGAATGGGAGGACAACCACCAATGTCACGAGTACCTGCATCATCATCCACATCTGTATCAGCAATCGGAAAGAACAACGCAGCCGCCTCGTCGAATTCATATCAAAAATTTTATAATTGTTTTCACAGTTATACTCCACCTTTAAAAGbet\u20101CTTGAAAAAAAAATCATCAAATTACTGGTAAATTTTTGTTAA ATGTCTGAGGGCAGCGGAGACCAATCACAACAACGACCATGGGCAAGTCCGCGACAGCAACCAATCAAAGGAATCGTACAGCCACGAGTACTTCCACCATTCGGAAAGCCAACACGACACACAAACAAACTGGACTACATTATGACAACAGTACTCAAAGAGGCTGGAAAACATAAACATGTCTGGCCGTTTCAGAAGCCCGTCGATGCGGTTGCTTTATGTATTCCTCTATATCACGAGAGAGTCGCCCGACCAATGGACTTGAAAACAATCGAGAATAGACTGAAAAGTACTTATTACACATGTGCTCAAGAATGCATTGATGATATCGAAACAGTTTTCCAAAACTGCTACACATTCAATGGGAAAGAGGACGACGTGACAATTATGGCCCAAAATGTGCACGAAGTGATAAAAAAGTCACTGGAACAAGCACCTCGCGAAGAGCATGATATGGATGTTTATTGGGGAAAAAATAAGAAAAAACCGGCAAAAAGTGACGGTGGATCGAAATCTTCGTCGAGCAAGAAGAATGATGCTCGTGGACCATCTGAAGCACCGTCAGAGGCTGGAAGTGAAGTTTCGTCTGTAACAACAGCATCAGCAGC AGCCCCGACGGTTTCTGAGTCTGCGAGTGTTGCCGCGAAGCCAGAACGAAAAGTGGCCGGAAAGAAGACGGGAAAACGAAAAGCCGAATCAGAAGATGACGAGAAGCCGGAACCTTTGA GAGCAAAACGAGAGGTGGCTGTTGTCAAAAAAGAAGTTCATCAGCCATTGCTCCCAAGTAT GAAGCCCTGTCTGAAGCTGCTCAATGATTTTTCTACAAAAAAATATCAGGAATTTGCTTGGCCATTCAACGAACCAGTAGACGCTGAACAACTGGGACTCCATGATTATCATAAAATTATCAAAGAACCAATGGATCTGAAATCAATGAAAGCAAAAATGGAAAGTGGAGCATACAAGGAACCTTCAGATTTCGAGCATGATGTTCGTTTAATGCTCAGGAATTGTTTTCTTTATAATCCAGTCGGTGATCCGGTTCACAGTTTTGGTCTTAGGTTTCAAGAAGTTTTTGATAGACGATGGGCTGAACTAGGTGATTCGAGTTCTCGTGCTTCATCAGTTGCACCTCAATCAGCTCCGATTGCTCCAACTCCGAAAGTAGCAAAATCAAGTGCTCCAAAAGAACCGAAAGAGTCTCGAAAAGAGCATAAAAAGGAGACGACTTTTGAAGCAAGCGGTGCAAAATCGGAGGATTTAATGCAGATAAACAACGCGTTGAGCATGATTCGAGAACGTGAGGAAAAGCTTAAAGCAGAGCTCGCCGCTGCACAAGCGATAAAGGATAAACTGACGAGTGTGAAGAATCGACGAGAAGATAATCCGAATGAGCCATTTCCGGAGAAGCTTATCAATGAGACAAGAGCCTTGTGCACGACGCAAGTTGGACAAAATGCTTCAAGTTCTTCAGCTTCTTCTGCTGCTTTGAGGAACGGACGAAGCAAAAAAGCAGCATCCGCACGTCTCTATGGTTACGAATTTGATTCGGATGATGAGGATAATAAGATGGCACTGACTTATGAGGAAAAACGAAACTTGAGCAATCTGATTAATAATTTACCCAACAATCAACTCAACACCATAATTTCGATTATTCAACGGAGAGAACGAAGCGCTCTGATGCAACAACAACTCGATGACAGTGAGGTTGAACTGGATTTCGAATCACTTGGAGATATGTGCCTGAGAGAAATGGGTGCATTTATCAAAACAATTCCAACATTAAACGGAAATGGCGATGATGAGAAGCCGAAAACGTCTTCGAAT CCGACATCTTCTGGAGCAACAGGATCAAAGGGTTCGTCGTCGTTGGAGAGCAAAAATGGAAAGAAAATAA4.6CACAGGTCTCTAGTGTATCCACTTCGAATGCGATGCCCGAAACCTCTTCATCCATCCGTCTCCTTCTCGCTCTCTCTCTCTCTCTCTCTTCTCCATCTCTCTCCACATTTTGCCTGCTATCTCGTGATTGTCGTCCCGTCCGTGTTCCGCCGCACACACTGCCTGTCTTCTCTTAACCGTGTGTCGATCAACTCCCAAACCGCTACGCTATTTCTCTCTCCCTCTCTCTCTCTCTTCGGCGGTGACATTTCTGACTAGATGGTCATACAAAACGCGTGCTGCGCGCGCGCTCCGCAAAAATCGACGCGAATCGATTAATGTGCGTCTCGTTTCTCTATCTCTGACCGCCCCCGCTTCAACCTAACACTATTTTTGAATGCTTTTCAACTGTAACTTGCAGCTAATTAGAAGTTGAGAGATAACCTGTTGCGATTGGCTCCGGGCAAGGGTTGGGAGGTCGCACCAGAAATTTTAGAGCTCTAGGATTTCAAATTTTTGGGTTTCAAGACCGTAACATGATTTTCTTGGAAATTTATCACAAATCATGTAGAAAATCGATATCAGTAAGAGGGAGTGAGTGATCTATCATTTTTTATCTTTCGATCTGAAATTCCACAGCGAAGGTTTTCTGCCGAAATTTCGAAATTGGTATTTTGAACTATCCGATAATTCGTAGAACATCAAGATAAAGTGTCAACCTATAGAAAATCACATGATTCGTCAGAAAATAACATTAATTTCATATGAAATAGTTGAGAAAGTGCTCAAAAATGGCCTAAAATTATCCAATAATCGACATTTGACAACTTTCAGCACACTTTTGAACCGTTTATCAATTGTTTCTGCTGAAATAGACGTATTTTTCGGACGAATCGAGTGATTTCCTATAGTTTTACACTGATTTTTGACAAAAAAATATTGATAGAACATGGTGCATTAGGCAATTTTTTAGAATTGCCGTCTACACCTGATTTCGATGGGTCCTCGTGACAAGACCCAAAATTTTATTATTTTTATCGTTGAAAAAAATCAAATCAATAACACCGCAATCAC CATTTGCAAAGTTTAATTAAATACAATTTTTATTAAAATATTTCAGGAATAAAAATATTAGTCAGAATAATCCCATGTTCTTCTAGCGATTTCAACTAATTCTTTGAAAATAACATTTCTTGGAATT TAAGAATACGAAAATAGTCACTTCTTTGTATTCTAGAAACGCTAATTCCTGCAACCGACAAA TTAAAAGTACAAAAAATGATACGGCAAGCGCGCTCCAATTCAAATCGAGTCTCCCGCCTTCCTTGACGTCATTGCTAACAGCTGCTTCGGTTTTTTCCTCCAAATTTCGTGGTTCAAATTTTAT TTTTAATTGAATTTTAACAAAATAGGAAGCTAGTTGAGTAACATTTATTATTAATTTTGTAAAATATTCTGCAAATTCGGCGTTTTCTTTTAATTCAAATAAAAGTTTTCAATAAAAAAAATCGATATTTTCAG4.7CATCTCGCGCCCGTGCCTCTGACTTCTAAGTCCAATTACTCTTCAACATCCCTACATGCTCTTTCTCCCTGTGCTCCCACCCCCTATTTTTGTTATTATCAAAAAACTTCTCTTAATTTCTTTGTTTTTTAGCTTCTTTTAAGTCACCTCTAACAATGAAATTGTGTAGATTCAAAAATAGAATTAATTCGTAATAAAAAGTCGAAAAAAATTGTGCTCCCTCCCCCCATTAATAATAATTCTATCCCAAAATCTACACAATGTTCTGTGTACACTTCTTATGTTTTTTACTTCTGATAAATTTTTTTGAAACATCATAGAAAAAACCGCACACAAAATACCTTATCATATGTTACGTTTCAGTTTATGACCGCAATTTTTATTTCTTCGCACGTCTGGGCCTCTCATGACGTCAAATCATGCTCATCGTGAAAAAGTTTTGGAGTATTTTTGGAATTTTTCAATCAAGTGAAAGTTTATGAAATTAATTTTCCTGCTTTTGCTTTTTGGGGTTTCCCCTATTGTTTGTCAAGATTTCGAGGACGGCGTTTTTCTTGCTAAAATCACAAGTATTGATGAGCACGATGCAAGAAAGATCGGAAGAAGGTTTGGGTTTGAGGCTCA GTGGAAG4.8ATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGgtaagtttaaacatatatatac taactaaccctgattatttaaattttcagCCAACACTTGTCACTACTTTCTGTTATGGTGTTCAATGCTTCTCGAGATACCCAGATCATATGAAACGGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTAT GTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACgtaagtttaaacagttcgg tactaactaaccatacatatttaaattttcagGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGA ATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATA CAACTATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTgtaagtttaaacatgattttactaactaactaatctgatttaaattttcagAACTTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAA4.9TATCCATATGACGTGCCGGACTACGCGTACCCGTATGATGTTCCAGACTACGCCTATCCGTACGACGTACCAGATTATGCA4.10CAGCAACCAATCAAAGGAATCGTACAGCCACGAGTACTTCCACCATTCGGAAAGCCAACACGACACACAAACAAACTGGACTACATTATGACAACAGTACTCAAAGAGGCTGGAAAACATAAACATGTCTGGCCGTTTCAGAAGCCCGTCGATGCGGTTGCTTTATGTATTCCTCTATATCACGAGAGAGTCGCCCGACCAATGGACTTGAAAACAATCGAGAATAGACTGAAAAGTACTTATTACACATGCGCTCAAGAATGCATTGATGATATCGAAACAGTTTTCCAAAACTGCTACACATTCAATGGGAAAGAGGACGACGTGACAATTATGGCCCAAAATGTGCACGAAGTGATAAAAAAGTCACTGGAACAAGCACCTCGCGAAGAGCATGATATGGATGTTTATTGGGGAAAAAATAAGAAAAAACCGGCAAAAAGTGACGGTGGATCGAAATCTTCGTCGAGCAAGAAGAATGATGCTCGTGGACCATCTGAAGCACCGTCAGAGGCTGGAAGTGAAGTTTCGTCTGTAACAACAGCATCAGCAGCAGCCCCGACGGTTTCTGAGTCTGCGAGTGTTGCCG4.11AAAAAAGCAGGCTTGACCGAGCCGAAGAAGGAGATTATAGAGGACGAAAATCATGGAATAT CCAAGAAAATACCAACAGATCCCAGGCAATACGAGAAAGTTACAGAGGGATGCCGGTTATTGGTCATGATGGCTTCACAAGAAGAAGAAAGATGGGCCGAAGTTATTTCAAGATGCCGAGCTGCAAATGGTTCAATTAAATTCTATGTCCATTATATCGATTGCAACCGAAGACTTGACGAATGGGTTCAGTCTGATAGGCTCAATTTAGCGTCGTGTGAGCTACCAAAAAAAGGAGGAAAGAAAGGAGCACACTTGCGGGAAGAAAATCGAGATTCGAATGAAAATGAAGGAAAGAAAAGCGGCCGAAAACGAAAGATTCCACTACTTCCGATGGATGATCTCAAGGCGGAATCCGTAGATCCATTACAAGCAATTTCAACGATGACCAGCGGATCTACTCCAAGTCTTCGAGGTTCCATGTCGATGGTCGGCCATAGTGAAGATGCAATGACAAGGATCCGAAATGTCGAATGCATTGAACTAGGAAGATCACGAATTCAGCCATGGTACTTTGCACCTTATCCACAACAATTGACAAGTTTGGATTGTATTTATATTTGCGAATTTTGTCTGAAATATCTAAAGTCGAAAACTTGTCTGAAACGGCACNTGGAAAAATGTGCAATGTGTCACCCACCTGGCAATCAAATCTACAGTCACGATAAACTTTCATTTTTTGAAATCGACGGCCGCAAAAACAAAAGCTATGCTCAGAATCTATGCCTGCTTGCCA AACTT4.12C. elegans orthologs of human genes from the cytochalasin screen were identified using Ortholist 2 , images were acquired on either a Zeiss AxioObserver.Z1 microscope equipped with a lumencor sola light engine and Ziess axiocam 506 camera driven by Zeiss ZenBlue software using a 63x/1.4 PlanApochromat objective, standard dSRed filter (Zeiss filter set 43), and a DFC9000 camera; or a Leica Thunder Imager equipped with a 63x/1.4 Plan AproChromat objective, standard dsRed filter (11525309), Leica DFC9000 GT camera, a Leica LED5 light source, and run on LAS X software. For confocal microscopy (gly\u201019p::LifeAct), imaging was performed on a Stellaris 5 confocal microscope equipped with a white light laser source and spectral filters, HyD detectors, 63x/1.4 Plan ApoChromat objective, and run on LAS X software.For high magnification live\u2010cell imaging, animals are picked off of plates and mounted directly onto a microscope slide containing M9\u2009+\u00a00.1\u00a0M sodium azide. For standard wide\u2010field microscopy floating in M9 in a rotator at 20\u00b0C, then washed 3x with M9 and plated on standard OP50 plates for 24\u2009h, then imaged.For imaging of fluorescent transcriptional reporters, animals were synchronized via bleaching and grown on standard RNAi plates and imaged at day 1 of adulthood. For imaging, animals were moved onto standard NGM plates without bacteria containing 5\u00a0\u03bcl of 100\u2009mM sodium azide to paralyze worms. Paralyzed worms were lined up and imaged immediately on a Leica M205FCA automated fluorescent stereoscope equipped with a standard GFP filter and Leica K5 camera and run on LAS X software. 3 biological replicates were performed per experiment with 4.14t\u2010test using Prism software.To measure gut bacteria invasion, animals were synchronized via bleaching and plated from hatch on RNAi of choice mixed with 20% HT115 bacteria expressing mCherry as previously described . The aqueous phase was mixed 1:1 with isopropanol then applied to a Qiagen RNeasy Mini Kit (74106) and RNA purification was performed as per manufacturer's directions.bet\u20101B overexpression, bet\u20101 RNAi, and bet\u20101(uth41) mutants were compared to N2 wild\u2010type control. In addition, bet\u20101B overexpressing worms were grown on mys\u20101 RNAi and compared to a mys\u20101 RNAi control.Library preparation was performed using a Kapa Biosystems mRNA Hyper Prep Kit sequencing was performed at the Vincent J Coates Genomic Sequencing Core at the University of California, Berkeley using an Illumina HS4000 mode SR100. Four biological replicates were measured per condition. Reads per gene were quantified using kallisto using 1\u00a0\u03bcg of RNA. RT\u2010PCR was performed using NEB Q5 DNA polymerase as per manufacturer's guidelines using primers listed below. Four biological replicates were performed per condition. Image quantification was performed using ImageJ by drawing an ROI 974 of equal size around each band and quantifying for integrated density. Data was normalized to a 975 4.16Thrashing assays were performed on animals synchronized via bleaching and aged on plates containing FUDR from day 1. 100\u2009\u03bcl of 10\u00a0mg/ml FUDR were spotted on the bacterial lawn. At the desired age, plates containing adult animals were flooded with 100\u2009\u03bcl of M9 solution, and 30\u2009sec videos were acquired on an M205FCA stereomicroscope equipped with a Leica K5 microscope and run on LAS X software. Thrashing was measured by eye over a 10\u00a0s period. A single trash is defined as bending of >50% of the animal's body in the opposite direction. Representative data of three independent biological replicates are presented. Dot plots were generated using Prism 7 software where every dot represents a single animal and lines represent median and interquartile range. All statistics were performed using nonparametric Mann\u2013Whitney testing.4.17A synchronized population of animals were collected via bleaching and 10 L4 animals were moved onto individual plates. Every 12\u2009h, animals were moved onto fresh plates and plates containing eggs were stored in a 15\u00b0C incubator for 2\u20133\u2009days. All live progeny on every egg\u2010lay plate were scored and summed to determine brood size. Dot plots were generated using Prism 7 software where every dot represents a single animal and lines represent median and interquartile range. All statistics were performed using non\u2010parametric Mann\u2013Whitney testing.4.18Caenorhabditis elegans life span assays were performed on standard RNAi plates and were all performed at 20\u00b0C as previously described (Bar\u2010Ziv et al.,\u00a0Caenorhabditis elegans experiments for manuscript revision. N.Dasgupta performed all senescent cell culture experiments. N.Dutta performed RTPCR analysis and assisted with life spans. H.Z., and W.F. performed all standard cell culture experiments. C.K.T., E.A.M., and O.S. performed all computational analysis for CRISPR\u2010Cas9 screening. D.M., A.A., S.H., and T.C.T. assisted with C. elegans experiments. P.D.A. supervised all senescent cell culture experiments. M.A.T. performed essential experiments that assisted in development of the manuscript. All authors edited the manuscript.G.G. and R.H.S. designed all experiments, performed or oversaw all experiments, and prepared the figures and manuscript. R.B.Z. performed computational analysis, figure construction, and writing for all transcriptomics data. M.A. performed all All authors of the manuscript declare that they have no competing interests.Figures S1\u2013S9Click here for additional data file.Table S1Click here for additional data file.Table S2Click here for additional data file.Table S3Click here for additional data file.Table S4Click here for additional data file.Table S5Click here for additional data file.Table S6Click here for additional data file.Table S7Click here for additional data file.Table S8Click here for additional data file."} +{"text": "Titin is the largest protein in humans, composed of more than one hundred immunoglobulin (Ig) domains, and plays a critical role in muscle\u2019s passive elasticity. Thus, the molecular design of this giant polyprotein is responsible for its mechanical function. Interestingly, most of these Ig domains are connected directly with very few interdomain residues/linker, which suggests such a design is necessary for its mechanical stability. To understand this design, we chose six representative Ig domains in titin and added nine glycine residues (9G) as an artificial interdomain linker between these Ig domains. We measured their mechanical stabilities using atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS) and compared them to the natural sequence. The AFM results showed that the linker affected the mechanical stability of Ig domains. The linker mostly reduces its mechanical stability to a moderate extent, but the opposite situation can happen. Thus, this effect is very complex and may depend on each particular domain\u2019s property. The giant muscle protein titin is a tandem modular construction designed polyprotein containing more than two hundred individually folded domains, such as immunoglobulin-like (Ig) and fibronectin-type III domains . These dTo examine the linker effect, we used atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS) to measure the mechanical stability of human Ig domains. SMFS can manipulate a single molecule mechanically ,20,21,22Thus, we choose six consecutive Ig domains of titin, including I27, I28, I29, I30, I31, and I32, as a representative unit (I27\u2013I31), to study the interdomain linker effect b. The stA high-precision AFM measurement system has been used for accurate measurement and comparison of Ig domains in titin ,65,66,67n = 1148, Thus, we built polyprotein Coh-I(27\u201332)/9G-NGL with a 9G linker for measurement. The 9G linker is present between each Ig domain except for the two end I27 and I32 domains a. By appn = 1808, Then, we used polyprotein Coh-I(27\u201332)-NGL with natural sequence for AFM measurement and comparison. The same cantilever used previously for the polyprotein with the linker was used here again to minimize the error. As expected, a similar unfolding pattern was observed b,d,e. Hon = 860). Then, AFM measurement on Coh-I(28\u201330)-NGL with natural sequence showed a force of 325 \u00b1 35 pN (n = 965).To confirm this effect, we chose three Ig domains only and constructed two shorter polyproteins, Coh-I(28\u201330)/9G-NGL and Coh-I(30\u201332)/9G-NGL, for measurement. Indeed, stretching these polyproteins resulted in a shorter force-extension curve with only two 28 nm-peaks from I28, I29, or I31, I32, and one 11 nm-peak from I30, as expected . For Cohn = 1120). However, AFM measurement on the natural sequence showed a different result. Two peaks were observed in the histogram, with a force of 276 pN and 345 pN, respectively (n = 1804), which has not been observed before. For Coh-I(30\u201332)/9G-NGL, the histogram of unfolding force from I31 and I32 showed a single peak with an average force of 320 \u00b1 33 pN , and 186 \u00b1 55 pN (n = 225) without linker. For I(28\u201330), 9G linker is only present on the N terminus. The force was 197 pN (n = 570), and 172 \u00b1 24 pN (n = 910) without linker. Finally, for I(30\u201332), 9G linker is only present on the C terminus. The force was 159 \u00b1 23 pN (n = 477), and 175 \u00b1 24 pN (n = 404) without the linker /9G-NGL, Coh-I(27\u201332)-NGL, Coh-I(28\u201330)/9G-NGL, Coh-I(28\u201330)-NGL, Coh-I(30\u201332)/9G-NGL, Coh-I(30\u201332)-NGL were obtained after Gibson assembly-based method [Escherichia coli strain BL21 (DE3) and cells were cultured overnight in LB medium at 18 \u00b0C by the addition of 1mM IPTG. The cells were pelleted by centrifugation and the polyprotein purification by Ni-NTA affinity. After using wash buffer to purify the target proteins, the polyproteins were eluted in elution buffer . Protein ligase AEP was obtained according to literature [d method . All platerature .Protein immobilization: The glass coverslips and probes were cleaned by plasma. Then, both probes and coverslips were immersed in 1% (v/v) APTES toluene solution for 1 h to add the NH2 group, followed by a reaction with Milli-Q water containing 2 mM ImSO2N3, 4 mM K2CO3, and 20 mM CuSO4 to add the N3 group. After flushing, they were further reacted with DBCO-PEG4-maleimide for at least 2 h to add the maleimide group. Peptide C-ELP20-NGL and GL-ELP20-C were respectively reacted onto the probe and coverslip. For AFM-SMFS measurement, 50 \u03bcL AFM buffer containing 100 \u03bcM Ig proteins and 50 \u03bcM AEP were pipetted on the glass slides for 40 min. The cantilevers were incubated with 50 \u03bcL solution of 60 \u03bcM GL-CBM-XDoc and 50 \u039cm AEP in the AFM buffer.AFM-SMFS Experiment: Measurements using Coh-Doc interaction of high rupture force as a standard were carried out Nanowizard4 (JPK) atomic force microscope. Using the equipartition theorem, the spring constant of ~30 pN nm\u22121 was obtained by calibrating the MLCT-Bio-DC (Bruker) cantilever in the AFM buffer solution. The functionalized cantilevers and glass coverslip immobilize polyproteins in AFM buffer at pH 7.4. All AFM experiments were performed at a constant pulling speed of 1000 nm\u00b7s\u22121.9G-I28-9G-I29-9G-I30-9G-I31-9G-I32)Protein sequence (I27-GGGGGGGGGPLIFITPLSDVKVFEKDEAKFECEVSREPKTFRWLKGTQEITGDDRFELIKDGTKHSMVIKSAAFEDEAKYMFEAEDKHTSGKLIIEGIGGGGGGGGGRLKFLTPLKDVTAKEKESAVFTVELSHDNIRVKWFKNDQRLHTTRSVSMQDEGKTHSITFKDLSIDDTSQIRVEAMGMSSEAKLTVLEGGGGGGGGGGDPYFTGKLQDYTGVEKDEVILQCEISKADAPVKWFKDGKEIKPSKNAVIKTDGKKRMLILKKALKSDIGQYTCDCGTDKTSGKLDIEDRGGGGGGGGGEIKLVRPLHSVEVMETETARFETEISEDDIHANWKLKGEALLQTPDCEIKEEGKIHSLVLHNCRLDQTGGVDFQAANVKSSAHLRVKPRGGGGGGGGGVIGLLRPLKDVTVTAGETATFDCELSYEDIPVEWYLKGKKLEPSDKVVPRSEGKVHTLTLRDVKLEDAGEVQLTAKDFKTHANLFVKEPLIEVEKPLYGVEVFVGETAHFEIELSEPDVHGQWKLKGQPLAASPDCEIIEDGKKHILILHNCQLGMTGEVSFQAANTKSAANLKVKEL"} +{"text": "The hub metabolite, nicotinamide adenine dinucleotide (NAD), can be used as an initiating nucleotide in RNA synthesis to result in NAD-capped RNAs (NAD-RNA). Since NAD has been heightened as one of the most essential modulators in aging and various age-related diseases, its attachment to RNA might indicate a yet-to-be discovered mechanism that impacts adult life-course. However, the unknown identity of NAD-linked RNAs in adult and aging tissues has hindered functional studies. Here, we introduce ONE-seq method to identify the RNA transcripts that contain NAD cap. ONE-seq has been optimized to use only one-step chemo-enzymatic biotinylation, followed by streptavidin capture and the nudix phosphohydrolase NudC-catalyzed elution, to specifically recover NAD-capped RNAs for epitranscriptome and gene-specific analyses. Using ONE-seq, we discover more than a thousand of previously unknown NAD-RNAs in the mouse liver and reveal epitranscriptome-wide dynamics of NAD-RNAs with age. ONE-seq empowers the identification of NAD-capped RNAs that are responsive to distinct physiological states, facilitating functional investigation into this modification. In SPAA (SPAAC) . As a re ethyl]-biotinamide, CAS: 717119-80-7, 5 mM, Amatek scientific, catalog: B-1328) under the catalysis of ADPRC in 100 \u03bcl of ADPRC reaction buffer was reacted with HEEB (4HCO3 in water and (B) acetonitrile at a flow rate of 0.4 ml/min. The injection volume was 2 \u03bcl. The gradient was as follows: 0 min 0% B, 2 min 0% B, 4 min 20% B, 17 min 40% B, 19 min 100% B, 24 min 100% B, and 30 min 0% B. The retention time of NAD and ADPR-Biotin (product) was 9.393 min and 9.929 min, respectively. The corresponding compounds were purified from HPLC and then analyzed by LC\u2013MS by Agilent Technologies 6120 Quadrupole with a C18 column . The mobile phase was composed of (A) 0.1% formic acid in water and (B) acetonitrile at a flow rate of 0.4 ml/min. The injection volume was 5 \u03bcl. The gradient was as follows: 0 min 10% B, 4 min 100% B, and 7 min 100% B.Samples were analyzed by an Agilent Technologies 1260 Infinity with a C18 column monitoring at 260 nm. The mobile phase was composed of (A) 100 mM NHTAATACGACTCACTATTACTGTGTCGTCGTCGTCTGCTGTCTCTCTCTCGCGGGC-3\u2032; boldface letters denote the sequence of T7 class II promotor (\u03d52.5)) and (anti-sense: 5\u2032-GCCCGCGAGAGAGAGACAGCAGACGACGACGACACAGTAATAGTGAGTCGTATTAGTGATC-3\u2032) ) and (anti-sense: 5\u2032-GGCAGCAAGCCGGAGGAGCAGAAGAACGACAGGGCCAGCGAGCCGCAGGCGGCAGCGCGCACGCCCACGCCCGCGAGAGAGAGACAGCAGACGACGACGACACAGTAATAGTGAGTCGTATTAGTGATC-3\u2032). To prepare spike-in ppp-RNA of 62 nt, oligonucleotide was synthesized (Genewiz) and annealed to make double-stranded DNA template ) and (anti-sense: 5\u2032-TGGCTGGCCGCATGCCCGCGGCAGAAGTGACATTATGGCTAAAGTTGTGGTAGCACTTCCCTAATAGTGAGTCGTATTA-3\u2032). To prepare spike-in NAD-RNA (500 nt) and m7Gppp-RNA (500 nt) with identical sequence, oligonucleotide without adenine was synthesized (Genewiz) and were subjected to polyadenylation for poly(A) tails elongation ) and (anti-sense: 5\u2032-AAGAAGAACACCAAGAAGCCGAACAACAGCAAGACGGCCAAGAAAAAGACGAAGAGGCAGAAGAAGAAGAACACCAGCAAGAGCCCCAGGAAGAAGCCGACCACCAAGAAGACGAAGCCCAACAGCACGAAGCGGAACACCAGGGAGACGCCCACGAACAACACCACGGCGCGGGACAAGAAGAAGCCGACGACCAAGAAGAAGAAGGAGCGCACCAGGACGAAGCCAACGGGCAAGGCGGACAAGAAGAAGACGAGCAGCAACAAGAGGACGGGGAAGCGGCAGAAGCACAGCACGCCGAAGGACAGGGAGGACACGAGGGAGGGCCAGGGCACGGGCAGCAAGCCGGAGGAGCAGAAGAACAACAGGGACAGCAAGCCGAAGGAGGCAACGCCCACGCCCACGCCGGACACGCAGAACAAGAGGCCGAAAACGACGCCGACCAGCACGACCAGGAAGGGCACCACCCCGGAGAACAGCACCACGCCCAAGCACACCATAATAGTGAGTCGTATTA-3\u2032). For in vitro transcription, 10 \u03bcM of double-stranded DNA (dsDNA) template in 100 \u03bcl transcription buffer , along with 1 mM of each of GTP, CTP and UTP, with 1mM ATP , 4 mM NAD (for NAD-RNA) or 4 mM m7GpppA (for m7G-RNA), 10 \u03bcl of T7 RNA polymerase , 5% DMSO, 5 mM DTT and 2.5-unit RNase inhibitor were added and the transcription mixture was incubated at 37\u00b0C for 4 h. The reaction was incubated with 11-unit DNase I at 37\u00b0C for 30 min to remove the DNA template. RNA was then extracted using acid phenol/chloroform and precipitated with isopropanol at -80\u00b0C overnight. The RNA pellet was washed twice with 75% ethanol, air-dried, redissolved in DEPC-treated H2O, and stored at \u201380\u00b0C. For spike-in RNAs used as internal controls for gene-specific qRT-PCR assessment, NAD-RNA is 106 nt as described above. To prepare ppp-RNA, 88 nt oligo nucleotide was synthesized and annealed to make double-stranded DNA template. Primer extension was used to increase the length of the template to encode 106 nt RNA ) and (anti-sense: 5\u2032-CAAGGCGTTGGTCGCTTCCGGATTGTTTACATAACCGGACATAATCATAGGACCTCTCACACACAGTTCGCCTCTTTGATTAACGCCCAGCGTTTTCCCGGTATCCAATAGTGAGTCGTATTAGTGATC-3\u2032). To prepare 45 nt m7Gppp-RNA, oligo nucleotide was synthesized (Genewiz) and annealed to make double-stranded DNA template. Primer extension was used to increase the length of the template to encode 45 nt RNA ) and (anti-sense: 5\u2032-CGACAGGGCCCGCGAGAGAGAGACAGCAGACGACGACGACACAGTAATAGTGAGTCGTATTAGTGATC-3\u2032).To prepare short RNA of 38 nucleotides (nt), 10 \u03bcM template oligo DNA (template sequence: 5\u2032-GATCACGATC-3\u2032) were com7G-RNA (500 nt) and ppp-RNA (500 nt) spike-ins with different sequences from that of NAD-RNA . The synthesis of m7G-RNA (500 nt) was as described in and were subjected to polyadenylation for poly(A) tails elongation ) and (anti-sense: 5\u2032- TGGCCTTGTAGGTGGTCTTGACCTCAGCGTCGTAGTGGCCGCCGTCCTTCAGCTTCAGCCTCTGCTTGATCTCGCCCTTCAGGGCGCCGTCCTCGGGGTACATCCGCTCGGAGGAGGCCTCCCAGCCCATGGTCTTCTTCTGCATTACGGGGCCGTCGGAGGGGAAGTTGGTGCCGCGCAGCTTCACCTTGTAGATGAACTCGCCGTCCTGCAGGGAGGAGTCCTGGGTCACGGTCACCACGCCGCCGTCCTCGAAGTTCATCACGCGCTCCCACTTGAAGCCCTCGGGGAAGGACAGCTTCAAGTAGTCGGGGATGTCGGCGGGGTGCTTCACGTAGGCCTTGGAGCCGTACATGAACTGAGGGGACAGGATGTCCCAGGCGAAGGGCAGGGGGCCACCCTTGGTCACCTTCAGCTTGGCGGTCTGGGTGCCCTCGTAGGGGCGGCCCTCGCCCTCGCCCTCGATCTCGAACTCGTGGCCGTTCACGGAGCCCTCCATGTAATAGTGAGTCGTATTAGTGATC-3\u2032). To prepare ppp-RNA, oligonucleotide was synthesized (Genewiz) and were subjected to polyadenylation for poly(A) tails elongation ) and (anti-sense: 5\u2032-TCGTTGGGGTCTTTGCTCAGGGCGGACTGGGTGCTCAGGTAGTGGTTGTCGGGCAGCAGCACGGGGCCGTCGCCGATGGGGGTGTTCTGCTGGTAGTGGTCGGCGAGCTGCACGCTGCCGTCCTCGATGTTGTGGCGGATCTTGAAGTTCACCTTGATGCCGTTCTTCTGCTTGTCGGCCATGATATAGACGTTGTGGCTGTTGTAGTTGTACTCCAGCTTGTGCCCCAGGATGTTGCCGTCCTCCTTGAAGTCGATGCCCTTCAGCTCGATGCGGTTCACCAGGGTGTCGCCCTCGAACTTCACCTCGGCGCGGGTCTTGTAGTTGCCGTCGTCCTTGAAGAAGATGGTGCGCTCCTGGACGTAGCCTTCGGGCATGGCGGACTTGAAGAAGTCGTGCTGCTTCATGTGGTCGGGGTAGCGGCTGAAGCACTGCACGCCGTAGGTCAGGGTGGTCACGAGGGTGGGCCAGGGCACGGGCAGCTTGCCGGTGGTGCAGATAATAGTGAGTCGTATTA-3\u2032).For boronate affinity experiment, we synthesized mribed in . To prep2) at 37\u00b0C for 1 h. Product was purified with Zymo column according to the instruction of manufacturer and analyzed by an 8% polyacrylamide TBE urea gel. Gel was stained by SYBR Gold and fluorescence was detected by Typhoon FLA 7000 fluorescent image analyzer (GE Life Science).NAD-RNA (1 \u03bcg) was reacted with 40 mM and 100 mM HEEB (1 M stock in DMSO) under the catalysis of ADPRC (25 \u03bcg/ml) with 0.4 U/\u03bcl of RNase Inhibitor in 100 \u03bcl of ADPRC reaction buffer were washed 3 times with immobilization buffer , and the254nm (0.18 J/cm2) twice. Membrane was blocked in 5% BSA in PBST (0.1% Tween20 in PBS) for 1 h, followed by incubation with Alexa Fluor\u00ae 790 Streptavidin at room temperature for 2 h in the dark. Membrane was washed with PBST and PBS for 3 times, respectively, and imaged on the Odyssey LiCor CLx scanner (Li-Cor Biosciences) with the software set to auto-detect the signal intensity at 800 nm channel. Finally, the membrane was stained with methylene blue solution (0.3% w/v methylene blue\u00a0+\u00a030% v/v ethanol\u00a0+\u00a070% v/v H2O) which was then cross-linked by UVv/v H2O) to visua7Gppp-RNA (45 nt) were incubated with 100 mM HEEB (1 M stock in DMSO) with ADPRC (25 \u03bcg/ml) in 100 \u03bcl of ADPRC reaction buffer at 37\u00b0C for 1 h. 100 \u03bcl of DEPC-treated H2O was then added and acid phenol/ether extraction was performed to stop the reaction and 0.4 U/\u03bcl of RNase Inhibitor at 25\u00b0C for 30 min. Beads were washed four times with streptavidin wash buffer (50 mM Tris\u2013HCl (pH 7.4) and 8 M urea), and three times with DEPC-treated H2O. The biotinylated RNA on the beads was extracted with Trizol LS and chloroform. Input (see above) RNAs and the biotinylated RNA on the beads were analyzed by an 8% polyacrylamide TBE urea PAGE gel. Gel was stained by SYBR Gold and fluorescence was detected by Typhoon FLA 7000 fluorescent image analyzer (GE Life Science).100 ng of spike-in NAD-RNA (38 nt) and 200 ng of mreaction . RNAs we7Gppp-RNA (38 nt) was performed with 1 \u03bcl of NudC in 25 \u03bcl of NudC reaction buffer at 37\u00b0C for 30 min. Product was purified with Trizol LS and analyzed by an 8% polyacrylamide TBE\u2013urea gel. Gel was stained by SYBR Gold and fluorescence was detected by Typhoon FLA 7000 fluorescent image analyzer (GE Life Science).De-capping of 200 ng NAD-RNA (38 nt) or m7Gppp-RNA (45 nt) were mixed together and incubated with 500 nM NudC in 25 \u03bcl of NudC reaction buffer at 37\u00b0C for 30 min. Product was purified with Trizol LS and analyzed by an 8% polyacrylamide TBE urea gel. Gel was stained by SYBR Gold and fluorescence was detected by Typhoon FLA 7000 fluorescent image analyzer (GE Life Science).100 ng of spike-in NAD-RNA (38 nt) and 200 ng of m2O was then added and acid phenol/ether extraction was performed to stop the reaction . Total RNAs (100 \u03bcg) was incubated with 100 mM HEEB (1 M stock in DMSO), ADPRC and 0.4 U/\u03bcl of RNase Inhibitor in 100 \u03bcl of ADPRC reaction buffer at 37\u00b0C for 1 h. 100 \u03bcl of DEPC-treated Hreaction . RNAs we2O. To ensure complete elution, biotin-conjugated RNAs were replaced from streptavidin beads by incubating with 1 mM biotin buffer at 94\u00b0C for 8 min, followed by incubation with 500 nM NudC in 25 \u03bcl of NudC reaction buffer at 37\u00b0C for 30 min. After NudC treatment, biotinylated-RNAs that are resistant to NudC catalysis, potentially derived from contaminating m7G-RNAs, were retained on beads by incubation with high-capacity streptavidin particle at 25\u00b0C for 30 min. Eluted RNAs in the supernatant were used for next step.After HEEB reaction, biotinylated RNAs were incubated with streptavidin bead particles and 0.4 U/\u03bcl of RNase Inhibitor at 25\u00b0C for 30 min. Beads were washed four times with streptavidin wash buffer (50 mM Tris\u2013HCl (pH 7.4) and 8 M urea), and three times with DEPC-treated H7Gppp-RNA (38 nt) was performed with or without 1 \u03bcl of yDcpS in 1X yDcpS reaction buffer in 20 \u03bcl total volume at 37\u00b0C for 1 h. Product was purified with Zymo column according to the instruction of manufacturer and analyzed by an 8% polyacrylamide TBE urea gel. Gel was stained by SYBR Gold and fluorescence was detected by Typhoon FLA 7000 fluorescent image analyzer (GE Life Science).De-capping of 200 ng NAD-RNA (38 nt) and m7Gppp-RNA (106 nt) were incubated with 100 mM HEEB (1 M stock in DMSO) with or without ADPRC (25 \u03bcg/ml) in 100 \u03bcl of ADPRC reaction buffer at 37\u00b0C for 1\u00a0h, followed by NudC-catalyzed NAD-RNA elution. Input (see above) and NudC-eluted RNAs from three biological replicates were used for qRT-PCR. Reverse transcription was performed with gene specific primers using SuperScript III SuperMix . qPCR was performed using SYBR Green master mix to detect NAD-RNA, ppp-RNA or m7Gppp-RNA from three biological replicates using specific primers described in t test.Total RNAs (100 \u03bcg) and 100 ng of spike-in NAD-RNA (106 nt), ppp-RNA (106 nt) or mInput (see above) and NudC-eluted RNAs from four biological replicates of livers from 2- and 18-month old C57BL/6 mice were used for NGS library construction, in accordance with manufacturer's instructions . Library quality was assessed by Bioanalyzer 2100 , and quantification was performed by qRT-PCR with a reference to a standard library. Libraries were pooled together in equimolar amounts to a final 2 nM concentration and denatured with 0.1 M NaOH . Libraries were sequenced on the Illumina NovaSeq 6000 system .7Gppp-RNA, 1% NAD-RNA/99% m7Gppp-RNA, 5% NAD-RNA/95% m7Gppp-RNA or 10% NAD-RNA/90% m7Gppp-RNA, respectively. The mixture of total RNAs and spike-in RNA (500 nt) were incubated with 100 mM HEEB (1 M stock in DMSO) with ADPRC (25 \u03bcg/ml) in 100 \u03bcl of ADPRC reaction buffer at 37\u00b0C for 1\u00a0h, followed by NudC-catalyzed NAD-RNA elution. Input (see above) and NudC-eluted RNAs from three biological replicates of livers from 18-month old C57BL/6 mice were subjected to polyA-selected RNA sequencing as mentioned above.Total RNAs (100 \u03bcg) were mixed with 1 ng of 3\u2032-end polyadenylated spike-in RNA (500 nt) that had 0% NAD-RNA/100% m7Gppp-RNA (38 nt) was ligated with 5 \u03bcM 3\u2032 adaptor oligo listed in 1 \u03bcg of NAD-RNA (38 nt) or mBoronic acid beads were washed 3 times with binding buffer , and then incubated with RNA oligonucleotide in binding buffer in a thermomixer at 37\u00b0C for 2 h. Beads were then washed 3 times with wash buffer ; RNA oligonucleotide bound by the beads was extracted with Trizol LS ). Purified RNA product was analyzed by an 8% polyacrylamide TBE\u2013urea PAGE gel. Gel was stained by SYBR Gold and fluorescence was detected by Typhoon FLA 7000 fluorescent image analyzer (GE Life Science).7Gppp-RNA, and 1 ng of ppp-RNA). RNAs were incubated with yDcpS in 50 \u03bcl of 1X yDcpS reaction buffer at 37\u00b0C for 1 h, to remove m7G cap. After purification with Trizol LS , yDcpS-treated RNAs were combined with 100 \u03bcmol oligo d(T)30VN in 100 \u03bcl 0.5\u00d7 SSC buffer , and incubated at 80\u00b0C for 5 min followed by 50\u00b0C for 60 min. Then, 1 \u03bcl RNase H and 5.6 \u03bcl 10\u00d7 Reaction buffer were added. The mixture was incubated at 37\u00b0C for 20 min, and purified with Trizol LS . This step aims to promote 3\u2032-end adaptor ligation by removing polyA sequence tract from the endogenous transcripts. Prior to 3\u2032-end ligation, polyA-depleted RNAs were treated with 2 U CIAP in the presence of 40 U RNaseOUT at 37\u00b0C for 1 h to remove 5\u2032-terminal phosphate. RNA was extracted with Trizol LS . 5 \u03bcg of CIAP-treated RNA products were ligated with 100 \u03bcM 3\u2032 adaptor oligo listed in 2O. Reverse transcription was performed with adaptor-based primer listed in 7G-RNA (500 nt) was used as a negative control. Primers were listed in t-test.Total RNAs were extracted from three biological replicates of livers from 2-month old C57BL/6 mice as described above. For each replicate, total RNAs (50 \u03bcg) were mixed with polyadenylated spike-in RNAs with theDESeq2 (v1.30.0) . NAD-RNAs were defined as fold change of normalized transcript counts\u00a0\u22652 and FDR\u00a0<0.05 in NudC-treated samples compared to those in input samples. Gene annotation information, such as chromosome, gene-types, gene-lengths and intron coordinates were retrieved from Gencode (M23) annotations. The violin plot, bar plot, line chart and scatter plot were generated by R package ggplot2 (v3.3.2) (Genes that had zero count in more than 25% (3 out of 12) sequencing libraries were removed before performing differential analysis. Principle component analysis (PCA) was performed with R function \u2018prcomp\u2019 using a transformed counts matrix by function \u2018vst\u2019. To identify NAD-RNA from total RNA-seq data, we used R package v1.30.0) to perfo(v3.3.2) .gprofiler2 (v0.2.1) in Cytoscape (v3.8.2) . Pathway(v0.2.1) and the (v0.2.1) without (v3.8.2) . Network(v3.8.2) .2) at 37\u00b0C for 1 h. 100 \u03bcl of DEPC-treated H2O was then added and acid phenol/ether extraction was performed to stop the reaction following the manufacturer's protocol with primers listed in Total RNA was extracted from four biological replicates of livers from 2-month old C57BL/6 mice as described above. For each replicate, 100 \u03bcg total RNA, mixed with 1 ng of NAD-RNA (106 nt) and 1 ng of ppp-RNA (106 nt), was incubated with 100 mM HEEB (1 M stock in DMSO) with ADPRC (25 \u03bcg/ml) in 100 \u03bcl of ADPRC reaction buffer ethyl]-biotinamide) as a reactant, allowing only one reaction to biotinylate NAD-capped RNAs. To avoid contaminating signals, we designed a NudC-based post-treatment to elute biotin-conjugated RNAs specifically derived from NAD, but not m7G-capped transcripts from streptavidin beads. Therefore, our method circumvented laborious steps to purify mRNAs followed by clearance of m7G-capped RNAs. With significantly simplified procedures, our method enabled NAD-RNA profiling directly from total RNA. Based on the same platform, qRT-PCR analysis on specific NAD-RNAs could be readily applied.Compared to previous methods that require multiple reactions, we introduced HEEB and NAD-RNA (38 nt) with 100 mM HEEB to the reaction. While NAD-RNA reacted with HEEB, as indicated by the presence of a biotinylated form, m7G-RNA had no such a product, reflecting specificity but not m7Gppp-RNA (38 nt) as shown by a lower-sized band corresponding to the de-capped product to the same reaction. The result clearly showed that NudC was able to selectively de-cap NAD-capped (38 nt) but not m7G-capped RNA (45 nt) but not m7Gppp-capped forms (38 nt) from streptavidin beads , but not NAD-RNA (38 nt) was sensitive to yDcpS treatment, a decapping enzyme that hydrolyzes the triphosphate linkage of m7G-capped RNA Figure . We tests Figure and\u00a0F. Apped RNA .7G-capped RNA (m7Gppp-RNA), respectively). HEEB reacted with NAD-RNA (106 nt) and m7Gppp-RNA (106 nt), but not ppp-RNA (106 nt), resulting in a band retained by the streptavidin beads (7Gppp-RNA (106 nt) could be detected on streptavidin beads; treatment of NudC, however, found no evidence to elute m7Gppp-RNA (106 nt) Figure .7G-cap, followed by polyA tails. Presumably, endogenous transcripts may contain both NAD and m7G-capped forms, though the percentage may differ for particular genes. The rationale of this design is to mimic endogenous genes that have either low (0% or 1%) or relatively high (5% or 10%) NAD modification. Total RNAs were mixed with spike-ins that had different ratios of NAD-RNA and m7G-RNA. The sample that contained 100% m7G-RNA spike-in represented a gene with no NAD capping, which allowed the assessment of the specificity of ONE-seq platform. Other samples that contained 1%, 5%, and 10% of NAD- relative to m7G-RNA spike-ins were used to determine the capture sensitivity. We subjected 100 \u03bcg total RNA from mouse livers mixed with 1 ng spike-in RNA to ONE-seq experiment, followed by polyA-selected RNA sequencing. In the sample mixed with 100% m7G-RNA spike-in, we found no enrichment but had either NAD or mt Figure . As demot Figure , represee Figure , suggeste Figure , reflect7G-capped RNAs.Fourth, we determined the noise-cancelling effect of NudC by comparative analysis of RNA-seq datasets. Total RNAs, after HEEB reaction, were captured by streptavidin beads, followed by either NudC-catalyzed elution (NudC+) or mock treatment (NudC-) . polyA-s7G and NAD-capping.We tested the utility of ONE-seq. We profiled NAD-RNAs from mouse liver tissues of young (2-month) and aged (18-month) cohorts. After quality control, we obtained in average \u223c12.3 million high-quality and uniquely mapped sequencing read pairs from each library . Assessmcis diols, occurring naturally at the 3\u2032-end of RNA, as well as in the 7-methylguanosine of m7G-cap and the nicotinamide riboside of NAD-cap at the terminal is ligated to the 3\u2032-end of RNA, such that the vicinal-diol moiety of the ribose at the 3\u2032-end of RNA no longer exists. At this step, affinity binding can only occur between the boronyl group from boronic acid and 1,2-cis diols from the nicotinamide riboside of NAD-cap. Importantly, we perform reverse transcription using an adaptor-specific primer to harvest RNAs with 3\u2032end blocker, thereby abolishing false signals from boronate binding of RNAs that are not properly ligated with adaptor. Consequently, only RNA transcripts that contain NAD-cap can be selectively enriched by boronate affinity.We validated newly identified NAD-RNAs by an ADPRC-independent method. To do this, we applied boronic acid beads. Our rationale lies at the fact that boronyl groups of boronic acid can form relatively stable complexes with 1,2- NAD-cap . Inspire NAD-cap ,38, we d NAD-cap . First, 7G, but not NAD, from RNA oligos (38 nt). In the presence of RNA ligase, 3\u2032adaptor was efficiently ligated to the spike-in RNAs (38 nt), yielding an upper band in the gel that were designed with different sequences. This experiment clearly demonstrated that NAD-RNA (500 nt) spike-ins, but not ppp-RNA (500 nt) and m7Gppp-RNA (500 nt), were selectively and significantly enriched by boronic acid beads and transcription (e.g. RNA polymerase II) were found to contain NAD-cap. Also, transcripts with 5\u2032-NAD modification were enriched in cell cycle (e.g. centromere protein and CDK) and DNA damage responses (e.g. ATM and TP53). Not just housekeeping genes, transcriptional factors (e.g. BMP5 and Interferon), epigenetic pathways , and histone-related genes were also subject to NAD modification Figure . In mitoIn line with an age-modulated decrease of NAD , both thAbove evidence supported the notion that ONE-seq platform permits epi-transcriptome-wide profiling directly from total RNA, prompting us to extend its application for gene-specific assessment by qRT-PCR. To do this, we included non-capped ppp-RNA (106 nt) as a baseline negative control, and NAD-RNA (106 nt) as a positive control. Total RNA and internal spike-in controls were subjected to ONE-seq experiment, followed by qRT-PCR. The relative abundance was calculated between NudC\u00a0+\u00a0and input samples Figure . Our datN-acetylglucosamine (UDP-GlcNAc), can be incorporated at the 5\u2032-end of RNA during transcription initiation in both prokaryotes and eukaryotes , flavin adenine dinucleotide (FAD), uridine diphosphate glucose (UDP-Glc) and uridine diphosphate karyotes . CapZymeNCIN cap . As a re7G) has been found to contaminate the NAD-RNA profile. As such, SPAAC-NAD-seq , Mup13 (http://genome.ucsc.edu/s/lda97/Mup13_ONE-seq), Mup19 (http://genome.ucsc.edu/s/lda97/Mup19_ONE-seq) and Mettl7b (http://genome.ucsc.edu/s/lda97/Mettl7b_ONE-seq) can be accessed at UCSC Genome Browser online sessions. All source codes for data analysis are available at Zenodo https://doi.org/10.5281/zenodo.7293917.All high-throughput RNA sequencing data as well as transcript quantifications have been deposited at the Gene Expression Omnibus under accession number GSE194271. Genome browser views for Cyp2c70 (gkac1136_Supplemental_FilesClick here for additional data file."} +{"text": "In this announcement, we present the set of putative terpene synthase (TS) gene fragments detected in a subseafloor sediment sample collected off Shimokita Peninsula, Japan. This data set contains sequences with 72 to 100% identity to TS from actinobacteria and cyanobacteria. Terpenoids (TRPs) are the largest class of specialized metabolites , and manSubseafloor sediments represent an environment with unique microbiological communities and metabolic activities . HoweverChikyu shakedown cruise of CK-06-06 and frozen at \u221280\u00b0C immediately after the sampling. DNA was extracted from 5 g of the frozen sediment as previously described (The sediment sample was collected during the D/V TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCATCGAGATGCGSCGCAAGG-3\u2032) and geosmin-rev (5\u2032-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGASCGSAKGTGCCACTCGTG-3\u2032) primers . The 2-MIB TS primers were mib-for (5\u2032-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGACGACDNBTACTGCGAGGAC-3\u2032) and mib-rev (5\u2032-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGGVCGGAAGTTGTTGAACTG-3\u2032) (331\u2009bp). The PCR mix consisted of 1\u00d7 EmeraldAmp Max PCR master mix (TaKaRa), 0.4 \u03bcM each primer, and 0.05\u2009ng of sediment DNA. The two-phase touchdown PCR protocol for increased specificity and sensitivity was used (The geosmin TS fragment (432\u2009bp) was amplified using primers geosmin-for . OTUs were filtered for chimeras using VSEARCH\u2019s implementation of UCHIME de novo (The obtained sequences were pro de novo training de novo .This data set can be used in studies on TS gene diversity and distribution in subseafloor environments.PRJNA846928. The GenBank accession numbers for OTUs are ON723903 to ON723912 (2-MIB) and ON723913 to ON723935 (geosmin).Raw reads were deposited in a BioProject at DDBJ/ENA/GenBank under the accession number"} +{"text": "In the published article, there were errors. In the original article, there was an error in the description of the Reverse primer sequence in Materials and methods.Materials and methods, \u201cDeep amplicon sequencing of CMV-UL40 genomic DNA\u201d.A correction has been made to This sentence previously stated:\u201cThe following forward and reverse primers were used: Forward, 5\u2032-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCAACAGTCGGCAGAATGAAC-3\u2032 and Reverse, 5\u2032-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCTGGAACACGACGCATA-3\u2019.\u201dThe corrected sentence appears below:\u201cThe following forward and reverse primers were used: Forward, 5\u2032-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCAACAGTCGGCAGAATGAAC-3\u2032 and Reverse, 5\u2032-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCTGGAACACGAGCGGACATA-3\u2019.\u201dMaterials and methods.In the original article, there was another error in the description of the concentration of the anti-HLA-E antibody in Materials and methods, \u201cImmunohistochemistry analysis\u201d.A correction has been made to This sentence previously stated:\u201cAfter antigen retrieval with boiling citrate buffer (pH 6.0), the sections were incubated with 5% skim milk for 1\u00a0h at room temperature to prevent nonspecific binding and stained with 10 \u03bcL/mL anti-HLA-E antibody [MEM-E/02] or IgG from mouse serum (Sigma-Aldrich) overnight at 4\u00b0C.\u201dThe corrected sentence appears below:\u201cAfter antigen retrieval with boiling citrate buffer (pH 6.0), the sections were incubated with 5% skim milk for 1\u00a0h at room temperature to prevent nonspecific binding and stained with 10 \u03bcg/mL anti-HLA-E antibody [MEM-E/02] or IgG from mouse serum (Sigma-Aldrich) overnight at 4\u00b0C.\u201dThe authors apologize for these errors and state that these do not change the scientific conclusions of the article in any way. The original article has been updated."} +{"text": "We report an approach of combining CRISPR-Cas9 gene editing with florescence cell sorting to tag and purify human endogenous protein complexes from HEK cells for structural studies by single-particle cryogenic electron microscopy. This procedure is demonstrated by applying the method to study human proteasomal complexes, enabling rapid determination of high-resolution structures of several proteasomal complexes. We envision this approach will enable structural, biochemical, and biophysical studies of many important endogenous human macromolecular complexes. The ability to produce folded and functional proteins is a necessity for structural biology and many other biological sciences. This task is particularly challenging for numerous biomedically important targets in human cells, including membrane proteins and large macromolecular assemblies, hampering mechanistic studies and drug development efforts. Here we describe a method combining CRISPR-Cas gene editing and fluorescence-activated cell sorting to rapidly tag and purify endogenous proteins in HEK cells for structural characterization. We applied this approach to study the human proteasome from HEK cells and rapidly determined cryogenic electron microscopy structures of major proteasomal complexes, including a high-resolution structure of intact human PA28\u03b1\u03b2\u201320S. Our structures reveal that PA28 with a subunit stoichiometry of 3\u03b1/4\u03b2 engages tightly with the 20S proteasome. Addition of a hydrophilic peptide shows that polypeptides entering through PA28 are held in the antechamber of 20S prior to degradation in the proteolytic chamber. This study provides critical insights into an important proteasome complex and demonstrates key methodologies for the tagging of proteins from endogenous sources. Escherichia coli), insect (Sf9), and mammalian (HEK) cells. Overexpression approaches frequently encounter issues with proteins that are misfolded, nonfunctional, or degraded by the host cells. Overexpression of certain proteins may also alter protein homeostasis within cells. Substantial optimization is often necessary to identify the right conditions to produce suitable proteins for study, which is time-consuming and challenging in many cases. The problem is compounded further by multiprotein complexes where multiple subunits need to be assembled in the correct order and stoichiometries, making the coexpression of multiple subunits and purification of protein assemblies even more challenging.Recent technological breakthroughs in single-particle cryogenic electron microscopy (cryo-EM) have greatly accelerated the pace of high-resolution structure determination of biological macromolecules. However, a major bottleneck in the structural study of important targets including membrane proteins, protein\u2013DNA complexes, and large protein assemblies is sample production. The challenge of producing functional proteins extends beyond structural biology and impacts many areas of biological sciences that currently rely on techniques of protein overexpression. Conventional methods of protein production involve overexpression of a target gene from a plasmid within heterologous systems, including bacterial . Selecting the \u03b24-StrepII cell population, which had the highest knock-in efficiency, we expanded this cell line and purified the human proteasome by StrepII pull-down.To extract the endogenous human proteasome for structural studies, we incorporated a mNG2[11]-StrepII tag onto the C terminus of the 20S \u03b21 (Psmb6), \u03b22 (Psmb7), and \u03b24 (Psmb2) B subunitSI Appendix, Fig. S2), followed by cryo-EM structure determination of singly capped proteasomal complexes of PA28\u201320S, PA200\u201320S, and 19S\u201320S at \u223c2.6 to \u223c3.2 \u00c5 resolution, with and without MG-132 inhibitor .Affinity purification of human endogenous 20S(\u03b24-StrepII) pulls down multiple proteasomal complexes, including 20S, singly capped with 19S, PA200, and PA28. The purified proteasomal complexes were first visualized by negative-stain EM . Furthermore, the resulting cryo-EM map of PA28\u201320S purified from 20S(\u03b24-StrepII) pull-down shows side-chain density that matches PA28\u03b2 in some regions while matching PA28\u03b3 in other regions , suggesting that the cryo-EM map is an average of both PA28\u03b1\u03b2 and PA28\u03b3 complexes. However, the high similarity between the two complexes prevented using computational classification to separate them (Endogenous PA28 subunits are known to form two different heptameric complexes: a heteroheptamer composed of PA28\u03b1 and PA28\u03b2 subunits and a homoheptamer composed of PA28\u03b3 subunits. Pull-down of 20S(\u03b24-StrepII) is expected to yield both PA28\u03b1\u03b2\u201320S and PA28\u03b3\u201320S complexes. Indeed, mass spectrometry analysis of the purified proteins reveal all three PA28 subunits to be present in the purified sample . Additionally, the tagging of PA28\u03b1 with sfCherry2[11]-ALFA tag was performed in the cell line that already contained mNG2[11]-StrepII\u2013tagged 20S \u03b24, demonstrating the ability to multiplex orthogonal tags onto different subunits of a larger protein assembly to increase target specificity. The cryo-EM map of PA28\u03b1\u03b2\u201320S purified by ALFA-PA28\u03b1 pull-down allowed robust modeling of the side-chain densities of PA28\u03b1\u03b2, allowing us to distinguish the subunit stoichiometry of the complex.To isolate a homogeneous PA28\u201320S complex for structural studies, we engineered an sfCherry2[11]-ALFA tag onto the N terminus of the PA28\u03b1 subunit using the method described above, except here we used a split sfCherry system rather tSI Appendix, Fig. S6 and Table S2), different from the 4\u03b1/3\u03b2 assembly observed previously.Previous crystallographic structural analysis of the mouse PA28\u03b1\u03b2 heteroheptamer found a stoichiometry of four \u03b1 and three \u03b2 subunits . The proSI Appendix, Fig. S9). There is one low-occupancy binding site situated between the 20S \u03b11 and \u03b12 subunits where a phenylalanine, 20S \u03b11 F29, may be sterically hindering the binding of the C-terminal tail of PA28\u03b2 . We suspect that this additional density does not represent endogenous substrates but rather is from ALFA peptide used to elute purified complex. ALFA peptide is present at high concentrations in the elusion buffer and likely enters 20S as substrates through the \u03b1-ring gate opened by PA28. Consistent with this argument, cryo-EM analysis of proteasomal complexes purified via StrepII pull-down of 20S(\u03b24-StrepII) did not show similar density inside any map of 20S, PA200\u201320S, or 19S\u201320S.Proteasome activators serve to open the 20S gate and facilitate the loading of unfolded polypeptides into the 20S inner chambers for degradation. Passing through the open gate in the \u03b1-ring, unfolded polypeptides first enter the antechamber formed between an \u03b1- and \u03b2-ring and then move into the proteolytic chamber formed between two \u03b2-rings for peptide cleavage. Interestingly, the cryo-EM map of PA28\u03b1\u03b2\u201320S purified by ALFA-PA28\u03b1 pull-down shows additional density inside the 20S antechamber that is proximal to the bound PA28 , which may serve to bind unfolded polypeptides. The ALFA peptide accumulates in the 20S antechamber and is likely degraded once it enters the proteolytic chamber. To visualize what might happen if proteolysis was blocked, we purified proteasome complexes via 20S(\u03b24-StrepII) by StrepII pull-down in the presence of the peptide-like inhibitor MG-132 and carried out cryo-EM analysis , consistent with a previous study of the yeast proteasome , and signal amplification using our recent tag-assisted split enzyme complementation method could fuOne exciting future direction stemming from the concepts described here is the study of endogenous proteins from animal models. This can allow the structural and biophysical characterization of proteins in their physiological states, which can provide important insights into how proteins function in\u00a0vivo . EfficieTAATACGACTCACTATAGAAAAAAAGCACCGACTCGGTGCAAAAAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTAAACTTGCTATGCTGTTTCCAGCATAGCTCTTAAACGATGTTAGGAGCCCTGTTTGGTTTAAGAGCTATGCTGGAATAATACGACTCACTATAGTTATTGCATACTAGAATCCCGTTTAAGAGCTATGCTGGAATAATACGACTCACTATAGGCCACCCACTGATGCCATTCGTTTAAGAGCTATGCTGGAATAATACGACTCACTATAGCCCGGTCATGGCCATGCTCAGTTTAAGAGCTATGCTGGAATAATACGACTCACTATAGACCGAGCTCAACTTCAAGGAGTGGCAAAAGGCCTTTACCGATATGATGGGGAGTGCGTGGTCCCACCCTCAATTCGAGAAGTAACATCATGTCCTCCCTCCCACTTGCCAGGGAACTTTTTTTTGATGGGCTCCTTTATTGACAAAAATGGCATCCATGACCTGGATAACATTTCCTTCCCCAAACAGGGCTCCGGCAGCACCGAGCTCAACTTCAAGGAGTGGCAAAAGGCCTTTACCGATATGATGGGGAGTGCGTGGTCCCACCCTCAATTCGAGAAGTGAATACTGGGATTCTAGTATGCAATAAGAGATGCCCTGTACTGATGCAAAATTTACAAGTACTTTTGGGAGACCAGATACCCAAATTCGCCGTTGCCACTTTACCACCCGCCGGCAGCACCGAGCTCAACTTCAAGGAGTGGCAAAAGGCCTTTACCGATATGATGGGGAGTGCGTGGTCCCACCCTCAATTCGAGAAGTGAATGGCATCAGTGGGTGGCTGGCCGCGGTTCTGGAAGGTGGTGAGCATTGAGGCATCACTCCTCTGGAGATTGAGGTGCTGGAAGAAACAGTCCAAACAATGGACACTTCCGGCAGCCCTTCTAGATTAGAAGAGGAATTGAGACGACGATTGACTGAACCAGGATCTTATACTATCGTTGAACAATACGAGAGAGCTGAGGCAAGACACTCTACAGGTTCTGGCGCCATGCTCAGGGTCCAGCCCGAGGCCCAAGCCAAGGTGAGCGCCGCCCACTCCACTCCTTGTGCGGCGCTAGGCCCCCCGTCCCGGTCATGSI Appendix, Fig. S1). The reactions were completed in a 100-\u03bcL reaction containing 50 \u03bcL 2\u00d7 Phusion master mix, 2 \u03bcL ML557\u2009+\u2009558 mix at 50 \u03bcM, 0.5 \u03bcL ML611 at 4 \u03bcM, 0.5 \u03bcL of each gene-specific oligo at 4 \u03bcM, and 47 \u03bcL diethyl pyrocarbonate (DEPC) H2O. PCR program: 95\u2009\u00b0C, 3 min; 20\u00d7 ; 72\u2009\u00b0C, 60 s. The PCR product was purified using a Zymo DNA Clean and Concentrator Kit (Zymo Research D4013) and DNA was eluted in 10 \u03bcL. The expected DNA concentration is around 100 ng/\u00b5L. DNA was stored at \u221220\u2009\u00b0C until used for gRNA synthesis.The IVT template for LMNA gRNA was made by PCR (In vitro transcription was carried out using the HiScribe T7 Quick High Yield RNA Synthesis Kit (NEB E2050S) with the addition of RNAsin (Promega N2111). In 20 \u03bcL, 300 ng of DNA template, 10 \u03bcL NTP buffer mix, 2 \u03bcL T7 polymerase, 1 \u03bcL RNAsin, and water was added. Addition of RNAsin is important to prevent RNA degradation. The reaction was incubated overnight and the RNA was purified using the RNA Clean and Concentrator Kit (Zymo Research R1017). RNA was eluted in 10 \u03bcL with an expected concentration >6 \u03bcg/\u03bcL. Single-guide RNA (sgRNA) was stored at \u221280\u2009\u00b0C immediately after measuring concentration and diluting to 130 \u03bcM.2O, and 4 \u03bcL sgRNA were mixed and incubated at 70\u2009\u00b0C for 5 min to refold the gRNA. During this step, 10-\u03bcL aliquots of purified Cas9 at 40 \u03bcM were thawed on ice and once thawed were slowly added to the diluted sgRNA in Cas9 buffer and incubated at 37\u2009\u00b0C for 10 min for ribonucleoprotein complex formation. Six microliters of HDR template donor was added to the ribonucleoprotein mix, and all samples were kept on ice until ready for nucleofection. For efficient recovery post-knock-in (KI), a six-well plate with 2 mL of Expi293F expression media (Thermo Fisher) per well was incubated at 37\u2009\u00b0C. An appropriate amount of supplemented Lonza Amaxa solution corresponding to the number of KIs to be performed was prepared at room temperature in the cell culture hood. For each sample, 65.6 \u03bcL of SF solution (Lonza Group Ltd.) and 14.4 \u03bcL of supplement was added to an Eppendorf tube for a total of 80 \u03bcL per KI. Lonza nucleofector instruments/computers were then turned on and kept ready for nucleofection. The Amaxa solution is toxic to cells, so it is important to make sure everything is ready to go beforehand.In a sterile PCR or microcentrifuge tube, 8 \u03bcL Cas9 Buffer, 12 \u03bcL DEPC Hg for 3 min.Supernatant was removed and cells were resuspended in 1 mL of phosphate-buffered saline to wash. The cells were centrifuged again at 500 \u00d7 g for 3 min. PCR tubes containing ribonucleoproteins were brought into the tissue culture hood. Cells were resuspended in 80 \u03bcL of supplemented Amaxa solution and the cell resuspension was added to the 40 \u03bcL of preformed ribonucleoprotein + HDR template mix. The 120-\u03bcL mixture was pipetted into the bottom of the nucleofection plate, avoiding air bubbles. The nucleofection was carried out in 100-\u03bcL cuvettes on a Lonza Nucleofector X Unit (Lonza AAF-1002X) attached to Lonza 4D Nucleofector Core Unit (Lonza AAF-1002B). Cells were nucleofected using the FS-100 program and immediately recovered using 400 \u03bcL of media from the prewarmed six-well plate and transferred to the corresponding well. Because the Amaxa solution is toxic to the cells, it is important to perform these steps quickly. The cells were incubated at 37\u2009\u00b0C and 8% CO2.Cells were harvested by centrifugation at 500 \u00d7 2. The next day, the cells were transferred to a 125-mL vented flat-bottom Erlenmeyer flask containing 20 mL Expi293F media and incubated at 37\u2009\u00b0C and 8% CO2 with shaking at 120 rpm until the cell density reached 1 million/mL.Cells were monitored over 5 to 7 d. Many cells will look unhealthy and die, and growth rate will slow dramatically. Cells will begin to recover after 5 d. Once cells expanded to the point where they began to detach from the well bottom, the plate was transferred to a shaker operating at 120 rpm and incubated at 37\u2009\u00b0C and 8% COTo prepare the transfection mixture, 20 \u03bcg of pSFFV-mNG2 (1-10) plasmid was added to 1 mL of Opti-MEM (Gibco) in one tube and 53 \u03bcL of ExpiFectamine 293 (Gibco) was added to 1 mL of Opti-MEM (Gibco) in a separate tube. The mixture was incubated at room temperature for 5 min. The Opti-MEM containing Expifectamine 293 was then added to the solution containing DNA, inverted multiple times to mix, and then incubated at room temperature for 25 min. The transfection mixture was then added to 20 million Expi293F cells in 20 mL of media in a 125-mL vented flat-bottom Erlenmeyer flask. The cells were incubated at 37\u2009\u00b0C for 2 d with shaking at 120 rpm. If the knock-in was successful, some cells will show green fluorescence the next day, which will get brighter on day 2.2 for 2 d with shaking at 120 rpm. Cells were transferred to 20 mL of Expi293F media in a 125-mL vented Erlenmeyer flask. When the cell density reached 2 \u00d7 106 per mL, cells were transferred to 100 mL of Expi293F media in a 500-mL vented Erlenmeyer flask. When cells reached a density of 2 \u00d7 106 per mL, cells were harvested and frozen in aliquots.FACS sorting and flow cytometry were performed on a BD FACSAria II in the Laboratory for Cell Analysis at the University of California, San Francisco (UCSF). mNG2 signal was measured with the 488-nm laser and 530/30 bandpass filter. One million cells per sample were sorted into Eppendorf tubes and then plated in a six-well plate. The six-well plate was incubated at 37\u2009\u00b0C and 8% COTotal RNA was extracted from 1 million cells using the Monarch Total RNA Miniprep Kit (NEB T2010S). cDNA was prepared from 1 \u00b5g of extracted RNA using LunaScript RT SuperMix Kit (NEB E3010). No Template and No Reverse Transcriptase controls (NTC and NRT) were performed in parallel to cDNA preparations. cDNA was analyzed in a 2% agarose gel. Sequencing confirmation of amplicons was completed by Elimbio.2, and shaking at 120 rpm. When cells reached a density of 3 million cells per mL, cells were harvested by centrifugation at 1,000 \u00d7 g for 5 min. Cells were resuspended in 20 mL Buffer A supplemented with SigmaFast protease inhibitor (Sigma). Cells were lysed by sonication and insoluble debris were removed by ultracentrifugation at 100,000 \u00d7 g for 20 min. For StrepII-tagged proteins, the supernatant was passed through a column containing 0.2 mL Strep-Tactin XT Sepharose resin (Cytiva) by gravity flow. The resin was washed with 2 mL Buffer A and the bound proteins were eluted with 1 mL Buffer A supplemented with 50 mM biotin . For ALFA-tagged proteins, the supernatant was passed through a column containing 0.2 mL ALFA Selector PE resin (NanoTag Biotechnologies) by gravity flow. The resin was washed with 2 mL Buffer A and the bound proteins were eluted at room temperature with 1 mL Buffer A supplemented with 200 \u03bcM ALFA peptide . To acquire 19S-bound proteasome complexes, 1 mM AMPPNP (Sigma) and 2 mM MgCl were included throughout the purification. To acquire MG-132 bound proteasomal complexes, cells were treated with 5 \u03bcM MG-132 for 15 min prior to harvesting and MG-132 was maintained throughout the purification, along with 1 mM ATP. The eluted proteins were concentrated in a 100 K centrifugal concentrating device.Expi293F cell lines were grown in 100 mL Expi293 expression medium (Thermo Fisher) in 500-mL flat-bottom Erlenmeyer flasks at 37\u2009\u00b0C, 8% CONegative-stain EM grids were prepared following established protocol . SpecificisTEM . For PSMB2-mNG2 (11)-strepII with MG132, the 19S-20S classes were grouped and subjected to another round of 3D classification using cryoSPARC multirefinement (20-\u00c5 resolution limit) with eight classes to separate the different conformations. The resulting classes were refined to high resolution using cryoSPARC homogeneous refinement . Maps were filtered by local resolution in cryoSPARC. Atomic models were built into the cryo-EM density maps using Coot -strepII and mCherry -strepII . Images cisTEM . Particling Coot , 39 and ing Coot , 41 withing Coot .Supplementary File"} +{"text": "The AKT kinases have emerged as promising therapeutic targets in oncology and both allosteric and ATP-competitive AKT inhibitors have entered clinical investigation. However, long-term efficacy of such inhibitors will likely be challenged by the development of resistance. We have established prostate cancer models of acquired resistance to the allosteric inhibitor MK-2206 or the ATP-competitive inhibitor ipatasertib following prolonged exposure. While alterations in AKT are associated with acquired resistance to MK-2206, ipatasertib resistance is driven by rewired compensatory activity of parallel signaling pathways. Importantly, MK-2206 resistance can be overcome by treatment with ipatasertib, while ipatasertib resistance can be reversed by co-treatment with inhibitors of pathways including PIM signaling. These findings demonstrate that distinct resistance mechanisms arise to the two classes of AKT inhibitors and that combination approaches may reverse resistance to ATP-competitive inhibition. How resistance to different classes of AKT inhibitors can emerge is unclear. Here, the authors show that resistance to allosteric inhibitors is mainly due to mutation of AKT1 while the ATP competitive resistance is driven by activation of PIM kinases in prostate cancer models. The AKT/PKB serine/threonine kinase functions as a central node in this pathway and is being investigated as a therapeutic target in oncology2. Three isoforms of AKT exist in humans that each contain a Plekstrin homology (PH) domain, a kinase domain, and a C-terminal hydrophobic regulatory region3. Activation of these isoforms is mediated by recruitment to PtdIns-3,4-P2 and PtdIns-3,4,5-P3 (PIP3) at the plasma membrane and subsequent phosphorylation of T308 and S473 by 3-phosphoinositide-dependent protein kinase 1 (PDPK1) and mTOR complex 2 (mTORC2), respectively. Aberrant activation of AKT in cancer may occur via several mechanisms including mutational activation of the catalytic subunit of PI3K, which generates PIP3 and, indirectly, PI3,4P2; loss of the PIP3 phosphatase PTEN; and, albeit less frequently, activating mutations in AKT4. Upon activation, AKT mediates various cellular processes including cell survival, metabolism and proliferation by regulating the activity of downstream proteins including proline-rich AKT substrate of 40\u2009kDa (PRAS40), glycogen synthase kinase 3 (GSK-3), Forkhead box class O (FoxO) transcription factors, tuberous sclerosis complex 2 (TSC2), Bcl-2 associated death promoter (BAD), mTOR complex 1 (mTORC1), eukaryotic translation inhibition factor 4E-binding protein 1 (4EBP1), and the S6 ribosomal protein kinase4.Enhanced activity of the\u00a0phosphoinositide 3-kinase (PI3K)/AKT/mechanistic target of rapamycin (mTOR) signaling pathway is among the most frequently observed changes in cancer and is associated with tumor invasiveness, survival, and proliferation2. Importantly, these inhibitors differentially exploit the on-off activity cycle of AKT. In its inactive state, AKT adopts a closed conformation in which the PH domain interacts with the kinase domain, also referred to as the PH-in state6. Upon recruitment to the membrane and phosphorylation at T308 and S473, the interaction between the PH and kinase domains is released, resulting in an open PH-out conformation conducive to ATP-binding5. Allosteric inhibitors preferentially bind to\u00a0the inactive PH-in conformation at a cavity formed between the PH and kinase domains, preventing phosphorylation and activation of AKT6. In contrast, ATP-competitive inhibitors selectively target the PH-out conformation, protecting AKT from dephosphorylation at T308 and S473 while simultaneously blocking ATP binding and kinase activity7. As a result, decreased AKT phosphorylation at both T308 and S473 is typically observed in allosteric inhibitor-treated cells while increased or sustained pAKT at both sites is characteristic of the ATP-competitive inhibitors.Two main classes of AKT inhibitors (AKTis) have entered clinical investigation in oncology: allosteric inhibitors such as MK-2206 and adenosine 5\u2032-triphosphate (ATP)-competitive inhibitors such as ipatasertib/GDC-00688, the therapeutic potential of AKTis is likely to be the greatest in indications associated with PI3K/AKT pathway activating alterations. One such indication is prostate cancer. Activation of the PI3K/AKT pathway is thought to comprise roughly 50% of metastatic castration-resistant prostate cancer (mCRPC), frequently via PTEN loss11. In fact, a randomized phase II study evaluating combined inhibition of AKT via ipatasertib and androgen signaling via abiraterone in patients with mCRPC showed superior antitumor activity of the combination compared to abiraterone alone, especially in patients with PTEN-loss tumors12. Additionally, in metastatic triple-negative breast cancer (mTNBC), another indication associated with frequent PI3K/AKT pathway activating alterations, the combination of ipatasertib and paclitaxel also improved progression-free survival compared to paclitaxel alone in a randomized phase II trial, with a more pronounced effect observed in patients with PIK3CA/AKT1/PTEN-altered tumors13. Phase III clinical trials are currently underway to further evaluate ipatasertib as a therapeutic agent in these indications.Given that intrinsic sensitivity to AKTis such as ipatasertib correlates with AKT pathway activation14. Although some mechanisms of intrinsic resistance to PI3K/AKT/mTOR pathway inhibitors have been described, including SGK1 signaling in breast cancer15, Wnt-\u03b2-catenin signaling in colon cancer16, androgen receptor signaling in prostate cancer17 and RAS/RAF pathway signaling across multiple cancers8, few studies have explored mechanisms of acquired resistance to AKTis following prolonged treatment. Further, it remains unknown whether overlapping or distinct mechanisms of resistance will arise to long-term treatment with allosteric vs. ATP-competitive inhibitors. Here, we aim to identify mechanisms of acquired resistance to both allosteric and ATP-competitive AKTis using an unbiased approach. We performed systematic analysis of cell lines with acquired AKTi-resistance (AKTi-R) using methods including RNA sequencing (RNA-seq) and whole exome sequencing (exome-seq) and explored potential functional dependencies and combination strategies using a chemical genetics screen. Our findings indicate that distinct mechanisms do arise to the two different classes of AKTis and that combination approaches may be taken to reverse this resistance.While treatment of tumors with targeted therapies can initially result in impressive clinical outcomes, resistance is likely to emerge over extended treatment times50s or W80C AKT1 , pPRAS40 (T246) and pS6 (S235/236) levels comparable to that observed following overexpression of WT AKT1 Fig.\u00a0. In cont50) Fig.\u00a0. We alsoAKT1S1)/PRAS40 were detected in multiple G-R clones \u2009Given that a high number of alterations in both gene expression and exome sequence were detected in G-R cells compared to parental LNCaP cells kinases models, respectivelylls Fig.\u00a0. Given tlls Fig.\u00a0. Consist36. Interestingly, while PIM3 transcript levels are not increased in G-R cells compared with parental cells by RNA-seq analysis , each potently targeted by the pan-PIM kinase inhibitors included in this study, LNCaP cells primarily express PIM3 protein , suggesting that the low level PIM3 expression in the parental cells can antagonize the effect of AKT inhibition to some extent, the impact of PIM3 knockdown on ipatasertib sensitivity was more pronounced in G-R cells -inducible overexpression experiments. While siRNA-mediated depletion of PIM3 resulted in some increase in sensitivity of parental cells to ipatasertib (~2.9-fold decrease in ICed) Fig.\u00a0. Therefoed) Fig.\u00a0. Furthered) Fig.\u00a0. AltogetAmong the prostate cancer cell lines characterized in Supplementary Fig.\u00a039), or a combination of ipatasertib and GDC-0339. Tumor growth inhibition was evident following treatment with ipatasertib in Par X1.6 but not G-R3 X1.2 tumor-bearing mice, demonstrating that G-R3 X1.2 retained AKTi resistance in vivo mediating resistance appear to be context-dependent. For example, in PC3-LN4 prostate cancer cells, intrinsic resistance to both allosteric and ATP-competitive AKTis has been demonstrated to involve AKTi-induced PIM1 upregulation followed by a PIM1-dependent increase in receptor tyrosine kinase expression via cap-independent translation51. In various breast cancer cell lines, intrinsic resistance to the PI3K\u03b1 or AKT inhibitors has been demonstrated to involve PIM1, with PIM3 likely playing a less prominent role in this setting52. PIM2 expression has been linked to the resistance of breast cancer and MM cell lines to PI3K inhibitors GDC-0941 or BKM12053. Interestingly, PIM3 upregulation has been reported as a feedback mechanism in response to mTORC1 inhibition by rapamycin through miR-33 mediated suppression encoded by the SREBP loci54. In the current study, we did not observe increased PIM3 transcripts in the LNCaP parental or G-R cells in the presence of ipatasertib , AKT1 , AKT2 , AKT3 , pAKT(T308) , pAKT(S473) , pPRAS40(T246) , PRAS40 , pS6(S235/236) , S6 , p4EBP1 (T37/46) , p4EBP1 (S65) (#9456 1:1000), 4EBP1 , PARP (#9532 1:1000), Cleaved PARP , PTEN , PIM2 , PIM3 , pGSK-3\u03b2 (S9) , GSK-3\u03b2 , pBAD (S112) , and BAD were obtained from Cell Signaling Technology. The PIM1 antibody was obtained from Abnova . An additional antibody to total PRAS40 was obtained from Invitrogen/ThermoFisher . Protein loading was assessed using antibodies to \u03b2-actin , \u03b2-Tubulin or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) .Further information and requests for reagents may be directed to, and will be fulfilled by, the corresponding author, Kui Lin (lin.kui@gene.com).56. LNCaP cells harbor a frameshift mutation (K6fs*4) (COSMIC # COSM4929) and loss of heterozygosity (LOH) in PTEN58. The presence of both alterations was confirmed in the LNCaP line in-house via exome sequencing and SNP array. AKTi-resistant (AKTi-R) cell lines were established by treating cells with gradually escalating doses of ipatasertib or MK-2206 until reaching a maximum dose of 5\u2009\u03bcM of AKTi. ipatasertib-resistant (G-R) and MK-2206-resistant (M-R) cell pools were then subjected to single cell sorting using FACSAria instrumentation and software (BD Biosciences) and surviving clones were expanded in the presence of AKTi at the maximum doses indicated above. MK-2206 or ipatasertib-resistant cell pools are denoted as M-Rpool or G-Rpool, respectively. Individual AKTi-R clones were assigned numbers, which are indicated following the M-R or G-R prefix. LNCaP G-RB cell lines were established following long-term exposure of LNCaP cells to 5\u2009\u03bcM ipatasertib, single cell sorting, and expansion of surviving clones in the presence of the AKTi. The Par X1.6 and G-R3 X1.2 selected lines used for in vivo studies were derived from LNCaP and LNCaP G-R clone 3 (G-R3) tumors that displayed growth in untreated male SCID.bg C.B-17 mice (Charles River Labs). The R0068 X1.2 line was established from mice bearing LNCaP tumors that had been treated with 50\u2009mg/kg ipatasertib over the course of 106 days. Whole tumors from individual mice were excised, minced in complete media, and plated in tissue culture flasks. Cell lines were established following one to two passages. After establishment in culture, G-R3X1.2 and R0068 X1.2 cells were maintained in the presence of 5\u2009\u03bcM ipatasertib. All cell lines were maintained at 37\u2009\u00b0C/5% CO2 in Roswell Park Memorial Institute medium (RPMI) 1640 supplemented with 10% fetal bovine serum (FBS) (Sigma), 2 mM L-Glutamine, and 0.01\u2009M HEPES, pH 7.2. Growth medium for AKTi-R cell lines was additionally supplemented with AKTi at the indicated concentration for cell line maintenance.Cell lines were originally obtained from the American Type Culture Collection (ATCC) and genotyped by Genentech\u2019s cell banking facility. LNCaP is an approximately tetraploid epithelial line derived from a prostate adenocarcinoma metastasis6 cells into male NOD scid gamma (NSG) mice . Testosterone pellets were implanted into the dorsal shoulder 5 days prior to cell inoculation. Animals were distributed into treatment groups (n\u2009=\u20099/group) when the tumors reached a mean volume of approximately 230 to 350 mm3. GDC-0068 and GDC-0339 were formulated in 0.5% methylcellulose/0.2% Tween-80 (MCT) and were administered once daily (QD) via oral formulation at 25 and 100\u2009mg/kg, respectively for 21 days. Tumor volumes were determined using digital calipers using the formula (L x W x W)/2. Tumor volumes and body weights were recorded twice weekly over the course of the study. Mice with tumor volumes\u2009>2000\u2009mm3 or recorded body weight loss of >20% from their start of treatment were euthanized per Institutional Animal Care and Use Committee guidelines.All in vivo efficacy studies were approved by Genentech\u2019s Institutional Animal Care and Use Committee and adhere to the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. Tumor xenografts derived from the LNCaP Par and LNCaP G-R3 X1.2 cell lines were established by subcutaneous injection of 10\u2009\u00d7\u20091059. In brief, as tumors generally exhibit exponential growth, tumor volumes were subjected to natural log transformation before analysis. A generalized additive mixed model (GAMM) was then applied to describe the change of transformed tumor volumes over time using regression splines with auto-generated spline bases as this approach addresses both repeated measurements from the same study subjects and moderate dropouts before study end. Estimates of group-level efficacy were obtained by calculating a growth contrast, the difference in AUC-based growth rates between the treatment and reference group fits. To calculate AUC-based growth rates, group AUC values are corrected for starting tumor burden and then subjected to slope equivalence \u201cnormalization\u201d. Slope equivalence \u201cnormalization\u201d of AUC results in the actual slope of a fit on the natural log (LN) scale in cases of log-linear growth. In the cases of non-log-linear growth, such \u201cnormalization\u201d results in the constant log-linear growth rate that would have been needed to yield the baseline-corrected AUC that was actually observed for a fit on the natural log scale. Mathematically, this \u201cnormalization\u201d is attained by dividing each estimated baseline-corrected AUC value by half of the square of the common study period resulting in units of natural log units per day. The more negative the Growth Contrast value, the greater the anti-tumor effect. The 95% confidence intervals are based on the fitted model and variability measures of the data.Analysis and comparison of tumor growth was performed as detailed previously using a package of customized functions in R v3.6.2 which integrate software from open source packages including lme4, mgcv, gamm4, multcomp, settings, plyr, and several packages from the tidyverse such as magrittr, dplyr, tidyr, and ggplot2A generalized additive mixed model (GAMM) was also employed to describe the change in raw body weights over time with regression splines. After data fitting, raw body weight data at each time point from all individual animals and all group fits were normalized to the starting weight and reported as a percentage to yield % body weight change.8. Briefly, cells were plated in black/clear bottom 384 well plates and incubated at 37\u2009\u00b0C under 5% CO2. The following day, cells were treated with a 9-point dose titration of indicated inhibitors or with DMSO control. All conditions were tested in quadruplicate within each experiment. Treated cells were then incubated for 4 days and viability was assessed using the CellTiter-Glo\u00ae (Promega) luminescent assay according to the manufacturer\u2019s instructions. Total luminescence was measured on a Wallac Multilabel Reader (PerkinElmer) and was considered to represent cellular viability. Dose response curves generated with Prism (GraphPad) depict mean % viability (% DMSO control), with error bars representing standard error of the mean (SEM), from quadruplicate samples (y-axis) versus concentration of inhibitor (x-axis) from a single representative experiment. The inhibitor concentration resulting in the half maximum inhibitor effect (IC50) was calculated from % viability values from quadruplicate wells using a 4-parameter curve analysis . Scatter plots depict absolute IC50 values from independent biological repeats, with bars denoting mean absolute IC50 values and standard error of the mean (SEM). Mean IC50 values are denoted below x-axis and sample size (n) is indicated for each cell line/condition above scatter plot data. Unless otherwise stated, at least 3 independent experiments were performed to assess reproducibility. Where applicable, statistical significance is indicated above scatter plot data used to make comparisons.Cellular viability was assessed 4 days after addition of inhibitors using the CellTiter-Glo\u00ae (Promega) luminescent assay as described previouslyCells were washed with cold 1X phosphate-buffered saline (PBS) and lysed in Cell Extraction Buffer (CEB) (Biosource/Thermo Fisher Scientific) supplemented with a protease inhibitor cocktail (Sigma) and phosphatase inhibitor cocktail (Roche). Protein concentrations were determined using the Lowry-based RC DC protein assay (Bio-Rad) and normalized for equal protein loading. Lysates were loaded onto Tris\u2013glycine gels (Invitrogen) and proteins were separated by electrophoresis. Proteins were then transferred onto nitrocellulose membranes using the iBlot\u00ae dry blotting system (Invitrogen), and membranes were blocked with blocking buffer for fluorescent Western blotting (Rockland or LI-COR). Primary antibodies (see below) were detected using IR Dye 800-conjugated (Rockland or LI-COR) or Alexa Fluor 680 (Invitrogen) or IR Dye 680 (LI-COR) species-selective secondary antibodies. Detection and quantification were conducted using an Odyssey infrared scanner (LI-COR) using the manufacturer\u2019s software. Protein loading was assessed using antibodies to \u03b2-actin, \u03b2-Tubulin, or GAPDH. Raw immunoblot images for cropped blots are provided in the Supplementary Information.Par or AKTi-R cells were plated in duplicate in complete RPMI medium and, the following day, treated with DMSO, 5\u2009\u03bcM ipatasertib, or MK-2206 for 14\u2009h. Cells were then subjected to total RNA extraction using the RNeasy kit (Qiagen) and RNA concentrations were read using a NanoDrop 8000 spectrophotometer (Thermo Scientific). Following confirmation of RNA integrity with the 2200 TapeStation system (Agilent Technologies), RNA-seq libraries were prepared using the TruSeq RNA Sample Preparation Kit v2 (Illumina) from 1\u2009\u03bcg of total RNA. Library size was determined using 2200 TapeStation and High Sensitivity D1000 screen tape (Agilent Technologies) and concentration was assessed by qPCR-based methodology . The libraries were multiplexed and sequenced on Illumina HiSeq2500 (Illumina) to generate 50 million paired-end 75 base pair reads. RNA-seq reads containing 30% or more bases with a Phred quality score of 23 or lower were excluded. The remaining high quality reads were then mapped to NCBI GRCh37 (hg19) using GSNAP and default settings. Multimapping reads were discarded. Gene expression levels were summarized into count and RPKM . Differential expression analysis was performed using limma. Genes with adjusted p-value < 0.05 and absolute value of log2FC\u2009>\u2009=\u20091 were considered to be differentially expressed. For the hierarchical clustering and heatmap of RNA-seq transcriptome analysis, the RPKM values for the top 100 most variably expressed genes were z-scored and clustered using Euclidean distance.Par or AKTi-R cells were plated in complete RPMI medium and the following day, DNA extraction was performed using the DNeasy kit (Qiagen). Prior to processing by whole exome sequencing, the concentration and integrity of DNA samples was determined using NanoDrop 8000 (Thermo Fisher Scientific) and 2200 TapeStation (Agilent Technologies), respectively. Exome capture was performed using 0.5\u2009\u03bcg of genomic DNA and SureSelectXT Human All Exon v5 kit (50 megabases [Mb]) according to manufacturer\u2019s protocol . Fragment size distribution of post-capture amplified libraries was determined with 2200 TapeStation using high sensitivity D1000 screen tape . Concentration of the libraries was measured by Qubit (Thermo Fisher Scientific). Exome capture libraries were sequenced on HiSeq 2500 to generate 75 million paired-end 75 base pair reads. High quality exome-seq reads were mapped to NCBI GRCh38 using GSNAP. Somatic SNVs and INDELs were called by comparing the treatment resistant clones against the parental clones using LoFreq with its default setting. Highly-confident variants were annotated using Ensembl Variant Effect Predictor and filtered with dbSNP 138, ExAC 0.3.1 and RepeatMasker 4.0.5. The functional consequences of somatic variants were annotated using SIFT, PolyPhen and Condel.60.DNA from parental or AKTi-R cells was extracted and assessed for quality and quantity as described above. Illumina HumanOnmi2.5-8 arrays were then used to assay genotype, DNA copy number, and loss of heterozygosity as described previouslyThe Lipofectamine RNAi Max (Life Technologies) transfection reagent was used to transfect cells with siRNA oligonucleotides (see table below) according to manufacturer\u2019s instructions. Briefly, siRNA oligonucleotides and Lipofectamine RNAi Max were each mixed with Opti-MEM\u00ae (Invitrogen) in separate microcentrifuge tubes or wells in multiwell plates. These mixtures were combined and incubated for 15\u2009min prior to mixing with cells in suspension. Cells were transfected with a final concentration of 25\u2009nM siRNA duplexes. ON-TARGETplus SMART pool and individual siRNA oligonucleotides are reported to be highly specific, as verified by microarray analysis, as a result of unique dual-strand modification patterns used to synthesize the reagents.LNCaP cells were transfected with a pCMV6-AC-GFP vector containing the human AKT3 sequence (NM_005465) (Origene) or with EV using Fugene HD FuGENE\u00ae HD (Promega Corporation). 48\u2009h later, cells were subjected to selection with 0.4\u2009mg/l G418/Geneticin (Invitrogen/Gibco) and surviving cells were expanded. Cells were then sorted FACSAria instrumentation and associated software (BD Biosciences) for positive GFP expression and were maintained in the presence of 0.4\u2009mg/l G418/Geneticin.The piggyBac transposon-based system (System Biosciences) was used to introduce a cumate-inducible version of AKT1 WT or W80C to LNCaP Par or M-R7 cells. Using NheI and BstBI, the following sequences were cloned into the B-Cuo-MCS-IRES-GFP-EF1-CymR-Puro Inducible cDNA Cloning and Expression Vector (System Biosciences #PBQM812A-1):AKT1 WTGGCGCCACCATGAGCGACGTGGCTATTGTGAAGGAGGGTTGGCTGCACAAACGAGGGGAGTACATCAAGACCTGGCGGCCACGCTACTTCCTCCTCAAGAATGATGGCACCTTCATTGGCTACAAGGAGCGGCCGCAGGATGTGGACCAACGTGAGGCTCCCCTCAACAACTTCTCTGTGGCGCAGTGCCAGCTGATGAAGACGGAGCGGCCCCGGCCCAACACCTTCATCATCCGCTGCCTGCAGTGGACCACTGTCATCGAACGCACCTTCCATGTGGAGACTCCTGAGGAGCGGGAGGAGTGGACAACCGCCATCCAGACTGTGGCTGACGGCCTCAAGAAGCAGGAGGAGGAGGAGATGGACTTCCGGTCGGGCTCACCCAGTGACAACTCAGGGGCTGAAGAGATGGAGGTGTCCCTGGCCAAGCCCAAGCACCGCGTGACCATGAACGAGTTTGAGTACCTGAAGCTGCTGGGCAAGGGCACTTTCGGCAAGGTGATCCTGGTGAAGGAGAAGGCCACAGGCCGCTACTACGCCATGAAGATCCTCAAGAAGGAAGTCATCGTGGCCAAGGACGAGGTGGCCCACACACTCACCGAGAACCGCGTCCTGCAGAACTCCAGGCACCCCTTCCTCACAGCCCTGAAGTACTCTTTCCAGACCCACGACCGCCTCTGCTTTGTCATGGAGTACGCCAACGGGGGCGAGCTGTTCTTCCACCTGTCCCGGGAGCGTGTGTTCTCCGAGGACCGGGCCCGCTTCTATGGCGCTGAGATTGTGTCAGCCCTGGACTACCTGCACTCGGAGAAGAACGTGGTGTACCGGGACCTCAAGCTGGAGAACCTCATGCTGGACAAGGACGGGCACATTAAGATCACAGACTTCGGGCTGTGCAAGGAGGGGATCAAGGACGGTGCCACCATGAAGACCTTTTGCGGCACACCTGAGTACCTGGCCCCCGAGGTGCTGGAGGACAATGACTACGGCCGTGCAGTGGACTGGTGGGGGCTGGGCGTGGTCATGTACGAGATGATGTGCGGTCGCCTGCCCTTCTACAACCAGGACCATGAGAAGCTTTTTGAGCTCATCCTCATGGAGGAGATCCGCTTCCCGCGCACGCTTGGTCCCGAGGCCAAGTCCTTGCTTTCAGGGCTGCTCAAGAAGGACCCCAAGCAGAGGCTTGGCGGGGGCTCCGAGGACGCCAAGGAGATCATGCAGCATCGCTTCTTTGCCGGTATCGTGTGGCAGCACGTGTACGAGAAGAAGCTCAGCCCACCCTTCAAGCCCCAGGTCACGTCGGAGACTGACACCAGGTATTTTGATGAGGAGTTCACGGCCCAGATGATCACCATCACACCGCCTGACCAAGATGACAGCATGGAGTGTGTGGACAGCGAGCGCAGGCCCCACTTCCCCCAGTTCTCCTACTCGGCCAGCGGCACGGCCTGAAKT1 W80CGGCGCCACCATGAGCGACGTGGCTATTGTGAAGGAGGGTTGGCTGCACAAACGAGGGGAGTACATCAAGACCTGGCGGCCACGCTACTTCCTCCTCAAGAATGATGGCACCTTCATTGGCTACAAGGAGCGGCCGCAGGATGTGGACCAACGTGAGGCTCCCCTCAACAACTTCTCTGTGGCGCAGTGCCAGCTGATGAAGACGGAGCGGCCCCGGCCCAACACCTTCATCATCCGCTGCCTGCAGTGTACCACTGTCATCGAACGCACCTTCCATGTGGAGACTCCTGAGGAGCGGGAGGAGTGGACAACCGCCATCCAGACTGTGGCTGACGGCCTCAAGAAGCAGGAGGAGGAGGAGATGGACTTCCGGTCGGGCTCACCCAGTGACAACTCAGGGGCTGAAGAGATGGAGGTGTCCCTGGCCAAGCCCAAGCACCGCGTGACCATGAACGAGTTTGAGTACCTGAAGCTGCTGGGCAAGGGCACTTTCGGCAAGGTGATCCTGGTGAAGGAGAAGGCCACAGGCCGCTACTACGCCATGAAGATCCTCAAGAAGGAAGTCATCGTGGCCAAGGACGAGGTGGCCCACACACTCACCGAGAACCGCGTCCTGCAGAACTCCAGGCACCCCTTCCTCACAGCCCTGAAGTACTCTTTCCAGACCCACGACCGCCTCTGCTTTGTCATGGAGTACGCCAACGGGGGCGAGCTGTTCTTCCACCTGTCCCGGGAGCGTGTGTTCTCCGAGGACCGGGCCCGCTTCTATGGCGCTGAGATTGTGTCAGCCCTGGACTACCTGCACTCGGAGAAGAACGTGGTGTACCGGGACCTCAAGCTGGAGAACCTCATGCTGGACAAGGACGGGCACATTAAGATCACAGACTTCGGGCTGTGCAAGGAGGGGATCAAGGACGGTGCCACCATGAAGACCTTTTGCGGCACACCTGAGTACCTGGCCCCCGAGGTGCTGGAGGACAATGACTACGGCCGTGCAGTGGACTGGTGGGGGCTGGGCGTGGTCATGTACGAGATGATGTGCGGTCGCCTGCCCTTCTACAACCAGGACCATGAGAAGCTTTTTGAGCTCATCCTCATGGAGGAGATCCGCTTCCCGCGCACGCTTGGTCCCGAGGCCAAGTCCTTGCTTTCAGGGCTGCTCAAGAAGGACCCCAAGCAGAGGCTTGGCGGGGGCTCCGAGGACGCCAAGGAGATCATGCAGCATCGCTTCTTTGCCGGTATCGTGTGGCAGCACGTGTACGAGAAGAAGCTCAGCCCACCCTTCAAGCCCCAGGTCACGTCGGAGACTGACACCAGGTATTTTGATGAGGAGTTCACGGCCCAGATGATCACCATCACACCGCCTGACCAAGATGACAGCATGGAGTGTGTGGACAGCGAGCGCAGGCCCCACTTCCCCCAGTTCTCCTACTCGGCCAGCGGCACGGCCTGAThese sequences include a silent mutation to confer resistance to the ON-TARGET plus siRNA oligonucleotide (Dharmacon) targeting AKT1 with the sequence CAUCACACCACCUGACCAA. The piggyBac expression vector includes a GFP expression cassette separated from the AKT1 sequence by an internal ribosome entry site (IRES), enabling independent expression of both genes from a single transcript. Using either the PureFection\u2122 (System Biosciences) or FuGENE\u00ae HD (Promega Corporation) transfection reagents, LNCaP Par or M-R7 cells were transfected with the piggyBac transposase (System Biosciences) combined with the empty piggyBac expression vector, AKT1 WT piggyBac vector, or AKT1 W80C piggyBac vector. Transfected cells were then incubated for 48\u2009h prior to selection with 1\u2009\u03bcg/ml puromycin and subsequently expanded. Leaky expression was minimized by using FACSAria instrumentation and associated software (BD Biosciences) to select for cells that do not express GFP in the absence of cumate. In an effort to obtain populations that express similar levels of AKT1 WT vs. AKT1 W80C, cells were treated with 10\u2009\u03bcg/ml cumate for 5 days and were subjected to sorting for specific GFP expression levels using FACSAria instrumentation and associated software (BD Biosciences).Cell sorting was performed on multiple FACSAria\u2122 Fusions running DIVASoftware v8.0.1 equipped with 5 lasers (BD Biosciences). The instruments were set up with a nozzle size of 100 micron at a frequency of 32\u2009kHz and pressure of 20\u2009psi. The \u201cFour-Way Purity\u201d sort mode was used for coincident discrimination.http://www.cbioportal.org/index.do?session_id=5b5e1288498eb8b3d5672636). All AKT1 mutation information reported in those selected studies was then retrieved. The frequency of each AKT1 mutation detected within the same indication was calculated from these studies . Data bases used include TCGA: The Cancer Genome Atlas, https://portal.gdc.cancer.gov; METABRIC: Molecular Taxonomy of Breast Cancer International Consortium (Nature 2012 & Nat Commun 2016), Pierra et al., 2016 https://www.ncbi.nlm.nih.gov/pubmed/27161491; MSK-IMPACT: Memorial Sloan Kettering Cancer Center\u2019s Integrated Mutation Profiling of Actionable Cancer Targets .Cancer genomics studies in which AKT1 W80 alterations were detected in patients were first identified using cBioPortal was constructed using standard PCR techniques and mutants were generated using the QuikChange Site-Directed Mutagenesis Kit (Stratagene/Agilent Technologies). Mutant or WT AKT1 was cloned into the pRetro-internal ribosome entry site (IRES)-GFP vector (Clontech). MEK1 N3 was constructed as previously described61 and cloned into the pMXs-puro retroviral vector (Cell Biolabs). Retroviral constructs expressing the WT or mutant protein were transfected into the Phoenix amphoteric packaging cell line using Fugene6 (Roche). Viral supernatant was harvested 48\u2009h after transfection and filtered using a 0.45-\u03bcM syringe filter. Ba/F3 cells were then infected with virus by spinoculation , and cells infected with WT or mutant AKT1 were sorted by flow cytometry based on GFP fluorescence. Infected cells were selected with 2\u2009\u03bcg/mL puromycin for 7 days. Pools of these cells were used for subsequent studies. All Ba/F3-derived cell lines were maintained in culture in the presence of 2\u2009ng/mL recombinant murine IL-3 (R&D Systems). For analysis of the impact of WT vs. mutant AKT1 on the response to AKT inhibition, Ba/F3 cells which stably co-express MEK1 N3 and WT or mutant AKT1 were washed three times with 1X PBS and plated in the absence of IL-3 in complete RPMI in 384 well plates (1000 cells per well). The next day, cells were treated with a 9-point dose-titration of MK-2206 or ipatasertib using a maximum dose of 20\u2009\u03bcM. DMSO controls were included and all conditions were tested in 4 replicate wells. Cellular viability was assessed 4 days after addition of inhibitors using the CellTiter-Glo\u00ae (Promega) luminescent assay as described previously8. For analysis of protein levels, lysates were prepared from cells cultured in the absence of IL-3 for 1 day and subjected to Western blotting as described above.The impact of WT or mutant AKT1 on sensitivity to MK-2206 or ipatasertib was assessed using the IL-3 independent viability assay in the Ba/F3 model. Survival of the Ba/F3 murine pro-B cell line is constitutively growth-factor dependent but can be rendered IL-3 independent via co-expression of AKT1 and an activated form of the MAP2 kinase mitogen-activated protein kinase (MAPK)/extracellular-signal- regulated kinase (ERK) kinase (MEK1) , termed MEK1 N362. The data was processed using Genedata Screener, Version 14 , with a four\u2011parameter Hill equation using compound dose-response data normalized to the median of 42 vehicle\u2011treated wells on each plate. A \u201cRobust Fit\u201d strategy was also employed by Genedata Screener, which is based on Tukey\u2019s biweight and is resistant to outlier data. The reported absolute IC50 is the dose at which cross\u2011run estimated inhibition is 50% relative to DMSO control wells. In addition to absolute IC50, mean fitted viability across the nine tested doses was also computed.A library consisting of 426 compounds including targeted agents, chemotherapeutics, and tool compounds was used to screen for inhibitors associated with enhanced sensitivity in AKTi-R cells compared with Par cell lines. Compounds were obtained from in-house synthesis or purchased from commercial vendors. Cells were plated in 384 well plates using seeding densities previous determined to achieve approximately 70\u201380% confluence at the final time point of the assay. AKTi-R cells were plated in the presence of inhibitor at the dose used to maintain the resistance and Par cells were plated in DMSO control media. The following day, cells were treated with a 9-point dose titration of each inhibitor or DMSO control and 4 days later, cell viability was assessed as described above. Screening drug management, quality control, and the calculation of drug response statistics were performed as described previously63 as well as Highest single agent (HSA) analysis64, as described previously65. Briefly, a Bliss expectation for a combined response (C) of drugs A and B was calculated by the equation: C\u2009=\u2009(A\u2009+\u2009B)\u2009\u2212\u2009(A\u2009\u00d7\u2009B) where A and B are the fractional growth inhibitions of each respective drug at a given dose. The difference between the Bliss expectation and the observed growth inhibition of the combination of drugs A and B at the same dose is the \u201cDelta.Bliss.\u201d Delta.Bliss scores were summed across the dose matrix to generate a Bliss sum. Bliss sum\u2009=\u20090 indicates that the combination treatment is additive (as expected for independent pathway effects); Bliss sum > 0 indicates activity greater than additive (synergy); and Bliss sum <0 indicates the combination is less than additive (antagonism). Using the Highest single agent (HSA) model64, scores were calculated at each dose matrix point based on the excess loss of viability in the combination in comparison to the highest single drug response. At least three independent experiments were performed to assess reproducibility unless otherwise indicated. Heatmaps depict % viability inhibition, Bliss score, or HSA score associated with each dose combination point following treatment of cells with the two agents. Mean Bliss sum values from three independent biological replicates are depicted in scatter plots, with mean Bliss sum values denoted below the x-axis and sample size (n) indicated for each cell line/condition above scatter plot data.Par or G-R cells were plated in 384 well plates and 24\u2009h later were treated with a 9-point dose titration of GDC-0068/ipatasertib, a second inhibitor, or a combination of the two. DMSO controls were also included. After a further 4 days, cell viability was assessed using the CellTiter-Glo\u00ae assay as described above. The combination effect of ipatasertib and other inhibitors was assessed by Bliss independence analysis66 were cloned into the BH1.4 Dox-inducible piggyBac vector with an IRES-turboGFP-nuclear expression marker . LNCaP Par cells were transfected with the piggyBac transposase combined with the empty BH1.4 vector or the PIM expression constructs. Transfected cells were then subjected to selection with puromycin and subsequently expanded. Leaky expression was minimized by FACS sorting to select for cells that do not express GFP in the absence of Dox. In an effort to obtain populations that express similar levels of PIM proteins, cells were treated with 100\u2009ng/ml Dox for 3 days and subjected to sorting for specific GFP expression levels.Sequences encoding the full length forms of human PIM1 (NP_001230115.1), PIM2 (NP_006866.2), PIM3 (NP_001001852.2), or a kinase-deficient PIM3 K69M mutantFlash frozen tumors were pulverized to powder by mechanical force (Covaris cryoPREP). Tumor powder was lysed with RIPA lysis buffer (Sigma) supplemented with a cocktail of phosphatase and protease inhibitors (Thermo Fisher Scientific) and 1\u2009mM PMSF (Sigma). Tumor lysates were homogenized by mechanical disruption (SPEX SamplePrep) and cleared by centrifugation at 14,000\u2009rpm for 10\u2009min at 4\u2009\u00b0C. Protein levels were quantified by the BCA Protein Assay Kit (Pierce Biotechnology) and normalized to equal concentrations. Equal amounts of protein lysates were resolved using NuPAGE Bis-Tris precast gels (Thermo Fisher Scientific) and transferred onto nitrocellulose membranes using the iBlot\u00ae dry blotting system (Thermo Fisher Scientific). Membranes were blocked with blocking buffer for fluorescent Western blotting (LI-COR). Primary antibodies were detected using IR Dye 800-conjugated (LI-COR) or IR Dye 680-conjuated (LI-COR) species-selective secondary antibodies. Detection and quantification were conducted using an Odyssey infrared scanner (LI-COR) using the manufacturer\u2019s software. Protein loading was assessed using antibodies to \u03b2-actin or GAPDH.Immunohistochemisty (IHC) for xenograft tumors were carried out using 5-\u03bcm paraffin sections of formalin-fixed tissue on a VentanaBenchmark XT instrument (VMSI) by deparaffinization, treatment with antigen retrieval buffer (VMSI) and incubation with primary antibodies against PTEN (#9559), pAKT (S473) (#4060), pPRAS40 (T246) (#2997), pS6 (S235/236) (#2211), cleaved caspase 3 (Asp175) (#9661) , or cyclin D1 at 37\u2009\u00b0C. Bound antibody was detected using DABMap technology (VMSI) and sections were counterstained with hematoxylin. IHC stained slides were scanned on a NanoZoomer XR whole slide imager at 200x magnification, and were used to quantify the tumor and IHC positive staining areas. Segmentation of tumor regions and DAB positive pixels was performed by a custom algorithm using standard morphological operations and global RGB color thresholds running on Matlab 2019a .A two-tailed Student\u2019s t-test or a one-way analysis of variance (ANOVA) was performed when comparing two groups or more than two groups, respectively. Statistical analysis was carried out using Prism v9.3.1 (GraphPad) and Microsoft Excel. Data are expressed as the mean\u2009\u00b1\u2009standard deviation (SD) or standard error of means (SEM) as described in the figure legends. Each experiment was repeated at least three times unless otherwise indicated.Further information on research design is available in the\u00a0Supplementary InformationInventory of Supporting InformationSupplementary Dataset 1Supplementary Dataset 2Supplementary Dataset 3Supplementary Dataset 4Supplementary Dataset 5Reporting Summary"} +{"text": "Ferroptosis has been identified to play a crucial role in the progression and treatment of cancers. KAT6B, as a histone acetyltransferase, is involved in multiple cancer development. However, the function of KAT6B in glioma is still elusive. Here, we aimed to evaluate the effect of KAT6B on ferroptosis in glioma cells and explored the potential mechanisms. We observed that the expression of KAT6B was enhanced in clinical glioma samples. The viability of glioma cells was repressed by erastin and the overexpression of KAT6B rescued the phenotype in the cells. Meanwhile, the apoptosis of glioma cells was induced by the treatment of erastin, while the overexpression of KAT6B blocked the effect in the cells. The levels of lipid ROS and iron were promoted by the treatment of erastin and the overexpression of KAT6B could reverse the effect in the cells. Mechanically, we identified that the expression of STAT3 was repressed by the KAT6B knockdown in glioma cells. The KAT6B was able to enrich on the promoter of STAT3 in glioma cells. Meanwhile, ChIP assay showed that the knockdown of KAT6B inhibited the enrichment of histone H3 lysine 23 acetylation (H3K23ac) and RNA polymerase II (RNA pol II) on STAT3 promoter in the cells. Depletion of STAT3 reversed KAT6B-regulated viability, apoptosis, and ferroptosis of glioma cells. Thus, we concluded that KAT6B contributes to glioma progression by repressing ferroptosis Glioma serves as a prevalent malignancy among brain tumors in adults, which is also highly aggressive and with grave prognosis . Cancer KAT6B is a major histone acetyltransferase, widely reported during the progression of various diseases, including cancer, cardiovascular diseases, and neurodegenerative diseases . KAT6B eIn this work, we studied the function of KAT6B in glioma cells. We aimed to evaluate the effect of KAT6B on ferroptosis in glioma cells and explored the potential mechanisms.n = 35) were collected from glioma patients hospitalized, who received no chemotherapy and radiotherapy before surgery. All tumor samples were clinicopathologically confirmed as glioma . All of glioma tissues were astrocytoma and IDH wildtype. Normal brain tissues were collected from patients undergoing brain tissue resection due to craniocerebral injury. All samples were immediately stored in liquid nitrogen with RNAhold (TransGen). All patients have signed the informed consent forms. All experiments were approved by the Clinical Ethics Committee of our hospital.The glioma samples , maintained in DMEM medium added with 10% FBS and 1% penicillin/streptomycin , and were incubated in a 37\u00b0C atmosphere with 5% CO5 cells per well and transfected with 50\u2009nmol oligonucleotides or their corresponding negative controls [The small interference RNA targeting KAT6B and STAT3 was designed and synthesized by Ribobio (China). Recombinant plasmid pcDNA-KAT6B was constructed by cloning KAT6B cDNA into pcDNA3.1 vectors . The U251 and LN229 cells were seeded in a 6-well plate with 4 \u00d7 10controls by using\u03bcl/well) was added to each well and incubated for another 2\u2009h at 37\u00b0C. The absorbance values were measured at 450\u2009nm using a microplate detector .Cell viability was evaluated by the cell counting kit-8 in accordance with the manufacturer's description. The cells were planted in a 96-well plate, incubated overnight, and treated with corresponding oligonucleotides. At indicated time point, the CCK-8 solution was added, and samples were incubated for 30\u2009min at 37\u00b0C, 5% CO2 and protected from light. Excess C11-BODIPY was removed by washing the cells twice with PBS. Oxidation of the polyunsaturated butadienyl portion of the dye results in a shift of the fluorescence emission peak from 590\u2009nm to 510\u2009nm in a manner proportional to lipid ROS generation. This shift was analyzed using a flow cytometer [2+ or total iron in each cell line. 2 \u00d7 106 of cells were rapidly homogenized in 4-10 volumes of Iron Assay buffer. Samples were centrifuged at 13,000\u2009g for 10 minutes at 4\u00b0C to remove insoluble material. To measure ferrous iron, 5\u2009\u03bcL of iron assay buffer was added to each well. To measure ferric iron, two sets of wells were set up. Then, 5\u2009\u03bcL of assay buffer was added to the samples in one set of wells and 5\u2009\u03bcL of iron reducer was added to the other set of wells. To measure total iron, add 5\u2009\u03bcL of Iron Reducer to each of the sample wells to reduce Fe3+ to Fe2+. Samples were mixed well using a horizontal shaker or by pipetting, and the reactions were incubated for 30 minutes at room temperature, protected from light. Then, 100\u2009\u03bcL of Iron Probe was added to each well-containing standard or test samples. Samples were mixed well using a horizontal shaker or by pipetting, and the reactions were incubated for 60 minutes at room temperature, protected from light. Finally, the absorbance was measured at 593\u2009nm (A593) [The lipid ROS was measured by flow cytometry analysis in the cells. For Lipid ROS, cells were treated as indicated, and then typsinezed and resuspended in medium plus 10% FBS. Then, 10\u2009ytometer . The lev\u0394\u0394Ct- method.Total RNAs were isolated from tissues or cells using Trizol reagent , reversely transcribed into cDNA by using the High Capacity cDNA Reverse Transcription Kit . The relative RNA levels were quantified by quantitative real-time PCR using the SuperScript III Platinum SYBR Green One-Step qRT-PCR Kit . GAPDH was adopted as internal control for normalization following the 2The primer sequences are as follows: KAT6B forward: 5\u2032- ATACGAGCGAATGGGTCAGAGTGATTTTGG-3\u2032, reverse: 5\u2032- GTTCACAGAGTTGACATTGTAGGCTGGCG-3\u2032; STAT3 forward: 5\u2032- AACTCTCACGGACGAGGAGCT-3\u2032, reverse: 5\u2032-AGTAGTGAACTGGACGCCGG-3\u2032; GAPDH forward: 5\u2032-AACGGATTTGGTCGTATTGGG-3\u2032, reverse: 5\u2032-CCTGGAAGATGGTGATGGGAT-3\u2032.6 cells were fixed with formaldehyde for 10\u2009min at room temperature. Fixation was stopped with the addition of 1/10 volume 1.25\u2009M glycine, and the samples were incubated for 5\u2009min at room temperature. The sonication step was performed in a Qsonica sonicator , and 200\u2009\u03bcg of the protein-chromatin complex was used in each immunoprecipitation. Antibody-protein complexes were captured with preblocked dynabeads protein G . ChIP DNA was analyzed by qPCR with SYBR Green (Biorad) on an ABI-7500 (Applied Biosystems) using the primers specified. The antibodies used are as follows: KAT6B , H3K23ac (Active motif), RNA pol II , and normal mouse IgG . The promoter sequence was shown as follows:ChIP assay was performed by using Pierce Magnetic ChIP Kit in accordance with the manufacturer's protocols. Antibodies against KAT6B, H3K23ac, and RNA polymerase II were used to precipitate the DNA-protein complex. The level of immunoprecipitated DNA was evaluated by qRT-PCR assay. Briefly, ChIP assays were essentially performed as previously described with sliTGCCCTGTAGATGCCTCTGTCCCTCATTGGATATGAGGTGGTACGAGCGGTCTGAATCTGTAGACTTAGACAGGCTTCAGGTATCTGGGGAACCATGTGAGTAAAACCTTGTATTGTTTCTGGGGCTAGAATCTAGTCTCCTTCAAACACTCAGAAGGATCTGTGACTGCCTTCAAAGGTTAAGAGTCATTGATTTTTCTCACTAGAAATTAATAAGAACAGCTATCCTTGGGGAGAAGGAATGATGGGGGTAGGGAAAGATTTTCATTTAATATTTTTGACCTTTCTGAACGGTCTGCATTTTCTAAACAGGAACATGTATTAGGTTCATTGAAAAAAAATATGTCAGGGGTTAGCTGAGCAGTGAGATAATGGGTCCTTTTTAGATTTTTGTCTTTATACCTGTTCGTATTAAACAAACACAAATATTAATAACATATTTTTAAAGACCCAAAAAAATAAAAATTAAAAACCCTGATAGTATCAGCACATACACAGAAATCACTCCATTATGCAAAGTTCATCCTCTATTATGAAAGGCAAAATGTCTACATTTCCTATCAACCACTGGCTTCAATTCAGTAAAACTTGCATACCAAGTAGGCAAGGTGGAAAAGAAAAAGGCAGAACATTTCATGTATTTCAATTCAGACGCATAAAAATGTCAAGCCCTACACGTTATCAGCTTTCGTATACACCGTCTTCTGCATTCGCCTGTACGGGCCAATGGGCTAGCTGGTCGGCGTTTGATGCTTGAAGTGATGGAACGGAGTACGGGGTTAAATCCACTACCCTCTCCCCACGCACTCTAGTAATTACTCTATTTCCACGTCATGTTTCCGGGTGTGTGTGTCCCTGCTCACGCAGAAACTGAAGTTCAAAGCAGGCGGAGTCACCCATGTTCTTTTTGTTGTCCCCAGAACCCAATTCAGGAGTTGGGTCCCCAGAGGATCTGGAGATACCTGGGGACTATCTAACTAGCTGATTCCCGCGTGGTAAGAGGCTCTCAACCTCGCCACCACGTGGTGCCAAGGGCCGGGAAAAGGGAGAGCGGGCAGGAGGGAGCTGTATCAGGGGCATTTAAAGTGCCTTGACGTCACGCACTGCCAGGAACTCAGCTGAGTTTTCAGCAGGACATTCCGGTCATCTTCCCTCCCTCCCCCCGGGCTTCTGTGCCCAAGTCCTCGGCTCTTCCCTCGCTGTGGCGGAGGGAGGAGCACCGAACTGTCGGAACAGCCAGCACAGGGGCGTATCAGTCTCCTCTTGGCTCCGCCCTTTCTCCTAGCTGCTCTCCTCATTGGTCAGTGGGCGGGGCTTCGGCTGTACCGCACACGCACTGGGACCTCTGGGTGGCCGAACGAGCTGGCCTTTCATGAATTATGCATGACGGCGTGCCTCGGCCAGGCTGGGGCTGGGCGAGGATTGGCTGAAGGGGCTGTAATTCAGCGGTTTCCGGAGCTGCGGCGGCGCAGACTGGGAGGGGGAGCCGGGGGTTCCGACGTCGCAGCCGAGGGAACAAGCCCCAACCGGATCCTGGACAGGCACCCCGGCTTGGCGCTGTCTCTCCCCCTCGGCTCGGAGAGGCCCTTCGGCCTGAGGGAGCCTCGCCGCCCGTCCCCGGCACACGCGCAGCCCCGGCCTCTCGGCCTCTGCCGGAGAAACAGGTGAAGGGGGTGCAGGGTGGGGCC.t test or one-way ANOVA test. A p value less than 0.05 was considered to be statistically significant.Data in this work are presented as mean \u00b1 SD. Statistical analysis was performed by using SPSS software (version 17.0). Statistical comparison between two or more groups was determined by student's To analyze the correlation of KAT2B with glioma, the expression of KAT2B was analyzed in the clinical glioma samples. We observed that the expression of KAT2B was enhanced in clinical glioma samples . To evalNext, we were interested in the correlation of KAT2B with ferroptosis in glioma cells. To this end, the U251 and LN229 cells were treated with ferroptosis activator erastin or cotreated with erastin and KAT6B overexpressing plasmid. We found that the viability and colony formation numbers of U251 and LN229 cells were repressed by erastin and the overexpression of KAT6B rescued the phenotype in the cells Figures . MeanwhiThen, we evaluated the effect of KAT6B on the levels of ferroptosis markers, including lipid ROS and iron, in the glioma cells. We observed that the levels of lipid ROS and iron were promoted by the treatment of erastin and the overexpression of KAT6B could reverse the effect in the cells Figures .We next tried to explore the underlying mechanism by which KAT6B regulated glioma. We observed that the expression of STAT3 was repressed by the KAT6B knockdown in U251 and LN229 cells . We idenNext, we validated the correlation of KAT6B and STAT3 in the regulation of glioma cells. The effectiveness of STAT3 depletion by shRNA was validated in U251 and LN229 cells Figure . We obseGlioma is a prevalent malignancy among brain tumors, and ferroptosis plays crucial roles in the progression and treatment of cancers. Histone acetyltransferase KAT6B is involved in multiple cancer development but the function of KAT6B in glioma remains unclear. In this study, we uncovered the effect and the potential mechanism of KAT6B on ferroptosis in glioma cells.\u03baB signaling in tongue squamous cell carcinoma [Previous studies have identified the function of KAT6B in cancer development. The inhibition of histone acetyltransferases KAT6A/B contributes to senescence and regulates tumor growth . KAT6B farcinoma . These rMoreover, it has been reported that STAT3 inhibits ferroptosis-mediated IIR-ALI by targeting SLC7A11 . miR-519via epigenetically inducing STAT3 (Therefore, we concluded that KAT6B contributes to glioma progression by repressing ferroptosis ng STAT3 . KAT6B m"} +{"text": "In the mouse embryo, the sensors responded to cytoskeletal relaxation and stretch applied by micro-aspiration. They reported organ-specific differences and a spatiotemporal tension gradient along the proximodistal axis of the limb bud, raising the possibility that mechanical mechanisms coregulate pattern formation. These mouse strains and software are potentially valuable tools for testing and refining mechanotransduction hypotheses in vivo.Nuclear mechanotransduction is a growing field with exciting implications for the regulation of gene expression and cellular function. Mechanical signals may be transduced to the nuclear interior biochemically or physically through connections between the cell surface and chromatin. To define mechanical stresses upon the nucleus in physiological settings, we generated transgenic mouse strains that harbour FRET-based tension sensors or control constructs in the outer and inner aspects of the nuclear envelope. We knocked-in a published esprin-2G sensor to measure tensions across the LINC complex and generated a new sensor that links the inner nuclear membrane to chromatin. To mitigate challenges inherent to fluorescence lifetime analysis Summary: We introduce transgenic and software tools to measure tensions across the outer and inner nuclear envelope in mice. These tools facilitate our ability to test mechanotransduction hypotheses in vivo. The cell nucleus experiences physical forces in a manner that has the potential to alter chromatin conformation , gene exin vitro studies complex that links f-actin, microtubules, and intermediate filaments to the nuclear lamina . Mammaliin vitro and the INM protein NEMP1 in mice. The former was previously generated for use in vitro , whereasWe wished to generate knock-in mouse strains that harbour tension sensors within the outer and inner aspects of the nuclear envelope. To measure force transmission across the LINC complex, we generated a mouse strain capable of conditionally expressing a previously published tension sensor (NespTS) . The conin vitro. Super resolution stimulated emission-depletion (STED) microscopy revealed appropriate co-localisation of NmpTS and controls with laminB1 (reflecting INM location), and that of NespTS external to laminB1 . We chosLIMvivo) . To maxiLIMvivo) . DiffereLIMvivo) . The proLIMvivo) .There are two principal sources of noise in our setting. A quick fluorescence decay that is likely due to autofluorescence can artificially decrease the measured fluorescent lifetime, and background, or scattered, light can artificially increase the fit value for measured lifetime unless addressed appropriately . In addi\u00a0We tested individual sensor responses in vivo using E9.5 wild-type (+/+Nemp1) distal limb bud mesoderm as a model system due to its accessibility and well-studied patterning characteristics in intact embryos under live conditions. Relative to NmpTS, NmpDO exhibited higher mean lifetime values, as expected. NmpTL and NespHL reported low lifetime values relative to NmpDO and previously published donor only mTFP measurements was a barrier to reliable FLIM analysis when we applied commercially available fitting software. Similar hurdles were encountered previously with an E-cadherin tension sensor in Drosophila, resulting in apparently poor sensitivity . Testing of constructs' proper subcellular localisation was done by Lipofectamine 2000 transient transfection in 293T cells. EBFP and IRES were removed from the pR26-CAG/BFP-Dest plasmid and each construct was cloned into this ROSA26 targeting vector via gateway cloning through pDONOR for mouseline generation. These constructs were sequenced to select reliable candidates for mouseline generation.NmpTS/DO/TL were generated by PCR amplification of the tension sensor module from the Vinculin Tension Sensor and ligaChimeras were generated by traditional homologous recombination through aggregation and implantation of electroporated embryonic stem cells (ESCs) in wild-type embryos . Final ESCs were selected by PCR genotyping and copy number analysis (TCAG at the Peter Gilgan Centre for Research and Learning). Founders were confirmed by PCR genotyping and fluorescence imaging of crossings with various Cre lines. Constitutively active lines were generated by crossings with pCX-NLS-Cre 1Nagy) or CMV-Cre and littermates were compared visually and by weight. These lines were then inbred or outcrossed with CD1 to assess fertility.in vitro. For immunofluorescence of embryonic tissue sections, 550\u2005nm or 488\u2005nm excitation wavelength with 592\u2005nm or 660\u2005nm depletion wavelength were used on a Leica SP8 Lightning Confocal. Huygens software was employed for deconvolution. Confocal images were captured on a Nikon A1R confocal microscope using Nikon NIS software for acquisition and Volocity for image processing. The FRET donor of the sensors is mTFP, so all FLIM data were acquired on a Nikon A1R confocal microscope connected to a PicoQuant pulse system with a 440\u2005nm laser and 520/35\u2005nm detector channel in Picoquant SymPhoTime 64 software. All FLIM images were acquired using transgenic mice in a wild-type background.All imaging was performed in the Sickkids Research Institute Imaging Facility. Super resolution STED microscopy was performed on a Leica SP8 confocal microscope using 458\u2005nm or 514\u2005nm excitation wavelength with 592\u2005nm or 660\u2005nm depletion wavelength for sensor constructs 2 held in place with cheese cloth. In roller culture experiments, embryos were first placed in roller culture consisting of DMEM with 50% rat serum, 5% DMSO, and specified final drug concentrations and rolled for 1\u2005h at 37\u00b0C in a 20% O2, 5% CO2 chamber. Aspiration experiments were performed on the A1R using a 90\u2005\u00b5m diameter glass needle in a previously published device fixation allowed for imaging of mTFP or Venus within sensor constructs, but longer fixation of in vivo samples required labelling of sensor constructs with mouse monoclonal \u03b1-GFP .In fixed sample imaging, \u03b1-aminB1 rabbit polyclonal antibody was used to label LaminB1 or \u03b1-O-GlcNAc mouse monoclonal antibody was used to label nuclear pores . In fixed FLIM data were segmented into isolated nuclear membrane regions. Unless clarified otherwise in the figure legends, each dataset presented is nuclei from at least three separate embryos. With the exception of VinTFP which was fitted to entire fields of view, 15 nuclei, or as many clear nuclei as were available, per image were segmented for each region of each embryo. Further, segments with low quality data were rejected prior to statistical analysis.P-values were calculated using the t-test, applying Welch's correction to comparisons with significantly different standard deviations for datasets expected to be normally distributed and using the Mann\u2013Whitney test for non-normally distributed datasets. For t-tests, four key assumptions were considered prior to test application. Pregnancy rate comparison P-values were calculated using the \u03c7-square test. Variance comparisons were performed using F-tests. P-values greater than 0.05 were considered insignificant. In all experiments where tissues from the same embryos were compared, the values were normalised to the mean of all embryos and tissues for that experiment.Statistical analysis was done in Prism. With the exception of pregnancy rate comparisons, regression analyses, and variance comparisons, https://www.github.com/HopyanLab/FLIMvivo. The basic FLIMvivo.py script takes a CSV data file and can perform either convolution or tail fitting applying either a mono-exponential or bi-exponential model. Segmentation masks for analysed FLIM images were generated using either the FLIMvivo or the FLIMfit , then calls upon FLIMvivo to fit the extracted data. The script uses FLIMvivo first to fit the full field with a model of a Gaussian instrument response convoluted with a bi-exponential in order to establish an autofluorescence lifetime C. It cheWe model the FLIM-FRET decay as a bi-exponential, with autofluorescent and signal components,For our fitting algorithms we use a maximum likelihood fitting scheme assuming Poisson statistics, minimizing the \u2018negative log-likelihood\u2019 (NLL),Finally, in fitting, we treat the end point (where the data is cut off) as fit variable, chosen by maximizing the ratio of best fit NLL to the square root of the number of data points. This way the data range is uniformly chosen by maximizing a measure of data contribution to our fit metric, thereby eliminating any potential bias introduced by choosing manually.All constructs were genotyped using these primers: Forward: CTCTGCTGCCTCCTGGCTTCT, WT Reverse: CGAGGCGGATCACAAGCAATA, Mutant Reverse: CCGCGAGCTGTGGAAAAAAAAGGG.ATGGCGGGAGGAATGAAAGTGGCGGTCTCGCCGGCAGTTGGTC CCGGGCCCTGGGGCTCGGGAGTCGGGGGC GGTGGGACAGTGC GGC TACTCTTGATCCTCTCCGGCTGCTTGGTCTACGGCACAGCT GAAACTGATGTAAATGTGGTCATGCTTCAGGAATCCCAA GTTTGTGAAAAGCGTGCCAGCCAACAATTCTGTTACACAAATGTGCTTATCCCAAAATGGCATGATATATGGACACGGATACAGATCCGAGTAAATAGTTCCAGATTGGTTCGAGTCACCCAGGTGGAGAATGAGGAGAAACTGAAGGAGCTAGAGCAGTTTAGTATCTGGAACTTTTTTTCCTCCTTTTTAAAAGAGAAATTGAATGACACCTATGTTAACGTGGGTCTATACAGCACAAAAACCTGCCTCAAAGTTGAGATTATAGAGAAGGACACCAAGTACAGTGTCATTGTGATCCGGAGATTTGATCCCAAACTCTTTCTTGTTTTCCTTCTTGGACTTATGCTATTTTTTTGTGGAGACTTGCTGAGCAGAAGTCAAATTTTCTACTACTCTACTGGGATGACTGTGGGAATTGTGGCCTCTCTGCTAATCATCATTTTTATACTATCTAAGTTTATGCCTAAGAAAAGTCCCATTTACGTCATCCTGGTGGGAGGCTGGTCTTTTTCTCTGTACCTCATTCAACTAGTTTTTAAAAATTTACAAGAGATCTGGAGGTGTTACTGGCAGTATCTTTTAAGTTATGTCCTCACAGTTGGATTCATGAGTTTTGCAGTATGTTACAAGTATGGGCCCTTGGAGAATGAACGAAGTATCAACCTGCTGACCTGGACCTTGCAGCTGATGGGCCTGTGTTTCATGTATTCTGGCATCCAGATACCACATATTGCCCTTGCCATTATCATCATTGCTCTTTGTACTAAGAACCTGGAACACGGATCCATGGTGAGCAAGGGCGAGGAGACCACAATGGGCGTAATCAAGCCCGACATGAAGATCAAGCTGAAGATGGAGGGCAACGTGAATGGCCACGCCTTCGTGATCGAGGGCGAGGGCGAGGGCAAGCCCTACGACGGCACCAACACCATCAACCTGGAGGTGAAGGAGGGAGCCCCCCTGCCCTTCTCCTACGACATTCTGACCACCGCGTTCGCCTACGGCAACAGGGCCTTCACCAAGTACCCCGACGACATCCCCAACTACTTCAAGCAGTCCTTCCCCGAGGGCTACTCTTGGGAGCGCACCATGACCTTCGAGGACAAGGGCATCGTGAAGGTGAAGTCCGACATCTCCATGGAGGAGGACTCCTTCATCTACGAGATACACCTCAAGGGCGAGAACTTCCCCCCCAACGGCCCCGTGATGCAGAAGAAGACCACCGGCTGGGACGCCTCCACCGAGAGGATGTACGTGCGCGACGGCGTGCTGAAGGGCGACGTCAAGCACAAGCTGCTGCTGGAGGGCGGCGGCCACCACCGCGTTGACTTCAAGACCATCTACAGGGCCAAGAAGGCGGTGAAGCTGCCCGACTATCACTTTGTGGACCACCGCATCGAGATCCTGAACCACGACAAGGACTACAACAAGGTGACCGTTTACGAGAGCGCCGTGGCCCGCAACTCCACCGACGGCATGGACGAGCTGTACAAGGGGCCAGGTGGTGCAGGGCCAGGTGGTGCAGGGCCAGGTGGTGCAGGGCCAGGTGGTGCAGGGCCCGGTGGTGCAGGTCCAGGTGGTGCAGGTCCAGGTGGTGCAGGTCCAGGTGGTGCTATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGCTGATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGGGCTACGGCCTGCAGTGCTTCGCCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCACCGCCGACAAGCAGAAGAACGGCATCAAGGCCAACTTCAAGATCCGCCACAACATCGAGGACGGCGGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCTACCAGTCCAAGCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGGAATTCCCTATTCAGTGGCTGTACATCACCTGCAGAAAGGTGTGTAAGGGAGCAGAAAAGCCTGTTCCCCCTCGTCTCCTGACAGAAGAAGAATATCGGATACAAGGAGAGGTAGAAACCCGAAAGGCTTTAGAGGAGCTCCGAGAATTTTGTAACAGTCCAGACTGCTCTGCTTGGAAGACTGTTTCTCGAATCCAGTCTCCAAAAAGATTTGCTGACTTTGTGGAAGGCTCTTCCCACCTCACGCCAAATGAAGTTTCTGTCCATGAGCAGGAGTATGGATTAGGGAGCATTATTGCCCAGGATGAAATCTATGAGGAAGCATCCTCTGAGGAGGAGGACTCATATTCTCGGTGTCCTGCTATCACACAGAACAACTTTCTAACCTGA.ATGGCGGGAGGAATGAAAGTGGCGGTCTCGCCGGCAGTTGGTCCCGGGCCCTGGGGCTCGGGAGTCGGGGGCGGTGGGACAGTGCGGCTACTCTTGATCCTCTCCGGCTGCTTGGTCTACGGCACAGCTGAAACTGATGTAAATGTGGTCATGCTTCAGGAATCCCAAGTTTGTGAAAAGCGTGCCAGCCAACAATTCTGTTACACAAATGTGCTTATCCCAAAATGGCATGATATATGGACACGGATACAGATCCGAGTAAATAGTTCCAGATTGGTTCGAGTCACCCAGGTGGAGAATGAGGAGAAACTGAAGGAGCTAGAGCAGTTTAGTATCTGGAACTTTTTTTCCTCCTTTTTAAAAGAGAAATTGAATGACACCTATGTTAACGTGGGTCTATACAGCACAAAAACCTGCCTCAAAGTTGAGATTATAGAGAAGGACACCAAGTACAGTGTCATTGTGATCCGGAGATTTGATCCCAAACTCTTTCTTGTTTTCCTTCTTGGACTTATGCTATTTTTTTGTGGAGACTTGCTGAGCAGAAGTCAAATTTTCTACTACTCTACTGGGATGACTGTGGGAATTGTGGCCTCTCTGCTAATCATCATTTTTATACTATCTAAGTTTATGCCTAAGAAAAGTCCCATTTACGTCATCCTGGTGGGAGGCTGGTCTTTTTCTCTGTACCTCATTCAACTAGTTTTTAAAAATTTACAAGAGATCTGGAGGTGTTACTGGCAGTATCTTTTAAGTTATGTCCTCACAGTTGGATTCATGAGTTTTGCAGTATGTTACAAGTATGGGCCCTTGGAGAATGAACGAAGTATCAACCTGCTGACCTGGACCTTGCAGCTGATGGGCCTGTGTTTCATGTATTCTGGCATCCAGATACCACATATTGCCCTTGCCATTATCATCATTGCTCTTTGTACTAAGAACCTGGAACACGGATCCATGGTGAGCAAGGGCGAGGAGACCACAATGGGCGTAATCAAGCCCGACATGAAGATCAAGCTGAAGATGGAGGGCAACGTGAATGGCCACGCCTTCGTGATCGAGGGCGAGGGCGAGGGCAAGCCCTACGACGGCACCAACACCATCAACCTGGAGGTGAAGGAGGGAGCCCCCCTGCCCTTCTCCTACGACATTCTGACCACCGCGTTCGCCTACGGCAACAGGGCCTTCACCAAGTACCCCGACGACATCCCCAACTACTTCAAGCAGTCCTTCCCCGAGGGCTACTCTTGGGAGCGCACCATGACCTTCGAGGACAAGGGCATCGTGAAGGTGAAGTCCGACATCTCCATGGAGGAGGACTCCTTCATCTACGAGATACACCTCAAGGGCGAGAACTTCCCCCCCAACGGCCCCGTGATGCAGAAGAAGACCACCGGCTGGGACGCCTCCACCGAGAGGATGTACGTGCGCGACGGCGTGCTGAAGGGCGACGTCAAGCACAAGCTGCTGCTGGAGGGCGGCGGCCACCACCGCGTTGACTTCAAGACCATCTACAGGGCCAAGAAGGCGGTGAAGCTGCCCGACTATCACTTTGTGGACCACCGCATCGAGATCCTGAACCACGACAAGGACTACAACAAGGTGACCGTTTACGAGAGCGCCGTGGCCCGCAACTCCACCGACGGCATGGACGAGCTGTACAAGGGGCCAGGTGGTGCAGGGCCAGGTGGTGCAGGGCCAGGTGGTGCAGGGCCAGGTGGTGCAGGGCCCGGTGGTGCAGGTCCAGGTGGTGCAGGTCCAGGTGGTGCAGGTCCAGGTGGTGCTATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGCTGATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGGGCTACGGCCTGCAGTGCTTCGCCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCACCGCCGACAAGCAGAAGAACGGCATCAAGGCCAACTTCAAGATCCGCCACAACATCGAGGACGGCGGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCTACCAGTCCAAGCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTGA.ATGGCGGGAGGAATGAAAGTGGCGGTCTCGCCGGCAGTTGGTCCCGGGCCCTGGGGCTCGGGAGTCGGGGGCGGTGGGACAGTGCGGCTACTCTTGATCCTCTCCGGCTGCTTGGTCTACGGCACAGCTGAAACTGATGTAAATGTGGTCATGCTTCAGGAATCCCAAGTTTGTGAAAAGCGTGCCAGCCAACAATTCTGTTACACAAATGTGCTTATCCCAAAATGGCATGATATATGGACACGGATACAGATCCGAGTAAATAGTTCCAGATTGGTTCGAGTCACCCAGGTGGAGAATGAGGAGAAACTGAAGGAGCTAGAGCAGTTTAGTATCTGGAACTTTTTTTCCTCCTTTTTAAAAGAGAAATTGAATGACACCTATGTTAACGTGGGTCTATACAGCACAAAAACCTGCCTCAAAGTTGAGATTATAGAGAAGGACACCAAGTACAGTGTCATTGTGATCCGGAGATTTGATCCCAAACTCTTTCTTGTTTTCCTTCTTGGACTTATGCTATTTTTTTGTGGAGACTTGCTGAGCAGAAGTCAAATTTTCTACTACTCTACTGGGATGACTGTGGGAATTGTGGCCTCTCTGCTAATCATCATTTTTATACTATCTAAGTTTATGCCTAAGAAAAGTCCCATTTACGTCATCCTGGTGGGAGGCTGGTCTTTTTCTCTGTACCTCATTCAACTAGTTTTTAAAAATTTACAAGAGATCTGGAGGTGTTACTGGCAGTATCTTTTAAGTTATGTCCTCACAGTTGGATTCATGAGTTTTGCAGTATGTTACAAGTATGGGCCCTTGGAGAATGAACGAAGTATCAACCTGCTGACCTGGACCTTGCAGCTGATGGGCCTGTGTTTCATGTATTCTGGCATCCAGATACCACATATTGCCCTTGCCATTATCATCATTGCTCTTTGTACTAAGAACCTGGAACACGGATCCATGGTGAGCAAGGGCGAGGAGACCACAATGGGCGTAATCAAGCCCGACATGAAGATCAAGCTGAAGATGGAGGGCAACGTGAATGGCCACGCCTTCGTGATCGAGGGCGAGGGCGAGGGCAAGCCCTACGACGGCACCAACACCATCAACCTGGAGGTGAAGGAGGGAGCCCCCCTGCCCTTCTCCTACGACATTCTGACCACCGCGTTCGCCTACGGCAACAGGGCCTTCACCAAGTACCCCGACGACATCCCCAACTACTTCAAGCAGTCCTTCCCCGAGGGCTACTCTTGGGAGCGCACCATGACCTTCGAGGACAAGGGCATCGTGAAGGTGAAGTCCGACATCTCCATGGAGGAGGACTCCTTCATCTACGAGATACACCTCAAGGGCGAGAACTTCCCCCCCAACGGCCCCGTGATGCAGAAGAAGACCACCGGCTGGGACGCCTCCACCGAGAGGATGTACGTGCGCGACGGCGTGCTGAAGGGCGACGTCAAGCACAAGCTGCTGCTGGAGGGCGGCGGCCACCACCGCGTTGACTTCAAGACCATCTACAGGGCCAAGAAGGCGGTGAAGCTGCCCGACTATCACTTTGTGGACCACCGCATCGAGATCCTGAACCACGACAAGGACTACAACAAGGTGACCGTTTACGAGAGCGCCGTGGCCCGCAACTCCACCGACGGCATGGACGAGCTGTACAAGGAATTCCCTATTCAGTGGCTGTACATCACCTGCAGAAAGGTGTGTAAGGGAGCAGAAAAGCCTGTTCCCCCTCGTCTCCTGACAGAAGAAGAATATCGGATACAAGGAGAGGTAGAAACCCGAAAGGCTTTAGAGGAGCTCCGAGAATTTTGTAACAGTCCAGACTGCTCTGCTTGGAAGACTGTTTCTCGAATCCAGTCTCCAAAAAGATTTGCTGACTTTGTGGAAGGCTCTTCCCACCTCACGCCAAATGAAGTTTCTGTCCATGAGCAGGAGTATGGATTAGGGAGCATTATTGCCCAGGATGAAATCTATGAGGAAGCATCCTCTGAGGAGGAGGACTCATATTCTCGGTGTCCTGCTATCACACAGAACAACTTTCTAACCTGA.10.1242/biolopen.059656_sup1Supplementary informationClick here for additional data file."} +{"text": "Previous studies have shown that aging promotes myocardial apoptosis. However, the detailed mechanisms remain unclear. Our recent studies revealed that aging not only activates apoptosis, but also activates some anti-apoptotic factors. By quantitative phosphoproteomics, here we demonstrated that aging increases cytochrome c (Cytc) phosphorylation at threonine 50 (T50), a post-translational modification with unknown functional impact. With point mutation and lentivirus transfection, cardiomyocytes were divided into four groups: empty vector group, WT (wild type), T50E (as a phosphomimic variant), and T50A (non-phosphorylatable). TUNEL staining and flow cytometry were used to determine the apoptosis ratio in different groups after hypoxic/reoxygenated (H/R) treatment. The results showed that T50-phosphorylated Cytc suppressed myocardial apoptosis induced by H/R. Furthermore, Western Blot and ELISA measurements revealed that Cytc T50 phosphorylation inhibited caspase-9 and caspase-3 activity without altering caspase-8, BCL-2, BCL-XL, and Bax expression. In our study, we demonstrated that aging increases phosphorylation Cytc at T50 and this aging-increasing phosphorylation site can suppress H/R-induced apoptosis. Acute myocardial infarction is one of the leading causes of death worldwide. Thanks to the rapid development of interventional technology, the number of people dying from acute myocardial infarction has decreased significantly. On the contrary, more and more people are suffering from post-ischemic heart failure . Heart fSubstantial evidence indicates that apoptosis is the leading cause of ischemia-induced heart failure . Our preMore interestingly, our previous work demonstrates that aging not only stimulates pro-apoptotic pathways in cardiomyocytes, but also activates some anti-apoptotic factors , 9, 10. In the present study, we performed proteomics on myocardial tissue obtained from young and old mice. Compared with the young group, the expression of 88 proteins was up-regulated, and 80 were down-regulated in old mice. At a post-translational modification level, phosphorylation levels were up-regulated at 445 sites and down-regulated at 1526 sites in the old group. Phosphorylation level changes were widespread and stable in each sample. More importantly, these changes occurred in many essential apoptotic regulatory proteins, including MAPK14, Akt1, mTORC2, and p21. Upon further analysis of these results, changes in Cytc phosphorylation at T50 caught our attention as Cytc phosphorylation is involved in mitochondrial function . SurprisWe found a new aging-increasing phosphorylation site by proteomics, which may be associated with aging-related myocardial apoptosis regulation.in vivo and is closely related to apoptosis [P-value<0.05 as a cut-off, the change in the amount of differential modification >1.3 was considered as significantly up-regulated and less than 1/1.3 as significantly down-regulated. and increased apoptosis in T50A group when compared with WT group is also closely related to apoptosis. Lee et al. isolated Cytc from cow heart and identified the phosphorylation site by immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry. Tyr-97 is the first phosphorylation site discovered on Cytc . The othIn conclusion, there are several innovations in this study. First, we observed that aging increases phosphorylation Cytc at T50. Second, this aging-increasing phosphorylation site can suppress H/R-induced apoptosis. In addition, this anti-apoptotic effect is mediated mainly through inhibition of downstream caspase-9 and caspase-3 activity. Based on our previous studies , T50-phoIn summary, our study proved a new phosphorylation site that is increased in the process of aging. More importantly, phosphorylation at this site can decrease the apoptosis ratio induced by H/R.Approved by the institutional ethics committee, this study was in compliance with the United States National Institutes of Health guidelines. Male C57 mice (aged eight weeks and 18 months) were anesthetized with sodium pentobarbital. Mouse hearts were harvested by exteriorizing the heart via a left thoracic incision.Heart tissue from young and old mouse were harvested and delivered to PTM-Biolab Co., Ltd. for phosphoproteomic analysis. The quantitative study of phosphorylated proteomics used TMT labeling and phosphorylated enrichment techniques and high-resolution liquid chromatography-mass spectrometry.http://www.ebi.ac.uk/GOA/). Firstly, converting identified protein ID to UniProt ID and then mapping to GO IDs by protein ID. If some identified proteins were not annotated by UniProt GOA database, the InterProScan soft was used to annotate protein\u2019s GO function based on the protein sequence alignment method. Then proteins were classified by Gene Ontology annotation based on three categories: biological process, cellular component, and molecular function.Gene Ontology (GO) annotation proteome was derived from the UniProt-GOA database .A special antibody was ordered from PTM-Biolab Co., Ltd. an antigen. Specific antibody response was examined with immunohistochemistry staining.Myocardial tissue and AC16 cells were homogenized in an ice-cold lysis buffer. After homogenization, the lysates were centrifuged. The supernatant was saved and separated by electrophoresis on SDS-PAGE and transferred onto polyvinylidene difluoride-plus membranes. After blocking buffer, the immunoblots were probed with anti-T50 phosphorylation of Cytc, anti-Cytc, anti-Cleaved Caspase-3, anti-Caspase-3, anti-Cleaved Caspase-9, anti-Caspase-9, anti-BCL-2, anti-BCL-XL, anti-Bax, and anti-GAPDH antibodies overnight at 4\u00b0 C, followed by incubation with fluorescent-conjugated secondary antibodies at room temperature for 1 hour.in situ by the use of 3, 3 diaminobenzidine kit .Paraffin sections made from young and aged mouse hearts were used for immunohistochemistry assays to detect protein expression levels of T50 phosphorylation of Cytc proteins. In accordance with the manufacturer\u2019s instruction, tissue sections stained immunohistochemically are determined separately by two pathologists using the indirect streptavidin peroxidase method. The primary antibodies against T50 and horseradish peroxidase-conjugated IgG were used in this study. Then, the proteins were visualized in vitro, we generated T50E for constant phosphorylation and T50A for non-phosphorylation. Three sequences of WT, T50E and T50A are as below. WT:ATGGGTGATGTTGAGAAAGGCAAGAAGATTTTTATTATGAAGTGTTCCCAGTGCCACACCGTTGAAAAGGGAGGCAAGCACAAGACTGGGCCAAATCTCCATGGTCTCTTTGGGCGGAAGACAGGTCAGGCCCCTGGATACTCTTACACAGCCGCCAATAAGAACAAAGGCATCATCTGGGGAGAGGATACACTGATGGAGTATTTGGAGAATCCCAAGAAGTACATCCCTGGAACAAAAATGATCTTTGTCGGCATTAAGAAGAAGGAAGAAAGGGCAGACTTAATAGCTTATCTCAAAAAAGCTACTAATGAGTAA. T50E:ATGGGTGATGTTGAGAAAGGCAAGAAGATTTTTATTATGAAGTGTTCCCAGTGCCACACCGTTGAAAAGGGAGGCAAGCACAAGACTGGGCCAAATCTCCATGGTCTCTTTGGGCGGAAGACAGGTCAGGCCCCTGGATACTCTTACGAGGCCGCCAATAAGAACAAAGGCATCATCTGGGGAGAGGATACACTGATGGAGTATTTGGAGAATCCCAAGAAGTACATCCCTGGAACAAAAATGATCTTTGTCGGCATTAAGAAGAAGGAAGAAAGGGCAGACTTAATAGCTTATCTCAAAAAAGCTACTAATGAGTAA. T5OA:ATGGGTGATGTTGAGAAAGGCAAGAAGATTTTTATTATGAAGTGTTCCCAGTGCCACACCGTTGAAAAGGGAGGCAAGCACAAGACTGGGCCAAATCTCCATGGTCTCTTTGGGCGGAAGACAGGTCAGGCCCCTGGATACTCTTACGCCGCCGCCAATAAGAACAAAGGCATCATCTGGGGAGAGGATACACTGATGGAGTATTTGGAGAATCCCAAGAAGTACATCCCTGGAACAAAAATGATCTTTGTCGGCATTAAGAAGAAGGAAGAAAGGGCAGACTTAATAGCTTATCTCAAAAAAGCTACTAATGAGTAA. With these sequences, construct lentivirus vectors, purchased from Shanghai Yibeirui Bio-pharmaceutical Technology Co., Ltd. .Three sequences of WT, T50E and T50A were synthesized after codon optimization, purchased from Sangon Biotech Co., Ltd. . To study the effect of T50 phosphorylation Human adult ventricular cardiomyocyte cell line AC16 was purchased from the American Type Culture Collection . Cells were cultured in Dulbecco\u2019s modified Eagle\u2019s medium containing 10% fetal bovine serum and 1% penicillin/streptomycin in a humidified incubator with 5% CO2 at 37\u00b0 C. In this study, four types of lentiviruses were used to infect AC16 cells. Infection efficiency was monitored by fluorescence microscopy 48 hours post lentivirus infection.In vitro cardiomyocyte H/R modelSeventy-two hours after cardiomyocyte transfection, all groups were treated with hypoxia (95% N2 and 5% CO2) for 6 hours , followed by normoxia for 2 hours.Cardiomyocytes were cultured directly upon coverslips. TUNEL staining was performed per the manufacturer\u2019s instructions . Total nuclei were stained by DAPI . Apoptotic index was determined via blinded manner.After incubation for two days, the harvested cells were used for apoptotic determination by two methods. First, the apoptotic rate of transfected cells was evaluated by using Annexin V APC/PI apoptosis detection kit (KeyGEN) by following the manufacturer\u2019s instruction and then analyzed using FACScan. All tests are carried out in triplicate. Second, by following the manufacturer\u2019s instructions, Caspase-9 and Caspase-3 levels were detected utilizing human Caspase-9 ELISA kit (BC3890), Caspase-3 ELISA kit (BC3830) and Caspase-8 ELISA kit (BC3880) from Beijing Solarbio Science and Technology Co., Ltd. .The data were analyzed with Prism 8.0 . All values in the text and figures are presented as mean \u00b1 SD. Statistical differences were determined by Student\u2019s t-test for comparison between 2 groups and ANOVA followed by Bonferroni multiple comparison test for comparison among \u22653 groups. Probabilities of.05 or less were considered statistically significant.The data used to support the findings of this study are available from the corresponding author upon request.in vivo or clinical significance should be explored in the future.In this study, phosphoproteomics were performed only in myocardial tissue from young and old mice, which reduces the targeting of the experiment. The relationship between T50 phosphorylation and mitochondrial release or caspase 9 interaction will be explored in the next step. In addition, the functional impact of Cytc T50 phosphorylation"} +{"text": "Early detection of tumor cells by identifying universal Tumor Associated Antigens (TAA) can drastically change our diagnostic, theranostic and therapeutic possibilities to cure cancer. Human Telomerase Reverse Transcriptase (hTERT), a hallmark of cancer, could act as an optimal TAA candidate. Here we report about the development of a monoclonal antibody against hTERT peptide (\u03b1-hTERT mAb) presented on the surface of cancer cells and its possible applications as a pan-cancer marker. Liquid biopsies, an innovative tool in precision oncology, comprising the noninvasive analysis of circulating tumor-derived material to counteract limitations associated with tissue biopsies. Within the tumor circulome, the US Food and Drug Administration already approved the use of circulating tumor cells (CTCs) as valid liquid biopsies. However, currently CTCs are being trapped using antibodies against specific cancer types, with anti EpCAM as the most common antibody, directed mainly against solid tumors. Moreover, the precision medicine approach is based on specific cancer type directed antibodies. Our novel mAb against the hTERT 16-mer peptide, corresponding to amino acids 611\u2013626, is capable of detecting various types of cancer cells both in vitro and ex vivo from tumors of patients with either hematological or solid tumors. This antibody does not bind to normal lymphocytes cells. Cleavage of our antibody to F(ab\u2019)2 fragments increased its binding specificity to the tested cancer cells. Future studies may point to the use of this antibody in the procedure of capturing CTCs. Therefore, their isolation should be very specific [We are in the midst of major advances in understanding cancer biology and in development of rationalized biological drugs against specific targets in cancer cells . Howeverspecific .The most common strategy for capturing CTCs is based on the expression of specific markers on their membrane. By using the EpCAM and CK markers, the commercial technology of CellSearch enables the isolation of epithelial derived tumor cells by magnetic based antibodies ,6,7. AltIn order to overcome these drawbacks, new isolation strategies were being developed based on differences in their properties such as size, adherence, density, electric charge of tumor versus normal cells ,15,16,17Currently, there is no validated universal marker that enables a successful identification of large variety of CTCs as an early diagnosis strategy. Here we describe our approach using telomerase peptide presented on the membrane of all cancer cells for the development of an anti-telomerase antibody-based tool for pan cancer cells CTC isolation. Telomerase is the hallmark of the cancer cell. It is highly expressed in almost all types of cancer cells and is repressed in most somatic cells . A panelThe specificity of the newly synthesized \u03b1-hTERT mAb (designed as \u03b1-hTERT) was tested by assessing its ability to bind specifically the 16-amino acid telomerase peptide of which it was designed against (not shown). The binding of the newly synthesized \u03b1-hTERT mAb to various cancer cell lines and fibroblasts (non-cancer cells) was tested using a flow cytometry. The following cells were assayed: Jurkat (T cell leukemia), MCF-7 (breast adenocarcinoma), U266 (multiple myeloma), MEL (melanoma), JeKo-1 (mantle cell lymphoma), A549 (lung carcinoma). Fibroblast cells served as a negative control. The ability of the antibody to bind these cells is presented in The binding of \u03b1-hTERT mAb to cancer cells isolated from the peripheral blood of patients with CLL (ex vivo) was tested as described in Materials and Methods. A total of 57 blood samples at different stages of the disease , including primary diagnosed and treated, were tested. The binding to 4 representative blood samples of CLL and 4 solid tumors samples of different origin are shown in The lack of \u03b1-hTERT mAb\u2019s binding to normal lymphocytes is depicted in 5 ovarian tumors and 9 ascites samples from ovary cancer patients were analyzed. The binding of \u03b1-hTERT mAb to PBMCs of a patient with Merkel carcinoma is presented in Ex vivo binding of the \u03b1-hTERT mAb to cells obtained from three patients with breast cancer is shown in The In addition, we were able to detect the binding of the \u03b1-hTERT mAb to a small subset of cells from two patients with lung carcinomas as presented by dot plots on In order to optimize the binding specificity of the \u03b1-hTERT mAb to the TERT antigen, the F(ab\u2019)2 fragment was cleaved off using ficin. The F(ab\u2019)2 fragment was detected by gel electrophoresis (SDS-PAGE), and its binding to CLL cells was analyzed by flow cytometry. The results of the binding assays of the F(ab\u2019)2 fragment obtained from two different preparations are demonstrated in SEQ ID #15\u2019-DVVMTQTPLTLSVTIGQPASISCKSSQNLLYSDGKTYLNWLLQRPGQSPKRLIYLVSKVDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYFCWQGTHLPYTFGGGTKLEIK-3\u2019SEQ ID #25\u2019VQLQQSGAELVRPGASVTLSCKASGYIFTDYEKHWVKQTPVHGLEWIGAIDPESGSTVYNQRFKGKATLTADKSSGTAYMELRSLTSEDSAVYFCFLLRLFAYWGQGTLVTVSA -3\u2019The amino acid sequences of the proteins comprising the light chain of the anti-hTERT mAb was assessed by Edman degradation, and the sequencing of the heavy chain was performed using an in-gel protein digestion followed by liquid chromatography-mass spectrophotometry (LC-MS) procedures, as described in Materials and Methods. The light chain and heavSEQ ID #35\u2019-GATGTTGTGATGACCCAGACTCCACTCACTTTGTCGGTGACCATTGGACAACCAGCCTCCATCTCTTGCAAGTCAAGTCAGAACCTCTTATATAGTGATGGAAAGACATATTTGAATTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAAGTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTTTTGCTGGCAAGGTACTCATCTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAGAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGTGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACATTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATCGTCAAGAGCTTCAACAGGAATGAGTGTTAA-3\u2019SEQ ID #45\u2019-caggtgCAGCTGCAGCAGTCTGGGGCTGAACTGGTGAGGCCTGGGGCTTCAGTGACGCTGTCCTGCAAGGCTTCGGGCTACATATTTACTGATTATGAAAAGCACTGGGTGAAGCAGACACCTGTGCATGGCCTGGAGTGGATTGGAGCTATTGATCCTGAAAGTGGTAGTACTGTCTACAATCAGAGATTCAAGGGCAAGGCCACACTGACTGCAGACAAATCTTCCGGCACAGCCTACATGGAACTCCGCAGCCTGACATCTGAGGATTCTGCCGTCTATTTCTGCTTTTTACTACGGCTATTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCC-3\u2019The nucleic acid sequencing was performed using the degenerate primer-based sequencing technique described in Materials and Methods. The light chain and heavy chain nucleic acids sequences of \u03b1-hTERT designated herein as SEQ ID #3 and SEQ ID #4, respectively, are:SEQ ID #65\u2019-GATGTTGTGATGACCCAGACTCCACTCACTTTGTCGGTGACCATTGGACAACCAGCCTCCATCTCTTGCAAGTCAAGTCAGAACCTCTTATATAGTGATGGAAAGACATATTTGAATTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAAGTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTTTTGCTGGCAAGGTACTCATCTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTC-3\u2019The variable region of the light (VL) chain comprises the nucleotide sequence of SEQ ID #6:Many signal transduction pathways which mediate malignant transformation have been deciphered. Accordingly, new efficient anti-cancer rationalized drugs were developed . HoweverRecently, a novel technology has been developed for this purpose which is based on liquid biopsies . Liquid The isolation of CTCs is evolving as a promising approach for genetic analysis and therapeutic decisions in cancer and as a possible tool for detection of tumors. The available methods for isolation of CTCs are specific for various types of cancer and therefore the hunting of these CTCs is done by binding to a specific antibody which serves as a cancer marker, which varies among the different malignant cells as well as lacks in others. Herein we describe a somewhat novel approach in which we use the peptide of telomerase, the hallmark of cancer cells, as a bait to trap all cancer cell types based on the definition of the hTERT as a pan-cancer marker . This deTelomerase containing cells present on their membrane telomerase driven peptides. Our strategy is based on one of them, termed GV1001, a 16 amino acid peptide (EARPALLTSRLRFIPK) which was used to construct an antibody for the identification of cancer cells expressing telomerase . Our conSince the \u03b1-hTERT antibody is present specifically on cancer cells membrane, it was shown to be successfully used as a cancer related antigen for developing anti-cancer vaccination in several studies to treat advanced pancreatic cancer, non-small cell lung cancer, melanoma, and other cancers ,30,31,32Next, we showed that CLL cells obtained from patients differing in their CLL cells concentrations, bound our antibody. When we labelled the cells firstly with our antibody and only then with \u03b1CD19 and \u03b1CD5 as CLL markers, we got similar results, thus emphasizing the capability of the antibody to recognize and bind CLL cells . More imOur study has several limitations. First, hTERT is also expressed in actively dividing normal somatic cells, including small intestine, colon, lymph nodes and also hematopoietic stem cells, although to a much lesser extent compared to malignant cells ,34. In t). The fact that the extent of the antibody binding differed in various cell lines may stem from the different expression of the enzyme in these cancer cells. We have subsequently sequenced our specific antibody and provide herein the full sequence of both heavy and light chains of it.Second, the specificity of our mAb was evaluated by a parallel assessment of the binding of our mAB to ex vivo samples obtained from oncological patients and from healthy volunteers. To calculate the specificity and sensitivity of our antibody, we currently collect more samples in order to analyze these parameters by a Receiver Operating Characteristic (ROC) curve in the coming future. Third, we did not report the efficacy of our antibody, since only in CLL cells and not in the cells originated from the solid tumors we could compare the extent of the hTERT antibody binding to the presence of CLL markers on the neoplastic cells. All in all, we have characterized an anti-telomerase specific mAb which was shown to be specific to telomerase positive cells, both in vitro and ex vivo . The facFinally, to improve the specificity of the \u03b1-hTERT mAb we have cleaved it to F(ab\u2019)2 fragment. As expected, the cleaved F(ab\u2019)2 fragment bound cancer cells specifically ex vivo , in lineThese results show that our anti-hTERT antibody-based system is able to identify and isolate circulating tumor cells of various origin.One major pitfall in our study might have been that the antibody would not identify all cancer cells expressing telomerase and therefore the number of isolated CTCs will not be maximal; This point already proven not to be a problem, as our antibody is highly sensitive and specific for cancer cells. However, the advantages in the development of this \u201cCTC trapping\u201d anti \u201cpersonalized medicine\u201d based approach are clear: it is an easy cheap and noninvasive one.Hopefully the approach that we developed will be thoroughly explored in future studies which may result in the development of a more beneficial method to trap CTCs for the benefit of patients with cancer.2. Jurkat and MEL cell media were supplemented with 10% FBS, U-266 was grown in the presence of 15% FBS and JeKo-1 with 20% FBS. Fibroblasts were propagated in DMEM with 20% FBS supplemented with L-glutamine and penicillin/streptomycin/nystatin.Cells were grown under standard growth conditions. The following cells were used: Jurkat (T cell leukemia), MCF-7 (breast adenocarcinoma), U266 (multiple myeloma), MEL (melanoma), JeKo-1 (mantle cell lymphoma), A549 (lung carcinoma). Human mononuclear cells (MNCs) from healthy volunteers and human fibroblasts served as a negative control. All cell lines were originated from the American Type Culture Collection (ATCC). U-266, Jurkat, JeKo-1 and MEL were cultured in RPMI-1640, A549 and MCF-7 in DMEM supplemented with 10\u201320% fetal bovine serum (FBS), 100 units/mL L-glutamine and 1% penicillin/streptomycin/nystatin at 37 \u00b0C with 5% COPatients\u2019 samples were obtained upon consent via protocol # 0038-12 approved by the IRB of Rabin Medical Center.Peripheral blood samples were collected and stored at room temperature and processed within 24 h of the collection. All participants signed an informed consent. Peripheral blood mononuclear cells (PBMCs) were isolated from the blood samples using standard protocol with Lymphoprep .The hTERT peptide GV1001, EARPALLTSRLRFIPK, was synthesized by Genemed Synthesis . Hybridomas were produced by ProteoGenix . The Screening of the various antibodies containing supernatants was performed by ELISA and Flow Cytometry. The antibodies and isotype controls for flow cytometry were purchased from BioXCell Company .F(ab\u2019)2 fragments of \u03b1-hTERT mAb were prepared according to the protocol provided by Pierce mouse IgG1 Fab and F(ab\u2019)2 preparation kit . The fragments were generated by using ficin in the presence of cysteine and analyzed by polyacrylamide gel electrophoresis (PAGE).The specificity of monoclonal antibodies (mAbs) to the hTERT peptide was assessed by sandwich ELISA, using a standard protocol, wherein the tested mAbs binds to the hTERT peptide and not to the carrier protein (KLH) immobilized to the assay plate. Unbound antibodies were removed by washing with PBS. Subsequently, the bounded antibodies were detected by horse radish peroxidase (HRP)-conjugated anti-mouse IgG antibody and a 3,3\u2032,5,5\u2032-tetramethylbenzidine (TMB) substrate. The optical density was measured at 450 nm by microplate reader .The binding of the \u03b1-hTERT antibodies to a panel of cancer cells from patients was assessed by flow cytometry analysis. Blood, tumor and ascites samples were first stained with primary \u03b1-hTERT peptide mAb or isotype control, then incubated with FITC or AF647-conjugated \u03b1-mouse secondary Ab for the detection of hTERT positive cells.Chronic lymphocytic leukemia (CLL) cells were marked as CD19+/CD5+ ; ovarian cancer cells were detected by following the marker CCCR5. Antibodies were purchased from ENCO, Jackson, Sigma and Abcam, CA, GB).All cells were prepared for the flow cytometry analysis according to a routinely used procedure. Cell lines: floating cells collected from the growth media by centrifugation, adherent cells were trypsinized. Peripheral blood mononuclear cells (PBMCs) obtained from whole blood were isolated by Lymphoprep .The flow cytometry data was acquired using a BD FACS Calibur flow cytometer , acquisition and analysis were performed using Cell Quest Pro software .PBMCs were centrifuged, the pellet was resuspended in PBS +2% FBS and equally split into tubes. Cells were subsequently stained with \u03b1-hTERT or isotype mAb, washed with Phosphate Buffer Saline (PBS) and incubated with fluorescently labeled secondary anti-mouse Ab . Following washing cells were stained with tumor markers antibodies and re-suspended in PBS for acquisition on a flow cytometer. The same setup and compensation were used for all CLL samples. Cell debris and aggregates were excluded on forward scatter vs. side scatter. Data is presented by dot plots, histograms and graphs.The sequencing of the light chain of the antibody was performed using Edman degradation, in gel protein digestion and liquid chromatography-mass spectrophotometry (LC-MS) conducted by the Mass Spectrometry (MS) biological services unit of the Weizmann Institute of Science, Rehovot.The heavy chain of the purified antibody was cleaved by trypsinization prior to LC-MS analysis.The nucleotide sequence was obtained by using degenerated primers based on predicted sequences designed at the Tel Aviv University. The Kapa HiFi HotStart PCR was used for the PCR reaction and the procedure was performed according to routinely used protocol. The nucleic acid sequencing was performed by the Instrumentation and Service Center at Tel Aviv University.Immunofluorescence analysis was conducted as described previously . Basical"} +{"text": "RNA polyadenylation plays a central role in RNA maturation, fate, and stability. In response to developmental cues, polyA tail lengths can vary, affecting the translation efficiency and stability of mRNAs. Here we develop Nanopore 3\u2032 end-capture sequencing (Nano3P-seq), a method that relies on nanopore cDNA sequencing to simultaneously quantify RNA abundance, tail composition, and tail length dynamics at per-read resolution. By employing a template-switching-based sequencing protocol, Nano3P-seq can sequence RNA molecule from its 3\u2032 end, regardless of its polyadenylation status, without the need for PCR amplification or ligation of RNA adapters. We demonstrate that Nano3P-seq provides quantitative estimates of RNA abundance and tail lengths, and captures a wide diversity of RNA biotypes. We find that, in addition to mRNA and long non-coding RNA, polyA tails can be identified in 16S mitochondrial ribosomal RNA in both mouse and zebrafish models. Moreover, we show that mRNA tail lengths are dynamically regulated during vertebrate embryogenesis at an isoform-specific level, correlating with mRNA decay. Finally, we demonstrate the ability of Nano3P-seq in capturing non-A bases within polyA tails of various lengths, and reveal their distribution during vertebrate embryogenesis. Overall, Nano3P-seq is a simple and robust method for accurately estimating transcript levels, tail lengths, and tail composition heterogeneity in individual reads, with minimal library preparation biases, both in the coding and non-coding transcriptome. Nano3P-seq presents a nanopore-based sequencing tool to profile polyA-tailed and non-polyA-tailed transcripts, as well as capture polyA tail length and composition. Polyadenylation of RNA is one such modification, which is known to affect the stability and translation efficiency of the RNA molecule4 and to play an essential role in determining the fate of RNA molecules in a wide range of biological processes6.RNA molecules are subject to multiple co- and post-transcriptional modifications, shaping them to their final mature form6. Indeed, in the first hours after fertilization, vertebrate embryos undergo major cellular reprogramming, a process known as the maternal-to-zygotic transition (MZT)7. During the MZT, maternally inherited RNA and proteins are responsible for activation of the zygotic genome and are later replaced by the zygotic program9. Because the MZT begins in a transcriptionally silent embryo, this transition relies heavily on post-transcriptional regulatory mechanisms7, including modulation of the polyadenylation status of the RNA molecules10. Therefore, characterizing the dynamics of RNA polyadenylation is key to understanding how these modifications regulate the fate and function of RNA molecules.One context in which polyadenylation has been shown to play a major role in determining RNA fate and decay is vertebrate embryogenesis11. While these methods have been successfully employed to characterize the dynamics of polyA tail lengths in various contexts, they have several caveats: (i) they provide a limited perspective on isoform\u2013tail relationships owing to the short-read-length nature of NGS-based technologies; (ii) they do not provide single-molecule resolution; (iii) they are severely affected by PCR amplification biases; and (iv) they can only measure tail lengths that are shorter than the read length.In the past few years, several transcriptome-wide methods have become available for studying the dynamics of polyadenylated tails (polyA tails) based on next-generation sequencing (NGS), such as PAL-seq or TAIL-seq13. To sequence native RNAs using dRNA-seq, polyA-tailed RNA molecules are ligated to a 3\u2032 adapter that contains an oligo(dT) overhang. Consequently, dRNA-seq libraries capture the full-length polyA tail; however, ligation occurs only on RNA molecules that anneal to the oligo(dT) overhang, thus exclusively capturing polyA transcripts with tail lengths greater than 10 nucleotides. A variation of this method consisting of in vitro poly(I)-tailing the transcriptome before library preparation has been proposed for studying nascent RNAs using dRNA-seq15, thus capturing both polyadenylated and non-polyadenylated mRNAs. However, major limitations to this variation include low sequencing yields compared with standard dRNA-seq (10\u201330%)14 and a lack of tools to distinguish poly(I) and poly(A) signals; therefore, polyA tail length information is lost in these datasets15. An alternative approach for studying the transcriptome using nanopore technologies is direct cDNA sequencing (dcDNA-seq), but this approach cannot sequence the polyA\u2212 transcriptome, nor can it capture polyA tail length information. Overall, both dRNA-seq and dcDNA-seq nanopore library preparation protocols are limited to the sequencing of polyadenylated transcripts and thus cannot provide a comprehensive view of both polyadenylated and deadenylated RNA molecules, in addition to being unable to capture RNA molecules with other types of RNA tails .To overcome these limitations, the direct RNA sequencing (dRNA-seq) platform offered by Oxford Nanopore Technologies (ONT) has been proposed as a means to study both the transcriptome and polyA tail lengths simultaneouslyHere we present a novel method that employs nanopore sequencing to simultaneously obtain per-isoform transcriptome abundance and tail lengths in full-length individual reads, with minimal library preparation steps, which we term Nanopore 3\u2032-end-capture sequencing (Nano3P-seq). Notably, Nano3P-seq uses template switching to initiate reverse transcription and, therefore, does not require 3\u2032 end adapter ligation steps, PCR amplification, or second-strand cDNA synthesis. We demonstrate that Nano3P-seq can capture any type of RNA molecule regardless of its 3\u2032 sequence, including polyA-tailed and non-tailed RNA. Moreover, we show that Nano3P-seq can accurately quantify RNA abundances in both the coding and non-coding transcriptome, as well as quantify the polyA tail lengths and tail composition of individual RNA molecules in a highly reproducible manner.+ RNA molecules and per-isoform (Pearson\u2019s R\u2009=\u20090.97) 93) Fig. and per-Next, we employed Nano3P-seq to examine the RNA dynamics that occur during the maternal-to-zygotic transition (MZT) at single-molecule resolution Fig. . To this8, with a decay of mRNA genes previously reported to have a \u2018maternal decay mode\u2019 . PolyA tail length estimates were highly reproducible across independent biological replicates sequenced in independent flow cells for all three time points studied (R\u2009=\u20090.85\u20130.95) Fig. . We obse95) Fig. , in agre21. We observed that the three groups of mRNAs that are known to decay showed a significant decrease in mRNA abundance , as previously describednce Fig. , as expence Fig. . SpecifiA major feature that distinguishes nanopore sequencing from NGS is its ability to produce long reads, allowing RNA polyadenylation dynamics to be studied at the isoform level. Therefore, we wondered whether Nano3P-seq could identify differentially polyadenylated transcript isoforms during the MZT.P\u2009<\u20090.05) during the MZT. Notably, we observed that analyses at the per-gene level often revealed a different picture compared with analyses at per-isoform level. For example, in khdrbs1a and syncrip, per-isoform analysis revealed opposite tail length dynamics among isoforms during the MZT, with one isoform decreasing and another isoform increasing in polyA tail length as the MZT progressed of analyzed transcripts varied significantly in polyA tail length of analyzed genes presented significant differences in their polyA tail lengths across isoforms Fig. . Altoget34. Some modifications occur in base positions that are involved in Watson\u2013Crick (WC) base pairing, causing a disruption during reverse transcription. Consequently, these modifications can be seen as increased \u2018errors\u2019 and drop-off rates in RNA-seq datasets38. One example is the hypermodified base m1acp3\u03a8, which is present in the eukaryotic small subunit (SSU) rRNA39 crucial for the final processing steps of precursor rRNA (pre-rRNA) into mature SSU rRNA41.RNA molecules are decorated with chemical modifications, which are essential for the stability, maturation, fate, and function of the RNA1acp3\u03a8-modified site was very high in mature rRNAs but not present in pre-rRNAs, suggesting that this modification is only present in mature rRNA populations. The presence of the hypermodification was also accompanied by a drop-off in sequencing coverage at the m1acp3\u03a8-modified site, in agreement with its ability to disrupt the Watson\u2013Crick base pairing. Analysis of the \u2018error\u2019 signatures along the SSU transcripts showed that this position was the only one position to change between precursor and processed rRNA molecules at this site in the pre-rRNAs, which also causes increased \u2018errors\u2019 in dRNA-seq datasets, but not in Nano3P-seq datasets and analyzed the sequencing error patterns in the two populations Fig. . We obseles Fig. . These r44. However, these methods cannot be used to detect tail modifications among the vast majority of tail nucleotides, as Illumina sequencing quality strongly deteriorates in homopolymeric stretches and with increased read length. By contrast, Pacific BioSciences (PacBio)-based approaches such as FLAM-seq can sequence through the entire tail, however, they cannot unambiguously identify 3\u2032 terminal modifications45.Recent works using TAIL-seq have reported that a number of terminal modifications in polyA tails, such as polyuridine stretches, play a role in mRNA decayTherefore, we explored whether Nano3P-seq could accurately identify nucleotide composition variations within polyA tails. To this end, we designed synthetic cDNA molecules with polyA tails ending with A (30A), U (1U), UUU (3U), UUUUU (5U), CCCCC (5C), and GGGGG (5G), as well as a polyA tail that contained several internal G nucleotides at fixed positions (IntG) Fig. ; MethodsNext, we analyzed the mRNA tail composition of zebrafish embryos from different developmental stages . Analysis of tail base abundance revealed that G was the most common non-A base in zebrafish mRNAs and that there was a significant decrease in non-A bases with progression of the MZT Fig. . Our ana46.Finally, we examined the relationship between tail length and the presence of non-A bases in the tails. Although the median tail length increases during the MZT Figs and 3g, + transcripts using oligo(dT) beads. Although these two approaches are often used interchangeably, their effects on the transcriptome composition are not equal. Nano3P-seq allows us to compare the effects of these two approaches on both the transcriptome composition and polyA tail length distribution. In terms of its effects on transcriptome composition, we found that ribodepletion captures a larger variety of RNA biotypes compared with polyA selection, including several non-polyA-tailed RNA biotypes, as expected ribodepletion of the sample using biotinylated oligonucleotides that are complementary to rRNAs or (ii) selective enrichment of polyAted Fig. . Howeverted Fig. , suggest12. In terms of sequencing output, the yields of Nano3P-seq runs were similar\u2014or slightly better\u2014to those observed in dRNA-seq runs, producing ~100,000\u2013200,000 reads in Flongle devices and ~500,000\u20132,000,000 in MinION devices, depending on the RNA input type and quality of the flowcell and TAIL-seq (Pearson\u2019s R\u2009=\u20090.19) on HeLa cell lines and to estimate polyA tail lengths . However, a major limitation of NGS-based methods is their inability to assign a given polyA tail length to a specific transcript isoform, which causes a loss of isoform-specific tail length information. In addition, NGS-based methods cannot measure tail lengths greater than the read length, thus biasing our view of polyA tail dynamics to those transcripts that display shorter tail lengths.In the past few years, a variety of NGS-based high-throughput methods have been developed to characterize the 3\u2032 ends of RNA molecules at a transcriptome-wide scale, including methods to reveal polyA tail sites , or molecules with polyA tails shorter than 10 nucleotides, thus biasing the view of the transcriptome toward polyadenylated molecules. Customized dRNA-seq methods involving in vitro poly(G/I)-tailing have been developed to overcome some of these limitations, but a lack of bioinformatic tools to distinguish polyI and polyA signals limits their applicability to study polyA tail length differences across transcripts in these datasets15. In addition, dRNA-seq requires 500\u2009ng RNA as input, whereas Nano3P-seq requires as little as 50\u2009ng, thus decreasing the required input material by tenfold. Nano3P-seq addresses the current limitations by offering a simple and robust solution for studying the coding and non-coding transcriptome simultaneously regardless of the presence or absence of polyA tails or 3\u2032 tail composition, without PCR or ligation biases, and with single-read and single-isoform resolution. Moreover, the use of thermostable group II intron reverse transcriptase (TGIRT) in the Nano3P-seq protocol not only maximizes the production of full-length cDNAs, but also ensures the inclusion of RNA molecules that are highly structured and/or modified, which would often not be captured (or their representation would be significantly biased) using standard viral reverse transcriptases57.Nanopore dRNA-seq has been proposed as an alternative long-read sequencing technology for studying polyA tail lengthsNano3P-seq also provides quantitative measurements of RNA abundances Fig. and captOverall, our work demonstrates that Nano3P-seq can simultaneously capture both non-polyA-tailed and polyA-tailed transcriptomes, making it possible to accurately quantify the RNA abundances and polyA tail lengths at per-read and per-isoform levels, while minimizing the amount of biases introduced during library preparation. These features set Nano3P-seq as a simple, potent, and robust method that can provide mechanistic insights into the regulation of RNA molecules and improve our understanding of mRNA tailing processes and post-transcriptional control.58 were in vitro transcribed using Ampliscribe T7-Flash Transcription Kit (Lucigen-ASF3507). Curlcake 2 was polyadenylated using Escherichia coli polyA Polymerase (NEB-M0276S). polyA-tailed RNAs were purified using RNAClean XP beads. The quality of the in vitro transcribed (IVT) products as well as the addition of polyA tail to the synthetic constructs was assessed using Agilent 4200 Tapestation were loaded on a 2.5% agarose gel and stained with GelRed . Product sizes determined using the GeneRuler 50-base-pair DNA ladder . Subsequently, tail- and gene-specific PCR products of mouse 16S rRNA were purified by gel-elution and sent for Sanger sequencing with the shared forward primer (5\u2032-GGTCGGTTTCTATCTATTTACGATTTCTC-3\u2032). Resulting chromatograms were analyzed using SnapGene (v.6.0.2). After confirming alignment to the reference sequence, the unclipped chromatograms were used to visualize 3\u2032 ends.Poly(A) Tail-Length Assay Kit was used according to the manufacturer\u2019s instructions. PCR products corresponding to the tail- and gene-specific primer combinations of mouse 16S rRNA and human Saccharomyces cerevisiae (strain BY4741) was grown at 30\u2009\u00b0C in standard YPD medium . Cells were then quickly transferred into 50-ml pre-chilled falcon tubes, and centrifuged for 5\u2009min at 3,000g in a 4\u2009\u00b0C pre-chilled centrifuge. Supernatant was discarded, and cells were flash frozen. Flash frozen pellets were resuspended in 700\u2009\u00b5l TRIzol (Life Technologies) with 350\u2009\u00b5l acid washed and autoclaved glass beads . The cells were disrupted using a vortex on top speed for 7 cycles of 15\u2009s (the samples were chilled on ice for 30\u2009s between cycles). Afterwards, the samples were incubated at room temperature for 5\u2009min and 200\u2009\u00b5l chloroform was added. After briefly vortexing the suspension, the samples were incubated for 5\u2009min at room temperature. Then they were centrifuged at 14,000g for 15\u2009min at 4\u2009\u00b0C and the upper aqueous phase was transferred to a new tube. RNA was precipitated with 2\u00d7 volume molecular grade absolute ethanol and 0.1\u00d7 volume sodium acetate . The samples were then incubated for 1\u2009h at \u221220\u2009\u00b0C and centrifuged at 14,000g for 15\u2009min at 4\u2009\u00b0C. The pellet was then washed with 70% ethanol and resuspended with nuclease-free water after air drying for 5\u2009min on the benchtop. Purity of the total RNA was measured with the NanoDrop 2000 Spectrophotometer. Total RNA was then treated with Turbo DNase (2\u2009\u03bcl enzyme for 50\u2009\u03bcl reaction of 200\u2009ng\u2009\u03bcl\u22121 RNA) at 37\u2009\u00b0C for 15\u2009min, with a subsequent RNAClean XP bead cleanup.2 and tissues were snap frozen in liquid nitrogen. Animals were kept on a 12:12\u2009h light:dark cycle and provided with water and food ad libitum.Experiments were performed with male mice aged between 8 and 10\u2009weeks. All mice were euthanized using COMus musculus) brain, we followed previously published protocols (10.17504/protocols.io.3fkgjkw) with minor changes. A quarter of a C57BL6/J mouse brain was used for this protocol, and all samples and reagents were kept on ice during the protocol. Brain tissue was mined with a razor blade into smaller pieces. Cold Nuclei EZ Lysis Buffer ) was added to the tissue in 1.5-ml eppendorf tube. The sample was homogenized using a 1-ml dounce (stroking ~10\u201320 times), and the homogenate was transferred into a 2-ml eppendorf tube. One milliliter of cold Nuclei EZ Lysis Buffer was added and mixed, followed by 4\u2009min incubation on ice. During the incubation, the sample was gently mixed a couple of times using a pipette. Homogenate was filtered using a 70-\u03bcm strainer mesh, and the flowthrough was collected in a polystyrene round-bottom FACS tube and subsequently transferred into a new 2-ml tube. The sample was centrifuged at 500g for 5\u2009min at 4\u2009\u00b0C and the supernatant was removed. The nuclei/mitochondria enriched sample was resuspended in another 1.5\u2009ml EZ Lysis buffer and incubated for 5\u2009min on ice. The sample was centrifuged at 500g for 5\u2009min 4\u2009\u00b0C and the supernatant was discarded (cytoplasm). Five-hundred microliters of nuclei wash and resuspension buffer ) was added to the sample and incubated for 5\u2009min without resuspending to allow buffer interchange. After incubation, 1\u2009ml nuclei wash and resuspension buffer was added and the sample was resuspended. The sample was centrifuged at 500g for 5\u2009min at 4\u2009\u00b0C. The supernatant was removed and only ~50\u2009\u03bcl was left. Using 1.4\u2009ml nuclei wash and resuspension buffer, the sample was resuspended and transferred to a 1.5-ml eppendorf tube. The last washing step was repeated and the pellet was resuspended in 300\u2009\u03bcL nuclei wash and resuspension buffer. RNA was extracted using TRIzol (Life Technologies) following the manufacturer\u2019s protocol.To isolate nuclear/mitochondrial-enriched RNA from the mouse (Danio rerio) embryos were obtained through natural mating of the TU-AB strain of mixed ages (5\u201318\u2009months). Mating pairs were randomly chosen from a pool of 60 males and 60 females allocated for each day of the month. Embryos and adult fish were maintained at 28\u2009\u00b0C.Wild-type zebrafish and total RNA was extracted using the manufacturer\u2019s protocol. Total RNA concentration was calculated by nanodrop.For polyA-selected RNA samples, polyadenylated RNAs were isolated with oligo(dT) magnetic beads according to the manufacturer\u2019s protocol and eluted in 30\u2009\u00b5l before nanodrop quantification.\u22121) to 37\u2009\u00b0C for hybridization. In the meantime, Dynabeads MyOne Streptavidin C1 beads were resuspended by carefully vortexing at medium speed. Eighty microliters of Dynabeads bead resuspension (10\u2009mg\u2009ml\u22121) was transferred into a tube, which then was placed on a magnetic rack. After aspirating the supernatant, 100\u2009\u03bcl of bead resuspension buffer was added to the sample and beads were resuspended in this buffer by agitating the tube. Sample was placed on a magnet and the supernatant was aspirated. This step was performed twice. Beads were then resuspended in 100\u2009\u03bcl bead wash buffer (0.1\u2009M NaCl) and placed on magnet to aspirate the supernatant. Beads were then resuspended in a 160\u2009\u03bcl depletion buffer . This suspension was then divided into two tubes of 80\u2009\u03bcl, which will be used consecutively. Twenty microliters of hybridized riboPOOL and total RNA was briefly centrifuged to spin down droplets and it was pipetted into the tube containing 80\u2009\u03bcl of beads in depletion buffer . The tube containing the mix was agitated to resuspend the solution well. Then the mix was incubated at 37\u2009\u00b0C for 15\u2009min, followed by a 50\u2009\u00b0C incubation for 5\u2009min. Immediately before use, the second tube containing 80\u2009\u03bcl of beads was placed on a magnetic rack and the supernatant was aspirated. After the incubation at 50\u2009\u00b0C, the first depletion reaction was placed on a magnet and the supernatant was transferred into the tube containing the other set of beads. The mix was incubated again at 37\u2009\u00b0C for 15\u2009min, followed by a 50\u2009\u00b0C incubation for 5\u2009min. After briefly spinning down the droplets, the mix was placed on a magnet for 2\u2009min. The supernatant was transferred into a different tube and cleaned up using RNA Clean & Concentrator-5 .Ribodepletion was performed on zebrafish total RNA using riboPOOL oligos following the manufacturer\u2019s protocol. In brief, 5\u2009\u03bcg total RNA in 14\u2009\u03bcl was mixed with 1\u2009\u03bcl resuspended riboPOOL oligos, 5\u2009\u03bcl hybridization buffer and 0.5\u2009\u03bcl SUPERase\u2022In RNase Inhibitor . The mix was incubated for 10\u2009min at 68\u2009\u00b0C, followed by a slow cool down (3\u2009\u00b0C\u2009minMycoplasma-tested in-house) were cultured in DMEM high glucose with 10% fetal bovine serum supplement . Pellets were obtained from 6 million cells and 1\u2009ml TRIzol (Life Technologies) was added to each pellet, Sample was incubated at room temperature for a few minutes after adding 200\u2009\u03bcl chloroform and vortexing briefly. After the incubation, the solution was centrifuged at 4\u2009\u00b0C with 14,000g. Aqueous phase from the previous step was transferred to another eppendorf tube and equal amount of absolute ethanol was added to the solution. This suspension was then transferred to an RNeasy Mini Spin Column from RNeasy Mini Kit , and total RNA was isolated following the manufacturer\u2019s protocol. Then, polyadenylated RNAs were isolated with Dynabeads Oligo(dT) 25 according to the manufacturer\u2019s protocol and eluted in 20\u2009\u00b5L. Concentration and quality of RNA was evaluated using a Nanodrop 2000 Spectrophotometer and an Agilent 4200 TapeStation, respectively.HeLa cell lines in order to pre-anneal the oligos. Then, 50\u2009ng RNA was mixed with 2\u2009\u00b5l pre-annealed R_RNA\u2009+\u2009D_DNA oligo, 1\u2009\u03bcl 100\u2009mM dithiothreitol, 4\u2009\u00b5l 5\u00d7 TGIRT Buffer , 1\u2009\u00b5l RNAse Inhibitor Murine , 1\u2009\u00b5l TGIRT (InGex) and nuclease-free water up to 19\u2009\u00b5l. We should note that if 50\u2009ng are used as input, only 1\u2009\u00b5l TGIRT is needed, whereas if 100\u2009ng is used as input, 2\u2009\u00b5l TGIRT enzyme is needed. The reverse transcription mix was initially incubated at room temperature for 30\u2009min before adding 1\u2009\u00b5l 10\u2009mM dNTP mix. Then the mix was incubated at 60\u2009\u00b0C for 60\u2009min and inactivated by heating at 75\u2009\u00b0C for 15\u2009min before moving to ice. RNAse Cocktail was added to the mix to digest the RNA, and the mix was incubated at 37\u2009\u00b0C for 10\u2009min. The reaction was then cleaned up using 0.8\u00d7 AMPure XP Beads . To be able to ligate the sequencing adapters to the first cDNA strand, 1\u2009\u00b5l 10\u2009\u00b5M CompA_DNA (5\u2032 GAAGATAGAGCGACAGGCAAGTGATCGGAAGA 3\u2032) was annealed to the 15\u2009\u00b5l cDNA in a tube with 2.25\u2009\u00b5l 0.1\u2009M Tris pH 7.5, 2.25\u2009\u00b5l 0.5\u2009M NaCl and 2\u2009\u00b5l nuclease-free water. The mix was incubated at 94\u2009\u00b0C for 1\u2009min and the temperature was ramped down to 25\u2009\u00b0C (\u22120.1\u2009\u00b0C\u2009s\u22121) to anneal the complementary to the first-strand cDNA. Then, 22.5\u2009\u00b5l first-strand cDNA was mixed with 5\u2009\u00b5l Adapter Mix (AMX), 22.5\u2009\u00b5l Rnase-free water and 50\u2009\u00b5l Blunt/TA Ligase Mix and incubated in room temperature for 10\u2009min. The reaction was cleaned up using 0.8\u00d7 AMPure XP beads, using WSB (Washing buffer) buffer for washing. The sample was then eluted in elution buffer and mixed with sequencing buffer and loading beads before loading onto a primed R9.4.1 flowcell. Libraries were run on either Flongle or MinION flow cells with MinKNOW acquisition software version v.3.5.5. A detailed step-by-step Nano3P-seq protocol is provided as a The protocol is based on the direct cDNA Sequencing ONT protocol (DCB_9091_v109_revC_04Feb2019), with several modifications to be able to perform TGIRT template switching. Before starting the library preparation, 1\u2009\u00b5l 10\u00b5M R_RNA (oligo: 5\u2032 rGrArArGrArUrArGrArGrCrGrArCrArGrGrCrArArGrUrGrArUrCrGrGrArArG/3SpC3/3\u2032) and 1\u2009\u00b5l 10\u2009\u00b5M D_DNA (5\u2032/5Phos/CTTCCGATCACTTGCCTGTCGCTCTATCTTCN 3\u2032) were mixed with 1\u2009\u00b5l 0.1\u2009M Tris pH 7.5, 1\u2009\u00b5l 0.5\u2009M NaCl, 0.5\u2009\u03bcl RNAse Inhibitor Murine and 5.5\u2009\u03bcl RNase-free water. The mix was incubated at 94\u2009\u00b0C for 1\u2009min and the temperature was ramped down to 25\u2009\u00b0C (\u22120.1\u2009\u00b0C\u2009s\u22121) to pre-anneal the oligonucleotides. Then, 50\u2009ng polyA-tailed RNA was mixed with 1\u2009\u00b5l pre-annealed VNP\u2009+\u2009CompA, 1\u2009\u03bcl 100\u2009mM dithiothreitol, 4\u2009\u00b5l 5\u00d7 TGIRT Buffer , 1\u2009\u00b5l RNAse Inhibitor Murine , 1\u2009\u00b5l TGIRT and nuclease-free water up to 19\u2009\u00b5l. The reverse transcription mix was initially incubated at room temperature for 30\u2009min before adding 1\u2009\u00b5l 10\u2009mM dNTP mix. Then the mix was incubated at 60\u2009\u00b0C for 60\u2009min and inactivated by heating at 75\u2009\u00b0C for 15\u2009min before moving onto ice. Furthermore, RNAse Cocktail was added to the mix to digest the RNA and the mix was incubated at 37\u2009\u00b0C for 10\u2009min. Then the reaction was cleaned up using 0.8\u00d7 AMPure XP Beads . To be able to ligate the sequencing adapters the the first strand, 1\u2009\u00b5l 10\u2009\u00b5M CompA was again annealed to the 15\u2009\u00b5l cDNA in a tube with 2.25\u2009\u00b5l 0.1\u2009M Tris pH 7.5, 2.25\u2009\u00b5l 0.5\u2009M NaCl and 2\u2009\u00b5l nuclease-free water. The mix was incubated at 94\u2009\u00b0C for 1\u2009min and the temperature was ramped down to 25\u2009\u00b0C (\u22120.1\u2009\u00b0C\u2009s\u22121) to anneal the complementary to the first-strand cDNA. Furthermore, 22.5\u2009\u00b5l first-strand cDNA was mixed with 5\u2009\u00b5l Adapter Mix (AMX), 22.5\u2009\u03bcl Rnase-free water and 50\u2009\u00b5l Blunt/TA Ligase Mix and incubated at room temperature for 10\u2009min. The reaction was cleaned up using 0.8\u00d7 AMPure XP beads, using WSB Buffer for washing. The sample was then eluted in elution buffer and mixed with sequencing buffer and loading beads before loading onto a primed R9.4.1 flowcell and run on a MinION sequencer with MinKNOW acquisition software version v.3.5.5.Some adjustments were made to the original Direct cDNA-Sequencing ONT protocol (SQK-DCS109), to be able to use TGIRT (InGex) as reverse transcription enzyme for nanopore sequencing, as this enzyme does not produce CCC overhang, which is typically exploited by the dcDNA-seq library preparation protocol Fig. . In brieA total of 12 synthetic cDNA standards were synthesized as ultramers by IDT (Integrated DNA Technologies) to assess the tail length estimation and tail composition quantification accuracy of Nano3P-seq.Synthetic cDNA standards designed to assess accuracy in tail length estimation:cDNA_pA_standard_0: /5Phos/CTTCCGATCACTTGCCTGTCGCTCTATCTTCGTAAATAGAAATAGACTAGCTCCACTTTTAAGAATTATTTATGCAATTAAATACATGGGTGACCAAAAGAGCGGGCGGATACACGCGTCACCACAAGCAGAATAAAAGGTAAACCTGAAATTGTTTTAACATAAAATGAAAAATGCTTGTTTGCAACCCTATATAGAAcDNA_pA_standard_15: /5Phos/CTTCCGATCACTTGCCTGTCGCTCTATCTTCTTTTTTTTTTTTTTTGTAAATAGAAATAGACTAGCTCCACTTTTAAGAATTATTTATGCAATTAAATACATGGGTGACCAAAAGAGCGGGCGGATACACGCGTCACCACAAGCAGAATAAAAGGTAAACCTGAAATTGTTTTAACATAAAATGAAAAATGCTTGTTTGcDNA_pA_standard_30: /5Phos/CTTCCGATCACTTGCCTGTCGCTCTATCTTCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGTAAATAGAAATAGACTAGCTCCACTTTTAAGAATTATTTATGCAATTAAATACATGGGTGACCAAAAGAGCGGGCGGATACACGCGTCACCACAAGCAGAATAAAAGGTAAACCTGAAATTGTTTTAACATAAAATGcDNA_pA_standard_60: /5Phos/CTTCCGATCACTTGCCTGTCGCTCTATCTTCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGTAAATAGAAATAGACTAGCTCCACTTTTAAGAATTATTTATGCAATTAAATACATGGGTGACCAAAAGAGCGGGCGGATACACGCGTCACCACAAGCAGAATAAAAGcDNA_pA_standard_90: /5Phos/CTTCCGATCACTTGCCTGTCGCTCTATCTTCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGTAAATAGAAATAGACTAGCTCCACTTTTAAGAATTATTTATGCAATTAAATACATGGGTGACCAAAAGAGCGGGCGGcDNA_pA_standard_120: /5Phos/CTTCCGATCACTTGCCTGTCGCTCTATCTTCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGTAAATAGAAATAGACTAGCTCCACTTTTAAGAATTATTTATGCAATTSynthetic cDNA standards designed to assess accuracy in tail composition analyses:cDNA_p29A_pU1_standard_30: /5Phos/CTTCCGATCACTTGCCTGTCGCTCTATCTTCATTTTTTTTTTTTTTTTTTTTTTTTTTTTTGTAAATAGAAATAGACTAGCTCCACTTTTAAGAATTATTTATGCAATTAAATACATGGGTGACCAAAAGAGCGGGCGGATACACGCGTCACCACAAGCAGAATAAAAGGTAAACCTGAAATTGTTTTAACATAAAATGcDNA_p27A_pU3_standard_30: /5Phos/CTTCCGATCACTTGCCTGTCGCTCTATCTTCAAATTTTTTTTTTTTTTTTTTTTTTTTTTTGTAAATAGAAATAGACTAGCTCCACTTTTAAGAATTATTTATGCAATTAAATACATGGGTGACCAAAAGAGCGGGCGGATACACGCGTCACCACAAGCAGAATAAAAGGTAAACCTGAAATTGTTTTAACATAAAATGcDNA_p25A_pU5_standard_30: /5Phos/CTTCCGATCACTTGCCTGTCGCTCTATCTTCAAAAATTTTTTTTTTTTTTTTTTTTTTTTTGTAAATAGAAATAGACTAGCTCCACTTTTAAGAATTATTTATGCAATTAAATACATGGGTGACCAAAAGAGCGGGCGGATACACGCGTCACCACAAGCAGAATAAAAGGTAAACCTGAAATTGTTTTAACATAAAATGcDNA_p25A_pC5_standard_30: /5Phos/CTTCCGATCACTTGCCTGTCGCTCTATCTTCGGGGGTTTTTTTTTTTTTTTTTTTTTTTTTGTAAATAGAAATAGACTAGCTCCACTTTTAAGAATTATTTATGCAATTAAATACATGGGTGACCAAAAGAGCGGGCGGATACACGCGTCACCACAAGCAGAATAAAAGGTAAACCTGAAATTGTTTTAACATAAAATGcDNA_p25A_pG5_standard_30: /5Phos/CTTCCGATCACTTGCCTGTCGCTCTATCTTCCCCCCTTTTTTTTTTTTTTTTTTTTTTTTTGTAAATAGAAATAGACTAGCTCCACTTTTAAGAATTATTTATGCAATTAAATACATGGGTGACCAAAAGAGCGGGCGGATACACGCGTCACCACAAGCAGAATAAAAGGTAAACCTGAAATTGTTTTAACATAAAATGcDNA_pA_internalG_standard_30: /5Phos/CTTCCGATCACTTGCCTGTCGCTCTATCTTCTTTTCTTTTCTTTTCTTTTTTTTTTTTTTTGTAAATAGAAATAGACTAGCTCCACTTTTAAGAATTATTTATGCAATTAAATACATGGGTGACCAAAAGAGCGGGCGGATACACGCGTCACCACAAGCAGAATAAAAGGTAAACCTGAAATTGTTTTAACATAAAATG18 with minimap2 with \u2018-ax splice -k14 -uf --MD\u2019 parameters59. For zebrafish dRNA-seq samples, reads were base-called with Guppy v.4.0. Base-called reads were first mapped to maternal and somatic zebrafish ribosomal RNA sequences taken from42 and then to the genome (GRCz11) with minimap259 with \u2018-ax splice -k14 -uf --MD\u2019 parameters. Mapped reads were intersected with ENSEMBL version 103 annotation (Danio_rerio.GRCz11.103.2.gtf) using bedtools v.2.29.1 intersect option60.dRNA-seq library preparations were prepared following manufacturer\u2019s recommendations, using 450\u2009ng (in the case of polyA-enriched RNA zebrafish run) or 500\u2009ng (in the case of in vitro transcribed \u2018sequins\u2019) as input material. Samples were sequenced in an R 9.4.1 MinION flowcell using a GridION sequencing device in the case of \u2018sequins\u2019, and in a R 9.4.1 PromethION flowcell using a PromethION sequencing device in the case of zebrafish RNA. For sequins, reads were base-called using stand-alone Guppy v.3.0.3 with default parameters and then the base-called reads were mapped to sequin sequences59 with the following \u2018-ax splice -k14 -uf --MD\u2019 parameters when mapping to genome and \u2018-ax map-ont -k14 --MD\u2019 when mapping to transcriptome, unless stated otherwise.All the Nano3P-seq runs were base-called and demultiplexed using stand-alone Guppy v.6.0.1 with default parameters. All runs were mapped using minimap258, and mapped reads were then intersected with annotations of Curlcake 1 and 2 sequences to filter out the incomplete reads using bedtools v.2.29.1 intersect option. For yeast total RNA, we first mapped the base-called reads to S. cerevisiae ribosomal RNAs and then mapped the rest of the reads to the S. cerevisiae genome (SacCer3). Mapped reads were then intersected with SacCer64 annotation exon ends, to filter out incomplete reads. For the analysis of nuclear/mitochondrial-enriched mouse brain RNA spiked in with sequins18, we first mapped the base-called reads to Mus musculus ribosomal RNAs and then mapped the rest of the rest of the reads to the M. musculus genome (GRCm38), supplemented with sequin chromosome (chrIS). Mapped reads were then intersected with ENSEMBL version 102 annotation (Mus_musculus.GRCm38.102.gtf) and sequin annotation (RNAsequins.v2.2.gtf) exon ends, to filter the incomplete reads. For zebrafish RNA (polyA-selected and ribodepleted), we first mapped the base-called reads to ribosomal RNAs and then mapped the rest of the reads to the genome (GRCz11). Mapped read starts were then intersected with ENSEMBL version 103 annotation (Danio_rerio.GRCz11.103.2.gtf) exon ends, to filter the incomplete reads. For HeLa mRNA, we first mapped the base-called reads to human ribosomal RNAs ; then, the rest of the reads (unmapped) were mapped to the Homo sapiens genome (GRCh38). Mapped read starts were then intersected with ENSEMBL version 107 annotation (Homo_sapiens.GRCh38.107.gtf) exon ends, to filter out incomplete reads . For cDNA standards, base-called reads were mapped to cDNA sequence reference using minimap2, except for the cDNA_pA_standard_120, which was mapped using bwa short-read aligner61 with the following parameters \u2018mem -xont2d\u2019. A different aligner was used for cDNA_pA_standard_120 because minimap2, which is a long-read mapping algorithm, did not yield any mapped reads for this standard owing to its short length62 (30 nucleotides once the pA tail length has been soft-clipped). We should note that the use of a different aligner should not affect the polyA tail length estimations of the reads, as these are done at the level of current intensity . We should note that fewer reads were base-called and mapped to cDNA_pA_standard_120, cDNA_pA_standard_90 and cDNA_pA_standard_60, in comparison to the other cDNA standards , base-called reads were mapped to Curlcake 1 and 2 sequenceshttps://github.com/ablab/IsoQuant) was used with Danio_rerio.GRCz11.103.2.gtf annotation using the following parameters \u2018--genedb gtf_file --complete_genedb --bam bam_file --data_type nanopore -o output\u2019. A complete step-by-step command line of the bioinformatic analysis done on Nano3P-seq datasets can be found in the GitHub repository . All reference sequences used to map the runs mentioned above are included in the Nano3P-seq GitHub repository.For the assignment of reads to isoforms, IsoQuant package ; rather, they estimate polyA tail lengths by comparing the relative duration of the current signal corresponding to the polyA tail region to the total duration of the sequenced read.For dRNA-seq reads, polyA tail length estimation was performed using NanoTail, a module from Master of Poreshttps://github.com/adnaniazi/tailfindr/tree/nano3p-seq). All code used to estimate polyA tail lengths and post-process Nano3P-seq data can be found at https://github.com/novoalab/Nano3P_Seq.For Nano3P-seq reads, polyA tail length estimation was performed using the Nano3P-seq version of tailfindR (https://github.com/rrwick/Porechop) with the following parameters \u2018--extra_end_trim 0 --end_threshold 50\u2019, to remove the adapter sequences. Because Porechop sometimes failed at removal of the adapter sequences, only reads containing more than 80% A bases in their tail composition were kept for downstream analyses, thus ensuring that untrimmed reads are not included in downstream analyses .Base-called reads mapped to the zebrafish genome and cDNA standards for tail composition quantification were first trimmed using the Porechop tool . Mice maintenance was approved by the Garvan/St Vincent\u2019s Hospital Animal Ethics Committee, in accordance with the guidelines of the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (Project No. 16/14 and 16/26). All animals were entered into the study in a randomized order and operators were blinded to genotype and treatments.Further information on research design is available in the Any methods, additional references, Nature Portfolio reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at 10.1038/s41592-022-01714-w.Supplementary InformationSupplementary Protocol, Supplementary Tables 1\u20133, and Supplementary Notes 1\u20132.Reporting SummaryPeer Review FileSupplementary SoftwareCode corresponding to TailfindR Nano3P-seq branch found in GitHub repository (attached as a separate zip file). This version incorporates the custom adapter sequences that are used in Nano3P-seq library preparation protocols."} +{"text": "The innate immune response controls the acute phase of virus infections; critical to this response is the induction of type I interferon (IFN) and resultant IFN-stimulated genes to establish an antiviral environment. One such gene, zinc finger antiviral protein (ZAP), is a potent antiviral factor that inhibits replication of diverse RNA and DNA viruses by binding preferentially to CpG-rich viral RNA. ZAP restricts alphaviruses and the flavivirus Japanese encephalitis virus (JEV) by inhibiting translation of their positive-sense RNA genomes. While ZAP residues important for RNA binding and CpG specificity have been identified by recent structural studies, their role in viral translation inhibition has yet to be characterized. Additionally, the ubiquitin E3 ligase tripartite motif-containing protein 25 (TRIM25) has recently been uncovered as a critical co-factor for ZAP\u2019s suppression of alphavirus translation. While TRIM25 RNA binding is required for efficient TRIM25 ligase activity, its importance in the context of ZAP translation inhibition remains unclear. Here, we characterized the effects of ZAP and TRIM25 RNA binding on translation inhibition in the context of the prototype alphavirus Sindbis virus (SINV) and JEV. To do so, we generated a series of ZAP and TRIM25 RNA binding mutants, characterized loss of their binding to SINV genomic RNA, and assessed their ability to interact with each other and to suppress SINV replication, SINV translation, and JEV translation. We found that mutations compromising general RNA binding of ZAP and TRIM25 impact their ability to restrict SINV replication, but mutations specifically targeting ZAP CpG-mediated RNA binding have a greater effect on SINV and JEV translation inhibition. Interestingly, ZAP-TRIM25 interaction is a critical determinant of JEV translation inhibition. Taken together, these findings illuminate the contribution of RNA binding and co-factor interaction to the synergistic inhibition of viral translation by ZAP and TRIM25. The type I interferon (IFN) response is one of the first lines of cellular defense against invading pathogens. The IFN-induced zinc finger antiviral protein (ZAP) is a potent inhibitor of diverse RNA and DNA viruses . ZAP encEarlier efforts to elucidate ZAP RNA binding activity showed that ZAP binds RNA with its four N-terminal CCCH ZnFs, and mutations of ZnFs 2 and 4 most dramatically reduce ZAP antiviral activity . The firWhile much attention has been given to characterizing ZAP RNA binding, less has been given to its critical co-factors such as TRIM25. TRIM25 was identified as a ZAP co-factor in the context of inhibiting alphavirus translation . HoweverWhile some have attempted to illuminate the contribution of ZAP or TRIM25 RNA binding to the ZAP-TRIM25 interaction, these studies have focused on only a few select mutations. Still, most agree that RNA binding in either ZAP or TRIM25 is not required for the ZAP-TRIM25 interaction. One group demonstrated not only that RNase A treatment has little effect on the ZAPL-TRIM25 interaction, but also that an example ZAPL RNA binding mutant retains and even increases its interaction with TRIM25 . AnotherIn light of the recent novel structural insights, we asked how different ZAP and TRIM25 RNA binding mutations affect the ability of ZAP and TRIM25 to interact with one another and to restrict SINV and JEV translation. We curated a panel of ZAP and TRIM25 RNA binding mutants from prior studies, including mutants with a range of RNA binding and antiviral capabilities . We firsTo investigate the role of ZAP and TRIM25 RNA binding in the context of viral translation, we generated a panel of constructs with mutations previously demonstrated to impact RNA binding. For ZAP, each of the following mutations was introduced in both ZAPS and ZAPL to probe potential isoform differences in RNA binding and antiviral function. These mutations fall into two general categories: 1) ZnF mutants that individually disrupt each CCCH motif and 2) CpG RNA binding cavity mutants. We made four individual mutations to disrupt each N-terminal CCCH ZnF: H86K, C88R, C168R, and H191R, which are located in ZnF 1, 2, 3, and 4, respectively . Two putFor TRIM25, we generated two constructs, one in which we replaced the lysine-rich motif in the L2 linker with alanines (TRIM25 7KA) and one in which we deleted the previously identified RNA binding domain in the PRY/SPRY domain (TRIM25 \u0394RBD) .in vitro RNA pull-down assay. We incubated lysates of cells transfected with ZAP or TRIM25 mutants with biotin-labeled SINV (Toto1101 strain) genomic RNA, allowing for RNA and bound protein to be immunoprecipitated using streptavidin beads and probed for the presence of bound ZAP or TRIM25. As a negative control, we also assessed the ability of the WT constructs to bind firefly luciferase (Fluc) RNA. We quantified the resultant ZAP and TRIM25 bound to RNA and normalized to input ZAP and TRIM25 protein levels with ImageJ . Becausee (Rluc) . We attee (Rluc) .We found that the ZAP and TRIM25 RNA binding mutants inhibit JEV translation to varying degrees Figure\u00a05Upon assaying TRIM25 variants, we found that TRIM25 \u0394RBD inhibits JEV translation similarly to TRIM25 WT and its parental 293T lines . TRIM25 Infections with SINV expressing firefly luciferase (Toto1101/Luc) and temperature-sensitive SINV (Toto1101/Luc:ts6) have been previously described . Each inBamHI and HindIII restriction sites. ZAPS and ZAPL were cloned into pcDNA3.1-3XFLAG from pTRIP-TagRFP-hZAPS and pTRIP-TagRFP-hZAPL \u201d, to replace wild-type sequence in TRIM25. The 7KA mutated sequence is bolded in the below gene block, and nonessential nucleotides on the 5\u2019 and 3\u2019 ends are written in lowercase.Point mutations in ZAPS and ZAPL were generated using the Q5 Site-Directed Mutagenesis Kit (New England Biolabs) and all plasmids were verified by sequencing (Genewiz). Primers for mutations were synthesized by Integrated DNA Technologies was genealanines , and utiTGTACAGTCAGATCAACGGGGCGTCGAGAGCACTGGATGATGTGAGAAACAGGCAGCAGGATGTGCGGATGACTGCAAACAGAAAGGTGGAGCAGCTACAACAAGAATACACGGAAATGAAGGCTCTCTTGGACGCCTCAGAGACCACCTCGACAAGGAAGATAAAGGAAGAGGAGAAGAGGGTCAACAGCAAGTTTGACACCATTTATCAGATTCTCCTCAAGAAGAAGAGTGAGATCCAGACCTTGAAGGAGGAGATTGAACAGAGCCTGACCAAGAGGGATGAGTTCGAGTTTCTGGAGAAAGCATCAAAACTGCGAGGAATCTCAACAAAGCCAGTCTACATCCCCGAGGTGGAACTGAACCACAAGCTGATAAAAGGCATCCACCAGAGCACCATAGACCTCAAAAACGAGCTGAAGCAGTGCATCGGGCGGCTCCAGGAGCCCACCCCCAGTTCAGGTGACCCTGGAGAGCATGACCCAGCGTCCACACACAAATCCACACGCCCTGTGGCAGCAGTCTCCGCAGAGGAAGCAGCATCCGCAGCACCTCCCCCTGTCCCTGCCTTACCCAGCAAGCTTCCCACGTTTGGAGCCCCGGAACAGTTAGTGGATTTAAAACAAGCTGGCTTGGAGGCTGCAGCCAAAGCCACCAGCTCACATCCGAACTCAACATCTCTCAAGGCCAAGGTGCTGGAGACCTTCCTGGCCAAGTCCAGACCTGAGCTCCTGGAGTATTACATTAAAGTCATCCTGGACTACAACACCGCCCACAACAAAGTGGCTCTGTCAGAGTGCTATACAGTAGCTTCTGTGGCTGAGATGCCTCAGAACTACCGGCCGCATCCCCAGAGGTTCACATACTGCTCTCAGGTGCTGGGCCTGCACTGCTACAAGAAGGGGATCCgttt-3\u20195\u2019gtttX-tremeGENE9 DNA Transfection Reagent (Roche Life Science) was used to transfect cells at a ratio of 3 \u03bcL to 1 \u03bcg DNA according to the manufacturer\u2019s instructions. To keep the total plasmid amount in co-transfections constant, empty vectors pcDNA3.1-myc or 3XFLAG were transfected as necessary .XhoI linearization of pToto1101 (in vitro by Sp6 RNA polymerase (New England Biolabs) in the presence of the cap analog [m7G(5\u2019)ppp(5\u2019)G] (New England Biolabs). Fluc DNA templates for transcription were amplified from the pGL3-Control plasmid (Promega). Fluc RNA was transcribed in vitro using the mMESSAGE mMACHINE T7 Transcription Kit (Invitrogen). Biotin-labeled RNAs were generated by adding 10mM biotin-16-UTP (Roche Life Science) to in vitro transcription reactions. JEV replicon DNA templates for transcription were generated by XhoI linearization and transcribed in vitro using the mMESSAGE mMACHINE T7 Transcription Kit (Invitrogen). Transcribed RNAs were purified using the Quick-RNA Miniprep Kit (Zymo Research) and biotinylation was confirmed by streptavidin dot blot and incubated with RNA probes in a final volume of 100 \u03bcL RNA binding buffer supplemented with 1 unit/\u03bcL RNAseOUT (Thermo Fisher Scientific), 1 \u03bcg/\u03bcL heparin (Sigma-Aldrich), and 100 ng/\u03bcL yeast tRNA (ThermoFisher) for 30\u00a0min at 30\u00b0C. Lysate-RNA mixtures were then incubated with 300 \u03bcL of Dynabeads M-280 Streptavidin (Invitrogen) for 30\u00a0min at room temperature on a shaker. Protein-RNA complexes were washed three times by RNA binding buffer, and proteins were eluted by incubation with 30 \u03bcL of 4x Laemmli Sample Buffer (Bio-Rad) for 5\u00a0min at 95\u00b0C. The proteins were further analyzed by immunoblot and quantified by ImageJ as previously described and 4-15% precast Mini-PROTEAN TGX Gels (Bio-Rad) before transferring to a PVDF membrane (Bio-Rad). Immunodetection was achieved with 1:2,500 anti-myc , 1:20,000 anti-FLAG (Sigma-Aldrich), and 1:20,000 anti-actin-HRP (Sigma-Aldrich). Primary antibodies were detected with 1:20,000 goat anti-mouse HRP (Jackson ImmunoResearch) or 1:20,000 goat anti-rabbit HRP (Thermo Fisher Scientific). Proteins were visualized on a ChemiDoc (Bio-Rad) using ProSignal Pico ECL Reagent (Genesee Scientific). ImageJ was used to quantify western blots as previously described . BrieflyTo assess ZAP or TRIM25 co-immunoprecipitation (co-IP) with RNA binding mutants of TRIM25 or ZAP, respectively, cells were transfected in 6-well plates, collected, and then lysed by rotating in FLAG IP buffer supplemented with a complete protease inhibitor cocktail (Roche Life Science) at 4\u00b0C for 30\u00a0min, before spinning down at 14,000 rpm at 4\u00b0C for 15\u00a0min. To equilibrate beads prior to use, anti-FLAG beads or anti-myc beads were washed 3 times in FLAG IP buffer. Three hundred \u03bcL of whole cell lysate (WCL) were incubated with 30 \u03bcL of anti-FLAG or -myc beads rotating at 4\u02daC for 45 minutes. FLAG IP buffer was used to wash immunoprecipitates 3 times before eluting bound proteins with SDS loading buffer, and boiling for 5 minutes for immunoblot analysis. Western blot ImageJ analysis was performed as previously described . BrieflyTransIT-mRNA Transfection Kit (Mirus Bio). Cells were lysed 4 hours post-transfection of replicon RNA and luciferase activity was measured by Dual-Luciferase Reporter Assay (Promega). Each independent experiment included triplicate wells of biological replicates per condition.Following transfection of ZAP or TRIM25 constructs into ZAP or TRIM25 KO 293T for 48 hours, JEV replicon RNA was transfected by Statistical analyses in The original contributions presented in the study are included in the article/EY, LN, and ML conceptualized and designed the study. CW and RK assisted in cloning ZAP and TRIM25 RNA binding mutants. LN performed RNA binding and JEV replicon experiments, while EY performed co-IP and SINV replication and translation experiments. LN performed the correlation analysis. EY and LN co-wrote the first draft of the manuscript. ML provided critical feedback. All authors contributed to manuscript revision, read, and approved the submitted version.This work was supported in part by NIH R01AI158704 (ML), UCLA AIDS Institute and Charity Treks 2019 Seed Grant (ML), Ruth L. Kirschstein Multidisciplinary Training Grant in Microbial Pathogenesis , Ruth L. Kirschstein Cellular and Molecular Biology Training Program , Warsaw Fellowship (EY), and Whitcome Fellowship .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."} +{"text": "Currently, a total of 81 corticalis group species are known worldwide, amongst them 67 were recorded from China. However, the diversity of this group in China is still insufficiently known.The Clubionaxianningsp. nov. is described as a new species of the C.corticalis species-group collected from Hubei Province, China. Clubiona Latreille, 1804 currently contains 518 catalogued species that are found worldwide, except for the Polar Regions and South America .OP675437; YHCLU0273, \u2640, GenBank OP675436.A DNA barcode was also obtained for species matching. A partial fragment of the mitochondrial cytochrome oxidase subunit I (CO1) gene was amplified and sequenced for two specimens, using the primers LCOI1490 (5\u2019-GGTCAACAAATCATAAAGATATTG-3\u2019) and HCOI2198 (5\u2019-TAAACTTCAGGGTGACCAAAAAAT-3\u2019) . For addAll measurements were obtained using an Olympus SZX7 stereomicroscope and given in millimetres. Eye diameters are taken at the widest point. The total body length does not include chelicerae or spinnerets length. Leg lengths are given as total length . Most of the terminologies used in text and figure legends follows All specimens are deposited Museum of Guizhou Normal University, Guiyang, Guizhou, China.Zhong & Yusp. n.B0CC6F23-8DFB-5B4A-BAD0-E8AC5114D6878DFF1CC4-1C1A-46A6-8300-FF36FFFA2644Type status:Holotype. Occurrence: recordedBy: Yang Zhong, Xusheng Gong, Qianle Lu; individualID: YHCLU0272; individualCount: 1; sex: male; lifeStage: adult; behavior: foraging; preparations: whole animal (ETOH); associatedSequences: OP675437GenBank: ; occurrenceID: 1312422A-3320-5AE6-9BA9-5DCB3013B14F; Taxon: order: Araneae; family: Clubionidae; genus: Clubiona; specificEpithet: xianning; scientificNameAuthorship: Zhong & Yu; Location: continent: Asian; country: China; countryCode: CHN; stateProvince: Hubei; county: Tongshan; municipality: Xianning; locality: Jiugongshan Nature Reserve; decimalLatitude: 29.39; decimalLongitude: 114.65; Identification: identifiedBy: Hao Yu; dateIdentified: 2022-05; Event: samplingProtocol: by hand; samplingEffort: 10 km by foot; year: 2020; month: 7; day: 4; Record Level: basisOfRecord: PreservedSpecimenType status:Holotype. Occurrence: recordedBy: Yang Zhong, Xusheng Gong, Qianle Lu; individualID: YHCLU0273; individualCount: 1; sex: female; lifeStage: adult; behavior: foraging; preparations: whole animal (ETOH); associatedSequences: OP675436GenBank: ; occurrenceID: 085B9104-F139-5EC1-9913-5A5DB9C7C9C6; Taxon: order: Araneae; family: Clubionidae; genus: Clubiona; specificEpithet: xianning; scientificNameAuthorship: Zhong & Yu; Location: continent: Asian; country: China; countryCode: CHN; stateProvince: Hubei; county: Tongshan; municipality: Xianning; locality: Jiugongshan Nature Reserve; decimalLatitude: 29.39; decimalLongitude: 114.65; Identification: identifiedBy: Hao Yu; dateIdentified: 2022-05; Event: samplingProtocol: by hand; samplingEffort: 10 km by foot; year: 2020; month: 7; day: 4; Record Level: basisOfRecord: PreservedSpecimenMale 5'TTCTGGTCAGCTATAGTTGGTACAGCTATAAGAGTTATAATTCGTATAGAATTAGGTCAATCTGGAGCTTTTTTAGGTGATGATCATTTGTATAATGTAGTAGTTACTGCTCATGCTTTTGTTATAATTTTTTTTATAGTAATACCTATTATAATTGGGGGGTTTGGAAATTGATTAGTTCCATTAATATTAGGGGCAGGTGATATAGCTTTTCCTCGTATAAATAATTTAAGTTTTTGACTTTTACCACCTTCATTAATATTATTAGTTATATCATCTATGGCTGAGATGGGAGTTGGGGCTGGATGAACAGTTTATCCCCCTCTTGCTTCTTTAGTAGGTCATACGGGAAGAGCAATGGATTTTGCTATTTTTTCATTACATTTAGCTGGGGCTTCTTCTATTATAGGAGCTGTTAATTTTATTACTACTATTATGAATATACGATCTTTTGGAATAATAATGGAAAAGATTTCATTATTTGTTTGGTCTGTTTTAATTACAGCTATTTTATTATTATTATCTTTGCCAGTTTTAGCCGGGGCTATTACTATATTATTAACTGATCGTAATTTTAATACGTCTTTTTTTGACCCTGCTGGGGGAGGTGATCCTATTTTATTTCAACATTTATTTTGATTTTTTGGTCACCC3' .5'TCTGGTCAGCTATAGTTGGTACAGCTATAAGAGTTATAATTCGTATAGAATTAGGTCAATCTGGAGCTTTTTTAGGTGATGATCATTTGTATAATGTAGTAGTTACTGCTCATGCTTTTGTTATAATTTTTTTTATAGTAATACCTATTATAATTGGGGGGTTTGGAAATTGATTAGTTCCATTAATATTAGGGGCAGGTGATATAGCTTTTCCTCGTATAAATAATTTAAGTTTTTGACTTTTACCACCTTCATTAATATTATTAGTTATATCATCTATGGCTGAGATGGGAGTTGGGGCTGGATGAACAGTTTATCCCCCTCTTGCTTCTTTAGTAGGTCATACGGGAAGAGCAATGGATTTTGCTATTTTTTCATTACATTTAGCTGGGGCTTCTTCTATTATAGGAGCTGTTAATTTTATTACTACTATTATGAATATACGATCTTTTGGAATAATAATGGAAAAGATTTCATTATTTGTTTGGTCTGTTTTAATTACAGCTATTTTATTATTATTATCTTTGCCAGTTTTAGCCGGGGCTATTACTATATTATTAACTGATCGTAATTTTAATACGTCTTTTTTTGACCCTGCTGGGGGAGGTGATCCTATTTTATTTCAACATTTATTTTGATTTTTTGGTCACCC3' , differ from C.altissimus by: (1) atrium large, nearly as wide as epigynal plate (cf. Fig. C.xianningsp. nov. also can by separated from C.caohai and C.altissimus by their habitus: abdomen without distinct colour pattern in C.xianningsp. nov. (Fig. C.caohai and C.altissimus (Male of the new species resembles that of Yu, 2020 : fig. 2DYu, 2020 : fig. 2Bov. Fig. E\u2013H, but The specific name refers to the type locality and is a noun in apposition.Known from the Mt. Jiugong, Hubei Province, China Fig. ."} +{"text": "P < 0.05). The relative expression of circHIPK3 in patients with TNM stage II/III was dramatically inferior to that in patients with tumor, node, and metastasis (TNM) stage I (P < 0.05). Expression of circHIPK3 in patients with lymph node metastasis was dramatically inferior to that in patients without lymph node metastasis (P < 0.05). Of the lung cancer tissues of patients with different TNM stages, only six patients had high expression, and the remaining 104 patients had low expression. Moreover, electrophoresis revealed that circHIPK3 can only be amplified in cDNA, but not in gDNA. Gefitinib-mediated apoptosis rate of lung cancer drug-resistant cell lines decreased notably. In summary, the circular RNA circHIPK3 may have a notably low expression in lung cancer tissues, whose low expression had a certain enhancement effect on the drug resistance of lung adenocarcinoma cells to gefitinib.To study the mechanism of circular ribonucleic acid (RNA) circHIPK3 involved in the resistance of lung cancer cells to gefitinib, 110 patients with lung cancer were recruited as the research objects, and the tumor tissue and para-cancerous tissue of each patient's surgical specimens were collected and paraffinized to detect the expression of circHIPK3 in different tissues. Gefitinib drug-resistant cell line of lung cancer was constructed with gefitinib to detect cell apoptosis under different conditions. As a result, the relative expression of circHIPK3 in patients with tumor diameter no less than 3\u2009cm was dramatically inferior to that in patients with tumor diameter less than 3\u2009cm ( Lung cancer is a malignant tumor, mainly originated from the mucosal epithelium of the bronchus, which is classified into small cell carcinoma and nonsmall cell carcinoma according to the histological changes. In recent years, with the serious air pollution and the gradual deterioration of the environment, the global mortality rate of lung cancer is on the rise, seriously threatening people's health . AccordicircRNA is a new type of RNA molecule that is characterized by a covalent closed loop and widely exists in eukaryotes . circRNAIn recent years, related studies found that using siRNA to knockdown circHIPK3 can inhibit cell proliferation. In addition, circHIPK3 can adsorb miR-124 in liver cancer and promote the proliferation of liver cancer cells by regulating the expression of target genes. However, its regulation and expression mechanism in lung cancer is still unclear. Therefore, this study was developed to explore the mechanism of the circular RNA circHIPK3 involved in the resistance of lung cancer cells to gefitinib.In this study, 110 lung cancer patients admitted to X Hospital from September 2019 to September 2020 were collected as the research objects. Paraffin specimens were collected from 58 male patients and 52 female patients. The study has been approved by the Medical Ethics Committee. Patients and their families understood the study content and methods of specimen acquisition and agreed to sign corresponding informed consent forms.Inclusion criteria: (i) patients diagnosed with lung cancer by pathology and imaging; (ii) patients aged between 45 and 75 years; (iii) there was no metastasis of lung lesions, mediastinal lymph node enlargement, or pleural hypertrophy; (iv) patients who were not recently treated with other drugs or antibiotics in the study; (v) patients with normal coagulation function and platelets.Exclusion criteria: (i) patients with diseases of other systems or organs; (ii) patients who had not received cooperative treatment due to personal or other factors; (iii) patients with incomplete clinical data and history information , etc.).\u03bcm/slice) and stored in an enzyme-free centrifuge tube containing xylene .Paraffin sections of tumor tissue and para-cancer tissue from surgical specimens of each included subject were collected. The collected paraffin samples were sliced with uniform thickness were added. After the tissue was dissolved, it was bathed in water at 95\u00b0C for 10\u2009min and centrifuged at 8,600\u2009rpm at room temperature for 5\u2009min. The supernatant was absorbed and transferred to a new Rnase-free centrifuge tube. 220\u2009\u03bcL buffer RB was added, shaken, and mixed, then added with anhydrous ethanol, and mixed again.Sectioning: the collected paraffin specimens were sectionalized with a thickness of 10\u2009\u03bcL solution and precipitate were absorbed and added to the adsorption column, centrifuged at high speed for 1\u2009min. The waste solution was filtered and repeated several times until the solution and precipitate passed through the adsorption column. 80\u2009\u03bcL DNase I working solution was added to the center of the adsorption column and left for 15\u2009min at room temperature. 500\u2009\u03bcL protein-free RW1 was added and centrifuged, then added with rinse solution, centrifuged again, and the waste solution was discarded. After drying, the adsorption column was transferred to a new RNase-free centrifuge tube. 35\u2009\u03bcL RNase-free ddH2O was added and centrifuged at 8,600\u2009rpm for 5\u2009min to obtain RNA solution. RNase R was used to process the extracted total RNA to avoid artifacts. RNase R processing reaction system: 2\u2009\u03bcg RNA, 1\u2009\u03bcL RiboLock, 2\u2009\u03bcL 10x RNase R reaction buffer, and 1\u2009\u03bcL RNase R, with sterile water supplementing the system to 20\u2009\u03bcL [\u03bcL 5\u2009\u00d7\u2009PrimeScript Buffer, 0.5\u2009\u03bcL enzyme mix I, 0.5\u2009\u03bcL random 6 mers, and 7\u2009\u03bcL total RNA were added to a 0.2\u2009mL enzyme-free EP tube to configure a 10\u2009\u03bcL system. The EP tube was closed and shaken for 15\u2009s to mix, centrifuged, and put in the reverse transcription machine. The reverse transcription conditions: 37\u00b0C for 15\u2009min, 85\u00b0C for 5\u2009sec. After the reaction, the synthesized cDNA was transferred to -20\u00b0C refrigerator for storage. The length of circHIPK3 searched on NCBI was 1,099\u2009bp, and the specific base sequence was as follows: GTATGGCCTCACAAGTCTTGGTCTACCCACCATATGTTTATCAAACTCAGTCAAGTGCCTTTTGTAGTGTGAAGAAACTCAAAGTAGAGCCAAGCAGTTGTGTATTCCAGGAAAGAAACTATCCACGGACCTATGTGAATGGTAGAAACTTTGGAAATTCTCATCCTCCCACTAAGGGTAGTGCTTTTCAGACAAAGATACCATTTAATAGACCTCGAGGACACAACTTTTCATTGCGACAAGTGCTGTTGTTTTGAAAAACACTGCAGGTGCTACAAAGGTCATAGCAGCTCAGGCACAGCAAGCTCACGTGCAGGCACCTCAGATTGGGGCGTGGCGAAACAGATTGCATTTCCTAGAAGGCCCCCAGCGATGTGGATTGAAGCGCAAGAGTGAGGAGTTGGATAATCATAGCAGCGCAATGCAGATTGTCGATGAATTGTCCATACTTCCTGCAATGTTGCAAACCAACATGGGAAATCCAGTGACAGTTGTGACAGCTACCACAGGATCAAAACAGAATTGTACCACTGGAGAAGGTGACTATCAGTTAGTACAGCATGAAGTCTTATGCTCCATGAAAAATACTTACGAAGTCCTTGATTTTCTTGGTCGAGGCACGTTTGGCCAGGTAGTTAAATGCTGGAAAAGAGGGACAAATGAAATTGTAGCAATCAAAATTTTGAAGAATCATCCTTCTTATGCCCGTCAAGGTCAAATAGAAGTGAGCATATTAGCAAGGCTCAGTACTGAAAATGCTGATGAATATAACTTTGTACGAGCTTATGAATGCTTTCAGCACCGTAACCATACTTGTTTAGTCTTTGAGATGCTGGAACAAAACTTGTATGACTTTCTGAAACAAAATAAATTTAGTCCCCTGCCACTAAAAGTGATTCGGCCCATTCTTCAACAAGTGGCCACTGCACTGAAAAAATTGAAAAGTCTTGGTTTAATTCATGCTGATCTCAAGCCAGAGAATATTATGTTGGTGGATCCTGTTCGGCAGCCTTACAGGGTTAAAGTAATAGACTTTGGGTCGGCCAGTCATGTATCAAAGACTGTTTGT TCAACATATCTACAATCTCGGTACTACAG.700\u2009to 20\u2009\u03bcL . The insThe designed upstream and downstream primers were as follows: circHIPK3-F: TAGACTTTGGGTCGGCCAGT; circHIPK3-R: TGGAATACACAACTGCTTGGC.The expression of circHIPK3 was detected by real-time fluorescence quantitative polymerase chain reaction (PCR). circHIPK3 is derived from the HIPK3 gene and consists of exon 2 head-to-tail splicing. Head-to-tail splicing can be generated by trans-splicing or genome rearrangement. The sequence is a circular sequence, which is assembled from the first position of the corresponding linear RNA, and a new unique sequence is generated at the reverse splicing site. The circRNA specific primers were designed with this as the target sequence, so as to specifically recognize circRNA and distinguish circular sequence from linear RNA. The identification and location of circHIPK3 was shown in Serum-free cell freezing medium (RPMI) 1640 cell culture medium consisted of 10% fetal bovine serum (FBS) , 100\u2009U/mL penicillin and 100\u2009mg/mL streptomycin . The cell cryopreservation tube was taken out of the liquid nitrogen and quickly melted in a constant temperature water bath at 37\u00b0C. The thawed cell cryopreservation solution was transferred to a centrifuge tube containing cell culture medium and centrifuged at high speed for 5\u2009min. Then, after cell culture medium was added to the lower sediment to resuspend the cells, they were transferred to RPMI medium and incubated at constant temperature. The medium was changed once the cells grew adherently.After the cell coverage in the culture dish reached more than 80%, passaging began. After the cell culture solution was aspirated, trypsin digestion solution was added, and cells were observed under a microscope. After culturing until the cells became round, culture medium was added to stop the digestion, and the cells were resuspended and passed to a culture dish.50) of gefitinib on sensitive cells was 1.78\u2009mol/L. Then, the culture medium containing 17.5\u2009\u03bcmol/L gefitinib was used for 24\u2009h, and the medium containing half of the inhibitory concentration gefitinib was immediately replaced for 5-10\u2009d, until the cells grew steadily and were subcultured for 3 times. IC50 was determined again. The medium containing 17.5\u2009\u03bcmol/L gefitinib was cultured for 24\u2009h, and the medium with gradually increased inhibitory concentration of gefitinib was replaced until the cells grew statically in the medium containing 17.5\u2009\u03bcmol/L gefitinib and were continuously passed for 3 times. Then, the IC50 of gefitinib was 34.5\u2009\u03bcmol/L and was named A549/GR.Gefitinib was dissolved in dimethyl sulfoxide (DMSO) (Abis (Shanghai) Biotechnology Co., Ltd.). The methyl tetrazolium (MTT) assay was used to determine the median lethal concentration of gefitinib on lung cancer cells . In this study, human lung adenocarcinoma cell line A549 was used to induce gefitinib-resistant cell lines. Resistant cells were induced by a combination of high-dose shocks and gradually increased doses. MTT assay was used to determine the 50% inhibitory concentration was added to each well for 4\u2009h, and the culture was terminated. The supernatant cultured in the hole should be carefully sucked and discarded. For suspended cells, the supernatant should be discarded after centrifugation. 150\u2009\u03bcL dimethyl sulfoxide (DMSO) was added to each well and shaken to dissolve completely. At 490\u2009nm, the light absorption value (OD) of each hole was measured by enzyme-linked immunosorbent assay (ELISA) and recorded. The cell growth curve was plotted with time as abscissa and light absorption value as ordinate.Single cell suspension was prepared with culture medium containing 10% fetal bovine serum and inoculated into 96-well plates with 1,000-10,000 cells per well, and volume of each well was 200\u2009P < 0.05.SPSS 19.0 was employed for data statistics and analysis. Mean \u00b1 standard\u2009deviation (P > 0.05). There was also no considerable difference in the relative expression of circHIPK3 between patients less than 60 years old and patients no less than 60 years old (P > 0.05).P < 0.05). The relative expression of circHIPK3 in patients with T stage II/III tumors was dramatically inferior to that in patients with T stage I (P < 0.05).P < 0.05). The relative expression of circHIPK3 in patients with lymph node metastasis was dramatically inferior to that in patients without lymph node metastasis (P < 0.05).The circular RNA circHIPK3 is widely and highly expressed in lung cancer cells, mainly located in the cytoplasm, but also in the nucleus. The expression of circHIPK3 in cancer tissues and normal lung tissues adjacent to cancer is notably different, so it is used for the diagnosis of lung cancer. With pathological and imaging findings as diagnostic criteria, The 110 lung cancer patients included in this study were graded into stage I, stage II, and stage III according to the TNM staging. Among them, 39 patients were in TNM stage I, 36 were in stage II, and 35 were in stage III. The circular RNA circHIPK3 was amplified by designing primers and then electrophoresed on agarose gel plate to get the electrophoresis, as shown in The surface of normal cells is composed of lipids distributed asymmetrically on the inner and outer lobes of the plasma membrane. Phosphatidylserine is mostly distributed in the inner lobes of the plasma membrane and exposed to the cytoplasm . HoweverGefitinib can specifically block a certain pathway of lung cancer cells, block the conduction, infiltration, and metastasis of lung cancer cells, locate and kill cancer cells, and cause little damage to normal human cells , 26. It P < 0.05). The relative expression of circHIPK3 in patients with TNM stage II/III was dramatically inferior to that in patients with TNM stage I (P < 0.05). In addition, the relative expression of circHIPK3 in patients with lymph node metastasis was dramatically inferior to that in patients without lymph node metastasis (P < 0.05). The results indicated that the relative expression of circHIPK3 in the tumor tissues of patients with lung cancer was closely related to the diameter of the patient's tumor, the TNM stage, and the occurrence of lymph node metastasis. However, there was no high correlation with the patient's age, gender, and other information. Among lung cancer tissues of patients with different TNM stages, only 6 patients had high expression, and the remaining 104 patients had low expression. Therefore, circHIPK3 may have a notably low expression in lung cancer tissues and related cell lines, which had a high correlation with the tumor stage of patients. Flow cytometry apoptosis detection results found that gefitinib-mediated apoptosis rate of lung cancer drug-resistant cell lines decreased notably, which was similar to Zhou et al.'s (2020) [In this study, the tumor tissues and para-cancerous tissues from surgical specimens of 110 study subjects were collected to detect differences in the expression of circHIPK3 in different tissues. Moreover, a lung cancer gefitinib drug-resistant cell line was constructed, and cell apoptosis was detected under different conditions, to study the mechanism of circRNA involved in the resistance of lung cancer cells to gefitinib. As a result, the relative expression of circHIPK3 in patients with tumor diameter no less than 3\u2009cm was dramatically inferior to that in patients with tumor diameter less than 3\u2009cm (s (2020) researchIn this study, tumor tissues and adjacent tissues were collected from surgical specimens of lung cancer patients to detect differences in the expression of circHIPK3 in different patients and different tumor tissues. In addition, a lung cancer gefitinib drug-resistant cell line was constructed to detect cell apoptosis under different conditions. The results showed that the circular RNA circHIPK3 may have a considerably low expression in lung cancer tissues, and its low expression had a certain enhancement effect on the drug resistance of lung adenocarcinoma cells to gefitinib. However, the sample size selected in this study is small, and the representativeness is low. Therefore, the selection of test sample size will be increased in subsequent experiments. Further study is required to clarify the mechanism of circRNA involved in the resistance of lung cancer cells to gefitinib. In short, this study provides a certain theoretical basis and data support for the drug treatment of lung cancer."} +{"text": "In chromatin, linker histone H1 binds to nucleosomes, forming chromatosomes, and changes the transcription status. However, the mechanism by which RNA polymerase II (RNAPII) transcribes the DNA in the chromatosome has remained enigmatic. Here we report the cryo-electron microscopy (cryo-EM) structures of transcribing RNAPII-chromatosome complexes (forms I and II), in which RNAPII is paused at the entry linker DNA region of the chromatosome due to H1 binding. In the form I complex, the H1 bound to the nucleosome restricts the linker DNA orientation, and the exit linker DNA is captured by the RNAPII DNA binding cleft. In the form II complex, the RNAPII progresses a few bases ahead by releasing the exit linker DNA from the RNAPII cleft, and directly clashes with the H1 bound to the nucleosome. The transcription elongation factor Spt4/5 masks the RNAPII DNA binding region, and drastically reduces the H1-mediated RNAPII pausing. The mechanism by which RNAPII transcribes the DNA in the chromatosome with H1 has remained enigmatic. Here the authors present the cryo-EM structures of the RNAPII-chromatosome complexes, and explain how RNAPII is regulated by H1 in chromatin. NCPs are connected by linker DNA segments, and appear as a beads-on-a-string fiber2.Chromatin compacts genomic DNA in eukaryotes, and the nucleosome is an elemental architecture. The nucleosome is composed of linker DNAs and the nucleosome core particle (NCP), which folds an approximately 150 base-pair DNA segment into a histone octamer, comprising the four core histones H2A, H2B, H3, and H4. In the NCP, the DNA is symmetrically wrapped 1.7-turns around the histone octamer, and the central region of the nucleosomal DNA is located on the dyad axis3, and promote further compaction of chromatin7. H1 consists of three domains, the N-terminal disordered region (\u223c35 residues), the central globular domain (\u223c80 residues), and the C-terminal disordered region (\u223c100 residues), and in the chromatosome, these domains mainly bind to a linker DNA, the DNA around the dyad axis, and another linker DNA, respectively11. This tripartite nucleosome binding by H1 is termed \u201con-dyad\u201d binding, and it compacts the nucleosome architecture by restricting the linker DNA orientation and flexibility14. H1 also binds the nucleosome in an \u201coff-dyad\u201d mode, in which the central globular domain mainly binds to a linker DNA16.Linker histones, such as H1, are major chromatin components that specifically bind to the nucleosomal DNA forming the chromatosome17. H1 binding to the nucleosome induces structural changes in chromatin, thus regulating transcription processes catalyzed by RNA polymerase II (RNAPII)5. For instance, a biochemical study with Drosophila egg extracts indicated that H1 binding to reconstituted chromatin represses RNAPII transcription18. A mouse knock-out study revealed that embryos with an H1 ratio reduced to approximately 50% cease development at mid-gestation19. H1 reportedly functions in gene regulation through chromatin compaction and 3D genome organization20. Consistently, acute depletion of H1 leads to perturbations of gene expression in the constitutive heterochromatin in mouse embryonic stem cells21. H1 alleles have been identified as genes with driver mutations in lymphoma, revealing that H1 is a bona fide tumor suppressor22. These facts indicate that H1 plays pivotal roles in development, differentiation, and tumorigenesis by appropriately reducing transcription efficiency according to the cellular status.H1 is the most abundant nucleosome binding protein, and exists in a 50\u2013130% amount relative to nucleosomes5. H1.2 is the major somatic linker histone. Genome-wide studies revealed that the genomic H1.2 binding loci negatively correlate with the RNAPII transcription activation, suggesting the repressor function of H1.225. In contrast, genetic and biochemical studies demonstrated that H1.2 potentially upregulates gene expression30. Therefore, H1.2 may function to both repress and activate RNAPII transcription5, probably depending on cell types and genomic loci. These different outcomes may be the results of complex transcription regulation at the levels of transcription initiation and elongation, but the direct effects of H1 on transcription are not well understood.Eleven non-allelic human H1 isoforms have been described, and they may have specific functions in altering the cellular transcription status, primarily by repressing transcription by RNAPII32. H1 causes RNAPII pausing at the 3\u2032 splice site, and regulates alternative splicing32. These observations suggested the specific involvement of linker histones in the regulation of transcription elongation, but the molecular details have remained elusive. In the present study, we investigate the basic properties of the chromatosome in transcription elongation by performing in vitro transcription experiments and cryo-EM analyses, which reveal how H1 accomplishes nucleosome repression in transcription elongation.Linker histones are associated with the gene-body regions of actively transcribed genesKomagataella pastoris was loaded onto the bubble, and the transcription reaction was conducted in the presence of the transcription elongation factor TFIIS , lacking half of the C-terminal region (amino acid residues 151-212) Fig.\u00a0. Interes12) Fig.\u00a0. The RNA12) Fig.\u00a0, probablSus scrofa domesticus36 analysis of the RNAPII transcribing the entry linker DNA , the exit linker DNA of the chromatosome sterically conflicts with the transcribing RNAPII. In the natural chromatin context, this exit linker DNA is followed by another nucleosome (or chromatosome). This led us to test whether a second, neighboring nucleosome/chromatosome affects the RNAPII pausing. To do so, we performed the chromatosome transcription assay with the di-nucleosome template in the presence of H1.2 Fig.\u00a0. In thisner Fig.\u00a0. The strner Fig.\u00a0. Therefo40. It binds and seals the RNAPII DNA-binding cleft41, and enhances the transcription elongation efficiency in the nucleosome43. Interestingly, when the Spt4/5-bound RNAPII structure was superimposed on the current RNAPII-chromatosome complex, Spt4/5 overlapped with the exit linker DNA in the form I complex is one of the transcription elongation factors that is tightly associated with transcribing RNAPIIlex Fig.\u00a0. This filex Fig.\u00a0. This sulex Fig.\u00a0, lane 4.This study has revealed that a chromatosome poses a higher transcriptional barrier than an H1-free nucleosome in vitro. H1 causes RNAPII pausing before it enters the chromatosome. This is because H1 brings the entry and exit linker DNAs close together, and the RNAPII on the entry linker DNA sterically conflicts with the exit linker DNA, hindering its progression. As shown in the form I complex, the exit linker DNA directly interacts with the RNAPII DNA-binding cleft, thus inducing the RNAPII pausing Fig.\u00a0. The for14. Therefore, the bound H1 may persist on the nucleosome while the RNAPII invades the nucleosomal DNA region strain and the JM109(DE3) strain (for H4) as N-terminally hexa-histidine (His6)-tagged proteins, and isolated under denaturing conditions. The His6-tagged proteins were purified by chromatography with nickel-nitrilotriacetic acid agarose (Ni-NTA) resin (QIAGEN). The His6-tag portion was then removed by thrombin protease, and the histones were purified by cation exchange chromatography on a MonoS column under denaturing conditions. The purified histones were dialyzed against water and lyophilized.Human histones H2A, H2B, H3.1, and H4 were expressed in E. coli cells and purified by the method described previously49. Briefly, H1.2 and H1.2 deltaC were produced in the BL21-CodonPlus(DE3)-RIL strain, as C-terminally His6-SUMO-tagged proteins. The His6-SUMO-tagged proteins were purified by Ni-NTA agarose (QIAGEN) chromatography. The His6-SUMO tag portion was then removed by PreScission protease treatment, and H1.2 and H1.2 deltaC were purified by MonoS cation exchange column chromatography. Purified H1.2 and H1.2 deltaC were stored at \u221280\u2009\u00b0C. K. pastoris RNAPII, composed of 12 subunits, was purified as described previously50. Briefly, K. pastoris encoding a TAP-tagged Rpb2 was cultured, and the tagged-RNAPII was purified by affinity column chromatography on anti-FLAG M2 affinity gel (Sigma-Aldrich), and anion-exchange column chromatography. K. pastoris TFIIS and Spt4/5 were purified as described previously40. These proteins were produced as His6-tagged proteins in the E. coli KRX strain. The His6-tagged proteins were then purified by chromatography on a COSMOGEL His-Accept column , and eluted by cleavage of the His6-tag portion by HRV-3C protease treatment. The eluted proteins were purified by Resource S cation-exchange column chromatography.Human H1.2 and H1.2 deltaC (H1.2(1-150)) were expressed in S. scrofa RNAPII preparation, fresh S. scrofa thymus was homogenized using a 2L blender (Waring) in buffer A (50\u2009mM Tris-HCl (pH 7.9), 1\u2009mM EDTA, 10 \u03bcM ZnCl2, 10% glycerol, 1\u2009mM DTT, 1\u2009mM phenylmethylsulfonyl fluoride, 1\u2009mM benzamidine, 0.6\u2009\u00b5M leupeptin, 2\u2009\u00b5M pepstatin A). The homogenized material was centrifuged at 12,200\u2009\u00d7\u2009g for 20\u2009min at 4\u2009\u00b0C, and the supernatant was filtered through Miracloth (Millipore). A 5% solution of polyethyleneimine (adjusted to pH 7.9 at 25\u2009\u00b0C) was added to a final concentration of 0.1%. After mixing for 10\u2009min at 4\u2009\u00b0C, the precipitate was collected by centrifugation at 15,750 xg for 20\u2009min. The pellet was washed twice with buffer A, and the RNAP II fraction was extracted from the pellet with buffer A containing 0.15\u20130.2\u2009M ammonium sulfate. A Q Sepharose (Cytiva) slurry was added to the eluted fraction, and the ammonium sulfate concentration was adjusted to 0.15\u2009M. After rotation at 4\u2009\u00b0C for 30\u2009min, the resin was washed with 6 volumes of buffer A containing 0.15\u2009M ammonium sulfate, followed by 3 volumes of buffer A containing 0.2\u2009M ammonium sulfate. RNAPII was eluted with buffer A containing 0.4\u2009M ammonium sulfate. The eluate was precipitated by 50% ammonium sulfate overnight. The precipitate was collected by centrifugation at 22,900\u2009\u00d7\u2009g for 20\u2009min at 4\u2009\u00b0C, resuspended in buffer A, and then dialyzed against 1\u2009L of buffer B (50\u2009mM Tris-HCl (pH 7.9), 150\u2009mM NaCl, 1\u2009mM EDTA, 10\u2009\u00b5M ZnCl2, 2\u2009mM DTT). The dialyzed sample was subjected to anion-exchange chromatography on a Mono Q (Cytiva) column. The RNAPII-containing fraction was concentrated with an Amicon Ultra-15 centrifugal filter unit (Merck) and purified by size exclusion chromatography on a Superose 6 column (Cytiva), equilibrated with buffer C (5\u2009mM HEPES-NaOH (pH 7.3), 150\u2009mM NaCl, 10\u2009\u00b5M ZnCl2, 10\u2009mM DTT). Peak fractions containing RNAPII were collected and stored as a 10% glycerol stock at \u221280\u2009\u00b0C. Judging from the electrophoretic band intensities, the purity of RNAPII in this fraction is about 30% buffer, containing 150\u2009mM NaCl, 1\u2009mM DTT, 5% glycerol, and 10\u2009\u03bcM zinc chloride, flash-frozen in liquid nitrogen, and stored at \u221280\u2009\u00b0C.For the E. coli DH5 strain. The 193\u2009bp Widom 601 fragment was excised from the plasmid DNA by EcoRV (Takara), and purified by polyethylene glycol precipitation. The ends of the DNA fragments were dephosphorylated by calf intestinal alkaline phosphatase (Takara). The 193\u2009bp Widom 601 DNA fragment was also produced by polymerase chain reaction. The purified 193\u2009bp Widom 601 DNA fragment was cleaved with HinfI, resulting in the 161 base-pair DNA with a 3\u2009bp overhang at the 5\u2032 end (Takara). This DNA fragment was further purified by DEAE-5PW anion-exchange column chromatography (TOSOH) or native polyacrylamide gel electrophoresis purification using a Prep Cell model 491 apparatus (Bio-Rad).For the preparation of the DNA fragment used in the mono-chromatosome transcription, the plasmid DNA containing the 193\u2009bp Widom 601 sequence was prepared from the The sequences of both strands of the resulting DNA fragment are as follows:non-template strand: 5\u2032:AATCCGGTGCCGAGGCCGCTCAATTGGTCGTAGACAGCTCTAGCACCGCTTAAACGCACGTACGCGCTGTCCCCCGCGTTTTAACCGCCAAGGGGATTACTCCCTAGTCTCCAGGCACGTGTCAGATATATACATCCAGGCCTTGTGTCGCGAAATTCATAGATtemplate strand: 5\u2032:ATCTATGAATTTCGCGACACAAGGCCTGGATGTATATATCTGACACGTGCCTGGAGACTAGGGAGTAATCCCCTTGGCGGTTAAAACGCGGGGGACAGCGCGTACGTGCGTTTAAGCGGTGCTAGAGCTGTCTACGACCAATTGAGCGGCCTCGGCACCGG.The purified DNA fragment was ligated with the double-stranded oligonucleotide 50-mer (purchased from Eurofins) by T4 DNA ligase (NIPPON GENE). The sequences of both strands of the double-stranded oligonucleotide 50-mer are as follows:non-template strand: 5\u2032:CCTTTAAAGCAATAGGAGCTTACGGTCCACTTGTGTTTGGTGTGTTTGGGtemplate strand: 5\u2032:ATTCCCAAACACACCAAACACAAGTGGACCGTAAGCTCCTATTGCTTTAA.For the preparation of the DNA fragment used in the di-chromatosome transcription, the 368\u2009bp DNA fragment containing the Widom 601 and 603 sequences connected with 48\u2009bp linker DNA was produced by polymerase chain reaction. The purified DNA fragment was cleaved with HinfI, resulting in the 354\u2009bp DNA with a 3\u2009bp overhang at the 5\u2032 end (Takara). This DNA fragment was further purified by native polyacrylamide gel electrophoresis, using a Prep Cell model 491 apparatus (Bio-Rad).The sequences of both strands of the resulting DNA fragment are as follows:non-template strand: 5\u2032:AATCCGGTGCCGAGGCCGCTCAATTGGTCGTAGACAGCTCTAGCACCGCTTAAACGCACGTACGCGCTGTCCCCCGCGTTTTAACCGCCAAGGGGATTACTCCCTAGTCTCCAGGCACGTGTCAGATATATACATCCAGGTCATTCCGGACGTGTTTGTCCTCTGCCTTTAAAGCAATAGGAGCTTACCCCCAGGGACTTGAAGTAATAAGGACGGAGGGCCTCTTTCAACATCGATGCACGGTGGTTAGCCTTGGATTGCGCTCTACCGTGCGCTAAGCGTACTTAGAAGCCCGAGTGACGACTTCACACGGTAGGTGGGCGCGCGAACTGAGCCTTGTGTCGCGAAATTCATGATtemplate strand: 5\u2032:ATCATGAATTTCGCGACACAAGGCTCAGTTCGCGCGCCCACCTACCGTGTGAAGTCGTCACTCGGGCTTCTAAGTACGCTTAGCGCACGGTAGAGCGCAATCCAAGGCTAACCACCGTGCATCGATGTTGAAAGAGGCCCTCCGTCCTTATTACTTCAAGTCCCTGGGGGTAAGCTCCTATTGCTTTAAAGGCAGAGGACAAACACGTCCGGAATGACCTGGATGTATATATCTGACACGTGCCTGGAGACTAGGGAGTAATCCCCTTGGCGGTTAAAACGCGGGGGACAGCGCGTACGTGCGTTTAAGCGGTGCTAGAGCTGTCTACGACCAATTGAGCGGCCTCGGCACCGG.The purified DNA fragment was ligated with the same double-stranded oligonucleotide 50-mer (purchased from Eurofins) as described above by T4 DNA ligase (NIPPON GENE).48. Briefly, the freeze-dried histones were mixed at an equal molar ratio under denaturing conditions. The histone octamer was refolded by dialysis against refolding buffer, containing 10\u2009mM Tris-HCl (pH 7.5), 1\u2009mM EDTA, 2\u2009M NaCl, and 5\u2009mM 2-mercaptoethanol. The histone octamer was purified by chromatography on a Superdex 200 gel filtration column . The resulting octamer was flash-frozen in liquid nitrogen, and stored at \u221280\u2009\u00b0C. The histone octamer and the 161 or 354\u2009bp DNA fragment were mixed, and the nucleosome was reconstituted by the salt dialysis method. The double-stranded oligonucleotide 47-mer containing a 3\u2009bp overhang at the 5\u2032 end was ligated to the cohesive DNA end of the reconstituted nucleosome. The oligonucleotide also contained a 9 base mismatched region (bubble) at the region 14\u201322\u2009bps from the 3\u2032 end. The resulting nucleosome with a bubble region was purified by non-denaturing gel electrophoresis, using a Prep Cell apparatus (Bio-Rad).The histone octamer was reconstituted as described previouslyThe sequences of both strands of the oligonucleotide 47-mer containing a bubble region are as follows:non-template strand: 5\u2032TTCTTAAATACCATGGCCATCTTCATTCCGGACGTGTTTGTCCTCTGtemplate strand: 5\u2032:AGGCAGAGGACAAACACGTCCGGAATGAGAGCTAATTTGGTATTTAAGAA.The complete DNA sequences of both strands for the mono-chromatosome transcription experiments are as follows:non-template strand: 5\u2032TTCTTAAATACCATGGCCATCTTCATTCCGGACGTGTTTGTCCTCTGCCTTTAAAGCAATAGGAGCTTACGGTCCACTTGTGTTTGGTGTGTTTGGGAATCCGGTGCCGAGGCCGCTCAATTGGTCGTAGACAGCTCTAGCACCGCTTAAACGCACGTACGCGCTGTCCCCCGCGTTTTAACCGCCAAGGGGATTACTCCCTAGTCTCCAGGCACGTGTCAGATATATACATCCAGGCCTTGTGTCGCGAAATTCATAGATtemplate strand: 5\u2032ATCTATGAATTTCGCGACACAAGGCCTGGATGTATATATCTGACACGTGCCTGGAGACTAGGGAGTAATCCCCTTGGCGGTTAAAACGCGGGGGACAGCGCGTACGTGCGTTTAAGCGGTGCTAGAGCTGTCTACGACCAATTGAGCGGCCTCGGCACCGGATTCCCAAACACACCAAACACAAGTGGACCGTAAGCTCCTATTGCTTTAAAGGCAGAGGACAAACACGTCCGGAATGAGAGCTAATTTGGTATTTAAGAA.The complete DNA sequences of both strands for the di-chromatosome transcription experiments are as follows:non-template strand: 5\u2032TTCTTAAATACCATGGCCATCTTCATTCCGGACGTGTTTGTCCTCTGCCTTTAAAGCAATAGGAGCTTACGGTCCACTTGTGTTTGGTGTGTTTGGGAATCCGGTGCCGAGGCCGCTCAATTGGTCGTAGACAGCTCTAGCACCGCTTAAACGCACGTACGCGCTGTCCCCCGCGTTTTAACCGCCAAGGGGATTACTCCCTAGTCTCCAGGCACGTGTCAGATATATACATCCAGGTCATTCCGGACGTGTTTGTCCTCTGCCTTTAAAGCAATAGGAGCTTACCCCCAGGGACTTGAAGTAATAAGGACGGAGGGCCTCTTTCAACATCGATGCACGGTGGTTAGCCTTGGATTGCGCTCTACCGTGCGCTAAGCGTACTTAGAAGCCCGAGTGACGACTTCACACGGTAGGTGGGCGCGCGAACTGAGCCTTGTGTCGCGAAATTCATGATtemplate strand: 5\u2032ATCATGAATTTCGCGACACAAGGCTCAGTTCGCGCGCCCACCTACCGTGTGAAGTCGTCACTCGGGCTTCTAAGTACGCTTAGCGCACGGTAGAGCGCAATCCAAGGCTAACCACCGTGCATCGATGTTGAAAGAGGCCCTCCGTCCTTATTACTTCAAGTCCCTGGGGGTAAGCTCCTATTGCTTTAAAGGCAGAGGACAAACACGTCCGGAATGACCTGGATGTATATATCTGACACGTGCCTGGAGACTAGGGAGTAATCCCCTTGGCGGTTAAAACGCGGGGGACAGCGCGTACGTGCGTTTAAGCGGTGCTAGAGCTGTCTACGACCAATTGAGCGGCCTCGGCACCGGATTCCCAAACACACCAAACACAAGTGGACCGTAAGCTCCTATTGCTTTAAAGGCAGAGGACAAACACGTCCGGAATGAGAGCTAATTTGGTATTTAAGAAK. pastoris RNAPII or approximately the corresponding amount of the partially purified S. scrofa RNAPII), TFIIS (0.14\u2009\u03bcM of K. pastoris TFIIS or 0.11\u2009\u03bcM of H. sapiens TFIIS), and the DY647-labeled primer RNA (5\u2032-DY647- AUAAUUAGCUC-3\u2032) (0.57\u2009\u03bcM) (Dharmacon), in the presence or absence of Spt4/5 (0.57\u2009\u03bcM), in a 7\u2009\u03bcL reaction at 30\u2009\u00b0C for 30\u2009min. For the experiment with K. pastoris RNAPII, the reaction solution contained 37\u2009mM HEPES-KOH (pH 7.5), 7.1\u2009mM MgCl2, 43\u2009mM potassium acetate, 0.29\u2009\u03bcM zinc acetate , 0.37\u2009mM HEPES-NaOH (pH 7.5), 7.1\u2009mM MgCl2, 37\u2009mM potassium acetate, 0.29\u2009\u03bcM zinc acetate, 29\u2009\u03bcM Tris(2-carboxyethyl)phosphine, 0.46\u2009mM DTT, 2.0% glycerol, 6.14\u2009mM NaCl, 0.29\u2009\u03bcM zinc chloride, 0.57\u2009mM UTP, 0.57\u2009mM GTP, and 0.57\u2009mM ATP. The reactions were then incubated for 15\u2009min at 4\u2009\u00b0C for transcription with K. pastoris RNAPII or 30\u2009\u00b0C for transcription with S. scrofa RNAPII. H1.2 was added to the reaction mixture in nucleosome:H1.2 ratios of 1:0, 1:3, 1:4.5, 1:6, and 1:9, and was bound to the nucleosome for 30\u2009min by an incubation at 4\u2009\u00b0C for transcription with K. pastoris RNAPII or at 30\u2009\u00b0C for transcription with S. scrofa RNAPII. H1.2 was dissolved in 10\u2009mM Tris-HCl buffer (pH 7.5), 0.05\u2009mM phenylmethylsulfonyl fluoride (PMSF), 0.25\u2009mM EDTA, 75\u2009mM NaCl, 10% glycerol, 10\u2009mM HEPES-KOH (pH 7.5), 0.5\u2009mM dithiothreitol, 50\u2009mM KCl, and 1\u2009mM 2-mercaptoethanol. CTP was then added to re-start the RNAPII progression. After the reaction was incubated with RNAPII phosphine, 0.14\u2009mM DTT, 0.7% glycerol, 0.57\u2009mM UTP, 0.57\u2009mM GTP, and 0.57\u2009mM ATP, at 30\u2009\u00b0C for 30\u2009min, followed by an incubation at 4\u2009\u00b0C for 15\u2009min. Afterwards, H1.2 was added to the reaction mixture at a nucleosome:H1.2 ratio of 1:6, and was bound to the nucleosome by an incubation at 4\u2009\u00b0C for 30\u2009min. CTP was then added to re-start the RNAPII progression. After a 5\u2009min reaction, 216\u2009\u03bcL of 0.5\u2009M EDTA was added to the reaction mixture. Under these conditions, a ~60-nt RNA product predominantly accumulated in the presence of H1.2. The resulting RNAPII-chromatosome complexes were fractionated by the GraFix method51. A gradient was prepared with low buffer (20\u2009mM HEPES-KOH (pH 7.5), 50\u2009mM potassium acetate, 0.2\u2009\u03bcM zinc acetate, 0.1\u2009mM Tris(2-carboxyethyl)phosphine, and 10% (w/v) sucrose) and high buffer (20\u2009mM HEPES-KOH (pH 7.5), 50\u2009mM potassium acetate, 0.2\u2009\u03bcM zinc acetate, 0.1\u2009mM Tris(2-carboxyethyl)phosphine, 25% (w/v) sucrose, and 0.1% glutaraldehyde). The sample was applied to the top of the gradient and centrifuged at 4\u2009\u00b0C for 16\u2009h at 124,779 \u00d7 g, using an SW41 rotor (Beckman Coulter). After centrifugation, the fractions containing the RNAPII-chromatosome complexes were collected and dialyzed twice against 20\u2009mM HEPES-NaOH (pH 7.5) buffer, containing 0.2\u2009\u03bcM zinc acetate and 0.1\u2009mM Tris(2-carboxyethyl)phosphine. The resulting RNAPII-chromatosome complexes were then concentrated with an Amicon Ultra 100\u2009K centrifugal filter unit (Millipore), until the DNA concentration of the sample reached 78.4\u2009\u03bcg/mL. The samples were applied to glow-discharged Quantifoil grids . Each grid was blotted at 4\u2009\u00b0C and 100% humidity in a Vitrobot Mark IV (Thermo Fisher Scientific) , and then immediately plunged into liquid ethane.The 6-FAM-labeled nucleosome (0.14\u2009\u03bcM) was mixed with RNAPII (0.14\u2009\u03bcM), TFIIS (0.14\u2009\u03bcM), and the DY647-labeled primer RNA (5\u2032-DY647- AUAAUUAGCUC-3\u2032) (0.57\u2009\u03bcM) (Dharmacon) in 1.5\u2009mL of reaction solution, containing 37\u2009mM HEPES-KOH (pH 7.5), 7.1\u2009mM MgCl\u2212/\u00c5, using the SerialEM software52.Cryo-EM images of the RNAPII-chromatosome complexes were collected by a Krios G3i electron microscope (Thermo Fisher Scientific), equipped with a K3 direct electron detector with a 25\u2009eV slit width of the BioQuantum energy filter (Gatan), and operated at 300\u2009kV at a pixel size of 1.07 \u00c5. Each image of the RNAPII-chromatosome complex was recorded with a 7\u2009s exposure time, and then fractionated into 40 frames with a total dose of 56.2 e53 and Gctf54, respectively. Preliminary RNAPII-chromatosome maps were then obtained after particle picking with gautomatch (http://www.mrc-lmb.cam.ac.uk/kzhang/) and quick 2D and 3D classifications with Relion3. Using the particle coordinates from these preliminary processing results, the neural networks for Topaz55 were trained, and then another round of particle picking was performed with Topaz. Finally, the coordinates from the Topaz picking and the initial gautomatch picking were merged, and served as the starting set for further image processing with Relion3.First, the motion correction and CTF estimation were performed with Relion3During the initial stage of image processing, the dataset was split in two batches that were processed independently. As the dataset was highly heterogeneous, refinements were mostly focused on the RNAPII region of the molecule, and to remove bad particles and particles centered on the nucleosome, 2D and 3D classifications were performed occasionally. After the initial 3D refinement using the RNAPII mask, the Bayesian polishing and CTF refinement were performed. After another 3D refinement, 3D classification was performed to remove RNAPII particles lacking downstream DNA. A 2.8\u2009\u00c5 resolution RNAPII map was obtained at this stage, and then another Bayesian polishing was performed to downscale particles to 1.49\u2009\u00c5/pix. Finally, 3D classifications with the mask enveloping RNAPII and nucleosome were done to select particles containing both RNAPII and nucleosome.56.Next, the particles from the two batches were merged and subjected to 3D classification, which revealed two different complexes of RNAPII transcribing a chromatosome (forms I and II). For each complex, RNAPII subtraction and 3D classification around the nucleosome were performed to select classes with high quality chromatosome density. A significant amount of continuous motion remained between the RNAPII and chromatosome, so local refinements for the RNAPII region and the chromatosome region were performed, in addition to the overall reconstruction. Composite maps were also calculated for each complex structure, using the phenix.combine_focused_maps tool in the Phenix package40, modeled manually with WinCoot (COOT57 for windows), and then refined against the consensus RNAPII reconstruction using phenix.real_space_refine tool in the Phenix package56. The atomic model for the chromatosome was based on previous crystal structures (PDB: 3LZ0 and 4QLC)58, modeled manually, and then refined against the nucleosome reconstruction of the form I complex. The RNAPII and nucleosome models were then fit into their respective overall reconstructions, and the interfaces between the two bodies and the connecting DNA were manually edited with WinCoot and ISOLDE59. Figures were prepared using UCSF Chimera60, ChimeraX61, and PyMOL.First, the RNAPII and the chromatosome were independently modeled. The atomic model for RNAPII was based on the previous crystal structure (PDB: 5XOG)Further information on research design is available in the\u00a0Supplementary informationPeer Review FileReporting Summary"} +{"text": "Earinus Wesmael, 1837 are transferred to a new genus, Chilearinus Sharkey gen. nov. Presently three Nearctic species of Earinus are recognized, i.e., Earinuserythropoda Cameron, 1887, Earinuslimitaris Say,1835, and Earinuszeirapherae Walley, 1935, and these are retained in Earinus. Earinuschubuquensis Berta, 2000 and Earinusscitus Enderlein, 1920 are transferred to Chilearinus, i.e., C.chubuquensis, and C.scitus, comb. nov. One other species is transferred to Chilearinus, i.e., Microgasterrubricollis Spinola, 1851, Chilearinusrubricollis, comb. nov. Two other Neotropical species, Earinushubrechtae Braet, 2002 and Earinusbourguignoni Braet, 2002 were described under the genus Earinus but are here transferred to Lytopylus, L.hubrechtae, and L.bourguignonicomb. nov. Two new species of Chilearinus are described, C.covidchronos and C.janbertspp. nov. The status of Agathislaevithorax Spinola,1851, Agathisrubricata Spinola,1851, and Agathisareolata Spinola, 1851 is discussed. A neotype is designated for Earinuslimitaris and diagnosed with a COI barcode. Earinusaustinbakeri and Earinuswalleyispp. nov. are described. The status of both Earinus and Chilearinus in the Americas is discussed. A revised key to the genera of Agathidinae of the Americas is presented.The Neotropical members formerly included in Earinus Wesmael, 1837 are transferred to a new genus, Chilearinus Sharkey gen. nov. Presently three Nearctic species of Earinus are recognized, i.e., Earinuserythropoda Cameron, 1887, Earinuslimitaris Say,1835, and Earinuszeirapherae Walley, 1935, and these are retained in Earinus. Earinuschubuquensis Berta, 2000 and Earinusscitus Enderlein, 1920 are transferred to Chilearinus, i.e., C.chubuquensis and C.scitus, comb. nov. One other species is transferred to Chilearinus, i.e., Microgasterrubricollis Spinola, 1851, Chilearinusrubricollis, comb. nov. Two other Neotropical species, Earinushubrechtae Braet, 2002, and Earinusbourguignoni Braet, 2002 were described under the genus Earinus but are here transferred to Lytopylus, L.hubrechtae, and L.bourguignoni comb. nov. Two new species of Chilearinus are described, C.covidchronos and C.janbert spp. nov. The status of Agathislaevithorax Spinola,1851, Agathisrubricata Spinola,1851, and Agathisareolata Spinola, 1851 is discussed. A neotype is designated for Earinuslimitaris and diagnosed with a COI barcode. Earinusaustinbakeri and Earinuswalleyi spp. nov. are described. The status of both Earinus and Chilearinus in the Americas is discussed. A revised key to the genera of Agathidinae of the Americas is presented.Neotropical species formerly included in c oxidase subunit I (COI) gene using standard insect primers LepF1 (5\u2032-ATTCAACCAATCATAAAGATATTGG-3\u2032) and LepR1 (5\u2032-TAAACTTCTGGATGTCCAAAAAATCA-3\u2032) (Molecular work was carried out at the CBG using standard protocols. A leg from each frozen-then-oven-dried specimen was destructively sampled for DNA extraction using a glass fiber protocol . ExtractATCA-3\u2032) . If inithttp://www.boldsystems.org/index.php/IDS_OpenIdEngine); (2) paste the COI sequence of the query organism (in forward orientation) into the query box and search against the appropriate library ; (3) the search results page shows the top hits based on percentage similarity starting with the closest matches ; (4) use the Tree-Based Identification button to generate a neighbor-joining tree and find the query taxon (name in red). This allows you to visualize how distant the query sequence is from the closest matches.The BOLD database can be used to identify specimens using the following steps: (1) navigate to the identification tab of the BOLD Systems database can also be employed to differentiate them. Members of Lytopylus differ most significantly in that they lack vein Cub in the hind wing. See couplet 25 in the key below.Notauli absent; hind coxal cavities open; tarsal claws with basal lobes; second submarginal cell quadrate, never petiolate; foretibia lacking sclerotized spines/pegs; hind wing Cub strong and emanating from an angle on the basal cell. Most similar morphologically to Head. Lateral carina on frons (as found in members of Alabagrus) absent; interantennal space slightly raised above antennal sockets; gena not extended ventroposteriorly into sharp prominence; mandible dorsoventrally flattened (twisted); labial palpus with 4 segments, third segment slightly more than \u00bd length of apical segment. Mesosoma. Propleuron lacking a sharp bump; notauli absent; mesoscutum smooth with a median pit (presumably a remnant of notauli), postscutellar depression absent; propodeum mostly smooth, sometimes with weak smooth sculpture medially; sclerite between hind coxal cavities and metasomal foramen absent. Precoxal groove absent or smooth and weakly impressed. Legs. Foretibia lacking dull pegs (unlike Earinus); mid- and hind tibia with blunt apical or preapical pegs; all tarsal claws with a rounded basal lobe. Wings. Forewing RS+Ma vein mostly present but not usually completely tubular; second submarginal cell large, quadrate and usually higher than long; RS of forewing complete to wing margin; hind wing r and r-m cross veins absent; hind wing vein Cub strong and emanating from an angle on the basal cell. Metasoma. First median tergite smooth, longer than apical width, lateral longitudinal carina absent or weak and short; remaining terga smooth; ovipositor ranging from as long as the body to twice the length of the body, but this is based on small sample of a few dozen species.Unknown.This is a species-rich genus with hundreds of species, based on specimens identified by MS. It is widespread in Chile and southern Argentina. A few species are found at high altitudes as far north as Ecuador and Colombia.Chilearinus in a broader concept of Earinus. Agathidinae from Chile. Since members of Chilearinus are by far the most species-rich of Chilean agathidines, and since his descriptions do not contradict membership in the genus, these species are probably members of Chilearinus, i.e., Agathislaevithorax, Agathisrubricata, and Agathisareolata. They certainly are not members of Agathis since this genus does not extend into the southern regions of South America. These specimens should be in the Hymenoptera collection of Maximilian Spinola whose collection is housed in the Museo Regionale di Scienze Naturali (MRSN) in Turin (Torino). One of us (MS) could not locate these specimens during a visit to MSRN in 1985, but a specimen of Chilearinus, Microgasterrubricollis Spinola, 1851, was present. Microgaster may seem an odd place for placement of what we now consider an agathidine, but such was the classification at the time. It is clear from the following that Spinola knew the species was closely related to Earinus, \u201cEste Microgastro habria pertenecido \u00e1 [sic] la primera seccion del G. Microdus, N. V. Es., y al sub-g\u00e9nero Earinus Wesm.\u201d .\u2640, Chile, Regi\u00f3n IX, PN Nahualbuta, COI barcode. BOLD sample ID H1145. BOLD BIN code BOLD:AAV0870. GenBank Accession Code OL702761.AATTTTATATTTTATATTTGGAATTTGATCGGGAATTTTAGGTTTATCAATAAGTTTAATTATTCGAATAGAATTAAGAGTAGGGGGTAATTTTATTGGTAATGATCAAATTTATAATAGAATTGTNGCTGCTCATGCTTTTATTATAATTTTTTTTATAGTTATACCAATTATAATTGGAGGATTTGGAAATTGATTAATTCCATTAATATTGGGGGGGCCAGATATAGCTTTCCCTCGAATAAATAATATAAGATTTTGATTATTAATTCCTTCATTATTATTATTAATTTTAAGGTCTTTAATTAATGTTGGGGTAGGTACTGGATGAACTGTTTATCCTCCTTTATCATTAAATATAAGTCATAGTGGTATATCTGTAGATTTAGCTATTTTTTCTTTACATATTGCTGGAATTTCTTCAATTATAGGTGCTATAAATTTTATTACAACTATTTTAAATATGTGAATAATTAATATTAAAATTGATAAAATACCTTTATTAGTTTGATCAATTTTAATTACGGCAATTTTATTATTATTATCTTTGCCAGTTTTAGCTGGAGCTATTACTATATTATTAACAGATCGTAATTTAAATACTAGATTTTTTGATCCTTCTGGAGGAGGAGATCCAATTTTATATCAACATTTATTTSee key.None.Named in acknowledgment of the covid pandemic occurring during the production of this manuscript.Taxon classificationAnimaliaHymenopteraBraconidae\ufeffSharkeysp. nov.AB9C892C-E5CA-5F6A-BE83-F0ED72EAEB87http://zoobank.org/AF4C4A3B-EBD8-4305-AF39-DC9176C868A837.493\u00b0S, 72.582\u00b0W, 1168 m, 8.ii.2005, Heraty, .\u2640, Chile, Regi\u00f3n IX, PN Nahualbuta, COI barcode. BOLD sample ID H12114. BOLD BIN: BOLD:AEM7846. GenBank Accession Code OL702760.TTTTAGGATTATCAATAAGTTTAATTATTCGAATAGAATTAAGAGTAGGTGGTAATTTTATTGGTAATGATCAAATTTATAATAGGATTGTNACTGCTCATGCTTTTATTATAATTTTTTTTATAGTTATACCAATTATAATTGGAGGATTTGGAAATTGATTAATTCCATTAATATTAGGGGGTCCAGATATAGCCTTCCCTCGAATAAATAATATAAGATTTTGATTATTAATTCCTTCATTATTATTATTAATTTTAAGATCTTTAATTAATGTTGGAGTAGGTACTGGATGAACTGTTTATCCTCCTTTATCATTAAATATAAGTCATAGTGGTATATCTGTAGATTTGGCTATTTTTTCTTTACATATTGCTGGAATTTCTTCAATTATAGGGGCTATAAATTTTATTACAACTATTTTAAATATATGAATAATTAATATTAAAATTGATAAAATACCTTTATTAGTTTGATCAATTTTGATTACAGCAATTTTATTATTATTATCTTTACCAGTTTTAGCTGGGGCTATTACTATATTATTAACAGATCGTAATTTAAATACTAGATTTTTTGATCCTTCTGGAGGGGGAGATCCAATTTTATATCAACATTTATTTTGATTTTTSee key.None.A conjunction of Paul Hebert and Dan Janzen in recognition of their enormous contributions towards the conservation of nature.Taxon classificationAnimaliaHymenopteraBraconidae\ufeffWesmael, 18373EB02370-41B3-561B-B842-5117B904EE03Earinus, i.e., E.erythropoda Cameron, 1887, E.limitaris , and E.zeirapherae Walley, 1935, and here we describe two more, Earinusaustinbakeri sp. nov. and Earinuswalleyi sp. nov. In the Nearctic, Earinus is common and widespread with the southernmost record being the sole recognized specimen of E.erythropoda from northern Sonora state, Mexico. Earinus differs from Chilearinus in the possession of pegs/spines in the foretibia and the characters given in the key.In the Americas, there are three previously recognized species of Hymenoptera Institute and borrowed specimens, there are probably between eight and 12 species in the Nearctic region. They are extremely similar in color, but there are obvious differences among specimens in body dimensions, degree of punctation, color of the hind coxae, ocellar configuration, ovipositor length, length and density of setae on the ovipositor sheath, and dimensions of the first metasomal tergum. Unfortunately, these are not sufficient to allow confident delineation of species limits. For example, the differences in the key between E.limitaris and E.erythropoda are trivial. There are numerous specimens scattered over the Nearctic region that will key to E.erythropoda, but they might all be E.limitaris, or the two nominal species may be conspecific, or there may be multiple cryptic species. Likewise, there are probably a number of undescribed Nearctic species that will key to either E.zeirapherae or E.austinbakeri. In other words, the key is sufficient to discriminate among the barcoded species and E.zeirapherae but not among these and the undescribed species. The key is presented in part to satisfy the code of Zoological Nomenclature to act as a diagnosis for E.austinbakeri and E.walleyi. Only dense sampling of COI barcodes and perhaps other genes will supply the information necessary to delimit Nearctic Earinus species.Based on the collection in the Taxon classificationAnimaliaHymenopteraBraconidae\ufeffSharkeysp. nov.107889AA-0F66-5D06-A568-CA774826DA26http://zoobank.org/D169A981-8A48-4E53-B1D1-CB072D89814744.2829\u00b0N, 77.7963\u00b0W, 131 m, 05\u201320.Jun.2014 . BOLD sample ID BIOUG33065-A05, BOLD BIN code BOLD:ADL5164. GenBank Accession Code OM158425.\u2640, Canada, Ontario, Ferris Provincial Park, Consensus barcode based on four specimens.ATTTTATATTTTATATTTGGGATTTGATCYGGAATTGTGGGKTTATCAATAAGTTTAATTATTCGTATGGARTTAAGAGTAGGGGGBAATTTAATTGGKAATGATCAAATTTATAATAGTATTGTTACTGCTCATGCATTTATTATAATTTTTTTTATAGTTATRCCAATTATAATTGGTGGGTTTGGTAATTGGTTAATTCCTTTAATATTAGGRGGTCCCGATATRGCTTTCCCTCGAATGAAYAATATAAGRTTTTGATTATTAATTCCTTCTTTATTATTATTAATTTTAAGATCTTTAATTAATATTGGGGTTGGAACTGGTTGAACGGTYTATCCTCCTTTATCATTRAATATAAGTCATAGTGGTATATCTGTTGATTTGGCTATTTTYTCTTTACATATTGCGGGRATTTCTTCTATTATAGGGGCAATAAATTTTATTACTACTATTTTAAATATATGAATAATAAATATTAAAGTTGATAAAATGTCTTTATTRATTTGATCAATTTTAATTACTGCTATTTTATTATTATTATCTTTACCTGTTTTAGCRGGRGCAATTACTATATTATTAACAGATCGTAATTTAAATACAAGATTTTTTGATCCTTCTGGAGGTGGGGATCCAATTTTATATCAACATTTATTTE.austinbakeri but differing by the characters given in the key as well as having the ovipositor sheath more setose. The COI barcodes of the two species differ by 6.29% (p-distance), reinforcing the conclusion that they are different species.Very similar to http://www.boldsystems.org).BIOUG01028-C01, BIOUG01028-F12, BIOUG32793-A05. These are sample IDs; the data for these specimens can be found by searching for these codes on BOLD .E.limitaris, or several more species may have similar morphologies. COI barcode data are needed. Several line drawings, modified from The sole identified specimen is the holotype. It differs little from many specimens that are widespread in the United States. It could be that they all belong to Taxon classificationAnimaliaHymenopteraBraconidae\ufeffEAE38190-F29E-5D79-A78E-22DCB293E7E9Bassuslimitaris Say, 1835.38\u00b055'N, 78\u00b049'W, 30.viii\u201319.ix.2005 . BOLD sample ID H1141. BOLD BIN code BOLD:AAU8493. GenBank Accession Code OM237775.\u2642, USA, West Virginia, Hardy County, 3 mi. NE Mathias, COI barcode based on 9 specimens.Consensus AATTTTATATTTTATATTTGGAATTTGATCAGGAATTTTAGGTTTATCAATAAGATTAATTATTCGAATAGAATTAAGDATAGGTGGTAATTTRATTGGTAATGATCAAATTTATAATAGTGTTGTTYCTGCTCATGCTTTTATTATAATTTTTTTTATAGTTATACCAATTATGATTGGRGGRTTTGGRAATTGATTAGTTCCTTTAATATTGGGRGGTCCTGATATAGCTTTYCCTCGAATAAATAATATAAGATTTTGATTATTAATTCCTTCTTTATTATTATTAATTTTGAGTTCTTTAATTAATATTGGGGTRGGGACTGGKTGAACAGTTTATCCTCCRTTATCTTTAAATATAAGRCATAGTGGAATATCAGTTGATTTAGCTATTTTTTCATTACATATYGCAGGAATTTCTTCAATTATAGGGGCAATAAATTTTATTACTACTATYATAAATATATGAATAATAAATATTAAAATTGATAAAATACCTTTATTAGTTTGATCAATTTTAATTACTGCTATTTTATTATTATTATCATTRCCAGTTTTAGCTGGRGCAATTACTATATTATTAACAGATCGAAATTTRAATACAAGATTTTTTGATCCTTCTGGAGGGGGGGATCCAATTTTATATCAACATTTATTTSee key.http://www.boldsystems.org).ASGLE-0444, ASGLE-0446, ASGLE-0449, ASGLE-0451, ASGLE-0452, ASGLE-0445, BIOUG01022-D11, BIOUG32892-B07. These are sample IDs; data on them can be found by searching for these codes on BOLD which differs by only 2.54% (p-distance) from E.limitaris , as well as either Missouri or Indiana, or both. It is unknown if Taxon classificationAnimaliaHymenopteraBraconidae\ufeffSharkeysp. nov.8DA974A7-E3F6-5E6F-A2B2-FF42AC77EBAAhttp://zoobank.org/BDFBEADA-2082-46A5-B648-EB181E09CBB558.3734\u00b0N, 94.1342\u00b0W, 3\u20137.vii.2007, Malaise trap . BOLD sample ID. 07PROBE-20853, BOLD BIN code BOLD:AAF9894. GenBank Accession Code FJ413805.\u2640, Canada, Manitoba, Churchill pump house, 15 km S Churchill, Goose Creek Road, Consensus barcode based on four specimens.TATTTTATATTTTATATTTGGAATTTGATCAGGTATTGTAGGTTTATCAATAAGATTAATTATTCGAATGGAATTAAGAGTGGGRGGTAATTTAATTGGRAATGATCAAATTTATAATAGTATTGTTACTGCTCATGCTTTTATTATAATTTTTTTTATAGTTATACCTATTATAATTGGGGGRTTTGGTAATTGATTARTCCCATTAATATTGGGAGGTCCTGATATAGCTTTCCCTCGTATAAATAATATGAGATTTTGATTATTAATCCCYTCTTTATTAATATTAATTTTAAGATCTTTAATTAATATTGGAGTAGGGACTGGTTGGACAGTTTATCCTCCKTTATCATTAAATATAAGTCATAGTGGAATATCTGTTGATTTGGCTATTTTTTCTTTACATATTGCGGGRGTTTCTTCTATTATAGGGGCAATAAATTTTATTACTACTATTTTAAATATRTGAATAATAAATATTAAAATTGATAAAATGTCTTTATTAATTTGATCAATTTTAATTACTGCTATTTTATTATTATTRTCTTTACCAGTTTTAGCAGGAGCTATTACTATATTATTAACAGATCGTAATTTAAATACAAGATTTTTTGATCCTTCYGGAGGGGGTGACCCAATTTTATATCAACATTTATTTE.zeirapherae, differing by the characters given in the key as well as having the ovipositor sheath less setose. The COI barcodes of the two species differ by 6.29% (p-distance) all but ensuring that they are different species.Very similar to http://www.boldsystems.org).All are from the same locality as the holotype, 07PROBE-23096, 07PROBE-23097, 09PROBE-A0304. These are specimen IDs; more data on the specimens can be found by searching for these codes on BOLD , former research scientist at the Canadian National Collection and author of Taxon classificationAnimaliaHymenopteraBraconidae\ufeffWalley, 1935766B1C04-99FA-52A3-8733-9263AE5C92E1\u2640, Grand River, Nova Scotia, 11.May.1932 (M. L. Prebble) No. 3847 .Tortricidae: Aclerishudsoniana, Choristoneurarosaceana, Rhyacioniaadana, Zeirapheracanadensis, Zeirapheragriseana, and Zeirapheraratzeburgiana. Since there are many species, including E.austinbakeri and E.walleyi, that are morphologically similar to E.zeirapherae, all hosts that do not belong to the genus Zeiraphera need confirmation.The following are all reported as hosts by Zeirapheraratzburgiana. Contrary to the image of the holotype in Figure The holotype Fig. is from (Modified from"} +{"text": "However, how cells interpret these post-translational microtubule modification codes to selectively regulate organelle positioning remains largely unknown. The endoplasmic reticulum\u00a0(ER) is an interconnected network of diverse morphologies that extends promiscuously throughout the cytoplasm3, forming abundant contacts with other organelles4. Dysregulation of endoplasmic reticulum morphology is tightly linked to neurologic disorders and cancer6. Here we demonstrate that three membrane-bound endoplasmic reticulum proteins preferentially interact with different microtubule populations, with CLIMP63 binding centrosome microtubules, kinectin (KTN1) binding perinuclear polyglutamylated microtubules, and p180 binding glutamylated microtubules. Knockout of these proteins or manipulation of microtubule populations and glutamylation status results in marked changes in endoplasmic reticulum positioning, leading to similar redistributions of other organelles. During nutrient starvation, cells modulate CLIMP63 protein levels and p180\u2013microtubule binding to bidirectionally move endoplasmic reticulum and lysosomes for proper autophagic responses.Organelles move along differentially modified microtubules to establish and maintain their proper distributions and functions The endoplasmic reticulum proteins CLIMP63, kinectin and p180 bind preferentially to subsets of microtubules with different post-translational modifications, thereby linking the \u2018tubulin code\u2019 to the intracellular distribution of membrane organelles. Although this code has been implicated in cargo selection and directed organelle movement2, how it is decoded to mediate transport and control distribution remains largely unknown.Eukaryotes compartmentalize cellular functions within distinct organelles, and regulation of organelle position is critical for cell health. Organelles are transported bidirectionally by motor and adaptor proteins along microtubules7. The ER is a compelling candidate for exploiting the complexity of the tubulin code, since it spreads throughout the cytoplasm in association with microtubules and makes abundant organelle contacts10. Most studies of ER shaping and organelle contacts have emphasized peripheral tubular ER. How the denser perinuclear ER is shaped and asymmetrically distributed remains largely unknown, although three ER membrane-bound proteins\u2014CLIMP63, p180 and KTN1\u2014localize prominently to perinuclear ER and are considered sheet-forming proteins11. Even so, depletion of CLIMP63 may paradoxically lead to\u00a0the expansion of ER matrices or\u00a0sheets in the periphery12, and perinuclear ER matrices or\u00a0sheets remain abundant even upon simultaneous knockdown of all three proteins, prefiguring more complex functional roles11.Endoplasmic reticulum (ER) comprises structurally and functionally divergent membrane compartments that include interconnected tubules, perinuclear matrices and sheets, and the nuclear envelope12, peripheral ER in CLIMP63-knockout cells is populated with increased numbers of dense matrices\u00a0or\u00a0sheets\u2014a \u2018dispersed\u2019 phenotype. KTN1 knockout also disperses ER, whereas p180-knockout cells exhibit a contrasting \u2018clustered\u2019 ER phenotype, with the peripheral network remaining tubular and perinuclear ER collapsing asymmetrically into a smaller area at one side of the nucleus to visualize their microtubule associations in cells ) that shape the tubules. The polygonal network is generated via atlastin-mediated tethering and fusion of tubules at three-way junctions and distributed via cytoskeletal interactions2. Modifications are dynamic and rapidly reversible, but evidence for how they affect microtubule-related functions has been limited2. We have shown here that KTN1 preferentially binds perinuclear polyglutamylated microtubules with long glutamate chains, whereas p180 binds glutamylated microtubules with either short or long chains. By contrast, CLIMP63 has a higher threshold for response to microtubule glutamylation. We cannot exclude that increased affinity\u00a0at higher glutamate numbers for TTLL7-modified microtubules stems from additional chains that TTLL7 initiates on tubulin tails, and not only from introduction of longer chains. Conversely, p180 is more sensitive to any increase in glutamate numbers on the tubulin tail and robustly binds both mono- and polyglutamylated microtubules. This differential effect on microtubule binding according to glutamylation state has previously been observed for the microtubule-severing ATPase spastin29 and may represent a general feature of this modification, enabling fine tuning of molecular interactions. Thus, a small difference in the number of glutamates added to tubulin side chains may exert a substantial qualitative effect on ER distribution. Other ER-localized, microtubule-binding proteins30 are likely to contribute to overall cellular ER positioning. Indeed, even in p180 and KTN1 double-knockout cells, TTLL7 overexpression still disperses ER, suggesting the involvement of other ER proteins. Moreover, tubular ER selectively moves along acetylated microtubules27, further indicating that ER distribution is broadly sensitive to microtubule modifications.Microtubule diversity can be achieved via different tubulin gene products, differential interactions with microtubule-associated proteins and numerous post-translational modifications31, and axonal microtubules are highly glutamylated2. Thus, p180 may affect microtubule remodelling by differentially recognizing glutamylated axonal microtubules. Of note, although dysregulation of ER shaping and microtubule polyglutamylation lead to different neurodegenerative diseases33, these diseases share some\u00a0similar cellular phenotypes, including mitochondrial distribution defects and axon degeneration, suggesting possible convergence.The ability of cells to dynamically control ER distribution through differential microtubule modifications has important functional implications. For instance, p180 regulates microtubule remodelling in axons1, and their ability to selectively distribute organelles relies on a tubulin code. Our results indicate that ER distribution is mediated via specific membrane-bound proteins with differential binding to different levels and types of microtubule glutamylation, broadly affecting distributions of most other organelles. ER thus interprets the tubulin code to regulate movement and positioning of cellular organelles. Rather than imbuing each organelle with its own sensing and response mechanisms, cells achieve organizational efficiency by using ER as a first-line sensor and responder. This role is exemplified during nutrient starvation, when cells increase CLIMP63 protein levels to move ER towards the perinuclear region, which also clusters lysosomes for efficient autophagic degradation. Then, cells harness enhanced p180\u2013microtubule binding to redistribute ER and lysosomes for a proper reset. There are likely to be other ER proteins that also decipher the tubulin code, with important implications for ER function in health and disease.When ER positioning is disrupted, distributions of other organelles are affected. Microtubules have key roles in organelle distribution21. pCMV6_p180s-myc-Flag (RC218816), pCMV6_KTN1-myc-Flag (RC219832), pCMV6_TTLL4-myc-Flag (RC205206) and pCMV6_CCP1-myc-Flag (RC220826) were obtained from Origene Technologies. pcDNA3.1_TTLL6-Flag (OHu07095), pcDNA3.1_CCP5-Flag (Ohu28493), pcDNA3.1_CCP6-Flag (OHu24335) and pcDNA3.1_p180L (OHu24745) were obtained from GenScript. Mutants of CLIMP63, p180s and KTN1 were generated in a pcDNA3.1(+) vector with a C-terminal HA-epitope tag. Specifically, Ser-to-Glu mutants of CLIMP63 were synthesized by GenScript. pcNDA3.1-KTN1, pN1-KTN1-mApple, pC1-sp-mScarlet-KTN1, pN1-KTN1-mNeonGreen, 2\u00d7Strep-p180s_29-381-mNeonGreen, 2\u00d7Strep-p180s_29-381-mNeonGreen and CLIMP63-mNeonGreen-2\u00d7Strep were also constructed using standard cloning procedures. 5\u2032 and 3\u2032 UTR of KTN1 were synthesized by Integrated DNA Technologies and ligated into the pN1-KTN1-mNeonGreen vector. Human TTLL4, TTLL6, TTLL7, CCP1, CCP5 and CCP6 inserts were also cloned into pCMV14-3\u00d7Flag and pN1-mApple. DNA oligonucleotides were synthesized by Integrated DNA Technologies.GFP-mCherry-LC3 was a gift from Juan S. Bonifacino. mEmerald-Sec61\u03b2, pcDNA3.1_CLIMP63-HA, mApple-SiT (Golgi apparatus), mKO-SKL (peroxisome), YFP-KDEL (ER) and CFP-LAMP1 (lysosome) were constructed as described previously34. Camptothecin (S1288), DC661 (S8808), etoposide (S1225) and MG132 (S2619) were purchased from Selleckchem. LysoSensor Green DND 189 (L7535), G418 (11811098) and puromycin (A1113802) were from Thermo Fisher Scientific. Centrinone B was from Tocris. GTP (G8877), ATP, Taxol (T7402), tunicamycin (SML1287), Earle\u2019s Balanced Salts and Duolink In Situ PLA kit were from Sigma-Aldrich. The Cathepsin L Activity Assay Kit (Fluorometric) (ab65306) was obtained from Abcam.LD540 was provided by C. Thiele2. HEK 293T cells were transfected with Avalanche-Everyday Transfection Reagent . U2OS and COS7 cells were electroporated using Cell Line Nucleofector Kit V and Cell Line Nucleofector Kit R following the manufacturer\u2019s instructions. Amounts of key plasmids transfected are (per 1\u2009\u00d7\u2009106 U2OS cells or 5\u2009\u00d7\u2009105 COS7 cells): 0.3\u2009\u03bcg CLIMP63-HA, 0.5\u2009\u03bcg p180s-HA, 2\u2009\u03bcg KTN1-mApple, 1\u2009\u03bcg CLIMP63-mEmerald (for overexpression), 2\u2009\u03bcg p180s-mEmerald, 4\u2009\u03bcg p180L-mEmerald, 4\u2009\u03bcg KTN1-mEmerald, as well as (in various vectors) 1\u2009\u03bcg TTLL4, 1\u2009\u03bcg TTLL6, 0.3\u2009\u03bcg TTLL7, 4\u2009\u03bcg CCP1, 0.5\u2009\u03bcg CCP5, and 1\u2009\u03bcg CCP6.All cell lines were obtained from the American Type Culture Collection: HEK 293T (CRL-11268), COS7 (CRL-1651), HeLa (CCL-2), RPE1 (CRL-4000) and U2OS (HTB-96) cells. HEK 293T, COS7 and HeLa cells were cultured in Dulbecco\u2019s Modified Eagle Medium , RPE1 cells were cultured in DMEM/F12 (1:1) Medium , and U2OS cells were cultured in McCoy\u2019s 5A medium (Thermo Fisher Scientific16600108); all were supplemented with 10% fetal bovine serum and 1\u00d7penicillin/streptomycin/amphotericin B (Thermo Fisher Scientific 15240112) at 37\u2009\u00b0C with 5%\u2009CO6 U2OS cells using Lonza Cell Line Nucleofector Kit V.For RNAi knock down of AKAP450, two siRNAs targeting ATATGAACACAGCTTATGA and AACTTTGAAGTTAACTATCAA were synthesized by Eurofins Genomics. Cells were transfected using Avalanche-Omni Transfection Reagent with 20\u2009pmol siRNA for 3 days. For RNAi knockdown of CCP5, ON-TARGETplus siRNA sets targeting human CCP5 were purchased from Horizon Discovery (LQ-009468-00-0005), and 60\u2009pmol siRNAs were transfected per 1\u2009\u00d7\u200910Primary antibodies used: mouse monoclonal anti-AKAP450 , rabbit polyclonal anti-Atlastin2 , rabbit polyclonal anti-Atlastin3 , rabbit monoclonal anti-Catalase , mouse monoclonal anti-Climp63 , mouse monoclonal anti-Flag M2 , rabbit polyclonal anti-GFP , mouse monoclonal anti-GM130 , rabbit polyclonal anti-GM130 , mouse monoclonal anti-HA , rabbit polyclonal anti-kinectin , rabbit monoclonal anti-kinectin , mouse monoclonal anti-Lamp1 , rabbit polyclonal anti-LC3 , rabbit polyclonal anti-Lunapark , mouse monoclonal anti-Myc , rabbit polyclonal anti-p180 , rabbit polyclonal anti-Pericentrin , rabbit polyclonal anti-polyglutamylation (polyE) , mouse monoclonal anti-glutamylation clone GT335 , rabbit polyclonal anti-REEP2 , rabbit polyclonal anti-REEP3 , rabbitpolyclonal anti-REEP4 , rabbit polyclonal anti-REEP5 , rabbit polyclonal anti-reticulon3 , rabbit polyclonal anti-reticulon4 , rabbit polyclonal anti-RPL3 , rabbit polyclonal anti-TOM20 , mouse monoclonal anti-TOM20 , rabbit polyclonal anti-TRAP\u03b1 , rat monoclonal anti-\u03b1-tubulin Alexa Fluor 647 , mouse monoclonal anti-\u03b1-tubulin , mouse monoclonal anti-\u03b2-tubulin . Alexa Fluor 405/488/568/633 conjugated goat anti-rabbit/mouse IgG (H+L) highly cross-adsorbed secondary antibodies were from Thermo Fisher Scientific. HRP-conjugated goat anti-mouse or anti-rabbit secondary antibodies were from Santa Cruz Biotechnology.7 and selected using 200\u20131,000\u2009\u03bcg\u00a0\u03bcl\u22121 G418 for two weeks; green-positive cells were sorted into mono-clones by flow cytometry using a MoFlo Astrios cell sorter (Beckman Coulter) and cultured in the presence of 200\u2009\u03bcg\u00a0\u03bcl\u22121 G418 for 2\u20133 weeks. Proliferated clones were verified by immunoblotting and fluorescence imaging.To generate U2OS cells stably expressing mEmerald-Sec61\u03b2, cells were transfected with the mEmerald-Sec61\u03b235. The targets used were: CLIMP63:GCCGCGCCCGCCATGCCCTCGG; p180 in U2OS:\u00a0GGTGTCGACTTTCTCCATGAAGG; p180 in COS7: GACACCAGGAAGATGCCAATGG; KTN1:\u00a0GAAAAGCCAGAAGAAGAGG and GTTAGGGAAAGAAAAAAGAAGG.All CRISPR\u2013Cas9 knockout assays used eSpCas9(1.1)CCAGCCCGCGGCCCGAGCCGCCGCCGCGCCCGCCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCTTGACCTACGGCGTGCAGTGCTTCGCCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAAGGTCTATATCACCGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGACCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCAAGCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGtccggactcagatctcgagctcaagcttcgaattctgcagtcgacggtaccgcgggcccgggatccCCCTCGGCCAAACAAAGGGGCTCCAAGGGCGGCCACG;For knock-in of CLIMP63, the same target as in CLIMP63 knockout was used, and a PCR fragment with 37\u2009bp homology arms on each side of the mEmerald-coding sequence was used as a homologous recombination template as follows:\u00a0GGCCTCCTCGGCTTGGCCGCCGTCGAGCCCGCCGTCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCTTGACCTACGGCGTGCAGTGCTTCGCCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAAGGTCTATATCACCGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGACCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCAAGCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGGAGCCCGCCGTCTACTTCAAGGAGCAGTTTCTGGAC. Note that amino acids 18\u201320 (EPA) were appended to both sides, acting as a linker..\u00a0To generate mEmerald-calreticulin knock-in COS7 cells, wild-type Cas9 with a gRNA targeting the end of the signal sequence of calreticulin (GAGCCCGCCGTCTACTTCAAGG) was selected, and a PCR fragment with 36\u2009bp homology arms on each side of the mEmerald-coding sequence was used as a homologous recombination template as follows: 15 before further analysis.To deplete the centrosome, cells were treated with 125\u2009\u03bcM CNB for 1 week as describedCells were quickly rinsed with PBS, directly lysed with sample buffer , and boiled for 5\u2009min. Proteins were then resolved by SDS\u2013PAGE using Mini-PROTEAN TGX Precast Protein Gels (Bio-Rad Laboratories) and transferred to nitrocellulose membranes using the Trans-Blot Turbo RTA Midi Nitrocellulose Transfer Kit (Bio-Rad Laboratories) following the manufacturer\u2019s instructions. Membranes were blocked with 4% milk in TBST , and incubated with primary antibody (diluted in blocking buffer) at 4\u2009\u00b0C overnight. After washing with TBST, membranes were incubated with secondary antibody at room temperature for 2\u2009h, followed by intensive washing with TBST. Immunoreactive proteins were visualized with GE Healthcare LS ECL Prime Western Blotting Detection Reagent (RPN2236) and imaged using a ChemiDoc XRS+ (Bio-Rad). Band intensities were quantified using Fiji software (NIH).\u22121 LD540 in PBS for 5\u2009min. Cells were mounted using Fluoromount-G (SouthernBiotech) and imaged using a Zeiss LSM880 confocal microscope in Airyscan mode equipped with a 63\u2009\u00d7\u20091.4 NA Plan-Apochromat oil objective (Carl Zeiss). Images were acquired using ZEN software (Carl Zeiss) and processed with ZEN software or Fiji (NIH).Cells were fixed with 4% paraformaldehyde in PBS (Lonza) for 30\u2009min at room temperature and permeabilized with 0.1% Triton X-100 in PBS for 10\u2009min. Alternatively, for immunostaining of glutamylation (GT335) and polyglutamylation (polyE), cells were fixed and permeabilized with cold methanol for 5\u2009min at \u221220\u2009\u00b0C. Then, after blocking with 3%\u2009BSA for 30\u2009min, cells were immunostained with polyE antibody at 4\u2009\u00b0C overnight, then with polyE and GT335 together at 4\u2009\u00b0C overnight, followed by secondary antibody staining at room temperature for 1\u2009h, and finally with anti-\u03b1-tubulin Alexa Fluor 647 at room temperature for 2.5\u2009h. For staining of lipid droplets with LD540 dye, cells were incubated with 0.1\u2009\u03bcg\u00a0mlr,\u03b8)-space representation of the cell\u2019s fluorescence distribution. For analysis referring to \u2018normalized\u2019 data, we account for the shape of the cytoplasm by finding the radius at each angle where the nuclear envelope and the edge of the cell are located. The fluorescence data were then rescaled to a normalized axis with the cytoplasm between the nuclear envelope and the cell periphery scaled from 0 to 100%. The nucleoplasm is scaled to stretch between \u221225 and 0, as a control. .Three-dimensional images were acquired using a Zeiss LSM880 confocal microscope in Airyscan mode and reconstructed using ZEN software (Zeiss Microscopy). Summed intensity projections were generated using floating point notation to carry precision. A custom macro in Fiji-ImageJ was used to define the centre of the nucleus and remove the signal of neighbouring cells to avoid perturbing the results. From the manually defined centre, a radius was drawn out past the furthest point on the cell and swept through 360\u00b0 in 0.1\u00b0 steps, taking a line profile each time and rescaling the data to correct for artifacts generated by the square shape of the pixels. The resulting data represents an for 30\u2009min on ice. Cell lysates were centrifugated twice at 20,000g for 20\u2009min at 4\u2009\u00b0C. The supernatant was supplemented with 1\u2009mM GTP and 40\u2009\u03bcM Taxol and incubated at 4\u2009\u00b0C or 37\u2009\u00b0C for 30\u2009min for tubulin polymerization before centrifugation at 20,000g for 30\u2009min at 4\u2009\u00b0C or 37\u2009\u00b0C, respectively. The resulting pellets (P) and supernatants (S) were collected and subjected to immunoblot analysis. In some experiments, only the pellets and supernatants of the 37\u2009\u00b0C samples are shown.To test the microtubule-binding affinities of CLIMP63, p180 and KTN1, cells were lysed in PIPES buffer was performed according to the manufacturer\u2019s instructions. Samples were observed under a Zeiss LSM880 confocal microscope with a 20\u2009\u00d7\u20091.0 NA objective using the Airyscan function. The total intensity of the PLA signal per cell was quantified using Fiji software.g at 4\u2009\u00b0C for 30\u2009min. Supernatants were combined with Strep-Tactin XT beads (IBA Lifesciences) and rotated gently for 3\u2009h. After extensive washing with lysis buffer (PBS plus 500\u2009mM NaCl and 1% Triton X-100) and then wash buffer (IBA Lifesciences), bound proteins were eluted with Strep-Tactin XT Elution Buffer (IBA Lifesciences). Eluted proteins were subjected to multiple rounds of PBS dilution and concentration using 10 kDa protein concentrators (Sigma-Aldrich), before being aliquoted and frozen in liquid nitrogen.Deletion fragments of p180 and KTN1 as well as full-length CLIMP63 were expressed as fusions with mNeonGreen-2\u00d7Strep in HEK 293T cells. 48\u2009h post-transfection, cells were lysed in PBS (Lonza) plus 500\u2009mM NaCl, 1% Triton X-100, and protease inhibitors and then centrifugated at 30,00036. TTLL4 and TTLL6 were expressed in Escherichia coli and purified as previously described18. TTLL7 was also expressed in E. coli and purified as previously described19\u00a0. Taxol-stabilized microtubules were polymerized out of 98.5% unmodified tubulin and 1.5% biotinylated brain tubulin29 (Cytoskeleton T333P). Unmodified microtubules were modified using TTLL4, TTLL7 or TTLL6 at 1:10 molar ratio of enzyme to tubulin at room temperature in 20\u2009mM HEPES (pH 7.0), 50\u2009mM NaCl, 10\u2009mM MgCl2, 1\u2009mM glutamate, 1\u2009mM ATP, 0.5\u2009mM TCEP, and 10\u2009\u03bcM Taxol for 4.5\u2009h for TTLL4, between 20\u2009min and 2\u2009h for TTLL7, and between 7.5 and 22\u2009h for TTLL6. Control microtubules were incubated with the enzymes under the same conditions but with aspartate, which is not a substrate for TTLL glutamylases, instead of glutamate. Enzymes were removed through a high-salt wash as previously described29. The extent of glutamylation was determined by liquid chromatography\u2013electrospray mass spectrometry29 (LC\u2013MS). The spectra display the characteristic distributions of masses with peaks separated by 129 Da, which corresponds to one glutamate . Next, a solution containing 60\u2009mM Pipes (pH 6.8), 0.7\u2009mM MgCl2, 0.7\u2009mM EGTA, 50\u2009mM KCl, 10\u2009mM 2-mercaptoethanol, 10\u2009\u03bcM Taxol, 1% F127 Pluronic, 1.4\u2009mg/ml casein, 20\u2009mM glucose, glucose oxidase, and catalase was flushed into the chamber, followed by the same solution containing 4.7\u2009nM mNeon-labeled p180, KTN1 or CLIMP63. Images were acquired after allowing for equilibration for 5\u2009min at room temperature using total internal reflection fluorescence (TIRF) microscopy at an exposure of 100\u2009ms for the GFP channel. Unlabelled microtubules were visualized using interference reflection microscopy38. Multiple fields of view were imaged. Background corrected line scan average intensities were measured using Fiji software. Multiple chambers were quantified for each condition.For microtubule-binding assays, microtubules were immobilized in chambers made of silanized glassTotal mRNA were extracted using TRIzol (Thermo Fisher Scientific 15596018) and Direct-zol RNA Miniprep , then reverse-transcribed using the SuperScript IV First-Strand Synthesis System . Real-time PCR primers, designed by a free online tool developed by Integrated DNA technologies, were as follows: CCP5-RT: GACTGCCAGGAACTGCTAAA and AGGAGCTCCCGATGGTAATA; GAPDH-RT: GGTGTGAACCATGAGAAGTATGA and GAGTCCTTCCACGATACCAAAG.Real-time PCR was performed using Applied Biosystems PowerUp SYBR Green Master Mix with Applied Biosystems QuantStudio 6 Flex real-time PCR instrument. Data were collected and analysed in QuantStudio Real-time PCR Software and Microsoft Excel using the 22. Images were acquired with a Zeiss LSM880 confocal microscope equipped with a 32-channel multi-anode spectral detector (Carl Zeiss) using a 63\u00d7/1.4 NA objective lens, at 37\u2009\u00b0C and with 5%\u2009CO2. Fluorophores were excited simultaneously using 458, 514 and 594\u2009nm lasers and a 458/514/594\u2009nm beam splitter, with images collected onto a linear array of 32 photomultiplier tube elements in \u03bb mode at 9.7\u2009nm bins from 468 to 687\u2009nm. Spectra were defined by imaging singly labelled cells for each of the fluorophore reporters, using the same acquisition and laser settings as for multiply labeled cells. Multispectral images were unmixed using the linear unmixing package in ZEN (Carl Zeiss).Multispectral imaging was performed as described previouslyz-projection was performed using maximum projection before quantification. The mCherry-positive vesicles indicate autophagosomes already fused with lysosomes, as the GFP signal would be quenched by the acidic environment of lysosomes; vesicles with both GFP and mCherry fluorescence indicate autophagosomes not yet fused with lysosomes. Quantifications of these two types of vesicles were performed manually using Fiji software.For autophagosome\u2013lysosome fusion assessments, U2OS cells were transfected with GFP-mCherry-LC3 for 24\u2009h, treated with EBSS for 2\u2009h before fixation with 4% paraformaldeyde in PBS, and imaged using a Zeiss LSM880 confocal microscope in Airyscan mode equipped with a 63\u2009\u00d7\u20091.4 NA Plan-Apochromat oil objective (Carl Zeiss). A For lysosome acidification assays, U2OS cells were labeled with 1 \u03bcM LysoSensor Green DND 189 for 4\u2009min and immediately imaged within one minute with a Zeiss Axio microscope using a 20\u00d7/0.4 NA objective. Images were captured with ZEN software, and total intensities of each cell were quantified in Fiji.6 cells were assayed in each sample.Cathepsin L activity assays were carried out using the Abcam Cathepsin L Activity Assay kit following the manufacturer\u2019s instructions; 1\u2009\u00d7\u200910P values are shown on top of the corresponding columns, as determined by one-way ANOVA followed by Dunnett\u2019s multiple comparisons test, Mann\u2013Whitney test, Kruskal\u2013Wallis test or by unpaired two-sided t-test as indicated in the figure legends. When representative images are shown, at least three repeats were performed except for Extended Data Figs. No statistical method was used to predetermine sample size. All groups were randomly assigned and every group represents a distincttreatment or condition. Data were not analysed in a double-blinded manner. All comparisons were performed using Graphpad Prism or Microsoft Excel software. Data are expressed as means \u00b1 s.d., Further information on research design is available in the\u00a0Any methods, additional references, Nature Research reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at 10.1038/s41586-021-04204-9.Supplementary InformationThis file contains Supplementary Text and Supplementary Figs 1\u20137.Reporting SummaryPeer Review FileSupplementary Video 1Wild-type or CLIMP63, p180 KO U2OS cells stably expressing mEmerald\u2013Sec61\u03b2 were labelled with Lysotracker Red, starved with EBSS, and recorded for 2.5\u00a0h with imaging at 1 min intervals. Scale bar, 10 \u00b5m."} +{"text": "This novel, state-of-the-art platform fundamentally advances the utility of RM to study protective and pathogenic T cell responses.T cell receptor (TCR) clonotype tracking is a powerful tool for interrogating T cell mediated immune processes. New methods to pair a single cell\u2019s transcriptional program with its TCR identity allow monitoring of T cell clonotype-specific transcriptional dynamics. While these technologies have been available for human and mouse T cells studies, they have not been developed for Rhesus Macaques (RM), a critical translational organism for autoimmune diseases, vaccine development and transplantation. We describe a new pipeline, \u2018RM-scTCR-Seq\u2019, which, for the first time, enables RM specific single cell TCR amplification, reconstruction and pairing of RM TCR\u2019s with their transcriptional profiles. We apply this method to a RM model of GVHD, and identify and track More icities) .To date, clonotype tracking technologies have been developed for both mouse and human TCRs, facilitating studies of T cell clonal dynamics at an unprecedented level of molecular sensitivity \u201311. HoweTo address these limitations, nonhuman primate (NHP) models (particularly using rhesus macaques (RM)) have been essential , and havThe identification and tracking of TCR clonotypes are of critical importance for each of the clinical settings described above, in order to understand the molecular mechanisms driving immune protection or disease pathogenesis. For acute GVHD (aGVHD), dissecting the link between T cell clonal architecture and disease is particularly relevant, given that donor-derived alloreactive T cells are the main culprits in the aGVHD-mediated destruction of the skin, intestine, and liver \u201334. Accuin vitro and in vivo identification and tracking of RM derived T cell clonotypes. Pairing of in vivo identified alloreactive clonotypes with their transcriptional profile revealed a highly activated cytotoxic CD8 T cell signature during NHP aGVHD.We now describe the design and validation of RM-specific primers compatible with the human 10x Genomics single cell sequencing platform, which accurately amplifies the RM TCR alpha and beta regions. Amplified fragments aligned to our custom-assembled and annotated RM TCR reference permit, for the first time, the This study was conducted in strict accordance with (USDA) United States Department of Agriculture regulations and the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. It was approved by the Massachusetts General Hospital and Biomere Animal Care and Use Committees. T cells were obtained from healthy colony RM or from animals who underwent allogenic HCT in the setting of previously published studies .137Cs) radiation. Responder PBMCs were stained with cell trace violet as per manufacturer\u2019s instructions. 2\u00d7105 T cell-enriched responder PBMCs along with an equal number of stimulator PBMCs were added to each well in a 96-well plate (Corning) in X-vivo-15 medium (BioWhitaker) supplemented with 10% FBS (Irvine Scietific) and incubated at 37\u00b0C for a total of 5 days. Cell culture media change was performed on day 3 of the culture. At the end of 5 days, cells were stained with an extracellular antibody for CD3 (clone SP34-2), CD20 (clone 2H7) and CD14 for 20min at 4\u00b0C, and high-, medium- and non-proliferating CD3+ T cells (identified based on the dilution of CTV) were sorted and processed for single cell sequencing using the Chromium Next GEM Single Cell 5\u2019 Reagent Kit v1 with optimized RM primers as described below.RM PBMCs were isolated from whole blood by Ficoll gradient centrifugation, and then used for MLR assays either immediately, or after liquid nitrogen cryopreservation in 10% dimethyl sulfoxide (DMSO)/90% fetal bovine serum (FBS). At the time of the MLR, stimulator PBMCs were irradiated with 3500 cGy of , we also validated primers and PCR conditions compatible with the newer Next GEM Single Cell 5\u2019 v2 Sample Index Plate TT (see below). https://assets.ctfassets.net/an68im79xiti/31W4aZOJ8C2ipxzTrcQIWn/72c5c2bc3dd7784f44f5218ca345ce04/CG000086_ChromiumSingleCellV_D_J_ReagentKits_UG_RevM.pdf). Primer sequences are listed in Single Cell V(D)J v1: GEMs were generated using v2 Gel Beads and the v1 Target Enrichment kit substituting the custom primers for the off-the-shelf human/mouse T Cell Mix 1 and 2 premixed primers. Sample Indexing was performed using the i7 Multiplex Kit plate . TCR alpha and beta primers were designed to perform in a nested PCR approach to optimize target enrichment of the respective constant regions Table\u00a01.PCR was performed with an initial denaturation temperature of 98\u00b0C for 45 seconds, followed by denaturation at 98\u00b0C for 20 seconds. Annealing was optimal at 67\u00b0C for 30 seconds with extension of 72\u00b0C for 1 min. A total of 20 PCR cycles were performed with a final extension step at 72\u00b0C for 1 min.https://assets.ctfassets.net/an68im79xiti/57JaTECQNBPSpyDz8oucdi/92da9f62521ea1f5c781ba234bd0a0f5/CG000331_ChromiumNextGEMSingleCell5-v2_UserGuide_RevC.pdf). TRAC inner and outer primers were 2996bp apart, and the beta chain inner and outer primers were 354bp apart. Primer sequences are listed in Next GEM Single Cell 5\u2019 v2: GEMs were also generated using v2 reagent kits for GEM generation and library preparation while substituting the custom RM primers for the off-the-shelf human/mouse T Cell Mix 1 and 2 premixed primers. Sample Indexing was performed using the Dual Index Kit TT (10x Genomics PN-1000215). Primers Table\u00a02 PCR was performed with an initial denaturation temperature of 98\u00b0C for 45 seconds, followed by denaturation at 98\u00b0C for 20 seconds. Annealing was optimal at 62\u00b0C for 30 seconds with extension of 72\u00b0C for 1 min. A total of 20 PCR cycles were performed with a final extension step at 72\u00b0C for 1 min. Sequencing results utilizing v2 primers are listed in The reference transcriptome Data S4 1. We first filtered Ensembl\u2019s gtf file for v104 of the macaca mulatta gene annotation using cellranger\u2019s mkgtf command with the following attributes:\u2013attribute=gene_biotype:protein_coding \u2013attribute=gene_biotype:lincRNA \u2013attribute=gene_biotype:miRNA \u2013attribute=gene_biotype:antisense \u2013attribute=gene_biotype:IG_LV_gene \u2013attribute=gene_biotype:IG_V_gene \u2013attribute=gene_biotype:IG_V_pseudogene \u2013attribute=gene_biotype:IG_D_gene \u2013attribute=gene_biotype:IG_J_gene \u2013attribute=gene_biotype:IG_J_pseudogene \u2013attribute=gene_biotype:IG_C_gene \u2013attribute=gene_biotype:IG_C_pseudogene \u2013attribute=gene_biotype:TR_V_gene \u2013attribute=gene_biotype:TR_V_pseudogene \u2013attribute=gene_biotype:TR_D_gene \u2013attribute=gene_biotype:TR_J_gene \u2013attribute=gene_biotype:TR_J_pseudogene \u2013attribute=gene_biotype:TR_C_gene2. We next edited cellranger\u2019s mkref program to use more than one thread using the command:sed -i \u201888s/num_threads/#num_threads/\u2019/cellranger-6.0.0/bin/rna/mkref3. We then created the reference transcriptome using cellranger\u2019s mkref command: cellranger-6.0.0/bin/rna/mkref \u2013genome=MMulatta_Ensembl_v104 \u2013fasta=MMulatta_Ensembl_v104_combined.fasta \u2013genes=MMulatta_Ensembl_v104_combined_filtered.gtf \u2013nthreads=32 \u2013memgb=1281. We used version 6.0.2 of Cellranger to create the initial VDJ reference based on IMGT , 36.2. We used the following command to generate the initial reference: cellranger-6.0.2/lib/bin/fetch-imgt \u2013genome vdj_IMGT_rhesus \u2013species \u201cMacaca mulatta\u201d.3. This version of the VDJ reference did not include constant regions, so we added the following sequences - obtained from IMGT and Ensembl - to the reference:>340|TRAC*01 IMGT|TRAC|C-REGION|TR|TRA|None|01ATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGGCTCTAAATCCAATGACACCTCTGTCTGCCTATTTACTGATTTTGATTCTGTAATGAATGTGTCACAAAGCAAGGATTCTGACGTGCATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTTAAGAGCAACGGTGCTGTGGCCTGGAGCAACAAATCCGATTTTGCATGTACAAGCGCCTTCAAGGACAGCGTTATTCCAGCAGACACCTTCTTCCCCGGCACAGAAAGTGTCTGTGATGCCAACCTGGTTGAGAAAAGCTTTGAAACAGATATGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGCTGA>341|TRAC*02 TRAC_205_Ensembl_CDS|TRAC|C-REGION|TR|TRA|None|02ATGAATGTGTCACAAAGCAAGGATTCTGACGTGCATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTTAAGAGCAACGGTGCTGTGGCCTGGAGCAACAAATCCAATTTTGCATGTACAAGCGCCTTCAAGGACAGCGTTATTCCAGCAGACACCTTCTTCCCCGGCACAGAAAGTGTCTGTGATGCCAACCTGGTTGAGAAAAGCTTTGAAACAGATATGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGCTGA>342|TRAC*03 TRAC_201_Ensembl_CDS|TRAC|C-REGION|TR|TRA|None|03ATGAATGTGTCACAAAGCAAGGATTCTGACGTGCATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTTAAGAGCAACGGTGCTGTGGCCTGGAGCAACAAATCCAATTTTGCATGTACAAGCGCCTTCAAGGACAGCGTTATTCCAGCAGACACCTTCTTCCCCGGCACAGAAAGTGTCTGTGATGCCAACCTGGTTGAGAAAAGCTTTGAAACAGATATGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGCTGA>343|TRAC*04 TRAC_202_Ensembl_CDS|TRAC|C-REGION|TR|TRA|None|04ATGAATGTGTCACAAAGCAAGGATTCTGACGTGCATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTTAAGAGCAACGGTGCTGTGGCCTGGAGCAACAAATCCAATTTTGCATGTACAAGCGCCTTCAAGGACAGCGTTATTCCAGCAGACACCTTCTTCCCCGGCACAGAAAGTGTCTGTGATGCCAACCTGGTTGAGAAAAGCTTTGAAACAGATATGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGCTGA>344|TRAC*05 TRAC_203_Ensembl_CDS|TRAC|C-REGION|TR|TRA|None|05ACTGGGGTAAACAACCTCTTCTTTGGGACTGGAACAAGACTCACCGTTCTTCCAGATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGGCTCTAAATCCAATGACACCTCTGTCTGCCTATTTACTGATTTTGATTCTGTAATGAATGTGTCACAAAGCAAGGATTCTGACGTGCATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTTAAGAGCAACGGTGCTGTGGCCTGGAGCAACAAATCCAATTTTGCATGTACAAGCGCCTTCAAGGACAGCGTTATTCCAGCAGACACCTTCTTCCCCGGCACAGAAAGTGTCTGTGATGCCAACCTGGTTGAGAAAAGCTTTGAAACAGATATGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGCTGA>345|TRAC*06 TRAC_204_Ensembl_CDS|TRAC|C-REGION|TR|TRA|None|06ATGAATGTGTCACAAAGCAAGGATTCTGACGTGCATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTTAAGAGCAACGGTGCTGTGGCCTGGAGCAACAAATCCAATTTTGCATGTACAAGCGCCTTCAAGGACAGCGTTATTCCAGCAGACACCTTCTTCCCCGGCACAGAAAGTGTCTGTGATGCCAACCTGGTTGAGAAAAGCTTTGAAACAGATATGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGCTGA>346|TRBC1*01 NW_001114291|TRBC1|C-REGION|TR|TRB|None|01AGGACCTGAAAAAGGTGTTCCCACCCAAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACGCTGGTGTGCCTGGCCACAGGCTTCTACCCCGACCACGTGGAGCTGAGCTGGTGGGTGAACGGGAAAGAGGTGCACAGTGGGGTCAGCACGGACCCACAGCCCCTCAAGGAGCAGCCCGCCCTCGAGGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCACAACCCCCGCAACCACTTCCGCTGCCAAGTCCAGTTCTATGGGCTCTCGGAGGATGACGAGTGGACCGAGGACAGGGACAAGCCCATCACCCAAAAGATCAGCGCCGAGGTCTGGGGTAGAGCAGACTGTGGCTTCACCTCGGTGTCCTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCCTGCTGGGGAAGGCCACCCTGTATGCTGTGCTGGTCAGTGCCCTCATGTTGATGGCCATGGTCAGAGGAAGGATTTC>347|TRBC1*02 IMGT000073|TRBC1|C-REGION|TR|TRB|None|02AGGACCTGAAAAAGGTGTTCCCACCCAAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACGCTGGTGTGCCTGGCCACAGGCTTCTACCCCGACCACGTGGAGCTGAGCTGGTGGGTGAACGGGAAAGAGGTGCACAGTGGGGTCAGCACGGACCCACAGCCCCTCAAGGAGCAGCCCGCCCTCGAGGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCAGCCACCTTCTGGCACAACCCCCGCAACCACTTCCGCTGCCAAGTCCAGTTCTATGGGCTCTCGGAGGATGACGAGTGGACCAGGACAGGGACAAGCCCATCACCCAAAAGATCAGCGCCGAGGTCTGGGGTAGAGCAGACTGTGGCTTCACCTCAGTGTCCTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCCTGCTGGGGAAGGCCTCCCTGTATGCTGTGCTGGTCAGTGCCCTCATGTTGATGGCCATGGTCAAGAGGAAGGATTTC>348|TRBC2*03 IMGT000073|TRBC2|C-REGION|TR|TRB|None|01AGGACCTGAAAAAAGTGTTCCCACCCAAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACGCTGGTGTGCCTGGCCACAGCTTCTACCCCGACCACGTGGAGTTGAGCTGGTGGGTGAACGGGAAAGAGGTGCACAGTGGGGTCAGCACGGACCCACAGCCCCTCAAGGAGCAGCCCACCCTCGAGGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCACAACCCCCGCAACCACTTCCGCTGCCAAGTCCAGTTCTATGGGCTCTCGGAGGATGACGAGTGGACCGAGGACAGGGACAAGCCCATCACCCAAAAGATCAGCGCTGAGGCCTGGGGTAGAGCAGACTGTGGCTTCACCTCTGAGTCTTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGGTCAGTGCCCTCGTGCTGATGGCCATGGTCAAGAGAAAGGATTCCThis resulted in an initial set of 348 sequences. We then checked these sequences for premature stop codons, frameshifts, and other potential problems using 10x\u2019s enclone software, v0.5.42. This identified 44 problematic sequences which were removed, leaving us with a final total of 304 VDJ segments.Samples were aligned using the Cellranger multi pipeline (v6.0.2), with VDJ libraries listed as \u201cvdj-t\u201d to indicate T cell libraries. Samples were aggregated with the Cellranger aggr pipeline (v6.0.2) with normalize=none. Our reference transcriptome was created with the Cellranger makeref command, and used genome assembly Mmul_10 as the reference genome , and EnsWe used FASTQC to assess the quality of our sequencing data. After alignment of this data, we loaded the filtered_feature_bc_matrix.h5 file from Cellranger multi into Seurat and removed genes which were present in less than 5 cells. This resulted in a dataset with 38,147 cells by 16,163 genes. Following the recommendations in Lucken and Theis to normaWe then limited the dataset to samples from the PBMC, the MLR experiments, and the GVHD target organs. We then removed genes present in less than 5 cells, resulting in a dataset with 23,618 cells by 15,195 genes. This dataset was normalized using the same pipeline previously described in this section, retaining 22 PC\u2019s from the PCA.Clonotypes used in the gene expression analysis were identified using cellranger\u2019s default settings in \u201ccellranger vdj\u201d for the scTCR-Seq libraries. Clonotypes used for the Shannon Diversity metric calculations were identified by grouping together T cells on the basis of the CDR3 region in the alpha chain or beta chain. This is to provide a more stringent test of the specificity of clonotype calling, as Cellranger\u2019s default settings do not allow for clonotypes to be shared across different donors. The Morisita index was calculated using the \u201cvegan\u201d package in R.As discussed in the Results section, the clonotypes used for the alpha chain and beta chain Morisita heatmaps . The primers targeting the beta chain were specifically designed to amplify both TRBC1 and TRBC2. Primer length, CG content and annealing temperatures were adjusted to be used with the forward primers supplied in the human 5\u2019 10x Genomics kit. Cycling conditions were modified from the original 10x protocol to allow optimal amplification of the targeted region in RM 2. We haRM-scTCR-Seq primers annealed to the alpha and beta RM constant region and amplified transcripts covering the complete alpha and beta loci including the variable V and J regions for the alpha as well as V, D and J regions for the beta locus. and can We assessed the quality of our primers using summary statistics from 10x\u2019s \u2018Cellranger\u2019 software , and found that our primers consistently amplify high-quality reads from the TCR alpha and beta chains. For this assessment, we prepared peripheral blood mononuclear cells (PBMCs) from three representative RMs and used these PBMCs to perform RM-scTCR-Seq. We observed that 49-52% of the aligned reads mapped to the alpha chain and 22-25% of the reads aligned to the beta chain Table S2In addition to demonstrating that the sequencing reads mapped to known TCR loci, we were also able to reconstruct the alpha and beta chains from these reads Table S2These data demonstrate, for the first time, efficient amplification of the RM TCR alpha and beta region by utilizing optimized RM primer pairs compatible with the human 5\u2019 10x Genomics single cell sequencing platform. Amplified reads enabled high quality beta chain reconstruction in almost all, and reconstruction of full TCRs in a significant number of sequenced cells.in vitro assays or in vivo disease models enables the evaluation of the clonal repertoire, clonal dynamics, and the tracking of specific T cells over time. To validate our single cell TCR sequencing and analysis pipeline, we first utilized an in vitro allo-proliferation assay: the mixed lymphocyte reaction (MLR) (Identification of T cell clonotypes using on (MLR) \u201347. To pon (MLR) (Table on (MLR) .Using the RM-scTCR-Seq pipeline, we were also able to track individual clonotypes throughout the three MLR peaks (To further assess our ability to accurately track T cell clonotypes samples) , 50 betw). Importantly we were also able to identify clonotypes that existed in both the Blood sample and the high-proliferating population or beta chain MI (MI <0.001), demonstrate the specificity of the RM-scTCR-Seq approach or not (termed `MLR-`). Each MLR+ cluster incorporated at least 37 cells Table\u00a03.ted gene , 52 was d VISION to scored VISION as well d VISION .in vivo utility of the RM-scTCR-Seq pipeline.These results demonstrate the feasibility of combining RM-scTCR-Seq and scRNA-Seq to identify and interrogate alloreactive T cell clonotypes in aGVHD target organs in RM, providing proof-of-concept of the TCR repertoire analysis has become a fundamental tool to understand the immune responses to infections and vaccines, as well as the immunopathogenesis of rejection after solid organ transplantation, and GVHD after HCT , 54\u201358. Prior to the work described herein, two major limitations significantly hindered the development of T cell clonotype tracking in RM. They were (1): The lack of optimized, single cell sequencing-compatible primers covering both the TCR alpha and beta region for RM; (2) The poor genomic annotation of the alpha and beta loci in RM, which resulted in inadequate reconstruction of amplified VDJ PCR reads. To address both of these technical barriers, we have designed a robust set of RM-specific TCR primer pairs which anneal to the constant region of the alpha and beta TCR loci, and which are compatible with the commonly used single cell sequencing platform from 10x Genomics. Optimizing PCR cycling and temperature conditions resulted in efficient amplification of the alpha and beta regions of the RM TCR and generated productive TCR reconstructions. Additionally, we created an updated reference for the TCR alpha and beta chain region, enhancing our ability to annotate the assembled RM TCR\u2019s.www.10xgenomics.com/resourses/dataset). The difference in the alignment rate may be explained by differing levels of annotation for these organisms: 10x provides human references which have been refined over time, but this resource does not exist for RM, which necessitated our creation of a new reference (see Methods) by combining elements of the Ensembl and IMGT references, and then carefully filtering out sequences identified by 10x\u2019s enclone software that contained errors . As annotation of the RM TCR improves, we anticipate some gains in read mapping. As mentioned earlier, we have noticed that UTR regions are annotated in the human and mouse VDJ references, and we expect that addition of these sequences to the RM reference will improve alignment rates. We additionally tested whether our reads adequately covered all genomic V(D)J regions and confirmed that in the 30 primary VDJ samples analyzed, reads mapped to 91% (179 of 196) of all V(D)J regions from our reference J gene. We note that this mapping efficiency is somewhat less than the ~91% of reads mapping to the human VDJ reference, in a human dataset provided by 10x and the National Cancer Center. VT is supported by an ASTCT New Investigator Award and the CIBMTR/Be The Match Foundation Amy Strelzer Manasevit Research Program Award. AS is supported by the Searle Scholars Program, the Beckman Young Investigator Program, a Sloan Fellowship in Chemistry, and the NIH . LK is supported by NIH grants U19 Al1051731, R01 HL095791, P01 HL158504, and by a Leukemia and Lymphoma Society TRP grant.AS reports compensation for consulting and/or SAB membership from Merck, Honeycomb Biotechnologies, Cellarity, Repertoire Immune Medicines, Hovione, Third Rock Ventures, Ochre Bio, FL82, and Dahlia Biosciences unrelated to this work. AS has received research support from Merck, Novartis, Leo Pharma, Janssen, the Bill and Melinda Gates Foundation, the Moore Foundation, the Pew-Stewart Trust, Foundation MIT, the Chan Zuckerberg Initiative, Novo Nordisk and the FDA unrelated to this work. LK is on the scientific advisory board for HiFiBio and Mammoth Biosciences. She reports research funding from Kymab Limited, Magenta Therapeutics, BlueBird Bio, and Regeneron Pharmaceuticals. She reports consulting fees from Equillium, FortySeven Inc, Novartis Inc, EMD Serono, Gillead Sciences, Vertex Pharmaceuticals, and Takeda Pharmaceuticals. LK reports grants and personal fees from Bristol Myers Squibb that are managed under an agreement with Harvard Medical School.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."} +{"text": "First, our study reveals a unified molecular genetic landscape of cortical cell types that integrates their transcriptome, open chromatin and DNA methylation maps. Second, cross-species analysis achieves a consensus taxonomy of transcriptomic types and their hierarchical organization that is conserved from mouse to marmoset and human. Third, in situ single-cell transcriptomics provides a spatially resolved cell-type atlas of the motor cortex. Fourth, cross-modal analysis provides compelling evidence for the transcriptomic, epigenomic and gene regulatory basis of neuronal phenotypes such as their physiological and anatomical properties, demonstrating the biological validity and genomic underpinning of neuron types. We further present an extensive genetic toolset for targeting glutamatergic neuron types towards linking their molecular and developmental identity to their circuit function. Together, our results establish a unifying and mechanistic framework of neuronal cell-type organization that integrates multi-layered molecular genetic and spatial information with multi-faceted phenotypic properties.Here we report the generation of a multimodal cell census and atlas of the mammalian primary motor cortex as the initial product of the BRAIN Initiative Cell Census Network (BICCN). This was achieved by coordinated large-scale analyses of single-cell transcriptomes, chromatin accessibility, DNA methylomes, spatially resolved single-cell transcriptomes, morphological and electrophysiological properties and cellular resolution input\u2013output mapping, integrated through cross-modal computational analysis. Our results advance the collective knowledge and understanding of brain cell-type organization The BRAIN Initiative Cell Census Network has constructed a multimodal cell census and atlas of the mammalian primary motor\u00a0cortex in a landmark effort towards understanding brain cell-type diversity,\u00a0neural circuit organization and brain function. Unique among body organs, the human brain is a vast network of information processing units, comprising billions of neurons interconnected through trillions of synapses. Diverse neuronal and non-neuronal cells display a wide range of molecular, anatomical, and physiological properties that together shape the network dynamics and computations underlying mental activities and behaviour. Brain networks self-assemble during development, leveraging genomic information shaped by evolution to build a set of stereotyped network scaffolds that are largely identical among individuals; life experiences then customize neural circuits in each individual. An essential step towards understanding the architecture, development, function and diseases of the brain is to discover and map its constituent elements of neurons\u00a0and other cell types.5. Neurons are remarkably complex and heterogeneous, both locally and in their long-range axonal projections, which can span the entire brain and connect to many target regions. Many conventional techniques analyse one neuron at a time, and often study only one or two cellular phenotypes in an incomplete way . As a result, despite major advances in past decades, phenotypic analyses of neuron types have remained severely limited in resolution, robustness, comprehensiveness and throughput. Complexities in the relationship between different cellular phenotypes have fuelled long-standing debates on neuronal classification6.The notion of a\u00a0\u2018neuron type\u2019, with similar properties among its members,\u00a0as the basic unit of brain circuits has been an important concept for over a century; however, rigorous and quantitative definitions have remained surprisingly elusive11. Similarly, single-cell DNA methylation and chromatin accessibility studies have begun to reveal cell-type-specific genome-wide epigenetic landscapes and gene regulatory networks in the brain15. Notably, the scalability and high information content of these methods enable comprehensive quantitative analysis and classification of all cell types, which are readily applicable to brain tissues across species and provide a quantitative means of comparative analysis17.Single-cell genomics technologies provide unprecedented resolution and throughput to measure the transcriptomic and epigenomic profiles of individual cells and have rapidly influenced many areas of biology including neuroscience, promising to catalyse a transformation from phenotypic description and classification to a mechanistic and explanatory molecular genetic framework for the cellular basis of brain organization. The application of single-cell RNA sequencing (scRNA-seq) to the neocortex and other brain regions has revealed a complex but tractable hierarchical organization of transcriptomic cell types that are consistent overall with knowledge from decades of anatomical, physiological and developmental studies but with an unmatched level of granularity19. Imaging-based single-cell transcriptomics and its combination with functional imaging, and integration of electrophysiology and single-cell sequencing, enable mapping of the spatial organization and key phenotypic properties of molecularly defined cell types24. Finally, molecular classification of cell types enables genetic access to specific cell types using transgenic mice27 and, more recently, enhancer-based viral vectors32. All of these methods have been applied to brain tissues in independent studies, but not yet in a coordinated fashion to establish how different modalities correspond with one another, and whether a molecular genetic framework is explanatory for other functionally important cellular phenotypes.Other recent technological advances provide the resolution and throughput to analyse whole-brain neuronal morphology and comprehensive projection mapping33. A key concept is the Brain Cell Census, similar conceptually to a population census, that defines the constituent neuronal and non-neuronal cell types and their proportions, spatial distributions and defining phenotypic characteristics. This cell-type classification, organized as a taxonomy, should aim for consensus across modalities and across mammalian species for conserved types. Beyond the cell census, a Brain Cell Atlas would be embedded in a 3D common coordinate framework (CCF) of the brain34, in which the precise location and distribution of all cell types and their multi-modal features are registered and displayed. This spatial framework facilitates integration, interpretation and navigation of various types of information for understanding brain network organization and function.The overarching goal of the BRAIN Initiative Cell Census Network (BICCN) is to leverage these technologies to generate an open-access reference brain cell atlas that integrates molecular, spatial, morphological, connectional and functional data for describing cell types in mouse, human and non-human primate36. We describe a synthesis of eleven companion studies through a coordinated multi-laboratory effort. In these studies, we derive a cross-species consensus molecular taxonomy of cell types using scRNA-seq or single-nucleus RNA sequencing (snRNA-seq), DNA methylation and chromatin accessibility data40. In mouse, we map the spatial cellular organization by multiplexed error-robust fluorescence in situ hybridization (MERFISH)41, characterize morphological and electrophysiological properties by multimodal profiling using patch clamp recording, biocytin staining and scRNA-seq (Patch-seq)43, describe the cellular input\u2013output wiring diagrams by anterograde and retrograde tracing44, identify glutamatergic neuron axon projection patterns by Epi-retro-seq45, Retro-MERFISH41 and single-neuron complete morphology reconstruction46, and describe transgenic driver lines targeting glutamatergic cell types on the basis of marker genes and lineages47. Finally, we integrate this information into a cohesive description of cell types in MOp. These datasets are organized by the BRAIN Cell Data Center (BCDC) and made public through the BICCN web portal (https://www.biccn.org). Key concepts and terms are described in Extended Data Table Here we present the cell census and atlas of cell types in the primary motor cortex of mouse, marmoset and human , suggesting another level of regulation in defining single-cell connectional specificity.Cell-type transcriptional and epigenetic signatures guide the generation of genetic tools for targeting glutamatergic pyramidal neuron types and fate mapping their progenitor types.Multi-site coordination within BICCN and data archives enabled a high degree of standardization, computational integration and creation of open data resources for community dissemination of data, tools and knowledge.Major findings:37. The combined sc/snRNA-seq datasets contained a\u00a0large number of cells profiled using both droplet-based and deep full-length sequencing methods and LIGER, to combine the transcriptomic and epigenomic datasets and derive an integrated molecular taxonomy consisting of 56 neuronal cell types 37 and transcriptional regulatory networks. Similarly, we established M1 cell-type taxonomies for human (127 t-types) and marmoset (94 t-types) by unsupervised clustering of snRNA-seq data, followed by integration with epigenomic datasets38.A mouse MOp molecular taxonomy was derived from seven scRNA-seq and snRNA-seq (sc/snRNA-seq) datasets and single-nucleus methylcytosine sequencing (snmC-seq2) and single-nucleus assay for transposase-accessible chromatin using sequencing (snATAC-seq) datasets)37 Fig. . This inLamp5, Sncg and Vip) and three medial ganglionic eminence (MGE)-derived subclasses ), layer and projection pattern in mouse for glutamatergic excitatory neurons , extratelencephalic (ET), corticothalamic (CT), near-projecting (NP) and layer 6b (L6b)), and non-neuronal functional subclasses .To establish a consensus classification of MOp and M1 cell types among mouse, human and marmoset, we integrated snRNA-seq datasets across species and identified 45 conserved t-types, including 24 GABAergic (\u03b3-aminobutyric acid-producing), 13 glutamatergic and 8 non-neuronal types , whereas others aligned several-to-several . This may reflect over- or under-clustering, limitations in aligning highly similar cell types, or species-specific expansion of cell-type diversity.The resolution of this cross-species consensus taxonomy was lower than that derived from each species alone, owing to variation in gene expression across species. The degree of species alignments varied across consensus types Fig. ; some tyn\u2009=\u200940 nuclei) were under-sampled compared with marmoset (n\u2009=\u2009463) and mouse , and average expression was not adequately estimated38.We expected that cell types from more recent common ancestors would share more similar gene expression profiles. Indeed, transcriptomic profiles of consensus cell types were more correlated between human and marmoset, and had 25\u201350% fewer differentially expressed genes than between primate and mouse Fig. . The oneGlutamatergic subclasses expressed 50\u2013450 marker genes and, unexpectedly, the majority of markers were species-enriched Fig. . This ev51, to identify cell types in situ and map their spatial organization. We selected a panel of 258 genes on the basis of prior knowledge of marker genes for major cortical cell types and genes identified using sc/snRNA-seq data, and we imaged approximately 300,000 individual cells across MOp and adjacent areas41.We used MERFISH, a single-cell transcriptome imaging methodLamp5, Sncg, Vip, Sst and Pvalb subclasses) and good correspondence at cluster level41.Clustering analysis of the MERFISH-derived single-cell expression profiles resulted in a total of 95 cell clusters in MOp Fig. , which s41. Notably, IT cells, the largest branch of neurons in the MOp, formed a largely continuous gradient of cells with correlated gradual changes between their expression profiles and their cortical depths41 identified projection targets of different neuron types in the MOp41 Fig. . Retrogr 41 Fig. . Overall42. We mapped these cells to the mouse MOp transcriptomic taxonomy37 and obtained their transcriptomes using Smart-seq2 sequencingy37 Fig. . Cells wy37 Fig. , therebyy37 Fig. .Fig. 3CoSst interneurons were often characterized by large membrane time constants, pronounced hyperpolarization sag, and rebound firing after stimulation offset. However, within each subclass, there was substantial variation in morpho-electric properties between t-types. This variation was not random but organized such that transcriptomically similar t-types had more similar morpho-electric properties than distant t-types. For example, excitatory t-types from the IT subclasses with more similar transcriptomes were also located at adjacent cortical depths, suggesting that distance in t-space co-varied with anatomical distance42, even within a layer , and cortico-subcortical projecting neurons in L5 ET. Many cortico-thalamic projecting neurons were also observed in L6 CT levels to integrate the L5 ET Epi-retro-seq cells with the ALM Retro-seq cells and observed enrichment of MY-projecting cells in the same cluster45.Enrichment of L5 ET neurons with Epi-retro-seq (40.2% versus 5.62% in unbiased profiling of MOp using snmC-seq2) enabled investigation of subtypes of L5 ET neurons known to project to multiple subcortical targets in TH, VTA+SN, pons and MYers Fig. . MY-projers Fig. , in agre12. We identified 511 differentially CH-methylated genes (CH-DMGs) and 58,680 CG-DMRs across the L5 ET clusters are reliable markers of regulatory elements such as enhancersers Fig. . We alsoers Fig. . For exa25. Here we present a genetic toolkit for dissecting and fate-mapping glutamatergic pyramidal neuron (PyN) subpopulations largely on the basis of their developmental genetic programs.Genetic access to specific neural subpopulations and progenitors is necessary for multi-modal analyses to validate t-types, fate-map their developmental trajectories, and study their function in circuit operation55 12.5 RGs in the dorsal neuroepithelium, distributed along a medial-high and lateral-low gradient, consistent with their mRNA expression at this stage60. These RGs generated PyNs across all cortical layers, suggesting multipotency generate PyNs either directly or indirectly through intermediate progenitors\u00a0(IPs)55 Fig. . Temporanes Fig. that faincy Fig. .Fig. 5GePlxnd1 (ITPlxnd1), L5b and L6 for ET-Fezf2 (ETFezf2), L6 for CT-Tle4 (CTTle4). Anterograde projection tracing in MOp of adult animals demonstrated that ITPlxnd1 projected to multiple ipsilateral and contralateral cortical areas and to STR/caudate putamen (CP); ETFezf2 projected robustly to several ipsilateral cortical sites, CP and numerous subcortical targets including TH, MY and corticospinal tract; CTTle4 projected specifically to a set of thalamic nuclei47 neurons resided in L5a and projected ipsilaterally to multiple cortical areas, contralaterally to homotypic and heterotypic areas, and bilaterally to CP neurons resided in L2; although they also projected to ipsilateral cortical and striatal targets, and to homotypic contralateral cortex, they extended minimal projections to heterotypic contralateral cortex and CP ) and retrograde (cholera toxin b (CTb)) tract tracing44 region using classic anterograde , ET and CT (Ntsr1 and Tle4) subclasses with distinct laminar distributions62.We generated a fine-grained areal and laminar distribution map of multiple MOp-ul projection neuron populations using retrograde tracing44 , ventral anterior-lateral complex (VAL) and ventral medial nucleus (VM)) and cortical areas such as MOs and SSp.Viral tracers were used to systematically examine MOp-ul cell subclass-specific inputs and outputs46, augmented with publicly available single-cell reconstructions from the Janelia Mouselight project18. Additional analysis was also conducted using BARseq64. This analysis revealed a rich diversity of projection patterns within the IT, ET and CT subclasses , Rspo1 for L4/5 IT (1), Htr2c for L4/5 IT (2-3), and Rorb for L4/5 IT and L5 IT and uniform manifold approximation and projection (UMAP) embedding of all IT neurons (excluding the highly distinct L6 IT Car3 cells) from 11 mouse molecular datasets, including 6 sc/snRNA-seq datasets, and the snmC-seq2, snATAC-seq, Epi-retro-seq, MERFISH and Patch-seq data Fig. . This repo1 Fig. , a L4 cevel Fig. , probabl9 after co-clustering all the SMART-seq glutamatergic transcriptomes from both regions ons Fig. . In UMAP42, cells from the L4/5 IT_1 type had no or minimal apical dendrites without tufts in L1, in contrast to cells from the L2/3 IT, L4/5 IT_2 and L5 IT types, which had tufted apical dendrites exhibited two local morphological features typical of L4 neurons from sensory cortices type and Npnt for SCF L5 ET (2-3) types display a largely gradual transition across cortical depth or layers and master transcription factors specific to neuronal subclasses in the combined transcriptomic and epigenomic datasets Fig. . We founUnderstanding the principles of brain circuit organization requires a detailed understanding of its basic components. The current effort combines a wide array of single-cell-based techniques to derive a robust and comprehensive molecular cell-type classification and census of the primary motor cortex of mouse, marmoset and human, coupled with a spatial atlas of cell types and an anatomical input\u2013output wiring diagram in mouse. We demonstrate the robustness and validity of this classification through strong correlations across cellular phenotypes, and strong conservation across species. Together these data comprise a cell atlas of the primary motor cortex that encompasses a comprehensive reference catalogue of cell types, their proportions, spatial distributions, anatomical and physiological characteristics, and molecular genetic profiles, registered into a CCF. This cell atlas establishes a foundation for an integrative study of the architecture and function of cortical circuits akin to reference genomes for studying gene function and genome regulatory architecture. Furthermore, it provides a map of the genes that contribute to cellular phenotypes and their epigenetic regulation. These data resources and associated tools enabling genetic access for manipulative experimentation are publicly available. This body of work provides a roadmap for exploring cellular diversity and organization across brain regions, organ systems and species.10, our multimodal cross-species study of the primary motor cortex suggests that a general principle of cortical cell-type organization is its hierarchical relationship, whereby high-level classes linked by major branches comprise progressively finer subpopulations connected by minor branches. In this scheme, the higher-level classes and subclasses are categorically and concordantly distinct from each other across modalities, are conserved across species, and probably arise from different developmental programs, such as GABAergic neuron derivatives of different zones of the ganglionic eminences or the layer-selective glutamatergic neurons derived sequentially from progenitors of the cortical plate. At the lower branch levels (types or clusters), however, while certain cell types are highly distinct , distinctions and boundaries among many other clusters can be ambiguous and vary among different modalities.Substantiating previous studies69, is the coexistence of discrete and continuous variations of cell features across modalities at the lower branch level. A compelling example is the continuous and concordant variation of transcriptomic, anatomical and physiological properties along cortical depth within multiple cell populations, including the glutamatergic L2/3\u2013L6 IT and GABAergic Sst and Pvalb subclasses. Although some of the variations may result from technical factors, such as differences in the resolution of measurements across data modalities (with transcriptomics providing the highest granularity at present), a major source of these continuous variations may reflect true biology, supported by the coordinated variation across transcriptomic, spatial, morphological and physiological properties as shown by MERFISH and Patch-seq. Therefore, another emerging principle of cell type organization is the coexistence of discrete and continuous variations that underlie cell-type diversity.In this context, another important finding, consistent with and building on multiple other studies11. Region-specific connectivity patterns of similar molecular types may be a major factor defining the functional specificity of the primary motor cortex.Together, the principles of hierarchical organization comprising discrete classes and types as well as continuum within and across subpopulations represent a more nuanced and biologically realistic description of cell-type landscape, with implications in cell classification and census. For example, the multimodal variations at finer granularity may preclude a fully discretized representation of cell types with consistency across cell phenotypes, and may explain some of the discrepancies in estimated numbers of cell types using different approaches. An intriguing question is whether continuous variations of cell features will increase further or become more discretized in the context of neural circuit operation, converging to a set of distinct functional elements from a more continuous cellular landscape. An example of this is regionalization. We identify a MOp-specific input\u2013output wiring diagram\u2014however, transcriptomic cell types are generally shared between MOp and its neighbouring cortical areasOur findings have major implications for understanding the biological basis of cellular identity towards a more rigorous, quantitative and satisfying definition and classification of cell types. First and foremost, our discovery of the compelling correspondence across molecular genetic, anatomical and physiological features of hierarchically organized cell populations, reflecting developmental origins and mainly conserved across mammalian species, demonstrates the biological validity and genomic underpinning of major cell types. These findings establish a unifying and mechanistic framework of cell-type classification that integrates multi-layered molecular genetic information with multi-faceted phenotypic properties. Thus, single-cell transcriptomics and epigenomics can serve as powerful approaches for establishing a foundational framework of cell types, owing to not only their unparalleled scalability but also to their representation of the underlying molecular genetic programs rooted in development and evolution. Physiological, morphological and connectional characterizations assign functional attributes to cells; their concordance with molecular identities provides strong validation to the molecularly defined cell types, whereas their differential variations reveal additional, probably network- and activity-driven factors that contribute to further refinement of cell types.37. Looking ahead, it is important to note that at more refined levels, the number of cell types that can be distinguished will probably change with additional cellular features characterized at greater breadth and depth using new methods and approaches.While the higher levels of the hierarchy comprise ~around 25 subclasses that are identified with remarkable consistency across multiple species and experimental modalities, many finer levels of cell properties do not neatly segregate into discrete and consistent sets of cell types with perfect correspondence among data modalities. These include aspects of continuous distributions, species specializations and mismatches between molecular and anatomical phenotypes that may result from developmental events no longer represented in the adult. Different methods provide somewhat different granularity of clustering, and thus different numbers of putative cell types. For example, single-cell transcriptomics identifies around 100 clusters representing the terminal leaves of this hierarchically branched organizationOverall, the landscape of cell types appears to be generated from a combination of specification through evolutionarily driven and developmentally regulated genetic mechanisms, and refinement of cellular identities through intercellular interactions within the network in which the cells are embedded. In this scenario, genetic mechanisms drive intrinsic or cell-autonomous determination of cell fate, as well as progressive temporal generation of cell types from common progenitor pools that explain global similarities and continuous features of cellular phenotypes reflecting developmental gradients. Network influences can drive further phenotypic refinement that may not be reflected in the adult genetic signature\u2014for example for axonal projection and synaptic connectivity that may reflect transient or stochastic developmental events, region or circuit-specific and/or activity or plasticity-dependent modification to form and reshape functionally specific circuits. Future studies focusing on these mechanisms and testing of the ensuing hypotheses will enable a deeper understanding of the nature of variability among related cell types in the mammalian brain.70.Evolutionary conservation is strong evidence of functional significance. The demonstrated conservation of cell types from mouse, marmoset, macaque and human suggests that these conserved types have important roles in cortical circuitry and function in mammals and even more distantly related species. We also find that similarity of cell types varies as a function of evolutionary distance, with substantial species differences that represent either adaptive specialization or genetic drift. For the most part, species specializations tend to appear at the finer branches of the hierarchical taxonomy. This result is consistent with a recent hypothesis in which cell types are defined by common evolutionary descent and evolve independently, such that new cell types are generally derived from existing genetic programs and appear as specializations at the finer levels of the taxonomic treeA surprising finding across all homologous cell types was the relatively high degree of divergence for genes with cell-type-specific expression in a given species. This observation provides a clear path to identify core conserved genes underlying the canonical identity and features of those cell types. Furthermore, it highlights the need to understand species adaptations superimposed on the conserved program, as many specific cellular phenotypes may vary across species including gene expression, epigenetic regulation, morphology, connectivity and physiological properties. As we illustrated in the Betz cells, there is clear homology across species in the L5 ET subclass, but variation in many measurable properties across species.Our findings have major implications for the consideration of model organisms to understand human brain function and disease. Despite major investments, animal models of neuropsychiatric disorders have often been characterized by \u2018loss of translation\u2019, fuelling heated debates about the utility of model organisms in the development of treatments for human diseases. Cell census information aligned across species will be highly valuable for making rational choices about the best models for each disease and therapeutic target. For example, the characterization of cell types and their properties shown in Fig. The approach we took to generate a cell census and atlas through a systematic dissection of cell types opens up numerous avenues for future work. The MOp census and atlas provides a foundational platform for the broad neuroscience community to accumulate and integrate cell-type information across species. Classification of cell types based on their molecular, spatial and connectional properties in the adult sets the stage for developmental studies to understand the molecular genetic programs underlying cell-type specification, maturation and circuit assembly. The molecular genetic information promises to deliver tools for genetic access to many brain cell types via transgenic and enhancer virus strategies. A combination of single-cell transcriptomics and functional measurements may further elucidate the roles of distinct cell types in circuit computation during behaviour, bridging the gap between molecular and functional definition of cell types. The systematic, multi-modal strategy described here can be extended to the whole brain, and major efforts are underway in the BICCN to generate a brain-wide cell census and atlas in the mouse with increasing coverage of human and non-human primates.Car3), the layer 5 neurons projecting to extratelencephalic targets (L5 ET), the CT-projecting neurons in layer 6 (L6 CT), the NP neurons found in layers 5 and 6, and the L6b neurons whose projection patterns remain largely unknown.In this manuscript we have adopted a nomenclature for major subclasses of cortical glutamatergic excitatory neurons, which have long-range projections both within and outside of the cortex, following a long tradition of naming conventions that often classify neurons based on their projection targets. This nomenclature is based on our de novo transcriptomic taxonomy Fig. that orgWhile the IT, CT, NP and L6b neurons have been consistently labelled as such in the field, the L5 ET neurons have not been named consistently in the literature, largely owing to their large variety of projection targets and other phenotypic features that vary depending on cortical areas and species. Here we use the term L5 ET to refer to this prominent and distinct subclass of neurons as a standard name that can be accurately used across cortical regions and across species, and we provide our rationale below.48. Accordingly, various names incorporating these features have been adopted to refer to L5 ET versus L5 IT cells, such as L5b versus L5a, thick-tufted versus thin-tufted and burst-firing versus regular-firing. The most common term used to refer to L5 ET cells residing in motor cortical areas has been PT, which refers to neurons projecting to the pyramidal tract. As accurately stated in Wikipedia, \u201cThe pyramidal tracts include both the corticobulbar tract and the corticospinal tract. These are aggregations of efferent nerve fibers from the upper motor neurons that travel from the cerebral cortex and terminate either in the brainstem (corticobulbar) or spinal cord and are involved in the control of motor functions of the body.\u201dIt has long been appreciated that cortical layer 5 contains two distinct populations of neurons that can be distinguished, not only based on the presence or absence of projections to ET targets (ET and IT cells), but also based on their predominant soma locations, dendritic morphologies and intrinsic physiology73 and prominently highlighted in this manuscript, it is now clear that PT represents only a subset of L5 ET cells and is thus unable to accurately encompass the entire L5 ET subclass. This realization is informed by comparisons across species and cortical areas, and by single-cell transcriptomics and descriptions of the projections of single neurons, as well as studies linking transcriptional clusters to projection targets.Owing to the past wide use of the term PT, we do not take the decision to use L5 ET rather than PT lightly. However, in the face of multiple lines of evidence that have accumulated over the last several years38 PT refers to motor neurons that project into MY or spinal cord, but in many cortical areas none of the L5 ET cells are motor neurons; and 2) even in the motor cortex many cells in the L5 ET subclass do not project to the pyramidal tract and instead project solely to the TH (or to TH and other non-PT targets). This is revealed by single-neuron reconstructions53 Figs. , 8, BARs even in 49. Given that the telencephalon is equivalent to the cerebrum, ET and subcerebral have the same meaning and the term L5-SCPN would be an accurate and equivalent alternative. But the \u2018L5\u2019 qualifier is crucial in either case to distinguish these cells from the L6 CT subclass. We favour the use of ET because SCPN has not been widely adopted and due to symmetry with the widely used \u2018IT\u2019 nomenclature. Alternatively, given their evidence that \u201cunlike pyramidal tract neurons in the motor cortex, these neurons in the auditory cortex do not project to the spinal cord\u201d, Chen et al64 used the term \u2018pyramidal tract-like\u2019 (PT- l). We also favour L5 ET over L5 PT-l which clings to an inaccurate and now outdated nomenclature.We recognize that another name that has been used to describe L5 ET cells is subcerebral projection neuron (SCPN)38. Code for generating Figs. http://data.nemoarchive.org/publication_release/Lein_2020_M1_study_analysis/Transcriptomics/flagship/. Analysis was performed in RStudio using R version 3.5.3, R packages: Seurat 3.1.1, ggplot2 3.2.1 and scrattch.hicat 0.0.22.To identify homologous cell types across species, human, marmoset and mouse 10x v3 snRNA-seq datasets were integrated using Seurat\u2019s SCTransform workflow. Each major cell class was integrated separately across species. Expression matrices were reduced to 14,870 one-to-one orthologues across the three species . Nuclei were downsampled to have approximately equivalent numbers at the subclass level across species. Marker genes were identified for each species cluster using Seurat\u2019s FindAllMarkers function with test.use set to \u2018roc\u2019, >\u00a00.7 classification power. Markers were used as input to guide alignment and anchor-finding during integration steps. For full methods see ref. https://github.com/AllenInstitute/BICCN_M1_Evo). The integrated space (from the previously mentioned Seurat integration) was over-clustering into small sets of highly similar nuclei for each class (about 500 clusters per class). Clusters were aggregated into metacells, then hierarchical clustering was performed based on the metacell gene expression matrix using Ward\u2019s method. Hierarchical trees were then assessed for cluster size, species mixing and branch stability by subsampling the dataset 100 times with 95% of nuclei. Finally, we recursively searched every node of the tree, and if certain heuristic criteria were not sufficient for a node below the upper node, all nodes below the upper node were pruned and nuclei belonging to this subtree were merged into one homologous group. We identified 24 GABAergic, 13 glutamatergic and 8 non-neuronal cross-species consensus clusters that were highly mixed across species and robust. For full methods see ref. 38. A final dendrogram of consensus cell types was constructed by transforming the raw unique molecular identifier (UMI) counts to log2(counts per million (CPM)) normalized counts. Up to 50 marker genes per cross-species cluster were identified by using the scrattch.hicat (v0.0.22) (https://github.com/AllenInstitute/scrattch.hicat) display_cl and select_markers functions with the following parameters; q1.th\u2009=\u20090.4, q.diff.th\u2009=\u20090.5, de.score.th\u2009=\u200980. Median cross-species cluster log2 CPM expression of these genes were then used as input for scrattch.hicat\u2019s build_dend function. This analysis was bootstrapped 10,000 times with branch colour denoting confidence. Branch robustness was assessed by rebuilding the dendrogram 10,000 times with a random 80% subset of variable genes across clusters and calculating the proportion of iterations that clusters were present on the same branch. Consensus taxonomy agreement in Fig. To establish a robust cross-species cell type taxonomy, we applied a tree-based clustering method on integrated class-level datasets . For a given cross-species cluster, each sample was split by species and donor, then a Wald test was performed between each species pair. Genes with adjusted P-values\u2009<\u20090.05 and log2 fold-changes greater than 2 in either direction were counted and reported in Fig. Expression matrices were subsetted to include one-to-one orthologous genes across all three species. Spearman correlations shown in Fig. 75 into a target region in INTACT mice76, which turned on Cre-dependent GFP expression in the nuclei of MOp neurons projecting to the injected target region. Individual GFP-labelled nuclei of MOp projection neurons were then isolated using fluorescence-activated nucleus sorting (FANS) of each single nucleus.We injected retrograde tracer rAAV2-retro-Cre45. Specifically, we quantified the ratio between the number of cells in expected on-target subclasses versus in expected off-target subclasses , denoted as rp, and compared the ratio with the one expected from the unbiased data without enrichment for specific projections, denoted as ru. This provides an estimation of signal-to-noise ratio of each FANS experiment. For IT projections, we used IT subclasses as on-target and L6 CT + inhibitory as off-target, and for ET projections, we used L5 ET as on-target and IT + inhibitory as off-target. For the MOp neurons without enrichment of projections, the expected ratio between cells in IT subclasses and in L6 CT + inhibitory are ru\u00a0=\u00a02,652:1,775, whereas the expected ratio between cells in L5 ET subclass and in IT + inhibitory are ru\u00a0=\u00a0202:3,434. The fold enrichment in the text was computed by rp/ru for each FANS run separately and averaged across IT or ET targets respectively.The methods used to evaluate contamination level and potential reasons are described in detail in ref. We want to point out that, in addition to this computational method, other methods are available to evaluate and minimize potential contamination in Epi-retro-seq. In cases in which differences in expected results from on- versus off-target populations are unknown, other available methods would need to be used to eliminate cases in which injections might have directly labelled cells outside the intended target region, such as examination of labelling along the injection electrode track.37. The read counts were normalized by the total read counts per cell and log transformed. Top 5,000 highly variable genes were identified with Scanpy78 (v1.8.1) and z-score was scaled across all the cells. For Epi-retro-seq, the posterior methylation levels of 12,261 genes in the 848 L5 ET cells were computed45. Top 5,000 highly variable genes were identified with AllCools79 and z-score was scaled across all the cells. The 1,512 genes as the intersection between the two highly variable gene lists were used in Scanorama80 (v1.7.1) to integrate the z-scored expression matrix and minus z-scored methylation matrix with sigma equal to 100.For snRNA-seq, the 4,515 cells from 10x v3 B dataset labelled as L5 ET by SCF were selected45, or from the integrated clustering of multiple datasets37. The integrated clustering and embedding of the 11 datasets are then generated by projecting all datasets into the 10x v2 scRNA-seq dataset using SingleCellFusion79. Genome browser views of IT and ET cell types . MERFISH data were analysed using custom Python code, which is available at https://github.com/ZhuangLab/MERlin.To integrate IT cell types from different mouse datasets, we first take all cells that are labelled as IT, except for L6_IT_Car3, from the 11 datasets as listed in Fig. es Figs. are take81 v2.2.7.1. using Python 3.6 with parameter: \u201c--nomodel --shift \u2212100 --ext 200 --qval 1e-2 \u2013B --SPMR\u201d. First, we extended peak summits by 250 bp on either side to a final width of 501 bp. Then, to account for differences in performance of MACS2 based on read depth and/or number of nuclei in individual clusters, we converted MACS2 peak scores ) to \u2018score per million\u201982. Next, a union peak set was obtained by applying an iterative overlap peak-merging procedure, which avoids daisy-chaining and still allows for use of fixed-width peaks. Finally, we filtered peaks by choosing a score per million cut-off of 5 as cCREs for downstream analysis.For peak calling in the snATAC-seq data, we extracted all the fragments for each cluster, and then performed peak calling on each aggregate profile using MACS283 with the following parameters: aggregation k\u2009=\u200950, window size\u2009=\u2009500 kb, distance constraint\u2009=\u2009250 kb. In order to find an optimal co-accessibility threshold, we generated a random shuffled cCRE-by-cell matrix as background and calculated co-accessible scores from this shuffled matrix. We fitted the distribution of co-accessibility scores from random shuffled background into a normal distribution model by using the R package fitdistrplus84. Next, we tested every co-accessible cCRE pair and set the cut-off at co-accessibility score with an empirically defined significance threshold of FDR\u00a0<\u00a00.01. The cCREs outside of \u00b11 kb of transcriptional start sites in GENCODE mm10 (v16) were considered distal. Next, we assigned co-accessibility pairs to three groups: proximal-to-proximal, distal-to-distal and distal-to-proximal. In this study, we focus only on distal-to-proximal pairs. We calculated the Pearson\u2019s correlation coefficient (PCC) between gene expression (scRNA SMART-seq) and cCRE accessibility across the joint clusters to examine the relationships between the distal cCREs and target genes as predicted by the co-accessibility pairs. To do so, we first aggregated all nuclei or cells from scRNA-seq and snATAC-seq for every joint cluster to calculate accessibility scores (log2 CPM) and relative expression levels (log2 transcripts per million). Then, PCC was calculated for every gene-cCRE pair within a 1-Mbp window centred on the transcriptional start sites for every gene. We also generated a set of background pairs by randomly selecting regions from different chromosomes and shuffling the cluster labels. Finally, we fit a normal distribution model on background and defined a cut-off at PCC score with an empirically defined significance threshold of FDR\u00a0<\u00a00.01, in order to select significant positively correlated cCRE-gene pairs.First, co-accessible cCREs are identified for all open regions in all neuron types (cell clusters with less than 100 nuclei from snATAC-seq are excluded) using Cicerocis-regulatory modules based on their relative accessibility across cell clusters. We adapted NMF (Python package: sklearn v.0.24.2) to decompose the cluster-by-cCRE matrix V into a coefficient matrix H and a basis matrix W (N\u00a0\u00d7\u00a0R), with a given rank R: V\u00a0\u2248\u00a0WH.We used nonnegative matrix factorization (NMF) to group cCREs into R . Several criteria have been proposed to decide whether a given rank R decomposes the occupancy profile matrix into meaningful clusters. Here we applied a measurement called sparseness85 to evaluate the clustering result. Median values were calculated from 100 times for NMF runs at each given rank with a random seed, which will ensure the measurements are stable. Next, we used the coefficient matrix to associate modules with distinct cell clusters. In the coefficient matrix, each row represents a module and each column represents a cell cluster. The values in the matrix indicate the weights of clusters in their corresponding module. The coefficient matrix was then scaled by column (cluster) from 0 to 1. Subsequently, we used a coefficient\u2009>\u20090.1 (~95th percentile of the whole matrix) as a threshold to associate a cluster with a module. Similarly, we associated each module with accessible elements using the basis matrix. For each element and each module, we derived a basis coefficient score, which represents the accessible signal contributed by all clusters in the defined module.The basis matrix defines module related accessible cCREs, and the coefficient matrix defines the cell cluster components and their weights in each module. The key issue to decompose the occupancy profile matrix was to find a reasonable value for the rank P-value\u2009<\u00a00.05, cluster average fold change\u2009>\u00a02). To perform the motif enrichment analysis, we used known motifs from the JASPAR 2020 database86 and the subclass specific hypo-CG-DMR identified in Yao et al.37. The AME software from the MEME suite (v5.1.1)87 was used to identify significant motif enrichment using default parameters and the same background region set as described37. All genes in Extended Data Fig. All analyses for this section were at the subclass level. For RNA expression, we used the scSMART-seq dataset and compared each subclass with the rest of the population through a one-tailed Wilcoxon test and FDR correction to select significantly differentially expressed transcription factors of Cold Spring Harbor Laboratory, University of California Berkeley and Allen Institute, in accordance with NIH guidelines. Mouse knockin driver lines are being deposited to the Jackson Laboratory for wide distribution.Tle4-2A-CreER, Fezf2-2A-CreER, PlexinD1-2A-CreER, PlexinD1-2A-Flp and Tbr2-2A-CreER were generated by inserting a 2A-CreER or 2A-Flp cassette in-frame before the STOP codon of the targeted gene. Targeting vectors were generated using a PCR-based cloning approach47. In brief, for each gene of interest, two partially overlapping BAC clones from the RPCI-23&24 library (made from C57BL/b mice) were chosen from the Mouse Genome Browser. 5\u2032 and 3\u2032 homology arms were PCR amplified using the BAC DNA as template and cloned into a building vector to flank the 2A-CreERT2 or 2A-Flp expressing cassette as described27. These targeting vectors were purified, tested for integrity by enzyme restriction and PCR sequencing. Linearized targeting vectors were electroporated into a 129SVj/B6 hybrid ES cell line (v.6.5). ES cell clones were first screened by PCR and then confirmed by Southern blotting using appropriate probes. DIG-labelled Southern probes were generated by PCR, subcloned and tested on wild-type genomic DNA to verify that they give clear and expected results. Positive v6.5 ES cell clones were used for tetraploid complementation to obtain male heterozygous mice following standard procedures. The F0 males and subsequent generations were bred with reporter lines and induced with tamoxifen at the appropriate ages to characterize the resulting genetically targeted recombination patterns. Drivers Tle4-2A-CreER, Fezf2-2A-CreER and PlexinD1-2A-CreER were additionally crossed with reporter Rosa26-CAG-LSL-Flp and Tbr2-2A-CreER;PlexinD1-2A-Flp with reporter dual-tTA, and induced with tamoxifen at the appropriate age to perform anterograde viral tracing, with Flp- or tTA-dependent AAV vector expressing EGFP (AAV8-CAG-fDIO-TVA-EGFP or AAV-TRE-3g-TVA-EGFP), to characterize the resulting axon projection patterns.Driver and reporter mouse lines were generated using a PCR-based cloning. Knockin mouse lines Npnt and Slco2a1, respectively, using ribonucleoprotein (RNP) complexes composed of SpCas9-NLS protein and in vitro transcribed sgRNA (Npnt: GATGATGTGAGCTTGAAAAG and Slco2a1: CAGTCTGCAGGAGAATGCCT). These RNP complexes were nucleofected into 106 v6.5 mouse embryonic stem cells along with repair constructs in which P2A-FlpO or P2A-Cre was flanked with the following sequences homologous to the target site, thereby enabling homology-directed repair.To generate lines bearing in-frame genomic insertions of P2A-FlpO or P2A-Cre, we engineered double-strand breaks at the stop codons of Npnt-P2A-FlpO: TGGCCCTTGAGCTCTAGTGTTCCCACTTGCCATAGAAATCTGATCTTCGGTTTGGGGGAAGGGTTGCCTTACCATGCTCCATGAGTGAGCACTGGGAAAAGGGGCAGAGGAGGCCTGACCAGTGTATACGTTCTCTCCCTAGGTCATCTTCAAAGGTGAAAAAAGGCGTGGTCACACGGGGGAGATTGGATTGGATGATGTGAGCTTGAAGCGCGGAAGATGTGGAAGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCTATGGCTCCTAAGAAGAAGAGGAAGGTGATGAGCCAGTTCGACATCCTGTGCAAGACCCCGCCGAAGGTGCTGGTGCGGCAGTTCGTGGAGAGATTCGAGAGGCCCAGCGGCGAAAAGATCGCCAGCTGTGCCGCCGAGCTGACCTACCTGTGCTGGATGATCACCCACAACGGCACCGCGATCAAGAGGGCCACCTTCATGAGTTATAACACCATCATCAGCAACAGCCTGAGTTTTGACATCGTGAACAAGAGCCTGCAGTTCAAGTACAAGACCCAGAAGGCCACCATCCTGGAGGCCAGCCTGAAGAAGCTGATCCCCGCATGGGAGTTCACGATTATCCCTTACAACGGCCAGAAGCACCAGAGCGACATCACCGACATCGTGTCCAGCCTGCAGCTGCAGTTCGAAAGCAGCGAGGAGGCCGACAAGGGGAATAGCCACAGCAAGAAGATGCTGAAGGCCCTGCTGTCCGAAGGCGAGAGCATCTGGGAGATTACCGAGAAGATCCTGAACAGCTTCGAGTACACCAGCAGATTTACCAAAACGAAGACCCTGTACCAGTTCCTGTTCCTGGCCACATTCATCAACTGCGGCAGGTTCAGCGACATCAAGAACGTGGACCCGAAGAGCTTCAAGCTCGTCCAGAACAAGTATCTGGGCGTGATCATTCAGTGCCTGGTCACGGAGACCAAGACAAGCGTGTCCAGGCACATCTACTTTTTCAGCGCCAGAGGCAGGATCGACCCCCTGGTGTACCTGGACGAGTTCCTGAGGAACAGCGAGCCCGTGCTGAAGAGAGTGAACAGGACCGGCAACAGCAGCAGCAACAAGCAGGAGTACCAGCTGCTGAAGGACAACCTGGTGCGCAGCTACAACAAGGCCCTGAAGAAGAACGCCCCCTACCCCATCTTCGCTATTAAAAACGGCCCTAAGAGCCACATCGGCAGGCACCTGATGACCAGCTTTCTGAGCATGAAGGGCCTGACCGAGCTGACAAACGTGGTGGGCAACTGGAGCGACAAGAGGGCCTCCGCCGTGGCCAGGACCACCTACACCCACCAGATCACCGCCATCCCCGACCACTACTTCGCCCTGGTGTCCAGGTACTACGCCTACGACCCCATCAGTAAGGAGATGATCGCCCTGAAGGACGAGACCAACCCCATCGAGGAGTGGCAGCACATCGAGCAGCTGAAGGGCAGCGCCGAGGGCAGCATCAGATACCCCGCCTGGAACGGCATTATAAGCCAGGAGGTGCTGGACTACCTGAGCAGCTACATCAACAGGCGGATCTGAAAGAGGTCGCTGCTGAGAAGACCCCTGGCAGCTCCCGAGCTAGCAGTGAATTTGTCGCTCTCCCTCATTTCCCAATGCTTGCCCTCTTGTCTCCCTCTTATCAGGCCTAGGGCAGGAGTGGGTCAGGAGGAAGGTTGCTTGGTGACTCGGGTCTCGGTGGCCTGTTTTGGTGCAATCCCAGTGAACAGTGACACTCTCGAAGTACAGGAGCATCTGGAGACACCTCCGGGCCCTTCTGSlco2a1-P2A-Cre:TGCCCCTGGGCCTCACCATACCTGTCTCTTCCTGCCTCATAGGTACCTGGGCCTACAGGTAATCTACAAGGTCTTGGGCACACTGCTGCTCTTCTTCATCAGCTGGAGGGTGAAGAAGAACAGGGAATACAGTCTGCAGGAGAATGCTTCCGGATTGATTGGAAGCGGAGCTACTAACTTCTCCCTGTTGAAACAAGCAGGGGATGTCGAAGAGAATCCTGGACCTATGGCTCCTAAGAAGAAGAGGAAGGTGATGAGCCAGTTCGACATCCTGTGCAAGACTCCTCCAAAGGTGCTGGTGCGGCAGTTCGTGGAGAGATTCGAGAGGCCCAGCGGCGAGAAGATCGCCAGCTGTGCCGCCGAGCTGACCTACCTGTGCTGGATGATCACCCACAACGGCACCGCCATCAAGAGGGCCACCTTCATGAGCTACAACACCATCATCAGCAACAGCCTGAGCTTCGACATCGTGAACAAGAGCCTGCAGTTCAAGTACAAGACCCAGAAGGCCACCATCCTGGAGGCCAGCCTGAAGAAGCTGATCCCCGCCTGGGAGTTCACCATCATCCCTTACAACGGCCAGAAGCACCAGAGCGACATCACCGACATCGTGTCCAGCCTGCAGCTGCAGTTCGAGAGCAGCGAGGAGGCCGACAAGGGCAACAGCCACAGCAAGAAGATGCTGAAGGCCCTGCTGTCCGAGGGCGAGAGCATCTGGGAGATCACCGAGAAGATCCTGAACAGCTTCGAGTACACCAGCAGGTTCACCAAGACCAAGACCCTGTACCAGTTCCTGTTCCTGGCCACATTCATCAACTGCGGCAGGTTCAGCGACATCAAGAACGTGGACCCCAAGAGCTTCAAGCTGGTGCAGAACAAGTACCTGGGCGTGATCATTCAGTGCCTGGTGACCGAGACCAAGACAAGCGTGTCCAGGCACATCTACTTTTTCAGCGCCAGAGGCAGGATCGACCCCCTGGTGTACCTGGACGAGTTCCTGAGGAACAGCGAGCCCGTGCTGAAGAGAGTGAACAGGACCGGCAACAGCAGCAGCAACAAGCAGGAGTACCAGCTGCTGAAGGACAACCTGGTGCGCAGCTACAACAAGGCCCTGAAGAAGAACGCCCCCTACCCCATCTTCGCTATCAAGAACGGCCCTAAGAGCCACATCGGCAGGCACCTGATGACCAGCTTTCTGAGCATGAAGGGCCTGACCGAGCTGACAAACGTGGTGGGCAACTGGAGCGACAAGAGGGCCTCCGCCGTGGCCAGGACCACCTACACCCACCAGATCACCGCCATCCCCGACCACTACTTCGCCCTGGTGTCCAGGTACTACGCCTACGACCCCATCAGCAAGGAGATGATCGCCCTGAAGGACGAGACCAACCCCATCGAGGAGTGGCAGCACATCGAGCAGCTGAAGGGCAGCGCCGAGGGCAGCATCAGATACCCCGCCTGGAACGGCATCATCAGCCAGGAGGTGCTGGACTACCTGAGCAGCTACATCAACAGGCGGATCTGACCTTCAGCTGGGACTACTGCCCTGCCCCAGAGACTGGATATCCTACCCCTCCACACCTACCTATATTAACTAATGTTAGCATGCCTTCCTCCTCCTTCCNpnt-P2A-FlpO left homology arm; GATTGAGGTCAGGCCAGAAG and TCGACATCGTGAACAAGAGC for Npnt-P2A-FlpO right homology arm; CTGGTGAAAGGGGAACTCTTGCT and GATCCCTGAACATGTCCATCAGG for Slco2a1-P2A-Cre left homology arm; TACAGCATCCCTGACAAACACCA and TAGCACCGCAGGTGTAGAGAAGG for Slco2a1-P2A-Cre right homology arm.Transfected cells were cultured and resulting colonies directly screened by PCR for correct integration using the following genotyping primers: flanking primer ATGCATTGCTTCATGCCATA and internal recombinase primer CCTTCAGCAGCTGGTACTCC for The inserted transgenes were fully sequenced and candidate lines were analysed for normal karyotype. Lines passing quality control were aggregated with albino morulae and implanted into pseudopregnant females, producing germline-competent chimeric founders which in turn were crossed with the appropriate reporter lines on the C57/BL6 background.All experimental procedures using live animals were performed according to protocols approved by Institutional Animal Care and Use Committees (IACUC) of all participating institutions: Allen Institute for Brain Science, Baylor College of Medicine, Broad Institute of MIT and Harvard, Cold Spring Harbor Laboratory, Harvard University, Salk Institute for Biological Studies, University of California Berkeley, University of California San Diego and University of Southern California. Macaque experiments were performed on animals designated for euthanasia via the Washington National Primate Research Center\u2019s Tissue Distribution Program.Postmortem adult human brain tissue collection was performed in accordance with the provisions of the United States Uniform Anatomical Gift Act of 2006 described in the California Health and Safety Code section 7150 (effective 1 January 2008) and other applicable state and federal laws and regulations. The Western Institutional Review Board reviewed tissue collection processes and determined that they did not constitute human subjects research requiring institutional review board (IRB) review. Before commencing the human Patch-seq, the donor provided informed consent and experimental procedures were approved by the hospital institute review board.Further information on research design is available in the\u00a0Any methods, additional references, Nature Research reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at 10.1038/s41586-021-03950-0.Reporting SummarySupplementary Table 1Confusion matrices corresponding to Extended Data Figure 5.Supplementary Table 2Cell type nomenclature.Peer Review File"} +{"text": "Deroplatys truncate, D. lobate, Amorphoscelis chinensis and Macromantis sp. belong to Deroplatyidae, Amorphoscelidae and Photinaidae family, respectively. Our results indicated that the ATP8 gene may be lost in D. truncate and D. lobata mt genome, and four tRNA genes have not been found in D. truncate, D. lobata and Macromantis sp. A dN/dS pair analysis was conducted and it was found that all genes have evolved under purifying selection. Furthermore, we tested the phylogenetic relationships between the eight families of the Mantodea, including 35 species of praying Mantis. Based on the complete mitochondrial genome data, it was also suggested as sister to Deroplatyidae + Mantidae, Metallyticus sp., the only representative of Metallyticidae, is sister to the remaining mantises. Our results support the taxonomic system of Schwarz and Roy and are consistent with previous studies.Praying mantises are distributed all over the world. Though some Mantodea mitogenomes have been reported, an evolutionary genomic and phylogenetic analysis study lacks the latest taxonomic system. In the present study, four new mitogenomes were sequenced and annotated. The praying mantis comprises up to 2,300 species having diversified morphological and ecological characteristics. These insects colonize at wider range of habitats, including arid and tropical rainforests, temperate regions and engaged in multiple hunting tactics , 2. PrayThe mitochondrial genome (mitogenome), as a robust molecular marker , has recAmorphoscelis belongs to the subfamily Amorphoscelinae, which can be distinguished from all other mantis through some characters such as: small, dorsoventrally flattened body, and adapted to a bark-living lifestyle Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1:\u00a0YesReviewer #2:\u00a0Partly**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1:\u00a0YesReviewer #2:\u00a0I Don't Know**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data\u2014e.g. participant privacy or use of data from a third party\u2014those must be specified.The Reviewer #1:\u00a0YesReviewer #2:\u00a0Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1:\u00a0NoReviewer #2:\u00a0No**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1:\u00a0In the manuscript titled 'Complete mitochondrial genomes of four species of praying mantises with ribosomal second structure, evolutionary and phylogenetic analyses' the author present the complete mitochondrial genome of four species of mantid. Overall the study is good and should be published but some revisions need to be addressed before publishing.1) The authors need to have a native English speaker review the document for grammar. It is not unintelligible but there are many spots where it is difficult to follow or does not sound scientific.2) Throughout the document, the species D. truncata and D. lobata are converted to truncate and lobate due to autcorrect from their word processor . It is annoying but an easy fix.3) I would like to see a table or tables that list the gene order and position for all ORFs for each species, this generally is a useful reference for other readers.Reviewer #2:\u00a0The authors sequence and annotate the mitogenomes of four mantises species and combine this new data with existing mantis data to estimate phylogenetic relationships in the group. They find a few missing genes in some of the four new mitogenomes, but tRNA secondary structure and codon usage was similar to other mantises. The phylogeny also tended to agree with current taxonomy.I think the study is definitely a worthwhile addition to insect mitogenomics. I particularly appreciate that you discuss your findings in the context of insect and mantis mitogenomics. However, I do have several issues that should be addressed, some more major then others:1. You need to re-work some of the structure in the Introduction. For example, the second paragraph introduced mitogenomes and then abruptly switches to introducing the taxonomy of mantises. I would separate these.2. Because the Methods comes last, I would include some brief methodological details in the Results and Discussion section.3. Please include more specific results. For example, you vaguely state that there is a high A+T% and list the most-used codons. Please provide specific values in the text.4. You begin discussing the utility of using cox1 as a barcoding gene (starting line 151), but this discussion appears abruptly. Why is this relevant? Is it related to your nucleotide diversity results? Please explain.5. I\u2019m a little confused by your sampling strategy. Did you sample the 35 mantis species mentioned in lines 189-190? Or were these taken from GenBank?6. I am not convinced you are actually missing the atp8 genes. In my experience, it can be difficult to annotate this gene, and you often have to look for it manually based on ORFs, etc. It also seems like there is a non-coding region on the 5\u2019 end of your atp6 genes when a \u201cmissing\u201d atp8 is reported. I would bet that is the atp8 gene. Also, were your missing genes confirmed with your PCR step? Finally, it looks like both atp6 and atp8 are missing from one species , but I didn\u2019t see any discussion of this. Please elaborate.7. Did you confirm that these assembled mitogenomes are in fact complete and circular? I recommend doing this, especially since you report missing genes.8. Please provide more methodological details. For example, how did you calculate Ka/KS? What were your PCR conditions? What GBlocks parameters did you use?9. There are several portions of the manuscript that are difficult to read. I recommend going through the text and again and edit for grammar, awkward phrasing, etc.Some minor comments:Line 25: There is no context for referencing the \u201cSchwarz and Roy (2019) taxonomic system.\u201d Perhaps just say \u201cthe latest taxonomy.\u201dLine 31: No need to say \u201crespectively.\u201d Same in line 82.Line 42: Be careful about using \u201cbugs\u201d when you mean \u201cinsects.\u201d Entomologists will strongly object to this usage\u2026Lines 74-78 should be in the IntroductionLine 100: Why do you rule out this second possibility? Please elaborate.Line 144: Please provide a brief overview of dN/dS ratio .Line 217: Confusing phrasing. Please re-write. It\u2019s not clear what \u201cminiscule to align means.\u201dFigure 1: It\u2019s not clear which mitogenomes go with each species. Please add names to the figure.**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article digital diagnostic tool,\u00a0 24 May 20214/5/2021Dear Editor, Thank you very much for your email message regarding our manuscript. We appreciated much the valuable comments from the reviewers for revising and improving our manuscript. Generally, we have incorporated all suggestions and comments of yours into this revision. More specifically, our corrections are as follows here under.We hope you can now accept this revised version. Most sincerely,All authorsReviewer #1: In the manuscript titled 'Complete mitochondrial genomes of four species of praying mantises with ribosomal second structure, evolutionary and phylogenetic analyses' the author present the complete mitochondrial genome of four species of mantid. Overall the study is good and should be published but some revisions need to be addressed before publishing.1) The authors need to have a native English speaker review the document for grammar. It is not unintelligible but there are many spots where it is difficult to follow or does not sound scientific.Reply: We thank this referee for his/her suggestions. We have polished the full text and further revised the corresponding parts. 2) Throughout the document, the species D. truncata and D. lobata are converted to truncate and lobate due to autcorrect from their word processor . It is annoying but an easy fix.Reply: Thank you. 3) I would like to see a table or tables that list the gene order and position for all ORFs for each species, this generally is a useful reference for other readers.Reply: We agree and have added the related information on the position of all ORFs. See Table S2. For the gene order, we refer to Figure 1 with the mitochondrial gene structure.Reviewer #2: The authors sequence and annotate the mitogenomes of four mantises species and combine this new data with existing mantis data to estimate phylogenetic relationships in the group. They find a few missing genes in some of the four new mitogenomes, but tRNA secondary structure and codon usage was similar to other mantises. The phylogeny also tended to agree with current taxonomy.I think the study is definitely a worthwhile addition to insect mitogenomics. I particularly appreciate that you discuss your findings in the context of insect and mantis mitogenomics. However, I do have several issues that should be addressed, some more major then others:1. You need to re-work some of the structure in the Introduction. For example, the second paragraph introduced mitogenomes and then abruptly switches to introducing the taxonomy of mantises. I would separate these.Reply: Agree. We have separated the two parts. Please check the line 53.2. Because the Methods comes last, I would include some brief methodological details in the Results and Discussion section.Reply: Thank you. We have added the brief methodological details in the Results and Discussion section. Please check lines 78-83. 3. Please include more specific results. For example, you vaguely state that there is a high A+T% and list the most-used codons. Please provide specific values in the text.Reply: Agree. We have added the whole genome, A+T-rich region and PCGs specific values of four mitochondrial genomes. Please check Table S2.4. You begin discussing the utility of using cox1 as a barcoding gene (starting line 151), but this discussion appears abruptly. Why is this relevant? Is it related to your nucleotide diversity results? Please explain.Reply: Thank you. The dN/dS pairwise analysis showed that cox1 is undergoing a robust purifying selection (0.028). That is why cox1 can be used as a molecular marker of barcoding entire Mantidae. We also reorganized the writing of this part. Please check lines 152-153.5. I\u2019m a little confused by your sampling strategy. Did you sample the 35 mantis species mentioned in lines 189-190? Or were these taken from GenBank?Reply: We have reorganized the writing of this part. Please check lines 189-193 and Table 1.6. I am not convinced you are actually missing the atp8 genes. In my experience, it can be difficult to annotate this gene, and you often have to look for it manually based on ORFs, etc. It also seems like there is a non-coding region on the 5\u2019 end of your atp6 genes when a \u201cmissing\u201d atp8 is reported. I would bet that is the atp8 gene. Also, were your missing genes confirmed with your PCR step? Finally, it looks like both atp6 and atp8 are missing from one species , but I didn\u2019t see any discussion of this. Please elaborate.Reply: Thank you for this advice. We know ATP8 which is difficult to annotate. We repeated to analysis the sequence for annotation of ATP8, and in addition we ran the PCR to amplify the corresponding part of about 1000bp to confirm the sequence. Unfortunately, we could not annotate the relevant homologous sequences. For the ATP6 of Macromantis sp. species, we have added this annotation name in Figure 1 and the Table S2. Please check Figure 1 and Table S2. 7. Did you confirm that these assembled mitogenomes are in fact complete and circular? I recommend doing this, especially since you report missing genes.Reply: We thank this referee for his/her comment. We received the assembled mitogenomes sequence and this was confirmed by PCR technology. The starting and the ending of sequence overlapped with 184bp, so we are sure that this is a complete and circular structure. We redesigned the primers for PCR and sequenced the PCR product to confirm the sequencing results. The primers are listed in Table S1. We also used ORF Finder and BLAST search engines against the GenBank database to identify protein-coding and rRNA genes. Please check the lines 206-208.8. Please provide more methodological details. For example, how did you calculate Ka/KS? What were your PCR conditions? What GBlocks parameters did you use?Reply: We agree and have added the methods of calculating Ka/KS. Please check lines 219-221. We also added the conditions of long PCR reactions. Please check lines 213-217. Also the GBlocks parameters were added in lines 237-239.9. There are several portions of the manuscript that are difficult to read. I recommend going through the text and again and edit for grammar, awkward phrasing, etc.Reply: Thanks for your suggestion. We polished the text and revised the corresponding parts. Some minor comments:Line 25: There is no context for referencing the \u201cSchwarz and Roy (2019) taxonomic system.\u201d Perhaps just say \u201cthe latest taxonomy.\u201dReply: Done; please check lines 26-27Line 31: No need to say \u201crespectively.\u201d Same in line 82.Reply: Done; please check lines 31, 84Line 42: Be careful about using \u201cbugs\u201d when you mean \u201cinsects.\u201d Entomologists will strongly object to this usage\u2026Reply: Done, please check line 42.Lines 74-78 should be in the IntroductionReply: Agree. We have moved this text to the Introduction. Please check line 64-68.Line 100: Why do you rule out this second possibility? Please elaborate.Reply: We apologize for this. Indeed the second possibility is also very likely. So, we have reorganized this text part. We have deleted our conclusion: \u201cWe concluded that a lack of the atp8 gene in D. truncate and D. lobate is most likely because of the former or the later reason.\u201d Please check lines 112-113. Thank you.Line 144: Please provide a brief overview of dN/dS ratio .Reply: Agree. Please check lines 151-152.Line 217: Confusing phrasing. Please re-write. It\u2019s not clear what \u201cminiscule to align means.\u201dReply: We thank this referee and have rephrased this part text, which means Nad4L is too short to align among the Mantodea species. ATP8 was not identified in D. truncate and D. lobate. So nad4L and atp8 were excluded in the phylogenetic analysis. Please check lines 238-241. Figure 1: It\u2019s not clear which mitogenomes go with each species. Please add names to the figure.Reply: Thanks and done.AttachmentR1-rebuttal letter-final.docxSubmitted filename: Click here for additional data file. 25 Jun 2021PONE-D-21-03889R1Complete mitochondrial genomes of four species of praying mantises with ribosomal second structure, evolutionary and phylogenetic analysesPLOS ONEDear Dr. Smagghe,Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE\u2019s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.Minor issues were raised which can be considered since they will improve the manuscript somewhat.plosone@plos.org. 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For instructions see:\u00a0We look forward to receiving your revised manuscript.Kind regards,Ben J Mans, PhDAcademic EditorPLOS ONEJournal Requirements:Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article\u2019s retracted status in the References list and also include a citation and full reference for the retraction notice.[Note: HTML markup is below. Please do not edit.]Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the \u201cComments to the Author\u201d section, enter your conflict of interest statement in the \u201cConfidential to Editor\u201d section, and submit your \"Accept\" recommendation.Reviewer #2:\u00a0(No Response)**********2. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #2:\u00a0Yes**********3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #2:\u00a0Yes**********4. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data\u2014e.g. participant privacy or use of data from a third party\u2014those must be specified.The Reviewer #2:\u00a0Yes**********5. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #2:\u00a0Yes**********6. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #2:\u00a0Thank you for your thorough responses and edits to your manuscript based on my comments. I think the manuscript has been improved. I do have a few comments that still should be addressed, based on my first round of comments:1. I still think the paragraphs in the Introduction are a little disconnected. Perhaps switch paragraph #2 (on the utility of mitogenomes for mantid phylogenetics) and paragraph #3 (on mantid taxonomy).2. I think you should add the AT% to the main text, not just in a supplement.3. I\u2019m still somewhat skeptical that the atp8 gene is actually missing, but your combination of NGS and PCR results are more convincing. It is difficult for me to fully evaluate this claim without access to the sequence data; but as is stated in the manuscript, atp8 and other genes are missing from other taxa. Is this the first case of missing mt genes in mantises?4. I recommend one more round of editing for writing clarity. For example, Lines 50-51 are a little confusing, and should probably be split up into two sentences.**********what does this mean?). If published, this will include your full peer review and any attached files.7. PLOS authors have the option to publish the peer review history of their article digital diagnostic tool,\u00a0 27 Jun 202126/06/2021Dear Editor, Thank you very much for your email message regarding our manuscript entitled \u201cComplete mitochondrial genomes of four species of praying mantises with ribosomal second structure, evolutionary and phylogenetic analyses\u201d. We appreciated the valuable comments from the reviewers and editor to revise and improving our manuscript. Generally, we have incorporated all suggestions and comments into this revision. More specifically, our adjustments are stated below in a point-by-point manner.We hope that this revised version can be accepted for publication. Most sincerely,All authorsReviewer #2: Thank you for your thorough responses and edits to your manuscript based on my comments. I think the manuscript has been improved. I do have a few comments that still should be addressed, based on my first round of comments:1. I still think the paragraphs in the Introduction are a little disconnected. Perhaps switch paragraph #2 (on the utility of mitogenomes for mantid phylogenetics) and paragraph #3 (on mantid taxonomy).Reply: We agree and have switched the paragraph #2 and paragraph #3. See lines 46-68.2. I think you should add the AT% to the main text, not just in a supplement.Reply: Indeed, we have added the AT% information to the main text; see in Table 2.3. I\u2019m still somewhat skeptical that the atp8 gene is actually missing, but your combination of NGS and PCR results are more convincing. It is difficult for me to fully evaluate this claim without access to the sequence data; but as is stated in the manuscript, atp8 and other genes are missing from other taxa. Is this the first case of missing mt genes in mantises?Reply: Thanks for your suggestion. Here we attach mitochondrial sequencing results for D. truncate species, and we really did not identify the atp8 gene. So, we imply that atp8 gene may have been lost. Yes, from our sequence of D. truncate and D. lobata, we can confirm the atp8 is lost. This is the first case of missing Mt gene in mantises. TAATTATCCCATGAAAGATTAGTATTAAACCAATAACTCTAAATTTATTTTAAATAATAGAGTAAAATGCCTGATTTAAAAGGAATATCATGATAGGATAAAATATGTAAATTAATTACTTTTACTAATATTATCATTACACATAACAGAGTTAAACTGTTTCCTAAAATATCAAAAATTTTTGTGCATCCTACACTAAAATATAGAAAAGTAAGCTAAATTTAAGCTAATGGGTTCATACCCCATATATAGAGTTTACACTCTCTTTTCTAGTGCCCAACAATTCGATAAAAATCCTCTTTTTAATAACTTTAATTAGAGGTGTGTTAATTTCATTATGTGCTAATTCATGAATAGGAGCATGAATAGGATTAGAAATTAACTTACTCTCCTTCATTCCTTTACTTTCTTCCAGAAAAAACTTGTTCTCAACTGAAGCTTCACTTAAATATTTTCTTATTCAAGCTATTTCATCATCTACATTTCTATTTCTAATTATGATAAAAATTAATATCCAAGAAATGTTTTATCTAATAAAATTTAATAATTGAAACACCTTAATTACAATTCCTATTCTAATGAAAATTGCATCTGCACCTTTTCATTGATGATTACCTTCCGTGATAGAAGGTTTATCTTGAATAAATTGTTTTATTATTCTGTCAATTCAAAAAGTAGCCCCATTAATATTCATTTCCTACTTAATCACTAATAACTTCTTTATTCAAATCATCATTACAATTTCTGCAATTATAGGAGCTATTGGAGGTCTCAATCAAATTTCTCTACGAAAAATTTTATCATTTTCATCTATCAACCATATTGGATGAATATTAACAACCATAATTATAGGTTCTAACTTTTGAATAACATACTTTATCATTTATACGGTAAATATTATCCCTATTATTTTTATGGTAAACAAAATAAACCTATCTTTTATTCCTCAGACCTTTAATTCCTTCAATAACAAAAAAATTATTAAATTCATTCTATTTATCTCCCTCCTTTCTTTAGGGGGCCTCCCTCCTTTTATTGGTTTTTTCCCAAAATGAATTATCATTCAAATTATAATCCAAAATTTAATAATTCTTACATCAATAACCCTAATTATATCCTCCCTTTTAACCCTTTATTATTATCTACGAATTATTTACACAACATTAATAATCACAAATTCAGAAATAACTTGAATAGCAATCTACTCAAATAACAACTTAGGAAAAAGAACATTTCTTTTCTTATTCATTCTTATTTTTGGTTTATCAATCTGTACACTTATCTTAACAATCTATTAAGATTTTAAGTTAACCAAACTAATATCCTTCAAAGTTATAAATAAAGTGATTCATCTTTAAGTCTTAGTATTACTTACACCTTTAGAATTGCATTCTAATATCATCTCCATGAATATAAAACTTTAGTAAAAGAGATAATAATCTCATTAATAAATTTACAATTTATTACCTAATATCAGCCATTTTACTTTTTTTTTTGCAACGATGATTATTCTCAACAAATCATAAGGATATTGGAACATTATATTTTATTTTTGGTGCATGGGCAAGTATATTAGGAACATCTTTAAGAATTTTAATTCGAACAGAGTTAAGACAACCCGGATCTTTAATTGGAGATGATCAAATTTATAATGTCATTGTAACAGCTCACGCTTTTATTATAATCTTCTTTATAGTTATACCCATTATAATTGGTGGATTTGGTAATTGATTAATTCCTTTAATACTTGGAGCTCCAGATATAGCCTTTCCTCGAATAAATAATATAAGATTTTGATTATTACCTCCTTCTATTCTATTTCTTATTATCGGAAGAGTTGTAGAAAGAGGAGCTGGTACAGGATGAACAGTCTATCCCCCTCTTTCAGCCAGAATTGCTCACGCCGGCCCCGCAATTGATTTAACCATTTTTTCCTTACACTTAGCTGGAATATCAAGAATTATAGGAGCTGTAAATTTTATCACAACTATAATTAACATAAAATCAATTCATATAAATCATACTCAAATTCCTTTATTTGTTTGATCTGTAGGAATTACAGCAATTCTTCTTCTTTTATCTCTCCCTGTACTTGCTGGGGCAATCACTATACTTCTAACTGATCGAAACTTAAATACATCCTTTTTCGACCCTGCTGGAGGGGGGGATCCTATTCTCTATCAACATCTATTTTGATTTTTTGGACATCCTGAAGTATATATTTTAATTTTACCAGGATTTGGTATAATTTCTCATGTCATTTCTCATGAAAGAGGAAAAAAAGAAGCTTTTGGAAATTATGGAATAATTTGAGCTATATCAGCCATTGGTTTTCTGGGGTTTATTGTATGAGCTCATCATATATTTACAGTAGGAATAGATGTAGATACACGAGCCTACTTTACAGGAGCTACTATAATTATTGCTGTTCCTACGGGTATTAAAATTTTTAGTTGACTCACAACTATATACGGAACAAAAATAATTTATAGTGTAGTATCTTTATGAGCATTAGGATTTATTTTTTTATTTACAGTTGGAGGTCTTACAGGAGTAATTTTAGCCAATTCAGCTATTGATATTATTCTTCATGACACTTATTATGTAGTAGCCCATTTTCACTATGTACTTTCAATAGGAGCCGTTTTTGCTATTATAGCAGGGTTCATTCATTGATATCCCTTATTCACTGGATTATCATTAAATCCTAATTGATTAAAAAGTCAATTTTTCACAATATTTGTAGGAGTTAATTTAACATTTTTTCCACAACATTTCTTAGGATTAGCTGGAATACCTCGACGCTATTCAGATTACCCTGACGCCTATAGATCATGAAACTTCTTATCATCTGTAGGAGCAATAATTTCTTTCGCTGCTGTTATCATATTCATTCTTATCTTATGAGAAAGAATTACCTTAAATCGATTTATATTATTTTCATCTCAAATAAATAGATCAATTGAATGAATTCATAATTTTCCCCCAGCTGAACACACCTACAATGAACTAACTCTAATTACAAATTAAAACCTAAATTGTTGATAATTTTCCTCATTTCTAATGTGGCAGAATAGTGCACTGGGTTTAAGCTCCAAAAATAAAGATAAACTTTTTTTAGAAGTTATTTTAATGGCTACAAATGCAAATTTAGGATTTCAAGATAGAGCCTCCCCCTTAATAGAACAACTCATGTATTTTCATGATCATTCCATATTTATTATTACTATAATTGTAATTACAGTAAGTTATATAATTATAGCTTTAATAGTAAATAAATTTTCTGATCGTCATGTTATAGATGGTCAATATTTAGAAATTTTTTGAACAGTTCTTCCAGCCATAGTTCTAGTCTTCATCGCTCTACCTTCTCTACGGATTTTATACCTAATTGATGAAAATACAAATCCAACATTAACCTTAAAAACAATTGGTCATCAATGGTATTGAAGTTACGAATATTCAGATTTTACCAATATTGAATTTAACTCTTACATAATTCCACAAAATGATTTAAATCTATTTAACATGCGTTCACTTGAAGTTGACAATCGAACATCATTACCCATAAATACCTTAACACGAATTTTAATCACCTCAGATGATGTTATTCATTCTTGAACAATTCCGAGAATTGGAGTAAAAGCTGATGCTACTCCGGGACGATTAAATCAAGCAACATTTTGATTTAATCGTCCCGGAGTATTTTATGGTCAATGTTCAGAAATTTGTGGAGCAAATCACAGATTTATACCTATTGTAATTGAAAGAACTTTGATTAATAATTTTCTTAGTTGAATCTTAAATTATATTGAATCACTAGATGACTGAAAATAAGTGATGGTCTCTTAAACCATATCATAGTAACATAATAACTACTTCTAGTGATTGACTAACAATTTATCAAGAAGTTAGTTAAAAAATAACATTAGTATGTCAAACTAAAGTCATTATCATTTAATACATCTTTATACCCCAAATAATACCCCTAAATTGGCTAATCCTATTTTACATTGTTTCTACTAGATTAATTTTTTTTAATGTAATAAATTTTTTTATATTTTCCCACAAAATCCCATTAACTTCAAATAAAATTTTACTAAAAACCCTAATTTGAAAATGATAACCAACCTATTTTCAATTTTTGATCCCTCTTCAAATTTTATAAACCTATCAATAAATTGACTTAGAATTTGAATCGGATTATTATTATTTCCTTCCTCGATATGGTTAATTTCATCACGAAATAAAACCCTTTGAAGTTTTATTTTAAGTAAACTTCATGAAGAATTTAAATTATTAATTGGTAAAAAAAAAATTAACAAAGGATCAACATTCATATTTATTTCAACATTTTTACTTATTATATATAATAATTGTATAGGATTATTTCCATATATTTTTACTGGGACAAGTCATATAGCTATAACTCTATCTTTTGCTTTACCTTTATGACTAAGATTTATACTCTTTGGTTGAATTAATAACTCTAATCACATATTTATTCATTTAGTTCCTCAAGGAACTCCAAATATATTAATACCTCTTATAGTTTGTATTGAAACAATTAGAAATTTAATTCGTCCCGGAACTTTAGCTATTCGACTCGCAGCAAATATAATTGCAGGACATTTATTAATAACCCTCCTAGGAAATTCTGGGAGAAACATTATAGATTCATTTTTACCCCTATTAATTTTAGTTCAAATTATACTTTTAACTCTAGAATCCGCAGTTGCTATTATCCAATCATATGTTTTTGCAGTATTAAGTACTTTATACTCTAGAGAAGTAAATTAATAATGATAATACACACTAATCACCCCTACCATTTAGTTACTTATAGTCCCTGACCTATTATAACTACTTTAAGAATCATAATCATAATATTAGGTTTTATTAAATTTTCTTATGAGTTTAGTGAAAAATTTATGCTATTAGGAACTTTAATTTTAATTTTAATTACTACTCAATGATGACGTGACGTTGTACGAGAAAGTACATATCAAGGATTACACACTAAAAAAGTAATCTTTGGACTACGATGAGGAATAATTTTATTTATTATTTCAGAAATTTTTTTCTTTGTATCTTTTTTTTGAACTTTTTATCATAGAAGCTTAACTCCTACTATTGAATTAGGGTCCTTTTGACCACCTCAAGGAATCTGACCCTTTAATGCTCTTCATGTTCCTCTTCTTAATACAACGGTACTTTTAGCATCAGGCATCACTATTACATGAAGTCACCATGGACTATTAATAAATAATTATAATCAAGCCACCCAAGGATTAATATTTACCATTATCCTTGGGATTTATTTTACCATCTTACAACTCTATGAATATTATGAAGCTCCGTTTACAATTGCGGATTCAGTTTTTGGGTCAATCTTTTTTATAGCAACTGGATTTCATGGACTTCATGTAATTATTGGAACTACATTCTTAGTTACATGCTTATTTCGAATAATTTATAAACATTTTACATCTATTCACCACTTTGGTTTCGAGGCAGCAGCCTGATATTGACATTTTGTGGACGTAGTATGATTATTCCTGTACATTTCTATTTATTGATGAGGGAGATAAATCCAATTTATTTAGTATAAAAGTACAATTGATTTCCAATCAAAAAGTCTATATTTAATTAGAATAAATAATTAAAATTTTAATTTTTATCTCATTCATTACTATATCAATTACCTTAACAATCATATTACTAACAAATTTCTTGTCAAAGAAAAAAATTGAAGACCGAGAAAAAAATTCACCTTTTGAATGTGGATTTGATCCGATTAGATCCTCGCGCCTTCCCTTTTCTTTACGTTTCTTTTTGATTGGAGTAATCTTTTTAATTTTTGATGTAGAAATCGCCTTTATCTTACCAATAATTATCATTCCTCTTACATCAAAAATAACATCTTGAATATCTACTAGAATTATATTCTTATTGATCTTAACAACTGGTTTATTTCATGAATGAAATCAAGGTTCTCTCGACTGAGCAACTTAACTTTATAAGGGTTATAGTTAAAAATAACATTTGACTTGCACTCAAAAAGTATTGAAATATCAATTTTCCTTATTATAAGTAAGAAGCAAATTCATTGTAATCAGTTTCGACCTGATAGTAAGATATTCATATCCTTATTTGTTGATTAATTGAAACCAAATAGAGGTATATCACTGTTAATGGTAAAATTGAAATTAATGCTTTCCAATTAAGAAAATGTGTAGATCGAATATAAGTTGCTAATTTATTATTCAAGTGGTTTAATCCCATTTACATTTTAATTTATATAGTTTAAATAAAACATTACATTTTCATTGTAAAAATAAATTTTTCAATTTTATTAATAGTAAGTACTCTATTTAAAGATAAATTAGTTATCTCAATAACAGCTTCAATGTTATACTCTCTATAAGATATTTAAATAAATACAAAATATTATTATAAGTAAAATACTAAAAAAAATCATTAAATAAGATTTTAAATCATTATATTGAAATCATTGATTAAAACATCTTAATTTTATCAAAATATAATATAAATTTTGTGCTCCAAAATATTCTCTTCAGCCTAAATCAATATATATCATTGAATTTAAACTAATAATTAAAGGTATTTTTCTTACTCCTTTAGTAAAAATTAAAGGTATATATCATATTGAAGCAAAAAAAATTGTCATTTTATAATACTTAAATGTTGAAAAATAATAATTTATTCTATACTTAAATATAATTCTTCCTAATCATAAACCTACAAATCTACCTAATAAAGGTATTATTTTCATTATTAATGTCATATAAATTATATAGGGAGTAAGAAAAATAATCCAATTTAATATCCCACCTCCAATAATTGCAAATAATATTAAACCTAATATTCCATATACTATTATTCAACTTTCATTTAATTTACATATTGATATTATATTAAAATCTCCTCATAATACATAATAGGATAAACGAAAAGAATAACTTACTGTTAAACCTGTTGAAAAAAAAAATAAAACATATATAAAGATATTTAAATTTCTTAAAGATAATATTTCTAAAATTATATCTTTTGAATAAAATCCTGCTAAAAAAGGTATACCACATAAAGCAAAATTTGATACTATAAAACAAGATGAAGTAAAAGGCATAAATATTACTAAATTTCCTATAAAACGAATATCCTGTAAATTTTTTATTCTATGGATTATTATTCCTGTACACATAAATAACAATGCCTTGAATAAAGCATGAGTTAATAAATGAAAAAAAGCCAAATCTGAAAAACCTAATGATAAAATTCTTATTATTAATCCTAATTGACTTAAAGTTGATAAAGCAACAATCTTTTTTAAATCATATTCAAAATTTGCTCCTAACCCTGATATAAACATTGTTAAAACAGATATTACTAGTAAAAACTTTAATAATCAATCCGGAAAAACCTTACAAAATCGGATTAATAAATAAACCCCAGCCGCAACCAAAGTTGATGAATGAACTAGGGCTGAGACAGGAGTTGGAGCAGCTATTGCTGCAGGCAATCAAGCTGAAAAGGGAATTTGTGCACTTTTTGTTATTCCTGCTAAAAGTACTAAAAATGAAATTAAATATATTTCAATTTCATTTAATGTACAATCTAAATAAAAAATATAATTTCATCTTCCAAAATTTAATATTCATGAAATTGCTATTAATAAAGCAACGTCCCCTACTCGATTAGATAAAGCTGTCAATATTCCAGCATTATAAGACTTAGTATTTTGATAATAAATTACAAGACAATAAGAAATTAAACCTAACCCATCTCAACCTAATAAAATTCTAATCAAATTTGGACTAATAATTAAAAACATTATAGACAATACAAATATTAATACTAAAAAAATAAACCGATTTAGTGATGAATCTCCAGTTATATAATCTTCTCTATATAAAATTACTAAGGATGAAATTAATAAAACAAAACTCATAAATATGAGAGACATTCAATCCAACAATATTGTTATAACAACTGAAGAAGTACTTAATCTAACAATTTCTCATTCAATAAAAATAATTAAATCATTTAAAATAAAAACCATTCTAGAAATAAATATTATTAAAGAAAATAATGCTAATAAAAAAAATCTAATAAAACATAATGATAAATAAACCATAACTTAAAATAACTATTCATTATATCCATGATACCACAAATCATAATTTTATGATAAACTATTTAAGTAAATTAACTTTTATTTAAAATCAAATTAAAACATAATCTCTCTTTAAAATTAATAAATTTAAGGGTAATCAATGAAGTATTATTAATAAATATTCACGTCTATACCCTCCAACTCTTCTATAAATGCCTGAATAATAAAATCCATGTTGACTATATGAATATAAATATAAAGTATAAGCAGCTCTAAAAAATGAAATTAATATAAGAAAAAATATAGATATTCATACCCAACTTACTATACTATTGAATAATCCAATTTCTCCTAATAAATTTAAAGTAGGGGGAGCAGCTATATTGCTAGATGAAAGTAAAAATCATCATAATGTTAAACTTGGCATCAAATTTAATAAACCCTTATTAATTAACAATCTTCGTCTTCCCAGACGCTCATAACTAATATTAGCTAAACAAAATAAACCAGATGAACATAAACCATGAGCAATTATTATTACAAAAGTTATATAAAACCCTCAAATATTTAATGTTATTAAGCCCCCAATAATTAAACCTATATGAACCACAGACGAATAGGCAATTAATGCTTTTACGTCTATTTGACGTAAACACATTAAACTAACTATAACCCCACCGATTAATCTAGTAGAAAATCAAAATACATTAACTAATATCCCAACTCTCTTCAAGAATTCATATACTCGTAATAAACCATAACCTCCTAACTTTAATAAAACTCCAGCTAAAATCATTGAACCTGAAACTGGAGCTTCAACATGAGCCTTAGGTAATCATAAATGCACTAAAAATATAGGCATCTTAATCAAAAAAGCTAAAATTATTCTAAAATATAAATATCAATTCAATAAATTATTTTTATAAACTAACGAAAAAACTAAATAGCCCATGTTGTTATAAAAATATATAATACCCATCAATAATGGTAATGAAGCTAAAAGAGTGTATAATAATAAATAAATACCAGCTTGCAATCGCTCAGGTTGATATCCTCACCCAAAAATTAAAAATAAAGTTGGAATCAAACTACCCTCAAAAAAAAAATAAAATGAAATGAAATTTATTCTACAAAAAGTACAAATTAATATCAATAACAATATTAAAATTATAAATATAAATAAATTATTATAAAATTTATATCGAATAACAGAATAACTAGCTAAGATTATTAAAACACAAATTCAAAATCTTAGCACAATAAGACCATATGATAAGTAATCATATCCAAATATATAACTCATTCTTATTCAATAAAAAAAATCCACTCCAATAAAAAATTTAAAAGATATAAAAAATAATAAATTCTGAATTAATAATCAATTTTTATTCATTAAACATAATGGAATCAAAAACATTAAACTTAAAATATATCTTAACATTGCAATAAGCTAAACGAATTAAAATAATCATTTCCATGAGTTCGAATTATAGAAACCAAAATTGATAATCCTAATACACCTTCACAAACTGCAAATGAAAGAAAAATTATAGTCATATATAACTCTCCTCTTATTATTAAATAAAAATACAAAATAATGAACAAAACTAAAACAATAAATTCTAAACTTAATAAAGTTACTAATAAATGTTTACGTCCAGAAGAAAAAACTCACAAACCACATAAAAATATAAAACAGAATATTATTAACATTTTTTAGTTTTAATAGTTTACTAAAAACACTGGTCTTGTAAACCAAAATTAAGAATAATTACTTTTAAAACTTCAAAGGAAAGAAACTCATCATTAATTCCCAAAATTAATATTTTATTATTAAACTACCCTTTGATATTTTATACTCAT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ATATAAACTTTCAAAAAAAAAATCCACTTTACCCAAAAAATAAGTATGCTTTTTTTAAAACGTTTTCCTCCCTAGAAGTAGGAATTAAGTGCCCAATTCAATTCGATTTAATGTTTTTTTTTAAACATTGGTCTACTAAAGTATATTTAGTATATAATAAATTTAATTATATATATGTCTTATCAAAGATTAATAAAAAAAATATTCTCAAACAGCGGTATACAAAAATATAAAGTAAGTAAGGTCCAACGCGGATTATCAATTAAATAATAGACTCCTCTAAATA4. I recommend one more round of editing for writing clarity. For example, Lines 50-51 are a little confusing, and should probably be split up into two sentences.Reply: Thanks for your suggestion. Lines 50-51 have been split up into two sentences. Please check lines 56-57.AttachmentR2-Reply letter-GS2.docxSubmitted filename: Click here for additional data file. 7 Jul 2021Complete mitochondrial genomes of four species of praying mantises with ribosomal second structure, evolutionary and phylogenetic analysesPONE-D-21-03889R2Dear Dr. Smagghe,We\u2019re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you\u2019ll receive an e-mail detailing the required amendments. When these have been addressed, you\u2019ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they\u2019ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact Kind regards,Ben J Mans, PhDAcademic EditorPLOS ONEAdditional Editor Comments :Reviewers' comments: 21 Oct 2021PONE-D-21-03889R2 Complete mitochondrial genomes of four species of praying mantises with ribosomal second structure, evolutionary and phylogenetic analyses Dear Dr. Smagghe:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. If we can help with anything else, please email us at Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staffon behalf ofDr. Ben J Mans Academic EditorPLOS ONE"} +{"text": "Drosophila melanogaster embryos, we observed that the nuclear distributions of transcription factors and histone modifications undergo a similar transformation of increasing heterogeneity. This spatial partitioning of the nucleus could lead to distinct local regulatory environments in space and time that are tuned for specific genes. Accordingly, transcription sites driven by different cis-regulatory regions each had their own temporally and spatially varying local histone environments, which could facilitate the finer spatial and temporal regulation of genes to consistently differentiate cells into organs and tissues. Thus, \u2018nuclear morphogenesis\u2019 may be a microscopic counterpart of the macroscopic process that shapes the animal body.An embryo experiences increasingly complex spatial and temporal patterns of gene expression as it matures, guiding the morphogenesis of its body. Using super-resolution fluorescence microscopy in Summary: The nuclear space becomes progressively heterogeneous during embryo development, forming distinct transcriptional sub-nuclear environments, paralleling the partitioning of the embryo body during morphogenesis. During embryogenesis, transcriptional regulation controls the expression of genes that pattern the animal body . This prDrosophila embryos at a late stage of development and that the developmental gene shavenbaby resided in regions enriched for TFs when it is transcriptionally active , rhomboid (rho) and snail (sna) enhancers to measure their local chromatin environment during different stages of development and at different locations in the embryo. In summary, we found that the distributions of TFs and epigenetic marks became progressively heterogeneous as development proceeded. Thus, the nuclear space may undergo a transformation that parallels morphogenesis, where the animal body becomes increasingly heterogeneous and segmented. Additionally, even for the same gene, the nuclear environments at transcription sites depended on the location and timing of expression. Organizing the nucleus to form multiple reconfigurable regulatory environments in a single nucleus could permit genes to perform different tasks at different times and facilitate the complicated regulatory patterns during later stages of embryogenesis.However, the origins and spatial-temporal behaviors of these transcriptional microenvironments during development remain unclear. When does the nuclear microenvironment form? How and when do genes interact with these microenvironments? Here, we imaged the distributions of TFs and histone modifications in Hunchback (Hb) is a TF that is a gap gene in the early embryo . We obsey-axis) when the image has been shifted by a given distance compared with the original (x-axis) indicates the abundance of structures at that length scale . There was a monotonous increase in autocorrelation as the embryo ages, suggesting that Hb environments spanning several hundred nanometers became more prevalent as development progressed. Indeed, stimulated emission depletion (STED) distributions across similar developmental stages with Airyscan and computed their autocorrelation . There was a similar upward trend, suggesting the formation of local TF environments in older embryos.To quantify the spatial distributions of Hb, we computed the spatial autocorrelation of Hb intensity G. The len (STED) imaging n (STED) H, bottomn (STED) H, middlen (STED) H, top, tFig.\u00a0S1F,G) and Engrailed , which are active within a narrower time frame during development, showed high autocorrelation when they are first expressed (stage 10) and when they reach maximum expression (stage 14) in nuclei on the ectoderm. At stage 16, when both are no longer expressed, their distributions became sparse with low autocorrelation. This two-state behavior for factors only expressed at later stages of development is unlike Hb and Kr, which showed a progressive increase in autocorrelation with intermediate states. This suggests that the general distributive properties of TFs may conform to the underlying nuclear architecture at the time of their expression.The TFs Ultrabithorax is expressed and imaged embryos at stages 5 and 10, in nuclei within 20\u2005\u00b5m of the ectoderm to preserve optical resolution. We stained for H3K4me1 . However, using RNA fluorescence in situ hybridization (FISH) to detect transcription sites degraded the signal from histone modifications . To improve quantitative image analysis, we detected transcription sites without the denaturing step in RNA FISH to preserve the signal from histone modifications (Drosophila melanogaster line expressing a reporter mRNA containing MS2 stem loops driven by 18\u2005kb of the cis-regulatory region of hb (hbBAC) with a l (hbBAC) . This sp embryos . The mRNhbBAC transcription sites between stages 5 and 10 showed changes that are distinct from the global trend in To quantify the spatial distribution of histone marks around transcription sites, we computed the average radial intensity distribution centered on the transcription site F-J acco. We obseFig.\u00a0S3A). We generated transgenic lines with MS2 reporter mRNA driven by enhancers from two genes active during this process: snail (sna) and rhomboid (rho). The constructs are named sna A2.2 W0.10 and rhoNEE, respectively. We observed that both were expressed in patterns corresponding to their endogenous genes : in an anterior-posterior band along the ventral side for sna A2.2 W0.10 . rhoNEE, on the other hand, showed no change in H3K4me1 and an increase in H3K4me3 only between early and mid cellularization . Thus, the regulatory environments around genes can change over relatively short time scales in a manner that depends on their cis-regulatory regions.To test whether we can resolve finer temporal changes in local histone environments, we focused on the 1 h cellularization process at end of the rapid nuclear division cycles at stage 5 , which wus genes , 2007 has a sharp ventral edge resulting from Snail repression and trails off gradually in the dorsal direction due to a decreasing gradient of the activator Dorsal but differential engagement from enhancers, depending on whether the repressor Snail is present. Together, these results imply that histone environments around genes depend on the properties and states of cis-regulatory elements. These differences could refine expression patterns over time, as is the case for many developmental genes of hunchback (hb) on a bacterial artificial chromosome (BAC) driving a yellow reporter mRNAs containing 24 MS2 stem loops generated by hb. The reporter constructs for rhoNEE and sna A2.2 W0.10 were derived from the plasmid pIB-hbP2-P2P-lacZ-\u03b1Tub3 UTR (hbP2 enhancer between the restriction sites HindIII and AscI replaced with the rhoNEE or the sna A2.2 W0.10 sequence. The hbP2 promoter was retained. The plasmids were synthesized by Genscript. The constructs were injected into Bloomington Drosophila Stock Center line 27388 and integrated using RMCE by GenetiVision. Virgin flies from a transgenic D. melanogaster line containing MS2 coat protein (MCP) fused to EGFP driven by the nanos promoter staining. The MCP-EGFP line does not contain a nuclear localization signal (NLS) to minimize MCP-EGFP forming aggregates in the nucleus in the absence of MS2 mRNAs.All Tub3 UTR with theter from were crorhoNEE, CTTGGGCAGGATGGAAAAATGGGAAAACATGCGGTGGGAAAAACACACATCGCGAAACATTTGGCGCAACTTGCGGAAGACAAGTGCGGCTGCAACAAAAAGTCGCGAAACGAAACTCTGGGAAGCGGAAAAAGGACACCTTGCTGTGCGGCGGGAAGCGCAAGTGGCGGGCGGAATTTCCTGATTCGCGATGCCATGAGGCACTCGCATATGTTGAGCACATGTTTTGGGGGAAATTCCCGGGCGACGGGCCAGGAATCAACGTCCTGTCCTGCGTGGGAAAAGCCCACGTCCTACCCACGCCCACTCGGTTACCTGAATTCGAGCT; and sna A2.2 W0.10, GTCGACCTAGTTCTGTTTTGTGACTCGGATTTACTATTTCGCATGGCTCCTCTTCGAACAATGTCAGTCGAGCTCTGTAGATCCCTGTGTTCCCTCTTCATTGTCAACTTGAACAAATGAGCCAGGGAACAAGGTGCAAAAATGGGACGGTCCTATTCTCAGCAAAAATTGACAAGAACAACAACAATGTCTATGGAAAATCGAACTTCATCCCAGCACCTGCAGAAATCCCGAGCGAGTCGGGGAAAAAGTATTTAACCCCCGAAAGGGTTTTCCCCAAAATAATGAAGTAATGAATGAAGCGGAAAACACTGGCCGCCAATCTACCTAATACTAATGAGCGGGCCAACCCGACCAGGAATTTTTGCAAGTCAGGTACTTCAACGGATATATGGGTTCGACAAGTGCGGATTTTCCCGCGACATCAATGAGGACTTGGCCGGGTTATCCGCGGTGCTCATCGGGCAATTCCGCGGCCGAGGACTTCATCGTAGTGATCATTAGGTAGATATGTGCATGGATGTGACATGGCGATCATTGCGCGGAATAACACACGTAATAACCGAGATATCCGGGATGACCCACCAGGTAGGATGTGAGGACATATAGAAAACCCCCAGCCAGTTTTTCCACTCGTCGTGGCTTGTTTTGCTTGAGTTTCGCTGACTGCGTAATTGGATAAGATGGGAAATTACTTTAAATCCTTCGCTGATCCACATCCGGACATTCGTCGAAGGAAAATCCATTGCAGGGAAATACGAAATGGAAATGCGGCTGGGTTATTGGCTCGACATTTCCCATCTTCCCTCACGCCATTGGTTGCAGGATCGCGGGGAATTGGAATTCCGCGCTGGAATTTTTTGTCACCTCTTGGGTTTATCAAAACTTTTGGGTTTGCTATGGATTTTTTCCAATTTTACCACCGCGCCTGGTTTTTTTTTTTTGACGACGCGGAAAATCGGACTTGGCTATGCGGGCTTGTCTGTTTTTCCGGGTACAAAGTCTGCATGTCAGCCTCCATGCGGGAGTGGGAGTTGGGAAAGTTTCCCATCGATAGTTGGAGGGGTGGCTTGAAAGTCTGGAGGTGCTAGCTGGGAAAGTTGTGTGTGCGCGATGAGGCAAGGAGTCAAAGATCAGGGGAGTTGGAAAGCGAGAATTGTGGGAATCGTCCAGGACTCAGCTGGATGCTGAGGGGCAGTATGATTTTTTTTACGTTATCAATCGAATTGATTTTAAGACAGCAGAACTTCACATACTAATAAGATGACCATGGGATTAGTTAAAATGTGTAACTCGTATTCGAATCGTCATTCTTTCACGGACCAATCGTGGGAACAGGAGATCTCTTCGATCCAAGCTCACAGGAGACTTGACACTCTTCGTCTATTCCTTGTCAAGTTTTTAATGACATCTCCTATGCCCTGAGCTATGTTTTCCTAGCTCTCATCGATCGCTGCCAATGAGCCACTGGAGATGATCCATAAGTCAGCGTAGAGTGCACCCCAGAGTTGACACTTGGTGTCTCGGAATTCGGCTCATTATCAGTGCTATTTTTGGAACACCTCTCTGCGAAGGTGTCATTTTTGTCAGTGCGTATCGCTCAGGTTCAACTCCCCACCAAAAACCGAATTTAGAGCATCGGCAGATGTACTTGAAGCACTCAATCTAAGTGAGGAAACCACCCCATGAACGAAGAGTACTAGGAGTCCTATTTGACTCGTGCTTAAAAATAGAAAATTACTTAGGGTGATCCATAGGTAGGGAGGCGATATTGTAACTTGCATTTCGGACCCGGACCTGCACGAGTTATTACGGGTGGGTTGTGAGCGTATCGGGAAATTGGAGAGCCACCAGATCTGTCATAACTTATACGGGGGATCCTTATTCCTGGGAGGGTGCGCCTGCGTCTGCTCTTCCGAGAGAGAGGTGGGAAATGGAGGAAGAGAGAGAGAGAGAGAGTGAGAGAGCAGGTAGAGGGAAGTGAGGGAAATACGCAATAAGGGTATGGGAAAAGTGCTGTTGTTGTTGCTAGGTAGCGACGCACACGTGCGAGTGTTTTTCTGTTTTGAAGAAGAACCACCACCAAATGG.The enhancer sequences were as follows: 1118w flies (for imaging TFs) and from crosses of the hbBAC, rhoNEE or sna A2.2 W0.10 reporter lines with the MCP-GFP line (for imaging transcription sites and histone modifications) were collected, fixed and IF stained using previously described protocols ;The secondary antibodies were as follows: Alexa488 donkey anti-mouse ; Alexa488 donkey anti-rabbit ; Alexa555 donkey anti-rabbit ; Alexa594 donkey anti-mouse ; and STAR RED goat anti-rabbit .in situ hybridization (FISH) and histone modifications using IF, we followed a previously described FISH protocol . One additional secondary antibody was used: Alexa488 donkey anti-sheep .To stain for transcription sites using RNA fluorescence protocol and the x-y and z direction of stacks used for quantification. The laser power and gain were adjusted to maximize the signal to noise ratio within the dynamic range of the PMT or Airyscan detector for each sample preparation condition (i.e. different antibodies). Within a given sample preparation condition, laser power and gain were kept the same across embryos. The acquired Airyscan stacks were processed with Zen 2.3 SP1 in 3D mode to obtain super-resolved images.Confocal and Airyscan imaging Drosophila embryos was carried out in ProLong Diamond without DAPI . Fixed embryos at the appropriate stage and orientation were imaged on a STEDYCON in 2D STED mode on an Olympus BX53 microscope. Excitation lasers with wavelengths of 594 and 640\u2005nm were used as appropriate for the specific fluorescent dyes. The wavelength of the STED laser was 775\u2005nm. All samples were imaged using an Olympus UPlanSApo 100\u00d7 Oil/1.4 objective. The resolutions used for the x-y and z directions were 25\u2005nm and 250\u2005nm, respectively. The laser power and gain were adjusted to maximize the signal-to-noise ratio within the dynamic range of the APD detector on the STEDYCON for each sample preparation condition. Within a given sample preparation condition, laser power and gain were kept the same across embryos.Mounting of fixed r = 0. Radially averaged auto-correlation functions were computed using an ImageJ macro . Airyscan images were used for the autocorrelation of Hb and Kr for their wider field of view compared with STED, in order to include a larger number of nuclei. Intensity distributions and autocorrelation functions were plotted using Matlab (MathWorks).Image processing was carried out using Fiji with the"} +{"text": "Aurkb is an essential gene, we conclude that this analog-sensitive allele is either catalytically inactive or not fully catalytically active in mouse.The mammalian genome encodes three Aurora protein kinase homologs (AURKA/B/C) which regulate chromosome segregation in nearly every cell type. AURKC expression is largely limited to meiotic cells. Because of the similarity in sequences between AURKB and AURKC, determining their separate functions during meiosis is challenging. We designed a chemical genetics approach to investigate AURKB function. Using Crispr/Cas9 genome editing in mouse, we replaced an ATP binding pocket amino acid to permit binding of cell-permeable ATP analogs. We also introduced a second site supressor mutation to tolerate the pocket enlargement. Heterozygous mice were fertile, but never produced homozygous analog-sensitive mice. Because Because AURKC shares high sequence homology with AURKB, standard approaches such as RNAi knockdown and small molecule inhibition to understand their roles do not provide the specificity required to assess non-overlapping functions. Furthermore, these kinases can compensate for one another in oocytes from mouse knockout strains , making phenotype assessments less clear. Therefore, alternative strategies to provide specificity are needed.The Aurora protein kinases (AURK) are essential regulators of chromosome segregation in mitotic and meiotic cell divisions (Carmena and Earnshaw 2003). Unlike mitotically dividing cells which express two AURKs (AURKA and AURKB), mammalian oocytes, which undergo meiosis, express three AURK homologs: AURKA/B/C . Although most protein kinases tolerate the ATP pocket enlargement, approximately 30% of protein kinases do not . A previous report demonstrates that human AURKB ATP-pocket enlargement (Leucine 154 to Alanine) abolishes its catalytic activity. Excitingly, this activity can be rescued by mutation of a second site suppressor in the C-terminus of the kinase (Histidine 250 to Tyrosine). When HeLa and U2OS cells were transiently transfected with the analog sensitive human AURKB allele, the kinase was active and cell division was not altered . Based on these data, we were keen to adopt this approach to study the potential unique functions of AURKB. We used Crispr/Cas9 genome editing to introduce the ATP-binding pocket point mutation (L159 in mouse) and second site suppressor mutation (H255 in mouse) into the mouse genome . After confirmation by restriction digestion and Sanger sequencing , we obtained 4 founders that were heterozygous for the analog-sensitive allele on the same chromosome. To expand the colony, we set up breeding cages using the heterozygous founders. Heterozygous animals were fertile and gave birth to pups. We also crossed heterozygous pups to increase chances of obtaining homozygous animals. After genotyping 80 pups that were born to 8 heterozygous mating pairs, we did not obtain any homozygous mice; wild-type and heterozygotes were born at altered Mendelian ratios . Because Aurkb is an essential gene , these data indicate that this analog-sensitive allele of Aurkb is either catalytically inactive or not fully catalytically active in mouse and that it cannot be used to determine specific AURKB functions in mouse oocytes.Chemical genetics is a strategy that documents superior specificity in inhibiting protein kinases. This approach involves enlarging the kinase ATP-binding pocket by introduction of an amino acid substitution. This enlargement allows specific acceptance of cell-permeable, bulky ATP analog inhibitors that cannot bind other kinases in the cell (Garske C57BL/6J mice: Jackson LaboratoryCas9 IDTL-A RNA guide (PAM sequence in parentheses) from Sigma: ACTACTTCTACGACCAGCAG (AGG)L-A donor oligo (mutations in lower case):CAGGCACTAGATCCCACACCAGGTAGGCCCTGACCGTGGCAGTCCGCTGCTCATCGAAGGTCCGACTCTTCTGCAGTTCCTTGTAGAGTTCCCCGCGAGGGGCGTATTCCgcGATTAAGTAGATtCTCTGCTGGTCGTAGAAGTAGTTGTAGAGTTGAAGGATGTTGGGATGTCTGGH-Y RNA guide (PAM sequence in parentheses) from Sigma: TGCCCCCAGAGATGATTGAG (GGG)H-Y donor oligo (mutations in lower case):TCCGACGATACGTCTCACTGTGGCTAGGGCTCTCGAAGGGTGGGTTCCCCACCATCAGTTCATAGCAGAGCACCCCGATGCACCATAGATCTACCATTTCATTATaCATGCGgCCCTCAATCATCTCTGGGGGCAGATAGTCCAGCGTGCCGCACATGGTCTTCCTCCTGGTGGGATGAGGenotyping Primers:L159A substitutionL159WT F: 5\u2019- CCAGCAGAGGATCTACTTAATCCT -3\u2019L159AS F: 5\u2019- CCAGCAGAGGATCTACTTAATCGC -3\u2019WT R: 5\u2019- ATGATctgtgagggtccacacaag -3\u2019H255Y substitutionH255WT F: 5\u2019- GAGATGATTGAGGGGCGCATGCAT -3\u2019H255Y F: 5\u2019- GAGATGATTGAGGGGCGCATGTAT -3\u2019WT R: 5\u2019- CTGGCTCCTACAGTACACAAAGG -3\u2019"} +{"text": "STX3 variants were previously reported in five individuals with the severe congenital enteropathy, microvillus inclusion disease (MVID). Here, we provide a significant extension of the phenotypic spectrum caused by STX3 variants. We report ten individuals of diverse geographic origin with biallelic STX3 loss-of-function variants, identified through exome sequencing, single-nucleotide polymorphism array-based homozygosity mapping, and international collaboration. The evaluated individuals all presented with MVID. Eight individuals also displayed early-onset severe retinal dystrophy, i.e., syndromic\u2014intestinal and retinal\u2014disease. These individuals harbored STX3 variants that affected both the retinal and intestinal STX3 transcripts, whereas STX3 variants affected only the intestinal transcript in individuals with solitary MVID. That STX3 is essential for retinal photoreceptor survival was confirmed by the creation of a rod photoreceptor-specific STX3 knockout mouse model which revealed a time-dependent reduction in the number of rod photoreceptors, thinning of the outer nuclear layer, and the eventual loss of both rod and cone photoreceptors. Together, our results provide a link between STX3 loss-of-function variants and a human retinal dystrophy. Depending on the genomic site of a human loss-of-function STX3 variant, it can cause MVID, the novel intestinal-retinal syndrome reported here or, hypothetically, an isolated retinal dystrophy.Biallelic The online version contains supplementary material available at 10.1007/s00439-021-02284-1. STX3, OMIM 600876) by us and by others . All affected individuals had homozygous loss-of-function For all clinical data presented including the molecular genetic studies appropriate informed consent was obtained from subjects or their parents in accordance with the guidelines of the respective institutes at which the subjects were seen and approved by the institutional review boards and ethics committees as required. Human cadaver tissue was donated to the Willed Body Program at the McGovern Medical School at the University of Texas Health Science Center in Houston. Use of cadaver specimens is deemed exempt by the Institutional Review Board.Animal procedures conformed to National Institutes of Health guidelines and were approved by the Animal Welfare Committee of the University of Texas Health Science Center at Houston. Mice were kept under standard housing conditions with unlimited access to food and water and with a 12\u00a0h light/dark cycle. Genotyping was performed by PCR using DNA isolated from tail snips as described with DNA extracted from peripheral blood leukocytes in P3, P4 and P5 as described . Variant designations are based on NCBI transcript reference NM_004177.5, using\u2009+\u20091 as the A of the ATG translation initiation codon. The DNA samples from all patients\u2019 parents, and from a number of additional family members were tested for the segregation and zygosity of STX3 variants identified in index patients .Novel pathogenic re Table . STX3 muSTX3 loss-of-function variants; STX3 variant identification by WES was reported for P1 and P2 or Alexa Fluor 488 where used for labeling. All antibodies were diluted with blocking solution . Specificity of immunolabeling was confirmed by processing a second set of sections in the absence of the primary antibodies, or by substituting normal rabbit serum for polyclonal antibodies, as appropriate.Images were captured with a ZEISS LSM 800 confocal microscope or with a Nikon A1R Confocal Laser Microscope at a thickness of 0.3\u20130.5\u00a0\u00b5m. Confocal images were processed using the manufacturer\u2019s software Zen 2.5 (Blue edition) or Nikon Imaging Software Element (Version 4.20) and with Adobe Photoshop.STX3 transcripts was analyzed by identifying matching reads from recent Illumina TruSeq\u00ae RNA-seq projects obtained with human retina samples . Probes of 120\u00a0bp length covering 60\u00a0bp upstream and downstream of the respective splice sites were analyzed. Default BLAST settings were used with the exception of the Expect threshold, which was set to 1000. Reads were identified as matching if they had at least 90\u00a0bp identity with the probe, corresponding to an E value of\u2009<\u200910\u201330. Reads per kilobase of transcript, per Million mapped reads (RPKM) where calculated for each probe.The expression of the different Sequences of probes used:HS STX3 8_9BAGCAGCATCAAGGAGCTTCACGACATGTTTATGGACATCGCCATGCTGGTGGAGAATCAGGGATCCATGATTGACCGTATTGAGAACAACATGGACCAGTCAGTGGGCTTTGTGGAGCGG:HS STX3 9B_10BGTGGAGCGGGCCGTGGCAGATACCAAAAAGGCTGTCAAGTATCAGAGTGAAGCCCGGAGGAAGAAGATCATGATCATGATCTGCTGTATTATCCTTGCGATCATCTTAGCTTCCACCATT:HS STX3 8_9AAGCAGCATCAAGGAGCTTCACGACATGTTTATGGACATCGCCATGCTGGTGGAGAATCAGGGTGAGATGTTAGATAACATAGAGTTGAATGTCATGCACACAGTGGACCACGTGGAGAAG:HS STX3 9A-10AAATAATCAATGCTAAAATGCCCAGCAACACAACTACTAGCACAATGATAATTATCAATTTCTTCCGGGCCTGACTCTGGTATTTCACAGCTTTTTTCGTTTCATCTCGTGCCTTCTCCAC:HS STX3 9A-11AGTGGAGAAGGCACGAGATGAAACGAAAAAAGCTGTGAAATACCAGAGTCAGGCCCGGAAGAAACTGATTTCACTCCAGACTGGTGTGGCCACCCTTGTCTTCAGATGAGAATGGAGTCTGHuman Tissue cDNA samples were purchased from Takara Bio USA, Inc. . Standard PCR reactions were performed with the OneTaq Hot Start Quick kit (New England Biolabs) using these primers:A: HS Syntaxin 3AB Sense exon 1:GGCTTCAGGATGAAGGACCGB: HS Syntaxin 3B Sense Exon 7:GCAACCCGGCCATCTTCACTTCTGC: HS Syntaxin 3A Antisense exon 10A:AGCCCAACGGAAAGTCCAATD: HS Syntaxin 3B Antisense Exon 11B:CCTGTCCCTGTCCTCCGCCCAATGAPDH1: ATGACATCAAGAAGGTGGTG,GAPDH2: CATACCAGGAAATGAGCTTGConditions: 35 cycles for each reaction, annealing temperatures STX3: 55\u00a0\u00b0C; GAPDH: 50\u00a0\u00b0C. PCR reactions were analyzed using agarose gel-electrophoresis and correctness of the amplified fragments was confirmed by direct sequencing.STX3 expression in the murine and non-mammalian retina and roles for Stx3 in protein trafficking in photoreceptors carried the same pathogenic variant that is located in exon 9A and thus spares STX3B.Given the high level of Severe visual impairment was seen and a diagnosis of EOSRD rendered in individuals P2-P9, as evidenced by the inability to respond to or track visual stimuli, locate or reach for objects. Half the subjects also exhibited nystagmus. Five subjects underwent fundoscopic examination, revealing pallor of the optic disks Fig.\u00a0 and fundSTX3A and STX3B, we asked whether both splice forms were present in the human retina. A GeneBank search identified a large number of ESTs and several full-length cDNA clones corresponding to STX3A. However, while no clones corresponding to STX3B were identified, analysis of the human STX3 gene sequence showed the presence of putative STX3B-specific exons 9B, 10B and 11B and inner plexiform layer (IPL) respectively, labeled for the major structural protein of the synaptic ribbon, ribeye, exhibited strong STX3 immunolabeling Fig.\u00a0a\u2013d, consStx3 was achieved with a mouse line that expresses an optimized iCre under the control of the rhodopsin promoter , as well as homozygous Stx3 floxed control mice and heterozygous Cre expressing control mice .To probe for an essential role of Stx3 in vision, we generated a cell-specific Stx3 mouse knockout line, as complete inactivation of the Stx3 gene in mice produces an embryonic lethal phenotype . Double-labeling for Stx3 and ribeye/CtBP2 and for Stx3 and cone opsin revealed that the remaining Stx3 signal was in cone photoreceptors and in the number of neuronal somata in the ONL was observed Fig.\u00a0a, c indiOPL Fig.\u00a0a. In somOPL Fig.\u00a0a, arrowsAt 8 and 12\u00a0weeks of age, an increasing cell loss and an increase in the ectopic expression of rhodopsin were observed. Cone photoreceptor loss and the ectopic expression of opsin in cones was also observed Fig.\u00a0b, c. TheSTX3 loss-of-function variants that affect both STX3A and STX3B transcript isoforms display visual impairment consistent with an EOSRD. We show that STX3B rather than STX3A is highly expressed in the human retina, where the protein is found in the inner and outer segments of rod and cone photoreceptors, and in both plexiform layers, where the synaptic endings of photoreceptors and bipolar cells reside. Inactivation of Stx3 in murine rod photoreceptors resulted in their rapid degeneration as shown here and in a very recent study . All the other data supporting the findings of this study are available within the article and from the corresponding authors upon request.The sequence of the Supplementary file1 Supplementary Fig.\u00a01. Simplified family trees, segregation and Sanger chromatograms relating to novel STX3 variants (TIFF 3415 KB)Below is the link to the electronic supplementary material."} +{"text": "Amputoearinus is given. Gnathopleurajosequesadai Sharkey, sp. nov. is reported as a hyperparasitoid of fly larvae, the first such record for the genus. The following new species are diagnosed primarily using COI barcode data; Sharkey is the authority for all: Agathidinae: Aerophilusdavidwagneri, Aerophilusfundacionbandorum, Aerophilusnicklaphami, Lytopylusdavidstopaki, Lytopylusdavidschindeli; Alysiinae: Gnathopleurajosequesadai; Braconinae: Braconandreamezae, Braconfranklinpaniaguai, Braconrafagutierrezi, Braconguillermoblancoi, Braconoscarmasisi, Braconpauldimaurai, Braconshebadimaurae, Saciremakarendimaurae; Cheloninae: Chelonusminorzunigai; Homolobinae: Homolobusstevestroudi; Macrocentrinae: Macrocentrusmichaelstroudi; Orgilinae: Stantoniagilbertfuentesi; Rhysipolinae: Rhysipolisstevearonsoni; Rogadinae: Aleiodeskaydodgeae, Aleiodeskerrydresslerae, Aleiodesjosesolanoi, Aleiodesjuniorporrasi, Aleiodesrocioecheverri, Aleiodesronaldzunigai, Choreborogasjesseausubeli, Triraphisdoncombi, and Yeliconesmayrabonillae.Twenty-nine species are treated, most of which have host caterpillar and food plant records, and all but one are new to science. The first host record for the agathidine genus Agathidinae, Braconinae, Cheloninae, Macrocentrinae, Orgilinae, Proteropinae, Rhysipolinae, and Rogadinae. Of these 28 species, 23 are reared from host caterpillars and their food plants are documented. This is an addendum to COI DNA barcode sequences. The purpose of this research is to diagnose and name 28 new species of Costa Rican braconids. We deal with braconid subfamilies: Alabagrus (Braconidae) and the Mesochorus (Ichneumonidae). We emphasize that in these two publications the keys and descriptions required enormous time, effort, and expense; they are little used; and when they are used, the results are usually erroneous. This makes them useless or even detrimental to the taxonomy and understanding of these groups.We briefly describe the fate of large monographs that treat small portions of hyper-diverse Neotropical ichneumonoids by exemplifying the Alabagrus revision has 32 citations and the Mesochorus revision has 39. The majority of the citations are geographical surveys that simply copy the distributional records that are in these papers. For example, Ichneumonidae occurring in Peru and included a number of species cited as being present in Peru by According to a search in Google Scholar (May 2021), the Alabagrus employed the key of Alabagrus albispina, A. imitatus, A. juchuy, A. kagaba, A. latisoma, A. latreillei, A. maya, A. mojos, A. nahuatl, A. nigrilitus, A. pachamama, A. paruyana, A. parvifaciatus, A. semialbus , A. tricarainatus, A. tripartitus, and A.warrau. Furthermore, five of the species reported by Alabagrus cocto, A. englishi, A.pecki, A.roibasi, and A. yaruro .Only four publications dealing with Neotropical . yaruro . There ian fauna . In thisAlabagrus, only one person other than Sharkey has used the key to arrive at a determination for Neotropical species, and in that instance most of the identifications are probably incorrect. It took Sharkey more seven years to produce the 1988 revision, and it is worse than useless because it is full of misleading information on species limits and species distributions, owing to misidentifications. Some might argue for an integrative approach, such as the revision of Alabagrus by COI barcode is the only reliable source for identification (barring much more expensive and complex multi-gene information)?In summary, in the 30-plus years since the publication of the morphology-based revision of Mesochorus , we estimate that there are approximately 688 species in Costa Rica. These species are almost exclusively from the Area de Conservaci\u00f3n Guanacaste, from rearings that have been conducted exclusively in the provinces of Guanacaste and Alajuela . To helptierrezi .Macrocentrusgeoffbarnardi . The specimens on C1 cannot be separated from those on C2 on morphological grounds. However, the entity (C1 + C2) can be separated on morphological grounds from all of the other specimens in BIN BOLD:ACK7466. Finally, no other specimens in the BIN are parasitoids of species of Neurophyseta. Therefore, we considered the entire cluster (C1 + C2) as one species. If further examination or data suggest that it is two, then one more will be also described. Similar arguments were used to delimit the other nine species in the BIN . The NJ tree produced by BOLD indicates slightly different relationships but, as with the Bayesian tree, the three morphologically indistinguishable species are not sister species nor are they nearest neighbors by any definition of that concept. We may have made different decisions if these lineages shared hosts or formed a monophyletic clade and were represented by very few sequences.Figure As with those of COI barcodes and submitting them to BOLD. Instructions on how to do this are included below.Identification of specimens to the subfamily level can be achieved using the key by Antaeotricha Janzen233 is identified to the genus Antaeotricha by classical morphology-based criteria and to Janzen233 by barcode and ecological information. However, no formal scientific species name is available until a barcode-match is obtained with an existing holotype or until it is described as new, or interim matched morphologically with a described species by a taxonomic specialist, which may take decades. Equally, Antaeotricharadicalis EPR03 is also an interim name based on what the species looks like, however, it is not a scientific name. It temporarily retains the information that this species is recognized by similarity with its look-alike, A.radicalis, before barcoding and associating it with other ecological data. Finally, a name such as gelJanzen01 Janzen407 signifies a caterpillar in the family Gelechiidae for which even a generic name is not obtainable at present.Some host species treated here are awaiting full identification and are given interim names. For example, c oxidase subunit I (COI) gene using standard insect primers LepF1 (5'-ATTCAACCAATCATAAAGATATTGG-3') and LepR1 (5'-TAAACTTCTGGATGTCCAAAAAATCA-3') (Molecular work was carried out at the CBG using their standard protocols. A leg of each frozen-then-oven-dried specimen was destructively sampled for DNA extraction using a glass fiber protocol . ExtractATCA-3') . If initBarcode sequences presented in the species descriptions herein are a consensus of the barcode sequences of all included individuals, meaning base pairs that differ between conspecific specimens are replaced by IUPAC ambiguity codes.http://janzen.sas.upenn.edu/caterpillars/database.lasso). Specimen voucher codes beginning with BIOUG are from the BOLD database (http://www.boldsystems.org), and most of the specimens obtained from ACG Malaise traps have this prefix. The DHJPAR and their associated SRNP codes can also be found on the BOLD database. The abundant collateral information obtainable from these two databases complements the species treatments. See Voucher codes are presented for all holotype specimens treated herein. All host caterpillars are individually vouchered to their individual records (yy-SRNP-xxxxx). Codes beginning with DHJPARxxxxxxx are for the parasite (or hyperparasite) specimens reared from the caterpillar; therefore, each wasp carries two voucher codes, one for the rearing (host) record and one for the wasp itself. The SRNP voucher codes are from the Janzen and Hallwachs\u2019 database (http://www.boldsystems.org/index.php/IDS_OpenIdEngine). 2. Paste the COI sequence of the query organism (in forward orientation) into the query box and search against the appropriate library . 3. The search results page shows the top hits based on % similarity starting with the closest matches. This page also provides additional information to help verify the identity of a match, such as links to the BIN where specimen data (including images) can be found, a distribution map, and a tree-based identification tool. 4. Use the Tree-Based Identification button to generate a neighbor-joining tree and find the query taxon (name in red). This allows you to visualize how distant the query sequence is from the closest matches.The BOLD database can be used to identify specimens using the following steps: 1. Navigate to the identification tab of the BOLD Systems database feeding on Desmopsisschippii (Annonaceae). Host caterpillar voucher code13-SRNP-972Hosts are all the same as that of the holotype: DHJPAR0054734, DHJPAR0055235, DHJPAR0051139, DHJPAR0051915, DHJPAR0055516, DHJPAR0054741, DHJPAR0055237, DHJPAR0054728, DHJPAR0055233.Aerophilusdavidwagneri is named in honor of David Wagner of the University of Connecticut, Storrs, Connecticut, USA, for his recent work as an environmental activist for a healthier global climate and wild biodiversity.Taxon classificationAnimaliaHymenopteraBraconidae?Sharkeysp. nov.EBD717AC-52CC-5638-BAA9-AA8747A70927http://zoobank.org/A73266E4-1332-4185-816A-4AD9EEDACEC4Figure BOLD:ACN0950; nearest neighbor: AerophiluscalcaratusBOLD:AAU4711; distance to nearest neighbor is 5.81%. Consensus barcode AATTTTATATTTTATTTTTGGAATTTGATCTGGTATTTTAGGATTATCAATAAGAATCATTATTCGTATAGAATTAAGATTAGGGGGTAATTTAATTGGTAATGATCAAATTTATAATAGAATTGTTCTGCTCATGCTTTTGTAATAATTTTTTTTATAGTTATACCGATTATAATTGGAGGATTTGGAAATTGATTAGTTCCTTTAATGTTAGGAGGTCCAGATATGGCCTTTCCACGRATAAATAATATAAGATTTTGATTATTAATTCCTTCATTAACTTTATTAATTTTAAGATCAATATTAAATGTTGGTGTAGGTACGGGATGAACTGTYTATCCTCCCTTATCATTAAATATAAGTCATAGAGGAATATCTGTAGATTTAGCAATTTTTTCTTTACATAYTGCTGGAATTTCTTCTATTATAGGAGCAATAAATTTTATTACTACAATTATTAATATATGRATAATAAATGTAAAAATTGATAAAATACCTTTATTGGTATGATCTATTTTTATTCTGCTATTTTATTATTATTATCTTTACCAGTATTAGCTGGGGCTATTCTATATTATTAACTGATCGAAATTTAAATCTAGATTTTTTGATCCTTCTGGAGGAGGAGATCCAATTTTATATCAACACTTATTTBIN: Aerophilusfundacionbandorum keys to A.macadamiae in Aerophilusfundacionbandorum differs in many ways. One of the most obvious is its wide, sharply angled, median propodeal areola feeding on Schnellaguianensis (Fabaceae), caterpillar voucher code13-SRNP-71694.Same host species as that of holotype DHJPAR0054547.Aerophilusfundacionbandorum is named in honor of the BAND Foundation of the USA, in recognition of its decades of support for growth and development of Costa Rica\u2019s \u00c1rea de Conservaci\u00f3n Guanacaste and most recently for adding 85 more hectares to ACG of original forest, Bosque Transici\u00f3n, lying on the nearly extinct fusion of dry forest with rain forest (http://www.gdfcf.org/content/introducing-bosque-transici\u00f3n).Taxon classificationAnimaliaHymenopteraBraconidae?Sharkeysp. nov.7B32CE61-1022-5440-8680-A2BCE450C6AEhttp://zoobank.org/5C52FDA0-10E1-4152-9EE5-4D7BFE64516EFigure BOLD:ACT7814; nearest neighbor: AerophiluscolleenhitchcockaeBOLD:ACA4890; distance to nearest neighbor is 5.16%. Consensus barcode: AATTTTATATTTTATTTTTGGAATTTGATCTGGAATTTTAGGATTATCAATAAGAATAATTATTCGTATAGAATTAAGATTAAGGGGCAATTTAATTGGAAATGATCAAATTTATAATAGAGTTGTT-CTGCTCATGCTTTTGTTATAATTTTTTTTATAGTTATACCAATTATGATTGGGGGTTTTGGTAATTGATTAATTCCTTTAATATTAGGAGGTCCAGATATAGCATTTCCTCGTATAAATAATATAAGATTTTGATTATTAATTCCTTCTTTATTTTTATTAATTTTAAGTTCAATATTAAATATTGGAGTAGGTACAGGATGAACTGTTTATCCTCCTTTATCATTAAATATAAGACACAGAGGAATATCTGTAGATTTAGCAATTTTTTCTTTACATATTGCTGGAATTTCTTCTATTATAGGGGCAATAAATTTTATTACTACAATTATTAATATATGAATAATAAACGTAAAAATTGATAAAATACCTTTATTAGTATGATCCATTTTTATT-CTGCTATTTTATTATTATTATCTTTACCAGTATTGGCTGGAGCTATT-CTATATTATTAACAGATCGAAATTTAAAT-CTAGATTCTTTGATCCTTCAGGGGGAGGAGATCCTATTTTATATCAACATTTATTTBIN: A.jessicadimauroae in A.nicklaphami differs in many ways. Two of the most obvious are the wide sharply angled median propodeal areola and the sharp lateral longitudinal carinae on the first metasomal median tergite. In A.jessicadimauroae the areola is gradually narrowed anteriorly and the carinae are not sharp.This species keys to Aerophiluscolleenhitchcockae, by having the hind coxa and femur entirely brown feeding on Ficuscitrifolia (Moraceae), caterpillar voucher code: 14-SRNP-41346.Aerophilusnicklaphami is named in honor of Nick Lapham of the BAND Foundation of the USA, in recognition of his decades of support for growth and development of Costa Rica\u2019s \u00c1rea de Conservaci\u00f3n Guanacaste, Costa Rica, and most recently adding 85 more hectares to ACG of original forest, Bosque Transici\u00f3n, lying on the nearly extinct fusion of dry forest with rain forest (http://www.gdfcf.org/content/introducing-bosque-transici\u00f3n).Taxon classificationAnimaliaHymenopteraBraconidae?Lindsay & Sharkey, 2006D52030DC-E128-5B95-8D89-CA6C069F82EDFigure There is no BIN for this specimen because the barcode is too short to merit a BIN code. The short barcode follows:ATATTTATTTAATTTTTGGAATTTGATCAGG-ATTTTAGGATTATCAATAAGAATAATTATTCGTATAGAATTAAGAATGGGGGGAAATTTTATTGGTAATGATCAAATTTATAATAGAATTGTT-CTGCTCATGCATTTATTATAATTTTTTTTAAAGTTATACCAATTATAATTGGAGGATTTGGAAATTGATTAATTCCTTTAATATTAGGGGGCCCAGAAAAAGCTTTCCCCCGAATAAATAATATAAAATTTTGATThis specimen was identified based on morphological criteria from the key in Reared specimen: ?: Costa Rica: Guanacaste, Area de Conservaci\u00f3n Guanacaste, Sector Del Oro, Puente Mena, 280 m, 11.04562 -85.45742; host caterpillar collection date: 11/07/2007, parasitoid eclosion: 27/07/2007; depository CNC, voucher code: DHJPAR0028287.Reared specimens host data: Dysodiaspissicornis (Thyrididae) a leaf-roller feeding on Heisteriaconcinna (Olacaceae), caterpillar voucher code: 07-SRNP-22487.This is the first host record for this wasp genus.Taxon classificationAnimaliaHymenopteraBraconidae?Sharkeysp. nov.9AA5EF7E-2B85-5723-B06A-C334F012820Bhttp://zoobank.org/B88988CF-D2D5-4B57-ACC5-B3D5A8E7A2DFFigure BOLD:ACJ2185; nearest neighbor: LytopylusdavidschindeliBOLD:ACB1289; distance to nearest neighbor is 2.56%. Consensus barcode: AATTTTATATTTTATATTTGGTATTTGATCAGGAATTTTAGGTTTATCATTAAGATTAATTATTCGAATAGAATTAAGAATTGGTGGAAATTTAATTGGAAATGATCAAATTTATAATAGAATTGTTACTGCTCATGCTTTTATTATAATTTTTTTTATAGTTATACCAATTATAATTGGAGGATTTGGTAATTGATTAATTCCTTTATTATTAGGAGGTCCTGATATAGCTTTCCCTCGAATAAATAATATAAGATTTTGATTATTAATTCCTTCATTATTATTATTAATTTTAAGATCTTTAATTAATATTGGTGTAGGTACAGGATGAACAGTTTATCCTCCATTATCTTTAAATATAAGTCATAGTGGTATATCTGTAGATATAGCAATTTTTTCTTTACATATTGCTGGAATTTCTTCAATTATAGGAGCTATAAATTTTATTACTACTATTATAAATATATGAATTTTAAATTTAAAATTTGATAAAATACCTTTATTAGTTTGATCAATTTTAATTACAGCAATTTTATTATTATTATCATTACCAGTTTTAGCTGGAGCTATTACTATATTATTAACTGATCGAAATTTAAATACAAGATTTTTTGATCCTTCAGGAGGAGGAGATCCAATTTTATATCAACATTTATTTBIN: L.youngcheae in L.youngcheae and entirely orange in L.davidstopaki. This species can be morphologically distinguished from its nearest neighbor, Lytopylusdavidschindeli, by having its mesosoma and coxae entirely tan feeding on Calatolacostaricensis (Metteniusaceae), caterpillar voucher code:13-SRNP-30082.elachJanzen01 Janzen527 , in recognition of his decades of editorial understanding and accepting the strange research emerging from the biodiversity inventory of Costa Rica\u2019s \u00c1rea de Conservaci\u00f3n Guanacaste.Taxon classificationAnimaliaHymenopteraBraconidae?Sharkeysp. nov.8C468F73-0A3F-557C-849D-396D447833BFhttp://zoobank.org/5006423F-2393-4963-9EE1-8423DC2CE954Figure BOLD:ACB1289; nearest neighbor: LytopylusdavidstopakiBOLD:ACJ2185; distance to nearest neighbor is 2.56%. Consensus barcode AATTTTATATTTTATATTTGGAATTTGATCAGGAATTTTAGGATTATCATTAAGATTAATTATTCGAATAGAATTAAGAATTGGAGGAAATTTAATTGGTAATGATCAAATTTATAACAGAATTGTAACTGCTCATGCTTTTATTATAATTTTTTTTATAGTTATACCAATTATAATTGGAGGATTTGGAAATTGATTAATTCCTTTAATATTAGGAGGTCCTGATATAGCTTTTCCTCGAATAAATAATATAAGATTTTGATTATTAATTCCTTCATTATTATTATTAATTTTAAGGTCTTTAATTAATATTGGAGTAGGAACAGGATGAACAGTTTATCCTCCTTTATCTTTAAATATAAGTCATAGTGGTATATCTGTAGATATGGCAATTTTTTCTTTACATATTGCTGGAATTTCTTCAATTATAGGAGCTATAAATTTTATTACTACTATTATAAATATATGAATTTTAAATTTAAAATTTGATAAAATACCTTTATTAATTTGATCAATTTTAATTACAGCAATTTTATTATTATTATCATTACCAGTTTTAGCTGGTGCTATTACTATATTATTAACTGATCGAAATTTAAATACAAGATTTTTTGATCCATCAGGAGGAGGAGATCCAATTTTATATCAACATTTATTTBIN: L.angelagonzalezae in Lytopylusdavidschindeli is almost completely smooth with the central areola barely indicated. This species can be morphologically distinguished from its nearest neighbor, Lytopylusdavidstopaki, by having the mesosoma and coxae entirely black or dark brown feeding on Prunusannularis (Rosaceae), caterpillar voucher code:12-SRNP-35088.elachJanzen01 Janzen185 which is a primary parasitoid of Pachyliaficus (Sphingidae) feeding on Macluratinctoria (Moraceae). Including the holotype, five specimens were reared from the fly puparia parasitizing the caterpillar, voucher code 08-SRNP-13289.The host flies were identified from their surviving sibs, one of which is DHJPAR0027924 of BIN BOLD:ACE9310.Hyperparasitoid of the fly Reared from the same caterpillar as the holotype DHJPAR0028038, DHJPAR0028039, DHJPAR0028040, DHJPAR0028041.Gnathopleurajosequesadai is named in honor of Jos\u00e9 Ram\u00f3n Quesada Mora, the manager of the 2020\u201321 BioAlfa Malaise traps for the Hacienda Baru Wildlife Refuge, Costa Rica.Gnathopleura confirmed to be a hyperparasitoid.This is the first species of Coleoptera and Lepidoptera, but use many other insect orders as well. A key to the Braconinae genera of the New World is in Braconines are mostly primarily idiobiont parasitoids of Taxon classificationAnimaliaHymenopteraBraconidae?Sharkeysp. nov.9281613C-E428-552C-94D0-B39D9FD09F6Chttp://zoobank.org/C8FDFE7C-5289-4EF6-A9F0-BC1C493AEBBDFigures BOLD:AAJ8891; nearest neighbor: BraconfranklinpaniaguaiBOLD:ACK6897; distance to nearest neighbor is 5.64%. Consensus barcode: TATATTATATTTTATACTTGGTATTTGATCTGGTATAATTGGTTTATCAATAAGTTTAATTATTCGGTTAGAATTAAGAATACCAGGAAGTTTATTAAGTAATGATCAAATTTATAATAGAATAGTTACAGCACATGCTTTTGTAATAATTTTTTTTATAGTTATACCAGTGATATTAGGAGGGTTTGGTAATTGATTAATCCCTTTAATATTGGGATCTCCTGATATAGCTTTTCCTCGAATAAATAATATAAGATTTTGATTATTAATTCCTTCATTAATTTTATTATTATTAAGAAGAATATTAAATGTAGGAGTGGGTACTGGTTGAACTATTTATCCTCCATTATCTTCTAACTTAGGGCATAGAGGTGTATCTGTTGATTTAGCTATTTTTTCTTTACATTTAGCTGGTATTTCATCTATTATAGGTTCAATTAATTTTATTTCTACAATTTTAAATATACATTTATTAATATTAAAATTAGATCAATTAACTTTATTTATTTGATCAATTTTTATTACAACTATTTTATTATTATTATCTTTACCTGTTTTAGCAGGAGCTATTACTATATTATTAACTGATCGAAATTTAAATACTTCATTTTTTGATTTTTCTGGAGGAGGGGATCCAATTTTATTTCAACATTTATTTBIN: B.franklinpaniaguai feeding on Chrysochlamysglauca (Clusiaceae). The species is a gregarious parasitoid; the holotype is one of 56 specimens that emerged from the host, caterpillar voucher code: 07-SRNP-30348.07-SRNP-30348 Ten specimens, reared from the same caterpillar as the holotype, were mounted and designated as paratypes (DHJPAR0066400 to DHJPAR0066409), depository CNC.Braconandreamezae is named in honor of Ministra Andrea Meza Murillo of the Ministerio de Recursos Naturales y Energ\u00eda de Costa Rica (MINAE) in recognition of her taking on this difficult ministerial task mid-government.Taxon classificationAnimaliaHymenopteraBraconidae?Sharkeysp. nov.3CC0B67E-7C11-5917-9AE6-2D3E0B4B56D3http://zoobank.org/45DC951E-E7E0-4C69-9518-E2628FAA99DEFigure BOLD:ACK6897; nearest neighbor: BraconalejandromasisiBOLD:AAA5367; distance to nearest neighbor is 4.49%. Consensus barcode: ATATTATATTTTTTATTTGGAATTTGAGCTGGAATAATTGGTTTATCAATAAGATTAATTATTCGTTTAGAATTAGGTATACCAGGTAGTTTATTAGGTAATGATCAAATTTATAATAGTATAGTTACAGCTCATGCTTTTGTAATAATTTTTTTTATAGTTATACCAGTAATATTAGGAGGATTTGGAAATTGATTAATTCCTTTAATATTAGGAGCTCCTGATATAGCTTTTCCTCGAATAAATAATATAAGATTTTGGTTATTAATTCCTTCATTAATTTTATTATTATTAAGAAGAATATTAAATGTTGGTGTAGGGACAGGTTGAACTATTTATCCTCCTTTATCATCTAGGTTAGGTCATGGAGGTATATCTGTTGATTTAATTATTTTTTCTTTACATTTAGCTGGTATTTCATCTATTATAGGATCTATTAATTTTATTACTACAATTTTAAATATGCATTTATTAATATTAAAGTTAGATCAATTAACTTTATTTATTTGATCAATTTTTATTACAACTATTTTATTATTATTATCTTTACCTGTATTAGCAGGAGCTATTACTATATTATTAACTGATCGAAATTTAAATACTTCATTTTTTGATTTTTCTGGAGGTGGGGATCCAATTTTATTTCAACATTTATTTBIN: B.alejandromasisi feeding on Crotonbillbergianus (Euphorbiaceae), caterpillar voucher code: 04-SRNP-56695.Eight specimens reared from the same host specimen as the holotype were mounted and designated as paratypes (DHJPAR0066410 to DHJPAR0066417), depository CNC.Braconfranklinpaniaguai is named in honor of Vice-Minister Franklin Paniagua Alfaro of the Ministerio de Recursos Naturales y Energ\u00eda de Costa Rica (MINAE) in recognition of his taking on this difficult task mid-government.Taxon classificationAnimaliaHymenopteraBraconidae?Sharkeysp. nov.4A5F25E2-2180-5096-8BC8-2B5996EE17B3http://zoobank.org/0093066D-6B8F-4674-9D40-9538CD2ECE5CFigure BOLD:ACB1290; nearest neighbor: Bracon sp.BOLD:AAV0639; distance to nearest neighbor is 2.24%. Consensus barcode: GATTTTATATTTTTTATTTGGTATATGAGCAGGTATAGTTGGTTTATCAATAAGGTTAATTATTCGATTAGAATTAGGTATACCTGGGAGTTTACTAGGTAATGATCAAATTTATAATAGTATAGTGACTGCTCATGCTTTTATTATAATTTTTTTTATAGTTATACCTGTAATATTAGGAGGATTTGGTAATTGATTAATTCCTTTAATATTAGGAGCTCCAGATATAGCTTTTCCTCGTTTAAATAATATAAGATTTTGGTTATTATTTCCTTCTTTAATTTTATTATTATTAAGAAGAATTTTAAATGTTGGTGTAGGTACTGGGTGAACAATATACCCACCTTTGTCATCTAGATTAGGTCATAGAGGGTTATCTGTTGATTTAGCTATTTTTTCTTTACATTTAGCTGGGGTTTCTTCTATTTTAGGTTCAGTTAATTTTATTACAACAATTTTAAATATACATTTATTAATATTAAAATTAGATCAATTAACTTTATTAATTTGATCAATTTTTATTACAACTATTTTATTATTATTATCTTTACCTGTTTTAGCTGGTGCTATTACTATATTATTAACAGATCGAAATTTAAATACTTCTTTTTTTGATTTTTCTGGTGGAGGGGATCCTATTTTATTTCAACATTTATTTBIN: This species can be morphologically distinguished from its nearest neighbor by having all femora dark brown and the metasoma dark brown dorsally starting at the third tergum Fig. comparedHolotype ?: Costa Rica: Alajuela, Area de Conservaci\u00f3n Guanacaste, Sector Rincon Rain Forest, Palomo, 96 m, 10.962 -85.28; host caterpillar collection date: 05/iii/2012, parasitoid eclosion: 18/iii/2012; depository CNC, holotype voucher code: DHJPAR0049049.Cosmorrhynchaalbistrigulana (Tortricidae) feeding on Dialiumguianense (Fabaceae). This is one of the only two species of Bracon reared by us that is solitary; the ten species treated by Braconrafagutierrezi is named in honor of SINAC Director Rafa Guti\u00e9rrez Rojas of the Ministerio de Recursos Naturales y Energ\u00eda de Costa Rica (MINAE) in recognition of his taking on this difficult task mid-government.Taxon classificationAnimaliaHymenopteraBraconidae?Sharkeysp. nov.CE2B1B46-C058-572B-A15B-75CF854B1A96http://zoobank.org/2E651361-5D12-419B-8A55-A2504505C00EFigure BOLD:AAT8852; nearest neighbor: Bracon sp. BOLD:AED7502; distance to nearest neighbor is 8.17%. Consensus barcode: TATTTTATATTTTTTATTTGGTATATGATCAGGAATAATTGGTTTATCAATAAGTTTAATTATTCGATTAGAATTAGGGATACCTGGAAGATTATTAGGTAATGATCAAATTTATAATAGAATAGTTACAGCTCATGCTTTTATTATAATTTTTTTTATAGTTATACCAATTATATTAGGAGGATTTGGGAATTGATTAATTCCTTTAATATTAGGAGCTCCTGATATAGCTTTTCCTCGTTTAAATAATATAAGATTTTGATTATTAATTCCTTCATTAACTTTATTATTATTAAGAAGAATTTTAAATGTAGGTGTAGGAACTGGGTGAACAATGTATCCTCCATTATCATCTAATTTAGGTCATAGAGGTTTATCTGTAGATTTAGCTATTTTTTCTTTACATTTAGCTGGAATTTCTTCTATTATAGGTTCAATAAATTTTATTACTACTATTTTAAATATACATTTAAAAATATTAAAACTTGATCAATTAACGTTATTAATTTGATCTATTTTTATTACTACAATTTTGTTATTATTATCTTTACCTGTTTTAGCTGGGGCAATTACTATACTATTAACTGATCGAAATTTAAATACTTCATTTTTTGATTTTTCTGGGGGAGGAGACCCAATTTTATTCCAACATTTATTT.BIN: There is only one low-quality image on BOLD for the nearest neighbor, but the p-distance makes it doubtful that it is conspecific.Holotype ?: Costa Rica: Guanacaste, Area de Conservaci\u00f3n Guanacaste, Sector Mundo Nuevo, Mamones, 365 m, 10.771 -85.429; host caterpillar collection date: 01/viii/2010, parasitoid eclosion: 12/viii/2010; depository CNC, holotype voucher code: DHJPAR0040470.Dysodiasica (Thyrididae) feeding on Pipermarginatum (Piperaceae). This is one of the only two species of Bracon reared by us that is solitary; the ten species treated by Braconguillermoblancoi is named in honor of Guillermo Blanco, the BioAlfa Malaise traps manager for Parque Nacional Isla del Coco, ACMIC (\u00c1rea de Conservaci\u00f3n Marino Isla del Coco), Costa Rica.Taxon classificationAnimaliaHymenopteraBraconidae?Sharkeysp. nov.CDA27D08-BB75-5E8B-9802-9C1D254958FEhttp://zoobank.org/48249926-80DE-4AB4-98E2-D204D3A3CFB7Figures BOLD:AAY4686; nearest neighbor: Bracon sp. BOLD:AEF4783; distance to nearest neighbor is 6.09%. Consensus barcode: AGTTTTGTATTTTTTATTTGGTATATGAGCTGGTATAGTTGGTTTATCAATAAGTTTAATTATTCGTTTAGAGTTAGGTATACCTGGAAGTTTATTAGGTAATGATCAAATTTATAATAGAATAGTTACAGCTCATGCTTTTGTTATAATTTTTTTTATAGTTATACCTGTTATAATTGGAGGATTTGGTAATTGATTAATTCCTTTAATATTAGGAGCTCCTGATATAGCTTTTCCTCGAATAAATAATATGAGATTTTGGTTATTAGTTCCTTCATTAACTTTATTATTATTAAGTAGAATTTTAAATGTAGGGGTAGGTACAGGTTGGACAATATATCCACCTTTATCTTCAAGTTTAGGTCATAGAGGGTTATCTGTTGATTTAGCTATTTTTTCTTTACATTTAGCTGGTGTTTCTTCAATTATAGGGGCAATAAATTTTATTACTACTATTTTAAATATGCATTTATTAATATTAAAATTAGATCAGTTAACTTTATTAGTTTGATCAATTTTTATTACTACTATTTTATTATTATTATCTTTACCTGTTTTAGCAGGAGCAATTACAATATTATTAACTGATCGAAATTTAAATACTTCTTTTTTTGATTTTTCAGGAGGTGGAGATCCTATTTTATTTCAACATTTATTT.BIN: This species can be morphologically distinguished from its nearest neighbor by having the hind femur dark brown Fig. comparedHolotype ?: Costa Rica: Guanacaste, Area de Conservaci\u00f3n Guanacaste, Sector Mundo Nuevo, Punta Plancha, 420 m, 10.742 -85.427; host caterpillar collection date: 17/x/2010, parasitoid eclosion: 28/x/2010; depository CNC, holotype voucher code: DHJPAR0041854.Anadasmus Janzen25 (Depressariidae) feeding on Mespilodaphneveraguensis (Lauraceae); four specimens emerged from the host, caterpillar voucher code: 10-SRNP-56886.Gregarious parasitoid of Two males, same data as holotype depository CNC.Braconoscarmasisi is named in honor of Oscar Masis, the BioAlfa Malaise traps manager for Parque Nacional Los Quetzales, ACOPAC , Costa Rica.Taxon classificationAnimaliaHymenopteraBraconidae?Sharkeysp. nov.6C04CB63-B7C7-59F2-AAC1-E1415E3F7692http://zoobank.org/723581AA-45CD-4F42-9AB1-CED38EBEA383Figure BOLD:AEF4305; nearest neighbor: Bracon sp. BOLD:ACG3693; distance to nearest neighbor is 9.62%. Consensus barcode: TGTTTTATATTTTTTATTTGGTATATGAGCTGGGATACTAGGTCTATCAATAAGATTAATTATCCGACTAGAGCTCGGAATACCGGGAAGTTTACTTGGTAATGACCAAATTTACAATAGAATAGTAACAGCTCATGCTTTTGTAATAATTTTTTTTATAGTTATACCTGTAATAGTAGGAGGATTTGGAAATTGACTATTACCTTTAATATTAGGAGCCCCTGATATAGCATTTCCTCGTTTAAATAATATAAGATTTTGATTACTTATTCCTTCCCTAACTTTATTATTAATAAGAAGAATTTTAAATGTAGGAGTAGGGACTGGATGAACAGTTTATCCTCCTTTATCCTCTTCACTAGGTCATAGAGGGTTATCAGTTGATTTGGCTATTTTTTCTTTACATATTGCAGGAATTTCCTCAATTTTGGGGGCTATTAACTTTATTTCAACTATTTTAAATATACATTTATTAATTTTAAAACTAGATCAACTAACATTATTAATTTGATCAATTTTTATTACAGCTATTTTATTATTATTATCTTTACCAGTATTAGCAGGAGCTATCACAATATTATTAACCGATCGAAATTTAAATACATCTTTTTTTGATTTTTCAGGAGGGGGTGACCCAATTTTATTTCAACATTTATTT.BIN: This species can be morphologically distinguished from its nearest neighbor by having large portions of the mesoscutum and mesepimeron brown Fig. comparedHolotype ?: Costa Rica: Guanacaste, Area de Conservaci\u00f3n Guanacaste, Sector Cacao, Sendero Cima, 1460 m, 10.933 -85.457, 23/iii/2009, Malaise trap, depository CNC, holotype voucher code: DHJPAR0051516.Braconpauldimaurai is named in honor of Paul Dimaura of Boston, Massachusetts for his decades of support of the University of Pennsylvania in general and D. H. Janzen\u2019s position as a professor of conservation biology specifically.Taxon classificationAnimaliaHymenopteraBraconidae?Sharkeysp. nov.D39CF64B-0CA1-5A5C-ABC6-CE4C0016B05Chttp://zoobank.org/E87C71B3-2C89-4EB5-AFE9-47FD75A1C533Figure BOLD:ADW8464; nearest neighbor: Bracon sp. BOLD:AEB6448; distance to nearest neighbor is 4.81%. Consensus barcode: TTTATATTTTTTATTTGGTATATGAGCAGGAATATTAGGATTATCACTAAGTTTAATTATTCGTTTAGAATTAGGGATACCTGGAAGATTATTAGGTAATGATCAAATTTATAATAGTATAGTTACAGCTCATGCATTTGTTATAATTTTTTTTATAGTTATACCAGTAATATTAGGAGGATTTGGTAATTGATTATTACCTTTAATATTAGGGGCTCCTGATATAGCTTTCCCACGAATAAATAATATAAGATTTTGGTTAATTATCCCTTCTTTAATTTTATTATTAATAAGAAGAATTTTAAATGTAGGAGTAGGAACAGGTTGAACAGTTTATCCTCCTTTATCATCTTCATTAGGGCATAGTGGGTTATCTGTTGATTTAGCTATTTTTTCTTTACATATTGCAGGAATTTCTTCAATTATAGGTTCAATTAATTTTATTACTACTATTTTTAATATACATTTATTTAAATTAAAATTAGATCAATTAACATTATTAATTTGATCTATTTTTATCACAACTATTTTACTTTTATTATCTTTACCAGTTTTAGCGGGGGGAATTACTATATTATTAACAGATCGTAATTTAAATACATCATTTTTTGATTTTTCTGGAGGGGGAGATCCTGTTTTATTTCAACATTTATT.BIN: No images of the unnamed nearest neighbor are available.Holotype ?: Costa Rica: Guanacaste, Area de Conservaci\u00f3n Guanacaste, Sector Pailas Dos, PL12-1, 828 m, 10.7642 -85.335, 14/v/2015, Malaise trap, depository CNC, holotype voucher code: BIOUG44786-F08, GenBank accession MW627534.Braconshebadimaurae is named in honor of Sheba Dimaura of Boston, Massachusetts for her decades of support of the University of Pennsylvania in general and D. H. Janzen\u2019s position as a Professor of Conservation Biology.Taxon classificationAnimaliaHymenopteraBraconidae?Sharkeysp. nov.1A4A7A9B-09BA-58BA-87AA-4A05BC58362Fhttp://zoobank.org/5BE4D1E3-9F52-444D-8F49-AC0130EEE9FFFigure BOLD:ADY0104; nearest neighbor: Sacirema sp. BOLD:AEH2057; distance to nearest neighbor is 2.24%. Consensus barcode: TTTATATTTTTTATTTGGGATATGATCTGGTATATTAGGTTTATCAATAAGTTTAATTATTCGATTAGAACTTGGAATACCATCAAGTTTATTAACAAATGATCAAATTTATAATAGAATAGTAACTGCCCATGCATTTGTCATAATTTTTTTTATAGTTATACCAATTATAATTGGTGGATTTGGAAATTGATTAATTCCTTTAATATTAAGAGCTCCAGATATAGCTTTCCCTCGTATAAATAATATAAGTTTTTGATTACTAATTCCTTCTTTAATAATATTAATTTTAAGAAGAATTATTAATACAGGTGTAGGTACTGGTTGAACAGTTTACCCTCCTTTATCTTCTTCTATAGGACATAGAGGAATTTCAGTTGATTTAGCAATTTTTTCTTTACATTTAGCTGGAGCTTCCTCAATTATAGGGTCTATTAATTTTATTTCAACTATTATTAATATACGACTTTATTTAATAAAAATAGATCAATTAACATTATTAATTTGATCTATTTTTATTACTACAATTTTATTATTATTATCATTACCAGTTCTAGCTGGGGCAATCACAATATTATTAACAGATCGAAATTTAAATACTACTTTTTTTGATTTTTCAGGAGGTGGGGATCCAATTTTATTCCAACACTTAT.BIN: Sacirema feeding on Hameliapatens (Rubiaceae), caterpillar voucher code:19-SRNP-35166.19-SRNP-35166 Same host data as holotype, DHJPAR0064500, DHJPAR0064502, depository CNC.Chelonusminorzunigai is named in honor of Minor Z\u00fa\u00f1iga Siles, the BioAlfa Malaise traps manager for Estaci\u00f3n Esquinas, Parque Nacional Tortuguero, ACTO (\u00c1rea de Conservaci\u00f3n Tortuguero), Costa Rica.Homolobinae are endoparasitoids of lepidopteran larvae. A key to the genera of the New World is included in Members of Taxon classificationAnimaliaHymenopteraBraconidae?Sharkeysp. nov.4D94064C-D823-5FDA-B2A0-49F2323CD7DFhttp://zoobank.org/D21DFA7D-C461-4F83-9521-79A1E9982771Figures BOLD:AAA7060. The nearest neighbor: Homolobus sp. BOLD:ACM2462 is separated by a p-distance of only 1.12%. Consensus barcode: TATTTTATATTTTATATTTGGAATTTGAGCTGGAATTTTAGGTATATCAATAAGAATTATTATTCGAATAGAATTAAGAATACCAGGTAATTTAATTGGTAACGATCAAATTTATAATAGTATTGTTACTGCTCATGCATTTATTATAATTTTTTTTATAGTTATACCAATTATAATTGGAGGGTTTGGAAATTGATTAATTCCTTTAATATTAGGATGTGTTGATATAGCTTTTCCTCGAATAAATAATATAAGATTTTGATTATTAATTCCATCATTAATTTTATTAATTTTAAGAAGAATTTTAAATGTTGGTGTTGGTACTGGATGAACTGTTTATCCTCCTTTATCTTTAAATATTGGTCATGGAGGTTTATCTGTTGATTTAGCTATTTTTTCTTTACATTTAGCTGGAATTTCTTCAATTATAGGAGCTATTAATTTTATTACTACTATTTTAAATATACGATCTAATTTAATTACAATAGATAAAATTTCTTTATTAAGTTGATCAATTTTAATTACTGTAATTTTATTATTATTATCTTTACCAGTTTTAGCTGGGGCTATTACAATATTATTAACTGATCGTAATTTAAATACATCTTTTTTTGATCCATCTGGTGGAGGGGATCCAATTTTATATCAACATTTATTT.BIN: Homolobusinfumator in H.stevestroudi and the size of the basal tooth of the hind tarsal claws, which are longer in H.stevestroudi. The convincing difference can be found by looking at the NJ tree produced from the BOLD website; H.stevestroudi is found in its own BIN, far removed from any other species of Homolobus and particularly distant from specimens identified as H.infumator from Norway. The type locality of H.infumator is England. All nine specimens in the unnamed nearest neighbor are from Canada. They might represent the same species as the Costa Rican specimens, but more sampling will need to be done between Canada and Costa Rica to confirm or refute. There are no obvious morphological differences based on the BOLD images of the Canadian specimens.The specimen keys to Holotype ?: Costa Rica: Guanacaste, Area de Conservaci\u00f3n Guanacaste, Sector Cacao, Sendero Toma Agua, 1140 m, 10.928 -85.467; host caterpillar collection date: 23/iv/2009, parasitoid eclosion: 18/v/2009; depository CNC, holotype voucher code: DHJPAR0035530, GenBank accession: MW627552.Pherotesiaminuisca (Geometridae) feeding on Zygiapalmana (Fabaceae), caterpillar voucher code: 09-SRNP-35488.Homolobusstevestroudi is named in honor of Steve Stroud as the primary supporter of the BioAlfa Malaise trapping at the Hacienda Bar\u00fa Wildlife Refuge, Savegre, ACOPAC, Costa Rica, as well as decades of support for the Area de Conservaci\u00f3n Guanacaste.Members of all genera are koinobiont endoparasitoids of caterpillars from a wide range of families. Most are solitary, but several gregarious species are known. A key to the genera of the New World is in Taxon classificationAnimaliaHymenopteraBraconidae?Sharkeysp. nov.C02F5F5C-22FE-545C-8AC9-DDF6868514D7http://zoobank.org/6E04139A-E5D6-4F29-BC63-DBD88D4EBADAFigure BOLD:ACK7466. Consensus barcode: TGTTTTATATTTTTTATTAGGTATTTGATGTGGATTAGTAGGTTTATCTTTAAGGTTACTTATTCGGTTAGAGTTGAGAAATTTAGGAAGATTATTAGGTAATGATCAAATTTATAATGTAGTTGTTACTATACATGCTTTTATTATAATTTTTTTTATGGTGATACCTATTATAATTGGAGGTTTTGGAAATTGATTAATTCCCTTAATATTAGGAGCTCCTGATATAGCTTTCCCTCGTATAAATAATATAAGATTTTGATTATTAATTCCTTCTTTGTTATTAATATTAATAAGAGGATTTATTATAGTTGGTAGTGGAACTGGGTGAACTATATATCCTCCTTTAAGTTCTTTAATTGGGCATAGAAGGTTTTCTGTTGATATAGTAATTTTTTCTTTACATTTAGCAGGGGTTTCTTCAATTATAGGAGCTATTAATTTTATTACAACAATTTTTAATATAAAATTAATTT---TAAAATTAGATCAGATTATATTATTTGTATGATCTGTATTAATTACTGCTTTTTTATTATTACTTTCTTTACCTGTTTTGGCAGGAGGAATTACTATATTATTAACAGATCGTAATTTAAATACTTCTTTTTTTGATTTTTCAGGAGGGGGAGATCCTGTTTTATTTCAACACTTATTT.This is the eighth species to be described in BIN Macrocentrusmichaelstroudi will key to M.gustavogutierrezi. Macrocentrusmichaelstroudi differs in its pale basal flagellomeres, contrasting with the melanic basal flagellomeres of M.gustavogutierrezi. The host caterpillar also differs from those of M.gustavogutierrezi. It belongs to the group of species with vein M+Cu of the forewing distinctly widened apically.In the morphological key to the species in this BIN , MacroceHolotype ?: Costa Rica: Guanacaste, Area de Conservaci\u00f3n Guanacaste, Sector Pitilla, Sendero Rotulo, 510 m, 11.0135 -85.4241; host caterpillar collection date: 22/i/2016, parasitoid eclosion: 01/iii/2016; depository CNC, holotype voucher code: DHJPAR0058830, GenBank accession: MW627584.Phaedropsis leialisDHJ03 (Crambidae) feeding on Gouanialupuloides (Rhamnaceae), caterpillar voucher code: 16-SRNP-30230.Macrocentrusmichaelstroudi is named in honor of Michael Stroud Bonilla as the primary supporter of the BioAlfa Malaise trapping at the Hacienda Baru Wildlife Refuge, Savegre, ACOPAC, Costa Rica, as well as decades of support for the Area de Conservaci\u00f3n Guanacaste.Members of all genera are koinobiont endoparasitoids of caterpillars. A key to the genera of the New World can be found in Taxon classificationAnimaliaHymenopteraBraconidae?Sharkeysp. nov.AC50A86E-2624-5E92-9CCD-172E1C60F06Bhttp://zoobank.org/6058EB69-E85F-4CFB-8771-92DD917FA191Figure BOLD:AEC3983; nearest neighbor: StantoniamiriamzunzaeBOLD:ACB1896; distance to nearest neighbor is 4.47%. Consensus barcode: TATATTGTATTTTTTGTTTGGTATATGGTCTGGGGTGTTAGGTTTATCACTAAGTTTAATTATTCGTATAGAATTAGGTCAAATTGGTTCATTTATTGGAAATGATCAAATTTATAATAGTATTGTTACTTCTCATGCTTTTATTATAATTTTTTTTATAGTTATGCCTATTATAATTGGGGGGTTTGGAAATTGATTGATTCCTTTAATATTAGGAAGTGTTGATATAGCTTTCCCTCGAATAAATAATATAAGATTTTGATTATTAATTCCTTCTTTAATATTATTAATTTTAAGAGGATTTATAAATATTGGTGTAGGTACAGGATGAACAGTTTACCCTCCTTTATCATTAAATGTTAGTCATATAGGAATTTCTGTAGATATAGCTATTTTTTCATTACATTTGGCTGGTATTTCTTCAATTATAGGTGCTATTAATTTTATTGTTACTATTATAAATATACGAAATTATGGGGTATTAATAGATAAAATTAGATTATTATCATGATCAATTTTAATTACAGCTATTTTATTATTGTTATCTTTACCTGTGTTAGCTGGTGCTATTACAATATTGTTAACTGACCGTAATTTAAATACATCCTTTTTTGATCCTGCTGGAGGAGGGGATCCTATTTTATATCAACATTTATTTBIN: S.miriamzunzae feeding on Vismiabaccifera (Hypericaceae), caterpillar voucher code:19-SRNP-46256. Erebidae is a new host-family record for Stantonia.Stantoniagilbertfuentesi is named in honor of Gilbert Fuentes of the Organizaci\u00f3n de Estudios Tropicales of Costa Rica in recognition of his decades of intensive management of the OET library of tropical publications.Members of the subfamily are thought to be solitary, koinobiont ectoparasitoids of caterpillars. A diagnosis for the subfamily is included in Taxon classificationAnimaliaHymenopteraBraconidae?Sharkeysp. nov.B58E733C-3E93-5BB7-918B-A9768C49D776http://zoobank.org/B5D5C71B-6810-4120-976F-D30A453FF262Figure BOLD:ADA0151; nearest neighbor: Rhysipolis sp. BOLD:ADL9389; distance to nearest neighbor is 2.91%. Consensus barcode: TGTATTATATTTTTTATTTGGAATTTGATCTGGAATAGTAGGTTTGTCTATGAGTTTAATTATTCGTTTAGAGTTAGGTATACCCGGTAGTTTGTTATTTAATGATCAGATTTATAATACGATAGTTACAGCTCATGCTTTTATTATAATTTTTTTTATAGTGATACCTGTAATGATTGGGGGGTTTGGTAATTGGTTAGTTCCATTAATGTTGGGGGCTCCTGATATAGCTTTTCCTCGTATGAATAATATAAGATTTTGATTATTAATTCCTTCTTTAATTTTATTATTTTTGAGGGGATTAGTAAATGTTGGGGTAGGTACTGGATGAACAGTTTATCCTCCTTTATCTTCTTCTATAGGTCATAGAGGTATTTCTGTTGATTTGGCTATTTTTTCTTTACATTTAGCTGGTATTTCTTCTATTATAGGAGCTATTAATTTTATTTCAACAATTTTTAATATGTGTTTATATTCAATTAATATAGATCAAATTAGTTTATTTATTTGATCTATTTTGATTACTGCTTTTTTATTATTGTTGTCTTTACCTGTTTTGGCAGGGGCTATTACTATATTGTTAACGGATCGAAATTTAAATACTTCATTTTTTGATTTTTCTGGBIN: This species can be morphologically distinguished from its nearest neighbor by having its mesoscutum entirely black Fig. comparedHolotype ?: Costa Rica: Guanacaste, Area de Conservaci\u00f3n Guanacaste, Sector San Cristobal, Estaci\u00f3n San Gerardo, 575 m, 10.8801 -85.389, 04/viii/2014, Malaise trap, depository CNC, holotype voucher code: BIOUG28483-E12, GenBank accession MW627555.BIOUG27682-G07.Rhysipolisstevearonsoni is named in honor of Steve Aronson of San Jose, Costa Rica, in recognition of decades of concern and involvement with the betterment of Costa Rica\u2019s positive relationship with its wild environment, and specifically with providing broadband internet to \u00c1rea de Conservaci\u00f3n Guanacaste as the first Costa Rican \u00c1rea de Conservaci\u00f3n to be so facilitated.Members of all genera are koinobiont endoparasitoids of caterpillars from a wide range of families. A key to the genera of the New World is in Sharkey (2021a).Taxon classificationAnimaliaHymenopteraBraconidae?Sharkeysp. nov.41DDCF67-4214-5C16-A53A-95B1E9F9DDE6http://zoobank.org/B8C158F4-07CA-4776-91B7-D7507870695FFigure BOLD:AEB2985; nearest neighbor: Aleiodes sp. BOLD:AAV6245; distance to nearest neighbor is 10.74%. Consensus barcode: AGTATTGTATTTTTTATTTGGTATATGAGCTGGAATAGTTGGTTTATCTATAAGATTAATTATTCGGTTAGAATTAAGAGTTGGGGGGAGAGTCTTAAAAAATGATCAGATTTATAAYGGTATAGTAACTTTGCATGCTTTTGTAATAATTTTTTTTATAGTTATACCTATTATATTAGGAGGGTTTGGAAATTGGTTAGTTCCTTTAATATTGAGAGCTCCAGATATAGCTTTCCCTCGAATAAATAATATAAGATTTTGATTATTAATTCCATCTTTTTTTTTATTATTGATTAGAGGTGTTATTAATTCAGGRGTAGGAACAGGTTGAACAATATATCCTCCTCTTTCTTTATTAATTGGTCATGATGGAATTTCTGTAGATATATCAATTTTTTCTTTACATTTAGCAGGAGCTTCTTCCATTATAGGTTCAATTAATTTTATTTCTACTATTTTTAATATAAAATTAAAAGATTTAAAATTAGATCAAGTTTCTTTATTTGTTTGATCTATTTTAATTACAACAATTTTATTATTGTTATCTTTACCTGTTTTAGCGGGGGCAATTACTATATTATTGACTGATCGAAACTTAAATACAAGATTTTTTGATTTTGCTGGAGGAGGGGATCCAATTTTATTTCAACATTTGTTT.BIN: This species can be morphologically distinguished from its nearest neighbor by having the base of the stigma brown Fig. , contrasHolotype ?: Costa Rica: Guanacaste, Area de Conservaci\u00f3n Guanacaste, Sector Brasilia, Gallinazo, 360 m, 11.0183 -85.372; host caterpillar collection date: 10/vii/2019, parasitoid eclosion: 23/vii/2019; depository CNC, holotype voucher code: DHJPAR0064529, GenBank accession: MW627545.Isogona Poole07 (Erebidae) feeding on Celtisiguanaea (Ulmaceae), caterpillar voucher code:19-SRNP-65351.DHJPAR0065341.Aleiodeskaydodgeae is named in honor of Kay Dodge of Costa Rica\u2019s Nicoya Peninsula today, original and decades long facilitator of ACG support from Peter Wege (RIP) of the Wege Foundation of Grand Rapids, Michigan, USA.Taxon classificationAnimaliaHymenopteraBraconidae?Sharkeysp. nov.3A4BC25A-6E30-5F9F-A290-6CBA3F6752E7http://zoobank.org/273165E1-C8BD-43E8-8EED-75004A28DD99Figure BOLD:AEF3944; nearest neighbor: Aleiodes sp. BOLD:AAG1309; distance to nearest neighbor is 8.29%. Consensus barcode: AGTATTATATTTTTTATTTGGAATATGAGCAGGAATAATTGGGATATCAATAAGTTTAATAATCCGATTAGAATTAAGAACAAATGGAAGAATCTTAAAAAATGATCAAATTTATAATGGTATGGTAACTTTACATGCCTTTATTATAATTTTTTTTATAGTAATACCAATTATAATTGGAGGATTTGGAAATTGATTAATTCCTTTAATATTAGGAGCTCCTGACATAGCTTTCCCACGTATAAATAATATAAGATTTTGATTACTAATACCTTCTTTAATACTTTTATTACTTAGAGGAATAATTAATACCGGGGTAGGAACAGGATGAACTATATATCCCCCTTTATCATCACTAATTGGACATAATGGAATTTCAGTAGATATATCTATTTTTTCTTTACACCTTGCAGGGGCTTCTTCAATTATAGGAGCAATCAACTTCATTTCAACTATTTTTAATATAAATATTATAACAATTAAAATAGATCAAATTATACTATTAATTTGATCTATTTTAATTACTACAATCCTTTTATTATTATCTTTACCAGTATTAGCAGGAGCAATTACTATATTACTAACAGATCGAAATTTAAATACAAGATTTTTTGACTTTTCAGGGGGAGGAGACCCTATTTTATTCCAACATCTTTTT.BIN: This species can be morphologically distinguished from its nearest neighbor by its pale stigma Fig. , which iHolotype ?: Costa Rica: Guanacaste, Area de Conservaci\u00f3n Guanacaste, Sector Pitilla, Bullas, 440 m, 10.98670 -85.38503; host caterpillar collection date: 25/i/2019, parasitoid eclosion: 13/ii/2019; depository CNC, holotype voucher code: DHJPAR0063995, GenBank accession: MW627548.Anomisgentilis (Erebidae) feeding on Peltaeaovata , caterpillar voucher code:19-SRNP-70250.DHJPAR0063970.Aleiodeskerrydresslerae is named in honor of Kerry Dressler for her life career of deep and intense work and interest in the taxonomy of orchids, and of supporting Bob Dressler\u2019s enthusiasm for the same.Taxon classificationAnimaliaHymenopteraBraconidae?Sharkeysp. nov.7B3CF334-BA1C-592A-952E-AC5502B0E972http://zoobank.org/2A3DC697-674E-4EB5-AE11-A15680375504Figure BOLD:AEB1913; nearest neighbor: Aleiodes sp. BOLD:AAM5681; distance to nearest neighbor is 2.24%. Consensus barcode: AATTTTATATTTTTTATTTGGTTTATGAGCAGGAATAATTGGGATATCAATAAGATTAATTATTCGATTAGAATTAAGAACTAGAGGTAGAATTTTAAAAAATGATCAAATTTATAACGGCATAGTAACTTTACATGCATTTATTATAATTTTTTTTATAGTAATACCCATTATAATTGGAGGGTTTGGAAATTGATTAATYCCTCTAATATTAGGAGCCCCTGATATAGCATTTCCTCGAATAAATAATATAAGATTTTGATTACTAATTCCATCATTAATATTTTTATTAATTAGAGGAATTATTAATACAGGTGTAGGAACAGGATGAACAATATATCCTCCATTATCTTCATTAATTGGACATAATAGAATTTCAGTTGATATATCAATTTTTTCTTTACATATAGCAGGTGCTTCATCAATTATAGGAGCTATTAATTTTATTTCAACAATTTTCAATATAAACTTAATAAAAATCAAAATAGAYCAAATTATATTATTAGTTTGATCAGTTTTAATTACAGCCATTTTATTATTACTTTCATTACCTGTTTTAGCAGGAGCAATCACTATATTRTTAACCGACCGTAACTTAAATACAAGTTTTTTTGATTTTTCAGGAGGAGGAGATCCTATTTTATTCCAACATTTATTT.BIN: The nearest neighbor is a sole specimen from Canada. There is no image available on BOLD but due to the distribution, conspecificity is doubtful.Holotype ?: Costa Rica: Guanacaste, Area de Conservaci\u00f3n Guanacaste, Sector Del Oro, Meteorologico, 590 m, 11.002 -85.4617; host caterpillar collection date: 25/vi/2019, parasitoid eclosion: 15/vii/2019; depository CNC, holotype voucher code: DHJPAR0064517, GenBank accession: MW627567.Herbitamedama (Geometridae) feeding on Dendropanaxarboreus , caterpillar voucher code:19-SRNP-20457.Prenestascyllalis feeding on Forsteroniaspicata (Apocynaceae), depository CNC.DHJPAR0064001 (DHJPAR0062033 not barcoded), host data Aleiodesjosesolanoi is named in honor of Jose Andr\u00e9s Solano, the BioAlfa Malaise traps manager for Estaci\u00f3n El Ceibo, Parque Nacional Braulio Carrillo, ACC, Costa Rica.Taxon classificationAnimaliaHymenopteraBraconidae?Sharkeysp. nov.7F53D704-D338-583F-AF41-E21CF4F5BA18http://zoobank.org/474FE466-BEF5-4451-8623-252D716F6B6FFigure BOLD:AAV7490; nearest neighbor: Aleiodes sp. BOLD:AAV6239, from French Guiana; distance to nearest neighbor is 8.65%. Consensus barcode. AATTTTATATTTTTTATTTGGTTTATGGTCAGGAATAATTGGCATGTCAATAAGATTAATTATTCGATTAGAATTAAGAACGAGAGGTAGAATTTTAAAAAATGACCAAATTTATAATGGCATAGTAACTTTACATGCATTTATTATAATTTTTTTTATAGTAATACCAATTATAATTGGTGGGTTTGGAAATTGATTAATTCCTTTAATATTAGGAGCCCCTGATATAGCATTTCCTCGTATAAATAATATAAGATTTTGATTATTAATCCCATCACTAATATTTTTATTGATTAGAGGTATTATTAATACAGGAGTAGGGACAGGATGAACTATATATCCTCCCCTATCTTCCTTAATTGGCCATAATAGAATATCAGTTGATATATCAATTTTTTCTCTCCATATAGCTGGAGCCTCATCAATCATAGGAGCAATTAATTTCATCTCAACAATTTTTAACATAAATCTAATAAAAATTAAAATAGACCAAATTATACTATTAGTATGGTCAGTTTTAATTACAGCTATTTTATTACTACTTTCATTACCTGTTTTAGCAGGAGCAATTACAATATTATTAACTGACCGTAATTTAAATACAAGATTTTTTGATTTTTCAGGAGGAGGGGACCCCATTTTATTCCAACATTTATTT.BIN: This species can be morphologically distinguished from its nearest neighbor by its uniformly colored hind legs Fig. , compareHolotype ?: Costa Rica: Alajuela, Guanacaste Area de Conservaci\u00f3n, Sector Rincon Rain Forest, Sendero Aura, 432 m, 10.9654 -85.3239; host caterpillar collection date: 04/vii/2019, parasitoid eclosion: 31/vii/2019; depository CNC, holotype voucher code: DHJPAR0064521, GenBank accession: MW627570.Geometridae) feeding on Serjaniaschiedeana (Sapindaceae), caterpillar voucher code:19-SRNP-27158.geoJanzen01 Janzen7158 .Aleiodesjuniorporrasi is named in honor of Junior Porras Quir\u00f3s, the BioAlfa Malaise traps manager for Estaci\u00f3n Altamira, Parque Nacional Chirripo, ACLAP, Costa Rica.Taxon classificationAnimaliaHymenopteraBraconidae?Sharkeysp. nov.55F61DB3-C558-584F-8E23-B05A51A872EBhttp://zoobank.org/E8FBEA44-9DE3-4E93-81F8-0CBAD405DB42Figure BOLD:AAM5673; nearest neighbor: Aleiodes sp. BOLD:AAH8820; distance to nearest neighbor is 4.03%. Consensus barcode: GTTTTATATTTTTTATTTGGGATATGAGCTGGTATATTAGGRTTATCTATAAGGTTAGTTATYCGTTTAGAATTAAGAAYTGTTGGRAGAGTTTTAAAAAATGATCAAATTTATAATGGKATGGTTACATTACATGCTTTTGTAATAATYTTTTTTATAGTTATACCTATTATAATTGGTGGGTTTGGAAATTGATTAATTCCTTTAATATTAGGGGCTCCTGATATAGCATTYCCTCGGATAAATAATATGAGATTTTGRTTATTAATTCCTTCATTTTTTTTATTATTAATTAGAGGTGTTATTAATTCAGGGGTAGGTACAGGTTGAACAATATACCCTCCCCTTTCTTTATTAATTGGTCATAATGGTTTATCAGTGGATATATCTATTTTTTCTTTACATTTAGCTGGRGCTTCTTCTATTATAGGATCAATTAATTTTATTTCAACTATTTTTAATATAAATTTATTTTATATTAAATTAGATCAGATTTCTTTATTAGTATGGTCAGTATTAATCACTACTATTTTATTATTATTATCTTTACCTGTTTTRGCAGGGGCTATTACTATATTATTGACTGATCGTAATTTAAATACAAGATTTTTTGATTTTTCGGGGGGAGGGGATCCAATTTTATTTCAACATTTA.BIN: There is no image on BOLD, but the p-distance makes it doubtful that it is conspecific.Holotype ?: Costa Rica: Guanacaste, Area de Conservaci\u00f3n Guanacaste, Sector San Cristobal, Estaci\u00f3n San Gerardo, 575 m, 10.88 -85.389, 18/xi/2013, Malaise trap, depository CNC, holotype voucher code: BIOUG20202-G10, GenBank accession: MW627543.Other material. BMNHE897799, from Belize deposited in the Natural History Museum (London), based on barcode, not viewed and lacking image on BOLD.Aleiodesrocioecheverri is named in honor of Rocio Echeverri of San Jose and Liberia, Costa Rica, in recognition of her lifetime of concern and involvement with the betterment of Costa Rica\u2019s positive relationship with its wild environment.Taxon classificationAnimaliaHymenopteraBraconidae?Sharkeysp. nov.01BDD77E-547F-5861-BF97-A6940D4496B8http://zoobank.org/A73F3D24-4F8D-42C3-94F3-EC56B5219C77Figure BOLD:AAH8707; nearest neighbor: Aleiodesnr.speciosusBOLD:AAM4342; distance to nearest neighbor is 2.4%. Consensus barcode: AATTTTATAYTTCATATTTGGAATATGAGCAGGTATAATTGGAATATCAATAAGATTAATTATTCGAATAGAATTAAGAACAAGAGGAAGAATTTTAAAAAATGACCAAATTTATAATGGTATAGTAACTTTGCATGCTTTTATTATAATTTTTTTTATAGTTATACCARTTATAATTGGGGGATTCGGAAATTGATTAATCCCCTTAATATTAGGGGCCCCTGATATAGCATTCCCTCGAATAAATAATATAAGATTTTGGTTATTAATTCCATCTTTATTATTTTTACTAATAAGAGGTATTATTAATACAGGAGTTGGGACAGGATGAACTATATACCCTCCTTTATCRTCTTTAATTGGACATAATAGAATTTCARTTGATATGTCAATTTTTTCTTTACATTTAGCAGGAGCTTCTTCTATTATAGGRGCAATTAATTTTATTTCAACAATTTTTAATATAAATTTAATAAAAATTAAATTAGACCAAATTTCATTGTTAATTTGATCAATTTTAATTACTACTATTTTATTATTATTATCTTTACCTGTACTAGCAGGAGCAATCACCATATTATTAACTGATCGTAACTTAAACACAAGATTTTTTGATTTTTCTGGAGGAGGAGAYCCAATTTTATTTCAACATTTATTT.BIN: No images are available on BOLD for the three specimens in the nearest neighbor, all from Ecuador.Holotype ?: Costa Rica: Guanacaste, Area de Conservaci\u00f3n Guanacaste, Sector Del Oro, Sendero Puertas, 400 m, 11.01087 -85.48816; host caterpillar collection date: 28/xii/2018, parasitoid eclosion: 07/i/2019; depository CNC, holotype voucher code: DHJPAR0064524, GenBank accession: MW627585.Geometridae) feeding on algae, caterpillar voucher code:18-SRNP-21223.geoJanzen01 19-SRNP-20029 , Costa Rica.Taxon classificationAnimaliaHymenopteraBraconidae?Sharkeysp. nov.236166A3-241E-5A99-9034-2FAAC25B4297http://zoobank.org/20C776D0-74D9-4B3D-B0E0-EE4E117B28CBFigure BOLD:AAM5951; nearest neighbor: Choreborogas sp. BOLD:ACG8400; distance to nearest neighbor is 2.71%. Consensus barcode:BIN: AGTATTGTATTTTTTTTTTGGTATATGATCAGGTATATTGGGYTTATCAATAAGGTTAATTATTCGGTTTGAATTAGGGGTTCCTGGATCATTTTTAGGTAATGATCAGATTTATAATAGAATTGTTACGGCYCATGCCTTGGTTATAATTTTTTTTATGGTTATACCTGTAATAATTGGGGGATTTGGTAATTGATTAATTCCTTTAATATTAGGRGCACCTGATATAGCTTTYCCTCGAATAAATAATATAAGATTTTGGTTATTAATTCCTTCTATTTTGTTATTGTTAGTTAGATCTTTAGTTAATGTTGGGGYAGGTACAGGATGAACAATTTATCCTCCTTTATCTTCRTTAATAGGTCATGGSGGGATTTCAGTTGATTTAGCTATTTTTTCTTTACATTTAGCTGGTGCATCATCAATTATAGGTGCAATTAATTTTATTTCTACAATTTTTAATATAAATTTATTTTCAATGAAAATAGATCAAATTATATTATTAGTTTGATCTGTATTAATCACTGCTTTTTTATTATTATTATCATTRCCTGTTTTGGCGGGGGCAATTACTATATTATTATTTGATCGTAAYATTAATAGAACTTTTTTTGATTTTTCAGGGGGAGGGGATCCTATTTTATTYCAGCATTTATTThis species can be morphologically distinguished from its nearest neighbor by its swollen hind basitarsus Fig. , which iHolotype ?: Costa Rica: Guanacaste, Area de Conservaci\u00f3n Guanacaste, Sector Pailas Dos, PL12-6, 853 m, 10.7637 -85.3331, 04/xii/2014, Malaise trap, depository CNC, holotype voucher code: BIOUG46391-F12, GenBank accession: MW627542.Malaise trapped, BIOUG46544-F12, BIOUG49790-H06, BIOUG07453-F05, BIOUG28810-A07, BIOUG29020-B09.Other material: BMNHE897774 from Belize is in the same BIN and likely conspecific. There is no image on BOLD and the specimen was not examined.Choreborogasjesseausubeli is named in honor of Jesse Ausubel of Rockerfeller University, New York, USA, for his very strong support of the germination and early development of DNA barcoding as an identification tool.Taxon classificationAnimaliaHymenopteraBraconidae?Sharkeysp. nov.BC1796D0-995C-5151-89B9-479740A8C5D5http://zoobank.org/8228960E-CF6E-4BF9-8C86-696F7EA67FE5Figures BOLD:AAH8815; nearest neighbor: Triraphis sp. BOLD:AAG5003 from Guyana. Distance to nearest neighbor is 6.28%. Consensus barcode:TGTTTTATATTTTTTATTTGGAATTTGAGCTGGTATAGTCGGGCTGTCTATAAGGTTAATTATTCGGTTAGAATTAAGTATACCAGGGAGATTATTGGGGAATGAYCAGATTTATAATGGTATAGTTACCGCTCATGCTTTTATTATAATTTTTTTTATGGTAATACCTATTATAATTGGTGGTTTTGGAAATTGATTAATTCCATTAATGTTGGGGGCYCCTGATATGGCTTTCCCTCGTATAAATAATATGAGGTTTTGGTTATTAATTCCYTCATTGACGTTATTAATTTTAAGGGCTGTAGTTAACGTTGGAGTAGGTACTGGGTGAACTTTATATCCYCCCTTATCTTCTTTAGTTGGTCATGGGGGTATATCTGTAGATATAGCTATTTTTTCTTTACATTTAGCTGGTGCCTCTTCTATTATAGGAGTTGTTAATTTTATTTCTACTATTTTTAATATAAAATTAATATCAATTAATTTAGATCAAATTAATTTATTTGTTTGATCAGTATTAATTACGGCTGTTTTATTATTATTATCTTTACCAGTATTAGCTGGTGCAATTACTATATTATTGACAGATCGTAATTTAAATACAACATTTTTTGATTTTTCTGGGGGGGGYGATCCTATTTTATTCCAACATTTATTTBIN: COI distance and geographic distribution suggest that they are not the same species.No image of the nearest neighbor is available on BOLD, but the Holotype ?: Costa Rica: Guanacaste, Area de Conservaci\u00f3n Guanacaste, Sector Pitilla, Sendero Naciente, 700 m, 10.98705 -85.42816; host caterpillar collection date: 09/ii/2010, parasitoid eclosion: 13/ii/2010; depository CNC, holotype voucher code: DHJPAR0038023. GenBank accession: HQ548697.Holotype host data.Eucleamesoamericana (Limacodidae) feeding on Thelypterisnicaraguensis (Thelypteridaceae), caterpillar voucher code: 10-SRNP-30444.BCLDQ0860.Triraphisdoncombi is named in honor of Dr. Don Comb (RIP), founder of the New England Biolabs and New England Biolabs Foundation, in recognition of his serious and ongoing support for the management and biodiversity conservation of \u00c1rea de Conservaci\u00f3n Guanacaste in northeastern Costa Rica (http://www.acguanacaste.ac.cr), through the Guanacaste Dry Forest Conservation Fund (http://www.acguanacaste.ac.cr).Taxon classificationAnimaliaHymenopteraBraconidae?Sharkeysp. nov.92179AC2-B0F2-536F-A9EC-A5CE15FADB3Fhttp://zoobank.org/8BE983A2-E1D9-4FB8-AC3C-6E2FB4B5F8AAFigures BOLD:AAT9450. Its nearest neighbor on BOLD is identified by D. Quicke as YeliconesartitusBOLD:AAM6954; distance to nearest neighbor is 9.2%. Consensus barcode: TGTTTTATATTTTTTATTAGGTATTTGATGTGGATTAGTAGGTTTATCTTTAAGGTTACTTATTCGGTTAGAGTTGAGAAATTTAGGAAGATTATTAGGTAATGATCAAATTTATAATGTAGTTGTTACTATACATGCTTTTATTATAATTTTTTTTATGGTGATACCTATTATAATTGGAGGTTTTGGAAATTGATTAATTCCCTTAATATTAGGAGCTCCTGATATAGCTTTCCCTCGTATAAATAATATAAGATTTTGATTATTAATTCCTTCTTTGTTATTAATATTAATAAGAGGATTTATTATAGTTGGTAGTGGAACTGGGTGAACTATATATCCTCCTTTAAGTTCTTTAATTGGGCATAGAAGGTTTTCTGTTGATATAGTAATTTTTTCTTTACATTTAGCAGGGGTTTCTTCAATTATAGGAGCTATTAATTTTATTACAACAATTTTTAATATAAAATTAATTT---TAAAATTAGATCAGATTATATTATTTGTATGATCTGTATTAATTACTGCTTTTTTATTATTACTTTCTTTACCTGTTTTGGCAGGAGGAATTACTATATTATTAACAGATCGTAATTTAAATACTTCTTTTTTTGATTTTTCAGGAGGGGGAGATCCTGTTTTATTTCAACACTTATTT.BIN: Y.vilawanae in the key of Yeliconesvilawanae has the apical 0.2 of hind tarsus and apical 0.8 of hind basitarsus brown. Yeliconesmayrabonillae has the basal four hind tarsomeres brown and the apical tarsomere yellow. This species can be morphologically distinguished from its nearest neighbor, Yeliconesartitus, by the color of the hind femur being entirely testaceous .This species keys to ous Fig. feeding on Vochysiaguatemalensis (Vochysiaceae), caterpillar voucher code:10-SRNP-42391.epipajanzen01 Janzen882 (Yeliconesmayrabonillae is named in honor of Mayra Bonilla as the primary supporter of the BioAlfa Malaise trapping at the Hacienda Baru Wildlife Refuge, Savegre, ACOPAC, Costa Rica, as well as decades of support for the Area de Conservaci\u00f3n Guanacaste."} +{"text": "Programmed cell death-ligand 1 (PD-L1)/PD-1 axis is critical for maintenance of immune homeostasis by limiting overactivation of effector T-cell responses. The impairment of PD-L1/PD-1 signals play an important role in the pathogenesis of inflammatory diseases, making this pathway an ideal target for novel therapeutics to induce immune tolerance. Given weakly acidic environment as a putative hallmark of inflammation, in this study we designed a new cargo by linking the ectodomain of murine PD-L1 to the N terminus of pHLIPs, a low pH-responding and membrane-insertion peptide, and demonstrated its potent immune-suppressive activity. Specifically, PD-L1-pHLIP spanned the cellular membrane and perfectly recognized its ligand PD-1 in acidic buffer. Immobile PD-L1-pHLIP actively inhibited T-cell proliferation and IFN-\u03b3 production. Importantly, soluble PD-L1-pHLIP retained its function to dampen T-cell responses under acidic condition instead of neutral aqueous solution. Overall, these data suggest that PD-L1-pHLIP has potentials to be a novel therapeutic avenue for T-cell-mediated inflammatory diseases. Genein vivo . In nonoin vivo . Of notein vivo , 10, indChronic inflammation is the hallmark of autoimmune diseases, characterized by massive infiltration and activation of immune cells , 12. NotThe amino acid sequences of recombinant proteins used in this study were indicated as below. All constructs carried a C-terminal histidine tag or IgG1 Fc fragment for purification and were cloned into pMT-puro vectors for expression in HEK293 cells. Stably transfected cells were selected with 6 \u03bcg/ml puromycin. The resulting proteins were purified using Ni-affinity or protein A agarose affinity chromatography column followed by further purification by SEC using a Superdex 200 (S200) column with 10 mM Tris and 150 mM NaCl, pH 7.5 (1\u00d7 TBS). The purity of recombinant proteins was determined by SDS-PAGE .Protein sequences used in this study:Murine PD-L1-WT pHLIP :MDAMKRGLCCVLLLCGAVFVSNSHHHHHHHHFTITAPKDLYVVEYGSNVTMECRFPVERELDLLALVVYWEKEDEQVIQFVAGEEDLKPQHSNFRGRASLPKDQLLKGNAALQITDVKLQDAGVYCCIISYGGADYKRITLKVNAPYRKINQRISVDPATSEHELICQAEGYPEAEVIWTNSDHQPVSGKRSVTTSRTEGMLek?>LNVTSSLRVNATANDVFYCTFWRSQPGQNHTAELIIPELPATHPPQNRTGGGGSGGGGSGGGGSAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTMurine PD-L1-mFc (denoted to PD-L1-Fc):MDAMKRGLCCVLLLCGAVFVSNSFTITAPKDLYVVEYGSNVTMECRFPVERELDLLALVVYWEKEDEQVIQFVAGEEDLKPQHSNFRGRASLPKDQLLKGNAALQITDVKLQDAGVYCCIISYGGADYKRITLKVNAPYRKINQRISVDPATSEHELICQAEGYPEAEVIWTNSDHQPVSGKRSVTTSRTEGMLLNVTSSLRVNATANDVFYCTFWRSQPGQNHTAELIIPELPATHPPQNRTDDDDKAVPRDSGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGKMurine PD-L1-mutated WT pHLIP (denoted to PD-L1-pHLIP(m)):MDAMKRGLCCVLLLCGAVFVSNSHHHHHHHHFTITAPKDLYVVEYGSNVTMECRFPVERELDLLALVVYWEKEDEQVIQFVAGEEDLKPQHSNFRGRASLPKDQLLKGNAALQITDVKLQDAGVYCCIISYGGADYKRITLKVNAPYRKINQRISVDPATSEHELICQAEGYPEAEVIWTNSDHQPVSGKRSVTTSRTEGML1LNVTSSLRVNATANDVFYCTFWRSQPGQNHTAELIIPELPATHPPQNRTGGGGSGGGGSGGGGSACEQNPIYWARYAKWLFTTPLLLLKLALLVDADEGTEBOV-secreted glycoprotein (sGP)-WT pHLIP (denoted to control protein):MDAMKRGLCCVLLLCGAVFVSNSHHHHHHIPLGVIHNSTLQVSDVDKLVCRDKLSSTNQLRSVGLNLEGNGVATDVPSATKRWGFRSGVPPKVVNYEAGEWAENCYNLEIKKPDGSECLPAAPDGIRGFPRCRYVHKVSGTGPCAGDFAFHKEGAFFLYDRLASTVIYRGTTFAEGVVAFLILPQAKKDFFSSHPLREPVNATEDPSSGYYSTTIRYQATGFGTNETEYLFEVDNLTYVQLESRFTPQFLLQLNETIYASGKRSNTTGKLIWKVNPEIDTTIGEWAFWETKGGGGSGGGGSGGGGSACEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTA total of 96-well plated were coated overnight with the indicated antigens at 2 or 5 \u03bcg/ml, washed three times with PBS-T, and blocked with 4% nonfat dried milk in PBS for 1 h at room temperature. Twofold serial dilution of biotin-labeled anti-mPD-L1 antibody from 6 \u03bcg/ml was measured. For mPD-1, which was biotinylated according to manufacturer\u2019s instructions , it was added at 25, 50, and 100 \u03bcg/ml and incubated for 1 h at room temperature. The wells were washed and avidin-conjugated HRP in 0.5% BSA was added and incubated for 1 h at room temperature. A TMB substrate kit was used for detection at 450 nm. IFN-\u03b3 levels in the supernatants were determined by sandwich ELSA assays .2 at 37\u00b0C. HEK293T cells were transiently transfected with mPD-1 and mPD-L1 expression plasmids using a PEI transfection protocol , respectively. 48 h later, the expression of PD-L1 and PD-1 on the surface of cells was detected by flow cytometry.Cell lines were purchased from ATCC and cultured in Dulbecco\u2019s modified Eagle\u2019s medium (DMEM) or RPMI 1640 medium supplemented with 10% fetal bovine serum and penicillin/streptomycin in a humidified atmosphere of 5% CORecombinant proteins were labeled by fluorescence dye (PE or AlexaFlour488) with Conjugation Kit, according to manufacturer\u2019s protocol . The HEK293T or HCT116 cell lines were placed in a laser confocal dish and cultured overnight. AlexaFlour488-conjugated protein was added at 10 \u00b5g/ml and cultured at 37\u00b0C for 1 h. Cells were washed twice with PBS with corresponding pH, and then the fluorescence on the surface of cell membrane was observed under confocal laser scanning microscope .5 cells/well) with stimulation of anti-CD3 and CD28 antibody cocktail for 72 h. BrdU cell proliferation assays were performed according to the manufacturer\u2019s protocol . In addition, lymphocytes were treated as described above with soluble proteins indicated in PBS with corresponding pH or containing lactic acid (10 or 20 mM). In some settings, neutralizing mAbs to murine PD-1 or PD-L1 were added.The splenocytes of BALB/c mice were isolated by gradient centrifugation. Human PBMC were isolated from whole blood of healthy donors with signed written informed consents. The indicated proteins at titrated concentrations were coated in 96-well plates at 4\u00b0C overnight. Lymphocytes were added (5 \u00d7 10The cells were incubated with PE-labeled proteins as indicated at 0.001\u201310 \u00b5g/ml for 0.5\u20134 h in PBS with corresponding pH or containing lactic acid (10 or 20 mM). In some settings, cells were incubated with the indicated proteins at 10 \u00b5g/ml for 1 h in PBS (pH 7.4 and 6.3). PE-labeled mPD-1 was added and incubated for another 30 min. Cells were washed twice with PBS with corresponding pH, and then detected by flow cytometry.For BrdU incorporation, mouse spleen lymphocytes were treated as described above. Six hours before the end of stimulation, BrdU solution with the final concentration of 10 \u00b5M was added. Cells were washed and incubated with APC-antimouse CD4/CD8 antibody at 4\u00b0C for 30 min. After washing, fixing, and permeabilizing , cells were incubated with PE-antimouse BrdU antibody in the dark at 4\u00b0C for another 30 min. The fluorescence intensity was measured by FACS Calibur II .Extracted total mRNA with TRIzol reagent, Single-strand cDNA was made from 1\u00b5g total RNA by reverse transcription (RT) using a TransScript II First-Strand cDNA Synthesis SuperMix . qPCR was conducted using SYBR Green qPCR mixture through 50 cycles in a IQ5 Real-Time PCR Detection Systems . The sequences of the primers used for qPCR were as follows: the forward primer of mouse IFN-\u03b3 is 5\u2019-ACAGCAAGGCGAAAAAGGATG-3\u2019 and the reverse primer is 5\u2019-TGGTGGACCACTCGGATGA-3\u2019. The forward primer of mouse GAPDH is 5\u2019-CATCAAGAAGGTGGTGAAGC-3\u2019 and the reverse primer is 5\u2019-CCTGTTGCTGTAGCCGTATT-3\u2019. Data were analyzed using the delta-delta Ct method.t-tests were used to compare experimental and control groups. Statistical significance is defined as p < 0.05.Data are expressed as the means \u00b1 SD according to at least 3 independent experiments. Two-tailed Student\u2019s 3 as a coupling linker and the fusion protein generated in the eukaryotic expression system. As well, PD-L1 (having His tags) and PD-L1-Fc protein were also expressed using the same methods. The identities of these proteins were validated by SDS-PAGE in vivo, pH was titrated by lactic acid (10 or 20mM). Similar effects were observed were incubated with fluorescence-labeled PD-L1-pHLIP for 1 h in pH 7.4 or 6.3 solutions and then detected by flow cytometry. No fluorescence was found in neutral aqueous solutions. In contrast, high magnitudes of fluorescence were visible in pH 6.3 buffer , indeed, potently inhibited the expansion of CD4+ and CD8+ T cells simultaneously . To determine whether PD-L1/PD-1 ligation was responsible for the inhibitory function of PD-L1-pHLIP, neutralizing antibodies to PD-L1 and PD-1 were added. Blocking PD-L1/PD-1 interaction using anti-PD-L1 or PD-1 antibody almost entirely abrogated the inhibitory ability of PD-L1-pHLIP, including suppressing lymphocyte proliferation and IFN-\u03b3 secretion . Overall, these results indicate that plate-bound PD-L1-pHLIP actively inhibits T lymphocyte expansion and cytokine production via binding to PD-1 receptor on the surface of T cells.Thereafter, we evaluated the function of plated-bound proteins to inhibit proliferation of lymphocytes at the intervals of 24\u201372 h following treatment with anti-CD3/CD28 mAbs. At 24 h poststimulation, none of them displayed inhibitory function. All of them, however, actively suppressed lymphocyte expansion at 48 and 72 h following TCR stimulation, with over 90% of inhibition rates at the soluble state in pH6.3 or 7.4 buffer respectively. As expected, these proteins at the soluble state did not suppress lymphocyte proliferation and IFN-\u03b3 secretion in neutral aqueous solution . In contrast, PD-L1 and PD-L1-Fc had no inhibitory effects on lymphocyte activation under the same condition . We further evaluated the function of these proteins in the solution containing 10mM lactic acid. Similarly, PD-L1-pHLIP displayed significantly inhibitory function on lymphocyte proliferation and IFN-\u03b3 production, instead of PD-L1 and PD-L1-Fc . Taken together, these data suggest that PD-L1-pHLIP actively suppresses T-cell activation under acidic condition.To test our hypothesis that under acidic conditions, PD-L1-pHLIP inserts and spans cellular membrane through conformational changes from unstructured coil to \u03b1 helix, thereby playing an immune-suppressive role in T-cell activation in vivo in the future. The reasons for choosing PD-L1 as a cargo are following: (a) PD-L1/PD-1 interaction is definitely critical to maintain T-cell tolerance and immune homeostasis. Long-term administration of PD-1 antibodies led to significant increase of risks for autoimmune diseases , which was due to that PD-1 mAb treatment abrogated endogenous signaling axis of PD-L1/PD-1, thereby pathogenically activated autoreactive T cells and ultimately caused the onset of autoimmune diseases , 31. Howdiseases \u201335; (b) diseases ; (c) PD-diseases , 38; (d)diseases , we specdiseases , 41; (f)in vitro and an inserted \u03b1-helical population (state III) (A recent study demonstrated that in NOD mice hematopoietic stem progenitor cells (HSPCs) were deficient in PD-L1 and transfusion of genetically engineered or pharmacologically modulated HSPCs overexpressing PD-L1 inhibited autoimmune response and reverted diabetes . Howeverate III) . In loweThe original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by The Ethics Committee of the Chinese PLA General Hospital. The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by The Ethics Committee of the Academy of Beijing Institute of Pharmacology and Toxicology.YiS and LH performed the experiments and prepared the manuscript. PY, MZ, and XW were involved in optimization of the experimental protocols. HX, CQ, JW, LL, JF, and YZ provided methodological support. YW, YaS, and GC conceived and guided the study. All authors contributed to the article and approved the submitted version.This work is supported by the National Natural Science Foundation of China .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."} +{"text": "Computational modeling approaches are evolving incredibly fast right now and are demonstrating great results in many applications, including de novo protein design. It suggests that the easier task of fine-tuning the fluorogen-binding properties of an already functional protein in silico should be readily achievable. To test this hypothesis, we used Rosetta for computational ligand docking followed by protein binding pocket redesign to further improve the previously described FAP DiB1 that is capable of binding to a BODIPY-like dye M739. Despite an inaccurate initial docking of the chromophore, the incorporated mutations nevertheless improved multiple photophysical parameters as well as the overall performance of the tag. The designed protein, DiB-RM, shows higher brightness, localization precision, and apparent photostability in protein-PAINT super-resolution imaging compared to its parental variant DiB1. Moreover, DiB-RM can be cleaved to obtain an efficient split system with enhanced performance compared to a parental DiB-split system. The possible reasons for the inaccurate ligand binding pose prediction and its consequence on the outcome of the design experiment are further discussed.The use of unnatural fluorogenic molecules widely expands the pallet of available genetically encoded fluorescent imaging tools through the design of fluorogen activating proteins (FAPs). While there is already a handful of such probes available, each of them went through laborious cycles of Computational approaches have recently made significant progress in the protein engineering field evolving from a tool for helping experimentalists to prioritize or short-list mutations for testing to being capable of making fully reliable predictions. However, not all the fields of protein modeling are evolving at a similar pace. That is why evaluating the capabilities of computational tools on different tasks is important to provide other scientists with up-to-date information on the state of the field. Here we tested the performance of Rosetta (one of the leading macromolecule modeling tools) in improving small molecule-binding proteins. We successfully redesigned a fluorogen binding protein DiB1 \u2013a protein that binds a non-fluorescent molecule and enforces its fluorescence in the obtained complex\u2013for improved brightness and better performance in super-resolution imaging. Our results suggest that such tasks can be already achieved without laborious library screenings. However, the flexibility of the proteins might still be underestimated during standard modeling protocols and should be closely evaluated. The biological role of many natural fluorogens is often directly connected to their ability to absorb light. For example, chlorophyll is used in photosystems of cyanobacteria, algae, and plants; flavins are essential parts of DNA photolyases [Fluorogenic molecules are compounds whose ability to fluoresce can be modulated, for example, by a chemical modification, change in the environment, or electronic structure . A numbetolyases and cryptolyases ; rhodopstolyases .A number of fluorescent probes has been created by mutating natural fluorogen-binding proteins to promote their fluorescence. For example, starting from bacterial light-oxygen-voltage\u2013sensing domains, such an effort yielded flavin-binding fluorescent proteins ; differeIn addition to natural fluorophores, fluorogenic compounds can be synthesized. The existing examples include but are not limited by BODIPY dyes, rhodamines, cyanines, and coumarins . The utiIn silico shortlisting with the further screening of the lipocalin Blc mutants launched DiBs FAPs tags family binding green [de novo ligand-binding \u03b2-barrel protein design using Rosetta followed by two additional rounds of modeling-directed mutagenesis of proteins selected by in vitro screening of a limited number of hits from the previous step yielded two DFHBI-binding proteins designated mFAP1 and mFAP2 [Several FAPs have been designed so far to use unnatural fluorogenic molecules. Screening of libraries of antibodies resulted in the discovery of binders for derivatives of thiazole orange, malachite green , and cyang green and red ng green BODIPY dng green . Finallynd mFAP2 .in silico modelling of more than 100 000 mutants followed by a rigid body docking of a library of green fluorescent protein (GFP) chromophore-like ligands. Nineteen mutants and ten fluorogens were shortlisted to be evaluated experimentally [in silico might be already possible.All FAP modeling projects described above involved screening of libraries of proteins to select the one with the desired properties, which is time and resource consuming. For example, the first-generation lipocalin Blc-based FAPs (DiBs) were obtained after mentally . HoweverHere we tested the performance of one of the leading macromolecules modeling suites, Rosetta , on the Kd [DiB1 protein was selected as a starting point for the protein design project. Among first generation Blc-based FAPs this mutant showed the lowest Kd indicatiKd . For thiKd . The besFig) was performed with gradually decreasing ligand sampling freedom until the solution converged on a single binding pose . First, 5 000 protein-ligand complexes were generated using a coarse protein binding pocket sampling strategy with 5 \u00c5 maximum ligand translation allowed per step and up to 360\u00b0 rotation. The top 500 models by total energy were further sorted based on their protein-ligand interface score and the 50 best were selected for a subsequent docking round. During the next step ligand translation and rotation were restricted to 1 \u00c5 and 45\u00b0, respectively. 100 structures were generated for each of 50 starting models resulting again in a total of 5 000 output structures. 50 best structures were selected as previously and the last round of docking was performed with 0.2 \u00c5 maximum translation and 5\u00b0 maximum rotation for fine-tuning of the ligand placement. The best scoring docking pose was further used for the ligand binding pocket design. In this model the ligand was located within an interaction distance from amino acids at positions 141 and 74, mutations in which were shown to influence the properties of the ligand:protein complex in our previous study [Docking of the M739 chromophore Fig was ing pose Fig. Firus study . While tus study . TherefoFig). The protocol was previously shown to perform better on apolar small molecules whose binding is dominated by van der Waals interactions [In fluorescent proteins, their ability to fluoresce and the brightness of the fluorescence highly depend on the capacity of the surrounding amino acids to stabilize the chromophore in a planar conformation . In the case of M739, the structure is already conformationally locked in the favorable planar conformation. Contrary, its fluorescence quantum yield (QY) was shown to vary dramatically depending on the solvent with an almost tenfold increase in dioxane compared to water . Thus, hractions .From the starting complex structure, we generated 5 000 designs. Amino acids with their C\u03b1 atoms within 6 \u00c5 of any ligand atom or with their C\u03b1 atoms within 8 \u00c5 of any ligand atom and with their C\u03b1-C\u03b2 vector pointing toward the ligand were allowed to be designed to any amino acid except cysteine. The residues with their C\u03b1 atoms within 10 \u00c5 of any ligand atom or with their C\u03b1 atoms within 12 \u00c5 of any ligand atom and their C\u03b1-C\u03b2 vector pointing toward the ligand were allowed to repack. The 2 000 best scoring designs were then sorted based on their ligand-protein interface score and the best 50 were selected for detailed analysis.Fig 1A and 1B). Mutations at positions 74, 108, 109, 116, 139, and 141 were relatively rare with the majority of the generated sequences retaining the native amino acid at those positions. In contrast, amino acids at positions 53, 76, 89, 90, and 107 were mutated in almost all 50 selected models. Four of these positions showed strong convergence toward one specific mutation: Phe at position 76, Tyr at position 89, Val at position 90, and Ala at position 107. Amino acid 53 was mutated to either alanine or glutamine with nearly equal frequency in the models.A total of 16 amino acid positions were found to be mutated at least in one of the selected designed models contained only mutations at these five positions. Four residues were converted to the most favorable amino acids for these positions. Alanine, the second most frequent amino acid in position 53 among 50 best-scored poses, was found in position 53 in that sequence. After additional manual examination, this sequence (designated DiB-RM) was selected for testing.Of note, one of the top-scoring models (in vitro (Table 1). In comparison with DiB1, the DiB-RM:M739 complex, with almost identical fluorescence spectra, demonstrated an increase in both quantum yield (QY) and extinction coefficient (\u025b) resulting in approximately 25% increased brightness. These changes were accompanied by some loss in the apparent binding affinity of the protein to the ligand.We first compared the photophysical properties of DiB-RM in complex with M739 chromophore with the parental protein DiB1 in cellulo . In concordance with the results obtained in vitro, DiB-RM demonstrated increased brightness compared to DiB1: ~ 1.38:1 versus ~ 1.29:1 observed in vitro.We next characterized the DiB-RM protein using transiently transfected living cells. We first generated a fusion protein of DiB-RM with histone H2B and blue fluorescent protein TagBFP, which was compared with a previously created H2B-TagBFP-DiB1 construct . Using TFig 2F and 2G).We next created DiB-RM fusions with various structural proteins and imaged transiently transfected mammalian cells using confocal fluorescence microscopy. The target intracellular structures were brightly fluorescent immediately upon the addition of the fluorogen and did not show any signs of mislocalization or aggregation and tested the behavior of the fusion protein composed of the newly obtained N fragment (designated DiB-RM-splitN1-109) with a fluorescent protein mNeonGreen in transiently transfected HEK cells. Interestingly, we did not observe aggregation as was seen previously for DiB2-split N fragment in a similar experiment. Inspired by this observation we also checked free DiB-RM-split C fragment distribution in cells. There is only one amino acid difference between DiB2 and DiB-RM proteins downstream to the split point (L141N) and it did not affect the performance of the DiB-RM-split C fragment: HEK cells transiently transfected with the DiB-RM-splitC110-177-TagBFP construct showed uniform labeling in the blue fluorescent channel . We then tested the self-assembly capability of the DiB-RM N and C fragments obtained via split between amino acid residues 109 and 110. Transient cotransfection of H2B-DiB-RM-splitN1-109 and TagBFP-DiB-RM-splitC110-177 constructs in living cells revealed successful attraction of TagBFP-DiB-RM-splitC110-177 fusion protein to the nuclei despite lacking a nuclear localization signal and staining nuclei in green upon the chromophore addition as a result of efficient self-assembly of split-DiB-RM.We previously created a self-assembling split system from the first generation of DiB proteins . Despiteregation Fig as w channel Fig. We in vitro (Table 1) using purified protein. As was previously observed for other self-assembling DiB-splits [We also characterized the DiB-RM-split:M739 complex B-splits , splittiS3 Movies). All three tags provided reconstructions of vimentin fibers with better resolution than widefield fluorescence microscopy . DiB1 showed an initial exponential decrease in the number of localizations that later turned into a linear decrease down to ~20% of the initial localization count. The DiB-RM signal decreased only linearly throughout the experiment. That allows for the accumulation of a much higher number of localizations during the same time period using DiB-RM. DiB-RM-split tag performed similarly to DiB1 . DiB-RM also demonstrated higher single-molecule brightness than DiB-RM-split and DiB1 . Both DiB1 and DiB-RM-split provided lower localization precision compared to DiB-RM .The main power of DiB FAPs lies in single-molecule localization microscopy of living cells ,14. We tapo form. The asymmetric unit contains two copies of the protein with the canonical lipocalin fold, a \u03b2-barrel with an \u03b1-helix . The two copies align well (0.77 \u00c5 rmsd across 158 C\u03b1 atoms) with the main difference found in the E/F loop and at the N terminus of the protein .To further characterize DiB-RM we obtained a number of crystal structures. We first successfully crystalized full-length DiB-RM in Fig 4C).Interestingly, during structure refinement, we discovered positive difference density map features in the binding pocket of the molecules. Based on the shape of the density we speculated that they belong to the dodecyl chains of n-Dodecyl-\u03b2-D-maltoside (DDM) present in the crystallization buffer . Unfortunately, we still observed the same long carbon chain-like density in the binding pocket of DiB-RM in these crystals. It might be explained by the higher affinity of DiB-RM to DDM than to M739 or by the requirement of the presence of DDM in the binding pocket of the protein for crystal formation in these conditions.We also obtained colored crystals in similar conditions using soaking or co-crystallization of DiB-RM with M739 Fig. UnfTo test the former hypothesis, we assessed the binding affinity of DDM to DiB-RM using tryptophan fluorescence quenching assay. Surprisingly, we have not detected any spectral changes upon the addition of up to 50x molar access of the DDM to the protein solution in normal buffer conditions. While this experiment cannot fully reproduce the processes and their dynamics that are happening during crystallization, the requirement of the presence of DDM for crystal formation in these conditions seems to be a more likely explanation.apo DiB-RM crystals in DDM-free conditions. This crystal also contains two protein molecules in the asymmetric unit . However, the relative orientation of the molecules differs: we observed approximately 50 degrees rotation . This observation along with the data from \u2019Protein interfaces, surfaces and assemblies\u2019 service PISA [Fig 4E).We later obtained other ice PISA suggestsFig 4F). Unsurprisingly, the crystals remained clear after the addition of the ligand in the drops.In comparison to the first structure, the main difference was observed in the conformation of the E/F loop of the protein. In the new structure, this loop is bent inwards and almost fully closes the entrance to the ligand binding pocket of the protein . Despite the backbone cleavage, the lipocalin fold is well preserved as it was previously observed for other DiB-derived split proteins [Fig 4I).We also obtained DiB-RM-split proteins Fig). The carbon chain-like density in the binding pocket was absent, which further supports our hypothesis that the previously observed density in full-length DiB-RM crystals indeed belongs to DDM and not to some other molecule co-purified with the protein. However, we were not able to locate any density for the M739 ligand either.Multiple colored crystals from DiB-RM-split:M739 mixture were obtained in 1.8 M lithium sulfate and 6% 2-propanol buffer in presence of different additives Fig. Thein vitro (except for the dissociation constant) and as a tag for protein-PAINT [Table 2) of the DiB-RM:M739 complex compared to the parental DiB1:M739 through the better protein:ligand interface packing, in the absence of the ligand these bulky amino acids might partially hinder the entrance of the ligand in the binding pocket.Using a fixed backbone Rosetta Design protocol, we predicted a set of mutations to improve the first generation lipocalin Blc-based FAP DiB1. The resulting protein, designated DiB-RM, performs better than its parental variant both in-PAINT with hig1-109 fragment, and the split protein successfully assembled in living cells without the need of including the additional overlapping \u03b2 strand in the C fragment. Regardless of lower brightness both in vitro and in super-resolution microscopy set up as well as less stable fluorescent signal compared to full-length DiB-RM tag, we speculate that the DiB-RM-split should be a scaffold of choice for further DiB-split system optimizations.Despite no intentional optimization for it, the DiB-RM-based split protein (DiB-RM-split) created analogously to the previously tested DiB2-split system also behapo Blc structures [Fig 5A) and served as an entry point for the vaccenic acid that was previously co-crystallized with this lipocalin [Fig 5B), the chromophore was found much deeper and angled . This became possible due to the rotation of the lipocalin E/F loop. While high flexibility of this loop was previously shown [Fig 5C).Recently, the structure of DiB1 in complex with the chromophore M739 has been solved . To our ructures ,25 Despite our inaccurate initial docking of the chromophore, it is obvious that the incorporated mutations nevertheless improved multiple photophysical parameters as well as the overall performance of the DiB tag. To further investigate this phenomenon, we ran a number of experiments.Figs S7A) confirming that steric clashes with the backbone most likely caused our wrong initial binding mode prediction.First, to test the possibility that using a starting position of the E/F loop incompatible with the binding mode observed in the crystal structure was responsible for the failure of Rosetta docking, we reran the docking using either the DiB1 crystal structure minimized in the absence of ligand, or the same DiB1 model that was generated for the initial docking but with deleted E/F loop (-6 amino acids). In both cases, all 50 top-scored structures had a crystal-like M739 placement . This observation together with our inability to obtain the DiB-RM:M739 co-crystal structure might indicate that we have stabilized an alternative binding site for the ligand by the introduced mutations but have not destroyed the other one. As a result, the ligand in DiB-RM might have multiple possible binding positions. This can also explain the larger apparent dissociation constant of this new complex.We then performed docking using the DiB1-based DiB-RM model generated the same way as the DiB1 model has been prepared before from the Table 2).To further investigate the role of the five introduced mutations we created and analyzed five reverse single mutant variants of DiB-RM , it is not surprising that this phenylalanine has been mutated by Rosetta. This substitution might be also important for the protein\u2019s performance in the crowded cell environment.All -exposed Fig, it Kd accompanies by the bathochromic shift of the fluorescence spectra and a decrease in both QY and \u025b. The side chain of the amino acid in position 76 packs against the ligand . These spectral changes upon introduction of the polar residue next to the ligand align well with the effects of the polarity of the environment on the chromophore M739 properties observed in the free ligand model [Reintroduction of asparagine at position 76 is the only mutation that results in tighter binding to the chromophore. That might be explained by the ability of bulkier phenylalanine to sample side chain conformations that are not compatible with the ligand\u2019s entrance to the binding pocket or its correct placement there. However, the lower e ligand Fig. Thend model .Fig). The observed changes might support the presence of an alternative ligand binding mode in DiB-RM where Y89 packs against the ligand. Alternatively, the introduction of tyrosine at position 89 can induce more complex rearrangements in the structure of the protein. For example, through interactions with the aromatic amino acids-rich flexible E/F loop.Reversing of the other introduced aromatic residue, DiB-RM-Y89S, results in an almost four-fold decrease in binding affinity as well as moderate spectral shifts and decrease in brightness similar to ones observed in DiB-RM-F76N mutant. The amino acid in position 89 is not in direct contact with the ligand, assuming DiB-RM interacts with the ligand similar to what is seen in the DiB1:M739 co-crystal structure Fig. TheFig). The most likely explanation of Rosetta\u2019s favoring of the E90V mutation is the better \u03b2-sheet propensity of valine compared to glutamate [Kd upon reversion of this mutation might indeed indicate its effect on the overall stability of the protein. Even less pronounced in vitro changes caused by A107S substitution located in a relatively flexible region of the protein (at the base of the E/F loop) make it difficult to propose its role, if any.The remaining two variants, DiB-RM-V90E and DiB-RM-A107S, carry substitutions at the positions that are pointing outside of the ligand-binding cavity Fig. Thelutamate . The obsOverall, it appears that the majority of the mutations proposed by Rosetta were beneficial for the DiB-RM performance with the polarity of the residues in the proximity of the ligand influencing the photophysical properties of the FAP the most. Hence, refining of the protein:ligand interface using a conventional RosettaLigand design protocol seems to be a possible option for optimization of FAPs with rigid, apolar ligands. The decrease in the ligand binding affinity might be further avoided in the future by employing a multistate design protocol with simapo form in different conditions further confirmed the already known high flexibility of the E/F lipocalin loop that was not properly addressed during our computational redesign. Rigidifying of this loop through shortening or designed interactions can provide better stabilization of the ligand in the binding pocket. This in turn can allow for expanding the compatible ligand libraries towards more flexible, conformationally unlocked chromophores [Here we explored the power and limitations of Rosetta for the redesign of a protein-ligand complex. Our work resulted in the creation of an improved FAP-based fluorescent tag, however, potentially through stabilization of an alternative ligand binding site. Future optimization of DiB-RM might be focused on disabling one of the two suggested binding sites of the ligand. Crystallization analysis of DiB-RM in the mophores ,30. SuchFive mutations suggested by computational modeling were introduced into the previously described DiB1-pBAD bacterial expression vector by self-The DiB-RM-split vectors for bacterial protein expression were created using plasmids pMRBad-Z-CspGFP (Addgene plasmid #40730), pET11a-Z-NspGFP (Addgene plasmid #40729) , and theThe H2B-TagBFP-DiB-RM plasmid was constructed by self-assembling cloning using DiAll other plasmids for mammalian expression were assembled using Golden Gate cloning following MoClo standard \u201336. EachThe resulted constructs\u2019 amino acid sequences are provided below. The linker sequence is underlined.>vimentin-DiB1DPPVATMASSPTPPRGVTVVNNFDCKRYLGTWYEIARFDHRFERGLEKVTATYSLRDDGGLNVINKGYNPDRGMWQQSEGKAYFTGAPTRAALKVSFFGPFYGGYNVIALDREYRHALVCGPDRDYLWINSRTPTISDEVKQEMLAVATREGFDVSKFIWVQQPGS*MSTRSVSSSSYRRMFGGPGTASRPSSSRSYVTTSTRTYSLGSALRPSTSRSLYASSPGGVYATRSSAVRLRSSVPGVRLLQDSVDFSLADAINTEFKNTRTNEKVELQELNDRFANYIDKVRFLEQQNKILLAELEQLKGQGKSRLGDLYEEEMRELRRQVDQLTNDKARVEVERDNLAEDIMRLREKLQEEMLQREEAENTLQSFRQDVDNASLARLDLERKVESLQEEIAFLKKLHEEEIQELQAQIQEQHVQIDVDVSKPDLTAALRDVRQQYESVAAKNLQEAEEWYKSKFADLSEAANRNNDALRQAKQESTEYRRQVQSLTCEVDALKGTNESLERQMREMEENFAVEAANYQDTIGRLQDEIQNMKEEMARHLREYQDLLNVKMALDIEIATYRKLLEGEESRISLPLPNFSSLNLRETNLDSLPLVDTHSKRTLLIKTVETRDGQVINETSQHHDDLEG>vimentin-DiB-RMDPPVATMASSPTPPRGVTVVNNFDCKRYLGTWYEIARFDHRAERGLEKVTATYSLRDDGGLNVIFKGYNPDRGMWQQYVGKAYFTGAPTRAALKVAFFGPFYGGYNVIALDREYRHALVCGPDRDYLWINSRTPTISDEVKQEMLAVATREGFDVSKFIWVQQPGS*MSTRSVSSSSYRRMFGGPGTASRPSSSRSYVTTSTRTYSLGSALRPSTSRSLYASSPGGVYATRSSAVRLRSSVPGVRLLQDSVDFSLADAINTEFKNTRTNEKVELQELNDRFANYIDKVRFLEQQNKILLAELEQLKGQGKSRLGDLYEEEMRELRRQVDQLTNDKARVEVERDNLAEDIMRLREKLQEEMLQREEAENTLQSFRQDVDNASLARLDLERKVESLQEEIAFLKKLHEEEIQELQAQIQEQHVQIDVDVSKPDLTAALRDVRQQYESVAAKNLQEAEEWYKSKFADLSEAANRNNDALRQAKQESTEYRRQVQSLTCEVDALKGTNESLERQMREMEENFAVEAANYQDTIGRLQDEIQNMKEEMARHLREYQDLLNVKMALDIEIATYRKLLEGEESRISLPLPNFSSLNLRETNLDSLPLVDTHSKRTLLIKTVETRDGQVINETSQHHDDLEG>vimentin-DiB-RM-split1-109DPPVATMASSPTPPRGVTVVNNFDCKRYLGTWYEIARFDHRAERGLEKVTATYSLRDDGGLNVIFKGYNPDRGMWQQYVGKAYFTGAPTRAALKVAFFS*MSTRSVSSSSYRRMFGGPGTASRPSSSRSYVTTSTRTYSLGSALRPSTSRSLYASSPGGVYATRSSAVRLRSSVPGVRLLQDSVDFSLADAINTEFKNTRTNEKVELQELNDRFANYIDKVRFLEQQNKILLAELEQLKGQGKSRLGDLYEEEMRELRRQVDQLTNDKARVEVERDNLAEDIMRLREKLQEEMLQREEAENTLQSFRQDVDNASLARLDLERKVESLQEEIAFLKKLHEEEIQELQAQIQEQHVQIDVDVSKPDLTAALRDVRQQYESVAAKNLQEAEEWYKSKFADLSEAANRNNDALRQAKQESTEYRRQVQSLTCEVDALKGTNESLERQMREMEENFAVEAANYQDTIGRLQDEIQNMKEEMARHLREYQDLLNVKMALDIEIATYRKLLEGEESRISLPLPNFSSLNLRETNLDSLPLVDTHSKRTLLIKTVETRDGQVINETSQHHDDLEG>DiB-RM-split110-177-TagBFPGDPPVATMSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKLVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCDLPSKLGHKLN*MGPFYGGYNVIALDREYRHALVCGPDRDYLWINSRTPTISDEVKQEMLAVATREGFDVSKFIWVQQPGS>mNeonGreen-DiB-RM-split1-109DPPVATMASSPTPPRGVTVVNNFDCKRYLGTWYEIARFDHRAERGLEKVTATYSLRDDGGLNVIFKGYNPDRGMWQQYVGKAYFTGAPTRAALKVAFFS*MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYEELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIISTFKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQKAFTDVMGMDELYKThe correctness of all obtained constructs was confirmed by sequencing.E. coli strain. Cells were grown in LB media or M9 minimal media supplemented with 100 \u03bcg/mL ampicillin (full-length DiB proteins) or 100 \u03bcg/mL ampicillin and 50 \u03bcg/mL kanamycin (split protein) at 37\u00b0C. Expression was induced by addition 0.04% L-arabinose (full-length DiB proteins) or 0.2% L-arabinose and 10 \u03bcM IPTG (split protein) at 0.8 OD. Cells were harvested after 3 hours of expression in LB or after overnight expression in minimal media at 37\u00b0C and were resuspended in PBS buffer, pH 7.4. Suspensions were frozen at -80\u00b0C and thawed at room temperature three times. DNA was destroyed by short sonication and the lysates were centrifuged to obtain cell-free extracts.All lipocalin proteins were expressed in XJb(DE3) Autolysis (Zymo Research) E. coli strain. Cells were grown in LB media supplemented with 100 \u03bcg/mL ampicillin at 37\u00b0C. Expression was induced by the addition of 500 \u03bcM IPTG at 0.8 OD. Cells were harvested after overnight expression at 18\u00b0C. Before purification cells were resuspended in PBS buffer, pH 7.4, and sonicated on ice. The lysates were centrifuged to obtain cell-free extracts.Fluorescent protein Venus was expressed in BL21(DE3) The proteins were first purified using gravity flow columns with TALON metal affinity resin (Clontech) and further purified by size-exclusion chromatography on a HiLoad 16/600 Superdex 75 pg column pre-equilibrated with 50 mM sodium phosphate buffer, pH 6.0.Protein concentrations were estimated using the Bradford dye-binding method-based colorimeKd). For each protein, the measurements were performed using at least two independent protein purifications and at least three technical replicates for each protein sample.Titrations were performed and analyzed as previously described using FlHoriba Jobin Yvon Fluoromax-3 fluorometer was used to detect full fluorescence excitation and fluorescence emission spectra for excitation/emission maxima evaluation.apo form and in the presence of ~0.5\u20133.0 \u03bcM of the M739 chromophore. FAP concentrations for experiments were chosen individually for each protein based on the previously calculated Kd values to ensure that at least 95% of the added chromophore is bound to the protein . Spectra of the corresponding apo FAP solutions were subtracted from the absorption spectra of the protein-fluorogen complexes. The absorption at 510 nm values from these corrected spectra was also plotted against the area under the corresponding fluorescence emission curves and the linear approximation of the correlation has been calculated. The QYs were then calculated as a ratio of the slopes of the protein of interest and standard curves multiplied by standard\u2019s QY. For each protein, the measurements were performed using protein aliquots from at least two independent protein purifications. Reported is a mean value.Fluorescence quantum yield (QY) was measured relative to the fluorescent protein Venus . First, FAP\u03b5 is the FAP-fluorogen complex extinction coefficient, FAPA\u2013the FAP-fluorogen complex absorption at maximum, AM739\u2013free chromophore absorption at the FAP-fluorogen complex absorption maximum, cM739\u2013total added chromophore concentration, and \u03b1\u2013a fraction of added chromophore that is bound to the protein calculated based on the previously determined FAP-fluorogen complex Kds. Reported is a mean value \u00b1 s.d. of measurements obtained for at least two independently expressed and purified protein samples using at least five data points per sample.Absorption spectra collected for QY calculations were also used for excitation coefficients calculations. For each of the FAP-fluorogen complexes, the complex absorption maximum has been determined. Free M739 chromophore spectra were used to define free chromophore contribution to absorption at the given wavelength and to calculated chromophore concentrations. The FAP-fluorogen complexes\u2019 extinction coefficients were calculated using the following equation:apo DiB-RM was crystalized at 21\u00b0C in 2 M ammonium sulphate, 2.5% 2-propanol supplemented with 5% w/v n-Dodecyl-b-D-maltoside according to the Hampton Research Additive Screen protocol using hanging drop vapor diffusion technique. Crystals grew within 1\u20133 days.Full-length apo DiB-RM were also obtained at 21\u00b0C in 1.5 M lithium sulfate, 0.1 M tris hydrochloride pH 7.0, and 5% 2-propanol, supplemented with 200 mM cesium chloride using hanging drop vapor diffusion technique. For this 2 \u03bcL of protein solution were mixed with 1.2 \u03bcL of crystallization buffer and 0.8 \u03bcL of 1 M cesium chloride from Hampton Research Additive Screen. Crystals grew within 2\u20133 weeks.Crystals of different morphology of full-length apo crystals were obtained at 21\u00b0C in 1.6 M ammonium sulfate, 0.1 M MES, pH 4.5 supplemented with 5% w/v n-Dodecyl-b-D-maltoside according to the Hampton Research Additive Screen protocol using hanging drop vapor diffusion technique. Crystals grew within 1 week.DiB-RM-split All crystals were flash-frozen in liquid nitrogen using Parabar 10312 oil as cryoprotectant.Table. The models have been deposited into the Protein Data Bank . Structure figures were prepared using PyMol .Diffraction data were collected at the Life Sciences Collaborative Access Team beamline 21-ID-G or 21-ID-F at the Advanced Photon Source, Argonne National Laboratory. The diffraction data were processed using the xia2 software suite . The cry2. For transient transfections, FuGENE HD reagent (Promega) was used. Immediately before imaging DMEM was replaced with HHBS media (Hanks Buffer (PanEco) supplemented with 20 mM HEPES (Sigma)).HEK293 and HeLa Kyoto cells were grown in Dulbecco\u2019s modification of Eagle\u2019s medium (DMEM) (PanEco) supplied with 50 U/ml penicillin and 50 \u03bcg/ml streptomycin (PanEco), 2 mM L-glutamine (PanEco), and 10% fetal bovine serum at 37\u00b0C and 5% COWidefield fluorescence microscopy was performed with the Leica DMI6000B inverted microscope equipped with HC PL Apo 100x NA 1.40 oil lens and HC PL Apo 40x NA 0.85 lens, CoolLED pE-300 light source, Zyla 5.5 sCMOS camera (Andor), using GFP and BFP filter sets.Confocal imaging was performed using an inverted Leica confocal microscope DMIRE2 TCS SP2 equipped with HCX PL APO lbd.BL 63.0x NA 1.40 OIL objective, excitation by 488 nm laser line (100 \u03bcW).-2 of 488 nm laser light, 33 ms frame exposure time, for 10,000 frames.Single-molecule localization super-resolution imaging of living cells was performed with Nanoimager S microscope at 37\u00b0C. The microscope was equipped with Olympus UPlanSApo 100x NA 1.40 oil immersion objective. Imaging experiments performed with 1.1 kW cmAll Rosetta runs were performed with weekly release 2015.12.57698.The A36C and L141N mutations were manually introduced into the crystal structure (PDB ID 1QWD). The structure was further minimized using Rosetta Relax application with the-flip_HNQ-no_optH false-relax:constrain_relax_to_start_coords-relax:ramp_constraints false-nstruct 50-ex1-ex2-use_input_scThe M739 chromophore geometry was optimized by the density functional method RB3LYP, using the 6\u2013311+G** basis set, a restricted hybrid HF-DFT SCF calculation was performed using Pulay DIIS + Geometric Direct Minimization to get a set of ideal bond lengths and angles. The conformers library for the ligand was further generated using BCL::Conf conformer generator .Docking of the M739 chromophore was performed in three steps. First, 5 000 structures were generated using 5 \u00c5 maximum ligand translation allowed per step and up to 360\u00b0 rotation. 50 structures were selected for the next docking round during which ligand translation and rotation were restricted to 1 \u00c5 and 45\u00b0 correspondingly. 100 structures were generated for each of the 50 starting models. Analogously 50 best structures from the second docking step were selected and the last round of docking was performed with 0.2 \u00c5 maximum translation and 5\u00b0 maximum rotation.The following RosettaScripts protocol has been used with the appropriate changes introduced to the Transform Mover during the second and the third docking rounds:\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0The following RosettaScripts protocol has been used to for DiB1 redesign:\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0S1 Table(XLSX)Click here for additional data file.S1 Fig(TIF)Click here for additional data file.S2 FigC).Each datapoint corresponds to one of 5 000 models generated for each round of the docking. Some outliers with high total and/or interface scores are not shown for better visibility of the majority of the data. Insets show overlays of the ligand positions in the 50 best models of the corresponding round of docking . After the third round, docking converged on a single binding pose solution ((TIF)Click here for additional data file.S3 FigThe protocol starts with a small ligand position perturbation followed by optimization of the protein:ligand interface. Amino acids are allowed to change their identity only if it results in a significant decrease in energy, defined through a \u201cfavor native\u201d bonus (1 REU in the used protocol). The sequence design step is followed by reevaluation of the ligand position using high resolution docking protocol. The final model is scored and saved for further analysis.(TIF)Click here for additional data file.S4 FigRepresented are data for 35\u201340 cells from the same field of view across the whole ligand concentration range. Box whiskers indicate standard deviation, horizontal lines within boxes indicate median values, circles indicate outliers.(TIF)Click here for additional data file.S5 Fig1-109 or (B) DiB-RM-split110-177-TagBFP constructs. The signal from green and blue channels, correspondingly. Scale bars are 25 \u03bcm.Widefield fluorescence images of HEK293 cells transiently transfected with (A) NeonGreen-DiB-RM-splitN(TIF)Click here for additional data file.S6 Fig(TIF)Click here for additional data file.S7 FigA) using the same DiB1 model that was generated for the initial docking but with deleted E/F loop (-6 amino acids) or (B) using the DiB1 crystal structure-based DiB-RM model starting from the initial (\u201cold\u201d) starting position of the ligand. The M739 chromophore from the co-crystal structure is shown as yellow sticks. All docked chromophores are shown as green lines.Rerun of the chromophore M739 docking ((TIF)Click here for additional data file.S8 Fig(TIF)Click here for additional data file.S1 MoviePlays at 30 fps (acquisition speed 30 Hz).(MP4)Click here for additional data file.S2 MoviePlays at 30 fps (acquisition speed 30 Hz).(MP4)Click here for additional data file.S3 MoviePlays at 30 fps (acquisition speed 30 Hz).(MP4)Click here for additional data file."} +{"text": "Since December 2019, a novel coronavirus responsible for a severe acute respiratory syndrome (SARS-CoV-2) is accountable for a major pandemic situation. The emergence of the B.1.1.7 strain, as a highly transmissible variant has accelerated the world-wide interest in tracking SARS-CoV-2 variants\u2019 occurrence. Similarly, other extremely infectious variants, were described and further others are expected to be discovered due to the long period of time on which the pandemic situation is lasting. All described SARS-CoV-2 variants present several mutations within the gene encoding the Spike protein, involved in host receptor recognition and entry into the cell. Hence, instead of sequencing the whole viral genome for variants\u2019 tracking, herein we propose to focus on the SPIKE region to increase the number of candidate samples to screen at once; an essential aspect to accelerate diagnostics, but also variants\u2019 emergence/progression surveillance. This proof of concept study accomplishes both at once, population-scale diagnostics and variants' tracking. This strategy relies on (1) the use of the portable MinION DNA sequencer; (2) a DNA barcoding and a SPIKE gene-centered variant\u2019s tracking, increasing the number of candidates per assay; and (3) a real-time diagnostics and variant\u2019s tracking monitoring thanks to our software RETIVAD. This strategy represents an optimal solution for addressing the current needs on SARS-CoV-2 progression surveillance, notably due to its affordable implementation, allowing its implantation even in remote places over the world. Early on 2020 major viral diagnostic efforts were deployed, including the development of news strategies aiming at (1) reducing the diagnostics time, (2) decrease the costs per assay, (3) providing population-scale solutions, but also (4) presenting a high sensitivity and specificity, notably to discriminate asymptomatic cases. While costs and required time per assay were early on addressed by the development of immune-based tests5. These efforts rely on (1) the incorporation of DNA molecular barcodes during sample preparation; (2) pooling barcoded samples for an \u201cin-one-run\u201d diagnostics assay by the use of massive parallel DNA sequencing; (3) stratification of diagnostics\u2019 outcome with the help of bioinformatics processing. While such strategies demonstrated to up-scale the diagnostics power beyond several thousand candidates per assay, they require to count with a DNA sequencing platform where large instruments (Illumina Sequencers) are driven by specialized technical engineers. As consequence, this strategy remains applicable to highly developed countries (due to the prohibitive costs of the required DNA sequencers), which in addition requires a dedicated pipeline strategy for transporting collected samples towards sequencing centers.With the aim of taking advantage of the viral nucleic acid targeting diagnostics but at the same time increasing the number of candidates\u2019 through-put, strategies based on the use of massive parallel DNA sequencing for population-scale diagnostics were proposed6.Beyond the diagnostics requirements, the identification of the highly transmissible SARS-CoV-2 variant B.1.1.7\u2014originated in the south of England\u2014has strongly accelerated the worldwide interest in tracking SARS-CoV-2 variants\u2019 occurrence. More recently, two other extremely infectious variants, namely the P.1 issued on the Brazilian city of Manaus and the B.1.351 initially detected In South Africa, were described, and unsurprisingly further others are expected to be discovered, notably due to the long period of time on which the pandemic situation is lasting7). Indeed, this technology is widely used for SARS-CoV-2 whole-genome sequencing, notably by the use of the amplicon sequencing protocol developed by the ARTIC Network (https://artic.network/ncov-2019)8. Furthermore, a Covid-19 diagnostics strategy combining Loop-mediated isothermal Amplification (LAMP) with Nanopore sequencing has been also described9.Herein, we describe a proof of concept study for population-scale diagnostics and variants\u2019 tracking using massive parallel DNA sequencing performed with the MinION Oxford Nanopore Technology (ONT). ONT MinION sequencers are well-known for their portability, their reduced cost, and their simplicity of use; thus, representing a suitable strategy for its implementation even in remote places over the world providing the outcome of the diagnostic in a real-time mode during sequencing; allowing to earn time for diagnostics report, essential in the current context of the pandemic situation. Finally, we have also demonstrated that our strategy is able to perform SARS-CoV-2 variants\u2019 detection across multiple candidates, notably by targeting the SPIKE gene instead of covering the whole viral genome Fig.\u00a0. This ta10.In summary, the proposed strategy satisfies the current need for counting with a population diagnostic assay, and discriminates between the various SARS-CoV-2 variants, which are recognized to potentially present variable infection properties as well as clinical outcomesWith the aim of performing SARS-CoV-2 diagnostics over multiple candidates with the MinION sequencer, we combined a regular reverse-transcription assay, with a particular PCR amplification allowing the generation of long DNA concatemers. This strategy allows to target short amplicons, like those used on quantitative real-time PCR strategies, but at the same time take advantage of the long fragments sequencing capabilities of the Oxford Nanopore technology.Reverse transcription (RT) is performed with a Covid-19 specific primer, presenting in addition, a unique molecular barcode (20nt length) and a common sequence adapter and a reverse-oriented sequence for the common Gibson sequence adapter (30nt length) is used together with a Gibson primer . Hence, we have developed a computational pipeline dedicated to (1) collect available fastq files; (2) retrieve Gibson adapter sequences within sequenced molecules; 3) split them into monomers; (4) evaluate the presence of cDNA & PCR molecular barcodes in their flanking regions; (5) align the inner monomer sequences towards the SARS-CoV-2 reference genome; and (6) display the aligned read counts per cDNA/PCR barcode combinations. This pipeline is setup to be reinitiated in an automated time interval (e.g. every 10\u00a0min) such that newly available sequences can be cumulated to the previous analyses, thus providing a real-time view of aligned read counts associated with cDNA/PCR barcode combinations were used for mimicking variable viral RNA copies per assay. Specifically, subsequent dilutions of the synthetic Wuhan-Hu-1 (GeneBankID: MN908947.3) SARS-CoV-2 RNA control sample has been used for reverse transcription, followed by a quantitative PCR assay targeting a region on the E gene of SARS-Cov-2, defined by primer sequences described previously (E-Sarbeco)For Nanopore DNA sequencing-based diagnostic, as described on Fig.\u00a0Subsequent dilutions of the synthetic SARS-CoV-2 RNA control sample were used for reverse transcription, performed with defined barcoded E-Sarbeco primers , in addition to a negative control sample devoid of viral RNA material (BC37) we can afford to combine diagnostic and variants\u2019 tracking into a single assay, which per se represents a major progress relative to the current strategies.In contrast to the aforementioned diagnostics effort targeting a short amplicon (E-Sarbeco), variants\u2019 tracking over the whole SPIKE gene uses a random-hexamer sequence for the reverse transcription step, and four primers targeting the SPIKE gene at different regions (~\u20091\u00a0kb distance among each primer) Fig.\u00a0A. Like i12 , human RNA\u2014issued from cell lines in culture\u2014has been combined with the synthetic viral RNA samples, such that the random-hexamer primers used for the reverse transcription assay could also provide means to count with a positive control for sample collection. Furthermore, for addressing variants tracking performance, in addition to the Wuhan-Hu-1 SARS-CoV-2 RNA control sample, we have used synthetic viral RNA samples corresponding to the highly transmissible SARS-CoV-2 variant B.1.1.7, first described in the UK in December 2020, as well as the strain HF2393 described as a particular clade circulating in France since January 202012 Fig.\u00a0C.To perform diagnostics and variants\u2019 tracking assay over multiple pseudo-candidates, we have used eight barcoded random-hexamer primers for reverse transcription BC37-BC44) in presence of different viral RNA titers (consecutive dilutions) and issued from the aforementioned synthetic viral RNA strains revealed a single common mutation, D614G, previously described within the French strain HF23936. As consequence, counting with methodologies for Covid-19 diagnostics but also variants\u2019 tracking on a population scale remains essential.Despite the current vaccination efforts, the pandemic situation anticipates the propagation of the recently described variants, as well as the emergence of new others, notably due to the major discrepancies between countries to access to optimal vaccination programsCurrently, diagnostics and variants\u2019 tracking are performed in two steps, which per se delays the time for obtaining a complete diagnostic. Furthermore, variants\u2019 tracking relies on full genome sequencing of SARS-CoV-2, which represent an expensive and labor-intensive effort, incompatible with population-scale assays. Finally, most of the variants\u2019 tracking efforts, relying on the use of large and expensive sequencing instruments, requires to centralize samples processing to dedicated institutions, which are rather absent on poor countries.To address all these pitfalls, we propose herein a methodology able to (1) perform SARS-CoV-2 diagnostic and variants\u2019 tracking at the same time; (2) shorten the time for accessing to the outcome of the diagnostic/variants\u2019 detection by the use of a real-time tracking system; (3) take advantage of the compact and affordable Oxford Nanopore MinION DNA sequencing instrument, notably for deploying the aforementioned diagnostics and variants\u2019 tracking platform anywhere over the world.Within this study we have validated the proposed Nanopore DNA sequencing-based diagnostic, by targeting a region on the E gene of SARS-Cov-2 (E-Sarbeco) on a total of 64 pseudo-candidate samples Fig.\u00a0. Importa13. Illumina instruments being a short fragments sequencing approach, HiSpike requires 42 primer pairs generating amplicons of 400 nucleotides of length for covering the SPIKE region. In contrary, our proposed strategy, based on the use of long DNA fragments, compatible with the Nanopore DNA sequencing technology, relies on 4 primers targeting the SPIKE region, one random primer for the cDNA step and a common Gibson sequence. In fact, a strategy for SARS-CoV-2 diagnostic and variants\u2019 surveillance, dedicated to be deployed anywhere on the world does not only requires to count with low cost instruments, but also involve a reduced number of reagents for allowing its use on a long-standing manner.The proposed variants\u2019 tracking focus on the analysis of the SPIKE gene instead of the whole SARS-CoV-2 genome. In fact, the SPIKE gene has shown to concentrate the majority of the currently discovered variants, which can be rationalized by the fact that it codes for the protein required for the interaction with the human cells. Noteworthy, most of the current vaccines against the Covid-19 are also based on the SPIKE region. Hence, concentrating variants\u2019 tracking towards this gene appears an optimal compromise to increase the number of candidates to screen at once. This philosophy is shared by a recent work describing the HiSpike method, performing variants\u2019 detection within the SPIKE gene with the help of the small Illumina MiSeq instrument14. In all cases, new mutations on the gene SPIKE were observed, further supporting our strategy for targeting this genomic region for variants surveillance.During the time of the final revision of this article, new variants derived from the lineage of B.1.1.7 has gained major attention notably due to the fact that they are vastly spreading in the UK, USA, and other locations For all these reasons, we are convinced that our proposed strategy, accompanied by the dedicated computational solution RETIVAD, can represent a game-changer approach for tracing variants\u2019 progression in the following months.Wuhan-Hu-1 (GeneBankID: MN908947.3); Twist Biosc. ID: Ctrl2.France/HF2393/2020 (EPI_ISL_418227); Twist Biosc. ID: Ctrl7.England/205,041,766/2020 (EPI_ISL_710528); Twist Biosc. ID: Ctrl14 (B.1.1.7).The following Synthetic SARS-CoV-2 RNA control samples, delivered at a concentration of 1 million copies per microliter has been used in this study:11, has been used:Primers used for reverse transcription are composed by (1) a fragment of the Gibson sequenced (redGibs: GAAGAACCTGTAGATAACTCGCTGT), (2) a barcode sequence issued from the ONT PCR Barcoding Expansion 1\u201396 kit (EXP-PBC096) and (3) the target sequence. For targeting the E-Sarbeco region within the SARS-CoV-2 genome, the reverse primer sequence provided by Corman et al.E_Sarbeco_R: ATATTGCAGCAGTACGCACACAFor SPIKE-centered variants tracing, the target sequence has been replaced by a random hexamer, notably to decrease the number of required primers during the assay.The following barcoded primers targeting the E-Sarbeco region were used for reverse transcription:redGibsBC33_E_Sarbeco_R: GAAGAACCTGTAGATAACTCGCTGTCAGACTTGGTACGGTTGGGTAACTATATTGCAGCAGTACGCACACAredGibsBC34_E_Sarbeco_R: GAAGAACCTGTAGATAACTCGCTGTGGACGAAGAACTCAAGTCAAAGGCATATTGCAGCAGTACGCACACAredGibsBC35_E_Sarbeco_R: GAAGAACCTGTAGATAACTCGCTGTCTACTTACGAAGCTGAGGGACTGCATATTGCAGCAGTACGCACACAredGibsBC36_E_Sarbeco_R: GAAGAACCTGTAGATAACTCGCTGTATGTCCCAGTTAGAGGAGGAAACAATATTGCAGCAGTACGCACACAredGibsBC37_E_Sarbeco_R: GAAGAACCTGTAGATAACTCGCTGTGCTTGCGATTGATGCTTAGTATCAATATTGCAGCAGTACGCACACAredGibsBC38_E_Sarbeco_R: GAAGAACCTGTAGATAACTCGCTGTACCACAGGAGGACGATACAGAGAAATATTGCAGCAGTACGCACACAredGibsBC39_E_Sarbeco_R: GAAGAACCTGTAGATAACTCGCTGTCCACAGTGTCAACTAGAGCCTCTCATATTGCAGCAGTACGCACACAredGibsBC40_E_Sarbeco_R: GAAGAACCTGTAGATAACTCGCTGTTAGTTTGGATGACCAAGGATAGCCATATTGCAGCAGTACGCACACAThe following barcoded random-hexamer primers were used for reverse transcription:redGibsBC37_Rhexamer_R: GAAGAACCTGTAGATAACTCGCTGTGCTTGCGATTGATGCTTAGTATCANNNNNNredGibsBC38_Rhexamer_R: GAAGAACCTGTAGATAACTCGCTGTACCACAGGAGGACGATACAGAGAANNNNNNredGibsBC39_Rhexamer_R: GAAGAACCTGTAGATAACTCGCTGTCCACAGTGTCAACTAGAGCCTCTCNNNNNNredGibsBC40_Rhexamer_R: GAAGAACCTGTAGATAACTCGCTGTTAGTTTGGATGACCAAGGATAGCCNNNNNNredGibsBC41_Rhexamer_R: GAAGAACCTGTAGATAACTCGCTGTGGAGTTCGTCCAGAGAAGTACACGNNNNNNredGibsBC42_Rhexamer_R: GAAGAACCTGTAGATAACTCGCTGTCTACGTGTAAGGCATACCTGCCAGNNNNNNredGibsBC43_Rhexamer_R: GAAGAACCTGTAGATAACTCGCTGTCTTTCGTTGTTGACTCGACGGTAGNNNNNNredGibsBC44_Rhexamer_R: GAAGAACCTGTAGATAACTCGCTGTAGTAGAAAGGGTTCCTTCCCACTCNNNNNN11, has been used:For PCR amplification a second set of barcoded primers were designed presenting the following structure: (1) A reverse-complementary Gibson fragment (revGibs: ACAGCGAGTTATCTACAGGTTCTTCAATGT), (2) a barcode sequence issued from the ONT PCR Barcoding Expansion 1\u201396 kit (EXP-PBC096), and (3) the target sequence. For targeting the E-Sarbeco region within the SARS-CoV-2 genome, the forward primer sequence provided by Corman et al.E_Sarbeco_F: ACAGGTACGTTAATAGTTAATAGCGTThe following barcoded primers targeting the E-Sarbeco region were used in this study:revGibs-_BC01_E_Sarbeco_F: ACAGCGAGTTATCTACAGGTTCTTCAATGTAAGAAAGTTGTCGGTGTCTTTGTGACAGGTACGTTAATAGTTAATAGCGTrevGibs-_BC02_E_Sarbeco_F: ACAGCGAGTTATCTACAGGTTCTTCAATGTTCGATTCCGTTTGTAGTCGTCTGTACAGGTACGTTAATAGTTAATAGCGTrevGibs-_BC03_E_Sarbeco_F: ACAGCGAGTTATCTACAGGTTCTTCAATGTGAGTCTTGTGTCCCAGTTACCAGGACAGGTACGTTAATAGTTAATAGCGTrevGibs-_BC04_E_Sarbeco_F: ACAGCGAGTTATCTACAGGTTCTTCAATGTTTCGGATTCTATCGTGTTTCCCTAACAGGTACGTTAATAGTTAATAGCGTrevGibs-_BC05_E_Sarbeco_F: ACAGCGAGTTATCTACAGGTTCTTCAATGTCTTGTCCAGGGTTTGTGTAACCTTACAGGTACGTTAATAGTTAATAGCGTrevGibs-_BC06_E_Sarbeco_F: ACAGCGAGTTATCTACAGGTTCTTCAATGTTTCTCGCAAAGGCAGAAAGTAGTCACAGGTACGTTAATAGTTAATAGCGTrevGibs-_BC07_E_Sarbeco_F: ACAGCGAGTTATCTACAGGTTCTTCAATGTGTGTTACCGTGGGAATGAATCCTTACAGGTACGTTAATAGTTAATAGCGTrevGibs-_BC08_E_Sarbeco_F: ACAGCGAGTTATCTACAGGTTCTTCAATGTTTCAGGGAACAAACCAAGTTACGTACAGGTACGTTAATAGTTAATAGCGTFor SPIKE-centered variants tracing, the reverse-complementary Gibson sequence has been excluded, notably because the PCR amplification performed over random-hexamer generated cDNA templates generate large fragments (>\u20091\u00a0kb). In contrary, the amplicon product issued from E-Sarbeco targeting oligonucleotides gives rise to short amplicons\u2014compatible with quantitative PCR assays, thus requiring concatemerization prior nanopore sequencing.With the aim of covering the SPIKE gene (~\u20093.8\u00a0kb), the following four primers targeting the SPIKE region with\u2009~\u20091\u00a0kb interval within them were designed:Spike1_F: AGGGGTACTGCTGTTATGTCTSpike2_F: TGCACTTGACCCTCTCTCAGSpike3_F: GCAGGCTGTTTAATAGGGGCSpike4_F: TGCAGACATATGTGACTCAACAThe following Spike targeting primers including the corresponding barcode sequences were used for this study:BC01_Spike_1_F: AAGAAAGTTGTCGGTGTCTTTGTGAGGGGTACTGCTGTTATGTCTBC02_Spike_1_F: TCGATTCCGTTTGTAGTCGTCTGTAGGGGTACTGCTGTTATGTCTBC03_Spike_1_F: GAGTCTTGTGTCCCAGTTACCAGGAGGGGTACTGCTGTTATGTCTBC04_Spike_1_F: TTCGGATTCTATCGTGTTTCCCTAAGGGGTACTGCTGTTATGTCTBC01_Spike_2_F: AAGAAAGTTGTCGGTGTCTTTGTGTGCACTTGACCCTCTCTCAGBC02_Spike_2_F: TCGATTCCGTTTGTAGTCGTCTGTTGCACTTGACCCTCTCTCAGBC03_Spike_2_F: GAGTCTTGTGTCCCAGTTACCAGGTGCACTTGACCCTCTCTCAGBC04_Spike_2_F: TTCGGATTCTATCGTGTTTCCCTATGCACTTGACCCTCTCTCAGBC01_Spike_3_F: AAGAAAGTTGTCGGTGTCTTTGTGGCAGGCTGTTTAATAGGGGCBC02_Spike_3_F: TCGATTCCGTTTGTAGTCGTCTGTGCAGGCTGTTTAATAGGGGCBC03_Spike_3_F: GAGTCTTGTGTCCCAGTTACCAGGGCAGGCTGTTTAATAGGGGCBC04_Spike_3_F: TTCGGATTCTATCGTGTTTCCCTAGCAGGCTGTTTAATAGGGGCBC01_Spike_4_F: AAGAAAGTTGTCGGTGTCTTTGTGTGCAGACATATGTGACTCAACABC02_Spike_4_F: TCGATTCCGTTTGTAGTCGTCTGTTGCAGACATATGTGACTCAACABC03_Spike_4_F: GAGTCTTGTGTCCCAGTTACCAGGTGCAGACATATGTGACTCAACABC04_Spike_4_F: TTCGGATTCTATCGTGTTTCCCTATGCAGACATATGTGACTCAACAReverse transcription assays were performed using SuperScript\u2122 IV Reverse Transcriptase ) with a standard reaction volume of 20ul including 0.5ul of super RNAseIn , 1ul DTT (0.1\u00a0M), 4 ul SSIV buffer (5X), 0.5 ul dNTP (10\u00a0mM each), 1 ul of synthetic SARS-CoV-2 RNA, 1 ul of 2uM gene-specific-barcoded oligonucleotide or 1 ul of 50uM random hexamer-barcoded oligonucleotide . For assays including random hexamer-barcoded primers, 1ul of human RNA were added to the assay together to the synthetic SARS-CoV-2 RNA and prior adding the random hexamers.Synthetic SARS-CoV-2 RNA was added at a copy number of 1 million , 100 thousand (d.f: 0.1), 10 thousand (d.f: 0.01) and 1 thousand (d.f: 0.001) per reaction respectively. Note that these consecutive dilutions give rise after downstream dilutions to the number of copies of 10 thousand, 1 thousand, 100 and 10 respectively, analyzed by quantitative PCR Fig.\u00a0B.Multiple reverse transcription assays\u2014each of them performed in presence of a defined barcoded primer\u2014were incubated at 50\u00a0\u00b0C during 30\u00a0min, then at 80\u00a0\u00b0C 10\u00a0min. Barcoded cDNA samples were pooled (8 samples at once) and cleaned with SPRIselect reagent with a ratio of 0.5x. Cleaned cDNA is recovered on 80ul . 40ul of this pooled & cleaned cDNA will be used for PCR amplification , while the remaining material is used for quantitative PCR evaluation.Barcoded-cDNA material has been validated by quantitative PCR assay with the following primer sequences:Primers targeting region on E gene of SARS-Cov-2 :Human_RNASE_P-F: AGATTTGGACCTGCGAGCGHuman_RNASE_P-R: GAGCGGCTGTCTCCACAAGTQuantitative PCR assays were performed with QuantiTect SYBR Green PCR Kit over a 1/10 diluted cDNA material.PCR amplification targeting the E-Sarbeco amplicon is performed on 25ul final volume including 12.5ul of Phusion Hot Start II High-Fidelity PCR Master Mix (ThermoFisher ref: F565), 2.5ul of 1uM barcoded-E-Sarbeco targeting primer (BC1-8), 2.5ul of 1uM short Gibson primer (AGAACCTGTAGATAACTCGCTGT) and 5ul of the pooled & cleaned cDNA.98\u00a0\u00b0C 30\u00a0s98\u00a0\u00b0C 10\u00a0s61\u00a0\u00b0C 20\u00a0s72\u00a0\u00b0C 30\u00a0sRepeat 2\u20134 steps 25\u00d798\u00a0\u00b0C 10\u00a0s61\u00a0\u00b0C 20\u00a0s72\u00a0\u00b0C 2\u00a0minRepeat 6\u20138 steps 20\u00d772\u00a0\u00b0C 10\u00a0minKeep at 12\u00a0\u00b0CFor short-amplicon E-Sarbeco PCR amplification the following thermal cycling program is used:For PCR amplification targeting SPIKE and E-Sarbeco on random-hexamers generated cDNA material the assay is performed with a pool of barcoded SPIKE primers and shortGibson. For 100ul mix 10ul of barcoded primer Spike1 (10uM), 10ul of Spike2 (10uM), 10ul of Spike (10uM), 10ul of Spike4 (10uM) and 40ul of ShortGibson (10uM) completed with distilled water is prepared. Then the PCR amplification is performed on 25ul final volume including 12.5ul of Phusion Hot Start II High-Fidelity PCR Master Mix (ThermoFisher ref: F565), 1.25ul of pooled barcoded-SPIKE & shortGibson primers and 5ul of pooled & cleaned cDNA.98\u00a0\u00b0C 30\u00a0s98\u00a0\u00b0C 10\u00a0s61\u00a0\u00b0C 45\u00a0s72\u00a0\u00b0C 2\u00a0minRepeat 2\u20134 steps 39\u00d772\u00a0\u00b0C 10\u00a0minKeep at 12\u00a0\u00b0CThe following thermal cycling program is used:In both cases (E-Sarbeco Concat-PCR or SPIKE PCR amplification), PCR products were verified by TapeStation automated electrophoresis .Multiple PCR assays are performed in presence of different barcoded primers, such that the combination with the barcoded cDNA material allows to generate a large complexity. Specifically, for E-Sarbeco targeting assay, 8 barcoded contact-PCR assays are pooled (8\u2009\u00d7\u200925\u2009=\u2009200ul) and cleaned with SPRIselect reagent with a ratio of 0.5\u00d7. Similarly, for SPIKE and E-Sarbeco PCR assays issued from random hexamer cDNA material, 4 barcoded PCR assays for each target regions were pooled (4\u2009\u00d7\u20092\u2009\u00d7\u200925\u2009=\u2009200ul) and cleaned with SPRIselect reagent with a ratio of 0.5x.Pooled & cleaned material is recovered on 50ul and used for Nanopore sequencing library preparation (Nanopore Ligation Sequencing Kit: SQK-LSK109). DNA libraries were sequenced on Nanopore FLO-MIN106D flowcells (R9) with the MinION Mk1C instrument .https://pypi.org/project/regex) with maximum 4 mismatches accepted by default); (2) split sequenced reads on monomers\u2014delimited by the presence of the identified Gibson sequences; (3) search for barcode sequences introduced during the reverse transcription ; (4) search for barcodes introduced during the PCR amplification ; (5) collect the sequences between the cDNA and PCR-associated barcodes for their alignment towards the SARS-CoV-2 reference genome . Considering that steps (3\u20135) are performed within the retrieved monomers in step (2), RETIVAD matches cDNA/PCR barcode combinations to aligned outcomes, thus providing a diagnostic outcome for the associated pseudo-candidate. Due to the low number of sequences retrieved at intervals of 10\u00a0min, and the relative short size of the SARS-CoV-2 genome (~\u200929\u00a0kb), RETIVAD is able to provide such diagnostic outcome within such 10\u00a0min interval. Hence, at the next round of data collection, RETIVAD reiterates on the aforementioned steps over the newly collected fastq files and appends the outcome to the previous results. The continuous gain on aligned read-counts per region of interest is visualized within scatterplot view (png file) updated during the processing software. RETIVAD collects each ten minutes the fastq files generated during sequencing from the Mk1C instrument and (1) search for Gibson sequences within the sequenced reads (regex query (racon package (version 1.4.20) is used for raw de novo DNA assembly, followed by the alignment with Minimap2 (version 2.17). Medaka (version 1.2.5) is used to create a consensus sequence and variant calls from the aligned data . RETIVAD generates BAM files per cDNA/PCR barcode combination harboring base conversion information and their associated coverage . Furthermore, summary files per cDNA/PCR barcode combination are also generated, providing the genomic position in which the base conversion has been detected, the identity of the converted bases as well as their associated coverage. Like in the case of the diagnostics processing, variants detection is performed in real time, thus providing the possibility to the user to visualize the results and decide whether sequencing requires still to be performed or whether the collected information is sufficient to conclude the diagnostics/variants tracing for the corresponding candidates.In addition to the diagnostic outcome, RETIVAD has been designed for performing real-time variants detection. For it, the RETIVAD generates a final summary report for the diagnostics outcome when no new fastq files are available after 40\u00a0min from the last data collection and processing.Supplementary Information."} +{"text": "HNF1B cause the complex syndrome renal cysts and diabetes (RCAD), characterized by developmental abnormalities of the kidneys, genital tracts and pancreas, and a variety of renal, pancreas and liver dysfunctions. The pathogenesis underlying this syndrome remains unclear as mice with heterozygous null mutations have no phenotype, while constitutive/conditional Hnf1b ablation leads to more severe phenotypes. We generated a novel mouse model carrying an identified human mutation at the intron-2 splice donor site. Unlike heterozygous mice previously characterized, mice heterozygous for the splicing mutation exhibited decreased HNF1B protein levels and bilateral renal cysts from embryonic day 15, originated from glomeruli, early proximal tubules (PTs) and intermediate nephron segments, concurrently with delayed PT differentiation, hydronephrosis and rare genital tract anomalies. Consistently, mRNA sequencing showed that most downregulated genes in embryonic kidneys were primarily expressed in early PTs and the loop of Henle and involved in ion/drug transport, organic acid and lipid metabolic processes, while the expression of previously identified targets upon Hnf1b ablation, including cystic disease genes, was weakly or not affected. Postnatal analyses revealed renal abnormalities, ranging from glomerular cysts to hydronephrosis and, rarely, multicystic dysplasia. Urinary proteomics uncovered a particular profile predictive of progressive decline in kidney function and fibrosis, and displayed common features with a recently reported urine proteome in an RCAD pediatric cohort. Altogether, our results show that reduced HNF1B levels lead to developmental disease phenotypes associated with the deregulation of a subset of HNF1B targets. They further suggest that this model represents a unique clinical/pathological viable model of the RCAD disease.Heterozygous mutations in Summary: A novel established mouse model carrying a heterozygous splicing human mutation in the Hnf1b gene exhibits phenotypes similar to those of patients with renal cysts and diabetes disease. HNF1B gene are the cause of a complex human syndrome known as renal cysts and diabetes , characterized by early onset of diabetes and developmental abnormalities of the kidney, genital tract and pancreas, as well as a variety of renal, liver, pancreas and biliary dysfunctions . Intragenic mutations map predominantly in the DNA binding domain (exon-2 and exon-4), while whole-gene deletions (1.3\u2005Mb deletion at chromosome 17q12) account for up to 50% of the HNF1B variants. HNF1B mutant carriers exhibit a highly variable phenotype, both between and within families. No clear genotype-phenotype correlations were observed for the type or location of mutations, and haploinsufficiency has been the main underlying disease proposed mechanism.More than 150 eletions . These mHNF1B mutations is severe non-diabetic renal disease. HNF1B mutations are also the most common monogenic causes of developmental kidney disease, being part of a spectrum of malformations known as congenital anomalies of the kidney and urinary tract branching and the induction of nephrogenesis . Moreoveutations , this moSp2/+Hnf1b] exhibited bilateral cysts and tubular dilatations as well as glomerular cysts from embryonic day (E)15, along with rare cases of genital tract abnormalities and extrarenal manifestations similar to those described in human HNF1B mutant carriers. Unlike previous mouse mutants heterozygous for a null allele, in which the HNF1B protein levels remained either unchanged or even increased ncreased , the HNFHnf1b ablation were relatively insensitive. Postnatal analyses revealed several renal abnormalities, ranging from few clusters of glomerular cysts and microcysts to hydronephrosis and, rarely, multicystic dysplasia. Further urine proteomic analyses uncovered a particular signature of differentially excreted peptides in Hnf1bSp2/+ mice, exhibiting several similarities to the urinary peptide signature reported in pediatric RCAD patients was sensitive to reduced HNF1B levels at different embryonic stages, whereas the previously identified targets upon patients .Hnf1b dosage in this mouse model differentially affects the expression of target genes, leading to the onset of disease phenotypes.Our results highlight that reduced Fig.\u00a0S1A), thus reproducing the c.544+1G>T (T) human mutation using RNA from Hnf1bSp2/+ heterozygotes and wild-type (WT) kidneys and primers located in exon-1 and exon-3. Sequence of the PCR products in Hnf1bSp2/+ mutants indicated the production of the two expected Hnf1b spliced isoforms A and B and four additional novel transcripts, present at low levels, corresponding to the isoforms A and B in which were deleted either exon-2 or the last 32\u2005bp of exon-2 through the activation of a near cryptic splice donor site . The same pattern of alternative splicing in Hnf1bSp2/+ heterozygous mice was observed at different stages .To define the consequences of this mutation on Fig.\u00a0S1C), variants lacking the last 32\u2005bp of exon-2 have not been described using primers located in the ATG translational site and the last 32\u2005bp of exon-2, thus allowing the detection of transcripts produced only by the normal Hnf1b allele (Table\u00a0S2). We found that, in Sp2/+Hnf1b, the Hnf1b transcripts were significantly decreased by 30-42% relative to WT levels, in particular during embryonic stages. In adults, the reduction in transcript levels was highly variable and modest without reaching significance . Thus, these truncated proteins are either very unstable or not produced as previously described in several dominant mutations associated with premature termination codons (PTCs) (see Discussion). Unlike previously characterized heterozygous mutants, we found a significant decrease in HNF1B protein levels in the Sp2/+Hnf1b mutants, not only at embryonic stages but also in adults , although in some cases mutant embryo kidneys exhibited dilated Bowman's capsules . Subsequently, from E15.5, we reproducibly observed bilateral cysts, including glomerular and tubular cysts in the cortico-medullar regions . Up to E14.5, heterozygous embryos developed apparently normally . A summary of histological renal phenotypes observed from E18.5 to postnatal day (P)0 is shown in Table\u00a0S1A.We then examined the influence of mouse strain susceptibility on the disease phenotype by backcrossing the om E15.5 G-N and a kidneys ; Fig.\u00a0S4Table\u00a0S1B). Heterozygous mutants in both backgrounds were able to reproduce, but they were less fertile than WT littermates. Further postnatal analysis revealed rare cases of genital tract abnormalities in either C57BL/6N or 129/sv backgrounds. Heterozygous mutants also exhibited several pancreatic dysfunctions, including glucose intolerance and pancreatitis, a phenotype that will be described elsewhere or attenuate (129/sv) the phenotype, further contributing to variability in phenotypic manifestations.These data show that Hnf1b is known to be required for early UB branching and initiation of nephrogenesis . Notably, the expression of Pax2, the transcription of which in the collecting system depends on Hnf1b , or at later stages . Nascent nephrons appeared normally induced as indicated by the presence of normally shaped comma- and S-shaped bodies and WT1-stained glomerular podocytes . Mutant kidneys without overt hydronephrosis were examined to visualize the collecting duct network and medullar nephron tubules. The expression of HNF4A, a marker of early PTs and a target of HNF1B , suggesting defective differentiation of PTs. Notably, HNF4A staining, which correlated with the reduced levels of HNF1B , in contrast to HNF4A expression, which showed only a partial restoration at E17.5 or P0 . Consistent with these observations, the mature PT marker LTA was not expressed up to E16.5 . However, Sp2/+Hnf1b PT clusters that were LTA+ both at E17.5 and P0 exhibited an unequal distribution and remained reduced in size . Interestingly, the Na-K-Cl co-transporter , expressed in the thick ascending limb (TAL) of the loop of Henle, stained most of Sp2/+Hnf1b medullar cysts and tubular dilatations at E17.5 and P0 . Distal tubules labeled by SLC12A3 (NCC) showed only rare and mild dilatations (data not shown).The expression of HNF1A, restricted to mature PTs, was similarly reduced at E16.5 B\u2032. Its eto E16.5 F\u2032 and beSp2/+Hnf1bcollecting ducts, stained either by AQP2 or pancytokeratin (CK) , were mainly devoid of dilatations. Intriguingly, they were not stained by Dolichos biflorus lectin (DBA), either during development or in postnatal life , uncovering impaired polarization of glycoconjugates in mutant collecting duct cells. This observation evoked an altered differentiation status of collecting duct cells, because previous studies have shown that UB cells begin to express DBA-binding glycoconjugates once they differentiate into stalks .Sp2/+Hnf1b non-cystic glomeruli displayed the normal layer of podocytes .Without evidence of urinary tract obstruction, we further examined HNF1B, HNF4A and WT1 immunostainings on serial embryonic sections, focusing on glomeruli with different degrees of Bowman's capsule expansion. We reproducibly observed that cystic glomeruli were surrounded by a decreased number of PTs that were often dilated or cystic H. Immunoodocytes F,I. In mHNF1B human mutant fetuses showing a very severe cystic renal phenotype exhibited a global decrease and partial disorganization in E15.5 heterozygous kidneys, both in dilated and non-dilated tubules K,M. In mFig. S6A). However, at later stages, the number of proliferating cells in Sp2/+Hnf1b normal tubular structures exhibited a 1.84-fold increase relative to WT tubules. Consistent with the enhanced proliferation associated with cystic expansion in autosomal dominant polycystic kidney disease . No changes were observed in the number of apoptotic cells assessed by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) (not shown).Further analysis of proliferation, using the mitosis marker phosphorylated histone H3, showed comparable numbers of proliferating cells in E14.5 renal tubules of heterozygous mutants and WT on WT and heterozygous mutant kidneys at E14.5 and at disease stages as detailed in the Materials and Methods.P-value <0.05. Remarkably, several genes were differentially expressed with log2FC>\u22121 at all stages (Table\u00a0S3), while only a few differentially expressed upregulated genes were detected. We found an increased number of downregulated genes from E14.5 to E17.5 followed by a partial decrease at P1, in agreement with a partial restoration of the expression of several PT markers. Accordingly, the numbers of downregulated genes shared between stages increased to reach 107 genes between E17.5 and P1 .Because heterozygous mutants still expressed HNF1B, we considered an absolute fold-change cutoff value of > or <0.5 log2FC, with an adjusted Table\u00a0S3, File\u00a06). At E17.5 and P1, downregulated genes included drug-metabolizing enzymes and more than 50 SLC-transmembrane-transporter genes .Downregulated genes with greater fold changes were predominantly expressed in early and maturing PTs and to a lesser extent in primitive loops of Henle and distal tubules (Table\u00a0S4).Gene ontology (GO)-term analyses highlighHnf4a, Tmem27 (Cltrn), Cubn, Spp1, Spp2, Pah, Kcnj1, Pdzk1 and several SLC-transmembrane transporter genes, representing the subset of targets that appeared particularly sensitive to reduced HNF1B protein levels (Table\u00a0S3). Interestingly, many of the genes downregulated at E17.5/P1 and regulated by HNF1B were also found strongly decreased in Hnf4a mutant P1 kidneys . By contrast, the expression of previously identified targets strongly reduced upon Hnf1b ablation, including Wnt9b, Pax2, Pkd2, Bicc (Bicc1), Tg737 (Ift88), Crb3, Kif12, Cys1, Glis2 and Glis3, were either modestly decreased or not affected. These results were further validated by qRT-PCR . Interestingly, the well-known targets Umod, Tmem27 and Pkhd1 (Table\u00a0S3).The regulatory sequences of most downregulated genes contained HNF1 consensus binding sites. Consistently, specific HNF1B recruitment to the regulatory sequences of many of them has been reported by chromatin immunoprecipitation (ChIP)-PCR or ChIP sequencing . This ocular phenotype remains undefined. It may be linked to the Rd8 mutation of the Crb1 gene present in the strain C57BL/6N , with the lacZ gene and the SV40 polyadenylation sequence and neomycin resistance cassette, replacing the first exon of Hnf1b (Hnf1btm1Sce) and C57BL/6N (C57BL/6N-Hnf1btm1Sce) backgrounds are available at EMMA together with the phenotype description. The +/\u2212Hnf1a mice were provided by Frank Gonzalez and mainAnimal care and the experimental protocols were approved by and conducted in accordance with French and European ethical legal guidelines and the local ethical committee for animal care , respecting the 3R rules.Paraffin-embedded human fetal tissues were obtained from family members with informed consent approved by the Ethics committee of Debr\u00e9 Hospital , as previously reported in Fbp and Spp2 cRNA probes were generated by PCR . Embryos and postnatal kidneys up to 2\u00a0months of age were fixed with 60% ethanol/11% formaldehyde and 10% acetic acid. Adult kidneys (>2\u00a0months) were fixed in alcoholic Bouin (Duboscq-Brasil) solution and paraffin sections were used for H&E histological analysis and immunohistochemistry. Antibody staining on paraffin sections was performed as described and washed in ice-cold PBS. WT and heterozygous mutant embryos were from the same litter. Adult kidneys were cut sagittal: one-half was used for histology, one-quarter for RNA extraction and the other quarter for protein extraction. Total RNA was extracted using an miRNeasy Mini Kit (Qiagen). Samples were treated with Dnase1 on the columns according to the manufacturer's instructions, and 250-500\u2005ng was reverse transcribed using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). RT-PCR was performed using Fast SYBR Green Master Mix (Applied Biosystems) and the Step-One Plus system (Applied Biosystems), as described . The priFig.\u00a0S1. PCR products were resolved in 2% agarose/TBE ethidium bromide gels and photographed using a GELDOC documentation system. Densitometry quantification was performed with ImageJ software. Primer sequences used were Gapdh for normalization and, for Hnf1b, vATG and v695 (Table\u00a0S2).Total RNA from microdissected kidneys was extracted and subjected to semiquantitative RT-PCR as described with theHnf1b allele are listed below:The cDNA sequences of spliced isoforms and the encoded truncated proteins from the mutated CGAGGAGGCCGCGGAGCAGCGGGCCGAGGTGGACCGGATGCTCAGCGAGGACCCGTGGAGGGCTGCCAAAATGATCAAGGGATACATGCAACAGCACAATATCCCCCAGAGGGAGGTGGTCGATGTCACAGGCCTGAACCAATCCCACCTCTCTCAACACCTCAACAAGGGCACCCCCATGAAGACCCAGAAGAGAGCTGCCCTGTACACTTGAgATGGTGTCCAAGCTCACGTCGCTCCAGCAAGAACTCCTGAGTGCCCTGCTGAGCTCCGGAGTCACCAAGGAAGTGCTGATCCAGGCCTTGGAGGAGTTACTGCCGTCCCCGAATTTCGGGGTGAAGCTGGAGACACTGCCCCTGTCCCCCGGGAGCGGGGCGGATCTCGACACCAAGCCGGTTTTCCATACTCTCACCAATGGCCACGCCAAGGGCCGCTTGTCTGGGGACGAGGGCTCAGAGGACGGCGACGACTATGACACTCCTCCCATCCTCAAAGAGCTCCAGGCGCTCAACACMVSKLTSLQQELLSALLSSGVTKEVLIQALEELLPSPNFGVKLETLPLSPGSGADLDTKPVFHTLTNGHAKGRLSGDEGSEDGDDYDTPPILKELQALNTEEAAEQRAEVDRMLSEDPWRAAKMIKGYMQQHNIPQREVVDVTGLNQSHLSQHLNKGTPMKTQKRAALYT*VQPDTAAaAGTTCAACCAGACAGTCCAGAGCTCTGGAAACATGACAGACAAAAGCAGTCAGGATCAGCTGCTGTTTCTCTTTCCAGAGTTCAGTCAACAGAACCAGGGGCCTGGGCAGTCGGAGGACACCTGCTCCGAGCCCACCAACAAGAAGATGCGCCGCAACCGGTTATGGTGTCCAAGCTCACGTCGCTCCAGCAAGAACTCCTGAGTGCCCTGCTGAGCTCCGGAGTCACCAAGGAAGTGCTGATCCAGGCCTTGGAGGAGTTACTGCCGTCCCCGAATTTCGGGGTGAAGCTGGAGACACTGCCCCTGTCCCCCGGGAGCGGGGCGGATCTCGACACCAAGCCGGTTTTCCATACTCTCACCAATGGCCACGCCAAGGGCCGCTTGTCTGGGGACGAGGGCTCAGAGGACGGCGACGACTATGACACTCCTCCCATCCTCAAAGAGCTCCAGGCGCTCAACACCGAGGAGGCCGCGGAGCAGCGGGCCGAGGTGGACCGGATGCTCAGMVSKLTSLQQELLSALLSSGVTKEVLIQALEELLPSPNFGVKLETLPLSPGSGADLDTKPVFHTLTNGHAKGRLSGDEGSEDGDDYDTPPILKELQALNTEEAAEQRAEVDRMLRVQPDSPELWKHDRQKQSGSAAVSLSRVQSTEPGAWAVGGHLLRAHQQEDAPQPV*TAAaAGTTCAGTCAACAGAACCAGGGGCCTGGGCAGTCGGAGGACACCTGCTCCGAGCCCACCAACAAGAAGATGCGCCGCAACCGGTTATGGTGTCCAAGCTCACGTCGCTCCAGCAAGAACTCCTGAGTGCCCTGCTGAGCTCCGGAGTCACCAAGGAAGTGCTGATCCAGGCCTTGGAGGAGTTACTGCCGTCCCCGAATTTCGGGGTGAAGCTGGAGACACTGCCCCTGTCCCCCGGGAGCGGGGCGGATCTCGACACCAAGCCGGTTTTCCATACTCTCACCAATGGCCACGCCAAGGGCCGCTTGTCTGGGGACGAGGGCTCAGAGGACGGCGACGACTATGACACTCCTCCCATCCTCAAAGAGCTCCAGGCGCTCAACACCGAGGAGGCCGCGGAGCAGCGGGCCGAGGTGGACCGGATGCTCAGMVSKLTSLQQELLSALLSSGVTKEVLIQALEELLPSPNFGVKLETLPLSPGSGADLDTKPVFHTLTNGHAKGRLSGDEGSEDGDDYDTPPILKELQALNTEEAAEQRAEVDRMLRVQSTEPGAWAVGGHLLRAHQQEDAPQPV*Note that the STOP codons of putative encoded proteins are followed by a purine, which is predicted to positively influence translational termination efficiency .GTACGTCAGAAAGCAACGGGAGATCCTCCGACCGAGGACCCGTGGAGGGCTGCCAAAATGATCAAGGGATACATGCAACAGCACAATATCCCCCAGAGGGAGGTGGTCGATGTCACAGGCCTGAACCAATCCCACCTCTCTCAACACCTCAACAAGGGCACCCCCATGAAGACCCAGAAGAGAGCTGCCCTGTACACTTGUnderlined text shows the 32\u2005bp of exon-2 spliced out of mouse exon-2.Human embryonic kidney (HEK) 293 cells were maintained and transiently transfected as described , with eig. The cellular pellet was resuspended into ice-cold HNB buffer , frozen and thawed four times in liquid N2, and centrifuged for 10\u2005min at 11,180 g. The supernatant containing the whole-cell extracts was frozen in liquid N2 and kept at \u221280\u00b0C.Transfected cells were washed with ice-cold PBS plus Roche protease inhibitors, scraped, transferred into Eppendorf tubes and centrifuged for 5\u2005min at 1006 n=3) different litters were pooled, frozen in liquid N2 and lysed in ice-cold lysis buffer using 23\u2005g and 26\u2005g syringes. Adult kidney pieces were frozen in liquid N2, reduced to a fine powder under liquid N2 and further lysed in lysis buffer using a syringe. Lysis buffer contained 15% glycerol, 10\u2005mM Tris-HCl pH 7.4, 150\u2005mM NaCl, 5\u2005mM EDTA, 1% NP40, 3\u2005mM sodium pyrophosphate and 50\u2005mM sodium fluoride, with complete protease inhibitor cocktail (Roche) added before using. Samples were centrifuged for 15\u2005min at 18,894 g at 4\u00b0C, and the supernatant was frozen in liquid N2 and kept at 80\u00b0C. Protein concentration was determined using a Pierce\u2122 BCA Protein Assay kit. Whole-cell extracts containing 20-30\u2005\u03bcg of protein were prepared in SDS sample buffer and subjected to SDS-PAGE . After the proteins were transferred onto a 0.2\u2005\u00b5m nitrocellulose membrane (Bio-Rad) and blocked with TBST , immunoblotting was performed by overnight incubation in TBST 1% skim milk buffer with a rabbit polyclonal antibody against HNF1B (1:500) previously validated (Table\u00a0S7). Positive HNF1B bands were detected by chemiluminescence . Western lightning Plus-ECL Perkin Elmer was used to detect \u03b1-tubulin. Images were captured with G-BOX Syngene Europe, Chemiluminescence Image Capture software and quantified by Image J. WT and HNF1B proteins normalized by \u03b1-tubulin expression were quantified from each litter. A WT sample was assigned a 100% value and the other samples of the same litter WT and heterozygotes were referred to as a percentage of this value. This allowed the comparison of different litters of a given embryonic stage. Statistical significance was determined using unpaired Student's t-test, using Prism 6.00 . P<0.05 was considered significant.The two kidneys of each embryo of at least three . Twenty-four-hour urine samples collected under mineral oil to avoid evaporation were obtained at baseline in individual metabolic cages, after 2-3\u2005days habituation. Each 24\u2005h, animals were weighed and food intake, water intake, urine volume and fecal weight recorded. Blood was sampled by retro-ocular puncture, in general 2\u2005weeks after being in the metabolic cages, and plasma samples were kept at \u221280\u00b0C. The urinary concentration ability was tested after 22\u2005h of water deprivation. Urinary creatinine, urea and electrolytes, plasma urea, creatinine, Mg2+, AST and ALT were measured on an Olympus AU400 Chemistry Analyzer . Osmolality was measured using a vapor pressure osmometer .Urine and plasma were obtained from age- and sex-matched heterozygous and WT mice . They were housed in a light- and temperature-controlled room with t-test or unpaired Student's t-test with Welch correction were used for statistical analysis. P<0.05 was considered significant.Data are presented as mean\u00b1s.e.m. Unpaired Student\u2019s Sp2/+Hnf1b and 18 WT mice from the age of 3, 8, 12 and 17\u2005months. Urine from WT and mutant mice was collected by spontaneous voiding or after animals were placed individually in metabolic cages as described. The total volume of 24-h urine was aliquoted and frozen at \u221280\u00b0C. A 150-\u00b5l sample of mouse urine was diluted with the same volume of urea buffer . Then, 150\u2005\u00b5l of the urine samples was ultrafiltrated, desalted, lyophilized and resuspended for proteome analysis as described online coupled to a micrOTOF II MS , as described . For norHnf1bSp2/+ mutants and WT embryos in the C57BL/6N background were microdissected from the same litter. This requirement limited the number of samples used in general to two WTs and two heterozygous mutants, independently of the sex. Note that we found a similar phenotype in males and females. The stages analyzed included E14.5 and at disease stages E15.5, E17.5 and P1. Note that at E15.5 we performed deep sequencing from pooled samples of three WT and three Sp2/+Hnf1b (six kidneys each sample). RNA-seq of E14.5, E15.5 and P1 samples was performed at the Alexander Fleming Institute, Genomics Facility, Greece; RNA-seq of E17.5 samples was performed at Fasteris, Switzerland (Table\u00a0S8).The two kidneys from heterozygous \u00ae mRNA DIRECT\u2122 Micro Kit (ThermoFisher Scientific). mRNA was digested with RNase III, purified, hybridized and ligated to Ion Adaptors (ThermoFisher Scientific), reverse transcribed, barcoded and amplified, using an Ion Total RNA-Seq Kit v2 (ThermoFisher Scientific). RNA-seq was performed on an Ion Proton\u2122 System (ThermoFisher Scientific), according to the manufacturer's instructions. The prepared libraries were quantified and pooled together in duplicates at the required concentration. The pools were then processed on a OneTouch 2 instrument and enriched on a OneTouch ES station. Templating was performed using an Ion PI\u2122 Template OT2 200 Kit (ThermoFisher Scientific) and sequencing with an Ion PI\u2122Sequencing 200 Kit on Ion Proton PI\u2122 chips (ThermoFisher Scientific) according to commercial protocols. The resulting RNA-seq BAM files were analyzed with the Bioconductor package metaseqR (http://www.bioconductor.org/packages/release/bioc/html/edgeR.html).RNA from microdissected kidneys was extracted by Tryzol, using a Qiagen miRNA mini kit for the extraction of total RNA and miRNAs. The quality of the RNA samples was assessed on an Agilent Bioanalyzer system using an RNA 6000 Nano Kit (Agilent Technologies) and RNA with a Ring higher than 8 was used. Then, 1-2\u2005\u03bcg of total RNA was used for mRNA isolation using a DynabeadsmetaseqR , applyinin vitro samples to generate a library of short inserts (the DNA Colonies Template Library). The library was sequenced on an Illumina HiSeq 2000. For each lane, 130-150 million DNA colonies producing pass filter sequences were assured. The read lengths are 1\u00d750\u2005bp or 1\u00d7100\u2005bp for single-read runs using the forward sequencing primer. The inserts can also be sequenced from both ends using a \u2018forward\u2019 and a \u2018reverse\u2019 sequencing primer, generating paired reads of 2\u00d7100\u2005bp. The data were processed using bioinformatics tools to extract biologically useful information. To homogenize mRNA-seq comparisons, RNA-seq files were all analyzed as described above (High-throughput DNA sequencing using Illumina technology consisted of processing ed above ."} +{"text": "Photoimmunotherapy is one of the most promising strategies in tumor immunotherapies, but targeted delivery of photosensitizers and adjuvants to tumors remains a major challenge. Here, as a proof of concept, we describe bone marrow mesenchymal stem cell-derived nanovesicles (NVs) displaying anti-PD-L1 antibodies (aPD-L1) that were genetically engineered for targeted drug delivery.The high affinity and specificity between aPD-L1 and tumor cells allow aPD-L1 NVs to selectively deliver photosensitizers to cancer tissues and exert potent directed photothermal ablation. The tumor immune microenvironment was programmed via ablation, and the model antigen ovalbumin (OVA) was designed to fuse with aPD-L1. The corresponding membrane vesicles were then extracted as an antigen\u2013antibody integrator (AAI). AAI can work as a nanovaccine with the immune adjuvant R837 encapsulated. This in turn can directly stimulate dendritic cells (DCs) to boast the body's immune response to residual lesions.aPD-L1 NV-based photoimmunotherapy significantly improves the efficacy of photothermal ablation and synergistically enhances subsequent immune activation. This study describes a promising strategy for developing ligand-targeted and personalized cancer photoimmunotherapy.The online version contains supplementary material available at 10.1186/s12951-022-01266-3. Immunotherapy is a novel strategy for tumor therapy that works via an activated immune system \u20133. HowevPhotoimmunotherapy has shown value versus conventional immunotherapies. Recent studies have shown that photothermal-based immunotherapy can induce tumor cell death via high temperatures while simultaneously activating the tumor immune response \u201316. The Actively targeting nanoplatforms to the tumor microenvironment often requires conjugation of targeting ligands. Chemical covalent linkage of receptors or ligands to deliver photosensitizers or adjuvants may cause a loss of tumor targeting and lead to limited photoimmunotherapy. Antibodies can facilitate immunoregulation and binding to specific markers on tumor cells \u201321. NatuCell membrane-derived nanovesicles (NVs) are biogenic nanocapsule structures obtained by crushing or squeezing selected cell membranes \u201327. NVs To optimize the curative effect of malignant tumors, we describe here a novel method based on cell membrane-derived biomimetic nanovesicles displaying aPD-L1 for photoimmunotherapy , and regulation of the immune checkpoint. Thus, it is an immunotherapy for residual and distal lesions. Accordingly, the model antigen ovalbumin (OVA) served as a tumor-associated antigen and was designed to be co-expressed with aPD-L1 on the membrane of MSCs and the corresponding membrane vesicles. OVA was extracted as an antigen\u2013antibody integrator (AAI) and is a promising multifunctional nanoplatform. With immune adjuvant (R837) encapsulated in AAI (AAI-R837), antigens and immune adjuvants are delivered to antigen-presenting cells (APCs) in a spatio-temporal way for follow-up immunotherapies. AAI can be a carrier for targeted delivery and can also carry antigen signal to APCs to promote the mature differentiation of DCs, thus boosting the immune response to tumors. Afterwards, AAI-R837 can work as a nanovaccine to facilitate immunotherapy. In this treatment strategy, the multi-functional nanoplatform was used in a combined way for photoimmunotherapy. The material achieved satisfactory inhibitory effects on malignant melanomas.A plasmid containing aPD-L1 fragments was first constructed to prepare aPD-L1 displaying MSCs. Then a lentiviral vector was used to infect MSCs extracted from the bone marrow cavity of C57BL/6 embryonic mice with the aPD-L1 displayed on the outside of the cell membrane Fig.\u00a0b. SubseqTo test the binding ability of aPD-L1 NVs to the B16F10 melanoma cells, NVs and aPD-L1 NVs labeling fluorescent dye FITC were incubated with B16F10 cells for 3\u00a0h and then imaged with confocal laser scanning microscopy (CLSM). Significant fluorescence was observed in the periphery of B16F10 cells after being incubated with FITC-aPD-L1 NVs: They were five-fold brighter than FITC-NVs Fig.\u00a0g. These 2, 5\u00a0min) was used to irradiate aPD-L1 NVs-ICG and free ICG at different concentrations; real-time detection used a NIR thermal imager was implemented on the B16F10 melanoma cells with or without aPD-L1 NVs-ICG. The aPD-L1 NVs-ICG had good photothermal killing effects on B16F10 melanoma cells test was used to verify the safety of different concentrations of aPD-L1 NVs-ICG on DC2.4 cells and B16F10 melanoma cells (a representative cancer cell). The aPD-L1 NVs-ICG have no obvious toxic effects on DC2.4 cells and B16F10 melanoma cells Fig.\u00a0d. In addlls Fig.\u00a0e. The sulls Fig.\u00a0f.Inflammation-induced PD-L1 propagates expression in the tumor microenvironment, thus inhibiting the antitumor cytotoxic T-cell response \u201341. TherThe drug distribution in vivo was monitored by an IVIS Lumina II imaging system. After 12\u00a0h and 24\u00a0h of administration, the aPD-L1 NVs-ICG had a significant fluorescence signal at the tumor site, and metabolites changed more slowly in vivo than free ICG and NVs-ICG was used to irradiate the initial tumor on the left side for photothermal treatment, and the NIR thermal imager was used to monitor the temperature change of tumor site in real time. The results show that tumors treated with aPD-L1 NVs-ICG after laser irradiation, plus the warming capacity of the melanoma itself, led to a surface temperature of 60 \u2103 that could ablate the tumor . The data indicated that the content of CD11c\u2009+\u2009CD86\u2009+\u2009increased compared to PBS-treated groups Fig.\u00a0e. HoweveFurthermore, after being co-incubated for 48\u00a0h, the cell culture medium was collected, and the content of immune-related cytokines was detected by ELSA Fig.\u00a0f\u2013i. The Encouraged by the results in vitro, we further investigated the effect of AAI-R837 on the immune response in vivo. First, we verified the process of AAI uptake by DCs and migration to lymph nodes in vivo. AAI, free NVs, and OVA labeled with fluorescent dye Cy5.5 were injected subcutaneously into the right leg of mice, and the migration process of drugs in vivo was observed by an IVIS Lumina II imaging system. The injected free Cy5.5 and Cy5.5-OVA rapidly diffused and metabolized in the body although there was a certain concentration of inguinal lymph nodes in the early stage. The product was metabolized quickly over time Fig.\u00a0a. Cy5.5-Next, photoimmunotherapy was done on the tumor. After that, various immunological materials were injected subcutaneously at the root of the tail on 13, 15, and 17 d as a vaccine for immunotherapy on remnant tumors Fig.\u00a0d. After The immune effect after treatment was also studied. Three days after the final immunization, distal tumors, serum, and lymph nodes were collected, and immune cells and cytokines related to the immune response were detected. A cell suspension was prepared by homogenized the material, and the content of mature DCs in lymph nodes, as well as CTLs in tumor was analyzed by FCM Fig.\u00a0g, h. CelIn summary, we constructed AAI that simultaneously express antibodies and tumor-related antigens using the cell membrane of bone marrow mesenchymal stem cells. Nano-vesicles expressing aPD-L1 can specifically bind to PD-L1 receptors on cancer cells and can be utilized to deliver photosensitizers to tumor sites for efficient photothermal therapy. Meanwhile, the aPD-L1 expressed on the modified nanocapsule can also specifically bind to the PD-L1 receptor on the DCs. In short, this design takes advantage of specific binding at the immune checkpoints. This provides a promising strategy for targeted drug delivery. Intriguingly, the immune environment in the tumor region was programmed via photothermal ablation. This led to improved immunotherapy to arrest the residual and distal tumor. Additionally, antibodies and antigens were co-expressed on AAI-R837 and delivered the immune adjuvant to the antigen presenting cells. Antigen signal was sent to the DCs correspondingly and the powerful immune response of the immune system was elicited. The experimental results indicated that the combined treatment of photoimmunotherapy on the melanoma model not only had a significant inhibiting effect on the initial tumor, but also inhibited distant tumor. The low immunogenicity of the materials extended the in vivo circulation time of the nanocarrier, and its biological safety suggests clinical applications.Imiquimod (R837) and Indooyanine gree (ICG) were purchased from Invivogen and BBI Life Science Corporation, respectively. Ovalbumin (OVA), calcein acetoxymethyl ester and propidium iodide (PI) were purchased from Sigma-Aldrich Inc. FITC was purchased from MedChemExpress LLC and Cy5.5 was purchased from APExBIO Technology LLC. Anti-mouse CD274 , FITC-anti-mouse CD86, PE-anti-mouse CD11c, TITC-anti-mouse CD8 and PerCP-anti-mouse CD3\u03b5 for flow cytometry were purchased from Biolegend Inc. Basement Membrane Matrix was purchased from Becton, Dickinson and Company. TNF-\u03b1, IL-6, IL-12 and IFN-\u03b3 ELSA kit were purchased from Beyotime Biotech Inc.2. To make aPD-L1 be expressed on membrane of MSCs, the aPD-L1 gene with a signal peptide, flag tags, aPD-L1 ScFv and transmembrane sequence were synthesized. While to make aPD-L1 and OVA be co-expressed on membrane of MSCs, plasmids was constructed containing signal peptide, flag tags, aPD-L1 ScFv, transmembrane sequence as well as an intramembrane fragment containing an OVA sequence.DNA sequence of membrane-located signal peptide (from integrin beta 1):ATGAATTTGCAACTGGTTTCCTGGATTGGATTGATCAGTTTGATTTGTTCTGTATTTGGCCAAACAGATAAADNA sequence of Flag tag:GACTACAAGGACGACGACGACAAGDNA sequence of aPD-L1 ScFv:GCCCAGGCCGCCCTGACCCAGCCCAGCAGCGTGAGCGCCAACCTGGGCGGCACCGTGAAGATCACCTGCAGCGGCGGCAGCGGCAGCTACGGCTGGTACCAGCAGAAGGCCCCCGGCAGCGCCCCCGTGAGCCTGATCTACGACAACACCAACAGGCCCAGCGACATCCCCAGCAGGTTCAGCGGCGCCCTGAGCGGCAGCACCGCCACCCTGACCATCACCGGCGTGCAGGCCGAGGACGAGGCCGTGTACTACTGCGGCAGCAGGGACAGCAGCAACGCCGGCAGCGTGTTCGGCGCCGGCACCACCCTGACCGTGCTGGGCCAGAGCAGCAGGAGCAGCGGCGGCGGCGGCAGCAGCGGCGGCGGCGGCAGCGCCCTGACCCTGGACGAGAGCGGCGGCGGCCTGCAGACCCCCGGCGGCGCCCTGAGCCTGGTGTGCAAGGCCAGCGGCTTCACCTTCAGCGACAGGGGCATGCACTGGGTGAGGCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGGGCGCCATCAGCAGGAGGGGCAGCACCACCACCTACGCCCCCGCCGTGAAGGGCAGGGCCACCATCACCAGGGACAACGGCCAGAGCACCGTGAGGCTGCAGCTGAACAACCTGACCGCCGAGGACACCGCCACCTACTTCTGCGCCAAGAACGACGACAGCGTGGGCATCGTGACCACCAGCACCATCGACGCCTGGGGCCACGGCACCGAGGTGATCGTGAGCAGCACCAGCGGCCAGGCCGGCCAGCACCACCACCACCACCACGGCGCCTACCCCTACGACGTGCCCGACTACGCCAGC.DNA sequence of transmembrane fragment:TTATGGGTCATCCTGCTGAGTGCTTTTGCCGGATTGTTGCTGTTAATGCTGCTCATTTTAGCACTGTGGDNA sequence of OVA:ATGGGCTCCATCGGCGCAGCAAGCATGGAATTTTGTTTTGATGTATTCAAGGAGCTCAAAGTCCACCATGCCAATGAGAACATCTTCTACTGCCCCATTGCCATCATGTCAGCTCTAGCCATGGTATACCTGGGTGCAAAAGACAGCACCAGGACACAGATAAATAAGGTTGTTCGCTTTGATAAACTTCCAGGATTCGGAGACAGTATTGAAGCTCAGTGTGGCACATCTGTAAACGTTCACTCTTCACTTAGAGACATCCTCAACCAAATCACCAAACCAAATGATGTTTATTCGTTCAGCCTTGCCAGTAGACTTTATGCTGAAGAGAGATACCCAATCCTGCCAGAATACTTGCAGTGTGTGAAGGAACTGTATAGAGGAGGCTTGGAACCTATCAACTTTCAAACAGCTGCAGATCAAGCCAGAGAGCTCATCAATTCCTGGGTAGAAAGTCAGACAAATGGAATTATCAGAAATGTCCTTCAGCCAAGCTCCGTGGATTCTCAAACTGCAATGGTTCTGGTTAATGCCATTGTCTTCAAAGGACTGTGGGAGAAAGCATTTAAGGATGAAGACACACAAGCAATGCCTTTCAGAGTGACTGAGCAAGAAAGCAAACCTGTGCAGATGATGTACCAGATTGGTTTATTTAG.AGTGGCATCAATGGCTTCTGAGAAAATGAAGATCCTGGAGCTTCCATTTGCCAGTGGGACAATGAGCATGTTGGTGCTGTTGCCTGATGAAGTCTCAGGCCTTGAGCAGCTTGAGAGTATAATCAACTTTGAAAAACTGACTGAATGGACCAGTTCTAATGTTATGGAAGAGAGGAAGATCAAAGTGTACTTACCTCGCATGAAGATGGAGGAAAAATACAACCTCACATCTGTCTTAATGGCTATGGGCATTACTGACGTGTTTAGCTCTTCAGCCAATCTGTCTGGCATCTCCTCAGCAGAGAGCCTGAAGATATCTCAAGCTGTCCATGCAGCACATGCAGAAATCAATGAAGCAGGCAGAGAGGTGGTAGGGTCAGCAGAGGCTGGAGTGGATGCTGCAAGCGTCTCTGAAGAATTTAGGGCTGACCATCCATTCCTCTTCTGTATCAAGCACATCGCAACCAACGCCGTTCTCTTCTTTGGCAGATGTGTTTCCCCTTAA.Bone marrow mesenchymal stem cells were extracted from the bone marrow of suckling C57BL/6 mice (Seven days after birthed) referring to the established method . The micThen, Lentivirus vector encoding corresponding sequence was applied to infect MSCs. Infected MSCs displaying aPD-L1 with or without OVA were cultured in C57BL/6 mouse bone marrow mesenchymal stem cell complete culture medium maintained in 20% FBS and 1% Penicillin and streptomycin.6 in a petri dish, the medium in the culture dish with MSCs was removed, then the MSCs were scraped away by cell scraping, and were collected into the centrifuge tube with phosphate buffer solution (PBS). After 5% deoxysodium cholate, a surfactant, was added to the cell suspension (v:v\u2009=\u20091:100), MSCs were broken by a low power ultrasound for 10 to 20\u00a0s using the ultrasonic crusher , and the protease inhibitor PMSF (10\u00a0mg/mL) was added (v:v\u2009=\u20091:200) to worked solution immediately. To remove the cytoplasm and nucleus, the suspending liquid was centrifuged at 3500\u00a0rpm (4\u00a0\u2103) for 5\u00a0min. Supernatant was collected for further centrifugation at 15,000\u00a0rpm(4\u00a0\u2103) for 20\u00a0min to acquire nanoscale membrane vesicles, then resulting vesicles were quantified by BCA kit (Beyotime Biotech Inc.) and dissolved in PBS and stored at 4\u00a0\u2103 for standby application.In the preparation of nanovesicles, when the number of cells reaches 5\u2573106 MSCs, the ultrasonic crushing instrument (20 w) was used for ultrasonic concussion for three times at 4\u00a0\u2103, each session lasts 5 to 10\u00a0s and interrupted at least 10\u00a0s. After resulting sample was centrifuged at 12,000\u00a0rpm (4\u00a0\u2103) for 20\u00a0min, the content of ICG in the supernatant was detected by a microplate analyzer, and the loading efficiency of ICG was calculated. NVs loading drugs were dispersed in PBS and stored at 4\u2103 for further study. When loading the immune adjuvant, 100 \u03bcL R837 (0.5\u00a0mg/mL) was added to NVs suspension, and the loading was carried out according to the same method mentioned above. The loading efficiency of R837 was determined by HPLC (Waters 1525)-UV visible detector at 325\u00a0nm. Acetonitrile was used as the mobile phase. ICG encapsulation and the UV absorption spectra of NVs-ICG were measured by microplate reader.After 200 \u03bcL ICG aqueous solution (1\u00a0mg/mL) was mixed with NVs suspension come from 5\u00d7102) for 5\u00a0min. During the heating of each solution, an NIR thermal imaging camera was used to record the temperature changes.The expression of aPD-L1 on the MSCs membrane was verified by immunofluorescence. The morphology and structure of NVs were characterized by fetenay transmission electron microscopy (F20). The dynamic particle size and electric potential of NVs was measured with Zeta sizer Nano-ZS . The existence of aPD-L1 on the NVs was detected with coimmunoprecipitation (CO-IP) assay and Western blot (WB) analysis. In order to study photothermal performance of acquired materials, aPD-L1 NVs-ICG and ICG with different concentrations were dispersed in ultrapure water, then the samples were irradiated with 808\u00a0nm laser (1\u00a0W/cm4 cells/well) and cultured for 24\u00a0h. FITC-labeled aPD-L1 NVs or NVs were incubated with the cancer cells for 3\u00a0h, and cells in the other group were incubated with antiPD-L1 antibodies (10\u00a0\u03bcg/mL) for 2\u00a0h before the addition of aPD-L1 NVs to the culture medium. The NVs that were not bound to the cells were washed with PBS for three times, then the nuclei were stained with DAPI for 15\u00a0min. After the excess dye was washed and added new medium to the wells, confocal microscopy was performed under a confocal laser scanning microscope (LSM780) with 60\u2009\u00d7\u2009oil-immersion lenses. The binding of AAI to DC2.4 cells was observed by the same method.To exam the ability of aPD-L1 NVs to binding to tumor cells, B16F10 cells were seeded in confocal culture dish (3\u2009\u00d7\u2009104 cells/well) for 24\u00a0h, respectively. After the cells were washed with PBS for three times, fresh medium containing different concentrations of aPD-L1 NVs-ICG and AAI-R837 was added for training another 24\u00a0h. The cell relative viability was detected by MTT assay and the cytotoxicity of the nanomedicine was determined.DC2.4 cells and B16F10 cells were selected to determine the cytotoxicity of the nanomedicine by standard MTT assay. DC2.4 cells and B16F10 cells were cultured in 96-well plates (1\u2009\u00d7\u2009104 cells/well) for 24\u00a0h. After washing the cells with PBS for three times, fresh medium containing 100\u00a0\u03bcg/mL of aPD-L1 NVs-ICG was added. Treated cells were irradiated with 808\u00a0nm laser (1.0\u00a0W/cm2) for 5\u00a0min. Viability of B16F10 cell was then assessed using standard MTT procedure. In order to intuitively evaluate the relative viability of B16F10 cells after different treatments, 2\u00a0\u03bcmol/L calcein acetoxymethyl ester and 4 \u03bc mol/L propidium iodide (PI) were used to stain the living and dead cells for 15\u00a0min, respectively. After washed with PBS, the cells were observed with inverted fluorescence microscope .To study the effect of photothermal therapy on B16F10 Cells with aPD-L1 NVs-ICG, B16F10 cells were cultured in 96-well plates(1\u2009\u00d7\u200910Bone marrow derived dendritic cells (BMDCs)were extracted from the bone marrow of 6\u2009~\u20098\u00a0weeks old C57BL/6 mice according to the established method . BMDCs w50\u00a0\u03bcg of ICG, 100\u00a0\u03bcg of NVs-ICG and aPD-L1 NVs-ICG (containing 50\u00a0\u03bcg of ICG) dispersed in 100 \u03bcL PBS were injected into subcutaneous melanoma burdened C57BL/6 mice via tail vein. Fluorescence imaging system was used to obtain fluorescence images at 0, 6, 12, 24 and 48\u00a0h after injection, respectively. At 48\u00a0h, the main organs and tumors of the mice were collected, and fluorescence imaging and intensity records were collected.NVs, OVA and AAI were labeled with Cy5.5, subsequently, and 50 \u03bcL normal saline with equivalent 10\u00a0\u03bcg of OVA or approximately 50\u00a0\u03bcg of vesicular proteins injected into unilateral foot pad of C57BL/6 mice. Fluorescence imaging in vivo was obtained carrying out the system of fluorescence images at different time points . The left and right inguinal lymph nodes of all mice were collected at 96\u00a0h after injected, fluorescence images were obtained and the fluorescence signal intensity was calculated using the imaging system.6 OVA-expressing B16F10 cells were suspended in PBS and subcutaneously injected into the left posterior back of C57BL/6 mice to challenged primary tumor. On 6 d, 3\u2009\u00d7\u2009106 OVA-expressing B16F10 cells were subcutaneously injected into the right posterior back of C57BL/6 mice to challenged distant tumor. On 9 d after first inoculation, primary tumor was about 200 mm3 in size, various formulations were injected into the tail vein containing equivalent 4.5\u00a0mg/kg of ICG or approximately 100\u00a0\u03bcg of vesicular proteins (five mice per group). After 12\u00a0h, the primary tumor was irradiated by 808\u00a0nm laser (0.75\u00a0W/cm2) for 8\u00a0min, and the temperature of tumor surface was monitored by near-infrared thermal imaging instrument. On 13, 15 and 17 d after challenge of the first tumor, mice treated with photothermal therapy were subcutaneously injected with different immune drugs containing equivalent 10\u00a0\u03bcg of OVA or 10\u00a0\u03bcg of R837 or approximately 50\u00a0\u03bcg of vesicular proteins for immunotherapy of distant tumor (five mice per group). The body weight of the mice was recorded using electronic balance, and the tumor size and volume were measured using a vernier caliper every other day. The calculation formula (V) was V\u2009=\u2009d2*D/2, d and D was mm in the shortest and longest diameters of the tumors respectively. Animals were euthanized when they showed signs of impaired health or tumors larger than 2cm3.Female C57BL/6 mice (6\u2009~\u20098\u00a0weeks) were purchased from Shanghai slake laboratory animal co., LTD and used according to the agreement approved by committee of Xiamen University Laboratory Animal Center. The mice were randomly divided into groups. 3\u2009\u00d7\u200910To test the immune response in the treated mice, the mice were sacrificed and serum, tumor, and lymph nodes were collected on 3 d after photothermal treatment or final immunization. The levels of TNF-\u03b1, IL-6, IL-12 and IFN-\u03b3 in serum were detected by ELISA kit according to the instructions. The tissues were homogenized to prepare single-cell suspension, and tumor cells were labeled with anti-mouse CD3a and CD8 antibodies, and lymph node cells were labeled with anti-mouse CD11c and CD86 antibodies. Then, the content of CD8\u2009+\u2009T lymphocytes and mature DC cells were detected by flow cytometry.The main organs and tumor tissues of mice in each treatment group were collected and fixed in 4% paraformaldehyde. Hematoxylin and eosin staining were performed before electron microscopy.All the statistical figures are mean\u2009\u00b1\u2009standard deviation, P values were calculated by Student's t test . All statistical analyses were performed using the IBM SPSS statistics 20.Additional file 1: Figure S1. (a) The standard curve of R837 was detected by HPLC; (b) The standard curve of ICG was measured by ultraviolet spectrophotometer; (c) Analysis of drug loading efficiency using nanometer delivery platform. Figure S2. The temperature change curve of the aPD-L1 NVs-ICG and ICG with corresponding concentration under the near infrared (NIR) laser irradiation at 808\u00a0nm wavelength . Figure S2. The temperature change curve of the aPD-L1 NVs-ICG and ICG with corresponding concentration under the near infrared (NIR) laser irradiation at 808\u00a0nm wavelength . Figure S4. (a) Temperature change curve of the tumor under different treatments under 808\u00a0nm laser irradiation . (b)Tumor volume growth curve for irradiated distant secondary tumors (in right flank) in mice after PTT with different drugs. (c) Survival curves of the mice after PTT with different drugs. Images of Hematoxylin and eosin (H&E) staining of irradiated primary tumors and distant secondary tumors sections after various treatments, respectively. Scale bar: 100\u00a0\u00b5m. (b)Weight curve after PTT with the different substances. Images of H&E staining of the main viscera after PTT, respectively. Scale bar: 100\u00a0\u00b5m. Figure S5. Productions of IL-6 (a), IL-12 (b), TNF-\u03b1 (c) in serum from the mouse determined by ELISA after treated with PTT for 3 d. P values were calculated by Student's t test . Figure S6. Cell viability of the different concentrations of AAI-R837 on B16 cells and DCs after co-incubation for 24\u00a0h. Figure S7. The curve of fluorescence signal intensity in inguinal lymph nodes with time after subcutaneous injection of various drugs. Figure S8. (a) Images of Hematoxylin and eosin (H&E) staining of distant secondary tumors sections after photoimmunotherapy, respectively. Scale bar: 100\u00a0\u00b5m. (b) Weight curve after photoimmunotherapy with the different substances. (c) Images of H&E staining of the main viscera after photoimmunotherapy, respectively. Scale bar: 100\u00a0\u00b5m."} +{"text": "Clubionazilla-group is a relatively small species group, distributed exclusively in East Asia, with only three species clearly documented so far.The Clubionahooda Dong & Zhang, 2016, which was previously placed in the C.trivialis-group, is assigned to the C.zilla-group in the present paper. A new spider of the C.zilla-group from Jiugong Mountain in China is described under the name of C.jiugong sp. nov. Detailed descriptions and photographs of the new species are provided. Clubionazilla D\u00f6nitz & Strand, 1906 was reported by C.zilla-group, was established to accommodate C.zilla. The C.zilla-group was rede\ufb01ned by Anaclubiona by Anaclubiona was rejected by zilla-group. The genus Anaclubiona Ono, 2010 is currently considered as a junior synonym of Clubiona by The female of zilla-group can be easily recognised by its tiny body (with body length 1.8\u20133.6 mm), in association with the characteristic genital organs. The male palp has a developed and occasionally branched embolic apophysis. The female epigyne has a pair of guide pockets (or hoods) or a transverse hood near the copulatory openings. The zilla-group is a relatively small taxon, with only three species having been clearly documented: C.zilla widespread in Japan, C.minima endemic to Honshu in Japan, C.tanikawai Ono, 1989 from Ryukyu Is. in Japan and Hunan and Taiwan in China. C.hooda Dong & Zhang, 2016 was assigned to the trivialis-group in the original publication and both sexes possess certain characters associated with the zilla-group. Therefore, it is very likely they are the opposite sexes of the same species. Based on that, as well as the DNA barcoding data, we matched the female and male together. This species is new to science and is described under the name of C.jiugong sp. nov. The aim of the current paper is to describe the new species, providing detailed morphological descriptions and illustrations.While examining spiders collected from Jiugong Mountain, Hubei Province, China Fig. A, we fouSpecimens in this study were collected by hand collecting from leaf-litter in Mt. Jiugong, Hubei. Spiders were fixed and preserved in 95% ethanol. Specimens were examined with an Olympus SZX7 stereomicroscope, details being studied with an Olympus CX41 compound microscope. Female epigynes and male palps were examined and illustrated after being dissected. Epigynes were removed and cleared in warm lactic acid before illustration. The vulva was also imaged after being embedded in Arabic gum. Photos were made with a Cannon EOS70D digital camera mounted on an Olympus CX41 compound microscope. The digital images were taken and assembled using Helifocus 3.10.3 software package . The disA DNA barcode was also obtained for the species matching. A partial fragment of the mitochondrial cytochrome oxidase subunit I (CO1) gene was amplified and sequenced for two specimens, using the primers LCOI1490 (5\u2019-GGTCAACAAATCATAAAGATATTG-3\u2019) and HCOI2198 (5\u2019-TAAACTTCAGGGTGACCAAAAAAT-3\u2019). For additional information on extraction, amplification and sequencing procedures, see All measurements were obtained using an Olympus SZX7 stereomicroscope and given in millimetres. Eye diameters are taken at the widest point. The total body length does not include chelicerae or spinnerets length. Leg lengths are given as total length . Most of the terminologies used in text and figure legends follow All specimens are deposited Museum of Guizhou Education University, Guiyang, Guizhou, China .Yu & Zhongsp. n.9B74C4BF-3DDC-5DB0-98D9-7D1EDCFDB51436f4d405-32d2-4119-97b6-9c3758c20f22Type status:Holotype. Occurrence: recordedBy: Qianle Lu; individualID: YHCLU0274; individualCount: 1; sex: male; lifeStage: adult; behavior: foraging; preparations: whole animal (EtOH); associatedSequences: MZ020606GenBank: ; Taxon: order: Araneae; family: Clubionidae; genus: Clubiona; specificEpithet: jiugong; scientificNameAuthorship: Yu & Zhong; Location: continent: Asian; country: China; countryCode: CHN; stateProvince: Hubei; county: Tongshan; locality: Jiugongshan Nature Reserve; decimalLatitude: 29.39; decimalLongitude: 114.65; Identification: identifiedBy: Hao Yu; dateIdentified: 2020-07; Event: samplingProtocol: by hand; samplingEffort: 10 km by foot; year: 2020; month: 7; day: 3; Record Level: institutionCode: MGEU; basisOfRecord: Preserved SpecimenType status:Holotype. Occurrence: recordedBy: Qianle Lu; individualID: YHCLU0275; individualCount: 1; sex: female; lifeStage: adult; behavior: foraging; preparations: whole animal (EtOH); associatedSequences: MZ020605GenBank: ; Taxon: order: Araneae; family: Clubionidae; genus: Clubiona; specificEpithet: jiugong; scientificNameAuthorship: Yu & Zhong; Location: continent: Asian; country: China; countryCode: CHN; stateProvince: Hubei; county: Tongshan; locality: Jiugongshan Nature Reserve; decimalLatitude: 29.39; decimalLongitude: 114.65; Identification: identifiedBy: Hao Yu; dateIdentified: 2020-07; Event: samplingProtocol: by hand; samplingEffort: 10 km by foot; year: 2020; month: 7; day: 4; Record Level: institutionCode: MGEU; basisOfRecord: Preserved SpecimenMale 5'CTTGATCTGCTATAGCAGGAACAGCTATAAGTGTTATAATTCGTATAGAATTAGGACAATCTGGAACATTTTTAGGAGATGATCATTTATATAATGTAGTAGTTACAGCTCATGCTTTTGTTATAATTTTTTTTATAGTAATACCTATTTTAATTGGAGGTTTTGGAAATTGAATAATTCCTATGATATTAGGAGCAGCTGATATAGCTTTTCCTCGTATAAATAATTTAAGTTTTTGATTATTACCTCCTTCGTTATTTATATTATTTATATCTTCTATAGCTGAAATAGGTGTGGGAGCAGGGTGAACTATTTATCCTCCTCTTGCATCTAGTATAGGTCATACAGGAAGAGCTATAGATTTTGCTATTTTTTCGTTACATCTAGCTGGAGCTTCTTCTATTATAGGGGCTGTAAATTTTATTACTACTATTATTAATATACGATATATTGGGATGAGAATAGAAAAAGTTCCATTATTTGTTTGGTCTGTTATAATTACTGCAGTACTCTTATTATTATCATTACCTGTATTAGCAGGTGCTATTACTATATTATTGACTGATCGAAATTTTAATACATCTTTTTTTGATCCAGCTGGAGGGGGAGATCCTATTTTATTTCAGCATTTATTTTGATTTTTTGG3' 5'TTTGATCTGCTATAGTAGGAACAGCTATAAGTGTTATAATTCGTATAGAATTGGGACAATCTGGAACATTTTTAGGAGATGATCATTTATATAATGTAGTAGTTACAGCTCATGCTTTTGTTATAATTTTTTTTATAGTAATACCAATTTTAATTGGAGGTTTTGGAAATTGAATAATTCCTATGATATTAGGAGCAGCTGATATAGCTTTTCCTCGTATAAATAATTTAAGTTTTTGATTATTACCCCCTTCGTTATTTATATTATTTATATCTTCTATAGCTGAAATAGGTGTGGGAGCAGGGTGAACTATTTATCCTCCTCTTGCATCTAGTATAGGTCATACAGGAAGAGCTATAGATTTTGCTATTTTTTCGTTACATCTAGCTGGAGCTTCTTCTATTATAGGGGCTGTAAATTTTATTACTACTATTATTAATATACGATATATTGGGATGAGAATAGAAAAAGTTCCATTATTTGTTTGGTCTATTATAATTACTGCAGTACTCTTATTATTATCATTACCTGTATTAGCAGGTGCTATTACTATATTATTGACTGATCGAAATTTTAATACATCTTTTTTTGACCCAGCTGGAGGAGGAGATCCTATTTTATTTCAGCATTTATTTTGATTTTTTGG3' , but is consistently separable by its genitalia. Male of the new species resembles that of C.hooda (C.hooda) and by the EPA originating from the prolateral portion of the tegulum, pointed to the retrolateral side and differ from C.zilla by: (1) copulatory openings closely spaced and partly fused, situated at the medial portion of epigynal plate posterior margin ; (2) the proximal half of copulatory ducts close together near the copulatory openings in female (C.zilla-group species with tiny bodies (less than 3.6 mm), C.trivialis-group is median sized clubionids . Although we have not examined the types of C.hooda, the species was well described with high-quality illustrations: developed embolar part apophysis in male and the paired epigynal hoods in female . The present study follows Anaclubiona as a synonym of Clubiona, rather than resurrecting the generic status of the zilla-group. Consequently, we temporarily place the new species in Clubiona sensu lato and assign them to C.zilla-group.The genus"} +{"text": "Our results show that Rad53 protects replication forks in part by antagonising Mrc1 stimulation of CMG unwinding.The Rad53 DNA checkpoint protein kinase plays multiple roles in the budding yeast cell response to DNA replication stress. Key amongst these is its enigmatic role in safeguarding DNA replication forks. Using DNA replication reactions reconstituted with purified proteins, we show Rad53 phosphorylation of Sld3/7 or Dbf4-dependent kinase blocks replication initiation whilst phosphorylation of Mrc1 or Mcm10 slows elongation. Mrc1 phosphorylation is necessary and sufficient to slow replication forks in complete reactions; Mcm10 phosphorylation can also slow replication forks, but only in the absence of unphosphorylated Mrc1. Mrc1 stimulates the unwinding rate of the replicative helicase, CMG, and Rad53 phosphorylation of Mrc1 prevents this. We show that a phosphorylation-mimicking Mrc1 mutant cannot stimulate replication in vitro and partially rescues the sensitivity of a Saccharomyces cerevisiae (ATR in humans) . Mec1 th humans) . In addi humans) .rad53 or mec1 mutant cells , and finally firing factors, DNA polymerases, and accessory factors were added to initiate DNA replication. We followed replication progression by separating the products on alkaline agarose gels to visualise incorporation of radiolabelled dCTP. Rad53 inhibits late origin firing in vivo by phosphorylating two substrates: Dbf4 and Sld3 . To detely shown , whilst ly shown . As showly shown . Howeverly shown .To test whether Rad53 also inhibited Sld3, we took a similar approach by pre-incubating Sld3/7 with Rad53 prior to addition to the replication reaction. Similar to DDK, pre-incubation of Sld3/7 with Rad53 and ATP resulted in reduced mobility of Sld3 in SDS-PAGE and inhiNext, we wanted to determine whether Rad53 could affect replication elongation. We pre-incubated the elongation factor mix with Rad53 and ATP, then added this to reactions after MCM loading, DDK phosphorylation, and firing factor addition. We stopped the reactions at early time points so that any effects of elongation could be more easily seen by the size of the leading strand replication products. KD are non-essential proteins known to directly stimulate replication fork rate . Their iKD , indicatecreased . This leecreased . These dKD was detected in Mrc1 immunoprecipitates as a measure of CMG helicase activity, Devbhandari and Remus have recently shown that more U* product is generated in reactions containing M/C/T than in reactions lacking M/C/T . This co17AQ protein, which cannot be phosphorylated by Mec1 that indeed exhibited faster replication than wild-type Mrc1 after Rad53 phosphorylation . Unfortunately, these mutants \u2014 especially Mrc119A \u2014 did not stimulate replication to the rate of unphosphorylated wild-type Mrc1 . This is likely due, in part, to the fact that these mutants exhibited defects in promoting faster replication in the absence of Rad53 indicating that they are not completely functional . Moreover, even Mrc119A was still inhibited slightly by incubation with Rad53 (compare lanes 5 and 6), suggesting that additional sites not mutated in this construct can still be phosphorylated by Rad53 and inhibit Mrc1 function. Cells expressing 19AMRC1 as the sole copy of MRC1 were not sensitive to the replication stress agent hydroxyurea (HU) . Another8D) was unable to stimulate replication even in the absence of Rad53 were identified in a phosphoproteomics screen of MMS-treated cells , whilst the mrc1\u2206, rad9\u2206 double mutant was completely defective in Rad53 phosphorylation (lanes 12\u201315). mrc1\u0394 rad9\u0394 mutants are inviable or MMS (0.006%), although they did not promote additional survival to higher concentrations (8 mM HU and 0.01% MMS) relative to MRC1 wild type . Similar01% MMS) arguing 1\u0394 cells . MRC18D ild type . These dOur results show that, in addition to its role in checkpoint activation upstream of Rad53, Mrc1 also has a role downstream of the checkpoint, as a substrate of Rad53. Phosphorylation of Mrc1 by Rad53 prevents Mrc1-dependent stimulation of CMG unwinding, leading to a reduced replication fork rate. We suggest that linking these two roles could allow Rad53 to slow replication speed specifically at a stressed or damaged fork, and, therefore, act efficiently at stochastic fork stalling events without initiating a global checkpoint response. But under more severe replication stress, where more Rad53 is active, Rad53 could phosphorylate Mrc1 at all forks to slow replication globally.Using a novel assay to measure DNA unwinding activity, we found that unwinding by CMG is not very synchronous, as evidenced by shallow unwinding curves. Moreover, sites as close as 1 kb from the origin were not completely unwound after 50 min. In the presence of Mrc1, unwinding was faster and the curves were steeper, suggesting more synchronous unwinding leading to even the site 2 kb from the origin approaching 100% unwinding by 30 min. In single-molecule experiments, CMG frequently paused and backtracked while unwinding DNA . The abi8D mutant showed normal checkpoint activation supporting the idea that it remains bound to replication forks. However, we note that Rad53 can target more than these eight sites and full phosphorylation may affect more functions and protein-protein interactions than those disrupted in the Mrc18D mutant.A recent structure of CMG bound to M/C/T suggests the C-terminus of Mrc1, containing the majority of its Rad53 phosphorylation sites, may contact Cdc45 and Mcm2 . Other wmrc1\u2206 cells treated with HU, suggesting that Mrc1 has some role in restraining CMG at stalled forks . The peptides were synthesised using FMOC for temporary \u03b1-amino group protection. Protecting groups used were Pbf for arginine, OtBu for glutamic acid and aspartic acid, Trt for asparagine, glutamine, histidine, and cysteine, tBu for serine, threonine and tyrosine, and Boc for lysine and tryptophan. Each amino acid was coupled by activating its carboxylic acid group with DIC in the presence of HOBT. Individual aliquots of amino acids were spotted on to a cellulose membrane which has been derivatised to have 8 to 10 ethylene glycol spacers between the cellulose and an amino group. Synthesis was accomplished by cycles of coupling of amino acids, washing then removal of the temporary \u03b1-amino protecting group by piperidine followed by more washing. Once the required number of cycles of coupling and deprotection and washing had been completed, the membranes were treated with a solution of 20 ml containing 95% TFA, 3% TIS, and 2% water for 4 hr. Following this treatment, membranes were washed four times with DCM, four times with ethanol, and twice with water to remove side chain protecting groups and TFA salts and once again with ethanol for easier drying. Just prior to kinase assay, membranes were washed extensively in reaction buffer, then incubated with 80 nM Rad53, 10 \u00b5M ATP, and 0.02 \u00b5Ci/\u00b5l \u03b3exo- , 20 nM GINS, 15 nM CDK, 20 nM Csm3/Tof1, 20 nM Mrc1, 20\u201330 nM Sld3/7, 20 nM Mcm10, 20\u201350 nM Sld2, and 350 nM RPA. At each time point, 2.5 \u00b5l of reaction was added to a tube containing 5 \u00b5l replication buffer and 1 \u00b5l MseI (NEB) for 3 min, and then the reaction was quenched with the addition of EDTA. Samples were then deproteinated with SDS and proteinase K followed by column clean-up (QIAquick PCR purification kit) according to manufacturer\u2019s instructions. The final elution was done with 300 \u00b5l 10 mM Tris pH 8. Then qPCR was performed in triplicate using 4 \u00b5l sample in 8\u20139 \u00b5l reaction with FastStart Universal SYBR Green Master Mix (Roche) and primers flanking each MseI cassette was then fit with a spline and normalised to the unwound DNA in the last timepoint of the closest site (200 bp from origin). The unwinding rate was then calculated by integrating the spline and averaging over the last three sites .AGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGGACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGGGGAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTGGCCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTCCTCGATTTTTTTATGTTTAGTTTCGCGGACGACGGTTTCGAGGTGGCGGTCTGGACCACGCCGGAGAGCGTCGAAGCGGAGGCGGTGTTCGCCGAGATCGGCTCGCGCAAAGCCGAGTTGAGCGAACTAAACATAAAAATACAGCATCAGATGGTAGGCCTCCTGGCGCCGCACCGGCCTCAGCATCCGGTACCTCAGCTGGCCACATCACTGTCTTTCTTATGACGGTACTACCGGTGTTCACTGCACCAAGGTAACACTCATTAAATTAAGGTTAAATTAATCTACACAATTCTCTTTTGCTATTGGTACCGGATTCTCCAGCTCTGACTTCAGCGTCTCTGAAGGAATCTTTGCAGGTGCTTACGCTTACTACCTAAACTACAATGGTGTTGTCGCTACTAGTGCCGCTTCTTCAACCACTGGATCTGGTCCTAGGGCTTCGGTCCGCCCCTACTTCAGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAATTCGGTTAAGTTAAGTTAAGGTTAAAGAAGCTAACGCCAGGGTTTTCCCAGTCACGATTCGGTGCTTCCGTTACCGGTTCAACTGCTTCCACTTCATGGGCTACTTTTTGGACCGGAACGGCTGGTACTATCGGCCTGGTATCATCCTTTACCGAAGCAACATCTGTTTACACTACAACACTAGACCAAGCACAGTCGTAGTTTCTTGTTCAGAGATGACTCCAATGGTAACGTCTATACCATTACCACAATCATTAAGTTAAATTAAGTTAATCAAACCGTTCCATGCTCATCCACTACCGCCACTATTACTTCTTGTGATGAAACTGGATGTCACGTTAGTACATCAACCGGTGCTGTTGTAACTGAAACCGTTTCTTCCAAGGCATACACAACTGCCAAAGTAACTCGTTGTGACGCCAATCAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCATGGTTAAAGTTAACTTAAATTAAGGTACGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAGGCTCAATGTAACTTAGCCACTGTCAATTGGGAATGTTCCAGGGATTCATGGACAACAACTGCAACTGGAGTATCATACACCACTGTCACCGTAACCCACTGTGACGACAATGGCTGTAACACCAAGACTAAGCTCCTGAAGCTACCACCACAACTATCGCCCACCAGGACCACCGTCACCTTTAGTGATGACAATGAAGGTAAGACCTTGGGTGAGTCTGGTCCAGCGGAGGGCCACTACTGTTTCTCCAAAGACATACACCACCGCTACTGTTACTCAGGGGGATAAAAATGCCTGCCTCACCAAGACTGTCACTTCTGAATGTCCTGAAGAAACTTCAGCAACTACTACTGTCACTTCTGAGGGTTCTAAAGCAACCTCATTGAGTCGACGCGGGGGCGACGATTAACCTTAACGTTAAGTTAAGCTAGCACGACTACGCATCCCTCTGACTACTTCTCGGGGTGGGACTATACTGGTACCGATACGGGCTGTGATGACAACGATGTGTAGAACTGGGACAATCAGATCTGAGGCCCCTGAAGCCACAACGGGTACTGTTTCTAACAACAGATACAACATGGAGGGCCAACATTGTCACAATAGAAGCTCCGCCAGAAACAGTAGAAACTTCAGAAACCAGTGCTGCCCCTAAGGACATACACTACTGCCACTGGTTACTCAATGGTTTAGAGGGTGGTTGCCACGTCAAGATAATCACCTCTAAAATACCTGAAGCTACTTCAACCGTCACGGGTGCTTCTCCAAAACGGCCTTACATAGCCGGATACAGTGACTTTGACAGGTTTGCGGGGCACAGCAATGACTTGCATAGCTGCGTGCGGGGGAAGGAACTCTTGCGTCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCATTATGGTTAACTTAAGTTAATTTAAGCTATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCAGTGAGTATCTCTGCTTGACGACCCCTTGGCGCAGAGGTGCTGGCCGCGTGCTAAGTTGAAGCGGCTGCACTGCTGCAAGGTCCGTCACGGAGGCGTCGGACCGGCAGGAGCACTAGCCCATCGACCCGTACGGGAACACTCTATATCGCTCTCGGACGGACATTCTGGATCCTCTAGAGTCGACCTGCAGGCATGCAAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTGGAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTG.600 0.5 and arrested with 20 \u00b5g/ml of alpha-factor for 2\u20133 hr at 25\u00b0C. Cells were washed two times with YPD and then resuspended in YPD + 200 mM HU. Cells were then harvested at the indicated times, and protein was extracted with 10% trichloroacetic acid. Extracts were then processed by 3\u20138% Tris-acetate SDS-PAGE, transferred to nitrocellulose, and immunoblotted with anti-Flag and anti-Rad53 antibodies.Log-phase yeast cultures in YPD were diluted to OD600 0.3 and arrested with 20 \u00b5g/ml of \u0251-factor for 3 hr at 25\u00b0C. Cells were washed two times with YPG and then resuspended in YPG + 200 mM HU + 200 \u03bcg/ml BrdU. After 1 hr, cells were washed two times with YPG and then resuspended in YPG + 2 mM thymidine. Cells were harvested at the indicated times, and DNA was extracted with phenol-chloroform. DNA was run on a 0.8% alkaline agarose gel, then alkaline transferred to a positively charged nylon membrane with the VacuGene XL Vacuum Blotting System (Amersham), and immunoblotted with anti-BrdU antibody .Log-phase yeast cultures in YP-raffinose were centrifuged and resuspended in YP-galactose to ODK227A,K339A was constructed by PCR with mutated oligos on pET21b-RAD53 and the 3xFLAG tag between the BamHI and NotI sites of pRS40N and then was cut with Blp1 to modify the already integrated codon-optimised MRC1 at the his3 locus. pVP14 (MRC117AQ) contains all S/T residues followed by Q sites mutated to A as in 14A) contains the following mutations: S911A, S918A, S920A, T952A, S957A, T996A, T997A, S1006A, S1033A, T1036A, S1039A, S1040A, S1043A, T1045A. pAWM25 (MRC119A) contains the following mutations: T882A, S911A, S918A, S920A, S924A, T932A, T952A, S957A, S958A, T996A, S997A, S1006A, S1010A, S1033A, T1036A, S1039A, S1040A, S1043A, T1045A. pAWM18 (Mrc141A) contains the following mutations: T882A, S911A, S918A, S920A, S924A, T932A, S937A, T952A, S957A, S958A, S961A, T963A, S965A, T967A, S969A, T970A, T971A, S972A, T974A, T977A, T996A, S997A, S1006A, S1010A, S1013A, T1015A, T1027A, S1033A, T1036A, S1039A, S1040A, S1043A, T1045A, T1050A, T1060A, T1063A, T1079A, T1081A, S1083A, S1089A, S1093A. pAWM17 (Mrc18D) contains the following mutations: S911D, S918D, S920D, T952D, S957D, T996D, S997D, S1006D. pAWM47 and pAWM48 contained base pairs 1424\u20133288 of MRC1 between XhoI and BamHI and 127 bp of the 3\u2019 UTR of MRC1 between BamHI and NotI. The 3xFLAG tag was added with flanking BamHI sites, and the plasmids were integrated into yeast after cutting with XbaI. The template used in the CMG helicase assay was made by modifying a plasmid with a pBlueScript vector and contained a synthetic origin with either two ORC binding sites 70 bp apart (pAWM36) or no ORC binding sites (pAWM37) (Yeast strains are listed in 1b-RAD53 . Mrc1 fr53 (MRC1 and modi(pAWM37) and a sy public reviews designed to be posted alongside the preprint for the benefit of readers; ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: i) Acceptance summary:S. cerevisiae in blocking both initiation of DNA replication and replication fork progression via inhibition of Mrc1 activation of the replicative DNA helicase. The paper elegantly utilizes the power of biochemical reconstitution of complete DNA replication with purified proteins and its regulation by checkpoint kinases.This paper addresses an important role for the DNA replication checkpoint kinase Rad53 in the yeast Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, including Bruce Stillman as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Kevin Struhl as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Oscar M Aparicio (Reviewer #3).Thank you for submitting your article \"Rad53 checkpoint kinase regulation of DNA replication fork rate via Mrc1 phosphorylation\" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.We all felt that this is a very strong paper that confirms the effect of Rad53 kinase on Sld3 and Dbf4 activities in initiation of DNA replication, but goes further and identifies Mrc1 and Mcm10 as substrates for the kinase in regulating replication fork progression and CMG activity. The results are clear and support the conclusions.Essential revisions:The reviewers made a number of suggestions, but only a subset of these are listed below and we ask that you consider adding the requested data.(1) Page 10 and Figure 5E. The observation that Mrc119A can still be inhibited by Rad53 kinase, albeit less than the wild type Mrc1 protein, might suggest (1) that there are additional phosphorylation sites (as suggested by the authors) or (2) that Rad53 binds directly to Mrc1 and this interaction may partially block its stimulation of CMG. Can the authors test if the Rad53 binds to purified Mrc1? The reason for asking this question is that it is possible that both Mrc1 and Rad53, which are both known to be located at active DNA replication forks, create a solid-state system, feedback control at the replication fork for Mec1 activating Rad53 kinase and Rad53 phosphorylating Mrc1.(2) How selective is the effect of Rad53 on the Mrc1-dependent replisome slowdown in the biochemical assays? It would be useful to exclude the possibility that any active kinase could phosphorylate Mrc1 and cause the same phenotype.(3) In addition to the viability assays shown in Figure 6, it would be useful if the authors could provide an assay that monitors replication fork progression in vivo to ensure that the Mrc1-8D protein restrains fork progression even in the absence of Rad53.Reviewer #1 (Recommendations for the authors):1. Page 10 and Figure 5E. The observation that Mrc119A can still be inhibited by Rad53 kinase, albeit less than the wild type Mrc1 protein, might suggest (1) that there are additional phosphorylation sites (as suggested by the authors) or (2) that Rad53 binds directly to Mrc1 and this interaction may partially block its stimulation of CMG. Can the authors test if the Rad53 binds to purified Mrc1? The reason for asking this question is that it is possible that both Mrc1 and Rad53, which are both known to be located at active DNA replication forks, create a solid-state system, feedback control at the replication fork for Mec1 activating Rad53 kinase and Rad53 phosphorylating Mrc1.Reviewer #2 (Recommendations for the authors):The key point that needs to be addressed, in my opinion, is whether the phenomena identified in the biochemical studies are reflective of the in vivo situation. I offer below a few comments that should help addressing this issue.(1) How selective is the effect of Rad53 on the Mrc1-dependent replisome slowdown in the biochemical assays? I ask this since recombinant Rad53 is highly active and it would be useful to exclude the possibility that any active kinase could phosphorylate Mrc1. Alternatively, does the effect of Rad53 on in vitro DNA replication dependent on its two FHA domains?(2) While I do have sympathy with the authors for their struggle in identifying the key Rad53 target sites on Mrc1, I feel they should nevertheless provide evidence that one or more of the sites mutated in the Mrc1-8D variant are indeed phosphorylated in cells, in a Rad53-dependent manner, in response to DNA replication stress.(3) In addition to the viability assays shown in Figure 6, it would be useful if the authors could provide an assay that monitors replication fork progression to ensure that the Mrc1-8D protein restrains fork progression even in the absence of Rad53.Reviewer #3 (Recommendations for the authors):I would really appreciate a figure showing the locations of the 8/14/19 mutations. I feel these should be added to the diagram in S3B.I suggest the authors construct and test an 8A version. If this allele exhibits more limited effects than 14A, it will strengthen the inference that the 8D changes are not just breaking the protein.I think a bit more in vivo analysis of these mutants would be useful and appreciated, especially by experts such as bulk analysis of DNA content during S phase progression. One would predict that the 8D allele would cause slow replication even without drugs. However, it might have little effect given the apparently intact checkpoint signaling and presumably intact origin firing control.One set of easy experiments that seems to be missing is more analysis of the 14A and 19A alleles in vivo. These alleles might be predicted to exhibit sensitivity to HU and MMS. Should effects be noted, it will probably be important to examine Rad53 activation in these mutants similar to analyses in 6B and C to rule out possible effects on transducing the signal to activate Rad53 as opposed to defects in receiving effector signals from activated Rad53. It is possible that the 14A and 19A mutations will have no sensitivities due to incomplete penetrance or redundant mechanisms, but worth a try. Essential revisions:The reviewers made a number of suggestions, but only a subset of these are listed below and we ask that you consider adding the requested data.(1) Page 10 and Figure 5E. The observation that Mrc119A can still be inhibited by Rad53 kinase, albeit less than the wild type Mrc1 protein, might suggest (1) that there are additional phosphorylation sites (as suggested by the authors) or (2) that Rad53 binds directly to Mrc1 and this interaction may partially block its stimulation of CMG. Can the authors test if the Rad53 binds to purified Mrc1? The reason for asking this question is that it is possible that both Mrc1 and Rad53, which are both known to be located at active DNA replication forks, create a solid-state system, feedback control at the replication fork for Mec1 activating Rad53 kinase and Rad53 phosphorylating Mrc1.KD does not slow replication. However, we have now included new data showing that neither wild type Rad53 nor Rad53KD co-immunoprecipitate appreciably with Mrc1. Under the same buffer conditions \u2014 identical to the buffer conditions in the replication reactions \u2014 Csm3/Tof1 co-immunoprecipitate nearly stoichiometrically. We think that, together, these results indicate that Rad53 inhibition of Mrc1 is likely via phosphorylation and not binding. We have added this point in the section \u201cRad53 inhibition of replication elongation via Mrc1 and Mcm10\u201d on page 6.The reviewer correctly points out that Rad53 might inhibit Mrc1 by binding and not just by phosphorylating Mrc1, as has been suggested for another Rad53 target, DDK . We think this is unlikely because pre-incubation of Mrc1 with Rad53(2) How selective is the effect of Rad53 on the Mrc1-dependent replisome slowdown in the biochemical assays? It would be useful to exclude the possibility that any active kinase could phosphorylate Mrc1 and cause the same phenotype.It is, of course, very difficult to exclude the possibility that some other kinase(s) may also inhibit Rad53. Nonetheless, we think this is an interesting point, especially given the fact that DDK has been implicated in Mrc1 regulation in fission yeast. We, therefore, tested whether the two main replicative kinases, DDK and CDK, can phosphorylate and inhibit Mrc1. In a new Figure 2 \u2014figure supplement 1B we show that neither DDK nor CDK were able to inhibit Mrc1 in replication elongation. We have added this point to section \u201cRad53 inhibition of replication elongation via Mrc1 and Mcm10\u201d on page 6.(3) In addition to the viability assays shown in Figure 6, it would be useful if the authors could provide an assay that monitors replication fork progression in vivo to ensure that the Mrc1-8D protein restrains fork progression even in the absence of Rad53.Drosophila melanogaster deoxyribonucleoside kinase (dmdNK) and the human equilibrative nucleoside transporter (hENT1) which together allow efficient BrdU incorporation and rapid chasing of BrdU from nucleotide pools . In these experiments, cells are released from \u03b1 factor into HU in medium containing BrdU. This results in a smear of short, labelled replication intermediates from early firing origins on alkaline agarose gels detected by \u2018western\u2019 blotting with anti-BrdU antibody. We can then estimate fork rates from the extension of this labelled smear to larger sizes after HU is washed out and BrdU is replaced with thymidine. We have done this in +RAD53 cells carrying wild-type MRC1 or 8D.MRC1 The replication intermediates of 8DMRC1 cells were shorter and were extended at a slower rate consistent with the idea that 8DMRC1 promotes slower replication forks in vivo . We have added this to the \u201cMrc18D slows fork rate in vitro and in vivo and partially rescues rad53 mutant sensitivity\u201d section on page 11. Unfortunately, this approach requires synchronisation with HU, and therefore cannot be used with rad53 mutants because of the extensive fork collapse and continued origin firing in HU . Rad53 becomes rapidly dephosphorylated after release from HU, so the latter part of the curve in the new Figure 6E represents forks moving in the absence of active Rad53. Although we recognise this does not exactly address the reviewer\u2019s question, it is probably the best we can do at the moment, and it shows that the 8D mutant does indeed have slower forks in vivo.This is a good point, but not a trivial request. We include new data that we believe at least partially addresses this issue. We previously showed that we could do \u2018pulse-chase\u2019 experiments in vivo using yeast strains that express the Reviewer #1 (Recommendations for the authors):1. Page 10 and Figure 5E. The observation that Mrc119A can still be inhibited by Rad53 kinase, albeit less than the wild type Mrc1 protein, might suggest (1) that there are additional phosphorylation sites (as suggested by the authors) or (2) that Rad53 binds directly to Mrc1 and this interaction may partially block its stimulation of CMG. Can the authors test if the Rad53 binds to purified Mrc1? The reason for asking this question is that it is possible that both Mrc1 and Rad53, which are both known to be located at active DNA replication forks, create a solid-state system, feedback control at the replication fork for Mec1 activating Rad53 kinase and Rad53 phosphorylating Mrc1.Addressed in Essential revisions.Reviewer #2 (Recommendations for the authors):The key point that needs to be addressed, in my opinion, is whether the phenomena identified in the biochemical studies are reflective of the in vivo situation. I offer below a few comments that should help addressing this issue.(1) How selective is the effect of Rad53 on the Mrc1-dependent replisome slowdown in the biochemical assays? I ask this since recombinant Rad53 is highly active and it would be useful to exclude the possibility that any active kinase could phosphorylate Mrc1. Alternatively, does the effect of Rad53 on in vitro DNA replication dependent on its two FHA domains?Addressed in Essential revisions.(2) While I do have sympathy with the authors for their struggle in identifying the key Rad53 target sites on Mrc1, I feel they should nevertheless provide evidence that one or more of the sites mutated in the Mrc1-8D variant are indeed phosphorylated in cells, in a Rad53-dependent manner, in response to DNA replication stress.8D have been detected by proteomics from cells treated with MMS showing they are indeed phosphorylated in response to replication stress. Another phosphoproteomics screen identified nearby residues that were also dependent on the presence of Rad53 . We have added this to the \u201cMrc18D slows fork rate in vitro and in vivo and partially rescues rad53 mutant sensitivity\u201d section on page 11. It would be very difficult/impossible to get the kind of mass spec coverage we would need to validate all of the sites, even in vitro.Apologies \u2013 we realise that we neglected to include some important references related to previous phosphoproteomic studies. Three of the eight sites in Mrc1(3) In addition to the viability assays shown in Figure 6, it would be useful if the authors could provide an assay that monitors replication fork progression to ensure that the Mrc1-8D protein restrains fork progression even in the absence of Rad53.Addressed in Essential revisions.Reviewer #3 (Recommendations for the authors):I would really appreciate a figure showing the locations of the 8/14/19 mutations. I feel these should be added to the diagram in S3B.We have now added figures with the location of the mutations to new Figure 5 \u2014figure supplement 1B.I suggest the authors construct and test an 8A version. If this allele exhibits more limited effects than 14A, it will strengthen the inference that the 8D changes are not just breaking the protein.14A mutant is nearly wild-type in its ability to promote replication speed in vitro, it would be very surprising if the Mrc18A mutant, which is effectively a subset of the 14A mutant wasn\u2019t also like wild type. Because we thought it would be highly unlikely the 8A mutant would work less well than the 14A mutant and because of time constraints we decided not to test this mutant.Because the Mrc1I think a bit more in vivo analysis of these mutants would be useful and appreciated, especially by experts such as bulk analysis of DNA content during S phase progression. One would predict that the 8D allele would cause slow replication even without drugs. However, it might have little effect given the apparently intact checkpoint signaling and presumably intact origin firing control.8D strains. Because S phase progression by FACS analysis reflects both the state of origin firing and replication fork speed, we instead used a system using BrdU incorporation and replication intermediate size measurements to determine replication fork rates in these cells. This experiment is described in Essential revisions.The reviewer makes a great suggestion to look at replication fork rate in the Mrc1One set of easy experiments that seems to be missing is more analysis of the 14A and 19A alleles in vivo. These alleles might be predicted to exhibit sensitivity to HU and MMS. Should effects be noted, it will probably be important to examine Rad53 activation in these mutants similar to analyses in 6B and C to rule out possible effects on transducing the signal to activate Rad53 as opposed to defects in receiving effector signals from activated Rad53. It is possible that the 14A and 19A mutations will have no sensitivities due to incomplete penetrance or redundant mechanisms, but worth a try.19A mutant in response to hydroxyurea and didn\u2019t detect any phenotype . As the reviewer points out, this could be due to incomplete penetrance or redundant mechanisms. We have added this to the section \u201cIdentification of Rad53 phosphorylation sites in Mrc1\u201d on page 10.We tested the sensitivity of the Mrc1"} +{"text": "Correction to: BMC Genomics 22, 771 (2021)https://doi.org/10.1186/s12864-021-08057-4Hsp23_E2 and Prx2540-1_E, were not entered correctly into this table.Following the publication of the original article , the corThe correct sequence of YB320 is 5\u2019-TAGTTGGGGATGTCTTCGAATGTACATATGTTCCAAATCG -3\u2019 and of YB362 is 5\u2019- TAGTTGGGGATGTCTTCCATTTAGCTCATCTCCACGCTAG -3\u2019.The correct Additional file Additional file 23: Table S10. Excel file with description of synthetic DNA fragments (oligos and gene blocks)."} +{"text": "Exosomes are extracellular vesicles of endosomal origin that are released by practically all cell types across metazoans. Exosomes are active vehicles of intercellular communication and can transfer lipids, RNAs, and proteins between different cells, tissues, or organs. Here, we describe a mechanism whereby proteins containing a KFERQ motif pentapeptide are loaded into a subpopulation of exosomes in a process that is dependent on the membrane protein LAMP2A. Moreover, we demonstrate that this mechanism is independent of the ESCRT machinery but dependent on HSC70, CD63, Alix, Syntenin-1, Rab31, and ceramides. We show that the master regulator of hypoxia HIF1A is loaded into exosomes by this mechanism to transport hypoxia signaling to normoxic cells. In addition, by tagging fluorescent proteins with KFERQ-like sequences, we were able to follow the interorgan transfer of exosomes. Our findings open new avenues for exosome engineering by allowing the loading of bioactive proteins by tagging them with KFERQ-like motifs. LAMP2A targets cytosolic proteins to extracellular vesicles to modulate interorgan communication. Exosomes are nanosized vesicles of 40 to 160 nm in diameter that are secreted by most cell types to the extracellular space , referred to as multivesicular bodies (MVBs) machinery the LAMP2A gene in a human cell line with normal chromosome number, ARPE-19. LAMP2A originates from the alternative splicing of the LAMP2 gene, which contains nine exons, including three different splice variants of the exon 9 . All splExtracellular vesicles (EVs) from the media supernatants of WT and LAMP2A KO ARPE-19 cells were isolated for mass spectrometry (MS)\u2013based proteome analysis. Note that there is still a lack of consensus on specific markers for exosomes, and the most common method used for exosome isolation, ultracentrifugation, is unable to separate exosomes from other populations of small EVs such as microvesicles. Following EV guidelines . TEM of 6 cells confirmed the presence of membrane proteins of endosomal origin, CD63 and LAMP2A, and the cytosolic proteins associated to EVs Flotillin-1 (FLOT1) and HSC70, as well as the absence of the endoplasmic reticulum marker calnexin and the plasma membrane marker Na+- and K+-dependent adenosine triphosphatase of 2.5 \u03bcg of whole-cell lysates and 100% of isolated sEVs secreted by 40.0 \u00d7 10P < 0.05) in LAMP2A KO sEVs and that 203 of the down-regulated proteins (67%) contained a putative KFERQ motif . Accordingly, the identified proteins were enriched in EV markers to the same level as in previously published EV studies (fig. S1D) . InitialRQ motif . These fTo address the mechanisms involved in the targeting of proteins containing KFERQ motifs to EVs, we used chimeric proteins consisting of a fluorescent protein, such as mCherry, and the KFERQ motifs of \u03b1-synuclein (VKKDQ) and ribonuclease A (KFERQ) separated by a peptide spacer , based oWe hypothesized that HSC70 is involved in the targeting of proteins into nascent EVs by binding and delivering proteins containing KFERQ-like motifs to endosomes. To assess whether HSC70 has a role in the loading of proteins into sEVs, we used Pifithrin-\u03bc (Pifi), a compound that acts by inhibiting the interaction of HSC70 with its substrates and a deWhile some EVs such as microvesicles originate from shedding of the plasma membrane, exosomes are formed by the inward invagination of the endosomal limiting membrane followed by fusion of the endosome with the plasma membrane. To investigate whether ExoSignal-tagged proteins are present in endosomes, we used the constitutively active mutant of Rab5 (Q79L) that blocks the conversion of early endosomes (EEs) into LEs, resulting in the formation of very large hybrid endosomes that accumulate large ILVs . LAMP2A Subsequently, we isolated endosomal compartments using a discontinuous sucrose gradient, according to protocol . The mat2 showed that HIF1A is present in sEVs only when cells express LAMP2A (fig. S4A). Isolation of sEVs from 769-P cells, KO for HIF1A and the ubiquitin ligase Von Hippel\u2013Lindau (AA) showed that only WT HIF1A is present in sEVs (fig. S4B). Moreover, subcellular fractioning using an OptiPrep linear gradient showed that endogenous HIF1A localizes to endosomal fractions and that LAMP2A KO decreases HIF1A presence in those fractions (fig. S4C). We next incubated endosomal fractions with glutathione S-transferase (GST) (no KFERQ motif) and HIF1A-GST, in the presence or absence of recombinant HSC70 and adenosine triphosphate (ATP), followed by trypsin treatment to degrade recombinant HIF1A protein unprotected by the endosomal membrane independently of LAMP2A , which expresses mCherry in endothelial cells, showed that GFP-ExoSignal was present in the lumen of the blood vessels and inside endothelial cells (Tg(mpeg1:mCherry), which expresses mCherry in macrophages, we observed that macrophages moving within the caudal plexus incorporated GFP-ExoSignal , expressing GFP in endothelial cells, with sEVs from WT cells, we were able to increase the number of vascular branches, including longer blood vessels with greater volume, in the retinal vascular network . The targeted proteins contain amino acid sequences, biochemically related to the KFERQ motif, a pentapeptide sequence previously involved in two selective forms of autophagy: chaperone-mediated autophagy (CMA) , 7.5 \u03bcM Pifi (Merck), and 50 nM bafilomycin A1 (Apollo Scientific).The human cell lines ARPE-19 and 769-P were cultured in Dulbecco\u2019s modified Eagle\u2019s medium (DMEM) with glutamine (Biowest) supplemented with 10% fetal bovine serum and penicillin-streptomycin . Cells were cultured at 37\u00b0C under 5% CO+,K+-ATPase, dilution of 1:1000 ; goat anti-GAPDH, dilution of 1:2000 ; mouse anti-ExoC2, dilution of 1:500 ; rabbit anti\u2013histone 3, dilution of 1:1000 ; anti-Vps4b, dilution of 1:500 , goat anti-GFP, dilution of 1:1000 , goat anti-Rab31, dilution of 1:500 ; mouse anti\u2013syntenin-1 ; and horseradish peroxidase (HRP)\u2013conjugated secondary goat anti-mouse , goat anti-rabbit , rabbit anti-goat , and goat anti-rat , dilution of 1:5000. The following were also used: Alexa Fluor 568\u2013conjugated donkey anti-goat , Alexa Fluor 488\u2013conjugated donkey anti-goat , Alexa Fluor 488\u2013conjugated donkey anti-rabbit , Alexa Fluor 546\u2013conjugated donkey anti-mouse , donkey anti-rabbit Cy3 , and donkey anti-mouse Cy5, dilution of 1:250. The following were also used: dextran, Alexa Fluor 488, 10,000 molecular weight (MW), anionic, fixable ; dextran, Alexa Fluor 647, 10,000 MW, anionic, fixable ; wheat germ agglutinin, Alexa Fluor 488 conjugate ; cathepsin B assay kit ; DAPI ; Protein G\u2013Sepharose ; Dynabeads Protein G ; nitrocellulose membranes ; ECL ; protease inhibitor cocktail ; OptiPrep iodixanol density media ; ONE-Glo Luciferase Assay System ; and Pierce BCA Protein Assay Kit .The following antibodies were used: mouse anti-LAMP2 clone H4B4, dilution of 1:1000 (WB) and 1:100 ; rabbit anti-LAMP2A, dilution of 1:500 (WB) and 1:100 ; rabbit anti-LAMP2B, dilution of 1:500 ; mouse anti-actin, dilution of 1:2000 ; goat anti-HIF1A, dilution of 1:1000 ; goat anti-CD63, dilution of 1:1000 ; mouse anti-CD63, dilution of 1:100 ; rabbit anti\u2013Flotillin-1, dilution of 1:500 ; goat anti-CTSB/cathepsin B clone S-12, dilution of 1:500 ; mouse anti-Alix, dilution of 1:500 ; mouse anti-TSG101, dilution of 1:500 ; goat anti-GST, dilution of 1:500 ; goat anti-EEA1, dilution of 1:500 ; goat anti-mCherry, dilution of 1:500 (WB) and 1:100 ; rabbit anti-DsRed, dilution of 1:100 ; mouse anti-m6pR, dilution of 1:100 ; rat anti-HSC70 clone 1B5, dilution of 1:1000 ; goat anti-Rab27, dilution of 1:500 ; goat anti-tubulin, dilution of 1:2000 ; mouse lamin B1, dilution of 1:500 ; goat anti-calnexin, dilution of 1:1000 ; goat anti\u2013NaThe sEV protein solution containing SDS and dithiothreitol (DTT) was loaded onto filtering columns and washed exhaustively with 8 M urea in Hepes buffer (m/z) was followed by higher-energy collisional dissociation (HCD) fragmentation and Orbitrap detection of the 15 most intense ions observed in the MS scan. Target value in the Orbitrap for MS scan was 1,000,000 ions at a resolution of 70,000 at m/z 200. Fragmentation in the HCD cell was performed at normalized collision energy of 31 eV. Ion selection threshold was set to 25,000 counts, and maximum injection time was 100 ms for MS scans and 300 and 500 ms for MS/MS scans. Selected sequenced ions were dynamically excluded for 45 s.Peptide samples were analyzed by nano\u2013LC-MS/MS (Dionex RSLCnano 3000) coupled to a Q-Exactive Orbitrap mass spectrometer (Thermo Scientific). Briefly, the samples (5 \u03bcl) were loaded onto a custom-made fused capillary precolumn with a flow of 5\u03bcl/min for 7 min. Trapped peptides were separated on a custom-made fused capillary column packed with ReproSil-PurC18 3-\u03bcm resin with a flow of 300 nl/min using a linear gradient from 92% A (0.1% formic acid) to 28% B (0.1% formic acid in 100% acetonitrile) over 93 min followed by a linear gradient from 28 to 35% B over 20 min at a flow rate of 300 nl/min. Mass spectra were acquired in positive ion mode, applying automatic data-dependent switch between one Orbitrap survey. MS scan in the mass range of 400 to 1200 mass/charge ratio transformation; (ii) removing common MS contaminants followed by log2 (x + 1) transformation and quantile normalization; and (iii) removing common MS contaminants followed by log2 (x + 1) transformation, quantile normalization, and abundance filtering to optimize overall Gaussian distribution of the quantitative values. For simplicity, only the quantile-normalized quantitative data are presented here. Statistical differences were calculated by using the R package limma (https://rshine.einsteinmed.org/).Statistical and bioinformatics analyses of MS data were performed in the statistical programming language R. Quantitative data from MaxQuant and VEMS were analyzed in R statistical programming language. IBAQ and protein spectral counts from the two programs were preprocessed by three approaches: (i) removing common MS contaminants followed by logFor this work, we used the plasmids pT81 HRE (x3)-Luciferase (https://zlab.bio/guide-design-resources).ARPE-19 cells were transduced with the lentiviral vector pCW-Cas9 protein] and psPAX2 (Rev and Pol proteins) into a producer cell line, 293STAR RDPro [American Type Culture Collection (ATCC)]. Recombinant viral particles were harvested 48 days later, cleared for cell debris by centrifugation at 3200The sequences were synthesized and cloned into pUC57 (GeneCust). PA-mCherry and PA-mCherry ExoSignal were subcloned into pLenti6 (Thermo Scientific) using a Gateway adapted vector using BP/LR Clonase II (Thermo Scientific) according to the manufacturer\u2019s instructions.Human LAMP2A was synthesized into pUC57 plasmid with the sequence 5\u2032-CAAGTTTGTACAAAAAAGCAGGCTCTCGAGCAatgGTGTGCTTCCGCCTCTTCCCGGTTCCGGGCTCAGGGCTCGTTCTGGTCTGCCTAGTCCTGGGAGCTGTGCGGTCTTATGCATTGGAACTTAATTTGACAGATTCAGAAAATGCCACTTGCCTTTATGCAAAATGGCAGATGAATTTCACAGTACGCTATGAAACTACAAATAAAACTTATAAAACTGTAACCATTTCAGACCATGGCACTGTGACATATAATGGAAGCATTTGTGGGGATGATCAGAATGGTCCCAAAATAGCAGTGCAGTTCGGACCTGGCTTTTCCTGGATTGCGAATTTTACCAAGGCAGCATCTACTTATTCAATTGACAGCGTCTCATTTTCCTACAACACTGGTGATAACACAACATTTCCTGATGCTGAAGATAAAGGAATTCTTACTGTTGATGAACTTTTGGCCATCAGAATTCCATTGAATGACCTTTTTAGATGCAATAGTTTATCAACTTTGGAAAAGAATGATGTTGTCCAACACTACTGGGATGTTCTTGTACAAGCTTTTGTCCAAAATGGCACAGTGAGCACAAATGAGTTCCTGTGTGATAAAGACAAAACTTCAACAGTGGCACCCACCATACACACCACTGTGCCATCTCCTACTACAACACCTACTCCAAAGGAAAAACCAGAAGCTGGAACCTATTCAGTTAATAATGGCAATGATACTTGTCTGCTGGCTACCATGGGGCTGCAGCTGAACATCACTCAGGATAAGGTTGCTTCAGTTATTAACATCAACCCCAATACAACTCACTCCACAGGCAGCTGCCGTTCTCACACTGCTCTACTTAGACTCAATAGCAGCACCATTAAGTATCTAGACTTTGTCTTTGCTGTGAAAAATGAAAACCGATTTTATCTGAAGGAAGTGAACATCAGCATGTATTTGGTTAATGGCTCCGTTTTCAGCATTGCAAATAACAATCTCAGCTACTGGGATGCCCCCCTGGGAAGTTCTTATATGTGCAACAAAGAGCAGACTGTTTCAGTGTCTGGAGCATTTCAGATAAATACCTTTGATCTAAGGGTTCAGCCTTTCAATGTGACACAAGGAAAGTATTCTACAGCTCAAGACTGCAGTGCAGATGACGACAACTTCCTTGTGCCCATAGCGGTGGGAGCTGCCTTGGCAGGAGTACTTATTCTAGTGTTGCTGGCTTATTTTATTGGTCTCAAGCACCATCATGCTGGATATGAGCAATTTTAGGGTACCACCCAGCTTTCTTGTACAAAGTGGACGCGT-3\u2032. Human LAMP2A was subcloned into pLenti6 (Thermo Scientific), a Gateway adapted vector using BP/LR Clonase II (Thermo Scientific), according to the manufacturer\u2019s instructions.P2A-GFP-ExoSignal was synthesized and subcloned into the p3E plasmid with the sequence 5\u2032-TACAGGTCACTAATACCATCTAAGTAGTTGATTCATAGTGACTGCATATGTTGTGTTTTACAGTATTATGTAGTCTGTTTTTTATGCAAAATCTAATTTAATATATTGATATTTATATCATTTTACGTTTCTCGTTCAACTTTCTTGTACAAAGTGGAGATCTatgGGAAGCGGACGTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCTGCTCGAGCAGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGGAAAGCTTTGTTAAAAAAGACCAAGCAGAACCACTACACCGAAAATTCGAACGACAATGAGGTACCGTCGACCAACTTTATTATACAAAGTTGGCATTATAAAAAAGCATTGCTTATCAATTTGTTGCAACGAACAGGTCACTATCAGTCAAAATAAAATCATTA-3\u2032. Subsequently, the sequence was subcloned into a pDestTol2pA5 plasmid (gift from C.-H. Yuhm\u2019s laboratory) according to the manufacturer\u2019s instructions (Thermo Scientific), along with p5E-Ubi and pME-mCherry (gift from C.-H. Yuhm\u2019s laboratory).g for 10 min, and used. For TSG101, VPS4b, CD63, and Alix, pLKO.1 plasmids with shRNA sequences were obtained from the RNAi Consortium . Control used was a nontargeting sequence . The target sequences used were as follows: TSG101 , VPS4b , CD63 , and Alix . For Syntenin-1 target, pLKO.1 plasmids with shRNA sequences were obtained from Sigma Mission library .All lentiviral particles were produced by cotransfection of pLenti6 and pMD 2.G (VSV-G protein) and psPAX2 (Rev and Pol proteins) into a producer cell line, 293STAR RDPro (ATCC). Recombinant viral particles were harvested 48 days later, cleared for cell debris by centrifugation at 3200For the knockdowns, Rab27a and Rab27b, as well as Rab31 microRNA (miRNA)\u2013expressing vectors, were constructed by inserting specific nucleotide sequences into pcDNA6.2-GW/EmGFP-miR plasmid harboring a Pol II promoter obtained from Thermo Scientific. These sequences are fused with GFP coding sequence. The synthesized oligonucleotides were annealed and ligated into pcDNA6.2-GW/EmGFP-miR according to the manufacturers\u2019 instructions. For Rab27a and Rab27b, tandem miRNA sequences were transferred into pAd adenoviral vector from Thermo Scientific using Gateway technology. Sequences used were as follows: Rab27a miRNA1, 5\u2032-AAACTTTGCTCATTTGTCAGG-3\u2032; Rab27a miRNA2, 5\u2032-TTAACTGATCCGTAGAGGCAT-3\u2032; Rab27b miRNA1, 5\u2032-ATTGACTTCCCTCTGATCTGG-3\u2032; and Rab27b miRNA2, 5\u2032-TTTCCCTGAAGATCCATTCGG-3\u2032. For Rab31, miRNA sequences were transferred into pAd adenoviral vector from Thermo Scientific using Gateway technology. Sequences used were as follows: miRNA1, 5\u2032-TTTCTTTGCAGGAAACGTCCC-3\u2032 and miRNA2, 5\u2032-TAAACTGAAGGCCATGTTGCG-3\u2032. Viral particles were produced, and cells were infected as described before in 0.1 M phosphate buffer (pH 7.4). Cells were processed for ultracryomicrotomy and contrasted as described . Samples were washed with phosphate-buffered saline (PBS) and fixed with 1% GA for 5 min. After washing with distilled water, grids were contrasted with uranyl oxalate (pH 7) for 5 min and transferred to methylcellulose-uranyl acetate for 10 min on ice. Observations were carried out using a Tecnai G2 Spirit BioTwin electron microscope (FEI) at 80 kV.g for 10 min, followed by centrifugation at 16,500g for 20 min. From this pellet, the microvesicle fraction was collected. To remove larger particles, the supernatant was filtered with a 0.22-\u03bcm filter unit, after which it was ultracentrifuged at 120,000g for 70 min, using an SW 32 Ti rotor. The resulting pellet was washed with PBS, and after ultracentrifugation, exosomes were resuspendedin PBS.Exosomes derived from 40 million cultured cells were isolated from 40 ml of conditioned medium. Cells were cultured in exosome-depleted medium for 48 hours, prepared in accordance with the work of Lasser and colleagues containing the exosomes was collected using a syringe with a 18-gauge needle. Exosomes were then transferred to a new ultracentrifuge tube, diluted in 35 ml of PBS, and centrifuged overnight at 100,000g and 4\u00b0C. The resulting pellet was resuspended in 100 \u03bcl of PBS, and protein quantification was performed.For MS, an extra step of purification was introduced using a 30% sucrose cushion . Cells were lysed by sonication (three rounds of 3 s at 4\u00b0C) and centrifuged at 1000g for 5 min (2\u00d7). Continuous 5 to 30% OptiPrep gradients were prepared in working solution using a gradient mixer. Postnuclear supernatant (PNS) was added to the top of 9-ml gradients and centrifuged at 100,000g for 16 hours using a 70.1 Ti rotor. Sequential fractions were collected . Fractions were recovered by ultracentrifugation at 100,000g for 30 min in an SW 32 Ti rotor. For WB, fractions were collected in PBS and further denaturated in Laemmli buffer.Continuous density gradients were performed as described previously with adaptations. Briefly, 8 \u00d7 10et al. was used (6), cultured in DMEM with 10% FBS, were washed with ice-cold PBS and collected with a cell scraper. Cells were collected to a 2-ml microfuge tube and centrifuged at 300g for 5 min at 4\u00b0C. Cell pellet was loosened using cold finger in a homogenization buffer . Samples were centrifuged at 1300g for 10 min at 4\u00b0C. Supernatant was discarded, and the pellet was gently resuspended with a wide-cut tip in three times the pellet volume of HB. The suspension was passed through a 25-gauge needle, attached to a 1-ml syringe, 10 times.For the separation of EEs and LEs, a protocol described by de Araujo g for 10 min at 4\u00b0C. The supernatant was collected and centrifuged again. The PNS that originated from the second centrifugation is used for organelle isolation. PNS (5%) is centrifuged at 4\u00b0C for 10 min at 16,000g. The recovered supernatant was collected for cytoplasmatic fraction, free of vesicles. For the remaining PNS, the sucrose concentration was adjusted to 40.6%. The PNS was then loaded at the bottom of an ultracentrifuge tube. The 40.6% solution was overlaid with 1.5 volumes of 35% sucrose solution and 1 volume of 25% sucrose solution, and the tube was filled to the top with HB. The gradient was centrifuged at 210,000g for 16 hours at 4\u00b0C, using a 70.1 Ti rotor. Each interface was collected. LEs are found in the 25%/HB interface. EEs are present in the 35%/25% interface. The endosomal fractions were diluted in 35 ml of HB solution and centrifuged at 100,000g for 1 hour in an SW 32 Ti rotor. The organelle pellets were resuspended in an appropriate buffer depending on the downstream procedures.The homogenate was then diluted in HB (1 part homogenate to 0.7 parts HB) and centrifuged at 16002PO4, pH 7.25 was adjusted with KOH) buffer according to protocol ] for 45 min at 37\u00b0C. After the incubation period, samples were cooled on ice for 1 min followed by a 30-min incubation with trypsin at 37\u00b0C. Samples were put back to ice and denaturated in Laemmli buffer, heated at 95\u00b0C for 5 min, and resolved on a 10% SDS\u2013polyacrylamide gel electrophoresis (PAGE) gel. The gel was transferred to a nitrocellulose membrane for 75 min at 100 V. The membranes were blocked with TBS-T containing 5% skim milk, followed by incubation with antibodies against the proteins of interest.Recombinant proteins GST (SICGEN) and GST-HIF1A were incubated with 5 \u03bcg of freshly isolated vesicles and recombinant protein HSC70 in Mops buffer and ATP regeneration buffer and sonicated three times, 1 s each at 4\u00b0C. Afterward, samples were centrifuged at 15,000g for 10 min, and pellets were discarded. All samples were incubated with 2 \u03bcg of the antibody against the protein of interest overnight at 4\u00b0C. Subsequently, 30 \u03bcl of Protein G\u2013Sepharose was added to the sample, and incubations proceeded at 4\u00b0C for 2 hours. Beads were washed three times with lysis buffer containing 0.15% NP-40, denatured with Laemmli buffer, and boiled at 95\u00b0C for 5 min. Samples were then analyzed by SDS-PAGE. The membranes were blocked with 5% nonfat milk in TBS-T and probed for the proteins of interest.Cells were collected from dishes with ice-cold PBS using a cell scraper and centrifuged at 15,0002PO4; pH 7.25 was adjusted with KOH). Magnetic beads were incubated with anti-LAMP2A or anti-LAMP2B antibodies for 1 hour. Subsequently, the endosomes were incubated with the magnetic beads for an additional hour at 4\u00b0C. Immunoprecipitates were then gently washed three times with KPBS. The samples were denatured with Laemmli buffer and boiled at 95\u00b0C for 5 min. Samples were then analyzed by SDS-PAGE. The membranes were blocked with 5% nonfat milk in TBS-T and probed for the proteins of interest.Afterward, endosomal immunoprecipitation was adapted from protocol , 150 mM NaCl, 10 mM iodoacetamide, 2 mM PMSF, and 1\u00d7 cocktail inhibitor from Sigma-Aldrich]. Samples were then centrifuged at 4\u00b0C for 10 min at 1000g. Supernatants were removed to a new 1.5-ml microfuge tube, and pellet (nuclear pellet) was lysed with Laemmli buffer. Supernatant was centrifuged at 4\u00b0C for 10 min at 16,000g. The resultant pellet (vesicular pellet) and the second supernatant (cytoplasm) were also lysed with Laemmli buffer. Samples were then analyzed by SDS-PAGE. The membranes were blocked with 5% nonfat milk in TBS-T and probed for the proteins of interest.To evaluate the presence of exosomal HIF1A in the nuclei of receiving cells, exosomes were isolated from ARPE-19 cells that were under hypoxia with 300 \u03bcM CoClHIF1A activity was measured by transducing cells with the reporter gene Luciferase under the control of the HRE, using ONE-Glo Luciferase Assay System (Promega), according to the manufacturer\u2019s specifications.casper, Tg(kdrl:mCherry), or Tg(mpeg1:mCherry) embryos, at 100-cell stage, were injected in the YSL with pUbi-mCherry-p2A-GFP-ExoSignal in the presence or absence of MOs at 150 nM concentration, with a microinjector under a stereomicroscope. We used a Syntenin-a MO oligonucleotide directed against the translation start site [5\u2032-ACAACGACATCCTTTCTGCTTTCA-3\u2032 (Tg(fli1a:EGFP) zebrafish with a microinjector under a stereomicroscope. At 5 dpf, embryos were fixed with 4% PFA overnight at 4\u00b0C. Embryos were washed in PBS and mounted in glycerol mounting media. Images were acquired in a Zeiss LSM 710 confocal microscope using a 40\u00d7 1.2 C-Apochromat water-immersion objective. When required, optical slices were acquired, and 3D reconstruction of the images and quantifications were performed using Imaris software.Zebrafish lines were maintained in a circulating system with 14-hour day and 10-hour night cycle periods at 28\u00b0C. The mating and spawning of zebrafish were incited by the change of dark to light. Embryos were collected before they start independent feeding, at 5 days postfertilization (dpf). Therefore, no ethical approval was necessary according to the Council Directive 2010/63/EU on the protection of animals used for scientific purposes with Tukey\u2019s multiple comparisons tests, using GraphPad Prism 8.0 software (GraphPad Software). For comparison between two groups, the paired"} +{"text": "Vagal sensory neurons (VSNs) form an important body-to-brain connection, navigating visceral organs along the rostral\u2013caudal axis of the body and crossing the surface\u2013lumen axis of organs into appropriate tissue layers6. The brain can discriminate numerous body signals through VSNs, but the underlying coding strategy remains poorly understood. Here we show that VSNs code visceral organ, tissue layer and stimulus modality\u2014three key features of an interoceptive signal\u2014in different dimensions. Large-scale single-cell profiling of VSNs from seven major organs\u00a0in mice using multiplexed projection barcodes reveals a \u2018visceral organ\u2019 dimension composed of differentially expressed gene modules that code organs along the body\u2019s rostral\u2013caudal axis. We discover another \u2018tissue layer\u2019 dimension with gene modules that code the locations of VSN endings along the surface\u2013lumen axis of organs. Using calcium-imaging-guided spatial transcriptomics, we show that VSNs are organized into functional units to sense similar stimuli across organs and tissue layers; this constitutes a third \u2018stimulus modality\u2019 dimension. The three independent feature-coding dimensions together specify many parallel VSN pathways in a combinatorial manner and facilitate the complex projection of VSNs in the brainstem. Our study highlights a multidimensional coding architecture of the mammalian vagal interoceptive system for effective signal communication.Interoception, the ability to timely and precisely sense changes inside the body, is critical for survival Single-cell profiling of vagal sensory neurons from seven organs in mice and calcium-imaging-guided spatial transcriptomics reveal that interoceptive signals are coded through three distinct dimensions, allowing efficient processing of multiple signals in parallel using a combinatorial strategy. As a key body\u2013brain axis in interoception, VSNs in the nodose and jugular ganglia transmit numerous signals from visceral organs in the respiratory, cardiovascular, gastrointestinal, endocrine and immune systems into the brainstem6. Signals communicated through VSNs are precisely discriminated in the brain for highly specific responses5, yet how this is achieved remains unclear. Extensive data show that VSNs are highly heterogeneous in multiple characteristics, including developmental origins, electrical properties, response patterns, molecular identities, terminal morphologies, sensory mechanisms, anatomical connections and physiological roles19. However, despite the accumulation of a large amount of data over the past seven decades describing the complexity of VSN characteristics, little is known about how such heterogeneity and diversity facilitates interoceptive coding at a systems level.Sensing the body\u2019s internal state is a critical life-ensuring process that maintains physiological homeostasis, provides motivational drivers and shapes our thoughts and emotions20. Similarly, the same bioactive signal (such as serotonin) released from different tissue layers within the intestine communicates different organ information to the brain21. We reasoned that a faithful coding of these features in VSNs is necessary for accurate signal discrimination in the brain. Here we developed several techniques to determine whether and how these essential features of interoceptive signals are coded in VSNs.The physiological role of an interoceptive signal can be specified by three important features: visceral organ; tissue layer; and stimulus modality. Stimuli that differ in these features represent different body changes. For example, stretching the arterial wall implicates an increase in blood pressure, whereas stretching the stomach wall signals food ingestion23. Whether a viscerotopic map exists in the nodose ganglion, where most visceral-organ-innervating VSNs are located6, is a subject of debate27. Using retrograde adeno-associated virus (AAVrg), we show that VSNs labelled from various visceral organs are largely non-overlapping along the spinal cordWe therefore hypothesize that visceral organs are coded by specific genes in VSNs. We developed a single-cell sequencing approach named \u2018Projection-seq\u2019 for unbiased, high-throughput genetic and anatomical dissection of complex neural circuits Fig. . EngineePhox2b+ placode-derived nodose VSNs18 were grouped into 12 subpopulations (A\u2013L) and 52 clusters (A1\u2013L2) on the basis of differentially expressed genes (DEGs) using Seurat and visualized using uniform manifold approximation and projection (UMAP) Fig. . VSNs laAP) Fig. . The disAP) Fig. . VSN cluAP) Fig. , showingAP) Fig. . VSNs thAP) Fig. . Togethe32. Of note, it seems that VSNs are primed for the functions of their target organs. For example, the stomach contains several anatomically and functionally distinct subregions: the proximal region similar to the oesophagus and the distal region similar to the intestine33. Accordingly, UPB-stomach+/UPB-oesophagus+ VSNs are genetically similar to oesophagus VSNs, whereas UPB-stomach+/UPB-duodenum+ VSNs are closer to duodenum VSNs , cardiovascular (heart), gastrointestinal and exocrine\u2013endocrine (pancreas) systems Fig. . Gut VSN6. Our discovery of a genetic \u2018visceral organ\u2019 trajectory is in accordance with this long-standing observation, suggesting that VSNs might follow morphogen gradients to their target spots. Indeed, signalling gradients formed by secreted morphogens help to establish an anterior\u2013posterior patterning34 and specify peripheral targets for vagal motor neurons35. Together, our data show that instead of a topographic organization, VSNs use a genetic trajectory to code visceral organs along the body\u2019s rostral\u2013caudal axis.Notably, we further identified a genetic trajectory that represents visceral organs along the body\u2019s rostral\u2013caudal axis Fig. . The relGpr65, Sst, Trpv1, Drd2 and Agtr1a and examined the innervation of tdTomato+ sensory fibres in whole-mount cleared organs. Notably, VSNs along this second trajectory project to different tissue layers Fig. and calced organ . The tis17, it is unclear how they contribute to interoceptive coding. Systematic analyses of VSN terminals within the lung, heart, oesophagus, stomach, duodenum and colon in Vglut2tdT mice revealed that there is a marked similarity between many stereotypical VSN ending types in the same tissue layer across visceral organs ; and (6) bud endings wrapped around specialized sensory epithelial cell clusters. Our data thus suggest that VSN ending structures are predominantly organ-independent but tightly associated with tissue layers.We then asked whether VSNs form organ-specific ending structures in various tissue layers. Although VSN endings have been extensively characterized in individual organsans Fig. , includi+ VSNs by nodose infection of AAV-FLEX-tdTomato in corresponding Cre mice; and (3) determined the sensory structures formed by DEG+ VSNs in the target organ or region using whole-organ clearing and volumetric imaging , we (1) identified enriched VSN clusters and their DEGs on the basis of Projection-seq data; (2) labelled DEGing Fig. . With thing Fig. , showing38. Together, our results reveal a two-dimensional genetic representation of the body\u2019s internal space in VSNs that codes the precise anatomical location of interoceptive signals biological processes for neuron development, cell\u2013cell signalling and synaptic signalling or ion transportation mice. VSN identity was then determined post hoc in cryo-sectioned nodose ganglia using RNAscope against 21 marker genes that together faithfully represent VSN subpopulations responsive VSNs were unambiguously registered from six mice Fig. . First, ice Fig. . VSNs reice Fig. . The vCaPiezo2+ VSNs (55.1%) were activated significantly faster than Piezo2\u2212 K/L-VSNs . Uts2b+Vip+Glp1r+Cckar+ G-VSNs represented a large fraction (39.2%) of intestinal stretch responders Fig. , suggestTo better quantify the relationship between VSN characteristics, we systematically mapped all characterized connections among the five VSN characteristics Fig. . In this47. Consistently, VSN central projections labelled using AAVrg-tdTomato from respiratory, cardiovascular and digestive systems were largely segregated, whereas vagal afferents from functionally related organs terminated in more overlapping areas guidelines. All mice were age- and gender-matched adults (older than 8 weeks) and no differences between sexes were observed.Glp1r-ires-Cre (029283), Gpr65-ires-Cre (029282), Npy2r-ires-Cre (029285), P2ry1-ires-Cre (029284), Agtr1a-Cre (030553), Calb2-ires-Cre (010774), Nts-Cre (017525), Vglut2-ires-Cre (016963), Sst-ires-Cre (013044), Twist2-Cre (008712), Vip-ires-Cre (010908), Piezo2-GFP-ires-Cre (027719), Trpv1-Cre (017769), Pvalb-Cre (017320), Vglut1-ires2-Cre (023527), Chat-ires-Cre (031661), lox-ChR2 (024109), lox-tdTomato (007914), and Snap25-2A-GCaMP6s-D (025111) were from the Jackson Laboratory. Drd2-Cre (032108-UCD) mice were from the Mutant Mouse Resource and Research Center (MMRRC). lox-L10-GFP mice were described before12.Wild-type C57BL/6J (000664), 12\u20131013 viral genomes per ml) were generated at the UNC Vector Core. Plasmids have been deposited to Addgene.UPB sequences were cloned from coding sequences of hChR2 , hM3Dq and hM4Di and inserted right before the SV40 poly (A) of AAVrg-CAG-tdTomato-WPRE-SV40 using the In-Fusion HD Cloning Kit . Projection-seq AAVs (at titre\u2009>\u200910UPB sequences (5\u2032\u20133\u2032):UPB-oesophagus :ACAGCACCATCCTCAACTCCACCAAGTTACCCTCATCGGACAACCTGCAGGTGCCTGAGGAGGAGCTGGGGATGGTGGACTTGGAGAGGAAAGCCGACAAGCTGCAGGCCCAGAAGAGCGTGGACGATGGAGGCAGTTTTCCAAAAAGCTTCTCCAAGCTTCCCATCCAGCTAGAGTCAGCCGTGGACACAGCTAAGACTTCTGACGTCAACTCCTCAGTGGGTAAGAGCACGGCCACTCTACCTCTGTCCTTCAAGGAAGCCACTCTGGCCAAGAGGTTTGCTCTGAAGACCAGAAGTCAGATCACTAAGCGGA.UPB-stomach :ATGGACTATGGCGGCGCTTTGTCTGCCGTCGGACGCGAACTTTTGTTCGTTACTAATCCTGTGGTGGTGAACGGGTCCGTCCTGGTCCCTGAGGATCAATGTTACTGTGCCGGATGGATTGAATCTCGCGGCACGAACGGCGCTCAGACCGCGTCAAATGTCCTGCAGTGGCTTGCAGCAGGATTCAGCATTTTGCTGCTGATGTTCTATGCCTACCAAACCTGGAAATCTACATGCGGCTGGGAGGAGATCTATGTGTGCGCCATTGAAATGGTTAAGGTGATTCTCGAGTTCTTTTTTGAGTTTAAGAATCCCTCTATGCTCTACCTT.UPB-duodenum :AATGGCAGCTCGGGCAATCAGTCCGTGCGCCTGGTCACGTCATCATCCCACAATCGCTATGAGACGGTGGAAATGGTCTTCATTGCCACAGTGACAGGCTCCCTGAGCCTGGTGACTGTCGTGGGCAACATCCTGGTGATGCTGTCCATCAAGGTCAACAGGCAGCTGCAGACAGTCAACAACTACTTCCTCTTCAGCCTGGCGTGTGCTGATCTCATCATAGGCGCCTTCTCCATGAACCTCTACACCGTGTACATCATCAAGGGCTACTGGCCCCTGGGCGCCGTGGTCTGCGACCTGTGGCTGGCCCTGGACTGCGTGGTGAGCAACGCCTCCGTCATGAACCTTCTCATCATCAGCTTTGACCGCTACTTCTGCGTCA.UPB-colon :ATCGATGGGCCTTAGGGAACTTGGCCTGTGACCTCTGGCTTGCCATTGACTGCGTAGCCAGCAATGCCTCTGTTATGAATCTTCTGGTCATCAGCTTTGACAGATACTTTTCCATCACGAGGCCGCTCACGTACCGAGCCAAACGAACAACAAAGAGAGCCGGTGTGATGATCGGTCTGGCTTGGGTCATCTCCTTTGTCCTTTGGGCTCCTGCCATCTTGTTCTGGCAATACTTTGTTGGAAAGAGAACTGTGCCTCCGGGAGAGTGCTTCATTCAGTTCCTCAGTGAGCCCACCATTACTTTTGGCACAGCCATCGCTGGTT.UPB-pancreas :AAGATGGCAGGCCTCATGATTGCTGCTGCCTGGGTACTGTCCTTCGTGCTCTGGGCGCCTGCCATCTTGTTCTGGCAGTTTGTGGTGGGTAAGCGGACGGTGCCCGACAACCAGTGCTTCATCCAGTTCCTGTCCAACCCAGCAGTGACCTTTGGCACAGCCATTGCTGGCTTCTACCTGCCTGTGGTCATCATGACGGTGCTGTACATCCACATCTCCCTGGCCAGTCGCAGCCGAGTCCACAAGCACCGGCCCGAGGGCCCGAAGGAGAAGAAAGCCAAGACGCTGGCCTTCCTCAAGAGCCCACTAATGAAGCAGA.UPB-lung :ACAGGACACCGGGTGCAGTGGCTGCGCTATGCAGAGTGGCTGCTCACTTGTCCTGTCATCCTTATCCGCCTGAGCAACCTCACCGGCCTGAGCAACGACTACAGCAGGAGAACCATGGGACTCCTTGTCTCAGACATCGGGACTATCGTGTGGGGGGCTACCAGCGCCATGGCAACCGGCTATGTTAAAGTCATCTTCTTTTGTCTTGGATTGTGCTATGGCGCGAACACATTTTTTCACGCCGCCAAAGCATATATCGAGGGTTATCATACTGTGCCA.UPB-heart :AGGACACTTCCAATGAGTCCAGCTCAGGCAGTGCCACCCAGAACACCAAGGAACGCCCAGCCACAGAGCTGTCCACCACAGAGGCCACCACGCCCGCCATGCCCGCCCCTCCCCTGCAGCCGCGGGCCCTCAACCCAGCCTCCAGATGGTCCAAGATCCAGATTGTGACGAAGCAGACAGGCAATGAGTGTGTGACAGCCATTGAGATTGTGCCTGCCACGCCGGCTGGCATGCGCCCTGCGGCCAACGTGGCCCGCAAGTTCGCCAGCATCGCTCGCAACCAGGTGCGCAAGAAGCGGCAGATGGCGGCCCGGGAGCGCAAAGTGACACGAACGATCTTTGCCATTCTGCTGGCCTTCATCCT.51 represents a powerful genetic tool in VSN studies54. AAVrg-tdTomato and AAVrg-GFP were purchased from the UNC vector core. AAVrg-FLEX-tdTomato (28306-AAVrg), AAVrg-CAG-FLEX-rc [Jaws-KGC-GFP-ER2] (84445-AAVrg) and pAAVrg-hSyn-Con/Fon hChR2(H134R)-EYFP (55645-AAVrg) were purchased from Addgene. All viruses contained 1012\u20131013 viral genomes per ml, and 0.05% Fast Green FCF was occasionally used to facilitate visualization.Retrograde adeno-associated virus (AAVrg)\u22121) and buprenorphine (1.5\u2009mg\u2009kg\u22121). Lung: virus was injected through a tracheal catheter into the lung using a Hamilton syringe. Heart: the mouse was ventilated using a mouse ventilator via an intubated angiocatheter. The heart was gently exposed via thoracotomy. Virus (5\u2009\u03bcl) was injected intramurally (20\u2009nl per second) using a Nanoject III injector at multiple sites covering most of the area of the heart. Stomach, duodenum, colon and pancreas: the target organ was gently exposed via an abdominal incision. Virus was injected intramurally at multiple sites covering most of the area . Oesophagus: the cervical oesophagus underneath the trachea was surgically exposed via a neck incision. The abdominal oesophagus and the oesophageal sphincter were gently exposed via an abdominal incision. Virus was injected into oesophageal muscularis and serosa layers .In all surgeries, mice were anaesthetized with 1\u20132% isoflurane on a heating pad, followed by subcutaneous injections of meloxicam were mixed and co-injected into the stomach in wild-type mice and reverse-transcribed into cDNA using the SuperScript IV First-Strand Synthesis System . Primer sets used : CATCGATACCGTCGACACAGGACACCGGGTGCAGTG (forward); CTGCTCGAAGCGGCCGCTGGCACAGTATGATAACCCTCG (reverse). UPB4 (5\u2032\u20133\u2032): CATCGATACCGTCGACATCGATGGGCCTTAGGGAAC (forward); TGCTCGAAGCGGCCGCAACCAGCGATGGCTGTGCC (reverse).17. Approximately 5,000\u201310,000 VSNs were loaded in each channel of the 10X microfluidic device to target 3,000\u20136,000 cells as an output of one sample. Single-cell cDNA libraries were prepared at the Yale Center for Genomic Analysis (YCGA) and sequenced using an Illumina NovaSeq S4 sequencer at 150\u2013300 million reads to achieve a fine sequencing depth of 30,000\u201350,000 reads per cell. For Projection-seq, 30 age- and gender-matched wild-type mice (divided into four samples) were first injected with different Projection-seq AAVs into thoracic and abdominal organs . Vagal ganglia were collected seven days later. Of the examined VSNs, 29.5% (102/346) were tdTomato+. VSNs were sequenced as mentioned above.For control scRNA-seq, vagal ganglia (left and right) were collected from 40 age- and gender-matched C57BL/6J wild-type mice (10 mice per sample) and VSNs were acutely isolated and enriched using previously described methods55, and 42 cell clusters identified using the top 30 principal components (PCs) were visualized using UMAP56 were re-clustered into 67 populations with the top 100 PCs were re-clustered into 52 clusters with the top 100 PCs and visualized with UMAP separately , and consistently more Sprr1a+ VSNs were observed after AAVrg injection or a custom mouse genome reference with additional sequence information of UPBs (Projection-seq) using the Cell Ranger software v.3.0.2 (10X Genomics). The following quality control metrics were applied to filter low-quality cells in scRNA-seq: number of genes per cell\u2009>\u2009500; number of genes per cell\u2009<\u20098,000; percentage of mitochondria genes\u2009<\u200910%. A total of 56,575 cells were sequenced . Control scRNA-seq and Projection-seq data were then integrated and processed using the R package Seurat v.3tely Fig.. DEGs fotely Fig.. VSN cluPrdm12+ (2.5%) neural-crest-derived jugular VSNs and 4,791 out of 14,590 (32.8%) Phox2b+ placode-derived nodose VSNs59 were recognized as UPB-positive (expression level\u2009>\u20090.8), suggesting that VSNs retrogradely labelled from the seven major visceral organs in our study mainly originate from the nodose but not the jugular ganglia. We therefore focused on nodose VSNs. After removing E-VSNs, 3,539 out of 4,609 UPB-marked nodose VSNs (76.8%) expressed a single barcode , and 740 out of 4,609 (16.1%) VSNs expressed were marked by two UPBs. A correlation matrix . DEGs for organ-specific VSNs, or between thoracic and abdominal VSNs were identified using the Wilcoxon rank-sum test . For example, Pou4f1, which is essential for DRG neuron specification32, is preferentially expressed in lung VSNs using the Upstream Analysis (Upstream Regulators) module. For cell\u2013cell interaction analysis between organ-innervating VSNs and various organ cell types , lung63 , colon64 , duodenum65 and pancreas60 . Cell\u2013cell interactions between organ-innervating UPB+ VSNs and indicated organ cell types were then analysed using the CellPhoneDB66 Python package. GO pathway analyses of DEGs in heart, lung, gut and pancreas VSNs, and along the tissue trajectory .Further analysis was performed for the Projection-seq dataset. A total of 42 out of 1,701 organ . The organ position score for VSN clusters as the weight value for each organ, expressed as \u03a3(Porgan-cluster\u2009\u00d7\u2009Positionorgan)/\u03a3Porgan-cluster. The organ trajectory score of an organ-specific VSN , and the tissue layer index for DEG+ VSNs \u2009\u00d7\u2009Indextissue) Fig. .12. In brief, mice were anaesthetized with 1\u20132% isoflurane and maintained on a heating pad. Both left and right vagal ganglia were surgically exposed. A virus mix containing a 1:1 dilution of AAV9-FLEX-tdTomato and AAV5-CAG-GFP with 0.05% (w/v) Fast Green FCF was injected using a Nanoject III injector (Drummond). Mice were euthanized four weeks after surgery for tissue collection (see \u2018Histology and immunochemistry\u2019).Vagal ganglia injection was performed as previously describedRNAscope HiPlex assays were performed following the manufacturer\u2019s protocol (Advanced Cell Diagnostics). Vagal ganglia were acutely dissected and freshly frozen in cryo-embedding medium (OCT). Cryosections were cut using a cryostat (Thermo Fisher Scientific), mounted onto Superfrost Plus slides (Thermo Fisher Scientific), and stored at \u221280\u2009\u00b0C until use. Slides were immediately immersed into fresh 4% paraformaldehyde (PFA) in RNase-free PBS for 60\u2009min at room temperature, followed by dehydration with 50%, 70% and 100% ethanol. Samples were then digested with protease IV for 30\u2009min at room temperature. After hybridization with designed probes , followed by 25\u2009ml cold 4% PFA. Fast Green FCF injected organs and transcardially perfused with 15\u2009ml cold PBS (pH 7.4) containing 10\u2009U\u2009mlFor brain samples, 40-\u03bcm cryosections were mounted onto Superfrost Plus slides. Cryosections were washed (3\u00d7 PBS), permeabilized , blocked ) and incubated with primary antibodies diluted in blocking buffer for 2\u2009h at room temperature. Then, the slides were washed (3\u00d7 PBST), and incubated with fluorophore-conjugated secondary antibodies , 1:1,000, Jackson ImmunoResearch) diluted in blocking buffer for 2\u2009h at room temperature. After incubation, the samples were washed (3\u00d7 PBST), and mounted with Fluoromount-G with DAPI before imaging with the Leica SP8 confocal microscope.72 and stained with the following protocol unless specifically mentioned. In brief, the dissected organ was immersed into 1/2-water-diluted reagent-1 with shaking at 37\u2009\u00b0C for 3\u20136\u2009h, followed by reagent-1 (R1) with shaking at 37\u2009\u00b0C for 7 days. R1 was replaced fresh every two days. Next, the tissue was washed (3\u00d7 PBS/0.01% NaN3), blocked and incubated with primary antibodies in blocking buffer with shaking for 7 days at room temperature. Samples were then washed and incubated with fluorophore-conjugated secondary antibodies diluted in blocking buffer with shaking for five days at room temperature. After antibody incubation, the samples were washed and immersed in 1/2-PBS-diluted reagent-2 overnight at room temperature, and then reagent-2 (R2) at 37\u2009\u00b0C for 2 days. The samples were finally immersed in oil for at least 1\u2009h and flattened to approximately 500\u2009\u03bcm in a custom-built imaging chamber and imaged using the Leica SP8 confocal microscope as described above, with a 10\u00d7 objective or a 40\u00d7 objective . Some heart samples were similarly processed but not flattened and imaged with a LaVision Vltramicroscope II light-sheet microscope at the CNNR Imaging Core at Yale University , 1:1,000) overnight, respectively, and treated with R2 for one day. Cleared ganglia were imaged with the Leica SP8 confocal microscope.tdT-GCaMP6s mice were continuously anaesthetized (1\u20132% isoflurane/oxygen) during the experiment. A tracheal tube was inserted for air injection. The upper oesophagus and pyloric sphincter were cannulated and flushed with saline multiple times to remove residual food particles. The duodenum (around 0.5\u2009cm below the pyloric sphincter) was cannulated with a bundle of six PE-10 tubing for separate delivery of saline, water and glucose (1\u2009M), Ensure , 10\u00d7 PBS and 150\u2009mM HCl (pH\u2009=\u20090.84). Left vagal ganglia were exposed and immobilized on a stable platform12. During calcium imaging, a series of stimuli were delivered to the same mice in the following sequence: (1) lung inflation for 20\u2009s with 600\u2009ml\u2009min\u22121 flow , twice with a 2-min interval; (2) small intestine stretch via fast injection of 600\u2009\u03bcl saline through the duodenal cannula; stomach stretch with 100\u2009\u03bcl, 300\u2009\u03bcl and 600\u2009\u03bcl saline through the oesophageal cannula for 30\u2009s (duration precisely controlled by closing or opening of the pyloric cannula); (4) small intestine infusion with 100\u2009\u03bcl saline, water, 1\u2009M glucose, Ensure, 10\u00d7 PBS and 150\u2009mM HCl in sequence with a 3-min interval between infusions. GCamp6s fluorescence was measured from two focal planes 15\u2009\u03bcm apart using a two-photon microscope . The imaging frequency for each plane was 1.72\u2009s per frame. At the end, electrical stimulation was applied to the vagus nerve and a z-stack of the ganglia was taken for both GCaMP and tdTomato signals for cell registration.Gpr65Trpa1 (R1T1), Runx3 (R1T2), Uts2b (R1T3), Gabra1 (R1T4), Slit2 (R1T5), Kcng1 (R1T6), Piezo2 (R1T7), Ddc (R1T8), Vip (R1T9), Trpv1 (R1T10), tdTomato (R1T11), Gpr65 (R1T12), Chodl (R2T1), Glp1r (R2T2), Grm5 (R2T3), Slc17a7 (R2T4), P2ry1 (R2T5), Tmc3 (R2T6), Car8 (R2T7), Nts (R2T8), Cckar (R2T9) and Calca (R2T11). The following criteria were used to categorize VSN subpopulations to generate successive in vivo imaging planes that resemble RNAscope sections. tdTomato\u2212 neurons were then registered on the basis of their relative distance and depth to their neighbouring tdTomato+ cells was defined as the average GCaMP6s fluorescence over a 10-frame period before stimulus induction and neuronal activity was calculated as \u0394F/F. Cells were coded as responsive to a given stimulus if the maximum GCaMP6s fluorescence was more than 100% above baseline during stimulus period . Peak response was identified as the maximum \u0394F/F within the stimulus period. To compare adaptation rates in lung stretch-sensitive VSNs, GCaMP6s traces were aligned at activation frame reached 10% of the peak response reached 10% of peak response.Regions of interest were manually extracted from GCaMP images. The stimulus induction frame was set as 0 unless specifically mentioned. Baseline signal . For quantitative analyses of MEs and IMAs in the indicated gastrointestinal regions; alveoli, longitudinal and patch-terminals in the lung; and varicose endings and IMAs in the heart .Four types of gut VSN ending types, three types of heart VSN ending types and five types of lung VSN ending types were classified on the basis of their morphologies and locations using whole-mount preparations in Vglut2ice Fig. ; n\u2009=\u20094\u20137Piezo2), H4 (Drd2) and I3/I4 (Agtr1a). In Piezo2tdT mice, most cardiac afferents were varicose surface endings. In Drd2tdT mice, cardiac afferents densely innervated myocardium with both branched and parallel IMAs. Agtr1a+ VSNs predominantly formed flower-spray endings in the heart and the aortic arch. Our results thus reveal the identity of various VSN cardiac ending types bud ending wrapping around NEBs near the bronchial bifurcation with branch endings and examined IMAs around pyloric sphincters in P2ry1tdT and Calb2tdT mice. Most (75.0%) of the P2ry1+ IMAs around the oesophageal sphincter were pIMAs and all Calb2+ IMAs were cIMAs , H2 (Vip) and C4 , whereas all afferent types in Vglut2tdT mice were enriched in region 8 around the pyloric sphincter. Both Sst+ and Gpr65+ VSNs formed MEs on the stomach, as reported16, with Sst+ endings mainly in the antrum and Gpr65+ endings more evenly distributed across the stomach, indicating that F1-VSNs form MEs. Both Glp1r+ and Piezo2+ VSNs predominantly formed IGLEs, suggesting that C4-VSNs form stomach IGLEs. Vip+ VSNs exhibited pIMA morphology in region 8, suggesting that H2-VSNs also form pIMAs for each ending type in each stomach region , calculated as the innervation intensity of corresponding ending types in various stomach regions normalized by the innervation intensity of the same ending type in the entire stomach in Vglut2tdT mice of each ending type in each stomach region was calculated as the percentage of corresponding dual-labelled VSNs forming this ending type normalized to the percentage of stomach-UPB single-labelled VSNs forming this ending type, expressed as \u03a3/\u03a3. We then calculated the total variance between anatomically and Projection-seq-derived innervation intensities in all stomach regions across all ending types, expressed as \u03a3 (SFnME- \u2212 AFnME-)2 + \u03a3 (SFnpIMA- \u2212 AFnpIMA-)2 + \u03a3 (SFncIMA- \u2212 AFncIMA-)2 + \u03a3 (SFnIGLE- \u2212 AFnIGLE-)2. The trial with the lowest variance was defined as the best fit. AFafferent type-stomach region and SFafferent type-stomach region for this condition were plotted together , Vip (H3/G2/G5), Glp1r (G2/G5), and Agtr1a (I5) , suggesting that both F- and G-VSN clusters form MEs. By contrast, Agtr1a+ neurons representing I5-VSNs primarily formed IGLEs in the duodenum , C5\u2013C8 VSNs also form oesophageal IMAs. Probably owing to low infection efficiency, no apparent oesophageal ME or IGLE clusters were revealed. Notably, among all Cre lines examined, afferents in NTStdT mice preferentially formed oesophageal IGLEs , expressed as (number of connections \u2212 minimum number of possible connections)/(maximum number of possible connections \u2212 minimum number of possible connections), for each pair of characteristics. We next calculated the statistical variance of connections per variable for both characteristics (v1 and v2). As both variances contribute to the correlation equally, the correlation index was finally calculated as (1\u2009\u2212\u2009C)/((v1\u2009+\u20091)\u2009\u00d7\u2009(v2\u2009+\u20091)).Correlations among various VSN characteristics, including 7 visceral organs , 4 tissue layer types , 6 VSN ending types , 11 VSN subpopulations and 4 response patterns , were plotted in Fig. Mouse line and organ combinations were selected on the basis of Projection-seq anterograde tracing results: lung alveoli ending , lung NEB ending , oesophageal IGLE , oesophageal IMA , heart IMA , stomach IGLE , stomach mucosal ending , stomach IMA , duodenal IGLE , duodenal mucosal ending and colon IGLE . Organ injection of AAVrgs is described in \u2018AAV infection of visceral organs\u2019. Brain processing and imaging is described in \u2018Histology and immunochemistry\u2019.Bregma-subnucleus) was calculated as average fluorescence in the sub-nucleus minus background fluorescence measured in a region in the sub-nucleus with no fluorescence-labelled vagal fibres; (2) the area of each sub-nucleus (ABregma-subnucleus) was measured; (3) the total fluorescence (TFBregma) was calculated as \u03a3 (FIBregma-subnucleus\u2009\u00d7\u2009ABregma-subnucleus); and (4) the percentage innervation (PI) of a sub-nucleus at a certain Bregma was calculated as FIBregma-subnucleus\u2009\u00d7\u2009ABregma-subnucleus/TF\u2009\u00d7\u2009100. The correlation variance between VSNs labelled from various visceral organs 2 and a phylogenetic tree was generated based on the correlation variance matrix using the seqlinkage function in MATLAB.Area innervated by vagal afferents retrogradely labelled from various visceral organs at different Bregma levels were measured using Fiji (ImageJ) , heart (n\u2009=\u20093); varicose: heart (n\u2009=\u20095), aorta (n\u2009=\u20096); pIMA: colon (n\u2009=\u20093), heart (n\u2009=\u20096); cIMA: stomach (n\u2009=\u20094), oesophagus (n\u2009=\u20093); epithelial: stomach (n\u2009=\u20094), lung (n\u2009=\u20095); bud: lung (n\u2009=\u20095), oesophagus (n\u2009=\u20093); Extended Data Fig. n\u2009=\u20093; Extended Data Fig. n\u2009=\u20092; Extended Data Fig. n\u2009=\u20093; Extended Data Fig. n\u2009=\u20093; Extended Data Fig. n\u2009=\u20092; Extended Data Fig. n\u2009=\u20093; Extended Data Fig. n\u2009=\u20092; Extended Data Fig. n\u2009=\u20094; Extended Data Fig. n\u2009=\u20095; Extended Data Fig. n\u2009=\u20092; Extended Data Fig. n\u2009=\u20094; Extended Data Fig. n\u2009=\u20094; Extended Data Fig. n\u2009=\u20094; Extended Data Fig. n\u2009=\u20093), stomach (n\u2009=\u20094), duodenum (n\u2009=\u20093), colon (n\u2009=\u20093), aorta (n\u2009=\u20096); Extended Data Fig. n\u2009=\u20093; Extended Data Fig. n\u2009=\u20094; Extended Data Fig. Piezo2 (n\u2009=\u20099), Drd2 (n\u2009=\u20096), Agtr1a (n\u2009=\u20093), Npr2r (n\u2009=\u20092); Extended Data Fig. n\u2009=\u20094; Extended Data Fig. Npy2r (n\u2009=\u20093), Trpv1 (n\u2009=\u20095), P2ry1 (n\u2009=\u20096), Agtr1a (n\u2009=\u20094), Piezo2 (n\u2009=\u20099), Vglut1 (n\u2009=\u20094), Pvalb (n\u2009=\u20093), Twist2 (n\u2009=\u20093); Extended Data Fig. P2ry1 (n\u2009=\u20094), Calb2 (n\u2009=\u20093), Glp1r (n\u2009=\u20095), Vip (n\u2009=\u20095), Gpr65 (n\u2009=\u20097), Sst (n\u2009=\u20095), Agtr1a (n\u2009=\u200912); Extended Data Fig. Glp1r (n\u2009=\u20093), Agtr1a (n\u2009=\u20094); Extended Data Fig. Trpv1 (n\u2009=\u20094), Agtr1a (n\u2009=\u20093); Extended Data Fig. Nts (n\u2009=\u20095), Trpv1 (n\u2009=\u20093), Agtr1a (n\u2009=\u20094), Gpr65 (n\u2009=\u20093); Extended Data Fig. n\u2009=\u20095; Extended Data Fig. n\u2009=\u20095; Extended Data Fig. n\u2009=\u20093), heart (n\u2009=\u20093), oesophagus (n\u2009=\u20092), stomach (n\u2009=\u20093), duodenum (n\u2009=\u20093), colon (n\u2009=\u20093), pancreas (n\u2009=\u20093); Extended Data Fig. n\u2009=\u20093 for all groups.Representative images and experiments were repeated independently in multiple mice with similar results: Fig. Further information on research design is available in the\u00a0Any methods, additional references, Nature Research reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at 10.1038/s41586-022-04515-5.Supplementary Figure 1Original source image for electrophoresis, related to Extended Data Fig. 2b. Red box indicates how the gel was cropped for the final figure.Reporting SummarySupplementary Table 1Detailed information for RNAscope probes."} +{"text": "However, the mechanisms underlying the regulation of FTO and its subsequent impact on the regulation of the epitranscriptome remain to be further elucidated. Here, we demonstrate that FTO expression is downregulated and inversely correlated with poor survival of lung adenocarcinoma patients. Mechanistically, Wnt signaling induces the binding of EZH2 to \u03b2-catenin. This protein complex binds to the LEF/TCF-binding elements at the promoter region of FTO, where EZH2 enhances H3K27me3 and inhibits FTO expression. Downregulated FTO expression substantially enhances the m6A levels in the mRNAs of a large number of genes in critical pathways, particularly metabolic pathway genes, such as MYC. Enhanced m6A levels on MYC mRNA recruit YTHDF1 binding, which promotes MYC mRNA translation and a subsequent increase in glycolysis and proliferation of tumor cells and tumorigenesis. Our findings uncovered a critical mechanism of epitranscriptome regulation by Wnt/\u03b2-catenin-mediated FTO downregulation and underscored the role of m6A modifications of MYC mRNA in regulating tumor cell glycolysis and growth.FTO removes the N6-methyladenosine (m The abundance and effects of m6A on RNA depend on the dynamic and integrated regulation by \u201cwriters\u201d and \u201cerasers\u201d, which add and remove the methylation, respectively, and \u201creaders\u201d, which recognize the modification8. The identified writers include methyltransferase-like (METTL) 3/14, Wilms tumor 1-associated protein (WTAP), RNA binding motif protein 15/15B (RBM15/15B), and KIAA1429, whereas erasers include fat mass and obesity-associated protein (FTO) and alkB homolog 5 (ALKBH5). YT521-B homology (YTH) domain-containing proteins , heterogeneous nuclear ribonucleoprotein (HNRNP) protein families, and IGF2 mRNA binding proteins (IGF2BP) families are regarded as readers9. FTO, as the first m6A demethylase identified11, regulates the m6A modification of critical genes in different types of cancer, such as glioblastoma12, acute myeloid leukemia (AML)14, cervical squamous cell carcinoma (CSCC)15, and breast cancer16. However, the mechanisms underlying the regulation of FTO and its subsequent impact on the regulation of the epitranscriptome remain to be further elucidated.N6-Methyladenosine : normal rabbit IgG (# 2729) (for immunoprecipitation and ChIP), \u03b2-catenin (#8480) (for immunoblotting and immunoprecipitation), FTO (#31687) (for immunoblotting and RIP), EZH2 (#5246) (for immunoprecipitation and ChIP), TCF4 (#2569) (for ChIP), H3K27me3 (#9733) (for ChIP), H3K9me2 (#4658) (for ChIP), HK2 (#2867) (for immunoblotting and IHC), and Ki-67 (#9449) (for IHC). A mouse monoclonal antibody against tubulin (T9026) (for immunoblotting) was purchased from Sigma-Aldrich . Rabbit antibodies recognizing c-Myc (ab32072) (for immunoblotting and IHC), Wnt-3a (ab219412) (for IHC) and FTO (ab124892) (for IHC) were purchased from Abcam . Rabbit antibody recognizing YTHDF1 (17479-1-AP) (for RIP and immunoblotting) was purchased from Proteintech . RIPA lysis and extraction buffer (89901) and Pierce IP lysis buffer (87787) were purchased from Thermo Fisher Scientific . Protein A/G plus-agarose (sc-2003) was purchased from Santa Cruz Biotechnology . Wnt-3a (5036-WN) was obtained from R&D Systems . Cycloheximide (CHX) (HY-12320) and nitro blue tetrazolium chloride (HY-15925) were purchased from MedChem Express . Agar (1182GR500) was purchased from BIO FROXX .http://xena.ucsc.edu/welcome-to-ucsc-xena/). Gene transcription estimates for each gene were analyzed with RNA-Seq using Expectation Maximization (RSEM) software.The clinical records and RNAseqV2 level 3 gene-level lung adenocarcinoma data were downloaded from TCGA (Forty pairs of frozen tissues for RNA extraction and 83 pairs of frozen tissues for immunohistochemistry (IHC) were obtained from patients with lung adenocarcinoma who underwent radical resections in the Department of Thoracic Surgery of the Cancer Hospital, Chinese Academy of Medical Sciences. The clinical features of the patients are summarized in Table S17. Section of lung adenocarcinoma TMA was stained with an antibody against FTO. The tissue sections were quantitatively scored according to the percentage of positive cells and staining intensity as described previously18. The following proportion scores were assigned to the sections: 1, 0\u20131%; 2, 2\u201310%; 3, 11\u201330%; 4, 31\u201370%; and 5, 71\u2013100%. The staining intensity was rated on a scale of 0\u20133: 0, negative; 1, weak; 2, moderate, and 3, strong. Then the proportion and intensity scores were combined to obtain a total score as described previously18.Eighty-three pairs of frozen tissues from lung adenocarcinoma patients were formalin-fixed and paraffin-embedded. The tissue microarray (TMA) was constructed as previously described2 at 37\u2009\u00b0C in a humidified incubator. And all these cells were routinely tested for mycoplasma. When cells were 50% confluent, the medium was replaced with a fresh medium containing 0.5% serum for 1 day, and then Wnt-3a was added at a final concentration of 60\u2009ng/ml for cell stimulation.H322 and HEK 293T cells were grown in Dulbecco\u2019s modified Eagle\u2019s medium (DMEM) supplemented with 10% fetal bovine serum (Invitrogen) and 1% penicillin-streptomycin. H358 cells were grown in RPMI 1640 supplemented with 10% FBS (Invitrogen) and 1% penicillin-streptomycin. Cells were cultured in 5% CO19. The relative mRNA expression levels were calculated by the 2\u2212\u0394\u0394Ct method with normalization to ACTB or GAPDH; the PCR primers are listed in Table STotal RNA was isolated with TRIzol reagent according to the manufacturer\u2019s instructions. RNA (1000\u2009ng) was reverse-transcribed into cDNA with a RevertAid First Strand cDNA Synthesis kit (Thermo); SYBR Green-based qRT-PCR was performed using a 7900HT fast real-time PCR system , as described previously20. The shRNA sequences are listed in Table SPlasmids containing transgenes and packaging plasmids were cotransfected into HEK 293T cells using Lipofectamine 3000 . Viruses were collected and concentrated after 48\u2009h. When tumor cells reached 50%-60% confluence, we infected the cells with concentrated virus and then selected them by antibiotic treatment21.Extraction of proteins with a modified buffer from cultured cells was followed by immunoprecipitation and immunoblotting with antibodies, as described previously22. The primers are listed as follows:ChIP assays were performed using the SimpleChIP\u00ae Enzymatic Chromatin IP kit according to the manufacturer\u2019s instructions, as described previouslyFTO promoter Forward: GTTATCCTTCTTTGCTCACTATGC;binding site within the FTO promoter Reverse: CTGAGGAAGTGAACTGAGCTC.binding site within the 6A reporter assays, the DNA fragments of MYC-CDS containing the wild-type m6A motifs, as well as the mutated motifs (m6A was replaced by T) were inserted into the Xhol site of the pMIR-REPORT luciferase reporter vector. Dual-luciferase reporter assays were performed, as described previously in HEK 293T cells23.For the promoter-reporter assay, the wild-type DNA oligos of the FTO promoter and the mutated oligos with three LEF/TCF-binding element (TBE) deletions were inserted into the upstream region of the firefly luciferase of the PGL4.1 vector. For mSequences of wild type FTO promoter:CTTTGCTCACTATGCTTCACTTA CATTATTCTTTACTTTCCTCGAACCCCCCATACCCTTGTCTTGCTCAAGG CCTTTGTATTAGCTGGTTCCTTAATCTTTGGAGCTCAGTTCACTTCCTCA GACAGGTTTTCCCTGACCATCCTATGTTAGAGTAGTCTTCCTTACATTTC TTCACTGTTTATTTCTTTTCTTTTCTTTTTTTTTTTTTTGAGACAGGGTC TTGCTATGTTGCCCGGGCTGGCCTTGAATTCATGGGTTCAAGTGATCCTC CCACCTCAGCCTCCCGAGTAGCTGGAACTACATGTGCGTGCCACCAAGCA TGGCTTGTATCTCTTATAGCAACTGCCTCTATCTGAAGTTATCAGATAAA ATTATTGTTTGTCTCCACTAAAAAAGGATAAACATCTTGAGACGGGTATT AGTCTTGTTCACAACTGTTCAGGAACAGTGCCTGGTACAGGGTGGGAACC AACATTAATATTTATTGAATGATTGGCTGTGCGTGGTGGCTCACACCTGT AATCCCAGCACTTTGCGAGGCCGAGACGGGCGGCTGACTTCAGGCCAGGA GTTCGAGACCAGCCTGGCCAACATGATGAAACCCTGTCTCTACTAAAAAT ACAAAAATTAGCTGGGTGTGGTGGCACACGCCTGTAATCCCAGCTACTCG GGAGGCTGAGGCAGAAGAATCGCTTGAACCTGGGAGGAGGAGTTTGCAGT AAGCTGAGGTCTTACCACTGCACTCCAGCCTGGGCAACGGAGCAAGAACC TGTCACACACACAAAAAAAAGAATAAAGAAAAAATATTTATTGAATGAAT AAATGAATATCAGGTACTGAGATTAAAATGGCAAGCAAAACCCCCGCCTT TATGAAGCTAGCAAGTTATGGAGGTAATCACATGATAAACAAATAATATA TAATTAAGCAAACAATAGACCACTCAGGAGGTTTAGGGTCTACCAACCAA CTCCTAATCCAGGGCAAATGAGCAAACTGTGTTAGGGACCTACAACTTGC AGGATCTGGATAGAGATGGCAATTAGCAGCATCAACTCTCACCTTCATGG CTGGGATATAACATTTCAAATTGGTCCTGGACGTGGGGATAAAGGGCGGC CTGTGATTCAGGCCTGAGGATGTGGAGGTGTCTTGGGCTGGGCTGCTTTC ACGCCAGCAGAACTCCAGGGCCAACTCCAGGGCCTTCTCCAGGCGGCAGA GCGGACCCTAGGACCCCGGCCCGCGCTGCAGTGGGGAGGGTCAGCAACCT CCACCCACCCTCATCCTCCCCCATCCTCCCGGGTACTCACCGTGCCACTG GCCCTGCAGCTAGCTACCGTTGCTATAGCGCCGACAGCGTGGCGGGCGGC TGGCCGAGAGGAGCACGGGAGAAACATGGCAGGCTCCCGTAGCCTCCTGG GAAATGTAGTTCTCCTTGGACTCTAGCCTGTTTGCTCGCGGGGTAGCGGA CTCATTTATGCTTGGTGTTATGATTGTAACTAAGAATCCTGGAGTGAGCT GGTTACAAAGTGAGCCCGACTTTCCATGGATGCACCATCCTAGAGTGCACGGAAGTACTCCTATAGAAAAGGTCAATTTTTAGGATCCTGTTGACACATA GGCCCGTGTATGAAAATGATTAGTTTTCCATGACAGAGTTAAGGTCACTT TAAAAATAATAATGATGATGATGATGATGATGGTGTTAACATTCATTGAA CGCTTACTATGTGCCAGGTACTGTTCTAAGTGTTCTGTTATAGGAATGAA GTGTCTCACCATATCCTTGTGAGGCTGTTACTCAAATGATTCCTGCTTTA CAAATGAGGAAGCTGAGACACAGATTAGTTAACTCACTTAAGGTGGTAGT TGAAAGTATTAATAGTTGTGTCTGGTGATATTTTTGGTTGTCACAACAAG GAAGCGGGATGCTACTGGCATCTAGTGAGTAGAGGCCATGGATGATGCTA AATATCTTACAGTGCTTAGGAGACATAATAATGAATTATCCAGCCCAAAA TGTTAATAATAGTGTAGAAGCTGAAAAACCCTGCACAATGCTGCAATGCC TCTCCAACACCATCTTATGTTATCCTTSequences of truncated FTO promoter:GGAAGTACTCCTATAGAAAAGGTCAATTTTTAGGATCCTGTTGACACATA GGCCCGTGTATGAAAATGATTAGTTTTCCATGACAGAGTTAAGGTCACTT TAAAAATAATAATGATGATGATGATGATGATGGTGTTAACATTCATTGAA CGCTTACTATGTGCCAGGTACTGTTCTAAGTGTTCTGTTATAGGAATGAA GTGTCTCACCATATCCTTGTGAGGCTGTTACTCAAATGATTCCTGCTTTA CAAATGAGGAAGCTGAGACACAGATTAGTTAACTCACTTAAGGTGGTAGT TGAAAGTATTAATAGTTGTGTCTGGTGATATTTTTGGTTGTCACAACAAG GAAGCGGGATGCTACTGGCATCTAGTGAGTAGAGGCCATGGATGATGCTA AATATCTTACAGTGCTTAGGAGACATAATAATGAATTATCCAGCCCAAAA TGTTAATAATAGTGTAGAAGCTGAAAAACCCTGCACAATGCTGCAATGCC TCTCCAACACCATCTTATGTTATCCTTACTATGCTTCACTTA CATTATTCTTTACTTTCCTCGAACCCCCCATACCCTTGTCTTGCTCAAGG CTAGCTGGTTCCTTAATCTCAGTTCACTTCCTCA GACAGGTTTTCCCTGACCATCCTATGTTAGAGTAGTCTTCCTTACATTTC TTCACTGTTTATTTCTTTTCTTTTCTTTTTTTTTTTTTTGAGACAGGGTC TTGCTATGTTGCCCGGGCTGGCCTTGAATTCATGGGTTCAAGTGATCCTC CCACCTCAGCCTCCCGAGTAGCTGGAACTACATGTGCGTGCCACCAAGCA TGGCTTGTATCTCTTATAGCAACTGCCTCTATCTGAAGTTATCAGATAAA ATTATTGTTTGTCTCCACTAAAAAAGGATAAACATCTTGAGACGGGTATT AGTCTTGTTCACAACTGTTCAGGAACAGTGCCTGGTACAGGGTGGGAACC AACATTAATATTTATTGAATGATTGGCTGTGCGTGGTGGCTCACACCTGT AATCCCAGCACTTTGCGAGGCCGAGACGGGCGGCTGACTTCAGGCCAGGA GTTCGAGACCAGCCTGGCCAACATGATGAAACCCTGTCTCTACTAAAAAT ACAAAAATTAGCTGGGTGTGGTGGCACACGCCTGTAATCCCAGCTACTCG GGAGGCTGAGGCAGAAGAATCGCTTGAACCTGGGAGGAGGAGTTTGCAGT AAGCTGAGGTCTTACCACTGCACTCCAGCCTGGGCAACGGAGCAAGAACC TGTCACACACACAAAAAAAAGAATAAAGAAAAAATATTTATTGAATGAAT AAATGAATATCAGGTACTGAGATTAAAATGGCAAGCAAAACCCCCGCCTT TATGAAGCTAGCAAGTTATGGAGGTAATCACATGATAAACAAATAATATA TAATTAAGCAAACAATAGACCACTCAGGAGGTTTAGGGTCTACCAACCAA CTCCTAATCCAGGGCAAATGAGCAAACTGTGTTAGGGACCTACAACTTGC AGGATCTGGATAGAGATGGCAATTAGCAGCATCAACTCTCACCTTCATGG CTGGGATATAACATTTCAAATTGGTCCTGGACGTGGGGATAAAGGGCGGC CTGTGATTCAGGCCTGAGGATGTGGAGGTGTCTTGGGCTGGGCTGCTTTC ACGCCAGCAGAACTCCAGGGCCAACTCCAGGGCCTTCTCCAGGCGGCAGA GCGGACCCTAGGACCCCGGCCCGCGCTGCAGTGGGGAGGGTCAGCAACCT CCACCCACCCTCATCCTCCCCCATCCTCCCGGGTACTCACCGTGCCACTG GCCCTGCAGCTAGCTACCGTTGCTATAGCGCCGACAGCGTGGCGGGCGGC TGGCCGAGAGGAGCACGGGAGAAACATGGCAGGCTCCCGTAGCCTCCTGG GAAATGTAGTTCTCCTTGGACTCTAGCCTGTTTGCTCGCGGGGTAGCGGA CTCATTTATGCTTGGTGTTATGATTGTAACTAAGAATCCTGGAGTGAGCT GGTTACAAAGTGAGCCCGACTTTCCATGGATGCACCATCCTAGAGTGCAC6A sites:MYC-CDS with wild-type mGGACTTGTTGCGGAAACGACGAGAACAGTTGAAACACAAACTTGAACAGCTACGGAACTCTTGTGCGGTAGTTATCCTTAAAAAAGCCACAGCATACATCCTGTCCGTCCAAGCAGAGGAGCAAAAGCTCATTTCTGAAGA6A sites:MYC-CDS with the mutated mGGTCTTGTTGCGGAAACGACGAGATCAGTTGAATCACAATCTTGATCAGCTACGGATCTCTTGTGCG.GTAGTTATCCTTAAAAAAGCCACAGCATACATCCTGTCCGTCCAAGCAGAGGAGCAAAAGCTCATTTCTGAAGA5 cells were seeded in a 6-well plate and maintained in a medium with 10% FBS for different periods of time. The cells were harvested and counted24.2\u2009\u00d7\u20091025. Briefly, for the bottom layer of agar, we deposited the mix of 1% agar and 2\u00d7 medium into each well of a six-well plate. For the upper layer of agar, we deposited the mix of 0.6% agar and a suspension of cells in each well (10000 cells/well). The cells were cultured for 21 days in a 37\u2009\u00b0C humidified cell culture incubator and then were stained with nitroblue tetrazolium chloride solution (200 \u03bcl/ well) and incubated overnight at 37\u2009\u00b0C.The assay was performed as described previously26.Migration and invasion assays were performed in the chamber coated with or without Matrigel matrix according to the manufacturer\u2019s instructions as previously described6 Cells were seeded in 6\u2009cm plates. After incubation for 24\u2009h, the culture medium was replaced and the cells were arrested at the S phase by adding thymidine (2\u2009mM) for 24\u2009h. The thymidine was then removed, and the cells were washed by PBS and cultured in a fresh culture medium for 3\u2009h to release cells before nocodazole treatment (100\u2009ng/ml) for 12\u2009h to arrest cells in the G2/M phase. Then, the cells were washed with PBS and cultured in a fresh medium to release cells27.2\u2009\u00d7\u200910g for 5\u2009min, followed by washing with PBS. The cells were then fixed in 1\u2009ml ice-cold 70% ethanol at 4\u2009\u00b0C for 24\u2009h and centrifuged at 500 \u00d7 g for 5\u2009min. After being washed with PBS, the fixed cells were fully resuspended in 1\u2009ml of 2\u2009M HCl/0.5% Triton X-100 solution and incubated at room temperature for 30\u2009min. The cells were spun down and neutralized in 1\u2009ml of 0.1\u2009M Na2B4O7 solution . Then, the cells were spun down and washed with cold PBS containing 1% BSA and incubated with mouse anti-BrdU at 4\u2009\u00b0C for 12\u2009h. Afterward, centrifuged cells were washed with cold PBS containing 1% BSA and incubated with Alexa Fluor 647-conjugated goat anti-mouse IgG in room temperature for 1\u2009h. Finally, the cells were spun down and washed with cold PBS containing 1% BSA, followed by resuspended in 300\u2009\u03bcl of PBS containing 5\u2009\u03bcg/ml DAPI for 15\u2009min at room temperature, and then cell cycle progression was analyzed by flow cytometry.One hour before harvesting cells at each time point, BrdU (20\u2009\u03bcM) was used to label the cells. The cells were collected by centrifugation at 500 \u00d7 18.Cells were seeded in culture dishes, and the medium was changed when cells reached 50% confluence. After incubation for 12\u201324\u2009h, the culture medium was collected. The glucose levels were detected by a glucose colorimetric assay kit , and the lactate levels were detected by a lactate colorimetric assay kit according to the manufacturer\u2019s instructions, and values were calculated as previously described6 cells were injected subcutaneously into the flank regions of female BALB/c nude mice (4\u20135 weeks). The width (W) and length (L) of the tumors were measured every three days, and the volume (V) of each tumor was calculated using the formula V\u2009=\u2009(W2\u2009\u00d7\u2009L/2). For lung colonization assays, 1\u2009\u00d7\u2009106 cells were injected into the tail vein of female NOD/SCID mice (6\u20137 weeks), and 6 weeks later the lung was removed and fixed with 10% formalin. Fixed lung tissues were embedded in paraffin and cut into consecutive sections. These sections were stained by hematoxylin and eosin (H&E)28. All animal experiments were approved by the Animal Care and Use Committee of the Cancer Hospital of the Chinese Academy of Medical Sciences.Mice were randomized into several groups. For the subcutaneous implantation model, 1\u2009\u00d7\u200910A Magna RIP kit was used to perform the RIP assays. A sufficient number of H322 cells were lysed by RIP lysis buffer, and the supernatant of the RIP lysate was incubated with specific antibodies on beads overnight at 4\u2009\u00b0C. After washing, RNA was extracted and analyzed by qRT-PCR.6A-seq was performed by Cloudseq Biotech Inc. according to the published procedure with slight modifications1. Briefly, fragmented mRNA was incubated with an anti-m6A polyclonal antibody in IPP buffer for 2\u2009h at 4\u2009\u00b0C. The mixture was then immunoprecipitated by incubation with protein-A beads (Thermo Fisher) at 4\u2009\u00b0C for an additional 2\u2009h. The bound RNA was eluted from the beads with N6-methyladenosine in IPP buffer, and then the RNA was extracted with TRIzol reagent (Thermo Fisher) following the manufacturer\u2019s instructions. Purified RNA was used for RNA-seq library generation with the NEBNext\u00ae Ultra\u2122 RNA Library Prep kit (NEB). Both the input sample without immunoprecipitation and the m6A IP samples were subjected to 150\u2009bp paired-end sequencing on an Illumina HiSeq 4000 sequencer.m29. After that, clean reads of all libraries were aligned to the reference genome by Hisat2 software (v2.0.4)30. Methylated sites on RNAs (peaks) were identified by MACS software31. Differentially methylated sites were identified by diffReps32. These peaks identified by both software and overlapped with exons of mRNAs were selected. Raw counts of mRNA sequencing were got by HTSeq software (v0.9.1) and normalized by edgeR software.Paired-end reads were harvested from Illumina HiSeq 4000 sequencer and were quality-controlled by Q30. After 3\u2032 adapter-trimming and low-quality reads removal by cutadapt software (v1.9.3), clean reads of input libraries were aligned to reference genome (UCSC HG19) by STAR software6A kit was used according to the manufacturer\u2019s instructions. m6A enrichment was analyzed by qPCR with specific primers, and data were normalized to input. Primer sequences were as follows:Total RNA was isolated with a miRNeasy kit with DNase I digestion. mRNA was extracted from total RNA using a GenElute mRNA Miniprep kit . Then, a Magna MeRIP mMYC Forward: TTGCGGAAACGACGAGAACA;MYC Reverse: TCATAGGTGATTGCTCAGGACA.33.Cycloheximide (CHX) was added to the cell culture at a final concentration of 100\u2009\u03bcg/ml for 10\u2009min to stop translation. Cells were washed with cold PBS and lysed with lysis buffer. The cell lysate was loaded onto the top of a 10\u201350% sucrose gradient tube immediately, and the tube was centrifuged at 36,000\u2009rpm for 3.5\u2009h at 4\u2009\u00b0C. The sample was separated into 12 fractions by a fraction collector and measured at 254\u2009nm. RNA was extracted by TRIzol and subjected to qPCR analysis. The relative expression of MYC in each fraction was normalized to GAPDH, as well as to the inputt-tests to compare means between two groups; p\u2009<\u20090.05 was considered significant.All data are expressed as the mean\u2009\u00b1\u2009SD. We used two-tailed Student\u2019s 35. As a major type of non-small-cell lung carcinoma, lung adenocarcinoma (LUAD) remains one of the most aggressive and fatal tumor types36. To determine the expression of FTO in lung adenocarcinoma, we analyzed TCGA data and revealed that FTO expression levels were much lower in lung adenocarcinoma tissues than in their adjacent normal tissues accounts for 85% of cases, is the most commonly occurring cancer and the leading cause of cancer deathues Fig. . To valiues Fig. . Consistues Fig. C, D. Kapues Fig. . These rTo determine the role of FTO in lung adenocarcinoma progression, we depleted FTO expression in H322 Fig. and H358We next subcutaneously injected H358 cells with or without FTO depletion into athymic nude mice. As shown in Fig. FTO promoter sequence with PROMO software and identified three potential LEF/TCF-binding elements (TBE) assays demonstrated that Wnt stimulation increased the binding of TCF4 to the promoter region of FTO Fig. S that areFTO Fig. . LuciferRNA Fig. and protRNA Fig. levels oion Fig. . These r42. ChIP analyses with anti-H3K27me3 and anti-H3K9me2 antibodies showed that Wnt stimulation significantly enriched H3K27me3 27 (H3K27me3) and dimethylation of histone H3 K9 (H3K9me2) are marks of transcription repressionme3 Fig. but not me3 Fig. in the Tnin Fig. and the ion Fig. , as deteion Fig. . These rWe next examined the clinical relevance of Wnt signaling component expression and FTO expression by TCGA analysis. We found that expression of a positive regulator of Wnt signaling Frizzled (FZD)9 and DVL1 was negatively correlated with FTO expression whereas expression of negative regulators of Wnt signaling APC, GSK3\u03b2, and AXIN2 was positively correlated with FTO expression in lung adenocarcinoma Fig. . These r6A RNA sequencing (m6A-seq) on H322 cells with or without FTO depletion. We showed that m6A peaks primarily occurred in a sequence context as GGAC (P\u2009=\u20093.5e10\u201341) Fig. S and were1) Fig. S, which aays Fig. , which iion Fig. . MethylaRNA Fig. , FTO ovels Fig. S. Consistlls Fig. . These r6A regulation on c-Myc expression, we constructed a luciferase reporter gene with an integrated CDS sequence containing the WT or mutated m6A sites from the 3\u2032 end of MYC mRNA , but not in subunits smaller than 40S, 40S, and 60S Table S. As expe) Table SA, B. Not) Table SC, D. Cor) Table S, S5E, la) Table S, S5F, ce) Table S, migrati) Table S, S5J of 48. Of note, the FTO depletion-enhanced these protein expressions were largely reduced by c-Myc depletion (Fig. We next subcutaneously injected H358 cells with or without FTO depletion or combined FTO and c-Myc depletion into athymic nude mice. We showed that FTO depletion-promoted tumor growth was blunted by c-Myc depletion Fig. . Immunohion Fig. . In addiion Fig. , furtherion Fig. . These r6A modification from mRNAs, plays instrumental roles in the regulation of many cellular functions7. However, how FTO is transcriptionally regulated, especially by oncogenic signaling, remains to be explored. We demonstrated here that FTO expression was downregulated in lung adenocarcinoma and positively correlated with overall survival. Depletion of FTO enhanced tumor cell proliferation, anchorage-independent growth, migration, invasion, and tumor formation in mice. Of special interest, we found that Wnt signaling, which is vital in lung adenocarcinoma tumorigenesis51, induced the binding of \u03b2-catenin/TCF/LEF to the TBEs of the FTO promoter region and suppressed FTO expression. Mechanistically, we showed that WNT signaling induced the binding of EZH2 to \u03b2-catenin, leading to an EZH2-dependent H3K27me3 increase at the FTO promoter region for inhibition of FTO expression. Downregulation of FTO expression enhanced m6A levels on mRNAs in a number of signaling pathways, including metabolic pathways, in which MYC is a master regulator of gene expression for glycolysis45. Enhanced m6A levels on MYC mRNA recruited the binding of YTHDF1 and enhanced MYC mRNA translation. FTO depletion and YTHDF1-dependent upregulation of c-Myc promoted tumor cell glycolysis, growth, migration, and invasion and accelerated tumor growth in mice and tumor metastasis to the lung (Fig. In addition to epigenetic regulation of gene expression, regulation of the epitranscriptome exerts another critical layer to regulate protein expression. FTO, a key demethylase that removes the mMYC gene transcription can be upregulated directly by \u03b2-catenin/TCF/LEF45. FTO downregulation can upregulate Wnt signaling by increasing m6A modification in FZD10 mRNA52. In addition, m6A-dependent and YTHDF2-dependent decreases in MYC transcript stability in leukemia cells were reported14. We showed that Wnt stimulation suppressed FTO expression, therefore inducing m6A-dependent and YTHDF1-dependent increases in MYC mRNA translation. Given that FTO globally regulates the expression of many genes, which can be signaling context-dependent and cancer type-dependent16, the differential and distinct regulation of m6A in mRNAs downstream of the FTO exhibit wide variation and may elicit distinct cellular activities. Our finding that FTO downregulation induced by Wnt/\u03b2-catenin signaling enhanced c-Myc expression through upregulation of m6A of MYC mRNA and subsequent YTHDF1 binding revealed a novel mechanism by which lung adenocarcinoma promotes glycolysis and growth. The discovery of the novel regulation of c-Myc expression may lead to an alternative approach for the therapeutic treatment of lung adenocarcinoma.Supplementary figure legendsSupplementary table 1Supplementary table 2Supplementary table 3Supplementary table 4Supplementary table 5Supplementary figure 1Supplementary figure 2Supplementary figure 3Supplementary figure 4Supplementary figure 5"} +{"text": "Accumulating evidences supported that knock-down of DHCR24 is linked to the pathological risk factors of AD, suggesting a potential role of DHCR24 in AD pathogenesis. However, the molecular mechanism link between DHCR24 and tauopathy remains unknown. Here, in order to elucidate the relationship between DHCR24 and tauopathy, we will focus on the effect of DHCR24 on the tau hyperphosphorylation at some toxic sites. In present study, we found that DHCR24 knock-down significantly lead to the hyperphosphorylation of tau sites at Thr181, Ser199, Thr231, Ser262, Ser396. Moreover, DHCR24 knock-down also increase the accumulation of p62 protein, simultaneously decreased the ratio of LC3-II/LC3-I and the number of autophagosome compared to the control groups, suggesting the inhibition of autophagy activity. In contrast, DHCR24 knock-in obviously abolished the effect of DHCR24 knock-down on tau hyperphosphrylation and autophagy. In addition, to elucidate the association between DHCR24 and tauopathy, we further showed that the level of plasma membrane cholesterol, lipid raft-anchored protein caveolin-1, and concomitantly total I class PI3-K (p110\u03b1), phospho-Akt (Thr308 and Ser473) were significantly decreased, resulting in the disruption of lipid raft/caveola and inhibition of PI3-K/Akt signaling in silencing DHCR24 SH-SY5Y cells compared to control groups. At the same time, DHCR24 knock-down simultaneously decreased the level of phosphorylated GSK3\u03b2 at Ser9 (inactive form) and increased the level of phosphorylated mTOR at Ser2448 (active form), leading to overactivation of GSK3\u03b2 and mTOR signaling. On the contrary, DHCR24 knock-in largely increased the level of membrane cholesterol and caveolin-1, suggesting the enhancement of lipid raft/caveola. And synchronously DHCR24 knock-in also abolished the effect of DHCR24 knock-down on the inhibition of PI3-K/Akt signaling as well as the overactivation of GSK3\u03b2 and mTOR signaling. Collectively, our data strongly supported DHCR24 knock-down lead to tau hyperphosphorylation and the inhibition of autophagy by a lipid raft-dependent PI3-K/Akt-mediated GSK3\u03b2 and mTOR signaling. Taking together, our results firstly demonstrated that the decrease of plasma membrane cholesterol mediated by DHCR24 deficiency might contribute to the tauopathy in AD and other tauopathies. Previous studies found that there is a significant reduction in the expression of new gene in specific vulnerable brain regions in Alzheimer's disease (AD) patients, which was named selective AD indicator 1 (Seladin-1), or 3\u03b2-hydroxysterol-\u039424 reductase (DHCR24) and mammalian target of rapamycin (mTOR) signaling, which are involved in the abnormal hyperphosphorylation of some tau sites in tauopathy. In our experiment, we will focus on the effect of DHCR24 on the tau hyperphosphorylation at some sites such as Thr181, Ser199, Thr231, Ser262, Ser256, Ser396, and autophagy, in order to elucidate the relationship between DHCR24 downregulation and tauopathy.DHCR24 is known as the key synthetase in cholesterol synthesis, which catalyzes the final step of cholesterol synthesis via the Bloch pathway, or converts lanosterol to cholesterol at the first step in the Kandutsch-Russell pathway , AKt (#4691), Phospho-Akt (T308) (#13038), Phospho-Akt (Ser473) (#4060), PI3 Kinase p110\u03b1 (#4249), GSK-3\u03b2 (#12456), Phospho-GSK-3\u03b2(Ser9) (#5558), mTOR (#2983), Phospho-mTOR (#2971), Tau (#46687), Phospho-Tau (Thr181) (#12885), Phospho-Tau (Ser199) (#29957), Phospho-Tau (Ser396) (#9632), LC3B (#3868), P62/SQSTM1 (#23214), GAPDH (#2118), secondary antibodies anti-rabbit IgG, HRP-linked antibody (#7074), Anti-rabbit IgG (H+L) (#5366), Anti-mouse IgG, HRP-linked antibody (#7076) were purchased from Cell Signaling Tech (USA), Primary antibodies against Caveolin-1 (ab2910), Phospho-Tau (Ser262) (ab131354) were purchased from British abcam Company, Primary antibodies against Phospho-Tau (Thr231) (4137) was purchased from China ABclonal Company. Primers were purchased from Sangon Biotech . Propidium Iodide (abs9105), Methyl-\u03b2-cyclodextrin (abs42021762), Filipin III (abs42018484) were purchased from China Absin Company.SH-SY5Y cells were obtained from Chinese Academy of Sciences and kept in DMEM F12 with 10% fetal bovine serum . Following ATCC protocols, all cells were cultured in a 5% CO2 humidified incubator at 37\u00b0C.With regard to lentivirus transfection, lentivirus at multiplicity of infection (MIO) about five mediated DHCR24 shRNA, DHCR24 cDNA, and NC infected into the SH-SY5Y cells. At 48 h after infection, the medium was replaced with fresh complete growth medium containing the appropriate concentration of puromycin (6 \u03bcg/mL). At 3 to 4 days after infection, the selected cells were used for experiments. Note: ShRNA target sequence was as follow: (5'-GCATCATCCCTGCCAAGAAGT-3'); The empty vector sequence was as follow: (5'-TTCTCCGAACGTGTCACGT-3'), cDNA sequence was as follow: (5' -ATGGAGCCCGCCGTGTCGCTGGCCGTGTGCGCGCTGCTCTTCCTGCTGTGGGTGCGCCTGAAGGGGCTGGAGTTCGTGCTCATCCACCAGCGCTGGGTGTTCGTGTGCCTCTTCCTCCTGCCGCTCTCGCTTATCTTCGATATCTACTACTACGTGCGCGCCTGGGTGGTGTTCAAGCTCAGCAGCGCTCCGCGCCTGCACGAGCAGCGCGTGCGGGACATCCAGAAGCAGGTGCGGGAATGGAAGGAGCAGGGTAGCAAGACCTTCATGTGCACGGGGCGCCCTGGCTGGCTCACTGTCTCACTACGTGTCGGGAAGTACAAGAAGACACACAAAAACATCATGATCAACCTGATGGACATTCTGGAAGTGGACACCAAGAAACAGATTGTCCGTGTGGAGCCCTTGGTGACCATGGGCCAGGTGACTGCCCTGCTGACCTCCATTGGCTGGACTCTCCCCGTGTTGCCTGAGCTTGATGACCTCACAGTGGGGGGCTTGATCATGGGCACAGGCATCGAGTCATCATCCCACAAGTACGGCCTGTTCCAACACATCTGCACTGCTTACGAGCTGGTCCTGGCTGATGGCAGCTTTGTGCGATGCACTCCGTCCGAAAACTCAGACCTGTTCTATGCCGTACCCTGGTCCTGTGGGACGCTGGGTTTCCTGGTGGCCGCTGAGATCCGCATCATCCCTGCCAAGAAGTACGTCAAGCTGCGTTTCGAGCCAGTGCGGGGC CTGGAGGCTATCTGTGCCAAGTTCACCCACGAGTCCCAGCGGCAGGAGAACCACTTCGTGGAAGGGCTGCTCTACTCCCTGGATGAGGCTGTCATTATGACAGGGGTCATGACAGATGAGGCAGAGCCCAGCAAGCTGAATAGCATTGGCAATTACTACAAGCCGTGGTTCTTTAAGCATGTGGAGAACTATCTGAAGACAAACCGAGAGGGCCTGGAGTACATTCCCTTGAGACACTACTACCACCGCCACACGCGCAGCATCTTCTGGGAGCTCCAGGACATTATCCCCTTTGGCAACAACCCCATCTTCCGCTACCTCTTTGGCTGGATGGTGCCTCCCAAGATCTCCCTCCTGAAGCTGACCCAGGGTGAGACCCTGCGCAAGCTGTACGAGCAGCACCACGTGGTGCAGGACATGCTGGTGCCCATGAAGTGCCTGCAGCAGGCCCTGCACACCTTCCAAAACGACATCCACGTCTACCCCATCTGGCTGTGTCCGTTCATCCTGCCCAGCCAGCCAGGCCTAGTGCACCCCAAAGGAAATGAGGCAGAGCTCTACATCGACATTGGAGCATATGGGGAGCCGCGTGTGAAACACTTTGAAGCCAGGTCCTGCATGAGGCAGCTGGAGAAGTTTGTCCGCAGCGTGCATGGCTTCCAGATGCTGTATGCCGACTGCTACATGAACCGGGAGGAGTTCTGGGAGATGTTTGATGGCTCCTTGTACCACAAGCTGCGAGAGAAGCTGGGTTGCCAGGACGCCTTCCCCGAGGTGTACGACAAGATCTGCAAGGCCGCCAGGCACTGA-3').Total RNA was extracted using RNAiso Plus according to the manufacturer's instructions. Reverse transcription was performed using a PrimeScript\u2122 RT Master Mix with specific primers see . ReactioSH-SY5Y cells seeded in twelve-well-plate were incubated for 12-20 h, followed by washing three times with PBS, then fixed with 4% paraformaldehyde at room temperature for 30 min, followed by washing three times with PBS.0.5% Triton X-100 was adopted to permeate cells for 20 min. Cells were blocked with 5% bovine serum Albumin (BSA) in PBS at room temperature for 1 h, then incubated with the primary antibody at 4\u00b0C overnight followed by three times washing with PBS. Samples were incubated in secondary antibody in the dark for 1 h followed by stained with DAPI (0.5 \u03bcg/mL) for 5 min at room temperature. Finally, cells were observed and the fluorescent images were captured by a confocal microscope .After designated treatment, lysates were generated by placing the cells in SDS lysis buffer containing a cocktail of protease and phosphatase inhibitors. BCA was performed to determine total protein concentrations, which were normalized to 1 mg/mL for all samples. Samples were then prepared in sample buffer and heated to 100\u00b0C for 8 min. Equal amounts of lysates were fractionated using 8\u201312% sodium dodecyl sulfate polyacrylamide gels and were then electrotransferred onto nitrocellulose membranes. Gels were run at a constant voltage (80V) for 2\u20133 h for maximum separation. Wet transfer was performed for 120 min at constant current (250 mA) using polyvinylidenedifluoride membrane presoaked in methanol. The membrane was blocked in 5% milk in 0.2% TBST. The membrane was then washed three times in TBST for 10 min each. After overnight incubation at 4\u00b0C with the primary antibodies the blots were washed and exposed for 1 h at room temperature to corresponding HRP-conjugated secondary antibodies. Chemiluminescent detection was then used to detect the expression of each protein; GAPDH level served as internal loading controls.Cells were grown on coverglass bottom dishes. In order to allow a better visualization of intracellular cholesterol pools, cells were treated with 10 mM methyl-\u03b2-cyclodextrin for 30 min at 37\u00b0C to remove cholesterol from the plasma membrane. Cells were then fixed for 10 min at RT with 4% paraformaldehyde in phosphate-buffered saline (PBS). Cells were incubated with a solution of filipin for 30 min. Finally, after two washes, cells were counterstained with propidium iodide for 5 min. Acquiring images with a confocal laser scanning microscope equipped with a 380 nm optically pumped semiconductor laser . Whole-cell cholesterol staining with filipin did not require M\u03b2CD treatment.SH-SY5Y cells were treated by stationary liquid which contained 3% glutaraldehyde and 0.22 mmol/L sucrose phosphate buffer for 4 h, then rinsed three times with 0.1 M phosphoric acid rinsing solution for 10\u201315 min, and fixed with 1% citric acid for 2 h. Next, stepwise ethanol dehydration, followed by epoxy resin embedding, overnight in a 30\u00b0C oven, then in a 60\u00b0C oven for 12 h. Using an ultra-thin slicer, 50-to 100-nm-thick slices were cut. Using uranium acetate and lead nitrate double staining, a transmission electron microscopy was used to examine autophagosomes in SH-SY5Y cells.P-values were determined using one-way ANOVA. Significance was defined as P < 0.05.Statistical analysis was performed using SPSS statistical software. All data were expressed as mean \u00b1 SD from at least three independent experiments. To explore the influence of DHCR24 on tauopathy in Alzheimer's disease, a transient knock-down of DHCR24 by lentivirus-mediated shRNA (knock-down group) and knock-in of DHCR24 by lentivirus-mediated cDNA (knock-in group) in the SH-SY5Y cell line was performed. DHCR24 shRNA, DHCR24 cDNA were well-expressed in SH-SY5Y cells, as indicated by GFP (green) under fluorescence microscopy . TransduSo far, it is still not to elucidate the potential relationship between DHCR24 and tau hyperphosphorylation. Increasing data supported that the hyperphosphorylation of tau at several sites, such as Thr181, Ser199, Thr231, Ser262, Ser356, and Ser396, which are tightly associated with tauopathy in AD and other tauopathies and western blot analysis. Autophagy is an intracellular degradation pathway essential for cellular and energy homoeostasis, involving in the clearance of misfolded proteins and damaged organelles and Phospho-Akt (Thr308) were also markedly lowered by DHCR24 knock-down in SH-SY5Y cells. In contrast, the DHCR24 knock-in obviously increased the expression level of total class I PI3-K and the Phospho-Akt (Ser473 and 308), leading to the enhancement of PI3-K/Akt signaling. Thus, we considered that the decrease of PI3-K expression induced by DHCR24 knock-down could directly inhibited the phosphorylation activation of Akt, leading to the inhibition of PI3-K/Akt signaling. In addition, previous study suggested that brain cholesterol deficiency induced by silencing DHCR24 was associated with reduce of membrane cholesterol and altered membrane cholesterol-rich lipid-raft composition including the dysfunction of caveolae in cholesterol-rich lipid-raft microdomains, resulting in the markedly Akt inactivation, which is partly consistent with our outcomes (Lu et al., /caveolae may lead to the alteration of raft-dependent functional protein kinase, such as PI3-K kinase (Noticeably, in our study, K kinase . TogetheIn present paper, we also further showed that the DHCR24 knock-down could induce overactivation of GSK3\u03b2 and mTOR kinase activity by the inhibition of PI3-K/Akt. Conversely, the DHCR24 knock-in could promote the activation of PI3-K/Akt signaling, and simultaneously inhibition of GSK3\u03b2 and mTOR signaling. Furthermore, GSK3\u03b2 was one of the first identified substrates of the heavily studied oncogenic kinase Akt, phosphorylation by which inhibits GSK3\u03b2 activity. Akt activation is highly dependent on the class I PI3-K, and in turn promte the GSK3\u03b2 at Ser9 phosphorylation and inhibition of GSK3\u03b2 activity (Buller et al., In addition, the alteration of GSK3\u03b2 and mTOR signaling plays a crucial role in tauopathy, which directly or indirectly participated in regulation of tau phosphorylation (Balaraman et al., Dysfunction of autophagy mechanism has been proposed to play a critical role in AD and other neurodegenerative disorders (Zare-Shahabadi et al., de novo synthetic ability and mainly rely on lipoprotein-conjugated cholesterol produced by Astrocytes (Dietschy and Turley, Noteworthily, Ledesma et al. revealed that the neuronal membrane cholesterol have a significant reduction, the loss of neuronal membrane cholesterol and anomalous raft microdomains, which contribute to excessive amyloidogenesis in AD patients (Abad-Rodriguez et al., In conclusion, in present study, our results further showed that DHCR24 could control the rate of cholesterol synthesis and cholesterol homeostasis by the Bloch pathway and/or K-R pathway. As a synthetase heavily involved in cholesterol synthesis, DHCR24 knock-down significantly lead to a reduction of cholesterol in plasma membrane and intracellular compartments, resulting in the cholesterol loss in SH-SY5Y cells. Furthermore, the decrease of plasma membrane cholesterol mediated by DHCR24 deficiency might contribute to the tauopathy in AD and other tauopathies, at least partly by membrane lipid raft-mediated signaling mechanism . Taken tThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.HZ designed the study and wrote the manuscript. XB contributed to the experimental work about tau phosphorylation and autophagy. JW and MZ contributed to the transfection work of SH-SY5Y cells. YX, LD, and KY provided their experimental assistance and TEM work. JZ and JB contributed to analysis of data. YZ and GX provided their technical assistance. All authors have read the manuscript and provided the input.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "The importance of histone variant H2A.Z in transcription regulation has been well established, yet its mechanism-of-action remains enigmatic. Conflicting evidence exists in support of both an activating and a repressive role of H2A.Z in transcription. Here we report cryo-electron microscopy (cryo-EM) structures of nucleosomes and chromatin fibers containing H2A.Z and those containing canonical H2A. The structures show that H2A.Z incorporation results in substantial structural changes in both nucleosome and chromatin fiber. While H2A.Z increases the mobility of DNA terminus in nucleosomes, it simultaneously enables nucleosome arrays to form a more regular and condensed chromatin fiber. We also demonstrated that H2A.Z\u2019s ability to enhance nucleosomal DNA mobility is largely attributed to its characteristic shorter C-terminus. Our study provides the structural basis for H2A.Z-mediated chromatin regulation, showing that the increase flexibility of the DNA termini in H2A.Z nucleosomes is central to its dual-functions in chromatin regulation and in transcription. Chromatin is a dynamic structure that has an important regulatory role in controlling the genomic DNA accessibility and various nuclear processes. One mechanism used by the cell to influence chromatin structure and function is through histone variant exchange, which confers the nucleosome with new chemical and physical properties. With the lack of details and insights into how histone variants influence chromatin structure and function, the molecular mechanism of variant-mediated gene expression is subject to much speculation.Drosophila, Tetrahymena and mouse dimer and the (H3\u2013H4) tetramer and in the nucleosome surface around the acidic patch while twle (NCP) ,15. An eic patch . The latThe DNA template containing twelve tandem repeats of 167-bp 601 Widom sequence was a kind gifts from Dr Craig Peterson. The DNA template containing twelve tandem repeats of 208-bp 601 Widom sequence was a kind gift from Dr Ed Luk. Large-scale plasmids were purified as previously described . To geneThe sequences for the 167-bp and 208-bp Widom sequence are listed as following with the 601 sequence underlined:CTGGAGAATCCCGGTGCCGAGGCCGCTCAATTGGTCGTAGACAGCTCTAGCACCGCTTAAACGCACGTACGCGCTGTCCCCCGCGTTTTAACCGCCAAGGGGATTACTCCCTAGTCTCCAGGCACGTGTCAGATATATACATCCTGTGCATGACTAGAT167-bp DNA repeat: ATCCCGCCCTGGAGAATCCCGGTGCCGAGGCCGCTCAATTGGTCGTAGACAGCTCTAGCACCGCTTAAACGCACGTACGCGCTGTCCCCCGCGTTTTAACCGCCAAGGGGATTACTCCCTAGTCTCCAGGCACGTGTCAGATATATACATCCTGTGCATGTATTGAACAGCGACCTTGCCGGAGT.208-bp DNA repeat: ACTTATGTGATGGACCCTATACGCGGCCGCCCTGGAGAATCCCGGTGCCGAGGCCGCTCAATTGGTCGTAGACAGCTCTAGCACCGCTTAAACGCACGTACGCGCTGTCCCCCGCGTTTTAACCGCCAAGGGGATTACTCCCTAGTCTCCAGGCACGTGTCAGATATATGCATCCTGTGCATGTATTGAACAGCGACCTTGCCGGAGT.Modified 208-bp DNA repeat with NsiI site introduced: ACTTATGTGATGGACCCTATACGCGGCCGCCXenopus laevis histones H2A, H2B, H3 and H4 were expressed in BL21 (DE3) pLysS E. coli cells and purified as previously described . The H2A.Z.1 gene was sub-cloned into pET LIC expression vector. The H2A.Z mutant in the acidic patch includes residue substitution D97N, S98K, G106Q and Glycine insertion after I100. The H2A.Z L1 loop mutant was constructed with residues in H2A.Z replaced by those from the H2A L1 loop . These mutations were introduced using Q5 site-directed mutagenesis kit . The H2A.Z M6\u2013M7 cassette mutant was constructed by swapping residues G93\u2013G120 with the corresponding H2A sequence (residue N90-P118). The H2A.Z C-terminus mutant was constructed by replacing H2A.Z amino acid G123-V128 with the corresponding H2A sequence (amino acid T121-K130). The H2A.Z C-terminus-extended mutant was constructed by replacing H2A.Z sequence from amino acid G93-V128 with the corresponding sequence in H2A from amino acid N90-K130. These mutants were obtained by gene synthesis (Synbio Technologies). Both wild-type and mutant H2A.Z were expressed in BL21 (DE3) E. coli cells and purified using the same procedure as the canonical histones.escribed . Mouse Hin vitro using salt dialysis as previously described of the chromatin fiber was screened using negative-stained EM.A 1:1 optimal ratio of octamer:DNA for nucleosome reconstitution and for array reconstitution was determined empirically through the electrophoretic mobility shift assay (EMSA). Induction of chromatin fiber formation and purification were done through the addition of Mgd method . BrieflyP\u00a0\u2264\u00a00.05.\u00a0Two-way Anova test and graphical representation was done using Prims 5 software.250 nM of 208 bp nucleosome was mixed with 45\u00a0U of restriction enzyme (HinfI or NsiI) in Cutsmart buffer (NEB) in total volume of 45ul and incubated at 37\u00b0C. Samples were collected every 15 min (5 ul), quenched with 8ul of stop buffer and incubated at 50\u00b0C for 20 min for deproteination. Samples were examined on an 8% Native-PAGE gel and stained with SYBR GOLD. Digestion results were analyzed using ImageJ software version 1.53e. Two-way Anova test was used to determine the level of significant difference at For the reactions mentioned in Figure P value.Fitting was done using the Solver add-in in Excel over 5 iterative cycles. The rate constants under 8\u00b0C and 100% humidity. Aliquots of 3.5 \u03bcl of the nucleosome were applied to glow-discharged QUANTIFOIL grids (R1.2/1.3 \u2013 400 mesh), blotted for 4\u20135 s\u00a0and plunged into liquid ethane cooled by liquid nitrogen. Grids were stored in liquid nitrogen until they were imaged.2 per micrograph in counting mode using EPU software, giving a pixel size of 1.12 \u00c5 at the specimen level. Defocus values range from 0.9 to 3 \u03bcm. Each movie was dose-fractionated to 78 frames with a dose rate of \u223c0.69 e/pixel/s. Total exposure time was 60 seconds, corresponding to a total dose of 33 e/ \u00c5crograph .2. For the H2A fiber, part of the dataset was collected with the same Titan Krios microscope using the condition mentioned above, and the rest of the dataset was collected using the Talos Arctica microscope operating at 200 keV with a Falcon 3EC direct electron detector at counting mode with a nominal magnification of 73 000\u00d7, giving a physical pixel size of 1.4 \u00c5/pixel. Each movie in the Arctica dataset was dose-fractionated to 78 frames with a dose rate of \u223c0.75 e/pixel/s. Total exposure time was 60 s, corresponding to a total dose of 30 e/ \u00c52 per micrograph. The micrographs from the Krios dataset were scaled to the same pixel size as the Arctica dataset, before the two datasets were merged . The histone core from mouse H2A.Z nucleosome structure (PDB: 1F66) and the 601 DNA from the NCP structure (PDB: 6FQ5) were combined to generate the initial template used for refinement. This template was manually fitted into the density map in UCSF Chimera . followeThe nominal resolution of the overall reconstructions of chromatin fibers is limited, due to the motions of individual nucleosome relative each other within the fiber, and thus it is intrinsically difficult to improve. However, the positions of nucleosome N3\u2013N10 in the multi-body-refined maps is sufficiently clear to fit the nucleosome crystal structures. The fiber models were built by rigid-body docking eight crystal structures of H2A.Z nucleosome (PDB ID: 1F66) or canonical nucleosome (PDB ID: 3LZ0) into the cryo-EM density map of the corresponding fibers using UCSF Chimera . The linIn Figure Xenopus histone H2A or mouse histone variant H2A.Z.1 were reconstituted using recombinant proteins , even though it appears faster than the H2A nucleosome control (P\u00a0= 0.015) Figure . Concurr) Figure is simil) Figure . In the ) Figure . For the) Figure . These ret\u00a0al. showed that variant histone H2A.Z facilitates the intramolecular folding of nucleosome arrays into 50S chromatin fibers in vitro, while simultaneously inhibiting the formation of a highly condensed structure and \u03b3 (27.5 \u00ba) are larger than those of the H2A fiber (\u03b2 = 12\u00ba and \u03b3 = 9.7\u00ba). The average shift d between units is shorter for H2A.Z fiber (59.5 \u00c5) than for the H2A fiber (61 \u00c5). Using these parameters, we built two 30-nm fiber models, each containing 24 tandem repeats of 167-bp 601 Windom sequence . Overall2+) , with a ) Figure , while t) Figure . Bendingin-vitro DNA accessibility assay. Several recent cryo-EM studies of histone variants such as CENP-A . Though the molecular details of these interactions and how their interplays contribute to chromatin higher-order folding are less clear, our model is generally consistent with an ic patch . The morLinker histones H1 are known as an important factor for stabilizing the higher-order chromatin structure and for in vitro. Based on these results, we propose a model for H2A.Z-mediated transcription regulation where the function of H2A.Z is context-dependent and the Protein Data Bank (https://www.rcsb.org/). EM maps are deposited at the Electron Microscopy Database for the canonical nucleosome (EMD-23632), H2A.Z nucleosome (EMD-23626), H2A fiber (EMD-23631), and H2A.Z fiber (EMD-23630) respectively. The protein coordinate of H2A.Z nucleosome is deposited at the Protein Data Bank (PDB ID: 7M1X).The data that support the findings of this study are openly available in the Electron Microscopy Database (gkab907_Supplemental_FileClick here for additional data file."} +{"text": "Tricholusia ni insect pupae as natural bioreactors, which are programmed by recombinant baculovirus vectors. Co-infecting the insect pupae with two baculovirus vectors expressing the RHDV GI.1- and RHDV GI.2-derived VP60 proteins, we obtained chimeric VLPs incorporating both proteins as determined by using serotype-specific monoclonal antibodies. The resulting VLPs showed the typical size and shape of this calicivirus as determined by electron microscopy. Rabbits immunised with the chimeric VLPs were fully protected against a lethal challenge infection with the two RHDV serotypes. This study demonstrates that it is possible to generate a dual cost-effective vaccine against this virus using a single production and purification process, greatly simplifying vaccine manufacturing.The VP60 capsid protein from rabbit haemorrhagic disease virus (RHDV), the causative agent of one of the most economically important disease in rabbits worldwide, forms virus-like particles (VLPs) when expressed using heterologous protein expression systems such as recombinant baculovirus, yeasts, plants or mammalian cell cultures. To prevent RHDV dissemination, it would be beneficial to develop a bivalent vaccine including both RHDV GI.1- and RHDV GI.2-derived VLPs to achieve robust immunisation against both serotypes. In the present work, we developed a strategy of production of a dual-serving RHDV vaccine co-expressing the VP60 proteins from the two RHDV predominant serotypes using CrisBio technology, which uses Lagovirus genus within the Caliciviridae [Rabbit haemorrhagic disease virus (RHDV) is the causative agent of a highly infectious and fatal disease affecting domestic and wild rabbits ,2,3. It iviridae . The posiviridae ,7. Additiviridae ,9. The viviridae .Commercially available vaccines are still traditional inactivated forms of the original pathogens , which aIn the course of its evolution, RHDV has thus far diverged to form six recognised genotypes , all higEscherichia coli [Saccharomyces cerevisiae [Trichoplusia ni pupae as natural bioreactors [The VP60 capsid protein expressed in heterologous recombinant systems forms characteristic VLPs, which are protective in immunised rabbits. VP60-derived VLPs have been obtained in hia coli , Saccharrevisiae , plants revisiae ,27, and revisiae ,29,30 anrevisiae ,32,33,34reactors to increSpodoptera frugiperda Sf21 cell line was cultured at 28 \u00b0C in TNM-FH culture media supplemented with 10% heat-inactivated foetal bovine serum , gentamicin at 50 \u00b5g/mL ), and Antibiotic-Antimicotic 1\u00d7 ). Recombinant baculoviruses (rBacs) were generated using the TopBac expression cassette [\u00ae II Reagent (Thermo Fisher Scientific) and following the manufacturer\u2019s instructions.The cassette ,37 as doBaculovirus stocks were titered on Sf21 cells using the 6-well plate format. Subconfluent Sf21 cells were infected with 10-fold serial dilutions of recBac stocks for 1 h at 28 \u00b0C. Subsequently, inocula were removed and cells were overlayed with an insect cell media-agarose mix at 1% agarose final concentration and further incubated for 3\u20135 days. Monolayers were stained with neutral red to facilitate plaque counting.The improved baculovirus vector system TopBac was used for the generation of recombinant baculoviruses. The construction of recombinant baculoviruses was carried out essentially as described in , with thRHDV GI.1 Isolate AST89 Genbank accession number Z29471MEGKARTAPQGEAAGTATTASVPGTTTDGMDPGVVATTSVVTAENSSASIATAGIGGPPQQVDQQETWRTNFYYNDVFTWSVADAPGSILYTVQHSPQNNPFTAVLSQMYAGWAGGMQFRFIVAGSGVFGGRLVAAVIPPGIEIGPGLEVRQFPHVVIDARSLEPVTITMPDLRPNMYHPTGDPGLVPTLVLSVYNNLINPFGGSTSAIQVTVETRPSEDFEFVMIRAPSSKTVDSISPAGLLTTPVLTGVGNDNRWNGQIVGLQPVPGGFSTCNRHWNLNGSTYGWSSPRFADIDHRRGSASYPGSNATNVLQFWYANAGSAIDNPISQVAPDGFPDMSFVPFNGPGIPAAGWVGFGAIWNSNSGAPNVTTVQAYELGFATGAPGNLQPTTNTSGSQTVAKSIYAVVTGTAQNPAGLFVMASGVISTPSANAITYTPQPDRIVTTPGTPAAAPVGKNTPIMFASVVRRTGDVNATAGSANGTQYGTGSQPLPVTIGLSLNNYSSALMPGQFFVWQLTFASGFMEIGLSVDGYFYAGTGASTTLIDLTELIDVRPVGPRPSKSTLVFNLGGTANGFSYV*RHDV GI.2\u2014previously RHDVb/2 isolate Nav10/11 accession number KM878681MEGKARTASQGETAGTATTASVPGTTTDGMDPGVVATTSVVTTENASTSIATAGIGGPPQQVDQQETWRTNFYYNDVFTWSVADAPGNILYTVQHSPQNNPFTAVLSQMYAGWAGGMQFRFIVAGSGVFGGRLVAAVIPPGIEIGPGLEVRQFPHVVIDARSLEPVTITMPDLRPNMYHPTGNPGLVPTLVLSVYNNLINPFGGSTSAIQVTVETRPSEDFEFVMIRAPSSKTVDSISPADLLTTPVLTGVGTDNRWNGEIVGLQPVPGGFSTCNRHWNLNGSTFGWSSPRFAAIDHDRGNASYPGSSSSNVLELWYASAGSAADNPISQIAPDGFPDMSFVPFSGTTVPTAGWVGFGGIWNSSNGAPFVTTVQAYELGFATGAPSNPQPTTTTSGAQIVAKSIYGVATGINQATAGLFVMASGVISTPNSSAITYTPQPNRIVNAPGTPAAAPIGKNTPIMFASVVRRTGDINAEAGSTNGTQYGAGSQPLPVTVGLSLNNYSSALMPGQFFVWQLNFASGFMELGLSVDGYFYAGTGASATLIDLSELVDIRPVGPRPSTSTLVYNLGGTTNGFSYV*8 pfu (plaque-forming units)/mL were reached. Baculovirus stocks were stored at \u221280 \u00b0C containing 5% glycerol. The correct expression of VP60 proteins from recombinant baculoviruses was confirmed by detection of proteins using specific sera by Western blot analysis.The recombinant baculoviruses produced were subjected to two rounds of clonal selection by plaque formation, and after virus propagation, infective titres of approximately 10Trichoplusia ni insects were reared following a previously described methodology [hodology . BrieflyThe crude protein extracts were obtained by homogenising the frozen biomass in the adequate protein extraction buffer. The resulting suspension was subjected to the downstream processing to obtaiw/v) aqueous uranyl acetate. Micrographs were recorded with an EM 2000 Ex microscope . For the quantitation analysis of VLP stability, samples obtained from GI.2 (N11-) and GI.1 (AST) infections or from co-infections with the two viruses under different conditions were analysed at day 0 and 60 of storage at 4 \u00b0C. Samples were diluted to a protein concentration of 25 mg/mL to obtain an even distribution of the capsids on the electron microscopy grid and an easily countable number of capsids per image at the suitable magnification. Three grids were prepared per sample and images were taken in four different areas of each grid. We quantified the capsids with ImageJ tools in 15 pictures.Electron microscopy analyses were performed by conventional means. Briefly, purified VLPs (approximately 5 \u00b5L) were applied to glow-discharged carbon-coated grids for 2 min. Samples were negatively stained with 2% was added. All washing steps were carried out in triplicate using PBS-Tween (0.05% PBS-Tw). For peroxidase-labelled antibodies, the Pierce\u2122 ECL Western Blotting Substrate system was used for detection (Thermo Fisher Scientific).2SO4. Absorbances were read at 450 nm using Varioskan Flash microplate reader and Skanit software.ELISA were carried out in flat bottomed 96-well plates . Plates were coated with 100, 50, or 10 ng of Mab (varying concentrations are indicated in the results section) in each well in PBS (1 mM a pH 7.3 with sodium azide 0.05% PBS-AS). Coating plates were incubated overnight at 4 \u00b0C. The next day the supernatant was removed, and plates were blocked with 1% BSA in PBS-AS for 1 h at 37 \u00b0C. Plates were washed 3 times with 200 \u00b5L PBS-Tw per well. Antibodies were added in volumes of 100 \u00b5L per well and incubated for 1 h at 37 \u00b0C followed by 3 washing steps as previously described. Secondary antibodies at the indicated dilutions were incubated for 1 h at 37 \u00b0C. Prior to revelation, plates were washed 3 times in PBS-Tw. For reaction development TMB (Sigma) (100 \u00b5L per well) was added and incubated at room temperature in the dark until the reaction was stopped using 100 \u00b5L 2M HNew Zealand White rabbits were supplied by San Bernardo Farm . Prior to vaccination, sera were collected from the marginal ear vein of all animals to confirm the absence of RHDV antibodies.Two protection experiments were carried out. In general terms, rabbits were vaccinated with chimeric RHDV VLPs and in experiment 1; 7 days post vaccination they were challenged with RHDV GI.2 and in experiment 2; 7 days post vaccination they were challenged with classic RHDV GI.1 isolate RHDV AST89.Group A: Non-vaccinated controls.Group B: 20 \u00b5g chimeric VLPs.Group C: 40 \u00b5g chimeric VLPs.Group D: 5 \u00b5g RHDV G1 VLPs + 5 \u00b5g RHDVb VLPs.Experimental challenge 1: 30-day-old rabbits were divided into 4 groups (Groups A\u2013D), each of 5 animals. Each group was vaccinated with the following VLPs administered in a total volume of 0.5 mL:Group E: Non-vaccinated controls.Group F: 40 \u00b5g chimeric VLPs.Experimental challenge 2: 2-month-old rabbits were divided into 2 groups (Groups E and F), each of 5 animals. Each group was vaccinated with the following VLPs administered in a total volume of 0.5 mL:Rabbits were kept under observation throughout the experiment and housed in pairs in a biosecurity level 2 laboratory. L. europaeus GI.2 virus, common name RHDV-Gal08/13, with 10 LD50. Groups E and F were challenged 7 days post vaccination with RHDV isolate L. europaeus RHDV GI.1 RHDV-Ast89 (Genbank accession number Z49271). Rabbits were infected on day 7 post vaccination with 150 hemagglutination units/0.5 mL (as determined using O- and B-type human erythrocytes). The virus was administered subcutaneously in the cervical area as a dilution of clarified infected liver homogenates. Euthanasia of surviving rabbits was carried out by intravenous injection with Dolethal following the manufacturer\u2019s instructions .Two virus isolates were used in this study. Groups A\u2013D were challenged 7 days post vaccination with isolate Symptoms of RHDV infection, such as nervous, respiratory, and digestive signs, were monitored and recorded daily. Blood samples were collected on days 0 and 7 post vaccination, and from all surviving rabbits 7 days post challenge. Serum was separated after clotting by centrifugation and stored at \u221220 \u00b0C until analysis. After euthanasia, rabbits were necropsied, and gross lesions were recorded. Samples of liver were directly frozen at \u221280 \u00b0C for total RNA extraction using the GenElute Mammalian Total RNA Miniprep Kit , and RHDV GI.2 VP60 detection and quantification were obtained by specific RT-qPCR .n = 5) from each group.Rabbit serum RHDV antibody levels were tested by enzyme-linked immunosorbent assay (ELISA) using the commercially available INgezim RHDV kit following the manufacturer\u2019s instructions. The absorbance was measured at 450 nm in a microplate reader and data were analysed using the Skanit software Version 2.4.5 (Thermo Fisher Scientific). Negative and positive control sera were included in all plates. Results are expressed as mean optical densities \u00b1 SD . To determine if VLPs obtained after co-infecting with the two baculoviruses were formed by a single VP60 protein or were chimeras of the two VP60 proteins, we designed ELISA and dot blot-based sandwich analyses using monoclonal antibodies capable of recognising one or the two VP60 proteins. To determine the consequences of the co-infection conditions on the chimeric VLP stability, we compared the VLP stability after 2 months of storage at 4 \u00b0C. For this analysis, VLPs were produced using GI.1/GI.2-derived particles at baculovirus infection ratios of 1:1, 1:3, and 3:1. In parallel, we performed the same analysis using GI.2 (N11-)- and GI.1 (AST)-derived VLPs produced independently. After this storage period, we observed a dramatic degradation (>70%) of GI.2 (N11)-derived VLPs, indicative of their low stability . This ra5 viral RNA copies per microliter\u2014one rabbit in group B), indicating a solid protection conferred by the chimeric VLPs. The negative control groups showed 80% mortality after infection with RHDV GI.1 or RHDV GI.2 viruses and all animals were positive for virus genome detection. Viral RNA detection ranged between 6.03 \u00d7 105 and 5.81 \u00d7 106 copies per microliter for animals in group A.To analyse the protection conferred by the chimeric VLPs against the two predominant RHDV serotypes, we used a mixture of GI.2 (N11-) and GI.1 (AST89)-derived VLPs at a single previously determined protective dose and two doses of chimeric VLPs to immunise rabbits. As expected, the mixture of 5 \u00b5g of both VLPs induced protection against a lethal dose of RHDV GI.2 virus, showing no interference of the GI.1 (AST89)-derived VLP in this protection. Two doses of chimeric VLPs of 20 and 40 \u00b5g were also used to determine the protection ratios against RHDV GI.2, and only 40 \u00b5g was used to protect against an RHDV GI.1 virus. A high protection rate of 80% against RHDV GI.2 was achieved with the 20 \u00b5g dose of chimeric VLP, and a rate of 100% was achieved with a dose of 40 \u00b5g against both lethal viruses , demonstAntibodies generated against RHDV were analysed using a commercial ELISA from sera extracted from all rabbits on day 0 (pre-vaccination) and from surviving animals on day 14 post vaccination . As expected, all sera were negative for anti-RHDV antibodies on day 0. Seven days post challenge, the majority of surviving animals elicited an immune response as indicated by rising Ab levels . OverallDevelopment of efficacious recombinant subunit vaccines is essential, especially for causal agents for which a cell culture capable of supporting its propagation has not been identified. This is the case for RHDV, which is the causative agent of one of the most economically important diseases in rabbits worldwide. Commercially available vaccines are still traditional inactivated viruses collected from organs of artificially infected rabbits, which is unacceptable from both the safety and ethical points of view. The production of a new-generation vaccine against RHDV needs to meet two main requirements: first, cost-efficiency in production to make it commercially viable, and second, the vaccine has to induce a broad protection against RHDV GI.1 and RHDV GI.2 viruses as cross-protection is only partial at best ,40.Caliciviridae. The productivity of this protein is generally good in most studied systems, but the development of a subunit vaccine based on the VLP that this protein forms is commercially difficult for the production and regulatory-associated costs, considering the limited number of vaccine doses applied in the domestic rabbit population worldwide. In addition, any vaccine against this virus has to be dual because of the potential of two circulating virus serotypes today, RHDV GI.1 and RHDV GI.2. In the present work, we attempted to produce RHDV VLPs in one of the most cost-efficient methods developed to date based on the use of Trichoplusia ni insect chrysalises (CrisBio technology), and also to develop a single production process (upstream and downstream) of a chimeric VLP-based vaccine containing the critical antigens from both predominant RHDVs circulating today.Virus-like particles have received considerable attention due to their potential application in human and veterinary vaccines against infectious diseases. VLPs are highly immunogenic and are extremely safe. In the case of RHDV, the capsid protein forms characteristic VLPs when expressed in different systems as for the other members of the RHDV has been previously described as a versatile platform for foreign B-cell epitope display, inducing protective humoral immune responses . It has In the absence of a widely available tissue culture system that can sustain replication of human noroviruses, VLPs have been used as a surrogate to study the capsid\u2019s structural features and as immunogens to elicit protective humoral responses . The mosTrichoplusia ni larvae [Trichoplusia ni Lepidoptera is highly susceptible to AcMNPV-derived vectors, and therefore, any common baculovirus can be used for industrial or experimental production. This technology is also linearly scalable, facilitating their implementation for large quantities of vaccine doses in a record time.RHDV GI.1 VLPs have been previously produced in i larvae and pupai larvae . Recentli larvae . The proIn the present work, we also used the TopBac expression cassette to generate the baculovirus vectors ,37. The In the present work, we demonstrated the feasibility of producing a vaccine against RHDV that incorporates two different capsid proteins derived from two virus subtypes and confers broad protection in immunised rabbits. The chimeric RHDV VLPs were produced in a single upstream and downstream production process, significatively reducing the complexity of a binary vaccine. In addition, the combination of TopBac baculovirus vector and CrisBio technology allows for a cost-efficient production required for a vaccine directed to domestic rabbits to make it commercially viable."} +{"text": "The authors wish to introduce the following corrections to their article .OLLAS::RPA-1 is incorrect. The correct sequence is:1) The DNA sequence for the TTCCCCAATTTTTATGTATCTGTTTCAGATAGTGAAAGATGTCCGGATTCGCCAACGAGCTCGGACCACGTCTCATGGGAAAGGCGGCAATTCACATCAATCACGATGTCTTCAATAArpa-1 was indicated to be placed on chromosome I, when its correct position is on chromosome II.2) In some places in the list of Strains The published article has been updated. These changes do not affect the results, discussion and conclusions presented in the article."} +{"text": "What is more, interfering with this process is an innovative strategy to overcome the chemo\u2010resistance of GBM cells.Glioblastoma multiforme (GBM) is a primary tumour of the central nervous system (CNS) that exhibits the highest degree of malignancy. Radiotherapy and chemotherapy are essential to prolong the survival time of patients. However, clinical work has demonstrated that sensitivity of GBM to chemotherapy decreases with time. The phenomenon of multi\u2010drug resistance (MDR) reminds us that there may exist some fundamental mechanisms in the process of chemo\u2010resistance. We tried to explore the mechanism of GBM chemo\u2010resistance from the perspective of energy metabolism. First, we found that the oxidative phosphorylation (OXPHOS) level of SHG44 and U87 cells increased under TMZ treatment. In further studies, it was found that the expression of PINK1 and mitophagy flux downstream was downregulated in GBM cells, which were secondary to the upregulation of TP53 in tumour cells under TMZ treatment. At the same time, we examined the mitochondrial morphology in tumour cells and found that the size of mitochondria in tumour cells increased under the treatment of TMZ, which originated from the regulation of AMPK on the subcellular localization of Drp1 under the condition of unbalanced energy supply and demand in tumour cells. The accumulation of mitochondrial mass and the optimization of mitochondrial quality accounted for the increased oxidative phosphorylation, and interruption of the mitochondrial fusion process downregulated the efficiency of oxidative phosphorylation and sensitized GBM cells to TMZ, which was also confirmed in the The progression\u2010free survival of patients with this condition after diagnosis is 6.2\u20137.5\u00a0months; the median survival is 14.6\u201316.7\u00a0monthsIn the studies of the chemo\u2010resistance, mitochondria are under spotlight for a long time. Mitochondria are involved in vital processes such as energy synthesis, regulation of calcium ion, ROS generation and apoptosis executionAs we all know, TMZ kills tumour cells by inducing DNA damage, and the survival cells are always with upregulated DNA damage response or multiple stress responses. Although these responses vary, energy support is essential.22.1TMZ, Compound C, AICAR, Mdivi1, MG132 and WY14643, were purchased from MedChemExpress company. MTT and N\u2010Acetyl\u2010L\u2010cysteine (NAC) were purchased from Sigma\u2010Aldrich. MitoTracker\u2122 Red FM (M22425), Hoechst 33342 (H1399) and anti\u2010Ubiquitin WB Antibody (13\u20131600) were purchased from Invitrogen . Anti\u2010phospho\u2010Ubiquitin (Ser65) (ABS1513\u2010I) was purchased from Merck KGaA company (1:1000). Anti\u2010P53 (21891\u20131\u2010AP), anti\u2010PINK1 (23274\u20131\u2010AP), anti\u2010Parkin (14060\u20131\u2010AP), anti\u2010Drp1 (12957\u20131\u2010AP), anti\u2010Opa1 (27733\u20131\u2010AP), anti\u2010Mfn1 (13798\u20131\u2010AP), anti\u2010Mfn2 (12186\u20131\u2010AP), anti\u2010Caspase\u20109 (10380\u20131\u2010AP), anti\u2010BAX (50599\u20132\u2010Ig), anti\u2010Caspase\u20103 (19677\u20131\u2010AP), anti\u2010VDAC1 (10866\u20131\u2010AP), anti\u2010Lamin B (12987\u20131\u2010AP), anti\u2010Cytochrome c (10993\u20131\u2010AP), and anti\u2010\u03b2\u2010actin (60008\u20131\u2010Ig) were purchased from ProteinTech Group, Inc., . Anti\u2010\u03b3\u2010H2A.X (ab81299) was purchased from Abcam (1:1000). Anti\u2010AMPK\u03b1 (2532) and p\u2010AMPK\u03b1 (50081) were purchased from Cell Signaling Technology, Inc. (1:1000). Anti\u2010phospho\u2010DRP1(Ser616) (DF2972) was purchased from Affinity Biosciences (1:1000). Anti\u2010phospho\u2010DRP1(Ser637) (6319S) was purchased from Cell Signaling Technology, Inc. .2.22\u00a0atmosphere.Human glioblastoma cell lines were obtained from the Chinese Academy of Medical Sciences and cultured in Dulbecco's modified Eagle's medium supplemented with 10% foetal calf serum, penicillin (50\u00a0U/ml) and streptomycin (50\u00a0\u00b5g/ml) purchased from Life Technologies and incubated at 37\u00b0C in a 5% CO2.36 U87MG cells were suspended in 100\u00a0\u00b5l of phosphate buffer saline (PBS) (Beyotime Institute of Biotechnology) and injected subcutaneously into the right armpit of the nude mice. The animals were raised in a specific pathogen\u2010free (SPF) environment, and tumour volume was observed regularly. One week later, when the tumour volume reached 50\u00a0mm3, the nude mice were randomly divided into four groups, with eight mice in each group. The treatment conditions for each group were as follows: control group: 200\u00a0\u00b5l of 5% Carboxymethyl Cellulose (CMC)\u2014daily gavage; TMZ group: 40\u00a0mg of TMZ suspended in 200\u00a0\u00b5l of 5% CMC\u2014daily gavage; WY14643\u00a0group: 10\u00a0mg of WY14643\u00a0suspended in 200\u00a0\u00b5l of 5% CMC\u2014daily gavage; TMZ+WY14641\u00a0group: TMZ (40\u00a0mg) + WY14643 (10\u00a0mg) suspended in 200\u00a0\u00b5l of 5% CMC\u2014daily gavage. The status of the nude mice was observed daily. Mice were weighed and the size of each tumour was measured every 2\u00a0days. The experiment was terminated when the longest diameter of the tumour reached 2\u00a0cm or signs of ulceration appeared. Upon termination of the experiment, the nude mice were sacrificed, and the tumour was stripped, photographed and weighed. The tumour tissue was used for the following experimental tests: (1) evaluating the ATP content of tissue cells; (2) the total protein was extracted from the tumour tissue for Western blotting (WB); (3) part of the tumour tissue was stored in 2.5% dialdehyde and analysed by transmission electron microscopy; and (4) part of the tumour tissue was stored in 4% paraformaldehyde for immunohistochemical analysis.The experimental protocol was approved by the local ethics committee (20200921\u20131). Nude mice were adapted to the experimental environment for a week prior to experimentation. Then, 5\u00a0\u00d7\u00a0102.4Cells were lysed in Prusiner's buffer , EDTA (5\u00a0mM), Triton \u00d7\u2010100 (0.5%), and deoxycholate and protease inhibitor cocktail). The homogenates obtained were then briefly sonicated. Aliquots of 10\u00b5g of total protein were then loaded onto 8%\u201316% SDS\u2010PAGE gels. After migration, proteins were wet\u2010transferred to PVDF membranes and immunoblotted using certain antibodies. Immunological complexes were detected with either anti\u2010rabbit or anti\u2010mouse IgG\u2010coupled peroxidase antibodies by the electrochemiluminescence detection method (Roche Diagnostics S.A.S).2.5Cells were grown on coverslips in 24\u2010well plates and treated with different agents. After staining with MitoTracker for 30\u00a0min, the coverslips were rinsed with PBS and fixed with 4% (w/v) paraformaldehyde for 25\u00a0min at room temperature followed by a PBS rinse. Permeabilization with 0.1% (v/v) Triton \u00d7\u2010100 for 7\u00a0min was followed by blockade with 10% horse serum for another 30\u00a0min. Following incubation with primary antibodies (1:100) at 4\u00b0C for 12\u00a0h, the coverslips were incubated with fluorescent secondary antibodies (1:200) for 30\u00a0min, rinsed with cold PBS and stained with Hoechst 33342 in accordance with the manufacturer's instructions for 7min before a final PBS rinse. A Revolve Hybrid Microscope (Discover ECHO) was used for image capture. Images were further processed by ImageJ Software version 1.52\u00a0s to assess the intensity of immunofluorescence (IF), as well as the mitochondrial parameters. For the evaluation of mitochondrial fusion, we evaluated three images in each treatment group from three independent experiments using ImageJ Software. Two parameters were used to assess the level of mitochondrial fusion: the mean size of the mitochondrial network (MS) and the mean length of mitochondria (ML).2.65 cells/well. Cells were then incubated overnight at 37\u00b0C, and the medium was replaced with fresh complete medium. After 24\u00a0h, the culture medium was collected and proteins were extracted by sonication. Extracted proteins were then quantified with a Bradford Protein Assay kit (Beyotime Institute of Biotechnology). Then, we measured the concentrations of glucose and lactate with glucose (RsBio) and lactate assay kits (Jiancheng Bio), respectively. The glucose consumption in each experimental group was calculated as follows: Glucose consumption\u00a0=\u00a0glucose concentration (fresh complete medium)\u2013glucose concentration .Cells were seeded in 6\u2010well plates at a density of 3\u00a0\u00d7\u00a0102.75 cells/well. Following overnight incubation at 37\u00b0C, the medium was replaced with fresh culture medium. After 6 or 24\u00a0h, in accordance with the manufacturer's instructions, cells were washed with PBS, and then, their ATP levels were determined using an ATP Assay Kit (Beyotime Institute of Biotechnology).Cells were seeded in 6\u2010well plates at a density of 5\u00a0\u00d7\u00a0102.8\u00ae Xtra\u2010Oxygen Consumption Assay (Luxcel Biosciences Cork) using a CLARIOstar microplate reader (BMG Labtech). In brief, cells were plated at a density of 8\u00a0\u00d7\u00a0104 cells/well in 96\u2010well plates and allowed to adhere overnight at 37\u00b0C using a plate block heater. The culture medium was removed from all wells and replaced with 160\u00a0\u03bcl of pre\u2010warmed reaction mixture, including 10\u00a0\u03bcl of reconstituted MitoXpress\u00ae Xtra reagent, 150\u00a0\u03bcl of fresh culture media with TMZ in each well. The wells were sealed by adding two drops of pre\u2010warmed HS Mineral Oil, and the plate was immediately analysed in a microplate reader.Cellular oxygen consumption rate (OCR) was measured by the MitoXpress2.94 cells per well. After exposure to TMZ and/or Mdivi1 or WY14643, for 24\u00a0h, the cells in each well were incubated with MTT solution (0.5\u00a0mg/ml) dissolved in PBS for 4\u00a0h at 37\u00b0C. Then, 150\u00a0\u00b5l of DMSO was added to each well. The absorbance was measured at 570\u00a0nm using a CLARIOstar microplate reader (BMG Labtech). Cell viability was then calculated as follows: cell viability\u00a0=\u00a0absorbance of experimental group/absorbance of control group\u00a0\u00d7\u00a0100%.Cells were seeded in 96\u2010well plates with 100\u00b5l of complete DMEM medium at a density of 1\u00a0\u00d7\u00a0102.10RNA was extracted using a RNeasy kit (Qiagen) in accordance with the manufacturer's protocol. Quantitative real\u2010time reverse transcription\u2010PCR (qRT\u2010PCR) was then performed using a two\u2010step method. Data analysis was based on the delt\u2010delt\u2010Ct method using \u03b2\u2010Actin as a normalization control. PCR was conducted in a QuantStudio5 real\u2010time PCR system (Thermo Fisher) and analysed using QuantStudio Design & Analysis software v1.3.1 (Thermo Fisher).In order to evaluate the mtDNA copy number, we extracted DNA from cells in each group and determined the copy number of the mtDNA gene ND1, as normalized to the 18S gene. In order to evaluate the extent of DNA damage, we selected two fragments of mtDNA (79bp and 230bp in length) as target templates for a qPCR array using the 18S gene as an internal reference. The extent of mtDNA damage was parallel to the value of 79bp/230bp. The primers used for the real\u2010time PCR are listed in Table\u00a02.11The Mitochondria and Nuclear Extraction Kit for Cells (Beyotime Biotechnology) was used to isolate nuclear and mitochondrial fractions from each experimental sample. In each case, we followed the manufacturer's guidelines.2.12A plasmid that overexpressed TP53, along with a negative control (NC) plasmid, were constructed by GenePharma Co., Ltd. Cells were transfected with the plasmids using the Thermo Scientific\u2122 TurboFect\u2122 Transfection Reagent in accordance with the manufacturer's protocol. In brief, cells were plated in 6\u2010well plates and transfected the next day with 4\u00a0\u03bcg of plasmid using 6\u00a0\u03bcl of transfection reagent. Cells were harvested 24\u00a0h after transfection, and whole\u2010cell lysates were isolated for Western blotting. The sequence of TP53 is as follows: ATGGAGGAGCCGCAGTCAGATCCTAGCGTCGAGCCCCCTCTGAGTCAGGAAACATTTTCAGACCTATGGAAACTACTTCCTGAAAACAACGTTCTGTCCCCCTTGCCGTCCCAAGCAATGGATGATTTGATGCTGTCCCCGGACGATATTGAACAATGGTTCACTGAAGACCCAGGTCCAGATGAAGCTCCCAGAATGCCAGAGGCTGCTCCCCCCGTGGCCCCTGCACCAGCAGCTCCTACACCGGCGGCCCCTGCACCAGCCCCCTCCTGGCCCCTGTCATCTTCTGTCCCTTCCCAGAAAACCTACCAGGGCAGCTACGGTTTCCGTCTGGGCTTCTTGCATTCTGGGACAGCCAAGTCTGTGACTTGCACGTACTCCCCTGCCCTCAACAAGATGTTTTGCCAACTGGCCAAGACCTGCCCTGTGCAGCTGTGGGTTGATTCCACACCCCCGCCCGGCACCCGCGTCCGCGCCATGGCCATCTACAAGCAGTCACAGCACATGACGGAGGTTGTGAGGCGCTGCCCCCACCATGAGCGCTGCTCAGATAGCGATGGTCTGGCCCCTCCTCAGCATCTTATCCGAGTGGAAGGAAATTTGCGTGTGGAGTATTTGGATGACAGAAACACTTTTCGACATAGTGTGGTGGTGCCCTATGAGCCGCCTGAGGTTGGCTCTGACTGTACCACCATCCACTACAACTACATGTGTAACAGTTCCTGCATGGGCGGCATGAACCGGAGGCCCATCCTCACCATCATCACACTGGAAGACTCCAGTGGTAATCTACTGGGACGGAACAGCTTTGAGGTGCGTGTTTGTGCCTGTCCTGGGAGAGACCGGCGCACAGAGGAAGAGAATCTCCGCAAGAAAGGGGAGCCTCACCACGAGCTGCCCCCAGGGAGCACTAAGCGAGCACTGCCCAACAACACCAGCTCCTCTCCCCAGCCAAAGAAGAAACCACTGGATGGAGAATATTTCACCCTTCAGATCCGTGGGCGTGAGCGCTTCGAGATGTTCCGAGAGCTGAATGAGGCCTTGGAACTCAAGGATGCCCAGGCTGGGAAGGAGCCAGGGGGGAGCAGGGCTCACTCCAGCCACCTGAAGTCCAAAAAGGGTCAGTCTACCTCCCGCCATAAAAAACTCATGTTCAAGACAGAAGGGCCTGACTCAGACTGA.2.13Tumour tissues were fixed in 4% paraformaldehyde for 24\u00a0h and embedded in paraffin. Paraffin sections were cut into sections (4\u00a0\u03bcm thick) and then deparaffinized and rehydrated prior to antigen retrieval. Sections were blocked with 10% bovine serum albumin in TBS\u2010Tween 20 (Sigma\u2010Aldrich) for 1\u00a0h at room temperature. Sections were then incubated overnight at 4\u2103 with primary antibodies against P53 (21891\u20131\u2010AP) and PINK1 (23274\u20131\u2010AP). The following morning, sections were incubated with secondary antibodies for 30\u00a0min at 37\u2103. Then, slides were incubated with diaminobenzidine for 5\u00a0min, and then counterstained with Gill's haematoxylin for 30\u00a0s. Images were captured on a microscope (Discover ECHO Company).2.14Tumour tissues from the different treatment groups were fixed in 2.5% glutaraldehyde and then embedded in an embedding agent. Next, specimens were sliced into sections (50\u201370\u00a0nm in thickness). The sections were then dyed with uranyl acetate and citrate dye solution. The TEM system (Thermo Fisher Scientific Company) operated at an acceleration voltage of 80\u00a0kV and an electron tomography voltage of 120\u00a0kV. Specimens were magnified by \u00d7150,000, and images were acquired. Three tumour specimens in each treatment group were randomly selected for TEM examination. And each specimen outputted a clear picture of which the mean length of mitochondria was measured directly by the software of TEM. Then, the data were analysed.2.15\u00ae easyCyte flow cytometer (Merck KGaA).Apoptosis assays were performed using an Apoptosis Detection Kit (BD Biosciences). Cells from different treatment groups were trypsinized with 0.25% trypsin, and assays were conducted in accordance with the manufacturer's protocol. In total, 10000 cells were counted in each treatment group using a Guava\u00ae easyCyte flow cytometer (Merck KGaA).For the analysis of mitochondrial mass, cells were grown in 6\u2010well plates and treated with the different agents. Cells were trypsinized with 0.25% trypsin and suspended cells were stained with MitoTracker\u2122 Red FM (Invitrogen) (1:10000) for 30\u00a0min at 37\u2103. Cells were then rinsed three times with PBS, and 10,000 cells were counted for each treatment group using a Guava\u00ae easyCyte flow cytometer (Merck KGaA).The production of ROS was measured by a ROS Assay Kit (Beyotime Biotechnology). In brief, cells were collected and resuspended in DMEM medium after treatment and then incubated with DCFH\u2010DA for 30\u00a0min at 37\u00b0C. Cells were then rinsed three times in PBS, and 10,000 cells were for each treatment group using a Guava2.16U87 cells were seeded at a density of 8000 cells/well and treated for 24\u00a0h with the respective treatments . On the day of the assay, cell culture medium was removed and replaced by assay medium supplemented with agents according to the protocol and the Seahorse XFe96 analyzer was used. In the test, basal cellular oxygen consumption was determined, followed by injection of the ATP synthase inhibitor oligomycin (5\u00a0\u00b5M), the uncoupler fluoro\u2010carbonyl cyanide phenylhydrazone (FCCP) (2\u00a0\u00b5M) and the complex I inhibitor rotenone (0.5\u00a0\u00b5M) combined with the complex III inhibitor antimycin A (0.5\u00a0\u00b5M). The oxygen consumption rate was detected, and each measurement was normalized to cell content.2.17t test. Analysis of variance (ANOVA) was also used to conduct multiple comparisons using GraphPad Prism 7.00 . Dunnett's post hoc multiple comparisons test was used after ANOVA. Data are presented as means\u00a0\u00b1\u00a0standard deviation, and p\u00a0<\u00a00.05 was considered to indicate a statistically significant difference.Data from three independent experiments were collected and analysed using SPSS 22.0 (IBM Corp.) and the Student's 33.1As an alkylating agent, TMZ can induce damage to nuclear DNA and mitochondrial DNA.In order to identify the mechanisms underlying the increased level of oxidative phosphorylation, we first detected the mass of mitochondria in the two cell lines, using three methods: (1) labelling mitochondria with specific fluorescent probes Figure\u00a0A, 2) by by2B) an3.2The mass of mitochondria within a cell is precisely controlled by mitophagy.3.3In order to further explore the mechanism by which mitophagy was downregulated, we focused on how the expression of PINK1 was regulated. We also investigated the elevated expression of intracellular nuclear transcription factor (NTF) in tumour cells under TMZ treatment. First, we found that TMZ treatment\u2010induced cellular DNA damage responses in both SHG44 and U87 cells; these responses involved high expression of \u03b3\u2010H2A.X and the increased aggregation of the protein in the nucleus establishing a cell model overexpressing TP53 by plasmid transfection; under this condition, we observed that PINK1 expression was downregulated in cells overexpressing TP53, and that the expression of Ser65 phosphorylated ubiquitin was correspondingly downregulated after TMZ treatment, the tumour cells showed reduced levels of ATP; thereafter, however, the levels of ATP increased. The curve created by plotting ATP concentration with time showed two troughs; one at 10\u00a0min and the other at 6\u00a0h Figure\u00a0A. FurtheWhat is more, to confirm that the AMPK regulated the function of mitochondria, we performed the mito stress test by using the seahorse analyzer and revealed that: 1.TMZ elevated the OXPHOS potential of mitochondria while repressing the activity of AMPK impaired the effect Figure D.3.6In order to verify that the mobilization of mitochondrial dynamics was involved in the metabolic reprogramming of tumour cells under chemotherapy stress and to explore whether interfering with mitochondrial dynamics could sensitize GBM to TMZ treatment, we used two different agents: WY14643 to promote mitochondrial fissionFinally, we found that WY14643 promoted mitochondrial fission and increased the efficacy of TMZ chemotherapy. This treatment also enhanced the efficacy of TMZ to induce apoptosis of SHG44 and U87 cells . Renxuan Huang: Software ; Visualization . Kunmeng Yang: Data curation . Yichun He: Writing \u2013 review & editing . Delu Dong: Supervision . Yufei Gao: Conceptualization (lead); Funding acquisition (lead).Fig S1Click here for additional data file."} +{"text": "Ephb4 and Coup-TFII (Nr2f2) vein-specific enhancers to demonstrate that ETS factors are equally essential for vein, arterial and angiogenic-specific enhancer activity patterns. Further, we show that ETS factor binding at these vein-specific enhancers is enriched by VEGFA signalling, similar to that seen at arterial and angiogenic enhancers. However, while arterial and angiogenic enhancers can be activated by VEGFA in vivo, the Ephb4 and Coup-TFII venous enhancers are not, suggesting that the specificity of VEGFA-induced arterial and angiogenic enhancer activity occurs via non-ETS transcription factors. These results support a model in which ETS factors are not the primary regulators of specific patterns of gene expression in different endothelial subtypes.Correct vascular differentiation requires distinct patterns of gene expression in different subtypes of endothelial cells. Members of the ETS transcription factor family are essential for the transcriptional activation of arterial and angiogenesis-specific gene regulatory elements, leading to the hypothesis that they play lineage-defining roles in arterial and angiogenic differentiation directly downstream of VEGFA signalling. However, an alternative explanation is that ETS binding at enhancers and promoters is a general requirement for activation of many endothelial genes regardless of expression pattern, with subtype-specificity provided by additional factors. Here we use analysis of \u2022Vein-specific enhancers can contain essential ETS motifs.\u2022VEGFA induced an increase in ETS binding at vein, arterial and angiogenic enhancers.\u2022VEGFA stimulation cannot induce vein-specific enhancer activity. The vascular system is subdivided into arteries, veins, lymphatics and capillaries, each comprised of genetically distinct ECs expressing specific fate-determining genes . The essE-26 transformation-specific) transcription factor family identified a group of ETS binding motifs required for arterial and angiogenic activity signalling may act via ETS transcription factors to enable specific activation of arterial and angiogenic genes. VEGFA signalling influences many processes during early vascular growth, and plays essential roles in vasculogenesis, arterial specification and angiogenesis . ETS traactivity . ERG binactivity . A similactivity , whilst activity .Despite their hypothesised role in arterial and angiogenic-specific patterns of gene expression, binding motifs for ETS factors are also a common feature of many pan-endothelial expressed gene promoters and enhancers . ETS1, Ein vivo. These results indicate that within the endothelium, VEGFA-stimulated ETS factor binding is a shared feature at enhancers associated with multiple different patterns of gene expression, and suggests that additional transcription factors may be primarily responsible for directing arterial, angiogenic and venous-specific gene expression patterns downstream of different growth factor signalling inputs.In this paper, we undertake a detailed analysis of two recently characterized vein-enriched gene enhancers. We demonstrate that, similar to arterial and angiogenic enhancers, ETS factor binding at these venous enhancers is necessary for enhancer activation and vein-specific patterns of reporter gene expression, and that this binding is also enriched by VEGFA signalling. However, unlike arterial and angiogenic enhancers, these venous enhancers cannot be directly activated by over-expression of VEGFA 22.1Ephb4 and Coup-TFII (Nr2f2) gene loci (Coup-TFII. Neither enhancer was active in the mature microvasculature (We have recently identified enhancers within the venous-enriched ene loci . Both enA/T) were able to compete for binding of either ETS1-DBD or ERG, or both proteins and Fig.A/T) A. Of theCGGAAG/A . We perfCGGAAG/A B\u2013C and fCGGAAG/A D\u2013E, bothproteins C while Eproteins E. The spproteins C and E. EPHB4 and COUP-TFII/NR2F2 were significantly enriched in these HUVECs . Similar to the venous enhancers investigated, ERG binding around these arterial-, angiogenic- and pan-endothelial-expressed enhancers was seen in both HUVEC and HUAEC cells, suggesting that binding of ERG to specific enhancer regions was not routinely restricted to the EC subtypes in which the associated genes are preferentially active . Taken together, these results demonstrate that functional ETS binding motifs can be present within vein EC-specific enhancers, and show that the ability to bind ERG and other ETS factors is not restricted to enhancers that are active in arterial and angiogenic ECs.Since the binding of ETS factors to vascular enhancers has been previously associated with arterial-specific and angiogenic-specific enhancers, we next investigate whether ETS factor binding was also a feature at venous-specific enhancers . We found significant binding for ERG, FLI1 and ETS1 at both Ephb4-2 and CoupTFII-965 venous enhancers . EMSA analysis confirmed that the mutated ETS binding motifs could not bind ETS proteins exclusively to the venous endothelium .In agreement with previous studies , our resproteins C and E. on (hpf) and Fig.on (hpf) A\u2013B. Howeon (hpf) A\u2013B. We sothelium and Fig.othelium C\u2013D, the The loss of Ephb4-2 and CoupTFII-965 vein enhancer activity after ETS motif mutation observed here is similar to the loss of activity seen when ETS motifs are mutated in the arterial and angiogenic Dll4in3 enhancer , in othe2.3erg and fli1. There are multiple ETS factors expressed in the developing zebrafish vasculature zebrafish was significantly reduced after morpholino knockdown of erg and fli1, with the strength of Ephb4-2:GFP transgene activity inversely correlated with the levels of erg/fli1 MO . Conversely, we saw a reduction in the expression of endogenous ephb4 and stab1l, a zebrafish venous marker, when assessed by whole mount in situ hybridization analysis . Similar reductions in the expression of some venous-enriched genes was also observed after ERG depletion in HUVECs transgenic zebrafish, although this was not as marked . As previously reported, erg/fli1 knockdown also resulted in reduced activity of the arterial/angiogenic Dll4in3 enhancer in transgenic zebrafish and tg(CoupTFII-965:GFP) zebrafish lines compared to the arterial (tg(Dll4in3:GFP) zebrafish line. At the lowest concentration of inhibitor, Dll4in3:GFP activity was more notably reduced than either venous enhancer, while higher SU5416 doses significantly reduced activity of all enhancers results in EC apoptosis, lower levels of VEGF inhibition result in reduced arterial and venous marker gene expression, although the reduction in venous genes was less pronounced and sometimes compensated by expansion of vein gene activity into the dorsal aorta . We therAlthough Dll4in3:GFP was more sensitive to inhibition of VEGFA signalling than either the venous Ephb4-2:GFP or the CoupTFII-965:GFP, our previous results show that changes in ETS factor binding after VEGFA stimulation can be seen at both venous and arterial enhancers. Consequently, it is unlikely that changes in ETS factor occupancy at enhancers can explain the differences between venous and arterial enhancer responses to VEGFA inhibition. An alternative explanation may be that additional transcription factors may be instead responsible for allowing arterial and angiogenic enhancers a greater sensitivity to VEGFA signalling.2.6in vivo. We first used an established model of VEGFA-stimulated blood vessel growth in mice, in which an adenovirus expressing VEGFA164 (Ad-VEGFA164) is injected intradermally into the ears of adult mice . In both enhancers, angiogenic expression was lost by 60 days after injection, a time-point known to be independent of VEGFA signalling.We next examined whether VEGFA over-expression, and subsequent increased ERG occupancy, is alone sufficient to initiate activity of venous-, arterial- and angiogenic-specific enhancers ult mice . This reult mice . Vessel ult mice . To deteenic ECs A. Ad-VEG164 injection can specifically re-activate the Dll4in3 and HLX-3 enhancers in their native EC sub-types. However, they cannot determine whether this occurs via a VEGFA-mediated increase in ERG binding, VEGFA-mediated increase in other transcription factors binding to these enhancers or VEGFA-mediated removal of repressive factor binding. If VEGFA-mediated activation of these enhancers occurs primarily via changes to ETS factors, we would expect a similar reactivation of venous enhancers. We therefore next determined if Ad-VEGFA164 injection was able to directly activate the Ephb4-2:lacZ and CoupTFII-965:lacZ transgenes. Unlike with the arterial and angiogenic enhancers, we observed no endothelial transgene activity at any time point in either Ephb4-2:lacZ or the CoupTFII-965:lacZ Ad-VEGFA164 injected ears, although occasional ectopic expression could be detected embryos (following protocol and concentration from tg(Ephb4-2:GFP) embryos . This did not change significantly when we increased the amount of vegfaa injected . This result therefore further indicates that increased VEGFA signalling does not increase Ephb4-2 enhancer activity. Additionally, at 50\u00a0\u200bpg vegfaa121 and vegfaa165 we saw no clear expansion of Ephb4-2:GFP expression beyond the venous endothelial expression pattern observed in control embryos (vegfaa121 and vegfaa165 mRNA into 1-cell stage tg(Dll4in3:GFP) embryos resulted in slightly increased GFP intensity and expansion into the caudal vein plexus, as previously described by vegfaa levels . Taken together, these results suggest that the failure of Ad-VEGFA164 to activate venous gene enhancers in the mouse ear is unlikely to be a result simply of developmental context. Furthermore, the Ephb4-2:lacZ transgenes can be reactivated in injured neonatal hearts , suggesting that absence of normal activity does not affect enhancer reactivation. Taken together, these results indicate that VEGFA stimulation is not sufficient to activate transcription from the Ephb4-2 venous EC-specific enhancer, despite its reliance on VEGFA-augmented ETS transcription factors.Although developmental arterial and angiogenic enhancers were reactivated in the adult mouse ear by Ad-VEGFA164 A and B, reduced . Furthern plexus . To dete3The role of ETS transcription factors in the regulatory hierarchy of endothelial cells has been unclear. The specification and maintenance of the endothelial cell lineage requires the shared activation and repression of many lineage-defining genes. However, differential gene expression within specific sub-populations of endothelial cells is also essential for vascular function. Consequently, spatial and temporal control of endothelial gene expression must involve multiple layers of regulation. While analysis of arterial and angiogenic-specific enhancers has supported a proposal that ETS factors play a specific role in the activation of arterial and angiogenic genes downstream of VEGFA signalling, ETS factors have also been implicated in the more general activation of genes, and their cognate regulatory elements, involved in endothelial identity and maintenance. However, the analysis of the precise roles played by ETS factors in the vasculature has been complicated by the abundance of different ETS factors in the endothelium coupled with extensive redundancy between different ETS family members. Parsing their two potential functions is further challenged by the multiple roles played by VEGFA signalling in the vasculature. Recent analysis in zebrafish concluded that low levels of VEGFA signalling promotes general endothelial identity and survival, while higher levels of VEGF signalling primarily promotes arterial specification . ConsequIn this paper, we have clearly demonstrated that vein-specific gene enhancers can be reliant on ETS factors for activation in venous ECs. This is similar to that previously reported in arterial-specific and angiogenic-specific enhancers, even though expression of venous genes is not induced to the same extent by high VEGFA signalling. Further, we have shown that VEGFA signalling can also increases ETS factor binding at venous enhancers, indicating that selective arterial and angiogenic gene activation is unlikely to be achieved through this mechanism. Because our analysis was predominantly restricted to two venous enhancers, the conclusions may not equally apply to all venous-expressed genes. Of note, Notch1 expression in arterial ECs , Tg(CoupTFII-965:lacZ) Tg(Dll4in3:lacZ) and Tg(HLX-3:lacZ) were generated as previously described containing 5\u00a0\u200bmM potassium ferrocyanide, 5\u00a0\u200bmM ferricyanide, 0.1% sodium deoxycholate, 0.2% Nonidet P-40, 2\u00a0\u200bmM MgCl2 and 1 X PBS. After staining, embryos were rinsed through a series of 1 X PBS washes, then fixed overnight in 4% paraformaldehyde at 4\u00a0\u200b\u00b0C. All embryos were imaged using a Leica M165C stereo microscope equipped with a ProGres CF Scan camera and CapturePro software (Jenoptik). In instances that images have been altered to improve quality and colour balance, each image within a set have been altered using the same parameters. This occasionally included to selective depletion of the yellow or red colour channel, in order to counteract issues from the X-gal stain solution (which is orange). All samples are stored in 4% PFA indefinitely and slowly become less orange. Consequently, samples stained more recently have a greater yellow/orange hue. An example of this alteration can be seen in and tg(CoupTFII-965:GFP) zebrafish lines were generated in at 28.5\u00a0\u200b\u00b0C. To image, all embryos were dechorionated and anesthetized with 0.01% tricaine mesylate. For analysis of F0 transgenic zebrafish, single embryos were transferred into a flat bottom 96-well plate, and GFP reporter gene expression screened with a Zeiss LSM 710 confocal microscope at 46\u201350 hpf. Whole zebrafish were imaged using the tile scan command, combined with Z-stack collection under a confocal microscope Zeiss LSM 710\u00a0\u200bMP (Carl Zeiss) at 488\u00a0\u200bnm excitation and 509\u00a0\u200bnm emission (EGFP).All animal procedures comply with all relevant ethical regulations, were approved by Clinical Medicine Local Ethical Review Committee, University of Oxford and licensed by the UK Home Office. Stable rated in . F0 mosarated in . EmbryosFor pharmacological inhibition of VEGF signalling, embryos were manually dechorionated and 0.625\u00a0\u200b\u03bcM, 1.25\u00a0\u200b\u03bcM, 2.5\u00a0\u200b\u03bcM and 5\u00a0\u200b\u03bcM of SU5416 (Stratech Scientific Ltd.) added at approximately 5ss as described in . Control4.3Ephb4-2mutETS and CoupTFIImutETS enhancer sequences were initially generated as custom-made, double-stranded linear DNA fragments . DNA fragments were cloned into the pCR8 vector using the pCR8/GW/TOPO TA Cloning Kit following manufacturer\u2019s instructions. Once cloning was confirmed, each enhancer was transferred from the pCR8/GW/enhancer entry vector to a suitable destination vector using Gateway LR Clonase II Enzyme mix following manufacturer\u2019s instructions. For mouse transgenesis, the enhancer was cloned into the hsp68-LacZ-Gateway vector (provided by N. Ahituv). For zebrafish transgenesis, the enhancer was cloned into the E1b-GFP-Tol2 vector (provided by N. Ahituv).4.3.1AATCAGTGCGTGCTCGTTAAGTCCTGGAGATCCACTGAGCGCGCAGCCTAACGCTGGAGAAAGTGGTTTGAAACCCAAAGTATAGAAAATGTAAATAAAAGGCAGGCGTGTCAGAGAGGGTGAGGGATCTCCGTAACACCTCATTTCATTTTTTTAAAGGAGGGGGACACTTCCCCGCCGCCTGCAGCCTTGACCTCCAAGGCGGGGGTAGGGACCGTTGTGGCTCTTTCCTGAGGCTGTTTCCTGTCTGGCTCCTGGGGGCCCTCGGGATGGCTGGGAGGGCCCTTCCTCTCATTTGCTAGCACCCCCTCTCATCCATCAGTTTGAGGGGAGGGTCCAGGAAAGACGGCCTCCTATCTACATCAGGGCACTGTGAGTGTGGGGCACGGGATGGTTGGATGAGAGAGGTGCTGTTCCCGAAGTCGGTCCTTTAAGGGCTGCGGTAAGGAGACTTTAATTTAAGGTAATTAGTACAGGGTCTGGAAACTCTGAGGTAGGAGTCTGGGGCACCTGGGAGTCTGCCAAATACCCTAAGGGCGCACACACACACCCCAGCGGGCGACCGGTGATGACCTCTTGTCCGCCTGCGCGCACACACACACCAGCGGGCGCGGGAGACCCGTGATGGCCTTTTGTCCCCGTGCACTTATCTTCCTGGCGCAAGTAGTGCTCCCCACCCCCTGCCCTTCCTCACAGCCCTGCCTGGGTCCCGCTCCGGGGTGGGTCAGCCAGGGCAGGAAACAGCCGGCTTGGCTGGAGCCAGGCTGACCGGCTAGATCTGGGAGTCCCCTCCTCCTTCCCCACGCAGACTCAGGCTCCCCTTCTCTTATCCACAGACACCCCCTTTTTTGCAGCTATCATTCTGCATCCGGGTCCCCCTGAATTTCTGAGTCGTGGCTTGTTCTCAC.4.3.2agCTGAGGCTGTTagCTGTCTGGCTCCTGGGGGCCCTCGGGATGGCTGGGAGGGCCCTagCTCTCATTTGCTAGCACCCCCTCTCATCCATCAGTTTGAGGGGAGGGTCCAGGAAAGACGGCCTCCTATCTACATCAGGGCACTGTGAGTGTGGGGCACGGGATGGTTGGATGAGAGAGGTGCTGTTCCCGAAGTCGGTCCTTTAAGGGCTGCGGTAAGGAGACTTTAATTTAAGGTAATTAGTACAGGGTCTGGAAACTCTGAGGTAGGAGTCTGGGGCACCTGGGAGTCTGCCAAATACCCTAAGGGCGCACACACACACCCCAGCGGGCGACCGGTGATGACCTCTTGTCCGCCTGCGCGCACACACACACCAGCGGGCGCGGGAGACCCGTGATGGCCTTTTGTCCCCGTGCACTTATCTagCTGGCGCAAGTAGTGCTCCCCACCCCCTGCCCTagCTCACAGCCCTGCCTGGGTCCCGCTCCGGGGTGGGTCAGCCAGGGCAGAGAACAGCCGGCTTGGCTGGAGCCAGGCTGACCGGCTAGATCTGGGAGTCCCCTCCTCCTTCCCCACGCAGACTCAGGCTCCCCTTCTCTTATCCACAGACACCCCCTTTTTTGCAGCTATCATTCTGCATCCGGGTCCCCCTGAATTTCTGAGTCGTGGCTTGTTCTCAC.AATCAGTGCGTGCTCGTTAAGTCCTGGAGATCCACTGAGCGCGCAGCCTAACGCTGGAGAAAGTGGTTTGAAACCCAAAGTATAGAAAATGTAAATAAAAGGCAGGCGTGTCAGAGAGGGTGAGGGATCTCCGTAACACCTCATTTCATTTTTTTAAAGGAGGGGGACACTTCCCCGCCGCCTGCAGCCTTGACCTCCAAGGCGGGGGTAGGGACCGTTGTGGCTCTT4.3.3GCTGAGACAAATGGAAAGCTGAAGATAAGGATCCTCTGAGGTGCGAACATACAGCTGTTGGGAATTGCCAGAGAATCGGACCAATAAAGGAAGTCACTATTTTTCCAGGCCTGAAGTGAGTTATAGGGCGAGACGGGTGTTGTATATTTATGTAAGGCAACAGCAGGGAGTTTAAGCGGCTGGATATTGCTGAAAGAGCATCATTCACATTCAGGCGGAGACAAAAGGTGGAAATGAAGCAACATCCTGGCCAAAGAAGGCCTCAAGACAGAATAATAACAGTTCAGAGAGGGGGGCTGTGTGCACGGCCGAGGGTCGGCCTCAAAACCAGGAAATGATCGAGATGCCTTGTCAGATCTTC.4.3.4GCTGAGACAAATGGAAAGCTGAAGATAAGGATCCTCTGAGGTGCGAACATACAGCTGTTGGGAATTGCCAGAGAATCGGACCAATAAAtcAAGTCACTATTTTTCCAGGCCTGAAGTGAGTTATAGGGCGAGACGGGTGTTGTATATTTATGTAAGGCAACAGCAGGGAGTTTAAGCGGCTGGATATTGCTGAAAGAGCATCATTCACATTCAGGCGGAGACAAAAGGTGGAAATGAAGCAACagCTTGGCCAAAGAAGGCCTCAAGACAGAATAATAACAGTTCAGAGAGGGGGGCTGTGTGCACGGCCGAGGGTCGGCCTCAAAACCAtcAAATGATCGAGATGCCTTGTCAGATCTTC.4.4Mouse and human sequences of putative enhancers were aligned using ClustalW . Binding4.5italic underlined nucleotides modified (GGA to TCA or TCC to TGA) in mutant version:Proteins were made using the TNT Quick Coupled Transcription/Translation system as described in the manufacturer\u2019s directions. The truncated ETS1 DNA binding domain (ETS-DBD) and full length ERG were in the pCITE2 plasmid, and transcribed using T7 polymerase. To make each probe, double stranded oligonucleotides with CTAG 5\u2032 overhangs were labelled with 32P-dCTP using Klenow (Promega) to fill in overhanging 5\u2032 ends, and purified on a non-denaturing polyacrylamide-TBE gel. 20\u00a0\u200b\u03bcl binding reactions consisted of 2\u20133\u00a0\u200b\u03bcl protein or lysate control, 2\u00a0\u200b\u03bcl 10X binding buffer and 0.5\u00a0\u200b\u03bcg poly dI-dC. For competitor lanes, a 100-fold excess of competitor DNA was added in a volume of 1\u00a0\u200b\u03bcl. Binding reactions were incubated at room temperature for 10\u00a0\u200bmin before the addition of radiolabelled probe, after which they were incubated an additional 20\u201330\u00a0\u200bmin. Gels were electrophoresed on a 10% non-denaturing polyacrylamide gel. Sequences of the probes and competitor regions are listed below, with tccccgccg;ETS control probe CTAGtaaatcctgaggctg.Ephb4-2 ETS-b CTAGgttgtggctctttcctgtctggc.Ephb4-2 ETS-c CTAGgaggctgttggatggctggga.Ephb4-2 ETS-d CTAGggccctcgtcctctcatttg.Ephb4-2 ETS-e CTAGagggccctggatggttgg.Ephb4-2 ETS-f CTAGgtggggcacgggatgagagaggtgc.Ephb4-2 ETS-g CTAGgatggtttcctggcgcaagta.Ephb4-2 ETS-h CTAGcacttatcttcctcacagcccEphb4-2 ETS-I CTAGcctgccctggaaacagcc.Ephb4-2 ETS-j CTAGagccagggcaggaaagctgaagataa.Coup-965 ETS-a CTAGacaaatggatcctctgag.Coup-965 ETS-b CTAGgctgaagataaggaattgccagaga.Coup-965 ETS-c CTAGagctgttgggaagtcactat.Coup-965 ETS-d CTAGcggaccaataaaggaaatgaagcaacatc.Coup-965 ETS-e CTAGaaggttcctggccaaag.Coup-965 ETS-f CTAGaagcaacaggaaatgatcgagatc.Coup-965 ETS-g CTAGcaaaaccatccaggcctg.Coup-965 ETS-h CTAGgtcactatttt4.6165 (PeproTech) at 25\u00a0\u200bng/ml for 1.5\u00a0\u200bh. Cells were then trypsinised and the cell pellet collected.For VEGF stimulation experiments in cells, human umbilical vein pooled endothelial cells were grown in Endothelial Cell Growth Medium 2 with BulletKit (PromoCell). Media was changed every 48\u00a0\u200bh. Four 80% confluent 15\u00a0\u200bcm dishes per condition were serum starved in 0.5% Foetal Bovine Serum (Gibco) overnight before being stimulated with VEGFAChromatin immunoprecipitation was carried out as previously described . BrieflyImmunoprecipitated DNA was analyzed by qPCR using TaqMan Custom Gene Expression Assay Probes (ThermoFischer) designed against 100bp regions of the Ephb4-2 enhancer, the CoupTFII-965 enhancer or a gene dessert region of Chromosome 7 as a negative control.4.6.1ACCCCTGCCCTTCCTTGCTGTTCTGCCTGGGTCCTGCGCCCGGGTTGGGGGGGGTGGGCCGGTCACCGAGGGCAGGAAACAGCCGGCTTCACTGGAGCCAGGCAGACCAG.4.6.2AGCGGCTGTATATTGCTGAAAGAGCATCATTCACATTCAGGCAGAGACAAAAGGTGGAAATGAAGTAACATCCTGGCTGAAGAAGGCCTCACGACAGAATA.4.6.3CCTCAGCCTCCCAAGTAGCTGGGATTACAGGTGTGTGCTACCATGCCTGGCTAATTTTTGTATTTTTAGTAGAGACAGGGTTTCACCATGTTGGCCAGGCTGGTCTCGAACTCCTGAACTCAGG TGATCTA.Each ChIP was performed on at least three biological replicates, with three technical replicates for each. Statistical analysis was performed in StepOne plus software, Microsoft Excel. Input was taken as the supernatant from the non-antibody control condition. Results are expressed as the mean of the % input defined as 100\u2217(2^(adjusted Input ct \u2013 ct IP)) across all replicates. Significant differences were calculated using ANOVA f test with p values subsequently derived from Tukey HSD test, accounting for multiple comparison correction. Graphs were produced using R statistical package.4.7https://ncbi.nlm.nih.gov/geo/) under accession GSE93030. The four ChIP-seq datasets used have accession numbers GSM2442775 to GSM2442778. Data consisted of ChIP-seq Model-based Analysis of ChIP-Seq (MACS) (https://CRAN.R-project.org/package = data.table and https://CRAN.R-project.org/package=ggpubr). GRCh37 ROIs: HLX-3 chr1:221,049,659\u2013221,050,776, Dll4in3 chr15:41,222,807\u201341,223,778, Ephb4-2 chr7:100,426,194\u2013100,427,393, and CoupTFII-965 chr15:95,908,708\u201395,909,301.ChIP-seq analysis was conducted on the published and publicly available data from q (MACS) regions q (MACS) . MACS pe4.8Antisense MOs were ordered from GebeTools LLC and dissolved in water before injected into 1\u20132\u00a0\u200bcell stage zebrafish embryos as previously described . Sequencfli1 MO (3 \u2013 6\u00a0\u200bng) TTTCCGCAATTTTCAGTGGAGCCCG (GGAGCCCG .erg MO (3 \u2013 6\u00a0\u200bng) CAGACGCCGTCATCTGCACGCTCAG .4.9in situ hybridization ephb4, efnb2 and stab1l probes were generated as custom-made, double-stranded linear DNA fragments , cloned into the pCR2 vector using the TOPO/TA cloning kit (Invitrogen 450641) and transcribed using SP6 and T7. The sequences are provided below. dll4 probe was kindly provided by R. Patient, University of Oxford, Oxford. Whole-mount in situ hybridization was conducted as previously described . Embryos were washed through a dilution series of 2 x SSC followed by 0.2 x SSC at 65\u00a0\u200b\u00b0C and thereafter taken through room temperature dilution washes of 100% MABT . Nonspecific sites were blocked with MAB block (MABT with 2% Boehringer block reagent) and the embryos incubated for 15\u00a0\u200bh with 1:4000 antiDIG antibody (Roche) at 4\u00a0\u200b\u00b0C, before washing in MABT. Prior to staining, embryos were washed in AP buffer and the in situ signal developed at room temperature with BM Purple (Sigma-Aldrich). Staining was stopped as appropriate, and embryos were bleached in 3% H2O2/0.5% KOH until pigmentation disappeared, then re-fixed in 4% PFA for 20\u00a0\u200bmin and washed 4 times with PBST. Embryos were transferred to 80% glycerol for imaging.For zebrafish whole-mount escribed . Brieflyin situ/condition. Experimental blinding was not used as phenotypes of control and treated were easily detectable due to morphological defects.Analysis was qualitative not quantitative, therefore no statistical analysis was applied to the observations of staining intensity and pattern. Numbers of zebrafish embryos was no less than 30/4.9.1TCTCAGCTCTGGACAAGCTGATCCGCAACCCGGCCTCACTCAAAATCACAGCGCAGGAGGGGGCGGGCCCCTCTCACCCTCTGCTGGACCAGCGGTCTCCACTCACGCCCTCATCCTGCGGGACAGTGGGTGACTGGCTGCGGGCCATCAAGATGGAGCGCTACGAGGAGACATTTCTGCAGGCGGGATACACGTCCATGCAGCTCGTCACCCACATCAACACGGAGGATCTGCTGCGTTTGGGAATAACTTTAGCAGGTCACCAGAAGAAGATTCTCTCCAGCATTGAGGCTCTCGGGATTCAAAACAAAGCACCAGGGAATGTGCTGTACTGA.4.9.2AAAACCAAGTCGATGAAAATCATCATGAAGGTTGGACAAAACCCCTCTGATCCCATTTCCCCCAAAGACTACCCTACCAGTTACCCTCCCAAACACCCTGACTTAGGGGGCAAGGACAGCAAATCGAATGAAGTACTTAAGCCAGATGCATCTCCTCATGGGGAAGATAAGGGAGATGGAAATAAATCCTCATCAGTCATTGGCTCAGAGGTGGCCCTGTTTGCCTGCATCGCCTCAGCAAGCGTCATCGTCATCATCATAATCATCATGCTAGTTTTCCTTCTCCTGAAGTATCGACGA.4.9.3GGATTCAGCAGCTACAGACACACCCAACCTCATCGACTAGCACAGACAGCAGCGTTAAACTCTCCCTTCATTTACCTGCAATCAGCTGACCGCTCTTAAAATAAAGGTTCTGTATTGGCATTGATGGTTCCGCGAAGAATCTTTATAAGCCATAACATCTTTCCATTTCCATGAGGTGTAAAAAGACTCTTTAGAATATTAAAATGTTACTTCATAAACATTTGATGTGTTTGATTGCAGATACTTCAGAGTGTTTAACTTCCACCCATTTATTTCTGCGTTTCACACATATTTTTGACTAAAAATGTTCTTTACATTAAGAAAAAATGGTGTACTCACCCTCAAGTAGGTCCAAACCTTCACGAGTTTCTTTTTTTGCCTTCTGTTGAACACAAACAAGAAAATATTTTGATAATGCGTTAAGCAAGGGGCCATTAGCTGTTTTGATCCAACTTTTTTCATGCGCATTTTAAATTATCGCATGTAAAAAAGCTTAATGGAAACCCAAGATGTGCTTAATTTGTCAAAATCTGCAT.4.10164 injections were performed on nude mice as described in (164 (provided by Lilly) diluted 1:30 in sterile 3% glycerol/PBS. At the required time-point after injection, ears were harvested and skin removed from the dorsal side. Ears were fixed in 2% paraformaldehyde and 0.2% glutaraldehyde in PBS for 20\u00a0\u200bmin at 4\u00a0\u200b\u00b0C, washed twice in PBS then X-gal stained overnight at room temperature. Ears were then placed in 4% paraformaldehyde for storage.Intradermal Ad-VEGFAribed in . Briefly164 injected ears, ears were harvested and X-gal stained as described above, then dehydrated through a series of ethanol washes, cleared by xylene and paraffin wax-embedded. 5 or 6-\u03bcm sections were prepared and de-waxed. For imaging of X-gal staining, slides were counterstained with nuclear fast red (Electron Microscopy Sciences).For histological analysis of Ad-VEGFA4.11vegfaa121 and pCS2+vegfaa165 plasmids were kindly provided by S. Sumanas, Cincinnati Children\u2019s Hospital Medical Center, Ohio, USA. vegfaa121 and vegfaa165 mRNA was synthesised in vitro using the mMessage mMachine SP6 transcription kit (ThermoFisher Scientific) and injected into 1\u00a0\u200bcell stage zebrafish embryos at a final concentration of 50\u00a0\u200bpg of mRNA per embryo. Analysis was qualitative not quantitative, therefore no statistical analysis was applied to the observations of staining intensity and pattern.pCS2+No competing interests declared."} +{"text": "The rat progression elevated gene-3 (PEG-3) promoter displays cancer-selective expression, whereas the rat growth arrest and DNA damage inducible gene-34 (GADD34) promoter lacks cancer specificity. PEG-3 and GADD34 minimal promoters display strong sequence homology except for two single point mutations. Since mutations are prevalent in many gene promoters resulting in significant alterations in promoter specificity and activity, we have explored the relevance of these two nucleotide alterations in determining cancer-selective gene expression. We demonstrate that these two point mutations are required to transform a non-cancer-specific promoter (pGADD) into a cancer-selective promoter (pGAPE). Additionally, we found GATA2 transcription factor binding sites in the GAPE-Prom, which regulates pGAPE activity selectively in cancer cells. This newly created pGAPE has all the necessary elements making it an appropriate genetic tool to noninvasively deliver imaging agents to follow tumor growth and progression to metastasis and for generating conditionally replicating adenoviruses that can express and deliver their payload exclusively in cancer.Progression-elevated gene-3 (PEG-3) and rat growth arrest and DNA damage-inducible gene-34 (GADD34) display significant sequence homology with regulation predominantly transcriptional. The rat full-length (FL) and minimal (min) PEG-3 promoter display cancer-selective expression in rodent and human tumors, allowing for cancer-directed regulation of transgenes, viral replication and in vivo imaging of tumors and metastases in animals, whereas the FL- and min-GADD34-Prom lack cancer specificity. Min-PEG-Prom and min-GADD34-Prom have identical sequences except for two single-point mutation differences (at \u2212260 bp and +159 bp). Engineering double mutations in the min-GADD34-Prom produce the GAPE-Prom. Changing one base pair (+159) or both point mutations in the min-GADD34-Prom, but not the FL-GADD34-Prom, results in cancer-selective transgene expression in diverse cancer cells vs. normal counterparts. Additionally, we identified a GATA2 transcription factor binding site, promoting cancer specificity when both min-PEG-Prom mutations are present in the GAPE-Prom. Taken together, introducing specific point mutations in a rat min-GADD34-Prom converts this non-cancer-specific promoter into a cancer-selective promoter, and the addition of GATA2 with existing AP1 and PEA3 transcription factors enhances further cancer-selective activity of the GAPE-Prom. The GAPE-Prom provides a genetic tool to specifically regulate transgene expression in cancer cells. Defining the steps necessary to convert a normal cell into a cancerous one has been aided by identification and interrogation of defined genetic elements that regulate these processes ,2,3. Usipeg-3 and gadd34 genes share 73% nucleotide and 59% amino acid sequence homology [peg-3 and the murine gadd34 genes [The N-terminal domain first 415 aa) of rat PEG-3 is identical to the rat GADD34 protein aa of ra,8. The p34 genes ,11,12,1334 genes . In cont34 genes . These speg-3 and gadd34 genes is chiefly transcriptional [Previous studies indicate that regulation of both the iptional . The fuliptional ,16. Promiptional ,15,16. Siptional . Furtheriptional ,15,16.Mutations are common events in gene promoters, which can result in dramatic changes in promoter specificity and activity ,19,20,21To determine why the cancer-specific min-PEG-Prom (pPEG) is functionally different than the pGADD, which shares almost identical sequence similarity, we compared the sequence of the promoter regions of the pPEG with the pGADD , which resulted in the identification of only two base pairs that were different . Using sUsing the luciferase assay, we compared the activity of the pGADD-, pPEG- and pGAPE-Luc in cancer vs. normal human cells. As shown in Previous studies have confirmed that when pPEG-Luc or pPEG-HSV-Tk were complexed with in vivo jetPEI a linear polyethylenimine (l-PEI), intravenous injection permitted imaging of both primary tumors and metastases . We initWe used in vivo jetPEI for delivery and imaging of the different promoters ,27. JetPAn important question was whether a single mutation in the pGADD was sufficient, or if both mutations were necessary, for pGAPE to display cancer-specific activity. To accomplish this goal, we generated a pGADD with a single mutation at bp \u2212260 and a second pGADD mutation at bp +159 . Using the same in vitro Luc-assay-based approach (described above), we analyzed the activity of pGADD, pPEG, pGAPE, pGADD1-1 and pGADD2-2 in the same series of human cancer, human immortalized normal cell lines, and primary normal cells shown in pPEG and pGAPE show tumor-specific in vivo imaging capabilities as well as enhanced tumor-specific expression in vitro, whereas the full-length GADD promoter, pT-GADD, does not show tumor specificity compared to normal cells. The same is true with the single mutation modification of bp \u2212260 (pGADD1-1), which lacks cancer specificity, whereas the change of bp +159 (pGADD2-2), shows cancer selectivity when engineered in pGADD (the minimum GADD34-Prom). To check the transcription factors involved in this cancer-selective activity, we analyzed the pGADD with and without mutations with the TFBIND software , which pChromatin immunoprecipitation (ChIP) assays confirmed an interaction between GATA2 and pGAPE in DU-145 and RWPE-1 cells A. DU-145We next determined whether changing GATA2 expression could modulate promoter binding to GATA2. RWPE-1 and DU-145 were transfected with GATA2 OE plasmid or shGATA-2 (inhibitory) plasmid for 48 h and then processed for ChIP assays. We predicted and confirmed that, following overexpression of GATA2, the binding to pGAPE/pPEG increased as compared to non-transfected cells, and conversely, GATA2 inhibition reduced binding to the pGAPE/pPEG. However, binding to the pGADD was not altered in either of these cell lines under these conditions C. CollecTo determine if a single mutation or both mutations (bp \u2212260 or bp +159) in the full-length GADD34-Prom could confer cancer selectivity, we developed total pT-GADD constructs with a single mutation at G-A, pT-GADD1-1-Luc or C-T, pT-GADD2-2-Luc, and with both the mutations, pT-GADD2-Luc . The CTV induces profound anticancer activity in vitro and in vivo in diverse cancer indications [Tissue- and cancer-selective/specific promoters also provide functional tools to potentially regulate virus replication in a conditional manner, including adenovirus, herpesvirus, human immunodeficiency virus, simian immunodeficiency virus, and others ,32,59,60-7/IL-24 , under cications ,30,31,32In summary, we document that two-point mutations are critical to convert a non-cancer-specific pGADD into a cancer-selective pGAPE. A single mutational change at bp +159 of pGADD to that of the pPEG also promotes cancer selectivity both in vitro and in vivo. Additionally, we define a new transcription factor, GATA2, to which binding to the GAPE-Prom is indispensable for cancer-selective expression and activity. This newly engineered pGAPE embodies all the necessary features that make it an ideal tool to regulate transgene expression in a cancer-selective manner to both deliver imaging agents noninvasively to follow tumor promotion and metastasis and create CRADs expressing replicative functions uniquely in cancers.2 at 37 \u00b0C. Cells were validated for authenticity in prior studies [Immortalized human prostate epithelial (RWPE-1) and prostate cancer cells, human immortalized pancreatic mesenchymal (LT-2) and pancreatic cancer cells, early passage primary human mammary epithelial (HMEC) and breast cancer (MDA-MB-231 and SUM159) cells, H-TERT immortalized primary human fetal astrocytes (IM-PHFA) and neuroblastoma cells were cultured as described previously ,64,65. S studies ,64,65 an studies ,64.\u00ae Plus Enzymatic Chromatin IP Kit (Magnetic Beads) was purchased from Cell signaling technology. Dual Luciferase Reporter Assay Kit was obtained from Promega and used in the study. Lipofectamine 2000 and Invivo JetPEI and transfection reagents, D-Luciferin Potassium Salt Bioluminescent Substrate , Agilent Quickchange II site directed mutagenesis kit were used in the study.Antibodies specific for: GATA2, Control Rabbit IgG , horseradish peroxidase (HRP)-conjugated secondary antibodies , and \u03b2-actin were used in this study. SimpleChIPRat progression-elevated gene-3 (PEG-3) was isolated from H5ts125-transformed rat embryo cancer cells by subtraction hybridization and is not expressed in normal rodent or human cells . The minpGADD34-FCGGGGTACCGAAAGAGAAAGAGAATGGGACAGCApGADD34-RCGGAAGATCT GGTCCGGTTCGGTTTGCCAAAAGCGGTCpGADD-FCGGGGTACCGAAAGAGAAAGAGAATGGGACGGCApGADD-RCGGAAGATCTGGTCCGGTTCGGTTTGCCAAAAGCGATCGAAAGAGAAAGAGAATGGGACAGCATGTGACTGCCTGATGAAGTTGGCGTGCTTGCTCAAAAGTTCTGCGAGATTGACGGCTCTCTGGATTTGAGCCAAGGACACGCCTGGGAAGCCACGGTGACCTCACAAGGCCCGGAATCTCCGCGAGAATTTCAGTGTTGTTTTCCTCTCTCCACCTTTCTCAGGGACTTCCGAAACTCCGCCTCTCCGGTGACGTCAGCATAGCGCTGCGTCAGACTATAAACTCCCGGGTGATCGTGTTGGCGCAGATTGACTCAGTTCGCAGCTTGTGGAAGATTACATGCGAGACCCCGCGCGACTCCGCATCCCTTTGCCGGGACAGCCTTTGCGACAGCCCGTGAGACATCACGTCCCCGAGCCCCACGCCTGAGGGCGACATGAACGCGCTGGCCTTGAGAGCAATCCGGACCCACGATCGCTTTTGGCAAACCGAACCGGACCGAAAGAGAAAGAGAATGGGACGGCATGTGACTGCCTGATGAAGTTGGCGTGCTTGCTCAAAAGTTCTGCGAGATTGACGGCTCTCTGGATTTGAGCCAAGGACACGCCTGGGAAGCCACGGTGACCTCACAAGGCCCGGAATCTCCGCGAGAATTTCAGTGTTGTTTTCCTCTCTCCACCTTTCTCAGGGACTTCCGAAACTCCGCCTCTCCGGTGACGTCAGCATAGCGCTGCGTCAGACTATAAACTCCCGGGTGATCGTGTTGGCGCAGATTGACTCAGTTCGCAGCTTGTGGAAGATTACATGCGAGACCCCGCGCGACTCCGCATCCCTTTGCCGGGACAGCCTTTGCGACAGCCCGTGAGACATCACGTCCCCGAGCCCCACGCCTGAGGGCGACATGAACGCGCTGGCCTTGAGAGCAATCCGGACCCACGACCGCTTTTGGCAAACCGAACCGGACCGAAAGAGAAAGAGAATGGGACAGCATGTGACTGCCTGATGAAGTTGGCGTGCTTGCTCAAAAGTTCTGCGAGATTGACGGCTCTCTGGATTTGAGCCAAGGACACGCCTGGGAAGCCACGGTGACCTCACAAGGCCCGGAATCTCCGCGAGAATTTCAGTGTTGTTTTCCTCTCTCCACCTTTCTCAGGGACTTCCGAAACTCCGCCTCTCCGGTGACGTCAGCATAGCGCTGCGTCAGACTATAAACTCCCGGGTGATCGTGTTGGCGCAGATTGACTCAGTTCGCAGCTTGTGGAAGATTACATGCGAGACCCCGCGCGACTCCGCATCCCTTTGCCGGGACAGCCTTTGCGACAGCCCGTGAGACATCACGTCCCCGAGCCCCACGCCTGAGGGCGACATGAACGCGCTGGCCTTGAGAGCAATCCGGACCCACGATCGCTTTTGGCAAACCGAACCGGACCGAAAGAGAAAGAGAATGGGACAGCATGTGACTGCCTGATGAAGTTGGCGTGCTTGCTCAAAAGTTCTGCGAGATTGACGGCTCTCTGGATTTGAGCCAAGGACACGCCTGGGAAGCCACGGTGACCTCACAAGGCCCGGAATCTCCGCGAGAATTTCAGTGTTGTTTTCCTCTCTCCACCTTTCTCAGGGACTTCCGAAACTCCGCCTCTCCGGTGACGTCAGCATAGCGCTGCGTCAGACTATAAACTCCCGGGTGATCGTGTTGGCGCAGATTGACTCAGTTCGCAGCTTGTGGAAGATTACATGCGAGACCCCGCGCGACTCCGCATCCCTTTGCCGGGACAGCCTTTGCGACAGCCCGTGAGACATCACGTCCCCGAGCCCCACGCCTGAGGGCGACATGAACGCGCTGGCCTTGAGAGCAATCCGGACCCACGACCGCTTTTGGCAAACCGAACCGGACCGAAAGAGAAAGAGAATGGGACGGCATGTGACTGCCTGATGAAGTTGGCGTGCTTGCTCAAAAGTTCTGCGAGATTGACGGCTCTCTGGATTTGAGCCAAGGACACGCCTGGGAAGCCACGGTGACCTCACAAGGCCCGGAATCTCCGCGAGAATTTCAGTGTTGTTTTCCTCTCTCCACCTTTCTCAGGGACTTCCGAAACTCCGCCTCTCCGGTGACGTCAGCATAGCGCTGCGTCAGACTATAAACTCCCGGGTGATCGTGTTGGCGCAGATTGACTCAGTTCGCAGCTTGTGGAAGATTACATGCGAGACCCCGCGCGACTCCGCATCCCTTTGCCGGGACAGCCTTTGCGACAGCCCGTGAGACATCACGTCCCCGAGCCCCACGCCTGAGGGCGACATGAACGCGCTGGCCTTGAGAGCAATCCGGACCCACGATCGCTTTTGGCAAACCGAACCGGACCSequences of the full-length GADD promoters with mutations are described in the The prostate cancer, pancreatic cancer, breast cancer, and neuroblastoma cell lines and primary or immortalized normal cells were plated in 24-well plates (BD Biosciences) and transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer\u2019s protocol. The indicated cells were transfected with Luc reporter constructs pPEG-Luc, pGADD-Luc, pGAPE-Luc or a empty vector (control). Luminescence was normalized for transfection efficiency by co-transfection with a vector expressing Renilla luciferase (Luc). After 48 h of transfection, the expression level of the Luc reporter was measured by the Dual Luciferase Reporter Assay Kit (Promega) ,16.Western blotting analysis was performed as described previously ,11,64. Rwt/vol) glucose. The glucose solutions of DNA and l-PEI polymer were then mixed together to give an N:P ratio (the number of nitrogen residues of in vivo jetPEI per number of phosphate groups of DNA) of 6:1 in a total volume of 400 \u03bcL. The DNA-PEI polyplex was injected intravenously as two 200 \u03bcL injections with a 5 min interval.Low-molecular-weight l-PEI\u2013based cationic polymer, in vivo jetPEI (Polyplus Transfection), was used for gene delivery ,27. The In vivo BLI was conducted at 24 and 48 h after the systemic delivery of reporter genes ,27. All 3 in 1% SDS. Primer pairs are listed below.ChIP assays were performed as described previously . BrieflyThe following primers were used in the ChIP assay:GADD F- GAA AGA GAA AGA GAA TGG GAC GGADD R- GTC CGG TTC GGT TTG CCA AAA GCG GGAPE F- GAA AGA GAA AGA GAA TGG GAC AGAPE R- GTC CGG TTC GGT TTG CCA AAA GCG AGAD1-1 F- GAA AGA GAA AGA GAA TGG GAC AGADD1-1 R- GTC CGG TTC GGT TTG CCA AAA GCG GGADD2-2 F- GAA AGA GAA AGA GAA TGG GAC GGADD2-2 R- GTC CGG TTC GGT TTG CCA AAA GCG AStatistical analysis was performed using SPSS 22.0 software , and statistical graphs were generated using GraphPad Prism . The generalized odds ratios (ORs) and 95% confidence interval (CI) of the promoters were calculated. The multiple comparisons were performed using false discovery rate (FDR) correction. False discovery rate (FDR) correction was used to analyze the promoter activity among the different promoters."} +{"text": "The results add significantly to our understanding of how BRMS1 interactions with Sin3/HDAC complexes regulate metastasis and expand insights into BRMS1\u2019s molecular role, as they demonstrate BRMS1 C-terminus involvement in distinct protein-protein interactions.Breast Cancer Metastasis Suppressor 1 (BRMS1) expression is associated with longer patient survival in multiple cancer types. Understanding BRMS1 functionality will provide insights into both mechanism of action and will enhance potential therapeutic development. In this study, we confirmed that the C-terminus of BRMS1 is critical for metastasis suppression and hypothesized that critical protein interactions in this region would explain its function. Phosphorylation status at S237 regulates BRMS1 protein interactions related to a variety of biological processes, phenotypes , and metastasis . Presence of S237 also directly decreased MDA-MB-231 breast carcinoma migration Metastasis is a multi-step process that occurs when cells disseminate from the primary neoplasm and eventually colonize distant organs. Successful completion of this complex cascade is associated with nearly all cancer-related morbidities and mortalities. Despite its causative role in cancer-specific mortality and morbidity, a complete understanding of the process and its mechanisms remain elusive. Metastasis is regulated by three types of genes: metastasis-promoting, -suppressing, and -efficiency modifying . The proBRMS1 was discovered in the year 2000 and its re-expression in multiple cell lines significantly decreases lung metastases in syngeneic and xenograft models \u20138. In adBRMS1 is a known member of Sin3 histone deacetylase (HDAC) transcriptional regulatory complexes in multiple eukaryotic cell types \u201313. It iIn order to translate BRMS1 into clinical practice, additional structural and interactome data are necessary. This study leverages previously described features of BRMS1 to clarify its molecular functions within a cell. Previous attempts to define BRMS1 structure-function have been met with uneven success. To date no one has successfully crystallized nor determined structure for full-length, wild-type BRMS1. Computer algorithms and NMR studies have identified structural information for BRMS1 domains . BRMS1 hin vivo studies demonstrating that alteration of the domain impacts overall metastatic burden [The C-terminus of the BRMS1 protein is of particular interest as it has been shown to play a critical role in metastatic suppression, due to c burden . Of notec burden . Due to The BRMS1 primers listed in 7) were cultured into a 15 cm tissue culture plates for 24 hours then Halo-BRMS1 constructs were transfected using Lipofectamine LTX (Thermo Fisher Scientific). After 48 hours cells were harvested and washed twice with ice-cold PBS. Cells were then resuspended in mammalian cell lysis buffer (Promega) (50 mM Tris\u00b7HCl (pH 7.5), 150 mM NaCl, 1% Triton\u00ae X-100, 0.1% sodium deoxycholate, 0.1 mM benzamidine HCl, 55 \u03bcM phenanthroline, 1 mM PMSF, 10 \u03bcM bestatin, 5 \u03bcM pepstatin A, and 20 \u03bcM leupeptin) followed by centrifugation at 21,000 \u00d7 g for 10 min at 4\u00b0C. To remove insoluble materials, cell extracts were diluted with 700 \u03bcl of TBS and centrifuged at 21,000 \u00d7 g for 10 min at 4\u00b0C. Next, cell extracts were incubated overnight at 4\u00b0C with magnetic beads . Before elution, magnetic beads were washed four times with wash buffer . Proteins bound to magnetic beads were eluted for 2 hours at room temperature using elution buffer containing 50 mM Tris-HCl pH 8.0, 0.5 mM EDTA, 0.005 mM DTT, and 2 Units AcTEV\u2122 Protease (Thermo Fisher Scientific). The eluate was further purified by passing through a Micro Bio-Spin column to remove residual beads prior to proteomic analyses.HEK293T cells . This application utilizes the average spectral counts of each bait across all baits to calculate the TopS score, which indicates a likelihood ratio of binding.Topological Scoring was completed for all proteins as previously described , 28. BriBRMS1 protein structure was predicted based upon its amino acid sequence retrieved from NCBI using I-TASSER , 30. BriThe primary sequence of BRMS1 was mutated at S237 to either Aspartic Acid (D) or Alanine (A). These mutants were subject to multiple predictive software applications. MuPro utilizesThree biological replicates were performed for each bait protein . The distributed normalized spectral abundance factor (dNSAF) was usedTo spatially map all significant 1175 proteins, we first applied a T-Distributed Stochastic Neighbor Embedding (t-SNE), a nonlinear visualization of the data followed by a k-means clustering on the two vectors generated from the t-SNE . The numTo compare the overall composition of SIN3A associated proteins between metastatic and normal breast cells, co-IP of SIN3A from thIn order to determine the biological enrichment of differentially expressed proteins in the mutants, we subjected 1175 proteins to Reactome pathway analysis. Enrichment was completed within the R environment and several packages such as clusterProfiler, ReactomePA, DOSE and enrichplot were used.http://www.ingenuity.com, Release date: December 2016). Visualization for overlapping significant proteins was completed in Venny 2.1 (https://bioinfogp.cnb.csic.es/tools/venny/). Heatmaps were generated within ClustVis (https://biit.cs.ut.ee/clustvis/).Significant proteins were subjected to multiple analyses. Disease enrichment was completed within Ingenuity Pathway Analysis cell culture medium (Thermo Fisher Scientific). MDA-MB-231 cells were cultured in 5% FBS, 0.5 mM NEAA 0 + DMEM/Sodium Bicarbonate (2.438 g/L) cell culture medium (Thermo Fisher Scientific). In both 293T and MDA-MB-231s, overexpression cells were first transduced with pENTR followed by pLENTI expression within 293FT cells according to manufacturer\u2019s instructions (Invitrogen). Clones were selected with blasticidin. Each construct contained a single or 3x-Flag epitope tag. Expression was quantified using validated BRMS1 1a5.7 antibody (as described previously ), FLAG-M5\u2019- GATCCATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGAAGGCTAGGGCAGCTGTGTCCCCTCAGAAGAGAAAATCGGATGGACCTTGATGATGAC -3\u2019, R: 5\u2019 -TCGAGTCATCATCAAGGTCCATCCGATTTTCTCTTCTGAGGGGACACAGCTGCCCTAGCCTTCTGTCATCGTCATCCTTGTAATCGATGTCATGATCTTTATAATCACCGTCATGGTCTTTGTAGTCCATG -3\u2019.F: Predicted amino acid sequences for each construct are:WTBRMS1DYKDDDDKMPVQPPSKDTEEMEAEGDSAAEMNGEEEESEEERSGSQTESEEESSEMDDEDYERRRSECVSEMLDLEKQFSELKEKLFRERLSQLRLRLEEVGAERAPEYTEPLGGLQRSLKIRIQVAGIYKGFCLDVIRNKYECELQGAKQHLESEKLLLYDTLQGELQERIQRLEEDRQSLDLSSEWWDDKLHARGSSRSWDSLPPSKRKKAPLVSGPYIVYMLQEIDILEDWTAIKKARAAVSPQKRKSDGP1-229BRMS1DYKDDDDKMPVQPPSKDTEEMEAEGDSAAEMNGEEEESEEERSGSQTESEEESSEMDDEDYERRRSECVSEMLDLEKQFSELKEKLFRERLSQLRLRLEEVGAERAPEYTEPLGGLQRSLKIRIQVAGIYKGFCLDVIRNKYECELQGAKQHLESEKLLLYDTLQGELQERIQRLEEDRQSLDLSSEWWDDKLHARGSSRSWDSLPPSKRKKAPLVSGPYIVYMLQEIDILEDWTAIS237ADYKDDDDKMPVQPPSKDTEEMEAEGDSAAEMNGEEEESEEERSGSQTESEEESSEMDDEDYERRRSECVSEMLDLEKQFSELKEKLFRERLSQLRLRLEEVGAERAPEYTEPLGGLQRSLKIRIQVAGIYKGFCLDVIRNKYECELQGAKQHLESEKLLLYDTLQGELQERIQRLEEDRQSLDLSSEWWDDKLHARGSSRSWDSLPPSKRKKAPLVSGPYIVYMLQEIDILEDWTAIKKARAAVAPQKRKSDGPS237DDYKDDDDKMPVQPPSKDTEEMEAEGDSAAEMNGEEEESEEERSGSQTESEEESSEMDDEDYERRRSECVSEMLDLEKQFSELKEKLFRERLSQLRLRLEEVGAERAPEYTEPLGGLQRSLKIRIQVAGIYKGFCLDVIRNKYECELQGAKQHLESEKLLLYDTLQGELQERIQRLEEDRQSLDLSSEWWDDKLHARGSSRSWDSLPPSKRKKAPLVSGPYIVYMLQEIDILEDWTAIKKARAAVDPQKRKSDGP230-246BRMS1MDYKDHDGDYKDHDIDYKDDDDKKARAAVSPQKRKSDGP293T cells were grown to 90% confluency, collected and RNA extracted an RNA Isolation kit (Zymo Research). Following RNA extraction cDNA completed with the iScript cDNA synthesis kit (Bio Rad). qRT-PCR was completed with a SYBER Green assay, and cDNA loaded at a concentration of 50 ng/uL. The following primers were utilized for analysis:https://imagej.nih.gov/ij/) was utilized for analysis, followed by statistical analysis in R (\u2018FAS\u2019 package), in which a Kruskal Wallis test followed by a Dunn\u2019s adjustment was completed (https://www.R-project.org/).Cells were cultured at a seeding density of 750,000 cells/well in 5% FBS, 0.5 mM NEAA + DMEM/Sodium Bicarbonate (2.438 g/L) media in 6-well plates (Thermo Fisher). After 48 hours cells were scratched, washed, and media replaced with serum-free DMEM/Sodium Bicarbonate (2.438 g/L) (Thermo Fisher). Cells were imaged at t = 18 to 24 hrs. Image J MDA-MB-231 cells were transduced with the construct as described above. For each construct, 3 clones were selected. 2x105 cells suspended in 100 uL of Hanks Balanced Salt Solution were intravenously injected within the tail vein of three week-aged athymic mice (Harlan HSD athymic nude-Foxn1nu), which were restrained in a tube to facilitate tail manipulation. The lung metastases grew for 6 weeks or until the mouse was moribund and required euthanasia. Lungs were removed and fixed in a mixture of Bouin\u2019s fixative and neutral buffered formalin (1:5 v/v), and macroscopic lung metastases were counted. This study was completed in 30 mice per construct, and 3 clones per construct. All animal studies were approved by the Institutional Animal Care and Use Committee at the University of Kansas Medical Center (#2014\u20132208). These experiments were performed in accordance with the PHS Policy on Humane Care and Use of Laboratory Animals, USDA regulations , the Federal Animal Welfare Act (7 USC 2131 et. Seq.), and the Guide for the Care and Use of Laboratory Animals, that were followed in attempts to alleviate animal suffering. A Kruskal Wallis test followed by a Dunn\u2019s adjustment was completed as per above for statistical analysis.To measure the successful colonization of the lungs by metastases for each construct [https://kmplot.com/analysis/) [The UALCAN web resource was utilized to assess the expression of BRMS1, BRMS1L, HDAC1, SIN3A, and SUDS3 in tumor compared to normal expression (ex.html) . All samalysis/) . PatientR packages Gplots, GGplots2, and RColor Brewer were utilized for image analysis of data.S237D ; BRMS1S237A ; BRMS11-229 ; and BRMS1230-246 . Regardl methods . These dGiven the lack of crystal structure for BRMS1 , we used the I-Tasser structure prediction method to predict the three-dimensional structure of BRMS1 using its amino acid sequence . The algin silico prediction methods, when combined with our previous findings that S237 near the C-terminus can be phosphorylated compelled us to posit that the phosphorylation site could regulate protein binding. To test this hypothesis, mass spectrometry was completed (WT), BRMS11-229, S237A, S237D, or BRMS1230-246 , applied to Z-statistics obtained from the QSPEC analysis [Mass spectrometry identified 1175 proteins whose binding was significantly (QSPEC Z-Score \u00a3-2) altered in at least one mutant compared to BRMS1analysis . This an1-229, and BRMS1230-246 cluster the most similarly. This finding may suggest that in order for BRMS1WT to interact with certain proteins it must include both phosphorylation at S237 as well as additional factors near the N-terminus that BRMS1230-246 lacks. That being said, BRMS1WT and S237D have little overlap, sharing only HDAC1, HDAC2, and SIN3A was employed . This mend SIN3A . This coTo further examine Sin3/HDAC complex binding with BRMS1 we utilized recently published crosslinking data of complex members . BRMS1 wTo examine the interaction between SIN3A and BRMS1 further, we utilized SIN3A-endogenously expressing cells. SIN3A was co-immunoprecipitated from the nuclear lysates of immortalized, but otherwise normal, MCF-10A breast cell line, as well as within the metastatic MDA-MB-231 breast cancer cell line. These precipitates were then subjected to mass spectrometry through MALDI-TOF analysis. Interactions with SIN3A were found to be context-dependent, i.e., normal breast cell line interactions differ from metastatic cancer cell interactions, though several of these interactions have been previously published and validated in many cell lines and systems , suggest\u22125) with SIN3B versus C-terminal (BRMS1230-246) binding were observed. Many of the significant pathways represented in the analysis were not represented in BRMS11-229, e.g., mRNA processing, rRNA processing, and immune responses (230-246), emphasizing the importance of the C-terminus in overall protein function and regulation of protein interactions. Importantly, many of the biological processes are tied to cancer-associated pathologies and further study into the BRMS1 C-terminus role in these pathways could identify the molecular role of BRMS1 in metastasis. To identify binding partners distinctly affected by particular domains of BRMS1, all protein interaction changes for each mutant were considered. To ensure that putative interacting proteins were truly associated with BRMS1, an additional criterion was added, i.e., the interactor must have a high TopS value in BRMS1WT, to demonstrate an increased likelihood of binding ratio . Several of the enriched pathologies are often associated with neoplasia, including cell cycle dysregulation, cell death and survival, RNA damage repair, embryonic development, immunological disease, and infectious disease. The latter findings compliment previously published results in which re-expression of BRMS1 in metastatic MDA-MB-435 cells were highly enriched for upregulated immune response genes [To further characterize how changes in interactor binding may play a role in the development of certain pathologies, disease enrichment within Ingenuity Pathway Analysis (IPA) was completed , Src, and Fascin \u201371. ThesWithin these two enriched disease pathways, several proteins overlap, including BReast CAncer gene 1 (BRCA1), mechanistic target of rapamycin (mTOR), and Cyclin Dependent Kinase Inhibitor 2A (CDKN2A) . SeveralWT and its mutants , we examined how BRMS1 interacting partners as well Firstly, we compared the expression of the Sin3/HDAC members from normal to breast tumor samples. BRMS1, HDAC1 and SUDS3 significantly increase expression within the tumor samples, while BRMS1L has significantly decreased expression . SIN3A iTo address this speculation, we also looked for an association between one of the Sin3/HDAC members and BRMS1 expression. To do this we first separated patients by expression of each Sin3/HDAC BRMS1-interacting member by expression greater than or less than the median expression of that particular member. Within those subsets, BRMS1 expression (high vs low) was examined for patient overall survival . When loin silico prediction methods, that the C-terminus functions in protein binding, and demonstrated through mass spectrometry that the C-terminus can impact BRMS1 associations with Sin3/HDAC complexes as well as proteins outside of the complexes. We model this hypothesis in This study investigates the role of BRMS1 C-terminus in protein function, with an emphasis on the function of the S237 phosphorylation site. We hypothesized, based upon It is interesting to speculate that once expression of either BRMS1L or SIN3A is lost, it appears that BRMS1 will then function to suppress metastasis and increase survival. Our data begin to define these interactions, in particular with SIN3A, but further studies are required in order to directly define those relationships, including whether the interactions are based on phosphorylation-status to regulate metastasis. The C-terminus and phosphorylation status appear to be vital to regulating the metastasis promoting miRNA miR-10b and corresponding reductions in motility and metastasis. The data also identify specific functionalities of protein complexes previously associated with BRMS1 and metastasis.This combination of results refine the molecular impact of BRMS1 on regulating metastasis and the potential development of therapeutics based upon BRMS1.S1 FigExpression of BRMS1WT, BRMS11-229, and phospho-mutants compared to parental and vector control 293T cells. (B) qPCR quantification of BRMS1230-246 expression in 293T cells. (C) Expression of BRMS1WT and phospho-mutants compared to vector control MDA-MB-231 cells. (D). Expression of BRMS11-229 within MDA-MB-231 cells. (E) qPCR quantification of BRMS1230-246 expression in MDA-MB-231 cells.(TIF)Click here for additional data file.S2 FigBRCA data was mined in which SIN3/HDAC members were separated by median expression into those greater than the median BRMS1 expression was then examined within these patients for overall survival, with high BRMS1 (indicated by Red) and low BRMS1 (indicated by black) were accounted for. This was completed in KM Plotter.(TIF)Click here for additional data file.S3 FigBRCA data was mined in which SIN3/HDAC members were separated by median expression into those less than the median BRMS1 expression was then examined within these patients for overall survival, with high BRMS1 (indicated by Red) and low BRMS1 (indicated by black) were accounted for. This was completed in KM Plotter.(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S1 Raw images(TIF)Click here for additional data file.S1 File(XLSX)Click here for additional data file.S1 Data(DOCX)Click here for additional data file."} +{"text": "EPAC1) regulate obligate intracellular parasitic bacterium rickettsial adherence to and invasion into vascular endothelial cells (ECs). However, underlying precise mechanism(s) remain unclear. The aim of the study is to dissect the functional role of the EPAC1-ANXA2 signaling pathway during initial adhesion of rickettsiae to EC surfaces. Methods: In the present study, an established system that is anatomically based and quantifies bacterial adhesion to ECs in vivo was combined with novel fluidic force microscopy (FluidFM) to dissect the functional role of the EPAC1-ANXA2 signaling pathway in rickettsiae\u2013EC adhesion. Results: The deletion of the EPAC1 gene impedes rickettsial binding to endothelium in vivo. Rickettsial OmpB shows a host EPAC1-dependent binding strength on the surface of a living brain microvascular EC (BMEC). Furthermore, ectopic expression of phosphodefective and phosphomimic mutants replacing tyrosine (Y) 23 of ANXA2 in ANXA2-knock out BMECs results in different binding force to reOmpB in response to the activation of EPAC1. Conclusions: EPAC1 modulates rickettsial adhesion, in association with Y23 phosphorylation of the binding receptor ANXA2. Underlying mechanism(s) should be further explored to delineate the accurate role of cAMP-EPAC system during rickettsial infection.Introduction: Intracellular cAMP receptor exchange proteins directly activated by cAMP 1 ( Rickettsia (R.), including R. rickettsii [R. parkeri [R. parkeri rickettsiosis [R. conorii, the causative agent of Mediterranean spotted fever [R. australis, which is a part of the transitional group (TRG) [R infections are frequently associated with severe morbidity and mortality [R infections, it only stops bacteria from reproducing, but does not kill the rickettsiae. A fatality rate as high as 32% has been reported in hospitalized patients with Mediterranean spotted fever [Rickettsioses are devastating human infections . These ackettsii ,3 and R. parkeri ,5,6 thatttsiosis , respected fever ; and R. up (TRG) and causup (TRG) . A licenup (TRG) ,2,9. Typup (TRG) ,12. Untrortality ,14,15. Aed fever ,17. Comped fever ,20,21,22EPAC1, not EPAC2, is the dominant isoform [EPAC1 protected mice from fatal rickettsioses [EPAC1 suppressed rickettsial adherence to and invasion into ECs [cAMP is one of the most common and universal second messengers. cAMP regulates a myriad of important biological processes under physiological and pathological conditions. Therefore, current pharmaceutical medications target the cAMP signaling pathway more than any other . cAMP tr isoform ,39,40. Wttsioses . Our ex into ECs . HoweverANXA2), a well-characterized plasminogen and plasminogen activator receptor [Staphylococcus aureus adhesion to EC surfaces [EPAC1 modulates ANXA2 tyrosine (Y) 23 phosphorylation, and inactivation of EPAC1 suppresses ANXA2 expression on the EC luminal surface by downregulating Y23 phosphorylation [EPAC1-ANXA2 signaling pathway is involved in the regulation of rickettsial adhesion to ECs.The mechanism(s) underlying bacterial adherence to vascular endothelial cells (ECs) under shear stress from flowing blood is critical to our understanding the initial stages of the pathogenesis of endovascular bacterial infections. The constitutively expressed native proteins on and/or in the mammalian host plasma membrane play crucial functional role(s) during the initial stage of infection, just prior to EC activation. Recently we reported that endothelial surface annexin A2 provides evidence to support that EPAC1 governs rickettsial adhesion to EC surfaces in association with regulation of ANXA2 Y23 phosphorylation.In the present study, an established, anatomically based, in vivo quantitative system that measures bacterial adhesion to ECs was combEPAC1 plays a critical regulatory role in the early stage of rickettsial invasion into nonphagocytic host cells [EPAC1-knock out (KO) mouse model [EPAC1 during rickettsial adhesion. Plaque assays reveal that the number of viable rickettsiae in circulating blood was higher in EPAC1-KO mice (n = 11) than wild-type (WT) mice (n = 10) at 30 min and 1 hr post-infection (p.i.) when the bacteria were given by the intravenous route , we found that rickettsiae mediate adhesion to the surface of ECs via host ANXA2 . CompareANXA2 .p < 0.01). To examine whether EPAC1 contributes to reOmp and BMEC binding, we compared the adhesion forces generated in WT versus EPAC1-KO BMECs; reduced adhesion forces between a reOmpB and a EPAC1-KO BMEC were recorded (p < 0.01) microscopy [EPAC1-KO BMECs at 15 min p.i. with 10 MOI rickettsiae . Furthercroscopy . All ECskettsiae .EPAC1 specific activator named I942 that competitively binds the cyclic nucleotide-binding domain of EPAC1 [p < 0.05) (ANXA2 in ECs diminished reOmpB binding forces to the ANXA2-KO BMEC surface (p < 0.01) , independent of its concentration in the culture medium . Depleti < 0.01) , corroboiving EC . Interese medium .EPAC1-ANXA2 pathway is involved in the interaction of OmpB with ECs during the initial stages of bacterial binding.Collectively, these data suggest that the EPAC1 regulates endothelial luminal surface ANXA2 expression by modulating the phosphorylation of Y23 [EPAC1-targeted site in the N-terminus of ANXA2 that leads to regulation of rickettsial adhesion, we transfected ANXA2-KO BMECs with mouse WT ANXA2 construct, phosphodefective ANXA2 mutant Y23F, or phosphomimic ANXA2 mutant Y23E, respectively, which were generated by site-directed mutagenesis using their respective mutant oligonucleotides [ANXA2 in these constructs-transfected ANXA2-KO BMECs were detected, respectively (Our previous study suggested that n of Y23 . To idenleotides . Expressectively .p = 5.36 \u00d7 10\u22128) and phosphomimic Y23E in ANXA2-KO BMECs rescued the diminished reOmpB and cell binding force, respectively, whereas the phosphodefective mutant Y23F (p = 0.90) failed to do so in the plasma or other major hematological parameters [ANXA2 phosphodefective mutant Y23A, not ANXA2 phosphomimic mutant Y23E, yields a predominantly negative effect for the translocation of ANXA2 to the membrane surface. The Y23A mutant can still bind to S100A10 but only in the cytosol, not in the plasma membrane [ANXA2 functions as a binding receptor for rickettsial OmpB, in the present study we further reveal that EPAC1 regulates rickettsial adhesion to EC surfaces by targeting the ANXA2 Y23. Depletion of ANXA2 in ECs reduced the enhanced binding force between reOmpB and ECs in the presence of I942. The WT ANXA2 construct rescued EPAC1-specific activator I942-induced enhancement of reOmpB binding forces to a ANXA2-KO BMEC surface. Furthermore, Y23E mutant rescued the diminished reOmpB and cell binding force in ANXA2-KO BMEC, whereas the Y23F mutant failed. However, there was no increasing in the binding force in the phosphomimic Y23E mutant-transfected ANXA2-KO BMEC in response to I942, further suggesting that EPAC1 modulates Y23 phosphorylation of the binding receptor ANXA2. Currently the accurate pathway how EPAC1 modulates the phosphorylation of specific residues(s) in ANXA2 remains to be identified. Furthermore, the potential crosstalk between cAMP-EPAC1 system and other identified factors involved in rickettsial adhesion and/or invasion should be investigated in future studies. and S25 ,56. Phosid rafts ,57 and tartments ,58. Afteechanism . We reporameters . Specifimembrane . Given tThe colloidal probe-based AFM assay was developed to study protein\u2013protein interactions and to quantify the interacting forces in the range from picoNewtons to nanoNewtons . During EPAC1-KO BMEC is still higher than the forces between BSA and EPAC1-KO BMEC. These data may suggest that intracellular EPAC1 is not the sole regulator on endothelial surface receptor for OmpB-mediated rickettsial binding. Such unknown host surface protein(s) remain to be identified. Furthermore, although BSA has been widely employed as a mock control using AFM or FluidFM to measure protein-protein interaction in studying bacterial adhesion [The binding forces between reOmpB and adhesion , identifEPAC1 modulates rickettsial adhesion, involving regulation of tyrosine 23 phosphorylation of the binding receptor ANXA2. This finding provides experimental and theoretical support for therapeutic strategies for rickettsiosis targeting the host cAMP-EPAC system. In conclusion, we have revealed a novel mechanism by which EPAC1-KO mice were derived as described previously [ANXA2-KO mice on the C57BL/6J background were a generous gift from Dr. Katherine Hajjar [R. australis. Therefore, this organism was chosen as the TRG rickettsial agent of choice [All animal experiments were performed according to protocols approved by the Institutional Animal Care and Use Committee of the University of Texas Medical Branch (UTMB). Wild-type (WT) mice (C57BL/6J) were obtained from The Jackson Laboratory . C57BL/6 eviously . ANXA2-KNY, USA) . All micf choice .R. australis (strain Cutlack) was prepared as described [escribed . UninfecEPAC1-KO, and ANXA2-KO mice using established protocol [Mouse brain microvascular endothelial cells (BMECs) were isolated from wild-type, protocol ,51,62.ANXA2, ANXA2 mutant Y23E, and ANXA2 mutant Y23F were purchased from GENEWIZ .A rabbit polyclonal antibody against SFG/TRG rickettsiae was obtained from the laboratory of Dr. David Walker . AlexaFluor 594-conjugated goat anti-rabbit IgG and ProLong Gold Antifade Reagent with DAPI were purchased from Invitrogen . Recombinant rickettsial OmpB (reOmpB) (amino acids 1363\u20131655) was purchased from MyBioSource . Unless otherwise indicated, all reagents were purchased from Thermo Fisher Scientific. Expression constructs for GFP-tagged mouse WT ANXA2, ANXA2 Y23E, and ANXA2 Y23F constructs (1 \u00b5g/500 \u00b5L for 1 \u00d7 105 cells), respectively, using Magnetofection PolyMag and PolyMag Neo . The efficacy of transfection was evaluated with GFP fluorescent microscopy [Transfections were performed using a published protocol . Cells wcroscopy .TACGGGTCAGTCAAACCCTACACCAACTTCGATGCTGAGAGGGATGCTCTGAACATTGAGACAGCAGTCAAGACCAAAGGAGTGGATGAGGTCACCATTGTCAACATCCTGACAAACCGCAGCAATGTGCAGAGGCAGGACATTGCCTTCGCCTATCAGAGAAGGACCAAAAAGGAGCTCCCGTCAGCGCTGAAGTCAGCCTTATCTGGCCACCTGGAGACGGTGATTTTGGGCCTATTGAAGACACCTGCCCAGTATGATGCTTCGGAACTAAAAGCTTCCATGAAGGGCCTGGGGACTGACGAGGACTCCCTCATTGAGATCATCTGCTCACGAACCAACCAGGAGCTGCAAGAGATCAACAGAGTGTACAAGGAAATGTACAAGACTGATCTGGAGAAGGACATCATCTCTGACACATCTGGAGACTTCCGAAAGCTGATGGTCGCCCTTGCAAAGGGCAGACGAGCAGAGGATGGCTCAGTTATTGACTACGAGCTGATTGACCAGGATGCCCGGGAGCTCTATGATGCCGGGGTGAAGAGGAAAGGAACCGACGTCCCCAAGTGGATCAGCATCATGACTGAGCGCAGTGTGTGCCACCTCCAGAAAGTGTTCGAAAGGTACAAGAGCTACAGCCCTTATGACATGCTGGAGAGCATCAAGAAAGAGGTCAAAGGGGACCTGGAGAACGCCTTCCTGAACCTGGTCCAGTGCATCCAGAACAAGCCCCTGTACTTCGCTGACCGGCTGTACGACTCCATGAAGGGCAAGGGGACTCGAGACAAGGTCCTGATTAGAATCATGGTCTCTCGCAGTGAAGTGGACATGCTGAAAATCAGATCTGAATTCAAGAGGAAATATGGCAAGTCCCTGTACTACTACATCCAGCAAGACACCAAGGGTGACTACCAGAAGGCACTGCTGTACCTGTGTGGTGGGGATGACTGA-3\u2019.5\u2019-ATGTCTACTGTCCACGAAATCCTGTGCAAGCTCAGCCTGGAGGGTGATCATTCTACACCCCCAAGTGCCANXA2 mutant Y23E, the sequence of bases encoding Y23 is replaced with GAA. For ANXA2 mutant Y23F, the sequence of bases encoding Y23 is replaced with UUC.For R. australis was injected through the tail vein, rickettsial virulence (by plaque assay) was measured in a 1 \u00b5L blood sample collected from the orbital venous sinus (OVS) at different times until 1 h post-infection (p.i.). Rickettsial antigens and mouse EPAC1 were detected with a rabbit polyclonal antibody against SFG/TRG rickettsiae (1:1000) and mouse monoclonal antibody against EPAC1 (1:500) , respectively, overnight at 4 \u00b0C, followed by incubation with AlexaFluor 594 goat anti-rabbit (1:1000) and AlexaFluor 488 goat anti-mouse (1:2000) antibodies for 15 min. Normal mouse or rabbit IgG was used as an antibody negative control during IF staining were coated with reOmpB using published protocols ,64. Brieg-poly(ethylene glycol) (PLL-g-PEG) to block nonspecific adhesions prior to calibration using the method of Sander et al. [The FluidFM system coupling Nanosurf Core AFM and Fluidic Pressure Controller was used for this assay. A micropipette (Cytosurge AG) was coated with 0.1 mg/ml poly(L-lysine)-r et al. and pre-r et al. . The forr et al. . Ten celt-test or One-Way ANOVA . p values are as follows: ** p < 0.01 and * p < 0.05. Statistical significance is considered as p < 0.05.Values are reported as mean \u00b1 SEM. The data were analyzed using the Student\u2019s"} +{"text": "The nematode Caenorhabditis elegans is one of the most intensely studied organisms in biology, offering many advantages for biochemistry. Using the highly active biotin ligase TurboID, we optimize here a proximity labeling protocol for C.\u00a0elegans. An advantage of TurboID is that biotin's high affinity for streptavidin means biotin-labeled proteins can be affinity-purified under harsh denaturing conditions. By combining extensive sonication with aggressive denaturation using SDS and urea, we achieved near-complete solubilization of worm proteins. We then used this protocol to characterize the proteomes of the worm gut, muscle, skin, and nervous system. Neurons are among the smallest C.\u00a0elegans cells. To probe the method's sensitivity, we expressed TurboID exclusively in the two AFD neurons and showed that the protocol could identify known and previously unknown proteins expressed selectively in AFD. The active zones of synapses are composed of a protein matrix that is difficult to solubilize and purify. To test if our protocol could solubilize active zone proteins, we knocked TurboID into the endogenous elks-1 gene, which encodes a presynaptic active zone protein. We identified many known ELKS-1-interacting active zone proteins, as well as previously uncharacterized synaptic proteins. Versatile vectors and the inherent advantages of using C.\u00a0elegans, including fast growth and the ability to rapidly make and functionally test knock-ins, make proximity labeling a valuable addition to the armory of this model organism.Proximity labeling provides a powerful Hin\u00a0vivo . Proximiin\u00a0vivo , 2, 7. T limited , 10 and limited , 12 and tissues or pretr tissues .Caenorhabditis elegans has proven a useful workhorse to investigate metazoan biology, offering powerful genetics, in\u00a0vivo cell biology, an anatomy described at electron micrograph resolution, and a defined number of cells whose gene expression can be profiled at single cell resolution tRNA synthetase (MuPheRS) , 27. MuPC.\u00a0elegans results in robust biotinylation signals in the intestine, with the strongest signals generated by TurboID , we generated transgenic animals expressing a TurboID-mNeongreen-3xFLAG (TbID-mNG) fusion protein in various tissues (B). We compared protein biotinylation levels in age-synchronized young adults expressing the neuronal rab-3p::TbID-mNG transgene with wild-type controls. In the absence of exogenously added biotin, transgenic animals showed only a slight increase in biotinylated proteins compared with controls. Adding exogenous biotin increased protein biotinylation specifically in animals expressing the rab-3p::TbID-mNG transgene (C). To optimize biotin availability to worm tissues, we treated animals with exogenous biotin for different time intervals, using biotinylation in neurons as a readout. A 2-h incubation was sufficient to achieve robust protein labeling (D). To identify an optimum biotin concentration for protein labeling, we treated animals with varying concentrations of biotin for 2\u00a0h. We observed a substantial increase in biotinylation in worms treated with 1\u00a0mM biotin but higher biotin concentrations did not appear to further increase biotinylation (E). We also used the E.\u00a0coli biotin auxotrophic strain MG1655 as a food source for worms instead of standard OP50 , indicating that TurboID is functional in all major C.\u00a0elegans tissues.Like pan-neuronal expression, intestinal, hypodermal, and muscle-specific expression of TurboID-mNG conferred robust biotinylation activity A, indicaC.\u00a0elegans (A\u2013C). A significant advantage of PL compared with IP is that extracts can be collected under strong denaturing conditions , since the biotin tag is covalently attached. This allowed us to achieve >95 % solubilization of proteins . After extensive washing and elution, we fractionated the affinity-purified biotinylated proteins by SDS-PAGE, visualized the proteins by Coomassie staining, and analyzed gel slices by LC-MS/MS analysis (B). Using a threshold of at least two unique peptides, we cumulatively identified >4000 proteins expressed in one or more tissues . In addition, we observed a strong correlation between the two replicates at the level of spectral counts (B). Consistent with previous findings, we identified most proteins in the intestine, followed by the hypodermis and muscle cells (B), (B), and 1274 proteins were detected in only one tissue (C). Proteins identified by MS/MS when TurboID was targeted to neurons included broadly expressed neuronal proteins, such as CLA-1, UNC-31, and proteins expressed in subsets of neurons, such as the glutamate decarboxylase UNC-25, which is known to be expressed in 26 neurons, and OSM-10, which is expressed in four pairs of neurons (D). Muscle-specific samples were enriched in CPNA-1, CPNA-2, PQN-22, and F21C10.7 (D); intestine-specific samples were enriched in ACOX-2, CGR-1, C49A9.9, and C49A9.3 (D), and hypodermis-specific samples included F17A9.5, PAH-1, R05H10.1, and CPN-2 (D). In addition, we identified tissue-specific enrichment for many proteins whose expression was previously uncharacterized . For some of these proteins we validated the tissue-selective expression highlighted by our MS/MS data by making transgenic reporters. The reporters confirmed the expression profile predicted by MS/MS, validating the method's specificity .We next optimized a protocol to extract and affinity purify biotinylated proteins from \u00a0elegans , A\u2013C. A ins A\u2013C, . We achiins A\u2013C, . Westernins A\u2013C, A. After analysis B. Using B and C, . Using tB and C, , we lookB and C, A. In addl counts B. Consisle cells B, 12). . C.\u00a0eleglls (B), B, and 12e tissue C. Protei neurons D. MuscleF21C10.7 D; intest C49A9.3 D, and hynd CPN-2 D. In addcterized , Fig.\u00a02EC.\u00a0elegans nervous system includes 118 classes of neurons, with most classes consisting of a single pair of neurons that form left/right homologs (gcy-8 promoter (A). Western blot analysis of extracts from these animals revealed a biotinylation signal significantly higher than that in extracts from wild-type controls (B). Correlation plots of mass spectrometry data obtained for affinity-purified extracts made from animals expressing the gcy-8p::TbID-mNG transgene showed reproducible results between replicates (A). As expected, these samples were enriched for proteins specifically or selectively expressed in AFD neurons when compared with similarly processed extracts from nontransgenic controls, or from animals expressing a rab-3p::TbID-mNG transgene (C and S4B). Enriched proteins included the transmembrane guanylate cyclases GCY-8 and GCY-18 and the homeobox transcription factor TTX-1 (D and S4B). Our MS data also identified other proteins enriched in the AFD-specific TurboID samples compared with the pan-neuronal TurboID controls (E and S4C). To examine if these proteins were selectively expressed in AFD neurons, we generated transgenic reporter lines (F). nex-4 and F37A4.6 were expressed specifically in AFD, albeit F37A4.6 at low levels; T06G6.3 was expressed in a small subset of neurons that included AFD (F). Our list of AFD-enriched proteins, identified by mass spectrometry, was consistent with AFD-specific gene expression identified by RNA Seq . ELKS-1 is expressed throughout the nervous system and localizes to the presynaptic active zone, in proximity to other presynaptic proteins such as \u03b1-liprin and RIM . Western blot analysis of extracts from ELKS-1::TbID animals did not show an increased biotinylation signal compared with wild-type controls (B). However, mass spectrometry analysis of streptavidin-purified proteins from this knock-in strain revealed enrichment of known synaptic proteins including UNC-10/RIM, SYD-1/SYDE1, SYD-2/\u03b1-liprin, SAD-1/BRSK1, CLA-1/CLArinet, C16E9.2/Sentryn, and the RIM-binding protein RIMB-1 (D). We measured enrichment by comparing mass spectrometry data obtained for control extracts processed in parallel from wild-type and from transgenic animals expressing free TurboID-mNG throughout the nervous system, rab-3p::TbID-mNG (C and S4E) or both nervous system and other tissues (G). Between-replicate correlation plots revealed reproducible results between experimental repeats (D). We also identified several previously uncharacterized proteins as enriched in ELKS-1::TbID-mNG samples . We expressed mNeongreen translational fusion transgenes for several of these proteins exclusively in the AFD neuron pair and showed that they colocalized with ELKS-1::mScarlet at presynaptic active zones where AFD is known to synapse with AIY interneurons . Together, our findings show that TurboID-mediated proximity labeling is an effective method to reveal protein interactors in C.\u00a0elegans at endogenous levels with specificity and sensitivity.We next asked if TurboID can highlight the interactome of a specific and RIM . To havesynapses A. Westercontrols B. Howeven RIMB-1 D. We meaTbID-mNG C and S4E tissues G. Betwee repeats D. We alsrneurons . Human ANKS1B, also known as AIDA-1, is a risk locus for autism and neurodevelopmental defects (C.\u00a0elegans ortholog of human KIAA0930. C16E9.2 was recently given the name sentryn (STRN-1). STRN-1 together with the SAD kinase and liprin-\u03b1 promote dense-core vesicle (DCV) pausing at presynaptic regions also suggest that these proteins are expressed in neurons. Such hypotheses raised by the MS data need to be directly tested, for example, using colocalization studies with fluorescently tagged reporters.Some proteins identified in the ELKS-1 proximity labeling experiments are usually considered nonneuronal. We speculate some of these proteins may also be expressed in neurons. For example, we find components of the muscle dense body, including the myotilin ortholog KETN-1/MYOT, the ALP-Enigma protein ALP-1, the sorbin homolog SORB-1, and ZYXin ZYX-1 enrichedC.\u00a0elegans, adding biotin to plates seeded with bacteria (E.\u00a0coli OP50 or NA22 strains) is sufficient to promote TurboID labeling, but long incubation times are required to achieve detectable protein biotinylation . We find that a 2-h incubation with exogenous biotin is sufficient for strong TurboID signals in worm neurons (D). Since in the lab C.\u00a0elegans is typically grown on E.\u00a0coli as a food source, feeding biotin-auxotrophic E.\u00a0coli to worms provides some control over the start of biotinylation by TurboID. For time-sensitive experiments, it may be possible to soak worms in a buffer containing biotin for shorter periods, e.g., 30 or 60\u00a0min and still achieve robust protein biotinylation, although further analysis using MS is required to confirm this.An important variable for TurboID protocols is the delivery of biotin or biotin derivatives. In cultured mammalian cells expressing TurboID or miniTurbo, adding biotin for as little as 10\u00a0min yields robust biotinylation . In C.\u00a0e neurons D. Since C.\u00a0elegans proteome comprises integral membrane proteins; however, only 10\u201311% of the proteins for which we detect peptides correspond to integral membrane proteins. Membrane protein identification is a long-standing problem in mass spectrometry: integral membrane proteins typically yield fewer tryptic peptides, due to a paucity of positively charged residues. In TurboID experiments underrepresentation is compounded, since biotinylation occurs on solvent exposed lysine residues. It will be interesting in future work to explore the recovery of membrane proteins when TurboID is targeted to plasma membrane of C.\u00a0elegans cells.About 27% of the A) and MS analysis (data not shown). These carboxylases; PCCA-1/PCCA , PYC-1/PC (pyruvate carboxylase), MCCC-1/MCCC (methylcronotoyl coenzyme A carboxylase), POD-2/ACACA (acetyl coenzyme A carboxylase) were the most abundantly detected proteins in our MS experiments in every condition tested, including controls. Depending on how broadly and strongly we expressed TurboID in C.\u00a0elegans tissues, these carboxylases account for 8%\u201385% of peptides identified in MS analysis. We aim to knock-in tags into each of these four genes , using CRISPR/Cas9 to allow their depletion by affinity purification, thereby increasing the sensitivity of proximity labeling, particularly when the TurboID-tagged protein is expressed in a small number of cells or at low levels.Four endogenously biotinylated carboxylases dominate the biotinylated proteome in TurboID expressing lines, as seen by both Western blot A and MS C.\u00a0elegans community to map the proteomes of specific cells and subcellular structures and to characterize the interactomes of proteins of interest.Despite extensive washing and use of denaturing conditions, streptavidin purified samples retain substantial nonspecific background. Notably, the mass spectrometer identifies many proteins even when we apply our affinity purification protocol to extracts made from control animals that do not express TurboID. These nonspecific contaminant proteins may be sticky and/or expressed at high enough levels to come through to our pipeline. Alternatively, there may be endogenous biotinylation of many proteins beyond the highly abundant carboxylases PCCA-1, PYC-1, POD-2, and MCCC-1. A future priority is to create a comprehensive list of such contaminants. In summary, our findings show that TurboID works well in a wide array of contexts in the worm. TurboID will be a reliable and useful tool for the E.\u00a0coli MG1655. C.\u00a0elegans husbandry otherwise followed standard laboratory culture conditions (Worms were grown at room temperature (22 \u00b0C) on nematode growth medium (NGM) plates seeded with the biotin auxotroph nditions .C.\u00a0elegans strains used in this study include:N2, the wild-type Bristol straindbIs37[rab-3p::mNeongreen::3XFLAG]AX7647 dbIs24[rab-3p::CeTurboID::mNeongreen::3XFLAG]AX7526 dbIs25[ges-1p::CeTurboID::mNeongreen::3XFLAG]AX7542 dbIs28[myo-3p::CeTurboID::mNeongreen::3XFLAG]AX7578 dbIs33[dpy-7p::CeTurboID::mNeongreen::3XFLAG]AX7623 AX7606 dbIs32[gcy-8p::CeTurboID::mNeongreen::3XFLAG]dbEx1234AX7917 dbEx1241AX7922 AX7928 dbEx1247AX8059 dbEx1266[R10E8.8p::mNeongreen::3XFLAG]AX7935 dbEx1251; dbEx1253AX7937 dbEx1255; dbEx1253dbEx1256[T06G6.3p::mNeongreen::3XFLAG]; dbEx1253AX7939 dbEx1296AX8110 dbEx1297AX8111 dbEx1300AX8117 elks-1(syb1710). The syb1710 allele is a knock-in that expresses ELKS-1 tagged C-terminally with CeTurboID::mNeongreen::3XFLAG.PHX1710 C.\u00a0elegans pEntry vector, which was a gift from Dr Dominique Glauser (University of Fribourg).DNA encoding N linker::CeTurboID::c-Myc and mNeongreen::3XFLAG were stitched together by fusion PCR using primer sequences that encoded a short Gly-Ser linker. The resulting PCR product (N linker::CeTurboID::c-Myc::mNeongreen::3XFLAG) was cloned into position 2 of a Multisite Gateway pDONR vector (ThermoFisher).https://www.addgene.org/.The codon-optimized TurboID plasmid is available at Addgene C.\u00a0elegans genomic DNA by PCR and cloned into position 1 of a Multisite Gateway Donor vector (Thermo Fisher). The resulting pEntryslot1 vectors containing these promoters were each mixed with the dg398 pEntryslot2_mNeongreen::3XFLAG::stop vector, a pEntryslot3 vector containing the let-858 or tbb-2 3\u2032UTR and pDEST (ThermoFisher) in an LR reaction. The resulting expression vectors were injected into the gonad of day 1 adult N2 worms at a concentration of 25\u00a0ng/uL.Promoters for B0379.1 (\u223c0.9\u00a0kbp), T01G5.1 (\u223c2.1\u00a0kbp), Y53G8AL.1 (\u223c2.5\u00a0kbp), nex-4 (\u223c1.6\u00a0kbp), F37A4.6 (\u223c2.5\u00a0kbp), T06G6.3 (\u223c2.2\u00a0kbp), and R10E8.8 (\u223c1.6\u00a0kbp) were amplified from C03H5.6 (\u223c2\u00a0kbp) and C11E4.6 (\u223c6\u00a0kbp) were amplified from C.\u00a0elegans genomic DNA by PCR and cloned into a position 2 Gateway donor vector. cDNA for H06I04.1 isoform a was synthesized by IDT and cloned into a position 2 Gateway donor\u00a0vector. The resulting pEntryslot2 vectors were mixed with dg397 pEntryslot3_mNeongreen::3XFLAG::stop::unc-54 3\u2032UTR entry vector and a pDEST vector in an LR reaction. elks-1 cDNA (\u223c2.5\u00a0kbp) was PCR-amplified from a C.\u00a0elegans cDNA library and cloned into a position 2 Gateway donor vector, mixed with gcy-8 promoter (position 1) and mScarlet (position 3) and a pDEST vector in an LR reaction. Resulting expression vectors were injected into the gonad of day 1 adult N2 worms at a concentration of 25\u00a0ng/\u03bcL.ORFs for Around 100 L4 stage transgenic animals expressing an extrachromosomal array were picked and transferred to unseeded NGM plates. The plates were placed in a UV cross-linker with lids removed and exposed to UV (energy setting 300). The irradiated worms were then transferred to seeded plates (10 worms/plate). In total, 200 of their F1 progeny expressing the transgene were picked and individually transferred to a seeded plate. Plates containing many transgenic F2 progeny, indicating potential integration of the transgene, were selected, and eight F2 animals from the candidate plates singled and examined for 100% transmission in the next generation. Integrants were outcrossed to N2 worms at least four times.F-myo-3p-Entry1-GGGGACAACTTTGTATAGAAAAGTTGTGTAGGCAATCAGTCAAACCGAATAAAAR-myo-3p-Entry1-GGGGACTGCTTTTTTGTACAAACTTGTTCTAGATGGATCTAGTGGTCGTGGGTTF-dpy-7p-Entry1-GGGGACAACTTTGTATAGAAAAGTTGAATCTCATTCCACGATTTCTCGCAACACATR-dpy-7p-Entry1-GGGGACTGCTTTTTTGTACAAACTTGTTATCTGGAACAAAATGTAAGAATATTCTTAF-gcy-8p-Entry1-GGGGACAACTTTGTATAGAAAAGTTGGGTTCAACAAGGGTATTGTATTGCAATCAGTGR-gcy-8p-Entry1-GGGGACTGCTTTTTTGTACAAACTTGTTTGATGTGGAAAAGGTAGAATCGAAAATCCF-T01G5.1p-Entry1- GGGGACAACTTTGTATAGAAAAGTTGCACCTGAACACAACATTTTCTGR-T01G5.1p-Entry1- GGGGACTGCTTTTTTGTACAAACTTGGACATGAAATTGTATCTGAAAGCF-Y53G8AL.1p-Entry1- GGGGACAACTTTGTATAGAAAAGTTGGTAGTTATGGAAAAGCAACGTCGGAGR-Y53G8AL.1p-Entry1- GGGGACTGCTTTTTTGTACAAACTTGTCTAAAATAGCATTGGTTCTGAAACTTTGF-B0379.1p-Entry1- GGGGACAACTTTGTATAGAAAAGTTGGAAACAATATTATTTTTGTTTCACAGR-B0379.1p-Entry1- GGGGACTGCTTTTTTGTACAAACTTGGTTGTTGTTGTCTCGATGGAAAAGF-nex-4p-Entry1- GGGGACAACTTTGTATAGAAAAGTTGCCTGAGAATTACTGAAGTTTAAGCR-nex-4p-Entry1- GGGGACTGCTTTTTTGTACAAACTTGCTCGAGTTACTTCAATGCTCACGF-F37A4.6p-Entry1- GGGGACAACTTTGTATAGAAAAGTTGGATTCAGAAAATTCAGAAAGGCATTCR-F37A4.6p-Entry1- GGGGACTGCTTTTTTGTACAAACTTGCATTATAGGAAGACTGAGATTCCAAGCF-T06G6.3p-Entry1- GGGGACAACTTTGTATAGAAAAGTTGCATTTTTTGCTCTAAAGGTGGAATAGR-T06G6.3p-Entry1- GGGGACTGCTTTTTTGTACAAACTTGCTTTGAAAAAAGTTCAGAGTAGTAGAGF-C03H5.6-Entry2- GGGGACAAGTTTGTACAAAAAAGCAGGCTtttcagaaaaATGTTAGAATGTATACATCCAACATR-C03H5.6-Entry2- GGGGACCACTTTGTACAAGAAAGCTGGGTATGTACGATTCATCAAACCACCTACF-C11E4.6-Entry2- GGGGACAAGTTTGTACAAAAAAGCAGGCTtttcagaaaaATGAGCCTCAAAGACTTTGTCATATCR-C11E4.6-Entry2- GGGGACCACTTTGTACAAGAAAGCTGGGTAATTTCTTTGGTTCTCAGTAGTTTGCTGF-H06I04.1-Entry2- GGGGACAAGTTTGTACAAAAAAGCAGGCTtttcagaaaaATGAGCAAAGAAACTGGAAAAATGGCGGR- H06I04.1-Entry2- GGGGACCACTTTGTACAAGAAAGCTGGGTAAAATGGTCCAGATCTTGCTTCATTGGTCF-R10E8.8p-Entry1- GGGGACAACTTTGTATAGAAAAGTTGCACATAAAATACGTTTTAGTAGCTGTCAGCACR-R10E8.8p-Entry1- GGGGACTGCTTTTTTGTACAAACTTGTTTTTGTCTGAAAATCGAACATTAAAAATAACAGGF-elks-1-cDNA-Entry2-GGGGACAAGTTTGTACAAAAAAGCAGGCTTTTCAGAAAAATGGCACCTGGTCCCGCACCATACAGCR-elks-1-cDNA-Entry2- GGGGACCACTTTGTACAAGAAAGCTGGGTAGGCCCAAATTCCGTCAGCATCGTCGTGrab-3 promoter (\u223c1.2 kb) and ges-1 promoter (\u223c3.4 kb) were used from de Bono lab plasmid collection.N/C terminus Gly-Ser rich linkersCeTurboIDIntronsC-mycmNeongreen3XFLAGGGAGGTGGTGGATCAGGCTCGGGAGGTCGAGGCTCAGGATCCGGTTCCGGCTCCGGCTCTGGTTCCGGTTCGGGTTCCGGTTCTGGAAAGGATAACACCGTTCCACTTAAGCTTATCGCCCTTCTTGCCAACGGAGAATTCCACTCTGGAGAGCAACTTGGAGAGACTCTTGGAATGTCCCGTGCTGCCATCAACAAGCATATCCAAACCCTTCGTGATTGGGGAGTTGATGTTTTCACTGTTCCAGGAAAGgtaagtttaaacatatatatactaactaaccctgattatttaaattttcagGGATACTCCCTTCCAGAGCCAATCCCACTTCTTAACGCCAAGCAAATCCTTGGACAACTTGATGGAGGATCCGTCGCTGTCCTTCCAGTTGTTGATTCCACCAACCAATACCTTCTTGACCGTATCGGAGAGCTTAAGTCTGGAGACGCCTGCATCGCTGAGTACCAACAAGCTGGACGCGGATCTCGCGGACGCAAGTGGTTCTCCCCATTCGGAGCCAACCTTTACCTTTCTATGTTCTGGCGTCTTAAGCGTGGACCAGCTGCTATCGGACTTGGACCAGTTATCGGAATCGTTATGGCTGAGGCCCTTCGTAAGCTTGGATATAAGgtaagtttaaacagttcggtactaactaaccatacatatttaaattttcagGTTCGTGTTAAGTGGCCAAACGATCTTTACCTTCAAGACCGTAAGCTTGCTGGAATCCTTGTCGAGCTTGCTGGAATCACCGGAGACGCCGCTCAAATCGTTATCGGAGCTGGAATCAACGTTGCCATGCGTCGTGTTGAGGAGTCTGTTGTTAACCAAGGATGGATCACTCTTCAAGAGGCTGGAATCAACCTTGATCGTAACACCCTTGCTGCCACCCTTATCCGTGAGCTTCGTGCTGCCCTTGAGCTTTTCGAGCAAGAGGGACTTGCCCCATACCTTCCACGCTGGGAGAAGCTTGACAACTTCATCAACCGCCCAGTTAAGCTTATCATCGGAGATAAGGAATCTTCGGAATCTCTCGCGGAATCGACAAGCAAGGAGCTCTTCTTCTTGAGCAAGATGGAGTCATTAAGCCATGGATGGGAGGAGAGATTTCCCTTCGTTCCGCTGAGAAGGCCGGAGGAGAACAGAAGCTTATAAGTGAGGAGGACCTGGGATCCGCTGGATCCGCTGCTGGATCCGGTGAGTTCATGGTGTCGAAGGGAGAAGAGGATAACATGGCTTCACTCCCAGCTACACACGAACTCCACATCTTCGGATCGATCAACGGAGTGGATTTCGATATGGTCGGACAAGgtaagtttaaacatatatatactaactaaccctgattatttaaattttcagGAACTGGAAACCCAAACGATGGATACGAGGAACTCAACCTCAAGTCGACAAAGGGAGATGCAATTCTCGCCATGGATTCTCGTGCCACACATCGGATACGGATTCCACCAATACCTCCCATACCCAGgtaagtttaaactgagttctactaactaacgagtaatatttaaattttcagATGGAATGTCACCATTCCAAGCTGCCATGGTGGATGGATCGGGATACCAAGTTCACCGAACAATGCAATTCGAGGATGGAGCCTCGCTCAGTGAACTACCGATACACATACGAGGGATCGCACATCAAGgtaagtttaaacagttcggtactaactaaccatacatatttaaattttcagGGAGAGGCTCAAGTTAAGGGAACAGGATTCCCAGCTGATGGACCAGTGATGACAAACTCACTCACAGCTGCTGATTGGTGCCGATCGAAAAAGACATACCCAAATGATAAGACAATATCTCGACATTCAAGTGGTCGTACACTACTGGAAACGGAAAGCGATACCGATCGACAGCCCGAACAACATACACATTCGCTAAGCCAATGGCCGCCAACTACCTCAAGgtaagtttaaacatgattttactaactaactaatctgatttaaattttcagAATCAACCAATGTACGTGTTCCGAAAGACAGAACTCAAGCACTCAAAGACAGAGCTGAACTTCAAAGAGTGGCAAAAGGCCTTCACAGATGTGATGGGAATGGATGAACTCTACAAGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGC.\u00a0elegans expressing fluorescent proteins were acquired using a Leica SP8 inverted laser scanning confocal microscope with 10\u00d7 0.3 NA dry, 63\u00d7 1.2 NA water or 63\u00d7 1.2 NA oil-immersion objectives, using the LAS X software platform (Leica). The Z-project function in Image J was used to obtain the figures used in the panels. Animals were mounted on 2% agarose pads and immobilized with 100\u00a0\u03bcM of sodium azide.Confocal microscopy images of transgenic C.\u00a0elegans grown on E.\u00a0coli MG1655 were harvested at L4 or young adult stage, washed three times in M9 buffer, incubated at room temperature (22\u00a0\u00b0C) in M9 buffer supplemented with 1\u00a0mM biotin, and E.\u00a0coli MG1655 for 2\u00a0h. Two hours later, the worms were washed three times in M9 buffer and flash frozen after the M9 buffer was completely aspirated and 4\u00d7 Bolt LDS sample buffer supplemented with fresh DTT. The samples were then thawed, boiled for 10\u00a0min at 90 \u00b0C, vortexed for 10\u00a0min, centrifuged for 30\u00a0min at 15,000\u00a0rpm at 4 \u00b0C, and the supernatant collected. Proteins were transferred to a PVDF membrane (Thermofisher Scientific) following electrophoresis using Bolt 4\u201312% Bis-Tris Plus gels (Thermofisher Scientific). Membranes were blocked for 1\u00a0h at room temperature with TBS-T buffer containing 5% BSA and incubated for 2\u00a0h at room temperature with HRP-conjugated or fluorescently labeled streptavidin or with HRP-conjugated antibodies. Membranes were then washed three times with TBS-T. The following antibodies or protein-HRP conjugates were used for this study: IRDye 800CW Streptavidin (1:7000 in TBS-T) (LI-COR Biosciences), Anti-FLAG M2-Peroxidase (1:5000 in TBS-T) (A8592 Sigma), anti-alpha tubulin-HRP (DM1A Abcam ab40742), Streptavidin-HRP (1:5000 in TBS-T) . Membranes were imaged using ChemiDoc the Imaging System .Synchronized populations of C.\u00a0elegans were bleached and the eggs transferred to NGM plates seeded with E.\u00a0coli MG1655 to obtain synchronized populations of worms. The animals were harvested at L4 or young adult stage, washed three times in M9 buffer, incubated at room temperature (22 \u00b0C) in M9 buffer supplemented with 1\u00a0mM biotin, and E.\u00a0coli MG1655 for 2\u00a0h unless stated otherwise. Two hours later, the worms were washed three times in M9 buffer and allowed to settle on ice after the last wash. After completely aspirating the M9 buffer, two volumes of RIPA buffer supplemented with 1\u00a0mM PMSF and cOmplete EDTA-free protease inhibitor cocktail (Roche Applied Science) were added to one volume of packed worms. The animals were again allowed to settle on ice and then added dropwise to liquid N2 to obtain frozen worm \u201cpopcorn.\u201d A Spex 6875D cryogenic mill was used to grind frozen C.\u00a0elegans to a fine powder, which was then stored at \u201380\u00a0\u00b0C. Worm powder was thawed by distributing it evenly along the length of a 50\u00a0ml falcon tube and rolling it on a tube roller at 4 \u00b0C. After the sample was completely thawed, it was centrifuged to collect the sample at the bottom of the tube. SDS and DTT were added to the sample to final concentrations of 1% and 10\u00a0mM respectively, from stock solutions of 20% SDS and 1M DTT. The tubes were gently inverted a few times and immediately incubated at 90 \u00b0C for 5\u00a0min. After heat treatment, the samples were sonicated continuously for 1\u00a0min twice, with brief cooling between the two sonication steps. Sonication used a probe sonicator microtip (MSE Soniprep 150 plus) and an amplitude setting of 16/max. The samples were cooled to room temperature following sonication and adjusted to 2 M urea using a stock solution . The samples were then centrifuged at 100,000g for 45\u00a0min at 22 \u00b0C, and the clear supernatant between the pellet and surface lipid layer transferred to a new tube.Gravid adult g for 5\u00a0min (or until the buffer was completely eluted from resin). Around 4\u00a0ml of clarified sample was then loaded onto the equilibrated spin column and desalted by centrifugation at 1000g for 5\u00a0min (or until the sample was completely eluted from resin) to remove free biotin. The desalting step was repeated once more using freshly equilibrated columns. Protein concentration in the samples was measured using Pierce 660\u00a0nm protein assay reagent supplemented with Ionic Detergent Compatibility Reagent (IDCR) (Thermo Fisher Scientific).Zeba spin desalting columns (7K MWCO) (Thermofisher) were equilibrated three times with 5\u00a0ml RIPA buffer containing 1% SDS and 2 M urea, freshly supplemented with protease inhibitors by centrifugation at 1000C.\u00a0elegans total protein lysate) were equilibrated in RIPA buffer by briefly incubating them three times in the buffer and using magnetic separation to retain beads while discarding buffer (note: we were able to reduce the amount of protein lysate to 5\u201310\u00a0mg in subsequent experiments without compromising the mass spectrometry data). Desalted, clarified samples were combined with Dynabeads in a 50\u00a0ml falcon tube and incubated overnight in a tube roller at room temperature. Beads were magnetically separated using a neodymium magnet taped to the tube and incubated on a rocking platform. Unbound lysate was removed and the Dynabeads transferred to 2\u00a0ml LoBind protein tubes (Eppendorf). Beads were washed five times with 2% SDS wash buffer and five times with 1M KCl wash buffer; beads were transferred to new tubes after each wash.Dynabeads MyOne streptavidin C1 to saturation in sample buffer (NuPAGE LDS sample buffer 4\u00d7) containing reducing agent (NuPAGE sample reducing agent 10\u00d7). Elution sample buffer was centrifuged at 13,000\u00a0rpm for 5\u00a0min to remove undissolved biotin and the elution sample buffer saturated with dissolved biotin was transferred to a new tube. Hundred microliter of elution buffer was applied to Dynabeads, vortexed gently, heated for 5\u00a0min at 90 \u00b0C, before the vortexing and heating steps were repeated. Dynabeads were separated magnetically and elution buffer containing biotinylated proteins was transferred to a new 1.5\u00a0ml LoBind protein tube (Eppendorf). Seventy microliter of sample was electrophoresed on a NuPAGE 4\u201312% Bis-Tris protein gel (Invitrogen), which was then stained with InstantBlue (Expedeon) for visualization. The gel was sliced into 20 fractions and sent for mass spectrometry analysis.via a nano-flow electrospray ionization source with a hybrid quadrupole orbitrap mass spectrometer . Data collection was performed in data-dependent acquisition (DDA) mode with an r\u00a0=\u00a070,000 (@ m/z 200) full MS scan from m/z 380\u20131600 with a target AGC value of 1e6 ions followed by 15\u00a0MS/MS scans at r\u00a0=\u00a017,500 (@ m/z 200) at a target AGC value of 1e5 ions. MS/MS scans were collected using a threshold energy of 27 for higher energy collisional dissociation (HCD) and a 30\u00a0s dynamic exclusion was employed to increase depth of coverage. Acquired raw files were then searched in MaxQuant (C.\u00a0elegans reference proteome from UniProt KB (including SwissProt and TrEMBL entries) downloaded on September 27, 2019. In total, 28,474 accessions were actually searched. We used trypsin, which cleaves peptides at the C-terminal side of lysine and arginine residues, to generate peptide fragments for mass spectrometry. Most parameters were kept at their default value. Carbamidomethyl (C) (+57.0214637236) was set as fixed modification. Variable modifications included were \u201cOxidation (M)\u201d (+15.9949146221), \u201cAcetyl (Protein N-term)\u201d (+42.0105646863), \u201cDeamidation (NQ)\u201d (+0.9840155848), \u201cGln->pyro-Glu\u201d (-17.0265491015), \u201cPhospho (STY)\u201d (+79.9663304084), as well as two custom modifications to account for TurboID-induced biotinylated peptides. Match Between Runs and Second Peptide Search were activated. All FDRs were set to 1%. Identified data was then reprocessed in R using the evidence.txt results table, but a decision was made to exclude indirect, MBR-based identifications to focus on higher confidence hits. The decoy used to establish the FDR was the reverted search database. MaxQuant applies a PSM-level FDR globally as well as per class of modified peptides (\"Site decoy fraction), and also at protein level. All three FDR levels were set to 1%.Polyacrylamide gel slices (1\u20132\u00a0mm) containing the purified proteins were prepared for mass spectrometric analysis using the Janus liquid handling system . Briefly, the excised protein gel pieces were placed in a well of a 96-well microtitre plate, destained with 50% v/v acetonitrile and 50\u00a0mM ammonium bicarbonate, reduced with 10\u00a0mM DTT, and alkylated with 55\u00a0mM iodoacetamide. After alkylation, proteins were digested with 6\u00a0ng/\u03bcl Trypsin overnight at 37 \u00b0C. The resulting peptides were extracted in 2% v/v formic acid, 2% v/v acetonitrile. The digest was analyzed by nano-scale capillary LC-MS/MS using an Ultimate U3000 HPLC (ThermoScientific Dionex) to deliver a flow of approximately 300\u00a0nl/min. A C18 Acclaim PepMap100 5\u00a0\u03bcm, 100\u00a0\u03bcm\u00a0\u00d7 20\u00a0mm nanoViper (ThermoScientific Dionex), trapped the peptides prior to separation on an EASY-Spray PepMap RSLC 2\u00a0\u03bcm, 100\u00a0\u00c5, 75\u00a0\u03bcm\u00a0\u00d7 250\u00a0mm nanoViper column (ThermoScientific Dionex). Peptides were eluted using a 60-min gradient of acetonitrile (2% to 80%). The analytical column outlet was directly interfaced MaxQuant (versionC.\u00a0elegans, for which there is no requirement for review and approval from an institutional animal care and use committee. Transgenic experiments were carried out following IST guidelines for such work.The work used the free-living nematode via the PRIDE partner repository at http://www.ebi.ac.uk/pride with the dataset identifier PXD027068. Submission details: Project Name: Interactome analysis of C.\u00a0elegans synapses by TurboID-based proximity labeling. Project accession: PXD027068. We have uploaded the mass-labeled MS/MS data on MS Viewer, available at https://msviewer.ucsf.edu/prospector/cgi-bin/msform.cgi?form=msviewer. The key to access the repository is bbfl7oetve. Only \u223c100,000 of the \u223c2.5 million spectra obtained in the course of our work are in the database, since replicates are omitted, and the best spectrum retained for each peptide. All remaining data are contained within the article.The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium This article contains The authors declare that they have no conflicts of interest with the contents of this article."} +{"text": "In the article titled \u201cLong Noncoding RNA TUG1/miR-29c Axis Affects Cell Proliferation, Invasion, and Migration in Human Pancreatic Cancer\u201d [Correct forward sequence should read as follows:5\u2032-gatccGCTTGGCTTCTATTCTGAATCCTTTCAAGAGAAGGATTCAGAATAGAAGCCAAGCTTTTTTG-3\u2032.Correct reverse sequence should read as follows:5\u2032-CAAAAAAGCTTGGCTTCTATTCTGAATCCTTCTCTTGAAAGGATTCAGAATAGAAGCCAAGCG-3\u2032."} +{"text": "KO-heart) die by 9 weeks of age due to extensive cardiomyocyte apoptosis and severe HF, which suggests that fortilin sustains cardiomyocyte viability. The lack of fortilin is also associated with drastic upregulation of p53 target genes in the hearts. The heart-specific deletion of p53 in fortilinKO-heart mice extends their life spans from 9 to 18 weeks by mitigating cardiomyocyte apoptosis. Our data suggest that fortilin is a novel cardiac p53 inhibitor and that its inadequate expression in failing hearts and subsequent overactivation of the p53 apoptosis pathway in cardiomyocytes exacerbates HF.Heart failure (HF) has reached epidemic proportions in developed countries, affecting over 20 million people worldwide. Despite modern medical and device therapies, 60\u201370% of HF patients still die within 5 years of diagnosis as it relentlessly progresses through pervasive apoptotic loss of cardiomyocytes. Although fortilin, a 172-amino-acid anti-p53 molecule, is one of the most expressed proteins in the heart, its precise role there has remained unknown. Also unclear is how cardiomyocytes are protected against apoptosis. Here, we report that failing human hearts express less fortilin than do non-failing hearts. We also found that mice lacking fortilin in the heart (fortilin Heart failure (HF)\u2014a complex clinical syndrome secondary to structural and functional impairments of the heart muscle\u2014has reached epidemic proportions in developed countries, currently affecting over 20 million people worldwide. Despite modern medical and device therapies, 30\u201340% of HF patients die within 1 year and 60\u201370% die within 5 years of diagnosis [Fortilin is a highly conserved, 172-amino-acid, 20\u2009kDa protein that blocks apoptosis \u201310, and To evaluate the role of fortilin in the heart and in human HF, we subjected tissue lysates of human hearts from subjects with non-failing hearts (NFHs) and HF patients with non-ischemic cardiomyopathy (NICM) and ischemic cardiomyopathy (ICM) from the Duke Human Heart Repository to an auflox/flox mice using standard homologous recombination techniques [fortilin KO mice by crossing fortilinflox/flox mice with mice over-expressing Cre-recombinase under the control of the cardiac-specific Myh6 promoter [+/+, Jackson Laboratory) mice as the control. As expected, fortilin was not detectable in the hearts of fortilinKO-heart mice, whereas it was normally expressed in all other tissues at both message Fig. . In cardces Fig. is excisces Fig. , while fces Fig. . We usedage Fig. and protage Fig. levels.KO-heart mice started to die as early as 6 weeks of age and all were dead by 9 weeks of age in fortilinKO-heart mice, but not in fortilinWT-heart mice than in fortilinKOp53WT and fortilinKOp53KO mice (the latter two had comparable expression), both at the message and protein levels. In addition, total p53 expression levels were significantly higher in fortilinKOp53WT than in fortilinKOp53KO mice, both at the message and protein levels . p53 expression levels also were significantly higher in fortilinKOp53WT than in fortilinWTp53WT mice, both at the message and protein levels . On the other hand, phosphorylated p53, an active form of p53, was most abundant in the hearts of fortilinKOp53WT mice, followed by those of fortilinWTp53WT and then by those of fortilinKOp53KO , suggesting that the lack of fortilin increases not only total p53 protein but also phosphorylated and activated p53 protein.To test the hypothesis that the lack of fortilin causes the heart to fail through overactivation of the p53 apoptotic pathway, we knocked out \ufeffp53 by crossing fortilinlox mice and geneice Fig. and S2b.protein levels of total p53 to be significantly higher in the heart of fortilinKOp53WT mice than in those of fortilinWTp53WT mice as we observed above. However, this process does not explain why the message levels of p53 were also significantly higher in the hearts of fortilinKOp53WT mice than in those of fortilinWTp53WT mice .Because fortilin binds p53 and facilitates its proteasome-mediated degradation at the protein level , it is ep53-pomoter-GLuc/SEAP cells). We then transiently silenced fortilin by treating the cells with lentiviral vector containing shRNA against fortilin (sh-fortilin). These cells were irradiated with a moderate X-ray dose (8\u2009Gy) and subjected to the GLuc-SEAP assay and the secreted alkaline phosphatase (SEAP) gene under the control of the constitutional CMV promoter . Echocardiography showed that the hearts of fortilinKOp53KO mice had better overall heart function . These data suggest that fortilin sustains heart function by negatively regulating p53 and that the lack of fortilin leads to inappropriate overactivation of the p53 pathway, apoptosis and loss of cardiomyocytes, dilated cardiomyopathy, HF, and death.Immunohistochemical staining of myocardium showed that the lack of p53 drastically reduced the expression levels of both BAX and cleaved lamin, which are apoptosis markers Fig. . ConsistKO-heart mice increased their survival by about 10 weeks .Although the deletion of p53 in the heart of fortilineks Fig. , it did ice Fig. . We founway Fig. , a vs. cKO-heart mice, we treated fortilinKOp53KO mice with either KIRA6, a selective IRE1\u03b1 inhibitor [KOp53KO mice to live modestly longer compared to vehicle treatment . At week 10 of treatment (about 18 weeks of age), both body weights and HW/BW ratios of KIRA6-treated fortilinKOp53KO mice were modestly but significantly greater than those of vehicle-treated fortilinKOp53KO mice level and by activation of the p53 promoter by knockdown of fortilin in the cardiomyocyte cell line . Because there are more endothelial cells and fibroblasts combined than cardiomyocytes in the murine hearts [KOp53KO heart. To evaluate the status of activated and functional p53 in the heart, we stained the tissue with anti-phospho-p53 antibody. We found that phosphorylated p53 were significantly less in fortilinKOp53KO mice than that in fortilinWTp53WT mice, suggesting that despite the similar total p53 signals between fortilinKOp53KO and fortilinWTp53WT hearts by both immunohistochemistry and Western blots , functional p53 is significantly less in fortilinKOp53KO hearts than in fortilinWTp53WT hearts.There were p53 signals detectable in the Western blot of the total heart lysates from fortiline hearts \u201332, it iBecause cardiomyocytes are not capable of proliferating in normal conditions, their continuous apoptotic loss leads to irreversible progression of HF. Protecting cardiomyocytes against apoptosis represents a new approach in HF gene therapy, distinct from the ones focusing on calcium metabolism and \u03b2-adrenergic receptor signal transduction . The cursh-fortilin) and random control sequence (lentivirussh-control) were purchased from Sigma . The lentivirussh-fortilin was experimentally shown to silence rat fortilin . Lentiviral particles that contained (a) the murine p53 promoter sequence fused to the GLuc cDNA, (b) the SEAP gene under the constitutional SV40 promoter, and (c) the puromycin resistance gene were obtained from GeneCopoeia . The actual promoter sequence is as follows: GGATCTGTGGCTAGCTGGGGTTGGTCATCACCACCGCATGGCGGAGGCACCGGTTCAAAGTCTGTATTTTTCTCCGCTGGGGAACCTTGGGGTACCGGAGCTGGGGCCAGGTCAGGAGGGAGGCTATCCGGAGCTAAGAGTCGCTCCTCCGACGTCTTCATTCTGTAGAGTAAGCCCCCGGAAGGCAGAGGTCGGGCAAGTCTCGCTGAGCCGGCTACCAGCTGCCGAGGCTAGAGTGCATTACCGTTCCCAGGGATGCTCAGAGACCGGAGTCCGCTTTCCTCTTCCGGAAAATGTAAGCCGAACCTAAAGCAATCACCAGGGAACGAGTGTCCAAAGCCAAGCGCCTAGGGTCGCTAGGCGCCGCCAGGGCTTCTTGCTCTCGCGGGAGTCGGGCCACCTTCCGATAGGCTCTCCGCATCCTCCTCCGATTCCGAGCGGGAAGGCGGGAAGGAACGACTTTGCCTACACCTCAAGCGCTGGAGAATTCCTAGAGGTTTCTGGGAGTTGTAGTCTGAACTCTGGGCCTTGGCGAAAACTACACGAGCGCCCCCTACCGTCCCCTGGGGGTAATTCTTAAAGCGCCTATCCTCCCTGGCCTGCAGAGGGCGCATAATTTCTACAGTTTTTGCCCCTCTTGACTATCTTGTTTTGAATCCCKinase-Inhibiting RNase Attenuator 6 (KIRA6) , an inhiGTAACCTCAGGTTTCCTTTCTCCCCATCTCTCCCCCCTTCTTGTTCCTCTCTTTCCCTTTCTCCCCCGCCCTCCCTTCATTCATTCGACATTTATTATCAAGTTCTTACTGCCTAACCCAGGACTATACAAGGCATTGGGAAAAAAATAGCAATGTTTTCTAGTTCTTAATCTCCATAAAGTTTTCGTTGCTGTGCAATTAAAGGCTGTGAAAACAGTCTTTACAGAGAGTGATAAGGACTGTACAGGAAATTAAACACGGTGGTGCGATACCAAGTATCTCGGAGAACACGTTAGATTGAGATACTATGAAAAGCCTTTCTAAAGTGACATTTTAGCTAATGAGGGGAAAAAGAACTTAGGGGCCCGTGTTGGTTCATCCCTGTACTTGGAAGGCCTAAAGCAGGAAGACGGCCGCGAATTCCAGGCCAGCCTTGGCTACAAAGACTCTGTCTTAAAAATCCAAAAAGATGGCTATGACTATCTAGCTGGATAGGAAAGAGCACAGAGCTCAGAACAGTGGCGGTCCACTTACGATAAAAACTTAATTCTTTCCACTCTTTATACTTGACACAGAGGCAGGAGTCCTCCGAATCGGTTTCCACCCATTTTGCCCTCACAGCTCTATATCTTAGACGACTTTTCACAAAGCGTTCCTGCTGAGGGCAACATCTCAGGGAGAATCCTGACTCTGCAAGTCCCCGCCTCCATTTCTTGCCCTCAACCCACGGAAGGACTTGCCCTTACTTGTTATGGCGACTATCCAGCTTTGTGCCAGGAGTCTCGCGGGGGTTGCTGGGATTGGGACTTTCCCCTCCCACGTGCTCACCCTGGCTAAAGTTCTGTAGCTTCAGTTCATTGGGACCATCCTGGCTGTAGGTAGCGACTACAGTTAGGGGGCAC.\u00ae CRL-1446\u2122) cell line (H9C2 hereafter) was directly obtained from American Type Culture Collection . Cells were maintained in Dulbecco\u2019s modified Eagle\u2019s medium with 10% fetal bovine serum (FBS) at 37\u2009\u00b0C in an atmosphere containing 5% CO2. H9C2 cells stably harboring the lentiviral construct that contained both the p53 promoter sequence driving the expression of GLuc and the SEAP gene under the control of the constitutional SV40 promoter were generated by transducing the cells with Lentivirusp53-promoter-GLuc/SEAP and selecting them with puromycin (H9C2p53-promoter-GLuc/SEAP). They were maintained in puromycin-containing (0.3\u2009\u00b5g/mL) media.MycoFluor\u2122 was used to detect mycoplasma contamination when appropriate. The H9C2(2-1) (ATCCp53-promoter-GLuc/SEAP cells were transiently transduced by either (lentivirussh-fortilin) or (lentivirussh-control) at the multiplicity of infection (MOI) of 1. The next day, cells were irradiated using the RS-2000 X-ray irradiator at 8\u2009Gy. Seventy-two hours after the irradiation, conditioned media were collected and subjected to both SEAP and luciferase assays using the Secrete-Pair Dual Luminescence Assay Kit according to the manufacturer\u2019s instruction (GeneCopoeia). The degree of activation of the p53 promoter was assessed by dividing the luciferase activity units by the SEAP activity units and expressing it in arbitrary units (A.U.).On the first day, H9C2Mice were given standard mouse chow and water ad libitum, maintained on a 12-h light\u2013dark cycle and seen by a scientist daily to observe their behavior and health. Any animal that displayed substantive signs of distress anytime during the protocol, including but not limited to poor grooming, hunched back posture, or a loss in weight exceeding 10% of the weights at the initiation of the experiments, were identified and treated accordingly, including removal from the experiment and euthanasia. For mouse experiments where grouping was based on pharmacological treatments, mice of the same genotype were randomly assigned to treatment and control groups.flox/flox mice, in which the fortilin gene was flanked by the LoxP sequence to allow tissue-specific deletion, were generated using the standard homologous recombination technique described previously: we micro-injected a mutant C57BL6 embryonic stem cell (ESC) line that contained two LoxP sequences flanking all six fortilin exons into C57BL6 blastocysts [flox/flox mice were fully in the C57BL/6J genetic background from the beginning. Fortilinflox/flox mice were then crossed with a transgenic flipase strain to remove the neomycin resistance gene cassette. To generate fortilinKO-heart mice, fortilinfloxflox mice were crossed with C57BL/6J mice overexpressing the Cre transgene under the control of cardiac-specific \u03b1-myosin heavy chain (Myh6) promoter [+/+, Jackson Laboratory, Ann Arbor, ME, Stock # 011038, already backcrossed to C57BL/6J for eight generations by the donating laboratory). Genotyping of fortilinKO-heart and fortilinWT-heart mice was performed on tail-derived genomic DNA using standard PCR-based methods. The presence of the \u03b1MHC-Cre transgene was detected using the following primer sets: 5\u2032-ATGACAGAC AGATCCCTCCTATCTCC-3\u2032 (M1) and 5\u2032-CTCATCACTCGTTGCATCATCGAC-3\u2032 (M2). \u03b1MHC-Cre+/\u2212 mice yielded a 300\u2009bp amplified fragment and \u03b1MHC-Cre\u2212/\u2212 mice yielded no amplicons. To determine the hemi- (\u03b1MHC-Cre+/\u2212) or homo- (\u03b1MHC-Cre+/+) zygosity of the MHC-Cre transgene, RT-qPCR was performed using the primers and probe sets described previously [\u2013\u0394\u0394CT method [Forward primer (oIMR1084): 5\u2019-GCGGTCTGGCAGTAAAAACTATC-3\u2019Reverse primer (oIMR1085): 5\u2019-GTGAAACAGCATTGCTGTCACTT-3\u2019Probe (13593): 5\u2019-FAM-AAACATGCTTCATCGTCGGTCCGG-IBFQ-3\u2019FAM, 6-carboyfluorescein; IBFQ, Iowa Black\u2122 double-quenched probe (Integrated Data Technologies (IDT), Coralville, IA)\u03b1MHC-Cre-transgene:Forward primer (oIMR1544): 5\u2019-CACGTGGGCTCCAGCATT-3\u2019Reverse primer (oIMR3580): 5\u2019-TCACCAGTCATTTCTGCCTTTG-3\u2019Probe (TmoIMR0105): 5\u2019-JOE-CCAATGGTCGGGCACTGCTCAA-IBFQ-3\u2019Apob (apolipoprotein B) [The target gene is otein B) .Internal control to calculate \u0394CT for the \u03b1MHC-Cre-transgene:Fortilinstocysts . The respromoter (\u03b1MHC-Creviously (see belT method to deterflox/flox mice yielded a 405\u2009bp fragment and wild-type fortilin mice (fortilinWT/WT mice) yielded a 280\u2009bp fragment.The presence of the LoxP-fortilin-LoxP knock-in construct was verified using the following primer sets: 5\u2032-TGGACCCTGACTTTCATCACCTC-3\u2032 (F1) and 5\u2032- GTCATCTAACCTTACCCCAGTAAGC-3\u2032 (F2): fortilinKO-heart mice were crossed with p53flox/flox mice available from the Jackson Laboratory . The status of the \u03b1MHC-Cre-transgene was evaluated by both PCR and RT-qPCR as described above, and the status of fortilin and p53 loci was determined using the following PCR-based genotyping strategies:Forward primer: 5\u2019-TGGACCCTGACTTTCATCACCTC-3\u2019Reverse primer: 5\u2019-GTCATCTAACCTTACCCCAGTAAGC-3\u2019Expected size of the mutant (floxed fortilin gene) allele: 405\u2009bpExpected size of the wildp-type allele: 280\u2009bpFortilin locus:Forward primer: 5\u2019-GGTTAAACCCAGCTTGACCA-3\u2019Reverse primer: 5\u2019-GGAGGCAGAGACAGTTGGAG-3\u2019Expected size of the mutant (floxed fortilin gene) allele: 390\u2009bpExpected size of the wild-type allele: 270\u2009bpP53 locus:To generate a strain of C57BL/6J mice that lack both fortilin and p53 in the heart, fortilinKOp53KO mice were daily and intraperitoneally injected with either 5\u2009mg/kg of KIRA6 in solution or the same solution without KIRA6 as vehicle (a) for the subsequent 10 weeks for the sacrifice group (N\u2009=\u20095 each) or (b) until death for the survival analysis group (N\u2009=\u20095 each). Mice were examined daily for their behavior and health. Mice in the sacrifice group were weighed and underwent transthoracic echocardiography before they were sacrificed, and their hearts were harvested and processed as described elsewhere in \u201cMethods\u201d section.For the KIRA6 rescue experiment, 8-week-old male fortilin2 and then by 0.5\u20133.0% isoflurane and placed on a heated platform, as described previously [KO-heart mice, the baseline echo was obtained when they were 7 weeks of age.Mouse transthoracic echocardiography was performed using a Vevo 2100 High-Resolution Imaging System on mice anesthetized initially by 4% isoflurane in 1\u2009L/min of Oeviously . We imagAfter being weighed, mice were sacrificed by isoflurane inhalation until effective, followed by exsanguination. Both the heart and lungs were routinely harvested. The liver, kidney, and spleen were harvested, when appropriate, for protein and RNA analyses. The harvested hearts and lungs were rinsed with phosphate-buffered saline (PBS), drained on absorbent paper, and weighed. The ratio of heart weight to body weight (HW/BW ratio) as well as that of lung weight to body weight (LW/BW ratio) was then calculated. For the sagittal cross-sectional analysis of the heart, the entire heart was fixed in 4% paraformaldehyde and subjected to paraffin embedding, sectioning, and hematoxylin and eosin staining according to the standard protocol. The right ventricle and atria were removed from the left ventricle (LV). The LV was then divided into three sections of equal size . The mid-ventricular part of the LV was fixed in 4% paraformaldehyde before being embedded in paraffin for immunohistochemistry. The apical portion of the LV was flash-frozen for subsequent protein extraction, and the basal portion of the LV was placed directly into Tri-Reagent and frozen for subsequent RNA extraction.\u2013\u0394\u0394CT method [\u00ae RT-PCR kit in the ABI Step One Plus Real-Time PCR system and the following primer and probe sets (Integrated DNA Technologies):Fortilin:Forward primer: 5\u2032-TCCGACATCTACAAGATCCGG-3\u2032,Reverse primer: 5\u2032-ATCTTGCCCTCCACCTCCA-3\u2032,Probe: 5\u2032-FAM-AGATCGCGG/ZEN/ACGGGCTGTGC-IBFQ-3\u2032ZEN\u2122\u2009=\u2009an internal quencher to enhance the quenching activity of the 3\u2019 quencher IBFQ (IDT)Mouse Col1:Forward primer: 5\u2032-GAAACCCGAGGTATGCTTGA-3\u2032,Reverse primer: 5\u2032-GTTGGGACAGTCCAGTTCTT-3\u2032,Probe: 5\u2032-FAM-TGTGCGATGACGTGCAATGCAATG-IBFQ-3\u2032Mouse Myh7Forward primer: 5\u2032-CCATCTCTGACAACGCCTATC-3\u2032,Reverse primer: 5\u2032-GGATGACCCTCTTAGTGTTGAC-3\u2032,Probe: 5\u2032-FAM-TCAGTCCATCCTCATCACCGGAGA-IBFQ-3\u2032Mouse ANFForward primer: 5\u2032-TCCGATAGATCTGCCCTCTT-3\u2032,Reverse primer: 5\u2032-CTCCAATCCTGTCAATCCTACC-3\u2032,Probe: 5\u2032-FAM-AAAGCAAAC/ZEN/TGAGGGCTCTGCTCG-IBFQ-3\u2032Mouse Acta1Forward primer: 5\u2032-CTCCCTGGAGAAGAGCTATGA-3\u2032,Reverse primer: 5\u2032-CGATAAAGGAAGGCTGGAAGAG-3\u2032,Probe: 5\u2032-FAM-CATCGGCAATGAGCGTTTCCGTTG-IBFQ-3\u2032Mouse Serca2Forward primer: 5\u2032-CATCAGTATGACGGGCTTGTAG-3\u2032,Reverse primer: 5\u2032-CTCGGTAGCTTCTCCAACTTTC-3\u2032,Probe: 5\u2032-FAM-AGCCACCATCTGTGCTCTGTGTAA-IBFQ-3\u2032Mouse p53Forward primer: 5\u2032-CAGCTTTGAGGTTCGTGTTTG-3\u2032,Reverse primer: 5\u2032-AGTTCAGGGCAAAGGACTTC-3\u2032,Probe: 5\u2032-FAM-TCTTCTTCT/ZEN/GTACGGCGGTCTCTCC-IBFQ-3\u2032Mouse BaxForward primer: 5\u2032-TTGCTGATGGCAACTTCAACTGGG-3\u2032,Reverse primer: 5\u2032-TGTCCAGCCCATGATGGTTCTGAT-3\u2032,Probe: 5\u2032- FAM-TTTGCTAGC/ZEN/AAACTGGTGCTCAAGGC-IBFQ-3\u2032Mouse PumaForward primer: 5\u2032-ATGGCGGACGACCTCAAC-3\u2032,Reverse primer: 5\u2032-AGTCCCATGAAGAGATTGTACATGAC-3\u2032,Probe: 5\u2032-FAM-AGCAGCATC/ZEN/GACACCGACCCTCAC-IBFQ-3\u2032Mouse NoxaForward primer: 5\u2032-TGCACCGGACATAACTGTGGTTCT-3\u2032,Reverse primer: 5\u2032-TGAGCACACTCGTCCTTCAAGTCT-3\u2032,Probe: 5\u2032-FAM-AAAGAGCAG/ZEN/GATGAGGAGCCCAAGC-IBFQ-3\u2032Mouse 18S rRNAForward primer: 5\u2032- GCCGCTAGAGGTGAAATTCT-3\u2032,Reverse primer: 5\u2032-TCGGAACTACGACGGTATCT-3\u2032,Probe: 5\u2032-JOEN-ACCAGAGCG/ZEN/AAA GCATTTGCCAAG-IBFQ-3\u2032JOEN\u2009=\u20096-carboxy-4\u2032,5\u2032-dichloro-2\u2032,7\u2032-dimethoxyfluoresceinMouse RT-qPCR was performed as described previously . We usedT method to calcuAnti-fortilin Anti-p53 Anti-GAPDH Anti-Bax Anti-PUMA Anti-NOXA Anti-Tropomyosin SDS\u2013PAGE and Western blot analyses were performed as described previously , 37\u201340 oAll antibodies were used with appropriate IRDye680LT- or IRDye800CW-conjugated secondary antibodies . The signal intensities of protein bands were quantified using the Odyssey Infrared Imaging System (LI-COR) and normalized to the signal intensity of the loading control protein (GAPDH or tropomyosin) and expressed in A.U. To quantify p53 expression in response to X-ray irradiation, 0.5% (v/v) TCE was added to a polyacrylamide gel before polymerization. After standard SDS\u2013PAGE, the gel was UV-irradiated on the Bio-Rad ChemiDoc MP Imaging System for 2\u2009min. The image was electronically captured, and the cumulative band densities were calculated to assess loading conditions as previously described . The sigTo evaluate the expression levels of fortilin in human hearts, we obtained de-identified tissue lysates of human hearts from patients with NFHs, non-ischemic cardiomyopathy (NICM), and ischemic cardiomyopathy (ICM) from the Duke Human Heart Repository . An automated capillary-based quantitative Western blot system called JESS\u2122 was used to (a) detect fortilin and (b) visualize total proteins loaded as described previously , 20. ComKOp53KO mice that were treated with either vehicle or KIRA6 were subjected to WES\u2122 (Protein Simple), an automated capillary-based quantitative Western blot system, to detect p-IRE1\u03b1 and glyceraldehyde 3-phosphate dehydrogenase ; the latter served as the loading control. JESS\u2122, but not WES\u2122, can visualize the total proteins loaded for normalization. Compass Software (v3.1) was used to calculate a fortilin expression index by dividing the area under the curve of a fortilin peak by that of a GAPDH peak loaded in the same capillary . The fortilin expression index was expressed in A.U.To quantify phosphorylated IRE1\u03b1, the mouse heart lysates from fortilinCleaved lamin A . The cleavage of lamin is a well-characterized event in apoptosis .BAX p-IRE1\u03b1 p53 phosphorylated p53 Mouse hearts were fixed in 4% paraformaldehyde and embedded in paraffin before they were sectioned at 5\u2009\u00b5m thickness. Immunohistochemistry of mouse hearts was performed as described previously using thTM DNA Fragmentation Detection Kit following the manufacturer\u2019s instructions. TUNEL-positive cells within about 0.145\u2009mm2 of the ROI were counted, and TUNEL indices were calculated as the number of TUNEL-positive cells per unit area (in mm2). Results are expressed in A.U.Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining of heart tissue was performed on formalin-fixed, paraffin-embedded samples as previously described , 46 usinTo assess the presence of cardiomyocyte hypertrophy, the heart tissue that had been formalin-fixed and paraffin-embedded was sectioned at 5\u2009\u00b5m thickness and stained with laminin . After stained sections were digitally captured using the EasyScan Digital Slide Scanner, cardiomyocyte areas were measured using ImageJ and expressed in A.U. as described previously . At leasThis study was performed in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All experiments involving animals were approved by the Institutional Animal Care and Use Committees of our institution. Human tissue samples used for this study were procured from the Duke Human Heart Repository (DHHR), which is a Duke University Health System Institutional Review Board (DUHS IRB) approved tissue repository. Samples were procured by the DHHR using written informed consent or a waiver of consent for discarded tissues. No HIPAA information was provided with any of the samples used in this study. Human myocardium was acquired from the left ventricular free wall of explanted hearts following cardiac transplantation. Non-failing (NF) left ventricular tissue was acquired from donors whose hearts were not utilized for transplant, thus becoming available for research.N) is indicated in either figures or the main text. The degree of the spread of data was expressed by the standard deviation (\u00b1SD). The difference between the control and study groups was analyzed using unpaired two-tailed Student\u2019s t-test for two groups or one-way analysis of variance (ANOVA) followed by Fisher\u2019s pairwise multiple comparisons for multiple groups. P\u2009<\u20090.05 was considered to be statistically significant. For survival analyses, Kaplan\u2013Meier survival curves were generated, and the Log-Rank (Mantel\u2013Cox) test was used to compare the curves. The numbers of mice used in in vivo experiments were determined by (i) power analysis, assuming an \u03b1 error rate of 0.05, a \u03b2 error rate of 0.20, and an expected difference of 25%, in Minitab 17 or (ii) our previous dataset and experience from similar experiments performed as part of past research. A similar variance was observed between the groups that were statistically compared. No data were excluded unless outliers were identified and verified by the outlier tests (Minitab 17). Although the scientists were not blinded to allocation during experiments and readouts evaluation, all readouts from the experiments were predetermined, highly objective, and obtained according to the validated protocols.All measurements were taken from distinct samples and the size of biological samples (SUPPLEMENTAL MATERIAL"} +{"text": "Surveying transcriptome-wide Igf2bp2 client mRNAs in white adipocytes reveals selective binding to mRNAs encoding adipogenic regulators and energy expenditure effectors, including adiponectin. These same target proteins are downregulated when either Igf2bp2 or lncRAP2 is downregulated, hindering adipocyte lipolysis. Proteomics and ribosome profiling show this occurs predominantly through mRNA accumulation, as lncRAP2-Igf2bp2 complex binding does not impact translation efficiency. Phenome-wide association studies reveal specific associations of genetic variants within both lncRAP2 and Igf2bp2 with body mass and type 2 diabetes, and both lncRAP2 and Igf2bp2 are suppressed in adipose depots of obese and diabetic individuals. Thus, the lncRAP2-Igf2bp2 complex potentiates adipose development and energy expenditure and is associated with susceptibility to obesity-linked diabetes.lncRAP2 is a conserved cytoplasmic lncRNA enriched in adipose tissue and required for adipogenesis. Using purification and \u2022lncRAP2 is a cytosolic lncRNA conserved in mouse and human needed for adipogenesis\u2022lncRAP2 complexes with mRNA stability and translation regulators, including Igf2bp2\u2022lncRAP2-Igf2bp2 stabilizes lipid metabolism mRNAs to potentiate energy expenditure\u2022lncRAP2-Igf2bp2 genetic and expression variation is linked to BMI, type 2 diabetes Biological sciences; Molecular physiology; Molecular biology; Omics Previous studies revealed that many lncRNAs are specifically enriched in white and/or brown adipocytes and play vital roles in adipocyte biology , includiin situ hybridization (smFISH) visually determines RNA abundance and location, revealing whether a lncRNA diffuses to trans sites beyond its chromosomal locus or remains tethered in cis in the nucleus (in\u00a0vivo (rather than in solution after cell lysis) . And fool lysis) .Here, we use these and other tools to interrogate the mode of action of lncRAP2, a white adipocyte-selective RNA that is essential for adipogenesis . We showin situ hybridization (smFISH) reveals that lncRAP2 diffuses from the nucleus, spreading throughout the cytoplasm at 14\u00a0\u00b1 3 transcripts per white and 9\u00a0\u00b1 3 transcripts per brown adipocyte in 3T3-Lvia posttranscriptional control of metabolically important proteins, impacting sensitivity to diet-induced obesity and type 2 diabetes risk (Igf2bp2 with those of depleting Ppar\u03b3 or linc-ADAL (lincRNA for adipogenesis and lipogenesis) in mature white adipocytes D and S3Fipocytes , including 138 whose mRNAs were found to be direct Igf2bp2 targets by RIP-seq. For both direct Igf2bp2 targets and non-targets, the changes in protein levels occurring upon lncRAP2 depletion closely correspond to those occurring upon t test) F. These depleted A, such adepleted G. By conIgf2bp2 depletion in mature adipocytes for 1,205 genes quantifiable by both RNA-seq and mass spectrometry. The correlation between the two types of responses was strong for direct Igf2bp2 targets with differentially regulated protein vs. non-targets, both after lncRAP2 depletion and after Igf2bp2 depletion I and S4B t test) . We founepletion J. In sum2 \u2265 0.8) with variants within lncRAP2, but not so with any other locus within 500kb, including the IDE gene associated with increased body mass index, higher fasting insulin levels, and insulin resistance in women with polycystic ovary syndrome in a Chinese population maps to IDE gene A and S5AIDE gene A, insulaIDE gene B.Figure\u00a0\u22128). Additional genetic variants mapping to the lncRAP2 transcription start and end sites and gene body (rs967878) also represent risk alleles for type 2 diabetes (p\u00a0< 109 to 10\u22128), thus evidencing association of multiple lncRAP2 alleles with diabetes (Igf2bp2 levels decrease as body mass index increases (Igf2bp2 trends lower in adipocytes of leptin-deficient (ob/ob) and leptin receptor-deficient (db/db) obese mice (The pathogenesis of type 2 diabetes can involve obesity-linked adipose dysfunction . To expl t test) E. Human ncreases D and S5Encreases F. We alsese mice F, which ese mice . We spec, ttest) G. Thus, Obesity has become pandemic , increasOur data show that multiple genetic variants associated with propensity to type 2 diabetes are confined to an lncRAP2 linkage disequilibrium and chromatin interaction domain. Analyzing hundreds of phenotypes interrogated by genome-wide association studies reveals that these variants, mapping to the lncRAP2 gene body and transcription start and end sites, are specifically associated with body fat mass, insulin secretion, and type 2 diabetes, implicating lncRAP2 alleles in the development of obesity and diabetes. Supporting this notion, lncRAP2 is suppressed in the white fat of mice and humans consuming a high-fat diet, and in humans lncRAP2 levels decrease progressively with increasing obesity, whereas lncRAP2 levels are restored upon weight loss.lncRAP2 is enriched in white relative to brown adipocytes and is critical for adipogenesis . AlthougIgf2bp2 levels in pancreatic islets .Further information and requests for resources and reagents should be directed to the lead contact, Marko Knoll (This study did not generate new unique reagents.C57BL/6J mice were bred in house or purchased from Jackson Laboratories (stock # 000664). All mice were housed under a 12\u00a0h light/dark cycle at constant temperature (20\u00b0C). All procedures were performed according to protocols approved by the Committee on Animal Care at the Massachusetts Institute of Technology.2 asphyxiation and interscapular brown adipose tissue (BAT) and subcutaneous white adipose tissue (scWAT) was harvested into room temperature plain DMEM . The fat pads were transferred into a well of a 6 well plate and minced with scissors for 5\u00a0min. Minced tissues were then transferred into a 50\u00a0mL conical tube with 3\u00a0mL Hank's balanced salt solution supplemented with 0.2% collagenase A and 2% BSA using a 1\u00a0mL pipet tip with the tip cut off to allow aspiration of larger pieces. The tissues were incubated agitating (350rpm) and repeated vortexing every 5\u00a0min for 10\u00a0s at 37\u00b0C for 30\u00a0min or 20\u00a0min for scWAT. Following collagenase digestion, 10\u00a0mL room temperature plain DMEM was added and cells were filtered through a 70\u00a0\u03bcm mesh filter . Mature adipocytes and the stromal vascular fraction (SVF) were separated by centrifugation at 700\u00a0g for 5\u00a0min. The supernatant was removed and the SVF resuspended in 10\u00a0mL room temperature plain DMEM followed by additional filtering through a 30\u00a0\u03bcm mesh filter (Miltenyi Biotec # 130-041-407) and subsequent centrifugation at 700\u00a0g for 5\u00a0min. The SVF from subcutaneous white fat pads (scWAT) of 8 mice were then resuspended in 10\u00a0mL DMEM supplemented with 10% heat inactivated new born calf serum and plated on two 10\u00a0cm dishes . The SVF from interscapular brown fat pads of 6\u20138 mice were then resuspended in 6\u00a0mL DMEM supplemented with 10% HI NCBS and plated on 3 wells of a six well plate . After 4 and 24 h, the medium was replaced by fresh, pre-warmed DMEM/10% HI NBCS at 37\u00b0C and with 5% CO2. Cells were grown to confluence and then passaged no more than two times before seeding the pre-adipocytes for differentiation.6\u20138 male 2\u20134\u00a0week old mice were sacrificed by COPre-adipocytes derived from BAT were cultured to confluence and then subsequently overgrown for 4\u20136 additional days until growth arrested. The cells were then induced to differentiate by culturing them for two days in induction medium consisting of DMEM supplemented with 10% fetal bovine serum and 850\u00a0nM insulin (Sigma-Aldrich #I1882), 0.5\u00a0\u03bcM dexamethasone (Sigma-Aldrich #D4902), 250\u00a0\u03bcM 3-isobutyl-1-methylxanthine , 1\u00a0\u03bcM rosiglitazone , 1\u00a0nM 3,3,5-triiodo-L-thyronine and 125\u00a0nM indomethacin (Sigma-Aldrich #I7378). Subsequently, the induction medium was replaced with DMEM supplemented with 10% FBS and 160\u00a0nM insulin and 1\u00a0nM T3 for another two days. The cells were then cultured in DMEM 10% FBS and 1\u00a0nM T3 until day 8 of differentiation, and the medium was replaced every other day. Pre-adipocytes from scWAT were cultured similar but the induction medium and following medium did not contain T3.Mature adipocyte cell layers were washed twice in plain pre-warmed DMEM and stimulated with 1\u00a0\u03bcM isoproterenol (Sigma-Aldrich I6504). After 6\u00a0h of stimulation, the cells were washed once with cold PBS and RNA was harvested using TRizol or QIAzol lysis reagent as described below. For immunoblotting, the cultures were harvested after stimulation with isoproterenol after washing with cold PBS on ice and adding 40\u00a0\u03bcL RIPA buffer per well of a 6 well plate or 100\u00a0\u03bcL RIPA buffer to a 10\u00a0cm dish.Was performed as described . BrieflyFollowing addition of fresh medium, cells were stimulated with isoproterenol. Cell culture medium was collected after 24h of stimulation and stored at\u221220\u00b0C. Glycerol release was measured using the Adipolysis Assay Kit following the instructions of the manufacturer.Total RNA was isolated from tissues or cells using TRizol or QIazol reagent (LifeTechnologies/Ambion) and a miRNAeasy kit (Qiagen). 300\u00a0ng were reverse transcribed using Superscript II reverse transcriptase (LifeTechnologies/Invitrogen) using random hexamers (LifeTechnologies/Invitrogen). The cDNA was diluted 1:10 and 2.5\u00a0\u03bcL for a 96 well plate or 1\u00a0\u03bcL for a 384 well plate were used for quantitative Real-time PCR. qPCR was carried out on an ABI7900HT Fast real-time PCR system (Applied Biosystems) and analyzed using the delta delta Ct method normalized to 18S if not stated otherwise. Results are shown as pooled data from 3\u20134 independent experiments displaying the average and SEM.Poly A+ RNA sequencing (TrueSeqStrandedPolyA) was performed on RNA samples using a Nextseq genome sequencer (Illumina). RNA-seq paired-end reads were aligned to the mouse genome using STAR v2.6.1 providedLysates were centrifuged for 10\u00a0min at 13,000\u00a0g to remove debris, and NuPAGE sample buffer and reducing buffer (LifeTechnologies) were added after measuring and adjusting the samples for protein concentration . 2\u201320\u00a0\u03bcg protein per sample were separated for 2\u20134\u00a0h at 60\u2013100\u00a0V using 8% 26 well NuPAGE Bis-Tris Midi gels in MOPS or MES buffer (LifeTechnologies) and in Criterion cells (Bio-Rad) using respective adapters (LifeTechnologies). Protein was wet transferred to polyvinylidene fluoride (PVDF) membranes in Criterion blotter cells (Bio-Rad) using 2 x NuPAGE Transfer buffer (LifeTechnologies) with 10% methanol for 25\u00a0min at 1 A. After blocking the membrane in filtered TBS-T with 3% BSA for 1 h, blots were incubated in primary antibody diluted in TBS-T 3% BSA sealed in hybridization bags and gently shaking at 4\u00b0C overnight, then washed three times for 10\u00a0min in TBS-T, incubated in secondary antibody diluted in TBS-T 3% BSA gently shaking for one hour at room temperature, and then washed again three times for 10\u00a0min in TBS-T. Antibody binding was visualized using ECL Plus Western Lightning reagent (PerkinElmer NEL 102) and blots were exposed to film . Films were scanned without adjustments using an Epson scanner. All immunoblotting data shown were reproduced with almost identical results in at least one and typically two to three additional independent experiments.Cells were washed in PBS and fixed in 3.7% formaldehyde solution for 1 h, followed by staining with Oil Red O for 1 h. Oil Red O was prepared by diluting a stock solution (0.5\u00a0g of Oil Red O (Sigma) in 100\u00a0mL of isopropanol) with water (6:4) followed by filtration. After staining, plates were washed twice in water and photographed.The 5\u2032 and-3\u2032 ends of lncRAP2 was determined using the FirstChoice RLM-Race Kit from Ambion following the manufacturers instructions. Primers were designed accordingly and can be found in To separate the nuclear from the cytoplasmic fraction differentiated 3T3-L1 adipocytes were harvested and 1 million cells were used to isolated the fractions using the PARIS kit (Life technologies) according to the manufacturers instructions. Separated fractions were then analyzed using Real-time PCR and gene specific primers.Single molecule RNA FISH and fluorescence microscopy were described previously . Brieflyhttp://hannonlab.cshl.edu/fastx_toolkit/index.html), discarding non-clipped reads or reads <25nt after linker clipping (\u201c-l 25 -c\u201d parameters), andRibosome profiling sequencing reads were clipped to remove 3\u2032 linkers using fastx_clipper from the FASTX-Toolkit using STAR v2.6.1 with default parameters and \u201c--seedSearchStartLmax 20 --sjdbOverhang 39 --outSJfilterOverhangMin 30 8 8 8 --sjdbScore 2\u201d. To measure gene-level and region-level expression from uniquely-aligned reads, we quantified read counts using HTseq-count. Only genes for which a read count could be obtained in each sample and replicate were retained, and counts were then normalized as counts per million mapped reads (cpm) using only reads from these genes.Gene-level translation efficiency was calculated as the ratio of normalized Ribo-seq read density (cpm) to the normalized RNA-seq read density (cpm).We computed the ribosome release score using the RRS program default. For mRN7 re-suspended in 2mL 1X PBS, then lysed in nuclear isolation buffer for 20\u00a0min. Nuclei were pelleted by centrifugation at 2,500\u00a0g for 15\u00a0min. Supernatant was discarded and nuclei were re-supended in 1mL RIP buffer containing the HALT protease and phosphatase inhibitor (Thermo scientific) and split into two fractions and mechanically sheared using a Dounce homogenizer with 20 strokes. Nuclear membrane and debris were pelleted by centrifugation at 13,000\u00a0rpm for 10\u00a0min at 4\u00b0C. The supernatant was pre-cleared by adding 30\u03bcL slurry of protein A/G beads and incubation for 2\u00a0h at 4\u00b0C on a rotator. Beads were removed by centrifugation at 2500\u00a0g for 1\u00a0min and 10% of the supernatant was removed to a new tube (10% input) and the rest was incubated with antibodies to Suz12 , IgG , Ezh2 , hnRNPU , CoREST or no antibody for 3h at 4\u00b0C on a rotator. Then 60\u03bcL slurry of protein A/G beads were added for 2h at 4\u00b0C on a rotator. Beads were pelleted by centrifugation at 2500\u00a0rpm for 30s and washed 3 times in 500\u03bcL RIP for 10\u00a0min each followed by one wash with 1X PBS. For the isolation of RNA, the beads were re-suspended in TRIzol after the last wash step and isolated according to the manufacturers instructions. The RNA pellet was re-suspended in 10\u03bcL dH2O and was directly used for reverse transcription using random hexamers and SuperScript II (Invitrogen). Analysis was done by real-time PCR and sequencing.RNA coimmunprecipitation was done as described . Mature RNA FISH probes were biotinylated using Biotin-XX, SSE (Thermo Fisher). As controls, we also used probes against H19 and BlooRaw reads (40bp unpaired and non-strand-specific) were mapped to mm9 using bowtie2 . Peak caChIRP-MS was performed as described with theMass spectrometry fragmentation spectra were correlated against a custom peptide database, formed by downloading all RefSeq species-specific (mm9) entries, and against a database of common contaminants using Mascot (Matrix Science) 2.5.1 and the Sequest algorithm (Thermo). The resulting Mascot search results were uploaded into Scaffold (Proteome Software), and a minimum of two peptides and peptide threshold of 95% and protein threshold of 99% were used for identification of peptides and positive protein identifications. Proteins enriched in ChIRP-MS experiments were identified based on several criteria. First, only proteins identified by 2 or more unique peptides in the differentiated sample were considered. Then proteins whose total peptide count was at least 10-fold greater in the exon probes versus intron probes were considered enriched. Finally, proteins introduced during sample preparation and purification were excluded from further analysis. To distinguish lncRAP2-specific interactors from proteins that generally bind RNA during its processing, we compared the proteins enriched by lncRAP2 with those enriched by Dleu2 , Neat1 aAfter proteins were separated on an Acryl-amid gel (see Western blot), the gel was fixed in 40% EtOH with 10% HAc for 1\u00a0h then washed 2\u00a0\u00d7 20\u00a0min in 30% EtOH and once for 20\u00a0min in water. The gel was sensitized for 1\u00a0min in 0.02% Na2S2O3 following 3\u00a0\u00d7 20\u00a0s wash in water and 20\u00a0min incubation in cold (4\u00b0C) 0.1% AgNO3 solution. The gel was washed 3\u00a0\u00d7 20\u00a0s in water and developed in a new chamber in 3% Na2CO3 with 0.05% formaldehyde. As soon as bands became visible the reaction was stopped by a short wash in water and then incubation in 5% HAc. All lanes were cut out and processed .Gene lists were analyzed for enrichment of genes grouped by biological process ontology or by curated annotations from the Molecular signatures database with GSEA using deGenomic sequences from regions of interest (e.g. UTRs) were searched for matches to a database of TF recognition sites for TFs Processed ChIA-PET, Hi-C, and capture Hi-C datasets were downloaded and visualized in triangle heatmap mode using the UCSC genome browser or in arc mode using the Washington University Epigenome browser, with default normalization and resolution settings and fixed display values.http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Genome-wide read density maps were generated by MACS2 using the \u201c--bdg\" option, normalized by RSeQC (http://CRAN.R-project.org/package=gplots).All sequencing reads were quality-checked with FastQC was assessed by a Fisher's exact test using the fisher.test R implementation with default parameters. For unpaired data, a Shapiro-Wilk normality test was first performed using the shapiro.test R implementation with default parameters; for normally distributed data we then used a two-sided ttest (t.test R implementation with default parameters) to assess confidence on the measured difference of their mean values. For unpaired data that don't follow a normal distribution, we used a non-parametric Wilcoxon rank-sum test to determine if they belong to the same distribution. Variance was represented as mean\u00a0\u00b1 SEM of n\u00a0= 3 replicates unless otherwise specified.>hg19_chr10: 94,176,182-94,180,397ATCCGTTACCCAACGGCTGTCGAAAGAGGAGCACGACGTGAACTTCCACCGACAATCGTTTAAATACTGCAGGCGAAGGACGGGTTCTTATTGTTCCTTCAGGCAAACCGGCAGCTGAGTCCAGCTTGTGAACTTGAACTTGAACTTGCTGAAGAAGCTCCCGGCGGCCCTCTGCTGGCCGCCGCCTTTGCCAGAGGGAGAGGCTGGAGTCACCCTGTTGGAACCCATTTCTGGGGCTGGCACCTGTTGGGCTGCCCGGCCGCGCGTACCTGGTCCCATCGGGGGCTCTGCCCACTCCGCTGATGACGCGGGTAGAAGGGAGGCCGCAGGGACACTCTGGGGGGACTGTGCCGGGCGGGCACCCCCCAGCTGCTCACTGTGGGGTGCGGCACCGAGGCCTGGTTGGGCTGCAAGGAGACCGACTGGGATTCCCGGGCTGGTGGCCGGGGAGACGGGGTAGAGGTGAGAAGCAAGAGCTCAGGAGGCCTCAGGCCCCAGCGCTGTGGGGCTGCCGTTGTCGTTCTGGGTGGAGGTCTGGCCAAACCGGCTTTTGCCCCGAGTGAGGAATTCCTGCTCATTTTGGTGTTAGTGGAGAGGTCGCTGTTCACAGCGGGGGTGGGGTTCGTCCCCTCCAGGCTAGTAGGGAGCTGGCTGGTGCATTGCTGTGTTGCTGCCCTTCTGCCCTGTCTCCTGATCCTGTTCAGACTCTGGGTGGCTCCTTTGAGTCTTCTCAGGCCGTGAAAGATGGGAAAGGCAGGTTGTTGAAACCACTGCTGCTCCGGGCTGGCAGAGACCCTGAGCTGTACCTCCTGGGATGAAAGGAGAACCCAGGAGCAGGCAGGTGACCACTGCTTCCTCATACTCCTCAGCAAGTCCTGGATCTCTTCCTGTCTGTAACCCACGGATACCATGAACTTAGTTTGCAGGGGGTCCTCCCAGTGGGAGAGTGGCTGCACACAGCGCTTAAGTTCCTCACCTTCATGACTCGCATTCATCCATGGACCCCTCACAGTGTGCTCTGAAGGGCCTCTCTTCCTGCAATTGACAATGAGAAATTTCTTCAGCAAGTTTTCACTCTCCGGGGACATGGGGGAGGGAATGGAATACATCCTGCTCAGTACCTGCTCCTATAGCTCCTTGAAGTTCTGTCCATCGAAAAATAAGGATCCATTTACCAGTATATAGAGGATAACTCGCAGACGCCATACATCCACCAGCTCATATTTGTGGCCCTAGAAGAATTCCAGGGCAGCCTAAGGGGACTGCCACAAAAGGTATCCAGCTTGTTGCGAAAGGCGAATTTATTCCTGAACCCAATCTGTGATGTTCATGTTAACATGGACAATACATTTCTGGTGACAATACACAATATCTGTGGACAATGCACTTCTGGTAACAGCTTGCATAGCAGACACTTTTGGTGGAATTTGCTTCAGGCCTCTTTCCTGCTGCTGTGAGCAGGTATTCACAAACTTCTCGCTAGCGTGCTTTGTGATGAGGCAGAGATTCCTCACTATCGATCACAGCAAATACTTCACTATGTTGGGGTGATCTGAGCCTTCATGATTCCTACTTTGTGGATGGTGTCTGGAGGCTGGAGGAGATCTGCTGAGTCTCATCAATGAACTTCACGGCGGCTGCTTTCCCAGTCAGGATGTCCCAGGCCAACCCCACCTTAGCCAGGTTGTCCTTGACGATGGTCTCAAGGATCCTCGCTGCCAATAAGGATCCCTCCTCAGCAGAGATGGCTGAGAGGCCCTGCGGCGTGTGGGACTTACTACTGGGCTTGGAGTCAAGGTGTCCCAGGTCGGCTTAATTTTGGGAAAAGCTGGTGAGAAGCTTGGTCCAAATCACCTGAAAAGAAAATCACTTGCATAGTTTACCTGAAGAACGGATTTCTATAATGGAATCAAAACACAATACTGGACAAAAATCAAATAAGCAAAAGCAACAAACTGAAAAGGAAATGCTGAGTAAAAAAGAACTTAAAAATTAGAAAAATAAGAGAAAAAAGAACAAGAAGAACCAGTAACCTCAGCCAAGCCAGGGACTTACATACGTGCTTGGAATTCCAAAGCAGCAGTTCCTTATGAGGAAGAACTAAGCCTAGTAACAAGGCTGAGGATAATCTATGTGGCTTTCTCATGCTTTGGTCTCAAGAACTCTTTACTCTTAAAGAAAATATATTGAGGACCACAAAGAGGATTTTTTATTGATATGGGTTACAGTTATGAATATTTACCTTATTAGAAATTAAAACCTCTAGGATGCTTCAATGGCCTTTTCTAGTTTGAAAAGATAACAGGCTGGGTGTGGTGGCTCACGCCTGTAATTCCAGCACTTTGGGAGGCCGAGGTGGGCAAATCACCTGAGCTCGGGAGTTCGAGAAAAGGTATAAAAATGTTTGGCTTTTAAAGAGCCCACAATATCTACACTTAAAATATTTCATTTTTTTCTTTAAACTCTAAATGATTGGTTTCAAAATGATGCCACAACTTAGCTGGCATTATGATAGTGTATAAGTATGTTCTGTTGTGTACGACACAATGAGCTTCATTATTACCATCTGCGACACTGAGGGGAAGATAGCTTTCATCATATTTTCTCATTTAAAGTTTTGCCCAGTTTCATTTGCATAGATTCCCTTTTTCCATGAGCTGCTATGTCAGTCTCAGCATCTTTCAATGTAGAGTTTGCAGCTATGAGTTGAGAAAGCACATTTTCTACTCTTTTTAAGTGAATAATCCACTGTGCCTGGTGTACCTCTCCTTCAGCATAGGATAGGGACATCCAGGTACTGGACCCGTCACTGGCACCTCAGTGGGGAGAACCCAGATGCCCCTACATGATGTTTAAAGATGCTTTATATACATAAAAGTGCACAAATCATCAGCCCACAGCTTGGTGACTGTTCACATATTGAACTCATCTATTTATCTAGTATCCAGGTCAAGAAACAGCCATTACAGCCCCCCAAGATCCCACACCCCTTTTCCAGTCACTTTCTCTGCAGTGATAACCACTCTTCTGTATTTTGACAGCATAGATTCATTTTGCTTATTTTTGAACTTTACATACATGGATTCATACAGTATTGGATCCTTTGTGTCTGCTTCCTTTGCTTAATTTGTTTTTGTTTGTTTGTTTGTTTGTTTGTTTTTCTTGAGACAGAGTCTTGCTCTTGTTGCCCAGGCTGGAGTGCAATGGCACGATCTCAGATCACTGCAACCTCCACCTCCTGGGTTCAAGCAATTCTCCTGCCTCAGCCTCCCAAGTAGCTGGGATTATAGGAGCTTGCCACCATGCCTGGCTAATTTTTGTATTTTTAGTTGAGACGGGGTTTCACCATGTTGGCCAGGCTGGTCTCGAACTCCTTACCTCATGTTCCGCCTGCCTCAGCCTCCCAAAGTGCTGGAATTACAGGCATGAACAACCACGCCTGGTCCTTTGCTCAATATTTTTGTGAGATCCATCCATATTGTTTATTATTCTAAATGCTCATTGTGTGACTGTAACACAATTTGTTAATTTGTTTATTCATTTTACTGTTACTGGGCAGTTGAGTAGTTCTCAGTTTTCAGATGCTATAGTGCTGCCATAAACATTCTTGTTCAGGTTTTTGGGGGACATATATATGGCTTTCTGTTGGATATATATAAATATATTAAGGGTGTGGCTGAACAACCATTTGACAGTTTATGCTAACAAGGTGACTCGTGGTAGGCCCCTTAGGCCAGGTGATATCAGCCTGACCTCCAGAGAGTGGGGTGGGGGCTGGAGACTGAGTTCAACCACATGGACAATAAGTCTATCATGTAATGAAGCCCCAGTAAAAACTCTGGATGCTGAAGCTCAGGTGAGTGTCCCTGATTGGCAGTACTCTATATGTGTTGTCTCACACATCCAAATCAGCAGGGTAATGCATTCTGAGGACCCCAGAGGCTTCACATTTGGAACCCTCTCAGACTCTGCTCTATCAATCTCTTTCTTTGGCTAATTTTGATCTCTATCCTTTCCCTGAAATAAACTGTAACTGTGAGTATAACAG."} +{"text": "Messenger RNA (mRNA) comprises three major parts: a 5'-UTR (UnTranslated Region), a coding region, and a 3'-UTR. The 3'-UTR contains signal sequences involved in polyadenylation, degradation and localization/stabilization processes. Some sequences in the 3'-UTR are involved in the localization of mRNAs in (e.g.) neurons, epithelial cells, oocytes and early embryos, but such localization has been most thoroughly studied in neurons. Neuronal polarity is maintained by the microtubules (MTs) found along both dendrites and axon and is partially influenced by sub-cellular mRNA localization. A widely studied mRNA is that for Tau protein, which is located in the axon hillock and growth cone; its localization depends on the well-characterized cis-acting signal (U-rich region) in the 3'-UTR.We compared the cis-acting signal of Tau with mRNAs in the axonal regions of neurons using the ClustalW program for alignment of sequences and the Mfold program for analysis of secondary structures.We found that at least 3 out of 12 mRNA analyzed have a sequence similar to the cis-acting signal of Tau in the 3'-UTR. This could indicate that these messengers are localized specifically in the axon. The Mfold program showed that these mRNAs have a similar \"bubble\" structure in the putative sequence signal.Hence, we suggest that a U-rich sequence in the 3'-UTR region of the mRNA could act as a signal for its localization in the axon in neuronal cells. Sequences homologous to the DTE sequence of BC1 mRNA could direct the messenger to the dendrites. Messengers with homologues of both types of sequence, e.g. \u03b2-actin, might be located in both dendrites and axon. A messenger RNA (mRNA) comprises three major parts, a 5'-UTR (UnTranslated Region), a coding region and a 3'-UTR. The 3'-UTR contains signal sequences involved in polyadenylation, degradation and localization/stabilization processes. Many studies have shown that certain sequences in the 3'-UTR are involved in localizing the mRNAs in different cells such as neurons, epithelial cells, oocytes and early embryos ,2. Such The mRNA of tau has been studied in detail . Tau is Recently, many mRNAs have been shown to be located in neuronal axons: \u03b2-actin, tropomyosin 3 (Tpm3), cofilin, vimentin, immunoglobulin heavy chain biding protein (Bip), heat shock protein 60 (HSP60), heat shock protein 70 (HSP70), heat shock protein 90 (HSP90), glucose regulated protein (grp75) and synuclein . The objThe 3'-UTR of tau mRNA contains 3884 bases; the U-rich region (in bold) is responsible for the localization of this mRNA in the axon hillock and growth cone.UUUUUUUUUUUUUACUUUAGCGGUUGCCUCCUAGGCCUGACUCCUUCCCAUGUUGAACUGGAGGCAGCCAAGUUAGGUGUCAAUGUCCUGGCAUCAGUAUGAACAGUC AGUAGUCCCAGGGCAGGGCCACACUUCUCCCAUCUUCUGCUUCCACCCCAGCUUGUGAUUGCUAGCCUCCCAGAGCUCAGCCGCCAUUAAGUCCCCAUGCACGUAAUCAGCCCUUCCAUA CCCCAAUUUGGGGAACAUACCCCUUGAUUGAAAUGUUUUCCCUCCAGUCCUAUGGAAGCGGUGCUGCCUGCCUGCUGGAGCAGCCAGCCAUCUCAGAGACGCAGCCCUUUCUCUCCUGUC CGCACCCUGCUGCGCUGUAGUCGGAUUCGUCUGUUUGUCUGGGUUCACCAGAGUGACUAUGAUAGUGAAAAGAAAAAGAAAAAGAAAAAAGAAAAAAGAAAAAAAAAAAAGGACGCAUGU UAUCUUGAAAUAUUUGUCAAAAGGUUGUAGCCCACCGCAGGGAUUGGAGGGCCUGAUAUUCCUUGUCUUCUUCGUGACUUAGGUCCAGGCCGGUCGAGUGCUACCCUGCUGGACAUCCCA UGUUUUGAAGGGUUUCUUCUUCAUCUGGGACCCCUGCAGACACUGGAUUGUGACAUUGGAGGUCUAUACAUUGGCCAAGGCUGAAGCACAGGACCCGUUAGAGGCAGCAGGCUCCGACUG UCAGGGAGAGCUUGUGGCUGGCCUGUUUCUCUGAGUGAAGAUGGUCCUCUCUAAUCACAACUUCAAGUCCCACAGCAGCCCUGGCAGACAUCUAAGAACUCCUGCAUCACAAGAGAAAAG GACACUAGUACCAGCAGGGAGAGCUGUGGCCCUAGAAAUUCCAUGACUCUCCACUACUAUCCGUGGGUCCUUUCCAAGCCUUGCCUCGUCACCAAGGGCUUGGGAUGGACUGCCCCACUG AUGAAAGGGACAUCUUUGGAGACCCCCUUGGUUUCCAAGGCGUCAGCCCCCUGACCUUGCAUGACCUCCUACAGCUGAAGGAUGAGGCCUUUAAAGAUUAGGAACCUCAGGCCCAGGUCG GCCACUUUGGGCUUGGGUACAGUUAGGGACGAUGCGGUAGAAGGAGGUGGCCAACCUUUCCAUAUAAGAGUUCUGUGUGCCCAGAGCUACCCUAUUGUGAGCUCCCCACUGCUGAUGGAC UUUAGCUGUCCUUAGAAGUGAAGAGUCCAACGGAGGAAAAGGAAGUGUGGUUUGAUGGUCUGUGGUCCCUUCAUCAUGGUUACCUGUUGUGGUUUUCUCUGUAUACCCCCAUUUACCCAU CCUGCAGUUCCUGUCCUUGAAUAGGGGUGGGGGUACUCUGCCAUAUCUCUUGUAGGCAGUCAGCCCCCAAGUCAUAGUUUGGAGUGAUCUGGUCAGUGCUAAUAGGCAGUUUACAAGGAA UUCUGGCUUGUUACUUCAGUGAGGACAAUCCCCCAAGGCCCUGGCACCUGUCCUGUCUUUCCAUGGCUCUCCACUGCAGAGCCAAUGUCUUUGGGUGGGCUAGAUAGGGUGUACAAUUUG CCUGGAACCUCCAAGCUCUUAAUCCACUUUAUCAAUAGUUCCAUUUAAAUUGACUUCAAUAUAAGAGUGUAUCCAUUUGAGAUUGCUUGUGUUGUGGGGUAAAGGGGGGAGGAGGAACAU GUUAAGAUAAUUGACAUGGGCAAGGGGAAGUCUUGAAGUGUAGCAGUUAAACCAUCUUGUAGCCCCAUUCAUGAUGUUGACCACUUGCUAGAGAGAAGAGGUGCCAUAAGGCUAGAACCU AGAGGCUUGGCUGUCCACCAACAGGCAGGCUUUUGCAAGGCAGAGGCAGCCAGCUAGGUCCCUGACUUCCCAGCCAGGUGCAGCUCUAAGAACUGCUCUUGCCUGCUGCCUUCUUGUGGU GUCCAGAGCCCACAGCCAAUGCCUCCUCAAAACCCUGGCUUCCUUCCUUCUAAUCCACUGGCACAUCAGCAUCACCUCCGGAUUGACUUCAGAUCCACAGCCUACACUACUAGCAGUGGG UAAGACCACUUCCUUUGUCCUUGUCUGUUCUCCAGAAAAGUGGGCAUGGAGGCGGUGUUAAUAACUAUAGGUCUGUGGCUUUAUGAGCCUUCAAACUUCUCUCUAGCUUCUGAAAGGGUU ACUUUUGGGCAGUAUUGCAGUCUCACCCUCCGAUGGCUGUAGCCUGUGCAGUUGCUGUACUGGGCAUGAUCUCCAGUGCUUGCAAGUCCCAUGAUUUCUUUGGUGUUUUGAGGGUGGGGG GAGGGACAUGAAUCAUCUUAGCUUAGCUUCCUGUCUGUGAAUGUCCAUAUAGUGUACUGUGUUUUAACAAACGAUUUACACUGACUGUUGCUGUACAAGUGAAUUUGGAAAUAAAGUUAU UACUCUGAUUAAACAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAUCAGGCCCCUGGGGCCGUCACUGAUCAUGGAGAGAAGAGAGAGUGAGAGUGUGGAAAAAAAAAAAAAAAGAAUGACCUGGCCCCUCACCCUCUGCCCUCCCCGCUGCUCCUCAUAGACAG GCUGACCAGCUUGUCACCUAACCUGCUUUUGUGGCUCGGGUUUGGCUCGGGACUUCAAAAUCAGUGAUGGGAAAAAGUAAAUUUCAUCUUUCCAAAUUGAUUUGUGGGCUAGUAAUAAAA UAUUUUUAAGGAAGGAAAAAAAAAACACGUAAAACCAUGGCCAAACAAAACCCAACAUUUCCUUGGCAAUUGUUAUUGACCCCGCCCCCCCCUCUGAGUUUUAGAGGGUGAAGGAGGCUU UGGAUAGAGGCUGCUUCUGGGGAUUGGCUGAGGGACUAGGGCAACUAAUUGCCCACAGCCCCAUCUUAGGGGCAUCAGGGACAGCGGCAGACAUGAAAGACUUGGGACUUGGUGUGUUUG UGGAGCCGUAAGGCGUAUGUUAACUUUGUGUGGGUUUGAGGGAGGACUGUGAUAGUGAAGGCUGAGAGAUGGGUGGGCUGGGAGUCAGAGGAGAGAGGUGAGGAAGACAGGUUGGGAGAG GGGGCAUUGCGUCCUUGCCAAGGAGCUUGGGAAGCACAGGUAGCCCUGGCUGCAGCAGUCUUAGCUAGCACAGAUGCCUGCCUGAGAAAGCACAGUGGGGUACAGUGGGUGUGUGUGCCC CUUCUGAAGGGCAGCCCAUGGGAGAAGGGGUAUUGGGCAGAAGGAAGGUAGGCCCCAGAAGGUGGCACCUUGUAGAUUGGUUCUCUGAAGGCUGACCUUGCCAUCCCAGGGCACUGCUCC CACCCUCCAGGAGGAGGUCUGAGCUGAGGAGCUUCCUUUUCGAUCUCACAGGAAAACCUGUGUUACUGAGUUCUGAAGUUUGGAACUACAGCCAUGAUUUUGGCCACCAUACAGACCUGG GACUUUAGGGCUAACCAGUUCUUUGUAAGGACUUGUGCCUCUUGCGGGAACAUCUGCCUGUUCUCAAGCCUGGUCCUCUGGCACUUCUGCAGUGGUGAGGGAUGGGGGUGGUAUUCUGGG AUGUGGGUCCCAGGCCUCCCAUCCCUCGCACAGCCACUGUAUCCCCUCUACCUGUCCUAUCAUGCCCACGUCUGCCACGAGAGCCAGUCACUGCCGUCCGUACAUCACGUCUCACCGUCC UGAGUGCCCAGCCUCCCAAGCCCAAUCCCUGGACCCCUGGGUAGUUAUGGCCAAUCUGCUCUACACUAGGGGUUGGAGUCCAGGGAAGGCAAAGAUUUGGGCCUUGGUCUCUAGUCCUAC GUUGCCAGAAUCCAACCAGUGUGCCUCCCACAAGGAACCUUACAACCUUGUUUGGUUUGCUCCAUCAGGCGUUUGGCGCCAUCGUGGAUGGAGUCCGUGUGUGCCUGGAGAUUACCCUGG ACACCUCUGCThis cis-acting signal of tau was compared base by base with the other afore mentioned mRNAs using simple alignment. We also made comparisons with another sequence that is specific for the localization of BC1 mRNA in dendrites, the Dendritic Target Element (DTE) .In the \u03b2-actin mRNA of chicken a cis-acting signal \"zipcode\" has been described; a zipcode binding protein binds to this sequence and this is a prerequisite for the localization of the mRNA. The sequence is a tandem repeat of an ACACCCACACCC motif. The mRNA of \u03b2-actin has been located in the axon of the neuron and in dendritic spines ,14. \u03b2-acCofilin is a cytoskeleton modulating protein; it is also known as actin depolymerizing factor (ADF). The potential role of cofilin is to modulate the changes of actin organization that accompany neurite initiation, axonogenesis and growth cone guidance . The posVimentin has been located by RT-PCR in the axons of dorsal root ganglia (DRG) neurons. A possible sequence signal in vimentin mRNA shares some U with tau but also contains more purines, which might indicate that the protein is not exclusive to the axonal region .Bip is a protein that binds to the immunoglobin heavy chains in pre-\u03b2 cells. Its mRNA shares some U with the tau sequence; nevertheless, its sequence suggests that this mRNa, like vimentin, is probably not exclusive to the axon .The heat shock proteins and grp75 messengers have similarities with the tau sequence, but once again they are probably not exclusive to the axon. They could interact with other proteins in different parts of the cell.Synuclein is a soluble unfolded protein that can aggregate into insoluble fibrils under several pathological conditions including Parkinson's and Alzheimer's diseases . The posThese analyses carried out by alignment allowed us to show that the cis-acting signals of the mRNAs examined have some homology with that of the tau messenger.The highest homology scores are:The secondary structures of these four mRNAs, which showed the closest homologies to the tau sequence, were analyzed using the program Mfold Figs. , 2, 3, 4The first messenger to be analyzed in the 3'-UTR with respect to its localization/stabilization was tau ,10. The Glucose regulated protein 75 (GRP75) is an important molecular chaperon belonging to the heat shock protein (HSP) family. It is highly expressed in conditions of glucose deprivation of glucose. Its messenger was located in the axon and it has a U-rich region. It might not be confined exclusively to the axon because this protein responds to a metabolic stress .Alfa-synuclein is involved in neurodegenerative diseases and its presence has been observed in the pre-synaptic and nuclear compartments, though the location in the nucleus has not been well documented. The synuclein messenger possesses a U-rich region; nevertheless a C interrupts the potential signal sequence. When it is wrongly folded, this protein may aggregate in the cell forming fibrils, typical of Alzheimer's and Parkinson's diseases. The aggregation of synuclein is similar to tau in Alzheimer patients, which could indicate similar intracellular behavior by both proteins .The 3'-UTR of the cofilin messenger has a U-rich region very similar to the signal sequence of tau, which on the face of it suggests that it might be located exclusively in the axon. Nevertheless, recent studies demonstrate that it participates in the shrinkage of dendritic spines associated with the long-term depression of hippocampal synapses, suggesting that it is also found in dendrites. Moreover, it is involved in neuronal development, axogenesis, guidance of the growth cone and dendrite formation. Although the cofilin messenger is present in axons, the possible participation of the protein in events related to the unplugging of synapses because of its association with actin further suggests that it is not confined to the axon but also occurs in the dendrites .The 3'-UTR of the \u03b2-actin messenger is very short and shows low homology when aligned with the tau cis-acting signal. However, when it was aligned with the dendritic target element, it showed better homology. The \u03b2-actin messenger was shown to possess a zipcode that leads it towards the dendrites instead of the axon .Molecular chaperones and their functions in protein folding have been implicated in several neurodegenerative conditions, including Parkinson's and Huntington's diseases, which are characterized by accumulation of protein aggregates . These aggregates have been shown in various experimental systems to respond to changes in levels of molecular chaperones, suggesting the possibility of therapeutic intervention and a role for chaperones in disease pathogenesis. It remains unclear whether chaperones also play a role in Alzheimer's disease, a neurodegenerative disorder characterized by \u03b2-amyloid and tau protein aggregates. In various cellular models, increased levels of Hsp70 and Hsp90 promote tau solubility and tau binding to microtubules, reduce insoluble tau and cause reduced tau phosphorylation. Conversely, lowered levels of Hsp70 and Hsp90 result in the opposite effects. A direct association between the chaperones and tau protein has been demonstrated. Many results suggest that the up-regulation of molecular chaperones may suppress the formation of neurofibrillary tangles by partitioning tau into a productive folding pathway and thereby preventing tau aggregation . When weOn the basis of the results we suggested a model for mRNA localization in the axon Fig. .The mRNAs containing the U-rich region could be complexed with a protein responsible for transport toward the axon, just as HuD complexes with and stabilizes the tau messenger . HuD itsThe mechanisms determining whether a messenger such as \u03b2-actin is transported preferentially to the axon or the dendrites are poorly understood. The existence of two potentially conflicting location signals in the 3'-UTR raises questions about how the final destination of such mRNAs is determined within the neuron.In the 3'-UTRs of some mRNAs in neurons there are cis-acting signals that direct mRNAs such as tau and GAP-43 to the axon. In general, these signals are rich in uridine and do not contain guanidine. Comparison of the Dendritic Target Element (DTE) with the 3'-UTRs of several axon-located messengers showed some homology in a specific region of the 3'-UTR. Most of the 3'-UTRs studied possess homologies with the signals involved in the localization of mRNAs in axons and dendrites. This might explain why as much \u03b2-actin is present in dendrites as in axons, though the distribution mechanisms in such cases are not understood. In addition, we found a DTE homology in the 3'-UTR of HSP70 and 90. The significance of this is not clear; some messengers are transported towards the axon or towards the dendrites as required.A sequence homologous to DTE in tau occurs near the end of the 3'-UTR, next to the polyadenylation site, which indicates that only the axon signal sequence is functional, because mRNA degradation starts at the poly(A) site. The 3'-UTR of MAP2 possesseVery U-rich sequences in the 3'-UTR might be signals that direct some mRNAs exclusively to the axon. If we understand which signals/sequences the neuronal cell uses for the correct location of its mRNAs, it might become possible to determine which factors lead to mislocalization of messengers and of proteins, as has recently been suggested in relation to certain neurodegenerative diseases such as Alzheimer's.Rattus norvegicus genome and were located using the following GeneBank accession numbers. \u03b2-actin; NM_031144, tropomyosin 3 (Tpm3); NM_057208, cofilin; NM_017147, vimentin; NM_031140, immunoglobulin heavy chain biding protein (Bip); M14050, heat shock protein 60 (HSP60); X53585, heat shock protein 70 (HSP70); L16764, heat shock protein 90 (HSP90); S45392, glucose regulated protein (grp75); s78556, synuclein; NM_031688; NM_057114 and NM_053576 and tau; X79321. The 3'-UTRs of the mRNAs were analyzed using the program ClustalW [All the mRNAs analyzed in this study belong to the ClustalW , and theClustalW .The author(s) declare that they have no competing interests."} +{"text": "Comprehensive delineation of complex cellular networks requires high-throughput interrogation of genetic interactions. To address this challenge, we describe the development of a multiplex combinatorial strategy to assess pairwise genetic interactions using CRISPR-Cas9 genome editing and next-generation sequencing. We characterize the performance of combinatorial genome editing and analysis using different promoter and gRNA designs and identified regions of the chimeric RNA that are compatible with next-generation sequencing preparation and quantification. This approach is an important step towards elucidating genetic networks relevant to human diseases and the development of more efficient Cas9-based therapeutics. Complex cellular processes that control cell state and decision-making are orchestrated through highly interconnected regulatory networks. Quantitative genetic interaction mapping enables the systematic discovery of how gene-gene interactions give rise to complex cellular processes . Uncovermultiplex strategy for assessing genetic interactions using CRISPR-Cas9 (MoSAIC).The CRISPR-Cas9 system enables efficient genome engineering of mammalian cells through a programmable guide-RNA (gRNA) that targets Cas9 to a desired locus for editing \u20138. Thus 2 in high-Glucose Dulbecco\u2019s modified Eagle\u2019s medium (DMEM) containing 10% fetal bovine serum and 1% Penicillin/Streptomycin (Life Technologies). HEK 293T cells containing eGFP were a gift from Stephen Goff (Columbia University). 293FT cells were obtained from Life Technologies and were maintained in the same medium formulation and supplemented with 0.1 mM non-essential amino acids, 2 mM L-glutamine and 500 ug/ml Geneticin.HEK 293T cells were obtained from the American Tissue Collection Center (ATCC) and grown at 37\u00b0C, 5% COhttp://www.broadinstitute.org/rnai/public/resources/protocols).Lentivirus was produced in 293FT cells and stable Cas9-eGFP cells were transduced as previously described inducible Cas9 cells were generated as follows. 293T cell clones stably expressing eGFP-Cas9 under dox inducible promoter were generated by transduction of PLX301-eGFP-Cas9/Bsd using LT1 transfection reagent (Mirus) followed by selection with 10mg/ml Blasticidin (Bsd). 293T cells were infected with lentiviral particles at MOI of 0.3 followed by clonal selection. We selected a clone with highest differential Cas9 expression following 48 hour induction using immunostaining of FLAG-tagged Cas9, followed by flow cytometry.The eGFP-Cas-9 clone was infected with lentivirus containing gRNA constructs targeting eGFP and STAT1 or eGFP-only. Twenty-four hours post-infection, the media was changed and supplemented with 10 ug/ml blasticidin (Life Technologies) and cells were selected for three days, prior to doxycycline induction of Cas9. Cells were harvested on days 14, 21, and 28 post-induction. Gene knockout efficiencies were measured by either flow cytometry or SURVEYOR assay. Flow cytometry was performed using a LSRII or LSR Fortessa to quantify fraction of eGFP positive cells.MV.1, MV.3, MV.5, MV.6, MV.7 originated from lentivector v_w0, originally called plxsgRNA (Addgene 50662). A point mutation was made in the PGK promoter to eliminate the BsmB1 restriction site for all down-stream cloning (v_w0).MV.2 originated from pLenticrispr (49535). Vs.d1 was amplified with primers containing eGFP gRNA 1 / STAT1 gRNA2 and cloned into the pLenticrispr vector to generate an all-in-one vector containing two gRNAs.To clone MV.1 backbone, pLenticrispr was used as a template with vs_p39(f) and vs_p40 (r) to amplify an insert containing the reverse direction chimeric RNA, filler region with BsmB1 restriction sites and a forward direction chimeric RNA sequence. The chimeric- filler-chimeric was cloned into v_w0. To clone in gRNAs, vs.d5 (dsDNA) containing reverse direction H1 promoter, LoxP site and forward direction U6 promoter, was amplified with primers containing eGFP gRNA 1 and STAT1 gRNA 2 as well as BsmB1 restriction sites. The PCR product containing both gRNAs and both promoters was cloned into the MV.1 backbone to generate MV.1.1 and MV.1.2.To clone MV.3 backbone, H1 promoter expressing short tracr RNA was cloned into v_w0 from px261 (Addgene 42337). To clone in gRNAs, vs.d11 (containing U6 promoter) was amplified with primers vs_p79 and vs_p80/ vs_p81 / vs_p82 and PCR products were cloned into MV.3 backbone.To clone MV.5 backbone, the U6 promoter, filler region and chimeric RNA was cloned into v_w0 from lenticrispr_v1 (Addgene #49535) using vs_p59/vs_p40 primers. To clone in gRNAs,v_w2 containing chimeric RNA-LoxP-site and H1 promoter were amplified with primers containing eGFP gRNA 1 and STAT1 gRNA 2 as well as BsmB1 restriction sites.To clone MV.6 backbone, U6 promoter, the filler region with BsmB1 restriction sites and the chimeric RNA v2, was cloned into v_w0 using vs_d10. To clone in gRNAs,v_w2 containing chimeric RNA-loxP-site and H1 promoter were amplified with primers containing eGFP gRNA 1 and STAT1 gRNA 2 as well as BsmB1 restriction sites.In MV.7, used the backbone established in MV.6. To clone in gRNAs, oligo pairs (vs_p75 / vs_p76 and vs_p77/vs_p78) containing -BsmB1 Overhang-gRNA1-chimeric RNA-gRNA 2 were synthesized, annealed and ligated into backbone.Unless noted, all DNA constructs and primers were obtained from IDT (Geneblocks) and used for PCR and assembly steps as described above.vs_d1: chimeric RNA\u2014LoxP site\u2014U6 promoter:GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTATAACTTCGTATAGCATACATTATACGAAGTTATGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCvs_d5: reverse H1 promoter\u2014LoxP site\u2014U6 promoter forward:TAGATCTGTGGTCTCATACAGAACTTATAAGATTCCCAAATCCAAAGACATTTCACGTTTATGGTGATTTCCCAGAACACATAGCGACATGCAAATATTGCAGGGCGCCACTCCCCTGTCCCTCACAGCCATCTTCCTGCCAGGGCGCACGCGCGCTGGGTGTTCCCGCCTAGTGACACTGGGCCCGCGATTCCTTGGAGCGGGTTGATGACGTCAGCGTTCGAATTATAACTTCGTATAGCATACATTATACGAAGTTATGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCv_w2: chimeric RNA\u2014LoxP site\u2014forward H1 promoter:GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTATAACTTCGTATAGCATACATTATACGAAGTTATAATTCGAACGCTGACGTCATCAACCCGCTCCAAGGAATCGCGGGCCCAGTGTCACTAGGCGGGAACACCCAGCGCGCGTGCGCCCTGGCAGGAAGATGGCTGTGAGGGACAGGGGAGTGGCGCCCTGCAATATTTGCATGTCGCTATGTGTTCTGGGAAATCACCATAAACGTGAAATGTCTTTGGATTTGGGAATCTTATAAGTTCTGTATGAGACCACAGATCTAvs_d10: Xho1 site\u2014forward U6 promoter\u2014BsmB1 filler region (36nt)\u2014Chimeric RNA version 2\u2014Nhe1:ATTCGAACTCGAGGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGGAGACGGTTTTCTTGCTCTTTTTTGTACGTCTCTGTTTTAGAGCCGGAAACGGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTGCTAGCGCTAACvs_d11: forward U6 promoter:GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCvs_p26 :AATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGvs_p64 :TATTTTAACTTGCCGTTTCCGGCvs_p59 (forward):ACGGACTCGAGGAGGGCCTATTTCCCATGATTCvs_p40 (reverse):GATCACGGAGCTAGCCTGCCATTTGTCTCAAGATCTAGAATTCvs_p75:CACCGGAGCTGGACGGCGACGTAAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTGAAGTTCGAGGGCGACACCCvs_p76:AAACGGGTGTCGCCCTCGAACTTCAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAACTTTACGTCGCCGTCCAGCTCCvs_p77:CACCGGAGCTGGACGGCGACGTAAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTCCCCGGGGAAGTTCGAGGGCGACACCCvs_p78:AAACGGGTGTCGCCCTCGAACTTCCCCGGGGAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAACTTTACGTCGCCGTCCAGCTCCvs_p79 (forward):AGGGATCCTGAGGGCCTATTTCCCATGAvs_p80 (reverse):GCGCTAGCTAAAAACAGCATAGCTCTAAAACGGGTGTCGCCCTCGAACTTCACAGCATAGCTCTAAAACTTTACGTCGCCGTCCAGCTCGGTGTTTCGTCCTTTCCACAAvs_p81 (reverse):TTAGCGCTAGCTAAAAGTTTTGGGACCATTCAAAACAGCATAGCTCTAAAACGGGTGTCGCCCTCGAACTTCGTTTTGGGACCATTCAAAACAGCATAGCTCTAAAACTTTACGTCGCCGTCCAGCTCGGTGTTTCGTCCTTTCCACAAGvs_p82 (reverse):AGCGCTAGCTAAAAGTTTTGGGACCATTCAAAACAGCATAGCTCTAAAACGGGTGTCGCCCTCGAACTTCGTTTTGGGACCATTCAAAACAGCATAGCTCTAAAACTTTACGTCGCCGTCCAGCTCGTTTTGGGACCATTCAAAACAGCATAGCTCTAAAACGGTGTTTCGTCCTTTCCACAeGFP gRNA 1:GAGCTGGACGGCGACGTAAAeGFP gRNA 2:GAAGTTCGAGGGCGACACCCSTAT1 gRNA 1:GATCATCCAGCTGTGACAGGSTAT1 gRNA 2: CCTGTCACAGCTGGATGATCeGFP sequence:GAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCWe used the crystal structure of the sgRNA-targetDNA-cas9 complex to model the UA31CG-AU32GC-sgRNA . The corIn order to comprehensively map genetic interactions in a gene network, all possible single and double knockouts (KO) need to be simultaneously interrogated. MoSAIC achieves this in a single step through PCR of a common DNA template with gRNA primer pools . The firTo optimize the system for simultaneous targeting of Cas9 to multiple loci, we designed and tested two MoSAIC-compatible strategies:1) dual promoter, dual gRNA transcripts, and 2) single promoter, single RNA transcript . We explored several designs that use RNA Pol III promoters U6 and H1 in different positions and orientations , having We began by benchmarking a previously described approach utilizing two unidirectional U6 promoters to express dual gRNAs designs. We founWe then explored whether the promoter choice and orientation impacted KO efficiency in a position-dependent fashion sequences is sufficient for multiplexed Cas9-mediated KO in the absence of the SpRNase III RNA cleavage enzyme [Previously, studies demonstrated that targeting Cas9 to multiple loci could be achieved by co-expressing the RNA cleavage enzyme ge sites . Anothere enzyme . In ordee enzyme Fig 4A)Csy4 alonWe further explored MV7 designs that incorporated transcripts containing two tandem gRNA-chimeric RNA sequences and thus did not require a tracrRNA . This deS. pyogenes CRISPR-Cas9 crystal structure (PDB-4OO8) [MoSAIC is designed such that gRNA pairs serve as barcodes that can then be PCR amplified and identified using next-generation sequencing. We achieved this by altering the second chimeric RNA sequence such that placement of a reverse sequencing primer results in PCR amplification of both gRNAs with an amplicon size that is NGS compatible . Primer DB-4OO8) to prediHere we report on the development of a generalizable strategy for multiplex targeting of Cas9 for mammalian genome engineering. We describe the implementation of this approach to combinatorially target two genes simultaneously and monitor the mutational efficiency of several gRNA designs. We find that gRNA pairs expressed from dual U6-H1 promoters lead to optimal Cas9-mediated genome editing, which can be combined with single transcript multiple gRNA designs (MV7) to increase editing efficiency. In addition, we find that a unidirectional single-transcript gRNA system resulted in low KO efficiency. While the precise reason for this observation has not been fully elucidated, we speculate that MV3.3 and 3.4 designs that lead to contiguous unprocessed gRNA products may cause a lower Cas9 targeting efficiency by virtue of Cas9 binding to either of the two gRNA positions, but not simultaneously. In addition, we observed that unidirectional dual gRNA designs resulted in increased KO efficiency for the second gRNA position. While further experiments are needed, we speculate that higher KO efficiency of the second gRNA may be the result of transcription read-through of first position gRNA, leading to increased levels of second gRNA transcripts and Cas9 targeting.To enhance multiplexing of our system, we identified specific chimeric RNA variants at positions 11\u201312 and 17\u201318 corresponding to the tetraloop repeat-anti-repeat duplex that are amenable to alteration. We showed that these chimeric RNA variants still maintained Cas9 targeting while also allowing compatible barcode amplification in a pooled format to enable multiplex assessment of cell populations using NGS. In this manner, the gRNA pairs serve as unique molecular barcodes linking their abundance in the cell population with their screened phenotype. This strategy is also compatible with the use of different Cas9 variants, including CRISPRi and CRISMoSAIC overcomes several key technical hurdles associated with high-throughput generation and measurement of dual loci perturbations in mammalian cells. Additionally, to facilitate subsequent iterative introduction of gRNA constructs and enable higher-order combinatorial genetic perturbations, the integrated lentiviral vector design includes loxP \u201clanding-pad\u201d sequences. The MoSAIC system expands the toolbox for genetic modification of mammalian genome and extends our knowledge of Cas9 targeting design parameters. Recently, Wong et al described the CombiGEM approach to generate combinatorial gRNA libraries for Cas9-based genomic interrogations by barcoded sequencing . The Com"} +{"text": "Herein we present the data necessary for generation of alternative means to produce equimolar mixtures of peptides Specifications TableValue of the data\u2022Excellent reproducibility of peptide concentration data produced by the analysis of tryptic digestions is the necessary for method comparisons. The costs associated with a particular approach in terms of reagents and time may be a pivotal factor.\u2022The Hi3 method is a very valuable approach in cases of relatively simple proteomes. The speed of method development for core laboratories can be critical. The Hi3 method is relatively straightforward, once reproducible results are obtained.\u2022The QConCAT design is very elegant but there are critical issues, such as the protein construct solubility and stability, which require careful attention to obtain optimal results. The database attached is necessary for analysis of the mass spectral results.1The data are compiled in the bar graphs for the quantitative proteome analyses of trivalent influenza vaccines and standards using three methods: Hi3, QconCAT and synthetic peptides.http://www.thegpm.org/crap/) and, since the viruses are grown in chicken eggs, the entire chicken proteome as well as selected full length influenza proteins (mostly from GISAID http://platform.gisaid.org/epi3/frontend#4f5b25, and the World Health Organization http://www.who.int/influenza/vaccines/virus/en/.Databases contained either the customized QconCAT protein sequence or the full sequences of the proteins represented therein, along with trypsin, several human keratins (cRAP from 2Data was obtained by the comparison of mass spectral signal from the three most intense fully tryptic peptides identified from the samples versus the internal standard protein. Experiments were designed to compare the mass spectral signal strengths from equimolar tryptic peptides identified by the Hi3 method by five different QconCAT designs as well as from synthetic peptides. The samples analyzed were commercial trivalent influenza vaccines as well as monovalent influenza reference standards. The mass spectra were obtained by reversed phase separation using a C18 UPLC column, Waters nanoAcquity UPLC, directly coupled to a Waters Synapt HDMS mass spectrometer. The mass spectrometer was programmed to carry out data-independent MSMS and incorporated a lock spray of glu-fibrinopeptide. The data was processed using Protein Lynx Global Server 3.0 for the identification of the three most intense peptides from a given protein identified in a custom database which has been attached. The intensities from all charge states of a peptide are included in the software calculations; however, the intensities resulting from in-source fragmentation and modified peptides must be added manually.2.12.1.1CATATGGATGACGATGATAAACTGGTGAACGAACTGACCGAATTCGCGAAACTGGGCGAATACGGCTTCCAGAATGCACTGATCGTTCGTCATCTGGTTGACGAACCACAAAACCTGATTAAAGACGCATTTCTGGGCTCCTTTCTGTACGAATACTCTCGCGTTGTTGGTCTGTCTACCCTGCCTGAGATTTACGAAAAACTGCCACTGGTCGGTGGTCATGAAGGCGCAGGCGTCGTCGTTGGTATGGGCGAAAACGTGAAATCCATCAGCATTGTAGGTTCCTACGTTGGCAACCGTGCCAATGAACTGCTGATCAACGTCAAATCCACCCAGAACGCGATCGACGAGATTACGAATAAAATGAACTACTACTGGACCCTGGTTGAGCCGGGCGATAAAGAACAGCTGTCCAGCGTGTCCTCTTTCGAACGTATGAACACGCAGTTTACCGCTGTAGGCAAAAGCACCCAGGCTGCCATCGACCAGATCAACGGTAAAATCGATCTGTGGTCCTATAATGCCGAGCTGCTGGTTGCCCTGGAAAATCAGCACACTATTGACCTGACTGATAGCGAAATGAACAAGGAGTTCTCTGAAGTGGAAGGTCGTTGGGACCTGTTCGTGGAACGTCTGTCTGGCGCGATGGACGAACTGCACAACGAAATCCTGGAACTGGATGAGAAACTGTCTACTCACAACGTTATCAACGCGGAAAACGCTCCGGGTGGCCCGTACAAAATTGTGGTGGACTACATGGTGCAGAAGAACCTGAACTCCCTGAGCGAGCTGGAAGTGAAAACTTTCTTCCTGACTCAGGGTGCCCTGCTGAACGACAAATACAACGGCATCATTACGGACACCATCAAATATGGTAACGGTGTTTGGATCGGTCGCGGTGACGTGTTCGTTATCCGCACGCTGCTGATGAACGAGCTGGGTGTTCCGTTCCACCTGGGCACCAAGCTGGTGGATTCTGTAGTCTCTTGGAGCAAAGTAATCGAGGGCTGGAGCAACCCGAAGATTCTGTTTATTGAAGAAGGCAAAGGTGTAACCCTGCTGCTGCCGGAACCGGAATGGACCTACCCGCGCCTGAACGTTGAAACTGATACTGCGGAAATTCGTTATGGTGAAGCGTATACCGACACCTATCACAGCTACGCTAACAAAGAGTGGACCTATATCGGTGTTGATGGTCCGGATAACAACGCTCTGCTGAAAGGCGGTCTGGAACCTATCAACTTCCAAACCGCAGCGGACCAGGCGCGTATCTCTCAAGCAGTACACGCTGCGCACGCAGAAATCAACGAAGCTGGCCGTCTGACCGAGTGGACTTCTTCCAATGTTATGGAAGAACGTAACGTTCTGCAGCCGTCTTCCGTAGATTCTCAGACCGCAATGGTTCTGGTAAACGCTATCGTTTTCAAGTAAGGATCC.2.1.2CATATGGACGATGACGACAAGCATGTCAAACTGGTGAACGAACTGACTGAATTCGCAAAAACTTGCGTGCGTTTCGAAAAACTGGGCGAATACGGTTTCCAGAACGCACTGATCGTGCGTTACACCCGTAAACTGAAACACCTGGTGGATGAACCTCAAAACCTGATCAAACAGAACTGCCGTCGTCCAATCAAAGTAGTGGGTCTGTCTACCCTGCCGGAGATTTACGAAAAAATGGAAAAGCGTCCGGTGAAGCTGCCTCTGGTCGGCGGTCACGAAGGCGCTGGTGTTGTTGTTGGTATGGGCGAGAACGTGAAAGGTTGGAAGGTTGTTAAATCTATTAGCATCGTTGGCTCTTACGTAGGTAACCGCGCCGACACTCGTGATCTGAAGAGCACCCAGAATGCCATCGATGAAATCACCAACAAAGTCAACTCTCGCGAAGGTCGTATGAACTACTATTGGACCCTGGTTGAACCGGGCGACAAGATTACCTTTCGTGAACTGCGCGAACAGCTGTCTTCTGTGTCCAGCTTCGAACGTTTTGAAATCCGTGAATGCCGCACGTTCTTTCTGACGCAGGGTGCACTGCTGAACGACAAACATTCCAACCGCGTACTGAAATACAACGGTATCATCACTGATACCATTAAATCCTGGCGCAGCTTTAAATACGGTAACGGCGTCTGGATCGGTCGTACGAAATCTGACCTGAAATCCACCCAGGCGGCCATTGATCAGATCAACGGCAAACTGAACCGTGATACCAAAATCGATCTGTGGTCCTACAACGCGGAACTGCTGGTGGCCCTGGAAAACCAACACACCATCGACCTGACCGACTCTGAAATGAACAAACTGTTCGAGCGTATTGAAAAAGAATTTTCCGAGGTGGAGGGTCGCATCCAAGATCGTCGCCCGTACCGCACTCTGCTGATGAATGAACTGGGTGTTCCATTCCACCTGGGTACCAAACAGGTTTGCCGCAACGGCCGTCTGGTTGACAGCGTAGTAAGCTGGTCTAAAGAAATCCTGCGTACCTTCAAAGTTATCGAAGGTTGGTCTAACCCGAAATCTAAACTGCTGCAGCGCCTGTCTGGTGCGATGGACGAACTGCACAACGAGATCCTGGAACTGGATGAGAAAGTTGACGACCGTCACATCCGCCTGTCCACCCACAATGTAATTAACGCAGAAAACGCTCCGGGCGGTCCGTACAAAATTGGTACTCGCTCCGGTCGTATTGTGGTTGACTATATGGTACAGAAGTCTGGCAAAGCAACTAAAGGCGTTACGCTGCTGCTGCCGGAACCGGAATGGACTTATCCGCGTCTGTCCTGTCGTTTCGTCAAGCTGAACGTTGAAACCGACACGGCGGAAATTCGTCTGATGTGTAAAGTGAAATATGGCGAAGCGTATACCGATACTTACCATTCTTACGCTAACAAGATCCTGCGCCTGTATCGTGGCGGCCTGGAACCGATTAATTTCCAGACTGCGGCGGATCAGGCTCGTGAGCTGATCCGTAGCCTGAAAATCTCCCAGGCAGTACACGCTGCTCACGCTGAAATCAACGAAGCGGGCCGTGAGGTAGTTCGTTTCGAGAAACTGACCGAATGGACCAGCAGCAATGTTATGGAGGAGCGTAAAATCAAATAAGGATCC.2.1.3CATATGGATGACGACGACAAAGCAAGCGGTAAACTGGTGAACGAGCTGACTGAGTTCGCTAAAGCGAGCGGTAAGCTGGGTGAATACGGTTTCCAGAACGCACTGATCGTACGTGCTTCTGGTAAACACCTGGTCGACGAGCCTCAAAACCTGATTAAAGCTAGCGGCAAAGACGCCTTCCTGGGTAGCTTCCTGTACGAGTACAGCCGTGCTTCTGGCAAAGTAGTAGGTCTGAGCACTCTGCCAGAAATCTACGAGAAAGCGTCTGGTAAGCTGCCTCTGGTCGGTGGTCATGAAGGTGCTGGTGTAGTAGTAGGTATGGGTGAGAACGTGAAAGCATCCGGTAAAAGCATCAGCATCGTCGGTTCCTACGTCGGTAACCGTGCAAGCGGTAAAGCTAACGAGCTGCTGATCAACGTCAAGGCAAGCGGCAAATCTACCCAGAACGCAATCGACGAGATCACTAACAAAGCATCTGGTAAAATGAACTACTACTGGACCCTGGTCGAGCCGGGCGACAAAGCCAGCGGTAAGGAGCAGCTGTCTTCTGTGTCTTCCTTCGAGCGTGCATCTGGTAAGATGAACACCCAGTTCACGGCAGTGGGTAAAGCATCCGGCAAGACTTTCTTCCTGACTCAGGGTGCACTGCTGAACGATAAAGCTTCCGGTAAATACAACGGCATCATCACGGACACCATCAAAGCGTCTGGCAAGTACGGCAACGGTGTGTGGATCGGTCGTGCTTCCGGTAAGGGTGATGTTTTCGTGATCCGTGCATCTGGCAAATCCACTCAGGCAGCAATCGACCAAATCAACGGTAAGGCTTCCGGCAAGATCGACCTGTGGTCTTACAACGCTGAACTGCTGGTTGCTCTGGAAAACCAGCATACTATCGACCTGACCGACTCTGAAATGAACAAAGCCTCCGGTAAAGAATTCTCCGAAGTGGAAGGCCGCGCGTCTGGTAAATGGGACCTGTTTGTGGAACGCGCCTCTGGTAAGACTCTGCTGATGAACGAACTGGGTGTGCCATTTCACCTGGGTACGAAAGCATCTGGCAAGCTGGTGGATTCTGTGGTATCCTGGTCTAAAGCCTCTGGTAAAGTTATCGAAGGCTGGTCCAACCCGAAAGCCAGCGGCAAGATCCTGTTTATTGAAGAAGGTAAAGCGTCCGGTAAACTGTCCGGCGCGATGGACGAACTGCACAACGAAATTCTGGAACTGGATGAAAAAGCTTCTGGCAAACTGTCCACCCACAACGTTATTAACGCGGAAAACGCCCCGGGCGGCCCGTACAAAGCTAGCGGTAAAATCGTTGTTGATTATATGGTTCAGAAAGCTTCCGGCAAAAACCTGAACAGCCTGTCCGAACTGGAAGTTAAAGCCTCCGGCAAAGGCGTTACCCTGCTGCTGCCGGAACCGGAATGGACCTACCCGCGTGCCAGCGGCAAACTGAATGTTGAAACCGATACCGCTGAAATTCGCGCCAGCGGTAAATATGGCGAAGCTTATACCGATACCTATCACTCCTATGCGAACAAAGCGAGCGGCAAAGAATGGACCTATATTGGCGTAGATGGCCCGGATAACAACGCGCTGCTGAAAGCCTCTGGCAAAGGCGGCCTGGAACCGATTAATTTTCAGACCGCGGCTGATCAGGCTCGTGCGAGCGGTAAAATTTCTCAGGCGGTTCATGCGGCGCACGCGGAAATTAATGAAGCGGGCCGTGCGTCTGGCAAACTGACCGAATGGACGTCTTCTAATGTTATGGAAGAACGCGCATCCGGCAAAAATGTTCTGCAACCGTCTAGCGTTGATTCCCAGACCGCGATGGTTCTGGTTAATGCGATTGTTTTCAAAGCGTCCGGCAAATAAGGATCC.2.1.4CATATGGCTGGTCGTGCGTCTGGTAAACTGGGTGAGTACGGTTTTCAGAACGCGCTGATCGTACGTGCGTCTGGTAAAGTAGTCGGTCTGTCTACCCTCCCGGAGATCTACGAAAAAGCGTCCGGTAAAGAGGTCCTGGTTCTGTGGGGTATTCACCACCCGTCTACTTCTGCAGATCAGCAGTCTCTGTACCAGAACGCAGACGCTTACGTATTCGTTGGCTCTTCTCGTGCGTCTGGCAAAATCGACCTGTGGTCTTACAACGCCGAACTGCTGGTTGCCCTGGAAAACCAGCACACTATCGACCTGACCGACTCCGAAATGAACAAAGCGTCCGGTAAACTGTCTGGTGCGATGGACGAACTGCACAACGAAATCCTGGAACTGGACGAGAAAGCCAGCGGTAAAACCTTCTTCCTGACTCAGGGTGCGCTGCTGAACGACAAAGCTTCTGGCAAAACCCTGCTGATGAACGAACTGGGTGTTCCGTTTCACCTCGGTACCAAAGCGTCTGGTAAAGGTGTTACCCTGCTGCTGCCGGAACCGGAATGGACTTATCCACGTGCCTCTGGTAAAGGTGGTCTGGAACCGATCAACTTTCAGACGGCCGCAGATCAGGCACGTGCTTCTGGTAAAATCTCTCAGGCTGTTCACGCCGCGCACGCAGAAATCAACGAAGCAGGTCGTGCTTCTGGCAAACTGAACGTTGAAACCGACACCGCGGAAATCCGTGCCTCTGGTAAACTGGTTGACAGCGTTGTTTCTTGGTCCAAAGCGTCCGGTAAATACAACGGTATCATCACCGACACCATCAAAGCCTCTGGTAAATTCACCTCCTCTGCCAACGGTGTTACGACCCACTACGTATCTCAGATCGGTGGTTTCCCGGATCAGACCGAAGACGGTGGTCTGCCGCAGTCTGGTCGTGCTTCTGGTAAATCTACCCAGGCGGCGATTGACCAGATCAACGGTAAAGCGTCCGGCAAATCTACGCAGAACGCGATCGACGAGATCACCAACAAAGCCTCTGGTAAACTGCCGCTGGTAGGTGGTCACGAAGGTGCAGGTGTTGTAGTGGGTATGGGTGAGAACGTGAAAGCGAGCGGTAAACTGGTTAACGAACTGACGGAGTTCGCCAAAGCCTCTGGTAAAAACCTGAACAGCCTCAGCGAACTGGAAGTGAAAGCCTCTGGCAAATACGGTAACGGTGTTTGGATCGGTCGTGCTAGCGGTAAATCTGGTTACAGCGGTATCTTCTCTGTTGAGGGTAAAGCCTCCGGTAAATACGGTGAAGCCTACACGGATACCTACCACTCTTACGCCAAAGCGAGCGGTAAACTGACCGAATGGACGAGCTCTAACGTTATGGAGGAACGTGCCTCTGGTAAAGACGCTTTCCTGGGCTCCTTCCTGTACGAATACTCTCGTGCGTCTGGTAAATCTATCTCTATCGTCGGCTCCTACGTAGGTAACCGTGCGTCTGGTAAAATGAACTACTACTGGACCCTGGTTGAACCGGGTGACAAAGCTTCCGGTAAATCTCAGCAGGCGGTTATCCCGAACATCGGTTTTCGTCCACGTGCTTCTGGCAAACTGAACTGGCTGACCCACCTGAACTTCAAAGCCTCCGGTAAAATGAACACCCAGTTCACGGCGGTTGGTAAAGCGTCTGGTAAAGCCAACGAACTGCTGATCAACGTGAAAGCCTCCGGTAAACACCTCGTAGACGAACCGCAGAACCTGATCAAAGCGTCTGGCAAAAACGTACTCCAGCCGTCTTCTGTTGACTCTCAGACCGCTATGGTTCTGGTCAACGCGATCGTATTCAAAGCCAGCGGTAAAGGTAACTCTGCCCCGCTGATCATTCGTGCCTCTGGTAAAGGTTGGGCTTTTGACGACGGTAACGACGTTTGGATGGGTCGTGCGTCTGGTAAAGGCGACGTATTCGTTATCCGTGCGTCTGGTAAAGCGGACACCATCTCTTCTCAGATCGAACTGGCCGTTCTGCTGTCTAACGAGGGTATCATCAACTCCGAAGACGAGCACCTGCTGGCTCTGGAACGTGCCTCTGGTAAACTGGCAGCGGCCCTGGAACACCACCACCACCACCACTAATAGGGATCC.2.22.2.12.2.2FASTA Protein Database used: 2015_2016_FluQuant.fasta"} +{"text": "The results indicate that transcriptional bursting is caused by interplay between RNA polymerases on DNA. The kinetics of in vitro transcriptional bursting is quantitatively consistent with the gene-nonspecific kinetics previously observed in noisy gene expression in vivo. Our kinetic analysis based on a cellular automaton model confirms that arrest and rescue by trailing RNA polymerase intrinsically causes transcriptional bursting.Cell-to-cell variability plays a critical role in cellular responses and decision-making in a population, and transcriptional bursting has been broadly studied by experimental and theoretical approaches as the potential source of cell-to-cell variability. Although molecular mechanisms of transcriptional bursting have been proposed, there is little consensus. An unsolved key question is whether transcriptional bursting is intertwined with many transcriptional regulatory factors or is an intrinsic characteristic of RNA polymerase on DNA. Here we design an Transcriptional bursting is a potential source of cell-to-cell variability but the molecular mechanisms are unclear. Here the authors use single molecule imaging to analyse the kinetics of bursting on DNA and observe that bursting is an intrinsic property of RNA polymerases on DNA. Our results show transcriptional bursting occurs with the minimum components of bacterial transcription, suggesting an intrinsic molecular mechanism of transcriptional bursting. Based on the kinetic analysis, we propose that transcriptional bursting is intrinsically caused by interplay between RNA polymerases (RNAPs) on DNA. Our analysis can quantitatively explain the gene-nonspecific kinetics previously observed in noisy gene expression \u22121 at our experimental conditions , to correct for the biased transcription rate for each nucleotide speciesin vitro transcription system -labelled RNAP holoenzyme and a DNA template containing a lacUV5 promoter and single- or six-target sites for the oligo probe (NTP] (in vitro single-molecule measurement (13kA) is \u223c0.2\u2009s\u22121 (in vitro single-molecule measurements originated from an arrest state. This was supported by atomic force microscopy (AFM) images of transcription elongation as well or RNAP enters an arrest state at a rate of kA/(kF+kA). In this model, one simulation time is consistent with 1/kF=100\u2009ms. Arrest state is rescued by pushing of a trailing RNAP (collision) at a rate of kR. When RNAP moves from the 71th and 72th box, the 57th box of the nascent mRNA is \u2018exposed' and the mRNA is \u2018visualized' and counted , we performed a simulation based on a cellular automaton model 1617. In 16.045\u2009s\u22121 . The treectively . On the ectively . Furtheron model as kR iskA) varies by a few orders of magnitude depending on measurement conditions not only in this study (kA=0.00005\u223c0.2\u2009s\u22121), but also in the previous study , is consistent with the suggested kinetic scheme under the gene-nonspecific constraint, in which only koff (10\u22125\u223c102\u2009s\u22121) is varied to change the gene expression level10kI). The kI is a critical parameter to control not only the mean of mRNA distribution but also the Fano factor as our experimental data suggest.To explain the discrepancy of the Hill coefficients between experimental data 1.9) and our model (1.0), however, we may need other sources of cooperativity such as indirect interplay via DNA stress generated by RNAP. As previously reported, RNAP generates local torsional stress on DNA, which is sufficiently long-lived to untwist the upstream region even on linear DNA is koff , was lar and our in vivo. In such models, where the regulatory proteins turn on the gene, the koff corresponds to their unbinding rate, which is generally constant as long as the binding sequence is unchanged, whereas in fact koff is largely changed in a gene-nonspecific mannerkoff in the previous models must be gene specific, which is contrary to the global feature of transcriptional bursting observed in vivo. On the other hand, to explain the gene-specific feature of transcriptional bursting as observed in yeastAt the same time, we note that models where the binding and unbinding of regulatory factors such as gyrase, and possibly enhancers in eukaryotic cells, cannot by themselves fully explain the gene-nonspecific kinetics of transcriptional bursting observed in vitro data, especially the trend between Fano factor and mean. However, we cannot fully rule out such effects by our in vitro experiments alone. Specifically, the sequence dependence of arrest rate during elongation was not fully examined, because our in vitro experiment .5\u2032-CCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATAaaagacgccttgttgttagccataaagtgataacctttaatcattgtctttattaatacaactcactataaggagagacaacttaaagagacttaaaagattaatttaaaatttatcaaaaagagtattgacttaaagtctaacctataggatacttacagccIn the above sequence, lower case letters indicate the T7A1 promoter region (\u2212163 to +38 from the transcription start site represented by the bold typed a). An underline indicates the oligo probe-binding site. The template sequence from the oligo probe-binding site to the 3\u2032-end (downstream region) is the same as in the original report5\u2032-CCACAACGGTTTCCCTCTAG-3\u2032 and5\u2032-/5Bio/G/Cy5/CCGGATAAAACTTGTGC-3\u2032 (synthesized by Integrated DNA Technologies).See In the Qdot tracking experiment with fastFISH , we usedaattgtgagcggataacaatttcacacaggaaacagctatggaccgcaagcttCCCTATCCCTTATCTTAACCACTCCAATTACATACACCCCCTATCCCTTATCTTAACCACTCCAATTACATACACCCCCTATCCCTTATCTTAACCACTCCAATTACATACACCCCCTATCCCTTATCTTAACCACTCCAATTACATACACCCCCTATCCCTTATCTTAACCACTCCAATTACATACACCAGCTTCCCTATCCCTTATCTTAACCACTCCAATTACATACACCTTTCAAAACTTCAAACTTCAAACTTCAAACTTCAAACTTCAAACTTCAAACTTCAAACTTCAAACTTCAAACTTCAAACCACCGTTGATATATCCCAATGGCTGCAGCTG GATATTACGGCCTTTTTAAAGACCGTAAAGAAAAATAAGCACAAGTTTTATCCGGC-3\u2032.Six-target template (641\u2009bp): 5\u2032-CCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATAtaatgcagctggcacgacaggtttctatgcttccggctcgtataatgtgtgglacUV5 promoter region (\u2212131 to +53 from the transcription start site represented by the bold typed a). Six underlines indicate the oligo probe-binding sites.In the above sequence, lower case letters indicate the Single-target template (517\u2009bp):aattgtgagcggataacaatttcacacaggaaacagctatgCCCTATCCCTTATCTTAACCACTCCAATTACATACACCTTTCAAAACTTCAAACTTCAAACTTCAAACTTCAAACTTCAAACTTCAAACTTCAAACTTCAAACTTCAAACTTCAAACCACCGTTGATATATCCCAATGGCTGCATTTCAAAACTTCAAACTTCAAACTTCAAACTTCAAACTTCAAACTTCAAACTTCAAACTTCAAACTTCAAACTTCAAACTGCAGCTGGATATTACGGCCTTTTTAAAGACCGTAAAGAAAAATAAGCACAAGTTTTATCCGGC-3\u20325\u2032-CCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATAtaatgcagctggcacgacaggtttcccgactggaaagcgggcagtga gcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtata atgtgtgglacUV5 promoter region (\u2212131 to +41 from the transcription start site represented by the bold typed a). Single underline indicates the oligo probe-binding sites. We prepared the templates for biotinylation at the both ends by PCR withIn the above sequence, lower case letters indicate the 5\u2032-/5Bio/CCACAACGGTTTCCCTCTAG-3\u2032 and5\u2032-/5Bio/GCCGGATAAAACTTGTGC-3\u2032.See lacZ (1-1179 when 1 indicates the first nucleotide of the gene) . The ups5\u2032-CCACAACGGTTTCCCTCTAG-3\u2032 and5\u2032-ATAATGCGAACAGCGCAC-3\u2032.PCR-amplified DNA fragments were purified by Wizard PCR Preps DNA Purification System (Promega). The sequence of the DNA template region in plasmids was verified by sequencing. Except as otherwise noted, the primers were synthesized by Hokkaido System Science Co., Ltd, Japan.Escherichia coli RNAP holoenzyme purchased from New England Biolabs. For the Qdot-tracking experiment, we designed the plasmid for co-overexpression of tagged E. coli RNAP subunitsrpoA-rpoB-rpoC-rpoZ between the NdeI and HindIII sites in pT7-7 by In-Fusion Cloning Kit (Clontech). The genes were provided by NBRP-E. coli at NIG . HaloTag and His-tag fragments were attached to the 3\u2032-end of rpoC. The Plasmid was transformed into BL21 \u03bbDE3. A single colony was inoculated into 1\u2009litre of Luria-Bertani liquid medium (LB) containing 100\u2009\u03bcg\u2009ml\u22121 ampicillin at 37\u2009\u00b0C until OD600 reached 0.3\u20130.5. The protein production was induced by the addition of isopropyl-\u03b2-D-thiogalactoside to 0.2\u2009mM and cells were grown overnight at 23\u2009\u00b0C. Cells were collected by centrifugation , rinsed with PBS and stored at \u221280\u2009\u00b0C. To proceed with protein purification, pellets were resuspended in 80\u2009ml lysis buffer and protease inhibitor (Roche) was added. Cells were disrupted by sonication, cleared by centrifugation and filtrated by the 0.22\u2009\u03bcm syringe filter (Millipore). The protein was purified by using 5\u2009ml HiTrap Heparin HP, 5\u2009ml HisTrap HP and Mono Q 5/50 GL, all purchased from GE HealthcarerpoD (\u03c370) gene was inserted in pT7-7 with amino terminus His tag. The Plasmid was transformed into BL21 \u03bbDE3. Cells were collected and disrupted as described above. The protein was purified by using TALON Metal Affinity Resin (Clontech) according to the product manual. The buffer of the eluted \u03c370 was changed to the storage buffer through a 50\u2009kDa MWCO filter and stored at \u221280\u2009\u00b0C.For the fastFISH and AFM imaging experiment, we used The fluorescent images were recorded by an Olympus IX71 inverted microscope. In the fastFISH experiment, illumination was provided by 532 and 640\u2009nm laser light (Coherent). The lasers were combined into one fibre output by OBIS Galaxy (Coherent). The laser light was expanded to a diameter of 8\u2009mm and focused by a 400\u2009mm focal-length lens into the back focal plane of the objective . The fluorescent photons were collected with a electron-multiplying charge-coupled device (CCD) camera . The effective pixel size was 74\u2009nm. The laser was steered by a piezo mirror (Physik Instrumente) and reflected by a dichroic mirror just below the objective lens. A dual-view apparatus (Hamamatsu Photonics) equipped with dichroic mirrors (Asahi Spectra) and emission filters was put in the Cy3 channel. In the Qdot-imaging experiment with fastFISH, illumination was provided by 488 and 532\u2009nm laser light (Coherent). Other optics were the same as described above. To maintain the temperature in the chamber, we warmed the objective by a lens heater .\u22121 PEG mixture solution (NHS-PEG-biotin and NHS-PEG were mixed at the ratio of 1:200 and dissolved in 0.45\u2009M K2SO4 and 0.1\u2009M NaHCO3 pH 9.0) was squeezed between two coverslips and incubated for 30\u2009min at 30\u2009\u00b0C. The NHS-PEG-biotin and NHS-PEG were purchased from NOF Corporation, Japan3) for 10\u2009min at room temperatureThe fluorescence imaging experiments were performed inside a sample chamber assembled with a PEG-coated glass. The PEG-coated glass was prepared as follows. Coverslips were cleaned by low-pressure plasma for 5\u2009min with a plasma system . The coverslips were then placed into a freshly prepared 3% solution of N-2-(aminoethyl)-3-aminopropyl-trimethoxysilane in acetone for 45\u2009min with gentle shaking. The amine-modified coverslips were then rinsed with MilliQ and dried by an air blower. A 20\u2009\u03bcl drops of 200\u2009mg\u2009mlin vitro transcription assays were performed in transcription buffer at the nucleotide concentration of 100[NTP] to correct for the biased transcription rate for nucleotide species\u22121 avidin solution was added into a sample chamber and incubated for 3\u2009min. After washing the chamber by transcription buffer, 1\u20132\u2009nM DNA template was added and washed again by transcription buffer. In fastFISH experiment . The fastFISH experiment was performed in 50\u2009nM self-quenched oligo probe, in which a hybridization rate is 0.2\u2009s\u22121 at 25\u2009\u00b0C . All single-molecule observations were performed within 30\u2009min. At least, the data within 30\u2009min have shown no time dependency.All periment and 4, sat 25\u2009\u00b0C . In Qdotat 25\u2009\u00b0C , a DNA t2 for 5\u2009min. The mica was then rinsed with MilliQ and dried by an air blower. Before acquisition of AFM images, in vitro transcription was performed at the concentration of 10\u201320\u2009nM DNA and 100\u2009nM RNAP in transcription buffer for 30\u2009min at room temperature. The reaction solution was diluted to 1\u20132\u2009nM DNA in 100[NTP] deposition buffer and added to freshly cleaved mica. After incubation for 2\u2009min, the mica was rinsed with MilliQ and dried by an air blower. AFM imaging was performed with a NanoWizard 3 operating in AC mode. Silicon cantilevers (OMCL-AC160TS) were purchased from Olympus. Images of 512 \u00d7 512 pixels were acquired with a scan size of <3\u2009\u03bcm at a scan rate of two lines per second.AFM images were acquired on mica in air. The mica was pretreated by incubation of 10\u2009mM MgClImages of 512 \u00d7 512 pixels were acquired by commercial software at 10\u2009Hz acquisition rate. In the fastFISH experiment, the positions of Cy5-labelled DNA and Qdot-labelled RNAP were decided by using a two-dimensional Gaussian distribution fitting3132The plots in m is the mean of mRNA number distribution, mmin and mmax are its minimum and maximum, respectively, kI is transcription initiation rate, khalf is the rate when m equals (mmax\u2212mmin)/2 and n is the Hill coefficient.Here, mmin=0.3\u00b10.1, mmax=3.9\u00b10.1, khalf=0.017\u00b10.002 and n =1.9\u00b10.2. The simulated data .The experimental data .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures 1-8, Supplementary Table 1 and Supplementary ReferencesA stochastic cellular automaton model of transcriptionA stochastic cellular automaton model with mRNA degradation"} +{"text": "AbstractSerenotheres Ahyong & Ng, 2005 (Pinnotheridae) is currently only represented by one species, Serenotheresbesutensis . A new species is now assigned to this genus, described from a date mussel Leiosolenusobesus Carpenter, 1857 collected in the Solomon Islands. Serenotheresjanussp. n. differs from Serenotheresbesutensis in possessing a conspicuously broader carapace, with the lateral margins of the dorsal lamellum distinctly produced and the posterolateral part deeply concave, the dorsal lamellum being highest at the median cleft, the rostrum is relatively more prominent, the surfaces of the anterolateral margin and hepatic region are less prominently pitted and eroded, the ischiomerus of the third maxilliped is relatively more rectangular, and the P2 merus is proportionately longer.The pea crab genus Durckheimia De Man, 1889 and Xanthasia White, 1846 , and established two new genera, namely Serenotheres Ahyong & Ng, 2005, for Durckheimiabesutensis Ser\u00e8ne, 1967; and Tridacnatheres Ahyong & Ng, 2005, for Xanthasiawhitei De Man, 1888. They commented that Serenotheres differed from all pinnotherid genera not only by the unusual carapace which has an additional large plate above its normal carapace surface which overhangs the frontal margin, but also by possessing a two-segmented third maxilliped palp , Smithsonian Institution, Washington D.C.The specimen examined is deposited in the = third maxillipedMXP3; = pereiopods 2\u20135P2\u2013P5 (first to fourth ambulatory legs), respectively. Measurements (in millimetres) are of the carapace width and length, respectively. The terminology used essentially follows that in The following abbreviations are used: PageBreakCATACTATTAACAGATCGTAATCTAAATACCTCATTCTTTGACCCAGCCGGTGGTGGAGATCCTGTTCTCTATCAACATTTATTTACCCTTATATTTTATCTTCGGAGCTTGGGCAGGTATAGTAGGAACTTCTTTAAGTTTAATAATTCGAGCTGAACTTAGACAACCAGGCAGACTTATTGGAAATGACCAAATTTATAATGTAATAGTTACAGCCCATGCTTTTGTTATAATTTTCTTTATAGTTATACCAATTATAATCGGAGGCTTCGGAAACTGATTAGTTCCTTTAATACTTGGGGCCCCAGATATAGCATTCCCTCGTATAAACAATATAAGATTTTGACTCTTACCTCCATCTTTATCACTCTTACTTACAAGAAGAATAGTTGAAAGTGGAGTAGGAACAGGATGAACTGTTTATCCTCCTCTAGCTTCAGCTATTGCCCATGCTGGAGCTTCTGTAGATTTAGGAATTTTCTCGCTTCATTTGGCCGGTGTATCGTCAATCTTAGGAGCAGTAAATTTTATTACTACTGTAATTAATATACGATCATATGGAATAATGATAGACCAAATACCACTATTTGTCTGATCAGTATTTATCACCGCAATCCTCCTACTTCTATCCCTACCGGTTCTAGCAGGAGCTATTAC. This record is deposited in Genbank under submission number KX949585.A mtDNA COI barcode was generated from this individual following standard Sanger sequencing protocols as outlined in Taxon classificationAnimaliaDecapodaPinnotheridaehttp://zoobank.org/FD849337-EB57-46F6-965D-8CECBADECD5FUSNM 1421642), in Leiosolenusobesus , from Njari Island, New Georgia, Solomon Islands, station SOLOM_026; 8.01374\u00b0S, 156.75649\u00b0E, coll. C. Meyer, 9 October, 2014.Holotype \u2640 (8.9 \u00d7 7.9 mm) large, subrectangular; articles 3\u20135 increasingly smaller, with flagellum very short, not extending beyond orbit symmetrical from left to right, generally similar in form; margins of carpus and propodus lined with dense, long setae, setation on merus less distinct and more spare; dactylus covered with sparse long setae Figs ; relativMXP3 in having the lateral margins of the dorsal carapace lamellum distinctly produced laterally to form a blunt angular lobe, with the posterolateral margin deeply concave (cf. Ng and Ahyong 2005) is similar to that of the holotype of Serenotheresjanus sp. n. (8.9 \u00d7 7.9 mm), so the differences observed cannot be explained by size.The type of PageBreakSerenotheresjanus sp. n. indicates a novel lineage among available Pinnotheridae sequences. The closest matches are 86\u201385% in sequence similarity to a handful of other pinnotherid genera including Zaops, Calyptraeotheres, Austinotheres, and Pinnixa Fig. . The fun"} +{"text": "Lysine acetylation is a common protein post-translational modification in bacteria and eukaryotes. Unlike phosphorylation, whose functional role in signaling has been established, it is unclear what regulatory mechanism acetylation plays and whether it is conserved across evolution. By performing a proteomic analysis of 48 phylogenetically distant bacteria, we discovered conserved acetylation sites on catalytically essential lysine residues that are invariant throughout evolution. Lysine acetylation removes the residue\u2019s charge and changes the shape of the pocket required for substrate or cofactor binding. Two-thirds of glycolytic and tricarboxylic acid (TCA) cycle enzymes are acetylated at these critical sites. Our data suggest that acetylation may play a direct role in metabolic regulation by switching off enzyme activity. We propose that protein acetylation is an ancient and widespread mechanism of protein activity regulation. Post-translational modifications can regulate the activity and localization of proteins inside the cell. Similar to phosphorylation, lysine acetylation is present in both eukaryotes and prokaryotes and modifies hundreds to thousands of proteins in cells. However, how lysine acetylation regulates protein function and whether such a mechanism is evolutionarily conserved is still poorly understood. Here, we investigated evolutionary and functional aspects of lysine acetylation by searching for acetylated lysines in a comprehensive proteomic data set from 48 phylogenetically distant bacteria. We found that lysine acetylation occurs in evolutionarily conserved lysine residues in catalytic sites of enzymes involved in central carbon metabolism. Moreover, this modification inhibits enzymatic activity. Our observations suggest that lysine acetylation is an evolutionarily conserved mechanism of controlling central metabolic activity by directly blocking enzyme active sites. Post-translational modifications (PTMs) play a critical role in regulating cellular function and response to external stimuli. PTMs, which include phosphorylation, glycosylation, and acetylation, are found in all cell types and lineages and thus likely arose early in evolution. Advances in mass spectrometry-based proteomics have greatly expanded the number of proteins that are known to be modified, but our ability to assign a functional role to specific modifications has lagged. Although acetylation has a well-known role in chromatin remodeling , it is n4\u2013In this paper, we investigated whether lysine acetylation is found on different enzymes and whether this modification is conserved throughout bacterial evolution by performing a comprehensive phyloproteomic analysis. We found that acetylation occurs in conserved lysine residues located in catalytic pockets of enzymes from the glycolytic pathway and the TCA cycle. Moreover, those modifications substantially changed the physical-chemical properties of the enzyme catalytic pocket inhibiting their activity.Proteobacteria, Firmicutes, Bacteroidetes, Actinobacteria, Cyanobacteria, and Fibrobacteres. Each bacterial strain was grown to early stationary phase database. Acetylated proteins were significantly enriched in glycolysis, pyruvate metabolism, ribosomes, and the TCA cycle, with median enrichment rganisms . AlthougDue to our interest in the mechanism of enzymatic regulation by acetylation, we narrowed our investigation to specific sites of acetylation and in particular those that are conserved across evolution. We focused on the set of enzymes involved in glycolysis and the TCA cycle and asked whether these enzymes contained lysines involved in substrate or cofactor binding and whether those lysines were identified as acetylated in diverse taxa, potentially pointing to a general regulatory mechanism common to all organisms. In glycolysis, there are nine enzymatic reactions for the conversion of glucose-6-phosphate to pyruvate. Seven of the nine enzymes contain catalytically important lysine residues in the active site . In four8\u2013We further investigated TCA cycle enzymes for catalytically essential and acetylated lysines. The transition from glycolysis (pyruvate) to the TCA cycle (acetyl-CoA) (part of the pyruvate metabolism pathway) involves a set of four enzymes that control the formation and therefore the pool of available acetyl-CoA. Three of the four enzymes contain lysine residues that participate in catalysis through substrate/cofactor binding. In both Acs and phos10.1128/mBio.01894-17.3TABLE\u00a0S3\u00a0TABLE\u00a0S3, XLS file, 0.03 MB.Lysine acetylation on conserved residues in enzyme catalytic regions. For enzymes in the glycolytic and TCA pathways, conserved sites of acetylation are listed. Each site is described by the gene, KEGG ortholog, protein accession number of one reference organism, acetylated sites, and the surrounding protein sequence. Acetylated sites are highlighted in yellow if they are known to bind substrate/cofactors. Download Copyright \u00a9 2017 Nakayasu et al.2017Nakayasu et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Synechococcus\u00a0elongatus enolase (Protein Data Bank [PDB] accession number 5J04) which contains the native substrate (phosphoenolpyruvate [PEP]), the two active site lysines are 2.4 and 2.8\u00a0\u00c5 from oxygen atoms in the substrate . The volume of the empty active site pocket was 162\u00a0\u00c53; with bound PEP, the volume was 70.2\u00a0\u00c53. After removing PEP and building acetyl groups on the K339 and K390 side chain amines, the volume of the pocket was 78.7\u00a0\u00c53. Thus, the acetylated lysines and the native substrate occupy a similar volume and position within the deeply buried active site were kept; all other nonconserved lysines were permuted with invariant lysines within their substrate binding site, whose acetylation was conserved and significantly upregulated in the presence of supplemented glucose , the lysine residues were conserved, suggesting that the use of lysine acetylation as a mechanism to control central metabolic activity is an ancient trait. We also note that acetylphosphate has been proposed to be the most primitive acetyl donor (before evolution of coenzyme A) and likely would have been present at the outset of the evolution of fundamental pathways, such as glycolysis and the TCA cycle and centrifuging under the same conditions. Cells were resuspended with 200\u00a0\u03bcl of 100\u00a0mM NH4HCO3 and lysed by a Bullet Blender (Next Advance) for 4\u00a0min at speed 8 in the presence of approximately 100\u00a0\u03bcl of 0.1-mm zirconia-silica beads at 4\u00b0C. Cell lysates were collected into fresh tubes, while the beads were washed with 200\u00a0\u03bcl of 100\u00a0mM NH4HCO3, and the supernatants were pooled together. The protein concentration was quantified by bicinchoninic acid (BCA) assay , and aliquots of 300\u00a0\u03bcg of proteins were denatured and reduced by adding 8 M urea and 5\u00a0mM dithiothreitol (DTT) and incubating at 60\u00b0C for 30\u00a0min with shaking at 850\u00a0rpm. The reaction mixtures were then diluted 10-fold in 100\u00a0mM NH4HCO3, and 1\u00a0M CaCl2 was added to a final concentration of 1\u00a0mM. Protein digestion was carried out for 3\u00a0h at 37\u00b0C with a 1/50 trypsin-protein ratio. Digested peptides were desalted in 50-mg C18 cartridges as previously described database (see \u201cData availability\u201d). Raw mass spectrometry files were converted to the PSI open format mzML . We tracked the functional enrichment for all pathways for all organisms. The data presented are the median enrichment of pathways across all organisms.All peptide spectrum matches were filtered for false-discovery rate using ostKoala . Figure\u00a0https://github.com/samuelpayne/TreeFigureForBiodiversityLibrary, utilizing the ETE 3 toolkit for phylogenomic data . Multiple-sequence alignments were performed using Muscle via a wemic data . This trmic data or the amic data . The annhttps://www.wwpdb.org/ website. The structures visualized in http://www.pymol.org). The enolase active site (PDB 5J04 structure) was analyzed with NACCESS 2.1.1 (5J04 structure have relative side chain atom accessibilities ranging from 0.6% to 94%. The distance from K390 N\u03b5 to phosphoenolpyruvate (PEP) O1\u2032 (one of the carboxylate O) is 2.4\u00a0\u00c5, while the distance from K339 N\u03b5 to PEP O2P (one of the phosphate O) is 2.8\u00a0\u00c5. The volume of the empty active site pocket in the PDB 5J04 structure was 162\u00a0\u00c53; with bound PEP, the volume was 70.2\u00a0\u00c53. After removing PEP and building acetyl groups on the K339 and K390 side chain amines, the volume of the pocket was 78.7\u00a0\u00c53.Protein structures from the Protein Data Bank (PDB) were identified via their UniProt accession number or manual search for a protein\u2019s functional annotation using the SS 2.1.1 and CASTSS 2.1.1 to gaugeLactobacillus\u00a0casei maps to K100 of E.\u00a0coli. For Eno, K339 from B.\u00a0subtilis maps to K342 of E.\u00a0coli. For Pgk, K201 of B.\u00a0subtilis maps to K197 of E.\u00a0coli. For LpdA, K56 of B.\u00a0subtilis maps to K54 of E.\u00a0coli. For Tpi, K11 of B.\u00a0subtilis maps to K11 of E.\u00a0coli. For Icd, K226 of B.\u00a0subtilis maps to K235 of E.\u00a0coli. Similar mapping was done for the sites identified by Kuhn et al. and MP043 (TGA CAA GCT TAG ATA GCG GCG GAT TTA GC). These primers were designed to remove the start codon and add a 5\u2032 NheI site and a 3\u2032 HindIII site. The B.\u00a0subtilis enolase was PCR amplified from B.\u00a0subtilis strain OI3269 using primers MP071 (GTC GGC TAG CCC ATA CAT TGT TGA TGT TTA TGC AC) and MP072 (TGA CCT CGA GGG TAA GGC TTT ATT TGG ATC TCT G). These primers were designed to remove the start codon and add a 5\u2032 NheI site and a 3\u2032 XhoI site. The mutant E.\u00a0coli 7xKtoQ enolase was made by ordering a gBlock from IDT (sequence below) in which lysines at positions 6, 62, 82, 85, 257, 328, and 419 were mutated to glutamines by changing the codons from AAA to CAA. The gBlock contained a 5\u2032 NheI site and a 3\u2032 HindIII site. The respective DNA fragment and the pET28a plasmid were then digested using the respective restriction enzymes, gel purified, and ligated together. The resulting plasmid constructs were then transformed in E.\u00a0coli BL21(DE3).Both the E.\u00a0coli 7xKtoQ enolase sequence (gBlock) follows (restriction sites and mutations are shown by lowercase letters): GTCGgctagcTCCAAAATCGTAcaaATCATCGGTCGTGAAATCATCGACTCCCGTGGTAACCCGACTGTTGAAGCCGAAGTACATCTGGAGGGTGGTTTCGTCGGTATGGCAGCTGCTCCGTCAGGTGCTTCTACTGGTTCCCGTGAAGCTCTGGAACTGCGCGATGGCGACAAATCCCGTTTCCTGGGTcaaGGCGTAACCAAAGCTGTTGCTGCGGTAAACGGCCCGATCGCTCAGGCGCTGATTGGCcaaGATGCTcaaGATCAGGCTGGCATTGACAAGATCATGATCGACCTGGACGGCACCGAAAACAAATCCAAATTCGGCGCGAACGCAATCCTGGCTGTATCTCTGGCTAACGCCAAAGCTGCTGCAGCTGCTAAAGGTATGCCGCTGTACGAGCACATCGCTGAACTGAACGGTACTCCGGGCAAATACTCTATGCCGGTTCCGATGATGAACATCATCAACGGTGGTGAGCACGCTGACAACAACGTTGATATCCAGGAATTCATGATTCAGCCGGTTGGCGCGAAAACTGTGAAAGAAGCCATCCGCATGGGTTCTGAAGTTTTCCATCACCTGGCAAAAGTTCTGAAAGCGAAAGGCATGAACACTGCTGTTGGTGACGAAGGTGGCTATGCGCCGAACCTGGGTTCCAACGCTGAAGCTCTGGCTGTTATCGCTGAAGCTGTTAAAGCTGCTGGTTATGAACTGGGCAAAGACATCACTTTGGCGATGGACTGCGCAGCTTCTGAATTCTACAAAGATGGTcaaTACGTTCTGGCTGGCGAAGGCAACAAAGCGTTCACCTCTGAAGAATTCACTCACTTCCTGGAAGAACTGACCAAACAGTACCCGATCGTTTCTATCGAAGACGGTCTGGACGAATCTGACTGGGACGGTTTCGCATACCAGACCAAAGTTCTGGGCGACAAAATCCAGCTGGTTGGTGACGACCTGTTCGTAACCAACACCAAGATCCTGcaaGAAGGTATCGAAAAAGGTATCGCTAACTCCATCCTGATCAAATTCAACCAGATCGGTTCTCTGACCGAAACTCTGGCTGCAATCAAGATGGCGAAAGATGCTGGCTACACTGCAGTTATCTCTCACCGTTCTGGCGAAACTGAAGACGCTACCATCGCTGACCTGGCTGTTGGTACTGCTGCAGGCCAGATCAAAACTGGTTCTATGAGCCGTTCTGACCGTGTTGCTAAATACAACCAGCTGATTCGTATCGAAGAAGCTCTGGGCGAAcaaGCACCGTACAACGGTCGTAAAGAGATCAAAGGCCAGGCATAAaagcttGTCA.The mutant 2, 0.1\u00a0mM dithiothreitol [DTT], 10% [vol/vol] glycerol) and then stored at \u221280\u00b0C.All enolase constructs were purified on a HisTrap column on an \u00c4KTA pure chromatography system using the denaturing protocol provided by the manufacturer. Fractions were collected and run on an SDS-polyacrylamide gel to check for the elution of the desired 6\u00d7His fusion protein. The pure-protein flowthrough was collected and dialyzed against four changes of 1\u00a0liter of TKMD , 10% glycerol, 10\u00a0mM MgCld-2-phosphoglycerate is converted to PEP, resulting in the formation of an intermediate that reacts with a peroxidase substrate, generating a colorimetric (570-nm) product proportional to the enolase activity present. The assay was repeated on three separate days using four dilutions of each enolase construct in acetylated and nonacetylated form to determine enolase activity.Enolase activity was measured using an enolase activity assay kit (Sigma-Aldrich). Briefly, this kit works via a coupled enzyme assay in which https://github.com/ or https://sourceforge.net/. Data format conversion and calibration were performed using msconvert, part of the proteowizard project (source code at http://proteowizard.sourceforge.net/). MSGF+ was used to annotate tandem mass spectral identification (source code at https://github.com/sangtaekim/msgfplus and webserver at https://massive.ucsd.edu/ProteoSAFe/static/massive.jsp). Postprocessing scripts as described above are available at https://github.com/samuelpayne/Biodiversity.Acetylation.Supplement.Coverage and https://github.com/samuelpayne/TreeFigureForBiodiversityLibrary.All data processing tools are publicly available at PXD005851.All primary mass spectrometry files and identification results are freely available at the proteomic data repository PRIDE under accession no."} +{"text": "The parasitic disease malaria remains a major global public health concern and no truly effective vaccine exists. One approach to the development of a malaria vaccine is to target the asexual blood stage that results in clinical symptoms. Most attempts have failed. New antigens such as P27A and P27 have emerged as potential new vaccine candidates. Multiple studies have demonstrated that antigens are more immunogenic and are better correlated with protection when presented on particulate delivery systems. One such particulate delivery system is the self-assembling protein nanoparticle (SAPN) that relies on coiled-coil domains of proteins to form stable nanoparticles. In the past we have used de novo designed amino acid domains to drive the formation of the coiled-coil scaffolds which present the antigenic epitopes on the particle surface.Here we use naturally occurring domains found in the tex1 protein to form the coiled-coil scaffolding of the nanoparticle. Thus, by engineering P27A and a new extended form of the coiled-coil domain P27 onto the N and C terminus of the SAPN protein monomer we have developed a particulate delivery system that effectively displays both antigens on a single particle that uses malaria tex1 sequences to form the nanoparticle scaffold. These particles are immunogenic in a murine model and induce immune responses similar to the ones observed in seropositive individuals in malaria endemic regions.Plasmodium species, these novel SAPNs may even provide cross-protection between Plasmodium falciparum and Plasmodium vivax the two major human malaria pathogens. As the SAPNs are also easy to manufacture and store they can be delivered to the population in need without complication thus providing a low cost malaria vaccine.We demonstrate that our P27/P27A-SAPNs induce an immune response akin to the one in seropositive individuals in Burkina Faso. Since P27 is highly conserved among different Plasmodium genus, resulted in at least 438,000 deaths worldwide in 2014 [Malaria, the mosquito-borne parasitic disease caused by members of the One of the most appealing, yet elusive, malaria vaccine candidates is one targeting the asexual blood stage, the clinical stage of the disease. Issues such as antigen polymorphism, lack of MHC I molecules on erythrocytes, and speed of erythrocyte infection all hamper the development of an asexual blood stage vaccine \u20139. Many P. falciparum in antibody-dependent cellular inhibition parasite-growth assay [In one such bioinformatic approach coiled-coil domains present in the asexual blood stage were targeted . Coiled-th assay . CurrentSubunit vaccines based solely on recombinant proteins are generally weakly immunogenic and will most likely not be successful for vaccination in humans. To be an effective vaccine an important consideration is the delivery system. Several recent effective vaccine candidates are based on particulate delivery systems that are able to repetitively express antigens . One proNormally, the sequence of amino acids that form the pentameric and trimeric oligomerization domains are de novo designed sequences that do not have homology to any human proteins to minimize the possibility of inducing an immune response that could be detrimental to the host receiving the vaccine. Here we detail the unusual step in the development of a SAPN vaccine candidate by using a parasite native coiled-coil sequence, the P27 epitope from the Tex1 protein, to form part of the core oligomerization domains. In combination with the intrinsically unstructured epitope P27A, this vaccine candidate is immunogenic in a murine model and induces antibodies that are recognizing the same antigens as sera from seropositive individuals in Burkina Faso, a malaria endemic region.Pept-P27-N (845\u2013871):KKRNVEEELHSLRKNYNIINEEIEEITPept-P27A (223\u2013326):HNNNEKNISYDKNLVKQENDNKDEARGNDNMCGNYDIHNERGEMLDKGKSYSGDEKINTSDN AKSCSGDEKVITSDNGKSYDYVKNESEEQEEKENMLNNKKRSPept-P27-NC (846\u2013893):KRNVEEELHSLRKNYNIINEEIEEITKEFEKKQEQVDEMILQIKNKELEPept-P27-C (872\u2013893):KEFEKKQEQVDEMILQIKNKELEPept-P27-N, Pept-P27A, Pept-P27-NC and Pept-P27-C present in the SAPN scaffold were synthesized by solid-phase Fmoc chemistry using Applied Biosystems 431A and 433A synthesizers as previously described [The single antigens escribed . PeptideSAPN-P27-1:MGHHHHHHASGSWEEWNAKWDEWIRAWVAWRAAWEKWKDDWAYWTLTWKYGELYSKLRNVEEELHSLRKNYNIINEEIEEITKEFEKKQEQVDEMIIQIKNKELESAPN-P27-2:MGHHHHHHASLIDYNKAALSKFKERGSWQTWNAKWDVWSNDWNAWRARWQAWVDDWAYWTLTWKYGELYSKLRNVEEELHSLRKNYNIINEEIEEITKEFEKKQEQVDEMIIQIKNKELESAPN-P27A-1:MGHHHHHHASGSWQTWNAKWDVWSNDWNAWRADWQAWVDDWAYWTLTWKYGELYSKLAEIERRVEANERALEELAQFVRALSMQAAELERRIEEIARGHNNNEKNISYDKNLVKQENDNKDEARGNDNMSGNYDIHNERGEMLDKGKSYSGDEKINTSDNAKSSSGDEKVITSDNGKSYDYVKNESEEQEEKENMLNNKKRSSAPN-P27A-2:MGHHHHHHASYYIPHQSSLPGSWQTWNAKWDVWSNDWNAWRADWQAWVDDWAYWTLTWKYGELYSKLAEIERRVEANERALEELAQFVRALSMQAAELSEDITKYFRHILYISFYFILVNRARGHNNNEKNISYDKNLVKQENDNKDEARGNDNMSGNYDIHNERGEMLDKGKSYSGDEKINTSDNAKSSSGDEKVITSDNGKSYDYVKNESEEQEEKENMLNNKKRSSAPN-Combo:MGDHHHHHHHHHHAAHAAHAAHAAHAAAARGHNNNEKNISYDKNLVKQENDNKDEARGNDNMCGNYDIHNERGEMLDKGKSYSGDEKINTSDNAKSCSGDEKVITSDNGKSYDYVKNESEEQEEKENMLNNKKRSASGSAKFVAAWTLKAAASGSWEEWNAKWDEWRNDQNDWREDWQAWRDDWAYWTLTWRYGELYSRLARIERRVEELRRLLQLIRHENRMVLQFVRALSMQARRLESKLRNVEEELHSLRKNYNIINEEIEEITKEFEKKQEQVDEMILQIKNKELEE. coli were synthesized by GeneScript USA Inc. containing the sequence of the constructs SAPN-P27-1, SAPN-P27-2, SAPN-P27A-1, SAPN-P27A-2 and SAPN-Combo. Each gene was cloned into a pPEP-T vector. The resulting constructs were transformed into Tuner (DE3) Competent Cells .Codon optimized genes for 600 of 0.8 cultures were induced with 1\u00a0mM Isopropyl-\u03b2-d-thiogalactopyranoside (IPTG) . Cultures were grown for 4\u00a0h, pelleted at 4000\u00d7\u00a0g and stored at \u221280\u00a0\u00baC until use.Protein expression was carried out in Luria\u2013Bertani (LB) Broth . Briefly, starter cultures were grown for 16\u00a0h at 36\u00a0\u00b0C and inoculated at 1/100 into 1L of LB and grown at 37\u00a0\u00b0C. At an OD2PO4, 20\u00a0mM Tris base, 5\u00a0mM tris(2-carboxyethyl)phosphine, pH 8.0), lysed by sonication, and centrifuged at 30,500\u00d7\u00a0g for 25\u00a0min to clarify the lysate. Purification was carried out on an \u00c4KTApurifier 100 using a 5\u00a0mL HisTrap HP column . Briefly columns were equilibrated with Binding Buffer phosphine, pH 8.0), lysate injected, and then washed with five column volumes of binding buffer. Columns were then washed with 5 column volumes of high phosphate buffer phosphine, pH 8.0), binding buffer, isopropanol wash to remove LPS [Cell pellets were thawed on ice and resuspended in Imidazole free buffer phosphine and resulting in refolded SAPN in 20\u00a0mM Tris base, 5% glycerol, pH 8.5.Refolding was monitored by dynamic light scattering using a Malvern Zetasizer Nano S equipped with a 633-nm laser. The hydrodynamic diameter was determined at 25\u00a0\u00b0C. Each sample was run 5 times, and the average result taken.0.025\u00a0mg/mL of each sample was adsorbed onto carbon coated grids that were subjected to a 10\u00a0s glow discharge, washed with distilled water three times, and negatively stained with 0.5% uranyl acetate . Electron micrographs were taken on an FEI Tecnai T12 Transmission Electron Microscope.Human plasma were collected during the malaria transmission season from adult donors living in Burkina Faso. Plasma from Swiss naive donors who had no malaria history were pooled and used as a negative control. Blood was taken by venipuncture into tubes containing EDTA according to the ethical clearance.BALB/c and C3H mice (5/group) were immunized with 10\u00a0\u03bcg of NP in PBS at the base of the tail three tiPrevalence (in humans) and titers (in mice) were determined by ELISA; antigens were diluted in PBS and used to coat ELISA plates. Antigen concentration for coating the 96-well flat plates was 1\u00a0\u03bcg/ml (50\u00a0\u03bcl) for peptides longer than 40 residues, and 5\u00a0\u03bcg/ml for peptides shorter or equal to 40 residues as previously described , 17. SecELISA plates were coated with the appropriate peptide as described above, washed and blocked. Serum samples at appropriate dilutions (at about 50\u201360% of maximum OD as determined by ELISA) were pre-incubated with appropriate competing synthetic peptides at increasing concentrations for 30\u00a0min at room temperature. 50\u00a0\u03bcl of the antibody-peptide mixture was added into the appropriate wells and incubated for 1\u00a0h at room temperature in a humid chamber. ELISA was then completed as described above.Plasmodium species \u201323. We bEach pentameric coiled-coiled domain will self-assemble with four other pentameric coiled-coiled domains, while each trimeric coiled-coiled domain will self-assemble with two other trimeric coiled-coil domains. To satisfy all of the assembly requirements 15 monomers will self-assemble into a structure known as a least common multiple unit (LCM). In a standard icosahedral structure four LCMs can further self-assemble into a full SAPN structure, meaning that 60 monomers make up each SAPN. However, recent biophysical studies combined with an analysis using viral tiling theory has shown that the SAPNs can possibly also assemble into higher order assemblies containing multiples of 60 chains per particle . Each SAP. falciparum.Another advantage of the SAPN design is that within the SAPN core universal T-helper epitopes can be engineered that ideally lead to more effective immune response. These epitopes range from de novo designed sequences that are engineered to bind to a wide range of MHC II haplotypes to sequences from known infectious agents that have been established to result as strong T-helper epitopes \u201337. TogeSAPN-P27-1, SAPN-P27-2, SAPN-P27A-1, SAPN-P27A-2 and SAPN-Combo is shown in Fig.\u00a0SAPN-P27-2 and SAPN-Combo are displayed in Fig.\u00a0SAPN-P27-1 and SAPN-P27-2) lead to severe aggregation, the constructs with the intrinsically unstructured epitope P27A (SAPN-P27A-1 and SAPN-P27A-2) formed very nice and soluble nanoparticles from thE. coli and purified under denaturing and reducing conditions using immobilized metal chromatography. Resulting protein was run on SDS-Page gel to verify that the correct protein was purified. The primary product was in the expected size range of 34.3\u00a0kDa.SAPN protein monomers were expressed in Tuner (DE3) SAPN-Combo has a hydrodynamic diameter of 26.4\u00a0nm, which falls into the expected range for refolded SAPNs in terms of % prevalence and average OD [The prevalence of the human antibody response specific to the so Table\u00a0. This reerage OD , 14.TablSAPN-Combo construct delivered in PBS buffer solution without addition of any adjuvant was tested in BALB/c and C3H mice. End point titers of about 1\u20132\u00a0\u00d7\u00a0106 were obtained when ELISA plates were coated with the homologous construct while partial inhibition (up to 50%) is observed when SAPN-Combo and single antigens (Pept-P27A and Pept-P27-NC) are used for coating and soluble inhibitors, respectively , thus improving its immunogenicity by coupling to a suitable carrier seems to be a highly promising approach.Pept-P27-C 62% is recognized by 62% of donor\u2019s sera from Burkina Faso induces a stronger immune response compared to the antibody production against the N-terminal region P27-N , rabbitIn summary, these new SAPNs represent a new vaccine strategy. The epitopes P27 and P27A are expressed primarily on the asexual blood stage of the parasite\u2019s life cycle, while CSP is expressed on the pre-erythrocytic stage. A CSP based vaccine will only prevent infection before clinical symptoms develop. An asexual blood stage vaccine will actually help deal with preventing parasitemia from further developing and reducing clinical symptoms. As both approaches continue to be developed they eventually can be admixed to generate a vaccine candidate that prevents the initial infection, and can eventually prevent any breakthrough infection.Plasmodium species. If confirmed in human clinical trials, it would allow the development of a robust vaccine easy to manufacture, store and deliver to the needed population. These vaccine characteristics have been and remain the \u201choly grail\u201d of vaccine development and formulation.Here we have demonstrated that SAPNs induce an immune response akin to the one in seropositive individuals in Burkina Faso. The SAPNs described in this manuscript represent a key improvement over previous designs. Modification of the nanoparticles allowed P27 to be presented, which was not possible with the first designs of the SAPNs. Since P27 is highly conserved, this might even provide cross-protection among different"} +{"text": "E-cadherin expression during porcine somatic cell reprogramming. The lipids supplement also makes a contribution to lipid droplets accumulation in the porcine iPSCs that resemble porcine preimplantation embryos. These findings may facilitate understanding of the lipid metabolism in porcine iPSCs and lay the foundation of bona fide porcine embryonic stem cell derivation.Large numbers of lipids exist in the porcine oocytes and early embryos and have the positive effects on their development, suggesting that the lipids may play an important role in pluripotency establishment and maintenance in pigs. However, the effects of lipids and their metabolites, such as fatty acids on reprogramming and the pluripotency gene expression of porcine-induced pluripotent stem cells (iPSCs), are unclear. Here, we generated the porcine iPSCs that resemble the mouse embryonic stem cells (ESCs) under lipid and fatty-acid-enriched cultural conditions (supplement of AlbuMAX). These porcine iPSCs show positive for the ESCs pluripotency markers and have the differentiation abilities to all three germ layers, and importantly, have the capability of aggregation into the inner cell mass (ICM) of porcine blastocysts. We further confirmed that lipid and fatty acid enriched condition can promote the cell proliferation and improve reprogramming efficiency by elevating cAMP levels. Interestingly, this lipids supplement promotes mesenchymal\u2013epithelial transition (MET) through the cAMP/PKA/CREB signal pathway and upregulates the Induced pluripotent stem cells (iPSCs) could be derived from somatic cell by the expression of transcription factors including OCT4, SOX2, KLF4 and c-MYC ,2. The i2+/PKC, PI3K/Akt, and MAPK signaling pathways [Currently, porcine iPSCs have been derived from several groups ,7,8. Howpathways . Lysophopathways . Sphingopathways ,13. Therpathways , but alspathways . Sphere pathways . In this study, porcine iPSCs were generated and cultured in the conditions containing AlbuMAX. The iPSCs show pluripotency and differentiation potential, and abilities of aggregation into both porcine parthenogenetic blastocysts and mouse early embryos. Genes associated with fatty acid metabolism were upregulated in the iPSCs. In addition, AlbuMAX upregulated cAMP levels and promoted mesenchymal\u2013epithelial transition (MET) through cAMP/PKA/CREB signal pathway, leading to enhance the reprogramming efficiency.During porcine embryonic fibroblasts (PEF) reprogramming, we counted the numbers of alkaline phosphatase-(AP) positive cells and analyzed the positive cells of pluripotency surface marker SSEA-1. AP-positive colonies in the medium including AlubMAX were more than that in control group A. BesideThe LpiPSCs that were morphologically similar to mouse ESCs were domed and three-dimensional A. In ordTo test the capability of aggregation of LpiPSCs into porcine embryos, we injected LpiPSCs into porcine parthenogenetic embryos at the eight-cell stage and examined when embryos developed to the blastocyst stage. The results indicated that LpiPSCs with green fluorescence incorporated into inner cell masses (ICM) of porcine embryos F. Cross-As shown in AP staining during reprogramming, more AP-positive cells were in LpiPSCs than the control. Therefore, AlbuMAX may promote cell proliferation. In order to confirm it, we performed the Bromodeoxyuridine immunofluorescent staining and flow cytometry analysis of BrdU positive cells in iPSCs. The results revealed that a lot of BrdU positive cells existed in LpiPSCs. The proportions were 35.31% A,B. As aNanog and EpCAM expression levels increased as compared to the control group , transport and metabolism were up-regulated significantly in LpiPSCs A, which E-cadherin and EpCAM were upregulated significantly at different stages , such as both sphingosine-1-phosphate and lysophosphatidic acid, which may activate the downstream cAMP/PKA signaling pathway . We hypoE-cadherin promoter. Therefore, we constructed a luciferase reporter and the mutation vector of E-cadherin promoter containing CREB binding sites and used for the teratoma assay. The animal study proposal in this study were approved by the Animal Care and Use Committee of China Agricultural University.Retroviral virus vectors (pMXs vector) separately carrying porcine Oct4, Sox2, Klf4 and c-Myc were used to generate iPSCs. The viral production was performed as the method described . RetroviThe reprogramming medium and the porcine iPSCs medium contained Dulbecco's Modified Eagle Medium (DMEM), 15% fetal bovine serum (FBS) , nonessential amino acids, Gluta-MAX, penicillin/streptomycin , \u03b2-mercaptoethanol , human LIF, 2i (CHIR99021 and PD0325901) and 1% AlbuMAX . The medium in the control group contained 15% fetal bovine serum (FBS), nonessential amino acids, Gluta-MAX, penicillin/streptomycin, \u03b2-mercaptoethanol (Sigma), human LIF, 2i (CHIR99021 and PD0325901) (Selleck) and 1% BSA . The iPSCs were passaged by TrypLE at a ratio of 1:6 every 2\u20133 days.2 incubator.For embryoid body formation, the iPSCs were digested and maintained in the medium mentioned above removal of human LIF, 2i (CHIR99021 and PD0325901) and AlbuMAX on a shaker (40 r/min) in a COTotal RNA was extracted using a Qiagen RNeasy mini RNA kit and then reverse-transcribed by Oligo-dT primer and M-MLV Reverse Transcriptase . The cDNAs were used for quantitative RT-PCR analysis with LightCycler 480 SYBR Green I Master Kit . The data was analyzed using the comparative CT method. Forward (5\u2032 to 3\u2032)Reverse (5\u2032 to 3\u2032)ReferencespMXs-Oct4GACGGCATCGCAGCTTGGATACACGAGAAGGCGAAGTCGGAAGpMXs-Sox2GACGGCATCGCAGCTTGGATACACGGCTGTTCTTCTGGTTGCpMXs-Klf4GACGGCATCGCAGCTTGGATACACGTCTTTGCTTCATGTGGGpMXs-MycGACGGCATCGCAGCTTGGATACACGAAATAAGGCTGCACCGAGTNanogCATCTGCTGAGACCCTCGACGGGCTTGTGGAAGAATCAGGEF-1\u03b1AATGCGGTGGGATCGACAAACACGCTCACGTTCAGCCTTTEndo-Oct4CAAACTGAGGTGCCTGCCCTTCCAAACTGAGGTGCCTGCCCTTCEndo-Sox2CATCAACGGTACACTGCCTCTCACTCTCCTCCCATTTCCCTCTTTEndo-Klf4CATGAGTTGGGGGAGGGAAGACTCACCAAGCACCATCGTTEndo-MycATCCAAGACCACCACCACTGATCCAAGACCACCACCACTGCdh1TGGGCCGAGTGAGTTTTGAATGACTGTAACCACACCGTCGSall4ATCGACGTTTATCCGAGCCCATCGACGTTTATCCGAGCCCEpCAMTGCTCTTTGAATGCGCTTGGAGAGCCCATCGTTGTTCTGGEsrrbCCGGACAAACTCTACGCCATGCTTGGCCCAACCAATGATGCdc2TTTTCAAAGCTGGCTCTGGGAGGGATGTGGTAGATCCCAGCTTACcncAGAAAGATGCCAGACGGTGGAGGAGGTTTTGGTTTCGGCACcnb1AGATCGCAGCAGGAGCTTTTCCTCGATTCACCACGACGATCcna2CTAACATTGCAGCAGACGGCATCCTTAAGAGGCGCAACCCAcox2AGGACTCAGGACGAGACACATTGAAGGACGGCATGCATCTPrkaa2ATTCTGTCACTGCGGAGAGCAATCCATGGTGTGACTGCCCAcadvlGAAGTCAAATGCCTGCCAGCATGTTGGCGCTCACCATGTAAcadsbACACCAAGTGGCTCATACGGTACCAATCTTCGCGTCTCGGFabp5AGGCACCAGTCCGCTTATTCTTTCGTAGGGCCATTCCCACThe primers were as bellows:The alkaline phosphatase (AP) staining was performed using an Alkaline Phosphatase Detection Kit according to the manufacturer\u2019s instructions.For immunofluorescence staining, cells were fixed in 4% PFA for 20 min, permeabilized with 0.2% Triton X-100 for 15 min, and blocked in the blocking buffer for 1 h. Primary and secondary antibodies were incubated overnight at 4 \u00b0C and for 1 h at room temperature, respectively. Membrane proteins were not permeabilized in the 0.2% Triton X-100. Nuclei were stained with Hoechst 333342 for 2 min at room temperature. The primary antibodies were as follows: OCT4 , SOX2 , SSEA-1 , Nanog , SOX17 , \u03b1-SMA , \u03b2-III-TUBULIN and BrdU . The secondary antibodies used here were as follows: goat anti-rabbit Alexa Fluor 594 , goat anti-mouse Alexa Fluor 594 , goat anti-mouse Alexa Fluor 594 , and goat anti-chicken Alexa Fluor 594 .For lipid droplets staining using Nile Red . The cells were fixed with 4% PFA for 20 min at room temperature and washed three times with Dulbecco's Phosphate Buffered Saline (DPBS) and then incubated with 5 ng/ml Nile Red for 5 min at room temperature.For flow cytometry analysis, single cells after digestion of colonies with Tryple were stained with primary antibody and secondary antibody for 1 h, respectively. These cells were then washed three times with DPBS and filtered through a 40-\u03bcm falcon. Fluorescence activated Cell Sorting (FACS) analysis was performed using the Moflo-XDP .7) were subcutaneously injected into the BALB/c nude mice. After two months, teratoma were harvested for histological analysis by hematoxylin and eosin (H&E) staining. For H&E staining, nuclei were stained with the alum hematoxylin and eosinophilic structures were stained with eosin.For teratoma formation, the porcine iPSCs (2 \u00d7 10The maturation and parthenogenetic activation (PA) of porcine oocytes were performed as described . Embryosg for 15 min (4 \u00b0C), supernatant was collected. The protein concentration of samples was measured using the BCA Protein Assay Kit (Beyotime). The samples were boiled at 99 \u00b0C for 5 min and were transferred on ice. The denatured protein samples were used for Sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride (PVDF) membranes. The PVDF membranes were in 5% nonfat milk for blocking and probed overnight at 4 \u00b0C with primary antibodies. Secondary antibodies were then probed for 1 h at room temperature. Protein bands were finally detected by SuperSignal West Dura Extended Duration Substrate .Cells were washed in cold DPBS and then lysed in sodium dodecyl sulfate (SDS) sample buffer on ice for 30 min. After centrifugation at 10,000\u00d7 \u00ae reagents were supplemented into all reactions and incubated for 10 min. The luminescence measurement values were used to calculate the cAMP concentration according to the kit manual.The cAMP levels of cells were detected using the cAMP-Glo assay kit according to the protocol (Promega). Cells were treated with an agonist or in the induction buffer for the desired length of time and then lysis buffer was added. The plate was incubated with shaking for 15 min. The cAMP detection solution was prepared and added to all wells, mixed and incubated for 20 min. Kinase-GloE-cadherin promoter were synthesized directly as shown below:Top chainCGTCAAGAGCCATCTGAAGGAGAGGCatcgagACTCTGTCTCAGTTATTTTTCCCTBottom chainagcttGCCTCTCCTTCAGATGGCTCTTGACGAGGGAAAAATAACTGAGACAGAGTcCREB binding cites in the Bases in lower-case were the cohesive ends. Bases in upper-case were the binding sites. In mutation vector construction, mutation cites were designed in the primers and mutation vectors were amplified by rolling circle amplification. CGTAC were transformed to CTGCA in mutation vectors.ForwardCTGCAAGAGCCATCTAGTTATTTTTCCCTReverseAGATGGCTCTTGCAGAGGGAAAAATAACTThe primers were shown as below:Luciferase activity analysis was performed following cell transfection 24 h using the Dual-Glo Luciferase Assay System (Promega)."} +{"text": "Ash1l upregulates Smad3 expression by directly targeting Smad3 promoter to increase local H3K4 trimethylation. Furthermore, we identify an lncRNA, namely lnc-Smad3, which interacts with the histone deacetylase HDAC1 and silences Smad3 transcription. After TGF-\u03b2 stimulation, activated Smad3 suppresses lnc-Smad3 transcription, thereby recovering the Smad3 promoter accessibility to Ash1l. By revealing the opposite regulatory functions of Ash1l and lnc-Smad3 in Smad3 expression, our data provide insights for the epigenetic control of Treg cell fate to potentially aid in the development of therapeutic intervention for autoimmune diseases.Regulatory T (Treg) cells are important for the maintenance of immune homoeostasis and prevention of autoimmune diseases. Epigenetic modifications have been reported to modulate autoimmunity by altering Treg cell fate. Here we show that the H3K4 methyltransferase Ash1l facilitates TGF-\u03b2-induced Treg cell polarization Smad3 locus.The transcriptional program activated by Smad2/Smad3 is critical for the induction and function of regulatory T cells. Here the authors show that the expression of Smad3 is modulated by the complementary functions of a methyltransferase Ash1l and an lncRNA lnc-Smad3 on the promoter accessibility of the mouse Foxp3 locus and promotes its expression, subsequently leading to Treg cell polarization6Regulatory T (Treg) cells are essential for immune homoeostasis by suppressing effector T cell responses during infection, inflammation and autoimmunity134In addition to the well-established functions of transcription factors and cytokines in Treg cell development, other cues, such as epigenetic modifications, are also involved in Treg cell fate810Tnfaip3 promoter region to induce its expression. Ash1l-silenced mice are more susceptible to bacterial infection and autoimmune diseasesLysine methylation is one of the most characterized histone modifications to date. In particular, H3K4 methylation associated with transcriptional activation is critical for the maintenance of cell fates+ T cells+CD8+ thymocytes in vitro, suggesting that Ash1l may regulate early thymic T cell developmentAsh1l gene also constitutes part of the Idd17 locus associated with increased susceptibility to autoimmune diabetes in NOD miceAsh1l is highly expressed in CD4in vitro. By contrast, a newly-identified long non-coding RNA (lncRNA), lnc-Smad3, recruits HDAC1 to the Smad3 promoter and selectively suppresses Smad3 but not Foxp3 expression. Interestingly, activation of the TGF-\u03b2/Smad3 axis suppresses lnc-Smad3 transcription, restoring accessibility of the Smad3 promoter to Ash1l. Ash1l-silenced mice are more susceptible to T cell-mediated colitis due to the impairment of Treg cell polarization. Lastly, ASH1L, FOXP3 and SMAD3 are downregulated in peripheral CD4+ T cells from patients with rheumatoid arthritis. Our results provide insights for the epigenetic control of Treg cell polarization during immune homoeostasis, and suggest a possible association between Ash1l and immune disorders.In this study, we show that Ash1l upregulates Smad3 expression by directly activating its promoter, and thereby promoting Foxp3 expression and induced Treg (iTreg) cell differentiation + T cells, which inspired us to further analyse the function of Ash1l in T cell development and differentiationAsh1l allele as compared with wild-type (WT) mice+ and CD8+ T cells in splenocytes from Ash1l-silenced mice was normal+ single-positive (CD4SP) and CD8SP thymocytes, activated CD44hiCD62Llo CD4+ T cells in the spleen and mesenteric lymph nodes (mLN) were also normal in Ash1l-silenced mice cell-skewing conditions to analyse the function of Ash1l in differentiation of CD4+ T cell subsets. Notably, we observed significantly impaired iTreg cell differentiation, but increased Th1 and Th17 differentiation in Ash1l-silenced CD4+ T cells as compared with WT CD4+ T cells . Accordingly, Ash1l-silenced Foxp3+ iTreg cells showed lower suppressive functions, with reduced expression of costimulatory molecules CD25, CTLA-4 and GITR on iTreg cells lentiviruses expressing 1\u2013880 amino acids (aa), 881\u20131,855 aa and 1,886\u20132,958 aa containing SET domain of Ash1l, respectively. We previously showed that the single amino acid point mutation Ash1ll\u0394N (N2212I) had abolished H3K4 methyltransferase activity T cells . Collectin vivo. We first investigated the susceptibility of Ash1l-silenced mice to the induction of 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis. Compared to WT mice, Ash1l-silenced mice developed more severe colitis upon TNBS induction, with a sharper loss of body weight, more exaggerated shortening of colon in appearance, and more severe colon inflammation as indicated by extensive infiltration of mononuclear cells, reduction of goblet cells and mucosa erosion in histology of patients with rheumatoid arthritis as compared to healthy controls located 165 kb upstream of Smad3 , and the occupancy of Pol II were downregulated at r region . This waSmad3 transcription. Since lncRNAs usually regulate neighbouring genes by recruiting specific chromatin remodelersSmad3 promoter, we transfected HepG2 cells with lnc-Smad3 expression vector or empty control vector, as well as Smad3 promoter luciferase-reporter construct under TGF-\u03b2 stimulation. The luciferase activity was lower in the presence of exogenous lnc-Smad3 than that with empty control ; CD45.1+ mice were from The Jackson Laboratory . All mice were maintained under specific pathogen-free conditions and used at 6\u20138 weeks of age. All animal experiments were approved by the Scientific Investigation Board of Second Military Medical University, Shanghai.Ash1l-silenced mice on FVB background were from Dr Tian Xu , and were backcrossed (12 polarizations) onto the C57BL/6J backgroundA total of 10 rheumatoid arthritis (RA) and 9 healthy control patients were recruited from Peking Union Medical College Hospital and Peking University People's Hospital and their peripheral blood samples were prepared from patients after informed consent was providedAnti-mouse CD11c, CD8 and B220-coated magnetic beads were from Miltenyi Biotech. Recombinant mouse GM-CSF and IL-4 were from Peprotech EC. APC-conjugated anti-mouse Foxp3 (FJK-16s), GITR (DTA-1), IFN-\u03b3 (XMG1.2), CTLA-4 (UC10-4B9), FITC-conjugated anti-mouse CD8a (53-6.7), IL-4 (BVD6-24G2), IL-10 (JES5-16E3), and anti-IFN-\u03b3 (R4-6A2), anti-IL-4 (11B11) mAbs were from eBioscience. FITC-conjugated anti-mouse Foxp3 (MF23), CD25 (7D4), PE-Cy7-conjugated anti-mouse CD4 (RM4-5), APC-conjugated anti-mouse CD62L (MEL-14), and anti-CD3 (145-2C11), anti-CD28 (37-51) mAbs, Golgi Plug were from BD PharMingen. FITC-conjugated anti-mouse CD44 (IM7), APC-conjugated IL-17A (TC11-18H10), recombinant mouse IL-2, IL-6 and IL-12 were from BioLegend. Recombinant human TGF-\u03b2 was from R&D Systems. Antibody specific to Ash1l (sc-98301X) was from Santa Cruz. Antibody specific to HDAC1 (ab7028) was from Abcam. The antibodies for cell staining were used at recommended dilution rates according to the manufacturers' instructions. TRIzol reagents were from Invitrogen. PrimeScript RT-PCR Kit (2641A) and SYBR Premix ExTaq Kit (RR420) were from Takara Bio Inc. RPMI1640 medium and FBS were from PAA laboratories.g, 30\u2009min). CD4+ T cells were sorted from fresh PBMCs with PerCP-conjugated anti-human CD4 antibody by FACS Aria II flow cytometer (BD Biosciences). The FACS sorting strategy was introduced in Human PBMCs were collected and purified by Ficoll-Paque PLUS density gradient centrifugation. In brief, collected whole-blood samples were diluted and mixed with Hanks' balanced salt solution (HBSS). Then blood mixture was slowly added to layer over Ficoll. PBMCs were collected at the interface of the upper layer and the Ficoll after centrifugation or thymus with mouse naive CD4+ T cell isolation kit and cultured under neutral (Th0) conditions with anti-CD3 (1\u2009\u03bcg\u2009ml\u22121), soluble anti-CD28 mAbs (1\u2009\u03bcg\u2009ml\u22121) and 10% (vol/vol) FBS in RPMI1640, unless otherwise indicated. For T cell polarization, CD4+ T cells were cultured in the neutral medium described above and stimulated with polarizing cytokines as follows: for iTreg, anti-IFN-\u03b3 (10\u2009\u03bcg\u2009ml\u22121), anti-IL-4 (10\u2009\u03bcg\u2009ml\u22121), IL-2 (5\u2009ng\u2009ml\u22121) and recombinant human TGF-\u03b2 (10\u2009ng\u2009ml\u22121); for Th1, anti-IL-4 (10\u2009\u03bcg\u2009ml\u22121), IL-2 (5\u2009ng\u2009ml\u22121) and IL-12 (10\u2009ng\u2009ml\u22121); and for Th17, anti-IFN-\u03b3 (10\u2009\u03bcg\u2009ml\u22121), anti-IL-4 (10\u2009\u03bcg\u2009ml\u22121), human IL-6 (10\u2009ng\u2009ml\u22121) and recombinant human TGF-\u03b2 (1\u2009ng\u2009ml\u22121). Differentiated iTreg cells were further enriched with mouse CD25 positive selection kit .Naive CD44 carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled naive CD4+ effector T cells from CD45.1 mice were cultured with 1 \u00d7 104 CD11c+ DC used as antigen-presenting cells in 96-well round-bottom plates. Overall, 2.5 \u00d7 104 differentiated and purified WT or Ash1l-silenced iTreg cells were added as suppressive cells. Cells were stimulated with anti-CD3 (1\u2009\u03bcg\u2009ml\u22121) for 3 days. T cell proliferation was determined by CFSE dilution with flow cytometry.A total of 5 \u00d7 10\u2212/\u2212 mice were injected intravenously with 4 \u00d7 105 CD4+CD25\u2212 cells from CD45.1 WT mice with or without 1 \u00d7 105 WT or Ash1l-silenced CD4+CD25+ differentiated iTreg cells. Weight changes were monitored weekly. Mice were killed at week 6 and subjected to examination of colon morphology and histology.Mice were presensitizated with 150\u2009\u03bcl of 1% (w/v) TNBS solution diluted in acetone/olive oil on the back. On day 7, mice were further given 100\u2009\u03bcl of 2.5% (w/v) TNBS solution (diluted in 50% ethanol) or ethanol only intrarectally through a catheter inserted into the colon 4\u2009cm proximal to the anus. Beginning from the second sensitization, inflammatory bowel disease development was monitored and recorded by measuring the body weight daily. Mice were killed 3 days after the second sensitization and subjected to examination of colon morphology and histology, as well as Treg cells proportion. T cell adoptive transfer colitis was performed as follows. Rag1\u2212/\u2212 mice were injected intravenously with 4 \u00d7 105 CD4+CD25\u2212 cells from WT or Ash1l-silenced mice. Monitored the weight changes and scarified the mice at week 4. T cells were isolated and Foxp3 expression was analysed with flow cytometry.Rag1For cell surface staining, the single-cell suspensions were incubated with the antibody cocktails for 20\u2009min at 4\u2009\u00b0C. For intracellular staining, cells were fixed and permeabilized with 4% paraformaldehyde for 20\u2009min at 4\u2009\u00b0C followed by intracellular staining with specific anti-cytokine antibodies. Data were obtained on an LSR II and analysed with FACSDiva software (both from BD Biosciences). The FACS gating strategies were represented in SYBR RT-PCR kit (Takara) and LightCycler (Roche) were used for quantitative RT-PCR analysisCells were lysed in 0.1% NP40 ice-cold PBS with protease inhibitor cocktail and Ribonucleoside Vanadyl Complex (10\u2009mM) (New England BioLabs), then after short centrifugation, the supernatant was collected as cytoplasmic fraction and the remainder with additional washing were considered as nuclear pellets.The activated Smad2/3 in nuclear extract or the whole-cell lysate was detected with Smad2/3 ELISA Kit according to the manufacture's protocol . In brief, 5\u2009\u03bcg nuclear extract or the whole-cell lysate was added in the 96-well clear plate pre-immobilized with the Smad2/3 consensus sequencing oligo, incubated and washed. Then the activated Smad2/3 binding to the oligo was detected with a specific antibody against Smad2/3 subunit and a HRP-conjugated secondary antibody. The optical density of each well was determined with a microplate reader at 450\u2009nm.+ T cells and then subcloned into the pcDNA3.1 eukaryotic expression vector (Invitrogen). Lnc-Smad3, Ash1l-fragment 1, 2, 3, and Ash1l-fragment3 mutant expression vectors were constructed by PCR-based amplification from cDNA of mouse CD4+ T cells and then subcloned into pCDH cDNA Cloning and Expression Lentivectors (System Biosciences). All constructs were confirmed by DNA sequencing. These plasmids transfected into HEK293T cell line (from American Type Culture Collection) with JetPEI reagents (Polyplus), and the protein coding capacity was tested by western blotting assay. The Smad3 promoter luciferase reporter was constructed via cloning the fragment of the Smad3 promoter into the pGL3 luciferase-reporter vector (Promega) and the DNA sequence of the insert was verified.Recombinant vectors encoding mouse Smad3 (NM_016769.4) were constructed by PCR-based amplification from cDNA of mouse CD44 cells per well, 96-well plate, from American Type Culture Collection) were transiently transfected with the lnc-Smad3 expression vector, or empty control vector as well as the Smad3 promoter firefly luciferase-reporter construct and Renilla luciferase-reporter vector (Promega) with Fugene HD (Promega). Cells were stimulated with TGF-\u03b2 24\u2009h after transfection. Twenty-four hours after stimulation luciferase expression was determined by measuring luminescence with the Dual-Luciferase Reporter Assay System (Promega). The firefly luciferase activity was normalized to renilla luciferase activity.HepG2 cells (3 \u00d7 10+ T cells were stimulated with plate-coated anti-CD3 (1\u2009\u03bcg\u2009ml\u22121), soluble anti-CD28 mAbs (1\u2009\u03bcg\u2009ml\u22121) and IL-2 (10\u2009ng\u2009ml\u22121) for 24\u2009h before knockdown or overexpression assay. For knockdown assay, the target sequence of lnc-Smad3 was 5\u2032-CATTGGACCATTTGATTCTTCCTAA-3\u2032 (bolded and underlined in the following lnc-Smad3 sequence); the control siRNA sequence was 5\u2032-CATCCAGTTTATTAGCTTCCGTTAA-3\u2032. siRNA was transfected into activated CD4+ naive T cells through the use of Lipofectamine 2000 according to the manufacture's protocol (Life Technologies). For overexpression experiments, virus solutions containing lentivirus and polybrene (8\u2009\u03bcg\u2009ml\u22121) were added to the activated CD4+ T cells, followed by centrifugation at 2,500\u2009r.p.m for 2\u2009h at room temperature. Virus supernatants were replaced with fresh culture medium at 6\u2009h after infection. Cells were cultured for an additional 18\u2009h and then sorted as enhanced green fluorescent protein (EGFP)-positive cells. Sorted cells were cultured for 3 days under iTreg cell conditions. Transduction efficiency was determined by EGFP expression and knockdown efficiency was measured by RT-PCR.Naive CD4CATTGGACCATTTGATTCTTCCTAAAGAAAGGCCAAACCACCTGTCAAACCCACGAGCAACACAGCAATGGCTAAGCTGAAGAAGCCAAGTTATATGTCTAGCTCCTCTGTGGGTGGCAGGATCCAGCTGTGGGGAGTCCAAGGTTCCTTGTGTTCTTCTGGCTTGACTATGATATTTCCAATTTGGAGGCACAAAGAATTGAGAACTTGGGGATCCACTGAGGTCTGAGGCATGGTGGTACCAGAAGAAATAAAGGGGCCATCAGCATGAAGCTGGTCTCCCATTGACAGCACCCTGACTTCTCCATCAGGTTCTATCAGCATGTCTACTGTGAGGTTGGTGACGCTGTCTGCAGGTGGGTATGCTTCAATCACGCCACCTGGTCAGGAAACCATGGAGTAGAGGAAAAGACACAGTCAAGGGAATGCTATGCTTATAGCAGGAGGAAATGCTCTGTGTTTTAGCAAGGGAACTTAGACTATCTCACACCTGTCATATCCTGGGAAACTTTGGGTGAGTAGTTCAACCTTTGGCCTCCTTTTCCTTATTTATAGAATGGAGGCATAATATCTACCTTGCCAAGCTTTTGTGAAGTTTGAGAAACTGAGTCTATGGTATTTAGCCTGGTACTTGGAACATGGGAGGGCTCAATGAACAGTAAGTAGTCTTTATTATGATCATAATTACAAATATTACAAATACTTTATAAAAGCCATACTTAGAACAACTCATTACGTTAATAAAGAATCCAAGGATTGTGGTC>mouse lnc-Smad3 transcript (GenBank Accession number: KY652933) CGGCGCGTGCGCACAGGACGGGACGGGAGGGCGGAGCGACTGCGCAGATCAGGAAAATTGTCACCTCTGGCCTCAGGGAAACTGAGGCTCTGAGCAGTTAAGAGGCCAACGATCCAGGTTTATGCTATCAGTGTCTGAGATACAATTAAGTCACCTTTTTGGGTGACATTTCCCTTGACTAGTACCTCAAGATTATGATCCAGGTGGACCATCCCTCCATTGCCTCCAGACATGTCTATGACGCTACCTAGGGATTGTGAAGATTTACCACCTGGTGGAAAATTAAAAAAAAAGACTTATTCTGCAAATTGACCAAGCTTAAGAGATACAGCACTGAGAATTCACCCACTACAGAGCTGGTAACCAGGCCTTCAGAGTAAGCTGTGATGTATAGCCATTCTCCTCAGCAGCCTGTTTGACTGAGGGATATAGGAATGACTGCCCTTACCCATGTTTTTGTTCAGATTTATGGTTCTAAATCTGATGGAAAATCTATCHIP analyses were performed with the following antibodies: RNA Polymerase II (Pol II), acetyl-Histone H3 (Lys27), trimethyl-Histone H3 (Lys4), Ash1l and HDAC1. Fold enrichment was quantified using quantitative RT-PCR and calculated as a percentage of Input chromatin (% input). Sequences of the primers for amplification of the Smad2, Smad3, Smad4, Foxp3 and lnc-Smad3 promoter regions are in \u22121, Promega) at 37\u2009\u00b0C for 30\u2009min and then stopped by EDTA (50\u2009mM). Genome DNA was extracted and subjected to quantitative RT-PCR. Data were presented as changed fold concluded with 2\u0394Ct, with relative to CD4+ T cells transduced with Lenti-CTR, set as 1.For chromatin accessibility analysis, nucleus were pretreated with DNase I .Total RNA extracted from mouse B220RNA immunoprecipitation (RIP) was performed with antibodies specific to mouse Ash1l, Smad3 and HDAC1 by using Magna RIP RNA-Binding Protein Immunoprecipitation Kit according to the manufacture's protocol .+T cells were fixed in 4% formaldehyde plus 10% acetic acid in PBS for 15\u2009min at room temperature, and then were permeabilized in PBS plus 0.2%\u20130.5% Triton X-100 and 5\u2009mM vanadyl ribonucleoside complex (10\u2009mM) (New England BioLabs) for 5\u2009min on ice, washed in PBS three times and rinsed once in 2 \u00d7 SSC bufferFluorescence-conjugated lnc-Smad3 probes were used for FISH assay. Naive CD4t test. P values of <0.05 were considered statistically significant .The statistical significance between two groups was determined by Student's All the data supporting the findings of this study are available within the article and its How to cite this article: Xia, M. et al. Ash1l and lnc-Smad3 coordinate Smad3 locus accessibility to modulate iTreg polarization and T cell autoimmunity. Nat. Commun.8, 15818 doi: 10.1038/ncomms15818 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations."} +{"text": "The Malarial parasite resides in the host RBC during its erythrocytic cycle. Plasmodium meets its entire nutritional requirement fromRBC. It scavenges the hemoglobin of RBCs to meet its amino acid requirement. The host hemoglobin is made of different chains and itis dependent on age. Hemoglobin F (HbF), which has two-alpha and two gamma chain persists in children upto six years, andhemoglobin A (HbA) made of two alpha and two beta chains dominates. Therefore, it is of interest to compare the compositionalfeatures of HbA with HbF. Isoleucine is present in hemoglobin of children (gamma chain of HbF) while it is absent in adulthemoglobin (HbA). The presence of Isoleucine (I) makes HbF ideally suitable for the growth of parasite, as it does not have to dependupon the exogenous supply of the isoleucine, which might be responsible for making children more vulnerable to malaria as comparedto adults. Malaria is one of the most prevalent diseases in developingcountries . MalariaMalaria affects all age groups. However, the children are affectedthe most. According to WHO report there were an estimated 438000 malaria deaths around the world in 2015, 69% of total deathsdue to malaria are known to occur in children aged from 6months to 5 years .Though the parasite culture in RBC containing adult Hb isroutinely performed The studIn this study, we show the abundance of different amino acids inPlasmodium falciparum 3D7 and distribution of different aminoacids according to its essential nature in host. We have alsocompared the amino acid composition of different chains ofhaemoglobin to determine the difference that leads to preferenceof RBC containing HbF, which might explain the disease severityin children.The FASTA format of all protein sequence, 5,369 proteins, ofPlasmodium was downloaded from PlasmoDB. Composition ofprotein sequences was completed using ProtParaman tool.ProtParaman tool is an online tool, which is freely available anddetermines the sequence composition and predicts other physicalparameters . Total aComplete protein sequences of 5369 were downloaded fromPlasmoDB for Plasmodium falciparum 3D7. The amino acid compositions of total 5369 proteins are represented by pie chart. As The The protein sequence of the haemoglobin chain was downloadedfrom NCBI and the composition of amino acid was determinedusing ProtParaman tool as descrThe fasta formats for different chain of the haemoglobins wereobtained from NCBI. The assertion number of different chains ofare as follows:MGHFTEEDKATITSLWGKVNVEDAGGETLGRLLVVYPWTQRFFDSFGNLSSASAIMGNPKVKAHGKKVLTSLGDATKHLDDLKGTFAQLSELHCDKLHVDPENFKLLGNVLVTVLAIHFGKEFTPEVQASWQKMVTAVASALSSRYHMVHFTAEEKAAVTSLWSKMNVEEAGGEALGRLLVVYPWTQRFFDSFGNLSSPSAILGNPKVKAHGKKVLTSFGDAIKNMDNLKPAFAKLSELHCDKLHVDPENFKLLGNVMVIILATHFGKEFTPEVQAA WQKLVSAVAI ALAHKYHMVHLTPEEKTAVNALWGKVNVDAVGGEALGRLLVVYPWTQRFFESFGDLSSPDAVMGNPKVKAHGKKVLGAFSDGLAHLDNLKGTFSQLSELHCDKLHVDPENFRLLGNVLVCVLARNFGKEFTPQMQAAYQKVVAGVANALAHKYHMVHLTPEEKSAVTALWGKVNVDEVGGEALGRLLVVYPWTQRFFESFGDLSTPDAVMGNPKVKAHGKKVLGAFSDGLAHLDNLKGTFATLSELHCDKLHVDPENFRLLGNVLVCVLAHHFGKEFTPPVQAAYQKVVAGVANALAHKYHMVLSPADKTNVKAAWGKVGAHAGEYGAEALERMFLSFPTTKTYFPHFDLSHGSAQVKGHGKKVADALTNAVAHVDDMPNALSALSDLHAHKLRVDPVNFKLLSHCLLVTLAAHLPAEFTPAVHASLDKFLASVSTVLTSKYMSLTKTERTIIVSMWAKISTQADTIGTETLERLFLSHPQTKTYFPHFDLHPGSAQLRAHGSKVVAAVGDAVKSIDDIGGALSKLSELHAYILRVDPVNFKLLSHCLLVTLAARFPADFTAEAHAAWDKFLSVVSSVLTEKYRMultiple sequence analysis of different chain of Hemoglobin wasdone using Clustal omega. The results show similarity betweendifferent chains of haemoglobin. Further, we looked at thedistribution of amino acid in different haemoglobin chain . We obsePlasmodium parasite, scavenges the haemoglobin to meets itsamino acid requirements while lipids are obtained from RBCsmembrane. The distribution of amino acids in the proteins ofPlasmodium shows incorporation of all the 20 amino acids.However, the amino acids, which are essential and conditionallyessential to the host, are present in higher percentage than nonessentialamino acids.Among the essential amino acids leucine is required mostfollowed by isoleucine. Among the conditionally essential aminoacids N is required the most. N is present in parasite as repeatwhich might have role in immune evasion by antigenic variation.Other amino acids like arginine, which is essential for polyaminesynthesis is important for robust growth of the parasite.I make up to 9% of total amino acids in Plasmodium falciparum hasto be obtained exogenously from blood, as it is absent in adulthaemoglobin. Istvan et al. has shown that the absence of I affectsparasite growth and I analogue inhibits the parasite growth .The gamAs Plasmodium solely depends upon the degradation ofhaemoglobin for its amino acid requirements it meets most of thenutritional requirements from haemoglobin. As HbF contains allthe amino acids including isoleucine (absent in HbA) required bythe parasite hence, this might be aid growth of parasite in HbFcontaining RBC. Besides at the age of 9 up to, 18% of RBCcontains HbF. Hence, we hypothesise that presence of HbFwould aid parasite growth leading to disease severity in children.Authors declare no conflict of Interest"} +{"text": "AbstractMastigodiaptomus Light, 1939, named Mastigodiaptomuscuneatussp. n. was found in a freshwater system in the City of Mazatl\u00e1n, in the northern region of Mexico. Morphologically, the females of this new species are distinguishable from those of its congeners by the following combination of features: the right distal corner of the genital double-somite and second urosomite have a wedge-shaped projection, the fourth urosomite has no dorsal projection and its integument is smooth. The males are distinct by the following features: the right caudal ramus has a wedge-shaped structure at the disto-ventral inner corner; the basis of the right fifth leg has one triangular and one rounded projection at the distal and proximal margins, respectively, plus one hyaline membrane on the caudal surface close to the inner margin; the aculeus length is almost the width of the right second exopod (Exp2); and the frontal and caudal surfaces of the right Exp2 are smooth. Furthermore, the analysis of the COI gene of Mastigodiaptomuscuneatus sp. n. has revealed that Mastigodiaptomusalbuquerquensis is its nearest congener, with 18.64% of genetic distance. A key for the identification of the known species of the genus is provided.A new species of the genus Diaptomidae G. O. Sars, 1903 is one of the most common families of freshwater copepods worldwide, some genera of this family have restricted distributional patterns and present endemic forms, as the genus Mastigodiaptomus Light, 1939. Recent studies of this genus in the Neotropical region have added new species of these diaptomids , Mastigodiaptomustexensis , Mastigodiaptomusamatitlanensis , Mastigodiaptomusalbuquerquensis , Mastigodiaptomuspatzcuarensis , Mastigodiaptomusmontezumae , Mastigodiaptomusnesus Bowman, 1986, Mastigodiaptomusmaya Su\u00e1rez-Morales & El\u00edas-Guti\u00e9rrez, 2000, Mastigodiaptomusreidae Su\u00e1rez-Morales & El\u00edas-Guti\u00e9rrez, 2000, and Mastigodiaptomussuarezmoralesi Guti\u00e9rrez-Aguirre & Cervantes-Mart\u00ednez, 2013.aptomids or the mr, 1938) . CurrentCyclopidae genus Mesocyclops; and Cyclopidae genus Eucyclops, particularly the Eucyclopsserrulatus species complex. Mastigodiaptomus genus using differences in the integument of prosomal wings and fifth legs of both sexes. Empirical evidence gathered from several species of freshwater crustaceans has shown that these morphological differences are consistent with reproductive isolation Chetumal, M\u00e9xico and the Colecci\u00f3n Nacional de Crust\u00e1ceos (CNCR) del Instituto de Biolog\u00eda, Universidad Nacional Aut\u00f3noma de M\u00e9xico.The type material was deposited at the Colecci\u00f3n de Referencia de El Colegio de la Frontera Sur were preserved after capture in 96% ethanol and prepared for barcoding following standard methods. DNA was extracted using the HOTSHOT method . A segmeBOLD) produced sequences of Mastigodiaptomusalbuquerquensis, Mastigodiaptomuspatzcuarensis, Mastigodiaptomuscf.albuquerquensis , Microcrustacean from Mexico (MCM), and Zooplankton II (ZPII) and compared with our sequence of Mastigodiaptomuscuneatus sp. n., this latter sequence is into the project MCM. In these project files, the electropherograms, sequence data, photographs, primers data, and collection details are available (on the Barcode of Life Data System http://www.boldsystems.org). Fifty-two COI gene sequences > 500 bp were used for the analysis, and BOLD Aligner and the ID Tree using the model Kimura 2 parameter were used as an outgroup.These sequences were downloaded from the ter K2P; were utiPageBreakTaxon classificationAnimaliaCalanoidaDiaptomidaehttp://zoobank.org/FADC3B97-FB6F-4559-B71B-EB6F76A3246FECOCH-Z-09339. Collected 28.VIII.2014. Collectors: A. Cervantes-Martinez, N. Hern\u00e1ndez L\u00f3pez, M. Bastidas, and J. Aguilar Rubio.One adult female dissected on one slide: PageBreakECOCH-Z-09340. Collected 28.VIII.2014. Same collectors.One adult male dissected on one slide: ECOCH-Z-09341. Collected 28.VIII.2014. Same collectors.Four adult females and five adult males preserved in 90% ethanol with a drop of glycerine. PageBreakCNCR-31861. Collected 28.VIII.2014. Same collectors.Two adult females and two adult males preserved in 90% ethanol: 23\u00b014'10\"N; 106\u00b026'18\"W.A lagoon called Laguna El Camar\u00f3n in Avenida Insurgentes, Mazatl\u00e1n, Sinaloa City, M\u00e9xico; The name of the species means \u201cwedged\u201d in Latin and refers to the chitinous protuberance present on the right disto-lateral corner of the first and second urosomites in females, and on the right caudal ramus on the ventral surface in males.Adult female: Cuticle surfaces of prosomal somites smooth dorsal and laterally Fig. . AntennuAdult male: The cuticle surfaces of prosomal somites are smooth dorsally and laterally Fig. . Right an = 6 with a movable seta at tip. Rectangular, nude coxa. Basis with 4 long setae. Enp two-segmented, Enp1 with 4 ely Fig. .PageBreakand 2-segmented endopodite: Enp1 and Enp2 with 5 and 4 setae, respectively. Basis rectangular with 4 setae; coxal endite quadrangular 4-setulated. Praecoxal arthrite with 15 spiniform setae, 11 anterior, 4 posterior.Maxillule Fig. : Coxal eMaxilla Fig. : First pMastigodiaptomusalbuquerquensis and Mastigodiaptomuspatzcuarensis (see Maxilliped (not figured): Same structure as described and illustrated for nsis see .Diaptomidae family with a smooth hyaline membrane.PageBreakAntennule, antenna, mandible, maxillule, maxilla, maxilliped, and P1-P4 as described for female.Right wing of fifth pediger with 1 tiny dorsal spinule and 1 ventral spine Fig. ; left wiPageBreakUrosome: Urosomites nude dorsally and ventrally. First urosomite with thin spine on right side and fold on left side Fig. . Fourth PageBreakP5: Coxal segments with strong spines on caudal view; left and right basis with a lateral seta Fig. .Left basis with a triangular protuberance on distal margin of frontal surface Fig. . Both leRight basis basally and distally projected: basal projection rounded whereas distal projection triangle-shaped; one semi-triangular sclerotization on caudal surface of right basis Fig. . Right EMastigodiaptomuscuneatus sp. n. is shown below:The nucleotide sequence (607 bp) obtained for specimen MAGA-0156 , identified as GGAGCCTGGTCAGGCATAGTAGGAACAGGCCTTAGAATGATTATTCGGATGGAGTTAGGACAAGCCGGGTCTTTAATTGGAGATGACCAAATTTATAATGTAGTAGTTACTGCTCATGCTTTTGTTATAATTTTTTTTATGGTGATACCTATTTTAATTGGGGGGTTTGGTAATTGGCTTGTTCCGTTAATATTAGGTGCAGCGGATATAGCTTTCCCTCGAATAAATAATATAAGATTTTGATTTTTATTGCCAGCTTTAGTCATATTGTTATCTAGGTCGCTTGTTGAAAGAGGGGCGGGAACAGGGTGAACTGTGTATCCCCCCCTGTCTAGCAACATTGCCCATGCTGGCAGGTCCGTTGATTTTGCTATTTTTTCGCTTCATTTAGCTGGGGTTAGGTCTATTTTGGGCGCAGTAAATTTTATTAGCACATTAGGAAATTTGCGGGCGTTTGGAATAATTTTAGATCGAATACCACTTTTTGCTTGAGCCGTTTTAATCACGGCTATCTTGTTATTGCTTTCTCTTCCTGTTTTAGCCGGGGCGATTACAATGCTTCTTACAGATCGGAACCTCAACTCAAGATTTTATGAT.K2P maximum distance between the surveyed species reaching 5.52% appeared to be morphologically close to Mastigodiaptomusamatitlanensis . Similarities between these species in females include the presence of one protrusion on the second urosomite and the bulbose lateral margins of genital double-somite. The similarities in males are the short aculeus and the lack of hyaline membrane on the right Exp2 of the fifth leg.Mastigodiaptomuscuneatus sp. n. can be separated from Mastigodiaptomusamatitlanensis by the following features: the dorsal projection on the last pediger absent vs. present; the genital double-somite with vs. without a protrusion on the distal right side; on the genital double-somite, the right spine located at a higher level than the left spine vs. both left and right spines placed at same level; and the endopod of fifth leg long and 2-segmented vs. short and 1-segmented.However, Mastigodiaptomuscuneatus sp. n. in comparison to the rectangular basis with a transversal, distal, cuneiform lamella in Mastigodiaptomusamatitlanensis. In addition, the distal margins of the left and right endopods bear short setules in Mastigodiaptomuscuneatus sp. n. whereas in Mastigodiaptomusamatitlanensis these distal margins bear one slender seta. The aculeus on the right Exp2 is clearly straight in Mastigodiaptomuscuneatus sp. n. but short, distal and curved in Mastigodiaptomusamatitlanensis. The Exp2 is smooth in Mastigodiaptomuscuneatus sp. n. but in Mastigodiaptomusamatitlanensis an oblique ridge on the caudal surface is present. Finally, we assume that the wedge on the right caudal ramus present in Mastigodiaptomuscuneatus sp. n. is absent in Mastigodiaptomusamatitlanensis because there is no mention of a similar feature in The males of these species show more morphological differences in the fifth leg: the caudal surface of the right basis is bulbose with the oblique, medial, angulose and curved cuticular process in Mastigodiaptomuscuneatus sp. n. is genetically closest to Mastigodiaptomusalbuquerquensis s. str. and the species recorded in Mexico with one sclerotization on the right basis of fifth leg of males such as Mastigodiaptomuspatzcuarensis. As previously discussed , which probably are cryptic species , Mastigodiaptomuspatzcuarensis, Mastigodiaptomusmontezumae , Mastigodiaptomusnesus (Caribbean and south eastern Mexico), and Mastigodiaptomustexensis (Texas and south eastern Mexico), whereas the species that are assumed to have restricted distribution or endemics are Mastigodiaptomusreidae, Mastigodiaptomusmaya, Mastigodiaptomuspurpureus, Mastigodiaptomusamatitlanensis, Mastigodiaptomussuarezmoralesi (see Mastigodiaptomuscuneatus sp. n.lesi see and, proMastigodiaptomuscuneatus sp. n. was compared with the ten known Mastigodiaptomus species, particularly in the female and male urosomes, the male right antennule and fifth legs, and in the COI gene sequence. This report increases the number of recognized species of Mastigodiaptomus to eleven. Mastigodiaptomuscuneatus sp. n. appears to be part of the Mastigodiaptomusalbuquerquensis complex.Morphological and genetic differences were found when MalesFemales"} +{"text": "Molecular characterization showed that lncR492 interacts with the mRNA binding protein HuR and facilitates its inhibitory function by activation of Wnt signaling. Thus, lncRNAs modulate the fate decision of pluripotent stem cells.RNA interference (RNAi) screens have been shown to be valuable to study embryonic stem cell (ESC) self-renewal and they have been successfully applied to identify coding as well as noncoding genes required for maintaining pluripotency. Here, we used an RNAi library targeting >640 long noncoding RNAs (lncRNA) to probe for their role in early cell differentiation. Utilizing a Sox1-GFP ESC reporter cell line, we identified the lncRNA Embryonic stem cells (ESC) are characterized by their ability of long-term self-renewal as well as their potential to differentiate into each cell type of the embryo proper. After the first isolation of embryonic stem cells from the mouse blastocyst , 2 the rRecent sequencing approaches have shown that the majority of the genome is transcribed . Among tTUNA was identified as sustainer of pluripotency but was also required for neural differentiation [Large-scale functional studies have identified numerous lncRNAs that play a regulatory role in the maintenance of pluripotency \u201310. It hlncR492 as an inhibitor of neural differentiation, which exerts its function by interacting with the mRNA binding protein HuR and activating Wnt signaling, thereby blocking ectodermal differentiation in a lineage specific manner.To identify specific neural differentiation regulating lncRNAs, we performed a large-scale loss-of-function RNAi screen in a Sox1-GFP ESC reporter line. We discovered the lncRNA Austin Smith and Konstantinos Anastassiadis kindly provided the Sox1-GFP and Oct4-GFP cell lines. The Foxa2-GFP and T-GFP cell lines were generated in the BAC TransgeneOmics project and kind\u2122) supplemented with 1x N2, 1x B27, 1x NEAA, 1x Penicillin/Streptomycin, 50 \u03bcM 2-mercaptoethanol , 3 \u03bcM CHIR99021, 1 \u03bcM PD0325901 and LIF (generated in house). For differentiation towards the ectoderm cells were cultured in N2B27 containing medium without the two inhibitors and LIF. For differentiation into endoderm (Foxa2-GFP ESC) and mesoderm (T-GFP ESC), the two inhibitors and LIF were replaced by 30 \u03bcg/ml ActivinA (MPI protein facility) or 10 \u03bcg/ml BMP4 (R&D Systems), respectively. The cells were seeded on gelatin-coated dishes with a density of 15000 cells/cm2 and grown for 4 to 5 days before they were harvested for experiments.Sox1-GFP, Foxa2-GFP, T-GFP ESC and R1/E ESC lines were cultured on gelatin-coated plates in DMEM supplemented with 10% Pansera ES FBS (PAN Biotech), 1x NEAA, 1x Penicillin/Streptomycin, 50\u03bcM 2-mercaptoethanol and LIF (MPI-CBG protein facility).Oct4-GFP ESC were additionally cultured on gelatin-coated plates in DMEM or the corresponding non-labeled amino acids, respectively. All other supplements were maintained as described. Cells were grown for five passages, lysed and checked for successful label incorporation by MS.For SILAC-labeling the standard DMEM was replaced with SILAC-DMEM medium supplemented with 40 mg/mL EsiRNAs were synthesized as described previously . For theNheI and XhoI restriction sites in the flanking regions. After sequencing, the PCR products were cloned into the pCGIT destination vector under the CAG promoter . pCGIT without insert was used as control (EV\u2014empty vector).The cDNAs for lncR492 and HuR were amplified by PCR introducing ESCs were transiently transfected with the overexpression or control plasmids using Lipofectamine 2000 (ThermoFisher Scientific). 50 ng of plasmid DNA were combined with 0.2 \u03bcl Lipfectamine 2000 and incubated in 50 \u03bcl OptiMEM for 10 minutes. The transfection mix was added to one well of a 96-well plate. 4500 cells per well in 200 \u03bcl N2B27 medium supplemented with 2i+LIF were seeded on top. After 24h differentiation was initiated as described above.18 primer. Standard RT-PCR reactions were performed with a MyTaq\u2122 Red DNA Polymerase (Bioline). Quantitative RT-RCRs (qRT-PCR) were run with the SYBR Green qPCR kit (Abgene) on an CFX96 Touch\u2122 Real-Time PCR Detection System (Bio-Rad). Measured transcript levels were normalized to Gapdh. Samples were run in duplicates. Primers used are listed below.Total RNA was isolated by using the RNeasy Mini kit (QIAGEN). For each RT reaction 1 \u03bcg of RNA was reverse transcribed with SuperScript III Reverse Transcriptase (ThermoFisher Scientific) utilizing oligo(dT)\u2122 Eukaryotic mRNA Isolation Kit, EpiCentre). Subsequent 500 ng of RNA were reverse transcribed as described above.For endonuclease treatment 5 \u03bcg RNA were treated with a 5\u2019-phosphate-dependent exonuclease according to manufactor\u2019s instructions (mRNA-ONLYThe forward and reverse primer sequences (written 5\u2019 to 3\u2019) were as follows:Gapdh, ACTCCACTCACGGCAAATTC, GGATGCAGGGATGATGTTCTlncR492, GCTGCTGTTTCACACCCAAG, TGACTAGGCGATCCTGACCASox1, CCTTGCTAGAAGTTGCGGTC, TCACTCAGGGCTGAACTGTGSrrm4, TCTCGTCGAAGTCCCAGCTA, CACTGGTTATCCTCCGAGCCOct4, AGAGGGAACCTCCTCTGAGC, TGATTGGCGATGTGAGTGATBrachyury (T), GAACAGCTCTCCAACCTATG, AGACTGGGATACTGGCTAGAGFoxa2, GCTGCAGACACTTCCTACTAC, GGACACAGACAGGTGAGACTPax6, CACCAGACTCACCTGACACC, TCACTCCGCTGTGACTGTTCNestin, GCAGGAGAAGCAGGGTCT, AGGTGCTGGTCCTCTGGTNanog, GGAAGCAGAAGATGCGGACT, ATGCGTTCACCAGATAGCCCMalat1, GTTTGTGATTGGAGCCGAG, AAGGGAGGGGAGAGAGAACAU1 snRNA, GCGCGATTTGGCAAGATGA, TTCTTCCCGGGTTTCTGCTCStar solution, exposed to Amersham Hyperfilm , which was developed using an OPTIMAX (PROTEC).The sequences of the gene-specific Northern blot probes are listed below (written 5\u2019 to 3\u2019). Two probes per gene were mixed together. Northern blot was performed according to the DIG Northern Starter Kit\u2019s (Roche) manual. Briefly, RNA was separated on a pre-cast 1% agarose gel at 35V for 3.5h in 1x MOPS buffer. The separated RNA was blotted on a nitrocellulose membrane and cross-linked by UV-light. The membrane was prehybridized with DIG easy Hyb for 30 minutes at 68\u00b0C, followed by an hybridization step with a DIG-labeled, gene-specific RNA probe mix diluted in DIG easy Hyb over night at 68\u00b0C. Unspecific binding was eliminated by several stringency washes. Thereafter the membrane was blocked, incubated with a DIG-specific antibody and washed. Finally, the membrane was incubated with CDP-lncR492 probe 1UCAGAACCCAGCACACUGUCAGCCUCAGAGCAUACAAUCUUUUGAGUGAGUGAGCUAGAUUUGACAACGAUGCUUGAUGCUUGCGAGCAUAUGAGGGGCUCGCAGCCUCUUCCUAGGCACUACUGUCUCCCUCCGGAGACGCCUCCUGGCCUCCUUGAUUAUGAACACCUUUGCUAAUGCUCACAGUCUUCUUGUUCUCAUGGCCCUUGGAGGAUAUGUGCAGUGGUGAACACAGCUlncR492 probe 2AUGGCUUUUUCCUCGCUACAGUUCAGAGUCGCCACAAUCACAGGGGGCCGGGGAAGAUCACCAGCAAUCAGUGUUCAAACGGCCCAAAGAGAUUGUUCUGAGUCUCCUUGCCACUCCGCUGAUGGAGUAGGGCCUUCUAAUUGGCCCUUCUGUCUCUCCUGCCUCGCCCUUCCAAUCUUUUGCCUUCUUGGCAGCUAGGUUCAUGCUUAUAGACCAUUUUUCGGUGAGUCAUUGCCUUGAUUUAAAACCCUUCGGCUUCCACUUGAGACUUGAGCCAUCCAGGCUCCCUGCCAUGGAGGGAGAGGGCAGUCAUGAUCUGGUUUUGGCCAUCCAUCUCUCUCCUCUAUCCUGUUCCCUGAGUCCUUGUUCUGAGCAUCUACCUGGGGCAUCCUUCCUUCCCACUAACUCCUGUAUCAUGUCCCUGGACCACUCCCCACCUGACUGGUCCUCUGUGUUUUCAGAGUAGCUGAUUUCUUUUGCACUGCCUAGCGCCACUGGGAACAGACAUCGUCUGUGAGCUUUGGGAAGGGapdh probe 1CAGCGAACUUUAUUGAUGGUAUUCAAGAGAGUAGGGAGGGCUCCCUAGGCCCCUCCUGUUAUUAUGGGGGUCUGGGAUGGAAAUUGUGAGGGAGAUGCUCAGUGUUGGGGGCCGAGUUGGGAUAGGGCCUCUCUUGCUCAGUGUCCUUGCUGGGGUGGGUGGUCCAGGGUUUCUUACUCCUUGGAGGCCAUGUAGGCCAUGAGGUCCACCACCCUGUUGCUGUAGCCGUAUUCAUUGUCAUACCAGGAAAUGAGCUUGACAAAGUUGUCAUUGAGAGCAAUGCCAGCCCCGGCAUCGAAGGUGGAAGAGUGGGAGUUGCUGUUGAAGUCGCAGGAGACAACCUGGUCCUCAGUGUAGCCCAAGAUGCCCUUCAGUGGGCCCUCAGAUGCCUGCUUCACCACCUUCUUGAGapdh probe 2AAGCAGUUGGUGGUGCAGGAUGCAUUGCUGACAAUCUUGAGUGAGUUGUCAUAUUUCUCGUGGUUCACACCCAUCACAAACAUGGGGGCAUCGGCAGAAGGGGCGGAGAUGAUGACCCUUUUGGCUCCACCCUUCAAGUGGGCCCCGGCCUUCUCCAUGGUGGUGAAGACACCAGUAGACUCCACGACAUACUCAGCACCGGCCUCACCCCAUUUGAUGUUAGUGGGGUCUCGCUCCUGGAAGAUGGUGAUGGGCUUCCCGUUGAUGACAAGCUUCCCAUUCUCGGCCUUGACUGUGCCGUUGAAUUUGCCGUGAGUGGAGUCAUACUGGAACAUGUAGACCAUGUAGUUGAGGUCAAUGAAGGGGUCGUUGAUGGCAACAAUCUCCACU6 cells were washed with ice-cold PBS. Cells were resuspended in ice-cold, protease inhibitor (Roche) containing HD-buffer and incubated on ice for 10 minutes. IGEPAL was added to a final concentration of 0.5%. Quickly citric acid (100 mM) was added to a final concentration of 1 mM. The cells were vortexed vigorously. The nuclei were separated from the cytoplasmic fraction by gentle centrifugation. Total RNA was isolated with the PARIS Kit (Ambion). 1 \u03bcg of RNA was reverse transcribed with SuperScript III Reverse Transcriptase (ThermoFisher Scientific) utilizing oligo(dT)18 primer. qRT-RCR were run as described above.Approx. 10x10lncR492 probe 2 was used as FISH probe.Sox1-GFP ESC were transfected with esiRNA using Lipofectamine2000 (Thermo Fisher Scientific) and seeded on gelatin-coated chambered slides (Ibidi). After 48 h RNA-FISH was performed as described earlier . The seqFor immunofluorescent stainings cells were fixed with 4% paraformaldehyde for 10 minutes at room temperature. All following steps were performed at room temperature. After washing with PBS cells were blocked and premeabilized with 10% FCS in staining buffer (0.3%TritonX-100 in PBS) for 30 minutes. The primary antibody rabbit-anti-Tubb3 was diluted 1:1000 in staining buffer and incubated for 1 h. After washing the cells were incubated with donkey-anti-rabbit-Cy3 secondary antibody (Jackson ImmunoResearch Laboratories) and DAPI (Sigma) for 30 minutes. Finally the cells were imaged using a Zeiss Axiovert 200M microscope equipped with an ApoTome (Zeiss). Images are projections of the maximum intensity. Total numbers of nuclei as well as Tubb3+ cells were counted manually.To create the 450 bp RNA baits, forward primers containing the T7 promoter sequence and reverse primers with the S1 aptamer sequence were used in a PCR amplification reaction on the pCAGGS plasmid. A fragment of the human PTPN13 mRNA of equal length (450 bp) was used as a control bait. The primer sequences are written 5\u2019 to 3\u2019.CGTTAATACGACTCACTATAGGCTCGAGATCCAGCTGGGCACAGGC.AK016992 (1\u2013450 bp) + T7 forward primer, CATGGCCCGGCCCGCGACTATCTTACGCACTTGCATGATTCTGGTCGGTCCCATGGATCCAACTCTGCTGATCGGATCTGTCCTC.AK016992 (1\u2013450 bp) + BioApt reverse primer, CGTTAATACGACTCACTATAGGGGCTCCCGGACCTTCCCAAAG.AK016992 (451\u2013900 bp) + T7 forward primer, CATGGCCCGGCCCGCGACTATCTTACGCACTTGCATGATTCTGGTCGGTCCCATGGATCCAGATTGTTCTGAGTCTCCTTGCCAC.AK016992 (451\u2013900 bp) + BioApt reverse primer, CGTTAATACGACTCACTATAGGCTTTGGGCCGTTTGAACACTG.AK016992 (901\u20131350 bp) + T7 forward primer, CATGGCCCGGCCCGCGACTATCTTACGCACTTGCATGATTCTGGTCGGTCCCATGGATCCTCTAGACGGGTACAATGCCTTC.AK016992 (901\u20131350 bp) + BioApt reverse primer, CGTTAATACGACTCACTATAGGAGGGCCATGAGAACAAGAAG.AK016992 (220\u2013670 bp) + T7 forward primer, CATGGCCCGGCCCGCGACTATCTTACGCACTTGCATGATTCTGGTCGGTCCCATGGATCCGTTTTGGCCATCCATCTCTCTC.AK016992 (220\u2013670 bp) + BioApt reverse primer, CGTTAATACGACTCACTATAGGCAGATCATGACTGCCCTCTCC.AK016992 (671\u20131120 bp) + T7 forward primer, CATGGCCCGGCCCGCGACTATCTTACGCACTTGCATGATTCTGGTCGGTCCCATGGATCCCGCTTGGGTGTGAAACAGCA.AK016992 (671\u20131120 bp) + BioApt reverse primer, CGTTAATACGACTCACTATAGG CAATATATTTTCTGCTATCAAGTC.PTPN13 + T7 forward primer, CATGGCCCGGCCCGCGACTATCTTACGCACTTGCATGATTCTGGTCGGTCCCATGGATCCCTTTATTAAAATATTGGAAAACATTTTTG.PTPN13 + BioApt reverse primer, in vitro transcription according to the manufacturer\u2019s protocol (Fermentas). Successful transcription was monitored by agarose gel electrophoresis and RNA concentration was quantified by A280 absorbance on a Nanodrop system (Peqlab). 25 \u03bcg of each S1-tagged RNA was coupled to paramagnetic streptavidin C1 beads in RNA binding buffer , 0.5% IGEPAL CA-620) and incubated on a rotation wheel for 30 min at 4\u00b0C. RNA-bound beads were washed 3 times with RNA washing buffer , 0.5% IGEPAL CA-620), prior to incubation with 400 \u03bcg nuclear extract, with 20 \u03bcg competitor yeast tRNA (Invitrogen) added, for 30 min at 4\u00b0C with gentle agitation. After mild washing, SILAC heavy and light fractions were combined 1:1 and samples were boiled in 1x LDS buffer (Invitrogen) and separated on a 4\u201312% NuPAGE Novex Bis-Tris precast gel (Life Technologies) at 180 V in 1x MOPS.The PCR products were used for 17703201) using two layers of C18material (Empore).Coomassie stained gels were cut in one slice and destained with 50% EtOH/25 mM ammonium bicarbonate (ABC). The resulting gel pieces were dehydrated with 100% acetonitrile (ACN) and dried for 5 min in a concentrator (Eppendorf). Samples were incubated with reduction buffer (10 mM DTT/50 mM ABC) for 30 min at 56\u00b0C and further alkylated for 30 min in the dark with iodoacetamide (50 mM IAA/50 mM ABC). Gel pieces were completely dehydrated with ACN and covered in trypsin solution (1 \u03bcg trypsin per sample). Proteins were digested over night at 37\u00b0C and peptides were extracted twice by incubation with extraction buffer (3% TFA and 30% ACN) for 15 min. The gel pieces were dehydrated with 100% ACN and the extracted volume reduced to aproximately 150 \u03bcl in a concentrator (Eppendorf). Extracted peptides were desalted in StageTips (PMID:17721543). MS full scans were obtained in the orbitrap at 70,000 resolution with a maximal injection time of 20 ms, while MS/MS scan resolution was set to 17,500 resolution and maximal injection for 120 ms. Unassigned and charge state 1 were excluded from MS/MS selection and peptide match was preferred. Raw files were processed with MaxQuant (version 1.5.2.8.) (PMID:19029910) and searched against human UNIPROT annotated protein database provided with MaxQuant using the Andromeda search engine (PMID:21254760). Carbamidomethylation was set as a fixed modification, while acetyl (N-term protein) and oxidation (Met) were considered as variable modifications. Trypsin (specific) was selected as enzyme specificity with maximal two miscleavages for MaxQuant analysis. Proteins were quantified with at least 2 ratio counts based on unmodified unique and razor peptides. Known contaminants and reverse hits were removed before plotting the protein ratios of the forward and reverse experiments in R (version 3.2.2).Eluted peptides were injected via an autosampler into an uHPLC and loaded on a 25 cm capillary packed in-house with Reprosil C18-AQ 1.9 \u03bcm resin (Dr. Maisch) for reverse-phase chromatography. The EASY-nLC 1000 HPLC system was directly mounted to a Q Exactive Plus mass spectrometer (Thermo). Peptides were eluted from the column with a 90 min optimized gradient from 2 to 40% ACN with 0.1% formic acid at a flow rate of 200 nL/min. Chromatography was stabilized with a column oven set-up operating at 40\u00b0C (Sonation). The heated capillary temperature was set to 250\u00b0C. Spray voltage ranged from 2.2\u20132.4 kV. The mass spectrometer was operated in data-dependent acquisition mode with one MS full scan and up to ten triggered MS/MS scans using HCD fragmentation (PMID:RNA pull-down experiments were performed 3 times\u2014once for mass spectrometry and twice for western blot to confirm the enrichment of HuR.\u00ae Reporter Assay (Promega) according to the manufacture\u2019s instruction and the EnVision Multilabel Reader (PerkinElmer).Cells were co-transfected with Super 8x TOPFlash or Super 8x FOPFlash luciferase plasmid together with either target-specific esiRNA or overexpression plasmid as well as pCMV renilla plasmid to correct for transfection efficiency. Cells were cultured in N2B27+2i+LIF. 48 h after transfection cells were lysed and bioluminescence was analysed using the Dual-LuciferaseTotal cell lysates were harvest 72h after transfection using RIPA lysis buffer. 20 \u03bcg of each protein sample were loaded and analysed by western blot using a mouse anti-HuR monoclonal antibody , rabbit anti-Tubb3 and mouse anti-Gapdh as primary antibodies. As secondary antibodies HRP-conjugated species-specific antibodies were used. For quantitative analysis species-specific secondary antibodies from LI-COR Biosciences have been used. Blots were scanned and analysed with Odyssey.t-test.The results are shown as mean \u00b1 standard deviation (SD) as indicated in the figure legends. Statistical significance was calculated using the unpaired Student\u2019s Sox1 is one of the earliest transcription factors marking the neural ectoderm and Sox1lncR492 (accession no. AK016992) had the strongest effect (Z-score (1st esiRNA) = 4.0 and Z-score (2nd esiRNA) = 5.4; lncR492. Both esiRNAs achieved a knock-down efficiency of >60% tail -tailed and capped , 6. cDNA(A) tail . Further capping . To furtock-down . Consistfraction .lncR492 as a non-coding nuclear transcript, which might play a role in regulating ectodermal cell fate commitment.Thus, the esiRNA-based screen in differentiating ESCs identified lncR492 acts as an inhibitor of ectodermal differentiation. To clarify its role and specificity in lineage commitment, we first tested its expression in pluripotent and differentiating ESCs. These results revealed that lncR492 has its highest expression in the pluripotent state. Interestingly, together with Oct4, lncR492 is down-regulated during neural differentiation , Brachyury-GFP+ and Foxa2-GFP+ (endoderm) cells after differentiation. In accordance with the reporter, the lineage specific genes were up-regulated when the cells were cultured under differentiation conditions in N2B27 alone (Sox1-GFP) or supplemented with 30 \u03bcg/ml Activin A (Foxa2-GFP) or 10 \u03bcg/ml BMP4 -positive cells in the lncR492 knock-down condition compared to cells treated with a control esiRNA as seen by immunofluorescent staining and obtained similar results , which was most enriched in pull downs with fragment 1 was identified as well as an 11 bp long sequence in the 3\u2019 region (HuR BS2) of the transcript mRNAs and RNA-anscript . The posern blot . ESC difock-down . Importantiation . Likewisntiation . In accopression . Howeverpression . Given tpression . Hence, lncR492 gene expression. Interestingly, knock-down and overexpression of HuR resulted in a respective decrease and increase of lncR492, whereas Srrm4 transcript levels were not affected Click here for additional data file.S2 FigLncR492 expression in differentiated ESCs. Therefore T-GFP and Foxa2-GFP reporter cells were differentiated N2B27-containing medium supplemented with 10 \u03bcg/ml BMP4 or 30\u03bcg/ml ActivinA for 4 days, respectively. Sox1-GFP ESCs were differentiated for 4 days in medium containing the serum replacement N2B27 only. GFP+ cells were sorted by FACS, RNA was isolated and lncR492 as well as lineage specific gene expression was analysed by qRT-PCR. Expression was normalized to undifferentiated ESC. Data presents mean \u00b1 SD of three independent experiments.(A) lncR492 and Srrm4 during the time course of differentiation. Data presents mean \u00b1 SD of three independent experiments.(B) QRT-PCR analysis of lncR492 knock-down or overexpression in T-GFP ESCs. Bar graph represents the quantification of three independent western blot experiments. Data presents the mean \u00b1 SD.(C) Western blot analysis of Tubb3 after lncR492 knock-down or overexpression. Cells were harvested after 4 days of differentiation in N2B27. Data presents the mean \u00b1 SD of three independent experiments.(D) Gene expression analysis of the T-GFP reporter ESC (R1/E) by qRT-PCR after lncR492 knock-down and overexpression in Oct4-GFP ESC cultured in N2B27+2i+LIF medium. Data represents mean \u00b1 SD of four independent experiments.(E) FACS analysis of GFP expression after lncR492 knock-down and overexpression in Oct4-GFP ESC cultured in medium supplemented with FCS+LIF. Knock-down of Rad21 was used as a positive control. Data represents mean \u00b1 SD of four independent experiments.(F) FACS analysis of GFP expression after * p<0.05; ** p<0.01; *** p<0.001; n.s.\u2013not significant.(TIF)Click here for additional data file.S3 FigSrrm4 expression after lncR492 knock-down and overexpression measured by qRT-PCR. Data represents mean \u00b1 SD of three independent experiments.(A) (B) Summary table of proteins detected by mass spectrometry analysis. The lncRNA transcript was split into five overlapping fragments of 450 bp length each. The top ten putative interaction proteins for each lncRNA fragment are listed according to their abundance.lncR492. Putative binding sides for HuR are highlighted in red based on the consensus sequence NNUUNNUUU.(C) Nucleic acid sequence (mRNA) of (D) Western blot of HuR knock-down and overexpression. Gapdh was used as loading control. EV\u2014empty vector.lncR492 and HuR knock-down or HuR overexpression. Cells were differentiated for 4 days in N2B27 supplemented with 30 ng/ml ActivinA. Data presents mean \u00b1 SD of three independent experiments.(E) FACS analysis of Foxa2-GFP expression after (F) FACS analysis of Oct4-GFP expression 48h after HuR knock-down and overexpression. Oct4-GFP cells were cultured in N2B27+2i+LIF medium. Data presents mean \u00b1 SD of three independent experiments.* p<0.05; ** p<0.01; *** p<0.001; n.s.\u2013not significant.(TIF)Click here for additional data file.S1 TableZ-scores of the primary and the validation screen are shown for each replicate. Hits of the primary screen with an average Z-score >3 are highlighted in green (increasing the number of Sox1-GFP positive cells) and hits with an average Z-score < -3 are highlighted in orange (decreasing the number of Sox1-GFP positive cells). In the validation screen a Z-score > 2 or <-2 are considered as hit and highlighted in green.(XLSX)Click here for additional data file.S2 TableIdentified proteins for each fragment used in the pull-down experiment are shown.(XLSX)Click here for additional data file."} +{"text": "Such integrations rendered cells sensitive to the cytotoxic drugs hydroxyurea and methyl methanesulfonate. We constructed dph3 and msh3 strains with mutated ATG start codons (ATGmut), which allowed investigating drug sensitivity without potential interference by marker insertions. The dph3-ATGmut and a dph3::loxP-ura4-loxM gene disruption strain, but not msh3-ATGmut, turned out to be sensitive to hydroxyurea and methyl methanesulfonate, likewise the strains with cassettes integrated at the msh3 locus. The fungicide sordarin, which inhibits diphthamide modified eEF2 of Saccharomyces cerevisiae, barely affected survival of wild type and msh3\u0394 S. pombe cells, while the dph3\u0394 mutant was sensitive. The msh3-ATG mutation, but not dph3\u0394 or the dph3-ATG mutation caused a defect in mating-type switching, indicating that the ura4 marker at the dph3 locus did not interfere with Msh3 function. We conclude that Dph3 is required for cellular resistance to the fungicide sordarin and to the cytotoxic drugs hydroxyurea and methyl methanesulfonate. This is likely mediated by efficient translation of proteins in response to DNA damage and replication stress.Dph3 is involved in diphthamide modification of the eukaryotic translation elongation factor eEF2 and in Elongator-mediated modifications of tRNAs, where a 5-methoxycarbonyl-methyl moiety is added to wobble uridines. Lack of such modifications affects protein synthesis due to inaccurate translation of mRNAs at ribosomes. We have discovered that integration of markers at the The online version of this article (doi:10.1007/s00294-017-0711-x) contains supplementary material, which is available to authorized users. Saccharomyces cerevisiae by Trm9\u2013Trm112 and to 5-carbamoylmethyl-uridine (ncm5U34) by an unknown enzymatic activity and methyl methanesulfonate (MMS). Msh3 is a eukaryotic homologue of bacterial MutS. MutS is a DNA mismatch binding protein that initiates removal of mismatched and unpaired nucleotides, which were incorporated into the nascent strand during replication that drug sensitivity was indeed due to an impaired dph3 function. Thus, Dph3 plays a role in response to DNA damage and replication stress, likely through modifications of tRNA and/or eEF2, which allow efficient biosynthesis of DNA damage response proteins.In this study, we discovered that replacement of the open reading frame of S. pombe msh3 and dph3 genes share an intergenic region of only 268 base pairs (bp) between the two ATG start codons . Data obtained by functional genomics further revealed that the intergenic region constitutes the 5\u2032 untranslated regions (5\u2032 UTR) of the msh3 and dph3 mRNAs, with divergent and likely overlapping promoters from here on of base\u2013base mismatches and loops of one to four nucleotides , demonstrating that the bands produced with the reverse-transcribed samples reflect amplification of cDNA derived from mRNA. In addition, PCR on a cDNA sample of a dph3::loxP-ura4-loxM strain yielded no bands , demonstrating that the amplified DNA was dph3 specific. We then used dph3q-F and seven reverse primers for PCR that prime at different positions, covering most of the dph3 mRNA. We could not detect any relevant differences between wild type and the msh3::kanMX mutant colonies, indicative for a switching defect. The h90dph3::loxP-ura4-loxM and h90dph3-ATGmut mutants formed iodine positive colonies, although less homogeneously stained than wild type colonies were not formed. Thus, replacement of dph3 by the ura4 marker did not disrupt the Msh3 function in mating-type switching. The phenotype of reduced sporulation of dph3 mutants is unrelated to Msh3 functions, as dph3-ATGmut does not have an integrated cassette that may interfere with msh3 expression.We noticed that colonies of the ies Fig.\u00a0a. Both dres Fig.\u00a0b. On thedph3 (kti11), like the other dph mutated genes in S. cerevisiae causes resistance to diphtheria toxin and sordarin due to the lack of the diphthamide modification of eEF2 mutants and unlike the other dph mutants for lysine have low protein levels in elp3 mutants after 3\u00a0days of incubation at 30\u00a0\u00b0C. Drugs used were HU (Formedium), MMS (ACROS Organics) and sordarin (Santa Cruz). Spot tests were carried out at least twice, with the exception of the experiments including the plates with 100\u2013150\u00a0\u03bcg/mL sordarin. To determine spore formation efficiency 10\u00a0\u03bcL of 1:10 dilutions in H2O of stationary phase cultures were spotted on MEA and incubated for 2\u00a0days at 30\u00a0\u00b0C. Means and standard deviations were calculated from six independent experiments, where 200 units (asci and zygotes) were counted for each of them. S. pombe strains are listed in Supplemental Table S1. The S. cerevisiae strain RCY2459 (MATa ho::LYS2 ura3 lys2 leu2::hisG in SK1 background) was a kind gift of Rita Cha .Yeast media were YEA (yeast extract agar), YEL (yeast extract liquid), MEA and MMA as described was obtained by transformation of the 972 wild type strain (h\u2212) with a PCR product obtained with primers D3kf 5\u2032-TTTTTAAAACGTCCGCCTATATTCATTTAGAAACTATAAATGCGTTACAAATCTATGTTGAGCAAATTTGATTGTACAGTTTTTATTTTCGCTTTATTACCGCCAGCTGAAGCTTCGTAC-3\u2032 and Dk3r 5\u2032-AGAACTTAAAACTGATGTATAGTTGCTTAAATTTATTTATACAGTAAAATCTTATGTGTTGAGTATCCTAATAAAGTAGTTAATTCAGTAATGCTCTCTCGGCCACTAGTGGATCTGATA-3\u2032 using plasmid pFA6a-kanMX was constructed by transformation of OL2137 (h\u2212ura4-D18) with a PCR fragment obtained with primers D3kf and Dk3r (see above) and pFA6a-hphMX6 derived from transformation of EH238 (smt-0 leu1-32 ura4-D18) with a PCR fragment obtained with primers msh3_pAW1_For 5\u2032-TTTTTAAAACGTCCGCCTATATTCATTTAGAAACTATAAATGCGTTACAAATCTATGTTGAGCAAATTTGATTGTACAGTTTTTATTTTCGCTTTATTACCGGATCCCCGGGTTAATTAA-3\u2032 and msh3_pAW1_Rev 5\u2032-AGAACTTAAAACTGATGTATAGTTGCTTAAATTTATTTATACAGTAAAATCTTATGTGTTGAGTATCCTAATAAAGTAGTTAATTCAGTAATGCTCTCTCGAATTCGAGCTCGTTTAAAC-3\u2032 using pAW1 is a transformant of OL2137 with a PCR fragment obtained with primers dph3_pAW1_For2 5\u2032-CTTAGCTTGTAGTTTTCATTATGGGCGGTTCCCACATATAAAACAAATTTTTGGTGGAGTGGCCACGCACCTTCTGCCAGTAGTGCATTGAAGCGGCAAAAGCTTAGCTACAAATCCCAC-3\u2032 and dph3_pAW1_Rev2 5\u2032-TATCACGATGTAAAGAGTAGCCCTCCTATCCTTCGTAATTTCAAGACATTTTGAGAATAAATAGAAGTAAAAAACCAAATAAGAAATTATAGGGAAAAAAGCTTGTGATATTGACGAAAC-3\u2032 and pAW1 as template.DE4 , a derivative of pAW8 6 tag followed by three glycine-encoding codons, added between the natural ATG start codon and the second codon of msh3. Nucleotides underlined in msh3_pAW8_Rev 5\u2032 changed the two last codons of msh3 from GAA ATC (encoding the amino acids glutamic acid and isoleucine) to GAG CTC (encoding glutamic acid and leucine), thereby introducing a SacI restriction site, allowing further manipulation if desired.Plasmid pAW8-msh3 was constructed by in vitro Cre recombination between the h\u2212dph3-ATGmut ura4-D18) was constructed by transformation of DE4 with a PCR fragment obtained with primers dph3_ATGmut_For 5\u2032-TAGCTTGTAGTTTTCATTATGGGCGGTTCCCACATATAAAACAAATTTTTGGTGGAGTGGCCACGCACCTTCTGCCAGTAGTGCATTGAAGCGGCAAA-TGATCATTTTACGACGAAATCG-3\u2032 and dph3_Rev 5\u2032-TCACGATGTAAAGAGTAGCCCTCCTATCCTTCGTAATTTCAAGACATTTTGAGAATAAATAGAAGTAAAAAACCAAATAAGAAATTATAGGGAAAAATTATGCTGCAATGATTATAGGTG-3\u2032 and as template genomic DNA of strain RO144 (smt-0). \u201c-\u201din primer dph3_ATGmut_For indicates deletion of an A of the wild type sequence, which causes an in frame TGA stop codon instead of the ATG start codon. By this procedure, the lox sites flanking dph3 in DE4 were replaced in DE5. Sequencing confirmed the correct mutation in DE5 and an additional T155A point mutation, which is in intron II of the dph3 gene.Strain DE5 (smt-0 msh3-ATGmut leu1-32 ura4-D18) originated from the transformation of KK83 with a PCR fragment obtained from a mutagenized pAW8-msh3 plasmid as template. This plasmid contains the msh3 gene, with the 31st codon mutagenized from ATG to ATC by site directed mutagenesis with a QuikChange lightning site-directed mutagenesis kit (Agilent Technologies) using primers msh3_M31I_S 5\u2032-GGAGCAATATCAGAAGATATCGTTGCCCTCAGTGGTCCAG-3\u2032 and msh3_M31I_AS 5\u2032-CTGGACCACTGAGGGCAACGATATCTTCTGATATTGCTCC-3\u2032. Nucleotides deviating from the wild-type sequence are underlined. Primers for amplifying the 5\u2032 part of the open reading frame of msh3 with ATG mutations at codons 1 and 4 were msh3-ATGmut_For2 5\u2032-TCCGCCTATATTCATTTAGAAACTATAAATGCGTTACAAATCTATGTTGAGCAAATTTGATTGTACAGTTTTTATTTTCGCTTTATTACTCGAGAGGATAGAGTTATAACATTACTCATG-3\u2032 and msh3_Rev2 5\u2032-CTTAAAACTGATGTATAGTTGCTTAAATTTATTTATACAGTAAAATCTTATGTGTTGAGTATCCTAATAAAGTAGTTAATTCAGTAATGCTCTCTCTCAGATTTCTTCGAAAGCGGTAAG-3\u2032. Primer msh3-ATGmut_For2 contained base substitutions (underlined), which changed the ATG start codon of msh3 to TCG and the fourth codon from ATG to TAG. After integration into the genome, the mutated msh3 gene was amplified by PCR and sequencing confirmed the three desired ATG mutations. In addition, we found A555G, C696T and T1112C mutations. The primers used for PCR to construct an ATG mutated msh3 strain were designed in such a way that the (His)6 tag, the glycine linker and the lox sites of plasmid pAW8-msh3-M31I were not present in the genome of the resulting DE7 strain.DE7 including DNase I treatment. 2\u00a0\u00b5g of RNA were reverse transcribed with oligo (dT)18 primers using a Tetro cDNA Synthesis kit (Bioline). The resulting cDNA was subjected to 22\u201335 cycles of 30\u00a0s at 94\u00a0\u00b0C, 30\u00a0s at 57\u00a0\u00b0C and 30\u00a0s at 72\u00a0\u00b0C and a final extension for 10\u00a0min at 72\u00a0\u00b0C. For amplification of dph3 cDNA the forward primer dph3q-F 5\u2032-AGATTTCACGTTTGACGCCG-3\u2032 and the reverse primer dph3q-R 5\u2032-CTTGGGCAACGAGCAACATC-3\u2032 was used. In a pilot experiment , we used primers dph3_cPCR_For2 5\u2032-CTATGATGAAGATGAATTCATGGAAGTTG-3\u2032 and dph3_cPCR_Rev2 5\u2032-GAACAACAAACGTACTATGCCAATACAAAACC-3\u2032. Other reverse primers for amplification of dph3 cDNAs of various sizes were dph3q-R#2 5\u2032-TCACCCGGACAATCAAGCTG-3\u2032, dph3q-R#3 5\u2032-AGGTGCTGTAGAAGCATCGT-3\u2032, dph3q-R#4 5\u2032-GTAGCCCTCCTATCCTTCGT-3\u2032, dph3q-R#5 5\u2032-TGGAAAAGTCGTACGCTCAA-3\u2032, dph3q-R#6 5\u2032-AGGTGGGCTTTTAGTTTCGAGT-3\u2032, dph3q-R#7 5\u2032-ACGTTGAGCACAAGTACGAA-3\u2032 and dph3q-R#8 5\u2032-CCACTGTGGTATGTCGCACT-3\u2032. Primers for amplification of act1 cDNA were act1-For 5\u2032-AAGTACCCCATTGAGCACGG-3\u2032 and act1-Rev 5\u2032-CAGTCAACAAGCAAGGGTGC-3\u2032. PCR products were separated on 1.5% agarose gels in Tris\u2013Borate-EDTA buffer and images taken with a Gel Doc 2000 system (Bio-Rad). Band intensities of PCR products were quantified with the ImageJ software (NIH). dph3-specific bands were normalised to blanks to subtract background and to act1 cDNA.10\u00a0mL cultures were grown in YEL at 30\u00a0\u00b0C to a density of approximately 10S. pombe cells Supplementary material 2 (DOCX 15\u00a0kb)Below is the link to the electronic supplementary material."} +{"text": "C. elegans VAP homolog VPR-1 is essential for gonad development. vpr-1 null mutants are maternal effect sterile due to arrested gonadogenesis following embryo hatching. Somatic gonadal precursor cells and germ cells fail to proliferate fully and complete their respective differentiation programs. Maternal or zygotic vpr-1 expression is sufficient to induce gonadogenesis and fertility. Genetic mosaic and cell type-specific expression studies indicate that vpr-1 activity is important in the nervous system, germ line and intestine. VPR-1 acts in parallel to Notch signaling, a key regulator of germline stem cell proliferation and differentiation. Neuronal vpr-1 expression is sufficient for gonadogenesis induction during a limited time period shortly after hatching. These results support the model that the secreted VPR-1 MSPd acts at least in part on gonadal sheath cell precursors in L1 to early L2 stage hermaphrodites to permit gonadogenesis.VAMP/synaptobrevin-associated proteins (VAPs) contain an N-terminal major sperm protein domain (MSPd) that is associated with amyotrophic lateral sclerosis. VAPs have an intracellular housekeeping function, as well as an extracellular signaling function mediated by the secreted MSPd. Here we show that the Highlighted Article:vpr-1 null mutants are sterile upon hatching, a defect rescued by the expression of MSPd from almost any tissue except for the somatic gonad itself. See also the companion paper by Schultz et al. The major sperm protein domain (MSPd) is an evolutionarily conserved immunoglobulin-like structure found in unicellular and multicellular eukaryotes . The nam2+ dynamics and other processes. In addition, the VAP MSPd is cleaved from the transmembrane domain and secreted in a cell type-specific fashion A Lev et. Althoug fashion . The sec fashion . Hence, C. elegans. The C. elegans genome encodes numerous proteins that contain an MSPd and mouse Vapb mutants null mutation eliminates the first two vpr-1 exons, which encode the MSPd and part of the coiled-coil motif (vpr-1(tm1411) mutants derived from P0 vpr-1(tm1411)/hT2 heterozygous hermaphrodites exhibit limited fertility, with an average brood size of \u223c30 F2 progeny (vpr-1(tm1411) hermaphrodites lacking maternal (M\u2212) and zygotic (Z\u2212) vpr-1 expression (vpr-1(tm1411) mutant adults with maternal vpr-1 mRNA (M+ Z\u2212) contain functional sperm and oocytes /+ progeny produce functional gametes, similar to wild-type and M+ Z\u2212 vpr-1 mutant hermaphrodites null mutants transgenically expressing vpr-1 from fosmid DNA contain \u223c10 sheath cells (vpr-1 M\u2212 Z\u2212 gonads contain severely reduced numbers of sheath (\u223c4) and spermathecal (\u223c6) cells blast cells formed during L1 and early L2 C Hubbar. To inveransgene . In adulransgene E. We obscomplete E. Furtheskeleton . vpr-1 mth cells F. SS blath cells C. The adth cells C cells H-J. The ntiation .vpr-1 is broadly expressed in most tissues (vpr-1 functions (i.e. in which cell type) to promote gonad development. Transgenic vpr-1(tm1411) null mutants were generated using a DNA fosmid containing the vpr-1 genomic locus and a plasmid containing the sur-5p::NLS-GFP lineage marker worms rescued the gonad defects or failed ovulation, two defects consistent with abnormal somatic gonad development or function in the germ line using the pie-1 promoter (pie-1p::vpr-1g). Single-copy integrants on chromosome II were used to avoid germline silencing mechanisms. Two independently generated integrated transgenes completely rescued the vpr-1(tm1411) gonad and body wall muscle mitochondrial defects .Genetic mosaic worms lacking fertile , consist defects A-D gonad defects background. A small percentage (<20%) of transgenic mutants exhibited the novel gonad phenotype described above. In addition to the neuronal promoters, overexpressing vpr-1g specifically in the intestine using the ges-1 promoter . These results show that vpr-1 overexpression in diverse cell types is sufficient to promote gonadogenesis.A caveat with integrated transgenes is that ectopic expression in unwanted tissues may result from surrounding regulatory sequences. To avoid this issue, we tested a panel of well-characterized neuron-specific promoters using high-copy extrachromosomal arrays . Overexppromoter largely defects B,F. Simirneurons , the unc neurons , and the neurons null and clr-1(e1745ts) temperature-sensitive mutants have degenerating gonads , genetic mosaic and transgenic expression studies indicate that this degenerative defect is a consequence of hypodermal clr-1 loss, which causes massive fluid accumulation and larval lethality (vpr-1(tm1411) clr-1 RNAi hermaphrodites and transgenic vpr-1(tm1411); clr-1(e1745ts) hermaphrodites expressing clr-1 in the hypodermis exhibit correct muscle mitochondrial localization, yet have arrested gonads . Therefore, the clr-1 muscle pathway is unlikely to influence gonadogenesis. The MSPd also antagonizes the VAB-1 Eph receptor, which is expressed throughout the nervous system (vab-1(dx31); vpr-1(tm1411) double-null mutants have arrested gonads , indicating that excess VAB-1 signaling does not cause arrested gonadogenesis. Collectively, the results are consistent with MSPd signaling being important for gonad development, but independent of excess CLR-1 or VAB-1 signaling individually.The VPR-1 MSPd has been shown to interact with two broadly expressed receptors, CLR-1 and VAB-1, as well as an unidentified receptor(s) . clr-1 esurvival . In adulsurvival . We consFig.\u00a0S1) . Adult vs system . vab-1; glp-1(ar202ts) double mutants. glp-1(ar202ts) is a temperature-sensitive (ts) gain-of-function mutation (vpr-1(tm1411) single-mutant, glp-1(ar202ts) single-mutant, and vpr-1(tm1411); glp-1(ar202ts) double-mutant hermaphrodites grown at the restrictive temperature (25\u00b0C) from L1 (vpr-1(tm1411); glp-1(ar202ts) gonads (glp-1(ar202ts) gonads (vpr-1(tm1411) gonads gonads D lack th) gonads C and ins) gonads B. This rglp-1(bn18ts) is a temperature-sensitive loss-of-function mutation (glp-1(bn18ts) and vpr-1(tm1411) single mutants are small, but with an important difference. Germ cells in glp-1(bn18ts) gonads differentiate as sperm, whereas germ cells in vpr-1(tm1411) gonads fail to differentiate. If GLP-1 signaling is independent of vpr-1, then vpr-1(tm1411); glp-1(bn18ts) double-mutant gonads should contain sperm. To test this prediction, we grew single- and double-mutant hermaphrodites at 25\u00b0C from L1, stained them with DAPI, and performed fluorescence deconvolution microscopy. Characteristic highly condensed sperm chromosomes are not observed in vpr-1(tm1411) gonads (glp-1(bn18ts) single-mutant (vpr-1(tm1411); glp-1(bn18ts) double-mutant (vpr-1 mutants. These genetic data support the model that vpr-1 and glp-1 act in independent genetic pathways to promote germ cell expansion and differentiation.mutation . Gonads ) gonads B, but are-mutant E and vpre-mutant F gonads.vpr-1 activity we used the Q system, a drug-inducible binary gene expression system , which is added to plates, inhibits QS repressor activity, thereby activating gene expression. We used the glr-5 promoter to drive QF and QS expression in \u223c56 interneurons mutants . Six lines grew robustly in the presence of QA. All seven lines exhibited minimal QA-independent vpr-1 expression, as indicated by gamete development in a small percentage of transgenic vpr-1(tm1411) hermaphrodites. The Q system was more tightly regulated than the heat shock promoter (data not shown). Providing QA produced functional VPR-1, as evidenced by rescue of the muscle mitochondrial defect . We selected line 3 for further characterization because it grew very slowly without QA but grew rapidly with QA. An advantage of this line is that sufficient numbers of transgenic vpr-1(tm1411) homozygotes could be generated for staging. Similar results were observed for lines that failed to grow without QA.SS blast cells form during L1 and early L2, but do not complete development until mid to late L4 . To deten system . The QF cription A. A tranrneurons A. Seven vpr-1 mutant gonads. Class 2 gonads contain visible oocytes or an expanded germ cell population, but are sterile with no fertilized eggs in the uterus. Class 3 gonads contain differentiated gametes and fertilized eggs. In the absence of QA, 57% of transgenic vpr-1 mutant gonads were class 1 and 19% were class 3 embryos is sufficient for gonad induction. As this maternal contribution is not renewable, we considered the possibility that vpr-1 is sufficient transiently, early in development. To investigate this idea further, we provided QA to transgenic vpr-1 mutant adults, let their progeny hatch on QA plates, and then moved the L2 worms to plates lacking QA. We found little difference in class 3 gonad percentage in these experiments compared with controls grown continuously on QA or P4 (germ line) lineage caused gonads to arrest, although gonad development often appeared more advanced than that of gonads in M\u2212 Z\u2212 vpr-1 animals. Intestinal vpr-1 loss also caused sterility in some animals. Gonads from these mosaics produced oocytes but exhibited ovulation defects or other defects consistent with an abnormal somatic gonad. Our mosaics used fosmid DNA containing the entire vpr-1 genomic locus and likely most gene regulatory sequences. Although transgenes are typically silenced in the germ line due to their repetitive nature /hT2 [bli-4(e937) let-?(q782) qIs48] ; JK2868 qIs56 [lag-2p::GFP+ unc-119(+)]; WS2170 opIs110 [lim-7p::YFP::act-5+unc-119(+)]; OD58 unc-119(ed3) III; ltIs38 [pie-1p::GFP::PH(PLC1delta1)+unc-119(+)]; DG1575 tnIs6 [lim-7p::GFP+rol-6(su1006)]; CB4108 fog-2(q71); CZ337 vab-1(dx31); GC833 glp-1(ar202); DG2389 glp-1(bn18); XM1101 vpr-1(tm1411)/hT2 [bli-4(e937) let-?(q782) qIs48]; clr-1(e1745ts); XM1102 clr-1(e2530)/mIn1 [dpy-10(e128) mIs14]; XM1103 vpr-1(tm1411)/hT2 [bli-4(e937) let-?(q782) qIs48]; vab-1(dx31); XM1104 vpr-1(tm1411)/hT2 [bli-4(e937) let-?(q782) qIs48]; glp-1(ar202); Xm1105 vpr-1(tm1411)/hT2 [bli-4(e937) let-?(q782) qIs48]; glp-1(bn18). E. coli . The folclr-1(e1745), glp-1(ar202) and glp-1(bn18) temperature-sensitive alleles were conducted at permissive (16\u00b0C) and restrictive (25\u00b0C) temperatures, as indicated. Strain construction was performed using PCR, sequencing and phenotypic analyses. vpr-1(tm1411) mutants are maternal effect sterile. Phenotypes were evaluated in vpr-1(tm1411) homozygous F2 progeny from vpr-1(tm1411)/hT2 heterozygotes (P0), unless otherwise indicated. vpr-1(tm1411) homozygous F1 progeny contain maternal vpr-1 mRNA. To investigate zygotic vpr-1 activity, progeny of fertile F1 vpr-1 mutants mated to wild-type males were examined. RNAi was performed by the feeding method . This vpr-1 genomic fragment enhanced rescue of the vpr-1 mutant gonadogenesis defect relative to the vpr-1 cDNA fused to the unc-54 3\u2032 UTR. PCR was used to amplify sequences from genomic DNA. The unc-119p::vpr-1g, unc-25p::vpr-1g, unc-17p::vpr-1g, glr-5p::vpr-1g and ges-1p::vpr-1g constructs were generated using PCR and restriction enzymes in a TOPO vector backbone. The rol-6p::vpr-1g and myo-3p::vpr-1g constructs were generated using Gibson assembly (New England Biolabs) in a pGEM backbone. Primers are shown below. The pie-1p::vpr-1g construct was generated by Knudra Transgenics. The pie-1 promoter sequence included 1095 bp upstream of the translational start site. The vpr-1 DNA sequence included exons and introns, as well as 745 bp of the 3\u2032 UTR.Pan-neuronal (2000 bp) , GABA mo1893 bp) , choline2003 bp) , head in0003 bp) , intesti2003 bp) , body wa2385 bp) and hypo2000 bp) promoterclr-1 genomic locus, Gibson assembly was used to construct a plasmid containing clr-1 2\u2005kb left homology arm::tdTomato::clr-1 3\u2032 UTR::C. briggsae unc-119::2\u2005kb right homology arm. The single guide RNA (sgRNA) plasmid was derived from Addgene plasmid 46169. Cas9 targeting sequence was 5\u2032-ACTATATCTCTAAGACATAT-3\u2032. PCR was used to amplify the entire sgRNA backbone, except for 20 bp from unc-119. DNA fusions were constructed using Gibson assembly. The rol-6p::clr-1 construct was made with clr-1 genomic DNA. 2\u2005kb upstream of the rol-6 start codon was amplified by PCR. All constructs were confirmed by sequencing (UAB Heflin Center for Genomics Sciences). Primers were: vpr-1 F1 sacII, GGGGACAACTTTCCGCGGAAAAAAATGTCTGAAAAGCACAGTCTTCTG; vpr-1 R1 kpnI, GGGGACTGCTTTGGTACCCCGAGATAATACGGCGAAAA; glr-5 F1 BHI NtI, GGGGACAACTTTGGATCCGCGGCCGCGTCACAATTTTCGGGTGTCGTAG; glr-5 R1 NeI ScII, GGGGACTGCTTTCCGCGGGCTAGCGATGCTTATTATTCACATGTTTCAAACC; unc-17 F1 BHI NEI, GGGGACAACTTTGGATCCAGCGGCCGCTTCACACAATTAAGAATTTTAAGATTTGGG; unc-17 R1 NeI ScII, GGGGACTGCTTTCCGCGGGCTAGCCTCTCTCTCTCCCCCTGGAATATT; ges-1 F1 BHI NtI, GGGGACAACTTTGGATCCAGCGGCCGCAAACTCCGAACTATGATGACGAA; ges-1 R1 NeI ScII, GGGGACTGCTTTCCGCGGGCTAGCCTGAATTCAAAGATAAGATATGTAATAGATTTTT; unc-25 F1 BHI NtI, GGGGACAACTGGATCCAGCGGCCGCGAGAAATAAGAAATAATTGTATAATTTTTTTTTC; unc-25 R1 ScII NeI, GGGGACTGCTTTCCGCGGGCTAGCTTTTGGCGGTGAACTGAGCTTTT; topo F1 KpnI, GGGGACAACTTTGGTACCCCTGAATGGCGAATGGAC; topo pR1 BHI, GGGGACTGCTTTGGATCCAGCTCACTCAAAGGCGGTAA; vpr-1 F1 NheI, GGGGACAACTTTGCTAGCAAAAAAATGTCTGAAAAGCACAGTCTTCTG; glr-5 R1 ScII NeI, GGGGACTGCTTTGCTAGCCCGCGGGATGCTTATTATTCACATGTTTCAAACC; F1 rol-6 pgem5, AGGTCGACCATATGGGAGAGCTAGAAAAACGATGGATTGAGTTATCTGG; R1 vpr-1 rol-6, AGACTGTGCTTTTCAGACATCTGGAAATTTTCAGTTAGATCTAAAGATATATCC; F2 rol-6 vpr-1, GATCTAACTGAAAATTTCCAGATGTCTGAAAAGCACAGTCTTCTG; R2 pgem vpr-1, CTATGCATCCAACGCGTTGGGAACCATAAACATCAAATTTTATTGTACCATATAC; F1 pmyo-3 pgem5, GGTCGACCATATGGGAGAGCTGGCTATAATAAGTTCTTGAATAAAATAATTTTCCC; R1 vpr-1 pmyo-3, GCAGAAGACTGTGCTTTTCAGACATTTCTAGATGGATCTAGTGGTCGTGG; F2 pmyo-3 vpr-1, CCACGACCACTAGATCCATCTAGAAATGTCTGAAAAGCACAGTCTTCTGC; R2 pmyo-3 pgem5, GCTATGCATCCAACGCGTTGGGAACCATAAACATCAAATTTTATTGTACCATATAC.To create the Cas9 DNA template for tdTomato insertion into the Microscopy images were taken by a motorized Zeiss Axioskop equipped with epifluorescence and AxioVision software version 4.8. For DAPI staining, worms were fixed in 10% neutral buffered formalin (Sigma-Aldrich), mixed with 0.5\u2005\u00b5g/ml DAPI, and incubated for 24\u2005h at 4\u00b0C. Worms were then washed five to eight times with sterile water and mounted for microscopy. To image gonads, axial scans were performed and out-of-focus light was removed with deconvolution software (AxioVision).C. elegans, plasmids (5-60\u2005ng/\u03bcl) were injected into wild-type or vpr-1(tm1411)/hT2 young adult hermaphrodite gonads. The myo-3p::mitoGFP or sur-5p::NLS-GFP constructs were used for selection by Knudra Transgenics. Integrated transgenes were crossed into the vpr-1(tm1411) background and maintained as transgenic vpr-1 mutant homozygotes.To generate transgenic election . Multiplvpr-1 genomic locus together with 10\u2005ng/\u00b5l pTG96 (sur-5p::NLS-GFP) plasmid into vpr-1(tm1411)/hT2 hermaphrodite gonads null hermaphrodites (vpr-1(tm1411) worms were screened from three independent lines. +vpr-1 loss in the AB, P1, P2, P3, P4, E, EMS and other lineages was scored as previously described worms. Progeny were screened for rescue of the unc-119 movement defect and loss of myo3p::mitoGFP. Individual worms were isolated repeatedly to ensure 100% segregation. PCR and sequencing were used to confirm tdTomato insertion. The clr-1::tdTomato Cas9 line did not exhibit the fluid accumulation phenotype caused by reduced clr-1 function . Primers were : XW08 F1, CGGTTTGAAACATGTGAATAATAAGCATCATGGGCGCGCCTCTAGAGGATC; XW08 R1, GTTCTACGACACCCGAAAATTGTGACGCATGCAAGCTTGGCGTAATC; XW08 glr-5 F1, GATTACGCCAAGCTTGCATGCGTCACAATTTTCGGGTGTCGTAGAAC; XW08 glr-5 R1, GATCCTCTAGAGGCGCGCCCATGATGCTTATTATTCACATGTTTCAAACCG; XW09 F1, GGTTTGAAACATGTGAATAATAAGCATCATGGGCGCGCCTCTAGAGGATCC; XW09 R1, CTACGACACCCGAAAATTGTGACGGCCGGCCCAGTCAGTGCG; XW09 glr-5 F1, CGCACTGACTGGGCCGGCCGTCACAATTTTCGGGTGTCGTAG; XW09 glr-5 R1, GGATCCTCTAGAGGCGCGCCCATGATGCTTATTATTCACATGTTTCAAACC; XW12 F1, GGGAAACTGCTTCAACGCATCATGAGTAAAGGAGAAGAACTTTTCACTG; XW12 R1, GCAGAAGACTGTGCTTTTCAGACATTTTTTCTACCGGTACCGTCGAC; VPR-1 F1, GTCGACGGTACCGGTAGAAAAAATGTCTGAAAAGCACAGTCTTCTGC; VPR-1 R1, AGGTGAAAGTAGGATGAGACAGCAACCATAAACATCAAATTTTATTGTACCATATACA; SL2 F1, TGTATATGGTACAATAAAATTTGATGTTTATGGTTGCTGTCTCATCCTACTTTCACCT; SL2 R1,CAGTGAAAAGTTCTTCTCCTTTACTCATGATGCGTTGAAGCAGTTTCCC.Q system plasmids ang Shen . Gibson C. elegans, glr-5p::QF-SL2::mCherry::3\u2032UTR-unc-54 (10\u2005ng/\u03bcl), glr-5p::QS-SL2::mCherry::3\u2032UTR-unc-54 (50\u2005ng/\u03bcl), QUASp::\u0394pes10::vpr-1-SL2::GFP::3\u2032UTR-unc-54 (10\u2005ng/\u03bcl) and myo-3p ::mito::GFP (30\u2005ng/\u03bcl) plasmids were injected into vpr-1(tm1411)/hT2 young adult hermaphrodite gonads. Transgenic lines were selected based on mCherry and myo3p::mito::GFP expression. Seven independent lines were created. Six responded well to quinic acid (QA) treatment. For treatment, 300\u2005\u00b5l 300\u2005mg/ml pH 6.5 QA (Sigma-Aldrich) was mixed with 40\u2005\u00b5l M9 buffer and added to NGM plates seeded with NA22 bacteria. All lines exhibited very low vpr-1 expression in the absence of QA, partially rescuing the vpr-1 mutant gonadogenesis defect in a small percentage of transgenic worms. Increasing the glr-5p::QS dosage in the transgenic arrays appeared to limit QA-independent vpr-1 expression.To generate transgenic t-tests were computed using Excel 2013 (Microsoft) without the assumption of equal variance.Two-tailed Student's"} +{"text": "Dual functions of a small regulatory subunit in the mitochondrial calcium uniporter complex. Published 21, April 2016D. melanogaster, sequence #13 in Supplementary file 1, was incorrect due to a mistaken copy-and-paste while preparing the manuscript. The correct D. melanogaster sequence is now entered in the corrected The protein sequence of EMRE from This error did not affect any results or conclusions of the paper.Incorrect sequence:MTSKTVFQNAFKTFLDFAINSLPSTQGGLNITATAPGGVGQRPFTNKAGVLKLIFVSASSLYIGGLIAHKGASYLEENEIFVPTDEDDDDCorrected sequence:MIVPRLALPISLALQRVSRRVAEHPHNLRILQRHMSSVYFRSGAIKPKPEEMPFGLLAIFCAVIPGLFVGATISKNVANFLEENDLFVPADDDDDEDThe article has been corrected accordingly."} +{"text": "The deubiquitylating enzyme USP15 plays significant roles in multiple cellular pathways including TGF-\u03b2 signaling, RNA splicing, and innate immunity. Evolutionarily conserved skipping of exon 7 occurs during transcription of the mRNAs encoding USP15 and its paralogue USP4, yielding two major isoforms for each gene. Exon 7 of USP15 encodes a serine-rich stretch of 29 amino acid residues located in the inter-region linker that connects the N-terminal putative regulatory region and the C-terminal enzymatic region. Previous findings suggested that the variation in the linker region leads to functional differences between the isoforms of the two deubiquitylating enzymes, but to date no direct evidence regarding such functional divergence has been published. We found that the long isoform of USP15 predominantly recognizes and deubiquitylates mysterin, a large ubiquitin ligase associated with the onset of moyamoya disease. This observation represents the first experimental evidence that the conserved exon skipping alters the substrate specificity of this class of deubiquitylating enzymes. In addition, we found that the interactomes of the short and long isoforms of USP15 only partially overlapped. Thus, USP15, a key gene in multiple cellular processes, generates two functionally different isoforms via evolutionarily conserved exon skipping. In previous work, we isolated a key gene involved in the cryptogenic cerebrovascular disorder moyamoya diseasein vivo.Here, we report the first example of isoform-specific bias in substrate preference of USP15. Biochemical evaluation of the association between USP15 and mysterin revealed that mysterin is predominantly recognized by the long isoform of USP15. Moreover, the USP15 long isoform potently deubiquitylated mysterin and maintained its basal expression level, indicating that alternative exon skipping biases the deubiquitylation substrate preference of USP15. In addition, we performed an extensive LC-MS/MS analysis of USP15-binding proteins and identified multiple isoform-specific binding proteins, suggesting that the two isoforms of USP15 play roles in distinct proteome networks and have overlapping but independent cellular activities To elucidate the cellular function of the moyamoya disease-associated protein mysterin, we sought to identify mysterin-binding proteins, reasoning that such proteins would include cofactors and/or substrates of mysterin. To this end, we transiently overexpressed mysterin harboring a FLAG tag at its C-terminus (mysterin-FLAG) in HEK293T human embryonic kidney cells. We precipitated mysterin-FLAG from cell lysates using anti-FLAG antibody and performed LC-MS/MS analysis on total co-precipitates without an isolation process such as SDS-PAGE. Four positive hits, including USP15, LYAR, hnRNPK, and IP3 receptor isoform 3 (IP3R3) in addition to mysterin itself, were reproducibly identified in four trials. In a replication study, we transiently expressed each candidate mysterin-binding protein in HEK293T cells, immunoprecipitated using specific antibodies, and then immunoblotted for mysterin. In this analysis, USP15 and LYAR were detectably physically associated with mysterin, whereas the other two proteins were not . However23We obtained cDNAs encoding both isoforms of USP15 from HEK293 cells by reverse transcription\u2013polymerase chain reaction (RT-PCR). To our surprise, the isoform of USP15 with longer linker region (hereafter USP15L) had significantly stronger affinity for mysterin than the other isoform with the shorter linker . Mysterin-3FLAG was precipitated using FLAG affinity beads, and its ubiquitylation state was examined by immunoblotting using anti-ubiquitin antibody. Overexpression of USP15L significantly decreased the polyubiquitin chain signal associated with mysterin-3FLAG, whereas overexpression of USP15S did not, suggesting that USP15L has the stronger deubiquitylation activity against mysterin . The pol0.5% SDS . Mutatio0.5% SDS , but did0.5% SDS , supportPrevious studies demonstrated that USP15 removes K48-linked polyubiquitin chains and thereby protects its substrates against proteasomal protein degradation triggered by K48-linked polyubiquitylation5How does the alternative variation in the linker region bias the affinity of USP15 for mysterin? Although the substrate-binding domain of USP15 has not been definitely identified, one simple model is that the linker region directly mediates mysterin binding. We evaluated the roles of structural regionss and domains in USP15, including the long linker region, in the mysterin-binding process using five truncated mutants of USP15, schematically represented in To further characterize the functional divergence of the two major isoforms of USP15, we performed an interactome analysis on each isoform, based on the expectation that each interactome will include substrates and/or cofactors specific for each isoform. We transiently overexpressed the long and short isoforms of USP15, C-terminally tagged with three tandem FLAG epitopes, in HEK293T cells; precipitated them from cell lysate using anti-FLAG antibody; and performed LC-MS/MS analysis on total co-precipitates without isolation. Positive hits reproducibly obtained in all four trials are shown in 927Alternative skipping of exon 7 in USP15 resulted in deletion of a short element consisting of 29 amino acids in the inter-region linker of USP15 , which s5The exon 7-encoded short element in the linker region of USP15 was necessary but not sufficient for the physical association with mysterin. Likewise, the DUSP domain and the C-terminal region were also essential but not sufficient. The C-region harbors the enzymatic core responsible for deubiquitylation activity12299279Sixma and colleagues recently reported that the DUSP domain of USP4 promotes the deubiquitylation cycle by discharging hydrolyzed free ubiquitin from the enzymatic core of USP4 through inter-domain interactionsVariation in the linker region is likely to have been maintained under natural selection. Phylogenic study suggested that USP4, 11, and 15 arose from a single ancestral USP, conserved between fungi and metazoans, following the emergence of vertebrates4in vivo. Despite the obvious phenotypes in mysterin knockdown/knockout zebrafish, the cellular and molecular functions of mysterin remain controversial. Recent studies proposed roles in insulin production in pancreatic \u03b2 cellsWe initiated this study based on our interest in the function and/or regulation of the moyamoya disease susceptibility factor mysterin. The resultant data suggest that USP15L stabilizes mysterin via removal of K48-linked polyubiquitin chains. These observations were obtained under conventional cell culture conditions, suggesting that USP15L maintains the basal expression level of this ubiquitin ligase, which was previously shown to have autoubiquitylation activity19NheI/NotI sites (Myc) or NheI/HindIII sites (3FLAG) into the mammalian expression vector pcDNA3.1+ . Restriction enzymes were purchased from New England Biolab or Fermentas . DNA ligase was purchased from Takara . The enzymatic mutant of USP15L (15Lmt), which harbors alanines instead of the cysteines at positions 269 and 812, and that of USP15S (15Smt), which harbors alanine instead of the cysteine at positions 269, were generated by mutagenesis PCR using the QuikChange Site-Directed Mutagenesis kit using primers USP15C298AF (GCCTCTGTGGCCTAAGTAACTTGGGAAATACGGCTTTCATGAACTCAGCTATTCAGTGTTTGAGCAACACACC), USP15C298AR (GGTGTGTTGCTCAAACACTGAATAGCTGAGTTCATGAAAGCCGTATTTCCCAAGTTACTTAGGCCACAGAGGC), USP15C812AF (GCTGAAGATCCCTGGTATTGTCCGAATGCTAAAGAACATCAGCAAGCCACAAAGAAATTGG), and USP15C812AR (CCAATTTCTTTGTGGCTTGCTGATGTTCTTTAGCATTCGGACAATACCAGGGATCTTCAGC). Deletion mutants were generated using seven primers USP15FNheI (described above), USP15F2NheI (ATGCGCTAGCATGCCATCATATACCGCTTATAAGAAC), USP15FdN2NheI (ATGCGCTAGCCGCCATGGGTCCTTCTACTCCTAAGTCC), USP15FdN3NheI (ATGCGCTAGCCGCCATGGTAAAGCACTGCAAAGTAGAA), USP15R13FLAGHindIII (ATGCAAGCTTCTACTTATCGTCGTCATCCTTGTAGTCGATGTCATGGTCTTTGTAGTCACCGTCATGATCCTTGTAATCAAGACAGTAATTTGAGTTTTTCACATT), USP15R23FLAGHindIII (ATGCAAGCTTCTACTTATCGTCGTCATCCTTGTAGTCGATGTCATGGTCTTTGTAGTCACCGTCATGATCCTTGTAATCAATAATCACGTGCTCTGTATCTTCTGTCC), and USP15R33FLAGHindIII (ATGCAAGCTTTTACTTATCGTCGTCATCCTTGTAGTCGATGTCATGGTCTTTGTAGTCACCGTCATGATCCTTGTAATCGTTAGTGTGCATACAGTTTTC). \u0394C1, \u0394C2, \u0394N1, \u0394N2, and \u0394N3 deletion mutants were generated by PCR using USP15FNheI and USP15R13FLAGHindIII, USP15FNheI and USP15R23FLAGHindIII, USP15F2NheI and USP15R33FLAGHindIII, USP15FdN2NheI and USP15R33FLAGHindIII, and USP15FdN3NheI and USP15R33FLAGHindIII, respectively.Full-length cDNAs for USP15L (NM_001252078.1) and USP15S (NM_006313.2) were obtained simultaneously by RT\u2013PCR using the OneStep RT-PCR kit with template RNA extracted from HEK293 cells using the RNeasy Mini Kit (QIAGEN). USP15L or S with Myc or three tandem FLAG (3FLAG) epitope tag at the C-terminus was generated by PCR using forward primer USP15FNheI (ATGCGCTAGCACCATGGCGGAAGGCGGAGCGGC) and reverse primers USP15RMycNotI (ATGCGCGGCCGCCTACAGATCCTCTTCTGAGATGAGTTTTTGTTCGTTAGTGTGCATACAG) or USP15R3FLAGHindIII (ATGCAAGCTTTTACTTATCGTCGTCATCCTTGTAGTCGATGTCATGGTCTTTGTAGTCACCGTCATGATCCTTGTAATCGTTAGTGTGCATACAGTTTTC). These PCR products, harboring exogenous restriction sites and epitope tag at the 3\u2032 terminus, were subcloned using KpnI, NotI, SalI, and BamHI. Exogenous NheI and XbaI sites and the Myc epitope sequence were introduced into the 5\u2032 and 3\u2032 termini by PCR using forward primer CloNheIIP3R3F1 (ATGCGCTAGCCGCCATGAGTGAAATGTCCAGCTTTCTTCACATCGG) and reverse primer CloXbaIIP3R3R53FLAG (TACGTCTAGATCAGATGTCATGATCTTTATAATCACCGTCATGGTCTTTGTAGTCCTTATCGTCGTCATCCTTGTAATCGCGGCTAATGCAGTTCTGG). The PCR product was subcloned into pcDNA3.1+ using the NheI/XbaI sites. Full-length cDNA of LYAR was obtained by RT-PCR using forward primer KpnILYARCloF (ATGCGGTACCGCCACCATGGTATTTTTTACATGC) and reverse primer NotI3FLAGLYARCloR (TACGGCGGCCGCTTAGATGTCATGATCTTTATAATCACCGTCATGGTCTTTGTAGTCCTTATCGTCGTCATCCTTGTAATCTTTCACAAGCTTGACTTTG), and then subcloned into pcDNA3.1+ using the KpnI/NotI sites.Mysterin-FLAG and -3FLAG were generated in our previous studies19HEK293 (used only for cloning of USP15) and HEK293T cells were maintained in DMEM containing 10% fetal bovine serum, 100\u2009U/mL penicillin, and 100\u2009\u03bcg/mL streptomycin. Plasmids were transfected into HEK293T cells using Lipofectamine LTX (Life Technologies) or PEI MAX . The amount of DNA was adjusted to obtain equivalent expression levels of the introduced proteins in each experiment. For detection of ubiquitylation, cells were treated with 1\u2009\u03bcM epoxomicin for 8\u2009h (or other indicated period) before harvest.Antibodies used for immunoprecipitation (IP), immunoblotting (IB), or immunostaining (IS) in this study include anti-FLAG antibody , anti-Myc antibody , andi-HA antibody , anti-ubiquitin antibody , anti-K48 linked ubiquitin antibody , and anti-GAPDH antibody . Anti-human mysterin mouse monoclonal antibody (1C9) was generated with the method previously reportedMysterin-FLAG or USP15L/S-3FLAG was expressed in HEK293T cells, and associated proteins were recovered from cell extracts by immunoprecipitation with anti-FLAG antibody. The associated protein complexes with mysterin-FLAG were immediately digested with lysyl endopeptidase . Protein complexes associated with USP15L/S-FLAG were reduced with 5\u2009mM TCEP, and then alkylated with 10\u2009mM monoiodoacetoamide. The alkylated protein complexes were digested with Lys-C and trypsin . The resultant peptides were analyzed using a direct nano-flow LC-MS/MS system, as described previouslyg, and the supernatant was incubated for 1\u2009hour with anti-FLAG M2 agarose beads (Sigma Aldrich) or anti-Myc antibody and protein G beads . At this point, aliquots of supernatant were resolved with Laemmli\u2019s sample buffer containing 1\u2009mM DTT as a control . The beads were washed three times with digitonin-containing HEPES buffer and resolved with Laemmli\u2019s sample buffer containing 1\u2009mM DTT.Protein-encoding or empty vector was transiently transfected into HEK293T cells. The cells were harvested 24 or 48\u2009hours after the transfection and lysed with HEPES buffer containing 1% NP40 (for ubiquitylation assay) or digitonin . The lysate was centrifuged at 13,000\u2009Samples were separated with SDS-PAGE using 7.5% or 3\u201310% acrylamide gel and transferred to nitrocellulose or PVDF membrane for 1.5\u2009hours at 75\u2009V at 4\u2009\u00b0C. Membrane was blocked using Blocking One for 1\u2009hour at room temperature, and then incubated sequentially with primary and secondary antibodies diluted in Can Get Signal . Detection was performed via the chemiluminescence or the alkaline phosphatase method.2HPO4 and 1.47\u2009mM KH2PO4, pH 7.4). After incubation with Blocking One for 4\u2009hours at room temperature, the cells were incubated sequentially with primary and fluorescence-conjugated second antibodies; each of the antibody incubations were followed by PBS washes. The nucleus was stained using Hoechst 33342. Fluorescence images were obtained by confocal microscopy .HEK293T cells expressing USP15L-Myc or S-Myc together with mysterin-3FLAG were immobilized with 4% paraformaldehyde for 10\u2009min at room temperature 24\u2009hours after transfection. The cells were permeabilized with 100\u2009ng/mL digitonin for 5\u2009minutes, and then washed with phosphate-buffered saline .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations."} +{"text": "Drosophila, we discovered a 29-bp consensus sequence that we designated as the Clock-Associated Transcriptional Activation Cassette or \u2018CATAC\u2019. To experimentally address the spatiotemporal expression information associated with this element, we generated constructs with four separate native CATAC elements upstream of a basal promoter driving expression of either the yeast Gal4 or firefly luciferase reporter genes. Reporter assays showed that presence of wild-type, but not mutated CATAC elements, imparted increased expression levels as well as rhythmic regulation. Part of the CATAC consensus sequence resembles the E-box binding site for the core circadian transcription factor CLOCK/CYCLE (CLK/CYC), and CATAC-mediated expression rhythms are lost in the presence of null mutations in either cyc or the gene encoding the CLK/CYC inhibitor, period (per). Nevertheless, our results indicate that CATAC's enhancer function persists in the absence of CLK/CYC. Thus, CATAC represents a novel cis-regulatory element encoding clock-controlled regulation.Circadian clocks are autonomous daily timekeeping mechanisms that allow organisms to adapt to environmental rhythms as well as temporally organize biological functions. Clock-controlled timekeeping involves extensive regulation of rhythmic gene expression. To date, relatively few clock-associated promoter elements have been identified and characterized. In an unbiased search of core clock gene promoters from 12 species of Drosophila melanogaster, a heterodimer consisting of CLOCK (CLK) and CYCLE (CYC), acts as the core transcription factor , Per-Arnt-Sim (PAS) domain transcription factors (TFs) for transcriptional activation . In the n factor ,3. CLK/Cn factor \u20136. The dn factor ,8.Drosophila, studies of the promoter regions of known clock genes, period (per) and timeless (tim), found that E-box-dependent enhancers are necessary for circadian transcriptional modulation. The per promoter has, arguably, the best studied circadian enhancer motif to date. The enhancer is a 69-bp sequence upstream of the transcription start site (TSS) that activates circadian gene expression boxes, which are a variant of the consensus E-box sequence and transcription factor binding sites (TFBSs) have provided more tools to determine the elements responsible for transcriptional regulation . As a continuation of this work, we, hereby, identified a second novel and independent 29-bp motif. The present study describes spatiotemporal expression information contributed by the latter motif, which was named Clock-Associated Transcriptional Activation Cassette or \u2018CATAC\u2019.Increasing knowledge of the gulation ,11. In pgulation , we identim, vrille, Pdp1 and cwo (stich1) in 12 Drosophila species . This identified a conserved set of CATAC sequences (Drosophila melanogaster genome (BDGP R5/dm3) is included in the The bioinformatics model used to predict the \u2018CATAC\u2019 element is identical to that previously described for the discovery of the E1\u2013E2 element . Brieflyequences . In ordeequences , we traiPdp1 or Slob promoters: Slob 4xCATAC enhancer, 5\u0384pGGCCGATAACGCGGCGTATGCGCAATGTCGAAGCATATTACGCATACGCCCCATCCGC; 5\u0384pGTTTGCTGTGGCGGATGGGGCGTATGCGTAATATGCTTCGACATTGCGCATACGCCGCGTTATC; 5\u0384pCACAGCAAACGCTGCGTATGCGTAATACTTTGTGCACACGTTGCGTATGAGTAATGTCCT; 5\u0384pAATTAGGACATTACTCATACGCAACGTGTGCACAAAGTATTACGCATACGCAGC, Slob mt4xCATAC enhancer, 5\u0384pGGCCGATATCCCGGCCTTTGCCCTATGTCGAAGCATATAAGGCAAGGCCCGAACCGC; 5\u0384pGATTGCTGTGGCGGTTCGGGCCTTTGCCTTATATGCTTCGACATAGGGCAAAGGCCGGGATATC; 5\u0384pCACAGCAATCCCTGCCTTTGCCT TATACTTTGTGCACTCCTTGCCTTTGACTTATGTCCT; 5\u0384pAATTAGGACATAAGTCAAAGGCAAGGAGTGCACAAAGTATAAGGCAAAGGCAGG, Pdp1 4xCATAC enhancer, 5\u0384pGGCCAGCACATTACGCATACGTCACGTGTTGCAAAAATAATACGTATACGACGCGTGTC; 5\u0384pTGGTACAGAAGACACGCGTCGTATACGTATTATTTTTGCAACACGTGACGTATGCGTAATGTGCT; 5\u0384pTTCTGTACCATGCGGCGTATGAGCAATCTGTTAATACGTTACCCATACGCCCCGTGGGC C; 5\u0384pAATTGGCCCACGGGGCGTATGGGTAACGTATTAACAGATTGCTCATACGCCGCA, Pdp1 mt4xCATAC enhancer, 5\u0384pGGCCAGCACATAAGGCAAAGGTCAGGAGTTGCAAAAATAAAAGGTAAAGGACGGGAGTC; 5\u0384pAGGTACAGAAGACTCCCGTCCTTTACCTTTTATTTTTGCAACTCCTGACCTTTGCCTTATGTGCT; 5\u0384pTTCTGTACCTTC CGGCCTTTGACCTATCTGTTAATACGTA AGCCAAAGGCCCGGAGGGCC; 5\u0384pA ATTGGCCCTCCGGGCCTTTGGCTTACGTATTAACAGATAGGTCAAAGGCCGGA, Pdp1 4xCATAC with Slob-like E-boxes enhancer, 5\u0384pGGCCAGCACATTACGCATACGTAACGTGTTGCAAAAATAATACGTATACGAAGCGTTTC; 5\u0384pTCGTACAGA AGAAACGCTTCGTATACGTATTATTTTTGCAACACGTTACGTATGCGTAATGT GCT; 5\u0384pTTCTGTACGATGGGGCGTATGAGCAATCTGTTAATACGTTACCCATACGCCGCGTTGGC C; 5\u0384pAATTGGCCAACGCGGCGTATGGGTAACGTATTAACAG ATTGCTCATACGCCCCA. Oligos were annealed to match their order and orientation in the native Pdp1 or Slob promoters. Flanking EagI and EcoRI sequences allowed for the 4xCATAC enhancers to be inserted upstream of the hsp70 basal promoter in the pPTGAL vector (luc) were created by blunt-end cloning to replace a 3193 bp fragment containing Gal4 flanked by PstI sites with a 1955 bp HindIII\u2013BamHI fragment containing the luciferase gene from pGL3-Basic (Promega) (luc can be requested online (http://www.addgene.org/Herman_Wijnen/). For each reporter construct, several independent lines were generated.Synthetic oligonucleotides were designed to match four separate, native occurrences of the CATAC element from either the L vector (obtainePromega) . Copies 1118w,\u00a0UAS-CD8::GFP, and 2eya mutations were obtained from the Bloomington Drosophila Stock Center. The 01, per01cyc and tim-luc alleles have been described previously and relative amplitude error (RAE) by an iterative, coupled fast Fourier transform-non-linear least squares (FFT-NLLS) multicomponent cosine analysis . RAE is Gal4 driven expression of membrane-tethered green fluorescent protein were dissected in Ringer's solution . Flies were harvested onto dry ice at time points CT0, CT6, CT12, CT18 and CT24, and adult heads were dissected on a chilled platform and transferred to guanidinium thiocyanate buffer. Four separate groups of flies (\u223c50 each) were used for each experimental condition. Total RNA was obtained from the heads using the RNAqueous4PCR kit (Ambion). Sample concentration and purity was analysed using a NanoDrop spectrophotometer. Samples exhibited OD 260/280 ratios between 1.8 and 2.1. Concentrations were adjusted to 25 ng/\u03bcl in 10 mM Tris\u2013HCl 0.1 mM EDTA pH 8.0 buffer and samples were frozen at \u201380\u00b0C in aliquots until further use. The RNA samples were then analyzed with the SensiFAST SYBR No-ROX One-Step qPCR Kit (Bioline) using experimental primer pairs designed to specifically amplify fragments of the circadian Pdp1 or Slob transcripts, the transgenic luciferase transcript or the EF1\u03b2 control transcript relative to EF1\u03b2 were determined using the comparative Cycle threshold (Ct) method . ript see . All pri,23. riptcis-acting elements associated with clock gene expression, we combined MEME for 12 species of Drosophila. As a result, a 29-bp motif designated the Clock-Associated Transcriptional Activation Cassette or \u2018CATAC,\u2019 was identified as over-represented in these promoters is provn = 97 genes) encoding transcripts predicted to exhibit circadian oscillations (Slob (Slowpoke binding protein) predicted to be induced upon activation of the circadian regulator CLK as well llations \u201328 (Suppllations . A promiprotein) . With th genomes .Based upon the sequence alignment, conservation, and subsequent CLK/CYC target gene enrichment analyses, CATAC was hypothesized to be a regulatory element involved in mediating spatiotemporal expression of clock-regulated genes.Pdp1 and Slob, that not only exhibit strong CLK/CYC-associated circadian oscillations, but also have an unusually high number of CATAC motifs. The Pdp1 gene encodes a clock component (PDP1-\u03b5) that impacts molecular circadian oscillations, particularly in the clock neurons, as well as circadian behaviour as well as the CRCGTG consensus for the E-box-like element within CATAC which allowed measurement of the transcriptional reporter constructs at relatively high frequency in vivo. Native Pdp1 and Slob 4xCATAC both showed strong luciferase expression and both mt4xCATAC constructs shared characteristically low level expression in which the orientation of Slob 4xCATAC in the vector was inverted and \u2018Pdp1 4xCATAC with Slob-like E-boxes\u2019 (PSE) in which the four E-boxes of Pdp1 4xCATAC were mutated such that their sequence fidelity matched that of the four E-boxes in Slob 4xCATAC , was significantly stronger than those for mt4xCATAC and empty vector controls, respectively and Slob (ZT 3\u20135). Heads showed luciferase expression in not only the compound eyes, but also the proboscis and antennae; each of which has been shown to possess a circadian oscillator genetic background , by and large there was little effect of the 01per mutation on their expression levels construct. PSE is a modified version of Pdp1 4xCATAC, which has E-box sequences that were mutated such that their deviation from the CACGTG consensus matched that of the four E-boxes in the Slob 4xCATAC enhancer , constructs. Thus, the considerable variation in quality of E-box sequences between these constructs did not obviously impact reporter gene rhythms. The sequence immediately flanking an E-box element has also been implicated in affecting regulation is known to bind E-boxes in clock-controlled genes (For all the similarities that CATAC shares with E-box motifs, CATAC is not simply another E-box element. Normally, an E-box regulated transcriptional element exhibits low reporter expression in a in per01 ,41. CATAed genes ,43 and mPdp1 and Slob. Of course, this conclusion comes with the caveat that the multimerized constructs do not represent the complexity of the native genomic environment. Mutational analysis of CATAC sequences in their natural context will constitute an important next step in the functional analysis of this cis-regulatory element. Nevertheless, the identification of CATAC contributes to our knowledge of circadian transcriptional elements, and may be used to further characterize the regulatory regions of clock and clock-regulated genes.Thus, we have discovered a novel circadian regulatory element that, although it possesses an E-box-like motif, exhibits non-E-box-like responses. Through use of multimerized CATAC element, we concluded that CATAC is capable of contributing to the rhythmicity of clock genes, such as Supplementary DataClick here for additional data file."} +{"text": "Open Biol. 7, 170139. (Published online 21 November 2017). (doi:10.1098/rsob.170139).A correction is required to the sequence of sgRNA_FD2 in .The correct sequence is as follows:sgRNA_FD2:TTCCAGAAGCCGCCTTTGCCCGGGTTTTAGAGCTAGAAATAGCAAG-3\u20325\u2032-TAATACGACTCACTATAAll the authors gave their consent to this correction."} +{"text": "Plasmodium falciparum presents serious challenges, as standard molecular techniques such as siRNA cannot be employed in this organism, and generating gene knock-outs of essential proteins requires specialized conditional approaches. In the study of protein kinases, pharmacological inhibition presents a feasible alternative option. However, as in mammalian systems, inhibitors often lack the desired selectivity. Described here is a chemical genetic approach to selectively inhibit Pfnek-2 in P. falciparum, a member of the NIMA-related kinase family that is essential for completion of the sexual development of the parasite.Examining essential biochemical pathways in Introduction of a valine to cysteine mutation at position 24 in the glycine rich loop of Pfnek-2 does not affect kinase activity but confers sensitivity to the protein kinase inhibitor 4-(6-ethynyl-9H-purin-2-ylamino) benzene sulfonamide (NCL-00016066). Using a combination of in vitro kinase assays and mass spectrometry, (including phosphoproteomics) the study shows that this compound acts as an irreversible inhibitor to the mutant Pfnek2 likely through a covalent link with the introduced cysteine residue. In particular, this was shown by analysis of total protein mass using mass spectrometry which showed a shift in molecular weight of the mutant kinase in the presence of the inhibitor to be precisely equivalent to the molecular weight of NCL-00016066. A similar molecular weight shift was not observed in the wild type kinase. Importantly, this inhibitor has little activity towards the wild type Pfnek-2 and, therefore, has all the properties of an effective chemical genetic tool that could be employed to determine the cellular targets for Pfnek-2.Allelic replacement of wild-type Pfnek-2 with the mutated kinase will allow for targeted inhibition of Pfnek-2 with NCL-00016066 and hence pave the way for comparative studies aimed at understanding the biological role and transmission-blocking potential of Pfnek-2. Despite the efficacy of artemisinin-based combination therapy (ACT) in the treatment of malaria, an estimated 214 million cases and 438,000 malaria deaths occurred globally in 2015 was added to each well on the plate. The plate was shaken for 10\u00a0min to ensure mixing and the absorbance at 595\u00a0nm was obtained using a CLARIOstar plate reader. .PF3D7_0525900) was purchased (with codon optimization for Escherichia coli expression) from GeneArt\u2122 Life Technologies. The codon optimized DNA sequence was as follows (the codon encoding V24 is highlighted in bold); ATGAGCAAACCGAAAATGATCGGTCCGTATGAAGTTGTGAAAAGCATTGGTCGTGGTAGCTTTGGTATTGTTACCGCAGTTAAAGATGAAAATGAAAAAATCTTTGTGATTAAAGAACTGGATATTAGCTGCATGAATAACAAAGAAAAAATGAATGTGGTGAATGAAATTCGTGCCCTGATTAAAATGAGCGTGCATCCGTTTATTGTGCGCTATAAAGAAGCCTTTGTTGAAGATTGCGTTCTGTATGTTGCGATGGATTATTGCATTAATGGTGATCTGGGCAAAGTGATCAAAAAACACAAAGAACTGGAAACCCCGATTCCTGAGAAAAAAATCAAACGTTGGCTGCTGCAGATTATTATGGCCATCAAATTCATTCATGATAAAAAACTGATCCATCGTGATCTGAAATGCAATAACATCTTCCTGGATGAAAAAGAACGTGCCAAAATTGGTGATTTTGGTCTGGCCAAATTTATCGAACAGACAGAACAGACCAATACCCTGTGTGGCACCATTGGTTATATGGCACCGGAAATTTGCAAAAATATCAATTACAGCTTCCCTGCCGATATTTGGAGCCTGGGTATTATTCTGTATGAACTGATTAGCCTGAAACCGCCTTTTAAAAGCAATAACAGCAATATGCTGAGCGTGGCCCAGAAAATTTGTGAAGATGAACCGGATCCGCTGCCGGATAGCTTTAGCAAAGATCTGATTAATCTGTGCTATTGGATGCTGAAAAAAGATTGGAAAGATCGTCCGACCATCTACGATATTATCAGCACCGATTATATCCAGGATGAACTGCAGCTGTTTAAACGTGAAATGCTGCAAGAACGTAACAGCCAGATT.The synthetic full-length coding sequence for Pfnek-2 and reverse (5\u2032-tctttaactgcggtacaaataccaaagctaccacgaccaatgc-3\u2032).E. coli BL21-Codon Plus(DE3)-RIPL strain (Agilent Technologies). Cells were treated with 1\u00a0mM IPTG when the optical density of the cell culture at 600\u00a0nm reached 0.6, and protein expression was induced overnight at 18\u00a0\u00b0C. For purification, cells were lysed in buffer [1 tablet per 10\u00a0ml (cOmplete\u2122 ULTRA Tablets by Roche)] by sonication for 10 cycles of 10\u00a0s bursts at 10\u00a0mA with a 30\u00a0s rest between each cycle. During sonication the cells were kept on ice. Lysates were then centrifuged at 20,000g for 30\u00a0min. The resulting supernatants were loaded onto a 0.5\u00a0ml pre-equilibrated Ni\u2013NTA Superflow resin (Qiagen) column, washed with buffer and proteins were eluted with the same buffer containing 400\u00a0mM Imidazole. Eluted proteins were dialysed against 50\u00a0mM bicine, pH 8.0, 150\u00a0mM NaCl, 1% glycerol and 1\u00a0mM DTT at 4\u00a0\u00b0C overnight. Dialysed Pfnek-2 protein was aliquoted and stored at \u221280\u00a0\u00b0C.Full-length Pfnek-2 as a 6-HIS-tagged protein was expressed in 2, 4\u00a0mM imidazole, 1\u00a0mM DTT) in the presence of 5\u00a0\u00b5g MBP. The reaction was started by the addition of 5\u00a0\u00b5l 200\u00a0\u00b5M ATP/32P-ATP (0.0185\u00a0MBq per reaction)\u2014total volume 20\u00a0\u00b5l. Following incubation of 30\u00a0min at 37\u00a0\u00b0C, the reactions were stopped by adding 10\u00a0\u00b5l 2\u00d7 Laemmli buffer and proteins were separated by SDS\u2013PAGE on 12% gels and stained with Coomassie blue. Dried gels were exposed to X-ray film.Recombinant His-tagged WT or V24C mutant Pfnek-2 were assayed for protein kinase activity using myelin basic protein (MBP) as a substrate. 1.8\u00a0\u00b5g of either Pfnek-2 WT or Pfnek-2 V24C were pre-incubated at room temperature for 40\u00a0min with/without NCL-00016066 in Pfnek-2 buffer was dissolved in DMSO to 10\u00a0mM and frozen in aliquots of 10\u00a0\u00b5l at \u221280\u00a0\u00b0C. These aliquots were kept in the dark and a fresh aliquot diluted prior to each kinase assay. All samples in each kinase assay were pre-incubated with NCL-00016066 for 40\u00a0min at room temperature and the reaction initiated by the addition of ATP/2, 1% glycerol, 4\u00a0mM imidazole, 1\u00a0mM DTT at room temperature for 30\u00a0min. 50\u00a0\u00b5l Ni\u2013NTA Superflow resin (Qiagen) was added and the samples made up to 500\u00a0\u00b5l in buffer and rotated for 1\u00a0h at room temperature. The beads were washed 3\u00d7 in 500\u00a0\u00b5l buffer for 5\u00a0min rotation and supernatants discarded. The in vitro kinase assay protocol described above was followed and the enzyme immobilized on beads (washed and supernatant removed) is assumed to be 5\u00a0\u00b5l for the purpose of the kinase assay. During incubation at 37\u00a0\u00b0C the beads were gently re-suspended every 10\u00a0min.Recombinant Pfnek-2 wild type and Pfnek-2 V24C were purified as previously described and incubated (1:1) without/with 20\u00a0\u00b5M NCL-00016066 diluted in 10\u00a0mM bicine, pH 8.0, 40\u00a0mM NaCl, 10\u00a0mM MgCl2, 4\u00a0mM imidazole, 1\u00a0mM DTT) at room temperature prior to mass spectrometry analysis.Recombinant Pfnek-2 wild type and Pfnek-2 V24C were purified as previously described except glycerol was absent from all buffers. The absence of glycerol had no effect on the activity of the enzyme and was only added to previous preparations to stabilize the enzyme. Glycerol however interferes with the mass spectrometry and so was omitted here. Both wild type and mutant (V24C) Pfnek-2 were incubated (1:1) without/with 100\u00a0\u00b5M NCL-00016066 diluted in buffer and molecular weight calculations made using ESIprot for 1\u00a0h. The gel was stained with four parts colloidal blue to one part methanol and destained with 10% acetic acid in 25% (v/v). Protein bands were excised from the gel and cut into small pieces and washed three times with 200\u00a0\u03bcl of buffer B [20\u00a0ml 400\u00a0mM ammonium bicarbonate (3.16\u00a0g/100\u00a0ml) with 100% acetonitrile in ratio 1:1]. The gel pieces were then washed twice with 200\u00a0\u03bcl acetonitrile (100%) and gel pieces air dried.Recombinant Pfnek-2 (4\u00a0\u00b5l of 13\u00a0mg/ml stock solution of enzyme) was incubated with/without 1\u00a0mM ATP in Pfnek-2 buffer followed by a further addition of acetonitrile for 10\u00a0min at room temperature followed by aspiration of the fluid and air drying. Trypsin solution (1\u00a0\u03bcg in 50\u00a0\u03bcl of 50\u00a0mM TEAB buffer per sample) was added and incubated at 37\u00a0\u00b0C overnight. The gel pieces were washed in 50\u00a0mM TEAB buffer for 5\u201310\u00a0min on a rotating platform. The digests and washes were combined and dried completely in a SpeedVac. The samples were then enriched for phosphopeptides as described below.g, and the flow-through (unbound peptides) collected and frozen. The IMAC beads were washed twice with 200\u00a0\u00b5l of IMAC load/wash buffer, once with 200\u00a0\u00b5l of HPLC grade water and eluted twice with 100\u00a0\u00b5l of solution containing 22\u00a0\u00b5l ammonia solution , 300\u00a0\u00b5l acetonitrile to a total volume of 1\u00a0ml with HPLC grade water. Eluates were concentrated to a small volume (15\u201320\u00a0\u00b5l) in a Speedvac centrifuge and submitted for mass spectrometry analysis.PHOS-Select iron affinity gel (Sigma) was equilibrated with 5\u00a0\u00d7\u00a01\u00a0ml IMAC load/wash buffer . 500\u00a0\u00b5l IMAC load/wash buffer was added to completely dry samples and 50\u00a0\u00b5l of 50% PHOS-Select slurry was added followed by rotating at room temperature for 1\u00a0h. Following incubation with IMAC beads, the samples were transferred onto Mobicol \u2018Classic\u2019 spin columns (2B Scientific Ltd), centrifuged for 30\u00a0s at 1000\u00d7This was carried out as previously described with one50 values of 23.95\u00a0\u00b1\u00a02.50 and 35.63\u00a0\u00b1\u00a03.03\u00a0\u00b5M as measured by SYBR Green I and LDH parasite death assays, respectively were resolved by SDS PAGE and appeared as a single band at approximately 38\u00a0kDa value, 6.30\u00a0\u00b1\u00a00.25 (n\u00a0=\u00a03)] by NCL-00016066, the mutant kinase was pre-incubated with NCL-00016066 (20\u00a0\u00b5M) prior to immobilization on Ni\u2013NTA beads, and unbound inhibitor were removed by extensive washing. As a control, Pfnek-2 was similarly incubated with high concentrations of NCL-00016066 (20\u00a0\u00b5M), which gave partial inhibition of kinase activity, which was reversed following washout Fig.\u00a0a. In conIn an effort to further characterize the nature of the inhibition of the mutant Pfnek-2 with NCL-00016066, wild type Pfnek-2 or Pfnek-2(V24C) were pre-incubated with or without 100\u00a0\u00b5M NCL-00016066 and the intact protein subjected to analysis by mass spectrometry.m/z ratio of Pfnek-2 (red trace Fig.\u00a0m/z ratio upon addition of NCL-00016066 (Fig.\u00a0Pfnek-2 without inhibitor (black trace Fig.\u00a0Table\u00a0Mass spectrometry analysis of the intact protein as described above indicated that Pfnek-2 undergoes multiple autophosphorylation events. Analysis of tryptic phospho-peptides unequivocally identified three autophosphorylation sites, Ser20, Ser215 and Ser219 Fig.\u00a0. InteresThe aim of the current study was to engineer Pfnek2 in a manner that would confer sensitivity of the kinase to a kinase inhibitor that otherwise would show low levels of activity to wild type Pfnek2. This was achieved by mutating Val24 in the wild type Pfnek-2 to cysteine. This mutation confers susceptibility to the kinase inhibitor NCL-00016066, and that the nature of this inhibition is likely through an irreversible covalent link of NCL-00016066 with the modified cysteine.Plasmodium berghei showed that parasites lacking the enzyme Pfnek-2 are able to undergo gametocytogenesis, gametogenesis and fertilization, but do not develop into ookinetes; this is likely due to a defect in meiosis, as pbnek-2\u2212 parasites appear to be unable to implement the pre-meiotic DNA replication that brings the DNA content to 4C in wild-type parasites, but remains 2C in the mutant parasites [Pfnek-2 is expressed predominantly in female gametocytes . Transmiarasites . The demarasites suggestsStructural analysis of the Hsnek2 in complex with NCL-00016066 demonstrated that this inhibitor acts in an irreversible manner by covalent linkage to cysteine 22 in the glycine rich loop. Interestingly, Pfnek-2 does not contain this cysteine, or any other cysteine, in this loop. Demonstrated here is the weak inhibitor activity of NCL-00016066 against wild-type Pfnek-2 likely due to a lack of a cysteine in this position. Consistent with this notion is that NCL-00016066 showed potent inhibitory activity if a cysteine residue is introduced into the glycine-rich loop of Pfnek-2. Furthermore, this inhibition appeared to be irreversible, and mass spectrometry studies indicated that this is likely due to covalent modification of the variant kinase at the substituted cysteine.Recent studies have deployed a chemical genetic approach to the study of PfPKG where parasites expressing a mutant form of the kinase that is resistant to inhibition by a selective PfPKG inhibitor was used to identify cellular targets and physiological roles of the kinase . The engIn this way, the data presented here opens the door to investigating the essential role that Pfnek-2 plays in the transmission of malaria from the host to the mosquito vector and in doing so might inform on the mode of action of potential transmission-blocking drugs targeting this kinase."} +{"text": "RIP (ribonucleoprotein immunoprecipitation) and RNA pull-down assays showed that Meg3 was co-immunoprecipitated with ATG3. In addition, Meg3 protected ATG3 mRNA from degradation following treatment with actinomycin D. Overall, our results suggest that the lncRNA Meg3 acts as a tumor suppressor in EOC by regulating ATG3 activity and inducing autophagy.Maternally expressed gene 3 (Meg3), a long non-coding RNA, has been reported to be associated with the pathogenesis of multiple malignancies. However, little is known regarding the role of Meg3 in epithelial ovarian cancer (EOC). In this study, we found that the expression of Meg3 was lower in epithelial ovarian carcinoma, and has potential to be considered as a biomarker for ovarian cancer. After transfecting the ovarian cancer cell lines OVCAR3 and A2780 with Meg3, phenotypic changes and autophagy-related molecules were examined. Upregulation of Meg3 inhibited cell proliferation, plate colony formation, induced cell cycle arrest in G2 phases, and promoted apoptosis. Observation of autophagosomes was performed by transmission electron microscopy. The expression levels of LC3-II, ATG3, LAMP1 were elevated, while SQSTM1/p62 expression declined. Upregulated expression of Meg3 also suppressed tumorigenesis Epithelial ovarian carcinoma (EOC) remains one of the most common gynecologic malignancies, with 21,290 new cases diagnosed and 14,000 deaths reported in the United States in 2015 [Long non-coding RNAs (lncRNAs) are a family of non-protein-coding RNAs greater than 200 nucleotides in length . LncRNAsMeg3 mRNA expression was lower in EOC tissue as compared to normal ovarian tissues and benign ovarian carcinomas. In metastatic omentum tumors, expression was found to be significantly lower as compared to all above assays were performed to confirm the functional interaction between MEG3 and autophagic protein ATG3. RNA from RIP assays with an antibody against ATG3 was used for qPCR analysis, which demonstrated an enrichment of the lncRNA Meg3 Figure . Next, wCells transfected with Meg3 were treated with actinomycin D to study the stability of ATG3 mRNA. Our results showed that ATG3 mRNA in cells transfected with Meg3 showed a slower rate of decay compared with control cells Figure .qRT-PCR and Western blot analysis were used to measure LC3, SQSTM1/P62, LAMP1 mRNA and protein expression after transfecting si-RNA to silence expression of ATG3 in OVCAR3 cells. Both mRNA and protein expression of LC3 Figure and LAMPRecently, a series of studies have focused on the molecular mechanism of pathogenesis and progression of tumors, and the action of lncRNAs has attracted intense research interest. Studies have revealed that lncRNAs are involved in various processes including development, cell proliferation, metastasis, fate decision, invasion and migration \u201315, whicTo investigate the action of Meg3 in EOC, we analyzed the mRNA expression of Meg3 in normal epithelial ovarian tissues, benign tumor tissues, primary ovarian carcinomas, and metastatic omentum tumors. We found that the expression of Meg3 was reduced significantly in primary tumors as compared to normal ovarian tissue and benign tumors. Furthermore, the expression of Meg3 in metastatic omentum tumors was even lower than in primary ovarian carcinoma. These results demonstrated that there is a close relationship between Meg3 and tumorigenesis, as well as the development of ovarian cancer.in vivo. In conclusion, Meg3 plays a suppressive role in EOC, which is consistent with the role of Meg3 in gastric cancer, cervical cancer, lung cancer and hepatocellular carcinoma.Transfection of ovarian cancer cell lines with Meg3 resulted in inhibition of cell proliferation, colony formation, migration and metastasis, and promotion of G2 phase arrest, as well as cell apoptosis, while after given Meg3-transfected cells with autophagy inhibitor 3MA, the cell viability and colony formation rate was improved as compared with Meg3-transfected cells. Nude mice xenograft assays showed that overexpression of Meg3 could reduce tumorigenesis of ovarian cancer cells in vivo and in vitro. Therefore, we suggest that Meg3 acts as a tumor suppressor in EOC, which may be achieved by initiating autophagy and causing type II cell death.Furthermore, autophagosomes observed under transmission electron microscopy indicated that Meg3 induced autophagy in EOC. Autophagy, in general, is a survival process. Autophagic (type II) cell death usually represents a failed attempt to overcome lethal stress, and disruption of this process promotes rather than inhibits cell death in many cases , 25. SevRecent studies have provided a significant perspective on the important role of lncRNAs in regulation of gene expression. Reports have revealed that lncRNAs may act as either scaffolds that contain distinct protein-interacting domains to bring specific protein into proximity of each other, and form unique functional complexes, or as guides to recruit proteins \u201332. UpreIn conclusion, the present study has shown for the first time that upregulated expression of Meg3 triggers autophagic flux through an interaction with ATG3 to suppress tumorigenesis and progression of EOC. Meg3 may serve as a potential biomarker of EOC. Our findings may provide a novel insight into the early diagnosis and treatment of ovarian cancer. And further research should delineate the role and regulation of the crosstalk between Meg3 and autophagy in pathogenesis and progression of EOC.EOC tissues (Ca), metastatic omentum tissues (Om), borderline tumor (Bo) tissues, benign tumor tissues (Be) and normal ovarian specimens (No) were obtained from patients who underwent surgical resection at the Department of Gynecology, the First Affiliated Hospital of China Medical University among 2005.01-2015.12. Tumor specimens were reviewed by microscopy by two independent pathologists and staged according to the FIGO staging system. Samples were frozen in liquid nitrogen immediately and stored at \u2212 80\u00b0C until use. No patients underwent chemotherapy or radiotherapy treatment prior to surgery. Informed consent was obtained from all subjects. Ethics board approval was obtained from the China Medical University Ethics Committee, and all specimens were handled and anonymized in an ethical and legal manner.2. The medium was changed every one to two days based on the culture state. MEG3-specific pcDNA overexpression vector (pcDNA-MEG3), ATG3-specific siRNA (si-ATG3) and corresponding control including empty pcDNA, and si-scramble were completed by Shanghai Genechem Co., Ltd., China. These recombinants were transfected into OVCAR3 or A2780 cells using Lipofectamine 2000 reagent according to the manufacturer's instructions. The sequence of Meg3 plasmid is CTCGAGCTAGCCCCTAGCGCAGACGGCGGAGAGCAGAGAGGGAGCGCGCCTTGGCTCGCTGGCCTTGGCGGCGGCTCCTCAGGAGAGCTGGGGCGCCCACGAGAGGATCCCTCACCCGGGTCTCTCCTCAGGGATGACATCATCCGTCCACCTCCTTGTCTTCAAGGACCACCTCCTCTCCATGCTGAGCTGCTGCCAAGGGGCCTGCTGCCCATCTACACCTCACGAGGGCACTAGGAGCACGGTTTCCTGGATCCCACCAACATACAAAGCAGCCACTCACTGACCCCCAGGACCAGGATGGCAAAGGATGAAGAGGACCGGAACTGACCAGCCAGCTGTCCCTCTTACCTAAAGACTTAAACCAATGCCCTAGTGAGGGGGCATTGGGCATTAAGCCCTGACCTTTGCTATGCTCATACTTTGACTCTATGAGTACTTTCCTATAAGTCTTTGCTTGTGTTCACCTGCTAGCAAACTGGAGTGTTTCCCTCCCCAAGGGGGTGTCAGTCTTTGTCGACTGACTCTGTCATCACCCTTATGATGTCCTGAATGGAAGGATCCCTTTGGGAAATTCTCAGGAGGGGGACCTGGGCCAAGGGCTTGGCCAGCATCCTGCTGGCAACTCCAAGGCCCTGGGTGGGCTTCTGGAATGAGCATGCTACTGAATCACCAAAGGCACGCCCGACCTCTCTGAAGATCTTCCTATCCTTTTCTGGGGGAATGGGGTCGATGAGAGCAACCTCCTAGGGTTGTTGTGAGAATTAAATGAGATAAAAGAGGCCTCAGGCAGGATCTGGCATAGAGGAGGTGATCAGCAAATGTTTGTTGAAAAGGTTTGACAGGTCAGTCCCTTCCCACCCCTCTTGCTTGTCTTACTTGTCTTATTTATTCTCCAACAGCACTCCAGGCAGCCCTTGTCCACGGGCTCTCCTTGCATCAGCCAAGCTTCTTGAAAGGCCTGTCTACACTTGCTGTCTTCCTTCCTCACCTCCAATTTCCTCTTCAACCCACTGCTTCCTGACTCGCTCTACTCCGTGGAAGCACGCTCACAAAGGCACGTGGGCCGTGGCCCGGCTGGGTCGGCTGAAGAACTGCGGATGGAAGCTGCGGAAGAGGCCCTGATGGGGCCCACCATCCCGGACCCAAGTCTTCTTCCTGGCGGGCCTCTCGTCTCCTTCCTGGTTTGGGCGGAAGCCATCACCTGGATGCCTACGTGGGAAGGGACCTCGAATGTGGGACCCCAGCCCCTCTCCAGCTCGAAATCCCTCCACAGCCACGGGGACACCCTGCACCTATTCCCACGGGACAGGCTGGACCCAGAGACTCTGGACCCGGGGCCTCCCCTTGAGTAGAGACCCGCCCTCTGACTGATGGACGCCGCTGACCTGGGGTCAGACCCGTGGGCTGGACCCCTGCCCACCCCGCAGGAACCCTGAGGCCTAGGGGAGCTGTTGAGCCTTCAGTGTCTGCATGTGGGAAGTGGGCTCCTTCACCTACCTCACAGGGCTGTTGTGAGGGGCGCTGTGATGCGGTTCCAAAGCACAGGGCTTGGCGCACCCCACTGTGCTCTCAATAAATGTGTTTCCTGTCTTAACAAAAAGGATCC. The sequence of the siRNA targeting ATG3 was as follows Sense: CAUUGAGACUGUUGCAGAAdtdt; Anti-sense:UUCUGCAACAGUCUCAAUGdtdt.The human ovarian carcinoma cell lines OVCAR3 and A2780 were obtained from the Tumor Cell Bank of the Chinese Academy of Medical Science . OVCAR3 cells were cultured in RPMI 1640 and A2780 cells were cultured in Dulbecco's Modified Eagle's Medium supplemented with 10% fetal bovine serum (FBS), and penicillin/streptomycin (100 U/mL). The cells were cultured in an incubator at 37\u00b0C and an atmosphere of 5% COThree thousand cells were seeded into each well of 96-well plate in 100 \u03bcL medium. At the time points of 0 h, 24 h, 48 h and 72 h, 20 \u03bcL of 5 mg/mL MTT reagent was added and plates incubated for 4 h. The medium was then removed and replaced with 150 \u03bcL DMSO. Absorbance at 490 nm was measured using a microplate spectrophotometer .3 cells in 100 \u03bcL medium were seeded into each well of the E-Plate. Following incubation at room temperature for 30 min, the E-Plates containing cells were placed on the RTCA SP/MP station positioned in a cell culture incubator. The CI values were measured automatically every 15 minutes to form a continuous proliferation curve.For real-time cell proliferation assays, 50 \u03bcL medium was added to each well of a 96-well E-Plate for background measurement. Subsequently, 5 \u00d7 10After washing with PBS, cells were treated with trypsin to be collected and washed, then fixed with 70% ice-cold ethanol at -20\u00b0C for at least 12 h. Cells were washed and re-suspended by centrifugation then stained with PI following the manufacturer's protocol for cell cycle analysis by flow cytometry.Cells were trypsinized, counted, suspended and seeded into six-well plates at a density of 500 cells per well, in regular culture medium, then cultured at 5% CO2 and 37\u00b0C for 2 weeks until visible clones appeared. The medium was discarded and the cells were carefully washed twice with PBS. After being fixed with methanol for 15 min, the cells were stained with Giemsa's solution for 15 min before washing with tap water and air-drying. The clone formation rate was calculated with the formula: Plate clone formation efficiency = (number of clones/number of cells inoculated) \u00d7 100%. All the experiments were repeated 3 times and the average values were reported.Cells were collected and centrifuged for 5 min and washed twice with cold PBS. Cells were then resuspended in 100 \u03bcL 1\u00d7buffer with 5 \u03bcL 7AAD and 5 \u03bcL PE-labeled annexin V (KeyGen) per sample in the dark. An additional 400 \u03bcL of 1\u00d7 buffer was added and the rate of apoptosis for each samples determined by flow cytometry within 1 h.TRIzol was used to extract the total RNA from EOC cell lines and tissues. Isolated RNA was transcribed into cDNA using an avian myeloblastosis virus reverse transcriptase and random primers . Amplification of the cDNA was performed by real-time quantitative PCR using the SYBR Premix Ex Taq\u2122 II kit . The expression level of each target gene was normalised to 18s mRNA. The data analysis was calculated according to the sample threshold cycle (Ct) value from three independent experiments, primers for RT-PCR could found in The complete proteome from ovarian cancer cells was extracted in RIPA buffer, preventing protease-mediated degradation of samples. Protein concentration was determined for each sample, and 40 \u03bcg of the denatured proteome was resolved by 15% SDS-polyacrylamide gel electrophoesis and then electro-transferred to Hybond membranes . After blocking the membranes with 5% fat-free milk at room temperature for 2 h, membranes were incubated with the primary antibodies primary antibodies targeting ATG3, LAMP1 , LC3 , and SQSTM1 at 4\u00b0C overnight. Membranes were washed three times with TBST then secondary antibodies (anti-rabbit), at dilutions of 1:5000 were added. Following incubation for 2 h at room temperature, the protein bands were visualized by enhanced chemiluminescence (ECL) following the manufacturer's instruction . GAPDH served as the loading control.The interaction between LncRNA MEG3 and ATG3 protein was examined using Pierce Magnetic RNA-Protein Pull-Down Kit (Thermo fisher) according to the manufacturer's protocols. Biotin-labeled MEG3 or antisense RNA was co-incubated with protein extract of OVCAR3 cells and magnetic bead. The generated bead-RNA-Protein compound was collected by low-speed centrifugation. After washed with Handee spin columns, bead compound was boiled in SDS buffer, and the retrieved protein was detected by western blot with expressional level of GAPDH as control.The Magna RIP RNA-Binding Protein Immunoprecipitation Kit was used for RIP assays following the manufacturer's protocol. Briefly, OVCAR3 cells were collected and lysed using RIP lysis buffer. Whole-cell extracts were incubated with RIP buffer containing magnetic beads conjugated to human anti-ATG3 antibody or the control IgG. Proteinase K was added to the samples to digest the protein, and the immunoprecipitated RNA isolated. Purified RNA was used for qRT-PCR analysis. Expressional level of GAPDH was as control.Control and transfected cells were treated with actD (dissolved in 100% ethanol) at a final concentration of 1-5 \u03bcM. The actD was added to cells 0 h, 1 h, 2 h, 4 h, 6 h or 12 h prior to RNA extraction with TRIzol reagent. Subsequently, qRT-PCR was used to analyze changes in RNA levels.7 were injected into the right flanks of the mice. The tumour volume was directly measured following inoculation and weight calculated using the formula: (length \u00d7 width2)/2.All animal studies were approved by the China Medical University Animal Care and Use Committee, and all experimentation on animals was performed in agreement with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Four-week-old female BALB/c nude mice with weights of approximately 20 g were obtained from Vital River Laboratories and housed in a pathogen\u2013free and temperature-controlled environment. Two hundred microliters of PBS containing approximately 1 \u00d7 102O2 to endogenous peroxidase activity. Next, the sections were heated in target retrieval solution (Dako) for 15 min in a microwave oven to retrieve the antigen. Non-specific binding was blocked by incubation with 10% goat serum for 2 h at room temperature. Slides were then incubated overnight at 4\u00b0C with anti-ATG3 primary antibody. An appropriate secondary antibody was added and incubated for 20 min at 37\u00b0C, and binding was visualized with 3,39-diaminobenzidine tetrahydrochloride (DAB). After each treatment, slides were washed three times with TBST for 5 min. Three independent observers randomly selected and counted 100 cells from five representative fields from each section. Any discrepancies were checked by three observers until a consensus was reached. Positive expression was graded as follows: 0 = negative; 1 = 1%-50%; 2 = 51%-74%; 3 \u2265 75%. The staining intensity was graded as follows: 1 = weak; 2 = intermediate; 3 = strong. The two grades were multiplied to obtain a final score: - = 0; + = 1-2; ++ = 3-4; +++ = 6-9).Paraffin-embedded tissue sections were deparaffinized in xylene and rehydrated in a graded series of ethanol solutions and then incubated for 20 min in 3% HTransfected or control OVCAR3 cell slides were fixed with 4% formaldehyde, blocked using 5% skimmed milk in 0.01 M PBS, pH 7.2 (PBS-M). Primary antibodies targeting LC3, SQSTM1 (P62) or ATG3 , were diluted 1:500 in PBS and added to the slides. After incubation overnight at 4\u00b0C, slides were washed once with 0.2% Tween 20 in PBS and twice with PBS, and then incubated for 30 min at room temperature with anti-rabbit IgG\u2013TRITC . The slides were washed three times with PBS, then fixed with glycerol/PBS , and observed and photographed using a laser confocal microscope .Data were analyzed using SPSS 17.0 software , and are presented as mean \u00b1 SD. Two groups were compared using an unpaired, two-tailed Student's t-test. Spearman rank correlation was used to test the association between Meg3 expression and FIGO stages. The ROC characteristics curve was used to calculate the sensitivity and specificity values of Meg3. and P < 0.05 was considered to be statistically significant."} +{"text": "Syringomyelia (SM) is a common condition affecting brachycephalic toy breed dogs and is characterized by the development of fluid-filled cavities within the spinal cord. It is often concurrent with a complex developmental malformation of the skull and craniocervical vertebrae called Chiari-like malformation (CM) characterized by a conformational change and overcrowding of the brain and cervical spinal cord particularly at the craniocervical junction. CM and SM have a polygenic mode of inheritance with variable penetrance.PCDH17 on CFA22 and ZWINT on CFA26. PCDH17 codes for a cell adhesion molecule expressed specifically in the brain and spinal cord. ZWINT plays a role in chromosome segregation and its expression is increased with the onset of neuropathic pain. Targeted genomic sequencing of these regions identified respectively 37 and 339 SNPs with significantly associated P values. Genotyping of tagSNPs selected from these 2 candidate loci in an extended cohort of 461 CKCS identified 2 SNPs on CFA22 that were significantly associated to SM strengthening the candidacy of this locus in SM development.We identified six cranial T1-weighted sagittal MRI measurements that were associated to maximum transverse diameter of the syrinx cavity. Increased syrinx transverse diameter has been correlated previously with increased likelihood of behavioral signs of pain. We next conducted a whole genome association study of these traits in 65 Cavalier King Charles Spaniel (CKCS) dogs . Two loci on CFA22 and CFA26 were found to be significantly associated to two traits associated with a reduced volume and altered orientation of the caudal cranial fossa. Their reconstructed haplotypes defined two associated regions that harbor only two genes: PCDH17 and ZWINT, significantly associated to two traits associated with syrinx transverse diameter. The locus on CFA22 was significantly associated to SM secondary to CM in the CKCS dog breed strengthening its candidacy for this disease. This study will provide an entry point for identification of the genetic factors predisposing to this condition and its underlying pathogenic mechanisms.We identified 2 loci on CFA22 and CFA26 that contained only 2 genes, The online version of this article (10.1186/s12863-018-0605-z) contains supplementary material, which is available to authorized users. Canine syringomyelia (SM) is a painful condition where fluid-containing cavities (syrinx or syringes) develop within the parenchyma of the spinal cord and which progress over time , 2. DepeIn the CKCS, risk of SM has been shown to be associated with increased brachycephaly with rostrocaudal doming i.e. a heightened cranium that slopes caudally and reduBMP3 (BONE MORPHOGENETIC PROTEIN 3) variations to skull diversity GGAATGGGAAGTGAGATGAACAGCATAGGAGATTTGAAAGGTAGCAAACAATCCAGGAGACCTACAGGCCCGTGATCAATGACTTATAAGATGATATTAAGGAAACATTATATATGATACTATATCCTCCTTTGAGAGTCTGTATGCATT and TTAATCTTCAAAACGGCCCAATGAGGATATCCCCATTTTGCAGATGAAGAATGAAGGAACGTTGAAGTTCAGTGATTTGATCCAGAATAAGTGCAGTGAGACTTCAGATCCAGCTATGGGGTTTGCCAAATTCAATCTCTGCCCTTCCTC[A/C]CTATTCTTAACCGCCAAATATTATTTATATTTGTAAGAATGCAGTTTTAAAGGTTGAAATTTTCAACTTCTCACACAGAGCAGATAGCTGGAGACGAAGATGGTAGGACTGCTCTCTCATTGCCTGCATTGTGCTCTCTGAGTAGTGAAA respectively. Two SNPs were also amplified on CFA26 at position 32,757,080 (rs23302138) and 32,797,595 with the probes TGTCCTCCTGGCTTCTGAGGGGGTGGGTGCGGGGCCTGGAGGCCCAGAGGGGAACAGGATGTGGCCACAGGATGGAGAGCTGACTTGTGCACAGGGGCCTGTGTGGGTCAGTCTGTGTCCCCGGCACCCCTGAAGCTGCAGGTGTCTCAG[T/C]AGAGCCCCTCAGTGGGTAACTCTGCCCCCAATTCCCTCCTTGGAGACTGCATCTCCTCCTGTGCCTCCTGCAAGTCGCTGTCAGCTTCCCTCCCCTGAGGTCTGACGCCTCCTGCAGGAAGTTCTCTGGGATTGGATCTCAAAATGGTGC and CATGAGTTGGAAGGGCAGTTAAGGGCAGAAGGACTTAGAGGCGGAGAGCATAGAGAAGGAAAAGGCACGATGGTGTGTTTGATTATCTCCCCCTCTCCATTCTCATGGTGCCACCTATCCTAATTCCAGTTCGTATTATCATAGGTCTCA[C/T]TCCACCAATAACGTCTCAATCACACACACCATGTCCTGTCTTCTCGTTGGTCTGTGCTATGATCTGTGTGGTTCTTCTCTTTCCCAGGAGGCCAGATCTGTATTTTGCTGATTACAATCTACTCTTTAATTCTGGATTGAATTGCTAACT respectively. Genotyping was performed using the TaqMan assay (Applied Biosystems) on 393 CKCS dogs .Tagging SNPs were selected to maximize coverage of each locus. They were identified using the tagger routine in the Haploview V4.2 software with a maximum rP values obtained from the association were corrected together for multiple testing using a storey\u2019s q value method [Initial GWAS analysis was done in 2 phases: association of the quantitative traits to disease, followed by association of SNPs to these quantitative traits. Association of the quantitative traits to the disease was done using a linear regression with a type III sum of squares in R V3.0.1 and age e method . Haplotye method and assoe method with ageL4\u2009+\u2009L7 all showed a significant association to STD size (Pbonferroni\u2009<\u20090.0019) and were therefore further investigated . This resulted in the identification of a group of 13 SNPs on CFA15 associated to ratio F-d/BC under a FDR of 0.05 and two SNPs on CFA26 , which were significantly associated to L4\u2009+\u2009L7 under a FDR of 0.05 (both P\u2009=\u20090.03754) and two on CFA26 (P\u2009=\u20090.01067 and 0.00231), that were significantly associated to ratio F-d/BC surrounding the SNPs significantly associated to PCDH17 (PROTO-CADHERIN 17) on CFA22 and ZWINT (ZW10 INTERACTING KINETOCHORE PROTEIN) on CFA26. In order to identify potential SM -predisposing mutations, both CFA22 and CFA26 candidate regions were submitted to targeted next generation sequencing. The reads of the sequences obtained from the 65 CKCS were aligned on CanFam 3.1 resulting in 5608 SNPs on CFA22 and 10,814 SNPs on CFA26. Except for one SNP (rs2305483), that was identified in the coding region of PCDH17 and that was non-conserved and synonymous, all identified SNPs resided in intergenic and deep intronic regions. Using improved coverage of the region, linkage disequilibrium blocks were reevaluated based on the significant SNPs in the regions. A total of 37 and 339 SNPs were defined as significantly associated to F-d/BC and L4\u2009+\u2009L7 respectively. Based on the hypothesis that causative SNPs would be significantly associated to their respective trait, these SNPs refined the regions of interest to 13,785,828-14,183,295\u00a0bp on CFA22 and 32,721,057\u00a0bp to 33,094,292\u00a0bp on CFA26.Each of the SM- associated regions harbored only one coding gene: P value\u2009=\u20090.7637 and 0.7614), the 2 selected TagSNPs on CFA22 at position 13,933,606\u00a0bp and 13,804,718\u00a0bp reached significance . Bonferroni corrected P value of the TagSNP at position 13,933,606\u00a0bp on CFA22, was still significant at a P value of 0.0104. Hence, we successfully identified a region on CFA22 associated to ratio F-d/BC and SM in the CKCS dogs.To investigate the potential association between the identified SNPs and SM, a cohort of 461 CKCS including 187 unaffected and 274 SM-affected were genotyped with two TagSNPs from each of the associated regions on CFA22 and CFA26. While the 2 selected TagSNPs on CFA26 at position 32,797,595\u00a0bp and 32,757,080\u00a0bp did not show any significant association to SM significantly associated to STD size. Genetic investigation of these traits identified significant association with SNPs on CFA15 with F-d/BC and on CFA26 with L4\u2009+\u2009L7. Both these traits represented a combination that demonstrated a reduction in the overall size of the caudal cranial fossa and rearrangement of the neural parenchyma identified a significant association of the candidate locus on CFA22 and SM. Hence, this candidate locus that was suggestive of association to the ratio F-d/BC in CKCS and to the line BC in a previous association study in GB dogs was found to be significantly associated to SM, strengthening its candidacy for SM. On the other hand, the tagSNPs on CFA26 that was significantly associated to PCDH17 and ZWINT (respectively). Targeted next generation sequencing of both CFA22 and CFA26 candidate loci identified a total of 37 and 339 significantly associated SNPs with ratio F-d/BC and L4\u2009+\u2009L7 respectively. No mutation in the coding region of either gene was detected, except for one synonymous mutation in PCDH17. We hypothesize that predisposing mutations in these two regions are most likely regulatory that would affect RNA expression of either PCDH17 or ZWINT or other unannotated transcripts. Alternatively, these regulatory mutations could have a long-range expression effect on transcripts residing outside the 2 candidate regions. RNA-sequencing or quantitative RT-PCR studies in affected tissues from dogs carrying the associated haplotypes are needed to test this hypothesis.The associated loci on CFA22 and CFA26 harboured each only one gene: PCDH17 (PROTOCADHERIN 17) belongs to the family of protocadherins that are involved in the adhesion and sorting of cells during tissue morphogenesis. It is expressed specifically in several regions of the developing and adult brain and spinal cord [PCDH17 in primary cortical neurons is associated with significantly decreased dendritic spine density and abnormal dendritic morphology [ZWINT (ZW10-INTERACTOR) has an important role in kinetochore assembly and proper chromosome segregation [ZWINT in pain development and in SM associated with CM.nal cord \u201341. It rnal cord . Overexprphology and it iregation . In ratsregation \u201346. We tCM with SM in the dog is very similar to a condition in humans called Chiari malformation I (CMI) with a reported frequency of 1 in 1280. As in dogs, the prevalence of SM secondary to CMI in humans is high reaching 65%\u201385% , 48. GenL4\u2009+\u2009L7 respectively. Genotyping of a larger cohort of CKCS dogs confirmed association of the locus on CFA22 with SM in this breed. Each of these 2 haplotypes harbored only one gene: PCDH17 on CFA22 that codes for a cell adhesion molecule specifically expressed in the brain and spinal cord and ZWINT that plays a role in proper chromosome segregation and whose expression is increased with the onset of neuropathic pain. Additional molecular genetic studies in larger CKCS cohorts from various affected brachycephalic breeds and in cell and animal models are needed to further investigate the role of the 2 associated loci and the genes they harbor in the pathogenesis of SM secondary to CM. Our study represents an essential step towards a better understanding of the complex genetics of this devastating condition and development of breeding strategies that aim at eliminating it from the affected dog breeds. It also provides an important model for studying CMI/SM in humans.In this study, we have used a genome-wide association study to decipher the genetics of SM secondary to CM in the CKCS breed. We identified 6 cranial T1-weighed sagittal MRI measurements that were associated with the syrinx transverse diameter. We next identified 2 haplotypes on CFA22 and CFA26 that were significantly associated to ratio F-d/BC and Additional file 1:Table S1. Characteristics and measurements of 96 CKCS of the cohort. This table includes the gender, age, clinical status and all MRI cranial measurements taken on the 96 CKCS dogs included in this study. (XLSX 41\u00a0kb)Additional file 2:Table S2. SNPs suggestive of association to SM in the CKCS breed. This table enlists all SNPs suggestive of association with FDR corrected scores between 0.05 and 0.1. These SNPs were identified following a GWAS using a mixed linear model with age as a covariate on the previously identified traits . (DOCX 15\u00a0kb)Additional file 3:Figure S1.P value distribution inside the CFA15 associated region. This region spans 1.7\u00a0Mb surrounding SNPs associated to ratio F-d/BC and was identified using Haploview V4.2. (PDF 54\u00a0kb)"} +{"text": "Schizosaccharomyces pombe does not play a role in MMR. To test microsatellite stability in S. pombe and hence DNA loop repair, we have inserted tetra-, penta-, and hepta-nucleotide repeats in the ade6 gene and determined their Ade+ reversion rates and spectra in wild type and various mutants. Our data indicate that loops with four unpaired nucleotides in the nascent and the template strand are the upper limit of MutS\u03b1- and MutL\u03b1-mediated MMR in S. pombe. Stability of hepta-nucleotide repeats requires Msh3 and Exo1 in MMR-independent processes as well as the DNA repair proteins Rad50, Rad51, and Rad2FEN1. Most strikingly, mutation rates in the double mutants msh3 exo1 and msh3 rad51 were decreased when compared to respective single mutants, indicating that Msh3 prevents error prone processes carried out by Exo1 and Rad51. We conclude that Msh3 has no obvious function in MMR in S. pombe, but contributes to DNA repeat stability in MMR-independent processes.Defective mismatch repair (MMR) in humans is associated with colon cancer and instability of microsatellites, that is, DNA sequences with one or several nucleotides repeated. Key factors of eukaryotic MMR are the heterodimers MutS\u03b1 (Msh2-Msh6), which recognizes base-base mismatches and unpaired nucleotides in DNA, and MutL\u03b1 (Mlh1-Pms1), which facilitates downstream steps. In addition, MutS\u03b2 (Msh2-Msh3) recognizes DNA loops of various sizes, although our previous data and the data presented here suggest that Msh3 of Repetitive DNA elements are widespread in genomes. They are located in centromeres, telomeres, rDNA genes, transposons, and intergenic regions . The newMSH2 and MLH1 causes microsatellite instability and a predisposition to colon and other types of cancer and MutS\u03b2 mutants exhibit instability of mono- and dinucleotide repeats and generate duplications of sequences flanked by repeats complex has single-stranded 3\u2032-exonuclease and endonuclease activities as well as structural functions in recombination processes . After 5nd MutS\u03b2 . S. cere repeats and repaSchizosaccharomyces pombe encodes the MutS homologs Msh1, Msh2, Msh3, and Msh6, the MutL homologs Mlh1 and Pms1, and the exonuclease Exo1. Based on homology with S. cerevisiae Msh1, S. pombe Msh1 likely acts in MMR of mitochondrial DNA. S. pombe Msh2, Msh6, Mlh1, and Pms1 are indispensable for repair of base-base mismatches and small loops with one or two nucleotides , yeast extract agar (YEA), and yeast extract liquid (YEL), and general genetic methods were used as described ; OL937 \u2212 mlh1h::kanMX his3-D1 ura4-D18 (\u2212 exo1h::ura4 ura4-D18 (\u2212 rad2h::ura4 ade6-704 leu1-32 ura4-D18 (smt-0 rad50::kanMX ura4-D18 (+ rad51h::kanMX ade6 leu1-32 ura4-D18 (The \u2212 msh2h::hphMX ura4-D18 and KK37 \u2212 msh6h::hphMX ura4-D18 originated from transformations of OL2137 (\u2212 ura4-D18h), with DNA fragments obtained by fusion PCRs. pFA6a-hphMX (smt-0) was used to amplify either 450 bp of the 3\u2032 UTR of the msh2 locus or 500 bp of the 5\u2032 UTR of the msh6 locus. Primers for the msh2 disruption were msh2_For 5\u2032-GAGGTTTTTTATTTATCCTTTTTGAGGACTTAACTGTGGCAAGGAGTTTCTTCTCCTGTTTTATACATTTCGCGTTCGCGCTTTAGAACATTCAATCAATCGGATCCCCGGGTTAATTAA; msh2_Rev 5\u2032-TTTCCTCGTTTTAGTAAAAAATTATTTTATTCATAAAATGCGCTTCCAAAAAACATGTACCTTGGTTGAATTCTTTCAATTAGTACCTTGCTCACATTCTGAATTCGAGCTCGTTTAAAC; msh2_For3 5\u2032-TTGAAAGAATTCAACCAAGG; and msh2_Rev3 5\u2032-GCTAAAACAAAATTATGCCG. Primers for the msh6 disruption were msh6_For 5\u2032-TATATATGTTATTTTGTGCTCTCATGTTAGCTTTGTTTACTATTAGAATGCTGCTTTTTGTAAATAACTGAACTTAGCCAAAACCAACACTTGTTCCAGTCGGATCCCCGGGTTAATTAA; msh6_Rev 5\u2032-ATAACGTAAGTAAATGGTAAATAAAAGCAAGCTTCCGCTTGCCAGCAAACGAAAGATATTGCTTTGAATAGTCATAAAACTGATAGAGTGTTGACAGTTAGAATTCGAGCTCGTTTAAAC; msh6_For2 5\u2032-CTCATCTTACCTAAACTCTC; and msh6_Rev2 5\u2032-GAACAAGTGTTGGTTTTGGC.Strains KK42 DraIII-HindIII fragment of the ade6 gene containing a (GACC)7 repeat near the DraIII site . The fragment was obtained by PCR with primers ade6-GACC7 5\u2032-TCCCACTTGGTGACCGACCGACCGACCGACCGACCGACCTTTATGTTGAAAAGTTCGTTC-3\u2032 and ade6-H 5\u2032-GGGCAAGCTTCAATGGTGTA-3\u2032, and subsequent digestion with DraIII and HindIII. PCR was performed under standard conditions using pCG162 as template (3 run immediately 3\u2032 to the (GACC)7 repeat was deleted. The 1.7-kb XhoI-EcoRI fragments of the plasmids containing either (GACC)7 or (GACC)7\u0394T were transformed into the S. pombe strain AM1 7 his3-D1 leu1-32 ura4-D18h and \u2013 ade6-(GACC)7h\u0394T his3-D1 leu1-32 ura4-D18. A strain with a (GACC)8 repeat was isolated during a fluctuation test with an h+msh2::7his3 ade6-(GACC) mutant.In pAN-K, a pUC18 derivative, the kanamycin resistance gene was replaced by a 340-bp \u2212 ade6h::ura4 ura4-D18) was constructed by transformation of strain OL2137 (\u2212 ura4-D18h) with a PCR fragment obtained with primers ade6_d_ura4_F 5\u2032-TCCTTTTGTACTGAAAAGTAAAACATTGGCTTACGACGGTCGTGGAAATTACGTTGTTCATCAACCATCTGAGATTCCTACTGCCATCAAAGCACTTGGTagcttagctacaaatcccac and ade6_d_ura4_R 5\u2032-GAATGGTCTCAGTTGTAGGATAAGCATAAACTTTTCCGTCTAAACTGCGTACTACCATCACTGCAATTTCCATGGAGAAAGGAACGAACTTTTCAACATagcttgtgatattgacgaaac, and pAW1 as template in ade6 were deleted and replaced by ura4 . After initial denaturation for 1 min at 94\u00b0, we applied two cycles with 30 sec at 94\u00b0, 30 sec at 45\u00b0, and 30 sec at 72\u00b0, followed by five cycles with 30 sec at 94\u00b0, 30 sec at 55\u00b0, and 30 sec at 72\u00b0. Reaction samples were transformed into strain DE1 using the method of 6ade6-(CTGCC). Primers Hepta-F 5\u2032-ACATTGGCTTACGACGGTCGTGGAAATTACGTTGTTCATCAACCATCTGAGATTCCTACTGCCATCAAAGCACTTGGTGATCGTCCATCGTCCATCGTCCATCGTCC ATCGTCCTTTATG and Hepta-R 5\u2032-AAGCATAAACTTTTCCGTCTAAACTGCGTACTACCATCACTGCAATTTCCATGGAGAAAGGAACGAACTTTTCAACATAAAGGACGATGGACGATGGACGATGGACGATGGACGATCACC were used for 5ade6-(ATCGTCC). Primers HeptadT-F 5\u2032-AACATTGGCTTACGACGGTCGTGGAAATTACGTTGTTCATCAACCATCTGAGATTCCTACTGCCATCAAAGCACTTGGTGATCGTCCATCGTCCATCGTCCATCGTCCATCGTCCTTATG and HeptadT-R 5\u2032-TAAGCATAAACTTTTCCGTCTAAACTGCGTACTACCATCACTGCAATTTCCATGGAGAAAGGAACGAACTTTTCAACATAAGGACGATGGACGATGGACGATGGACGATGGACGATCACC were used for 5ade6-(ATCGTCC)\u0394T. Here, a single T immediately downstream of the repeats has been deleted \u0394T, repeat tract changes were also analyzed by inspection of the color of Ade+ revertants 7\u0394T, (CTGCC)6, and (ATCGTCC)7, insertions of one repeat unit and deletions of two repeat units are the major events detectable. The opposite is the case with (GACC)8 and (ATCGTCC)7\u0394T, where deletions of one repeat unit and insertions of two repeat units are the principal events that can be detected, although in the case of (ATCGTCC)7\u0394T, we identified exclusive deletions as described below.We have previously reported that in-frame nucleotide insertions and codon changes in a region around nucleotide 1397 of the mutants . Inserti7of ade6-(GACC)\u0394T, deletions and insertion can be distinguished by the color of revertants \u0394T repeat in the various genetic backgrounds revealed deletions and sequencing of 23 7(GACC)\u0394T revertants revealed insertions (7(GACC)\u0394T repeat can be easily determined from a large number of revertants. Analyzing revertants of the other ade6 repeats did not allow a distinction by color, probably because Ade+ originating from deletions and insertions were both not fully functional. In these cases, PCR products of independent Ade+ revertants were subjected to sequencing to identify the number of repeats.In the case vertants . Deletiosertions . All revmsh2, msh3, msh6, mlh1, or exo1. In wild type, 8ade6-(GACC) and 7ade6-(GACC)\u0394T reverted to Ade+ with 1.2 \u00d7 10\u22125 and 2.1 \u00d7 10\u22125 reversions per cell division, respectively (8ade6-(GACC) reversion rates increased 39\u201344-fold in msh2, msh6, and mlh1 mutants and slightly decreased in msh3 and exo1 mutants. Similarly, 7ade6-(GACC)\u0394T reversion rates increased 17\u201322-fold in msh2, msh6, and mlh1 mutants and decreased 3\u20135-fold in msh3 and exo1 mutants. None of the differences between msh3, exo1, and wild type was statistically significant.We first analyzed stability of GACC tetra-nucleotide repeats in wild type, and in mutants deleted for either ectively . The ade7ade6-(GACC)\u0394T can be determined by colony color as described above and in Materials and Methods, and as illustrated in msh3, and exo1 strains, reversions mainly occurred by deletions revealed that in wild type, all 10 had two repeat insertions 6 repeat appeared to be more stable in the msh3 mutant. It reverted mostly through gain of one repeat unit in all strain backgrounds, with the possible exception of mlh1 to Ade+ . Rates w of mlh1 . We conc5ade6-(ATCGTCC)\u0394T and 5ade6-(ATCGTCC) reverted to Ade+ at rates of 7.2 \u00d7 10\u22126 and 7.5 \u00d7 10\u22126, respectively \u0394T (not significant) and 2.8\u20132.9-fold for 5ade6-(ATCGTCC) (significant) (5ade6-(ATCGTCC)\u0394T revertants contained four hepta-nucleotide repeats and thus originated from deletion of one repeat (ade6 (+. 5ade6-(ATCGTCC) reverted mainly by insertion of one repeat and less frequently by deletion of two repeats without any significant differences between the spectra of wild type and any of the mutants . All seqe repeat . The facat 5 repeat 5 repeat instability in msh3 and exo1 is not due to a defect in MMR. We therefore wanted to analyze the genetic context of msh3 and exo1 defects in microsatellite stability. To do this, we measured reversion rates of the hepta-nucleotide repeat in FEN1rad2, rad50, and rad51 mutants and in various double mutants 5 repeat when msh3 is mutated.The 5 repeat . Since d mutants . FEN1 anon rates . The msh mutants . Like wieat unit . In the S. cerevisiae, msh3 and msh6 mutants show little to moderate increases of mutation rates in mono- and dinucleotide repeats 7 and (GACC)8 tetra-, (CTGCC)6 penta-, and (ATCGTCC)5 hepta-nucleotide repeats 7\u0394T, were the predominant reversion events in msh2 and msh6 mutants, slippage of one repeat can occur in the template and in the nascent strand during replication, and both types of events are corrected by MMR mediated by MutS\u03b1 and MutL\u03b1.The assay with the (GACC)vertants . We founsertions . Thus, t6 in msh2, msh6, mlh1, and exo1 mutants were similar to that of wild type, but decreased in msh3 5\u0394T and (ATCGTCC)5 (5\u0394T and mainly by insertion of one repeat in (ATCGTCC)5. We conclude that loops in penta- and hepta-nucleotide repeats are not substrates of MutS\u03b1 and MutL\u03b1 in S. pombe.Mutation rates of the penta-nucleotide repeat (CTGCC) in msh3 . All stre repeat . InactivTCGTCC)5 . Like wimsh3 mutants had no significant defects in repair of base-base mismatches and of loops with one unpaired nucleotide in a T6 repeat and in nonrepetitive DNA (msh3 mutations caused some instability of a (GT)8 dinucleotide repeat, which was mostly evident by a reversion spectrum different to wild type (msh3 mainly reverted by two-nucleotide deletions in the (GT)8 repeat. However, this was clearly less frequent than in msh2, msh6, and pms1 mutants 8 repeat t strand . Rates wS. pombe Msh6, as part of MutS\u03b1, recognizes base-base mismatches and loops with one to four unpaired nucleotides, while Msh3 does not play a significant role in MMR, but rather maintains repeat stability independently of MMR. Consequently, S. pombe MMR cannot repair loops with five or more nucleotides, in contrast to human MMR (S. pombe microsatellites. It is therefore critical for humans, but not for S. pombe, to have an MMR system that can deal with larger loops.We conclude from our studies that"} +{"text": "Various types of stem cell lines have been derived from preimplantation or postimplantation mouse embryos: embryonic stem cell lines, epiblast stem cell lines, and trophoblast stem cell lines. It is not known if extraembryonic endoderm stem (XEN) cell lines can be derived from postimplantation mouse embryos. Here, we report the derivation of 77 XEN cell lines from 85 postimplantation embryos at embryonic day E5.5 or E6.5, in parallel to the derivation of 41 XEN lines from 69 preimplantation embryos at the blastocyst stage. We attain a success rate of 100% of XEN cell line derivation with our E5.5 whole-embryo and E6.5 disaggregated-embryo methods. Immunofluorescence and NanoString gene expression analyses indicate that the XEN cell lines that we derived from postimplantation embryos (post-XEN) are very similar to the XEN cell lines that we derived from preimplantation embryos (pre-XEN) using a conventional method. After injection into blastocysts, post-XEN cells contribute to extraembryonic endoderm in chimeras at E6.5 and E7.5. Mouse preimplantation embryonic development culminates in the blastocyst stage. A blastocyst consists of three cell lineages: epiblast, trophectoderm, and primitive endoderm (PrE). The epiblast develops into most of the embryo proper, the amnion, and the extraembryonic mesoderm of the yolk sac; the trophectoderm gives rise ultimately to the fetal portion of the placenta; and the primitive endoderm forms the two extraembryonic endoderm lineages \u2013 the visceral endoderm (VE) and the parietal endoderm (PE) of the yolk sac134in vitro model to identify patterning activities of the extraembryonic endoderm such as factors involved in cardiac induction17Stem cell lines have been derived from these three cell lineages91114Gata4 or Gata6 . We find that this and other pre-XEN cell lines are immunoreactive for XEN cell markers GATA4, GATA6, SOX7, SOX17, and DAB2, but negative for ES cell markers OCT4 and NANOG, and negative for TS cell marker CDX2.Xist locus on the X-chromosome; Sox17 and Gata6 are XEN-cell markers; and Cdx2 is a marker for trophoblast stem cells. We removed the ectoplacental cone of the embryos as much as possible, and transferred each embryo separately into a well of 4-well dish coated with 0.1% gelatin and covered with MEF in TS cell medium including 25\u2009ng/ml FGF4 and 1\u2009\u03bcg/ml heparin (referred to as F4H). One day later, the embryos had attached to the surface and started to form an outgrowth. The embryos had formed a large outgrowth after 5 days. We used TrypLE Express to disaggregate the outgrowths and passaged cells into a well of a 4-well dish. After cells reached 70\u201380% confluency, they were passaged into a well of a 12-well dish. After they reached 70\u201380% confluency again, cells were passaged into a well of a 6-well dish, and we then obtained stable post-XEN cell lines. The intrinsic red fluorescence of mTomato produced from the Gata6 promoter in the transgene was sufficiently high to detect it in the whole embryo and outgrowth, but not in the established post-XEN cell line at day 60 by immunofluorescence for GATA4 and counterstaining with DAPI. Sets of fluorescence images were captured for each line visualizing the intrinsic fluorescence of GFP, DAPI, and GATA4 immunoreactivity . The imaWe asked if post-XEN cells can differentiate into a VE identity by incubation with BMP438Dab2, Gata4, Gata6, Pdgfra, Sox7, and Sox17, versus low or no expression of ES cell-specific genes such as Nanog, Pou5f1/Oct4, Sox2, and Nr0b1. There is no expression of EpiSC-specific genes such as Cer1 and Fgf5 (data not shown). Thus, the NanoString gene expression analysis confirms and extends the immunofluorescence profiles.We applied the NanoString multiplex platform for gene expression4041in vivo. We injected into blastocysts cells of two R26-tauGFP41+ pre-XEN cell lines (X-ICM-4 and X-ICM-5) and one PDGFRa-GFP+ pre-XEN cell line (X47) that we had derived from isolated ICMs or blastocysts, and cells from four R26-tauGFP41+ post-XEN cell lines and one PDGFRa-GFP+ post-XEN cell line (X-E6.5-Z0663-9) that we had derived from E5.5 embryos or disaggregated E6.5 embryos that are at the origin of the post-XEN cell lines.Sox17 locus, we cannot detect GFP expression in embryos and cell lines derived from them.) The Xist1loxGFP strainThe PDGFRa-GFP strainAdvanced RPMI-1640 was supplemented with 20% (vol/vol) FBS , 2\u2009mM GlutaMAX Supplement (Gibco #35050), 1% penicillin/streptomycin , 0.1\u2009mM \u03b2-mercaptoethanol (Gibco #21985-023), 1\u2009mM sodium pyruvate (Gibco #11360-039), supplemented with 25\u2009ng/ml FGF4 (Peprotech #100-31) and 1\u2009\u03bcg/ml heparin (Sigma #H3149).ES cell lines were maintained on MEF-coated, pregelatinized tissue culture dishes in DMEM supplemented with 15% FBS (HyClone #SH30071.03), 2\u2009mM GlutaMAX Supplement, 1% penicillin/streptomycin, 1% \u03b2-mercaptoethanol , 0.1\u2009mM nonessential amino acids (Gibco #11140-035), 1\u2009mM sodium pyruvate, and 1000\u2009IU/ml leukemia inhibitory factor (LIF) (Millipore #ESG1107).Embryos were collected at the 2\u20138 cell stage by flushing oviducts using M2 medium (Sigma #M7167), and cultured in KSOM medium (Millipore #MR-106-D) to the blastocyst stage. The zona pellucida of blastocysts was then removed using acid Tyrode solution (Sigma #T1788). Blastocysts were transferred separately into a well of a 96-well dish coated with 0.1% gelatin and covered with MEF in ES medium supplemented with LIF and 1\u2009\u03bcM PD0325901 (Axon #1408) and 3\u2009\u03bcM CHIR99021 (Axon #1386), a combination of chemicals that is typically referred to as \u201c2i\u201d. After 4 days TrypLE Express (Gibco #12604-013) was used to disaggregate the embryonic outgrowths, and cells were passaged to a well of 24-well dish to derive ES cell lines.Embryos were collected at the 2\u20138 cell stage embryos and cultured in in KSOM medium to the blastocyst stage. The zona pellucida of blastocysts was removed using acid Tyrode solution. Blastocysts were transferred separately into a well of a 4 well-dish (Nunc #176740) coated with 0.1% gelatin and covered with MEF in ES medium supplemented with LIF. The XEN lines were derived as describedThe ectoplacental cone was removed with forceps or needles. A whole embryo was placed in a well of a 4-well dish (Nunc #176740) coated with 0.1% gelatin and covered with MEF in TS medium including F4H. After the embryos formed a large outgrowth, TrypLE Express was used to disaggregate the outgrowths and passage cells into a 4-well dish. When cells reached 70\u201380% confluency, they were passaged into a well of a 12-well dish until XEN cell lines were obtained, which were then passaged into a well of a 6-well dish. If the outgrowth of an E5.5 embryo grew well and XEN cells thrived, we continued to culture cells in TS medium including F4H. But if the outgrowth grew slowly and XEN cells were surrounded by trophoblast-derived cells, we cultured cells in ES medium supplemented with LIF, culture conditions that would inhibit trophoblast-derived cells; when XEN cells started to become abundant, we switched to TS medium including F4H. To prepare XEN cells for RNA extraction, cells were cultured in dishes coated with 0.1% gelatin but without MEF in TS medium including F4H.The ectoplacental cone was removed with forceps or needles. A whole embryo was treated with 0.1\u2009mg/ml collagenase (Gibco #17104-019) and 0.01\u2009mg/ml deoxyribonuclease (Gibco #D5025) for 20\u201330\u2009min at room temperature, followed by 0.2\u2009mg/ml TrypLE for 5\u2009min at room temperature. The embryo was disaggregated into single cells using a glass pipette with a diameter of 50\u201360\u2009\u03bcm. Cells were transferred into a well of a 4-well dish (Nunc #176740) coated with 0.1% gelatin and covered with MEF in TS medium including F4H. Three days later XEN colonies appeared. We picked these colonies, disaggregated them with a glass pipette or by TrypLE Express for 5\u2009min at 37\u2009\u00b0C, and passaged them into a well of a 4-well dish. When cells reached 70\u201380% confluency, they were passaged into a well of a 12-well dish until XEN cell lines were obtained, which were then passaged into a well of a 6-well dish.http://cellprofiler.org/ . The DAPI signal was used to segment individual cells by thresholding, de-clumping, and applying size and roundness filter in an effort to evaluated single cells that are not MEFs. After filtering, the GFP and GATA4 signals were evaluated in each cell, and the population characteristics for these two markers were quantified.Images representing Fields of View of post-XEN cell lines were imaged for intrinsic fluorescence of GFP, fluorescence of DAPI, and GATA4 immunoreactivity. Automated cell population characteristics were determined with CellProfiler Gelatin-coated plates were prepared by coating with 0.1% gelatin overnight at room temperature. XEN cells were cultured in TS medium with F4H and with or without 10 ng/mL BMP4 on gelatin-coated plates for four days.Cell lines X42, X44, X47 (PDGFRa-GFP\u2009\u00d7\u2009CAG::mRFP1); X35, X36 (B2D6F1\u2009\u00d7\u2009D4/EGFP); X97, X107 (D4/EGFP\u2009\u00d7\u2009DBA2/N); X-E6.5-81346-8 (ROSA-STOP-taulacZ x Sox17-Cre); X-E6.5-Z0617-2, X-E6.5-Z0617-5, X-E6.5-Z0617-8 (R26-tauGFP41 x Sox17-Cre); X-E6.5-78097-4 (Xist1loxGFP\u2009\u00d7\u2009DBA/2\u2009N); X-E6.5-82278-4 (Gata6-mTomato\u2009\u00d7\u2009Cdx2-GFP); X-E6.5-Z0663-12 (CD1\u2009\u00d7\u2009PDGFRa-GFP); and X-E5.5-6, X-E5.5-8, X-E5.5-9, X-E5.5-10, X-E5.5-13 (R26-tauGFP41\u2009\u00d7\u2009Sox17-Cre) were cultured in 4-well or 24-well dishes. Cells were fixed in 4% paraformaldehyde at 4\u2009\u00b0C overnight or room temperature for 30\u2009min, permeabilized with 0.1% Triton X-100 in 1\u00d7 PBS (1\u00d7 PBST) for 30\u2009min and blocked with 5% normal donkey serum diluted in 1\u00d7 PBST (blocking solution) for 1\u2009hr. Primary antibodies were diluted at 1:50\u20131:200 in blocking solution and samples incubated at 4\u2009\u00b0C rotating overnight. After three 10-min washes in 1\u00d7 PBST 10\u2009min, samples were incubated for 1\u20131.5\u2009hr at room temperature in a 1:500 dilution of secondary antibody in blocking solution, then washed and covered with 1\u00d7 PBST containing DAPI. Images were taken with an AMG EVOS (Life Technologies), Zeiss LSM 710 confocal microscope, and Nikon SMZ25 stereofluorescence microscope.Primary antibodies from Santa Cruz Biotechnology were against GATA4 (#SC-1237), DAB2 (#SC-13982), OCT3-4 (#SC-5279), NANOG (#SC-376915), and CDX2 (#SC-166830). Primary antibodies from R&D Systems were against GATA6 (#AF1700), SOX7 (#AF2766), SOX17 (#AF1924), and PDGFRa (#AF1062). Primary antibodies against E-cadherin (ECCD2) were from Invitrogen (#13-1900). Secondary antibodies from Jackson ImmunoResearch Laboratories were Cy5\u2122 AffiniPure Donkey anti-goat IgG (H+L) (#705-175-147), and Cy5\u2122 AffiniPure Donkey anti-rabbit IgG (H+L) (#711-175-152). Secondary antibodies from Invitrogen were Donkey anti-rabbit IgG (H+L) with Alexa Fluor 546 (#A10040), Goat anti-rat IgG (H+L) with Alexa Fluor 546 (#A11081), Donkey anti-mouse IgG with Alexa Fluor 546 (#A10036), Donkey anti-mouse IgG with Alexa Fluor 488 (#A21202), and Goat anti-chicken IgY (H+L) with Alexa Fluor 488 (#A11039).Actb and Gapdh. Normalized counts are displayed in a heatmap generated by the nSolver Analysis Software v2.5, using agglomerative clustering.Cells were cultured in 12-well plates with TS medium including F4H or ES medium supplemented with LIF. Dissociated cells were collected by trypsinization and centrifugation. Cell pellets were dispensed directly in RNAlater Stabilization Solution (Qiagen) and stored in \u221280\u2009\u00b0C for later use. Cell pellets were lysed in RLT Lysis Plus Buffer using a TissueLyser LT (Qiagen) at 40\u2009Hz for 2\u2009min. Total RNA extraction was performed using RNeasy Plus Micro kit (Qiagen) according to manufacturer\u2019s protocol. The custom NanoString CodeSet \u201cExtra\u201d was used. 100\u2009ng of total RNA samples were hybridized at 65\u2009\u00b0C for 18\u2009hr and processed with the nCounter Analysis System GEN1 (NanoString Technologies). The reporter counts were processed using nSolver Analysis Software v2.5 (NanoString). Two normalizations were performed to the counts, the first normalization to the generic positive controls, followed by normalization to the reference genes, Below are the nucleotide sequences of the NanoString probes; first line is the capture probe, second line the reporter probe.ActbACGATGGAGGGGCCGGACTCATCGTACTCCTGCTTGCGGGTGTAAAACGCAGCTCAGTAACAGTCCGCCTAGAAGCACTTGCGGTGCDab2GTCTCCTCGAGCATCAGGCACATCATCAATACCGATTAGCTTGGCCCAGCTGCTGCCATTCCCTTGAGTTTCATCATAGAATCCTGACTCATTTTDnmt3lGAGGCAGCGCATACTGCAGGATCCGGTGGAACTGGAACATGCCATGAATATCCAGAAGAAGGGCCGCTGACTCTCCTGGCDppa4CAAGTCTTTACAGTTGACTGCTGAACTGGTTATGACGCCCGTTGTGCTGGCACTACAACCCAGGGAAGAGGACATGCATGCGGAGGCTACAGGTATAAGCDppa5aCGCACGGCCCACAGCTCCAGGTTCAGGAAGTTTTAGTACCCTGCCAAGGAACCAGACTTCAGGGAAGACGAGATCAAGCTTATCCACCAEsrrbTTTCCAGAATGAACCGCTTCATCTTTAGGACATCCTGTCAACCCAAACCCGAGCAGGTAAAGCCGGAGGACTTGTCATGAAAGTGGCGTGTCCATFgf4ATTCTGGTAACAAAATTCCAAAGATACAGTCTTGTCCCTGGGCGCAGGAAACAGACCGACTCGGTAACAGTGGCAGATACAGAGCAGAAACATCAAACCCFoxa2CACAGACAGGTGAGACTGCTCCCTTGAGGCCTGAAGTCCCTTCCCTATTTAGAATGACAGATCACTGTGGCCCATCTATTTAGGGAFoxq1GCTGTCCTTACTCCGAGGTTTAGAGACTTTGAGCGGAAGACAAGCGTTTTGATTGTTGGGTGAAGTGAGGAGTGGAGTGATAGAAGTTGGTGCAGTFstAGGACTTTGTGATACACTTTCCCTCATAGGCTAATCCAATGGATCTGCCCCTTGGAATCCCATAGGCATTTTTTCCCGCCGCCACACTGGATATCTTCACFxyd3TGAGCCCGCCGACTCGGAGGCTGTACCAATCATAGTAGAAAGGATCATTTGCCACTCATAAGGACTATAATGCCCAGGGCACAGAGAATCCCTGCACAAAGapdhATACTTGGCAGGTTTCTCCAGGCGGCACGTCAGATCCACGACGCCTCAGATGCCTGCTTCACCACCTTCTTGATGTCATCGata4AGAGCCAGGTAACTGTCTGACTTAAGAGGGCTTGGCTTGGGCAAACAGTCTGTATTTTCTAGACAAAGGATCTGTGCTGGAGAAAGTCCCGata6ATCTGGACTGCTGGACAATATCAGACACAAGTGGTATGAGGCCTTCAGAGGGCACAGAAATCACGCATCGAAGGAATGTTATGTCTGCATTTTTGCTGCCKrt8TTCCCATCTCGGGTTTCAATCTTCTTCACAACCACAGCCTTGCAGTGGCCATTCACTTGGACACGACATCAGAAGACTCGGACACCAGCLama1CATTGGCTAAATCGGCATGGCGGTCATCCTTGATACAGACAGAACTCAGACATAACCTTTCCTACATGGACACTGACCTGGCCACTTTCLamb1CCAGGAAGGAATGCGGTCCTGAATGTACTGCCTTTCCACCACAATGACAAAAACTCCAAATAAGCCCCTTCAGGCACCCGGACGAACCCAGGTCCTGTNanogCATATTTCACCTGGTGGAGTCACAGAGTAGTTCAGGAATAATTCCAAGGCGAAGGAACCTGGCTTTGCCCTGACTTTAAGCCCAGATGTTGCGTAAGTCTNr0b1CACTTGAAAAAGAAACTCTTGATGGCCTGGACCGCAGCAGCTGGGAGCAACCTTTCAGATAGGCATACTCTTTGGTGTCAATGTTCAGACTCCAGPdgfraTATGGAGTAAGTCGCTCTCACACACTTACCACACCACCATGTTGGGAACAAAACATGAACAGGGGTATCTGGAAGCCATCTTGTATTGGAAGACCCTTCCPou5f1ACATGGTCTCCAGACTCCACCTCACACGGTTCTCAATGAGTGATCTGCTGTAGGGAGGGCTTCGGGCACTTCAGAAPth1rCCTGGGCACGGTGCAGCAGGAAAATCTGTTCCTCTTTGGTAAAGACATCGTGCTGTGTGCAGAACTTCCTTGAGCAGCTTGTCACATTGCGSox2CCCCGCCGCCCTCAGGTTTTCTCTGTACAAAAATAGTCCCCCAAAAAGAATGCGTAGTTTTTTTCCTCCAGATCTATACATGGTCCGATTCCCCCGCCCTSox7AGAAATCAGCACACCCCAACACTTTTGTGGACAGACGTTTTAGGTTTCTATCATCTTTTGCATCTGTAGATAAGAGTATGCTACAGCTCTGCTCTSox17GGCAGATACTGTTCGAATTCCGTGCGGTCCACCTCTGTCCCTGGTAGGGAAGACCCATCTCGGGCTTATACACAAAGTet3TTTTCAAAGAGCTGAATGAATGCACCAGGATTTTAGGATGGGCGTGTTTCGTCAGGCCCAACCTCTCAATGTCACAGAAATTAAAGCACCAACGTTCTCAAll animal studies were carried out in accordance with the German Animal Welfare Act, European Communities Council Directive 2010/63/EU, and institutional ethical and animal welfare guidelines of the Max Planck Institute of Biophysics and the Max Planck Research Unit for Neurogenetics. All experimental protocols were approved by the Regierungspr\u00e4sidium Darmstadt and the Veterin\u00e4ramt of the City of Frankfurt.How to cite this article: Lin, J. et al. Efficient derivation of extraembryonic endoderm stem cell lines from mouse postimplantation embryos. Sci. Rep.6, 39457; doi: 10.1038/srep39457 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations."} +{"text": "Here we have addressed the hypothesis that a barrier element in vertebrates should be capable of defending a gene from silencing by DNA methylation. Using an established stable reporter gene system, we find that HS4 acts specifically to protect a gene promoter from de novo DNA methylation. Notably, protection from methylation can occur in the absence of histone acetylation or transcription. There is a division of labor at HS4; the sequences that mediate protection from methylation are separable from those that mediate CTCF-dependent enhancer blocking and USF-dependent histone modification recruitment. The zinc finger protein VEZF1 was purified as the factor that specifically interacts with the methylation protection elements. VEZF1 is a candidate CpG island protection factor as the G-rich sequences bound by VEZF1 are frequently found at CpG island promoters. Indeed, we show that VEZF1 elements are sufficient to mediate demethylation and protection of the APRT CpG island promoter from DNA methylation. We propose that many barrier elements in vertebrates will prevent DNA methylation in addition to blocking the propagation of repressive histone modifications, as either process is sufficient to direct the establishment of an epigenetically stable silent chromatin state.There is growing consensus that genome organization and long-range gene regulation involves partitioning of the genome into domains of distinct epigenetic chromatin states. Chromatin insulator or barrier elements are key components of these processes as they can establish boundaries between chromatin states. The ability of elements such as the paradigm \u03b2-globin gene locus can protect a gene promoter from DNA methylation. Protection from DNA methylation is separable from other insulator activities and is mapped to three transcription factor binding sites occupied by the zinc finger protein VEZF1, a novel chromatin barrier protein. VEZF1 is a candidate factor for the protection of promoters from DNA methylation. We found that VEZF1-specific binding sites are sufficient to mediate demethylation and protection of the APRT gene promoter from DNA methylation. We propose that barrier elements in vertebrates must be capable of preventing DNA methylation in addition to blocking the propagation of silencing histone modifications, as either process is sufficient to direct the establishment of an inactive chromatin state.DNA sequences known as chromatin insulator or barrier elements are considered key components of genome organization as they can establish boundaries between transcriptionally permissive and repressive chromatin domains. Here we address the hypothesis that barrier elements in vertebrates can protect genes from transcriptional silencing that is marked by DNA methylation. We have found that the HS4 insulator element from the It has been proposed that genes and gene clusters are organized into chromatin domains that are maintained independent of their surroundings through the establishment of boundaries \u03b2-globin genes are clustered within a thirty kilobase domain of nuclease accessible chromatin, the 5\u2032 boundary of which is marked by a constitutive DNaseI hypersensitive site called HS4 and adult chicken red blood cells (an abundant source of nuclear protein for purification purposes). Complexes of similar mobility and intensity are observed between the two nuclear extracts and each of the FI, FIII and FV sites and determined whether it interacts with the G-rich footprinted sites of the HS4 insulator, as it was previously reported that human VEZF1 interacts with similar G-rich sites FV sites . The comA-globin\u03b2 promoter that contains a (dG-dC)16 string. A nuclear factor called Beta Globin Protein 1 (BGP1) was previously characterized as interacting with this site in vitro, but was considered to indirectly assist in activation by directing nucleosome placement A promoter site with an identical specificity to that of erythrocyte nuclear protein(s) (A promoter site A site is competed by unlabelled FI which contains a (dG-dC)9 string 6 string .The (dG-dC) strings present in the VEZF1 sites at HS4 are reminiscent of a site in the chicken s FV see , which mA sites analyses were performed to analyze the binding of VEZF1 at the chicken analyses . VEZF1 bxpressed . None ofin vivo, as all three VEZF1 binding sites are required for protection from DNA methylation. We found that this was not the case, as VEZF1 remains tightly bound at HS4 when any one of its binding sites is deleted (in vitro (data not shown). We have attempted to disrupt VEZF1 function at HS4 following knockdown by RNAi. We strived to achieve substantial knockdown of VEZF1 to disrupt its binding to the high affinity sites at HS4. Prolonged knockdown was also required as we have previously found that the de novo DNA methylation of these transgenes is a gradual process that takes many days to establish de novo DNA methylation of the HS4 element and no change in HS4's ability to protect a transgene from silencing during this period (data not shown). The inadequacy of RNAi to strip constitutive transcription factor binding from high affinity sites has also been observed for CTCF Vezf1 null ES cells, as we recently found that they are defective for de novo DNA methylation due to the requirement of Vezf1 for full transcriptional activity of the Dnmt3b gene in these cells We tested whether VEZF1 requires all three of its sites for binding to HS4 deleted . VEZF1 aAPRT gene promoter and its effects on methylation. SP1-like binding elements have been shown to be required to prevent methylation of the mouse and hamster APRT CpG island elements: two earlier papers have shown that deletion of the SP1-like elements is sufficient to induce methylation in these islands APRT CGI that contain three SP1-like elements are subject to demethylation upon integration into mouse ES cells APRT gene remaining unmethylated and expressed normally in Sp1 null ES cells and embryos APRT CGI elements (APRT CGI stably integrated into mouse ES cells (in vitro (CCCCCCTTTCCCC) that is reminiscent of the VEZF1-specific bipartite footprint III site found at the HS4 insulator element (CCCCCCGCATCCCC).To address whether VEZF1 elements also protect CpG island (CGI) promoters from DNA methylation, we investigated VEZF1 binding to the elements . We perfES cells . We founin vitro . Site 3 APRT CGI from methylation, we replaced each of the three SP1-like elements with the VEZF1-specific FIII element from the HS4 insulator a promoter can be protected from DNA methylation even when it lacks active histone modifications and transcriptional activity, and 6) short DNA elements bound by VEZF1 mediate the demethylation and protection of a CpG island from DNA methylation.Here we have studied the paradigm HS4 element to address our hypothesis that a barrier element in vertebrates must be capable of defending a gene from silencing by DNA methylation and have identified a novel CpG island factor. We have presented six findings: 1) a vertebrate barrier element protects a gene promoter from DNA methylation-mediated silencing, 2) the essential transcription factor VEZF1 is a barrier/anti-methylation factor, 3) there is a modular division of labor at the compound HS4 insulator as VEZF1-mediated protection from methylation is separable from CTCF-mediated enhancer blocking and USF-mediated recruitment of active histone modifications, 4) the de novo DNA methylation.We have previously demonstrated that the HS4 insulator acts as a barrier to the spread of histone methylation marks associated with repressive chromatin bona fide activity that is protective against DNA methylation.It was previously shown that the transgenes used in this study become marked by dense promoter DNA methylation upon chromosomal position effect silencing de novo DNA methylation is key to our understanding of how VEZF1 binding at HS4 could protect a promoter from epigenetic silencing. Previous studies using the same transgene system studied here found that non-insulated transgenes, regardless of integration site, are consistently subject to promoter methylation upon chromosomal silencing, and that flanking with HS4 elements can shield transgenes from this methylation \u03b2-globin locus, the spreading of repressive histone modifications is observed upon perturbation of active histone modification recruitment at the HS4 barrier Determining the source of de novo DNA methylation arise via spreading from the chromosomal integration site in our transgene system, we would expect to see high levels of methylation at compromised mutant insulators either coincident with, or prior to promoter methylation. However, we observe that promoters become methylated prior to the insulators, which remain unmethylated or become partially methylated. The observed independence of methylation states between insulator and promoter argue against spreading and clearly show that there can be no single mechanism that controls the methylation state of both the insulator and promoter. It remains possible that VEZF1 elements at HS4 are acting as a barrier to the spreading of a DNA methylation mechanism, but that additional processes prevent the accumulation of methylation at HS4 itself. An alternative possibility is that DNA methylation does not result from spreading, and that the insulator directly interacts with the promoter to deliver VEZF1 co-factors that prevent promoter methylation. In this model, the promoter itself would have its own program to recruit de novo DNA methylation, and VEZF1 would act as a factor that mediates inhibition of methylation. This would distinguish the activity of VEZF1 from those of USF1/USF2, which bind elsewhere in the insulator element and recruit a number of enzymes that deliver active histone modifications to the reporter gene Should Igf2/H19 domain de novo DNA methylation of HS4. It has previously been shown that DNA binding proteins can prevent the methylation of their binding sites simply by steric hindrance of de novo DNA methyltransferases (DNMTs) The 275 bp \u201ccore\u201d HS4 element comprises a CpG island (CGI) that is free of DNA methylation regardless of neighboring gene expression de novo methylation during early development, and how they remain hypomethylated irrespective of transcriptional status. Recent epigenomic profiling studies have begun to reveal a significant portion of CGIs that are subject to varying degrees of tissue-specific methylation in human somatic tissues de novo methylation, which can be selectively inactivated during development and may become defective during cancer progression cis-regulatory elements and trans-acting factors that control CGI methylation status is key to unraveling these processes.All constitutively expressed genes and \u223c40% of genes with tissue-restricted expression have CGI promoters APRT gene CGI promoter. It was previously shown that SP1-like binding elements are required to prevent CGI methylation APRT CGI. A promoter-less APRT CGI fragment containing only site 3 remains protected from DNA methylation de novo DNA methylation. Thus, VEZF1 binding elements can protect a CGI from DNA methylation. We attempted to definitively address the requirement for VEZF1, but discovered that global de novo DNA methylation mechanisms are defective in Vezf1 null ES cells DHFR gene were cultured and assayed for IL-2R expression by FACS as described previously Chicken 6C2 erythroleukemia cells carrying Gel mobility shift assays were performed as described previously in vitro translation using rabbit reticulocyte lysate (Promega).FI- and FIII-binding proteins were purified from adult chicken red blood nuclear protein extracts by ion exchange chromatography. Throughout the purification, eluate fractions were analyzed for FI- and FIII-binding activity with gel mobility shift assays. The binding specificity of partially purified proteins was checked by competition analysis after each purification step. FI- and FIII-binding activities co-fractionated following ion exchange chromatography with SP XL and Heparin sepharose . Phosphocellulose, Q and Sephacryl S300 columns were used in early purification attempts to resolve FI- and FIII-binding activities but they co-fractionated in each case (data not shown). FI- and FIII-binding activities both eluted in two distinct fractions of approximately 200 and 400 kDa following gel filtration (data not shown). The active fractions eluted from heparin sepharose were pooled then split into two and fractionated in parallel by FI or FIII DNA affinity as described previously 5\u2032CATGCCGCTCGAGCGGTTTTTTTTTTTTTTTTT was used in first strand cDNA synthesis with Superscript II reverse transcriptase (Invitrogen). The primers VEZF1_5\u2032, 5\u2032CCATGACCCATGGGCAGAGCCAAAGT and Adaptor 5\u2032CATGCCGCTCGAGCGG were used to amplify a full length chicken VEZF1 cDNA by PCR which was TA cloned into pCRII (Invitrogen). VEZF1 cDNA was sub-cloned into pCITE4b (Novagen) to generate p4bVEZFfull for the purpose of in vitro transcription. cDNA encoding chicken ZF5 was isolated by RT-PCR from 6C2 cell total RNA using primers designed from the published sequence (U51640). We found that the bases CpG 1306-7 in the published sequence were GpC in our clone, causing codon 436 to translate as alanine instead of arginine. We obtained the vectors pCDNA3-ZF5 and pEVRFO-ZF5 and we also found the CpG to GpC conflict with the published sequence. Full length chicken SP1 and SP3 cDNAs cloned in the pBluescript-based vectors pH-SP1 and pH-SP3-3 were a kind gift from Marc Castellazzi .Chicken VEZF1/BGP1 cDNA was cloned following RT-PCR from 6C2 cell total RNA based on an assumption of conservation of 5\u2032 cDNA sequence with human VEZF1/DB1. The oligonucleotide Adaptor-A E.coli . Peptides were produced in M15 [pREP4] E. coli followed by rapid lysis with B-PER reagent (Pierce). ZF5 peptide was soluble and purified on Ni-NTA agarose (Qiagen). VEZF1 peptide was insoluble and resulting inclusion bodies were prepared using B-PER reagent (Pierce), solubilized with 6M guanidium hydrochloride and immobilized on TALON Sepharose resin (Clontech) at pH 7. VEZF1 peptide was renatured in a stepwise manner with 6, 4, 3, 2, 1 and 0.5 M guanidium hydrochloride prior to elution. VEZF1 and ZF5 polyclonal IgG antibodies were purified from rabbit serum using PROSEP-A media . Anti-full length chicken VEZF1 antibodies were raised previously Polyclonal antibodies were raised against chicken VEZF1 (Ser376-Ala547) and chicken ZF5 peptides (Ser131-Lys248) produced in 7 cells/ml) and fixed with a final concentration of 0.25% formaldehyde at room temperature for 30 seconds. 6C2 cells were harvested in mid-exponential growth phase, divided into 30 ml aliquots containing 1\u00d7108 cells in fresh media and fixed with a final concentration of 0.8% formaldehyde at room temperature for 5 minutes. Reactions were stopped by adding glycine to a final concentration of 0.125 M. The cells were washed in PBS and resuspended in cell lysis buffer to isolate nuclei. Chromatin was prepared following washing and lysis of nuclei. Crosslinked chromatin was fragmented by sonicaton (Misonix) for a total time of 10 minutes in regular 10 second pulses at 4\u00b0C. Debris was removed by centrifugation at 15000 g for 10 minutes and chromatin was diluted in 10 volumes of X-ChIP buffer . Agarose gel electrophoresis was used to confirm that chromatin fragments were \u223c500 bp in length on average.ChIP analysis of transcription factor binding in chicken cells was performed using formaldehyde crosslinked chromatin prepared from chicken 10 day embryonic erythrocytes isolated from fertilized White Leghorn eggs or cultured 6C2 erythroleukemia cells. 10 day erythrocytes were washed and resuspended in 25 mls of PBS . Incubation with antibodies was performed overnight at 4\u00b0C on a rotating wheel. Between 10 and 30 \u00b5g of specific antibodies or 10 \u00b5g of normal rabbit IgG (Santa Cruz) were used per ChIP. Chromatin was precipitated with protein A agarose for 4 hours at 4\u00b0C with rotation. Immunoprecipitated chromatin was collected, washed, eluted and crosslinks reversed. DNA was then extracted by phenol-chloroform and ethanol precipitated in the presence of 10 \u00b5g glycogen.Chromatin was pre-cleared with 100 \u00b5l of protein A agarose and 50 \u00b5g of normal rabbit IgG (Santa Cruz) for 3 hours at 4\u00b0C on a rotating wheel. Aliquots of chromatin were taken to generate input DNA and protein for western analysis. Individual ChIPs were performed using chromatin from 1\u00d710\u03b2-globin locus as described previously Relative DNA enrichments were quantified by TaqMan real-time qPCR using the comparative Ct method relative to input DNA and normalized to the primer set 10.35 within the 16 kb condensed chromatin region upstream of the chicken GGAACAAGTTGGCAAGGTCCTAT10.35_For:\u2003TCTTCTGCCCTGCCCGTAT10.35_Rev:\u2003FAM-TGCAGTTCCCTGTTCATGTGCTTTTCG-TAMRA10.35_TM:\u2003TCCTGGAAGGTCCTGGAAG21.54_For:\u2003CGGGGGAGGGACGTAAT21.54_Rev:\u20036FAM-CCCAAAGCCCCCAGGGATGT-TAMRA21.54_TM:\u2003CTGTGGTCTCCTGCCTCACA39.8_For:\u2003AGGCTGGGTGCCCCTC39.8_Rev:\u2003FAM-CAATGCAGAGTGCTGTGGTTTGGAACTG-TAMRA39.8_TM:\u2003CACAGGAAACAGCTATGACATGATTPGI_5\u2032_For\u2003TCTGCCTTCTCCCTGATAACGPGI_5\u2032_Rev\u20036FAM-AATTCCTGCCCACACCCTCCTGC-TAMRAPGI_5\u2032_TM\u20039 cells treated with 1% formaldehyde for 5 minutes. Chromatin was prepared as described above, where fragments sizes ranged from 500\u2013700 bp. Semi-quantitative PCR was performed using the following primersChIP analysis of transcription factor binding in murine E14Tg2A.4 ES cell lines was performed using formaldehyde crosslinked chromatin prepared from 1\u00d710AAAGGCGTGCGGGAGCCAGAAATAPRT1/2_For: CCTTGGTAGGTGGGGAPRT1/2_Rev: CCCTGTTCCTGGGCTCCAPRT3_For: TGACTGGCCAGGAGGAPRT3_Rev: ChIP analysis from human embryonic kidney 293-T cell line SD5 that contains a stably integrated copy of the 275 bp HS4 core chicken insulator was performed as described for cultured chicken cells above. SD5 cells were crosslinked with 1.6% formaldehyde for 5 minutes. The following primers were used in SYBR quantitative PCR analysis:CGGGATCGCTTTCCTCTGAHS4_21.726_F: CCGTATCCCCCAGGTGTCTHS4_21.726_R: TCGCCTGCACAAATAGGGACP_DHFR_F: AGAACGCGCGGTCAAGTTTP_DHFR_R: ControlGACAGCAGCCGAACTTCGTTVEZF1_CDS_F: TGGTGCCCGAGGAAGATGVEZF1_CDS_R: APRT elements. The following elements were amplified from Hamster liver genomic DNA:AvaII and HpaII restriction sites in bold.Sp1-like motifs in red. CTAGAGGATCCGGACAACACCCACACCGGCCCCTCCAGGTCCAGAAAGCTGGCCCTGCGAGAAGCGGGACTGAAAAGGCGTGCGGGAGCCAGAAATCCAAAAGGGTGCCAAGGCATGCGTCCTTTTTCCACCCAGAAATAACCCCAGGCTTTCAATTTGAGGTTATTTCAATATCCAGCAAATGCGTTACTTCCTGCCAAAAGCCAGCCTCCCCGCAACCCACTCTCCCAGAGGCCCCGCCCCGTCCCGCCCCCTCCCGGCCTCTCCTCGTGCTGGATCGCTCCCTAAGGACGCCCCGCTCCAGAAGCCCCACCTACCAAGGACGCCCCACCCTTGTTCCCGGACTGGTATGACCCCAGCCTGCTGACATCCCTCCGCCCTTTCTCGTGCACGCGGCTATGGCGGAATCTGAGTTGCAGCTGGTGGCGCAGCGATCCGCAGTTTCCCCGACTTCCCCATCCCCGGCGTGCTGTTTAGGTGAGATCACGAGCCAGCAAGGCGTTGGAGCCCTGTTCCTGGGCTCCCGGCGAGGCGCATGGGCAGTCTCGGGGATCTTGTGGGGTCTCCGCCCCCCTTTCCCCGGCCACCAGCCTCTCCTTGTTCCCAGGGATATCTCGCCCCTCCTGAAGGACCCCGCCTCCTTCCGAGCTTCCATCCGCCTCCTGGCCAGTCACCTTAAGTCCACGCATGGCGGCAAGATCGACTACATCGCAGGTCTAAvaII and HpaII restriction sites in bold.Mutated bases in blue were introduced by site directed mutagenesis. Overall CpG content increased from 46 to 48 following these mutations. CTAGAGGATCCGGACAACACCCACACCGGCCCCTCCAGGTCCAGAAAGCTGGCCCTGCGAGAAGCGGGACTGAAAAGGCGTGCGGGAGCCAGAAATCCAAAAGGGTGCCAAGGCATGCGTCCTTTTTCCACCCAGAAATAACCCCAGGCTTTCAATTTGAGGTTATTTCAATATCCAGCAAATGCGTTACTTCCTGCCAAAAGCCAGCCTCCCCGCAACCCACTCTCCCAGAGACCCCCCGCATCCCCGACGCTACCCCCCGCATCCCCGATCTCCTCGTGCTGGATCGCTCCCTAAGGACGCCCCGCTCCAGAAGCCCCACCTACCAAGGACGCCCCACCCTTGTTCCCGGACTGGTATGACCCCAGCCTGCTGACATCCCTCCGCCCTTTCTCGTGCACGCGGCTATGGCGGAATCTGAGTTGCAGCTGGTGGCGCAGCGATCCGCAGTTTCCCCGACTTCCCCATCCCCGGCGTGCTGTTTAGGTGAGATCACGAGCCAGCAAGGCGTTGGAGCCCTGTTCCTGGGCTCCCGGCGAGGCGCATGGGCAGTCTCGGGGATCTTGTGGGGTCCGCTCCCCCCGCATCCCCGACACCAGCCTCTCCTTGTTCCCAGGGATATCTCGCCCCTCCTGAAGGACCCCGCCTCCTTCCGAGCTTCCATCCGCCTCCTGGCCAGTCACCTTAAGTCCACGCATGGCGGCAAGATCGACTACATCGCAGGTCTAHpaII site in bold.TCTAGATTGCTAGGAGTAGCACCTAAGATGAACTAGATGCTAAAAAATGCTGTATCTTTGGGGCACACGAGGGCATGCCTGGGCAGGCTTAGAGCCTGGTAGTCTCAGGGGCTGCACCAAAGTGTAATTCTTGTGCTAAATAACTTTCACTTACCAGTGCCAAGCACGGGCTTCAGAAACACCCTAGGGTCGCTGAATGTCCACCAGGGGAGTCAGACATGTCCAGAGGGTGAGAACCCCAGAGAATTCGGTAGCCCTGACATGTGCTACAATTACTGATGCCCACTTCCTACTGGTTCCTCCTGGCCATACCTCAGGAATTAGGGCATGCTTTCTGCCTGCTACAGTAGCTCATCCTCCCTGGAAGTGACCCCAGACATATACCCTGAACTGTAACCGATAAAGTGCGCCTGGGCAGATGTATTTGAGAGGTGGCAAAAGTAAACCATAGGTGTCCCCGAGCTAGATACAGAAGGCAGATAACATCCCCAAGGCTAAGCTGCTGCCCCAATAGCCATCAGCCTTCTAGTTATAGCTAGTAAGACCTAGTATTCCTGGTCAATACTATTCACTCAATCCTTACACCTCAGCCCTAACACGCCCCCTCTCTCATCCTAACAGGCCTAGACTCCAGGGGATTCTTGTTTGGCCCCTCCCTAGCTCAGGAGCTGGGCCTGGGCTGTGTGCTCATCCGGAAGCGAGGGAAGCTGCCAGGCCCCACAGTGTCAGCCTCCTATGCTCTCGAGTATGGCAAGGTAAGCAGGCAGTGGGTAGCTGTCTAGGAGTAAATGTGGGGGCTCAGAGAGGTTAAGTCATCAGGCCAGGTTTATACCACCAGGAAACATGGAGAAGCTAGGGGTGGTGGTTCTAGAAPRT CpG islands and an 870 bp non-island region of the APRT gene were cloned into pUC19. Each element was PCR amplified, half of which was subject to in vitro CpG methylation by M.SssI methyltransferase(New England Biolabs). In vitro methylation was validated by HpaII and MspI digestion. 1 \u00b5g of each APRT element was transfected into murine E14Tg2A.4 ES cells (BayGenomics) with 1 \u00b5g of the XbaI fragment of pREP7 (Invitrogen). Hygromycin resistant clones were grown in LIF without feeder co-culture. Genomic DNA was extracted after two weeks of culture. Probes for Southern blotting were generated using Ready-to-go dCTP beads .\u223c720 bp wild type and mutant Oligonucleotides were generated on an ABI 394 DNA synthesizer.The top strand sequences for each of the duplexes used for gel mobility shift analyses were:GACAGCCCCCCCCCAAAGCCCCCAGGGA5\u2032 GGAGCTCACGGG,FI wt CGCTCCCCCCGCATCCCCGAGCCGGggcgcgcct5\u2032 aggcgcgccCCGGTCCGG,FIII wt CCTGGGGGGATACGGGGAAAAAGCTTTAGG5\u2032 CCTGCAGACA,FV wt 5\u2032 ATTCGATCGGGGCGGGGCGAGC,Sp1 5\u2032 AATTGCAGAGCTGGGAATCGGGGGGGGGGGGGGGGCGGGTGGTGGTGTGG,glo wt 5\u2032 AATTGCAGAGCTGGGAATCGGGGGGGCGGGTGGTGGTGTGG,glo 7G 5\u2032 AATTGCAGAGCTGGGAATCGGGGGGCGGGTGGTGGTGTGG.glo 6G Asc I restriction sites used for cloning FIII sites in an earlier study are shown as lower case. All FI and FIII oligonucleotides were identical to the wt sequences above except for mutations indicated in DNA affinity columns were prepared using the following oligonucleotides5\u2032 gatcTCACGGGGACAGCCCCCCCCCAAAGCCCCCAFI-DA TOP 5\u2032 gatcTGGGGGCTTTGGGGGGGGGCTGTCCCCGTGAFI-DA BOTTOM 5\u2032 gatcGGTCCGGCGCTCCCCCCGCATCCCCGAGCCGGCAFIII-DA TOP 5\u2032 gatcTGCCGGCTCGGGGATGCGGGGGGAGCGCCGGACCFIII-DA BOTTOM PCR primers used in bisulfite sequencing were5\u2032 GGTATTAGAGTAGATTGTATTGAGAGTGTAHS4 5\u2032 double forward\u20035\u2032 CATAACTATTTCCTATATAAATCCCCHS4 5\u2032 double reverse\u20035\u2032 AGAGTAGATTGTATTGAGAGTGTATTATAHS4 5\u2032 single forward\u20035\u2032 ACATCCCTAAAAACTTTAAAAAAAAHS4 5\u2032 single reverse\u20035\u2032 GTTAAGGTTGGGGGTTTTTTIL-2R forward\u20035\u2032 AAAACTCTACCTAACAACCAAACACIL-2R reverse\u2003PCR primers used in RT-PCR gene expression analysis were5\u2032CAGTGCACGTTTGGCATTTGAAGGgVEZF805anti\u20035\u2032GAAAAGGCTTCTCGAGGCCTGATCGgVEZF716sense\u20035\u2032-6FAM-TCCAGGAGCGTGACCCCAGCA-TAMRAGgGAPDH-247T\u20035\u2032 ATGGGCACGCCATCACTATCGgGAPDH-226F\u20035\u2032 AACATACTCAGCACCTGCATCTGGgGAPDH-302R\u20035\u2032 TGCTGCGCTCGTTGTTGAGgB-ACTIN_F\u20035\u2032 CATCGTCCCCGGCGAGgB-ACTIN_R\u20035\u2032-6FAM-TGGCTCCGGTATGTGCAAGGCC-TAMRAGgB-ACTIN_T\u2003APRT non-island element were:The primers used for methylation specific PCR analysis of the 5\u2032\u2003TCTAGATTGCTAGGAGTAGCAPRTNI_3\u2032\u2003TCTAGAACCACCCCTAGCAPRTNI_Figure S1SacI - HindIII fragment originally defined as the \u201c250 bp core\u201d HS4 element in vitro DNaseI footprints AscI cloning sites present in all functional assays of the HS4 core are shown in italics.Core HS4 sequences. (A) The 244 bp (0.87 MB TIF)Click here for additional data file.Figure S2Identification of FI- and FIII-binding proteins. (A) Peptide sequences obtained from tandem MS sequencing of proteins isolated by FI- and FIII-DNA affinity. (B) Alignment of the amino acid sequences of chicken VEZF1/BGP1, human VEZF1/DB1, and mouse Vezf1 . Peptides obtained from tandem MS sequencing that match VEZF1 are indicated by lines above the alignment. Six C2H2 zinc fingers motifs are boxed. (C) Schematic representation of the domain structure of VEZF1. Only the zinc finger motifs share homology with factors other than VEZF1 orthologs, the nearest relative being the MAZ transcription factor.(4.96 MB TIF)Click here for additional data file.Figure S3A-globin\u03b2 promoter. Gel mobility supershift assays using 32P-labelled FI (A), FIII (B), FV (C), and glo wt (D) oligonucleotide duplexes. Adult chicken erythrocyte nuclear extract (ery) and recombinant chicken VEZF1, SP1, or SP3 used in the reactions are indicated by brackets above the lanes. Proteins were pre-incubated with antibodies (indicated above each lane) prior to incubation with DNA. Supershifts are evidenced by abrogation of specific complexes (asterisks) and/or formation of low mobility ternary complexes (SS). Antibodies alone do not give rise to complexes with any of the duplexes used (not shown).Supershift analysis of VEZF1, SP1, SP3, and ZF5 interactions with HS4 footprints and the (3.49 MB TIF)Click here for additional data file.Figure S432P-labelled FI and FIII oligonucleotide duplexes. Unlabelled competitor duplexes (indicated above each lane) were added at 50 fold molar excess. Recombinant chicken SP1, SP3, and ZF5 used in the reactions are indicated by brackets above the lanes. The competition profile of these proteins does not match that of red blood cell nuclear extract Click here for additional data file.Figure S5in vivo. (A) ChIP analysis of transcription factor interactions at the \u03b2-globin locus in 6C2 erythroid progenitor cells. DNA enrichments at the {lower case beta}A promoter or the HS4 and 3\u2032HS insulators were normalized to a negative control located in the 16 kb condensed chromatin region upstream of the \u03b2-globin locus. (B) ChIP analysis of transcription factor interactions with a stably integrated HS4 element in transgenic human 293 cells. Interactions with the DHFR CpG island promoter are shown as a positive control for SP1 and SP3 binding. DNA enrichments are normalized to an AT-rich negative control locus.SP1, SP3, and ZF5 do not interact with HS4 (0.49 MB TIF)Click here for additional data file.Figure S6IL-2R transgene showing location of QPCR primer sets used to analyze the interaction of VEZF1 with the transgenic HS4 and promoter elements. (B\u2013D) ChIP analysis of (B) VEZF1, (C) CTCF, or (D) USF1 interactions at the endogenous HS4 insulator (eHS4) and the transgenic HS4 (tHS4) and promoter (\u03b2-IL2Rpro) elements in the same 6C2 cell lines used for DNA methylation Schematic representation of the hylation and hist(1.09 MB TIF)Click here for additional data file.Figure S7\u03b2-globin locus.VEZF1 binding to HS4 is not significantly affected following VEZF1 RNAi. (A) Quantitative RT-PCR analysis following 48 hours of doxycycline-induction of two chicken 6C2 cell lines harboring lentiviral vectors that express VEZF1-specific miRNA. Expression levels are normalized to those of \u03b2-actin (ACTB) and untreated cells. VEZF1 mRNA levels are reproducibly knocked down by 70%. (B) Western blot analysis of chicken VEZF1 protein levels in one of the above lines with and without 14 days of doxycycline induction of VEZF1-specific miRNA. TBP levels were monitored as a loading control. VEZF1 and TBP band intensities visualized on a FUJI LAS3000 imager were quantified using AIDA software (shown below). Following normalization to TBP levels, VEZF1 protein levels were quantified to be knocked down by \u223c97%. VEZF1-specific miRNA may cause translational inhibition in addition to the mRNA degradation observed by RT-PCR (C) ChIP analysis of VEZF1 interaction with the endogenous HS4 element following 14 days of induced VEZF1 knockdown. DNA enrichments were normalized to a negative control located in the 16 kb condensed chromatin region upstream of the (1.64 MB TIF)Click here for additional data file.Figure S8APRT CpG island elements. VEZF1 and SP1 interact with the wild type APRT CpG island, but the FIII mutant APRT element is only bound by VEZF1. Gel mobility supershift assays using 32P-labelled oligonucleotide duplexes containing either the wild type APRT SP1 sites 1&2 (lanes 1\u20134) and site 3 (lanes 5\u20138) or the mutant sites 1&2 (lanes 9\u201312) and site 3 (lanes 13\u201316). The core sequences of the duplexes are as shown in Transcription factor interactions with (1.78 MB TIF)Click here for additional data file.Table S1VEZF1 sites in the HS4 barrier protect a promoter from DNA methylation. CpG methylation of transgene promoters flanked by wild type or mutant HS4 insulators after 30 or 90 days of culture. The scoring of individual CpG bases from each clone subject to bisulfite sequencing is shown. Methylated bases are marked as \u20181\u2019 and shaded blue. Average CpG methylation values are shown in (0.02 MB PDF)Click here for additional data file.Table S2de novo methylation of HS4. CpG methylation of wild type (WT) or mutant (\u0394I - \u0394V) HS4 insulators after 30 or 90 days of culture. Both copies of HS4 were sequenced, except for \u0394II and \u0394V, where only the outermost copy was sequenced (see Mutations of insulator protein binding sites result in the nced see . The sco(0.03 MB PDF)Click here for additional data file.Text S1DNA binding activities of candidate HS4-binding proteins; RNA interference methods; western blotting methods.(0.04 MB DOC)Click here for additional data file."} +{"text": "The vector was used to track reprogramming of HFF to iPSC. HFFs co-transduced with this reporter vector and vectors encoding 4 reprogramming factors were mostly positive for EGFP (67%) at an early stage of hiPSC formation. EGFP expression gradually disappeared and mCherry expression increased indicating less miRNAs specific to differentiated cells and expression of miRNAs specific to hESCs. Upon differentiation of the hiPSC into embryoid bodies, a large fraction of these hiPSCs regained EGFP expression and some of those cells became single positive for EGFP. Further differentiation into neural lineages showed distinct structures demarcated by either EGFP or mCherry expression. These findings demonstrate that a miRNA dependent reporter vector can be a useful tool to monitor living cells during reprogramming of hiPSC and subsequent differentiation to lineage specific cells.Human induced pluripotent stem cells (hiPSCs) provide new possibilities for regenerative therapies. In order for this potential to be achieved, it is critical to efficiently monitor the differentiation of these hiPSCs into specific lineages. Here, we describe a lentiviral reporter vector sensitive to specific microRNAs (miRNA) to show that a single vector bearing multiple miRNA target sequences conjugated to different reporters can be used to monitor hiPSC formation and subsequent differentiation from human fetal fibroblasts (HFFs). The reporter vector encodes EGFP conjugated to the targets of human embryonic stem cell (hESC) specific miRNAs ( OCT4, SOX2, KLF4, cMYC, LIN28 and NANOGHuman embryonic stem cells (hESCs) have significant therapeutic potential for various diseases, but the generation of these cells from individual patients raises ethical concerns. Recently, a technological breakthrough where somatic cells from mouse and human can be reprogrammed into hESC-like pluripotent cells, termed induced pluripotent stem cells (iPSCs), was made possible through ectopic expression of combinations of reprogramming factors including MicroRNAs (miRNAs) are small non-coding RNAs which regulate gene expression post-transcriptionally . The miR-302 gene encodes a cluster of eight miRNAs on chromosome 4 (miR-302b*-b-c*-c-a*-a-d-367) that are preferentially expressed in embryonal carcinoma cells, hESCs and hiPSCs miR-223, miR-155, and miR-142-3p are enriched in differentiated cells miR-223 and miR-142-3p are found in cells of primarily hematopoietic origin miR-155 is found in hematopoietic cells as well as in many types of lymphoma and solid cancers miR-155 is also expressed 20\u201350 fold higher in fibroblasts than in hESCs and hiPSCs We constructed a bidirectional vector whereby the reporter gene mCherry is conjugated with perfectly complementary miRNA target sites for miR-302a, miR-302b, miR-302c, and miR-302d or miR-155 results in ablation of EGFP or mCherry expression, respectively, demonstrating sensitivity of the vector to specific miRNAs . In hematopoietic lineage cells mCherry expression is entirely ablated whereas EGFP is maintained . After a prolonged period of culture on feeder cells, a fraction (<0.01%) of the cells reprograms and appears as hiPSC colonies The reprogramming of HFFs to hiPSCs can be achieved by the introduction of four transcription factors . Expression levels of EGFP and mCherry were monitored over the four week course of reprogramming to assess the activity of the reporter vector during generation of hiPSC and one hiPSC clone that was not transduced with this reporter vector (hiPSC #19) for characteristics of pluripotent stem cells. These hiPSC clones transduced with the reporter vector stably maintained mCherry expression and lack of EGFP expression for over 20 generations . Althougin vitro into EBs comprising the three embryonic germ layers miR-302a/d expression as the cells differentiated. Concomitantly, mCherry expression was slightly reduced in the cells, reflecting expression of one or more of the differentiation specific miRNAs miR-223, miR-155, or miR-142-3p. However, since the EBs represent multiple lineages of differentiated cells, we were unable to conclude which cells and to what extent these miRNAs are being expressed.hESC as well as hiPSC can be differentiated SOX1, SOX3 and PAX6, markers of neuroectodermal differentiation DNMT3B, REX1 and endogenous OCT4 which are assumed to be expressed in fully-reprogrammed hiPSCs DNMT3B, REX1 and endogenous OCT4 were downregulated, whereas SOX1, SOX3 and PAX6 were upregulated in the differentiated population compared to the undifferentiated population had reduced mCherry expression compared to two other hiPSC clones transduced with the reporter vector . AlthougThe development of this vector adds to the tools available to monitor hiPSC generation and subsequent differentiation of the hiPSC into different lineages. In addition to taking advantage of differential miRNA expression, other investigators have utilized differential promoters/enhancer expression in different lineages miR-223, miR-142-3p, and miR-155 are enriched primarily in cells of hematopoietic origin The work presented here indicates the potential great utility and flexibility of miRNA-regulatable lentiviral vectors to monitor various stages of reprogramming and the subsequent differentiation into lineage specific cells and tissues. For example, CCCGGGCGAGCAAGCTCAGTTTACACCGAATTCGGATCCTCACCAAAACATGGAAGCACTTAAGTCTCACCAAAACATGGAAGCACTTAAGTCTCACCAAAACATGGAAGCACTTAAGTCTCACCAAAACATGGAAGCACTTAGGCCTACACTCAAACATGGAAGCACTTAGTACACACTCAAACATGGAAGCACTTAGTACACACTCAAACATGGAAGCACTTAGTACACACTCAAACATGGAAGCACTTAGATATCGTCGAC) and differentiated cell specific-miRNA targets (CCCGGGTCGAATTCGGTACCAGATCTGGCGCGCCGTACGTGGGGTATTTGACAAACTGACAAGTCTGGGGTATTTGACAAACTGACAAGTCTGGGGTATTTGACAAACTGACAAGTCTGGGGTATTTGACAAACTGACAGGCCTACCCCTATCACGATTAGCATTAAAGTCACCCCTATCACGATTAGCATTAAAGTCACCCCTATCACGATTAGCATTAAAGTCACCCCTATCACGATTAGCATTAATTTAAATTCCATAAAGTAGGAAACACTACAGTACTCCATAAAGTAGGAAACACTACAGTACAGTTCCATAAAGTAGGAAACACTACAGTACTCCATAAAGTAGGAAACACTACAGATATCTGCATGCTTCGAAGCTAGCGGGCCC) were synthesized by Genescript . The synthesized fragment of differentiated cell specific-miRNA targets was conjugated to mCherry coding sequence amplified by PCR from pmCherry with primers (sense: ACGCACCGGTGGATCCAAGCTTGCCACCATGGTGAGCAAGGGCGAGGA and reverse: CTGCGAATTCTCACTACTTGTACAGCTCGTCCATGCCGCCGG) and exchanged with EGFP coding sequence in FG12 lentiviral vector AGTCAGCTAGCGCCACCATGGTGAGCAAGGGCGAG and reverse: CATCGACCCGGGAATTCTCATTACTTGTACAGCTCGTCCATG), and cloned into a pAAV-MCS vector (pAAV-EGFP). The synthesized fragment of hESC specific-miRNA targets was inserted into the pAAV-EGFP between stop codon of EGFP and hGH poly A signal. The fragment containing CMV minimal promoter, \u03b2-globin intron, EGFP, hESC specific-miRNA targets, and hGH poly A signal was amplified by PCR with primers (sense: GTACTCTCGAGCCCCATTGACGCAAATGGGCGGTAGG and reverse: CTCGTCTAGAAGGACAGGGAAGGGAGCAGTGGT) and cloned into the FG12 lentiviral vector deleted EGFP (EGFP-T/FG12) or into the mCherry-T/FG12 (EGFP-T/mCherry-T/FG12).For construction of the reporter vector, whole sequences of hESC specific-miRNA targets were substituted with EGFP coding sequence in the FRh11. The infectious titer was determined in 293T cells by infecting with the FRh11 encoding EGFP in the presence of 8 \u00b5g/ml polybrene. Reporter gene expression was monitored by flow cytometry.For reprogramming of HFFs, we substituted the ubiqutin C promoter of FG12 lentiviral vector with the RhMLV promoter (FRh11). The RhMLV promoter is derived from the long terminal repeat (LTR) region of Moloney murine leukemia virus (MLV) in the serum of one rhesus macaque monkey that developed T-cell lymphoma following autologous transplantation miR-302a, miR-302b, miR-302c, and miR302-d (PMIRH302abcdPA-2) and miR-155 (PMIRH155PA-1) from System biosciences . CopGFP sequence was eliminated from the vector.For ectopic expression of miRNAs, we purchased miRNA expressing lentiviral vectors for 2.293T hESCs (H1 clone) HFFs were isolated from the skin of 16 week-old fetus with DMEM supplemented10% FBS and 2 mM Glutamax (fibroblast medium) as reported previously CD34+ HPSCs were prepared from the liver of 16 weeks-old fetus as previously described Lentiviral vector stocks were generated using a vector plasmid, a packaging plasmid pCMV R8.2 \u0394Vpr, and a VSV-G envelope protein-coding plasmid by calcium phosphate-mediated transient transfection as previously described 4 cells per well of 6-well plates and infected with vectors encoding each reprogramming factor with or without a lentiviral vector encoding EGFP-T/mCherry-T at 300 ng (around multiplicity of infection of 3\u20135) of p24 per each virus. The cells were cultured for 3 days in fibroblast medium and replated at 5\u00d7104 cells per 60 mm dish on irradiated mouse embryonic fibroblast (iMEF) feeder cells. On the next day, the medium was replaced with KO-DMEM (Invitrogen) supplemented with 20% Knockout Serum Replacer , 2 mM Glutamax (Invitrogen), 0.1 mM non-essential amino acids (Invitrogen), 0.1 mM \u03b2-mercaptoethanol , and 50 ng/ml of recombinant human basic fibroblast growth factor (Invitrogen) (hiPSC medium). The medium was changed on a daily basis. To increase a reprogramming efficiency, the cells were treated with 0.5 mM valproic acid and 10 \u00b5M Y27632 for first 14 days The day before lentiviral vector transduction, HFFs (passage 1\u20133) were seeded at 5\u00d710Size-controlled EBs (3000 hESCs/EB) were formed using AggreWell\u2122 400 plates following the manufacturer's protocol. Briefly, hESCs and hiPSCs were incubated with 10 \u00b5M Y-27632 for 24 hrs before EB formation. Cells were harvested with Accutase as a single-cell suspension and used for EB formation. EBs were harvested into ultra low attachment plates and maintained in Iscove's Modified Dulbecco's Medium containing 10% FBS, 2 mM Glutamax, and 0.1 mM \u03b2-mercaptoethanol for differentiation into EBs. The medium was changed every 3 days.Induction of differentiation into neural lineages was performed as previously described with some modifications For detection of EGFP and mCherry expression, single-cell suspensions from 293T, HFF, hESC, and hiPSC were prepared using 0.25% trypsin-EDTA and collected in FACS buffer (2% FBS and 0.01% sodium azide in PBS). U937, Ramos and CEM cells were also collected in FACS buffer. For detection of hESC-specific markers, hESCs and hiPSCs were dissociated with 0.25% trypsin-EDTA into a single cell suspension. Cells were adjusted to 100,000 per sample in 100 \u00b5l of FACS buffer and then labeled with monoclonal antibodies conjugated with a fluorescent dye . For detection of iMEFs, cells were stained with an antibody specific with mouse CD29 conjugated with PE-Cy7 (eBioscience). Data were collected on a Cytomics FC500 and analyzed using FCS express .hESC and hiPSC colonies were grown on poly-L-lysine and Matrigel coated glass coverslips. Cells were fixed with 1.0% formaldehyde/PBS and permeabilized with 0.2% Triton-X 100 for 5 min on ice. Cells were then incubated with anti-human Nanog antibody and subsequently with DyLight488 conjugated donkey anti-rabbit IgG (BioLegend) and 7-amino-actinomycin D (7-AAD) (Invitrogen) for nuclear staining. After washing, cells were visualized with a LEICA DM IRB equipped with a SPOT camera and software .RNA extraction from hESC and hiPSC was performed using QIAGEN's RNeasy Mini kit following the manufacture's protocol . Total RNA (250 ng) was reverse-transcribed using QIAGEN's Omniscript RT-kit with a 0.5 ng/ml oligo dT primer (Invitrogen) in 20 \u00b5l reaction. PCR was performed with the HotMaster Taq DNA polymerase , using 0.5 \u00b5l of cDNA template and primers at a concentration of 3 pmol/\u00b5l. Five \u00b5l of PCR products was loaded in a 2% agarose gel containing ethidium bromide. All primer sequences were listed in Table 1."} +{"text": "Drosophila melanogaster by combining long-read sequencing, chromatin immunoprecipitation for the centromeric histone CENP-A, and high-resolution chromatin fiber imaging. Contrary to previous models that heralded satellite repeats as the major functional components, we demonstrate that functional centromeres form on islands of complex DNA sequences enriched in retroelements that are flanked by large arrays of satellite repeats. Each centromere displays distinct size and arrangement of its DNA elements but is similar in composition overall. We discover that a specific retroelement, G2/Jockey-3, is the most highly enriched sequence in CENP-A chromatin and is the only element shared among all centromeres. G2/Jockey-3 is also associated with CENP-A in the sister species D. simulans, revealing an unexpected conservation despite the reported turnover of centromeric satellite DNA. Our work reveals the DNA sequence identity of the active centromeres of a premier model organism and implicates retroelements as conserved features of centromeric DNA.Centromeres are essential chromosomal regions that mediate kinetochore assembly and spindle attachments during cell division. Despite their functional conservation, centromeres are among the most rapidly evolving genomic regions and can shape karyotype evolution and speciation across taxa. Although significant progress has been made in identifying centromere-associated proteins, the highly repetitive centromeres of metazoans have been refractory to DNA sequencing and assembly, leaving large gaps in our understanding of their functional organization and evolution. Here, we identify the sequence composition and organization of the centromeres of Drosophila melanogaster centromeres, which have long remained elusive despite the high quality of this species\u2019 genome. assembly.Long-read sequencing, CENP-A ChIP, and chromatin fiber imaging reveal the composition and organization of Drosophila and centromeric histone H3 [CenH3] in plants), which is necessary and sufficient for kinetochore activity \u00d7 (1 pmol/330 pg) \u00d7 (1/Oligopaint length in nt) = Oligopaint molarity [\u03bcM]G2/Jockey-3 oligo against the consensus of the 3\u2032 region of G2/Jockey-3 elements found within most centromere contigs. A 5\u2032 addition containing 5\u2032-CAGT-3\u2032 followed by universal forward primer binding sites separated by an XhoI cut site (5 \u2032-cacactccggtacgcacctgctcgagcagtgctcgttggcccacac-3\u2032). A 3\u2032 addition containing 5\u2032-ACTG-3\u2032 followed by universal reverse primer binding sites separated by a SpeI cut site (5\u2032-agggtagtcgttgtagctcgactagtggtacgcccagaagcatccc-3\u2032). The G2/Jockey-3 sequence was ordered as a \u201ccustom gene\u201d (IDTDNA.com) and synthesized in the pUCIDT (AMP) vector (pUCIDT-G2). The sequence of the insert is as follows .We designed a 1,643-bp cacactccggtacgcacctgctcgagcagtgctcgttggcccacacCGGACGGCTCTTGGTGCCGCTCTGAAGCCGAAAGAGCTGAAGCGTTTGCAGATCACCTCCAGAATGCATTCACACCATTTGACAGATGCACTGGCGAAGAGCGTGCTGCAACCACCAGGTTCCTAGAGAGTCCATGTCCTCCTAGCCTGCCCATAGAGCCCGTCACCCCAGAAGAGGTTGCGCAAGAGTCGCCTCACTAAAGGCTAGCAAATCCCCAGGACTGGATCGCATCGACGCCACATCCCTTAAAATGCTGCCACCTCCCTGTTCCCAGTTGCTGGCCAACATATACAACAGATGCTTCTCACTAGGGTACTTCCCGAGATCATGGAAACGTGCAGAAGTCATTCTCATCCTCAAACCTGGAAAACCTGAAGCCAATCTTGCCTCATATAGACCGATTAGTCTGCTGGCAATCCTCTCCAAAATACTCGAAAGAGTATTTCTGCGCAGAGTGTTGCCAGTACTGGACGAGGCTGGACTGATCCCTGATCACCAGTTTGGCTTCAGGCGATCCCACGGAACACCCGAGCAATGCCACCGGCTCGTAGCACGCATCCTAGATGCATTCGAGAACAAACGATACTGTTCGGCCGTATTCCTGGATGTCAAGCAGGCGTTCGACAGAGTGTGGCATCCTGGACTCCTCTACAAACTCAAGTCCCACCTTCCCAGTTCCCACTATGCCCTACTCAAATCGTATACTGAAGGAAGAGAGTTCCAAGTGCGATGCGGTTCCTCAACCAGCACGACAAGGCCTATACGAGCCGGAGTACCTCAAGGCAGCGTCCTTGGTCCCATCCTCTACACCCTGTTTACAGCAGACCTCCCTATCATACCCTCCCGTTACCTCACAGCAGCCACCTATGCAGATGACACGGCGTTCCTTGCCACCGCAACAAACCCTCAACTAGCATCAGCCATCATCCAGAGGCAACTGGATGCATTGGATCCATGGCTGAAACGCTGGAACATCGTGATCAACGCTGATAAATCCTCCCACACCACCTTCTCTCTGCGCAGAGGAGAATGCCCCCCGGTCTCACTCGACGGCGACACAATCCCTACCTCCAGCACCCCCAAATATTTAGGGCTGACCCTGGACAGAAGGCTGACTTGGGGCCCCCACATCAACAGAAAGCGTATCCAGGCCAACATACGCCTAAAGCAACTCCACTGGCTCATCGGTAAAAAGTCCAAGCTGCGAGAGAAACTAAAGATTCTCGTCTACAAGACTATTCTCAAGCCAATCTGGACGTACGGAATTCAGCTGTGGGGCACTGCAAGCACATCACATAGAAGGAAGATCCAGCGATTTCAAAACAGATGTTTGAGAATAGTCTCCAACGCCCATCCCTACCACGAAAATTCCGCCATCCACGAGGAGCTCGGGATTCCATGGGTAGACGACGAAATCTACAGACACAGTGTGAGATATGCTAGCAGACTGGAGAACCACCACAACCACCTGGCCGTCAACCTTCTAGACCATAGCCAATCCCTAAGACGCCTGCAGAGAACGCACCCGCTTGACCTTACTCAACATACTTAATCATACTTAACCCCTACCCAAGTACACTCGATGTACTCCCCTTAAGTTAATGTTTCCCTCCAAAAAATTTAATTATTGTCCACTAGGACAGgggatgcttctgggcgtaccactagtcgagctacaacgactaccctcagt-3\u20325\u2032-cagtG2 (500 ng) was digested using SpeI and XhoI restriction enzymes in 1x Cutsmart Buffer for 1 h at 37\u00b0C. The digest was run on a 1.0% SeaPlaque GTG agarose gel (Lonza), and a 1,689-bp band containing the G2 sequence was gel extracted and purified using the PureLink Quick Gel Extraction Kit (Invitrogen). DIG-labeled G2 probes were generated via PCR in 50-\u03bcl reactions consisting 0.09 ng of gel extracted G2 DNA, 0.5 \u03bcM of forward and reverse primers from the Universal_2 primer set to provide additional support for our candidate centromeric contigs . We mappnogaster to our anogaster and the We calculated the significance between different categories using a Kruskal-Wallis test by ranks with Dunn\u2019s test for post hoc analysis and the pairwise Wilcoxon rank sum test with false discovery rate (FDR) correction of type G2/Jockey-3 sequences based on RepeatMasker annotations and custom scripts. We aligned and manually inspected G2/Jockey-3 and IGS alignments using Geneious v8.1.6 [https://doi.org/10.5061/dryad.rb1bt3j [G2/Jockey-3 and IGS using RAxML v.8.2.11 with settings \u201c-m GTRGAMMA -T24 -d -p 12345 -# autoMRE -k -x 12345 -f a\u201d [We extracted all IGS elements from the genome using BLAST v2.7.1 with sets v8.1.6 (see Dry.rb1bt3j ). We con45 -f a\u201d . We used45 -f a\u201d to plot G2/Jockey-3 non-LTR retroelements have evidence for recent activity based on insertion polymorphism and expression. We examined RNA-seq reads from testes for evidence of G2/Jockey-3 because of the enrichment of these elements on the Y chromosome. We mapped poly-A [We investigated whether d poly-A and totad poly-A (S6 Tabld poly-A and estid poly-A ).S1 FigPlot of normalized CENP-A/input for simple tandem repeats for each ChIP-seq replicate, sorted by median (red lines). Shown are only the simple tandem repeats with median CENP-A/input > 1 in all four CENP-A ChIP replicates (see details in (TIF)Click here for additional data file.S2 FigG2 and Jockey-3 sequences in D. melanogaster genome and closely related species in the simulans clade (D. simulans and D. sechellia) and D. yakuba. In D. melanogaster, G2 and Jockey-3 are interleaved across the phylogeny and thus likely correspond to the same repeat type. We therefore refer to these elements collectively as G2/Jockey-3 throughout the manuscript. (See Dryad repository files 13 and 15: https://doi.org/10.5061/dryad.rb1bt3j [A maximum-likelihood phylogenetic tree showing the relationship between .rb1bt3j ). LTR, l(TIF)Click here for additional data file.S3 FigLocations of the top 100 strongest peaks for each ChIP experiment. (A) Plot of the location of top 100 strongest peaks for each ChIP experiment on the diagonal (see details in (TIF)Click here for additional data file.S4 FigJockey elements are shown in one color even though they are distinct elements). The normalized CENP-A enrichment over input is shown for three replicates (replicate 2 is in Organization of each CENP-A-enriched island corresponding to centromere candidates: (A) X centromere, (B) centromere 4; (C) Y centromere; (D) centromere 3; (E) centromere 2. Different repeat families are color coded Click here for additional data file.S5 FigRpL32 promoter region as a noncentromeric control. (C) Graph showing our ChIP-qPCR results using primers targeting other regions that showed CENP-A enrichment but that were not in our contigs. Again, the enrichment is calculated relative to the input and is normalized by RpL32 promoter as a noncentromeric control. We did not observe a robust CENP-A enrichment at these sites. The underlying data can be found in (A) Diagram showing putative centromere contigs showing the locations of CENP-A ChIPtigs in black and CENP-A MACS peaks in orange as in (TIF)Click here for additional data file.S6 FigPacBio reads were mapped to the genome using Minimap (v 2.11) and the setting \u201c-ax map-pb.\u201d Shown are (A) X centromere, (B) centromere 4, (C) Y centromere, (D) centromere 3, and (E) centromere 2. The depth of only the high-quality mapped reads (mapped Q \u2265 30) was estimated for each position and normalized by the median depth of other genomic regions (98.32\u00d7 for autosomes and 49.16\u00d7 for sex chromosomes) to get relative depth. The relative depths of the TE-rich islands are close to 1, whereas the depth of the flanking simple satellites is uneven, with some regions > 1 and some < 1. We therefore exclude simple repeats from any assembly-based analyses and color these regions gray in (TIF)Click here for additional data file.S7 FigProdsat (magenta) and AAGAG (blue); (C) AATAG (magenta) and AAGAG (blue) with AATAG blocks identified by white and yellow (large block) arrows; (D) Prodsat (magenta) and dodeca (blue); (E) AATAT (magenta) and SATIII (blue); (F) AATAT (magenta) and AAGAG (blue); (G) AATAG (magenta) and Prodsat (blue) with AATAG blocks identified by white and yellow (large block) arrows. DAPI is shown in gray. The underlying data can be found in Prodsat, Prod satellite.IF-FISH using an anti-CENP-C antibody (green) and satellite FISH probes in the following combinations: (A) AAGAT (magenta) and AAGAG (blue) with a high-contrast inset of AAGAT on the X chromosome; (B) (TIF)Click here for additional data file.S8 FigSATIII (blue); (B) dodeca (magenta) and Prodsat (blue); (C) AATAG (magenta) and Prodsat (blue) with a high-contrast inset of AATAG and Prodsat on cf(2R); (D) AAGAG (magenta) and Prodsat (blue); (E) AAGAG (magenta) and AATAG (blue) with a high-contrast inset of AATAG on chromosome 3; (F) AAGAT (magenta) and AAGAG (blue). DAPI is shown in gray. See also Prodsat, Prod satellite; S2, Schneider 2.IF-FISH using an anti-CENP-A antibody (green) and satellite FISH probes in the following combinations: (A) AATAT (magenta) and (TIF)Click here for additional data file.S9 FigG2/Jockey-3 consensus element obtained from mapping total and poly-A RNA-seq data from testes [G2/Jockey-3 copies surveyed on centromere (\u201cCen\u201d) X, 4, and 3 but not Y and 2 show low levels of transcription compared to the housekeeping gene Actin. Although the primers (G2/Jockey-3 copies not included in our assembly. Error bars = SD. The underlying data for this figure can be found in (A) Shown is the plot of the normalized reads depth from uniquely mapped reads across the m testes , 122 to primers are spec(TIF)Click here for additional data file.S10 FigD. melanogaster genome with related outgroups are similar to the IGS at 3Giglio but are on small contigs, tig00022795 and id = 102159_0. Contig tig00022795 is also moderately enriched in CENP-A. CENP-A, centromere protein A; IGS, intergenic spacer of the ribosomal genes.Maximum-likelihood phylogenetic tree of all individual IGS sequences found in the .rb1bt3j ). Node s.rb1bt3j ). All ce(TIF)Click here for additional data file.S11 FigPROTOP). PROTOP are DNA transposons that have not been recently active, and their distribution is primarily in heterochromatin. TART elements are non-LTR retroelements highly enriched at telomeres and are also moderately CENP-A enriched. DM1731 is a retroelement moderately enriched for CENP-A but not enriched in the centromere islands. Doc2, G, Jockey-1, and G2/Jockey-3 are CENP-A enriched non-LTR retroelements abundant in the centromere islands along the following chromosomes: (A) chromosome 2, (B) chromosome 3, (C) chromosome 4, (D) chromosome X, and (E) chromosome Y. Contigs from each chromosome were concatenated in order with an arbitrary insertion of 100 kb of \u201cN.\u201d Distances along the x-axis are approximate. The order and orientation of the Y chromosome contigs are based on gene order (see ). Each tands see Tables. (TIF)Click here for additional data file.S12 FigMaupiti and AAGAG (blue); (B) Lampedusa and AAGAT (blue); (C) Lipari and AATAT (blue); (D) Giglio and dodeca (blue). White boxes show the separate signals at the targeted centromeres. Yellow boxes show centromeric hybridizations at other centromeres. DAPI is shown in gray. Bar 5 \u03bcm. The underlying data for this figure can be found in IF-FISH using an antibody for CENP-C (green), centromere Oligopaint FISH probes (magenta), and FISH probes for centromeric satellites (blue) in the following combinations: (A) (TIF)Click here for additional data file.S13 FigMaupiti (X), (B) Lampedusa (4), (C) Lipari (Y), (D) Giglio (3). The \u201cSignal Adjusted\u201d panels in (A) and (B) show high-contrast Oligopaint hybridization for visualization of weak foci. Bar 5 \u03bcm. See also IF-FISH using an antibody for CENP-A (green) and centromere Oligopaint FISH probes designed to target centromere contigs (magenta). (A) (TIF)Click here for additional data file.S14 FigP < 0.0001; *adjusted P < 0.02, pairwise Wilcoxon rank sum test with FDR correction; Kruskal-Wallis test by ranks with Dunn\u2019s test for post hoc analysis. The underlying data for this figure can be found in Plots showing intra- and interchromosomal interactions between regions in Hi-C data from: (A) stage 16 embryos (end of embryogenesis) and (B) embryonic cycles 1\u20138 . The di(TIF)Click here for additional data file.S15 FigRsp locus ; (B) 100-kb Oligopaint for a heterochromatic region on chromosome 3L ; (C) 100-kb Oligopaint for a euchromatic region approximately 600 kb from the telomere of chromosome 3L . Arrows show the region of the fiber that was measured. Bar 5 \u03bcm. (D) Scatterplot showing the quantification of fiber lengths. Mean lengths were used to estimate the size in kb (approximately 10 kb/1 \u03bcm). Error bars show the standard deviation. P = 0.085 (n.s.) for each pair of measurements compared (two-tailed t test). The underlying data can be found in Rsp, Responder.Stretched chromatin fibers from female third instar larval brain cells using the following probes: (A) (TIF)Click here for additional data file.S16 FigMaupiti (magenta), and AAGAG probe (cyan) on female third instar larval brain cells. DAPI is shown in gray. CENP-A occupies Maupiti and the AAGAG satellite. We observed some variation in FISH signals and Maupiti and CENP-A domain lengths, likely because of the efficiency of Oligopaint binding and variable stretching in this region. Arrows show the region of the fiber that was measured. (H) Scatterplot showing the quantification of the length of Maupiti FISH and CENP-A IF signals. Error bars show the standard deviation. N = 24 fibers. Bar 5 \u03bcm. The underlying data for this figure can be found in (A-G) Examples of fibers visualized with IF with anti-CENP-A antibody (green), FISH with Oligopaints for (TIF)Click here for additional data file.S17 FigLampedusa (magenta), and AAGAT probe (cyan). DAPI is shown in gray. CENP-A occupies predominantly the island Lampedusa. Arrows show the region of the fiber that was measured. (H) Scatterplot showing the quantification of the length of Lampedusa FISH and CENP-A IF signals. Error bars show the standard deviation. N = 25 fibers. Bar 5 \u03bcm. The underlying data for this figure can be found in (A-G) Examples of fibers visualized by IF with anti-CENP-A antibody (green), FISH Oligopaint FISH for (TIF)Click here for additional data file.S18 FigLipari (magenta). DAPI is shown in gray. We did not include satellite FISH because no centromeric satellites are known for the Y. Note that the Oligopaints only target part of Lipari Examples of fibers visualized by IF with anti-CENP-A antibody (green), FISH with Oligopaints for pari see . CENP-A (TIF)Click here for additional data file.S19 FigGiglio (magenta), and a probe for the centromere 3\u2013specific dodeca satellite (cyan). DAPI is shown in gray. CENP-A occupies primarily Giglio and a small stretch of dodeca satellite. Note that the binding of the dodeca (an LNA probe) is quite variable between fibers and results in several gaps that could be a result of the higher stringency conditions needed for Giglio Oligopaint FISH. Arrows show the region of the fiber that was measured. (F) Scatterplot showing the quantification of the length of Giglio FISH and CENP-A IF signals. Error bars show the standard deviation. N = 30 fibers. Bar 5 \u03bcm. The underlying data can be found in (A-E) Examples of fibers visualized by IF with anti-CENP-A antibody (green), FISH with Oligopaints for (TIF)Click here for additional data file.S20 Figdodeca from the experiment in Giglio (magenta), and FISH with dodeca probe (cyan). DAPI is shown in gray. Note the presence of Giglio signal on the dodeca CENP-A region. Multiple, overlapping panels were often acquired to follow an individual fiber. Panels were then cropped and juxtaposed in the figure, with white lines showing the separate images. White boxes show the CENP-A domain on Giglio, and yellow boxes show the smaller domain on dodeca. N = 5 (these are rare fibers to find in our preparations because of their length). Bar 5 \u03bcm. CENP-A, centromere protein A; FISH, fluorescence in situ hybridization; IF, immunofluorescence.(A-D) Examples of longer fibers tracked along (TIF)Click here for additional data file.S21 FigProdsat (magenta) and AATAG (cyan). (C-D) Examples of fibers with AAGAG (cyan) and Prodsat (magenta). (E) Example of fiber with AAGAG (magenta) and AATAG (cyan). We propose that Capri is located between flanking blocks of AAGAG and AATAG satellites that reside very close to where the Prodsat begins. Arrows show the region that was measured for each fiber. (F) Scatterplot of CENP-A IF signal lengths. (G) Model for the organization of centromere 2 showing a possible location of Capri. Error bars show the standard deviation. N = 18 fibers. Bar 5 \u03bcm. The underlying data can be found in Prodsat, Prod satellite.(A-D) Examples of fibers visualized with IF with anti-CENP-A antibody (green) and FISH with satellites. DAPI is shown in gray. (A-B) Examples of fibers showing colocalization of CENP-A (green) with (TIF)Click here for additional data file.S1 TableWe used kseek to estim(XLSX)Click here for additional data file.S2 TableRows correspond to complex repeat families (TEs and complex satellites), with the counts per family in the ChIP and input reads from every dataset. We calculated enrichment for each repeat type by normalizing by total mapped reads for each dataset and taking the ratio of normalized values for each ChIP and its corresponding input. ChIP, chromatin immunoprecipitation; TE, transposable element.(XLSX)Click here for additional data file.S3 TableWe mapped all ChIP-seq data to the de novo assembled ChIPtigs and called peaks using MACS with high-quality reads . We also mapped ChIPtigs to the genome to determine its genomic location and assigned repeat IDs based on BLAST results.(XLSX)Click here for additional data file.S4 TableWe mapped the ChIP and input reads to our genome assembly and used the high-quality reads to call ChIP peaks with MACS. We show the peak locations for each dataset. ChIP, chromatin immunoprecipitation.(XLSX)Click here for additional data file.S5 TableWe used IDR to compare MACS peaks from different ChIP-seq replicates. We show the statistics for shared peaks from each comparison. ChIP-seq, chromatin immunoprecipitation sequencing; IDR, irreproducible discovery rate; OreR, Oregon-R.(XLSX)Click here for additional data file.S6 TableWe list reads and mapping summaries of all Illumina and long-read datasets generated in this paper or downloaded from NCBI\u2019s SRA.(XLSX)Click here for additional data file.S7 TableList of primers used for qPCR in this study. The centromere contig that each target is associated with is designated in the \u201cCentromere\u201d column. Note that in silico PCR for the 3_G2 primers predicted three specific products from centromere 3 as well as two products on contig tig00022795 and additional nonspecific products from the X chromosome when three or more mismatches are allowed all of the same 145-bp size. qPCR, quantitative PCR.(XLSX)Click here for additional data file.S8 TableG2/Jockey-3 outside of the centromeric islands. ChIP, chromatin immunoprecipitation; IDR, irreproducible discovery rate; OreR, Oregon-R.We listed peaks outside canonical centromeres with any agreement between replicate ChIP experiments (IDR \u2264 0.05). We also report any genes or repeat annotations that overlap the MACS peaks. Note that there is no general enrichment in (XLSX)Click here for additional data file.S9 TableP values. G2/Jockey-3, G, Doc2, and Jockey-1 are significantly enriched in centromeres relative to other heterochromatic regions and to the whole genome. Asterisk signs show that TART and ProtoP are significantly underrepresented in centromeres relative to other heterochromatic regions. FDR, false discovery rate; FISH, fluorescence in situ hybridization; TART, Telomere-associated retrotransposon; TE, transposable element.We show the copy numbers of TEs in different genomic regions. The sums of base pairs in the assembly size in centromeres , pericentromeric heterochromatin , and other regions were used to compute the distribution statistics of TEs. We created a 2-by-2 contingency table for each TE comparing observed to expected (based on the sum of bp) for each comparison: centromere to heterochromatin (\u201ccen-het\u201d) regions or centromeres to whole genome (\u201ccen-genome\u201d). We computed a Fisher\u2019s exact test with FDR correction to get adjusted (XLSX)Click here for additional data file.S10 TableHybridization conditions used for FISH with specific Oligopaints. FISH, fluorescence in situ hybridization.(XLSX)Click here for additional data file.S11 TableInformation on the fluors used and sequences of satellite FISH probes used in this report. * = \u201c+N\u201d designates the incorporation of an LNA. FISH, fluorescence in situ hybridization; LNA, locked nucleic acid.(XLSX)Click here for additional data file.S12 TableInformation on the 5\u2032 secondary oligo adapter site and sequence of satellite probes used in this report.(XLSX)Click here for additional data file.S13 TableSequence and fluors of secondary oligo probes used for fluorescence detection of Oligopaints and unlabeled satellite probes.(XLSX)Click here for additional data file.S14 TableList of primer sets used for library amplification and G2 probe synthesis.(XLSX)Click here for additional data file.S15 TableList of primer sets used for sublibrary amplification and Oligopaint synthesis.(XLSX)Click here for additional data file.S16 TableWe assigned contigs from the assembly to a chromosome and a chromatin status Click here for additional data file.S17 TableG2/Jockey-3. CENP-A, centromere protein A; IDR, irreproducible discovery rate; S2, Schneider 2.We compared the MACS peaks shared between \u201cnormal\u201d S2 (this study) and S2 with CENP-A overexpression using the IDR test. Some noncentromeric regions should have more CENP-A enrichment after CENP-A overexpression; however, only four peaks have IDR \u2264 0.05. None of these peaks have (XLSX)Click here for additional data file.S18 TablePercentage of probe signals that overlap with different cytological locations in S2 cells. The underlying data can be found in (XLSX)Click here for additional data file.S19 Tables, using an anti-CENP-A antibody to mark the centromere. Locations were designated as centromeric (\u201cCen\u201d), pericentric (\u201cPeri\u201d), or heterochromatic (\u201cHet\u201d). See also s, small chromosome 4; CENP-A, centromere protein A; cf(2L), centric fragment of chromosome 2L; cf(2R), centric fragment of chromosome 2R; S2, Schneider 2; X;4, Robertsonian translocation between chromosomes X and 4.Summary of the locations of satellite repeats determined by IF-FISH on S2 cell chromosomes X, X;4, 2, cf(2R), cf(2L), 3, 4, and 4(XLSX)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.S2 Data(XLSX)Click here for additional data file.S3 DataThe columns indicate the centromere contig ID, start and end coordinates of sequence, followed by the oligo sequence, and the melting temperature . Included are also the same Oligopaint sequences with 5' and 3' extensions containing the universal primer followed by library-specific barcodes (oligos.with.adaptors).(XLSX)Click here for additional data file.S1 Text(DOCX)Click here for additional data file."} +{"text": "Concerns were raised that some of the sequences reported in this article may not In Table 1, the sequences are written as 5\u2019 to 3\u2019, and for shRNA sequences, both rows in the first white band comprise a single sequence per column. The shRNA sequences were designed according to the polyclonal loci of pGenesil-1 vector, with the shRNA structure: BamHI+ Sense + Loop (TTCAAGACG) + Antisense+ Termination signal+ Sal I+ Hind III.As noted in Table 1, the COMMD7 shRNA Sense sequence is as follows:CTCTGGGTCTTAGTGAGGATTCAAGACGTCCTCACTAAGACCCAGAGTTTTTTGTCGACA-3\u20195\u2019-GATCCGFor clarity, the targeting sequences are in bold. The targeting sequence aligns to nucleotides 910\u2013928 of COMMD7 transcript variant 1 mRNA sequence (NM_053041.2).There is an error in Table 1 in that the COMMD7 shRNA Antisense sequence is reported in 3\u2019 to 5\u2019 orientation. The COMMD7 shRNA antisense sequence used in the study with targeting sequences in bold is as follows:GAGACCCAGAATCACTCCTAAGTTCTGCAGGAGTGATTCTGGGTCTCAAAAAACAGCTGTTCGA-5\u20193\u2019-GCIn Table 1, the Sense and Antisense sequences for COMMD7 (for PCR) are switched. The 5\u2019 to 3\u2019 sequence listed in Table 1 for COMMD7 Antisense (for PCR) is actually the Sense sequence, corresponding to nucleotides 758\u2013779 of COMMD7 transcript sequence NM_053041.2. The sequence listed for COMMD7 Sense is actually the Antisense sequence, corresponding to nucleotides 938\u2013915 of COMMD7 transcript sequence NM_053041.2.A 2009 report , as a ce3 are reported. As noted in the Methods, tumor sizes were estimated using the equation, V = L x W2 . This resulted in reported tumor volume estimates twice as large as would be calculated using the standard technique for this type of experiment in which tumor volumes are instead estimated as V = (L x W2)/2. [In Fig 5, tumor sizes up to 5000 mmx W2)/2. .The authors also provide here additional information about the controls used for the reported experiments:\u201cControl\u201d indicates HepG2 cells without plasmid transfection.\u201cScramble\u201d group was transfected with an shRNA construct generated using the following oligonucleotides:GACTTCATAAGGCGCATGCTTCAAGACGGCATGCGCCTTATGAAGTCTTTTTTGTCGACA-3\u2019(Sense) 5\u2019-GATCCCTGAAGTATTCCGCGTACGAAGTTCTGCCGTACGCGGAATACTTCAGAAAAAACAGCTGTTCGA-5\u2019(Antisense) 3\u2019-GThe targeting sequence (bold) for the scramble construct includes a 14 nucleotide stretch that is 100% identical to the human ODF2 transcript (variant X34).For electrophoretic mobility shift assays, the core \u03baB binding sequence in the rThe data underlying results in this article are no longer available."} +{"text": "This article presents data on initial experiments that were carried out to investigate new thermostable transketolase (TK) activities with l-arabinose. Transaminase (TAm) sequences from an in-house library of thermophilic strains were analyzed to compare homologies to characterized TAms with desired activity. DNA and amino acid sequences are presented for all the enzymes investigated. Calibration curves for products of the TK and TAm reactions are also presented along with chromatographic analysis of the various one-pot reactions.The dataset presented in this article is related to the research article entitled \u201cOne-pot, two-step transaminase and transketolase synthesis of Specifications TableValue of the data\u2022The data presented in this article gives new insight into the activities of thermostable enzymes not published before.\u2022The data represents a rationale behind why TKs and TAms were selected for the one-pot reaction.\u2022l-gluco-heptulose, is a pharmaceutically-relevant compound.Product of one-pot reaction, 1l-Arabinose is a major monosaccharide of sugar beet pulp (SBP), a by-product of sucrose extraction which is currently produced and sold as a low value animal feed l-gluco-heptulose, a high value, pharmaceutically relevant compound from l-arabinose using a two-step thermostable enzyme cascade. A thermostable TK catalyzed the synthesis of l-gluco-heptulose from l-arabinose and \u03b2-hydroxypyruvate (HPA) in which the latter was produced in situ from l-serine and \u03b1-ketoglutaric acid using a thermostable TAm.l-arabinose via the Seliwanoff assay l-gluco-heptulose.22.1E.coli BL21 DE3. Cell lysates were used to determine activity towards l-arabinose using the colorimetric assay, Seliwanoff assay. The Seliwanoff assay distinguishes between ketoses and aldoses using 6\u2009M HCl and resorcinol (Seliwanoff\u05f3s reagent) l-arabinose, Seliwanoff reagent was added to the reaction and heated at 100\u2009\u00b0C. Colour formation due to the presence of the ketose, l-gluco-heptulose, was observed within 15\u2009min equipped with a Dionex AminopacTM PA1 anion exchange column 4\u00d7250\u2009mm2 fitted with a Dionex AminopacTM PA1 guard column 4\u00d750\u2009mm2, an electrochemical detector system, and an eluent generator with a KOH 500 cartridge. The elution times of each compound can be observed in Dgeo. Standard calibration curves of l-gluco-heptulose and HPA were used for quantification purposes (Quantitative analysis of purposes , Fig. 5.2.4>TKDgeoDNA sequences were retrieved from the NCBI database >TKDgeoATGAGTCCCGAACAGCAGGCCGTGCGTCAGGATGTCGATCAGCTGAGCATCAACACCATCAGGACGCTTGCCATCGATGCGGTGCAGCGGGCCAACAGTGGCCACCCCGGCGCGCCGCTCGGCATGGCCCCGATGGGCTACGTGCTGTGGCAGCGCTTCCTGCGCCACAATCCGAAACATCCCGAGTGGCCGGGCCGCGACCGCTTCGTGCTGTCGGCAGGGCACGCCAGCATGCTGATCTACTCGCTGCTGCACCTCACCGGCTACGACCTGCCGCTGGAGGACATCAAGAACTTCCGCCAGTGGGGCAGCAAGACGCCTGGGCATCCCGAGTTCTTCCACACCCCAGGCCTAGACGCCACCACCGGCCCGCTCGGTCAGGGTGCGGCGATGACGGTGGGCATGGCGATGGCCGAAGCGCACCTCGCCGCACGCTACAACCGCCCCGGCTTCAAGGTCTTTGACAACTACACCTACGCGATCTTGGGGGACGGCGACCTGCAAGAAGGCGTCAACCACGAGGCCGCGTCGCTGGCAGGGCACCTCAAGCTGGGCAAGCTGATCTGGCTGCACGACGACAACCAGGTGCAGCTGGACACCGCCACGTTCAAGGCGGCCAACGAGGATACTGCGGAGCGTTACCGCGCCTACGGCTGGGAAGTTCTGCGTGTGCAAGACGGCAACAATCTCACGGAGATCGAGAACGCGATCCGCCAGGCACGGATGAACACCGAGCAGCCCACCCTGATCCAGGTTCGCACGGTGATCGGCTTCGGCAGTCCCCGTGCGGGCACCAGCAAGGCGCACGGCGAGCCGCTGGGCGAGGAAGGCGTGCAGGAGACCAAGGCGGCCCTGGGCTGGGACTACCCGCCCTTCACGGTGCCCGACGAGGTCAAGGCGCATATGGACGCGACTGAGCGTGGCGCGGAGTGGGAGGCCGACTGGAACGCGCTGATGGAGCGCTACCGTGCCGAGTACCCCGATCTCGCGGCGGAGGTTGACGCGCTGCTGGCGCGCGAACTGCCCGCCAATCTCGCCGAAGTGCTCCCCTCCTACGAAGTGGGCAGCAAGGCCATCGCCACCCGCAACGCGAGCGGTGAAGTCATCAATGCGCTGGCGCAGGTGGTGCCGGGCCTGATGGGGGGCAGTGCGGATCTCTCCGGCAGCACCAAAACCACCATCAAGGACGGCGGCGAGTTTCTGCCAGGAAACTACGGGGGCCGCAACGTCTACTTTGGCGTCCGCGAGTTTGGGATGGCCGCAGCGGGCAATGGCCTTTCGCTCTACGGAGGTGTTCGGCCCCTGGTGGGGACCTTCCTGGTGTTTGCGGACTACCTCAAGCCCGCCTTCCGCCTCTCCGCCCTTCAGTTCCAGCCGGTTACCTATGTCCTGACCCATGACTCCATTGGCCTGGGCGAAGACGGCCCAACCCACCAGCCTATTGAGCAGCTCGCCATGCTGCGCGCCGTGCCGGGTGCCCACGTGATTCGCCCCGCCGACGCCAACGAGACGGCGGCGGCCTGGCAGATGGCGCTGGAGTACGACAAGGGACCAACCGCTCTGGCCCTCTCCCGCCAGGATCTCCCAGTGCTGCCCCGCAACCACGCGGGCGTGAAGAAGGGCGCCTACGTGGTTCGCGACGCCGAAGGGGGGCCGGCACAGATCATCTTGATCGCCACCGGCTCGGAGGTGAGCCTGGCGCTGGATGCTGCCCAAGCGCTGGCGGAGGAAGGCATCCAGGCTCGGGTCGTCTCAATGCCCTGCATGGAAGTCTTCCGCCAGCAGGACGCCAGTTATCGGGACAGCGTGCTCACCCCCGGCGTGAAACGCGTGGCCATCGAGGCTGCCAGCCCGCTCCCCTGGTATGAGTGGGTGGGCTTTGACGGCGCGGTGATCGGAATGACCACCTTTGGCGCCTCGGCCCCAGCCAAAGTCCTCTTTGAGAAATTCGGCTTCAACGTGCCGAACGTCGTGCAGGTCGTCAAGGGCGTTTTGCAGAGGTGA>TKDradMSPEQQAVRQDVDQLSINTIRTLAIDAVQRANSGHPGAPLGMAPMGYVLWQRFLRHNPKHPEWPGRDRFVLSAGHASMLIYSLLHLTGYDLPLEDIKNFRQWGSKTPGHPEFFHTPGLDATTGPLGQGAAMTVGMAMAEAHLAARYNRPGFKVFDNYTYAILGDGDLQEGVNHEAASLAGHLKLGKLIWLHDDNQVQLDTATFKAANEDTAERYRAYGWEVLRVQDGNNLTEIENAIRQARMNTEQPTLIQVRTVIGFGSPRAGTSKAHGEPLGEEGVQETKAALGWDYPPFTVPDEVKAHMDATERGAEWEADWNALMERYRAEYPDLAAEVDALLARELPANLAEVLPSYEVGSKAIATRNASGEVINALAQVVPGLMGGSADLSGSTKTTIKDGGEFLPGNYGGRNVYFGVREFGMAAAGNGLSLYGGVRPLVGTFLVFADYLKPAFRLSALQFQPVTYVLTHDSIGLGEDGPTHQPIEQLAMLRAVPGAHVIRPADANETAAAWQMALEYDKGPTALALSRQDLPVLPRNHAGVKKGAYVVRDAEGGPAQIILIATGSEVSLALDAAQALAEEGIQARVVSMPCMEVFRQQDASYRDSVLTPGVKRVAIEAASPLPWYEWVGFDGAVIGMTTFGASAPAKVLFEKFGFNVPNVVQVVKGVLQRATGACAGACCAGAGCGTTTCCCAAAACGTGGCGCGGCTGAGTGTGAACACCATTCGCACGCTCGCCATTGACGCGGTGCAGGCCGCCAACTCGGGCCACCCCGGTGCGCCGCTGGGCATGGCCCCGATGGGCTACGTGCTGTGGCACAAGTTCCTGCGCCACAACCCCGCGCACCCTGAGTGGCCGGGCCGCGACCGCTTCGTGCTGTCGGCGGGGCACGCCTCCATGCTGATCTACAGCCTGCTGCACCTGACCGGCTACCAGGAAATGACGCTCGACGACCTGCGCCACTTCCGGCAGTGGGGCTACCACACCCCCGGCCACCCCGAGTTTTTCCACACCAAGGGTCTGGACGCGACCACCGGCCCGCTTGGGCAGGGCGCGGCGATGACGGTGGGCATGGCGATGGCCGAAGCACACCTCGCCGCCCGCTACAACCGCGAAGGCTTTCCGATTTTCGACAACCGCACCTACGCCATCATGGGCGACGGCGATCTGCAAGAAGGCATCAACCACGAAGCCGCCGCGCTCGCCGGGCACCTGAAACTCGGCAAGCTGATCTGGCTGCACGACGACAACCACATCCAGCTCGACACGCCCACGAACAAGGCCGAGTCCGAGGACACCGCCGCCCGCTTCCGCGCCTATGGCTGGAACGTGCTGAAGGTGGAAGACGGCGACAATCTGGACGAAATTGAAAAGGCGATTGCCGAGGCCCGCAGCCAAAGCGAGCGGCCCACGCTGATTCAGGTGCGCACCATCATCGGCTTCGGCAGCCCGCGCGCCGGCACGAGCAAGGCGCACGGCGAGCCGCTCGGCGAAGAGGGCGTGGCCGAGACGAAGAAAGCGTTGGGCTGGGAGTACCCCGCTTTTACCGTGCCCGACGAAGTGGCTGCGCACATGAACGCTCGCGCTAAGGGTGCTCAACTCGAAGCCGACTGGGAAAAACTGATGGCCGACTACCGCACCGCGCACCCCGACCTCGGCAAGGAAGTGGACGCGCTGCTCGCCCGTGAACTGCCCGCCAACCTCGCCGACCTGCTGCCCAAGTACGAAGTCGGCGGCAAGGCGGCGGCCACCCGCAACGCGAGCGGCGAAGTCATCAACGCGCTGGCGAAGGTGCTTCCCGGTTTGATGGGCGGCAGCGCGGACCTCTCGGGCTCGACCAAGACCACCATCAAGGACGGCGGCGAGATGGAAGCGGGCACCATGGGCGGGCGCAACGTGCTGTTCGGCGTGCGCGAGTTCGGCATGAGCGCCGCGGGCAATGGCCTGAGCCTCTACGGCGGCCTGCACCCGATGGTAGGCACCTTCCTGGTATTCGCCGACTACCTCAAGCCGGCTTTCCGCCTCTCGGCGCTGCAAATGCAGCCGGTGACTTACGTGCTGACCCACGACTCCATCGGTCTGGGCGAAGACGGGCCGACCCACCAGCCGGTGGACCAGCTCGCCATGCTGCGAGCGGTGCCGGGCGCCCACGTCATTCGCCCCGCCGACGCCAACGAAACCGCCGCCGCGTGGCTGATGGCGCTGGAATACGACAAGGGCCCCACCGCGCTCGCCCTCTCGCGCCAGGATCTGCCGATTCTGCCCGCCAACATCGAAGGCGTGAAGAAGGGCGCGTATGTCCTCCGAGATGTGGACGGTGCCGATGGTCAGGGGGCTCAAGTCATCCTGATC>TKDradGCCAGCGGCTCGGAAGTCGCCCTGGCCCTGAGCAGCGCCGAGCGGCTGGCCGAAGAGGGCGTGCAGGCCCGCGTGGTGTCCATGCCGTGCATGGAGGTCTTTCGCCAGCAGGAGCAGAGCTACCGCGACAGCGTGCTGACCCCCGGCGTGAAGCGCGTCGCCATCGAGGCCGCCAGCCCGCAGCCCTGGTACGAGTGGACGCTCGGCGGCCCAGTCATCGGCATGACGACCTTCGGTGCGTCGGCCCCGGCCAAGGTGCTGTTTGAGAAGTTCGGCTTCAGCGTGGAAAACGTGGTGAAGGTGGTCCACTCCGTGCTGTAA>pQR1743MTDQSVSQNVARLSVNTIRTLAIDAVQAANSGHPGAPLGMAPMGYVLWHKFLRHNPAHPEWPGRDRFVLSAGHASMLIYSLLHLTGYQEMTLDDLRHFRQWGYHTPGHPEFFHTKGLDATTGPLGQGAAMTVGMAMAEAHLAARYNREGFPIFDNRTYAIMGDGDLQEGINHEAAALAGHLKLGKLIWLHDDNHIQLDTPTNKAESEDTAARFRAYGWNVLKVEDGDNLDEIEKAIAEARSQSERPTLIQVRTIIGFGSPRAGTSKAHGEPLGEEGVAETKKALGWEYPAFTVPDEVAAHMNARAKGAQLEADWEKLMADYRTAHPDLGKEVDALLARELPANLADLLPKYEVGGKAAATRNASGEVINALAKVLPGLMGGSADLSGSTKTTIKDGGEMEAGTMGGRNVLFGVREFGMSAAGNGLSLYGGLHPMVGTFLVFADYLKPAFRLSALQMQPVTYVLTHDSIGLGEDGPTHQPVDQLAMLRAVPGAHVIRPADANETAAAWLMALEYDKGPTALALSRQDLPILPANIEGVKKGAYVLRDVDGADGQGAQVILIASGSEVALALSSAERLAEEGVQARVVSMPCMEVFRQQEQSYRDSVLTPGVKRVAIEAASPQPWYEWTLGGPVIGMTTFGASAPAKVLFEKFGFSVENVVKVVHSVL> pQR1743ATGGCGCATTCGATCGAAGAATTAGCGATTACCACCATTCGAACGCTGTCGATTGACGCGATCGAAAAAGCGAAATCCGGGCACCCGGGCATGCCGATGGGCGCGGCCCCGATGGCGTATACGCTCTGGACGAAATTTATGAACCATAATCCAGCGAATCCCAACTGGTTTAACCGCGACCGGTTTGTTTTGTCCGCTGGGCACGGGTCGATGCTGCTTTACAGCCTGCTTCATCTAAGCGGCTACGATGTCACGATGGACGACTTGAAACAGTTCCGCCAATGGGGAAGCAAAACGCCGGGCCATCCGGAATACGGCCATACGCCAGGGGTGGAGGCAACGACCGGCCCGCTCGGCCAAGGGATTGCGATGGCGGTCGGCATGGCGATGGCGGAACGGCATTTGGCGGCTGCATACAATCGCGATGGATTTGACATTATCAACCACTACACGTATGCGATTTGCGGCGACGGCGATTTGATGGAAGGAGTGGCGAGCGAAGCGGCGTCACTCGCCGGCCACTTGAAGCTCGGCCGTCTGATCGTCCTGTATGACTCGAACGACATTTCGCTGGACGGCGAGCTCAACTTGTCGTTTTCGGAAAACGTCGCCCAACGTTTCCAAGCGTACGGCTGGCAATATTTGCGCGTTGAGGACGGCAACAATATTGAAGAAATCGCCAAAGCGCTCGAGGAGGCGCGGACGGACCTCAGCCGGCCGACGCTCATTGAAGTAAAAACGACGATTGGCTACGGCGCGCCAAATAAAGCGGGCACGTCCGGCGTCCACGGCGCTCCGCTCGGCGCCCAAGAGGCGAAGTTGACGAAAGAAGCGTACCGCTGGACGTTTTCCGAAGATTTCTACGTGCCGGATGAAGTGTACGCTCATTTCCGGGAAACGGTGCAAGAAGCCGGAGCGAGAAAAGAAGCGGAGTGGAATGAGCGCTTCGTTGCTTACGAGCGGGCGCATCCGGAATTGGCCGCCGAGCTGAAGCAGGCGATTGAAGGGAAGCTTCCGGATGGCTGGGAAACATCGCTGCCGGTGTATGAAGCGGGCAAAAGCTTGGCGACCCGCTCATCGTCCGGGGAAGTGATCAACGCCATCGCCAAAGCGGTGCCGCATTGTTTGGCGGTTCGGCGGACTTGGCAAGCTCGAATAAAACGCTTATCAAAGGCGGCGGCAACTTCTTGCCGGACAGCTACGAAGGGCGCAACATTTGGTTTGGCGTGCGCGAGTTTGCCATGGGCGCGGCGTTAAACGGCATGGCGCTTCACGGCGGGTTGAAAGTGTTCGGCGGCACGTTCTTCGTGTTCTCCGACTACTTGCGCCCGGCGATTCGGCTGGCGGCGCTCATGGGCTTGCCGGTGACGTACGTGCTGACGCACGACAGCATCGCCGTCGGGGAAGACGGCCCGACGCATGAGCCGGTCGAGCATCTCGCTTCACTTCGGGCGATGCCGAACTTGTCAGTCATCCGGCCGGCTGACGCAAACGAAACGGCGGCCGCCTGGCGGCTGGCGCTCGAGTCGACGAACAAGCCGACTGCGCTCGTCTTGACGCGTCAAGATGTGCCGACATTGCCGACAACCGCTCAGTTGGCGTATGAAGGCGTGAAAAAAGGCGCGTACGTCGTTTCACCGGCGAAAAACGGCGCTCCGGAGGCGCTGTTGTTGGCGACTGGCTCGGAAGTCGGTCTGGCCGTCAAAGCGCAAGAAGCGCTCGCCGCTGAGGGCATCCATGTCTCCGTCATCAGCATGCCATCGTGGGACCGCTTCGAAGCGCAGCCAAAATCGTACCGCGATGAAGTGCTGCCGCCGGCCGTGACGAAGCGGCTCGCCATTGAAATGGGCGCCTCGCTCGGTTGGGAGCGCTACGTCGGCGCCGAGGGCGACATTTTGGCCATCGACCGATTCGGTGCTTCCGCTCCGGGAGAGAAAATCATGGCCGAGTATGGCTTTACGGTTGACAACGTCGTCCGCCGCACAAAAGCGCTGCTCGGCAAGTAA>pQR1744MAHSIEELAITTIRTLSIDAIEKAKSGHPGMPMGAAPMAYTLWTKFMNHNPANPNWFNRDRFVLSAGHGSMLLYSLLHLSGYDVTMDDLKQFRQWGSKTPGHPEYGHTPGVEATTGPLGQGIAMAVGMAMAERHLAAAYNRDGFDIINHYTYAICGDGDLMEGVASEAASLAGHLKLGRLIVLYDSNDISLDGELNLSFSENVAQRFQAYGWQYLRVEDGNNIEEIAKALEEARTDLSRPTLIEVKTTIGYGAPNKAGTSGVHGAPLGAQEAKLTKEAYRWTFSEDFYVPDEVYAHFRETVQEAGARKEAEWNERFVAYERAHPELAAELKQAIEGKLPDGWETSLPVYEAGKSLATRSSSGEVINAIAKAVPQLFGGSADLASSNKTLIKGGGNFLPDSYEGRNIWFGVREFAMGAALNGMALHGGLKVFGGTFFVFSDYLRPAIRLAALMGLPVTYVLTHDSIAVGEDGPTHEPVEHLASLRAMPNLSVIRPADANETAAAWRLALESTNKPTALVLTRQDVPTLPTTAQLAYEGVKKGAYVVSPAKNGAPEALLLATGSEVGLAVKAQEALAAEGIHVSVISMPSWDRFEAQPKSYRDEVLPPAVTKRLAIEMGASLGWERYVGAEGDILAIDRFGASAPGEKIMAEYGFTVDNVVRRTKALLGK>pQR1744ATGAACACCGGCACCCCAAAGACCCTGGACTGGTCTGATCTCGATAGACGTACCGTAGACGTGGTTCGTGCCCTGGCGATGGACGCGGTCGAAGAAGCGGGATCCGGGCACCCTGGAACCGCGATGAGTCTGGCGCCTGTGGCCTACCTGCTCTTCCAGAAGGTGATGCGGCACGATCCGACAGATCCGAAGTGGATCGGCCGCGACCGCTTCGTCCTGTCCTGCGGGCACTCCAGCCTCACGCTCTACATCCAGCTCTACCTGGCTGGCTACGGGCTGAGCCTGAACGACATCAAGCGGCTGCGCCAGTGGGGCAGCCTCACCCCGGGCCACCCCGAATACGGGCACACCGCCGGGGTGGAAACCACCACCGGCCCCTTGGGGCAGGGCATCGGCAACGCGGTCGGCATGGCCATGGCCGCCCGCCGGGAGCGGGGCCTGTTCGACCCGGACACCCCGATCGGGGAAAGCCCGTTCGACCACTACATCTACGTCCTGTGCTCTGACGGCGACGTCCAGGAGGGCATCAGCCACGAAGTAAGTGCCCTCGCCGGCACGCAGAAGCTCGGCAACCTCATCGTCATCTGGGACGACAACCGCATCTCCATCGAAGACGACACCCAGATCGCATTCACCGAAGACGTCGTCGCCCGCTACGCCGCCTACGGCTGGCACGTCCAAGAGGTCGAGTGGGTCGGCGAGGACGGCTCCTACCACGAAGACGTGGCGGCGCTGTACGACGCGATCCGGGCCGCCCAGGCGGAGACGGAACGTCCCTCTTTCATCCGGCTGCGCACCATCATCGGCTGGCCGTCCCCGAACAAGCAGAACACGGGGGCGATCCACGGCGCCGCGCTGGGGGCTGAAGAGGTCGCCGCCACCAAGCGGGTGCTGGGCCTCAACCCTGAGGCGCAGTTCGACGTGCCCAACGAGCTGCTGGAGCACGCCCGGGGCGTGGTGGCGCGGGGCCGCGCCGCCCGCCAGGAATGGGAGGCCTTGTTCGCCAAGTGGCGGGCCAACGCGGGCGAGCGTGCCGAACTGTTCGACCGGCTGATGGCAGGCTCGCTCCCGGACGGTTGGGAGAAGGCGATCCCGACCTTCGAGCCCAGCGCTAAGGGCATGGCCACCCGGAAAGCGTCCGGTGAGGTGCTGAGCGCGATCGCCCCGGTGCTGCCGGAGCTGTGGGGCGGCTCGGCGGACTTGGCCGGATCCAACAACACCACGCCTAAGGGCGAGCCGTCGTTCATCCCCGAGGAGCGGTCCACGAAGGCGTTCTCCGGCCACCGCTACGGCCGGGTGCTGCACTTCGGGATCCGTGAACACGGCATGGGGGCGATCCTCAACGGGATCGCGCTGCACGGCCCCACCCGCCCCTACGGTGGCACCTTCCTCGTGTTCAGCGACTACATGCGGCCGTCGGTGCGGCTGGCTGCCCTGATGAAGCTGCCGGTCACGTACGTGTGGACCCACGACTCGATCGGTCTGGGCGAAGACGGACCCACCCACCAGCCGGTGGAGCACCTGTGGTCGCTGCGCGCCATCCCCGGCCTGGCGGTGGTGCGTCCCGCCGACGCCAACGAGACGGCAGTGGCCTGGCGCACCATCCTGGAACGCAATGACGGCCCGGTGGCGCTCGCGCTGACCCGGCAGTCGGTTCCGGTTCTGGACCGCTCCGAGCTCGCCTCTGCGGAGCTGGTCTCCCGCGGCGGGTACATCCTGGCCGAAGCCAGCAACGGCCGTCCGGAGGCGATCATCATCGCCACCGGAAGTGAGGTGCAGATCGCGTTGGAGGCGCGTTCCCGCCTGGAGGAGTCGGGTACTCCTACCCGTGTGGTGTCGATGCCGTGCCTGGAGTGGTTCAACGAGCAGGACGACGCCTACCGCCAGCAGGTGCTTCCACCGTCGGTCCGGGTCCGGGTCTCCGTGGAAGCCGGGGTCGCCTTGGGCTGGCGCGAGCTGGTGGGCGAGTATGGCGAGTCGGTGAGTCTGGAACACTTCGGCGCTTCGGCTCCGTACGCGACTCTCTACGAGCAGTTCGGGCTCACCGCCGACCGGGTAGTGGCAGCCGTACACTCCAGCGCTGCCAAGCTCGGCGGTGACCGTGGATCAACGACCGGCAACTGA>pQR1745MNTGTPKTLDWSDLDRRTVDVVRALAMDAVEEAGSGHPGTAMSLAPVAYLLFQKVMRHDPTDPKWIGRDRFVLSCGHSSLTLYIQLYLAGYGLSLNDIKRLRQWGSLTPGHPEYGHTAGVETTTGPLGQGIGNAVGMAMAARRERGLFDPDTPIGESPFDHYIYVLCSDGDVQEGISHEVSALAGTQKLGNLIVIWDDNRISIEDDTQIAFTEDVVARYAAYGWHVQEVEWVGEDGSYHEDVAALYDAIRAAQAETERPSFIRLRTIIGWPSPNKQNTGAIHGAALGAEEVAATKRVLGLNPEAQFDVPNELLEHARGVVARGRAARQEWEALFAKWRANAGERAELFDRLMAGSLPDGWEKAIPTFEPSAKGMATRKASGEVLSAIAPVLPELWGGSADLAGSNNTTPKGEPSFIPEERSTKAFSGHRYGRVLHFGIREHGMGAILNGIALHGPTRPYGGTFLVFSDYMRPSVRLAALMKLPVTYVWTHDSIGLGEDGPTHQPVEHLWSLRAIPGLAVVRPADANETAVAWRTILERNDGPVALALTRQSVPVLDRSELASAELVSRGGYILAEASNGRPEAIIIATGSEVQIALEARSRLEESGTPTRVVSMPCLEWFNEQDDAYRQQVLPPSVRVRVSVEAGVALGWRELVGEYGESVSLEHFGASAPYATLYEQFGLTADRVVAAVHSSAAKLGGDRGSTTGNATGGAAAGGTTTCCCTATGAAAAACTTCCAGAAAGCGAACTCAAAGAGTTGAAAGAACTCGGAAGGCTCTGCCGTGGCGACATACTGAAAATGACCTACATAGCTAACTCAGGCCATCCTGGAGGATCCATGTCTTCGATCGATCTTTATCTTACCGTCTTCAAGTACGCAAAACTCAGACCCGTCGATGATCCTGCAAGAGACAGAATCGTGATCAGCCATGGACACACTTCTCCGGGTGTCTACGCAGCTATGGCTCGTTTGGGGTTTGTCGATCTCGATGAAGTCCTCGCAGGATTCAGACACCCCGCTTCCGTTTTTGAAGGACACGTGACCCGAGGTGTTGGGATCATCGACTGGACAACCGGAAACCTCGGTCAGGGTCTTTCAGCCGGACTCGGTTTTGCCCTCGCATCCAGGTTCACAGGAAAAGATTACCACGTCTTTGTTCTCATGAGTGACGCAGAACAGGCAAAAGGACAGGTGGCGGAGGCAAGAAGAGTGGCGAAAAAGTACGGTGTCACGAATCTCACAGTGATCATCGACTACAACGACGCCCAGATCAGTGGCCGTGCCAGAGACGTCATGCCCGTGAACATAAAGGAAAACTACTTAGCGGACGGCTGGAGGGTCATCGAGATCGATGGGCACGACTACGAACAGATCTATCTCGCACTGAAAGAAGCGGTAGAAGACGAAC> pQR1745TGAATCCCGTTGCCATAATCGCCAAAACGGTCATGGGAAAAGGCGTATCTTTCATGGAAAACGAGGTGAAATACCACGGAAAGCCTTTGAACAGAGAAGAACTCGAAAAAGCCCTCGCGGAACTCGGAATTGAAAACGATGTTGATGTGTACATCGAAAAAAGAAAACAACTTCCAGTGGAAAAACACAAGAAAGTCTACAAAACTTACCCGATCAAGATCGACACGGGAGAGCCCATCACCTACACCTCACCCACTGACAACAGAAGCGCATTCGGAAAAGCTATTCTGGATCTGGTGAAGAAGAACGTAAACAATCCAGAAACCACACCCATCGTCGCTGTGGACTGCGACCTGAAGGGATCGGTCAAACTCGACCTGCTCGACAAAGAGTTCCCTGAGAGACTCCTGGAAGTGGGCGTTCAGGAACACAACGCTGCCGCTATGGCGGGGGCACTCTCCGCAGAGGGTGTGATCACGTTCTTCGCTGATTTTGGTGTTTTTGGAATTTCTGAAACCTACAACCAGCACAGGCTGAACGCCATCAATGGAACGAACCTCAAAGTCGTTGTCACACACTGCGGACTCAACGTGGGAGAGGACGGAAAAACTCATCACGGACTCGACTACGTTTCCGGGCCGATGAACTGGTACGGTTTCAAAGTGATCGTCCCTGGTGATCCCAACCAGACGGATAGAGTTGTCAGATACGCCGCGAAGGAATACGGGAACTTCGTAATCGCCATGGGAAGATCTAAGCTTCCCATCATCCTCGATGAAAACGGGAAACCTTTCTTCGGAGAGGGTTACACCTTCGAATATGGGAAGATCGATGTCGTTAGAAAAGGTGACGACGCGGTGATCATAACTTACGGTTCTACACTCTGTGAAGCCGTAAATGCCGCAGACGAACTCAAGAAAGAAGGAGTAAACGTAGCCGTTCTGAATGTCTCCTGTCCGGTGGATCTCGATATAGAGACCTTGAAGATGGTCGATGGAAAACCCGTTCTCGTTGTGGAGGATCACAACGTTTTCACAGGACTTGGAAGCTTCCTTGGAACCACCCTTCTTGAAAACGGCATCATCCCGAAGAAATACGTGAGAGTAGGTGTTCCAGAATTCGCCGTGTCCGGCAGTTACACGATGCTCTACAAACTCTACGGCCTGGATAAAGATGGAATAATTTCCAGACTCAGAGAGATGCTCTAAMERFPYEKLPESELKELKELGRLCRGDILKMTYIANSGHPGGSMSSIDLYLTVFKYAKLRPVDDPARDRIVISHGHTSPGVYAAMARLGFVDLDEVLAGFRHPASVFEGHVTRGVGIIDWTTGNLGQGLSAGLGFALASRFTGKDYHVFVLMSDAEQAKGQVAEARRVAKKYGVTNLTVIIDYNDAQISGRARDVMPVNIKENYLADGWRVIEIDGHDYEQIYLALKEAVEDELNPVAIIAKTVMGKGVSFMENEVKYHGKPLNREELEKALAELGIENDVDVYIEKRKQLPVEKHKKVYKTYPIKIDTGEPITYTSPTDNRSAFGKAILDLVKKNVNNPETTPIVAVDCDLKGSVKLDLDKEFPERLLEVGVQEHNAAAMAGALSAEGVITFFADFGVFGISETYNQHRLNAINGTNLKVVVTHCGLNVGEDGKTHHGLDYVSGPMNWYGFKVIVPGDPNQTDRVVRYAAKEYGNFVIAMGRSKLPIILDENGKPFFGEGYTFEYGKIDVVRKGDDAVIITYGSTLCEAVNAADELKKEGVNVAVLNVSCPVDLDIETLKMVDGKPVLVVEDHNVFTGLGSFLGTTLLENGIIPKKYVRVGVPEFAVSGSYTMLYKLYGLDKDGIISRLREML2.5>TAmGsteDNA sequences were retrieved from the NCBI database >TAmGsteATGAAATTGGCAAAACGGGTGGCGTCGCTGACGCCATCGGCGACTTTGGCCATTACGGAGAAAGCAAAAGAACTAAAAGCGGCCGGGCATGACGTGATTGGTCTCGGAGCTGGCGAACCGGATTTCAACACGCCACAGCACATTCTTGATGCCGCCATCAAGGCAATGAACGAAGGACATACGAAATATACACCATCGGGCGGTTTGCCGGCGTTAAAGGAGGAAATTATAAAAAAATTCGCCCGCGACCAAGGCTTGGATTATGAGCCGGCTGAAGTGATTGTATGCGTCGGAGCGAAGCACGCCCTTTACACGCTGTTCCAAGTATTGCTCGATGAAGGCGACGAAGTGATCATTCCGACGCCATACTGGGTGAGCTATCCGGAACAAGTGAAACTGGCGGGCGGTGTTCCGGTTTACGTCGAAGGGCTTGAACAAAATCATTTTAAAATTACGCCGGAGCAGCTGAAACAGGCAATCACGCCGCGGACGAAAGCGGTTATCATCAACTCGCCGAGCAACCCGACTGGCATGATTTATACAGCCGAAGAGTTGAAGGCGCTTGGTGAGGTGTGCCTAGCGCATGGTGTATTGATCGTGTCAGATGAAATTTACGAAAAATTGACTTACGGCGGGGCGAAGCATGTGTCCATCGCTGAGTTGTCGCCGGAGCTGAAGGCGCAGACAGTCATCATTAACGGCGTGTCAAAGTCGCATTCGATGACGGGCTGGCGCATTGGTTATGCGGCGGGGCCGAAAGATATTATTAAGGCAATGACAGATTTGGCGAGCCACAGCACGTCCAACCCGACGTCAATCGCCCAATACGCGGCCATCGCTGCTTACAGCGGGCCGCAGGAGCCGGTCGAACAAATGCGCCAAGCGTTTGAACAACGGCTCAATATCATTTACGACAAGCTCGTGCAAATTCCAGGATTCACGTGCGTTAAGCCACAAGGGGCGTTTTATTTGTTCCCGAACGCCCGCGAAGCGGCTGCAATGGCCGGCTGCCGCACGGTCGACGAGTTCGTCGCTGCCTTGTTGGAGGAAGCGAAAGTCGCGCTTGTGCCCGGCTCTGGGTTTGGAGCGCCGGATAACGTTCGCTTGTCATACGCGACATCGCTCGATGCACTGGAAACCGCCGTGGAACGCATCCACCGGTTTATGGAAGCGCGCGCTTAA>TAmDGeoMKLAKRVASLTPSATLAITEKAKELKAAGHDVIGLGAGEPDFNTPQHILDAAIKAMNEGHTKYTPSGGLPALKEEIIKKFARDQGLDYEPAEVIVCVGAKHALYTLFQVLLDEGDEVIIPTPYWVSYPEQVKLAGGVPVYVEGLEQNHFKITPEQLKQAITPRTKAVIINSPSNPTGMITAEELKALGEVCLAHGVLIVSDEIYEKLTYGGAKHVSIAELSPELKAQTVIINGVSKSHSMTGWRIGYAAGPKDIIKAMTDLASHSTSNPTSIAQYAAIAAYSGPQEPVEQMRQAFEQRLNIIYDKLVQIPGFTCVKPQGAFYLFPNAREAAAMAGCRTVDEFVAALLEEAKVALVPGSGFGAPDNVRLSYATSLDALETAVERIHRFMEARA>TAmDGeoATGTTCGAGGACACGCCCGCACCCTTTCCACCGCACATTCTGCTGACGCCCGGTCCGACACCGATTCACCCCCGGGCCCAGCGGGCGCTGCTGCGCGAGATGCTGGGGCACATGGACCCTGAGGTGTTCGCCCTGAACCGCGAGATCCAGGCGGACTTGCGGGTGATGTACGGGACGGGGCCCCAGACCTTTACGGCGCTGCTGGCGGGCACCGGGAGCCTGGGCATGGAGGCGGGCTTCGCCAACTTGGTGGAGAGGGGAGACGACGTGCTGATCTGCGTCAATGGTGCCTTTGGTCAGCGCATGGCCGAGATGGCGGCGCGCTACGGTGCGAATGTACGGCGGGTGACCGCGCCGCTGGGCGAGCCGATCGACCCGGCCGACGTGGCTGCGCGGTTGAGCGGCGCGCGGCTGGTGGCGGTGGTGCATGGGGAGACGAGCACGGGTGTGCTCAATCCGCTTCCGGAGATTGCCGAGGCCGTGCGCGGGAGCGGGGCATTGCTGGCCGTGGACGCCGTGACGACCGCCGGGATGGAACCCTTCCATATGGCGGACTGGGGCGTGGACTACGCCTATACCGGCGCGCAGAAGTGCCTCTCGGCACCGCCCGGCCTGGCCCCGGTGGCGATCAGCGACCGTGCTCTCGCTCGCCACGCGGCCCGCCGCACGCCCACGCCGCTGTGGTACTGCGATTTTGAGGGCCTGCGCGACTACTGGGACCGGCACAGCTACCACCACACGGTCCCGGTGAATCTGCACTACGCCTTCCACGCCGCCCTGCGCGCCGCACTCGAAGAAGGCCTCCAAGCCCGGCAGGCCCGCGTGCGCGACCTTGGCCAGGCGGTGCTGGCGGCCCTGACGCCGCTGGGCTTCACGCCGTATGTGGCCGATCCCGCCGCGCGGCTGCCCACCGTCTTGGCCCTGCGTCTTCCTCCCGGCTTCGACGACGCGGGCGTTCGCCAGGCCCTACGGGAACGCGGGATCAGCGTGACCGGCGGCCTGGGACCGACGGCAGGGCTGATCTGGCGTCTGGGCCTGATGGGGGAAGCGGCTCGCCCCGCGCCCTACCGCGCGCTGATGCTCGCCCTGGAAGACCTGCTGGGCGAGCGGGGCTTGGTGGCGCGCTTCGAGGAGGCGCTGGGCGTCGCGGCCTGA>pQR1746MFEDTPAPFPPHILLTPGPTPIHPRAQRALLREMLGHMDPEVFALNREIQADLRVMYGTGPQTFTALLAGTGSLGMEAGFANLVERGDDVLICVNGAFGQRMAEMAARYGANVRRVTAPLGEPIDPADVAARLSGARLVAVVHGETSTGVLNPLPEIAEAVRGSGALLAVDAVTTAGMEPFHMADWGVDYAYTGAQKCLSAPPGLAPVAISDRALARHAARRTPTPLWYCDFEGLRDYWDRHSYHHTVPVNLHYAFHAALRAALEEGLQARQARVRDLGQAVLAALTPLGFTPYVADPAARLPTVLALRLPPGFDDAGVRQALRERGISVTGGLGPTAGLIWRLGLMGEAARPAPYRALMLALEDLLGERGLVARFEEALGVAA>pQR1746ATGACCTCTCCTTTCCGCCTCTCCGCCCGCGCCCAGAGCCTCAAGCCGTCTGCGACAGTGGCGGTCACGTCCCGCGCCCTGGAACTCCAGCGTCAGGGCCTGGACGTGATTTCCATGAGCGTGGGCGAGCCGGATTTCGACACGCCGCCACATGTCAAGGCCGCCGGCATCGCCGCCATCGAGGAAGGCAAGACCAAATACACCCCGGTCAGCGGCATTCCCGAACTGCGCGAGGCCATCAGCGCCAAGTTTCGGCGCGAAAACGGCCTGGACTACGCGCCGAACGCCGTGACGGTAACGAGCGGCGGTAAACAGGCGCTGTTCAACGCCTTTTTCGCGTTGCTGAACCCCGGCGACGAGGTGCTGATTCCCGCGCCCCACTGGGTCAGCTACCCCGAAATGGTCGCGCTGACCGGCGCGGTGCCGGTAACCGTACCCACTACGCCGCAGCAGGGCTTTCAACTCGACCCGGACGCCCTCGCCGCCGCCATCACGCCGCGCACCCGCATGGTGATTCTCAACAGCCCCGGCAACCCGACGGGCGCGGTGTTTCCGCCGGAAACCTTGCGGGCGGTGGCCGACCTCGCCACGCAGCACGGCTTGATGATCGTCACCGACGAAATCTACGAGCACCTCGTCTACGACGCCGAGCAGGTCAGCATCGGCACCTACGCGCCGGAGCACACCCTGACCATCAATGGCGCGAGCAAAGCGTATGCCATGACCGGCTGGCGCATCGGCTACGCGGGCGGGCCGCGCGAGGTGATTGCCGCCATGAACGCGCTGCAATCGCAAAGCACCAGCAACGCCAGCAGCGTCAGCCAGTACGCCGCCCTCGCCGCGCTGGAACAGCACGAGGAAACCATGCGCTTCATCGACAGGGCCCGCACCGCCTACCGCGAACGGCGCGACCGCATCGTGGCGGGCCTCAACGCGCTGGGGTTGCCCACGCCCACGCCGCAAGGGGCCTTTTACGTGATGGCCGACACCCGCGCCATTCACACCGACGAACTCGAAGCCGCCCGCATCATTCTGGATGAGGCGCAGGTCGCCGTCGTGCCCGGCACCGATTTCGCCGCGCCGGGACAGGTGCGCCTGAGCTACGCGACCAGCATGGACAACATCGAGGAAGTGCTGCGGCGGCTGGAAGGGGTCGTGCGGCGCTAA>pQR1748MTSPFRLSARAQSLKPSATVAVTSRALELQRQGLDVISMSVGEPDFDTPPHVKAAGIAAIEEGKTKYTPVSGIPELREAISAKFRRENGLDYAPNAVTVTSGGKQALFNAFFALLNPGDEVLIPAPHWVSYPEMVALTGAVPVTVPTTPQQGFQLDPDALAAAITPRTRMVILNSPGNPTGAVFPPETLRAVADLATQHGLMIVTDEIYEHLVYDAEQVSIGTYAPEHTLTINGASKAYAMTGWRIGYAGGPREVIAAMNALQSQSTSNASSVSQYAALAALEQHEETMRFIDRARTAYRERRDRIVAGLNALGLPTPTPQGAFYVMADTRAIHTDELEAARIILDEAQVAVVPGTDFAAPGQVRLSYATSMDNIEEVLRRLEGVVRR>pQR1748ATGACTGACCGACCTCGTATCTCCGCACGCATCGGCGGTATCTCCGAGTCAGCGACCCTGGCGGTGGACGCCAAGGCCAAGGCCCTGAAGGCCGCTGGGCATCCCGTGATCGGCTTCGGCGCCGGGGAGCCTGACTTCCCCACGCCCGACTACATCGTGGAGGCAGCGGTCGCCGCCTGCCGCGACTCGCGCTTCCACCGCTACACCCCGGCGGGAGGCCTCCCCGAACTCAAGGAAGCCATCGCGGCTAAGACGCTGCGCGACTCCGGCTACCGGGTGGAGCCGAACCAAGTCCTGGTCACCAACGGCGGCAAGCAAGCGATCTACGAGGCGTTCGCCACGCTGCTCGATCCGGGCGACGAGGTCATCGTGATCGCGCCCTACTGGACCACCTACCCTGAATCGATCCGGCTGGCCGGAGGAACCCCGGTCTACGTGGTCACCGACGAGTCCACTGGCTACCTGGCCACGGTCGAGCAGCTGGAGGCGGCCCGCACCGACCGCACCAAGGTGCTGCTGTTCGTCTCCCCCTCGAACCCGACCGGCGCGGTGTACTCGCCCGAGCAGGTCCGCGAGATCGGCCGGTGGGCCCTCGAACACAACCTGTGGGTGCTCACCGACGAGATCTACGAGCACCTCGTCTACGGGGACGCCCGGTTCTCCTCGATGCCGGTGGAAGTTCCGGAACTGGCCGACCGCACCGTGGTGGTCAACGGGGTGGCCAAGACCTACGCCATGACCGGGTGGCGGGTCGGCTGGCTCATCGGCCCCGTGGACGTGGTCAAGGCTGCGACCAACCTGCAGTCGCACGCCACCTCCAATGTGGCCAACGTCTCGCAGGCCGCGGCTCTGGCAGCGGTCTCCGGCGACCTGTCGGCCGTGGAGGAGATGAAGCAGGCCTTCGACCGGCGGCGGCAGACCATTGTGCGGATGCTCAACGAGATCCCCGGTGTGGTGTGCCCCGAGCCCCAGGGCGCGTTCTACGCCTACCCGTCGGTCAAGGAGATCCTCGGCAAGGAGATCCGCGGTCAGCGTCCGCAGACCTCCAGCGAGCTGGCGTCGCTGATCCTGGAGCACGCCAAGGTCGCGGTGGTCCCGGGCGAGGCGTTCGGCACTCCGGGCTACCTGCGGTTGTCCTACGCGTTGAGCGACGCCGATCTGGTCGAAGGGGTCAGCCGGATCGCCAAGCTGCTGAGCGAAGCCCACTGA>pQR1749MTDRPRISARIGGISESATLAVDAKAKALKAAGHPVIGFGAGEPDFPTPDYIVEAAVAACRDSRFHRYTPAGGLPELKEAIAAKTLRDSGYRVEPNQVLVTNGGKQAIYEAFATLLDPGDEVIVIAPYWTTYPESIRLAGGTPVYVVTDESTGYLATVEQLEAARTDRTKVLLFVSPSNPTGAVYSPEQVREIGRWALEHNLWVLTDEIYEHLVYGDARFSSMPVEVPELADRTVVVNGVAKTYAMTGWRVGWLIGPVDVVKAATNLQSHATSNVANVSQAAALAAVSGDLSAVEEMKQAFDRRRQTIVRMLNEIPGVVCPEPQGAFYAYPSVKEILGKEIRGQRPQTSSELASLILEHAKVAVVPGEAFGTPGYLRLSYALSDADLVEGVSRIAKLLSEAH>pQR1749ATGGTATCCAGGAGAATATCAGAGATTCCCATATCGAAAACCATGGAACTCGACGCGAAGGCCAAAGCCCTCATAAAAAAGGGAGAAGACGTGATCAATCTAACGGCTGGTGAGCCGGATTTTCCCACACCGGAACCCGTCGTGGAAGAAGCGGTGAGATTTCTCCAGAAAGGAGAAGTGAAATACACAGATCCTCGTGGTATCTACGAACTCAGAGAGGGTATAGCGAAAAGGATAGGCGAGAGATACAAAAAAGATATCTCACCGGATCAGGTCGTGGTGACGAATGGAGCGAAACAGGCTCTGTTCAATGCTTTCATGGCCCTTCTCGATCCCGGTGACGAAGTGATCGTGTTTTCTCCCGTCTGGGTCAGCTACATTCCTCAGATCATCCTTGCTGGTGGCACGGTGAACGTGGTTGAGACGTTCATGAGTAAAAATTTCCAGCCCAGTCTGGAAGAGGTGGAAGGGCTTCTTGTTGGGAAAACGAAAGCCGTTCTTATCAACTCGCCGAACAATCCCACTGGTGTGGTGTACAGAAGAGAGTTCCTTGAAGGACTTGTGAGACTTGCCAAGAAGAGGAATTTCTACATAATCAGCGACGAAGTCTACGATTCCCTTGTTTACACGGATGAATTCACATCGATACTCGATGTTTCTGAAGGATTCGACCGGATAGTTTACATAAACGGCTTCTCGAAGTCTCACTCCATGACCGGCTGGAGGGTGGGTTACCTGATATCGAGCGAAAAAGTAGCGACCGCTGTGTCGAAGATCCAGTCTCACACCACCTCCTGTATCAACACGGTAGCACAGTACGCCGCCTTGAAGGCTCTGGAAGTGGACAACTCTTACATGGTTCAGACCTTTAAAGAAAGAAAAAATTTCGTGGTGGAAAGATTGAAAAAGATGGGTGTTAAGTTCGTGGAACCAGAAGGTGCGTTCTACCTCTTTTTCAAAGTCCGGGGTGACGATGTGAAATTCTGTGAAAGGCTCCTCGAAGAAAAGAAGGTTGCACTCGTTCCAGGATCCGCTTTTCTGAAGCCTGGATTTGTGAGGCTTTCTTTTGCCACATCTATAGAAAGACTTACGGAGGCGCTGGATAGAATTGAAGACTTCCTCAATTCTCGTTGA>pQR1751MVSRRISEIPISKTMELDAKAKALIKKGEDVINLTAGEPDFPTPEPVVEEAVRFLQKGEVKYTDPRGIYELREGIAKRIGERYKKDISPDQVVVTNGAKQALFNAFMALLDPGDEVIVFSPVWVSYIPQIILAGGTVNVVETFMSKNFQPSLEEVEGLLVGKTKAVLINSPNNPTGVVYRREFLEGLVRLAKKRNFYIISDEVYDSLVYTDEFTSILDVSEGFDRIVYINGFSKSHSMTGWRVGYLISSEKVATAVSKIQSHTTSCINTVAQYAALKALEVDNSYMVQTFKERKNFVVERLKKMGVKFVEPEGAFYLFFKVRGDDVKFCERLLEEKKVALVPGSAFLKPGFVRLSFATSIERLTEALDRIEDFLNSR>pQR1751ATGGCACCTGACCTGCGCCACCTGCACACCTTCGGCGAACTGGATCCGCCGCAACGCCTGTTGATGGGCCCCGGCCCGGTCAATGCGCATCCACGCGTGCTGCGTGCGATGGCGGCCGACCTGCTTGGCCAGTTCGACCCGGAAATGACCACCTACATGAACGAGGTGATGGCGCTGTACCGCCCCTTGTTCGGCACCCAGAACCGCTGGACCTTTCTGGTCGATGGCACGGCGCGCGCCGGCATCGAAGCCGCGCTGGTGTCGCTGGTGCAGCCGGGCGACCGTGTGCTGGTGATCAACTTCGGCCGCTTCGGTTTGTTGCTGACCGAAATCCTTGGCCGGCTCGGCGCCGACGTCCACACCGTGGATGCGCCGTGGGGCGAGGTGGTGCCGCTGGCGGCGATTGCCGAGGCGATCGCAAGCGTGGCACCCAAGCTGGTGGCCACCGTGCACGGCGACACCTCCACCACCATGGCGCAGCCGCTCGATGGCCTAGGCGCGCTATGCCGGGCGGCCGGCGCGCTGAGTTACGTAGACGCCACAGCCACCATCGGCGGCATGGACATCGCCAGCGACCGCTGGGAGGTGGACGTGGTCACCGCGGGGCTGCAGAAATGCCTGGGCGGGCCGTCCGGCTCGGCGCCGATCACTGTCTCTGCCGCGGCAGCGGAGGCGATCTTTGCGCGGCGGCATGTCGAACGCGGCATCGTGCGCGAGGACATCGCCAACGGCAGCGGCCCACGCATCGCCTCGAATTATTTCGACCTGGCGATGATCATGGATTACTGGTCCGACAAGCGCCTCAATCACCACACCGAAGCCACCACCATGCTGTACGGCGCGCGCGAATGCGCACGCGTGGCCTTGCAGGAAGGCCTGGAGGCGCGCTACGCCCGGCATGCGGCTGCCGGCCGCGCGGTCAGCGCCGGCGTGCGCGCACTGGGGCTGGAGGTGTTCGGCGACGATGCGCACCGCATGAGCAATGTCACCGGCGTGGTGATCCCGCACGGCGTCGACAGTGAAGCAGTGCGGCGGCGCATGCGCGAGGATTTCGAAATCGAGATCGGCACCGCGTTCGGCCCGCTGCAAGGCAGGATCTGGCGCATCGGTGCGATGGGCTACAACGCGATGAAGCACAAGGTGCTGCTCACCCTGGCCGCACTGGAAGCGGTGCTGCGCGCCGAGGGCTACGCGTGCACCCAAGGCCTGGCGGTCGAAGCCGCACGCGCCGCCTGGCATGCGGAGCCGGCTGCATGA>pQR1752MAPDLRHLHTFGELDPPQRLLMGPGPVNAHPRVLRAMAADLLGQFDPEMTTYMNEVMALYRPLFGTQNRWTFLVDGTARAGIEAALVSLVQPGDRVLVINFGRFGLLLTEILGRLGADVHTVDAPWGEVVPLAAIAEAIASVAPKLVATVHGDTSTTMAQPLDGLGALCRAAGALSYVDATATIGGMDIASDRWEVDVVTAGLQKCLGGPSGSAPITVSAAAAEAIFARRHVERGIVREDIANGSGPRIASNYFDLAMIMDYWSDKRLNHHTEATTMLYGARECARVALQEGLEARYARHAAAGRAVSAGVRALGLEVFGDDAHRMSNVTGVVIPHGVDSEAVRRRMREDFEIEIGTAFGPLQGRIWRIGAMGYNAMKHKVLLTLAALEAVLRAEGYACTQGLAVEAARAAWHAEPAA>pQR1752ATGGGAAAGTTTCTTAAGAAACACTACATAATGGCACCTGGACCAACACCAGTCCCAAACGATATTTTAACAGAAGGAGCGAAGGAAACAATACACCACAGAACACCTCAGTTTGTTTCCATAATGGAAGAGACCCTCGAAAGTGCAAAGTACATCTTTCAGACAAAACACAACGTGTACGCCTTTGCTTCCACAGGAACTGGCGCTATGGAAGCGGCGGTGGCGAATCTTGTGAGCCCTGGAGACAAAGTGATCGTGGTTGTGGCTGGAAAGTTCGGTGAAAGATGGAGAGAGCTCTGTCAGGCTTACGGTGCTGATATCGTAGAAATCGCCCTCGAATGGGGAGACGCGGTCACACCTGAACAGATCGAAGAGGCTCTCAACAAAAACCCCGATGCGAAGGTCGTCTTCACCACCTACAGTGAAACATCGACGGGTACAGTCATAGACCTCGAAGGAATTGCCAGAGTCACGAAGGAAAAAGACGTTGTTCTTGTGACAGACGCTGTCAGCGCTCTTGGAGCAGAACCACTGAAGATGGATGAATGGGGTGTGGATCTCGTTGTCACAGGTTCACAGAAGGGTTTGATGTTACCTCCAGGACTGGCGCTCATCTCTCTCAACGACAAAGCGTGGGGGCTCGTGGAAAAATCCAGATCTCCAAGGTACTACTTCGATCTGAGGGCCTACAGGAAATCTTACCCCGACAATCCTTACACCCCCGCAGTAAACATGATATACATGTTGAGAAAGGCTCTTCAGATGATAAAAGAGGAAGGCATAGAAAACGTATGGGAAAGGCACAGAATACTGGGAGACGCAACAAGAGCAGCGGTGAAAGCACTTGGACTGGAACTCCTCTCGAAAAGACCGGGAAACGTTGTAACAGCCGTGAAAGTGCCTGAGGGCATCGATGGAAAACAGATTCCCAAGATCATGAGAGACAAGTACGGTGTGACCATCGCCGGTGGACAGGCTAAACTCAAGGGAAAAATATTCAGGATAGCACACCTCGGATACATGTCACCTTTCGACACCATAACTGCCATTTCCGCTCTTGAATTAACCTTGAAGGAACTCGGTTATGAGTTCGAACTCGGAGTCGGTGTTAAGGCAGCCGAAGCTGTCTTCGCTAAAGAATTCATTGGGGAGTGA>pQR1755MGKFLKKHYIMAPGPTPVPNDILTEGAKETIHHRTPQFVSIMEETLESAKYIFQTKHNVYAFASTGTGAMEAAVANLVSPGDKVIVVVAGKFGERWRELCQAYGADIVEIALEWGDAVTPEQIEEALNKNPDAKVVFTTYSETSTGTVIDLEGIARVTKEKDVVLVTDAVSALGAEPLKMDEWGVDLVVTGSQKGLMLPPGLALISLNDKAWGLVEKSRSPRYYFDLRAYRKSYPDNPYTPAVNMIYMLRKALQMIKEEGIENVWERHRILGDATRAAVKALGLELLSKRPGNVVTAVKVPEGIDGKQIPKIMRDKYGVTIAGGQAKLKGKIFRIAHLGYMSPFDTITAISALELTLKELGYEFELGVGVKAAEAVFAKEFIGE>pQR1755ATGACTCAGATTTTTAATTTTAGCGCCGGTCCAGCAATGCTGCCGGTTGAAGTACTGCGTCGTGCTGAACAGGAATTGTGTAATTGGAATGGCCTGGGCACATCGGTTATGGAAATCAGCCACCGCAGTAAAGAGTTTATGCAGGTTGCCGCTGAATCCGAACAGGATCTGCGTGATTTGCTGAAAATCCCCTCCAACTACAAAGTGCTCTTTTGCCACGGCGGTGCTCGTGCGCAATTCGCCGCAGTGCCGTTAAATCTTCTGGGCGAACGCTCAACGGCCGACTACATCGACGGCGGGTATTGGGCGCACAGCGCAATCAATGAAGCAGAAAAATACTGCACGCCTAACGTGATTGACGTGAAAATGCGCGTGGGCGAACTGCGTGGCATTAAGCCGATGCGTGAATGGAAATTGTCTGATGACGCGGCGTTTGTGCATTACTGCCCGAATGAAACCATCGACGGTATTGCGATCGAAGAAGAGCCGGACTTTGGCGATAAAATTGTGGTCGCCGACTATTCTTCCAGCATCCTGTCTCGTCGTATTGATGTCAGCCGTTACGGCGTGATCTATGCCGGTGCGCAGAAAAATATCGGCCCTGCCGGCCTGACGCTGGTTATCGTACGTGAAGATTTGCTGGGCAAGGCGCGCCGTGAGCTGCCATCGATTCTGGATTACCAGGTTCTGGCGGACAATGACTCCATGTTTAACACGCCACCGACCTTTGCCTGGTACCTGTCCGGTATGGTCTTCAAATGGCTGAAAGAGTACGGCGGTCTGGCTGAAATGGAAAAACGTAACCAGGAGAAGGCTGACCTGCTGTATAGCGCGATTGACGGTAACGATTTCTATCGTAATGACGTTGCGGTAGCGAACCGTTCTCGCATGAATGTGCCATTCCTGTTGGCGGATTCTGCGCTGGATAAAGTCTTCCTGGAAGAATCAGTCGCTGCAGGTCTGCACGCGCTGAAAGGCCATCGCGTAGTAGGCGGCATGCGTGCCTCTATCTACAATGCGATGCCGTTGGAAGGCGTGAAAGTGCTGACGGAATTTATGGCTGACTTCGCTCGTCGCCACGGTTGAMTQIFNFSAGPAMLPVEVLRRAEQELCNWNGLGTSVMEISHRSKEFMQVAAESEQDLRDLLKIPSNYKVLFCHGGARAQFAAVPLNLLGERSTADYIDGGYWAHSAINEAEKYCTPNVIDVKMRVGELRGIKPMREWKLSDDAAFVHYCPNETIDGIAIEEEPDFGDKIVVADYSSSILSRRIDVSRYGVIYAGAQKNIGPAGLTLVIVREDLLGKARRELPSILDYQVLADNDSMFNTPPTFAWYLSGMVFKWLKEYGGLAEMEKRNQEKADLLYSAIDGNDFYRNDVAVANRSRMNVPFLLADSALDKVFLEESVAAGLHALKGHRVVGGMRASIYNAMPLEGVKVLTEFMADFARRHG"} +{"text": "To produce synthetic nucleotides of notifiable dengue virus (1\u20134 types), Japanese encephalitis, yellow fever and Zika flaviviruses. These notifiable flaviviruses, particularly dengue and Zika, are problematic mosquito-borne infections in the Philippines, as well as in those countries with tropical and subtropical climates.An algorithmic design formulation of overlap extension \u2013 polymerase chain reaction (OE-PCR) was performed to propagate 50\u201360 oligomer lengths of select notifiable flaviviral RNAs to DNA nucleotides via the two-step process of OE-PCR.Algorithmic OE-PCR design formulation efficiently produced 253\u2013256\u00a0bp of notifiable flaviviruses. Comparing the newly designed algorithmic OE-PCR with existing executable programs demonstrated it to be efficient and useful in generating accurate sequences of synthetic flaviviral nucleotides.The efficiently and accurately produced novel synthetic nucleotides of notifiable dengue virus 1\u20134,\u00a0Japanese encephalitis, yellow fever and Zika flaviviruses using OE-PCR is useful in understanding the dynamics of flaviviral species and holds potential for the development of synthetic nucleotide-based immunogens. Dengue virus (1\u20134), Japanese encephalitis and Zika fevers are notable mosquito-borne infections that continue to be problematic in the Philippines and its southeast Asian neighbors. Flaviviral yellow fever affects other tropical countries. This study aimed to effectively and efficiently synthesize flaviviral nucleotides through a predictively accurate algorithmic overlap extension-polymerase chain reaction design. The cost-saving arithmetic formulation has enabled the advanced production of 253\u2013256\u00a0bp flaviviral nucleotide lengths. The newly synthesized and sequenced products are now ready for further experimentation and open the door for immunological exploration. Dengue and Zika fevers, including Japanese encephalitis (JEV), are particularly burdensome mosquito-borne infections in the Philippines and are also selectively reported in Japan . The chaThe OE-PCR in this experiment was performed via two processes: the first one is using the reference or original OE-PCR protocol ,12 and t1.Preparation of the first-stage primer mixed solution(A) Both ends primer 2.5\u00a0\u03bcl(B) Central primer (2\u20135) 0.5\u00a0\u03bcl(U) Deionized water (DW) 18\u00a0\u03bcl2.Preparation of reaction solution for the first-step PCR(A) First-stage primer mixed solution \u00d7 6\u00a0\u03bcl(A) GoTaq 12.5\u00a0\u03bcl(U) DW 6.5\u00a0\u03bcl3.First-stage PCR reaction(A) 95\u00b0C 2\u00a0min21(C) 72\u00b0C - 2\u00a0min4.Preparation of the second-step PCR solution(A) First-step PCR reaction solution 1\u00a0\u03bcl(B) Both ends primer (stock solution) 1\u00a0\u03bcl\u00a0\u00d7 2(C) GoTaq 12.5\u00a0\u03bcl(D) 9.5\u00a0\u03bcl of DW5.Second-step PCR reaction(A) 95\u00b0C 2\u00a0min(A) (95\u00b0C - 1\u00a0min \u2192 72\u00b0C - 2\u00a0min) \u00d7 35-times (C) 72\u00b0C - 2\u00a0min1. Preparation of the first-stage primer-mixed solution(A) Both ends primer 2.5\u00a0\u03bcl(B) Central primer (2\u20135) 0.5\u00a0\u03bcl(U) DW 193\u00a0\u03bcl(A) GoTaq 12.5\u00a0\u03bcl(U) DW 6.5\u00a0\u03bcl3. First-stage PCR reaction(A) 95\u00b0C 2\u00a0min(A) (95\u00b0C - 1\u00a0min \u2192 48\u00b0C - 1\u00a0min \u2192 72\u00b0C - 1\u00a0min) \u00d7 20(C) 72\u00b0C - 2\u00a0min4. Preparation of the second-step PCR solution(A) First-step PCR reaction solution 1\u00a0\u03bcl(B) Both ends primer (stock solution) 1\u00a0\u03bcl \u00d7 2(C) GoTaq 12.5\u00a0\u03bcl(D) 9.5\u00a0\u03bcl of DW5. Second-step PCR reaction(A) 95\u00b0C 2\u00a0min(B) (95\u00b0C. -1\u00a0min \u2192 72\u00b0C. -2\u00a0min) \u00d7 25(C) 72\u00b0C. -2\u00a0minDepicted in In the second PCR, the most outside oligomers work for PCR primers and amplified the whole size of target DNA.An electrophoresis of OE-PCR products was done for PCR product-size prediction after amplification of both OE-PCR and pathogen-specific PCR. Single product was confirmed on 1.5% agarose gel electrophoresis with 1\u00a0\u00d7 Tris/Borate/EDTA (TBE) buffer and UV-visualized with 0.7 micro-g/ml-ethidium bromide . The PCRThe following notable flaviviruses,\u00a0namely dengue (4 types), YF, JEV and Zika, were specifically processed for production of artificial DNAs. The algorithmic formulated OE-PCR was subsequently carried out in two-step cycles, with each cycle's DNA products depicted via UV-illuminated bands following gel electrophoresis . In the DNA sequencing was carried out in similar steps as previously described ,7 Eurof.Approaches to determine efficiency and cost\u2013effectiveness of the current algorithmic OE-PCR tool versus the existing programs are presented in It must be noted that the price of oligomers varies greatly depending on the length in base and according to the viral species involved. Owing to this, an algorithm was designed to achieve the maximum length within the lowest cost range in base size. Unfortunately, this is difficult to realize using the currently available reported design algorithms. In the stated design algorithms, the difficulty lies in how to control the length of the oligomer. This is because the setting of overlapping region of the oligomer has been designed first. With the current study, the basic length has been decided to begin with; coupled with a lowered priority on the thermodynamic stability of the overlapping region. This has resulted in fewer problems in the conduct of OE-PCR.The ongoing dilemma of largely problematic and imperfectly developed flaviviral immunogens prompted us to responsibly contribute on the need to develop an efficient algorithmic OE-PCR program design for synthetic nucleotide production of select notifiable dengue (1\u20134), JEV, YF and Zika flaviviruses. To this end, we developed an OE-PCR program that can be utilized as reference tool in the furtherance of flaviviral research. It can be deduced that the program formulation was found to be demonstrably reproducible, cost-effective and efficient in yielding substantial nucleotides of select notifiable flaviviral DNAs, which can be sourced out at the databases of DDBJ/NCBI with the respective accession numbers of LC227563, LC227561, LC227562, and LC227568 (dengue 1\u20134); LC227560 (JEV); LC227567 (YF); and LC227589 (Zika). The OE-PCR is a reliable method that can be utilized when designed and accurately predicted using algorithmic manipulation.\u00a0Henceforth, we finally conclude that the DNA primer design using algorithmic OE-PCR is a valuable tool in producing synthetic nucleotide products of pathogenic species of flaviviruses.in vivo would result in pathological or immunological response.The novel nucleotide sequences generated from this study can be utilized for studies looking to further understand the genomic diversity of a variety of flaviviral strains, including dengue (1\u20134), YF, JEV and Zika. In addition, clinical trials involving initial utilization of whole nucleotide sequence followed by booster immuno-stimulation of antigenic protein may serve as a starting point for generating long-term immune responses for flaviviral infections such as dengue and Zika, where trials for DNA-based vaccines are ongoing, yet faced with challenging dose-dependency concerns. Further experiments should be conducted to determine whether administration of synthetic nucleotides Box 1.\u2002flavi |2|1: ATGTATGCCGATGACACCGCAGGATGGGATACAAGAATCACACTAGAAGACCTAAAAAAT: 60flavi |2|2: TTGTGTTCTCCTTCCATGTGGTTTGTTACCATTTCTTCATTTTTTAGGTCTTCTAGTGT: 59flavi |2|3: CATGGAAGGAGAACACAAGAAACTAGCCGAGGCCATTTTCAAACTAACGTACCAAAACA: 59flavi |2|4: TGTGCCTCTTGGTGTTGGTCTTTGCACACGCACCACCTTGTTTTGGTACGTTAGTTTG: 58flavi |2|5: CAACACCAAGAGGCACAGTAATGGACATCATATCGAGAAGAGACCAAAGAGGTAGTGGA: 59flavi |2|6: TGGTGAAAGTATTGAGTCCATAGGTGCCAACTTGTCCACTACCTCTTTGGTCTC: 54flavi |3|1: ATGTATGCTGATGACACAGCAGGCTGGGACACAAGAATCACTGAGGATGACCTTCAAA: 58flavi |3|2: TGGTGGGGAGCCATCTGTTCCGTGATCAGTTCCTCATTTTGAAGGTCATCCTCAGT: 56flavi |3|3: TGGCTCCCCACCACAAGATCCTAGCCAAAGCCATTTTCAAACTAACCTATCAAAACAA: 58flavi |3|4: TCCCCGCGGTGTGGGTCTGAGGACTTTCACCACTTTGTTTTGATAGGTTAGTTTG: 55flavi |3|5: CACCGCGGGGAGCGGTGATGGATATCATATCCAGGAAAGACCAAAGAGGTAGTGGA: 56flavi |3|6: TGGTGAATGTGTTCAAACCATATGTTCCAACTTGTCCACTACCTCTTTGGTCTT: 54flavi |5|1: ATGTATGCAGATGACACAGCCGGATGGGACACAAGAATAACAGAGGATGATCTTCAGAA: 59flavi |5|2: CATGTTCAGGTTCCATGATGTCAGTGATTTTGGCCTCATTCTGAAGATCATCCTCTGT: 58flavi |5|3: CATGGAACCTGAACATGCCCTATTGGCCACGTCAATCTTTAAGCTAACCTACCAAAACA: 59flavi |5|4: GTTCCATTTTTCGCTGGTCTCTGCACCCTTACTACCTTGTTTTGGTAGGTTAGCTTA: 57flavi |5|5: AGCGAAAAATGGAACCGTGATGGATGTCATATCCAGACGTGACCAGAGAGGAAGTGG: 57flavi |5|6: TGGTGAAGGTGTTTAAGCCATAGGTTCCAACCTGTCCACTTCCTCTCTGGTCA: 53flavi |8|1: ATGTATGCTGATGACACAGCTGGTTGGGACACAAGAATAACAGAAGATGACCTGCACA: 58flavi |8|2: TGTGTTCAGGGTCCATTTGCTGTATGATCTTTTCCTCATTGTGCAGGTCATCTTCTG: 57flavi |8|3: AATGGACCCTGAACACAGGCAGTTAGCGAACGCTATATTCAAGCTCACATACCAAAACA: 59flavi |8|4: TGCCCGTTGGAGTCGGTCGTTGAACTTTGACCACTTTGTTTTGGTATGTGAGCTTG: 56flavi |8|5: ACTCCAACGGGCACGGTAATGGATATTATATCTAGGAAAGACCAAAGGGGCAGT: 54flavi |8|6: TGGTGAATGTATTCAGGCCATAAGTTCCCAGTTGTCCACTGCCCCTTTGGTC: 52flavi |15|1: ATGTACGCAGATGACACTGCTGGCTGGGACACCCGCATTAGTAAGTTTGATCTGGAGAA: 59flavi |15|2: GTGCCCTTCCTCCATTTGGTTGGTAATCAGAGCTTCATTCTCCAGATCAAACTTACT: 57flavi |15|3: TGGAGGAAGGGCACAGAACTCTGGCGTTGGCCGTGATTAAATACACATACCAAAACAA: 58flavi |15|4: TTTTTCCTCCTTCAGCTGGTCTGAGAACCTTCACCACTTTGTTTTGGTATGTGTATTTA: 59flavi |15|5: CAGCTGAAGGAGGAAAAACAGTTATGGACATCATTTCAAGACAAGACCAGAGAGGGAG: 58flavi |15|6: TGGTGAATGTGTTGAGAGCATAAGTGACAACTTGTCCACTCCCTCTCTGGTCTTGTC: 57flavi |25|1: TTCTACGCGGATGACACCGCTGGATGGGACACGCGCATCACAGAGGCAGACCTTGAT: 57flavi |25|2: TGTGATGTGGGCTCATGTAGTTCAAGATCTCCTGTTCATCATCAAGGTCTGCCTCTG: 57flavi |25|3: ATGAGCCCACATCACAAAAAACTGGCACAAGCAGTGATGGAAATGACATACAAGAACA: 58flavi |25|4: TTCCCTCCTGGGGCTGGTCTCAACACTTTCACCACTTTGTTCTTGTATGTCATTTCC: 57flavi |25|5: GCCCCAGGAGGGAAAGCCTACATGGATGTCATAAGTCGACGAGACCAGAGAGGAT: 55flavi |25|6: TGGTGATGGTGTTCAGAGCATAAGTCACTACCTGCCCGGATCCTCTCTGGTCTCGT: 56flavi |27|1: ATGTACGCTGATGACACCGCCGGATGGGACACTAGAATTACCAGAACTGATTTAGAAAA: 59flavi |27|2: GGTGTTCACCGTCTAGGAGCTCCAGCACCTTGGCTTCATTTTCTAAATCAGTTCTGGT: 58flavi |27|3: CCTAGACGGTGAACACCGCATGCTCGCTCGAGCCATAATTGAACTGACTTACAGGCACA: 59flavi |27|4: CTTTCCTTCTGCTGCAGGTCTCATGACCTTGACCACTTTGTGCCTGTAAGTCAGTTCA: 58flavi |27|5: TGCAGCAGAAGGAAAGACCGTAATGGACGTGATATCAAGAGAAGATCAAAGGGGGAG: 57flavi |27|6: TCGTGAAAGTGTTAAGGGCATAAGTGACCACCTGTCCACTCCCCCTTTGATCTTCT: 56This project aimed to develop synthetic nucleotides using overlap extension-polymerase chain reaction (OE-PCR), particularly of flaviviral organisms that are notorious for their ability to cause both animal and human diseases.A method is provided for generating template designs that could help to address epidemics caused by select flaviviral dengue, Zika and related species.Expansion of OE-PCR as a tool in developing templates for viral studies is presented, addressing concern regarding the lack of reported commercial production of artificial nucleotides of these flaviviruses.This research has delivered a reproducibly effective and efficient algorithmic OE-PCR design that is suitable for yielding substantial nucleotides of these flaviviruses.These synthetic nucleotides could pave the way for future collaborative research exploring the possibility of generating synthetic flaviviral nucleotide-based immunogens."} +{"text": "Tmem119 gene to generate knock-in mice expressing EGFP (JAX#031823) or CreERT2 (JAX#031820) for the identification and manipulation of microglia, respectively. Genetic characterization of the locus and qPCR-based analysis demonstrate correct positioning of the transgenes and intact expression of endogenous Tmem119 in the knock-in mouse models. Immunofluorescence analysis further shows that parenchymal microglia, but not other brain macrophages, are completely and faithfully labeled in the EGFP-line at different time points of development. Flow cytometry indicates highly selective expression of EGFP in CD11b+CD45lo microglia. Similarly, immunofluorescence and flow cytometry analyses using a Cre-dependent reporter mouse line demonstrate activity of CreERT2 primarily in microglia upon tamoxifen administration with the caveat of activity in leptomeningeal cells. Finally, flow cytometric analyses reveal absence of EGFP expression and minimal activity of CreERT2 in blood monocytes of the Tmem119-EGFP and Tmem119-CreERT2 lines, respectively. These new transgenic lines extend the microglia toolbox by providing the currently most specific genetic labeling and control over these cells in the myeloid compartment of mice.Microglia are specialized brain-resident macrophages with important functions in health and disease. To improve our understanding of these cells, the research community needs genetic tools to identify and control them in a manner that distinguishes them from closely related cell types. We have targeted the recently discovered microglia-specific Tools that specifically label and manipulate only microglia are currently unavailable, but are critically needed to further our understanding of this cell type. Complementing and significantly extending recently introduced microglia-specific immunostaining methods that have quickly become a new standard in the field, we generated two mouse lines that label and control gene expression in microglia with high specificity and made them publicly available. Using these readily accessible mice, the research community will be able to study microglia biology with improved specificity.Tie2, Runx1, Csf1r, Aif1, Lyz2, Itgam, Sall1, or CX3cr1 is highly and exclusively expressed in microglia in the brains of mice and humans and Tmem119-CreERT2 (JAX#031820), where microglia express EGFP and CreERT2, respectively, while preserving endogenous Tmem119 expression. We demonstrate that EGFP is expressed throughout the brain and that the tag is confined to microglia only, without significantly labeling other brain macrophages. We further provide evidence that the inducible Cre is primarily active in microglia by crossing to the conditional fluorescent reporter mouse line Ai14. In these mice, we also observe activity in leptomeningeal cells that line the surface of the brain and penetrate deep into the brain ensheathing some large blood vessels. Finally, we demonstrate minimal to absent transgene expression in monocytes of the mice. These publicly available mouse lines provide valuable tools for the functional study of bona fide microglia.Recent advances in RNA sequencing and other cell profiling technologies have enabled the discovery of cell-type-specific signature genes . Among td humans . AlthougAll animal procedures were performed in accordance with MIT\u2019s animal care regulations. Overall, >30 animals of >10 litters were generated per line and mice were crossed to C57BL/6J up to generation N3. For all experiments, at least three independent mice were analyzed, which included both sexes and no apparent sex differences were observed.Tmem119 knock-in mice, donor DNA templates encoding ribosome-skipping peptide porcine teschovirus-1 polyprotein and EGFP (P2A-EGFP) or CreERT2 (P2A-CreERT2) were synthesized. These sequences were flanked by sequences of 55\u2013300 bp for EGFP and 1.5 kb for CreERT2 homologous to 5\u2032 and 3\u2032 regions around the Tmem119 stop codon. These templates were injected into fertilized mouse oocytes together with a single crRNA that cuts at the stop codon.To generate AACTGGTCCTCCTGAAA-3\u2032 and 5\u2032-CAAAGCCTGTGAAGGGTGGG-3\u2032, respectively; * denoting phosphorothioate) and a reverse primer for a 55 bp right homology arm . Using these primers, large scale PCR with Takara PrimeStar was performed to obtain 40\u201360 \u03bcg of product. The PCR product was highly purified with the Qiagen PCR purification kit and subject to digestion of the antisense strand. Lambda exonuclease (New England Biolabs) was used to digest 20 \u03bcg of dsDNA at 37\u00b0C for 60 min. Complete digestion of dsDNA was confirmed by agarose gel electrophoresis and Sanger sequencing with sense- and antisense-binding primers.Donor DNA templates were generated by digesting pAAV-P2A-EGFP (sequence below) with XbaI and EcoRI (New England Biolabs) and inserting three gblocks using Gibson cloning according to the manufacturers\u2019 protocols. Resulting plasmids were purified and sequenced. For CreERT2, the highly purified dsDNA plasmid was directly used as donor DNA in injections. For EGFP, single strand DNA (ssDNA) was produced using PCR with forward primers for left homology arms between 55 and 300 bp and heated to 95\u00b0C for 5 min. Heated mixtures were cooled to room temperature for 10 min and 30 ng/\u03bcl EnGen Cas9 protein (New England Biolabs) was added. Mixtures were incubated at 37\u00b0C for 15 min to form Cas9 to crRNA-tracrRNA complexes. Final concentrations of 5 ng/\u03bcl donor DNA (P2A-EGFP ssDNA or P2A-CreERT2 plasmid dsDNA) and 10 ng/\u03bcl recombinant RAD51 protein were added. Mixtures were kept on ice until use, when they were incubated at 37\u00b0C for 15 min followed by centrifugation at 10,000 rpm for 1 min to prevent clogging of the micropipette.Mixtures for injection of zygotes were prepared freshly on the morning of the day of injection. Briefly, water to a final volume of 100 \u03bcl was mixed with final concentrations of 10 mInjection of mixtures was conducted using standardized protocols of the transgenics facility in zygotes obtained from pure C57BL/B6N mice (Taconic).GCCTCTGTCACTTAAGTTGG) and P2A-R (GCTTCAGCAGGCTGAAGTTA). A second amplicon of \u223c 2.5\u20133.5 kb (CAGTGTCGGAAGCGGAGCTA) and 3\u2032ofRHA-R (GAAAGAGGAAGCTAGAAGGG). Both amplicons were purified and then sequenced using primers spanning the entire length using Sanger sequencing (GENEWIZ). Trace files were aligned to in silico assemblies and analyzed using Snapgene.Founder mice were genetically examined by amplifying sequences spanning the 5\u2032 and 3\u2032 junction and including the entire inserted transgene. Specifically, high-quality DNA was obtained from ear punches using a tissue DNA extraction kit according to the manufacturer\u2019s instructions (Macherey-Nagel). One amplicon spanning \u223c1.5 kb at 95\u00b0C for 30 min. The lysed tissue solution was neutralized using an acidic buffer . PCR using primers WT-F GTCAGGAGGAGGCCCAGGAA, EGFP-F CTGCTGCCCGACAACCACTA, CreERT2-F ACCGCCTACATGCGCCCACT, and Common-R GTTTCCTGGGGTGCACCAGA yielded products of 400 bp for the wild-type allele and 320 bp for the EGFP or CreERT2 allele.To genotype mice, ear tissue was prepared using an alkaline buffer mice were obtained from JAX (stock 007914) and maintained in house. Homozygous Ai14 mice were bred to heterozygous Tmem119-EGFP+/\u2212 mice were dissected and snap-frozen in liquid nitrogen. Highly pure RNA was isolated using an RNeasy purification kit (Qiagen) following the manufacturer\u2019s instructions. Equal amounts of RNA (1.5 \u03bcg per sample) were reverse transcribed using an iScript Advanced cDNA synthesis kit (Bio-Rad). Resulting cDNA was diluted 1:50 in ultrapure water. Quantitative PCR (qPCR) was conducted using SsOAdvanced Universal SYBR Green Supermix (Bio-Rad) on a CFX96 real-time system. Primers used were exon-spanning whenever possible and of the following sequences: Tmem119-F CCTTCACCCAGAGCTGGTTC, Tmem119-R GGCTACATCCTCCAGGAAGG, GAPDH-F GCCTTCCGTGTTCCTACC, GAPDH-R CCTCAGTGTAGCCCAAGATG, b-actin-F CTAAGGCCAACCGTGAAAAG, b-actin-R ACCAGAGGCATACAGGGACA. Differential gene expression analysis was performed using built-in software for the Bio-Rad CFX96 real-time system.Brain hemispheres from adult Tamoxifen was dissolved in corn oil at 20 mg/ml under agitation for several hours in the dark and kept at room temperature for 2\u20133 d. Using needles from Harvard Apparatus (52-4025), separately housed adult animals were fed via oral gavage for 3 d. Needles were rinsed between days and syringes were replaced. At time of administration, the mice weighed \u223c19\u201326 g and received 250\u2013400 \u03bcl of the 20 mg/ml tamoxifen solution, corresponding to 0.2 mg/g body weight. Postnatal day (P)2 mice weighing \u223c2 g received 5 \u03bcl of the 20 mg/ml solution, corresponding to 50 \u03bcg/g body weight per day for 3 consecutive days. The health of the mice was monitored daily. At least 7 d week after the last dose, mice were killed by transcardiac perfusion.Adult mice were deeply anesthetized and perfused with 25 ml PBS followed by 25 ml 4% paraformaldehyde (PFA) in PBS. Early postnatal mice (P3) were perfused with 8 ml PBS and 8 ml PFA. Brains were surgically removed and postfixed in the fixative at 4\u00b0C for 24 h. Fixed brains were washed once in PBS and sliced into 100-\u03bcm-thick sagittal slices using a Leica VT1000S. Slices were washed twice in PBS, permeabilized in 1.2% TX100 in PBS for 15 min, washed twice in PBS, and subject to incubation in blocking solution . Blocked sections were incubated with primary antibodies for IBA1 , GFP , TMEM119 , Olig2 , NeuN , GFAP , or CD163 for 24 h at 4\u00b0C. Primary antibody incubation was followed by three washes in PBS and incubation with species-matched and Alexa fluorophore-conjugated secondary antibodies raised in goat for 2 h. DAPI (1:10.000) was included in a washing step or secondary antibody incubation. Slices were washed three times in PBS and mounted and coverslipped using VECTASHIELD H-1000 mounting medium.m Sodium citrate, pH 8.5) for 5 min, followed by incubation in the retrieval buffer at 80\u00b0C for 30 min. Sections were cooled to room temperature and washed twice in PBS. Blocking and immunostaining were then conducted as described for other antibodies in the immunofluorescence staining and imaging methods section.For immunostaining of CD163, an antigen retrieval step was included. Briefly, vibratome slices were washed twice in PBS, incubated in a retrieval buffer were counted manually.For imaging, slides were scanned on an Olympus FluoView FV1000 fixed stage confocal microscope (high-power magnifications) or Olympus BX61 epifluorescence microscope using built-in software. For coexpression analysis in the z-stacks were acquired at 20\u00d7 magnification with 2\u00d7 digital zoom and 1.2 \u03bcm step size. Images were imported as stacks into Neurolucida software. Cell bodies in the middle of the stacks with complete process arbors were selected and manually traced. Raw trace files were imported into Neurolucida explorer software and convex hull area, number of processes and total process length were determined using the convex hull analysis and branched structure analysis functions. Rendered traces were exported as monochrome vector graphics. Cell body areas were separately determined in FIJI using the polygon tool.For microglia morphology analysis, g for 5 min at 4\u00b0C, resuspended in 10 ml ice-cold HBSS, and pelleted again at 300 \u00d7 g for 5 min at 4\u00b0C. WBCs were resuspended in 500 \u03bcl ice-cold FACS buffer and subject to staining. In parallel with the WBC enrichment, brains were rapidly dissected into 2 ml ice-cold HBSS and cerebella and brainstem were removed. Brains were minced into small pieces and transferred to a Dounce homogenizer containing 5 ml ice-cold HBSS with 20 \u03bcg/ml DNase I . Tissue chunks were homogenized with 15 loose and 15 more tight strokes and the homogenate was transferred to a 50 ml falcon tube through a pre-wet 70 \u03bcm strainer. The strainer was rinsed with HBSS to top off the volume of each sample to 10 ml. Filtered homogenates were transferred to 15 ml falcon tubes and spun at 300 \u00d7 g for 5 min at 4\u00b0C. Supernatants were carefully removed, and pellets resuspended in 10 ml of ice-cold 40% Percoll in HBSS. Samples were spun at 500 \u00d7 g for 30 min at 4\u00b0C with full acceleration and deceleration. Myelin and debris from the supernatant were carefully removed and pellets resuspended in 10 ml ice-cold HBSS. Following another spin at 300 \u00d7 g for 5 min at 4\u00b0C, the supernatant was removed and the microglial pellet resuspended in 1 ml FACS buffer .Microglia and blood monocytes for single-cell suspensions for flow cytometry were prepared as follows. Mice were deeply anesthetized with isoflurane and transcardially perfused with ice-cold HBSS. During perfusion, 2 ml of whole blood was collected from the right atrium into tubes containing 40 \u03bcl of 10% (w/v) EDTA. The 2 ml of whole blood were added to 40 ml of red blood cell lysis buffer and incubated for 10 min at room temperature to lyse red blood cells. The suspension containing lysed RBCs and white blood cells was spun down at 300 \u00d7 m EDTA) and spun down at 300 \u00d7 g for 5 min at 4\u00b0C. Pellets were resuspended and incubated with 1:200 Mouse Fc Block on ice for 15 min. Samples were incubated with 1:200 rat anti CD45-APC/Cy7 and rat anti CD11b-PE/Cy5 at 4\u00b0C. Tubes were topped off to 2 ml with ice-cold FACS buffer and microglia pelleted at 300 \u00d7 g for 5 min at 4\u00b0C. Supernatants were removed and microglia resuspended in 500 \u03bcl. Resuspended microglia were filtered through corning strainer polystyrene tubes . Flow cytometry data were acquired on Aria II, Fortessa HTS, and LSRII HTS, and analyzed using FlowJo.Suspensions of white blood cells and microglia were transferred to 2 ml Eppendorf microcentrifuge tubes, and a small fraction of sample was removed for single-color controls. To stain dead cells, live/dead violet was added and incubated for 5 min on ice. Tubes with live/dead-stained cells were topped off to 2 ml with FACS buffer . Not assuming Gaussian distribution of the data, a nonparametric test (Mann\u2013Whitney) was used to compare median expression in both groups.Quantitative data from the qPCR, immunofluorescence, and microglia morphology experiments were analyzed using GraphPad prism. Testing for normality was not possible because pAAV-P2A-EGFPCCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGCGGCCGCACGCGTTTAATTAAGTGTCTAGACTGCAGAGGGCCCTGCGTATGAGTGCAAGTGGGTTTTAGGACCAGGATGAGGCGGGGTGGGGGTGCCTACCTGACGACCGACCCCGACCCACTGGACAAGCACCCAACCCCCATTCCCCAAATTGCGCATCCCCTATCAGAGAGGGGGAGGGGAAACAGGATGCGGCGAGGCGCGTGCGCACTGCCAGCTTCAGCACCGCGGACAGTGCCTTCGCCCCCGCCTGGCGGCGCGCGCCACCGCCGCCTCAGCACTGAAGGCGCGCTGACGTCACTCGCCGGTCCCCCGCAAACTCCCCTTCCCGGCCACCTTGGTCGCGTCCGCGCCGCCGCCGGCCCAGCCGGACCGCACCACGCGAGGCGCGAGATAGGGGGGCACGGGCGCGACCATCTGCGCTGCGGCGCCGGCGACTCAGCGCTGCCTCAGTCTGCGGTGGGCAGCGGAGGAGTCGTGTCGTGCCTGAGAGCGCAGTCGAGAAACCGGCTAGAGGATCCTTCGAAACCGGTGCTAGCAGCGCTGTTAACGGAAGCGGAGCCACTAACTTCTCCCTGTTGAAACAAGCAGGGGATGTCGAAGAGAATCCCGGGCCAATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCTTCGGCTACGGCCTGCAGTGCTTCGCCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCTACCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAGGCGCGCCCCTGCAGGGAATTCGATATCAAGCTTATCGATAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTATGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCATCGATACCGAGCGCTGCTCGAGAGATCTACGGGTGGCATCCCTGTGACCCCTCCCCAGTGCCTCTCCTGGCCCTGGAAGTTGCCACTCCAGTGCCCACCAGCCTTGTCCTAATAAAATTAAGTTGCATCATTTTGTCTGACTAGGTGTCCTTCTATAATATTATGGGGTGGAGGGGGGTGGTATGGAGCAAGGGGCAAGTTGGGAAGACAACCTGTAGGGCCTGCGGGGTCTATTGGGAACCAAGCTGGAGTGCAGTGGCACAATCTTGGCTCACTGCAATCTCCGCCTCCTGGGTTCAAGCGATTCTCCTGCCTCAGCCTCCCGAGTTGTTGGGATTCCAGGCATGCATGACCAGGCTCAGCTAATTTTTGTTTTTTTGGTAGAGACGGGGTTTCACCATATTGGCCAGGCTGGTCTCCAACTCCTAATCTCAGGTGATCTACCCACCTTGGCCTCCCAAATTGCTGGGATTACAGGCGTGAACCACTGCTCCCTTCCCTGTCCTTCTGATTTTGTAGGTAACCACGTGCGGACCGAGCGGCCGCAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGGGGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATACGTCAAAGCAACCATAGTACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTTGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGGCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTLHAGCGGCCGCACGCGTTTAATTAAGTGTCTAATTGGACAGGGAGATGGCTCAGGTGTGGAAAGCCAATATAGCGGTGCATGCTTGCAAGGCCAGGAGTAATGATGGGGACATAGGAGGATTCTGGGAGCCTGGCCGGATAACCTATCCCCTAACACATAAGCACCAGGTCCCACCCAGTAAGAGGCCCTGCCTCGAAGAAGAAGTGGAGGGTCCCTGAGGAGGAAGGGCATTTGGGGTTATCCTCTGGTCTCCAAATGCATCCCTCCTCCCACACAAACACACAAAGACACTTTCACTTGTATCTGATGCCTGGTTAGCCATGAGCGTAAGCTAGGATTGTGAATTCAGCCACCCTGGTACAGCAACTATTGAGTGCTTAGTGTATACTCTGGGTTCGAAACACCGAGGACAGAAATGAATAAGATGCAGTTTCTGTCCTCAGGACCACCAGAAGGAAACCAGGACGGGTGGCATCCTGTGTAGACAGACCCCGGAGTGTGTGCGTGTTGCTTAGCTAAGTGATTTTCTGGAGTGTGTTCTGGCTCCCCGGCCGTCCTCCCGAACTAGCAGACCACTCGTGCTGCCGGAGTCCTGGAGCGACAGGAGACTCAGCCTGACTTCTCTCTTAACTCTCCCTCCGCGCAGGTTCCATAGCTCAACATGGTCCCCTGGTTCCTCCTGTCTCTGCTGCTACTTGCGAGGCCTGTGCCTGGGGTGGCCTACTCTGTGTCACTCCCGGCCTCCTTCCTGGAGGATGTAGCCGGCAGCGGGGAAGCTGAGGGTTCTTCAGCCTCTTCCCCGAGCCTGCCGCCGCCTGGGACTCCAGCCTTCAGTCCCACACCGGAGAGACCCCAGCCCACAGCTCTGGACGGCCCCGTGCCACCCACCAACCTCCTGGAAGGGATCATGGATTTCTTCCGGCAGTACGTGATGCTCATCGCGGTGGTGGGCTCGCTGACCTTCCTCATCATGTTCATAGTCTGCGCCGCCCTCATCACGCGCCAGAAGCACAAGGCCACAGCCTACTACCCATCCTCGTTCCCTGAAAAGAAGTATGTGGACCAGAGAGACCGGGCTGGGGGACCCCGTACCTTCAGCGAGGTCCCTGACAGGGCACCTGACAGCCGGCATGAAGAAGGCCTGGACACCTCCCATCAGCTCCAGGCTGACATTCTGGCTGCTACCCAGAACCTCCGGTCTCCAGCTAGAGCCCTGCCAGGCAATGGGGAGGGAGCAAAGCCTGTGAAGGGTGGGTCGGAGGAGGAGGAGGAAGAGGTGCTCAGCGGTCAGGAGGAGGCCCAGGAAGCCCCAGTATGTGGGGTCACTGAAGAGAAGCTGGGGGTCCCAGAGGAGTCGGTCTCAGCAGAGGCTGAAGGGGTTCCTGCCACCAGTGAGGGCCAAGGGGAAGCAGAAGGGTCTTTCTCCTTAGCCCAGGAATCCCAGGGAGCAACTGGTCCTCCTGAAAGTCCCTGTGCCTGCAACAGAGTCTCTCCCAGTGTCP2A-CreERT2TGTGCCTGCAACAGAGTCTCTCCCAGTGTCGGAAGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCTATGGCCAATTTACTGACCGTACACCAAAATTTGCCTGCATTACCGGTCGATGCAACGAGTGATGAGGTTCGCAAGAACCTGATGGACATGTTCAGGGATCGCCAGGCGTTTTCTGAGCATACCTGGAAAATGCTTCTGTCCGTTTGCCGGTCGTGGGCGGCATGGTGCAAGTTGAATAACCGGAAATGGTTTCCCGCAGAACCTGAAGATGTTCGCGATTATCTTCTATATCTTCAGGCGCGCGGTCTGGCAGTAAAAACTATCCAGCAACATTTGGGCCAGCTAAACATGCTTCATCGTCGGTCCGGGCTGCCACGACCAAGTGACAGCAATGCTGTTTCACTGGTTATGCGGCGGATCCGAAAAGAAAACGTTGATGCCGGTGAACGTGCAAAACAGGCTCTAGCGTTCGAACGCACTGATTTCGACCAGGTTCGTTCACTCATGGAAAATAGCGATCGCTGCCAGGATATACGTAATCTGGCATTTCTGGGGATTGCTTATAACACCCTGTTACGTATAGCCGAAATTGCCAGGATCAGGGTTAAAGATATCTCACGTACTGACGGTGGGAGAATGTTAATCCATATTGGCAGAACGAAAACGCTGGTTAGCACCGCAGGTGTAGAGAAGGCACTTAGCCTGGGGGTAACTAAACTGGTCGAGCGATGGATTTCCGTCTCTGGTGTAGCTGATGATCCGAATAACTACCTGTTTTGCCGGGTCAGAAAAAATGGTGTTGCCGCGCCATCTGCCACCAGCCAGCTATCAACTCGCGCCCTGGAAGGGATTTTTGAAGCAACTCATCGATTGATTTACGGCGCTAAGGATGACTCTGGTCAGAGATACCTGGCCTGGTCTGGACACAGTGCCCGTGTCGGAGCCGCGCGAGATATGGCCCGCGCTGGAGTTTCAATACCGGAGATCATGCAAGCTGGTGGCTGGACCAATGTAAATATTGTCATGAACTATATCCGTAACCTGGATAGTGAAACAGGGGCAATGGTGCGCCTGCTGGAAGATGGCGATCTCGAGCCATCTGCTGGAGACATGAGAGCTGCCAACCTTTGGCCAAGCCCGCTCATGATCAAACGCTCTAAGAAGAACAGCCTGGCCTTGTCCCTGACGGCCGACCAGATGGTCAGTGCCTTGTTGGATGCTGAGCCCCCCATACTCTATTCCGAGTATGATCCTACCAGACCCTTCAGTGAAGCTTCGATGATGGGCTTACTGACCAACCTGGCAGACAGGGAGCTGGTTCACATGATCAACTGGGCGAAGAGGGTGCCAGGCTTTGTGGATTTGACCCTCCATGATCAGGTCCACCTTCTAGAATGTGCCTGGCTAGAGATCCTGATGATTGGTCTCGTCTGGCGCTCCATGGAGCACCCAGTGAAGCTACTGTTTGCTCCTAACTTGCTCTTGGACAGGAACCAGGGAAAATGTGTAGAGGGCATGGTGGAGATCTTCGACATGCTGCTGGCTACATCATCTCGGTTCCGCATGATGAATCTGCAGGGAGAGGAGTTTGTGTGCCTCAAATCTATTATTTTGCTTAATTCTGGAGTGTACACATTTCTGTCCAGCACCCTGAAGTCTCTGGAAGAGAAGGACCATATCCACCGAGTCCTGGACAAGATCACAGACACTTTGATCCACCTGATGGCCAAGGCAGGCCTGACCCTGCAGCAGCAGCACCAGCGGCTGGCCCAGCTCCTCCTCATCCTCTCCCACATCAGGCACATGAGTAACAAAGGCATGGAGCATCTGTACAGCATGAAGTGCAAGAACGTGGTGCCCCTCTATGACCTGCTGCTGGAGGCGGCGGACGCCCACCGCCTACATGCGCCCACTAGCCGTGGAGGGGCATCCGTGGAGGAGACGGACCAAAGCCACTTGGCCACTGCGGGCTCTACTTCATCGCATTCCTTGCAAAAGTATTACATCACGGGGGAGGCAGAGGGTTTCCCTGCCACAGCTTGACATGCCCCAGAACTGCTGGGACCCGAATGTP2A-EGFPTGTGCCTGCAACAGAGTCTCTCCCAGTGTCGGAAGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCTATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAACATGCCCCAGAACTGCTGGGACCCGAATGTRHACATGCCCCAGAACTGCTGGGACCCGAATGTTGGGTCCTTGAGGGTCACCTCTTTGGTCAAGAAAGGCATTCAGCTCTAACTGCTCCTTGATACCACGTGGCTTGGCCATTGCTGGTGCCAAGGCTGACCCCGAACTGGCAGAGCCGATGCCCTCTGGTGCACCCCAGGAAACATCTCCCCAAGTTCCAGCGCCCTTAATGACTCTTGCCACCCTGGGGGCTTCACCCTAACGCACCACTTCTCTGGAAGGGGAAGGCCAGACACATGCCAGTTGGGGCTGCATGAGGCAGTCCTCAGAGCAGAAGGGGACCAGGCCAGAGGCCACCTGTGACGGGGCAAACTGCATCTCGGCTGTGGAGACCAGAGGGGCTGTTAGATTTGGAAGACATCAATGACTGGGCCTGCGGCGCAGCCCGTGTCTGGTAATACCAGGGACGGCAGAGGCGTTTGCATCTTCCCATCACCTGCAATGTCGCTGTCACTCTGCCCCTGTTCAGTGGACTTGATTAGTTAGGAAACTTCTGGAAGGGGCCCCCTACTTTATATCACAGAGTTTGCCCTAGACACCCCGTGGAAACACAAACTCAAAATCAAGTGGCTTTAGGAGCCGCTGTGCCCCTCCACAAGCTGACATGGCTACTCTAAGGGTTCCTGCTGGGCTGGCTTTGCTACGCTTTCCTCAAGCTGCTTTCTTATTACCAGGATGCCTCACAGCTACAAAGTCCAATCTCACAGCACCCGCACTGGAAAATACTGTTCTCCATTTTTTTCCAAGGCACCCTGCTTTATGATTGGCTCAGAGTTGGAATATGGGATGCAGGGCGTCTGGCTCTCTCAAGTGTCAAGCAACCCTGGCTTCCGCATGTGGGGCGAGGGGAGTGAGCAGAACTCTTCTCTGTCAGTCCACGAAGTAGACTAGAAGCCAACGGGTGCCTGGGGGCAACTGACGGTCTGGATTCTGACGCCCAGCCTGGAGCAGGGGCCTGGCCCATCCTATGCTCACACAGTGGTCTGGCAGCCTGAGCCCACACAACTCCTCAGTCCTTGACAATGCTTAGGCTCTGTTCTGAGGGACTCCCCACACCTCCATTCAGGGCTCCCCGAGTCCTCAGCTCTTCGGATGTGGATATATGACACACTAGCATAATAAATCTTGATCTGGCTTGAGTCTCTCTGGATTATCTGCACAGGTCTGCAGGAATGGGGTGTTCATGGGCTGGGGGCAGCATTGGGGGGAGGGCATCAGCCAGGTCACAGAGCTGAGACTGTGTGTGGGTGCTCACAGGTGTTGGACCCACGTACCTGAGGAGTCCTGGAGGTGGTGTGTCAAACGTACAGACACTTTGCTGCCTGGCTCTGCAGCTGGTGCAGCCATGCCACAGTCACTTCTTTGTGCTGTGGGACAAAGCTGGGGCAAAGTCATGGCTGCCAATTCCCTACAGATGGGTGAGAACTGACTGAGCCTCAGGGCGATATCAAGCTTATCGATAATCAACCTCTTmem119 gene to generate such mouse lines , into the stop codon of murine Tmem119 animals B. This ie 3\u2032 UTR C,D. AddiTmem119 is currently unknown, both its temporally distinct pattern and high expression level suggest functional importance of this gene . We further examined TMEM119 protein expression using immunofluorescence and found no difference between WT and Tmem119-EGFP+/\u2212 knock-in mice Together, these data support the conclusion that Tmem119 knock-in mice were generated with precision.Although the function of endogenous his gene . We thusTmem119-EGFP+/\u2212 knock-in mice , number of processes , convex hull area , and total process length are comparable between microglia from control and knock-in mice. These data suggest that the genetic modification does not affect basic microglial properties.To further test whether the genetic engineering could affect the properties of microglia, we examined basic morphologic features of reconstructed immunostained microglia in WT and -in mice H,I. UsinTmem119-EGFP line, we used confocal microscopy on brain slices. Native EGFP fluorescence and antibody-enhanced signal were visible across the brain in sagittal brain sections prepared from P25 Tmem119-EGFP+/\u2212 mice . We determined that all CD11b+CD45lo microglia corresponding to non-microglial cells express EGFP to all parenchymal microglia (IBA1+) revealed very high efficiency of recombination (+IBA1+) among all tdTomato-labeled cells (tdT+) in different parenchymal regions, and found high specificity close to 100% .In this study, we addressed the critical need for transgenic mouse lines that specifically label or control microglia, but not other closely related cell types with overlapping signature gene expression . We targTmem119 out through insertion of EGFP or CreERT2 into the Tmem119 ORF and instead chose to preserve expression by using a polycistronic knock-in approach with ribosomal skipping peptide P2A +/\u2212 mice show tdTomato expression in IBA1-expressing microglia, indicating that this line is suitable for Cre-dependent manipulation of genes and transgene expression in microglia without affecting other major neuronal or glial cell types in the CNS (Cx3cr1 mouse line (Tmem119-CreERT2 line a highly useful tool for the conditional study of microglia, while also suggesting potential application for leptomeningeal cells.Our experiments using the CNS . In this the CNS K,L. The use line , and miguse line N\u2013P. Thisuse line , which muse line Q\u2013V. Althuse line Q\u2013S. Togeuse line . TogetheTmem119-EGFP and Tmem119-CreERT2; Ai14 mice revealed complete absence and minimal expression, respectively (Tmem119-EGFP knock-in mice was expected based on previous observations, tdTomato expression in 3% blood monocytes of the Tmem119-CreERT2; Ai14 mice was rather surprising. Similar to other brain macrophages discussed above, blood monocytes may express Tmem119 at very low, almost undetectable levels, which may nonetheless be sufficient to cause occasional all-or-none Cre recombination events. Together, these data render the Tmem119 knock-in lines powerful tools to investigate microglia in health and disease without significant confounds from monocyte contribution.Examination of EGFP and tdTomato expression in blood monocytes of ectively . While, Tmem119-EGFP line might be of great value (Tmem119-EGFP line may enable cell sorting without the need for antibodies, possibly even at early developmental stages before the onset of TMEM119 appearance on the membrane, as well as in vivo imaging approaches. Furthermore, the absence of EGFP fluorescence in blood monocytes renders this line uniquely useful for the study of microglia in brain disorders that involve monocyte infiltration. The Tmem119-CreERT2 line will significantly facilitate conditional control of gene expression selectively in microglia, especially in cases when monocytes, but not leptomeningeal cells, could likely confound interpretations. Compared with constitutive Cre lines, which cumulatively recombine throughout development and thus deny temporal control, the CreERT2 line will allow precise control of gene expression to examine the role of microglia in temporally restricted developmental processes. In this capacity, dose-dependency of CreERT2 and potentially resulting mosaic expression of recombined alleles using low doses may allow the study of candidate genes in adjacent microglia populations sharing the same microenvironment. Beyond the application of Tmem119 knock-in and other currently available mouse lines, genetic approaches such as targeting other microglia-signature genes, engineering highly specific artificial promoters and regulatory regions, and using intersectional split-Cre approaches hold great promise to produce even more specific tools to study microglia in the future.During the past decade, available microglia lines have been instrumental in advancing our understanding of microglia. These lines remain powerful tools to study microglia function. At the same time, it is widely acknowledged that ontogenetic differences, as well as differences in localization and microenvironment between parenchymal microglia and their closely related cells from the monophagocytic system warrant more discerning observation and manipulation . Recentlat value B. In add"} +{"text": "Abs targeting the HA head of influenza viruses are often associated with protection from influenza virus infections. These Abs typically have limited breadth, since mutations frequently arise in HA head epitopes. New vaccines targeting the more conserved HA stalk domain are being developed. Abs that target the HA stalk are protective in animal models, but it is unknown if these Abs exist at protective levels in humans. Here, we completed experiments to determine if Abs against the HA head and stalk were associated with protection from naturally acquired human influenza virus infections during the 2015\u20132016 influenza season. in vitro antibody-dependent cellular cytotoxicity activity. In passive transfer experiments, sera from participants with high HAI activity efficiently protected mice, while sera with low HAI activity protected mice to a lower extent. Our data suggest that HA head Abs are more efficient at protecting against H1N1 infection than HA stalk Abs.Seasonal influenza viruses are a major cause of human disease worldwide. Most neutralizing antibodies (Abs) elicited by influenza viruses target the head domain of the hemagglutinin (HA) protein. Anti-HA head Abs can be highly potent, but they have limited breadth since the HA head is variable. There is great interest in developing new universal immunization strategies that elicit broadly neutralizing Abs against conserved regions of HA, such as the stalk domain. Although HA stalk Abs can provide protection in animal models, it is unknown if they are present at sufficient levels in humans to provide protection against naturally acquired influenza virus infections. Here, we quantified H1N1 HA head- and stalk-specific Abs in 179 adults hospitalized during the 2015\u20132016 influenza virus season. We found that HA head Abs, as measured by hemagglutinin inhibition (HAI) assays, were associated with protection against naturally acquired H1N1 infection. HA stalk-specific serum total IgG titers were also associated with protection, but this association was attenuated and not statistically significant after adjustment for HA head-specific Ab titers. We found slightly higher titers of HA stalk-specific IgG1 and IgA Abs in sera from uninfected participants than in sera from infected participants; however, we found no difference in serum IMPORTANCE Abs targeting the HA head of influenza viruses are often associated with protection from influenza virus infections. These Abs typically have limited breadth, since mutations frequently arise in HA head epitopes. New vaccines targeting the more conserved HA stalk domain are being developed. Abs that target the HA stalk are protective in animal models, but it is unknown if these Abs exist at protective levels in humans. Here, we completed experiments to determine if Abs against the HA head and stalk were associated with protection from naturally acquired human influenza virus infections during the 2015\u20132016 influenza season. Seasonal influenza viruses cause annual epidemics worldwide. Although seasonal influenza vaccines usually provide moderate protection against circulating strains, vaccine effectiveness can be low when there are antigenic mismatches between vaccine strains and circulating strains , 2. Addiin vitro (\u2013Antibody (Ab)-mediated immunity is important for protecting against influenza virus infections . The vir5\u2013in vitro \u201317.Conventional influenza vaccines effectively elicit HA head-reactive Abs but not HA stalk Abs . As a reHA stalk Abs protect animals from group 1 and group 2 influenza A virus infections , 23\u201329. 23\u201330\u2013Despite the recent interest in developing new HA stalk-based vaccines, the amount of HA stalk Abs required to protect humans from influenza virus infections and influenza-related disease has not been established. A recent human pH1N1 challenge study demonstrated that HA stalk Ab titers are associated with reduced viral shedding but are not independently associated with protection against influenza infection . While h(This article was submitted to an online preprint archive .)We analyzed sera collected from 179 participants enrolled in a hospital-based study during the 2015\u20132016 influenza season . Adults We quantified serum titers of HA head-specific Abs against the predominant 2015\u20132016 H1N1 strain using hemagglutination inhibition (HAI) assays . HAI assWe next quantified relative titers of H1 stalk IgG Abs using enzyme-linked immunosorbent assays (ELISAs) coated with headless H1 proteins . SimilarAlthough both HAI titers and HA stalk IgG titers were associated with H1N1 protection in unadjusted models , only HAWe completed several experiments to validate our HA stalk IgG data. Headless H1 proteins are engineered to possess only the HA stalk domain and not the HA globular head domain . We compSome HA stalk Abs mediate protection through nonneutralizing mechanisms that involve processes such as ADCC . IgG1 anWe next evaluated serum HA stalk IgA Abs, since IgA Abs can be important for controlling respiratory infections. For example, mucosal IgA potently reduces the risk of influenza transmission events in guinea pigs in a dose-dependent manner and suppin vitro and in vivo -reporter influenza viruses and HA head (EM-4C04). For these assays, we incubated HA-expressing 293T cells with serum and then added human peripheral blood mononuclear cells (PBMCs). We then measured CD107a (LAMP1) expression on CD3\u2212 CD56+ NK cells by flow cytometry. CD107a is a sensitive NK cell degranulation marker whose expression levels strongly correlate with cytokine production and cytotoxicity by NK cells in response to Ab-Fc receptor engagement and uninfected individuals that had low (\u226440) HAI titers (abbreviated as uninfected HAIlow). We also passively transferred pooled sera from infected individuals, all of whom had low (\u226440) HAI titers (abbreviated as infected HAIlow). For these experiments, equal volumes of human sera were transferred for each experimental condition. Mice were challenged with a sublethal dose of H1N1 4 h after serum transfer, and body weights were monitored for 15\u2009days was larger than the effect size of HA stalk Ab-associated protection (14.2% reduced risk of infection for every 2-fold increase in titer). In our study, HAI titers were independently associated with protection in adjusted models; however, HA stalk Abs were not. However, the effects of both HAI and HA stalk Ab titers were only slightly attenuated in our adjusted model, and it is possible that our relatively small sample size limited our ability to detect an independent association between HA stalk titers and protection.There are several limitations to our study. Since our sample size was relatively small, we only evaluated the contribution of Abs to the HA head and stalk. Larger studies will be required to independently evaluate other immune correlates of protection. For example, it will be important for future studies to evaluate the relationship between HA head and stalk Ab-associated protection and neuraminidase (NA) Ab-associated protection. Recent studies have shown that NA Abs are associated with protection in an H1N1 challenge cohort , and NA de novo Ab responses to H1N1 infection, which could convolute the analyses of Ab types associated with protection. While this is a possibility, it is less of a concern since we found that all infected individuals have very low HAI titers. If our infected participants were making de novo Ab responses, we would anticipate that some of them would have high HAI titers to the infecting H1N1 virus. In addition, Ab titers do not typically increase as days from symptom onset to blood specimen collection increases , we cannot exclude that some participants in our studies were infected for a prolonged period of time before being admitted to the hospital. This raises the possibility that some participants have already mounted ncreases , which sin vitro neutralization titers contributed to protection in these experiments.It is interesting that n titers , but notn titers , were ashead Abs \u20139, whereead Abs \u2013. It shouead Abs \u2013, which cead Abs \u2013, and theead Abs \u2013 and D. ITaken together, our findings provide important new insights into the prevalence and functionality of HA head and stalk Abs in humans. Future studies that tease out the interdependence of HA head and stalk Abs, as well as Abs and T cells against other viral antigens, will be useful in guiding the development of new universal influenza vaccine antigens.in vitro neutralization assays, ADCC assays, and passive transfers) were completed at the University of Pennsylvania using deidentified sera.During the 2015\u20132016 influenza season, adult (\u226518\u2009years) patients hospitalized for treatment of acute respiratory illnesses at the University of Michigan Hospital in Ann Arbor, MI, and Henry Ford Hospital in Detroit, MI, were prospectively enrolled in a case test-negative design study of influenza vaccine effectiveness. All participants provided informed consent and were enrolled \u226410\u2009days from illness onset during the period of influenza circulation (January to April of 2015 and 2016). Participants completed an enrollment interview and had throat and nasal swab specimens collected and combined for influenza virus identification. Influenza vaccination status was defined by self-report and documentation in the electronic medical record and Michigan Care Improvement Registry (MCIR). When available, clinical serum specimens collected as early as possible after hospital admission were retrieved; all specimens were collected \u226410\u2009days from illness onset based on the enrollment case definition. Studies involving humans were approved by the Institutional Review Boards of the University of Michigan and University of Pennsylvania. All experiments ethyl chloromethyl ketone (TPCK)-treated trypsin, HEPES, and gentamicin. Virus was isolated from the infected MDCK cells 3\u2009days later. We extracted viral RNA and sequenced the HA gene of A/HUP/04/16.Viruses possessing A/California/07/2009 HA and NA or A/HUP/04/2016 HA and NA were generated by reverse genetics using internal genes from A/Puerto Rico/08/1934. Viruses were engineered to possess the Q226R HA mutation, which facilitates viral growth in chicken eggs. Viruses were grown in fertilized chicken eggs, and the HA gene was sequenced to verify that additional mutations did not arise during propagation. We isolated the A/HUP/04/2016 virus from respiratory secretions obtained from a patient at the Hospital of the University of Pennsylvania in 2016. For this process, deidentified clinical material from the Hospital of the University of Pennsylvania Clinical Virology Laboratory was added to Madin-Darby canine kidney (MDCK) cells in serum-free medium with 2HCO3, 300\u2009mM NaCl, and 20\u2009mM imidazole, and then eluted using pH 8 buffer containing 50\u2009mM Na2HCO3, 300\u2009mM NaCl, and 300\u2009mM imidazole. Purified protein was buffer exchanged into phosphate-buffered saline . Following purification, the headless HA stalk proteins were biotinylated using the Avidity BirA-500 kit (no. BirA500) and stored in aliquots at \u221280\u00b0C. Plasmids encoding the recombinant chimeric (c6/H1) HA were provided by Florian Krammer (Mt. Sinai). The detailed protocol for expression of this protein is published elsewhere in 5-ml polypropylene columns , washed with pH 8 buffer containing 50\u2009mM Nalsewhere . In briePlasmids encoding the human MAb EM-4C04, 70-1F02, and CR9114 IgG1 isotypes were provided by Patrick Wilson at the University of Chicago. The heavy-chain constant regions for IgG2, IgG3, and IgA (sequences listed below) were synthesized as a gBlock by IDT and cloned into the pSport6 vector containing the heavy chain of CR9114. All MAbs were expressed in 293T cells and purified 4 days postinfection using NAb protein A/G spin kits for the IgG isotypes or using peptide M agarose for the IgA isotype.The IgG2 sequence is CGCATGATGCGTCGACCAAGGGTCCTAGCGTTTTCCCGCTCGCACCTTGTAGTCGGAGCACCTCCGAATCTACGGCGGCGCTCGGATGTCTGGTTAAGGATTACTTTCCTGAACCTGTTACTGTATCTTGGAATTCAGGAGCACTGACATCTGGTGTACATACTTTTCCAGCGGTTTTGCAGTCATCTGGTCTTTATTCCCTGTCCAGTGTGGTAACAGTACCATCCTCAAACTTTGGAACTCAGACCTATACCTGCAATGTGGACCACAAGCCATCCAATACAAAAGTCGATAAGACTGTCGAGCGGAAGTGCTGTGTCGAATGCCCTCCCTGCCCCGCTCCGCCGGTTGCAGGGCCAAGTGTATTTCTTTTTCCACCAAAACCAAAAGATACGCTTATGATATCTCGCACGCCTGAAGTAACCTGCGTAGTCGTTGATGTAAGTCACGAGGATCCCGAAGTTCAATTCAATTGGTATGTAGATGGCGTTGAAGTGCATAATGCAAAGACCAAACCTAGAGAAGAACAATTCAATAGTACCTTTCGCGTGGTTAGCGTACTCACAGTCGTCCACCAGGATTGGCTGAATGGGAAGGAGTACAAATGCAAGGTCTCTAACAAAGGTCTTCCGGCCCCCATAGAAAAAACGATCAGTAAGACCAAGGGGCAGCCCAGAGAGCCACAGGTTTATACGTTGCCTCCGTCTCGCGAGGAAATGACTAAAAACCAGGTCAGCCTGACTTGTTTGGTGAAAGGGTTTTACCCGAGCGATATTGCTGTGGAATGGGAGAGTAACGGGCAACCGGAGAACAATTACAAAACGACACCGCCCATGCTTGATAGTGATGGTTCCTTCTTCTTGTACAGCAAGTTGACGGTTGATAAATCCAGGTGGCAGCAAGGAAATGTTTTCTCTTGTTCAGTGATGCATGAGGCGCTCCACAACCATTATACGCAAAAATCACTCTCACTTTCACCGGGGAAATGAAGCTTGAGCAGGGCCT.The IgG3 sequence is CGCATGATGCGTCGACCAAAGGGCCGTCAGTCTTTCCCTTGGCGCCGTGCTCCAGGAGTACCAGCGGCGGCACCGCGGCGTTGGGATGTCTTGTCAAGGATTATTTTCCCGAACCCGTCACCGTAAGCTGGAACAGTGGGGCATTGACGTCTGGCGTTCATACTTTTCCGGCAGTACTTCAGAGTTCCGGCCTTTATTCTTTGTCAAGCGTTGTTACCGTACCATCCAGTAGCCTTGGCACCCAGACCTACACCTGTAATGTTAATCACAAACCAAGTAACACCAAGGTTGATAAGAGGGTTGAGCTTAAAACACCGCTTGGTGACACAACCCATACGTGTCCAAGATGTCCGGAGCCGAAGAGTTGTGATACCCCGCCGCCGTGTCCTCGCTGTCCGGAACCAAAGAGCTGTGATACCCCCCCACCTTGTCCCAGATGTCCTGAACCGAAATCATGTGACACGCCACCACCTTGCCCAAGATGTCCCGCGCCAGAGCTGCTGGGTGGGCCCAGCGTATTTCTTTTTCCACCCAAACCGAAGGATACCCTTATGATAAGCAGGACTCCCGAGGTTACCTGCGTGGTGGTTGACGTAAGTCACGAAGACCCCGAAGTCCAATTCAAATGGTATGTTGATGGGGTCGAAGTACACAACGCGAAGACTAAACCGAGAGAGGAACAGTATAATAGCACATTCCGGGTTGTTTCCGTACTTACAGTACTTCATCAGGACTGGCTTAATGGCAAGGAGTACAAGTGCAAAGTCAGTAACAAGGCACTCCCTGCTCCGATTGAAAAGACAATATCAAAGACGAAAGGTCAACCCAGAGAGCCGCAGGTCTACACACTCCCTCCGTCCAGAGAAGAGATGACGAAAAACCAAGTTTCATTGACGTGCCTCGTTAAAGGATTCTACCCAAGCGACATAGCTGTTGAGTGGGAGAGCAGCGGCCAGCCTGAGAACAATTATAATACTACCCCCCCCATGCTCGACTCTGATGGTAGTTTTTTTCTGTACTCCAAGCTGACGGTAGACAAAAGTAGATGGCAGCAAGGCAACATCTTCAGTTGCTCTGTTATGCACGAGGCGTTGCACAACCGATTCACACAGAAGTCACTGAGCCTGTCTCCGGGTAAATGAAGCTTGAGCAGGGCCT.The IgA sequence is CGCATGATGCGTCGACTTCTCCAAAAGTGTTTCCCCTCAGTTTGTGTTCCACTCAACCGGATGGTAACGTGGTGATTGCTTGTCTCGTGCAAGGTTTTTTCCCACAGGAACCGCTGAGTGTTACATGGTCAGAGTCAGGCCAAGGTGTAACCGCGCGCAACTTTCCCCCTTCACAGGACGCTAGTGGCGATCTGTATACTACCTCCTCTCAGCTCACTCTTCCCGCCACACAATGCCTCGCTGGGAAATCTGTAACCTGCCACGTTAAACATTACACTAATCCATCACAGGACGTTACCGTGCCGTGCCCTGTACCATCCACGCCGCCTACGCCGTCACCGTCAACTCCTCCTACTCCCTCACCCTCTTGTTGTCACCCGCGCCTCTCTCTTCACAGACCGGCCTTGGAGGACCTTCTCCTTGGGTCTGAGGCGAATTTGACTTGCACGCTCACGGGGTTGCGGGACGCTAGTGGGGTTACGTTTACATGGACACCTTCATCAGGGAAGTCTGCCGTTCAGGGCCCCCCAGAGCGCGATTTGTGCGGGTGTTACAGCGTATCTTCTGTGCTGCCTGGGTGCGCTGAGCCCTGGAATCACGGCAAAACGTTTACCTGCACCGCTGCTTACCCAGAGAGCAAAACCCCTCTGACGGCTACATTGTCCAAGTCAGGCAACACATTTCGCCCCGAAGTCCACCTCTTGCCACCTCCATCCGAAGAACTCGCCCTGAACGAACTCGTGACGCTGACGTGCCTTGCACGCGGCTTTTCCCCGAAAGACGTTCTCGTCCGGTGGCTTCAAGGTTCTCAGGAACTCCCACGGGAGAAGTACCTGACCTGGGCTTCACGCCAGGAACCTTCACAAGGGACGACCACTTTCGCAGTCACGTCAATTCTGAGAGTTGCCGCTGAGGACTGGAAGAAGGGAGATACTTTCAGTTGTATGGTAGGTCACGAAGCACTGCCGCTGGCATTTACGCAGAAAACCATCGATCGGCTTGCCGGAAAGCCTACTCATGTTAACGTTTCCGTAGTGATGGCGGAGGTAGATGGCACATGTTACTGAAGCTTGAGCAGGGCCT.Serum samples were pretreated with receptor-destroying enzyme followed by hemadsorption, in accordance with WHO recommended protocols . HAI titHeadless HA ELISAs were performed on 96-well Immulon 4HBX flat-bottom microtiter plates coated with 0.5\u2009\u03bcg/well of streptavidin . We completed total IgG headless HA ELISAs with all serum samples and isotype headless HA ELISAs with serum samples that had sufficient volumes. Biotinylated headless HA protein was diluted in biotinylation buffer containing 1\u00d7 Tris-buffered saline , 0.005% Tween , and 0.1% bovine serum albumin to 0.25\u2009\u03bcg/ml, and 50\u2009\u03bcl was added per well and incubated on a rocker for 1 h at room temperature. Each well was then blocked for an additional 1 h at room temperature using biotinylation blocking buffer containing 1\u00d7 TBS , 0.005% Tween 20 , and 1% bovine serum albumin . Each serum sample was serially diluted in biotinylation buffer , added to the ELISA plates, and allowed to incubate for 1 h at room temperature on a rocker. As a control, we added the human CR9114 stalk-specific MAb, starting at 0.03\u2009\u03bcg/ml, to verify equal coating of plates and to determine relative serum titers. Peroxidase-conjugated goat anti-human IgG , peroxidase-conjugated mouse anti-human IgG1 , peroxidase-conjugated mouse anti-human IgG2 , peroxidase-conjugated mouse anti-human IgG3 , or peroxidase-conjugated goat anti-human IgA was incubated for 1 h at room temperature on a rocker. Finally, SureBlue TMB peroxidase substrate was added to each well, and the reaction was stopped with the addition of 250\u2009mM HCl solution. Plates were extensively washed with PBS and 0.1% Tween 20 between each step using a BioTek 405 LS microplate washer. Relative titers were determined using a consistent concentration of the CR9114 MAb for each plate and reported as the corresponding inverse of the serum dilution that generated the equivalent optical densities (OD). Each type of ELISA was performed twice.Chimeric HA ELISAs were performed on 96-well Immulon 4HBX flat-bottom microtiter plates . HA proteins were diluted in PBS to 2\u2009\u03bcg/ml and coated at 50\u2009\u03bcl per well overnight at 4\u00b0C. Plates were blocked using an ELISA buffer containing 3% goat serum , 0.5% milk , and 0.1% Tween 20 in PBS 1\u00d7 for 2 h at room temperature. Each serum sample was serially diluted in the ELISA buffer (starting at 1:100 dilutions), added to the ELISA plates, and allowed to incubate for 2 h at room temperature. As a control, we added the human CR9114 stalk-specific MAb, starting at 0.03\u2009\u03bcg/ml, to verify equal coating of plates and to determine relative serum titers. Peroxidase-conjugated goat anti-human IgG next was incubated for 1 h at room temperature. Finally, SureBlue TMB peroxidase substrate was added to each well, and the reaction was stopped with the addition of 250\u2009mM HCl solution. Plates were extensively washed with PBS and 0.1% Tween 20 between each step using a BioTek 405 LS microplate washer. Relative titers were determined using a consistent concentration of the CR9114 MAb for each plate and reported as the corresponding inverse of the serum dilution that generated the equivalent OD. Each ELISA was performed twice.Competition ELISAs were performed on 96-well Immulon 4HBX flat-bottom microtiter plates . HA proteins were diluted in 1\u00d7 Dulbecco\u2019s PBS to 2\u2009\u03bcg/ml and coated at 50\u2009\u03bcl per well overnight at 4\u00b0C. Plates were blocked using the biotinylation blocking buffer, described earlier, for 2 h at room temperature. Each serum sample was serially diluted in biotinylation buffer (starting at 1:10 dilution), added to the ELISA plates, and allowed to incubate for 1 h at room temperature before adding human 70-1F02 MAb that haIn vitro neutralization assays were completed using a subset of samples. We excluded samples that had limited amounts of sera. Plasmids encoding pH1N1 viruses possessing genes encoding eGFP in place of most of the PB1 gene segment were provided by Jesse Bloom at The Fred Hutchinson Cancer Research Center. The eGFP segment retained the noncoding and 80 terminal coding nucleotides, allowing this segment to be efficiently and stably packaged into the virions. Detailed protocols for the reverse genetics, expression, and in vitro neutralization assays using the recombinant viruses have been published elsewhere 24 h before transfection. 293T cells were then transfected using 20\u2009\u03bcl Opti-MEM , 1\u2009\u03bcl Lipofectamine 2000 , and 500\u2009ng plasmids encoding the HA gene from A/California/07/09 per well and incubated at 37\u00b0C for approximately 30 h before performing the ADCC assay. Approximately 12 h before performing the ADCC assay, frozen PBMCs from four separate donors (obtained through the University of Pennsylvania Human Immunology Core) were thawed at 37\u00b0C and then washed 3\u00d7 using 15 ml of warmed complete RPMI medium . Each aliquot of PBMCs was then transferred to a 50-ml conical tube and rested overnight in 23 ml of complete RPMI medium at a 5\u00b0 angle, with the cap loosened to allow for gas exchange. On the day of the assay, serum samples were diluted in Dulbecco\u2019s modified Eagle\u2019s medium supplemented with 10% FBS at a 1:10 dilution. As a control for this assay, the human CR9114 HA stalk-specific MAb was included at a concentration of 5\u2009\u03bcg/ml to ensure efficient activation of ADCC. Transfected 293T cells were loosened by pipetting and transferred to a 96-well U-bottom plate and spun down for 1 min at 1,200 rpm, and the medium was flicked out. The serum/MAb dilutions were transferred to the plates containing the transfected 293T cells and were mixed with the transfected cells by gentle pipetting and incubated at 37\u00b0C for 2 h. PBMC aliquots were combined, spun down, and counted, and a master mix of 2e7 cells/ml was set up using complete RPMI medium. Aliquots of the PBMC master mix were set up for the live/dead and unstained control wells. Phycoerythrin-conjugated mouse anti-human CD107a was added at a 1:50 dilution. Brefeldin A was added to 10\u2009\u03bcg/ml. Monensin was added to 5\u2009\u03bcl per 1\u2009ml of PBMC master mix concentration. An aliquot of 200\u2009\u03bcl was made, phorbol myristate acetate was added to a 5\u2009\u03bcg/ml concentration, and ionomycin was added to a 1\u2009\u03bcg/ml concentration. Serum/cell suspensions were spun down at 1,200 rpm for 1 min, and medium was flicked out. The PBMC master mix and the aliquots for the various controls were plated at 50\u2009\u03bcl per well and mixed gently by pipetting, followed by incubation at 37\u00b0C for 4 h. Cells were then stained in the following manner. Live/dead fixable near-infrared stain was diluted 1:50 in DPBS and 1% bovine serum albumin for 30 min in the dark at 4\u00b0C. Human FcR blocking reagent was diluted 1:25 in DPBS and 1% bovine serum albumin and incubated in the dark for 10 min at 4\u00b0C. Alexa Fluor 647-conjugated mouse anti-human-CD3 and BV421-conjugated mouse anti-human CD56 were diluted 1:200 in DPBS plus 1% bovine serum albumin and incubated in the dark for 30 min at room temperature. Cells were then fixed using 10% paraformaldehyde diluted in MilliQ water for 6 min at room temperature. Cells were extensively washed with DPBS and 1% bovine serum albumin between each step. Cells were stored overnight at 4\u00b0C in 100\u2009\u03bcl/well of DPBS and 1% bovine serum albumin . Flow cytometry was performed using and LSRII . Compensation controls were set up using anti-mouse Ig\u03ba beads and run for each antibody for every experiment, and voltages were adjusted accordingly. All data were analyzed in FlowJo by gating on single cells that were CD3+/mFcgR alpha chain \u2212/\u2212) that were provided by Jeffrey Ravetch at the Rockefeller University (high (>40 HAI titer), uninfected HAIlow (\u226440 HAI titer), and infected HAIlow (\u226440 HAI titer), and then heat treated for 30 min at 55\u00b0C. Serum or sterile PBS was then transferred into mice by intraperitoneal injection. Four hours posttransfer, mice were bled by submandibular puncture, anesthetized using isoflurane, and challenged intranasally using 50\u2009\u03bcl of sterile PBS containing a sublethal dose (9e4 50% tissue culture infective dose units) of A/California/07/09. ELISAs were run on the sera collected from each animal to verify that the passive transfer was successful. Mice were weighed on the day on infection and then daily for 15 days postinfection. Weight loss was reported as percent weight loss relative to the starting weight of each mouse. For the passive transfer normalized by volume, two independent experiments were performed using a mix of male and female humanized FcR mice for a total of 6 mice per group per experiment. For the passive transfer normalized by HA antibody titer, a single experiment was performed using a mix of male and female humanized FcR mice for a total of 6 mice per group, since we had limited amounts of sera available for this study. One-way analysis of variance (ANOVA) was performed for each day postinfection between groups using GraphPad Prism software.All mouse experiments were reviewed and approved by the IACUCs of the Wistar Institute and University of Pennsylvania. All passive transfer experiments were performed in humanized FcR mice .Fisher\u2019s exact tests, one-way ANOVAs, and Welch\u2019s"} +{"text": "Little is known about miRNA decay. A target-directed miRNA degradation mechanism (TDMD) has been suggested, but further investigation on endogenous targets is necessary. Here, we identify hundreds of targets eligible for TDMD and show that an endogenous RNA (Serpine1) controls the degradation of two miRNAs (miR-30b-5p and miR-30c-5p) in mouse fibroblasts. In our study, TDMD occurs when the target is expressed at relatively low levels, similar in range to those of its miRNAs (100\u2013200 copies per cell), and becomes more effective at high target:miRNA ratios (>10:1). We employ CRISPR/Cas9 to delete the miR-30 responsive element within Serpine1 3'UTR and interfere with TDMD. TDMD suppression increases miR-30b/c levels and boosts their activity towards other targets, modulating gene expression and cellular phenotypes . In conclusion, a sophisticated regulatory layer of miRNA and gene expression mediated by specific endogenous targets exists in mammalian cells. Via the target-directed miRNA degradation process, RNAs can induce degradation of miRNAs by binding with extensive complementarity. Here, the authors show Serpine1 mRNA as one such RNA that can control the levels of the endogenous miRNA miR-30b/c-5p by modulating miRNA degradation. Targets are bound through base paring between the miRNA and their miRNA responsive elements (MREs), usually located in the 3\u2032 untranslated region (3\u2032UTR)2. To act as such, any MRE usually presents complementarity to bases 2\u20137 (the seed) of miRNAs; however, other sequences, usually located near the miRNA 3\u2032 end, may also form additional base pairs and thus participate in target recognition. Due to the low levels of complementarity between miRNAs and their RNA targets, from hundreds to thousands RNAs could interact with the same miRNA sequence, as demonstrated by high-throughput experimental studies4. For the interaction with their targets to take place, miRNAs must be loaded onto Argonaute proteins (AGO) and form the core of the RNA-induced silencing complex (RISC). Within RISC, miRNAs induce silencing by target destabilisation and/or translational repression6. Computational methods, such as TargetScan7 and others8, are able to predict miRNA targets and their MREs based on seed type hierarchy (8-mer\u2009>\u20097-mer\u2013m8\u2009>\u20097-mer\u2013A1\u2009>\u20096-mer) and on sequence conservation of orthologous mRNAs as found by comparative genome analysis. Usually, target expression changes slightly when miRNA levels are perturbed10; however, the resulting phenotypic effect can be profound as targets often converge towards the same pathway or biological process.MicroRNAs (miRNAs) are an evolutionarily conserved class of small (about 18\u201322 nt long) non-coding RNAs that function in post-transcriptional regulation of gene expression11 and the target-directed miRNA degradation (TDMD) mechanism12. The ceRNA theory postulates that endogenous targets compete with each other for binding to a shared miRNA; therefore, a sudden change in the expression of a competing endogenous target might influence miRNA activity on other targets13. Most evidence in favour of the ceRNA hypothesis comes from over-expression approaches, so that the impact of ceRNAs on miRNA-mediated mechanisms in physiological settings is still debated16. In the TDMD mechanism, the RNA target (the TDMD target) promotes degradation of its miRNA18, accompanied by post-transcriptional modification of the miRNA sequence, i.e., tailing (addition of nucleotides at the 3\u2032 end) and trimming (shortening)19, and unloading from AGO20. Studies performed using artificial targets showed that extended complementarity to miRNAs 3\u2032 regions combined with a central bulge of\u2009\u2264\u20095 nt, promotes miRNA degradation21. However, TDMD molecular basis and physiological role are still obscure. Endogenous RNA targets implicated in TDMD and the role they play in modulating miRNA activity need to be further investigated, especially in non-neuronal cells. So far, the evidence for accelerated miRNA decay comes from studies on viral targets and on artificial transcripts, both characterised either by a central bulge or by perfect complementarity24. Indeed, it has been shown that, in physiological conditions, miRNA decay can be accelerated by a rapid change in gene expression , suggesting the existence of a post-transcriptional mechanism able to control miRNA levels. However, precise molecular details remain obscure. We and others have recently shed light on the dynamics of miRNA decay in mammalian cells by using new tailored approaches based on in vivo RNA labelling28. In our study, different pools of miRNAs were identified on the basis of their decay pattern: \u201cslow\u201d miRNAs, very stable (T1/2\u2009>\u200924\u2009h), and apparently downregulated through dilution by cell division; and \u201cfast\u201d miRNAs, quickly turned over in the cell (T1/2 from 4 to 14\u2009h). Intriguingly, we found a significant association between miRNA type of decay (\u201cfast\u201d or \u201cslow\u201d) and number and expression levels of targets bearing an additional complementarity to miRNA 3\u2032 ends (3C-targets27), as required for TDMD. On these bases, we hypothesised that TDMD is a general mechanism, with specific endogenous targets able to control the dynamics of degradation of their miRNAs.Intriguingly, target:miRNA interactions have been suggested to act as a bidirectional control mechanism, with targets in turn affecting miRNAs activity. Two mechanisms have been reported: the competing endogenous RNA (ceRNA) hypothesisIn this study, we provide evidence that the endogenous target Serpine1 is able to control the levels of the two miRNAs miR-30b-5p and -30c-5p by inducing their degradation during cell cycle re-entry of quiescent fibroblasts stimulated with serum. Following an in-depth investigation of the interaction between Serpine1 and miR-30b/c, we describe TDMD as a post-transcriptional mechanism that controls levels and activity of miRNAs.29) on top of the canonical seed pairing low, with 3C-scores between \u22120.01 and -0.03; (ii) mid, scoring between \u22120.03 and \u22120.05; and (iii) high, with 3C-score lower than \u22120.05 , 18,856 pairs were found, with 1083 falling in the 3C-high class ; and (b) the max contribution (over the entire time course) of each single target to the total pool of 3C-targets of a given miRNA . A few 3C-targets stood out in the analysis , a very high 3C-score for miR-30b-5p and -30c-5p, and a significant contribution to the 3C-target pool .Using the TargetScan database as a source of miRNA:target pairs, we identified endogenous RNAs that could trigger miRNA degradation and focused our analysis on targets presenting a 3\u2032 pairing contribution . Using the IsomiRage tool to map isomiRs32, we found that 3\u2032 tailing of both miR-30b/c had substantially increased, mirroring their downregulation patterns belong to two different genetic units and, therefore, are not co-transcribed and 30b-5p . To this aim, a titration curve was obtained using synthetic miR-30 RNA oligonucleotides and a Serpine1 plasmid DNA template Fig.\u00a0, suggest80) Fig.\u00a0. To rule80) Fig.\u00a0. In MRE-80) Fig.\u00a0. We also80) Fig.\u00a0, with a 80) Fig.\u00a0. In MRE-80) Fig.\u00a0. While t80) Fig.\u00a0. The lev80) Fig.\u00a0, further18, four mismatches in the 3\u2032 end were sufficient to nearly abolish TDMD fused to Serpine1 3\u2032UTR Fig.\u00a0. We used15. Conversely, TDMD suppression (as in MRE-KO cells) should result in increased miRNA activity. Using a highly sensitive reporter system to measure miR-30c activity at single-cell level , we isolated miR-30 targets based on: (i) predictions by computational approach ; (ii) experimentally supported interactions (n\u2009=\u20091037 for miR-30\u2009s) obtained by High-throughput Sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP-from TarBase7.035); and (iii) miRNA:target chimaeras (n\u2009=\u20091037 for miR-30c-5p), obtained by sequencing of Covalent Ligation of Endogenous Argonaute-bound RNAs but significantly more repressed in mutant cells and IV [genes repressed in mutant cells at late (8h-10h-12h) time-points] were significantly enriched with miR-30 direct targets and of the circadian clock , which in turn is coupled with the cell cycle40. In summary, the Serpine1:miR-30b/c interaction profoundly affects cell behaviour by increasing sensitivity to apoptosis and by accelerating the G1/S transition during cell cycle re-entry of quiescent cells.We next investigated whether the increased miR-30 activity observed when the Serpine1:miR-30b/c interaction is lost, might also cause phenotypical alterations Fig.\u00a0. Previoulls Fig.\u00a0, it appells Fig.\u00a0. Analysills Fig.\u00a0. BrdU inlls Fig.\u00a0, suggestlls Fig.\u00a0. Throughlls Fig.\u00a0, which mlls Fig.\u00a0. In partlls Fig.\u00a0. Notablyets Fig.\u00a0. In part18. Finally, it only affects two members (miR-30b and 30c) of the conserved miR-30 family that are not co-transcribed, thus coupling them post-transcriptionally while uncoupling them from the other three miR-30 members (miR-30a/d/e). All miR-30s can interact with Serpine1 via their seed sequence , but only miR-30b/c present an extended 3\u2032 end complementarity (3C pairing) to Serpine1 transcript. Therefore, as previously observed in vitro and with artificial targets20, 3C pairing is essential in the case of a TDMD mechanism triggered by endogenous targets. Accordingly, Serpine1 has stronger effects on miR-30c than on miR-30b, as miR-30c forms additional 3\u2032 base pairings with Serpine1. The importance of 3C pairing is highlighted also by the fact that it is required in reconstitution experiments. Additional mismatches can be tolerated close to the bulge, but completely impair miRNA degradation if located in proximity of miRNA 3\u2032 end. Complementarity is not the only requisite for TDMD, which also presents some context-dependent quantitative requirements. Therefore, we performed absolute measurements of miRNA and target expression (copies per cells) and computed the corresponding target-per miRNA value (TPM) to investigate the stoichiometry of their interaction. Upon serum stimulation of quiescent cells, Serpine1 is strongly induced so to largely exceed the levels of miR-30b/c (TPM\u2009>\u200910) and very rapidly halve their levels . No changes in transcription or processing are concomitantly observed and in range with. the number of miRNA copies (about 70\u201380\u2009cpc for miR-30c and 140 for miR-30b). Nevertheless, miR-30b and miR-30c are subjected to TDMD also in growing cells, as in MRE-KO cells the levels of the two miRNAs almost doubled. Therefore, endogenous TDMD takes place even for targets that are present at relatively low levels of expression, provided that they are in a stoichiometric range with their cognate miRNAs (TPM\u2009\u2265\u20091). The reason being that every time the miRNA interacts with the target, it is irreversibly subtracted from the miRNA pool by degradation16. It is worth mentioning that in reconstitution experiments, where targets are provided ectopically, TDMD requirements differ slightly as Serpine1-MRE acts mostly on miR-30c-5p and, in order to do so, must reach higher levels of expression (at least ~1000\u2009cpc). In these experiments, miRNA levels were analysed at 36\u2009h post infection. It is conceivable that a longer time interval is necessary to observe a significant reduction in miR-30b abundance, as this is expressed at relatively higher levels than miR-30c. It should be also stressed that, at low MOI, some cells were uninfected and did not ectopically express Serpine1-MRE (as determined by monitoring RFP). This might explain why ectopically expressed Serpine1 triggered TDMD at higher levels (at least ~1000 cpc) when compared to endogenously expressed Serpine1 (170\u2009cpc).Here, we provide molecular evidence that in mammalian cells a target-directed miRNA degradation mechanism exists and can control miRNA activity and cell functions. We found hundreds of targets that are potentially capable of TDMD. Focusing our study on Serpine1, we provide a proof-of-principle characterisation of TDMD at endogenous levels of expression, as opposed to acute expression of artificial targets or ectopic over-expression. Indeed, we show that under physiological conditions Serpine1 post-transcriptionally regulates two closely related miRNAs, miR-30b, and miR-30c, by inducing their degradation. Degradation promoted by Serpine1 is characterised by some peculiar features. It involves specific post-transcriptional modifications at the miRNA 3\u2032 end, with higher-than-normal levels of adenylation but no uridylation involved, as previously reported17. In their study, the NREP transcript promotes degradation of miR-29b in neuronal cells. The NREP:miR-29b and Serpine1:miR-30b/c pairs share the same complementarity features. Significantly, NREP is almost exclusively expressed in the brain (where TDMD is very effective) and imparts a spatial restriction to miR-29b expression in the cerebellum. Loss of the NREP:miR-29b interaction generates an in vivo phenotype characterised by impaired motor functions in fish and mice. A relevant role for TDMD in animal behaviour has thus been claimed. However, the impact of NREP on shared miR-29 targets was not investigated. Endogenous targets involved in TDMD are expected to influence miRNA-mediated repression. By investigating the Serpine1:miR-30 case, we provide compelling evidence in favour of such hypothesis. MRE-KO cells show a significant increase in miR-30 activity, as confirmed using both a synthetic reporter directly monitoring miRNA activity (miR-sensor) and endogenous targets identified by prediction algorithms or experimental approaches. In growing conditions (at steady-state), effects on endogenous targets are mild (with a ~5% increase in repression) and no major changes in phenotype are observed. Gene expression alterations observed in this context are likely to be a consequence of clone adaptation to the new (increased) levels of miR-30b/c. On the contrary, MRE-KO cells show remarkable phenotypes when exposed to acute stresses. In particular, upon serum stimulation mutant cells fail to properly regulate miR-30b/c levels, enter more quickly into S-phase and display an increased mitotic and cell death rate. In this context, strong (~20%) and specific repression of miR-30 targets is observed, with a certain degree of specificity towards miR-30b/c preferential targets (as opposed to shared miR-30 targets). Indeed, two independent studies mapping miRNA:target interactions suggest that miR-30b/c preferentially bind those targets that possess supplementary pairing in the 3\u2032 region37. In future, it would be worth trying to identify such specific targets and characterise them in serum-stimulated cells , as they might help clarify miR-30b/c function in cell cycle re-entry. In conclusion, we have provided evidence that in mammalian cells endogenous targets can control miRNA levels post-transcriptionally and thus modulate miRNA activity on all their targets.How many endogenous TDMD targets do exist? We chose to study Serpine1 as it was one of the top hits in a list of candidate targets displaying ideal structural characteristics: a high-affinity seed (8-mer or 7-mer-m8), a central bulge (\u2009>\u20092 and\u2009<\u20095\u2009nts) and a high 3C-score (high degree of complementarity at the 3\u2032 end). Based on our preliminary observations, we can anticipate that at least 1000 target:miRNA pairs present molecular characteristics that make them suitable for TDMD. While this work was under revision, a different case of endogenous TDMD was provided by Bitetti and co-workers17) or impart temporal-restriction to miRNA activity in dynamic settings (regulation in time\u2014as for Serpine1:miR-30b/c during cell cycle re-entry).We can no longer consider miRNA targets as being all equal. The way has now been opened to the identification of new endogenous TDMD targets that might be involved in different physiological or pathological processes. TDMD confers a further level of flexibility to miRNA regulation. It can restrict miRNA expression to a specific subset of cells and statistics were produced using JMP 12 (SAS) software. Microsoft Excel was used to generate bar graphs with average and standard deviation (s.d.) or standard error mean (s.e.m.) of repeated experiments. The number of replicates and the statistical tests used are indicated in the figure legends.Mouse 3T9 fibroblasts a kind gift of Bruno Amati. Cells were cultured in DMEM (Dulbecco\u2019s modified Eagle\u2019s medium) supplemented with 10% fetal bovine serum (FBS), 100\u2009U/ml penicillin and 100\u2009mg/ml streptomycin. Cells were routinely checked for mycoplasma contamination and resulted negative. 3T9 cells were induced to quiescence in a serum-deprived medium (0.1% FBS) for 3 days. Cell cycle re-entry was stimulated by adding fresh medium with 10% FBS. For cell cycle analysis, cells were treated with 33\u2009\u00b5M 5-bromo-2\u2032-deoxyuridine (BrdU) for 30\u2009min, harvested by trypsinisation, and ethanol-fixed. Staining was performed with anti-BrdU primary antibody and anti-mouse FITC-conjugated secondary antibody . DNA was stained with overnight incubation with 2.5\u2009\u00b5g/ml propidium iodide (Sigma). Samples were acquired on a FACS-CALIBUR (BD Biosciences) flow cytometer and analysed using FlowJo 10 software. Live time-lapse analysis of mitosis and apoptosis during cell cycle re-entry was performed with a high-content screening station equipped with a microscope incubation chamber, imaging cells in bright field with a 20\u2009\u00d7\u2009/NA 0.45 objective (Nikon Eclipse TE2000-E inverted microscope) every 15\u2009min starting at 12\u2009h after serum stimulation and reconstructed with ImageJ software. For each sample, mitosis and cell deaths were manually annotated at each time frame: ~200 cells were followed (in three independent replicas) and data reported as number of mitosis (or cell deaths) occurring every 100 cells.Cells were grown in normal conditions or under starvation, and treated or not with 1\u2009\u03bcM of doxorubicin (Sigma) for 48\u2009h to induce apoptosis. After treatment, adherent as well as detached cells were harvested and labelled with Annexin-V-APC for 45\u2009min. Next, cells were labelled with propidium iodide and immediately analysed by MACSQuant Flow cytometer (Miltenyi). Data analysis was performed by FlowJo software (TreeStar).Cells were plated in 96-well plate and analysed at 24, 48, 72, and 96\u2009h by Cell Titre GLO (Promega) following the manufacturer\u2019s protocol., and RNA was extracted with the miRNeasy Micro Kit and then eluted in 14\u2009\u03bcl of RNase-free water. 4sU-RNA quantification was conducted with Qubit\u00ae (Life Technologies).Starting from 40\u2009\u00b5g of total RNA diluted in 100\u2009\u00b5l of RNase-free water, 100\u2009\u00b5l of biotinylation buffer and 50\u2009\u00b5l of EZ-link biotin-HPDP were added and incubated for 3\u2009h at room temperature (RT). Unbound biotin-HPDP was removed by adding chloroform/isoamyl alcohol (24:1) and purifying the mix using MaXtract high-density tubes (Qiagen). RNA was precipitated by adding NaCl (5\u2009M) at 1/10 volume and isopropanol at equal volume. Biotinylated RNA was resuspended in water , quantified with a Nanodrop 1000 spectrophotometer (to keep track of the yield after the first phase of the procedure) and then purified using 50\u2009\u00b5l of Dynabeads MyOne Streptavidin T1 (Invitrogen). Before mixing with RNA, beads were washed twice in wash buffer A and once in wash buffer B (100\u2009mM NaCl) and then resuspended in 100\u2009\u00b5l of buffer C to a final concentration of 5\u2009\u03bcg/\u03bcl. RNA was added in an equal volume and rotated at RT for 15\u2009min. Beads were washed three times with wash buffer D . RNA was eluted from the beads in 100\u2009\u00b5l of 10\u2009mM EDTA in 95% formamide . The eluted fraction was diluted in TRIzol\u00ae LS Reagent IsomiRage workflow, as previously described32. Raw data together with detailed description of the procedures are available in GEO database (GSE104650). Normalisation was performed with the library size (reads-per-million) and then (only for the serum stimulation experiments) further corrected for serum-induced RNA amplification as previously reported26.Total RNA, including small species, was isolated through the miRNeasy Mini Kit (Qiagen). When the expected yield was\u2009<\u20091\u2009\u00b5g, the miRNeasy Micro Kit (Qiagen) was used. Small RNA sequencing (sRNA-seq) libraries were prepared using 1000\u2009ng of total RNA with the Illumina TruSeq\u2122 Small RNA Kit, following the manufacturer\u2019s instructions. Sequencing was performed on an Illumina HiSeq 2000 at 50\u2009bp single-read mode and 20 million read depth (8\u00d7). Sequencing quality was checked in the FASTQC report, considering only experiments with Q30 or above . Analysis was performed with the q value relative to the control was lower than 0.05 and whose maximum expression was higher than RPKM of 1. For the identification of genes differentially expressed in MRE-KO cells upon serum stimulation and then 1000\u2009ng were purified with Ribozero rRNA removal kit (Illumina). Libraries were generated with the TruSeq RNA Library Prep Kit v2 (Illumina). Next, sequencing was performed on an Illumina HiSeq 2000 at 50\u2009bp single-read mode and 50 million read depth (3\u00d7). Reads were aligned to the mm9 mouse reference genome using the TopHat aligner (version 2.0.6) with default parameters. Differentially expressed genes . In particular, miR-30c-5p and miR-30b-5p were detected using miScript Primer Assay MS00001386 and MS00011725. Primary transcripts of miRNAs (pri-miRNAs) were evaluated by quantitative PCR on total RNA. RT-qPCR was performed using SuperScript\u00ae VILO cDNA Synthesis Kit (Life Technologies cat.no. 11754050) and Fast SYBR green master mix (Life Technologies). About 25\u2009ng of cDNA were used as the input to detect pri-miRNAs, and 5\u2009ng to detect mRNAs. Primer pairs were designed using computer-assisted primer design software (Primer3), preferentially in the 500\u2009bp upstream of the sequence of the mature miRNA according to mm10 genome. The complete list of RT-qPCR primers used in this study is in Supplementary Table\u00a0Absolute quantification of miR-30c was performed by droplet-digital PCR on a QX200 system (Bio-Rad), following the manufacturers\u2019 instructions. Briefly, 10\u2009ng of RNA was reverse-transcribed with miRCURY LNA Universal RT microRNA PCR kit (Exiqon). Then, ddPCR reactions were prepared replacing Exiqon PCR master mix with QX200 EvaGreen ddPCR Supermix (Bio-Rad) and diluting 1:4 Exiqon PCR primer mix for miR-30c-5p (cat. 204783). Serial dilutions of cDNA (0.4/0.2/0.1/0.05\u2009ng) were used as to measure assay linearity. Droplets were generated and PCR amplification performed following EvaGreen cycling conditions. Finally, we read the droplets on a QX200 droplet reader. We typically obtained ~20,000 droplets for each sample.Mouse miRNA targets were downloaded from TargetScan 6.2 . 3C-targets were selected according to criteria outlined in Fig.\u00a034. SgRNAs were subsequently cloned in PX458 vector and sequence-verified. 3T9 fibroblasts were co-transfected with two PX458 vectors (sgRNA_1 and sgRNA_2) harbouring single sgRNAs sequence (5\u2009\u00b5g of DNA for each construct). At 24\u2009h and 48\u2009h post-transfection, cells were harvested for FACS sorting. The top 2%\u20133% of GFP\u2009+\u2009cells were sorted as individual cells into 96-well plates. After 2 weeks, 96-well plates were duplicated and deletions in clonal cells were screened by PCR , using the CRISPR Design ToolgAAGCAAGCTGTGTCAAGGGAsgRNA_1(-) Sense: cacccAntisense: aaacTCCCTTGACACAGCTTGCTTgTCTCCCAGTGGGGGGGCCCTsgRNA_2 (\u2009+\u2009) Sense: cacccAntisense: aaacAGGGCCCCCCCACTGGGAGANote that the efficient transcription from the U6 promoter requires a 5\u2032 g.Serpine1 protein expression in MRE-KO clones was analysed by western blot .KpnI-NotI sites at the extremities. After homologous recombination in BJ5183-AD-1 cells (Agilent), positive recombinants (isolated by PacI digestion) were used to produce adenoviruses in 239Ad cells. Viruses were finally concentrated with Adeno-X maxi purification kit (Takara).In order to re-express miR-30 MRE, recombinant adenoviruses were produced using the AdEasy System (Agilent), following manufacturers\u2019 instructions. Briefly, gene synthesis (GenScript) generated a mCherry-Serpine1 (3\u2032UTR) construct (see below for the complete sequence) that was cloned into pShuttle vector (Agilent) by adding mCherry-Serpine 3\u2032UTR construct (underlined with a solid line the miR-30 MRE):ACGCGTAGAGTGTAGGTGACTTGTTTACAGAGCTCCAGCTTTTTTCGACCCACAAACTTTTTTCATTTGGAAAGGGTGTAAGAAAAGTCGGACGTGTGTGTGCCTGGCTCTTCGTCCCCAGTCTCCCAGTGGGGGGGCCCTGGGGAGATTCCAGGGGTGTGATTGAATATTTATCTCTTGCTCTTGTATGTTTGTTGGGGAGAAGAAGCACTTTTAAGGAAAATGCTTCTTATTTAAACCGTGGCATACGGCATCCCATTTGGGGTCTGCATCCCTGTATGTCAGGGGTGCATCACTCCACAAACCTGCCCCTCTGGGTAGCCTCGTGATGGGGCTCACACTGCCGCCTAGTGGCAGCCGAACACACCCTTACCCGGTCCCTCCCTCCCTCCCCCCCCCCCCCCCCCCCCCCCGTGGCTCTTTTCCTTAGGGACCTTGCCAAGGTGATGCTTGGCAACCCACGTTAAAGGAAGGGGGGAAAAAAGATTAGATGGAAGAGAGAGAGATTTGAGAGAGGGCAAAGTGGTTTCAAATTTTTCCAAGGCATTCAGAAGCAGAGAGGGAAAAGGGGCTGTGTGACCTAACAGGACAGAACTTTCTCCAATTACTGGGTGAGTCAGAGCTGCACTGGTGACTCACTTCAATGTGTCATTTCCGGCTGCTGTATGTGAGCAGTGGACACGTGGGGGGGCGGGGGGGGGATGAAAGAGACAGCAGCTCCTGGTCAACCACCTTAGTTAGATAATCTTTTTTGAAAGCTTCCTAGCTGGAGGTATGATCAGAAAACCAATTTACTGAAAAACTGCACAAGAAGGTACGGTGAATGTAATTTCCTAGCAGGCCACTCTGCATCTGTTATGTCTCCACCGGAAAAAAAATAATCATGTTGGTGTTTTTGCTTTTCTCTCTCTCCCTCTTTCTCTCTGATTTTTTTTTCCTCTCTTTTCATTATGCACTGGACAGCCACACACCGTGTACCCATAGGGCCCCAAATGTGGGGTCACATGGTCTTGAATTTTGTTGGTTACATATGCCTTTTTGTTGTTGTTTGTCTTCACTTTTGATATATAAACAGGTAAATATGTTTTTTAAAAAATACTAAATATAGAGAATATGCAAACAAAAGCGGCCGC-3\u20325\u2032-GGTACCCGCCACCATGGTGAGCAAGGGCGAGGAGGATAACATGGCCATCATCAAGGAGTTCATGCGCTTCAAGGTGCACATGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGTGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTCATGTACGGCTCCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGACTACTTGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGGCGAGTTCATCTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCTCCGACGGCCCCGTAATGCAGAAGAAGACCATGGGCTGGGAGGCCTCCTCCGAGCGGATGTACCCCGAGGACGGCGCCCTGAAGGGCGAGATCAAGCAGAGGCTGAAGCTGAAGGACGGCGGCCACTACGACGCTGAGGTCAAGACCACCTACAAGGCCAAGAAGCCCGTGCAGCTGCCCGGCGCCTACAACGTCAACATCAAGTTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAACAGTACGAACGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTGTACAAGTAGCTCGAGCAGTGGGAAGAGACGCCTTCATTTGGGACGAAACTGGAGATGTTATAAGCAGAAACTCTGAAGAAAAGGATTATTTAAAGGACTCTATGGGGAGAAAGAGAAGGCAACTCCTCCTTACCCCCCACACTGGTAATCTTTCCAACCAGCATCCCAGACCTCGGACTCTTGAAGGGAAAAGAGTCTAACTCCCTCCTCCCTAGGGATTCCTACCCCACAAAGGTCTCATGGACCATAGAACTCACAGTACCTGGATCTGCCCAGCATGCCCTTTGGACCCAGTTCCCACCGAGGCCCCAGCAGAGTGGAGGGCACAACACTTTCATTCAGCAAAATCGTTTGTGTTCCAGTCACACTGTGGGCACCTCTTGCATCGCCTGCCATTGCTGTGGAGGGTGGCCATGGGCCAAAGGAAAAAGCACTGTCCTATCTCAAGGTCCACTGTGGAAATGTCCACCTTGCCCACCTCCAAGGGGCAACGGATAGACAGATCAAATGGTGGCCCAATAGCGAGCCTTCTCCCTGCTCCCTCCCTTGACACAGCTTGCTTATGTTATTTCKpnIGGTACC MluIACGCGT SacIGAGCTC Not1GCGGCCGC In order to produce other adenoviral constructs, the following oligonucleotides were subcloned into MluI-SacI sites flanking the miR-30 MRE:Ad-RFP-SE1(MUT)FW: cgcgtACTCTGAAAGTGAAAAGCCAATGgagctREV: cCATTGGCTTTTCACTTTCAGAGTaAd-RFP-SE1(SEED)FW: cgcgtGATATTGTCCTGACTTGTTTACAgagctREV: cTGTAAACAAGTCAGGACAATATCamiR-30_MRE in MRE-KO cells, we cloned MRE\u2009+\u2009~20 surrounding bases (Supplementary Table\u00a0EcoRI-XhoI sites that were put after the stop codon of a eGFP cloned in a pcDNA3.1(\u2009+\u2009) mammalian expression vector (Thermo Fisher Scientific).In order to re-express different variants of Serpine1Vectors were transfected in MRE-KO cells with Lipofectamine 3000 reagent (Thermo Fisher Scientific). Cells were collected for analysis after 48\u2009h.XbaI/XmaI) into the 3\u2032UTR of a destabilised GFP (dGFP) in a bidirectional lentiviral vector (BdLV_1370), which also expresses \u0394NGFR. The vector . Data analysis was performed by FlowJo software (TreeStar): fluorescent levels of \u0394NGFR and dGFP for each cell were log transformed and divided in 100 bins (each of 1000 cells) based on \u0394NGFR level. The mean \u0394NGFR or dGFP expression was calculated in each bin and repression folds were calculated, at similar \u0394NGFR mean expression, as the ratio: [dGFP(miR-30)/[dGFP(control)].Sequences harbouring four complementary repeats for miR-30c-5p or a control sequence (see below) were purchased from TwinHelix and cloned (miR-30c-5p sensor sequence:TCTAGATAAGCTGAGAGTGTAGGATGTTTACACGATGCTGAGAGTGTAGGATGTTTACAACGCGTGTCGACGCTGAGAGTGTAGGATGTTTACATCACGCTGAGAGTGTAGGATGTTTACACCCGGGControl (scrambled) sensor sequenceFW TCTAGAGGAGCTCCACCGCGGTGGCATCREV CCGGGATGCCACCGCGGTGGAGCTCCThttp://www.ncbi. nlm.nih.gov/geo/) under the accession number GSE104650. The 'serum stimulation data set', which contains miRNA expression analysis of WT cells, was previously submitted under accession number GSE72655. Data within the manuscript is available from the authors upon reasonable request.The small RNA sequencing (sRNA-seq) and the RNA-sequencing data set for this study have been submitted to the NCBI Gene Expression Omnibus (GEO; Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Data 1Supplementary Data 2Supplementary Data 3"} +{"text": "EBNA1 mRNA and highlight the importance of in-cellulo screening assays for targeting RNA structure-dependent interactions.Protein-RNA interactions (PRIs) control pivotal steps in RNA biogenesis, regulate multiple physiological and pathological cellular networks, and are emerging as important drug targets. However, targeting of specific protein-RNA interactions for therapeutic developments is still poorly advanced. Studies and manipulation of these interactions are technically challenging and in vitro drug screening assays are often hampered due to the complexity of RNA structures. The binding of nucleolin (NCL) to a G-quadruplex (G4) structure in the messenger RNA (mRNA) of the Epstein-Barr virus (EBV)-encoded EBNA1 has emerged as an interesting therapeutic target to interfere with immune evasion of EBV-associated cancers. Using the NCL-EBNA1 mRNA interaction as a model, we describe a quantitative proximity ligation assay (PLA)-based in cellulo approach to determine the structure activity relationship of small chemical G4 ligands. Our results show how different G4 ligands have different effects on NCL binding to G4 of the Accumulating evidence indicates an ever-expanding role for RNAs in regulating most aspects of cell biology that range from small non-coding RNAs to messenger RNAs (mRNAs). The more traditional role of mRNAs as \u201conly messengers\u201d is changing and new knowledge is emerging showing how the encoding sequences are taking on more diverse functions and can influence the activity of the encoded proteins. Most, if not all aspects of mRNAs, and this is presumably true also for non-coding RNAs, are governed by interactions with cellular proteins. These ribonucleoproteins (RNP) complexes control RNA metabolism and form scaffolds to orchestrate and organize protein networks and complex functional units . Thus, iEBNA1 mRNA (EBNA1 mRNA translation and thereby the production of EBNA1-derived antigenic peptides for the major histocompatibility (MHC) class I pathway, allowing EBV-infected cells to evade the immune system [EBNA1 mRNA-NCL interaction represents an interesting target for developing drugs that aim to induce an immune response against EBV-related diseases. It also serves as a broader model for developing techniques required to study structured RNA-protein interactions. Here, we use the NCL-EBNA1 mRNA interaction to illustrate how different compounds binding to the same G4 have specific effects on the interaction with NCL in cellulo. This illustrates a so far unknown role of G4 structures in mediating specific interactions with proteins, indicating that particular G4-protein interactions can be targeted specifically. We also report how to generate quantitative data using proximity ligation to verify the capacity of different G4 ligands to prevent NCL-EBNA1 mRNA interactions and their role in controlling EBNA1 mRNA translation.G-quadruplexes (G4) are non-canonical nucleic acid structures based on the stacking of several G-quartets, further stabilized by cations positioned in the central channel of the G4 helix ,8. Thesee system . As EBV l-glutamine, 100 U/mL penicillin, and 100 \u00b5g/mL streptomycin. H1299 transient transfections were performed using Genejuice reagent according to manufacturer\u2019s protocol. All cells were cultured at 37 \u00b0C with 5% CO2. For drug treatments, 105 B95-8 cells were incubated with 5 \u00b5M of PhenDC3 [The human lung carcinoma cell line H1299 and the EBV-producing marmoset B-cell line B95-8 were cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS), 2 mM v/v) ethanol at 4 \u00b0C. After rehydration in PBS for 30 min, samples were permeabilized with PBS 0.4% Triton X-100, 0.05% CHAPS for 10 min at room temperature, and pre-treated with hybridization buffer for 30 min at room temperature. Samples were then incubated overnight with 50 ng of an EBNA1-digoxigenin DNA probe (5\u2032 CTTTCCAAACCACCCTCCTTTTTTGCGCCTGCCTCCATCAAAAA-digoxigenin 3\u2032) in a humidified chamber at 37 \u00b0C. To avoid secondary structures, the probe was diluted in 5 \u00b5L of water, denaturated at 80 \u00b0C for 5 min, chilled on ice for 5 min, and resuspended in 35 \u00b5L of hybridization buffer. After hybridization, samples were serially washed for 20 min with 2X SSC 10% formamide, hybridization buffer (twice), 2X SSC, and PBS. Washes were carried out at room temperature, except with hybridization buffer (37 \u00b0C). Samples were saturated with PBS 3% BSA for 30 min and incubated with a mouse anti-digoxigenin for 2 h at room temperature. A goat anti-mouse immunoglobulin G (IgG) secondary antibody conjugated to Alexa Fluor\u00ae 568 (Sigma) was used to detect immunocomplexes (1 h at 37 \u00b0C) and DAPI was used for nuclear counterstaining under standard conditions. For IF, fixed samples were saturated with PBS 3% BSA for 30 min, incubated with rabbit polyclonal antibody anti-NCL for 2 h and goat anti-rabbit Ig secondary antibody conjugated to Alexa Fluor\u00ae 488 (Sigma) for 1 h at 37 \u00b0C. DAPI was used for nuclear counterstaining.H1299 cells were plated on 12-mm-diameter coverslips in 24-well plates and transfected with 200 ng of EBNA1 construct . At 24 hThe sequence of EBNA1 cDNA (GenBank: MG021311.1) is as follows (the GAr-encoding sequence which forms G4 is highlighted in cyan):GGAGCAGGAGGAGGGGCAGGAGCAGGAGGGGCAGGAGCAGGAGGAGGGGCAGGAGCAGGAGGAGGGGCAGGAGCAGGAGGAGCAGGAGGAGGGGCAGGAGCAGGAGGAGGGGCAGGAGCAGGAGGAGGGGCAGGAGCAGGAGGAGGGGCAGGAGCAGGAGGAGGGGCAGGAGCAGGAGGAGGGGCAGGAGGAGGAGGAGGGGCAGGAGCAGGAGGAGGGGCAGGAGCAGGAGGAGGGGCAGGAGCAGGAGGAGGGGCAGGAGGGGCAGGAGCAGGAGGAGGGGCAGGAGCAGGAGGAGGGGCAGGAGCAGGAGGAGGGGCAGGAGCAGGAGGGGCAGGAGCAGGAGGAGGGGCAGGAGCAGGAGGGGCAGGAGCAGGAGGAGGGGCAGGAGCAGGAGGAGGGGCAGGAGCAGGAGGGGCAGGAGCAGGAGGGGCAGGAGCAGGAGGGGCAGGAGCAGGAGGGGCAGGAGGAGGAGGAGCAGGAGGGGCAGGAGGGGCAGGAGCAGGAGGGGCAGGAGGGGCAGGAGCAGGAGGAGGGGCAGGAGGGGCAGGAGCAGGAGGAGGGGCAGGAGGGGCAGGAGCAGGAGGGGCAGGAGGGGCAGGAGCAGGAGGGGCAGGAGGGGCAGGAGCAGGAGGGGCAGGAGGGGCAGGAGCAGGAGGAGGGGCAGGAGCAGGAGGGGCAGGAGCAGGAGGTGGAGGCCGGGGTCGAGGAGGCAGTGGAGGCCGGGGTCGAGGAGGTAGTGGAGGCCGGGGTCGAGGAGGTAGTGGAGGCCGCCGGGGTAGAGGACGTGAAAGAGCCAGGGGGGGAAGTCGTGAAAGAGCCAGGGGGAGAGGTCGTGGACGTGGTGAAAAGAGGCCCAGGAGTCCCAGTAGTCAGTCATCATCATCCGGGTCTCCACCGCGCAGGCCCCCTCCAGGTAGAAGGCCATTTTTCCACCCTGTAGGGGAAGCCGATTATTTTGAATACCACCAAGAAGGTGGCCCAGATGGTGAGCCTGACATGCCCCCGGGAGCGATAGAGCAGGGCCCCGCAGATGACCCAGGAGAAGGCCCAAGCACTGGACCCCGGGGTCAGGGTGATGGAGGCAGGCGCAAAAAAGGAGGGTGGTTTGGAAAGCATCGTGGTCAAGGAGGTTCCAACCAGAAATTTGAGAACATTGCAGAAGGTTTAAGAACTCTCCTGGCTAGGTGTCACGTAGAAAGGACTACCGATGAAGGAACTTGGGTCGCCGGTGTGTTCGTATATGGAGGTAGTAAGACCTCCCTTTACAACCTCAGGCGAGGAATTGCCCTTGCTATTCCACAATGTCGTCTTACACCATTGAGTCGTCTCCCCTTTGGAATGGCCCCTGGACCCGGCCCACAACCTGGCCCACTAAGGGAGTCCATTGTCTGTTATTTCATTGTCTTTTTACAAACTCATATATTTGCTGAGGGTTTGAAGGATGCGATTAAGGACCTTGTTATGCCAAAGCCCGCTCCTACCTGCAATATCAAGGCGACTGTGTGCAGCTTTGACGATGGAGTAGATTTGCCTCCCTGGTTTCCACCTATGGTGGAAGGGGCTGCCGCGGAGGGTGATGACGGAGATGACGGAGATGAAGGAGGTGATGGAGATGAGGGTGAGGAAGGGCAGGAGTGAATGTCTGACGAGGGACCAGGTACAGGACCTGGAAATGGCCTAGGACAGAAGGAAGACACATCTGGACCAGACGGCTCCAGCGGCAGTGGACCTCAAAGAAGAGGGGGGGATAACCATGGACGAGGACGGGGAAGAGGACGAGGACGAGGAGGCGGAAGACCAGGAGCTCCGGGCGGCTCAGGATCAGGGCCAAGACATAGAGATGGTGTCCGGAGACCCCAAAAACGTCCAAGTTGCATTGGCTGCAAAGGGGCCCACGGTGGAACAl-lysine 0.01% (Sigma) for 30 min and air-dried for 5 min in 24-well plates. B-cells were then resuspended in PBS, plated on pre-treated coverslips and incubated for 2 h at room temperature. After a wash with PBS for 5 min, cells were fixed with PBS 4% paraformaldehyde for 20 min and re-washed with PBS for 10 min. Following fixation, both cell lines were processed for in situ hybridization according to the rISH-IF protocol. Samples were then saturated with PBS 3% BSA for 30 min and incubated for 2 h at room temperature with a mix of primary antibody containing the mouse anti-digoxigenin and the rabbit anti-nucleolin previously described. Subsequently, PLA was carried out using the Duolink PLA in situ kit (Sigma), anti-rabbit plus and anti-mouse minus probes (Sigma) following the manufacturer\u2019s protocol. H1299 cells were plated and fixed as previously described. For experiments using B95-8, 12-mm-diameter coverslips were coated with poly-Samples were examined with an LSM 800 confocal laser microscope . ImageJ was usedEBNA1 mRNA translation and, as a consequence, by minimizing the production of antigenic peptides for the major histocompatibility (MHC) class I pathway [EBNA1 mRNA and this is essential for suppressing the translation of the EBNA1 mRNA and the production of antigenic peptides was originally conceived to detect proteins in close proximity. For this, one pair of primary antibodies raised in two different species is used to target the two proteins of interest C. AfterwEBNA1 mRNA interactions in situ using either transfected cells or EBV-infected cells. PLA complexes are depicted as white dots and each dot represents an interaction between NCL and EBNA1 mRNA. In line with rISH-IF and IF results, interactions were uncovered in or at the close vicinity of the nuclear compartment of EBNA1-transfected cells \u2248 0.47 \u00b5M); DC50 (PhenDC3) \u2248 0.26 \u00b5M. This was further confirmed by the thermal stabilization values (\u0394Tm) measured by the FRET-melting assay on EBNA1 G4 , and anti-G4 antibodies [EBNA1 mRNA [An estimated 2\u20133% of cancers are associated with EBV, making GAr-based EBNA1 immune evasion an important target for therapeutic approaches against EBV-related cancers ,21. TherBNA mRNA , we treaeraction A. This iin vitro ,22. In ain vitro , while Pin vitro . Altogeteraction C. It has 20.5 \u00b0C . Althoug 20.5 \u00b0C . Howevertibodies , whereasNA1 mRNA . All theProtein-mRNA interactions influence multiple aspects of cellular function. However, in spite of progress experienced over the last decades, the study of these interactions has been restricted to assays technically demanding and unable to provide accurate information at subcellular levels. In this context, PLA represents an attractive alternative to overcome these limitations, opening new avenues for the study of protein-mRNA interactions in cellulo. We provided evidences that PLA can be coupled to quantitative analysis and be successfully employed to screen molecules interfering with target protein-mRNA interactions. Additionally, the approach described here can be adapted to other technologies, like flow cytometry, for medium to high throughput drug screening. This highlights the potential of mRNA-protein PLA as a tool for efforts focused not only on targeting specific G4 structures, but more generally for drug development based on disruption of specific protein-RNA structure complexes."} +{"text": "The most abundant polyproline-rich peptides were derived from acrosin, homeobox protein HoxB4, lysine-specific demethylase 6B, proline-rich protein 12, and proline-rich membrane anchor 1 (PRiMA). The research article associated with the data in this report can be found in Saxena et al. (2018). The Data in Brief report lists all the polyproline-rich peptides identified in PoBChE tetramers.Milk of the domestic pig has 10 times more butyrylcholinesterase (BChE) per mL than porcine serum. We purified BChE from porcine milk by affinity chromatography on Hupresin-Sepharose. The pure porcine BChE (PoBChE) was a tetramer with a molecular weight of 340,000, similar to that of human BChE tetramers. The C-terminal 40 residues of PoBChE constitute the tetramerization domain. The glue that holds the 4 BChE subunits together is a polyproline-rich peptide. Mass spectrometry analysis of trypsin-digested PoBChE identified a variety of polyproline-rich peptides originating from 12 different proteins. The donor proteins exist in the nucleus or cytoplasm of cells and contribute their polyproline-rich peptides after a cell is degraded. The secreted PoBChE scavenges the polyproline-rich peptides and incorporates one polyproline peptide per PoBChE tetramer, where the polyproline peptide is bound noncovalently but very tightly with an estimated dissociation constant of 10 Specifications tableValue of the data\u2022The finding that PoBChE is abundant in porcine milk \u2022The set of polyproline-rich peptides in PoBChE tetramers is different from the set in human BChE tetramers \u2022Are excess polyproline-rich peptides toxic to cells? Does BChE incorporate polyproline peptides from degraded cells because degraded cells are a convenient source? If excess polyproline-rich peptides are toxic to cells, BChE might be protecting the organism by scavenging these peptides.\u2022The BChE tetramer incorporates not only short polyproline-rich peptides, but also long proteins that contain a polyproline-rich region. An example is the C5 variant of human BChE whose tetrameric structure includes a 60\u202fkDa lamellipodin protein \u2022The ability of BChE subunits to assemble into stable, long-lived tetramers by binding the polyproline-rich region of a protein, suggests that BChE could serve as a delivery vehicle for any protein that has been engineered to include a polyproline-rich tag.1We present all the polyproline-rich peptides associated with PoBChE tetramers that were identified by mass spectrometry. In addition we show the complete amino acid sequence of the full-length protein that donated each polyproline-rich peptide, the location of the polyproline-rich peptide within the full-length protein sequence, and the abundance of the polyproline-rich peptide relative to PoBChE peptides. A brief description is given of the function of the donor protein and its location within a cell.22.1PoBChE was purified from defatted porcine milk (1200\u202fmL) by affinity chromatography on procainamide-Sepharose 2.2The Hupresin-purified PoBChE was reduced in volume from 7.3\u202fmL to 0.17\u202fmL in a Centricon YM-30 spin filter. After the buffer was changed to 10\u202fmM ammonium bicarbonate pH 8, the PoBChE was deglycosylated with 1\u202f\u00b5l of PNGaseF for 1\u202fh. Noncovalently bound polyproline-rich peptides were released from PoBChE by denaturing the protein in a boiling water bath for 5\u202fmin. The denatured 170\u202f\u00b5g of PoBChE protein in 170\u202f\u00b5L of 10\u202fmM ammonium bicarbonate pH 8 was digested with 2\u202f\u00b5g of trypsin for 20\u202fh at 37\u202f\u00b0C in a humidified chamber. Particles that could clog the small diameter tubing in the Ultra High Pressure Liquid Chromatography column were removed by centrifuging the digest for 30\u202fmin at 14,000\u202frpm in a microfuge. A 10\u202f\u00b5L aliquot from the top of the centrifuged digest was transferred to an autosampler vial. The protein concentration in the digest was estimated at 1\u202f\u00b5g/\u00b5L.2.3Sus Scrofa proteins using the Paragon algorithm from Protein Pilot (AB Sciex) (The protocol for liquid chromatography tandem mass spectrometry (LC\u2013MS/MS) is described in detail in B Sciex) .Table 1AImage 1VSAKPPQALSFAKRLQQLIEALKGTAFSSGRSYYETETTDLQELPAS.>sp|P08001|ACRO_PIG Acrosin OS=Sus scrofa GN=ACR PE=1 SV=5 MLPTAVLLVLAVSVAARDNATCDGPCGLRFRQKLESGMRVVGGMSAEPGAWPWMVSLQIFMYHNNRRYHTCGGILLNSHWVLTAAHCFKNKKKVTDWRLIFGANEVVWGSNKPVKPPLQERFVEEIIIHEKYVSGLEINDIALIKITPPVPCGPFIGPGCLPQFKAGPPRAPQTCWVTGWGYLKEKGPRTSPTLQEARVALIDLELCNSTRWYNGRIRSTNVCAGYPRGKIDTCQGDSGGPLMCRDRAENTFVVVGITSWGVGCARAKRPGVYTSTWPYLNWIASKIGSNALQMVQLGTPPRPSTPAPPVRPPSVQTPVRPPWYFQRPPGPSQQPGSRPRP27 residues PAPPPAPPPPPPPPPPPPPPPPPPPQQ MW 2648.4.https://www.gtexportal.org/home/gene/ACR The human acrosin gene is expressed in testis and to a small extent in mammary breast tissue, lung, spleen, adipose tissue, and tibial artery.Acrosin is the major proteinase present in the acrosome of mature spermatozoa. It is a typical serine proteinase with trypsin-like specificity. It is stored in the acrosome in its precursor form, proacrosin. The active enzyme functions in the lysis of the zona pellucida, thus facilitating penetration of the sperm through the innermost glycoprotein layers of the ovum. The mRNA for proacrosin is synthesized only in the postmeiotic stages of spermatogenesis. In humans proacrosin first appears in the haploid spermatids. Image 2SPRAPAPPPSGALLPEPGQRCEAVSSSPPPPPCAQNPLHPSPSHSACKEPVVYPWMRKVHVSTVNPNYAGGEPKRSRTAYTRQQVLELEKEFHYNRYLTRRRRVEIAHALCLSERQIKIWFQNRRMKWKKDHKLPNTKIRSGGPASAAGGPPGRPNGGPPAL.>XP_003131596.1 PREDICTED: homeobox protein Hox-B4 [Sus scrofa] MAMSSFLINSNYVDPKFPPCEEYSQSDYLPSDHSPGYYAGGQRRESSFQPEAGFGRRAACTVQRYAAC21 residues RDPGPPPPPPPPPPPPPPPGL MW 2069.1.The HOXB4 gene is a member of the Antp homeobox family and encodes a nuclear protein with a homeobox DNA-binding domain. It is included in a cluster of homeobox B genes located on human chromosome 17. The encoded protein functions as a sequence-specific transcription factor that is involved in development. Intracellular or ectopic expression of this protein expands hematopoietic stem and progenitor cells in vivo and in vitro, making it a potential candidate for therapeutic stem cell expansion.Image 3SPPFQLTKPGLWSTLHGDAWGPERKGTAPPERQEQRHSLPRHPYPYPAPAYPSRTPWPRLVPAAPPGPGPXPPGAESHGCPPATRPPGSDLRESRVQRSRMDSSVSPAATTACVPYAPSRPPALPGTTTSSSSSSSNTGLRGVEPSPGIPGADHYQTPALEVSSHQGRLGPSAHSSRKPFLAAPAATPHLSLPPGPPSPPPPPCPRLLRPPPPPAWLKGPACRAAREDGEILEELFFGAEGRPRPPPPPLPHREGFLGPPAPRFSVGTQDSHTPPTPPTTSSSSSNNGSHSSSPTGSVSFPPPPYLARSMDPLPRPPSPTLSPQDPPLAPLSLALPPAPPSSCHQNTSGSFRRPESPRPRVSFPKTPEVGPGPSPGPLNKAPQPVPSRVGELPARGPRLFDFPPTPLEDQFEEPAEFKILPDGLANIMKMLDESIRKEEEQQQQEAGVVPPPPLKEPFASLQPPFPTDTAPATTAATTAATTTATQEEEKKPPPALPPPPPLAKFPPPPQPQPPPPPLPPPASPASLLKSLASVLEGQKYCYRGTGAAVATRPGPLPTTQYSPGPPSGATAPPPTSAAPSAQGSPQPSASSSSQFSTSGGPWARERRAGEEPAPGPTTPAPPPPPLPLPPARSESEVLEEISRACETLVERVGRGATDPADPADTADPVDTGAERLLPPAQAKEEAGGASAVAAAAAGPGSSKRRQKEHQKEHRRHRRACKDSVGRRPREGRAKAKAKAPKEKSRRVLGNLDLQSEEIQGREKARPDLGGASKAKPPTAPAPLPAPAPSTQSTPPSAPVPGKKAREEAPGPPGVSRADMLKLRSLSEGPPKELKIRLIKVESGDKETFIASEVEERRLRMADLTISHCAADVVRASKNAKVKGKFRESYLSPAQSVKPKINTEEKLPREKLNPPTPSIYLESKRDAFSPVLLQFCTDPRNPITVIRGLAGSLRLNLGLFSTKTLVEASGEHTVEVRTQVQQPSDENWDLTGTRQIWPCESSRSHTTIAKYAQYQASSFQESLQEEKESEDEESEEPDSTTETPPSSAPDPKNHHIIKFGTNIDLSDAKRWKPQLQELLKLPAFMRVTSTGNMLSHVGHTILGMNTVQLYMKVPGSRTPGHQENNNFCSVNINIGPGDCEWFAVHEHYWETISAFCDRHGVDYLTGSWWPILDDLYASNIPVYRFVQRPGDLVWINAGTVHWVQATGWCNNIAWNVGPLTAYQYQLALERYEWNEVKNVKSIVPMIHVSWNVARTVKISDPDLFKMIKFCLLQSMKHCQVQRESLVRAGKKIAYQGRVKDEPAYYCNECDVEVFNILFVTSENGSRNTYLVHCEACARRRSAGLQGVVVLEQYRTEELAQAYDAFTLAPASTSR.>XP_005657086.1 PREDICTED: LOW QUALITY PROTEIN: lysine-specific demethylase 6B [Sus scrofa] MHRAVDPPGARTAREAFALGGLSCAGAWSSCPPHPPPRSAWLPGGRCSASIGQPPLSAPLPPSHGSSSGHPNKLYFAPGTPNPRPLHGKLESLHGCVQALLREPAQPGLWEQLGQLYESEHDSEEAIRCYHSALRYGGSLAELGPRIGRLQQAQLWNFHAGSCQHRPKVLPPLEQVWNLLHLEHKRNYGAKRGGPPVKRAAEPPVVQPVPPAALSGPSGEEGLSPGGKRRRGCNSEQTGLPPGL28 residues PLPPPPLPPPPPPPPPPPPPPPLPGLAT MW 2737.5.Histone demethylase specifically demethylates \u05f3Lys-27\u05f3 of histone H3, thereby playing a central role in histone code . Demethylates trimethylated and dimethylated H3 \u05f3Lys-27\u05f3 . Plays a central role in regulation of posterior development, by regulating HOX gene expression (PubMed:17851529). Involved in inflammatory response by participating in macrophage differentiation in case of inflammation by regulating gene expression and macrophage differentiation (PubMed:17825402). Plays a demethylase-independent role in chromatin remodeling to regulate T-box family member-dependent gene expression by acting as a link between T-box factors and the SMARCA4-containing SWI/SNF remodeling complex.Image 4PPRVQLPVSLDLPLFPSIMMQPVQHPALPPQLALQLPQMDTLSADLTQLCQQQLGLDPNFLRHSQFKRPRTRITDDQLKILRAYFDINNSPSEEQIQEMAEKSGLSQKVIKHWFRNTLFKERQRNKDSPYNFSNPPITVLEDIRIDPQPSSLEHYKSDASFSKRSSRTRFTDYQLRVLQDFFDTNAYPKDDEIEQLSTVLNLPTRVIVVWFQNARQKARKSYENQAEAKDNEKRELTNERYIRTSNMQYQCKKCNVVFPRIFDLITHQKKQCYKDEDDDAQDESQTEDSMDATDQVVYKHCTVSGQTEAAKNAPVAAASSGSGASTPLLPSPKPEPEKTSPKPEYPTEKPKQSDPSPPSQGTKPALPLASTSSEPPQAAAAQPQPQPPKQPQLIGRPPSASQTPIPSSPLQISMTSLQNSLPPQLLQYQCDQCTVAFPTLELWQEHQHMHFLAAQNQFLHSPFLERPMDMPYMIFDPNNPLMTGQLLSGFLTQMPPQNASSQTPASATVAASLKGNWDDKEDTNCSEKEGGNSGEDQHRDKRXRTTITPDKLEILYEKYLLDSNPTRKMLDHIAREVGLKKRVVQVWFQNTRARERKGQFRAVGPAQSHKRCPFCRALFKAKSALESHIRSRHWNEGKQAGYSLPPSPLIATEDGGESPQKYIYFDYPSLPLTKIDLSSENELASTVSTPVSKTAELSPKNLLSPSSFKAECSEDVENLNAPPAEAGYDQNKPDFDETSSINTAISDATTGDEGNAEMESTTGSSGDVKPALSPKEPKTLDTLAKTATTPTTEVCDEKFLFSLTSPSIPFNDKDGDHDQSFYITDDPDDNADRSETSSIADPSSPNPFGSSNPFKSKSNDRPGHKRFRTQMSNLQLKVLKACFSDYRTPTMQECEMLGNEIGLPKRVVQVWFQNARAKEKKFKINIGKPFMINQSGTEGTKPECTLCGVKYSARLSIRDHIFSKQHISKVRETVGSQLDREKDYLAPTTVRQLMAQQELDRIKKASDVLGLAVQQPSMMDSSSLHGISLPAAYPGLPGLPPVLLPGMNGPSSLPGFPQNSNTLTPPGAGMLGFPTSATSSPALSLSSAPTKPLLQImage 5SGQQTEPQNKESEKKQTKPNKVKKIKEEELEATKPEKHPKKEEKISSALSVLGKVVGETHVDPSQLQALQNAIAGDPASFLGGQFLPYFIPGFASYFTPQLPGTVQGGYLPPVCGMESLFPYGPTMPQTLAGLSPGALLQQYQQYQQNLQDSLQKQQKQQQQEQPQKPGQAKTSKGESEPPQNASDASETKEDKSTATESTKEEPQLESKSADFSDTYVVPFVKYEFICRKCQMMFTDEDAAVNHQKSFCYFGQPLIDPQETVLRVPVSRYQCLACDVAISGNEALSQHLQSSLHKEKTIKQAMRNAKEHVRLLPHSVCSPNPNTTSTSQSAASSNTYPHLSCFSMKSWPNILFQASARRAASSPSSPPSLSLPSTVTSSLCSTSGVQTSLPTESCSDESDSELSQKLEDLDNSLEVKAKPASGLDGNFNSIRMDMFSV.>XP_005663076.1 PREDICTED: LOW QUALITY PROTEIN: zinc finger homeobox protein 4 [Sus scrofa] METCDSPPISRQENGQSTSKLCGTAQLDNEVPEKVAGMEPDRENSSTDDNLKTDERKSEVLLGFSVENAAATQVTSAKEIPCNECATSFPSLQKYMEHHCPNARLPVLKDDNESEISELEDSDVENLTGEIVYQPDGSAYIIEDSKESGQNAQTGANSKLFSTAMFLDSLASAGEKSDQSASAPMSFYPQIINTFHIASSLGKPFTADQAFPNTSALAGVGPVLHSFRVYDLRHKREKDYLTSDGSAKNSCVSKDVPNNVDLSKFDGCVSDGKRKPVLMCFLCKLSFGYIRSFVTHAVHDHRMTLNEEEQKLLSNKCVSAIIQGIGKDKEPLISFLEPKKSTSVYPHFSTTNLIGPDPTFRGLWSAFHVENGDSLPAGFAFLKGSAGTSGSAEQPLGITQMPKAEVTLGGLSSLVVNTPITSVSLSNASSESSKMSESKDQENDCERPKESNALHPNGECPVKSEPTEAGDEDEEDAYSNELEDEEVLGELTDSIGNKDFPLLNQSISPLSSSVLKFIEKGPSSSSASVTDDAEKKKPTAAVRASGGVANSYGIGGKDFAEASASKDGATAAHSSEPARGDEDSSATPHQHGFTPSAPGTPGPGGDGSPGSGIECPKCDTVLGSSRSLGGHMTMMHSRNSCKTLKCPKCNWHYKYQQTLEAHMKEKHPEPGGSCVYCKTGQPHPRLARGESYTCGYKPFRCEVCNYSTTTKGNLSIHMQSDKHLNNVQNLQNGNGEQVFGHSAPAPNTSLSGCGTPSPSKPKQKPTWRCEVCDYETNVARNLRIHMTSEKHMHNMMLLQQNMKQIQHNLHLGLAPAEAELYQYYLAQNIGLTGMKLENPGDPQLMLNPFQLDPATAAALAPGLVNNELPPEIRLASGQLMGDDLSLLTAGELSPYISDPALKLFQCAVCNKFTSDSLEALSVHVSSERSLPEEEWRAVIGDIYQCKLCNYNTQLKANFQLHCKTDKHMQKYQLVAHIKEGGKSNEWRLKCIAIGNPVHLKCNACDYYTNSVDKLRLHTTNHRHEAALKLYKHLQKQEGAVNPESCYYYCAVCDYSTKVKLNLVQHVRSVKHQQTEGLRKLQLHQQGLAPEEDNLSEIFFVKDCPPNELETASLGARTCEDDLLEQQLRAPSEEQSEETEGASRPTAVAEDDEKDTSERDNNEGKNSNKDTGIITPEKELKVSVAGGTQPLLLAKEEDVATKRSKPTEDSKFCHEQFYQCPYCNYNSRDQSRIQMHVLSQHSVQPVICCPLCQDVLSNKMHLQLHLTHLHSVSPDCVEKLLMTVPVPDVMMPNSLLLPAAASEKSERDTPAAITAEGPGKYSGESPMDDKSMAGLDDSKAIMEIKSEEQKPTKEPTEASEWNKNSSKDGKISDPLQDQLSEQQKRQPLSVSDRHVYKYRCNHCSLAFKTMQKLQIHSQYHAIRAATMCNLCQRSFRTFQALKKHLEAGHPELSEAELQQLYASLPVNGELWAESETMAQDDHALEQEMEREYEVDHEGKASPVGSDSSSIPDDMGSEPKRTLPFRKGPNFTMEKFLDPSRPYKCTVCKESFTQKNILLVHYNSVSHLHKLKKVLQEASSPVPQETNSSTDNKPYKCSICNVAYSQSSTLEIHMRSVLHQTKARAAKLEPSSHVVSGHSAANVSSPGQGMLDSMSLAGVSSKDTHLDAKELNKKQTPELISAQPAHHPPQSPAQIQMQLQHELQQQAAFFQPQFLNPAFLPHFPMTPEALLQFQQPQFLFPFYIPGTEFSLGPDLGLPGSAAFGMPGMTGMAGSLLEDLKQQIQTQHHVGQTQLQILQQQAQQYQATQPQLQSQKPQQQPQPQPQQQQASKLLKQEQTTLASAECPIVKDIPSFKEAEEMAKKQDKPKQEVXSEGEGLKEGKDEKKQKSSEPSILPPRIASGARGNAAKALLENFGFELVIQYNENRQKVQKKGKSGEGESTEKLECGTCGKLFSNVLILKSHQEHVHGQFFPYGALEKFARQYREAYDKLYPISPSSPETPPPPPPPPPLPPAPPQPASLGPVKLPSTVSTPIQAPPP22 residues TPPPPPPPPPPPPPPPPPPPSA MW 2121.1.18 residues TPPPPPPPPPPPPPPSSL MW 1764.9.May play a role in neural and muscle differentiation. May be involved in transcriptional regulation.Image 6PMPVHEPLSPQQLQQQQDMYNKKIPSLFEIVVRPTGQLAEKLGVRFPGPGGPPGPMGPGPNMGPPGPMGGPMHPDMHPDMHPDMHPDMHPDMPMGPGMNPGPPMGPGGPPMMPYGPGDSPHSGMMPPIPPAQNFYENFYQQQEGMEMEPGLIGDTEDYGHYEELPGEPGEHLFPEHPLEPDSFSEGGPPGRPKPGAGVPDFLPSAQRALYLRIQQKQQEEEERARRLAESSKQDRENEEGDPGNWYSSDEDEGGSSVTSILKTLRQQTSSRPQASGGELSSSGLGDPRLQKGHPTGGRLADPRLSRDPRLSRHAEASGGSGPGDTGPSDPRLARSLPTPRPKGGLHSSPGGPSGSKGSGPPPAEEEEGERALREKAVNIPLDPLPGHPLRDPRSQLQQFSHIKKDVTLSKPSFARTVLWNPEDLIPLPIPKQDAVPPVPVALQSMPALDPRLHRTTTTSGPPNPRQRPGTSTDPSASGSNLPDFELLSRILKTVNATGPSAAPGPGDKPSDPRVRKTPTDPRLQKPADSATSSRAAKPGSTEVSPSASPSGESSPPATAPYDPRVLAAGGLGQGSGSGQSSVLSGISLYDPRTPNAGGKATEPAADTGTQPKGPEGNGKSAATKAKEPPFVRKSALEQPESGKPGADGGAAAATDRYNSYNRPRPKATPAAAASGTPPPEGASPQPGVHNLPVPTLFGTVKQAPKTGSGSPFAGNSPAREGEQDAGSLKDVFKGFDPTASPFCQ.>XP_005664683.1 PREDICTED: LOW QUALITY PROTEIN: zinc finger CCCH domain-containing protein 4 [Sus scrofa] MEAAPGTPPPPPSESPPPPSPPLPSTPSPPPCSPDACPATPHLLHHRLPLPDDREDGELEEGELEDDGAEETQDTSGGPERSRKEKGDKHHSDSDEEKSHRRLKRKRKKEREKEKRRSKKRRKSKHKRHASSSDDFSDFSDDSDFSPSEKGHRKYREYSPPYAPSHQQYPPSHTTPLPKKAYSKMDSKGYSMYEDYENEQYGEYEGDEEEDMGKDDYDDFTKELNQYRRAKEGSSRGRGSRGRGRGYRGRGSRGGSRGRGMGRGSRGRGRGSMGGDHPEDDEDFYEEEMEYGESEEPMGDEDYDDYSKELNQYRRSKDGRGRGLSRGRGRGSRGRGKGMGRGRGRGGSRGGMNKGGMNEDDDFYDEDMGDGGGGGGSYRRSDHDKPHQQSDKKGKVICKYFVEGRCTWGDHCNFSHDIELPKKRELCKFYITGFCARAENCPYMHGDFPCKLYHTTGNCINGDDCMFSHDPLTEETRELLDKMLADDAEAGAEDEKEVEELKKQGINPLPKPPPGVGLLPTPPRPPGPPAPTSPNGRPLQ19 residues GGPPPPPPPPPPPPGPPQM MW 1806.9.NCBI and UniProt have ZC3H4 zinc finger CCCH-type containing protein 4 (Homo sapiens).This gene encodes a member of a family of CCCH (C-x8-C-x5-C-x3-H type) zinc finger domain-containing proteins. These zinc finger domains, which coordinate zinc finger binding and are characterized by three cysteine residues and one histidine residue, are nucleic acid-binding. Other family members are known to function in post-transcriptional regulation.Image 7GRTPPTLLSTLQYPRPSSGTLASASPDWAGPGARLRQQSSSSKGDSPELKPRAVHKQGPSPVSPNALDRTAAWLLTMNAQLLEDEALGPDPPHRDRLRSKEELSQAEKDLAVLQDKLRISTKKLEEYETLFKCQEETTQKLVLEYQARLEEGEERLRRQQEDKDIQMKGIISRLMSVEEELKKDHAEMQAAVDSKQKIIDAQEKRIASLDAANARLMSALTQLKERYSMQARNGISPTNPTKLQITENGEFRNSSNC.>XP_003353684.3 PREDICTED: LOW QUALITY PROTEIN: disabled homolog 2-interacting protein-like isoform 1 [Sus scrofa] MSAGGSARKSTGRPSYYYRLLRRPRLQRQRSRSRSRTRPARESPQERPGSRRSLPGSLSEKSPSMEPSAATPFRVTGFLSRRLKGSIKRTKSQPKLDRNHSFRHILPGFRSAAAAAAAASAADNERSHLMPRLKESRSHESLLSPSSAVEALALSMEEEVVIKPVHSSILGQDYCFEVTTSSGSKCFSCRSAAERDKWMENLRRAVHPNKDNSRRVEHILKLWVIEAKDLPAKKKYLCELCLDDVLYARTTGKLKTDNVFWGEHFEFHNLPPLRTVTVHLYRETDKKKKKERNSYLGLVSLPAASVAGRQFVEKWYPVVTPNPKGGKGPGPMIRIKARYQTITILPMEMYKEFAEHITNHYLGLCAALEPILSAKTKEEMASALVHILQSTGKVKDFLTDLMMSEVDRCGENEHLIFRENTLATKAIEEYLKLVGQKYLQDALGEFIKALYESDENCEVDPSKCSAADLPEHQGNLKMCCELAFCKIINSYCVFPRELKEVFASWRQECSSRGRPDISERLISASLFLRFLCPAIMSPSLFHLLQEYPDDRTARTLTLIAKVTQNLANFAKFGSKEEYMSFMNQFLEHEWTNMQRFLLEISNPETISNTAGFEGYIDLGRELSSLHSLLWEAVSQLEQSIVSKLGPLPRILRDVHTALSTPGSGQLTGTNDLASTPGSGSSSISAGLQKMVIENDLSGLIDFTRLPSPTPENKDLFFVTRSSGVQPSPARSSSYSEANEPDLQMANGGKSLSMVDLQDARALDGEAGSPAGPDALAADGQVPTAQLVAGWPARAAPVSLAGLATVRRAGQTPTTPGTSEGAPGRPQLLAPLSFQNPVYQMAAGLPLSPRGLGDSGSEGHSSLSSHSNSEELAAAAKLGSFSS;ATAAAAEDLGRRPGELARRQMSLTEKGGQPTVPRQNSAGPQRR16 residues IDQPPPPPPPPPPAPR MW 1668.9.Functions as a scaffold protein implicated in the regulation of a large spectrum of both general and specialized signaling pathways.Image 8LALSEDTEPSSSESRTGLCSPEDNSLTPLLDEVAAPEGRAATVPRGRGRSRGDSSRSSASELRRDSLTSPEDELGAEVGDEAGDKKSPWQRREERPLMVFNVK.>XP_005668832.1 PREDICTED: protein FAM171A2 [Sus scrofa] MPPPSGPSVLARLLPLLGLLLGGASRAPGKSPPEPPSPQEILIKVQVYVSGELVPLARASVDVFGNRMLLAAGTTDSEGVATLPLSYRLGTWVLVTAARPGFLTNSVPWRVDKLPLYASVSLYLLPERPATLILYEDLVHILLGSPGARSQPWVQFQRRAARLPVSSTYSQLWASLTPASTQQEMRAFPAFLGTDASSSGNGSWLELMPVAAVSVHLLAGNGTEVPLSGPIHLSLPVPSEPRALAVGTSIPAWRFDPKSGLWVRNGTGVIRKEGRQLYWTFISPQLGYWAAAMASPTSGLVTITSGIQDIGTYHTIFLLTILAALALLVLILLCLLIYYCRRRCLKPRQQHRKLQLSGPSDGNKRDQATSMSQLHLICGGPLEPAASGDPEAPPPGPLHSAFSSSRDLAASRDDFFRAKPRSASRPAAEPAGARGGEGAGLKGARSVEGPGGLEPGLEEYRRGPPGTATFLQEPPSPPPPFEHYLGHKGAAESKTPDFLLSQSVDQLERPPSLSQAGQLIFCGSIDHHSQVRHSYIDLQAGGGGRSTDASLDSGVDVHEARPARRRPLREERERAA17 residues AAAPPPPPPPPPPAPPR MW 1622.9.Image 9CPYSGAGFPPAPPPPPPPPLPGGPPVPPPPPGLPPPSHLNGYSHLGKKKRMRSFFWKTIPEEQVRGKTNIWTLAARQQHHYQIDTKTIEELFGQQEESAKSSPSRRGGPLNSSFREAREEITILDAKRSMNIGIFLKQFKKSPPSIVEDIHQGKSEHYGSETLREFLKLLPESEEIKKLKAFSGDVAKLSLADSFLHCLIQVPNYSLRIEAMVLKKEFLPSCSSLYTDMTILRTATKELMSCEELHSILHLVLQAGNIMNAGGYAGNAVGFKLSSLLKLADTKANKPGMNLLHFVAQEAQKKDAVLLNFSEKLHHVQEAARLSLDNTEAELHSLFVRTRSLKENIQRDGELCQQMEDFLQFAVEELSELERWKQELLAEAHTLIDFFCEDKDTVKLDECLQIFRDFCIKFNKAVKDNHDREVQELKQLQRLKEQEQKRRSWAAGELGFSRSSSENDVELLTKRGAEDPFLHSRPISPSHRPPNTRRSRLSLGASADRELLTFLESSTGNPEELKFNSLPRSCPRQAPPSRAWMESGEQRDQDSSQAHRLPASKDQEEATDPPSTWQSQLLAPRLEEPATALPRVRRSGVSILRKRNSEPLGLGPVRSPPLSPLALGIKEHELVTGLAQFDLQAPKGPEEPARLTMNDFSPMELMSVVGESPQAPRAPNDHRCEGLIPPCFSNEDLGNILLYVRAHAASRPYRESRAPSRSSFRKPSVKPLRNVPRPKPDEDKMCRSSSQGPESPEEAPRAPAAPSAPRGPAPVPSFARNTVASSSRCLRTDSPAVARPPGLTRTVSQRQLRAKGGPEEAAPKDGGALRRASSARGPRKGPELPEGPRAGSEASPKGRGAGERASVRLKDASRPALGKGLHPLRK.>XP_005666867.1 PREDICTED: FH2 domain-containing protein 1 [Sus scrofa] MHVMNCVSSVSDKGNGNIAPAAGFMIGQT14 residues PPPPSPPPPPPPPP MW 1366.7.Formin-homology-domain-containing protein FHOD1 is involved in cell migration and adhesion, acting as a regulator of stress fibers organization, maturation of integrin-based adhesion sites, and podosome-associated contractility.Image 10GGLTSPIFCSTKPKKLLKTSSFHLLRRRDPPFQTPKKLYAQEYEFEADEDKADVPADIRLNPRRLPDLVSSCRSRPALSPLGDIDFCPPNPGPDGPRRRGRKPTKAKRDGPPRPRGRPRIRPLEGPATAGPALASTPTDGAKKPRGRGRGRGRKAEEAGGTRLEPLKPLKIKLSVPKAGEGLGASSGEAVSGADPNSLDSSLTREKIEAKIKEVEEKQPEMKSGFMASFLDFLKSGKRHPPLYQAGLTPPLSPPKSVPPSVPARGLQPQPPSTPAVPHPPPAGAFGLGGALEAAESEGLGLGCPSPCKRLDEELKRNLETLPSFSSDEEDSVAKNRDLQESISSAISALDDPPLAGPKDTSTPDGPPLAADAAVPGPPPLPGLPSASSNGTPEPPLLEEKPPPSPPPVPTPQPPPPPPALPSPPPLVAPAPSSPPPQImage 11PPAPAPAPAPPALPSPPAPPPPPAAAAAPPPEEPAAPSPDDSEPPDARPLHLAKKQETAAVCGETDEEAGESGGEGIFRERDEFVIRAEDIPSLKLALQTGREPPPIWRVQKALLQKFTPEIKDGQRQFCATSNYLGYFGDAKNRYQRLYVKFLENVNKKDYVRVCARKPWHRPPVPVRRSGQAKGPSSSGGSSAPPPKAPAPPPKPETPDKMASEKPPEQTPETAVPEPPAPEKPSPPRLVEKEKEKERTPRGERPLRGERGTGGRQIRPDRGLTTGQPATSRLPKSRPTKVKAEPPPKKRKKWLKEAAGNASAGGGPPGSSSDSESSPGAPSEDERAVPGRLLKTRAMREMYRSYVEMLVSTALDPDMIQALEDTHDELYLPPMRKIDGLLNEHKKKVLKRLSLSPALQDALHTFPQLQVEQSGEGSPEEGAVRLRPAGEPYNRKTLSKLKRSVVRAQEFKVELDKSGYYTLYHSLHHYKYHTFLRCRDQTLAIEGGAEDLGQEEVVQQCMRNQPWLEQLFDSFSDLLAQAQAHSRCG.>XP_003127395.2 PREDICTED: proline-rich protein 12 isoform X1[Sus scrofa] MDRNYPSAGFGDPLGAGAGWSYERSAKASLVYGSSRTSHPETDILHRQAYAAPHPLQSYATNHHPAGLSGLFDTGLHHAGSAGPDASVMNLISALESRGPQPGPSASSLLSQFRSPSWQTAMHTPGPTELFISGALPGSSTFPSSSALSAYQHPASFGSRPFPVPSSLSLQDPPFSPPANGLLSPHDVLHLKPSQAPTVPSSLGFERLAGGGVLGPAGLGPAQTPPYRPGPPDPPPPPRHLPTQFNLLASSSAAAAAAAEQSSPQLYNFSGAAPGPPPPERALPRQDTVIKHYQRPASAQPPPPPPPAHALQHYLSCGGSYPSMGHRANLACSPLGGGEPSPGAGEPSKAGPSGATAGASGRAAGPEAAGGGGAGGGGGGYRPIIQSPGYKTGKGGYGAAAGGANRPPPPRSTATPKCQSLGGPAAAYATGKASGAGGAGGQAYSPGQPQGLLGPQAYGQGFGGGQAQDLSKGPSYSGGPQQPPNGPPPPGLATCQSYSPDQLQGQLYGVQGEPYPGPAAHSQGLPTASPSLSYSTGHSPALSGHGGGWGPSSLGGGGEASPSHIIRPLQSPPAPGRPPGVGSPGAPGKYLSSVLASAPFLAPPGAGSYAAGAGGYKGKGDGSELLAGPGGPPAERTEDEEFLIQHLLQAPSPPRTSGADGLVGEDGAADASKGLGGSGGAGGPPGTPYELAKEDPQRYHLQSVIRTSASLDEGATAALELGLGRLKEKKKGPERGGETPEGLATSVVHYGAGAKELGAFLQKSPPPPPPTAQSAQPTPHGLLLEAGGPDLPLVLPPPPPQLLPSVLSHAPSPSSSAPKVGVHLLEPAARDGAPPPPPPPPPPPMPLQLEAHLRSHGLEPGAPSPRLRPEESLEPPGAMQELLGALEPLPPGPGDTGVGPPTAEGKDPSGAYRSPSPQGTKAPRFVPLTSICFPDSLLQDEERSFFPTMEEMFGGGPADDYGKAGPPEDEGDPKAGAGPPPGPPAYDPYGPYCSSRASGAGPETPGLGLDPSKPPELPSTVNAEPLGLIQSGPHQA19 residues APPPPPPPPPPPPPASEPK MW 1863.0.20 residues LPPPPPPPPPPPPPPPPPPP MW 1975.1.Image 12NQANTELPKLSRDEQRGRGALLQDICKGTKLKKVTNINDRSAPILEKPKGSSGGYGPGAAALQPKGGLFQGGVPKLRPVGAKDGSENLAGKPALQVPSSRAAAPRPPVSTASGRPQDDTDSNRASLPELPRTQRPSLPDLSRPHATSSTGMKHSSSAPPPPPPGRRANAPPTPLAMHSNKAPAYNREKPLPPTPGQRLHPGREGPSAPPPVKPPPSPVNIRTGPSGQSLAPPPPPYRQPPGVPNGPSSPTNESAPELPQRHNSLHRKTPGPVRGLAPPPPTSASPSLQSNRPPPPARDPPSRGAAPPPPPPMIRNGARDAPPPPPPYRMHGSEPLSRGKPPPPPSRTPAGPPPPPPPPLRNGHRDSITTVRSFLDDFESKYSFHPVEDFPAPEEYKHFQRVYPSKTNRAARGAPPLPPILR.>NP_001231241.1 WAS/WASL-interacting protein family member2 Sus scrofa 19 residues MPIPPPPPPPPGPPPPPTF MW 1926.0.Plays an active role in the formation of cell surface protrusions downstream of activated PDGFB receptors. Plays an important role in actin-microspike formation through cooperation with WASL. May cooperate with WASP and WASL to induce mobilization and reorganization of the actin filament system.Image 13LTPVKCEDPQRVVPTVNPVKTNGTLLRNGGFPGAPNKIPNGDICCKPGSIVDKAPVQPLMHRPEKDRCPQAGPRERVRFNEKVQYHGYCPDCDTRYNIKNREVHLHSEPVHPPGKLPPQGPHHPPPPHLPPFPLENGGLGISHSNSFPPLRPATVPPPTAPKPQKTILRKSTTTTV.>XP_005655053.1 PREDICTED: proline-rich protein 16 [Sus scrofa] MTDSSKTDTLNSSSSGTTASSIEKIKVQANAPLIKPPAHPSAILTVLRK9 residue PNPPPPPPR MW 967.5.Regulator of cell size that promotes cell size increase independently of mTOR and Hippo signaling pathways. Acts by stimulating the translation of specific mRNAs, including those encoding proteins affecting mitochondrial functions. Increases mitochondrial mass and respiration.Image 14LLSAPAPNATSCPAEESWWSGLAIVIAVCCASLVFLTVLVIICYKAIKRKPLRKEENGTSVAEYPMTSSQSNKGVDVNSAVV.>XP_020955308.1 proline-rich membrane anchor 1 isoform X2 [Sus scrofa] MLLRDLVLRRGCCWPSLLLHCALHPLWGFVQVAHGEPQKSCSKVTDSCQHICQCR16 residues PPPPLPPPPPPPPPPR MW 1645.9.Required to anchor acetylcholinesterase (ACHE) to the basal lamina of the neuromuscular junction and to the membrane of neuronal synapses in brain. Also able to organize ACHE into tetramers."} +{"text": "Dumbbell-shaped DNA minimal vectors represent genetic vectors solely composed of the gene expression cassette of interest and terminal closing loop structures. Dumbbell vectors for small hairpin RNA or microRNA expression are extremely small-sized, which is advantageous with regard to cellular delivery and nuclear diffusion. Conventional strategies for the generation of small RNA-expressing dumbbell vectors require cloning of a respective plasmid vector, which is subsequently used for dumbbell production. Here, we present a novel cloning-free method for the generation of small RNA-expressing dumbbell vectors that also does not require any restriction endonucleases. This new PCR-based method uses a universal DNA template comprising an inverted repeat of the minimal H1 promoter and the miR-30 stem. The sequences coding for small RNA expression are introduced by the PCR primers. Dumbbells are formed by denaturing and reannealing of the PCR product and are covalently closed using ssDNA ligase. The new protocol generates plus- and/or minus-strand dumbbells, both of which were shown to trigger efficient target gene knockdown. This method enables fast, cheap production of small RNA-expressing dumbbell vectors in a high throughput-compatible manner for functional genomics screens or, as dumbbells are not prone to transgene silencing, for knockdown studies in primary cells. For shRNA gene delivery, researchers explore viral or non-viral delivery vectors. While viral vectors are costly and often trigger immune responses or pose the risk of genomic vector integration, many non-viral delivery vectors involve non-nucleic acid helper functions that can be toxic to the cells.Here, we report a cloning-free method for the generation of shRNA-expressing dumbbell vectors. This PCR-based method uses a universal template and sequences coding for a specific shRNA are introduced by the PCR primers. This novel protocol produces size-minimized hairpin-template transcribing dumbbells, does not require any restriction or nicking endonucleases, and is high throughput compatible.5), and (3) the hsa-miR-30 precursor stem in which the self-complementary sequence portions were separated from each other A\u2013S1C. Paequenced D and S1ENext, we aimed to PCR-amplify the universal template using primers that introduced the sequence coding for a published firefly luciferase-targeting shRNA.With the decision to use either a 5\u2032-phosphorylated forward primer, a 5\u2032-phosphorylated reverse primer, or two phosphorylated primers for PCR, only (1) plus-strand-derived dumbbells, (2) minus-strand-derived dumbbells, or (3) a mix of both can be generated and S2. Employing the above protocol using either phosphorylated forward or reverse primers, we generated both plus- and minus-strand-derived luciferase- or lamin A/C-targeting dumbbells in separate reactions\u00a0 and 4. TThe protocol described here combines all the advantages of previously reported protocols for dumbbell vector production. It represents (1) a cloning-free protocol that (2) does not involve any restriction or nicking endonucleases, (3) employs an efficient intra-molecular ligation reaction, and (4) allows production of extremely small hairpin template-transcribing dumbbell vectors. The previously described gap-primer PCR protocol also involves an intra-molecular ligation but requires a cloning step for the generation of every new vector, and it is not suitable to generate hairpin template-transcribing vectors due to the presence of abasic sequence positions.We demonstrate the proof-of-principle that this new method can generate partly mismatched shRNA-expressing dumbbell vectors, indicating the technology might also be explored for the generation of miRNA-expressing dumbbells. Mismatches in dumbbell vectors were reported earlier and demonstrated not to impair vector activity.We observed that among the luciferase- or lamin A/C-targeting dumbbells, the minus- or plus-strand-derived dumbbell exhibited a stronger target gene knockdown activity, respectively. This difference might be assigned to differences with regard to the efficiency and accuracy of endogenous shRNA processing by Dicer, which depends on the sequence and structure of shRNA loops and stems. Here, we employed\u00a0the hsa-miR-30 stem, as miRNA stems were reported to facilitate shRNA processing and knockdown activities in most of the cases.In conclusion, this novel method efficiently generates size-minimized hairpin template-transcribing dumbbells in a short period of time and at low costs and can be explored for the parallelized production of shRNA or miRNA expression vectors for functional genomics screens or drug development.AGCTTCGCGCTCACTGAGAAGATTTTTCTGTGCTCTCATACAGAACTTATAAGATTCCCAAATCCAAAGACATTTCACGTTTATGGTGATTTCCCAGAACACATAGCGACATGCAAATATGAATTGTCCAGTT-3\u2032; oligo UT2, 5\u2032P-GGACAATTCATATTTGCATGTCGCTATGTGTTCTGGGAAATCACCATAAACGTGAAATGTCTTTGGATTTGGGAATCTTATAAGTTCTGTATGAGAGCACAGAAAAATCTTCTCAGTGAGCGCGA-3\u2032; oligo UT3, 5\u2032P-TTCTGGACAATTCATATTTGCATGTCGCTATGTGTTCTGGGAAATCACCATAAACGTGAAATGTCTTTGGATTTGGGAATCTTATAAGTTCTGTATGAGAGCACAGAAAAATCTTCTCAGTAGGCAAAG-3\u2032; oligo UT4, 5\u2032-GATCCTTTGCCTACTGAGAAGATTTTTCTGTGCTCTCATACAGAACTTATAAGATTCCCAAATCCAAAGACATTTCACGTTTATGGTGATTTCCCAGAACACATAGCGACATGCAAATATGAATTGTCCAGAAAACT-3\u2032. Bold indicates HindIII and BamHI compatible overhangs; underlined indicates dumbbell loop-forming tetranucleotide. The 5\u2032 phosphorylated oligos UT2 and UT3 were hybridized with the complementary oligos UT1 and UT4, respectively. The resulting UT1/UT2 and UT3/UT4 duplexes were ligated to form the universal template sequence bearing HindIII and BamHI-compatible 5\u2032 overhangs, which was subsequently cloned into pVax1 , yielding the universal template vector pVax1-UT endonucleolytic cleavage followed by analytical agarose gel electrophoresis, which yielded the expected insert size of 262\u00a0bp, and by sequencing of the ligation sites A and S1BpVax1-UT B. For clon sites C and S1DtgaaggctcctcagaaacagctcCGCGCTCACTGAGAAGATTT-3\u2032; FP_Lamin 5\u2032-tgaaagcccagatcgtcaccacccgcCGCGCTCACTGAGAAGATTT-3\u2032; reverse primers, RP_Luciferase 5\u2032-agagaggctcctcagaaacagctcTTTGCCTACTGAGAAGATTTTTCTGT-3\u2032. RP_Lamin 5\u2032-agagaagcccagatcgtcaccaccttTTTGCCTACTGAGAAGATTTTTCTGT-3\u2032.Luciferase- or lamin A/C-specific primers were synthesized by AITbiotech (Singapore) or IDT (Singapore). Uppercase letters indicate the universal template binding sites, and lowercase letters indicate the shRNA coding sequences in which the loop-forming nucleotides are underlined: forward primers, FP_Luciferase 5\u2032-Two blocking ODNs were added to the PCR to suppress refolding and self-priming of the universal template strands: Block_1, 5\u2032-GGACAATTCATATTTGCATGTCGCTATGTGTTCTGGGAAATCACCATAAACGTGAAATGTCTTTGGATTTGGGAATCTTATAAGTTCTGTATGAGAGCACAGAAAAATCTTCTCAGTGAGCGCGA-3\u2032; Block_2, 5\u2032-TTCTGGACAATTCATATTTGCATGTCGCTATGTGTTCTGGGAAATCACCATAAACGTGAAATGTCTTTGGATTTGGGAATCTTATAAGTTCTGTATGAGAGCACAGAAAAATCTTCTCAGTAGGCAAAG-3\u2032.Primers for the quantification of luciferase and \u03b2-actin mRNA levels were synthesized by AITbiotech (Singapore). PCR forward primers are as follow: qPCR_FP_Luciferase 5\u2032-CGCTGGGCGTTAATCAAAGA-3\u2032; qPCR_RPb-actin 5\u2032-CTGGCACCCAGCACAATG-3\u2032. Reverse transcription and PCR reverse primers are as follows: qPCR_RP_Luciferase 5\u2032-GTGTTCGTCTTCGTCCCAGT-3\u2032; qPCR_RPb-actin 5\u2032- GCCGATCCACACGGAGTACT-3\u2032.For the generation of strand-specific dumbbell vectors, either the forward or the reverse primers were 5\u2032-phosphorylated. Each 50 pmol primer was incubated with 10\u00a0U T4 polynucleotide kinase in the presence of 1\u00a0mM ATP at 37\u00b0C for 20\u00a0min followed by heat inactivation of the enzyme at 75\u00b0C for 10\u00a0min.Taq DNA polymerase (Invitrogen),\u00a01.0\u00a0\u03bcM of each primer and blocking ODNs, 0.2\u00a0mM of each 2\u2019-deoxyribonucleoside 5\u2019-triphosphate , 100\u00a0ng of HindIII/BamHI cleaved pVax1-UT, 5% v/v DMSO in a reaction volume of 30\u201350\u00a0\u03bcL in 1\u00d7 Taq DNA polymerase buffer (Invitrogen). Linearization of pVAX1-UT usually improves the PCR yields but is not essential. Thermal cycling was carried out as follows: initial denaturation at 96\u00b0C for 5\u00a0mins 27 cycles of denaturation , annealing , and extension ; and final extension at 72\u00b0C for 10\u00a0mins. A 50-\u03bcL PCR reaction yielded about 10\u00a0\u03bcg DNA.PCR amplification of the universal template and appendage of shRNA encoding DNA was carried out using 1\u00a0U 2, and 200\u00a0mM Tris-HCl, pH 7.4), heat-denatured\u00a0at 96\u00b0C for 5\u00a0min followed by gradual cooling to room temperature to allow for intramolecular folding of plus- and/or minus-strand dumbbell vectors. The resulting DNA was concentrated using ethanol precipitation, pelleted by centrifugation, and resuspended in nuclease-free water.PCR products were purified through silica-membrane-based spin columns . Purified products were diluted to 400\u00a0\u03bcL in 1\u00d7 hybridization buffer , and 50 to 100\u00a0U CircLigaseII ssDNA ligase in 1\u00d7 CircLigaseII reaction buffer at 60\u00b0C for 16 h, followed by heat inactivation of the ligase at 80\u00b0C for 10\u00a0min. Highest conversion yields were observed when ligating 6\u00a0\u03bcg DNA with 100\u00a0U CircLigase.1 to 6\u00a0\u03bcg (\u223c10 to 60 pmol) of DNA was incubated with 2.5\u00a0mM MnClAfter ligation, products were treated with 10\u00a0U of T7 DNA polymerase at 37\u00b0C for 1\u00a0h followed by heat inactivation at 80\u00b0C for 10\u00a0min. Products were assessed on 10% native polyacrylamide gels or 1% agarose gels, stained with ethidium bromide post-electrophoresis, and/or purified using phenol-chloroform-isoamylalcohol (25:24:1) extraction (1\u00d7), chloroform-isoamylalcohol (24:1) re-extraction (3\u00d7), and ethanol precipitation.4 cells/well were seeded in a 96-well plate. Cells were co-transfected with 100\u00a0ng of luciferase expression plasmid pGL3 and 1.5 pmol or 0.5 pmol of either plus- or minus-strand dumbbell vector DNA using Lipofectamine 2000 and a reagent:DNA ratio of 1:2.5. For the positive control (pGL3 only), empty pVAX1 was used as feeder DNA to ensure all cells received the same quantity of DNA. 48\u00a0h post-transfection, cells were washed with sterile PBS and lysed in 20\u00a0\u03bcL passive lysis buffer for 20\u00a0min, employing gentle shaking. 10\u00a0\u03bcL of lysate was treated with 50\u00a0\u03bcL of LARII reagent , and luminescence was quantified on the Biotek reader .HEK293T cells were maintained in DMEM supplemented with 10% (v/v) fetal bovine serum and 1% penicillin-streptomycin antibiotic solution . 24\u00a0h prior to transfection, 2\u00a0\u00d7 10HEK293T cells were cultivated and seeded in 96-well plates 24\u00a0h prior to transfection as described above. Cells were transfected with 0.1, 0.5, or 2.5 pmol dumbbell vector DNA or 3 pmol siGENOMELamin A/C control siRNA using Lipofectamine 3000 according to the manufacturer\u2019s protocol. Medium was changed 24\u00a0h post-transfection, and cells were harvested after 48 h. For FACS analyses, the media was aspirated, and the cells were rinsed once with PBS before trypsinization with 50\u00a0\u03bcL of 1\u00d7 trypsin-EDTA (Gibco). Trypsinized cells were collected by centrifugation at 4,200\u00a0rpm for 6\u00a0min in 200\u00a0\u03bcL media. Pelleted cells were resuspended in 100\u00a0\u03bcL media, fixed and permeabilized with intracellular fixation and permeabilization buffer set according to manufacturer\u2019s protocol prior to intracellular staining. To assess lamin A/C knockdown, cellular lamin A/C was stained by anti-lamin A+C antibody (ab133256) (1/200) and donkey anti-rabbit IgG H&Ls AF647 (ab150075) (1/200) . FACS was performed on LSRFortessa cell analyzer, and FACSDiva software v6.1.3 was used for the acquisition of the samples. FlowJo software V10.5.2 was used for data analyses.mfold and/or RNAfold.18Minimum free energy secondary structures of DNA and RNA were folded using the algorithms Diagrams represent mean values\u00a0\u00b1 SEM of three independent experiments.\u00a0The statistical analysis was performed using repeated one-way ANOVA with Tukey\u2019s post hoc multiple comparison\u2019s test (luciferase knockdown data) or using Student\u2019s t test (lamin A/C knockdown data). The GraphPad Prism version 6 software was used for the statistical analysis. p values are as indicated.V.P. developed the concept. V.P., S.L.C., A.G., P.S.L., and G.S.X.T. designed the experiments. S.L.C., A.G., P.S.L., and G.S.X.T. carried out the experiments, and all authors analyzed the data. V.P., S.L.C., A.G., and P.S.L. wrote the manuscript.The authors declare no competing interests."} +{"text": "Memory consolidation theory suggests that once memory formation has been completed, memory is maintained at a stable strength and is incapable of further enhancement. However, the current study reveals that even long after formation, contextual fear memory could be further enhanced. Such unexpected enhancement is possible because memory is dynamically maintained at an intermediate level that allows for bidirectional regulation. Here we find that both Rac1 activation and expression of \u03b12-chimaerin are stimulated by single-trial contextual fear conditioning. Such sustained Rac1 activity mediates reversible forgetting, and \u03b12-chimaerin acts as a memory molecule that reverses forgetting to sustain memory through inhibition of Rac1 activity during the maintenance stage. Therefore, the balance between activated Rac1 and expressed \u03b12-chimaerin defines dynamic long-term memory maintenance. Our findings demonstrate that consolidated memory maintains capacity for bidirectional regulation. Memory consolidation theory suggests that memory is maintained at a stable strength after formation. The authors show that memory is dynamically maintained at an intermediate level allowing a bidirectional regulation which is mediated by a balance between activated Rac1 and expressed \u03b12-chimaerin. Guided by such widely accepted theory, the study of memory maintenance is understandably devoted to the identification of learning-stimulated self-sustainable protein synthesis regulatory mechanisms5. Indeed, a number of candidate mechanisms for such a simple maintenance have been reported, including the autophosphorylation of calcium/calmodulin-dependent protein-kinase type II (CAMKII)7, and the persistent protein synthesis regulated via self-perpetuating prion-like translational factors such as the cytoplasmic polyadenylation binding protein-3 (CPEB-3)8. It is a common experience, however, when recalling an episode that happened some time ago, the memory is vivid at one time but vague at others. Such flexible recalling experiences seem to be counterintuitive to memory consolidation theory. In addition to common experiences, it is noteworthy that recent publications have reported a number of intriguing observations that appear inconsistent with the notion of stabilized memory. First, synaptic connections involved in maintaining LTM are highly dynamic5. Second, overexpression of the self-autonomous protein-kinase-M-zeta (PKM\u03b6)9 was reported to enhance memory long after memory formation, but mechanisms underlying such enhancement remain unexplained10.Memory consolidation theory suggests that a newly acquired labile memory is gradually consolidated or stabilized through gene-regulation-based persistent modifications in synaptic structures within neural circuits and the resulting consolidated long-term memory (LTM) is maintained at a stable strength11. The recently proposed concept of active forgetting describes an intrinsic mechanism, i.e., learning or training evokes active forgetting to accelerate the decay of a formed memory. Multiple mechanisms are attributed to forgetting, including through activation of Rac1 or Cdc42, as well as through neurogenesis11. Recent progress in study of forgetting reveals that suppression of forgetting, inhibition of Rac1-dependent forgetting in particular, is capable of prolonging an hour-long labile memory to days14 in a range of tasks from invertebrates to vertebrates16. Moreover, a study reports that LTM decayed away during infancy can be recovered in adulthood17, suggesting that neurogenesis-based forgetting does not lead to memory erasure. These observations got us interested in investigating whether forgetting mechanisms, Rac1-dependent forgetting in particular, play any roles in memory maintenance. One possible candidate molecule to inhibit Rac1 activity comes from recent studies in mice during developmental stages21. It reveals that the negative regulator of Rac1 activity, \u03b12-chimaerin, acts as a brake through its GTPase-activating function to constrain Rac1 activity in regulating hippocampal dendrite and spine morphogenesis22 for establishment of normal cognitive ability23. However, the roles of interaction between Rac1 activity and \u03b12-chimaerin in memory maintenance in adulthood remain to be unraveled.Forgetting, the flip side of memory consolidation, is also an important feature for different types of memoriesThe present study provides a novel mechanism for dynamic memory maintenance that affords an intuitive explanation as to why LTM is still capable of being enhanced or faded long after completing formation. By integrating genetic, pharmacological, and optogenetic manipulations during behavioral paradigms, we demonstrate that the interplay between learning-evoked Rac1-dependent forgetting and learning-induced expression of a memory molecule \u03b12-chimaerin enables consolidated contextual fear memory and is maintained at an intermediate level so that memory could be enhanced through expression of more \u03b12-chimaerin while attenuated through elevated Rac1 activity. Moreover, forgetting induced by enhanced Rac1 activity is reversible through suppression of Rac1 activity. Thus, our results reveal that consolidated memory is maintained through the learning-evoked Rac1-dependent reversible forgetting mechanism balanced by learning-evoked expression of a memory maintenance molecule, \u03b12-chimaerin.14, we were interested in studying whether Rac1-dependent forgetting mechanisms play roles in LTM maintenance. In an effort to investigate this issue, we assayed Rac1 activity in hippocampal extracts of mice trained in the contextual fear conditioning (CFC) during the maintenance period of memory. Wild-type (WT) mice were subjected to single- or three-trial CFC and were tested for memory retention at different time points Fig.\u00a0. To avoiays Fig.\u00a0 and thenays Fig.\u00a0. Consideays Fig.\u00a0. Consistays Fig.\u00a0. In addiays Fig.\u00a0. Howeverays Fig.\u00a0.Fig. 1In15. Mice were bilaterally injected with adeno-associated viruses (AAVs), carrying mutant transgenes that encoded either dominant-negative Rac1 (Rac1-DN) to inhibit endogenous Rac1 activity or constitutively active Rac1 (Rac1-CA) to increase Rac1 activity, targeted to hippocampal excitatory neurons15 and AAV9-tetracycline response element (TRE)-Tandem dimer Tomato (tdTomato) to the CA1 region of WT mice was assessed by the percentage of Rac1-GTP+/c-fos+ colabeled cells among c-fos+ cells (engram cells). We found that 79% of c-fos+ cells were labeled with activated Rac1 positive (Rac1-GTP+) at 24\u2009h following training in CA1 engram cells and the control (tdTomato) mice were injected with AAV9-c-fos:tTA and AAV9-TRE-tdTomato mice were injected bilaterally into the CA1 with a virus cocktail of AAVato Fig.\u00a0, top. Inato Fig.\u00a0, bottom.ato Fig.\u00a0 at day 4ato Fig.\u00a0. Next, wThe findings from the above two experiments led us to make a third prediction that the naturally faded memory for several days . Additional injections of ANI further supported the finding Fig.\u00a0. This ex23. For knockdown, we generated AAVs that carry a short hairpin RNA (shRNA) sequence22 that targets \u03b12-chimaerin (AAV-\u03b12 shRNA), fused to tdTomato, under the control of the cytomegalovirus (CMV) early enhancer/chicken \u03b2-actin (CAG) promoter induced via a single tetanus (weak stimulation) at the Schaffer collaterals of the hippocampal CA1 region. Knockdown of \u03b12-chimaerin in slices expressing AAV-\u03b12 shRNA led to a reduction of potentiation and accelerated LTP decay Fig.\u00a0. In cont 14 Fig.\u00a0, because 14 Fig.\u00a0 while ac 14 Fig.\u00a0.Fig. 6\u03b1215. Thus, interplay between activated Rac1 and expressed \u03b12-chimaerin defines the intermediate level of memory during maintenance period and allows dynamic memory maintenance were purchased from the Vital River Laboratory and were housed in groups under standard conditions according to the Tsinghua University animal facility. Mice were maintained on a 12\u2009h light/dark cycle and were tested during the light phase of the cycle. All animal work complied with ethical regulations for animal testing and research, and was done in accordance with Institutional Animal Care and Use Committee (IACUC) approval by Tsinghua University and followed by all Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) guidelines.All plasmids were constructed by standard molecular biology procedures and subsequently verified by double-strand DNA sequencing. The \u03b11-chimaerin and \u03b12-chimaerin were synthesized by the GENEWIZ company (sequence are showed below). The pAAV-CaMKII\u03b1-EGFP was a gift from Bryan Roth (Addgene plasmid #50469). The \u03b11-chimaerin and \u03b12-chimaerin were sub-cloned into AAV backbone pAAV-CaMKII\u03b1-EGFP using the BsrGI/EcoRI restriction enzymes.\u03b11-chimaerin (Sequence ID: NM_001206602.1)5\u2032-ATGCCATCCAAAGAGTCTTGGTCAGGGAGGAAAACTAATAGGGCTGCAGTTCACAAATCAAAACAAGAGGGCCGTCAGCAAGATTTATTGATAGCAGCCTTGGGAATGAAACTGGGTTCTCCAAAGTCGTCTGTGACAATCTGGCAACCTCTGAAACTCTTTGCTTATTCGCAGTTGACATCACTTGTTAGAAGAGCAACTCTGAAAGAAAACGAGCAAATTCCAAAATATGAAAAGATTCACAATTTCAAGGTGCATACATTCAGAGGGCCACACTGGTGTGAATACTGTGCCAACTTTATGTGGGGTCTCATTGCTCAGGGAGTGAAATGTGCAGATTGTGGTTTGAATGTTCATAAGCAGTGTTCCAAGATGGTCCCAAATGACTGTAAGCCAGACTTGAAGCATGTCAAAAAGGTCTACAGCTGTGACCTTACGACGCTCGTGAAAGCACATACCACTAAGCGGCCAATGGTGGTAGACATGTGCATCAGGGAGATTGAGTCTAGAGGTCTTAATTCTGAAGGACTATACCGAGTATCAGGATTTAGTGACCTAATTGAAGATGTCAAGATGGCTTTCGACAGAGATGGTGAGAAGGCAGATATTTCTGTGAACATGTATGAAGATATCAACATTATCACTGGTGCACTTAAACTGTACTTCAGGGATTTGCCAATTCCACTCATTACATATGATGCCTACCCTAAGTTTATAGAATCTGCCAAAATTATGGATCCGGATGAGCAATTGGAAACCCTTCATGAAGCACTGAAACTACTGCCACCTGCTCACTGCGAAACCCTCCGGTACCTCATGGCACATCTAAAGAGAGTGACCCTCCACGAAAAGGAGAATCTTATGAATGCAGAGAACCTTGGAATCGTCTTTGGACCCACCCTTATGAGATCTCCAGAACTAGACGCCATGGCTGCATTGAATGATATACGGTATCAGAGACTGGTGGTGGAGCTGCTTATCAAAAACGAAGACATTTTATTTTAA-3\u2032\u03b12-chimaerin (Sequence ID: NM_001822.5)5\u2032-ATGGCCCTGACCCTGTTTGATACAGATGAATATAGACCTCCTGTTTGGAAATCTTACTTATATCAGCTACAACAGGAAGCCCCTCATCCTCGAAGAATTACCTGTACTTGCGAGGTGGAAAACAGACCAAAGTATTATGGAAGAGAGTTTCATGGCATGATCTCCAGAGAAGCAGCCGACCAGCTCTTGATTGTGGCTGAGGGGAGCTACCTCATCCGGGAGAGCCAGCGGCAGCCAGGGACCTACACTTTGGCTTTAAGATTTGGAAGTCAAACCAGAAACTTCAGGCTCTACTACGATGGCAAGCACTTTGTTGGGGAGAAACGCTTTGAGTCCATCCACGATCTGGTGACTGATGGCTTGATTACTCTCTATATTGAAACCAAGGCAGCAGAATACATTGCCAAGATGACGATAAACCCAATTTATGAGCACGTAGGATACACAACCTTAAACAGAGAGCCAGCATACAAAAAACATATGCCAGTCCTGAAAGAGACACATGATGAGAGAGATTCTACAGGCCAGGATGGGGTGTCAGAGAAAAGGTTGACATCACTTGTTAGAAGAGCAACTCTGAAAGAAAACGAGCAAATTCCAAAATATGAAAAGATTCACAATTTCAAGGTGCATACATTCAGAGGGCCACACTGGTGTGAATACTGTGCCAACTTTATGTGGGGTCTCATTGCTCAGGGAGTGAAATGTGCAGATTGTGGTTTGAATGTTCATAAGCAGTGTTCCAAGATGGTCCCAAATGACTGTAAGCCAGACTTGAAGCATGTCAAAAAGGTCTACAGCTGTGACCTTACGACGCTCGTGAAAGCACATACCACTAAGCGGCCAATGGTGGTAGACATGTGCATCAGGGAGATTGAGTCTAGAGGTCTTAATTCTGAAGGACTATACCGAGTATCAGGATTTAGTGACCTAATTGAAGATGTCAAGATGGCTTTCGACAGAGATGGTGAGAAGGCAGATATTTCTGTGAACATGTATGAAGATATCAACATTATCACTGGTGCACTTAAACTGTACTTCAGGGATTTGCCAATTCCACTCATTACATATGATGCCTACCCTAAGTTTATAGAATCTGCCAAAATTATGGATCCGGATGAGCAATTGGAAACCCTTCATGAAGCACTGAAACTACTGCCACCTGCTCACTGCGAAACCCTCCGGTACCTCATGGCACATCTAAAGAGAGTGACCCTCCACGAAAAGGAGAATCTTATGAATGCAGAGAACCTTGGAATCGTCTTTGGACCCACCCTTATGAGATCTCCAGAACTAGACGCCATGGCTGCATTGAATGATATACGGTATCAGAGACTGGTGGTGGAGCTGCTTATCAAAAACGAAGACATTTTATTTTAA-3\u2032\u03b11-chimaerin was 5\u2032-GCTT TCAGCAATGTGTCAT-3\u203239. The \u03b12-chimaerin shRNA sequence binding to the mouse \u03b12-chimaerin was 5\u2032-GCACATGGCAGTCCTGAAA-3\u2032, whereas the negative control scrambled shRNA (ctrl shRNA) was 5\u2032-GTTCATATGTTCACCTATT-3\u203222. The U6 promoter sequence was 5\u2032- GATCCGACGCCGCCATCTCTAGGCCCGCGCCGGCCCCCTCGCACAGACTTGTGGGAGAAGCTCGGCTACTCCCCTGCCCCGGTTAATTTGCATATAATATTTCCTAGTAACTATAGAGGCTTAATGTGCGATAAAAGACAGATAATCTGTTCTTTTTAATACTAGCTACATTTTACATGATAGGCTTGGATTTCTATAAGAGATACAAATACTAAATTATTATTTTAAAAAACAGCACAAAAGGAAACTCACCCTAACTGTAAAGTAATTGTGTGTTTTGAGACTATAAATATCCCTTGGAGAAAAGCCTTGTTT-3\u2032.The U6 promoter-driven \u03b11-chimaerin shRNA (\u03b11 shRNA), \u03b12-chimaerin shRNA (\u03b12 shRNA), or negative control scrambled shRNA (ctrl shRNA) constructs were synthesized (GENWIZ company) and sub-cloned into AAV backbone pAVV-CAG-tdTomato (Addgene plasmid # 59462) using the EcoRI/HindIII and EcoRV/HindIII restriction enzymes, respectively. The \u03b11-chimaerin shRNA sequence binding to the mouse 39. Keyhole limpet hemocyanin-coupled synthetic peptides for \u03b11-chimaerin was MPSKESWSGRKANR and keyhole limpet hemocyanin-coupled synthetic peptides for \u03b12-chimaerin was HDEKEATGQDGVSEKR. Anti-Rac1-GTP (active Rac1) monoclonal antibody was from the NewEast company (Cat. No. 26903). Anti-Rac1 antibody was from BD Transduction Laboratories (Cat. No. 610650). Anti-phospho-PAK1/2/3 antibody was from novusbio company (Cat. No. NB100-82131). Anti-Cleaved Caspase-3 antibody was from Cell Signaling (Cat. No. 9661). Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Cat. No. #7072) and HRP-conjugated goat anti-rabbit IgG (Cat. No. #7071) were from Cell Signaling Technology. Alexa Fluro\u00ae 488 affinipure donkey anti-mouse IgM was from the Jackson ImmunoResearch Laboratories company (Cat. No. #711-545-140). Actin antibody was from the Merck Millipore company (Cat. No. #MAB1501).The following antibodies were used: primary antibodies for \u03b11-chimaerin and \u03b12-chimaerin were raised in rabbits immunized with keyhole limpet hemocyanin-coupled synthetic peptides as previously reported15. ANI was dissolved in 0.9% saline and the pH was adjusted with 1\u2009N HCl to 7.0\u20137.4. Mice were subcutaneously injected with 150\u2009mg per kg of ANI or an equivalent volume of saline immediately after training. This amount of ANI was shown to effectively inhibit cerebral protein synthesis in mice (~96%)40.Ehop016 was dissolved in a solution containing 1% dimethylsulfoxide/30% PEG/1% Tween-80. Mice were intraperitoneally injected with 20\u2009mg per kg of Ehop016 solution or an equivalent volume of saline. At the dose used, Ehop016 has high efficiency to block Rac1 activity9-c-fos:tTA (300\u2009nl of AAV9-c-fos:tTA) and 300\u2009nl of the AAV9-TRE-tdTomato into the CA1 . For the activation of Rac1 activity in CA1 engram cells, a virus cocktail AAV9-c-fos:tTA (300\u2009nl of AAV9-c-fos:tTA and 300\u2009nl of AAV9-TRE:photoactivatable Rac1 (PaRac1)-tdTomato41 or the AAV9-c-fos-EGFP (as the control group) were injected into the mice CA1 . Following the injections, the needle stayed for 10\u2009min before slowly withdrawn and the wound sutured. After surgery, animals were allowed to recover for two weeks prior to the performance of all subsequent experiments.Mice were anesthetized with 0.2% sodium pentobarbital (5\u2009ml/kg) and fixed to a stereotaxic frame. Their body temperature was kept at 36\u2009\u00b0C by a heating pad and their skull was exposed. Furthermore, holes in the skull were drilled by a micromotor drill. The injection of the virus was performed using a 10\u2009ml nanofil syringe under controlled by the UMP3 and Micro4 system (WPI), with a speed of 50\u2009nl/min. The dorsal hippocampus injections were bilaterally targeted to \u22122.0\u2009mm AP, \u00b11.5\u2009mm\u2009ML, and \u22121.5\u2009mm DV; the DG injections were bilaterally targeted to -2.0\u2009mm AP, \u00b11.5\u2009mm\u2009ML, and \u22122.0\u2009mm DV. The viral volumes of the AAV-Rac1-CA, AAV-Rac1-DN, and AAV-control were 400\u2009nl for DG and 600\u2009nl for CA1. Mice were bilaterally injected with 1\u2009\u00b5l of either AAV-\u03b11-chimaerin, AAV-\u03b12-chimaerin, AAV-control, AAV-\u03b11-chimaerin shRNA, AAV-\u03b12-chimaerin shRNA, or AAV-control shRNA. For labeling of the CA1 engram cells, a virus cocktail AAV9-c-fos:tTA) and 500\u2009nl of the AAV9-TRE:PaRac1-tdTomato41 or the AAV9-c-fos-EGFP (as the control group) into the CA1. Furthermore, they were bilaterally implanted with optical fibers into the CA1 . Mice were allowed to recover for two weeks under the ON-Dox condition prior to the start of the behavioral experiments. The cohorts of mice used for the optogenetic engram stimulation, mice were fed regular food, without Dox, for 24\u201330\u2009h until contextual fear-conditioning training. After CFC, mice were returned to their home cage under the ON-Dox condition. Following behavioral testing, brain sections were examined to confirm efficient virus-mediated labeling of the target areas.Mice were bilaterally injected with a virus cocktail (500\u2009nl of AAV2) was measured at the tip of the fiber. A blue light from a laser was ON for 30\u2009s to stimulate the photoactivatable Rac1 for five trials, with trial intervals of 120\u2009s31. For all behavioral procedures, the freezing behavior was measured in light stimulated mice at 1.5\u2009h after stimulation. Successively, animals were put back to their home cage.Mice were intra-CA1 implanted with optic fiber after AAV injection. The optic fibers of mice were connected to a 473\u2009nm blue laser diode via a FC/PC adaptor. Light intensity had a stainless-steel rod floor which was connected to a shock generator in a sound-attenuating box. In single-trial CFC sessions, mice were individually placed in the conditioning and freely explored the area for 3\u2009min. Then, mice were exposed to one footshock and were returned to their home cage 30\u2009s after. In the three-trial CFC task, the footshock was delivered at 180, 240, and 300\u2009s. Mice remained in the conditioning chamber for a total of 330 s. In only context or only shock training, mice were exposed in a context for 3\u2009min without shock or one footshock in the absence of context. After that, mice were placed back in their home cage. During testing, mice were placed back in the conditioning chamber for 4\u2009min at different times . For all procedures, the animal freezing behaviors were monitored using a manufacturer\u2019s software.The single- and three-trial CFC procedures were based on those of Denny et al.Subject mice were individually placed in the three-chamber apparatus for 10\u2009min to habituate to the environment (day 1). On day 2, a 6-week-old male C57BL/6J mouse was placed in the left grid enclosure for familiarization. Successively, the test mouse was located in the center compartment of the three-chamber apparatus for 10-min social training. During experimental testing (1\u2009h and day 3), a novel 6-week-old male C57BL/6J mouse was placed in the right grid enclosure and the familiar mouse in the left grid enclosure. And then, test mouse was placed into the social chamber and was allowed to explore for 10\u2009min. The amount of time for the test mouse spent in close interaction was recorded using the Any maze system. The discrimination index (DI) was calculated using the following formula: (time exploring the novel mouse\u2009\u2212\u2009time exploring the familiar mouse)/(time exploring the novel mouse\u2009+\u2009time exploring the familiar mouse) * 100.15. For the testing phase, one of the objects was exchanged with a new one, and the time spent exploring the two objects was separately recorded using ANY-MAZE software. The DI was calculated using the following formula: (time exploring the novel object\u2009\u2212\u2009time exploring the familiar object)/(time exploring the novel object\u2009+\u2009time exploring the familiar object)\u2009\u00d7\u2009100. For the memory decay curve, time intervals between sampling and testing were 1 and 24\u2009h.For the habituation phase, individual adult male mice were placed in a chamber (50\u2009cm\u2009\u00d7\u200950\u2009cm\u2009\u00d7\u200940\u2009cm) and allowed to freely explore the context for 10\u2009min while being recorded by an overhead camera. This phase was also conducted as the open field test to assess the animal\u2019s basal locomotor activity indicated by the average speed and anxiety levels indicated by the percentage of time spent in the middle arena (30\u2009cm\u2009\u00d7\u200930\u2009cm). For the sampling phase, the animal was placed in the same chamber containing two different objects for 5\u2009min and allowed to explore the object. To saturate the odor left by the previous mice and to make the mice more relaxed, we placed standard animal beddings in the chamberIsolated hippocampi were homogenized in cell lysis buffer with protease inhibitors. The protein concentration was determined using the BCA protein assay kit . For the detection of the levels of \u03b11-chimaerin, \u03b12-chimaerin, total Rac1 and actin, 20 \u03bcg of homogenates was separated by 15% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes . Membranes were blocked in milk solution (5% milk in TBS and 0.1% Tween 20) for one hour at room temperature. Subsequently, membranes were individually incubated with primary antibodies against \u03b11-chimaerin , \u03b12-chimaerin , total Rac1 , and actin overnight at 4\u2009\u00b0C. All the HRP-conjugated secondary antibodies were used at 1:2000 dilutions for membranes incubation at room temperature for one hour. For the relative level of the Rac-GTP assay, a GST-tagged PAK-PBD beads were incubated with the lysate mix (300\u2009\u03bcg) for overnight at 4\u2009\u00b0C. The beads were washed three times with lysis buffer at room temperature. Then, bound proteins were eluted with loading buffer and analyzed by western blotting. Image quantification analysis in bands of the western blots was calculated by the ImageJ software .Mice were anesthetized with 0.2% sodium pentobarbital (5\u2009ml/kg) and perfused intracardially with 30\u2009ml of 4% paraformaldehyde. The brains were fixed in 4% paraformaldehyde overnight at 4\u2009\u00b0C and serial coronal sections (30\u2009\u03bcm) were then taken throughout the hippocampus using a vibratome. The floating sections were collected and rinsed in a 0.1\u2009M phosphate-buffered saline (PBS) (pH 7.4) solution with 0.1% Triton X-100 and incubated with blocking solution (10% donkey serum and 0.1% Triton X-100) for 2\u2009h at room temperature. The slices were then incubated with the following primary antibodies at 4\u2009\u00b0C for overnight: anti-active Rac1 , anti-Cleaved Caspase-3 , and anti-phospho-PAK1/2/3 . Following three times rinsed (30\u2009min each) in the PBS solution, slices were incubated with the secondary antibodies conjugated with dyes. Then sections were washed three times with PBS solution and mounted on slides with antifade mounting medium . The images of the immunohistochemistry were captured on a Zeiss LSM 710.Cell counting of c-Fos-positive cells and Rac1-active cells was performed in the CA1 by utilizing imaging analysis function of Zeiss software (Zen blue 2.3). The quantification of the number of c-Fos-positive cells was performed by thresholding c-Fos immunoreactivity above background levels automatically. The quantification of the number of Rac1-GTP cells was performed by setting the threshold manually from 1000 to 4096. The quantification of the number of phospho-PAK cells was performed by setting the threshold manually from 692 to 4096. The quantification of the number of cells with 4\u2032,6-diamidino-2-phenylindole was performed by setting the threshold manually from 616 to 4096.2PO4, 1\u2009mM MgSO4, 2\u2009mM CaCl2, 26\u2009mM NaHCO3, and 10\u2009mM glucose) for at least 1\u2009h. Following the recovering at room temperature, the slices were transferred to the field excitatory post-synaptic potentials (EPSPs) recording chamber with ACSF (flow rate 1.5\u2009ml/min) for at least 20\u2009min prior to the recording. Filed EPSPs were evoked from CA3-CA1 synapses through the stimulation of the Schaffer collateral axons and recorded in CA1 extracellular fEPSPs. The stimulation intensity was adjusted to elicit a 30% maximal response. The LTP protocol for the 100\u2009Hz consisted in 10\u2009\u00d7\u2009100\u2009Hz bursts (four pulses per burst) with a 200\u2009ms interval between bursts. Data acquisition and analysis were performed using the multielectrode MED64 hardware and software packages (Panasonic).Transverse hippocampal slices were prepared from 4-month-old mice. Animals were killed by decapitation in accordance to the institutional regulations. Hippocampi were sliced (300\u2009\u03bcm) by a VF-300 microtome (Precisionary Instruments) and kept in an interface chamber with oxygenated artificial cerebrospinal fluid or two-way ANOVA where appropriate. The data are shown as the mean\u2009\u00b1\u2009SEM and NS indicates non-significance (p\u2009>\u20090.05). The significant levels were set to P\u2009=\u20090.05. Significant for comparison: *p\u2009<\u20090.05; **p\u2009<\u20090.01; ***p\u2009<\u20090.001; ****p\u2009<\u20090.0001. The specific statistical tests used and the details of p-values for each experiment can be found in Supplementary Table\u00a0Statistical analyses were performed in GraphPad Prism. All data were analyzed with an unpaired Further information on research design is available in the\u00a0Supplementary InformationTransparent Peer Review FileReporting Summary"} +{"text": "Correction to: Nature Communications 10.1038/s41467-018-04901-6; published online 27 June 2018In the original version of the Supplementary Information file associated with this Article, the sequence \u20181x MS2 scRNA.b2\u2019 was incorrectly given as \u2018GAAGATCCGGCCTGCAGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCGCACATGAGGATCACCCATGTGCTTTTTT\u2019 and should have read \u2018GAAGATCCGGCCTGCAGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACATGAGGATCACCCATGTGCTTTTTTT\u2019. The error has now been fixed and the corrected version of the Supplementary Information PDF is available to download from the HTML version of the Article."} +{"text": "Crucial steps in the miniaturisation of biosensors are the conversion of a biological signal into an electrical current as well as the direct sampling of bodily fluids. Here we show that protein sensors in combination with a nanopore, acting as an electrical transducer, can accurately quantify metabolites in real time directly from nanoliter amounts of blood and other bodily fluids. Incorporation of the nanopore into portable electronic devices will allow developing sensitive, continuous, and non-invasive sensors for metabolites for point-of-care and home diagnostics. Protein nanopores are emerging as sensors for a variety of biomolecules. Here the authors develop a nanopore based on the bacterial toxin ClyA, in conjunction with binding proteins for glucose and asparagine, to detect these biomolecules simultaneously from a variety of unprocessed, diluted body fluids. The ability to monitor the health of individuals that exchange information through the Internet without requiring human intervention would undoubtedly bring about a revolution in personalised healthcare. Over the past decades, the miniaturisation of electronic devices combined with recent advances in the wireless transmission of data and microfluidics have opened up new possibilities for making such devices. However, one key area that is preventing their breakthrough is the production of sensing elements for biologically relevant molecules, which can be easily interfaced with silicon-based electronics. Such elements have long lagged behind the electronic industry in terms of miniaturisation and cost reduction.1. However, such oxidase enzymes are not ubiquitous, and today\u2019s commercially available home diagnostic and implantable devices are entirely limited to one well-known example: the glucose sensor. However, despite the sensor has seen many improvements since it was first introduced in the early 1960s2, it still suffers from limitations including enzyme instability, complicated surface immobilisation procedures, baseline shifts, and does not tolerate well changes in micro-environmental factors such as changes in pH and ionic strength5.At the moment, biosensors that can be integrated in electronic devices rely mostly on the electric signal that is generated by the oxidation of a target analyte catalysed by an immobilised enzyme6. Nanopores can also detect polypeptide8 biomarkers. However, their use in diagnostic devices is complicated by the low, typically picomolar, concentration of such biomarkers in biological fluids. By contrast, metabolites are highly abundant in bodily fluids, and their variation in concentration is linked to many diseases9. Nanopores equipped with cyclodextrins10, cucurbiturils11, or cyclic peptides12 may recognise metabolites. However, the detection and quantification of such analytes directly from a biological sample cannot be easily implemented because the recognition elements lack selectivity.Nanopores, which are nanometre-sized water conduits spanning biological or artificial membranes, are emerging as a new class of biosensors. When a potential is applied, the flux of ions across an individual nanopore generates an electrical signal that allows direct interfacing with electronic devices. Most notably, nanopores can be integrated into electronic devices and are now used for low-cost, high-speed, and portable sequencing of DNA16. Here, using proteins from a protein family that in cells recognise an enormous variety of molecules, we show that ClyA nanopores can report the concentration of glucose and asparagine directly from samples of blood, sweat, and other bodily fluids. Conveniently, no sample preparation is required and the concentration of the metabolite can be monitored continuously.Recently, we have shown that the biological nanopore cytolysin A (ClyA) can be used to monitor the function of proteins when they are lodged inside the nanopore Fig.\u00a013\u201316. He17. GBP belongs to the class of substrate-binding proteins which, with minor structural differences, recognise hundreds of different metabolites in cells18. The structure of the GBP has been solved in the ligand-free state (open or apo form) configuration and in the closed, ligand-bound, configuration. Interestingly, NMR studies revealed that in solution, apo-GBP adopts both configurations even in the absence of glucose19. In our experimental setup, GBP (44\u2009nM) was added to the cis side of a ClyA-AS20 nanopore . Two current levels 19.In this work, we used a glucose-binding protein (GBP) that binds glucose with high affinity and specificitytrans side of the nanopore increased the number of events corresponding to the closed configuration and opening rate constants could be determined from the inverse of L1 and L2 dwell times 21. The dependence of the fractional time of GBP in the closed configuration with the glucose concentration fitted well to a Hill function with the Hill coefficient set to one corresponded well to the value measured in bulk (2\u20135\u2009\u00d7\u200910\u22127\u2009M)23. A GBP variant (GBP-A213R) with reduced affinity for glucose25 showed only one single level in the absence of glucose . Extrapolation from the calibration curve , sweat (2\u2009\u00b5L), urine (200\u2009nL), and saliva (8\u2009\u00b5L) were added to the rve Fig.\u00a0 allowed 0\u2009nL, swe26, while its abnormal concentration in blood has been associated to Parkinson\u2019s disease27. Under \u221290\u2009mV, the blocked pore level of apo-SBD1 contained one main current level corresponding to the open protein configuration . As observed before for GBP, the binding constants measured by the nanopore were similar to the ones measured in bulk28. The percentage of bound fraction was used to extrapolate the concentration of asparagine in the trans solution was markedly different than the open protein configuration of GBP (Ires%\u2009=\u200969.1\u2009\u00b1\u20090.2%). Thus, when GBP (44\u2009nM) and SBD1 (120\u2009nM) were simultaneously present in the cis solution, blockades corresponding to each protein adaptor could be readily distinguished , enzymes from Thermo Fisher Scientific, and lipids from Avanti Polar Lipids.Escherichia coli at the beginning of the gene . To maintain the open-reading frame, two extra bases were inserted after the NcoI site, resulting in an additional alanine residue after the starting methionine. For purification, a His6-tag was attached at the C-terminus of GBP, via a flexible glycine\u2013serine\u2013serine linker and the open-reading frame was terminated by two consecutive stop codons, followed by a Hind III restriction site (3\u2032-end). The attachment of the His6-tag was carried out in two consecutive PCR reactions. During the first PCR reaction, the GBP gene was amplified directly from a single BL21 (DE3) E. coli (Lucigen) colony using Phire Hot Start II DNA polymerase, 0.4\u2009\u00b5M dNTPs, 6\u2009\u00b5M of MS-Glu_F (TATATATCCATGGCTaataagaaggtgataaccctgtctgctgtg), and MS-Glu_r1 (gatggtgatgGCTGCTGCCtttcttgctgaattcagccaggttgtctttatctacgcc) primers in a 50\u2009\u00b5L reaction volume. The PCR reaction cycling protocol was as follows: pre-incubation step at 98\u2009\u00b0C for 30\u2009s and then 30 cycles of denaturation at 98\u2009\u00b0C for 5\u2009s and extension at 72\u2009\u00b0C for 1\u2009min. The amplified product was purified using QIAquick PCR Purification Kit (Qiagen) and served as a template for the second PCR reaction, which used ~100\u2009ng of the purified PCR product amplified by Phire Hot Start II DNA polymerase using 0.4\u2009\u00b5M dNTPs, 6\u2009\u00b5M of MS-Glu_F, and MS-Glu_r2 (atatatataagctttcattaatggtGATGGTGATGGCTGCTGCCTTTCTTGCTGAATTC) primers in 300\u2009\u00b5L volume. The cycling protocol was the same as in the previous step. The resulting PCR product encoding for the His6-tagged GBP gene was purified with QIAquick PCR Purification Kit (Qiagen) and digested with NcoI and HindIII . The gel purified insert was cloned under control of the T7 promoter into the pT7-SC1 expression plasmid29 using sticky-end ligation via NcoI (5\u2032) and HindIII (3\u2032) sites. Ligation mixture of 0.6\u2009\u00b5L was transformed into 50\u2009\u00b5L of E. cloni\u00ae 10G cells (Lucigen) by electroporation. The transformed bacteria were grown overnight at 37\u2009\u00b0C on ampicillin-containing (100\u2009\u00b5g/mL) LB agar plates. The identity of the clones was confirmed by sequencing.To allow cloning, a NcoI site (CCATGG) was introduced into the sequence of the wild-type GBP from MANKKVITLSAVMASMLFGAAAHAADTRIGVTIYKYDDNFMSVVRKAIEQDAKAAPDVQLLMNDSQNDQSKQNDQIDVLLAKGVKALAINLVDPAAAGTVIEKARGQNVPVVFFNKEPSRKALDSYDKAYYVGTDSKESGIIQGDLIAKHWAANQGWDLNKDGQIQFVLLKGEPGHPDAEARTTYVIKELNDKGIKTEQLQLDTAMWDTAQAKDKMDAWLSGPNANKIEVVIANNDAMAMGAVEALKAHNKSSIPVFGVDALPEALALVKSGALAGTVLNDANNQAKATFDLAKNLADGKGAADGTNWKIDNKVVRVPYVGVDKDNLAEFSKKGSSHHHHHCCATGGCTAATAAGAAGGTGATAACCCTGTCTGCTGTGATGGCCAGCATGTTATTCGGTGCCGCTGCACACGCTGCTGATACTCGCATTGGTGTAACAATCTATAAGTACGACGATAACTTTATGTCTGTAGTGCGCAAGGCTATTGAGCAAGATGCGAAAGCCGCGCCAGATGTTCAGCTGCTGATGAATGATTCTCAGAATGACCAGTCCAAGCAGAACGATCAGATCGACGTATTGCTGGCGAAAGGGGTGAAGGCACTGGCAATCAACCTGGTTGACCCGGCAGCTGCGGGTACGGTGATTGAGAAAGCGCGTGGGCAAAACGTGCCGGTGGTTTTCTTCAACAAAGAACCGTCTCGTAAGGCGCTGGATAGCTACGACAAAGCCTACTACGTTGGCACTGACTCCAAAGAGTCCGGCATTATTCAAGGCGATTTGATTGCTAAACACTGGGCGGCGAATCAGGGTTGGGATCTGAACAAAGACGGTCAGATTCAGTTCGTACTGCTGAAAGGTGAACCGGGCCATCCGGATGCAGAAGCACGTACCACTTACGTGATTAAAGAATTGAACGATAAAGGCATCAAAACTGAACAGTTACAGTTAGATACCGCAATGTGGGACACCGCTCAGGCGAAAGATAAGATGGACGCCTGGCTGTCTGGCCCGAACGCCAACAAAATCGAAGTGGTTATCGCCAACAACGATGCGATGGCAATGGGCGCGGTTGAAGCGCTGAAAGCACACAACAAGTCCAGCATTCCGGTGTTTGGCGTCGATGCGCTGCCAGAAGCGCTGGCGCTGGTGAAATCCGGTGCACTGGCGGGCACCGTACTGAACGATGCTAACAACCAGGCGAAAGCGACCTTTGATCTGGCGAAAAACCTGGCCGATGGTAAAGGTGCGGCTGATGGCACCAACTGGAAAATCGACAACAAAGTGGTCCGCGTACCTTATGTTGGCGTAGATAAAGACAACCTGGCTGAATTCAGCAAGAAAGGCAGCAGCCATCACCATCACCATTAATGAAAGCTT30 on a circular plasmid template DNA (pT7-SC1 containing GBP gene) with Phire\u00ae Hot Start Polymerase (Finnzymes) by using two complementary primers. Six \u00b5M forward (GGTTATCGCCAACAACGATAGGATGGCA) and 6\u2009\u00b5M reverse primer (GCGCCCATTGCCATCCTATCGTTGTT) containing a codon encoding the mutation were used in 50\u2009\u00b5L final volume to amplify the gene . The PCR product was incubated with DpnI (1 FDU) for 1\u2009h at 37\u2009\u00b0C to digest the plasmid template. The remaining DpnI enzyme was deactivated at 55\u2009\u00b0C. The mixture was then incorporated into E. cloni\u00ae 10G cells (Lucigen) by electroporation. Transformants containing the plasmid were grown overnight at 37\u2009\u00b0C on LB agar plates supplemented with 100\u2009\u03bcg/mL ampicillin and 1% glucose. Single colonies were picked and inoculated into 10\u2009mL LB medium supplemented with 100\u2009mg/L of ampicillin for plasmid DNA preparation. The presence of the mutation was confirmed by sequencing of the plasmid.The GBP-A213R was constructed using the QuickChange protocol for site-directed mutagenesisE. coli strain MC1061 and purified as described previously.28 The cell lysate was mixed with 50\u2009mM potassium phosphate, pH 8.0, 200\u2009mM KCl, 20% glycerol (buffer A) supplied with 20\u2009mM imidazole and incubated with Ni2+-sepharose resin for 1\u2009h at 4\u2009\u00b0C. Next, the resin was washed with 20 column volumes of buffer A supplied with 50\u2009mM imidazole, followed by protein elution with 500\u2009mM imidazole in buffer A. To prevent protein aggregation, 5\u2009mM EDTA was added immediately after elution. Afterwards the histidine-tag was cleaved off by adding 2.5% w/w His-TEV (S219V)31 and dialyzing overnight against 50\u2009mM Tris-Cl, 0.5\u2009mM DTT, 0.5\u2009mM EDTA, pH 8.0 to remove imidazole. The histidine-tagged tobacco etch virus (TEV) protease and residual uncut protein were removed by loading it on a small 0.5\u2009mL Ni2+-sepharose column collecting the flow-through. Next, proteins were further purified with size-exclusion chromatography on a Superdex-200 column equilibrated with 50\u2009mM Tris-HCl, 200\u2009mM NaCl, pH 7.5. Peak fractions were collected in aliquots and were stored at \u221280\u2009\u00b0C after flash-freezing in liquid nitrogen.Substrate-binding domains carrying a histidine-tag were expressed in E. cloni\u00ae EXPRESS BL21 (DE3) cells were transformed with the pT7-SC1 plasmid containing the ClyA-AS gene. ClyA-AS contains eight mutations relative to the S. Typhi ClyA-WT: C87A, L99Q, E103G, F166Y, I203V, C285S, K294R, and H307Y. Transformants were selected after overnight growth at 37\u2009\u00b0C on LB agar plates supplemented with 100\u2009mg/L ampicillin and 1% glucose. The resulting colonies were inoculated into 400\u2009mL 2xYT medium containing 100\u2009mg/L of ampicillin. The cells were grown at 37\u2009\u00b0C (200 r.p.m. shaking) until an OD600 of ~0.8 was reached. The expression of ClyA-AS was induced by addition of 0.5\u2009mM IPTG. The cell cultures were further grown overnight at 25\u2009\u00b0C. The next day, the bacteria were harvested by centrifugation at 6000\u00d7g at 4\u2009\u00b0C for 25\u2009min and the pellets were stored at \u221280\u2009\u00b0C. The pellets containing monomeric ClyA-AS were thawed and solubilized with 20\u2009mL lysis buffer and incubated for 20\u2009min at 37\u2009\u00b0C. The bacteria were then lysed by sonication (Branson). The lysate was subsequently centrifuged at 6000\u00d7g at 4\u2009\u00b0C for 20\u2009min and the cellular debris discarded. The supernatant was mixed with 150\u2009\u03bcL of Ni-NTA resin (Qiagen) in wash buffer . After 1\u2009h, the resin was loaded into a column and washed with ~5\u2009mL of the wash buffer. The protein was eluted with ~100\u2009\u03bcL elution buffer . Type I ClyA-AS oligomers were obtained by incubation of ClyA-AS monomers with 0.2% \u03b2-dodecylmaltoside (DDM) for 30\u2009min at 37\u2009\u00b0C. The oligomers were separated from monomers by blue native polyacrylamide gel electrophoresis using 4\u201320% polyacrylamide gels20. The band corresponding to type I ClyA-AS were cut out from the gel and was extracted in 150\u2009mM NaCl, 15\u2009mM Tris-HCl, pH 7.5 supplemented with 0.2% DDM and 10\u2009mM EDTA.E. cloni\u00ae EXPRESS BL21 (DE3) cells were transformed with the pT7-SC1 plasmid containing the His6-tagged GBP gene. Transformants were selected after overnight growth at 37\u2009\u00b0C on LB agar plates supplemented with 100\u2009mg/L ampicillin and 1% glucose. The resulting colonies were inoculated into 400\u2009mL 2xYT medium containing 100\u2009mg/L of ampicillin. The cells were grown at 37\u2009\u00b0C (200 r.p.m. shaking) until an OD600 of ~0.8 was reached. The expression of GBP was induced by addition of 0.5\u2009mM IPTG. The cell cultures were further grown overnight at 25\u2009\u00b0C. The next day, the bacteria were harvested by centrifugation at 6000\u00d7g at 4\u2009\u00b0C for 25\u2009min. The protein was purified directly afterwards by osmotic shock. The pellets were thoroughly dissolved in 20% ice-cold sucrose solution and incubated, while shaking at 4\u2009\u00b0C for 10\u2009min. The cell suspension was centrifuged at 6000\u00d7g at 4\u2009\u00b0C for 10\u2009min and cells collected. The cells were re-suspended in 4\u00d7 volume of ice-cold MilliQ water supplemented with 5\u2009mM MgCl2 and incubated, while shaking, on ice for 20\u2009min. The cells collected again by centrifugation at 6000\u00d7g at 4\u2009\u00b0C for 20\u2009min. The supernatant was mixed with 150\u2009\u03bcL of Ni-NTA resin (Qiagen) in wash buffer . After 1\u2009h, the resin was loaded into a column and washed with ~5\u2009mL of the wash buffer. The protein was eluted with ~500\u2009\u03bcL elution buffer . The protein (500\u2009\u03bcL) was then dialysed exhaustively overnight against dialysis buffer , whereby the dialysis buffer was replaced for fresh buffer at least three times. Protein concentration was measured by using the Nanodrop 2000C at 280\u2009nm (Thermo Fisher Scientific). Protein was stored with 10% glycerol in \u221280\u2009\u00b0C after flash-freezing until further use.sn-glycero-3-phosphocholine (DPhPC) in pentane (10\u2009mg/mL) was then added to the buffer present in both compartments. After evaporation of the pentane, a lipid monolayer at the air\u2013water interface can self-assemble spontaneously by lowering and raising the buffer across the aperture.\u00a00.01\u20130.1 ng of purified ClyA-AS were added to the grounded cis compartment. The reconstitution of single nanopores was monitored electrically by applying a \u221235\u2009mV bias. ClyA-AS nanopores display a higher open pore current at positive than at negative applied potentials, which provides a useful tool to determine the orientation of the pore. After insertion of a single pore, GBP or SBD1 were added to the cis compartment. This was followed by addition of a glucose or asparagine sample or bodily fluid sample in the trans compartment.The setup consisted of two chambers separated by a 25\u2009\u03bcm thick polytetrafluoroethylene film (Goodfellow Cambridge Limited) containing an aperture of approximately 100\u2009\u03bcm in diameter. The aperture was pre-treated with a solution of hexadecane [10% (v/v) in pentane] to leave a hydrophobic coating to support bilayer formation. 1,2-diphytanoyl-All traces were routinely recorded at \u221290\u2009mV or \u221260\u2009mV applied transmembrane potential by using an Axopatch 200B patch clamp amplifier (Axon Instruments) and digitised with a DigiData 1440\u2009A/D converter (Axon Instruments). The data was sampled by applying a 2\u2009kHz low-pass Bessel filter and at a frequency of 10\u2009kHz. Obtained traces were filtered digitally with a Gaussian low-pass filter with a 100\u2009Hz cutoff. Data were recorded by using the Clampex 10.4 software (Molecular Devices) and the subsequent analysis was carried out with the Clampfit software (Molecular Devices).\u03c4on) and ligand-independent opening (\u03c4off) of GBP were obtained from single exponential fits to histograms of the inter-event times and dwell times, respectively. The mean lifetime of the open state (\u03c4on) was converted to the on-rate constants by using kon\u2009=\u20091/\u03c4on[glucose], where [glucose] is the concentration of glucose added to the trans side. The \u03c4off was converted to the off-rate by using koff\u2009=\u20091/\u03c4off. The equilibrium binding constant was calculated by plotting the fraction of closed configuration against the concentration of substrate and the data fitted to a Hill function . Events shorter than 2\u2009ms were ignored. The mean dwell times for the ligand-induced closing were used to determine the glucose in the bodily fluids. The solution of the glucose assay kit contains a hexokinase enzyme that uses adenosine triphosphate (ATP) as a co-factor to phosphorylate glucose. The glucose-6-phosphate (G6P) is then oxidised to 6-phospho-gluconate in the presence of oxidised nicotinamide adenine dinucleotide (NAD) in a reaction catalysed by glucose-6-phosphate dehydrogenase (G6PD). The generation of NADH was spectrophotometrically observed by measuring absorbance at 340\u2009nm. Under these conditions, the final concentration of produced NADH corresponds to the glucose concentration in the sample. In order to perform the experiment, a dilution of bodily fluid was added to the reaction mixture. The reaction was incubated for 15\u2009min at room temperature and then the absorbance was measured at 340\u2009nm (\u03b5\u2009=\u20096220\u2009M\u22121\u2009cm\u22121). For each sample, three different dilutions were measured. The commercial glucose metre Accu-Chek\u00ae uses a mutant variant of quinoprotein glucose dehydrogenase and has a measurement range between 0.6 and 33.3\u2009mmol/L32. The Accu-Chek\u00ae is considered to be a reliable device for monitoring glucose levels in blood from finger pricks33.A glucose assay kit (HK) from Sigma-Aldrich and a commercial glucose metre . The blood samples were collected shortly before the experiments to minimise ex vivo artefactual changes. Sweat, urine, and saliva samples were either freshly collected before, or stored at \u221220\u2009\u00b0C, before the start of experiments. The solutions were kept on ice for the duration of an experiment. The blood samples were diluted 500-folds and the urine sample 10-folds with buffer before adding 5 and 2\u2009\u00b5L, respectively, to the kopening\u2009=\u20090.75\u2009\u00b1\u20090.12\u2009s\u22121, 500\u2009nM glucose), compared to the binding of asparagine to SBD1 , thus longer integration times are required for accurate reading. It follows that (GBP-A213R), which shows reduced affinity for glucose .All measurements were made by adding small volumes of biological samples to an electrophysiology chamber (500\u2009\u00b5L). We did not observe a particular instability of the bilayer . After mixing, a concentrator was used to evaporate all liquids by vacuum evaporation at 60\u2009\u00b0C and 240\u00d7g, and the sample was reconstituted in an identical volume of water as the former fixed amount of supernatant. A final 5-min-long centrifugation at 20,000\u00d7g step was performed and the supernatant was stored at 4\u2009\u00b0C before analysis.Proteins in 150\u2009\u00b5L saliva and 60\u2009\u00b5L serum samples were precipitated by incubation with 8% trichloroaceticacid (CASnr 76\u201303\u20139) for 5\u2009min at 60\u2009\u00b0C and 450 r.p.m. using a thermomixer followed by cooling on ice for 30\u2009min and centrifugation for 10\u2009min at 20,000\u00d7HPLC quantification of asparagine was performed on an Agilent 1100 HPLC binary system equipped with an 1100 Fluorescence detector (FLD) and a Gemini C18 column while a 40\u2009mM sodium phosphate buffer, pH 7.8 (solvent A) and a mixture of 45:45:10 acetonitrile/methanol/water (solvent B) were used. The fluorogenic labelling of amino acids was performed automatically by the auto sampler of the HPLC machine using a mixture of 10\u2009mg o-phtalaldehyde and 10\u2009\u00b5L 3-mercaptapropionic acid in water as reagent. The auto sampler mixed 0.5\u2009\u00b5L sample with 1.3\u2009\u00b5L 0.4\u2009N borate buffer, pH 10.2 followed by 0.5\u2009\u00b5L reagent and 7.6\u2009\u00b5L water right before each injection. After the sample injection, amino acids were separated by applying solvent B at 1.5%/min increase for 25\u2009min using a flow rate of 250\u2009\u00b5L/min . Thereafter, the column was cleaned for 5\u2009min at 66% solvent B followed by another 12\u2009min at 100% solvent B. Then the column was equilibrated back to 5% solvent B. Fluorescence was measured at an excitation wavelength of 340\u2009nm and an emission wavelength of 450\u2009nm.Quantifications were performed using the method of standard addition at three different concentrations of analyte . The concentration of the analyte was obtained by measuring the peak areas, while each sample was analysed in triplicate. Citrulline was added as internal standard at 200\u2009\u00b5M to correct for saliva and serum sample handling volume differences next to control run-to-run retention time shifts regarding all samples.Supplementary InformationPeer Review File"} +{"text": "DNA replication commences at eukaryotic replication origins following assembly and activation of bidirectional CMG helicases. Once activated, CMG unwinds the parental DNA duplex and DNA polymerase \u03b1-primase initiates synthesis on both template strands. By utilizing an origin-dependent replication system using purified yeast proteins, we have mapped start sites for leading-strand replication. Synthesis is mostly initiated outside the origin sequence. Strikingly, rightward leading strands are primed left of the origin and vice versa. We show that each leading strand is established from a lagging-strand primer synthesized by the replisome on the opposite side of the origin. Preventing elongation of primers synthesized left of the origin blocked rightward leading strands, demonstrating that replisomes are interdependent for leading-strand synthesis establishment. The mechanism we reveal negates the need for dedicated leading-strand priming and necessitates a crucial role for the lagging-strand polymerase Pol \u03b4 in connecting the nascent leading strand with the advancing replisome. \u2022S.\u00a0cerevisiae DNA replication originsMapping of leading-strand start sites at two \u2022Leading-strand synthesis is established from \u201clagging-strand\u201d primers\u2022Pol \u03b4 likely plays a key role in establishing all nascent leading strands\u2022Replisomes remain interdependent until new leading strands are established Aria and Yeeles describe the mechanism by which leading-strand replication is established at eukaryotic DNA replication origins. \u201cLagging-strand\u201d primers, synthesized by replisomes on opposite sides of the origin, are elongated back across the origin by Pol \u03b4 until the 3\u02b9 ends are coupled to Pol \u03b5 at the advancing replication forks. Bidirectional DNA replication is initiated from specific regions of the genome, termed origins. In eukaryotes, assembly of the DNA replication machinery (replisome) begins in the G1 phase of the cell cycle when the ATP-dependent motor component of the replicative helicase, the hexameric Mcm2\u20137 complex (MCM), is loaded at origins by the origin recognition complex (ORC), Cdc6 and Cdt1 . The MCMin\u00a0vivo studies in budding yeast have reached conflicting conclusions regarding the location of leading-strand start sites relative to an origin. For example, one study concluded that the ARS1 origin contains a single leading-strand start site (Once sufficient single-stranded DNA (ssDNA) has been exposed at origins, synthesis of leading and lagging strands is initiated by the DNA polymerase \u03b1-primase complex (Pol\u00a0\u03b1). Lagging-strand synthesis requires repeated cycles of Pol \u03b1-dependent priming and subsequent primer extension by Pol \u03b4. Pol \u03b1 first synthesizes 7\u201312 nucleotide (nt) RNA primers before transferring them to the DNA polymerase domain, where further extension to about 20\u201325 nt takes place . Evidencart site . The sitart site , and theart site . Howeverart site . Consequin\u00a0vivo C. Subseq (PIP is very similar to the 35% rate reduction that we observed previously when PCNA was omitted from reactions (PIP contributes significantly to leading-strand synthesis.At early time points (6 and 9\u00a0min), Pol \u03b5eriments E and S1Aeriments . In reacents . Moreoveeactions . These fPIP in leading-strand replication, we created two additional Pol \u03b5 mutants: one in which catalysis was disrupted by a point mutation in the polymerase active site (Pol \u03b5Cat) (PIP/Cat). Replication with Pol \u03b5PIP/Cat proceeded at a similar rate to Pol \u03b5Cat and a muPol \u03b5Cat F. In con. We reasoned that initiation via pathway 1 should be refractory to challenge by this mutant, whereas it should block initiation via pathway 2 , a potent block to replicative polymerases, 16 nt to the left of the ACS in the lagging-strand template A. The CPBased on our previous work , preventS.\u00a0cerevisiae origins. Synthesis is predominantly initiated outside the origin sequence; Left leading strands are started to the right, and Right leading strands are started to the left .Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Joseph Yeeles containing integrated expression constructs; or from Escherichia coli RosettaTM 2(DE3) cells (Novagen) (genotype: F\u2013 ompT hsdSB(rB\u2013 mB\u2013) gal dcm (DE3) pRARE2 (CamR)) transformed with plasmids for protein overexpression. Plasmids details are reported in the Proteins were purified from The ARS306 templates used in this study were modified from the plasmid ZN3 using stTo generate linear replication templates all plasmids were purified with CsCl gradients, linearized at unique restriction sites and processed as previously indicated . ExperimCTGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGTCCTTCAATGAAACATCGTTGGCCACTAATTTGGCCAGTGCAAAGTAGAACAAATCGGCAGCCTCCCAAGAAAGCTCCTTCTTACCCTTTGCCTCAGTCAGTTCTTCAGCTTCTTCCTTGATCTTGGCATCTAACAATGCAGAGTCGTTGAATAGTCTTCTAGTATAAGATTCCTCTGGAGCGTCCTGTAGCCTTTGTTTTAGTAAAGATTCTAGCCCCACCAAACCATGCTTGAATTCACCAAAGCAAGACATGGTCTCCAAGTGGCAAAATCCAACGTTTTCTTGTTCAACGATAAACTTTAAGGCATCCGAATCACAGTCAGTAGAGATTTGTAAAAGCTTTTGGCCATTGCCAGAAGTTTCACCCTTGATCCAGATTTCATTCCTAGAACGAGAATAATAAACGCCACGACCCAATTCGATGGCCTTTGCTATAGATTTCTTCGAAGAATACACCAACCCTAGACAACGCTCATATTGGTCCACAACTAGGGTGGTATATAAACCGTCAGGACGGTCTGTACGTACTTCACCAAGCACTTCTTTGGTCAACATATCCTTGCTTAATTTCTTTATGGACACAATTTTATCTTGCGAGAATTTTTGTTTTACCATGAATTGATTGGAGAAAACACCGTTCTCTTCCACAACAACACGCTCCTTTGGTACATTCAATTGTTCAACCAAGTGTTCGGCTGTTTTAGCATCTTGGCTTGCAATGAACAGAGAAGAAACTCCGTTGTTCAAGAAGGCAATGATTTCATCATCGCTGAATTTACCACTTGGCAAGGACAAAGCCACCAATGGAACTTCTTCCTCTTTGGAGAACTGGAGAATCTCTTCATTACTCAGGCTCGAGCCATCCAAAAGTACCTGACCAACAAGTGAAACGTATTCCTTCTTACTATTCCATGAGGCCAGATCATCAATTAACGGTAGAATCGGCAAAACCATTATTCAGAAAAAAAATTTTGTAAACTATTGTATTACTATTACACAGCGCAGTTGTGCTATGATATTAAAATGTATCCAGAACACACATCGGAGGTGAATATAACGTTCCATATCTATTATATACACAGTATACTACTGTTCATAGTCATATCCTTTTCTTACCTTCTATATCGAATGACTGATAATGCAACGTGAGTCACTGTGCATGGGTTTAGCAATTATTAAACTAATTTACCGGAGTCACTATTAGAGTCAGTTCGACTGCCTAGAAGAACTGCTGGTTGTCAGGATTGTGATGGGGGCATTCTGCTGTATTATGACCCATCGTATCGCAATGCTCACACCACTGTTGTCTTCCTGCCGTGGTATCGACTGGTGCAGGGGGGTCGAAAATTGGCAACGATTCCACGGCTGTTTGTGCTTGAGCCTGTTCCAACTGTTTGAACCTTTCATTAGCCTCTTCAAGTTTTTTCGTTAAGGATGCCACCTCTTCCGATGAGGAATCTTGTGGTTTTGTCAAAAATAGTTCCTTGCTCAAATTTTGGTATTCTTTACTGAGCGAATCGTTATGCATTTTCAATTGTTCGCGTTCTTTAGCCCACTTTGTCTTGTGTAACTCAAATTGGTCTTCTATGTTGCGTAATTGTTCCAGCTGTTTTTTCAGGAGTTCGACATCTTCGTTGGCACCAGTGGGTTGATTATGAGAAAGATTTCTCTCTTCGTTTTCTTTGATCTCTTCGTGTAGTTGGCTTACGACAGCAAGTAGCTGTTCATTCTCAGCGTCAAAAAACTGCTTTTGTTTGGCTTGCTGTCTGCGTTCGAGCAGACATTGTTGCTTGAGATGGTCTATCTCTTTCTCTCTTTCTTGTATTGTGGCTTCATACCTATCAAAAGTCGGTTGCACTTCTTCGAGGACCATTCTTTGGTCATCGAGTAGCCTTTTGTAGTGTAGTTGTTTCCTTTGTAGCTTTTCGATGGTCAATTGGCGATCGCGTAATTCAATTGTAACTTCGCTGCTATTGAGGTCATTCATGTGGCCATTGTCCGGTTTCCAATCGCTGGTGGTGTTGTGATTAGCCTTTCTGTCTGATGACAGGATAGAGTCCACCTCCATTCTGTCTTCTCTGTTATCGTAACCAAATTCTTGCTGTTGATGGTGATCCGATGCCTCCTGGTCCATCGACTGTTGATTACCGCTGTGCCGACTGGTGATCCGGAAACTTCTCATGGGTGTGGGGGATTTAGGATCATCCATGGGAGAGAAGCGCTTAGTGAGCCTCACAATAGATCTGTTCACGGGTATTGATAGCGGTTCCATTGTCGTTCTTCTCGAGGTTTGCCATATCGGTCCGTTCTCGATCAATGATGCGACTTTTTGCAACTGAATAAATAGTCCACTTTGAGGATACTCCGTTTGAAAATACTTCTTCCCCTAGGAATGATCCATCGTTCTTACCAATGTTGGCAAGTAAGTCTACACCAGCAAACATTCCACGCGTCGTGTCCACTGGACCCACGTATTTCAGTTGTCCGCGGCCGAAATTTGGGATTTGGTTTAAACATCCTATCTTTCTTTGATATCTATCCATGGTATATTAAGCGCATACGGCGCCAGCCACTAGTCAACGCCTTTTACCTTGTCCTTTGATGCATGCCTCGTCCAAACGTTTTTGGTGTCTTGGCCAATTGCCCTTCTGAAAAATCTCACTGTCCGCAACTCATTAAAAGATACCCAAGCAAGCTACACGATAAAGAAAGGAGAAAGTTCATTACTGGAACGTACATATAGCGATACAAACGTATAGCAAAGATCTGAAATGGATACGGATAAGTTAATCTCAGAGGCTGAGTCTCATTTTTCTCAAGGAAACCATGCAGAAGCTGTTGCGAAGTTGACATCCGCAGCTCAGTCGAACCCCAATGACGAGCAAATGTCAACTATTGAATCATTAATTCAAAAAATCGCAGGATACGTCATGGACAACCGTAGTGGTGGTAGTGACGCCTCGCAAGATCGTGCTGCTGGTGGTGGTTCATCTTTTATGAACACTTTAATGGCAGACTCTAAGGGTTCTTCCCAAACGCAACTAGGAAAACTAGCTTTGTTAGCCACAGTGATGACACACTCATCAAATAAAGGTTCTTCTAACAGAGGGTTTGACGTAGGGACTGTCATGTCAATGCTAAGTGGTTCTGGCGGCGGGAGCCAAAGTATGGGTGCTTCCGGCCTGGCTGCCTTGGCTTCTCAATTCTTTAAGTCAGGTAACAATTCCCAAGGTCAGGGACAAGGTCAAGGTCAAGGTCAAGGTCAAGGACAAGGTCAAGGTCAAGGTTCTTTTACTGCTTTGGCGTCTTTGGCTTCATCTTTCATGAATTCCAACAACAATAATCAGCAAGGTCAAAATCAAAGCTCCGGTGGTTCCTCCTTTGGAGCACTGGCTTCTATGGCAAGCTCTTTTATGCATTCCAATAATAATCAGAACTCCAACAATAGTCAACAGGGCTATAACCAATCCTATCAAAACGGTAACCAAAATAGTCAAGGTTACAATAATCAACAGTACCAAGGTCGCGACGGTGGTTACCAACAACAACAGGGACAATCTGGTGGTGCTTTTTCCTCATTGGCCTCCATGGCTCAATCTTACTTAGGTGGTGGACAAACTCAATCCAACCAACAGCAATACAATCAACAAGGCCAAAACAACCAGCAGCAATACCAGCAACAAGGCCAAAACTATCAGCATCAACAACAGGGTCAGCAGCAGCAACAAGGCCACTCCAGTTCATTCTCAGCTTTGGCTTCCATGGCAAGTTCCTACCTGGGCAATAACTCCAATTCAAATTCGAGTTATGTGTACACGCAACAGGCTAATGAGTATGGTAGACCGCAACAGAATGGTCAACAGCAATCCAATGAGTACGGAAGACCGCAATACGGCGGAAACCAGAACTCCTAAGGACAGCACGAATCCTTCAATTTTTCTGGCAACTTTTCTCAACAGAACAATAACGGCGCGCCGAACCGCTACTGAACGATGATTCAGTTCGCCTTCTATCCTAAGTTTACGTATTTGCTAGCGCATATAACTTAGCGGGAAATTATTAATTGACCGGTAGGACAATTTTGTTGCACGTGATGCCTCAATCGTCTGCTTGCTTCCATAGTTAACATGAGGATCCGCAGTACCAACCTCAGCACTTAAGTCCTCAGCGCAGTACCAACTGCAGGATGCCCTTTTTGACGTATTGAATGGCATAATTGCACTGTCACTTTTCGCGCTGTCTCATTTTGGTGCGATGATGAAACTTTCATGAAACGTCTGTAATTTGAAACAAATAACGTAATTCTCGGGATTGGTTTTATTTAAATGACAATGTAAGAGTGGCTTTGTAAGGTATGTGTTGCTCTTAAAATATTTGGATACGACATCCAAAATCTTTTTTCCTTTAAGAGCAGGATATAAGTCGACAAGTTTCTGAAAATCAAAATGGTAGCAACAATAATGCAGACGACAACAACTGTGCTGACGACAGTCGCCGCAATGTCTACTACCTTAGCATCCCATTACATATCTTCGCAAGCTAGTTCCTCGACGAGTGTAACAACAGTAACGACAATAGCGACATCAATACGCTCTACACCGTCTAATCTACTCTTTTCTAATGTGGCGGCTCAGCCAAAATCATCTTCAGCAAGCACAATTGGGCTTTCAATCGGACTTCCCATCGGAATATTCTGTTTCGGATTACTTATCCTTTTGTGTTATTTCTACCTTAAAAGGAATTCGGTGTCCATTTCAAATCCACCCATGTCAGCTACGATTCCAAGGGAAGAGGAATATTGTCGCCGCACTAATTGGTTCTCACGGTTATTTTGGCAGAGTAAGTGTGAGGATCAGAATTCATATTCTAATCGTGATATTGAGAAGTATAACGACACCCAGTGGACCTCGGGTGATAACAAGTCTTCAAAAATACAGTACAAAATTTCCAAACCCATAATACCGCAGCATATACTGACACCTAAGAAAACGGTGAAGAACCCATATGCTTGGTCTGGTAAAAACATTTCGTTAGACCCCAAAGTGAACGAAATGGAGGAAGAGAAAGTTGTGGATGCATTCCTGTATACTAAACCACCGAATATTGTCCATATTGAATCCAGCATCCCCTCGTATAATGATTTACCTTCTCAAAAAACGGTGTCCTCAAAGAAAACTGCGTTAAAAACGAGTGAGAAATGGAGTTACGAATCTCCACTATCTCGATGGTTCTTGAGGGGTTCTACATACTTTAAGGATTATGGCTTATCAAAGACCTCTTTAAAGACCCCAACTGGGGCTCCACAACTGAAGCAAATGAAAATGCTCTCCCGGATAAGTAAGGGTTACTTCAATGAGTCAGATATAATGCCTGACGAACGATCGCCCATCTTGGAGAGCATACGACTACCGCCGTATAATAACACGCCTCTGGATGCAAATGACAGTGTGAATAACTTGGGTAATACCACGCCAGATTCACAAATCACATCTTATCGCAACAATAACATCGATCTAATCACGGCAAGACCCCATTCAGTGATATACGGTACTACTGCACAACAAACTTTGGAAACCAACTTCAATGATCATCATGACTGCAATAAAAGCACTGAGAAACACGAGTTGATAATACCCACCCCATCAAAACCACTAAAGAAAAGGATATAAAGAAGACAAAGTAAAATGTATCAGCATTTACAACATTTGTCACGTTCTAAACCATTGCCGCTTACTCCAAACTCCAAATATAATGGGGAGGCTTGCGTCCAATTAGGGAAGACATATACAGTTATTCAGGATTACGAGCCTAGATTGACAGACGAAATAAGAATCTCGCTGGGTGAAAAAGTTAAAATTCTGGCCACTCATACCGATGGATGGTGTCTGGTAGAAAAGTGTAATACACAAAAGGGTTCTATTCACGTCAGTGTTGACGATAAAAGATACCTCAATGAAGATAGAGGCATTGTGCCTGGTGACTGTCTCCAAGAATACGACTGATGAAAATAATATTGACGTTCGCATTTAATCTATACCTATAATTCTGTACTTATATACTGTTCCTTAATTGAAGATTTCAACATCGTTTTTGATGTAGGTCTTTTCACCTGGAGGTGCGGCTGGGCTACCGAAGACTAATTGAGCTTGTACGGTCCAAGACTCAGGGATTTTGCTTGGCAAAGCAGCTTTTATGTAACCATTGTAGTGTTGTAGGTGACCACCCAGGCCCATTGCCTCCAAGGCAACCCACGAGTTGATTTGAGCGGCACCAGAGGTATGGTCCGCGAAACTAGGGAATGCAGCTGCGTACGCTGGGAAGTCAGCCTTTAGCTTTTCAGTTACCTTGTCGTCGGTGAAGAAGATTACAGAACCAAAGGCCTCATCCCTTGCTGAAGCAGGCCTCTTTTGACCGGCAGGGCTTTCTATAGCCTTAGTCACTTCGTCCCAAACTTTTTTGTGAGTTTCACCAGTCAAGATAACAGCGCGATTTGGCTGGGAGTTGAAAGCGGTGGGTGTGGCTCGAATGATGGTTTGGACGACGGATTGGATGTCGTTGATAGTAATTTCACCAGGTGCGGCCGCTTTCAAAGCGTAAATAGTACGACGAGCAGTTAAAGTTTTCAAATAAGTTGCAACAGCAGACATGATATTGGATTGCCGGAATGGCGATATGTTGATCCCGGATACTTCAGTCTACGAAAAAAGTACAAATTATGTGTCAGTTCCTTCAGTATGGTGTCCTTATATACTGTAGTTTGGACAAGGTGCAAATGCCAAGACCCTAGCCCGAAAAGCTCGAGGCACCCCAGGATCTTCCCCTTTACGTAATTTTCACGTAAAACGCCACAGTCCGATTTTTCTAGAATAATCATTAGTAAAAGCGGTATACTGGATTATTGTACGATAACAAGGTAGAGCTTTATTACTAAGCTAAGACGTTCTTACATCAATAGTGCTGTTCGTTATTGACGTCAGGAGAAGGAGCGGGTCTGGTGAATAGTGTAAGCAGTGTTTCTGAACTTTTTCTTCGTCTAAGTCCTTGTAATGTAAGGTAAGAATGCAAGCATCTTGTTTGTAACCCGGGTGTACGTTGACGTTAGTAAGGGGTGTACGTTGACGTTAGTAAGTCACAAACCCAAGCTTAACTTCTTCGTGAGGAAGGAAAGTGTTGTCTCCTACTTTTTTCAAATTTTCGAATTGTATTTATATTTATTTAGTACTTCTTGAGTTTACATATCCTTCGTAAAAATGCAACTTTTGTCGAAAAACACTTCCAAAAAAAAATAATAATGAATTTATGAAGCATACTAACGAGCGAGCACATCGCTGACCTATCATTACTTCATGAGATAAATTAAGATCTCCTCATATGCGAATTTCCTGTTCAGTGATAAACGTTGATTACGTTATTGATAAAAGTCTTTTCTTCTGGCAAGGGGTACCTGGAACACCAAAGACCAATTGAGATTGTACAGTCCACGCAATAGGAACATCTTGAGGCAAAGCAGATTTGACGTAGTCATTATAGTGTTGCAAATTAGCCCCCAATCCCAATAGTTCGAGGGCAGTCCAAGACTGAATTTGCACAGCACCGGTCGTATGAGCTCGCATGTTGGGAAAGCGGCTGCCAAGGCTGGAAATCTCTTTGCAGTTTTTCAGTTGGTCCTTCATCAGTGAAGAAAATGACTGAACCGTAAGCCTCATCTCTGCAAGACTCTGGTCTCTTATAAAGCAGTTGGCATTGCGCTCGCAACAGCATCCCATATCCTTTTGTGTGTATCACCAACGATAATGACAGCGCGATTCACTTGTGAGTTAAAAGCTGTTGGCGTATTCTTGAGAATAACGTGTACAGTTCTCTTTACATCATCCAAACCGACACCTTGTGGTAATTCGGGCTTCAAATTGTAGATGGTACGACGGTTTGTAATAGCGTTTAAGTAGTTTCCAGTTGGGGACATTTCTTTGGCTTGGAGGTCTGGTGTTCTTGATTTTGATGGTGTATATAGCTTTAAAAAACCAAAAATGATCAACCTTTATATCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGTCCTTCAATGAAACATCGTTGGCCACTAATTTGGCCAGTGCAAAGTAGAACAAATCGGCAGCCTCCCAAGAAAGCTCCTTCTTACCCTTTGCCTCAGTCAGTTCTTCAGCTTCTTCCTTGATCTTGGCATCTAACAATGCAGAGTCGTTGAATAGTCTTCTAGTATAAGATTCCTCTGGAGCGTCCTGTAGCCTTTGTTTTAGTAAAGATTCTAGCCCCACCAAACCATGCTTGAATTCACCAAAGCAAGACATGGTCTCCAAGTGGCAAAATCCAACGTTTTCTTGTTCAACGATAAACTTTAAGGCATCCGAATCACAGTCAGTAGAGATTTGTAAAAGCTTTTGGCCATTGCCAGAAGTTTCACCCTTGATCCAGATTTCATTCCTAGAACGAGAATAATAAACGCCACGACCCAATTCGATGGCCTTTGCTATAGATTTCTTCGAAGAATACACCAACCCTAGACAACGCTCATATTGGTCCACAACTAGGGTGGTATATAAACCGTCAGGACGGTCTGTACGTACTTCACCAAGCACTTCTTTGGTCAACATATCCTTGCTTAATTTCTTTATGGACACAATTTTATCTTGCGAGAATTTTTGTTTTACCATGAATTGATTGGAGAAAACACCGTTCTCTTCCACAACAACACGCTCCTTTGGTACATTCAATTGTTCAACCAAGTGTTCGGCTGTTTTAGCATCTTGGCTTGCAATGAACAGAGAAGAAACTCCGTTGTTCAAGAAGGCAATGATTTCATCATCGCTGAATTTACCACTTGGCAAGGACAAAGCCACCAATGGAACTTCTTCCTCTTTGGAGAACTGGAGAATCTCTTCATTACTCAGGCTCGAGCCATCCAAAAGTACCTGACCAACAAGTGAAACGTATTCCTTCTTACTATTCCATGAGGCCAGATCATCAATTAACGGTAGAATCGGCAAAACCATTATTCAGAAAAAAAATTTTGTAAACTATTGTATTACTATTACACAGCGCAGTTGTGCTATGATATTAAAATGTATCCAGAACACACATCGGAGGTGAATATAACGTTCCATATCTATTATATACACAGTATACTACTGTTCATAGTCATATCCTTTTCTTACCTTCTATATCGAATGACTGATAATGCAACGTGAGTCACTGTGCATGGGTTTAGCAATTATTAAACTAATTTACCGGAGTCACTATTAGAGTCAGTTCGACTGCCTAGAAGAACTGCTGGTTGTCAGGATTGTGATGGGGGCATTCTGCTGTATTATGACCCATCGTATCGCAATGCTCACACCACTGTTGTCTTCCTGCCGTGGTATCGACTGGTGCAGGGGGGTCGAAAATTGATATACGGTACTACTGCACAACAAACTTTGGAAACCAACTTCAATGATCATCATGACTGCAATAAAAGCACTGAGAAACACGAGTTGATAATACCCACCCCATCAAAACCACTAAAGAAAAGGATATAAAGAAGACAAAGTAAAATGTATCAGCATTTACAACATTTGTCACGTTCTAAACCATTGCCGCTTACTCCAAACTCCAAATATAATGGGGAGGCTTGCGTCCAATTAGGGAAGACATATACAGTTATTCAGGATTACGAGCCTAGATTGACAGACGAAATAAGAATCTCGCTGGGTGAAAAAGTTAAAATTCTGGCCACTCATACCGATGGATGGTGTCTGGTAGAAAAGTGTAATACACAAAAGGGTTCTATTCACGTCAGTGTTGACGATAAAAGATACCTCAATGAAGATAGAGGCATTGTGCCTGGTGACTGTCTCCAAGAATACGACTGATGAAAATAATATTGACGTTCGCATTTAATCTATACCTATAATTCTGTACTTATATACTGTTCCTTAATTGAAGATTTCAACATCGTTTTTGATGTAGGTCTTTTCACCTGGAGGTGCGGCTGGGCTACCGAAGACTAATTGAGCTTGTACGGTCCAAGACTCAGGGATTTTGCTTGGCAAAGCAGCTTTTATGTAACCATTGTAGTGTTGTAGGTGACCACCCAGGCCCATTGCCTCCAAGGCAACCCACGAGTTGATTTGAGCGGCACCAGAGGTATGGTCCGCGAAACTAGGGAATGCAGCTGCGTACGCTGGGAAGTCAGCCTTTAGCTTTTCAGTTACCTTGTCGTCGGTGAAGAAGATTACAGAACCAAAGGCCTCATCCCTTGCTGAAGCAGGCCTCTTTTGACCGGCAGGGCTTTCTATAGCCTTAGTCACTTCGTCCCAAACTTTTTTGTGAGTTTCACCAGTCAAGATAACAGCGCGATTTGGCTGGGAGTTGAAAGCGGTGGGTGTGGCTCGAATGATGGTTTGGACGACGGATTGGATGTCGTTGATAGTAATTTCACCAGGTAACTCCGGTTTCAAAGCGTAAATAGTACGACGAGCAGTTAAAGTTTTCAAATAAGTTGCAACAGCAGACATGATATTGGATTGCCGGAATGGCGATATGTTGATCCCGGATACTTCAGTCTACGAAAAAAGTACAAATTATGTAGTCAGTTCCTTCAGTATGGTGTCCTTATATACTGTAGTTTGGACAAGGTGCAAATGCCAAGACCCTAGCCCGAAAAGCTCGAGGCACCCCAGGATCTTCCCCTTTACGTAATTTTCACGTAAAACGCCACAGTCCGATTTTTCTCGAATAATCATTAGTAAAAGCGGTATACTGGATTATTGTACGATAACAAGGTAGAGCTTTATTACTAAGCTAAGACGTTCTTACATCAATAGTGCTGTTCGTTATTGATGTTAGGAGAAGGAGCGGGTCTGGTGAATAGTGTAAGCAGTGTTTCTGAACTTTTTCTTCGTCTAAGTCCTTGTAATGTAAGGTAAGAATGCAAGCATCTTGTTTGTAACCCGGGTGTACGTTGACGTTAGTAAGTCACAAACCCAAGCTTAACTTCTTCGTGAGGAAGGAAAGTGTTGTGCTGAGGACTTAAGTGCTGAGGGAATTGTATTTATATTTATTTAGTACTTCTTGAGTTTACATATCCTTCGTAAAAATGCAACTTTTGTCGAAAAACACTTCCAAAAAAAAATAATAATGAATTTATGAAGCATACTAACGAGCGAGCACATCGCTGACCTATCATTACTTCATGAGATAAATTAAGATCTCCTCATATGCGAATTTCCTGTTCAGTGATAAACGTTGATTACGTTATTGATAAAAGTCTTTTCTTCTGGCAAGGGGTACCTGGAACACCAAAGACCAATTGAGATTGTACAGTCCACGCAATAGGAACATCTTGAGGCAAAGCAGATTTGACGTAGTCATTATAGTGTTGCAAATTAGCCCCCAATCCCAATAGTTCGAGGGCAGTCCAAGACTGAATTTGCACAGCACCGGTCGTATGAGCTCGCATGTTGGGAAAGCGGCTGCCAAGGCTGGAAATCTCTTTGCAGTTTTTCAGTTGGTCCTTCATCAGTGAGAAAATGACTGAACCGTAAGCCTCATCTCTGCAAGACTCTGGTCTCTTATAAAGCAGTTGGCATTGCGCTCGCAACAGCATCCCATATCCTTTTGTGTGTATCACCAACGATAATGACAGCGCGATTCACTTGTGAGTTAAAAGCTGTTGGCGTATTCTTGAGAATAACGTGTACAGTTCTCTTTACATCATCCAAACCGACACCTTGTGGTAATTCGGGCTTCAAATTGTAGATGGTACGACGGTTTGTAATAGCGTTTAAGTAGTTTCCAGTTGGGGACATTTCTTTGGCTTGGAGGTCTGGTGTTCTTGATTTTGATGGTGTATATAGCTTTAAAAAACCAAAAATGATCAACCTTTATATCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGTCCTTCAATGAAACATCGTTGGCCACTAATTTGGCCAGTGCAAAGTAGAACAAATCGGCAGCCTCCCAAGAAAGCTCCTTCTTACCCTTTGCCTCAGTCAGTTCTTCAGCTTCTTCCTTGATCTTGGCATCTAACAATGCAGAGTCGTTGAATAGTCTTCTAGTATAAGATTCCTCTGGAGCGTCCTGTAGCCTTTGTTTTAGTAAAGATTCTAGCCCCACCAAACCATGCTTGAATTCACCAAAGCAAGACATGGTCTCCAAGTGGCAAAATCCAACGTTTTCTTGTTCAACGATAAACTTTAAGGCATCCGAATCACAGTCAGTAGAGATTTGTAAAAGCTTTTGGCCATTGCCAGAAGTTTCACCCTTGATCCAGATTTCATTCCTAGAACGAGAATAATAAACGCCACGACCCAATTCGATGGCCTTTGCTATAGATTTCTTCGAAGAATACACCAACCCTAGACAACGCTCATATTGGTCCACAACTAGGGTGGTATATAAACCGTCAGGACGGTCTGTACGTACTTCACCAAGCACTTCTTTGGTCAACATATCCTTGCTTAATTTCTTTATGGACACAATTTTATCTTGCGAGAATTTTTGTTTTACCATGAATTGATTGGAGAAAACACCGTTCTCTTCCACAACAACACGCTCCTTTGGTACATTCAATTGTTCAACCAAGTGTTCGGCTGTTTTAGCATCTTGGCTTGCAATGAACAGAGAAGAAACTCCGTTGTTCAAGAAGGCAATGATTTCATCATCGCTGAATTTACCACTTGGCAAGGACAAAGCCACCAATGGAACTTCTTCCTCTTTGGAGAACTGGAGAATCTCTTCATTACTCAGGCTCGAGCCATCCAAAAGTACCTGACCAACAAGTGAAACGTATTCCTTCTTACTATTCCATGAGGCCAGATCATCAATTAACGGTAGAATCGGCAAAACCATTATTCAGAAAAAAAATTTTGTAAACTATTGTATTACTATTACACAGCGCAGTTGTGCTATGATATTAAAATGTATCCAGAACACACATCGGAGGTGAATATAACGTTCCATATCTATTATATACACAGTATACTACTGTTCATAGTCATATCCTTTTCTTACCTTCTATATCGAATGACTGATAATGCAACGTGAGTCACTGTGCATGGGTTTAGCAATTATTAAACTAATTTACCGGAGTCACTATTAGAGTCAGTTCGACTGCCTAGAAGAACTGCTGGTTGTCAGGATTGTGATGGGGGCATTCTGCTGTATTATGACCCATCGTATCGCAATGCTCACACCACTGTTGTCTTCCTGCCGTGGTATCGACTGGTGCAGGGGGGTCGAAAATTGATATACGGTACTACTGCACAACAAACTTTGGAAACCAACTTCAATGATCATCATGACTGCAATAAAAGCACTGAGAAACACGAGTTGATAATACCCACCCCATCAAAACCACTAAAGAAAAGGATATAAAGAAGACAAAGTAAAATGTATCAGCATTTACAACATTTGTCACGTTCTAAACCATTGCCGCTTACTCCAAACTCCAAATATAATGGGGAGGCTTGCGTCCAATTAGGGAAGACATATACAGTTATTCAGGATTACGAGCCTAGATTGACAGACGAAATAAGAATCTCGCTGGGTGAAAAAGTTAAAATTCTGGCCACTCATACCGATGGATGGTGTCTGGTAGAAAAGTGTAATACACAAAAGGGTTCTATTCACGTCAGTGTTGACGATAAAAGATACCTCAATGAAGATAGAGGCATTGTGCCTGGTGACTGTCTCCAAGAATACGACTGATGAAAATAATATTGACGTTCGCATTTAATCTATACCTATAATTCTGTACTTATATACTGTTCCTTAATTGAAGATTTCAACATCGTTTTTGATGTAGGTCTTTTCACCTGGAGGTGCGGCTGGGCTACCGAAGACTAATTGAGCTTGTACGGTCCAAGACTCAGGGATTTTGCTTGGCAAAGCAGCTTTTATGTAACCATTGTAGTGTTGTAGGTGACCACCCAGGCCCATTGCCTCCAAGGCAACCCACGAGTTGATTTGAGCGGCACCAGAGGTATGGTCCGCGAAACTAGGGAATGCAGCTGCGTACGCTGGGAAGTCAGCCTTTAGCTTTTCAGTTACCTTGTCGTCGGTGAAGAAGATTACAGAACCAAAGGCCTCATCCCTTGCTGAAGCAGGCCTCTTTTGACCGGCAGGGCTTTCTATAGCCTTAGTCACTTCGTCCCAAACTTTTTTGTGAGTTTCACCAGTCAAGATAACAGCGCGATTTGGCTGGGAGTTGAAAGCGGTGGGTGTGGCTCGAATGATGGTTTGGACGACGGATTGGATGTCGTTGATAGTAATTTCACCAGGTGCGGCCGCTTTCAAAGCGTAAATAGTACGACGAGCAGTTAAAGTTTTCAAATAAGTTGCAACAGCAGACATGATATTGGATTGCCGGAATGGCGATATGTTGATCCCGGATACTTCAGTCTACGAAAAAAGTACAAATTATGTGTCAGTTCCTTCAGTATGGTGTCCTTATATACTGTAGTTTGGACAAGGTGCAAATGCCAAGACCCTAGCCCGAAAAGCTCGAGGCACCCCAGGATCTTCCCCTTTACGTAATTTTCACGTAAAACGCCACAGTCCGATTTTTCTAGAATAATCATTAGTAAAAGCGGTATACTGGATTATTGTACGATAACAAGGTAGAGCTTTATTACTAAGCTAAGACGTTCTTACATCAATAGTGCTGTTCGTTATTGACGTCAGGAGAAGGAGCGGGTCTGGTGAATAGTGTAAGCAGTGTTTCTGAACTTTTTCTTCGTCTAAGTCCTTGTAATGTAAGGTAAGAATGCAAGCATCTTGTTTGTAACCCGGGTGTACGTTGACGTTAGTAAGGGGTGTACGTTGACGTTAGTAAGTCACAAACCCAAGCTTAACTTCTTCGTGAGGAAGGAAAGTGTTGTCTCCTACTTTTTTCAAATTTTCGAATTGTATTTATATTTATTTAGTACTTCTTGAGTTTACATATCCTTCGTAAAAATGCAACTTTTGTCGAAAAACACTTCCAAAAAAAAATAATAATGAATTTATGAAGCATACTAACGAGCGAGCACATCGCTGACCTATCATTACTTCATGAGATAAATTAAGATCTCCTCATATGCGAATTTCCTGTTCAGTGATAAACGTTGATTACGTTATTGATAAAAGTCTTTTCTTCTGGCAAGGGGTACCTGGAACACCAAAGACCAATTGAGATTGTACAGTCCACGCAATAGGAACATCTTGAGGCAAAGCAGATTTGACGTAGTCATTATAGTGTTGCAAATTAGCCCCCAATCCCAATAGTTCGAGGGCAGTCCAAGACTGAATTTGCACAGCACCGGTCGTATGAGCTCGCATGTTGGGAAAGCGGCTGCCAAGGCTGGAAATCTCTTTGCAGTTTTTCAGTTGGTCCTTCATCAGTGAAGAAAATGACTGAACCGTAAGCCTCATCTCTGCAAGACTCTGGTCTCTTATAAAGCAGTTGGCATTGCGCTCGCAACAGCATCCCATATCCTTTTGTGTGTATCACCAACGATAATGACAGCGCGATTCACTTGTGAGTTAAAAGCTGTTGGCGTATTCTTGAGAATAACGTGTACAGTTCTCTTTACATCATCCAAACCGACACCTTGTGGTAATTCGGGCTTCAAATTGTAGATGGTACGACGGTTTGTAATAGCGTTTAAGTAGTTTCCAGTTGGGGACATTTCTTTGGCTTGGAGGTCTGGTGTTCTTGATTTTGATGGTGTATATAGCTTTAAAAAACCAAAAATGATCAACCTTTATATCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACpol2 gene was modified in yJF1 (PIP) and D640A (Pol \u03b5Cat) were obtained by site directed mutagenesis, and subsequently sub-cloned into pRS304 (Pol2\u00a0+ Dpb4-CBP) (The endogenous in yJF1 strain t in yJF1 as templpb4-CBP) . The Polpb4-CBP) was mutaWith the exception of the Pol \u03b5 and Pol \u03b4 mutants (see details below), all proteins were purified as described previously . A list Pol \u03b5 mutants were expressed and purified in S.\u00a0cerevisiae essentially as for wild-type Pol \u03b5 . Protein elution was carried out with 10 CV of Buffer T\u00a0+ 200\u00a0mM NaCl, supplemented with 2\u00a0mM EDTA and 2\u00a0mM EGTA. Eluted protein was diluted 1:2 in Buffer\u00a0T\u00a0lacking NaCl to reduce the salt concentration tot 100\u00a0mM, and then loaded on MonoQ column . The column was washed with Buffer T containing 100\u00a0mM NaCl and Pol \u03b4Cat was eluted with 20 CV of 100-600\u00a0mM NaCl. The peak fraction was dialysed against storage buffer .Pol \u03b4 protein , with so2; 50\u00a0mM potassium glutamate; 0.5\u00a0mM ATP; 60\u00a0\u03bcM dATP; 60\u00a0\u03bcM dGTP. Protein mix containing PCNA and Pol \u03b5 or Pol \u03b5PIP was added to RPA-bound template. The two protein-DNA mixtures were split and increasing concentrations of RFC were added. The reactions were incubated at 30\u00b0C for 5\u00a0minutes and DNA synthesis started by adding nucleotide mix (60\u00a0\u03bcM of each dTTP and dCTP and 1\u00a0\u03bcCi [\u03b1-32P]-dCTP). After 12\u00a0minutes at 30\u00b0C, the reactions were stopped with stop solution and separated on 0.7% alkaline agarose gels.Primer extension reactions were essentially performed as described previously but using M13-mp18 ssDNA (NEB), rather than \u03d5X174 . Singula2; 0.1\u00a0mg/ml BSA; 3\u00a0mM ATP; 5\u00a0nM DNA template; 75\u00a0nM Cdt1-Mcm2-7; 40\u00a0nM Cdc6; 20\u00a0nM ORC; 25\u00a0nM DDK. After a 10\u00a0min incubation S-CDK was added to 100\u00a0nM and incubation prolonged for a further 5\u00a0min. The loading reaction was 4-fold diluted into replication buffer containing the following components, reported in their final concentrations: 25\u00a0mM HEPES-KOH, pH 7.6; 250\u00a0mM potassium glutamate; 0.01% NP-40-S; 1\u00a0mM DTT; 10\u00a0mM Mg(OAc)2; 0.1\u00a0mg/ml BSA; 3\u00a0mM ATP; 200\u00a0\u03bcM CTP; 200\u00a0\u03bcM GTP; 200\u00a0\u03bcM UTP; 30\u00a0\u03bcM dATP; 30\u00a0\u03bcM dCTP; 30\u00a0\u03bcM dGTP; 30\u00a0\u03bcM dTTP; 1\u00a0\u03bcCi [\u03b1-32P]-dCTP. Samples were pre-warmed at 30\u00b0C for 1\u00a0minute and reactions were initiated by addition of replication proteins to the following concentrations: 30\u00a0nM Dpb11; 100\u00a0nM GINS; 30\u00a0nM Cdc45; 10\u00a0nM Mcm10; 20\u00a0nM Ctf4; 20\u00a0nM Csm3/Tof1; 20\u00a0nM RFC; 20\u00a0nM PCNA; 20\u00a0nM Pol \u03b5 (or\u00a0mutants); 10\u00a0nM Pol \u03b4 (or Pol \u03b4Cat); 100\u00a0nM RPA; 20\u00a0nM Pol \u03b1; 10\u00a0nM Topoisomerase I; 10\u00a0nM Mrc1; 12.5\u00a0nM Sld3/7; 30\u00a0nM Sld2. The samples were incubated at 30\u00b0C for the indicated times and were processed and imaged as described through \u223c400\u00a0\u03bcL Sephacryl-S400 High Resolution columns, previously equilibrated in 25\u00a0mM HEPES-KOH, pH 7.6; 100\u00a0mM potassium glutamate; 0.01% NP-40-S; 1\u00a0mM DTT; 10\u00a0mM Mg(OAc)2; 40\u00a0mM KCl. MCM loading and phosphorylation was performed by adding 3\u00a0mM ATP; 0.1\u00a0mg/ml BSA; 75\u00a0nM Cdt1-Mcm2-7; 40\u00a0nM Cdc6; 25\u00a0nM DDK. After 10\u00a0minutes at 24\u00b0C, S-CDK was added to 25\u00a0mM and incubation continued for 5\u00a0minutes. The nucleotide components were added and the mix was pre-warmed at 30\u00b0C for 1\u00a0minute. Reactions were initiated by addition of replication proteins as described for standard replication reactions. Nhp6 and FACT were also added to final concentrations of 400\u00a0nM and 40\u00a0nM respectively. For the experiments on the ARS1 template the DDK concentration was 50\u00a0nM, the Sld3/7 concentration was 25\u00a0nM, the GINS concentration was 200\u00a0nM, the Mcm10 concentration was 5\u00a0nM and the RPA concentration was 60\u00a0nM. After quenching by addition of an equal volume of 50\u00a0mM EDTA, proteins were removed from the replication mix by proteinase K \u2013 SDS (0.1%) treatment, for 15\u00a0minutes at 37\u00b0C, followed by phenol-chloroform-isoamyl alcohol extraction (Sigma-Aldrich). Unincorporated nucleotide was removed using Illustra G-50 columns . Samples were analyzed as described in Cut Smart buffer, for 30\u00a0minutes at 37\u00b0C. Digests were stopped by adding EDTA to final concentration of 50\u00a0mM, followed by deprotinization with proteinase K - SDS treatment and phenol-chloroform extraction as described above. Sample aliquots were analyzed on 1% alkaline and 0.8% native agarose gels, where required. The remaining digested products were ethanol precipitated, washed with 70% ethanol, air-dried and resuspended in 10\u00a0mM Tris-HCl, pH 8; 1\u00a0mM EDTA. For the RNase HII experiments, digestion products were further treated with RNase HII enzyme (NEB) for 1 hour at 37\u00b0C. The reactions were stopped with 50\u00a0mM EDTA and processed as described above for the restriction digests. For polyacrylamide gel analysis an equal volume of 2x loading dye was added to the samples. Samples were incubated for 3\u00a0minutes at 95\u00b0C, promptly transferred to ice, before being applied to a 40\u00a0cm x 20\u00a0cm denaturing 4% polyacrylamide (Bis-Acrylamide 19:1 \u2013 Fisher scientific), 7\u00a0M Urea, in 1x Tris-Borate-EDTA buffer (TBE) gel. Gels were run for 170\u00a0minutes at constant 40 Watt.Native agarose gels and acrylamide gels were dried directly onto 3MM chromatography paper . Alkaline agarose gels were fixed with two 15\u00a0min incubations at 4\u00b0C in 5% trichloroacetic acid solution before drying on 3MM chromatography paper . For quantification, gels were exposed on BAS-IP MS Storage Phosphor Screens and imaged on a Typhoon phophorimager . Gels were also autoradiographed using Amersham Hyperfilm MP for presentation.Quantification and data analysis were performed with ImageJ software and Prism 7. For pulse-chase experiments to determine maximum leading-strand synthesis rates and S1 lFor the initiation site mapping experiments lanes profiles were generated in ImageJ. The migration positions of the replication products were converted to product lengths using standard curves generated from the molecular weight markers. To generate the standard curve the migration position of the marker bands were plot against the Log10 of their length and data were fit to a second order polynomial. Signal from the no enzyme lanes was subtracted from the profiles and data were normalized by dividing by the maximum peak value for a given profile. Initiation site positions relative to the 5\u0374 end of the ACS were then derived by subtracting the distance between the enzyme cleavage site and the 5\u0374 end of the ACS."} +{"text": "CRISPR\u2013Cas9 enables recombination between homologous chromosome arms at predefined sites and also underscores the need for caution when applying CRISPR technologies in translational medicine. Drosophila, the detected Cas9-mediated editing events frequently resulted in germline-transmitted exchange of chromosome arms\u2014often without indels. These findings demonstrate the feasibility of using the system for generating recombinants and also highlight an unforeseen risk of using CRISPR-Cas9 for therapeutic intervention.CRISPR\u2013Cas9\u2013based genome editing has transformed the life sciences, enabling virtually unlimited genetic manipulation of genomes: The RNA-guided Cas9 endonuclease cuts DNA at a specific target sequence and the resulting double-strand breaks are mended by one of the intrinsic cellular repair pathways. Imprecise double-strand repair will introduce random mutations such as indels or point mutations, whereas precise editing will restore or specifically edit the locus as mandated by an endogenous or exogenously provided template. Recent studies indicate that CRISPR-induced DNA cuts may also result in the exchange of genetic information between homologous chromosome arms. However, conclusive data of such recombination events in higher eukaryotes are lacking. Here, we show that in CRISPR\u2013Cas9\u2013based genome editing has revolutionized genetic research, triggering the development of a plethora of technologies and applications that provide unprecedented control over genes in a growing list of model species , 6, 7, 8Drosophila. Although these findings expand the tool-box of CRISPR-based genome manipulation in research, they also raise concerns about the use of gene editing in therapeutic settings.Here, we set out to examine the occurrence and frequency of genetic exchanges between homologous chromosome arms initiated by Cas9-induced DSBs. We show that Cas9-triggered DSBs induce germline-transmitted recombination between homologous chromosome arms in up to 39% of the CRISPR events in Drosophila , is in-frame with the downstream ORF and resides within the unique 20-nt gRNA target, starting 4 nt upstream of the PAM sequence. Activation of CIGAR is achieved by Cas9-induced DNA cleavage within the STOPT codon. The induced DSBs will then be mended by NHEJ-mediated repair concomitantly severing or eliminating the STOPT codon. The resulting indel leads to repositioning of the upstream ATGs relative to the ORF, that is, causing one of the ATGs to be \u201cshifted in-frame\u201d with the ORF.NHEJ is a major repair mechanism triggered by CRISPR\u2013Cas9\u2013induced DSBs in osophila , 30. Levosophila . CIGAR cery cell , (ii) a ery cell , and /+), we found that in some of the F1 animals, the CRISPR target sequence of the CIGARmCherry reporter became located 5\u2032 of the eGFP ORF and vice versa. Sequence analysis of 84 animals from different crosses revealed a total of 26 animals in which the sequences on one side of the DSB had been exchanged ; molecular analysis of the target site. Deleted bp shown as: \u2013. Inserted/exchanged bp shown as: N. Bold, grey sequence numbers show un-rearranged (un-CRISPRed) CIGAR reporters. To facilitate the detection of recombination, the first 4 nt of the shifter of CIGAReGFP (CGGC) and CIGARmCherry (CCCC) are highlighted. The PAM site is shown in bold letters. In addition, we highlight the PCR setup used to amplify the shifter sequences of CIGAReGFP (green) or CIGARmCherry (red). The fwd primer anneals within the ubiquitin promoter , and the reverse primers are either specific for eGFP or mCherry, respectively. The used primers for CIGAReGFP: CIGAR-fwd: CAACAAAGTTGGCGTCGATA and CIGAReGFP-rev: GAACTTCAGGGTCAGCTTGC. The used primers for CIGARmCherry: CIGAR-fwd: CAACAAAGTTGGCGTCGATA and CIGARmCherry-rev: AAGCGCATGAACTCCTTGATG.Shown are the readable sequences (n = 84) for recombination on the X chromosome . Such individuals were crossed to yw animals, and their offspring were scored for recombination events ; molecular analysis of the target site. Deleted bp shown as: \u2013. Inserted/exchanged bp shown as: N. Bold, grey sequence numbers show un-rearranged (un-CRISPRed) CIGAR reporters. To facilitate the detection of recombination, the first 4 nt of the shifter of CIGAReGFP (CGGC) and CIGARmCherry (CCCC) are highlighted. The PAM site is shown in bold letters. In addition, we highlight the PCR setup used to amplify the shifter sequences of CIGAReGFP (green) or CIGARmCherry (red). The fwd primer anneals within the ubiquitin promoter , and the reverse primers are either specific for eGFP or mCherry, respectively. The used primers for CIGAReGFP: CIGAR-fwd: CAACAAAGTTGGCGTCGATA and CIGAReGFP-rev: GAACTTCAGGGTCAGCTTGC. The used primers for CIGARmCherry: CIGAR-fwd: CAACAAAGTTGGCGTCGATA and CIGARmCherry-rev: AAGCGCATGAACTCCTTGATG.Shown are the readable sequences (n = 156) for recombination on the fourth chromosome differed. However, the recombination events could only be demonstrated by sequence analysis of the immediate vicinity of the CRISPR site; more distant phenotypic markers were not present on these chromosomes. Therefore, we could not rule out that, at least in some cases, other mechanisms, such as gene conversion, were responsible for the observed sequence exchange between the two CIGAR reporters in trans .w+-marked CIGARmCherry,102F, w+ and the recessive viable mutation svspa-pol close to the tip of the right arm on chromosome 4. We selected the Cas9 target site, targeted with sgRNA-3, in the 3\u2032UTR of the toy gene residing about 18 kb downstream of the CIGARmCherry,102F, w+ transgene insertion site. To induce recombination at the target site 1021,y+, svspa-pol embryos with active Cas9-sgRNA RNP complexes containing recombinant Cas9 and in vitro\u2013translated sgRNA-3 1021,y+, svspa-pol flies to visually detect recombination events in the offspring , the frequency of CRISPR-mediated targeted recombination in this experiment is estimated to be \u223c1.1%. This frequency is at least four orders of magnitude higher than that of the rare spontaneous recombination rate predicted for chromosome 4 , 41w+-mamosome 4 .Recombination between two visible markers.Table S4 mCherry 102F, w+/Dp(1021) y+, svspa-pol injected with Cas9.List of recombinants recovered from yw; CIGARTable S5 mini-white gene (+w), used as transgene reporter, is juxtaposed to heterochromatic regions via chromosomal rearrangements or translocations 1021,y+, svspa-pol animals exhibiting PEV were crossed to yw; ciD,svspa-pol/sv\u0394122 animals. Without exception, the tested PEV chromosomes were unable to complement the sv\u0394122 mutation, indicating that they must have lost distal parts of chromosome 4.The remaining 36 (out of 57) putative recombinants recovered from the above experiment were homozygous lethal and exhibited position effect variegation PEV; in the aocations , 44, 45.d to PEV . We, theromosome . To assey+ marker on the short left arm of chromosome 4 and the w+-marked CIGARmCherry,102F transgene inserted at 102F of the right arm 1021, y+, svspa-pol were injected with RNPs containing Cas9 protein complexed with in vitro\u2013translated sgRNA-3. The RNPs induce DSBs 3\u2032 of toy located distal of CIGARmCherry,102F but should not have any influence on the recombination between the y+ and the w+ markers. G0 animals were crossed with yw animals to score recombination events between the y+ and the w+ marker on the fourth chromosome , we repeated the experiment using the same experimental conditions, but assessed recombination between a + marker . We scre+ marker , TR wereIn summary, our experiments involving chromosome 4 revealed that germ-line\u2013transmitted recombinants can be recovered at frequencies ranging from about 1.1% (recombination between visible markers) to 26% . These percentages represent the number of recombinants recovered from the total number of animals analyzed. When only the detected Cas9-mediated events are taken into consideration, the frequency of recombination was even 39%.Drosophila, however, the first meiotic divisions occur at much later timepoints , likelysgRNAs are provided via transgenes (nanos (nos) promoter and the Cas9 transgene contains the nos 3\u2032UTR recapitulating germline-specific nos expression, transcript localization, and translational control -EcoRI fragment from pCaSpR3-Up2-RX polyA was subcloned into a pBluescript (pBS) vector between the EcoRI and XhoI sites using blunt-end ligation. The EcoRI-ubi-Acc65I fragment from the resulting plasmid was subcloned into pEPattB , a unique gRNA target sequence (referred to as sgRNA-1), the linker sequence, and the eGFP gene were designed (as shown below), synthesized by GenScript, and delivered ligated into the pUC57-Kan vector (pCIGAR-D0).To create the The pCIGAR-D0 insert:5\u2032-KpnI_CAACATGGTGCAACATGGTGCAACATGGTGCGGCGACAGCAGA ACGTAGCGGGACGATAGGCTGCAGATCCTTGGCGCGCCTTCAGGAGGCGGTGCTACTGCTGGCGCTGGTGGAGCCGGTGGACCTGCGGGGTTAATTGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCATAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAGAATTC_EcoRI-3\u2032.KpnI and EcoRI and ligated into pre\u2013double-digested pKB342 vector in line with a tubulin 3\u2032 trailer, transformed, and purified, resulting in pKB342_CIGAR. To remove the CIGAR sequence including the tubulin trailer, pKB342_CIGAR was then digested with KpnI and XbaI, and the fragment was ligated into a KpnI- and XbaI-digested pUbiattB vector containing an ubiquitin-p63E promoter (see above). This resulted in the final product referred to as pUbiattB-CIGAReGFP.The synthetic sequence was excised with pUbiattB-CIGAReGFP were used for the construction of the pUbiattB-CIGARmCherry. The only difference was in the design of the target site; the gRNA (sgRNA-2) has a different unique target sequence and contains the mCherry gene as the fluorescent marker. The insert ligated into the pUC57-Kan vector was ordered from GenScript.The same sequential digestions and ligation as for the making of the CIGARmCherry insert:The 5\u2032-KpnI_CAACATGGTGCAACATGGTGCAACATGGTGCCCCGAGACAAGCACCTGACGGGACGATAGGCTGCAGATCCTTGGCGCGCCTTCAGGAGGCGGTGCTACTGCTGGCGCTGGTGGAGCCGGTGGACCTGCGGGGTTAATTGTGAGCAAGGGCGAGGAGGACAACATGGCCATCATCAAGGAGTTCATGCGCTTTAAGGTGCACATGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGCGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTCATGTACGGCTCCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGACTACTTGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGGCGAGTTCATCTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCTCCGACGGCCCCGTAATGCAGAAGAAGACCATGGGCTGGGAGGCCTCCTCCGAGCGGATGTACCCCGAGGACGGCGCCCTGAAGGGCGAGATCAAGCAGAGGCTGAAGCTGAAGGACGGCGGCCACTACGACGCCGAGGTCAAGACCACCTACAAGGCCAAGAAGCCCGTGCAGCTGCCCGGCGCCTACAACGTCAACATCAAGCTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAACAGTACGAGCGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTGTACAAGTAA_EcoRI-3\u2032.pCFD5 vector was a gift from Fillip Port following the protocol described in the supplementary methods of pCFD5 cloning protocol.The Addgene) . pCFD5 ipCFD5 internal tRNA multi-gRNA Scaffold:5\u2032-GTCGGGGCTTTGAGTGTGTGTAGACATCAAGCATCGGTGGTTCAGTGGTAGAATGCTCGCCTGCCACGCGGGCGGCCCGGGTTCGATTCCCGGCCGATGCAGGGTCTTCGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCAACAAAGCACCAGTGGTCTAGTGGTAGAATAGTACCCTGCCACGGTACAGACCCGGGTTCGATTCCCGGCTGGTGCAGAAGACCTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTT-3\u2032.sgRNA-1 and sgRNA-2 were inserted into the pCFD5 vector using primer (5\u2032-GCGGCCCGGGTTCGATTCCCGGCCGATGCACGGCGACAGCAGAACGTAGCGTTTTAGAGCTAGAAATAGCAAG-3\u2032) for sgRNA-1 and (5\u2032-ATTTTAACTTGCTATTTCTAGCTCTAAAACGTCAGGTGCTTGTCTCGGGGTGCACCAGCCGGGAATCGAACCC-3\u2032) for sgRNA-2.Through the process of Gibson cloning, both our sgRNAs were performed as described in reference IVT of sgRNA-3:IVT oligo used specific for CTGTTGATAAGCACGCAATCGTTTTAGAGCTAGAAATAGC-3\u20325\u2032-GAAATTAATACGACTCACTATAGG.sgRNA-R used for template PCR:IVT oligo used AAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC-3\u2032.5\u2032-sgRNA primer and the sgRNA-R are shown in bold letters.Complementary sequences of the specific sgRNA . This corresponds roughly to a 1:2 ratio (SpCas9: sgRNA-3). SpCas9 has about 5\u00d7 the molecular weight of the sgRNA.The concentration of SpCas9 injected was about 800 ng/\u03bcl SpCas9 and about 300 ng/\u03bcl of sgRNA, ddH2O, and 10\u00d7 incubation buffer NEB and mix thoroughly. Gently add the SpCas9 and mix again thoroughly by pipetting up and down. It is mandatory to add SpCas9 as the last ingredient because low salt concentrations may cause SpCas9 to precipitate : add cipitate .sgRNA .X \u03bcl 1 \u03bcl 10\u00d7 NEB incubation buffer.2O (RNase free) to a total of 10 \u03bcl (X + Y = 6.5 \u03bcl).Y \u03bcl ddH2.5 \u03bcl SpCas9 .Mix gently and incubate the mix at 37\u00b0C for 2 min.Load the mix onto a column (Ultrafree-MC-HV 0.45 \u03bcm [Ref: UFC30HV00]).g.Spin for 1 min in a table-top centrifuge @14,000Reincubate the flow-through at 37\u00b0C for 2 min.Let the mix equilibrate at RT for \u223c30 min before injection. Never put the mix back on ice.Drosophila Stock Center (see the Acknowledgments section).Crosses were done at 25\u00b0C. Unless noted otherwise, fly lines were obtained from the Bloomington ZH-attP 5D [X chromosome] or ZH-attP 102F [fourth chromosome]) were generated by phiC31 integrase-mediated transgenesis ; CIGAR-fwd: CAACAAAGTTGGCGTCGATA and CIGARmCherry-rev: AAGCGCATGAACTCCTTGATG , respectively. PCR settings were as follows: 95\u00b0C, 5 min; 35 cycles of 95\u00b0C, 25 s; 60\u00b0C, 25 s; and 72\u00b0C, 30 s); final elongation of 72\u00b0C, 10 s. The same PCR setting was used to analyze the shifter sequence of the CIGAR reporters by single fly PCR.Transgenic CIGAR fly lines was inserted into the attP 40 site .The The images were taken on Axio Zoom V16 (Zeiss) and were processed in Adobe Photoshop or Adobe Illustrator.CIGAReGFP or CIGARmCherry construct on the fourth chromosome at position 102F ). To test if CRISPR-Cas9\u2013mediated DSBs also result in recombination, the fourth chromosome, nos-Cas9/Y; U6:3-sgRNACIGAR/+; CIGAReGFP,102F, w+/CIGARmCherry,102F,w+ G0 males were crossed to yw females /+; CIGAReGFP,102F, w+/CIGARmCherry,102F, w+ G0 females to yw males). The offspring of such crosses were first scored at the larval stage to identify animals with either an activated CIGAReGFP,102F or CIGARmCherry,102F reporter. This preselection was made to ascertain that DSBs occurred in both constructs enhancing the likelihood of detecting recombination events on the fourth chromosome in case they occur. 17 vials containing GFP and mCherry-positive larvae were selected for further analysis. A total of 172 y,w+ F1 animals harboring either a CIGAReGFP,102F or CIGARmCherry,102F reporter but lacking the sgRNA plasmid U6:pCFD5 (to avoid mosaic flies) from 13 of these crosses were randomly picked right after hatching. Single fly PCR of the target as well as part of the fluorophore region for the CIGAR reporters was performed for these 172 animals (Tables S2 and S3). PCR products and readable sequences were obtained in 156 cases.To determine if we could also induce CRISPR/Cas9\u2013mediated recombination on the fourth chromosome, we first generated flies harboring either a ion 102F . These aFor the recombination experiments using the CIGAR transgenes on chromosome 4, we estimate the recombination frequency to be 26%: 41 recombinants identified/156 flies analyzed \u00d7 100 located downstream of the w+ marker near the tip of chromosome 4 and is visible if the mutation is homozygous or over a null allele of sv such as sv\u0394122 .To confirm that indeed recombination between sister chromatids occurs after CRISPR\u2013Cas9\u2013induced DSBs, we tested if we could induce recombination between two visible markers that are separated by about 100 kb and S3. mosome 4 . The Casion site . The rouyw; CIGARmCherry,102F, w+/Dp1021,y+, svspa-pol embryos were injected with recombinant Cas9 protein complexed with in vitro\u2013translated sgRNA-3 targeting the 3\u2032UTR of the toy gene. From the eclosing G0 animals, a total of 135 crosses were set up. Either five G0 females (29 crosses) or single G0 males (106 crosses) were crossed with homozygous Dp1021,y+, svspa-pol flies. F1 offspring were either yw; CIGARmCherry,102F, w+/Dp1021,y+, svspa-pol or homozygous Dp1021,y+, svspa-pol animals 1021,y+, svspa-pol and would be scored as having rough, red eyes . The second recombination event (TR-B) that could occur would be yw; Dp1021,y+, sv+/Dp1021,y+, svspa-pol flies having white and smooth eyes . From a total of 8,604 scored F1 animals, 253 putative recombinant flies were recovered: 216 had rough red eyes and 37 animals had white and smooth eyes (five independent G0 crosses). Notably, in one cross, 31 w\u2212, y+sv+ animals were present in one tube corresponding to about 25\u201330% of the offspring. 70 of the 253 putative recombinants were backcrossed to homozygous Dp1021,y+, svspa-pol flies to establish stocks. 61 of the 70 putative recombinants were recovered from independent crosses . 13 of the 70 crosses remained without offspring. Most importantly, from 21 putative recombinants, we could generate homozygous viable lines. 17 of the 21 lines showed the y, w+, svspa-pol phenotype, whereas four lines were w, y+, sv+ marking all 21 lines as true recombinants. The remaining 36 putative recombinants were homozygous lethal .spa-pol) . Recombiy, w+, svspa-pol phenotype, the other was phenotypically w, y+, sv+, indicating that we may have recovered both chromosomes from the same recombination event. A third recombined animal from the same cross showed no CRISPR mark and thus can be counted as independent recombination event.We then investigated these animals by PCR specific for the CRISPR target site. Sequence analysis revealed that with the exception of two animals all CRISPR sites were without any indel . Interestingly, two recombinants from the same cross showed the same CRISPR mark (6-bp deletion). However, they had the complementary phenotype: Whereas one fly showed the y+ marker on the short, left arm of chromosome 4 and the w+-marked CIGARmCherry,102F inserted at 102F 1021,y+, svspa-pol) were injected with Cas9 protein complexed with in vitro\u2013translated sgRNA-3. This again would induce DSBs 3\u2032 of toy located distal of CIGARmCherry,102F but should not have any influence on the recombination between the y+ and the w+ markers. G0 animals were this time crossed with yw animals to be able to score crossover events between the y+ and the w+ marker , we backcrossed such animals again against yw flies. Spontaneous recombination between y+ and w+ could be excluded as the above-mentioned backcross would have only revealed phenotypically y+w+ or yw flies. Instead, nondisjunction could be confirmed in all 11 cases because the backcross revealed phenotypically y+w+, yw, y+w, and yw+ flies. We determined the non-disjunction rate for the fourth chromosome to be 1 in 1,200, which is similar to the one observed for the X chromosome (To assess if CRISPR\u2013Cas9\u2013induced DSBs in general would enhance the frequency of recombination away from the Cas9 target site, we repeated the experiment and assessed the recombination frequency between a + marker . As experomosome ."} +{"text": "The replisome must overcome DNA damage to ensure complete chromosome replication. Here, we describe the earliest events in this process by reconstituting collisions between a eukaryotic replisome, assembled with purified proteins, and DNA damage. Lagging-strand lesions are bypassed without delay, leaving daughter-strand gaps roughly the size of an Okazaki fragment. In contrast, leading-strand polymerase stalling significantly impacts replication fork progression. We reveal that the core replisome itself can bypass leading-strand damage by re-priming synthesis beyond it. Surprisingly, this restart activity is rare, mainly due to inefficient leading-strand re-priming, rather than single-stranded DNA exposure or primer extension. We find several unanticipated mechanistic distinctions between leading- and lagging-strand priming that we propose control the replisome\u2019s initial response to DNA damage. Notably, leading-strand restart was specifically stimulated by RPA depletion, which can occur under conditions of replication stress. Our results have implications for pathway choice at stalled forks and priming at DNA replication origins. \u2022Reconstitution of collisions between a eukaryotic replisome and DNA damage\u2022Leading-strand damage specifically causes fork stalling and uncoupling\u2022The eukaryotic replisome can re-initiate leading-strands downstream of DNA damage\u2022Multiple mechanistic differences exist between leading- and lagging-strand priming To study the earliest events following DNA polymerase stalling during replication, Taylor and Yeeles reconstituted collisions between a yeast replisome assembled with purified proteins and template DNA damage. Surprisingly, re-priming of leading-strand synthesis beyond damage is inefficient but is promoted by RPA depletion, whereas lagging-strand priming occurs robustly after damage. Accurate and efficient DNA replication is vital to ensure faithful and timely transmission of genetic information. This task is accomplished by a complex and highly regulated molecular machine known as the replisome. Replisomes frequently encounter a wide variety of obstacles to their progression, including template DNA damage, DNA secondary structures, and the transcription machinery . ConsideEscherichia coli found that, following UV irradiation of nucleotide excision repair (NER)-defective cells, replication continued and nascent DNA was synthesized as small fragments interspersed with single-stranded DNA (ssDNA) gaps (E.\u00a0coli proteins (E.\u00a0coli replisome has the inherent capacity to \u201cskip\u201d over multiple leading-strand lesions via re-priming (Early studies in NA) gaps . Based oproteins . Howeverproteins . Mechani-priming .Experiments in eukaryotic cells also found that replication was discontinuous following UV irradiation . More rein\u00a0vivo following treatment of cells with DNA-damaging agents by which it might operate, how efficiently it occurs, or how it might be regulated. In\u00a0this study, we sought to address these outstanding questions by analyzing the response of a reconstituted core eukaryotic replisome to site-specific DNA damage.More generally, how the in\u00a0vivo rate, DNA polymerase epsilon (Pol \u03b5) catalyzes the bulk of leading-strand synthesis in conjunction with PCNA, DNA polymerase delta (Pol \u03b4) is required for complete lagging-strand synthesis, and Mrc1 and Csm3/Tof1 are critical for maximum replication rates , which is one of the primary lesions generated by UV irradiation, at a specific location in plasmid DNA B and devLAG) with a similar efficiency to those synthesized from an undamaged template C. The retemplate D, nativents. LAG E, as pronts. LAG G illustrin\u00a0vitro studies by linearizing plasmids with AhdI required for the chase. Compared to an undamaged template, replication forks were almost universally delayed for several min by CPDLEAD composed of stalled leading strands, Okazaki fragments, and a distinct population migrating at the expected position for leading-strand restart were only visible after AvrII digestion in reactions lacking RPA but containing Pol \u03b1 in step 2 , \u223c60\u00a0bp ynthesis . It is ain\u00a0vivo occurs following treatment with DNA damaging agents, but it had not been possible to exclude other indirect effects of genotoxins or the involvement of DNA repair complexes and the transcription machinery. Because fork progression can be rapidly and efficiently rescued by artificially mimicking leading-strand \u201cre-priming\u201d with an oligonucleotide, we propose that the underlying basis for prolonged fork stalling and continued uncoupled synthesis may simply be a failure to re-prime the leading-strand template. If correct, then observations of stalled, slow-moving, and uncoupled replication forks in\u00a0vivo , isolated stalled forks should be rapidly rescued by forks from neighboring origins. We propose that this may be the principal mechanism by which stalled forks are rescued when DNA damage is infrequently encountered, which would generate daughter-strand gaps for processing by DDT pathways.In striking contrast to E.\u00a0coli , our resin\u00a0vivo. It is notable that some eukaryotes encode an additional primase, PrimPol promote PrimPol . PrimPol PrimPol .in\u00a0vitro raises the possibility that re-priming might fulfill a more significant role under conditions of catastrophic global fork arrest, which can have a dramatic effect on free RPA levels in\u00a0vivo .Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Joseph Yeeles modified to overexpress proteins of interest as detailed in the Key Resources table by transforming with linearized plasmids using standard genetic procedures; or Escherichia coli Rosetta\u2122 2(DE3) cells (Novagen) (genotype: F\u2013\u00a0ompT hsdSB(rB\u2013 mB\u2013) gal dcm (DE3) pRARE2 (CamR)) transformed with plasmids for overexpression of proteins of interest as detailed in the Proteins were purified from Escherichia coli are listed in the ORC \u2013 ORC, carrying a CBP-TEV tag on the Orc1 subunit, was purified using affinity purification with Calmodulin Sepharose 4B , followed by gel filtration through a Superdex 200 column.Cdc6 \u2013 GST-tagged Cdc6 was bound to Glutathione Sepharose 4B and released by cleavage with GST-tagged 3C protease. The eluted protein was further purified using a Bio-Gel HT hydroxyapatite column (Bio-Rad).Cdt1-Mcm2-7 \u2013 Cdt1-Mcm2-7 with a CBP-TEV tag on the N terminus of Mcm3 was purified using a Calmodulin Sepharose 4B column, followed by Superdex 200 gel filtration.DDK \u2013 DDK carrying a CBP tag on Dbf4 was bound to and eluted from Calmodulin Sepharose 4B resin. The eluted protein was dephosphorylated with lambda protein phosphatase and was then further purified by gel filtration (Superdex 200).S-CDK \u2013 The S-CDK complex carrying a N-terminal CBP-TEV tag on Clb5 was bound to Calmodulin Affinity Resin (Agilent). Protein was eluted by cleavage with Tobacco Etch Virus protease and was further purified by gel filtration (Superdex 200).Cdc45 \u2013 Cdc45 carrying a double internal FLAG tag was immunoprecipitated using Anti-FLAG M2 affinity gel (Sigma). The resulting eluate was further purified using a Bio-Gel HT hydroxyapatite column.Sld3/7 \u2013 Sld3/7 carrying a C-terminal TCP tag on Sld3 was first bound to IgG Sepharose 6 Fast Flow resin and the protein eluted by cleavage with Tobacco Etch Virus protease. The His-tagged protease was removed by passing the eluate over Ni-NTA resin (QIAGEN) and the flow through was further purified through a Superdex 200 gel filtration column.Sld2 \u2013 3xFLAG-tagged Sld2 was precipitated from cell lysate with ammonium sulfate, resuspended in buffer, and then immunoprecipitated with Anti-FLAG M2 affinity gel. Protein was eluted with 3xFLAG peptide (Sigma) and the peptide was removed by binding and eluting the protein from a HiTrap SP HP column .Dpb11 \u2013 Dpb11-3xFLAG was purified using Anti-FLAG M2 affinity gel followed by MonoS chromatography.Pol \u03b5 \u2013 Pol \u03b5, tagged with a C-terminal CBP tag on Dpb4, was purified sequentially over Calmodulin Sepharose 4B, HiTrap Heparin HP and Superdex 200 columns.GINS \u2013 The GINS complex, modified with a 6xHis tag at the N terminus of Psf3, was bound to Ni-NTA and eluted with increasing imidazole concentration. Eluted protein was further purified over MonoQ and Superdex 200 columns, followed a second Ni-NTA column.Pol \u03b1 \u2013 Pol alpha \u2013 primase, modified with an N-terminal CBP tag on Pri1 was purified sequentially over Calmodulin Sepharose 4B, MonoQ and Superdex 200 columns.Mcm10 \u2013 6xHis-Mcm10 was purified over Ni-NTA followed by two rounds of MonoS chromatography.Ctf4 \u2013 CBP-TEV-Ctf4 was purified by Calmodulin Sepharose 4B chromatography, followed by MonoQ and Superdex 200 gel filtration columns.Mrc1 \u2013 Mrc1-2xFLAG was purified by FLAG immunoprecipitation followed by MonoQ chromatography.Topo I \u2013 CBP-TEV-Topo I was bound to Calmodulin Sepharose 4B. Protein was eluted with Tobacco Etch Virus protease. His-tagged protease was removed by passing the eluate over a TALON column (Clontech) and the flow through was concentrated and separated through a Superdex 200 column.Csm3/Tof1 \u2013 Csm3/Tof1 carrying a N-terminal CBP-TEV tag on Csm3 was bound to Calmodulin Sepharose 4B. Protein was eluted with Tobacco Etch Virus protease. His-tagged protease was removed by passing the eluate over a TALON column and the flow through was concentrated and separated through a Superdex 200 column.RFC \u2013 The RFC complex containing an N-terminal CBP tag on Rfc3 was purified by sequential chromatography over Calmodulin Sepharose 4B, MonoS and Superdex 200 columns.PCNA \u2013 Native PCNA was purified following overexpression in Escherichia coli. Nucleic acids were precipitated from the cell lysate with polymin P and then proteins were selectively precipitated with ammonium sulfate. Precipitated material was resuspended in buffer and then applied to HiTrap SP HP and HiTrap Heparin HP columns assembled in tandem. The flow through containing PCNA was further purified over HiTrap DEAE Fast Flow and MonoQ columns.Pol \u03b4 \u2013 Pol \u03b4 with a C-terminal TEV-CBP tag on Pol32 was purified over Calmodulin Sepharose 4B, HiTrap Heparin HP and Superdex 200 columns.RPA \u2013 Native RPA was purified following overexpression in Escherichia coli. Protein was purified over HiTrap Blue HP , ssDNA cellulose (Sigma) and MonoQ columns.Isw1a \u2013 Isw1a carrying a C-terminal 3xFLAG tag on Ioc3 was immunoprecipitated from cell lysate using Anti-FLAG M2 affinity gel. Protein was eluted with 3xFLAG peptide and was further purified by MonoQ chromatography.Histones \u2013 Native histones were purified following overexpression in Escherichia coli by HiTrap Heparin HP and Superdex 200 chromatography.Nap1 \u2013 GST-Nap1 was bound to Glutathione Sepharose 4B and eluted from the resin with GST-3C protease. Eluted protein was further purified on a MonoQ column.FACT \u2013 His-tagged FACT was purified by TALON and MonoQ chromatography.Nhp6 \u2013 Native Nhp6 was purified following overexpression in Escherichia coli. Proteins were precipitated from the cell lysate with trichloroacetic acid. The precipitated protein was resuspended in buffer and further purified over a HiTrap SP HP column.Proteins were purified as described previously . All yeaThe DNA replication template was produced by direct synthesis of a modified version of the genomic DNA surrounding the early-firing origin ARS306 and subsequent cloning and modification in pBluescript II KS(\u2013). The complete sequence of the resulting two plasmids used for replication of damaged DNA introduced at two different sites is given below.Saccharomyces Genome Database. Modifications were made to (1) introduce restriction enzyme sites to facilitate cloning of three different fragments of this region into pBluescript II KS(\u2013) ; (2) introduce several unique diagnostic restriction enzyme sites in proximity to both sites of DNA damage cloning, used in a variety of different assays; (3) introduce two closely spaced restriction enzymes for cloning of cassettes containing BbvCI nicking endonuclease sites for the subsequent cloning of DNA damage at two different locations approximately 3 kb (PstI and BamHI) or 4.5 kb (SphI and SpeI) left of ARS306; (4) mutate any sequences outside of the origin with a strong ORC binding consensus, to facilitate tight origin specificity of replication initiation; (5) introduce other unique restriction enzyme sites for other purposes, including for asymmetric template linearization with respect the origin. The modified sequence was synthesized by Invitrogen GeneArt Gene Synthesis in three segments flanked by (1) KpnI and SalI; (2) SalI and EagI; (3) EagI and SacI. These segments were cloned using these restriction sites into the multiple cloning site of pBluescript II KS(\u2013). The resulting plasmids were then digested with either PstI and BamHI, or SphI and SpeI, for introduction of cassettes for the subsequent cloning of DNA damage at two different positions.A region of genomic DNA on chromosome III spanning approximately 7.5 kb left and 0.2 kb right of ARS306 was chosen from the \u02c6 indicates position of top strand nicking), was designed: CC\u02c6TCAGCACTTAAGTCC\u02c6TCAGC. This sequence and its reverse complement were incorporated into oligonucleotides flanked by overhangs compatible with the combination of either PstI and BamHI . To achieve this, the following cassette for cloning DNA damage, comprising an AflII restriction enzyme site (underlined) flanked by two Nt.BbvCI sites ) and ZraI (in the far left flank of the modified genomic DNA region) to excise approximately 1 kb DNA and the two blunt ends ligated to give a final plasmid size of approximately 9.7 kb. For the plasmid containing a cassette 4.5 kb from the origin, an additional two restriction enzyme sites, SacI and PsiI, were introduced approximately 1 kb and 1.6 kb respectively downstream of the cassette by site directed mutagenesis to facilitate characterization of the stalled fork. The final plasmids are hereafter referred to as ZN3 (plasmid with a cassette 3 kb from the origin) and ZN5SP1 (plasmid with a cassette 4.5 kb from the origin) and their sequences are given below:CTGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGTCCTTCAATGAAACATCGTTGGCCACTAATTTGGCCAGTGCAAAGTAGAACAAATCGGCAGCCTCCCAAGAAAGCTCCTTCTTACCCTTTGCCTCAGTCAGTTCTTCAGCTTCTTCCTTGATCTTGGCATCTAACAATGCAGAGTCGTTGAATAGTCTTCTAGTATAAGATTCCTCTGGAGCGTCCTGTAGCCTTTGTTTTAGTAAAGATTCTAGCCCCACCAAACCATGCTTGAATTCACCAAAGCAAGACATGGTCTCCAAGTGGCAAAATCCAACGTTTTCTTGTTCAACGATAAACTTTAAGGCATCCGAATCACAGTCAGTAGAGATTTGTAAAAGCTTTTGGCCATTGCCAGAAGTTTCACCCTTGATCCAGATTTCATTCCTAGAACGAGAATAATAAACGCCACGACCCAATTCGATGGCCTTTGCTATAGATTTCTTCGAAGAATACACCAACCCTAGACAACGCTCATATTGGTCCACAACTAGGGTGGTATATAAACCGTCAGGACGGTCTGTACGTACTTCACCAAGCACTTCTTTGGTCAACATATCCTTGCTTAATTTCTTTATGGACACAATTTTATCTTGCGAGAATTTTTGTTTTACCATGAATTGATTGGAGAAAACACCGTTCTCTTCCACAACAACACGCTCCTTTGGTACATTCAATTGTTCAACCAAGTGTTCGGCTGTTTTAGCATCTTGGCTTGCAATGAACAGAGAAGAAACTCCGTTGTTCAAGAAGGCAATGATTTCATCATCGCTGAATTTACCACTTGGCAAGGACAAAGCCACCAATGGAACTTCTTCCTCTTTGGAGAACTGGAGAATCTCTTCATTACTCAGGCTCGAGCCATCCAAAAGTACCTGACCAACAAGTGAAACGTATTCCTTCTTACTATTCCATGAGGCCAGATCATCAATTAACGGTAGAATCGGCAAAACCATTATTCAGAAAAAAAATTTTGTAAACTATTGTATTACTATTACACAGCGCAGTTGTGCTATGATATTAAAATGTATCCAGAACACACATCGGAGGTGAATATAACGTTCCATATCTATTATATACACAGTATACTACTGTTCATAGTCATATCCTTTTCTTACCTTCTATATCGAATGACTGATAATGCAACGTGAGTCACTGTGCATGGGTTTAGCAATTATTAAACTAATTTACCGGAGTCACTATTAGAGTCAGTTCGACTGCCTAGAAGAACTGCTGGTTGTCAGGATTGTGATGGGGGCATTCTGCTGTATTATGACCCATCGTATCGCAATGCTCACACCACTGTTGTCTTCCTGCCGTGGTATCGACTGGTGCAGGGGGGTCGAAAATTGGCAACGATTCCACGGCTGTTTGTGCTTGAGCCTGTTCCAACTGTTTGAACCTTTCATTAGCCTCTTCAAGTTTTTTCGTTAAGGATGCCACCTCTTCCGATGAGGAATCTTGTGGTTTTGTCAAAAATAGTTCCTTGCTCAAATTTTGGTATTCTTTACTGAGCGAATCGTTATGCATTTTCAATTGTTCGCGTTCTTTAGCCCACTTTGTCTTGTGTAACTCAAATTGGTCTTCTATGTTGCGTAATTGTTCCAGCTGTTTTTTCAGGAGTTCGACATCTTCGTTGGCACCAGTGGGTTGATTATGAGAAAGATTTCTCTCTTCGTTTTCTTTGATCTCTTCGTGTAGTTGGCTTACGACAGCAAGTAGCTGTTCATTCTCAGCGTCAAAAAACTGCTTTTGTTTGGCTTGCTGTCTGCGTTCGAGCAGACATTGTTGCTTGAGATGGTCTATCTCTTTCTCTCTTTCTTGTATTGTGGCTTCATACCTATCAAAAGTCGGTTGCACTTCTTCGAGGACCATTCTTTGGTCATCGAGTAGCCTTTTGTAGTGTAGTTGTTTCCTTTGTAGCTTTTCGATGGTCAATTGGCGATCGCGTAATTCAATTGTAACTTCGCTGCTATTGAGGTCATTCATGTGGCCATTGTCCGGTTTCCAATCGCTGGTGGTGTTGTGATTAGCCTTTCTGTCTGATGACAGGATAGAGTCCACCTCCATTCTGTCTTCTCTGTTATCGTAACCAAATTCTTGCTGTTGATGGTGATCCGATGCCTCCTGGTCCATCGACTGTTGATTACCGCTGTGCCGACTGGTGATCCGGAAACTTCTCATGGGTGTGGGGGATTTAGGATCATCCATGGGAGAGAAGCGCTTAGTGAGCCTCACAATAGATCTGTTCACGGGTATTGATAGCGGTTCCATTGTCGTTCTTCTCGAGGTTTGCCATATCGGTCCGTTCTCGATCAATGATGCGACTTTTTGCAACTGAATAAATAGTCCACTTTGAGGATACTCCGTTTGAAAATACTTCTTCCCCTAGGAATGATCCATCGTTCTTACCAATGTTGGCAAGTAAGTCTACACCAGCAAACATTCCACGCGTCGTGTCCACTGGACCCACGTATTTCAGTTGTCCGCGGCCGAAATTTGGGATTTGGTTTAAACATCCTATCTTTCTTTGATATCTATCCATGGTATATTAAGCGCATACGGCGCCAGCCACTAGTCAACGCCTTTTACCTTGTCCTTTGATGCATGCCTCGTCCAAACGTTTTTGGTGTCTTGGCCAATTGCCCTTCTGAAAAATCTCACTGTCCGCAACTCATTAAAAGATACCCAAGCAAGCTACACGATAAAGAAAGGAGAAAGTTCATTACTGGAACGTACATATAGCGATACAAACGTATAGCAAAGATCTGAAATGGATACGGATAAGTTAATCTCAGAGGCTGAGTCTCATTTTTCTCAAGGAAACCATGCAGAAGCTGTTGCGAAGTTGACATCCGCAGCTCAGTCGAACCCCAATGACGAGCAAATGTCAACTATTGAATCATTAATTCAAAAAATCGCAGGATACGTCATGGACAACCGTAGTGGTGGTAGTGACGCCTCGCAAGATCGTGCTGCTGGTGGTGGTTCATCTTTTATGAACACTTTAATGGCAGACTCTAAGGGTTCTTCCCAAACGCAACTAGGAAAACTAGCTTTGTTAGCCACAGTGATGACACACTCATCAAATAAAGGTTCTTCTAACAGAGGGTTTGACGTAGGGACTGTCATGTCAATGCTAAGTGGTTCTGGCGGCGGGAGCCAAAGTATGGGTGCTTCCGGCCTGGCTGCCTTGGCTTCTCAATTCTTTAAGTCAGGTAACAATTCCCAAGGTCAGGGACAAGGTCAAGGTCAAGGTCAAGGTCAAGGACAAGGTCAAGGTCAAGGTTCTTTTACTGCTTTGGCGTCTTTGGCTTCATCTTTCATGAATTCCAACAACAATAATCAGCAAGGTCAAAATCAAAGCTCCGGTGGTTCCTCCTTTGGAGCACTGGCTTCTATGGCAAGCTCTTTTATGCATTCCAATAATAATCAGAACTCCAACAATAGTCAACAGGGCTATAACCAATCCTATCAAAACGGTAACCAAAATAGTCAAGGTTACAATAATCAACAGTACCAAGGTCGCGACGGTGGTTACCAACAACAACAGGGACAATCTGGTGGTGCTTTTTCCTCATTGGCCTCCATGGCTCAATCTTACTTAGGTGGTGGACAAACTCAATCCAACCAACAGCAATACAATCAACAAGGCCAAAACAACCAGCAGCAATACCAGCAACAAGGCCAAAACTATCAGCATCAACAACAGGGTCAGCAGCAGCAACAAGGCCACTCCAGTTCATTCTCAGCTTTGGCTTCCATGGCAAGTTCCTACCTGGGCAATAACTCCAATTCAAATTCGAGTTATGTGTACACGCAACAGGCTAATGAGTATGGTAGACCGCAACAGAATGGTCAACAGCAATCCAATGAGTACGGAAGACCGCAATACGGCGGAAACCAGAACTCCTAAGGACAGCACGAATCCTTCAATTTTTCTGGCAACTTTTCTCAACAGAACAATAACGGCGCGCCGAACCGCTACTGAACGATGATTCAGTTCGCCTTCTATCCTAAGTTTACGTATTTGCTAGCGCATATAACTTAGCGGGAAATTATTAATTGACCGGTAGGACAATTTTGTTGCACGTGATGCCTCAATCGTCTGCTTGCTTCCATAGTTAACATGAGGATCCGCAGTACCAACCTCAGCACTTAAGTCCTCAGCGCAGTACCAACTGCAGGATGCCCTTTTTGACGTATTGAATGGCATAATTGCACTGTCACTTTTCGCGCTGTCTCATTTTGGTGCGATGATGAAACTTTCATGAAACGTCTGTAATTTGAAACAAATAACGTAATTCTCGGGATTGGTTTTATTTAAATGACAATGTAAGAGTGGCTTTGTAAGGTATGTGTTGCTCTTAAAATATTTGGATACGACATCCAAAATCTTTTTTCCTTTAAGAGCAGGATATAAGTCGACAAGTTTCTGAAAATCAAAATGGTAGCAACAATAATGCAGACGACAACAACTGTGCTGACGACAGTCGCCGCAATGTCTACTACCTTAGCATCCCATTACATATCTTCGCAAGCTAGTTCCTCGACGAGTGTAACAACAGTAACGACAATAGCGACATCAATACGCTCTACACCGTCTAATCTACTCTTTTCTAATGTGGCGGCTCAGCCAAAATCATCTTCAGCAAGCACAATTGGGCTTTCAATCGGACTTCCCATCGGAATATTCTGTTTCGGATTACTTATCCTTTTGTGTTATTTCTACCTTAAAAGGAATTCGGTGTCCATTTCAAATCCACCCATGTCAGCTACGATTCCAAGGGAAGAGGAATATTGTCGCCGCACTAATTGGTTCTCACGGTTATTTTGGCAGAGTAAGTGTGAGGATCAGAATTCATATTCTAATCGTGATATTGAGAAGTATAACGACACCCAGTGGACCTCGGGTGATAACAAGTCTTCAAAAATACAGTACAAAATTTCCAAACCCATAATACCGCAGCATATACTGACACCTAAGAAAACGGTGAAGAACCCATATGCTTGGTCTGGTAAAAACATTTCGTTAGACCCCAAAGTGAACGAAATGGAGGAAGAGAAAGTTGTGGATGCATTCCTGTATACTAAACCACCGAATATTGTCCATATTGAATCCAGCATCCCCTCGTATAATGATTTACCTTCTCAAAAAACGGTGTCCTCAAAGAAAACTGCGTTAAAAACGAGTGAGAAATGGAGTTACGAATCTCCACTATCTCGATGGTTCTTGAGGGGTTCTACATACTTTAAGGATTATGGCTTATCAAAGACCTCTTTAAAGACCCCAACTGGGGCTCCACAACTGAAGCAAATGAAAATGCTCTCCCGGATAAGTAAGGGTTACTTCAATGAGTCAGATATAATGCCTGACGAACGATCGCCCATCTTGGAGTATAATAACACGCCTCTGGATGCAAATGACAGTGTGAATAACTTGGGTAATACCACGCCAGATTCACAAATCACATCTTATCGCAACAATAACATCGATCTAATCACGGCAAGACCCCATTCAGTGATATACGGTACTACTGCACAACAAACTTTGGAAACCAACTTCAATGATCATCATGACTGCAATAAAAGCACTGAGAAACACGAGTTGATAATACCCACCCCATCAAAACCACTAAAGAAAAGGATATAAAGAAGACAAAGTAAAATGTATCAGCATTTACAACATTTGTCACGTTCTAAACCATTGCCGCTTACTCCAAACTCCAAATATAATGGGGAGGCTTGCGTCCAATTAGGGAAGACATATACAGTTATTCAGGATTACGAGCCTAGATTGACAGACGAAATAAGAATCTCGCTGGGTGAAAAAGTTAAAATTCTGGCCACTCATACCGATGGATGGTGTCTGGTAGAAAAGTGTAATACACAAAAGGGTTCTATTCACGTCAGTGTTGACGATAAAAGATACCTCAATGAAGATAGAGGCATTGTGCCTGGTGACTGTCTCCAAGAATACGACTGATGAAAATAATATTGACGTTCGCATTTAATCTATACCTATAATTCTGTACTTATATACTGTTCCTTAATTGAAGATTTCAACATCGTTTTTGATGTAGGTCTTTTCACCTGGAGGTGCGGCTGGGCTACCGAAGACTAATTGAGCTTGTACGGTCCAAGACTCAGGGATTTTGCTTGGCAAAGCAGCTTTTATGTAACCATTGTAGTGTTGTAGGTGACCACCCAGGCCCATTGCCTCCAAGGCAACCCACGAGTTGATTTGAGCGGCACCAGAGGTATGGTCCGCGAAACTAGGGAATGCAGCTGCGTACGCTGGGAAGTCAGCCTTTAGCTTTTCAGTTACCTTGTCGTCGGTGAAGAAGATTACAGAACCAAAGGCCTCATCCCTTGCTGAAGCAGGCCTCTTTTGACCGGCAGGGCTTTCTATAGCCTTAGTCACTTCGTCCCAAACTTTTTTGTGAGTTTCACCAGTCAAGATAACAGCGCGATTTGGCTGGGAGTTGAAAGCGGTGGGTGTGGCTCGAATGATGGTTTGGACGACGGATTGGATGTCGTTGATAGTAATTTCACCAGGTAACTCCGGTTTCAAAGCGTAAATAGTACGACGAGCAGTTAAAGTTTTCAAATAAGTTGCAACAGCAGACATGATATTGGATTGCCGGAATGGCGATATGTTGATCCCGGATACTTCAGTCTACGAAAAAAGTACAAATTATGTAGTCAGTTCCTTCAGTATGGTGTCCTTATATACTGTAGTTTGGACAAGGTGCAAATGCCAAGACCCTAGCCCGAAAAGCTCGAGGCACCCCAGGATCTTCCCCTTTACGTAATTTTCACGTAAAACGCCACAGTCCGATTTTTCTCGAATAATCATTAGTAAAAGCGGTATACTGGATTATTGTACGATAACAAGGTAGAGCTTTATTACTAAGCTAAGACGTTCTTACATCAATAGTGCTGTTCGTTATTGATGTTAGGAGAAGGAGCGGGTCTGGTGAATAGTGTAAGCAGTGTTTCTGAACTTTTTCTTCGTCTAAGTCCTTGTAATGTAAGGTAAGAATGCAAGCATCTTGTTTGTAACCCGGGTGTACGTTGACGTTAGTAAGTCACAAACCCAAGCTTAACTTCTTCGTGAGGAAGGAAAGTGTTGTCTCCTACTTTTTTCAAATTTTCGAATTGTATTTATATTTATTTAGTACTTCTTGAGTTTACATATCCTTCGTAAAAATGCAACTTTTGTCGAAAAACACTTCCAAAAAAAAATAATAATGAATTTATGAAGCATACTAACGAGCGAGCACATCGCTGACCTATCATTACTTCATGAGATAAATTAAGATCTCCTCATATGCGAATTTCCTGTTCAGTGATAAACGTTGATTACGTTATTGATAAAAGTCTTTTCTTCTGGCAAGGGGTACCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTGCGCGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGTCCTTCAATGAAACATCGTTGGCCACTAATTTGGCCAGTGCAAAGTAGAACAAATCGGCAGCCTCCCAAGAAAGCTCCTTCTTACCCTTTGCCTCAGTCAGTTCTTCAGCTTCTTCCTTGATCTTGGCATCTAACAATGCAGAGTCGTTGAATAGTCTTCTAGTATAAGATTCCTCTGGAGCGTCCTGTAGCCTTTGTTTTAGTAAAGATTCTAGCCCCACCAAACCATGCTTGAATTCACCAAAGCAAGACATGGTCTCCAAGTGGCAAAATCCAACGTTTTCTTGTTCAACGATAAACTTTAAGGCATCCGAATCACAGTCAGTAGAGATTTGTAAAAGCTTTTGGCCATTGCCAGAAGTTTCACCCTTGATCCAGATTTCATTCCTAGAACGAGAATAATAAACGCCACGACCCAATTCGATGGCCTTTGCTATAGATTTCTTCGAAGAATACACCAACCCTAGACAACGCTCATATTGGTCCACAACTAGGGTGGTATATAAACCGTCAGGACGGTCTGTACGTACTTCACCAAGCACTTCTTTGGTCAACATATCCTTGCTTAATTTCTTTATGGACACAATTTTATCTTGCGAGAATTTTTGTTTTACCATGAATTGATTGGAGAAAACACCGTTCTCTTCCACAACAACACGCTCCTTTGGTACATTCAATTGTTCAACCAAGTGTTCGGCTGTTTTAGCATCTTGGCTTGCAATGAACAGAGAAGAAACTCCGTTGTTCAAGAAGGCAATGATTTCATCATCGCTGAATTTACCACTTGGCAAGGACAAAGCCACCAATGGAACTTCTTCCTCTTTGGAGAACTGGAGAATCTCTTCATTACTCAGGCTCGAGCCATCCAAAAGTACCTGACCAACAAGTGAAACGTATTCCTTCTTACTATTCCATGAGGCCAGATCATCAATTAACGGTAGAATCGGCAAAACCATTATTCAGAAAAAAAATTTTGTAAACTATTGTATTACTATTACACAGCGCAGTTGTGCTATGATATTATAATGTATCCAGAACACACATCGGAGGTGAATATAACGTTCCATATCTATTATATACACAGTATACTACTGTTCATAGTCATATCCTTTTCTTACCTTCTATATCGAATGACTGATAATGCAACGTGAGTCACTGTGCATGGGTTTAGCAATTATTAAACTAATTTACCGGAGTCACTATTAGAGTCAGTTCGACTGCCTAGAAGAACTGCTGGTTGTCAGGATTGTGATGGGGGCATTCTGCTGTATTATGACCCATCGTATCGCAATGCTCACACCACTGTTGTCTTCCTGCCGTGGTATCGACTGGTGCAGGGGGGTCGAAAATTGGCAACGATTCCACGGCTGTTTGTGCTTGAGCCTGTTCCAACTGTTTGAACCTTTCATTAGCCTCTTCAAGTTTTTTCGTTAAGGATGCCACCTCTTCCGATGAGGAATCTTGTGGTTTTGTCAAAAATAGTTCCTTGCTCAAATTTTGGTATTCTTTACTGAGCGAATCGTTATGCATTTTCAATTGTTCGCGTTCTTTAGCCCACTTTGTCTTGTGTAACTCAAATTGGTCTTCTATGTTGCGTAATTGTTCCAGCTGTTTTTTCAGGAGCTCGACATCTTCGTTGGCACCAGTGGGTTGATTATGAGAAAGATTTCTCTCTTCGTTTTCTTTGATCTCTTCGTGTAGTTGGCTTACGACAGCAAGTAGCTGTTCATTCTCAGCGTCAAAAAACTGCTTTTGTTTGGCTTGCTGTCTGCGTTCGAGCAGACATTGTTGCTTGAGATGGTCTATCTCTTTCTCTCTTTCTTGTATTGTGGCTTCATACCTATCAAAAGTCGGTTGCACTTCTTCGAGGACCATTCTTTGGTCATCGAGTAGCCTTTTGTAGTGTAGTTGTTTCCTTTGTAGCTTTTCGATGGTCAATTGGCGATCGCGTAATTCAATTGTAACTTCGCTGCTATTGAGGTCATTCATGTGGCCATTGTCCGGTTTCCAATCGCTGGTGGTGTTGTGATTAGCCTTTCTGTCTGATGACAGGATAGAGTCCACCTCCATTCTGTCTTCTCTGTTATCGTAACCAAATTCTTGCTGTTGATGGTGATCCGATGCCTCCTGGTCCATCGACTGTTGATTACCGCTGTGCCGACTGGTGATCCGGAAACTTCTCATGGGTGTGGGGGATTTAGGATCATCCATGGGAGAGAAGCGCTTAGTGAGCCTCACAATAGATCTGTTCACGGGTATTGATAGCGGTTCCATTGTCGTTCTTCTCGAGGTTTGCCATATCGGTCCGTTCTCGATCAATGATGCGACTTTTTGCAACTGAATAAATAGTCCACTTTGAGGATACTCCGTTTGAAAATACTTCTTCCCCTAGGAATGATCCATCGTTCTTACCAATGTTGGCAAGTAAGTCTACACCAGCAAACATTCCACGCGTCGTGTCCACTGGACCCACGTATTTCAGTTGTCCGCGGCCGAAATTTGGGATTTGGTTTAAACATCCTATCTTTCTTTGATATCTATCCATGGTATATTAAGCGCATACGGCGCCAGCCACTAGTGCAGTACCAACCTCAGCACTTAAGTCCTCAGCGCAGTACCAAGCATGCCTCGTCCAAACGTTTTTGGTGTCTTGGCCAATTGCCCTTCTGAAAAATCTCACTGTCCGCAACTCATTAAAAGATACCCAAGCAAGCTACACGATAAAGAAAGGAGAAAGTTCATTACTGGAACGTACATATAGCGATACAAACGTATAGCAAAGATCTGAAATGGATACGGATAAGTTAATCTCAGAGGCTGAGTCTCATTTTTCTCAAGGAAACCATGCAGAAGCTGTTGCGAAGTTGACATCCGCAGCTCAGTCGAACCCCAATGACGAGCAAATGTCAACTATTGAATCATTAATTCAAAAAATCGCAGGATACGTCATGGACAACCGTAGTGGTGGTAGTGACGCCTCGCAAGATCGTGCTGCTGGTGGTGGTTCATCTTTTATGAACACTTTAATGGCAGACTCTAAGGGTTCTTCCCAAACGCAACTAGGAAAACTAGCTTTGTTAGCCACAGTGATGACACACTCATCAAATAAAGGTTCTTCTAACAGAGGGTTTGACGTAGGGACTGTCATGTCAATGCTAAGTGGTTCTGGCGGCGGGAGCCAAAGTATGGGTGCTTCCGGCCTGGCTGCCTTGGCTTCTCAATTCTTTAAGTCAGGTAACAATTCCCAAGGTCAGGGACAAGGTCAAGGTCAAGGTCAAGGTCAAGGACAAGGTCAAGGTCAAGGTTCTTTTACTGCTTTGGCGTCTTTGGCTTCATCTTTCATGAATTCCAACAACAATAATCAGCAAGGTCAAAATCAAAGCTCCGGTGGTTCCTCCTTTGGAGCACTGGCTTCTATGGCAAGCTCTTTTATGCATTCCAATAATAATCAGAACTCCAACAATAGTCAACAGGGCTATAACCAATCCTATCAAAACGGTAACCAAAATAGTCAAGGTTACAATAATCAACAGTACCAAGGTCGCGACGGTGGTTACCAACAACAACAGGGACAATCTGGTGGTGCTTTTTCCTCATTGGCCTCCATGGCTCAATCTTACTTAGGTGGTGGACAAACTCAATCCAACCAACAGCAATACAATCAACAAGGCCAAAACAACCAGCAGCAATACCAGCAACAAGGCCAAAACTATCAGCATCAACAACAGGGTCAGCAGCAGCAACAAGGCCACTCCAGTTCATTCTCAGCTTTGGCTTCCATGGCAAGTTCCTACCTGGGCAATAACTCCAATTCAAATTCGAGTTATGTGTACACGCAACAGGCTAATGAGTATGGTAGACCGCAACAGAATGGTCAACAGCAATCCAATGAGTACGGAAGACCGCAATACGGCGGAAACCAGAACTCCTAAGGACAGCACGAATCCTTCAATTTTTCTGGCAACTTTTCTCAACAGAACAATAACGGCGCGCCGAACCGCTACTGAACGATGATTCAGTTCGCCTTCTATCCTAAGTTTACGTATTTGCTAGCGCATATAACTTAGCGGGAAATTATTAATTGACCGGTAGGACAATTTTGTTGCACGTGATGCCTCAATCGTCTGCTTGCTTCCATAGTTAACATGAGGATCCTTTCAAAACAGAGTTGTATCTCTGCAGGATGCCCTTTTTGACGTATTGAATGGCATAATTGCACTGTCACTTTTCGCGCTGTCTCATTTTGGTGCGATGATGAAACTTTCATGAAACGTCTGTAATTTGAAACAAATAACGTAATTCTCGGGATTGGTTTTATTTAAATGACAATGTAAGAGTGGCTTTGTAAGGTATGTGTTGCTCTTAAAATATTTGGATACGACATCCAAAATCTTTTTTCCTTTAAGAGCAGGATATAAGTCGACAAGTTTCTGAAAATCAAAATGGTAGCAACAATAATGCAGACGACAACAACTGTGCTGACGACAGTCGCCGCAATGTCTACTACCTTAGCATCCCATTACATATCTTCGCAAGCTAGTTCCTCGACGAGTGTAACAACAGTAACGACAATAGCGACATCAATACGCTCTACACCGTCTAATCTACTCTTTTCTAATGTGGCGGCTCAGCCAAAATCATCTTCAGCAAGCACAATTGGGCTTTCAATCGGACTTCCCATCGGAATATTCTGTTTCGGATTACTTATCCTTTTGTGTTATTTCTACCTTAAAAGGAATTCGGTGTCCATTTCAAATCCACCCATGTCAGCTACGATTCCAAGGGAAGAGGAATATTGTCGCCGCACTAATTGGTTCTCACGGTTATTTTGGCAGAGTAAGTGTGAGGATCAGAATTCATATTCTAATCGTGATATTGAGAAGTATAACGACACCCAGTGGACCTCGGGTGATAACAAGTCTTCAAAAATACAGTACAAAATTTCCAAACCCATAATACCGCAGCATATACTGACACCTAAGAAAACGGTGAAGAACCCATATGCTTGGTCTGGTAAAAACATTTCGTTAGACCCCAAAGTGAACGAAATGGAGGAAGAGAAAGTTGTGGATGCATTCCTGTATACTAAACCACCGAATATTGTCCATATTGAATCCAGCATCCCCTCGTATAATGATTTACCTTCTCAAAAAACGGTGTCCTCAAAGAAAACTGCGTTAAAAACGAGTGAGAAATGGAGTTACGAATCTCCACTATCTCGATGGTTCTTGAGGGGTTCTACATACTTTAAGGATTATGGCTTATCAAAGACCTCTTTAAAGACCCCAACTGGGGCTCCACAACTGAAGCAAATGAAAATGCTCTCCCGGATAAGTAAGGGTTACTTCAATGAGTCAGATATAATGCCTGACGAACGATCGCCCATCTTGGAGTATAATAACACGCCTCTGGATGCAAATGACAGTGTGAATAACTTGGGTAATACCACGCCAGATTCACAAATCACATCTTATCGCAACAATAACATCGATCTAATCACGGCAAGACCCCATTCAGTGATATACGGTACTACTGCACAACAAACTTTGGAAACCAACTTCAATGATCATCATGACTGCAATAAAAGCACTGAGAAACACGAGTTGATAATACCCACCCCATCAAAACCACTAAAGAAAAGGATATAAAGAAGACAAAGTAAAATGTATCAGCATTTACAACATTTGTCACGTTCTAAACCATTGCCGCTTACTCCAAACTCCAAATATAATGGGGAGGCTTGCGTCCAATTAGGGAAGACATATACAGTTATTCAGGATTACGAGCCTAGATTGACAGACGAAATAAGAATCTCGCTGGGTGAAAAAGTTAAAATTCTGGCCACTCATACCGATGGATGGTGTCTGGTAGAAAAGTGTAATACACAAAAGGGTTCTATTCACGTCAGTGTTGACGATAAAAGATACCTCAATGAAGATAGAGGCATTGTGCCTGGTGACTGTCTCCAAGAATACGACTGATGAAAATAATATTGACGTTCGCATTTAATCTATACCTATAATTCTGTACTTATATACTGTTCCTTAATTGAAGATTTCAACATCGTTTTTGATGTAGGTCTTTTCACCTGGAGGTGCGGCTGGGCTACCGAAGACTAATTGAGCTTGTACGGTCCAAGACTCAGGGATTTTGCTTGGCAAAGCAGCTTTTATGTAACCATTGTAGTGTTGTAGGTGACCACCCAGGCCCATTGCCTCCAAGGCAACCCACGAGTTGATTTGAGCGGCACCAGAGGTATGGTCCGCGAAACTAGGGAATGCAGCTGCGTACGCTGGGAAGTCAGCCTTTAGCTTTTCAGTTACCTTGTCGTCGGTGAAGAAGATTACAGAACCAAAGGCCTCATCCCTTGCTGAAGCAGGCCTCTTTTGACCGGCAGGGCTTTCTATAGCCTTAGTCACTTCGTCCCAAACTTTTTTGTGAGTTTCACCAGTCAAGATAACAGCGCGATTTGGCTGGGAGTTGAAAGCGGTGGGTGTGGCTCGAATGATGGTTTGGACGACGGATTGGATGTCGTTGATAGTAATTTCACCAGGTAACTCCGGTTTCAAAGCGTAAATAGTACGACGAGCAGTTAAAGTTTTCAAATAAGTTGCAACAGCAGACATGATATTGGATTGCCGGAATGGCGATATGTTGATCCCGGATACTTCAGTCTACGAAAAAAGTACAAATTATGTAGTCAGTTCCTTCAGTATGGTGTCCTTATATACTGTAGTTTGGACAAGGTGCAAATGCCAAGACCCTAGCCCGAAAAGCTCGAGGCACCCCAGGATCTTCCCCTTTACGTAATTTTCACGTAAAACGCCACAGTCCGATTTTTCTCGAATAATCATTAGTAAAAGCGGTATACTGGATTATTGTACGATAACAAGGTAGAGCTTTATTACTAAGCTAAGACGTTCTTACATCAATAGTGCTGTTCGTTATTGATGTTAGGAGAAGGAGCGGGTCTGGTGAATAGTGTAAGCAGTGTTTCTGAACTTTTTCTTCGTCTAAGTCCTTGTAATGTAAGGTAAGAATGCAAGCATCTTGTTTGTAACCCGGGTGTACGTTGACGTTAGTAAGTCACAAACCCAAGCTTAACTTCTTCGTGAGGAAGGAAAGTGTTGTCTCCTACTTTTTTCAAATTTTCGAATTGTATTTATATTTATTTAGTACTTCTTGAGTTTACATATCCTTCGTAAAAATGCAACTTTTGTCGAAAAACACTTCCAAAAAAAAATAATAATGAATTTATGAAGCATACTAACGAGCGAGCACATCGCTGACCTATCATTACTTCATGAGATAAATTAAGATCTCCTCATATGCGAATTTCCTGTTCAGTGATAAACGTTGATTACGTTATTGATAAAAGTCTTTTCTTCTGGCAAGGGGTACCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTGCGCGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACOligonucleotides for undamaged DNA or DNA containing an abasic site or CPD were synthesized and purified with 21\u00a0\u03bcL Nt.BbvCI at 37\u00b0C for 2 h. A further 5\u00a0\u03bcL Nt.BbvCI (50\u00a0U) were then added per tube and digested for a further 1 h. Complete nicking to open circular DNA from covalently closed DNA was confirmed by agarose gel electrophoresis in the presence of ethidium bromide , contain2 with 110\u00a0U T4 DNA ligase (New England Biolabs M0202M) per \u03bcg plasmid at 16\u00b0C overnight in the dark or AleI (for studying a lagging strand template CPD and undamaged controls), with the following exceptions. In Template linearization was performed in all cases by mixing 8-10\u00a0\u03bcg plasmid DNA in 50\u00a0\u03bcL final reaction volume containing 1X\u00a0CutSmart Buffer (New England Biolabs B7204), 2.5\u00a0\u03bcL each restriction enzyme and incubating at\u00a037\u00b0C for 3 h. The DNA was extracted with phenol:chloroform:isoamyl alcohol 25:24:1 saturated with TE (Sigma-Aldrich P2069) and the aqueous phase collected and DNA precipitated with 0.3\u00a0M NaCl\u00a0+ 2.8 volumes ice cold 100% ethanol in dry ice. The pellet\u00a0was harvested, washed with room temperature 70% ethanol, harvested, air-dried and resuspended in 15-20\u00a0\u03bcL TE for use in replication assays.2 with 233\u00a0U T4 DNA ligase (New England Biolabs M0202M) per \u03bcg plasmid at 16\u00b0C overnight in the dark. The following day, SDS (to 0.1%) and proteinase K (1/100 volumes) were added and incubated at 37\u00b0C for 20\u00a0min. The DNA was extracted with phenol:chloroform:isoamyl alcohol 25:24:1 saturated with TE (Sigma-Aldrich P2069) and the aqueous phase collected. Ligated template DNA was isolated from oligonucleotides by passing the aqueous phase over Sephacryl S-400 High Resolution matrix columns (prepared by applying 800\u00a0\u03bcL 50% slurry to 0.8\u00a0mL Pierce Centrifuge Columns (Thermo Scientific 89868) and washing 2\u00a0\u00d7 250\u00a0\u03bcL TE by centrifuging at 700 g for 1\u00a0min). 35-40\u00a0\u03bcL was applied per column and collected by centrifuging at 700 g for 2\u00a0min. DNA was pooled, frozen, lyophilized in a vacuum concentrator and resuspended to 250-500\u00a0ng/\u03bcl in TE.Undamaged (maxi prep) and CPD-containing plasmid DNA were linearized with SapI restriction enzyme (New England Biolabs R0569), which cuts approximately 0.5 kb right of ARS306. Biotinylated oligonucleotides , 2\u00a0M NaCl, 1\u00a0mM EDTA) then mixed with 4\u00a0\u03bcg DNA\u00a0+ buffer A to a total volume of 80\u00a0\u03bcl, and incubated at 25\u00b0C for 30\u00a0min, shaking at\u00a0500\u00a0rpm. The supernatant was discarded and the beads washed with 2\u00a0\u00d7 100\u00a0\u03bcL buffer B (10\u00a0mM HEPES-KOH (pH 7.6), 1\u00a0M KCl, 1\u00a0mM EDTA), 2\u00a0\u00d7 100\u00a0\u03bcL buffer C (10\u00a0mM HEPES-KOH (pH 7.6), 1\u00a0mM EDTA), resuspended in 75\u00a0\u03bcL buffer C and stored at 4\u00b0C in the dark.32P]-ATP using 10\u00a0U T4 Polynucleotide Kinase (New England Biolabs M0201S) in total volume 20\u00a0\u03bcL at 37\u00b0C for 1 h. The DNA was extracted with phenol:chloroform:isoamyl alcohol 25:24:1 saturated with TE (10\u00a0mM Tris-HCl (pH 8.0), 1\u00a0mM EDTA) (Sigma-Aldrich P2069) and the buffer exchanged and unincorporated nucleotides were removed from the aqueous phase with illustra MicroSpin G-50 columns . These markers were run as size standards in all gels and cropped from the final image for presentation.Molecular weight standards were prepared by first dephosphorylating 12.5\u00a0\u03bcg (25\u00a0\u03bcl) \u03bb DNA-HindIII Digest (New England Biolabs) with 10\u00a0U Antarctic Phosphatase (New England Biolabs M0289S) in total volume 30\u00a0\u03bcL at 37\u00b0C for 1 h. The DNA was then purified using a QIAquick PCR Purification Kit (QIAGEN) and eluted in 30\u00a0\u03bcL water. 1\u00a0\u03bcg of this DNA was labeled with \u03b3--dCTP, 12.5\u00a0nM Cdt1/Mcm2-7, 7.5\u00a0nM Cdc6, 3.3\u00a0nM ORC, 8.3\u00a0nM DDK, 20\u00a0nM S-CDK. In reactions omitting Pol \u03b4, 117\u00a0mM potassium glutamate was used in all experiments , 100\u00a0mM potassium glutamate, 0.01% NP-40-S, 1\u00a0mM DTT, 10\u00a0mM Mg(OAc)eriments , except eriments F or 4\u00a0mieriments F and S5Geriments were addFor reactions not requiring post-quenching restriction enzyme digests or native gel analysis, unincorporated nucleotides were removed from the quenched reaction mix with illustra MicroSpin G-50 columns , which also exchanged the buffer to TE. Samples were supplemented with 20\u00a0mM EDTA, and 1/10 volume alkaline loading dye added. For all other reactions, SDS (to 0.1%) and proteinase K (1/100 volumes) were added and incubated at 37\u00b0C for 20\u00a0min. The DNA was extracted with phenol:chloroform:isoamyl alcohol 25:24:1 saturated with TE (10\u00a0mM Tris-HCl (pH\u00a08.0), 1\u00a0mM EDTA) (Sigma-Aldrich P2069) and the buffer exchanged and unincorporated nucleotides were removed from the aqueous phase with illustra MicroSpin G-50 columns . Except where indicated, samples for native gels were not digested with restriction enzymes, and mixed with native loading dye (20\u00a0mM Tris-HCl (pH 8.0), 50\u00a0mM EDTA, 10% Ficoll 400, 2% N-Lauroylsarcosine sodium salt solution) without additional processing. For restriction enzyme digests for the products of a standard 5-10\u00a0\u03bcL replication assay sample, 0.5-1\u00a0\u03bcL enzyme was added in 1X CutSmart (New England Biolabs B7204). Enzyme (2-4\u00a0\u03bcl) and buffer volumes were scaled up for larger samples for 2D gels. Digests were performed at 37\u00b0C for 30\u00a0min, except SmaI which was incubated at 25\u00b0C. Digests were quenched with EDTA, samples split where appropriate, and added to native or alkaline loading dye.Denatured samples were analyzed in denaturing 0.6% agarose gels run at 24\u00a0V overnight in 30\u00a0mM NaOH, 2\u00a0mM EDTA. Native samples were analyzed in vertical 1% agarose gels run at 21-24\u00a0V overnight in modified 1X TAE (50\u00a0mM Tris-HCl (pH 7.9), 40\u00a0mM NaOAC, 1\u00a0mM EDTA (pH 8.0)), except for 2, 100\u00a0mM KOAc, 0.1% NP-40-S, 5% glycerol, 0.1\u00a0mg/ml BSA) on ice for 10\u00a0min. ATP (3\u00a0mM), creatine phosphate (40\u00a0mM) and creatine phosphate kinase (140\u00a0\u03bcg/ml) were added and chromatin assembly initiated by addition of DNA and transferring the reactions to 30\u00b0C for 1 h.Chromatin was assembled on 3\u00a0nM (20\u00a0ng/\u03bcl) AhdI- or AleI-linearized DNA in 40\u00a0\u03bcL total reaction volume. Nap1 (3\u00a0\u03bcM), histone octamers (370\u00a0nM) and ISW1 (30\u00a0nM) were pre-incubated in chromatin assembly buffer (25\u00a0mM HEPES-KOH (pH 7.6), 10\u00a0mM Mg(OAc)2, 40\u00a0mM KCl) and collecting by centrifuging at 700 g for 2\u00a0min. For helicase loading and phosphorylation, a volume of chromatin corresponding to the total reaction volume less the contribution from ATP, BSA, Cdt1/Mcm2-7, Cdc6, ORC and DDK was used. Chromatin replication was otherwise performed as for naked DNA templates except where noted in the figures and with the following exceptions: Sld2 was used at 30\u00a0nM; Pol \u03b4 was used at 10\u00a0nM in To prepare chromatin for replication assays, chromatin was exchanged to replication assay buffer by applying 40\u00a0\u03bcL chromatin assembly reaction to Sephacryl S-400 High Resolution matrix columns (prepared by applying 800\u00a0\u03bcL 50% slurry to 0.8\u00a0mL Pierce Centrifuge Columns (Thermo Scientific 89868) and washing with 3\u00a0\u00d7 250\u00a0\u03bcL 25\u00a0mM HEPES-KOH (pH 7.6), 100\u00a0mM potassium glutamate, 0.01% NP-40-S, 1\u00a0mM DTT, 10\u00a0mM Mg(OAc)2 was added to 5\u00a0mM. 20\u00a0\u03bcL was collected and 1\u00a0\u03bcL 20-fold diluted MNase (100\u00a0U) added and the mixture incubated at 37\u00b0C for 5\u00a0min. The reaction was stopped with 20\u00a0mM EGTA. 5 volumes of buffer PB were added and the mixture applied to a QIAquick spin column, washed with buffer PE and eluted in 35\u00a0\u03bcL water. Products were analyzed by 1.5% agarose gel electrophoresis.After chromatin assembly CaCl32P]-dCTP. In 2, 0.1\u00a0mg/ml BSA, 3\u00a0mM ATP, 400\u00a0\u03bcM CTP, GTP, UTP, 30\u00a0\u03bcM dATP, dCTP, dGTP, dTTP, 66\u00a0nM \u03b1-[32P]-dCTP, 20\u00a0nM Pol \u03b5, 5\u00a0nM Mcm10, 20\u00a0nM Ctf4, 20\u00a0nM Csm3/Tof1, 20\u00a0nM Mrc1, 20\u00a0nM RFC, 20\u00a0nM PCNA, 10\u00a0nM TopoI, 20\u00a0nM Pol \u03b1. In Helicase loading and phosphorylation was performed by incubating equal volumes of DNA-bound Dynabead slurry in buffer C (prepared as described above) with a mixture containing reaction buffer components, proteins and ATP to give a final reaction mix as used for helicase loading and phosphorylation on a standard soluble template, but without KCl. After treatment with S-CDK as described above, an appropriate volume of the mixture was collected such that it would be diluted 5-fold in the final reaction mix. Replication was initiated as described above but in the absence of \u03b1--dCTP, 20\u00a0nM Pol \u03b1 and RPA (0-400\u00a0nM). RPA was first pre-bound to the template in the absence of Pol \u03b1 for 10\u00a0min. Reactions were initiated by addition of Pol \u03b1 and were incubated for 20\u00a0min. Samples were processed and run through alkaline agarose gels as described for soluble replication reactions.Primed template was prepared by annealing oligonucleotide JY180 (500\u00a0nM) (sequence: Denaturing gels were fixed with two changes of cold 5% trichloroacetic acid and dried onto chromatography paper (Whatman). Native gels were dried directly onto chromatography paper. Most gels were autoradiographed with Amersham Hyperfilm MP for presentation. For quantification, gels were exposed on BAS-IP MS Storage Phosphor Screens and screens were developed on a Typhoon phosphorimager .Replication product quantification was performed in ImageJ after converting the .gel files to 16-Bit Tiff files using the Linearize GelData command. Lane profiles shown in In all cases, graphs of quantified data show the mean and errors (SEM) from n (stated in the figure legends) independent experimental repeats."} +{"text": "DEDD), which is overexpressed in\u2009>\u200960% of\u00a0TNBCs, drives a mitogen-independent G1/S cell cycle transition through cytoplasm localization. The gain of cytosolic DEDD enhances cyclin D1 expression by interacting with heat shock 71\u2009kDa protein 8 (HSC70). Concurrently, DEDD interacts with Rb family proteins and promotes their proteasome-mediated degradation. DEDD overexpression renders TNBCs vulnerable to cell cycle inhibition. Patients with\u00a0TNBC have been excluded from CDK 4/6 inhibitor clinical trials due to the perceived high frequency of Rb-loss in TNBCs. Interestingly, our study demonstrated that, irrespective of Rb status, TNBCs with DEDD overexpression exhibit a DEDD-dependent vulnerability to combinatorial treatment with\u00a0CDK4/6 inhibitor and EGFR inhibitor in vitro and in vivo. Thus, our study provided a rationale for the clinical application of CDK4/6 inhibitor combinatorial regimens for patients with\u00a0TNBC.Lacking targetable molecular drivers, triple-negative breast cancer (TNBC) is the most clinically challenging subtype of\u00a0breast cancer. In this study, we reveal that\u00a0Death Effector Domain-containing DNA-binding protein ( The use of of CDK4/6 inhibitors to treat patients with\u00a0TNBC\u00a0is limited by loss of\u00a0Rb. Here, the authors show that a combination of CDK4/6 inhibitor and EGFR inhibitor is effective against DEDD-overexpressing\u00a0TNBC, independent of Rb status. Despite the heterogeneous nature of the tumor, targeted anti-cancer therapies have achieved clinical success through the exploitation of functionally essential genomic alternations, or tumor-specific vulnerabilities3. In the past decade, a compendium of the conceptual frameworks explaining mechanisms of tumor vulnerabilities has been proposed and experimentally validated, including oncogene/non-oncogene addiction4, synthetic lethality5, collateral lethality6, and synthetic essentiality7. A successful clinical translation of cancer genome studies project)) for effective targeted anti-cancer regimens relies on two prerequisites: (1) tumor-specific genetic or epigenetic events that confer unique vulnerabilities (the Achilles\u2019 heel of cancer) to therapeutic targeting; (2) prognostic biomarkers coupled with an accessible clinical assay for selecting patients who will most likely respond to the targeted therapy.Cancer is a highly heterogeneous genetic disease8. Estrogen receptor (ER)-targeting agents and human epidermal growth factor receptor 2 (HER2)-targeting therapeutics have become first-line treatments for ER-positive breast cancer and HER2-positive breast cancer, respectively. Extensive pre-clinical and clinical studies have demonstrated that biomarker-based patient selection for targeted therapies has led to a significant improvement in cancer treatment with reduced side effects compared to traditional chemotherapies9. Despite such clinical success, targeted therapy options for the most aggressive breast cancer subtype, triple-negative breast cancer (TNBC), remains limited. Because TNBC lack established therapeutic targets , non-specific chemotherapy remains the primary treatment option for TNBC patients. TNBC exhibits a higher level of genome instability and a distinct mutational landscape11. Despite the highly heterogeneous nature of TNBC genome landscapes, some TNBC-specific tumor vulnerabilities are emerging as clinically translatable targets for TNBC patients. For example, poly-ADP ribose polymerase (PARP) was identified to have a synthetic lethal dependency on BRCA1 mutation and has been targeted in TNBC clinical trials, ultimately resulting in recent Food and Drug Administration (FDA) approval of PARP inhibitors for TNBC12. Additionally, bromodomain and extra-terminal (BET) protein13 and the pyrimidine synthesis pathway14 have shown promise to be targetable TNBC vulnerabilities.Breast cancer targeted therapies are among the most successful examples of applying the concept of targeting tumor-specific vulnerabilitiesIn this study, we employed a pooled whole-genome RNA interference (RNAi) functional screen in TNBC cells to explore potential synthetic lethal pathways that synergize with epidermal growth factor receptor (EGFR)/HER2 inhibitor lapatinib (LAP).DEDD) as a potential vulnerability from our screen. Interestingly, DEDD is significantly upregulated in >60% of TNBC tumors. While DEDD has been known to function as a pro-apoptotic protein in the nucleus15, we found that DEDD is strongly expressed in the cytosol of tumor cells. Mechanistically, cytosolic DEDD promotes G1/S cell cycle transition through multiple mechanisms. First, DEDD interacts with heat-shock cognate 71\u2009kDa protein (HSC70) to enhance cyclin D1 expression. Second, overexpressed cytosolic DEDD promotes the proteasome-mediated degradation of retinoblastoma (Rb) family proteins to enable G1/S transition. Addicted to an accelerated G1/S cell cycle progression, tumor cells with DEDD overexpression exhibit an increased susceptibility to the combinatorial treatment of cyclin-dependent kinases 4/6 (CDK4/6) and EGFR inhibitors. Furthermore, a combinatorial regimen of CDK4/6 and EGFR inhibitors synergistically inhibited the progression of TNBC xenografts and patient-derived xenograft (PDX) in vivo. These pre-clinical results provide a strong rationale to extend recently FDA-approved CDK4/6 inhibitors to TNBC patients.Unexpectedly, we identified death effector domain-containing protein were potentially critical for cell survival are dysregulated (amplification and messenger RNA (mRNA) upregulation) in more than 40% of TNBC tumors are\u00a0located close together on chromosome 1q23.3\u201342.1 -containing proteins. Without known enzymatic activity, DEDD executes its biological function primarily through protein\u2013protein interactions via its DED domain18. Previous studies suggested that DEDD may interact with cyclin B1, decrease Cdk1/cyclin B1 activity, and regulate cell size during pre-mitosis phases by facilitating the G1-phase rRNA synthesis19. However, most studies have focused on the capacity of DEDD to promote apoptosis through partnering with other DED-containing proteins20. Since DEDD is involved in pro-apoptotic processes, it is thought to have tumor suppressor activities21. Paradoxically, DEDD is aberrantly overexpressed in TNBC than TNBC cells with moderate DEDD expression (HCC1806) incorporation assay. We found that knocking down DEDD significantly decreased EdU incorporation in HCC38 and MDA-MB-468 TNBC cells for 18\u2009h, and then released cell cycle inhibition by replacing nocodazole media with a fresh culture medium. We found that DEDD protein peaked in cells at the G1 phase, but not other cell cycle phases Fig.\u00a0, suggestlls Fig.\u00a0, suggestion Fig.\u00a0, suggestDEDD nuclear localization leads to caspase activation, which induces apoptosis, and abolishing the nuclear translocation of DEDD decreases the apoptosis-promoting potential of the protein20. Interestingly, immunohistochemistry (IHC) staining for DEDD in TNBC tissue samples showed that DEDD primarily localizes to the cytoplasm of tumor cells that have high DEDD expression DEDD or \u0394NLS-DEDD in DEDD-diploid normal cell lines including 293FT and normal human breast epithelial cells (HMECs) 22. Ectopic overexpression of WT DEDD promoted G1\u2013S transition in 293FT cells, which could be significantly diminished by preventing DEDD nuclear-cytosol shuttling using nuclear export inhibitor leptomycin (lepto) was sufficient to override the G1/S checkpoint and increase the proportion of cells in S phase independent of mitogen activation (serum starvation) as determined by increased EdU incorporation Fig.\u00a015. Then,to) Fig.\u00a0. This obion Fig.\u00a0. Additioion Fig.\u00a0. Cyclin wal Fig.\u00a0. InteresDEDD executes its functions\u00a0through protein-protein interactions23. To explore interaction partners of DEDD in the cytosol, we immunoprecipitated the \u0394NLS-DEDD protein complex and analyzed co-immunoprecipitated proteins by mass spectrometry. Interestingly, multiple peptides representing the HSC70 were identified expression, which phenocopied the Rb protein expression pattern in Rb-WT TNBC cell lines and RBL2 (p130), were reported to be CDK4/6 substratesnes Fig.\u00a0. MoreoveEDD Fig.\u00a0. We furtDEDD, we hypothesized that gain-of-function DEDD in TNBC promotes mitogen-independent G1/S transition and confers addiction to an accelerated cell cycle program, thereby creating a tumor-specific vulnerability for therapeutic targeting. CDK4/6 inhibitors show significant clinical efficacy in cancer treatment by targeting the central G1/S transition machinery29. Three CDK4/6 inhibitors have recently received FDA approval for ER\u2009+\u2009breast cancer, including albociclib (PALBO)\u00a0, ribociclib (RIBO)\u00a0, and abemaciclib (ABE) . Expression of WT Rb, the perceived CDK4/6 downstream target, is currently considered a prerequisite for the clinical anti-cancer efficacy of CDK4/6 inhibitors30.Based on the multifaceted cell cycle-promoting mechanisms of 31, TNBC patients are excluded from CDK4/6 inhibitor clinical trials. Interestingly, we found that targeting CDK4/6 in combination with LAP in DEDD-overexpressed TNBC significantly and synergistically inhibited cell proliferation in both Rb-WT and Rb-deficient (MDA-MB-468) TNBC cells rendered the KTB cells resistant to LAP mono treatment, confirming the proliferation-driving function of cytoplasmic DEDD. Interestingly, KTB cells with overexpressed DEDD were more responsive to CDK4/6 inhibitor treatment and Combo treatment could further shift the sensitivity of TNBC cells to combo treatment. Overexpression of \u0394NLS-DEDD in MDA-MB-436 cells, which moderately express DEDD is a transcription factor and has been shown to be frequently upregulated in multiple cancers and linked to sustained Akt signalingnes Fig.\u00a0. We nextent Fig.\u00a0. Ectopicent Fig.\u00a0, right. ent Fig.\u00a0. This fuEDD Fig.\u00a0, signifiEDD Fig.\u00a0, Left, slls Fig.\u00a0, right. lls Fig.\u00a0. To testlls Fig.\u00a0. Notablylls Fig.\u00a0.Fig. 6CyDEDD expression and localization as well as the sensitivity of TNBC cells to Combo treatment in vivo. First, we examined DEDD expression and localization in xenograft breast tumors derived from two TNBC cell lines and two TNBC PDXs that were described previously35. IHC analysis of DEDD showed heterogeneous but strong DEDD expression that was localized predominately to the cytoplasm of epithelial cells within the tumors transformation in soft-agar assays (data not shown). Likewise, DEDD did not influence overall survival and disease-free survival of TNBC patients who received traditional chemotherapies /AKT pathway through its lipid phosphatase activity, while a nuclear pool of PTEN maintains chromosomal stability41. DEDD has been well documented to be a nuclear-targeting protein through its three nuclear localization sequences18. DEDD functions as an apoptosis facilitator in the nucleus by interacting with caspase-8 and caspase-10, and stimulating caspase-dependent cell death42. Surprisingly, once overexpressed in TNBC cells, DEDD primarily resides in the cytoplasm phosphorylates Rb on Ser567 in response to genotoxic stress. Hyperphosphorylation of Rb leads to its interaction with E3 ligases Hdm248. However, the functional relevance of the above in relation to observations to tumorigenesis in vivo has not been addressed. In this study, we discovered that aberrant overexpression of DEDD greatly accelerated ubiquitination and proteasomal degradation of Rb family proteins, including Rb protein and Rb family protein RBL1 in TNBC cells is a transcription target of FoxM1, and FoxM1 promotes breast cancer tumorigenesis by transcriptionally activating PDGFA, which led to further Akt phosphorylation in breast cancer cells54. Supporting such findings, we observed that phosphorylation and total expression of FoxM1 were effectively blocked by CDK4/6 inhibitors and palbociclib (P-7766) were purchased from LC Laboratories. Abemaciclib (HY-16297), ribociclib (HY-15777), and nocodazole (HY-13520) were purchased from MedchemExpress Inc. Leptomycin B (9676S) was purchased from Cell Signaling Technology. MG-132 (80053-196) was purchased from VWR. CHX (01810) was purchased from Sigma-Aldrich. The 293FT (R70007) cells were purchased from Thermo Fisher Scientific Company. BT-20, BT-474, HCC1806, HCC1937, HCC38, MDA-MB-231, MDA-MB-436, and MDA-MB-468 cell lines were obtained from ATCC . The 293FT cell line was cultured in Dulbecco\u2019s modified Eagle\u2019s medium (DMEM) high glucose supplemented with 10% FBS and 1% penicillin\u2013streptomycin (Pen-Strep). BT-20, BT-474, MDA-MB-231, MDA-MB-436, and MDA-MB-468 cell lines were cultured in DMEM/Ham\u2019s F-12 (1:1) supplemented with 10% FBS and 1% Pen-Strep. HCC1806, HCC1937, and HCC38 cell lines were cultured in RPMI-1640 medium supplemented with 10% FBS and 1% Pen-Strep. The MCF-10A was originally purchased from ATCC and cultured in DMEM/F-12 medium supplemented with 5% horse serum (Invitrogen), 20\u2009ng/mL EGF, 10\u2009\u00b5g/mL insulin, 0.5\u2009\u00b5g/mL hydrocortisone, 100\u2009ng/mL cholera toxin, and 1% Pen-Strep. Normal human mammary epithelial lines from Komen Tissue Bank (KTB) were obtained from our collaborator Dr. Nakshatri and cultured in DMEM/F-12 supplemented with 10% FBS, 0.4\u2009\u03bcg/mL hydrocortisone, 5\u2009\u03bcg/mL insulin, 20\u2009ng/mL EGF, 24\u2009mg/L adenine, and 5\u2009\u03bcM ROCK inhibitor (Y-27632). All cell lines were maintained at 37\u2009\u00b0C in a 5% CODEDD complementary DNA (cDNA) sequence It was obtained from the UCSC Genome Browser.Human DEDD coding sequence:5\u2032-ATGGCGGGCCTAAAGCGGCGGGCAAGCCAGGTGTGGCCAGAAGAGCATGGTGAGCAGGAACATGGGCTGTACAGCCTGCACCGCATGTTTGACATCGTGGGCACTCATCTGACACACAGAGATGTGCGCGTGCTTTCTTTCCTCTTTGTTGATGTCATTGATGACCACGAGCGTGGACTCATCCGAAATGGACGTGACTTCTTATTGGCACTGGAGCGCCAGGGCCGCTGTGATGAAAGTAACTTTCGCCAGGTGCTGCAGCTGCTGCGCATCATCACTCGCCACGACCTGCTGCCCTACGTCACCCTCAAGAGGAGACGGGCTGTGTGCCCTGATCTTGTAGACAAGTATCTGGAGGAGACATCAATTCGCTATGTGACCCCCAGAGCCCTCAGTGATCCAGAACCAAGGCCTCCCCAGCCCTCTAAAACAGTGCCTCCCCACTATCCTGTGGTGTGTTGCCCCACTTCGGGTCCTCAGATGTGTAGCAAGCGGCCAGCCCGAGGGAGAGCCACACTTGGGAGCCAGCGAAAACGCCGGAAGTCAGTGACACCAGATCCCAAGGAGAAGCAGACATGTGACATCAGACTGCGGGTTCGGGCTGAATACTGCCAGCATGAGACTGCTCTGCAGGGCAATGTCTTCTCTAACAAGCAGGACCCACTTGAGCGCCAGTTTGAGCGCTTTAACCAGGCCAACACCATCCTCAAGTCCCGGGACCTGGGCTCCATCATCTGTGACATCAAGTTCTCTGAGCTCACCTACCTCGATGCATTCTGGCGTGACTACATCAATGGCTCTTTATTAGAGGCACTTAAAGGTGTCTTCATCACAGACTCCCTCAAGCAAGCTGTGGGCCATGAAGCCATCAAGCTGCTGGTAAATGTAGACGAGGAGGACTATGAGCTGGGCCGACAGAAACTCCTGAGGAACTTGATGCTGCAAGCATTGCCCTGA-3\u2032.DEDD DEL-NLS coding sequence:5\u2032-ATGGCGGGCCTAGCGGCAGCAGCAAGCCAGGTGTGGCCAGAAGAGCATGGTGAGCAGGAACATGGGCTGTACAGCCTGCACCGCATGTTTGACATCGTGGGCACTCATCTGACACACAGAGATGTGCGCGTGCTTTCTTTCCTCTTTGTTGATGTCATTGATGACCACGAGCGTGGACTCATCCGAAATGGACGTGACTTCTTATTGGCACTGGAGCGCCAGGGCCGCTGTGATGAAAGTAACTTTCGCCAGGTGCTGCAGCTGCTGCGCATCATCACTCGCCACGACCTGCTGCCCTACGTCACCCTCAAGCTGAGACTCGCTGTGTGCCCTGATCTTGTAGACAAGTATCTGGAGGAGACATCAATTCGCTATGTGACCCCCAGAGCCCTCAGTGATCCAGAACCAAGGCCTCCCCAGCCCTCTAAAACAGTGCCTCCCCACTATCCTGTGGTGTGTTGCCCCACTTCGGGTCCTCAGATGTGTAGCAAGCGGCCAGCCCGAGGGAGAGCCACACTTGGGAGCCAGCGAATCCTGCGGATCTCAGTGACACCAGATCCCAAGGAGAAGCAGACATGTGACATCAGACTGCGGGTTCGGGCTGAATACTGCCAGCATGAGACTGCTCTGCAGGGCAATGTCTTCTCTAACAAGCAGGACCCACTTGAGCGCCAGTTTGAGCGCTTTAACCAGGCCAACACCATCCTCAAGTCCCGGGACCTGGGCTCCATCATCTGTGACATCAAGTTCTCTGAGCTCACCTACCTCGATGCATTCTGGCGTGACTACATCAATGGCTCTTTATTAGAGGCACTTAAAGGTGTCTTCATCACAGACTCCCTCAAGCAAGCTGTGGGCCATGAAGCCATCAAGCTGCTGGTAAATGTAGACGAGGAGGACTATGAGCTGGGCCGACAGAAACTCCTGAGGAACTTGATGCTGCAAGCATTGCCCTGA-3\u2032.DEDD cDNA coding sequence was cloned into lentivirus mammalian expression vector pLOVE empty (Addgene #15948) or pLenti-CMV-puro-DEST (Addgene #17452) with an N-terminal 3\u00d7-FLAG tag. DEDD mutant entry vector was assembled through GeneArt Gene Synthesis (Thermo Fisher Scientific). For lentiviral production, a lentiviral expression vector was co-transfected with second-generation lentivirus packing vectors psPAX2 and pMD2.G (Addgene) into 293FT cells using CalFectin . Forty-eight hours after transfection, breast cancer cell lines were stably infected with viral particles. pLOVE-GFP served as packaging and infection efficiency control. Lentiviral-based pLKO.1 DEDD shRNA vectors were purchased from Sigma-Aldrich Co. . The control shRNA vector (pLKO.1 scrambled shRNA) was from Addgene Inc. .Human DEDD-targeting shRNA sequences used in the lentiviral constructs were: shDEDD\u2212493, 5\u2032-CCGGGAGGAGACATCAATTCGCTATCTCGAGATAGCGAATTGATGTCTCCTCTTTTTTG-3\u2032 (targeting CDs); shDEDD-867, 5\u2032-CCGGCATCATCTGTGACATCAAGTTCTCGAGAACTTGATGTCACAGATGATGTTTTTTG-3\u2032 (targeting CDs); shDEDD1056, 5\u2032-CCGGGAAACTCCTGAGGAACTTGATCTCGAGATCAAGTTCCTCAGGAGTTTCTTTTTTG-3\u2032.The Stable expression or knockdown clones were selected by culturing cells with puromycin (1\u20135\u2009\u03bcg/mL) for at least 48\u2009h. The uninfected cells were also treated with puromycin as a control. Infected cells have a multiplicity of infection (MOI) around 0.3\u20130.6.DEDD or 3\u00d7-FLAG-Del-NLS-DEDD:Primers for constructing 3\u00d7-FLAG-DEDD\u2014forward primer: melting temperature (TM): 72.4\u2009\u2103N-3\u00d7-FLAG-5\u2032-CACCATG GAC TAC AAA GAC CAC GAC GGC GAC TAC AAA GAC CAC GAC ATC GAC TAC AAA GAC GAT GAC GAC AAGATGGCGGGCCTA.DEDD\u2014reverse primer: TM: 72.1\u2009\u2103N-3\u00d7-FLAG-5\u2032-TCA GGG CAA TGC TTG CAG CAT CAA GTT CCT CAG GAG TTT CTG TCG GCC CAG CTC ATA GTC.Primers for detecting gene mRNA level:DEDD\u2014forward primer: TM: 56.6\u2009\u21035\u2032-ATGGACGTGACTTCTTATTG-3\u2032.DEDD\u2014reverse primer: TM: 56.4\u2009\u21035\u2032-ATACTTGTCTACAAGATCAGGG-3\u2032.RB1\u2014forward primer: TM: 57.2\u2009\u21035\u2032-CAGAAATGACTTCTACTCGAAC-3\u2032.RB1\u2014reverse primer: TM: 58.9\u2009\u21035\u2032-AATGTGGCCATAAACAGAAC-3\u2032.ABCB10\u2014forward primer: TM: 62.1\u2009\u21035\u2032-CCATATTTCAGGATTTCAGCC-3\u2032.ABCB10\u2014reverse primer: TM: 54.8\u2009\u21035\u2032-GACTAATAGTTCCAGAAGCAG-3\u2032.UAP1\u2014forward primer: TM: 56.7\u2009\u21035\u2032-GATAGTCAGAATGGGAAAGAC-3\u2032.UAP1\u2014reverse primer: TM: 56.3\u21035\u2032-GCATAGGAGATAAGAGGAGAG-3\u2032.17.In vitro pooled drop-out screening by infect HCC1806 cells at 0.3\u2009MOI with DECIPHER shRNA lentiviral library , which includes about 27,000 shRNA constructs targeting 5043 genes on human signaling pathways. After 8 days of puromycin (1\u2009\u00b5g/mL) selection, infected cells were treated with either dimethyl sulfoxide (DMSO) or LAP (2\u2009\u00b5M) for 14 days with replacing fresh medium containing drug every 48\u2009h. After 14 days of treatment, the cells are collected and sent for barcodes sequencing by Illumina sequencing. The row barcode counts were analyzed for the \u201cdrop-out\u201d hits using the MAGeCK software as described beforeN, N-dimethyl formamide) , 10% SDS (sodium dodecyl sulfate) ) overnight. The 96-well plate was read at 570\u2009nm absorbance by a microplate reader. We used the wells with cells untreated as 100% control and the wells with only medium as 0% control. Percentage of inhibition of cell proliferation was calculated as [100\u2009\u2212\u2009((OD treated\u2009 \u2212\u2009OD empty)/(OD untreated\u2009\u2013\u2009OD empty)\u2009\u00d7\u2009100]. For direct cell counting, cells (3000/well) were seeded in at least triplicate in 96-well plates and treated with various concentrations of the single treatment or combination treatment for designated times. Cell proliferation was determined by directly counting cell numbers in each well using hemacytometer and compared using the wells with untreated cells as 100% control and the wells with only medium as 0% control. For the clonogenic assay, cells were plated approximately 3000 cells/well in 24-well plates in 10% FBS. After 24\u2009h, cells were treated in triplicate with various concentrations of a single or combination treatment for approximately 2 weeks with media replacement and treatment every 48\u2009h. Cells were washed with phosphate-buffered saline (PBS), fixed, and stained with a solution containing 6% glutaraldehyde and 0.5% crystal violet. Plates were dried, and each well was photographed and quantified using ImageJ 1.43u .For MTT -2,5-diphenyltetrazolium bromide) assays, cells (3000/well) were seeded in at least triplicate in 96-well plates and treated with various concentrations of a single treatment or combination treatment for designated times. Cell proliferation was determined by incubating with MTT for 3\u2009h and then in lysing buffer (50% DMF (\u00ae Green Supermix (Bio-Rad). Relative expression levels were calculated using the 2\u2212\u0394\u0394CT method.Total cell RNA will be extracted using TriZol reagent (Invitrogen) following the manufacturer\u2019s instructions. One microgram of total RNA was subjected to reverse transcription to synthesize cDNA using the Verso cDNA Kit (Thermo Fisher Scientific). A 20\u2009\u03bcL volume reaction consisted of 2\u2009\u03bcL reverse transcription product and 20\u2009nM of both forward and reverse primer. The samples were incubated with 2\u00d7 iTaq\u2122 Universal SYBR\u00ae Plus EdU Alexa Fluor\u00ae 488 Flow Cytometry Assay Kit or by Click-iT\u2122 EdU Alexa Fluor\u2122 594 Imaging Assay. In the Click-iT\u00ae Plus EdU Alexa Fluor\u00ae 488 Flow Cytometry Assay Kit (Thermo Fisher Scientific) plus 2\u2009\u03bcg/mL propodium podide staining (Thermo Fisher Scientific), cells were rehydrated in 1% bovine serum albumin-PBS at a density of 1\u2009\u00d7\u2009106 cells/mL, transferred into flow cytometry analysis tubes, and stored overnight at 4\u2009\u00b0C prior to samples analysis by Beckman Coulter FC500 Flow Cytometer. EdU signal was detected in FL1 (515\u2013536\u2009nm), and PI signal was detected in FL3 (590\u2013615\u2009nm). Twenty thousand events will be recorded and analyzed using the FlowJo software. In the Click-iT\u2122 EdU Alexa Fluor\u2122 594 Imaging Assay, cells were mounted in ProLong\u2122 Gold Antifade Mountant for 24\u2009h before imaging using the Leica fluorescence microscope in HCRI Tissue Core Facility . All experiments were repeated three times, and quantitative results were analyzed by one-way analysis of variance (ANOVA) or t test, where P\u2009<\u20090.05 was considered statistically significant.Twenty-four hours before the experiment, the culture medium was replaced with medium without FBS. This would synchronize the cells in G0. After 24-h serum starvation, the cells were treated with 1\u2009ng/mL EGFs to induce cell cycle reentry. Cells were treated with 10\u2009\u00b5M of EdU for 1\u20136\u2009h. Cells were collected and processed following the protocol of either Click-iTDEDD plasmid. Forty-eight hours later, cells were treated with 1\u2009ng/mL of EGF and then with 1\u2009\u00b5M MG-132 for 6\u2009h before collection. Harvested cells were re-suspended in 500\u2009\u00b5L of M2 lysis buffer . After incubation for 1\u2009h on ice, cell lysates were incubated with 1\u2009\u00b5g of Rb (4H1) antibody overnight (18\u2009h). One percent of cell lysates were saved as input control. After that, the samples were incubated with 50\u2009\u00b5L of protein A/G agarose beads (Santa Cruz) for 2\u2009h at 4\u2009\u00b0C. Immunocomplexes were washed with M2 lysis buffer three times before immunoblotting. For immunoblotting, proteins were extracted using M2 lysis buffer and proteinase plus phosphatase inhibitor cocktail . Protein extracts were resolved using 4\u201320% gradient SDS-polyacrylamide gel electrophoresis gels and transferred to nitrocellulose membranes (Thermo Fisher Scientific #88018). Membranes were probed with primary antibodies overnight on a 4\u2009\u00b0C shaker and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies. Signals were visualized with SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific #34078).For immunoprecipitation, cells first were transfected with either empty or DEDD (Sc-271192) and HSC70 (sc-7298) antibodies were purchased from Santa Cruz Biotechnology. \u03b2-Actin (ab8226) was purchased from Abcam. FLAG M2 (F1804) was purchased from Sigma-Aldrich. Cyclin B1 (#4138), cyclin D1 (#2978), cyclin A2 (#4656), cyclin E1 (#4129), p-Erk (#4370), Erk (#4695), p-EGFR (#3777), EGFR (#4267), p-Akt (#4060), Akt (#4691), Rb (#9309), p-Rb (#9308), RBL1 (#89798), Ubi (#3933), p-FoxM1 (#14655), and FoxM1 (#5436) were purchased from Cell Signaling Technology. For immunoprecipitation assay, antibodies were diluted into 50\u2009ng/\u00b5L. For immunoblotting assay, antibodies were diluted into 2.5\u2009ng/\u00b5L. For immunofluorescence assay, antibodies were diluted into 25\u2009ng/\u00b5L. All immunoblotting original uncropped images were provided as Supplementary materials (Source Data (pptx)) with the manuscript.For immunofluorescence staining, cells were serum starved overnight before stimulation with 1\u2009ng/mL of EGF and treated with 1\u2009\u00b5M MG-132 for 6\u2009h before fixation. Cells were fixed with 4% paraformaldehyde (PFA) and permeabilization in 0.2% Triton X-100 (PBS) and followed by anti-FLAG and Rb antibodies (dilution 1:50) incubation for 18\u2009h at 4\u2009\u00b0C. After incubation with tetramethylrhodamine-conjugated or fluorescein isothiocyanate-conjugated secondary antibody for 1\u2009h at 25\u2009\u00b0C, images were captured with an Olympus FV1000 2-photon confocal microscope.DEDD plasmids for 72\u2009h before puromycin (1\u2009\u03bcg/mL) treatment for 48\u2009h. The cells were further cultured for two more doublings. The harvested cells were washed with 1\u00d7 PBS and re-suspended in 1:9 ratio of cell mass to extraction buffer (Dynabeads\u00ae Co-Immunoprecipitation (Co-IP) Kit, Thermo Scientific) with protease inhibitors. The cell lysates were incubated on ice for 30\u2009min and then centrifuged at 2600\u2009\u00d7\u2009g for 5\u2009min at 4\u2009\u00b0C to remove large cell debris and nuclei. BCA (bicinchoninic acid) assay was used to quantify the protein concentration in the supernatant. About 5000\u2009\u03bcg of protein was incubated with 1\u2009\u03bcg of anti-FLAG antibody -coupled Dynabeads (10\u2009\u03bcg of antibody) on a roller at 4\u2009\u00b0C for 1\u2009h. Another 2000\u2009\u03bcg of protein was incubated with 1\u2009mg of anti-mouse IgG1 -coupled Dynabeads (10\u2009\u03bcg of antibody) for non-specific control immunoprecipitation. One percent of cell lysates were saved as input control. The Dynabeads\u00ae Co-IP complexes were washed with extraction buffer for three times. The proteins bound to beads were released in 60\u2009\u03bcL of elution buffer provided by the kit. The samples were added five volumes of cold (\u221220\u2009\u00b0C) acetone overnight at \u221220\u2009\u00b0C.Cells were infected with lentiviruses containing pLenti-CMV-puro-3\u00d7-FLAG-\u0394NLS-41. When the tumor reached the volume of approximately 100\u2009mm3, the mice were randomly divided into groups with an even distribution of tumor sizes (10 mice/group). LAP was given every 48\u2009h at a dose of 100\u2009mg/kg in the vehicle (0.5% hydroxypropylmethylcellulose with 0.1% Tween-20) via oral gavage. Abemaciclib was given every 48\u2009h at a dose of 25\u2009mg/kg in the vehicle (0.5% hydroxypropylmethylcellulose with 0.1% Tween-20) via oral gavage. Tumor volume was calculated as: tumor volume\u2009=\u20091/2 (length\u2009\u00d7\u2009width2). As designated time point post treatment, tumors were collected and fixed in 4% PFA for 24\u2009h followed by 70% ethanol for another 24\u2009h before formalin-fixed paraffin-embedded embedding for histology analysis.All animal experiments were performed in strict accordance with IACUC guidelines. Rag1 knockout (Rag1\u2212/\u2212) and nonobese diabetic/severe combined immunodeficiency (NOD SCID) mice were obtained from Taconic Farms . For TNBC xenograft experiments, 8-week-old female Rag1\u2212/\u2212 mice were transplanted with one million cancer cells suspended in 0.1\u2009ml of Matrigel mixture (1:1 mixture ratio of Matrigel and DMEM/F-12 medium without FBS) orthotopically into contralateral sides of the fourth mammary fat pads of the mice. For TNBC PDXs, 3.5-week-old female NOD SCID mice have received PDX tumor pieces as previously describedDEDD, Ki-67, or cleaved caspase-3 were detected by DEDD antibody (Santa Cruz Biotechnology #sc-271192), Ki-67 antibody (Ki-67 (D2H10), rabbit monoclonal antibody (IHC-specific) , or cleaved caspase-3 antibody (cleaved caspase-3 (Asp175) antibody, Cell Signaling Technology #9661) through IHC using the Polink-2 Plus HRP Rabbit with DAB Kit (GBI Labs) after 20\u2009min of antigen retrieval. The stained slides were scanned using the Aperio scanner in the HCRI Tissue Core Facility . Images were scored for the percentage of Ki-67 tumor cell nuclei per total tumor cell nuclei in each captured field using the ImageScope software. For DEDD and cleaved caspase-3, images were scored as the percentage of positive staining area per captured total area. All quantification was performed in a fashion that was blinded to the treatment group.PFA-fixed tumor samples were paraffin embedded and cut into 5\u2009\u03bcm thin slices and fixed on glass slides. The raw data for drug synergism analysis were generated by the MTT assay. The cells were evenly plated 24\u2009h before administration of drug dosages. Both single and combination treatments were completed in triplicate at each dosage (4\u20136 different dosages in a series dilution). Then, the absorbance reading of each well was calculated as FA by the treatment. The wells treated with DMSO served as non-affected controls. The wells with medium only served as full-affected control. The mean FAs at different concentrations were then analyzed by the CompuSyn software to generate simulation for the drug synergism analysis. The CI were plotted along with indicated FA for each cell line. CI\u2009>\u20091, antagonism; CI\u2009=\u20091, addition; CI\u2009<\u20091, synergism.http://www.cbioportal.org]. The oncoprint heatmap was generated by including mutations, putative copy-number changes, and mRNA expression z-scores (RNA-seq V2 RSEM with z threshold\u2009\u00b1\u20092). The survival data were extracted from the same dataset for Kaplan\u2013Meier plot in R using \u201cSurvival\u201d and \u201cSurvminer\u201d packages. The boxplot of the TCGA segmented copy-number changes without germline CNV (n\u2009=\u20091099) was generated using the University of California, Santa Cruz (UCSC) Cancer Browser. Gene expression data of commonly used cells lines were extracted from Broad Institute Cancer Cell Line Encyclopedia (CCLE). The heatmap of relative gene expression was generated in R using pHeatmap package. RNA-seq profiling of DEDD knockdown cells was conducted by standard Illumina TruSeq library preparation followed by Illumina sequencing using miSeq. The differential gene expression analysis was performed using DE-seq2 package. An adjusted P value of <0.05 was considered statistically significant. We performed GSEA analysis using GenePattern interface [http://www.broadinstitute.org/gsea/] using C6 v5.2 symbol (oncogenic signature) sub-datasets. Network analysis was conducted through the web-based bioinformatics package NetworkAnalyst [https://www.networkanalyst.ca/]-based on recommended protocol. All quantitative data were analyzed and plotted using the GraphPad Prism software. P values were generated using either two-sided t test or Fisher\u2019s exact test, or one-way ANOVA test.TCGA breast-invasive carcinoma provisional dataset (1105 samples) was sourced from cBioportal [Further information on research design is available in the\u00a0Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Reporting SummarySource Data"} +{"text": "Third, it uses adenovirus for the capacity to package the all-in-one vector, and for its high efficiency of transduction. We tested the all-in-one adenoviral CRISPR-Cas9 in a circadian clock model cell line U2OS, and demonstrated that essential clock genes such as Bmal1 and Per1 were knocked out so efficiently that functional assays could be performed from the heterogenic population without any clonal selection and expansion. This streamlined approach may prove invaluable for rapid functional assays of candidate genes in diverse biological pathways, including the circadian clock.CRISPR-Cas9 is a powerful gene editing technique that can induce mutations in a target gene of interest in almost any mammalian cell line. However, its practicality can be limited if target cell lines are difficult to transfect and do not proliferate. In the current study, we have developed a streamlined approach for CRISPR-based gene knockouts with three key advantages, which allows phenotypic assay of gene knockouts without clonal selection and expansion. First, it integrates into a single, all-in-one vector transgenes for Cas9, sgRNA, and a fluorescence marker. Second, we used the Gateway system to rapidly clone specific sgRNAs into the all-in-one vector through PCR and Unlike the siRNA approach, CRISPR-Cas can permanently alter the function of genes by mutating them. CRISPR-Cas9 derived from Streptococcus pyogenes is the most widely used system because of its ease of use and effectiveness4. The system requires introducing two components: the nuclease Cas9 and a single-guide RNA (sgRNA) which targets the nuclease to a specific genomic locus based on base pairing at a 20-nt target sequence. The target sequence must be followed immediately by the motif 5\u2032-NGG-3\u2032, which is called PAM (protospacer adjacent motif)4. The sgRNA-guided Cas9 generates a double strand break (DSB) in the target site, which may modify the genome in two different ways. The first is random insertion/deletion (indel) mutagenesis which occurs when the DSB is repaired by the error-prone non-homologous end-joining (NHEJ) pathway, often resulting in a knockout or loss-of-function mutation4. The second is precise allele editing through the high-fidelity homology-directed repair (HDR) pathway, which may occur in a small percentage of the cells (<5%)6. More definitive than siRNA, more precise than older transgene insertion methods, and more efficient than older homologous recombination methods8, CRISPR has become one of the most widely used methods to alter a gene to interrogate its function or for medical applications10.The prokaryotic defense system CRISPR-Cas was discovered as an adaptive immunity against foreign DNA molecules and converted to a revolutionary genome editing tool16, but clonal selection and expansion would be still required unless the transduction efficiency and expression of the viral vectors are very high. Among the most widely used viral vectors, lentivirus and adeno-associated virus (AAV) are preferred for human gene therapy applications, but adenovirus (AV) has important advantages as a research tool18. AV has the highest transduction efficiency in diverse target cells20; unlike lentivirus, its genome does not integrate into the host genome, thus reducing off-target mutations; and AV has a substantially higher genome capacity than AAV21. We have used this capacity to co-package three CRISPR-Cas9 components along with their promoters: Cas9, sgRNA and a fluorescent protein to aid in sorting for transduced cells. A fluorescent protein such as GFP or mCherry is also used to monitor active viral production in packaging cell lines such as 293\u2009A and 91121. When active adenoviral particles are made and released from the cells, they infect neighboring cells forming comet-like plaques which can be monitored under fluorescent microscopy. Others have previously demonstrated the feasibility of using AV to deliver Cas9 and sgRNA into diverse target cells, including primary and immortalized cells in vitro, and in vivo18. Most have (like us) used a replication-incompetent AV5 vector lacking E1 and E3; one group used a new AV vector devoid of all viral genes14, but with the trade-off of a more cumbersome production process and less infectable AV. In this study, we demonstrate the efficacy and versatility of an all-in-one adenovirus CRISPR-Cas9 system to target a specific biological pathway, the circadian clock.However, CRISPR has limitations. Transfection of the Cas9 and sgRNA in plasmids typically only succeeds for a minority of cells, and in certain cell lines which do not proliferate or are not easily transfected with plasmids, clonal selection and expansion from the transfected heterogenic population may not be practical or possible. Various CRISPR viral vectors have been employed to try to overcome these problems24. Adverse consequences from having a faulty clock include compromised sleep quality, poor work performance, and increased risk for accidents in the short-term, as well as metabolic diseases and cancer in the long-term. Because the circadian clock is cell autonomous, and rhythms can be measured in cells in real-time using a luciferase reporter under control of a clock promoter, clock mechanisms can be studied in cell culture models such as mouse embryonic fibroblasts (MEFs) or human cell line U2OS27. Clock gene regulation, protein rhythms, and protein-protein interactions in cultured cells are comparable to those in in vivo tissues29. The molecular backbone of the circadian clock is a transcriptional negative feedback loop with interacting positive and negative elements31. CLOCK and BMAL1 are the positive elements, activating transcription of many downstream genes, including behavior-regulating genes and the main negative elements, Period and Cryptochrome , whose products form an auto-inhibitory complex31. Unlike other core clock genes, Bmal1 has no redundant paralog. Disruption of Bmal1 results in complete arrhythmicity while disruption of a single Cry or Per family member produces subtle circadian phenotypes34.The circadian clock is a molecular circuit that controls daily rhythms in physiology and behavior, such as metabolic oscillations and wake-sleep cycles35, and adenovirus-mediated transduction is ~100% efficient and not cytotoxic, clonal selection and expansion may not be necessary to study the phenotype of knockouts if targeting is properly designed. We showed that a complete disruption of the circadian clock can be achieved in cell culture without clonal selection and expansion by targeting a splicing site in the essential clock gene Bmal1 using the all-in-one adenoviral CRISPR-Cas9 system. We believe interrogation of gene function can be performed in diverse cell lines in a rapid manner by using our all-in-one adenoviral CRISPR-Cas9 system since it does not involve conventional cloning, clonal selection and expansion.In the current study, we developed an all-in-one adenoviral vector where sgRNA can be easily cloned by the Gateway cloning system without labor-intensive conventional cloning and selection. Our vector achieves packaging of sgRNA, the SpCas9 transgene, and a fluorescent protein transgene into the same viral particle. Since indel efficiency of CRISPR-Cas9 is very high, up to ~100%4, we adopted the Gateway cloning technology. Gateway technology was developed by Invitrogen to transfer DNA fragments between plasmids using the bacteriophage lambda site-specific recombination system36. Briefly, a DNA fragment of interest is first cloned into an Entry plasmid where the DNA fragment becomes flanked by specific recombination sequences called attL1 and attL2. The DNA fragment in the Entry plasmid can then be transferred to any Destination vector by LR recombinase. Since only positive recombinants are selected, the subcloning procedure is highly efficient and rapid.To improve upon the conventional procedure of cloning sgRNA into plasmids using enzyme digestion and ligationCbh promoter (pShuttle-Cas9-DEST) Fig.\u00a0. BrieflyST) Fig.\u00a0. Recombiase Fig.\u00a0. It has Bmal1 promoter27. This cell line is widely used as a model system to study clock mechanisms, and bioluminescence signal from Bmal1-dLuc (or a similar reporter) is widely used as a real-time clock reporter since it faithfully reports the activity of the endogenous clock mechanism.Before packaging the vector into adenovirus, we first tested the functionality of the all-in-one vector construct itself. We used it to edit essential clock genes\u2014whose null phenotypes were well characterized\u2014by transfecting it into a human cell line, U2OS, expressing a rapidly degraded luciferase (dLuc) under a Bmal1. In the mammalian system, Bmal1 is the only essential clock gene without redundancy32. Thus, if CRISPR-Cas9 generates frame-shifting mutations (via NHEJ) in early exons of both alleles of Bmal1, the C-terminal transcriptional activation domain of BMAL1 will be deleted, resulting in circadian arrhythmicity37. Aided by an in silico design tool (http://chopchop.cbu.uib.no), we identified sgRNA sequences targeting four exons of Bmal1 based on the 20 nt sequence immediately preceding SpCas9 PAM sequences in those exons clones were detected as well as indels , and then the positive cells are expanded before subjected to functional assays. This approach can be applied to many cell lines, but it cannot be used in many primary cell lines that do not proliferate or cell lines that are very difficult to transfect. Moreover, clonal selection and expansion by FACS or drug selection may be labor-intensive and time-consuming, and demands a certain level of expertise.We thus confirmed the functionality of our all-in-one plasmid, but also confirmed the limitations of the typical paradigm of CRISPR-Cas9 mutagenesis21. Thus we hypothesized that high-titer infection of all-in-one CRISPR-Cas9 adenovirus into U2OS cells can achieve ~100% transduction efficiency and generate knockouts in the majority of cells, thus making the downstream clonal selection and expansion unnecessary. To test this possibility, we generated a new adenoviral shuttle vector expressing GFP and Cas9 from two different promoters (pAdTrack-Cas9-DEST) Fig.\u00a0. The pShncy Fig.\u00a0. The BmaBmal1 exon 6 because our previous transfection experiments found that targeting exon 6 produced more arrhythmic clones (62%) than targeting other Bmal1 exons. Considering significant wt contamination (15\u201330%), targeting near this specific splicing site machinery was revolutionary in editing the genome to produce precisely engineered knockout and knockin mice. The functional insights from these genetically engineered mouse models cannot be overstated. However, it is also important to recognize the mechanistic insights gained by studying mutant (and wt) cells in culture, as the clock mechanism is cell-autonomous and largely conserved across cell types42. Cell culture models will continue to be critically important for future advances in circadian biology, given their time- and cost-efficiencies. As our understanding of a biological pathway is most significantly advanced by identification of relevant new genes, a next frontier in circadian biology would be to identify new clock genes. We also have much to learn still from the hundreds of natural polymorphisms in known clock genes that lack a corresponding animal model. It has been already demonstrated that some of these polymorphisms are associated with circadian disorders such as Familial Advanced Sleep Phase Syndrome44. In the pre-CRISPR era, mutant cells were usually derived from mutant animals, as cell genomes could not be easily modified in culture. Now, however, cell culture models can be precisely edited using CRISPR-Cas9.As with most biological fields, our current understanding of the circadian clock has been shaped by genetic manipulations that have led to identification of a dozen essential clock genes to date19, but adenovirus can be easily concentrated by density gradient ultracentrifugation to achieve higher titers21. Adenoviral titer can be adjusted to maximize infection rate without cytotoxicity by visual inspection of fluorescence and cell morphology. We routinely measure robust bioluminescence rhythms from ~100% AV-infected MEFs and U2OS cells for several days, demonstrating that the physiology of these cells are not compromised45. Since AV transduction can be 100% efficient in delivering Cas9:sgRNA, whereas FACS sorting following transfection still includes non-transfected cells, AV is a more efficient way to achieve knockouts even when FACS sorting is feasible. A simple serial dilution without FACS sorting would be enough for clonal isolation and expansion for downstream assays , and not possible for all cell types. We show that CRISPR-Cas9 combined with AV can remove the need for clonal selection and expansion, thus addressing all of those issues in AV-infectible cells Fig.\u00a0. We and ays Fig.\u00a0.Bmal1-E7 and Per2-E15 in Fig.\u00a0Bmal1-E6. As a future refinement, we propose that the efficiency can be further increased by using two different adenoviruses targeting two different early exons. We have shown that MEFs could be doubly transduced by AV26. Although AV has been tested to deliver Cas9 and/or sgRNA into cells and showed knockdown of target proteins up to 90% in some cases15, to our knowledge, our present study is the first one to demonstrate a functional knockout without clonal selection by an all-in-one AV. All AV viral genes can be removed in the AV vector to increase packaging capacity and reduce immune response in vivo14. However, when the AV vector devoid of all viral genes is used, packaging efficiency suffers, resulting in vector titers ~two orders of magnitude lower, even after multiple rounds of amplification, compared to ours14. AV can also be employed to deliver catalytically-inactive Cas9 as a synthetic transcription factor. It has been shown that a catalytically-inactive Cas9 can be targeted to promoters to activate or inhibit transcription of their downstream genes10. AV is superior to lentivirus for that purpose because expression levels of transgenes are much higher by AV than lentivirus.Even if transduction of Cas9-sgRNA is near 100% and if Cas9-sgRNA can induce indels in ~100% of cells, one may expect frame-shifting mutations to occur only in 2/3 of cases. However, based on our results, the NHEJ repair process is not random, but rather locus-specific was PCR-amplified with primers with attL1 and attL2 sequence and subcloned into pEAR A1A .pEntry-sgRNA was generated by Mutagenex as follows. U6-sgRNA fragment from pU6-(BbsI)_Cbh-Cas9-T2A-mCherry plasmid (Addgene #6432421). Then, R1-CmR-ccdB-R2 fragment from the Gateway pDEST was subcloned into the intermediate vector _Cbh-Cas9-T2A-mCherry plasmid (Addgene #64324) was subcloned into XbaI and NotI sites of pShuttle , respectively _Cbh-Cas9-T2A-mCherry plasmid (Addgene #64324), and R1-Cmet al.4 The final PCR products were purified and mixed with either pShuttle-Cas9-DEST or pAdTrack-Cas9-DEST along with LR clonase (Thermo Fisher Scientific #11791020) to generate final all-in-one vectors. Bacterial colonies transformed with the reaction mixture were screened by colony PCR that amplifies DNA region flanked by R1 and R2 (negative clones), or B1 and B2 (positive recombinants). Two different sets of primers were used for pShuttle and pAdTrack as follows.The first round of PCR was performed using two sets of primers as described in Table\u00a0pShuttle-Cas9-DEST:attR1 Up Fwd: 5\u2032-GAGCCCACTGCTTACTGGCTTATC-3\u2032Cbh Rev: 5\u2032-CGTACTTGGCATATGATACACTTGA-3\u2032Size of PCR amplicons: 1,966\u2009bp negative clone and 691\u2009bp for positive recombinant.pAdTrack-Cas9-DEST:KpnIattR1F: 5\u2032-ATAGGTACCCCCACTGCTTACTGGCTTATCGAAATTAATAC-3\u2032KpnIattR2R: 5\u2032-CCGTAAGTTATGTAACGGGTACCTCTAGATCAACCAC-3\u2032Size of PCR amplicons: 1820 bp for negative clone and 545\u2009bp for positive recombinant.Bmal1 promoter was described previously. For transfection with the all-in-one plasmids, cells were plated into 6\u2009cm dishes to be approximately 60% confluent on the day of transfection. Cells were transfected with Polyfect according to the manufacturer\u2019s protocol and incubated for 2 days before they were subjected to trypsinization and FACS sorting using BD FACSAria SORP equipped with an Automated Cell Deposition Unit (ACDU) for sorting into 96 well plates. Cells were trypsinized with Trypsin (0.25%)-EDTA (2.21\u2009mM) for 3\u2009min and were filtered through mesh with 50um pores. FACS-sorted cells were collected into three groups: negative, intermediate and bright mCherry, and plated into 35\u2009mm dishes and grown for 2 days before harvest and genomic DNA extraction. Before the cells were harvested, they were inspected for mCherry expression. Even the bright mCherry group included 10\u201330% of non-mCherry cells. Harvested cell pellets were homogenized in 250 ul solution A containing 0.1\u2009M Tris-HCl pH\u2009=\u20099.0, 0.1\u2009M EDTA, and 1% SDS, and incubated at 70\u2009\u00b0C for 30\u2009minutes. 35 ul 8\u2009M potassium acetate was added and incubated at room temperature for 5\u2009minutes. The samples were centrifuged at 13,000\u2009rpm for 15\u2009minutes and genomic DNA was purified by subjecting the supernatant to phenol-chloroform extraction and ethanol precipitation. The extracted genomic DNA pellet was dissolved in 100 ul water and used as a template to PCR amplify the target genomic locus with a set of primers . Digestion products were resolved on 8% acrylamide/bis TBE gel and visualized with EtBr.A U2OS cell line expressing luciferase under a Bmal1 exon 7 and Per2 exon 15. Wt contamination was confirmed: 4 out of 16 were wt for Bmal1 exon 6 and 2 out of 15 were wt for Per2.PCR amplicons obtained above were cloned into plasmids using the TOPO-PCR cloning kit (Invitrogen #K2800), and inserts were sequenced from 20 colonies each for Bmal1 exon 6, 8 and 9, and Per2 exon 5 were set up for bioluminescence monitoring as described previously19. For immunoblotting for BMAL1, a previously described anti-BMAL1 antibody (GP3) was used47. For human PER2, novel polyclonal anti human PER2 antibodies were generated in guinea pigs using aa 1\u2013200 peptide by Cocalico Biologicals, Inc . hP2-GP49 was used in the current study. The antibody was validated by oscillations of human PER2 in U2OS cells and a novel monoclonal antibody against human PER2 (hP2-C6A3). The monoclonal antibody was selected from clones of antibodies raised against aa 1\u2013200 of human PER2 by Boreda Biotech . The monoclonal antibody was also able to detect oscillations of PERs in U2OS cells similarly . When compared with 293 cells, expression forming units (efu) of the adenovirus was 5\u201310 fold lower for U2OS cells. However, U2OS cells could be infected with ~100% efficiency without further purification, unlike infection for MEFs19. To ensure 100% infection in U2OS cells with the all-in-one adenovirus, the cells were infected at MOI of 50 twice, two days apart.Adenoviruses expressing mutant BMAL1 lacking the DNA-binding domain or wt BMAL1 were described previouslyCACCTGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTTCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTGTAATACGACTCACTATAGGGCGAATTGGGTACCGGGCCCCCCCTCGAGGTCGACGGTATCGATAAGCTTGATATCGAATTCCTGCAGCCCGGGGGATCCACTAGTTCTAGAGCGGCCGCCACCGCGGTGGAGCTCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTTCGAGCTTGGCGTAATCATGGTCATATGCCATGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGAACCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCNNN: MCST7-F 5\u2032-TAATACGACTCACTATAGGGT3-F 5\u2032-ATTAACCCTCACTAAAGGGA1 Seq-F 5\u2032-GATTAAGTTGGGTAACGCCAGGGA1 Seq-R 5\u2032-CTTGAGCGTCGATTTTTGTGATGCA1 Flk-F 5\u2032-GTAATACGACTCACTATAGGGC.Supplementary figures"} +{"text": "Chronic exposure of pancreatic \u03b2-cells to excess free fatty acids is thought to contribute to type 2 diabetes pathogenesis in obesity by impairing \u03b2-cell function and even leading to apoptosis. In \u03b2-cells, lipid droplet-associated protein perilipin 5 (PLIN5) has been shown to enhance insulin secretion by regulating intracellular lipid metabolism; the roles of PLIN5 in response to lipotoxicity remain poorly understood.INS-1 \u03b2-cells were transfected with PLIN5-overexpression adenovirus (Ad-PLIN5) and treated with palmitate. C57BL/6\u2009J male mice were fed with high fat diet and tail intravenous injected with adeno-associated virus overexpressing PLIN5 (AAV-PLIN5) in \u03b2-cells.Our data showed that palmitate and PPAR agonists including WY14643 (PPAR\u03b1), GW501516 (PPAR\u03b2/\u03b4), rosiglitazone (PPAR\u03b3) in vitro all induced PLIN5 expression in INS-1 cells. Under palmitate overload, although upregulating PLIN5 promoted lipid droplet storage, it alleviated lipotoxicity in INS-1 \u03b2-cells with improved cell viability, cell apoptosis and \u03b2-cell function. The protection role of PLIN5 in \u03b2-cell function observed in cell experiments were further verified in in vivo study indicated by mitigated glucose intolerance in high fat diet fed mice with \u03b2-cell-specific overexpression of PLIN5. Mechanistic experiments revealed that enhanced FAO induced by elevation of PLIN5, followed by decreased ER stress may be a major mechanism responsible for alleviation of lipotoxicity observed in the present study.Our finding substantiated the important role of PLIN5 in protection against lipotoxicity in \u03b2-cells.The online version of this article (10.1186/s12986-019-0375-2) contains supplementary material, which is available to authorized users. The prevalence of type 2 diabetes (T2DM), a chronic metabolic disorder, has been increasing steadily, partially due to the rising obesity rates. Excess adiposity and dyslipidemia commonly seen in obesity not only induce insulin resistance but also directly damage \u03b2-cell function, a fundamental defect in diabetes . Indeed,Excessive FFA are mainly stored in the form of triglyceride (TG) in lipid droplet (LD) in most of mammalian cells. LD consist of a score of neutral lipids surrounded by a phospholipid monolayer in which are embedded proteins named LD-associated proteins . In mammThe perilipin family proteins (PLINs) were the major LD-associated proteins that encompass five members, from PLIN1 to PLIN5. In contrast to PLIN1, which is mostly highly expressed in white adipose tissue, PLIN5 is mainly expressed in tissues with high oxidative capacity such as red muscle, heart, liver and brown adipose . As a LD12 viral genome/ml. After 5-month dietary manipulation, AAV-PLIN5 or AAV-MIP was delivered to HFD mice by tail vein injection at 5\u2009\u00d7\u20091011 viral genome per mouse. CHOW mice were injected with saline as the control group. 1\u2009week after injection, fasting (12\u2009h) and fed glucose (2\u2009h) levels were determined from tail vein blood with a glucometer . 10\u201314\u2009days after injection, glucose tolerance tests (GTT) and insulin tolerance tests (ITT) were conducted. For GTT, mice were fasted for overnight (16\u2009h) and then injected i.p. with D-glucose at 1.5\u2009g/kg of body weight. For ITT, mice were performed after 6\u2009h fasting and injected i.p. with human regular insulin at 0.75\u2009IU/kg of body weight. Blood glucose was measured from tail vein blood at the indicated time interval after the injection using a glucometer (FreeStyle). The areas under the curves for blood glucose in GTT (AUCg) and ITT (AUCitt) were calculated. Serum insulin at 0, 15, 30\u2009min after GTT and fasting serum FFA were measured according to manufacturers\u2019 instructions. At the end of the study, mice pancreatic islets were obtained as described previously reacting with mitochondrial dehydrogenase to produce an orange water soluble formazan dye. Prior to the analysis, the INS-1 cells were plated in 24-well cell culture plates at a density of 5\u2009\u00d7\u200910Cell apoptosis was measured by flow cytometric analysis. Briefly, after treatments, the cells were trypsinized, washed with PBS, and suspended in 195\u2009\u03bcl binding buffer. Then, cells were incubated with 5\u2009\u03bcl Annexin V-FITC for 15\u2009min and 5\u2009\u03bcl propidium iodide for 5\u2009min at 4\u2009\u00b0C in the dark. The cells were then analyzed on a FAC Scan flow cytometer to quantify apoptosis.INS-1 cells were treated with adenovirus for 24\u2009h in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), after which 0.5\u2009mM PA was added to the medium for another 12\u2009h or 48\u2009h. Cells were starved for 2\u2009h in glucose-free RPMI 1640 and then were washed twice with glucose-free Krebs-Ringer bicarbonate (KRB) buffer (pH\u20097.4). GSIS was investigated as described previously, with minor modifications . BrieflyS6K, Bcl-2, phospho-mTOR (p-mTOR), total mTOR which were all purchased from Cell Signaling and PLIN5 from Progen Biotechnik, ACO from Abcam, CHOP from Immunoway, CPT-1 and BiP from Santa Cruz. All the secondary antibodies and internal reference GAPDH were all purchased from Santa Cruz. Immunoreactive bands were visualized with an ECL reagent kit (Millipore). Optical densities of each band were calculated and analyzed by using Image J analysis software.Proteins were extracted and immunoblotted as described previously . The folCyclin D1, Cyclin D2, spliced XBP-1 (XBP-1(s)), sterol-regulatory element binding protein-1c (SREBP-1c), CHOP, BiP, and PLIN5 were detected using primers indicated in Table\u00a0Total RNA from INS-1 cell line was isolated using TRIzol reagent . First-strand cDNA was synthesized with Moloney murine leukemia virus (M-MLV) reverse transcriptase and random hexamer primers . Gene expressions were analyzed using the SYBR Green PCR system, following the manufacturer\u2019s recommendations . t-tests. To compare data sets of more than two groups, we used one-way ANOVA followed by Bonferroni\u2019s multiple comparison tests. Two-way ANOVA determined significance for GTT and ITT. P\u2009<\u20090.05 (two-tailed) was considered significant.All quantitative data were presented as means \u00b1 SEM and cell experiments were performed at least three times. Differences of numeric parameters between two groups were assessed using Student\u2019s PLIN5. Hence, the upregulation of PLIN5 by PA may dependent on PPAR\u03b1 activation in INS-1 cells. And the induction role of PLIN5 expression by PPAR\u03b2/\u03b4 and/or PPAR\u03b3 activation is indirect which needs to be investigated in the future study.Recently, PLIN5 was confirmed to be an LD protein in both human and murine islets and altering expression level of PLIN5 can impact insulin secretion by regulating lipolysis . However. In fasted islets, elevated PLIN5 expression and high TG content was observed recently [SREBP-1c . However, upregulating PLIN5 compromised this induction effect of PA which suggesting the mitigation of ER stress induced by PA overload . Furthermore, overexpressing CPT-1A in PLIN5 deficient cells alleviated the induction of BiP, CHOP by PA incubation and GFP, with sites for ECORI, BamHI, NdeI, SacI and ApaI. The size of AAV2/8 vector, MIP and PLIN5 was 4,492\u2009bp, 874\u2009bp and 1,392\u2009bp respectively. Left: map of AAV-PLIN5; Right: map of AAV-MIP. Sequences of MIP: 5\u2032-TTGTAGCTGGAATAGAGCATGCACTAACAGATGGAGACAGCTGGCTTTGAGCTCTGAAGCAAGTATTACATATGGAGACTTGCTGGCCTTCAGGTGCTTATCTTGTTATTGGATACTGCAGGAGGATGTACCACAGGGCTTCAGCTCAGCTGACCCCCAAGTGGGATATGGAAAGAGAGATAGAGGAGGAGGGACCATTAAGTGCCTTGCTGCCTGAATTCTGCTTTCCTTCTACCTCTGAGAGAGAGCTGGGGACTCGGCTGAGTTAAGAACCCAGCTATCAATTGGAACTGTGAAACAGTCCAAGGGACAAAGATACTAGGTCCCCAACTGCAACTTCCTGGGGAATGATGTGGAAAAATGCTCAGCCAAGGACAAAGAAAGCATCACCCACTCTGGAACAATGTCCCCTGCTGTGAACTGGTTCATCAGGCCATCAGGGCCCCTTGTTAAGACTCTAATTACCCTAGGACTAAGTAGAGGTGTTGACGTCCAATGAGCGCTTTCTGCAGACCTAGCACCAGGGAAGTGTTTGGAAACTGCAGCTTCAGCCCCTCTGGCCATCTGCTGACCTACCCCACCTGGAGCCCTTAATGGGTCAAACAGCAAAGTCCAGGGGGCAGAGAGGAGGTGCTTTGGTCTATAAAGGTAGTGGGGACCCAGTAACCACCAGCCCTAAGTGATCCGCTACAATCAAAAACCATCAGCAAGCAGGAAGGTACTCTTCTCAGTGGGCCTGGCTCCCCAGCTAAGACCTCAGGGACTTGAGGTAGGATATAGCCTCCTCTCTTACGTGAAACTTTTGCTATCCTCAACCCAGCCTATCTTCCAGGTTATTGTTTCAACA-3\u2032 Sequences of PLIN5: 5\u2032-ATGTCTGAAGAAGAGGCGGCTCAGATCCCCAGATCCAGTGTGTGGGAGCAGGACCAGCAGAACGTGGTGCAGCGTGTGGTGGCTCTGCCCCTGGTCAGGGCCACGTGCACCGCGGTCTGCGATGTTTACAGTGCAGCCAAGGACAGGCACCCGCTGCTGGGCTCCGCCTGCCGCCTGGCTGAGAACTGCGTGTGCGGCCTGACCACCCGTGCCCTGGACCACGCCCAGCCGCTGCTCGAGCACCTGCAGCCCCAGCTGGCCACTATGAACAGCCTCGCCTGCAGGGGCCTGGACAAGCTGGAAGAGAAGCTTCCCTTTCTCCAGCAACCTTCGGAGACGGTGGTGACCTCAGCCAAGGACGTGGTGGCCAGCAGTGTCACGGGTGTGGTGGACCTGGCCCGGAGGGGCCGGCGCTGGAGCGTGGAGCTGAAGCGCTCCGTGAGCCATGCTGTGGATGTTGTACTGGAAAAATCAGAGGAGCTGGTGGATCACTTCCTGCCCATGACGGAGGAAGAGCTCGCGGCACTGGCGGCTGAGGCTGAAGGCCCTGAAGTGGGTTCGGTGGAGGATCAGAGGAGACAGCAGGGCTACTTTGTGCGCCTCGGCTCCCTGTCAGCACGGATCCGCCACCTGGCCTACGAGCACTCTGTGGGGAAACTGAGGCAGAGCAAACACCGTGCCCAGGACACCCTGGCCCAGCTGCAGGAGACGCTGGAGCTGATAGACCACATGCAGTGTGGGGTGACCCCCACCGCCCCGGCCCGCCCTGGGAAGGTGCACGAGCTGTGGGGGGAATGGGGCCAGCGCCCTCCGGAGAGCCGCCGCCGGAGCCAGGCAGAGCTGGAGACGCTGGTGCTGTCCCGCAGCCTGACCCAGGAGCTGCAGGGCACGGTAGAGGCTCTGGAGTCCAGCGTGTGGGGCCTGCCCGCCGGCGCCCAGGAGAAGGTGGCTGAGGTGCGGCGCAGTGTGGATGCCCTGCAGACCGCCTTCGCTGATGCCCGCTGCTTCAGGGACGTGCCAGCGGCCGCGCTGGCCGAGGGCCGGGGTCGCGTGGCCCACGCGCACGCCTGCGTGGACGAGCTGCTGGAGCTGGTGGTGCAGGCCGTGCCGCTGCCCTGGCTGGTGGGACCCTTCGCGCCCATCCTTGTGGAGCGACCCGAGCCCCTGCCCGACCTGGCGGACCTGGTGGACGAGGTCATCGGGGGCCCTGACCCCCGCTGGGCGCACCTGGACTGGCCGGCCCAGCAGAGAGCCTGGGAGGCAGAGCACAGGGACGGGAGTGGGAATGGGGATGGGGACAGGATGGGTGTTGCCGGGGACATCTGCGAGCAGGAACCCGAGACCCCCAGCTGCCCGGTCAAGCACACCCTGATGCCCGAGCTGGACTTCTGA-3\u2032 (EPS 980 kb)"} +{"text": "Clerodendron cyrtophyllum, was isolated from root of Clerodendron cyrtophyllum Turcz, which was able to produce the highest activity of \u03b2-glucosidase up to 33.72 U/mL at 144 h during fermentation on Potato Dextrose Broth (PDB). The obtained fungus was grown on isoflavones-rich soybean extract to produce genistein and daidzein, achieving the conversion rate of 98.7%. Genistein and daidzein were isolated and purified by column chromatography using hexane/acetone (29:1/1:1), reaching purities of over 90% of total isoflavones, as identified and determined by TLC, LC-MS/MS, and 1H and 13C NMR spectroscopy. These results imply that the isolated P. citrinum is a potential fungal strain for industrial-scale production of genistein and daidzein from isoflavones-containing soybean extracts. These products may serve as potential raw materials for manufacture of functional foods that are based on aglycones.Soybeans offer an abundant source of isoflavones, which confer useful bioactivities when existing in aglycone forms. The conversion of isoflavones into aglycones via fermentation of soybean products is often realized by \u03b2-glucosidase, an enzyme produced by fungi. In this study, a filamentous fungus, Isoflavones are polyphenolics that exert estrogen-like effects and have been widely utilized in manufacture of foods and cosmetics . IsoflavGlycine max) ; ; with a flow rate of 1.0 mL/min. Mass spectra of Electron Spray Ionisation (ESI) were recorded on the Agilent 1100 Series mass spectrometer connected with Varian 320-MS . 1H-NMR spectra were recorded by 500 MHz with DMSO-d6 or acetone-d6 as solvents and tetramethylsilane as an internal standard. 13C-NMR spectra were recorded at 125 MHz (Bruker XL-500) with DMSO-d6 or acetone-d6 as solvents and as internal standard.To perform TLC (thin layer chromatography) analysis, silica gel 60 Fp < 0.05 via Student\u2019s t-test. Statistical analysis was performed in the JMP Pro 13.2 software.Experiments were carried out in triplicate for the accuracy of data. Statistical significant differences were realized at Clerodendron cyrtophyllum Turcz sp. were used as microbial sources to isolate fungi with \u03b2-glucosidase activity on PDA medium. At 10\u22125 dilution, colonies were isolated separately. They were then subcultured on sterilized PDA plates several times to obtain pure culture for identification and \u03b2-glucosidase enzyme assay. After several subcultures at 30 \u00b0C for 72 h, 10 fungi were isolated with different morphological characteristics of colony. Five fungi were isolated from fresh samples (designated as BC) of C. cyrtophyllum Turcz\u2019s while five other fungi were isolated from dried plant samples (designated as BSD).Roots from For enzymatic screening and fungal selection, the ten fungal isolates were tested for their capability of enzyme production. The \u03b2-glucosidase activity of these fungi is shown in the Aspergillus species.On SA medium, colonies of BC2 grew fast and displayed a compact green or yellow basal felt enclosed by a layer of white of erect conidiophores. The diameter of colonies was approximately measured as 2.4 cm. Microscopically, conidiophore stipes are long and smooth-walled and the color of hyaline turned dark towards the vesicle. Conidial heads were biseriate, large, globose, and showed tendency to radiate, splitting into several loose columns with age. Additionally, phialides, usually in the form of septate metulae, were also observed. These morphological characteristics affirm that this fungus belongs to the Penicillium species.The shade of colonies of BSD5 growing on SA medium was green in color and a dense felt of conidiophores was observed. The average diameter of colonies after three days of incubation was 1.2 cm. Observing under a microscope, conidiophores were hyaline and smooth-walled. In addition, terminal verticils, carrying 3\u20135 metulae each, were observed on conidiophores. In each metula, around 3\u20137 phialides were recognized. Regarding conidia, they are smooth-walled and their shape appeared to be globose or subglobose. In addition, the production of conidia was basipetal from the phialides. Therefore, the described fungus belonged to the Penicillium sp. was identified to ensure that this fungus belonged to Penicillium sp. Penicillium sp. was sequenced as:After identification by morphological characteristics, genomic DNA of the \u201cCATGCTCCGGCCGCCATGGCGGCCGCGGGAATTCGATTTCCGTAGGTGAACCTGCGGAAGGATCATTACCGAGTGCGGGCCCCTCGGGGCCCAACCTCCCACCCGTGTTGCCCGAACCTATGTTGCCTCGGCGGGCCCCGCGCCCGCCGACGGCCCCCCTGAACGCTGTCTGAAGTTGCAGTCTGAGACCTATAACGAAATTAGTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAACTAATGTGAATTGCAGAATTCAGTGAATCATCGAGTCTTTGAACGCACATTGCGCCCTCTGGTATTCCGGAGGGCATGCCTGTCCGAGCGTCATTGCTGCCCTCAAGCCCGGCTTGTGTGTTGGGCCCCGTCCCCCCCGCCGGGGGGACGGGCCCGAAAGGCAGCGGCGGCACCGCGTCCGGTCCTCGAGCGTATGGGGCTTCGTCACCCGCTCTAGTAGGCCCGGCCGGCGCCAGCCGACCCCCAACCTTTAATTATCTCAGGTTGACCT\u201d.Penicillium citrinum existing in the database, as evidenced by the excellent bootstrap results. Therefore, the examined fungus presumably belonged to the Penicillium genus and firmly aligned with the P. citrinum species, as demonstrated by the the strong sequence similarities with the said species.To gain insights into the evolutionary relationship, two methods for creation of the phylogenetic trees, namely Neighbor Joining (NJ) and Maximum Parsimony (MP), were employed, resulting in almost identical topologies. The aligned dataset consisted of 42 taxa and 100 characters. Our sequence (marked as unknown) achieved 100% matching with those of The optimum incubation time is the time interval at which the highest \u03b2-glusosidase activity was attained. After incubation for 24, 48, 72, 96, 120, 144, and 168 h, \u03b2-glusosidase activity was 0.23, 2.56, 8.16, 21.25, 23.55, 33.37, and 23.23 U/mL , respectP. citrinum was tested to grow in fermentation medium containing various carbon sources under the temperature of 30 \u00b0C, agitation speed of 200 rpm, and for six days. Carbon source is a critical factor affecting the production of enzymes. Therefore, v/v) volumetric ratio of P. citrinum inoculum to CDN substrate, the transformation into aglycones from glucoside forms complete took place during 72 h of fermentation (v/v) of P. citrinum enzyme to CDN with fermentation time of 72 h at 30 \u00b0C.entation . This in15H10O4, daidzein) with molecular weight of 254.23 (15H10O4). Data indicates that daidzein obtained after extraction and purification from fermentation broth is of high purity. Similarly, genistein ion fragments appeared at m/z 269.0 ) appeared as yellow-brown with 5% FeCl3 and dark green with Ce(SO4)2 (data not shown). 1H-NMR : \u03b4 (ppm) 6.28 , 6.41 , 6.90 , 7.46 , 8.163 , 13.03 . 13C-NMR : \u03b4 (ppm) 94.15 , 99.52 , 105.87 , 115.65 , 122.75 , 123.76 , 130.86 , 153.98 , 163.64 , 158.76 , 158.13 , 164.70 , 181.35 .For genistein , light yellow needle crystals, Rf = 0.30 ) appeared as dark-green with Ce(SO4)2 and colorless with 5% FeCl3 (data not shown). 1H-NMR : \u03b4 (ppm) 6.89 , 6.98 , 7.47 , 8.06 , 8.14 . 13C-NMR : \u03b4 (ppm) 102.04 , 115.07 , 116.58 , 123.43 , 127.23 , 129.99 , 125.74 , 157.11 , 157.37 , 162.45 , 174.63 .For daidzein ; white needle crystals, RPenicillium funiculosum was 30\u201336 U/mL, achieved on 3% rice bran or defatted oil cakes after 288 h fermentation. By stark contrast, the peak activity was found at a much lower level of 2.8 U/mL with Penicillium miczynskii cultured on 3% pineapple peel within 216 h of fermentation to their aglycones by ectively , on soy hila J18 . MoreoveIsoflavone aglycones can be derived from soybean waste by extraction with ethanol/water solvent followed by acid hydrolysis and purification , reachinP. citrinum fungus strain that produces the highest \u03b2-glucosidase activity of 33.72 U/mL, thus placing it amongst the most active fungi in this regard, has been successfully isolated from roots of Clerodendron cyrtophyllum Turcz. The fungus demonstrated a catalytic capacity to hydrolyze isoflavones-rich soybean extract into aglycones with a hydrolysis yield of 98.7% after 72 h of fermentation at 30 \u00b0C. Purification of the hydrolyzed mixture (rich in genistein and daidzein) by column chromatography using hexane/acetone (29:1/1:1) resulted in aglycone products with purity of over 90%. These results imply that soybean extract is a promising raw material for manufacture of functional foods derived from aglycones. Our study demonstrated a potential pathway for the production of aglycones from residual soybean extract via fermentation with an isolated fungi as a biocatalyst, with applciations in the pharmaceutical and functional food industries.The"} +{"text": "The engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technology enables purification of specific genomic regions interacting with their associated molecules. In enChIP, the locus to be purified is first tagged with engineered DNA-binding molecules. An example of such engineered DNA-binding molecules to tag the locus of interest is the clustered regularly interspaced short palindromic repeats (CRISPR) system, consisting of a catalytically-inactive form of Cas9 (dCas9) and guide RNA (gRNA). Subsequently, the tagged locus is subjected to affinity purification for identification of interacting molecules. In our previous studies, we developed enChIP systems for analysis of mammalian genome functions. Here, we developed an enChIP system to analyze bacterial genome functions.Streptococcus pyogenes dCas9 fused to a 3xFLAG-tag (3xFLAG-dCas9) in bacteria. Inducible expression of 3xFLAG-dCas9 in Escherichia coli was confirmed by immunoblot analysis. We were able to purify specific genomic regions of E. coli preserving their molecular interactions. The system is potentially useful for analysis of interactions between specific genomic regions and their associated molecules in bacterial cells to understand genome functions such as transcription, DNA repair, and DNA recombination.We generated a plasmid inducibly expressing To understand the regulatory mechanisms underlying genome functions such as transcription, it is essential to identify the molecules associated with a genomic region of interest in vivo. The engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technology we developed recently, enables specific isolation of genomic regions of interest interacting with their associated molecules , 2. ExamStreptococcus pyogenes dCas9 fused to a 3xFLAG-tag (3xFLAG-dCas9) and gRNA in bacteria. The developed enChIP system isolated target genomic regions from Escherichia coli. The system might enable identification of molecules associated with a specific genomic region in bacteria, and thus help to elucidate their genome functions.Previously, we developed enChIP systems for analysis of mammalian genome functions. Here, we report development of an in-cell enChIP system for bacterial cells Fig.\u00a0a. The syBglII and BstZ17I and treated with bacterial alkaline phosphatase (E. coli C75) . The double-strand DNA (dsDNA) containing the coding sequence of the 3xFLAG-tag and the N-terminal portion of dCas9 (agatctaaagaggagaaaggatctATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGCTCATGGATAAGAAATACTCAATAGGCTTAGCTATCGGCACAAATAGCGTCGGATGGGCGGTGATCACTGATGAATATAAGGTTCCGTCTAAAAAGTTCAAGGTTCTGGGAAATACAGACCGCCACAGTATCAAAAAAAATCTTATAGGGGCTCTTTTATTTGACAGTGGAGAGACAGCGGAAGCGACTCGTCTCAAACGGACAGCTCGTAGAAGGTATAC) was synthesized (Invitrogen) and digested with BglII and BstZ17I. The cleaved pdCas9-bacteria and the synthetic DNA fragment were purified by agarose gel electrophoresis and ligated.To construct the doxycycline (Dox)-inducible 3xFLAG-dCas9 expression plasmid 3xFLAG-dCas9/p-bacteria (Addgene #64325), pdCas9-bacteria (Addgene #44249) was digeSpeI and HindIII and treated with bacterial alkaline phosphatase (E. coli C75). The dsDNA for targeting the lacZ gene (ACTAGTGCTCTGCTACCTGCGCCAGCGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTGAAGCTT) (underlined: the target sequences) was synthesized (Invitrogen) and digested with SpeI and HindIII. The cleaved pgRNA-bacteria and the synthetic DNA fragment were purified by agarose gel electrophoresis and ligated. For construction of gRNAs targeting the promoter region of the rpoH gene, oligodeoxyribonucleotides was digeDH5\u03b1 was transformed with 3xFLAG-dCas9/p-bacteria alone or together with gRNA expression plasmids, and transformed bacteria were selected with chloramphenicol (Cam) (25\u00a0\u00b5g/ml) for 3xFLAG-dCas9/p-bacteria alone or a combination of Cam (25\u00a0\u00b5g/ml) and ampicillin (Amp) (50\u00a0\u00b5g/ml) for 3xFLAG-dCas9/p-bacteria plus gRNA expression plasmid.DH5\u03b1 transformed with 3xFLAG-dCas9/p-bacteria was cultured in 2\u00a0ml of LB media containing Cam (25\u00a0\u00b5g/ml) at 37\u00a0\u00b0C overnight with shaking. One hundred microliters of the culture liquid was mixed with 900\u00a0\u00b5l of LB media containing Cam (25\u00a0\u00b5g/ml) and incubated for 1\u00a0h with shaking. Subsequently, Dox (2\u00a0\u00b5M) was added to the culture media for induction of expression of 3xFLAG-dCas9. After incubation with shaking for 4\u00a0h, 400\u00a0\u00b5l of the culture liquid was centrifuged at 5000\u00a0rpm for 1\u00a0min, and the bacterial pellets were suspended in 100\u00a0\u00b5l of 4\u00d7\u2009SDS buffer. After boiling at 100\u00a0\u00b0C for 5\u00a0min, 10\u00a0\u00b5l of the sample was subjected to SDS-PAGE with a 5\u201320% gradient gel followed by immunoblot analysis with anti-FLAG M2 antibody (Ab) .600 of the culture media reached 0.5, Dox (2\u00a0\u00b5M) was added for induction of 3xFLAG-dCas9 expression. After incubation at 37\u00a0\u00b0C for 4.5\u00a0h with shaking, the bacterial cells were fixed with 1% formaldehyde at 37\u00a0\u00b0C for 5\u00a0min and neutralized with glycine at room temperature for 10\u00a0min. After centrifugation, the cell pellets were suspended in 800\u00a0\u00b5l of modified lysis buffer 3 , and DNA was fragmented by sonication using Ultrasonic disruptor UD-201 (TOMY SEIKO) with conditions: Output, 3; Duty, 100% (continuous); Time, Free; 6 cycles of sonication for 10\u00a0s and cooling on ice for 20\u00a0s (the average length of chromatin fragments was 1 kbp). The sonicated chromatin was subjected to enChIP-real-time PCR analysis as described previously . First, we designed two gRNAs targeting the promoter region of the rpoH gene was markedly lower than that of the rpoH promoter region factor . This gee sigma 3 factor [e sigma 3 factor [e sigma 3 factor [E. coli genome while preserving their chromatin structures, and potentially contributes to the understanding of bacterial genome functions such as transcription and DNA repair.In this study, we developed an enChIP system for analysis of bacterial genomes. This system enables efficient isolation of specific genomic regions from the Further studies might be necessary to assess the utility of this system combined with MS and NGS to identify molecules associated with the target genomic regions in bacteria."} +{"text": "The circadian oscillator is a molecular feedback circuit whose orchestration involves posttranslational control of the activity and protein levels of its components. Although controlled proteolysis of circadian proteins is critical for oscillator function, our understanding of the underlying mechanisms remains incomplete. Here, we report that JmjC domain\u2013containing protein 5 (JMJD5) interacts with CRYPTOCHROME 1 (CRY1) in an F-box/leucine-rich repeat protein 3 (FBXL3)-dependent manner and facilitates targeting of CRY1 to the proteasome. Genetic deletion of JMJD5 results in greater CRY1 stability, reduced CRY1 association with the proteasome, and disruption of circadian gene expression. We also report that in the absence of JMJD5, AMP-regulated protein kinase (AMPK)-induced CRY1 degradation is impaired, establishing JMJD5 as a key player in this mechanism. JMJD5 cooperates with CRY1 to repress circadian locomotor output cycles protein kaput (CLOCK)\u2013brain and muscle ARNT-like protein 1 (BMAL1), thus linking CRY1 destabilization to repressive function. Finally, we find that ablation of JMJD5 impacts FBXL3- and CRY1-related functions beyond the oscillator. In mammals, circadian rhythms are generated by a molecular oscillator in which the circadian locomotor output cycles protein kaput (CLOCK)\u2013brain and muscle ARNT-like protein 1 (BMAL1) transcription factors drive expression of the genes coding for their own repressors, the CRYPTOCHROME (CRY) and PERIOD (PER) proteins. A key feature of the oscillator is that the protein stability of its components is highly regulated. Previous studies had implicated the JmjC domain\u2013containing protein 5 (JMJD5) in regulation of the circadian clock in plants and flies. Here, we show that cells and livers that lack JMJD5 exhibit dysregulation of circadian gene expression. Mechanistically, JMJD5 is required for CRY1 degradation, including its destabilization by AMP-regulated protein kinase (AMPK), by facilitating its interaction with the proteasome. We found that JMJD5 is needed for normal CRY1-mediated transcriptional repression, thereby uncovering an inverse relationship between CRY1 stability and circadian repression. Finally, we showed that JMJD5 impinges on non-clock roles of F-box/leucine-rich repeat protein 3 (FBXL3) and CRY1. Altogether, our studies demonstrate that JMJD5 is a novel link between the oscillator and other physiological processes. Bmal1 gene transcription, which contributes to robust amplitude in circadian rhythms. However, the function of the circadian oscillator involves a much larger repertoire of factors that include other transcription regulators, kinases, phosphatases, ubiquitin ligases and peptidases, and chromatin regulators. Together, this large cohort of molecules acts in concert to generate circadian rhythms, coordinate the clock with other physiological processes, and enable environmental information to be integrated into its function.Circadian rhythms are endogenous, approximately 24-hour oscillations in behavior and physiology that evolved as an adaptation to the day\u2013night cycle. These rhythms are generated by a cell-autonomous timekeeping mechanism known as the molecular circadian oscillator. At its most basic, the oscillator is a transcription\u2013translation circuit formed by two interlocked delayed negative feedback loops . In one \u03b2-TRCP1/2 ubiquitin ligase complex and 300 mM NaCl) and incubated on ice for 45\u201360 minutes. This was then centrifuged at 3,000 rpm for 10 minutes at 4 \u00b0C. The resulting supernatant was the nuclear extract and was used in subsequent applications. Nuclear extracts from the MEFs were prepared using this protocol, and confluent 35-mm dishes of cells were used.Whole-cell lysates from cells and livers were prepared using lysis buffer containing 150 mM NaCl, 50 mM Tris-HCl, 0.5% TX-100, 0.5% NP-40, 0.25% Sodium Deoxycholate 0.025% SDS along with EDTA-free protease inhibitor cocktail and phosphatase inhibitors . Briefly, cells or crushed tissue was incubated with lysis buffer for 30 minutes on ice and then spun at 10,000 rpm for 10 minutes at 4 \u00b0C. To prepare the nuclear lysates, liver tissue was homogenized in a hypotonic buffer , cells were lysed using a lysis buffer containing 200 mM NaCl, 50 mM Tris-HCl, 1% TX-100, and 1% NP-40 supplemented with phosphatase and protease inhibitors. HEK293T cells were seeded out in 6-well plates and transfected the next day with a total of 2.5 \u03bcg of DNA . Forty-eight hours post transfection, the cells were lysed, incubated on ice for 30\u201345 minutes, and spun at 10,000 rpm for 10 minutes at 4 \u00b0C. The supernatant was incubated with the M2 beads overnight at 4 \u00b0C while tumbling. Subsequently, the beads were washed 3 times with chilled 1X TBS for 5 minutes each. The protein was eluted from the beads with equal volume of 3X flag peptide (Sigma Cat# F4799-4MG). The eluate was boiled in NuPAGE LDS Sample Buffer and reducing buffer and subjected to SDS-PAGE-immunoblot analysis.HEK293T cells were transfected with 1.5 \u03bcg V5-FBXL3, 400 ng HA-CRY1K:R, and 600 ng of V5-JMJD5. Forty-eight hours post transfection, the cells were lysed using the lysis buffer described above and immunoprecipitated with HA antibody bound to protein G beads for 1 hour at 4 \u00b0C. The beads were washed 3 times with chilled 1X TBS for 5 minutes with mild tumbling. The bound proteins were eluted by boiling in sample buffer and subjected to SDS-PAGE-immunoblot analysis.Jmjd5+/+ and Jmjd5\u2212/\u2212 MEFs were transfected with HA-RPN1 and 48 hours later were lysed with buffer containing 400 mM NaCl, 50 mM Tris-HCl, 1% TX-100, and 0.25% Sodium Deoxycholate, supplemented with phosphatase and protease inhibitors. The lysates were incubated on ice for 30\u201345 minutes and spun at 10,000 rpm for 10 minutes at 4 \u00b0C. Protein G beads were prebound with anti-HA tag antibody. The lysates were incubated with the antibody\u2013bead complexes for 1 hour at 4 \u00b0C and washed 5 times with 1X TBS containing 0.5% Triton X-100. The bound proteins were eluted by boiling in sample buffer and subjected to SDS-PAGE-immunoblot analysis to detect endogenous CRY1 levels bound to RPN1.FLAG M2 (Sigma), Anti-HA (12CA5), V5 (Abcam), and Anti-CRY1 were the antibodies used in this study. Anti-CRY1 (687) antibody was a kind gift from Satchin Panda.Jmjd5+/+ and Jmjd5\u2212/\u2212 MEFs were seeded out in 6-well plates at 350,000 cells per well and shocked 48 hours later with 100 nM of Dexamethasone for 2 hours before supplementing with fresh medium. Thirty-six hours post shock, the cells were harvested using 1 ml TRIzol (Fisher Scientific Cat# 15596026) and stored at \u221280 \u00b0C every 4 hours. Total RNA was then prepared using the manufacturer\u2019s instructions. For real-time qPCR, 1 \u03bcg of RNA was reverse transcribed to cDNA using qScript cDNA SuperMix (Quanta Biosciences Cat# 95048\u2013025). FastStart Universal SYBR Green Master (Rox) (Roche Cat# 4913850001) was used to perform the qPCR reaction in a BIO-RAD CFX384 Touch Real-Time PCR System.mDbp-F: GAG CCT TCT GCA GGG AAA CAmDbp-R: GCC TTG CGC TCC TTT TCCmCLOCK-F: AGAACTTGGCATTGAAGAGTCTCmCLOCK-R: GTCAGACCCAGAATCTTGGCTBmal1-F: GCC CCA CCG ACC TAC TCTmBmal1-R: TGT CTG TGT CCA TAC TTT CTT GGmCry1-F: ATC GTG CGC ATT TCA CAT ACmCry1-R: TCC GCC ATT GAG TTC TAT GATmCry2-F: GCA GAG CCT GGT TCA AGCmCry2-R: GCC ACT GGA TAG TGC TCT GGmPer1-F: GCT TCG TGG ACT TGA CAC CTmPer1-R: TGC TTT AGA TCG GCA GTG GTmPer2-F: TCC GAG TAT ATC GTG AAG AAC GmPer2-R: CAG GAT CTT CCC AGA AAC CAmNr1d1-F: GGA GCT GGG CCT ATT CAC CGCmNr1d1-R: GCT GCT CCA CCG AAG CGG AA.mJmjd5-F: CGCAGTCCTCCAGACACACCmJmjd5-R: CAAGATCACAGGCCTCCCAGmmMrpl46-F: GGTCCGGTCATTTTTTTTGTCAmMrpl46-R: GGGAGCAGGCATTCCTACAGmRORA-F: GTGGAGACAAATCGTCAGGAATmRORA-R: TGGTCCGATCAATCAAACAGTTCmABCG5-F: AGG GCC TCA CAT CAA CAG AGmABCG5-R: GCT GAC GCT GTA GGA CAC ATmGSTM3-F: CCC CAA CTT TGA CCG AAG CmGSTM3-R: GGT GTC CAT AAC TTG GTT CTC CAmLPL-F: GGG AGT TTG GCT CCA GAG TTTmLPL-R: TGT GTC TTC AGG GGT CCT TAGmLIPG-F: ATG CGA AAC ACG GTT TTC CTGmLIPG -R: GTA GCT GGT ACT CCA GTG GGmPCK1-F: CTG CAT AAC GGT CTG GAC TTCmPCK1-R: CAG CAA CTG CCC GTA CTC CmANGPTL4-F: CAT CCT GGG ACG AGA TGA ACTmANGPTL4-R: TGA CAA GCG TTA CCA CAG GChJMJD5-F: GGC CCG TGA TCC TGA AAG GhJMJD5-R: GGC TCA TTC ACG ATG TAT TTG ChCRY1-F: ACA GGT GGC GAT TTT TGC TTChCRY1-R: TCC AAA GGG CTC AGA ATC ATA CThFBXL3-F: GCA GCT TGT GAT ATA CTA TCG CAhFBXL3-R: TGG TCG AGC AGT TGA AAT AAG TChHPRT1-F: CCT GGC GTC GTG ATT AGT GAThHPRT1-R: AGA CGT TCA GTC CTG TCC ATA AAll the shRNA and siRNA knockdowns were performed using transient cotransfection of the constructs along with overexpression constructs of FLAG-CRY1, HA-JMJD5, and V5-FBXL3 in HEK293T cells in 6-well plates.shRNA hFBXL3 Sigma MISSION shRNA TRCN0000369031 (5\u2032-CCGGCTGATCAGTGTCACGGCTTAACTCGAGTTAAGCCGTGACACTGATCAGTTTTTG-3\u2032), shRNA hCRY1 Sigma MISSION shRNA TRCN0000231065 (5\u2032-CCGGGGAACGAGACGCAGCTATTAACTCGAGTTAATAGCTGCGTCTCGTTCCTTTTTG-3\u2032), siRNA hJMJD5 Sigma MISSION siRNA EHU149061, siRNA universal negative control Sigma MISSION siRNA SIC001.Jmjd5LKO mice between 6 and 9 weeks old were entrained in a light tight chamber to a 12-hour light:12-hour dark cycle for 10 days and released into constant darkness for 24 hours. Mice were then killed every 4 hours for a 24-hour period.Control or C57BL/6NTac-Jmjd5tm1a(EUCOMM)Wtsi frozen embryos were obtained from the European Conditional Mouse Mutagenesis Program (EM:04155), and embryo reconstitution was performed at Charles River. Born heterozygotes were crossed with the FLP deleter strain B6.129S4-Gt(ROSA)26Sortm1(FLP1)Dym/RainJ (Jackson Laboratory 009086) to excise the lacZ-neo cassette. Thereby, conditional Jmjd5flox knockouts were created, which were bred for several generations onto a C57BL/6 background. To generate liver-specific JMJD5-null animals, Jmjd5flox/flox mice were bred with a transgenic mouse line that specifically expresses CRE in the liver at high levels /Cre+ littermates were used as controls.The following gBLOCKS were ordered from IDT and cloned into pGL3 BASIC (Promega).Wild-type E-boxes: AGTGCTAGCCATCACCCACTCACCCCTTAACGACACGTGGGCCCTCAATTGCCCTTCTCTCAGGATCTGAAGGGTCAGAGGAAAGGGTTGGATTCTTTATAACAAGGCTGGGGAGAGGCCAGGGAATGTCAGTCTAGGTTTTTCTCTCTCCCACTTCCCTTGGGTAGCAGACATTTCATTCACCCGGCACCAGGACAGGTGTCTTGTTCTGCCAAGCTGGTCAGTTTAGGAAGTAGGTTTCTCTTGAGCACTTCCTGTGGCCCAGGTATCCTCCCTGAAAAGGGGTAGTTTCCCTCCCTCACTTCCCTTTCATTATTGACGGTGTGAGACATCCTGATCGCATTGGCTGACTGAGCGGTGTCTGAGGCCCTTCAGCCCAGCACCAGCACCCAAGTCCACGTGCAGGGATGTGTGTGACACAGCCCTGACCTCAGTGGGGGCCAGTAGCCAATCAGATGCCAGGAAGAGATCCTTAGCCAACCGGGGGCGGGGCCTGCGGCTCTTCGGGCAGAAGGCCAATGAGGGGCAGGGCCTGGCATTATGCAACCCGCCTCCCAGCCTCGCGGAGCTTCTGGGTTGCAAGCTTAGC.E-boxes mutated: AGTGCTAGCCATCACCCACTCACCCCTTAACGACAgGTcGGCCCTCAATTGCCCTTCTCTCAGGATCTGAAGGGTCAGAGGAAAGGGTTGGATTCTTTATAACAAGGCTGGGGAGAGGCCAGGGAATGTCAGTCTAGGTTTTTCTCTCTCCCACTTCCCTTGGGTAGCAGACATTTCATTCACCCGGCACCAGGACAGGTGTCTTGTTCTGCCAAGCTGGTCAGTTTAGGAAGTAGGTTTCTCTTGAGCACTTCCTGTGGCCCAGGTATCCTCCCTGAAAAGGGGTAGTTTCCCTCCCTCACTTCCCTTTCATTATTGACGGTGTGAGACATCCTGATCGCATTGGCTGACTGAGCGGTGTCTGAGGCCCTTCAGCCCAGCACCAGCACCCAAGTCCAgGTcCAGGGATGTGTGTGACACAGCCCTGACCTCAGTGGGGGCCAGTAGCCAATCAGATGCCAGGAAGAGATCCTTAGCCAACCGGGGGCGGGGCCTGCGGCTCTTCGGGCAGAAGGCCAATGAGGGGCAGGGCCTGGCATTATGCAACCCGCCTCCCAGCCTCGCGGAGCTTCTGGGTTGCAAGCTTAGC.S1 Fign = 3) and mice livers (mean \u00b1 SEM n = 4) were determined by qPCR analysis. JMJD5, JmjC domain\u2013containing protein 5; MEF, mouse embryo fibroblast; qPCR, quantitative PCR.mRNA levels of JMJD5+/+ and JMJD5\u2212/\u2212 MEFs (mean \u00b1 SEM (TIF)Click here for additional data file.S2 FigPer2-Luc promoter in Jmjd5+/+ and Jmjd5\u2212/\u2212 MEFs . JMJD5, JmjC domain\u2013containing protein 5; MEF, mouse embryo fibroblast.Real-time bioluminescence measurement from overexpressed (TIF)Click here for additional data file.S3 FigJmjd5LKO, JmjC domain\u2013containing protein 5 liver knockout; loxP, locus of X-over P1.Schema of targeting vector and resulting floxed allele. Exons shown as solid boxes; Frt and loxP sites are respectively shown as green and purple triangles. Frt, flippase recognition target; (TIF)Click here for additional data file.S4 FigH321A (MUT) in the real-time luciferase assay in (A) Relative protein levels of FLAG-JMJD5 and FLAG-JMJD5(TIF)Click here for additional data file.S5 Fig(A) FLAG-CLOCK and BMAL, (B) FLAG-PER1, and (C) FLAG-PER2. BMAL, brain and muscle ARNT-like protein; CLOCK, circadian locomotor output cycles protein kaput; JMJD5, JmjC domain\u2013containing protein 5; PER, PERIOD.(TIF)Click here for additional data file.S6 FigJmjd5LKO, JmjC domain\u2013containing protein 5 liver knockout; WLE, whole liver extract.Total protein stain was used as loading control. CRY1, CRYPTOCHROME 1; (TIF)Click here for additional data file.S7 FigShown are the inputs for the coimmunoprecipitations of FLAG-tagged CRY1 and mutant CRY1s with (A) V5-FBXL3 and (B) HA-JMJD5. CRY1, CRYPTOCHROME 1; FBXL3, F-box/leucine-rich repeat protein 3; JMJD5, JmjC domain\u2013containing protein 5.(TIF)Click here for additional data file.S8 FigHEK293T, human embryonic kidney 293T; HPRT1, hypoxanthine phosphoribosyltransferase 1 gene; qPCR, quantitative PCR; shRNA, short hairpin RNA; siRNA, small interfering RNA.(TIF)Click here for additional data file.S9 FigImmunoprecipitation of FLAG-CRY2 in HEK293T cells shows no interaction with HA-JMJD5 both in the absence and presence of V5 FBXL3. FLAG-CRY1 interaction with HA-JMJD5 is shown as a positive control. * denotes an unknown band observed only when FLAG-CRY2 is transfected. CRY, CRYPTOCHROME; FBXL3, F-box/leucine-rich repeat protein 3; HEK293T, human embryonic kidney 293T; JMJD5, JmjC domain\u2013containing protein 5.(TIF)Click here for additional data file.S10 Fig(A and B) Shown are the areas of the blot used to quantify the accumulation of CRY1. (C) Quantification of the main band (non-ubiquitylated CRY1) reveals a similar pattern of accumulation as quantifying all the forms of CRY1 as shown in (TIF)Click here for additional data file.S11 Fig71A/280A; CRY1, CRYPTOCHROME 1; JMJD5, JmjC domain\u2013containing protein 5.Real-time luciferase assays from (TIF)Click here for additional data file.S12 FigCRY1, CRYPTOCHROME 1; MEF, mouse embryo fibroblast.(TIF)Click here for additional data file.S1 Data(XLSX)Click here for additional data file."} +{"text": "Auxiliary subunits of the CKAMP family differentially modulate AMPA receptor properties. Published 1, December 2015In the Materials and Methods subsection \"In situ hybridization\" the sequences of radiolabeled oligodeoxyribonucleotide probes used for CKAMP52 and CKAMP59 were mixed up. These are the correct sequences:Ckamp52ins1 = 5\u2019AATGTCAGCCAGAGCCCTGTGGATGTTCATCTCTCGCGGACkamp59ins1 = 5\u2019GCGGCATAGCACGCCAGTCGAGGTTGGAGGGCTTCATGGTGTTWe apologize for the mistake.The article has been corrected accordingly."} +{"text": "Utilizing both in situ hybridization and quantitative mRNA analysis, we investigated the changes in the gene network state caused by the removal of one or both of the early acting enhancers. brk5\u2019 deletion generally phenocopied the gene mutant, including expansion of the BMP ligand decapentaplegic (dpp) as well as inducing variability in amnioserosa tissue cell number suggesting a loss of canalization. In contrast, brk3\u2019 deletion presented unique phenotypes including dorsal expansion of several ventrally expressed genes and a decrease in amnioserosa cell number. Similarly, deletions were made for two enhancers associated with the gene short-gastrulation (sog), sog.int and sog.dist, demonstrating that they also exhibit distinct patterning phenotypes and affect canalization. In summary, this study shows that similar gene expression driven by coacting enhancers can support distinct, and sometimes complementary, functions within gene regulatory networks and, moreover, that phenotypes associated with individual enhancer deletion mutants can provide insight into new gene functions.Developmental genes are often regulated by multiple enhancers exhibiting similar spatiotemporal outputs, which are generally considered redundantly acting though few have been studied functionally. Using CRISPR-Cas9, we created deletions of two enhancers, brinker (brk) and short-gastrulation (sog) from the genome of D. melanogaster fruit fly using CRISPR-Cas9 genome editing. Surprisingly, opposite phenotypes relating to some target genes are associated with the enhancer deletions. Deletion of one enhancer generally exhibits phenotypes in early embryo patterning similar to respective gene mutants; whereas, in contrast, deletion of the other presents unique phenotypes including change in cell number for a particular tissue in the embryo, the amnioserosa. In summary, this study shows that coacting enhancers driving similar expression outputs can support distinct, and sometimes complementary, functions to differentially impact the development of embryos and that the individual mutation of these enhancers can provide insight into new gene functions.Genes expressed during development are often regulated by multiple cis-regulatory sequences, non-coding DNA, also known as enhancer sequences. Many instances have been found where two or more distinct enhancer sequences support similar spatiotemporal outputs relating to a single gene. These enhancers have generally been considered redundantly acting, or overlapping in activity, though few have been studied functionally. We created deletions of coacting enhancer pairs associated with the genes Drosophila embryos, brinker (brk) and short gastrulation (sog), which are each associated with two enhancers that control their expression in the early embryo when both enhancers should be active (yw), brk\u03945\u2019, brk\u03943\u2019, and brk\u03945\u2019\u03943\u2019. First, as proof of principle, total counts of brk mRNA were measured and, as expected, the brk\u03945\u2019 data is similar to wildtype (as the brk3\u2019 enhancer is predominantly acting at late stage 5), whereas counts are decreased for the brk\u03943\u2019 and brk\u03945\u2019\u03943\u2019 datasets and level (NanoString) of expression allowed us to more fully investigate the changes in gene regulation in these mutant embryos or antagonistic effects (i.e. opposite phenotypes) on target gene expression outputs. NanoString allows us to detect mRNA abundance, which is complementary to the spatial data provided by stainings. For example, dization , and levdization . During s mutant . The brkgenotype . However embryos .brk encodes a transcription factor that acts as a transcriptional repressor to limit BMP signaling and support patterning in the early embryo and at other stages of development including in the wing disc .All flies were reared at 23\u00b0C on standard fly media. ftz-lacZ , sogY506ftz-lacZ , brk 3\u2019-vep-lacZ , sog.intvep-lacZ , sog.disvep-lacZ , and Hishttp://flycrispr.molbio.wisc.edu/tools) was used to identify the PAM sequences with no predicted off target hits. The gRNA plasmids were then injected into either y2 cho2 v1 P{nos-phiC31\\int.NLS}X; attP2 (III) (NIG-Fly #TBX-0003) or y1 v1 P{nos-phiC31\\int.NLS}X; attP40 (II) (NIG-Fly #TBX-0002). Stable gRNA transgenic lines were created and then crossed to a Cas9 expressing line . Individuals from the next generation were screened by PCR for the deletions. Alternatively, for the sog\u0394int and sog\u0394dist, a homologous recombination cassette (HRC) was created by adding 1kb homology arms to the plasmid pDsRed-attP . Lines were screened for RFP in the eyes and the RFP cassette was subsequently removed in the same way as for the single mutants. All lines represent isogenic backgrounds differing only in the gRNA line used to create the specific deletion.For CRISPR-Cas9 deletions within the genome, gRNA constructs were created by modifying the pCFD4 plasmid to targsog, aos, zen, sna, and rho were transcribed from cDNA subcloned into pGEM-T vector or full-length cDNA as for brk . For fluorescent in situ hybridization (FISH), probes were detected using Sheep anti-digoxigenin (Life Technologies PA185378), Mouse anti-Bio (Invitrogen 03\u20133700), and Rabbit anti-FITC (Invitrogen A889). Fluorescently labelled secondary antibodies were all from ThermoFisher (used at 1:400). Enzymatic detection was performed using digoxigenin labelled probes followed by detection with Anti-Digoxigenin-AP antibody and standard AP staining using NBT/BCIP. Other antibodies used in this study were Hnt , pSMAD1/5 , and dpErk .Embryos were fixed and stained following standard protocols. Antisense RNA probes labeled with digoxigenin, biotin, or FITC-UTP were used to detect reporter or in vivo gene expression as described previously . Probes brk\u03945\u2019 and the brk\u03945\u2019\u03943\u2019), there were no obvious defects in cellularization seen in any of the mutants. Using nuclear shape and size combined with percent cellularization to characterize developmental stage was judged to be an adequate method to synchronously stage embryos that is independent of the genotype.Specific staging of embryo images is indicated in the associated figure legend. For early and late stage 5, the shape and length of the nucleus as well as the membrane front of the cellularizing cells was used to define either early or late stage 5. Early stage 5 (nuclear cycle (nc)14 A and B) included all embryos in nc14 that arThe following primers were used to generate riboprobes:dppi-f ccagaactagaaaaccggaagcdppi-t7-r gaaatTAATACGACTCACTATAgggCGCCTGTGCTAAAGACCCTGstumps-f TGGCCCAGAACATCGTCAGTTTstumps-r-t7 gaaatTAATACGACTCACTATAgggATGAGACTTCACCTGCTCCTGGATtld B-f ATGTGGATGAGTGTTCAATtld B-r-T7 gaaatTAATACGACTCACTATAgggTCCCTTCGCTGGACCTCTCATRace-f ATGAGACTGTTTCTGCTAGCCCTGCRace-T7-r GAAAATTAATACGACTCACTATAGGGACGCAAGCAGAAGGCACAGATAnetA\u2013f ATGATCCGTGGAATCTTGCTCCTGCnetA-T7-r AATTTAATACGACTCACTATAGGGCTTTGCACTCATTGGCTTCCTTGGCpnr-f ATCTCAAACCCTCGCTCAGCpnr-T7-r aagtaatacgactcactatagggagaCGAGGTGGCCATCAGTTTGGsog ex1-f TCAGGTTCAGTCGCTCTTGAsog ex1-T7-r AATTTAATACGACTCACTATAGGGGTGTCGGACTCCTCGAACATdpp and rho was measured using ImageJ. First, an ellipse was fit to the outside of the embryo image, and the perimeter of this ellipse was measured; then, an arc was manually drawn along this ellipse matching the extent of fluorescent signal for each gene measured. The length of the arc was measured, and a ratio of arc length to full circumference was determined. The width of zen at stage 6 was measured using chopped sections that were co-stained with DAPI to mark each of the nuclei. The cells expressing zen were counted manually. The amnioserosa cell number was counted using stage 10\u201313 stage embryos stained with Hnt antibody. Nuclei on the lateral side of the embryo only were counted manually in ImageJ by planing through the z stacks and using the Cell Counter plug-in. Box plots were created using BoxPlotR (http://shiny.chemgrid.org/boxplotr/).Embryos were sectioned along the anterior-posterior axis manually using a razor blade, and cylindrical mid-embryo sections were mounted on the cut side allowing for imaging of the axial plane. Percentage of embryo circumference for \u03c72 [The width of the pMad gradient was measured as described with the\u03c72 , we used\u03c72 . The scastumps were made in ImageJ. Z-stack projections were created using the sum slices function and then the heat map was applied using the LUT function.Heatmap representation of images for pMad and Grids were drawn on standard 10 cm apple juice collection plates, dividing each into 160 quadrants. One embryo was transferred from a two hour collection into each of the quadrants of the grid. The plates were aged at 25\u00b0C in a humidified chamber for 30 hours, and the number of hatched and unhatched embryos was manually counted. This was repeated for a minimum of three times on different days for each genotype.brk mutant lines and nuclear morphology was observed on a Zeiss LSM800 microscope. Embryos were collected at nc14C for each genotype. This specific stage was determined to be when the nuclei were elongated but not yet disorganized, and the membrane front fully encapsulates the nuclei [WT, brk\u03945\u2019\u03943\u2019) or 5 individual embryos were analyzed for each genotype.NanoString was run as described . H2A-RFPe nuclei . Once exbrk\u03945\u2019 and brk\u03943\u2019 were verified by crossing deletions made with different gRNA lines in trans to check for offsite effects. For brk\u03945\u2019\u03943\u2019, two independent mutant lines made using the same gRNA pair was crossed in the same manner. No differences were seen between the single mutant embryos and the trans-heterozygous embryos in the expression of brk, dpp, Race, or zen so all analyses were done using a single mutant line for each deletion.Individual deletion lines for brk or sog gene expression, specifically. For brk analysis, males of the genotype YW, brk\u03945\u2019, brk\u03943\u2019, or brk\u03945\u2019\u03943\u2019 were crossed to females of genotype brkM68/FM7 ftz-lacZ. To identify trans-heterozygous embryos and assay for changes in amnioserosa, embryos were stained with a combination of antibodies: anti-\u03b2Gal to detect the balancer, anti-Sxl to identify female embryos, and anti-Hnt to detect amnioserosa. Similar trends were seen as for homozygous enhancer mutants :3\u2019grna-f attttaacttgctatttctagctctaaaacCGCCTCGGCCGGCGTCGCTGCgacgttaaattgaaaataggtc3\u2019grna-r attttaacttgctatttctagctctaaaacCGGGGTGGAAAAGCGACCCGCgacgttaaattgaaaataggtc5\u2019grna-f tatataggaaagatatccgggtgaacttcGTACCTGTTCGATCCTTCATgttttagagctagaaatagcaag5\u2019grna-r attttaacttgctatttctagctctaaaacTTGGGCTTTCGTTGCACAACgacgttaaattgaaaataggtcSog.int grna-f tatataggaaagatatccgggtgaacttcGTCAAAATCTTTAGTTAAAGgttttagagctagaaatagcaagSog.int grna-r: attttaacttgctatttctagctctaaaacTAGATCCCGGGATTTGTGCCgacgttaaattgaaaataggtcSog.distgrna-f:tatataggaaagatatccgggtgaacttcGATGATAGGTGGACTCTTGATgttttagagctagaaatagcaagSog.distgrna-r:attttaacttgctatttctagctctaaaacAGCGAATACGTGGAATTCCTCgacgttaaattgaaaataggtcSet 2 (second independent generation of deletions used to test for 2nd site mutations):3\u2019grna2-f tatataggaaagatatccgggtgaacttcGTTCCAAAACTTTAATCTGTTgttttagagctagaaatagcaag3\u2019grna2-r attttaacttgctatttctagctctaaaac GCAATCATTCTTCAATTCATCgacgttaaattgaaaataggtc5\u2019grna2-f tatataggaaagatatccgggtgaacttcGACCCGATGAAGGATCGAACgttttagagctagaaatagcaag5\u2019grna2-r attttaacttgctatttctagctctaaaacGCTTTCGTTGCACAACTTTAgacgttaaattgaaaataggtcHDR templates:Sog.int LA-DSR-f gtacgtgaattccgaaactcgcgtgtgttatctaSog.int LA-DSR-r ctagcggcggccgctaactaaagattttgactagtSog.int RA-DSR-f gtacgtggcgcgccatcccgggatttgtgccSog.int RA-DSR-r ctagcgctgcaggcggcagacagttgaataaaSog.dist LA-DSR-f gtacgtcccgggtccatccccacaccatttatSog.dist LA-DSR-r ctagcggcggccgcgaatacgtggaattcctttcgSog.dist RA-DSR-f gtacgtactagtaagagtccacctatcatcccagtSog.dist RA-DSR-r ctagcgggcgcgccagatgcgccagaagtacgCRISPR-Cas9 Deletions:Deleted base pairs capitalized. Indel/added sequence shown in bold italics.brk\u03945\u2019:tggtattaaaactgaaaatcaatctaaaaatcaaccattgataacattttattgaatcaaaccaaaagccaaattgattcctgaatccaaaagacccgatgaaggatcgaacaggtactacgatgatattggtcgGAAAATACCTGCGCATCCTGGTGGTTTATGGTGCGGCCGTAAATGCAAGCCAAGTTCTTTACGGCTTCTCTGGCACAAACCCTAAATGTGGATTACGCTAATATTGCCCCCCCTAATAAAAACGGTCGTTGTCCAGGGCCGAATATTGCGTCTGATTGGTTTTTCCCACGATTACAATTAGCCGGACGGACACAAACTGACCTGAGCTGACCCGCAAAAAGACACGGTTGTCCGGCAGTCGGAACTGAAGGAAACTAAAGGAAACTGAGGGCAGGTCAGCGCTATGGATTGTGCACTAAGTTGCTTAATCCGACGGGAAATCCAAAACACAACCCGAGCCCGATCCTTCGCTCCTTCGATTTAAGCCAAAGTTAGAGGCACAGGCACACATGTGTGTTTGGTTTGAACGGGAAAGCCCCATTTTAAAGCTGGCCAACCAACGGCAACACATGTTCATGTTAGGACCGATACAGGTTGACATTCCCTGGAAGGATGCACCTCTGGGAGATTCCCACAACCGGCAGCAGGTCATGTCCAACCGATCGTTGCGGGAGCCACTTGTCCCGAAAAAATCCAAAGAAACTATCAAGTGGCGTTTAGGGAAACTCAAAACTTTCCAACCACACCATATCTTTCTAACGCCACACAATAAACTGGTATGATCACTGTTAAGATCAAAATGGGCTAAATAAAGCGATCATGAATCATTTACTATAAACTGAAGAGTTTTCTATTCTTTAATATAAAAGAAAAGATGAGTAACACCCTACAAAAATTTTAGGACTACAGATCTACCCACCGATGATGATCAACCCTTCCCAAAAAAAAGTCACAGCTTGTATTTCGTAAAGATTTCAGATTTCTTAAGACACGATCACCAACTGATCTATGGACTTCTTAAACCATTGGGCTTTCGTTGCACAACTTTAGGGATTTGTTTTTTTTTTTTTTTTTTTGATTCCACATCCGATTTCTTCTTCGAAGCACTTCGATCTTTTCTCacatcagtttgatcgacaatcgacgatcgatggctgtgtgaaaacaagtttcgtaactatgtaaattaacctccabrk\u03943\u2019:GGAGAGCAGAGAGCAAGCAGGAGCCAAGAGGCAAAGCCAAAAGAAATGCGTTTCTTTTATTTTTGACGCGTCTTAGATTCTTCCTCTGCCGCTTCTTCTCTCGTTTTTTTCTTTCACTTTTTTGTTTTTTTTTTTTTTGTTTTTGATTTCTTGGAATCGCATCTACTTTGAACCTTCCACGTTCCGACGGAAGACTCAGAGACTCGACTGCATTGTATATTTGAATTTCATTTCAATTTAAATACGATTTTCTATGAAAATGCACGCACCACAAATACGTGCAATCGTTATCCAAACCATCTACCCCTCCCCTCCTCCCCTTTATCACCATGCTCTTTGCCTCCGCCGTCCTCTGCTTTTTTTTTGTTTTTTACCCCATACATGTACATAATACGATCCAAGTTTCATGAAAAAAACCGGAGAAACAAATTGATCATGTTTTGAATGCATTTCTGTTTGATTTCAATTTCGATTTGTGTACTCCATAAATGCGAAAGAATCTTGGATTGGAGTTGGGGGGATAAAATGGATGGATATATGGGGTGGGGTAAATCAGAATTATACGGCATATGCGGATCTAATCTAATAATATACAATATTACTGAGTCGCATACCCTAGAAATCGCACATTGATTAGCACAACCTGTCCAAATTATCCGAGCAAAATCCCCCCAAATCACGCAACACTCTAAACTGATCCGAAAGATCCTAACAGTTGAAAATCCACCTAGTCGAGCTAAAACCAAATTGTCAGAGGATTGCCTTGAATGGCCCACTTAAGCCAGGCGTGCGGCTGAAAGGAGCCGAGGATTCCTTGCCGAATCCGAATCGGAATCCTGTGCCAAGGACAGCATGCAACGCACCGATGCGAGTGCCGAATGACCTTTGGGAAAATACGTGTTGATCGACATCCATCGGCAATTGTTTGAGATCTTCGGAAATCTCTCACCTTTCGAATGGGAAATTCCTTAGGATCATCGAGCTAGCTGGTCAGAGAAGGCCGTTCATAAGCATGCAAATAAAGATTTCACACCTCGGCCAGCGCACTAGGGTGCATGGTGCAGCATAGGGCTCTCAGCCAAAGGACACGAAACGAAATGCATGGAGATCGGGACACCTGGGAAATACCCAACCTGCCCGAAACATATGGACGCACAACGCACAGGAGGGATCTCTCGCTCTGAATGGAAAGCAGAAACTCTCTATAGGCACTAGCTATCCATCCAAGTGGTTCCCGAAAAAAAAAACCCTCACAATATAATTCGTAAATCCCAGCCGCAGGTATTTAGTTCCGCCTTTGTCCATCTTTGGAATTCCCAATGCTATTATTGCACCTAAGCCACTGGAGTGGGAGGTTTTGCAGTGTGTGTGCGCGTTGCAAGTTGCTGCCGGTGAGCAACACATCGAATTCGTCCCATCTCTGTCTGGCCGCGACTCGTCATCTCTCTCTCTCTCTCTCTCTCTCTGATCTCTCTGTCCCGCTCTGGTTCGTGCTCTTGCTGTCGCCGGCGCCGCAGGTCAACAGCGATGTGTGGCATTTTCTAACAGTTGCTGCTGCTGTCGCAGGTTGCAGGTTGCATGTTTTGTTGCAGTTGCTGCAGCTTATATGACGCTTACATTTGTTATATGTATCTGCCACTGGGGGTTTTCCGTCAATATTGCAGGTTGCATAGCTTATGCTGCAGCTTCTATTGATGACTAATTTATTGTTGCTTGCTTGCTTGAGCAAGTTGCTTTAGGTTTACATTAAAACGGCACATTTCTTGGCATCAGTTGCACTCTCTGTTGGTATAATTGTTGCTGTTTCTAGTATCAAGTGTACTAATGTTGCAGGTGCTGCGCATGCATATGTTGCAAGTCTGTTGTTTATTGTAATGTACCCATAAAACTGCATATATTTATACATAGGTGTTTAGATTTCTACTATTATCGCTGAAAACTTGTTTTTCTATTCAATCTTGCTCATAAAATGGTTCAAATATATATAGATTTGCTGCACTCCCGACTTAATTCTTCCAAAATGGCTGTTAAATTTCAAACTATTGCCCATGTTGTTTTCGTTTAAATCGCTTATGGTTTTTCGGATGTTGTTTTTCGTTCAATCGCTGGCTGATTTTCTTGCTCTCCCGAAAATCCACACGCACATTTTCATTGTCGACAAATTTTCAGACGGCAGGACAAAGAATTTTTCATTCGTTTGTTTTCCACGCACTCGGTCTGAATGGCCAAAAAGGAAAATGTGACGAGCTAGAGGGAAATAGGGCACATTTTTGGGGGCAAGCGAGATGACCAAAAAAGACCTGCGATCCGTTCCGATCCGCTCCGGGTCGCTTTTCCACCCCgggggcattgttttcagaccgagagtatatagtagaatctcctattatatcttctattcctatttgtatttgtatttgtacgagacgggcagaaagaacgagaggaatggagatagagggbrk3\u2019 in brk\u03945\u2019\u03943\u2019:gaagcgaatgccaatgcacaaggagctaaacgagtgcgagcgagacgggcagaaagaacgagaggaatggagatagagggAGCAGGAGCCAAGAGGCAAAGCCAAAAGAAATGCGTTTCTTTTATTTTTGACGCGTCTTAGATTCTTCCTCTGCCGCTTCTTCTCTCGTTTTTTTCTTTCACTTTTTTGTTTTTTTTTTTTTTGTTTTTGATTTCTTGGAATCGCATCTACTTTGAACCTTCCACGTTCCGACGGAAGACTCAGAGACTCGACTGCATTGTATATTTGAATTTCATTTCAATTTAAATACGATTTTCTATGAAAATGCACGCACCACAAATACGTGCAATCGTTATCCAAACCATCTACCCCTCCCCTCCTCCCCTTTATCACCATGCTCTTTGCCTCCGCCGTCCTCTGCTTTTTTTTTGTTTTTTACCCCATACATGTACATAATACGATCCAAGTTTCATGAAAAAAACCGGAGAAACAAATTGATCATGTTTTGAATGCATTTCTGTTTGATTTCAATTTCGATTTGTGTACTCCATAAATGCGAAAGAATCTTGGATTGGAGTTGGGGGGATAAAATGGATGGATATATGGGGTGGGGTAAATCAGAATTATACGGCATATGCGGATCTAATCTAATAATATACAATATTACTGAGTCGCATACCCTAGAAATCGCACATTGATTAGCACAACCTGTCCAAATTATCCGAGCAAAATCCCCCCAAATCACGCAACACTCTAAACTGATCCGAAAGATCCTAACAGTTGAAAATCCACCTAGTCGAGCTAAAACCAAATTGTCAGAGGATTGCCTTGAATGGCCCACTTAAGCCAGGCGTGCGGCTGAAAGGAGCCGAGGATTCCTTGCCGAATCCGAATCGGAATCCTGTGCCAAGGACAGCATGCAACGCACCGATGCGAGTGCCGAATGACCTTTGGGAAAATACGTGTTGATCGACATCCATCGGCAATTGTTTGAGATCTTCGGAAATCTCTCACCTTTCGAATGGGAAATTCCTTAGGATCATCGAGCTAGCTGGTCAGAGAAGGCCGTTCATAAGCATGCAAATAAAGATTTCACACCTCGGCCAGCGCACTAGGGTGCATGGTGCAGCATAGGGCTCTCAGCCAAAGGACACGAAACGAAATGCATGGAGATCGGGACACCTGGGAAATACCCAACCTGCCCGAAACATATGGACGCACAACGCACAGGAGGGATCTCTCGCTCTGAATGGAAAGCAGAAACTCTCTATAGGCACTAGCTATCCATCCAAGTGGTTCCCGAAAAAAAAAACCCTCACAATATAATTCGTAAATCCCAGCCGCAGGTATTTAGTTCCGCCTTTGTCCATCTTTGGAATTCCCAATGCTATTATTGCACCTAAGCCACTGGAGTGGGAGGTTTTGCAGTGTGTGTGCGCGTTGCAAGTTGCTGCCGGTGAGCAACACATCGAATTCGTCCCATCTCTGTCTGGCCGCGACTCGTCATCTCTCTCTCTCTCTCTCTCTCTCTGATCTCTCTGTCCCGCTCTGGTTCGTGCTCTTGCTGTCGCCGGCGCCGCAGGTCAACAGCGATGTGTGGCATTTTCTAACAGTTGCTGCTGCTGTCGCAGGTTGCAGGTTGCATGTTTTGTTGCAGTTGCTGCAGCTTATATGACGCTTACATTTGTTATATGTATCTGCCACTGGGGGTTTTCCGTCAATATTGCAGGTTGCATAGCTTATGCTGCAGCTTCTATTGATGACTAATTTATTGTTGCTTGCTTGCTTGAGCAAGTTGCTTTAGGTTTACATTAAAACGGCACATTTCTTGGCATCAGTTGCACTCTCTGTTGGTATAATTGTTGCTGTTTCTAGTATCAAGTGTACTAATGTTGCAGGTGCTGCGCATGCATATGTTGCAAGTCTGTTGTTTATTGTAATGTACCCATAAAACTGCATATATTTATACATAGGTGTTTAGATTTCTACTATTATCGCTGAAAACTTGTTTTTCTATTCAATCTTGCTCATAAAATGGTTCAAATATATATAGATTTGCTGCACTCCCGACTTAATTCTTCCAAAATGGCTGTTAAATTTCAAACTATTGCCCATGTTGTTTTCGTTTAAATCGCTTATGGTTTTTCGGATGTTGTTTTTCGTTCAATCGCTGGCTGATTTTCTTGCTCTCCCGAAAATCCACACGCACATTTTCATTGTCGACAAATTTTCAGACGGCAGGACAAAGAATTTTTCATTCGTTTGTTTTCCACGCACTCGGTCTGAATGGCCAAAAAGGAAAATGTGACGAGCTAGAGGGAAATAGGGCACATTTTTGGGGGCAAGCGAGATGACCAAAAAAGACCTGCGATCCGTTCCGATCCGCTCCGGGTCGCTTTTCCACCCCggcattgttttcagaccgagagtatatagtagaatctcctattatatcttctattcctatttgtasog\u0394int:GCGGCCGCGGACATATGCACACCTGCGATCATAACTTCGTATAATGTATGCTATACGAAGTTATAGAAGAGCACTAGTAAAGATCTCCATGCATAAGGCGCGCCAAGGGGTATTAACTGTTGCCTGTTGCTTTCGACATTTCTACCTCCGCTGCGATTGCATAAGTTGCCAATGCCATTGCGCATACGCCGTGTCGTCTATATGGCTATATGGCTATATGGCTGTATGGTGCGGGGAAATCCCCGTAATCGCAGGTAGAATTCCAGCCGGTGCCGAGGCGGGACCTGCTCGCACCTCTAATCCCGCCAGGGTTTTCGGGACATGGGATATTCCCGACGGCACAGCATAGCACTCCGTTTTCTTTTTTTTTTTTTATTATTATTGTGTCCAGTTTTAATCCGGAAAGCGGGAATTCCCTTCCGCTCGCTGCCTGCACTGCGCTGCGCAGACGCATCGGCGTCCGTAAGCCGCTTACCAAAAAGATACGGGTATACCCAAATGGATGCCTGCCCATGTATATAGACCATTGGGTGGTATGGACCATGGACCATAAAGCGGCACCCAATTGCAATTTGTTGCAACTCACACGCTTGATTAGCTGCATTTCCTGTTTGCAAACCCCCCTCCCAATACTCCCTACACTCATATATTATATATGCATATAAATGTATGTGTATATTGCATACTATTATGTTCCCGAGTTGCAGTTTTTTTTTCCTTTTTTTTCCACTATTTCTTTCTTGGTTCGCTGCCGTTGTTGCATTTTCAATTAAAAACAGATTGCGGTTTACGCTGCAGCGACTTTGAAACGTTACTCGGCTTACACACAAAATGCAGCGCGATAGTAATGATGATTAACAGGTTATGTTAGGGCTTAGTTGATACGCCGTAGatcccgggatttgtgccggaaccggaacgaatatcgaatcaatcgcatatccttaaagttcgaactagtcaaaatctttagttasog\u0394dist:GGCCGCGGACATATGCACACCTGCGATCGTAGTGCCCCAACTGGGGTAACCTTTGAGTTCTCTCAGTTGGGGGCGTAGATAACTTCGTATAATGTATGCTATACGAAGTTATAGAAGAGCACTAGTGGGTAGTCCACATTCCATTCCATCGAAAGGAATTCCACGTATTCGCTGGGCGTTTCGGGGGCGGACTGGGCATATGGGCGTGGTGGGAGGGGGTGGGCGTCACGGGGCGTGGCCACACGTGAAACGGCTAATTTGTTGATGCAAATGCGGGTGTGGCCTGTTCTCGGATCGGAGTCCCCAGCACGCGCCCACAAATAAATAAATGGCTGGAGATGGAAATGGAAACCGATGACGGGGAATAGGGAATGACGGCTGGGTAGCGGGGGAGACGGGGATGGGAACTGGGGGGCTGGGGTCTTGGACCGAGATCGCAGCCAATGGCGACAATTGGCATTCACCGTCGCAATTAATCATCATCACCGTCCGATTGACAATCGGGCCACAAATAAATCAAAATCAAAGGCATAAGAAAGCGAATGCAGCGTCGGCATACAACTGCACTGAAAGAAAAATATGTATGAAGCTTCTGGTCAAGCAAACCGCAAGAAGCGCATTTAAGCTCAAGTATGCTCAAGTATTTGTTGTGTAACTTTAGATGATTCAAAATCGCTTTCTTATGTCTTTTTTGACTGCACTTGCCCTGAATTTCGTCGAGTGTAGTTCCCAATCATTGCAACAATTCATCAAGTATGCAAACACACGCGGCCGAATCAATCAATGGCGAGTAGTAGGAGTGCATGGAGGATGAGAAGGAGGAGAAGTTGGTTGAGAGGTCATCGTTGCGATTCTGCGATTCAGCAGTTCCACAGAAGGTGTCGTAATCCTGGACGCAAGGGTGCACGGACCAACTGACAGGGGCAAGTGCGTCCTGTGCCACCAGATGACGCACGATGCGGCCGGAAAAACCCAAAATCAAAAACCGAAAACCGAAAACCTGGTCAGAGTTTCCGAAAACCAAAGAGCCAACATCGAATGCGGCACAATAACCCGATTGTCTGCGAATACCCACGATGATCTAGAATCGCACGGAGAGCACTCTCACGCATCCGTGGCCATATGGGTGCGGCCAAATCGGAAATTCCCAGGACAGGTAGAATGCATTGGATATACGGGTATACGGATTGGAATTGGGATTGGGATTGGGACTAGCACCAGGTTGCAACGCCCGCCAAGAAGCCAATTTAAATAAGCAGCATAAACAAAAGCGACAGCGTTTTATGATCCCCGCTCCTTATCCTTGCACAAGGATATCGCCATGGCCACGCAGGTAGGAATAGCAGATATGGCGGCAATGATGCGCCAACCGCACTGCTTCGTCCTGGTCCTGGTCGGATGGGCTTTTCCCACGCAACCGCGACCTTATCTGCGCCCCTTTTATGAGGCTGCATCTGTTTTCGCACCTCGATGCCGTTGGCATTATAGCCACATGTGTATGGTGGGAATTTCCGATCGACCAGCCTACCTGTTCCGCTGAAACCCGGGAATCTGTCCATCCTGAGCTTCCACACACACACACACACACACACAGGTCAGTCGGCATCAATTGGCTGCCATAAACATATAACAATCAATATTGAATCCTTTATCGTAGAATTTGTTGTATATGCCCATTGCAGTCCTTCGATTAAATGATGGCTAAAATGAATAAAATGAGTTGCTAGGTTCGAAGTAGGTGAAAACTGGTCAATAAGCCATATCCTTTATGATTAGATATTCTAGTAATCTGGCATGAAATACACAAATATAATAGCGAGTGATTAGTTTTATATAAGTATTTCTATTAAACCTCTAATAATTACCGCTTTACATATAGATTTTTTTAGTTGGATGCGGGCTTTAGTACCATATAGCTTTACCTGAATCTTATCGTTTGCTTTGGCTGATCCCCCAGGATCTGCGACATAATGCCAAACACAAGCAACTTGTTCGCCTGATCTTCGCAATCCCAGCAGGTTAACTACCCGATTTTATCGAGCAACAGCAAACTCTTTGAACTGATTTGCTATATTTCATTTATGGCCGATTCCAAGTCCATGTCCATTTCCCATTTCCATGTTCGTGTCCAAGTCCGTTGTTCGTTGTCCATTTCGATGTCAGTCATTCATTTTTGTGCTCGACTGCTAATGCCTTAAAGCCAAAATCCATTTCGTCGTTTGGCCATATAATCCAACATTATACATTAAATATGGCACTTGAAAGCACCGCGCGTCTGCATGCCAATTTCAAGGCCCCTTTTCCTCCACTTCACCACCCACAGCATACCATCCCATATCCCACCGGAATTTTCCGATTTTCCCCATTTTCCCCCAACAACACCACCACCTTGCTGCGATCGCACTTGTCGCTCGCGCCGGCGCCTTCAGGGTCGTCAATTTTTTGAGCGACATTTCGAAACGGTGGCTAAAATCACAAAAGCCAACAGAAAAAAATAAAAATCAACTATGGCGAAAAAGTAATACAAATGAAATCCGAAAACAAATAAGCGATTTTGTTTGTGGGGAGAAATGATGATGATCGGCGCCAGACGGACATAAAAACGCCAAAAGAAACGGAAAGGAAGCCTGGGAAAGGCGCATTTCCACTTTGTTTATTCGGAGCTCTGAAGCTCTTTTTAATGGTTTCTTTTTTTTTTTTTTTTTGGCCAAATGGCCAATTAATGCGATTTACTTGATACGCTTTTGCATTTTGTTGTTGCTTTACGACGGCGGATATTAATTAATTGGCCCGTCTCTGGGGAGCAATTCCCATTGTGCCCATCaagagtccacctatcatcccagttcgcagtccaaacctgacaatcgattaaaattacctctccagttgcacagatcaacggggtagtccacattccattccatcgaaaggaattccacgtattcgcS1 Fig(A) Dark field images of lateral view of cuticle preps from first instar larvae. (B) Bright field images taken with 40X objective of dentical bands in the A2 abdominal segment. Ventral view with anterior to the left.(TIF)Click here for additional data file.S2 Figbrk\u03945\u2019\u03943\u2019) or 5 individual embryos are shown by black bars. WT shown in black, brk\u03945\u2019 in gold, brk\u03943\u2019 in blue and brk\u03945\u2019\u03943\u2019 in grey. Genes broken into groups based on expression level for ease of display.Results for 70 genes assayed by NanoString in nc14C (late stage 5) embryos see . The res(TIFF)Click here for additional data file.S3 FignetA and (B) stumps gene loci. Brk binding is significant only in late stage 5. Flybase defined protein coding regions for each gene shown in blue under Brk ChIP-seq tracks.(A-B) Screen shots from database of Brk ChIP-seq data showing (TIF)Click here for additional data file.S4 Figtsg and (B) vn, or immunostained with an antibody to (C) dpErk. (D) Cross section of stage 6 embryos showing zen expression (green). Magnified image shows only dorsal one-third of embryo. Representative images for each genotype, further quantified in E. (E) Box plot of width, in number of cells, expressing zen. P-values determined by Welsh\u2019s t-test comparing brk\u03945\u2019, brk\u03943\u2019, and brk\u03945\u2019\u03943\u2019 to WT for zen were P = 0.4, P = 5.5x10-5, P = 0.06, respectively. Significance indicated in graph by *P<0.05, ***P<0.0001. (F) Percentage of embryos showing normal (blue) vs disrupted (orange) expression of Race in early stage 6 embryos. Number of embryos counted for each graph in this figure indicated under genotype.(A-C) Dorsal view of stage 6 embryos hybridized with riboprobes to (A) (TIF)Click here for additional data file.S5 Figbrk and egr. egr expression is diminished or lost in the brk\u03945\u2019 and brk\u03945\u2019\u03943\u2019 embryos. (B) In situ hybridization of late stage 5 embryos, dorsal views, with riboprobes to egr. egr expression remains low in the brk\u03945\u2019 but is expanded in the brk\u03943\u2019 embryos. (C) FISH staining of late stage 5 embryos, lateral views, with riboprobes to Cv-2. White arrows indicate expanded Cv-2 expression in the brk\u03945\u2019 and brk\u03945\u2019 \u03943\u2019 embryos. (D) Model of canalization loop acting to regulate amnioserosa cell number, reproduced from [egr, (F) Cv-2, and (G) zen loci.(A) FISH staining of early stage 5 embryos, lateral views, with riboprobes to ced from . (E-G) Sced from showing (TIF)Click here for additional data file.S6 Figtld (green), ind and sna (both purple). All embryos are trans-heterozygous females of the genotypes indicated. Consistent with the patterns seen in the homozygous brk CRISPR mutants, tld is expanded ventrally, beyond the domain of ind expression in the trans-heterozygous embryos with brk\u03945\u2019 and brk\u03945\u2019 \u03943\u2019 but not significantly in brk\u03943\u2019. (F) Comparison of number of amnioserosa cells in homozygous brk enhancer mutants to trans-heterozygous combinations with brk- gene mutant. Homozygous mutant data is reproduced from (A-E) FISH staining of late stage 5 embryos, lateral views, with riboprobes to (TIF)Click here for additional data file.S1 DatasetExcel file containing raw counts for all graphically represented data depicted in Figs (XLSX)Click here for additional data file."} diff --git a/PMC_clustering_410.jsonl b/PMC_clustering_410.jsonl new file mode 100644 index 0000000000000000000000000000000000000000..dbc172e4bd2fa50e4072ec1e2f51b56c508e9d05 --- /dev/null +++ b/PMC_clustering_410.jsonl @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:8eb8a50728c6d831cd4e5ccc130efd4006cf9b4ada25c08b435d1b22265ba3c3 +size 30257516 diff --git a/PMC_clustering_411.jsonl b/PMC_clustering_411.jsonl new file mode 100644 index 0000000000000000000000000000000000000000..0c1a4f754d152cd13b768327db9506cf3dbec769 --- /dev/null +++ b/PMC_clustering_411.jsonl @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:43287e38a9717400fa6675426e2145b3947e4e7611c71f0c79ee6fe4570402c0 +size 113586298 diff --git a/PMC_clustering_412.jsonl b/PMC_clustering_412.jsonl new file mode 100644 index 0000000000000000000000000000000000000000..4ca9d79f6a24a36844ec1893ac8d22a5656d86b0 --- /dev/null +++ b/PMC_clustering_412.jsonl @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:bf9317caeb47f7c58aa69a345181bdc62de19320c25166619e4d34fcd9e3b187 +size 50757258 diff --git a/PMC_clustering_413.jsonl b/PMC_clustering_413.jsonl new file mode 100644 index 0000000000000000000000000000000000000000..2ff06a9f31c281a25b12a9d8e5a404c8add45366 --- /dev/null +++ b/PMC_clustering_413.jsonl @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:a8dff4b4423a5f5a272daa89d05429a484b959c483183183388726c667440651 +size 69819462 diff --git a/PMC_clustering_414.jsonl b/PMC_clustering_414.jsonl new file mode 100644 index 0000000000000000000000000000000000000000..1c068dbcaf940bf893623bf788be3c9083718138 --- /dev/null +++ b/PMC_clustering_414.jsonl @@ -0,0 +1,1105 @@ +{"text": "The correct spelling is: Maralyn Foureur. The correct citation is: 1. Asefa F, Cummins A, Dessie Y, Foureur M, Hayen A (2020) Midwives\u2019 and obstetricians\u2019 perspectives about pregnancy related weight management in Ethiopia: A qualitative study. PLoS ONE 15(12): e0244221."} +{"text": "The Pan African Medical Journal. 2018;30:266. doi:10.11604/pamj.2018.30.266.13209.Ce Corrigendum modifie l'article original Dans l'article original archiv\u00e9 sur Pubmed, le nom de l'auteur Abdelkader Jalil El Hangouche est archiv\u00e9 Hangouche AJE au lieu de El Hangouche AJ ."} +{"text": "Genes [The authors wish to make the following correction to their paper published in Genes :In the original article, the supplementary materials on page 14 were mistakenly listed as follows:Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4425/10/2/74/s1, Supplementary File 1: Table S1. Full lists of DEGs; Table S2. L6_HR_DAVID; Table S3. Viral read counts; Table S4. Differentially expressed viral genes; Table S5. L7_HR_DAVID; Table S6. MDV QTL candidates; Table S7. MDV QTL data; Table S8. Genes only expressed in L6; Table S9. Genes only expressed in L7. Supplementary File 2: Figure S1. MAC_inherent_pathways; Figure S2. MAC_inherent_biofunctions.This should be replaced with:Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4425/10/2/74/s1, Supplementary File 1: Table S1. MDV QTL data; Table S2. Full lists of DEGs; Table S3. Viral read counts; Table S4. Differentially expressed viral genes; Table S5. L6_HR_DAVID; Table S6. L7_HR_DAVID; Table S7. Genes only expressed in L6; Table S8. Genes only expressed in L7; Table S9. MDV QTL candidates. Supplementary File 2: Figure S1. MAC_inherent_pathways; Figure S2. MAC_inherent_biofunctions.https://www.mdpi.com/2073-4425/10/2/74.The authors apologize for any inconvenience caused and the change does not affect the scientific results. The manuscript will be updated, and the original will remain online on the article webpage at"} +{"text": "Scientific Reports 10.1038/s41598-020-59286-8, published online 12 February 2020Correction to: This Article contains a typographical error in Table 2 in the Parameter column where,Portal vein enlargement(diameter \u22651.2\u2009cm)should read:Portal vein enlargement(diameter \u22651.5\u2009cm)"} +{"text": "Anthuroidea is presented, based on previous taxonomic or ecological literature and recent collections. The present study, a part of the subproject on the Biodiversity of Sultan Iskandar Marine Park, recognised 24 species in 12 genera and 5 families from Peninsular Malaysia. An extensive list of bibliographical references, detailed information on habitat and distributional records, museum locations of type material are provided for each species. Amongst the listed species, 11 are recently discovered Malaysian species belonging to the genera Amakusanthura Nunomura, 1977, Apanthura Stebbing, 1900, Expanathura W\u00e4gele, 1981, Leptanthura G. O. Sars, 1897, Kupellonura Barnard, 1925, Pendanthura Menzies & Glynn, 1968 and Tinggianthura Chew, Rahim & bin Haji Ross, 2014. Our records were limited to shallow subtidal reefs of peninsular Malaysian coast, suggesting that the number of species in the list may rise with an extensive survey.An up-to-date checklist of the Peninsular Malaysian marine Anthuroidea of the Peninsular Malaysia comprises 24 species in 12 genera and 5 families, including some new distributional data.The up-to-date checklist of marine Isopoda is one of the most successful and rapidly evolving malacostracan orders, displaying a tremendous diversity in marine, terrestrial and continental waters. Although isopods have colonised almost every niche and are the most important group in terms of specific diversity, they have been largely ignored in studies dealing with conservation strategies. According to Tropical reefs are known to support enormous biodiversity and portray a high priority for conservation action amongst marine ecosystems . A fauna6\u00b021'56.05\"N, 99\u00b040'31.13\"E), Pulau Payar , Pulau Pangkor and Port Dickson . The following collection localities were from the east coast of MP: Pulau Perhentian , Pulau Tioman , Pulau Seri Buat , Pulau Aur , Pulau Dayang and Pulau Tinggi ; amongst coral rubble. Littoral 0.5 to 15 m depth (present study)In fine sand to medium sand from around \u2018; eventRemarks: C. Melvin; Record Level: collectionCode: UKMMZ-1571HONG KONG\u2015Tolo Channel , Conic Island Cave. MALAYSIA\u2015Pahang State: Pulau Tioman (new record).Fig. M\u00fcller, 199290B7893E-BE01-5EAE-A2FF-59F056823BC0http://www.marinespecies.org/aphia.php?p=taxdetails&id=255383ApanthurabruscaiType status:Holotype. Location: island: Pulau Babi Besar; country: Malaysia; stateProvince: Johor; verbatimCoordinates: N2\u00b050.483', E104\u00b09.566'; Event: habitat: Fringing-reef and reef-flat under coral rocks mainly covered with coralline algae, intertidal (M\u00fcller 1992a); Record Level: modified: male, 3.4 mm; datasetID: ZMB 26953Only known from type locality .This species record is based on literature only. No specimen was collected during the present study.Negoescu, 19971717BC2B-3042-50A1-850A-4EC97FD0B98Chttp://www.marinespecies.org/aphia.php?p=taxdetails&id=255402MF680510ApanthurapariensisType status:Other material. Location: island: Pulau Pangkor; country: Malaysia; stateProvince: Perak; verbatimCoordinates: N4\u00b011'22.32\", E100\u00b032'50.22\"; Event: eventDate: 15 Apr 2014; habitat: Littoral 0.5 to 3 m depth, amongst coral rubble ; Record Level: collectionCode: UKMMZ-1583; UKMMZ-1584; UKMMZ-1585Type status:Other material. Location: island: Pulau Langkawi; country: Malaysia; stateProvince: Kedah; verbatimCoordinates: N6\u00b021'56.05\" E99\u00b040'31.13\"; Event: eventDate: 4 Nov 2013; habitat: Intertidal, amongst coral rubble; Record Level: collectionCode: UKMMZ-1586Type status:Other material. Location: island: Pulau Langkawi; country: Malaysia; stateProvince: Kedah; verbatimCoordinates: N6\u00b021'56.05\" E99\u00b040'31.13\"; Event: eventDate: 8 Mac 2015; habitat: Intertidal, amongst coral rubble; Record Level: collectionCode: UKMMZ-1587INDONESIA\u2015Pari Island, Java Sea . MALAYSIA\u2015Perak State: Pulau Pangkor (new record).\u2015Kedah State: Pulau Langkawi (new record).Fig. 6D116825-BF79-58DD-A5B2-6C9FFB7E06AFhttp://www.marinespecies.org/aphia.php?p=taxdetails&id=255410MF680509AmakusanthurastockiApanthurastockiType status:Other material. Occurrence: recordedBy: C. Melvin; Location: island: Mentinggi, Pulau Tinggi; country: Malaysia; stateProvince: Johor; verbatimCoordinates: N2\u00b016'21.67\", E104\u00b07'18.61\"; Event: eventDate: 19 April 2013; habitat: Littoral 0.5 to 3 m depth, amongst coral rubble ; Record Level: collectionCode: UKMMZ-1576Type status:Other material. Occurrence: recordedBy: C. Melvin; Location: island: Kg Pasir Panjang, Pulau Tinggi; country: Malaysia; stateProvince: Johor; verbatimCoordinates: N2\u00b017'37.96\", E104\u00b06'1.97\"; Event: eventDate: 18 Dec 2012; habitat: Littoral 0.5 to 3 m depth, amongst coral rubble ; Record Level: collectionCode: UKMMZ-1577Type status:Other material. Occurrence: recordedBy: C. Melvin; Location: island: Sebirah Kechil, Pulau Tinggi; country: Malaysia; stateProvince: Johor; verbatimCoordinates: N2\u00b018.622', E104\u00b005.616'; Event: eventDate: 18 Apr 2013; habitat: Littoral 0.5 to 3 m depth, amongst coral rubble ; Record Level: collectionCode: UKMMZ-1578Type status:Other material. Occurrence: recordedBy: C. Melvin; Location: island: Pulau Seri Buat; country: Malaysia; stateProvince: Pahang; verbatimCoordinates: N2\u00b041'13.59\", E103\u00b055'25.99\"; Event: eventDate: 19 Apr 2014; habitat: Littoral 0.5 to 7 m depth, amongst coral rubble ; Record Level: collectionCode: UKMMZ-1579Type status:Other material. Occurrence: recordedBy: C. Melvin; Location: island: Kg Pasir Panjang, Pulau Tinggi; country: Malaysia; stateProvince: Johor; verbatimCoordinates: N2\u00b017'37.96\", E104\u00b06'1.97\"; Event: eventDate: 15 Jun 2015; habitat: Littoral 0.5 to 3 m depth, amongst coral rubble ; Record Level: collectionCode: UKMMZ-1580Type status:Other material. Occurrence: recordedBy: C. Melvin; Location: island: Pulau Dayang; country: Malaysia; stateProvince: Johor; verbatimCoordinates: N2\u00b028'40.90\", E104\u00b030'19.12\"; Event: eventDate: 26 Jul 2016; habitat: Littoral 0.5 to 3 m depth, amongst coral rubble ; Record Level: collectionCode: UKMMZ-1581Type status:Other material. Occurrence: recordedBy: C. Melvin; Location: island: Pulau Aur; country: Malaysia; stateProvince: Johor; verbatimCoordinates: N2\u00b028'17.24\", E104\u00b030'53.14\"; Event: eventDate: 26 Jul 2016; habitat: Littoral 0.5 to 3 m depth, amongst coral rubble ; Record Level: collectionCode: UKMMZ-1582SRI LANKA\u2015Indian Ocean ; MALAYSIA\u2015Johor State: Pulau Besar; Pulau Tinggi; Pulau Seri Buat (new record).Fig. M\u00fcller, 199248B4900A-914E-5D55-8559-2B11DD147ABFhttp://www.marinespecies.org/aphia.php?p=taxdetails&id=255413ApanthuratiomanaeType status:Other material. Occurrence: recordedBy: C. Melvin; Location: island: Pulau Tioman; country: Malaysia; stateProvince: Pahang; verbatimCoordinates: N2\u00b054'15.44\", E104\u00b0 6'1.08\"; Event: eventDate: 18 April 2014; habitat: Amongst coral rubble, littoral 0.5 to 7 m depth; Record Level: collectionCode: UKMMZ-1572MALAYSIA\u2015Pahang State: Pulau Tioman .Fig. M\u00fcller, 1991A1DC35BE-90E0-5F31-897B-C41D039A2F67http://www.marinespecies.org/aphia.php?p=taxdetails&id=255423CyathurabentotaeType status:Holotype. Event: habitat: Fringing-reef and sabellid reef; dead coral substratum, 1 to 3 m depth; Record Level: collectionCode: ZMB 26953; 1 cr (ZMB 26956)SRI LANKA\u2015Indian Ocean ; MALAYSIA\u2015Pahang State: Pulau Tioman.This species record is based on literature only. No specimen was collected during the present study.Barnard, 1925D8243EE3-31A8-5BF4-8F3E-CE91F61E8334http://www.marinespecies.org/aphia.php?p=taxdetails&id=258320MesanthuraalbolineataType status:Holotype. Location: country: Singapore; Event: habitat: Acropora sp. and Pocilloporadamicornis .Fringing reef, outer reef-flat, reef-margin and upper coral-slope; dead coral substratum, ; Record Level: collectionCode: ZMB 26938SINGAPORE\u2015; MALAYSIA\u2015Johor State: Pulau Babi Besar.This species record is based on literature only. No specimen was collected during the present study.M\u00fcller, 1993C91FB4BC-D900-5EF9-A294-F49DAD7B2F93http://www.marinespecies.org/aphia.php?p=taxdetails&id=258321Mesanthuraasiatica : 20\u201325, figs. 1\u201325.Type status:Holotype. Event: habitat: Acropora sp. and Pocilloporadamicornis Fringing reef, outer reef-flat, reef-margin and upper coral-slope; dead coral substratum, ; Record Level: collectionCode: ZMB 26939a.Type status:Paratype. Record Level: collectionCode: ZMB 26939bType status:Other material. Location: island: Pulau Tioman; country: Malaysia; stateProvince: Pahang; verbatimCoordinates: N2\u00b054'15.44\", E104\u00b06'1.08\"; Event: eventDate: 18 April 2014; habitat: Amongst coral rubble, littoral 0.5 to 7 m depth; Record Level: collectionCode: UKMMZ-1588; UKMMZ-1589; UKMMZ-1590Type status:Other material. Location: island: Kg Pasir Panjang, Pulau Tinggi; country: Malaysia; stateProvince: Johor; verbatimCoordinates: N2\u00b017'37.96\", E104\u00b06'1.97\"; Event: eventDate: 16 Jun 2015; habitat: Amongst coral rubble, littoral 0.5 to 3 m depth; Record Level: collectionCode: UKMMZ-1591MALAYSIA\u2015Johor State: Pulau Babi Besar ; Pulau Tinggi (new record);\u2015Pahang State: Pulau Tioman (new record).Fig. M\u00fcller, 199362D3AD96-641F-5D52-A376-92B6FA074481http://www.marinespecies.org/aphia.php?p=taxdetails&id=258340MesanthurakilianiType status:Holotype. Location: island: Pulau Tioman; country: Malaysia; Event: habitat: Acropora sp. and PocilloporadamicornisFringing reef, outer reef-flat, reef-margin and upper coral-slope; dead coral substratum, ; Record Level: collectionCode: ZMB 26940aType status:Paratype. Location: island: Pulau Tioman; country: Malaysia; Event: habitat: Acropora sp. and PocilloporadamicornisFringing reef, outer reef-flat, reef-margin and upper coral-slope; dead coral substratum, ; Record Level: collectionCode: ZMB 26940b.MALAYSIA\u2015Pahang State: Pulau Tioman;\u2015Johor State: Pulau Babi Besar.This species record is based on literature only. No specimen was collected during the present study.Kensley, 1980A525220C-59DC-51C9-9B6F-BB52C2E29BBChttp://www.marinespecies.org/aphia.php?p=taxdetails&id=211377MesanthuraproteiType status:Other material. Location: island: Batu Malang, Pulau Tioman; country: Malaysia; stateProvince: Pahang; verbatimCoordinates: N2\u00b054'15.44\", E104\u00b0 6'1.08\"; Event: eventDate: 18 April 2014; habitat: Amongst coral rubble, littoral 0.5 to 7 m depth; Record Level: institutionCode: UKMMZ-1592; UKMMZ-1593MOZAMBIQUE\u2015South of Inhambane ; MADAGASCAR\u2015Abrolhos Islands; THAILAND\u2015Ko-Sichang; KENYA; MALAYSIA\u2015Johor State: Pulau Babi Besar;\u2015Pahang State: Pulau Tioman, (new record).Fig. Kensley & Schotte, 200068F3E9AF-BF62-5C43-A90F-7D82386902EBhttp://www.marinespecies.org/aphia.php?p=taxdetails&id=211379MesanthuraquadrataType status:Other material. Location: island: Pulau Seri Buat; country: Malaysia; stateProvince: Pahang; verbatimCoordinates: N2\u00b041'13.59\", E103\u00b055'25.99\"; Event: eventDate: 19 April 2014; habitat: Amongst coral rubble, littoral 0.5 to 7 m depth; Record Level: collectionCode: UKMMZ-1594, UKMMZ-1595Type status:Other material. Location: island: Batu Malang, Pulau Tioman; country: Malaysia; stateProvince: Pahang; verbatimCoordinates: N2\u00b054'15.44\", E104\u00b06'1.08\"; Event: eventDate: 18 April 2014; habitat: Amongst coral rubble, littoral 0.5 to 7 m depth; Record Level: collectionCode: UKMMZ-1596Type status:Other material. Location: island: Kg Pasir Panjang, Pulau Tinggi; country: Malaysia; stateProvince: Johor; verbatimCoordinates: N2\u00b017'37.96\", E104\u00b06'1.97\"; Event: eventDate: 15 June 2015; habitat: Amongst coral rubble, littoral 0.5 to 3 m depth; Record Level: collectionCode: UKMMZ-1597Type status:Other material. Location: island: Sebirah Kechil, Pulau Tinggi; country: Malaysia; stateProvince: Johor; verbatimCoordinates: N2\u00b018.622', E104\u00b005.616'; Event: eventDate: 15 June 2015; habitat: Amongst coral rubble, littoral 0.5 to 3 m depth; Record Level: collectionCode: UKMMZ-1598Type status:Other material. Location: island: Kg Pasir Panjang, Pulau Tinggi; country: Malaysia; stateProvince: Johor; verbatimCoordinates: N2\u00b017'37.96\", E104\u00b06'1.97\"; Event: eventDate: 13 Oct 2012; habitat: Assoc. artificial substrate unit at 3 m depth; Record Level: collectionCode: (29 juveniles) UKMMZ-1599Type status:Other material. Location: island: Sebirah Kechil, Pulau Tinggi; country: Malaysia; stateProvince: Johor; verbatimCoordinates: N2\u00b018.622', E104\u00b005.616'; Event: eventDate: 18 April 2013; habitat: Amongst coral rubble, littoral 0.5 to 3 m depth; Record Level: collectionCode: UKMMZ-1560SEYCHELLES\u2015Mah\u00e9 beach, Mah\u00e9 Island ; MALAYSIA\u2015Pahang State: Pulau Seri Buat, (new record); Pulau Tioman, (new record);\u2015Johor State: Pulau Tinggi, (new record).Fig. Chew, bin Abdul Rahim & Mohd Yusof, 201608F7B939-508A-539F-9E42-66584690E57Chttp://www.marinespecies.org/aphia.php?p=taxdetails&id=882015MF680512PendanthuratinggiensisType status:Other material. Location: island: Mentinggi, Pulau Tinggi; country: Malaysia; stateProvince: Johor; verbatimCoordinates: N2\u00b016'21.67\", E104\u00b0 7'18.61\"; Event: eventDate: 19 April 2013; habitat: Amongst coral rubble. Littoral 0.5 to 3 m depth; Record Level: collectionCode: UKMMZ-1541; UKMMZ-1542; UKMMZ-1543; UKMMZ-1544; ZRC 2016.0013; ZRC 2016.0014Type status:Other material. Location: island: Kg Pasir Panjang, Pulau Tinggi; country: Malaysia; stateProvince: Johor; verbatimCoordinates: N2\u00b017'35.08\", E104\u00b0 6'7.13\"; Event: eventDate: 16 Aug 2012; habitat: Amongst coral rubble. Littoral 0.5 to 3 m depth; Record Level: collectionCode: UKMMZ-1544Type status:Other material. Location: island: Sebirah Kechil, Pulau Tinggi; country: Malaysia; stateProvince: Johor; verbatimCoordinates: N2\u00b018.622', E104\u00b0 05.616'; Event: eventDate: 18 April 2013; habitat: Amongst coral rubble. Littoral 0.5 to 3 m depth; Record Level: collectionCode: UKMMZ-1545Type status:Other material. Location: island: Kg Pasir Panjang, Pulau Tinggi; country: Malaysia; stateProvince: Johor; verbatimCoordinates: N2\u00b017'35.08\", E104\u00b0 6'7.13\"; Event: eventDate: 15 June 2015; habitat: Amongst coral rubble. Littoral 0.5 to 3 m depth; Record Level: collectionCode: UKMMZ-1546Type status:Other material. Location: island: Sebirah Kechil, Pulau Tinggi; country: Malaysia; stateProvince: Johor; verbatimCoordinates: N2\u00b018.622', E104\u00b0 05.616'; Event: eventDate: 15 June 2015; habitat: Amongst coral rubble. Littoral 0.5 to 3 m depth; Record Level: collectionCode: UKMMZ-1621Only known from type locality Chew, bin Abdul Rahim & Mohd Yusof, 2016DF907F7D-6A68-5B81-A3B9-B35B00D74847http://www.marinespecies.org/aphia.php?p=taxdetails&id=882016PendanthuratiomanensisType status:Other material. Location: island: Batu Malang, Pulau Tioman; country: Malaysia; stateProvince: Pahang; verbatimCoordinates: N2\u00b054'15.44\", E104\u00b0 6'1.08\"; Event: eventDate: 18 April 2014; habitat: Amongst coral rubble. Littoral 0.5 to 7 m depth; Record Level: collectionCode: UKMMZ-1547; UKMMZ-1549; ZRC 2016.0015Only known from type locality Chew, bin Abdul Rahim & bin Haji Ross, 201462B3AE2C-BF38-5B25-B68D-2B7A33B78618http://www.marinespecies.org/aphia.php?p=taxdetails&id=1054956TinggianthuraalbaType status:Paratype. Location: island: Kampung Pasir Panjang, Pulau Tinggi; country: Malaysia; stateProvince: Johor; verbatimCoordinates: N2\u00b017'35.08\", E104\u00b0 6'7.13\"; Event: eventDate: 16 August 2012; habitat: Amongst coral rubble. Littoral 0.5 to 3 m depth; Record Level: collectionCode: UKMMZ-1481; UKMMZ-1482; UKMMZ-1483Type status:Other material. Location: island: Kampung Pasir Panjang, Pulau Tinggi; country: Malaysia; stateProvince: Johor; verbatimCoordinates: N2\u00b017'35.08\", E104\u00b0 6'7.13\"; Event: eventDate: 18 Dec 2012; habitat: Amongst coral rubble. Littoral 0.5 to 3 m depth; Record Level: collectionCode: UKMMZ-1622Type status:Other material. Location: island: Kampung Pasir Panjang, Pulau Tinggi; country: Malaysia; stateProvince: Johor; verbatimCoordinates: N2\u00b017'35.08\", E104\u00b0 6'7.13\"; Event: eventDate: 28 Feb 2013; habitat: Amongst coral rubble. Littoral 0.5 to 3 m depth; Record Level: collectionCode: UKMMZ-1623Only known from type locality M\u00fcller 19921C60CB7B-7966-5A03-8094-CC89A0E23536http://www.marinespecies.org/aphia.php?p=taxdetails&id=255500EisothistosbesarType status:Holotype. Location: island: Pulau Babi Besar; country: Malaysia; stateProvince: Johor; verbatimCoordinates: N2\u00b050.483', E104\u00b09.566'; Event: habitat: Acropora sp. and PocilloporadamicornisFringing reef, outer reef-flat, reef-margin and upper coral-slope; dead coral substratum, ; Record Level: collectionCode: MHNG.Only known from type locality This species record is based on literature only. No specimen was collected during the present study.Chew, bin Abdul Rahim & binti Mohd Yusof, 2018E19DF907-90D4-5E2B-BE2F-052703486B5Fhttp://www.marinespecies.org/aphia.php?p=taxdetails&id=1056465EisothistostiomanensisType status:Other material. Location: island: Labas, Pulau Tioman; country: Malaysia; stateProvince: Pahang; verbatimCoordinates: N2\u00b053'13.71\", E104\u00b0 3'54.65\"; Event: eventDate: 18 April 2014; habitat: Amongst coral rubble. Littoral 0.5 to 15 m depth; Record Level: collectionCode: UKMMZ-1559Only known from type locality .301CB3BA-E0F8-5CB3-94D1-2A3C59E4CE8Dhttp://www.marinespecies.org/aphia.php?p=taxdetails&id=260392MF680511PanathuracollarisExpanathuracollarisType status:Other material. Location: island: Sebirah Kechil, Pulau Tinggi; country: Malaysia; stateProvince: Johor; verbatimCoordinates: N2\u00b018.622\u2019, E104\u00b0 5.616\u2019; Event: eventDate: 16 May 2013; habitat: Amongst coral rubble, littoral 0.5 to 3 m; Record Level: collectionCode: UKMMZ-1565; UKMMZ-1566; UKMMZ-1567Type status:Other material. Location: island: Mentinggi, Pulau Tinggi; country: Malaysia; stateProvince: Johor; verbatimCoordinates: N2\u00b016\u201921.67\", E104\u00b0 7\u201918.61\"; Event: eventDate: 19 April 2013; habitat: Amongst coral rubble, littoral 0.5 to 3 m; Record Level: collectionCode: UKMMZ-1568Type status:Other material. Location: island: Batu Malang, Pulau Tioman; country: Malaysia; stateProvince: Pahang; verbatimCoordinates: N2\u00b054\u201915.44\", E104\u00b0 6\u20191.08\"; Event: eventDate: 18 April 2014; habitat: Amongst coral rubble, littoral 0.5 to 7 m; Record Level: collectionCode: UKMMZ-1569Type status:Other material. Location: island: Labas, Pulau Tioman; country: Malaysia; stateProvince: Pahang; verbatimCoordinates: N2\u00b053\u201913.71\", E104\u00b03\u201954.65\"; Event: eventDate: 18 April 2014; habitat: Amongst coral rubble, littoral 0.5 to 15 m; Record Level: collectionCode: UKMMZ-1570Type status:Other material. Location: island: Pantai Kok, Pulau Langkawi; country: Malaysia; stateProvince: Kedah; verbatimCoordinates: N6\u00b021\u201956.05\", E99\u00b040\u201931.13\"; Event: habitat: Intertidal coral rubble; Record Level: collectionCode: UKMMZ-1625Type status:Other material. Location: island: Batu Bonchek, Pulau Dayang; country: Malaysia; stateProvince: Johor; verbatimCoordinates: N2\u00b028\u201940.90\", E104\u00b0 30\u201919.12\"; Event: eventDate: 26 July 2016; habitat: Amongst coral rubble, littoral 0.5 to 3 m; Record Level: collectionCode: UKMMZ-1568FIJI ; COOK ISLAND; MOOREA\u2015Chesterfield and Melish Reefs, Coral Sea; LORD HOWE ISLAND\u2015Tasman Sea; PAPUA NEW GUINEA; AUSTRALIA\u2015Northern Territory, Queensland; MALAYSIA\u2015Pulau Dayang, Pulau Tinggi, (New record); Pulau Tioman, Malaysia (New record); Pulau Langkawi (New record).Fig. Poore & Lew Ton, 198869F151D3-EB7F-5E87-A197-7ABCEC315E74http://www.marinespecies.org/aphia.php?p=taxdetails&id=255527KupellonuragidgeeType status:Other material. Location: island: Batu Malang, Pulau Tioman; country: Malaysia; stateProvince: Pahang; verbatimCoordinates: N2\u00b054'15.44\", E104\u00b0 6'1.08\"; Event: eventDate: 18 April 2014; habitat: Amongst coral rubble, littoral 0.5 to 7 m depth; Record Level: collectionCode: UKMMZ-1602AUSTRALIA\u2015Lizard Island ; MALAYSIA\u2015Pulau Tioman (new record).Fig. E97A59EE-122C-5D8D-B6CC-368918E7A019http://www.marinespecies.org/aphia.php?p=taxdetails&id=258455KatanthurabarnardiAccalathurabarnardiType status:Other material. Location: island: Batu Malang, Pulau Tioman; country: Malaysia; stateProvince: Pahang; verbatimCoordinates: N2\u00b054'15.44\", E104\u00b0 6'1.08\"; Event: eventDate: 18 April 2014; habitat: Amongst coral rubble, littoral 0.5 to 7 m depth; Record Level: collectionCode: UKMMZ-1603; UKMMZ-1604; UKMMZ-1605Type status:Other material. Location: island: Labas, Pulau Tioman; country: Malaysia; stateProvince: Pahang; verbatimCoordinates: N2\u00b053'13.71\", E104\u00b0 3'54.65\"; Event: eventDate: 18 April 2014; habitat: Amongst coral rubble, littoral 0.5 to 15 m depth; Record Level: collectionCode: UKMMZ-1606Type status:Other material. Location: island: Pulau Seri Buat; country: Malaysia; stateProvince: Pahang; verbatimCoordinates: N2\u00b041'13.59\", E103\u00b0 55'25.99\"; Event: eventDate: 19 April 2014; habitat: Amongst coral rubble, littoral 0.5 to 7 m depth; Record Level: collectionCode: UKMMZ-1607Type status:Other material. Location: island: Batu Bonchek, Pulau Dayang; country: Malaysia; stateProvince: Johor; verbatimCoordinates: N2\u00b028'40.90\", E104\u00b0 30'19.12\"; Event: eventDate: 26 July 2016; habitat: Amongst coral rubble, littoral 0.5 to 3 m depth; Record Level: collectionCode: UKMMZ-1608Type status:Other material. Location: island: Teluk Rha, Pulau Aur; country: Malaysia; stateProvince: Johor; verbatimCoordinates: N2\u00b028'17.24\", E104\u00b0 30'53.14\"; Event: eventDate: 27 July 2016; habitat: Amongst coral rubble, littoral 0.5 to 3 m depth; Record Level: collectionCode: UKMMZ-1609INDONESIA\u2015Solo Strait, Java Sea ; MALAYSIA\u2015Pulau Tioman; Pulau Seri Buat (new record).Fig. 784B7E0C-E030-598C-942E-6D89B075715Dhttp://www.marinespecies.org/aphia.php?p=taxdetails&id=258457MF680508CalathuraborradaileiAccalathuraborradaileiType status:Other material. Location: island: Kampung Pasir Panjang, Pulau Tinggi; country: Malaysia; stateProvince: Johor; verbatimCoordinates: N2\u00b017'37.96\", E104\u00b0 6'1.97\"; Event: eventDate: 28 February 2013; habitat: coral rubble, intertidal; Record Level: collectionCode: UKMMZ-1610; UKMMZ-1611; UKMMZ-1612Type status:Other material. Location: island: Kampung Pasir Panjang, Pulau Tinggi; country: Malaysia; stateProvince: Johor; verbatimCoordinates: N2\u00b017'37.96\", E104\u00b0 6'1.97\"; Event: eventDate: 18 December 2012; habitat: coral rubble, intertidal; Record Level: collectionCode: UKMMZ-1613Type status:Other material. Location: island: Kampung Pasir Panjang, Pulau Tinggi; country: Malaysia; stateProvince: Johor; verbatimCoordinates: N2\u00b017'37.96\", E104\u00b06'1.97\"; Event: eventDate: 15 June 2015; habitat: coral rubble, intertidal; Record Level: collectionCode: UKMMZ-1614Type status:Other material. Location: island: Sebirah Kechil, Pulau Tinggi; country: Malaysia; stateProvince: Johor; verbatimCoordinates: N2\u00b018.622', E104\u00b0 05.616'; Event: eventDate: 15 June 2015; habitat: coral rubble, intertidal; Record Level: collectionCode: UKMMZ-1615Type status:Other material. Location: island: Batu Bonchel, Pulau Dayang; country: Malaysia; stateProvince: Johor; verbatimCoordinates: N2\u00b028'40.90\", E104\u00b0 30'19.12\"; Event: eventDate: 26 July 2015; habitat: coral rubble, 0.5 to 3 m depth; Record Level: collectionCode: UKMMZ-1616MALDIVES\u2015Fadifolu ; THAILAND; INDIA\u2015Chilka Lake; Kollam; MALAYSIA\u2015Pulau Dayang, Pulau Tinggi (new record).Fig. M\u00fcller, 1992AF83FC82-276E-54E6-A13F-60CB2DAE714Ahttp://www.marinespecies.org/aphia.php?p=taxdetails&id=255547LeptanthuracoralliophilaType status:Other material. Location: island: Pulau Seri Buat; country: Malaysia; stateProvince: Pahang; verbatimCoordinates: N2\u00b041'13.59\", E103\u00b055'25.99\"; Event: eventDate: 19 April 2014; habitat: Amongst coral rubble, intertidal to 7 m depth; Record Level: collectionCode: UKMMZ-1617; UKMMZ-1618MALAYSIA\u2015Pulau Babi Besar ; Pulau Seri Buat.Fig. Kensley, 19795C575653-D82F-5FF2-B70B-B83DF055C22Dhttp://www.marinespecies.org/aphia.php?p=taxdetails&id=255571ParanthuraastrolabiumType status:Other material. Location: island: Batu Malang, Pulau Tioman; country: Malaysia; stateProvince: Pahang; verbatimCoordinates: N2\u00b054'15.44\", E104\u00b0 6'1.08\"; Event: eventDate: 18 April 2014; habitat: Amongst coral rubble, intertidal to 7 m; Record Level: collectionCode: UKMMZ-1619; UKMMZ-1621Type status:Other material. Location: island: Pulau Seri Buat; country: Malaysia; stateProvince: Pahang; verbatimCoordinates: N2\u00b041'13.59\", E103\u00b055'25.99\"; Event: eventDate: 19 April 2014; habitat: Amongst coral rubble, intertidal to 7 m; Record Level: collectionCode: UKMMZ-1622FIJI\u2015Great Astrolabe Barrier Reef ; Suva Reef; MALAYSIA\u2015Pulau Tioman, Malaysia; Pulau Seri Buat (new record).Fig. Negoescu, 19973282B05D-7D7F-5C46-B4E7-00DDFA5AFFD7http://www.marinespecies.org/aphia.php?p=taxdetails&id=255615ParanthurasetigeraType status:Other material. Location: island: Kampung Pasir Panjang, Pulau Tinggi; country: Malaysia; stateProvince: Johor; verbatimCoordinates: N2\u00b017'37.96\", E 104\u00b0 6'1.97\"; Event: eventDate: 18 December 2012; habitat: coral rubble, intertidal; Record Level: collectionCode: UKMMZ-1623; UKMMZ-1624; UKMMZ-1625Type status:Other material. Location: island: Kampung Pasir Panjang, Pulau Tinggi; country: Malaysia; stateProvince: Johor; verbatimCoordinates: N2\u00b017'37.96\", E104\u00b0 6'1.97\"; Event: eventDate: 28 Feb 2013; habitat: coral rubble, intertidal; Record Level: collectionCode: UKMMZ-1626INDONESIA\u2015Southeast Bali Island, Sanur beach, Bali Island ; MALAYSIA\u2015Pulau Tinggi (new record).Fig. Kensley & Schotte, 2000A15F5BC0-FD4C-5880-BEFB-5FED3C68858Dhttp://www.marinespecies.org/aphia.php?p=taxdetails&id=211339ParanthuraseychellensisType status:Holotype. Location: island: Labas, Pulau Tioman; country: Malaysia; stateProvince: Pahang; verbatimCoordinates: N2\u00b053'13.71\", E104\u00b0 3'54.65\"; Event: eventDate: 18 April 2014; habitat: Amongst coral rubble, littoral 0.5 to 15 m depth; Record Level: collectionCode: UKMMZ-1627; UKMMZ-1628; UKMMZ-1629Type status:Holotype. Location: island: Labas, Pulau Tioman; country: Malaysia; stateProvince: Pahang; verbatimCoordinates: N2\u00b053'13.71\", E104\u00b0 3'54.65\"; Event: eventDate: 18 April 2014; habitat: Amongst coral rubble, littoral 0.5 to 15 m depth; Record Level: collectionCode: UKMMZ-1630SEYCHELLES\u2015Mah\u00e9 Beach Mah\u00e9 Island ; MALAYSIA\u2015Pulau Tioman (new record).Fig. Anthuroidea are known from the Malaysian Peninsular, comprising 12 genera and 24 species. Of these, 10 species are recently discovered Malaysian species and they contribute about 1.5% of all known Anthuroidea of the world. Clearly, this number is only a fraction of their true diversity, given that habitats like shallow reefs, seagrasses and mangroves have not received much taxonomic attention. More species are yet to be discovered, especially after exploring inaccessible habitats, revealing of cryptic species using molecular methods and exploring east Malaysian (Sabah and Sarawak) waters. The latter is a region of high known marine biodiversity.Currently five families of the marine Anthuridae species with 58.3% in the Malaysian region and 45.8% in the Indian Ocean region. It is followed by a small number of Leptanthuridae , Paranthuridae , Expanathuridae and Hyssuridae . Having highlighted the similarity, a minor but crucial difference was also noticed, particularly on the occurrence of Antheluridae species in the Indian Ocean. According to Antheluridae species are restricted to deep-water, cool-temperate or polar regions. Although several species are found to inhabit the warmer and shallower seas, particularly the eight species of Anthomuda Schultz, 1979, they are mostly captured from the Indian Ocean (Anthuridae (17 species), can be attributed to the fact that the family is the largest and most widespread (Apanthura and Mesanthura, are recorded with the highest species diversity amongst the Malaysian anthuroid fauna. On the other hand, there is no record of any Antheluridae species occurring in the Malaysian region, most probably due to the fact that they are rare in the warmer seas (The anthuroids of Peninsular Malaysia are similarly taxonomically represented as the adjacent thoroughly studied Indian Ocean region by an Ocean . The higdespread . Some ofmer seas .Eisothistos species are rare and, if reported, they have usually been collected from breaking open serpulid tubes (Amakusanthurakoonyumae, a species previously known to inhabit sandy substratum (Currently, all known Malaysian anthuroids are found to be associated with coral rubble. Other habitats, such as sandy and muddy sediments and algal beds, have not been sampled. Due to the fact that they are very well represented in the high spatial complexity coral rubble , coral rid tubes . Moreovebstratum , is now"} +{"text": "Correction to: Basic Clin Androlhttps://doi.org/10.1186/s12610-018-0076-0Following publication of the original article , the autFanny (Given name) Jumeau (Family name)Julien (Given name) Sigala (Family name)Francisco-Jose (Given name) Fernandez-Gomez (Family name)Sabiha (Given name) Eddarkaoui (Family name)Sophie (Given name) Duban-Deweer (Family name)Luc (Given name) Bu\u00e9e (Family name)H\u00e9l\u00e8ne (Given name) B\u00e9hal (Family name)Nicolas (Given name) Sergeant (Family name)Val\u00e9rie (Given name) Mitchell (Family name)"} +{"text": "This article has been corrected: Due to errors in image selection, the pictures of H1299 cell morphology treated with recombinant TGF-\u03b2 and TGF-\u03b2 receptor inhibitor in 99801-99815. https://doi.org/10.18632/oncotarget.21068Original article: Oncotarget. 2017; 8:99801\u201399815."} +{"text": "There are errors in the Author Contributions. The publisher apologizes for the errors. The correct contributions are:Conceptualization: Stefania Titton, Deborah BarskyData curation: Stefania Titton, Deborah Barsky, Am\u00e8lia Bargall\u00f3Formal analysis: Stefania Titton, Alexia Serrano-RamosFunding acquisition: Juan Manuel Jimenez ArenasInvestigation: Stefania Titton, Deborah Barsky, Josep Maria Verg\u00e8s, Jos\u00e9 Garc\u00eda SolanoMethodology: Stefania TittonProject administration: Isidro Toro-Moyano, Robert Sala-Ramos, Juan Manuel Jimenez ArenasResources: Isidro Toro-MoyanoSoftware: Alexia Serrano-RamosSupervision: Deborah Barsky, Am\u00e8lia Bargall\u00f3Visualization: Stefania TittonWriting\u2014original draft: Stefania TittonWriting\u2014review & editing: Deborah Barsky, Am\u00e8lia Bargall\u00f3, Juan Manuel Jimenez Arenas"} +{"text": "Correction to: BMC Infect Dis (2020) 20:603https://doi.org/10.1186/s12879-020-05271-5After publication of the original article , the autThe incorrect author name is:Stefanie von Felten \u22c4 given name: von Felten; family name: StefanieMirjam Faes Hesse \u22c4 given name: Mirjam Faes; family name: HesseThe correct author name is:Stefanie von Felten \u22c4 given name: Stefanie; family name: von FeltenMirjam Faes Hesse \u22c4 given name: Mirjam; family name: Faes HesseThe original article has been corrected."} +{"text": "Correction to: Trials (2020) 21:80https://doi.org/10.1186/s13063-019-3965-4Original name spelling:Charlotte Fl\u00fcheCorrect name spelling:Charlotte Fl\u00fchAfter publication of our article the authThe original article has been corrected."} +{"text": "Scientific Reports 10.1038/srep37783, published online 25 November 2016Correction to: This Article contains a typographical error in Equation 1.should read:"} +{"text": "Scientific Reportshttps://doi.org/10.1038/s41598-019-43716-3, published online 16 May 2019.Correction to: This Article contains a typographical error in the Acknowledgements section.\u201cNatural Environmental Research Council grant number [NE/L002531/1]\u201dshould read:\u201cNatural Environment Research Council: NE/N012070/1\u201d."} +{"text": "Chaetodon lunulatus) that avoids gill monogenean parasites while living amongst closely related parasitized species. The metabolome and microbiome of several sympatric butterflyfish species from the island of Moorea (French Polynesia) were previously described. In this study, we used the previously generated datasets in an attempt to identify metabolites and bacteria potentially involved in parasite defense mechanisms. We investigated the interplay between the gill mucus metabolome and microbiome of the non-susceptible C. lunulatus versus sympatric butterflyfish species that were always found parasitized in the Central and Eastern Indo-Pacific. After observing significant differences between the metabolome and bacteria of susceptible versus non-susceptible fish, we obtained the discriminant metabolites and operational taxonomic units (OTUs) using a supervised analysis. Some of the most important discriminant metabolites were identified as peptides, and three new peptides derived from \u03b2-subunit hemoglobin from C. lunulatus were purified, characterized and synthesized to confirm their structures. We also identified specific bacterial families and OTUs typical from low-oxygen habitats in C. lunulatus gill mucus. By using a correlation network between the two datasets, we found a Fusobacteriaceae strain exclusively present in C. lunulatus and highly correlated to the peptides. Finally, we discuss the possible involvement of these peptides and Fusobacteriaceae in monogenean avoidance by this fish species.Understanding natural defense mechanisms against parasites can be a valuable tool for the development of innovative therapies. We have previously identified a butterflyfish species ( Parasites are crucial for a well-balanced ecosystem, contributing to its organization by modifying competitive and trophic interactions and driving natural selection . HoweverEnterobacter bacterium from mosquito inhibited the development of malaria parasites by the production of reactive oxygen species (ROS), rendering mosquitoes resistant to infection 5+ (CLHb\u03b2-1) and 644.8738 [M + 2H]2+ (CLHb\u03b2-2) pre-purification of the acidic extract from CLHb\u03b2-2) . Another + 3H]3+ .C. reticulatus and C. ornatissimus) [m/z 679.7758 [M + 5H]5+ (CLHb\u03b2-4) and 702.3929 [M + 5H]5+ (CLHb\u03b2-5) in the mucus samples of all analyzed species (Acidic extractions of two heavily parasitized species (issimus) were don species . The mas species , Table 1+m/z values would match the expected average MH+m/z values for the five peptides discussed above eluted fraction showed the presence of several potential peptides, including five peaks whose MHed above . After ned above , Table 1http://www.uwm.edu.pl/biochemia/index.php/pl/biopep) showed no significant sequence identity to any sequence in these databases. Homology search with the Swissprot\u2013UniprotKB database using Mascot and Sequest highlighted peptides derived from the hemoglobin subunit beta-B of the Japanese amberjack Seriola quinqueradiata with high scores (see Chaetodon austriacus +m/z 1288.74177; [M + 2H]2+m/z 644.87479; [M + 3H]3+m/z 430.1914; found m/z: 644.8743.CLHb\u03b2-2: VKWTDAERAAITSLWGKIDVGEIGPQALTR Crude peptide . ESI-HRMS: Calculated for C147H237N41O44: [M+H]+m/z 3281.76464; [M + 2H]2+m/z 1641.38623; [M + 3H]3+m/z 1094.5934; [M + 4H]4+m/z 821.19703; [M + 5H]5+m/z 657.1592 found m/z: 657.1589.CLHb\u03b2-3: AAITSLWGKIDVGEIGPQALTR Crude peptide . ESI-HRMS: Calculated for C103H170N28O31: [M + H]+m/z 2296.26651; [M + 2H]2+m/z 1148.6372; [M + 3H]3+m/z 766.0941; found m/z: 766.0927.CLHb\u03b2-4: VKWTDAERAAITSLWGKIDVGEIGPQALTRL Crude peptide . ESI-HRMS: Calculated for C153H248N42O45: [M + H]+m/z 3394.84871; [M + 2H]2+m/z 1697.92827; [M + 3H]3+m/z 1132.28757; [M + 4H]4+m/z 849.46802; [M + 5H]5+m/z 679.77600 found m/z: 679.7773.CLHb\u03b2-5: VKWTDAERAAITSLWGKIDVGEIGPQALTRLL Crude peptide . ESI-HRMS: Calculated for C159H259N43O46: [M + H]+m/z 3507.93277; [M + 2H]2+m/z 1754.47029; [M + 3H]3+m/z 1169.98281; [M + 4H]4+m/z 877.73906; [M + 5H]5+m/z 702.3928 found m/z: 702.3931.C. lunulatus, C. ornatissimus, C. reticulatus, and C. vagabundus) were studied. Gill mucus DNA was extracted, and DNA samples were sent for 454 pyrosequencing. Multiplex raw SFF (standard flowgram format) files were analyzed using a hybrid analysis pipeline using Usearch5 and Qiime1, and non-chimeric sequences were unweighted and grouped into operational taxonomic units (OTUs) with a cut-off of 98% . Variable importance in projection (VIP) was obtained (package RVAiedeMemoire for R), and features and OTUs with VIP scores higher than 1 were retained. Kruskal\u2013Wallis test was used to test for a significant difference in the intensity of metabolites and relative abundance of OTUs between parasitized and non-parasitized fish. In order to explore the correlation between VIP metabolites and OTUs, a correlation network was constructed using the software Cytoscape v3.4.0 [Multidimensional matrices obtained from the metabolome and microbiome data were analyzed equally. Principal coordinate analysis was used to visualize the differences between the parasitized and non-parasitized fish metabolome and microbiome. Analysis of similarities was used to detect significant differences between the PCoA groups. Partial least squares discriminant analysis was used to select the metabolites and OTUs driving the differences between the two fish groups. PLS-DA model accuracy was calculated using two-fold cross-validation and the resulting number of misclassifications (NMC) was compared to its permutated null distribution (999 permutations) to test for model significance (e v3.4.0 . Spearma"} +{"text": "AdamowiczKarthikeyan AdhimoolamJoshua N. AdkinsMatthew AglerEric AguiarStephen Dela AhatorHajar Fauzan AhmadIrfan AhmadWarish AhmedPalok AichOmid AkbariBegum AlaybeyogluMichaeline B. N. AlbrightDelphine AldebertGajender AletiNabil-Fareed AlikhanDavid AllandJacob AllenEmma Allen-VercoeAlexandre AlmeidaLauren Victoria AlteioJohn AlverdyKatherine R. AmatoCarol Viviana Amaya GomezOle Herman AmburJan AmendAlongkorn AmnuaykanjanasinAmitesh AnandAnnette Carola AndersonGregory G. AndersonAnand AndiappanAndres Andrade DominguezNana Y. D. AnkrahSten AnslanRebecca AnsorgeHaike AntelmannVijay C. AntharamMaciek R. AntoniewiczAndr\u00e9 AntunesL. Caetano M. AntunesVictor Hugo AquinoManuel ArandaGabriele ArcariCatherine R. ArmbrusterZach ArmstrongJames T. ArnoneCarol ArnostiGunjan AroraMario L. Arrieta-OrtizMelinda Marie AshcroftMarina Elisabeth AspholmLaetitia AttaiechJennifer M. AuchtungFikri Y. AvciMian Muhammad AwaisSelcan AydinM. Andrea Azcarate-PerilOliver BaarsPaul BabitzkeTaeok BaeChengrong BaiJonathon L. BakerPaul William BakerSreejata BandopadhyayCarlos BarajasWendy S. BarclayMohammed BarigouLars BarquistRodolphe BarrangouFrederic J. BarrasJonathan BarrattFrancois-Xavier BarreRyan P. BartelmeLuther A. BarteltMirko BasenChristine Marie BassisFlorian F. BauerAnthony D. BaughnJose M. BautistaSean K. BayJagadeesh BayryWilliam N. BeaversChristine BeemelmannsOded BejaPedro Belda-FerreLisa K. BeldenAlfonso Ben\u00edtez-P\u00e1ezSean BenlerSarah Ben MaamarGian Maria Niccolo BenucciMaureen BergBeatrice BergerMegan BergkesselRenaud BerlemontEmma BerminghamAnthony BertagnolliAndrew David BertiKatja BettenbrockOliver K. I. BezuidtKyle BibbyEthan BierSarah BigotElisabeth M. BikSteven BillerJordan T. BirdKyle BittingerAlain BlanchardJeffrey BlanchardJake BledsoeAlissa BleemRobert M. BlumenthalSaid BogatyrevNicholas Andrew BokulichJennifer M. BombergerIvo G. BonecaCarla Yaneth BonillaErika BonsagliaElizabeth BoonRicardo M. BorgesOlivier BorkowskiGuillaume BorrelJo\u00e3o BotelhoMichael J. BouchardPaul BoudreauAlice BoulangerBob BourettJoel BozueG\u00fcnter BraderPatrick H. BradleyKristoffer BrandvoldChristopher Br\u00e4senMya BreitbartMichael R. BrentVanessa BrissonIlana Lauren BritoMark Br\u00f6nstrupAmanda BrownDaniel R. BrownSebastian BruchmannHarald BruessowSascha BrunkeJames BrunnerJason BubierDaniel H. BuckleyMichelle Monique Conni BucknerSara Ashley BumgardnerCatherine M. BurgessSara BurgessAlita R. BurmeisterJeremy P. BurtonSusheel Bhanu BusiLuc\u00e9lia CabralDaniel CaddellJianfeng CaiPaolo CalligariMagdalena CalusinskaAntonio CamargoBarbara J. CampbellH\u00fcseyin CanXinyun CaoAndr\u00e9s M. Caraballo-Rodr\u00edguezValerie Jean CarabettaAlessandra CarattoliNicholas H. CarbonettiJane M. CarltonAdrian CarperTyler J. CarrierJohn R. CaseySantiago Castillo-Ram\u00edrezDuccio CavalieriYunrong ChaiStephane ChaillouKok Gan ChanSiu H. J. ChanChristopher Peter ChanwayYanjie (Jay) ChaoMatthew R. ChapmanGina M. ChaputBenoit ChassaingJonathan ChekanBaodong ChenCasey ChenFusheng ChenJen-Tsung ChenJun ChenLiang ChenStanley H. ChenTingtao ChenWei ChenMarc G. ChevretteLoo Wee ChiaJason ChinByung-Kwan ChoSanghyun ChoAh Reum ChoiJessica ChopykMaria ChuvochinaNico J. ClaassensDennis ClaessenThomas ClavelJos\u00e9 Francisco Cobo D\u00edazAna Cl\u00e1udia CoelhoLuis Pedro CoehloAidan CoffeyNatalie CohenDevin Coleman-DerrJames CollinsGeorg ConradsLydia M. ContrerasTyrrell ConwayRene CortesePaul Cos\u00d6mer CoskunZachary CostliowJason CrawfordAlexander Crits-ChristophDaniel CrollSean CrossonSean Andrew CroweDavid E. CrowleyZhongli CuiMichael CunliffeCameron R. CurrieJohanna Patricia DailyBenjamin DainatMartina Dal BelloRobert E. DanczakWashington DaSilvaMaude DavidSeana K. DavidsonShawn DavidsonThomas DawsonAllan DegenHidde de JongTom DelmontJill DembrowskiXiangyu DengXin DengYijie DengJonathan J. DennisTodd DeSantisMegan De Ste. CroixSuzanne DevkotaTravis J. De WolfeJonathan D. D\u2019GamaAvantika DhabariaGeorge C. diCenzoGregory J. DickChristian DienerChristina Diez-VivesStephen P. DiggleYousong DingAlden Conlan DirksJocelyne DiRuggieroRay DixonUlrich DobrindtDaryl DommanRodolpho Martin do PradoMert D\u00f6\u015fkayaRyan S. DosterNir DraymanPavel DrevinekAlexandre DrouinGeorge L. DrusanoMelissa DsouzaChao DuRebbeca DuarVikash Kumar DubeyDaniel Christopher DucatLesley L. DuffyTim J. DumonceauxColum DunneSanjucta DuttaRachel J. DuttonMohammed DwidarTom EdlindMartinique Lefevre EdwardsEleonora EgidiGisli EinarssonDamian EkiertMikros EmmanuelAlexandra EmmonsStephanie EngelMelinda Anne EngevikTobias EnglBrendan EpsteinCarmen Escudero-MartinezAlessandra S. EustaquioSarah Elisabeth EwaldJeremiah FaithAili FanKunkun FanSeamus FanningSara FedericiConor FeehilyAdam M. FeistTam\u00e1s Felf\u00f6ldiFengqin FengJean-Luc FeratJane FergusonPedro Eduardo FerreiraElvira Fiallo-Oliv\u00e9Daniel FigeysCelio Fernando Figueiredo AngoliniGilberto E. FloresStelios FodelianakisHerrison FontanaLarry J. ForneyJane FosterLeonard J. FosterHugo FragaMichael T. FranceEmanuela FrangipaniEric A. FranzosaLori FrappierDavid N. FredricksKelle C. FreelIddo FriedbergJens Christian FrisvadTakashi FujishiroTak FungChikara FurusawaIwona GabrielAna Cristina GalesMichael G. GanzleFerran Garcia-PichelJulia M. GauglitzPatrice GaurivaudJennifer Geddes-McalisterEdward GeisingerMikhail S. GelfandDavid GellLandon John GetzBenjamin GewurzRaad GharaibehSteven R. GillCaitlin GionfriddoJorge A. GironAnum GlasgowBettina GlaslSydney GlassmanErin S. GloagMatthew Robert GoddardBen GoldIsabel G\u00f3mez-HurtadoAlicia Gonz\u00e1lezClaire L. GorrieCelia W. 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KiedrowskiPatrick KieferMogens KilstrupJae Su KimYoung-Mo KimJonathan KlassenManuel KleinerStefan KlumppJen KniesPetr KohoutTyler KokjohnCarolin KolmederBethany KolodyAleksandra KolodziejczykYahya KoochJanet Cheruiyot Kosgey\u00c1kos T. Kov\u00e1csRosa Krajmalnik-BrownTino KrellArjun KrishnanAndrew M. KropinskiJia-Liang KuangJessica Z. Kubicek-SutherlandLouise KuhnAnand KumarAshwani KumarRoshan KumarDeepak KumaresanRanjith KumavathYakov KuzyakovMatthias LabrenzChristophe LacroixDaniel A. LafontaineAriadna LagunaLeo LahtiHsin-Chih LaiJohanna LampePawel LaniewskiRafael Laso-P\u00e9rezChristian L. LauberAonghus LavellePedro Humberto LebreChristopher T. LefevreLaura E. Lehtovirta-MorleyVanessa LeoneRuth E. LeyBailiang LiHoukai LiHui LiMeng LiShiming LiXiangrui LiYong LiZhiyong LiChen LiaoXin-Di LiaoTami LiebermanGeorge LiechtiZachary LiechtyRoland LillShaun LimJonathan Y. LinAbigail LindFangqiong LingNing LingJohn J. LipumaSara LittleGuangliang LiuGuodong LiuJinxin LiuJun LiuLong LiuWei LiuAndrew LongAna LongoAllison LopatkinNorberto Peporine LopesGabriel L. LozanoCatherine LozuponeXin M. LuoAndr\u00e9anne LupienLongxian LvMichael LyuBin MaYingfei MaRoderick I. MackieHannah MacLeodKaren L. MadsenRick M. MaizelsPiotr MajewskiJuraj MajtanLucie Anne MalardJacob MaloneSerena ManaraNicasio ManciniChristopher MancusoRabindra Kumar MandalNathan P. ManesRen MaoRamona MarascoJulian R. MarchesiMaria L. MarcoAbelardo MargollesMartin G. MarinusJeffrey MarlowIan P. G. MarshallMaria MartellJulia E. MartinVeronica MartinBeatriz Mart\u00ednezJos\u00e9 Luis Mart\u00ednezEsteban Mart\u00ednez-Garc\u00edaManuel Martinez-GarciaMaria Elena MartinoJon MartinsonVincent G. MartinsonPrince Peter MathaiAlison MatherDespoina MavridouXavier MayaliMelinda Jane MayerChase MayersCameron McBrideMark J. McBrideJohn P. McCutcheonAlice C. McHardyAoife J. McHughMichael J. McLarenLynn M. McMullenConor MeehanLuis C. MejiaJohn Scott MeschkeJoseph R. MihaljevicChristian MilaniIan J. MillerRobert H. MillsTanja MimmoKiwamu MinamisawaSamuel MinotBiswapriya Biswavas MisraCaroline MitchellSuparna MitraSara MitriJennifer M. MobberleyLuke A. MoeAndrew MoellerWendy MokBabak MomeniDenise MonackJonathan MonkKyung MoonJamie MortonAlexander MoschenKathy MouQu\u00e9zia MouraEnock MpofuQinghui MuC. S. MukhopadhyayCarly Muletz-WolzIssa Atanda MurainaMario MuscarellaKevin S. MyersCarey D. NadellMaya NadimpalliRavinder K. NagpalRyohei Thomas NakanoJesus Navas-CastilloDan NaylorEvgeniya V. NazarovaAndrew L. NealDavid M. NeedhamJulia NeilsonRoy NeilsonEric Jorge NelsonJiajia NiTimothy J. NiceMartina NieblerKang NingBen NiuMasaru Konishi NobuCecilia NoeckerMichael James NotoAkos NyergesSpencer V. NyholmDarren ObbardIngrid ObernostererAmanda G. OglesbyAnil OjhaAnders OmslandMarc OngenaFabini D. OrataMehmet A. OrmanDaniel Barros OrtegaCarlos R. OsorioGeorge O\u2019TooleMarc OuelletteKalon OverholtWill A. OverholtAnne-Marie OverstreetBen O. OysermanEgon Anderson OzerMarton PalatinszkyBrian PalenikFernando Lucas PalhanoRobert J. PalmerBernhard O. PalssonKevin Panke-BuisseIvan Parra-IzquierdoRaghuveer ParthasarathyLaila Partida-MartinezSally R. PartridgeJavier PascualEdoardo PasolliAlessandro PasseraVinay PathakKiran Raosaheb PatilRob PatroSushmita PatwardhanIan T. PaulsenSara PaverSamuel H. PayneTalima PearsonRoger D. PechousChristie PeeblesItsik Pe\u2019erYousong PengRita de Cassia PessottiKevin PetheWilliam A. Petri, Jr.Vjacheslav PetrovHoang Cong PhanCaroline C. PhilpottAzul Pinochet-BarrosGabin PitonAnna PlantingaMircea PodarLaurent PoirelAlessandra PolissiPhillip B. PopeWelkin H. PopeSteven L. PorterAshok PrasadAkbar Adjie PratamaSarah Pacocha PreheimMorgan N. PriceJohn R. PringleSambhawa PriyaMaraike ProbstLita ProctorBirgit M. PruessAlexandra E. 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Samuelson\u00c1lvaro San MillanAlyson SantoroAlfonso Santos-LopezChristian Santos-Medell\u00ednRumakanta SapkotaMartin SappAlain SarniguetGuillaume SarrabayrouseYuko SasakiLakkakula SatishKarin SauerUwe SauerAlexei SavchenkoJimmy H. SawNeha SawhneyPatrick D. SchlossStephan Schmitz-EsserJochen SchneiderKarin SchnetzRebecca A. SchomerCarla Bianca SchubigerMonika Stefanie Sch\u00fctzBianca SclaviNinecia ScottKimberley D. SeedAnna Maria SeekatzLeopoldo SegalStephanie SeifertShlomo SelaAditi SenguptaLauren Marie SeylerYongqi ShaoMichael ShapiraGil SharonAlaullah SheikhQirong ShenZike ShengLiat ShenhavAhmed A. ShiblMichael U. ShilohWilliam ShoemakerFrancesca L. ShortSteven M. ShortNachshon SiboniJessica R. SieberPaul SiegelMitchell SingerSteven SingerSonia SinghalCarolyn M. SlupskyYounes SmaniBarth F. SmetsNicholas M. SmithSarah SmithTrever C. SmithTheo H. M. SmitsAndrea S\u00f6llingerPacifica SommersMike Seong SonUtkarsh SoodJorg SoppaPatrick SorensenDiana Zita SousaPeter SpeckSanjeeva SrivastavaEric V. StabbBlake W. StampsKenneth M. StedmanAndrew D. SteenAshley A. StegelmeierBlaire StevenLisa StinsonStephen Robert StockdaleJuergen StolzD. G. StoreyGilles StouvenakersFrancesco StratiWolfgang R. StreitGiannis StringlisRhona K. StuartXiaoquan SuZhengchang SuTatsuki SugiMuhammad SulemanSnorre SulheimChristopher S. SullivanAnne O. SummersChaomin SunZheng SunAntonet SvircevKelly S. SwansonMarc A. Sze\u00c9va SzentirmaiKazuhiro TakemotoLi TanAzuma TaokaJulien TapSuvi TaponenFlorence TardyMartin T\u00e4ubelMartin TaubertLeho TedersooNicolas TerraponHerve TettelinBas TeusinkRobert Th\u00e4nertOlivier ThasCasey M. TheriotNikhil A. ThomasMitchell G. ThompsonJ. Cameron ThrashRuth E. TimmeAnna D. TischlerHirokazu TojuMagaly ToroM\u00f3nica Torres Beltr\u00e1nMoritz TreeckLaura TreuStacey Trevathan-TackettJean-Pierre TrezziAnupriya TripathiFran\u00e7ois TrotteinAndrew W. TrumanRenee M. TsolisKieran TuohyTomasz Wojciech TurowskiJane F. TurtonShipra VaishnavaJesus ValdesJake ValenzuelaLizah Van der AartMarjan W. van der WoudeJordi van GestelMarc Warwick Van GoethemDaria Van TyneJose Vargas-Mu\u00f1izSuzan Pantaroto VasconcellosJoachim VaterBalaji VeeraraghavanNic M. VegaAlain VeilleuxNadine VeithHelianthous VermaMarco A. R. VinoloMarie-Joelle VirolleEmily VogtmannChris VoigtMartin von BergenLela VukovicJoseph Thomas WadeDavid M. WagnerIrene Wagner-DoblerJacob WaldbauerKevin John WaldronElizabeth WalkerAaron WalshDavid A. WalshWilliam A. WaltersGuanghua WangHarris WangJianjun WangJinfeng WangJun WangShulei WangSishuo WangWenmei WangXiaoxue WangXueshan WangYan WangZhenyuan WangZhong WangKenneth WasmundKarrie A. WeberTilmann WeberYulong WeiRebecca WeiserMartin WelchMalcolm F. WhiteRichard Allen White IIITimothy A. WhiteheadWilliam B. WhitmanTabor WhitneyMatthias WietzRoland Conrad WilhelmPeter WilliamsonTomasz WilmanskiJennifer R. WilsonCharles WimpeePhitchayapak WintachaiJeffrey H. WitheyPatricia G. WolfAlan J. WolfeMatthew C. WolfgangStephen WoloszynekJeffrey A. WoodsColin WorbyFelipe Christoff WoutersKelly C. WrightonFuqing WuWeihui WuSander WuytsWilliam Wenwei XiangBingbing XiaoShichao XingLing XuZhenjiang Zech XuZhi-Xiang XuKatherine XueAixin YanDongsoo YangJin-Kui YangJun YangVicky YaoMin YapLaxmi YeruvaRyland F. YoungYing YuNatalya YutinLivia S. ZaramelaKarsten ZenglerMusu ZhaKateryna ZhalninaHaoran ZhangJiachao ZhangJunling ZhangQuan-Guo ZhangRuifu ZhangJiangchao ZhaoKun ZhaoNi ZhaoYanlin ZhaoJusheng ZhengJiyong ZhouPing ZhouShungui ZhouWenhao ZhouZhemin ZhouQiyun ZhuYan ZhuStefan ZimmermannErik R. ZinserZhiyong ZongJackie ZorzThe extraordinary complexity and challenges that faced us all in 2020 fell hardest on those who are traditionally underserved, marginalized, and experiencing prejudice. The unprecedented lockdown reshaped our work environment and changed how we report the results of our scientific investigation, but it also caused huge pressure on certain groups, often disproportionately impacting early-career scientists, women, and minorities. Therefore, as 2020 finally draws to a close and we look to a brighter new year, it is a good time to extend our thanks to all the reviewers who have gone \u201cabove and beyond\u201d to ensure the integrity of scientific research reporting in the face of extreme pressure due to isolation, homeschooling, and limited Wi-Fi. Your sacrifice ensures that our truth will not be eroded and that we can still trust research reported in"} +{"text": "The correct name is: Rani Cathrine Chellappa. The correct citation is: Chellappa RC, Lukose B, Rani P (2020) G82S RAGE polymorphism influences amyloid-RAGE interactions relevant in Alzheimer\u2019s disease pathology. PLoS ONE 15(10): e0225487."} +{"text": "In the manuscript \u201cTemporal and spatial distribution of polio vaccine coverage in Brazil between 1997 and 2021\u201d, DOI: https://doi.org/10.1590/1980-549720230037, published in the Rev Bras Epidemiol. 2023; 26: e230037:Page 1, where it reads:H\u00e9vila Medeiros Ferreira Gomes BragaIt should read:H\u00e9vila Ferreira Gomes Medeiros BragaPage 1, how to cite this articleWhere it reads:Braga HMFGIt should read:Braga HFGMPage 9, authors\u2019 contributionsWhere it reads:Braga HMFGIt should read:Braga HFGM"} +{"text": "Scientific Reports 10.1038/s41598-023-42062-9, published online 15 September 2023Correction to: The original version of the Article contained an error in the Author Information statement.\u201cThese authors contributed equally: Ivan Coronado, Samiksha Pachade, Roomasa Channa, Sunil A. Sheth and Luca Giancardo.\u201dnow reads:\u201cThese authors contributed equally: Ivan Coronado\u00a0and Samiksha Pachade.\u201d\u201cThese authors jointly supervised this work: Roomasa Channa, Sunil A. Sheth and Luca Giancardo.\u201dThe original Article has been corrected."} +{"text": "DOI: 10.1111/jcmm.13119\u2009J Cell Mol Med. 2017 Sep;21(9):1989\u20131999.[2] ShRNA knock\u2010down of CXCR7 inhibits tumour invasion and metastasis in hepatocellular carcinoma after transcatheter arterial chemoembolization. DOI: Referencehttps://doi.org/10.1002/1878-0261.126031 Gao J, Dai C, Yu X, Yin X\u2010B, Zhou F. Circ\u2010TCF4.85 silencing inhibits cancer progression through microRNA\u2010486\u20105p\u2010targeted inhibition of ABCF2 in hepatocellular carcinoma. Mol Oncol. 2020;14:447\u201361. DOI: 10.1111/jcmm.13119\u2009J Cell Mol Med. 2017 Sep;21(9):1989\u20131999.[2] ShRNA knock\u2010down of CXCR7 inhibits tumour invasion and metastasis in hepatocellular carcinoma after transcatheter arterial chemoembolization. DOI: Referencehttps://doi.org/10.1002/1878-0261.126031 Gao J, Dai C, Yu X, Yin X\u2010B, Zhou F. Circ\u2010TCF4.85 silencing inhibits cancer progression through microRNA\u2010486\u20105p\u2010targeted inhibition of ABCF2 in hepatocellular carcinoma. Mol Oncol. 2020;14:447\u201361."} +{"text": "Erratum zu:HNO 202310.1007/s00106-023-01313-xIn diesem Artikel wurde die Bildunterschrift von Abb.\u00a0Die Abbildungen sollten wie folgt aussehen:Der Originalbeitrag wurde korrigiert."} +{"text": "Erratum zu:Chirurgie 202310.1007/s00104-023-01820-1In diesem Artikel fehlten die Adressdaten bzw. die Daten zur institutionellen Zugeh\u00f6rigkeit f\u00fcr die Autoren J\u00f6rg Andreas M\u00fcller, Simon Trommer, Katharina Lampe, Roland S. Croner, Dirk Vordermark und Daniel Medenwald. Bitte beachten Sie die oben genannten Adressdaten. Der Originalbeitrag wurde korrigiert."} +{"text": "Correction: Psicologia: Refex\u00e3o e Cr\u00edtica 36, 25 (2023)https://doi.org/10.1186/s41155-023-00263-1Following publication of the original article (Zanini et al., The original article (Zanini et al.,"} +{"text": "Scientific Reports 10.1038/s41598-023-44125-3, published online 09 October 2023Correction to: The original version of this Article contained an error in the Author Information section.\u201cThese authors contributed equally: Brian P. Scottoline and Jamie O. Lo.\u201dnow reads:\u201cThese authors contributed equally: Lyndsey E. Shorey-Kendrick and B. Adam Crosland.\u201d\u201cThese authors jointly supervised this work: Brian P. Scottoline and Jamie O. Lo.\u201dThe original Article has been corrected."} +{"text": "Correction: BMC Med 19, 235 (2021) https://doi.org/10.1186/s12916-021-02108-zIn Fig.\u00a0In Fig.\u00a0In our original article the multThe correct figures are given ahead.We regret the error."} +{"text": "Sci., 2023, 14, 3982\u20133989, https://doi.org/10.1039/D3SC00132F.Correction for \u2018Molybdenum chloride double perovskites: dimensionality control of optical and magnetic properties\u2019 by Devesh Chandra Binwal"} +{"text": "Caption: Grow\u2010out ponds for GIFT Nile tilapia, Malaysia.Credit: Trong Trinh (WorldFish)."} +{"text": "Fabaceae; courtesy of Caian S. Gerolamo.\u2019Upon original publication, Figure 1E legend was given as follows: \u2018(E) Asymmetrical stem resulting from atypical cambial activity (single cambium); The legend has been corrected to read:Schnella sp. Fabaceae; courtesy of Caian S. Gerolamo.\u2019(E) Non-cylindrical stem resulting from atypical cambial activity (single cambium);"} +{"text": "Correction: Psicologia: Reflex\u00e3o e Cr\u00edtica 36, 28 (2023)https://doi.org/10.1186/s41155-023-00269-9Following publication of the original article (Gupta & Kumari, The abstract has been added; the author\u2019s name Aarzoo has been corrected to Aarzoo GuptaThe original article (Gupta & Kumari,"} +{"text": "Correction: BMC Trials 24, 440\u00a0(2023)https://doi.org/10.1186/s13063-023-07462-2Following publication of the original article , we haveOriginally published Fig.\u00a01:Corrected Fig.\u00a01:The original article has been corrected."} +{"text": "DOI: 10.1107/S1600536810042455/sj5040Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Weak inter\u00admolecular C\u2014H\u22efO hydrogen bonds stabilize the crystal structure.In the title compound, [Ni(C DOI: 10.1107/S1600536811009056/hy2415Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the crystal, O\u2014H\u22efO and N\u2014H\u22efO hydrogen bonds link the components into a three-dimensional network.In the title complex, (CH DOI: 10.1107/S1600536812011439/lh5430Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "Figure 1-7 were placed incorrectly in this article. The legends are placed correctly.The correct Figures are:Figure 1: Figure 2: Figure 3: Figure 4: Figure 5: Figure 6: Figure 7:"} +{"text": "The mol\u00adecules are linked by O\u2014H\u22efO hydrogen bonds, forming a chain developing parallel to [101].In the title compound, [Zn(C DOI: 10.1107/S1600536811012244/dn2671Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the crystal, weak C\u2014H\u22efN hydrogen bonds between the DMAP and azide ligands link these discrete complex mol\u00adecules into a three-dimensional supra\u00admolecular network.In the title complex, [Zn(N DOI: Click here for additional data file.10.1107/S1600536813004686/xu5676Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "The two aromatic rings are almost coplanar, making a dihedral angle of 4.73\u2005(5)\u00b0. The crystal structure is stabilized by N\u2014H\u22efO hydrogen bonds, which link the mol\u00adecules into chains along the b axis.In the title compound, C DOI: 10.1107/S1600536811051944/ds2157Isup2.hkl Structure factors: contains datablock(s) I. DOI: 10.1107/S1600536811051944/ds2157Isup3.cml Supplementary material file. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The bis-chelating dimethyl\u00adglyoximate ligands, which occupy equatorial sites, are linked by strong intra\u00admolecular O\u2014H\u22efO hydrogen bonds.In the title compound, [Co(C DOI: 10.1107/S1600536812000967/is5041Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "The CoII ion is in a tetra\u00adhedral coordination geometry. The cations exhibit chair-shaped conformations. A three-dimensional supra\u00admolecular architecture is formed through N\u2014H\u22efCl and C\u2014H\u22efCl hydrogen bonds between the dianions and the cations.The title compound, (C DOI: 10.1107/S1600536811053128/pk2366Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S160053681002725X/jh2179Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The tetra\u00adaza\u00adcyclo\u00adtetra\u00addecane ligand inter\u00adacts with the NiII atom in the cis V configuration and the final two ligand binding sites are occupied by water.The crystal structure of the title complex, [Ni(C DOI: 10.1107/S160053681203276X/pk2429Isup2.molSupplementary material file. DOI: 10.1107/S160053681203276X/pk2429Isup3.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "It is further assembled by O\u2014H\u22efO hydrogen-bond inter\u00adactions involving both the coordinated and uncoordinated water molecules. The latter exhibits half-occupancy.The title compound, [Cu DOI: 10.1107/S1600536811022756/jh2295Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the crystal, the dications and perchlorate anions are linked through N\u2014H\u22efO hydrogen bonding and weak C\u2014H\u22efO hydrogen bonding into a three-dimensional supra\u00admolecular network.In the title compound, C DOI: 10.1107/S1600536811033976/xu5296Isup2.hkl Structure factors: contains datablock(s) I. DOI: 10.1107/S1600536811033976/xu5296Isup3.cml Supplementary material file. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The pyridine-2,4-dicarb\u00adoxy\u00adlic acid ligands exhibit a new coordination mode. Adjacent metal centers are linked by the O atoms of the pyridine-2,4-dicarb\u00adoxy\u00adlic acid ligands, and then form a three-dimensional supra\u00admolecular polymeric framework.In the title complex, [Ba(C DOI: 10.1107/S1600536810023457/pb2031Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S160053681102890X/ng5198Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The morpholine ring adopts a chair conformation.In the title complex, [CuBr(C DOI: 10.1107/S160053681002670X/hb5543Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The chain motifs are oriented parallel to the c-axis direction. Individual chains are connected into a supra\u00admolecular network via O\u2014H\u22efO hydrogen bonding involving the aqua ligands.In the title compound, {[Cd(C DOI: 10.1107/S1600536811047842/ds2153Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "A symmetry-related pair of six-membered hydrogen-bonded rings [graph-set motif S11(6)] are present for the terminal vanillin\u2013imine moieties. Two lattice methanol solvent mol\u00adecules are present per formula unit (Z\u2032 = 1/2), which form hydrogen-bonded chains along [010] with two orientations due to disorder of the methanol H-atom.The title compound, C DOI: 10.1107/S1600536812034940/hg5235Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "DOI: 10.1107/S160053681103306X/gw2107Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The structure shows a \u03bc2-O:O\u2032-bridging mode of the four 3,4,5-tri\u00admeth\u00adoxy\u00adbenzoate ligands finally stabilizing the two ZnII atoms in the dinuclear complex in a distorted square-pyramidal environment. The fifth coord\u00adin\u00adation site in the apical position of the pyramid is occupied by a coordinating dimethyl sulfoxide solvent mol\u00adecule equally disordered over two positions.The colourless title complex, [Zn DOI: 10.1107/S1600536813023118/kj2227Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S1600536813023118/kj2227Isup3.molSupplementary material file. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "The complete solid-state structure can be described as a three-dimensional supra\u00admolecular framework, stabilized by extensive hydrogen-bonding inter\u00adactions involving the coordinated water mol\u00adecules, uncoordin\u00adated imidazole N atom, protonated pyridine N and carboxyl\u00adate O atoms of the H2PIDC\u2212 ligands.In the title complex, [Zn(C DOI: 10.1107/S1600536810031855/jh2196Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The cobalt(II) cation is coordinated by two terminal N-bonded thio\u00adcyanate anions and two N-bonded 1,3-bis\u00ad(pyridin-4-yl)propane ligands, as well as two O atoms of methanol mol\u00adecules in a slightly distorted octa\u00adhedral coordination mode. Adjacent cations are connected into chains parallel to [10The asymmetric unit of the title compound, [Co(NCS) DOI: 10.1107/S1600536812000499/wm2581Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "In the crystal, mol\u00adecules are linked by O\u2014H\u22efO hydrogen bonds involving carboxyl groups, forming one-dimensional ladders. Two-dimensional layers are formed by inter\u00adpenetration of these one-dimensional ladders.In the title compound, C DOI: 10.1107/S1600536812012275/bq2343Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S1600536812012275/bq2343Isup3.cmlSupplementary material file. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "The benzene mol\u00adecule lies about a threefold rotoinversion axis.In the title compound, [Sn(C DOI: 10.1107/S1600536811055474/is5011Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "In the crystal, molecules are linked via pairs of O\u2014H\u22efO hydrogen bonds, forming inversion dimers.In the title compound, C DOI: 10.1107/S1600536811052275/fj2486Isup2.hkl Structure factors: contains datablock(s) I. DOI: 10.1107/S1600536811052275/fj2486Isup3.cml Supplementary material file. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The bridging function of the cis ligands leads to a helical chain structure along [100].In the title coordination polymer, [HgBr DOI: 10.1107/S1600536811028066/hg5063Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536811046022/hy2482Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The (pyridin-3-yl)(pyridin-4-yl)methanone ligand acts in a \u03bc2-bridging mode, linking the metal atoms, leading to an infinite chain along [-110]. O\u2014H\u22efO hydrogen bonds involving the lattice water mol\u00adecules connect these chains into a three-dimensional network.In the title complex, {[Cd(CH DOI: 10.1107/S1600536812019101/bt5902Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "In the crystal, mol\u00adecules are linked by C\u2014H\u22efN hydrogen bonds into chains along the c-axis direction. The asymmetric unit of the title compound, [Fe(C DOI: 10.1107/S1600536813031310/rn2116Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supporting information:"} +{"text": "The Zn\u22efZn distance is 3.076\u2005(1)\u2005\u00c5. Each Zn atom is five-coordinated by two O and two N atoms from two Schiff base ligands, and by one azide N atom, forming a square-pyramidal geometry.The title compound, [Zn DOI: 10.1107/S1600536811043455/qm2038Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The crystal packing is stabilized by O\u2014H\u22efO and C\u2014H\u22efO hydrogen bonds.In the title compound, [Cu(C DOI: 10.1107/S1600536811056145/hy2502Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "J. Am. DOI: 10.1107/S1600536811043820/bh2385Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The aromatic rings of the L ligand form a dihedral angle of 2.1\u2005(1)\u00b0.In the title compound, [Sn(C DOI: Click here for additional data file.10.1107/S1600536812047216/cv5361Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "No classic hydrogen bonds or inter\u00admolecular inter\u00adactions are observed in the crystal.In the title compound, [FeSn(C DOI: 10.1107/S1600536810022488/zq2044Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the crystal, mol\u00adecules are linked into a two-dimensional network parallel to (011) by O\u2014H\u22efO hydrogen bonds.In the title complex, [Cu(C DOI: 10.1107/S1600536812022702/bx2410Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "The figures in the article were corrupted. The correct figures are listed below.Figure 1: [^]Figure 2: [^]Figure 3: [^]Figure 4: [^]Figure 5: [^]Figure 6: [^]"} +{"text": "The pyridine and the pyrrole rings are nearly perpendicular to each other, making a dihedral angle of 84.83\u2005(7)\u00b0.In the title compound, [Zn(C DOI: 10.1107/S1600536810028503/dn2587Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The title complex, [CuBr(C DOI: 10.1107/S1600536811001814/hg2780Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In each, the 1,3-dioxane ring adopts an envelope conformation with the dimethyl-substituted C atom forming the flap. The crystal structure is stabilized by weak inter\u00admolecular C\u2014H\u22efO hydrogen bonds.The asymmetric unit of the title compound, C DOI: 10.1107/S1600536811002285/lh5196Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "There is a strong intra\u00admolecular O\u2014H\u22efN hydrogen bonds (which induces planarity of the structure). In the crystal, mol\u00adecules are linked by pairs of O\u2014H\u22efN hydrogen bonds, forming inversion dimers.In the title compound, C DOI: 10.1107/S1600536812001213/hg5155Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S1600536812001213/hg5155Isup3.cmlSupplementary material file. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "Bull. S DOI: 10.1107/S1600536814000361/pj2008Isup2.hklStructure factors: contains datablock(s) I. DOI: CCDC reference: crystallographic information; 3D view; checkCIF reportAdditional supporting information:"} +{"text": "In the crystal, weak inter\u00admolecular C\u2014H\u22efO hydrogen bonds link the complex cations and perchlorate anions.In the title compound, [Mn(C DOI: 10.1107/S1600536811023531/hy2441Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The ZnII atoms are bridged by terephthalate ligands, generating an infinite zigzag chain along [101].In the title coordination polymer, [Zn(C DOI: 10.1107/S1600536810022385/is2548Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the crystal, numerous O\u2014H\u22efO hydrogen bonds link the mol\u00adecules into a three-dimensional network.The binuclear title complex, [Co DOI: Click here for additional data file.10.1107/S1600536813000196/tk5188Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "In the crystal, the solvent mol\u00adecules and anions are linked by weak O\u2014H\u22efO hydrogen bonding.In the title solvated mol\u00adecular salt, [CuCl(C DOI: 10.1107/S1600536812017941/hb6747Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536812035337/vn2048Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "In the crystal, inter\u00admolecular O\u2014H\u22efO hydrogen bonds form a one-dimensional left-handed helical structureextending parallel to [001]. The water molecule of crystallization shows half-occupancy.In the title complex, [Cu(C DOI: 10.1107/S1600536811012049/hy2419Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The borohydride anions are situated on special positions with 4mm site symmetry and show rotational disorder around the fourfold axis.In the crystal structure of the title compound, C DOI: 10.1107/S1600536811029291/cv5120Isup2.cml Supplementary material file. DOI: 10.1107/S1600536811029291/cv5120Isup2.rtv Rietveld powder data: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials: interactive version of Fig. 1 Enhanced figure:"} +{"text": "The coordinating pyridine rings are oriented in an almost perpendicular fashion, making a dihedral angle of 83.7\u2005(5)\u00b0. In the title compound, [ZnI DOI: Click here for additional data file.10.1107/S1600536813001736/lr2093Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "The crystal structure features a three-dimensional hydrogen-bonding network based on a strong and distinctive pattern of O\u2014H\u22efO hydrogen-bonding inter\u00adactions.In the centrosymmetric title molecule, [Mn DOI: 10.1107/S1600536811008063/pb2059Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The distance between the CuI atoms within the dinuclear unit is 2.6723\u2005(11)\u2005\u00c5.In the centrosymmetric dinuclear title complex, [Cu DOI: 10.1107/S1600536812020843/kp2406Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "The K atoms occupy the The title compound, KSn DOI: 10.1107/S1600536811035604/fi2111Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536811045624/bt5698Isup2.hkl Structure factors: contains datablock(s) I. DOI: 10.1107/S1600536811045624/bt5698Isup3.cml Supplementary material file. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the crystal, adjacent mol\u00adecules are linked by inter\u00admolecular O\u2014H\u22efO inter\u00adactions, generating a helical hydrogen-bonded chain running along the b axis.The five-coordinate Sn atom in the title compound, [Sn(C DOI: 10.1107/S1600536810033623/bt5330Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "These ligands bridge the NiII complex units, forming chains extending along the [110] and [In the title compound, {[Ni(C DOI: 10.1107/S1600536811007021/zs2096Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The VV atom is five-coordinated by the two O and one N donor atoms of the Schiff base ligand, one methano\u00adlate O atom and one oxido O atom, forming a distorted square-pyramidal geometry.The title oxidovanadium(V) complex, [V(C DOI: 10.1107/S1600536811040207/qm2033Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the crystal, mol\u00adecules are connected into a three-dimensional architecture through O\u2014H\u22efO hydrogen bonds involving water mol\u00adecules and carboxyl\u00adate groups.In the title compound, [Mn(NCS) DOI: 10.1107/S1600536814000701/bt6955Isup2.hklStructure factors: contains datablock(s) I. DOI: CCDC reference: crystallographic information; 3D view; checkCIF reportAdditional supporting information:"} +{"text": "The ligands connect the metal atoms, forming layers parallel to the ab plane. O\u2014H\u22efO hydrogen bonds further assemble adjacent layers into a three-dimensional supra\u00admolecular network.In the title coordination polymer, {[Dy(C DOI: 10.1107/S1600536810041784/rz2501Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The 1,3-dioxane ring is in a slightly distorted boat conformation. The crystal structure is stabilized by weak inter\u00admolecular C\u2014H\u22efO hydrogen bonds.The title compound, C DOI: 10.1107/S1600536811026201/lh5270Isup2.hkl Structure factors: contains datablock(s) I. DOI: 10.1107/S1600536811026201/lh5270Isup3.cml Supplementary material file. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the crystal, the complex exhibits a chain structure parallel to the b axis.In the title polymeric coordination compound, [Sn(CH DOI: 10.1107/S1600536810054243/rz2544Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "One of the meth\u00adoxy side chains of the ligand is disordered over two orientations in a 0.700\u2005(6):0.300\u2005(6) ratio.In the centrosymmetric title compound, [OsCl DOI: 10.1107/S1600536811048926/hb6479Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The butane-1,2,3,4-tetra\u00adcarboxyl\u00adate ligands lie on inversion centers and bridge SrII ions, forming a three-dimensional network. Within the three-dimensional structure, there are O\u2014H\u22efO hydrogen bonds involving the water mol\u00adecules and carboxyl\u00adate O atoms.In the title compound, [Sr DOI: 10.1107/S1600536811046265/lh5368Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The purpose of this table is to provide the community with a citable record of publications of ongoing genome sequencing projects that have led to a publication in the scientific literature. While our goal is to make the list complete, there is no guarantee that we may have omitted one or more publications appearing in this time frame. Readers and authors who wish to have publications added to subsequent versions of this list are invited to provide the bibliographic data for such references to the SIGS editorial office. Thermogladius cellulolyticus\u201d 1633, sequence accession CP003531 [\u201cMethanomassiliicoccus luminyensis, sequence accession CAJE01000001 through CAJE01000026 [Pyrococcus sp. Strain ST04, sequence accession CP003534 [Leptospirillum ferrooxidans Strain C2-3, sequence accession AP012342 [Prochlorococcus marinus MED4, sequence accession BX548174 [Acinetobacter sp. Strain HA, sequence accession AJXD00000000 [00000000 Acinetobacter venetianus RAG-1T, sequence accession AKIQ00000000 [00000000 Aeromonas aquariorum, sequence accession BAFL01000001 through BAFL01000036, and AP012343 [AP012343 Agrobacterium tumefaciens CCNWGS0286, sequence accession AGSM00000000 [Alcaligenes faecalis subsp. faecalis NCIB 8687, sequence accession AKMR01000001 through AKMR01000186 [01000186 Alishewanella aestuarii Strain B11T, sequence accession ALAB00000000 [00000000 Alishewanella agri BL06T, sequence accession AKKU00000000 [00000000 Bartonella birtlesii strain IBS 135T, sequence accession AKIP00000000 [00000000 Brucella abortus A13334, sequence accession CP003176.1 (Chromosome I), CP003177.1 (Chromosome II) [Brucella canis Strain HSK A52141, sequence accession CP003174.1 (chromosome I), and CP003175.1 (chromosome II) [Brucella melitensis 16M13w, sequence accession AHWE00000000 [00000000 Brucella melitensis 16M1w, sequence accession AHWD00000000 [00000000 Brucella melitensis S66, sequence accession AHWB00000000 [00000000 Brucella melitensis16M, sequence accession AHWC00000000 [00000000 Burkholderia sp. Strain KJ006, sequence accession CP003514 (chromosome I), CP003515 (chromosome II), CP003516 (chromosome III), and CP003517 (plasmid pKJ006) [Burkholderia terrae Strain BS001, sequence accession AKAU00000000 [00000000 Burkholderia thailandensis MSMB43, sequence accession AJXB00000000 [00000000 CandidatusSulfurovum sediminum, sequence accession AJLE00000000 [00000000 Catenovulum agarivorans YM01T, sequence accession AJWM00000000 [00000000 Citrobacter sp. Strain A1, sequence accession AKTT00000000 [00000000 Citreicella aestuarii Strain 357, sequence accession AJKJ00000000 [00000000 Cronobacter sakazakii ES15, sequence accession CP003312 [CP003312 Dickeya zeae Strain ZJU1202, sequence accession AJVN00000000 [00000000 Enterobacter cloacae GS1, sequence accession AJXP00000000 [00000000 Enterobacter radicincitans DSM16656T, sequence accession AKYD00000000 [00000000 Enterobacter sp. Isolate Ag1, sequence accession AKXM00000000 [00000000 Escherichia coli J53, sequnce accession AICK00000000 [00000000 Escherichia coli LCT-EC106, sequence accession [ccession Escherichia coli NCCP15647, sequence accession AJMB00000000 [00000000 Escherichia coli W26, sequence accession AGIA00000000 [00000000 Gluconobacter oxydans WSH-003, sequence accession AHKI00000000 [00000000 Halomonas stevensii S18214T, sequence accession AJTS00000000 [00000000 Helicobacter cinaedi Strain PAGU611, sequence accession AP012344 (chromosome) and AP012345 (plasmid) [Helicobacter pylori hpEurope Strain N6, sequence accession CAHX01000001 to CAHX01000054 [01000054 Herbaspirillum lusitanum P6-12, sequence accession AJHH00000000 [00000000 Herbaspirillum sp. Strain GW103, sequence accession AJVC00000000 [00000000 Hydrocarboniphaga effusa strain AP103T, sequence accession AKGD00000000 [00000000 Hydrogenophaga sp. Strain PBC, sequence accession AJWL00000000 [00000000 Klebsiella oxytoca E718, sequence accession CP003683 [CP003683 Methylobacterium extorquens sp. strain 4-46, sequence accessions NC_010511, NC_010373, NC_010374 [C_010374 Methylobacterium extorquens strain BJ001, sequence accessions NC_010725, NC_010727, NC_010721 [C_010721 Methylobacterium extorquens strain CM4, sequence accessions NC_011757, NC_011758, NC_011760 [C_011760 Methylobacterium extorquens strain JCM 2831, sequence accessions NC_010510, NC_010509, NC_010514, NC_010517, NC_010518, NC_010502, NC_010504, NC_010507 [C_010507 Methylobacterium extorquens strain ORS 2060, sequence accessions NC_011894,NC_011892, NC_011887, NC_011893, NC_011895, NC_011888, NC_011889, NC_011890 [C_011890 Methylobacterium extorquens strain PA1, sequence accessions NC_010172 [C_010172 Methylobacterium sp. Strain GXF4, sequence accession AKFK00000000 [00000000 Methylophaga sp. Strain JAM1, sequence accession CP003390 [CP003390 Methylophaga sp. Strain JAM7 sequence accession CP003380 (chromosome), CP003381 (plasmid) [plasmid) Modestobacter marinus Strain BC501, sequence accession FO203431 [FO203431 Mycobacterium massiliense M18, sequence accession AJSC00000000 [00000000 Neisseria meningitidis Capsule Null Locus Strain, sequence accession CAJS01000001 through CAJS01000042 [01000042 Novosphingobium sp. Strain Rr 2-17, sequence accession AKFJ00000000 [00000000 Providencia stuartii Clinical Isolate MRSN 2154, sequence accession CP003488 [Pseudaminobacter salicylatoxidans KCT001, sequence accession CAIU00000000 [00000000 Pseudoalteromonas issachenkonii PAMC 22718, sequence accession AJTK00000000 [00000000 Pseudomonas aeruginosa Strain SJTD-1, sequence accession AKCM00000000 [00000000 Pseudomonas aeruginosa Strain XMG, sequence accession AJXX00000000 [00000000 Pseudomonas fuscovaginae CB98818, sequence accession ALAQ00000000 [00000000 Pseudomonas pseudoalcaligenes KF707, sequence accession AJMR00000000 [00000000 Pseudomonas putida Strain ND6, sequence accession CP003588 [CP003588 Pseudomonas putida Strain SJTE-1, sequence accession AKCL00000000 [00000000 Pseudomonas sp. Strain HYS, sequence accession AJJP00000000 [00000000 Pseudomonas sp. Strain M47T1, sequence accession AJWX00000000 [00000000 Pseudomonas stutzeri TS44, sequence accession AJXE00000000 [00000000 Pseudomonas stutzeri Strain JM300, sequence accession CP003725 [CP003725 Ralstonia sp. strain PBA, sequence accession AJWL00000000 [00000000 Rhodanobacter DSM 17631, sequence accession AJXT00000000 [00000000 Rhodanobacter DSM 18449, sequence accession AJXU00000000 [00000000 Rhodanobacter DSM 18863, sequence accession AJXW00000000 [00000000 Rhodanobacter DSM 24678, sequence accession AJXV00000000 [00000000 Rhodanobacter strain 115, sequence accession AJXS00000000 [00000000 Rhodanobacter strain DSM 23569, sequence accession AGIL00000000 [00000000 Rickettsia australis strain PhillipsT, sequence accession AKVZ00000000 [00000000 Rickettsia conorii subsp. caspia, sequence accession AJUR00000000 [00000000 Rickettsia conorii subsp. israelensis, sequence accession AJVP00000000 [00000000 Rickettsia sp. Strain MEAM1, sequence accession AJWD00000000 [00000000 Salmonella enterica serotype Newport CVM19443, sequence accession AHUB00000000 [00000000 Salmonella enterica serotype Newport CVM19470, sequence accession AHUE00000000 [00000000 Salmonella enterica serotype Newport CVM19593, sequence accession AHUD00000000 [00000000 Salmonella enterica serotype Newport CVM21538, sequence accession AHTV00000000 [00000000 Salmonella enterica serotype Newport CVM21550, sequence accession AHTT00000000 [00000000 Salmonella enterica serotype Newport CVM33953, sequence accession AHTM00000000 [00000000 Salmonella enterica serotype Newport CVM35185, sequence accession AHTJ00000000 [00000000 Salmonella enterica serotype Newport CVM37978, sequence accession AHUC00000000 [00000000 Salmonella enterica serovar Typhi UJ308A, sequence accession AJTD00000000 [00000000 Salmonella enterica Serovar Typhi UJ816A, sequence accession AJTE00000000 [00000000 Serratia marcescens strain LCT-SM213, seqeunce accession AJUV00000000 [00000000 Serratia plymuthica Strain PRI-2C, sequence accession AJTB00000000 [00000000 Serratia sp. Strain M24T3, sequence accession [ccession Sinorhizobium fredii USDA257, sequence accession CP003563 through CP003582 [CP003582 Sphingobium indicum B90A, sequence accession AJXQ00000000 [00000000 Stenotrophomonas maltophilia PML168, sequence accession CAJH01000001 through CAJH01000097 [01000097 Sulfuricella denitrificans skB26, sequence accession BAFJ01000001 through BAFJ01000023 [01000023 Xanthomonas campestris JX, sequence accession AJVO00000000 [00000000 Yersinia pestis Strain 2501, sequence accession AKVQ00000000 [00000000 Geobacillus thermoglucosidans\u201d TNO-09.020, sequence accession AJJN00000000 [\u201c00000000 Aerococcus viridans LL1, sequence accession AJTG00000000 [00000000 Bacillus anthracis H9401, sequence accession CP002091.1 ( chromosome), CP002092.1.1 (plasmid pXO1), and CP002093.1 (plasmid pXO2) [id pXO2) Bacillus atrophaeus C89, sequence accession AJRJ00000000 [00000000 Bacillus cereus NC7401, sequence accession AP007209 (chromosome), AP007210 (plasmid pNCcld), AP007211 , AP007212 , AP007213 , and AP007214 [4, 3 kb) Bacillus methanolicus MGA3, sequence accession ADWW00000000 [00000000 Bacillus methanolicus PB1, sequence accession AFEU00000000 [00000000 Bacillus siamensis KCTC 13613T, sequence accession AJVF00000000 [00000000 Bacillus sp. Strain 5B6, sequence accession AJST00000000 [00000000 Bacillus sp. Strain 916, sequence accession AFSU00000000 [00000000 Clostridium beijerinckii Strain G117, sequence acceession AKWA00000000 [00000000 Corynebacterium pseudotuberculosis Strain 1/06-A, sequence accession CP003082 [CP003082 Enterococcus faecalis D32, sequence accession CP003726 through CP003728 [CP003728 Enterococcus faecalis strain NP-10011, sequence accession AB712291 [Enterococcus faecium Clinical Isolate LCT-EF128, sequence accession AJUP00000000 [00000000 Enterococcus hirae ATCC 9790, sequence accession CP003504 (chromosome), NC_015845 (plasmid pTG9790) [pTG9790) Lactobacillus mucosae LM1, sequence accession AHIT00000000 [00000000 Lactobacillus rossiae DSM 15814, sequence accession AKZK00000000 [00000000 Lactococcus garvieae IPLA 31405, sequence accession AKFO00000000 [00000000 Paenibacillus polymyxa OSY-DF, sequence accession AIPP00000000 [00000000 Pediococcus pentosaceus strain IE-3, sequence accession CAHU01000001 through CAHU01000091 [01000091 Pelosinus fermentans A11, sequence accession AKVM00000000 [00000000 Pelosinus fermentans B4, sequence accession AKVJ00000000 [00000000 Pelosinus fermentans JBW45, sequence accession AKVO00000000 [00000000 Pelosinus fermentans R7, sequence accession AKVN00000000 [00000000 Planococcus antarcticus DSM 14505, sequence accession AJYB00000000 [00000000 Rhodococcus sp. strain DK17, sequence accession AJLQ00000000 [00000000 Staphylococcus aureus Strain LCT-SA112, sequence accession AJLP00000000 [00000000 Staphylococcus capitis QN1, sequence accession AJTG00000000 [00000000 Staphylococcus equorum subsp. equorum Mu2, sequence accession CAJL01000001 to CAJL01000030 [01000030 Staphylococcus hominis ZBW5, sequence accession AKGC00000000 [00000000 Staphylococcus saprophyticus subsp. saprophyticus M1-1, sequence accession AHKB00000000 [00000000 Streptococcus mutans GS-5, sequence accession CP003686 [CP003686 Streptococcus pyogenes M1 476, sequence accession AP012491 [AP012491 Streptococcus salivarius PS4, sequence accession AJFW00000000 [00000000 Streptococcus thermophilus Strain MN-ZLW-002, sequence accession CP003499 [CP003499 Ureibacillus thermosphaericus Strain Thermo-BF, sequence accession AJIK00000000 [00000000 Mycoplasma leachii Strain PG50T, sequence accession CP002108.1 [002108.1 Mycoplasma mycoides subsp. mycoides, sequence accession CP002107.1 [002107.1 Mycoplasma wenyonii Strain Massachusetts, sequence accession CP003703 [CP003703 Actinomyces massiliensis Strain 4401292T, sequence accession AKIO00000000 [00000000 Bifidobacterium animalis subsp. lactis B420, sequence accesion CP003497 [CP003497 Bifidobacterium animalis subsp. lactisBi-07, sequence accesion CP003498 [CP003498 Bifidobacterium bifidum strain BGN4, sequence accession CP001361 [CP001361 Brevibacterium massiliense Strain 541308T, sequence accession CAJD00000000 [00000000 Corynebacterium bovis DSM 20582, sequence accession AENJ00000000 [00000000 Corynebacterium diphtheriae Biovar Intermedius NCTC 5011, sequence accession AJVH00000000 [00000000 Corynebacterium pseudotuberculosis strain 1/06-A, sequence accession CP003082 [CP003082 Corynebacterium pseudotuberculosis strain 3/99-5 sequence accession CP003152.1 [003152.1 Corynebacterium pseudotuberculosis strain 42/02-A, sequence accession CP003062 [CP003062 Microbacterium yannicii, sequence accession CAJF01000001 through CAJF01000067 [01000067 Micromonospora lupini Lupac 08, sequence accession CAIE01000001 [01000001 Mycobacterium bolletii Strain M24, sequence accession AJLY00000000 [00000000 Mycobacterium intracellulare Clinical Strain MOTT-36Y, sequence accession CP003491 [CP003491 Mycobacterium massiliense M18, sequence accession AJSC00000000 [00000000 Mycobacterium massiliense strain GO 06, sequence accession CP003699 [CP003699 Mycobacterium massiliense strain M154, sequence accession AJMA00000000 [00000000 Mycobacterium tuberculosis RGTB327, sequence accession CP003233 [CP003233 Mycobacterium tuberculosis MTB423, sequence accession CP003234 [CP003234 Parascardovia denticolens IPLA 20019, sequence accession AKII00000000 [00000000 Saccharothrix espanaensis DSM 44229T, sequence accession HE804045 [HE804045 Streptomyces auratus Strain AGR0001, sequence accession AJGV00000000 [00000000 Streptomyces cattleya\u201d DSM46488T, sequence accession FQ859185 and FQ859184 [\u201cFQ859184 Streptomyces globisporus C-1027, sequence accession AJUO00000000 [00000000 Streptococcus mutans GS-5, sequence accession CP003686 [CP003686 Streptomyces sp. Strain AA1529, sequence accession ALAP00000000 [00000000 Streptomyces sulphureus L180, sequence accession AJTQ0000000 [Q0000000 Borrelia crocidurae, sequence accession CP003426 (chromosome), CP003427 to CP003465 (plasmids) [lasmids) Treponema sp. Strain JC4, sequence accession JQ783348 [JQ783348 Flavobacterium sp. Strain F52, sequence accession AKZQ00000000 [00000000 Fusobacterium nucleatum subsp. fusiforme ATCC 51190T, sequence accession AKXI00000000 [00000000 Imtechella halotolerans\u201d\u201c K1T, sequence accession AJJU00000000 [00000000 Actinophage PIS136, sequence accession JX006077 Aeromonas hydrophila Phage CC2, sequence accession JX123262 [JX123262 Bacteriophage BC-611, sequence accession AB712291 Bacteriophage SSU5, sequence accession JQ965645 Blattabacterium sp. strain BGIGA, sequence accession [ccession Caulobacter crescentus Bacteriophage \u03c6CbK, sequence accession JX163858 [JX163858 Celeribacter Bacteriophage P12053L, sequence accession JQ809650 [JQ809650 Croceibacter Bacteriophage P2559S, sequence accession JQ867099 [JQ867099 Cronobacter sakazakii Temperate Bacteriophage phiES15 JQ780327 [JQ780327 Marinomonas Bacteriophage P12026, sequence accession JQ867100 [JQ867100 Pectobacterium carotovorum subsp. carotovorum Bacteriophage PP1, sequence accession JQ837901 [JQ837901 Persicivirga bacteriophages P12024L, sequence accession JQ823123 [JQ823123 Persicivirga bacteriophages P12024S, sequence accession JQ823122 [JQ823122 phage clP1, sequence accession JN051154 Pseudomonas aeruginosa Siphophage MP1412, sequence accession JX131330 [JX131330 Pseudomonas aeruginosa Temperate Phage MP29, sequence accession EU272036 [EU272036 Pseudomonas aeruginosa Temperate Phage MP42, sequence accession JQ762257 [JQ762257 Pseudomonas Phage \u03a6-S1, sequence accession JX173487 [JX173487 Siphophage MP1412, sequence accession JX131330 Staphylococcus aureus Bacteriophage GH15, sequence accession JQ686190 [JQ686190 Vibrio vulnificus Bacteriophage SSP002, sequence accession JQ692107 [JQ692107 African bovine rotaviruses RVA/Cow-wt/ZAF/1603/2007/G6P, sequence accession S9(VP7) JN831209, S4(VP4) JN831210, S6(VP6) JN831211, S1(VP1) JN831212, S2(VP2) JN831213, S3(VP3) JN831214, S5(NSP1) JN831204, S8(NSP2) JN831205, S7(NSP3) JN831206, S10(NSP4) JN831207, S11(NSP5) JN831208 African bovine rotaviruses RVA/Cow-wt/ZAF/1604/2007/G8P, sequence accession S9(VP7) JN831220, S4(VP4) JN831221, S6(VP6) JN831222, S1(VP1) JN831223, S2(VP2) JN831224, S3(VP3) JN831225, S5(NSP1) JN831215, S8(NSP2) JN831216, S7(NSP3) JN831217, S10(NSP4) JN831218, S11(NSP5) JN831219 African bovine rotaviruses RVA/Cow-wt/ZAF/1605/2007/G6P, sequence accession S9(VP7) JN831231, S4(VP4) JN831232, S6(VP6) JN831233, S1(VP1) JN831234, S2(VP2) JN831235, S3(VP3) JN831236, S5(NSP1) JN831226, S8(NSP2) JN831227, S7(NSP3) JN831228, S10(NSP4) JN831229, S11(NSP5) JN831230 Avian Leukosis Virus, sequence accession JX254901 Avian Influenza Virus H3N2, sequence accession JX175250 through JX175257 Avian Influenza Virus H5N2, sequence accession JQ990145 through JQ990152 Avian-Like H4N8 Swine Influenza, sequence accession JX151007 through JX151014 Avian Paramyxovirus, sequence accession JQ886184 Avian Tembusu-Related Virus Strain WR, sequence accession JX196334 Bluetongue Virus Serotype 9, sequence accession JX003687 to JX003696 Bluetongue Virus Serotype 16, sequence accession Bombyx mori Nucleopolyhedrovirus, sequence accession JQ991009 Bovine Viral Diarrhea Virus 2, sequence accession JF714967 Bovine Foamy Viruses, sequence accession JX307861 Canine Noroviruses, sequence accession FJ692500 and FJ692501 Chicken Anemia Virus, sequence accession JX260426 Chikungunya Virus, sequence accession JX088705 Chinese Virulent Avian Coronavirus GX-YL5, sequence accession HQ848267 Chinese Virulent Avian Coronavirus GX-YL9, sequence accession HQ850618 Coxsackievirus B4, sequence accession JX308222 Enterovirus C (HEV-C117), sequence accession JX262382 Genotype 4 Hepatitis E Virus Strain, sequence accession JQ993308 H10N8 Avian Influenza Virus, sequence accession JQ924786 to JQ924793 H9N2 Subtype Influenza Virus FJG9, sequence accession JF715008.1, JN869514.1 through JN869520.1 .Herpes Simplex Virus 1 Strain McKrae, sequence accession JX142173 Human Coronavirus NL63, sequence accession JX104161 Human G10P Rotavirus, sequence accession AB714258 through AB714268 Ikoma Lyssavirus, sequence accession JX193798 Korean sacbrood viruses AmSBV-Kor19, sequence accession JQ390592 Korean sacbrood viruses AmSBV-Kor21, sequence accession JQ390591 , sequence accession JN835456 [Mitochondrion of Frankliniella occidentalisJN835456 New Circular DNA Virus from Grapevine, sequence accession JQ901105 Novel Porcine Epidemic Diarrhea Virus, sequence accession JX112709 Pararetrovirus, sequence accession JQ926983 Parechovirus, sequence accession JX050181 Peste des Petits Ruminants Virus, sequence accession JX217850 Polyomavirus, sequence accession JQ412134 Porcine Circovirus 2b Strain CC1, sequence accession JQ955679 Porcine circovirus type 2 (PCV2), sequence accession JX294717 Porcine Epidemic Diarrhea Virus Strain AJ1102, sequence accession JX188454 Porcine Sapelovirus Strain YC2011, sequence accession JX286666 [Respiratory Syndrome Virus Strain QY2010, sequence accession JQ743666 SAT 2 Foot-and-Mouth Disease Virus, sequence accession JX014255 SAT 2 Foot-and-Mouth Disease Virus PAT, sequence accession JX014256 Street Rabies Virus, sequence accession HQ450386 Waterfowl aviadenovirus goose adenovirus 4, sequence accession JF510462 , sequence accession NC_015104 [cpDNA of Smilax chinaC_015104 , sequence accession JQ310743 [Elodea canadensisJQ310743 Ogura-type mitochondrial genome, sequence accession AB694743 Aspergillus oryzae Strain 3.042, sequence accession AKHY00000000 [00000000 Rhodosporidium toruloides MTCC 457, sequence accession AJMJ00000000 [00000000 Helicoverpa armigera, sequence accession HQ613271 [HQ613271 plasmidIncN plasmid pRSB201, sequence accession JN102341 plasmidIncN plasmid pRSB203, sequence accession JN102342 plasmidIncN plasmid pRSB205, sequence accession JN102343 plasmidIncN plasmid pRSB206, sequence accession JN102344"} +{"text": "In the crystal, the components are linked through N\u2014H\u22efO and O\u2014H\u22efO hydrogen bonds, forming [100] chains of alternating hydrazone and methanol mol\u00adecules.In the title compound, C DOI: 10.1107/S1600536811029394/hb6324Isup2.hkl Structure factors: contains datablock(s) I. DOI: 10.1107/S1600536811029394/hb6324Isup3.cml Supplementary material file. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Neighbouring pairs of CuII atoms are linked by four basally coordinating bridging acetate ligands as in the crystal structure of copper acetate monohydrate. The fifth, apically coordinating ligand links two of the dicopper tetra\u00adacetate paddlewheel-units together, thus building a linear coordination polymer which extends along [10-1]. Each apical acetate ligand is linked by an N\u2014H\u22efO hydrogen bond to a triethyl\u00adammonium cation. Weak C\u2014H\u22efO hydrogen bonding interactions also occur.In the title compound, {[(C DOI: 10.1107/S1600536812033405/hp2044Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S1600536812033405/hp2044Isup3.molSupplementary material file. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "One of the two independent Cl\u2212 counter-anions sits on a special position (site symmetry In the title complex, [CrCl DOI: 10.1107/S1600536811039328/wm2515Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536811018228/rn2085Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the crystal, the mol\u00adecule is located on an inversion center. The Mo atom is equally disordered over two positions; the range of Mo\u2014C distances is 2.2244\u2005(19)\u20132.3400\u2005(17)\u2005\u00c5 for both components of the disorder.The title compound, [Mo(C DOI: 10.1107/S1600536812002504/tk5041Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S1600536812002504/tk5041Isup3.molSupplementary material file. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "In the crystal, water O\u2014H\u22efCl and imidazole N\u2014H\u22efCl hydrogen bonds give rise to a three-dimensional structure.In the title compound, [Mg(C DOI: 10.1107/S1600536813021478/zs2271Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "The benzoyl and aniline benzene rings are tilted relative to each other by 82.8 (1)\u00b0. In the crystal, inter\u00admolecular N\u2014H\u22efO hydrogen bonds link the mol\u00adecules into infinite chains running along the c-axis direction.In the title compound, C DOI: 10.1107/S1600536811041651/bt5668Isup2.hkl Structure factors: contains datablock(s) I. DOI: 10.1107/S1600536811041651/bt5668Isup3.cml Supplementary material file. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The crystal structure features N\u2014H\u22efO hydrogen bonds, which link the mol\u00adecules into C(4) chains running along the a axis.In the title compound, C DOI: 10.1107/S1600536811045107/bt5692Isup2.hkl Structure factors: contains datablock(s) I. DOI: 10.1107/S1600536811045107/bt5692Isup3.cml Supplementary material file. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The bipyridine ligands also lies on a twofold rotation axis and bridge the CoII ions, forming chains extending along [010]. An intra\u00adchain O\u2014H\u22efO hydrogen bond is observed.In the title compound, [Co(C DOI: 10.1107/S1600536811046149/lh5365Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536810047793/hg2742Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the crystal, there are no identifiable directional inter\u00adactions between cations and anions except for Coulombic forces.In the title mol\u00adecular salt, (C DOI: 10.1107/S1600536812023008/hb6797Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "Eight NdIII ions and 12 pda ligands form a large [Nd8(pda)12] ring, and four NdIII ions and six pda ligands form a small [Nd4(pda)6] ring. These rings are further connected by the coordination inter\u00adactions of pda ligands and NdIII, generating a three-dimensional supra\u00admolecular framework.In the title coordination polymer, [Nd DOI: 10.1107/S1600536811006817/wm2461Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The two CuII atoms are separated by 2.6850\u2005(7)\u2005\u00c5, and together with the four formate ligands they form a paddle-wheel unit. The hexa\u00admine ligand uses only two of its four N atoms to link Cu2 cluster units, affording a zigzag chain running along the b-axis direction. The hexa\u00admine ligand lies on a mirror plane.In the title polymeric compound, [Cu DOI: 10.1107/S160053681303184X/ng5345Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S160053681303184X/ng5345Isup3.molSupplementary material file. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "Extensive O\u2014H\u22efO hydrogen bonding connects the mol\u00adecules into a three-dimensional supra\u00admolecular structure.In the centrosymmetric binuclear title compound, [Ni DOI: 10.1107/S1600536810049718/ds2071Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "An intra\u00admolecular N\u2014H\u22efO hydrogen bond occurs. The hy\u00addroxy group is hydrogen bonded to the double-bond S atom of an inversion-related mol\u00adecule, generating a hydrogen-bonded dimer in the crystal structure.In the title compound, C DOI: 10.1107/S1600536811012736/xu5185Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Each PrIII ion is nine-coordinated by one 1,10-phenanthroline mol\u00adecule, one bidentate carboxyl\u00adate group and four bridging carboxyl\u00adate groups in a distorted PrN2O7 monocapped square-anti\u00adprismatic geometry. The title compound is isotypic with its terbium- and dysprosium-containing analogues.In the centrosymmetric binuclear title complex, [Pr DOI: 10.1107/S1600536811034702/hb6378Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the crystal, N\u2014H\u22efBr hydrogen bonds link the N,N-di\u00admethyl\u00adanilinium cations and both Br\u2212 anions and [SnBr6]2\u2212 dianions into a layered arrangement parallel to (001). In the title compound, (C DOI: Click here for additional data file.10.1107/S1600536813012403/nk2205Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S1600536813012403/nk2205Isup3.cdxSupplementary material file. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "The compound has crystallographic mirror symmetry with both the cation and the tetrahedral anion located across a mirror plane. The cation and anion are linked by a C\u2014H\u22efO hydrogen bond.The title compound, (C DOI: 10.1107/S1600536811050677/ez2273Isup2.hkl Structure factors: contains datablock(s) I. DOI: 10.1107/S1600536811050677/ez2273Isup3.cdx Supplementary material file. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The coordination around the RuII atom can thus be considered as octa\u00adhedral with slight trigonal distortion.In the title complex, [RuCl DOI: 10.1107/S1600536811035379/im2311Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Weak C\u2014H\u22efN inter\u00adactions contribute to the crystal packing stability.The structure of the title compound, [Mn(NCS) DOI: 10.1107/S1600536811051282/hg5141Isup2.hkl Structure factors: contains datablock(s) I. DOI: 10.1107/S1600536811051282/hg5141Isup3.cdx Supplementary material file. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The six-coordinate metal atom displays a distorted octa\u00adhedral geometry.In the crystal structure of the title compound, [Ir(C DOI: 10.1107/S1600536811034222/si2371Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536813019570/lr2111Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "The CoIII atom is coordinated by a methyl group, an N-bonded pyridine and two N,N\u2032-bidentate dimethyl\u00adglyoximate ligands in a distorted octa\u00adhedral geometry. The glyoximate ligands exhibit intra\u00admolecular O\u2014H\u22efO hydrogen bonds, which is very common in cobaloxime derivatives.The title compound, [Co(C DOI: 10.1107/S1600536812001092/fk2049Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "The structure contains voids of 113\u2005(19)\u2005\u00c53, but no solvent mol\u00adecule could reasonably be located there.The crystal structure of the title salt, [Sn(C DOI: 10.1107/S1600536810039450/xu5034Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the crystal, mol\u00adecules are linked by inter\u00admolecular C\u2014H\u22efO hydrogen bonds into chains parallel to the b axis.In the title compound, C DOI: 10.1107/S1600536811018332/rz2596Isup2.hkl Structure factors: contains datablocks I. DOI: 10.1107/S1600536811018332/rz2596Isup3.cml Supplementary material file. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The cyclo\u00adhexyl group is disordered over two orientations with site-occupancy factors of 0.600\u2005(14) and 0.400\u2005(14).In the title compound, [Bi(C DOI: 10.1107/S1600536811002510/lx2186Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Weak inter\u00admolecular C\u2014H\u22efO hydrogen bonds link cations and anions into a three-dimensional structure.The asymmetric part of the title compound, (C DOI: 10.1107/S1600536811043091/cv5168Isup2.hkl Structure factors: contains datablock(s) I. DOI: 10.1107/S1600536811043091/cv5168Isup3.cml Supplementary material file. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "AbstractMicatagla Argaman (Bradynobaenidae: Apterogyninae) is reviewed from Egypt, based on specimens collected from Wadi Allaqi and Kom Osheim (Fayoum) and those deposited in Egyptian insect collections as well as recorded data from the literature. A single species, Micatagla klugi (Andr\u00e9), was previously recorded from Egypt. Micatagla allaqiensissp. n., Micatagla ezzatisp. n. and Micatagla pseudoraineriisp. n. are described here. Micatagla antropovi Pagliano is also newly recorded from the Egyptian fauna. An illustrated key and a faunistic list comprising all Micatagla species recorded from Egypt are given.The genus PageBreakMicatagla Argaman, 1994 is a relatively large genus in the subfamily Apterogyninae, with 47 recorded species from Namibia. Members of the genus are widely distributed in Africa; only two species were recorded from Asia of female without basal tegumental yellow spot; eyes small, distant from occipital carina at least by their own diameter; hind trochanter with ventral lamella; forewing with closed brachial cell (except open in Micatagla noorti). Both sexes are normally quite colourful as they consist of red mixed with ferruginous to black, only few species have individuals all black.Members of the genus Micatagla is represented by a single species, Micatagla klugi . In the Micatagla species in the different Egyptian localities is plotted and Kom Osheim (Fayoum) and those deposited in the Egyptian insect collections as well as previous records from Egypt. Sampling was done by means of pitfall traps. Morphological terms are based on plotted using DICollection sites. Aswan: 24\u00b005'26\"N, 32\u00b054'00\"E; Bir Um Reiga: 29\u00b032'28\"N, 32\u00b021'45\"E; Kafr Hakim: 30\u00b004'54\"N, 31\u00b007'00\"E; Kom Osheim: 29\u00b033'46\"N, 30\u00b054'36\"E; Massara: 30\u00b004'15\"N, 31\u00b014'43\"E; Mokattam: 30\u00b001'00\"N, 31\u00b017'16\"E; Wadi Allaqi: 22\u00b050'20\"N, 33\u00b011'54\"E; Wadi Assiouti: 27\u00b012'30\"N, 31\u00b018'50\"E; Wadi Digla: 29\u00b057'30\"N, 31\u00b020'06\"E; Wadi el-Tih: 29\u00b009'00\"N, 33\u00b032'00\"E; Wadi Garawi: 29\u00b048'02\"N, 31\u00b027'39\"E. Wadi Hoff: 29\u00b053'22\"N, 31\u00b020'25\"E.Collection repositories (BMNH).Egypt, 1 \u2640, Kom Osheim (Fayoum), 30.v.2013 (leg. Ahmad M. Soliman) [CUE].Saudi Arabia, Yemen ; Egypt http://species-id.net/wiki/Micatagla_klugiApterogyna klugi Andr\u00e9, 1899: 69, holotype \u2640: Aswan (Egypt), (probably in MNHN).Apterogyna klugimocsaryi). : Apterogyna gridelliiMicatagla klugi by Invrea, 1959: 117, holotype \u2640: Egypt; synonymized with Utapitoca klugi : Egypt, 8 \u2640, Wadi Assiouti (Assiout), 1\u20132.iv.1917 (leg. Andr\u00e9) [PPDD]; 1\u2640, Cairo, with no date (leg. Adair) [PPDD]; 1\u2640, Mokattam (Cairo), April (leg. Innes Bey) [ESEC]; 1\u2640, without locality or date (leg. Ferrant) [ESEC].Wadi Hoff (Helwan), Bir Um Reiga (Red Sea), Massara (Cairo), Wadi Digla (Cairo), Wadi Garawi (Helwan), Wadi el-Tih (Sinai) .Egypt; Saudi Arabia .PageBreakGadallah & Solimansp. n.http://zoobank.org/EACEA2EE-E24B-49DB-8F48-64465128B1D5http://species-id.net/wiki/Micatagla_pseudorainerii27\u00b012'30\"N, 31\u00b018'50\"E], 13.iv.1934 (leg. ?) [CUE].Holotype \u2640: Egypt, Wadi Assiouti, Assiout .Holotype \u2640: Southern Egypt, Wadi Allaqi, Aswan , 8.v.1932 (leg.?) [CUE].Holotype \u2640: Egypt, Kafr Hakim, Giza [Female (Holotype). Body length 6 mm.Colour. Red, except mandible reddish brown distally, maxillary and labial palpi pale; mid and hind tibial spurs waxy white; 2nd & 3rd metasomal segments blackish red , T4 & T5 pale red, T6 reddish brown.Pubescence. Body including legs clothed with fine erect to recumbent whitish hairs, relatively longer on mesopleuron and distinctly denser on face and vertex of head, T4 and T5 than elsewhere. Posterior margin of T1 with fringe of irregular and inwardly directed whitish hairs; T2 and T3 with apical fascia of silvery inwardly directed hairs that are much denser than that of T1.Head ."} +{"text": "DOI: 10.1107/S1600536810023378/rk2207Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The mol\u00adecule lies on a sixfold inversion axis.In the crystal structure of the title compound, [Hf(C DOI: 10.1107/S1600536811014516/ng5148Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the crystal, the components are linked by C\u2014H\u22efCl inter\u00adactions.In the title solvated complex, [NiCl DOI: 10.1107/S1600536811042759/hb6452Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The [SnCl6]2\u2212 anion exhibits almost perfect octa\u00adhedral geometry. The 3-aza\u00adniumylpyridin-1-ium and chloride ions are connected via medium\u2013strong charge-supported N\u2014H\u22efCl hydrogen bonds, forming undulating layers in the (110) plane. The [SnCl6]2\u2212 ions are located between these layers and occupy cavities formed by two facing layer puckers.The asymmetric unit of the title compound, (C DOI: Click here for additional data file.10.1107/S1600536813006806/pk2471Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536813030286/fj2648Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "Each 1,3-di-4-pyridyl\u00adpropane ligand displays a monodentate coordinating mode. In the crystal, there exist O\u2014H\u22efO, O\u2014H\u22efN and C\u2014H\u22efO hydrogen bonds. The perchlorate anions and the coordinated and lattice water mol\u00adecules play an important role in the formation of these hydrogen bonds. One of the two lattice water molecules shows half-occupancy.In the title complex, [Mn(C DOI: 10.1107/S1600536811041511/pv2456Isup2.cdx Supplementary material file. DOI: 10.1107/S1600536811041511/pv2456Isup3.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The anion bridges adjacent metal atoms, forming zigzag polymeric chains parallel to [011] and [0In the title coordination polymer, [Sn(CH DOI: 10.1107/S1600536811055176/rz2693Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "The ligands link the CdII ions into a zigzag chain extending along [0In the title compound, {[CdBr DOI: 10.1107/S160053681101662X/hy2426Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The ZnII atom is five-coordinated in a distorted square-pyramidal geometry by five O atoms from four bpdc ligands. The dihedral angle between the benzene rings is 52.32\u2005(12)\u00b0.The crystal structure of the polymeric title complex, [Zn(C DOI: 10.1107/S1600536812038901/hy2581Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "The m-BDC ligand is located over a twofold rotation axis. The CdII atoms are bridged by the m-BDC ligands, leading to a wave-shaped chain structure along [010]. N\u2014H\u22efO hydrogen bonds connect the chains.In the title coordinaltion polymer, [Cd(C DOI: 10.1107/S1600536811001243/hy2397Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the crystal, O\u2014H\u22efO and O\u2014H\u22efN hydrogen bonds help to establish the packing.In the title compound, [Mg(C DOI: 10.1107/S1600536810039437/hb5638Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The presence of two bulky tert-butyl groups on the carboxyl\u00adate prevents any hydrogen-bonding inter\u00adactions involving the hy\u00addroxy group.The title compound, [Sn(C DOI: 10.1107/S1600536810021884/hy2317Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the crystal, C\u2014H\u22efBr inter\u00adactions link the mol\u00adecules.The asymmetric unit of the title compound, [ZnBr DOI: 10.1107/S1600536810028692/hb5556Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The tetra\u00adhedral angles are in the range 104.93\u2005(9)\u2013118.81\u2005(9)\u00b0.In the title compound, [Zn(C DOI: 10.1107/S1600536811054821/bv2196Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536812016777/zs2193Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "The word \"Factor\" is misspelled in the article title. The correct title is: Intranasal \"painless\" Human Nerve Growth Factor Slows Amyloid Neurodegeneration and Prevents Memory Deficits in App X PS1 Mice. The correct citation is: Capsoni S, Marinelli S, Ceci M, Vignone D, Amato G, et al. (2012) Intranasal \u201cpainless\u201d Human Nerve Growth Factor Slows Amyloid Neurodegeneration and Prevents Memory Deficits in App X PS1 Mice. PLoS ONE 7(5): e37555. doi:10.1371/journal.pone.0037555"} +{"text": "The bridging ligand, which is completed by crystallographic twofold symmetry, links the CoII atoms into [10In the title one-dimensional coordination polymer, [Co(C DOI: 10.1107/S160053681004715X/hb5708Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The Sb and N atoms are trans to each other.In the title compound, [FePdCl(C DOI: 10.1107/S1600536813015109/vn2072Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "DOI: Click here for additional data file.10.1107/S1600536812046648/xu5650Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "C. R. H DOI: 10.1107/S1600536813018588/br2229Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536810049007/bq2246Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Each AlO4 unit shares four O atoms with four adjacent PO4 units, leading to an anionic chain along [100]. The negative charge of the chain is compensated by doubly protonated camphoric amine cations. N\u2014H\u22efO hydrogen bonds connect the cations and the anionic chains. O\u2014H\u22efO hydrogen bonds are present in the chain.In the title compound, {(C DOI: 10.1107/S1600536812028826/hy2556Isup2.molSupplementary material file. DOI: 10.1107/S1600536812028826/hy2556Isup3.cdxSupplementary material file. DOI: 10.1107/S1600536812028826/hy2556Isup4.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S1600536812028826/hy2556Isup5.cdxSupplementary material file. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "In the crystal, mol\u00adecules are linked by weak C\u2014H\u22efO inter\u00adactions.In the mononuclear title complex, [Cu(C DOI: 10.1107/S1600536811045636/ff2036Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The dihedral angles between the benzene rings and the pyrazine ring are 14.66\u2005(8) and 49.76\u2005(12)\u00b0.In the title complex, [Ir(C DOI: 10.1107/S1600536812031546/hy2570Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "The cage consists of a mononuclear CuII unit [Cu(Hm-dtc)2], three \u03bc4-bridging Hm-dtc\u2212 ligands, eight CuI ions with distorted tetra\u00adhedral or trigonal pyramidal coordination geometries and four \u03bc2-bridging bromide anions. The incorporated central bromide anion inter\u00adacts with nine Cu ions with shorter Cu\u2014Br separations than the sum of the van der Waals radii for Cu and Br.The reaction of Cu(Hm-dtc) DOI: Click here for additional data file.10.1107/S1600536813009938/is5263Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S1600536813009938/is5263Isup4.cdxSupplementary material file. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536811004466/jj2075Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The NiII ion is coordinated by four S atoms of two mnt2\u2212 ligands and exhibits a square-planar coordination geometry.The asymmetric unit of the title complex, (C DOI: 10.1107/S1600536812008161/tk5062Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536812033004/lr2073Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "The NaI atom has a six-coordinate distorted-octa\u00adhedral environment. Isatine-3-oximate O atoms and water mol\u00adecules bridge adjacent Na atoms, forming a one-dimensional polymeric structure parallel to [100]. Each isatine-3-oxime dimerizes through N\u2014H\u22efO interactions and in addition each oxime is linked to a coordination polymer. Thus, coordination polymers are linked by O\u2014H\u22efO and O\u2014H\u22efN interactions from isatine-3-oxime dimers, building a two-dimensional network parallel to [110]. The reaction of hydroxyl\u00adamine hydro\u00adchloride with isatin in ethanol, catalysed with HCl and neutralized with Na DOI: 10.1107/S1600536811018290/bt5520Isup2.mol Supplementary material file. DOI: 10.1107/S1600536811018290/bt5520Isup3.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The CuII ion adopts a CuO2N3 distorted square-pyramidal coordination. An O\u2014H\u22efO hydrogen bond is formed between the methanol solvent mol\u00adecule and the hydrazide O atom of the L 2\u2212 ligand.The title mononuclear complex, [Cu(C DOI: 10.1107/S1600536811054316/kp2374Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The mol\u00adecules are connected into a three-dimensional architecture by O\u2014H\u22efO hydrogen bonds. The perchlorate anion is disordered over two positions; the major component has a site-occupancy factor of 0.525\u2005(19).In the structure of the title complex, [Ni(CH DOI: 10.1107/S1600536812007970/tk5060Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S1600536812007970/tk5060Isup3.molSupplementary material file. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "The crystal structure is stabilized by van der Waals inter\u00adactions.The Sn atom in the title compound, [Sn(CH DOI: 10.1107/S160053681102561X/bx2356Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Pairs of CdII ions are bridged by two end-to-end inversely bridging \u03bc-NCS-N:S thio\u00adcyanate groups, forming a two-dimensional network with the remaining two trans positions of the octa\u00adhedrally coordinated CdII ions occupied by the N atoms of two neutral 2-meth\u00adoxy\u00adaniline ligands. The crystal structure is stabilized by intra\u00adlayer N\u2014H\u22efS hydrogen bonds.The structure of the title compound, [Cd(NCS) DOI: Click here for additional data file.10.1107/S1600536813010738/rz5058Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S1600536813010738/rz5058Isup3.cdxSupplementary material file. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "The phenyl ring is rotated by 62.16\u2005(4)\u00b0 with respect to the plane of the quinoline system. In the crystal, O\u2014H\u22efO hydrogen bonds link mol\u00adecules into infinite chains running along the b-axis direction.In the title compound, C DOI: Click here for additional data file.10.1107/S1600536813000226/fy2079Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S1600536813000226/fy2079Isup3.cmlSupplementary material file. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "The two benzene rings of the biphenyl unit form a dihedral angle of 49.08\u2005(11)\u00b0. In the crystal, mol\u00adecules are linked into [100] chains by C\u2014H\u22efO hydrogen bonds.In the title compound, C DOI: 10.1107/S1600536812018053/hb6749Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S1600536812018053/hb6749Isup3.cmlSupplementary material file. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "The 4-methoxybenzenesulfonate anions act as bis-chelating and bridging ligands, forming a two-dimensional polymer parallel to (001), which is further linked into a three-dimensional network by weak C\u2014H\u22efO hydrogen bonds.In the title complex, [Na(C DOI: 10.1107/S1600536813025919/lh5648Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536810051342/bt5431Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In contrast to the first polymorph , the current study revealed monoclinic symmetry for the second polymorph. The configuration of the tetra\u00adhedral mol\u00adecule shows approximate sC symmetry. Strong O\u2014H\u22efO hydrogen bonds connect the mol\u00adecules to infinite zigzag chains along [010], which are further connected by weak inter\u00admolecular C\u2014H\u22efO contacts into a three-dimensional network.The title compound, C DOI: 10.1107/S1600536811025505/wm2504Isup2.cdx Supplementary material file. DOI: 10.1107/S1600536811025505/wm2504Isup3.hkl Structure factors: contains datablock(s) I. DOI: 10.1107/S1600536811025505/wm2504Isup4.cml Supplementary material file. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the crystal, extensive O\u2014H\u22efO and N\u2014H\u22efO hydrogen bonds link the complex cations, nitrate anions and lattice water mol\u00adecules into a three-dimensional network.In the centrosymmetric title compound, [Co(C DOI: Click here for additional data file.10.1107/S1600536813012968/hy2611Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "Adjacent SnIV atoms are bridged by the ligands, thereby forming a chain propagating in [010]. In the title polymeric coordination compound, [Sn(CH DOI: 10.1107/S1600536811049713/hb6523Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The crystal structure displays O\u2014H\u22efO and N\u2014H\u22efO hydrogen bonds, which connect the components into an extended three-dimensional network.In the title complex, [Ni(C DOI: 10.1107/S1600536813032169/jj2177Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supporting information:"} +{"text": "The bridging ligand, which is completed by crystallographic inversion symmetry, links the ZnII atoms into zigzag chains propagating in [101]. Within the ligand, the dihedral angle between the central benzene ring and terminal imidazole ring is 27.82\u2005(13)\u00b0.In the title one-dimensional coordination polymer, [ZnCl DOI: 10.1107/S1600536810044429/hb5712Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The ligands link the CdII ions into a ribbon-like structure running along [201]. One O atom of the nitrate anion is disordered over two positions with site-occupancy factors of 0.59\u2005(2) and 0.41\u2005(2).In the title compound, [Cd(NO DOI: 10.1107/S1600536811032697/hy2453Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the crystal, inter\u00admolecular O\u2014H\u22efO hydrogen bonds link mol\u00adecules into a two-dimensional supramolecular structure parallel to (001).In the title complex, [Zn(C DOI: 10.1107/S1600536811021623/hg5046Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The two perchlorate anions are disordered over two sites with a refined occupancy ratio of 0.528\u2005(19):0.472\u2005(19).In the title compound, [Ni(C DOI: 10.1107/S1600536810051627/nk2079Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Coordinated and uncoordinated water mol\u00adecules form O\u2014H\u22efO hydrogen bonds, leading to a three-dimensional framework.In the title compound, [Zn(C DOI: 10.1107/S1600536810017538/is2541Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Adjacent mol\u00adecules are linked by O\u2014H\u22efO hydrogen bonds, forming a chain running along [001].In the title compound, [Sn(C DOI: 10.1107/S1600536813031401/ng5348Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "N\u2014H\u22efCl and O\u2014H\u22efCl hydrogen bonding leads to the formation of layers parallel to (100).The title salt, [Ru(Tp)(CH DOI: Click here for additional data file.10.1107/S1600536812042110/ng5297Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536811011226/ru2002Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The organic ligands link inorganic MnII nodes, forming a zigzag chain along the c axis.In the title compound, [Mn(C DOI: 10.1107/S1600536810045587/jh2224Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The mol\u00adecule, which has non-crystallographic C2-symmetry, consists of a core structure of two CuI ions, bridged by two iodide ions. Each CuI ion is also coordinated by one equivalent of the chiral bidentate (R)-BINAP ligand . Thus, both cations show a distorted tetra\u00adhedral geometry being surrounded by two I atoms and two P atoms from the (R)-BINAP ligands. The complex consists of isolated butterfly-shaped mol\u00adecules featuring an angle of 146.11\u2005(2)\u00b0 between adjacent CuI2 planes. The structure displays intra\u00admolecular C\u2014H\u22efI hydrogen bonding and contains disordered water. The absolute configuration of this chiral complex was determined by anomalous dispersion effects.The structure of the title compound, [Cu DOI: 10.1107/S1600536812011051/bt5845Isup2.molSupplementary material file. DOI: 10.1107/S1600536812011051/bt5845Isup3.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S1600536812011051/bt5845Isup4.molSupplementary material file. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "In the guanidinate-bridged THF-stabilized dimer the Li\u22efLi separation is short at 2.479\u2005(8)\u2005\u00c5.In the dinuclear centrosymmetric title complex, [Li DOI: 10.1107/S1600536810046477/jj2061Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the structure, centrosymmetrically related mol\u00adecules are linked into dimers by pairs of C\u2014H\u22efN hydrogen bonds.In title compound, C DOI: 10.1107/S1600536813031152/rz5092Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S1600536813031152/rz5092Isup3.cmlSupplementary material file. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "Each YbIII ion is eight-coordinated by two O atoms from two bridging L ligands, four O atoms from two chelating L groups and two N atoms from one chelating phen mol\u00adecule in a distorted YbN2O6 dodeca\u00adhedral geometry.In the centrosymmetric binuclear title complex, [Yb DOI: 10.1107/S1600536811036105/wm2528Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the crystal, a dimeric structure is observed as a result of N\u2014H\u22efCl inter\u00adactions between two symmetry-related mol\u00adecules.The single-crystal X-ray structure analysis of [RuCl DOI: 10.1107/S1600536811043170/ff2034Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The methyl group is disordered over two equally occupied positions. In the crystal, N\u2014H\u22efO hydrogen bonds link the mol\u00adecules into infinite C(4) chains running along the a axis.In the title compound, C DOI: 10.1107/S1600536811043315/bt5681Isup2.hkl Structure factors: contains datablock(s) I. DOI: 10.1107/S1600536811043315/bt5681Isup3.cml Supplementary material file. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the crystal, mol\u00adecules are linked by N\u2014H\u22efO hydrogen bonds into a C(4) chain propagating in the b-axis direction.In the title compound, C DOI: 10.1107/S1600536811037123/hb6394Isup2.hkl Structure factors: contains datablock(s) I. DOI: 10.1107/S1600536811037123/hb6394Isup3.cml Supplementary material file. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The crystal structure is stabilized by weak inter\u00admolecular C\u2014H\u22efO hydrogen bonds.In the title complex, [Co(C DOI: 10.1107/S160053681102410X/lh5253Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Each L ligand is situated on an inversion center and bridges two CoII atoms, forming a zigzag polymeric chain propagating in [10In the title coordination polymer, [Co(C DOI: 10.1107/S1600536810053742/cv5012Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The InIII coordination centers are bridged head-to-head via In\u2014O bonds, yielding four-membered In2O2 rings and zigzag polymeric chains along [001].The title compound, [In DOI: 10.1107/S1600536813028985/lh5663Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S1600536813028985/lh5663Isup3.cdxSupplementary material file. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "The crystal structure shows intermolecular N\u2014H\u22efO hydrogen bonds.The title mononuclear complex, [Ni(C DOI: 10.1107/S1600536812019502/im2371Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "The bridging mode of the O atoms results in the formation of a three-dimensional framework, stabilized by two O\u2014H\u22efO hydrogen-bonding inter\u00adactions.The title compound, Pr(SO DOI: 10.1107/S1600536811000298/mg2113Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The \u03bc3-2-fluoro\u00adbenzoate ligand bridges three symmetry-related SrII atoms, giving rise to a chain structure extending along [010]. The polymeric chains are connected via O\u2014H\u22efO hydrogen bonds into a two-dimensional supra\u00admolecular structure parallel to (100).In the title compound, {[Sr(C DOI: 10.1107/S1600536811008397/hy2410Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The sixth author's name was written incorrectly. The correct name is: Jalal Nourlil. In the author contributions, \"Performed the experiments\" should read: CT PL MAS EA ME JN AF JEE SVM AR NC AJT ECH HB; \"Analyzed the data\" should read: CT PL MAS EA ME JN AF JEE SVM AR NC AJT ECH HB; \"Contributed reagents/materials/analysis tools\" should read: CT PL MAS EA ME JN AF JEE SVM AR NC AJT ECH HB; and \"Wrote the paper\" should read: CT PL MAS EA ME JN AF JEE SVM AR NC AJT ECH HB."} +{"text": "The eight-membered ring has a slightly distorted boat conformation.The title mol\u00adecule, C DOI: 10.1107/S1600536811014851/lh5233Isup2.hkl Structure factors: contains datablocks I. DOI: 10.1107/S1600536811014851/lh5233Isup3.cml Supplementary material file. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The crystal structure is stabilized by N\u2014H\u22efO and C\u2014H\u22efO hydrogen-bonding inter\u00adactions.In the title complex, [Cd(C DOI: 10.1107/S160053681104596X/pv2474Isup2.hkl Structure factors: contains datablock(s) I. DOI: 10.1107/S160053681104596X/pv2474Isup3.cdx Supplementary material file. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The Carene\u2014Si distances are significantly longer than in the analogous non-fluorinated compound. The packing of the mol\u00adecules results in a herringbone motif in the ac plane.The asymmetric unit of the title compound, C DOI: 10.1107/S1600536812010677/mw2055Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S1600536812010677/mw2055Isup3.cmlSupplementary material file. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "The two aromatic rings make a dihedral angle of 88.5\u2005(3)\u00b0. In the crystal, N\u2014H\u22efO hydrogen bonds link the mol\u00adecules into C(4) chains propagating in [010].In the title compound, C DOI: 10.1107/S1600536811047805/ds2154Isup2.hkl Structure factors: contains datablock(s) I. DOI: 10.1107/S1600536811047805/ds2154Isup3.cml Supplementary material file. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The MnIII atom possesses a distorted trans-MnO4N2 octa\u00adhedral coordination environment. The bridging ligands lead to [010]-chain polymeric cations {[Mn(HL)2]+}n in the crystal. The charge-balancing iodide ions are disordered over two sites in a 0.690\u2005(2):0.310\u2005(2) ratio and a weak O\u2014H\u22efI hydrogen bond occurs. The crystal studied was found to be a racemic twin.In the title one-dimensional coordination polymer, {[Mn(C DOI: Click here for additional data file.10.1107/S1600536813012695/hb7060Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "One nitro O atom of one picrate ion is disordered over two sites with occupancies of 0.54\u2005(5) and 0.46\u2005(5).In the title complex, [Mn(C DOI: 10.1107/S1600536812026037/bh2434Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "The anions bridge adjacent TbIII ions into double chains. Adjacent chains are further connected into sheets parallel to (10In the title coordination polymer, {[Tb(C DOI: 10.1107/S1600536811005447/wm2449Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Each DyIII ion is nine-coordinated by one 1,10-phenanthroline mol\u00adecule, one bidentate carboxyl\u00adate group and four bridging carboxyl\u00adate groups in a distorted DyN2O7 monocapped square-anti\u00adprismatic geometry. The title compound is isotypic with its terbium-containing analogue.In the centrosymmetric binuclear title complex, [Dy DOI: 10.1107/S1600536811034696/hb6365Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The correct Reference 45 is:45. Lawson ND, Weinstein BM (2002) In vivo imaging of embryonic vascular development using transgenic zebrafish. Dev Biol 248:307\u2013318."} +{"text": "The HPAA ligands coordinate in a bridging tridentate mode. In the crystal, inter\u00admolecular O\u2014H\u22efO hydrogen bonds form a three-dimensional network which consolidates the packing.In the title compound, {[Ce(C DOI: 10.1107/S1600536810047239/cv2794Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Such dimers are connected via bidentate hydrogen squarate groups [HC4O4]\u2212, leading to chains that propagate along the b axis. Inter- and intra\u00admolecular O\u2014H\u22efO hydrogen bonds maintain the crystal packing through a three-dimensional network.The title structure, {[Ba DOI: 10.1107/S1600536811002996/bq2274Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The C atoms of the THF molecule are disordered over two positions in a 0.55\u2005(2):0.45\u2005(2) ratio.In the mononuclear title compound, [MoCl DOI: 10.1107/S1600536810021690/ez2211Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "These chains are further linked through N\u2014H\u22efO, O\u2014H\u22efO and C\u2014H\u22efO hydrogen bonds to the nitrate anions, forming well-separated infinite planar layers parallel to (001).In the crystal structure of the title compound, C DOI: 10.1107/S1600536811027978/lx2193Isup2.hkl Structure factors: contains datablock(s) I. DOI: 10.1107/S1600536811027978/lx2193Isup3.cml Supplementary material file. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The perchlorate O-atoms are disordered over two positions in a 0.584\u2005(14):0.416\u2005(14) ratio.In the title complex, [Ag(C DOI: 10.1107/S1600536810025171/su2189Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S160053681104520X/bt5685Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Difference electron density maps indicate a disorder of the Br atom over two positions in an approximate 0.95:0.05 ratio. However, this disorder could not be resolved satisfactorily with the present data.In the title compound, [Rh(C DOI: 10.1107/S1600536812011944/mw2054Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "The ZnII atom shows trigonal\u2013prismatic coordination.In the title coordination polymer, [Zn(C DOI: 10.1107/S1600536812019836/ng5269Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "In the crystal, the mol\u00adecules are linked into a zigzag chain along the a axis via N\u2014H\u22efO hydrogen bonds.In the title compound, C DOI: 10.1107/S1600536812009257/is5084Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S1600536812009257/is5084Isup3.cmlSupplementary material file. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "Dissertauer 2007. Dissert DOI: 10.1107/S1600536811041237/hp2013Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the crystal, strong N\u2014H\u22efO and weak C\u2014H\u22efO hydrogen bonds link the mol\u00adecules into chains running parallel to [010].In the title mol\u00adecule, C DOI: 10.1107/S1600536814003936/hg5385Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S1600536814003936/hg5385Isup3.cmlSupporting information file. DOI: 987953CCDC reference: crystallographic information; 3D view; checkCIF reportAdditional supporting information:"} +{"text": "The nitrate anions form a ring around one pair of double-stacked dications. An intricate three-dimensional N\u2014H\u22efO and N\u2014H\u22ef hydrogen-bonding network exists in the crystal structure.In the title molecular salt, C DOI: 10.1107/S1600536811042917/ez2262Isup2.hkl Structure factors: contains datablock(s) I. DOI: 10.1107/S1600536811042917/ez2262Isup3.mol Supplementary material file. DOI: 10.1107/S1600536811042917/ez2262Isup4.cml Supplementary material file. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The ZnII atom and the two Br atoms are located on a threefold axis.In the title compound, [ZnBr(C DOI: 10.1107/S1600536811011809/rn2081Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The apical positions are occupied by two C atoms of 4-fluoro\u00adbenzyl groups.In the title complex, [Sn(C DOI: 10.1107/S1600536811054274/hp2022Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the crystal, the components are linked by C\u2014H\u22efBr inter\u00adactions, thereby generating a three-dimensional network.In the title mol\u00adecular salt, (C DOI: 10.1107/S1600536811034830/hb6385Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Each CuI atom displays a tetra\u00adhedral coordination environment, formed by one S atom and three N atoms from one 2-(pyridin-2-yldisulfan\u00adyl)pyridine and two dicyanamide ligands. The crystal structure is stabilized by C\u2014H\u22efN hydrogen bonds, forming a three-dimensional network.In the title compound, [Cu(C DOI: 10.1107/S1600536811002728/zq2085Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The structure represents a second crystal form of the salt, the first being an acetonitrile solvate [Watton :0.1385\u2005(17).The title compound, [Cu(CHtton 2009. Acta Cr DOI: 10.1107/S1600536810042285/fj2351Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The chains are arranged along the b-axis direction, forming a layer structure extending in the bc plane. O\u2014H\u22efO hydrogen bonding between the layers results in the formation of a three-dimensional supra\u00admolecular framework. The structure is isotypic with the Zn analogue .The water-coordinated Ni DOI: 10.1107/S1600536812001900/hp2024Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S1600536812001900/hp2024Isup4.cdxSupplementary material file. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "The complex polymeric chains are inter\u00adconnected via inter\u00admolecular water O\u2014H\u22efN hydrogen bonds into a three-dimensional network. In the title compound, [Cd(C DOI: 10.1107/S1600536812014626/kp2399Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "The linear mol\u00adecule L acts as a bidente bridging ligand, connecting the metal atoms into a chain along [101].In the title compound, [Cu(CHO DOI: 10.1107/S1600536811024019/rn2086Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The methyl-bearing C atom in the cyclo\u00adpentane ring is disordered over two positions with a site-occupation factor of 0.899\u2005(8) for the major occupied site.The exocyclic C=C double-bond in the title compound, C DOI: 10.1107/S1600536811033708/bt5619Isup2.hkl Structure factors: contains datablock(s) I. DOI: 10.1107/S1600536811033708/bt5619Isup3.cml Supplementary material file. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Each terephthalate ligand acts as a bis-monodentate ligand that binds two CuII atoms, thus forming two unique chains extending parallel to [110]. The imidazole ligands are attached on both sides of the chains.In the title compound, [Cu(C DOI: 10.1107/S1600536811024822/jh2303Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The purpose of this table is to provide the community with a citable record of publications of ongoing genome sequencing projects that have led to a publication in the scientific literature. While our goal is to make the list complete, there is no guarantee that we may have omitted one or more publications appearing in this time frame. Readers and authors who wish to have publications added to this subsequent versions of this list are invited to provide the bibliometric data for such references to the SIGS editorial office. CrenarchaeotaPhylum \u201cMetallosphaera cuprina\u201d Ar-4, sequence accession CP002656 [CP002656 Thermoproteus uzoniensis 768-20, sequence accession CP002590 [\u201cVulcanisaeta moutnovskia\u201d 768-28, sequence accession CP002529 [CP002529 EuryarchaeotaPhylum Methanosaeta concilii, sequence accession CP002565 (chromosome), CP002566 (plasmid) [plasmid) Pyrococcus sp. NA2, sequence accession CP002670 [CP002670 Thermococcus barophilus MP, sequence accession CP002372 (chromosome) and CP002373 plasmid) [plasmid) ChloroflexiPhylum Oscillochloris trichoides DG-6, sequence accession ADVR00000000 [00000000 ProteobacteriaPhylum Achromobacter xylosoxidans A8, sequence accession CP002287 (chromosome), CP002288 (plasmid pA81), and CP002289 (plasmid pA82) [Acinetobacter baumannii 3990, sequence accession AEOY00000000 [Acinetobacter baumannii ST78, sequence accession AEPA00000000 [00000000 Acinetobacter baumannii, sequence accession AEPA00000000 [00000000 Acinetobacter baumannii MDR-TJ, sequence accession AEOE00000000 [00000000 Acinetobacter baumannii TCDC-AB0715, sequence accession CP002522 (chromosome), CP002523 (p1ABTCDC0715) and CP002524 (p2ABTCDC0715) [CDC0715) Salmonella enterica serovar Typhimurium UK-1 ATCC 68169, sequence accession CP002614 (chromosome), CP002615 (plasmid) [plasmid) Acinetobacter calcoaceticus PHEA-2, sequence accession CP002177 [CP002177 Aeromonas caviae Ae398, sequence accession CACP01000001 to CACP01000149 [Aeromonas veronii B565, sequence accession CP002607 [CP002607 Bordetella pertussis CS, sequence accession CP002695 [CP002695 Brucella melitensis M111, sequence accession AFFB00000000 [00000000 Brucella melitensis M28-12, sequence accession AFFA00000000 [00000000 Brucella melitensis M5, sequence accession AFEZ00000000 [00000000 Brucella suis S2, sequence accession AFFC00000000 [00000000 Brucella melitensis M5-90, sequence accession CP001851 and CP001852 [Brucella melitensis M28, sequence accession CP002459 and CP002460 [CP002460 Burkholderia gladioli BSR3, sequence accession CP002599 to CP002604 [CP002604 Burkholderia phytofirmans, sequence accession CP001052 to CP001054 [CP001054 Burkholderia rhizoxinica HKI 0454, sequence accession FR687359 (chromosome), FR687360 (pBRH01), FR687361 (pBRH02) [(pBRH02) Campylobacter jejuni S3, sequence accession CP001960 (chromosome) CP001961 (plasmid) [plasmid) Liberibacter solanacearum, sequence accession CP002371 [Candidatus \u201cCitromicrobium sp.\u201d JLT1363, sequence accession AEUE01000000 [01000000 Cronobacter turicensis LMG 23827, sequence accession NC_013282 to NC_013285 [C_013285 Desulfovibrio africanus Walvis Bay, sequence accession AFHE00000000 [00000000 Desulfovibrio desulfuricans ND132, sequence accession AEUJ00000000 [00000000 Dickeya dadantii 3937, sequence accession CP002038 [CP002038 \u201cEnterobacter mori\u201d LMG 25706, sequence accession AEXB01000000 [01000000 Erwinia amylovora, sequence accession [ccession Erwinia sp. Ejp617, sequence accession CP002124 (chromosome), CP002125 (pJE01), CP002126 (pJE02), CP002127 (pJE03), CP002128 (pJE04), and CP002129 (pJE05) [ (pJE05) Escherichia coli AA86 KACC15541, sequence accession AFET00000000 [00000000 Escherichia coli UM146, sequence accession CP002167 [CP002167 \u201cGallibacterium anati\u201d UMN179, sequence accession CP002667, CP002668 [CP002668 Gluconacetobacter sp. SXCC-1, sequence accession AFCH0000000 [H0000000 Haemophilus para\t SH0165 serovar 5, sequence accession CP001321 [CP001321 Herbaspirillum seropedicae SmR1, sequence accession [ccession Ketogulonicigenium vulgare Y25, sequence accession CP002224 (chromosome), CP002225 (plasmid), and CP002226 (plasmid) [plasmid) Methylocystis sp. sp. Rockwell, sequence accession AEVM00000000 [00000000 Methylophaga thiooxydans DSM010, sequence accession ABXT00000000 [00000000 Methylovorus sp. MP688, sequence accession CP002258 [CP002258 Neisseria gonorrhoeae TCDC-NG08107, sequence accession CP002440 and CP002441 [CP002441 Neisseria meningitidis H44/76, sequence accession AEQZ00000000 [00000000 Neisseria meningitidis WUE2594, sequence accession FR774048 [FR774048 Oceanicaulis sp. HTCC2633, sequence accession AAMQ00000000 [00000000 Paracoccus sp. sp. TRP, sequence accession AEPN00000000 [00000000 Parvularcula bermudensis HTCC2503T, sequence accession CP002156 [CP002156 \u201cPhotobacterium mandapamensis\u201d svers. 1.1, sequence accession BACE01000001 to BACE01000031 [01000031 \u201cPolymorphum gilvum\u201d SL003B-26A1T LMG 25793T, sequence accession CP002568, CP002569 [CP002569 Pseudomonas savastanoi pv. glycinea (Psg) B076, sequence accession AEGG01000000 [01000000 Pseudomonas savastanoi pv. glycinea (Psg) race 4, sequence accession AEGH01000000 [01000000 Pseudomonas sp. S9, sequence accession AFFX00000000 [00000000 Pseudomonas stutzeri DSM4166, sequence accession CP002622 [CP002622 Pusillimonas sp. T7-7, sequence accession [ccession Rhizobium etli CNPAF512, sequence accession AEYZ00000000 [00000000 Rhodobacter sphaeroides WS8N, sequence accession AFER00000000 [00000000 Roseobacter sp. HTCC2038, sequence accession ABXE00000000 [00000000 Rubrivivax benzoatilyticus JA2T, sequence accession AEWG00000000 [00000000 Ruegeria TW15, sequence accession AEYW01000000 [01000000 Salmonella enterica serovar Choleraesuis SCSA50, sequence accession CM001062-CM001063 [CM001063 Salmonella enterica serovar Dublin SD3246, sequence accession CM001151-CM001152 [CM001152 Salmonella enterica serovar Typhimurium 4/74, sequence accession CP002487 - CP002490 [CP002490 Sphingomonas sp. S17, sequence accession AFGG01000000 [01000000 Taylorella equigenitalis MCE9, sequence accession CP002456 [CP002456 Unnamed strain IMCC1989, sequence accession AEVK00000000 Unnamed strain IMCC2047, sequence accession AEGL00000000 Unnamed strain IMCC3088, sequence accession AEIG00000000 Unnamed strain IMCC9063 SAR11 subgroup 3, sequence accession CP002511 Variovorax paradoxus S110, sequence accession [ccession Vibrio anguillarum pJM1, sequence accession AEZA00000000, AEZB00000000, AEZC00000000 [00000000 Vibrio furnissii NCTC 11218, sequence accession CP002377 (chromosome I) and CP002378 (chromosome II) [some II) Vibrio parahaemolyticus clinical O4:K12 serotype, sequence accession AFBW00000000 [00000000 Vibrio rotiferianus strain DAT722, sequence accession [ccession Vibrio vulnificus MO6-24/O, sequence accession CP002469 and CP002470 [CP002470 Yersinia pestis KIM D27, sequence accession ADDC00000000 [00000000 Yersinia enterocolitica 3/O:9, sequence accession CP002246 (chromosome)_CP002247 (pYV plasmid) [plasmid) Yersinia enterocolitica subsp. palearctica 2 serogroup O:3, sequence accession FR729477 (chromosome) FR745874 (plasmid) [FirmicutesPhylum Bacillus amyloliquefaciens LL3, sequence accession CP002634, CP002635 [CP002635 Bacillus amyloliquefaciens TA208, sequence accession CP002627 [CP002627 Bacillus subtilis BSn5, sequence accession CP002468 [CP002468 Bacillus subtilis subsp. spizizenii gtP20b, sequence accession AEHM00000000 [00000000 Bacillus thuringiensis YBT-020, sequence accession CP002508 (chromosome), CP002509 (plasmid pBMB26), CP002510 , CP002654 (pLBU02), CP002655 (pLBU03) [(pLBU03) Lactobacillus casei (EP 164209630B1), sequence accession CP002616 and CP002617 [ EP 1642030B1, seqLactobacillus casei BD-II, sequence accession CP002618 and CP002619 [CP002619 Lactobacillus coryniformis subsp. coryniformis KCTC 3167, sequence accession AELK00000000 [00000000 Lactobacillus delbrueckii subsp. bulgaricus, sequence accession CP000156 [CP000156 Lactobacillus delbrueckii subsp. bulgaricus ND02, sequence accession CP002341 and CP002342 [CP002342 Lactobacillus farciminis KCTC 3681, sequence accession AEOT00000000 [00000000 Lactobacillus helveticus H10, sequence accession CP002429 (chromosome) and CP002430 (plasmid) [plasmid) Lactobacillus plantarum ST-III, sequence accession CP002222 [CP002222 Lactobacillus reuteri ATCC 53608, sequence accession CACS02000000 [02000000 Lactococcus garvieae 21881, sequence accession AFCC01000000 [01000000 Lactococcus garvieae UNIUD074, sequence accession AFHF01000000 [01000000 Lactococcus lactis subsp. lactis CV56, sequence accession CP002365 through CP002370 [CP002370 Leuconostoc fallax KCTC 3537, sequence accession AEIZ00000000 [00000000 Leuconostoc gelidum KCTC 3527, sequence accession AEMI00000000 [00000000 Leuconostoc inhae KCTC 3774, sequence accession AEMJ00000000 [00000000 Listeria monocytogenes J1-220, sequence accession AFBU00000000 [00000000 Listeria monocytogenes J1816, sequence accession AFBU00000000 [00000000 Listeria monocytogenes HCC23, sequence accession CP001175 [CP001175 Melissococcus plutonius ATCC 35311, sequence accession AP012200 (chromosome), AP012201 (plasmid) [plasmid) Ornithinibacillus TW25, sequence accession AEWH00000000 [00000000 Paenibacillus polymyxa SC2, sequence accession CP002213 (chromosome) and CP002214 (plasmid) [plasmid) Staphylococcus aureus O11, sequence accession AEUQ01000000 [01000000 Staphylococcus aureus straub O46, sequence accession AEUR01000000 [01000000 Staphylococcus aureus T0131, ST239-MRSA-SCCmec type III, sequence accession CP002643 [CP002643 Staphylococcus pseudintermedius ED99, sequence accession CP002478 [CP002478 Staphylococcus pseudintermedius HKU10-03, sequence accession CP002439 [CP002439 Streptococcus parauberis KCTC11537BP, sequence accession CP002471 [CP002471 Streptococcus suis JS14, sequence accession CP002465 [CP002465 Streptococcus thermophilus ND03, sequence accession CP002340 [CP002340 Turicibacter sanguinis PC909, sequence accession ADMN00000000 [00000000 Weissella cibaria KACC 11862, sequence accession AEKT01000000 [01000000 TenericutesPhylum Mycoplasma alligatoris A21JP2T, sequence accession NZ_ADNC01000000 [01000000 Mycoplasma crocodyli MP145T, sequence accession CP001991 [CP001991 Mycoplasma bovis PG45 (ATCC 25523), sequence accession CP002188 [CP002188 Mycoplasma haemofelis, sequence accession FR773153 [FR773153 Mycoplasma haemofelis Ohio2, sequence accession AEVA00000000 [00000000 Mycoplasma suis Illinois, sequence accession ADWK01000001 [01000001 Mycoplasma suis KI3806, sequence accession FQ790233 [FQ790233 ActinobacteriaPhylum Bifidobacterium bifidum S17, sequence accession CP002220 [CP002220 Bifidobacterium longum subsp. longum BBMN68, sequence accession CP002286 [CP002286 Corynebacterium pseudotuberculosis I19, sequence accession CP002251 [CP002251 Janibacter sp. HTCC2649, sequence accession AAMN00000000 [00000000 Microbacterium testaceum StLB037, sequence accession AP012052 [AP012052 Mycobacterium bovis BCG, sequence accession later [on later Nocardioides sp. JS614, sequence accession CP000509, CP000508 [CP000508 Saccharopolyspora spinosa NRRL 18395, sequence accession AEYC00000000 [00000000 Streptomyces griseoaurantiacus, sequence accession AEYX01000000 [01000000 Streptomyces griseus XyelbKG-1, sequence accession ADFC00000000 [00000000 Streptomyces sp. PP-C42, sequence accession AEWS01000000 [01000000 Verrucosispora maris AB-18-032, sequence accession CP002638, CP002639 [CP002639 ChlamydiaePhylum Chlamydia pecorum E58, sequence accession CP002608 [CP002608 Chlamydia psittaci 6BC, sequence accession CP002586 (chromosome), CP002587 (plasmid) [plasmid) Chlamydia psittaci Cal10, sequence accession AEZD00000000 [00000000 Chlamydophila psittaci RD1, sequence accession FQ482149 (chromosome) FQ482150 (plasmid) [plasmid) SpirochaetesPhylum Borrelia burgdorferi, sequence accession ABJZ02000001-5 (chromosome) CP001519 (Ip17) CP001518 (IP28-2) CP001523 (Ip28-4) CP001524 (Ip54) CP001522 (for cp26)_CP001517 (cp32-3) CP001520 (cp32-4) ABJZ02000006-7(Ip32-6) CP001521 (cp32-7)_CP001516 (cp32-12) [cp32-12) Treponema paraluiscuniculi Cuniculi A, sequence accession CP002103 [CP002103 FibrobacteresPhylum Fibrobacter succinogenes S85 S85, sequence accession CP001792 [CP001792 BacteroidetesPhylum Algoriphagus sp. PR1, sequence accession AAXU01000000 [01000000 Bacteroides vulgatus PC510, sequence accession ADKO01000000 [01000000 Kordia algicida OT-1, sequence accession ABIB00000000 [00000000 Maribacter sp. HTCC2170, sequence accession CP002157 [CP002157 Riemerella anatipestifer RA-GD, sequence accession CP002562 [CP002562 Riemerella anatipestifer RA-YM, sequence accession AENH00000000 [00000000 VerrucomicrobiaPhylum Akkermansia muciniphila ATCC BAA-835, sequence accession NC_010655 [C_010655 Opitutus terrae PB90-1, sequence accession CP001032 [CP001032 \u201cChthoniobacter flavus\u201d Ellin428, sequence accession ABVL00000000 [00000000 \u201cPedosphaera parvula\u201d Ellin514, sequence accession ABOX00000000 [00000000 LentisphaeraePhylum Victivallis vadensis ATCC BAA-548, sequence accession ABDE02000001-ABDE02000027 [02000027"} +{"text": "Two L ligands coordinate the GdIII ion in a monodentate mode, while the third coordinates it in a bidentate\u2013chelating coordination mode. An extensive three-dimensional O\u2014H\u22efO hydrogen-bonding network consolidates the crystal packing.In the title compound, [Gd(C DOI: 10.1107/S1600536811016163/cv5069Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Both the S and Br atoms act as bridging ligands, connecting pairs of CuI atoms and generating chains propagating in [100]. Inter-chain N\u2014H\u22efN hydrogen bonds generate layers in the ac plane. Weak intra-chain N\u2014H\u22efBr inter\u00adactions also occur. In the title coordination polymer, [CuBr(C DOI: 10.1107/S1600536812014444/hb6718Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "Inter\u00admolecular N\u2014H\u22efO hydrogen bonding forms a one-dimensional motif parallel to the cell ab diagonal.In the title complex, [Co(NCS) DOI: 10.1107/S1600536811029060/qm2016Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The crystal packing is stabilized by N\u2014H\u22efCl hydrogen bonds, forming a layered arrangement parallel to (1-10). In the title compound, [CoCl(C DOI: Click here for additional data file.10.1107/S1600536813004650/bt6888Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "Both types of DCT ligands act as bridging, forming a one-dimensional polymeric structure propagating parallel to [10In the title compound, {(C DOI: 10.1107/S1600536810051366/lh5182Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The binuclear anion exhibits a pseudo-threefold symmetry and contains two six-coordinate Mn atoms. Each metal atom is coordinated by three facially oriented CO ligands and three doubly-bridging phenolate ligands. The average O\u2014Mn\u2014O bond angle is 74.9\u2005(7)\u00b0 in the Mn2O3 metal\u2013phenolate dimeric core, yielding a distorted octa\u00adhedron for each metal.The title compound, (C DOI: 10.1107/S1600536811029266/bg2415Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Aeromonas sp. MDS8 strain MCC2167) with the ability to produce ammonia during 16 h of growth at 37\u00b0C, isolated from dairy sludge, with a size of 4,841,753 bp and a G+C content of 63.1%, is reported here.The draft genome sequence of an amylase-, protease-, lipase-, oxidase-, and catalase-producing Gram-negative bacillus ( The contigs were submitted to the Rapid Annotations using Subsystems Technology (RAST) ; type IV pilus biogenesis protein PilF; phosphoribosylformylglycinamidine synthase, synthetase subunit (EC 6.3.5.3)/phosphoribosylformylglycinamidine synthase, glutamine amidotransferase subunit (EC 6.3.5.3); small-subunit (SSU) ribosomal protein S1p (accession no. AOTK0100001); DNA mismatch repair protein MutS; ammonium transporter (accession no. AOTK01000007); decarboxylase family protein; DNA polymerase IV; DNA polymerase I (accession no. AOTK01000008); pantothenate:Na+ symporter (TC 2.A.21.1.1) (accession no. AOTK01000018); arsenic resistance protein ACR3 (accession no. AOTK01000020); nitrite reductase [NAD(P)H] large subunit; nitrite reductase [NAD(P)H] small subunit; nitrite transporter NirC (accession no. AOTK01000057); proteinase inhibitor (accession no. AOTK01000070); tRNA binding protein YgjH (accession no. AOTK01000074); putative plasmid replication protein RepB (accession no. AOTK01000104); alkaline phosphatase; NrfC protein; and NrfD protein (accession no. AOTK01000006).The important genes contained in the different contigs are as follows: genes for the ribosome hybernation protein YfiA; ferric uptake regulation protein FUR; NaAOTK01000000. The version described in this paper is the first version, AOTK01000001.This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession no."} +{"text": "The cyclo\u00adpentyl ring in L is disordered over two conformations in a 0.640\u2005(19):0.360\u2005(19) ratio.In the title compound, [Co(C DOI: 10.1107/S1600536811002194/cv5039Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The seven-membered ring adopts a distorted half-chair conformation.In the crystal structure of the title compound, C DOI: 10.1107/S1600536810052888/bt5422Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536813025464/hp2061Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "The term \"A\u03b240/42\" is incorrect in the article title.The correct title is: Differential Regulation of Amyloid Precursor Protein/Presenilin 1 Interaction during A\u03b240/42 Production Detected Using Fusion ConstructsThe correct citation is: Sato N, Okochi M, Shinohara M, Thinakaran G, Takeda S, et al. (2012) Differential Regulation of Amyloid Precursor Protein/Presenilin 1 Interaction during A\u03b240/42 Production Detected Using Fusion Constructs. PLoS ONE 7(11): e48551. doi:10.1371/journal.pone.0048551"} +{"text": "DOI: 10.1107/S1600536810025225/hb5520Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The structure exhibits disorder in one of the ethyl chains, which was refined using a two-site model with 0.70\u2005(6):0.30\u2005(6) occupancy.In the title complex, [Co(NCS) DOI: 10.1107/S1600536812021988/ez2290Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "The dihedral angle between the mean planes of the two bipyridine ligands is 87.67\u2005(6)\u00b0.In the title compound, [Cd(C DOI: Click here for additional data file.10.1107/S1600536812044108/lh5540Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "The mol\u00adecule contains two fused six-membered rings both in chair conformations. In the crystal, mol\u00adecules are linked into chains running parallel to the a axis by O\u2014H\u22efO hydrogen bonds.The title compound, C DOI: 10.1107/S1600536811053712/bt5751Isup2.hkl Structure factors: contains datablock(s) I. DOI: 10.1107/S1600536811053712/bt5751Isup3.cml Supplementary material file. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The Supporting Information Tables S1-S4 were erroneously omitted. Please see the missing Tables below.Table S1: Click here for additional data file.Table S2: Click here for additional data file.Table S3: Click here for additional data file.Table S4: Click here for additional data file."} +{"text": "The crystal packing exhibits no classical inter\u00admolecular contacts.The asymmetric unit of the title compound, C DOI: 10.1107/S1600536810048981/cv5005Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The 3,5-dinitropyridin-4-oxido N-oxide ligand is formally a zwitterionic anion; the anion binds to the metal atom through the N-oxide O atom. The chains are connected into a three-dimensional network by O\u2014H\u22efO hydrogen bonds involving the coordinated and uncoordinated water mol\u00adecules.In the title coordination polymer, {[Nd(C DOI: 10.1107/S1600536810041139/xu5029Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Z. Anor DOI: 10.1107/S1600536812027857/wm2643Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536811031199/tk2775Isup2.hkl Structure factors: contains datablock(s) I. DOI: 10.1107/S1600536811031199/tk2775Isup3.cml Supplementary material file. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536810024773/ng2792Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Each anion acts as a \u03bc3-bridge, linking symmetry-related CdII ions into a layer parallel to (010). In the crystal, numerous O\u2014H\u22efO and C\u2014H\u22efO hydrogen bonds occur. The coordinated water mol\u00adecules and carboxyl\u00adate O atoms act as donors or acceptors in the formation of these hydrogen-bonding inter\u00adactions.In the title polymer, [Cd(C DOI: 10.1107/S1600536811044187/pv2458Isup2.hkl Structure factors: contains datablock(s) I. DOI: 10.1107/S1600536811044187/pv2458Isup3.cdx Supplementary material file. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In \u201cSerogroup W-135 Meningococcal Disease during the Hajj, 2000,\u201d p. 665, author Sahar Makki was inadvertently not included. The correct author list should read as follows:Jairam R. Lingappa,* Abdullah M. Al-Rabeah,\u2020 Rana Hajjeh,* Tajammal Mustafa,\u2020 Adel Fatani,\u2020 Tami Al-Bassam,\u2020 Amira Badukhan,\u2020 Abdulhafiz Turkistani,\u2020 Sahar Makki,\u2020 Nassen Al-Hamdan,\u2020 Mohamed Al-Jeffri,\u2020 Yaqoub Al Mazrou,\u2020 Bradley A. Perkins,* Tonja Popovic,* Leonard W. Mayer,* and Nancy E. Rosenstein**Centers for Disease Control and Prevention, Atlanta, Georgia, USA\u2020\u2020Saudi Arabian Ministry of Health, Riyadh, Kingdom of Saudi Arabia"} +{"text": "In the crystal, anions and cations are inter\u00adconnected by N\u2014H\u22efBr hydrogen bonds, forming ribbons parallel to [0-11].The structure of the title salt, (C DOI: 10.1107/S1600536812011117/bh2418Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "Intra\u00admolecular O\u2014H\u22efO hydrogen bonds are observed in the DHB ligands.In the mononuclear title complex, [Nd(C DOI: 10.1107/S1600536810042583/hy2364Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The anions bridge adjacent HoIII ions into double chains. Adjacent chains are further connected into sheets. O\u2014H\u22efO hydrogen bonds involving both coordinated and uncoordinated water mol\u00adecules generate a three-dimensional supra\u00admolecular framework.In the title coordination polymer, {[Ho(C DOI: 10.1107/S1600536811016953/sj5136Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The PbII atom is in a \u03a8-square-anti\u00adprismaic coordination.In the centrosymmetric binuclear title compound, [Pb DOI: 10.1107/S1600536810032125/ci5151Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the title complex, [Ni(C DOI: 10.1107/S1600536812024749/ru2036Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "Some formatting errors occurred in Tables 1, 2, and 3. Please review the correctly formatted tables here:Table 1: [^]Table 2: [^]Table 3: [^]"} +{"text": "The major contributions to the cohesion and the stability of this two-dimensional polymeric structure are the covalent Cd\u2014S,N bonds and one weak intra\u00adlayer N\u2014H\u22efS hydrogen bond.The structure of the title polymeric compound, [Cd(SCN) DOI: 10.1107/S1600536813031255/vn2078Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S1600536813031255/vn2078Isup3.cdxSupplementary material file. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "The Na+ ion is eight-coordinated by four ligand O atoms and four nitrate O atoms. The ligand links the CuII and Na ions, forming a layered arrangement extending parallel to (001). In the title heterodinuclear complex, [CuNa(C DOI: 10.1107/S1600536811040025/ng5236Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "\u00c5 between pairs of inversion-related mol\u00adecules.The title compound, [Mn(C al. 2006. Inorg. DOI: 10.1107/S1600536810048270/lh5164Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the crystal, mol\u00adecules are linked via pairs of N\u2014H\u22efO hydrogen bonds, forming inversion dimers.In the title compound, [Zn(CH DOI: 10.1107/S1600536812017564/su2407Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "Weak cation-to-anion C\u2014H\u22efCl inter\u00adactions generate a three-dimensional network.In the crystal structure of the title mol\u00adecular salt, (C DOI: 10.1107/S1600536811048823/hb6492Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The piperazine-1,4-dicarbodithio\u00adate linker has an almost ideal chair conformation. The geometry about the gold atoms is severely distorted tetra\u00adhedral punctuated by a very acute S\u2014Au\u2014S bite angle.In the title compound, [Au DOI: 10.1107/S1600536811044229/ng5253Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Its structure is isotypic with the MnII, FeII, CoII, NiII, CuII and ZnII analogues. The MgII cation has a slightly distorted octa\u00adhedral geometry containing four N atoms from two 1,10-phenanthroline mol\u00adecules and two N atoms from two thio\u00adcyanate anions. The asymmetric unit contains one-half mol\u00adecule, and the complete complex has 2 symmetry.The title compound, [Mg(NCS) DOI: 10.1107/S1600536810027054/bh2298Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Inter\u00admolecular C\u2014H\u22ef\u03c0 inter\u00adactions are observed in the crystal structure.In the title compound, [Cd(C DOI: 10.1107/S1600536812038433/hy2583Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S1600536812038433/hy2583Isup3.cdxSupplementary material file. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "The benzene solvent mol\u00adecule is located about a crystallographic inversion centre. The title complex is isostructural with trans-dichloridobis(triphenyl\u00adphosphane)\u00adpalladium(II) 1,4-dichloro\u00adbenzene sesquisolvate .The title complex, [PdCl al. 1983. Acta Cr DOI: 10.1107/S1600536812012494/tk5074Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "The central six-membered ring in both mol\u00adecules has a chair conformation with equatorial substituents. In the crystal, mol\u00adecules are linked into [100] C(4) chains of alternating A and B mol\u00adecules by N\u2014H\u22efO hydrogen bonds.The title compound, C DOI: 10.1107/S160053681103515X/hb6389Isup2.hkl Structure factors: contains datablock(s) I. DOI: 10.1107/S160053681103515X/hb6389Isup3.cml Supplementary material file. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The TbIII ion adopts a doubly-capped square-anti\u00adprismatic coord\u00adination environment defined by three chelating nitrate anions and two N,O-bidentate nitronyl nitroxide radical ligands. Weak C\u2014H\u22efO hydrogen bonds connect the molecules into a three-dimensional framework. The title structure is isotypic with the Ho analogue .The title compound, [Tb(NO [Li 2012. Acta Cr DOI: Click here for additional data file.10.1107/S1600536812040287/mw2078Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "The bridging ligands link metal centres, forming a three-dimensional network which is stabilized by inter\u00admolecular O\u2014H\u22efN hydrogen-bonding inter\u00adactions.In the title polymeric complex, [Cd(C DOI: 10.1107/S1600536810027406/rz2474Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536812011749/ff2059Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "The ferrocenyl\u00adimine moieties are trans to each other.The title compound, [Fe DOI: 10.1107/S1600536812009191/pk2391Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "The two benzene rings of the biphenyl unit form a dihedral angle of 55.99\u2005(8)\u00b0. There are no significant hydrogen bonds observed in the crystal of this compound.In the title compound, C DOI: 10.1107/S1600536812018065/hb6748Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S1600536812018065/hb6748Isup3.cmlSupplementary material file. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "The octa\u00adhedra are connected by malonate anions, forming chains along the c-axis direction. O\u2014H\u22efO hydrogen bonds link these chains into a three-dimensional network.In the title compound, [Mg(C DOI: 10.1107/S1600536813034193/kj2236Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supporting information:"} +{"text": "The crystal structure is characterized by polymeric zigzag chains running parallel to [2-10] and is stabilized by O\u2014H\u22efO hydrogen bonds.In the title complex, {[Zn(C DOI: 10.1107/S1600536812025068/bt5933Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "A NiP2N rhombus occurs within the chelating ligand.In the title complex, [NiCl DOI: 10.1107/S1600536811042760/hb6453Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The crystal structure exhibits alternating organic and inorganic layers parallel to and 3.9340\u2005(15)\u2005\u00c5].In the title compound, (C DOI: 10.1107/S1600536811007252/hy2408Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The cation is connected to the anion via three-center N\u2014H\u22efCl hydrogen bonds.In the title compound, (C DOI: 10.1107/S1600536811025347/gk2387Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536811001759/ng5084Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the anion, three BiIII ions adopt an octa\u00adhedral coordination constructed by six I\u2212 ligands. The three BiI6 octa\u00adhedra are fused together through trans face-sharing.In the title complex, [Co(C DOI: 10.1107/S1600536811033460/om2460Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The morpholine ring adopts a chair conformation.In the title complex, [ZnBr DOI: 10.1107/S1600536811002753/su2250Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The dihedral angle between the fluoro\u00adbenzene rings is 69.10\u2005(15).In the title mol\u00adecule, C DOI: 10.1107/S1600536812010744/hb6677Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S1600536812010744/hb6677Isup3.cmlSupplementary material file. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "Weak inter\u00admolecular C\u2014H\u22efO hydrogen bonding is present in the crystal structure.In the title compound, [Bi(C DOI: 10.1107/S1600536811021039/xu5208Isup2.hkl Structure factors: contains datablock(s) I. DOI: 10.1107/S1600536811021039/xu5208Isup3.cml Supplementary material file. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "These ligands bridge the NiII complex units, forming zigzag chains along the c axis. Adjacent chains are linked by O\u2014H\u22efO hydrogen bonds, forming a three-dimensional supra\u00admolecular network.In the title compound, [Ni(CHO DOI: 10.1107/S1600536811012281/bq2289Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536810027236/lh5081Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Hydrogen bonding between hy\u00addroxy H and carboxyl\u00adate O atoms results in a layer structure parallel to the ab plane.In the title compound, [Cu(C DOI: 10.1107/S1600536811023038/om2435Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the crystal, the components are connected by O\u2014H\u22efN hydrogen bonds, forming a chain in the b-axis direction.The title co-crystal, C DOI: 10.1107/S1600536811029308/vm2111Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The asymmetric unit comprises one Zr, one La and four S atoms. The Zr and three S atoms are situated on mirror planes. The structure consists of LaS8 face-sharing bicapped distorted trigonal prisms and ZrS7 edge-sharing monocapped octa\u00adhedra.Zirconium(IV) dilanthanum(III) penta\u00adsulfide, ZrLa DOI: 10.1107/S1600536811045193/wm2549Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the crystal, the molecules are linked by C\u2014H\u22efO and C\u2014H\u22efBr inter\u00adactions.The non-H atoms of the title compound, C DOI: 10.1107/S1600536811030960/bt5592Isup2.hkl Structure factors: contains datablock(s) I. DOI: 10.1107/S1600536811030960/bt5592Isup3.cml Supplementary material file. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the crystal, complex mol\u00adecules are linked via inter\u00admolecular O\u2014H\u22efO hydrogen bonds, forming chains along [100].In the title complex, [Sn(C DOI: 10.1107/S1600536811011184/lh5223Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "III atom in the anion of the title complex, (C9H14N)[Ni(C4N2S2)2], is coordinated by four S atoms of two maleonitrile\u00addithiol\u00adate ligands, and exhibits a square-planar coordination geometry.The Ni DOI: 10.1107/S1600536811042103/tk2798Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the crystal, the mol\u00adecules pack in layers parallel to (10The title compound, C DOI: 10.1107/S1600536810024311/fk2020Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the crystal, the 2-[(propan-2-yl\u00adoxy)\u00adcarbon\u00adyl]quinolin-1-ium cation is linked to the Sn complex anion by an N\u2014H\u22efO hydrogen bond.In the title salt, (C DOI: 10.1107/S1600536812019496/xu5530Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "The overall coordination sphere of the Hg+ atom is a considerably distorted octa\u00adhedron. The crystal specimen under investigation was twinned by non-merohedry with a refined twin domain fraction of 0.853\u2005(14):0.147\u2005(14).The crystal structure of mercurous iodide, Hgurst 1926. J. Am. DOI: 10.1107/S1600536811056339/wm2566Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "In the crystal, the mol\u00adecules are connected by O\u2014H\u22efN hydrogen bonds into centrosymmetric dimers. The amino H atom is not involved in hydrogen bonding.The dihedral angle between the two phenyl rings in the title compound, C DOI: 10.1107/S1600536812002012/bt5789Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S1600536812002012/bt5789Isup3.cmlSupplementary material file. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "In the crystal, the carboxyl\u00adate groups act as bridging ligands, forming a two-dimensional polymer parallel to (001). The aqua ligand, which lies on a twofold rotation axis, forms inter\u00admolecular O\u2014H\u22efO hydrogen bonds within these layers. In the title compound, [K DOI: 10.1107/S1600536812008963/lh5422Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "Conventional O\u2014H\u22efO hydrogen bonds link the complex mol\u00adecules, forming layers parallel to the ab plane.In the monomeric title complex, [Cu(C DOI: 10.1107/S160053681100064X/bt5450Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Each GdIII ion is nine-coordinated by one 1,10-phenanthroline mol\u00adecule, one bidentate chelating carboxyl\u00adate group and four bridging carboxyl\u00adate groups in a distorted GdN2O7 monocapped square-anti\u00adprismatic geometry.In the centrosymmetric binuclear title complex, [Gd DOI: 10.1107/S1600536811036130/wm2524Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536811042061/ng5233Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The complete dianionic ligand is generated by crystallographic twofold symmetry. In the crystal, a two-dimensional supra\u00admolecular structure parallel to (001) is formed through O\u2014H\u22efO hydrogen-bond inter\u00adactions between the coordinated water mol\u00adecules and the O atoms of nearby carboxyl\u00adate groups.In the title compound, [Ni(C DOI: 10.1107/S1600536811016400/hb5864Isup2.hkl Structure factors: contains datablocks I. DOI: 10.1107/S1600536811016400/hb5864Isup3.cdx Supplementary material file. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S160053681100849X/jh2269Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The crystal structure is stabilized by inter\u00admolecular N\u2014H\u22efO hydrogen bonds, which link the mol\u00adecules into chains running along the c axis.In the title compound, C DOI: 10.1107/S1600536811044904/bt5690Isup2.hkl Structure factors: contains datablock(s) I. DOI: 10.1107/S1600536811044904/bt5690Isup3.cml Supplementary material file. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Each TbIII ion is nine-coordinated by one 1,10-phenanthroline mol\u00adecule, one bidentate carboxyl\u00adate group and four bridging carboxyl\u00adate groups in a distorted TbN2O7 monocapped square-anti\u00adprismatic geometry.In the centrosymmetric binuclear title complex, [Tb DOI: 10.1107/S1600536811032041/hb6349Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the crystal, mol\u00adecules are linked via O\u2014H\u22efO hydrogen bonds into a two-dimensional network parallel to (10In the title complex, [Ni(C DOI: 10.1107/S1600536811041134/lh5343Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536811033800/lh5314Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Within the two-dimensional complex polymer which is parallel to (100), coordinating water mol\u00adecules form inter\u00admolecular O\u2014H\u22efO hydrogen bonds with carboxyl\u00adate and phen\u00adoxy O-atom acceptors, as well as with the partial-occupancy solvent water mol\u00adecules.In the title compound, {[Mg(C DOI: 10.1107/S1600536812035246/lh5512Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S1600536812035246/lh5512Isup3.cmlSupplementary material file. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536812018831/aa2053Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "In the crystal, weak C\u2014H\u22efO hydrogen bonds between the meth\u00adoxy groups connect adjacent mol\u00adecules, giving chains which extend along [001].In the title compound, C DOI: 10.1107/S1600536812036288/zs2220Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S1600536812036288/zs2220Isup3.cmlSupplementary material file. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "The solvent mol\u00adecules are linked by hydrogen bonds.The title dinuclear centrosymmetric complex, [Hg DOI: 10.1107/S160053681102040X/kp2329Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "A supra\u00admolecular chain aligned along [101] mediated by charge-assisted O/N\u2014H\u22efCl\u2212 hydrogen bonds features in the crystal packing. Chains are connected into a three-dimensional architecture by C\u2014H\u22efO(hy\u00addroxy) inter\u00adactions.In the title salt, C DOI: 10.1107/S1600536814004565/hg5387Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S1600536814004565/hg5387Isup3.cmlSupporting information file. DOI: 988939CCDC reference: crystallographic information; 3D view; checkCIF reportAdditional supporting information:"} +{"text": "The resulting cationic complex and the 3,5-dimethyl\u00adbenzoate anion are inter\u00adconnected by N\u2014H\u22efO hydrogen bonds.In the title compound, [Pd(C DOI: 10.1107/S1600536810028369/bt5293Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536812013049/mw2060Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "In the crystal N\u2014H\u22efN hydrogen bonds link the mol\u00adecules into chains. Weak C\u2014H\u22efN inter\u00adactions are also present.In the title compound, C DOI: 10.1107/S1600536812037816/xu5598Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S1600536812037816/xu5598Isup3.cmlSupplementary material file. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "The metal atoms each show a distorted square-pyramidal coordination geometry. Intra\u00admolecular O\u2014H\u22efO hydrogen bonds occur. In the crystal, O\u2014H\u22efO hydrogen bonds join the components into a chain extending along the a axis.In the title compound, [Cu DOI: 10.1107/S1600536811041481/gk2407Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Inter\u00admolecular O\u2014H\u22efO hydrogen bonds form an extensive three-dimensional hydrogen-bonding network, which consolidates the crystal packing.In the dinuclear title complex, [Fe DOI: 10.1107/S1600536810041966/cv2763Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The mol\u00adecule contains two fused (trans) six-membered rings which both exibit a chair conformation. In the crystal, mol\u00adecules are linked into chains along [100] by weak C\u2014H\u22efO hydrogen bonds involving the methyl and carbonyl groups.The title compound, C DOI: 10.1107/S1600536812029303/fj2573Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S1600536812029303/fj2573Isup3.cmlSupplementary material file. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "The zinc cation is coordinated by four N atoms of two terminal N-bonded thio\u00adcyanate anions and of two symmetry-related 3-methyl\u00adpyridine co-ligands, defining a slightly distorted tetra\u00adhedral coordination polyhedron.The asymmetric unit of the title compound, [Zn(NCS) DOI: 10.1107/S1600536811024561/wm2500Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the crystal, the constituent units are linked into a two-dimensional network parallel to the ab plane by N\u2014H\u22efO and O\u2014H\u22efO hydrogen bonds.In the title compound, C DOI: 10.1107/S1600536811035537/ci5198Isup2.hkl Structure factors: contains datablock(s) I. DOI: 10.1107/S1600536811035537/ci5198Isup3.cml Supplementary material file. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the crystal, the complex mol\u00adecules are linked by O\u2014H\u22efO hydrogen bonds between the water and sulfato ligands, forming a three-dimensional network.The title complex, [Mn al. 1995. Inorg. al. 1996. Polyhed DOI: 10.1107/S160053681103604X/is2772Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The carboxyl\u00adate group and the hy\u00addroxy group form O\u2014H\u22efO hydrogen bonds, generating a head-to-tail chain along the b axis. Neighbouring hydrogen-bonded chains are linked by the water mol\u00adecule, generating two independent O\u2014H\u22efO donor hydrogen bonds. The carboxyl\u00adate group thus constructs a hydrogen-bonded host layer parallel to (10In the title compound, C DOI: 10.1107/S1600536811024044/fj2433Isup2.hkl Structure factors: contains datablock(s) I. DOI: 10.1107/S1600536811024044/fj2433Isup3.cml Supplementary material file. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The nuclei are 3.122\u2005(2)\u2005\u00c5 apart. The mol\u00adecule exhibits a twofold rotation axis.The title compound, [Au DOI: 10.1107/S1600536811028856/bg2412Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The NiII atom is coordinated by three N atoms and one O atom [Ni\u2014O = 1.859\u2005(2)\u2005\u00c5], resulting in a pseudo-square-planar coordination environment.The title compound, [Ni(C DOI: 10.1107/S1600536812026827/im2378Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536811011974/gk2354Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The five-membered chelate ring adopts a twisted conformation. In the crystal, weak C\u2014H\u22efCl hydrogen bonds link the mol\u00adecules into (001) sheets.In the title complex, [PtCl DOI: Click here for additional data file.10.1107/S1600536812048295/hb7001Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "In the complex, the two coordinated benzotriazole rings rings are nearly perpendicular, the dihedral angle between their planes being 87.08\u2005(6)\u00b0.In the crystal structure of the title coordination compound, [Ag(NO DOI: 10.1107/S1600536812006368/vn2026Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "The TiIV atom is coordinated by two fully deprotonated O,N,O\u2032-tridentate phen\u00adoxy\u00adamine ligands in a distorted octa\u00adhedral environment. Within this arrangement the O atoms occupy the equatorial sites and the N atoms the axial sites. One of the toluene mol\u00adecules is disordered over two sets of sites in a 0.628\u2005(18):0.372\u2005(18) ratio.The title compound, [Ti(C DOI: Click here for additional data file.10.1107/S1600536813007022/br2221Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S1600536813007022/br2221Isup3.cdxSupplementary material file. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "The methyl groups are disordered over two equally occupied positions. In the crystal, inter\u00admolecular N\u2014H\u22efO hydrogen bonds link the mol\u00adecules into infinite chains running along the a axis.In the title compound, C DOI: 10.1107/S1600536811040967/bq2308Isup2.hkl Structure factors: contains datablock(s) I. DOI: 10.1107/S1600536811040967/bq2308Isup3.cml Supplementary material file. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "One of the thienyl groups and the bound THF are disordered with 0.5:0.5 occupancy. The free THF is disordered around the threefold axis.In the mononuclear title compound, [Tb(C DOI: 10.1107/S160053681104623X/nc2251Isup2.cdx Supplementary material file. DOI: 10.1107/S160053681104623X/nc2251Isup3.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The coordination around the Rh atom shows a slightly distorted square-planar arrangement.In the title compound, DOI: 10.1107/S1600536811038505/mw2021Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536810047628/zq2074Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Mol\u00adecules are packed in positions of least steric hindrance, with the phosphane ligands positioned above and below the Rh\u2013acetyl\u00adacetonate backbone.The title compound, [Rh(C DOI: 10.1107/S1600536811050483/mw2037Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the crystal, weak C\u2014H\u22efO inter\u00adactions link the mol\u00adecules into zigzag chains propagating in [100].In the title compound, C DOI: 10.1107/S160053681303033X/cv5435Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S160053681303033X/cv5435Isup3.cmlSupplementary material file. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "The VV atom is five-coordinated by N,O,O\u2032-donor atoms of the Schiff base ligand, one meth\u00adoxy O atom and one oxide O atom, forming a square-pyramidal geometry.The title oxidovanadium(V) complex, [V(C DOI: 10.1107/S1600536811050173/qm2035Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Adjacent Sn atoms are bridged by the two O atoms of the carboxylate ligand, forming a chain structure along the a-axis direction.In the title polymeric coordination compound, [Sn(CH DOI: 10.1107/S160053681104918X/kp2369Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The bond lengths and bond angles are within normal ranges. The dihedral angle between the least-squares planes of the aromatic rings within each ligand is 82.76\u2005(17)\u00b0.In the title Schiff base complex, [Zn(C DOI: Click here for additional data file.10.1107/S1600536812043930/ds2215Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536812011786/bt5848Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "The mol\u00adecules assume an approximate propellar shape, with the three aromatic rings being bent with respect to the plane formed by the C atoms that are connected to the methine C atom .The title compound, C DOI: 10.1107/S1600536812003698/bt5804Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S1600536812003698/bt5804Isup3.cmlSupplementary material file. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "Extensive hydrogen bonds establish a layered network structure extending parallel to (001).In the three-dimensional tetra\u00adchloro\u00adphthalate-bridged title complex [Er(C DOI: 10.1107/S1600536812016923/bt5856Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536810047501/nc2197Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Each LaIII ion is nine-coordinated by one 1,10-phenanthroline mol\u00adecule, one bidentate chelating carboxyl\u00adate group and four bridging carboxyl\u00adate groups in a distorted LaN2O7 monocapped square-anti\u00adprismatic geometry.In the centrosymmetric binuclear title complex, [La DOI: 10.1107/S1600536811036117/wm2525Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Correction to:British Journal of Cancer (2011) 104, 188\u2013192; doi:10.1038/sj.bjc.6605911Upon publication in early 2011, the authors noticed a small error in the Methods section regarding the ATC codes for genotoxic and non-genotoxic TCA drugs, which may have caused some confusion for readers. The corrected text is presented below:The list of ATC codes for TCA drugs should read:TCAs (genotoxic) : desipramine (N06AA01), imipramine (N06AA02), imipramine oxide (N06AA03), clomipramine (N06AA04), opipramol (N06AA05), trimipramine (N06AA06), doxepine (N06AA12), amoxapine (N06AA17), lofepramine (N06AA07); TCAs (non-genotoxic) : amitriptyline (N06AA09), nortriptyline (N06AA10), protriptyline (N06AA11), dosulepine (N06AA16)\u2019.\u2018"} +{"text": "In the crystal structure, symmetry-related mol\u00adecules are linked by N\u2014H\u22efO hydrogen bonds, generating a three-dimensional network.In the title complex, [Ni(C DOI: 10.1107/S1600536810041577/lh5153Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The resulting graph-set descriptor of this ring system is R22(10). The nitrate counter-anions connect the dicationic dimers via N\u2014H\u22efO hydrogen bonds, forming two-dimensional networks in the bc plane.In the crystal of the title salt, C DOI: 10.1107/S1600536813027694/lh5656Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S1600536813027694/lh5656Isup3.cmlSupplementary material file. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536811043972/ff2038Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the crystal, the complex exhibits a zigzag chain structure running parallel to the c axis. An intra\u00adchain C\u2014H\u22efO hydrogen bond is observed.In the polymeric title coordination compound, [Sn(C DOI: 10.1107/S1600536811049245/rz2663Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The asymmetric unit comprises one Eu3+ cation, two aqua ligands and one and a half thiophene-2,5-dicarboxylate anions . The Eu3+ cation is eight-coordinated in a distorted dodeca\u00adhedral geometry. The crystal structure features O\u2014H\u22efO hydrogen bonds.The three-dimensional coordination polymer, [Eu DOI: 10.1107/S1600536812029546/rk2364Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "A weak intra\u00admolecular C\u2014H\u22efO inter\u00adaction is noted. The crystal studied was a racemic twin.The structure of the title compound, [Pd(C DOI: 10.1107/S1600536812040068/tk5150Isup2.molSupplementary material file. DOI: 10.1107/S1600536812040068/tk5150Isup3.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "In the crystal structure, C\u2014H\u22efCl hydrogen-bonding inter\u00adactions connect the mol\u00adecules into a three-dimensional network.In the title compound, [CdCl DOI: 10.1107/S1600536810043163/pv2344Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The coordination geometry at the RhI ion is distorted square-planar. The toluene solvent mol\u00adecule is disordered over two different orientations, with site-occupation factors of 0.810\u2005(2) and 0.190\u2005(2).The reaction of (\u03b7 DOI: 10.1107/S1600536812002851/bt5795Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536810021859/hg2685Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The water mol\u00adecules and the organic cations inter\u00adact with the [ZnCl4]2\u2212 complex anions, building up a two-dimensional hydrogen-bonded network parallel to (100).In the title compound, (C DOI: 10.1107/S1600536811017478/dn2685Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The NiII atom is coordinated in a slightly distorted square-planar geometry by two O atoms and two N atoms from two 2-[imino\u00ad(phen\u00adyl)meth\u00adyl]-5-meth\u00adoxy\u00adphenolate ligands. The dihedral angle between the symmetry-unique phenyl and benzene rings is 73.2\u2005(1)\u00b0.The title complex, [Ni(C DOI: Click here for additional data file.10.1107/S1600536812043061/lh5543Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S1600536812043061/lh5543Isup3.cdxSupplementary material file. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "The crystal structure exhibits parallel stacking of the diammonium dication in its packing arrangement, together with inorganic\u2013organic layering that is typical of these n-alkyl\u00addiammonium salts. An intricate three-dimensional hydrogen-bonding network exists in the crystal structure where the hydrogen bonds link the cation and anion layers together through the sulfate anions and the water mol\u00adecules.The crystal structure of the title compound, C DOI: 10.1107/S1600536811030030/zk2015Isup2.hkl Structure factors: contains datablock(s) I. DOI: 10.1107/S1600536811030030/zk2015Isup3.mol Supplementary material file. DOI: 10.1107/S1600536811030030/zk2015Isup4.cml Supplementary material file. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The crystal structure features polymeric chains running along the b axis which are stabilized by N\u2014H\u22efN hydrogen bonds.In the title coordination polymer, [Hg(NCS) DOI: 10.1107/S1600536812016790/bt5873Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "In the chain, the Cd2+ cation is coordinated by four bridging Cl\u2212 ligands in equatorial positions and two N atoms from symmetry-related and likewise bridging 1,3-bis\u00ad(imidazol-1-yl)propane ligands in axial positions, forming a distorted CdCl4N2 octa\u00adhedron.The title complex, [CdCl DOI: 10.1107/S1600536812021083/wm2624Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "Each L ligand also lies across an inversion center and bridges two CdII ions, forming infinite two-dimensional recta\u00adngular layers running parallel to (010).In the title coordination polymer, [CdCl DOI: 10.1107/S1600536811027036/gw2103Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The Cd atom in the complex [Cd(Se4)2]2\u2212 anion is tetra\u00adhedrally coordinated by two chelating tetra\u00adselenide ligands, and both CdSe4 rings exhibit an envelope conformation.The title compound, (EtMe DOI: 10.1107/S1600536811007227/zl2349Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the crystal, O\u2014H\u22efO hydrogen bonds link the mol\u00adecules into a layer structure parallel to (-101).In the title compound, [Zn(C DOI: Click here for additional data file.10.1107/S1600536812045357/vn2058Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "The crystal structure is stabilized by intermolecular O\u2014H\u22efN hydrogen bonds involving the coordinated water mol\u00adecules and the N atoms of the tetra\u00adzolide group.In the title complex, [Mn(C DOI: 10.1107/S1600536811047428/pv2481Isup2.hkl Structure factors: contains datablock(s) I. DOI: 10.1107/S1600536811047428/pv2481Isup4.cdx Supplementary material file. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The TbIII atom displays a dodecahedral geometry, while the MoV ion exhibits a distorted square-anti\u00adprismatic geometry. The Tb atoms are located on a special position of site symmetry In the title compound, [Tb(C DOI: 10.1107/S1600536811020022/bt5505Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Two H2mbidc ligands bridge two ZnII atoms, generating a double-chain along [In the title compound, {[Zn(C DOI: 10.1107/S1600536811006532/hy2396Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The pyrazole rings have a 21.09\u2005(5)\u00b0 angle between their mean planes and exhibit a trans-like geometry in which the in-plane lone pairs of electrons on the 2-N nitrogen atoms point in opposite directions. The title compound, C DOI: 10.1107/S1600536812032801/zq2175Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S1600536812032801/zq2175Isup3.cmlSupplementary material file. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "Multiple figures in the article contain errors. Please see corrected versions of those figures here: Figure 1: Figure 2: Figure 3: Figure 4: Figure 5: Figure 6: Figure 7: Figure 8: Figure 9:"} +{"text": "The 4,4\u2032-bpy ligand has an inversion center at the mid-point of the central C\u2014C bond. The PbII atoms are linked by bidentate bridging 4,4\u2032-bpy into a chain along [101]. These chains are further connected into layers via C\u2014H\u22efO hydrogen bonds.In the title complex, [Pb(C DOI: 10.1107/S1600536811035021/hy2458Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536812035362/tk5138Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S1600536812035362/tk5138Isup3.cdxSupplementary material file. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "In the crystal structure, carboxyl\u00adate bridges link the metal atoms, forming zigzag chains parallel to the b axis.In the title polymeric coordination compound, [Sn(CH DOI: 10.1107/S1600536810047835/rz2525Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The mol\u00adecular structure is comprised of two hydro\u00adtris\u00ad(1-pyrazol\u00adyl)borate ligands (Tp\u2212) and a central FeII ion, which is coordinated by six pyrazole N atoms from two two Tp\u2212 ligands, yielding a distorted bipyramidal FeN6 geometry. The complete molecule exhibits symmetry 2/m.The title compound, [Fe(C al. 1980. Inorg. DOI: 10.1107/S1600536811025839/hg5058Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Both Ni atoms are located on crystallographic centres of inversion.The title compound, [Ni(C DOI: 10.1107/S1600536811028510/im2294Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The bridging ligands generate a three-dimensional structure.In the title coordination polymer, [Zn(C DOI: 10.1107/S1600536810042406/ng5050Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Each heptanedioato ligand bridges three Mg atoms, generating polymeric layers parallel to the bc plane. The polymeric layers related by translation along the a axis inter\u00adact further via O\u2014H\u22efO hydrogen bonds, which consolidate the crystal packing.In the title compound, [Mg DOI: 10.1107/S1600536811015492/cv5076Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the crystal, O\u2014H\u22efCl hydrogen bonds link the mol\u00adecules into [100] chains.In the title compound, [NiCl DOI: 10.1107/S160053681103128X/hb6336Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536810038298/hb5649Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "C\u2014H\u22efO\u2014H inter\u00adactions.The title compound, [Fe(C al. 2006. Acta Cr DOI: 10.1107/S1600536811047775/tk5015Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "An intramolecular C\u2014H\u22efO hydrox\u00adyl\u2013carboxyl hydrogen bond occurs in the anion.In the structure of the title salt, C DOI: 10.1107/S1600536810024888/ez2219Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The mol\u00adecular conformation is stabilized by intra\u00admolecular O\u2014H\u22efO hydrogen bonds.In the title compound, [Ni(HCOO) DOI: 10.1107/S1600536811050859/vm2139Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The dihedral angle between the pyridine and the cyclo\u00adpropyl rings is 95.4\u2005(8)\u00b0.In the title compound, [ZnBr DOI: 10.1107/S1600536810025201/hb5526Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the crystal, weak C\u2014H\u22efF hydrogen bonds link the complex cations and hexa\u00adfluorido\u00adphosphate anions into a three-dimensional supra\u00admolecular structure.In the title compound, [CoRu(C DOI: 10.1107/S1600536813028195/hy2638Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "The dihedral angle between the mean planes of the two coordinating ligands is 83.65\u2005(5)\u00b0. The crystal packing is consolidated by weak C\u2014H\u22efN hydrogen-bonding inter\u00adactions.In the title compound, [Zn(C DOI: 10.1107/S1600536812009592/wm2598Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "In the crystal, extensive O\u2014H\u22efO hydrogen-bonding inter\u00adactions result in a three-dimensional supra\u00admolecular architecture.In the title compound, [Ce(C DOI: 10.1107/S1600536811052688/ez2275Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The 1,1\u2032-methyl\u00adenebis(1H-imidazole) ligands adopt a bis-monodentate bridging mode linking two CoII atoms.The title compound, [Co DOI: 10.1107/S1600536811010610/jh2275Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Inter\u00admolecular O\u2014H\u22efO hydrogen bonds generate a three-dimensional hydrogen-bonding network, which consolidates the crystal packing.The asymmetric unit of the title compound, [Co(C DOI: 10.1107/S1600536810023810/cv2723Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The dihedral angle between the mean planes of the two coordinating ligands is 85.65\u2005(5)\u00b0. Weak C\u2014H\u22efS hydrogen bonds are also observed.The title compound, [Zn(C DOI: 10.1107/S1600536812013529/wm2612Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "The octahedral [Zn(H2O)6]2+ cations, solvent water mol\u00adecules and anions inter\u00adact via O\u2014H\u22efO hydrogen bonds, forming a three-dimensional network.The title compound, [Zn(H DOI: 10.1107/S1600536810027972/jh2181Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The 4,4\u2032-diazenediyldibenzoate anions do not coordinate to Ag. O\u2014H\u22efO hydrogen bonds stabilize the crystal structure.In the title compound, [Ag DOI: 10.1107/S1600536811008816/bt5474Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The purpose of this table is to provide the community with a citable record of publications of ongoing genome sequencing projects that have led to a publication in the scientific literature. While our goal is to make the list complete, there is no guarantee that we may have omitted one or more publications appearing in this time frame. Readers and authors who wish to have publications added to this subsequent versions of this list are invited to provide the bibliometric data for such references to the SIGS editorial office. CrenarchaeotaPhylum EuryarchaeotaPhylum Pyrococcus yayanosii CH1, sequence accession CP002779 Pantoea ananatis LMG20103, sequence accession CP001875 [Helicobacter bizzozeronii strain CIII-1, sequence accession FR871757 (chromosome) and FR871758 (HBZ-1) [ (HBZ-1) Vibrio anguillarum 775, sequence accession CP002284 to CP002285 [CP002285 Zymomonas mobilis subsp. pomaceae, sequence accession CP002865 (chromosome), CP002866 (p29192_1), CP002867 (p29192_2) [29192_2) Agrobacterium sp. strain ATCC 31749, sequence accession AECL01000000 [01000000 Xanthomonas spp. strain Xrc, sequence accesssion CP002789 [CP002789 Xanthomonas spp. strain Xoc, sequence accesssion AAQN00000000 [00000000 Glaciecola sp. Strain 4H-3-7+YE-5, sequence accession CP002526 (chromosome) and CP002527 (plasmid) [plasmid) Escherichia coli Strain HM605, sequence accession CADZ01000001 through CADZ01000154 [01000154 Salinisphaera shabanensis, sequence accession AFNV00000000 [00000000 Methyloversatilis universalis FAM5T, sequence accession AFHG00000000 [00000000 Alicycliphilus denitrificans Strain BC, sequence accession CP002449 (chromosome), CP002450 (megaplasmid), CP002451 (plasmid) [plasmid) .Alicycliphilus denitrificans K601T, sequence accession CP002657 (chromosome) and CP002658 (plasmid) [plasmid) Oligotropha carboxidovorans Strain OM4, sequence accession CP002821 (chromosome), CP002822 (pHCG3b), CP002823 (pOC167B) [pOC167B) Oligotropha carboxidovorans Strain OM5, sequence accession CP002826 (chromosome), CP002827 (pHCG3), and CP002828 (pOC167) Bradyrhizobiaceae strain SG-6C, sequence accession AFOF01000000 [01000000 Hyphomicrobium sp. Strain MC1, sequence accession FQ859181 [FQ859181 Shewanella sp. Strain HN-41, sequence accession AFOZ01000000 [01000000 Myxococcus fulvus HW-1, sequence accession CP002830 [CP002830 Nitrosomonas sp. Strain AL212, sequence accession NC_015222 (chromosome), NC_015223 pNAL21201), NC_015221 (pNAL21202) [AL21202) Ruegeria sp. Strain KLH11, sequence accession ACCW00000000 [00000000 Acidovorax avenae subsp. avenae RS-1, sequence accession AFPT01000000 [01000000 Escherichia coli (ExPEC), sequence accession AFAT00000000 [00000000 Vibrio mimicus SX-4, sequence accession ADOO01000000 [01000000 Agrobacterium tumefaciens Strain F2, sequence accession AFSD00000000 [00000000 Pasteurella multocida subsp. gallicida AFRR01000001 to AFRR01000489 [01000489 Pseudomonas aeruginosa 138244, sequence accession AEVV00000000 [00000000 Pseudomonas aeruginosa 152504, sequence accession AEVW00000000 [00000000 Campylobacter jejuni strain 305, sequence accession ADHL00000000 [00000000 Campylobacter jejuni strain DFVF1099, sequence accession ADHK00000000 [00000000 Xanthomonas campestris pv. raphani strain 756C, sequence accession CP002789 [CP002789 Xanthomonas campestris pv. raphani strain BLS256, sequence accession AAQN01000001 [01000001 Rickettsia heilongjiangensis, sequence accession CP002912 [CP002912 Acidiphilium sp. Strain PM (DSM 24941), sequence accession AFPR00000000 [00000000 Pseudomonas putida Strain S16, sequence accession CP002870 [CP002870 Acinetobacter lwoffii, sequence accession AFQY01000000 [01000000 FirmicutesPhylum Caldalkalibacillus thermarum strain TA2.A1, sequence accession AFCE00000000 [00000000 Listeria monocytogenes Scott A, sequence accession AFGI00000000 [00000000 Lactococcus garvieae 8831, sequence accession AFCD00000000 [00000000 Natranaerobius thermophilus JW/NM-WN-LF, sequence accession CP001034 (chromosome), CP001035 (plasmid) [plasmid) Melissococcus plutonius ATCC 35311, sequence accession AP012200 (chromosome) and AP012201 (plasmid) [plasmid) Lactobacillus buchneri NRRL B-30929, sequence accession CP002652 (chromosome), CP002653 (plasmid pLBU01), CP002654 (plasmid pLBU02), and CP002655 (plasmid pLBU03) [Lactobacillus kefiranofaciens ZW3 , sequence accession CP002764 (chromosome), CP002765 (plasmid), and CP002766 (plasmid) [plasmid) Bacillus megaterium strain QM B1551, sequence accession CP001983 (chromosome), CP001984 to CP001990 (plasmids pBM100 through pBM700) [Bacillus megaterium strain DSM319, sequence accession CP001982 (chromosome) [omosome) Listeria monocytogenes serovar 4a strain M7, sequence accession CP002816 [CP002816 Bacillus coagulans 2-6, sequence accession CP002472 [CP002472 Streptococcus salivarius strain CCHSS3, sequence accession FR873481 [FR873481 Paenibacillus elgii B69, sequence accession AFHW01000000 [01000000 Lactobacillus pentosus MP-10, sequence accession FR871759 through FR871848 [FR871848 Leuconostoc pseudomesenteroides KCTC 3652, sequence accession AEOQ00000001 through AEOQ00001160 [00001160 Lactobacillus mali KCTC 3596, sequence accession BACP01000001 through BACP01000122 [01000122 Paenibacillus polymyxa Type Strain ATCC 842T, sequence accession AFOX01000000 [01000000 Streptococcus salivarius strain JIM8777, sequence accssion FR873482 [FR873482 Lactobacillus cypricasei KCTC 13900, sequence accession BACS01000001 to BACS01000487 [01000487 Lactobacillus zeae KCTC 3804, sequence accession BACQ01000001 to BACQ101000113 [01000113 Listeria monocytogenes Serovar 4a Strain M7, sequence accession CP002816 [CP002816 Lactobacillus salivarius GJ-24, sequence accession AFOI00000000 [00000000 Lactobacillus johnsonii PF01, sequence accession AFQJ01000000 [01000000 Clostridium acetobutylicum DSM 1731, sequence accession CP002660 through CP002662 [CP002662 Lactobacillus suebicus KCTC 3549, sequence accession BACO01000000 [01000000 Brevibacillus laterosporus LMG 15441, sequence accession AFRV00000000 [00000000 Lactobacillus salivarius NIAS840, sequence accession AFMN00000000 [00000000 Bifidobacterium animalis subsp. lactis CNCM I-2494, sequence accession CP002915 [CP002915 Megasphaera elsdenii, sequence accession HE576794 [HE576794 Lactobacillus versmoldensis KCTC 3814, sequence accession BACR01000001 to BACR01000102 [01000102 Lactobacillus pentosus IG1, sequence accession FR874848 to FR874860 [FR874860 Alicyclobacillus acidocaldarius Strain Tc-4-1, sequence accession CP002902 [CP002902 Streptococcus thermophilus Strain JIM8232, sequence accession FR875178 [FR875178 Streptococcus equi subsp. zooepidemicus Strain ATCC 35246, sequence accession CP002904 [CP002904 Bacillus amyloliquefaciens XH7, sequence accession CP002927 [CP002927 Leuconostoc kimchii Strain C2, sequence accession CP002898 [CP002898 Lactobacillus malefermentans KCTC 3548, sequence accession BACN01000001 to BACN01000172 [01000172 Weissella koreensis KACC 15510, sequence accession CP002900 [CP002900 TenericutesPhylum Mycoplasma bovis Strain Hubei-1, sequence accession CP002513 [CP002513 Mycoplasma fermentans Strain M64, sequence accession NC_014921 [C_014921 Haloplasma contractile, sequence accession AFNU00000000 [00000000 Mycoplasma ovipneumoniae Strain SC01, sequence accession AFHO01000000 [01000000 ActinobacteriaPhylum Kocuria rhizophila P7-4, sequence accession AFID00000000 [00000000 Streptomyces S4, sequence accession CADY01000000 [01000000 Corynebacterium nuruki S6-4T, sequence accession AFIZ00000000 [00000000 Propionibacterium humerusii, sequence accession AFAM00000000.1 [000000.1 Strain JDM601, sequence accession CP002329 Streptomyces sp. strain T\u00fc6071, sequence accession AFHJ01000000 [01000000 Bifidobacterium breve UCC2003, sequence accession CP000303 [CP000303 Propionibacterium acnes, sequence accession CP002815 [CP002815 Amycolicicoccus subflavus DQS3-9A1T, sequence accession CP002786 (chromosome), CP002787 (plasmid pAS9A-1), and CP002788 (plasmid pAS9A-2). [AS9A-2). Gordonia neofelifaecis NRRL B-59395, sequence accession AEUD01000000 [01000000 Pseudonocardia dioxanivorans strain CB1190, sequence accession NC_015312-4 and CP002595-7 [002595-7 Bifidobacterium longum subsp. longum KACC 91563, sequence accession CP002794 to CP002796 [CP002796 Streptomyces cattleya NRRL 8057, sequence accession FQ859185 (chromosome) and FQ859184 (megaplasmid) [plasmid) Rhodococcus sp. Strain R04, sequence accession AFAQ01000000 [01000000 Mycobacterium bovis BCG Moreau, sequence accession [ccession Saccharopolyspora spinosa NRRL 18395, sequence accession [ccession Mycobacterium tuberculosis CCDC5079, sequence accession [ccession Mycobacterium tuberculosis CCDC5180, sequence accession [ccession Amycolatopsis mediterranei S699, sequence accession CP002896 [CP002896 Nesterenkonia sp. Strain F, sequence accession AFRW01000000 [01000000 Streptomyces xinghaiensis NRRL B24674T, sequence accession AFRP01000000 [01000000 ChlamydiaePhylum Chlamydophila abortus variant strain LLG, sequence accession AFHM01000000 [01000000 Chlamydia psittaci 6BC, sequence accession CP002586 (chromosome), CP002587 (plasmid) [plasmid) Chlamydia psittaci Cal10, sequence accession AEZD00000000 (draft chromosome and plasmid) [plasmid) Chlamydia trachomatis, sequence accession CP002024 [CP002024 SpirochaetesPhylum Spirochaeta thermophila DSM 6192, sequence accession CP001698 [CP001698 Brachyspira intermedia, sequence accession CP002874 (chromosome) and CP002875 (plasmid) [plasmid) FibrobacteresPhylum BacteroidetesPhylum Porphyromonas gingivalis TDC60, sequence accession AP012203 [AP012203 Krokinobacter sp. strain 4H-3-7-5, sequence accession CP002528 [CP002528 Lacinutrix sp. strain 5H-3-7-4, sequence accession CP002825 [CP002825 Bacterium HQM9, sequence accession AFPB00000000 [00000000 Anaerophaga sp. Strain HS1, sequence accession AFSL00000000 [00000000 Capnocytophaga canimorsus Strain 5, sequence accession CP002113 [CP002113 Mesoflavibacter zeaxanthinifaciens strain S86, sequence accession AFOE00000000 [00000000 VerrucomicrobiaPhylum LentisphaeraePhylum ThermotogaePhylum Kosmotoga olearia Strain TBF 19.5.1, sequence accession CP001634 [CP001634 ArchaeaDomain Nitrosoarchaeum koreensis\" MY1, sequence accession AFPU00000000 [\"Candidatus 00000000 Cucumis sativus L., sequence accession FI132140\u2013FI136208, GS765762\u2013GS766880, GS815969\u2013GS874855 [North-European Cucumber GS874855 Ricinus communis organelle genome, sequence accession JF937588(chloroplast), HQ874649 (mitochondria) [Castor bean hondria) Umatilla, sequence accession HQ842619 through HQ842628 [Stretch Lagoon Orbivirus HQ842628 Gadus morhua, sequence accession CAEA01000001 through CAEA01554869 [Atlantic cod 01554869 Solanum tuberosum L., sequence accession GS025503 through GS026177 [Potato GS026177 \u03a6CA82, sequence accession HQ264138 Paramecium caudatumreveals mitochondria, sequence accession NC001324 [NC001324 bacteriophage IME08, sequence accession NC_014260 virus (ILTV), sequence accession HQ_630064 Macropus eugenii, sequence accession ABQO000000000 [Australian kangaroo 00000000 Aichi virus, sequence accession FJ890523 \"Candidatus Tremblaya princeps\" Strain PCVAL, sequence accession CP002918"} +{"text": "The [SnCl6]2\u2212 anion undergoes a slight distortion from octa\u00adhedral symmetry as the result of the formation of four unforked charge-supported N\u2014H\u22efCl hydrogen bonds. The hydrogen bonds between the cations and anions form layers perpendicular to [101]. These layers are built by 24-membered rings which can be classified with an R88(24) graph-set descriptor. According to this hydrogen-bonding motif, the title compound is isostructural with (C6H16N)2[IrCl6].The title compound, (C DOI: Click here for additional data file.10.1107/S1600536812043371/mw2089Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S1600536812043371/mw2089Isup3.molSupplementary material file. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "Two trans-N:S-bridging thio\u00adcyanates complete the N5S donor set around the Cd atom. In the crystal, adjacent CdII ions are linked by the thio\u00adcyanate N:S-bridges into polymeric chains along the c axis.In the title compound, [Cd(NCS) DOI: 10.1107/S1600536811010063/om2412Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The complex displays a nano-sized porous metal\u2013organic framework that belongs to a topological network.The solvothermal reaction of Co(NO DOI: 10.1107/S1600536811017661/tk2740Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536810027595/bt5296Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "N\u2014H\u22efBr hydrogen bonds link the organic cations and bioctahedral face-sharing anions into a three-dimensional network.In the title compound, (C DOI: Click here for additional data file.10.1107/S1600536813014335/vn2071Isup2.cdxSupplementary material file. DOI: 10.1107/S1600536813014335/vn2071Isup3.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "The complex mol\u00adecules are stabilized by intra\u00admolecular O\u2014H\u22efCl hydrogen bonds.In the title compound, [NdCl DOI: 10.1107/S1600536812014055/zs2190Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "The solvent water mol\u00adecules are lodged in the channels.In the crystal structure of the title monohydrate salt, [ZnCl(C DOI: 10.1107/S1600536810052682/bg2383Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The dihedral angle between the two bromo-substituted benzene rings is 10.1\u2005(3)\u00b0.In the title complex, [Ni(C DOI: 10.1107/S1600536812004321/lh5411Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "One of the bridging Hg\u2014Cl bonds is significantly longer than the other.In the centrosymmetric dinuclear title complex, [Hg DOI: 10.1107/S1600536811004703/gk2347Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The purpose of this table is to provide the community with a citable record of publications of ongoing genome sequencing projects that have led to a publication in the scientific literature. While our goal is to make the list complete, there is no guarantee that we may have omitted one or more publications appearing in this time frame. Readers and authors who wish to have publications added to subsequent versions of this list are invited to provide the bibliographic data for such references to the SIGS editorial office. Thermogladius cellulolyticus\u201d 1633, sequence accession CP003531 [\u201cMethanomassiliicoccus luminyensis, sequence accession CAJE01000001 through CAJE01000026 [Pyrococcus sp. Strain ST04, sequence accession CP003534 [Leptospirillum ferrooxidans Strain C2-3, sequence accession AP012342 [Prochlorococcus marinus MED4, sequence accession BX548174 [Acinetobacter sp. Strain HA, sequence accession AJXD00000000 [00000000 Acinetobacter venetianus RAG-1T, sequence accession AKIQ00000000 [00000000 Agrobacterium tumefaciens CCNWGS0286, sequence accession AGSM00000000 [Alcaligenes faecalis subsp. faecalis NCIB 8687, sequence accession AKMR01000001 through AKMR01000186 [01000186 Alishewanella agri BL06T, sequence accession AKKU00000000 [00000000 Alishewanella aestuarii Strain B11T, sequence accession ALAB00000000 [Bacillus methanolicus MGA3, sequence accession ADWW00000000 [00000000 Bacillus methanolicus PB1, sequence accession AFEU00000000 [00000000 CandidatusSulfurovum sediminum\u201d, sequence accession AJLE00000000 [\u201c00000000 Bartonella birtlesii strain IBS 135T, sequence accession AKIP00000000 [00000000 Brucella abortus A13334, sequence accession CP003176.1 (Chromosome I),CP003177.1 (Chromosome II) [some II) Brucella canis Strain HSK A52141, sequence accession CP003174.1 (chromosome I), and CP003175.1 (chromosome II) [some II) Brucella melitensis S66, sequence accession AHWB00000000 [00000000 Brucella melitensis16M, sequence accession AHWC00000000 [00000000 Brucella melitensis 16M1w, sequence accession AHWD00000000 [00000000 Brucella melitensis 16M13w, sequence accession AHWE00000000 [00000000 Burkholderia sp. Strain KJ006, sequence accession CP003514 (chromosome I), CP003515 (chromosome II), CP003516 (chromosome III), and CP003517 (plasmid pKJ006) [ pKJ006) .Burkholderia terrae Strain BS001, sequence accession AKAU00000000 [00000000 Burkholderia thailandensis MSMB43, sequence accession AJXB00000000 [00000000 Catenovulum agarivorans YM01T, sequence accession AJWM00000000 [00000000 Citrobacter sp. Strain A1, sequence accession AKTT00000000 [00000000 Cronobacter sakazakii ES15, sequence accession CP003312 [CP003312 Dickeya zeae Strain ZJU1202, sequence accession AJVN00000000 [00000000 Enterobacter cloacae GS1, sequence accession AJXP00000000 [00000000 Enterococcus faecium Clinical Isolate LCT-EF128, sequence accession AJUP00000000 [00000000 Enterobacter radicincitans DSM16656T, sequence accession AKYD00000000 [00000000 Escherichia coli J53, sequnce accession AICK00000000 [00000000 Escherichia coli LCT-EC106, sequence accession [ccession Escherichia coli NCCP15647, sequence accession AJMB00000000 [00000000 Escherichia coli W26, sequence accession AGIA00000000 [00000000 Gluconobacter oxydans WSH-003, sequence accession AHKI00000000 [00000000 Halomonas stevensii S18214T, sequence accession AJTS00000000 [00000000 Helicobacter cinaedi Strain PAGU611, sequence accession AP012344 (chromosome) and AP012345 (plasmid) [plasmid) Helicobacter pylori hpEurope Strain N6, sequence accession CAHX01000001 to CAHX01000054 [01000054 Herbaspirillum lusitanum P6-12, sequence accession AJHH00000000 [00000000 Herbaspirillum sp. Strain GW103, sequence accession AJVC00000000 [00000000 Hydrocarboniphaga effusa strain AP103T, sequence accession AKGD00000000 [00000000 Hydrogenophaga sp. Strain PBC, sequence accession AJWL00000000 [00000000 Klebsiella oxytoca E718, sequence accession CP003683 [CP003683 Methylobacterium extorquens sp. strain 4-46, sequence accessions NC_010511, NC_010373, NC_010374 [C_010374 Methylobacterium extorquens strain BJ001, sequence accessions NC_010725, NC_010727, NC_010721 [C_010721 Methylobacterium extorquens strain CM4, sequence accessions NC_011757, NC_011758, NC_011760 [C_011760 Methylobacterium extorquens strain JCM 2831, sequence accessions NC_010510, NC_010509, NC_010514, NC_010517, NC_010518, NC_010502, NC_010504, NC_010507 [C_010507 Methylobacterium extorquens strain ORS 2060, sequence accessions NC_011894,NC_011892, NC_011887, NC_011893, NC_011895, NC_011888, NC_011889, NC_011890 [C_011890 Methylobacterium extorquens strain PA1, sequence accessions NC_010172 [C_010172 Methylobacterium sp. Strain GXF4, sequence accession AKFK00000000 [00000000 Methylophaga sp. Strain JAM1, sequence accession CP003390 [CP003390 Methylophaga sp. Strain JAM7 sequence accession CP003380 (chromosome), CP003381 (plasmid) [plasmid) Modestobacter marinus Strain BC501, sequence accession FO203431 [FO203431 Mycobacterium massiliense M18, sequence accession AJSC00000000 [00000000 Neisseria meningitidis Capsule Null Locus Strain, sequence accession CAJS01000001 through CAJS01000042 [01000042 Novosphingobium sp. Strain Rr 2-17, sequence accession AKFJ00000000 [00000000 Providencia stuartii Clinical Isolate MRSN 2154, sequence accession CP003488 [CP003488 Pseudaminobacter salicylatoxidans KCT001, sequence accession CAIU00000000 [00000000 Pseudomonas aeruginosa Strain SJTD-1, sequence accession AKCM00000000 [00000000 Pseudomonas aeruginosa Strain XMG, sequence accession AJXX00000000 [00000000 Pseudomonas fuscovaginae CB98818, sequence accession ALAQ00000000 [00000000 Pseudoalteromonas issachenkonii PAMC 22718, sequence accession AJTK00000000 [00000000 Pseudomonas sp. Strain HYS, sequence accession AJJP00000000 [00000000 Pseudomonas pseudoalcaligenes KF707, sequence accession AJMR00000000 [00000000 Pseudomonas putida Strain ND6, sequence accession CP003588 [CP003588 Pseudomonas putida Strain SJTE-1, sequence accession AKCL00000000 [00000000 Pseudomonas sp. Strain M47T1, sequence accession AJWX00000000 [00000000 Pseudomonas stutzeri TS44, sequence accession AJXE00000000 [00000000 Ralstonia sp. strain PBA, sequence accession AJWL00000000 [00000000 Rhodanobacter strain DSM 23569, sequence accession AGIL00000000 [00000000 Rhodanobacter strain 115, sequence accession AJXS00000000 [00000000 Rhodanobacter DSM 17631, sequence accession AJXT00000000 [00000000 Rhodanobacter DSM 18449, sequence accession AJXU00000000 [00000000 Rhodanobacter DSM 18863, sequence accession AJXW00000000 [00000000 Rhodanobacter DSM 24678, sequence accession AJXV00000000 [00000000 Rickettsia conorii subsp. caspia, sequence accession AJUR00000000 [00000000 Rickettsia australis strain PhillipsT, sequence accession AKVZ00000000 [00000000 Rickettsia sp. Strain MEAM1, sequence accession AJWD00000000 [00000000 Rickettsia conorii subsp. israelensis, sequence accession AJVP00000000 [00000000 Serratia plymuthica Strain PRI-2C, sequence accession AJTB00000000 [00000000 Serratia marcescens strain LCT-SM213, seqeunce accession AJUV00000000 [00000000 Sinorhizobium fredii USDA257, sequence accession CP003563 through CP003582 [CP003582 Sphingobium indicum B90A, sequence accession AJXQ00000000 [00000000 Stenotrophomonas maltophilia PML168, sequence accession CAJH01000001 through CAJH01000097 [01000097 Salmonella enterica serotype Newport CVM35185, sequence accession AHTJ00000000 [00000000 Salmonella enterica serotype Newport CVM33953, sequence accession AHTM00000000 [00000000 Salmonella enterica serotype Newport CVM21550, sequence accession AHTT00000000 [00000000 Salmonella enterica serotype Newport CVM21538, sequence accession AHTV00000000 [00000000 Salmonella enterica serotype Newport CVM37978, sequence accession AHUC00000000 [00000000 Salmonella enterica serotype Newport CVM19593, sequence accession AHUD00000000 [00000000 Salmonella enterica serotype Newport CVM19443, sequence accession AHUB00000000 [00000000 Salmonella enterica serotype Newport CVM19470, sequence accession AHUE00000000 [00000000 Salmonella enterica serovar Typhi UJ308A, sequence accession AJTD00000000 [00000000 Salmonella enterica Serovar Typhi UJ816A, sequence accession AJTE00000000 [00000000 Serratia sp. Strain M24T3, sequence accession [ccession Sulfuricella denitrificans skB26, sequence accession BAFJ01000001 through BAFJ01000023 [01000023 Xanthomonas campestris JX, sequence accession AJVO00000000 [00000000 Yersinia pestis Strain 2501, sequence accession AKVQ00000000 [00000000 Aeromonas aquariorum, sequence accession BAFL01000001 through BAFL01000036, and AP012343 [AP012343 Aerococcus viridans LL1, sequence accession AJTG00000000 [00000000 Bacillus anthracis H9401, sequence accession CP002091.1 ( chromosome), CP002092.1.1 (plasmid pXO1), and CP002093.1 (plasmid pXO2) [id pXO2) Bacillus atrophaeus C89, sequence accession AJRJ00000000 [00000000 Bacillus cereus NC7401, sequence accession AP007209 (chromosome), AP007210 (plasmid pNCcld), AP007211 , AP007212 , AP007213 , and AP007214 [4, 3 kb) Bacillus siamensis KCTC 13613T, sequence accession AJVF00000000 [00000000 Bacillus sp. Strain 5B6, sequence accession AJST00000000 [00000000 Bacillus sp. Strain 916, sequence accession AFSU00000000 [00000000 Citreicella aestuarii Strain 357, sequence accession AJKJ00000000 [00000000 Clostridium beijerinckii Strain G117, sequence acceession AKWA00000000 [00000000 Corynebacterium pseudotuberculosis Strain 1/06-A, sequence accession CP003082 [CP003082 Enterobacter sp. Isolate Ag1, sequence accession AKXM00000000 [00000000 Enterococcus faecalis D32, sequence accession CP003726 through CP003728 [CP003728 Enterococcus faecalis strain NP-10011, sequence accession AB712291 [Enterococcus hirae ATCC 9790, sequence accession CP003504 (chromosome), NC_015845 (plasmid pTG9790) [pTG9790) Geobacillus thermoglucosidans\u201d TNO-09.020, sequence accession AJJN00000000 [\u201c00000000 Lactococcus garvieae IPLA 31405, sequence accession AKFO00000000 [00000000 Lactobacillus mucosae LM1, sequence accession AHIT00000000 [00000000 Lactobacillus rossiae DSM 15814, sequence accession AKZK00000000 [00000000 Paenibacillus polymyxa OSY-DF, sequence accession AIPP00000000 [00000000 Pediococcus pentosaceus strain IE-3, sequence accession CAHU01000001 through CAHU01000091 [01000091 Pelosinus fermentans A11, sequence accession AKVM00000000 [00000000 Pelosinus fermentans B4, sequence accession AKVJ00000000 [00000000 Pelosinus fermentans JBW45, sequence accession AKVO00000000 [00000000 Pelosinus fermentans R7, sequence accession AKVN00000000 [00000000 Planococcus antarcticus DSM 14505, sequence accession AJYB00000000 [00000000 Pseudomonas stutzeri Strain JM300, sequence accession CP003725 [CP003725 Rhodococcus sp. strain DK17, sequence accession AJLQ00000000 [00000000 Staphylococcus aureus Strain LCT-SA112, sequence accession AJLP00000000 [00000000 Staphylococcus capitis QN1, sequence accession AJTG00000000 [00000000 Staphylococcus equorum subsp. equorum Mu2, sequence accession CAJL01000001 to CAJL01000030 [01000030 Staphylococcus hominis ZBW5, sequence accession AKGC00000000 [00000000 Staphylococcus saprophyticus subsp. saprophyticus M1-1, sequence accession AHKB00000000 [00000000 Streptococcus mutans GS-5, sequence accession CP003686 [CP003686 Streptococcus pyogenes M1 476, sequence accession AP012491 [AP012491 Streptococcus salivarius PS4, sequence accession AJFW00000000 [00000000 Streptococcus thermophilus Strain MN-ZLW-002, sequence accession CP003499 [CP003499 Ureibacillus thermosphaericus Strain Thermo-BF, sequence accession AJIK00000000 [00000000 Mycoplasma leachii Strain PG50T, sequence accession CP002108.1 [002108.1 Mycoplasma mycoides subsp. mycoides, sequence accession CP002107.1 [002107.1 Mycoplasma wenyonii Strain Massachusetts, sequence accession CP003703 [CP003703 Actinomyces massiliensis Strain 4401292T, sequence accession AKIO00000000 [00000000 Bifidobacterium animalis subsp. lactis B420, sequence accesion CP003497 [CP003497 Bifidobacterium animalis subsp. lactisBi-07, sequence accesion CP003498 [CP003498 Bifidobacterium bifidum strain BGN4, sequence accession CP001361 [CP001361 Brevibacterium massiliense Strain 541308T, sequence accession CAJD00000000 [00000000 Corynebacterium bovis DSM 20582, sequence accession AENJ00000000 [00000000 Corynebacterium diphtheriae Biovar Intermedius NCTC 5011, sequence accession AJVH00000000 [00000000 Corynebacterium pseudotuberculosis strain 1/06-A, sequence accession CP003082 [CP003082 Corynebacterium pseudotuberculosis strain 3/99-5 sequence accession CP003152.1 [003152.1 Corynebacterium pseudotuberculosis strain 42/02-A, sequence accession CP003062 [CP003062 Microbacterium yannicii, sequence accession CAJF01000001 through CAJF01000067 [01000067 Micromonospora lupini Lupac 08, sequence accession CAIE01000001 [01000001 Mycobacterium bolletii Strain M24, sequence accession AJLY00000000 [00000000 Mycobacterium intracellulare Clinical Strain MOTT-36Y, sequence accession CP003491 [CP003491 Mycobacterium massiliense M18, sequence accession AJSC00000000 [00000000 Mycobacterium massiliense strain GO 06, sequence accession CP003699 [CP003699 Mycobacterium massiliense strain M154, sequence accession AJMA00000000 [00000000 Mycobacterium tuberculosis RGTB327, sequence accession CP003233 [CP003233 Mycobacterium tuberculosis MTB423, sequence accession CP003234 [CP003234 Parascardovia denticolens IPLA 20019, sequence accession AKII00000000 [00000000 Saccharothrix espanaensis DSM 44229T, sequence accession HE804045 [HE804045 Streptomyces auratus Strain AGR0001, sequence accession AJGV00000000 [00000000 Streptomyces cattleya\u201d DSM46488T, sequence accession FQ859185 and FQ859184 [\u201cFQ859184 Streptomyces globisporus C-1027, sequence accession AJUO00000000 [00000000 Streptococcus mutans GS-5, sequence accession CP003686 [CP003686 Streptomyces sp. Strain AA1529, sequence accession ALAP00000000 [00000000 Streptomyces sulphureus L180, sequence accession AJTQ0000000 [Q0000000 Borrelia crocidurae, sequence accession CP003426 (chromosome), CP003427 to CP003465 (plasmids) [lasmids) Treponema sp. Strain JC4, sequence accession JQ783348 [JQ783348 Flavobacterium sp. Strain F52, sequence accession AKZQ00000000 [00000000 Fusobacterium nucleatum subsp. fusiforme ATCC 51190T, sequence accession AKXI00000000 [00000000 Imtechella halotolerans\u201d\u201c K1T, sequence accession AJJU00000000 [00000000 Actinophage PIS136, sequence accession JX006077 Aeromonas hydrophila Phage CC2, sequence accession JX123262 [JX123262 Bacteriophage BC-611, sequence accession AB712291 Bacteriophage SSU5, sequence accession JQ965645 Blattabacterium sp. strain BGIGA, sequence accession [ccession Caulobacter crescentus Bacteriophage \u03c6CbK, sequence accession JX163858 [JX163858 Celeribacter Bacteriophage P12053L, sequence accession JQ809650 [JQ809650 Croceibacter Bacteriophage P2559S, sequence accession JQ867099 [JQ867099 Cronobacter sakazakii Temperate Bacteriophage phiES15 JQ780327 [JQ780327 Marinomonas Bacteriophage P12026, sequence accession JQ867100 [JQ867100 Pectobacterium carotovorum subsp. carotovorum Bacteriophage PP1, sequence accession JQ837901 [JQ837901 Persicivirga bacteriophages P12024L, sequence accession JQ823123 [JQ823123 Persicivirga bacteriophages P12024S, sequence accession JQ823122 [JQ823122 phage clP1, sequence accession JN051154 Pseudomonas aeruginosa Siphophage MP1412, sequence accession JX131330 [JX131330 Pseudomonas aeruginosa Temperate Phage MP29, sequence accession EU272036 [EU272036 Pseudomonas aeruginosa Temperate Phage MP42, sequence accession JQ762257 [JQ762257 Pseudomonas Phage \u03a6-S1, sequence accession JX173487 [JX173487 Siphophage MP1412, sequence accession JX131330 Staphylococcus aureus Bacteriophage GH15, sequence accession JQ686190 [JQ686190 Vibrio vulnificus Bacteriophage SSP002, sequence accession JQ692107 [JQ692107 African bovine rotaviruses RVA/Cow-wt/ZAF/1603/2007/G6P, sequence accession S9(VP7) JN831209, S4(VP4) JN831210, S6(VP6) JN831211, S1(VP1) JN831212, S2(VP2) JN831213, S3(VP3) JN831214, S5(NSP1) JN831204, S8(NSP2) JN831205, S7(NSP3) JN831206, S10(NSP4) JN831207, S11(NSP5) JN831208 African bovine rotaviruses RVA/Cow-wt/ZAF/1604/2007/G8P, sequence accession S9(VP7) JN831220, S4(VP4) JN831221, S6(VP6) JN831222, S1(VP1) JN831223, S2(VP2) JN831224, S3(VP3) JN831225, S5(NSP1) JN831215, S8(NSP2) JN831216, S7(NSP3) JN831217, S10(NSP4) JN831218, S11(NSP5) JN831219 African bovine rotaviruses RVA/Cow-wt/ZAF/1605/2007/G6P, sequence accession S9(VP7) JN831231, S4(VP4) JN831232, S6(VP6) JN831233, S1(VP1) JN831234, S2(VP2) JN831235, S3(VP3) JN831236, S5(NSP1) JN831226, S8(NSP2) JN831227, S7(NSP3) JN831228, S10(NSP4) JN831229, S11(NSP5) JN831230 Avian Leukosis Virus, sequence accession JX254901 Avian Influenza Virus H3N2, sequence accession JX175250 through JX175257 Avian Influenza Virus H5N2, sequence accession JQ990145 through JQ990152 Avian-Like H4N8 Swine Influenza, sequence accession JX151007 through JX151014 Avian Paramyxovirus, sequence accession JQ886184 Avian Tembusu-Related Virus Strain WR, sequence accession JX196334 Bluetongue Virus Serotype 9, sequence accession JX003687 to JX003696 Bluetongue Virus Serotype 16, sequence accession Bombyx mori Nucleopolyhedrovirus, sequence accession JQ991009 Bovine Viral Diarrhea Virus 2, sequence accession JF714967 Bovine Foamy Viruses, sequence accession JX307861 Canine Noroviruses, sequence accession FJ692500 and FJ692501 Chicken Anemia Virus, sequence accession JX260426 Chikungunya Virus, sequence accession JX088705 Chinese Virulent Avian Coronavirus GX-YL5, sequence accession HQ848267 Chinese Virulent Avian Coronavirus GX-YL9, sequence accession HQ850618 Coxsackievirus B4, sequence accession JX308222 Enterovirus C (HEV-C117), sequence accession JX262382 Genotype 4 Hepatitis E Virus Strain, sequence accession JQ993308 H10N8 Avian Influenza Virus, sequence accession JQ924786 to JQ924793 H9N2 Subtype Influenza Virus FJG9, sequence accession JF715008.1, JN869514.1 through JN869520.1 .Herpes Simplex Virus 1 Strain McKrae, sequence accession JX142173 Human Coronavirus NL63, sequence accession JX104161 Human G10P Rotavirus, sequence accession AB714258 through AB714268 Ikoma Lyssavirus, sequence accession JX193798 Korean sacbrood viruses AmSBV-Kor19, sequence accession JQ390592 Korean sacbrood viruses AmSBV-Kor21, sequence accession JQ390591 , sequence accession JN835456 [Mitochondrion of Frankliniella occidentalisJN835456 New Circular DNA Virus from Grapevine, sequence accession JQ901105 Novel Porcine Epidemic Diarrhea Virus, sequence accession JX112709 Pararetrovirus, sequence accession JQ926983 Parechovirus, sequence accession JX050181 Peste des Petits Ruminants Virus, sequence accession JX217850 Polyomavirus, sequence accession JQ412134 Porcine Circovirus 2b Strain CC1, sequence accession JQ955679 Porcine circovirus type 2 (PCV2), sequence accession JX294717 Porcine Epidemic Diarrhea Virus Strain AJ1102, sequence accession JX188454 Porcine Sapelovirus Strain YC2011, sequence accession JX286666 [Respiratory Syndrome Virus Strain QY2010, sequence accession JQ743666 SAT 2 Foot-and-Mouth Disease Virus, sequence accession JX014255 SAT 2 Foot-and-Mouth Disease Virus PAT, sequence accession JX014256 Street Rabies Virus, sequence accession HQ450386 Waterfowl aviadenovirus goose adenovirus 4, sequence accession JF510462 , sequence accession NC_015104 [cpDNA of Smilax chinaC_015104 , sequence accession JQ310743 [Elodea canadensisJQ310743 Ogura-type mitochondrial genome, sequence accession AB694743 Aspergillus oryzae Strain 3.042, sequence accession AKHY00000000 [00000000 Rhodosporidium toruloides MTCC 457, sequence accession AJMJ00000000 [00000000 Helicoverpa armigera, sequence accession HQ613271 [HQ613271 plasmidIncN plasmid pRSB201, sequence accession JN102341 plasmidIncN plasmid pRSB203, sequence accession JN102342 plasmidIncN plasmid pRSB205, sequence accession JN102343 plasmidIncN plasmid pRSB206, sequence accession JN102344"} +{"text": "The asymmetric unit comprises one half-mol\u00adecule, the structural dimeric unit being generated by inversion symmetry with an Mg\u22efMg distance of 3.469\u2005(2)\u2005\u00c5. The Mg(II) atom exhibits a distorted tetrahedral coordination geometry. The crystal packing is defined by van der Waals inter\u00adactions only.The title benzyl Grignard reagent, [Mg DOI: 10.1107/S1600536812025445/kp2423Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "The gold(I) atoms are 3.4873\u2005(7)\u2005\u00c5 apart. The mol\u00adecule exhibits a crystallographic twofold rotation axis.The title compound, [Au DOI: 10.1107/S1600536810050506/br2152Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The diphosphadiborane mol\u00adecule consists of an ideal planar four-membered B2P2 ring with an additional phenyl and a \u2013PPh2 group attached to each B atom.In the title compound, C DOI: 10.1107/S1600536812011361/yk2048Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "DOI: Click here for additional data file.10.1107/S1600536812045461/sj5274Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "The absolute configuration at the stereogenic S-atom center was determined as S. The crystal structure is stabilized by inter\u00admolecular C\u2014H\u22efO contacts.In the title compound, C DOI: 10.1107/S1600536811041420/bt5660Isup2.hkl Structure factors: contains datablock(s) I. DOI: 10.1107/S1600536811041420/bt5660Isup3.cdx Supplementary material file. DOI: 10.1107/S1600536811041420/bt5660Isup4.cml Supplementary material file. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the crystal, pairs of bifurcated O\u2014H\u22ef hydrogen bonds link adjacent mol\u00adecules, forming centrosymmetric dimers. The acetonitrile solvent mol\u00adecule shows 0.5 occupancy.In the title complex, [Co(C DOI: 10.1107/S160053681105330X/xu5401Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "An iodide ligand completes the coordination sphere, with the seven-coordinate BiIII atom adopting a highly distorted monocapped octa\u00adhedral geometry.The title compound, [Bi(C DOI: 10.1107/S1600536813033515/sj5375Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supporting information:"} +{"text": "DOI: 10.1107/S1600536811013274/bt5511Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The CuII atom is four-coordinated by the phenolate O atoms and imine N atoms from two Schiff base ligands, in a highly distorted square-planar geometry. The O- and N-donor atoms are mutually trans and the dihedral angle between the two benzene rings is 55.8\u2005(3)\u00b0.The title compound, [Cu(C DOI: 10.1107/S1600536810025481/sj5030Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The asymetric unit consists of an [Fe(phen)3]2+ cation, two [Cr(phen)(NCS)4]\u2212 anions, three acetonitrile solvent mol\u00adecules and a water mol\u00adecule. The Fe and Cr atoms both show a slightly distorted octa\u00adhedral FeN6 and CrN6 coordination geometry with adjacent angles in the range 79.67\u2005(12)\u201395.21\u2005(12)\u00b0. No classical hydrogen bonding involving the water molecule is observed.Single crystals of the title heterometallic compound, [Fe(C DOI: 10.1107/S1600536812012949/ru2031Isup2.cdxSupplementary material file. DOI: 10.1107/S1600536812012949/ru2031Isup3.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "Extensive O\u2014H\u22efO hydrogen-bonding inter\u00adactions between the cations, anions and water mol\u00adecules stabilize the three-dimensional network.In the title compound, [Mn(H DOI: 10.1107/S1600536810022300/hy2318Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The Br aroms are trans-disposed as a result of symmetry.The title complex, [RuBr DOI: Click here for additional data file.10.1107/S1600536813000871/hy2613Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "The ligands are coordinated in a chelate fashion, forming three five-membered rings. In the crystal, translationally related complex molecules are organized into columns along [001] via C\u2014H\u22efO hydrogen bonds.In the title compound, [Co(C DOI: 10.1107/S1600536811043303/hy2479Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The crystal packing is characterized by a C\u2014H\u22efO hydrogen-bonded ribbon structure approximately parallel to [10In the title compound, [Zn(C DOI: 10.1107/S1600536811019854/ng5159Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the crystal, the 3-methyl\u00adanilinium cations link with the complex antimonate anions and Cl\u2212 anions via N\u2014H\u22efCl hydrogen bonds.In the title compound, (C DOI: 10.1107/S1600536811049087/xu5391Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The crystal studied was a non-merohedral twin with the minor domain being in a 15.8\u2005(1)% proportion.The Sn(IV) atom in the title compound, [Sn(C DOI: 10.1107/S1600536810048300/hg2755Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Each purinate ligand chelates two ZnII cations through two imidazole N atoms of the purinate anion ligand, leading to the formation of a three-dimensional network.In the title compound, [Zn(C DOI: 10.1107/S1600536812011245/vn2034Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "The mol\u00adecule contains two fused six-membered rings, which both display a chair conformation. In the crystal, mol\u00adecules are linked into chains propagating along the b axis by inter\u00admolecular O\u2014H\u22efO hydrogen bonds.The title compound, C DOI: 10.1107/S1600536811004454/fj2393Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Each 5-amino\u00adisophthalate anion acts as a \u03bc3-bridge linking symmetry-related ZnII ions into a layered polymeric structure parallel to (100). The asymmetric unit also comprises a disordered crystal water molecule located on an inversion centre with 0.25 occupancy. In the crystal, N\u2014H\u22efO hydrogen bonds form a three-dimensional network.In the title coordination polymer, {[Zn(C DOI: 10.1107/S1600536811050045/kp2352Isup2.hkl Structure factors: contains datablock(s) I. DOI: 10.1107/S1600536811050045/kp2352Isup3.cdx Supplementary material file. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the crystal, N\u2014H\u22efO hydrogen bonds link the mol\u00adecules into chains running along the b-axis direction.In the mol\u00adecule of the title compound, C DOI: 10.1107/S160053681302401X/ff2117Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S160053681302401X/ff2117Isup3.cmlSupplementary material file. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "Each pair of double-stacked dications is surrounded by a ring of ten nitrate anions. An intricate three-dimensional N---H...O and N---H... hydrogen-bonding network exists in the crystal structure.The crystal structure of the title compound, C DOI: 10.1107/S1600536811042929/lr2031Isup2.hkl Structure factors: contains datablock(s) I. DOI: 10.1107/S1600536811042929/lr2031Isup3.mol Supplementary material file. DOI: 10.1107/S1600536811042929/lr2031Isup4.cml Supplementary material file. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "A three-dimensional supra\u00admolecular network is built from the uncoordinated nitrate anion, the water mol\u00adecule and the cation through O\u2014H\u22efO hydrogen bonds.In the title compound, [Ag(C DOI: 10.1107/S1600536810020945/ng2780Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The presence of a PF6 \u2212 anion promotes the existence of an extensive network of weak C\u2014H\u22efX inter\u00adactions.The title compound, [RuBr(C DOI: 10.1107/S1600536811002662/cv5041Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "This compound is isostructural with KCuFe(PO4)2 [Badri et al. iron(III) bis\u00ad(phosphate), RbCuFe(PO al. 2011, J. Soli DOI: 10.1107/S1600536813019569/br2226Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "In the crystal, O(water)\u2014H\u22efO and C\u2014H\u22efO hydrogen bonds form a three-dimensional-network.In the title compound, [Cu(C DOI: Click here for additional data file.10.1107/S1600536812044625/qm2087Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "The O atom of the nitrate anion occupies the apical site. The crystal structure features intra\u00admolecular N\u2014H\u22efO hydrogen bonds.Copper nitrate in methanol solution cleaves the N\u2014C DOI: 10.1107/S160053681201241X/xu5488Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "Each arsine-substituted Os atom bears one equatorial and two axial terminal carbonyl ligands, whereas the unsubstituted Os atom bears two equatorial and two axial terminal carbonyl ligands. The dihedral angles between the two benzene rings in the diphenyl\u00adarsino groups are 67.42\u2005(16) and 61.99\u2005(16)\u00b0. In the crystal, mol\u00adecules are linked via C\u2014H\u22efO hydrogen bonds into zigzag chains propagating along [010].The title compound, [Os DOI: 10.1107/S1600536812024208/hb6822Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "Classical O\u2014H\u22efO hydrogen bonds link both methanol solvent mol\u00adecules with the nitrate anion.In the title compound, [Co(C DOI: 10.1107/S1600536810032241/si2285Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The metal complex lies on a fourfold rotation axis that passes through the Os, N, O and Cl atoms. The NO and Cl ligands are disordered in an 0.511\u2005(12):0.486\u2005(12) ratio.The title compound, [OsCl(NO)(C DOI: 10.1107/S1600536811001401/ng5100Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The 4,4-bi\u00adpyridine and Cl\u2212 anions bridge the AgI cations, forming polymeric chains running along [21-1]. In the crystal, weak C\u2014H\u22efCl inter\u00adactions link the polymeric chains into a three-dimensiona supra\u00admolecular architecture.In the title coordination polymer, [Ag DOI: Click here for additional data file.10.1107/S160053681301413X/xu5704Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "We confirm the previous studies, but to modern standards of precision and with all H atoms located. The central Ni atom (site symmetry A preliminary X-ray study of the title mol\u00adecular salt, [Ni(CH DOI: 10.1107/S1600536810026668/hb5527Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "There are no close C\u2014H\u22efCl contacts.The two mol\u00adecules in the asymmetric unit of the title compound, C DOI: 10.1107/S1600536812014924/bt5833Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S1600536812014924/bt5833Isup3.cmlSupplementary material file. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "A weak intra\u00admolecular C\u2014H\u22efS inter\u00adaction generates an S(6) ring. No inter\u00admolecular hydrogen bonds are observed in the crystal structure.The mol\u00adecule of the title compound, C DOI: 10.1107/S1600536812035003/hb6910Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S1600536812035003/hb6910Isup3.cmlSupplementary material file. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536811040402/bg2419Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "An intra\u00admolecular Bi\u22efN nonbonding inter\u00adaction is observed in the distorted trigonal triaryl\u00adbis\u00admuth coordination of the title compound. The title compound, [Bi(C DOI: 10.1107/S1600536810043655/si2298Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "AbstractXylographus Melli\u00e9 based on type specimens deposited in the Museum of Comparative Zoology (USA), Museum f\u00fcr Naturkunde Berlin (Germany), the Natural History Museum (UK), Mus\u00e9um d\u2019Histoire Naturelle de la Ville de Gen\u00e8ve (Switzerland), Mus\u00e9um National d\u2019Histoire Naturelle (France), Naturhistoriska Riksmuseet (Sweden) and Naturhistorisches Museum Wien (Austria). We designate lectotypes for the following species: Cis fultoni Broun, 1886, Xylographus anthracinus Melli\u00e9, 1849, X. bicolor Pic, 1916, X. brasiliensis Pic, 1916, X. ceylonicus Ancey, 1876, X. contractus Melli\u00e9, 1849, X. corpulentus Melli\u00e9, 1849, X. dentatus Pic, 1922, X. gibbus Melli\u00e9, 1849, X. hypocritus Melli\u00e9, 1849, X. javanus Pic, 1937, X. lemoulti Pic, 1916, X. longicollis Pic, 1922, X. madagascariensis Melli\u00e9, 1849, X. nitidissimus Pic, 1916, X. perforatus Gerstaecker, 1871, X. porcus Gorham, 1886, X. punctatus Melli\u00e9, 1849, X. ritsemai Pic, 1921, X. rufescens Pic, 1921, X. rufipennis Pic, 1934, X. rufipes Pic, 1930, X. seychellensis Scott, 1926, X. subopacus Pic, 1929, X. subsinuatus Pic, 1916, X. suillus Gorham, 1886, X. testaceitarsis Pic, 1916 and X. tomicoides Reitter, 1902. We propose the following syn. n. (senior synonym listed first): X. anthracinus = X. testaceitarsis, X. brasiliensis = X. lucasi Lopes-Andrade & Zacaro, X. corpulentus = X. lemoulti and X. richardi Melli\u00e9, X. madagascariensis = X. eichelbaumi Reitter, X. rufipennis, X. seychellensis Scott and X. tarsalis F\u00e5hraeus, X. nitidissimus = X. longicollis, X. subsinuatus = X. rufescens. We exclude three species from Xylographus: Cis renominatus, nom. n. , Paratrichapus fultoni , comb. n. and P. javanus , comb. n.We designate lectotypes and propose nomenclatural changes in Xylographus Melli\u00e9 is a genus of minute tree-fungus beetles with 36 described species, occurring in most continental and insular lands of tropical and subtropical regions :We examined 195 type specimens of Museum of Comparative Zoology, Harvard University MCZ Museum f\u00fcr Naturkunde Berlin MFNB Mus\u00e9um d\u2019histoire naturelle de la ville de Gen\u00e8ve MHNG Mus\u00e9um National d\u2019Histoire Naturelle MNHN The Natural History Museum NHM Naturhistorisches Museum Wien NHMW Naturhistoriska Riksmuseet NHRSXylographus cited by Cis, and the original description of Paratrichapus by Paratrichapus was described as having a 3-3-3 tarsal formula, but after studying its type material and images of microscope slide preparations by Hugh Scott, we observed that it was certainly 4-4-4 as in all other ciids. Xylographus and Paratrichapus are morphologically similar, so we propose the characteristics stated on We used the generic features of Xylographus bostrichoides and Xylographus richardi. And we did not have access to type material of Xylographus scheerpeltzi. In the case of Xylographus bostrichoides, we had at hand several named historical specimens, including those used for its redescription by Xylographus richardi, we had only a named specimen for examination. The description of Xylographus scheerpeltzi is adequately detailed and includes information on the morphology of sclerites of male abdominal terminalia. In all other cases, we had access to the original type series and dissected male abdominal terminalia whenever necessary and possible. The morphology of sclerites of male abdominal terminalia of Ciidae is stable intraspecifically and distinctly varies interspecifically, even between closely related species , subtle variations of male secondary sexual characteristics or based only on females. A single author, Maurice Pic, was responsible for half of the names here recognized as junior synonyms. He is known for having proposed thousands of new names of beetles based mostly on anecdotal descriptions and small type-series. Lack of access to type material was also a great problem. Xylographus seychellensis stating that he did it with some hesitation, because he has not examined possible conspecifics, as Xylographus madagascariensis and Xylographus eichelbaumi, the senior and a junior synonym proposed here, respectively. The same was true to Xylographus lucasi, whose authors separates individual labels. Data in square brackets were added for clarification. Remarks are provided for some species.A complete list of Xylographus anthracinus Melli\u00e9, 1849Xylographus testaceitarsis Pic, 1916, syn. n.PageBreakXylographus bicolor Pic, 1916Xylographus bostrichoides Cis cribatus Lucas, 1849Xylographus bostrichoides var. aubei Melli\u00e9, 1849Xylographus brasiliensis Pic, 1916Xylographus lucasi Lopes-Andrade & Zacaro, 2003, syn. n.Xylographus bynoei Blair, 1940Xylographus ceylonicus Ancey, 1876Xylographus contractus Melli\u00e9, 1849Xylographus corpulentus Melli\u00e9, 1849Xylographus lemoulti Pic, 1916, syn. n.Xylographus richardi Melli\u00e9, 1849, syn. n.Xylographus gibbus Melli\u00e9, 1849Xylographus globipennis Reitter, 1911Xylographus hypocritus Melli\u00e9, 1849Xylographus madagascariensis Melli\u00e9, 1849Xylographus eichelbaumi Reitter, 1908, syn. n.Xylographus rufipennis Pic, 1934, syn. n.Xylographus seychellensis Scott, 1926, syn. n.Xylographus tarsalis F\u00e5hraeus, 1871, syn. n.Xylographus nitidissimus Pic, 1916Xylographus longicollis Pic, 1922, syn. n.Xylographus perforatus Gerstaecker, 1871Xylographus porcus Gorham, 1886Xylographus punctatus Melli\u00e9, 1849Xylographus ritsemai Pic, 1921Xylographus rufipes Pic, 1930Xylographus scheerpeltzi Nobuchi & Wada, 1956Xylographus subopacus Pic, 1929Xylographus subsinuatus Pic, 1916Xylographus rufescens Pic, 1921, syn. n.Xylographus suillus Gorham, 1886Xylographus tomicoides Reitter, 1902Cis renominatus, nom. n.Xylographus dentatus Pic, 1922, not Cis dentatus Melli\u00e9, 1849.Paratrichapus fultoni , comb. n.Cis fultoni Broun, 1886Paratrichapus javanus , comb. n.Xylographus javanus Pic, 1937PageBreakMelli\u00e9, 1849http://species-id.net/wiki/Xylographus_anthracinusXylographus anthracinusXylographus testaceitarsissyn. n. Type-locality: Mahatsinjo, Madagascar. Pic 1916: 13., Anthracinus Dup. Madagascar.[handwritten] \\ Ex-Mus\u00e6o Mniszech [printed] \\ [red label] LECTOTYPE Xylographus anthracinus Melli\u00e9 [handwritten]\u201d; 2 female paralectotypes (MNHN), labeled: \u201canthracinus (ex coll. Chev.) [handwritten] \\ Melli\u00e9 vidit [handwritten] \\ [yellow label] PARALECTOTYPE Xylographus anthracinus Melli\u00e9 [handwritten]\u201d; 2 male paralectotypes (MHNG), labeled: \u201cColl. Melly [printed] \\ [yellow label] PARALECTOTYPE Xylographus anthracinus Melli\u00e9 [handwritten]\u201d.MADAGASCAR: male lectotype (MNHN), here designated, labeled: \u201cXylographus testaceitarsis Pic 1916, here designated, labeled: \u201cMAHATSINJO pr\u00e8s Tananarive [printed] \\ Type [handwritten] \\ testaceitarsis Pic [handwritten] \\ [red label] LECTOTYPE Xylographus testaceitarsis Pic [handwritten]\u201d; 1 male and 3 female paralectotypes (MNHN), labeled: \u201cMAHATSINJO pr\u00e8s Tananarive [printed] \\ [yellow label] PARALECTOTYPE Xylographus testaceitarsis Pic [handwritten]\u201d.MADAGASCAR: male lectotype (MNHN) of Xylographus anthracinus and the lectotype of Xylographus testaceitarsis. They are males of about the same size and with secondary sexual characteristic similarly developed. We have also dissected and compared sclerites of their abdominal terminalia and noted no difference.There is no morphological difference between the lectotype of Pic, 1916http://species-id.net/wiki/Xylographus_bicolorXylographus bicolor Pic 1916: 13. Type-locality: Mahatsinjo, Madagascar.bicolor Pic [handwritten] \\ [red label] LECTOTYPE Xylographus bicolor Pic [handwritten]\u201d.MADAGASCAR: male lectotype (MNHN), here designated, labeled: \u201cMAHATSINJO pr\u00e8s Tananarive [printed] \\ Type [handwritten] \\ http://species-id.net/wiki/Xylographus_bostrichoidesCis bostrichoides : 93. TypCis cribatusXylographus bostrichoidesaubei var. PageBreakaubei, and dozens of specimens that do fit the currently accepted species limits. Xylographus bostrichoides. However, after studying this specimen, we determined it is a member of Scolytinae (Curculionidae) and fits neither the original description by Unfortunately we did not find the type material of Dufour in the MNHN. We have found only specimens used by Pic, 1916http://species-id.net/wiki/Xylographus_brasiliensisXylographus brasiliensis Pic 1916: 13. Type-locality: Rio Verde, Brazil.Xylographus lucasisyn. n. Type-locality: Venda Nova do Imigrante, Esp\u00edrito Santo, Brazil.Xylographus [handwritten] \\ Type [handwritten] \\ Brasiliensis Pic [handwritten] \\ [red label] LECTOTYPE Xylographus brasiliensis Pic [handwritten]\u201d.BRASIL: female lectotype (MNHN), here designated, labeled: \u201cBresil. Goyaz. Rio Verde [printed] \\ See Xylographus lucasi, the authors did not have access to type specimens of Xylographus brasiliensis and stated that its description was vague (Lopes-Andrade and Lawrence 2003). After we examined the available type of Xylographus brasiliensis, a female located in the MNHN, we observed there is no difference between it and female paratypes of Xylographus lucasi. We have located in the MNHN a male specimen collected in \u201cGoyaz\u201d (which may correspond to the current state of Goi\u00e1s or to Tocantins), a historical specimen but not from the original type series of Xylographus brasiliensis. We dissected it and compared the sclerites of abdominal terminalia to those of male paratypes of Xylographus lucasi, and they are exactly the same. The species is widespread in the tropical South America and the type localities of both names are within its known range (pers. obs.).In the description of Blair, 1940http://species-id.net/wiki/Xylographus_bynoeiXylographus bynoeiXylographus bynoei Blair Type det. K.G. Blair 1939 [handwritten]\u201d; 2 male and 2 female paratypes (NHM), labeled: \u201c[yellow disc] Paratype [printed] \\ N.W. Australia pres. By B. Bynoe, R.N. Surgeon on H.M.S. Beagle. See Stokes, Voyage of Discoveries. 1846 [handwritten]\u201d; 4 female paralectotypes (NHM), labeled: \u201cAustralia 44.4 [handwritten] \\ [yellow disc] Paratype [printed]\u201d.AUSTRALIA: male holoype (NHM), labeled: \u201c[faded blue disc] N. Holl. [above] 44.4 [below] [handwritten] \\ [red disc] Holotype [printed] \\ PageBreakAncey, 1876http://species-id.net/wiki/Xylographus_ceylonicusXylographus ceylonicusXylographus ceylonicus Ancey, n. sp. Ceylan Types [handwritten] \\ [red label] LECTOTYPE Xylographus Ceylonicus Ancey [handwritten]\u201d; 6 males, 1 female and 7 specimens of undetermined gender, all paralectotypes (MNHN), labeled: \u201cXylographus Ceylonicus Ancey, n. sp. Ceylan Types [handwritten] \\ [yellow label] PARALECTOTYPE Xylographus ceylonicus Ancey [handwritten]\u201d; 3 male and 1 female paralectotypes (MNHN), labeled: \u201cCEYLAN [printed] \\ type [handwritten] \\ Syntypes [handwritten] \\ Ceylan Pointe de Galles [handwritten] \\ Xylographus ceylonicus Ancey [handwritten] \\ [yellow label] PARALECTOTYPE Xylographus ceylonicus Ancey [handwritten]\u201d; 7 paralectotypes of undetermined gender (MNHN), labeled: \u201cXylographus Ceylonicus Ancey Ceylan [handwritten] \\ Ex. Coll. REITTER [printed] \\ [yellow label] PARALECTOTYPE Xylographus ceylonicus Ancey [handwritten]\u201d; 2 paralectotypes of undetermined gender (MFNB), labeled: \u201cXylographus Ceylonicus Ancey Ceylon Ancey Type [handwritten] \\ Coll. L.W. Schaufuss [printed] \\ [red label] ? SYNTYPUS Xylographus ceylonicusXylographus ceylonicus Ancey [handwritten]\u201d; 2 paralectotypes of undetermined gender (MFNB), labeled: \u201cXylographus Ceylonicus Ancey Ceylan [handwritten] \\ [red label] ? SYNTYPUS Xylographus ceylonicusXylographus ceylonicus Ancey [handwritten]\u201d; 2 paralectotypes of undetermined gender (MFNB), labeled: \u201cXylographus Ceylonicus Ancey Ceylan [handwritten] \\ ex Coll. Hiller [handwritten] \\ [red label] ? SYNTYPUS Xylographus ceylonicusXylographus ceylonicus Ancey [handwritten]\u201d.SRI LANKA: male lectotype (MNHN), here designated, labeled: \u201cMelli\u00e9, 1849http://species-id.net/wiki/Xylographus_contractusXylographus contractusXylographus Contractus Bresil Lap. Cast. 72 [handwritten] \\ [red label] LECTOTYPE Xylographus contractus Melli\u00e9 [handwritten]\u201d; 1 female paralectotype (MNHN), labeled: \u201c[green disc] Xylographus contractus Bresil [unreadable] 83 [handwritten] \\ [yellow label] PARALECTOTYPE Xylographus contractus Melli\u00e9 [handwritten]\u201d.BRASIL: female lectotype (MNHN), here designated, labeled: \u201c[green disc] PageBreakMelli\u00e9, 1849http://species-id.net/wiki/Xylographus_corpulentusXylographus corpulentusXylographus lemoultisyn. n. Type-locality: St-Laurent du Maroni, French Guiana. Pic 1916: 4. Xylographus richardisyn. n. Type-locality: Cayenne, French Guiana.Xylographus corpulentus Melli\u00e9 [handwritten]\u201d; 1 female paralectotype (MNHN), labeled: \u201c[green label] \u2640 [handwritten] \\ Melli\u00e9 vidit [handwritten] \\ [yellow label] PARALECTOTYPE Xylographus corpulentus Melli\u00e9 [handwritten]\u201d; 1 female paralectotype (MNHM), labeled: \u201c[green label] \u2640 [handwritten] \\ Melli\u00e9 vidit [handwritten] \\ Corpulentus Mell. (Coll. Chevrolat) [handwritten] \\ [yellow label] PARALECTOTYPE Xylographus corpulentus Melli\u00e9 [handwritten]\u201d; 1 male and 2 female paralectotypes (MNHN), labeled: \u201c[green disc] Xylographus Corpulentus Perou Lap. Cast. 72 [handwritten] \\ [yellow label] PARALECTOTYPE Xylographus corpulentus Melli\u00e9 [handwritten]\u201d; 1 male paralectotype (MNHN), labeled: \u201c[green disc] Xylographus Corpulentus Kunze Perou T. Cast. 83 [handwritten] \\ [yellow label] PARALECTOTYPE Xylographus corpulentus Melli\u00e9 [handwritten]\u201d; 1 male and 2 female paralectotypes (MNHN), labeled: \u201c[green label] Kunze 19 [handwritten] \\ [green label] Cis Corpulentus Kunze Perou [handwritten] \\ [yellow label] PARALECTOTYPE Xylographus corpulentus Melli\u00e9 [handwritten]\u201d.PERU: male lectotype (MNHN), here designated, labeled: \u201c[green label] \u2642 [handwritten] \\ Melli\u00e9 vidit [handwritten] \\ [red label] LECTOTYPE PageBreakXylographus lemoulti Pic, 1916, here designated, labeled: \u201cNOVEMBRE [printed] \\ [green label] GUYANE FRAN\u00c7AISE St-LAURENT du MARONI [printed] \\ [green label] COLL LE MOULT [printed] \\ Type [handwritten] \\ [red label] LECTOTYPE Xylographus lemoulti Pic [handwritten]\u201d; 3 male and 2 female paralectotypes (MNHN), labeled: \u201cNOVEMBRE [printed] \\ [green label] GUYANE FRAN\u00c7AISE St-LAURENT du MARONI [printed] \\ [green label] COLL LE MOULT [printed] \\ [yellow label] PARALECTOTYPE Xylographus lemoulti Pic [handwritten]\u201d; 2 male and 1 female paralectotypes (MNHN), labeled: \u201cNOVEMBRE [printed] \\ GUYANE FRAN\u00c7AISE St-LAURENT du MARONI [printed] \\ COLL LE MOULT [printed] \\ [yellow label] PARALECTOTYPE Xylographus lemoulti Pic [handwritten]\u201d; 1 male paralectotype (MNHN), labeled: \u201cNOVEMBRE [printed] \\ GUYANE FRAN\u00c7AISE St-LAURENT du MARONI [printed] \\ COLL LE MOULT [printed] \\ [yellow label] PARALECTOTYPE Xylographus lemoulti Pic [handwritten]\u201d; 3 male and 4 female paralectotypes (MNHN), labeled: \u201cOCTOBRE [printed] \\ GUYANE FRAN\u00c7AISE St-LAURENT du MARONI [printed] \\ COLL LE MOULT [printed] \\ [yellow label] PARALECTOTYPE Xylographus lemoulti Pic [handwritten]\u201d; 1 male and 3 female paralectotypes (MNHN), labeled: \u201cJUIN [printed] \\ [green label] GUYANE FRAN\u00c7AISE St-LAURENT du MARONI [printed] \\ [green label] COLL LE MOULT [printed] \\ [yellow label] PARALECTOTYPE Xylographus lemoulti Pic [handwritten]\u201d; 1 male paralectotype (MNHN), labeled: \u201cMAI [printed] \\ GUYANE FRAN\u00c7AISE St-LAURENT du MARONI [printed] \\ COLL LE MOULT [printed] \\ [yellow label] PARALECTOTYPE Xylographus lemoulti Pic [handwritten]\u201d.FRENCH GUIANA: male lectotype (MNHN) of Xylographus richardi in the Chevrolat collection of MNHN. We located a female specimen from Colombia in the Melly collection of MHNG named as Xylographus richardi. Xylographus corpulentus and Xylographus lemoulti and they are exactly the same. Xylographus corpulentus and Xylographus richardi, stating that they resemble each other \u201cpour la taille et la forme\u201d, with Xylographus richardi being more punctate. We believe the description of Xylographus richardi was based on a female specimen, because the pronotal surface between punctures is described as being finely rugose. We have observed that it is common in female Xylographus species to have pronotal surface distinctly more rugose than that of males. The type of Xylographus corpulentus was described as being black, while the one of Xylographus richardi was described as reddish. It is a common variation found in Xylographus corpulentus, in which teneral adults may be reddish (pers. obs.). Xylographus lemoulti differs from Xylographus richardi in the coloration and pronotal shape, again a consequence of the fact that the description of Xylographus richardi was based in a teneral adult female. The type-localities of Xylographus lemoulti and Xylographus richardi are approximately 200 Km apart and both are in the coast of French Guiana.There are several type specimens of species described by Melli\u00e9 deposited in historical collections of the MHNG and the MNHN. In the MHNG, these types are in the A. Melly collection, who has a surname similar to that of J. Melli\u00e9 but shall not be confounded. We did not find type material of Melli\u00e9, 1849http://species-id.net/wiki/Xylographus_gibbusXylographus gibbusXylographus gibbus Klg. Mell. Colomb. T. Cast. 72 [handwritten] \\ [green label] Cis gibbus Klug [handwritten] \\ Xylographus gibbus Reiche \\ MUSEUM PARIS COLL. DE MARSEUL 2842-00 [printed] \\ [red label] LECTOTYPE Xylographus gibbus Melli\u00e9 [handwritten]\u201d; 1 male paralectotype (MHNG), labeled: \u201cGibbus Klug Colombie Klug Melli\u00e9 [handwritten] \\ [yellow label] PARALECTOTYPE Xylographus gibbus Melli\u00e9 [handwritten]\u201d.COLOMBIA: female lectotype (MNHN), here designated, labeled: \u201c[green disc] PageBreakReitter, 1911Xylographus globipennisSee Melli\u00e9, 1849http://species-id.net/wiki/Xylographus_hypocritusXylographus hypocritusHypocritus Dup. Madagascar. [handwritten] \\ Ex-Mus\u00e6o Mniszech [printed] \\ [red label] LECTOTYPE Xylographus hypocritus Melli\u00e9 [handwritten]\u201d; 1 female paralectotype (MNHN), labeled: \u201cMadagascar [handwritten] \\ Ex-Mus\u00e6o Mniszech [printed] \\ [yellow label] PARALECTOTYPE Xylographus hypocritus Melli\u00e9 [handwritten]\u201d.MADAGASCAR: male lectotype (MNHN), here designated, labeled: \u201cMelli\u00e9, 1849http://species-id.net/wiki/Xylographus_madagascariensisXylographus madagascariensisXylographus eichelbaumisyn. n. Type-locality: Amani, Tanzania.Xylographus rufipennissyn. n. Type-locality: Gura, Kenya.Xylographus seychellensissyn. n. Type-locality: Mahe, Seychelles.Xylographus tarsalissyn. n. Type-locality: Caffraria (Eastern Cape of South Africa).Madagascariensis Dup. Madagascar.[handwritten] \\ Ex-Mus\u00e6o Mniszech [printed] \\ [red label] LECTOTYPE Xylographus madagascariensis Melli\u00e9 [handwritten]\u201d.MADAGASCAR: male lectotype (MNHN), here designated, labeled: \u201cPageBreakXylographus rufipennis Pic, 1934, here designated, labeled: \u201c[red disc]Type [printed] \\ R. E, DENT GURA R, 7500 AUG 1929 [printed] \\ Xylographus rufipennis n. sp. [handwritten] \\ Pres. By Imp. Inst. Ent B. M. 1934-42. [printed] \\ [red label] LECTOTYPE Xylographus rufipennis Pic [handwritten]\u201d; 1 female paralectotype (MNHN), labeled: \u201cR. E, DENT GURA R, 7500 AUG 1929 [printed] \\ Xylographus rufipennis n. sp [handwritten] \\ ex. British museum [handwritten] \\ [yellow label] PARALECTOTYPE Xylographus rufipennis Pic [handwritten]\u201d. SEYCHELLES: male lectotype (NHM) of Xylographus seychellensisXylographus seychellensis, Scott TYPE. \u2642. [handwritten] \\ Figured specimen [printed] (outline whole vis) [handwritten] \\ TYPE [printed] \\ [red label] LECTOTYPE Xylographus seychellensis Scott [handwritten]\u201d. SOUTH AFRICA: male lectotype (NHRS) of Xylographus tarsalisXylographus tarsalis F\u00c5HR. Det. Julio Ferrer 1995 [handwritten] \\ [green label] Riksmuseum Stockholm [printed]\u201d; 1 male and 1 female paralectotypes (NHRS), labeled: \u201cCaffraria [printed] \\ J. Wahlb. [printed] \\ [red label] Paralectotype Xylographus tarsalis F\u00c5HR. Det. Julio Ferrer 1995 [handwritten] \\ [green label] Riksmuseum Stockholm [printed]\u201d. TANZANIA: female holotype (NHMW) of Xylographus eichelbaumiFomes nigrolachatus [handwritten] \\ Amani. Deutsch Ostafr. [handwritten] \\ Xylographus eichelbaumi m. Typ. 1907. [handwritten] \\ Eichelbaumi Usambara Reitt. [handwritten] \\ [red label] HOLOTYPE Xylographus eichelbaumi Reitt. [handwritten]\u201d.KENYA: female lectotype (NHM) of Xylographus madagascariensis and that he described Xylographus seychellensis with some hesitation. If he had examined the known male type of Xylographus madagascariensis, he would have observed that it was just slightly more elongate than the specimens he had at hand, with no other differences. Such a small difference in body elongation is expected to occur in Xylographus species with broad geopraphical distribution , labeled: \u201ctype [handwritten] \\ [yellow label] PARALECTOTYPE Xylographus nitidissimus Pic [handwritten]\u201d; 4 male and 4 female paralectotypes (MNHN), labeled: \u201cSan Thom\u00e9 [handwritten] \\ [yellow label] PARALECTOTYPE Xylographus nitidissimus Pic [handwritten]\u201d.S\u00c3O TOM\u00c9 AND PR\u00cdNCIPE: male lectotype (MNHN) of Xylographus longicollis Pic, 1922, here designated, labeled: \u201c? Dahomey [handwritten] \\ Provenance ? [handwritten] \\ longicollis n. sp. [handwritten] \\ [red label] LECTOTYPE Xylographus longicollis Pic [handwritten]\u201d.BENIN: female lectotype (MNHN) of Xylographus longicollis is a female of Xylographus nitidissimus.We observed that the female lectotype of Gerstaecker, 1871http://species-id.net/wiki/Xylographus_perforatusXylographus perforatusperforatus Gerst.* Sansibar Cooke [handwritten] \\ 56743 [printed] \\ [blue label] Hist.-Coll. (Coleoptera) Nr. 56743 (1. Ex) Xylographus perforatus Gerst. Sansibar, Cooke Zool. Mus. Berlin [printed] \\ [red label] SYNTYPUS Xylographus perforatusXylographus perforatus Gerstaecker [handwritten]\u201d; 2 male and 3 female paralectotypes (MFNB), labeled: \u201c[blue label] Hist.-Coll. (Coleoptera) Nr. 56743 (1.-5. Ex) Xylographus perforatus Gerst. Sansibar, Cooke Zool. Mus. Berlin [printed] \\ [red label] SYNTYPUS Xylographus perforatusXylographus perforatus Gerstaecker [handwritten]\u201d; 1 female paralectotype (MNHN), labeled: \u201cZanzibar, C. Cooke. [printed] \\ [blue label] MUSEUM PARIS Collection L\u00e9on Fairmaire 1906 [printed] \\ Xylographus perforatus Gerst. [handwritten] P. Lesne vid. [printed] \\ [yellow label] PARALECTOTYPE Xylographus perforatus Gerstaecker [handwritten]\u201d; 1 female paralectotype (MNHN), labeled: \u201cZanzibar, C. Cooke. [printed] \\ Bragance (Para) M. de Mathan [printed] \\ Kein Scolytide [handwritten] \\ [yellow label] PARALECTOTYPE Xylographus perforatus Gerstaecker [handwritten]\u201d; 1 male and 1 female paralectotype (MNHN), labeled: \u201cZanzibar, C. Cooke. [printed] \\ Xylographus perforatus Gerst. Zanzib. [handwritten] \\ [yellow label] PARALECTOTYPE Xylographus perforatus Gerstaecker [handwritten]\u201d; 1 male and 1 female paralectotypes (MCZ), labeled: \u201cZanzibar, C. Cooke. [printed] \\ [black disc] \\ Xylographus perforatus 140 Gerstaecker [printed] \\ [yellow label] PARALECTOTYPE Xylographus perforatus Gerstaecker [handwritten]\u201d.TANZANIA: male lectotype (MFNB), here designated, labeled: \u201c[green label] Gorham, 1886http://species-id.net/wiki/Xylographus_porcusXylographus porcusPageBreakCis, porcus [handwritten] \\ B.C.A., Col., III (2). Xylographus porcus. [printed] \\ [red label] LECTOTYPE Xylographus porcus Gorh. [handwritten]\u201d; 1 male and 2 female paralectotypes (MNHN), labeled: \u201cTeleman, Vera Paz. Champion. [printed] \\ Xylographus porcus, Gorh [handwritten] \\ [yellow label] PARALECTOTYPE Xylographus porcus Gorh. [handwritten]\u201d; 2 female paralectotypes (MNHN), labeled: \u201cZapote, Guatemala. G. C. Champion. [printed] \\ [yellow label] PARALECTOTYPE Xylographus porcus Gorh. [handwritten]\u201d; 1 male and 2 female paralectotypes (MNHN), labeled: \u201cPantaleon, 700 ft. Champion. [printed] \\ [yellow label] PARALECTOTYPE Xylographus porcus Gorh. [handwritten]\u201d.GUATEMALA: male lectotype (NHM), here designated, labeled: \u201c[purple disc] LECTOTYPE [printed] \\ Teleman, Vera Paz. Champion. [printed] \\ Type. [printed] \\ Melli\u00e9, 1849http://species-id.net/wiki/Xylographus_punctatusXylographus punctatusXylographus punctatus Mellie [handwritten]\u201d; 1 male and 3 female paralectotypes (MNHN), labeled: \u201cNouv. Grenada [handwritten] \\ Coll. Chevrolat [handwritten] \\ [yellow label] PARALECTOTYPE Xylographus punctatus Mellie [handwritten]\u201d; 1female paralectotype (MNHN), labeled: \u201cCarthagene [handwritten] \\ Collect. Chevrolat [handwritten] \\ [yellow label] PARALECTOTYPE Xylographus punctatus Mellie [handwritten]\u201d; 1 male paralectotype (MNHN), labeled: \u201c[green disc] Xylographus punctatus Mell. Colomb. T. Cast. 72 [handwritten] \\ [pink disc] punctatus [handwritten] \\ punctatus Chev. Colombia [handwritten] \\ MUSEUM PARIS COLL DE MARSEUL [printed] \\ [yellow label] PARALECTOTYPE Xylographus punctatus Mellie [handwritten]\u201d; 1 male paralectotype (MNHN), labeled: \u201c[green disc] Xylographus punctatus Colomb. Lap. Cast. 72 [handwritten] \\ [green label] Chevt. Colomb [handwritten] \\ [yellow label] PARALECTOTYPE Xylographus punctatus Mellie [handwritten]\u201d.COLOMBIA: male lectotype (MNHN), here designated, labeled: \u201c[green label] \u2642 [handwritten] \\ Nouv. Grenada [handwritten] \\ Coll. Chevrolat [handwritten] \\ [red label] LECTOTYPE Pic, 1921http://species-id.net/wiki/Xylographus_ritsemaiXylographus ritsemaiPageBreakRitsemai Pic [handwritten] \\ type [handwritten] \\ Ceylon. Ancey. [handwritten] \\ [red label] LECTOTYPE Xylographus ritsemai Pic [handwritten]\u201d; 1 male paralectotype (MNHN), labeled: \u201cRitsemai Pic [handwritten] \\ type [handwritten] \\ [yellow label] PARALECTOTYPE Xylographus ritsemai Pic [handwritten]\u201d.SRI LANKA: male lectotype (MNHN), here designated, labeled: \u201cPic, 1930http://species-id.net/wiki/Xylographus_rufipesXylographus rufipesXylographus rufipes n. sp. [handwritten] \\ [red label] LECTOTYPE Xylographus rufipes Pic [handwritten]\u201d; 1 male paralectotype (NHM), labeled: \u201cArgentina Tucum\u00e1n 1 Oct. 1929 [printed] \\ H. E. Box leg. [printed] \\ 2709 [printed] \\ Xylographus rufipes, Pic sp. nov. [handwritten] \\ Brit. Mus. 1948-460. [printed] \\ [yellow label] PARALECTOTYPE Xylographus rufipes Pic [handwritten]\u201d.ARGENTINA: male lectotype (MNHN), here designated, labeled: \u201cArgentina Tucum\u00e1n 1 Oct. 1929 [printed] \\ H. E. Box leg. [printed] \\ 2705 [printed] \\ Nobuchi & Wada, 1956http://species-id.net/wiki/Xylographus_scheerpeltziXylographus scheerpeltziPic, 1929http://species-id.net/wiki/Xylographus_subopacusXylographus subopacusPageBreakXylographus subopacus Pic [handwritten]\u201d; 1 female paralectotype (MNHN), labeled: \u201cBELGIAN CONGO. 18 m. S. W. of Elizabethville. 1928. Dr. H. S. Evans. [printed] \\ Xylographus subopacus n. sp. [handwritten] \\ [yellow label] PARALECTOTYPE Xylographus subopacus Pic [handwritten]\u201d.DEMOCRATIC REPUBLIC OF THE CONGO: female lectotype (NHM), here designated, labeled: \u201c[purple disc] LECTOTYPE [printed] \\ BELGIAN CONGO. 18 m. S. W. of Elizabethville. 24.iii.1928. Dr. H. S. Evans. [printed] \\ Pres. by Imp. Inst. Ent. Brit. Mus. 1932-147. [printed] \\ [red label] LECTOTYPE Pic, 1916http://species-id.net/wiki/Xylographus_subsinuatusXylographus subsinuatus Pic 1916: 4. Type-locality: Madagascar.Xylographus rufescenssyn. n. Type-locality: Bourbon Island (Reunion).subsinuatus Pic [handwritten] \\ [red label] LECTOTYPE Xylographus subsinuatus Pic [handwritten]\u201d; 6 males, 3 females, 17 specimens of undetermined gender, all paralectotypes (MNHN), labeled: \u201cMADAGASCAR Plantations du Sambirano COLLECTION LE MOULT [printed] \\ [red label] Coll. C [handwritten] \\ [yellow label] PARALECTOTYPE Xylographus subsinuatus Pic [handwritten]\u201d.MADAGASCAR: male lectotype (MNHN), here designated, labeled: \u201cMADAGASCAR Plantations du Sambirano COLLECTION LE MOULT [printed] \\ [red label] Coll. C [handwritten] \\ Type [handwritten] \\ Xylographus rufescensrufescens Pic [handwritten] \\ [red label] LECTOTYPE Xylographus rufescens Pic [handwritten]\u201d.REUNION: male lectotype (MNHN) of Xylographus rufescens is a small teneral male of Xylographus subsinuatus. We dissected the types and compared the sclerites of abdominal terminalia, which are identical.We observed that the lectotype of Gorham, 1886http://species-id.net/wiki/Xylographus_suillusXylographus suillusXylographus suillus, Gorh. [handwritten] \\ B.C.A., Coll., III (2). Xylographus suillus, Gorh. [printed] \\ [red label] LECTOTYPE Xylographus suillus Gorh. [handwritten]\u201d; 2 male and 4 female paralectotypes (MNHN), labeled: \u201cTeleman, Vera Paz. Champion. [printed] \\ [pink label] In boleti attached to manaca palm [handwritten] \\ Xylographus suillus, Gor. [handwritten] \\ [yellow label] PARALECTOTYPE Xyloraphus suillus Gorh. [handwritten]\u201d.GUATEMALA: male lectotype (NHM), here designated, labeled: \u201c[purple disc] LECTOTYPE [printed] \\ Type. Sp. figured [printed] \\ Teleman, Vera Paz. Champion. [printed] \\ PageBreakReitter, 1902http://species-id.net/wiki/Xylographus_tomicoidesXylographus tomicoidestomicoides m. 1902 [handwritten] \\ [red label] LECTOTYPE Xylographus tomicoides Reitter [handwritten]\u201d.RUSSIA: male lectotype (MNHN), here designated, labeled: \u201cAmur [handwritten] \\ Xylographus dentatusCis dentatus Melli\u00e9, 1849. Type-locality: Republic of the Congo.dentatus n. sp. [handwritten] \\ Cis sp. A. Kompantsev det. 2010 [handwritten] \\ [red label] LECTOTYPE Xylographus dentatus Pic [handwritten] \\ [green label] Cis renominatus Sandoval-G\u00f3mez, L\u00f3pes-Andrade & Lawrence nom. n.\u201d; 6 male and 2 female paralectotypes (MNHN), labeled: \u201cFranz. Congo [printed] \\ [yellow label] PARALECTOTYPE Xylographus dentatus Pic [handwritten] \\ [green label] Cis renominatus Sandoval-G\u00f3mez, L\u00f3pes-Andrade & Lawrence nom. n.\u201d.REPUBLIC OF THE CONGO: male lectotype (MNHN), here designated, labeled: \u201cFranz. Congo [printed] \\ Xylographus dentatus Pic, 1922 is transferred to the genus Cis, but Cis dentatus becomes a junior secondary homonym of Cis dentatus Melli\u00e9, 1849. The replacement name proposed here means \u201crenamed\u201d.comb. n.http://species-id.net/wiki/Paratrichapus_fultoniCis fultoniXylographus fultoni PageBreakCis fultoni [handwritten] \\ Rhopalodontus fultoni Broun. K. Paviour-Smith det. 1966 [handwritten] \\ [red label] LECTOTYPE Cis fultoni Broun [handwritten]\u201d; 1 female paralectotype (NHM), labeled: \u201c1614 [handwritten] \\ Taieri [printed] \\ New Zealand. Broun Coll. Brit. Mus. 1922-482 [printed] \\ Cis fultoni [handwritten] \\ Rhopalodontus fultoni Broun. K. Paviour-Smith det. 1966 [handwritten] \\ [yellow label] PARALECTOTYPE Cis fultoni Broun [handwritten]\u201d.NEW ZEALAND: male lectotype (NHM), here designated, labeled: \u201c1614 [handwritten] \\ Taieri [printed] \\ New Zealand. Broun Coll. Brit. Mus. 1922-482 [printed] \\ comb. n.http://species-id.net/wiki/Paratrichapus_javanusXylographus javanusXylographus javanusrufomarginatus var. Fomes melanopurus Mont. [printed] \\ n. sp. diff\u00e8re de Xylographus ceylonicus Ancey par la forme plus allong\u00e9e, le thorax moins court, plus fortement r\u00e9tr\u00e9ci en avant, les \u00e9lytres sans pli hum\u00e9ral brillant [handwritten] \\ [red label] LECTOTYPE Xylographus javanus Pic [printed] \\ Paratrichapus javanus comb. n. Sandoval-G\u00f3mez, Lopes-Andrade & Lawrence [handwritten]\u201d; 1 male paralectotype (MNHN), labeled: \u201cF. C. DRESCHER G. Tangkoeban Prahoe 4000.5000 Voet. Preanger. Java 31.x.1934 [printed] \\ ex Fomes melanopurus Mont. [printed] \\ [yellow label] PARALECTOTYPE Xylographus javanus Pic [printed] \\ Paratrichapus javanus comb. n. Sandoval-G\u00f3mez, Lopes-Andrade & Lawrence [handwritten]\u201d; 1female paralectotype (MNHN), labeled: \u201cF. C. DRESCHER G. Tangkoeban Prahoe 4000.5000 Voet. Preanger. Java 22.i.1935 [printed] \\ ex Fomes melanopurus Mont. [printed] \\ Xylographus javanus n. sp. [handwritten] \\ [yellow label] PARALECTOTYPE Xylographus javanus Pic [printed] \\ Paratrichapus javanus comb. n. Sandoval-G\u00f3mez, Lopes-Andrade & Lawrence [handwritten]\u201d.INDONESIA: male lectotype (MNHN), here designated, labeled: \u201cF. C. DRESCHER G. Tangkoeban Prahoe 4000.5000 Voet. Preanger. Java 31.x.1934 [printed] \\ ex"} +{"text": "The coordination polyhedron shows a slightly distorted Archimedean square-anti\u00adprismatic geometry. The crystal packing is stabilized by weak C\u2014H\u22efO inter\u00adactions.In the title compound, [Hf(C DOI: 10.1107/S1600536810030400/bg2349Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The cation adopts a chair conformation. The crystal structure is stabilized by inter\u00admolecular N\u2014H\u22efCl hydrogen bonds.In the complex anion of the title compound, (C DOI: 10.1107/S1600536810047689/bx2322Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The metal atoms are linked by the dianions into zigzag chains running parallel to [11The asymmetric unit of the title compound, {[Ni(C DOI: 10.1107/S1600536810045794/rz2513Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536810049081/pk2286Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536811022598/mg2119Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the crystal, mol\u00adecules are linked by N\u2014H\u22efO hydrogen bonds, forming C(4) chains propagating in [001].In the title compound, C DOI: 10.1107/S160053681101035X/hb5818Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the crystal, numerous N\u2014H\u22efCl and bifurcated N\u2014H\u22ef hydrogen bonds link the components.The asymmetric unit of the title compound, (C DOI: 10.1107/S1600536811049464/hb6519Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The Zn2+ cation is surrounded by two N atoms and two O atoms from L2\u2212, in a nearly planar configuration, and one methanol O atom, forming a slightly distorted square-pyramidal geometry. The methanol molecule is disordered over two sets of sites in a 0.5:0.5 ratio. In the crystal, O\u2014H\u22efO hydrogen bonds link the mol\u00adecules into chains parallel to [001].In the title complex, [Zn(C DOI: 10.1107/S1600536813016863/jj2167Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S1600536813016863/jj2167Isup3.cdxSupplementary material file. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "The crystal structure is stabilized by O\u2014H\u22efN and N\u2014H\u22efN hydrogen bonds, forming a three-dimensional network.In the title compound, [Cu(C DOI: 10.1107/S1600536810050178/bt5405Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Therefore the asymmetric unit contains one crystallographically independent half-mol\u00adecule. The Cd atom adopts a tetra\u00adhedral coordination geometry, coordinated by two I atoms and two N atoms from the symmetry-related 4-(2-methyl\u00adstyr\u00adyl)pyridine ligands.In the title complex, [CdI DOI: 10.1107/S1600536811037573/bq2302Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The metal ions are linked by the EG and L ligands, forming two-dimensional coordination networks, which are associated into the three-dimensional structure through O\u2014H\u22efO hydrogen bonds.In the title complex, {[Mn(C DOI: 10.1107/S1600536810045411/fk2028Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the crystal, adjacent mol\u00adecules are linked by N\u2013H\u22efNcyano hydrogen bonds, forming a chain running along [110].In the title compound, C DOI: 10.1107/S1600536813029589/ng5344Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "In the crystal structure, mol\u00adecules pack via weak C\u2014H\u22efN and C\u2014H\u22efCl inter\u00adactions.In the title compound, [MnCl DOI: 10.1107/S1600536811043352/su2330Isup2.hkl Structure factors: contains datablock(s) I. DOI: 10.1107/S1600536811043352/su2330Isup3.mol Supplementary material file. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The hpc ligands bridge adjacent EuIII ions, forming infinite double chains. Adjacent chains are further connected by hpc ligands into sheets. O\u2014H\u22efO hydrogen bonds then generate a three-dimensional supra\u00admolecular framework.In the title coordination polymer, {[Eu(C DOI: 10.1107/S1600536810048518/hb5735Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The dihedral angle between the two Cp rings is 3.20\u2005(17)\u00b0. The crystal packing is mainly stabilized by van der Waals forces.In the title mol\u00adecule, [Fe(C DOI: 10.1107/S1600536811014796/mw2007Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The PrIII ions are bridged by the oxalate ligands, forming a layer parallel to (001). O\u2014H\u22efO hydrogen bonds connect the layers.In the title complex, [Pr DOI: 10.1107/S1600536812011014/hy2521Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "Two Cl atoms complete a distorted square-pyramidal geometry around the metal atom. In the crystal, a C\u2014H\u22efCl inter\u00adaction connects pairs of mol\u00adecules into centrosymetric dimers.In the title compound, [CdCl DOI: 10.1107/S1600536811005538/pv2387Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "O\u2014H\u22efO hydrogen-bonding inter\u00adactions involving the water mol\u00adecules form infinite chains parallel to [010].In the title compound, [Zn(C DOI: 10.1107/S1600536810050865/dn2633Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The coordination sphere around Cu(II) ion can be described as tetragonally distorted octa\u00adhedral with two perchlorate O atoms occupying the apical positions and four N atoms from two N 1-(2-furyl\u00admethyl)ethane-1,2-diamine ligands in the basal plane.In the title complex, [Cu(ClO DOI: 10.1107/S1600536811012232/nk2094Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The C atoms of the propan-1-aminium cation are disordered over two sets of sites in a 0.65\u2005(3):0.35\u2005(3) ratio. The crystal structure is stabilized by N\u2014H\u22efO hydrogen bonds.In the anion of the title solvated molecular salt, C DOI: 10.1107/S1600536811016850/bt5538Isup2.hkl Structure factors: contains datablocks I. DOI: 10.1107/S1600536811016850/bt5538Isup3.cml Supplementary material file. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The two bpds ligands of the same axial chirality bridge ZnII atoms, generating repeated rhomboidal chains, which are linked by O\u2014H\u22efO hydrogen bonds into a ladder structure.In the title compound, {[Zn(C DOI: 10.1107/S1600536810021331/er2078Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the crystal, mol\u00adecules are linked via O\u2014H\u22efO and O\u2014H\u22efN hydrogen bonds, forming double-stranded chains propagating along [010].In the title compound, [Sn(CH DOI: Click here for additional data file.10.1107/S1600536813012622/su2595Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536810023214/pv2294Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The anions bridge adjacent TmIII ions into double chains. Adjacent chains are further connected into sheets. O\u2014H\u22efO hydrogen bonds involving both coordinated and uncoordinated water mol\u00adecules generate a three-dimensional supra\u00admolecular framework.In the title coordination polymer, {[Tm(C DOI: 10.1107/S1600536811007628/pv2392Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Each TMIPA anion bridges three CdII cations, forming polymeric complex sheets parallel to (001). Weak C\u2014H\u22efO hydrogen bonding occurs between adjacent sheets.In the crystal structure of the polymeric title complex, [Cd(C DOI: 10.1107/S1600536811054183/xu5394Isup2.hkl Structure factors: contains datablock(s) I. DOI: 10.1107/S1600536811054183/xu5394Isup3.mol Supplementary material file. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The pyridinium NH H atom is disordered over the two ligands. Adjacent mononuclear clusters are linked through N\u2014H\u22efO and N\u2014H\u22efN hydrogen-bonding inter\u00adactions, generating layers in the (102) plane.In the title compound, [Nd(NO DOI: 10.1107/S1600536812011397/bt5847Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "The chain structure is constructed from square-pyramidally coordinated CuII atoms linked through l-methio\u00adnate ligands. The chains propagate along the a-axis direction and are linked to perchlorate anions via N\u2014H\u22efO hydrogen bonds.The structure of the title compound, {[Cu(C DOI: 10.1107/S1600536811014000/ff2004Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Intra\u00admolecular N\u2014H\u22efO hydrogen bonds help to establish the configuration.In the title compound, [Ni(NO DOI: 10.1107/S1600536810044624/si2301Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the crystal, N\u2014H\u22efO hydrogen bonds link the mol\u00adecules into infinite chains running along the b axis.In the title compound, C DOI: 10.1107/S1600536811050732/bt5725Isup2.hkl Structure factors: contains datablock(s) I. DOI: 10.1107/S1600536811050732/bt5725Isup3.cml Supplementary material file. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The Figure 3 caption does not include the correct legend colors. The corrected Figure 3 caption reads:Figure 3. Diversity measures at the site scale in each estuary: \u03b1-site , \u03b3- site (green bars) and \u03b2-site (blue bars). Sites: 1 to 10. Significant Spearman correlation (r) between diversity measures at each estuary is included in left corner of graphs. doi:10.1371/journal.pone.0065575.g003"} +{"text": "Phylogenetic comparison of bacteriophages requires whole genome approaches such as dotplot analysis, genome pairwise maps, and gene content analysis. Currently mycobacteriophages, a highly studied phage group, are categorized into related clusters based on the comparative analysis of whole genome sequences. With the recent explosion of phage isolation, a simple method for phage cluster prediction would facilitate analysis of crude or complex samples without whole genome isolation and sequencing. The hypothesis of this study was that mycobacteriophage-cluster prediction is possible using comparison of a single, ubiquitous, semi-conserved gene. Tape Measure Protein (TMP) was selected to test the hypothesis because it is typically the longest gene in mycobacteriophage genomes and because regions within the TMP gene are conserved.A single gene, TMP, identified the known Mycobacteriophage clusters and subclusters using a Gepard dotplot comparison or a phylogenetic tree constructed from global alignment and maximum likelihood comparisons. Gepard analysis of 247 mycobacteriophage TMP sequences appropriately recovered 98.8% of the subcluster assignments that were made by whole-genome comparison. Subcluster-specific primers within TMP allow for PCR determination of the mycobacteriophage subcluster from DNA samples. Using the single-gene comparison approach for siphovirus coliphages, phage groupings by TMP comparison reflected relationships observed in a whole genome dotplot comparison and confirm the potential utility of this approach to another widely studied group of phages.TMP sequence comparison and PCR results support the hypothesis that a single gene can be used for distinguishing phage cluster and subcluster assignments. TMP single-gene analysis can quickly and accurately aid in mycobacteriophage classification. Mycobacterium tuberculosis and the nonpathogenic M. smegmatis. Mycobacteriophages are the most studied of all bacteriophages with 2,413 mycobacteriophages isolated, more than 344 genomes fully sequenced (http://phagesdb.org/) and approximately 223 full phage genome sequences available on GenBank, making the analysis of these phages a model for bacteriophage research. The number of mycobacteriophages isolated and sequenced in recent years has led to the identification of genetic relationships and subsequent assignment of phages into 17 clusters and 30 subclusters based on whole genome comparison for 74 of the 79 phages included: Acadian (B5) , Adephagia (K1) , Airmid (A5) , Angelica (K1) , Arbiter , Ares (B2) , Avani (F2) [JQ809702], Babsiella (I1) , Backyardigan (A4), Baka (J) , Barnyard (H2) , Benedict (A5) , Bongo (M) , BPs (J), Brujita (I1) , Bxz2 (A3) , Charlie (N) , Che12 (A2), Che9c (I2), Che9d (F2), ChrisnMich (B4) , Colbert (B1), Cooper (B4) , Corndog (O) , CrimD (K1) , Cuco (A5) , Daisy (B3) , DaVinci (A6) , DotProduct (F1) , Eagle (A4) , Faith1 , Firecracker (O), Fruitloop (F1), Gladiator (A6), Gumball (D1) , Halo (G) , Hammer (A6), Harvey (B1) , Hedgerow (B2) , HelDan (A3) , Henry (E) , Hertubise (B1) , Hope (G) , island3 (I1) , JC27 (A1) , JHC117 , JoeDirt (L1) , Konstantine (H1) , Kostya (E) , KSSJEB , Larva (K5) , LeBron (L1) , LHTSCC (A4) , Lilac (E) , LittleE (J) , MacnCheese (K3) [JX042579], Omega (J) , PBI1 (D1) , Phlyer (B3) , Pipefish (B3) , Pixie (K3) , PLot (D1) , Predator (H1) , Redi (N) , RedRock (A2) , Rey (M) , RockyHorror (F1) , Rumpelstiltskin (L2) , Switzer , TM4 (A1) , Trixie (A2) , UPIE (L1) , Yoshi (F2) . Five mycobacteriophage genomes for the 79-phage comparison were downloaded from http://phagesdb.org, and included Archie (L2), Catdawg (0), Frederick (B4), Kratio (K) and Xerxes (N). The genomes from phagesdb.org were unannotated; therefore, DNA Master (http://cobamide2.bio.pitt.edu) was used to auto-annotate the genomes and identify TMP and MCP. For the 247-mycobacteriophage comparison, genomes included the previous 79 along with 157 sequences from GenBank and 11 sequences from the phagesdb.org website. The sequences from phagesdb.org included Bernardo, Hawkeye, HotShotFirst, JAMaL, Mendokysei, Mosby, Odin, Pegleg, Squirty, TA17A, and Whirlwhind. Fasta files of whole genome sequences were downloaded from the http://phagesdb.org website and TMP sequences were identified by Blast searches of the genomes. The 157 mycobacteriophage TMP sequences gathered from GenBank were as follows (cluster) [GenBank Accession number]: 244 (E) [DQ398041], ABU (B1) [JF704091], Adjutor (D1) [EU676000], Aeneas (A1) [JQ809703], Akoma (B3) [JN699006], Alice (C1) [JF704092], Alma (A9) [JN699005], Anaya (K1) [JF704106.1], Angel (G) [NC_012788.1], AnnaL29 (A1) [JN572060], Ardmore (F1) [NC_013936.1], Athena (B3) [JN699003], Ava3 (C1) [JQ911768], Avrafan (G) [JN699002.1], BarrelRoll (K1) [JN643714.1], Bask21 (E) [JF937091.1], Bethlehem (A1) [AY500153], BigNuz (P) [JN412591.1], BillKnuckles (A1) [JN699000], Blue7 (A6) [JN698999], Boomer (F1) [NC_011054.1], BPBiebs31 (A1) [JF957057], Bruns (A1) [JN698998], Butterscotch (D1) [FJ168660], Bxb1 (A1) [AF271693], Bxz1 (C1) [AY129337], Cali (C1) [EU826471], Catera (C1) [DQ398053], Chah (B1) [FJ174694], Che8 (F1) [NC_004680.1], Cjw1 (E) [AY129331], Courthouse (J) [JN698997.1], D29 (A2) [AF022214], Dandelion (C1) [JN412588], DD5 (A1) [EU744252], DeadP (F1) [JN698996.1], DLane (F1) [JF937093.1], Doom (A1) [JN153085], Dori (Singleton) [JN698995.1], Drago (F1) [JN542517.1], Drazdys (C1) [JF704116], Dreamboat (A1) [JN660814], DS6A (Singleton) [JN698994.1], Elph10 (E) [JN391441.1], EricB (A6) [JN049605], ET08 (C1) [GQ303260.1], Euphoria (A1) [JN153086], Eureka (E) [JN412590.1], Fang (B1) [GU247133], Flux (A4) [JQ809701], Gadjet (B3) [JN698992], George (A5) [JF704107], Ghost (C1) [JF704096], Giles (Q) [NC_009993.2], GUmbie (F1) [JN398368.1], Ibhubesi (F1) [JF937098.1], ICleared (A4) [JQ896627], IsaacEli (B1) [JN698990], JacAttac (B1) [JN698989], Jasper (A1) [EU744251], JAWS (K1) [JN185608.1], Jebeks (P) [JN572061.1], Jeffabunny (A6) [JN699019], Kamiyu (B3) [JN699018], KBG (A1) [EU744248], Kikipoo (B1) [JN699017], KLucky39 (B1) [JF704099], Kugel (A1) [JN699016], L5 (A2) [Z18946], Lesedi (A1) [JF937100], Liefie (G) [JN412593.1], LinStu (C1) [JN412592], Llij (F1) [NC_008196.1], Lockley (A1) [EU744249], LRRHood (C1) [GQ303262.1], Marvin (S) [JF704100.1], MeeZee (A4) [JN243856], Microwolf (A3) [JF704101], MoMoMixon (C1) [JN699626], Morgushi (B1) [JN638753], Mozy (F1) [JF937102.1], MrGordo (A1) [JN020140], Murdoc (B1) [JN638752], Museum (A1) [JF937103], Mutaforma13 (F1) [JN020142.1], Myrna (C2) [EU826466], Nappy (C1) [JN699627], Nigel (B4) [EU770221], Nova (D1) [JN699014], Oline (B1) [JN192463], Oosterbaan (B1) [JF704109], Optimus (J) [JF957059.1], Orion (B1) [DQ398046], OSmaximus (B1) [JN006064], Pacc40 (F1) [NC_011287.1], PackMan (A9) [JF704110], Patience (Singleton) [JN412589.1], Peaches (A4) [GQ303263.1], Perseus (A1) [JN572689], PG1 (B1) [AF547430], Phaedrus (B3) [EU816589], Phipps (B1) [JF704102], Pio (C1) [JN699013], Pleione (C1) [JN624850], PMC (F1) [NC_008205.1], Porky (E) [NC_011055.1], Puhltonio (B1) [GQ303264.1], Pukovnik (A2) [EU744250], Pumpkin (E) [GQ303265.1], Qyrzula (B2) [DQ398048], Rakim (E) [JN006062], Ramsey (F1) [NC_011289.1], RidgeCB (A1) [JN398369], Rizal (C1) [EU826467], Rockstar (A3) [JF704111], Rosebush (B2) [AY129334], Saintus (A8) [JN831654], Scoot17C (B1) [GU247134], ScottMcG (C1) [EU826469], Sebata (C1) [JN204348], Send513 (R ) [JF704112.1], Serendipity (B1) [JN006063], SG4 (F1) [JN699012.1], Shaka (A4) [JF792674], Shauna1 (F1) [JN020141.1], ShiLan (F1) [JN020143.1], SirDuracell (E) [JF937106.1], SirHarley (D1) [JF937107], SkiPole (A1) [GU247132], Solon (A1) [EU826470a], Spud (C1) [EU826468], Stinger (B4) [JN699011], Taj (F1) [JX121091.1], TallGrassMM (B1) [JN699010], Thibault (J) [JN201525.1], Thora (B1) [JF957056], ThreeOh3d2 (B1) [JN699009], Tiger (A5) [JX042578], Timshel (A7) [JF957060], TiroTheta9 (A4) [JN561150], Toto (E) [JN006061], Troll4 (D1) [FJ168662], Turbido (A2) [JN408460], Tweety (F1) [NC_009820.1], Twister (A10) [JQ512844], U2 (A1) [AY500152], UncleHowie (B1) [GQ303266.1], Violet (A1) [JN687951], Vista (B1) [JN699008], Vix (A3) [JF704114], Vortex (B1) [JF704103], Wally (C1) [JN699625], Wee (F1) [NC_014901.1], Wildcat (Singleton) [NC_008206.1], Wile (A4) [JN243857], Yoshand (B1) [JF937109], Zemanar (B4) [JF704104].Seventy-nine full genomes were collected from GenBank representing a large extent of diversity of phages infecting Mycobacterium spp. The phage genome, TMP and MCP sequences were collected from GenBank and from An additional 24 TMP sequences from coliphages were used which included HK578 [NC_019724], mEp213 [NC_019720], vB_EcoS_Rogue1 [NC_019718], HK446 [NC_019714], HK140 [NC_019710], mEp235 [NC_019708], mEp043 c-1 [NC_019706], mEpX2 [NC_019705], HK630 [NC_019723], HK633 [NC_019719], HK225 [NC_019717], mEp234 [NC_019715], HK629 [NC_019711], mEpX1 [NC_019709], mEp237 [JQ182730], JL1 [NC_019419], HK022 [NC_002166], lambda [NC_001416], JK06 [NC_007291], T1 [NC_005833], HK97 [NC_002167], N15 [NC_001901], and Escherichia phages ADB-2 [NC_019725], and HK75 [NC_016160].http://tree.bio.ed.ac.uk/software/). The confidence interval of percent clustered and subclustered phage based on TMP comparison of 247 sequences was determined using a Confidence Interval for Proportions with an alpha level of 0.05 (95% confidence level).Gepard was usedBacillus cereus PBC1 phage as outgroup. In order to infer a phylogeny the neighbor-joining method was used and the phylogeny bootstrapped 10,000 times to assess nodal support. A 50% majority-rule consensus tree was obtained using Paup* 4.0 [http://www.genealogicalsorting.org) [For the alignment-free phylogeny, feature frequency profiles were useaup* 4.0 and annoaup* 4.0 ) metric aup* 4.0 . The gening.org) .The authors have no competing interests to declare.KCS, DPB, JHG and SHB were responsible for the design and coordination of the research. KCS conducted the laboratory research. SHB drafted the manuscript, JHG drafted figures and edited extensively. KCS, ECN, JNBF, JHG and DPB participated in genomic and gene analysis. All authors contributed to editing of the manuscript. All authors read and approved the final manuscript."} +{"text": "In the crystal, O\u2014H\u22efO hydrogen bonds are observed between the coordinated carboxyl\u00adate O atoms and the solvent water mol\u00adecule.In the title compound, [Cu(C DOI: 10.1107/S1600536812020193/jj2130Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "Each oxalate ligand bridges two CdII atoms, generating a zigzag chain structure propagating along [100]. The packing of the structure is consolidated by non-classical C\u2014H\u22efO hydrogen-bonding inter\u00adactions.In the title complex, [Cd(C DOI: 10.1107/S1600536810040341/wm2410Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The dihedral angles between the piperidine groups and the NCN chelate rings are 51.5\u2005(1) and 52.3\u2005(1)\u00b0.In the mononuclear title complex, [Ti(C DOI: 10.1107/S1600536810053262/hg2762Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The diacetate and 4,4\u2032-bipyridine ligands also lie across inversion centers. The bridging ligands form layers parallel to (11via O\u2014H\u22efO hydrogen bonds between the coordinated water mol\u00adecules and the carboxyl\u00adate O atoms, giving a three-dimensional supra\u00admolecular architecture.In the title compound, [Cd(C DOI: 10.1107/S1600536811031400/zs2132Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Complex mol\u00adecules are connected by C\u2014H\u22efO hydrogen bonds into a three-dimensional network.In the title compound, [Ho(NO DOI: 10.1107/S1600536812012445/is5078Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "In the crystal, a two-dimensional framework parallel to (010) is formed by N\u2014H\u22efO and O\u2014H\u22efO hydrogen bonds.In the title compound, [Zn(C DOI: 10.1107/S1600536811012992/nk2096Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The mol\u00adecule has approximate C 2 point symmetry. The dihedral angles between the phenyl and benzene rings on either side of the ligand are 64.56\u2005(14) and 65.61\u2005(13)\u00b0.In the title compound, [FeCl DOI: 10.1107/S1600536811053773/lh5395Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "II atom in the mononuclear title compound, [Ni(C10H10NS2)(NCS)(C18H15P)], exists within a S2PN donor set that defines a distorted square-planar geometry. A significant asymmetry in the Ni\u2014S bond lengths support the less effective trans effect of SCN\u2212 over PPh3.The Ni DOI: 10.1107/S1600536811050550/bt5724Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The sulfonate groups doubly bridge symmetry-related NiII centers, forming polymeric chains along the a axis.In the title polymeric complex, [Ni(C DOI: 10.1107/S1600536810020325/bh2285Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The Schiff base ligand acts as a chelating ligand and coordinates to the ZnII atom via two N atoms.In the title compound, [ZnBr DOI: 10.1107/S1600536812027997/bt5947Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "The 4-pybpy ligand, having a twofold rotation axis, functions in a bridging coordination mode, connecting the CoII ions into a corrugated chain along [In the title complex, [Co(CHO DOI: 10.1107/S1600536811021118/hy2424Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The molecules are packed as two monomeric mol\u00adecules with one acetone solvent mol\u00adecule sitting at the centre.The title compound, [Rh(C DOI: 10.1107/S1600536812008148/hg5181Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536811036373/vm2118Isup2.hkl Structure factors: contains datablock(s) I. DOI: 10.1107/S1600536811036373/vm2118Isup3.cml Supplementary material file. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The CoII atom, in an octa\u00adhedral enviroment, is coordinated by four N atoms from two 2-methyl\u00adsulfanyl-4-(pyridin-2-yl)pyrimidine ligands and two Cl atoms.The asymmetric unit of the title compound, [CoCl DOI: 10.1107/S1600536811030881/vm2113Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "A two-dimensional polymer is generated, lying parallel to (100), within which there are water\u2013carboxyl\u00adate O\u2014H\u22efO hy\u00addro\u00adgen-bonding inter\u00adactions.In the structure of the title complex, [Cs(C DOI: 10.1107/S1600536813029395/wm2781Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S1600536813029395/wm2781Isup3.cmlSupplementary material file. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "Each 1,10-phenanthroline ligand displays a bidentate chelating coordinating mode and the 4-sulfonato\u00adbenzotriazolide ions act as \u03bc2-bridges, linking different Zn2+ cations into a chain along the b axis. The crystal structure is consolidated by C\u2014H\u22efO hydrogen-bonding inter\u00adactions.In the title complex, [Zn(C DOI: 10.1107/S160053681104743X/pv2482Isup2.hkl Structure factors: contains datablock(s) I. DOI: 10.1107/S160053681104743X/pv2482Isup3.cdx Supplementary material file. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The two aromatic rings make a dihedral angle of 77.4\u2005(1)\u00b0. In the crystal, the molecules are linked by N\u2014H\u22efO hydrogen bonds, forming C(4) chains propagating in [010].In the mol\u00adecular structure of the title compound, C DOI: 10.1107/S1600536811047271/bt5712Isup2.hkl Structure factors: contains datablock(s) I. DOI: 10.1107/S1600536811047271/bt5712Isup3.cml Supplementary material file. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "A three-dimensional network is generated by inter\u00admolecular N\u2014H\u22efO and O\u2014H\u22efO hydrogen-bonding interactions involving the cation, the complex anion and the lattice water molecule.In the title compound, (C DOI: 10.1107/S1600536813022058/mw2113Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "The Mo6+ centre adopts a highly distorted octa\u00adhedral geometry, being surrounded by four chloride and two terminal oxide groups. The oxide ligands are mutually cis.In the title compound, (C DOI: 10.1107/S1600536810023950/sj5018Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The complex mol\u00adecule has an inversion center lying at the mid-point of the Cu\u2014Cu bond.The binuclear title compound, [Cu DOI: 10.1107/S1600536811029837/hy2450Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The five-membered chelate ring is puckered on the C\u2014C bond. In the title compound, [RuCl DOI: 10.1107/S1600536811022227/hb5901Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The CoII atom occupies a special position on a twofold rotation axis. In the crystal, mol\u00adecules are linked via weak C\u2014H\u22efBr inter\u00adactions. In the title complex, [Co(C DOI: 10.1107/S160053681104459X/br2176Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The amide N atom is a hydrogen-bond donor to the uncoordinated carboxyl\u00adate O atom. The geometry at the five-coordinate Sn atom is trans-C3SnO2 trigonal-bipyramidal.In the title polymeric complex, [Sn(C DOI: 10.1107/S1600536810026978/xu2796Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "One of the coordinated nitrate anions is disordered over two set of sites in a 0.85:0.15 ratio.The crystal structure of the title compound, [Cu(NO DOI: 10.1107/S1600536811005290/nc2216Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The asymmetric unit comprises one nickel cation, two thio\u00adcyanate anions and two actonitrile mol\u00adecules. In the crystal, the NiII cations are connected by bridging thio\u00adcyanate anions into a three-dimensional coordination network.In the title compound, [Ni(NCS) DOI: 10.1107/S1600536811004132/im2264Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the crystal, the almost symmetrically bridging \u03bc2-C=N-R ligand (neglecting the different atomic radii of Fe and Pt) is strongly bent towards the Fe(CO)3 fragment, with a C=N-R angle of only 121.1\u2005(4)\u00b0.The title compound, [FePt(C DOI: 10.1107/S1600536812004023/fi2121Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "An intra\u00admolecular O\u2014H\u22efN hydrogen bond is present between the quinazoline and hy\u00addroxy groups. In the crystal, mol\u00adecules are stacked along the b-axis direction.In the mol\u00adecule of the title compound, C DOI: 10.1107/S1600536814019990/gg2141Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S1600536814019990/gg2141Isup3.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S1600536814019990/gg2141fig1.tif17 16 2 . DOI: 17H16N2OS with atom labels and 50% probability displacement ellipsoids for nonhydrogen atoms.A mol\u00adecule of CClick here for additional data file.10.1107/S1600536814019990/gg2141fig2.tifb . DOI: b axis.Crystal structure packing viewed down the 1022918CCDC reference: crystallographic information; 3D view; checkCIF reportAdditional supporting information:"} +{"text": "Scientific Reports5: Article number: 1278410.1038/srep12784; published online: 08052015; updated: 10052015In this Article, Fig. 6H is a duplication of Supplementary Figure 4. The correct Fig. 6H appears below as"} +{"text": "Scientific Reports5: Article number: 12437; 10.1038/srep12437 published online: 07202015; updated: 01202016.Chapitre 13: L\u2019orbite et ses muscles. In Optique Physiologique. Tome Troisi\u00e8me: l\u2019Espace Visuel 28\u201341 \u2019. The correct reference is listed below:This Article contains errors in Reference 40 which was incorrectly given as \u2018Le Grand, Y. Chapitre 13: L\u2019orbite et ses muscles. In Optique Physiologique. Tome premier : la dioptrique de l\u2019\u0153il et sa correction. 153\u2013163 .Le Grand, Y."} +{"text": "The C\u2014O bond length of 1.3157\u2005(13)\u2005\u00c5 shows double-bond character, indicating charge delocalization within the NCO plane of the iminium ion. In the crystal, C\u2014H\u22efO hydrogen bonds between H atoms of the cations and O atoms of neighbouring ethyl sulfate anions are present, generating a three-dimensional network.In the title salt, C DOI: 10.1107/S2056989015020678/hb7521Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989015020678/hb7521Isup3.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989015020678/hb7521fig1.tif. DOI: The structure of the title compound with displacement ellipsoids at the 50% probability level.Click here for additional data file.10.1107/S2056989015020678/hb7521fig2.tifac . DOI: ac view).C\u2014H\u22efO hydrogen bonds (black dashed lines) between H atoms of the cations and oxygen atoms of the ethyl sulfate ions (1434493CCDC reference: crystallographic information; 3D view; checkCIF reportAdditional supporting information:"} +{"text": "The full contentscan be found at http://onlinelibrary.wiley.com/doi/10.1111/bph.13352/full. Nuclearhormone receptors are one of the eight major pharmacological targets into which theGuide is divided, with the others being: G protein\u2010coupled receptors, ligand\u2010gatedion channels, voltage\u2010gated ion channels, other ion channels, catalytic receptors,enzymes and transporters. These are presented with nomenclature guidance and summaryinformation on the best available pharmacological tools, alongside key references andsuggestions for further reading. The Concise Guide is published in landscape formatin order to facilitate comparison of related targets. It is a condensed version ofmaterial contemporary to late 2015, which is presented in greater detail andconstantly updated on the website www.guidetopharmacology.org, superseding datapresented in the previous Guides to Receptors & Channels and the Concise Guide toPHARMACOLOGY 2013/14. It is produced in conjunction with NC\u2010IUPHAR and provides theofficial IUPHAR classification and nomenclature for human drug targets, whereappropriate. It consolidates information previously curated and displayed separatelyin IUPHAR\u2010DB and GRAC and provides a permanent, citable, point\u2010in\u2010time record thatwill survive database updates.The Concise Guide to PHARMACOLOGY 2015/16provides concise overviews of the key properties of over 1750 human drug targets withtheir pharmacology, plus links to an open access knowledgebase of drug targets andtheir ligands ( The authors state that there are no conflictsof interest to declare.Nuclear hormone receptors are specialisedtranscription factors with commonalities of sequence and structure, which bind ashomo\u2010 or heterodimers to specific consensus sequences of DNA (response elements) inthe promoter region of particular target genes. They regulate (either promoting orrepressing) transcription of these target genes in response to a variety ofendogenous ligands. Endogenous agonists are hydrophobic entities which, when bound tothe receptor promote conformational changes in the receptor to allow recruitment (ordissociation) of protein partners, generating a large multiprotein complex.Two major subclasses of nuclear receptors withidentified endogenous agonists can be identified: steroid and non\u2010steroid hormonereceptors. Steroid hormone receptors function typically as dimeric entities and arethought to be resident outside the nucleus in the unliganded state in a complex withchaperone proteins, which are liberated upon agonist binding. Migration to thenucleus and interaction with other regulators of gene transcription, including RNApolymerase, acetyltransferases and deacetylases, allows gene transcription to beregulated. Non\u2010steroid hormone receptors typically exhibit a greater distribution inthe nucleus in the unliganded state and interact with other nuclear receptors to formheterodimers, as well as with other regulators of gene transcription, leading tochanges in gene transcription upon agonist binding.Selectivity of gene regulation is brought aboutthrough interaction of nuclear receptors with particular consensus sequences of DNA,which are arranged typically as repeats or inverted palindromes to allow accumulationof multiple transcription factors in the promoter regions of genes.TRs,nomenclature as agreed by theNC\u2010IUPHARSubcommittee on Nuclear Hormone Receptors [41]) are nuclear hormone receptors of the NR1A family, with diverse rolesregulating macronutrient metabolism, cognition and cardiovascular homeostasis. TRsare activated by thyroxine (4T) and thyroid hormone (triiodothyronine). Onceactivated by a ligand, the receptor acts as a transcription factor either as amonomer, homodimer or heterodimer with members of the retinoid X receptor family.NH\u20103 has been described as anantagonist at TRs with modest selectivity for TR\u03b2[111]. Thyroid hormone receptors Minireview: cracking themetabolic code for thyroid hormone signaling. J. Clin. Invest.122: 3035\u201043 [PMID:22945636]Brent GA. (2012) Mechanisms of thyroid hormoneaction. et al. (2006)International Union of Pharmacology. LIX. The pharmacology and classification of thenuclear receptor superfamily: thyroid hormone receptors. Pharmacol.Rev.58: 705\u201011 [PMID:17132849]Flamant F et al. (2011) Roleof thyroid receptor \u03b2 in lipid metabolism. Biochim. Biophys. Acta1812: 929\u201037 [PMID:21194564]Pramfalk C et al. (2011) Thethyroid hormones and their nuclear receptors in the gut: from developmental biologyto cancer. Biochim. Biophys. Acta1812: 938\u201046 [PMID:21194566]Sirakov M et al. (2011)Thyromimetics: a journey from bench to bed\u2010side. Pharmacol. Ther.131: 33\u20109 [PMID:21504761]Tancevski I nomenclature asagreed by theNC\u2010IUPHARSubcommittee on Nuclear Hormone Receptors [46]) are nuclear hormone receptors of the NR1B family activated by thevitamin A\u2010derived agonists tretinoin (ATRA) and alitretinoin, and theRAR\u2010selective synthetic agonists TTNPB and adapalene. BMS493 is a family\u2010selectiveantagonist [47]. Retinoic acid receptors Proteinkinases and the proteasome join in the combinatorial control of transcription bynuclear retinoic acid receptors. Trends Cell Biol.17: 302\u20109 [PMID:17467991]Bour G et al. (2011) Themolecular physiology of nuclear retinoic acid receptors. From health to disease.Biochim. Biophys. Acta1812: 1023\u201031 [PMID:20970498]Duong V et al. (2006)International Union of Pharmacology. LX. Retinoic acid receptors. Pharmacol.Rev.58: 712\u201025 [PMID:17132850]Germain P Nat. Rev.Neurosci.8: 755\u201065 [PMID:17882253]Maden M. (2007) Retinoic acid in thedevelopment, regeneration and maintenance of the nervous system. et al. (2006) Functionof retinoid nuclear receptors: lessons from genetic and pharmacological dissectionsof the retinoic acid signaling pathway during mouse embryogenesis. Annu. Rev.Pharmacol. Toxicol.46: 451\u201080 [PMID:16402912]Mark M PPARs, nomenclature as agreed by theNC\u2010IUPHARSubcommittee on Nuclear Hormone Receptors[101]) are nuclear hormone receptors of the NR1C family, with diverse rolesregulating lipid homeostasis, cellular differentiation, proliferation and the immuneresponse. PPARs have many potential endogenous agonists , including \u039412,14\u2010PGJ215\u2010deoxy\u2010, prostacyclin (2PGI), many fatty acids and their oxidation products, lysophosphatidic acid(LPA) [98], 13\u2010HODE, 15S\u2010HETE, Paz\u2010PC, azelaoyl\u2010PAF and leukotriene B4(4LTB). Bezafibrate acts as anon\u2010selective agonist for the PPAR family [159]. These receptors also bindhypolipidaemic drugs (PPAR\u03b1) and anti\u2010diabetic thiazolidinediones (PPAR\u03b3), as well asmany non\u2010steroidal anti\u2010inflammatory drugs, such as sulindac and indomethacin. Once activated bya ligand, the receptor forms a heterodimer with members of the retinoid X receptorfamily and can act as a transcription factor. Although radioligand binding assayshave been described for all three receptors, the radioligands are not commerciallyavailable. Commonly, receptor occupancy studies are conducted using fluorescentligands and truncated forms of the receptor limited to the ligand binding domain.Peroxisome proliferator\u2010activated receptors. Agonists with mixedactivity at PPAR\u03b1 and PPAR\u03b3 have also been described .As with the estrogen receptor antagonists, manyagents show tissue\u2010selective efficacy PPAR\u2010\u03b3as a therapeutic target in cardiovascular disease: evidence and uncertainty.J. Lipid Res.53: 1738\u201054 [PMID:22685322]Huang JV et al. (2006)International Union of Pharmacology. LXI. Peroxisome proliferator\u2010activatedreceptors. Pharmacol. Rev.58: 726\u201041 [PMID:17132851]Michalik L et al. (2008) PPARsMediate Lipid Signaling in Inflammation and Cancer. PPAR Res2008: 134059 [PMID:19125181]Michalik L et al. (2012) Therole of peroxisome proliferator\u2010activated receptors in carcinogenesis andchemoprevention. Nat. Rev. Cancer12: 181\u201095 [PMID:22318237]Peters JM et al. (2012)Targeting peroxisome proliferator\u2010activated receptors (PPARs): development ofmodulators. J. Med. Chem.55: 4027\u201061 [PMID:22260081]Pirat C et al. (2011) PPARsare a unique set of fatty acid regulated transcription factors controlling both lipidmetabolism and inflammation. Biochim. Biophys. Acta1812: 1007\u201022 [PMID:21382489]Varga T et al. (2011)Peroxisome proliferator\u2010activated receptors and cancer: challenges and opportunities.Br. J. Pharmacol.164: 68\u201082 [PMID:21449912]Youssef J nomenclature as agreedby theNC\u2010IUPHARSubcommittee on Nuclear Hormone Receptors [7]) have yet to be officially paired with an endogenous ligand, but arethought to be activated by heme. Rev\u2010erb receptors International Union of Pharmacology. LXVI. Orphan nuclear receptors.Pharmacol. Rev.58: 798\u2010836 [PMID:17132856]Benoit G et al. (2009)Rev\u2010erb\u2010alpha: an integrator of circadian rhythms and metabolism. J. Appl.Physiol.107: 1972\u201080 [PMID:19696364]Duez H et al. (2006)Overview of nomenclature of nuclear receptors. Pharmacol. Rev.58: 685\u2010704 [PMID:17132848]Germain P et al. (2010)Structural overview of the nuclear receptor superfamily: insights into physiology andtherapeutics. Annu. Rev. Physiol.72: 247\u201072 [PMID:20148675]Huang P et al. (2010)Structure of Rev\u2010erbalpha bound to N\u2010CoR reveals a unique mechanism of nuclearreceptor\u2010co\u2010repressor interaction. Nat. Struct. Mol. Biol.17: 808\u201014 [PMID:20581824]Phelan CA Mol. Cell. Endocrinol.334: 3\u201013 [PMID:20615454]Sladek FM. (2011) What are nuclear receptorligands? et al. (2010) Nuclearreceptor Rev\u2010erbalpha: a heme receptor that coordinates circadian rhythm andmetabolism. Nucl Recept Signal8: e001 [PMID:20414452]Yin L nomenclature as agreed by theNC\u2010IUPHARSubcommittee on Nuclear Hormone Receptors [7]) have yet to be assigned a definitive endogenous ligand, although ROR\u03b1may be synthesized with a \u2018captured\u2019 agonist such as cholesterol. Retinoic acid receptor\u2010related orphan receptorsInternational Union of Pharmacology. LXVI. Orphan nuclear receptors.Pharmacol. Rev.58: 798\u2010836 [PMID:17132856]Benoit G nomenclature as agreed by theNC\u2010IUPHARSubcommittee on Nuclear Hormone Receptors[105]) are members of a steroid analogue\u2010activated nuclear receptor subfamily,which form heterodimers with members of the retinoid X receptor family. Endogenousligands for LXRs include hydroxycholesterols (OHC), while FXRs appear to be activatedby bile acids. Liver X and farnesoid X receptors Liver X receptors as regulators of macrophage inflammatory and metabolic pathways.Biochim. Biophys. Acta1812: 982\u201094 [PMID:21193033]A\u2010Gonz\u00e1lez N et al. (2010) Liverx receptor signaling pathways and atherosclerosis. Arterioscler. Thromb.Vasc. Biol.30: 1513\u20108 [PMID:20631351]Calkin AC et al. (2011) Nuclearbile acid receptor FXR in the hepatic regeneration. Biochim. Biophys.Acta1812: 888\u201092 [PMID:21167938]Chen WD et al. (2011) Roleof nuclear receptors for bile acid metabolism, bile secretion, cholestasis, andgallstone disease. Biochim. Biophys. Acta1812: 867\u201078 [PMID:21194565]Claudel T et al. (2011)Liver X receptors, lipids and their reproductive secrets in the male.Biochim. Biophys. Acta1812: 974\u201081 [PMID:21334438]El\u2010Hajjaji FZ et al. (2010) Bileacids and their nuclear receptor FXR: Relevance for hepatobiliary andgastrointestinal disease. Biochim. Biophys. Acta1801: 683\u201092 [PMID:20399894]Gadaleta RM et al. (2011)Proteomics for the discovery of nuclear bile acid receptor FXR targets.Biochim. Biophys. Acta1812: 836\u201041 [PMID:21439373]Gardmo C Biochim. Biophys. Acta1812: 842\u201050 [PMID:21130162]Kemper JK. (2011) Regulation of FXRtranscriptional activity in health and disease: Emerging roles of FXR cofactors andpost\u2010translational modifications. et al. (2013) FXRsignaling in the enterohepatic system. Mol. Cell. Endocrinol.368: 17\u201029 [PMID:22609541]Matsubara T et al. (2006)International Union of Pharmacology. LXII. The NR1H and NR1I receptors: constitutiveandrostane receptor, pregnene X receptor, farnesoid X receptor alpha, farnesoid Xreceptor beta, liver X receptor alpha, liver X receptor beta, and vitamin D receptor.Pharmacol. Rev.58: 742\u201059 [PMID:17132852]Moore DD nomenclature as agreed by theNC\u2010IUPHARSubcommittee on Nuclear Hormone Receptors[105]) are members of the NR1I family of nuclear receptors, which formheterodimers with members of the retinoid X receptor family. PXR and CAR areactivated by a range of exogenous compounds, with no established endogenousphysiological agonists, although high concentrations of bile acids and bile pigmentsactivate PXR and CAR [105]. Vitamin D (VDR), Pregnane X (PXR) andConstitutive Androstane (CAR) receptors (Exp. Dermatol.20: 7\u201013 [PMID:21197695]Bikle DD. (2011) Vitamin D: an ancient hormone.et al. (2010) Theyin and yang of vitamin D receptor (VDR) signaling in neoplastic progression:operational networks and tissue\u2010specific growth control. Biochem.Pharmacol.79: 1\u20109 [PMID:19737544]Campbell FC et al. (2012) Nuclearreceptors in the multidrug resistance through the regulation of drug\u2010metabolizingenzymes and drug transporters. Biochem. Pharmacol.83: 1112\u201026 [PMID:22326308]Chen Y et al. (2012) PregnaneX receptor as a target for treatment of inflammatory bowel disorders. TrendsPharmacol. Sci.33: 323\u201030 [PMID:22609277]Cheng J et al. (2011)Nuclear receptor PXR, transcriptional circuits and metabolic relevance.Biochim. Biophys. Acta1812: 956\u201063 [PMID:21295138]Ihunnah CA et al. (2011)Constitutive androstane receptor (CAR) is a xenosensor and target for therapy.Biochemistry Mosc.76: 1087\u201097 [PMID:22098234]Kachaylo EM et al. (2006)International Union of Pharmacology. LXII. The NR1H and NR1I receptors: constitutiveandrostane receptor, pregnene X receptor, farnesoid X receptor alpha, farnesoid Xreceptor beta, liver X receptor alpha, liver X receptor beta, and vitamin D receptor.Pharmacol. Rev.58: 742\u201059 [PMID:17132852]Moore DD et al. (2010) VitaminD, disease and therapeutic opportunities. Nat Rev Drug Discov9: 941\u201055 [PMID:21119732]Plum LA et al. (2013)Nuclear\u2010receptor\u2010mediated regulation of drug\u2010 and bile\u2010acid\u2010transporter proteins ingut and liver. Drug Metab. Rev.45: 48\u201059 [PMID:23330541]Staudinger JL NC\u2010IUPHARSubcommittee on Nuclear Hormone Receptors [7]. While linoleic acid has been identified as the endogenous ligand forHNF4\u03b1 its function remains ambiguous [167]. HNF4\u03b3 has yet to bepaired with an endogenous ligand. The nomenclature of hepatocyte nuclear factor\u20104receptors is agreed by the et al. (2006)International Union of Pharmacology. LXVI. Orphan nuclear receptors.Pharmacol. Rev.58: 798\u2010836 [PMID:17132856]Benoit G et al. (2006)Overview of nomenclature of nuclear receptors. Pharmacol. Rev.58: 685\u2010704 [PMID:17132848]Germain P et al. (2010)Structural overview of the nuclear receptor superfamily: insights into physiology andtherapeutics. Annu. Rev. Physiol.72: 247\u201072 [PMID:20148675]Huang P et al.(2010) HNF4\u03b1\u2013role in drug metabolism and potential drug target? Curr OpinPharmacol10: 698\u2010705 [PMID:20833107]Hwang\u2010Verslues WW Mol. Cell. Endocrinol.334: 3\u201013 [PMID:20615454]Sladek FM. (2011) What are nuclear receptorligands? nomenclature asagreed by theNC\u2010IUPHARSubcommittee on Nuclear Hormone Receptors [45]) are NR2B family members activated by alitretinoin and theRXR\u2010selective agonists bexarotene and LG100268, sometimes referred toas rexinoids. UVI3003[109] and HX 531 [37] have been described as apan\u2010RXR antagonists. These receptors form RXR\u2010RAR heterodimers and RXR\u2010RXR homodimers. Retinoid X receptors Proteinkinases and the proteasome join in the combinatorial control of transcription bynuclear retinoic acid receptors. Trends Cell Biol.17: 302\u20109 [PMID:17467991]Bour G et al. (2011) Themolecular physiology of nuclear retinoic acid receptors. From health to disease.Biochim. Biophys. Acta1812: 1023\u201031 [PMID:20970498]Duong V et al. (2006)International Union of Pharmacology. LXIII. Retinoid X receptors. Pharmacol.Rev.58: 760\u201072 [PMID:17132853]Germain P et al. (2010)Retinoid X receptors: common heterodimerization partners with distinct functions.Trends Endocrinol. Metab.21: 676\u201083 [PMID:20674387]Lefebvre P Nat. Rev.Neurosci.8: 755\u201065 [PMID:17882253]Maden M. (2007) Retinoic acid in thedevelopment, regeneration and maintenance of the nervous system. et al. (2006) Functionof retinoid nuclear receptors: lessons from genetic and pharmacological dissectionsof the retinoic acid signaling pathway during mouse embryogenesis. Annu. Rev.Pharmacol. Toxicol.46: 451\u201080 [PMID:16402912]Mark M et al. (2011)Modulation of RXR function through ligand design. Biochim BiophysActa[PMID:21515403]P\u00e9rez E nomenclature asagreed by theNC\u2010IUPHARSubcommittee on Nuclear Hormone Receptors [7]) have yet to be officially paired with an endogenous ligand, althoughtesticular receptor 4 has been reported to respond to retinoids. Testicular receptors International Union of Pharmacology. LXVI. Orphan nuclear receptors.Pharmacol. Rev.58: 798\u2010836 [PMID:17132856]Benoit G et al. (2009)Rev\u2010erb\u2010alpha: an integrator of circadian rhythms and metabolism. J. Appl.Physiol.107: 1972\u201080 [PMID:19696364]Duez H et al. (2006)Overview of nomenclature of nuclear receptors. Pharmacol. Rev.58: 685\u2010704 [PMID:17132848]Germain P et al. (2010)Structural overview of the nuclear receptor superfamily: insights into physiology andtherapeutics. Annu. Rev. Physiol.72: 247\u201072 [PMID:20148675]Huang P et al. (2010)Structure of Rev\u2010erbalpha bound to N\u2010CoR reveals a unique mechanism of nuclearreceptor\u2010co\u2010repressor interaction. Nat. Struct. Mol. Biol.17: 808\u201014 [PMID:20581824]Phelan CA et al. (2010)Minireview: steroidogenic factor 1: its roles in differentiation, development, anddisease. Mol. Endocrinol.24: 1322\u201037 [PMID:20203099]Schimmer BP Mol. Cell. Endocrinol.334: 3\u201013 [PMID:20615454]Sladek FM. (2011) What are nuclear receptorligands? et al. (2010) Nuclearreceptor Rev\u2010erbalpha: a heme receptor that coordinates circadian rhythm andmetabolism. Nucl Recept Signal8: e001 [PMID:20414452]Yin L et al. (2011) Role ofnuclear receptor SHP in metabolism and cancer. Biochim. Biophys.Acta1812: 893\u2010908 [PMID:20970497]Zhang Y et al. (2010) NR4Aorphan nuclear receptors: transcriptional regulators of gene expression in metabolismand vascular biology. Arterioscler. Thromb. Vasc. Biol.30: 1535\u201041 [PMID:20631354]Zhao Y nomenclature asagreed by theNC\u2010IUPHARSubcommittee on Nuclear Hormone Receptors [7]) have yet to be officially paired with an endogenous ligand. Tailless\u2010like receptors International Union of Pharmacology. LXVI. Orphan nuclear receptors.Pharmacol. Rev.58: 798\u2010836 [PMID:17132856]Benoit G et al. (2006)Overview of nomenclature of nuclear receptors. Pharmacol. Rev.58: 685\u2010704 [PMID:17132848]Germain P et al. (2011) A tale oftailless. Dev. Neurosci.33: 1\u201013 [PMID:21124006]Gui H et al. (2010)Structural overview of the nuclear receptor superfamily: insights into physiology andtherapeutics. Annu. Rev. Physiol.72: 247\u201072 [PMID:20148675]Huang P Mol. Cell. Endocrinol.334: 3\u201013 [PMID:20615454]Sladek FM. (2011) What are nuclear receptorligands? nomenclature asagreed by theNC\u2010IUPHARSubcommittee on Nuclear Hormone Receptors [7]) have yet to be officially paired with an endogenous ligand. COUP\u2010TF\u2010like receptors International Union of Pharmacology. LXVI. Orphan nuclear receptors.Pharmacol. Rev.58: 798\u2010836 [PMID:17132856]Benoit G et al. (2006)Overview of nomenclature of nuclear receptors. Pharmacol. Rev.58: 685\u2010704 [PMID:17132848]Germain P et al. (2010)Structural overview of the nuclear receptor superfamily: insights into physiology andtherapeutics. Annu. Rev. Physiol.72: 247\u201072 [PMID:20148675]Huang P et al. (2011) Coupd'Etat: an orphan takes control. Endocr. Rev.32: 404\u201021 [PMID:21257780]Lin FJ Mol. Cell. Endocrinol.334: 3\u201013 [PMID:20615454]Sladek FM. (2011) What are nuclear receptorligands? nomenclatureas agreed by theNC\u2010IUPHARSubcommittee on Nuclear Hormone Receptors [7]) have yet to be officially paired with an endogenous ligand. Estrogen\u2010related receptors International Union of Pharmacology. LXVI. Orphan nuclear receptors.Pharmacol. Rev.58: 798\u2010836 [PMID:17132856]Benoit G et al. (2011)Functional and physiological genomics of estrogen\u2010related receptors (ERRs) in healthand disease. Biochim. Biophys. Acta1812: 1032\u201040 [PMID:21172432]Deblois G et al. (2013)Oestrogen\u2010related receptors in breast cancer: control of cellular metabolism andbeyond. Nat. Rev. Cancer13: 27\u201036 [PMID:23192231]Deblois G et al. (2006)Overview of nomenclature of nuclear receptors. Pharmacol. Rev.58: 685\u2010704 [PMID:17132848]Germain P et al. (2010)Structural overview of the nuclear receptor superfamily: insights into physiology andtherapeutics. Annu. Rev. Physiol.72: 247\u201072 [PMID:20148675]Huang P et al.(2010) HNF4\u03b1\u2013role in drug metabolism and potential drug target? Curr OpinPharmacol10: 698\u2010705 [PMID:20833107]Hwang\u2010Verslues WW Mol. Cell. Endocrinol.334: 3\u201013 [PMID:20615454]Sladek FM. (2011) What are nuclear receptorligands? nomenclature as agreed by theNC\u2010IUPHARSubcommittee on Nuclear Hormone Receptors [7]) have yet to be officially paired with an endogenous ligand. Nerve growth factor IB\u2010like receptorsInternational Union of Pharmacology. LXVI. Orphan nuclear receptors.Pharmacol. Rev.58: 798\u2010836 [PMID:17132856]Benoit G et al. (2006)Overview of nomenclature of nuclear receptors. Pharmacol. Rev.58: 685\u2010704 [PMID:17132848]Germain P et al. (2010)Structural overview of the nuclear receptor superfamily: insights into physiology andtherapeutics. Annu. Rev. Physiol.72: 247\u201072 [PMID:20148675]Huang P et al. (2011)Inflammation: a role for NR4A orphan nuclear receptors? Biochem. Soc.Trans.39: 688\u201093 [PMID:21428963]McMorrow JP et al. (2012)Molecular pathways: the role of NR4A orphan nuclear receptors in cancer.Clin. Cancer Res.18: 3223\u20108 [PMID:22566377]Mohan HM et al. (2010)Minireview: Nuclear hormone receptor 4A signaling: implications for metabolicdisease. Mol. Endocrinol.24: 1891\u2010903 [PMID:20392876]Pearen MA Mol. Cell. Endocrinol.334: 3\u201013 [PMID:20615454]Sladek FM. (2011) What are nuclear receptorligands? et al. (2010) NR4Aorphan nuclear receptors: transcriptional regulators of gene expression in metabolismand vascular biology. Arterioscler. Thromb. Vasc. Biol.30: 1535\u201041 [PMID:20631354]Zhao Y et al. (2012)NR4All in the vessel wall. J. Steroid Biochem. Mol. Biol.130: 186\u201093 [PMID:21277978]van Tiel CM nomenclature as agreed by theNC\u2010IUPHARSubcommittee on Nuclear Hormone Receptors [7]) have yet to be officially paired with an endogenous ligand. Fushi tarazu F1\u2010like receptorsInternational Union of Pharmacology. LXVI. Orphan nuclear receptors.Pharmacol. Rev.58: 798\u2010836 [PMID:17132856]Benoit G et al. (2012)Steroidogenic factor 1 and the central nervous system. 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(2010)The role of the orphan receptor SF\u20101 in the development and function of the ovary.Reprod Biol10: 177\u201093 [PMID:21113200]Mlnynarczuk J et al. (2010)Minireview: steroidogenic factor 1: its roles in differentiation, development, anddisease. Mol. Endocrinol.24: 1322\u201037 [PMID:20203099]Schimmer BP Mol. Cell. Endocrinol.334: 3\u201013 [PMID:20615454]Sladek FM. (2011) What are nuclear receptorligands? nomenclature as agreed by theNC\u2010IUPHARSubcommittee on Nuclear Hormone Receptors [7]) have yet to be officially paired with an endogenous ligand. Germ cell nuclear factor receptorsInternational Union of Pharmacology. LXVI. Orphan nuclear receptors.Pharmacol. Rev.58: 798\u2010836 [PMID:17132856]Benoit G et al. (2006)Overview of nomenclature of nuclear receptors. Pharmacol. Rev.58: 685\u2010704 [PMID:17132848]Germain P et al. (2010)Structural overview of the nuclear receptor superfamily: insights into physiology andtherapeutics. Annu. Rev. Physiol.72: 247\u201072 [PMID:20148675]Huang P Mol. Cell. Endocrinol.334: 3\u201013 [PMID:20615454]Sladek FM. (2011) What are nuclear receptorligands? nomenclature asagreed by theNC\u2010IUPHARSubcommittee on Nuclear Hormone Receptors [7]) have yet to be officially paired with an endogenous ligand. Dax\u2010like receptors International Union of Pharmacology. LXVI. Orphan nuclear receptors.Pharmacol. Rev.58: 798\u2010836 [PMID:17132856]Benoit G et al. (2008)Molecular basis of endocrine regulation by orphan nuclear receptor Small HeterodimerPartner. Endocr. J.55: 253\u201068 [PMID:17984569]Chanda D et al. (2012)Ligand\u2010independent actions of the orphan receptors/corepressors DAX\u20101 and SHP inmetabolism, reproduction and disease. J. Steroid Biochem. Mol. Biol.130: 169\u201079 [PMID:21550402]Ehrlund A et al. (2011) Roleof DAX\u20101 (NR0B1) and steroidogenic factor\u20101 (NR5A1) in human adrenal function.Endocr Dev20: 38\u201046 [PMID:21164257]El\u2010Khairi R et al. (2006)Overview of nomenclature of nuclear receptors. Pharmacol. Rev.58: 685\u2010704 [PMID:17132848]Germain P et al. (2010)Structural overview of the nuclear receptor superfamily: insights into physiology andtherapeutics. Annu. Rev. Physiol.72: 247\u201072 [PMID:20148675]Huang P et al. (2010)The role of the orphan receptor SF\u20101 in the development and function of the ovary.Reprod Biol10: 177\u201093 [PMID:21113200]Mlnynarczuk J Mol. Cell. Endocrinol.334: 3\u201013 [PMID:20615454]Sladek FM. (2011) What are nuclear receptorligands? et al. (2011) Role ofnuclear receptor SHP in metabolism and cancer. Biochim. Biophys.Acta1812: 893\u2010908 [PMID:20970497]Zhang Y nomenclatureas agreed by theNC\u2010IUPHARSubcommittee on Nuclear Hormone Receptors ) are nuclear hormone receptors of the NR3 class, with endogenousagonists that may be divided into 3\u2010hydroxysteroids (estrone and 17\u03b2\u2010estradiol) and3\u2010ketosteroids . These receptorsexist as dimers coupled with chaperone molecules and immunophilinFKBP52:FKBP4, Q02790), which are shed onbinding the steroid hormone. Although rapid signalling phenomena are observed, the principal signallingcascade appears to involve binding of the activated receptors to nuclear hormoneresponse elements of the genome, with a 15\u2010nucleotide consensus sequenceAGAACAnnnTGTTCT as homo\u2010 orheterodimers. They also affect transcription by protein\u2010protein interactions withother transcription factors, such as activator protein 1 (AP\u20101) and nuclear factor\u03baB (NF\u2010\u03baB). Splice variants of each of thesereceptors can form functional or non\u2010functional monomers that can dimerize to formfunctional or non\u2010functional receptors. For example, alternative splicing of PR mRNAproduces A and B monomers that combine to produce functional AA, AB and BB receptorswith distinct characteristics [150].Steroid hormone receptors has been described. Humanorthologues of 7TM 'membrane progestin receptors' , initially discovered in fish , appear to localize tointracellular membranes and respond to 'non\u2010genomic' progesterone analoguesindependently of G proteins [137].A 7TM receptor responsive to estrogen activity regulatesdiverse physiological processes R,R\u2010THC exhibits partialagonist activity at ER\u03b1. Estrogen receptors may beblocked non\u2010selectively by tamoxifen and raloxifene and labelled by3H]17\u03b2\u2010estradiol[and 3H]tamoxifen.3H]dexamethasone. Mutations in AR underlietesticular feminization and androgen insensibility syndromes, spinal and bulbarmuscular atrophy (Kennedy's disease)."} +{"text": "The correct name is: Vera L\u00facia Garcia Calich. The correct citation is: Ersching J, Basso AS, Calich VLG, Bortoluci KR, Rodrigues MM (2016) A Human Trypanosome Suppresses CD8+ T Cell Priming by Dendritic Cells through the Induction of Immune Regulatory CD4+ Foxp3+ T Cells. PLoS Pathog 12(6): e1005698. doi:"} +{"text": "The other five-membered ring adopts a twisted conformation with the twist being about the methine\u2013methyl\u00adene C\u2014C bond. The seven-membered ring is based on a twisted boat conformation. No specific inter\u00adactions are noted in the the crystal packing.The lactone ring in the title mol\u00adecule, C DOI: 10.1107/S2056989015002510/tk5358Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989015002510/tk5358Isup3.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989015002510/tk5358fig1.tif. DOI: A mol\u00adecule showing atom labels and 50% probability displacement ellipsoids for non-H atoms.1047797CCDC reference: crystallographic information; 3D view; checkCIF reportAdditional supporting information:"} +{"text": "Scientific Reports6: Article number: 20842; 10.1038/srep20842 published online: 02162016; updated: 04292016.In this Article, the Figure labels were omitted from Figure 5b. The correct Figure 5b appears below as"} +{"text": "The complex cation consists of two inversion-related [Co(C21H24N4)]2+ moieties bridged by a pair of fluoride ligands. The CoII cation is six-coordinated in a distorted octa\u00adhedral geometry and forms a +II high-spin state. In the crystal, the complex cation and the BF4\u2212 anion are connected by C\u2014H\u22efF hydrogen bonds, forming a three-dimensional network. An intra\u00admolecular C\u2014H\u22efF hydrogen bond is also observed.Reaction of Co(BF DOI: 10.1107/S1600536814021631/is5368Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S1600536814021631/is5368Isup3.docSupporting information file. DOI: Click here for additional data file.10.1107/S1600536814021631/is5368fig1.tif. DOI: Perspective view of the complex showing 50% displacement ellipsoids. Hydrogen atoms are omitted for clarity.1027138CCDC reference: crystallographic information; 3D view; checkCIF reportAdditional supporting information:"} +{"text": "Nucl. Acids Res. (2007) 35 (5): e29 doi: 10.1093/nar/gkl1134The panels were inadvertently duplicated in Figure"} +{"text": "The 4H-pyran ring fused with the naphthalene ring system has a flattened boat conformation. In the crystal, N\u2014H\u22efN hydrogen bonds generate chains along the b-axis direction. Further N\u2014H\u22efN hydrogen bonds link these chains into sheets parallel to (010). The crystal packing also features C\u2014H\u22ef\u03c0 inter\u00adactions. The crystal studied was an inversion twin with a 0.557\u2005(16):0.443\u2005(16) domain ratio.In the title compound, C DOI: 10.1107/S2056989015011159/sj5465Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989015011159/sj5465Isup3.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989015011159/sj5465fig1.tif. DOI: View of the title compound with the atom-numbering scheme. Displacement ellipsoids for non-H atoms are drawn at the 50% probability level.Click here for additional data file.10.1107/S2056989015011159/sj5465fig2.tifa . DOI: a axis, with hydrogen bonds drawn as dashed lines.Crystal packing of the title compound viewed along the Click here for additional data file.10.1107/S2056989015011159/sj5465fig3.tifb . DOI: b axis.A view of the packing showing mol\u00adecules stacked along the 1405640CCDC reference: crystallographic information; 3D view; checkCIF reportAdditional supporting information:"} +{"text": "Nature Communications6: Article number: 7347 10.1038/ncomms8347 (2015); Published 06122015; Updated 09082015This Article contains an error in"} +{"text": "In the anion, the sulfonic acid group is deprotonated and the dihedral angle between the planes of the carb\u00adoxy\u00adlic acid group and the benzene ring is 12.41\u2005(11)\u00b0. In the crystal, the anions are linked into inversion dimers by pairs of O\u2014H\u22efO hydrogen bonds, which generate R22(16) loops. The dications link the anion dimers into [10-2] chains via N\u2014H\u22efO hydrogen bonds.In the title mol\u00adecular salt, C DOI: 10.1107/S1600536814022673/hb7296Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S1600536814022673/hb7296Isup3.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S1600536814022673/hb7296fig1.tif. DOI: View of the asymmetry unit of (I) showing displacement ellipsoids at the 50% probability level. Symmetry code: (i) 2\u2013x, \u2013y, 1\u2013z.Click here for additional data file.10.1107/S1600536814022673/hb7296fig2.tif. DOI: The hydrogen-bonded chain of (I).1029402CCDC reference: crystallographic information; 3D view; checkCIF reportAdditional supporting information:"} +{"text": "Nature Communications6: Article number: 7868 10.1038/ncomms8868 (2015); Published: 07282015; Updated: 09182015The image in Supplementary Figure 1"} +{"text": "The publisher apologizes for this error. The correct citation is: Kory Westlund J, D\u2019Mello SK, Olney AM (2015) Motion Tracker: Camera-Based Monitoring of Bodily Movements Using Motion Silhouettes. PLoS ONE 10(6): e0130293. doi:"} +{"text": "Weak C\u2014H\u22efO inter\u00adactions help to direct the packing, forming sheets lying parallel to (020).In the title compound, C DOI: 10.1107/S2056989015008324/lr2135Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989015008324/lr2135Isup3.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989015008324/lr2135fig1.tif. DOI: The title mol\u00adecule with labeling scheme and 50% probability ellipsoids.Click here for additional data file.10.1107/S2056989015008324/lr2135fig2.tif . DOI: Packing viewed towards the (101062089CCDC reference: crystallographic information; 3D view; checkCIF reportAdditional supporting information:"} +{"text": "Scientific Reports6: Article number: 20144; 10.1038/srep20144 published online: 02042016; updated: 05202016.In this Article, Figure 3 is incorrect. The correct Figure 3 appears below as"} +{"text": "The deprotonated hydroxyl groups of the piperidineethanolate anions bridge CuII cations, forming the tetra\u00adnuclear complex. All piperidine rings display a chair conformation. In the crystal, there are no significant inter\u00admolecular inter\u00adactions present. The crystal studied was an inversion twin refined with a minor component of 0.18\u2005(5). In the title tetra\u00adnuclear compound, [Cu DOI: 10.1107/S1600536814009052/xu5784Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S1600536814009052/xu5784Isup3.cdxSupporting information file. DOI: 998731CCDC reference: crystallographic information; 3D view; checkCIF reportAdditional supporting information:"} +{"text": "The line through the I atoms forms an angle of 78.39\u2005(16)\u00b0 with the normal to the pyridine ring.In the title adduct, C DOI: 10.1107/S2056989015010518/rz5157Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989015010518/rz5157Isup3.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989015010518/rz5157fig1.tif. DOI: The mol\u00adecular structure of the title compound, with 50% probability displacement ellipsoids for non-H atoms.1404151CCDC reference: crystallographic information; 3D view; checkCIF reportAdditional supporting information:"} +{"text": "AbstractGyponareversa DeLong & Martinson, 1972 has its ovipositor described and illustrated based on the examination of specimens from its type locality. This is the first species of Gypona Germar, 1821 to have the female genitalia detailed description published. Gypona Germar, 1821 (Cicadellidae: Iassinae: Gyponini) includes about 200 described species (Gypona (G.) reversa DeLong & Martinson, 1972 was described based on male holotype and paratype . Coelho ae e.g., , we herewww.hadleyweb.pwp.blueyonder.co.uk). Terminalia were stored in a small vial with glycerin pinned below the specimen.The descriptive terminology adopted herein follows mainly The specimen was collected in a fragment of Atlantic Forest inserted on the Universidade Federal de Vi\u00e7osa (UFV) campus. UFV is located in Vi\u00e7osa municipality, \u201cZona da Mata\u201d of Minas Gerais State, southeastern Brazil and has The specimen examined belongs to Cole\u00e7\u00e3o Entomol\u00f3gica Jos\u00e9 Alfredo Pinheiro Dutra, Departamento de Zoologia (DZRJ), Instituto de Biologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.DeLong & Martinson, 1972Type status:Other material. Occurrence: recordedBy: Luci B. N. Coelho; individualCount: 1; sex: female; lifeStage: adult; Taxon: taxonID: Native; scientificNameID: lsid:zoobank.org:act:8B3CBB30-41F7-4CE3-ADD5-8C00FE4D4D6A; acceptedNameUsage: Gypona reversa DeLong & Martinson, 1972; kingdom: Animalia; phylum: Arthropoda; class: Hexapoda; order: Hemiptera; family: Cicadellidae; genus: Gypona; subgenus: Gypona; specificEpithet: reversa; scientificNameAuthorship: DeLong & Martinson, 1972; Location: continent: South America; country: Brazil; countryCode: BR; stateProvince: Minas Gerais; municipality: Vi\u00e7osa; locality: Mata da Biologia, Recanto das Cigarras; verbatimElevation: 650 m; verbatimCoordinates: 20\u00b045'31.8\"S 42\u00b051'40.5\"W; Identification: identifiedBy: Luci B. N. Coelho; dateIdentified: 1993; Event: eventDate: 08/09/1993; Record Level: language: en; institutionCode: DZRJ; collectionCode: Insects; ownerInstitutionCode: Cole\u00e7\u00e3o Entomol\u00f3gica Professor Jos\u00e9 Alfredo Pinheiro Dutra; basisOfRecord: PreservedSpecimenType status:Other material. Occurrence: recordedBy: Luci B. N. Coelho; individualCount: 1; sex: female; lifeStage: adult; Taxon: taxonID: Native; scientificNameID: lsid:zoobank.org:act:8B3CBB30-41F7-4CE3-ADD5-8C00FE4D4D6A; acceptedNameUsage: Gypona reversa DeLong & Martinson, 1973; kingdom: Animalia; phylum: Arthropoda; class: Hexapoda; order: Hemiptera; family: Cicadellidae; genus: Gypona; subgenus: Gypona; specificEpithet: reversa; Location: continent: South America; country: Brazil; countryCode: BR; stateProvince: Minas Gerais; municipality: Vi\u00e7osa; locality: Mata da Biologia, Recanto das Cigarras; verbatimElevation: 650 m; verbatimCoordinates: 20\u00b045'31.8\"S 42\u00b051'40.5\"W; Identification: identifiedBy: Luci B. N. Coelho; dateIdentified: 1993; Event: eventDate: 06/28/1993; Record Level: language: en; institutionCode: DZRJ; collectionCode: Insects; ownerInstitutionCode: Cole\u00e7\u00e3o Entomol\u00f3gica Professor Jos\u00e9 Alfredo Pinheiro Dutra; basisOfRecord: PreservedSpecimenGyponareversa DeLong & Martinson, 1972Redescription of femaleLength 7.8 mm. General color green with black and brown spots on pronotum and wings Figs , 2a. Cro2222Forewings translucent, apical region smoky, black spot at the insertion point; brown spot at the apex of each claval vein, inner discal cell with two brown spots, one basal and other apical; irregular brown band from clavus apex to median portion of fifth apical cell; about 3.3 times longer than wide; appendix brown, vestigial.Sternite VII Fig. a, c 2.4 Pygofer, in lateral view, approximately triangular shaped, with apex rounded; scattered macrosetae at posterior margin Fig. d. Ovipos3345545667676739General color green with black and brown spots on pronotum and wings Figs , 2a. Ste2337Brazil and Mexico .Wedeliapaludosa DC (Compositae).Specimens were collected in a grove of Atlantic forest, feeding on Studied specimens20\u00b045'31.8\"S 42\u00b051'40.5\"W , L.B.N. Coelho leg., 1 \u2640, 27/vi/1993; 1 \u2640, 09/viii/1993 (DZRJ).BRAZIL, Minas Gerais, Vi\u00e7osa, Universidade Federal de Vi\u00e7osa, Recanto das Cigarras, Gypona species. Gyponaverticalis St\u00e5l, 1864, which is quite shorter than in G.reversa (Fig. 6G.hiata DeLong and Freytag, 1967. Compared with G.hiata, the sternite VII of G.reversa has a more convex lateral margin, and posterior margin with two \"teeth\" separated by shallow concavity (Fig. 6This is the first published description of the ovipositor valvulae of any ity Fig. a, c; pygity Fig. a, d; venity Fig. b; apicalClinonana Osborn, 1988. Nevertheless, the authors believe that researchers should always make an effort to describe the female genitalia in as much detail as possible, so hopefully in the future it might become a source of useful taxonomic characteristics.According to"} +{"text": "The paper:VASOPRESSIN VS TERLIPRESSIN IN TREATMENT OF REFRACTORY SHOCK.Scarpati G., Piazza O.Transl Med UniSa. 2013 Jan 4;5:22\u20137. has been withdrawn after authors request.The paper:THE NOVEL THERAPEUTHIC TARGETS IN THE TREATMENT OF CHRONIC PAIN.Palomba R., Bonaccia P., Graffi M., Costa F. Transl Med UniSa. 2012 Apr 30;3:57\u201361. has been withdrawn after authors request."} +{"text": "Scientific Reports6: Article number: 2340310.1038/srep23403; published online: 03212016; updated: 05312016In this Article, Figure 5E and 5F were omitted. The Figure legends are correct. The correct Figure 5 appears below as"} +{"text": "Nature Communications6: Article number: 7721 10.1038/ncomms8721 (2015); Published 07132015; Updated 09082015The image in"} +{"text": "BMC Ear, Nose and Throat Disorders would like to thank all of our reviewers who have contributed to the journal in Volume 14 (2014).The editors of Ilona AndersonAustriaMinakshi BhardwajIndiaMarc BraemBelgiumItzhak BrookUSAJos\u00e9 Cameselle-TeijeiroSpainPeter W. DettmarUKY.P. Peter DiUSAWouter DreschlerNetherlandsCaterina FiniziaSwedenElizabeth FitzpatrickCanadaNoriyuki FujimaJapanKaryn GalvinAustraliaMiguel GoncalvesUKHasantha GunasekeraAustraliaHerv\u00e9 HaasFranceStefan HegemannSwitzerlandSebastian HothGermanyHiroaki IchijoJapanTakao ImaiJapanFikret KasapogluTurkeyTejs KlugDenmarkMasahiro KomoriJapanKenji KondoJapanSeung-Han LeeSouth KoreaDavid J LimUSAFernanda MarianoBrazilElismauro MendoncaBrazilRanko MladinaCroatiaMarkus MoessnerGermanyTsutomu NakashimaJapanGeorge NoussiosGreeceToyoaki OhbuchiJapanBolajoko O. OlusanyaNigeriaFredrik PeterssonSingaporeAntonio SchindlerItalyAiden ShearerUSASivakumaran Theru ArumugamUSAValentino ValentiniItalyFabiana ValeraBrazilKatrien VermeireBelgiumPetros VlastarakosUKChe-Ming WuTaiwanHasan YasanTurkeyB\u00fclent YaziciTurkey"} +{"text": "An intra\u00admolecular C\u2014H\u22efBr hydrogen bond forms an S(5) ring motif. In the crystal, weak C\u2014H\u22efBr inter\u00adactions link the mol\u00adecules into helical chains along the b-axis direction.In the title compound, [SbBr(C DOI: 10.1107/S1600536814020066/is5373Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S1600536814020066/is5373Isup3.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S1600536814020066/is5373fig1.tif. DOI: The mol\u00adecular structure of the title compound, showing 50% probability displacement ellipsoids. The dashed line indicates the C\u2014H\u22efBr hydrogen bond.Click here for additional data file.10.1107/S1600536814020066/is5373fig2.tif. DOI: A crystal packing view of the title compound. Dashed lines indicate hydrogen bonds. H atoms not involved in the hydrogen bonds have been omitted for clarity.1023098CCDC reference: crystallographic information; 3D view; checkCIF reportAdditional supporting information:"} +{"text": "The Li and In atoms are in distorted tetra\u00adhedral N2O2 and C2O2 bonding environments, respectively. The Li atom is further chelated by a tmeda group, yielding a spiro\u00adcyclic structure.The mixed bimetallic title compound, [InLi(CH DOI: 10.1107/S2056989015023476/lh5799Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989015023476/lh5799Isup4.cdxSupporting information file. DOI: Click here for additional data file.10.1107/S2056989015023476/lh5799fig1.tifI . DOI: I), with displacement ellipsoids drawn at the 50% probability level. H atoms have been omitted for clarity.The mol\u00adecular structure of (1440726CCDC reference: crystallographic information; 3D view; checkCIF reportAdditional supporting information:"} +{"text": "Scientific Reports6: Article number: 23027; 10.1038/srep23027 published online: 03142016; updated: 04292016.In this Article, Figure 4 is a duplication of Figure 6. The correct Figure 4 appears below as"} +{"text": "Scientific Reports5: Article number: 1643410.1038/srep16434; published online: 11102015; updated: 01222016This Article contains an error in Fig. 2, where the y-axis \u2018Herbage mass ln\u2009+\u20091(t/ha)\u2019 was incorrectly given as \u2018Herbage mass ln\u2009+\u20091(kg/ha)\u2019. The correct Fig. 2 appears below as"} +{"text": "The deprotonated ligands act as N,S-donors, forming five-membered metalla-rings. The NiII atom is four-coordinated in a slightly distorted square-planar environment. In the crystal, the discrete complex mol\u00adecules are linked by weak N\u2014H\u22efO hydrogen bonds, generating chains along [110]. The chains are further connected via weak O\u2014H\u22efN inter\u00adactions into a layered network extending parallel to (001).In the title compound, [Ni(C DOI: 10.1107/S1600536814009027/lr2126Isup2.hklStructure factors: contains datablock(s) I. DOI: 998671CCDC reference: crystallographic information; 3D view; checkCIF reportAdditional supporting information:"} +{"text": "Nature Communications 6: Article number: 8191 doi: 10.1038/ncomms9191 (2015); Published 09042015; Updated 10132015An incorrect version of the Supplementary Figure 1-4, Supplementary Table 1, Supplementary Methods and Supplementary References"} +{"text": "Nature Communications6: Article number: 10116 10.1038/ncomms10116 (2015); Published: 12022015; Updated: 04202016In Fig. 6f of this article, the histogram label \u2018Lsd1' should read \u2018IAP'. The correct version of Fig. 6 appears below.Figure 6"} +{"text": "AbstractIdiopyga Savchenko, 1987 is a northern hemisphere group of short-palped crane flies . In the current article we describe a new species, Dicranomyia (I.) boreobaltica Salmela sp.n., and redescribe the male and female post-abdomen of a closely related species, D. (I.) intricata Alexander. A standard DNA barcoding fragment of 5\u2032 region of the cytochrome c oxidase I (COI) gene of the new species is presented, whilst the K2P minimum distances between the new species and 10 other species of the subgenus were found to range from 5.1 to 15.7 % (mean 11.2 %). Phylogenetic analyses (parsimony and maximum likelihood) based on COI sequences support the identity of the new species and its close relationship with D. (I.) intricata and D. (I.) esbeni (Nielsen). The new species is known from the northern Baltic area of Finland. The new species has been mostly collected from Baltic coastal meadows but an additional relict population is known from a calcareous rich fen that was estimated to have been at sea level circa 600-700 years ago. Dicranomyia (I.) intricata (syn. D.suecica Nielsen) is a Holarctic species, occurring in the north boreal and subarctic vegetation zones in Fennoscandia.The subgenus D. (I.) magnicauda Lundstr\u00f6m, D. (I.) murina (Zetterstedt) and D. (I.) ponojensis Lundstr\u00f6m. A few species, or taxa that are recognised as subspecies, may have very restricted ranges (e.g. D. (I.) melleicaudastenoptera Savchenko [D. (I.) lulensis (Tjeder) [D. (I.) esbeni (Nielsen) [Dicranomyia (Idiopyga) species are characterised by a complicated structure of male hypopygium, having appendages on their ventral gonostylus and gonocoxite. The female cerci are very short in species such as D. (I.) intricata Alexander and D. (I.) lulensis whereas a normal length in e.g. D. (I.) halterella Edwards and D. (I.) ponojensis. Species of the subgenus occur around temperate and subarctic wetlands (Tjeder) ) or disjNielsen) ). Dicrannds e.g. , and somnds e.g. .Dicranomyia (I.) intricata was reported from Finland by Nieminen (http://www.finbol.org/eng/ENG_finbol.html), an abnormal intraspecific divergence was noted among the sequenced D. (I.) intricata specimens. Followed up by morphological examinations of voucher specimens, it was obvious that instead of one species there were two, distinguishable on both molecular and morphological basis (D. (I.) intricata, D.suecica (Nielsen)) allowed us to associate our morphospecies to taxonomic species. In the current article we describe a new Dicranomyia species from Finland and redescribe male and female post-abdomen of D. (I.) intricata. The new species is hitherto known from the northern Baltic area whereas D. (I.) intricata has a Holarctic range. To summarize, the approach we use is integrative was sequenced from a total of 22 Dicranomyia specimens and one Metalimnobia specimen. Legs or 2\u20133 abdominal segments of the specimens were placed in 96% ethanol in a 96-well lysis microplate and dispatched to the Canadian Centre for DNA Barcoding, Biodiversity Institute of Ontario where DNA was extracted and sequenced using standard protocols and primers (D. (I.) esbeni for which the last 51 basepairs were missing due to sequencing problems. The new sequences are deposited in GenBank under accession numbers KP064165-KP064187 analysis in TNT version 1.1 . Nodal sMaximum likelihood approachMaximum likelihood analysis was conducted with RAxML ver. 8.0.22 in the CSalmelasp. n.urn:lsid:zoobank.org:act:5537A033-CC12-41C0-869C-F603D8775005Type status:Holotype. Occurrence: catalogNumber: JES-20120094; recordedBy: T. Nieminen; individualCount: 1; sex: male; Taxon: genus: Dicranomyia; subgenus: Idiopyga; specificEpithet: boreobaltica; scientificNameAuthorship: Salmela; Location: country: Finland; stateProvince: Ostrobothnia borealis pars ouluensis; verbatimLocality: Oulunsalo, Papinkari; verbatimLatitude: 64.9060; verbatimLongitude: 25.3764; verbatimCoordinateSystem: decimal degrees; verbatimSRS: WGS84; Event: samplingProtocol: Malaise trap; eventDate: 2005-8-11/10-8; habitat: Baltic coastal meadow; Record Level: institutionCode: ZMUTType status:Paratype. Occurrence: recordedBy: T. Nieminen; individualCount: 1; sex: male; Taxon: genus: Dicranomyia; subgenus: Idiopyga; specificEpithet: boreobaltica; scientificNameAuthorship: Salmela; Location: country: Finland; stateProvince: Ostrobothnia borealis pars ouluensis; verbatimLocality: Hailuoto, P\u00f6k\u00f6nnokka; verbatimLatitude: 65.0790; verbatimLongitude: 24.8883; verbatimCoordinateSystem: decimal degrees; verbatimSRS: WGS84; Event: samplingProtocol: Malaise trap; eventDate: 2005-8-11/10-8; habitat: Baltic coastal meadow; Record Level: institutionCode: JSOType status:Paratype. Occurrence: recordedBy: T. Nieminen; individualCount: 1; sex: male; Taxon: genus: Dicranomyia; subgenus: Idiopyga; specificEpithet: boreobaltica; scientificNameAuthorship: Salmela; Location: country: Finland; stateProvince: Ostrobothnia borealis pars ouluensis; verbatimLocality: Hailuoto, P\u00f6k\u00f6nnokka; verbatimLatitude: 65.0790; verbatimLongitude: 24.8883; verbatimCoordinateSystem: decimal degrees; verbatimSRS: WGS84; Event: samplingProtocol: Malaise trap; eventDate: 2005-8-11/10-8; habitat: Baltic coastal meadow; Record Level: institutionCode: ZMUTType status:Paratype. Occurrence: catalogNumber: DIPT-JS-2014-0248; recordedBy: J. Salmela; individualCount: 1; sex: male; Taxon: genus: Dicranomyia; subgenus: Idiopyga; specificEpithet: boreobaltica; scientificNameAuthorship: Salmela; Location: country: Finland; stateProvince: Ostrobothnia borealis pars borealis; verbatimLocality: Tornio, Isonkummunj\u00e4nk\u00e4 Mire Conservation Area, Kusiaiskorpi; verbatimLatitude: 65.8880; verbatimLongitude: 24.4792; verbatimCoordinateSystem: decimal degrees; verbatimSRS: WGS84; Event: samplingProtocol: Malaise trap; eventDate: 2013-8-1/9-26; habitat: Rich fen, rusty spring; Record Level: institutionCode: JESType status:Paratype. Occurrence: catalogNumber: DIPT-JS-2014-0251; recordedBy: J. Salmela; individualCount: 1; sex: female; Taxon: genus: Dicranomyia; subgenus: Idiopyga; specificEpithet: boreobaltica; scientificNameAuthorship: Salmela; Location: country: Finland; stateProvince: Ostrobothnia borealis pars borealis; verbatimLocality: Tornio, Isonkummunj\u00e4nk\u00e4 Mire Conservation Area, Kusiaiskorpi; verbatimLatitude: 65.8880; verbatimLongitude: 24.4792; verbatimCoordinateSystem: decimal degrees; verbatimSRS: WGS84; Event: samplingProtocol: Malaise trap; eventDate: 2013-8-1/9-26; habitat: Rich fen, rusty spring; Record Level: institutionCode: JESType status:Paratype. Occurrence: recordedBy: J. Salmela; individualCount: 1; sex: female; Taxon: genus: Dicranomyia; subgenus: Idiopyga; specificEpithet: boreobaltica; scientificNameAuthorship: Salmela; Location: country: Finland; stateProvince: Ostrobothnia borealis pars borealis; verbatimLocality: Tornio, Isonkummunj\u00e4nk\u00e4 Mire Conservation Area, Kusiaiskorpi; verbatimLatitude: 65.8880; verbatimLongitude: 24.4792; verbatimCoordinateSystem: decimal degrees; verbatimSRS: WGS84; Event: samplingProtocol: Malaise trap; eventDate: 2013-8-1/9-26; habitat: Rich fen, rusty spring; Record Level: institutionCode: ZMUTType status:Other material. Occurrence: catalogNumber: DIPT-JS-2014-0114; recordedBy: J. Salmela; individualCount: 7; sex: 4 females, 3 males; Taxon: genus: Dicranomyia; subgenus: Idiopyga; specificEpithet: boreobaltica; scientificNameAuthorship: Salmela; Location: country: Finland; stateProvince: Ostrobothnia borealis pars borealis; verbatimLocality: Tornio, Isonkummunj\u00e4nk\u00e4 Mire Conservation Area, Kusiaiskorpi; verbatimLatitude: 65.8880; verbatimLongitude: 24.4792; verbatimCoordinateSystem: decimal degrees; verbatimSRS: WGS84; Event: samplingProtocol: Malaise trap; eventDate: 2013-8-1/9-26; habitat: Rich fen, rusty spring; Record Level: institutionCode: JESDicranomyia (Idiopyga) intricataDicranomyia (Idiopyga) cf.intricataHead. Vertex dark brown, with short black setae. Rostrum light brown with a few short dark setae. Palpus 5-segmented; first palpomere very short, globular, 1.5 times wider than long; other palpomeres elongated, p2 length 140 \u00b5m, p3 100 \u00b5m, p4 100 \u00b5m and p5 120 \u00b5m. First palpomere with a long ventral seta, approximately 2 times longer than width of palpomere. Second and third palpomeres with 5 setae, arranged in the apical half of segments. Fourth palpomere bearing ca. 12 setae and p5 with 13-15 setae, most of these on the apices of the segments. Antennae 14-segmented, dark brown, segments bearing black setae mostly exceeding width of respective segment; setae straight on scape (ca. 10 setae) and pecidel (ca. 15 setae), straight or curved on flagellomeres (ca. 5 setae on each flagellomere). Scape cylindrical, length 200 \u00b5m, width 75 \u00b5m, pedicel wider apically than basally, length 115 \u00b5m, width 75 \u00b5m. Flagellomeres oval, longer than wide; f1 length 120 \u00b5m, width 65 \u00b5m, f2 length 8 \u00b5m, width 5 \u00b5m, f10 length 110 \u00b5m, width 40 \u00b5m. Thorax mainly dark brown. Prescutum dark brown, only small yellowish spots on hind lateral corners. Scutum dark brown with longitudinal yellow median line and yellow lateral spots near wing base. Mediotergite and anepisternum dark brown, mediotergite sometimes with narrow yellowish anterior margin. Laterotergite and anepimeron yellowish brown. Katepisternum bicolored: anterior half dark brown, posterior half yellowish brown. Fore coxa brown, mid and hind coxae yellowish brown. Femorae light brown or brown, tibiae and tarsi dark brown. Length of fore femora 4500 \u00b5m, tibia 5250 \u00b5m, t1 3500 \u00b5m, t2 1100 \u00b5m, t3 875 \u00b5m, t4 300 \u00b5m, t5 175 \u00b5m, claw 130 \u00b5m. Length of mid femora 5575 \u00b5m, tibia 5625 \u00b5m, t1 3200 \u00b5m, t2 1150 \u00b5m, t3 625 \u00b5m, t4 275 \u00b5m, t5 175 \u00b5m, claw 130 \u00b5m. Length of hind femora 5600 \u00b5m, tibia 5750 \u00b5m, t1 3050 \u00b5m, t2 1150, t3 650 \u00b5m, t4 275 \u00b5m, t5 178 \u00b5m, claw 130 \u00b5m. Halter grayish-brown. Wing clear, veins light brown - brown, pterostigma brown intricata. Ventrobasal lobe of ventral gonostylus sinuous, apex oval. Inner appendage of gonocoxite apically rounded. Rostral prolongantion of ventral apically rather narrow and subrostral prolongation simple, not bilobed, bearing dark stout spines. Female infra-anal plate with strong caudal peak.Brownish, small species, very close to borealis, Latin)= north, baltica (Latin)= referring to the Baltic Sea. The species is so far known from the northern Baltic area. The species name is deemed to be a latinized adjective in nominative singular.Boreo intricata). These coastal meadows are produced by a phenomenon called land uplift, that is, the rebound of earth's crust after the retreating of the ice sheet; in the Bothnian Bay the rate of land uplift is about 8 mm/year (D. (I.) boreobaltica Salmela sp.n. is a recent relict species in the fen. It should be noted that some plants typical for the Baltic shores or brackish water have isolated populations on calcareous ponds or mires far from coastal areas boreobaltica Salmela sp.n. was absent from the samples. This and other negative records (i.e. absence) from >500 Malaise trapping sites in Finnish wetlands and insect species in the Baltic coastal areas (D. (I.) boreobaltica Salmela sp.n. could either be i) a recently evolved allopatric species that survived Pleistocene glaciations and is currently only present in the Baltic area or ii) a disjunct species having populations in other areas.Due to its apparent rarity, that is, small area of occupancy and extent of occurrence, the species could most likely be assessed as a threatened species according to IUCN criteria. Habitats of this species are highly endangered, usually small and isolated. There are a total of ca. 4200 ha of Baltic coastal meadows along the Finnish coast, and all such habitat types are red-listed . Also spal areas . Hence, D. (I.) intricata. As already stated in the title of this article, the new species is cryptic, meaning that it is hard to distinguish from its sister species by morphological characters. Strictly speaking, cryptic species may mean taxa that are morphologically indisguishable boreobaltica Salmela sp.n. and D. (I.) intricata are not separated by a distinct barrier, they have non-overlapping ranges perhaps because of biogeographic factors driven by climate boreobaltica Salmela sp.n. and D. (I.) intricata are practically identical. The most important differences in male and female post-abdomen between the species are summarized in Table D. (Idiopyga) species, the new species is quite close to D. (I.) esbeni. Besides other details, the ventrobasal lobe of gonocoxite in D. (I.) esbeni is sinuous (see D. (I.) boreobaltica Salmela sp.n. Dicranomyia (I.) melleicauda complicata de Meijere is also quite close to the new species, but has rather stout iagx and apically wide lvg sequence of Dicranomyia (I.) boreobaltica Salmela sp.n. BOLD Sample ID JES-20120094, holotype specimen:Standard 5\u2032 region (658 bp) of the cytochrome TACCTTATACTTTATTTTTGGAGCTTGAGCAGGAATAGTGGGAACTTCATTAAGTATTATTATTCGAGCAGAATTAGGACACCCAGGTGCATTAATTGGAGACGACCAGATTTATAATGTGGTAGTTACTGCCCATGCTTTTATTATAATTTTCTTTATAGTTATACCAATTATAATTGGAGGATTCGGTAATTGATTAGTTCCTTTAATATTAGGAGCCCCAGATATAGCTTTCCCTCGAATAAATAATATAAGTTTTTGAATACTTCCCCCTTCTTTAACTTTATTATTAGCTAGAAGCATAGTTGAAAACGGGGCAGGAACTGGCTGAACAGTATACCCTCCCCTTTCTTCTGGAATTGCCCATTCAGGGGCTTCTGTAGATTTAGCTATTTTTTCTCTTCACCTAGCAGGTATTTCTTCTATTTTAGGAGCTGTTAATTTTATTACAACTGTTATTAATATACGTTCAGCAGGAATTTCATTTGATCGAATACCATTATTTGTTTGATCAGTAGTAATTACTGCTATTTTATTGCTTTTATCACTTCCTGTTTTAGCCGGAGCTATTACAATATTATTAACAGATCGAAACTTAAATACTTCATTTTTTGATCCCGCAGGTGGAGGAGACCCTATTTTATATCAGCATTTATTTD. (I.) intricata (K2P distance 5.13 %), D. (I.) esbeni (7.16 %) and D. (I.) magnicauda (9.51 %); other distances within examined D. (Idiopyga) species range between 10.76 and 15.70 %.Based on K2P distanceAlexander, 1927Type status:Holotype. Occurrence: recordedBy: O. Bryant; individualCount: 1; sex: male; Taxon: genus: Dicranomyia; subgenus: Idiopyga; specificEpithet: intricata; scientificNameAuthorship: Alexander; Location: country: Canada; stateProvince: Alberta; verbatimLocality: Lesser Slave Lake; verbatimLatitude: 55.35; verbatimLongitude: -115.09; verbatimCoordinateSystem: decimal degrees; verbatimSRS: WGS84; Event: eventDate: 1924-8-1; Record Level: institutionCode: USNMType status:Paratype. Occurrence: recordedBy: O. Bryant; individualCount: 1; sex: male; Taxon: genus: Dicranomyia; subgenus: Idiopyga; specificEpithet: intricata; scientificNameAuthorship: Alexander; Location: country: Canada; stateProvince: Alberta; verbatimLocality: Lesser Slave Lake, Grizzly mt.; minimumElevationInMeters: 914; Event: eventDate: 1924-8-15; Record Level: institutionCode: USNMType status:Holotype. Occurrence: catalogNumber: 855; recordedBy: H. Frantz; individualCount: 1; sex: male; Taxon: genus: Dicranomyia; subgenus: Idiopyga; specificEpithet: suecica; scientificNameAuthorship: Nielsen; Location: country: Sweden; stateProvince: Abisko; verbatimLatitude: 68.35; verbatimLongitude: 18.79; verbatimCoordinateSystem: decimal degrees; verbatimSRS: WGS84; Event: eventDate: unknown; Record Level: institutionCode: ZMUCType status:Other material. Occurrence: recordedBy: P.T. Bruggemann; individualCount: 1; sex: male; Taxon: genus: Dicranomyia; subgenus: Idiopyga; specificEpithet: intricata; scientificNameAuthorship: Alexander; Location: country: Canada; stateProvince: Yukon; verbatimLocality: Dawson; minimumElevationInMeters: 335; Event: eventDate: 1949-8-6; Record Level: institutionCode: USNMType status:Other material. Occurrence: recordedBy: W.W. Moss; individualCount: 1; sex: male; Taxon: genus: Dicranomyia; subgenus: Idiopyga; specificEpithet: intricata; scientificNameAuthorship: Alexander; Location: country: Canada; stateProvince: British Columbia; verbatimLocality: Telegraph Creek; minimumElevationInMeters: 335; Event: eventDate: 1960-8-28; Record Level: institutionCode: USNMType status:Other material. Occurrence: recordedBy: W.W. Moss; individualCount: 1; sex: male; Taxon: genus: Dicranomyia; subgenus: Idiopyga; specificEpithet: intricata; scientificNameAuthorship: Alexander; Location: country: Canada; stateProvince: British Columbia; verbatimLocality: Telegraph Creek, Sawmill Lake; Event: eventDate: 1960-8-18; Record Level: institutionCode: USNMType status:Other material. Occurrence: recordedBy: O. Bryant; individualCount: 1; sex: male; Taxon: genus: Dicranomyia; subgenus: Idiopyga; specificEpithet: intricata; scientificNameAuthorship: Alexander; Location: country: Canada; stateProvince: Northwest Territories; verbatimLocality: Aklavik; Event: eventDate: 1931-8-27; Record Level: institutionCode: USNMType status:Other material. Occurrence: catalogNumber: JES-20120082; recordedBy: J. Salmela; individualCount: 1; sex: female; Taxon: genus: Dicranomyia; subgenus: Idiopyga; specificEpithet: intricata; scientificNameAuthorship: Alexander; Location: country: Finland; stateProvince: Lapponia kemensis pars occidentalis; verbatimLocality: Kittil\u00e4, Mustaoja-Nunaravuoma Mire Conservation Area, Mustaoja W; verbatimLatitude: 67.6390; verbatimLongitude: 25.4277; verbatimCoordinateSystem: decimal degrees; verbatimSRS: WGS84; Event: samplingProtocol: sweep net; eventDate: 2009-8-19; habitat: rich flark fen; Record Level: institutionCode: ZMUTType status:Other material. Occurrence: catalogNumber: DIPT-JS-2014-0336; recordedBy: J. Salmela; individualCount: 32; sex: 29 male, 3 female; Taxon: genus: Dicranomyia; subgenus: Idiopyga; specificEpithet: intricata; scientificNameAuthorship: Alexander; Location: country: Finland; stateProvince: Lapponia enontekiensis; verbatimLocality: Enonteki\u00f6, Tarvantovaara Wilderness Area, Tomuttirova W; verbatimLatitude: 68.6369; verbatimLongitude: 22.5381; verbatimCoordinateSystem: decimal degrees; verbatimSRS: WGS84; Event: samplingProtocol: sweep net; eventDate: 2009-8-26; habitat: swampy flark fen; Record Level: institutionCode: JESType status:Other material. Occurrence: catalogNumber: JES-20110082; recordedBy: J. Salmela; individualCount: 1; sex: male; Taxon: genus: Dicranomyia; subgenus: Idiopyga; specificEpithet: intricata; scientificNameAuthorship: Alexander; Location: country: Finland; stateProvince: Lapponia enontekiensis; verbatimLocality: Enonteki\u00f6, Tarvantovaara Wilderness Area, Tomuttirova W; verbatimLatitude: 68.6369; verbatimLongitude: 22.5381; verbatimCoordinateSystem: decimal degrees; verbatimSRS: WGS84; Event: samplingProtocol: sweep net; eventDate: 2009-8-26; habitat: swampy flark fen; Record Level: institutionCode: ZMUTType status:Other material. Occurrence: catalogNumber: DIPT-JS-2014-0337; recordedBy: J. Salmela; individualCount: 10; sex: 5 male, 5 female; Taxon: genus: Dicranomyia; subgenus: Idiopyga; specificEpithet: intricata; scientificNameAuthorship: Alexander; Location: country: Finland; stateProvince: Lapponia enontekiensis; verbatimLocality: Enonteki\u00f6, Tarvantovaara Wilderness Area, Tomuttirova N; verbatimLatitude: 68.6391; verbatimLongitude: 22.5518; verbatimCoordinateSystem: decimal degrees; verbatimSRS: WGS84; Event: samplingProtocol: sweep net; eventDate: 2009-8-26; habitat: intermediate rich flark fen; Record Level: institutionCode: JESType status:Other material. Occurrence: catalogNumber: DIPT-JS-2014-0182; recordedBy: J. Salmela; individualCount: 1; sex: male; Taxon: genus: Dicranomyia; subgenus: Idiopyga; specificEpithet: intricata; scientificNameAuthorship: Alexander; Location: country: Finland; stateProvince: Lapponia kemensis pars orientalis; verbatimLocality: Sodankyl\u00e4, Pomokaira-Tenni\u00f6aapa Mire Conservation Area, Syv\u00e4kuru; verbatimLatitude: 67.8718; verbatimLongitude: 26.2126; verbatimCoordinateSystem: decimal degrees; verbatimSRS: WGS84; Event: samplingProtocol: Malaise trap; eventDate: 2013-8-15/9-19; habitat: spring fen; Record Level: institutionCode: JESType status:Other material. Occurrence: catalogNumber: DIPT-JS-2014-0338; recordedBy: J. Salmela; individualCount: 4; sex: 1 male, 3 female; Taxon: genus: Dicranomyia; subgenus: Idiopyga; specificEpithet: intricata; scientificNameAuthorship: Alexander; Location: country: Finland; stateProvince: Ostrobothnia borealis pars borealis; verbatimLocality: Kemij\u00e4rvi, Salmiaavanhete; verbatimLatitude: 66.9929; verbatimLongitude: 27.0578; verbatimCoordinateSystem: decimal degrees; verbatimSRS: WGS84; Event: samplingProtocol: sweep net; eventDate: 2009-8-15; habitat: rich flark fen; Record Level: institutionCode: JESType status:Other material. Occurrence: catalogNumber: DIPT-JS-2014-0340; recordedBy: J. Salmela; individualCount: 1; sex: 1 female; Taxon: genus: Dicranomyia; subgenus: Idiopyga; specificEpithet: intricata; scientificNameAuthorship: Alexander; Location: country: Finland; stateProvince: Lapponia inariensis; verbatimLocality: Inari, Kaunisp\u00e4\u00e4; verbatimLatitude: 68.4461; verbatimLongitude: 27.4351; verbatimCoordinateSystem: decimal degrees; verbatimSRS: WGS84; Event: samplingProtocol: sweep net; eventDate: 2013-8-16; habitat: alpine wetland; Record Level: institutionCode: JESDicranomyiaintricataLimonia (Dicranomyia) suecicaLimonia (Dicranomyia) suecicaDicranomyia (Idiopyga) intricataD. (I.) intricata intricata by D.suecica is lost , but based on Tjeder's detailed illustrations this nomenclature can be verified. Alexander's original description is good, and there is no need to thoroughly re-describe this species. However, male and female genitalia are illustrated here and diagnostic characters are discussed under D. (I.) boreobaltica Salmela sp.n.The holotype specimen of ata Fig. a is in gata Fig. , fig. 1 ata Fig. b. The hoy Tjeder and the D. (I.) boreobaltica Salmela sp.n., see Fig. 101010Male hypopygium. 9th tergite and proctiger as in Fig. 10Female postabdomen. Cerci and hypogynial valves, see Fig. D. (I.) intricata is known from the north boreal ecoregion, both from the zone of coniferous forests and from the subarctic fell area in the northernmost part of the country , Sweden intricata (Sphagnum mosses (http://en.wikipedia.org/wiki/Muskeg). Most of the Finnish sampling sites are aapamires, that is, minerotrophic fens with wet, usually moss covered, flarks (hollows) and drier hummock-level strings. Most of the sites are intermediate rich or rich fens, characterised by brown mosses . The species was especially abundant on two closely lying intermediate rich, Sphagnum dominated aapamires in Enonteki\u00f6, NW Finnish Lapland, but single specimens were also caught along a spring and a headwater stream intricata is red-listed in Finland intricata is absent from the northern Baltic coastal area and is replaced there by a sibling species (D. (I.) boreobaltica sp.n. Salmela). Thus the range (\"extent of occurrence\") of D. (I.) intricata is actually smaller that was thought in the 2010 assessment. However, the species is not extremely rare and there are most likely hundreds of square kilometres of suitable breeding sites for the species in Finnish Lapland. Nevertheless, the species may be jeopardized by climate change and it may also be used as an indicator of pristine boreal mires.land NT, . At the Dicranomyia (I.) boreobaltica Salmela sp.n.See c oxidase I (COI) gene of Dicranomyia (I.) intricata :Standard 5\u2032 region (658 bp) of the cytochrome TACCTTATACTTTATTTTTGGAGCTTGAGCAGGAATAGTAGGAACTTCACTAAGTATTATTATTCGAGCAGAATTAGGACACCCAGGAGCATTAATTGGAGATGACCAAATTTATAATGTAGTAGTTACTGCCCATGCTTTTATTATAATTTTTTTTATAGTTATACCAATTATAATTGGTGGATTCGGTAATTGATTAGTTCCTTTAATATTAGGAGCCCCAGATATAGCTTTCCCTCGAATAAATAATATAAGTTTTTGAATACTTCCCCCTTCTTTAACCTTATTATTAGCTAGAAGTATAGTTGAAAACGGGGCAGGAACTGGTTGAACAGTTTACCCTCCCCTTTCTTCTGGAATTGCTCATTCAGGAGCTTCTGTAGACTTAGCTATTTTTTCTCTTCATTTAGCAGGTATTTCTTCTATTTTAGGAGCTGTTAACTTTATTACAACTGTTATTAATATACGTTCAGCAGGAATTTCATTCGACCGAATACCATTATTTGTTTGATCAGTAGTAATTACTGCTATTCTATTACTCTTATCACTCCCTGTTTTAGCTGGAGCTATTACAATATTATTAACAGATCGAAACTTAAACACTTCATTTTTTGACCCTGCAGGTGGAGGAGATCCTATTTTATACCAACACTTATTTL = -3501.956818) in Fig. 13D. (I.) boreobaltica Salmela sp.n. as sister to D. (I.) intricata and these two species grouping together with D. (I.) esbeni. Nodal supports in both results are good giving strong indications that D. (I.) boreobaltica Salmela sp.n. forms its own group being in a sister group relationship with D. (I.) intricata. There are altogether 31 bases in COI that differ between D. (I.) boreobaltica Salmela sp.n. and D. (I.) intricata, resulting in 4.7 % difference between these species. Although nodal supports in the deeper, evolutionary older nodes are low, separate species are clearly distinct.The phylogenetic tree (length 622 steps) resulting from the parsimony analysis is shown in Fig. 13Supplementary material 1Diptera, Limoniidae), USNMDicranomyia (I.) intricata Alexander, 1927 (Data type: imagesDicranomyia (I.) intricata Alexander, males, permanently slide-mounted by C.P. Alexander, deposited in USNM . Digital photos of the slides.Brief description: Non-type material of File: oo_33643.pdfJukka SalmelaSupplementary material 2COI sequences of Dicranomyia (Idiopyga) species and three out-group speciesData type: GenomicBrief description: COI 5' standard DNA barcoding fragmentFile: oo_33852.txtJukka Salmela"} +{"text": "The incidence of oropharyngeal cancer is increasing in the developed world. This has led to a large rise in research activity and clinical trials in this area, yet there is no consensus on which outcomes should be measured. As a result, the outcomes measured often differ between trials of comparable interventions, making the combination or comparison of results between trials impossible. Outcomes may also be \u2018cherry-picked\u2019, such that favourable results are reported, and less favourable results withheld. The development of a minimum outcome reporting standard, known as a core outcome set, goes some way to addressing these problems. Core outcome sets are ideally developed using a patient-centred approach so that the outcomes measured are relevant to patients and clinical practice. Core outcome sets drive up the quality and relevance of research by ensuring that the right outcomes are consistently measured and reported in trials in specific areas of health or healthcare.This is a mixed methods study involving three phases to develop a core outcome set for oropharyngeal cancer clinical trials. Firstly, a systematic review will establish which outcomes are measured in published oropharyngeal cancer randomised controlled trials (RCTs). Secondly, qualitative interviews with patients and carers in the UK and the USA will aim to establish which outcomes are important to these stakeholders. Data from these first two stages will be used to develop a comprehensive list of outcomes to be considered for inclusion in the core outcome set. In the third stage, patients and clinicians will participate in an iterative consensus exercise known as a Delphi study to refine the contents of the core outcome set. This protocol lays out the methodology to be implemented in the CONSENSUS study.A core outcome set defines a minimum outcome reporting standard for clinical trials in a particular area of health or healthcare. Its consistent implementation in oropharyngeal cancer clinical trials will improve the quality and relevance of research.13823 (17 January 2013).This study is registered at the National Institute for Health Research (NIHR) Clinical Research Network (CRN) portfolio, ID Around 1,500 cases of oropharyngeal squamous cell carcinoma (OPSCC) are diagnosed in the UK every year 2. (\u201cHead and Neck Neoplasms\u201d[Mesh:NoExp])3. \u201cOtorhinolaryngologic Neoplasms\u201d[Mesh:NoExp]4. \u201cPharyngeal Neoplasms\u201d[Mesh:NoExp]5. \u201cNeoplasms\u201d[Mesh]6. 9. (#5 OR #6)10. \u201cOropharynx\u201d[Mesh]11. 12. (#10 OR #11)13. (#9 AND #12)14. (HNSCC ORSCCHN OR OP-SCC OR OPSCC)15. (#1 OR #2 OR #3 OR #4 OR #13 OR #14)16. ) OR ) OR ) OR OR OR OR OR OR OR OR OR (\u201cMulticenter Study\u201d[Publication Type]) OR (\u201cMulticenter Studies as Topic\u201d[Mesh]) OR (\u201cRandom Allocation\u201d[Mesh]) OR (\u201cDouble-Blind Method\u201d[Mesh]) OR (\u201cSingle-Blind Method\u201d[Mesh]) OR (\u201cCross-Over Studies\u201d[Mesh]) OR (\u201cPlacebos\u201d[Mesh]) OR ) OR (blind[tiab] OR blinding[tiab] OR blinded[tiab] OR mask[tiab] OR masking[tiab] OR masked[tiab] OR placebo[tiab] OR placebos[tiab] OR rct[tiab] OR random[tiab] OR randomised[tiab] OR randomized[tiab] OR randomly[tiab] OR randomisation[tiab] OR randomization[tiab]) OR OR (divided[tiab] AND (group[tiab] OR groups[tiab])) OR (crossover[tiab]) OR (\u201ccross over\u201d[tiab]) OR (multicentre[tiab] OR multicentred[tiab] OR multicentric[tiab]) OR (versus[ti] OR vs[ti]) OR (\u201ctreatment arm\u201d[tiab]) OR (\u201cphase III\u201d[tiab] OR \u201cphase three\u201d[tiab] OR \u201cphase 3\u201d[tiab]) OR (\u201clatin square\u201d[tiab]) NOT NOT AND (\u201cHumans\u201d[Mesh])))))17. (#15 AND #16)18. \u201c\u201cCochrane Database Syst Rev\u201d\u201c[Journal]19. 20. (#19 OR #18)21. (#17 NOT #20)Cochrane Central Register of Controlled Trials (CENTRAL): 1 January 2003 to 14 May 20131. MeSH descriptor: [Oropharyngeal Neoplasms] explode all trees2. MeSH descriptor: [Head and Neck Neoplasms] this term only3. MeSH descriptor: [Pharyngeal Neoplasms] this term only4. MeSH descriptor: [Otorhinolaryngologic Neoplasms] this term only5. MeSH descriptor: [Neoplasms] explode all trees6. cancer* OR carcinoma* OR neoplas* OR tumor* OR tumour* OR malignan* OR SCC7. #5 OR #68. MeSH descriptor: [Oropharynx] explode all trees9. oropharyn* OR mesopharyn* OR tonsil* OR \u201chead and neck\u201d OR \u201chead neck\u201d OR \u201chead-neck\u201d OR \u201chead-and-neck\u201d OR pharyn* OR \u201ctongue base\u201d OR \u201csoft palate\u201d10. #8 OR #911. #7 AND #1012. HNSCC OR SCCHN OR OP-SCC OR OPSCC13. #1 OR #2 OR #3 OR #4 OR #11 OR #12Embase: 1 January 2003 to 14 May 20131. Exp Oropharynx tumor/2. Pharynx cancer/3. Neoplasm/4. .tw.5. #3 OR #46. exp OROPHARYNX/OR exp OROPHARYNX CANCER/OR exp OROPHARYNX CARCINOMA/7. .tw.8. #6 OR #79. #5 AND #810. (HNSCC OR SCCHN OR OP-SCC OR OPSCC).tw.11. #1 OR #2 OR #9 OR #1012. .mp. OR crossover*.tw. 13. (cross adj over*).tw.14. (cross adj over*).tw.15. ((blind* OR mask*) and (single OR double OR triple OR treble)).tw.16. (treatment adj arm*).tw.17. (control* adj group*).tw.18. (phase adj III).mp. OR three.tw. 19. (versus OR vs).tw.20. rct.tw.21. CROSSOVER PROCEDURE/22. DOUBLE BLIND PROCEDURE/23. SINGLE BLIND PROCEDURE/24. RANDOMIZATION/25. PLACEBO/26. exp CLINICAL TRIAL/27. PARALLEL DESIGN/28. LATIN SQUARE DESIGN/29. #12 OR #13 OR #14 OR #15 OR #16 OR #17 OR #18 OR #19 OR #20 OR #21 OR #22 OR #23 OR #24 OR #25 OR #26 OR #27 OR #2830. #11 AND #2931. limit #30 to human32. \u201csystematic review\u201d.tw.33. #31 NOT #32CENTRAL: Cochrane Central Register of Controlled Trials; CNS: Clinical nurse specialist; COMET: Core Outcome Measures in Effectiveness Trials; CRT: Chemoradiotherapy; HPV: Human papillomavirus; HPV-16: Human papillomavirus type 16; HTA: Health Technology Assessment; ICF: International Classification of Functioning, Disability and Health; NHS: National Health Service; NIHR: National Institute for Health Research; OMERACT: Outcome Measures in Rheumatology; OPSCC: Oropharyngeal squamous cell carcinoma; PI: Principal investigator; PRO: Patient-reported outcome; RA: Rheumatoid arthritis; RCT: Randomised controlled trial; RT: Radiotherapy; SCCHN: Squamous cell carcinoma of the head and neck; SLT: Speech and language therapist.The authors declare that they have no competing interests.AW conceived and designed the study, and wrote the manuscript. CTS conceived and designed the study, and provided critical revision of the manuscript. BY conceived and designed the study, and provided critical revision of the manuscript. TMJ conceived and designed the study, and provided critical revision of the manuscript. All authors read and approved the final manuscript."} +{"text": "Scientific Reports6: Article number: 2977910.1038/srep29779; published online: 07152016; updated: 08162016This Article contains typographical errors in Table 1. In the column \u2018Slip (twinning) system\u2019, the seventh {332} <113> twin value \u201c(3"} +{"text": "The correct name is: Petr Stastny. The correct citation is: Petr M, Stastny P, Pecha O, \u0160teffl M, \u0160eda O, Kohl\u00edkov\u00e1 E (2014) PPARA Intron Polymorphism Associated with Power Performance in 30-s Anaerobic Wingate Test. PLoS ONE 9(9): e107171. doi:"} +{"text": "The second half is gererated by inversion symmetry. The crystal structure has a layered arrangement built from distorted edge-sharing LiO3(OH)2 tetra\u00adhedra parallel to (100), with naphthalenedi\u00adcarboxyl\u00adate bridging the LiO3(OH)2 layers along the [100] direction. Hydrogen bonding between the water molecule and adjacent carboxylate groups consolidates the packing.The title compound, [Li DOI: 10.1107/S1600536814013130/pj2011Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S1600536814013130/pj2011Isup3.molSupporting information file. DOI: 1006973CCDC reference: crystallographic information; 3D view; checkCIF reportAdditional supporting information:"} +{"text": "In the crystal, C\u2014H\u22efO hydrogen-bond inter\u00adactions link the mol\u00adecules into chains running parallel to the b axis.The mol\u00adecule of the commercially available title compound, C DOI: 10.1107/S2056989015008816/rz5158Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989015008816/rz5158Isup3.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989015008816/rz5158fig1.tif. DOI: The mol\u00adecular structure of title compound with displacement ellipsoids drawn at the 30% probability level.Click here for additional data file.10.1107/S2056989015008816/rz5158fig2.tifb via . DOI: b axis via C\u2014H\u22efO hydrogen bonds (dashed lines). H atoms not involved in hydrogen bonding are omitted.Crystal packing of the title compound, showing the formation of chains parallel to the 1063415CCDC reference: crystallographic information; 3D view; checkCIF reportAdditional supporting information:"} +{"text": "Abdominal infections are frequent causes of sepsis and septic shock in the intensive care unit (ICU) and are associated with adverse outcomes. We analyzed the characteristics, treatments and outcome of ICU patients with abdominal infections using data extracted from a one-day point prevalence study, the Extended Prevalence of Infection in the ICU (EPIC) II.EPIC II included 13,796 adult patients from 1,265 ICUs in 75 countries. Infection was defined using the International Sepsis Forum criteria. Microbiological analyses were performed locally. Participating ICUs provided patient follow-up until hospital discharge or for 60\u00a0days.Escherichia coli, methicillin-sensitive Staphylococcus aureus and anaerobic isolates, and fewer enterococci than patients who had been in the ICU longer. ICU and hospital mortality rates were 29.4% and 36.3%, respectively. ICU mortality was higher in patients with abdominal infections than in those with other infections . In multivariable analysis, hematological malignancy, mechanical ventilation, cirrhosis, need for renal replacement therapy and SAPS II score were independently associated with increased mortality.Of the 7,087 infected patients, 1,392 (19.6%) had an abdominal infection on the study day . Microbiological cultures were positive in 931 (67%) patients, most commonly Gram-negative bacteria (48.0%). Antibiotics were administered to 1366 (98.1%) patients. Patients who had been in the ICU for \u22642\u00a0days prior to the study day had more The characteristics, microbiology and antibiotic treatment of abdominal infections in critically ill patients are diverse. Mortality in patients with isolated abdominal infections was higher than in those who had other infections.The online version of this article (doi:10.1186/1471-2334-14-420) contains supplementary material, which is available to authorized users. Abdominal infection is a common indication for admission to the intensive care unit (ICU) and the abdomen is the second most common site of invasive infection among critically ill patients in epidemiological \u20133 and thMulticenter data on the clinical features and microbiology of abdominal infections in the critically ill are rare, and often limited to a single region or country. In recent years, an increase in abdominal infections due to nosocomial and resistant organisms has been reported \u201314, but The Extended Prevalence of Infection in the ICU (EPIC) II study was a large one-day point-prevalence study of infections in the ICU. The study showed that half of all ICU patients were infected on the study day and 71% were being treated with antibiotics ), median (interquartile range [IQR]), or number (%) as appropriate. To identify factors associated with mortality, a multivariable logistic regression model was constructed using variables for which the P-value was <0.1 in univariable analysis. Goodness of fit was assessed by the Hosmer-Lemeshow statistic. All tests were two-tailed, and a P\u2009<\u20090.05 was considered statistically significant.Of the 7,087 infected patients, 1,392 (19.6%) were diagnosed as having an abdominal infection on the study day , methicillin-resistant staphylococci in 59 patients (6.3%). Candida species were isolated in 156 patients (16.8%), 75.6% of these isolates were Candida albicans.Microbiological data were available for 931 patients (67%), with a total of 1,289 microorganisms isolated were receiving antibiotics: penicillins and other beta-lactam antibiotics were used most frequently and hospital lengths of stay (LOS) were similar in patients with abdominal infections and those with other infections. Overall ICU and hospital mortality rates were 29.4% and 36.3%, respectively , which was not found in previous studies. In an analysis of patients from the Sepsis Occurrence in Acutely Ill Patients (SOAP) study, Volakli et al. reported no differences in mortality rates among patients with abdominal infections and those with respiratory infections . The higAs expected, comorbidities, such as cirrhosis and hematological cancer, were associated with increased mortality, as was found in the EPIC II patients in general . Most noE. coli being the most prevalent pathogen; typical nosocomial microorganisms, such as P. aeruginosa and Enterobacter spp. were also frequently isolated. Indeed, P. aeruginosa was the second most frequently isolated Gram-negative microorganism, which may in part be due to the design of the study, but the percentages are comparable to other studies in this field [P. aeruginosa and Stenotrophomonas maltophilia isolated twice and four times more often, respectively, in non-survivors than in survivors. These pathogens may have a greater degree of pathogenicity , or may be more difficult to treat. Detailed data regarding antibiotic resistance, including information regarding resistance to specific antibiotics, were not collected so we are unable to comment further on these aspects.The microbiology patterns were diverse, and quite different from those in the overall EPIC II study population, in which staphylococci were isolated most frequently, and Gram-positive and Gram-negative organisms were equally present . In the is field . Among tThe findings in this study suggest that physicians around the world seem to comply with international guidelines in this field as most patients receive broad-spectrum antibiotics, often in combination with agents aimed at fungi or even at resistant Gram-positive microorganisms. We also found that patients who had stayed in the ICU for 2\u00a0days or less on the study day had different characteristics to those who had been longer on the ICU. Microbiological isolates and antibiotic treatments were remarkably different between these groups with fewer carbapenems, glycopeptides and antifungals used in patients with shorter stays. Current guidelines for the selection of antibiotic therapy in critically ill patients do not mention length of stay in the hospital as a consideration for empirical treatment in patients with high-severity non-nosocomial infections. Nevertheless, this group represents a category of patients, presumably with community-acquired infection, who could potentially be treated with narrower spectrum antibiotics when local ecology allows. This hypothesis warrants further evaluation.Candida isolation has been identified as an independent predictor of mortality in some studies [Candida non-albicans isolates was lower than frequently reported in invasive candidiasis studies or other studies in patients with Candida peritonitis. For example, Montravers et al. reported that only 58% of patients with Candida peritonitis had C. albicans isolated from intraoperative cultures [C. albicans[Candida peritonitis accounted for only 1% of the patients in this review, however. It is not clear whether the infecting Candida species or its susceptibility plays a major role in determining outcome [Fungal infections have received considerable attention in the last decade. Although the debate continues as to whether fungi are relevant in community-acquired disease, the situation is different in nosocomial infections and in severely ill patients . Candida studies , 26, 27, studies , should cultures . In pati albicans; patient outcome .This study has a number of limitations. Because the study was not primarily focused on abdominal infections, the exact source and extent of infection were not recorded and the efficacy of source control and appropriateness of antimicrobial therapy could not be evaluated. The rate of superinfection and/or tertiary peritonitis could not be assessed. Data on the community-acquired versus nosocomial nature of infections were also not available and we, therefore, used the length of stay as a surrogate marker, but acknowledge that this has its limitations. Finally, as in all point-prevalence studies, patients who are admitted for a long period of time may skew the findings with more data collected on those who stay in the ICU for a longer period of time.In conclusion, this study found that abdominal infections were present in about one fifth of ICU patients on the study day and concomitant infections were common. Microbiology patterns and choice of antibiotic therapy were diverse and differed in patients who had stayed in the ICU for 2\u00a0days or less compared to those with longer stays. Abdominal infections carry a poor prognosis, with higher mortality rates than in patients with infections from other sources. Disease severity, need for organ support and presence of comorbidities were independently associated with mortality in our cohort.Andorra: Hospital Nostra Senyora de Meritxell (A Margarit); Argentina: Centro de Educaci\u00f3n M\u00e9dica E Investigaciones Cl\u00ednicas ; Clinica de Especialidades Villa Maria (AJ Zazu); Cl\u00ednica Modelo de Mor\u00f3n (C Bevilacqua); Clinica Y Maternidad Suizo (M Curone); CMIC (R Rabuffetti); Hospital Aleman (P Comignani); Hospital Argerich (M Torres Boden); Hospital Britanico (F Chertcoff); Hospital Central de San Isidro (G Cardonatti); Hospital de Ni\u00f1os Dr. H\u00e9ctor Quintana (F Ad\u00e9n); Hospital del Ni\u00f1o Jes\u00fas (L Marcos); Hospital Dr Pedro Ecay (M D\u00f3nofrio); Hospital Espa\u00f1ol de Mendoza (R Fern\u00e1ndez); Hospital Espa\u00f1ol Medical Plaza (R Lamberghini); Hospital Internacional General de Agudos \"Jos\u00e9 de San Mat\u00edn\" ; Hospital Interzonal Dr. O. Alende (J Teves); Hospital Italiano de Buenos Aires ; Hospital Juan A. Fern\u00e1ndez (D Ceraso); Hospital Municipal de Chivilcoy (D Curcio); Hospital Profesor Alejandro Posadas (L Aguilar); Hospital Provincial de Rosario (C Weller); Hospital Provincial del Centenario (L Cardonnet); Hospital Regional Rio Gallegos (R Santa Cruz); Hospital Regional Ushuaia (E Manrique); Hospital Universitario Austral ; Hospital Universitario Universidad Abierta Interamericana (G Chiappero); Instituto Privado del Quemado Med-Inter (D Curcio); Nuevo Hospital El Milagro (P Ramos); Ramos Mejia Hospital (J Vergara); Sanatorio Agote (I Moine); Sanatorio de la Trinidad Mitre (S Ilutovich); Sanatorio de Los Arcos (G Jannello); Sanatorio Dupuytren (M Waschbusch); Sanatorio Frangioli de Salud 2000 Srl (G Rios Picaza); Sanatorio Mater Dei (A Raimondi); Sanatorio Otamendi Y Miroli (M Miriam); Sanatorio Parque (L Carlos); Sanatorio San Jos\u00e9 (D Curcio); Armenia: Centro Gallego de Buenos Aires (M Caridi); Australia: Alfred Hospital (T Leong); Barwon Health (N Orford); Blacktown Hospital (G Reece); Box Hill Hospital (D Ernest); Cabrini Hospital (F Hawker); Concord Repatriation General Hospital (J Tan); Epworth Eastern Private Hospital (C Giannellis); Epworth Hospital Richmond (B Ihle); Flinders Medical Centre (A Bersten); Frankston Hospital (J McInnes); Gold Coast Hospital ; John Hunter Hospital (B Mcfadyen); Joondalup Health Campus (J Vibert); Liverpool Hospital, Sydney South West Area Health Service (M Parr); Logan Hospital (K Tran); Mater Health Services (J Sutton); Mount Hospital (S Webb); Nambour General Hospital (N Groves); Nepean Hospital, NSW (L Cole); Prince Charles Hospital (D Long); Prince of Wales Hospital (F Bass); Princess Margaret Hospital For Children (S Erickson); Royal Brisbane and Womens' Hospital (J Lipman); Royal Children\u2019s Hospital, Brisbane (D Long); Royal Children\u2019s Hospital, Melbourne (C Delzoppo); Royal Darwin Hospital (J Thomas); Royal Perth Hospital (G Dobb); Royal Prince Alfred Hospital ; Sir Charles Gairdner Hospital (B Roberts); St John of God Hospital, Subiaco (S Webb); St Vincent\u2019s Hospital, Melbourne (J Santamaria); Sydney Children\u2019s Hospital (J Young); The Children\u2019s Hospital at Westmead, Sydney (M Festa); The John Flynn Private Hospital (R Holland); The Prince Charles Hospital (D Mullany); The Queen Elizabeth Hospital (P Williams); The Townsville Hospital (M Corkeron); The Wollongong Hospital ; Westmead Hospital (A Banerjee); Women\u2019s and Children\u2019s Hospital, Adelaide (M Yung); Austria: University Hospital Innsbruck ; General Hospital (P Faybik); Hospital Hietzing ; Krankenhaus Barmherzige Br\u00fcder Linz (F Firlinger); Krankenhaus Der Barmherigen Brueder Wien (G Zasmeta); Krankenhaus Der Barmherzigen Br\u00fcder St. Veit (M Zink); Krankenhaus Der Barmherzigen Schwestern Linz (W Sieber); Krankenhaus Steyr (J Hildegard); Landeskrankenhaus Klagenfurt (R Bakondy); Landeskrankenhaus Stolzalpe (J Schlieber); Landeskrankenhaus Deutschlandsberg (G Filzwieser); Medical University Innsbruck ; Medical University of Vienna (T Staudinger); Otto-Wagner Hospital (R Schuster); Unfallkrankenhaus Meidling Der Auva (W Scherzer); University Hospital (K Smolle); Wilhelminenspital ; Bangladesh: Central Hospital Limited (R Manzoor); Belgium: A.I.T. (J Brunain); Ambroise Par\u00e9 (A Dive); Asz-Aalst (G Huylenbroeck); Az Groeninge Kortrijk (M Van der Schueren); Az Maria Middelares (H 't kindt); Az Sint Jozef Malle (E Slock); Az Sint Lucas (D Rijckaert); Az St Augustinus (J Raemaekers); Az St Jan Av (M Bourgeois); Az Vesalius (I Van Cotthem); Az Damiaan Oostende (G Nackaerts); C.H.N.D.R.F. (D Gusu); Centre Hospitalier de Mouscron (P Gadisseaux); CH Libramont (V Olivier); Chirec - Braine-L'Alleud (H Lignian); CHPLT Verviers (P Michel); CHR Citadelle (V Fraipont); CHR Haute Senne Soignies (M Van der Stappen); CHR St Joseph Mons-Warquignies (F For\u00eat); CHU Brugmann ; CHU Charleroi (P Biston); CHU Saint-Pierre (A Roman); CHU Sart Tilman, Li\u00e8ge (B Lambermont); Clinique Sainte Elisabeth (A De Meulder); Clinique Notre Dame (V Frederic); Clinique Notre-Dame de Gr\u00e2ce (T Sottiaux); Clinique Saint Luc, Bouge (P Ruyffelaere); Cliniques de L'Europe, St-Michel (V Collin); Cliniques de L'Europe, Ste Elisabeth (S Anane); H\u00f4pital Francais (P Kleiren); H\u00f4pital Saint-Joseph (M Simon); Hornu (S Machayekhi); Imeldaziekenhuis (E Frans); Institut Jules Bordet ; Jan Yperman Hospital (R Joseph); Olv Ter Linden Ziekenhuis, Knokke (J Eerens); Saint Luc University Hospital (PF Laterre); Sint Augustinus, Veurne (B Lagrou); St Vincent (R Rutsaert); St-Jozefkliniek Bornem-Willebroek (W Pisarek); UCL Mont-Godinne (A Dive); Universitair Ziekenhuis Gent (J De Waele); University Hospital Brussels (H Spapen); University Hospital of Liege (P Damas); Erasme University Hospital (JL Vincent); ZNA Stuivenberg ; Belize: Universal Health Services, Medical Center ; Brazil: Bandeirantes Hospital (M Baptista); Barra Dor Hospital ; Biocor Instituto (M Braga); Casa de Saude Sao Jose Caxias (C Avila); Centro Hospitalar Unimed ; Centro Integrado de Aten\u00e7a\u00f5 \u00c0 Sa\u00fade -Unimed Vit\u00f3ria (E Caser); Cl\u00ednica S\u00e3o Vicente da G\u00e1vea (A Alves); Complexo Hospitalar Santa Casa de Porto Alegre (G Friedman); Erasto Gaertner Hospital (M Luz); Federal University of Sao Paulo (M Assuncao); Fundacao Hospital de Clinicas Gaspar Vianna (H Reis); Funda\u00e7\u00e3o Hospitalar Do Estado de Minas Gerais - Fhemig (A Gomes); Funda\u00e7\u00e3o Pio Xii (U Silva); UNIFESP (W Nogueira Fh); Hopital das Cl\u00ednicas - FMUSP (S El-Dash); Hospital Padre Albino-Faculdade de Medicina de Catanduva ; Hospital Alberto Cavalcanti (A Barbosa); Hospital Badim (C Coelho); Hospital Cardiotrauma Ipanema (M Knibel); Hospital Carlos Fernando Malzoni (C Minelli); Hospital Da Cidade de Passo Fundo (J Caovilla); Hospital das Cl\u00ednicas da Faculdade de Medicina de Ribeir\u00e3o Preto da Universidade de S\u00e3o Paulo (G Teixeira); Hospital das Cl\u00ednicas, Universisty of S\u00e3o Paulo (A Hovnanian); Hospital das Nacoes (A Rea-Neto); Hospital de Base-Famerp (SL Lobo); Hospital de Cl\u00ednicas Mario Lioni (M Lugarinho); Hospital de Cl\u00ednicas Niter\u00f3i (P Souza); Hospital de Doen\u00e7as Tropicais de Goi\u00e2nia (D Ferreira); Hospital do Cancer/Uopeccan (P Duarte); Hospital do Trabalhador (M Oliveira); Hospital dos Servidores do Estado Rio de Janeiro (J Marques); Hospital E Maternidade S\u00e3o Jos\u00e9 (R Machado); Hospital Estadual Diadema (P Rehder); Hospital Estadual do Grajau-Unisa ; Hospital Evangelico (M Grilo); Hospital Evangelico do Rio de Janeiro (P Quesado); Hospital Geral de Pedreira (M Moock); Hospital Geral de S\u00e3o Mateus (F Ferreira); Hospital Geral Roberto Santos (J Teles); Hospital Israelita Albert Einstein (E Silva); Hospital Israelita Albert Sabin (C Coelho); Hospital J\u00falia Kubitschek (A Morais); Hospital Mater Dei ; Hospital Memorial Arthur Ramos (M Wanderley); Hospital Meridional (M Velasco); Hospital Moinhos de Vento (N Brand\u00e3o da Silva); Hospital Municipal S\u00e3o Jos\u00e9 (J Feij\u00f3); Hospital Nossa Senhora Da Salete (P Duarte); Hospital Pasteur (V Souza Dantas); Hospital Portugu\u00eas (J Teles); Hospital Pr\u00f3-Card\u00edaco (R Costa Filho); Hospital Quinta D'Or (A Japiassu); Hospital Regional Ant\u00f4nio Dias (D Villela); Hospital Regional de Barbacena (C Santos); Hospital Salvador (R Passos); Hospital Samaritano (R Alheira-Rocha); Hospital Santa Izabel (R Silva); Hospital Santa Paula (J Houly); Hospital Sao Cristovao (J Aldrighi); Hospital S\u00e3o Lucas (R Hatum); Hospital S\u00e3o Lucas da PUCRS (F Suparregui Dias); Hospital S\u00e3o Luiz - Unidade Itaim (L Ferreira); Hospital S\u00e3o Rafael (L Ferro); Hospital S\u00e3o Vicente de Paulo (J Gomez); Hospital Universit\u00e1rio Clementindo Fraga Filho - Ufrj (R Fleury); Hospital Universitario da Universidade Federal do Rio de Janeiro (C David); Hospital Universit\u00e1rio de Santa Maria (T Resener); Hospital Universit\u00e1rio do Oeste do Paran\u00e1 (P Duarte); Hospital Universit\u00e1rio Lauro Wanderley - UTI Adulto (C Mendes); Hospital Universitario Regional de Maringa (A Germano); Hospital Vita Curitiba (M Oliveira); Hospital Vivalle (F De marco); Instituto de Espquisa Clinica Evandro Chagas (A Japiassu); Instituto Do Cora\u00e7\u00e3o - HC-FMUSP (S Lage); Instituto Nacional de Cancer ; Irmandade Santa Casa de Misericordia de Porto Alegre (A Torelly); Luxemburgo Hospital (R Sad); Mternidade Odete Valadares (A Barbosa); Prontocor Lagoa (G Oliveira); Samaritano Hospital (R Lima); Santa Casa Da Miseric\u00f3rdia de S\u00e3o Jo\u00e3o del Rei (J Paranhos); Santa Casa de Misericordia de Passos (M Oliveira); Santa Casa de Porto Alegre (M Rocha); S\u00e3o Sebasti\u00e3o Hospital (W Bitencourt); Universidade Federal Do Parana (A Rea-Neto); University of Londrina (C Grion); University of Sao Paulo (D Forte); Uti Da Disciplina de Cl\u00ednica M\u00e9dica-Unifesp (H Guimar\u00e3es); Vit\u00f3ria Apart Hospital (C Piras); Bulgaria: Mbal Ruse (L Stephanova); Multiprofile Hospital of Active Treatment, Ruse (L Lyubenov); Uh St. Ekaterina (G Tsarianski); Univerisity Hospital (G Dimov); Canada: Capital Health-Queen Elizabeth II Health Sciences Centre (R Green); Centre Hospitalier R\u00e9gional de Lanaudi\u00e8re (J Levasseur); Children\u2019s Hospital of Eastern Ontario (R Ward); CHU Sherbrooke (O Lesur); H\u00f4pital Charles Lemoyne (G Poirier); Mount Sinai Hospital (R Wax); Royal Jubilee Hospital (G Wood); St. Joseph\u2019s Healthcare (D Cook); St. Michael\u2019s Hospital ; Toronto General Hospital (M Herridge); Toronto Western Hospital (N Ferguson); Victoria General Hospital (G Wood); Chile: Clinica Alemana de Santiago (M Espinoza); Clinica las Condes ; Hospital Cl\u00ednico de la Pontificia Universidad Cat\u00f3lica de Chile (A Bruhn); Hospital del Trabajador (J Micolich); Hospital Dr G. Fricke ; Hospital El Pino (I Escamilla Leon); China: Beijing Chaoyang Hospital (Q Zhan); Beijing Tongren Hospital (Y Xu); Chinense Pla General Hospital (Y Zhao); Fuxing Hospital, Capital Medical University (L Zhang); Guangdong Provincial People\u2019s Hospital (T Qin); Peking Union Medical College Hospital (B Du); Peking University People\u2019s Hospital (M Li); Ren Ji Hospital,Shanghai Jiao Tong University (X Wang); The Affiliated Hospital of Ningxia Medical College of China (Y Jing); The First Affiliate Hospital of China Medical University (Z Zhang); The First Affiliated Hospital of Dalian Medical University (W Xianyao); The First People\u2019s Hospital of Nantong, Jiangsu (F Li); Zhong-Da Hospital and School of Clinical Medicine, Southeast University (Y Congshan); Colombia: Clinica General del Norte (C Rebolledo); Clinica Central del Quindio (D Diaz); Clinica Medellin (R Murillo Arboleda); Clinica Saludcoop (C Rebolledo); Clinica Santa Isabel de Valledupar. 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(S Smith); Newcastle General Hospital (A Vincent); Newham University Hospital Trust (P Withington); NHS Tayside (C Macmillan); Northampton General Hospital (R Webster); Papworth Hospital (A Vuylsteke); Peterborough Hospitals (B Appadu); Princess Royal Hospital (C Barrera groba); Queen Alexandra Hospital, Portsmouth (P McQuillan); Queen Elizabeth Hospital (M Blunt); Queen Elizabeth Medical Center, Birmingham (N Parekh); Rotherham DGH (D William); Royal Berkshire Hospital (C Jones); Royal Blackburn Hospital (A Krige); Royal Bournemouth NHS Foundation Trust (M Schuster-Bruce); Royal Cornwall Hospital (J Boyden); Royal Devon & Exeter NHS Foundation Trust (C Boulanger); Royal Infirmary of Edinburgh (D Swann); Royal Liverpool University Hospital ; Royal Marsden Hospital (T Wigmore); Royal Shrewsbury Hospital (R Law); Royal Sussex County Hospital ; South Tyneside Hospital (C Muench); Southmead Hospital (S Robinson); St George\u2019s Hospital ; St Helens and Knowsley Hospital (T Mahambrey); St John\u2019s Hospital (L Cameron); St. James\u2019s University Hospital (J Thornton); St. Mary\u2019s Hospital (M Stotz); St. Peters Hospital (M Russell); Stirling Royal Infirmary (A Longmate); Tameside General Hospital (R Kitson); Taunton & Somerset NHS Trust (B Browne); The Hillingdon Hospital (A Thorniley); The James Cook University Hospital ; Torbay Hospital (M Swart); University College Hospital (M Singer); University Hospital Birmingham (N Gautam); University Hospital of South Manchester (V Prasad); University Hospitals Coventry & Warwickshire Nhs Trust (D Watson); West Middlesex University Hospital (T Szakmany); West Suffolk Hospital (J Cardy); Western Infirmary Glasgow (A Binning); Wexham Park Hospital (R Loveland); Wirral Hospitals Nhs Trust (J Gannon); Wolverhampton Hospital (G Martinelli); Wythenshawe Hospital ; Yeovil District Hospital (J Howes) United States: Baystate Medical Center (J Steingrub); Denver Health Medical Center (L Ammons); Emory University Hospital - MICU (M Fisher); Englewood Hospital Medical Center (N Gandhi); Grady Memorial Hospital-Emory University (G Martin); Hospital of The University of Pennsylvania (C Deutschman); LDS Hospital, Salt Lake City (N Dean); Inova Fairfax Hospital (C Michetti); Los Angeles County\u2009+\u2009University of Southern California Medical Center (H Belzberg); LSUHSC Shreveport (K Hutchinson); Maine Medical Center (T Van der kloot); Mayo Clinic (B Afessa); Mount Sinai School of Medicine (D Kaufman); Nassau University Medical Center ; New York University (D Ost); Northwestern University ; Northwestern University Feinberg School of Medicine Critical Care (S Afifi); Olive View Ucla Medical Center (S Stein); Oregon Health & Science University (D Hagg); Orlando Regional Medical Center (E Jimenez); Penn State Hershey Medical Center (S Blosser); Rochester General Hospital (S Chhangani); Rush University Medical Center (R Kleinpell); Rutland Regional Medical Center (H Reich); Scott and White Hospital (E Fileds); Sharp Memorial Hospital (D Willms); South Texas Veterans Health Care System (P Castellanos-Mateus); St. Joseph Medical Center Fhs (L Melnik); Texas Tech University Health Sciences Center (L Oud); Cardiovascular ICU, The Methodist Hospital, Houston ; Medical ICU, The Methodist Hospital, Houston ; The University of Mississippi Medical Center (A Badr); The University of Texas Health Science Center at San Antonio ; University of Chicago (A Pohlman); University of Cincinnati (R Branson); University of Kansas Hospital (S Simpson); University of Miami School of Medicine (D Kett); University of Michigan ; University of Texas Health Science Center at San Antonio (C Patricia); University of Toledo Medical Center ; University of Virginia (R Sawyer); Uthsc At Memphis - Regional Medical Center (Site) (A Freire); Veterans Caribbean Healthcare System (W Rodriguez); Washington Hospital Center (A Ryan); West Suburban Medical Center (B Margolis); Winthrop University Hospital (M Groth) Uruguay: Amecom (H Escanda); Comta (J Baraibar); Hopital Policial (D Paciel); Hospital Maciel (H Bagnulo); Hospital Tacuarembo (F Hitta); M\u00e9dica Uruguaya Corporaci\u00f3n de Asistencia M\u00e9dica ; Sanatorio Americano - Femi (H Albornoz) Venezuela: Hospital Dr Luis Ortega ; Hospital Unversitario de Caracas (C Pacheco) Vietnam: Bach Mai Hospital (T Bui); Franco Vietnamese Hospital (F Potie); Ninh Thuan Hospital (C Nguyen Huu)Table S2. Antibiotic use in patients with abdominal infections. Table S3. Microbiology and antibiotic use in survivors and non-survivors. (DOC 117 KB)Additional file 1: Table S1: Sites of infection."} +{"text": "S7 FigRegression analyses of biochemical measures against population doublings. Regressions were made combining all data from all 3 donors (n \u2265 23). A-E Atmospheric oxygen tension, F-J Low (5%) oxygen tension; A, F) Wet weight vs. population doublings; B,G) Total GAG (per aggregate) vs. population doublings; C,H) Total HP (per aggregate) vs. population doublings; D, I) Normalized GAG (GAG/DNA) vs. population doublings; E,J) Normalized HP (HP/DNA) vs. population doublings.(TIF)Click here for additional data file."} +{"text": "The Editors would like to thank the following reviewers for their continuous support during the year of 2014:Franck AccadbledMichael D. AionaZaid Al-AubaidiJavier AlbinanaCristina AlvesAntonio AndreacchioDavid D. AronssonElhanan Bar-OnJeremy BauerJames BeatyMichael J. BellMohan V. BelthurMichael K.D. BensonErnesto BersuskyVictor BialikRainer Georg BiedermannJohn G. BirchRobert Dale BlasierStephanie BoehmThomas BoeniSilvio BoeroGerard BolliniDieter BolzChristopher F. BradishAmichai BreznerReinald BrunnerMichael T. BuschCarlo CamathiasFederico CanaveseKelly CarmichaelNorris C. CarrollPablo Casta\u00f1edaAnthony CatterallHenry ChambersTae-Joon ChoFranck ChotelAur\u00e9lien CourvoisierJuan Carlos CoutoAlvin Howell CrawfordRobert Jay CummingsPeter John CundyJaroslaw CzubakCharles d\u2019AmatoLuciano DiasJohn DimitriouMatthew B. DobbsJohn P. DormansDenis S. DrummondDeborah EastwoodEric W. EdmondsJohn EmansTomas EpeldeguiThomas ErbG. Ulrich ExnerSharon EylonEli EzraGuy FabryDavid S. FeldmanFrank FitoussiEdilson ForlinNiklaus F. FriederichBruno FuchsPatricia Moraes Barros FucsToshio FujiiJulia F. FunkRudolf GangerMark GastonYael GelferIsmat GhanemWerner GirschJoe Eric GordonReinhard GrafH. Kerr GrahamAlfred D. GrantFranz GrillRichard Henri GrossGunnar H\u00e4gglundCarol HaslerDorian HaukeShlomo HayekHanne Bastrup HedinBernhard HeimkesAnna Kathrin HellYoram HemoJohn Anthony HerringJohn E. HerzenbergSevan HopyanGamal HosnyJames B. HunterIvan HvidTalal IbrahimErnesto IppolitoRoger JawishAndrea JesterCharles E. JohnstonBenjamin JosephJean-Luc JouveAndre KaelinLori KarolHidehiko KawabataDerek M. KellyDavid KeretSam KhamisMininder S. KocherJoseph Ivan KrajbichRuediger KrauspeAndreas KriegKen N. KuoRon LamdanErica LamprechtMario LampropulosLennart A. LandinPierre LascombesDavid LebelSeok Hyun LeeWallace LehmanJoseph LessingLynn LindamanDavid G. LittleRandall LoderJohn E. LonsteinScott J. LuhmannNicolas LutzWilliam G. MackenzieEran MamanHans Michael MannerJohannes MayrTerence McGuireCharles T. MehlmanA. Ludwig MeissCarlo MilaniFreeman MillerMichael B. MillisKan MinBjarne Moeller-MadsenGuy MolenaersJose MorcuendeVincent MoscaScott J. MubarakAndreas Marc MuellerKishore MulpuriLucas MurnaghanYasuharu NakashimaMarek NapiontekYrj\u00e4n\u00e4 NietosvaaraIulian NusemAvi OhryHakan OmerogluNorman Y. OtsukaDror OvadiaKlaus ParschLaura Montserrat P\u00e9rez L\u00f3pezPeter PizzutilloThomas PresselTamir PritschGeorge T. RabChristof RadlerLeonhard E. RamseierJames E. RobbAndreas RoposchYishai RosenblattErich RutzRalph SakkersWudbhav N SankarDietrich SchlenzkaPerry L. SchoeneckerAstrid SchulzeEitan Nathan SegevDalia A. Sep\u00falvedaSheldon R. SimonDavid L. SkaggsTheddy SlongoHae Ryong SongPaul D. SponsellerCarl L. StanitskiRobert P. StantonDaniel StuderMichael D. SussmanMarek SynderTerje TerjesenTim TheologisStephen J. TredwellChristian TschaunerCosimo TudiscoAnja Van CampenhoutJohannes A. van der SluijsMichael G. VitaleAndrew WainwrightEric WallStewart WalshJohn H. WedgeDennis S. WeinerStuart L. WeinsteinDennis R. WengerBettina WesthoffRoger F. WidmannKaye E. WilkinsR. Baxter WillisThomas WirthJames G. WrightHaruhisa YanagidaMoshe YanivMuharrem YaziciReinhard D. ZellerKai Ziebarth"} +{"text": "The CuII atom is tetra\u00adcoordinated by two N,S-bidentate ligands, forming a highly distorted tetra\u00adhedral environment. The structure displays two intra\u00admolecular N\u2014H\u22efO hydrogen bonds. The title complex, [Cu(C DOI: 10.1107/S2056989015005022/lr2133Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989015005022/lr2133fig1.tif. DOI: The mol\u00adecular structure of (I), with atom labels and 50% probability displacement ellipsoids for non-H atoms.1039799CCDC reference: crystallographic information; 3D view; checkCIF reportAdditional supporting information:"} +{"text": "The correct reference is: Bosc N, Wroblowski B, Aci-S\u00e8che S, Meyer C, Bonnet P (2015) A Proteometric Analysis of Human Kinome: Insight into Discriminant Conformation-Dependent Residues. ACS Chem Biol 10: 2827\u20132840. doi:"} +{"text": "Scientific Reports6: Article number: 2269010.1038/srep22690; published online: 03112016; updated: 04202016This Article contains errors in the legend of Fig. 5.Diap3-Tg mutant mice.\u201d\u201cPost-weaning dietary supplement slowed or reversed hearing loss in should read:Diap3-Tg mutant mice.\u201d\u201cPost-weaning dietary supplement accelerated hearing loss in"} +{"text": "Nature Communications 6: Article number: 745110.1038/ncomms8451 (2015); Published: 06162015; Updated: 10222015The original version of the Supplementary Figures 1-15, Supplementary Table 1, Supplementary Note 1 and Supplementary Reference."} +{"text": "In the crystal, O\u2014H\u22efN hydrogen bonds lead to the formation of infinite chains along [101] which are further linked by N\u2014H\u22efO hydrogen bonds, forming (010) sheets.The title compound, C DOI: 10.1107/S2056989015009172/ff2137Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989015009172/ff2137Isup3.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989015009172/ff2137fig1.tif. DOI: Mol\u00adecular structure of the title compound, showing the atom-labelling scheme. Displacement ellipsoids are drawn at the 50% probability level. H atoms are drawn as spheres of arbitrary radius.Click here for additional data file.10.1107/S2056989015009172/ff2137fig2.tif. DOI: Mol\u00adecular packing of the title compound with hydrogen bonding shown as dashed lines.1400729CCDC reference: crystallographic information; 3D view; checkCIF reportAdditional supporting information:"} +{"text": "The central coordination angles with the PdII atom range from 83.14\u2005(10) to 97.25\u2005(12)\u00b0.In the title compound, [Pd(CF DOI: 10.1107/S1600536814007855/vn2082Isup2.hklStructure factors: contains datablock(s) I. DOI: 996158CCDC reference: crystallographic information; 3D view; checkCIF reportAdditional supporting information:"} +{"text": "The correct sentence is: Transfusion recipients received the human umbilical cord-derived mesenchymal stem cells (hUC-MSC; 4 [2.87\u201310]\u00d710"} +{"text": "In the crystal, the solvent mol\u00adecule is linked to the cation via a weak C\u2014H\u22efO hydrogen bond.In the tetra\u00adhydro\u00adfuran solvate of the title salt, C DOI: 10.1107/S1600536814011258/lh5704Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S1600536814011258/lh5704Isup3.cdxSupporting information file. DOI: 1003430CCDC reference: crystallographic information; 3D view; checkCIF reportAdditional supporting information:"} +{"text": "AbstractHemiptera: Adelgidae) is presented. Six family-group names are listed, five being synonyms of Adelgidae. Twenty-two genus-group names, of which nine are subjectively valid and in use, are presented with their type species, etymology, and grammatical gender. One hundred and six species-group names are listed, of which 70 are considered subjectively valid.A taxonomic and nomenclatural Catalogue of the adelgids ( Adelgidae is a small family of Hemiptera with 65 species, closely related to Aphididae. They exhibit a two-year life cycle, with some species alternating hosts between spruce (Picea) one year and species of another conifer genus the next. Other species or populations do not alternate hosts, feeding only on Picea or one of the other conifer genera. Like other Aphidomorpha, Adelgidae exhibit cyclical parthenogenesis, although they are oviparous unlike the viviparous Aphididae. Some adelgid species are important forestry pests, most notably the hemlock woolly adelgid, Adelgestsugae (Annand) and the balsam woolly adelgid, Adelgespiceae (Ratzeburg). Adelgidae are two extinct families, Elektraphididae and Mesozoicaphididae, the three families comprising the superfamily Adelgoidea and several subgenera, although some have preferred to treat the latter as full genera , two and seven valid genera and subgenera , and 65 and five valid species and subspecies . We include one genus-group nomen dubium, four suppressed genus-group names, three species-group nomina dubia, and two unavailable species-group names; the many such names still in combination with Chermes are not listed here geniculatus (Chermes)iculatus :202 (Cheisedakii Eichhorn in Adelges)japonicus (Chermes)aponicus :71 lapponicus (Chermes)pponicus :390 (Chepraecox (Chermeslapponicus Cholodkovsky)=praecox :28 (varilariciatus (Chermes)riciatus :137 (ChelaricisAdelges) (nomen protectum with respect to Coccuslaricis Bouch\u00e9)laricisVallot 1836subspecies atratus (Chermes)=atratus :39 (Chercoccineus (Chermes)=occineus :202 (Checonsolidatus (Chermes)=olidatus :137 (Chehamadryas (Anisophleba)=amadryas :320 (Anilariceti (Chermes)=lariceti :279 (Chelaricis (Coccus) =laricis :22 (Cocclaricis (Chermes)=laricis :644 (Cheobtectus (Chermes)=obtectus :200 (Chestrobilobius (Chermes)=bilobius :203 subspecies tardoides ardoides :175 (vartardus (Chermes)tardus :3 (=affinis :167 (Cnaniger (Chermes)=niger :41 (CherANNANDINASubgenus AdelgestsugaeType species Etymology. (Percy Nicol) Annand [American entomologist] + -inaGender. FemininetsugaeAdelges)APHRASTASIASubgenus ChermespectinataeType species. Etymology. Greek aphrastos \u2018unnoticed\u2019 + -iaGender. Femininepectinatae (Chermes)ctinatae :47 (Cherishiharai (Chermes)subspecies shiharai :75 (Cherpectinatae (subspecies ctinatae CHOLODKOVSKYASubgenus ChermesviridanusType species. Etymology. (Nikolai Alexandrovitsch) Cholodkovsky [Russian entomologist] + -aGender. FeminineoregonensisAdelges)viridanus (Chermes)iridanus :39 (Cherlaricicola (Chermes)=ricicola :151 (Cheviridulus (Chermes)iridulus :175 (CheDREYFUSIASubgenus ChermespiceaeType species. Etymology. (Ludwig Theodor) Dreyfus [German entomologist] + -iaGender. Feminineabietispiceae (Chermes) nomen dubium indicates that the species is probably real but that the description is insufficient to confirm its identity; PageBreakcompare with their description of Dreyfusiaknucheli Schneider-Orelli and Schneider. Dreyfusiaknucheli, but qualified it with a question mark)ispiceae :57 nomen dubium (nitectus :6 (Chermn dubium joshii (Dreyfusia)joshii :260 (Dreknucheli (Dreyfusia)knucheli :416 (Dremerkeri (Dreyfusia)merkeri :312 (Drenebrodensis (Dreyfusia)rodensis :246 (Drenordmannianae (Chermes)annianae :90 (Chernuesslini (Dreyfusia)=uesslini :739 (Drepiceae (Chermes)piceae :204 (Checanadensis (Dreyfusiapiceae (Ratzeburg))subspecies nadensis :454 )subspecies piceae (subspecies piceae bouvieri (Chermespiceae Ratzeburg)=bouvieri :10 (varipindrowiAdelges)prelli (Dreyfusia)prelli :822 (Dreschneideri (Dreyfusia)hneideri :684 (Dretodomatsui (Dreyfusia)domatsui :73,92 (DGILLETTEELLASubgenus ChermescooleyiType species. Etymology. (Clarence Preston) Gillette [American entomologist] + -ella [diminutive suffix]Gender. FeminineGilletteaB\u00f6rner 1909dNote. Replacement name for GILLETTEA=ChermescooleyiType species. Etymology. (Clarence Preston) Gillette [American entomologist] + -aGender. FeminineGilletteaHymenoptera, Cynipidae)Note. Junior homonym of cooleyi (Chermes)cooleyi :3 (Chermcoweni (Chermescooleyi Gillette)coweni :10 (varicummingae (Gilletteella) nomen nudumummingae :10,22,42glandulae (Zhang in Gilletteella)Zhang in :382 abietis :454 (Cheabietislaricis (Chermes) based on Larix-associated form of Chermesabietis=slaricis :88 =eviridis :39 nomen dubium=mabietis :99 (Aphilaricifoliae (Chermes)=cifoliae :752 (ChediversisAdelges)karafutonisAdelges)kitamiensis (Sacchiphantes)amiensis :341 segregis (Sacchiphantes)segregis :67 (Sacctorii (Eichhorn in Sacchiphantes)viridis (Chermes)viridis :202 =dentalis :281 Gistel [German entomologist] + -i + ella [diminutive suffix]PageBreakGender. FeminineAphanusNote. Replacement name for APHANUS=ChermeslapidariusType species. Etymology. Greek aphan\u0113s \u2018invisible\u2019Gender. MasculineAphanusHemiptera, Lygaeidae)Note. Junior homonym of lapidaria (Chermes) nomen dubium (placement unknown)apidaria :306 (ChePINEUSPINEODESSubgenus ChermespinifoliaeType species. Pine(us) [Hemiptera: Adelgidae]+ Greek -\u014dd\u0113s \u2018resembling\u2019Etymology. Gender. Masculinepinifoliae (Chermes)nifoliae :741 (Cheabieticolens (Chermes)=ticolens :156 (Chearmiger (Chermes)=armiger :5 (Chermmontanus (Chermes)=montanus :14 (CherPINEUSSubgenus CoccuspinicorticisType species. Etymology. Latin pineus \u2018of or pertaining to pine\u2019Gender. MasculineCHERMAPHIS=Kermaphispinivar.laevisType species. Cherm(es) [Hemiptera] + Aphis [Hemiptera: Aphididae]Etymology. Gender. FeminineCNAPHALODES=ChermespiniType species. Etymology. Greek knaphallon \u2018pillow\u2019 + Greek -\u014dd\u0113s \u2018resembling\u2019Gender. MasculineEOPINEUS=ChermesstrobiType species. Pineus [Hemiptera: Adelgidae]Etymology. Greek \u0113\u014ds \u2018dawn\u2019, \u2018early\u2019 + Gender. MasculineKERMAPHIS=AnisophlebapiniType species. Kerm(es) [Hemiptera: Coccoidea] + Aphis [Hemiptera: Aphididae]Etymology. Gender. FemininePageBreakPITYOPSYLLA=ChermespiniType species. Psylla [Hemiptera: Psyllidae]Etymology. Greek pitys \u2018pine\u2019 + Gender. FeminineCnaphalodesAmyot and Audinet Serville 1843Note. Suppressed and juniabietinusPineus)armandicolaPineus)boerneriPineus) (Pineuslaevis (Maskell), but iPineus) :11, InouiPineus) :36, and iPineus) :8 list tboyceiPineus)cembrae (Chermes)cembrae :47 (Chercembrae (subspecies cembrae sibiricus (Chermes)=ibiricus :388 (ChepinikoreanusPineuscembrae (Cholokovsky))subspecies cladogenousPineus)coloradensis (Chermes)radensis :16 (ChercortecicolusPineus)engelmanniiPineus)floccus (Chermes)floccus :137 (CheghaniiPineus)harukawaiPineus)havrylenkoiPineus)hosoyaiPineus)konowashiyaiPineus)laevis (Kermaphispini (Koch))laevis :16 (varimatsumuraiPineus)orientalis (Chermes)ientalis :3,6 (ChepatchaePineus)pineoides (Chermespini (Koch))ineoides :263 (varpini (Chermes)pini :328 (Checoniferarum (Chermes)=iferarum :222 (Chepini (Anisophleba)=pini :322 (Anipinicola (Chermespini (Koch))=pinicola :57 (varipiniyunnanensisPineus)sichunanus Zhang in Pineus)similis (Chermes)similis :15 (ChersimmondsiPineus)strobi (Coccus)strobi :643 =rticalis :197 (Chepinicorticis (Coccus)=corticis :871 (CocPageBreakstrobi (Coccus)=strobi :174 (Cocstrobi (Chermes)=strobi :203 (ChesylvestrisPineus)wallichianaePineus)ADELGESVallot 1836abieticolensPineus (Pineodes) pinifoliaeabietinusPineus (Pineus)abietisAdelges (Sacchiphantes)abietislaricisAdelges (Sacchiphantes) abietisabietispiceaeAdelges (Dreyfusia)aenigmaticusAdelges (Adelges)affinisAdelges (Adelges) tardusalaeviridisAdelges (Sacchiphantes) abietisANISOPHLEBAAdelges (Adelges)ANNANDINAAdelgesAPHANUSGisteliellaAPHRASTASIAAdelgesarmandicola Zhang et al. in Chen 1992 \u2013 Pineus (Pineus)armigerPineus (Pineodes) pinifoliaeatratusAdelges (Adelges) laricislariciboerneriPineus (Pineus)bouvieriAdelges (Dreyfusia) piceaepiceaeboyceiPineus (Pineus)canadensisAdelges (Dreyfusia) piceaecembraePineus (Pineus)CHERMAPHISPineus (Pineus)CHERMESLinnaeus 1758CHOLODKOVSKYAAdelgescladogenousPineus (Pineus)CNAPHALODESPineus (Pineus)coccineusAdelges (Adelges) laricislariciscoloradensisPineus (Pineus)coniferarumPineus (Pineus) piniconsolidatusAdelges (Adelges) laricislariciscooleyiAdelges (Gilletteella)cortecicolusPineus (Pineus)corticalisPineus (Pineus) strobicoweniAdelges (Gilletteella)cummingaeAdelges (Gilletteella)PageBreakdiversisAdelges (Sacchiphantes)DREYFUSIAAdelgesELATIPTUSAdelges (Sacchiphantes)engelmanniiPineus (Pineus)EOPINEUSPineus (Pineus)floccusPineus (Pineus)funitectusAdelges (Dreyfusia)gallarumabietisAdelges (Sacchiphantes) abietisgeniculatusAdelges (Adelges)ghaniiPineus (Pineus)GILLETTEAAdelges (Gilletteella)GILLETTEELLAAdelgesGISTELIELLAStrand 1928glandulae Zhang in Adelges (Gilletteella)hamadryasAdelges (Adelges) laricislaricisharukawaiPineus (Pineus)havrylenkoiPineus (Pineus)himalayensisAdelges (Dreyfusia) abietispiceaehosoyaiPineus (Pineus)isedakii Eichhorn in Adelges (Adelges)ishiharaiAdelges (Aphrastasia) pectinataejaponicusAdelges (Adelges)joshiiAdelges (Dreyfusia)karafutonisAdelges (Sacchiphantes)karamatsuAdelges (Adelges)KERMAPHISPineus (Pineus)kitamiensisAdelges (Sacchiphantes)knucheliAdelges (Dreyfusia)konowashiyaiPineus (Pineus)laevisPineus (Pineus)lapidariaGisteliellalapponicusAdelges (Adelges)LARICETHUSAdelges (Adelges)laricetiAdelges (Adelges) laricislaricislariciatusAdelges (Adelges)laricicolaAdelges (Cholodkovskya) viridanuslaricifoliaeAdelges (Sacchiphantes) abietislaricisAdelges (Adelges) laricislaricislaricisAdelges (Adelges) laricislaricislaricisAdelges (Adelges)matsumuraiPineus (Pineus)merkeriAdelges (Dreyfusia)montanusPineus (Pineodes) pinifoliaePageBreaknebrodensisAdelges (Dreyfusia)nigerAdelges (Adelges) tardusnordmannianaeAdelges (Dreyfusia)nuessliniAdelges (Dreyfusia) nordmannianaeobtectusAdelges (Adelges) laricislaricisoccidentalisAdelges (Sacchiphantes) viridisoccidentalisAdelges (Dreyfusia) piceaeoregonensisAdelges (Cholodkovskya)orientalisPineus (Pineus)patchaePineus (Pineus)pectinataeAdelges (Aphrastasia)PHLOEOPHTHIRIDIUMAdelges (Sacchiphantes)piceae Ratzeburg 1944 \u2013 Adelges (Dreyfusia)pindrowiAdelges (Dreyfusia)PINEODESPineuspineoidesPineus (Pineus)PINEUSShimer 1869piniPineus (Pineus)piniPineus (Pineus) pinipinicolaPineus (Pineus) pinipinicorticisPineus (Pineus) strobipinifoliaePineus (Pineodes)pinikoreanusPineus (Pineus) cembraepiniyunnanensisPineus (Pineus)PITYOPSYLLAPineus (Pineus)potaninilaricis Zhang in Adelges (Adelges) laricispraecoxAdelges (Adelges) lapponicusprelliAdelges (Dreyfusia)roseigallisAdelges (Sacchiphantes)SACCHIPHANTESAdelgesschneideriAdelges (Dreyfusia)segregisAdelges (Sacchiphantes)sibiricusPineus (Pineus) cembraecembraesichunanus Zhang in Pineus (Pineus)similisPineus (Pineus)simmondsiPineus (Pineus)strobiPineus (Pineus) strobistrobiPineus (Pineus)strobiPineus (Pineus) strobistrobilobiusAdelges (Adelges) laricislaricissylvestrisPineus (Pineus)tardoidesAdelges (Adelges)tardusAdelges (Adelges)PageBreaktodomatsuiAdelges (Dreyfusia)torii Eichhorn in Adelges (Sacchiphantes)tsugaeAdelges (Annandina)viridanusAdelges (Cholodkovskya)viridisAdelges (Sacchiphantes)viridulusAdelges (Cholodkovskya)wallichianaePineus (Pineus)"} +{"text": "Paenibacillus larvae is a Firmicute bacterium that causes American Foulbrood, a lethal disease in honeybees and is a major source of global agricultural losses. Although P. larvae phages were isolated prior to 2013, no full genome sequences of P. larvae bacteriophages were published or analyzed. This report includes an in-depth analysis of the structure, genomes, and relatedness of P. larvae myoviruses Abouo, Davis, Emery, Jimmer1, Jimmer2, and siphovirus phiIBB_Pl23 to each other and to other known phages.P. larvae phages Abouo, Davies, Emery, Jimmer1, and Jimmer2 are myoviruses with ~50 kbp genomes. The six P. larvae phages form three distinct groups by dotplot analysis. An annotated linear genome map of these six phages displays important identifiable genes and demonstrates the relationship between phages. Sixty phage assembly or structural protein genes and 133 regulatory or other non-structural protein genes were identifiable among the six P. larvae phages. Jimmer1, Jimmer2, and Davies formed stable lysogens resistant to superinfection by genetically similar phages. The correlation between tape measure protein gene length and phage tail length allowed identification of co-isolated phages Emery and Abouo in electron micrographs. A Phamerator database was assembled with the P. larvae phage genomes and 107 genomes of Firmicute-infecting phages, including 71 Bacillus phages. Phamerator identified conserved domains in 1,501 of 6,181 phamilies (only 24.3%) encoded by genes in the database and revealed that P. larvae phage genomes shared at least one phamily with 72 of the 107 other phages. The phamily relationship of large terminase proteins was used to indicate putative DNA packaging strategies. Analyses from CoreGenes, Phamerator, and electron micrograph measurements indicated Jimmer1, Jimmer2, Abouo and Davies were related to phages phiC2, EJ-1, KC5a, and AQ113, which are small-genome myoviruses that infect Streptococcus, Lactobacillus, and Clostridium, respectively.Paenibacillus genus and the first organization of P. larvae phages based on sequence and structure. This analysis provides an important contribution to the field of bacteriophage genomics by serving as a foundation on which to build an understanding of the natural predators of P. larvae.This paper represents the first comparison of phage genomes in the The online version of this article (doi:10.1186/1471-2164-15-745) contains supplementary material, which is available to authorized users. Paenibacillus larvae (P. larvae) is a sporulating Firmicute. It is the causative agent of American Foulbrood (AFB), a disease that infects and destroys the larvae of honeybees (Apis mellifera). The first eight P. larvae phages were reported between 1955 and 1999 and included BLAPaenibaciuded BLA, PBL0.5uded BLA, and PPLuded BLA. These p. larvae\u201310. Phagbacteria, 12.Recent advances in DNA sequencing technology have made it possible to sequence many bacteriophage genomes. When these sequences are analyzed, putative protein functions can be determined. Other studies have used comparative genomics to organize phages into related clusters, correlaComparative genomics can be accomplished using software specialized for phage genomes such as the computer program, Phamerator. PhameraP. larvae phages were isolated. These phages were fully sequenced and their genomes published, 0305phi8-36 (B. thuringiensis) [NC_009760], 250 (B. cereus) [GU229986], Andromeda (B. pumilus) [KC330684], AP50 (B. anthracis) [NC_011523], B103 (B. subtilis) [NC_004165], B4 (B. cereus) [JN790865], B5S (B. cereus) [JN797796], Bam35c (B. thuringiensis) [NC_005258], Basilisk (B. cereus) [KC595511], Bastille (B. cereus) [NC_018856], BCD7 (B. cereus) [NC_019515], BceA1 (B. cereus) [HE614282], BCJA1c (B. clarkii) [NC_006557], BCP78 (B. cereus) [NC_018860], BCU4 (B. cereus) [JN797798], BMBtp2 (B. thuringiensis) [JX887877], BPS13 (B. cereus) [NC_018857], BtCS33 (B. thuringiensis) [NC_018085], CAM003 (B. thuringiensis) [NC_024216.1], Cherry (B. anthracis) [NC_007457], Curly (B. pumilus) [KC330679], Eoghan (B. pumilus) [KC330680], Evoli, (B. thuringiensis) [NC_024207.1], Fah (B. anthracis) [NC_007814], Finn (B. pumilus) [KC330683], G (B. megaterium) [JN638751], GA-1 (Bacillus sp.) [NC_002649], Gir1 (Bacillus sp.) [Bacillus_Draft], Gamma 51 (B. cereus) [DQ222853], Gamma 53 (B. anthracis) [DQ222855], Gamma isolate d\u2019Herelle (B. cereus) [DQ289556], Gemini (B. pumilus) [KC330681], GIL16c (B. thuringiensis) [NC_006945], Hakuna (B. thuringiensis) [NC_024213.1], Hoody T (B. thuringiensis) [NC_024205], IEBH (B. thuringiensis) [NC_011167], JL (B. cereus) [KC595512], JPB9 (B. thuringiensis) [Bacillus_Draft], Megatron (B thuringiensis) [NC_024211.1], MG-B1 (B. weihenstephanensis) [NC_021336], Nf (B. subtilis) [EU622808], Pappano (B. pumilus) [Bacillus_Draft], PBC1 (B. cereus) [NC_017976], Pegasus (Bacillus sp.) [bacillus.phagesdb.org], pGIL01 (B. thuringiensis) [AJ536073], phi105 (B. subtilis) [NC_004167], phi29 (B. Subtilis) [NC_011048], phiAGATE (B. pumilus) [JX238501], phiNIT1 (B. subtilis) [NC_021856], phIS3501 (B. thuringiensis) [NC_019502], Pleiades (B. pumilus) [Bacillus_Draft], PM1 (B. subtilis) [NC_020883], Polaris (B. pumilus) [Bacillus_Draft], PZA (B. subtilis) [M11813], Riley (B. thuringiensis) [KJ489402], Shanette (B. cereus) [KC595513], SP10 (B. subtilis) [NC_019487], SPbeta (SPBc2) (B. subtilis) [NC_001884], SPO1 (B. subtilis) [NC_011421], SPP1 (B. Subtilis) [NC_004166], Stitch (Bacillus sp.) [Bacillus_Draft], Taylor (B. pumilus) [KC330682], TP21-L (B. cereus) [NC_011645], Troll (B. thuringiensis) [KF208639], W.Ph. (B. cereus) [NC_016563], WBeta (B. cereus) [NC_007734], Wip1 (B. anthracis) [KF188458], phBC6A51 (B. cereus) [NC_004820], phBC6A52 (B. cereus) [NC_004821], Bacillus virus 1 (Bacillus. sp. 6\u00a0k512) [NC_009737]; Clostridium phage phiC2 (C. difficile) [NC_009231]; Enterococcus phages phiEf11 [NC_013696], phiEF24C [NC_009904], phiFL3A [NC_013648]; Geobacillus phages GBSV1 (Geobacillus sp. 6\u00a0k51) [NC_008376], E2 (Geobacillus) [NC_009552]; Lactobacillus phages A2 (L. casei) [NC_004112], KC5a (L. gasseri) [NC_007924], Lb338-1 (L. paracasei) [NC_012530], Lc-Nu (L. rhamnosus) [NC_007501], LP65 (L. plantarum) [NC_006565], phiAT3 (L. casei) [NC_005893], phig1e (Lactobacillus) [NC_004305]; Listeria phages P100 (L. monocytogenes) [DQ004855], A118 (L. monocytogenes) [NC_003216], A500 (L. monocytogenes) [NC_009810], A511 (L. monocytogenes) [NC_009811], B054 (L. monocytogenes) [NC_009813], P40 (L. monocytogenes) [NC_011308]; Paenibacillus phages Abouo (P. larvae) [KC595517], Davies (P. larvae) [KC595518], Emery (P. larvae) [KC595516], Jimmer1 (P. larvae) [KC595515], Jimmer2 (P. larvae) [KC595514], PG1(P. glucanolyticus) [HQ332138], phiIBB_Pl23 (P. larvae) [KF010834]; Staphylococcus phages phi 12 (S. aureus) [AF424782], 37 (S. aureus) [NC_007055], 3A (S. aureus) [NC_007053], 47 (S. aureus) [NC_007054], 55 (S. aureus) [NC_007060], 88 (S. aureus) [NC_007063], G1 (S. aureus) [NC_007066], K (S. aureus) [NC_005880], PH15 (S. epidermidis) [NC_008723], phiSLT (S. aureus) [NC_002661], SA11-v (S. aureus) [NC_019511], SpaA1 (S. pasteuri) [NC_018277], Twort (S. aureus) [NC_007021], X2 (S. aureus) [NC_007065], tp310-2 (S. aureus) [NC_009762]; Streptococcus prophage EJ-1 (S. pneumoniae) [NC_005294].The neighbor-joining phylogenetic tree . The same files are also available atPhamerator database containing the 113 phage genomes.(ZIP 7 MB)Additional file 1:Spreadsheet exported from the Phamerator database to report all phage gene products in the database, the phams to which the gene products are assigned, and the conserved domains found in gene products in those phams.(XLSX 1 MB)Additional file 2:"} +{"text": "Scientific Reports5: Article number: 13827; 10.1038/srep13827 published online: 09082015; updated: 10162015.In this Article, Figure 4 is incorrect. The correct Figure 4 appears below as"} +{"text": "Nature Communications7 Article number:1110310.1038/ncomms11103 (2016); Published 03222016; Updated 06082016In"} +{"text": "In comparison with the previous report . The HgS4 polyhedra share a common S atom, building up chains extending parallel to [010]. All S atoms of the resulting 1\u221e[HgS2/1S2/2] chains are also part of SCN\u2212 anions that link these chains with the K+ cations into a three-dimensional network. The K\u2014N bond lengths of the distorted KN7 polyhedra lie between 2.926\u2005(2) and 3.051\u2005(3)\u2005\u00c5.The crystal structure of the room-temperature modification of K[Hg(SCN)adze 1952. Zh. Fiz DOI: 10.1107/S1600536814013403/hb0013Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S1600536814013403/hb0013fig1.tif3 4 . DOI: 3] in a projection along [010]. Displacement ellipsoids are drawn at the 90% probability level. Colour code: Hg green, S yellow, C blue, N magenta, K grey. The HgS4 polyhedron is shown in red.The crystal structure of K[Hg(SCN)1006909CCDC reference: crystallographic information; 3D view; checkCIF reportAdditional supporting information:"} +{"text": "In the crystal, pairs of O\u2014H\u22efO hydrogen bonds between carb\u00adoxy\u00adlic groups link mol\u00adecules, related by a twofold rotation axis, into supra\u00admolecular dimers.In the title compound, C DOI: 10.1107/S2056989014028254/xu5833Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989014028254/xu5833Isup3.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989014028254/xu5833fig1.tif. DOI: A mol\u00adecule showing atom labels and 50% probability displacement ellipsoids for non-H atoms.Click here for additional data file.10.1107/S2056989014028254/xu5833fig2.tif. DOI: Crystal packing in the structure with H atoms omitted and hydrogen bonds shown as dotted lines.1041493CCDC reference: crystallographic information; 3D view; checkCIF reportAdditional supporting information:"} +{"text": "Scientific Reports5: Article number: 10378; 10.1038/srep10378 published online: 05212015; updated: 01202016.The Acknowledgements section is incomplete.\u201cThis work was supported by SUTD-MIT international design center (IDC) grant.\u201dshould read:\u201cThis work was supported by SUTD-MIT international design center (IDC) grant and SUTD Temasek Seed funding (IGDSS1402031).\u201d"} +{"text": "The dioxane ring is in a half-chair conformation. An intra\u00admolecular C\u2014H\u22efO hydrogen forms an S(6) ring. The amine N atom is sp2-hybridized.In the title mol\u00adecule, C DOI: 10.1107/S1600536814013191/lh5709Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S1600536814013191/lh5709Isup3.cmlSupporting information file. DOI: crystallographic information; 3D view; checkCIF reportAdditional supporting information:"} +{"text": "In the crystal, the complex mol\u00adecules stack at a distance of 4.6738\u2005(3)\u2005\u00c5 along the a axis, which exclude any significant inter\u00adactions between the aromatic rings.In the title complex, [Ni(C DOI: 10.1107/S2056989015000328/ds2244Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989015000328/ds2244fig1.tifORTEP L 2 . DOI: ORTEP drawing (ellipsoid probability at 50%) of the centrosymmetric NiL2 complex.Click here for additional data file.10.1107/S2056989015000328/ds2244fig2.tif. DOI: Crystal packing of the complex.1035820CCDC reference: crystallographic information; 3D view; checkCIF reportAdditional supporting information:"} +{"text": "Correction: BMC Plant Biol 22, 269 (2022)\u00a0 \u00a0https:// doi. org/ 10. 1186/ s13063-\u200b021-\u200b05592-zFollowing the publication of the original article , we wereIncorrect tagging:GivenName: Lorena Fern\u00e1ndez.FamilyName: de la Cruz.Correct tagging:GivenName: Lorena.FamilyName: Fern\u00e1ndez de la Cruz.The original article has been corrected."} +{"text": "Erratum zu:Herzschr Elektrophys 202210.1007/s00399-022-00855-xIn der Originalversion des Beitrags stand der Hinweis zu Teil\u00a02 im Untertitel. Dieser Hinweis wurde nun in den Haupttitel eingef\u00fcgt.Der Originalbeitrag wurde korrigiert."} +{"text": "Erratum zu:Herzschr Elektrophys 202210.1007/s00399-022-00917-0In diesem Artikel wurde Abb.\u00a0Der Originalbeitrag wurde korrigiert."} +{"text": "Erratum zu:HNO 202110.1007/s00106-021-01143-9Dinh Nam Pham f\u00e4lschlicherweise als Nam Dinh Pham wiedergegeben.In diesem. Beitrag wurde versehentlich der Name des Autors Die Tab.\u00a0The original article has been corrected."} +{"text": "Correction: SN Bus Econ (2022) 2:166 10.1007/s43546-022-00347-7In this article the author family and given names have been interchanged:Tjulin \u00c5saVinberg StigLandstad Bodil JHedlund MarianneNordenmark MikaelThey should be read:\u00c5sa TjulinStig VinbergBodil J. LandstadMarianne HedlundMikael NordenmarkThe original article has been corrected."} +{"text": "This article was published with incorrect corresponding author details.sunshine1d1@126.comCurrently reads: * Corresponding author: Xian Sun, email sunshine1d1@126.com and Wei-Jan Wang, email: cvcsky@gmail.comThis should read: * Corresponding authors: Xian Sun, email"} +{"text": "Erratum zu:Herzschr Elektrophys 202210.1007/s00399-022-00854-yIn der Originalversion des Beitrags stand der Hinweis zu Teil\u00a01 im Untertitel. Dieser Hinweis wurde nun in den Haupttitel eingef\u00fcgt.Der Originalbeitrag wurde korrigiert."} +{"text": "Erratum zu:Der Nervenarzt 202110.1007/s00115-021-01108-xDer Beitrag enth\u00e4lt eine fehlerhafte Information. Bitte beachten Sie: Sigmund Freud (1856\u20131939) hat auch den Nobelpreis f\u00fcr Literatur nicht erhalten.Please note: Sigmund Freud (1856\u20131939) did not even receive the Nobel Prize for Literature."} +{"text": "Augochloropsis and other shiny green Halictinae have had various taxonomic issues and are often misidentified. One prevailing taxonomic issue is that Augochloropsismetallica (Fabricius) has two subspecies, that have long been recognized as morphologically distinct , but the subspecies are inconsistently applied in the literature. Here, we review the Augochloropsis of the Midwest and further address the Augochloropsis species in the broader United States to resolve the outstanding taxonomic issues with the midwestern species. We provide identification keys and diagnoses for the genera and species of the shiny green Halictinae of the midwestern United States, which includes the genera Agapostemon, Augochlora, Augochlorella, and Augochloropsis. This work results in taxonomic changes to Augochloropsis. Augochloropsissumptuosa (Smith) is split into two species, with the name Augochloropsissumptuosa retained for the eastern form, and Augochloropsishumeralis (Patton), stat. nov., reinstated for the western form. Augochloropsismetallica is split into five species, with two of those species occurring in the midwestern United States: Augochloropsismetallica and Augochloropsisviridula (Smith), stat. nov. Examination of the holotype of Augochloropsisfulgida (Smith) revealed that it does not agree with the prevailing concept of Augochloropsismetallicafulgida; it is reinstated as Augochloropsisfulgida, stat. nov., but is currently known only from the holotype female from Florida. Augochloropsiscuprea (Smith), long considered to be a synonym of Augochloropsismetallica, is also distinct, and we are reinstating Augochloropsiscuprea, stat. nov., though the range of this species is unclear. We further recognize Augochloropsisfulvofimbriata (Friese), stat. nov., from South and Central America, as distinct. These changes result in a total of three Augochloropsis species in the Midwest and seven named species in the United States. We are aware of additional species from the southern and southwestern United States that are undescribed, and we highlight additional taxonomic work that remains to be done. Augochloropsis Cockerell contains approximately 140 species, recognizable by their metallic coloration and distinctly-shaped tegula and Augochloropsiscuprea (Smith). Augochloropsiscuprea was in fact two species in his region and split it into Augochloropsiscuprea and Augochloropsisviridula (Smith). Augochloropsiscaerulea with the earlier name of Augochloropsishumeralis (Patton). After examination of the Fabricius types, and apparently unaware of the work by Dreisbach, Augochloropsiscuprea with the older name Augochloropsismetallica (Fabricius). Augochloropsismetallica into subspecies, suggesting Augochloropsismetallicafulgida (Smith) for the \u201csouthern variety.\u201d Moure\u2019s classification was followed by the most recent treatment performed by Augochloropsismetallica, replaced the name Augochloropsishumeralis with Augochloropsissumptuosa (Smith) based on correspondence with Moure, and recognized a third species, Augochloropsisanonyma Cockerell.The species of Augochloropsis in the United States in recent bee diversity studies, primarily with the usage of the subspecies of Augochloropsismetallica, with some researchers recognizing them and others not. As a result, when papers refer to \u201cAugochloropsismetallica\u201d it is often impossible to know whether they were referring to \u201cAugochloropsismetallicametallica\u201d or \u201cAugochloropsismetallicafulgida,\u201d especially given that many publications fail to cite the identification resources or taxon concepts they used to full species .NHMUKThe Natural History Museum, London, United Kingdom (J. Monks).CNBL The collection of the Cariveau Native Bee Lab, St. Paul Minnesota, USA (Z. Portman).CRC Catherine Reed Collection. Currently resides in the Cariveau Native Bee Lab and will be accessioned into the UMSP.EERC Elaine Evans Research Collection (E. Evans). Housed at the Cariveau Native Bee Lab (CNBL) and will be accessioned into the UMSP.IDNP Indiana Dunes National Park. Examined specimens deposited at the UMSP.iNat Selected high-quality records from the community science portal iNaturalist.com were examined for Augochloropsishumeralis. All record information is included in the material examined section.MASR Mike Arduser specimen record. Includes a combination of specimens in Mike Arduser\u2019s personal collection, as well as specimens Mike Arduser has personally identified but no longer has in hand.MNDNR The Minnesota Department of Natural Resources, St. Paul, MN, USA (J. Petersen and N. Gerjets). These are primarily deposited in the UMSP except for a small synoptic collection.NHMD Natural History Museum of Denmark, Copenhagen, Denmark (L. Vilhelmsen).USNMSmithsonian National Museum of Natural History, Washington D.C., USA.OSUC C.A. Triplehorn Insect Collection, Ohio State University Columbus, Ohio, USA (L. Musetti).OUMNH University Museum of Natural History, Oxford, United Kingdom (J. Hogan).UMSPUniversity of Minnesota Insect Collection, St. Paul, Minnesota, USA (R. Thomson).ggplot2\u2019 package . For county level records, points were mapped to the county centroids. Specimen images were taken using an Olympus DP27 camera mounted on an Olympus SZX16 stereo microscope, with the images stacked using CombineZP software . Images package and the package . State a package .Halictinae of the midwestern United States.Identification of the shiny green Taxon classificationAnimaliaHymenopteraHalictidae\ufeffGenusGu\u00e9rin-M\u00e9neville8D7C203F-8557-5AD3-B9B4-F4D1076EF99DAgapostemon are diagnosed by the complete carina on the rear face of the propodeum east of the central part of the state, with the easternmost records from Ellsworth and Rice counties. Recent survey efforts in eastern Nebraska have not found any Agapostemonangelicus east of the Grand Island area . No confirmed recent records are known from Minnesota, Missouri, or Iowa, despite extensive collection efforts in those states. However, care should be taken to look for it and more work needs to be done to confirm the eastern extent of the range of Agapostemonangelicus. In areas where the two species may overlap, it is recommended that females identified morphologically as Agapostemonangelicus or Agapostemontexanus be treated as a single morphospecies. The two species can also be separated by DNA barcodes.1Taxon classificationAnimaliaHymenopteraHalictidae\ufeffCresson2BE9D4EE-91AC-5E04-8770-7C82FF03F895Agapostemonmelliventris can be recognized by having the apex of the clypeus yellow as well as their non-metallic, light-colored metasoma. The terga are generally amber-colored but can be dark enough F638DA21-69D3-5C5F-B8A3-860D7F7E3F27Agapostemonsericeus can be recognized by the combination of the metallic green metasoma e.g., but was Agapostemonfemoratus Crawford, primarily a western species not recorded east of New Mexico, Colorado and Wyoming by Agapostemonsericeus, though the males are quite distinct, possessing a grossly enlarged hind femur, its width and length equal or nearly so. The key in Agapostemonsericeus is more distinctly punctate than Agapostemonfemoratus, but we do not consider this a reliable separating character. Curiously, there are several Missouri records of Agapostemonfemoratus from the 1960s identified by Roberts in separate online databases, 93483CC2-EAA6-59D6-BDCF-9FE3D3F52DF9Agapostemonsplendens can be recognized by the combination of the metallic green metasoma .The female of ate Figs , 6A. Agadge Fig. , the uprdge Fig. , and it Agapostemonsplendens can be recognized from all other midwestern Agapostemon by their very enlarged hind femur, with the length twice the width have used the shape of the ridges of the propodeal triangle to separate female Agapostemonsplendens from Agapostemonsericeus , but we have found the character variable and it can be quite subtle, particularly in smaller Agapostemonsplendens. Agapostemonsplendens is largely restricted to areas of deep sands. We have examined material from throughout the range of Agapostemonsplendens, and there are many individuals, especially in the southern US, that have the scutal sculpturing more reticulate, similar to Agapostemonsericeus. More work is needed to determine whether this represents normal variation or is potentially due to a cryptic species complex.Some previous works .The females of een Fig. and can een Fig. . FemalesAgapostemontexanus have S3 and S4 with a low transverse swelling and generally have distinct yellow marks on the apical sterna Agapostemonsericeus but can be distinguished based on the relative lengths of F1 and F2: Agapostemontexanus has F1 about three-fourths the length of F2 .Taxon classificationAnimaliaHymenopteraHalictidae\ufeff(Fabricius)13AFF97E-73A0-502B-A92D-F6E0847010F0Agapostemonvirescens are the only midwestern species that has the metasoma dark .Females of ark Fig. , rather Agapostemonvirescens can be recognized by the lack of a transverse swelling on S4 is a Great Plains species which occurs as far east as eastern Kansas, though Agapostemoncoloradinus is usually noticeably larger than Agapostemonvirescens with finer, closer striations on the hypostomal area on the underside of the head 70239F90-EDEB-5503-B9E2-E93B9396FAD0Augochlorellaaurata is very similar to Augochlorellapersimilis. Female Augochlorellaaurata can be recognized by having the striations of the propodeum continuing to the posterior margin A9FE1A28-2F27-53EF-BB6F-B41BD5CF0890Augochlorellapersimilis is very similar to Augochlorellaaurata. Females can be distinguished by the lack of rugae at the rear of the propodeal triangle, though this character can often be subtle 08C86848-7F67-5DD9-9487-910CFC28984DAugochlorapura is most similar to Augochlorellaaurata and Augochlorellapersimilis. Both sexes of Augochlorapura can be recognized by the distinct and prominent facial lobes and they have distinct punctures on the rear of the propodeum stat. nov.325809A4-9F43-55EC-9FC5-06CFB5C1657FAugochlorahumeralisLectotype: \u2640 USA, North-western Kansas, 8 Sep 1877 leg. S.W. Williston, on goldenrod [ANSP]. Images examined by ZP and MA. New lectotype designation. . Patton, 1879: 365 \u2640\u2642. AgapostemoncaeruleusHolotype: \u2642 USA, Colorado, Denver [USNM ENT 00536769]. Images examined by ZP and MA. Online record: http://n2t.net/ark:/65665/320b8ee01-69e8-40bd-ab90-fcb717151953. (Labels read: \u201cCol. // [illegible symbol] Type / No 5516 / U.S.N.M. [red label] // Ashmead / Collection // Collection / Ashmead // Augochlora (Agapostemon) / \u2640 coerulea Ash // USNM ENT / 00536769 [yellow label with barcode]\u201d). Ashmead, 1890: 7 \u2642 (not \u2640) syn. . HolotypAugochlorasumptuosabollianaAugochloropsiscaerulea by New synonym. Syntype(s?): USA, Texas, Lee Co. [USNM Type No. 23306 barcode #: 00536763]. Online record: http://n2t.net/ark:/65665/32fdc8c3b-b5b4-4cec-968e-4040825fa92d (Labels read: \u201cLee Co. / TX. 06 / VI. 0 [illegible symbol] // [red label] Type No. / 23306 / U.S.N.M. // A. sumptuosa / bolliana Ckll / TYPE // USNM ENT / 00536763 [yellow label with barcode]\u201d). Cockerell, 1909: 31 \u2640 pattonihumeralis Patton; syn. by Vachal, 1903: 132 (proposed replacement name for Augochlora (Augochloropsis) humeralis sumptuosa , whereas Augochloropsissumptuosa has the punctures always well-separated (about 3\u20135 puncture widths apart). In addition, females of Augochloropsissumptuosa have a weak but distinct semicircular carina around the propodeal triangle were located in the ANSP collection, where Augochlorahumeralis n.s.\u201d and \u201cAugochlorahumeralis n.sp.\u201d. The combination of the little-used name, the \u201cn. sp.\u201d, the type locality, and the fact that A.humeralis Patt., marked \u2018N. W. Kans., Williston\u2019\u201d. However, our inquiries to the Smithsonian have received no answer.Two syntypes of Augochloropsiscaerulea (Ashmead) to have priority because the name humeralis is a secondary homonym in the genus Halictus. However, humeralis is not a secondary homonym in the genus Augochloropsis and the substitute name is no longer in use, so following IZCN Article 59.3, the name humeralis is available and has priority.Augochlorasumptuosabolliana Cockerell is from Texas and was synonymized with Augochloropsissumptuosa by Augochloropsishumeralis, but a more critical evaluation of the specimen, with additional Texas material, should be performed.Augochloropsishumeralis occurs throughout the prairie region, ranging from North Dakota and Minnesota south to New Mexico and Texas, extending to Colorado in the west and Indiana in the east . The sociality and the specifics of the nesting biology are unknown.Colorado: Adams Co.: Denver : 1 \u2642 [iNat], 29 Aug 2019, @francesco167 leg.; Douglas Co.: : 1 \u2640 [iNat], Jul 2020, @calebcam leg.; Logan Co.: : 1 \u2642 [iNat], 22 Aug 2014, R. Webster leg. Illinois: Hancock Co.: Warsaw : 1 \u2642 [iNat], 14 Aug 2016, A. Moorehouse leg., Monardapunctata; Madison Co.: [MASR]; Mason Co.: : 1 \u2640 [iNat], 18 Jun 2019, A. Moorehouse leg., Asclepias sp. Indiana: Lake Co.: Indiana Dunes NP, Marquette Trail : 1 \u2640 [IDNP], 19 Jun 2019, McGill leg., blue pan; Indiana Dunes NP, Miller woods : 1 \u2642 [IDNP], 12 Sep 2018, McGill leg., white pan; 1 \u2640 [IDNP], 4 Jun 2019, McGill leg., yellow pan; 1 \u2640 [IDNP], 23 Jul 2019, McGill leg., yellow pan; Indiana Dunes NP, Miller woods : 1 \u2640 [IDNP], 23 Jul 2019, McGill leg., yellow pan; Newton Co.: Kankakee Sands : 1 \u2640 [iNat], 24 May 2018, D. Lucas leg.; Porter Co.: Indiana Dunes National Lakeshore, Mnoke Prairie : 1 \u2640 [IDNP], 29 Jun 2017, J. Villalpando leg., bee bowl. Minnesota: Faribault Co.: : 1 \u2640 [UMSP], 18 Sep 1911; Fillmore Co.: Pin Oak SNA : 1 \u2640 [MNDNR], 24 Jul 2017, bowl; Hennepin Co.: : 1 \u2642 [UMSP], date unknown; Norman Co.: Agassiz Dunes SNA : 1 \u2642 [MNDNR], 24 Aug 2015, bowl; Sherburne Co.: Sherburne National Wildlife Refuge : 2 \u2640 [EERC], 15 Aug 2016, E. Evans leg., bowl; Wabasha Co.: Weaver Dunes : 1 \u2640 [UMSP], 28 May 2015, M.J. Hatfield leg., Ceanothusherbaceus; Weaver Dunes TNC/SNA : 21 \u2640 [MNDNR], 6 May 2017, bowl; 15 \u2640 [MNDNR], 26 Jun 2017, bowl; 8 \u2642 [MNDNR], 24 Jul 2017, bowl; 3 \u2642 [MNDNR], 19 Aug 2017, bowl; 1 \u2640 [MNDNR], 21 Sep 2017, bowl; Washington Co.: Belwin Conservancy : 1 \u2640 [EERC], 4 Sep 2015, J. Gardner leg., net, Solidagonemoralis; Belwin Conservancy : 1 \u2640 [CRC], 12 Jun 1995, C.C. Reed leg., net, Penstemongrandifloris; 1 \u2640 [UMSP], 12 Jun 1995, C.C. Reed leg., net, P.grandiflorus; 9 \u2640 [CRC], 15 Jun 1995, C.C. Reed leg., net, P.grandifloris; 6 \u2640 [CRC], 16 Jun 1995, C.C. Reed leg., net, P.grandifloris; 1 \u2640 3 \u2642 , 15 Aug 1995, C.C. Reed leg., net, Daleapurpurea; 3 \u2640 [CRC], 13 Jun 1997, C.C. Reed leg., net, P.grandifloris; Gray Cloud Dunes : 1 \u2640 4 \u2642 [UMSP], 9 Jul 1988; Grey Cloud Dunes : 1 \u2642 [CNBL], 23 Jul 2018, J. Petersen leg., net; Grey Cloud Dunes : 1 \u2642 [iNat], 14 Sep 2018, A. Birkey leg.; Grey Cloud Dunes SNA : 1 \u2642 [MNDNR], 9 Oct 2018, net, S.nemoralis; Grey Cloud Dunes SNA : 1 \u2642 [MNDNR], 31 Jul 2018, net, D.villosa; Winona Co.: Whitewater WMA : 1 \u2640 [MNDNR], 6 May 2017, bowl; 1 \u2640 [MNDNR], 26 Jun 2017, bowl. Missouri: Clark Co.: [MASR]; Scott Co.: [MASR]. Nebraska: Hooker Co.: [MASR]; Rock Co.: : 1 \u2642 [iNat], Sep 2018, @allysond leg.; Thomas Co.: Neb Ntl For, near Halsey, 1 \u2642 [WRME], 9 Aug 1991, Arduser leg.; Neb. Ntl For. Nr Whitetail campground: 1 \u2640 [WRME], 10 Aug 1991, Arduser leg., Helianthuspetiolarus. New Mexico: Chaves Co.: [MASR]. North Dakota: Ransom Co.: : 1 \u2642 [iNat], 9 Aug 2021, E. Wood leg.. Oklahoma: Ellis Co.: [MASR]. South Dakota: Clay Co.: Missouri National Recreation River : 1 \u2642 [iNat], 7 Jul 2021, @stenthesnake leg. Texas: Taylor Co.: : 1 \u2640 [OSUC], 18 Jun 1952, J.N. Knull, D.J. Knull leg.Taxon classificationAnimaliaHymenopteraHalictidae\ufeff(Fabricius)B0093FDA-70B2-5EFB-A0E8-DF014DEA3C33AndrenametallicaHolotype: \u2640 \u201cAmerica\u201d [NHMD 308680]. Images examined by ZP and MA . Fabricius, 1793: 309 \u2640. MA Fig. (Labels Augochlorafervidacuprea] by Holotype: \u2642 North America [NHMUK014024969] Images examined by ZP and MA. Online record: https://data.nhm.ac.uk/object/3429259d-5af9-4c5f-9062-96a4a2770077 (Labels read: \u201cType / H.T. [label is circular with red border] // B.M. TYPE / HYM / 14.a.1230 // B.M. TYPE / HYM. / augochlora / fervida / NHMUK 014024969 [label with QR code]\u201d). Smith, 1853: 81 \u2642 (syn. ; Faulkner Co.: [MASR]; Franklin Co.: [MASR]; Jackson Co.: [MASR]; Monroe Co.: [MASR]; White Co.: [MASR]; Woodruff Co.: [MASR]. Illinois: Calhoun Co.: [MASR]; Carroll Co.: [MASR]; Jasper Co.: [MASR]; Madison Co.: [MASR]; Marion Co.: [MASR]; Randolph Co.: [MASR]; Williamson Co.: [MASR]. Iowa: Jasper Co.: [MASR]. Kansas: Barton Co.: [MASR]; Bourbon Co.: [MASR]; Butler Co.: [MASR]; Chase Co.: [MASR]; Coffey Co.: [MASR]; Dickinson Co.: [MASR]; Douglas Co.: : 1 \u2640 [UMSP], 11 Jun 1919, W.F. Hoffman leg.; 1 \u2640 [UMSP], 2 Jul 1919, W.F. Hoffman leg.; Geary Co.: [MASR]; Gove Co.: [MASR]; Greenwood Co.: [MASR]; Hodgeman Co.: [MASR]; Lane Co.: [MASR]; Lyon Co.: [MASR]; Morris Co.: [MASR]; Osage Co.: [MASR]; Pawnee Co.: [MASR]; Pottawatomie Co.: [MASR]; Reno Co.: [MASR]; Rice Co.: [MASR]; Riley Co.: [MASR]; Sheridan Co.: [MASR]; Thomas Co.: [MASR]; Trego Co.: [MASR]. Minnesota: Anoka Co.: Bunker Hills Reg. Pk. : 1 \u2640 [EERC], 8 Jun 2015, J. Gardner leg., net, Tradescantiaoccidentalis; Bunker Hills Regional Park : 1 \u2640 [EERC], 13 Jul 2016, E. Evans leg., bowl; Bunker Pr. Dunes : 1 \u2640 [UMSP], 20 Jun 1947; Cedar Creek Ecosystem Science Reserve : 2 \u2640 [EERC], 22 May 2015, J. Gardner leg., bowl trap; Cedar Creek Nat. Hist. : 1 \u2642 [CRC], 1 Aug 1991, C.C. Reed leg., net, Daleapurpurea; 1 \u2642 [UMSP], 20 Aug 1991, C.C. Reed leg.; Cedar Creek Natural History Area : 1 \u2640 [UMSP], 23 Jul 1986; 1 \u2642 [UMSP], 30 Jul 1990; 1 \u2640 [UMSP], 21 Sep 1992; 1 \u2640 [UMSP], 15 Aug 1995; Cedar Creek Ecosystem Science Reserve : 1 \u2642 [EERC], 12 Aug 2015, J. Gardner leg., net, D.villosa; Helen Allison Savanna SNA : 1 \u2640 [MNDNR], 6 May 2017, bowl; Rum River Cent. Reg. Pk. : 1 \u2640 [EERC], 12 Jun 2015, E. Evans leg., net, Amorphafruticosa; Anoka/Isanti Co.: Cedar Creek Natural History Area : 1 \u2640 [UMSP], 15 Aug 2000; Hennepin Co.: Crow Hassan Park Reserve : 2 \u2640 [UMSP], 13 Jul 1995, C.C. Reed leg., net, Astersericeus; Isanti Co.: Cedar Creek Natural History Area : 1 \u2640 [UMSP], 29 Aug 1981; 1 \u2640 [UMSP], 13 Jul 1991; 1 \u2640 [UMSP], 14 Jul 1992; 1 \u2640 [UMSP], 30 Sep 1992; 1 \u2640 [UMSP], 11 Aug 1993; Irving & John Anderson County Park : 1 \u2642 [EERC], 20 Jul 2015, E. Evans leg., net, Asclepiastuberosa; Lincoln Co.: Hole in the Mountain : 1 \u2640 [UMSP], 15 Jun 2016, N. Pennarolla, J. Leone leg., bowl; 1 \u2640 [UMSP], 27 Jun 2017, N. Pennarolla, J. Leone leg., bowl; Hole-in-the-Mountain TNC : 2 \u2640 [MNDNR], 6 Jun 2016, bowl; 1 \u2640 [MNDNR], 27 Jun 2016, bowl; Murray Co.: : 1 \u2640 [CNBL], 29 Jun 2019, Bee Bowls; Pipestone Co.: Prairie Coteau SNA : 1 \u2640 [MNDNR], 6 Jun 2016, bowl; Stearns Co.: St. Cloud : 1 \u2640 [UMSP], 22 Jun 1967; Yellow Medicine Co.: Mound Spring Prairie SNA : 6 \u2640 [MNDNR], 6 Jun 2016, bowl. Missouri: Barry Co.: [MASR]; Barton Co.: [MASR]; Benton Co.: [MASR]; Boone Co.: Columbia : 1 \u2640 [OSUC], 19 Oct 1955, W.A. Dimmitt leg.; Camden Co.: [MASR]; Douglas Co.: [MASR]; Franklin Co.: [MASR]; Harrison Co.: [MASR]; Howard Co.: Fayette : 9 \u2642 [UMSP], 25 Sep 1966, D.B. Crockett leg.; Jackson Co.: [MASR]; Jasper Co.: [MASR]; Jefferson Co.: [MASR]; Laclede Co.: [MASR]; Lafayette Co.: [MASR]; Linn Co.: [MASR]; Macon Co.: [MASR]; Mercer Co.: [MASR]; Miller Co.: [MASR]; Monroe Co.: [MASR]; Newton Co.: [MASR]; Pettis Co.: [MASR]; Ray Co.: [MASR]; Reynolds Co.: [MASR]; Saline Co.: [MASR]; Scott Co.: [MASR]; St. Clair Co.: [MASR]; St. Louis Co.: [MASR]; Ste. Genevieve Co.: [MASR]; Stoddard Co.: [MASR]; Sullivan Co.: [MASR]; Taney Co.: [MASR]. Nebraska: Co.: Halsey : 1 \u2640 [UMSP], 3 Sep 1924, R.W. Dawson leg.; Lancaster Co.: [MASR]; Richardson Co.: [MASR]. North Carolina: Wake Co.: Raleigh : 1 \u2640 [UMSP], 26 May 1940; 1 \u2642 [UMSP], 17 Nov 1940. Ohio: Gallia Co.: : 1 \u2640 [OSUC], 23 Aug 1942, C.H. Kennedy leg.; Jackson Co.: : 1 \u2642 [OSUC], 9 Aug 1942, J.E. Gillaspy leg.; 1 \u2640 [OSUC], 9 Aug 1942, R.W. Strandtmann leg.; Lawrence Co.: : 1 \u2642 [OSUC], 8 Aug 1942, R.W. Strandtmann leg.; 4 \u2640 1 \u2642 [OSUC], 9 Aug 1942, R.W. Strandtmann leg.; 6 \u2640 1 \u2642 [OSUC], 23 Aug 1942, C.H. Kennedy leg.; Muskingum Co.: New Concord : 1 \u2640 [OSUC], 22 May 1975, C. Dasch leg. Oklahoma: Ellis Co.: [MASR]. South Dakota: Co.: Black Hills : 1 \u2640 [UMSP], 15\u201330 Jun 1931, F. Miller leg. Texas: Dallas Co.: : 1 \u2640 [UMSP], 14 May 1937, H.C. Knutson leg., Marshalliacaespitosa; Smith Co.: : 1 \u2640 [UMSP], May 1947, Barr leg. Virginia: Arlington Co.: : 2 \u2642 [UMSP], 20 Jul 1929, C.E. Michel leg.; Fauquier Co.: Warrentown : 1 \u2642 [UMSP], 28 Jul 1929, C.E. Michel leg.Taxon classificationAnimaliaHymenopteraHalictidae\ufeff(Smith)stat. nov.79050CF9-D859-50A6-963B-8F9B9609075BAugochloraviridulaHolotype: \u2642 USA, New York, Trenton Falls [NHMUK 014024971]. Images examined by ZP and MA. Online record: https://data.nhm.ac.uk/object/10fb10b0-58d6-448c-b1b8-d3807ca35e0e (Labels read \u201cType / H.T. [label is circular with red border] // B.M. TYPE / HYM / 14.a.1232 // B.M. TYPE / HYM. / augochlora / viridula / NHMUK 014024971 [label with QR code]\u201d). Smith, 1853: 81 \u2642. AugochloralucidulaHolotype: \u2640 North America. Images examined by ZP and MA. Online record: https://data.nhm.ac.uk/object/9195d66b-dde0-4554-a11e-8352601fa232 (Labels read \u201cType / H.T. [label is circular with red border] // B.M. TYPE / HYM / 14.a.1233 // B.M. TYPE / HYM / augochlora / lucidula / NHMUK 014024972 [label with QR code]\u201d). Smith, 1853: 81 \u2640 syn. . HolotypHalictus (Augochlora) viridissimusviridula Smith, preoccupied in Halictus). Viereck, 1910: 688 (in Augochloropsis (Paraugochloropsis) metallicafulgida in : biologyAugochloropsis (Paraugochloropsis) fulgida and the interspaces between the punctures have slight tessellation, though they are still somewhat shining , which was previously used by Augochloropsisfulgida is reinstated as a separate species (see remarks for that species).This species has historically been referred to as ida Fig. , we founAugochloropsismetallica and Augochloropsisviridula recognition of ded Fig. , the gonded Fig. , the latded Fig. , and theded Fig. , in compwer Fig. , the gonwer Fig. , the latwer Fig. , and thewer Fig. . The shawer Fig. appears Augochloropsisviridula (Smith) and Augochloropsislucidula (Smith) were different sexes of the same species was recognized by Augochloropsiscuprea by Augochloropsisviridula was then correctly applied by viridula and lucidula conspecific as he considered Augochloropsisviridula a junior synonym of Augochloropsismetallicametallica and Augochloropsislucidula a junior synonym of Augochloropsismetallicafulgida. viridula and lucidula junior synonyms of Augochloropsismetallica. Here, after examination of the primary types, we agree with the interpretation of Augochloropsisviridula and Augochloropsislucidula as both conspecific and a true species.That Augochloropsismetallica were not recognized. However, the illustrations of the genitalia and other characters are clearly of Augochloropsisviridula based on the apically diverging lateral margins of the gonostyli.In the generic revision of augochlorine bees by USA: Alabama: Hale Co.: [MASR]. Arkansas: Lawrence Co.: [MASR]; Monroe Co.: [MASR]; White Co.: [MASR]; Woodruff Co.: [MASR]. Georgia: Catoosa Co.: [MASR]. Illinois: Carroll Co.: [MASR]; Jasper Co.: [MASR]; Madison Co.: [MASR]; Marion Co.: [MASR]; Ogle Co.: : 1 \u2640 [NACH], 1 Jul 2017, B. Bruninga-Socolar leg., net, Partheniumintegrifolium; : 1 \u2640 [NACH], 13 Jun 2017, B. Bruninga-Socolar leg., net, Trifoliumpratense; Randolph Co.: [MASR]; Williamson Co.: [MASR]. Indiana: Lake Co.: Indiana Dunes NP, Miller woods : 1 \u2640 [IDNP], 23 Aug 2019, McGill leg., blue pan. Iowa: Clayton Co.: [MASR]; Jasper Co.: [MASR]; Pottawattamie Co.: [MASR]; Story Co.: Ames : 2 \u2640 [UMSP], 16 Jun 1930, B.A. Haws leg., Swept from sweet clover. Kansas: Johnson Co.: [MASR]; Linn Co.: [MASR]. Maine: Knox Co.: : 1 \u2640 [OSUC], 15 Jul 1956, D.J. Borror leg. Michigan: Cheboygan Co.: : 1 \u2640 [OSUC], date unknown, C.H. Kennedy leg.; Gladwin Co.: [MASR]. Minnesota: Anoka Co.: Bunker Hills Regional Park : 1 \u2640 [EERC], 24 Jun 2016, J. Gardner leg., net, Crepistectorum; Cedar Creek Nat. Hist. : 1 \u2642 [UMSP], 1 Aug 1991, C.C. Reed leg.; Cedar Creek Natural History Area : 1 \u2640 [UMSP], 10 May 1993; Rum River Cent. Reg. Pk. : 1 \u2640 [EERC], 12 Jun 2015, E. Evans leg., net, Rosaarkansana; Rum River Cent. Reg. Pk. : 2 \u2640 [EERC], 12 Jun 2015, E. Evans leg., net, Ziziaaurea; Rum River Cent. Reg. Pk. : 7 \u2640 [EERC], 12 Jun 2015, E. Evans leg., net, Amorphafruticosa; Anoka/Isanti Co.: Cedar Creek Natural History Area : 1 \u2640 [UMSP], 17 Sep 2004; Blue Earth Co.: Gilfillan Lake WMA : 1 \u2640 [MNDNR], 3 Oct 2016, net, Symphyotrichumlanceolatum; Maple River WMA : 1 \u2642 [MNDNR], 14 Aug 2015, net, Solidagoaltissima; Carver Co.: Schneewind WMA : 1 \u2640 [MNDNR], 8 Aug 2018, net, Melilotusalba; Schneewind WMA : 1 \u2640 [MNDNR], 16 Jul 2018, bowl; Chisago Co.: Wild River SP : 1 \u2640 [MNDNR], 22 Jun 2020, N. Gerjets leg., pantrap; Douglas Co.: StaffansonTNC : 1 \u2640 [CNBL], 5 Jun 2018, G. Pardee leg., net, Z.aptera; 1 \u2640 [CNBL], 5 Jun 2018, I. Lane leg., net, Z.aptera; Fillmore Co.: : 1 \u2640 [UMSP], 24 May 1937, G. Kohls leg.; Goodhue Co.: Frontenac : 1 \u2640 [UMSP], 29 May 1930, C.E. Michel leg.; Spring Creek Prairie SNA : 1 \u2642 [MNDNR], 11 Aug 2017, net, Asclepiasverticillata; Goodhue/Wabasha Co.: E Frontenac, Lake Pepin : 1 \u2640 [UMSP], 29 May 1941, M.W. Wing leg.; Frontenac, Lake Pepin : 1 \u2640 [UMSP], 29 May 1941, M.W. Wing leg., net; Hennepin Co.: : 1 \u2640 [UMSP], 27 May 1922, A.A. Nichol leg.; Crow-Hassan Park Reserve : 1 \u2640 [MNDNR], 25 Aug 2015, bowl; Minnesota Valley National Wildlife Refuge : 1 \u2640 [MNDNR], 17 Jul 2017, net, Solanumdulcamara; St Bonifacius: 6 Mile Marsh : 1 \u2640 [CNBL], 28 Jul 2018, Z. Portman leg., net, M.alba; St Bonifacius: 6 Mile Marsh : 1 \u2640 [CNBL], 5 Jun 2020, Z. Portman leg., net, Z.aurea; Houston Co.: : 3 \u2640 [UMSP], 21 May 1938, H.E. Milliron leg.; : 2 \u2640 [UMSP], 23 May 1936, C.E. Michel leg.; 4 \u2640 [UMSP], 23 May 1936, D. Murray leg.; 1 \u2640 [UMSP], 23 May 1936, O. Elster leg.; 3 \u2640 [UMSP], 23 May 1936, R. Cottrell leg.; 1 \u2640 [UMSP], 22 May 1937, H.S. Telford leg.; 2 \u2640 [UMSP], 24 May 1937, C.E. Michel leg.; 1 \u2640 [UMSP], 20 May 1938, P. Nicholson leg.; 3 \u2640 [UMSP], 21 May 1938, C.E. Michel leg.; 2 \u2640 [UMSP], 21 May 1938, H.E. Milliron leg.; 1 \u2640 [UMSP], 21 May 1938, R. Anderson leg.; 1 \u2640 [UMSP], 22 May 1938, R. Anderson leg.; 1 \u2640 [UMSP], 21 Jun 1938, C.E. Michel leg.; 1 \u2640 [UMSP], 26 May 1940, I. Tarshie leg.; Beaver Crk. Valley St. Park : 2 \u2640 [UMSP], 4 Jul 1973, Malaise trap; Eitzen : 1 \u2640 [UMSP], 23 May 1936; Mississippi Bluff, 1\u20132 m N State Line : 1 \u2640 [UMSP], 30 May 1941, J.H. Hughes leg.; 1 \u2640 [UMSP], 27 May 1950; 1 \u2640 [UMSP], May 1957; Mississippi Bluffs 1 mi N. New Albin, Ia. : 1 \u2640 [UMSP], 29 May 1960; Mound Prairie SNA : 1 \u2640 [MNDNR], 26 Jun 2017, bowl; S.E. tip of county : 1 \u2640 [UMSP], 24 May 1935, H. Dodge leg.; Winnebago Cr. Vy., 2\u20134 m NE Eitzen : 1 \u2640 [UMSP], 27 Jun 1956; Isanti Co.: Cedar Creek Natural History Area : 1 \u2640 [UMSP], 1 Aug 1985; 2 \u2640 [UMSP], 21 May 1987, Rubus sp; 1 \u2642 [UMSP], 30 Sep 1992; 1 \u2640 [UMSP], 1 Sep 1993; 2 \u2640 [UMSP], 27 Jul 1994; 1 \u2642 [UMSP], 17 Sep 1994; 1 \u2640 [UMSP], 19 Aug 2000; 1 \u2640 [UMSP], 19 Aug 2001; Jackson Co.: Des Moines River SNA : 6 \u2640 [MNDNR], 6 Jun 2016, bowl; Kanabec Co.: Rice Creek WMA : 1 \u2640 [MNDNR], 30 Jun 2020, D. Drons leg., net, Rhusglabra; Kandiyohi Co.: Brenner : 1 \u2640 [CNBL], 7 Jun 2018, G. Pardee leg., net, Z.aptera; Brenner Lake WPA : 2 \u2640 [MNDNR], 6 Jul 2016, bowl; Nelson : 1 \u2640 [CNBL], 26 Jun 2017, R. Tucker leg., net, Cirsiumarvense; 1 \u2640 [CNBL], 4 Jun 2018, I. Lane leg., net, Z.aurea; 3 \u2640 [CNBL], 4 Jun 2018, S. Marconie leg., net, Z.aurea; 1 \u2640 [CNBL], 4 Jun 2018, T. Eicholz leg., net, Z.aurea; Rudningen : 1 \u2640 [CNBL], 26 Jun 2017, C. Herron-Sweet leg., net, Achilleamillefolium; Le Sueur Co.: Dove Lake WMA : 1 \u2640 [MNDNR], 1 Sep 2017, net, So. Sp; Kasota Prairie SNA : 1 \u2640 [MNDNR], 6 May 2017, bowl; Lyon Co.: Glynn Prairie SNA : 1 \u2640 [UMSP], 20 Jul 2017, N. Pennarolla, J. Leone leg., bowl; Mille Lacs Co.: Kunkel WMA : 1 \u2640 [MNDNR], 24 Jun 2020, D. Drons leg., pantrap; Princeton : 1 \u2640 [UMSP], 3 Oct 1994, A. Johnson leg.; Murray Co.: : 1 \u2640 [CNBL], 29 Jun 2019, Bee Bowls; Olmsted Co.: Oronoco Prairie SNA : 1 \u2640 [MNDNR], 13 Sep 2013, bowl; Pine Co.: Chengwatana State Forest : 1 \u2642 [MNDNR], 16 Jul 2020, N. Gerjets leg., net, Veranicastrumvirginicum; St. Croix SP : 1 \u2640 [MNDNR], 25 Aug 2020, N. Gerjets leg., net, So. Sp.; Pope Co.: Glacial Lakes State Park : 1 \u2642 [UMSP], 25 Jul 1973, Malaise trap; Ramsey Co.: Bald Eagle Otter Lk. Reg. Pk. : 1 \u2640 [EERC], 5 Sep 2015, E. Evans leg., bowl trap; Battle Creek Reg. Pk. : 1 \u2640 [EERC], 10 Jun 2015, J. Gardner leg., net, Cornussericea; Battle Creek Reg. Pk. : 2 \u2640 [EERC], 27 May 2015, J. Gardner leg., net, Geraniummaculatum; Battle Creek Regional Park : 1 \u2640 [EERC], 17 May 2016, E. Evans leg., bowl trap; Battle Creek Regional Park : 2 \u2640 [EERC], 8 Jun 2016, J. Gardner leg., net, Ru. Allegheniensis; Roseville, 3035 Fairview Avenue N : 1 \u2640 [UMSP], 5\u20137 Sep 2014, R.W. Holzenthal leg.; St Anthony Park : 1 \u2640 [UMSP], Jun year unknown; Redwood Co.: Cedar Mountain SNA : 12 \u2640 [MNDNR], 6 Jul 2016, bowl; Renville Co.: Morton Outcrops SNA : 1 \u2640 [MNDNR], 6 Jul 2016, bowl; 1 \u2640 [MNDNR], 18 Jul 2016, bowl; Sherburne Co.: Sherburne National Wildlife Refuge : 1 \u2640 [EERC], 10 Jun 2016, E. Evans leg., net, R.arkansana; Uncas Dunes SNA : 1 \u2640 [MNDNR], 11 Jun 2013, net; Stearns Co.: Avon Hills Forest SNA : 1 \u2642 [MNDNR], 13 Sep 2018, net, So.altissima; St. Cloud : 2 \u2640 [UMSP], 25 May 1968; 1 \u2642 [UMSP], 30 Jul 1968; Stevens Co.: Freeman WMA : 1 \u2640 [MNDNR], 21 Jun 2015, net, R. sp; Verlyn Marth Memorial Prairie SNA : 3 \u2640 [MNDNR], 6 Jul 2016, bowl; Swift Co.: Rice WPA : 1 \u2640 [UMSP], 26 Jun 2016, N. Pennarolla, J. Leone leg., bowl; Wabasha Co.: Reads Landing : 1 \u2640 [UMSP], 22 Jun 1934, C.E. Michel leg.; Washington Co.: : 1 \u2640 [UMSP], 9 May 1959; Afton State Park : 1 \u2640 [CRC], 11 Sep 1992, C.C. Reed leg., net; Arcola Bluffs SAC : 1 \u2640 [CNBL], 31 May 2018, K. Friedrich leg., vac, G.maculatum; 1 \u2640 [CNBL], 14 Jun 2018, K. Friedrich leg., vac, Erigeronphiladelphicus; Big Marine Park Res. : 6 \u2640 [EERC], 7 Jun 2016, J. Gardner leg., net, R.woodsii; Lost Valley Prairie SNA : 1 \u2640 [MNDNR], 13 Sep 2013, bowl; Lost Valley SNA : 1 \u2640 [UMSP], 19 Sep 1990, C.C. Reed leg.; 1 \u2640 [CRC], 19 Sep 1990, C.C. Reed leg., net; 1 \u2640 [UMSP], 28 Jul 1992, C.C. Reed leg.; St. Croix Savanna SNA : 1 \u2640 [MNDNR], 16 Sep 2013, bowl; St. Croix Savanna SNA : 1 \u2640 [MNDNR], 13 Sep 2013, bowl; St. Croix Savanna SNA : 1 \u2640 [UMSP], 5 Aug 1994, C.C. Reed leg., Monardafistulosa; Winona Co.: Great River Bluffs SP : 1 \u2640 [MNDNR], 19 Aug 2017, bowl; Wright Co.: Lake Maria SP : 1 \u2640 [MNDNR], 6 May 2017, bowl. Mississippi: Bolivar Co.: Cleveland : 1 \u2640 [UMSP], 21 Apr 1937, R.W. Dawson leg. Missouri: Atchison Co.: [MASR]; Barry Co.: [MASR]; Barton Co.: [MASR]; Benton Co.: [MASR]; Bollinger Co.: [MASR]; Callaway Co.: [MASR]; Camden Co.: [MASR]; Crawford Co.: [MASR]; Dallas Co.: [MASR]; Dent Co.: [MASR]; Douglas Co.: [MASR]; Franklin Co.: [MASR]; Greene Co.: [MASR]; Grundy Co.: [MASR]; Harrison Co.: [MASR]; Jackson Co.: [MASR]; Jasper Co.: [MASR]; Jefferson Co.: [MASR]; Johnson Co.: [MASR]; Laclede Co.: [MASR]; Lafayette Co.: [MASR]; Lewis Co.: [MASR]; Lincoln Co.: [MASR]; Macon Co.: [MASR]; Madison Co.: [MASR]; Mercer Co.: [MASR]; Monroe Co.: [MASR]; Montgomery Co.: [MASR]; Pemiscot Co.: [MASR]; Pettis Co.: [MASR]; Putnam Co.: [MASR]; Randolph Co.: [MASR]; Ray Co.: [MASR]; Reynolds Co.: [MASR]; Saline Co.: [MASR]; Shannon Co.: [MASR]; St. Francis Co.: [MASR]; St. Louis Co.: [MASR]; Ste. Genevieve Co.: [MASR]; Stoddard Co.: [MASR]; Taney Co.: [MASR]; Warren Co.: [MASR]. New York: Tompkins Co.: Ithaca : 1 \u2640 [OSUC], 27 Aug 1950, J. Cillie leg. North Carolina: Sampson Co.: Ivanhoe : 1 \u2640 [UMSP], 3 May 1945, T.B. Mitchell leg. Ohio: Champaign Co.: : 1 \u2642 [OSUC], 24 Jul 1954; 1 \u2640 [OSUC], 8 Jun 1994, N.F. Johnson leg., Malaise trap; Delaware Co.: : 1 \u2640 [OSUC], 2 Aug 1942, R.W. Strandtmann leg.; Fairfield Co.: : 1 \u2640 [OSUC], 16 Jun 1994, A. Sharkov leg.; Franklin Co.: : 1 \u2642 [OSUC], 21 Aug 1942; 1 \u2640 [OSUC], 18 Jun 1952; Greene Co.: [MASR]; : 1 \u2640 [OSUC], 6 Jun 1956, J.N. Knull leg.; 1 \u2640 [OSUC], 20 Jun 1957, J.N. Knull, D.J. Knull leg.; Hocking Co.: : 1 \u2640 [OSUC], 10 May 1935, R.C. Osburn leg.; 1 \u2640 [OSUC], 14 Jun 1943, R.C. Osburn leg.; 1 \u2640 [OSUC], 23 May year unknown, J.N. Knull, D.J. Knull leg.; 1 \u2640 [OSUC], 14 Jun year unknown, R.C. Osburn leg.; Jackson Co.: : 1 \u2640 [OSUC], 9 Aug 1942, R.W. Strandtmann leg.; Lawrence Co.: : 1 \u2642 [OSUC], 8 Aug 1942, R.W. Strandtmann leg.; Logan Co.: : 1 \u2640 [UMSP], 16 Jul 1930, J. Patton leg.; Lucas Co.: [MASR]; : 1 \u2640 [OSUC], 19 May 2003, M. Arduser leg., Lupinusperennis; Ottawa Co.: Catawba Island : 1 \u2640 [OSUC], 27 Jun 1902, J.G. S. leg.; Put-in-Bay : 1 \u2640 [OSUC], 20\u201330 Jun 1924; 1 \u2640 [OSUC], 14 Jul 1935, R.C. Osburn leg.; 1 \u2640 [OSUC], 22 Aug 1941, R.C. Osburn leg.; 1 \u2640 [OSUC], date unknown, C.H. Kennedy leg.; Paulding Co.: Charloe : 1 \u2640 [OSUC], 12 May 1951, H.F. Price leg.; Scioto Co.: : 1 \u2640 [OSUC], 6 Aug 1942, R.W. Strandtmann leg.; 1 \u2640 [OSUC], 9 Jun 1943, J.N. Knull, D.J. Knull leg.; Summit Co.: Ira : 1 \u2640 [OSUC], date unknown, J.S. Hine leg.; Vinton Co.: : 1 \u2640 [OSUC], 20 Jun 1901; Williams Co.: Bryan : 2 \u2640 [OSUC], date unknown. Tennessee: Davidson Co.: Nashville: [MASR]. Wisconsin: Burnett Co.: : 1 \u2640 [UMSP], M. Sabourin leg.; Crawford Co.: Barnum : 1 \u2640 [UMSP], 2 Aug 1922, A.M. Holmquist leg.; Dane Co.: Madison : 1 \u2640 [OSUC], 25 Jun 1916; La Crosse Co.: [MASR]; Oconto Co.: Lakewood : 1 \u2640 [UMSP], 15 Jul 1948, H.E. Milliron leg.; Polk Co.: Tewksbury SACN : 1 \u2640 [CNBL], 25 May 2017, K. Friedrich leg., vac, Barbareavulgaris; 2 \u2640 [CNBL], 8 Jun 2017, K. Friedrich leg., vac, Ru. Sp.; 1 \u2640 [CNBL], 4 Jun 2018, K. Friedrich leg., vac, Ru. Sp. Canada: Ontario: Middlesex Co.: London: [MASR].Taxon classificationAnimaliaHymenopteraHalictidae\ufeffCockerellBB67D262-7F85-5D6A-9BB1-DF7C093E0DAEAugochloraanonymaHolotype: \u2640 USA, Florida, No Name Key . Images examined by ZP and MA. Online record: http://n2t.net/ark:/65665/347b15a43-e8d1-4195-8eaf-f8ac9cbbec94 (labels read \u201cNo Name / Key 3.98 Fla // GN Collins / Collector // CL Pollard / Collector // TypeNo. / 24890 / U.S.N.M. [red label] // Augochlora / anonyma / Ckll. TYPE.\u201d). Cockerell, 1922: 15 \u2640. Augochloropsiscuprea (in Augochloropsis (Paraugochloropsis) anonyma in : catalogAugochloropsisanonyma can be recognized by the short propodeal triangle, which is impressed and narrower than the metanotum than the medial length of the metanotum . However, the narrow propodeal triangle of Augochloropsisanonyma stat. nov.0562824D-D2EC-5427-97DB-7290F59096AEAugochloracupreaHolotype: \u2640 North America [OUMNH]. / J.S. Moure 1957 // Probably the Holotype as labelled. No specimen labelled Type in B.M. / C.D. Michener in litt. 13 VIII 1965\u201d). Smith, 1853: 79 \u2640. Images examined by ZP and MA Fig. . HolotypAugochloropsiscuprea stat. nov.978E8C30-228E-50E9-8C5D-A504A558E484AugochlorafulgidaHolotype: \u2640 USA, Florida, St. John\u2019s Bluff, East Florida [NHMUK014024970]. Images examined by ZP and MA . Smith, 1853: 79 \u2640. MA Fig. . Online Augochlorafulgida A9E7120B-EDC0-533B-B6E7-A20A3FCE01D6AugochlorasumptuosaSyntype(s?): \u2640 North America. Type or types missing and presumed lost. Smith, 1853: 82 \u2640. AugochloralacustrisHolotype: \u2640 USA, Florida, Lakeland [USNM Type no. 24888]. Images examined by ZP and MA. Online record: http://n2t.net/ark:/65665/32a505d56-7e7d-4fea-ba47-574f3858121f . Cockerell, 1922: 14 \u2640 syn. . HolotypAugochlorafloridicaHolotype: \u2642 USA, Florida, Monticello [USNM Type no. 24889]. Images examined by ZP and MA. Online record: http://n2t.net/ark:/65665/3053e0fc2-b95d-49b6-9630-b55645b3e89d . Cockerell, 1922: 14 \u2642 syn. . HolotypAugochlorasumptuosa sumptuosa .Augochloropsissumptuosa in a more restricted sense than previous authors because we have split it into two species: Augochloropsissumptuosa and Augochloropsishumeralis. Now, Augochloropsissumptuosa refers to the species occurring in the southeastern United States though the exact range is unknown at this time, and it remains to be seen to what degree, if any, the range of the two species overlaps.We use Augochloropsissumptuosa have been lost. The type could not be located by Augochloropsissumptuosa can be determined from the original description. Specifically, the original description states \u201cthe base of the metathorax enclosed by an arched ridge, the enclosed space granulated, the sides of the truncation margined by sharp carinae.\u201d This description matches the southeastern species , the types of which were collected in western Kansas.The type or types of Augochloropsissumptuosa because we have not critically evaluated the status of two synonyms: Augochloralacustris Cockerell and Augochlorafloridica Cockerell. They were originally synonymized with Augochloropsissumptuosa by lacustris or floridica.\u201d We have examined images of the types, which are clear enough for us to tentatively agree. However, given that we have split Augochloropsissumptuosa into two species and there is potentially a third similar species in Florida, these types should be critically reexamined as part of a reevaluation of the Florida fauna.More work remains to be done on the taxonomy of Augochloropsis in the United States. We are listing them here in order to alert readers to their presence, as many have been incorrectly lumped together under existing species, particularly Augochloropsismetallica. However, we do not treat them further. We lack sufficient material of these species, and it is unknown whether they are undescribed or not, as they may be described from Mexico or they may be one of the many poorly known species described by Cockerell. The potential species and their locations include:We are aware of at least four additional potential species of Arizona: A species with a broad and shining propodeum in the female .Augochloropsissumptuosa seen in material from Archbold Biological Station .Florida: A species similar toAugochloropsismetallica andAugochloropsisviridula . This species may have contributed to the confusion by previous authors who believed thatAugochloropsismetallica andAugochloropsisviridula were a single variable species.Texas: A species that has a unique propodeal triangle Fig. and an iAugochloropsishumeralis .Texas: A species similar toAugochloropsis of the Midwest and made additional changes to the Augochloropsis of the broader United States. This work will allow for the confident identification of the species in the midwestern United States and allow the species\u2019 ranges to be better understood. However, there are areas of the southern United States (particularly Florida and Texas) where any Augochloropsis identifications must be undertaken with great care due to the number of undescribed or unknown species. We estimate there are an additional four species of Augochloropsis in the United States that are unknown or undescribed, not counting Augochloropsisfulgida, which is only known from the type and has the male now unknown. In addition, more work needs to be done to check the status of some of the current synonyms of Augochloropsissumptuosa and Augochloropsishumeralis from Texas and Florida. Even the genus name may change at some point, as Paraugochloropsis to genus level.Here, we have revised the Augochloropsis and the other shiny green Halictinae. However, similar to the situation in Augochloropsis, more taxonomic work is still needed in the other shiny green Halictinae. For example the Agapostemon of the United States were last revised 50 years ago are contributing to the high rates of misidentifications in bees.The taxonomic changes and identification resources provided here will allow for more accurate identification of ears ago , the Aug complex . Given tmens see . In addiAugochloropsis species in the United States and split what was formerly Augochloropsismetallica into five species , particularly since prior taxonomic research on those groups predates molecular tools and high-resolution images.Our work also demonstrates the difficulty, indeed the futility, of attempting to monitor many bee groups that are in taxonomic disarray . Here, wes Table . This wies Table , and it Epeolus ; 20 new oglossum ). This h"} +{"text": "Brain magnetic resonance imaging (MRI) showed multiple white matter T2-lesions with incomplete peripheral enhancement . Conside"} +{"text": "Vision was able to uphold its high standards for published papers due to the outstanding efforts of our reviewers. Thanks to the efforts of our reviewers in 2022, the median time to first decision was 30.5 days and the median time to publication was 65 days. Regardless of whether the articles they examined were ultimately published, the editors would like to express their appreciation and thank the following reviewers for the time and dedication that they have shown Vision:Ahmed AbdelghanyJuliette E. McGregorAkira ObanaKang-Ming ChangAlessandro MeduriKatarzyna NowomiejskaAlessio FacchinKazuo IchikawaAlfonso Vasquez-PerezKevin PatersonAlice Verticchio VercellinKjetil Falkenberg HansenAlicia Ruiz-PomedaKristopher R. GenschmerAlina GheorgheKrzysztof SchabowiczAmedeo D\u2019AngiulliKwok Ping ChanAmir KheradmandLaurence HarrisAna Tajadura-JimenezLei WanAndr\u00e9 MermoudLeo FleishmanAndreas EichlerLuca BattagliniAndrzej GrzybowskiLuigi Tame\u00c1ngel De-Juanas OlivaLuke ChongAnita Lyssek-Bor\u00f3nMael LeverAnna PiroManfred MarschallAnna Przekoracka-KrawczykManuel PereaArjen AlinkMarco BertaminiArne Lykke LarsenMare KoitArne OhlendorfMarek HamplArthur ZhaoMarialuisa MartelliBaingio PinnaMarina GregoricBenjamin KnierMario DalmasoBhagyashri BurguteMark J. SimcoeBiswajit PadhyMark W. GreenleeBrad BarnettMartin HowBruno RichardMarty BanksCalvin EiberMassimo BusinCarlos Ferr\u00e1s SextoMaurizio CammalleriCecilia PerinMaxim BakaevChong-Bin TsaiMd Manjurul AhsanChris OrietMelinda Y. ChangChristoph FaschingerMengxi ShenChristoph M. FriedrichMichael D. CrosslandClara Martinez-PerezMichael HoffmannClaudio BucoloMichael J. LipsonCl\u00e9mence BonnetMicha\u0142 S. NowakCord HuchzermeyerMichele LanzaDanial RoshandelMiguel TeusDaniel Josef LindeggerMile Bo\u0161njakDaniel L\u00f3pez MaloMinglang YinDavid Manzano S\u00e1nchezMohammad Shokrolah ShiraziDmitrii S. MaltsevMohsen Mosayebi-SamaniDoreen SchmidlMuneeswar NittalaDoug D. ChungNehal ElsherbinyDu\u0161ica PahorNicola Di StefanoEleftherios AnastasopoulosNicolae GicaElena DinteNicoleta AntonEleonora MauriziNikos KonstantinouEliya LevingerNingli WangEmily HandNorman M. SpivakEvan PalmerOlaia Lucas-Jim\u00e9nezEwald LindnerPadmanabhan PattabiramanFrancesco LenaPastore MassimilianoFrancisco AlonsoPatrick CavanaghFrank H. DurginPaul HibbardGaganpreet KaurPaul LintonGeorges DellatolasPavol Bo\u017eekGeorgios LabirisPeter KoulenGerd GigerenzerPushpa Raj JoshiGerd U. AuffarthQi FangGiacomo CollettiRafael IribarrenGiancarlo MontaniRalph WeidnerGiovanni William OliverioRan DuGiuseppe CirilloRebecca FullerGrainne ScanlonRicardo Bernardez-VilaboaGregory DeAngelisRicardo F. FraustoGrzegorz \u0141abuzRobert J. EnnisGuido MaielloRobert L. WhitwellGysbert van SettenRobert S. AllisonHafiz Tayyab RaufRoberto BellucciHakan KaymakRohan Bir SinghHaluk OgmenRosa BocciaHamdy AbdelkaderRuobing QianHarold BedellRupesh Kumar ChikaraHaruka AbeSamantha R. De SilvaHe-Sheng WangSerenella D\u2019IngeoHirokazu SakaguchiSergio MorraHortensia T. Sanchez-TocinoShao-Bin WangHuei-Jane LeeSinwan CheungHun-Ju YuSiyi ChenIoannis HalkiadakisSreelakshmi VasudevanIrene SperandioTae Keun YooIvan MatveevTatu RojalinIvana MravicicTejas KarhadkarJames J. WolffsohnThomas A. BuseyJamie M. BogleThomas SchmidtJared CoburnTimothy J. VickeryJavier Mart\u00ednTina McKayJee Taek KimTomislav KuzmanJeffrey BaraTsung-Jen WangJeffrey J. HuangVasile-Gabriel IanaJer\u00f3nimo Gonz\u00e1lez-BernalVida DemarinJin BaiWaldemar SwiderskiJinjun XiaWladimir KirschJoanna Konopi\u0144skaYokrat TonJovana Bjeki\u0107Yuntao HuJukka HyonaZhong-Lin LuHigh-quality academic publishing is built on rigorous peer review."} +{"text": "Article title: N-oleoylethanolamine_phosphatidylcholine complex loaded, DSPE-PEG integrated liposomes for efficient strokeAuthors: Xiangrui Yang and Shichao WuJournal:Drug DeliveryBibliometrics: Volume 28, Number 1, pages 2525\u20132533DOI:https://doi.org/10.1080/10717544.2021.2008058As per author\u2019s suggestions the figure 3 and 4 are replaced in the published article."} +{"text": "Correction To: BMC Medical Education (2022) 22:81210.1186/s12909-022-03881-y.Following publication of the original article , the aut\u201cOn average, Norwegian schools had stricter policies (mean score: 0.9) followed by Sweden (mean score: 0.7) and Denmark (mean score: 0.5)\u201d.Previous version: \u201cOn average, Norwegian schools had stricter policies (mean score: 0.8) followed by Sweden (mean score: 0.7) and Denmark (mean score: 0.5)\u201d.Amended version:"} +{"text": "Erratum zu:Chirurg 202110.1007/s00104-021-01450-5Dieser Beitrag zum Thema \u201eRobotische Hernienchirurgie. Rv-TAPP und r\u2011Rives/r-TARUP\u201c ist der zweite Teil einer Reihe von mehreren Beitr\u00e4gen zum Thema. Titel und Untertitel wurden angepasst. Die Originalversion dieses Beitrags wurde korrigiert."} +{"text": "Correction: Stem Cell Research & Therapy (2017) 8:17https://doi.org/10.1186/s13287-017-0476-7When sorting out the original data, the authors noted the representative masson images for MI\u2009+\u2009MET/CDC and MI\u2009+\u2009MET\u2009+\u2009MET/CDC groups in Fig."} +{"text": "Scientific Reportshttps://doi.org/10.1038/s41598-022-27290-9, published online 03 January 2023Correction to: The Acknowledgments section in the original version of this Article was incomplete.\u201cF.N. thanks the ERC Starting Grant n. 307322.\u201dnow reads:\u201cF.N. thanks the ERC Starting Grant n. 307322. D.B. warmly acknowledges financial support by the Italian MIUR PRIN2017\u00a0(Project Number 2017KY5ZX8).\u201dThe original Article has been corrected."} +{"text": "Correction: Antimicrobial Resistance & Infection Control (2020) 9:76https://doi.org/10.1186/s13756-020-00741-6The original article containsThe current, incorrect value is \u2018y (31.5%)\u2019 whereas the correct value is instead \u201917 (31.5%)\u2019."} +{"text": "DOI: 10.20945/2359-3997000000465Arch Endocrinol Metab. 2022;66(2):139-51Where you read:Table 2. Newly proposed classification based on two clinical casesShould read:Table 2. Newly proposed classification based on two clinical cases"} +{"text": "A two-microRNA signature predicts the progression of male thyroid cancer. Open Life Sci. 2021;16(1):981\u201391,"} +{"text": "Antibodies was able to uphold its high standards for published papers due to the outstanding efforts of our reviewers. Thanks to the efforts of our reviewers in 2022, the median time to first decision was 21 days and the median time to publication was 51 days. Regardless of whether the articles they examined were ultimately published, the editors would like to express their appreciation and thank the following reviewers for the time and dedication that they have shown Journal:A.J. RosenspireKenneth A. AndreoniAdam BarbKenta HarayaAdam WisnewskiKevin J. SelvaAfua Adjeiwaa MensahKevin M CoombsAlan SchmaljohnKira V. VyatkinaAlastair LawsonKisung KoAlexey TeplyakovKRISHNA CHAITANYAAli R. JazirehiKristine M. KimAlireza ZarebidokiKumiko Nakada-TsukuiAlvise SernicolaLaura TiberioAmita Datta-MannanLaureline BerthelotAmy XuLong Thanh PhamAndrea L. J. MarschallLuca VaraniAnisuz ZamanLuigi R. CeciAnthony CheungLuis Alvarez-VallinaAnton SholukhM. Quadir SiddiquiArtur ZalevskyMarc HilhorstAtheer ZgairMarco PalmaAvneesh GautamMaria RodriguezBernardo VegaMarie-Alix PoulBingchun ZhaoMark S. CraggBrian D. AdamsMartina JonesBruce WalcheckMatthias ScheweCarlo Cosimo CampaMehdi Arbabi GhahroudiCaterina Maria GambinoMichael G. WellerCharles DumontetMichael KundiChinh Tran-To SuMohamed FodaChiuan LeowMonika Christine Brunner-WeinzierlChristiane MoogNeil GreenspanCiesla MaciejNicolas SabarthColin H. QuinnOhad MazorCzeslaw RadziejewskiOlga BorgesDaisuke TsurutaParamita ChakrabortyDario GenovesePaulina \u017barnowiecDelphine Le RouxPenelope M. DrakeDeng-Ho YangPeter KorstenDenise ToscaniP\u00e9ter Ol\u00e1hDennis R. GouletPetros ChristopoulosDiana Panayotova-DimitrovaPrabakaran PonrajDiego MoricoliQun ZhouDominique LesuisseRaffaele FrazziDoreen M. FlossRahul MannanE. F. KolesanovaRenata Von Glehn PonsirenasEdwin J. Vazquez-CintronRicardo GiordanoElena CsernokRitthideach YorsaengElena SavvateevaRonald P. TaylorElizabeth Helen KempRoni NugrahaElizabeth J. PriceRosa Rodr\u00edguez-P\u00e9rezEmilia Bia\u0142opiotrowiczSafder GanaieEmilia CirilloSaketh ChemuruEmmanuel VictorSanjay DeyErika OrbanScott WetzelErwan MortierSebastian YuFaraz A. ChoudhurySebastien CalboFeliciana Real-Fern\u00e1ndezSelami Aykut TemizFlavio Di PisaSeshi SompuramFloris L. Van DelftSharath MadasuFrank BreitlingShin-Ichi OsadaFrank KrauseSina TaefehshokrFrederick J. KaskelSrinath ThirumalairajanG\u00e1bor KonczSukmook LeeGalia Ram\u00edrez-TolozaSyed AhmedGarima ArvikarTae Hyun KangGestur VidarssonTakahiro AnzaiGiorgia ManniTaku KouroGonca E. KarahanTakuya TadaGouveia Zelia LiciniaTaru S DuttHeather BaxTarun KeswaniHyosang BaeTheam Soon LimHyunbo ShimTimm AmendtItai BenharUrszula LisieckaItzel Amaro-EstradaV. Ashutosh RaoIzabela ChmielewskaVeera Venkat Shivaji Rama Rao EdupugantiJamshid TanhaVictoria PolonisJeremy SchmitVladimira BoyadzievaJerome E. TannerWakiro SatoJianlong LouWanessa C. LimaJingzhong GuoWei SunJohn De KruifWibke BayerJos\u00e9 Germano De SousaWilliam David TolbertJuan C. AlmagroXavier Fulladosa OliverasJuli\u00e1n Esteban Barahona-CorreaXingyi JiangJulius GrzeschikYinzhi LangJurgen SanesYoshiyuki NakamuraKarina Kubiak-OssowskaYutaka MatsudaKasturi BanerjeeZahra RattrayKatharine M. Lodge\u017divka DikaHigh-quality academic publishing is built on rigorous peer review."} +{"text": "Correction: BMC Pregnancy Childbirth 22, 456 (2022)https://doi.org/10.1186/s12884-022-04749-1Following publication of the original article , the autThe incorrect author name is: GivenName: Rikke, FamilyName: Damkj\u00e6r MaimburgThe correct author name is: GivenName: Rikke Damkj\u00e6r, FamilyName: MaimburgThe author group has been updated above and the original article has been"} +{"text": "Peer reviewing is a complex and often thankless task. The editors at mSystems are all specialists in their fields, but they are also all peer reviewers and authors. Having an author, reviewer, and editor all in the same brain can often create cognitive dissonance, as I have mentioned previously (https://doi.org/10.1128/mSystems.00797-19). However, all of us are committed to providing robust assessment, through constructive feedback, for every paper that we send out for peer review. Over the last 3\u2009years, it has become increasingly hard to find reviewers willing to perform peer review. While I have no hard data as to why this trend is occurring, like most things, I blame it on the pandemic. However, there has also been a concomitant maturing of many different microbiome-focused journals, which has taxed a relatively small group of researchers, who are often asked to review many different manuscripts simultaneously. I routinely receive 10 to 15 peer-review requests per week. Obviously, no one has the time to assess anything rigorously with that kind of workload, so I end up saying \u201cno\u201d\u2014a lot! How do I choose which ones I say yes to? Well, in the last 2 to 3\u2009years I have been focusing on performing peer review only for society journals. The American Society for Microbiology, Applied Microbiology International, International Society for Microbial Ecology, and many others provide substantial resources to their membership that are in part supported by revenues from the journals. Therefore, by supporting the journals as an editor, reviewer, or an author, you are generating revenue that provides support for your scientific community, and maybe even yourself. What could be better? Thank you to all the reviewers who made peer review at mSystems such a success this last year, and thank you for supporting ASM and your community.Ruth Chingcuangco AbanadorGhulam AbbasWafaa Abd El-GhanyMahmoud M. AbdelsattarLuis A. ActisZaky AdamBeth M. AdamowiczOehmen AdrianInnocent AfekeLivnat Afriat-JurnouHerve AgaisseAshwani AggarwalSurya D. AggarwalStephen Dela AhatorDin Ahmad UdTae-Hyuk AhnLianzhong AiSulaiman M. AlajelM. Tauqeer AlamBegum AlaybeyogluVirginia Helena Albarrac\u00ednMuhammad AliCeleste AllabandJonathan AllenRichard C. AllenCharlotte J. AlsterHassan M. Al-TameemiLauren Victoria AlteioEmrah AltindisJohn AlverdyDani AmorShivanthi AnandanMark T. AndersonMatthew Zack AndersonMichael AndersonAngel Andrade TorresJulien AndreaniCristina Andr\u00e9s-BarraoHaike AntelmannVijay C. AntharamMuzaffer ArikanJean ArmengaudCourtney R. ArmourRavi P. AryaAdrien AssieJennifer M. AuchtungLucas AuerSandrine AugerFrank O. AylwardDerya Aytan-AktugSheyda AzimiReha O. AzizogluJin-Woo BaeHarsh BaisJonathon L. BakerScott E. BakerVimal BalasubramanianAreen BanerjeeKalins BannerjeeMatthieu BarbierRoman Alfredo BarcoLars BarquistFranco BasileAshley C. BatemanBuzz BaumAndreas J. B\u00e4umlerClifford J. BeallPascale B. BeauregardAshley E. BeckChristine BeemelmannsTerrence BellDaniel Bellieny-RabeloMichael BenisraelMariana BenitezTrude BennettFabienne BenzAnne-Sophie BergotAnna BernasconiAnthony BertagnolliMartin BeukemaMohini BhattacharyaDerek Martens BickhartJoseph BielawskiJordan E. BisanzPradeep BistNidhan K. BiswasRyan Andrew BlausteinRan BlekhmanMark BlennerSamuel J. BloomfieldLouis-Marie BobayNick BohmannDavid BolamLuis M. Bola\u00f1osAlte BonesJoanna-Lynn C. BorgognaChiara BorsettoKeith Bouma-GregsonTravis BourretCara C. BoutteRobert BowersPatrick H. BradleyTimothy S. BretonCorinna BreusingSylvain BrisseAmanda M. V. BrownBianca Regina Palmer BrownChris M. BrownAllen BrowneKirsteen BrowningVincent BrunoGregory A. BuckMichal BukowskiAaron BurberryAlejandro BuschmannSusheel Bhanu BusiYuming CaiDouglas R. CallBenjamin J. CallahanAndrew CamilliFran\u00e7ois-Xavier Campbell-ValoisZoe CardonCarlo R. CarereAllison F. CareyJeffrey CareyMichael CarlsonTyler J. CarrierCatherine CarrilloSantiago Castillo-Ram\u00edrezLindsay J. CaverlyYunrong ChaiDipshikha ChakravorttyApostolos ChalkisRishi ChanderrajHao-Xun ChangBenoit ChassaingKausik ChattopadhyayNarendrakumar ChaudhariLianqiang CheTanya CheekeDing-Qiang ChenHuaihai ChenLiang ChenSongcan ChenWen-Hsiang ChenShu ChengLoo Wee ChiaAlexandra ChiaveriniMariana Carmen ChifiriucJason ChinByung-Kwan ChoDavide CiccareseGerard ClarkeChristine ClaytonAna G. Cobi\u00e1n-G\u00fcemesJos\u00e9 Francisco Cobo D\u00edazMaureen L. ColemanJames CollinsFabian CommichauIan Frank ConnertonLydia M. ContrerasGregory M. CookFernando CorralesClaire CouchFelipe H. CoutinhoBrett C. CovingtonDon A. CowanJohn V. CoxKeith A. CrandallAlex Crits-ChristophJie CuiJulia CuiRoss CunningChris CurtinTal DaganZhongmin DaiAjai A. DandekarKristen M. DeAngelisBrian DeFelicePatrick H. DegnanMarcus de GoffauStephen De LisleFrancesco DeloguIlenne Del ValleXin DengYijie DengLaura De NiesMahesh S. DesaiLes DethlefsenRae DevanNeha DhasmanaRishu DheerMuhe DiaoDulce Maria Diaz-Monta\u00f1oGeorge C. DicenzoChristian DienerNicholas DillonTatiana DimitriuFrancisco DionisioRay DixonUlrich DobrindtAlex DornburgGavin M. DouglasRichard G. DouglasSimon L. DoveGlen D\u2019SouzaRebbeca DuarCaroline DubeLeonard DuboisFrank DucaBreck A. DuerkopKarine DufresnePeter F. DunfieldAshley M. DunganJanani DurairajAvishek DuttaJonathan DworkinDavid DyerStefan DyksmaEmily EbelAnna EdlundMoamen ElmassryAkintunde EmiolaMelinda Anne EngevikTobias EnglHannah E. EpsteinMadeleine ErnstOier EtxebesteJay D. EvansDamien EveillardN. L. FahrenfeldYanhua FanAlonso FavelaNicolas FeauSara FedericiMichael J. FederleKelli FeeserJie FengXiaowen FengTimothy G. FerdelmanGabriel da Rocha FernandesMaria-Luz FernandezVal Fern\u00e1ndez-LanzaJustyna Fert-BoberCelio Fernando Figueiredo AngoliniTaicia Pacheco FillHelmut FischerAmaranta FocardiKristen FoustEdward M. FoxParaskevi C. FragkouGad FrankelKyle FrantzShiri FreilichAyari Fuentes Hern\u00e1ndezJed FuhrmanHeather FullertonCesare FurlanelloChikara FurusawaGiovanni GalloMaria Alessandra GammoneSukirth GanesanRaidah GangjiMichael G. GanzleXiang GaoXiaoyu GaoMelanie G. GareauMatthew J. GebertJennifer Geddes-McalisterStephen J. GiovannoniCatherine GirardAlain P. GobertFilipa Godoy-VitorinoAntonio Gonzalez PenaMilena GonzaloDavid R. GoodlettTobias GorisClaire L. GorrieMarcin GrabowiczMatti GralkaPeter L. GraumannMichael Jeffrey GrayChristen GrettenbergerAnne GroveAlyssa GrubeCarsten GrupstraMaria-Eugenia GuazzaroniEric Gu\u00e9donSohini GuhaFengbiao GuoLanping GuoVinod GuptaElaine M. HaaseDaniel H. HaftTatsuro HagiAria S. HahnMatthias HahnYang HaiSarah HainesLarry J. HalversonJoshua HammBrian K. HammerJens Andre HammerlLeendert W. HamoenNancy D. HansonJianjun HaoYanbin HaoJanani HariharanMatthew J. HarkeJoshua HarrisonErica M. HartmannNur A. HasanChristiane Hassenr\u00fcckRoland Hatzenpichler\u00a0Juliette HayerAnna HayesBridget HegartyJohann HeiderCaitlin HeilJack A. HeinemannNoelle HeldV\u00e9ronique HelferNicholas C. K. HengCarly HenkelMichael HensonMelissa M. Herbst-KralovetzAaron D. HerndayRachel HestrinRobert L. HettichTeppo HiltunenChris HineYing-Ning HoWouter D. HoffCasandra L. HoffmanWilliam HofstadterDeborah A. HoganJames F. HoldenSuzie HoopsJulie HorvathPaul A. HoskissonAlice HotoppXiaoyu HuChao-Li HuangEn HuangDavid Andrew-Essman HufnagelWenwen HuoValentina Hurtado-MccormickDouglas L. HusebyFilip HusnikFatima HussainKevin HybiskeS. HyodoFrancesco ImperiKeiichi InaoueIkbal Agah InceRichard E. IsaacsonElliot Walter JacksonScott A. JacksonWilliam R. JacobsCarsten Suhr JacobsenCholsoon JangJayanth JawaharFrancis E. JenneyZhongjun JiaHongchen JiangJuquan JiangSizun JiangYong JiangZiyun JiangShuo JiaoMona JohannessenAnders JohanssonZackary JohnsonEric JonesStuart JonesPeter JorthJayadev JoshiJyoti JoshiAri JumpponenSheryl JusticeLindsay KalanMarina G. KalyuzhnayaBrandi KamermansVinayak KapatralVera KarlicicLisa KarstensKazuyuki KasaharaTakao KasugaKathryn KauffmanShanlin KeChristina A. KelloggPatrick KemmerenMichelle A. KennedyJannigje Gerdien KersShahrbanoo Keshavarz Azizi RaftarChetan KeswaniBrandon KieftKristopher KieftMin-Soo KimTaegyu KimJeffrey Alan KimbrelClaas KirchchelleKwan Soo KoHitoshi KomatsuzawaWeidong KongKonstantinos T. KonstantinidisBon-Sang KooChristian KostAleksandar D. KosticK\u00e1roly Kov\u00e1csSascha KrauseJens KrethNicolas KrinkArianna KrinosLee KroosLee R. KrumholzAnand KumarRolf K\u00fcmmerliBenoit KunathJahnavi KurasamViola KurmKyohei KurodaKirsten K\u00fcselSimon LabartheSteve LabrieLeo LahtiRegina LamendellaLefu LanZachary LandryYelena LapidotGisele LapointeBeatrice LarocheChristian LauberDonald W. LawrenceChristopher E. LawsonChih-Ying LayFrancois LebretonAngelica LebronAlice LeddaCharles Kai-Wu LeeEun Yeol LeeGrace C. LeeJean-Luc LegrasOwen P. LeiserDanielle G. LemayCammie F. LesserPetra Anne LevinJanina P. LewisDan LiFu-Li LiFuyong LiMeng LiXian-Zhi LiXiaogang LiZi-Bo LiGuanxiang LiangChen LiaoJosie LibertucciIan Dennis Edmund Alan LidburyTami LiebermanVirginia LioyBin LiuGang LiuJia LiuJinxin LiuXingyin LiuYa-Jun LiuYu-Rong LiuYannan LiuJames LockeBrett LomanAllison LopatkinStilianos LoucaBrian Michael LunaJustin LundElaine LuoYuheng LuoZhao-Qing LuoJonathan LynchMeinan LyuJing Mei MaLiyuan MaMary MachadoRoderick I. MackieD. Mitch MageeJennifer MahonyRabindra Kumar MandalSubhrangshu MandalAmee MangesMichael ManhartShrikant Subhash MantriAlejandro Manzano-Mar\u00ednRamona MarascoMaria L. MarcoPierre MarijonIgor Mar\u00edn de MasJessica L. Mark WelchClarisse (Lisa) MarotzWilliam MartinJos\u00e9 Luis Mart\u00ednezEsteban Mart\u00ednez-Garc\u00edaAndreza F. MartinsAdam MartinyCristina Marzach\u00ecJen MatthewsFlorent MazelBonita McCuaigJoy A. McKennaKatelyn McNairAlan McNallyStephen J. McSorleyDaniel R. MendeAlessio MengoniSebastian MenkeHayden MetskyEisha MhatreLuciana MiglioreAndrew D. MillardYusuke MinatoJeremiah J. MinichSara MitriJennifer M. MobberleyOmkar Satyavan MohiteMariano A. MolinaKaren M. MollDenise M. MonackShirin MoossaviDaniela Morales-SanchezNancy A. MoranKaren MoreauChristopher E. MorganMorigen MorigenJennifer L. MorrowJamie MortonBenjamin Douglas MoserLuis Mota-BravoThabiso Eric MotaungIwona MrukRyan Sean MuellerArunika MukhopadhayaSunil MundraBrian T. MurphyMario E. MuscarellaVivek K. MutalikJillian MyersJatin NagpalDipti D. NayakStephen NayfachAndrew L. NealBrittany NeedhamWilliam C. NelsonTrang T. H. NguyenJens NielsenVincent NietoYosuke NishimuraCecilia NoeckerJuan NogalesTeresa NogueiraDaniel R. NogueraNoelle NoyesDele OgunremiMitsuo OguraNorio OhmagariKinji OhnoAnil OjhaAngela OliverioElin OrgWilliam D. OrsiOrla O\u2019SullivanPaul W. O'TooleHong-Yu OuAnne-Marie OverstreetMustafa \u00d6z\u00e7amAlan Roberto PachecoC\u00e1tia Pac\u00edficoJon E. PaczkowskiMelinda PaholcsekSepideh PakpourMarton PalatinszkyRobert J. PalmerMeichen PanGaurav PandharikarJason A. PapinTanya ParishTansol ParkElizabeth ParkinsonD. Joshua ParrisEdoardo PasolliAlessandro PasseraNastassia V. PatinKathryn A. PatrasAmaury PayellevilleJos\u00e9 PenadesLuciana PereiraAraceli Perez-LopezEverett C. PesciRita de Cassia PessottiJason M. PetersScott PetersonHrvoje PetkovicCatherine Ann PfisterC\u00e9cile PhilippeAurore PicotFlavia PinzariMircea PodarClaudia PogoreutzPhillip B. PopeDavide PorcellatoJan PostbergBradley E. PoulsenAkbar Adjie PratamaEva C. PreisnerDavid PriceG. W. PriceJohn G. PurdyLeah PyterFahd QadirWeigang QiuJohn QualeRobert Andrew QuinnLilliana RadoshevichSadequr RahmanTracy Lyn RaivioGeeta RamGordon RamageThandavarayan RamamurthyKarthik RamanKelly RamirezDavid RaskoTamar RatishviliChristoph RatzkeSamiksha RautKasie RaymannTimothy D. ReadMar\u00eda Rebolleda G\u00f3mezNicole RedversAndreas ReisnerTaylor Elaine ReiterLinta RejiZe RenAlejandro ReyesTodd B. ReynoldsFran\u00e7ois RibaletCharlie RiceJason M. RidlonAlice RiselyJohn RobertsFrancisco Rodriguez-ValeraRoderich RoemhildGeraint B. RogersRebekah RogersJohn R. RohdeVeronica Roman-ReynaDavid RomeroS\u00f8ren RosendahlCorinna RossCharl\u00e8ne RousselDenis RoyDaniel RozenAlbane RuaudPeter RubbensJohn RubinsteinLorena RuizAlistair RussellJason W. SahlSaaz Sakrikar\u00c1lvaro San MillanMagdalena San RomanTasha Marie Santiago-RodriguezAlyson E. SantoroRumakanta SapkotaGuillaume SarrabayrouseMariana Gabriela SartorioGoor SassonMatthew J. ScarboroughBernhard H. SchinkDirk SchneiderHannah Doris SchweitzerAlison J. ScottNichollas E. ScottJulie A. SegreH. Steven SeifertJana SeifertYuji SekiguchiAlberto SeveriniHeike SeyboldLauren Marie SeylerVahid ShahrezaeiMigun ShakyaHongmei ShangYongqi ShaoZongze ShaoJason W. ShapiroAshok SharmaSaadlee ShehreenCody SheikJiaxian ShenPaul SheridanZhou Jason ShiShota ShibasakiAmanda ShoreJoshua D. ShroutWeitao ShuaiElla T. SieradzkiAnna SimpsonAbhijeet SinghBaneshwar SinghSurender SinghGeorge SiopisClayton M. SmallChuck Randall SmallwoodMichael SmanskiPaul SmithChristian SohlenkampMichael B. SohnVirtu Solano ColladoKevin V. SolomonVincent SomervilleBokai SongUtkarsh SoodPatrick SorensenRogerio R. Sotelo-MundoVenky SoundararajanDiana Zita SousaBarbara SpellerbergPierre StallforthAdi SternNejc StopnisekJorg StulkeJian-Qiang SuXiaoquan SuZhengchang SuSmitha SukumarAnne O. SummersChaomin SunHui-Zeng SunJian SunJun SunMangesh SuryavanshiDouglas SweeneyKazuhiro TakemotoChandni TalwarYinjie J. TangGerald W. TannockHannah TavalireTegwin TaylorAnna TaylorEva TeiraBenno Herman Ter KuileJanne Gesine Th\u00f6mingVivek Thumbigere-MathXiaojun TianCurtis TilvesAnna D. TischlerAnanda TiwariFranklin ToapantaVittorio TracannaEmmanuel TreinerBrian K. TrevellineGareth TrublHein TunTomasz Wojciech TurowskiSilvia TurroniVaibhav UpadhyayBlake UshijimaBal\u00e1zs VajnaAriena H. C. van BruggenJustin J. J. van der HooftJanneke van de WijgertSimon vanVlietWillem J. B. van WamelJan-Willem VeeningClaire Veneault FourreyMarco VenturaBen VezinaPedro Vieira-BaptistaSara Vieira SilvaFlora Julie VincentJoseph M. VinetzMarie-Joelle VirolleJay VornhagenWillem WaegemanIrene Wagner-DoblerBrett Wagner MackenzieDavid W. WaiteSteffen WaldherrAaron WalshMarina Resendes de Sousa Walther-AntonioHai-Hong WangMinggui WangNancy WangQuanxi WangWenjie WangXueshan WangYong WangZhang WangZhong WangAlex D. WashburneKenneth WasmundSamantha Che WaterworthThomas D. WattsShasta WebbLucy A. WeinertMaggie WeinrothBrian James WerthNicole WheelerLeann WhitneyStefanie WidderLutz WiehlmannClaire E. WilliamsTomasz WilmanskiAmanda Marie WilsonMichael R. WilsonEtthel Martha WindelsNathan WisnoskiRobyn J. WrightWei-Li WuZhihong XieYunhe XuZhihui XuJason H. YangJun YangLiang YangQian YangYuyi YangZhaomin YangMin YapSimon YeWeimin YeAvihu YonaNicholas D. YoungblutLoubna YoussarHongwei D. YuKaifan YuRichard YuMin YueOlivier ZablockiJoseph P. ZackularArsalan H. ZaidiRemi ZallotFabio ZaniniLisa Zeigler AllenQinglu ZengQixiao ZhaiKateryna ZhalninaCong ZhangDao-Feng ZhangJie ZhangLei ZhangLiangliang ZhangMingzi ZhangRong ZhangRui ZhangTianyu ZhangXiaobo ZhangYongjun ZhangZheng ZhangJiangchao ZhaoJinbiao ZhaoWenjing ZhaoXilin ZhaoBeiwen ZhengHao ZhengHong ZhengJianqiu ZhengXiang ZhengXueyun ZhengXiang ZhongZengtao ZhongZhi-Ping ZhongXingang ZhouYu ZhouBaoli ZhuLei ZhuLifeng ZhuLong-Ji ZhuQiyun ZhuWenhan ZhuYongzhang ZhuRyan M. ZielsNadine ZiemertMichael ZimmermannFranz Georg ZinglErik R. ZinserMoreno ZolfoQiyun ZuBang \u51afEvery year I write a brief statement of thanks for the hundreds to thousands of reviewers that make peer review possible at"} +{"text": "British Journal of Nutrition/Volume 125/Issue 9/14 May 2021Published online by Cambridge University Press: 29 June 2020, pp. 1034-1042Print publication: 14 May 2021Details of correction: reformatted Table 1 suppliedExisting text:See Table 1Corrected text should read:See updated and reformatted Table 1"} +{"text": "Scientific Reports 10.1038/srep07002, published online 11 November 2014Correction to: This Article contains errors.In Figure"} +{"text": "The correct title is: Strategies to reduce COVID-19 risk perception among grocery shoppers in the US: A survey study. The correct citation is: Li J, Verteramo Chiu LJ, G\u00f3mez MI, Bills NL (2021) Strategies to reduce COVID-19 risk perception among grocery shoppers in the US: A survey study. PLoS ONE 16(4): e0251060."} +{"text": "International Journal of Neonatal Screening was able to maintain its standards for the high quality of its published papers. Thanks to the contribution of our reviewers, in 2021, the median time to first decision was 18 days and the median time to publication was 44 days. The editors would like to extend their gratitude and recognition to the following reviewers for their precious time and dedication, regardless of whether the papers they reviewed were finally published:Aaron GoldenbergLisa M. GehtlandAlessandra EvaLuca BrunelliAlex R. KemperM. Elske Van Den Akker-van MarleAlexander AsamoahMaartje BlomAllan Meldgaard LundMalcolm DonaldsonAmy CunninghamMarelle J. BouvaAmy E. Wiberley-BradfordMaria ContaldoAmy GaviglioMaria Francisca CoutinhoAna Argudo Ram\u00edrezMariaAnna MessinaAngela ScheuerleMarie AudrainAnita BoelenMark R. De HoraAnna SedivaMarleen JansenAntonella OlivieriMatthew HendersonAntonia RibesMay Ee PngArindam BhattacharjeeMei BakerBaba InusaMichele CagganaBeth A. TariniMiguel Angel Alc\u00e1ntara-OrtigozaBradford TherrellMimi S. KimCan FiciciogluMirjam D. Van Der BurgCarla CuthbertNatalya KashirskayaCarmencita PadillaNatasha HeatherCarol JohnsonNenad BlauCatharina SchuetzNikolas BoyChloe Miu MakNobuyuki IshigeColin KennedyNorma P. TavakoliCynthia F. HintonPamela A. Harris-HamanDavid CheillanPaolo CavorettoDavid WengerPatrice K. HeldDawn A LaneyPatrick DucoroyDeborah MarsdenPatrick JayDebra FreedenbergPaul Van TrotsenburgDenise Margaret HarrisonPaula J. WatersDietrich MaternPeter ClementsEduardo TizzanoP\u00e9ter MonostoriEnzo RanieriPeter SchielenErnest M. PostPhilip J. YoungFabian HauckPim W. J. VergeerFlorian Sebald EichlerPin-Wen ChenFran\u00e7ois BoemerR. Rodney HowellFrancois EyskensRajendra SinghFrancyne KubaskiRalph FingerhutFr\u00f6mmel ClaudiaReinson KaritGeorge E. HogansonRichard B. ParadGeorge TavartkiladzeRobert CurrierGo TajimaRoberto GiuglianiGustavo J. C. BorrajoRolf Zetterstr\u00f6mGuy Van VlietRose E. MaaseHanneke A. HaijesRui VitorinoHarvey LevySamantha A. VerganoHisahide NishioScott D. GrosseIsabelle Durand-ZaleskiScott ShoneJ. Gerard LoeberSerena GasperiniJames B. GibsonSerwet DemirdasJames PittShozo TomonagaJennifer TaylorShunji TomatsuJim BonhamStephan KempJoe OrsiniStuart MoatJos\u00e9 A. CochoStuart Paul AdamsJovanka KingSvetoz\u00e1r Dluholuck\u00fdKanshi MinamitaniTudor Lucian PopKarin Engstr\u00f6mUlrika Von D\u00f6belnKarl R. WhiteV\u011bra Frankov\u00e1Katarina Trebu\u0161ak-Podkraj\u0161ekVeronica WileyKate E. ArmstrongVeroniqa Lundb\u00e4ckKaterina S. KuceraVijaya KnightKatja EggermannVineet DhimanKazumichi FujiokaVito TerlizziKendra JonesWuh-Liang HwuKenji YamadaYvonne A. DanielKonstantinos PetritisYvonne Kellar-GuentherLeah HechtZenon Pogoreli\u0107Lennart Hammarstr\u00f6mZheng Feei MaRigorous peer-reviews are the basis of high-quality academic publishing. Thanks to the great efforts of our reviewers,"} +{"text": "This article has been corrected: Due to errors during figure assembly, the image for panel 3, column 1 in Original article: Oncoscience. 2018 Feb 23;5(1-2):21\u201338PMCID: PMC5854290PMID: 29556515DOI: 10.18632/oncoscience.395"} +{"text": "Lepidoptera, Papilionoidea) of Sulaymaniyah Province, in Kurdistan Region, Iraq. Investigations were carried out between April 2016 and April 2021, during which butterfly specimens were collected from 34 different localities throughout Sulaymaniyah Province. The collected butterflies belonged to 103 species within five families: five species of Papilionidae, 19 species of Hesperiidae, 18 species of Pieridae, 25 species of Lycaenidae and 36 species of Nymphalidae.This study investigates the butterfly fauna Pieriskrueperi Staudinger, 1860, Coliaserate Esper, 1803, Polyommatusthersites , Brenthismofidii Wyatt, 1968 and Pseudochazaramamurra Herrich-Sch\u00e4ffer, 1852 have been added as new records to the fauna of Iraqi butterflies.Eight species, Sulaymaniyah Province is a mountainous area located in the northeast of Iraq and the southeast part of the Iraqi Kurdistan Region. As a part of the Iraqi Kurdistan Region biogeographically, Sulaymaniyah is situated in the Irano-Tauranian in the southeast of the western Palearctic realm . The ProPapilionoidea 47E5BD06-C0D7-5380-93D4-01CCFD8684A9Type status:Other material. Location: county: Pishdar; locality: Sh\u00ean\u00ea Village; verbatimCoordinates: 36\u00b017'00\"N, 45\u00b016'01\"EType status:Other material. Location: county: Dukan; locality: Upper Dukan; verbatimCoordinates: 35\u00b056'59\"N, 44\u00b057'38\"EType status:Other material. Location: county: Chamchamal; locality: Goptapa Village; verbatimCoordinates: 35\u00b051'00\"N, 44\u00b050'07\"EType status:Other material. Location: county: Dukan; locality: Chami Razan Valley; verbatimCoordinates: 35\u00b048'03\"N, 44\u00b058'38\"EType status:Other material. Location: county: Sulyamaniyah; locality: Qlyasan; verbatimCoordinates: 35\u00b034'41\"N, 45\u00b022'01\"EType status:Other material. Location: county: Bakrajo; locality: Hazarmerd; verbatimCoordinates: 35\u00b029'56\"N, 45\u00b018'54\"EType status:Other material. Location: county: Bazyan; locality: D\u00eal\u00eazha; verbatimCoordinates: 35\u00b027'36\"N, 45\u00b011'26\"EType status:Other material. Location: county: Halabja; locality: Byara; verbatimCoordinates: 35\u00b013'47\"N, 46\u00b007'13\"EType status:Other material. Location: county: Kalar; locality: Awa Khwery; verbatimCoordinates: 34\u00b053'30\"N, 45\u00b033'29\"EReverdin, 1913CB23C105-8CA9-5362-9FF4-D64743141542Type status:Other material. Location: county: Chuarta; locality: Upper D\u00ear\u00ea Village; verbatimCoordinates: 35\u00b056'08\"N, 44\u00b057'38\"EType status:Other material. Location: county: Bakrajo; locality: Hazarmerd; verbatimCoordinates: 35\u00b029'56\"N, 45\u00b018'54\"EReverdin, 1913C346BA24-6621-51DE-96F8-39371D13F584Type status:Other material. Location: county: Chwarta; municipality: Basn\u00ea; locality: Upper D\u00ear\u00ea; verbatimCoordinates: 35\u00b056'08\"N, 44\u00b057'38\"E; Identification: identifiedBy: Farhad A. Khudhur; identificationReferences: Tshikolovets et.al. 2014; Event: eventDate: 21-Jun-20; Record Level: basisOfRecord: PreservedSpecimenType status:Other material. Location: county: Dukan; locality: Z\u00eaw\u00ea (Piramagroon Mount.); verbatimCoordinates: 35\u00b045'41\"N, 45\u00b014'17\"E; Identification: identifiedBy: Farhad A. Khudhur; identificationReferences: Tshikolovets etal. 2014; Event: eventDate: 10-Aug-20; Record Level: basisOfRecord: PreservedSpecimenFirst record for Iraq2D313E12-204D-58A1-9BAC-312E4545FA4AType status:Other material. Location: county: Dukan; locality: Qamchukha Village; verbatimCoordinates: 35\u00b053'51\"N, 45\u00b000'51\"EType status:Other material. Location: county: Mawat; locality: Mawat; verbatimCoordinates: 35\u00b053'10\"N, 45\u00b023'59\"EType status:Other material. Location: county: Dukan; locality: Sargalw ; verbatimCoordinates: 35\u00b052'44\"N, 45\u00b009'49\"EType status:Other material. Location: county: Sulyamaniyah; locality: Hawary Shar Park; verbatimCoordinates: 35\u00b036'41\"N, 45\u00b025'48\"EType status:Other material. Location: county: Sulyamaniyah; locality: Qlyasan; verbatimCoordinates: 35\u00b034'41\"N, 45\u00b022'01\"E721F97B5-6EF6-5A92-ABCA-27C68D40B96EType status:Other material. Location: county: Mawat; locality: Mawat; verbatimCoordinates: 35\u00b053'10\"N, 45\u00b023'59\"EType status:Other material. Location: county: Dukan; locality: Z\u00eaw\u00ea (Piramagroon Mount.); verbatimCoordinates: 35\u00b045'41\"N, 45\u00b014'17\"EE50C6867-41F1-52B8-97B5-C68CEB82B944Type status:Other material. Location: county: Chuarta; locality: Shanakhs\u00ea Village; verbatimCoordinates: 35\u00b058'37\"N, 45\u00b031'11\"EType status:Other material. Location: county: Mawat; locality: Galala Village; verbatimCoordinates: 35\u00b053'58\"N, 45\u00b019'51\"EType status:Other material. Location: county: Chuarta; locality: Little Bar\u00ea Village; verbatimCoordinates: 35\u00b053'02\"N, 45\u00b040'07\"EC3742920-EC1C-5681-83A1-B2F7AC4D6307Type status:Other material. Location: county: Dukan; locality: Qamchukha Village; verbatimCoordinates: 35\u00b053'51\"N, 45\u00b000'51\"EType status:Other material. Location: county: Sulyamaniyah; locality: Hawary Shar Park; verbatimCoordinates: 35\u00b036'41\"N, 45\u00b025'48\"EType status:Other material. Location: county: Qareh Dagh; locality: Qareh Dagh Mount.; verbatimCoordinates: 35\u00b014'27\"N, 45\u00b022'12\"E980DA679-1E1B-54FE-AEB1-955E9340269DType status:Other material. Location: county: Qareh Dagh; locality: Qareh Dagh Mount.; verbatimCoordinates: 35\u00b014'27\"N, 45\u00b022'12\"EType status:Other material. Location: county: Halabja; locality: Sargat Village; verbatimCoordinates: 35\u00b017'34\"N, 46\u00b006'18\"ECF4DFB30-CC29-5F02-B5A2-CDD0B2AF055AType status:Other material. Location: county: Bakrajo; locality: Hazarmerd; verbatimCoordinates: 35\u00b029'56\"N, 45\u00b018'54\"E0F4F4752-AA89-5478-BC8A-4B2967C2FE35Type status:Other material. Location: county: Bakrajo; locality: Hazarmerd; verbatimCoordinates: 35\u00b029'56\"N, 45\u00b018'54\"E9E6F9750-53A1-5D73-B484-75591F23ED05Type status:Other material. Location: county: Chuarta; locality: Upper D\u00ear\u00ea Village; verbatimCoordinates: 35\u00b056'08\"N, 44\u00b057'38\"EE02D5E0F-AD44-51E8-93AF-B87575CD9A0AType status:Other material. Location: county: Chuarta; locality: Upper D\u00ear\u00ea Village; verbatimCoordinates: 35\u00b056'08\"N, 44\u00b057'38\"EBC816D21-13B0-5A54-96E9-03494F635B44Type status:Other material. Location: county: Pishdar; locality: Sh\u00ean\u00ea Village; verbatimCoordinates: 36\u00b017'00\"N, 45\u00b016'01\"EType status:Other material. Location: county: Mawat; locality: Mawat; verbatimCoordinates: 35\u00b053'10\"N, 45\u00b023'59\"EDD1AA476-3466-5D23-8E1C-1670835741B1Type status:Other material. Location: county: Sulyamaniyah; locality: Azady Park; verbatimCoordinates: 35\u00b034'02\"N, 45\u00b025'51\"EF62984C9-9DE4-5349-B0C1-109301E60BBAType status:Other material. Location: county: Dukan; locality: Sargalw ; verbatimCoordinates: 35\u00b052'44\"N, 45\u00b009'49\"EC8B2A13B-05DD-5667-994E-D63F726806D7Type status:Other material. Location: county: Dukan; locality: Sargalw ; verbatimCoordinates: 35\u00b052'44\"N, 45\u00b009'49\"EType status:Other material. Location: county: Qareh Dagh; locality: Qareh Dagh Mount.; verbatimCoordinates: 35\u00b014'27\"N, 45\u00b022'12\"EF50B8E52-B0AC-5882-85DF-12A28A4CCA0AType status:Other material. Occurrence: recordedBy: F. A. Khudhur; sex: 2 males; Location: county: Bakrajo; municipality: Bakrajo; locality: Hazarmerd; verbatimCoordinates: 35\u00b029'56\"N, 45\u00b018'54\"E; Identification: identifiedBy: Farhad A. Khudhur; identificationReferences: Tshikolovets et al. 2014 & Bayta\u015f 2007; Event: eventDate: 14-May-20; Record Level: basisOfRecord: PreservedSpecimenFirst record for IraqAB50C740-1013-5567-86BD-3124B95D6F93Type status:Other material. Location: county: Chuarta; locality: Shanakhs\u00ea Village; verbatimCoordinates: 35\u00b058'37\"N, 45\u00b031'11\"EType status:Other material. Location: county: Dukan; locality: Sargalw ; verbatimCoordinates: 35\u00b052'44\"N, 45\u00b009'49\"EType status:Other material. Location: county: Qareh Dagh; locality: Qareh Dagh Mount.; verbatimCoordinates: 35\u00b014'27\"N, 45\u00b022'12\"E9A467315-09EF-5FB8-9A94-0B1F2B91E7E4Type status:Other material. Location: county: Sulyamaniyah; locality: Hawary Shar Park; verbatimCoordinates: 35\u00b036'41\"N, 45\u00b025'48\"EType status:Other material. Location: county: Sulyamaniyah; locality: Goyzha; verbatimCoordinates: 35\u00b034'57\"N, 45\u00b028'09\"EType status:Other material. Location: county: Bakrajo; locality: Hazarmerd; verbatimCoordinates: 35\u00b029'56\"N, 45\u00b018'54\"EType status:Other material. Location: county: Halabja; locality: Zalm Village; verbatimCoordinates: 35\u00b018'53\"N, 46\u00b005'07\"E08518401-1FDC-5CD5-9657-D8F9779F5016Type status:Other material. Location: county: Dukan; locality: Upper Dukan; verbatimCoordinates: 35\u00b056'59\"N, 44\u00b057'38\"EType status:Other material. Location: county: Chamchamal; locality: Goptapa Village; verbatimCoordinates: 35\u00b051'00\"N, 44\u00b050'07\"EType status:Other material. Location: county: Dukan; locality: Chami Razan Valley; verbatimCoordinates: 35\u00b048'03\"N, 44\u00b058'38\"EType status:Other material. Location: county: Bakrajo; locality: Hazarmerd; verbatimCoordinates: 35\u00b029'56\"N, 45\u00b018'54\"EA1BC242D-8C3E-58AD-A76B-1C6BB1CA8872Type status:Other material. Location: county: Dukan; locality: Upper Dukan; verbatimCoordinates: 35\u00b056'59\"N, 44\u00b057'38\"EType status:Other material. Location: county: Chamchamal; locality: Goptapa Village; verbatimCoordinates: 35\u00b051'00\"N, 44\u00b050'07\"EType status:Other material. Location: county: Dukan; locality: Chami Razan Valley; verbatimCoordinates: 35\u00b048'03\"N, 44\u00b058'38\"EType status:Other material. Location: county: Sulyamaniyah; locality: Qlyasan; verbatimCoordinates: 35\u00b034'41\"N, 45\u00b022'01\"EType status:Other material. Location: county: Bakrajo; locality: Hazarmerd; verbatimCoordinates: 35\u00b029'56\"N, 45\u00b018'54\"E9E7FA5B6-8FF2-52F2-8F73-355160292BCFType status:Other material. Location: county: Ranya; locality: Sarkapkan; verbatimCoordinates: 36\u00b021'04\"N, 44\u00b046'24\"EType status:Other material. Location: county: Pishdar; locality: Sh\u00ean\u00ea Village; verbatimCoordinates: 36\u00b017'00\"N, 45\u00b016'01\"EType status:Other material. Location: county: Chuarta; locality: Shanakhs\u00ea Village; verbatimCoordinates: 35\u00b058'37\"N, 45\u00b031'11\"EType status:Other material. Location: county: Chuarta; locality: Upper D\u00ear\u00ea Village; verbatimCoordinates: 35\u00b056'08\"N, 44\u00b057'38\"EType status:Other material. Location: county: Mawat; locality: Galala Village; verbatimCoordinates: 35\u00b053'58\"N, 45\u00b019'51\"EType status:Other material. Location: county: Penjwen; locality: Sya Gwez Village; verbatimCoordinates: 35\u00b048'37\"N, 45\u00b047'33\"EType status:Other material. Location: county: Dukan; locality: Z\u00eaw\u00ea (Piramagroon Mount.); verbatimCoordinates: 35\u00b045'41\"N, 45\u00b014'17\"EType status:Other material. Location: county: Qareh Dagh; locality: Qareh Dagh Mount.; verbatimCoordinates: 35\u00b014'27\"N, 45\u00b022'12\"EType status:Other material. Location: county: Halabja; locality: Byara; verbatimCoordinates: 35\u00b013'47\"N, 46\u00b007'13\"ELinnaeus, 17584CB87121-22CD-5697-9FEE-3CBD4EB2723DType status:Other material. Location: county: Sulyamaniyah; locality: Hawary Shar Park; verbatimCoordinates: 35\u00b036'41\"N, 45\u00b025'48\"EType status:Other material. Location: county: Sulyamaniyah; locality: Qlyasan; verbatimCoordinates: 35\u00b034'41\"N, 45\u00b022'01\"EType status:Other material. Location: county: Sulyamaniyah; locality: Azady Park; verbatimCoordinates: 35\u00b034'02\"N, 45\u00b025'51\"EType status:Other material. Location: county: Kalar; locality: Awa Khwery; verbatimCoordinates: 34\u00b053'30\"N, 45\u00b033'29\"ELinnaeus, 17588CECF23E-2EEF-527C-892A-8DB984EC6C94Type status:Other material. Location: county: Mawat; locality: Galala Village; verbatimCoordinates: 35\u00b053'58\"N, 45\u00b019'51\"EType status:Other material. Location: county: Dukan; locality: Z\u00eaw\u00ea (Piramagroon Mount.); verbatimCoordinates: 35\u00b045'41\"N, 45\u00b014'17\"EType status:Other material. Location: county: Sulyamaniyah; locality: Azady Park; verbatimCoordinates: 35\u00b034'02\"N, 45\u00b025'51\"EType status:Other material. Location: county: Qareh Dagh; locality: Qareh Dagh Mount.; verbatimCoordinates: 35\u00b014'27\"N, 45\u00b022'12\"E0A4FBE97-C41A-521A-972B-2FC9AA6B7A19Type status:Other material. Location: county: Ranya; locality: Sarkapkan; verbatimCoordinates: 36\u00b021'04\"N, 44\u00b046'24\"EAA45CC9D-29FF-5838-8A85-C202FF1F4D73Type status:Other material. Location: county: Bakrajo; locality: Hazarmerd; verbatimCoordinates: 35\u00b029'56\"N, 45\u00b018'54\"E1587F301-609C-5138-985D-4371CD78E4B6Type status:Other material. Location: county: Chamchamal; locality: Goptapa Village; verbatimCoordinates: 35\u00b051'00\"N, 44\u00b050'07\"EType status:Other material. Location: county: Bakrajo; locality: Hazarmerd; verbatimCoordinates: 35\u00b029'56\"N, 45\u00b018'54\"EType status:Other material. Location: county: Bazyan; locality: D\u00eal\u00eazha; verbatimCoordinates: 35\u00b027'36\"N, 45\u00b011'26\"EB59C59C9-282B-5A7A-9654-504290F3355BType status:Other material. Location: county: Dukan; locality: Qamchukha Village; verbatimCoordinates: 35\u00b053'51\"N, 45\u00b000'51\"EType status:Other material. Location: county: Sulyamaniyah; locality: Qlyasan; verbatimCoordinates: 35\u00b034'41\"N, 45\u00b022'01\"EType status:Other material. Location: county: Bakrajo; locality: Kany Pan; verbatimCoordinates: 35\u00b033'03\"N, 45\u00b018'00\"E005550A4-19F0-5FD5-842A-B651B275CA20Type status:Other material. Location: county: Pishdar; locality: Sh\u00ean\u00ea Village; verbatimCoordinates: 36\u00b017'00\"N, 45\u00b016'01\"EType status:Other material. Location: county: Dukan; locality: Qamchukha Village; verbatimCoordinates: 35\u00b053'51\"N, 45\u00b000'51\"EType status:Other material. Location: county: Mawat; locality: Mawat; verbatimCoordinates: 35\u00b053'10\"N, 45\u00b023'59\"EType status:Other material. Location: county: Chuarta; locality: Little Bar\u00ea Village; verbatimCoordinates: 35\u00b053'02\"N, 45\u00b040'07\"EType status:Other material. Location: county: Mawat; locality: Mokaba; verbatimCoordinates: 35\u00b045'26\"N, 45\u00b025'41\"EType status:Other material. Location: county: Sulyamaniyah; locality: Hawary Shar Park; verbatimCoordinates: 35\u00b036'41\"N, 45\u00b025'48\"EType status:Other material. Location: county: Sulyamaniyah; locality: Azady Park; verbatimCoordinates: 35\u00b034'02\"N, 45\u00b025'51\"EType status:Other material. Location: county: Qareh Dagh; locality: Qareh Dagh Mount.; verbatimCoordinates: 35\u00b014'27\"N, 45\u00b022'12\"E8CA274E5-AEB3-5FC8-84E7-D9215F75699CType status:Other material. Occurrence: recordedBy: F. A. Khudhur; sex: 1 male, 2 females; Location: county: Qareh Dagh; locality: Qareh Dagh Mount.; verbatimCoordinates: 35\u00b014'27\"N, 45\u00b022'12\"E; Identification: identifiedBy: Farhad A. Khudhur; identificationReferences: Tshikolovets etal. 2014; Event: eventDate: 12-Sep-20; Record Level: basisOfRecord: PreservedSpecimenFirst record for IraqHerrich-Sch\u00e4ffer, 1850FFD6851D-DEFB-55B2-9E87-4DF57BC44A5FType status:Other material. Location: county: Mawat; locality: Galala Village; verbatimCoordinates: 35\u00b053'58\"N, 45\u00b019'51\"EType status:Other material. Location: county: Sulyamaniyah; locality: Hawary Shar Park; verbatimCoordinates: 35\u00b036'41\"N, 45\u00b025'48\"E38F8DA10-E271-56A7-8D90-0E9D7D0C256DType status:Other material. Location: county: Dukan; locality: Qamchukha Village; verbatimCoordinates: 35\u00b053'51\"N, 45\u00b000'51\"EType status:Other material. Location: county: Sulyamaniyah; locality: Qlyasan; verbatimCoordinates: 35\u00b034'41\"N, 45\u00b022'01\"EType status:Other material. Location: county: Bakrajo; locality: Kany Pan; verbatimCoordinates: 35\u00b033'03\"N, 45\u00b018'00\"EType status:Other material. Location: county: Darbandikhan; locality: Sartak; verbatimCoordinates: 34\u00b056'45\"N, 45\u00b046'32\"EType status:Other material. Location: county: Kalar; locality: Awa Khwery; verbatimCoordinates: 34\u00b053'30\"N, 45\u00b033'29\"EA17472A5-6CBE-533A-A89E-F46024C37844Type status:Other material. Location: county: Penjwen; locality: Penjwen; verbatimCoordinates: 35\u00b036'09\"N, 45\u00b057'48\"ED6DA5F04-D749-5DDA-917F-9626F54DAFECType status:Other material. Occurrence: recordedBy: F. A. Khudhur; sex: 2 males; Location: county: Dukan; locality: Z\u00eaw\u00ea (Piramagroon Mount.); verbatimCoordinates: 35\u00b045'41\"N, 45\u00b014'17\"E; Identification: identifiedBy: Farhad A. Khudhur; identificationReferences: Tshikolovets etal. 2014; Event: eventDate: 4-May-21; Record Level: basisOfRecord: PreservedSpecimenFirst record for IraqABCEF433-40CF-5887-869B-D19D147E6FC9Type status:Other material. Location: county: Chuarta; locality: Upper D\u00ear\u00ea Village; verbatimCoordinates: 35\u00b056'08\"N, 44\u00b057'38\"EType status:Other material. Location: county: Mawat; locality: Galala Village; verbatimCoordinates: 35\u00b053'58\"N, 45\u00b019'51\"EType status:Other material. Location: county: Qareh Dagh; locality: Qareh Dagh Mount.; verbatimCoordinates: 35\u00b014'27\"N, 45\u00b022'12\"E81DCDB13-81C9-59F7-AE24-E02FC9EEF4D8Type status:Other material. Location: county: Halabja; locality: Sargat Village; verbatimCoordinates: 35\u00b017'34\"N, 46\u00b006'18\"EStaudinger, 1860D9D64BCB-8AEB-5EB5-9F65-091424FB3838Type status:Other material. Occurrence: recordedBy: F. A. Khudhur; sex: 1 male; Location: county: Said Sadiq; locality: Naw\u00ea Village; verbatimCoordinates: 35\u00b024'42\"N, 45\u00b057'59\"E; Identification: identifiedBy: Farhad A. Khudhur; identificationReferences: Tshikolovets etal. 2014; Event: eventDate: 27-Oct-17; Record Level: basisOfRecord: PreservedSpecimenFirst record for Iraq501E98B6-55BB-5735-9C4E-6400BADECD71Type status:Other material. Location: county: Sulyamaniyah; locality: Qlyasan; verbatimCoordinates: 35\u00b034'41\"N, 45\u00b022'01\"EType status:Other material. Location: county: Halabja; locality: Byara; verbatimCoordinates: 35\u00b013'47\"N, 46\u00b007'13\"E652E4B83-81F8-555E-BB7B-42D9B82F4A2DType status:Other material. Location: county: Ranya; locality: Sarkapkan; verbatimCoordinates: 36\u00b021'04\"N, 44\u00b046'24\"EType status:Other material. Location: county: Pishdar; locality: Sh\u00ean\u00ea Village; verbatimCoordinates: 36\u00b017'00\"N, 45\u00b016'01\"EType status:Other material. Location: county: Dukan; locality: Upper Dukan; verbatimCoordinates: 35\u00b056'59\"N, 44\u00b057'38\"EType status:Other material. Location: county: Mawat; locality: Mawat; verbatimCoordinates: 35\u00b053'10\"N, 45\u00b023'59\"EType status:Other material. Location: county: Chamchamal; locality: Goptapa Village; verbatimCoordinates: 35\u00b051'00\"N, 44\u00b050'07\"EType status:Other material. Location: county: Dukan; locality: Chami Razan Valley; verbatimCoordinates: 35\u00b048'03\"N, 44\u00b058'38\"EType status:Other material. Location: county: Mawat; locality: Mokaba; verbatimCoordinates: 35\u00b045'26\"N, 45\u00b025'41\"EType status:Other material. Location: county: Mawat; locality: Qaiwan Village; verbatimCoordinates: 35\u00b042'01\"N, 45\u00b025'03\"EType status:Other material. Location: county: Penjwen; locality: Penjwen; verbatimCoordinates: 35\u00b036'09\"N, 45\u00b057'48\"EType status:Other material. Location: county: Sulyamaniyah; locality: Qlyasan; verbatimCoordinates: 35\u00b034'41\"N, 45\u00b022'01\"EType status:Other material. Location: county: Bakrajo; locality: Kany Pan; verbatimCoordinates: 35\u00b033'03\"N, 45\u00b018'00\"EType status:Other material. Location: county: Bazyan; locality: D\u00eal\u00eazha; verbatimCoordinates: 35\u00b027'36\"N, 45\u00b011'26\"EType status:Other material. Location: county: Halabja; locality: Byara; verbatimCoordinates: 35\u00b013'47\"N, 46\u00b007'13\"E59902F06-7A2D-507F-B056-AF9855F85BCBType status:Other material. Location: county: Barzinja; locality: Basak Village; verbatimCoordinates: 35\u00b033'30\"N, 45\u00b042'57\"EKlug, 18296E7F946C-A1E1-5B44-83D0-FECBE97F577AType status:Other material. Location: county: Dukan; locality: Sargalw ; verbatimCoordinates: 35\u00b052'44\"N, 45\u00b009'49\"EType status:Other material. Location: county: Bakrajo; locality: Hazarmerd; verbatimCoordinates: 35\u00b029'56\"N, 45\u00b018'54\"EBBE7AEBC-D119-570B-A4FA-112A6EA15CB1Type status:Other material. Location: county: Ranya; locality: Sarkapkan; verbatimCoordinates: 36\u00b021'04\"N, 44\u00b046'24\"EType status:Other material. Location: county: Pishdar; locality: Sh\u00ean\u00ea Village; verbatimCoordinates: 36\u00b017'00\"N, 45\u00b016'01\"EType status:Other material. Location: county: Mawat; locality: Galala Village; verbatimCoordinates: 35\u00b053'58\"N, 45\u00b019'51\"EType status:Other material. Location: county: Mawat; locality: Mokaba; verbatimCoordinates: 35\u00b045'26\"N, 45\u00b025'41\"EType status:Other material. Location: county: Bakrajo; locality: Kany Pan; verbatimCoordinates: 35\u00b033'03\"N, 45\u00b018'00\"EType status:Other material. Location: county: Bakrajo; locality: Hazarmerd; verbatimCoordinates: 35\u00b029'56\"N, 45\u00b018'54\"EType status:Other material. Location: county: Qareh Dagh; locality: Qareh Dagh Mount.; verbatimCoordinates: 35\u00b014'27\"N, 45\u00b022'12\"E9348651C-D295-5D87-B5D3-87EF8948C866Type status:Other material. Location: county: Mawat; locality: Mawat; verbatimCoordinates: 35\u00b053'10\"N, 45\u00b023'59\"EType status:Other material. Location: county: Sulyamaniyah; locality: Qlyasan; verbatimCoordinates: 35\u00b034'41\"N, 45\u00b022'01\"EType status:Other material. Location: county: Barzinja; locality: Basak Village; verbatimCoordinates: 35\u00b033'30\"N, 45\u00b042'57\"EType status:Other material. Location: county: Bakrajo; locality: Kany Pan; verbatimCoordinates: 35\u00b033'03\"N, 45\u00b018'00\"EType status:Other material. Location: county: Bakrajo; locality: Hazarmerd; verbatimCoordinates: 35\u00b029'56\"N, 45\u00b018'54\"EType status:Other material. Location: county: Bazyan; locality: D\u00eal\u00eazha; verbatimCoordinates: 35\u00b027'36\"N, 45\u00b011'26\"EType status:Other material. Location: county: Said Sadiq; locality: Naw\u00ea Village; verbatimCoordinates: 35\u00b024'42\"N, 45\u00b057'59\"EType status:Other material. Location: county: Halabja; locality: Zalm Village; verbatimCoordinates: 35\u00b018'53\"N, 46\u00b005'07\"EType status:Other material. Location: county: Halabja; locality: Byara; verbatimCoordinates: 35\u00b013'47\"N, 46\u00b007'13\"EBC12FB61-8309-50C2-A7A1-EF4A006E71DBType status:Other material. Location: county: Dukan; locality: Z\u00eaw\u00ea (Piramagroon Mount.); verbatimCoordinates: 35\u00b045'41\"N, 45\u00b014'17\"EStaudinger, 1901DA743377-A90C-5A4C-8D3E-A88794DEAF9DType status:Other material. Location: county: Mawat; locality: Mawat; verbatimCoordinates: 35\u00b053'10\"N, 45\u00b023'59\"E4314CC83-00E0-5672-9455-146F46EBDF82Type status:Other material. Location: county: Mawat; locality: Mawat; verbatimCoordinates: 35\u00b053'10\"N, 45\u00b023'59\"EType status:Other material. Location: county: Mawat; locality: Balkha Village; verbatimCoordinates: 35\u00b047'09\"N, 45\u00b022'47\"E290E6791-658D-5FDF-B4F7-32AA42D064A3Type status:Other material. Location: county: Dukan; locality: Z\u00eaw\u00ea (Piramagroon Mount.); verbatimCoordinates: 35\u00b045'41\"N, 45\u00b014'17\"E0EAB179E-3C96-56FD-9ED5-F49CBE2DD4A3Type status:Other material. Location: county: Mawat; locality: Mokaba; verbatimCoordinates: 35\u00b045'26\"N, 45\u00b025'41\"EType status:Other material. Location: county: Bakrajo; locality: Hazarmerd; verbatimCoordinates: 35\u00b029'56\"N, 45\u00b018'54\"E2B9088EE-D9AB-5011-8157-98B0373534ACType status:Other material. Location: county: Mawat; locality: Mawat; verbatimCoordinates: 35\u00b053'10\"N, 45\u00b023'59\"EType status:Other material. Location: county: Penjwen; locality: Goll\u00ea Resort; verbatimCoordinates: 35\u00b046'31\"N, 45\u00b049'54\"EType status:Other material. Location: county: Sulyamaniyah; locality: Goyzha; verbatimCoordinates: 35\u00b034'57\"N, 45\u00b028'09\"EType status:Other material. Location: county: Bakrajo; locality: Kany Pan; verbatimCoordinates: 35\u00b033'03\"N, 45\u00b018'00\"EType status:Other material. Location: county: Bazyan; locality: D\u00eal\u00eazha; verbatimCoordinates: 35\u00b027'36\"N, 45\u00b011'26\"EType status:Other material. Location: county: Kalar; locality: Awa Khwery; verbatimCoordinates: 34\u00b053'30\"N, 45\u00b033'29\"E17B59791-23B4-5F59-96A1-288EE0DACAFBType status:Other material. Location: county: Sulyamaniyah; locality: Hawary Shar Park; verbatimCoordinates: 35\u00b036'41\"N, 45\u00b025'48\"EType status:Other material. Location: county: Sulyamaniyah; locality: Azady Park; verbatimCoordinates: 35\u00b034'02\"N, 45\u00b025'51\"EType status:Other material. Location: county: Bakrajo; locality: Hazarmerd; verbatimCoordinates: 35\u00b029'56\"N, 45\u00b018'54\"ECED16726-EA1D-5F03-BF05-86D3DB990573Type status:Other material. Location: county: Sulyamaniyah; locality: Hawary Shar Park; verbatimCoordinates: 35\u00b036'41\"N, 45\u00b025'48\"EType status:Other material. Location: county: Sulyamaniyah; locality: Qlyasan; verbatimCoordinates: 35\u00b034'41\"N, 45\u00b022'01\"EType status:Other material. Location: county: Sulyamaniyah; locality: Azady Park; verbatimCoordinates: 35\u00b034'02\"N, 45\u00b025'51\"EType status:Other material. Location: county: Bakrajo; locality: Kany Pan; verbatimCoordinates: 35\u00b033'03\"N, 45\u00b018'00\"E1CBDED23-CFAC-505D-8D33-55C42F7B13FCType status:Other material. Location: county: Pishdar; locality: Sh\u00ean\u00ea Village; verbatimCoordinates: 36\u00b017'00\"N, 45\u00b016'01\"EType status:Other material. Location: county: Dukan; locality: Z\u00eaw\u00ea (Piramagroon Mount.); verbatimCoordinates: 35\u00b045'41\"N, 45\u00b014'17\"EC068E71C-7327-53FD-9030-0BAF3FDAE6D0Type status:Other material. Location: county: Pishdar; locality: Sh\u00ean\u00ea Village; verbatimCoordinates: 36\u00b017'00\"N, 45\u00b016'01\"EType status:Other material. Location: county: Mawat; locality: Mawat; verbatimCoordinates: 35\u00b053'10\"N, 45\u00b023'59\"EType status:Other material. Location: county: Dukan; locality: Z\u00eaw\u00ea (Piramagroon Mount.); verbatimCoordinates: 35\u00b045'41\"N, 45\u00b014'17\"EType status:Other material. Location: county: Sulyamaniyah; locality: Qlyasan; verbatimCoordinates: 35\u00b034'41\"N, 45\u00b022'01\"E8B89871B-CC3E-5978-B598-0721B3B22C30Type status:Other material. Location: county: Chuarta; locality: Upper D\u00ear\u00ea Village; verbatimCoordinates: 35\u00b056'08\"N, 44\u00b057'38\"EType status:Other material. Location: county: Sulyamaniyah; locality: Goyzha; verbatimCoordinates: 35\u00b034'57\"N, 45\u00b028'09\"E201352C4-0BDC-5668-88BE-5C00AB616FC5Type status:Other material. Location: county: Chuarta; locality: Upper D\u00ear\u00ea Village; verbatimCoordinates: 35\u00b056'08\"N, 44\u00b057'38\"EType status:Other material. Location: county: Penjwen; locality: Goll\u00ea Resort; verbatimCoordinates: 35\u00b046'31\"N, 45\u00b049'54\"EKlug, 18346FD55369-A778-572E-A173-C1AABA0BAD9FType status:Other material. Location: county: Mawat; locality: Mawat; verbatimCoordinates: 35\u00b053'10\"N, 45\u00b023'59\"EAA1E221F-0CC2-55EA-BB93-8FD64D33B241Type status:Other material. Location: county: Chuarta; locality: Upper D\u00ear\u00ea Village; verbatimCoordinates: 35\u00b056'08\"N, 44\u00b057'38\"EType status:Other material. Location: county: Sulyamaniyah; locality: Hawary Shar Park; verbatimCoordinates: 35\u00b036'41\"N, 45\u00b025'48\"EType status:Other material. Location: county: Sulyamaniyah; locality: Goyzha; verbatimCoordinates: 35\u00b034'57\"N, 45\u00b028'09\"Evon Rottemburg, 177548698AF7-2915-59F6-BC34-DE237D84E18CType status:Other material. Location: county: Chuarta; locality: Shanakhs\u00ea Village; verbatimCoordinates: 35\u00b058'37\"N, 45\u00b031'11\"EType status:Other material. Location: county: Qareh Dagh; locality: Qareh Dagh Mount.; verbatimCoordinates: 35\u00b014'27\"N, 45\u00b022'12\"E3335AE52-2404-556B-A74B-508B04D5474FType status:Other material. Location: county: Penjwen; locality: Sya Gwez Village; verbatimCoordinates: 35\u00b048'37\"N, 45\u00b047'33\"EType status:Other material. Location: county: Dukan; locality: Z\u00eaw\u00ea (Piramagroon Mount.); verbatimCoordinates: 35\u00b045'41\"N, 45\u00b014'17\"ED33AB2A0-BD05-56F0-8CF3-06E220EBDCB2Type status:Other material. Location: county: Pishdar; locality: Sh\u00ean\u00ea Village; verbatimCoordinates: 36\u00b017'00\"N, 45\u00b016'01\"EType status:Other material. Location: county: Chuarta; locality: Upper D\u00ear\u00ea Village; verbatimCoordinates: 35\u00b056'08\"N, 44\u00b057'38\"E67CCC776-F211-509D-A227-5FD01D4BD5F8Type status:Other material. Occurrence: recordedBy: F. A. Khudhur; sex: 1 male; Location: county: Barzinja; locality: Basak Village; verbatimCoordinates: 35\u00b033'30\"N, 45\u00b042'57\"E; Identification: identifiedBy: Farhad A. Khudhur; identificationReferences: Tshikolovets etal. 2014; Event: eventDate: 5-May-16; Record Level: basisOfRecord: PreservedSpecimenFirst record for IraqE44CEE20-A1F4-567F-BBDF-E9435B4B30CBType status:Other material. Location: county: Pishdar; locality: Sh\u00ean\u00ea Village; verbatimCoordinates: 36\u00b017'00\"N, 45\u00b016'01\"E(Fabricius 1787).D42B3D57-1B27-5CF1-BD84-0D944F7A35D3Type status:Other material. Location: county: Dukan; locality: Z\u00eaw\u00ea (Piramagroon Mount.); verbatimCoordinates: 35\u00b045'41\"N, 45\u00b014'17\"E4AAC4DC0-6C1F-5A63-8632-AE63BE318C8DType status:Other material. Location: county: Mawat; locality: Galala Village; verbatimCoordinates: 35\u00b053'58\"N, 45\u00b019'51\"EType status:Other material. Location: county: Qareh Dagh; locality: Qareh Dagh Mount.; verbatimCoordinates: 35\u00b014'27\"N, 45\u00b022'12\"E8F581935-81D0-595F-801A-F132493814C5Type status:Other material. Location: county: Dukan; locality: Sargalw ; verbatimCoordinates: 35\u00b052'44\"N, 45\u00b009'49\"EType status:Other material. Location: county: Dukan; locality: Z\u00eaw\u00ea (Piramagroon Mount.); verbatimCoordinates: 35\u00b045'41\"N, 45\u00b014'17\"E81AAF4DD-94AD-509E-A6E9-99E11C445BCCType status:Other material. Location: county: Pishdar; locality: Sh\u00ean\u00ea Village; verbatimCoordinates: 36\u00b017'00\"N, 45\u00b016'01\"EType status:Other material. Location: county: Mawat; locality: Mawat; verbatimCoordinates: 35\u00b053'10\"N, 45\u00b023'59\"EType status:Other material. Location: county: Dukan; locality: Sargalw ; verbatimCoordinates: 35\u00b052'44\"N, 45\u00b009'49\"EType status:Other material. Location: county: Bakrajo; locality: Hazarmerd; verbatimCoordinates: 35\u00b029'56\"N, 45\u00b018'54\"EType status:Other material. Location: county: Bazyan; locality: D\u00eal\u00eazha; verbatimCoordinates: 35\u00b027'36\"N, 45\u00b011'26\"EType status:Other material. Location: county: Qareh Dagh; locality: Qareh Dagh Mount.; verbatimCoordinates: 35\u00b014'27\"N, 45\u00b022'12\"E9BB4A2A6-C5BF-5E85-901F-C65DBC83B4F9Type status:Other material. Location: county: Dukan; locality: Qamchukha Village; verbatimCoordinates: 35\u00b053'51\"N, 45\u00b000'51\"EType status:Other material. Location: county: Sulyamaniyah; locality: Azady Park; verbatimCoordinates: 35\u00b034'02\"N, 45\u00b025'51\"Evon Rottemburg, 1775166A5327-4E20-5D07-A8C6-73617D6A5B95Type status:Other material. 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Location: county: Qareh Dagh; locality: Qareh Dagh Mount.; verbatimCoordinates: 35\u00b014'27\"N, 45\u00b022'12\"EType status:Other material. Location: county: Halabja; locality: Byara; verbatimCoordinates: 35\u00b013'47\"N, 46\u00b007'13\"EWyatt, 1969ED90C0D3-E44D-59B6-8F06-749028AC093FType status:Other material. Occurrence: recordedBy: F. A. Khudhur; sex: 1 male; Location: county: Chuarta; locality: Upper D\u00ear\u00ea Village; verbatimCoordinates: 35\u00b056'08\"N, 45\u00b057'38\"E; Identification: identifiedBy: Farhad A. Khudhur; identificationReferences: Tshikolovets etal. 2014; Event: eventDate: 21-Jun-20; Record Level: basisOfRecord: PreservedSpecimenFirst record for Iraq.E9ABA8D2-C1F8-5196-A48A-443C1A41EF3BType status:Other material. Location: county: Chuarta; locality: Upper D\u00ear\u00ea Village; verbatimCoordinates: 35\u00b056'08\"N, 44\u00b057'38\"EType status:Other material. Location: county: Mawat; locality: Galala Village; verbatimCoordinates: 35\u00b053'58\"N, 45\u00b019'51\"EType status:Other material. Location: county: Sulyamaniyah; locality: Hawary Shar Park; verbatimCoordinates: 35\u00b036'41\"N, 45\u00b025'48\"EType status:Other material. Location: county: Qareh Dagh; locality: Qareh Dagh Mount.; verbatimCoordinates: 35\u00b014'27\"N, 45\u00b022'12\"E2267F894-F92F-576E-B26D-0D16FE3BC8E5Type status:Other material. Location: county: Chuarta; locality: Upper D\u00ear\u00ea Village; verbatimCoordinates: 35\u00b056'08\"N, 44\u00b057'38\"EType status:Other material. Location: county: Halabja; locality: Sargat Village; verbatimCoordinates: 35\u00b017'34\"N, 46\u00b006'18\"E5F5F3A7F-628C-5C49-A6D7-2FC79030A572Type status:Other material. Location: county: Chuarta; locality: Upper D\u00ear\u00ea Village; verbatimCoordinates: 35\u00b056'08\"N, 44\u00b057'38\"EType status:Other material. Location: county: Mawat; locality: Galala Village; verbatimCoordinates: 35\u00b053'58\"N, 45\u00b019'51\"EType status:Other material. Location: county: Mawat; locality: Mawat; verbatimCoordinates: 35\u00b053'10\"N, 45\u00b023'59\"EType status:Other material. Location: county: Dukan; locality: Sargalw ; verbatimCoordinates: 35\u00b052'44\"N, 45\u00b009'49\"EKollar, [1849]8A30FD5C-C1BC-54C4-866F-3641CF155BFBType status:Other material. Location: county: Chuarta; locality: Little Bar\u00ea Village; verbatimCoordinates: 35\u00b053'02\"N, 45\u00b040'07\"EType status:Other material. Location: county: Dukan; locality: Sargalw ; verbatimCoordinates: 35\u00b052'44\"N, 45\u00b009'49\"EType status:Other material. Location: county: Dukan; locality: Z\u00eaw\u00ea (Piramagroon Mount.); verbatimCoordinates: 35\u00b045'41\"N, 45\u00b014'17\"EType status:Other material. Location: county: Bakrajo; locality: Hazarmerd; verbatimCoordinates: 35\u00b029'56\"N, 45\u00b018'54\"EType status:Other material. Location: county: Qareh Dagh; locality: Qareh Dagh Mount.; verbatimCoordinates: 35\u00b014'27\"N, 45\u00b022'12\"E4CC45272-1102-5FF8-9FF2-8A70EB09D3E5Type status:Other material. Location: county: Sulyamaniyah; locality: Qlyasan; verbatimCoordinates: 35\u00b034'41\"N, 45\u00b022'01\"EE4B8572F-03BA-5B1E-845E-2677EBA8482CType status:Other material. Location: county: Sulyamaniyah; locality: Hawary Shar Park; verbatimCoordinates: 35\u00b036'41\"N, 45\u00b025'48\"ED1107299-105A-561C-8E3A-F848026D1634Type status:Other material. Location: county: Dukan; locality: Z\u00eaw\u00ea (Piramagroon Mount.); verbatimCoordinates: 35\u00b045'41\"N, 45\u00b014'17\"EType status:Other material. Location: county: Halabja; locality: Sargat Village; verbatimCoordinates: 35\u00b017'34\"N, 46\u00b006'18\"EType status:Other material. Location: county: Qareh Dagh; locality: Qareh Dagh Mount.; verbatimCoordinates: 35\u00b014'27\"N, 45\u00b022'12\"EB5367D38-EE07-56FF-8B59-4633EF0BE996Type status:Other material. 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Location: county: Penjwen; locality: Penjwen; verbatimCoordinates: 35\u00b036'09\"N, 45\u00b057'48\"EType status:Other material. Location: county: Halabja; locality: Sargat Village; verbatimCoordinates: 35\u00b017'34\"N, 46\u00b006'18\"EType status:Other material. Location: county: Qareh Dagh; locality: Qareh Dagh Mount.; verbatimCoordinates: 35\u00b014'27\"N, 45\u00b022'12\"EType status:Other material. Location: county: Halabja; locality: Byara; verbatimCoordinates: 35\u00b013'47\"N, 46\u00b007'13\"EC31719C5-CCE6-5E7D-BCC5-458941AC7CEAType status:Other material. Location: county: Dukan; locality: Z\u00eaw\u00ea (Piramagroon Mount.); verbatimCoordinates: 35\u00b045'41\"N, 45\u00b014'17\"E5C05DEC3-AA00-5A31-A986-33FA6E5FC134Type status:Other material. Location: county: Chuarta; locality: Upper D\u00ear\u00ea Village; verbatimCoordinates: 35\u00b056'08\"N, 44\u00b057'38\"ECC5D312E-F3EA-50A4-BFFC-EA273396B719Type status:Other material. 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Location: county: Halabja; locality: Sargat Village; verbatimCoordinates: 35\u00b017'34\"N, 46\u00b006'18\"E9D457FD5-DF32-5383-8AFE-E9E788DD78E6Type status:Other material. Location: county: Chuarta; locality: Upper D\u00ear\u00ea Village; verbatimCoordinates: 35\u00b056'08\"N, 44\u00b057'38\"EType status:Other material. Location: county: Dukan; locality: Sargalw ; verbatimCoordinates: 35\u00b052'44\"N, 45\u00b009'49\"EType status:Other material. Location: county: Bakrajo; locality: Kany Pan; verbatimCoordinates: 35\u00b033'03\"N, 45\u00b018'00\"EC5900C19-FFD3-57B6-9CEC-D4D1D909DB0CType status:Other material. Location: county: Pishdar; locality: Sh\u00ean\u00ea Village; verbatimCoordinates: 36\u00b017'00\"N, 45\u00b016'01\"EType status:Other material. Location: county: Chuarta; locality: Upper D\u00ear\u00ea Village; verbatimCoordinates: 35\u00b056'08\"N, 44\u00b057'38\"EType status:Other material. Location: county: Mawat; locality: Galala Village; verbatimCoordinates: 35\u00b053'58\"N, 45\u00b019'51\"EType status:Other material. Location: county: Mawat; locality: Mawat; verbatimCoordinates: 35\u00b053'10\"N, 45\u00b023'59\"EType status:Other material. Location: county: Penjwen; locality: Sya Gwez Village; verbatimCoordinates: 35\u00b048'37\"N, 45\u00b047'33\"EE7B37E1F-366C-53E7-BCB0-08DA2A29A21AType status:Other material. Location: county: Mawat; locality: Galala Village; verbatimCoordinates: 35\u00b053'58\"N, 45\u00b019'51\"EType status:Other material. Location: county: Mawat; locality: Mawat; verbatimCoordinates: 35\u00b053'10\"N, 45\u00b023'59\"EType status:Other material. Location: county: Dukan; locality: Z\u00eaw\u00ea (Piramagroon Mount.); verbatimCoordinates: 35\u00b045'41\"N, 45\u00b014'17\"E2FE3B218-B836-58DE-AC69-F6FF9BD36AC7Type status:Other material. 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Location: county: Penjwen; locality: Sya Gwez Village; verbatimCoordinates: 35\u00b048'37\"N, 45\u00b047'33\"EType status:Other material. Location: county: Penjwen; locality: Penjwen; verbatimCoordinates: 35\u00b036'09\"N, 45\u00b057'48\"EType status:Other material. Location: county: Halabja; locality: Sargat Village; verbatimCoordinates: 35\u00b017'34\"N, 46\u00b006'18\"EType status:Other material. Location: county: Halabja; locality: Byara; verbatimCoordinates: 35\u00b013'47\"N, 46\u00b007'13\"EE089A69D-8DA2-5638-A4E9-AA8245939297Type status:Other material. Location: county: Dukan; locality: Z\u00eaw\u00ea (Piramagroon Mount.); verbatimCoordinates: 35\u00b045'41\"N, 45\u00b014'17\"EE042A080-CA6C-59AF-9ABD-B82954F82FDFType status:Other material. Location: county: Dukan; locality: Sargalw ; verbatimCoordinates: 35\u00b052'44\"N, 45\u00b009'49\"EType status:Other material. Location: county: Qareh Dagh; locality: Qareh Dagh Mount.; verbatimCoordinates: 35\u00b014'27\"N, 45\u00b022'12\"E5EC545B3-0B9A-53ED-A5E3-AECA5596B004Type status:Other material. Location: county: Mawat; locality: Mawat; verbatimCoordinates: 35\u00b053'10\"N, 45\u00b023'59\"E75F215EC-866A-56D4-B6EB-F4B1DCD65E9AType status:Other material. Occurrence: recordedBy: F. A. Khudhur; sex: 1 female; Location: county: Mawat; locality: Galala Village; verbatimCoordinates: 35\u00b053'58\"N, 45\u00b019'51\"E; Identification: identifiedBy: Farhad A. Khudhur; identificationReferences: Eckweiler 2012, Tshikolovets et al. 2014 & Tikhonov et al. 2020; Event: eventDate: 11-Jun-20; Record Level: basisOfRecord: PreservedSpecimenType status:Other material. Occurrence: recordedBy: F. A. Khudhur; sex: 1 male; Location: county: Dukan; locality: Z\u00eaw\u00ea (Piramagroon Mount.); verbatimCoordinates: 35\u00b045'41\"N, 45\u00b014'17\"E; Identification: identifiedBy: Farhad A. Khudhur; identificationReferences: Eckweiler 2012, Tshikolovets et al. 2014 & Tikhonov et al. 2020; Event: eventDate: 6-Jun-20; Record Level: basisOfRecord: PreservedSpecimenFirst record for Iraq.109B9937-BFAA-51A4-BE90-4451C747E842Type status:Other material. Location: county: Chuarta; locality: Upper D\u00ear\u00ea Village; verbatimCoordinates: 35\u00b056'08\"N, 44\u00b057'38\"EType status:Other material. Location: county: Dukan; locality: Z\u00eaw\u00ea (Piramagroon Mount.); verbatimCoordinates: 35\u00b045'41\"N, 45\u00b014'17\"E52046FE5-7042-5DE8-9264-E0C0038125AFType status:Other material. Location: county: Dukan; locality: Z\u00eaw\u00ea (Piramagroon Mount.); verbatimCoordinates: 35\u00b045'41\"N, 45\u00b014'17\"EType status:Other material. Location: county: Qareh Dagh; locality: Qareh Dagh Mount.; verbatimCoordinates: 35\u00b014'27\"N, 45\u00b022'12\"E2D94AB28-2EC8-51AE-8DB2-585A5870637AType status:Other material. Location: county: Dukan; locality: Upper Dukan; verbatimCoordinates: 35\u00b056'59\"N, 44\u00b057'38\"EType status:Other material. Location: county: Sulyamaniyah; locality: Hawary Shar Park; verbatimCoordinates: 35\u00b036'41\"N, 45\u00b025'48\"EType status:Other material. Location: county: Sulyamaniyah; locality: Azady Park; verbatimCoordinates: 35\u00b034'02\"N, 45\u00b025'51\"EType status:Other material. Location: county: Bakrajo; locality: Hazarmerd; verbatimCoordinates: 35\u00b029'56\"N, 45\u00b018'54\"E773664AD-8BEB-5940-A347-049425582107Type status:Other material. Location: county: Ranya; locality: Sarkapkan; verbatimCoordinates: 36\u00b021'04\"N, 44\u00b046'24\"EType status:Other material. Location: county: Pishdar; locality: Sh\u00ean\u00ea Village; verbatimCoordinates: 36\u00b017'00\"N, 45\u00b016'01\"EType status:Other material. Location: county: Dukan; locality: Qamchukha Village; verbatimCoordinates: 35\u00b053'51\"N, 45\u00b000'51\"EType status:Other material. Location: county: Chamchamal; locality: Goptapa Village; verbatimCoordinates: 35\u00b051'00\"N, 44\u00b050'07\"EType status:Other material. Location: county: Dukan; locality: Chami Razan Valley; verbatimCoordinates: 35\u00b048'03\"N, 44\u00b058'38\"EType status:Other material. Location: county: Dukan; locality: Z\u00eaw\u00ea (Piramagroon Mount.); verbatimCoordinates: 35\u00b045'41\"N, 45\u00b014'17\"EType status:Other material. Location: county: Mawat; locality: Mokaba; verbatimCoordinates: 35\u00b045'26\"N, 45\u00b025'41\"EType status:Other material. Location: county: Sulyamaniyah; locality: Hawary Shar Park; verbatimCoordinates: 35\u00b036'41\"N, 45\u00b025'48\"EType status:Other material. Location: county: Sulyamaniyah; locality: Azady Park; verbatimCoordinates: 35\u00b034'02\"N, 45\u00b025'51\"EType status:Other material. Location: county: Barzinja; locality: Basak Village; verbatimCoordinates: 35\u00b033'30\"N, 45\u00b042'57\"EType status:Other material. Location: county: Bakrajo; locality: Kany Pan; verbatimCoordinates: 35\u00b033'03\"N, 45\u00b018'00\"EType status:Other material. Location: county: Bazyan; locality: D\u00eal\u00eazha; verbatimCoordinates: 35\u00b027'36\"N, 45\u00b011'26\"EType status:Other material. Location: county: Said Sadiq; locality: Naw\u00ea Village; verbatimCoordinates: 35\u00b024'42\"N, 45\u00b057'59\"EType status:Other material. Location: county: Darbandikhan; locality: Sartak; verbatimCoordinates: 34\u00b056'45\"N, 45\u00b046'32\"EPieridae; Pieriskrueperi, Gonepteryxrhamni and Coliaserate. The five others were two skippers, Carcharodusstauderi and Thymelicushyrax; two Nymphalids Brenthismofidii and Pseudochazaramamurra and a lycaenid butterfly Polyommatusthersites. All of these new records were predicted to be found here, since Sulaymaniyah is located near to the known distribution range of these species (Carcharodusstauderi, Gonepteryxrhamni and Pseudochazaramamurra. Furthermore, several other rarely found species were collected from Piramagroon Mountain, including Favoniusquercus, Satyriumgerhardi, Hyponephelewagneri, Hyponephelelupine, Parargeclimene and Erynnismarloyi. Therefore, this mountain deserves further investigations and will be the next focus for our future work.This study is first intensive faunistic study of butterfly in Sulaymaniyah Province. The diverse topography and ecosystems in Sulaymaniyah Province contributes to the richness in its biodiversity . In part species . Several species indicate"} +{"text": "P\u00fcschmann This led to some of the HRMS data being incorrect for compounds reported in the manuscript. The correct values for the affected compounds are listed below:The authors regret that the HRMS data in the original manuscript were erroneously reported as being acquired in FD4a) HRMS: (LIFDI\u2212) calcd for [C38H44GeO2]\u2212 (M\u2212): 606.25586. Found: 606.3292.Dimesityldimesitoylgermane (4b) HRMS: (LIFDI\u2212) calcd for [C32H32GeO2]\u2212 (M\u2212): 522.16196. Found: 522.2216.Dimesityldibenzoylgermane (o-toluoyl)germane (4c) HRMS: (LIFDI\u2212) calcd for [C34H36GeO2]\u2212 (M\u2212): 550.19326. Found: 550.2034.Dimesityldi(4e) HRMS: (LIFDI\u2212) calcd for [C36H32O2S2Ge]\u2212 (M\u2212): 634.10555. Found: 634.1927.Dimesityldibenzothiophenegermane (\u00a0The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "In deze studie werd dit model getest in een cohort van pati\u00ebnten die systematische biopten, MRI-gerichte biopten of beide ondergingen.Pati\u00ebnten met prostaatkanker (PCa) kunnen geselecteerd worden voor zenuwsparende robotgeassisteerde radicale prostatectomie (RARP) middels voorspellingsmodellen voor extraprostatische extensie (EPE). In 2019 hebben Soeterik et al. een H. Veerman, M.W. Heymans, P.J. van Leeuwen, A.N. Vis en H.G. van der PoelNederlands Kanker Instituut \u2013 Antoni van Leeuwenhoek, Amsterdameasy-to-use voorspellingsmodel met MRI-tumorstadium ontwikkeld en extern gevalideerd voor zijdespecifieke EPE. In deze studie werd dit model getest in een cohort van pati\u00ebnten die systematische biopten, MRI-gerichte biopten of beide ondergingen.Pati\u00ebnten met prostaatkanker (PCa) kunnen geselecteerd worden voor zenuwsparende robotgeassisteerde radicale prostatectomie (RARP) middels voorspellingsmodellen voor extraprostatische extensie (EPE). In 2019 hebben Soeterik et al. een area under the curve (AUC), calibration-in-the-large en calibratiecurves.Een prospectief verzameld cohort van 1170 opeenvolgende pati\u00ebnten die RARP ondergingen in twee hoogvolumecentra tussen 2018 en augustus 2021 werd retrospectief onderzocht. Alle pati\u00ebnten ondergingen een MRI-scan voor de operatie. De diagnose PCa werd gesteld met systematische biopten, MRI-targeted biopten of beide. Met het Soeterik-nomogram werd de zijdespecifieke kans op EPE berekend voor elke prostaathelft met complete data . Modeldiscriminatie en -kalibratie werden onderzocht middels de Pathologische EPE werd vastgesteld in 30% van de prostaathelften. De gemiddelde voorspelde kans op EPE was ook 30%. De onderscheidende waarde van het model was goed ). De voorspelde kans kwam goed overeen met het geobserveerde percentage van EPE met een intercept van -0,02 en een helling van 1,053. Er was onderschatting van het geobserveerde percentage EPE vanaf een voorspelde kans van 70%. De klinische consequentie van deze onderschatting is discutabel aangezien zenuwsparende chirurgie doorgaans wordt afgeraden bij deze drempelwaarde in verband met de kans op positieve snijvlakken. Zie figuur Het Soeterik-nomogram heeft een goede fit op een grote groep Nederlandse pati\u00ebnten. Het nomogram kan binnen Nederland goed gebruikt worden om pati\u00ebnten te selecteren voor zenuwsparende prostatectomie.D. Meijer, A.N. Vis, M.J. Roberts, P.M. van de Ven, H.G. van der Poel, M.L. Donswijk, T.N. Boellaard, I.G. Schoots, D.E. Oprea-Lager en P.J. van LeeuwenAmsterdam Universitair Medische Centra, locatie VUmc, AmsterdamHet preoperatief voorspellen van de kans op pelviene lymfe-kliermetastasen (pN1-ziekte) is cruciaal om pati\u00ebnten te selecteren die in aanmerking komen voor een uitgebreide pelviene lymfeklierdissectie (ePLND), ten tijde van de radicale prostatectomie (RALP). Het doel van deze studie was een nieuw prognostisch model te ontwikkelen om de kans op pN1-ziekte te voorspellen bij pati\u00ebnten met gelokaliseerd prostaatkanker, bestaande uit klinische en histologische parameters, alsmede de uitkomsten van preoperatieve beeldvormende technieken, zoals de MRI- en de PSMA PET-scan.area under the curve (AUC). Deze werd vergeleken met de prognostische waarde van bestaande nomogrammen, zoals het MSKCC- en het Briganti-nomogram.Alle 680 pati\u00ebnten die een PSMA PET- en MRI-scan ondergingen, voorafgaand aan de RALP met ePLND werden ge\u00efncludeerd. Door middel van een logistische regressieanalyse werd een prognostisch model ontwikkeld. De voorspellende waarde van dit nieuwe nomogram werd onderzocht met behulp van de In totaal hadden 175/680 pati\u00ebnten (26%) pelviene lymfekliermetastasen bij histopathologische evaluatie. Het nieuwe nomogram bestond uit de initi\u00eble PSA-waarde, radiologisch T-stadium op MRI, hoogste biopsie Grade Group (GG), biopsietechniek (MRI-targeted versus systematisch), percentage systematische biopten met klinisch significant prostaatkanker (GG \u2265 2) en bevindingen op de PSMA PET-scan. De AUC voor het voorspellen van pN-ziekte was 0,80 met het nieuwe model, vergeleken met 0,69 met zowel het MSKCC-nomogram en het Briganti-nomogram. Zie figuur Het nieuwe nomogram voor het voorspellen van pN1-ziekte is ontwikkeld in pati\u00ebnten met gelokaliseerd prostaatkanker, en bestaat uit klinische en histologische parameters, aangevuld met de resultaten van de MRI-scan en de PSMA PET-scan. Het nieuwe nomogram blijkt superieur aan alle bestaande nomogrammen en zorgt voor het (terecht) nalaten van een pelviene lymfeklierdissectie bij een substantieel deel van de pati\u00ebnten.A.P. Lambregts, A.J. Nieuwhof-Leppink, A.J. Klijn en R.P.J. SchroederUniversitair Medisch Centrum Utrecht, UtrechtEen overactieve blaas met urine-incontinentie bij kinderen heeft een grote impact op de dagelijkse activiteiten en kwaliteit van leven. In sommige gevallen reageert een overactieve blaas niet op urotherapie en anticholinergica. Vanwege de succesvolle uitkomsten bij de behandeling van een neurogene blaas, bieden intravesicale botulinetoxineinjecties een mogelijke oplossing bij kinderen die ongevoelig zijn voor therapie. In deze studie analyseren we de uitkomsten van botulinetoxine-injecties op blaasvolume en incontinentie bij kinderen met een overactieve blaas.Van 50 kinderen die waren gediagnosticeerd met een uitbehandelde niet-neurogene overactieve blaas die botulinetoxine-injecties ontvingen werden de dossiers retrospectief geanalyseerd. Het functionele blaasvolume wordt uitgedrukt als percentage van de verwachte blaascapaciteit voor de leeftijd. Respons wordt beschreven als verbetering in urge-incontinentie na botulinetoxine-injecties. Er werd een multivariate analyse uitgevoerd om voorspellers aan te tonen voor de respons.p = 0,000). 72% van de kinderen liet een verbetering in urinecontinentie zien op de korte termijn en 46% op de lange termijn (> 6 maanden). Het mannelijk geslacht en een klein uitgangsblaasvolume voorspelden een goede uitkomst op continentie op de lange termijn. De meest voorkomende complicatie was een urineweginfectie, die optrad bij zes kinderen (12%). Zie figuur Er werden 50 kinderen ge\u00efncludeerd met een mediane leeftijd 9,9 jaar met een man-vrouwratio van 1:4. Op de korte termijn (< 6 maanden) werd een significante groei van het functionele blaasvolume gezien met een mediaan van 52,9 naar 70% op dag 6-10 na behandeling werd een non-inferieure marge van 10% gekozen. De studie was geregistreerd in Trialregister.nl (NTR6449).Een dubbelblind gerandomiseerde, placebogecontroleerde studie werd uitgevoerd in 15 Nederlandse ziekenhuizen. Volwassen vrouwen ontvingen 2-5 dagen empirische intraveneuze antibiotica voor een Na inclusie van 97 pati\u00ebnten tussen 2017 en 2020, eindigde de studie voortijdig vanwege de COVID-19-pandemie. 50% van de pati\u00ebnten hadden een bacteriemie. De primaire uitkomstmaat werd bereikt bij 36/48 pati\u00ebnten in de fosfomycinegroep en 30/46 pati\u00ebnten in de ciprofloxacinegroep. Secundaire uitkomstmaten waren microbiologische genezing (negatieve urinekweek) op dag 6-10, klinische genezing op dag 30-35 en bijwerkingen van de therapie. Zie figuur 4.1.E. coli bij vrouwen. Fosfomycinegebruik was geassocieerd met meer gastro-intestinale bijwerkingen.Fosfomycine is non-inferieur aan ciprofloxacine voor de uitbehandeling van gecompliceerde urineweginfecties met L.P.W. Witte, M.T. Forss, K. Bolsunovskyi, T.P. Kilpel\u00e4inen, Y. Lee, Y. Aoki, S. Gudjonsson, F. Herv\u00e9, P. J\u00e4rvinen, S. Malde, J. Sairanen, L. Sander, G.H. Guyatt en K.A.O. TikkinenOndanks de hoge incidentie van hydroceles bij volwassenen, hebben urologische beroepsverenigingen geen formele richtlijnen voor de behandeling hiervan. Ons doel was om internationale praktijkvariatie in de behandeling van een hydrocele bij volwassenen in kaart te brengen. Postoperatieve complicaties komen vaak voor na hydrocelectomie en recidieven komen vaak voor na aspiratie (met of zonder sclerotherapie).We hebben een internationaal onderzoek uitgevoerd naar de behandeling van een hydrocele onder urologen in Belgi\u00eb, Denemarken, Finland, IJsland, Japan en Nederland van september 2020 tot december 2020. We identificeerden urologen uit de registers van de landelijke urologische beroepsverenigingen en selecteerden willekeurig 170 urologen en urologen in opleiding uit elk deelnemend land . Behalve in Finland en Japan (postenqu\u00eate), vulden alle deelnemers een e-mailenqu\u00eate in.Van de 864 gecontacteerde urologen deden er 437 (51%) mee. Van de respondenten was 28% vrouw en 19% uroloog in opleiding en had 52% zowel hydrocelectomie\u00ebn, als aspiraties uitgevoerd. In Belgi\u00eb (83%), Denemarken (55%) en Nederland (75%), voerden urologen het vaakst hydrocelectomie\u00ebn uit, terwijl in Finland (84%), Japan (61%) en IJsland (91%) de meeste urologen zowel hydrocelectomie\u00ebn als aspiraties uitvoerden. Urologen gaven de voorkeur aan een hydrocelectomie voor een grote hydrocele (80% vs. 38% voor kleine), jongere pati\u00ebnten (67% voor pati\u00ebnten < 50 jaar vs. 42% voor 70 jaar of meer), pati\u00ebnten met weinig of geen comorbiditeit (65% vs. 24% voor pati\u00ebnten met multipele comorbiditeit) en pati\u00ebnten zonder bloedverdunners (55% vs. 37% voor pati\u00ebnten met bloedverdunners). Zie figuur We vonden een grote variatie in de klinische praktijk met betrekking tot de behandeling van een hydrocele bij volwassenen binnen en tussen landen. Toekomstige studies en richtlijnen zijn nodig om deze variatie te verminderen en de behandeling van een hydrocele te standaardiseren wereldwijd.T. van Doeveren, B.K. Somani en S.M. HaenselFranciscus Gasthuis, RotterdamPediatric Urology 2021 voorziet in adviezen, maar er blijven veel onduidelijke elementen bestaan over diagnostiek en behandeling. Om navolging van de richtlijn in kaart te brengen en internationale routines te vergelijken, werd een online vragenlijst afgenomen.Torsio testis is een acute diagnose en de meest voorkomende oorzaak van irreversibele testiculaire ischemie bij kinderen en adolescenten. Snelle diagnostiek en een juiste behandeling kan blijvende schade voorkomen. De EAU richtlijn respondenten). De vragenlijst bestond uit 18 vragen over diagnostiek, therapie en demografie en werd ingeleid met een casus.De online vragenlijst werd via de NVU en een internationaal netwerk verspreid onder urologen en a(n)ios en 30,4% controleert dit met echo-Doppler. Torsio testis wordt door 76,5% van de respondenten gezien als een indicatie voor spoedchirurgie; 57% verricht een Winkelmann-plastiek. Fixatie van de getordeerde testis wordt door 77,2% verricht, waarbij 29,4% aangeeft de contralaterale testis alleen te fixeren bij een bewezen torsie. Opvallend is dat 28,0% van de Nederlandse respondenten een torsio testis niet als spoedoperatie behandelt, tegen slechts 12,4% internationaal. Verder blijkt 31,4% van de Nederlandse respondenten de testis niet te fixeren; tegenover 3,4% van de buitenlandse urologen. Zie tabel 6.1.De respondenten volgen in de diagnostiek en behandeling van torsio testis meestal de EAU-richtlijn. Wel blijkt er sprake van uiteenlopende meningen over manuele detorsie, gebruik van echografie in de diagnostiek, de timing van operatie en het fixeren van de contralaterale testis. Gezien het belang van de juiste behandeling is het belangrijk om de richtlijn op sommige punten aan te passen en uit te breiden, en deze vervolgens zo goed mogelijk op te volgen voor een eensgezind beleid.B.J. Bos, N.A.M. van Merode, M.S. Steffens en L.P.W. Witte Isala Ziekenhuis, ZwolleEr zijn verschillende behandelopties voor mannen met klachten van een chronische urineresidu (CUR). Wij hebben in kaart gebracht welke behandelingen pati\u00ebnten ondergingen en wat de gerelateerde complicaties waren.single-center, retrospectieve studie werden alle mannen ge\u00efncludeerd met klachten van een niet-neurogeen CUR van > 150 ml gedurende ten minste vier maanden tussen 2014 en 1 september 2020.Voor deze Curatieve behandeling. Van de 50 pati\u00ebnten die met een curatieve intentie werden behandeld, kreeg 1 pati\u00ebnt SNM en 49 pati\u00ebnten (28%) prostaatdesobstructie. Hierna kon 33% stoppen met katheteriseren . Palliatieve behandeling. Als eerste behandelstap werd gekozen voor CIC, CAD, SPC of watchful waiting (WW) in respectievelijk 20, 74, 6 en 0% van de gevallen. Als definitieve behandeling werd gekozen voor CIC, CAD, SPC en WW in respectievelijk 31, 21, 35 en 13% van de gevallen. Pati\u00ebnten ondergingen een mediaan van drie behandelstappen (range 1-18) tot de definitieve behandeling werd bereikt. Complicaties. Katheterisatie geeft significant meer kans op een urineweginfectie en macroscopische hematurie dan WW jaar. Het mediane residu na mictie was 158 ml. De gemiddelde IPSS was 17 \u00b1 8 en de IPSS-QoL was 3 \u00b1 1,5. De mediane follow-up was 68 (1-319) maanden waarin acht (1-51) ziekenhuiscontacten plaatsvonden. \u03b1-blokkers en 5-\u03b1-reductaseremmers werden gebruikt door 67 en 38% van de mannen. Katheterisatie bij CUR kan gepaard gaan met complicaties. Een geselecteerde groep pati\u00ebnten met een CUR zou voordeel kunnen hebben van een prostaatdesobstructie en daardoor kunnen stoppen met katheteriseren.A.J. Seinen, R. Elburg, L.M. Hollegien, M.H. Blanker en L.P.W. WitteIsala Ziekenhuis, ZwolleIn de dagelijkse praktijk kunnen de effectiviteit en de tolerantie van behandelingen voor overactieve blaas (OAB) per pati\u00ebnt verschillen. Daarnaast duurt het vaak weken tot maanden voordat het effect van een behandeling kan worden beoordeeld. Dit leidt niet tot een snelle vermindering van klachten, maar juist tot een verminderde kwaliteit van leven en extra kosten. Het doel van dit onderzoek was inzicht te krijgen in de behandelingen die pati\u00ebnten met OAB ondergaan, van diagnose tot uiteindelijke behandeling.single-center, retrospectieve cohortstudie van vrouwelijke pati\u00ebnten, < 18 jaar, met de diagnose OAB. De inclusieperiode liep van 1 januari 2014 en 30 september 2016. De follow-up eindigde wanneer een pati\u00ebnt een bevredigend behandeleffect ondervond en geen verdere behandeling nodig had, of op 1 januari 2020. De keuze voor een behandeling werd gemaakt door de pati\u00ebnt en de uroloog samen. Het aantal, de volgorde en de duur van de aangeboden behandelstappen werden geanalyseerd.Het betrof een Er werden 120 pati\u00ebnten ge\u00efncludeerd. De aangeboden behandelingen en de volgorde van behandelingen is weergeven in figuur De meeste pati\u00ebnten proberen ten minste twee behandelingen voordat ze een bevredigende verlichting van OAB ervaren. Evaluaties van de behandeling vergen tijd, omdat de duur tot therapeutisch effect verschilt per pati\u00ebnt en per behandeling. Onze bevindingen kunnen helpen bij het scheppen van re\u00eble verwachtingen voor pati\u00ebnten bij het zoeken naar een behandeling voor OAB.D. Meijer, R.M. Mohede, W.S.C. Eppinga, B.G.L. Vanneste, P. Meijnen, O.W.M. Meijer, L.A. Daniels, R.C.N. van den Bergh, A.P. Lont, R.H. Ettema, F.H.K. Oudshoorn, P.J. van Leeuwen, H.G. van der Poel, M.L. Donswijk, D.E. Oprea-Lager, E.E. Schaake en A.N. VisAmsterdam Universitair Medisch Centrum, AmsterdamDe prostaatspecifiek membraanantigeen (PSMA) PET/CT heeft zijn diagnostische waarde voor het beter detecteren van ziektelokalisaties van prostaatkanker bij pati\u00ebnten met een biochemisch recidief (BCR) na robotgeassisteerde radicale prostatectomie (RALP) inmiddels ruimschoots bewezen. De pati\u00ebntselectie die op dit moment nog lokale salvagebehandeling ondergaat, zoals salvage radiatietherapie (SRT), is daarmee ook veranderd. Hypothetisch gezien zouden pati\u00ebnten die PSMA-geleide SRT ondergaan mogelijk betere oncologische uitkomsten hebben dan pati\u00ebnten die \u2018blinde\u2019 SRT ondergaan. Het doel van deze studie was dan ook om de oncologische uitkomsten na SRT te vergelijken tussen pati\u00ebnten die wel, en pati\u00ebnten die geen PSMA PET/CT-scan hebben ondergaan voor BCR-prostaatkanker.case-control matching toegepast.Om deze oncologische uitkomsten te vergelijken tussen beide groepen, werd een historisch non-PSMA-cohort (2010-2015) vergeleken met een PSMA-cohort (2016-2020). Alle pati\u00ebnten ondergingen SRT zonder hormonale therapie. De primaire uitkomstmaat was biochemische progressie een jaar na SRT, gedefinieerd als een PSA-waarde van 0,2 ng/ml of hoger boven de nadir na SRT. Om de uitkomsten beter te kunnen vergelijken, werd p = 0,018).Van de 610 pati\u00ebnten die werden ge\u00efncludeerd in deze studie, bleven er na het matchen nog 106 over in elk cohort, met een mediane PSA-waarde ten tijde van SRT van 0,3 ng/ml. In totaal hadden 30/212 pati\u00ebnten (14%) biochemische progressie van de ziekte, een jaar na SRT. In het historisch cohort was dit 20% (21/106 pati\u00ebnten) vergeleken met 8% (9/106 pati\u00ebnten) in het PSMA-cohort komen voor bij 60% van de pati\u00ebnten. Het optimale behandelschema om blaaskrampen te voorkomen is onbekend.transverse abdominis plane (TAP)-blok, met clonidine of ketamine, of een penisblok, of perivesicale infiltraties, en/of periurethrale infiltraties met ropivaca\u00efne 20 ml 0,25%. De groepsgrootte (n = 42) was gepowered op 50% reductie van blaaskrampincidentie. Middels logistische regressie en linear mixed models werden verschillen tussen de blaaskrampincidentie (ja/nee), ernst en algehele pijn op de verkoeverkamer onderzocht.Er werd een prospectieve cohortanalyse uitgevoerd. Pati\u00ebnten met bioptbewezen prostaatkanker, die waren behandeld met RARP tussen januari 2017 en april 2020 werden ge\u00efncludeerd. Combinaties werden vergeleken van algehele anesthesie en een p = 0,001). Ten opzichte van het baselineprotocol werd een lagere incidentie van blaaskrampen gevonden in de groep met TAP-blok en perivesicale injecties en de groep met TAP-blok en periurethrale injecties . Er werden geen significante verschillen gevonden tussen alle protocollen met perivesicale en/of periurethrale injecties. Wanneer alle protocollen met perivesicale en/of periurethrale injecties werden vergeleken met alle protocollen zonder deze injecties, werd een reductie van 23% van de blaaskramp in de eerdere groep gevonden Er werden geen verschillen in pijnscore gevonden tussen de protocollen. Zie figuur 391 pati\u00ebnten werden onderzocht in acht opeenvolgende cohorten. Een combinatie van TAP-blok, perivesicale en periurethrale injecties leidde tot de laagste incidentie van blaaskramp en vergeleken met het baselineprotocol (TAP-blok) leidde deze combinatie tot een daling van 49% beelden verbeteren het begrip van de precieze locatie van de tumor in de prostaat ten opzicht van tweedimensionale beelden. Deze studie onderzocht of op MRI gebaseerde 3D-prostaatmodellen de preoperatieve planning van zenuwsparing (ZS) bij robotgeassisteerde radicale prostatectomie (RARP) be\u00efnvloeden.Van 20 pati\u00ebnten met bioptbewezen, gelokaliseerd prostaatkanker (< cT3b) die een RARP hadden ondergaan werden 3D-modellen gemaakt. Een uro-radioloog teken-de de PC, het kapsel, de urethra en vesicula seminales in op axiale T2-gewogen 3 Tesla MRI-coupes. Hiervan werden virtuele en 3D-geprinte modellen gemaakt. Zeven RARP-urologen schatten de mate van mogelijke circumferenti\u00eble ZS per prostaathelft (0-6) in op basis van MRI, virtuele en 3D-prints. Een relevant verschil in planning tussen MRI en de 3D-modellen werd gedefinieerd als een verschil van \u2265 3. Middels de intraclass correlatiecoeffici\u00ebnt (ICC) werd de overeenstemming in geschatte ZS tussen urologen onderzocht. Tevens werd de locatie van de indexlaesie (grootste laesie of hoogste Gleason-score) en de locatie van mogelijke extraprostatische extensie (EPE) volgens radiologische intekening vergeleken met het pathologieverslag. Zie figuur Een relevant verschil in planning van ZS werd gevonden in 70/280 (25%) van de gevallen tussen MRI en virtuele 3D-modellen en in 73/280 (26%) van de gevallen tussen MRI en 3D-prints. De overeenstemming tussen urologen in geschatte ZS was hoger met de 3D-modellen dan met MRI ; ICC virtuele modellen 0,52 ; ICC 3D-prints 0,58 ). De locatie van de indexlaesie van de 3D-modellen en het radicale prostatectomiepreparaat kwam overeen bij 19/20 (95%) van de pati\u00ebnten. De locatie van EPE kwam overeen bij 7/7 (100%) pati\u00ebnten.3D-modellen zorgen voor beter inzicht in de tumorlokalisatie en planning van zenuwsparing bij RARP. De 3D-modellen veranderen de zenuwsparing in 1/4 van de gevallen en zorgen voor een hogere overeenstemming tussen urologen.D.J.H. Baas, W. Vreuls, V. Gorden, B. van Uijthoven, M. Wouters, J.P.M. Sedelaar, H.J.E.J. Vrijhof, R.J. Hoekstra, M.A.J. van Zanten, F. Mange, J.P.A. van Basten en D.M. SomfordCanisius Wilhelmina Ziekenhuis, NijmegenRadicale resectie bij mannen die een robotgeassisteerde radicale prostatectomie (RARP) ondergaan, is essentieel om het risico op een recidief te minimaliseren. Behoud van de neurovasculaire bundels (NVB) geeft een grotere kans op positieve snijvlakken, maar vergroot de kans op behoud van erectiele functie. In enkele centra wordt intraoperatieve beoordeling van de snijvlakken middels vriescoupe (NeuroSAFE) aangeboden. Het doel van dit onderzoek was te evalueren of intraoperatieve snijvlakbeoordeling middels confocale lasermicroscopie (CLM) een geschikt alternatief is voor NeuroSAFE.Tussen mei en augustus 21 werden 20 NeuroSAFE-pati\u00ebnten gelijktijdig ge\u00ebvalueerd middels CLM. De Histolog\u00ae-scanner werd gebruikt om prostaatweefsel te scannen. Na de prostatectomie werden aan de posterolaterale zijde van de prostaat twee kapjes gesneden, na bewerking gescand met de Histolog\u00ae-scanner en beoordeeld op beeldkwaliteit door een getrainde pathologielaborant. Vervolgens werden de kapjes in 4-6 lamellen gesneden en ook gescand. De lamellen werden vervolgens ge\u00efnkt en verder verwerkt voor NeuroSAFE volgens protocol. De snijvlakken in vriescoupes werden als positief of negatief beoordeeld door een uro-patholoog. De CLM-beelden werden beoordeeld door \u00e9\u00e9n getrainde uropatholoog.Er werden 40 kapjes geanalyseerd. Drie pati\u00ebnten hadden extracapsulaire extensie op MRI. Zes pati\u00ebnten hadden een enkelzijdig positief snijvlak volgens NeuroSAFE. Vier van de zes NeuroSAFE-positieve pati\u00ebnten waren ook positief bij CLM-beoordeling. Bij \u00e9\u00e9n pati\u00ebnt kon het CLM-beeld niet worden beoordeeld vanwege cauterisatie-effect. Bij de andere pati\u00ebnt was het prostaatweefsel niet volledig gescand. De mediane proceduretijd voor NeuroSAFE bedroeg 45 minuten en voor CLM 22 minuten.CLM is een veelbelovende techniek voor intraoperatieve snijvlakbeoordeling. Een gerandomiseerde studie is nodig om de niet-inferioriteit ten opzichte van intraoperatieve vriescoupes vast te stellen en om het langetermijneffect op de oncologische resultaten te evalueren.J.M. Moll, W.J. Teubel, S.E. Erkens, A. Jozefzoon-Agai, N.F. Dits, A. van Rijswijk, W.M. van Weerden en G.W. Jenster Franciscus Gasthuis & Vlietland, RotterdamMaximale androgeenblokkade (MAB) met eerstegeneratie antiandrogenen geeft bij uitgezaaid prostaatcarcinoom (PC) slechts een minimale winst ten opzichte van androgeendeprivatie (ADT) alleen. Tweedegeneratie antiandrogenen tonen wel een klinisch betekenisvolle meerwaarde. In deze studie hebben wij vier hormoongevoelige PC-modellen onderworpen aan ADT of MAB met eerste of tweedegeneratie antiandrogeen om verschillen in het ontstaan van CRPC en het biologisch gedrag bij progressie te bestuderen.n = 10) of MAB met bicalutamide (n = 10), flutamide (n = 10) of RD162 (n = 5), een analogon van enzalutamide en apalutamide. Karakterisering van CRPC-cellijnen vond plaats met behulp van AR-groeirespons door middel van blootstelling aan een titratiereeks androgeen met of zonder antiandrogenen en qPCR voor kritieke genen in de cascades van AR, AR-V7, hormoonproductie, glucocortico\u00efdreceptor, EMT en WNT.4 AR positieve cellijnen werden na verdeling over individuele kweekbakken blootgesteld aan ADT . De AR groeirespons van CRPC verschilde per originele cellijn: VCaP was sterk AR-responsief, DuCaP was AR-hypersensitief, PC346C was zwak AR-responsief en LAPC4 was AR-onafhankelijk. Gestratificeerd naar originele cellijn, was er geen verschil in AR-groeirespons tussen CRPC na ADT of MAB. Expressie van SNAI1 en WNT5A is hoger bij onbehandelde PC346C en stijgt in de andere modellen bij het ontstaan van CRPC. In LAPC4 gaat de AR-cascade verloren.Het ontstaan van een CRPC-lijn verschilde per cellijn: VCaP 19/35, DuCaP 15/35, PC346C 34/35, LAPC4 15/35, Zowel de eigenschappen van de primaire tumor als het gebruik van tweedegeneratie antiandrogenen zijn bepalend voor de kans op het ontstaan en het biologisch gedrag van CRPC. Resistentie tegen bicalutamide en flutamide ontstaat makkelijker dan tegen RD162. Er zijn echter geen verschillen gemeten in biologisch gedrag tussen ADT en MAB.O.P.J. Vrooman en M.R. van BalkenRijnstate ziekenhuis ArnhemDe behandeling van benigne prostaathyperplasie met transprostatische implantaten is tot op heden veelal onder algehele anesthesie of sedatie toegepast. Met de toegenomen druk op OK-faciliteiten en om de behandeling maximaal minimaal invasief te maken, onderzochten we of deze behandeling ook onder lokale anesthesie goed kon worden uitgevoerd.Numeric Rating Scale (NRS) met range 0-10 de pijnscore gemeten. Alle mannen die behandeld werden tot 20 april 2021 werden daarnaast ge\u00efncludeerd ter analyse van de verbetering ten aanzien van de initi\u00eble mictieklachten, zodat de resultaten na drie maanden follow-up beschikbaar waren. De behandelde mannen werden terug gezien na zes weken en drie maanden met een IPSS/QoL-score.Alle mannen die werden behandeld tussen 3 november 2020 en 1 juni 2021 werden ge\u00efncludeerd ter analyse van hun pijnbeleving tijdens de ingreep onder lokale anesthesie uitgevoerd in dagopname. Premedicatie betrof ciprofloxacine (500 mg), paracetamol (1000 mg), naproxen (250 mg) en midazolam 7,5 mg (< 70 jaar) of 3,75 mg (> 70 jaar). 20 minuten voor de start van de behandeling werd gekoelde (4 \u00b0C) chloorhexidine/lidoca\u00efne gel (22 ml) ingebracht en met een penisklem werd de urethra afgesloten. Na afloop van iedere behandeling werd met behulp van een Er werden 29 mannen behandeld. De leeftijd bedroeg 69,0 jr. \u00b1 9,5 (mean \u00b1 SD). Het prostaatvolume bedroeg 37 \u00b1 9,8 ml. Het aantal gebruikte implantaten was 3,1 \u00b1 1,0. Bij vier mannen (14%) werd een katheter geplaatst na de procedure, 27 van de 29 mannen (93%) gingen dezelfde dag naar huis, twee bleven langer vanwege hematurie. De NRS van de 29 mannen bedroeg 3,8 . De verbetering van de IPSS-scores en toename van de QoL toonden ook onder lokale anesthesie een significante verbetering. Zie tabel Ondanks dat er nog sprake is van een leercurve blijkt dat het goed mogelijk is om transprostatische implantaten te plaatsen onder lokale anesthesie. Dit maakt de kosten lager zonder dat dit ten koste gaat van de kwaliteit van de behandeling en er wordt minder gebruikgemaakt van schaarse operatiekamer en/of sedatiecapaciteit.J. Bosveld, P.A. Hornung, A.J. Klijn en R.P.J. Schroeder Universitair Medisch Centrum Utrecht, UtrechtPhimosis is een aandoening van de voorhuid waarbij de voorhuid niet teruggetrokken kan worden. Bij de geboorte is dit een fysiologisch verschijnsel, maar op latere leeftijd kan phimosis pathologisch zijn. Traditioneel is besnijdenis, na topicaal corticostero\u00efdgebruik, de operationele behandeling van eerste keuze. De laatste jaren is er in verschillende landen een toenemende weerstand tegen het besnijden van kinderen. Bijgevolg is een toenemende tendens om in de plaats daarvan een preputiumplastiek uit te voeren. De huidige literatuur geeft geen uitsluitsel over welke operatieve benadering de voorkeur verdient. Daarom wordt in deze studie gekeken naar de langetermijneffecten van een preputiumplastiek bij kinderen met phimosis.dorsal slit. Kinderen die waren gediagnosticeerd met hypospadie of een begraven penis, evenals pati\u00ebnten met een eerdere voorhuidoperatie werden ge\u00ebxcludeerd. Het primaire resultaat van de preputiumplastiek was positief wanneer de voorhuid maanden na de operatie teruggetrokken kon worden.Een retrospectieve cohortstudie werd uitgevoerd bij jongens jonger dan 18 jaar met phimosis, die in ons ziekenhuis een preputiumplastiek ondergingen tussen 1 januari 2011 en 1 januari 2020. De uitgevoerde preputiumplastiektechnieken bestonden uit een meervoudige Z-plastiek, een (meervoudige) Y-V-plastiek en een (meervoudige) In totaal werden 176 pati\u00ebnten in onze studie opgenomen. Er waren 40 pati\u00ebnten met klachten van (recidiverende) balanitis. 62 van alle pati\u00ebnten hadden een ernstige phimosis. 21 van hen werden gediagnosticeerd met lichen sclerose. 139 pati\u00ebnten waren primair behandeld met topische corticostero\u00efden. Het resultaat van de preputiumplastiek was positief bij 163 pati\u00ebnten (93%). Twee pati\u00ebnten ontwikkelden wonddehiscentie en \u00e9\u00e9n pati\u00ebnt kreeg een infectie na de uitgevoerde plastiek. Van de 13 pati\u00ebnten (7%) met een negatieve uitkomst werd in acht gevallen een circumcisie verricht en in vier gevallen nogmaals een preputiumplastiek uitgevoerd.Preputiumplastiek is een haalbare operatieve behandeloptie bij pathologische phimosis.A. van Uitert, E.C.J. van de Wiel, J. Ramjith, J. Deinum, H.J.L.M. Timmers, J.A. Witjes, L.J. Schultze Kool en J.F. Langenhuijsen Radboudumc, Nijmegen2 is. Om de pati\u00ebntselectie verder te verbeteren, hebben we een preoperatief nomogram ontwikkeld om de operatieduur en complexiteit te voorspellen bij pati\u00ebnten die een indicatie hebben voor PRA.Posterieure retroperitoneale adrenalectomie (PRA) heeft meerdere voordelen ten opzichte van de transabdominale laparoscopische benadering op het gebied van operatieduur, bloedverlies, postoperatieve pijn en herstel. Het kan echter een technisch uitdagende procedure zijn. Volgens de internationale consensus komen pati\u00ebnten in aanmerking voor PRA als ze geopereerd moeten worden aan een goedaardige bijniertumor \u2264 7 cm en hun BMI < 35 kg/m2 was. De primaire uitkomstmaat was operatieduur als surrogaat voor chirurgische complexiteit. Door middel van 10 variabelen werd een predictiemodel gemaakt. Vervolgens werd door mid del van best-subsets regressieanalyse het beste \u00e9\u00e9n-variabeletot zeven-variabelenmodel gezocht.Alle pati\u00ebnten die een unilaterale PRA hebben ondergaan tussen februari 2011 en maart 2020 zijn ge\u00efncludeerd in de studie. Pati\u00ebnten kwamen in aanmerking voor PRA als ze geopereerd moesten worden aan een goedaardige bijniertumor van \u2264 7 cm en de BMI < 35 kg/m2 van 38,6. In dit model werden geslacht, feochromocytoom, BMI en peri-nefrisch vet meegenomen, alle significante voorspellers voor de operatieduur.Er werden 215 pati\u00ebnten ge\u00efncludeerd, met een gemiddelde leeftijd van 52 jaar en een gemiddelde tumorgrootte van 2,4 cm. Na de best-subsets regressieanalyse werd het vier-variabelenmodel geselecteerd en gekalibreerd, die de beste balans liet zien tussen voorspellende kracht en toepasbaarheid met een ROm de preoperatieve pati\u00ebntselectie voor PRA te verbeteren, hebben we een vier-variabelennomogram ontwikkeld om de operatieduur te voorspellen. Als het nomogram een langere operatieduur voorspelt en dus een complexere operatie, zou de transabdominale benadering overwogen moeten worden, aangezien deze meer werkruimte geeft. Tevens kan het nomogram gebruikt worden voor trainingsdoeleinden om pati\u00ebnten te selecteren met gunstige kenmerken voor urologen die PRA willen leren.S.M.H. Einerhand, N. van Dijk, J. van Dorp, J.M. de Feijter, M.L. van Montfoort, M.W. van de Kamp, T.N. Boellaard, K. Hendricksen, M.S. van der Heijden en B.W.G. van Rhijn Antoni van Leeuwenhoek \u2013 Nederlands Kanker Instituut, AmsterdamDe slechte uitkomsten van pati\u00ebnten met stadium III-urotheelcarcinoom (d.w.z. cT3-4aN0M0 of cT1-4N+M0 UC), zelfs na neoadjuvante/inductie chemotherapie (NAIC), tonen het belang van effectievere systemische behandeling. We vergeleken de effectiviteit van NAIC met combinatie-immuuntherapie (ICI) bij pati\u00ebnten die in ons instituut werden behandeld in 2018 en 2019.n = 24) of gemcitabine-carboplatin. Uitkomsten waren complete pathologische respons , complete pathologische downstaging , progressievrije overleving en overleving.Data van pati\u00ebnten die waren behandeld met NAIC werden verzameld uit onze prospectieve database. Pati\u00ebnten die niet in aanmerking kwamen voor cisplatine of dit weigerden, werden behandeld met ICI (ipilimumab-nivolumab) in de NABUCCO-studie of gemcitabine-carboplatin (n = 10). Pati\u00ebnt- en tumorkarakteristieken \u2013 onder andere leeftijd, Charlson Comorbidity Index, ASA-score en nierfunctie \u2013 verschilden niet significant tussen de behandelgroepen. NAIC werd bij 11 pati\u00ebnten (13%) vroegtijdig afgebroken vanwege progressie (n = 6) of toxiciteit (n = 5). ICI werd bij zes pati\u00ebnten (25%) na twee cycli afgebroken vanwege toxiciteit . Na NAIC ondergingen pati\u00ebnten chirurgie of chemoradiatie en konden zeven pati\u00ebnten (10%) geen consoliderende behandeling ondergaan vanwege progressie (n = 5) of toxiciteit (n = 2). Na ICI ondergingen alle pati\u00ebnten chirurgie. Na chirurgie (n = 74) werd pCR door respectievelijk 11 (22%) NAIC- en 11 (48%) ICI-pati\u00ebnten bereikt . pCD werd door respectievelijk 17 (35%) NAIC- en 14 (58%) ICI-pati\u00ebnten bereikt . Pati\u00ebnten die waren behandeld met NAIC kregen vaker progressie . Mediane (IQR) follow-up was 26 (20-32) maanden. In het gehele cohort (n = 107) was ICI geassocieerd met betere PFS en overleving . Zie figuur NAIC bestond uit een cisplatinegebaseerd regime om prostaatvolume te bepalen vergeleken met een prostatectomiepreparaat.Het gebruik van multiparametrische MRI en Een prospectieve studie met pati\u00ebnten die MRI en TRUS hadden ondergaan alvorens een robotgeassisteerde prostatectomie tussen januari 2020 en mei 2021 in Nederland. Prostaatmetingen werden verkregen door TRUS en MRI met de ellipso\u00efde formule. Resultaten werden vergeleken met het prostaat preparaat. Maximale interval tussen MRI, TRUS en prostatectomie was 6 maanden.concordance correlation coefficient (CCC) van 0,81 en 0,83. Regressieanalyse en Bland-Altman-analyse toonden acceptabele accuraatheid vergeleken met het prostaatpreparaat: R2 = 0,83 voor TRUS en R2 = 0,77 voor MRI. Metingen waren accurater in kleinere prostaten < 50 g: 31,2 cc (TRUS), 32,4 cc (MRI) en 40,9 g (preparaat) met 97% (TRUS) en 90% (MRI) van de metingen binnen 20 cc afwijking van het preparaat. Voor grotere prostaten, \u2265 50 g, waren gemiddelden 62,2 cc (TRUS), 67,8 cc (MRI) en 79,6 g (preparaat). Accuraatheid was minder met 58% (TRUS) en 69% (MRI) binnen 20 cc afwijking van het prostaatgewicht.214 pati\u00ebnten werden ge\u00efncludeerd met een gemiddelde leeftijd van 66,2 jaar. Het gemiddelde prostaatvolume werd onderschat met zowel TRUS als MRI vergeleken met het prostaatpreparaat . Dit resulteerde in een 22% onderschatting en 16% onderschatting voor respectievelijk TRUS en MRI. Correlatie was goed met een Pearsons correlatieco\u00ebffici\u00ebnt van 0,91 (TRUS) en 0,88 (MRI) en een Prostaatvolume gemeten door MRI en TRUS hebben vergelijkbare uitkomsten en een acceptabele voorspelling van het prostaatvolume. Zowel TRUS als MRI toonde onderschattingen van het prostaatvolume. MRI-metingen waren dichter bij het daadwerkelijke prostaatvolume, echter, het verschil met TRUS was klein. Beide modaliteiten kunnen ingezet worden en toegespitst worden op de voorkeur van de arts, beschikbaarheid en het lokale protocol of de voorkeur.H.A. de Barros, M.L. Donswijk, M.N. van Oosterom, J.J.M.A. Hendrikx, F.W.B. van Leeuwen, H.G. van der Poel en P.J. van Leeuwen Antoni van Leeuwenhoek, Amsterdam, Prostaatkankernetwerk NederlandOp prostaatspecifieke membraanantigeen (PSMA) gebaseerde radiogeleide chirurgie (RGC) is een veelbelovende techniek voor de detectie van prostaatkanker (PK)-laesies tijdens open salvagechirurgie. Toepassing van RGC bij robotgeassisteerde PK-chirurgie vraagt echter om nieuwe technologie. Doel van dit onderzoek was te evalueren of de geminiaturiseerde DROP-IN-gammaprobe robotgeassisteerde PSMA-RGC mogelijk maakt bij mannen met een recidief PK.99mTechnetium-gelabeld PSMA ligand (99mTc-PSMA-I&S). Primair werd de haalbaarheid van robotgeassisteerde PSMA-RGC onderzocht. Ook vond vergelijking van radioactiviteitmetingen en histologie plaats. Het PSA-gehalte werd 6-8 weken na chirurgie bepaald.In dit eerste prospectieve, in vivo haalbaarheidsonderzoek naar robotgeassisteerde RGC bij recidiverend PK werden 20 pati\u00ebnten met \u2264 2 PK-recidieven in het kleine bekken op PSMA PET/CT ge\u00efncludeerd (NCT03857113). Robotgeassisteerde PSMA-RGC met de DROP-IN-probe vond plaats 19-23 uur na intraveneuze toediening van een Ten tijde van chirurgie bedroeg de mediane leeftijd 68 jaar (IQR 66-72) en was het mediane PSA 1,02 ng/ml . Met behulp van de DROP-IN-probe konden 19 van de 21 (90%) preoperatief ge\u00efdentificeerde laesies robotgeassisteerd gereseceerd worden met een mediane operatieduur van 128 min (IQR 103-157). Op laesieniveau bedroeg de sensitiviteit van PSMA-RGC 86% en de specificiteit 100%. Een PSA-daling > 50% en een PSA < 0,2 ng/ml werd waargenomen bij respectievelijk 12 van de 18 (67%) en drie van de 18 (17%) pati\u00ebnten. Zie figuur De DROP-IN-probe maakt robotgeassisteerde PSMA-RGC mogelijk. Middels deze procedure kunnen PK-laesies zeer specifiek gedetecteerd en gereseceerd worden.J.A. van der Leun, R.P.J. Schroeder, G. Tsachouridis, L. Hermsen en E. de BruijnWilhelmina Kinderziekenhuis, Utrechtclean intermittent catheterisation (CIC), antimuscarinica en antibioticaprofylaxe is dan ge\u00efndiceerd. Momenteel is het onbekend hoeveel pati\u00ebnten met SBO langdurige CIC nodig hebben. Het doel van deze studie was om dit aantal te identificeren. Daarnaast worden eventuele voorspellende factoren onderzocht.Spina bifida occulta (SBO) is een aangeboren aandoening van de wervelkolom waarbij het neurale weefsel alleen bedekt is met huid. De ernst van urologische symptomen is hierbij zeer heterogeen. Het slechtste scenario is een neurogene blaas met detrusor-sfincterdyssynergie, wat kan leiden tot nier schade. Preventieve behandeling middels Deze retrospectieve cohortstudie evalueert alle SBO-pati\u00ebnten die waren behandeld in het Wilhelmina Kinderziekenhuis tussen 1990 en 2020. Inclusiecriteria waren: minderjarige (0-18) SBO-pati\u00ebnten met minstens vier jaar follow-up. Alle pati\u00ebnten van wie gegevens over de diagnose, de uitkomst of de initi\u00eble behandeling ontbraken, werden ge\u00ebxcludeerd. Pati\u00ebnten werden verdeeld in twee groepen op basis van de primaire uitkomst: wel of geen gebruik van CIC aan het eind van de follow-up. Baselinekenmerken en data van drie UDO\u2019s werden uit het elektronisch medisch dossier verzameld en vergeleken. Analyse werd uitgevoerd met gebruik van de chi-kwadraattoets, de Fisher-exact-, de Mann-Whitney-U- of de T-test.Er werden 36 pati\u00ebnten ge\u00efncludeerd. De mediane (IQR) leeftijd bij diagnose was 2,00 maanden. De mediane (IQR) follow-up was 13,4 jaar. Aan het einde van de follow-up gebruikten 13 pati\u00ebnten CIC. Van de pati\u00ebnten die geen CIC gebruikten, waren 11 pati\u00ebnten nooit begonnen en 12 patienten gestopt. Op baseline waren er geen significante verschillen tussen de groepen. Het tweede UDO, bij een mediane (IQR) leeftijd van 29,27 maanden, toonde een significant hoger mediaan postmictie residuvolume in de CIC-groep: 72,00 ml vs. 11,00 ml .Ten minste een derde van alle SBO-pati\u00ebnten heeft langdurige CIC-behandeling nodig. Daarnaast is er een significant hoger postmictieresidu in de CIC-groep.J.G. Heetman, P.D. Polm, T.F.W. Soeterik, J. Lavalaye, P.E.F. Stijns, L. Wever, H.H.E. van Melick en R.C.N. van den BerghSt. Antonius Ziekenhuis, Nieuwegeinactive surveillance (AS) blijft een uitdaging. Bij het gebruik van de huidige klinische parameters zoals MRI en gerichte biopten vindt er in 30-40% van de gevallen Gleason-upgrading plaats na prostatectomie. Het gebruik van 68GA-PSMA-PET/CT kan mogelijk lokale informatie toevoegen op moleculair niveau, en zo mogelijk de risico-inschatting verbeteren.Pati\u00ebntselectie voor 68Ga-PSMA-PET/CT. Er werden aanvullende biopten afgenomen bij een PSMA-laesie (SUVmax > 4) indien die eerder niet zichtbaar was op de MRI en/of niet was gesampled met de systematische en/of gerichte biopten. De studie is gepowered om een upgrading van 10% te detecteren. Hiervoor zijn 141 pati\u00ebnten nodig.De prospectieve cohortstudie PASPoRT includeert pati\u00ebnten met een diagnose prostaatkanker < 6 maanden met een indicatie voor AS. Alle deelnemers hebben een mpMRI ondergaan en vervolgens zijn er systematische en bij een zichtbare laesie, gericht biopten afgenomen. Vervolgens kregen alle pati\u00ebnten een max is 4,60. Aanvullende biopten werden bij 25 pati\u00ebnten (29%) afgenomen. Van deze pati\u00ebnten werden er 9 (10%) ge\u00fcpgraded . De gemiddelde SUVmax van de ge\u00fcpgrade groep is 7,81 en 5 pati\u00ebnten hadden een PIRADS 1 of 2 op de mpMRI. In een multivariabele regressieanalyse zijn de SUVmax en een lage PIRADS-score geassocieerd met Gleason-upgrading. PSA, prostaatvolume en prostaatdensity hebben geen significant verband. In deze groep zijn op de PSMA PET/CT-scan geen metastasen gevonden.Tot nu toe zijn er 86 pati\u00ebnten ge\u00efncludeerd. De gemiddelde leeftijd is 67 jaar en het gemiddelde PSA is 6,66. Van alle pati\u00ebnten hadden 17 (20%) een cT2 bij toucher en 5 (6%) een ISUP GG2. De gemiddelde SUVOnze voorlopige resultaten laten zien dat de toevoeging van PSMA-PET/CT, met aanvullende biopten van eerder niet zichtbare laesies, in potentie een toegevoegde waarde heeft bij pati\u00ebnten met laagtot gemiddeld-risico prostaatkanker die eerder al een mpMRI hebben gehad, met name bij pati\u00ebnten bij wie op de MRI geen afwijking zichtbaar was.L. Cools Paulino Pereira, A. Kums, P.M. Hennus en J. Beck Diakonessenhuis, UtrechtHoewel eerder is aangetoond dat het plaatsen van stents voorafgaand aan een ureterorenoscopie veilig is met weinig complicaties, wordt plaatsing geassocieerd met postoperatieve gecompliceerde urineweginfecties. Eerdere studies hebben de correlatie onderzocht tussen preoperatief geplaatste dubbel-J-stents en toename van bacteri\u00eble kolonisatie en bacteriurie; de associatie met toename van postoperatieve gecompliceerde urineweg-infecties blijft echter onduidelijk. Het doel van deze studie is te onderzoeken of preoperatieve dubbel-J-stents en de verblijfsduur van deze stents de kans op postoperatieve gecompliceerde urineweginfecties vergroten.Deze retrospectieve studie vond plaats in een ziekenhuis in Nederland. Alle volwassen pati\u00ebnten die een ureterorenoscopie ondergingen in 2019 kwamen in aanmerking voor inclusie. Een deel van deze pati\u00ebnten kreeg preoperatief een dubbel-J-stent, die vervolgens werd verwijderd of vervangen tijdens ureterorenoscopie. Gegevens over pati\u00ebnten, verblijfsduur van de stents en de aanwezigheid van gecompliceerde urineweginfecties werden verzameld. Potenti\u00eble risicofactoren werden ge\u00ebvalueerd middels univariate en multivariate logistische regressiemodellen.p = 0,034). Echter, multivariate logistische regressieanalyse toonde identieke risicofactoren aan voor beide groepen, namelijk: vrouwelijk geslacht, preoperatieve positieve urinekweken en recidief van urolithiasis.83 van de 195 ge\u00efncludeerde pati\u00ebnten hadden preoperatief een dubbel-J-stent gekregen. 41,5% van alle patienten was vrouw; de mediane leeftijd was 56 jaar oud (IQR 38-67). 16,9% van de pati\u00ebnten met een stent werd gediagnosticeerd met een postoperatieve gecompliceerde urineweginfectie, in vergelijking tot 7,1% in de groep zonder preoperatieve stent of niersparende chirurgie voor urotheelcarcinoom van de hogere urinewegen (UTUC) wordt gebaseerd op preoperatieve factoren, zoals spontane en selectieve urinecytologie en tumorgrootte. Literatuur over de voorspellende waarde hiervan is echter beperkt. Deze studie onderzoekt of deze variabelen bruikbaar zijn om de histologische uitkomst na nefro-ureterectomie te voorspellen en om te beoordelen of er vaker gekozen kan worden voor niersparende chirurgie.Alle pati\u00ebnten die een RNU ondergingen voor UTUC tussen 2010 tot 2020 werden ge\u00efncludeerd. De urinecytologie werd geclassificeerd als benigne (TPS \u2264 3) of maligne (TPS 4 of 5) en vergeleken met de histologische bevindingen van de RNU, zijnde laaggradig (graad 1 en 2a) en hooggradig (graad 2b en 3) en stadi\u00ebring .Van de 100 ge\u00efncludeerde RNU-preparaten bleek 65% hooggradig en 46% spierinvasief. Maligne cytologie had een sensitiviteit en specificiteit voor hooggradig UTUC van respectievelijk 60 en 69% en voor spierinvasief UTUC van 67 en 65%. De positief en negatief voorspellende waarde was resp. 78 en 48% voor hooggradig UTUC en 62 en 70% voor spierinvasief UTUC. De gemiddelde tumorgrootte was 4,2 cm bij pati\u00ebnten met een hooggradige UTUC en 3,6 cm bij een laaggradige UTUC. Een grotere tumor (per centimeter) had een odds ratio van 1,35 voor hooggradig UTUC en 1,16 voor spierinvasief UTUC.European Association of Urology worden mogelijk kansen gemist om meer pati\u00ebnten niersparend te behandelen. Deze data kan een argument zijn om in selecte gevallen toch te kiezen voor niersparende behandeling, ondanks de aanwezigheid van hooggradige cytologie of een tumor > 2 cm.Urinecytologie heeft een matige voorspellende waarde voor gradering en invasiviteit van UTUC. Bij pati\u00ebnten met maligne cytologie was in 40% van de gevallen sprake van een laaggradig UTUC en in 33% van niet-spierinvasief UTUC. Tumorgrootte was geassocieerd met tumorgradering, maar niet met tumorstadi\u00ebring. Met de huidige criteria van de A.M.T.J. Heemels, M.R. van Balken, P.C. Weijerman, R. Koot, B. Hendrikx en B.K. KroonRijnstate Ziekenhuis, ArnhemSinds 2014 worden pati\u00ebnten met peniskanker in ons ziekenhuis behandeld vanuit het streven zorg waar mogelijk dichtbij de pati\u00ebnt te organiseren. Een essentieel onderdeel van deze behandeling is de schildwachtklierbiopsie. Hierbij kunnen occulte metastasen in cN0-liezen worden opgespoord. Het doel van deze studie was om de resultaten van de schildwachtklierbiopsie in ons ziekenhuis te evalueren.Vanaf 2014 tot 2021 werden 94 opeenvolgende pati\u00ebnten met een hoog stadium (> T1aG1) peniscarcinoom prospectief in deze studie ge\u00efncludeerd. De gemiddelde leeftijd was 71 jaar (41-99). 88 pati\u00ebnten hadden beiderzijds klinisch onverdachte liezen (cN0). Zes pati\u00ebnten hadden enkelzijdig een onverdachte lies en contralateraal een klierpositieve lies (cN1). Schildwachtklierbiopsie werd verricht in de 182 onverdachte liezen. Behandeling van de primaire tumor werd voorafgaand of in dezelfde sessie verricht. Preoperatief werd een lymfoscintigram gemaakt na injectie met 99mTechnetium-nanocllo\u00efd rondom de tumor of het litteken. De schildwachtklier werd intraoperatief ge\u00efdentificeerd met behulp van een blauwe speurstof en een gammaprobe. Alleen bij een tumorpositieve schildwachtklier werd later een liesklierdissectie uitgevoerd.De mediane follow-up was 23 maanden (2-81). Lymfoscintigrafie visualiseerde ten minste 1 schildwachtklier bij 93 pati\u00ebnten (99% detectie). Er werden 219 schildwachtklieren gevisualiseerd en 238 verwijderd. De schildwachtklier bleek positief bij 13 pati\u00ebnten (15 liezen). Complicaties traden op bij 19% (18/94) van de geopereerde pati\u00ebnten. Bij \u00e9\u00e9n pati\u00ebnt werd na een negatieve schildwachtklierprocedure een tumorpositieve klier in de lies aangetroffen na een follow-up van vijf jaar. Dit resulteerde in een foutnegatief percentage van 7% (1/14). Dit is lager dan gerapporteerd wordt in de literatuur .Schildwachtklierbiopsie bij peniskanker in ons ziekenhuis verloopt succesvol en is veilig: het detectiepercentage is hoog, de sensitiviteit is hoog en de morbiditeit is acceptabel.M. Huijben, M.T.W.T. Lock, V.F. de Kemp, L.M.O. de Kort en H.M.K. van BredaUniversitair Medisch Centrum UtrechtSubfertiliteit bij mannen is een veel voorkomend en wereldwijd probleem. Clomifeencitraat (CC) is een selectieve oestrogeenreceptorblokker en kan de spermakwaliteit verbeteren door de hormoonsynthese en spermatogenese te stimuleren. Er is gebrek aan bewijs over de werkzaamheid van CC als therapie voor mannelijke subfertiliteit. Het doel van deze studie was de effectiviteit en veiligheid van CC voor subfertiele mannen te onderzoeken.single-center studie zijn mannen die met CC werden behandeld vanwege subfertiliteit retrospectief geanalyseerd. De primaire uitkomst was verandering in semenparameters. Secundaire uitkomstenmaten waren evaluatie van totaal testosteron (TT), lute\u00efniserend hormoon (LH) en follikelstimulerend hormoon (FSH), zwangerschap, hemoglobine (Hb), hematocriet (Ht), prostaat specifiek antigeen (PSA), bijwerkingen, potentiele voorspellers voor biochemische en/of klinische respons.In deze p < 0,05). Er waren geen veranderingen in Hb, Ht en PSA tijdens de behandeling. Bij 8% van de pati\u00ebnten traden milde bijwerkingen op tijdens CC-behandeling . Laagnormaal FSH v\u00f3\u00f3r CC-behandeling was voorspellend voor semenverbetering tijdens behandeling.In totaal werden 52 subfertiele mannen behandeld met CC. Bij 6/52 pati\u00ebnten vermeldde de anamnese testosterongebruik of misbruik (2 op medische indicatie). Bij 13/46 pati\u00ebnten (28%) zonder testosterongebruik in de voorgeschiedenis was er sprake van een verbetering van zaadcelconcentratie, bij 22 pati\u00ebnten (48%) van de totale zaadcelmotiliteit en bij 20 pati\u00ebnten (43%) een verbetering van de VCM (volume x progressieve concentratie x motiliteit). Bij 7/13 pati\u00ebnten met verbetering in spermaconcentratie (54%) kwam een zwangerschap tot stand (spontaan of met behulp van inseminatie). Bij alle zes mannen met testosterongebruik in de voorgeschiedenis trad een verbetering van semenkwaliteit op. TT-, FSH- en LH-waarden stegen tijdens behandeling is een alternatieve Er is gebrek aan studies over het gebruik van CC voor mannen met hypogonadisme. Het doel van deze retrospectieve studie was om de effectiviteit en veiligheid van CC voor hypogonadale mannen te evalueren.single-center studie werden mannen die werden behandeld met CC voor hypogonadisme retrospectief geevalueerd. De primaire uitkomst waren hormonale parameters, inclusief totaal testosteron (TT), vrij testosteron (FT), lute\u00efniserend hormoon (LH) en follikelstimulerend hormoon (FSH). Secundaire uitkomstmaten waren hypogonadale symptomen, metabole en lipidenparameters, hemoglobine (Hb), hematocriet (Ht), prostaatspecifiek antigeen (PSA), bijwerkingen en mogelijke voorspellers voor de biochemische en/of klinische respons.In deze In totaal werden 153 hypogonadale mannen behandeld met CC. De mediane behandelduur was 10 maanden (range 1-96). De gemiddelde TT-, FT-, LH- en FSH-waarden stegen tijdens de behandeling. TT steeg van 9 naar 16 nmol/l , waarbij slechts beperkte informatie bekend is over de impact van de leercurve op perioperatieve uitkomsten. In deze studie werden de resultaten sinds de introductie van de RARC in ons ziekenhuis ge\u00ebvalueerd.Alle pati\u00ebnten die tussen februari 2014 en april 2021 een RARC voor blaaskanker ondergingen, werden ge\u00efncludeerd. Pati\u00ebnten ondergingen een in opzet intracorporele urinedeviatie (ICUD) en werden geopereerd door in totaal vier urologen. Primaire uitkomstmaten waren de 30- en 90-dagen-mortaliteit (30dM/90dM), heropnamepercentage (30dH/90dH) en complicatiepercentage volgens Clavien-Dindo (30dC/90dC). Secundaire uitkomstmaten waren snijvlakstatus, operatieduur (OT), bloedverlies (EBL), opnameduur (LoS) en ziektespecifieke overleving (DSS).177 pati\u00ebnten werden ge\u00efncludeerd. De 30dM en 90dM was 1,1% en 5,1%. Het percentage complicaties > 2 volgens Clavien-Dindo was 24,3% zowel na 30 als 90 dagen. De 30dH en 90dH was 22,6% en 23,7%, met minder heropnames in 2019 en 2020. 12 van de 173 ICUD\u2019s moesten worden geconverteerd. Na 2019 werd deze conversie niet meer gezien. Positief snijvlakpercentage was 2,6%. Het mediaan EBL was 400 ml (IQR 250-700), mediane OT was 450 min (IQR 411-490), mediane LoS was 9 dagen (IQR 7-12) en mediane lymfeklieropbrengst was 15 (IQR 11-21). DSS was 31,6% bij een mediane follow-up van 20 maanden. Er werd een afname gezien van de mediane OT van 520 naar 420 minuten. Overige secundaire uitkomstmaten verbeterden minimaal of bleven gelijk bij het doorlopen van de leercurve.De oncologische en perioperatieve resultaten van RARC zijn grotendeels in lijn met de resultaten van grote open radicale cystectomieseries. De 30dC, maar niet de 90dC, lijkt wat hoger te zijn. Verder is de operatieduur langer in vergelijking met de open radicale cystectomie, maar deze nam wel af tijdens de studieperiode. Een evidente impact van de leercurve op de resultaten anders dan operatieduur werd niet geobjectiveerd.A.E. van der West, D. van de Kerkhof, E.L. Koldewijn en T.J.N. HermansCatharina Ziekenhuis, EindhovenDe urine-MCM5-analyse (ADXBLADDER) zou kunnen worden ingezet in de follow-up van het niet-spierinvasief urotheelcelcarcinoom van de blaas (NMIBC). Wij delen de eerste resultaten uit onze praktijk aangaande pati\u00ebnten in de follow-up van hooggradig (HG) NMIBC.Gedurende zes maanden zijn in \u00e9\u00e9n centrum 43 pati\u00ebnten in de follow-up van HG NMIBC geselecteerd middels controle van spreekuren. Pati\u00ebnten werden ge\u00efncludeerd binnen de eerste drie jaar follow-up. Behandelbeleid werd enkel gebaseerd op cystoscopie \u00e9n cytologisch urineonderzoek. Er werd extra urine verzameld voor de ADX-BLADDER-test. Bij klinische verdenking op een recidief werd zo nodig een transurethrale resectie tumor (TURT) verricht. Exclusiecriteria waren een katheterisatie en/of urineweginfectie binnen twee weken voor de ADXBLADDER-test.n = 7), pT1HG (n = 26), CIS (n = 6) en CIS + pTa/1HG (n = 4). Bij 34 pati\u00ebnten werd een re-TURT verricht . Alle pati\u00ebnten kregen een adjuvante behandeling middels chemospoelingen (n = 4), BCG-spoelingen (n = 31) of BCG- en chemospoelingen (n = 8). De gemiddelde follow-up na diagnose was 14 maanden. 10 pati\u00ebnten hadden een positieve ADXBLADDER, waarvan twee klinisch de verdenking hadden op een recidief blaastumor (positief-voorspellende waarde 20%). Hierbij was er een verdenking op persisterend CIS bij cystoscopie en positieve urinecytologie (TPS 5). Omdat er geen klinische consequenties waren, werd er geen TURT verricht (= limitatie). 33 pati\u00ebnten hadden een negatieve ADXBLADDER, waarvan er drie klinisch de verdenking hadden op een recidief blaastumor. Echter, TURT toonde geen carcinoom (negatief-voorspellende waarde 100%). Het lage recidiefpercentage in dit specifieke HG NIMBC-cohort beperkt echter vooralsnog de power van de voorspellende waarden. Zie tabel De primaire pathologie bestond uit: pTaHG (In dit cohortonderzoek had 77% (33/43) van de cystoscopie\u00ebn gereduceerd kunnen worden middels ADXBLADDER. De beperkte power noodzaakt echter tot verdere validatie in een groter HG NMIBC-cohort.B.K. Kroon, M.R. van Balken en H.M. KroonRijnstate ArnhemIn 2016 en 2019 is onderzoek verricht naar de levensverwachting van algemeen chirurgen in Nederland. Deze bleek duidelijk lager dan de gemiddelde levensverwachting van andere hoogopgeleide Nederlanders en ook lager dan die van de gemiddelde Nederlandse medisch-specialist. In deze studie onderzoeken we de levensverwachting van de Nederlandse uroloog.Via de NVU werden geboorte- en sterftedata opgevraagd.De gemiddelde sterfteleeftijd werd berekend. Daarnaast werd de gemiddelde sterfteleeftijd berekend van urologen die hun pensioengerechtelijke leeftijd haalden. Deze cijfers zijn vergeleken met de online beschikbare cijfers van het CBS en verstrekte cijfers van de SPMS.n = 44) werden gemiddeld 81,3 jaar oud . De levensverwachting van de Nederlandse uroloog is 5,1 jaar korten dan van de gemiddelde Nederlandse man . In vergelijking met de hoger opgeleide Nederlandse man is de levensverwachting van de Nederlandse uroloog 9,1 jaar korter en 6,1 jaar korter dan van de hoger opgeleide man die de leeftijd van 65 bereikt heeft . Urologen worden zelfs nog minder oud dan chirurgen: 75,1 vs. 78,1. Gepensioneerde urologen worden gemiddeld 6,6 jaar minder oud dan hun gepensioneerde collega medisch-specialisten . Verdere uitsplitsing van de cijfers per medisch specialisme werd door de SPMS niet verstrekt.Sinds 2010 worden sterftecijfers door de NVU bijgehouden. Op basis van 58 overleden urologen was de gemiddelde levensduur 75,1 jaar . De urologen die hun pensioen haalden (De levensverwachting van Nederlandse urologen is duidelijk lager dan die van andere Nederlanders, hoogopgeleide Nederlanders en medisch-specialisten in het algemeen. Urologen zijn dus net als algemeen chirurgen weinig duurzame medisch-specialisten. Een vervolgstap stap naar mogelijke oorzaken en uiteindelijk verbetering van deze verontrustende uitkomsten zou een vergelijkbare analyse per specialisme zijn om de levensverwachting voor elk specialisme in kaart te brengen."} +{"text": "PMCID: PMC4162138\rPMID: 25221644\rDOI: 10.18632/genesandcancer.24\rThis article has been corrected: The authors agree that Figure"} +{"text": "Scientific Reports 10.1038/s41598-021-89861-6, published online 18 May 2021Correction to: f) legend,The original version of this Article contained an error in the Figure\u00a03 (f) OCR vs ECAR from the mitochondrial stress test assay. Squares: SW948; Circles: SW116.\u201d\u201c(now reads:f) OCR vs ECAR from the mitochondrial stress test assay. Squares: SW1116; Circles: SW948.\u201d\u201c(The original Article has been corrected."} +{"text": "Acta Cryst. (2007), E63, m2536.Corrigendum to et al. , four imidazole H atoms are missing in the refinement.In the article by Li The revised crystal data, data collection and structure refinement details are summarized in Table\u00a01The structure of chlorido\u00adtetra\u00adkis\u00ad(imidazole)\u00adcopper(II) chlo\u00adride, reported in the article by Li al. 2007, has bee10.1107/S2056989021002267/me6126sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989021002267/me6126Isup2.hklStructure factors: contains datablock(s) I. DOI: 2066475CCDC reference: crystallographic information; 3D view; checkCIF reportAdditional supporting information:"} +{"text": "Anoplophora Hope, 1839 (Coleoptera: Cerambycidae: Lamiinae: Lamiini) includes 47 species (without subspecies) occurring in East, South and Southeast Asia. Amongst them, 38 species are known from CHINA. Members of this genus are familiar to Chinese people with a widely-used common name: \u201c\u661f\u5929\u725b [starry longhorn beetle]\u201d. Anoplophora species have great economic importance, attacking and damaging numerous hardwood trees and some coniferous trees.The genus Anoplophorahuangjianbinisp. n. (Coleoptera: Cerambycidae: Lamiinae: Lamiini) is described from Fujian and Guangxi, CHINA. Diagnostic characters of the new species are illustrated and comparison with closely-related congeners is provided.A new species of starry longhorn beetle, Anoplophora Hope, 1839 (Coleoptera: Cerambycidae: Lamiinae: Lamiini) can be separated from allied genera by a combination of the following characters: female sternite VII with lateral notches approximately at the level where the ventral apodeme of sternite VIII attaches; mesotergum consisting of two overlapping plates (as in Monochamus and Eupromus), but overlap evenly and broadly convex laterally, with small notches extending laterally anterior to the base of scutellum; antennal scape with small to large apical cicatrix; and pronotum with posteromedial callus in most species (Anoplophora (Coleoptera: Cerambycidae)\u201d by Anoplophora. Anoplophorasiderea Bi, Chen & Ohbayashi, 2020, A.fanjingensis Yang, Yang & Tian, 2020 and A.puxian Wang & He, 2021 are the latest three species contributing to the genus presented us with a pair of specimens from Fujian, CHINA, which were identified as an unkown species of Anoplophora. Later, more specimens of this species were available to us from different sources, including those from Guangxi, CHINA. Herein, we describe and illustrate it under the name of Anoplophorahuangjianbinisp. n. Therefore, the number of the Anoplophora species from CHINA comes to 39 (without subspecies) (The genus species . Since the genus . Shortlyspecies) . ImportaSpecimens were relaxed and softened in a HH-2 digital homoeothermic water bath at 44.4\u2103 for 14 hours, then transferred to distilled water to clean, observe and dissect. In order to examine the genitalia, the abdomen was detached and treated with a 10% solution of potassium hydroxide (KOH) for 12 hours, then transferred to distilled water to remove the remaining KOH and stop any further bleaching. After examination, the body parts were mounted on a glass slide with Euparal Mounting Medium for future studies. Habitus images were taken using a Canon 50D DSLR with a Canon EF 100 mm f/2.8L IS USM lens and a Canon MT-24EX Macro Twin Lite Flash was used as the light source. Images of the morphological details were taken using a Canon macro photo lens MP-E 65 mm on a Canon 5DsR. Images of the same specimen/structure at different focal planes were combined using Zerene Stacker 1.04 stacking software. Adobe Photoshop CS6 was used for post-processing. The terminology adopted in this paper for external features of the body and genitalia follows CCZC: collection of Chao Zhou, Chengdu, CHINA; CLGS: Collection of Liang Guo, Sanming, CHINA; CLHC: Collection of Li He, Chengdu, CHINA; CJBH: Collection of Jian-Bin Huang, Nanping, CHINA; CPYL: Collection of Peng-Yu Liu, Nanping, CHINA; CTLH: Collection of Tian-Long He, Huainan, CHINA; MYNU: insect collection of Mianyang Normal University, Mianyang, CHINA.The material examined for this study is deposited in the following institutional and private collections: Anoplophorachiangi Hua & Zhang, 1991. CHINA: 1\u2642. CHINA: 1\u26421\u2640, Yunnan, Honghe Hani & Yi Autonomous Prefecture, Lvchun County, Huanglianshan National Nature Reserve [\u9ec4\u8fde\u5c71\u56fd\u5bb6\u7ea7\u81ea\u7136\u4fdd\u62a4\u533a], alt. 1850 m, VI.2019, Tian-Long He leg. (CTLH); 1\u2640, Yunnan, Xishuangbanna Dai Autonomous Prefecture, Mengla County [\u52d0\u814a\u53bf], alt. 1200 m, VIII.2020, Zhong-Xiong Fu leg. (CLHC); 1\u2640, ditto except alt. 800 m, Yi Li leg. (CLHC); 1\u2642, Yunnan, Baoshan City, Tengchong, Yunfeng mountain [\u4e91\u5cf0\u5c71], alt. 971 m, 15.VII.2021, Chen-Yan Jin leg. (CLHC); 3\u2640\u2640, Guangxi, Fangchenggang City, Shangsi County, Shiwandashan National Forest Park [\u5341\u4e07\u5927\u5c71\u56fd\u5bb6\u68ee\u6797\u516c\u56ed], VI.2021, local people leg. (CLHC); LAOS: 1\u2642. Anoplophoraimitator. CHINA: 1\u2642. CHINA: 1\u2642.The following material was studied for comparison: : 1\u2642Fig. D, Guizhoantennal length: length between the base and the apex of antenna; body length: length between the head vertex and the elytral apex along the mid-line; elytral length: length between the basal border and the apex of elytra along suture; head length: length between the anterior apex of clypeus and the posterior margin of occiput along the midline; head width: widest part of head (including eyes); humeral width: width across elytral humeri; pronotal length: length of the pronotum along the mid-line; pronotal apical width: width across the apical margin of pronotum; pronotal basal width: width across the basal margin of pronotum; pronotal maximum width: widest part of pronotum .Measurement criteria in millimetres (mm) are as follows: Wang & He, 2021sp. n.C8622FE1-9F27-5969-9896-860DA13036655DF3AD6C-7A89-487E-9684-DFF86E318C61Type status:Holotype. Occurrence: recordedBy: Jian-Bin Huang; individualCount: 1; sex: male; Location: country: CHINA; stateProvince: Fujian; verbatimLocality: Sanming City, Sha County, Luoboding [\u4e09\u660e\u5e02\u6c99\u53bf\u9523\u94b9\u9876]; verbatimElevation: 1360 m; verbatimLatitude: N26.25843\u00b0; verbatimLongitude: E117.73736\u00b0; Event: year: 2018; month: 7; Record Level: institutionCode: MYNUType status:Paratype. Occurrence: recordedBy: Jian-Bin Huang; individualCount: 1; sex: female; Location: country: CHINA; stateProvince: Fujian; verbatimLocality: Sanming City, Sha County, Luoboding [\u4e09\u660e\u5e02\u6c99\u53bf\u9523\u94b9\u9876]; verbatimElevation: 1360 m; verbatimLatitude: N26.25843\u00b0; verbatimLongitude: E117.73736\u00b0; Event: year: 2018; month: 7; Record Level: institutionCode: MYNUType status:Paratype. Occurrence: recordedBy: Yong Li; individualCount: 1; sex: male; Location: country: CHINA; stateProvince: Fujian; verbatimLocality: Sanming City, Sha County, Luoboding [\u4e09\u660e\u5e02\u6c99\u53bf\u9523\u94b9\u9876]; verbatimElevation: 1360 m; verbatimLatitude: N26.25843\u00b0; verbatimLongitude: E117.73736\u00b0; Event: year: 2018; month: 7; Record Level: collectionCode: CJBHType status:Paratype. Occurrence: recordedBy: Liang Guo; individualCount: 1; sex: female; Location: country: CHINA; stateProvince: Fujian; verbatimLocality: Sanming City, Tianbaoyan Nature Reserve [\u4e09\u660e\u5e02\u5929\u5b9d\u5ca9\u81ea\u7136\u4fdd\u62a4\u533a]; verbatimElevation: 1100 m; Event: year: 2015; month: 6; day: 19; Record Level: collectionCode: CLGSType status:Paratype. Occurrence: recordedBy: Jian-Bin Huang; individualCount: 1; sex: male; Location: country: CHINA; stateProvince: Fujian; verbatimLocality: Ningde City, Gutian County, Shitashan [\u5b81\u5fb7\u5e02\u53e4\u7530\u53bf\u77f3\u5854\u5c71]; verbatimElevation: 1340 m; verbatimLatitude: N26.84247\u00b0; verbatimLongitude: E118.63524\u00b0; Event: year: 2020; month: 7; day: 9; Record Level: collectionCode: CJBHType status:Paratype. Occurrence: recordedBy: Peng-Yu Liu; individualCount: 1; sex: male; Location: country: CHINA; stateProvince: Fujian; verbatimLocality: Ningde City, Gutian County [\u5b81\u5fb7\u5e02\u53e4\u7530\u53bf]; verbatimElevation: 1550 m; Event: year: 2021; month: 7; day: 18; Record Level: collectionCode: CPYLType status:Paratype. Occurrence: recordedBy: Jian-Bin Huang; individualCount: 1; sex: female; Location: country: CHINA; stateProvince: Fujian; verbatimLocality: Quanzhou City, Daiyun Mountain, hiking trail [\u6cc9\u5dde\u5e02\u6234\u4e91\u5c71\u767b\u5c71\u6b65\u9053]; verbatimElevation: 1520 m; Event: year: 2021; month: 7; day: 4; Record Level: collectionCode: CLHCType status:Paratype. Occurrence: recordedBy: local people; individualCount: 1; sex: male; Location: country: CHINA; stateProvince: Guangxi; verbatimLocality: Nanning City, Wuming County, Damingshan [\u5357\u5b81\u5e02\u6b66\u9e23\u53bf\u5927\u660e\u5c71]; verbatimElevation: 1600 m; Event: year: 2005; month: 7; Record Level: collectionCode: CJBHType status:Paratype. Occurrence: recordedBy: local people; individualCount: 1; sex: male; Location: country: CHINA; stateProvince: Guangxi; verbatimLocality: Laibin City, Jinxiu County, Changtong Township, Daojiang Village, Pingbantun [\u6765\u5bbe\u5e02\u91d1\u79c0\u53bf\u957f\u578c\u4e61\u9053\u6c5f\u6751\u5e73\u529e\u5c6f]; verbatimElevation: 1375 m; verbatimLatitude: N24.09509\u00b0; verbatimLongitude: E110.18344\u00b0; Event: year: 2015; month: 7; day: 8; Record Level: collectionID: CLHCType status:Paratype. Occurrence: recordedBy: Chun-Fu Feng; individualCount: 1; sex: female; Location: country: CHINA; stateProvince: Guangxi; verbatimLocality: Laibin City, Jinxiu County, Changtong Township [\u6765\u5bbe\u5e02\u91d1\u79c0\u53bf\u957f\u578c\u4e61]; Event: year: 2020; month: 5; Record Level: collectionCode: CCZCType status:Paratype. Occurrence: recordedBy: local people; individualCount: 1; sex: female; Location: country: CHINA; stateProvince: Guangxi; verbatimLocality: Laibin City, Jinxiu County, Shengtangshan [\u6765\u5bbe\u5e02\u91d1\u79c0\u53bf\u5723\u5802\u5c71]; verbatimElevation: 1500 m; Event: year: 2018; month: 7; Record Level: collectionCode: CJBHType status:Paratype. Occurrence: recordedBy: Huang-Shun Xi; individualCount: 1; sex: male; Location: country: CHINA; stateProvince: Guangxi; verbatimLocality: Laibin City, Jinxiu County, Dayaoshan Mountain [\u6765\u5bbe\u5e02\u91d1\u79c0\u53bf\u5927\u7476\u5c71]; verbatimElevation: 1350 m; Event: year: 2018; month: 7; day: 2; Record Level: collectionCode: CTLHHolotype male. Body 28.6 mm long, widest just after elytral humeri (10.8 mm). Length of different body parts (mm): head (3.3), antenna (54.5), pronotum (5.1), elytra (20.4); width: head (5.9), pronotal apex (6.2), pronotal base (6.6), elytral humeri (10.1).Habitus . Due to the condition of different specimens, whitish or white pubescence or maculae may be distinct, vague or absent.Female paratypes. Body 34.8\u201335.6 mm long, widest just after elytral humeri (13.8 mm). Length of different body parts : head (3.9), antenna (56.1), pronotum (6.2), elytra (25.3); width: head (7.3), pronotal apex (7.6), pronotal base (8.2), elytral humeri (13.3). Antennomeres with length ratio from base to tip: 5.24 \u2013 1.00 \u2013 6.78 \u2013 5.88 \u2013 5.02 \u2013 4.39 \u2013 4.35 \u2013 4.11 \u2013 3.84 \u2013 3.66 \u2013 5.24.Similar to male in general appearance, but distinct by the following characters: body much larger Fig. C and D; Anoplophorahuangjianbinisp. n. is similar to A.imitator by the granules (or granulation) lacking on the anterior part of elytra much larger/longer and more dense on pronotum, elytra, metasternum and abdominal sternites III\u2013VII; besides, it also has large maculae around gena and frons and on mesepisternum (absent in A.huangjianbinisp. n.). Moreover, the new species is distinguished from its congeneric species by a combination of the following characters: abdominal tergite VII almost simply rounded at posterior margin Fig. A, A.eleg88) Fig. B, A.simi00) Fig. C, A.chia991 Fig. D and A.sgin Fig. A; sternigin Fig. B; spiculgin Fig. F; mediangin Fig. A short, gin Fig. A and B.The new species is dedicated to the collector of most type specimens, Mr. Jian-Bin Huang , an enthusiastic amateur entomologist. The name is a noun in the genitive case. \u201c\u5251\u658c\u661f\u5929\u725b (Pinyin: Jian Bin Xing Tian Niu)\u201d is proposed for the Chinese common name of this new species.CHINA .Habitat with broad-leaved mixed forest at Luoboding (Fujian) is shown in Fig."} +{"text": "Scientific Reports 10.1038/srep41564, published online 27 January 2017Correction to: Pantoea alhagisp. nov\u2019The Article contains an error in the Discussion section under subheading \u2018Description of T\u00a0(=CCTCC M 2016052T=KCTC 52262T), was isolated from surface-sterilized leaves of\u00a0Alhagi sparsifolia\u00a0collected from Taklamakan Desert in Xinjiang Uyghur Autonomous Region, north-west China. The 16S rRNA, ATP synthase beta subunit (atpD), DNA gyrase B subunit (gyrB), initiation translation factor 2 (infB) and RNA polymerase beta subunit (rpoB) gene sequences of strain LTYR-11ZT\u00a0have been deposited in GenBank/EMBL/DDBJ under the accession numbers KX494924-KX494928, respectively.\u2019\u2018The type strain, LTYR-11Zshould read:T\u00a0(=CCTCC AB 2019082T=KCTC 52262T), was isolated from surface-sterilized leaves of\u00a0Alhagi sparsifolia\u00a0collected from Taklamakan Desert in Xinjiang Uyghur Autonomous Region, north-west China. The 16S rRNA, ATP synthase beta subunit (atpD), DNA gyrase B subunit (gyrB), initiation translation factor 2 (infB) and RNA polymerase beta subunit (rpoB) gene sequences of strain LTYR-11ZT\u00a0have been deposited in GenBank/EMBL/DDBJ under the accession numbers KX494924-KX494928, respectively.\u2019\u2018The type strain, LTYR-11Z"} +{"text": "The correct name is: Emil F. Kendziorra. The correct citation is: Gillett CR, Brame T, Kendziorra EF (2021) Comprehensive survey of United States internet users\u2019 sentiments towards cryopreservation. PLoS ONE 16(1): e0244980."} +{"text": "Non-Coding RNA was able to maintain its standards for the high quality of its published papers. Thanks to the contribution of our reviewers, in 2021, the median time to first decision was 15 days and the median time to publication was 37 days. The editors would like to extend their gratitude and recognition to the following reviewers for their precious time and dedication, regardless of whether the papers they reviewed were finally published:Abhishek DeyLei ShiAlan DombkowskiLeonard LipovichAlessandro SammarcoLetizia PittoAlex De LencastreLin HuangAlexey NikulinLorenzo FarinaAlfonso M. CayotaLuca AgnelliAndr\u00e9 GerberLuca Lo PiccoloAndrea CaporaliLucia NatarelliAngel Vizoso-V\u00e1zquezLuke SelthAnisha GuptaMai HazekawaAnna RiesterMarco GaviraghiAntoine M. DujonMargarida Gama-CarvalhoAntonio MusioMaria DuhagonAria BaniahmadMaria Elizabeth Rossi Da SilvaArijita SarkarMaria HondeleArjen E. Van\u2019t HofMaria SundvallArturo L\u00f3pez CastelMarina CristoderoAssam El-OstaMartin SztachoBabu V. SajeshMartina KirchnerBei ChengMartina RossiBodhisattwa BanerjeeMatthias S. LeisegangByron BaronMatus SykoraCameron BrackenMd. Motiar RahmanCecilia BattistelliMichael TellierCelia Fern\u00e1ndez RubioMichele De BortoliCesar Lopez-CamarilloMohammed L. AbbaCharles Bou-NaderMunekazu YamakuchiChristine HappelNatalia SimionescuChristopher HellenNicholas DelihasChristos K. KontosNicolas CasadeiChristos PapaneophytouNicoletta BianchiClaudia KutterNithyananda ThorenoorCristian TaccioliOliver M\u00fchlemannDaniela GradiaOvidiu SirbuDavid C. ZappullaPablo SmircichDavid LeavesleyPanagiotis AlexiouDeepanjan PaulPaola OstanoEdoardo BertoliniPatricia Mirella ScarduaElena L\u00f3pez-Jim\u00e9nezPavel IvanovErica ScottPeter BaiEstanis NavarroPraveen AranyEsteban OrellanaPrzemys\u0142aw NucFederico Dajas-BailadorQinyu SunFrancisco J. EnguitaRajneesh SrivastavaFranck MartinRobert WeinzierlGaetano SantulliRocio T. Martinez NunezGianpiero Di LevaRoland KlassenGiulia MatacchioneRui FuGiuseppe PalmieriRussell HamiltonGiuseppina E. GriecoRyuya FukunagaGrasiele SausenSabyasachi DashGunter MeisterSamuel Pushparaj Robert JeyasinghHelen VincentSamuela PasqualiHenrik NielsenSatya KotaHiroji AibaS\u00e9bastien PfefferHisashi MeraShahrokh GhobadlooHua-Sheng ChiuShizuka UchidaHui LiShu-Ling TzengIlaria GuerrieroSilvia LeeIzabella Slezak-ProchazkaSimon ConnJan Nov\u00e1kSimon RaynerJarmila Hnilicov\u00e1Simona ZaamiJens HahneStefan Ernst SeemannJianhua WangStefano CagninJiao Jiao LiStephan BernhartJijun HuangStephan HamperlJoanna Sztuba-SolinskaSuba RajendrenJoanna WilliamsSumit MukherjeeJoe ChihadeTadashi YoshidaJos\u00e9 AndradeTanja KunejJuan Jos\u00e9 Gonz\u00e1lez-PlazaVasiliki TsigkouKatherine McJunkinXucheng HouKathrin TyryshkinYork MarahrensKausik ChakrabartiYoshihiro TaguchiLasse LindahlRigorous peer-reviews are the basis of high-quality academic publishing. Thanks to the great efforts of our reviewers,"} +{"text": "Correction to:BMC Genomics22, 618 (2021)https://doi.org/10.1186/s12864-021-07909-3Following publication of the original article , it was The correct versions of Fig. Additional file 1: Table S1. IEI Candidate Genes without Filter.Additional file 2: Table S2. IEI Candidate Genes with high (>.9) pLI Filter.Additional file 3: Table S3. IEI Candidate Genes with Average in all Immune Cells >\u20090 TPM Filter.Additional file 4: Table S4. IEI Candidate Genes with Average in B Cells >\u20091 TPM Filter.Additional file 5: Table S5. IEI Candidate Genes with Average in DCs\u2009>\u20091 TPM Filter.Additional file 6: Table S6. IEI Candidate Genes with Average in Myeloid Cells >\u20091 TPM Filter.Additional file 7: Table S7. IEI Candidate Genes with Average in NK Cells >\u20091 TPM Filter.Additional file 8: Table S8. IEI Candidate Genes with Average in T Cells >\u20091 TPM Filter."} +{"text": "Correction to: BMC Biology 20, 1\u201320 (2021)https://doi.org/10.1186/s12915-021-01206-xThe original article contained minor errors in Figs.\u00a01 and 3 which have both since been corrected."} +{"text": "Abstract, Results, final sentence: This sentence, which previously read:\u201cSport-specific lighter weighted bat swings and swing-specific isometrics resulted in improved subsequent competition weight bat swing velocities, ranging from ~\u20091.3\u20133.3%; ES\u2009=\u20090.16\u20130.57.\u201dshould read:\u201cSport-specific lighter weighted bat swings and swing-specific isometrics resulted in improved subsequent competition weight bat swing velocities, ranging from ~\u20091.3\u20134.9%; ES\u2009=\u20090.16\u20130.57.\u201dThe original article has been corrected."} +{"text": "Following publication of the original article , two typOriginal formula 6: Correct formula 6: Original formula 19: Correct formula 19: The original article has been"} +{"text": "Erratum zu:Hautarzt 202210.1007/s00105-022-04954-1Im Originalbeitrag war Tab.\u00a010\u202f%.Die Filterkonzentration von Tris-biphenyl Triazine (nano) (TBPT) mEk ist Die korrigierte Tabelle finden Sie im Folgenden.Der Originalbeitrag wurde korrigiert."} +{"text": "Scientific Reportshttps://doi.org/10.1038/s41598-021-04610-z, published online 11 January 2022Correction to: The original version of this Article contained an error in Funding information.\u201cThe RELICO study has been sponsored by Menarini Hellas S.A.\u201dnow reads:\u201cThe SLEPICO study has been sponsored by Menarini Hellas S.A.\u201dThe original Article has been corrected."} +{"text": "Erick H. Turner notes th"} +{"text": "Enterobacter sakazakii is an opportunistic pathogen that can cause infections such as necrotizing enterocolitis, bacteraemia, meningitis and brain abscess/lesions. When the species was defined in 1980, 15 biogroups were described and it was suggested that these could represent multiple species. In this study the taxonomic relationship of strains described as E. sakazakii was further investigated.E. sakazakii were divided into separate groups on the basis of f-AFLP fingerprints, ribopatterns and full-length 16S rRNA gene sequences. DNA-DNA hybridizations revealed five genomospecies. The phenotypic profiles of the genomospecies were determined and biochemical markers identified.Strains identified as E. sakazakii and proposes a reclassification of these organisms.This study clarifies the taxonomy of Enterobacter sakazakii was defined as a new species in 1980 by Farmer et al , adaptor: 5'-CTC GTA GAC TGC GTA CC-3'; 3'-CTG ACG CAT GGT TAA-5'. Restriction enzyme: Taq I [tetracutter], adaptor: 5'-GAC GAT GAG TCC TGA C-3'; 3'-TAC TCA GGA CTG GC-5'. Selective amplification of the restriction fragments was performed using the following primer combination: E01: 5'-GAC TGC GTA CCA ATT CA-3'; T01: 5'-CGA TGA GTC CTG ACC GAA-3'.DNA was prepared using the method of Gevers et al . PurifieAmplicons were separated according to their length on a high resolution polyacrylamide gel using a DNA sequencer (ABI 377) and visualized by the 5'-end labelling of the 6-bp cutter with the fluorescent dye FAM. The resulting electrophoretic patterns were tracked and normalized using the GeneScan 3.1 software . Normalized tables of peaks, containing fragments of 50 to 536 base pairs, were transferred into the BioNumerics\u2122 4.5 software . For numerical analysis, data intervals were delineated between the 75- and 500-bp bands of the internal size standard. Clustering of the BioNumerics\u2122 4.5 software generated AFLP\u2122 DNA fingerprint patterns was performed using the Dice coefficient and the UPGMA algorithm with an optimization of 0% and position tolerance of 0.2%.EcoR1 restriction enzyme. Riboprint patterns were downloaded to Bionumerics v.4.5, a UPGMA dendogram was constructed using a DICE coefficient with an optimization of 1% and a position tolerance of 1.5%.Ribotyping was performed using the automated RiboPrinter\u2122 Microbial Characterization System . Isolates were grown on TSA and prepared according to standard procedures using th2O. PCR-ready DNA was prepared by washing 10 \u03bcl of the concentrated culture with 200 \u03bcl H2O and resuspending the pellet in 50 \u03bcl Prepman Ultra (Applied Biosystems). The cells were heated at 99\u00b0C for 10 min. Ribosomal 16S rRNA gene was amplified from 1 \u03bcl of PCR-ready DNA using the high-fidelity polymerase PrimeStar (Takara) and the primers P0 and P6 in the presence of betaine: 3 min at 95\u00b0C; 30 cycles of 30 sec at 95\u00b0C, 30 sec at 56\u00b0C, 2 min at 72\u00b0C; 5 min at 72\u00b0C. P0 \u2013 5'-AGA GTT TGA TCC TGG CTC AG-3'; P6 \u2013 5'-GTA CGG CTA CCT TGT TAC GA-3'. The PCR was carried out in 3 \u00d7 15 \u03bcl, which were subsequently pooled. After purification the amplified fragments were sequenced using the primers P0 and P6. Some templates produced a double sequence close to the P0 site, these were sequenced using another primer that binds in the reverse orientation to a conserved region in the middle of the 16S region (095P: 5'-TAC GGC GTG GAC TAC CAG-3'). The new reactions produced a double sequence less than 80 bases from the end of the PCR fragment (P0 site). Additional sequences were downloaded from EMBL database.16S rRNA gene sequencing was performed by Fasteris SA, Switzerland. Isolates were grown 18 h in 5 ml BHI at 37\u00b0C and 1 ml centrifuged at 13,000 g for 5 min, the pellet was washed with 1 ml HFull-length 16S rRNA gene sequences (> 1300 bases) were added to an ARB alignment of about 28,000 small-subunit rRNA sequences by using the alignment tool ARB_EDIT of the ARB program package . AlignmeEF059844], ATCC 29004 [GenBank: EF059868], ATCC 29544T [GenBank: EF059843], ATCC 51329 [GenBank: EF059845], CDC 1716-77 [GenBank: EF059883], CDC 3128-77 [GenBank: EF059882], CDC 3523-75 [GenBank: EF059875], CDC 5960-70 [GenBank: EF059874], E265 [GenBank: AY803191], E266 [GenBank: AY803190], E269 [GenBank: EF059819], E271 [GenBank: EF059820], E272 [GenBank: EF059821], E274 [GenBank: EF059822], E280 [GenBank: EF059823], E283 [GenBank: EF059824], E292 [GenBank: EF059825], E302 [GenBank: EF059826], E309 [GenBank: EF059827], E314 [GenBank: EF059828], E328 [GenBank: EF059829], E393 [GenBank: AY752941], E413 [GenBank: EF059830], E423 [GenBank: EF059831], E429 [GenBank: EF059832], E436 [GenBank: EF059833], E440 [GenBank: EF059834], E444 [GenBank: EF059835], E450 [GenBank: EF059836], E456 [GenBank: EF059837], E465 [GenBank: EF059839], E468 [GenBank: AY752942], E488 [GenBank: EF059840], E531 [GenBank: EF059842], E604 [GenBank: EF059846], E607 [GenBank: EF059847], E609 [GenBank: EF059848], E616 [GenBank: EF059849], E620 [GenBank: EF059850], E621 [GenBank: EF059851], E622 [GenBank: EF059852], E624 [GenBank: EF059853], E625 [GenBank: EF059854], E626 [GenBank: EF059855], E627 [GenBank: EF059856], E632 [GenBank: EF059857], E639 [GenBank: EF059858], E644 [GenBank: EF059859], E676 [GenBank: EF059860], E680 [GenBank: EF059861], E681 [GenBank: EF059862], E688 [GenBank: EF059863], E694 [GenBank: EF059864], E717 [GenBank: EF059865], E739 [GenBank: EF059867], E757 [GenBank: EF059869], E761 [GenBank: EF059870], E768 [GenBank: EF059871], E769 [GenBank: EF059872], E775 [GenBank: EF059873], E814 [GenBank: EF059880], E872 [GenBank: EF059884], E883 [GenBank: EF059886], E888 [GenBank: EF059887], E890 [GenBank: EF059888], E895 [GenBank: EF059889], E904 [GenBank: EF059890], CFS237 LMG 23823 [GenBank: EF059892], LMG 23824 [GenBank: EF059841], E464 LMG 23825 [GenBank: EF059838], CDC 1058-77 LMG 23826 [GenBank: EF059881], 3032 LMG 23827 [GenBank: EF059891], M609 [GenBank: EF059885], NCTC 8155 [GenBank: EF059866], NCTC 9238 [GenBank: EF059876], NCTC 9529 [GenBank: EF059877], NCTC 9844 [GenBank: EF059878], NCTC 9846 [GenBank: EF059879], z1084 [GenBank: AY803192], z759 [GenBank: AY752939], z858 [GenBank: AY752936], z954 [GenBank: AY752938], zES11 [GenBank: AY803187], zES4 [GenBank: AY803186], zESVO7 [GenBank: AY803189].The accession numbers of the 16S rRNA genes included in this study are as follows: ATCC 12868 [GenBank: CI performed the biochemical profiling and collated the data. AL and RS analyzed the 16S rRNA gene sequences. NM and SF performed the statistical analysis of the phenotypic data. EB and JM performed the Ribotyping and analysis. IC is responsible for the f-AFLP analysis at the BCCM/LMG Bacteria Collection, where the AFLP analyses were performed. HJ managed the project and edited the manuscript. All authors contributed to the text and read and approved the final manuscript.E. sakazakii biogroups.Description of Click here for file"} +{"text": "Life maintains high quality standards for its published papers. In 2017, a total of 51 papers were published in the journal. Thanks to the cooperation of our reviewers, the median time to first decision was 21 days and the median time to publication was 51 days. The editors would like to express their sincere gratitude to the following reviewers for their time and dedication in 2017:Peer review is an essential part in the publication process, ensuring that Aaron EngelhartLiam M. LongoAaron S. BurtonLinda McGownAdam KunLisa SteinAddy ProssLoren WilliamsAlexander BolshoyLuc JaegerAna CrnkovicLukasz DziewitAndre BrackMarc TesseraAndr\u00e9s de la EscosuraMarc T. FacciottiAndrew J. SurmanMarie-Christine, MaurelArmen MulkidjanianMarkus RalserArturo BecerraMartha A. GroverBal\u00e1zs K\u00f6nny\u0171Massimo Di GiulioBarry SavilleMatthew PasekBonnie BaxterMatthew PownerBrian J. CaffertyMichael BlaberBrian R. FrancisMichael HechtCarl DevitoMichael RussellCarl TrindleMichael SchweizerChandra WickramasingheMichele FioreCharles LiottaMike Dyall-SmithCharles Jr. CarterMikhail M. Vorob\u2019evChristian MayerMin GuoChristoph A. WeberMV JoseChristophe LavelleNam-Joon ChoChunliang LiuNediljko BudisaConstantinos StathopoulosNiles LehmanCynthia M. MortonNils HolmDave SpeijerNobuhiko SuematsuDavid PennyPasquale StanoDavid S. RossPatrick O\u2019DonoghueDennis G\u00f6rlichPaul ClarkeDiego Luis GonzalezPaul HiggsDino MorasPeter StrazewskiDuncan ForganPeter UnrauEliana Beltr\u00e1n-PardoPeter WaldeElias ChatzitheodoridisPeter R. WillsEmanuele ZonaroRafal M. WieczorekEnver Cagri IzguRaffaele SaladinoFalcinelli StefanoRainer MerklFengjie SunRalf M\u00f6llerFilipa L. SousaRichard EgelFilippo CascheraRichard P. FahlmanGabriel ZentnerRob M. De GraafGeoff WildRobert PascalGeorge FoxRobert Root-BernsteinGeorgy KarevRomeu GuimaraesGreg SpringsteenRosanna del GaudioGregory FournierRussell F. DoolittleHarold S. BernhardtSandra PizzarelloHelen HansmaSatoshi AkanumaHerv\u00e9 SeligmannSheref MansyHodaka FujiiShin-ichi YokoboriHubert Dominique BeckerSimone GianneriniHussein KaddourSolveig TosiHyman HartmanSreejith RamakrishnanIgor ShuryakStefan FoxJack T. O\u2019Malley-JamesStuart BartlettJason RaymondSzymon WasikJiann-Ruey HongTakahiro HohsakaJiri \u0160ponerTetsuo KushiroJoanne K. HobbsTetsushi SakumaJohn YinThomas GeorgelinJordi Pi\u00f1eroThomas SimonsonJuan Manuel Garc\u00eda-RuizThomas C. BoothbyKaren BellThomas M. OrlandoKelly SheppardTony JiaKenji IkeharaUlrich MullerKenneth A. DillVeronica VaidaKensaku SakamotoWilliam BainsKepa Ruiz-MirazoWilliam H. McClainKoichiro MatsunoYoshihiro FurukawaKumar Saurabh SinghYu-Fen ZhaoKunio KawamuraA.V. KorotayevLeonardo Sorci"} +{"text": "Nature Communications8 Article number:14193 ; DOI: 10.1038/ncomms14193 (2017); Published 01312017; Updated 04062017This Article contains errors in"} +{"text": "Scientific Reports6: Article number: 3605510.1038/srep36055; published online: 11032016; updated: 12192016This Article contains an error in the legend of Table 1.\u201cValues indicate absolute log2-FC.\u201dshould read:\u201cValues indicate absolute fold-change.\u201d"} +{"text": "Scientific Reports4: Article number: 7621; 10.1038/srep07621 published online: 12232014; updated: 06012017.This article was originally published under a CC BY-NC-SA 4.0 license, but has now been made available under a CC BY 4.0 license. The PDF and HTML versions of the paper have been modified accordingly."} +{"text": "Scientific Reports7:205; doi:10.1038/s41598-017-00324-3; Article published online 16 March 2017This Article contains an error in Figure 5, where \u2018T1 (true negative)\u2019 should read:\u201cCountry reports LOWER ecological status & model predicts LOWER ecological status\u201d.The correct Figure 5 appears below as Figure"} +{"text": "Nucleic Acids Res (2015) 43 (6): 2993\u20133011. doi:10.1093/nar/gkv162The Authors wish to apologise for an error in Figure"} +{"text": "Scientific Reports6: Article number: 34023; 10.1038/srep34023 published online: 09302016; updated: 06212017.k should read k.This Article contains typographical errors, where all instances of \\bm"} +{"text": "Scientific Reports4: Article number: 5703; 10.1038/srep05703 published online: 07162014; updated: 01312017.This article was originally published under a CC BY-NC-SA 4.0 license, but has now been made available under a CC BY 4.0 license. The PDF and HTML versions of the paper have been modified accordingly."} +{"text": "Scientific Reports6: Article number: 2285810.1038/srep22858; published online: 03092016; updated: 02132017L) and right (\u03b8R) angles are incorrectly defined. The correct This Article contains errors in"} +{"text": "Scientific Reports6: Article number: 23473; 10.1038/srep23473 published online: 04012016; updated: 12142016.In this Article, the legend of Figure 5 is incorrect:a) BlCel5B in the crystallographic and closed configuration; (b) Bacillus halodurans Cel5B (BhCel5B) (PDB id: 4V2X) (c) Piromyces rhizinflata GH5 endoglucanase (PDB id: 3AYR); (d) Clostridium cellulolyticum GH5 endoglucanase (PDB id: 1EDG); (e) Clostridium cellulovorans GH5 endoglucanase (PDB id: 3NDY); (f) Bacteroides ovatus GH5 xyloglucanase (PDB id: 3ZMR); (g) Paenibacillus pabuli GH5 xyloglucanase (PDB id: 2JEP); (h) Prevotella bryantii GH5 endoglucanase (PDB id: 3VDH); (i) Ruminiclostridium thermocellum multifunctional GH5 cellulase, xylanase and mannase (PDB id: 4IM4); (j) Bacteroidetes bacterium AC2a endocellulase (PDB id: 4YHE). BlCel5B in the crystallographic and closed configuration; (b) Bacillus halodurans Cel5B (BhCel5B) (PDB id: 4V2X) (c) Piromyces rhizinflata GH5 endoglucanase (PDB id: 3AYR); (d) Clostridium cellulolyticum GH5 endoglucanase (PDB id: 1EDG); (e) Clostridium cellulovorans GH5 endoglucanase (PDB id: 3NDY); (f) Bacteroides ovatus GH5 xyloglucanase (PDB id: 3ZMR); (g) Paenibacillus pabuli GH5 xyloglucanase (PDB id: 2JEP); (h) Prevotella bryantii GH5 endoglucanase (PDB id: 3VDH); (i) Ruminiclostridium thermocellum multifunctional GH5 cellulase, xylanase and mannase (PDB id: 4IM4); (j) Bacteroidetes bacterium AC2a endocellulase (PDB id: 4YHE).("} +{"text": "Scientific Reports6: Article number: 2818610.1038/srep28186; published online: 06242016; updated: 09222016D (micromolar)\u2019 is incorrectly given as \u2018KD (millimolar)\u2019. The correct Figure 10D appears below as This Article contains an error in Figure 10D, where the y-axis \u2018K"} +{"text": "The correct name is: C. Raina MacIntyre. The correct citation is: Bui CM, Gardner L, MacIntyre CR, Sarkar S (2017) Influenza A H5N1 and H7N9 in China: A spatial risk analysis. PLoS ONE 12(4): e0174980."} +{"text": "In the original publication two authThe original article was updated to rectify these errors.Incorrect author name 1:Stephanie Combs.Correct author name 1:Stephanie E. Combs.Incorrect author name 2:Andra Krauze.Correct author name 2:Andra V. Krauze."} +{"text": "Nature Communications5: Article number: 3359; DOI: 10.1038/ncomms4359 (2014); Published: 02282014; Updated: 04202017This Article was originally published under a CC BY-NC-ND 3.0 license, but has now been made available under a CC BY 4.0 license. The PDF and HTML versions of the paper have been modified accordingly."} +{"text": "Scientific Reports 10.1038/s41598-017-02143-y, published online 18 May 2017Correction to: This Article contains typographical errors in the Supplementary Information file.In Equation S1,should read:In Equation S8,should read:In Equation S13,should read:And finally,\u2018By using equation (S5), Should read:\u2018By using equation (S5),"} +{"text": "Scientific Reports6: Article number: 35462; 10.1038/srep35462 published online: 11202016; updated: 03222017.This Article contains errors in Figure 5B where the CFU values for T0 and T1 are incorrect. The correct Figure 5B appears below as"} +{"text": "Scientific Reports7: Article number: 4596110.1038/srep45961; published online: 04122017; updated: 03092017This Article contains a typographical error in the Methods section under subheading \u2018Data Availability. Associated Article\u2019,\u201cProtocol Exchange doi: 1038/Protex.2017.039 (2017)\u201d.should read:\u201cProtocol Exchange doi: 10.1038/protex.2017.039\u201d."} +{"text": "MRI revealed unilateral basal ganglia and brainstem lesions . Eightee1 . A heter"} +{"text": "Scientific Data4:170078 doi: 10.1038/sdata.2017.78 (2017); Published 20 June 2017; Updated 17 April 2018."} +{"text": "Int J Epidemiol, 2016, doi: https://doi.org/10.1093/ije/dyw233First published online: 5 November 2016, A funding statement was missing for author David Stuckler. The funding statement should have included \u201cDS is funded by ERC HRES 313590.\u201d"} +{"text": "Correction to:Translational Psychiatry (2017) 7, e1164; doi:10.1038/tp.2017.142; published online 4 July 2017The ninth author\u2019s name was presented incorrectly. It should appear as J Quevedo."} +{"text": "Virus-triggered effects were associated with an enhanced activation of CD4+ T helper cells and CD8+ cytotoxic T lymphocytes and augmented cytokine expression. By contrast, (iii) IL-10R neutralization during chronic TMEV-infection was not associated with enhanced peripheral immunopathology but an increased CD3+ T cell influx in the spinal cord. IL-10R neutralization causes a breakdown in peripheral immune tolerance in genetically predisposed mice, which leads to immune-mediated colitis, resembling inflammatory bowel disease. Hyperactive immune state following IL-10R blockade is enhanced by central nervous system-restricted viral infection in a disease phase-dependent manner.Theiler\u00b4s murine encephalomyelitis virus (TMEV)-infection is a widely used animal model for studying demyelinating disorders, including multiple sclerosis (MS). The immunosuppressive cytokine Interleukin (IL)-10 counteracts hyperactive immune responses and critically controls immune homeostasis in infectious and autoimmune disorders. In order to investigate the effect of signaling via Interleukin-10 receptor (IL-10R) in infectious neurological diseases, TMEV-infected SJL mice were treated with IL-10R blocking antibody (Ab) in the acute and chronic phase of the disease. The findings demonstrate that (i) Ab-mediated IL-10 neutralization leads to progressive colitis with a reduction in Foxp3 Briefly\u00ae performance tests for quantifying motor coordination deficits were performed by two examiners in a non-blinded manner as previously described and group \u201cIL10R\u2193late/TMEV\u201d [experiment II]). TMEV = Theiler\u00b4s murine encephalomyelitis virus; dpi = days post infection; IL = interleukin; TNF = tumor necrosis factor; TGF = transmissible growth factor; INF = interferon; Foxp3 = forkhead box P3 protein; MBP = myelin basic protein; np-NF = non-phosphorylated neurofilament; gMFI = geometric mean of fluorescence intensity; ND = not determined; units: 1 = rounds per minute; 2 = points (semiquantitative scoring); 3 = gram; 4 = number of labelled cells in the spinal cord; 5 = percentage [%] of MBP-unstained (demyelinated) area; 6 = number of labelled axons in the spinal cord; 7 = copy numbers; 8 = percentage [%] of labelled cells. Table D. Summary of data obtained from animals receiving TMEV-infection and intraperitoneal application of IgG1-specific isotype control (group \u201cisotypeearly/TMEV\u201d [experiment I] and group \u201cisotypelate/TMEV\u201d [experiment II]). TMEV = Theiler\u00b4s murine encephalomyelitis virus; dpi = days post infection; IL = interleukin; TNF = tumor necrosis factor; TGF = transmissible growth factor; INF = interferon; Foxp3 = forkhead box P3 protein; MBP = myelin basic protein; np-NF = non-phosphorylated neurofilament; gMFI = geometric mean of fluorescence intensity; ND = not determined; units: 1 = rounds per minute; 2 = points (semiquantitative scoring); 3 = gram; 4 = number of labelled cells in the cord; 5 = percentage [%] of MBP-unstained (demyelinated) area; 6 = number of labelled axons in the spinal cord; 7 = copy numbers; 8 = percentage [%] of labelled cells. Table E. Summary of data obtained from animals receiving mock-infection and intraperitoneal application of anti-IL-10R antibody (group \u201cisotypeearly/mock\u201d [experiment I] and group \u201cisotypelate/mock\u201d [experiment II]). TMEV = Theiler\u00b4s murine encephalomyelitis virus; dpi = days post infection; IL = interleukin; TNF = tumor necrosis factor; TGF = transmissible growth factor; INF = interferon; Foxp3 = forkhead box P3 protein; MBP = myelin basic protein; np-NF = non-phosphorylated neurofilament; gMFI = geometric mean of fluorescence intensity;ND = not determined; units: 1 = rounds per minute; 2 = points (semiquantitative scoring); 3 = gram; 4 = number of labelled cells/axons in the spinal cord; 5 = percentage [%] of MBP-unstained (demyelinated) area; 6 = copy numbers; 7 = percentage [%] of labelled cells. experiment II: effects of IL-10R blockade during chronic Theiler\u2019s murine encephalomyelitis.Table F. Summary of statistical analyses of arrow = significant up(\u2191)- or downregulation (\u2193) in TMEV-infected SJL mice receiving anti-IL-10R Ab compared with mice receiving IgG1 specific isotype control in the chronic phase of the disease; administration of Ab or isotype control respectively was performed at 35 and 42 dpi; * bold p-values = significant difference between both groups, p\u2009\u2264\u20090.05, determined by Wilcoxon\u00b4s rank sum-tests. TMEV = Theiler\u00b4s murine encephalomyelitis virus; dpi = days post infection; IL = interleukin; TNF = tumor necrosis factor; TGF = transmissible growth factor; INF = interferon; Foxp3 = forkhead box P3 protein; MBP = myelin basic protein; np-NF = non-phosphorylated neurofilament; gMFI = geometric mean of fluorescence intensity; ND = not determined. experiment II: effects of chronic Theiler\u2019s murine encephalomyelitis virus infection upon IL-10R blockade.Table G. Summary of statistical analyses of arrows = significant up-regulation (\u2191) or down-regulation (\u2193) in infected SJL mice receiving IL-10R Ab compared to non-infected mice receiving anti-IL-10R Ab during the chronic infection phase (35 and 42 dpi); * bold p-values display significant differences between both groups (p\u2009\u2264\u20090.05) determined by Wilcoxon\u00b4s rank sum-tests. TMEV = Theiler\u00b4s murine encephalomyelitis virus; dpi = days post infection; Foxp3 = forkhead box P3 protein; MBP = myelin basic protein; np-NF = non-phosphorylated neurofilament; gMFI = geometric mean of fluorescence intensity. experiment III: effects of IL-10R blockade in non-infected animals.Table H. Summary of statistical analyses of arrow = significant up(\u2191)- or downregulation (\u2193) in SJL mice receiving anti-IL-10R Ab compared with mice receiving IgG1 specific isotype only; administration of Ab or isotype control, respectively, was performed at day 0, 7 and 14; * bold p-values = significant difference between both groups, p\u2009\u2264\u20090.05, determined by Wilcoxon\u00b4s rank sum-tests. Foxp3 = Iba-1 = ionized calcium binding adaptor molecule 1; forkhead box P3 protein; gMFI = geometric mean of fluorescence intensity. Table I. Summary of data obtained from non-infected animals receiving intraperitoneal application of anti-IL10R Ab . * given are median values (minimum and maximum); gMFI = geometric mean of fluorescence intensity; units: 1 = points (semiquantitative scoring); 2 = number of labelled cells in the lamina propria per high power field, 3 = percentage [%] of labelled cells. Table J. Summary of data obtained from non-infected animals receiving intraperitoneal application of IgG1-specific isotype control . * given are median values (minimum and maximum); gMFI = geometric mean of fluorescence intensity; units: 1 = points (semiquantitative scoring); 2 = number of labelled cells in the lamina propria per high power field; 3 = percentage [%] of labelled cells.(DOC)Click here for additional data file."} +{"text": "Scientific Reports7: Article number: 4261610.1038/srep42616; published online: 02172017; updated: 05092017This Article contains errors in Figure 7. The correct Figure 7 appears below as"} +{"text": "Scientific Reports5: Article number: 937810.1038/srep09378; published online: 03232015; updated: 12092016This acknowledgments section in this Article is incomplete:\u201cThis work was supported by National Science Centre (project grant no. UMO-2011/01/B/NZ1/03686 and N N301 668540)\u201d.should read:\u201cThis work was supported by National Science Centre (project grant no. UMO-2011/01/B/NZ1/03686 and N N301 668540). Marta Moskot was funded by National Science Centre project grant no. UMO-2014/12/T/NZ2/00538\u201d."} +{"text": "Nature Communications6: Article number: 7021; DOI: 10.1038/ncomms8021 (2015); Published 04242015; Updated 02072017This Article was originally published under a CC BY-NC-ND 4.0 license, but has now been made available under a CC BY 4.0 license. The PDF and HTML versions of the paper have been modified accordingly."} +{"text": "Scientific Reports6: Article number: 21788; 10.1038/srep21788 published online: 02232016; updated: 09132017.Bi (eV/K)\u2019 should read \u2018Bi (meV/K)\u2019.This Article contains an error in Table 1, where \u2018"} +{"text": "Scientific Reports6: Article number: 31896; 10.1038/srep31896 published online: 07222016; updated: 07272017.This Article contains errors. In the \u2018Thermal Stability: Non-Ambient X-ray Diffraction\u2019 section, under subheading \u2018Thermodynamic stability: Knudsen effusion mass spectrometry and Knudsen effusion mass loss\u2019,3, MAPbBr3 and MAPbI3, respectively\u201d.\u201cThe resulting values of should read:3, MAPbBr3 and MAPbI3, respectively\u201d.\u201cThe resulting values of In Equation (13),\u201cshould read:\u201cIn Equation (14),\u201cshould read:\u201cIn Equation (15),\u201cshould read:\u201c"} +{"text": "Healthcare-associated infections and antibiotic resistance\u00a0Point Prevalence Survey of Hospital Acquired Infections and Antimicrobial Use in Ireland 2017Epi-Insight 2017;18(3)March 2017, Irelandhttp://ndsc.newsweaver.ie/epiinsight/1kxgb6ek1nx?a=1&p=51521198&t=17517774\u00a0Staphylococcus aureus (LA-MRSA) leads to better patient careAdaptation of isolation measures in case of livestock-associated multiresistant Infectieziekten Bulletin 2017; 28(2)24 February 2017, the Netherlandshttp://www.rivm.nl/dsresource?objectid=ab0ce6b4-98d2-4a37-a5e0-be92adfab4f8&type=pdf&disposition=inline\u00a0Staphylococcus aureus on pig farmsMultiresistant Infectieziekten Bulletin 2017; 28(2)24 February 2017, the Netherlandshttp://www.rivm.nl/dsresource?objectid=fcaeeac9-5fde-4d54-87d9-e521780685e5&type=pdf&disposition=inline\u00a0Staphylococcus aureusGenetic exchange between multiresistant Infectieziekten Bulletin 2017; 28(2)24 February 2017, the Netherlandshttp://www.rivm.nl/dsresource?objectid=f6a62f77-9997-4930-8dca-febd780fe65c&type=pdf&disposition=inline\u00a0Antibiotic resistance in gastrointestinal bacteriaInfectieziekten Bulletin 2017; 28(1)24 January 2017, the Netherlandshttp://www.rivm.nl/dsresource?objectid=9d23345f-3449-45f7-9934-021076cf3bd9&type=pdf&disposition=inline\u00a0National Annual Antimicrobial Point Prevalence Survey 2016Epi-Insight 2016;17(12)December 2016, Irelandhttp://ndsc.newsweaver.ie/epiinsight/kgky01jxfir?a=1&p=51164834&t=17517774\u00a0Food- and waterborne diseases\u00a0Authorities are investigating new cases of EHEC infectionFolkh\u00e4lsomyndigheten website13 March 2017, Swedenhttps://www.folkhalsomyndigheten.se/nyheter-och-press/nyhetsarkiv/2017/mars/myndigheter-utreder-nya-fall-av-ehec-infektion/\u00a0Clostridium difficile ribotype 176 in GermanyOccurrence of Epidemiologisches Bulletin 10, 20179 March 2017, Germanyhttps://www.rki.de/DE/Content/Infekt/EpidBull/Archiv/2017/Ausgaben/10_17.pdf?__blob=publicationFile\u00a0Campylobacter in chicken meatNeed to reduce Folkh\u00e4lsomyndigheten website10 February 2017, Swedenhttps://www.folkhalsomyndigheten.se/nyheter-och-press/nyhetsarkiv/2017/februari/campylobacter-pa-kycklingkott-behover-minska/\u00a0Salmonella and Campylobacter infectionsAnnual summary of HPS Weekly Report 2017; 51(05)7 February 2017, Scotlandhttp://www.hps.scot.nhs.uk/documents/ewr/pdf2017/1705.pdf\u00a0Campylobacter infection among a group of youngstersA cluster of Infectieziekten Bulletin 2017; 28(1)24 January 2017, the Netherlandshttp://www.rivm.nl/dsresource?objectid=71b5ca01-ba17-42f9-9d27-12460ee4b38e&type=pdf&disposition=inline\u00a0Listeria monocytogenes in the Netherlands in 2015Surveillance of Infectieziekten Bulletin 2017; 28(1)24 January 2017, the Netherlandshttp://www.rivm.nl/dsresource?objectid=47e55962-7d01-4ae6-8b2d-edba4f88a40e&type=pdf&disposition=inline\u00a0Salmonella typhimurium infection in infants after a trip to a farmVlaams Infectieziektebulletin; 4/2016December 2016, Belgiumhttps://www.zorg-en-gezondheid.be/sites/default/files/atoms/files/2016-4-salmon-T-CH.%20Lambrechts.pdf\u00a0Hepatitides\u00a0Acute and chronic Hepatitis C, 2016EPI-NEWS 11, 201715 March 2017, Denmarkhttp://www.ssi.dk/Aktuelt/Nyhedsbreve/EPI-NYT/2017/Uge%2011%20-%202017.aspx\u00a0Acute and chronic Hepatitis B 2016EPI-NEWS 9, 20171 March 2017, Denmarkhttp://www.ssi.dk/Aktuelt/Nyhedsbreve/EPI-NYT/2017/Uge%209%20-%202017.aspx\u00a0Hepatitis B infection in Scotland: 2015HPS Weekly Report 2017; 51(02)17 January 2017, Scotlandhttp://www.hps.scot.nhs.uk/documents/ewr/pdf2017/1702.pdf\u00a0Vaccine-preventable diseases\u00a0Invasive meningococcal disease (laboratory reports in England): England: October to December 2016Health Protection Report; 11(8)24 February 2017, United Kingdomhttps://www.gov.uk/government/uploads/system/uploads/attachment_data/file/594800/hpr0817_imd.pdf\u00a0Pertussis vaccination programme for pregnant women update: vaccine coverage in England, October to December 2016Health Protection Report; 11(8)24 February 2017, United Kingdomhttps://www.gov.uk/government/uploads/system/uploads/attachment_data/file/594799/hpr0817_prntl-prtsss-VC.pdf\u00a0Examination of a pertussis outbreak in children with high vaccination rates in Kiel, October 2015-March 2016Epidemiologisches Bulletin 6, 20179 February 2017, Germanyhttps://www.rki.de/DE/Content/Infekt/EpidBull/Archiv/2017/Ausgaben/06_17.pdf?__blob=publicationFile\u00a0Increase in the incidence of invasive meningococcal disease caused by group W135EPI-NEWS 6, 20178 February 2017, Denmarkhttp://www.ssi.dk/Aktuelt/Nyhedsbreve/EPI-NYT/2017/Uge%206%20-%202017.aspx\u00a0Shingles vaccine coverage report, England, September to November 2016Health Protection Report; 11(4)27 January 2017, United Kingdomhttps://www.gov.uk/government/uploads/system/uploads/attachment_data/file/586837/hpr0417_shngls.pdf\u00a0Sudden increase of invasive meningococcal disease serogroup W in 2015 and 2016Infectieziekten Bulletin 2017; 28(1)24 January 2017, the Netherlandshttp://www.rivm.nl/dsresource?objectid=9fcfee07-c3dc-4af8-9437-7e328a66469c&type=pdf&disposition=inline\u00a0Measles, mumps and rubella and childhood vaccine uptakeHPS Weekly Report 2017; 51(01)10 January 2017, Scotlandhttp://www.hps.scot.nhs.uk/documents/ewr/pdf2017/1701.pdf\u00a0New childhood immunisation scheduleEpi-Insight 2017;18(1)January 2017, Irelandhttp://ndsc.newsweaver.ie/epiinsight/uc4nexz6bh2?a=1&p=51284091&t=17517774\u00a0Cases of measles in North ZealandEPI-NEWS 1/2, 201711 January 2017, Denmarkhttp://www.ssi.dk/Aktuelt/Nyhedsbreve/EPI-NYT/2017/Uge%201-2%20-%202017.aspx\u00a0Laboratory confirmed cases of pertussis reported to the enhanced pertussis surveillance programme in England during July to September 2016Health Protection Report; 10(44)16 December 2016, United Kingdomhttps://www.gov.uk/government/uploads/system/uploads/attachment_data/file/578804/hpr4416_prtsss.pdf\u00a0Invasive meningococcal disease (laboratory reports in England): England: July to September 2016Health Protection Report; 10(44)16 December 2016, United Kingdomhttps://www.gov.uk/government/uploads/system/uploads/attachment_data/file/578806/hpr4416_imd.pdf\u00a0National measles outbreak in Ireland declared overEpi-Insight 2016;17(12)December 2016, Irelandhttp://ndsc.newsweaver.ie/epiinsight/y461qge49cz?a=1&p=51143038&t=17517774\u00a0Respiratory diseases\u00a0Legionella pneumonia and Pontiac fever at a campsite in CroatiaInfectieziekten Bulletin 2017; 28(3)23 March 2017, the Netherlandshttp://www.rivm.nl/dsresource?objectid=b2fe27ea-1c7a-4378-9be2-8d44f0262d04&type=pdf&disposition=inline\u00a0Tuberculosis epidemiology in France in 2015. Impact of the suspension of BCG mandatory vaccination on child tuberculosis, 2007-2015Bulletin \u00e9pid\u00e9miologique hebdomadaire, 721 March 2017, Francehttp://invs.santepubliquefrance.fr/beh/2017/7/pdf/2017_7.pdf\u00a0Anti-tuberculosis drug resistance in France in 2014-2015Bulletin \u00e9pid\u00e9miologique hebdomadaire, 721 March 2017, Francehttp://invs.santepubliquefrance.fr/beh/2017/7/pdf/2017_7.pdf\u00a0Status of influenza season 2016/17EPI-NEWS 5, 20171 February 2017, Denmarkhttp://www.ssi.dk/Aktuelt/Nyhedsbreve/EPI-NYT/2017/Uge%205%20-%202017.aspx\u00a0Influenza vaccine uptake among healthcare workers \u2013 interim survey results mid season 2016-2017Epi-Insight 2017;18(1)January 2017, Irelandhttp://ndsc.newsweaver.ie/epiinsight/1p50tjai5xn?a=1&p=51284092&t=17517774\u00a0Respiratory bacteria quarterly report. Quarter three: 1 July to 31 September 2016HPS Weekly Report 2016; 50(50)13 December 2016, Scotlandhttp://www.hps.scot.nhs.uk/documents/ewr/pdf2016/1650.pdf\u00a0Sexually transmitted diseases\u00a0ANSWER. HIV infection and AIDS: Quarterly report to 31 December 2016HPS Weekly Report 2017; 51(11)21 March 2017, Scotlandhttp://www.hps.scot.nhs.uk/documents/ewr/pdf2017/1711.pdf\u00a0New HIV preventive drug\u2019s acceptance and constraints of African and Caribbean population living in Ile-de-France: the use of Truvada\u00ae in pre-exposure prophylaxis (PrEP). An exploratory surveyBulletin \u00e9pid\u00e9miologique hebdomadaire, 67 March 2017, Francehttp://invs.santepubliquefrance.fr/beh/2017/6/pdf/2017_6.pdf\u00a0Increase in gonorrhoea cases in the South-East: heterosexual transmission a featureEpi-Insight 2017;18(3)March 2017, Irelandhttp://ndsc.newsweaver.ie/epiinsight/83681u1ih1x?a=1&p=51521199&t=17517774\u00a0Shigella among MSM in DublinRecent outbreak of ciprofloxacin-resistant Epi-Insight 2017;18(2)February 2017, Irelandhttp://ndsc.newsweaver.ie/epiinsight/y9rvan81q6z10gkzp9yxn5?a=2&p=51399875&t=17517804\u00a0Persons living with HIV in Italy: data from the second national surveyNot Ist Super Sanit\u00e0 2017;30(1)January 2017, Italyhttp://www.iss.it/binary/publ/cont/ONLINE_GENNAIO.pdf\u00a0Further increase in syphilis infections in men who have sex with menEpidemiologisches Bulletin 50, 201619 December 2016, Germanyhttp://www.rki.de/DE/Content/Infekt/EpidBull/Archiv/2016/Ausgaben/50_16.pdf?__blob=publicationFile\u00a0Zoonoses and vector-borne diseases\u00a0Malaria and illegal gold mining in French Guiana: a major public health challengeBulletin \u00e9pid\u00e9miologique hebdomadaire, 67 March 2017, Francehttp://invs.santepubliquefrance.fr/beh/2017/6/pdf/2017_6.pdf\u00a0Creutzfeldt-Jakob disease (CJD) biannual update (February 2017)Health Protection Report; 11(6)10 February 2017, United Kingdomhttps://www.gov.uk/government/uploads/system/uploads/attachment_data/file/591266/hpr0617_cjd.pdf\u00a0Several cases of psittacosis among people who cleaned bird feedersFolkh\u00e4lsomyndigheten website26 January 2017, Swedenhttps://www.folkhalsomyndigheten.se/nyheter-och-press/nyhetsarkiv/2017/januari/flera-fall-av-papegojsjuka-bland-personer-som-rengjort-fagelbord/\u00a0Other\u00a0Increase in reporting of \u2018polio-like\u2019 diseases in 2016Epidemiologisches Bulletin 9, 20172 March 2017, Germanyhttps://www.rki.de/DE/Content/Infekt/EpidBull/Archiv/2017/Ausgaben/09_17.pdf?__blob=publicationFile\u00a0How long is scabies crustosa contagious in a deceased patient?Infectieziekten Bulletin 2017; 28(1)24 January 2017, the Netherlandshttp://www.rivm.nl/dsresource?objectid=a0d3b667-4f75-4b94-ba63-01f4fafec1d9&type=pdf&disposition=inline\u00a0Antenatal screening for infectious diseases in England: summary report for 2015Health Protection Report; 11(2)13 January 2017, United Kingdomhttps://www.gov.uk/government/uploads/system/uploads/attachment_data/file/583576/hpr0217_naism.pdf"} +{"text": "Scientific Reports6: Article number: 3478610.1038/srep34786; published online: 10102016; updated: 01122017In this Article, Figure 2 is duplicated as Figure 4. The correct Figure 4 appears below as"} +{"text": "Brain Sciences maintains high quality standards for its published papers. In 2017, a total of 160 papers were published in the journal. Thanks to the cooperation of our reviewers, the median time to first decision was 26 days and the median time to publication was 58.5 days. The editors would like to express their sincere gratitude to the following reviewers for their time and dedication in 2017:Abhishek DesaiJohn MagnottiAdel HelmyJohn RolstonAdrian ParkeJon HennerAgnieszka FiszerJoohyung LeeAkassoglou KaterinaJos\u00e9 RouillardAkiko TamasakiJosep Esteve-RomeroAlan DorvalJosephine BarnesAlan G. FinkelJoshua ParkAlan WinstonJudith S. MillerAlcy R. TorresJulie A. GriecoAlejandro L\u00f3pez Tob\u00f3nJulie Gros-LouisAlessandro DidonnaJulio OrtegaAlessandro TonacciJyoti MishraAlexander U. BrandtKarolina MoustopoulouAllen L. HoKate KarelinaAmanda M. BrownKathryn M. YorkstonAmanda SeidlKathy CarterAmy MandavilleKathy MountjoyAna LedoKatsuhiko NishimoriAndr\u00e9 A. FentonKatsura TakanoAndre StrydomKavitha YaddanapudiAndreas LuftKaylah LalondeAndreas SchulzeKevin C. BrennanAndrej KralKevin J. BlackAndres M. LozanoKevin SchalinskieAndrew BagshawKirsten R. M\u00fcller-VahlAndrew C. TalkKylie BaileyAndrew J. SolomonLaraine WinterAndrew PostLarisa PoluektovaAndriy TemkoLarry Abel\u00c1ngel Romero Mart\u00ednezLee CampbellAngela NarayanLeena MohapatraAnita Riecher-R\u00f6sslerLeher SinghAnn B. RaginLeonard Verhagen MetmanAnna Fogdell-HahnLesley JonesAnna J. EsbensenLeslie SwansonAnne BeemelmannsLeyan XuAnne DezetterLili-Naz HazratiAnouk Y. J. M. SmeetsLin ZhuAnthony AkkariLindsay D. NelsonAnthony R. MawsonLisa Wise-FaberowskiAntonella ScorzielloLise EliotAntonino F. GermanoLitofsky N. ScottAntonio De TantiLora Talley WattsApit HemakomLoren MarulisArmin FuchsLori-Ann R. SacreyArthur A. VandenbarkLuca CasartelliAshraf S. GorgeyLuis R. PerazaAthanasia MouzakiLyn O\u2019GradyAtsuhiro NakagawaLynn UlatowskiBalapal S. BasavarajappaMadelyn RayBarbara S. KoppelMarcel GiezenBarbara TettenbornMarcello CiaccioBengt K\u00e4ll\u00e9nMarco CarotenutoBilal KhokharMarcus ChenBoris KotchoubeyMarcus GrimmBrendan RooneyMaria CabelloBrent KiousMaria ChahrourBrian ChristieMaria Chiara BuscarinuBrian Nils LundstromMaria Christina SergakiBrooke N. MacnamaraMaria HernandezBruce LyethMaria LucaBruno MeloniMariko SaitoBryan DuckhamMarion EhrichBryan HowellMarita PruessnerCalvin LiMark B. DetweilerCameron B. JeterMark R. N. KotterCamila AquinoMarkus WettsteinCarlotta Zanaboni DinaMartha MichalkiewiczCarmela BravaccioMartijn BaartCarmelo Lucio SturialeMartin Gerbert FraschCaroline JungeMartin Sp\u00fclerCatherine S. Tamis-LeMondaMartina ArdizziCecilia GiuliviMARVIN R. DiazC\u00e9dric GaleraMary MotzCharles A. NelsonMATTHEW ROBSONChi-Chang HuangMatthias HohmannChloe LaneMeera E. ModiChristian P. M\u00fcllerMelanie MorrisonChristian SalvatoreMelanie PinaChristine WinterMelissa BarberChristopher MillerMelissa RavenChristopher-James HarveyMichael A. Ben-AvieClaire O\u2019CallaghanMichael D. RawlinsClare HarropMichael JohnstonClaudia MaennelMichael PerlisClaudio LiguoriMichael SchaeferClea WarburtonMichael YoongClement HamaniMichal MielcarekClodagh NolanMichel BilliardCynthia R. GrossMila VulchanovaDamian A. StanleyMohd. Farooq ShaikhDamian JenkinsMohtashem SamsamDamir JanigroMontserrat Zurr\u00f3nDaniel CohenMonty SilverdaleDaniel PetersonMyra FernandesDaniel SwingleyNafisa M. JadavjiDario D. LofrumentoNathanael J. YatesDario SiniscalcoNavkiran KalsiDavid Bar-OrNawei SunDavid FacalNesha BurghardtDavid M. FergussonNiccol\u00f2 MoraDavid M. SchnyerNicholas AltieriDavid P. GavinNicole OsierDavid R. ShprecherNikolaos ZiogasDavid T. DenhardtNnamdi PoleDavid W. EvansNo\u00e9mie Auclair OuelletDavid WagnerNoman NaseerDavid ZieglerNora Vanegas ArroyaveDavide BarbagalloN\u00faria Esteve-GibertDeane AikinsOlaf HaukDeAnna L. AdkinsOliver QuarrellDeborah FergusonOnintza SagredoDenise GobertOwen CarmichaelDenise SharonOzgul GokDennis W. VanePaisit PaueksakonDevanshi SethPanagiotis BamidisDevin W. McBridePaola RusminiDhanabalan MuraliPaolo CalabresiDiego Torres-RussottoPatricia BroderickDomenico De BerardisPatricia K. CoyleDomenico ServelloPatr\u00edcia MacielDominic ChengPatrizia GiannoniDora LozsadiPaul E. GreeneDouglas WeldonPaul GorczynskiDwight PiercePaul WilliamsonE. Richard StanleyPeik GustafssonEberhard UhlPeter C. Van Der EndeEdward V. QuadrosPeter E. WaisEdward VulPeter N. SteinmetzEgilius L. H. SpieringsPeter RumneyEiichi SuehiroPhilip HazellEling De BruinPhilippe SchuchtElizabeth A. StricklandPhillip TsengEmily D. RickettsPhu HoangEmily EdmondsPierre DuquetteEmily S. NicholsPierre RoubertouxEric BravermanPieter J. HoekstraEric GlasgowPranela RameshwarEric VerinQingzhong RenErik SistermansRadwa BadawyErika SkoeRakesh Kumar TiwariErwin B. MontgomeryRamon Y. BirnbaumEugene ParkRany MakaryusFabiana GeraciRasheda Arman ChowdhuryFabio CampanellaRay MeddisFabio Di DomenicoRebecca FortgangFarid RahimiRebecca ParkFelix Jimenez-RondanRemy BationFelix SeptiantoRenee HollingsworthFeng GengReto StockerFeng GuRicardo S. OsorioFeng Vankee LinRichard C. DethFernando L. ValeRichard E. MainsFilmer ChuRichard FryeFiras BannoutRichard J. YouleFlorence GressierRifai ChaiFlorence LevyRobert B. MichaelFlorien BoeleRobert G. MairFlorin DespaRobert GonzalezFrancesco BartoliRobert KotloskiFrancesco Carlo MorabitoRobert LalondeFrancesco RigoliRobert R. HanseboutFrancisca S RodriguezRobert T. MalletFrancisca S. RodriguezRocco Salvatore CalabroFranco GemignaniRodger J. ElbleFrank FlemischRodrigo NosedaFr\u00e9d\u00e9ric CauseretRomain Vuillefroy De SillyFrederik SteynRosella AbetiFredrik KarlssonRosemary SheehanFriedemann PaulRupsa DattaFulvio LauretaniRussell SchacharGabriel G. De La TorreRyan A. StevensonGabriela G. WernerSamuel FrankGanesh NaikSandy ShultzGarnett P. McMillanSanjay MaggirwarGary SimonSarah BaylessGayann\u00e9e KediaSarah HellewellGeon Ho BahnSarah LagemanGeorg WidhalmSarah RabbittGeorge ChrousosScott R. SchroederGeorge OpieSebastian GluthGeorgios PaslakisShashi S. SeshiaGholson LyonShervin AssariGiacomo VivantiShraddha RegeGianluca SerafiniShyam SeetharamanGiordano D'UrsoSigny SheldonGiovanni AssenzaSiobhain M. O'mahonyGiri Kumar ChandakaSoledad ZarateGlen BergeronSonja ScholzGordon HarperSonya L. JakubecGregory FricchioneSophia FrangouGregory HeathStan KutcherGregory P. MuellerSt\u00e9phanie Chambaron-GinhacGuennadi SaikoStephen HollandGurudutt PendyalaStephen HoneybulH. Lee SwansonStephen R. JacksonHans Van Der SteenStephen SawcerHans-Gert BernsteinSteven A. J. ChamuleauHarrison C. WalkerSteven PassmoreHasse WalumSusan J. NicolsonHeather BowlingSusan JergerHideya KodamaSusann M. Brady-KalnayHiran ThabrewSu-Youne ChangHiroaki WakimotoSven KroenerHirokazu DoiT. Dianne LangfordHolly BowenTaku HatanoHon Wah LeeTatjana AueHong JiaoTerri PogodaHovagim BakardjianThanh Dang-VuHoward ChertkowTh\u00e9r\u00e8se M. JayHoward PrenticeThomas HeinbockelIlya BezprozvannyThomas W. WeickertInga D. NeumannTilman HenschIngvild Saksvik-LehouillierTimothy J. CrowIsaac T. SchieferTimothy RittmanItoh YasuhiroTobias DerfussJ. Leigh LeasureTobias S. AndersenJacqueline E. ReznikT\u00f6res TheorellJaishankar BharatharajTrinidad Montero-MelendezJake NeumannTrishna PatelJames AdamsUlrich PalmJames O'CallaghanVicki L. KristmanJames R. CouchVictor RiveraJannette Rodr\u00edguez-PallaresVictor V. DyakinJared W. YoungVictoria M. LeavittJavier Ortu\u00f1o-SierraVincent R. HarleyJayashri GhoshVincenzo GuidettiJean R. HughesVincenzo NataleJean-Philippe LangevinVirginia V. W. McIntoshJennifer BeerVishwanath SankarasubramanianJennifer L. LyonsWenchao ZhouJennifer RodgerWendy O. KalbergJeroen L. A. PenningsWilhelm MistiaenJ\u00e9r\u00f4me BrunelinWilliam D. KearnsJ\u00e9r\u00f4me DevyWissam DeebJerzy BodurkaWolfgang MannJessica RobinWoo-Yang KimJia ChengXiaobing YuanJie ShiXueni PanJill CrittendenYadvinder AhiJinyuan WangYang ZhangJoachim FandreyYe XiongJodi M HeapsYuan-Pin LinJoerg WischhusenYuri BozziJohanna BickYvonne HoellerJohn GruzelierZhuo XingJohn L. WoodardPeer review is an essential part in the publication process, ensuring that"} +{"text": "Scientific Reports6: Article number: 3275310.1038/srep32753; published online: 09082016; updated: 03162017This Article contains errors in Figure 5F\u2013J, where the right-hand y axes \u2018Density (lncRNAs)\u2019 are incorrectly given as \u2018Density (except lncRNAs)\u2019. The correct Figure 5 appears below as"} +{"text": "Scientific Reports6: Article number: 34490; 10.1038/srep34490 published online: 10042016; updated: 05032017.This Article contains a typographical error. In the Results section, under the heading \u2018The most affected protein interfaces in cancer\u2019,\u2018EGFR-ERRB2\u2019should read:\u2018EGFR-ERBB2\u2019."} +{"text": "Title: Generalized Tetanus Initially Presenting with DysmasesisAuthors:\u00a0Shah Faisal Ahmad Tarfarosh,\u00a0Omar Farooq,\u00a0Ishrat H. Dar,\u00a0Samia Rashid,\u00a0Ummer YaseenOriginal Citation: Zunga P M, Tarfarosh S, Farooq O, et al. Generalized Tetanus Initially Presenting with Dysmasesis . Cureus 8(6): e644. doi:10.7759/cureus.644The authors were originally placed in the incorrect order and Ummer Yaseen was erroneously added as an author instead of Mohd Iqbal Dar. The author list should be corrected as follows.Corrected Author List:\u00a0Omar Farooq,\u00a0Ishrat H. Dar, Mohd Iqbal Dar, Samia Rashid, Parvaiz M. Zunga, Shah Faisal Ahmad Tarfarosh"} +{"text": "Scientific Reports7: Article number: 45991; 10.1038/srep45991 published online: 04112017; updated: 09252017.In this Article, Figure 4 is incorrect. The correct Figure 4 appears below as"} +{"text": "Int J Epidemiol 2016; 45 (2): 554-564. doi: https://doi.org/10.1093/ije/dyv359First published online: 10 April 2016, A funding statement was missing for author David Stuckler. The funding statement should have included \u201cD.S. is funded by ERC HRES 313590.\u201d"} +{"text": "Scientific Reports4: Article number: 611510.1038/srep06115; published online: 08192014; updated: 05122017This article was originally published under a [CC BY-NC-SA 4.0/CC BY-NC-ND 4.0] license, but has now been made available under a [CC BY 4.0] license. The PDF and HTML versions of the paper have been modified accordingly."} +{"text": "Nature Communications8: Article number: 14158; DOI: 10.1038/ncomms14158 (2017); Published 02132017; Updated 11292017.In this Article, the clinical isolate MAD/GM3_10/14 of respiratory syncytial virus (RSV) is incorrectly described as an RSV A isolate. MAD/GM3_10/14 has been identified as an RSV B isolate on the basis of its fusion protein sequence. The fusion protein sequences of the clinical isolates used in this study have been deposited in GenBank under accession codes MF361899 (MAD/GM2_2/12), MF361900 (MAD/GM2_12/12), MF361901 (MAD/GM2_13/12), MF361902 (MAD/GM2_14/12), MF361903 (MAD/GM3_10/14), MF361904 (MON/9/92), and MF361905 (MAD/GM3_7/13)."} +{"text": "Scientific Reports7:459; doi:10.1038/s41598-017-00540-x; Article published online 28 March 2017This Article contains an incorrect version of Figure 7. The correct Figure\u00a07 appears below as Figure"} +{"text": "Scientific Reports6: Article number: 33830; 10.1038/srep33830 published online: 09222016; updated: 01192017.\u2212tg\u201d. The correct This Article contains an error in panel C of"} +{"text": "The records can be retrieved from the nucleotide database by searching Entrez Nucleotide using the accession numbers below:More detailed citations for the Chloroplast SNP data are also now available (* chloroplast_region1: KX556608 - KX556920* chloroplast_region2: KX556921 - KX557233* chloroplast_region3: KX556295 - KX556607* chloroplast_region4: KX555982 - KX556294"} +{"text": "Scientific Reports6: Article number: 2659110.1038/srep26591; published online: 05262016; updated: 10062016.This Article contains errors in Figure 2D where the Hodgkin lymphoma \u2018TET1-MSP\u2019 methylated and unmethylated MSP bands are incorrect. The correct Figure 2D appears below as"} +{"text": "Adenocarsinoma macroscopic view.Figure 4. Adenocarsinoma microscopy view.Figure 6. Adenocarsinoma spot samples in MALDI spectrometry. MALDI\u200a = \u200amatrix assisted laser desorption ionization."} +{"text": "The correct name is: Khan Bahadar Khan. The correct citation is: Bahadar Khan K, A Khaliq A, Shahid M (2016) A Morphological Hessian Based Approach for Retinal Blood Vessels Segmentation and Denoising Using Region Based Otsu Thresholding. PLoS ONE 11(7): e0158996. doi:"} +{"text": "Scientific Reports6: Article number: 3029810.1038/srep30298; published online: 07222016; updated: 09022016In this Article, reference 15 is duplicated as reference 13. The correct reference 15 appears below:Mol. Neurobiol. 50, 1\u20135 (2014).Bazan, N. G. Is there a molecular logic that sustains neuronal functional integrity and survival? Lipid signaling is necessary for neuroprotective neuronal transcriptional programs."} +{"text": "In our recent article , it has Additional file 1:Table S2. Gene count table for 48 RNA-seq nasal brushing subjects from GALA cohort. (XLSX 5732 kb)"} +{"text": "Scientific Reports6: Article number: 38442; 10.1038/srep38442 published online: 12022016; updated: 02162017.This Article contains an error in Table 4. The text in the first row \u2018CON_PiiA (strain P3UCB1)\u2019 was incorrectly given as \u2018CON_PiiB (strain H17O-S1).\u2019"} +{"text": "Scientific Reports6: Article number: 2454910.1038/srep24549; published online: 04152016; updated: 02092017This Article contains an error in Figure 4, where Figure 4d was inadvertently duplicated in Figure 4h. The correct Figure 4 appears below as"} +{"text": "Scientific Reports7: Article number: 40537; 10.1038/srep40537 published online: 01162017; updated: 04072017.This Article contains errors in Table 1. The correct Table 1 appears below as"} +{"text": "AbstractCulicoides biting midges are small insects that are proven vectors of pathogens that cause disease in animals and humans. There are 1,368 species of Culicoides in the world, including 149 species in Brazil and 122 species in the Brazilian Amazon Basin. This study documents specimens that were collected between 2013 and 2015 in the municipalities of Alvorada d\u2019Oeste, Buritis, Cacoal, Costa Marques, Espig\u00e3o d\u2019Oeste, Guajar\u00e1-Mirim, Pimenta Bueno, Porto Velho and S\u00e3o Francisco Guapor\u00e9. Collections were performed using HP light traps in forest, pasture and peridomicilie environments.Culicoidescarpenteri Wirth & Blanton, 1953; C.dasyophrus Macfie, 1940; C.eublepharus Macfie, 1948; C.galindoi Wirth & Blanton, 1953; C.heliconiae Fox & Hoffman, 1944; and C.ignacioi Forattini, 1957. This is the first record in Brazil of C.darlingtonae Wirth & Blanton, 1971.Species newly recorded in Rond\u00f4nia State include Culicoides Latreille, 1809 are small insects; their blood-feeding females bite birds and mammals, including humans, and they sometimes feed in large swarms. The bites are painful and can cause severe reactions. These insects can also transmit pathogens that cause disease in humans and domestic animals of economic importance and peridomicilies .During entomological studies carried out between 2013 and 2015, in the municipalities of Alvorada d\u2019Oeste, Buritis, Cacoal, Costa Marques, Espig\u00e3o d\u2019Oeste, Guajar\u00e1-Mirim, Pimenta Bueno, Porto Velho and S\u00e3o Francisco Guapor\u00e9 Culicoides specimens were preserved in 70% ethanol, and then dissected and slide-mounted in phenol-balsam as described by Culicoides species was based on Fox & Hoffman, 1944Culicoides (Hoffmania) heliconiaeC.rozeboomi Barbosa, 1947 Synonyms: Type status:Other material. Occurrence: recordedBy: J.F. Medeiros; individualCount: 1; sex: male; lifeStage: adult; Location: country: Brazil; stateProvince: Rond\u00f4nia; municipality: Cacoal; verbatimCoordinates: 11\u00b025'53\"S; 61\u00b026'52\"W11\u00b025'53\"S; 61\u00b026'52\"W; Event: samplingProtocol: HP light traps; eventDate: 2014-11-12/21; habitat: fragmented forestType status:Other material. Occurrence: recordedBy: L.P.C. Carvalho; individualCount: 1; sex: female; lifeStage: adult; Location: country: Brazil; stateProvince: Rond\u00f4nia; municipality: Buritis; verbatimCoordinates: 10\u00b012'47\"S; 63\u00b053'46\"W10\u00b012'47\"S; 63\u00b053'46\"W; Event: samplingProtocol: HP light traps; eventDate: 2015-05-26; habitat: forest3 which is narrow and transverse, r3 with pale spot present anterior to base of M1, a single pale spot crosses the second radial cell, apices of CuA1 and CuA2 pale; spermathecae with short, slender necks , Grenada and Trinidad and Tobago .This species was here recorded for the first time in Rond\u00f4nia State.Forattini, 1957Culicoides (Hoffmania) ignacioiC.saintjusti Tavares & Ruiz, 1980 Synonyms: Type status:Other material. Occurrence: recordedBy: L.P.C. Carvalho; individualCount: 1; sex: male; lifeStage: adult; Location: country: Brazil; stateProvince: Rond\u00f4nia; municipality: Porto Velho; locationRemarks: Pesque e Pague Farm; verbatimCoordinates: 08\u00b040'02\"S; 63\u00b042'14\"W08\u00b040'02\"S; 63\u00b042'14\"W; Event: samplingProtocol: HP light traps; eventDate: 2015-05-13/14; habitat: pastureType status:Other material. Occurrence: recordedBy: J.F. Medeiros; individualCount: 3; sex: female; lifeStage: adult; Location: country: Brazil; stateProvince: Rond\u00f4nia; municipality: Guajar\u00e1-Mirim; verbatimCoordinates: 10\u00b047'18\"S; 65\u00b020'32\"W10\u00b047'18\"S; 65\u00b020'32\"W; Event: samplingProtocol: HP light traps; eventDate: 2013-10; habitat: forestC.fernandoi, but can be distinguished by the mandible which has 20\u201322 teeth (14\u201315 in C.fernandoi), vein R3 pale , distal pale spot in cell r3 large, transverse (crescent-shaped or subdivided in C.fernandoi) .Brazil and Paraguay .This species was here recorded for the first time in Rond\u00f4nia State.Wirth & Blanton, 1953Type status:Other material. Occurrence: recordedBy: J.F. Medeiros; individualCount: 1; sex: female; lifeStage: adult; Location: country: Brazil; stateProvince: Rond\u00f4nia; municipality: Costa Marques; verbatimCoordinates: 12\u00b024'25\"S; 64\u00b014'26\"W12\u00b024'25\"S; 64\u00b014'26\"W; Event: samplingProtocol: HP light traps; eventDate: 2015-05-22; habitat: forest3 broadly extending across cell near apex from anterior wing margin to vein M1; apices of veins M1, M2 and CuA1 dark; subapical pale spot in cell m2, distal pale spot in cell m1 broadly meeting wing margin; two pale spots in distal part of cell m2, the distal one broadly meeting wing margin; the proximal one connected by a pale area extending to base of cell and including the pale spots lying in front of mediocubital fork and behind medial fork; pale area in cell CuA1 nearly filling entire cell; anal cell pale except for a large dark area centering on middle of stem of mediocubital vein; halter pale .This species was here recorded for the first time in Rond\u00f4nia State.Macfie, 1940Type status:Other material. Occurrence: recordedBy: J.F. Medeiros; individualCount: 1; sex: female; lifeStage: adult; Location: country: Brazil; stateProvince: Rond\u00f4nia; municipality: Cacoal; verbatimCoordinates: 11\u00b025'53\"S; 61\u00b026'52\"W11\u00b025'53\"S; 61\u00b026'52\"W; Event: samplingProtocol: HP light traps; eventDate: 2014-11-12/21; habitat: forest3 more or less fused, the posterior one located slightly proximad of the anterior one, distal pale spot in cell r3 small, only one small pale spot in distal part of anal cell and one pale spot in distal part of cell m2; halter whitish; spermatheca one, pyriform .This species was here recorded for the first time in Rond\u00f4nia State.Macfie, 1948CulicoideseublepharusC.transferrans Ortiz, 1953 Synonyms: Type status:Other material. Occurrence: recordedBy: J.F. Medeiros; individualCount: 1; sex: female; lifeStage: adult; Location: country: Brazil; stateProvince: Rond\u00f4nia; municipality: Cacoal; verbatimCoordinates: 11\u00b025'53\"S; 61\u00b026'52\"W11\u00b025'53\"S; 61\u00b026'52\"W; Event: samplingProtocol: HP light traps; eventDate: 2014-11-12/21; habitat: forest fragment3, the distal pair usually fused and broadly attaining wing margin anteriorly; two pale spots each in cells m1 and apices of cells m2 and anal cell; pale spots present behind medial fork and in front of mediocubital fork; halter yellowish; spermatheca one, oval with a short portion of the duct sclerotized .This species was here recorded for the first time in Rond\u00f4nia State.Wirth & Blanton, 1953Type status:Other material. Occurrence: recordedBy: J.F. Medeiros; sex: 1 female, 1 male; lifeStage: adult; Location: country: Brazil; stateProvince: Rond\u00f4nia; municipality: Cacoal; verbatimLocality: Ch\u00e1cara do Gerson and Rancho Cassiano; verbatimCoordinates: 11\u00b025'53\"S; 61\u00b026'52\"W11\u00b025'53\"S; 61\u00b026'52\"W; Event: samplingProtocol: HP light traps; eventDate: 2014-11-12/21; habitat: forest fragment3 rounded distally, leaving a small dark area in apex of cell; halter pale; spermathecae two, pyriform, subequal .This species was here recorded for the first time in Rond\u00f4nia State.Wirth & Blanton, 1971Type status:Other material. Occurrence: recordedBy: J.F. Medeiros; sex: 2 females, 3 males; lifeStage: adult; Location: country: Brazil; stateProvince: Rond\u00f4nia; municipality: Espig\u00e3o do Oeste; verbatimCoordinates: 11\u00b067'63\"S; 60\u00b069'90\"W11\u00b067'63\"S; 60\u00b069'90\"W; Event: samplingProtocol: HP light traps; eventDate: 2014-04; habitat: forestType status:Other material. Occurrence: recordedBy: J.F. Medeiros; sex: 3 females, 5 males; lifeStage: adult; Location: country: Brazil; stateProvince: Rond\u00f4nia; municipality: Pimenta Bueno; verbatimLocality: Usina Hidrel\u00e9trica Rondon II; verbatimCoordinates: 11\u00b096'48\"S; 60\u00b069'90\"W11\u00b096'48\"S; 60\u00b069'90\"W; Event: samplingProtocol: HP light traps; eventDate: 2014-10-10/11; habitat: fragment forestType status:Other material. Occurrence: recordedBy: J.F. Medeiros; individualCount: 1; sex: female; lifeStage: adult; Location: country: Brazil; stateProvince: Rond\u00f4nia; municipality: Cacoal; verbatimLocality: Rancho Cassiano; verbatimCoordinates: 11\u00b025'53\"S; 61\u00b026'52\"W11\u00b025'53\"S; 61\u00b026'52\"W; Event: samplingProtocol: HP light traps; eventDate: 2014-11-12/21; habitat: fragment forest3 with contiguous poststigmatic pale spots, cell m1 with two pale spots, cell m2 with small pale spot; two spermathecae and rudimentary third, with sclerotized ring .This species was here recorded for the first time in Brazil.Culicoides species collected in this study are distributed throughout the Brazilian Amazon (C.dasyophrus, C.eublepharus, C.galindoi and C.ignacioi are found from Acre State to Maranh\u00e3o State (C.heliconiae herein were collected in a forest environment, which is the same habitat as the specimens collected by Fittkau in 1970 in APEG Forest, Par\u00e1 State (The major n Amazon ; C.dasy\u00e3o State . The sper\u00e1 State . The mair\u00e1 State .Culicoidesignacioi is distributed throughout Paraguay and Brazil (Culicoides from many points of collection and this species was captured in highest abundance in Cerrado forest, in the State of Maranh\u00e3o.d Brazil . In Brazd Brazil . The speCulicoideseublepharus has been recorded in Mexico, Costa Rica, Venezuela, Ecuador and Brazil. It has been found in the Brazilian Amazon in states of Amazonas, Par\u00e1 and Roraima ( Roraima . The speCulicoides still is understood in Brazil. This study shows that species diversity is high among Amazonian Culicoides. Furthermore, this study will be helpful in knowledge of Culicoides fauna in the Amazon, and may contribute to a better understanding of the medical importance and vector epidemiology these insects.This study increases the number of species recorded in Rond\u00f4nia State to 40. Of the seven species here recorded, all are new records in Rond\u00f4nia state, and one is also a new record in Brazil; underlining how poorly the distribution of even medically important insects such as"} +{"text": "Nature Communications6: Article number: 6549; DOI: 10.1038/ncomms7549 (2015); Published: 03202015; Updated: 04202017This Article was originally published under a CC BY-NC-ND 4.0 license, but has now been made available under a CC BY 4.0 license. The PDF and HTML versions of the paper have been modified accordingly."} +{"text": "Nature Communications 7:12321 doi:10.1038/ncomms12321 (2016); Published 5 Aug 2016; Updated 13 Sep 2016In"} +{"text": "The European Centre for Disease Prevention and Control (ECDC) invites applications for the following position:Scientific Officer Epidemic Intelligencehttps://ecdc.europa.eu/en/work-us/vacancies?f%5B0%5D=deadline_date%3A1The application deadline expires on 22 March 2018. For more information, please visit the ECDC website:"} +{"text": "Lathyrus sativus L.) Genotypes under Salinity Stress\u201d [Lathyrus sativus) Genotypes, Differing in Arsenic Tolerance. Agric Res (2013) 2: 330. doi:10.1007/s40003-013-0085-3 in Figure 6.International Scholarly Research Notices has retracted the article titled \u201cGrowth Responses and Leaf Antioxidant Metabolism of Grass Pea ( Stress\u201d . The art"} +{"text": "Scientific Reports7: Article number: 4219710.1038/srep42197; published online: 02142017; updated: 03232017This Article contains an error in the Acknowledgements section:\u201cMatthieu Raoelsion\u201dshould read:\u201cMatthieu Raoelison\u201d"} +{"text": "Scientific Reports7: Article number: 46709; 10.1038/srep46709 published online: 04272017; updated: 09072017.In this Article, Figure 2b is a duplication of Figure 2c. The correct Figure 2b appears below as"} +{"text": "The two phen planes are oriented at a dihedral angle of 57.48\u2005(11)\u00b0. The perchlorate anion links with the Mn complex cation via weak C\u2014H\u22efO hydrogen bonding.In the title complex, [Mn(CHO DOI: 10.1107/S1600536809049277/xu2641Isup2.hklStructure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "The ZnII atom (site symmetry trans-ZnN2O4 octa\u00adhedral geometry defined by two monodentate N-bonded and two bidentate O,O-bonded ppa monoanions. The extended two-dimensional structure arising from this connectivity is a square grid and the disordered uncoordinated water mol\u00adecules occupy cavities within the grid. An N\u2014H\u22efO hydrogen bond occurs.The title compound, {[Zn(C DOI: 10.1107/S1600536809036939/hb5062Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The hydrogen-bonding scheme gives rise to a three-dimensional network.In the crystal structure of the title compound, C DOI: 10.1107/S1600536808014347/tk2271Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The mol\u00adecules are linked by N\u2014H\u22efO hydrogen bonds, forming a layer network.In the mononuclear title compound, [Cd(C DOI: 10.1107/S1600536808009409/ng2436Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The remaining coordination sites are occupied by two N atoms of a 1,10-phenanthroline ligand and two O atoms of a 6-hydroxy\u00adnaphthalene-1-carboxyl\u00adate ligand. The crystal packing is stabilized by O\u2014H\u22efO hydrogen-bonding inter\u00adactions.The title complex, [Cd DOI: 10.1107/S1600536809043475/bt5107Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Adjacent Sn atoms are bridged by coordination to the two O atoms of each 1,1\u2032-binaphthyl-2,2\u2032-diyl phospho\u00adnate ligand, forming a one-dimensional chain structure parallel to the b axis.In the title polymeric coordination compound, [Sn(CH DOI: 10.1107/S160053680905291X/sj2705Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The two imidazole rings are in an anti configuration. The anion has a trigonal planar coordination geometry.The title compound, (C DOI: 10.1107/S1600536808021521/cf2209Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The heterocyclic ring exhibits a twisted conformation.In the crystal structure of the title compound, C DOI: 10.1107/S1600536808007186/ng2433Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the title compound, C DOI: 10.1107/S1600536808012452/sg2233Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "It is isotypic with K2Bi(PO4)(WO4). The three-dimensional framework is built up from [Bi(PO4)(WO4)] nets, which are organized by adhesion of [BiPO4] layers and [WO4] tetra\u00adhedra above and below of those layers. The inter\u00adstitial space is occupied by Cs atoms. Bi, W and P atoms lie on crystallographic twofold axes.Dicaesium bis\u00admuth(III) phosphate(V) tungstate(VI), Cs DOI: 10.1107/S160053680903147X/br2113Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536809052015/rz2395Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the crystal structure, inter\u00admolecular O\u2014H\u22efO hydrogen bonds link the complex and solvent mol\u00adecules into infinite chains.In the monomeric title compound, [Sn(C DOI: 10.1107/S1600536808016140/hb2726Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The packing is governed by inter\u00admolecular O\u2014H\u22efO hydrogen-bonding inter\u00adactions.In the title coordination polymer, [Sm(C DOI: 10.1107/S1600536808031036/rz2243Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The Cl\u2212 anions take part in the formation of weak C\u2014H\u22efCl hydrogen bonds, which contribute to the crystal packing stabilization.In the title compound, [Cr(C DOI: 10.1107/S1600536808016784/cv2410Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the crystal structure, inter\u00admolecular O\u2014H\u22efO hydrogen bonds link complex and solvent mol\u00adecules into a three-dimensional network.The binuclear title complex, [Hg DOI: 10.1107/S1600536809026221/lh2830Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Intra\u00admolecular N\u2014H\u22efCl hydrogen bonding is observed in the dimeric complex.In the title complex, [Cu DOI: 10.1107/S1600536809044997/xu2644Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The ferrocene-1,1\u2032-diyl units exhibit a regular geometry with negligible tilting and balanced Fe\u2013ring centroid distances, and with the attached substituents assuming conformations close to ideal synclinal eclipsed.The title compound, [Fe(C DOI: 10.1107/S1600536809036459/dn2483Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The Cd atom lies on an inversion centre. The distorted octa\u00adhedral Cd environment contains two planar trans-related N,N-chelating S-2-(pyrrolidin-2-yl)-1H-1,3-benzimidazole ligands in one plane and two monodentate nitrate ligands. N\u2014H\u22efO hydrogen bonds involving a nitrate O atom build up an infinite chain parallel to the a axis.The title compound, [Cd(NO DOI: 10.1107/S1600536808006454/dn2315Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Three methyl\u00adene groups of the ligand are disordered over two orientations in a 0.555\u2005(6):0.445\u2005(6) ratio.In the title compound, [Zn(C DOI: 10.1107/S1600536808005060/hb2702Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the crystal packing, complex mol\u00adecules are linked by hydrogen bonds into a two-dimensional layer.The asymmetric unit of the title compound, [Zn(C DOI: 10.1107/S1600536809001688/kp2199Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The Zn atom is coordinated by two chelating acetyl\u00adacetonate groups and one oxazoline ligand in the apical position of a slightly distorted square-pyramidal metal\u2013ligand geometry.The title material, [Zn(C DOI: 10.1107/S1600536808042712/kj2105Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536809045930/br2124Isup2.hklStructure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536809007399/ng2554Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536810003879/hb5301Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The solvent mol\u00adecule is linked to the 28-atom ring by two hydrogen bonds.In the dinuclear centrosymmetric title complex, [Hg DOI: 10.1107/S1600536808028754/ng2488Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials: interactive version of Fig. 3 Enhanced figure:"} +{"text": "In the crystal structure, mol\u00adecules are linked through inter\u00admolecular N\u2014H\u22efS hydrogen bonds, forming chains along the b axis.In the mononuclear title compound, [Zn(NCS) DOI: 10.1107/S1600536810007300/ci5046Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The asymmetric unit consists of one Cd2+ cation, one succinate anion and two aqua ligands. The Cd atoms present a distorted penta\u00adgonal bipyramidal coordination and are bridged into layers parallel to (201) by succinate ligands.The title compound, [Cd(C DOI: 10.1107/S1600536809011593/bg2243Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536808006417/cs2069Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The BPTC anions link the CdII atoms, forming a one-dimensional chain structure. The L ligands are attached on both sides of the chain. A twofold rotation axis passes through the complex molecule. The crystal structure involves O\u2014H\u22efO hydrogen bonds.In the title compound, {[Cd DOI: 10.1107/S1600536808013676/rz2212Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The Pd atom displays a slightly distorted square-planar geometry, with the N- and C-atom donors in a cis arrangement.The centrosymmetric title compound, [Pd DOI: 10.1107/S1600536808010301/cs2073Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The dihedral angles between the pyridine and benzene rings are 23.9\u2005(6) and 70.7\u2005(1)\u00b0.In the title compound, [CrCl DOI: 10.1107/S1600536810034689/ng5023Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The In atom has a six-coordinate octa\u00adhedral environment, being bonded to the N atoms of six thio\u00adcyanate groups. The bond lengths and angles show normal values. The crystal packing exhibits no significantly short inter\u00admolecular contacts.The title complex, [Na(C DOI: 10.1107/S1600536809039282/ds2003Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The Cu atom -1H-1,2,4-triazole ligands [dihedral angle between the triazole and pyridine rings = 68.08\u2005(8)\u00b0] result in a two-dimensional 44 sheet structure in the crystal.The title coordination polymer, [CuCl DOI: 10.1107/S160053680900645X/hb2898Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The centrosymmetric dimer contains two Jahn\u2013Teller-distorted mangan\u00adese(III) ions, each in an octa\u00adhedral geometry, connected through two phen\u00adoxy bridges from two ligands.The title compound, [Mn DOI: 10.1107/S1600536808000226/hy2115Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The dihedral angle between the cyano\u00adphenyl and phenolate rings is 47.62\u2005(7)\u00b0.In the title complex, [Ni(C DOI: 10.1107/S1600536808004765/dn2310Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536809051629/ci2979Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The CoII atom (site symmetry trans-CoN2O4 octa\u00adhedral geometry defined by two monodentate N-bonded and two bidentate O,O\u2032-bonded ppa anions. The extended two-dimensional structure is a square grid, which is consolidated by N\u2014H\u22efO hydrogen bonds. The disordered uncoordinated water mol\u00adecules occupy cavities within the grid.The title compound, {[Co(C DOI: 10.1107/S1600536809040185/hb5124Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "There are two distinct Mn2+ cations in the chain, both with site symmetry 4]2\u2212 tetra\u00adhedra occupy the spaces between the chains.The title compound, [Mn(C DOI: 10.1107/S1600536808028912/hb2779Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Inter\u00admolecular hydrogen bonds of the type C\u2014H\u22efX link the complexes into a two-dimensional network.In the title complex, [Ni(C DOI: 10.1107/S1600536810007336/jh2131Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The thio\u00adcyanate ligand acts as bridging ligand, forming chains along [100]. A crystallographic mirror plane runs through the CuI ion, the thio\u00adcyanate ligand and the middle of the phenanthroline ligand.In the title complex, [Cu(NCS)(C DOI: 10.1107/S1600536810035002/lh5123Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The hydrogen-bonding inter\u00adactions give rise to a two-dimensional array.In the title compound, C DOI: 10.1107/S1600536809009544/tk2398Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S160053680900422X/tk2359Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The Rh atoms in the cations and anion exhibit square-planar coordination and are separated without any unusual inter\u00adactions.In the title complex, [Rh(C DOI: 10.1107/S1600536808032509/pv2102Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The CuII ions are linked into a one-dimensional coordination polymer by tethering 1,3-di-4-pyridylpropane ligands, whose central methyl\u00adene C atoms are situated on twofold rotation axes. The chains are oriented parallel to the c axis, and stack into a supra\u00admolecular three-dimensional structure through O\u2014H\u22efO hydrogen-bonding inter\u00adactions.In the title compound, [Cu(C DOI: 10.1107/S1600536809031821/tk2526Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The bond distances in the coordinated i-mnt ligands indicate some delocalization of the \u03c0-system.In the title compound, (C DOI: 10.1107/S1600536808036726/pk2127Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The CuII atom lies on an inversion center and is four-coordinated in a square-planar geometry by two N and two O atoms from two 4-bromo-2-(cyclo\u00adpentyl\u00adimino\u00admeth\u00adyl)phenolate Schiff base ligands.The title compound, [Cu(C DOI: 10.1107/S1600536809006606/su2099Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Electron delocalization occurs within the chelating rings.In the title compound, [Al(C DOI: 10.1107/S1600536809049812/xu2684Isup2.hklStructure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "The crystal structure involves intermolecular C\u2014H\u22efS hydrogen bonds.In the dinuclear title compound, [Zn{S DOI: 10.1107/S1600536808010003/sj2481Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "An N\u2014H\u22efO hydrogen bond links the mononuclear complex to the DMF solvent mol\u00adecules.In the structure of [Cu(C DOI: 10.1107/S1600536808043808/tk2349Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536809010587/tk2401Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Acta Cr DOI: 10.1107/S1600536808022952/bx2160Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The carboxyl\u00adate anion is equally disordered over two positions.The tin atom in the title compound, [Sn(C DOI: 10.1107/S1600536810005921/xu2728Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The coordination polyhedron shows a slightly distorted Archimedean square antiprismatic geometry. The asymmetric unit contains a toluene solvent mol\u00adecule. The crystal structure involves C\u2014H\u22ef.F hydrogen bonds.In the title compound, [Hf(C DOI: 10.1107/S1600536808015237/hy2134Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The crystal structure is stabilized by inter\u00admolecular N\u2014H\u22efO hydrogen bonding.In the cation of the title compound, C DOI: 10.1107/S1600536809039026/xu2591Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The mol\u00adecule adopts a classical gauche conformation.In the title compound, C DOI: 10.1107/S1600536810036676/bt5357Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The mol\u00adecules are linked together via hydrogen bonds involving the water mol\u00adecules, forming a three-dimensional network.In the title complex, [La(C DOI: 10.1107/S1600536808001955/hg2369Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The two independent tin atoms show distorted trigonal-bipyramidal SnC2O3 and SnC2O2Br coordination geometries.In the centrosymmetric title compound, [Sn DOI: 10.1107/S1600536809051691/hb5234Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536809045413/cv2637Isup2.hklStructure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "Adjacent CuII atoms are connected via the bridging tetra\u00adfluoro\u00adterephthalate ligands into a one-dimensional chain running along the [101] direction.In the title compound, [Cu(C DOI: 10.1107/S1600536808018977/hy2139Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536809035272/tk2532Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The crystal structure involves intra- and intermolecular O\u2014H\u22efO hydrogen bonds.In the title compound, [Mg(SO DOI: 10.1107/S1600536808009938/im2060Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The DMPA ligands coordin\u00adate in the bis-chelate, bridging and bridging tridentate modes.In the title centrosymmetric dinuclear complex, [Tm DOI: 10.1107/S1600536809051435/lh2956Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the crystal structure, mol\u00adecules form a layered network linked by O\u2014H\u22efO hydrogen bonds.In the title compound, [Zn(C DOI: 10.1107/S1600536808042530/hb2880Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The mononuclear mol\u00adecule lies on a twofold axis that passes through the pyridine N atoms. The toluene solvent mol\u00adecule is equally disordered about a twofold axis.In the title chromium silanethiol\u00adate, [Cr(C DOI: 10.1107/S1600536809021680/ng2589Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The dihedral angle between the aromatic rings of the ligand is 67.9\u2005(4)\u00b0. In the crystal, inversion dimers linked by pairs of N\u2014H\u22efO hydrogen bonds occur, generating R 2 2(8) loops.In the title complex, [Cu(C DOI: 10.1107/S1600536810010305/hb5365Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Adjacent layers are linked through weak van der Waals inter\u00adactions, resulting in a three-dimensional supra\u00admolecular network.In the title complex, [Ni(C DOI: 10.1107/S1600536809033820/at2863Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536808020679/nc2107Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Weak C\u2014H\u22efO inter\u00adactions join the mol\u00adecules together into an infinite three-dimensional network.The asymmetric unit of the title compound, C DOI: 10.1107/S1600536809031924/jh2093Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536808020424/bi2288Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536810010147/bt5216Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The NiII atom lies on a center of inversion.The dithio\u00adcarbamate anions in the title compound, [Ni(C DOI: 10.1107/S1600536810006677/sj2734Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The four-coordinate CuII ion \u20132.881\u2005(3)\u2005\u00c5], which link the chains into a three-dimensional structure.The title compound, [CuEu DOI: 10.1107/S1600536808038476/pv2118Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The NiII ions are connected via the tetra\u00adfluoro\u00adterephthalate anions into a one-dimensional chain running along the crystallographic [011] direction.In the title compound, [Ni(C DOI: 10.1107/S1600536808016619/cv2417Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Inter\u00admolecular N\u2014H\u22efO hydrogen bonds link the mol\u00adecules related by translation along the b axis into two independent hydrogen-bonded chains. The crystal studied exhibited inversion twinning.The title compound, C DOI: 10.1107/S1600536808014232/cv2412Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Mol\u00adecules are linked by Cl atoms to form a zigzag chain extending in the c direction.In the title compound, [Bi(C DOI: 10.1107/S1600536808032820/su2063Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Both cations are completed by crystallographic inversion symmetry, generating almost regular ZnO6 octa\u00adhedra. In the crystal, the cations, anions and uncoordinated water mol\u00adecules are linked by O\u2014H\u22efO hydrogen bonds, forming a three-dimensional network.The asymmetric unit of the title hydrated mol\u00adecular salt, [Zn(H DOI: 10.1107/S1600536809048375/hb5229Isup2.hklStructure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "The crystal structure is stabilized by N\u2014H\u22efBr hydrogen bonds.The tin(IV) atom in the salt, (C DOI: 10.1107/S1600536808013561/bt2709Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Each AgI ion shows a linear AgN2 coordination. The ions are linked by N\u2014H\u22efO hydrogen bonds.The asymmetric unit of the title compound, [Ag(C DOI: 10.1107/S1600536809051459/ci2975Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the title compound, [Co(C DOI: 10.1107/S1600536809007387/ng2552Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536808002687/su2042Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The ligands bridge the Zn atoms to form a two-dimensional -network.In the title compound, [Zn(C DOI: 10.1107/S1600536808023982/bt2755Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In addition, the crystal structure is stabilized by numerous O\u2014H\u22efO and N\u2014H\u22efO hydrogen bonds.In the title three-dimensional coordination polymer, {[Fe(C DOI: 10.1107/S1600536808006326/gk2125Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The Schiff base ligand coordinates via the imine N and pyridine N atoms. The CoII atom lies on a crystallographic twofold rotational axis. Non-classical inter\u00admolecular C\u2014H\u22efO hydrogen bonds link the complex mol\u00adecules into chains along [001].In the title complex, [Co(NCS) DOI: 10.1107/S1600536809029006/pv2186Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536810038043/gk2299Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the crystal, cations associate via N\u2014H\u22efO hydrogen bonds involving the ammonium and carbonyl residues, forming a 14-membered {\u22efHNC2NCO}2 synthon. The cations and anions are arranged in alternating layers arranged along the a-axis direction, the major association between them being N\u2014H\u22efBr contacts.In the title salt, (C DOI: 10.1107/S160053680904687X/hb5214Isup2.hklStructure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536809006370/ez2159Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536809043359/hb5097Isup2.hklStructure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "The Pd atom is located on a crystallographic twofold rotation axis: thus, there is just one half-mol\u00adecule in the asymmetric unit. The structure is isomorphous with the platinum analogue cis-[PtCl2{P(OMe)2Ph}2].The title compound, [PdCl DOI: 10.1107/S1600536810006471/bt5195Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The asymmetric unit contains one half mol\u00adecule in which the Pd atom lies on a centre of symmetry.In the title complex, [Pd(C DOI: 10.1107/S1600536809016390/rk2144Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536809004243/tk2371Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The ZnII atom lies on an inversion center in a distorted octa\u00adhedral environment with two planar trans-related N,N\u2032-chelating 2-[5-(2-pyrid\u00adyl)-2H-tetra\u00adzol-2-yl]acetic acid ligands in the equatorial plane and two water mol\u00adecules in the axial positions. In the crystal, O\u2014H\u22efO hydrogen bonds generate an infinite three-dimensional network.The title compound, [Zn(C DOI: 10.1107/S1600536809023940/dn2467Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the title compound, C DOI: 10.1107/S1600536808025312/pv2092Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536809030700/hy2214Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The structure has been published previously but H atoms were not included in the model [Raston & White \u00b0. One ethyl group is disordered over two positions of equal occupancy.The title compound, [Sb(Chite 1976. Chem. S DOI: 10.1107/S1600536809005303/fi2068Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The crystal structure involves inter\u00admolecular C\u2014H\u22efN hydrogen bonds.In the centrosymmetric title dinuclear zinc(II) compound, [Zn DOI: 10.1107/S1600536808013184/bq2072Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536808025531/ez2133Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The NiII atom (site symmetry trans-NiN2O4 octa\u00adhedral geometry defined by two monodentate N-bonded and two bidentate O,O\u2032-bonded ppa monoanions. The extended two-dimensional structure is a square grid. An inter\u00admolecular N\u2014H\u22efO hydrogen bond occurs.The title compound, [Ni(C DOI: 10.1107/S1600536809055408/hb5292Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The complex mol\u00adecule has a \u03bc-dialkoxo-bridged binuclear structure with both CuII centers exhibiting a square-planar coordination geometry.The title complex, [Cu DOI: 10.1107/S1600536808024707/gk2162Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Each bia ligand acts as bridge between CdII ions, forming one-dimensional coordination polymers along [010], with a shortest Cd\u22efCd distance of 4.27\u2005(2)\u2005\u00c5.In title compound, [Cd(C DOI: 10.1107/S1600536808041044/bi2328Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The dihedral angle between adjacent benzene rings is 70.33\u2005(12)\u00b0.In the title compound, [Cu(C DOI: 10.1107/S160053680900796X/bq2120Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The dihedral angle between the pyridine and benzene rings is 8.04\u2005(17)\u00b0.In the title complex, [ZnBr DOI: 10.1107/S1600536809025653/lh2845Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536809031596/hb5040Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The Br- counter- anion is also located on an inversion centre.In the title compound, [CoBr DOI: 10.1107/S160053680904166X/is2468Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The two phenolate rings form a dihedral angle of 10.53\u2005(2)\u00b0.In the title complex, [Co(C DOI: 10.1107/S1600536808034144/gw2053Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The Zn atom is five coordinated by two azide anions and three nicotinate N-oxide ligands. Each nicotinate N-oxide bridges three Zn atoms, whereas the azide bridges two Zn atoms, resulting in the formation of a two-dimensional metal\u2013organic polymer developing parallel to (100).The title compound, [Zn(C DOI: 10.1107/S1600536808039275/dn2407Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Two of the three Lu atoms occupy special positions (Wyckoff positions 3a and 3b, site symmetry c axis, with a repeat period of 12 PO4 tetra\u00adhedra, joined with LuO6 octa\u00adhedra.A new polymorph of lutetium polyphosphate, Lu(PO DOI: 10.1107/S1600536808021995/fi2065Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536807067773/hg2363Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The structure contains isolated neutral complexes, which are linked by O(water)\u2014H\u22efN hydrogen bonds generating chains along [010].In the title compound, [Mn(C DOI: 10.1107/S1600536808041755/fj2176Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the crystal structure, inter\u00admolecular N\u2014H\u22efO hydrogen bonds link mol\u00adecules into one-dimensional chains propagating along the b axis direction.In the title complex, [Cu(C DOI: 10.1107/S1600536808001803/lh2590Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The Zn atom is four-coordinated by two O and two N atoms from two Schiff base ligands, forming a severely distorted square-planar geometry. The central C atom of the propyl group is disordered over two positions about the twofold axis.The title mononuclear zinc(II) complex, [Zn(C DOI: 10.1107/S1600536808015602/rz2218Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the crystal structure, mol\u00adecules are linked through inter\u00admolecular N\u2014H\u22efO hydrogen bonds, forming chains running along the a axis.In the title centrosymmetric mononuclear nickel(II) complex, [Ni(C DOI: 10.1107/S1600536808023684/sj2526Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The ligand L adopts an N:N\u2032-bidentate bridging mode in a trans configuration, bridging the Cu atoms via translation symmetry, forming a two-dimensional layer-like structure. The perchlorate ions serve as acceptors for inter\u00admolecular C\u2014H\u22efO hydrogen bonds, which link the layers into a three-dimensional network. The ClO4 \u2212 anion is disordered with an occupation ratio of 0.658:0.342.In the title compound, {[Cu(C DOI: 10.1107/S1600536809008708/su2101Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536809019795/xu2530Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The metal atom lies on a special position of The metal atom of the title salt, [Co(C DOI: 10.1107/S1600536809051423/ci2949Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the crystal, mol\u00adecules are packed in layers parallel to the (101) plane.In the title molecular salt, C DOI: 10.1107/S1600536809054026/vm2016Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The complex has a bridging bis-bidentate carboxyl\u00adate group resulting in a zig-zag chain structure parallel to [001]. The Sn atom is six-coordinated and displays a distorted octa\u00adhedral geometry.The title complex, [Sn(CH DOI: 10.1107/S1600536809004097/bx2192Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the crystal, mol\u00adecules are linked by N\u2014H\u22efBr hydrogen bonds, generating sheets containing R 2 2(8) loops.In the title compound, [ZnBr DOI: 10.1107/S1600536809043694/hb5146Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Non-classical inter\u00admolecular C\u2014H\u22efO hydrogen bonds link the complex into a three-dimensional supra\u00admolecular framework.In the title compound, [Zn(C DOI: 10.1107/S160053680904001X/lx2113Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The two cyclo\u00adhexane rings assume the typical chair conformation. No hydrogen bonding is observed in the crystal structure.In the title compound, [Ti(C DOI: 10.1107/S1600536808002870/xu2396Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "\u00c5], giving a three-dimensional supra\u00admolecular framework.The title compound, {[Cd(C al. 2009. J. Coor DOI: 10.1107/S1600536810036147/bg2362Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The three independent halogen atoms are each a mixture of bromine and chlorine atoms . An N\u2014H\u22ef hydrogen bond is present.The Sn atom in the title salt, (C DOI: 10.1107/S1600536809019734/tk2458Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The crystal structure involves O\u2014H\u22efO hydrogen bonds.In the title compound, [Zn(C DOI: 10.1107/S1600536808015742/hy2133Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S160053680803643X/at2661Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "II atom in the title compound, [Pb(C10H8NO)2], is chelated by two oxine (2-methyl\u00adquinolin-8-olate) anions in a \u03a8-trigonal\u2013bipyramidal geometry; the N atoms occupy the axial sites. The mol\u00adecule lies about a twofold rotation axis.The Pb DOI: 10.1107/S1600536810012742/bt5241Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Adjacent Sn atoms are bridged by coordination to the two O atoms of each 2-chloro\u00adphenyl\u00adacetato ligand, forming a chain structure. In the title polymeric coordination compound, [Sn(CH DOI: 10.1107/S1600536809038872/sj2651Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536809033479/hb5057Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "A symmetry-generated cpp ligand completes the CoN2O3 trigonal-bipyramidal geometry for the metal ion, with the N atoms occupying both equatorial and axial sites. The bridging cpp ligands form chains propagating in [110] and O\u2014H\u22efO hydrogen bonds consolidate the packing.In the title compound, [Co(C DOI: 10.1107/S1600536809035089/hb5085Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The four water O atoms in the equatorial plane around the ZnII centre (The asymmetric unit of the title compound, [Zn(C DOI: 10.1107/S1600536809031766/hk2732Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The 4,4\u2032-bipyridine (bipy) ligands are highly twisted with respect to each other, the dihedral angle between the two pyridyl ring planes being 38.9\u2005(2)\u00b0. The bipy ligands act as bridging ligands between [Cu(ClO4)2] units, generating an infinite one-dimensional zigzag chain along [010]. Intra- and intermolecular C\u2014H\u22efO hydrogen bonds are present in the crystal structure.The central CuN DOI: 10.1107/S1600536808021296/im2074Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536808015080/sj2502Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536808020928/rk2094Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the crystal structure, mol\u00adecules are linked through inter\u00admolecular N\u2014H\u22efO hydrogen bonds, forming dimers.In the title complex, [ZnI DOI: 10.1107/S1600536809040495/om2281Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The crystal structure is stabilized by inter\u00admolecular C\u2014H\u22efO hydrogen bonds.The title compound, [Zn(C DOI: 10.1107/S1600536809032784/hg2551Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The crystal packing is stabilized by hydrogen bonds involving deprotonated HCt ligands, coordinated water mol\u00adecules and uncoordinated solvent water mol\u00adecules.The title mononuclear copper(II) complex, [Cu(C DOI: 10.1107/S160053680802549X/bg2201Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The pyridyl H atoms form strong N\u2014H\u22efO hydrogen bonds with the carbonyl O atoms of the adjacent mol\u00adecules, generating a chain structure propagating in [100].In the title complex, [Mn(C DOI: 10.1107/S160053681001514X/pv2275Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536808001487/xu2400Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "O\u2014H\u22efO hydrogen bonds link the mol\u00adecules together.In the title compound, [Pd(C DOI: 10.1107/S1600536808016097/cf2196Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The Jahn\u2013Teller-distorted manganese(III) centre has an octa\u00adhedral geometry. Extensive O\u2014H\u22efO hydrogen-bonding inter\u00adactions generate a two-dimensional sheet structure parallel to (103).The title compound, [Mn(C DOI: 10.1107/S1600536809032553/bt5032Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536809006771/xu2482Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The structure is stabilized by inter\u00admolecular O\u2014H\u22efO hydrogen bonds.The complete molecule of the title compound, C DOI: 10.1107/S160053680903997X/sj2660Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The asymmetric unit onsists of two half-molecules; each Ni atom lies on a centre of symmetry. The NiII ions are coordinated by four O atoms from two deprotonated 3-ethoxy\u00adsalicylaldehyde ligands in a slightly distorted square-planar coordination environment.The title compound, [Ni(C DOI: 10.1107/S160053680800809X/lh2604Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The Cu atom is located on a twofold crystallographic symmetry axis and is coordinated by four N atoms in a slightly distorted square-planar geometry. Inter\u00admolecular N\u2014H\u22efO hydrogen bonds are present.The title complex, [Cu(C DOI: 10.1107/S1600536809001664/hg2468Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The bridging norf anion results in one-dimensional chains propogating along [010]. A network of O\u2014H\u22efO and N\u2014H\u22efO hydrogen bonds helps to establish the crystal structure.In the title compound, {[Cu(C DOI: 10.1107/S1600536808026469/hb2782Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The dihedral angle between the pyridine and triazole rings is 41.73\u2005(8)\u00b0. In the crystal structure, mol\u00adecules are hydrogen bonded via triazole NH groups and pyridyl N atoms, forming chains parallel to the a axis.The title compound, C DOI: 10.1107/S1600536808011744/lh2615Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The CoII atoms are bridged by ligands, generating a two-dimensional -network. Adjacent fishnet planes are linked to the nitrate anions via O\u2014H\u22efO hydrogen bonds, forming a three-dimensional supra\u00admolecular structure.In the title compound, {[Co(C DOI: 10.1107/S1600536809005881/ng2547Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The zinc(II) metal centre has a slightly distorted tetra\u00adhedral coordination geometry.In the title compound, (C DOI: 10.1107/S1600536809014822/rz2310Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The structure contains two distinct octa\u00adhedral Ga(OH2)6 units (each of The title compound, [Ga(H DOI: 10.1107/S1600536809028086/mg2076Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S160053680800425X/hg2378Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S160053681000680X/hb5336Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The NiII atom is six-coordinated by the phenolate O, imine N and pyridine N atoms from two tridentate Schiff base ligands in a distorted NiN4O2 octa\u00adhedral geometry. The dihedral angles between the noncoordinated pyridyl rings of each ligand are 72.95\u2005(8) and 69.59\u2005(7)\u00b0.The title compound, [Ni(C DOI: 10.1107/S160053680900244X/hb2899Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The bridging bidentate di-4-pyridyl sulfide ligands link the CoII centres into a three-dimensional network. The four coordinating pyridine groups are donors and acceptors (N atoms) for intra\u00admolecular C\u2014H\u22efN and C\u2014H\u22efCl hydrogen bonds. In the title compound, [CoCl DOI: 10.1107/S1600536809001676/si2149Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Each 2,6-dimethyl\u00adpyridinium-3,5-dicarboxyl\u00adate anion bridges two Cu atoms, forming a two-dimensional coordination polymer. A three-dimensional supra\u00admolecular network is built from N\u2014H\u22efO hydrogen bonds involving the pyridinium NH and the carboxyl COO groups.In the title coordination polymer, [Cu(C DOI: 10.1107/S1600536808035873/ng2511Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The central ring adopts a chair conformation and the dihedral angle between the aromatic rings is 56.69\u2005(4)\u00b0.The complete molecule of the title compound, C DOI: 10.1107/S1600536809049617/hg2598Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536809010204/hb2924Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536808010878/hb2722Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The benzene-1,3,5-tricarboxyl\u00adate anion bridges two ZnII atoms and two NaI atoms, resulting in the formation of a two-dimensional layer structure. Inter\u00admolecular O\u2014H\u22efO hydrogen-bonding inter\u00adactions generate a three-dimensional superamolecular structure.In the title compound, [NaZn(C DOI: 10.1107/S1600536810009232/jj2022Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The compound is an example of a five-coordinate silver complex containing a bidentate ligand.The title compound, [Ag(C DOI: 10.1107/S1600536809000890/ng2535Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536810011670/pb2026Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Both Cu(II) atoms show a similar slightly distorted square-planar coordination, resulting from four O atoms of two 2-methoxyphenolate anions.In the title compound, [Cu(C DOI: 10.1107/S160053680903582X/gw2068Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The chains constructed through the trans-related acetate groups of the ligand are inter-connected via O\u2014H\u22efO hydrogen bonds involving coordinated aqua ligands, the nitrate anions and the partial-occupancy (0.25) water mol\u00adecule of solvation, forming a three-dimensional structure.In the title polymeric coordination complex, {[Cd(C DOI: 10.1107/S1600536810036317/zs2059Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "N\u2014H\u22efN hydrogen bonds between the ligand and azide ion link the complex mol\u00adecules into a three-dimensional network.In the title mononuclear complex, [Zn(N DOI: 10.1107/S1600536809016055/hy2194Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the crystal structure, mol\u00adecules are linked into a two-dimensional network parallel to the bc plane by C\u2014H\u22efO hydrogen bonds.In the title centrosymmetric mononuclear nickel(II) complex, [Ni(C DOI: 10.1107/S1600536809004279/ci2768Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the crystal structure, inter\u00admolecular C\u2014H\u22efN hydrogen bonds link the molecules into a one-dimensional chain in the a+c direction.In the title compound, [Fe(C DOI: 10.1107/S1600536808023106/kj2092Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The compound is composed of extended polymeric chains in which two BTA N atoms bridge [Zn(BTA)2] fragments along [001]. Cations and anions are linked by N\u2014H\u22efN hydrogen-bond inter\u00adactions along [010].In the title compound, {(C DOI: 10.1107/S160053680902563X/bx2217Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the crystal structure, symmetry-related mol\u00adecules are linked by an N\u2014H\u22efS hydrogen bond.In the title mononuclear copper(II) complex, [Cu(C DOI: 10.1107/S1600536808016589/su2051Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S160053680901719X/lh2807Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536809035661/ng2628Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Two neighboring Co centers are bridged by an oxalate ligand, forming a one-dimensional chain structure.In the one-dimensional title coordination polymer, [Co(C DOI: 10.1107/S1600536809012690/hy2191Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The geometry around the AlIII atom is distorted tetra\u00adhedral, defined by two methyl groups, one N and one O atom from the Schiff base ligand.The title compound, [Al(CH DOI: 10.1107/S1600536808041858/hy2172Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The two O and two S atoms are cis to each other.In the title compound, [Ni(C DOI: 10.1107/S160053680900302X/gk2187Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the crystal structure, ions are linked by O\u2014H\u22efO hydrogen bonds.In the title compound, [Zn(C DOI: 10.1107/S1600536808025622/bt2766Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The structure contains pairs of gallium-centered tetra\u00adhedra connected through a shared oxygen vertex. Orthoborate triangles connect the basal vertices of the tetra\u00adhedra, forming a three-dimensional network with voids occupied by rubidium ions.The title compound, Rb DOI: 10.1107/S1600536808005783/mg2048Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The chains are cross-linked by O\u2014H\u22efO hydrogen bonds, forming sheets.In the title compound, [Zn(C DOI: 10.1107/S1600536808006764/hb2701Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The agents blocked L1210 leukemic cell DNA and RNA syntheses by inhibitingmultiple enzyme activities for nucleic acid synthesis, e.g. PRPP amido transferase, IMP dehydrogenase, DNApolymerase \u03b1, thymidine kinase, and TMP kinase. The copper (II) complex 3 demonstrated improved abilityto inhibit L1210 partially purified DNA topoisomerase II compared to the parent compound while the sodiumsalt was inactive at 100 \u03bcM.Sodium N-[(trimethylamineboryl)-carbonyl]-L-phenylalanine"} +{"text": "The two Ni atoms are bridged by two phenolate O atoms, forming a four-membered Ni2O2 ring.In the title centrosymmetric dinuclear nickel complex, [Ni DOI: 10.1107/S1600536809010174/hy2188Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The chains aggregate into pseudo-layers parallel to the (101) crystal planes by N\u2014H\u22efO hydrogen bonding. Unligated perchlorate anions and water mol\u00adecules of crystallization provide additional hydrogen bonding between pseudo-layers.In the title compound, {[Cu(C DOI: 10.1107/S1600536808036490/lh2730Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Each L ligand bridges symmetry-related ZnII ions, forming a two-dimensional layer with a grid. In the crystal structure, inter\u00admolecular O\u2014H\u22efO hydrogen bonds connect perchlorate counter-anions to the layers.In the title compound, {[Zn(C DOI: 10.1107/S1600536809013130/lh2801Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the crystal structure, the metal atoms are linked by carboxyl\u00adate bridges into polymeric chains extending along the b axis.In the title compound, [Sn(CH DOI: 10.1107/S1600536808040798/rz2273Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The resulting structure is a two-dimensional polymer with layers in the (100) plane.In the the title compound, [Co(C DOI: 10.1107/S160053681000646X/bt5196Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The dinuclear mol\u00adecule lies across a centre of inversion. The solvent dichloro\u00admethane mol\u00adecule is disordered about a centre of inversion. The dinuclear centrosymmetric title compound, [Sn DOI: 10.1107/S1600536808025890/ng2481Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Inter\u00admolecular O\u2014H\u22efO hydrogen bonding is observed between the terminal hydr\u00adoxy groups in the crystal structure.In the title compound, [Cu(C DOI: 10.1107/S160053680904999X/xu2681Isup2.hklStructure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "Two such MnIII ions are linked by a square-planar Ni(CN)4 unit, which lies on an inversion centre. A two-dimensional network is formed by O\u2014H\u22efO and O\u2014H\u22efN hydrogen bonds.In the title compound, [Mn DOI: 10.1107/S1600536808012749/cf2192Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The Eu atom and one Br atom each lie on a twofold rotation axis.The title compound crystallizes with the GdCl DOI: 10.1107/S1600536808014359/mg2051Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The Tm and both Cu atoms lie on mirror planes. The Tm atom is seven-coordinate with a capped distorted trigonal\u2013prismatic coordination geometry, while the Cu atoms adopt trigonal CuBrN2 and tetra\u00adhedral CuBr3N coordination modes, respectively. The Cu atom in the trigonal coordination environment is disordered over two sites of equal occupancy. The crystal structure is constructed from two distinct units of dimeric [Tm2(\u03bc2-OH(IN)6(H2O)4] cores (IN = isonicotinate) and one-dimensional inorganic [Cu4Br3]n chains, which are linked together, forming heterometallic Cu\u2013halide\u2013lanthanide\u2013organic layers.A new thulium(III)\u2013copper(I) heterometallic coordination polymer, [Cu DOI: 10.1107/S1600536808028675/sj2536Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536808010763/cv2399Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536809034916/pv2195Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The zinc(II) metal centre displays a distorted octa\u00adhedral coordination environment provided by N atoms of two bidentate chelating and two monodentate 5-(2-amino\u00adphen\u00adyl)tetra\u00adzolate ligands. These ligands act as bridges, linking adjacent Zn atoms into polymeric criss-crossed chains parallel to the [110] and [The polymeric title compound, [Zn(C DOI: 10.1107/S1600536808021946/rz2234Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The ribbons are connected by C\u2014H\u22efO hydrogen bonds and van der Waals inter\u00adactions.In the title structure, [Pb(C DOI: 10.1107/S1600536810002199/kp2247Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536809032413/hb5048Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The four O atoms belonging to two 2,4-penta\u00adnedionate anions lie in the equatorial plane and the two N atoms occupy the axial coordination sites.In the title compound, [Mn(C DOI: 10.1107/S1600536808038440/hb2842Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536809042160/pv2218Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The 1,1\u2032-diimidazole ligand and the cyclo\u00adhexane-1,4-dicarboxyl\u00adate dianion bridge metal centres, forming a two-dimensional network. The network is consolidated by O\u2014H\u22efO hydrogen bonds between the statistically occupied water molecules and O atoms of the two carboxylate groups.In the title coordination polymer, {[Ni(C DOI: 10.1107/S1600536809004024/bt2863Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "One C atom of one ethyl group is disordered with site occupancies of 0.61\u2005(3):0.39\u2005(3).The Sn atom of the title compound, [Sn(C DOI: 10.1107/S1600536808032388/tk2313Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The cations lie in sheets parallel to (The asymmetric unit of the title compound, (C DOI: 10.1107/S1600536808043523/bt2834Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536809034795/hb5080Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The coordination bonds give rise to a layer structure parallel to (010).In the title coordination polymer, [CuI(C DOI: 10.1107/S1600536808031188/ng2496Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536808007472/cs2070Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The zigzag chains, extending parallel to [011], are further packed into a three-dimensional network by hydrogen bonds.The title compound, {[Zn(C DOI: 10.1107/S1600536808035514/bq2094Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Zh. Str DOI: 10.1107/S1600536808009719/fi2061Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Individual [Zn(N3)2(dpa)]n chains are connected into supra\u00admolecular layers via N\u2014H\u22efN hydrogen bonding between the central amine groups of the dpa ligands and terminal unligated azide N atoms. The azide ligands in one supra\u00admolecular layer penetrate through the neighboring layers above and below, allowing stacking into a three-dimensional structure.In the title compound, [Zn(N DOI: 10.1107/S1600536809051599/zl2257Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the crystal packing, the arrays are linked by adjacent ring motifs, together with additional inter\u00admolecular O\u2014H\u22efO inter\u00adactions, into a three-dimensional network.A monoclinic polymorph of the title compound, [Na(Chino 1993. Bull. C DOI: 10.1107/S1600536810000681/tk2611Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The two Cu atoms, one with an N2P2 ligand set and the other with an NP2 ligand set, are bridged by two bis\u00ad(diphenyl\u00adphosphino)methane ligands, forming an eight-membered ring.The title dinuclear copper(I) complex, {[Cu DOI: 10.1107/S1600536809040896/jh2105Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536809032048/hb5044Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Two cations are linked into a centrosymmetric dimer via three bridging carboxyl\u00adate groups of 2,4-difluoro\u00adbenzoate ligands. Each Gd3+ ion is nine-coordinated by seven O atoms and two N atoms.In the title compound, [Gd DOI: 10.1107/S1600536808004431/cf2184Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Each cation links two [PMo12O40]3\u2212 anions, which link three cations through N\u2014H\u22efO hydrogen bonds, generating an infinite supra\u00admolecular chain-like structure.In the title compound, (C DOI: 10.1107/S1600536808014463/cs2077Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536810003727/sj2718Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The Mn atom lies on an inversion centre. The distorted octa\u00adhedral Mn environment contains two planar trans-related N,N\u2032-chelating 5-(2-pyrid\u00adyl)\u00adtetra\u00adzolate ligands in the equatorial plane and two axial water mol\u00adecules. O\u2014H\u22efN hydrogen bonds generate an infinite three-dimensional network.The title compound, [Mn(C DOI: 10.1107/S1600536808010106/dn2338Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The CuII ion is in a slightly distorted square-pyramidal coordination environment. In the crystal structure, inter\u00admolecular O\u2014H\u22efO hydrogen bonds connect complex mol\u00adecules into chains along [001].The title complex, [Cu(C DOI: 10.1107/S1600536808029449/lh2688Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The two 4,4\u2032-ethyl\u00adenedibenzoate dianions are located on inversion centres bridging two neighboring CdII centres. O\u2014H\u22efO hydrogen-bonding inter\u00adactions further stabilize the crystal structure. The DMF molecule is equally disordered about a center of inversion.In the title compound, [Cd(C DOI: 10.1107/S1600536809024714/bt2984Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The new \u03b2-polymorph is 1.05 times denser than the previously known polymorph [Rae et al. was obtained for an active pharmaceutical ingredient, bis\u00admuth tribenzoate, [Bi(C al. 1998. Acta Cr DOI: 10.1107/S1600536810035543/rk2224Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the crystal, O\u2014H\u22efO hydrogen bonds help to establish the packing.In the title compound, [Zn(C DOI: 10.1107/S1600536809034473/hb5073Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The coordination around mercury is completed by two bromido ligands resulting in a distorted tetra\u00adhedral arrangement.In the title polymeric complex, [HgBr DOI: 10.1107/S1600536808030055/si2110Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Each NiII atom is chelated by two oxalate ligands and one 2,2\u2032-bipyridine, forming a slightly distorted octa\u00adhedral geometry. Oxlate acts as a bridge to link neighbouring pairs of NiII cations, forming a one-dimensional wave-like chain. The crystal showed partial inversion twinning.The title compound, [Ni(C DOI: 10.1107/S1600536808028389/cf2213Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the crystal, associations of two cations and one anion linked by N\u2014H\u22efBr hydrogen bonds occur.In the title compound, (C DOI: 10.1107/S160053680901705X/hb2965Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The dihedral angle between rings of anion is 86.1\u2005(1)\u00b0.In the crystal of the title hydrated salt, C DOI: 10.1107/S1600536809033856/ng2622Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536809020340/tk2467Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536809041919/fi2085Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536809030943/bh2240Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The Nb atom is situated on a site of symmetry 6 \u2212 anion has crystallographic fourfold symmetry.The title complex, [Nb(C DOI: 10.1107/S1600536808026627/lx2067Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the crystal structure, O\u2014H\u22efO hydrogen bonds lead to associations of one metal complex and two diglyme mol\u00adecules.In the title 1:2 adduct, [SnCl DOI: 10.1107/S1600536808029917/bt2792Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In addition, the dinuclear species is stabilized by two hydrogen-bonded perchlorate anions.Each Cu atom in the dinuclear centrosymmetric title complex, [Cu DOI: 10.1107/S1600536807068663/tk2238Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Inter\u00admolecular N\u2014H\u22efN hydrogen bonds link neighboring molecules into extended chains parallel to [100].In the title complex, [Ni(C DOI: 10.1107/S1600536808028171/bv2105Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The axial thio\u00adcyanate ligands act in a \u03bc1,3-bridging mode to connect symmetry-related CdII ions into one-dimensional chains along [010]. In addition, there are inter\u00admolecular C\u2014H\u22efO contacts between chains.In the title crystal structure, [Cd(NCS)(NO DOI: 10.1107/S1600536808032297/lh2703Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The four metal ions in the cluster are held together by four bridging hydroxide groups. Each NiII atom adopts a distorted octa\u00adhedral geometry. The title complex, [Ni DOI: 10.1107/S1600536810003697/rz2413Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The NiII atoms are linked to each other, forming an infinite chain parallel to (In the title complex, [Ni(C DOI: 10.1107/S1600536808030377/dn2372Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "This results in alternating corrugated inorganic and organic layers in the crystal.In the title mol\u00adecular salt, (C DOI: 10.1107/S160053681001812X/hb5451Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The ZnII ion is coordinated by two bpp and two ip ligands in a distorted tetra\u00adhedral environment. Each ligand coordinates in a bridging mode to connect ZnII ions into a three-dimensional diamondoid-type structure.The title compound, [Zn(C DOI: 10.1107/S1600536808001621/lh2583Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The Cu atom is 0.1801\u2005(11)\u2005\u00c5 above the N3O mean basal plane.The Cu atom of the title complex, [Cu(C DOI: 10.1107/S1600536809014573/rk2135Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "One of the sulfonate O atoms is disordered over two positions [ratio 0.70\u2005(5):0.30\u2005(5)].In the title compound, [Pd(C DOI: 10.1107/S1600536809032760/bx2229Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S160053680902234X/bh2230Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The ReI atoms are in a slightly distorted octa\u00adhedral environment, whereas the ReVII atoms show the typical tetra\u00adhedral coordination mode. The dihedral angle between the two bipyridine groups is 34.3\u2005(7)\u00b0.The title compound, [Re DOI: 10.1107/S160053680800490X/at2546Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The ZnII atoms show distorted tetrahedral coordination environments. Adjacent mol\u00adecules are linked by N\u2014H\u22efCl hydrogen bonds, forming a three-dimensional network.The title compound, [ZnCl DOI: 10.1107/S1600536809052738/bt5133Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The P and one S atom of each trimethyl trithio\u00adphosphite ligand are employed for coordination. The mol\u00adecular structure exhibits the rare motif of copper(I) bridged by two trifluoro\u00admethane\u00adsulfonate anions generating eight-membered rings.The title compound, [Cu DOI: 10.1107/S1600536808041809/lh2742Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The tethering 3-bpmp ligands promote the formation of [ZnCl2(3-bpmp)]n chains situated parallel to (via C\u2014H\u22efCl inter\u00adactions to form supra\u00admolecular layers, which in turn stack to construct the three-dimensional crystal structure.In the title compound, [ZnCl DOI: 10.1107/S1600536809013877/ng2573Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The coordinated water mol\u00adecule plays an important role in crystal packing consolidation via O\u2014H\u22efO hydrogen bonding.In the title compound, [Sn DOI: 10.1107/S1600536808043614/cv2496Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Each metal atom is chelated by two Schiff base anions in a distorted square-planar coordination environment.The asymmetric unit of the crystal structure of the title compound, [Ni(C DOI: 10.1107/S1600536809020790/xu2537Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The N-heterocyclic carbene (NHC) metallacrown ether ligand binds to the Pd atom in a trans orientation through the carbene C atoms of the two imidazole rings and generates a 25-membered chelate ring. Two mutually trans S-bound thio\u00adcyanate ligands complete the coordination.The coordination geometry of the Pd atom in the title compound, [Pd(SCN) N-heterocyclic carbene ligands and their complexes, see: Herrmann ] = 0.066 wR(F 2) = 0.218 S = 1.05 8672 reflections566 parameters61 restraintsH-atom parameters constrainedmax = 0.78 e \u00c5\u22123 \u0394\u03c1min = \u22121.31 e \u00c5\u22123 \u0394\u03c1 DIFRAC used to solve structure: SHELXS97 (Sheldrick, 2008SHELXL97 (Sheldrick, 2008ORTEP-3 for Windows (Farrugia, 1997SHELXL97.Data collection: 10.1107/S1600536809014615/sj2617sup1.cif Crystal structure: contains datablocks I, global. DOI: 10.1107/S1600536809014615/sj2617Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Three of the C atoms of the n-butyl group are disordered over two sites with equal occupancies.The Sn atom in the title compound, [Sn(C DOI: 10.1107/S1600536810005830/lh2995Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The oxalate and pyrazine-2-carboxyl\u00adate ligands bridge the CeIII ions, forming a two-dimensional structure. In addition, inter\u00admolecular O\u2014H\u22efO and O\u2014H\u22efN hydrogen bonds connect the two-dimensional structure into a three-dimensional network.In the hydro\u00adthermally synthesized title compound, [Ce(C DOI: 10.1107/S1600536808029164/lh2687Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The Mn2+ cation is surrounded by six N atoms from three chelating 3-(2-pyrid\u00adyl)-1H-pyrazole ligands in a distorted octa\u00adhedral coordination. In the heteropolyanion, two O atoms of the central PO4 group (The asymmetric unit of the title compound, [Mn(C DOI: 10.1107/S160053681000320X/wm2301Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The structural parameters compared with those in [(\u03b75-Me5Cp)2Zr(TePh)2] [Howard, Trnka & Parkin and by more acute Zr\u2014Te\u2014C (\u223c5\u00b0) angles, although the Zr\u2014Te distances are essentially the same. The crystal studied exhibited some inversion twinning.The structure of the title compound, [Zr(Crkin 1995. Inorg. DOI: 10.1107/S1600536808009574/sj2480Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The imino NH groups participate in inter\u00admolecular N\u2014H\u22efO hydrogen bonds.The title complex, [Zn(NO DOI: 10.1107/S1600536809037672/fj2244Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The eight-coordinate geometry approximates a square anti\u00adprism. Hydrogen bonds of the O\u2014H\u22efO type connect the uncoordinated water mol\u00adecules to the dinuclear species, forming a three-dimensional network.The title dinuclear compound, [Tb DOI: 10.1107/S1600536809052489/xu2706Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536808013792/im2062Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Two of the water mol\u00adecules are disordered over three sites in a 0.68:0.55:0.77 ratio.The crystal structure of the title compound, (C DOI: 10.1107/S1600536810018325/bt5270Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "A slightly distorted trigonal-bipyramidal SnC3O2 coordination geometry arises for the metal, with the O atoms in the axial sites. Weak C\u2014H\u22efO hydrogen bonds help to stabilize the packing.The polymeric structure of the title compound, [Sn(CH DOI: 10.1107/S1600536809051587/hb5258Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536808002341/dn2314Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The Sn atom is six-coordinate with a distorted octa\u00adhedral geometry. Additional O\u2014H\u22efO hydrogen bonding leads to stabilization of the crystal structure.The title compound, [Sn DOI: 10.1107/S1600536808032832/sg2266Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Each Sb atom exhibits a slightly distorted trigonal-bipyramidal geometry, with the O atom in the apical site. The crystal structure is stabilized by inter\u00admolecular C\u2014H\u22efO hydrogen bonds, forming a three-dimensional network.The asymmetric unit of the title compound, [Sb(C DOI: 10.1107/S1600536808040816/rz2272Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The complex features a Pt(II) atom coordinated by two Cl and two P atoms, yielding a slightly distorted cis square-planar geometry.The title compound, [PtCl DOI: 10.1107/S1600536809042226/fi2086Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536809000270/at2697Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The five-membered chelating glycinate ring assumes an envelope conformation. The complex mol\u00adecules are assembled by inter\u00admolecular N\u2014H\u22efO hydrogen bonding.In the crystal structure of the title compound, [Co(C DOI: 10.1107/S1600536808013135/xu2418Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials: interactive version of Fig. 3 Enhanced figure:"} +{"text": "The Zn atom lies on a special position of site symmetry 2.The Zn atom in the title compound, [Zn(C DOI: 10.1107/S1600536808002213/hg2370Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The crystal packing is consolidated by additional O\u2014H\u22efO and N\u2014H\u22efO hydrogen bonds.The novel pentaborate with a transition-metal complex as counter-cation and with water of crystallization, tris(ethylenediamine)cobalt(II) bis dihydrate, [Co(C DOI: 10.1107/S1600536809007296/pv2140Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "All interatomic distances, angles and the hydrogen bond geometry are very similar for the three structures.The title compound, [Tb(C DOI: 10.1107/S1600536809001494/at2682Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The dihedral angle between the planes of the two NiNC3O chelate rings is 14.37\u2005(12)\u00b0.In the title complex, [Ni(C DOI: 10.1107/S1600536809019965/bt2968Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The hydro\u00adcarbon chains are in the fully extended all-trans conformation and are arranged in a tail-to-tail double bilayer.The structure of the title compound, poly[di-\u03bc-penta\u00adnoato-zinc(II)], [Zn{CH DOI: 10.1107/S1600536808008283/cf2188Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536810010895/bt5207Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the crystal, mol\u00adecules are linked by N\u2014H\u22efCl hydrogen bonds, generating (100) sheets containing R 2 2(8) loops.In the title compound, [ZnCl DOI: 10.1107/S1600536810012274/hb5384Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In common with the monoclinic polymorph [Howie et al. hydrogen-bonding inter\u00adactions generate a three-dimensional network.The asymmetric unit of the title hydrate, 2[Sn(H al. 2005. Inorg. DOI: 10.1107/S1600536810006021/hb5334Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S160053680900988X/bx2197Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials: interactive version of Fig. 1 Enhanced figure:"} +{"text": "The acetate bridges link adjacent CuII cations, forming a chain. The crystal structure involves O\u2014H\u22efO hydrogen bonds.In the title compound, [Cu(CH DOI: 10.1107/S1600536808016073/cf2197Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The Cd atoms are connected by two dicyanamide ligands, resulting in a neutral chain propagating parallel to [010].In the title compound, [Cd(C DOI: 10.1107/S1600536809046364/pv2226Isup2.hklStructure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "The molecules are assembled via inter\u00admolecular N\u2014H\u22efO hydrogen-bonding inter\u00adactions into a three-dimensional network.In the title mononuclear complex, [Co(C DOI: 10.1107/S1600536808005722/ng2407Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Weak C\u2014H\u22efO contacts generate dimeric units via crystallographic inversion centres.In the title complex, [Sb(C DOI: 10.1107/S1600536808029632/si2109Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The Mn^II^ cation has a distorted octahedral coordination geometry. The 1,3-di(tetra\u00adzol-5-yl)benzene ligand is planar. All H atoms bonded to O atoms participate in hydrogen bonds, which link the mol\u00adecules into a framework structure.The title compound, [Mn(C Data collection: 10.1107/S1600536808028316/ez2139sup1.cif Crystal structure: contains datablocks global. DOI: 10.1107/S1600536808028316/ez2139Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The trans-N2S4 donor set defines a distorted octa\u00adhedral geometry.In the title complex, [Ni(C DOI: 10.1107/S1600536809050223/tk2572Isup2.hklStructure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "The word \"Mediates\" was misspelled in the article title. The correct title is: CX\u2083CR1 Is Expressed by Human B Lymphocytes and Mediates CX\u2083CL1 Driven Chemotaxis of Tonsil Centrocytes. The correct citation is: Corcione A, Ferretti E, Bertolotto M, Fais F, Raffaghello L, et al. (2009) CX\u2083CR1 Is Expressed by Human B Lymphocytes and Mediates CX\u2083CL1 Driven Chemotaxis of Tonsil Centrocytes. PLoS ONE 4(12): e8485. doi:10.1371/journal.pone.0008485"} +{"text": "The coordination of Cu(I) is slightly distorted tetrahedral.The title compound, [CuBr(C DOI: 10.1107/S1600536808012439/dn2345Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The isophthalate ligands bridge the metal ions to form polymeric zigzag chains extending along the b axis. Weak C\u2014H\u22efO inter\u00adactions contribute to the crystal packing stability.In the title compound, [Co(C DOI: 10.1107/S1600536808030778/rz2249Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Both Sn centres exhibit similar distorted trigonal\u2013bipyramidal [C3SnO2] coordination, with the O atoms of the carboyxlate ligands in trans positions.The title compound, [Sn(C DOI: 10.1107/S160053680802028X/wm2181Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The coordination geometry of the ZnII ion is slightly distorted square-planar, formed by two N atoms and two O atoms from the L ligand.The title compound, [Zn(C DOI: 10.1107/S1600536809038616/hg2565Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the crystal structure, weak bifurcated C\u2014H\u22efO hydrogen bonds involving the carbonyl O atoms as acceptors result in R 2 2(7) ring motifs.In the title compound, C DOI: 10.1107/S1600536810005404/lh2990Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The mol\u00adecules are linked together via hydrogen bonds involving the solvent water and methanol mol\u00adecules.In the title complex, [Gd(C DOI: 10.1107/S1600536808001931/om2208Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The SnO4NC2 centre has a distorted penta\u00adgonal\u2013bipyramidal geometry with the C atoms in the axial positions.The title compound, [Sn(CH DOI: 10.1107/S1600536807067165/hb2677Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The fully deprotonated PDC anion acts a \u03bc3-bridging ligand, establishing a chain structure along the a axis. These polymeric chains are connected into a three-dimensional framework via several inter\u00admolecular O\u2014H\u22efO hydrogen bonds.In the title coordination polymer, {[Mn(C DOI: 10.1107/S1600536808029413/sg2261Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Acta Cr DOI: 10.1107/S1600536810006306/hy2284Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The dihedral angle between the pyridine and benzene rings is 15.15\u2005(13)\u00b0.In the title compound, [ZnI DOI: 10.1107/S1600536808020680/rz2225Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The compound crystallizes with one CH2Cl2 mol\u00adecule per asymmetric unit. The benzyl\u00adamine ligand and the ReI centre form a non-planar six-membered chelate ring.In the crystal structure of the title compound, [ReCl(C DOI: 10.1107/S160053680706802X/zl2094Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The bond distances in the coordinated i-mnt ligands indicate some delocalization of the \u03c0-system.In the title compound, (C DOI: 10.1107/S1600536808037616/pk2131Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the title compound, C DOI: 10.1107/S1600536808025300/fj2138Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "C\u2014H\u22efO hydrogen bonds connect the complex mol\u00adecules into a three-dimensional supra\u00admolecular structure.In the title compound, [Zn(C DOI: 10.1107/S1600536808009483/hy2125Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Concomitant treatment in addition to intervention may influence the primary outcome, especially in complex interventions such as surgical trials. Evidence-based standards for perioperative care after distal pancreatectomy, however, have been rarely defined. This study's objective was therefore to identify and analyse the current basis of evidence for perioperative management in distal pancreatectomy.A standardised questionnaire was sent to 23 European centres recruiting patients for a randomized controlled trial (RCT) on open distal pancreatectomy that would compare suture versus stapler closure of the pancreatic remnant . Perioperative strategies were assessed. Moreover, a systematic literature search in the Medline database was performed and retrieved meta-analyses and RCTs were reviewed.All 23 centres returned the questionnaire. Consensus for thoracic epidural catheters (TECs), pain treatment and transverse incisions was found, as well as strong consensus for the placement of intra-abdominal drainages and perioperative single-shot antibiotics. Also, there was consensus that bowel preparation, somatostatin application, postoperative nasogastric tubes and intravenous feeding might not be beneficial. The literature search identified 16 meta-analyses and 19 RCTs demonstrating that bowel preparation, somatostatin therapy and nasogastric tubes can be omitted. Early mobilisation, feeding and TECs seem to be beneficial for patients. The value of drainages remains unclear.Most perioperative standards within the centres participating in the DISPACT trial are in accordance with current available evidence. The need for drainages requires further investigation.Clinical trial registration: ISRCTN 18452029 Short-term outcomes after major abdominal surgery are influenced by indication, surgical intervention, the surgeons' expertise, hospital volume and perioperative management -5. In orA recent study of 617 consecutive pancreatic resections demonstrated local complications to be more frequent than systemic complications . Thus, tTwenty-three European centres participating in the DISPACT trial were evaluated using a standardized questionnaire to evaluate perioperative management OR doubl* [tw] OR tripl* [tw]) AND (mask* [tw] OR blind* [tw])) OR placebos [MESH] OR placebo* [tw] OR random* [tw] OR research design [mh:noexp] OR Meta-Analysis [ptyp] OR systematic review [tw] NOT NOT case reports [pt] NOT Comment [PT] NOT letter [PT] NOT child [MESH] NOT infant [MESH] NOT child [MESH] NOT infant [MESH]) AND AND OR ((singl* [tw] OR doubl* [tw] OR tripl* [tw]) AND (mask* [tw] OR blind* [tw])) OR placebos [MESH] OR placebo* [tw] OR random* [tw] OR research design [mh:noexp] OR Meta-Analysis [ptyp] OR systematic review [tw] NOT NOT case reports [pt] NOT Comment [PT] NOT letter [PT] NOT child [MESH] NOT infant [MESH]) AND (somatostatin [tw] OR octreotide [tw] OR vapreotide [tw] OR lanreotide [tw]) AND OR ((singl* [tw] OR doubl* [tw] OR tripl* [tw]) AND (mask* [tw] OR blind* [tw])) OR placebos [MESH] OR placebo* [tw] OR random* [tw] OR research design [mh:noexp] OR Meta-Analysis [ptyp] OR systematic review [tw] NOT NOT case reports [pt] NOT Comment [PT] NOT letter [PT] NOT child [MESH] NOT infant [MESH])(Surgery [MESH]) AND AND OR ((singl* [tw] OR doubl* [tw] OR tripl* [tw]) AND (mask* [tw] OR blind* [tw])) OR placebos [MESH] OR placebo* [tw] OR random* [tw] OR research design [mh:noexp] OR Meta-Analysis [ptyp] OR systematic review [tw] NOT NOT case reports [pt] NOT Comment [PT] NOT letter [PT] NOT child [MESH] NOT infant [MESH]) AND AND OR ((singl* [tw] OR doubl* [tw] OR tripl* [tw]) AND (mask* [tw] OR blind* [tw])) OR placebos [MESH] OR placebo* [tw] OR random* [tw] OR research design [mh:noexp] OR Meta-Analysis [ptyp] OR systematic review [tw] NOT NOT case reports [pt] NOT Comment [PT] NOT letter [PT] NOT child [MESH] NOT infant [MESH] NOT child [MESH] NOT infant [MESH]) AND (Nasogastric decompression [tw] OR nasogastric tube [tw]) AND OR ((singl* [tw] OR doubl* [tw] OR tripl* [tw]) AND (mask* [tw] OR blind* [tw])) OR placebos [MESH] OR placebo* [tw] OR random* [tw] OR research design [mh:noexp] OR Meta-Analysis [ptyp] OR systematic review [tw] NOT NOT case reports [pt] NOT Comment [PT] NOT letter [PT] NOT child [MESH] NOT infant [MESH] NOT child [MESH] NOT infant [MESH])(Surgery [MESH]) AND AND OR ((singl* [tw] OR doubl* [tw] OR tripl* [tw]) AND (mask* [tw] OR blind* [tw])) OR placebos [MESH] OR placebo* [tw] OR random* [tw] OR research design [mh:noexp] OR Meta-Analysis [ptyp] OR systematic review [tw] NOT NOT case reports [pt] NOT Comment [PT] NOT letter [PT] NOT child [MESH] NOT infant [MESH] NOT child [MESH] NOT infant [MESH]) AND AND OR ((singl* [tw] OR doubl* [tw] OR tripl* [tw]) AND (mask* [tw] OR blind* [tw])) OR placebos [MESH] OR placebo* [tw] OR random* [tw] OR research design [mh:noexp] OR Meta-Analysis [ptyp] OR systematic review [tw] NOT NOT case reports [pt] NOT Comment [PT] NOT letter [PT] NOT child [MESH] NOT infant [MESH] NOT child [MESH] NOT infant [MESH]) AND (mobilisation [tw] OR mobilization [tw] OR postoperative mobilisation [tw] OR patient mobilisation [tw] OR postoperative mobilization [tw] OR patient mobilization [tw]) AND OR ((singl* [tw] OR doubl* [tw] OR tripl* [tw]) AND (mask* [tw] OR blind* [tw])) OR placebos [MESH] OR placebo* [tw] OR random* [tw] OR research design [mh:noexp] OR Meta-Analysis [ptyp] OR systematic review [tw] NOT NOT case reports [pt] NOT Comment [PT] NOT letter [PT] NOT child [MESH] NOT infant [MESH] NOT child [MESH] NOT infant [MESH])Survey on peri-operative standards in distal pancreatectomy. A copy of the research instrument used for the survey.Click here for fileMeta-analyses and randomized controlled trials on perioperative management. Appendix 1 summarizes relevant data from the meta-analyses and randomized controlled trials found by the literature search of best practice.Click here for file"} +{"text": "The triiodide anions are located in general positions, whereas the cations are located on centres of inversion.The title compound, [Sr(C DOI: 10.1107/S1600536808012373/nc2102Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536810015758/hy2302Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Three dinucleating O,N:N\u2032,O\u2032-donor ligands provide three diazine (=N\u2014N=) bridges between the metal ions, yielding a binuclear triple helicate structure with crystallographic C 2 symmetry, the rotation axis bis\u00adecting one N\u2014N bond.In the title binuclear iron(III) complex, [Fe DOI: 10.1107/S1600536808027438/cf2210Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In addition, each AgI atom is coordin\u00adated by one chelating 2,2\u2032-bipyridine ligand, giving a distorted trigonal coordination environment.The title complex, [Ag DOI: 10.1107/S1600536809014871/at2766Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The ligand bridges two AgI atoms, forming a polymeric zigzag chain propagating parallel to [001]. The uncoordinated water mol\u00adecule is involved in hydrogen bonds with sulfonate O atoms.In the title compound, {[Ag(C DOI: 10.1107/S1600536810012596/hy2295Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The SnIV atom is in a distorted trans-C3SnO2 trigonal-bipyramidal geometry. The presence of two bulky tert-butyl groups on the benzene ring prevents any hydrogen-bonding inter\u00adactions involving the hydroxyl substituents.The title compound, [Sn(C DOI: 10.1107/S1600536809023150/tk2461Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536809027044/ng2598Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Tetra\u00adchloridoferrate(III) anions lie between these chains, accepting N\u2014H\u22efCl hydrogen bonds from both H atoms of the picolinamidium \u2013NH2 group.The title compound, (C DOI: 10.1107/S1600536809040148/zq2012Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The polymeric three-dimensional structure is stabilized by inter\u00admolecular O\u2014H\u22efO hydrogen bonds.In the title coordination polymer, [La(C DOI: 10.1107/S1600536809020479/rz2328Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "These aggregate into the three-dimensional structure via N\u2014H\u22efO hydrogen-bonding mechanisms imparted by 4,4\u2032-imino\u00addipyridinium dications situated between the layers.In the title salt, {(C DOI: 10.1107/S1600536808038348/tk2326Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536810005829/ci5035Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The crystal packing exhibits weak inter\u00admolecular C\u2014H\u22efBr inter\u00adactions.In the title compound, [PdBr DOI: 10.1107/S1600536809001986/cv2489Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The ZnII atom is coordinated in a distorted tetra\u00adhedral environment by four O atoms from four 2,4-dichloro\u00adphenoxy\u00adacetate ligands. Each ligand bridges two ZnII atoms, forming a polymeric chain along the a axis. Adjacent chains are connected via C\u2014H\u22efCl hydrogen bonds.The title polymeric compound, [Zn(C DOI: 10.1107/S1600536810015977/ci5084Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The twisted mol\u00adecules pack in parallel regions (ab plane) which then form a herringbone pattern along c.The racemic title compound, C DOI: 10.1107/S1600536808014803/bq2079Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The methyl group of the benzoic acid mol\u00adecule is disordered over two sites in a 0.563\u2005(17):0.437\u2005(17) ratio. In the title compound, [Ni(C DOI: 10.1107/S1600536809044705/hb5141Isup2.hklStructure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "In the unique cation, the dihedral angle between the benzene and pyridine rings is 7.1\u2005(2)\u2005\u00c5.In the title complex, (C DOI: 10.1107/S1600536809015360/lh2806Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the crystal structure, inter\u00admolecular N\u2014H\u22efI hydrogen bonds link cations and anions into a three-dimensional network.In the title compound, [Cd(dien) DOI: 10.1107/S1600536808008040/lh2585Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536809031389/hb5029Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The 4,4\u2032-bipyridine ligands bridge CoII ions into a one-dimensional chain structure. In the crystal structure, inter\u00admolecular O\u2014H\u22efO hydrogen bonds link cations and anions into a three-dimensional network. The dianions are completely disordered about an inversion center.In the title complex, {[Co(C DOI: 10.1107/S1600536809021552/lh2836Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The amino group forms a hydrogen bond to the pyrazine N-4 atom of an adjacent mol\u00adecule, forming a chain motif.The two aromatic systems in the title compound, C DOI: 10.1107/S160053680803729X/sg2277Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The ligands bridge CdII ions, forming one-dimensional chains along [001], which are connected by N\u2014H\u22efN and N\u2014H\u22efS hydrogen bonds into a three-dimensional network.In the title compound, DOI: 10.1107/S160053680902371X/pk2170Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536808004248/su2044Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The metal atoms are bridged by the pySO3 ligands to form a one-dimensional chain. The chains are further connected into a three-dimensional architecture via hydrogen bonds.In the title polymeric complex, [Mn(C DOI: 10.1107/S1600536808012282/gk2137Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The crystal structure displays the classical O\u2014H\u22efO inter\u00admolecular hydrogen bonding typical for carboxylic acids forming dimers. Symmetry-related dimers are, in turn, linked through head-to-tail pairs of inter\u00admolecular N\u2014H\u22efO inter\u00adactions, giving rise to a zigzag chain along the c axis.The title compound, C DOI: 10.1107/S1600536808035721/lh2721Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The Co centre is in a distorted tetrahedral coordination by three N atoms of three different triazole ligands and one O atom of the 3,5-dinitrobenzoate anion.The title compound, [Co(C DOI: 10.1107/S160053680804155X/bt2826Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536810013759/zs2034Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The NaI cation is located on a threefold rotation axis and is surrounded by six O atoms from three opc anions. The opc anions link the Mn and Na cations, forming a three-dimensional polymeric structure. The uncoordinated water mol\u00adecule, located on a threefold rotation axis, is equally disordered over two sites. The three-dimensional network is consolidated by N\u2014H\u22efO hydrogen bonds.In the crystal structure of the title compound, {[MnNa(C II and CoII complexes, see: Zhang et al. 3]\u00b7H2O = 0.033 wR(F 2) = 0.099 S = 1.16 1315 reflections103 parameters1 restraintH-atom parameters constrainedmax = 0.35 e \u00c5\u22123 \u0394\u03c1min = \u22120.49 e \u00c5\u22123 \u0394\u03c1Absolute structure: Flack 1983, 649 FriFlack parameter: \u22120.01 (3) PROCESS-AUTO (Rigaku, 1998PROCESS-AUTO; data reduction: CrystalStructure (Rigaku/MSC, 2002SIR92 (Altomare et al., 1993SHELXL97 (Sheldrick, 2008ORTEP-3 (Farrugia, 1997WinGX (Farrugia, 1999Data collection: 10.1107/S1600536810002953/ng2724sup1.cif Crystal structure: contains datablocks I, global. DOI: 10.1107/S1600536810002953/ng2724Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536809027196/dn2466Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The crystal packing is stabilized by O\u2014H\u22efO hydrogen bonds.In the title compound, [Cu(C DOI: 10.1107/S1600536808009495/bt2691Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536808041408/hg2447Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The bridging function of the iodide ions leads to a chain structure propagating in [001].In the title coordination polymer, [CdI DOI: 10.1107/S1600536810014091/hb5403Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The thio\u00adcyanate anion bridges the CuI atoms, forming a zigzag chain along [101]. The Schiff base ligand adopts an E,E configuration and the dihedral angle between the terminal benzene rings is 53.68\u2005(8)\u00b0.In the cyrstal structure of the title compound, [Cu(NCS)(C DOI: 10.1107/S1600536808041925/lh2744Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The edta ligands link the neodymium metal centres, forming polymeric chains running along the a axis of the unit cell. These chains are further assembled via inter\u00admolecular O\u2014H\u22efO hydrogen-bonding inter\u00adactions into a three-dimensional supra\u00admolecular network.In the title complex, [Nd(C DOI: 10.1107/S1600536808026445/bg2202Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536809035041/jh2099Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the crystal packing, a three-dimensional network is constructed via hydrogen-bonding involving the water mol\u00adecules, uncoordinated imidazole N atom, protonated pyridine N and carboxyl\u00adate O atoms.In the title complex, [Fe(C DOI: 10.1107/S160053680902337X/kp2221Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536808015390/sj2506Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536808012828/dn2343Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials: interactive version of Fig. 1 Enhanced figure:"} +{"text": "The crystal structure is stabilized by a network of O\u2014H\u22efO hydrogen bonds, resulting in a two-dimensional supra\u00admolecular structure.In the neutral title complex, [Fe(C DOI: 10.1107/S1600536808042529/ng2527Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Regarding the coordinating C=C bonds as occupying a single coordination site each, the geometry around each Ir atom is square-planar.The title complex, [Ir DOI: 10.1107/S1600536808007216/cf2181Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536808035460/tk2320Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The non-H atoms of the equatorial plane are coplanar, with a mean deviation of 0.0355\u2005(2)\u2005\u00c5. The FeII cation lies on an inversion centre. Thus, the asymmetric unit comprises one half-mol\u00adecule.In the title compound, [Fe(N DOI: 10.1107/S1600536808018539/kp2177Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The Ni2+ cation is surrounded in a slightly distorted octa\u00adhedral coordination by six N atoms from three chelating 3-(2-pyrid\u00adyl)-1H-pyrazole ligands. In the heteropolyanion, two O atoms of the central SiO4 group (The asymmetric unit of the title compound, [Ni(C DOI: 10.1107/S1600536810000978/wm2297Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536808020850/ng2470Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the crystal, the molecules form hydrogen bonded chains propagating in [100] linked by O\u2014H\u22efO interactions. Further O\u2014H\u22efO bonds cross-link the chains.The title compound, C DOI: 10.1107/S1600536809017656/fl2248Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536808002201/lh2588Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The LaIII atom is coordinated by nine O atoms from three carboxyl\u00adate, three sulfonate and two hydroxyl groups, and one water mol\u00adecule, forming a distorted trigonal-prismatic square-face tricapped geometry.The title compound, [La(C DOI: 10.1107/S1600536809007879/is2373Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The carboxyl\u00adate ligands bridge pairs of gadolinium(III) ions, forming a zigzag chain along [100]. Hydrogen bonds link the chains into sheets parallel to (001).In the title compound, [Gd(C DOI: 10.1107/S1600536808012981/om2225Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the crystal structure, N\u2014H\u22efN hydrogen bonds help to consolidate the packing.In the title compound, [Mn(N DOI: 10.1107/S1600536808017984/hb2744Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536809018364/fi2078Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The four-coordinate AgI ion adopts a tetra\u00adhedral geometry, being bonded to two N atoms from two bridging isonicotinate ligands and two O atoms from two acetate ligands. These metal coordination units are connected by bridging isonicotinate and acetate ligands, generating a three-dimensional network.In the title homochiral three-dimensional heterometallic complex, [AgSm(C DOI: 10.1107/S1600536809048430/pv2235Isup2.hklStructure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "In the crystal structure, cations and anions are linked by inter\u00admolecular N\u2014H\u22efCl hydrogen bonds, forming a three-dimensional network.In the title compound, (C DOI: 10.1107/S1600536810007877/rz2412Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536809022417/hb2992Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials: interactive version of Fig. 1 Enhanced figure:"} +{"text": "The three N atoms span the axial\u2013equatorial\u2013axial sites of the trigonal-bipyramidal coordination polyhedron; the geometry of the CuII atom is 31% distorted from trigonal-bipyramidal .The title compound, [CuCl DOI: 10.1107/S1600536810037025/xu5030Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The metallabicyclic ring system is essentially planar (maximum deviation 0.059\u2005\u00c5).The crystal structure of the title compound, [Ru(C DOI: 10.1107/S1600536808040944/lh2738Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The two N-heterocycles both chelate the metal atom.The chloride and chloro\u00addifluoro\u00adacetate anions occupy DOI: 10.1107/S1600536808010829/sg2236Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The Ni2+ cation is in a distorted octa\u00adhedral environment, coordinated by six N atoms from three chelating 3-(2-pyrid\u00adyl)-1H-pyrazole ligands. In the one-electron reduced heteropolyanion, two O atoms of the central PO4 group (M\u2014N bond lengths, whereas all other bond lengths, angles and the hydrogen-bonding motifs are very similar.The hydro\u00adthermally prepared title compound, [Ni(C DOI: 10.1107/S1600536810005945/wm2306Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The carboxyl\u00adate groups of pyrazine-2-carboxyl\u00adate and oxalate ligands link the lanthanum metal centres, forming layers parallel to (10In the title complex, [La(C DOI: 10.1107/S1600536808041664/dn2413Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536808025452/hy2145Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the anion, the chloro substituent is disordered over two rings (occupancy ratio 0.81:0.19); the two chloro\u00addifluoro\u00admethyl groups are also disordered over two sites for their halogen atoms (occupancy ratios 0.72:0.28 and 0.70:0.30).In the title salt, [Ag(C DOI: 10.1107/S1600536808014128/bq2076Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The three O atoms are arranged fac, as are the three S atoms.In the title compound, [Co(C DOI: 10.1107/S1600536808011598/hy2130Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Corner-sharing PbCl6 octa\u00adhedra extend parallel to the ac plane. Adjacent layers are staggered relative to one another, with diammonium cations separating these layers. The cations exhibit via N\u2014H\u22efCl hydrogen bonding in the right-angled halogen sub-type of the terminal halide hydrogen-bonding motif.The title compound, {(C DOI: 10.1107/S1600536810016818/wm2339Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The dihedral angle between the two essentially planar quinoline ring systems is 45.02\u2005(9)\u00b0. In the crystal structure, an extensive O\u2014H\u22efO hydrogen-bonding network forms layers parallel to the ab plane.In the title compound, [Cu(SO DOI: 10.1107/S1600536808001980/lh2587Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The resulting geometry is distorted octa\u00adhedral within a ClN2O3 donor set.In the title complex, [Fe(C DOI: 10.1107/S1600536809031432/tk2520Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the chain, the ZnII atom adopts a slightly distorted octa\u00adhedral coordination geometry involving four water mol\u00adecules at the equatorial positions. The noncoordinated benzene-1,4-dicarboxyl\u00adate anion, which is also located on a twofold rotation axis, bridges adjacent chains through O\u2014H\u22efO hydrogen bonds, forming a three-dimensional supra\u00admolecular network.In the title compound, {[Zn(C DOI: 10.1107/S1600536809019412/is2415Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The hydroxyl group acts as a donor, forming an intra\u00admolecular O\u2014H\u22efN hydrogen bond.In the title compound, [Zn(C DOI: 10.1107/S1600536809001949/hy2173Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Both PbBr6 octa\u00adhedra and the organic cation exhibit via N\u2014H\u22efBr hydrogen bonding in the right-angled halogen sub-type of the terminal halide hydrogen-bonding motif.The title compound, {(C DOI: 10.1107/S1600536810016806/wm2338Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S160053680904495X/hb5192Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the crystal structure, mol\u00adecules are linked by bifurcated N\u2014H\u22ef hydrogen bonds, generating [001] chains.In the title compound, C DOI: 10.1107/S1600536810010810/hb5367Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536809024015/tk2481Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The piperidine ring adopts a distorted boat conformation in both mol\u00adecules, in which the N atom assumes an almost planar configuration.The asymmetric unit of the title compound, C DOI: 10.1107/S1600536809045358/ci2956Isup2.hklStructure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "The Na atoms are disordered over three sites .The crystal structure of the title compound, 2Na DOI: 10.1107/S1600536808024331/bt2752Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The bridging dca anions lead to a polymeric chain propagating in [100].In the title compound, [Cd(C DOI: 10.1107/S1600536808044000/hb2881Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "O\u2014H\u22efO hydrogen bonds link the complexes and uncoordinated water mol\u00adecules into two-dimensional networks parallel to (001).In the title compound, [Ni(C DOI: 10.1107/S1600536809022028/bi2378Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the crystal, adjacent mol\u00adecules are linked via pairs of N\u2014H\u22efO hydrogen bonds into chains propagating in [010].In title compound, [Cu(C DOI: 10.1107/S1600536809044262/hb5128Isup2.hklStructure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "The polyoxoanions are linked together via the bipyridyl cations, acting as hydrogen-bond donors, generating a two-dimensional supra\u00admolecular network. The asymmetric unit contains 1.5 4,4\u2032-bipyridinium (H2bpy) units, with an inversion centre in the central bond of the second H2bpy unit. The site symmetry of the anion is The title compound, tris\u00ad diarsenoocta\u00addeca\u00admolybdate(VI), (C DOI: 10.1107/S1600536809050260/fi2091Isup2.hklStructure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "GdIII ions are bridged by bidentate and tridentate 3-PYA ligands, resulting in a two-dimensional structure.In the title compound, [Gd(C DOI: 10.1107/S1600536808012270/lh2617Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The mol\u00adecular structure displays a cobalt(I) center in a distorted trigonal-planar coordination environment, with one Cp and two phosphane ligands. There are two crystallographically independent mol\u00adecules in the asymmetric unit besides the disordered solvent molecules.The title compound, [Co(C DOI: 10.1107/S1600536808042268/im2092Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536810016466/pk2244Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the structure, the oxalate and 5-amino\u00adisophthalate ligands link the dysprosium ions, building up a two-dimensional metal\u2013organic framework parallel to the (10The title complex, [Dy(C DOI: 10.1107/S1600536809019199/dn2450Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Acta Cr DOI: 10.1107/S1600536809051903/cv2664Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The structure displays weak intra\u00admolecular C\u2014H\u22efBr hydrogen-bonding inter\u00adactions.In the title complex, [PtBr DOI: 10.1107/S160053680803208X/rz2250Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the crystal structure, two inversion-related cations are linked to the anion via weak N\u2014H\u22efBr hydrogen bonds.In the title compound, (C DOI: 10.1107/S1600536809030323/lh2842Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The CoII centre exists in an all cis-octa\u00adhedral coordination geometry.In the title compound, [Co(C DOI: 10.1107/S1600536809037878/bt5061Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "All interatomic distances, angles and the hydrogen bond geometry are very similar for the three structures..The title compound, [Pr(C DOI: 10.1107/S1600536809001500/at2681Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The Cd atom has a distorted tetrahedral coordination. Inter\u00admolecular C\u2014H\u22efI hydrogen bonds link the monomeric units, generating a one-dimensional supra\u00admolecular chain along the a axis.In the title compound, [CdI DOI: 10.1107/S1600536808030225/bq2097Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The cation and anion are linked by an N\u2014H\u22efBr hydrogen bond.The anion in the title compound, (C DOI: 10.1107/S1600536808032248/tk2315Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536809032425/hb5049Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Symmetry-related Sn atoms are bridged by diisopropyl\u00addithio\u00adcarbamoylacetato ligands, forming a one-dimensional polymer along [001].In the title compound, [Sn(C DOI: 10.1107/S1600536810005933/lh2996Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The complex mol\u00adecules are linked into layers parallel to (001) by N\u2014H\u22efN hydrogen bonds, with the H atoms disordered over four symmetry-equivalent non-coordinated N atoms.In the title compound, [Mn(C DOI: 10.1107/S1600536809008411/bi2351Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The metal centre is also chelated by three deprotonated 1,3-diphenyl\u00adpropane-1,3-dione (dbm) ligands, forming enanti\u00adomerically pure [Tb(dbm)3 RRL]. The TbIII atom is located in a distorted square anti\u00adprism of eight coordinating atoms (six O and two N atoms).In the title compound, [Tb(C DOI: 10.1107/S1600536809019783/ng2576Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The mean planes of two naphthyl systems of the ligand make a dihedral angle of 40.32\u2005(11)\u00b0.In the title complex, [Cu(C DOI: 10.1107/S1600536809030694/hy2212Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The LaIII ions are bridged by \u03bc4-isophthalate ligands, forming two-dimensional layers. In the crystal structure, these layers are connected by inter\u00admolecular O\u2014H\u22efO hydrogen bonds into a three-dimensional network.In the title coordination polymer, [La DOI: 10.1107/S1600536809054543/lh2972Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536808009707/at2559Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The geometry about the Pd atom is distorted square-planar. The pyrazole rings are almost perpendicular, forming a dihedral angle of 86.6\u2005(6)\u00b0 to each other, to mitigate steric conflict between their methyl groups.The title compound, [PdCl(C al. 2006. Acta Cr DOI: 10.1107/S1600536810035427/su2207Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the crystal structure, adjacent ZnII atoms are linked by two H2nba ligands, forming one-dimensional ribbons along the c axis. These ribbons are further assembled into layers parallel to the bc plane via O\u2014H\u22efO hydrogen bonds.In the title coordination complex, [Zn(C DOI: 10.1107/S1600536809051836/ng2697Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The compound crystallizes with the Pd(II) atom on a twofold rotation axis. The palladium center has a slightly distorted square-planar environment, with the two P\u2014S chelating ligands adopting a cis configuration. The present structure is a pseudo-polymorph of [Pd(C18H14PS)2]\u00b7CH2Cl2.The title compound, [Pd(C DOI: 10.1107/S1600536808026718/bt2741Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Both dithio\u00adcarbamate and the N-heterocyclic ligands function in a chelating mode.In the title compound, [Eu(C DOI: 10.1107/S160053680904135X/xu2634Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The title centrosymmetric coordination polymer, {[Cu(C DOI: 10.1107/S1600536810014510/su2168Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The Sn atoms in the two independent mol\u00adecules adopt distorted trans-C3SnO2 trigonal\u2013bipyramidal geometries. The repeat distance of the polymeric chain is b/2.The 4-nitro\u00adcinnamate anion in the title compound, [Sn(C DOI: 10.1107/S1600536808032236/tk2314Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "II atom in the title Schiff base complex, [Cu(C15H11N3O5)(C5H5N)], is O,N,O\u2032-chelated by the doubly deprotonated Schiff base ligand. The metal centre is in a square-planar coordination geometry.The pyridine-coordinated Cu DOI: 10.1107/S1600536808042803/bt2838Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536808008957/bt2692Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The mol\u00adecule lies across a twofold rotation axis, around which two 1,10-phenanthroline ligands are arranged. There are short contacts between the 1,10-phenanthroline groups and the O atoms of the croconate ligand, which probably stabilize the crystal structure via weak C\u2014H\u22efO interactions.In the title compound, [Zn(C DOI: 10.1107/S1600536808033709/sg2254Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Two independent mol\u00adecules comprise the asymmetric unit, each of which lies on a mirror plane that passes through the C2Sn unit.In the title compound, [Sn(CH DOI: 10.1107/S1600536809030062/tk2508Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the crystal structure, inter\u00admolecular N\u2014H\u22efCl hydrogen bonds link the organic cations and the uncoordinated chloride ion.In the title compound, (C DOI: 10.1107/S1600536810002886/hb5291Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Each pair of adjacent MnII atoms is bridged by a CH3O-ip ligand, forming a helical chain running along a crystallographic 21 axis in the c-axis direction. These chains are decorated with 2,2\u2032-bipy ligands on alternating sides. O\u2014H\u22efO hydrogen bonding involving the water molecules stabilizes the crystal structure.In the title compound, {[Mn(C DOI: 10.1107/S1600536809035466/bg2299Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The water solvent mol\u00adecule is disordered over two positions in a 1:1 ratio.In the crystal structure of the title compound, [Ni(N DOI: 10.1107/S1600536809029407/rn2055Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536808033102/at2636Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "One 2-dph ligand functions in a bridging mode and connects Pb ions into a linear chain. The crystal packing is governed by intra- and inter\u00admolecular O\u2014H\u22efO hydrogen bonds.In the title complex, {[Pb(C DOI: 10.1107/S1600536810004162/hy2276Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Two disordered dichloro\u00admethane solvent mol\u00adecules are each 0.25-occupied on a twofold rotation axis.In the title compound, [FePt(C DOI: 10.1107/S1600536808043493/hy2171Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Each FeIII center in the complex has a six-coordinated distorted cis-FeN2O4 octa\u00adhedral geometry.The structure of the title compound, [Fe DOI: 10.1107/S1600536809005091/hg2478Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The nickel(II) center is coordinated by two P atoms of the chelating PNP ligand, Ph2PN(iPr)PPh2, and two bromide ions in a distorted square-planar geometry.The title compound, [NiBr DOI: 10.1107/S1600536809003936/ci2764Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536809038045/hb5109Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The other two imino N atoms act as hydrogen-bond acceptors from phenolate OH groups. The toluene solvent mol\u00adecule is disordered about a centre of inversion.In the toluene hemisolvated tripodal tris\u00ad(2-amino\u00adethyl)amine Schiff base, C DOI: 10.1107/S1600536809002906/bt2855Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The Fe atom is six-coordinated by three phenolic O and three imine N atoms from three Schiff base ligands in an octa\u00adhedral geometry.The title complex, [Fe(C DOI: 10.1107/S1600536808014165/sj2500Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The compound adopts a linear chain structure.In the title compound, [CoCl DOI: 10.1107/S1600536808006685/ng2432Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Its non-centrosymmetric polymorph structure, (II), was known previously and has been redetermined at 193\u2005(2)\u2005K -\u03bcassa 2002. Acta Cr DOI: 10.1107/S1600536809009088/si2150Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Hydrogen bonds exist between the OH group of the pyridine-4-carbaldehyde oxime ligand and the two O atoms of the acetyl\u00adacetonate ions.The title compound, [U(C DOI: 10.1107/S1600536808012889/im2063Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The cation is bowed due to crystal packing effects, and the dihedral angle between the xylyl rings is 52.3\u2005(7)\u00b0. In the title compound, [Au(C DOI: 10.1107/S1600536808027116/hb2785Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The bridging ligand results in an infinite chain. A network of O\u2014H\u22efO hydrogen bonds helps to establish the crystal structure.In the title compound, {[Cu(C DOI: 10.1107/S1600536808025439/hb2747Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The two O atoms are cis, as are the two S atoms.In the title compound, [Ni(C DOI: 10.1107/S1600536808017145/hy2137Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536808005382/dn2320Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Methanol\u2013acetate O\u2014H\u22efO hydrogen bonds link the dinculear units into a linear supra\u00admolecular chain extending parallel to [100].The reaction of zinc acetate and 2-methyl-8-hydroxy\u00adquinoline in methanol yielded the centrosymmetric dinuclear title compound, [Zn DOI: 10.1107/S1600536809014214/tk2424Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The linear butadiyne substituent has alternating C\u2014C triple and single bonds, while the unsubstituted cyclo\u00adpenta\u00addiene ring is slightly positionally disordered and retains a close to eclipsed conformation.The title compound, [Fe(C DOI: 10.1107/S1600536809005522/hg2474Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The Ni\u22efNi distance is 3.2398\u2005(12)\u2005\u00c5.In the title azide-bridged dinuclear centrosymmetric nickel(II) complex, [Ni DOI: 10.1107/S1600536808037902/ci2712Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Two cations are linked into dimers via three bridging carboxyl\u00adate groups from three 2,5-difluoro\u00adbenzoic acid units. The GdIII ion is nine-coord\u00adinated by seven O atoms and two N atoms.In the centrosymmetric title compound, [Gd DOI: 10.1107/S1600536808023507/er2055Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The Pd atom is located on a centre of inversion.The title compound, [PdCl DOI: 10.1107/S1600536808008337/bt2685Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Two short intra\u00admolecular C\u2014H\u22efO contacts occur in the mol\u00adecule.In the centrosymmetric title compound, [Zn(C DOI: 10.1107/S1600536809031109/hb5031Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The ferrocenyl unit deviates from an eclipsed geometry with tilted cp rings; the inter\u00adplanar angle between the cp and imidazole rings is 114.11\u2005(4)\u00b0.In the title compound, [Fe(C DOI: 10.1107/S1600536808029231/dn2373Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Extensive O\u2014H\u22efO hydrogen-bonding inter\u00adactions connect the supra\u00admolecular chains into a three-dimensional network.The title supra\u00admolecular polymer, [Cu(S DOI: 10.1107/S1600536809047096/hb5217Isup2.hklStructure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "The MgII ion is hexa\u00adcoordinated to four tetra\u00adhydro\u00adfuran (THF) ligands, and two AlH4 \u2212 anions through bridging H atoms. The Al\u2014H distances are more precise compared to those previously determined . The mol\u00adecule has twofold rotation symmetry.The structure of the title compound, [Mg(AlH al. 1995. Chem. BFuhr 2002. J. Allo DOI: 10.1107/S1600536810014200/ci5044Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The SnIV atom is six-coordinate within a distored octa\u00adhedral geometry defined by a C2Cl2O2 donor set.The mol\u00adecule of the title compound, [Sn(C DOI: 10.1107/S1600536809039269/tk2538Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The two carboxyl\u00adate groups of each L ligand, which adopt a syn-anti coordination mode, combine with four MnII atoms, yielding one-dimensional chains extending along [010].The title compound, [Mn DOI: 10.1107/S1600536810008123/fi2094Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The alkyl chains of the ligands are equally disordered over two sets of sites.In the title compound, [Zn(C DOI: 10.1107/S1600536810002825/hb5312Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the crystal, inter\u00admolecular O\u2014H\u22efO hydrogen bonds form an extensive three-dimensional network, which consolidates the crystal packing.In the title compound, [Ni(C DOI: 10.1107/S1600536809028190/cv2585Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The TBDC ligands act as bridging ligands, forming chains along [110]. These chains are further linked into a two-dimensional network via inter\u00admolecular O\u2014H\u22efO hydrogen bonds. The solvent water mol\u00adecule lies on a twofold rotation axis.In the title complex, {[Cu(C DOI: 10.1107/S1600536810036913/lh5099Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The ions are linked via N\u2014H\u22efCl hydrogen bonds into chains running along the b axis.The crystal structure of the title compound, (C DOI: 10.1107/S1600536808007368/ya2069Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Each Mo atom is coordinated by an \u03b75-cyclo\u00adpenta\u00addienyl ligand, two carbonyl ligands and a diphenyl\u00adphosphine ligand in a piano-stool fashion.The title compound, [Mo DOI: 10.1107/S1600536808017996/hg2402Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The symmetry-equivalent VV atoms exhibit coordination geometries defined by cis-terminal fluoride and oxide groups, unsymmetrically bridging oxide groups and the N-atom donors of the phenanthroline ligands. The crystal packing is stabilized by weak inter\u00admolecular C\u2014H\u22efO and C\u2014H\u22efF hydrogen bonds.The title compound, [V DOI: 10.1107/S1600536810037232/lh5133Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the crystal structure, mol\u00adecules are linked through inter\u00admolecular N\u2014H\u22efO hydrogen bonds, forming chains running along the b axis.In the title complex, [ZnI DOI: 10.1107/S1600536809037210/om2276Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The metal centres are connected via the tridentate L ligands into a three-dimensional polymeric structure.In the title compound, [Cd(C DOI: 10.1107/S160053680904255X/cv2629Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "J. Soli DOI: 10.1107/S1600536808033394/wm2190Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536809003584/hb2904Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The title compound, (C DOI: 10.1107/S1600536810010196/dn2549Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536808036131/pv2114Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The distorted square\u2013pyramidal N2O3 coordination geometry is completed by an O atom derived from a perchlorate anion.In the centrosymmetric and dinuclear title complex, [Mn DOI: 10.1107/S1600536808035551/tk2317Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536809032061/hb5046Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536810013541/hb5404Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The bridging phenyl\u00adcyanamide anions generate an infinite chain propagating in [001].In the title coordination polymer, [Co(C DOI: 10.1107/S160053681000557X/hb5302Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the complex cations, the NaI centre is coordinated by five O atoms of the 2,3-naphtho-15-crown-5 ligand and one water O atom. The complex mol\u00adecules form a two-dimensional network via weak O\u2014H\u22efS inter\u00adactions between adjacent cations and anions The title complex, [Na(C DOI: 10.1107/S1600536809038641/pk2189Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Inter\u00admolecular O\u2014H\u22efCl and O\u2014H\u22efO hydrogen bonds form an extensive three-dimensional hydrogen-bonding network, which consolidates the crystal packing.In the title compound, [CoCl(C DOI: 10.1107/S1600536808042591/cv2481Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The ZnII atom is five coordinate with a distorted trigonal\u2013bipyramidal geometry, coordinating with three N atoms of the Schiff base (2-morpholinoeth\u00adyl)(2-pyridylmethyl\u00adidene)amine and two N atoms from two thio\u00adcyanate ligands. The morpholine ring adopts a chair configuration.The title compound, [Zn(NCS) DOI: 10.1107/S1600536808044061/su2087Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The metal center is five-coordinate in a trans-Br2SnC3 trigonal bipyramidal geometry. The cation is disordered about a center of inversion.The anion in the title salt, (C DOI: 10.1107/S1600536808011094/sg2227Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The dinuclear complex mol\u00adecules are assembled into one-dimensional supra\u00admolecular chains extending in the [100] direction by hydrogen bonds. Inter\u00adchain hydrogen bonds further link these chains into layers perpendicular to [001].In the title compound, [Cu DOI: 10.1107/S1600536808034570/cs2084Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "A crystallographic twofold axis bisects the molecule.The title compound, C DOI: 10.1107/S1600536808015377/fj2094Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The word \"Initiation\" was incorrectly entered as \"Initiator.\" The correct title is: \"A Novel Target of Action of Minocycline in NGF-Induced Neurite Outgrowth in PC12 Cells: Translation Initiation Factor eIF4AI\". The correct citation is: Hashimoto K, Ishima T (2010) A Novel Target of Action of Minocycline in NGF-Induced Neurite Outgrowth in PC12 Cells: Translation Initiator Factor eIF4AI. PLoS ONE 5(11): e15430. doi:10.1371/journal.pone.0015430"} +{"text": "In the crystal structure no obvious hydrogen bonding is observed.The five-coordinate Sn atom in the title salt, [(CH DOI: 10.1107/S1600536809019722/tk2456Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The metal centres are linked in an unusual triple-bridged mode into chains parallel to [101].In the title compound, [Cu(C DOI: 10.1107/S1600536809030050/pv2188Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The S atom from the other NCS\u2212 ion completes the distorted square-pyramidal coordination.In the title complex, [Cu(NCS) DOI: 10.1107/S1600536809048594/kj2135Isup2.hklStructure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "In the crystal structure, inter\u00admolecular O\u2014H\u22efO hydrogen bonds link the mol\u00adecules into layers parallel to the bc plane.In the title mononuclear complex, [Cu(C DOI: 10.1107/S1600536808010593/cv2398Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536810036202/wm2402Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The complete ligand is generated by crystallographic twofold symmetry, resulting in an infinite one-dimensional architecture along [101].In the title coordination polymer, [FeCl DOI: 10.1107/S1600536810002837/hb5310Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "N\u2014H\u22efO hydrogen bonding is present in the crystal structure.In the crystal structure of the title compound, [Cu(C DOI: 10.1107/S1600536809004577/xu2458Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The five-membered imidazole ring is disordered over two positions with the major component having a site occupancy of 0.712\u2005(4); the N-bound methyl substituents are ordered. The imidazole ring is approximately perpendicular to the six-membered phenyl\u00adene ring .The title imidazolium-based ionic-liquid salt, C DOI: 10.1107/S1600536809026324/tk2494Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The diethyl ether ligand adopts a nearly planar W-type conformation.In the title compound, [Li(C DOI: 10.1107/S1600536809011556/dn2437Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The crystal structure displays weak inter\u00admolecular N\u2014H\u22efCl hydrogen bonding.In the title compound, [PdCl DOI: 10.1107/S1600536809008472/rk2132Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The NiII ion, lying on an inversion centre, is four-coordinated in a square-planar geometry by two phenolate O and two imine N atoms from two symmetry-related 2-imino\u00admethyl-5-methoxy\u00adphenolate ligands. In the crystal, mol\u00adecules are linked into corrugated layers parallel to (100) by N\u2014H\u22efO hydrogen bonds.The title compound, [Ni(C DOI: 10.1107/S1600536809039233/ci2923Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "DOI: 10.1107/S1600536808023799/tk2286Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Correction to: Chiropr Man Therap (2019) 27:63https://doi.org/10.1186/s12998-019-0283-6Original name tagging:After publication of our article the authAx\u00e9n IbenHestbaek LiseCorrect name tagging:Leboeuf-Yde CharlotteIben Ax\u00e9nLise HestbaekCharlotte Leboeuf-YdeThe original article has been corrected."} +{"text": "The correct reference is: Salusso A, Raimunda D. Pseudomonas Aeruginosa CDF Transporters CzcD and YiiP are Involved in Zn2+ Efflux, Outer Membrane Permeability and Antibiotic Resistance. Biophys J.; 2017; 112: 3, 16a."} +{"text": "The Pan African Medical Journal. 2018;30:266. doi:10.11604/pamj.2018.30.266.13209.Ce Corrigendum modifie l'article original Dans l'article original archiv\u00e9 sur Pubmed, le nom de l'auteur Abdelkader Jalil El Hangouche est archiv\u00e9 Hangouche AJE au lieu de El Hangouche AJ ."} +{"text": "GigaScience, 6, 2017; doi: 10.1093/gigascience/gix077.A formula was incorrect upon initial publication. The formula \u201cp-value = binomial_cdf (T(v)-Kv(s), T(v), PNull (s))\u201d should instead be \u201cp-value = binomial_cdf (T(v)-Kv(s), T(v), 1-PNull (s)).\u201dThis has now been corrected."} +{"text": "Scientific Reports 10.1038/s41598-017-18331-9, published online 21 December 2017Correction to: This Article contains typographical errors in the Acknowledgements section.\u201cHorizon 2020 Framework Programme (H2020) (792720)\u201dshould read:\u201cHorizon 2020 Framework Programme (H2020) (732720)\u201d"} +{"text": "Nature Communications; 10.1038/ncomms15287; published online 16 May 2017.Correction to: This Article contains errors in Fig."} +{"text": "Correction to: Br J Cancer (2018) 117, 1819\u20131827; 10.1038/bjc.2017.353; published online 12 October 2017Supplementary Table Supplementary Table 1Supplementary Figure Legends"} +{"text": "Scientific Reports5; Article number: 1533810.1038/srep15338; published online: 10212015; updated: 07022018In the original version of this Article, the author Karthiga Santhana Kumar was incorrectly indexed.In addition, this Article contained an error in the Results section, under the subheading \u2018Automated quantification of cell dissemination\u2019,http://www.infozentrum.ethz.ch/uploads/user_upload/Software/.\u201d\u201cA program suite referred to as automated Cell Dissemination counter (aCDc) consisting of three open source, executable Java programs (.jar) files can be downloaded through the following web link now reads:https://urldefense.proofpoint.com/v2/url?u=http-3A__www.infozentrum.ethz.ch_uploads_user-5Fupload_Software_ImageAnalysisSoftware.zip&d=DwIGaQ&c=vh6FgFnduejNhPPD0fl_yRaSfZy8CWbWnIf4XJhSqx8&r=Q0HWMVGq_tlDBf7wAxJCv189FbTXBj2Fzg4N_lVZ7NM&m=y4-cT7GLvN-bRkjsbfl2iBNdNwRNEYQ4YmdXP3tZjas&s=o6yob3XKrSnFjEfuAxXQN7WgIuJhyqBCi8hBAm94hFg&e=.\u201d\u201cA program suite referred to as automated Cell Dissemination counter (aCDc) consisting of three open source, executable Java programs (.jar) files can be downloaded through the following web link"} +{"text": "This article has been corrected: The correct affiliation information is given below:1 Josep Carreras Leukemia Research Institute, Universitat Aut\u00f2noma de Barcelona, Spainhttps://doi.org/10.18632/oncotarget.12497Original article: Oncotarget. 2016; 7:72057-72066."} +{"text": "Scientific Reports 10.1038/s41598-017-06977-4, published online 08 August 2017Correction to: This Article contained an error in the legend of Table 1 where,\u201cFor each interactors, the interacting TANC protein, the detection method and the binding region are here listed. Y2H: Yeast two hybrid; Co-IP: Co-immunoprecipitation; SPR: Surface plasmon resonance HTS: High-Throughput System; AC: Affinity Capture; PL: Proximity Label; MS: Mass spectrometry; CLIP: Cross-Linking ImmunoPrecipitation SF-TAP/MS: systematic tandem affinity purifications coupled to mass spectrometry. LIG_PDZ_Class_1: PDZ-binding motif; LIG_EVH1_1: Proline-rich motif binding to signal transduction class I EVH1 domains; DEG_SCF_TRCP1: SCF-betaTrCP1 complex target site; MOD_LATS_1: phosphorylation motif recognised by the LATS kinases; DOC_PP1_RVXF_1: PP1 docking motif; LIG_14-3-3_2: phospho-motif mediating the interaction with 14-3-3 proteins; LIG_Actin_WH2_2: Actin-binding motif; TRG_NES_CRM1_1: Nuclear Export Signal.\u201dnow reads:3) and helium (0.03\u2009g/cm3) were kept fixed during the fitting procedure, and their thicknesses were infinite. Error estimates were obtained for every parameter by varying the parameter until the \u03c7 based R-value is changed by 5% . The method does not reveal correlations between the parameters. On the right side, theoretical bulk density and the molecular length of the molecules are given for comparison.\u201d\u201cThe tabulated parameters are obtained from fitting the reflectivity data with box models. For models with more than one layer, the layer closest to the calcite surface is indexed 1 (e.g. Methanol-1). The density for calcite (2.71\u2009g/cm"} +{"text": "G. Tuberoso."} +{"text": "Scientific Data 2:150050 doi: 10.1038/sdata.2015.50 (2015); Published 29 September 2015; Updated 3 July 2018.Panels a-e were accidentally omitted from"} +{"text": "Correction to: BMC Genomics (2019) 20:262https://doi.org/10.1186/s12864-019-5576-6Following publication of the original article , the autPampon\u00e9t Vanessa Carvalho CayresOriginally the author name was published as:Vanessa Carvalho Cayres Pampon\u00e9tThe correct author name is:"} +{"text": "The European Centre for Disease Prevention and Control (ECDC) invites applications for the following positions:Disease Programme Coordination ManagerThe application deadline expires on 03 September 2018. Financial assistantThe application deadline expires on 03 September 2018.https://ecdc.europa.eu/en/work-us/vacancies?f%5B0%5D=deadline_date%3A1&f%5B1%5D=vacancy_type%3A1700For more information on either position, please visit the ECDC website:"} +{"text": "The original publication containsThe correct version:Medina-Pradas et al. (2011): \u201cEFA/CV/CtV/ICR\u201d"} +{"text": "Pulmonaria officinalis L., Boraginaceae) is considered to possess therapeutic properties and it has been traditionally used as a remedy against various lung disorders in many countries. Nevertheless, very few data concerning its phytochemical composition are available. This research aims to provide a detailed description of specialized metabolites from the aerial parts of lungwort. Nine previously undescribed and 36 known phenolic compounds were detected in the 50% methanolic extract. Following multistep preparative procedures, structures of newly discovered compounds were determined using one- and two-dimensional techniques of NMR spectroscopy. Among the identified compounds were caffeic acid esters with aliphatic hydroxycarboxylic acids, conjugates of dicaffeic acid with rosmarinic acid, and previously unknown isomers of isosalvianolic acid A and yunnaneic acid E, as well as other lignans. Concentrations of all identified phenolic derivatives in the investigated herbal material were estimated using a method based on liquid chromatography with high-resolution mass spectrometry detection. Seasonal changes in the concentration of metabolites were also investigated using targeted and untargeted metabolomics techniques.Lungwort ( Pulmonaria officinalis L (lungwort), belonging to the Boraginaceae family, is a herbaceous perennial plant, widely spread in Europe and western Asia. It has a long tradition of use in folk medicine of many countries as a remedy against various respiratory diseases including asthma, chronic bronchitis, tuberculosis, laryngitis, and coughs. It also has expectorant, antitussive, and diaphoretic properties photocycloaddition of two olefinic moieties \u2212 at m/z 353. Compound 6 was identified as 3-O-caffeoylquinic acid, whereas compound 9 was identified as 4-O-caffeoylquinic acid, by comparisons of MS/MS fragmentation patterns and retention times with that of authentic standards. The last isomer was only tentatively identified as a 5-O-caffeoylquinic acid (13) by its fragmentation pattern alone \u2212 ion of digoxin at RT 17.48 min). At this stage of data processing, standard t-test and fold-change analyses were carried out to provide a preliminary overview of features potentially characteristic for the two phenological stages under study. Next, all missing intensity values and intensities that were equal to zero in the data matrix were replaced by the half of the minimum positive value that was found within the data, and multivariate PCA was applied to investigate systematic variation in the data matrix and to identify potential groups in an unsupervised manner.For untargeted metabolomic analyses, data obtained from LC-QTOF-MS/MS runs were processed using Find Molecular Features function of Bruker DataAnalysis ver. 4.4 SR1 software. The following parameters were used: a signal-to-noise threshold of 9, correlation coefficient threshold of 0.7, and the minimum compound length of seven spectra. For each detected compound, in addition to the deprotonated ion, all typical adduct and composite ions were grouped into a single feature. Next, features from the entire dataset were subjected to advanced bucket generation with Bruker ProfileAnalysis ver. 2.3 software, using retention time range 1.2\u201322.0 min and oAnalyst . Data weThe bias related to instrumental drift was minimized by randomization of the sample list injection order. It was achieved using an in-house-developed VBA script in Microsoft Excel.P. officinalis that had been isolated in the course of this study, and 25 \u00b5g/mL of the internal standard. This QC sample was used to monitor the quality and stability of the data acquisition and was analyzed in replicate after each block of 20 analyses.The performance of the LC-MS and the data processing systems were monitored with two types of quality control (QC) samples. Class-specific QC samples were prepared by mixing 10 \u00b5L aliquots of each sample within the group and diluting them 10 times, just like normal samples. Class QC samples were analyzed after every six analyses of normal samples. During data processing, features detected in less than 75% of class-specific QC samples were removed from the dataset. The frequency distribution of the relative standard deviation for the peak intensities and peak numbers in the QC samples are given in 1H, 13C DEPTQ, 1H\u201313C HSQC, 1H\u201313C H2BC, 1H\u201313C HMBC, 1H\u201313C F2-coupled perfect-CLIP HSQC, 1H\u201313C HSQC-TOCSY, 1H\u20131H COSY DQF, 1H\u20131H TOCSY, 1H\u20131H NOESY, 1H\u20131H TROESY, 1D\u2013TOCSY, 1D-TROESY, CSSF-1D-NOESY, and CSSF-1D-TOCSY \u2212 .Danshensu lactic acid (2); light brownish amorphous powder; UV \u03bbmax (nm) 260; HR-QTOF-MS (neg.) m/z 312.1086 [M \u2212 H]\u2212 .Menisdaurin (O-(E)-Caffeoyl-threonic acid (3); white amorphous powder; UV \u03bbmax (nm) 325; \u22125 M, MeOH): [\u0398]228 \u2212 3330, [\u0398]252 \u2212 32, [\u0398]259 \u2212 170, [\u0398]297 + 2385, [\u0398]306 + 2250, [\u0398]315 + 1877, [\u0398]322 + 2185, [\u0398]325 + 2034, [\u0398]333 + 2338, [\u0398]342 + 1962, [\u0398]347 + 2091, [\u0398]372 + 284, [\u0398]377 + 340; HR-QTOF-MS (neg.) m/z 297.0619 [M \u2212 H]\u2212 . 1H and 13C-NMR spectroscopic data (3-pic data .O-(E)-Caffeoyl-l-threonic acid (4); white amorphous powder; UV \u03bbmax (nm) 325; \u22125 M, MeOH) [\u0398]237 + 1647, [\u0398]338 \u2212 1574; HR-QTOF-MS (neg.) m/z 297.0611 [M \u2212 H]\u2212 .2-5); white amorphous powder; UV \u03bbmax (nm) 275; HR-QTOF-MS (neg.) m/z 215.0825 [M \u2212 H]\u2212 .Lycoperodine-1 (6); white amorphous powder; UV \u03bbmax (nm) 325; HR-QTOF-MS (neg.) m/z 353.0882 [M \u2212 H]\u2212 .Chlorogenic acid (7); white amorphous powder; UV \u03bbmax (nm) 325; HR-QTOF-MS (neg.) m/z 405.2126 [M \u2212 H]\u2212 .Actinidioionoside (8); white amorphous powder; UV \u03bbmax (nm) 325; HR-QTOF-MS (neg.) m/z 179.0262 [M \u2212 H]\u2212 .Caffeic acid (9); white amorphous powder; UV \u03bbmax (nm) 325; HR-QTOF-MS (neg.) m/z 353.0882 [M \u2212 H]\u2212 .Cryptochlorogenic acid (O-(E)-Feruloyl-\u03b1-sorbopyranosyl-(2\u2032\u21921)-\u03b1-glucopyranoside (10); white amorphous powder; UV \u03bbmax (nm) 325; m/z 517.1572 [M \u2212 H]\u2212 . 1H and 13C-NMR spectroscopic data (3\u2032-pic data .O-(E)-Caffeoyl-d-glyceric acid (11); white amorphous powder; UV \u03bbmax (nm) 325; \u22125 M, MeOH) [\u0398]265 \u2212 144, [\u0398]317 \u2212 5806, [\u0398]380 + 296; HR-QTOF-MS (neg.) m/z 267.0508 [M \u2212 H]\u2212 .2-O-(E)-Caffeoyl-l-threonic acid (12); white amorphous powder; UV \u03bbmax (nm) 215, 325; \u22125 M, MeOH) [\u0398]261\u2212169, [\u0398]283 \u2212 593, [\u0398]300 \u2212 509, [\u0398]325 \u2212 1349, [\u0398]384 + 80, [\u0398]380 + 296; HR-QTOF-MS (neg.) m/z 297.0616 [M \u2212 H]\u2212 .4-13); tentative identification; UV \u03bbmax (nm) 325; HR-QTOF-MS (neg.) m/z 353.0878 [M \u2212 H]\u2212 .Neochlorogenic acid (O-(E)-Caffeoyl- glyceric acid (14); white amorphous powder; UV \u03bbmax (nm) 215, 325; \u22125 M, MeOH) [\u0398]231 \u2212 1125, [\u0398]253 \u2212 207, [\u0398]273 \u2212 277, [\u0398]293 + 208, [\u0398]305 + 429, [\u0398]324 + 1234, [\u0398]343 + 76, [\u0398]358 \u2212 204, [\u0398]373 \u2212 286; HR-QTOF-MS (neg.) m/z 267.0509 [M \u2212 H]\u2212 .1H and 13C-NMR spectroscopic data (3-pic data . O-p-Coumaroylquinic acid (15); white amorphous powder; UV \u03bbmax (nm) 225, 310; m/z 337.0924 [M \u2212 H]\u2212 .3-O-p-Coumaroylquinic acid (16); tentative identification; UV \u03bbmax (nm) 225, 310; HR-QTOF-MS (neg.) m/z 337.0926 [M \u2212 H]\u2212 .4-O-p-Coumaroylquinic acid (17); tentative identification; UV \u03bbmax (nm) 225, 310; HR-QTOF-MS (neg.) m/z 337.0924 [M \u2212 H]\u2212 .5-18); white amorphous powder; UV \u03bbmax (nm) 255, 345; \u22125 M, MeOH) [\u0398]254 \u2212 14186, [\u0398]292 + 3337, [\u0398]313 \u2212 4443, [\u0398]351 + 12263; HR-QTOF-MS (neg.) m/z 537.1034 [M \u2212 H]\u2212 . 1H and 13C-NMR spectroscopic data (Globoidnan B (pic data .19) yellow amorphous powder; UV \u03bbmax (nm) 255, 355; HR-QTOF-MS (neg.) m/z 609.1464 [M \u2212 H]\u2212 .Rutin (20); tentative identification; UV \u03bbmax (nm) 345; HR-QTOF-MS (neg.) m/z 593.1501 [M \u2212 H]\u2212 .Nicotiflorin isomer (O-\u03b2-glucoside (21); yellow amorphous powder; UV \u03bbmax (nm) 255, 355; HR-QTOF-MS (neg.) m/z 463.0892 [M \u2212 H]\u2212 .Quercetin 3-22); white amorphous powder; UV \u03bbmax (nm) 265; m/z 571.1092 [M \u2212 H]\u2212 .Yunnaneic acid E -\u03b2-glucoside (23); yellow amorphous powder; UV \u03bbmax (nm) 255, 355; HR-QTOF-MS (neg.) m/z 549.0876 [M \u2212 H]\u2212 .Quercetin 3-24); yellow amorphous powder; UV \u03bbmax (nm) 265, 345; HR-QTOF-MS (neg.) m/z 593.1503 [M \u2212 H]\u2212 .Nicotiflorin (25); yellow amorphous powder; UV \u03bbmax (nm) 265, 345; HR-QTOF-MS (neg.) m/z 447.0940 [M \u2212 H]\u2212 .Astragalin (26); light cream amorphous powder; UV \u03bbmax (nm) 285; HR-QTOF-MS (neg.) m/z 719.1607 [M \u2212 H]\u2212 .Shimobashiric acid C (27); light yellowish amorphous powder; UV \u03bbmax (nm) 220, 330; CD [\u0398]232 \u2212 9423, [\u0398]254 \u2212 1004, [\u0398]275 \u2212 3233, [\u0398]302 + 7406, [\u0398]313 + 6634, [\u0398]326 + 7255; HR-QTOF-MS (neg.) m/z 359.0773 [M \u2212 H]\u2212 .Rosmarinic acid -\u03b2-glucoside (28); yellow amorphous powder; UV \u03bbmax (nm) 265, 345; HR-QTOF-MS (neg.) m/z 533.0940 [M \u2212 H]\u2212 .Kaempferol 3-29); white amorphous powder; UV \u03bbmax (nm) 310; \u22125 M, MeOH) [\u0398]253 \u2212 70887, [\u0398]290 + 6650, [\u0398]332 \u2212 7130, [\u0398]386 + 290; HR-QTOF-MS (neg.) m/z 537.1035 [M \u2212 H]\u2212 .Monardic acid A (30); white amorphous powder; UV \u03bbmax (nm) 265; HR-QTOF-MS (neg.) m/z 509.1094 [M \u2212 H]\u2212 . 1H and 13C-NMR spectroscopic data -2-((3-(3-(carboxycarbonyl)-3\u2032,4\u2032-dihydroxy--4-yl) propanoyl)oxy)-3-propanoic acid (pic data .31); white amorphous powder; UV \u03bbmax (nm) 310; \u22125 M, MeOH) [\u0398]253 + 28451, [\u0398]281 \u2212 3444, [\u0398]330 + 8800; HR-QTOF-MS (neg.) m/z 537.1054 [M \u2212 H]\u2212 .Lithospermic acid A (32); white amorphous powder; UV \u03bbmax (nm) 325; \u22125 M, MeOH) [\u0398]237 + 16506, [\u0398]246 + 15155, [\u0398]253 + 15786, [\u0398]247 \u2212 4592, [\u0398]294 + 15809, [\u0398]341 \u2212 47198; HR-QTOF-MS (neg.) m/z 999.2766 [M \u2212 H]\u2212 . 1H and 13C-NMR spectroscopic data (Pulmonarioside A (pic data .O-(8\u2033-Z-caffeoyl) rosmarinic acid) (33); white amorphous powder; UV \u03bbmax (nm) 325; m/z 537.1034 [M \u2212 H]\u2212 .Salvianolic acid H, (3\u2032-34); white amorphous powder; UV \u03bbmax (nm) 250; 288; 330; \u22125 M, MeOH) [\u0398]235 + 14042, [\u0398]255 + 25042, [\u0398]280 \u2212 2869; [\u0398]303 + 9014, [\u0398]334 + 11379; HR-QTOF-MS (neg.) m/z 717.1444 [M \u2212 H]\u2212 .Lithospermic acid B (35); white amorphous powder; UV \u03bbmax (nm) 325; \u22125 M, MeOH) [\u0398]253 + 32447, [\u0398]274 \u2212 4384, [\u0398]296 + 20685, [\u0398]340 \u2212 56660; HR-QTOF-MS (neg.) m/z 1013.2946 [M \u2212 H]\u2212 . 1H and 13C-NMR spectroscopic data (Pulmonarioside B (pic data .36); white amorphous powder; UV \u03bbmax (nm) 280; \u22125 M, MeOH) [\u0398]231 \u2212 13436, [\u0398]252 \u2212 9737, [\u0398]269 \u2212 11309, [\u0398]300 + 41370; HR-QTOF-MS (neg.) m/z 1093.2255 [M \u2212 H]\u2212 .Yunnaneic acid B (37); white amorphous powder; UV \u03bbmax (nm) 260, 320; m/z 491.0979 [M \u2212 H]\u2212 .Globoidnan A (38); white amorphous powder; UV \u03bbmax (nm) 325; \u22125 M, MeOH) [\u0398]246 + 10304, [\u0398]284 \u2212 24134, [\u0398]324 + 26434; HR-QTOF-MS (neg.) m/z 551.1190 [M \u2212 H]\u2212 .1H and 13C-NMR spectroscopic data (Pulmitric acid A (pic data .39); white amorphous powder; UV \u03bbmax (nm) 320; m/z 535.0885 [M \u2212 H]\u2212 ; 1H and 13C-NMR spectroscopic data (Pulmitric acid B (pic data .40); white amorphous powder; UV \u03bbmax (nm) 320; \u22125 M , MeOH) [\u0398]230 \u2212 7505, [\u0398]255 + 5772, [\u0398]276 \u2212 1307, [\u0398]305 + 15195; HR-QTOF-MS (neg.) m/z 493.1134 [M \u2212 H]\u2212 ; 1H and 13C-NMR spectroscopic data ; white amorphous powder; UV \u03bbmax (nm) 320; \u22125 M, MeOH) [\u0398]248 \u2212 6367, [\u0398]305 + 3958; HR-QTOF-MS (neg.) m/z 493.1134 [M \u2212 H]\u2212 ; 1H and 13C-NMR spectroscopic data ; tentative identification; UV \u03bbmax (nm) 320; HR-QTOF-MS (neg.) m/z 493.1133 [M \u2212 H]\u2212 .Isosalvianolic acid A isomer (43); white amorphous powder; UV \u03bbmax (nm) 330; m/z 373.0932 [M \u2212 H]\u2212 . Rosmarinic acid methyl ester (44); white amorphous powder; UV \u03bbmax (nm) 325; HR-QTOF-MS (neg.) m/z 551.1189 [M \u2212 H]\u2212 . Salvianolic acid H-9\u2033-methylester (3\u2032-O-(8\u2033-Z-caffeoyl)rosmarinic acid-9\u2033-methylester) (45) tentative identification; UV \u03bbmax (nm) 220; HR-QTOF-MS (neg.) m/z 519.0926 [M \u2212 H]\u2212 .Lycopic acid C (Pulmonaria officinalis. The presented research may provide insights for the potential applications of lungwort as a dietary supplement or a nutraceutical, and may it also contribute to the broader application of Pulmonariae Herba. Extracts of P. officinalis may serve as a prominent supply of rosmarinic acid and related compounds, as well as a source of several others metabolites. Among the 45 identified metabolites, we found many compounds with well-established therapeutic properties, although none of them alone can be directly associated with the ethnomedicinal use of lungwort. Our results also show progressive changes in the phytochemical composition of P. officinalis during the phenological cycle, presumably reflecting both changes in the physiological state of plants, as well as varying intensity of different abiotic factors.To the best of our knowledge, this is the first comprehensive study of specialized metabolites in the aerial parts of"} +{"text": "Chem. Commun., 2018, DOI: 10.1039/c7md00575j.Correction for \u2018Novel valdecoxib derivatives by ruthenium("} +{"text": "Retraction Note: Stem Cell Res Therhttps://doi.org/10.1186/scrt443This article has been"} +{"text": "Correction to: Antimicrob Resist Infect Controlhttps://doi.org/10.1186/s13756-018-0389-yThe original article contains\u201cTPB has been employed extensively in pharmacy practice research.\u201d"} +{"text": "Enterobacteriaceae, which they named Edwardsiella (for CDC microbiologist Philip R. Edwards) tarda . These o"} +{"text": "This article has been corrected: The correct Title information is given below:ARF\u2013Mdm2\u2013P53-dependent cellular senescenceNLRP6 targeting suppresses gastric tumorigenesis via P14111597-111607. https://doi.org/10.18632/oncotarget.22876Original article: Oncotarget. 2017; 8:"} +{"text": "EGFR mediates LPA\u2010induced proteolytic enzyme expression and ovarian cancer invasion: Inhibition by resveratrol. Mol Oncol7, 121\u2013129.23127547"} +{"text": "Although the Concise Guide represents approximately 400 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point\u2010in\u2010time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/10.1111/bph.14747. In addition to this overview, in which are identified Other protein targets which fall outside of the subsequent categorisation, there are six areas of focus: G protein\u2010coupled receptors, ion channels, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid\u20102019, and supersedes data presented in the 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the International Union of Basic and Clinical Pharmacology Committee on Receptor Nomenclature and Drug Classification (NC\u2010IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.The Concise Guide to PHARMACOLOGY 2019/20 is the fourth in this series of biennial publications. The Concise Guide provides concise overviews of the key properties of nearly 1800 human drug targets with an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands ( Thyroid hormone receptorsS231 1B. Retinoic acid receptorsS232 1C. Peroxisome proliferator\u2010activated receptorsS233 1D. Rev\u2010Erb receptorsS234 1F. Retinoic acid\u2010related orphansS234 1H. Liver X receptor\u2010like receptorsS235 1I. Vitamin D receptor\u2010like receptorsS236 2A. Hepatocyte nuclear factor\u20104 receptorsS237 2B. Retinoid X receptorsS238 2C. Testicular receptorsS238 2E. Tailless\u2010like receptorsS239 2F. COUP\u2010TF\u2010like receptorsS239 3B. Estrogen\u2010related receptorsS240 4A. Nerve growth factor IB\u2010like receptorsS241 5A. Fushi tarazu F1\u2010like receptorsS241 6A. Germ cell nuclear factor receptorsS242 0B. DAX\u2010like receptorsS242 Steroid hormone receptorsS243 3A. Estrogen receptorsS244 3C. 3\u2010Ketosteroid receptorsS248 Cytokine receptor familyS249 IL\u20102 receptor familyS251 IL\u20103 receptor familyS252 IL\u20106 receptor familyS254 IL\u201012 receptor familyS255 Prolactin receptor familyS256 Interferon receptor familyS257 IL\u201010 receptor familyS258 Immunoglobulin\u2010like family of IL\u20101 receptorsS259 IL\u201017 receptor familyS259 GDNF receptor familyS260 IntegrinsS264 Pattern recognition receptorsS264 Toll\u2010like receptor familyS266 NOD\u2010like receptor familyS268 RIG\u2010I\u2010like receptor familyS269 Receptor Guanylyl Cyclase (RGC) familyS269 Transmembrane quanylyl cyclasesS270 Nitric oxide (NO)\u2010sensitive (soluble) guanylyl cyclaseS271 Receptor tyrosine kinases (RTKs)S272 Type I RTKs: ErbB receptor familyS273 Type II RTKs: Insulin receptor familyS274 Type III RTKs: PDGFR, CSFR, Kit, FLT3 receptor familyS275 Type IV RTKs: VEGF receptor familyS275 Type V RTKs: FGF (broblast growth factor) receptor familyS276 Type VI RTKs: PTK7/CCK4S277 Type VII RTKs: Neurotrophin receptor/Trk familyS278 Type VIII RTKs: ROR familyS278 Type IX RTKs: MuSKS279 Type X RTKs: HGF (hepatocyte growth factor) receptor familyS279 Type XI RTKs: TAM receptor familyS280 Type XII RTKs: TIE family of angiopoietin receptorsS280 Type XIII RTKs: Ephrin receptor familyS281 Type XIV RTKs: RETS282 Type XV RTKs: RYKS282 Type XVI RTKs: DDR (collagen receptor) familyS283 Type XVII RTKs: ROS receptorsS283 Type XVIII RTKs: LMR familyS284 Type XIX RTKs: Leukocyte tyrosine kinase (LTK) receptor familyS284 Type XX RTKs: STYK1S286 Receptor serine/threonine kinase (RSTK) familyS286 Type I receptor serine/threonine kinasesS287 Type II receptor serine/threonine kinasesS287 Type III receptor serine/threonine kinasesS287 RSTK functional heteromersS289 Receptor tyrosine phosphatase (RTP) familyS291 Tumour necrosis factor (TNF) receptor familyS301 Acetylcholine turnoverS302 Adenosine turnoverS303 Amino acid hydroxylasesS304 L\u2010Arginine turnoverS304 2.1.1.\u2010 Protein arginine N\u2010methyltransferasesS305 ArginaseS305 Arginine:glycine amidinotransferaseS305 Dimethylarginine dimethylaminohydrolasesS306 Nitric oxide synthasesS307 Carbonic anhydrasesS308 Carboxylases and decarboxylasesS308 CarboxylasesS309 DecarboxylasesS311 Catecholamine turnoverS313 Ceramide turnoverS313 Serine palmitoyltransferaseS314 Ceramide synthase4\u2010desaturaseS314 Sphingolipid \u0394S315 Sphingomyelin synthaseS315 Sphingomyelin phosphodiesteraseS316 Neutral sphingomyelinase coupling factorsS316 Ceramide glucosyltransferaseS316 Acid ceramidaseS317 Neutral ceramidasesS317 Alkaline ceramidasesS318 Ceramide kinaseS319 Chromatin modifying enzymesS319 2.1.1.\u2010 Protein arginine N\u2010methyltransferasesS320 3.5.1.\u2010 Histone deacetylases (HDACs)S321 Cyclic nucleotide turnover/signallingS321 Adenylyl cyclases (ACs)S323 Exchange protein activated by cyclic AMP (EPACs)S323 Phosphodiesterases, 3\u2019,5\u2019\u2010cyclic nucleotide (PDEs)S327 Cytochrome P450S327 CYP2 familyS328 CYP2 familyS329 CYP3 familyS330 CYP4 familyS331 CYP5, CYP7 and CYP8 familiesS332 CYP11, CYP17, CYP19, CYP20 and CYP21 familiesS333 CYP24, CYP26 and CYP27 familiesS333 CYP39, CYP46 and CYP51 familiesS334 DNA topoisomerasesS335 Endocannabinoid turnoverN\u2010Acylethanolamine turnoverS336 S337 2\u2010Acylglycerol ester turnoverS338 Eicosanoid turnoverS338 CyclooxygenaseS339 Prostaglandin synthasesS341 LipoxygenasesS342 Leukotriene and lipoxin metabolismS343 GABA turnoverS344 Glycerophospholipid turnoverS345 Phosphoinositide\u2010specific phospholipase C2S346 Phospholipase AS348 Phosphatidylcholine\u2010specific phospholipase DS349 Lipid phosphate phosphatasesS349 Phosphatidylinositol kinasesS350 1\u2010phosphatidylinositol 4\u2010kinase familyS351 Phosphatidylinositol\u20104\u2010phosphate 3\u2010kinase familyS351 Phosphatidylinositol 3\u2010kinase familyS351 Phosphatidylinositol\u20104,5\u2010bisphosphate 3\u2010kinase familyS352 1\u2010phosphatidylinositol\u20103\u2010phosphate 5\u2010kinase familyS353 Type I PIP kinases (1\u2010phosphatidylinositol\u20104\u2010phosphate 5\u2010kinase family)S353 Type II PIP kinases (1\u2010phosphatidylinositol\u20105\u2010phosphate 4\u2010kinase family)S356 Phosphatidylinositol phosphate kinasesS356 Haem oxygenaseS358 Hydrogen sulphide synthesisS358 HydrolasesS360 Inositol phosphate turnoverS360 Inositol 1,4,5\u2010trisphosphate 3\u2010kinasesS360 Inositol polyphosphate phosphatasesS361 Inositol monophosphataseS361 Kinases (EC 2.7.x.x)S362 Rho kinaseS362 Protein kinase C (PKC) familyS363 Alpha subfamilyS363 Delta subfamilyS364 Eta subfamilyS364 FRAP subfamilyS365 Cyclin\u2010dependent kinase (CDK) familyS365 CDK4 subfamilyS366 GSK subfamilyS367 Polo\u2010like kinase (PLK) familyS367 STE7 familyS368 Abl familyS368 Ack familyS369 Janus kinase (JakA) familyS369 Src familyS370 Tec familyS371 RAF familyS372 Lanosterol biosynthesis pathwayS374 Nucleoside synthesis and metabolismS376 Paraoxonase (PON) familyS377 Peptidases and proteinasesS377 A1: PepsinS377 A22: PresenilinS378 C14: CaspaseS378 M1: Aminopeptidase NS379 M2: Angiotensin\u2010converting (ACE and ACE2)S379 M10: Matrix metallopeptidaseS380 M12: Astacin/AdamalysinS380 M28: Aminopeptidase YS381 M19: Membrane dipeptidaseS381 S1: ChymotrypsinS382 T1: ProteasomeS382 S8: SubtilisinS383 S9: Prolyl oligopeptidaseS383 Poly ADP\u2010ribose polymerasesS384 Prolyl hydroxylasesS384 Sphingosine 1\u2010phosphate turnoverS385 Sphingosine kinaseS386 Sphingosine 1\u2010phosphate phosphataseS387 Sphingosine 1\u2010phosphate lyaseS387 Thyroid hormone turnoverS388 1.14.13.9 Kynurenine 3\u2010monooxygenaseS389 2.5.1.58 Protein farnesyltransferaseS390 3.5.1.\u2010 Histone deacetylases (HDACs)S391 3.5.3.15 Peptidyl arginine deiminases (PADI)S391 3.6.5.2 Small monomeric GTPasesS391 RAS subfamilyS392 RAB subfamilyS399 ATP\u2010binding cassette transporter familyS399 ABCA subfamilyS401 ABCB subfamilyS403 ABCC subfamilyS404 ABCD subfamily of peroxisomal ABC transportersS405 ABCG subfamilyS406 F\u2010type and V\u2010type ATPasesS406 F\u2010type ATPaseS407 V\u2010type ATPaseS407 P\u2010type ATPases+/K+\u2010ATPasesS407 Na2+\u2010ATPasesS408 Ca+/K+\u2010ATPasesS408 H+\u2010ATPasesS408 CuS409 Phospholipid\u2010transporting ATPasesS409 SLC superfamily of solute carriersS410 SLC1 family of amino acid transportersS410 Glutamate transporter subfamilyS412 Alanine/serine/cysteine transporter subfamilyS413 SLC2 family of hexose and sugar alcohol transportersS413 Class I transportersS414 Class II transportersS415 Proton\u2010coupled inositol transporterS415 SLC3 and SLC7 families of heteromeric amino acid transporters (HATs)S415 SLC3 familyS416 SLC7 familyS417 SLC4 family of bicarbonate transportersS417 Anion exchangers3\u2212 transportersS418 Sodium\u2010dependent HCOS418 SLC5 family of sodium\u2010dependent glucose transportersS419 Hexose transporter familyS420 Choline transporterS421 Sodium iodide symporter, sodium\u2010dependent multivitamin transporter and sodium\u2010coupled monocarboxylate transportersmyo\u2010inositol cotransporter transportersS422 Sodium S423 SLC6 neurotransmitter transporter familyS423 Monoamine transporter subfamilyS424 GABA transporter subfamilyS425 Glycine transporter subfamilyS427 Neutral amino acid transporter subfamilyS428 SLC8 family of sodium/calcium exchangersS429 SLC9 family of sodium/hydrogen exchangersS429 SLC10 family of sodium\u2010bile acid co\u2010transportersS431 SLC11 family of proton\u2010coupled metal ion transportersS431 SLC12 family of cation\u2010coupled chloride transportersS433 SLC13 family of sodium\u2010dependent sulphate/carboxylate transportersS434 SLC14 family of facilitative urea transportersS435 SLC15 family of peptide transportersS437 SLC16 family of monocarboxylate transportersS438 SLC17 phosphate and organic anion transporter familyS438 Type I sodium\u2010phosphate co\u2010transportersS439 Sialic acid transporterS439 Vesicular glutamate transporters (VGLUTs)S440 Vesicular nucleotide transporterS440 SLC18 family of vesicular amine transportersS442 SLC19 family of vitamin transportersS443 SLC20 family of sodium\u2010dependent phosphate transportersS443 SLC22 family of organic cation and anion transportersS444 Organic cation transporters (OCT)S445 Organic zwitterions/cation transporters (OCTN)S446 Organic anion transporters (OATs)S446 Urate transporterS447 Atypical SLC22B subfamilyS448 SLC23 family of ascorbic acid transportersS449 SLC24 family of sodium/potassium/calcium exchangersS450 SLC25 family of mitochondrial transportersS450 Mitochondrial di\u2010 and tri\u2010carboxylic acid transporter subfamilyS451 Mitochondrial amino acid transporter subfamilyS452 Mitochondrial phosphate transportersS452 Mitochondrial nucleotide transporter subfamilyS453 Mitochondrial uncoupling proteinsS454 Miscellaneous SLC25 mitochondrial transportersS454 SLC26 family of anion exchangersS454 Selective sulphate transportersS455 Chloride/bicarbonate exchangersS455 Anion channelsS456 Other SLC26 anion exchangersS457 SLC27 family of fatty acid transportersS458 SLC28 and SLC29 families of nucleoside transportersS458 SLC28 familyS459 SLC29 familyS461 SLC30 zinc transporter familyS461 SLC31 family of copper transportersS462 SLC32 vesicular inhibitory amino acid transporterS463 SLC33 acetylCoA transporterS464 SLC34 family of sodium phosphate co\u2010transportersS465 SLC35 family of nucleotide sugar transportersS466 SLC36 family of proton\u2010coupled amino acid transportersS468 SLC37 family of phosphosugar/phosphate exchangersS468 SLC38 family of sodium\u2010dependent neutral amino acid transportersS469 System A\u2010like transportersS469 System N\u2010like transportersS470 Orphan SLC38 transportersS470 SLC39 family of metal ion transportersS471 SLC40 iron transporterS472 SLC41 family of divalent cation transportersS473 SLC42 family of Rhesus glycoprotein ammoniumtransportersS473 SLC43 family of large neutral amino acid transportersS474 SLC44 choline transporter\u2010like familyS475 SLC45 family of putative sugar transportersS475 SLC46 family of folate transportersS477 SLC47 family of multidrug and toxin extrusion transportersS477 SLC48 heme transporterS478 SLC49 family of FLVCR\u2010related heme transportersS479 SLC50 sugar transporterS479 SLC51 family of steroid\u2010derived molecule transportersS480 SLC52 family of riboflavin transportersS481 SLC53 Phosphate carriersS481 SLC54 Mitochondrial pyruvate carriersS482 SLC55 Mitochondrial cation/proton exchangersS482 SLC56 SideroflexinsS483 SLC57 NiPA\u2010like magnesium transporter familyS483 SLC58 MagT\u2010like magnesium transporter familyS484 SLC59 Sodium\u2010dependent lysophosphatidylcholine symporter familyS484 SLC60 Glucose transportersS485 SLC61 Molybdate transporter familyS485 SLC62 Pyrophosphate transportersS486 SLC63 Sphingosine phosphate transportersS486 SLC64 Golgi Ca2+/H+ exchangersS487 SLC65 NPC\u2010type cholesterol transportersS488 SLCO family of organic anion transporting polypeptideshttps://www.guidetopharmacology.org/). This database is supported by the British Pharmacological Society (BPS), the International Union of Basic and Clinical Pharmacology (IUPHAR), the University of Edinburgh and previously the Wellcome Trust. Data included in the Guide to PHARMACOLOGY are derived in large part from interactions with the subcommittees of the Nomenclature Committee of the International Union of Basic and Clinical Pharmacology (NC\u2010IUPHAR). A major influence on the development of the database was Tony Harmar (1951\u20102014), who worked with a passion to establish the curators as a team of highly informed and informative individuals, with a focus on high\u2010quality data input, ensuring a suitably validated dataset. The Editors of the Concise Guide have compiled the individual records, in concert with the team of Curators, drawing on the expert knowledge of these latter subcommittees. The tables allow an indication of the status of the nomenclature for the group of targets listed, usually previously published in Pharmacological Reviews. In the absence of an established subcommittee, advice from several prominent, independent experts has generally been obtained to produce an authoritative consensus on nomenclature, which attempts to fit in within the general guidelines from NC\u2010IUPHAR. This current edition, the Concise Guide to PHARMACOLOGY 2019/20, is the latest snapshot of the database in print form, following on from the Concise Guide to PHARMACOLOGY 2017/18. It contains data drawn from the online database as a rapid overview of the major pharmacological targets. Thus, there are many fewer targets presented in the Concise Guide compared to the online database. The priority for inclusion in the Concise Guide is the presence of quantitative pharmacological data for human proteins. This means that often orphan family members are not presented in the Concise Guide, although structural information is available on the online database. The organisation of the data is tabular (where appropriate) with a standardised format, where possible on a single page, intended to aid understanding of, and comparison within, a particular target group. The Concise Guide is intended as an initial resource, with links to additional reviews and resources for greater depth and information. Pharmacological and structural data focus primarily on human gene products, wherever possible, with links to HGNC gene nomenclature and UniProt IDs. In a few cases, where data from human proteins are limited, data from other species are indicated. Pharmacological tools listed are prioritised on the basis of selectivity and availability. That is, agents are included where they are both available AND the most selective. The Concise Guide is divided into seven sections, which comprise pharmacological targets of similar structure/function. These are G protein\u2010coupled receptors, ion channels , catalytic receptors, nuclear hormone receptors, enzymes, transporters and other protein targets. We hope that the Concise Guide will provide for researchers, teachers and students a state\u2010of\u2010the art source of accurate, curated information on the background to their work that they will use in the Introductions to their Research Papers or Reviews, or in supporting their teaching and studies. We recommend that any citations to information in the Concise Guide are presented in the following format: Alexander SPH et al. (2019). The Concise Guide to PHARMACOLOGY 2019/20: Introduction and Other Protein Targets. Br J Pharmacol 176: S1\u2013S20. In this overview are listed protein targets of pharmacological interest, which are not G protein\u2010coupled receptors, ion channels, nuclear hormone receptors, catalytic receptors, transporters or enzymes.In order to allow clarity and consistency in pharmacology, there is a need for a comprehensive organisation and presentation of the targets of drugs. This is the philosophy of the IUPHAR/BPS Guide to PHARMACOLOGY presented on the online free access database also supported the initiation and expansion of the database. We are also tremendously grateful to the long list of collaborators from NC\u2010IUPHAR subcommittees and beyond, who have assisted in the construction of the Concise Guide to PHARMACOLOGY 2019/20 and the online database http://www.guidetopharmacology.org.We are extremely grateful to the British Pharmacological Society and the International Union of Basic and Clinical Pharmacology, for financial support of the website and for advice from the NC\u2010IUPHAR subcommittees. We thank the University of Edinburgh, who host the The authors state that there are no conflicts of interest to disclose.http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=1021\u2013 http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=982\u2013 http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=970\u2013 http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=983\u2013 http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=971\u2013 http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=910\u2013 http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=866\u2013 http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=966\u2013 http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=997\u2013 http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=998\u2013 http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=986\u2013 http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=901\u2013 http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=916\u2013 http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=930\u2013 http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=915\u2013 http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=946\u2013 http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=935\u2013 http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=784\u2013 http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=954\u2013 http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=949\u2013 http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=985\u2013 http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=987\u2013 http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=984\u2013 http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=868\u2013 http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=889\u2013 http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=876\u2013 http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=888\u2013 http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=934\u2013 http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=924\u2013 http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=919\u2013 http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=921\u2013 http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=991\u2013 http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=941\u2013 http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=942\u2013 http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=945\u2013 http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=929\u2013 http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=1001\u2013 http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=906\u2013 http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=907\u2013 http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=932\u2013 http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=905\u2013 http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=875\u2013 http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=1000\u2013 http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=989\u2013 http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=972\u2013 http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=1019\u2013 http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=973\u2013 http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=974\u2013 http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=990\u2013 http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=995\u2013 http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=996\u2013 http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=904\u2013 http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=903\u2013 provisional nomenclature, http://www.ensembl.org/Homo_sapiens/Gene/Family/Genes?family=ENSFM00500000270960) respond to the 30 kDa complement\u2010related protein hormone adiponectin originally cloned from adipocytes [http://www.ncbi.nlm.nih.gov/pubmed/8619847?dopt=AbstractPlus]. Although sequence data suggest 7TM domains, immunological evidence indicates that, contrary to typical 7TM topology, the carboxyl terminus is extracellular, while the amino terminus is intracellular [http://www.ncbi.nlm.nih.gov/pubmed/12802337?dopt=AbstractPlus]. Signalling through these receptors appears to avoid G proteins; modelling based on the crystal structures of the adiponectin receptors suggested ceramidase acivity, which would make these the first in a new family of catalytic receptors [http://www.ncbi.nlm.nih.gov/pubmed/25855295?dopt=AbstractPlus].Adiponectin receptors has also been suggested to be a receptor for (hexameric) adiponectin [http://www.ncbi.nlm.nih.gov/pubmed/15210937?dopt=AbstractPlus].T\u2010Cadherin Adiponectin: a manifold therapeutic target for metabolic syndrome, diabetes, and coronary disease? Cardiovasc Diabetol13: 103 [https://www.ncbi.nlm.nih.gov/pubmed/24957699?dopt=AbstractPlus]Fisman EZ et al. (2018) Structure and function analysis of adiponectin receptors toward development of novel antidiabetic agents promoting healthy longevity. Endocr J65: 971\u2010977 [https://www.ncbi.nlm.nih.gov/pubmed/30282888]Okada\u2010Iwabu M et al. (2016) Adiponectin signaling and function in insulin target tissues. J Mol Cell Biol8: 101\u20109 [https://www.ncbi.nlm.nih.gov/pubmed/26993044?dopt=AbstractPlus]Ruan H et al. (2017) Cardiovascular Adiponectin Resistance: The Critical Role of Adiponectin Receptor Modification. Trends Endocrinol. Metab.28: 519\u2010530 [https://www.ncbi.nlm.nih.gov/pubmed/28473178?dopt=AbstractPlus]Wang Y et al. (2014) Adiponectin and insulin cross talk: the microvascular connection. Trends Cardiovasc. Med.24: 319\u201024 [https://www.ncbi.nlm.nih.gov/pubmed/25220977?dopt=AbstractPlus]Zhao L http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=760), degranulation and aggregation of platelets, as well as proteins circulating in the plasma. The coagulation cascade involves multiple proteins being converted to more active forms from less active precursors, typically through proteolysis (see http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=759&familyType=ENZYME). Listed here are the components of the coagulation cascade targetted by agents in current clinical usage.Coagulation as a process is interpreted as a mechanism for reducing excessive blood loss through the generation of a gel\u2010like clot local to the site of injury. The process involves the activation, adhesion (see Blood125: 2045\u201051 [https://www.ncbi.nlm.nih.gov/pubmed/25712994?dopt=AbstractPlus]Astermark J. (2015) FVIII inhibitors: pathogenesis and avoidance. et al. (2017) New clotting disorders that cast new light on blood coagulation and may play a role in clinical practice. J. Thromb. Thrombolysis44: 71\u201075 [https://www.ncbi.nlm.nih.gov/pubmed/28251495?dopt=AbstractPlus]Girolami A et al. (2016) Blood flow and mass transfer regulation of coagulation. Blood Rev.30: 357\u201068 [https://www.ncbi.nlm.nih.gov/pubmed/27133256?dopt=AbstractPlus]Rana K Bromodomains bind proteins with acetylated lysine residues, such as histones, to regulate gene transcription. Listed herein are examples of bromodomain\u2010containing proteins for which sufficient pharmacology exists.et al. (2017) Functions of bromodomain\u2010containing proteins and their roles in homeostasis and cancer. Nat. Rev. Mol. Cell Biol.18: 246\u2010262 [https://www.ncbi.nlm.nih.gov/pubmed/28053347?dopt=AbstractPlus]Fujisawa T Biochem Pharmacol159: 40\u201051 [https://www.ncbi.nlm.nih.gov/pubmed/30414936]Myrianthopoulos V & Mikros E. (2019) From bench to bedside, via desktop. Recent advances in the application of cutting\u2010edge in silico tools in the research of drugs targeting bromodomain modules. et al. (2017) BET bromodomain proteins and epigenetic regulation of inflammation: implications for type 2 diabetes and breast cancer. Cell. Mol. Life Sci.74: 231\u2010243 [https://www.ncbi.nlm.nih.gov/pubmed/27491296?dopt=AbstractPlus]Nicholas DA Drug Discov Today23: 76\u201089 [https://www.ncbi.nlm.nih.gov/pubmed/28943305]Ramadoss M & Mahadevan V. (2018) Targeting the cancer epigenome: synergistic therapy with bromodomain inhibitors. et al. (2019) Small\u2010molecule PROTAC degraders of the Bromodomain and Extra Terminal (BET) proteins \u2010 A review. Drug Discov Today Technol31: 43\u201051 [https://www.ncbi.nlm.nih.gov/pubmed/31200858]Yang CY http://www.ncbi.nlm.nih.gov/pubmed/23716704?dopt=AbstractPlus]. These amyloidogenic mutants are linked to the development of pathological amyloidoses, including familial amyloid polyneuropathy (FAP) , familial amyloid cardiomyopathy (FAC) [http://www.ncbi.nlm.nih.gov/pubmed/9017939?dopt=AbstractPlus], amyloidotic vitreous opacities, carpal tunnel syndrome [http://www.ncbi.nlm.nih.gov/pubmed/10403814?dopt=AbstractPlus] and others. In old age, non\u2010mutated TTR can also form pathological amyloid fibrils [http://www.ncbi.nlm.nih.gov/pubmed/7016817?dopt=AbstractPlus]. Pharmacological intervention to reduce or prevent TTR dissociation is being pursued as a theapeutic strategy. To date one small molecule kinetic stabilising molecule (http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=8378) has been approved for FAP, and is being evaluated in clinical trials for other TTR amyloidoses.Transthyretin (TTR) is a homo\u2010tetrameric protein which transports thyroxine in the plasma and cerebrospinal fluid and retinol (vitamin A) in the plasma. Many disease causing mutations in the protein have been reported, many of which cause complex dissociation and protein mis\u2010assembly and deposition of toxic aggregates amyloid fibril formation [et al. (2019) Hereditary transthyretin amyloidosis: a model of medical progress for a fatal disease. Nat Rev Neurol15: 387\u2010404 [https://www.ncbi.nlm.nih.gov/pubmed/31209302]Adams D et al. (2017) Is transthyretin a good marker of nutritional status? Clin Nutr36: 364\u2010370 [https://www.ncbi.nlm.nih.gov/pubmed/27381508?dopt=AbstractPlus]Delli\u00e8re S et al. (2017) Transthyretin amyloidosis: an under\u2010recognized neuropathy and cardiomyopathy. Clin. Sci.131: 395\u2010409 [https://www.ncbi.nlm.nih.gov/pubmed/28213611?dopt=AbstractPlus]Galant NJ Biol Pharm Bull41: 979\u2010984 [https://www.ncbi.nlm.nih.gov/pubmed/29962408]Yokoyama T & Mizuguchi M. (2018) Inhibition of the Amyloidogenesis of Transthyretin by Natural Products and Synthetic Compounds. et al. (2019) Transthyretin Amyloid Cardiomyopathy: JACC State\u2010of\u2010the\u2010Art Review. J Am Coll Cardiol73: 2872\u20102891 [https://www.ncbi.nlm.nih.gov/pubmed/31171094]Ruberg FL http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=1232) or receptors . Many CDs are targeted for therapeutic gain using antibodies for the treatment of proliferative disorders. A full listing of all the Clusters of Differentiation proteins is not possible in the Guide to PHARMACOLOGY; listed herein are selected members of the family targeted for therapeutic gain.Cluster of differentiation refers to an attempt to catalogue systematically a series of over 300 cell\u2010surface proteins associated with immunotyping. Many members of the group have identified functions as enzymes ) and programmed cell death 1 ligand 2 . These ligands are cell surface peptides, normally involved in immune system regulation. Expression of PD\u20101 by cancer cells induces immune tolerance and evasion of immune system attack. Anti\u2010PD\u20101 monoclonal antibodies are used to induce immune checkpoint blockade as a therapeutic intervention in cancer, effectively re\u2010establishing immune vigilance. http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=7499 was the first anti\u2010PD\u20101 antibody to be approved by the US FDA.The endogenous ligands for human PD\u20101 are programmed cell death 1 ligand 1 The glycobiology of the CD system: a dictionary for translating marker designations into glycan/lectin structure and function. Trends Biochem. Sci.40: 360\u201076 [https://www.ncbi.nlm.nih.gov/pubmed/25981696?dopt=AbstractPlus]Gabius HJ et al. (2019) CD markers variations in chronic lymphocytic leukemia: New insights into prognosis. J Cell Physiol. 234: 19420\u201039 [https://www.ncbi.nlm.nih.gov/pubmed/31049958]Vosoughi T Methyllysine reader proteins bind to methylated proteins, such as histones, allowing regulation of gene expression.et al. (2018) Histone methylation and acetylation in macrophages as a mechanism for regulation of inflammatory responses. J Cell Physiol. 233: 6495\u20109507 [https://www.ncbi.nlm.nih.gov/pubmed/29574768]Daskalaki MG et al. (2019) Epigenetic interplays between DNA demethylation and histone methylation for protecting oncogenesis. J Biochem. 165: 297\u2010299 [https://www.ncbi.nlm.nih.gov/pubmed/30605533]Furuya K Cell Mol Life Sci76: 2873\u201083 [https://www.ncbi.nlm.nih.gov/pubmed/31123776]Levy D. (2019) Lysine methylation signaling of non\u2010histone proteins in the nucleus. et al. (2019) Understanding histone H3 lysine 36 methylation and its deregulation in disease. Cell Mol Life Sci in press [https://www.ncbi.nlm.nih.gov/pubmed/31147750]Li J et al. (2019) Role of histone modification and DNA methylation in signaling pathways involved in diabetic retinopathy. J Cell Physiol.234: 7839\u20107846 [https://www.ncbi.nlm.nih.gov/pubmed/30515789]Shafabakhsh R e.g. in plasma) or transport these agents; to the nucleus to interact with nuclear receptors or for interaction with metabolic enzymes. Although sequence homology is limited, crystallographic studies suggest conserved 3D structures across the group of binding proteins.Fatty acid\u2010binding proteins are low molecular weight (100\u2010130 aa) chaperones for long chain fatty acids, fatty acyl CoA esters, eicosanoids, retinols, retinoic acids and related metabolites and are usually regarded as being responsible for allowing the otherwise hydrophobic ligands to be mobile in aqueous media. These binding proteins may perform functions extracellularly (http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=6735 exhibits high affinity for FABP4 (pIC50 8.8) compared to FABP3 or FABP5 (pIC50 <6.6) . http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=6736 is reported to interfere with FABP4 action [http://www.ncbi.nlm.nih.gov/pubmed/19754198?dopt=AbstractPlus]. Ibuprofen displays some selectivity for FABP4 (pIC50 5.5) relative to FABP3 (pIC50 3.5) and FABP5 (pIC50 3.8) [http://www.ncbi.nlm.nih.gov/pubmed/24248795?dopt=AbstractPlus]. Fenofibric acid displays some selectivity for FABP5 (pIC50 5.5) relative to FABP3 (pIC50 4.5) and FABP4 (pIC50 4.6) [http://www.ncbi.nlm.nih.gov/pubmed/24248795?dopt=AbstractPlus]. Multiple pseudogenes for the FABPs have been identified in the human genome.Although not tested at all FABPs, et al. (2015) Enterocyte fatty acid\u2010binding proteins (FABPs): different functions of liver and intestinal FABPs in the intestine. Prostaglandins Leukot. Essent. Fatty Acids93: 9\u201016 [https://www.ncbi.nlm.nih.gov/pubmed/25458898?dopt=AbstractPlus]Gajda AM Prostaglandins Leukot Essent Fatty Acids93: 45\u20109 [https://www.ncbi.nlm.nih.gov/pubmed/25154384?dopt=AbstractPlus]Glatz JF. (2015) Lipids and lipid binding proteins: a perfect match. et al. (2015) Metabolic functions of FABPs\u2013mechanisms and therapeutic implications. Nat Rev Endocrinol11: 592\u2010605 [https://www.ncbi.nlm.nih.gov/pubmed/26260145?dopt=AbstractPlus]Hotamisligil GS et al. (2016) Fatty acid binding proteins and the nervous system: Their impact on mental conditions. Neurosci. Res.102: 47\u201055 [https://www.ncbi.nlm.nih.gov/pubmed/25205626?dopt=AbstractPlus]Matsumata M et al. (2016) Heart lipid droplets and lipid droplet\u2010binding proteins: Biochemistry, physiology, and pathology. Exp. Cell Res.340: 198\u2010204 [https://www.ncbi.nlm.nih.gov/pubmed/26524506?dopt=AbstractPlus]Osumi T http://www.ncbi.nlm.nih.gov/pubmed/20971825?dopt=AbstractPlus]. As the Notch ligands are also membrane bound, cells have to be in close proximity for receptorligand interactions to occur. Cleavage of the intracellular domain (ICD) of activated Notch receptors by \u03b3\u2010secretase is required for downstream signalling and Notch\u2010induced transcriptional modulation . This is why \u03b3\u2010secretase inhibitors can be used to downregulate Notch signalling and explains their anticancer action. One such small molecule is http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=7338 [http://www.ncbi.nlm.nih.gov/pubmed/19773430?dopt=AbstractPlus], although development of this compound has been terminated following an unsuccessful Phase II single agent clinical trial in metastatic colorectal cancer [http://www.ncbi.nlm.nih.gov/pubmed/22445247?dopt=AbstractPlus].The canonical Notch signalling pathway has four type I transmembrane Notch receptors (Notch1\u20104) and five ligands . Each member of this highly conserved receptor family plays a unique role in cell\u2010fate determination during embryogenesis, differentiation, tissue patterning, proliferation and cell death , with http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=8451 and http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=8453 identified as antibody inhibitors of ligand:receptor binding [http://www.ncbi.nlm.nih.gov/pubmed/25388163?dopt=AbstractPlus].Aberrant Notch signalling is implicated in a number of human cancers [et al. (2018) Notch signaling and neuronal death in stroke. Prog. Neurobiol.165\u2010167: 103\u2010116 [https://www.ncbi.nlm.nih.gov/pubmed/29574014?dopt=AbstractPlus]Arumugam TV et al. (2016) The Notch intracellular domain integrates signals from Wnt, Hedgehog, TGF\u03b2/BMP and hypoxia pathways. Biochim. Biophys. Acta1863: 303\u201013 [https://www.ncbi.nlm.nih.gov/pubmed/26592459?dopt=AbstractPlus]Borggrefe T et al. (2015) Ligand\u2010Independent Mechanisms of Notch Activity. Trends Cell Biol.25: 697\u2010707 [https://www.ncbi.nlm.nih.gov/pubmed/26437585?dopt=AbstractPlus]Palmer WH et al. (2015) Molecular pathways: translational and therapeutic implications of the Notch signaling pathway in cancer. Clin. Cancer Res.21: 955\u201061 [https://www.ncbi.nlm.nih.gov/pubmed/25388163?dopt=AbstractPlus]Previs RA et al. (2015) Targeting Notch, Hedgehog, and Wnt pathways in cancer stem cells: clinical update. Nat Rev Clin Oncol12: 445\u201064 [https://www.ncbi.nlm.nih.gov/pubmed/25850553?dopt=AbstractPlus]Takebe N http://www.ncbi.nlm.nih.gov/pubmed/8756726?dopt=AbstractPlus, http://www.ncbi.nlm.nih.gov/pubmed/9108480?dopt=AbstractPlus, http://www.ncbi.nlm.nih.gov/pubmed/9417641?dopt=AbstractPlus], leading to a termination of GPCR signalling. Interactions through protein: protein interactions of many RGS proteins have been identified for targets other than heteromeric G proteins. Sequence analysis of the 20 RGS proteins suggests four families of RGS: RZ, R4, R7 and R12 families. Many of these proteins have been identified to have effects other than through targetting G proteins. Included here is RGS4 for which a number of pharmacological inhibitors have been described.Regulators of G protein signalling (RGS) proteins display a common RGS domain that interacts with the GTP\u2010bound G\u03b1 subunits of heterotrimeric G proteins, enhancing GTP hydrolysis by stabilising the transition state [http://www.ncbi.nlm.nih.gov/pubmed/16765607?dopt=AbstractPlus]. It consists of RGS17 , RGS19 and RGS20 . All members contain an N\u2010terminal cysteine string motif [http://www.ncbi.nlm.nih.gov/pubmed/17183362?dopt=AbstractPlus] which is a site of palmitoylation and could serve functions in membrane targeting, protein stability or aid protein\u2010protein interactions [http://www.ncbi.nlm.nih.gov/pubmed/17126529?dopt=AbstractPlus]. However, the function in the case of RZ family RGS proteins is not yet fully understood. Members of the RZ family of RGS proteins are the only RGS proteins that have selective GTPase activating\u2010protein (GAP) activity for G\u03b1z, a function that resulted in the name of the family . However, the members of the RZ family are able to also GAP G\u03b1i/o members with varying selectivity.The RZ family of RGS proteins is less well characterized than the other families [This is the largest family of RGS proteins.This family of RGS proteins shows some selectivity for Gai/o proteins.http://www.ncbi.nlm.nih.gov/pubmed/26123306?dopt=AbstractPlus]. The G\u03b1i/o\u2010Loco (GoLoco) motif in RGS12 and 14 has GDI activity (for Guanine nucleotide Dissociation Inhibitor) towards G\u03b1i1, G\u03b1i2 and G\u03b1i3 . Through this activity RGS12 and RGS14 can inhibit G protein signaling both by accelerating GTP hydrolysis and by preventing G protein activation. Splice variants of RGS12 and RGS14 also contain membrane targeting and protein\u2010protein interaction domains .The R12 family consists of RGS10, 12 and 14. RGS12 and 14 are large proteins with additional domains that can participate in protein\u2010protein interactions and other functions. In contrast, RGS10 is a small protein consisting of the RGS domain and small N\u2010 and C\u2010termini, similar to members of the R4 family. However, sequence homology of the RGS10 RGS domain clearly places it in the R12 family Alqinyah M et al. (2002) Regulators of G\u2010protein signalling as new central nervous system drug targets. Nat Rev Drug Discov1: 187\u201097 [https://www.ncbi.nlm.nih.gov/pubmed/12120503?dopt=AbstractPlus]Neubig RR et al. (2010) Non\u2010canonical functions of RGS proteins. Cell. Signal.22: 1274\u201081 [https://www.ncbi.nlm.nih.gov/pubmed/20363320?dopt=AbstractPlus]Sethakorn N Br. J. Pharmacol.174: 427\u2010437 [https://www.ncbi.nlm.nih.gov/pubmed/28098342?dopt=AbstractPlus]Sj\u00f6gren B. (2017) The evolution of regulators of G protein signalling proteins as drug targets \u2010 20 years in the making: IUPHAR Review 21. et al. (2010) Thinking outside of the \u2018RGS box\u2019: new approaches to therapeutic targeting of regulators of G protein signaling. Mol. Pharmacol.78: 550\u20107 [https://www.ncbi.nlm.nih.gov/pubmed/20664002?dopt=AbstractPlus]Sj\u00f6gren B http://www.ncbi.nlm.nih.gov/pubmed/27042935?dopt=AbstractPlus] suggests a trimeric structure of a single short transmembrane domain traversing the endoplasmic reticulum membrane, with the bulk of the protein facing the cytosol. A wide range of compounds, ranging from psychoactive agents to antihistamines, have been observed to bind to these sites.Although termed \u2019receptors\u2019, the evidence for coupling through conventional signalling pathways is lacking. Initially described as a subtype of opioid receptors, there is only a modest pharmacological overlap and no structural convergence with the G protein\u2010coupled receptors; the crystal structure of the sigma1 receptor [http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=1606 also shows activity at opioid receptors. The sigma2 receptor has recently been reported to be http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=2553 [http://www.ncbi.nlm.nih.gov/pubmed/28559337?dopt=AbstractPlus], a 4TM protein partner of NPC1, the Niemann\u2010Pick C1 protein, a 13TM cholesterol\u2010binding protein.et al. (2016) Biochemical Pharmacology of the Sigma\u20101 Receptor. Mol. Pharmacol.89: 142\u201053 [https://www.ncbi.nlm.nih.gov/pubmed/26560551?dopt=AbstractPlus]Chu UB et al. (2015) Sigma\u20101 receptor and inflammatory pain. Inflamm. Res.64: 377\u201081 [https://www.ncbi.nlm.nih.gov/pubmed/25902777?dopt=AbstractPlus]Gris G et al. (2016) Sigma receptors [\u03c3Rs]: biology in normal and diseased states. J. Recept. Signal Transduct. Res.36: 327\u2010388 [https://www.ncbi.nlm.nih.gov/pubmed/26056947?dopt=AbstractPlus]Rousseaux CG et al. (2018) The sigma\u20101 receptor as a regulator of dopamine neurotransmission: A potential therapeutic target for methamphetamine addiction. Pharmacol Ther186: 152\u2010167 [https://www.ncbi.nlm.nih.gov/pubmed/29360540]Sambo DO et al. (2016) The Sigma\u20101 Receptor as a Pluripotent Modulator in Living Systems. Trends Pharmacol. Sci.37: 262\u2010278 [https://www.ncbi.nlm.nih.gov/pubmed/26869505?dopt=AbstractPlus]Su TP et al. (2019) Allosteric Modulators of Sigma\u20101 Receptor: A Review. Front Pharmacol10: 223 [https://www.ncbi.nlm.nih.gov/pubmed/30941035]Vavers E Tubulins are a family of intracellular proteins most commonly associated with microtubules, part of the cytoskeleton. They are exploited for therapeutic gain in cancer chemotherapy as targets for agents derived from a variety of natural products: taxanes, colchicine and vinca alkaloids. These are thought to act primarily through \u03b2\u2010tubulin, thereby interfering with the normal processes of tubulin polymer formation and disassembly.et al. (2019) Current advances of tubulin inhibitors as dual acting small molecules for cancer therapy. Med Res Rev39: 1398\u20101426 [https://www.ncbi.nlm.nih.gov/pubmed/30746734]Arnst KE Proc Natl Acad Sci U S A116: 10366\u201010371 [https://www.ncbi.nlm.nih.gov/pubmed/31072936]Eshun\u2010Wilson L. (2019) Effects of alpha\u2010tubulin acetylation on microtubule structure and stability. et al. (2017) The tubulin code at a glance. J. Cell. Sci.130: 1347\u20101353 [https://www.ncbi.nlm.nih.gov/pubmed/28325758?dopt=AbstractPlus]Gadadhar S et al. (2018) Tubulin Posttranslational Modifications and Emerging Links to Human Disease. Cell173: 1323\u20101327 [https://www.ncbi.nlm.nih.gov/pubmed/29856952]Magiera MM et al. (2017) Anti\u2010mitotic agents: Are they emerging molecules for cancer treatment? Pharmacol. Ther.173: 67\u201082 [https://www.ncbi.nlm.nih.gov/pubmed/28174095?dopt=AbstractPlus]Penna LS"} +{"text": "This article has been corrected: The correct author information is given below:Pin Li2,1,*and Xu Zhang1,2https://doi.org/10.18632/oncotarget.19934Original article: Oncotarget. 2017; 8:62489-62499."} diff --git a/PMC_clustering_415.jsonl b/PMC_clustering_415.jsonl new file mode 100644 index 0000000000000000000000000000000000000000..8856fddc226e9c8d6fe23c014ae2bcd5252f29f5 --- /dev/null +++ b/PMC_clustering_415.jsonl @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:2d4d3e9411de2433bde8d36b967fe9d180f7d2990ee0608a138affc114b86ca6 +size 73162760 diff --git a/PMC_clustering_416.jsonl b/PMC_clustering_416.jsonl new file mode 100644 index 0000000000000000000000000000000000000000..78db7da6215a1abe194cac3cef99577769993d55 --- /dev/null +++ b/PMC_clustering_416.jsonl @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:1bd0beef131ec064a9b9f458eed7968c045829467894f0bc8e0833346f6c03e5 +size 18996099 diff --git a/PMC_clustering_417.jsonl b/PMC_clustering_417.jsonl new file mode 100644 index 0000000000000000000000000000000000000000..c999c7a97c9e311d4755e7b424a9c77edc8fee7e --- /dev/null +++ b/PMC_clustering_417.jsonl @@ -0,0 +1,797 @@ +{"text": "Recent advances in the field of molecular pathology and the publication of the revised fourth edition of the WHO Classification of central nervous system (CNS) tumors have significantly reshaped the approach to both diagnosis and therapy of brain tumors . Due to This Research Topic entitled \u201cMolecular Advances in Diagnosis and Treatment of CNS Tumors\u201d includes 22 original research articles and 3 review articles that cover several important themes:Glioblastoma (GBM) is the most common and devastating primary brain tumor in adults. It is therefore essential to identify novel and effective biomarkers or risk signatures for GBM patients. Wang et al. examined differentially expressed genes between GBM and low-grade glioma (LGG) and selected five genes to construct a risk signature to independently predict the outcome of GBM patients, as well as stratified by radio-chemotherapy, isocitrate dehydrogenase 1 (IDH1) and O6-methylguanine-DNA methyltransferase (MGMT) promoter status. Zhang et al. evaluated the expression level of integrin beta 5 (ITGB5) and the relationship of its elevated expression with glioma progression and poor survival in GBM patients. It appears ITGB5 plays important regulatory roles in angiogenesis and the immune response, and is required for invasion and migration of neoplastic cells and endothelial proliferation in GBM. Zusman et al. discussed how harvesting GBM tissue using traditional surgical approach and the automated resection NICO Myriad\u2122 system may impact the translational research value of the sample. Their study further supports the need to harvest and analyze multiple specimens for each tumor, in order to capture the genomic diversity and maximize the benefits of molecularly-based therapeutics. Zhang et al. demonstrated that Forkhead Box P2 (FOXP2) was the target protein of miR-9-5p. In addition, high expression of miR-9-5p and low expression of FOXP2 were related to better outcome in GBM patients, whereas down regulated FOXP2 expression was capable of inhibiting glioma proliferation through cell cycle arrest. Liu et al. determined the candidate genes that may function as biomarkers to further distinguish patients with IDH-wildtype GBM. The investigators developed a seven-gene-based signature, which allocated each patient to a risk group (low or high). Subsequent bioinformatics analysis predicted that the seven-gene signature was involved in the immune response, inflammatory response, cell adhesion, and apoptotic process. Marchi et al. attempted to correlate the biomolecular aspects of MGMT methylation status in relation to the maximal surgical extent of resection. Interestingly, a positive prognostic value exists only in case of the presence of residual tumor tissue. Dent et al. explored whether a multiple sclerosis drug, Fingolimod would synergize with dimethyl fumarate and its plasma breakdown product MMF to kill GBM neoplastic cells. Indeed, the data demonstrated that the above combination produced reactive oxygen species and killed tumor cells more effectively via death receptor signaling and autophagy induction. Hu et al. conducted a systematic analysis of survival-associated alternative splicing event. The nomogram with age, pharmaceutical and radiation therapy, alternate donor site, and exon skip signatures provided excellent prognostic predictive value.Treatment effectiveness and overall prognosis for glioma patients depend heavily on the genetic and epigenetic factors in each individual tumor. Gates et al. discovered that primary brain tumors are genetically heterogeneous, and the physical distance within a given glioma positively correlates to genomic distance in number of genes, copy number variations, and methylation profiles. They further derived quantitative linear relationships between physical and genomic distances. Su et al. showed that \u03b3Klotho is highly expressed in gliomas epigenetically and its expression is significantly associated with high tumor aggressiveness and poor outcomes for glioma patients. Mechanistically, LCTL might play an important immunosuppressive role via FGF signaling in glioma. Yang et al. performed weighted gene co-expression network analysis in a large public database of glioma samples. The derived brown co-expression module and the biomarker TNFRSF1A were strongly related to glioma grading. Furthermore upregulated TNFRSF1A was tightly associated with clinical features. Zhang et al. systematically analyzed the relationship between methyltransferase-related gene expression profiles and clinical outcomes in glioma patients and identified a novel methyltransferase-related risk signature for predicting the prognosis of gliomas.Recently non-coding types of RNA have been shown to play a vital role in glioma tumorigenesis. Jin et al. characterized a novel non-coding RNA, lipocalin-2-derived circular RNA, in glioma tumorigenesis. The investigators demonstrated that it facilitated glioma progression by sponging miR-661 to increase RAB3D expression. Similarly, Zheng et al. characterized non-coding competitive RNA networks as alternative therapeutic targets in the treatment of GBM. Sun et al. explored the expression profiles and potential relationship between long non-coding RNAs (lncRNAs) and mRNAs in glioma patients. Both lncRNAs and mRNAs exhibited dynamic differential expression profiles, consistent with their roles in critical biological processes and pathways associated with tumor pathogenesis.Several manuscripts cover some of the most fascinating developments in the field. Zhang et al. conducted a meta-analysis to evaluate the prognostic role of connexin protein Cx43 in glioma. The results showed that Cx43 expression was a clearly negative factor with tumor grades and beneficial for survival time, offering evidence that Cx43 is generally a tumor suppressor. Deng et al. explored the influence of IDH1 mutation on the immune microenvironment and developed an IDH1-associated immune prognostic signature to help classify LGG patients into subgroups with distinct outcomes and immunophenotypes. Liu et al. discussed the correlations of soluble PD-L1 (sPD-L1) with clinical features in brain tumors and assessed its diagnostic value in gliomas. Both serum and CSF sPD-L1 showed significant value, but serum sPD-L1 rather than blood-based inflammatory markers had the best diagnostic performance in the diagnosis and stratification of glioma. In addition, a descending trend in the level of serum sPD-L1 was observed in postoperative patients. Hung et al. studied the important question of glioma stem-like cells contributing to drug resistance and tumor recurrence. Their study suggests that a sonic hedgehog (Shh) inhibitor could induce autophagy of CD133+ GSCs through mTOR independent pathway. Therefore, targeting the Shh signal pathway may overcome chemoresistance and provide a therapeutic strategy for patients with malignant gliomas.Informative Review Articles:Tang et al. reviewed the advantages and possible limitations of mRNA-based gene therapy including the in vitro synthesis of mRNA, the feasible methods for synthetic mRNA delivery and clinical therapeutic prospects of mRNA-based gene therapy for glioblastoma. Yu et al. reviewed the regulation of MGMT expression and its role in chemotherapy, especially in glioma. Targeting MGMT seems to be a promising approach to overcome chemoresistance. Hu et al. reviewed the relationship between ferroptosis, a new type of cell death, and temozolomide (TMZ) resistance. Importantly, targeted ferroptosis can be used to reverse TMZ resistance.In summary, management of CNS tumor patients has undergone a molecular revolution driven by the development of high throughput molecular techniques. Molecular testing has become an essential part for the optimal CNS tumor patient workup. At the current stage, a combination of FISH, copy number array, NGS panel and genome-wide methylation profiling can be used to detect molecular alterations in order to provide the best possible patient care. It is true that our ability of amassing molecular data currently surpasses our ability to utilize this information for treatment; however, it is clear that informative molecular biomarkers will guide future clinical trials and lead to the development of new therapeutic strategies.Z-HZ, M-TL, and LC are the coeditors for this Research Topic. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Recently, Johansson and colleagues published an article about the mechanisms causing female reproductive disorders with a special focus on the ovaries (Johansson et al. In recent years, stem cell-based tests have been established where precursor cells are exposed to test compounds during the differentiation period (Krug et al."} +{"text": "In 2015, the World Health Organization (WHO) published the guideline \u201cSugars intake for adults and children\u201d [The paper presented by Anderson et al. studied While still more research is needed on the association of both pure fruit juice and ASB consumption with all-cause mortality risk, Anderson et al. provide an important contribution to the current scientific evidence in this field by finding different associations for all-cause mortality between consumption of SSBs and consumption of pure fruit juice. Since the association between SSB consumption and all-cause mortality is well established , 5, 6, i"} +{"text": "Short (<200 nt) and long (>200 nt) non-coding (nc) RNAs account for majority of mammalian transcriptional output and encompass RNA species critical for various aspects of development and disease Ambros, . We haveDeeper insight into these enigmatic RNA species clearly requires efforts from both wet-lab and computational avenues of research . One of them, miR-30c, directly repressed cyclin-dependent kinase 12 (CDK12) through a complementary site in the 3\u2032 UTR . Several DNA damage response (DDR) genes were significantly downregulated after introducing miR-30c or repressing CDK12, suggesting that miR-30c regulates embryo development through the DDR pathway .Zhao et al. examined deregulated miRNAs, lncRNAs and circRNAs in the hair follicle cycle of Angora Rabbit (Oryctolagus cuniculus) and provides comprehensive repository of ncRNAs potentially relevant to this process.Mature hair follicles in mammals undergo periodic self-renewal processes called hair follicle cycles. Understanding the molecular regulatory mechanisms of the renewal cycle is important in medicine and developmental biology. Wang et al. profiled lncRNAs in the CD4+ T cells in the mouse model of acute asthma. They found 36 up- and 98 down-regulated lncRNAs in the disease compared with the control samples . The potential functions of deregulated lncRNA were analyzed by performing miRNA binding analysis .Xun et al. proposed an efficient experimental method to find miRNA binding sequences in genomic DNA in vivo, thus potentially identifying miRNA binding sites in the regulatory regions of genes.It has been well-established that miRNAs work by guiding RNA-induced silencing complex (RISC) to their target RNA binding sites in cytoplasm Bartel, . HoweverOu-Yang et al. proposed a novel method called two-side sparse self-representation (TSSR) for predicting lncRNA-disease associations. TSSR significantly outperformed other tested methods and identified some candidate lncRNA-disease associations .Zhang et al. proposed a method called CRlncRC2 for predicting associations between lncRNAs and cancers. More than four hundred cancer-related lncRNA candidates were identified, which were evaluated by examining the Lnc2Cancer database, reviewing literature, and performing statistical analysis of multiple relevant data sources containing information on mutations and differential gene expression in cancers . These results demonstrated that CRlncRC2 is an effective and accurate method for identification of cancer-related lncRNAs .Shen et al. proposed a new method for identifying lncRNA-protein interactions by employing Kernel Ridge Regression, based on Fast Kernel Learning (LPI-FKLKRR). LPI-FKLKRR demonstrated a superior performance compared with a series of other methods as judged by area under precision recall curve.LncRNAs are assumed to realize their functions by interacting with other molecules, such as proteins, chromatin and other RNA species. Huang et al. introduced a computational method to predict interactions between lncRNAs and miRNAs leveraging the information of expression profile data for these transcripts and the graph convolution technique. The proposed model is based on the assumption that the interaction between an lncRNA and a miRNA could be deciphered from their co-expression pattern. Compared with the conventional miRNA-target prediction algorithms based on sequence matching, their work presents a new approach to predict lncRNA:miRNA interactions.Fukunaga et al. introduced a web server, called LncRRIsearch, for predicting lncRNA:lncRNA and lncRNA:mRNA interactions in human and mouse. The tissue-specific expression and cellular localization data of lncRNAs are integrated in this web server to explore tissue-specific or subcellular-localized lncRNA interactions .Li and Liu summarizing recent evidences suggesting that coding and non-coding properties are inherent to both coding and non-coding transcripts. In other words, some lncRNAs and circRNAs could be used to produce short peptides, i.e., have coding capabilities. On the other hand, 3\u2032 and 5\u2032 UTRs of coding genes have non-coding functions such as recruiting RNA-binding proteins (Li and Liu).Smith et al. reviewed the miRNAs and lncRNAs that play key roles in the initiation and progression of pediatric solid tumors. Pediatric tumors, due to lower mutation load compared with adult ones, are assumed to arise from mis-regulation of networks normally functioning during development at transcriptional level . The authors summarized accumulating evidence of involvement of miRNAs and lncRNAs in the regulatory networks functioning during oncogenesis.Watson et al. explored small RNAs in neurodegenerative diseases. This comprehensive review discusses roles of various small RNAs in multiple neurodegenerative diseases, including Alzheimer's, Parkinson's, multiple sclerosis, Amyotrophoic lateral sclerosis, and Huntington's disease.Ram\u00f3n y Cajal et al. proposed that the interactions between miRNAs and lncRNAs might contribute to the cell-type specific outcomes and to the determination of cell fate. In one model, miRNAs could be competitively sequestered by tissue-specific lncRNAs. In another context, miRNAs released to extracellular space as ligands could interact with lncRNAs in different organs as receptors to either sequester the miRNAs or induce degradation of the miRNAs or the lncRNAs.Recent evidences show that ncRNAs, both miRNAs and lncRNAs, could serve as communication factors between cells (Bayraktar et al., Non-coding RNAs have been associated with various biological processes and human diseases. These phenomena were further expanded and reviewed by several studies in this Research Topic. A number of wet lab and computational methods as well as database resources reported in the Topic should help to refine the connections between ncRNAs and diseases and identify the mechanisms of actions of the former, thus further contributing to the advancement of the ncRNA field.YZ and PK conceived of the work and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Floral plantings are promoted to foster ecological intensification of agriculture through provisioning of ecosystem services. However, a comprehensive assessment of the effectiveness of different floral plantings, their characteristics and consequences for crop yield is lacking. Here we quantified the impacts of flower strips and hedgerows on pest control (18 studies) and pollination services (17 studies) in adjacent crops in North America, Europe and New Zealand. Flower strips, but not hedgerows, enhanced pest control services in adjacent fields by 16% on average. However, effects on crop pollination and yield were more variable. Our synthesis identifies several important drivers of variability in effectiveness of plantings: pollination services declined exponentially with distance from plantings, and perennial and older flower strips with higher flowering plant diversity enhanced pollination more effectively. These findings provide promising pathways to optimise floral plantings to more effectively contribute to ecosystem service delivery and ecological intensification of agriculture in the future. Our quantitative synthesis of the impacts of floral plantings on pest control, crop pollination and yield reveals that flower strips, but not hedgerows, enhanced pest control services in adjacent fields by 16% on average, while effects on crop pollination and yield were more variable. Our synthesis identifies several important drivers of this variability in effectiveness of plantings: pollination services declined exponentially with distance from plantings, and perennial and older flower strips with higher flowering plant diversity enhanced pollination more effectively, with important implications for the the design and implementation of these measures to effectively promote ecological intensification of agriculture in the future The \u2018exporter\u2019 hypothesis the extent to which flower strips and hedgerows enhance pollination and pest control services in adjacent crops; (2) how service provisioning changes with distance from floral plantings; (3) the role of plant diversity and time since establishment of floral plantings in promoting pollination and pest control services; (4) whether simplification of the surrounding landscape modifies the responses; and (5) whether floral plantings enhance crop yield in adjacent fields.Our synthesis reveals general positive effects of flower strips but not hedgerows on pest control services in adjacent crop fields. Effects on crop pollination, however, depended on flowering plant diversity and age since establishment, with more species\u2010rich and older plantings being more effective. However, no consistent impacts of flower strips on crop yield could be detected, highlighting the need for further optimisations of plantings as measures for ecological intensification.To identify data sets suitable to address our research questions, we performed a search in the ISI Web of Science and SCOPUS (records published until 31.12.2017 were considered). To minimise potential publication bias because (1) average z\u2010scores follow a normal distribution, and (2) the variability present in the raw data is not constrained as in other indices that are bound between 0 and 1 therefore focus on flower strip effects on pollination and pest control services. Information on plant species richness was available in 12 out of 18 pest control studies and 10 out of 17 pollination studies. Whenever available, the species richness of flowering plants was used. Otherwise, for some flower strip studies, the number of sown, potentially flowering plant species (excluding grasses) was used. Time since establishment of flower strips, that is the time span between seeding or planting and data sampling, was available for all studies ranging from 3 to 122\u00a0months.et al., et al., The proportional cover of arable crops was available and analysed as a proxy for landscape simplification . Only 10 studies measured services in several years since establishment linear mixed\u2010effect models with planting (field with or without planting) were separately fitted for flower strips and hedgerows for the response variables pollination service and pest control service. To test how the effects on service provisioning change with distance from plantings (question 2) and with landscape simplification (question 4) these explanatory variables and their interactions with the fixed effects described earlier were included in the models. Exploratory analyses showed that neither distance nor landscape simplification effects differed between flower strips and hedgerows; that is no significant interactive effects of planting type with any of the tested fixed effects. We therefore pooled flower strip and hedgerow data in the final models, excluding planting type and its two or three\u2010way interactions as fixed effects. In addition to linear relationships we tested for an exponential decline of measured response variables from the border of the field by fitting log10(distance) in the linear mixed\u2010effect models described earlier. In this case, field nested within study was included as a random effect. To test the intermediate landscape complexity hypothesis, we tested for linear as well as hump\u2010shaped relationships between landscape context, and its interaction with local floral plantings by fitting landscape variables as a quadratic fixed predictor in the models described earlier . To present the ranges covered by the agricultural landscape gradients, we did not standardise measures of landscape simplification within studies recommended for testing significant effects of , et al. . All staThe provisioning of pest control services in crop fields adjacent to flower strips was enhanced by 16% on average compared to fields without flower strips. On average, pest control services were also increased in crops adjacent to hedgerows, but effects were more variable and overall not statistically significant Fig.\u00a0; Table\u00a01Crop pollination effects were more variable across studies and overall not significantly different between crops with or without adjacent floral planting across all studies and within\u2010field distances Fig\u00a0; Table\u00a01Crop pollination services, but not pest control services, tended to increase with flowering plant species richness of the adjacent flower strip . These results indicate that floral plantings have great potential to benefit ecosystem service provision, but to do so will need to be carefully tailored for functioning at specific spatial scales. Flower diversity and strip age are important drivers through which this can be achieved and they should be considered integrally before floral plantings can make a significant contribution to the ecological intensification of agricultural production.sensu Morandin and Kremen, et al., et al., We found positive effects of flower strips on ecosystem service provisioning in support of the \u2018exporter\u2019 hypothesis . We fou, et al, .et al., et al., et al., et al., et al., et al., et al., et al., Consistent with previous studies (e.g. Dainese et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., Crop yield is affected by a complex interplay of a multitude of agricultural management practices such as fertilisation, level of pesticide use, pest pressures, soil cultivation and other factors such as local soil and climatic conditions (e.g. Bartomeus Our synthesis demonstrates enhanced natural pest control services to crops adjacent flower strips plantings, across a broad suite of regions, cropping systems and types of flower strips studied. However, it also reveals inconsistent and highly variable effects of flower strips and hedgerows on crop pollination services and yield. This highlights a strong need to identify the key factors driving this variability and the effectiveness of different types of floral plantings in contributing to ecosystem service delivery. Informed by such improved understanding, the design, implementation and management of floral plantings can increase their effectiveness as measures for ecological intensification. This synthesis identifies several promising pathways towards more effective floral plantings for the provision of ecosystem services and ecological intensification: the modelled exponential distance\u2010decay function of pollination service provisioning by floral plantings into crop field helps to predict service provision in crop fields; together with the lack of a strong planting area effect, our findings suggest that a dense spatial network of relatively small plantings will be more effective than a few large ones to optimise pollination service provisioning. Moreover, it identifies important drivers of the effectiveness of floral plantings for delivery of crop pollination services: flowering plant diversity and age. Based on these findings we strongly encourage the establishment, adequate management and restoration of existing perennial floral plantings that ensure the availability of high floral diversity across several years as promising pathways towards optimised measures for ecological intensification.MA and LS designed the study. MA, DK, MT, BRB, RB, AJC, MD, FD, MHE, DG, ADG, DG, HG, HH, FH, RI, KJ, PJ, MJ, EK, CK, DAL, GML, LM, MMK, LM, SCP, SGP, MR, HS, AS, CT, TT, EV, EV, IMGV, AW, DBW, FW, KW, NMW, MW, SW and LS contributed data. MA compiled the dataset. LS and MA analysed the data. MA, LS, DK, MG, SGP and MR interpreted results. MA wrote the paper and all authors contributed to revisionFig S1Click here for additional data file.Table S1Click here for additional data file.Table S2Click here for additional data file.Table S3Click here for additional data file.Table S4Click here for additional data file.Table S5Click here for additional data file."} +{"text": "Cachexia is a metabolic mutiny that directly reduces life expectancy in chronic conditions such as cancer. The underlying mechanisms associated with cachexia involve inflammation, metabolism, and anorexia. Therefore, the need to identify cachexia biomarkers is warranted to better understand catabolism change and assess various therapeutic interventions. Among inflammatory proteins, growth differentiation factor-15 (GDF15), an atypical transforming growth factor-beta (TGF-\u03b2) superfamily member, emerges as a stress-related hormone. In inflammatory conditions, cardiovascular diseases, and cancer, GDF15 is a biomarker for disease outcome. GDF15 is also implicated in energy homeostasis, body weight regulation, and plays a distinct role in cachexia. The recent discovery of its receptor, glial cell line-derived neurotrophic factor (GDNF) family receptor \u03b1-like (GFRAL), sheds light on its metabolic function. Herein, we critically review the mechanisms involving GDF15 in cancer cachexia and discuss therapeutic interventions to improve outcomes in people living with cancer. Almost eighty percent of advanced cancer patients are affected by involuntary wasting, which is inversely related to handgrip strength, high toxicities of anti-cancer drugs, quality of life, and survival.18Since cachexia preferentially causes loss of LBM more than FM, there is a need for a biomarker for the early diagnosis of cachexia, especially the atrophy in skeletal muscle.et al. demonstrated reversal of cancer cachexia by injecting anti-bodies against Act A and Mstn in mice model leading to prolong survival.et al., demonstrated that GDF15 inhibitors reversed the loss of LBM and FM in mice.In recent years, several markers have been reported to be involved in skeletal muscle atrophy in cancer cachexia.et al. found that cancer-related weight loss was linked to changes in the diet and inflammatory cytokines.Table .In 2005, Fouladiun et al.'s recommendation in 2000, the protein was coined growth differentiation factor 15 (GDF15).49Almost two decades ago GDF15 was identified as a member of the TGF-\u03b2 superfamily.et al. injected GDF15 subcutaneously into mice which caused a rapid decrease in food intake and hence, a decline in weight.et al. in 2012 further demonstrated that the overexpression of GDF15 in tumors led to the inhibition of food intake in an animal model which subsequently causes loss of both LBM and FM.58Several studies demonstrated the overexpression of GDF15 in many advanced cancers such as prostate, urothelial, breast, gastric, colorectal, and esophagus.in-vivo and in-vitro studies which were responsible for its anti-tumorigenic effect.et al. demonstrated a strong association of elevated plasma GDF15 levels with metastasis in prostate cancer.46GDF15 may have a context-dependent role in cancers: an antitumorigenic role of GDF15 was observed in early cancer while an association of GDF15 with the induction of tumor growth was observed in advanced cancer depending on a tumor type and its micro-environment.In one animal study, GDF15 was identified as the only biomarker for the loss of skeletal muscle and weight in cancer.65et al. showed an absence of a postprandial increase in GDF15 serum levels in healthy participants suggesting that it is unlikely that this hormone acts as a 'satiety factor'.et al., in 2017 showed that GDF15 serum levels induced a minimal change in response to caloric surpluses or deficits in both mice and humans, differentiating its action from intestinally derived satiety hormones and leptin. Furthermore, the same group showed that GDF15 expression was regulated by the tissue stress response in mice, and its administration triggered conditioned taste aversion suggesting that GDF15 induces an aversive response to nutritional stress.et al. in 2019, also demonstrated that GDF15 administration triggered conditioned taste aversion via GFRAL, while Borner et al. showed that GDF15 Induces anorexia through nausea and emesis in the mouse model. Globally, these findings indicate that GDF15 does not act as an 'anti-hunger' hormone or has any 'satiety effect' but induces an aversive response to nutritional stress. In 2017, the GDF15 receptor was identified as the glial cell-derived neurotrophic factor (GDNF) family receptor alpha-like (GFRAL) expressed in the brainstem.et al. identified novel characteristics of GDF15 in disease tolerance in the context of infection. This group showed in a mouse model that GDF15 is induced in inflammatory diseases through the central induction of metabolic adaptation and contribute to a protective effect in organ damage. This inflammation-induced tolerance effect is achieved by metabolic reprogramming and the production of triglycerides via hepatic sympathetic outflow, hence preventing cardiac damage after an LPS endotoxemic challenge.73It was established in the 1980s that the \u03b2 adrenergic stimulation causes increase lipolysis and inhibit the activity of the lipoprotein lipase resulting in increased plasma triglycerides.In August 2020, Suriben et al. were the first to link the GDF15/GRFAL sympathetic pathway with the hepatic lipid oxidation in a cancer cachexia mouse model.Despite the breakthrough in inhibiting the GDF15/GFRAL pathway for the treatment of cachexia in the mouse model, several issues concerning interplay between host and tumor must be addressed for cachectic cancer patients.65For most patients with metastatic cancers, the cause of death is due to cachexia more than the direct effect of tumor bulk and organ failure. Based on the absence of any therapy for improving cachexia and on similar mechanistic evidence in the animal model and human, clinical trials testing anti-GDF15/GFRAL treatments appear promising, specifically in patients with elevated circulating GDF15 levels.Beyond its cachectic effect, GDF15 has been also implicated in tumor cell apoptosis and the development of metastasis. In addition, GDF15 can also modulate the tumor microenvironment, innate immune surveillance, and response to immunetherapy.47Globally, considering both host and cancer factors, only the completion of clinical trials using an inhibitor of the GDF15/GFRAL pathway will provide clinical evidence on the merit of reversing this \u201cmetabolic signature\u201d to improve the lives of those living with advanced cancer.81GDF15 plasma level correlates with tumor progression and has been considered as a tumor biomarker."} +{"text": "TheFinally, safety is not guaranteed for nutrients, given the known issues with intakes above tolerable upper intake levels (UL). Adverse events were inadequately assessed in the context of purported benefits. Calder et al.\u2019s recommended intakes do not adequately define amounts or intended population given differences in UL by age/sex . The systematic review of omega-3 fatty acids and antioxidants that they cited could not rule out adverse events .Though recommendations by Calder et al. are made in the context of the COVID-19 pandemic, we note that this review offers no direct evidence to support the claim for dietary supplements for prevention or treatment of COVID-19, per se ["} +{"text": "Despite efficient virological suppression on antiretroviral therapy (ART), people living with HIV (PLWH), experience an increased burden of premature co-morbidities, such as cancer and end-organ disease. With remaining challenges in terms of access to therapy, adherence and potential long-term drug toxicity, improving their long-term healthcare outcome, including new strategies for HIV clearance, remains a global priority. There is, therefore, an ongoing need to better characterize and harness the immune response in order to develop new strategies and supplement current therapeutic approaches for a \u201cfunctional\u201d cure. Current efforts toward HIV eradication to enhance immune recognition and elimination of persistently infected cells have highlighted the need for an optimized \u201ckill\u201d approach. Natural killer (NK) cells play an important role in antiviral defense and by virtue of their innate and adaptive features hold great promise as a focus of \u201ckill\u201d efforts. Galvanized by advances in the cancer field, NK cell exploitation, represents a transformative approach to augment HIV therapeutic modalities, circumventing many of the limitations inherent to T cell approaches. In this review we will discuss recent advances in our understanding of the development of NK cell adaptive/memory responses in HIV infection and highlight new and exciting opportunities to exploit the beneficial attributes of NK cells for HIV immunotherapy. NK cells are multipotent innate effector cells that play pivotal roles in antiviral and tumor immunity that target the interaction between MHC class I and NK cell inhibitory receptors represents one strategy that is currently used to enhance NK cell anti-tumor activity .Following the rise of single cell cloning techniques, next-generation anti-HIV-1 broadly neutralizing antibodies (bNAbs) with greater potency/breadth and the ability to suppress viral replication and potential for Fc-mediated clearance of virus-infected cells have now entered the clinical arena could differentiate into effector NK cells mediating an innate antiviral response and protection against HIV -engineered NK cells offer great promise as a new immunotherapeutic tool in the HIV field. The recent success of CAR NK cells derived from cord blood transduced with a retroviral vector, expressing the genes encoding anti-CD19, IL-15, and a safety switch (inducible caspase 9), in patients with refractory or relapsed CD19 positive cancers, represents a remarkable achievement in the field (Wang et al., In addition to the influence of CMV co-infection, a recent study demonstrated that the pro-inflammatory milieu in HIV infected patients drives the expansion of a defined NK subset with memory-like properties, characterized by CD94loCD69+T-betloEomeshi signature (Stegmann et al., Antigen-specific NK cells have been reported in rhesus macaques infected with SIV/SHIV (Reeves et al., in vivo persistence they are endowed with enhanced capacity for ADCC (Schlums et al., Adaptive NK cells make a significant proportion of the peripheral NK cell pool in HIV infection (Zhou et al., A concern with utilizing bulk NK cells for immunotherapy is their potential for unwanted immunoregulatory functions, such as regulation of T cell responses (Peppa et al., in vitro expansion capability with predictable selectivity as an alternative or combination strategy for a functional cure. Utilizing HLA-E expressing transfectants has been a successful strategy for obtaining robust proliferation of functional adaptive NK cells, with profound skewing toward a single self KIR, and enhanced NKG2C effector potential against allogeneic acute lymphoblastic leukemia primary blasts (Liu et al., Importantly adaptive NK cells have Indeed, adoptive transfer of cytokine-induced adaptive NK cells are being tested in phase I clinical trials in AML patients (Romee et al., ex vivo expansion for clinical application represents an exciting new avenue for the development of novel therapeutic interventions in the field of HIV infection. These approaches can be combined with therapeutic antibodies improving their efficacy. In addition, the generation of memory NK cell represents a novel goal of new vaccination approaches incorporating targeted adjuvants or through enhancing presentation via HLA-E. Future innovative strategies for cure include manipulation of the metabolic machinery of immune cells and attempts to intrinsically rewire NK cells to improve their immunotherapeutic potential.The study of adaptive NK cell subpopulations and Despite the considerable amount of progress, additional work is required to fully unravel the unique properties of specialized and memory NK cells subsets, especially within key effector sites, along with their potential for functional exhaustion. This knowledge would be critical in order to leverage their distinct features and maximize their therapeutic use in chronic viral infections while offsetting any detrimental effects to adaptive immunity and the host.AA, AO, and EM contributed to writing specific sections. DP edited the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "This special issue is dedicated to entropy-based fatigue, fracture, failure prediction and structural health monitoring. The unification of laws of thermodynamics and Newtonian mechanics has been a pursuit of many scientists since the mid-19th century. Distinguished scientists from around the world who contributed to this special issue all show that unification of Newtonian mechanics with thermodynamics using entropy as a link eliminates the need for phenomenological continuum mechanics, where the second law of thermodynamics is usually imposed only as an external constraint, but is not satisfied at the material level, because derivative of displacement with respect to entropy is assumed to be zero. For example, the theory of elasticity assumes that there is no entropy generation at the material level. As a result, everything is reversible, which violates the second law of thermodynamics.Group from Indian Institute of Technology Madras and University at Buffalo used unified mechanics theory for low cycle fatigue life prediction of Ti-6Al-4V alloys. Bin Jamal et al. show thaScientists from Belarus State University contributed a noteworthy paper with their recent advances on mechanothermodynamics, which is essentially a theory almost identical to the unified mechanics theory. They both use entropy generation rate for degradation and unification of Newtonian mechanics and thermodynamics laws. Sosnovskiy and Sherbakov formulatA Purdue University group contributed their outstanding work on using maximum entropy models for fatigue damage in metals with application to low-cycle fatigue of aluminum 2024-T351. Young and Subbarayan [University of Maryland, College Park scientists contributed an excellent study on measures of entropy to characterize fatigue damage in metallic materials. Yun and Modarres show thaUniversity of Texas at Austin researchers contributed an excellent paper on degradation-entropy generation methodology for system and process characterization and failure analysis. Osara and Bryant formulatScientists from Northwestern Polytechnical University and Xi\u2019an University of Architecture and Technology contributed an exceptional study titled an entropy-based failure prediction model for the creep and fatigue of metallic materials. Wang and Yao state th\u22127 m/cycle. In conclusion, they prove that entropy generation can accurately predict the fatigue crack growth rate of dual-phase steels under spectrum loading.Universiti Kebangsaan Malaysia group, contributed a great study on prediction of fatigue crack growth rate based on entropy generation. Idris et al. present Researchers from Beihang University and Beijing Aeronautical Science & Technology Research Institute contributed a very interesting study on using copula entropy for quantifying dependence among multiple degradation processes. Sun et al. studied Scientists from Beihang University and North China University of Water Resources and Electric Power contributed an indirectly related paper on intelligent analysis algorithm for satellite health under time-varying and extremely high thermal loads. Li et al. present 2FeCoMo0.5V0.2 medium entropy alloy by rotationally accelerated shot peening. Liang et al. [2FeCoMo0.5V0.2 medium-entropy alloy by rotationally accelerated shot peening (RASP). Transmission electron microscopy analysis revealed that deformation twinning and dislocation activities are responsible for the effective grain refinement of the high-entropy alloy. In order to reveal the effectiveness of surface nano-crystallization on the Ni2FeCoMo0.5V0.2 medium-entropy alloy, a common model material, Ni, is used as a reference.Nanjing University of Science and Technology and City University of Hong Kong teams participated with their paper titled effective surface nano-crystallization of Nig et al. reported"} +{"text": "Major depressive disorder and neuroticism (Neu) share a large genetic basis. We sought to determine whether this shared basis could be decomposed to identify genetic factors that are specific to depression.We analysed summary statistics from genome-wide association studies (GWAS) of depression and compared them with GWAS of Neu (from UK Biobank). First, we used a pairwise GWAS analysis to classify variants as associated with only depression, with only Neu or with both. Second, we estimated partial genetic correlations to test whether the depression's genetic link with other phenotypes was explained by shared overlap with Neu.We found evidence that most genomic regions (25/37) associated with depression are likely to be shared with Neu. The overlapping common genetic variance of depression and Neu was genetically correlated primarily with psychiatric disorders. We found that the genetic contributions to depression, that were not shared with Neu, were positively correlated with metabolic phenotypes and cardiovascular disease, and negatively correlated with the personality trait conscientiousness. After removing shared genetic overlap with Neu, depression still had a specific association with schizophrenia, bipolar disorder, coronary artery disease and age of first birth. Independent of depression, Neu had specific genetic correlates in ulcerative colitis, pubertal growth, anorexia and education.Our findings demonstrate that, while genetic risk factors for depression are largely shared with Neu, there are also non-Neu-related features of depression that may be useful for further patient or phenotypic stratification. Major depressive disorder (MDD) is a leading cause of morbidity worldwide, currently affecting approximately 4% of the world's population , is a robustly and consistently replicated dimension of personality that is relatively stable over time Eysenck, . Neu feaet al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., Twin, family and genomic studies have shown that population variation in Neu and liability to depression are conferred by both genetic and environmental risk factors . We compared the depression summary statistics with summary statistics for Neu .We used depression summary statistics from the Psychiatric Genomics Consortium (PGC) ; for Neu we used summary statistics from the UK Biobank sample . We used the munge_sumstats.py tool . Using these filtered summary statistics, the gwas-pw program and within 3\u00a0Mb, then used MAGMA genes and conducted GWAS catalogue lookups using FUMA . We defined segments that were associated with neither trait as those that had a total posterior probability of association <0.2 and that did not contain any genome-wide hits for either trait. To examine the depression-only signal, we excluded segments associated with Neu or with both traits from the depression summary statistics, clumped single-nucleotide polymorphisms (SNPs) that were in linkage disequilibrium (LD) with either MDD or Neu. These included psychiatric disorders, personality traits, cardiovascular and inflammatory diseases, anthropometric and life-history traits, education and lifestyle factors. We supplemented this list of traits with BMI since obesity has been identified as a potential stratifying factor for depression but not with Neu after adjusting for depression (Neu\u00b7adjMD). Finally, we identified traits with the opposite patterns of having genetic correlations that were specific to Neu or to Neu after adjusting for depression. We tested whether unadjusted and adjusted correlations were different from zero and whether adjusted correlations were smaller than their unadjusted counterparts and corrected for multiple testing using False Discovery Rate explained by the genetic architecture shared between depression and Neu (\u2018neurotic depression\u2019) or (2) specific to depression and independent of Neu (\u2018non-neurotic depression\u2019) (and vice versa for \u2018depressive neuroticism\u2019 and \u2018non-depressive neuroticism\u2019). We estimated the genetic covariance among MD, Neu and each trait using GenomicSEM (We used pairwise GWAS (Pickrell ability) , Table 1et al., p\u00a0<\u00a02.77\u00a0\u00d7\u00a010\u22126) with the partitioned genomic segments. There were 30 genes significantly associated with depression only (online Supplementary Table S3), 203 genes associated with Neu only (online Supplementary Table S4) and 104 genes associated with both depression and Neu (online Supplementary Table S5). We used FUMA and to coronary heart disease (p\u00a0=\u00a03.2\u00a0\u00d7\u00a010\u22123). Neu-only gene sets were related to traits such as intracranial volume, Parkinson's disease and high-density lipoprotein cholesterol. The gene sets shared by depression and Neu were related to cross-disorder psychiatric traits, coffee consumption and epilepsy, among other traits (online Supplementary S6).We used MAGMA and with Neu where overlap with depression has been removed (Neu\u00b7adjMD) , schizophrenia, bipolar disorder, coronary artery disease and age of first birth. There were three additional traits that showed a similar pattern of full and partial correlations with MD, but where the effect sizes overlapped with those for Neu, and thus their status of having a specific relationship was less well supported. Finally, there were three traits that showed specific relationships with Neu after removing shared overlap with MD.RFWD2 (ring finger and WD repeat domain 2), a gene that can promote tumour growth which is involved in the Krebs cycle; rs4143229 is in an intron of the ecto-NOX disulphide-thiol exchanger 1 gene (ENOX1) which is expressed in the nervous systems and has been implicated in autoimmune disorders and immune systems gene, which has been implicated in childhood asthma gene. A second association signal (rs12129573) was in the intron of an uncharacterised non-protein coding RNA (LOC105378800). In a further region, a signal was found (97.8\u2013100.6\u00a0Mb on chromosome 6) that showed a clear separation between the association signals for each trait . The SNP associated with depression (rs12202410) was near the F-box and leucine-rich repeat protein 4 (FBXL4) gene which is related to energy homoeostasis contained two association signals for depression. The first signal (rs1460942) was shared with Neu and was close to the neuronal growth regulator 1 (et al., Using a GWAS catalogue lookup with FUMA (Watanabe et al., et al., et al., et al., Differential association of MD with other traits, once shared overlap with Neu was accounted for, was also shown in our analysis of LD score genetic correlations. We found that, unlike Neu, MD was genetically correlated with BMI, triglyceride levels and coronary artery disease, suggesting the atypical depressive subtype related to cardio-metabolic traits (Lasserre et al., et al., et al., et al., et al., et al., et al., et al., One limitation of our study is that by using summary statistics from GWAS, we were only able to assess the overlap in genetic architecture between depression and Neu that arises from common variants. Even biobank-sized samples of millions of participants can be underpowered for detecting associations with rare variants unless such variants have very large effect sizes (Visscher et al., et al., et al., et al., et al., et al., just noisy measures of the same underlying liability. If these differences represent distinct genetic subtypes of depression, then most cases of depression will stem from this Neu\u2013depression nexus, while a smaller proportion may have aetiologies that are distinct from Neu. Some of these associations, such as between depression, chronotype and metabolic phenotypes, are suggestive of endogenous depression's features but do not point to all the characteristics of this previously described subtype. Confirming known depression subtypes and identifying new subtypes will be useful for phenotypic and clinical stratification. The unique associations of triglyceride levels and BMI with depression, but not at all with Neu, confirms that depression with comorbid obesity and other metabolic factors should be studied as a subtype when exploring aetiology and testing treatment efficacy. The partial genetic correlations that ADHD, schizophrenia and bipolar disorder have with depression after adjusting for Neu imply that polygenic risk scores for these other disorders may be useful to screen or stratify participants even if they do not manifest these other disorders.Neu is a major risk factor for depression (Bagby et al., et al., et al., et al., et al., Neither depression (Kendler"} +{"text": "Due to their enormous surface area compared to other cell types, neurons face unique challenges in properly handling supply and retrieval of the plasma membrane (PM)\u2014a process termed PM turnover\u2014in their distal areas. Because of the length and extensiveness of dendritic branches in neurons, the transport of materials needed for PM turnover from soma to distal dendrites will be inefficient and quite burdensome for somatic organelles. To meet local demands, PM turnover in dendrites most likely requires local cellular machinery, such as dendritic endocytic and secretory systems, dysregulation of which may result in dendritic pathology observed in various neurodegenerative diseases (NDs). Supporting this notion, a growing body of literature provides evidence to suggest the pathogenic contribution of dysregulated PM turnover to dendritic pathology in certain NDs. In this article, we present our perspective view that impaired dendritic endocytic and secretory systems may contribute to dendritic pathology by encumbering PM turnover in NDs. Dendrites are neuronal compartments essential for receiving electrochemical signals from presynaptic neurons through formed synapses. Accurate neuronal wiring relies critically on the proper establishment of the dendritic field that is achieved by both structural build-ups of dendritic arbors and functional maturation of synapses . This process of PM turnover is mediated primarily by endocytic and secretory pathways. However, due to its highly elaborate dendrites, a typical neuron has a 10,000 times larger surface area than does a typical epithelial cell , such as Alzheimer\u2019s disease (AD), Parkinson\u2019s disease (PD), polyglutamine (polyQ) diseases, and amyotrophic lateral sclerosis expand or reduce its size; (2) alter its shape; and (3) insert or remove from its PM the membranous lipids and proteins needed to convey both intra- and extra-cellular signals.N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes required for the membrane fusion do not form neurons, defects in exocytosis-mediated dendritic growth were mitigated by blocking clathrin-mediated endocytosis (CME) using a temperature-sensitive dominant-negative allele of shibire , has a specialized membrane-stacked morphology similar to that of Golgi Gray, ,b. BasedDrosophila , which was first defined in cultured hippocampal neurons GTPase proteins are among the most compelling candidate molecular machinery that may play crucial roles in (dendritic) PM turnover. Rabs, which were first found in rat brains , Rab GTPase-activating proteins (Rab GAPs), and Rab-GDP dissociation inhibitors (Rab GDIs) control the guanine nucleotide status of Rab complexes initiate vesicle budding from PM at clathrin-coated pits pathway, through which membrane vesicles are transported directly from EEs to PM; and the slow recycling (10\u201320 min) pathway, through which membrane vesicles are transported to PM Rab35 , where membrane fission actively occurs neurons that mutation of a dynein subunit gene, dlic, led to proximally \u201cbushy\u201d dendrites and that dlic and Rab5 double mutation resulted in greatly simplified dendritic morphology. Interestingly, this double mutant phenotype was similar to those seen in neurons with Rab5 mutation only. These data indicate that Rab5, in a co-operation with dlic, plays a regulatory role in dendrite morphogenesis. Another study on the genetic interaction between Protein Kinase A (PKA) and Rab5 in C4 da neurons showed that PKA could also contribute to the dendritic arbor development by altering Rab5-endosomal transport in dendrites , in primary cortical neurons increased dendritic branches and decreased endolysosomal trafficking in dendrites seems to be involved. In this section, we will briefly outline the generalized characteristics of the early secretory pathway by describing some of its key regulators and make extensions to the dendritic secretory pathway and NDs where appropriate.Drosophila and yeast are mediated by coat protein complex II (COPII) vesicles (Brandizzi and Barlowe, cis-Golgi (Suda et al., cis-Golgi (Sztul and Lupashin, The secretory pathway comprises the transport of secretory and membranous materials from ER to Golgi and ultimately to PM. ER-to-ERGIC and ERGIC-to-Golgi in mammals and ER-to-Golgi transport in other less-developed species such as via Dsl1 tethering complex in yeast (Andag and Schmitt, The transport process between the ER and Golgi is not unidirectional. The best characterized retrograde transport process from Golgi to ER is the COPI pathway Spang, . COPI coAlthough the origins of dendritic secretory units are mostly unknown, we suspect that they are not entirely discrete from the canonical secretory units in the soma. Indeed, a study reported that GOPs may originate from somatic Golgi in rat hippocampal neurons (Quassollo et al., Drosophila has also provided a link between the dendritic secretory pathway and dendritic pathology. Chung et al. (CrebA mRNA level. Because CrebA is the master regulator of the secretory pathway (Abrams and Andrew, Sec31 (Chung et al., Sar1 in Drosophila da neurons (Ye et al., A recent study on polyQ toxicity in g et al. showed tLrrk2, Sec16A detached from the dendritic ERES, which led to the impairment of ER-to-Golgi transport and NMDA receptor trafficking in mouse primary hippocampal neurons (Cho et al., Drosophila ortholog of LRRK2, co-localized with somatic Golgi and GOPs in Drosophila da neurons, and that overexpression of a PD-linked mutant form of LRRK2, LRRK2 G2019S, suppressed anterograde movements of GOPs marked by ManII-eGFP. This GOP transport defect may underlie the dendrite degeneration observed in LRRK2 G2019S-expressing Drosophila da neurons (Lin et al., Glutamatergic excitotoxicity involving the NMDA receptor is often observed in animal models of NDs (Lewerenz and Maher, Neuronal dendrites seem to be highly vulnerable to neurotoxic insults, including those that arise in NDs (Luebke et al., Sec31 and nuclear polyQ expression lead to the loss of GOPs, but not somatic Golgi (Chung et al., Sec31, Rab1, and Sar1, all lead to impaired arborization of dendrites, but normal morphology of axons in Drosophila da neurons (Ye et al., Drosophila model for progressive myoclonus epilepsy (Praschberger et al., Drosophila motoneurons (Ryglewski et al., Although the dendritic and the canonical pathways occur in distinct areas of the neuron, they share many of the regulatory molecules. Also, pieces of evidence show that at least parts of the dendritic secretory system, such as GOPs, may be derived from the canonical somatic secretory system (Quassollo et al., In this review article, we presented our perspective view that impaired PM turnover involving dysregulation of the dendritic endocytic and secretory pathways may contribute to dendritic pathology in NDs. Although there is a growing body of evidence for the potential link between impaired PM turnover and dendritic pathology in NDs, our understanding of the exact pathogenic mechanisms remains largely elusive. We propose that dendritic pathology in NDs may involve dysregulation of the regulatory machinery, such as Rab GTPases and COPI/COPII, for the dendritic endocytic and secretory pathways described above. Dysregulation of the dendritic pathways appears to complement cytoskeleton impairment as underlying pathogenic mechanisms for dendritic pathology. Because dendritic defects are often early features of ND, future studies to elucidate the pathogenic mechanisms by which impaired PM turnover contributes to dendritic pathology in NDs will deepen our understanding of the early pathogenesis of NDs.JP, CC, SP and SL conceptualized the theme of the review and wrote the manuscript together. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Alzheimer\u2019s disease (AD) is a neurodegenerative disorder characterized by amyloid-\u03b2 (A\u03b2) plaques and the formation of neurofibrillary tangles (NFTs) composed of hyperphosphorylated tau. In response to A\u03b2 and tau aggregates, microglia, the primary innate immune cells of the central nervous system (CNS), facilitate A\u03b2 and tau clearance and contribute to neuroinflammation that damages neurons. Microglia also perform a wide range of other functions, e.g., synaptic pruning, within the CNS that require a large amount of energy. Glucose appears to be the primary energy source, but microglia can utilize several other substrates for energy production including other sugars and ketone bodies. Recent studies have demonstrated that changes in the metabolic profiles of immune cells, including macrophages, are important in controlling their activation and effector functions. Additional studies have focused on the role of metabolism in neuron and astrocyte function while until recently microglia metabolism has been considerably less well understood. Considering many neurological disorders, such as neurodegeneration associated with AD, are associated with chronic inflammation and alterations in brain energy metabolism, it is hypothesized that microglial metabolism plays a significant role in the inflammatory responses of microglia during neurodegeneration. Here, we review the role of microglial immunometabolism in AD. Alzheimer\u2019s disease (AD) is an age-related neurodegenerative disorder associated with memory loss and impaired cognitive abilities. AD is a major cause of disability and dependency in the United States and worldwide, causing a significant impact on not only the individual patient, but also their family, community, and the healthcare system , account for 10\u201315% of the adult glial cell population in the brain inflammasome is activated by glycolytic enzymes activity compared to wild-type cells; decreases in mTOR signaling were associated with increased autophagy. Additionally, the metabolic deficiency, and lack of microglial responsiveness, was restored in Trem2\u2212/\u2212 5XFAD mice by increasing microglial energy capacity with cyclocreatine and glucose metabolism in neurodegeneration has been identified , and acetoacetate Laffel, . Levels To date AD drug discovery research has focused on tauopathy or A\u03b2 reduction. As discussed above, glycolysis is a major factor in maintaining activated microglia, while non-activated microglia rely more on oxidative metabolism (Bernhart et al., Microglia shift to an anti-inflammatory phenotype in response to BHB (Huang et al., APOE4 allele do not see the improvement in cognitive function as subjects administered CT who are not carriers of the APOE4 allele (Reger et al., Medium-chain triglyceride diets have been developed to provide a more palatable alternative to the ketogenic diet (Huttenlocher et al., TREM2 deficiency results in decreased neuroinflammation and protects against neurodegeneration (Leyns et al., TREM2 increases tau pathology (Bemiller et al., Another approach to AD treatment is to target microglial genes important in microglial metabolism. As previously discussed, TREM2 is vital to microglial metabolic fitness (Ulland et al., The emerging field of immunometabolism has provided significant progress in our understanding of how cellular and systemic metabolism affects immune responses. More importantly, these data suggest that targeting immune cell metabolism may be a valuable strategy for the development of advanced therapeutics to treat human disease (Bettencourt and Powell, DS drafted the manuscript. TU reviewed and edited the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Minimal hepatic encephalopathy (MHE), which shows mild cognitive impairment, is a subtle complication of cirrhosis that has been shown to affect daily functioning and quality of life. However, until 2014, relevant guidelines do not give much attention to the diagnosis and treatment of MHE, resulting in patients being ignored and denied the benefits of treatment. In this review, we summarize recent cognition-based research about (1) alteration of nerve cells, including astrocytes, microglial cells and neurons, in mild cognitive impairment in MHE; (2) comparison of methods in detecting cognitive impairment in MHE; and (3) comparison of methods for therapy of cognitive impairment in MHE. We hope to provide information about diagnosis and treatment of cognitive impairment in patients with MHE. Minimal hepatic encephalopathy (MHE), which affects 30\u201355% of cirrhosis patients, is a subtle complication of cirrhosis that may have a detrimental effect on daily functioning. In the MHE phase, patients show mild cognitive impairment, which lead to deficits of attention, and psychomotor slowing diagnostic criteria have not been standardized; (2) there is no overt clinical manifestation in patients with MHE; and (3) hyperammonemia is present with inflammation and certain levels of ammonemia the relationship between cognitive impairment and nerve cell injury; (2) comparison of diagnostic methods for MHE; and (3) comparison of therapeutic methods for MHE.During MHE, the main contributor to cognitive impairment is hyperammonemia. In patients with liver disease, ammonia accumulates in blood due to deficient activity of hepatic urea cycle enzymes. In the brain, nerve cells take up ammonia and cause cognitive impairment. For example, astrocytes take up ammonia and glutamine synthetase detoxifies ammonia into glutamine. The rapid accumulation of glutamine creates an osmotic gradient that results in astrocyte swelling secretion, play important roles in nerve cell impairment. For example, hyperammonemia significantly reduces long-term potentiation and alters mRNA for DA receptors, which cause deficits in disturbed synaptic plasticity and novelty acquisition in hippocampal and corticostriatal pathways involved in goal-directed and learning behavior -\u03b1. Astrocytic TNF-\u03b1, which triggers neurodegenerative progression consequently or indirectly, resulting in cognitive impairment. Ding et al. found that low-dose DA (10\u00a0\u03bcM) produced TNF-\u03b1 in primary astrocytes, and co-culture of these astrocytes and neurons exhibited neuronal apoptosis compared with control group , which often occurs in cirrhosis patients, contributes to the MHE pathogenesis. Ding et al. found that the final production of neurotrophic factors, including PI3K/AKT signaling pathway to the phosphorylation of N-Methyl-d-Aspartate receptors and downstream activation of the CaMKIV/CREB pathway, was impaired in MHE rats -1\u03b2, Toll-like-receptor (TLR)-4 agonist lipopolysaccharide, and TNF-\u03b1. These pro-inflammatory microglia are characterized by immune-potentiating abilities, antigen-presenting and microbicidal -E2 and IL-6. And hyperammonemia also caused reduced Anti-inflammatory IL-10 and microglia activation in hippocampus. Increased TNF-\u03b1 and IL-1\u03b2 levels and phosphorylation (activity) of p38 cause GABAergic and glutamatergic neurotransmission. For example, membrane expression of GluR2 of AMPA receptor and subunits \u03b11 of etc. GABAA receptor is increased, while expression of subunits GluR1 of AMPA receptors and NR1 and NR2a of NMDA receptors is reduced. These altered membrane expression receptors are associated with hyperammonemia-induced microglial activation. And are responsible for impairment of spatial learning and altered neurotransmission in the radial maze. In turn, these inflammatory injuries contribute to microglial differentiation from anti-inflammatory M2 to pro-inflammatory M1 phenotype is involved in the perturbation of neural synaptogenesis. Cholinesterase (CHO) overload in combination with DA burden elicits memory loss and cognitive decline via the peroxisome proliferator-activated receptor (PPAR) \u03b3/extracellular signal-regulated kinase (ERK)/CREB pathway in MHE. In turn, DA triggers CHO biosynthesis via activation of the C-Jun N-terminal kinase 3/sterol regulatory element-binding protein 2 signaling pathway in primary cultured astrocytes. Zhuge et al. found that CHO secreted from astrocytes stimulated secretion of DA from primary cultured neurons. PPAR\u03b3/pERK/pCREB expression was decreased by DA-induced synergistic leads to synergistic synaptic impairment in represents the ability to suppress irrelevant motor or cognitive processes, and is useful for diagnosis of MHE (Di Lemma and Field The novel electronic number connection test (eNCT) has test\u2013retest reliability to detect cognitive function and monitor cognitive impairment in patients with cirrhosis. Wuensch et al. found that the eNCT performance was negatively correlated with PHES performance in patients with cirrhosis. Control participants showed significantly faster eNCT completion times compared with cirrhosis patients in the transition from an unimpaired mental state to HE applies electromagnetic waves emitted by a graded magnetic field to acquire the internal structure of the objects (Lockwood et al. Different kinds of MRI play an important role in the detection of MHE. Kooka et al. demonstrated magnetic resonance spectroscopy, which shows that reduced magnetization transfer ratio in the whole brain field and an increase in glutamate/glutamine or taurine in chronic HE patients contribute to early and objective diagnosis of MHE. The levels of brain glutamine were significantly lower and the levels of brain myo-inositol were significantly higher in the control group compared with the MHE group (Kale et al. In addition to hyperammonemia, inflammation also modulates neuropsychological function in patients with MHE. For example, Circulating IL-6 is negatively associated with memory function in low-dose endotoxemia (Krabbe et al. Paper-and-pencil test used in PHES is the gold standard for diagnosis of MHE. But the process of diagnosis of PHES is inconvenient and affected by many factors. The diagnostic methods take time, and are affected by demographic factors, and lack ecological validity and language functions, such as verbal memory. PHES focuses on only two cognitive domains but it is not sensitive enough to detect early neurological alterations. Patients classified as without MHE by PHES have a high risk of suffering overt HE. Around 40% of patients without MHE according to PHES fail two other psychometric tests Bajaj . Other kCFF is a noninvasive, rapid, simple test for diagnosis of MHE. Compared with PHES, CFF has a positive predictive value of 93.2\u2009\u00b1\u20097.44%, specificity of 92.7\u2009\u00b1\u20097.96%, negative predictive value of 90.4\u2009\u00b1\u20098.91% and sensitivity of 91.1\u2009\u00b1\u20098.32%. CFF is excellent for diagnosis of MHE, with an area under the curve of 0.937 (Metwally et al. Neuroimaging studies can detect diffuse abnormal metabolic activity of nerve cells, which is a typical feature of patients with MHE. MRI can provide objective and reliable imaging biomarkers that are necessary to help diagnose or identify MHE (Zhang et al. In addition to hyperammonemia, there is a parallel relationship between inflammatory cytokines and MHE, or a significant correlation between proinflammatory cytokines with MD values on diffusion tensor imaging (Srivastava et al. Recent guidelines suggest that either alternative techniques, such as computerized tests, neurophysiological testing or EEG should be used alongside PHES for multicenter studies (Morgan et al. As mentioned above, nerve cell injury plays an important role in MHE progression, so protection of nerve cells is one way to prevent and treat MHE. Good nutritional status is an important way to relieve nerve cell damage. Myosteatosis and sarcopenia, probably by reducing the handling of ammonia in the muscle, are independently associated with MHE. Venous ammonia is significantly higher in patients with sarcopenia and myosteatosis and inversely correlated with both parameters (Nardelli et al. Reducing neuroinflammation is also an important way to relieve nerve cell damage. Malaguarnera et al. demonstrated that bicuculline decreases anxiety and improves working memory and spatial learning in hyperammonemic rats. Bicuculline can reduce activation of GABAA receptors, which contributes to neuroinflammation. Meanwhile, bicuculline reduces astrocyte activation and not microglial activation. Bicuculline reverses the changes in membrane expression of AMPA and NMDA receptor subunits (Malaguarnera et al. Disorder of intestinal flora and bacterial translocation, which increase production and absorption of intestinal toxins, are closely related to HE. There are different degrees of intestinal flora disorder in patients with chronic liver disease. Beneficial bacteria such as bifidobacteria are decreased while urease-producing bacteria are the source of gut-derived toxins. Production and absorption of intestinal toxins significantly increase, but the liver cannot metabolize these toxins completely, which leads to toxin retention (McPhail et al. Enterococcus and Enterobacteriaceae were significantly decreased while the predominant bacteria were significantly enriched (Xia et al. MHE is also associated with individual microbiota signatures. For example, the relative abundance of Lactobacillaceae is higher in MHE, whereas abundance of autochthonous Lachnospiraceae is higher in those without MHE (Bajaj et al. Probiotics are well-tolerated, natural and safe and appropriate for long-term treatment of MHE (Jiang et al. Rifaximin is a non-absorbed, gut-selective antibiotic with a low resistance profile that is commonly used to treat HE. It achieves high concentrations in the human intestine, where it is active against many enteropathogens (Goel et al. Rifaximin has always been a second-line drug in the treatment of HE, but there are no unified conclusions for treatment of MHE. Bajaj et al. demonstrated that over the 8-week period, MHE patients treated with rifaximin showed significant improvements in avoiding total driving errors, speeding, and illegal turns. Rifaximin also made improvements in the psychosocial dimension of the sickness impact profile and the anti-inflammatory cytokine IL-10 levels (Bajaj et al. LOLA may be administered orally or parenterally. The benefits of LOLA for the treatment of HE have been known for 50\u00a0years (Buyeverov et al. Lactulose is a disaccharide composed of galactose and fructose. As early as 1957, the prebiotic properties of lactulose were reported in both adults and infants. Due to promising outcomes, low price and high availability, Lactulose does not undergo cleavage by human gastrointestinal enzymes (Ruszkowski and Witkowski Psychometric tests improved in 75% of MHE patients after treatment with lactulose (El-Karaksy et al. Probiotics, rifaximin, LOLA and lactulose can improve MHE, but they also have some weaknesses Table . ProbiotStreptococcus abundance in the gut (Zuo et al. In recent years, many scientists have compared the efficacy of these drugs in preventing and treating MHE, and there was no difference between probiotics and lactulose (Jiang et al. Given the adverse effects of lactulose, cost of rifaximin and the safety of probiotics, LOLA appears to have beneficial effects in MHE, although its role in therapy is not clearly defined Table .MHE has serious consequences for quality of life, increasing the number of accidents, falls, hospitalizations and associated costs (Llansola et al. Timely and accurate discovery of cognitive impairment is the key to diagnosis of MHE. Psychometric tests, CFF, EEG and MRI are useful to evaluate cognitive function in an intuitive or abstract way. Psychometric tests are irreplaceable now. Compared with other psychometric tests, ANT is little influenced by age and education level. MRI, which can more accurately reflect changes in cognitive function, may be the best option in the future, if the problem of lack of detection accuracy of the measured signal can be resolved. CFF and EEG should be used alongside PHES Table .Timely correction of cognitive impairment is the key to treatment of MHE. One method is to prevent nerve cell injury, through improving the nutritional status of nerve cells, and blocking their injury by inflammatory mediators. Another method is to improve intestinal flora and reduce serum ammonia level. Among the drugs to improve intestinal flora, LOLA has few adverse effects and low cost, which may become the ideal choice in the future Table ."} +{"text": "Forster et al. performeThe median-joining network approach employed in Forster et al. is a metAs an evolutionary biologist working in a developing country, I have experienced firsthand how sensational findings can influence decision-making processes by diverting time and resources to control virus strains deemed to be \u201cmore aggressive.\u201d In the fog of war, scarce resources are allocated in haste, and the developing world does not have well-informed science advisers sitting in every key meeting to help provide balanced scientific viewpoints. The scientific community, as a whole, needs to be extra cautious in interpreting new findings related to coronavirus disease 2019 (COVID-19), and any potential misinformation must be promptly addressed. Scientific discourse is the basic foundation of science, and high-profile publications, especially controversial ones, require constructive dialogues for advancement of science and betterment of society, particularly during an ongoing war against a global pandemic."} +{"text": "Hair cells are the mechanosensory receptors of the inner ear and can be damaged by noise, aging, and ototoxic drugs. This damage often results in permanent sensorineural hearing loss. Hair cells have high energy demands and rely on mitochondria to produce ATP as well as contribute to intracellular calcium homeostasis. In addition to generating ATP, mitochondria produce reactive oxygen species, which can lead to oxidative stress, and regulate cell death pathways. Zebrafish lateral-line hair cells are structurally and functionally analogous to cochlear hair cells but are optically and pharmacologically accessible within an intact specimen, making the zebrafish a good model in which to study hair-cell mitochondrial activity. Moreover, the ease of genetic manipulation of zebrafish embryos allows for the study of mutations implicated in human deafness, as well as the generation of transgenic models to visualize mitochondrial calcium transients and mitochondrial activity in live organisms. Studies of the zebrafish lateral line have shown that variations in mitochondrial activity can predict hair-cell susceptibility to damage by aminoglycosides or noise exposure. In addition, antioxidants have been shown to protect against noise trauma and ototoxic drug\u2013induced hair-cell death. In this review, we discuss the tools and findings of recent investigations into zebrafish hair-cell mitochondria and their involvement in cellular processes, both under homeostatic conditions and in response to noise or ototoxic drugs. The zebrafish lateral line is a valuable model in which to study the roles of mitochondria in hair-cell pathologies and to develop therapeutic strategies to prevent sensorineural hearing loss in humans. IP3Rs in the ER are involved in Ca2+ release into the cytosol and are coupled with mitochondrial VDAC in MAMs to transiently elevate intracellular Ca2+ levels, they observed increased mitochondrial Ca2+ uptake that corresponded with an increase in mitochondrial transmembrane potential (\u0394\u03a8m), suggesting that even transient increases in mitochondrial Ca2+ can affect mitochondrial activity in hair cells. Cumulatively, these results show that under non-pathological conditions mitochondria take up Ca2+ released from the ER and that changes in mitochondrial Ca2+ can alter mitochondrial activity.In conjunction with the mitochondrion, the endoplasmic reticulum (ER) is also a critical regulator of Cah Vance, . In zebrg et al. combined2+ indicators described earlier, Pickett et al. collapses, followed by a spike in cytosolic Ca2+ wild-type strain of zebrafish were less vulnerable to gentamicin-induced hair-cell loss than the ABOne consequence of oxidative phosphorylation is the generation of ROS. Mitochondrial ROS are generally produced in the form of superoxide or hydrogen peroxide due to oxidation of metabolic intermediates in the electron transport chain complexes I and III Brand, . Hydroxym and mitochondrial oxidation, as reflected by the indicator mitoSOX (2+ uptake; blocking entry of Ca2+ into the mitochondria with Ru360 reduced ROS levels and mitochondrial oxidation after neomycin exposure. Cumulatively, these data suggest that mitochondrial Ca2+ uptake is an event upstream of neomycin ototoxicity, with ROS playing an additional role.Mitochondrial ROS production may play a role in aminoglycoside ototoxicity. It has been shown that aminoglycosides bind to iron salts and stimulate the production of free radicals by Fenton chemistry (Priuska and Schacht, mitoSOX . These oin vitro, also protected against neomycin-induced hair-cell loss (Hirose et al., 2DCFDA labeling (Exogenous antioxidants have shown promising otoprotective effects in zebrafish lateral line and mammalian cochlear explants (Ton and Parng, labeling .d-methionine prevented this sonic-induced hair-cell loss, suggesting a role for oxidative stress in this model. Because zebrafish can be exposed to drugs by bath application, they are an optimal system in which to screen for protective or harmful drugs. By screening a redox library for compounds that protected against damage, glutathione, baicalein, d-\u03b1-tocopherylquinone, and ferulic acid ethylester were identified as protective agents (Uribe et al., In mammals, it has been shown that ROS are generated in the cochlea after noise exposure, and that antioxidants administered before or after exposure can potentially ameliorate noise-induced damage (Yamane et al., It has been suggested, specifically in the context of aminoglycoside ototoxicity, that hair cells \u201cfind a way to die\u201d such that inhibition of one death pathway will lead to the activation of other death pathways, and that it may be necessary to target multiple pathways to fully protect hair cells from ototoxins, such as by using drug cocktails or by using drugs that have multiple modes of action (Vandenabeele et al., Cisplatin is an anti-cancer chemotherapeutic drug that is commonly used to treat a number of different cancers. Notably, cisplatin treatment causes hearing loss in up to 80% of patients (Frisina et al., Similar to work with aminoglycosides, protection against cisplatin ototoxicity using antioxidants and ROS scavengers has been an intriguing topic of recent study. Epicatechin, a ROS scavenger derived from tea leaves, has been shown to protect zebrafish from cisplatin-induced lateral-line hair-cell loss (Kim C. H et al., While acquired hearing loss can be caused by exposure to noise or ototoxic drugs, some hearing loss is inherited (Lenz and Avraham, Zebrafish have been a popular model for genetic studies due to their high fecundity and ease of genetic manipulation. Forward genetic screens in zebrafish have proven useful for identifying genes required for hearing and balance as well as in pathways involved in aminoglycoside-induced death (Nicolson et al., pappaa, which encodes pregnancy-associated plasma protein-aa (Wolman et al., pappaap170 mutant zebrafish were more susceptible to neomycin-induced hair-cell death and had elevated ROS levels in their hair cells. In addition, pappaap170 mutant hair cells had increased mitochondrial Ca2+, hyperpolarized \u0394\u03a8m, and reduced expression of the mitochondrial antioxidant genes gpx, sod1, and sod2, all of which could contribute to increased ROS levels. Treatment with the ROS scavenger mitoTEMPO rescued pappaap170 mutant susceptibility to neomycin-induced hair-cell death, suggesting that elevated ROS underlies the enhanced hair-cell death in pappaap170 mutants. The study supports the utility of zebrafish forward genetic screens in identifying novel genes involved in mitochondrial function and hair-cell vulnerability.Forward genetic screens have been particularly useful for identifying novel gene function in the zebrafish lateral line. One such gene identified using forward genetics is mtu1 to study its function in hair cells (Zhang et al., mtu1-deficient zebrafish had deficient thiolation of mitochondrial tRNA, as well as decreased levels of mitochondrial tRNA and mitochondrial proteins (Zhang et al., mtu1\u2212/\u2212 zebrafish also had deficient oxidative phosphorylation and reduced ATP. In the lateral line, mtu1\u2212/\u2212 zebrafish had fewer hair cells per neuromast. These results support a role for mitochondrial tRNA modification in deafness and demonstrate the value in using reverse genetics to study gene function in hair cells.Another study used CRISPR/Cas9 technology to delete the gene in vivo. Studies utilizing this system have shed light on the roles of mitochondria in calcium homeostasis and synapse regulation as well as supported roles of mitochondria in cell death pathways, particularly in response to ototoxic drugs like aminoglycosides. The strides made from zebrafish studies contribute to the understanding of hearing loss in humans and will lead to development of preventative or protective therapies in the future.The zebrafish lateral line is a valuable model system in which to study hair-cell mitochondria and offers unique tools such as the ability to visualize mitochondrial dynamics MH and LS wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "The importance of training in regulating body mass and performance is well known. Physical training induces metabolic changes in the organism, leading to the activation of adaptive mechanisms aimed at establishing a new dynamic equilibrium. However, exercise can have both positive and negative effects on inflammatory and redox statuses. In recent years, attention has focused on the regulation of energy homeostasis and most studies have reported the involvement of peripheral signals in influencing energy and even inflammatory homeostasis due to overtraining syndrome. Among these, leptin, adiponectin, ghrelin, interleukin-6 (IL6), interleukin-1\u03b2 (IL1\u03b2) and tumour necrosis factor a (TNFa) were reported to influence energy and even inflammatory homeostasis. However, most studies were performed on sedentary individuals undergoing an aerobic training program. Therefore, the purpose of this review was to focus on high-performance exercise studies performed in athletes to correlate peripheral mediators and key inflammation markers with physiological and pathological conditions in different sports such as basketball, soccer, swimming and cycling. In elite athletes, there is large disparity among the training protocols; the effects on oxidative stress (OS) and inflammatory cytokines are still not well known. Is important to highlight that excessive training loads are able to trigger the syndrome known as overtraining syndrome (OTS), a phenomenon in which there is an increase in pro-inflammatory markers and consequently a decrease in sport performance . TherefoPeripheral mediators could be used to monitor both long- and short-term effects in elite athletes during training exercise. Leptin, adiponectin and ghrelin exert important effects on the hormonal regulation in response to acute exercise and chronic training and are related to each other. It is interesting to evaluate the effects of specific training on these molecules and on metabolic state in athletes since cytokine responses are linked to changes in physical performance. J\u00fcrim\u00e4ea et al. found thProfessional cyclists are a class of highly trained athletes subjected to different schedules of training and often to ultra-endurance training. Serrano et al. reportedNieman et al. reportedThey suggest a possible link between the increase in adiponectin and the training improvement due to the endurance or resistance training in cyclists. C\u00f3rdova Mart\u00ednez et al. determinIn young well-trained female basketball players, Plinta et al. found, aThe study of Souglis et al. reportedUnal et al. carried Mart\u00edn-S\u00e1nchez et al. , using aIn conclusion, in elite soccer players, more than in other athletes, an intensive and inadequate training program modifies the inflammatory status, and this may be associated with a reduction in performance.Karamouzis et al. reportedAerobic and anaerobic exercises result in alterations of redox homeostasis in untrained, trained and well-trained athletes. Following intensive physical activity in elite athletes, the source of systemic oxidative stress (OS) is not fully understood but it is proposed that skeletal muscle is the main contributor to the exercise-induced ROS . In this2O2, glutathione disulfide (GSSG) level and catalase (CAT) activity in well-trained female athletes who practice anaerobic/aerobic sports. They conclude that these results could be related to the adaptation mechanisms of antioxidative defense that depends on the type of sport. The results of a study by Souglis et al. [The influence of seasons on training has been analyzed by Balog et al. measurins et al. showed ts et al. , we suggs et al. reportedAltitude exposure can increase markers of OS after acute exercise and this is observed in endurance-trained cyclists. McGinnis et al. simulateDifferent studies demonstrated damage associated with OS in DNA following exhaustive/high-intensity endurance exercise, eccentric exercise and unaccustomed exercise. However, this effect is recovered after adequate rest. DNA damage is measured with an increase in 8-hydroxy-2\u2032-deoxyguanosine (8-OHdG) plasma levels because the activity of several DNA-repairing enzymes (among them: human 8-oxoguanine DNA glycosylase1 and oxidized purine-nucleoside triphosphate) is increased to protect against exercise-induced DNA damage. Yasuda et al. determinIn a short report, Lekhi et al. analyzedIn a recent study, Maleki et al. reportedDuring the season, elite basketball players train twice a day and play one or two games per week. Even though both aerobic and anaerobic systems are activated, several studies have demonstrated that anaerobic metabolism is the primary energy pathway activated in basketball players because basketball requires movements with variable velocity, continuous accelerations, decelerations, stopping and sudden changes of direction. These authors reported that elite basketball players show good anaerobic power and modeThe overall duration of a typical basketball match is 40\u201348 min, in which an athlete carries out a combination of high-intensity actions interspersed with low-intensity activities and active or passive recovery . In one 2\u2013 and H2O2 remained unchanged. On the other hand, SOD and CAT activity increased, while GSH decreased. Le Moal et al. [The physical demands activity required for soccer players includes: sprints, change of direction and high-intensity running involving both aerobic and anaerobic energy pathways leading to alterations of biological processes such as inflammation, muscle damage and OS. Jakovljevi\u0107 et al. investigl et al. followedIn summary, oxidative stress is markedly upregulated after a soccer game and it is correlated with cumulated training loads during the football season.Competitive swimming combines factors such as the simultaneous contribution of arms and legs to propulsion, water immersion and prone position and allows muscles to work in harmony and in accordance with each other, since gravity drops down to almost zero in water. Unlike other popular sports, the OS that occurs in swimming is largely affected by the type of competitions, and swimmers require a sophisticated training for both aerobic (endurance exercises) and anaerobic (short high-effort exercises below 200 m) exercise to increase performance. Several studies indicate that swimming is involved in OS homeostasis changing but the duration and intensity of the stress are strictly correlated with the effort . The stuThe purpose of this review was to focus on high-performance exercise studies performed in athletes to correlate peripheral mediators and key inflammation markers with physiological and pathological conditions in different sports such as basketball, soccer, swimming and cycling. The main source of discrepancies among reviewed studies indeed mainly concerns differences in the type, duration and intensity of training . In well"} +{"text": "Many nematodes show context-dependent, experience-dependent and/or life-stage-dependent behavioural responses to CO2, suggesting that CO2 plays crucial roles throughout the nematode life cycle in multiple ethological contexts. Nematodes also show a wide range of physiological responses to CO2. Here, we review the diverse responses of parasitic and free-living nematodes to CO2. We also discuss the molecular, cellular and neural circuit mechanisms that mediate CO2 detection in nematodes, and that drive context-dependent and experience-dependent responses of nematodes to CO2.Carbon dioxide (CO The initial repulsion from CO2 experienced by Heligmosomoides polygyrus iL3s on feces may enable them to disperse off of feces and into the environment to host seek. Following a prolonged period without feces, CO2 attraction may drive them towards new hosts or fresh host feces to increase their chances of host entry through ingestion in passively ingested ruminant parasites such as Haemonchus contortus are parasites that infect and kill insects , suggesting that jumping is highly sensitive to environmental CO2 in Steinernema scapterisci is in fact regulated by ambient CO2 levels.Like some mammalian-parasitic nematodes, some EPNs exhibit plasticity in their olfactory responses to COof weeks are a major cause of agricultural crop damage throughout the world. It has been estimated that PPNs are responsible for approximately 100 billion dollars of crop loss per year worldwide are repelled by CO2. CO2 repulsion by JIVs plays an important role in dispersal from its insect vector, the pine sawyer beetle, into the pine tree levels, as well as immediate O2 context. For example, animals exposed to elevated CO2 levels (2.5% CO2) become robustly attracted to CO2 over the course of hours in a reversible manner inhibits egg-laying behaviour, at least transiently mutations in npr-1 and animals carrying the natural variant of npr-1 avoid CO2 under low O2 conditions but do not respond to CO2 at normal atmospheric O2 levels (21% O2) mutation or a natural variant of npr-1, the URX neurons are tonically active under high O2 conditions and inhibit CO2 avoidance at high O2. The RIA interneurons appear to act downstream of URX to partially mediate its effects on the CO2 circuit (Kodama-Namba et al., glb-5 also acts via the URX neurons to modulate CO2 responsiveness as a function of ambient O2 levels (McGrath et al., et al., The extent to which CO2 responsiveness in C. elegans have been elucidated in some detail. The shift in CO2 response from repulsion to attraction when animals are moved from low CO2 to high CO2 cultivation conditions results from the differential activity of a single set of interneurons downstream of the BAG sensory neurons (Guillermin et al., 2, CO2 exposure inhibits the AIY interneurons and activates the RIA and RIG interneurons. In contrast, in animals that have been cultivated at high CO2, CO2 exposure activates AIY and inhibits RIA. Moreover, RIG is silenced such that it no longer responds to CO2 (2 response is not determined by whether an \u2018attractive\u2019 or \u2018repulsive\u2019 pathway is activated; rather, it is determined by experience-dependent modulation of interneuron activity in a single pathway (Guillermin et al., 2 responsiveness in animals cultured under high vs low CO2 conditions (et al., The mechanisms underlying experience-dependent modulation of COs to CO2 . Thus, Cnditions (Guiller et al., .2 repulsion to CO2 attraction that occurs during starvation also arises due to the differential activities of the AIY and RIG interneurons (Rengarajan et al., 2 evokes activating and inhibiting responses with approximately equal frequency (2 is attractive or repulsive is regulated by biogenic amine signalling. Dopamine promotes CO2 avoidance in well-fed animals by promoting activation of RIG and inhibition of AIY, while octopamine promotes CO2 attraction in starved animals by promoting activation of AIY (et al., 2 circuit is modulated during starvation by opposing biogenic amine signals. Neuropeptide signalling also regulates CO2 responsiveness during starvation (et al., 2 attraction in dauer larvae is less well understood but is regulated at least in part by neuropeptide signalling (Lee et al., The shift from COrequency . At the n of AIY (Rengara et al., . Thus, tarvation (Rengara et al., . Finally2 on other behaviours in C. elegans have also been elucidated. For instance, CO2-evoked activity in the AWC sensory neurons triggers a cGMP signalling pathway that ultimately inhibits the activity of the HSN neurons, resulting in the inhibition of egg-laying behaviour (Fenk and de Bono, C. elegans, resulting in increased longevity in BAG-ablated animals (Liu and Cai, jnk-1 and kgb-2 suppress CO2-induced fertility defects, indicating that JNK signalling may be involved in regulating fertility in response to CO2 (Vadasz et al., Some of the molecular and cellular mechanisms that mediate the effects of CO2 responsiveness in C. elegans have been elucidated in appreciable detail, several questions remain unexplored. For example, more information is needed to fully understand how the differential flow of information from BAG neurons to downstream interneurons generates experience-dependent plasticity of CO2-evoked behaviour. One intriguing possibility is that the BAG neurons express or release different neurotransmitters or neuropeptides in response to CO2 under varying conditions. Consistent with this possibility, the BAG neurons modulate the expression of FLP-19 neuropeptides as a function of their CO2-evoked activity (Rojo Romanos et al., 2-sensing neurons have not been identified. Finally, the CO2 microcircuit that drives CO2 attraction in dauers remains poorly understood, although it appears to involve dauer-specific, gap-junction-mediated signalling between the BAG neurons and the downstream AIB interneurons (Bhattacharya et al., 2 attraction in dauers. A better understanding of the neural circuits and signalling pathways that regulate CO2 responsiveness as a function of experience, context and life stage will provide important insights into how a single sensory cue can give rise to diverse behavioural responses in an ethologically-appropriate manner.Although the mechanisms underlying COet al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., 2 responsiveness in C. elegans as a starting point for launching investigations into the mechanisms of CO2 responsiveness in parasitic nematodes. In the case of both the necromenic nematode Pristionchus pacificus and the EPNs Heterorhabditis bacteriophora and Steinernema carpocapsae, BAG neurons were identified on the basis of conserved neuroanatomical position and shown to be required for behavioural responses to CO2 by laser ablation analyses (Hallem and Sternberg, et al., Pristionchus pacificus adults do not show acute CO2 avoidance, and BAG-ablated Heterorhabditis bacteriophora and Steinernema carpocapsae IJs do not show CO2 attraction (Hallem et al., 2-evoked jumping behaviour in Steinernema carpocapsae requires the BAG neurons (Hallem et al., 2 response are at least partly conserved across nematode species. However, the interneurons that operate downstream of BAG neurons to mediate CO2 responsiveness in other nematode species have not yet been identified. Moreover, nothing is currently known about the neural circuits and molecular signals that promote CO2 responsiveness in mammalian-parasitic nematodes. In future studies, it will also be interesting to determine whether similar or distinct mechanisms operate in C. elegans and parasitic nematodes to modulate CO2 responses depending on context, previous experience or life stage.The anatomy and function of nematode sensory neurons are generally conserved across species (Ashton 2 responsiveness in mammalian-parasitic nematodes. The identification of the neural mechanisms that drive or regulate the CO2 responses of mammalian-parasitic nematodes both inside and outside the host could lead to the identification of new drug targets or new strategies for nematode control. Until recently, investigations into the mechanisms underlying sensory behaviours in parasitic nematodes were limited to laser ablation analysis due to the dearth of resources and tools required for the genetic manipulation of these parasites. Laser ablation analysis has been used to establish the function of a number of different sensory neurons in mammalian-parasitic nematodes, including Strongyloides stercoralis, hookworms and Haemonchus contortus (Ashton et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., A major focus going forward will be on elucidating the cellular and molecular mechanisms underlying COStrongyloides stercoralis and Strongyloides ratti. Strongyloides stercoralis and Strongyloides ratti are more readily amenable to genetic manipulation than other parasitic nematodes because they can undergo one free-living generation (Viney, C. elegans (Evans, Strongyloides stercoralis is a human parasite that infects approximately 370 million people worldwide (Page et al., Strongyloides stercoralis is of interest as a model for other human-parasitic nematodes such as hookworms that cannot be genetically manipulated.The most genetically tractable parasitic nematodes are n Viney, . Foreigns Evans, . Most ot1 progeny of the microinjected adults (Lok and Massey, et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., Strongyloides has been aided by the identification of several promoters that drive expression in single cells or subsets of cells (Junio et al., et al., et al., C. elegans are stably expressed across generations, extrachromosomal arrays in Strongyloides are silenced after the F1 generation by as-yet-unknown mechanisms (Junio et al., et al., Strongyloides by methods that promote genomic integration of transgenes, such as transposon-mediated random integration (Shao et al., et al., Transgenic nematodes can be generated by introducing plasmid DNA containing exogenous genes; these genes are then expressed as extrachromosomal arrays in the FStrongyloides stercoralis and Strongyloides ratti. The recent development of an approach for CRISPR/Cas9-mediated targeted gene disruption in these species provided the first insights into the genetic mechanisms that drive sensory behaviours (et al., et al., et al., Strongyloides stercoralis infective larvae (Bryant et al., Strongyloides ratti. RNAi approaches using both dsRNA and siRNA have been used to study the effects of transcriptional knockdown of genes in several parasitic nematode species, although with varying efficacy (Geldhof et al., et al., et al., et al., et al., et al., Strongyloides ratti, a recent study demonstrated the first successful knockdown of multiple mRNAs using an siRNA approach (Dulovic and Streit, Strongyloides ratti iL3s, although mapping the locations of these mutations has not been possible yet (Viney et al., et al., Methods for disrupting gene function are also now available for haviours (Gang etStrongyloides stercoralis to CO2. For example, it will be interesting to determine whether the BAG neurons, which sense CO2 and promote behavioural responses to CO2 in C. elegans, play a similar role in Strongyloides stercoralis. It will also be important to elucidate the neural circuitry that operates downstream of the CO2-sensing neurons to mediate or modulate CO2-evoked behaviours in Strongyloides stercoralis. An intriguing possibility is that while sensory neuron function may be generally conserved across species, interneuron function may be less well conserved and may instead reflect species-specific behavioural and physiological responses to CO2. In addition, through the systematic screening of candidate genes known to be involved in CO2 responsiveness in C. elegans, it might be possible to uncover molecular signals that regulate parasite\u2013host interactions or that are required for successful parasitism. In the long run, a better understanding of the molecular and cellular bases of CO2-evoked behaviours in parasitic nematodes may lead to new avenues for nematode control. It may also shed light on some of the unique sensory mechanisms that operate in parasitic nematodes to shape parasite-specific behavioural responses.Using a combination of the above approaches, it should be possible to identify the neural mechanisms and molecular pathways that are involved in driving behavioural and physiological responses of"} +{"text": "We read with great interest the recent article by Shimazui et al. who concluded that in septic patients, mortality in non-elderly patients was increased with hypothermia and decreased with fever, while mortality in elderly patients was not associated with body temperature (BT) . We woul We read with great interest the letter by Honore et al., concerning the potential confounding effect of continuous replacement therapy (CRRT) on body temperature (BT) measurement. The generally higher risk of developing acute kidney injury (AKI) in elderly patients might also influence the results.2 ). However, non-elderly patients were more frequently initiated on CRRT within first 24\u2009h compared to elderly patients ; non-elderly patients had higher chance of temperature alteration by CRRT. In addition, adding \u201cCRRT\u201d as covariates including previously reported analysis in the Cox regression for the hazard of death over 90\u2009days by the BT adjusted with potential imbalances of age (per year), sex, chronic steroid use, and APACHE II score yielded results similar to the primary findings .As Honore et al. have pointed out, the morbidity of AKI on admission was significantly higher in elderly patients compared to non-elderly patients (elderly 82.5%, non-elderly 73.8%, According to these results, we conclude that the BT, especially hypothermia, may be associated with mortality in non-elderly septic patients including those treated with CRRT, while the association between BT and mortality remains inconsistent in elderly septic patients even after adjusting the CRRT effects."} +{"text": "This editorial summarizes eight research articles included in this supplement issue for the 2020 International Conference on Intelligent Biology and Medicine (ICIBM 2020) conference, that was held on August 9-10, 2020 ,with a topic on data-driven analytics in biomedical genomics. These articles cover a wide range of topics in medical genomics that focus on integrative analysis of genomics data together with other types of data toward understanding complex human diseases,including cancer. With the growing importance of data analytics in biomedical science, we expect this collection of research articles provides scientific discussions in this direction. The 2020 International Conference on Intelligent Biology and Medicine (ICIBM 2020), the official conference of the International Association for Intelligent Biology and Medicine (IAIBM), was held virtually from August 9th to 10th, 2020 due to the COVID-19 pandemic. Established in 2012, the ICIBM conference has grown as a venue to cultivate interdisciplinary research and education at the intersection of bioinformatics, intelligent computing, systems biology, and medical informatics. The first virtual conference of ICIBM (ICIBM 2020) had approximately 300 attendees, with 41 oral presentations scheduled in four live sessions selected from 75 original submissions. With rigorous review for ICIBM 2020 followed by second-round reviews for journal submissions, eight high-quality manuscripts were selected to be published in the ICIBM 2020 BMC Medical Genomics special issue.This supplementary issue includes eight manuscripts that cover a variety of topics in medical genomics, with a focus on data-driven analytics in biomedical genomics research. Here we summarize the contribution of each of these eight manuscripts.In the manuscript titled \u201cA pan-kidney cancer study identifies subtype specific perturbations on pathways with potential drivers in renal cell carcinoma\u201d, Zhan et al. introducIn the study of \u201cPinpointing miRNA and genes enrichment over trait-relevant tissue network in Genome-wide Association Studies\u201d, Li et al. combinedIn the manuscript titled \u201cCharacterization of genome-wide association study data reveals spatiotemporal heterogeneity of mental disorders\u201d, Dai et al. presenteIn \u201cNetwork-based Drug Sensitivity Prediction\u201d, Ahmed et al. exploredIn the study of \u201cDifferential alternative splicing (AS) between hepatocellular carcinoma with normal and elevated serum alpha-fetoprotein\u201d, Jin et al. reportedIn the manuscript titled \u201cIntegrative analysis of histopathological images and chromatin accessibility data for estrogen receptor-positive breast cancer\u201d, Xu et al. providedIn the manuscript titled \u201cThe circular RNA expression profile in ovarian serous cystadenocarcinoma reveals a complex circRNA-miRNA regulatory network\u201d, Zhuang et al. studied In the manuscript titled \u201cConditional transcriptional relationships may serve as cancer prognostic markers\u201d, Yu et al. incorporThis supplementary issue includes a collection of eight manuscripts that are focused on various perspectives of integrating data across multiple data types , to understand the etiology, prognostics and progression of diseases. With the emerging big data with regards to its volume, velocity, and variety in biomedical science, we anticipate integrative analytics will significantly propel future biomedical advances in realizing the full potentials of big data in biomedicine."} +{"text": "Recently, a growing attention has been observed toward potential advantages of stem cell (SC)-based therapies in regenerative treatments. Mesenchymal stem/stromal cells (MSCs) are now considered excellent candidates for tissue replacement therapies and tissue engineering. Autologous MSCs importantly contribute to the state-of-the-art clinical strategies for SC-based alveolar bone regeneration. The donor cells and immune cells play a prominent role in determining the clinical success of MSCs therapy. In line with the promising future that stem cell therapy has shown for tissue engineering applications, dental stem cells have also attracted the attention of the relevant researchers in recent years. The current literature review aims to survey the variety and extension of SC-application in tissue-regenerative dentistry. In this regard, the relevant English written literature was searched using keywords: \u201ctissue engineering\u201d, \u201cstem cells\u201d, \u201cdental stem cells\u201d, and \u201cdentistry strategies\u201d. According to the available database, SCs application has become increasingly widespread because of its accessibility, plasticity, and high proliferative ability. Among the growing recognized niches and tissues containing higher SCs, dental tissues are evidenced to be rich sources of MSCs. According to the literature, dental SCs are mostly present in the dental pulp, periodontal ligament, and dental follicle tissues. In this regard, the present review has described the recent findings on the potential of dental stem cells to be used in tissue regeneration. Losingal., 2010). One ofal., 2010; Abou Neal., 2010). It canal., 2010). SC-basal., 2010). In thial., 2010). AccordThe SC types ever investigated for application in regenerative medicine can be divided into two categories: embryonic stem cells (ESCs) and adult stem cells (ASCs). ESCs are pluripotent stem cells originating from the inner cell mass of the blastocyst-stage embryos they are much higher scalable in the undifferentiated state compared to other SC types is a more general terminology for pluripotent human embryonic stem (hES) cells with stem cell-like developmental quality dda, 2009). The hEdda, 2009), 3) thedda, 2009). Findindda, 2009; Sternbedda, 2009). The cldda, 2009). While dda, 2009). Previodda, 2009). Using dda, 2009).Dental pulp pluripotent stem cells (DPPSCs or DPSCs) originate from the cranial neural crest in the embryonic stage . The isin-vitro or in-vivo, SHED populations can successfully differentiate into various specialized cell populations such as odontoblasts, osteoblasts, chondrocytes, adipocytes, and neural cells or the primary teeth are among the most studied SC types and the most valuable source of stem cells in tissue engineering studies and cell-based regenerative medicine therapies . The real., 2016). In 200al., 2016). This ral., 2016). The SHal., 2016). Fortunal., 2016). In 200al., 2011). The odal., 2011). More ral., 2016). In thial., 2016; Rosa etal., 2016). The scal., 2016). Tetracal., 2016). in-vitro , drug developments, and inventing novel therapies have already shown great promises in animal model trials for regenerative treatment of Parkinson's disease and sickle cell anemia . Human al., 2010), and soal., 2012). The iPal., 2012). The hial., 2012). Despital., 2012). Anotheal., 2012). TherefThe stem cells from the apical papilla (SCAPs) belong to a unique SC line locating at the apical tissues of the growing tooth roots when at least two-thirds of the root have formed were first obtained from the bone marrow of the iliac crest . HoweveEmersion and recovery of dental diseases such as deterioration, is substantially under the effect of the teeth microenvironment, so that any pathologic alteration that can affect the endogenous MSCs' functions and their regeneration capacity may lead to substantial bone loss. Similarly, transplanted exogenous MSCs are highly influenced by the microenvironment of both donor and recipient niches that creates a major challenge to using MSCs for therapeutic regeneration purposes in diseased microenvironments .Adipose tissue-derived stem cells (ASCs) are considered an abundant MSC source that can be obtained through lipectomy or lipoaspiration from different adipose tissues such as the chin, hips, upper arms, and abdomen . The ASEpithelial stem cells and MSC-like cells are reported to locate within particular niches in dental pulp, dental follicle, and periodontal tissues (ligament stem cells) . Dentalal., 2007). Furtheal., 2007). The \u201ctal., 2018). The dental follicle stem cells (DFSCs) are located within the dental follicle or bilayered Hertwig's epithelial root sheath (HERS); they originate from the ectomesenchymal progenitor cell population and differentiate into cementoblasts or osteoblasts (cementogenesis) during tooth root formation . DFSCs in-vitro and in-vivo studies are a few mesenchymal progenitor cells within the PDL that remain proliferative, and their differentiation potential provides great promises for SC-based regenerative therapies in dentistry . Yet, tal., 2015). PDLSCsal., 2016). PDLSCsal., 2016; Bright al., 2016). In perGingival mesenchymal stem cells (GMSCs) originate from gingival connective tissue, mostly referred to as lamina propria . The giSalivary gland-derived stem cells (SGSCs) were firstly isolated from a rat submandibular gland . Similain-vitro and in-vivo researches imply the mediation of cell chemo-attraction, differentiation, and proliferation in the GFs capability of increasing tissue regeneration capacity are natural biological molecules with growth-promoting activities that usually have been initially identified for their functions as mediators and regulators in cellular events . Based al., 2011). Despital., 2011; \u00d6zdemiral., 2011). Next tal., 2011). Anotheal., 2011). EMD isal., 2011). Considal., 2011). Severaal., 2011). BMP-2,al., 2011). Therefin-vivo and the ex-vivo methods. During the in-vivo strategy, natural healing potential of pulp tissue is elevated by injecting BMP proteins or BMP genes, while in the ex-vivo strategy, DPSCs are isolated and differentiated in the laboratory using BMP proteins or BMP genes. The induced SCs are then transplanted into the tooth are multi-functional growth factors from the transforming growth factor-beta (TGF\u03b2) superfamily . The gral., 2011).In-vivo study of VEGF in immuno-deficient mice has shown its potential for angiogenesis induction and enhances the survival of subcutaneously transplanted dental pulp cells . The moal., 2008).In-situ hybridization technique has shown that four highly homologous genes encode FGFR family isoforms, which differentially express in dental epithelium and mesenchyme family consists of 18 receptor-binding members that regulate cellular activities such asal., 2013, 2017). ). ex-vivIn these SC-based therapeutic strategies that use DPSCs for regenerating dentin-pulp complex, GF concentrations in the dentin matrix should be precisely considered, because they are directly in contact with the pulp and consequently, have a significant effect on DPSCs proliferation as the first stage of pulp repair . The de\u03b2-tricalcium phosphate/type I collagen, gelatin sponge, HA disks, etc.) and successfully achieve regenerated periodontium tissues that pivotally contribute to embedding the tooth in the jaw, maintaining the tooth homeostasis, repairing, feeding, and also harboring progenitor cell populations called PDLSCs . PDLSCsal., 2011; Han et al., 2011; Ninomiyal., 2011). These al., 2011; Tsumanual., 2011). The tral., 2011; Han et al., 2011); howeveal., 2011; Ninomiyal., 2011). As an al., 2011). In anoal., 2011).2+, and actuate the protein kinase C (PKC) cascade. These mechanisms lead to the migration and preferential adhesion of progenitor cells. The cementum-specific proteins (CEMPs) associated with these activities contain cementum-derived growth factor (CDGF), cementum attachment protein (CAP), and cementum protein-1 (CEMP1). The CEMPs' activity induces the differentiation of periodontal ligament stem cells (PDLSCs), dental follicle stem cells (DFSCs), and, adipose-derived stem cells (ADSCs) into cementoblasts and osteoblasts resulting in periodontium regeneration, PDL fibers formation, periodontal angiogenesis, and creation of a cementum-like mineralized extracellular matrix (ECM) cells are considered to be cementogenetic and able to produce a thin layer of acellular cementum around the root neck. HERS also can cover the lower part of the root by thicker cellular cementum. A challenge in periodontal regeneration researches using cellular intrinsic fiber cementum (CIFC) is achieving an adequate attachment function. The low-quality attachment of the newly-formed cementum in CIFC is due to lack of acellular extrinsic fiber cementum (AEFC), low density of the inserting fibers, and weak interfacial tissue bonding . In damal., 2019). in-vitro floating sphere assays, and two-dimensional (2D) or 3D cultures of salivary gland cells . The pral., 2017). The SGal., 2017). The ocal., 2017; Paz et al., 2017). Entire-tooth loss can lead to several physical and mental sufferings which may extensively compromise the life quality and self-esteem of the patient. Even, in some wildlife species, losing the complete dentition can end their life. The regeneration of the entire tooth has come to the focus of many pieces of research after successful results of regenerating tooth elements. As a major organ, a tooth is constructed from multiple hard and soft tissues. Enamel, dentin, and cementum are the hard tissues that surround the dental pulp as the only vascularized tissue in teeth . A releal., 2008). Beforeal., 2008). In somal., 2008; Botelhoal., 2008). In a cal., 2008). In anoal., 2008). Also, al., 2008; Kim et al., 2008). Then, al., 2008). Considex-vivo cell manipulation makes cell homing more easily adjustable and more potential to be commercialized for clinical processes. Therefore, this under-recognized strategy can be offered as an alternative to cell-delivery-based tooth regeneration . Cell hal. (201060]) use) useex-val. (2010[60]). Inal. (2010). de novo regeneration of the whole tooth structures . Howeveani, 2014). De noval., 2011). Howeveal., 2011). In craal., 2011). Regardal., 2011). In thial., 2011). Anotheal., 2011). Despital., 2011), and thal., 2011). \u03b3 secretion, and promote cytotoxic effects on the virus-infected cells . MSCs cal., 2016). Their al., 2016). MSCs cal., 2016). MSCs cal., 2016). MSCs cal., 2016). MSCs aal., 2016). Regenerative dentistry is increasingly recognized as a state-of-the-art field of medicine among dental clinicians during dental treatments as a procedure of acquiring stem cells and storing them for potential future autologous treatments. Adult stem cells have been successfully acquired from sources such as oral and maxillofacial regions. For achieving the ultimate purpose, which is craniofacial regeneration, there has remained a long way to be paved for identifying the effective factors in immunomodulatory functions of adult MSCs, BMSCs, and pluripotent stem cells. Such information is required for a more effective outcome of SC-based bone and periodontal tissue restoration, especially for transplanting at the inflamed sites. Knowing the immunomodulatory properties of adult stem cells used in dental and periodontal regeneration is vital for reaching optimal tissue regeneration and controlling the local immune responses during transplantation. Among different strategies in the regenerative dentistry field, tissue engineering and chair-side cellular grafting approaches are more promising because of their more predictable regenerative results. The randomized controlled type of clinical trials with long follow-ups is the most required type of scientific evidence for a comprehensive establishing of reconstructive dental therapeutics. Another necessary field to be elaborated is understanding the biological processes underlying both graft donor and recipient during bone regeneration. Despite all unrecognized aspects of stem cell-based bone and periodontal tissue reconstruction, prosthodontists are increasingly being attracted to stem cell biology because of its successful results as well as the unresolved inefficiencies of implant dentistry. The mechanisms that control the fates and functions of the transplanted stem cells need to be studied. Despite multiple SC-based studies on the dental pulp and periodontal regeneration in animal models, serious considerations, and clinical trials with long-term follow-up are necessary to speed up the translation of basic and preclinical SC-based studies to the dental clinics in terms of regulation, immunity, technology, ethics, and any other possible clinical restriction. These fundamental concerns need to be sorted out to make regenerative treatments practically applicable and beneficial for patients with pathological or injured dental pulp and/or periodontal tissues. Autologous stem cells are already started to be used in some clinical trials for regenerating pulp and periodontal tissues; however, their approval as well as outcomes have not yet been broadcasted and transmitted to the guidelines or indications. Stem cell-based regenerative approaches can help many people around the world who suffer from dentistry complications that strongly warrant further studies. Fortunately, the technologies of modern imaging systems, nanotechnology, and mathematical modeling are increasingly developing and coming to help stem cell-based regenerative studies achieve more reliable and qualitative outcomes in a much shorter time. Mohsen Yazdanian and Hamid Tebyanian equally contributed as corresponding authors.The authors declare that this work was done by the persons named in this article. Armin Soudi, Mohsen Yazdanian, Reza Ranjbar, Hamid Tebyanian, and Alireza Yazdanian were involved in study design and data collections. Armin Soudi, Mohsen Yazdanian, Reza Ranjbar, Hamid Tebyanian, Elahe Tahmasebi, Ali Keshvad, and Alexander Seifalian were involved in critically reviewing the data and writing the review article.The authors would like to acknowledge the useful comments were given by colleagues at the Research Center for Prevention of Oral and Dental Diseases, Baqiyatallah University of Medical Sciences, Tehran, Iran.There was no financial support.This article is a review and does not contain any experiment on humans or animals performed by any of the authors.The authors declare that they have no competing interests."} +{"text": "Several studies suggested that depression might worsen the clinical outcome of diabetes mellitus; however, such association was confounded by duration of illness and baseline complications. This study aimed to assess whether depression increases the risk of diabetes complications and mortality among incident patients with diabetes.This was a population-based matched cohort study using Taiwan's National Health Insurance Research Database. A total of 38 537 incident patients with diabetes who had depressive disorders and 154 148 incident diabetes patients without depression who were matched by age, sex and cohort entry year were randomly selected. The study endpoint was the development of macrovascular and microvascular complications, all-cause mortality and cause-specific mortality.Among participants, the mean (\u00b1SD) age was 52.61 (\u00b112.45) years, and 39.63% were male. The average duration of follow-up for mortality was 5.5 years, ranging from 0 to 14 years. The adjusted hazard ratios were 1.35 for macrovascular complications and 1.08 for all-cause mortality. However, there was no association of depression with microvascular complications, mortality due to cardiovascular diseases or mortality due to diabetes mellitus. The effect of depression on diabetes complications and mortality was more prominent among young adults than among middle-aged and older adults.Depression was associated with macrovascular complications and all-cause mortality in our patient cohort. However, the magnitude of association was less than that in previous studies. Further research should focus on the benefits and risks of treatment for depression on diabetes outcome. Patients with type 1 diabetes mellitus were identified if their ICD-9 diagnostic code was 250.x1 or 250.x3 and they never used oral antidiabetic drug; the patients who remained were categorised into type 2 diabetes mellitus. The first date of antidiabetic prescription was defined as the cohort entry date. Given that some patients delayed treatment and had complications when diabetes was diagnosed, we excluded those with macro- or microvascular complications before or on the cohort entry date (n\u00a0=\u00a0758\u00a0041). We did not include patients with a diagnosis of schizophrenia or bipolar disorders (n\u00a0=\u00a05664). In addition, those with information errors regarding sex, age or mortality date were also excluded (n\u00a0=\u00a013\u00a0259). The study population included 1\u00a0087\u00a0125 incident patients with diabetes.Using the NHIRD, we identified incident patients with diabetes aged 20 years or more who were firstly prescribed antidiabetic agents and had at least a diagnosis of diabetes mellitus code: 250.x) between 2001 and 2014 ; major depressive disorder, single episode (ICD-9 code: 296.2x); dysthymic disorder (ICD-9 code: 300.4) or depressive disorder not otherwise specified (ICD-9 code: 311). If a patient has two or more different diagnostic codes of depression, the types of depression were categorised based on the above-mentioned hierarchy. Finally, we identified a total of 38\u00a0537 patients with diabetes who had depressive disorders.We identified 154\u00a0148 eligible comparison participants with diabetes mellitus, who had no preexisting complications and no diagnosis of severe mental illnesses. For each patient with diabetes who had a depressive disorder, we randomly selected four comparison participants matched by age (year of birth), sex and the cohort entry year. The comparison participants were censored if they were diagnosed with a depressive disorder after the cohort entry date. The details of the selection procedure are shown in et al., et al., The study outcomes included macro- and microvascular diabetes complications, which were identified based on ICD-9-CM diagnostic and procedural codes and the NHI procedural codes from outpatient and inpatient claims records. Macrovascular complications included hospitalisation for acute coronary syndrome (ICD-9-CM: 410.x-411.x) and stroke (ICD-9-CM: 430.x-438.x). Validation of the inpatient diagnosis of the acute coronary syndrome and stroke has been well documented : I00\u2013I99) or diabetes . To test the sensitivity of our methods, we included unnatural death and suicide .Patients' demographic variables included age, sex and the year of cohort entry. The duration between the first diagnosis of diabetes and the initiation of pharmacotherapy was also measured and categorised into <1 and \u2a7e1 year. Potential confounders, which were associated with both diabetes complications and depressive disorders, were assessed in the year preceding the cohort entry date, which included comorbid conditions of hypertension (ICD-9 code: 401.x-405.x), dyslipidemia (ICD-9 code: 272.x), chronic pulmonary disease , chronic liver disease and alcohol- and substance-related disorders . In addition, medication use that reflected underlying medical conditions was assessed, including angiotensin-converting enzyme inhibitor (ACEI) and angiotensin receptor blocker (ARB) (ATC code: C09), beta blockers (ATC code: C07), calcium channel blockers (ATC code: C08), lipid-lowering agents (ATC code: C10) and nonsteroidal anti-inflammatory drugs (ATC code: M10).We calculated the incidence of macro- and microvascular diabetes complications and mortality rates based on the event number divided by follow-up person-years. Multivariate Cox proportional hazards models were used with adjustment for the abovementioned confounders, to estimate the hazard ratios (HRs) of depressive disorders for the study outcomes. Subgroup analyses were conducted to evaluate whether the risks of depression for diabetes complications and mortality were modified by types of depression, age, sex and duration between diabetes diagnosis and initiation of pharmacotherapy.post-hoc analyses to evaluate whether there were differences in the quality of diabetes care during the follow-up period between patients who had diabetes, with and without depressive disorder. Indicators of care quality included medication adherence for antidiabetic drugs, frequency of receiving blood sugar testing (HbA1c or fasting blood sugar), lipid profiles, serum creatinine, retina examinations and electrocardiograms during the follow-up period. Antidiabetic drug adherence was measured using the medication possession ratio (MPR), which was defined as the total days of prescribed antidiabetic medication supply divided by the follow-up period. Antidiabetic drug adherence was categorised as poor, irregular and good, based on the MPR .The quality of diabetes care might be a mediator in the association of depression with diabetes complications and mortality; therefore, we did not include these variables in our model. We used p values <0.05.All statistical analyses were conducted using SAS version 9.4 . The statistical significance of relationships was assessed using 95% confidence intervals (CI) or There were 38\u00a0537 patients with diabetes who had depressive disorders and 154\u00a0148 patients with diabetes with no depressive disorders, as comparison participants. The average follow-up period was 5.5 years (ranging from 0 to 14 years). The mean (\u00b1SD) age of participants was 52.61 (\u00b112.45) years, and 39.63% were male. Most of the patients have type 2 diabetes mellitus 99.4%. Among patients with depressive disorders, 26.21% had major depressive disorder, recurrent episode, 15.96% had major depressive disorder, single episode; 48.11% had dysthymia and 9.72% had depressive disorder, not otherwhere classified. Patients with diabetes who had depressive disorders had a higher proportion of comorbid conditions and medication use, except for ACEI/ARB use. The percentage of duration between diabetes diagnosis and initiation of pharmacotherapy \u2a7e1 year was 17% for patients with diabetes and depression and 24% for comparison subjects see .Table 1v. 2.29 per 1000 person-years), unnatural mortality (2.46 v. 0.77 per 1000 person-years), suicide (1.41 v. 0.27 per 1000 persona-years) and all-cause mortality (21.91 v. 15.96 per 1000 person-years) between patients who had diabetes, with and without depressive disorders. However, there was no difference with respect to microvascular complications and mortality due to diabetes mellitus for comparison participants. The same was true for the crude incidence of mortality due to cardiovascular diseases , unnatural mortality , suicide and all-cause mortality than comparison participants. However, there was no statistically significant difference in microvascular complications and death owing to cardiovascular diseases. We found that depression was associated with a reduced risk of death owing to diabetes .Table 3In subgroup analyses, we found that the magnitude of associations with developing macrovascular complications, unnatural death, suicide and all-cause mortality was higher among those with major depression with recurrent episode than those with dysthymia or depressive disorder, not otherwise specified (the 95% CIs were not overlapping). In terms of the modifying effect of sex, we found that the associations between depression and developing macrovascular complications and unnatural mortality were stronger among women than among men. Otherwise, sex did not have a modifying effect on the incidence of microvascular complications and all-cause and other cause-specific mortality. In terms of age, there were effect modifications on diabetes complications and mortality. HRs in young adults (aged 20\u201344 years) with depressive disorders for all diabetes complications, all-cause mortality and all cause-specific mortality were higher than those among middle-aged (aged 45\u201364 years) and older (aged 65 years or more) adults. Furthermore, we found that duration between diabetes diagnosis and initiation of pharmacotherapy had a significant modifying effect on the associations with microvascular complications, diabetes-related death and all cause-mortality; the associations were stronger among patients with duration \u2a7e1 year than those with duration >1 year.Assessment of the quality of diabetes care showed that patients with diabetes who had depressive disorders had slightly higher proportions of good antidiabetic compliance and higher rates of undergoing screening tests; however, the difference was quite small .Table 4In this study, we found that depression was associated with an increased risk of macrovascular complications, unnatural mortality, suicide and all-cause mortality among patients with incident diabetes mellitus. In contrast, there was no association between microvascular complications and mortality owing to diabetes mellitus and cardiovascular diseases. We found that age had a multiplicative effect modification on such associations. The magnitude of association was higher among young adults than middle-aged and older adults. Regarding the quality of care, we found that patients with diabetes who had depressive disorders had slightly better antidiabetic medication compliance and a slightly higher proportion of undergoing screening tests.et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., The findings of a positive association between depression and macrovascular complications were consistent with those of several previous studies (Black et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., We also found that depression was related to an increased all-cause mortality rate; however, the magnitude was smaller than that found in previous studies (Black et al., et al., et al., et al., et al., et al., et al., et al., We found there was no association between depression and microvascular complications. This finding was against several studies (Black et al., In the subgroup analysis, major depression with recurrent episodes had greater associations with macrovascular complications and all-cause mortality than mild depression. These severity-response relationships further support the hypothesis between depression and diabetes complications. In addition, we found that women had a greater risk for mortality and macrovascular complications associated with depression than men. Previous studies have shown that the effect of diabetes on the risk of coronary disease is significantly greater for women than men (Lee et al., et al., Several limitations should be considered in this study. First, we identified patients with depressive disorders based on claims records. However, some patients with depression were not diagnosed or treated and could be misclassified into the comparison group; therefore, it would introduce bias towards the null. The effect of depression on diabetes complications and mortality might be underestimated. The duration between the diagnosis of depression and diabetes mellitus was unknown; therefore, we could not explore the effect of duration of depression on the study association. Second, only a few patients with type 1 diabetes mellitus were included because the age of onset was generally before 20 years. Our findings could not be concluded for those with type 1 diabetes mellitus due to the limited statistical power. Third, the accuracy of the diagnosis of microvascular complications of diabetes was not yet validated in the NHIRD; however, we used procedural claims data to confirm ambulatory diagnostic codes, or we only used inpatient claims data to minimise the possibility of misclassification. Fourth, several important factors, such as smoking, body weight, exercise, diet control and family history of depression and/or diabetes are not available in the NHIRD. Although we tried to use chronic pulmonary disease, dyslipidemia, hypertension and alcohol or substance use disorders as proxy measures for smoking, obesity and unhealthy lifestyle behaviours, there were still residual confounding effects. Especially, the lifestyle behaviours are time-variant and would be influenced by depression; therefore, these factors need to be adjusted by advanced methodology (Fewell The strengths of this study are the novelty of analysing the associations between depression and diabetes complications among incident patients with diabetes, the elimination of confounding by duration of illness and pre-existing complications, the use of a nationwide representative cohort with clear temporal relationships, a very large sample and a well-defined method for identifying complications of diabetes.In conclusions, we found that patients with diabetes mellitus had a higher rate of macrovascular complications and all-cause mortality when they had comorbid depressive disorders. However, we found a lower adverse effect of depression than the findings of previous studies. This might be owing to the inclusion in our study sample of only incident patients with diabetes, thereby eliminating confounding owing to the duration of illness and preexisting complications. Depression is a modifiable risk factor of diabetes outcome. Further research should focus on evaluating the effect of depression treatment on advanced complications and mortality among patients with diabetes mellitus."} +{"text": "Protein quality control (PQC) systems play essential roles in the recognition, refolding and clearance of aberrant proteins, thus ensuring cellular protein homeostasis, or proteostasis. Especially, continued proliferation and differentiation of stem cells require a high rate of translation; therefore, accurate PQC systems are essential to maintain stem cell function. Growing evidence suggested crucial roles of PQC systems in regulating the stemness and differentiation of stem cells. This review focuses on current knowledge regarding the components of the proteostasis network in stem cells, and the importance of proteostasis in maintaining stem cell identity and regenerative functions. A complete understanding of this process might uncover potential applications in aging intervention and aging-related diseases. Stem cells serve as the origin of a multicellular organism. They can divide to give rise to daughter cells that remain as stem cells or become differentiated with a specific function. The multi-differentiation potential gives stem cells unparalleled advantages in regenerative medicine. Originally, stem cells can be categorized into two main groups: embryonic stem cells (ESCs) and adult stem cells (ASCs). Yet with the development of reprogramming technologies, somatic cells can also be reprogrammed into ESC-like cells, termed as induced pluripotent stem cells (iPSCs). Collectively, ESCs and iPSCs are referred to as pluripotent stem cells (PSCs) because of their high capacity for self-renewal and their ability for multipotent differentiation, offering far-reaching potential in disease modeling and transplant therapies , while such high levels of expression diminish during differentiation is activated to cope with misfolded proteins, either facilitating their proper re-folding or delivering them for degradation via the proteasome or autophagy pathways is a central cellular organelle in proteostasis. It is involved in the synthesis, modification, and delivery of proteins to their target sites in the secretory pathway and the extracellular space is a central regulator, as it plays a vital role in protein folding, ER calcium binding, and regulating the activities of transmembrane ER stress sensors. Consistently, Grp78 homozygous knockout mouse embryos failed to hatch from zona pellucida, and exhibited proliferation defects and extremely high levels of apoptosis in the inner cell mass, demonstrating that Grp78 is crucial for embryonic cell growth and pluripotent cell survival enhances ER protein folding, thereby increasing the repopulation capacity of hematopoietic stem cells (HSCs) in xenograft assays, connecting the UPR to the maintenance of HSC properties also plays an important role in the maintenance of stem cell characteristics and homeostasis increase plays a crucial role in the self-renewal and differentiation of HSCs and NSCs (Matsumoto et al., Sel1L, reduced self-renewal and resulted in HSC depletion (Liu et al., Taken together, the UPS is essential for maintaining stemness in both PSCs and ASCs, and dysfunction of the UPS may contribute to perturbed stem cell function and fate control. The main differences between UPS-related cellular reactions in stem cells and those in differentiated cells were summarized in Fig. Misfolded and aggregated proteins can also be degraded by a separate autophagy-mediated pathway. Autophagy is a highly conserved intracellular process, in which damaged or unwanted proteins, cytosolic fractions, and organelles are degraded by the lysosome (He and Klionsky, Despite extensive research on autophagy in somatic cell physiology, relatively little is known about the roles of autophagy in stem cell biology. Recent studies report that autophagy is crucial for ESCs and for various ASCs, i.e. HSCs, MSCs, NSCs, and gut stem cells, although with different requirements for its activity (He et al., Apg5 in mESCs causes defects in autophagosome formation and consequent accumulation of proteins in the cytoplasm (Mizushima et al., beclin-1, a mammalian ortholog of the yeast autophagy-related gene 6, die early in embryogenesis, and the autophagic response in mESCs is significantly altered as a result of the beclin-1 deficiency (Yue et al., Ambra1, an autophagy gene, are impaired for neuronal generation (Vazquez et al., In contrast to the high levels of proteasome activity observed during self-renewal, high levels of\u00a0autophagy activity was exhibited during early differentiation of ESCs\u00a0and NSCs (Tra et al., Atg7 or Fip200 in the hematopoietic system results in the loss of normal HSC function and death of the mice, suggesting that both autophagy genes are necessary for adult HSC maintenance (Liu et al., Atg3 or Atg7 can inactivate autophagy and restore the expression of 6-photofructo-2-kinase/frutose-2,6-biphosphatase 3, which can in turn restart cell proliferation (Flynn et al., Atg5 plays a key role in the maintenance of HSCs, and the reconstitution ability of Atg5-deficient HSCs in bone marrow of chimeric mice is significantly impaired (Jung et al., Atg5, Atg7, and Atg12 can mediate the self-renewal, differentiation, and regeneration of the muscle and hematopoietic system, and the overexpression of Atg7 can rejuvenate aged satellite cells and HSCs and restore their regeneration ability (Garc\u00eda-Prat et al., Different from ESC sand NSCs, in which autophagy activity is required during differentiation, autophagy activity is decreased during the differentiation of MSCs, HSCs, dermal stem cells, and epiblast stem cells (Mortensen et al., Likewise, autophagy is essential for iPSC reprogramming. A distinguishing feature of iPSCs is that the existing number and mass of mitochondria in the somatic cell origins are strikingly diminished during reprogramming. Hence, their metabolic pattern is switched from oxidative phosphorylation to glycolysis and this is considered as an important mechanism in iPSC reprogramming. ATG3-dependent autophagy can act as an executor for mitochondrial clearance during somatic cell reprogramming (Liu et al., Oct4 mRNA (Bradley et al., Previous studies have revealed that genomic and epigenetic stability is essential for stem cell identity. Yet recent years have seen increasing evidence that supported a pivotal role for proteostasis in regulating pluripotency and differentiation of stem cells as well. Under physiological conditions, the proteostasis network is equipped with high versatility in response to distinct stimuli (Balch et al., Furthermore, defects in proteostasis lead to the dysfunction of somatic stem cells, and eventually result in impairment of organismal development and aging, which encourages studies of PQC in search of treatment of aging and aging-related diseases. Pathological conditions, environmental and metabolic stresses, and aging contribute to the production of aberrant proteins in addition to the normal and physiological sources of misfolded proteins (Haigis and Yankner, Study of the regulatory network in proteostasis will also provide new aspects in understanding embryonic development, aging, and pathogenesis. For instance, misfolded proteins have been linked to many neurodegenerative diseases such as Huntington\u2019s disease, Parkinson\u2019s disease, and Alzheimer\u2019s disease, in which aberrant protein aggregates overwhelm the cellular clearance machinery (Bosco et al.,"} +{"text": "Small interfering RNA (siRNA) is a class of nucleic acid-based drugs (NABDs) able to block gene expression by interaction with mRNA before its translation. Small interfering RNAs (siRNAs) therefore present extraordinary potential due to their ability to silence the expression of disease-causing genes. Even if the mechanism of action has been successfully investigated (Nobel Prize in Physiology or Medicine 2006 to Andrew Z. Fire and Craig C. Mello \u201cfor their discovery of RNA interference \u2013 gene silencing by double-stranded RNA\u201d) and siRNA drugs can be candidates to fight, in principle, any diseases. However, the practice of siRNA-based therapies is restricted because of relevant inconveniences. SiRNAs are negatively charged large macromolecules and this entails difficult crossing of cell membranes; they undergo rapid degradation by plasma enzymes and are easily subjected to fast hepatic/renal clearance sequestration. These features seriously hinder siRNAs\u2019 usability in therapeutics. Currently, the scientific community focused on gene therapy research is developing studies to overcome the obstacles related to siRNA\u2019s features.Pharmaceutics titled \u201cDrug Delivery of siRNA Therapeutics\u201d aims to present the state of the art of siRNA delivery, embracing investigation strategies of international research groups with different experiences and skills. The Special Issue will thus be devoted to presenting the current connections between experimental and in silico approaches for therapies based on siRNA delivery, accounting for all the most promising techniques based on liposomes, polymeric and inorganic nanoparticles, aptamers, chemical modification of siRNAs, and so on.This Special Issue of Reviews (five) and research papers (eight) constitute this Special Issue. A representative international scientific community focused on gene-therapies researches is represented by 12 different countries involving 75 scientists with multidisciplinary skills.In the reviews, different research activities cover several disciplines of investigation mainly focused on approaches of siRNA therapies to combat several kinds of cancer in laboratory conditions and the current state of siRNA\u2013lipid delivery systems in clinical trials.In Marson et al., studies Research papers deal with experimental new strategies to design and develop innovative suitable and effective vectors for siRNA delivery such as liposomes, dendrimers, aptamers, polymer\u2013lipid systems, polymeric, co-polymeric and magnetic nanoparticles.3O4 nanoparticles prepared for the delivery of therapeutic siRNAs to contrast oral cancer cells\u2019 growth. Craparo et al., [Stiina Kontturi et al., aimed th et al., studied"} +{"text": "The factors associated with suicidal ideation among adolescents have been extensively characterised, but the mechanisms underlying the complexities of the relationship between experiences of childhood trauma and suicidal ideation have been less studied. This study examined the direct effect of childhood trauma on suicidal ideation on the one hand and whether school bullying victimisation and Internet addiction mediate the association between childhood trauma and suicidal ideation on the other hand.This school-based mental health survey was carried out in Qinghai Province in Northwest China in December 2019. We employed standardised questionnaires to collect sociodemographic and target mental health outcomes. Hierarchical multiple logistic regression and structural equation modelling were performed for the data analyses.p < 0.001). School bullying victimisation and Internet addiction mediated the relationship between childhood trauma and suicidal ideation. Internet addiction played a mediating role between school bullying and suicidal ideation.This study included 5864 university students. The prevalence of lifetime suicidal ideation and Internet addiction were 34.7% and 21.4%, respectively. Overall, 16.4% and 11.4% of participants reported experiences of childhood trauma and school bullying victimisation, respectively. There were direct effects of childhood trauma, school bullying victimisation and Internet addiction on suicidal ideation. The total effect of childhood trauma on suicidal ideation was 0.201 (Childhood trauma had both direct and indirect effects on suicidal ideation; these effects were mediated by school bullying victimisation and Internet addiction in Chinese university students. Elucidating these relationships will therefore be useful in developing and implementing more targeted interventions and strategies to improve the mental well-being of Chinese university students. In each university or college, a stratified (according to the majors) random sampling method was used to select the classes, and cluster sampling was then used in each class.Questionnaires were distributed to participants and collected after completion by our study investigators who were uniformly trained prior to the on-site survey. Students who were fully enrolled in the universities were included. A total of 6500 questionnaires were distributed, and 6200 questionnaires were returned, yielding a response rate of 95.4%. Students from Qinghai University, Qinghai Nationalities University, Qinghai Institute Of Health Sciences and Xining Urban Vocational & Technical College accounted for 30.0%, 27.5%, 26.9% and 15.7% of the sample, respectively. Finally, data from 5864 participants were analysed in this study after cases with \u2a7e 20% missing data were deleted.et al., The Ethics Committee of the Medical College of Qinghai University approved the study protocol. The survey process followed the principles of anonymity and voluntariness, and all university students involved in this survey provided the informed consent. We followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelines to report this study , sex , place of residence prior to entering the university (non-plateau/plateau area), ethnicity (Han/others), self-perceived family economic level , only-child status (no/yes), self-perceived weight , self-perceived health , whether in a dating relationship (no/yes) and relationships with classmates, teachers and family (poor/fair/good), was collected.et al., et al., et al., Suicidal ideation (SI) was assessed using the fourth and fifth items of the Beck Scale for Suicidal Ideation (BSS) , which has satisfactory psychometric properties (Cronbach's ) Young, , 2008, hn) and percentage (%) or the mean and standard deviation (s.d.), as appropriate. Hierarchical multiple logistic regression was carried out to examine the associations between experiences of childhood trauma and suicidal ideation. In step 1, the model was unadjusted by setting suicidal ideation as the dependent variable and childhood trauma as the independent variable. In step 2, adjustments were made for age (years), sex, place of residence prior to entering university, ethnicity, self-perceived family economic level, only-child status, self-perceived weight, self-perceived health status, whether in a dating relationship, relationships with classmates, relationships with teachers or relationships with family. In step 3, school bullying victimisation was added, and Internet addiction was added in the last step. At each step, the R2 change (\u0394R2) was used to indicate the predictive power of each group of predictor(s) when adjustments were made for previous predictor(s). A post hoc analysis was performed by reversing steps 3 and 4. The results were expressed with odds ratios (ORs) and their 95% confidence intervals (CIs).The sociodemographic and clinical characteristics were described with the number .We performed a structural equation model (SEM) to evaluate the hypothesis of the mediating effects of Internet addiction and school bullying victimisation in the relationship between childhood trauma and suicide ideation. Sociodemographic and clinical characteristics that showed statistical significance in step 4 in hierarchical multiple logistic regression were adjusted in the SEM. We used the R lavaan package Rosseel, , and a cs.d.\u00a0=\u00a01.52) were included in this study. Among the participants, 62.4% were (3657) female, 44.8% (2629) were of Han ethnicity and 79.4% (4656) lived in high-altitude areas prior to entering the university. A total of 5864 university students with an average age of 19.9 years and 21.4% , respectively. Overall, 16.4% and 11.4% of university students reported experiences of childhood trauma and school bullying, respectively.R2\u00a0=\u00a00.201, \u0394R2\u00a0=\u00a00.148). School bullying victimisation, tested in step 3, captured an additional 0.8% of variance in suicidal ideation beyond the effects of basic sociodemographic and clinical factors and the experiences of childhood trauma . When Internet addiction was added in the last step, it yielded an additional 0.8% of the variance , showing that experiences of childhood trauma , Internet addiction and school bullying victimisation were positively associated with suicidal ideation. When we reversed the order of entry in the regression model, entering Internet addiction in the third step, school bullying victimisation predicted suicide ideation over and above Internet addiction in the fourth step .\u03b2\u00a0=\u00a00.160, p\u00a0<\u00a00.001), school bullying victimisation and Internet addiction on suicidal ideation. The total effect of childhood trauma on suicidal ideation was 0.201 (p\u00a0<\u00a00.001). The final SEM also revealed the mediating effects of school bullying victimisation and Internet addiction on the association between childhood trauma and suicidal ideation . School bullying victimisation also had an indirect effect on suicidal ideation which was mediated by Internet addiction . Goodness-of-fit indices indicated satisfactory fit of the SEM.This study, based on a sample of 5864 university students from parts of the Chinese Tibetan Plateau (i.e. Qinghai Province), allowed us to identify the following: (1) our mental health problems of interest were common among Chinese university students; (2) childhood trauma, school bullying victimisation and Internet addiction had associations with suicidal ideation among the population of interest; (3) there were indirect effects of childhood trauma on suicidal ideation, which were mediated by school bullying victimisation and Internet addiction; and (4) Internet addiction played a mediating role in the relationship between school bullying victimisation and suicidal ideation.et al., et al., et al., et al., et al., et al., At present, suicidal ideation among adolescents is widely concerned around the world (Mortier et al., et al., et al., et al., et al., After adjustments were made for the control variables, hierarchical regression models indicated that childhood trauma, school bullying victimisation and Internet addiction increased the likelihood of having suicidal ideation. We thus conducted SEM by adjusting for sociodemographic factors, personal health factors and dating status, and we identified the direct effect as well as the indirect effect of childhood trauma on suicidal ideation, the latter of which was mediated by school bullying victimisation. Consistently, the direct effect of childhood trauma on suicidal ideation was demonstrated in another Chinese study including 922 freshmen (Shi et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., Internet addiction also played a mediating role in the relationship between childhood trauma and suicidal ideation. Childhood trauma and its subtypes, such as emotional, physical and sexual abuse, were reported as factors associated with Internet addiction or Internet gaming disorders in different populations (Dalbudak et al., et al., The findings underscore the importance and necessity of implementing suicide intervention strategies and preventing adverse childhood events and invisible or visible on-campus bullying and Internet addiction. Professional levels of psychological counselling and guidance, mental health education courses, campus safety management and other interventions should be considered and practically implemented (Jimerson and Furlong, In conclusion, this study extended the findings of previous literature by elucidating the direct effects of childhood trauma, school bullying victimisation and Internet addiction on suicidal ideation among university students, as well as the mediating roles of school bullying victimisation and Internet addiction in the relationship between childhood trauma and suicidal ideation. Integrally targeted interventions and strategies that can eliminate and alleviate school bullying events, Internet addiction and the influences of childhood trauma should be developed and implemented to reduce the risk of suicidal ideation and improve the comprehensive mental well-being of Chinese university students."} +{"text": "P < 0.01) and a significant dilution effect (P < 0.01). When considering a subsample of horses treated with macrocyclic lactones only, young horses grazed with cattle had 50% fewer strongyle eggs excreted in their faeces than horses grazed in equine-only pastures (P < 0.01). This is the first evidence of the benefits of mixed grazing with cattle as an alternative to control strongyle infection in horses, although this promising alternative remains largely unknown by horse breeders.Strongyle infection is an important issue in horse breeding. It impairs horse health and performance, with young horses being the most sensitive. Strongyle control has long relied on the systematic use of chemical treatments. However, expanding anthelmintic resistance among strongyles calls for alternative options. Mixed grazing is assumed to reduce strongyle load on the pasture as the result of a dilution effect. This has been shown in small ruminants grazing with cattle, but the putative benefits of co-grazing between horses and cattle have not yet been evaluated. Here, we conducted field surveys and face-to-face interviews on 44 farms from two contrasted saddle-horse production areas, Normandy and northern Massif Central, to compare equine strongyle management practices between specialized systems and mixed horse-cattle systems. Our goals were (i) to quantify breeders\u2019 awareness of the putative benefits associated with the co-grazing of horses and cattle, (ii) to establish whether mixed farming was associated with different strongyle management strategies and (iii) to test whether strongyle egg excretion was reduced in horses grazed with beef cattle. Every breeder relied on systematic calendar treatments, and only 8 out of the 23 mixed breeders were aware that co-grazing of horses with cattle could be used as part of their strongyle control strategy. Management practices were similar across both systems in Normandy. In Massif Central, mixed breeders formed a distinct cluster from their specialized counterparts: deworming was less frequent and stocking density was higher in mixed farms, while specialized breeders seemed more willing to integrate herd and plot management into control strategies. Faecal egg counts measured in horses from Massif Central were significantly reduced when horses were grazed with cattle. This was the result of an increased reliance on macrocyclic lactones in mixed farms ( Horse breeders are increasingly challenged by drug resistance when controlling strongyle infection. Although largely unknown by mixed breeders, alternate grazing or co-grazing with horses and cattle is assumed to reduce strongyle load in pastures as the result of a dilution effect. Here, we reveal a decrease in strongyle egg excretion in young saddle horses grazing with beef cattle. This dilution effect is likely to decrease treatment frequency and thus veterinary costs and environmental side-effects of drug metabolites on dung beetle assemblages.et al., et al., et al., et al., Parasitic infection by strongyle nematodes (mostly cyathostomins) is common in grazing horses. High levels of infection affect horse welfare and performance and can eventually lead to death to establish if horse breeders integrate herd and grassland management as part of their strongyle control strategy, (ii) to analyse if horse deworming and grazing management differ between mixed horse-cattle and specialized horse farms, and (iii) to quantify strongyle egg excretion in both types of system to determine putative benefits of co-grazing horses with beef cattle.The study was conducted on 44 farms producing saddle horses with or without beef cattle that were selected according to three additional criteria: at least three mares were kept on a farm, farms were located in lowlands and grasslands represented more than 80% of the agricultural area. Normandy and northern Massif Central were considered as two contrasted case studies: in Normandy, high-level sport horses are bred and grazed on productive grasslands, while in Massif Central, horses are mainly bred for leisure and grazed on less productive grasslands.Face-to-face interviews were carried out on 23 farms from Normandy and 21 farms from northern Massif Central . Interviews (1 to 2.5-h long according to farm size and the complexity of pasture management) focused on farmer beliefs and practices related to grazing, plot cleaning and animal health management.(i)LU) per hectare, intermediate between 0.6 and 1.0 LU/ha and high between 1.0 and 1.4 LU/ha . To obtain three balanced classes for statistical analyses, annual mean stocking rate was considered low between 0.2 and 0.6 livestock unit ((ii)The proportion of total grassland area in both cut and grazed management was partitioned into three categories: lower than 30%, intermediate (between 30% and 45%) or higher than 45% of grassland area. Grazing horses on mixed grasslands can strongly decrease numbers of infective larvae in pastures none when strongyle control only relied on a systematic calendar treatment; yes_livestock when additional practices were based on herd management, for example, reducing stocking rate ); (iii) the person in charge of parasite control and (iv) the anthelmintic class given to young horses and mares.The implemented deworming strategy was also addressed during the interviews. Four variables were covered: (i) the number of anthelmintic treatments administered to young horses , or mares ; (ii) the strategy used for deworming , or systematic plus additional treatments based on faecal egg counts (ERP) indeed varies across anthelmintic classes . A farm was considered of excellent genetic merit when at least one of the mares was registered with a score higher than +9 for one of the genetic indices. A farm was considered of high genetic merit when at least one of the mares was registered with a positive score for one index. All other farms were considered to produce horses for leisure.To test whether FEC was reduced in horses from mixed systems, faeces were sampled in autumn in horses at greater risk of infection (18 to 42 months old). For logistical reasons and according to breeders\u2019 willingness to participate, the sampling took place on Massif Central farms only.n = 23 horses, 6 farms) or alone in a specialized farm . Fresh individual faeces were collected on the ground and kept at 4\u00b0C for less than 48 h; eggs were counted (test sensitivity: 15 eggs/g) at the official local veterinary services (DDCSPP du Puy de D\u00f4me), based on sedimentation and concentration by a flotation technique on farming system (two classes), the number of treatments (three classes), the proportion of time each horse spent on mixed grasslands, grassland stocking rate and time since the end of the ERP. A variable selection procedure was implemented with the stepAIC function of the MASS package that retained the farming system, the time since the end of ERP and stocking rate.To establish whether FEC was significantly associated with the farming system, a linear regression was performed. We built a full model regressing FEC and mixed systems (n = 19). For all statistical analyses, effects were considered significant when the P-value was lower than 0.05.In a second analysis, we considered a subsample of 28 horses treated with macrocyclic lactones only. A two-sample n = 33 farms; moxidectin n = 22 farms). Additional herd and grassland management practices were implemented and considered as part of breeders\u2019 strongyle control strategy in a total of 18 farms, 11 from Massif Central and 7 from Normandy.Face-to-face interviews were also undertaken to evaluate the general beliefs and awareness regarding parasite control management in both mixed and specialized systems. Strongyle control relied on a systematic calendar treatment in all 44 farms surveyed. The most commonly used anthelmintics were fenbendazole (in 36 farms) and macrocyclic lactones and included pasture liming (n = 5), use of rotary slashers (n = 5) or dung-spreading harrows (n = 4), avoiding rapidly returning herds to the same pasture (n = 3) and removing dung from pastures (n = 1). The sum is higher than 14, as some breeders used 2 of these grassland management practices.Plot-cleaning practices were the most common , two-thirds of which were mixed cattle-horse farms and one-third specialized horse farms. Remaining clusters, named MC-mix and MC-spe, were mostly composed of mixed (70%) or specialized (83%) farms from northern Massif Central, respectively. This suggests that practices were different between mixed and specialized systems in Massif Central, whereas practices were much more similar across systems in Normandy.Following the analysis of breeders\u2019 awareness, we attempted to summarize their strategies in order to establish whether mixed farming systems were associated with particular practices. Multiple correspondence analysis applied to management practice data revealed three clusters separated along two axes explaining 13.0% and 10.6% of total variance, respectively. The first cluster, named Nor, included 22 farms, mainly in Normandy but not stocking rate . Farming system was also associated with a significant difference in FEC , with horses from the mixed farming systems excreting half as many eggs than their counterparts from specialized farms.FEC was conducted on 46 horses from mixed and specialized farms to establish whether farming system was impacting horse excretion and what variables underpinned this variation. Our regression model found that time since the end of ERP was significantly driving observed variation in FEC (P < 0.01; Figure A second analysis was performed on a subsample of 28 horses last treated with macrocyclic lactones. It confirmed that young horses grazed with cattle had 50% fewer strongyle eggs excreted in their faeces than horses grazed in equine-only pastures (et al., et al., et al., The purpose of our study was to compare specialized saddle horse farms with mixed horse-cattle farms and to establish whether any difference in parasite control could be found. The limited awareness of the putative beneficial effects associated with the co-grazing strategy was striking, especially because mixed grazing is a key pillar of integrated parasite management in agroecological grassland-based systems (Dumont et al., et al., et al., et al. This lack of awareness regarding mixed grazing was also associated with the widespread use of fenbendazole, despite high prevalence of resistant strongyle populations in France (Traversa et al., et al., A key finding from our surveys is that specialized horse breeders from Massif Central seemed more willing than the mixed breeders to integrate herd and plot management into their control strategies. In these farms, the proportion of grasslands under both cut and grazed management was also the highest, and early cuts under mixed management are known to strongly decrease the number of infective larvae in pastures (Martin-Rosset, et al., et al., et al., et al., et al., et al., While our results are thus likely influenced by regional context, they also provide the first indication that the co-grazing of horses and cattle has beneficial effect for equine strongyle control. Our results are consistent with previous observations in ruminant farming, showing that simultaneous grazing by cattle and sheep (Southcott and Barger, We provide the first evidence of a decrease in the parasite burdens of young saddle horses grazing the same pastures as cattle in mixed farms, compared with horses grazing alone in specialized systems. This opens a promising alternative for controlling horse parasitic infection that remains largely unknown by horse breeders. Association between horses and cattle at pasture is facilitated through the use of the same type of fencing for the two species, with possible limitations due to injury risks which could be solved through alternate grazing. This suggests a diversity of more sustainable agroecological parasite management strategies in horse farms as alternatives to repeated chemical treatment, which raises serious resistance issues."} +{"text": "The current status and ongoing development of 3D electron diffraction and microcrystal electron diffraction in macromolecular crystallography are reviewed. Microcrystal electron diffraction (MicroED) has recently emerged as a promising method for macromolecular structure determination in structural biology. Since the first protein structure was determined in 2013, the method has been evolving rapidly. Several protein structures have been determined and various studies indicate that MicroED is capable of (i) revealing atomic structures with charges, (ii) solving new protein structures by molecular replacement, (iii) visualizing ligand-binding interactions and (iv) determining membrane-protein structures from microcrystals embedded in lipidic mesophases. However, further development and optimization is required to make MicroED experiments more accurate and more accessible to the structural biology community. Here, we provide an overview of the current status of the field, and highlight the ongoing development, to provide an indication of where the field may be going in the coming years. We anticipate that MicroED will become a robust method for macromolecular structure determination, complementing existing methods in structural biology. However, the rate-limiting step in macromolecular crystallography inhibiting structure determination is often crystallization can make use of such small protein microcrystals by outrunning the radiation damage using a diffraction-before-destruction scheme. SFX data are collected using an X-ray free-electron laser (XFEL), which generates brief and highly intense X-ray pulses on a femtosecond timescale, taking single still diffraction snapshots of partial reflections from individual hydrated microcrystals before the sample is destroyed micrometre-sized crystals can be obtained from proteins that are well below 50\u2005kDa was determined in 2013 occur more frequently with increasing crystal thickness and will affect structure determination milling cannot be separated from kinematic scattering. Because of dynamical scattering, weaker reflections on average will appear more intense. As a result, the first-order kinematic approximation that is used in structure refinement is no longer valid. An effective way to minimize dynamical scattering is to carefully select thin crystals for data collection. The crystal thickness ideally should not exceed much more than about 200\u2005nm (Subramanian 4.3.et al., 2018et al., 1999et al., 2015et al., 1996et al., 1999et al., 2015Electrons are charged particles and interact with the electrostatic potential of the crystal (Cowley, 19954.4.ab initio phasing of short peptide fragments (Sawaya et al., 2016et al., 2018et al., 2020de novo phasing of protein structures using electron diffraction has not yet been achieved. The anomalous signal may be too insignificant for experimental phasing using anomalous dispersion (Cowley, 1995To date, all structural models of proteins determined by MicroED have been phased using molecular replacement. Direct methods can be used for electron diffraction data (Dorset & Hauptman, 19764.5.et al., 1999et al., 2005et al., 1984et al., 1990et al., 1992et al., 1996et al., 1998et al., 2003et al., 2013et al., 2020In 2D electron crystallography, reconstructions from TEM images can be combined with high-resolution electron diffraction patterns for structure determination (Unwin & Henderson, 19754.6.et al., 2019et al., 2020et al., 2018et al., 2020et al., 2019et al., 2020\u2212\u2005\u00c5\u22122 was found to have an optimal signal-to-noise ratio (B\u00fccker et al., 2020In macromolecular crystallography, the total electron dose limits the maximum attainable resolution and data quality. Using the continuous-rotation method in MicroED, the critical accumulated dose is spread out over an entire tilt series to sample a substantial wedge in reciprocal space. In contrast, in SFX only single snapshots are recorded from tens of thousands of individual crystals (Schlichting, 2015et al., 2013et al., 2020et al., 2019In SerialED, only a single still diffraction pattern of a randomly orientated crystal is acquired, lacking information about unit-cell parameters owing to the large Ewald sphere. This limitation can be overcome by automation, collecting and integrating data from many thousands of randomly oriented crystals (White 5.de novo phasing through experimental phasing and/or high-resolution imaging may be realized in the years to come. We anticipate that with these advances, MicroED will play an ever more important role in macromolecular crystallography.Here, we illustrate the various achievements made by MicroED in the past years, and discuss the novel opportunities that it may bring for structural biology. Recent successes include determining increasingly challenging structures, resolving ligand-binding interactions and enabling structure determination of membrane proteins from microcrystals embedded in lipidic mesophases. The field is still evolving, and improvements in specimen preparation, optimization of TEM hardware and accurate modelling of the electrostatic potential are needed. Even more so,"} +{"text": "Antimicrobial resistance (AMR) is a threat to public health O'Neil, . AMR occDrug\u2010resistance infections contribute to longer hospital stays, higher medical costs and increased incidence of morbidity , 23,000 deaths and two million illnesses were caused by antibiotic\u2010resistant bacterial infections (Centers for Disease Control, (Zetts et al., . Researchers have found that approximately 154 million primary care visits in the United States result in an antibiotic prescription and approximately 30 per cent, or 47 million, of these prescriptions are not necessary (PEW, One of the main risk factors for AMR in the United States is inappropriate prescription of antimicrobial therapies by primary care providers ary PEW, .(Zetts et al., . However, people in extremely rural communities in the United States tend to wait longer before seeing a provider for treatment for upper respiratory symptoms (Morgan & Hart, Researchers have shown that patients\u2019 demand for antimicrobial therapies varies depending on cultural background and geographic location (Larson et al., 2Interventions at the primary level are needed long term to prevent the spread of AMR (Centers for Disease Control, Appropriate nursing interventions at the primary level are also needed to overcome cultural and geographic barriers (Hicks et al., 3Researchers have demonstrated that the primary level of prevention of AMR has a clear impact and is cost\u2010effective (Ball, Dains, Flynn, Solomon, & Stewart, All authors contributed equally to this article."} +{"text": "Breast cancer is the most frequently diagnosed cancer type and the second leading cause of cancer-related deaths among women worldwide. The causes of breast cancer are not yet fully known, although a number of risk factors have been identified. Tumor biomarker is a term used to describe potential markers of cancer development and progression. As we explore these biomarkers further, we must try to understand the underlying mechanisms of tumor development, as we move along the path to discovery of novel therapies that will increase our ability to offer personalized patient care in the future. With the migration of advanced high throughput technologies, such as Next Generation Sequencing from clinical practice, biomarker research and discovery are poised to explode once again. Translation of novel biomarkers into clinical practice and diagnostic laboratories is coupled with regulatory and administrative requirements that must be met, while collaboration between research institutions, industry, and the private sector drives further advancements in the field of breast cancer biomarker discovery and application.N-Acetyltransferase 1 Knockout Elevates Acetyl Coenzyme A Levels and Reduces Anchorage-Independent Growth in Human Breast Cancer Cell Lines\u201d by Stepp et al., \u201cOverexpression of Kynurenine 3-Monooxygenase Correlates with Cancer Malignancy and Predicts Poor Prognosis in Canine Mammary Gland Tumors\u201d by Chiu et al., \u201cNotch Signaling Activation as a Hallmark for Triple Negative Breast Cancer Subtype\u201d by Giuli et al., and \u201cHuman Mitotic Centromere-Associated Kinesin is Targeted by MicroRNA 485-5p/181c and Prognosticates Poor Survivability of Breast Cancer\u201d by Lu et al. contributed different biomarkers of breast cancer which may help to assess prognosis or predictive indicators. The focus of the third group of articles is on genetic mutations in breast cancer. The paper titled \u201cAssociation of ESR1 Mutations and Visceral Metastasis in Patients with Estrogen Receptor-Positive Advanced Breast Cancer from Brazil\u201d by Reinert et al. observed an association of ESR1 mutations with metastasis. The paper titled \u201cRole of Four ABC Transporter Genes in Pharmacogenetic Susceptibility to Breast Cancer in Jordanian Patients\u201d by AL-Eitan et al. proposed the ABCB1 mutation associated with breast cancer in Jordanian Arabs. The paper titled \u201cA Review of the Hereditary Component of Triple Negative Breast Cancer: High- and Moderate-Penetrance Breast Cancer Genes, Low-Penetrance Loci and the Role of Nontraditional Genetic Elements\u201d by Ellsworth et al. described genes and genetic elements, which have been associated with increased risk of triple negative breast cancer. The paper titled \u201cPregnancy Hypertension and a Commonly Inherited IGF1R Variant (rs2016347) Reduce Breast Cancer Risk by Enhancing Mammary Gland Involution\u201d by Powell et al. observed that statistically significant decrease in terminal duct lobular unit counts signifies increased breast epithelial involution in women with prior hypertension who inherited the TT genotype of IGF1R SNP (rs2016347).The aim of this special issue is to provide new findings regarding molecular pathways and biomarkers that could improve the diagnosis and the prognostic classification of breast cancers, their application in the clinical setting, and their potential utility in personalized patient therapy. The total of submissions is 50. After single-blind peer review by at least two reviewers, 14 papers were finally accepted to be published. The accepted rate is 28%. The average number of authors for each accepted paper is 7. The affiliated institutes of the authors are from Brazil, China, Italy, Iraq, Jordan, Portugal, Spain, Saudi Arabia, Taiwan, the UK, and the USA. These accepted papers can be organized in different groups. The focus of the first group of articles is on prognosis and therapy in breast cancer. The findings of the paper titled \u201cExploring the Role of Breast Density on Cancer Prognosis among Women Attending Population-Based Screening Programmes\u201d by Domingo et al. reveal that increased breast density was associated with worse survival outcomes among women participating in breast cancer screening. The paper titled \u201cAttenuated Total Reflection-Fourier Transform Infrared (ATR-FTIR) Spectroscopy Analysis of Saliva for Breast Cancer Diagnosis\u201d by Ferreira et al. showed ATR-FTIR spectroscopy can be used in saliva samples to discriminate breast cancer patients from benign patients and healthy subjects. The paper titled \u201cDovitinib Triggers Apoptosis and Autophagic Cell Death by Targeting SHP-1/p-STAT3 Signaling in Human Breast Cancers\u201d by Chiu et al. has provided the evidence for anticancer effect of dovitinib to suggest it as a potential target for breast cancer therapy. The focus of the second group of articles is on biomarkers in breast cancer. The paper titled \u201cA Novel Role for Cathepsin S as a Potential Biomarker in Triple Negative Breast Cancer\u201d by Wilkinson et al. investigated the expression profile of Cathepsin S in breast cancer patients. The paper titled \u201cIdentification of Cell-Free Circulating MicroRNAs for the Detection of Early Breast Cancer and Molecular Subtyping\u201d by Souza et al. identified the molecular signature miRNA as noninvasive biomarkers in patients with breast cancer. The paper titled \u201cCD133 in Breast Cancer Cells: More Than a Stem Cell Marker\u201d by Brugnoli et al. reviewed the value of CD133 as prognostic factor of malignant progression of breast cancer. The other four papers titled \u201cIn summary, the research papers cover a wide range of applications including potential diagnostics, predictions, and treatment. Furthermore, this special issue also includes regional genetic mutation, breast density on cancer prognosis, small RNA as a biomarker, surface marker of cancer stem cells, potential marker of cancer metastasis, and spectroscopy predicting the rate of cancer. This may be helpful in assessing prognostic or predictive indicators, as well as developing new therapies and new insights aimed at improving breast cancer.Chia-Jung LiHui-Ming ChenJi-Ching Lai"} +{"text": "Critical comment on the review by Okada et al. on the effect of early versus delayed mobilization because of their definition of early mobilization as mobilization within a week of ICU admission in contrast to current evidence. In their systematic review and meta-analysis, Okada et al. investigate the impact of early versus delayed mobilization for in-hospital mortality and health-related quality of life among critically ill patients, including 11 studies in their meta-analysis [Aware that there is no uniform definition of \u201cearly mobilization\u201d in the ICU yet, to use 1\u2009week as cut-off point seems unreasonable for various reasons. So far, only studies starting early mobilization within 72\u2009h have been able to improve patient outcomes, as summarized in published narrative reviews . SchweicAnother current meta-analysis using different definitions was able to show an effect of early mobilization . FinallyIn conclusion, as timing seems crucial for patient-centered outcomes, early mobilization should be consistently defined as mobilization within 72\u2009h of ICU admission."} +{"text": "M\u0101ori and Pacific Islander people are a priority population originating from Australasia. M\u0101ori and Pacific Islander children exhibit greater risk of obesity and associated morbidities compared to children of other descent, secondary to unique cultural practices and socioeconomic disadvantage. Despite these known risk factors, there is limited synthesised evidence for preventing and treating childhood obesity in this unique population. The objective of this systematic review was to identify and evaluate global prevention or treatment interventions for overweight or obesity that targeted M\u0101ori and Pacific Islander children and adolescents (aged 2\u201317\u2009years).The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines were followed. The databases PubMed, EMBASE, Scopus, Web of Science and CINAHL were searched from inception to August 2018. Study quality and risk of bias was assessed using a modified Downs and Black Quality Checklist for Health Care Intervention Studies. Studies were included if RCT/intervention/case control/ or prevention study designs. The study group was defined under the search term \u2018Oceanic Ancestry Group\u2019.Of the initial 94 articles identified, six were included describing two prevention and three treatment interventions. Interventions were heterogenous in setting, design, length and outcomes. Four interventions were implemented in New Zealand. Most studies were of \u2018fair\u2019 quality. One study recruited an exclusive population of M\u0101ori and Pacific Islander participants. In the five studies that recruited mixed populations, one performed sub-group analysis on M\u0101ori and Pacific Islander participants. No study reported an improvement in anthropometric outcomes post-intervention in complete or sub-group analysis. Improvements in cardiometabolic or psychological secondary outcomes were inconsistent across all studies.There is a lack of evidence to recommend specific intervention characteristics to optimise obesity prevention or treatment outcomes for M\u0101ori and Pacific Islander children. Future research requires greater consideration of cultural values and beliefs, community engagement, exclusive targeting of M\u0101ori and Pacific Islander children and families, and sub-group analyses for mixed-population studies. Incorporating co-design principles during study design and implementation can maximise the cultural specificity of interventions and may contribute to improved health and weight-related outcomes for this at-risk, priority population.CRD42019121790 (26 March 2019).PROSPERO The authors concluded that a co-design approach empowered the target population by enabling active, participatory action throughout each phase of design [In their recently published article, Verbiest et al. describef design .Whilst standalone trials are essential in building an evidence-base for appropriate childhood overweight/obesity prevention and intervention characteristics in M\u0101ori and Pacific Islander peoples, a coordinated whole-of-government, policy-level and healthcare systems response is paramount in contributing to a reduction in prevalence. In response to the growing prevalence of obesity and congruent with the World Health Organization (WHO) Report of the Commission on Ending Childhood Obesity , New ZeaA localised example of a culturally-tailored, integrated health system approach to improving M\u0101ori and Pacific Islander child and adolescent health is the \u2018Good Start Program\u2019, a statewide, schools-based, nutrition and physical activity initiative embedded within the Queensland Health system, Australia. A qualitative evaluation of its implementation concluded that building a system-wide workforce of M\u0101ori and Pacific Islander Health Workers was a significant contributor to enabling high-level community engagement, cultural specificity of interventions, community member satisfaction and trust with the Good Start Program, and ultimately positively shifting M\u0101ori and Pacific Islander community members\u2019 attitudes to and knowledge of health .There were a number of observed strengths of included studies. Project Energize, the New Zealand schools-based prevention intervention described by Rush et al. , 26 in tn\u2009=\u200918) and the participants of SWITCH trial of Maddison et al. [n\u2009=\u00a0116), which may have limited the generalisability of findings to the larger population of Hawaii. Most interventions were described as stand-alone, and there was little evidence of attempted integration within a broader, health system to maximise reach, impact, and sustainability.The primary limitation of the current literature is the lack of exclusive recruitment of M\u0101ori and Pacific Islander participants and limited sub-group analysis in mixed participant demographics. Descriptions of intervention intensity and intervention fidelity could have been strengthened; this may have contributed to the modesty of reported results, especially anthropometric outcomes. The intervention of Chansavang et al. was impln et al. were offn et al. . Anothern et al. , 25\u201327 an et al. and 5-yen et al. analysedThe primary strength of this review is its novelty, being the first known to synthesise global evidence of prevention and treatment interventions for the unique, at-risk population of M\u0101ori and Pacific Islander children and adolescents. Findings of this review are potentially transferable to priority populations from various developed countries, including the USA and Canada, where young Alaska Native and American Indian children, and First Nations and Inuit peoples, respectively, exhibit high obesity prevalence rates . As an en\u2009=\u00a06) to prevent or treat obesity in M\u0101ori and Pacific Islander children and adolescents generated minimal impact in improving anthropometric indicators of weight or improving cardiometabolic or psychological secondary outcomes. There is a lack of evidence to recommend specific intervention characteristics that will optimise overweight/obesity prevention or treatment interventions in M\u0101ori and Pacific Islander children and adolescents. These results are possibly secondary to a lack of intervention intensity and specificity to M\u0101ori and Pacific Islander peoples. The authors propose the following recommendations for future research:Cultural-tailoring of interventions, preferably utilising a co-design approach, with adequate methodological reporting;Implementation of interventions that exclusively target M\u0101ori and Pacific Islander children and adolescents; fostering community engagement, leadership and ownership at every stage of the proposed intervention i.e. from conception to evaluation;Performing intervention sub-group analysis on M\u0101ori and Pacific Islander participants in mixed-population studies; andIntegrating and evaluating, where possible, long-term, mixed-methods interventions within an existing healthcare system to maximise reach and sustainability for policy- and population-level impactOverall, previously reported studies (Consideration of these recommendations in future research will optimise interventions to tackle childhood overweight/obesity in the unique priority population of M\u0101ori and Pacific Islanders, who exhibit a significantly higher prevalence of childhood overweight/obesity in Australia and New Zealand, as well as demonstrate substantial socioeconomic and health disadvantage that inherently increases population risk for long-term overweight/obesity and its comorbidities.Additional file 1. PRISMA checklist.docxAdditional file 2. Search strategy in EMBASE according to the PICO format.docxAdditional file 3. Individual quality assessment and risk of bias results for included studies.docx"} +{"text": "Cannabis Sativa plant, interacts with the endocannabinoid system by inhibiting fatty acid amide hydrolase (FAAH) activity (the rate-limiting enzyme for anandamide hydrolysis), allosterically modulating CB1 and CB2 receptors, and activating components of the \u201cextended endocannabinoid system.\u201d Congruently, CBD has shown prominent pro-neurogenic effects, and, unlike \u03949-tetrahydrocannabinol, it has the advantage of being devoid of psychotomimetic effects. Here, we first review pre-clinical studies supporting the facilitating effects of CBD on adult hippocampal neurogenesis and available data disclosing cannabinoid mechanisms by which CBD can induce neural proliferation and differentiation. We then review the respective implications for its neuroprotective, anxiolytic, anti-depressant, and anti-reward actions. In conclusion, accumulating evidence reveals that, in rodents, adult neurogenesis is key to understand the behavioral manifestation of symptomatology related to different mental disorders. Hence, understanding how CBD promotes adult neurogenesis in rodents could shed light upon translational therapeutic strategies aimed to ameliorate psychiatric symptomatology dependent on hippocampal function in humans.During the last decades, researchers have investigated the functional relevance of adult hippocampal neurogenesis in normal brain function as well as in the pathogenesis of diverse psychiatric conditions. Although the underlying mechanisms of newborn neuron differentiation and circuit integration have yet to be fully elucidated, considerable evidence suggests that the endocannabinoid system plays a pivotal role throughout the processes of adult neurogenesis. Thus, synthetic, and natural cannabinoid compounds targeting the endocannabinoid system have been utilized to modulate the proliferation and survival of neural progenitor cells and immature neurons. Cannabidiol (CBD), a constituent of the During the last half-century, considerable progress has been made to understand, prevent, and treat such conditions. However, treatment options are still far from optimal in terms of efficacy and specificity, and there remain important untreatable maladaptive phenotypes and treatment-resistant patients. To solve this issue, basic and applied research has tried to identify new altered neuropsychological mechanisms suitable to promote new therapeutic strategies , CBD is devoid of psychotomimetic and rewarding effects , transient potential vanilloid 1 (TRPV1), G-protein 55 (GPR55) and peroxisome proliferator-activated gamma (PPAR\u03b3) receptors, as well as the antagonism of adenosine reuptake of the dentate gyrus . Moreover, the authors reported an interesting opposition to the effects of THC on this measure. Months later, Demirakca et al. (Considering that the endocannabinoid (eCB) system exerts important functions in the regulation of neuronal generation and survival and BrdU/NeuN. Due to the prolonged presence of both markers in different stages of the neurogenesis process (for a review see Kempermann et al., in vitro, Campos et al. (in vivo. Inverted U-shaped dose-response curves usually suggest the participation of multiple pharmacological mechanisms. In this way, it has already been described that CBD also exerts a similar anxiolytic dose-response curve (for a review see Jurkus et al., 1A and TRPV1 mechanisms (Campos and Guimar\u00e3es, 1A receptors. However, CBD-induced proliferation in HiB5 hippocampal progenitor cells was not blocked by a 5-HT1A antagonist (Campos et al., CBD pro-neurogenesis also shows great consistency across doses. Literature findings report increases in neuronal proliferation and differentiation after CBD doses ranging from 3 to 30 mg/kg, usually after prolonged treatments (\u226510 days; s et al. describes et al. showed t\u2212/\u2212 mice (Aguado et al., The eCB system stands out as a key regulator of newborn neuron generation, survival, maturation, and functional integration in the adult hippocampus. Neural progenitor cells, and their descendants, express a functional eCB system and are subject to the effects of endocannabinoid signaling (Prenderville et al., in vitro and in vivo evidence has suggested such interplay. The first evidence was given by Wolf et al. (\u2212/\u2212 mice. The seminal work of Campos et al. (in vitro. CB1 and CB2 antagonists prevented the pro-neurogenic effect of CBD in hippocampal HiB5 progenitor cells. Furthermore, CB1 and CB2 receptor agonists, as well as eCB degradation inhibitors mimicked the pro-neurogenic effects of CBD. Interestingly, CBD effects were abrogated when the FAAH was inhibited. Combined, these results imply that the pro-neurogenic effects of CBD depending on the increase of AEA concentration. Crucially, CBD is an inhibitor of the FAAH and is well known to increase AEA concentration (Bisogno et al., in vivo. After a CBD treatment in chronically stressed mice, neuronal differentiation, and late survival were found to be increased in CBD-treated mice (Foga\u00e7a et al., Given the mechanistic interactions between CBD and eCB system, a plausible hypothesis originated stating that CBD increases adult neurogenesis by modulating the eCB system. Accordingly, f et al. . In theis et al. further A considerable number of studies have reported the pro-neurogenic effects of CBD, and some among them have even related these with an eCB mechanism of action. But, can the pro-neurogenic effects of CBD account for some of its therapeutic applications? Answering this question requires specialized experimental strategies designed to rule out CBD pro-neurogenesis, leaving intact its other pharmacological mechanisms and so, fewer experiments have been conducted. Nonetheless, a handful of studies have addressed this question, presenting evidence for a potential implication in the protection against neurodegenerative diseases (Esposito et al., Neurodegenerative and ischemic conditions are among the circumstances in which hippocampal function can manifest greater impairments (Shah et al., A significant amount of animal and human data has emerged relating the neuro-modulatory role of adult hippocampal neurogenesis, its interactions with broader hippocampal circuits, and its implications on altered behaviors in different neuropsychiatric disorders (Beckervordersandforth and Rolando, ML and OV were responsible for the study concept and design. Both authors drafted the manuscript and approved the final version for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Cell death (CD) is a fundamental biological process that is indispensable in all living organisms Ameisen, . Phloem + outward rectifying conductances (KORC) was recorded in response to various CD-inducing microbe-derived molecules, such as harpins , cell shrinkage (Yekkour et al., + efflux channel were also shown to have fewer autophagosomes compared to the wild-type plant upon ROS-induced CD (Demidchik, + efflux through KORC is supposed to result in dramatic K+ loss from plant cells and promotes CD (Demidchik et al., + loss was effectively shown to be involved in tobacco cell death induced by palmitoleic acid and ceramide (Peters and Chin, Nicotiana benthamiana plants undergoing oxidative stress and transiently expressing CED-9, an anti-apoptotic gene from the bcl-2 family (Craig, + efflux and maintaining intracellular K+ homeostasis (Shabala et al., An increase in Ky Craig, , are capIn response to ozone or DON, plant cells rapidly activate anion currents, followed by a delayed activation of KORC (Tran et al., In plant the vacuolar collapse seems a key step in cell shrinkage but to our knowledge the role of vacuolar conductances is poorly documented. Therefore, further studies are needed to decipher the role of vacuolar channels in this process.+ channels and is prevented by application of blockers of Cl\u2212 or K+ channels (Okada and Maeno, As a whole, these data are reminiscent of those described in numerous studies in animal cells and show that plant CD could involve specific modifications of ion transporter activities that could be significant and crucial in the successful propagation of CD . ActivatFB, DR, DT, and PL conceived and wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Anorexia nervosa and bulimia nervosa are two severe eating disorders associated with high premature mortality, suicidal risk and serious medical complications. Transition between anorexia nervosa and bulimia nervosa over the illness course and familial co-aggregation of the two eating disorders imply aetiological overlap. However, genetic and environmental liabilities to the overlap are poorly understood. Quantitative genetic research using clinical diagnosis is needed.N = 782 938) of randomly selected full-sisters and maternal half-sisters born in Sweden between 1970 and 2005. Structural equation modelling was applied to quantify heritability of clinically diagnosed anorexia nervosa and bulimia nervosa and the contributions of genetic and environmental effects on their overlap.We acquired a clinical diagnosis of anorexia nervosa and bulimia nervosa in a large population-based sample (36\u201350%)] and 41% (31\u201352%), respectively, in the study population, with the remaining variance explained by variance in unique environmental effects. We found statistically significant genetic and unique environmental correlations [0.55 (0.43\u20130.66)] between the two clinically diagnosed eating disorders; and their overlap was about equally explained by genetic and unique environmental effects [co-heritability 47% (35\u201358%)].Our study supports shared mechanisms for anorexia nervosa and bulimia nervosa and extends the literature from self-reported behavioural measures to clinical diagnosis. The findings encourage future molecular genetic research on both eating disorders and emphasize clinical vigilance for symptom fluctuation between them. Anorexia nervosa is characterized by significantly low body weight and intense fear of weight gain of bulimia nervosa compared to the same types of relatives of individuals without anorexia nervosa, and In the current work, we used clinical diagnoses of anorexia nervosa and bulimia nervosa in a large Swedish female cohort containing full-sisters and maternal half-sisters . The application of diagnostic information considerably expands the clinical significance of the results. Using non-twin siblings significantly increased sample size and improved precision in estimating the underlying morbidity liabilities. We quantified the contribution of genetic and environmental effects on anorexia nervosa and bulimia nervosa and their overlap and estimated the genetic and environmental correlations between the two eating disorders.The current study was approved by the Regional Ethics Review Board in Stockholm, Sweden.et al., et al., et al., et al., et al., We acquired information from several Swedish national registers linked by the unique individual identification number. The Swedish Population Register provided birth year and month, death date and migration information from the treatment quality registers. We were unable to distinguish between restricting and binge-eating and purging anorexia nervosa from the ICD diagnosis in the NPR. Bulimia nervosa diagnosis was identified with ICD-10 codes F50.2 or F50.3 from the NPR, or meeting DSM-IV-TR criteria for bulimia nervosa or sub-threshold bulimia nervosa in EDNOS from the treatment quality registers. Bulimia nervosa was not an independent eating disorder category in the Swedish version of ICD-9 . Therefos.d.)\u00a0=\u00a09.3]. We then randomly selected one pair of full-sisters per mother in the remaining mothers after excluding mothers of the selected maternal half-sisters. We excluded twin pairs from full-sister pairs, resulting in 334\u00a0433 pairs of full-sisters for analysis . No individual was included in more than one pair. The selected full-sisters and maternal half-sisters comprised the study population, where 6104 (0.91%) individuals among the full-sisters and 938 (0.82%) among the maternal half-sisters had been diagnosed with anorexia nervosa; 3142 (0.47%) among the full-sisters and 579 (0.51%) among the maternal half-sisters had been diagnosed with bulimia nervosa; 679 (0.10%) among the full-sisters and 122 (0.11%) among the half-sisters had been diagnosed with both disorders and maternal half-sisters (sisters born to the same mother but different fathers) who were born in Sweden between 1970 and 2005. We excluded individuals who emigrated or died before age 6, adopted individuals and individuals whose biological parents were not identifiable from the registers. We did not have access to information on race and ethnicity; however, being born in Sweden might suggest that our study population was comprised primarily of individuals of Scandinavian/Nordic ancestry. We then randomly selected one pair of maternal half-sisters per mother in full-sisters and 0.03 (s.e.\u00a0=\u00a00.06) in maternal half-sisters; the cross-sister-within-trait correlation for bulimia nervosa was estimated as 0.20 (s.e.\u00a0=\u00a00.03) in full-sisters and 0.13 (s.e.\u00a0=\u00a00.07) in maternal half-sisters . The AE model was selected for interpretation because it had the lowest AIC , illustrating the diagnostic transition between the two eating disorders within individuals regardless of the type of siblings. The cross-sister-cross-trait correlation further characterizes the nature of the association between the disorders across the individuals in a pair. A higher cross-sister-cross-trait correlation was observed in full-sister pairs than in maternal half-sisters , suggesting genetic effects on the overlap between the two eating disorders ] of the phenotypic correlation of the two disorders was explained by their genetic covariance and the remaining 54% [95% CI (42\u201365%)] was explained by their environmental covariance . We founet al., s.e. 1%) in the most updated genome-wide association study (GWAS) ] and environmental correlations [0.55 (0.43\u20130.66)] suggest an aetiological overlap between anorexia nervosa and bulimia nervosa and underscore the importance of vigilance for transitions between the two eating disorders during treatment. Using large population data, we significantly improved the precision of the estimates. We reported heritability for clinically diagnosed anorexia nervosa as 43% (36\u201350%) and bulimia nervosa as 41% (31\u201352%), which was slightly lower than, but within the confidence interval of, the heritability found in the twin study based on self-reported questionnaire data [57% (0\u201381%) for anorexia nervosa and 62% (8\u201370%) for bulimia nervosa] ] and between anorexia nervosa and major depression (around 0.5 in multiple studies) (Wade et al., et al., et al., et al., In addition, we reported significant unique environmental effects, both disorder-specific and common to the two eating disorders. Previous research suggested various potential environmental risk factors for eating disorders, such as dieting and events that trigger mood change and thin-ideal internalization (Chua et al., et al., et al., Using extended familial relatedness drastically increased the available sample size of the models in our study, and the use of clinical diagnostic information increased the clinical significance of the results. Our results are consistent with the prior twin study on the overlap between anorexia nervosa and bulimia nervosa based on self-reported measures of eating behaviours (Bulik et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., Limitations of the current study would arise if there are violations of basic assumptions of the models, such as the assumption of equal shared environmental factors across different types of siblings [equal environment assumption, EEA (Plomin To our knowledge, this is the first quantitative genetic study on clinically diagnosed anorexia nervosa and bulimia nervosa and their overlap. The moderate and statistically significant genetic and environmental correlations between the two eating disorders might suggest aetiological overlaps and emphasize clinical vigilance of the transition between the two disorders."} +{"text": "In modern biotechnological and medical research, RNA-guided nucleases (RGNs) continue to be highly effective in targeted modification of genomes and the manipulation of gene expression (Sander and Joung CRISPR-Cas proteins are non-specific endonucleases that bind a protospacer adjacent motif (PAM) located in the proximity of the genomic target nuclease concentration and features and (ii) target site accessibility, functionality, and uniqueness. The occurrence of complementary sites within the genome, which form highly stable duplexes to guide oligonucleotide, is one of the important determinants of off-target activity. Cas9 and Cas12a (Cpf1) activity can be modulated by chromatin states to varying degrees. Off-targeting is expected to be context dependent because chromatin states and DNA accessibility is tissue/cell- or condition-dependent RISC complex formation, several key measurements including association kinetics, equilibrium binding energies, and single turnover cleavage rates allows revealing of important rules for binding and cleavage of targets. Becker and co-authors (Becker et al. A comparison of Cas9- and Cas12a-binding experiments showed variable binding kinetics responses to target sequence mutations, which explained why Cas12a enables the selection of DNA sequences more precisely than Cas9 (Strohkendl et al. For Cas nucleases, targeting rules were empirically established by different groups (Klein et al. Several models and approaches based upon thermodynamics and kinetics have the potential to explain off-targeting patterns for CRISPR-Cas and AGO2, as well as for other systems. To go beyond binding energetics, Klein and co-authors (Klein et al. Bisaria and co-authors (Bisaria et al. Attenuating DNA cleavage kinetics can be successfully applied for enhancing gene editing specificity and reducing off-targeting not only to CRISPR systems but also to different engineered nucleases (Becker et al."} +{"text": "Ichihashi et al reported that 43% of patients had antipsychotic polypharmacy. Number of antipsychotics used in patients with schizophrenia in Japan was the greatest among Asian countries. However, the antipsychotic polypharmacy rate in Japan decreased gradually. Recent systematic review, meta\u2010analysis and meta\u2010regression analysis demonstrated that antipsychotic augmentation was superior to monotherapy. However, several cohort studies have suggested a significant association between antipsychotic daily dose and mortality. In addition, most pharmacokinetic interactions with antipsychotics occur at the metabolic level and usually involve changes in the activity of the major drug\u2010metabolizing enzymes involved in their biotransformation. Thus, avoidance of unnecessary polypharmacy, knowledge of the interaction profiles of individual agents, and careful individualization of dosage based on close evaluation of clinical response and possibly plasma drug concentrations are essential to prevent and minimize potentially adverse drug interactions in patients receiving antipsychotics. Ichihashi et alAntipsychotics are not always effective in schizophrenia patients. There are still patients with treatment\u2010resistant schizophrenia (TRS), which has led to a growing trend of resorting to atypical antipsychotic polypharmacy due to fewer side effects.Number of antipsychotics used and psychotropic drug loading in patients with schizophrenia in Japan was the greatest among Asian countries.A recent systematic review, meta\u2010analysis and meta\u2010regression analysis demonstrated that antipsychotic augmentation was superior to monotherapy regarding total symptom reduction, although the superiority was only apparent in open\u2010label and low\u2010quality trials and not in double\u2010blind and high\u2010quality trials.On the other hand, several guidelines consistently recommend monotherapy of antipsychotic agents. For example, the Maudsley Prescribing Guidelines in Psychiatry states that we should concern ourselves with the addition of an antipsychotic to another antipsychotic solely to increase efficacy. From a theoretical point of view, since all antipsychotics block D2 receptors , there is limited rationale for additional antipsychotics. In addition, many clinical trials studying the side effects of polypharmacy have associated polypharmacy with an increased incidence of side effects, although most have been uncontrolled studies or observational studies.Several cohort studies have suggested a significant association between antipsychotic daily dose and mortality.Mortality associated with antipsychotic polypharmacy has not yet been concluded. Previous reports have suggested that significant mortality risks are 2.46 Thus, although evident disadvantage of antipsychotic polypharmacy has not yet been concluded, avoidance of unnecessary polypharmacy, knowledge of the interaction profiles of individual agents, and careful individualization of dosage based on close evaluation of clinical response and possibly plasma drug concentrations are essential to prevent and minimize potentially adverse drug interactions in patients receiving antipsychotics.Norio Yasui\u2010Furukori has been a speaker for Otsuka Pharmaceutical Co., Ltd., Mochida Pharmaceutical Co., Ltd., Dainippon\u2010Sumitomo Pharmaceutical Co., and MSD Co. Kazutaka Shimoda has received research support from Novartis Pharma KK, Dainippon Sumitomo Pharma Co., Astellas Pharma Inc, Meiji Seika Pharma Co., Ltd., Eisai Co., Ltd., Pfizer Inc, Otsuka Pharmaceutical Co., Ltd., Daiichi Sankyo Co., and Takeda Pharmaceutical Co., Ltd., and honoraria from Eisai Co., Ltd., Mitsubishi Tanabe Pharma Corporation, Takeda Pharmaceutical Co., Ltd., Meiji Seika Pharma Co., Ltd., Janssen Pharmaceutical KK, Shionogi & Co., Ltd., Dainippon Sumitomo Pharma Co., Daiichi Sankyo Co., and Pfizer Inc The funders did not have any role in data collection or in the study design, analysis, decision to publish, or preparation of the manuscript. The remaining authors declare that they have no competing interests to report.NYF designed the study and wrote the initial draft of the manuscript. KS contributed to supervision of the draft."} +{"text": "On page 288, \u2018recent reports have shown that CAII can also reduce nitrite (NO2\u2212) to nitric oxide (NO), and thus, may play a role in vasodilation and regulation of blood pressure to nitric oxide (NO), and thus, may play a role in vasodilation and regulation of blood pressure On pages 288\u2013299, \u2018However, when dialyzed with ethylenediaminetetraacetic acid (EDTA), the enzyme retained its carbonic anhydrase activity yet lost its nitrite reductase activity And on page 291, \u2018... indicating that a metal cofactor within the bovine blood was needed for the CAII-dependent nitrite reductase activity (Andring et al., 2018et al., 2018et al. (2018et al., 2018( al. 2018 and shou"} +{"text": "A discussion of recent discoveries in the etiolation/de-etiolation field, focusing on post-transcriptional processes and ultrastructural changes, along with comments on usage of the term \u2018etiolation\u2019, and of common etiolation/de-etiolation systems. The state of etiolation is generally defined by the presence of non-green plastids (etioplasts) in plant tissues that would normally contain chloroplasts. In the commonly used dark-grown seedling system, etiolation is coupled with a type of growth called skotomorphogenesis. Upon illumination, de-etiolation occurs, marked by the transition from etioplast to chloroplast, and, at the seedling level, a switch to photomorphogenic growth. Etiolation and de-etiolation systems are therefore important for understanding both the acquisition of photosynthetic capacity during chloroplast biogenesis and plant responses to light\u2014the most relevant signal in the life and growth of the organism. In this review, we discuss recent discoveries (within the past 2\u20133 years) in the field of etiolation and de-etiolation, with a particular focus on post-transcriptional processes and ultrastructural changes. We further discuss ambiguities in definitions of the term \u2018etiolation\u2019, and benefits and biases of common etiolation/de-etiolation systems. Finally, we raise several open questions and future research possibilities. Etiolation involves prolonged growth in the absence of light that results in the development of etioplasts in tissue that would have chloroplasts if subjected to light. Etioplasts do not contain chlorophyll or stacked thylakoid membranes, but rather have a paracrystalline lipid\u2013pigment\u2013protein structure known as the prolamellar body (PLB). The PLB consists largely of the plastid lipids monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), and an association of the chlorophyll precursor protochlorophyllide (Pchlide), the light-dependent protochlorophyllide oxidoreductase (LPOR) that is responsible for its conversion, and the cofactor NADPH germination and growth of seedlings in complete darkness, the term \u2018etiolated\u2019 is commonly defined additionally by the presence of a skotomorphogenic phenotype of elongated hypocotyls, shortened roots, and small, closed cotyledons . In thesThe majority of the data discussed in this Expert View refer to work undertaken in such seedling-based etiolation/de-etiolation systems. The various limitation of these systems and possible alternative or complementary systems are also discussed (in the section \u2018New systems required and new lessons learned\u2019).\u00e9tiolier (i.e. straw), is still used as a descriptor for a range of pale or yellowing phenotypes. These include nitrogen-deficient rice albostrians mutant are 20\u201324 nt-long molecules that regulate gene expression via RNA-dependent DNA methylation, translation inhibition, or mRNA cleavage (reviewed in Zea mays) to understand the establishment of photosynthesis in C3 versus C4 species. These studies, which defined several specific sRNA roles, such as the repression of photomorphogenic growth by miR396 via members of the Growth Regulating Factors family is an evolutionarily conserved protein kinase that acts as a central hub to control cellular- and organism-level development : RNA\u2013protein complexes that are conserved in eukaryotes and regulate gene expression by degradation or translational arrest of mRNA (reviewed in Genomes uncoupled (gun) mutants defective in plastid-to-nucleus retrograde signaling (gun2\u20136) have defects in genes for enzymes involved in tetrapyrrole biosynthesis. More recently, a role for the enigmatic GUN1 in regulating protein import via the cytosolic heat shock protein 90 (HSP90) chaperone was clarified using a de-etiolation system is produced early during greening as a by-product of tetrapyrrole biosynthesis and lipid to protein ratios change with greening . The rolin vitro system recently used to show the requirement for MGDG and charged lipids in regulating LPOR complex formation and activity . These issues argue for alternative systems, such as the ues e.g. .Phaseolus coccineus) and pea (Pisum sativum) (Landoltia punctate), a flat-leafed aquatic monocot requires limited growth time and space yet provides sufficient material compared with other experimental systems such as the shoot apical meristem, and (ii) is highly customizable by use of different timing and lighting regimes and introduction of different substances to the growth medium (sativum) . PLBs hasativum) . The pro monocot , and in Cucumis sativus; Persea americana; Phaseolus vulgaris) (Nicotiana tabacum) thylakoid membranes, early studies in cucumber (ulgaris) , varioustabacum) . These fThe benefits of the standard seedling etiolation and/or de-etiolation systems means that they have been used often in recent years to study diverse topics including gravitropism changes during greening in Arabidopsis , acts downstream of the COP1\u2013auxin cascade during de-etiolation in the dark . Soluble1O2; 1O2 retrograde signaling mediates de-etiolation via the EXECUTER1 pathway (The early assembly of the PSII oxygen evolving complex results in the (damaging) formation of singlet oxygen (Three recent studies investigated the effect of decreased MGDG ("} +{"text": "Recent years have seen the idea of a close association between nutrition and the modulation of cancer development/progression reinforced. In fact, an increasing number of experimental and epidemiological evidence has been produced, supporting the concept that many different bioactive components of food may be implicated in either the promotion of or the protection against carcinogenesis. At the cellular level, such compounds can have an impact on different but sometimes intertwined processes, such as growth and differentiation, DNA repair, programmed cell death, and oxidative stress. In addition, compelling evidence is starting to build up of the existence of primary epigenetic targets of dietary compounds, such as oncogenic/oncosuppressor miRNAs or DNA-modifying enzymes, which in turn impair gene expression and function. This editorial aims to summarize the themes of the 31 papers published in the Special Issue \u201cRole of Natural Bioactive Compounds in the Rise and Fall of Cancers\u201d presenting the latest findings on the intracellular pathways and mechanisms affected by selected natural molecules influencing the fine-tuning of cancer phenotype.Plant polyphenols have been among the most studied natural compounds by the contributors to this issue.In the original article group, Polonio-Alcal\u00e0 et al. showed tOther natural phenolic compounds whose activity is discussed in the original articles of this issue are:\u2013Gallic acid , widely distributed in natural plants, fruits, and green tea, whose tumor-suppressive effect via the p53-mediated downregulation of the transmembrane protein PD-L1 was demonstrated by Kang et al. [\u2013Olea europaea L., whose presence in enriched extracts from olive leaves was proven to reduce the glycolytic rate of a wide range of solid and liquid tumor cells via the downregulation of the three key effectors of the glycolytic pathway, GLUT-1, PKM2 and MCT4, likely resulting in a decreased glucose entrance and biomass production [Oleuropein, the main bioactive phenolic component of oduction ;\u2013Oleacein , the main secoiridoid contained in extra virgin olive oil, able to elicit significant anti-tumor activity by promoting cell cycle arrest and apoptosis in multiple myeloma cells due to its histone deacetylase inhibitory properties ;\u2013Dadzein , present in soybeans, whose 4-sulphate metabolite produced by gut microbiota was found to exert an anti-estrogenic effect on ER\u03b1-positive breast cancer cells via the downregulation of the anti-apoptotic neuroglobin protein thus rendering cells more prone to the paclitaxel treatment [\u2013Gigantol, a bibenzyl compound from orchid species, whose ability to destabilize tumor integrity via the suppression of the PI3K/AKT/mTOR and JAK/STAT pathways was demonstrated by Losuwannarak et al. in non-s\u2013Lonchocarpus sericeus, proven to be a powerful inhibitor of the Wnt/\u03b2-catenin pathway able to selectively suppress the migration and proliferation of a panel of colorectal cancer cell lines in vitro and in a preclinical colorectal cancer mouse model [Lonchocarpin, a chalcone isolated from se model ;\u2013Isorhamnetin, , a flavonol aglycone found in some medicinal plants, able to exert an anti-proliferative effect on human bladder cancer cells via the induction of cell cycle arrest during the G2/M phase and apoptosis, accompanied by the activation of the AMPK signaling pathway and ROS overproduction ;\u2013Piper genus plants, whose ability to inhibit XIAP protein, involved in the regulation of caspase-dependent/independent cell death pathways, was reported by Mu\u00f1oz et al. [Erioquinol, eriopodol A and gibbilimbol B, derived from z et al. in breas\u2013Calocedrus formosana Florin leaves extract, proven to interfere with cell cycle and microtubule dynamics in lung adenocarcinoma cells, also inhibiting tumor growth in a xenograft mouse model [Vatein, isolated from se model .Magnolia-derived polyphenol honokiol based upon its ability to impair cell cycle progression, inhibit epithelial\u2013mesenchymal transition, and suppress cell motility, invasion, metastasis and angiogenesis. Zhou et al. [In the Review section, Perrone et al. discusseu et al. summarizu et al. reviewedu et al. focused \u2013Malva pseudolavatera leaves, which showed a promising selective anti-proliferative and pro-apoptotic effect on acute myeloid leukemia cell lines, determining PARP cleavage, cytochrome-c release, Bax/Bcl-2 ratio increase and ROS overproduction [The methanolic extract of oduction ;\u2013Eicosapentaenoic acid, an \u03c9-3 polyunsaturated fatty acid, which played a protective role, both alone and in combination with angiotensin-converting enzyme inhibitors, in attenuating adipocyte-induced proinflammatory cytokine expression and the migration of breast cancer cells in an in vitro model of obesity-induced breast cancer ;\u2013Fucoidan, a sulphated polysaccharide derived from brown seaweed, whose combination with gemcitabine determined an enhanced pro-apoptotic and cell cycle-inhibitory activity on selected uterine carcinosarcoma and stromal sarcoma cell lines ;\u2013Nicotin, whose mechanisms underlying the promotion of melanoma cell proliferation and migration mediated through \u03b19-nAChR-initiated carcinogenic signaling and PD-L1 expression were reported by Nguyen et al. ;\u2013Streptomyces sp. MUM256, isolated from mangrove soil in Malaysia, and the cyclic dipeptides contained whose ability to induce cell cycle arrest and apoptosis was demonstrated by Tan et al. [The ethyl acetate fraction of the crude extract of n et al. in colon\u2013Luffariella variabilis, which preferentially inhibits the proliferation of oral cancer cells inducing apoptosis and DNA damages via oxidative stresses, such as intracellular ROS and MitoSOX/MitoMP [Manoalide, an antibiotic sesterterpenoid isolated from the marine sponge X/MitoMP ;\u2013\u03bb-carrageenan, a family of linear sulfated polysaccharides, proven to enhance the effect of radiotherapy by suppressing the survival and invasiveness of different cancer cell lines in vitro and in vivo through the Rac GTPase-activating protein 1 pathway ;\u2013Ethanol, which was found to trigger a pro-survival autophagic response following the induction of oxidative and endoplasmic reticulum stress in colon cancer cells, and the activation of Nrf2 and HO-1 also leading to the acquisition of a more aggressive phenotype ;\u2013Colchicum autumnale, whose enhanced anticancer effects and reduced cytotoxicity on colon cancer cells when delivered in the nanoformulated form was reported by AbouAitah et al. [Colchicine, an alkaloid present in the medicinal plant h et al. .The other articles and reviews addressed further cancer-related issues relevant to types of compounds of a different nature, specifically:In the Review section, Del Corn\u00f2 et al. discusseThe number of manuscripts published in this Special Issue indicates an active interest in research about the molecular/pharmacological mechanisms used by natural products exerting anti-tumoral effects which deserve further and deeper studies. I wish to thank all the contributors of this issue for sharing with us their experimental or reviewed data which will surely attract readers\u2019 attention and encourage the publication of other high-quality papers in this field."} +{"text": "This Special Issue containing seminal contributions from international experts highlights the current understanding of Rho GTPases in cancer, with an emphasis on recognizing their central importance as critical targets for cancer therapy and for chemosensitization of current therapeutic strategies. A comprehensive review by Jung et al. gives anNew regulatory mechanisms for Rho GTPases are presented in a review by Humphries et al. , who desNovel directions for Rho GTPase guanine nucleotide exchange factors (GEFs) are also highlighted in a number of peer-reviewed research articles. Using in silico analyses and in vitro experimental studies in keratinocytes, Lorenzo-Martin et al. implicatTwo articles by Dr. Hendrick Ungefroren\u2019s group contribute to the role of the constitutively active Rac1B splice variant in transforming growth factor (TGF\u03b2) signaling ,9. The aFinally, Dyberg et al. implicate the downstream effector of Rho, Rho kinase (ROCK), in EMT and medulloblastoma growth by demonstrating that ROCK mRNA is preferentially expressed in metastatic tumors . They usIn summary, herein, you will find timely articles on the ubiquitous role of Rho GTPases in cancer and how understanding their mechanisms of action can lead to the design and development of targeted therapeutic strategies."} +{"text": "The worldwide increase of human life expectancy (\u201clifespan\u201d) along with the concomitant rapid population aging represents the major social phenomena of the last century. This has substantially impacted our societies by virtue of the huge economic implications and public health challenges definition of reference values; (2) identification of reference standards specific for the disease of interest ; (3) proper inclusion and contextualization within the diagnostic process; and (4) statistical processes supporting the whole framework. They concluded that various methodological issues remain to be addressed in order to perform an adequate and complete clinical validation of candidate CSF biomarkers for AD.Using cerebrospinal fluid (CSF) biomarkers for AD as a paradigmatic example, D'Anca et al. explored the current understanding of role of exosomes in physiological aging and age-related neurodegenerative diseases such as AD, Parkinson's Disease (PD) and frontotemporal dementia. Insights from the study of exosomes and their genetic cargo have elevated their role beyond mere waste disposal function to fundamental mediators of intercellular communication, akin to the proverbial double-edge sword serving as Trojan horses of neurodegeneration vis-\u00e0-vis providing neuroprotection from neurodegeneration. Exosomes can be detected in many biological fluids, enhancing their appeal as potential sources of biomarkers suitable for use in clinical practice.Five papers in this Research Topic shed precious insights into emerging biomarkers from readily available biological samples. In their comprehensive review, Tay et al. prospectively studied serum levels of Dickkopf-1 (Dkk-1) in older adults with mild cognitive impairment (MCI) and mild-to-moderate AD. The findings revealed that Dkk-1 increased significantly from baseline amongst progressors, while non-progressors exhibited decremental Dkk-1 at 1 year, alluding to the putative role in MCI and AD progression of dysfunctional Wnt signaling through Dkk-1 antagonism. Sun et al. demonstrated that cofilin 2 expression was significantly increased in AD patients and different AD models , with good discriminatory ability that distinguish AD from healthy subjects and in differential diagnosis of AD from vascular dementia. By studying 391 cognitively normal subjects aged 23\u201391 years from Asia, USA and Europe, Lue et al. characterized the relationship between age and three plasma AD core biomarkers , provided the normal ranges of A\u00df species and t-Tau in plasma, and explicated the development of a dynamic relationship between these biomarkers from middle to old age. Lastly, Falconi et al. investigated the transcriptional regulation of the Adenosine A2A receptors (A2ARs) gene in human peripheral blood mononuclear cells obtained from PD patients and in the striatum of the 6-hydroxydopamine-induced PD mouse model. They reported an increase in A2AR mRNA expression and protein levels in both human cells and mice that is accompanied by histone acetylation and DNA methylation, paving the way for therapeutic interventions in future.Similarly, Li et al. shed light on the neural mechanisms of working memory deficits in MCI. By combining static and temporal dynamic examination of amplitude of low-frequency fluctuations (ALFF) from functional magnetic resonance imaging, they reported background network changes especially in the parietal and temporal lobes during working memory states in MCI. Similarly, Pur et al. enhanced understanding by demonstrating the moderating effect of cortical thickness on blood oxygen level-dependent (BOLD) signal variability age-related changes and highlight the importance of considering these effects when evaluating BOLDSD alternations across the lifespan. Kobayashi et al. examined whether dementia with Lewy Bodies (DLB) follows an AD-type trajectory whereby amyloid-\u00df deposition begins considerably before onset of dementia. They observed a low amyloid load in REM sleep behavioral disorder, a prodromal symptom of DLB, suggesting that this phenomenon does not always precede the onset of cognitive decline in DLB. Lastly, through the use of MRI voxel studies to examine neural substrates, Wu, Geng et al. implicated the left-precentral cortex and left inferior frontal gyrus areas that accounted for olfactory impairment in a cohort of AD and MCI patients in the Chinese Han population.Another prominent theme in this Research Topic was the contribution of advanced neuroimaging biomarkers. For instance, in terms of explicating mechanisms that underpin underlying pathophysiological processes, Wang, Hatashita et al. examined [18F]-flutemetamol (FMM), a fluorinated derivative of the prototypic [11C]-Pittsburgh Compound B (PIB), and demonstrated that [18F]-FMM PET imaging can track longitudinal changes in A\u03b2 deposition across the AD spectrum, similarly to [11C]-PIB PET. Notably, they reported that the increase in A\u03b2 deposition is not constant across the AD spectrum but faster in the predementia stage . Using dynamic [18F] florbetapir PET, Verfaillie et al. investigated the possibility that self-perceived cognitive decline (SCD), generally associated with a three- to six-fold increased risk of AD, may reflect an early symptom of A\u03b2 related pathology. They concluded that A\u03b2 load was associated with SCD related worries rather than subjective cognitive functioning per se .Other papers in this Research Topic illuminated the knowledge gap in our understanding about the role of misfolded protein accumulation in neurodegenerative diseases. The extracellular accumulation of amyloid beta (A\u03b2) peptide is the paradigmatic example, with recent neuroimaging capabilities in tracking A\u03b2 deposition a fascinating way to distinguish if the observed A\u03b2 accumulation is characteristic of the aging process or conversely, an etio-pathogenetic mechanism of AD . Furthermore, the study by Wu, Xu et al. provides proof-of-concept evidence that a topological examination of the structural connectivity networks with different parcellation schemes can provide important complementary AD-related information and thus contribute to a more accurate and earlier diagnosis of AD. Another promising technique involved amide proton transfer (APT) imaging as an imaging modality to detect tissue protein. In fact, Wang, Chen et al. adopting a modified APT method in animal models, demonstrated that APT imaging could potentially provide molecular biomarkers for non-invasive diagnosis of AD. Using direct non-invasive brain network electrophysiological imaging, Zifman et al. established that this new technique can be used both to monitor brain aging and for early detection of abnormal changes leading to neurodegeneration.Other papers highlight advances in neuroimaging techniques that pave the way for fresh perspectives in early diagnosis of AD. Utilizing sophisticated measurements of cortical thickness with 3.0-Tesla MRI in a large population of cognitively normal individuals and patients with AD continuum, Lorio et al. described how the combination of these imaging modalities can be used as biomarkers of PD severity and prognosis which can be potentially useful for clinical trials. Likewise, Pelizzari et al. proposed the concomitant use of DTI to detect brain tissue microstructural alterations, together with arterial spin labeling (ASL) MRI to analyse abnormal cerebral perfusion patterns in PD. The study showed that DTI is a more sensitive technique than ASL to detect alterations in the basal ganglia in the early phase of PD, suggesting that a relationship between microstructural integrity and perfusion changes in the caudate may be present .Bioimaging techniques are also useful for the differential diagnosis of PD. Dopamine-transporter SPECT (DAT-SPECT), diffusion tensor imaging (DTI), and structural magnetic resonance imaging (sMRI) provide unique information about neurotransmitters and microstructural properties in PD. The longitudinal study of Chen et al. developed an innovative predictive model to evaluate white matter burden in hypertensive patients using metrics in 24-h ambulatory BP monitoring (systolic blood pressure [SBP] and daytime SBP standard deviation) and carotid ultrasound IMT. Leveraging upon the diagnostic potential of diabetic retinopathy (DR) afforded by its time-course which precedes the occurrence of T2DM cognitive impairment, Lu et al. verified the correlation between DR from fundus examination and T2DM cognitive impairment. They further established using magnetic resonance spectroscopy (1H-MRS) that this may be attributed to bilateral changes in hippocampal brain metabolism, alluding to the potential role of 1H-MRS for early diagnosis of T2DM cognitive impairment .Papers in this Research Topic also addressed the issue of age-associated co-morbidities, particularly cardiovascular diseases, and related risk factors such as Type II diabetes mellitus (T2DM), which often confound the measurement and interpretation of neurodegenerative biomarkers. Exploring the association between white matter hyperintensities with higher Intima-media thickness (IMT) and blood pressure variability, Bandera et al. suggested an omics approach to accelerate the discovery of reliable biomarkers of HIV-associated neurocognitive disorders in the current era of virological suppression with modern anti-retroviral therapy. Similarly, persistent inflammation has been implicated as a cardinal mechanism of neurocognitive impairment and neurodegenerative features in Multiple Sclerosis (MS). Jakimovski et al. reported the novel finding that almost half the elderly subjects with MS are impaired on tests of cognitive processing speed or verbal fluency. Since the deficit in verbal fluency is not a typical hallmark of the cognitive profile associated with MS, it may represent a unique trait of old persons with MS neurocognitive profile .A major pathogenetic influence of co-morbidities or their treatment that have emerged in recent years is that of concomitant inflammatory processes that may alter disease manifestation and confound biomarker interpretation. An example is the cognitive decline observed in some HIV subjects during ART-mediated viral suppression, a phenomena which has been ascribed to cytokine-mediated inflammation. Barha et al. underlined the role of biological sex as a potential moderator in the relationship between physical activity and brain health in older adults. They suggested that different neurobiological mechanisms as well as physiological adaptations to physical activities could be responsible for the differences in trajectories of decline observed in men and women . This highlights the importance of taking into consideration the potent moderating influence of sex differences for both cognitive and neural outcomes in future therapeutic and rehabilitative interventions involving physical activity in older adults.Lastly, there is growing interest in the role of physical activity and exercise as \u201cmedicine\u201d that can be prescribed for brain health. In their comprehensive review, As guest editors for this Research Topic, we commend this collection of 23 articles to our readers as a timely addition and important contribution to the field. We are confident that this will spur further discourse and open avenues for further research into the rapidly evolving and fascinating area of biomarkers to disentangle physiological from pathological brain aging.FG, WL, and BA conceived the manuscript. FG and WL drafted the paper. BA critically appraised and edited the manuscript. All authors read and approved the final version of the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Background: Growing evidence points out that a disturbance of gut microbiota may also disturb the gut\u2013brain communication. However, it is not clear to what extent the alteration of microbiota composition can modulate brain function, affecting host behaviors. Here, we investigated the effects of gut microbiota depletion on emotional behaviors.Methods: Mice in the experimental group were orally administered ceftriaxone sodium solution for 11 weeks. The open-field test and tail-suspension test were employed for the neurobehavioral assessment of the mice. Fecal samples were collected for 16s rDNA sequencing. The serum levels of cytokines and corticosterone were quantified using enzyme-linked immunosorbent assays. The immunohistochemistry method was used for the detection of brain-derived neurotrophic factor (BDNF) and c-Fos protein.Results: The gut microbiota for antibiotic-treated mice showed lower richness and diversity compared with normal controls. This effect was accompanied by increased anxiety-like, depression-like, and aggressive behaviors. We found these changes to be possibly associated with a dysregulation of the immune system, abnormal activity of the hypothalamic-pituitary-adrenal axis, and an alteration of neurochemistry.Conclusions: The findings demonstrate the indispensable role of microbiota in the gut\u2013brain communication and suggest that the absence of conventional gut microbiota could affect the nervous system, influencing brain function. Gut microbiota, known as a reservoir of bacteria, not only plays an essential role in host digestion and energy metabolism but shapes host immunity and given either sterile saline solution or ceftriaxone sodium solution intragastrically once a day for 11 consecutive weeks were maintained (ten mice per cage) under a specific-pathogen-free (SPF) condition at 22\u201326\u00b0C, 40\u201360% humidity, and 12-h light-dark cycle. The mice were given 1 week to acclimate. All mice were fed with adequate food and clean water. At the end of adaptive phase, all mice were randomly divided into two groups : open-field test (OFT) \u2192 tail-suspension test (TST). The OFT, which involves a low stress level, preceded the TST, which involves a high stress level was used to extract fecal DNA. Then, the extraction was eluted using elution buffer and stored at \u221280\u00b0C until PCR amplification detection by LC-Bio . The V3-V4 region of the prokaryotic 16S rRNA gene was amplified with primers 338F (5\u2032-ACTCCTACGGGAGGCAGCAG-3\u2032) and 806R (5\u2032-GGACTACHVGGGTWTCTAAT-3\u2032) axis in rodents. Rising corticosterone levels suggest increases in HPA axis activity in the hippocampus and c-Fos in the amygdala. The whole process consisted of brain collection, sectioning, and immunolabeling. The details referred to previous literatures . The staining extent was divided into five grades according to the percentage of positive cells in the region: negative, 0\u201325, 26\u201350, 51\u201375, and 76\u2013100% regions of the hippocampus . Similarly, semi-quantification of c-Fos was performed by calculating the intensity score and fraction score in the CeC, CeL, and CeM regions of the amygdala. The immunohistochemical analysis was performed blind.P-values less than 0.05 were considered statistically significant.Data were expressed as the mean \u00b1 standard deviation or median (IQR) and analyzed by one-way ANOVA or Wilcoxon rank sum test in SPSS 22.0 software . p < 0.05) .OFT is usually performed to assess locomotor activity and exploratory behavior after gavage for 11 weeks in the AB group . In addiA slight decrease of BDNF in the CA1, CA3, and DG regions of the hippocampus was observed in the AB group compared to the CT group , Table 3The gut, a vulnerable but vital organ, is affected by different factors easily. Antibiotics are one of the common causes leading to gut disturbance, especially given the broad spectrum of antibiotics. Consistently, little is known about adverse effects of these antibiotics on health except for drug resistance. But, recently, medications with antibiotic have been reported to enhance the risk of allergies, inflammatory bowel diseases, obesity, and even mental diseases (Harris and Baffy, Ceftriaxone administration caused significant weight loss in the study. This is consistent with the previous finding that the weight gain of mice was delayed significantly following the ceftriaxone treatment (Miao et al., Proteobacteria became a dominant phylum, and the abundance of Bacteroidetes, Firmicutes, Actinobacteria, and Deferribacteres decreased. This result is supported by studies that ceftriaxone could characteristically decrease the alpha-diversity of the fecal microbiota accompanied with more Proteobacteria and less Bacteroidetes (Cheng et al., Proteobacteria is perceived as a diagnostic characteristic since it is closely related to colon epithelial oxygenation as well as the disruption of the gut anaerobic environment (Zhu et al., Firmicutes has become a controversial strain as some studies identified an increase in Firmicutes after ceftriaxone treatment, but others have demonstrated a declined Firmicutes in the ceftriaxone group (Cheng et al., Firmicutes (Huang et al., Firmicutes led to a reduction in short-chain fatty acids, which are an important physiological basis for low-level inflammation during depression (Huang et al., Bacteroidetes, as an important microbe for short-chain fatty acids, almost disappeared from the feces of the mice during exposure to ceftriaxone (Miao et al., Porphyromonadaceae, Escherichia, and Parabacteroides dominated the gut microbiota of the AB group mice, while Lactobacillus, Clostridiales, Acetatifactor, Bacteroidetes, Barnesiella, Helicobacter, Prevotella, Bacteroidales, and Alistipes were lowered. In line with this, some researchers have proposed that decreased Barnesiella after ceftriaxone gavage is a common and sensitive gut microbiota of the BALB/c mice and can be used as an indicator for assessing the balance of the gut microbiota (Zhao et al., Bacteroidetes is closely associated with digestion and interacts with the host's immune system, affecting the growth of other bacteria (Karlsson et al., Escherichia prevalence after oral antibiotic treatment has been reported for vancomycin and imipenem (Stokes, Escherichia could be beneficial or harmful as Escherichia is both a commensal and pathogenic inhabitant of a host's gastrointestinal tract. But most of the time, Escherichia is considered a potential pro-inflammatory bacteria (Liu et al., Porphyromonadaceae associates with mental deficits and cognitive disorders as well as anxiety-like behaviors in mice (Scott et al., Lactobacillus is known as a protective species against long-lasting metabolic disturbances and prevents gut dysbiosis, but was suppressed by ceftriaxone (Robles-Vera et al., Parabacteroide relates to the etiology of depression (Cheung et al., Ceftriaxone could result in a significant gut microbiota dysbiosis by killing most of the normal flora and providing the living space for other potential pathogens (Cheng et al., Stokes, , amoxici Stokes, , and met Stokes, . It is dBifidobacterium and Lactobacillus (Logan and Katzman, Lactobacillus fermentum strain NS9 administration not only normalized the composition of gut microbiota but reduced the anxiety-like behavior induced by ampicillin (Wang et al., Bifidobacterium infantis has also been identified in the rat separation model of depression (Desbonnet et al., Alterations in gut microbiota were accompanied by behavioral changes in the mice, including anxiety-like, depression-like, and aggressive behaviors. These behavioral changes cannot necessarily be a result of the direct toxic effect of ceftriaxone on the brain, since ceftriaxone is a non-absorbable antibiotic and usually given by injection. Previous studies have demonstrated the complex interaction between gut microbiota and the CNS; this is what is known as the MGB axis (Wang and Wang, Schistosoma mansoni showed a reduction in behaviors such as exploration and grooming (Sulaiman et al., Immune dysregulation was demonstrated by high levels of serum cytokines. This is supported by the evidence that inflammatory factors associate with a profile of behavioral changes (Capuron and Miller, Bifidobacterium species, have been demonstrated to be efficient in restoring HPA axis function (Moya-P\u00e9rez et al., Elevated corticosterone, one marker of HPA axis activation, was observed in the mice of the AB group (Borrow et al., Bifidobaterium adolescentis shows a promising anxiolytic and antidepressant property as it up-regulated BDNF expression by restoring the balance of gut microbiota (Guo Y. et al., Campylobacter jejuni (Goehler et al., The BDNF level showed a decreasing trend in the hippocampus of the AB group. According to the previous study, BDNF can maintain and promote development, differentiation and regeneration of neurons as well as affect learning and memory (Bercik et al., In general, we found that mice exposed to 11 weeks of ceftriaxone sodium treatment had a lower diversity and abundance of gut microbiota and showed more behavioral changes as compared to mice that were given normal saline. Dysregulation of the nerve-endocrine-immunological network may be a potential mechanism underlying abnormal behaviors induced by impaired gut microbiota. The study revealed the unknown side effects of antibiotics to a certain extent. Follow-up studies rebalancing the gut dysbacteriosis are required to further confirm the relationship between gut microbiota and brain function.PRJNA592623.The datasets generated for this study can be found in the NCBI, SRA accession: The animal study was reviewed and approved by Animal Care Advisory Committee of Sichuan University.ZZ and HY put forward the hypothesis, and BW, CT, LZ, and ML guided and supervised the experimental investigation and practices. LM, HW, and JL were responsible for data collection and analysis. YY provided pathologic diagnosis. ZZ was the main executer and involved in the entire research process, from proposal, planning and execution to implementation and composition. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Alzheimer's disease (AD) is the most common form of dementia. Its prevalence will significantly increase in the coming decades, whereas no efficient treatment is currently available. AD has two main neuropathological lesions: amyloid plaques and neurofibrillary tangles (NFTs). Amyloid plaques are composed of amyloid \u00df (A\u00df) peptides cleaved from the Amyloid Precursor Protein (APP) . Although genetic factors are estimated to represent 60% of the risk to develop LOAD, they remained largely unknown for a long time except for APOE accounts for <5% of AD cases and is well characterized by mutations in three genes (The endolysosomal proteins listed above play critical roles at various steps of dynamin-dependent endocytosis and further steps . EndocytDynamin-dependent endocytosis is regulated by the interplay of the interactions between PRD of dynamin and SH3 domain-containing proteins (Ferguson and De Camilli, in vitro (Talaga et al., post-mortem brain tissues of AD and other tauopathies (Nishikawa et al., In vitro phosphorylation of tau by GSK3\u00df, a kinase abnormally activated in AD brains (Leroy et al., Given that endocytic proteins such as SHIP1, SHIP2, and SYNJ1 are PI-5-phosphatases involved in PI metabolism (Ramos et al., Many of the endocytic machinery proteins implicated in AD risk possess SH2, SH3 domains, and/or PRD and are involved in actin dynamics as well as in regulation of PIs. Because the endocytic machinery needs fine-tuned regulation of PIs and endocytic protein-protein interactions, the endocytic pathway must be highly vulnerable. Dysregulation of even one of these endocytic proteins could lead to significant endocytic abnormalities. Hyperphosphorylation of tau may further accelerate endocytic dysregulation. Genetic risk factors and tau pathology might well have profound impacts on synaptic functions, endolysosomal/autophagic pathways, and APP processing via dysfunction of endocytosis, actin network, and PI metabolism . EndolysKA constructed the main concept of the manuscript by exchanging opinions with the other authors. All authors participated in writing the manuscript. All authors contributed to manuscript revision, read and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Ion channels are expressed throughout nervous system development. The type and diversity of conductances and gating mechanisms vary at different developmental stages and with the progressive maturational status of neural cells. The variety of ion channels allows for distinct signaling mechanisms in developing neural cells that in turn regulate the needed cellular processes taking place during each developmental period. These include neural cell proliferation and neuronal differentiation, which are crucial for developmental events ranging from the earliest steps of morphogenesis of the neural tube through the establishment of neuronal circuits. Here, we compile studies assessing the ontogeny of ionic currents in the developing nervous system. We then review work demonstrating a role for ion channels in neural tube formation, to underscore the necessity of the signaling downstream ion channels even at the earliest stages of neural development. We discuss the function of ion channels in neural cell proliferation and neuronal differentiation and conclude with how the regulation of all these morphogenetic and cellular processes by electrical activity enables the appropriate development of the nervous system and the establishment of functional circuits adapted to respond to a changing environment. Nervous system development is a complex process in which neural cells undergo a transformation from neural stem cells to highly specialized neurons and glia to form different brain structures and spinal cord and establish circuitry that facilitates simple to advanced neural functions.Many cues have been recognized as drivers of the first steps in nervous system development. Morphogenetic proteins and growth factors regulate the number and type of neural cells as well as the morphogenesis of the neural tube. These include Sonic hedgehog (Shh), Bone Morphogenetic Proteins (BMPs), Wnts and trophic factors such as EGF, IGF, NGF, BDNF to mention few. Most of these factors are not exclusive to the organogenesis of the brain and spinal cord but instead support growth and act as morphogens of every tissue and organ in the developing embryo. Mechanistically, these developmental cues trigger a transcriptional combinatorial code that accompanies cells through their cell cycle progression and exit, differentiation and maturation and IP3 receptor-operated Ca2+ release from stores in ventral domains of the spinal cord and CaNeural activity is a feature of the maturing and mature nervous system, which during development facilitates the refinement of neural connections. The expression of ion channels in mature neurons is intrinsic to neuronal function. Diverse ion conductances are indispensable for neurotransmission, thus, the roles of different ion channels in synaptic function and neuronal excitability have been extensively studied. In contrast, the neurophysiological features of neural cells before synapse formation and before neuronal differentiation has not been as strong a focus of attention as those of mature neurons. Nevertheless, studies have argued that other forms of neural activity are present in neural cells throughout nervous system development Spitzer, .This activity may not be structured under a classical chemical synapse, but it is certainly dependent on ion channels gated by diverse mechanisms. Expression of voltage- and neurotransmitter-gated ion channels as well as transient receptor potential (TRP) channels, among others, is apparent in neural stem cells as early as neural plate stages (Abdul-Wajid et al., Here, we review studies addressing the pattern of expression of ion channels during development in neural cells before and during synapse formation. We compile investigations demonstrating a role for ion channels in neural cell proliferation, neural tube formation, and neuronal differentiation and discuss the consequences of having neural activity functioning in the early stages of nervous system development.Xenopus laevis spinal cord neurons are first recorded 8 h after exiting the cell cycle, when, these events manifest spontaneously, are Ca2+-dependent and long in duration (Spitzer and Lamborghini, + channel contributes to shorten the action potential duration and shifts it from Ca2+- to Na+-mediated (Barish, 2+, Na+ and K+ voltage-gated channel subunits for which their expression is developmentally regulated have been investigated (Harris, + current as development advances (Gurantz et al., 2+ currents are dominant at the earliest embryonic stage of chick limb motor neuron development, while later T currents decrease and N and L Ca2+ currents increase (McCobb et al., + and K+ currents in these motor neurons during embryonic development result in changes in action potential amplitude and duration, respectively, which in turn, modify the instructions of motor neurons to the muscle (McCobb et al., The excitable nature of neurons and muscle cells is dependent on the specific expression of ion channels and their subcellular localization in these cells. Seminal studies have investigated the developmental appearance of excitability in neurons and muscle cells through the progressive and differential expression of ion channels. Embryonic spinal cord neurons have served as a powerful model for the study of the ontogeny of excitability during development. Action potentials in Barish, . The ide Harris, . In partXenopus spinal cord neurons and their activation contributes to the spontaneous Ca2+ spike activity in these cells before and during synapse formation (Root et al., In addition to voltage-gated ion channels and their fundamental role in contributing to the excitability of developing neurons and muscle cells, other types of ion channels are also present at the early stages of embryonic development. These channels are gated by diverse mechanisms, including notably, neurotransmitter-operated channels. GABA and glutamate receptors are expressed in immature Xenopus embryo (Spencer et al., Our recently published study shows that the cold-sensitive channel TRPM8 is expressed in the developing Xenopus neuroectodermal cells exhibit Ca2+ transients (Abdul-Wajid et al., 2+ channels (Abdul-Wajid et al., Expression of these diverse types of channels appears to start at even earlier stages of neural development, before the neural tube is formed. At neural plate stages, Transcripts and proteins for glutamate (Root et al., The process of neural tube formation consists of transforming a flat layer of cells known as the neural plate into a tubular structure from which the brain and spinal cord originate. The cellular events encompassing neural tube morphogenesis, all of which are tightly regulated, include neural plate cell proliferation, apicobasal polarization, apical constriction, elongation, cell intercalation, migration and differentiation (Wallingford et al., Xenopus laevis embryos. We demonstrated that during neural tube formation neural plate cells exhibit Ca2+ transients partly mediated by NMDA receptors. Inhibiting glutamate signaling, through pharmacological inhibition of NMDA receptors or downregulation of the GluN1 subunit, induces NTDs (2+ dynamics in the neural plate to a similar extent as inhibition of NMDA receptors. Moreover, preincubating embryos with NMDA partially rescues both the number of Ca2+ transients in the folding neural plate and the valproic acid-induced NTD phenotype (Sequerra et al., Our recently published study (Sequerra et al., ces NTDs . Valproices NTDs , also in2B interferes with mouse neural tube closure and morphogenesis (Choi et al., Many other neurotransmitter signaling systems have been identified as participants in the process of neural tube formation. Inhibiting serotonin receptors 5HTA and GABAB receptor ligands to pregnant rats alters embryos\u2019 neural tube formation leading to NTDs (Briner, A receptors. Another chloride conductance-mediated neurotransmitter system that affects neural tube formation is glycine. Blocking glycinergic signaling by administering strychnine to pregnant rats results in embryos with anencephaly, anterior NTD (Garc\u00eda-Alcocer et al., Administering GABA Briner, . The fac Briner, , which mSimilarly, enhancing or inhibiting NO levels by enhancing BMP signaling or inhibiting NO synthase in chicken embryos induces NTDs (Traister et al., Noradrenaline promotes neuronal differentiation by upregulating expression of N-tubulin in noggin-expressing neural plate cells, which is prevented by inhibiting \u03b1-adrenergic receptors (Messenger et al., 2+ signaling is a plausible common denominator for the action of diverse neurotransmitter systems on neural tube formation. Sources of Ca2+ can be intracellular from stores or extracellular through Ca2+ influx. Early studies in cultured rat embryos during cephalic neural fold elevation and neural tube closure assessed the role of Ca2+ influx and found that reducing it causes opening of the elevated neural folds (Smedley and Stanisstreet, 2+ transients in the neural plate, suggesting that additional mechanisms other than these ionotropic glutamate receptors contribute to neural plate cell Ca2+ dynamics during folding (Sequerra et al., Xenopus laevis embryos consisting of a Na+-dependent inward current which is stronger in the mid-lateral neural plate and decreases near the midline of the neural groove (Robinson and Stump, 2+ currents with characteristic kinetic parameters to transduce the signaling required for neural tube morphogenesis. Indeed, T-type Ca2+ channels are involved in neural tube formation and loss of them impairs neural fold closure in Xenopus laevis and Ciona embryos. Ca2+ influx through these channels is necessary for the regulation of cell adhesion during neural tube formation by ephrin signaling (Abdul-Wajid et al., 2+ transients seem to regulate apical actin dynamics in superficial neural plate cells of Xenopus laevis embryos, which, in turn, regulates neural plate cell apical constriction necessary for neural tube formation (Christodoulou and Skourides, Ca2+ signaling and downstream effectors recruited for neural plate folding and neural tube formation. The elucidation of these mechanisms will contribute to the delineation of safe therapies for the treatment of epilepsy during pregnancy.Further investigation is needed to identify the molecular mechanisms eliciting CaThe generation of the appropriate number of neurons and glial cells is essential not only during nervous system development but also in the adult brain where neurogenesis occurs in the hippocampus and olfactory bulb, and the peripheral nervous system during regeneration and remodeling. Thus, this is a highly regulated process because the dysregulated proliferation of neural stem cells can lead from tumors to neurodevelopmental disorders and birth defects like NTDs.2+ channels have all been implicated in regulating neural plate cell proliferation.The expression of ion channels during the early stages of development supports a role for them in the relevant cellular processes pertinent to these stages including neural cell proliferation. Different types of ion channels including voltage-gated, neurotransmitter-gated, TRPC and store-operated CaXenopus embryos, and, likely as a consequence, impairs lateromedial migration leading to NTDs. An increase in neural plate cell proliferation is also apparent by incubating embryos with the AED valproic acid (Sequerra et al., The action of glutamate-mediated regulation of neural plate cell proliferation is apparent as early as neural plate stages. We found that blocking NMDA receptor signaling increases neural plate cell proliferation in 2+ channels (LoTurco et al., In vitro studies on embryonic rat hippocampal neural progenitors reveal that stimulating glutamate signaling enhances neural cell proliferation or neuronal differentiation depending on the temporal pattern of NMDA receptor activation (Joo et al., Regulation of neural plate cell proliferation by glutamate-gated ion channels is present at later developmental stages during corticogenesis in the rodent brain. The effects of glutamate signaling on neural progenitor proliferation vary depending on the nervous system structure, the developmental stage and the type of model system and manipulation used in specific studies. Glutamate decreases the number of proliferating embryonic rat cortical cells through an AMPA/Kainate receptor-dependent mechanism that leads to depolarization of neural progenitors in the ventricular zone and activation of voltage-gated CaA receptor. In vivo studies in the neonatal mouse subventricular zone (Young et al., in vitro studies in cerebellar granule cells (Fiszman et al., A receptor-induced depolarization enhances neural progenitor cell proliferation. In contrast, activation of this receptor in the ventricular zone of the rat embryonic neocortex (LoTurco et al., 2+ channels and inhibits cell cycle progression. Even in the adult mouse brain, the GABAA receptor seems crucial in regulating neurogenesis by controlling hippocampal neural progenitors. GABAA receptor \u03b32 subunit-mediated signaling is responsible for controlling the experience-dependent transition between quiescence vs. proliferative states of the mouse hippocampal neural stem cell niche (Song et al., A receptor favors the quiescence of adult neural stem cells (Song et al., A receptor signaling (Dumitru et al., Another important neurotransmitter-gated ion channel that participates in neural progenitor cell proliferation is the GABAvia membrane depolarization. In particular, voltage-gated Ca2+ channels are pivotal for the regulation of neural progenitor proliferation and mouse embryonic cortical layer formation (Malmersj\u00f6 et al., 2+ channels enable Ca2+ transients that propagate through a network of neural progenitors connected by gap junctions. Inhibiting this electrotonic transmission decreases neural progenitor proliferation suggesting that correlated Ca2+ transients are necessary for the regulation of neural progenitor proliferation (Malmersj\u00f6 et al., 2+ channels in embryonic neural cells increases neural cell proliferation that has detrimental consequences to brain morphogenesis (Pai et al., 2+ channel-mediated signaling has, depends on developmental timing and location.Other ion channels directly involved in regulating neural cell proliferation include the voltage-gated ion channels. Moreover, some actions of the neurotransmitter receptor-gated ion channels seem to converge into the recruitment of voltage-gated ion channels 2+ channels, voltage-gated K+ and Na+ channels are involved in regulating neural cell proliferation. The voltage-gated Na+ channel \u03b21 subunit is necessary for inhibiting granule cell precursor proliferation during the first week of mouse postnatal dentate gyrus development (Brackenbury et al., + channels was discovered recently in Drosophila larvae, where the single pore-forming voltage-gated Na+ channel \u03b1 subunit, paralytic, regulates neural progenitor proliferation and survival (Piggott et al., + channel regulates neural stem cell proliferation in the subependymal zone and consequently neurogenesis in the mouse olfactory bulb (Petrik et al., + channel \u03b1-subunit Kv6.4 regulates the proliferation of cells lining the embryonic brain ventricles (Shen et al., v6.4 increases it. Moreover, the Kv6.4 action appears antagonized by the expression of a homolog to the delayed rectifier K+ channel subunit Kv2.1, for which gain and loss of function manipulations cause the opposite effects on neural progenitor proliferation and ventricular brain development (Shen et al., In addition to Ca2+-dependent mechanism (Fiorio Pla et al., 2+ release-activated Ca2+ channels that enable store-operated Ca2+ entry in these cells mediated by Orai1 and STIM1. Downregulating the expression of these molecules decreases in vitro and in vivo proliferation of neural stem cells (Somasundaram et al., Non-voltage-gated ion channels have also been implicated in regulating neural cell proliferation. For instance, TRPC1 participates in bFGF/FGFR1-mediated proliferation of embryonic rat neural stem cells through a Ca+ channels and membrane depolarization, oligodendrocyte progenitor proliferation is regulated by controlling the progression of the cell cycle at the G1 phase, likely through a cAMP and cyclin-dependent kinase inhibitors p27Kip1 and p21CIP1 mechanism (Ghiani et al., A receptor-triggered hyperpolarization in embryonic and neural crest stem cells, S-phase checkpoint kinases of the phosphatidylinositol-3-OH kinase-related kinase family and, subsequently the histone variant H2AX, regulate proliferation (And\u00e4ng et al., + channel by fluid flow elicits Ca2+ dynamics through Ca2+ release-activated channels that activate ERK, regulating neural cell proliferation in the mouse subependymal zone in the olfactory bulb (Petrik et al., in vivo activates ERK in neural plate cells during Xenopus neural tube formation. In turn, constitutive activation of ERK during neural plate folding completely rescues the NTD phenotype induced by dysregulated neural plate cell proliferation due to blocking NMDA receptors or incubation with the AED valproic acid (Sequerra et al., The specific effect that is triggered downstream of all these ion channels on cell proliferation varies among these studies. This is likely due to differences in downstream signaling, recruitment of molecular partners, environmental milieu and maturational status of the cells subjected to these ion channel-triggered signaling mechanisms. There are many downstream signaling effectors reported to mediate ion channel-dependent regulation of neural cell proliferation. For instance, downstream of voltage-gated K+ channel, which in turn assigns a functional neuronal identity to developing nervous system cells (Maue et al., 2+ spikes generated by voltage-gated Ca2+ and Na+ channels in embryonic neurons (Gu et al., Xenopus laevis development (Gu and Spitzer, 2+ spikes that embryonic neurons exhibit is important for the type of neurotransmitter spinal cord neurons express, following a homeostatic rule by which higher spike frequency leads to expression of inhibitory neurotransmitters while suppression of these spikes results in expression of excitatory neurotransmitters (Borodinsky et al., 2+ waves, dependent on both extracellular Ca2+ and Ca2+ release from ion channels present in intracellular Ca2+ stores such as ryanodine receptors, are apparent in growth cones of developing neurons (Gu et al., It has been long recognized that expression of ion channels and prominently voltage-gated ion channels, their density, clustering, and subcellular localization are at the core of what distinguishes a neuron from other cell types. This fundamental question was addressed by pioneering studies from the Mandel lab when they cloned the transcription factor REST and identified it as a silencer element active in nonneuronal cells. In contrast, the absence of REST in neurons allows for the expression of the type II Na+ channels have been at the center of the process of neuronal differentiation in a variety of central nervous system structures and species. Weaver mice carry a mutation in a G-protein coupled inward rectifier K+ channel, GIRK2, that affects the pore-forming domain of the protein and impairs cerebellar granule neuron differentiation immediately after cell cycle exit (Patil et al., + channels, in turn, is a key determinant of the spontaneous Ca2+ activity that developing neurons exhibit. For instance, K+ channels like the small conductance Ca2+-activated K+ channel SK2 are expressed in immature Purkinje cells in the postnatal rat cerebellum and establish a feedback loop with Ca2+ influx and activation of Ca2+ channels to regulate the spatiotemporal features of Ca2+ transients in developing neurons (Cingolani et al., + channel during a critical period of development (Ribera and Spitzer, K2+ activity that differentiating neurons exhibit during development either directly or indirectly. Another instance of direct regulation of Ca2+ activity in developing neurons that connects the process of differentiation with the environment in which embryos develop is represented by the role of TRPM8, cold-sensitive channel, in spinal cord neuron differentiation (Spencer et al., 2+ spike frequency in ventral spinal cord neurons of Xenopus laevis embryos. In turn, Ca2+ spike activity regulates the expression of Hb9 (Spencer et al., +, Ca2+, and K+ channels and the downstream interaction between Ca2+ and cAMP dynamics (Peng et al., While all these studies are focused on specific ion channels and their role in different aspects of neuronal differentiation, they converge on shaping the spontaneous Ca2+ dynamics downstream of neurotransmitter receptor activation. NMDA receptor function is necessary for the development of dendritic arbors in differentiating retinotectal neurons and to establish appropriate retinotectal topographic maps (Cline and Constantine-Paton, 2+ channels to regulate neuritogenesis (Maric et al., Neurotransmitter-gated channels that regulate neuronal differentiation also involve Ca2+ channel-dependent mechanism that results in a higher number of calbindin-expressing mouse hippocampal pyramidal neurons when Neurotrophin-3 signaling is enhanced and lower when it is decreased (Boukhaddaoui et al., 2+]i in growth cones of rat cerebellar granule cells and Xenopus spinal cord neurons through the recruitment of TRPC channel activity (Li et al., Neurotransmitter modulation of neuronal differentiation through direct or indirect regulation of ion channel activity is a function shared by neurotrophic factors. Neurotrophin 3 signaling regulates the specification of neuronal phenotype through a voltage-gated CaXenopus retinal ganglion cells that direct their growth towards softer tissue (Koser et al., Ion channels can be mechanically gated and participate in the differentiation of developing neurons. Piezo 1, a mechanosensitive channel, mediates axonal growth and pathfinding of + channels are involved in regulating axonal outgrowth and morphology. In zebrafish, knockdown of the Nav1.6a alters axonal outgrowth and morphology of dorsally and ventrally projecting secondary motor neurons (Pineda and Ribera, Voltage-gated Na2+ transients recruit activity-dependent transcription factors like CREB (Belgacem and Borodinsky, 2+ channels are recruited downstream of the clustering of the neural cell adhesion molecule 2, inducing submembrane Ca2+ spikes that in turn activate the protein tyrosine kinase c-Src, which results in CaMKII activation and increases in filopodia density, neurite outgrowth and branching (Sheng et al., Downstream of channel activity local and whole-cell effectors are recruited to change, for example, directionality and growth rate of neurites and neurotransmitter specification, respectively. Cav1.2 that translocates to the nucleus and acts as a transcription factor controlling rat neuronal differentiation (Gomez-Ospina et al., 2+ permeation and is dependent on localized RhoA activation (Krey et al., 2+-dependent imbalance in the numbers of subtypes of differentiated cortical neurons (Panagiotakos et al., Alternatively, ion channels may trigger downstream signaling relevant for neuronal differentiation independently of ion permeation. For example, in some instances, the channel itself regulates transcription, as shown for the C-terminal fragment of Ca2+ dynamics in developing neural cells. The participation of neural activity via ion channel expression throughout neural development poses the question of whether this makes the developing nervous system more vulnerable to \u201chijacking\u201d of the necessary signaling mechanisms by exogenous unwanted factors. For instance, we have shown that incubating embryos with the AED valproic acid interferes with Ca2+ dynamics in neural plate cells and results in NTDs (Sequerra et al., 2+ activity-dependent mechanism operating in embryonic spinal cord neurons, exhibit an enhanced escape locomotor performance compared to siblings grown at warm temperatures when tested at cold temperatures (Spencer et al., The studies presented demonstrate that ion channels are expressed from the very first stages of neural development. Furthermore, the signaling mechanisms triggered by these ion channels participate in all the relevant cellular processes of early development including neural cell proliferation and neuronal differentiation, mostly through imprinting specific spatiotemporal CaRG, KS and LB wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Dear Editor,Hi CD8+ peripheral T-cells for a cytolytic gene signature. Expression of cytotoxic genes in the peripheral blood was associated with worse prognosis, which may be explained by persistent micrometastatic disease that finally leads to recurrence . The aual., 2020). Numerous studies have analyzed the association of breast cancer with outcome, including the spontaneous course of disease and response to chemotherapy (Sparano et al., 2018; Wang etThe author declares no conflict of interest."} +{"text": "By utilizing high-throughput technologies, precision medicine is being developed as a preventative, diagnostic and treatment tool to combat complex human diseases. It is therefore necessary to investigate how to integrate these multi-scale \u2018omics datasets to distinguish the novel individual-specific disease causes from conventional cohort-common disease causes. Currently, machine learning plays an important role in biological and biomedical research, especially in the analysis of big \u2018omics data. This Research Topic focuses on the application of wet \u2018omics technology and dry machine learning approaches together to further develop precision medicine.Liu, R. et al. proposed a single-sample-based hidden Markov model approach to detect the dynamical differences between a normal and a pre-disease states, to detect the immediately upcoming critical transition from the pre-disease state. Lee et al. implemented a deep learning-based python package for multimodal longitudinal data integration, especially the numerical data including time series and non-time series data. Yu et al. implemented an adjusted individual-specific edge-network analysis (iENA) method when a limited number of samples from one individual are available, and made a proof-of-concept study on individual-specific disease classification based on microbiota compositional dynamics.Chen et al. analyzed the miRNA expression profiles in whole plasma, Extracellular Vesicle (EV) and EV-free plasma of lung cancer patients and identified several discriminative miRNAs and classification rules as potential non-invasive biomarkers by Monte-Carlo feature selection method and Repeated Incremental Pruning to Produce Error Reduction method. Liu, Z. et al. conducted a genome-wide analysis of allele-specific expression (ASE) in colorectal cancer patients, providing a systematic understanding of how ASE is implicated in both tumor and normal tissues. Hu et al. used RNA sequencing data to identify and quantify the circRNAs in atrial fibrillation (AF) by bioinformatics analysis and characterized their potential functions through the competing endogenous RNA network and protein-protein interaction network. Shi, X. et al. screened a cohort of Total anomalous pulmonary venous connection cases and healthy controls for rare copy number variants by whole exome sequencing, providing candidate genes associated with rare congenital birth defect. Wu et al. performed whole exome sequencing on seven members of an HSCR family, making a first report on the in-frameshift variant p.Phe147del in RET responsible for heritable HSCR. Xie et al. investigated rare Copy number variants (CNVs) in a recruited cohort of unrelated patients with pulmonary atresia and a population-matched control cohort of healthy children by whole-exome sequencing, helping elucidate critical disease genes and new insights of pathogenesis. Meng et al. made a brief research report on the driver gene mutations in Chinese patients with non-small cell lung cancer by target sequencing and Hotspot3D computational approach together.Ho et al. provided a review of polygenic risk scoring and machine learning in complex disease risk prediction with tissue-specific targets, expecting their power to manage complex diseases for customized preventive interventions. Li et al. identified target genes at Juvenile idiopathic arthritis risk loci in neutrophils by an integrated multi-omics approach, constructing a protein-protein interaction network on the basis of a machine learning approach. Dai et al. applied the mega-analysis of Odds Ratio (MegaOR) method to prioritize candidate genes of Crohn's Disease, based on a comprehensive collected multi-dimensional data. Wang, C.H. et al. detected differentially expressed lncRNAs and mRNAs in atherosclerosis by analyzing public datasets with the weighted gene co-expression network analysis, and this bioinformatics study would provide potential novel therapeutic and prognostic targets for atherosclerosis. Jiang, S. et al. collected and profiled the circRNA expressions of heart tissues from Atrial fibrillation patients and healthy controls, providing new insights of the circRNA roles in AF with highly potential interaction mechanisms among circRNAs, microRNAs, and mRNAs.Gu et al. reused the Surveillance, Epidemiology, and End Results registry database to conduct stratification analyses, univariable and multivariable analyses, indicating surgery is an important component of multidisciplinary treatment and sublober resection is not inferior to lobectomy for the specific patients. Zhang, J. et al. exploited the largest crohn's disease dataset and ulcerative colitis dataset by a two-step approach, exhaustively searching for epistasis with dense markers and exploiting marker dependencies. Du et al. analyzed the genome-wide splicing data in 16 cancer types with normal samples by a network-based and modularized approach and captured the pan-cancer splicing and modularized perturbation, which support the dominant patterns of cancer-associated splicing. Zhao et al. assessed the prognostic value of Apolipoprotein E and explored the potential relationship with tumor progression in colorectal cancer (CRC), by collecting the microarray data from the Gene Expression Omnibus and exploring the gene with prognostic significance from the TCGA database. Tang et al. proposed an effective data integration framework HCI (High-order Correlation Integration) to realize high-dimensional data feature extraction with extensive flexibility and applicability on sample clustering with RNA-seq data on bulk and single-cell levels. Chang et al. identified new susceptibility genes and causal sub-networks in schizophrenia by an integrated network-based approach, and reported the N-methyl-D-aspartate receptor interactome highly targeted by multiple types of genetic risk factors. Wang and Liu recognized potential diagnostic biomarkers of Alzheimer's disease by integrating gene expression profiles from six brain regions in a machine-learning manner and validating marker genes in multiple cross-validations and functional enrichment analyses. Xu et al. provided an effective way for the annotation of nuclear non-coding and mitochondrial genes and the identification of new steady RNAs, making a pan RNA-seq analysis to suggest the ubiquitous existence of both 5' and 3' end small RNAs.Yang et al. presented a new pathogen detection and strain typing method UltraStrain for Salmonella enterica based on whole genome sequencing data, which includes a noise filtering step, a strains identification step on the basis of statistical learning, and a final refinement step. Tan et al. conducted comprehensive and systematic experiments, including in vitro genetic assessments and an in vivo acute toxicity study, aiming to study safety issues associated with Bacteroides ovatus ELH-B2. Qiu et al. set up an in-silico model emerging or re-emerging dengue virus (DENV) based on possible antigenicity-dominant positions of envelope (E) protein, so that, the DENV serotyping may be re-considered antigenetically rather than genetically. Zhang, B. et al. collected and re-analyzed the published fecal 16S rDNA sequencing datasets to identify biomarkers to classify and predict colorectal tumors by random forest method, and the trained random forest model has good AUC performance for CRC when combined all samples, although the predication performed poorly for advance adenoma and adenoma.Luo et al. proposed a manifold learning-based method to predict disease-gene associations by assuming that the geodesic distance of related disease-gene pairs should be shorter than that of non-associated disease-gene pairs. Tkachev et al. proposed a heuristic technique termed FLOating Window Projective Separator (FloWPS) for data trimming with SVM and applied it for personalized predictions based on molecular data. Wang, W. et al. developed a new multiple-instance leaning algorithm derived from AdaBoost and accessed this algorithm on annotating proteins that bind DNA and RNA. Xiao et al. proposed a method called BPLLDA to predict lncRNA-disease associations from a heterogeneous lncRNA-disease association network assuming the association paths on network with fixed lengths. Zou et al. used decision tree, random forest and neural network to predict diabetes mellitus by the hospital physical examination data, and the best prediction could be achieved by random forest after dimensionality reduction by principal component analysis and minimum redundancy maximum relevance.Guo et al. proposed a new approach SGL-LMM for mining multivariate associations of quantitative traits by combining sparse group lasso and linear mixed model together, which can consider confounding effects and groups of SNPs simultaneously. Zhang, W. et al. developed a new calling method for differentially expressed genes as DECtp by integrating tumor purity information into a generalized least square procedure and a follow-up Wald test. Cheng et al. utilized a Mendelian randomization (MR) to test the influence of body mass index (BMI) on the risk of T2DM based on GWAS data, validating the causal effect of high BMI on the risk of T2DM. Feng et al. utilized one analysis procedure of feature selection and classification on both transcriptomes and methylomes cancer data, suggesting age should be an essential factor rather than confounding factor in the training and optimization of disease diagnosis model.Qin et al. developed a new joint gene set analysis statistical framework, aiming to improve the power of identifying enriched gene sets by integrating multiple similar disease datasets when the sample size is limited. Shi, Q. et al. proposed a new computational framework of \u201cMulti-view Subspace Clustering Analysis\u201d to capture the underlying heterogeneity of samples from multiple data types, by first measuring the local similarities of samples in the same subspace and then extracting the global consensus sample patterns. Jiang, P. et al. developed a new variants mining algorithm based on trio-based sequencing data, and applied this method on a Ventricular septal defect (VSD) trio and identified several genes and lncRNA highly related to VSD.Finally, we sincerely thank the reviewers for their great efforts to ensure the high quality of all contributing articles, and we hope this Research Topic can attract wide attention in these topics of precision medicine based on machine learning and omics data.TZ drafted the manuscript. TZ, TH, and CL revised the manuscript.This study was supported by the National Natural Science Foundation of China (11871456), the Shanghai Municipal Science and Technology Major Project (2017SHZDZX01), and the Natural Science Foundation of Shanghai (17ZR1446100).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "In this review, we discuss the prevalence, molecular mechanisms, and functional evidence for androgen-regulated prostate cancer fusion genes and transcripts. We also discuss the clinical relevance of especially the most common prostate cancer fusion gene TMPRSS2-ERG, as well as present open questions of prostate cancer fusions requiring further investigation.Androgens are steroid hormones governing the male reproductive development and function. As such, androgens and the key mediator of their effects, androgen receptor (AR), have a leading role in many diseases. Prostate cancer is a major disease where AR and its transcription factor function affect a significant number of patients worldwide. While disease-related AR-driven transcriptional programs are connected to the presence and activity of the receptor itself, also novel modes of transcriptional regulation by androgens are exploited by cancer cells. One of the most intriguing and ingenious mechanisms is to bring previously unconnected genes under the control of AR. Most often this occurs through genetic rearrangements resulting in fusion genes where an androgen-regulated promoter area is combined to a protein-coding area of a previously androgen-unaffected gene. These gene fusions are distinctly frequent in prostate cancer compared to other common solid tumors, a phenomenon still requiring an explanation. Interestingly, also another mode of connecting androgen regulation to a previously unaffected gene product exists via transcriptional read-through mechanisms. Furthermore, androgen regulation of fusion genes and transcripts is not linked to only protein-coding genes. Pseudogenes and non-coding RNAs (ncRNAs), including long non-coding RNAs (lncRNAs) can also be affected by androgens and Androgens are steroid hormones governing the development of male reproductive tract organs and secondary male sex characteristics, as well as functioning in the regulation of muscle mass, fat deposition, and function of steroid hormone-sensitive neurons , also referred to as prostein, as a 5\u2032 partner . The KLK4-KLKP1 fusion is formed either by a trans-splicing mechanism or an in-frame fusion due to a microdeletion, leading to the fusion of the first two exons of KLK4 with exon 4 and 5 of KLKP1. The resulting chimeric sequence predicts a 164\u2013amino acid protein, of which the latter third is derived from KLKP1, leading to a conversion of the non-coding pseudogene to a protein-coding gene. Utilizing cell culture and chicken chorioallantoic membrane (CAM) assay, the expression of KLK4-KLKP1 fusion transcript was shown to affect cell proliferation, cell invasion, intravasation, and tumor formation in prostate cancer. LncRNAs are >200 bp long RNAs that do not encode for protein end-products. They are known to play important roles in the regulation of gene expression and to be dysregulated in several types of human malignancies, including prostate cancer was shown to be strongly correlated with ERG expression in ERG-positive primary tumors, as well as CRPCs developed prostatic adenocarcinoma, whereas Pten+/\u2212 mice only showed HGPIN lesions. Moreover, two genes involved in promoting cell migration and invasion (CXCR4 and ADAMTS1) were found to be upregulated in the context of ERG overexpression , showed a significant reduction in tumorigenicity, concomitant reduction in the expression of the oncogene C-MYC and upregulation of prostate epithelial differentiation genes KLK3 and SLC45A3 in LNCaP and VCaP cells. The results revealed that ERG and AR can co-occupy the same target genes and ERG functions as a repressor of AR-driven lineage-specific differentiation program. ERG also directly regulates the expression of the histone methyltransferase EZH2, by binding to its promoter and activating EZH2-mediated cell de-differentiation program transcriptional program, whereas ETV1 was found to upregulate genes involved in AR signaling and cooperates in its activation.Carver and colleagues also showed that in vitro and in vivo and pointed to a role of the ETV1 transcriptional program in the development of more aggressive disease and poorer clinical outcome . These authors, in contrast to what reported by Tomlins and colleagues is a direct target of ERG in VCaP cells and that PAI-1 inhibited the invasion of VCaP cells treatment of VCaP and LNCaP cells showed increased expression of both ERG and CXCR4 in VCaP, but not LNCaP, suggesting that, indeed, the androgen-mediated ERG overexpression caused by the TMPRSS2:ERG fusion drives CXCR4 expression in VCaP cells, confirmed by the lack of CXCR4 induction when siERG-VCaP cells were treated with R1881. Androgen-induced CXCR4 overexpression was also shown to increase invasiveness of VCaP cells . Moreover, ERG overexpression was shown to activate the Wnt signaling pathway via increased expression of the Wnt receptor FZD4 and upregulation of the EMT mediator integrin-linked kinase (ILK) and its downstream effectors Snail and LEF-1 lesions and TMPRSS2:ERG has been shown to improve the sensitivity for prostate cancer diagnosis , derived from PC-3, to assess the role of the TMPRSS2:ERG fusion transcript in the formation of bone metastases. They used PC3c clones that overexpress the most common TMPRSS2:ERG transcript variant (TMPRSS2 exon 1 and ERG exon 4) at variable levels and also including the 72 bp exon 11 previously shown to be associated with more advanced stages of the disease . These genes were confirmed to be directly regulated by ERG overexpression. Knock-down experiments confirmed that PLXNA2 is directly involved in the increased migration and invasion capabilities of prostate cancer cells and Endothelin-1 (ET-1), responsible for improved acquisition of a bone-like phenotype in cancer cells (osteomimicry), helping the cancer cells survive in the bone microenvironment in separated foci of prostate cancers revealed interfocal heterogeneity and intrafocal homogeneity, indicating that individual foci are the result of clonal expansion (Mehra et al., Prostate cancer is a heterogeneous disease which very often harbors multiple cancer foci within the same gland. It is well-established that different foci are histologically and molecularly heterogeneous, suggesting that they are clonally independent (Wise et al., SPINK1 had been reported in a subset of ETS-negative prostate cancer samples exclusively (Tomlins et al., ERG and SPINK1 overexpression were mutually exclusive in all tumor foci (Fontugne et al., ERG and SPINK1 overexpression within different regions of either the same tumor focus or different foci, but not in the same tumor cells (Lu et al., CHD1 and MAP3K7, and mutations in SPOP, FOXA1, and IDH1 were also found to be associated with the ETS-fusion negative subtype (Liu et al., SKIL fusions described in Annala et al. (ERG. For example, correlative analysis with other ETS gene fusions showed that KLK4-KLKP1 expression is associated with ERG but not ETV1, ETV4, or ETV5 (Chakravarthi et al., Profiling studies of fusion genes in multifocal disease are also important to evaluate co-occurrence of these alterations. Other FISH analyses of recurrent ETS gene rearrangements in multifocal prostates showed complex patterns of alterations, with both rearranged and un-rearranged foci and multiple ETS rearrangements within the same gland (Clark et al., a et al. and the a et al. were idea et al. , the ERGTumor-specific gene fusions can serve as diagnostic biomarkers or help define molecular subtypes of tumors. For example, gene fusions involving ETS transcription factors have been utilized in diagnostic applications, such as with detection of TMPRSS2-ERG fusion transcripts in urine samples or CTCs from patients or ERG protein by immunostaining in biopsies [reviewed recently in Kumar-Sinha et al. , Berg 2, and Garin silico methods, demonstrating that a small molecule targeting the ERG-ETS domain suppressed its transcriptional activity and reverse transformed the characteristics of prostate cancers aberrantly expressing ERG (Butler et al., in vivo and in vitro, impaired the expression of genes enriched in ERG and prostate cancer relevant gene signatures, and inhibited growth of ERG-positive tumors in mouse xenograft models (Wang et al., As the TMPRSS2-ERG fusion is the most common alteration in prostate cancer, molecular targeting of it has gained attraction as a potential therapeutic strategy. Recent examples include the work of Wang and colleagues, who identified a series of peptides that interact specifically with the DNA binding domain of ERG, leading to proteolytic degradation of the ERG protein, and attenuation of ERG-mediated transcription, chromatin recruitment, protein-protein interactions, cell invasion and proliferation, and tumor growth (Wang et al., TNIK as a potential therapeutic target in ERG-fusion gene positive prostate cancer (Lee et al., On the basis of the interaction of ERG and other ETS fusions with the DNA repair proteins PARP1 and DNA-PKc, use of PARP inhibitors has shown initial promise and is being tested in ETS fusion-positive prostate cancers [reviewed in Kumar-Sinha et al. and PedeSLC45A3 with FGFR2 in which the SLC45A3 non-coding exon 1 is fused to the intact coding region of FGFR2 has been found from a brain metastasis of a prostate cancer patient (Wu et al., While the therapeutic targeting of transcription factor oncogenes remains challenging, tumors with fusions involving therapeutically targetable genes, most often kinases, often have the strongest implications in personalized treatment of cancer patients. Amongst prostate cancer fusion genes, especially the effects of androgen-regulated SLC45A3-BRAF and a non-androgen-regulated ESRP1-RAF1 are targetable. The effects of ectopic expression of these fusion genes were studied in RWPE benign immortalized prostate epithelial cells and resulted in increased proliferation, invasion and anchorage-independent growth, which were sensitive to RAF and MEK inhibitors (Palanisamy et al., in vivo environment and is thus valuable in developing new drugs and selecting appropriate treatment strategies for prostate cancer patients. In terms of prostate cancer fusion genes, the expression of ERG has been shown to be retained in the PDXs along with other molecular, histopathologic, and genomic characteristics (Palanisamy et al., Prostate cancer xenografts play a central role in pharmacological testing of potential drugs. The VCaP cell line, due to the TMPRSS2-ERG fusion gene it harbors, has been widely utilized in xenograft drug studies. For example, TMPRSS2-ERG harboring VCaP bone xenograft models were shown to better respond to enzalutamide treatment, suggesting that ERG expression status in tumors could help stratify patients for enzalutamide therapy (Semaan et al., de novo expression of a cancer driver protein, for some fusions the advantage seems not as straightforwardly explained nor convincingly supported by functional data. Especially, despite a lot of effort, the field has yet to pinpoint why and how TMPRSS2-ERG fusion is an early event in prostate cancer development, yet the most significant functions of it seem concentrated in the phase of metastatic disease. The PCAWG Consortium recently reported that, amongst their 3,540 fusion events identified in 1,188 pan-cancer samples studied, 82% were associated with specific genomic rearrangements (PCAWG Transcriptome Core Group et al., The frequent gene fusions in prostate cancer are a curiosity amongst solid tumors. Why and how this particular tumor type benefits so much from these rearrangements for them to be so frequent are still open questions. While the benefit with certain fusions may clearly result from de novo structures and functions, it is possible that also some of the other fusion transcripts may have non-coding functions yet to be discovered. This is supported by the notion that up to 20% of expressed prostate cancer fusion transcripts are non-canonical, with one or both transcripts in antisense orientation (Vellichirammal et al., The case of SLC45A3-ELK4 fusion has proven that it is possible for a chimeric RNA to function as a ncRNA, even though the 3\u2032 fusion partner is initially protein-coding. Considering that chimeric transcripts may have acquired MS and LL wrote the manuscript. SK designed the figures. MS, LL, and SK edited the manuscript. All authors have approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Circulating tumor cells (CTCs) are shed into the bloodstream from primary tumors and metastatic lesions and provide significant information about tumor progression and metastasis. CTCs contribute to tumor metastasis through the epithelial-to-mesenchymal transition (EMT). CTC clusters and stem-like phenotypes lead to a more aggressive and metastatic potential. CTCs retain the heterogeneity and imitate the nature of corresponding primary tumors. Therefore, it is important to use single-cell based analysis to obtain information on tumor heterogeneity and biology. CTCs are also good candidates for building preclinical models for drug screening, disease modeling, genome editing, tumor immunity research, and organ-like biobank establishment. In this article, we summarize the current CTC capture technology, dissect the phenotypes associated with CTC metastasis, and review the progress in single-cell based analysis and preclinical modeling of the pattern and kinetics of CTCs. In particular, we discuss the use of CTCs to assess the progression of hepatocellular carcinoma (HCC). Circulating tumor cells (CTCs), which carry valuable tumor information, arise from the hematogenous diffusion of metastatic tumors in vitro and in vivo CTC culture approaches have provided significant insights into tumor development and metastasis With emerging technological developments, CTC research is not limited to enumeration. Different CTC phenotypes have been analyzed, including the epithelial, mesenchymal, and stem cell types and CTC clusters, which were associated with distinct kinetics and functions Hepatocellular carcinoma (HCC) is the seventh most common cancer and the second leading cause of cancer-related deaths worldwide In recent years, a series of \u201cliquid biopsy\u201d techniques have been developed to evaluate HCC biomarkers Figure 1.CTCs, first discovered by Thomas Ashworth 150 years ago, can be obtained multiple times from tumor patients without an invasive approach. The recent development of new and powerful technologies has remarkably facilitated the precise capture of CTCs et al.et al. developed a novel Labyrinth microfluidic device, offering unique features over other inertial devices to efficiently isolate CTCs from the peripheral blood of HCC patients CTCs from HCC patients are primarily isolated based on their unique biological or physical properties. The biological methods include immune magnetic bead capture and nucleic acid aptamer capture. Court et al., \u201cCTC-iChip\u201d, an inertial focusing-enhanced microfluidic CTC capture platform, is capable of isolating CTCs with or without tumour membrane epitopes et al. proposed an on\u2010chip strategy combining multiplex SERS nano-vectors and multivariate analysis for in situ profiling of circulating tumor cell phenotypes on microfluidic chips et al. reported a magnetically assisted surface\u2010enhanced Raman scattering (SERS) biosensor for HCC CTC detection et al.Currently, nanomaterials and microfluidic chips are widely used for CTC capture. According to the study by Ozkumur et al. showed that tumor cells undergo gradual or complete EMT that is associated with slow proliferation, loss of epithelial cell adhesion molecule EpCAM expression, and increased migration et al. showed that mixed CTCs might be vital for intrahepatic metastasis, and mesenchymal CTCs could predict extrahepatic metastasis et al. revealed that total CTC count and the proportion of M-CTCs are negative factors for postoperative HCC recurrence et al. mapped the distribution and characterized the biological features of HCC CTCs along the transportation route EMT is a process that initiates metastasis et al. that CTC clusters originate from oligoclonal tumor cell groupings rather than cell aggregation in the blood vessels et al. prospectively measured CTCs at five key vascular sites in patients with localized HCC et al.Besides EMT, CTC clusters have been proposed to indicate the initiation of tumor metastasis et al. showed that ICAM-1 is a marker of HCC stem cells. They quantified circulating CD45-ICAM-1+ tumor cells from 60 HCC patients using flow cytometry and found that higher frequencies of circulating CD45-ICAM-1+ cells in HCC patients correlated with more aggressive tumor behavior and worse clinical outcomes et al. found that EpCAM+ CTCs show stem cell characteristics and indicate poor prognosis of HCC after curative resection It has been more than a century since Cohnheim proposed the \u201cembryonic theory\u201d of cancer. The cancer stem cell (CSC) hypothesis argues that cancers arise from a subset of malignant cells that possess stem cell characteristics et al.et al.et al.In animal models, CD90+CXCR4+ HCC cells could be CTSCs, as reported by Zhu et al. systematically investigated the clinical significance of diverse subtypes of CTCs and showed that the presence of EpCAM+ multiploid CTSCs (cut-off: \u22651 cell in 6 ml of blood), EpCAM- small triploid CTCs (\u22655 cells), CTM (\u22651), and increased triploid CTCs, were highly relevant for poor prognosis Although the tumor metastasis mechanism remains unclear Figure 2.Single-cell sequencing technology has been a major breakthrough in sequencing history. CTCs can be processed and analyzed as single cells and then subjected to single-cell sequencing. Given the considerable heterogeneity of CTCs, it is essential to analyze the molecular and genetic characteristics of single CTCs et al. developed a new technology combining image flow cytometry and high-density single-cell mRNA sequencing to identify CTCs in HCC patients et al. collected blood from the peripheral vein, peripheral artery, hepatic vein, inferior hepatic vena cava (IHIVC) and portal vein (PoV) before tumor resection. They analyzed the EMT phenotypes of CTCs using the 4-channel immunofluorescence CellSearch assay and microflow quantitative RT-PCR. The study demonstrated the heterogeneity of EMT status in CTCs across different vascular compartments With advances in the next-generation sequencing (NGS) and single-cell sequencing (SCS) technology, it is possible to obtain the complete genomes of CTCs and compare them with the corresponding primary and metastatic tumors. Several clinically relevant genomic alterations have been discovered, such as single nucleotide variations (SNV), microsatellite instability, and copy-number variations, providing valuable information for the companion diagnostics et al. established a CTC line using the peripheral blood from a mouse HCC model Epigenetic events, including histone modification and DNA base modifications (such as methylation and hydroxy-methylation), also contribute to tumor cell heterogeneity. ChIP-seq (Illumina) is used for studying histone modifications at the single-cell level et al. developed a microchip-assisted single-cell proteomic method for profiling CTC surface antigens facilitating the phenotypic and functional analysis of single CTCs Single-cell protein analysis identifies the heterogeneity of seemingly similar tumor cells, providing critical insights into the mechanisms underlying tumor heterogeneity et al. isolated CTCs from 31 patients and cultured them into spheroids Although the techniques mentioned above have provided abundant information about CTCs, their functions still need to be validated in preclinical models. CTC-based preclinical models include 2D cultures, CTC spheroids, CTC-derived organoids, and xenografts in vivo model in which CTCs enriched from patient blood are injected into immunocompromised mice to generate tumors and expand the number of original tumor cells et al. had developed a xenograft assay and shown that primary human luminal breast cancer CTCs contain metastasis-initiating cells (MICs) that cause metastasis in the bone, lung, and liver. These MIC-containing CTCs expressed EpCAM, CD44, CD47, and MET +CD44+CD47+MET+ CTCs correlated with increased metastatic sites and a poor survival rate. Recently, Vishnoi et al. established a novel triple- negative breast cancer (TNBC) liver metastasis\u2010specific CDX model that selectively recapitulates CTC biology for four sequential generations of mice CTC-derived xenograft (CDX) is an et al. was a significant advance et al.in situ capture method for ex vivo CTC expansion in a 3D co-culture model simulating tumor microenvironment. They successfully expanded CTCs from 14 out of 19 early-stage lung cancer patients and revealed several mutations, including TP53 in both cultured CTCs and primary lung cancer. This strategy sets the stage to further characterize CTC biology and metastatic factors in patients with early-stage cancers. Although CDOs have not been established from HCC patients yet, we propose a strategy for analyzing HCC CDOs .The establishment of CDX models is time-consuming and inapplicable to large-scale drug screening. CDO is the alternative that allows quick molecular analysis and high-throughput drug screening. An organoid is a special 3D culture model harboring a semisolid extracellular matrix supplemented with growth factors for tissues et al. used an advanced CanPatrol CTC-enrichment technique and in situ hybridization to sort and classify CTCs from blood samples and found 90.18% of HCC patients to be CTC positive, even at the early stage of HCC et al. implicated CTCs in tumor staging There is considerable evidence for a crucial role of CTCs as initiators of metastasis, suggesting CTCs as a biomarker for the early detection of HCC. In previous CTC studies, early detection of HCC was mainly based on the assessment of CTC numbers, showing a significant positive correlation between the CTC number and the standard Barcelona Clinic Liver Cancer (BCLC) stage as well as the serum AFP level et al.et al. also reported the association of increased postoperative CTC numbers with worse prognosis of HCC patients et al., CTCs were classified using EMT markers and the presence of mesenchymal CTCs, together with mixed phenotypic and epithelial CTCs predicted the shortest relapse-free survival In patients diagnosed with HCC, CTCs not only contribute to neoplasm staging but are also useful for prognosis et al. used CanPatrol\u2122 system to count CTCs 1 day prior to and 30 days after surgical excision of HCC et al. to detect the preoperative levels of EpCAMmRNA+ CTCs and CD4+CD25+Foxp3+ Treg cells in 49 HCC patients. The data showed that elevated CTC/Treg levels implied a higher risk of postoperative recurrence Since CTC numbers may change after anti-tumor treatment, they could be used for predicting or evaluating therapeutic efficacy before or after treatments. Ye et al. presented a novel system to provide quantitative information of sorafenib-related targets through simultaneously detecting phosphorylated ERK (pERK) and Akt (pAkt) in HCC CTCs Mutation profiles and drug-resistant molecular expression profiles have been widely identified in many solid tumors. In this context, CTC analysis allows the determination of therapeutic targets and resistance mechanisms to cancer therapies at the DNA, RNA, and protein levels and has great potential to identify the patient population most likely to respond to specific treatments. Li et al. that evaluation of PD-L1+ CTCs discriminated between HCC patients with early-stage and advanced/metastatic disease Table Immune checkpoint inhibitors have launched a new era in immunotherapy, with exceptional long-term remissions in some patients across diverse tumor entities. However, only a few patients respond to this modality, with many experiencing severe side effects Supplementary Figure.CTCs offer valuable diagnostic and therapeutic information on HCC, although some challenges still exist in their identification, quantification, and/or characterization. Currently, new CTC capture technologies are emerging, while the CellSearch system is still the only FDA-approved CTC detection and quantification method. So, we urgently need novel and certified tools for quick isolation and characterization of CTCs. There are various sources of CTCs including primary tumors and metastatic lesions. Identifying of the sources of captured CTCs is beneficial for a more in-depth analysis. For a comprehensive understanding of CTC biology, multiple omic disciplines should be combined for single-cell analysis. With the development of CDO approaches, we can co-culture CTCs with immune cells to simulate the tumor microenvironment to monitor tumor progression and associated molecular events. Furthermore, as part of liquid biopsy, CTC testing should be combined with other liquid biopsies such as analysis of ctDNA and exosomes to promote the efficiency of clinical CTC tests. Ongoing and future research on CTC capture technology, molecular profiling, sing-cell analysis, and preclinical models are expected to considerably improve CTC testing for early diagnosis, efficacious treatment, and effective prognostic management of HCC. The overview of CTC research is showed in the Supplementary figures and tables.Click here for additional data file."} +{"text": "Tau is a microtubule-associated protein (MAPT) that is highly expressed in neurons and implicated in several cellular processes. Tau misfolding and self-aggregation give rise to proteinaceous deposits known as neuro-fibrillary tangles. Tau tangles play a key role in the genesis of a group of diseases commonly referred to as tauopathies; notably, these aggregates start to form decades before any clinical symptoms manifest. Advanced imaging methodologies have clarified important structural and functional aspects of tau and could have a role as diagnostic tools in clinical research. In the present review, recent progresses in tau imaging will be discussed. We will focus mainly on super-resolution imaging methods and the development of near-infrared fluorescent probes. Tauopathy is a general term referring to a group of disorders characterized by the accumulation of misfolded tau protein. Highly heterogeneous from a clinical perspective, tauopathies can be further divided into primary tauopathies (where tau is the leading cause of neurodegeneration) and secondary tauopathies (where a tauopathy is associated with other pathologies). Progressive supranuclear palsy (PSP), Pick\u2019s disease (PiD), and fronto-temporal dementia (FTD) are examples of primary tauopathies while, Alzheimer\u2019s disease (AD), Lewy\u2019s body disorder (LBD), and Down\u2019s syndrome (DS) belong to the second group STED experiments, revealed that in FTD neurons, an aberrant microtubular organization was responsible for the deformation of the nuclear lamina , and [11C]PBB3. These compounds represented a \u201cproof of concept\u201d in the process of tau PET imaging and their properties have extensively been examined in the whole brain; in addition, MAO-B correlated with glial fibrillary acid protein (GFAP) a marker for astrocytes, and therefore they concluded that [18F]THK5351 PET could be useful to evaluate tau-associated neuroinflammatory states in AD rather than tau pathology. PET scanning using the tracer [18F]AV-1451 was performed on a 76-year-old female patient carrying the MAPT R406W mutation. This mutation leads to the presence of tau aggregates similar to those found in AD. Clinically, the patient presented a long history of cognitive deficits and behavioral abnormalities. Postmortem immunohistochemical staining for hyperphosphorylated tau protein showed a strong positive correlation between in vivo [18F]AV-1451 retention and the density of tau aggregates . In this study, no significant correlation was reported between in vivo PET and autoptic examination of tau deposits; therefore, the authors conclude that [18F]-AV-1451 may not be suited to detect tau aggregates in these non-AD tauopathies . Another big obstacle is represented by the heterogeneous structural organization of tau aggregates in AD and non-AD-related tauopathies. The need for dyes with better selectivity, affinity, and specificity is opening the door for the development of a second generation of tau tracers . In vitro experiments confirmed its ability to label intracellular tau aggregates; however, no experiments on mouse models of tauopathies nor on postmortem human AD tissues have been performed with better fluorescence emission upon tau aggregates binding (\u03bbem = 650 nm) have been proposed and a large Stokes shift (110 nm). When incubated with AD human brain slices, the probe 2e displayed a staining pattern that resembled that which was obtained with an antibody against ptau epitopes experiments allowed a strong and stable signal from the brain of both JNPL3 and htau/PS1 mice to be detected. Very little signal was observed in control animals. Moreover, older mice from both genotypes showed higher signals compared with younger mice further suggesting the specificity of the scFv 235 antibody (Krishnaswamy et al., Several research groups are actively involved in developing affordable probes/methodologies that could be potentially used for the diagnosis of tau-based neurodegenerative disorders. A plethora of approaches using different chemical strategies have been proposed (Verwilst et al., in vitro aggregation of misfolded proteins in particular (Cosentino et al., in vitro and in vivo. Tau aggregates have recently been described in the retina of AD patients (Sch\u00f6n et al., Advanced imaging methodologies are providing valuable information about tau structure, interaction with proteins and organelles, localization, and mechanisms of self-aggregation. Far from being definitive, these techniques are still under active development as several limitations need to be overcome. A critical step for improvement is represented by protein labeling with fluorescent probes (Wang et al., All authors reviewed and discussed relevant literature, wrote sections, and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "In this editorial, we briefly summarized the International Conference on Intelligent Biology and Medicine 2019 (ICIBM 2019) that was held on June 9\u201311, 2019 at Columbus, Ohio, USA. We further introduced the 19 research articles included in this supplement issue, covering four major areas, namely computational method development, genomics analysis, network-based analysis and biomarker prediction. The selected papers perform cutting edge computational research applied to a broad range of human diseases such as cancer, neural degenerative and chronic inflammatory disease. They also proposed solutions for fundamental medical genomics problems range from basic data processing and quality control to functional interpretation, biomarker and drug prediction, and database releasing. The International Conference on Intelligent Biology and Medicine 2019 (ICIBM 2019) was co-hosted by the International Association for Intelligent Biology and Medicine (IAIBM) and the Department of Biomedical Informatics at The Ohio State University on June 9\u201311, 2019 in Columbus, OH. A total of 164 researchers attended the conference, of which 79 were faculty/staff and 84 were trainees. The conference included four keynote lectures, four eminent scholar talks, five tutorials and workshops, twelve concurrent regular scientific sessions, and one poster session. It covered a board range of topics, including but not limited to next-generation sequencing, single cell analyses, deep learning, metabolomics, genomics, and other omics research, systems biology, medical applications and translational research involving high-throughput data, computational methods and novel applications of computational tools, and others. Among 105 original manuscript submissions, 19 research articles of interests to computational method development and applications in medical genomics were selected for the ICIBM 2019 BMC Medical Genomics Supplement Issue after with careful peer reviews. The proposed computational methods and applications cover a broad range of biomedical topics and are innovative with significant biological and clinical implications. A more detailed summary of the conference arrangement, scientific programs, and achievements were published elsewhere [This supplement issue includes five papers that propose computational methods to predict Li et al. developed a new computational method, namely SIMCCDA (Speedup Inductive Matrix Completion for CircRNA-8 Disease Associations prediction), which is the first work that apply the recommendation system based inductive matrix completion to predict circRNA-disease associations . The cirLi et al. developed a novel computational workflow for customized neoantigen prediction and selection . The worMostavi et al. conducted a novel application of convolutional neural network model for cancer type prediction by using gene expression data . Novel CCai et al. developed a new association test, namely weighted Adaptive Fisher (wAF) to test the association of common and rare SNVs and detect dense and sparse signals in GWAS . wAF is Foroughi pour et al. developed a new computational capability to conduct binary classification based on high dimensional SNP data . Binary Three genomics analysis are included in this supplement issue, two utilized network-assisted methods to explore new SNPs in multiple sclerosis and Cleft lip with/without cleft palate, while the other focused on somatic mutations in genetic regulatory elements in melanoma. Each of the two genomics data based study identified significant sets of biological and clinical associated genetic events.Zhang et al. explored the landscape of somatic synonymous mutations in genetic regulatory elements in melanoma . This stManuel et al. conducted a network-assisted search in two multiple sclerosis GWAS data sets to identify the gene networks that were associated with the disease . With apYan et al. conducted a integrative network-assisted GWAS study of the Cleft lip with/without cleft palate (CL/P) . By usinFour biological network based studies, including two differential co-expression analysis and two network based drug prediction, are included in this supplement issue. Two studies identified variations in co-expression networks through different disease stages of Alzheimer\u2019s disease and chronic kidney disease, which can be potentially used as biomarkers of disease progression. Two novel network based drug prediction method was developed by predicting variations in signaling pathway activities in patient subgroups and applied to identify novel drug and targets in pancreatic and ovarian cancer.Upadhyaya et al. conducted a differential gene co-expression analysis of gene expression data collected from patients\u2019 plasma samples of Alzheimer\u2019s disease . The autnuclear SMAD2/3 signaling and signaling events mediated by focal adhesion kinase, 27 including Regulation of nuclear SMAD2/3 signaling. The study also identified a list of vanishing hub genes and disrupted correlations within and between key signaling pathways, on the pathophysiological mechanisms of CKD progression.Yu et al. conducted a differential co-expression analysis of the RNA-seq data of chronic kidney disease (CKD) collected from patients at different disease stages . Kidney Liu et al. conducted a network based study to identify possible drug targets in pancreatic ductal adenocarcinoma . A novelZhang et al. conducted an integrative network analysis to identify potential drug targets of ovarian cancer . In theiA substantial part of this supplement issue is composed by biomarker predictions, including seven works with utilizing deep leaHuang et al. developed a novel auto-encoder based model, namely AECOX, to identify prognostic marker genes from cohort transcriptomics data . CompariLiu et al. developed a novel computational pipeline to predict colon cancer prognosis by using gene expression level of transcriptional factors . UnivariDong et al. developed a new computational pipeline to identify breast cancer patients\u2019 prognosis associated transcriptional regulatory factors (TFs), by using estimated activity level of TFs . Data seSmerekanych et al. conducted a systematic identification of gene expression, pseudogene expression, miRNA expression, and pseudogene-gene interactions and clinical factors that were predictive to breast cancer patients\u2019 prognosis . Based oAdnan et al. established a comparative evaluation of network features to predict the metastasis of breast cancer on data collected in 12 cohorts . The autZhang et al. conducted a pan-cancer study to demonstrate the clinical implication of class-3 semaphorins in the treatment of cancer . By compByun et al. conducted a study of the differential alternative splicing in HIV infected T cells . ApplicaICIBM conferences provide a friendly forum for researchers to present and publish cutting-edge biomedical studies. Stimulated by state-of-the-art biotechnologies, therapeutic strategies, biological hypotheses and interdisciplinary capabilities, computational scientists developed models to link biomedical data with biological hypothesis from different perspectives. Here, the 16 selected works illustrating innovative computational ideas or applications of new computational methods on important biomedical questions. Even though the high complexity of some models may limit their application in a broader domain and some observations still need further experimental validations, we anticipate some of these biomedical studies and computationally derived results can contribute in real clinical applications, as is discussed in this supplemental issue."} +{"text": "Oxidative stress plays an important role in the pathogenesis of several different neurodegenerative diseases (NDDs), such as Alzheimer\u2019s disease (AD), Parkinson\u2019s disease (PD), Huntington\u2019s disease, amyotrophic lateral sclerosis (ALS) and multiple sclerosis (MS) ,2. In paLin et al. performeOther manuscripts reviewed the emerging role of oxidative stress-responsive proteins, such as sestrins, in various neurological diseases, protein DJ-1 in PD and mitofusin-2 (Mfn2) in AD. Chen et al. reportedAmong neurodegenerative events, chronic inflammation can exacerbate the oxidative stress in the cells, leading to oxidation and damage of cellular components, increased inflammation and activation of neuronal death pathways . In thisAs previously highlighted, the identification and use of oxidative stress biomarkers in NDDs could be useful for the development of new drugs as well as for the early diagnosis and demonstration of drug efficacy in clinical studies. Padureanu et al. evaluateAmong the research manuscripts of this Special Issue, some papers evaluated the antioxidant and anti-inflammatory effects of different molecules in several in vivo models of neurodegeneration. Hong et al. evaluateIn conclusion, the manuscripts published in this Special Issue highlight recent advances in knowledge of the oxidative stress\u2019s contribution to various NDDs as well as novel antioxidant strategies of neuroprotection for NDDs."} +{"text": "Recently, Ebmeyer and colleagues published a 28-day feeding study with six pyrrolizidine alkaloids in rats (Ebmeyer et al. Hepatotoxicity represents a major focus in current toxicological research (Jansen et al. Often risk evaluation is hampered by a lack of carefully performed subchronic or chronic animal studies with human relevant doses. Therefore, the present study of Ebmeyer et al. represents an important milestone in the current research on the hepatotoxicity of pyrrolizidine alkaloids."} +{"text": "Ficus elastica using transmission electron microscopy. As depicted in a model, an inner polysaccharide-rich layer and an outer cutin (or cutan)-rich layer may support the composite, heterogeneous concept of the leaf cuticle.Two distinct layers in terms of texture and electron density were observed in the leaf cuticle of The cuticle represents the outermost surface structure of a variety of organisms such as plants and insects. As the interface between plants and their environment, the plant cuticle plays a number of roles mostly associated with\u00a0protection against biotic and abiotic stresses including pathogen infection and water loss (Dom\u00ednguez et al., Ficus elastica were fixed, dehydrated, and embedded in LR white resin (Kim, Leaves of the rubber tree sin Kim, . Energy-sin Kim, . Two difThe heterogeneous cuticle structure was strikingly similar to that of the model depicted by Heredia-Guerrero et al. . They pr"} +{"text": "The ability to rapidly detect viable pathogens in food is important for public health and food safety reasons. Culture-based detection methods, the traditional means of demonstrating microbial viability, tend to be laborious, time consuming and slow to provide results. Several culture-independent methods to detect viable pathogens have been reported in recent years, including both nucleic acid\u2013based (PCR combined with use of cell viability dyes or reverse-transcriptase PCR to detect messenger RNA) and phage-based methods. Some of these newer methods, particularly phage-based methods, show promise in terms of speed, sensitivity of detection and cost compared with culture for food testing. This review provides an overview of these new approaches and their food testing applications, and discusses their current limitations and future prospects in relation to detection of viable pathogens in food.\u2022 Cultural methods may be \u2018gold standard\u2019 for assessing viability of pathogens, but they are too slow.\u2022 Nucleic acid\u2013based methods offer speed of detection but not consistently proof of cell viability.\u2022 Phage-based methods appear to offer best alternative to culture for detecting viable pathogens. Salmonella spp., Campylobacter spp., Listeria monocytogenes, Staphylococcus aureus and pathogenic Escherichia coli are the main pathogens that cause the highest number of outbreaks linked to food sources , for example. Tests for foodborne pathogens have historically been culture-based, which is still considered the gold standard methods.As stated earlier, culture-based methods are generally regarded as the \u2018gold standard\u2019 for microbiological analysis of food. Traditional culture relies on the ability of bacteria to grow and multiply on laboratory media and form visible colonies. These methods still represent the first choice for many food testing laboratories as they are sensitive, inexpensive, easy to use, and give either qualitative or quantitative information on the number and type of viable microorganisms present in the food samples Doyle . HoweverThere are essentially two culture-independent approaches that represent promising alternatives to culture-based approaches for detection of viable foodborne pathogens, namely nucleic acid\u2013based and bacteriophage-based detection methods. The advantages and limitations of these culture-independent methods are summarised in Table Nucleic acid\u2013based methods operate by detecting specific DNA or RNA sequences of the target pathogenic organism. Polymerase chain reaction, or PCR, is the most commonly used nucleic acid amplification method for detecting pathogenic microorganisms, and over the last two decades, many different advances on the original PCR protocol have been described is considered a better indicator of cell viability than DNA, since this molecule is only present in metabolically active cells (Sheridan et al. The high specificity and natural affinity of bacteriophages, or simply phages, for their host cells make phage-based methods an attractive proposition. Bacteriophages can only replicate inside living cells, meaning that phage-based methods can be tests to demonstrate cell viability (Richter et al. . In this method samples are incubated with seed bacteriophages to start the lytic cycle. Just before the end of the latent period, a chemical virucide (McNerney et al. Salmonella Typhimurium and Staphylococcus aureus (Stewart et al. Salmonella Enteritidis and Escherichia coli 0157:H7 (Favrin et al. Listeria monocytogenes (Oliveira et al. Mycobacterium avium subsp. paratuberculosis (Foddai et al. Most phage-based tests employ lytic phages as lysing agents, and detection of the new progeny phages or intracellular material released from target bacterial cells provides the indication of cell viability. One of the simplest lytic phage-based tests is called the phage amplification assay or simply the plaque assay (Stewart et al. Listeria monocytogenes within 8\u00a0h (Stambach et al. Faster phage-based detection can be achieved by combining the lytic part of the plaque assay and an alternative end-point detection method, such as immunological (Stewart et al. A third type of lytic phage\u2013based method to detect intracellular components released from bacteria also exists. After phage lysis, the quantity of released compounds is monitored through a bioluminescence assay using an enzyme and a substrate. The amount of light generated is proportional to the quantity of intracellular compound released and to the bacterial concentration originally present in samples. Examples of intracellular markers are adenosine-5 triphosphate or ATP (Griffiths More rapid and sensitive tests for detection of viable pathogens in food are continually being sought. Culture-based methods are becoming too laborious and time consuming to apply, and might have limited detection capability if pathogens in a VBNC state are present in food. Molecular tests, particularly mRNA-based tests, represent a potential solution for the rapid detection of living microorganisms. However, the perishable nature of mRNA still represents a barrier to the large-scale use of reverse transcriptase PCR for food testing purposes. A range of lytic phage\u2013based methodologies have emerged over the last two decades, which are exhibiting high detection sensitivity for several foodborne pathogens in many different matrices including food and water. The combination of phage amplification and lysis with PCR/qPCR, immunoassay or enzyme assay endpoint detection approaches seems to be the most promising rapid alternative to cultural methods for detection of viable pathogens in food. Providing host cell metabolism is occurring, phage amplification will take place and pathogen cells will eventually burst to release measurable intracellular components such as ATP, enzymes, host DNA or progeny phages."} +{"text": "Irisin is a PGC-1\u03b1-dependent myokine that causes increased energy expenditure by driving the development of white adipose tissue into brown fat-like tissue. Exercise can improve irisin levels and lead to its release into the blood. In ischemic stroke, neurons are always sensitive to energy supply; after a series of pathophysiological processes, reactive oxygen species that are detrimental to cell survival via mitochondrial dysfunction are generated in large quantities. As a protein associated with exercise, irisin can alleviate brain injury in the pathogenesis of ischemic stroke. It is thought that irisin can upregulate the levels of brain-derived neurotrophic factor (BDNF), which protects nerve cells from injury during ischemic stroke. Furthermore, the release of irisin into the blood via exercise influences the mitochondrial dynamics crucial to maintaining the normal function of nerve cells. Consequently, we intended to summarize the known effects of irisin during ischemic stroke. The incidence of stroke has increased rapidly over the past few decades, causing it to become one of the main causes of death and long-term disability worldwide in various ways by regulating sugar, lipid, and protein metabolism (Febbraio and Pedersen, The role of FNDC5/irisin in learning and memory is mediated by BDNF expression, which plays an important role in neural remodeling in conditions such as Alzheimer's disease (Wrann et al., in vivo (Peng et al., in vitro (Yu et al., A recent study has reported that irisin protects the blood-brain barrier from ischemic injury by decreasing the expression of MMP-9 (Guo et al., Notably, although Li et al. have fouAs previously described, the discovery of irisin provides an alternative direction for studying the potential treatment methods for ischemic stroke. In 2017, Lidongjie et al. found that irisin synthesis reduces the infarct volume and the degree of brain edema and improves the neurobehavioral score in an oxygen-glucose deprivation model (Li et al., Mitochondrial dynamics mainly consist of fission and fusion. Fission is mediated by the proteins Drp1, Fis1, and MFF. Drp1 is recruited from the cytosol to the outer membrane of mitochondria and interacts with its receptor proteins MFF and Fis1 to create the fission complex. Drp1 is then oligomerized into filaments that wrap around mitochondria, leading to mitochondrial constriction and sequential separation of the inner and outer membrane. Drp1 reportedly has a crucial role in ischemic stroke; brain edema, the infarct area, and other neuronal injuries are alleviated following Drp1 downregulation (Anzell et al., Three different GTPases mediate fusion, including Opa1 and Mfn1/2. Mfn1/2 are anchored to the outer membrane of mitochondria, while inner membrane fusion is mediated by Opa1. A lack of mitofusins prevents fusion of both the outer and inner membrane of the mitochondria, while the loss of Opa1 only blocks fusion of the inner membrane. Mitochondrial fusion proteins are less studied in ischemic stroke. Mfn2 is reported to exert an anti-apoptotic effect, and its expression decreases in the presence of ROS. Opa1 can attenuate infarct volume in ischemic stroke, and its expression is increased after exercise (Anzell et al., In ischemic stroke, cell survival and pathobiology are involved in mitochondrial dynamics. As mitochondrial dynamics processes, fission and fusion are crucial to mitochondrial function. Fusion is presumed to be beneficial to cell survival, but studies show that fission facilitates cell death (Li and Liu, It is reported that mitochondrial homeostasis is closely related to AMPK upregulation associated with altered cell energy metabolism (Siteneski et al., AMPK is a heterotrimer including an \u03b1-subunit and two regulatory subunits, \u03b2 and \u03b3. The \u03b1-subunit is the main catalytic part of AMPK, containing a kinase domain and the key residue Thr172. When the ratio of ATP-AMP decreases, the AMPK complex is activated by phosphorylation on Thr172 in the \u03b1-subunit. The activated AMPK affects the mitochondrial dynamics by activating downstream substrates. When stresses, such as ischemia or hypoxia, are applied, the phosphorylated AMPK directly phosphorylates MFF. MFF then recruits Drp1 to the mitochondrial membrane, selectively causing fission of the damaged mitochondria and protecting normal mitochondrial function (Wang and Youle, It has been reported that exercise is a potential activator of AMPK, demonstrating the possibility that AMPK can affect mitochondrial dynamics via exercise (Trewin et al., Although treatment strategies for stroke have been explored over several decades, intravenous thrombolysis remains the primary and most effective method (Keizman et al., Irisin is reportedly induced by physical exercise to augment energy expenditure, according to the initial report (Bostr\u00f6m et al., Irisin protects against endothelial injury and ameliorates atherosclerosis in Apo-E knockout mice (Lu et al., Many scholars have also suggested that irisin is an exercise-induced muscle factor, with exercise promoting its large-scale expression in skeletal muscles, the heart, and the brain. These brain regions include Purkinje cells in the cerebellum (Varela-Rodr\u00edguez et al., Evidence suggests that irisin levels are affected by a large number of stressors. It is well-established that acute exercise increases the levels of blood irisin (Jedrychowski et al., All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Childless older adults may be at risk for poorer health cross-nationally, yet most studies on this topic analyze only a small number of countries and only 1 or 2 health outcomes. To our knowledge, two papers exist that explore associations between childlessness and multiple indicators of health using data from a large number of regionally diverse countries , but neither study includes an examination of socioeconomic resources. The level of health risk faced by childless older adults is likely to be distinctly shaped by older adults\u2019 socioeconomic resources . Associations between childlessness, socioeconomic resources, and health may also differ by country context. Using harmonized, cross-national data for adults aged 50 and older across 20 high- and middle-income countries (United States (HRS), European Union (SHARE), Mexico (MHAS), and China (CHARLS) from the Gateway to Global Aging data repository), we explore if and how individual-level socioeconomic resources moderate associations between childlessness and five health indicators . Results suggest that associations between childlessness and health outcomes vary by individual socioeconomic resources in some country contexts, but not in others. We discuss these findings in light of the impact of individual-level socioeconomic resources on older adults\u2019 support options and health outcomes cross-nationally."} +{"text": "B. Ngor et al.; Scientific Reports 10.1038/s41598-018-27340-1 (2018).3.As one of the richest sources of fisheries-related data in the lower Mekong basin, the Tonle Sap dai fishery has received considerable attention in the literature in recent years as concerns grow over the impacts of hydropower dams on fisheries, which are important for livelihoods and food security4 reported a decline since 2000 in the catch of larger species which tend to occupy higher trophic levels; compensatory increases in the catch of smaller species; and declines in the mean body weight (and length) of common species in the Tonle Sap dai fishery, as evidence of the effects of indiscriminate fishing or \u201cfishing-down\u201d of the multi-species fish assemblage in the lower Mekong basin. We provide evidence below that suggest that these apparent recent changes are more likely to reflect changing hydrological conditions than fishing-down effects, possibly caused by climate change and recently also by hydropower development.Ngor et al.6 of monitoring data which include one of the driest fishing seasons on record (1998\u201399). The authors thereby created a time series beginning with the three wettest seasons (largest floods) since monitoring began (2000\u20131 to 2002\u20133) that were followed by 12 seasons of variable, but decreasing flows caused by hydropower dam construction, low rainfalls possibly resulting from climate change, and abstractions for agricultureWithout reference to hydrological variation, Ngor et al. reported a temporal decline in the catch of larger, high-trophic level species; compensatory increases in the catch of smaller species; and declines in the mean body weight (and length) of six common species. The authors reported these findings as evidence of the effects of indiscriminate fishing (fishing-down) in the lower Mekong basin, without considering alternative explanations for the observed patterns.2. Moreover, the fishery itself is not standardised, with net types, mesh sizes and flow rates through the gears varying inter and intra-annually. Catches may therefore not reflect changes in underlying abundance7. Despite this, we re-examined the analysis of Ngor et al. at face value.Ngor et al. describe their dataset as being generated from a \u201cstandardized biological catch assessment\u201d. In reality, this assessment is complex having undergone numerous changes through time, including to sampling efforte-transformed catch time series of larger species (TL\u2009>\u200945\u00a0cm) excluding those with zero catch in any year. These 28 species formed approximately 16% of the total catch during the study period. We also found negative regression coefficients for all 28 species, supporting the findings of Ngor et al. However, the combined annual catch of these 28 species did not decline significantly through time .Using the 15-season dataset that accompanied Ngor et al., we re-fitted regression models to the log2\u2009=\u20090.46; p-value\u2009<\u20090.01) \u2014a measure of flood extent and duration with the FI. Rle Table . The fivHenicorhynchus, that formed 42% of the total catch during the study period, exhibited no significant trend through time .Contrary to Ngor et al. we found no clear evidence of a compensatory response by small species. The annual catch of the three most prolific small species of the genus 2. The time series analysis of mean fish weight illustrated in Figure\u00a04 of Ngor et al. was subject to sampling-related bias because the number of observations of fish weight in each month varied significantly each year.The growth of species of fish caught by the dai fishery is non-linear and seasonale-transformed body weight using \u2018year\u2019 and the FI as alternative independent explanatory variables. To aid model comparisons we first standardised both independent variables.To avoid this bias, and minimise any gear selectivity effects, we examined how the mean body weight of the six common species examined by Ngor et al. varied in both December and January each year corresponding to the end of the flood, when any flood-related effects on fish growth would manifest. We compared regressions of mean logThe FI explained more of the variation in mean fish weight than \u2018year\u2019 in 9 of the 12 regressions compared in Cambodia in 201213, we suspect that since then, an already fished-down assemblage has been further impacted by periods of low flow and limited flooding, possibly caused by climate change and hydropower dams6.While fishing pressure undoubtedly caused some fishing-down between 1970 and the 1990s corresponding to strong population growth and the spread of modern fishing methods"} +{"text": "N-acylethanolamines (NAEs) and N-acyl amino acids belonging to the complex lipid signaling system termed endocannabinoidome. These molecules exert a variety of biological activities in the central nervous system, as they modulate physiological processes in neurons and glial cells and are involved in the pathophysiology of neurological and psychiatric disorders. Their effects on dopamine cells have attracted attention, as dysfunctions of dopamine systems characterize a range of psychiatric disorders, i.e., schizophrenia and substance use disorders (SUD). While canonical endocannabinoids are known to regulate excitatory and inhibitory synaptic inputs impinging on dopamine cells and modulate several dopamine-mediated behaviors, such as reward and addiction, the effects of other lipid neuromodulators are far less clear. Here, we review the emerging role of endocannabinoid-like neuromodulators in dopamine signaling, with a focus on non-cannabinoid N-acylethanolamines and their receptors. Mounting evidence suggests that these neuromodulators contribute to modulate synaptic transmission in dopamine regions and might represent a target for novel medications in alcohol and nicotine use disorder.The family of lipid neuromodulators has been rapidly growing, as the use of different -omics techniques led to the discovery of a large number of naturally occurring N-arachidonoylethanolamide family, also termed fatty acid ethanolamides. NAEs differ in the length and saturation of the hydrocarbon chain and their receptor affinity are emerging as an intriguing class of neuromodulators, although largely uncharacterized so far at physiologically relevant concentrations, the others display an affinity for peroxisome proliferator-activated receptor-\u03b1 .Very little is known about the biosynthesis of N-acyl amino acids are hydrolyzed to free fatty acids and ethanolamine or amino acids are synthesized following activation of metabotropic glutamate, muscarinic, or dopamine D2 receptors . Very little is known about the functional relevance of N-acyl amino acids and their receptors such as GPR18, GPR55, or GPR92; this topic is discussed in Burstein evokes an increase in Ca2+ permeability and a rise in intracellular Ca2+, which is necessary for the activity of the Ca2+-dependent NAT isoform .An extensive literature substantiates the role of the dopamine system in addiction and SUD. Dopamine facilitates the development of long-lasting forms of synaptic adaptations that determine the effectiveness of reward and reward predictors to control subsequent seeking behavior depends on increased eCB levels within mesolimbic dopamine regions were elevated in the plasma (Best et al., The role of NAEs in alcohol dependence has been extensively explored by studying the catabolic enzyme FAAH, both in rodents and humans. Several studies stress out the importance of FAAH genetic variants (Zhou et al., PPAR\u03b1 is upstream of diverse genes that are modulated by ethanol or involved in ethanol-induced effects (Ferguson et al., Tobacco use is associated with high morbidity and mortality, it being the most preventable cause of death in the world (World Health Organization, Both tobacco smoke and nicotine can affect the eCB system. Tobacco smoke alters FAAH, NAPE-PLD, and MAGL levels in the striatum (Torres et al., The involvement of the eCB system in nicotine dependence was demonstrated by the effect of FAAH inhibitors. FAAH inhibitors suppress many reward-related effects of nicotine in rats and non-human primates, such as nicotine self-administration and reinstatement of nicotine seeking (Scherma et al., via \u03b17-nAChRs, and subsequent increase of intracellular Ca2+ (Melis et al., in vivo and in vitro strategies, Melis et al. (N-oleoyl glycine was shown by Donvito et al. (N-acyl amino acid is synthesized in dopamine cells and acts as an endogenous neuromodulator in a similar fashion of other NAEs with a dopamine moiety such as N-arachidonoyldopamine or N-oleoyldopamine (Ferreira et al., Besides the effect of the major eCBs, there is increasing evidence of the involvement of PEA and OEA in nicotine addiction, as they have a crucial role as endogenous modulators of cholinergic transmission (Melis et al., s et al. confirmeo et al. to countBased on the mechanisms described, the suppression of nicotine-induced responses of dopamine neurons by PPAR\u03b1 agonists raised the interest on these ligands as a promising strategy to prevent nicotine relapse (Melis and Pistis, A way to circumvent the limited brain permeability of fibrates is to increase brain levels of endogenous PPAR\u03b1 agonists, such as PEA and OEA. The recent development of brain-permeant selective NAAA inhibitors offers the advantage to modulate levels of PEA and OEA selectively, and not AEA, therefore concurrently limiting psychiatric side effects due to eCB-CB1 alteration. Similar to direct PPAR\u03b1 agonists, also NAAA inhibitors display potential as anti-smoking medications, as they block nicotine-induced excitation of dopamine cells, dopamine elevations in the nucleus accumbens, and conditioned place preference in a PPAR\u03b1-dependent manner (Sagheddu et al., The expanded eCB system, the \u201cendocannabinoidome,\u201d is a hotbed for a large number of lipid signaling molecules, enzymes, and receptors and represents a Pandora\u2019s box for drug discovery (Cristino et al., This review article summarizes evidence suggesting that NAE/PPAR\u03b1 signaling shows promise as a target in the treatment of SUD, particularly alcohol and nicotine use disorder. A parsimonious unifying hypothesis for this effect is NAE/PPAR\u03b1\u2019s ability to modulate dopamine cell activity by specifically dampening stress-evoked excitatory drive from cholinergic afferents on VTA dopamine cells. Hence, a heightened cholinergic transmission has long been postulated to contribute to detrimental effects induced by stress, such as depression (Janowsky et al., N-acyl amino acid family suggest that analogs of these lipid neuromodulators could become potential drug candidates.SUD represents an unmet clinical need, with drugs currently in use that show limited efficacy or untoward side effects. Indeed, results reported for members of the NAE and CS, LT, TM, and MP wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Genes. The topics of the selected manuscripts cover a wide range of current topics in biomedical research including cancer informatics, transcriptomic, computational algorithms, visualization and tools, deep learning, and microbiome research. In this editorial, we briefly introduce each of the manuscripts and discuss their contribution to the advance of science and technology.The International Association for Intelligent Biology and Medicine (IAIBM) is a nonprofit organization that promotes intelligent biology and medical science. It hosts an annual International Conference on Intelligent Biology and Medicine (ICIBM), which was established in 2012. The ICIBM 2019 was held from 9 to 11 June 2019 in Columbus, Ohio, USA. Out of the 105 original research manuscripts submitted to the conference, 18 were selected for publication in a Special Issue in Genes were further reviewed by a minimum of two additional external reviewers. In the end, 18 manuscripts were selected for publication in the Special Issue, which covered the topics of cancer research, gene expression, single cell sequencing, novel computational algorithms, and microbiome research. In this editorial, we introduce the 18 selected research manuscripts.The International Conference on Intelligent Biology and Medicine ICIBM 2019) was organized and hosted by the International Association for Intelligent Biology and Medicine (IAIBM) and the Department of Biomedical Informatics at Ohio State University from 9 to 11 June 2019 in Columbus, Ohio, USA. The detailed description of the conference and its organization and achievements is summarized in [9 was orgChen et al. published \u201cComputational Cancer Cell Models to Guide Precision Breast Cancer Medicine\u201d . In thisA pathway is a summary of a set of genes that can be connected via their biological process, regulation, mechanism. or phenomenon. Pathways of important function can be alternatively activated in cancer. Wang et al. published \u201cIdentification of Alternatively-Activated Pathways between Primary Breast Cancer and Liver Metastatic Cancer Using Microarray Data\u201d, in which the authors proposed an alternatively-activated pathway mining method based onCirculating tumor DNA (ctDNA) has been found in the bloodstream which originated from cancerous cells. Research on ctDNA has been expanding over the last decade resulting in substantial advancement in the identification of single nucleotide variants from ctDNA. Copy number variation (CNV), which is also considered to be an important cancer biomarker, has been very difficult to detect from ctDNA due to the low amount and complex CNV features. Peng et al. published \u201cCNV Detection from Circulating Tumor DNA in Late-Stage Non-Small Cell Lung Cancer Patients\u201d to address the critical issue of CNV identification in ctDNA . Their mDNA methylation plays a variety of roles in cancer, including a critical role in the control of gene activity, which helps to convert gene expression in normal tissue to a cancerous pattern. Utilizing deep learning techniques, Liu et al. published \u201cDNA Methylation Markers for Pan-Cancer Prediction by Deep Learning\u201d, in which they studied the prognostic value of DNA methylation . Using dFOXO1 and PAX3/7 and observed substantial restructuring of co-expression networks related to fusion status and fusion type.Gene fusion describes hybrid genes that are formed from two independent genes. Gene fusion has been a common feature in cancer genomes and has served as a molecular target in therapeutic development. In Helm et al.\u2019s \u201cGene Co-Expression Networks Restructured Gene Fusion in Rhabdomyosarcoma Cancers\u201d, the authors studied gene fusion features in rhabdomyosarcoma . In thisTumor-infiltrating leukocytes (TILs) are immune cells surrounding tumor cells, and several studies have shown that TILs are potential survival predictors in several types of cancers including liver cancer, which is highly associated with a hepatitis virus. Hsiao et al. studied TIL abundance and compositions concerning hepatocellular carcinomas survival in their manuscript, entitled \u201cTumor-Infiltrating Leukocyte Composition and Prognostic Power in Hepatitis B- and Hepatitis C-Related Hepatocellular Carcinomas\u201d . The autNetwork and biomarker analyses have been heavily utilized for cancer research. Liu et al. combined the concept of both a network and biomarker approach in their paper entitled \u201cNetwork as a Biomarker: A Novel Network-Based Sparse Bayesian Machine for Pathway-Driven Drug Response Prediction\u201d . The autIn the paper entitled \u201cKinetic Modeling of DUSP Regulation in Herceptin-Resistant HER2-Positive Breast Cancer\u201d, Buiga et al. focused on the analysis of dual-specificity phosphatases (DUSPs) in HER2-positive breast cancer , a highlCompelling evidence has shown that microRNAs (miRNAs) can regulate genes and be associated with various cancers through a post-transcriptional suppression regulation mechanism. The dysregulation of miRNA can substantially alter the landscape of the transcriptome level of messenger RNAs (mRNAs). Dai et al. published \u201cIdentifying Interaction Clusters for miRNA and mRNA Pairs in TCGA Network\u201d, which describes a novel cluster scoring method to identify mRNA and miRNA interaction pairs . Their aCryogenic electron microscopy (cryoEM) is an electron microscopy technique applied on samples cooled to cryogenic temperatures and embedded in an environment of vitreous water. It is often used to study structural biology. The analysis of cryoEM often involves a clustering algorithm. Al-Azzawi et al. published \u201cA Super-Clustering Approach for Fully Automated Single Particle Picking in Cryo-EM\u201d, a manuscript that describes a newly developed fully automated super-clustering algorithm for single particle picking in cyroEM micrographs. The authors focused on identifying, detecting, and picking particles of the complex and irregular shapes in micrographs with extremely low signal-to-noise ratio .While single cell sequencing has quickly emerged as a powerful technology for measuring DNA variants or transcriptome abundance at the single cell resolution, numerous challenges currently remain in this new field. One of the major shortcomings of single cell RNA-seq is the excessive zero counts, because only a small fraction of the transcripts sequenced in each cell. This problem can be alleviated by sequencing more, but it is at a high financial cost and also a lack of enough cells available for sequencing. The sparsity of the gene expression for each cell creates additional downstream analysis challenges such as cell type identification. Zand et al. introduced a network-based method, netImpute to battlIn the era of big data, data visualization tools are essential for analyzing massive amounts of information and making data-driven decision. This is no difference in transcriptomic data analysis. Al-Ouran et al. published \u201cA Portal to Visualize Transcriptome Profiles in Mouse Models of Neurological Disorders\u201d, in which they described a new web-based platform for visualizing mouse transcriptome data . The webNon-coding RNA has been the focus of many research studies over the last decade. Porto et al. published \u201cLong Non-Coding RNA Expression Levels Modulate Cell-Type-Specific Splicing Patterns by Altering Their Interaction Landscape with RNA-Binding Proteins\u201d, a study in whichLepisosteus oculatus)\u201d [nfil3 and cry families are different between spotted gar and humans. These findings help decipher the repertoires of the spotted gar\u2019s circadian system and shed light on how the vertebrate circadian clock systems have evolved.A circadian rhythm is a natural internal process that regulates the sleep-wake cycle. While the canonical circadian clock genes and their regulatory mechanisms appear highly conserved, the evolution of clock gene families is still unclear due to several rounds of whole genome duplication in vertebrates. Sun et al. studied circadian clock genes in spotted gar, a non-teleost ray-finned fish, and published their findings in the manuscript \u201cThe Molecular Evolution of Circadian Clock Genes in Spotted Gar (ulatus)\u201d . PhylogeIn data science, a large dataset is often assembled from multiple smaller datasets with heterogeneity. The missing variable has become a common problem when combining datasets, which poses a major challenge for downstream analysis. Bartlett et al. published \u201cForming Big Datasets through Latent Class Concatenation of Imperfectly Matched Databases Features\u201d, to address this problem . The autGenotyping data has been aiding researchers for large genetic association studies for the last two decades. Imputation is an important preprocessing step for combining genotyping data or increasing coverage. Traditional genotype imputation methods are typically based on haplotype-clustering algorithms, hidden Markov models (HMMs), and statistical inference. Chen et al. described their new deep learning-based imputation method in theirThe microbiome studies many microorganisms in a particular environment. Microbiome research has been greatly enhanced by the advancement of 16S rRNA high throughput sequencing. Liu et al. published \u201cChanges in the Microbial Community Diversity of Oil Exploitation\u201d, a study on microbiome in several offshore petroleum production sites . The autICIBM is an annual international conference, which has been held every year since 2012 (except 2017). It promotes a highly interactive and friendly platform for both young and senior researchers to exchange their research, foster collaboration, as well as expand educational activities. Approximately one hundred and seventy researchers and trainees from around the world joined the 2019 conference and contributed to a rich conference program, which included four keynote lectures, four eminent scholar talks, five tutorials and workshops, twelve concurrent sessions, a poster session, and other conference activities. Among the 105 original research manuscripts, we selected 18 for the Special Issue after two rounds of peer reviews. These 18 manuscripts describe innovative, computational works in the field. We expect these manuscripts to promote further investigation in the same or similar topics, and lead to more research toward translational clinical applications."} +{"text": "Objective: Mild cognitive impairment (MCI) is an important risk state for dementia, particularly Alzheimer\u2019s disease (AD). Depression, anxiety, and apathy are commonly observed neuropsychiatric features in MCI, which have been linked to cognitive and functional decline in daily activities, as well as disease progression. Accordingly, the study\u2019s objective is to review the prevalence, neuropsychological characteristics, and conversion rates to dementia between MCI patients with and without depression, anxiety, and apathy.Methods: A PubMed search and critical review were performed relating to studies of MCI, depression, anxiety, and apathy.Results: MCI patients have a high prevalence of depression/anxiety/apathy; furthermore, patients with MCI and concomitant depression/anxiety/apathy have more pronounced cognitive deficits and progress more often to dementia than MCI patients without depression/anxiety/apathy.Conclusions and Implications: Depression, anxiety, and apathy are common in MCI and represent possible risk factors for cognitive decline and progression to dementia. Further studies are needed to better understand the role and neurobiology of depression, anxiety, and apathy in MCI. Mild cognitive impairment (MCI) represents a transitional stage between healthy aging and dementia. Subjects with MCI complain about cognitive impairments, have documented cognitive deficits relative to age- and education-matched controls\u2014although they are less impaired than patients with dementia\u2014and have largely intact activities of daily living Sanford, . Dependiin vivo by biomarkers in the National Institute on Aging and Alzheimer\u2019s Association Research Framework in Jack et al. presented a reanalysis of subpopulations already included in other studies; (b) reported a patient population of less than 10 patients; or (c) were commentaries, technical notes, or review articles summarizing the results of previous studies.We included original studies from tertiary referrals and population-based studies written in English that examined populations with MCI, depression, anxiety and apathy. A systematic literature search was performed using the PubMed database. Search terms were via depression compared to MCI patients without depression (13.5%; Lee G. J. et al., However, there is research that demonstrated contrary conclusions. A 3-year prospective study of MCI outpatients demonstrated no increased risk of AD in patients with symptoms of depression (Palmer et al., Vascular factors play an important role in depression within preclinical dementia. In MCI patients, new onset of depression was associated with deep subcortical cerebral white matter hyperintensity severity (Kim et al., The reported prevalence of apathy in MCI is between 10.7% and 44.8% (Palmer et al., Patients with MCI with apathy have an increased risk of dementia, independent of depression. A systematic review found that apathy was associated with an approximately two-fold increased risk of dementia in memory clinic patients (van Dalen et al., Anxiety symptoms have been studied less than depression, and the relationship between anxiety and cognition is complex. The reported prevalence of anxiety in MCI patients ranged between 9.9%\u201352% (Lyketsos et al., Generalized anxiety disorder is the main anxiety disorder associated with poor global cognitive functioning, and this association is moderated by sex but not by the presence of depressive episodes (Potvin et al., Anticipatory anxiety is significantly associated with earlier conversion to AD, but this association does not remain significant following an adjustment for cognitive status at the baseline; anxiety for upcoming events and purposeless activity frequently co-occur, which indicates anticipatory anxiety may be a marker of severity rather than an independent predictor of disease progression (Gallagher et al., Other investigators have failed to find an association between anxiety symptoms in patients with MCI and an increased risk of conversion to AD (Robert et al., Depression, anxiety, and apathy are common in MCI patients and are important indicators in the progression to dementia in MCI patients, which emphasizes the importance of assessing depressive symptoms as well as anxiety and apathy in the early stages of cognitive impairment. Further studies are needed to better understand the role and neurobiology of depression, anxiety, and apathy in MCI. Indeed, further studies on observation of larger patient populations and long follow-up are needed.LM designed and wrote the manuscript.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Meanwhile, the Editors became aware of adenouncement published by independent journalists from the \u201cFor Better Science\u201d websiteincluding this paper. This denouncement consisted of potential data falsification and/orinaccuracy of results in western blots and flow cytometry plots.As per consensus between the Authors and the Editors-in-Chief of the Brazilian Journal ofMedical and Biological Research (BJMBR), the article titled \u201cEffect of lncRNA HULCknockdown on rat secreting pituitary adenoma GH3 cells\u201d that was published in year 2019,volume 52, issue 4, has been retracted."} +{"text": "APOE) is the best-known and has the strongest association with AD development. AD probability decreases in carriers of the e2 variant of the APOE gene (APOE-e2), whereas APOE-e4 is believed to be a strong risk factor is a neurodegenerative condition that inevitably impairs cognitive functions and influences a patient's behavior, mood, and self-reliance. Due to demographic changes, AD and other age-associated diseases have become increasingly common and burdensome for families, as well as entire societies. It is extremely important that we learn more about specific mechanisms that can be linked to the development of the disease. The main symptoms of AD, observed in the central nervous system, are brain atrophy and loss of neurons and synapses. They are believed to result from excessive aggregation of tau protein and amyloid plaques (composed of \u00df-amyloid). However, neither the initial cause nor the detailed chain of events that lead to this type of neurodegeneration are known. No deterministic genes were identified for late-onset Alzheimer's disease (LOAD), but several risk genes seem to be involved in its pathogenesis. The gene coding apolipoprotein E of negative impact on human brain function in AD. Results indicating enhanced APP synthesis are in agreement with studies showing higher levels of A\u03b2 in brains of APOE-e4 carriers, examined post-mortem and other risk-genes. Moreover, it was shown that neurons need astrocytes and microglia to eliminate redundant synapses homeostasis and synaptic stability maintenance, with APOE-e4 having the most negative impact on the brain. APOE-e4 limits the astrocytes' ability to recycle and clear extracellular cholesterol (Fernandez et al., APOE-e4 showed accumulation of cholesterol and could not efficiently fulfill their role related to clearance of A\u03b2 (Lin et al., The question arises: how is it possible to link these cell-level studies with the same ranking (g et al. , and preg et al. , showed g et al. . APOE haAPOE-e4 that leads to an increase in toxic A\u03b2 forms and impairs astrocytes' function, which can initiate the whole cascade of changes related to later loss of synapses and cognitive functions. It may indicate that, in the brains of APOE-e4 carriers, AD risks begin to accumulate from early developmental stages when too many synapses are formed and not enough of them are pruned (Chung et al., APOE-e4 risk related to loss of cognitive functions is predominant for persons older than 50 years of age, people homozygous for APOE-e4 may experience the risk much earlier, just after 40 years of age (Liu et al., APOE-e4 could affect the brain even earlier, changing its structure, function and neurochemistry (see DiBattista et al., APOE-e4 young carriers perform equally well or even much better in a variety of cognitive tasks compared to non-carriers (Mondadori et al., APOE-e4 on neuronal signaling pathways throughout the lifespan may help us to identify early biomarkers and target therapy against AD in the future.Perhaps it is the initial higher number of synapses and APP in neurons with PD wrote the first draft. EK critically edited and improved the manuscript. PD and EK read and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Exosomes (EXs) and extracellular microvesicles (EMVs) represent a diverse assortment of plasma membrane-derived nanovesicles, 30\u20131000 nm in diameter, released by all cell lineages of the central nervous system (CNS). They are examples of a very active and dynamic form of extracellular communication and the conveyance of biological information transfer essential to maintain homeostatic neurological functions and contain complex molecular cargoes representative of the cytoplasm of their cells of origin. These molecular cargoes include various mixtures of proteins, lipids, proteolipids, cytokines, chemokines, carbohydrates, microRNAs (miRNA) and messenger RNAs (mRNA) and other components, including end-stage neurotoxic and pathogenic metabolic products, such as amyloid beta (A\u03b2) peptides. Brain microglia, for example, respond to both acute CNS injuries and degenerative diseases with complex reactions via the induction of a pro-inflammatory phenotype, and secrete EXs and EMVs enriched in selective pathogenic microRNAs (miRNAs) such as miRNA-34a, miRNA-125b, miRNA-146a, miRNA-155, and others that are known to promote neuro-inflammation, induce complement activation, disrupt innate\u2013immune signaling and deregulate the expression of neuron-specific phosphoproteins involved in neurotropism and synaptic signaling. This communication will review our current understanding of the trafficking of miRNA-containing EXs and EMVs from astrocytes and \u201cactivated pro-inflammatory\u201d microglia to target neurons in neurodegenerative diseases with an emphasis on Alzheimer\u2019s disease wherever possible. As sucl., 2014 ; Rodrigul., 2014 ).(i) were first characterized about ~40 years ago as a heterogeneous group of cell cytoplasm-derived intracellular micro-particles (MPs) of endosomal origin ; (ii) are variable in their content and morphology, ranging in size from about ~30 to ~100 nm in diameter; (iii) are released by a variety of central nervous system (CNS) cells whose function was originally described as a cytological basis for intercellular signaling and communication : al. 1981 ; Columboal. 1981 ; Jiang eal. 1981 ; Mathewsal. 1981 ; Arbo etal. 1981 ; Martinsal. 1981 ). EXs oral. 1981 ; Arbo etal. 1981 ). Extracal. 1981 ; Webers al. 1981 ). Similaal. 1981 . Astrocyal. 1981 ; Leidal al. 1981 ). Some oal. 1981 ; Serpental. 1981 ; Vanherlal. 1981 ; Mathieual. 1981 ; Arbo etal. 1981 ; Seyedraal. 1981 ; Upadhyaal. 1981 ). As mosal. 1981 ; Urbanelal. 1981 ; Federical. 1981 ; Groot aal. 1981 ; Stahl eal. 1981 ; Upadhyaal. 1981 ; VanherlEscherichia coli and Bacteroides fragilis, species of the family Brassicaceae such as Arabidopsis thaliana, Protists, protozoa such as Amoeba proteus and slime molds such as Dictyostelium discoideum, nematodes such as Caenorhabditis, and up the evolutionary scale to a wide range of mammals including humans. The biogenesis, secretion and the release of EXs, MPs and/or EMVs into the extracellular space or external environment for the purpose of mediating intercellular communication or transmitting DNA- or RNA-encoded genetic information between different cell types and the environment is therefore a very ancient and conserved evolutionary process tract bacteria such as l., 2014 ; Arbo etl., 2014 ). From wl., 2014 ; Cong etl., 2014 ; Avsar el., 2014 ).(i) are released under normal physiological conditions, but are also discharged from parent cells upon cellular activation, hypoxia and/or hyperoxia, senescence, apoptosis and disease via a paracrine- and endocrine-type type action to their target cells; (ii) represent one of the major biological mechanisms for genetic exchange, immune signaling and the spread of inflammation and disease between cells of the host; (iii) EX and EMV trafficking in the mammalian CNS is a particularly robust, active and dynamic process . The hul., 2007 ; Seyedral., 2007 ; Song etl., 2007 ). Indeedl., 2007 ; Vanherll., 2007 ).extracellular directional strategy\u201d for vesicular sorting and translocation over both short and long distances . Many ol., 2015 ; Serpentl., 2015 ).(i) that these vesicular organelles consist of a unique repertoire of cytoplasmic components representing cellular, molecular and genetic information that is a direct reflection of the unique biological condition of the parent cell\u2019s cytoplasm at the time of vesicular release; (ii) that these microparticles play important roles as enveloped proteolipids, a nucleic acid-enriched \u201cinformation packet\u201d in a complex extracellular communication network; (iii) that EXs and EMVs may reprogram recipient, adjacent cells and/or distant tissues as CNS-resident cells involved in immune-surveillance and the maintenance of normal cellular homeostasis, while also contributing to neuropathology during disease; (iv) that the molecular content and rates of production and secretion of EXs and EMVs vary greatly depending on the cell-type and physiological state of the cells of vesicular origin . Attestl., 2018 ; Barnes l., 2018 ; Leidal l., 2018 ).Atropa belladonna) and higher animals (Homo sapiens) over many billions of years of evolution are the smallest known gene information-carrying nucleic acids yet discovered; (ii) are important posttranscriptional and epigenetic regulators of mRNA abundance, speciation and complexity in aging, development, neurological health and disease processes; (iii) play pivotal roles in the initiation, development and propagation of many human CNS disorders including progressive terminal cancers and lethal, age-related neurological syndromes with an inflammatory component; (iv) are loaded into both EXs and EMVs and are a typical component of their vesicular cargoes are soluble, amphipathic, single-stranded non-coding RNAs (sncRNAs) 18- to 25-ribonucleotides (nt) in length whose RNA sequences have been very highly selected. Some miRNAs, such as the neurologically relevant miRNA-378, are very highly conserved, and their core ribonucleotide sequence has remained virtually unchanged in plants . Abundal., 1990 ; Lukiw 2l., 1990 ; Lukiw 2l., 1990 ; Zhao etl., 1990 ; Hosaka l., 1990 ; Slota el., 1990 ; Swarbril., 1990 ; Briand Homo sapiens while only about 25\u201330 individual miRNA species, or fewer, are abundant and easily detected in the human brain and retina . The upl., 2016 ; Lukiw 2l., 2016 ; Zhao etl., 2016 ; Wang etl., 2016 ; summaril., 2016 ). Importl., 2016 ; Zhao anl., 2016 ).pro-inflammatory miRNAs\u201d, including miRNA-34a, miRNA-125b, miRNA-146a, miRNA-155 and others in the parenchyma of the temporal lobe neocortex (Lukiw 2007 [(i) from the microarray analysis of mast cells involved in innate and adaptive immunity, autoimmunity, and inflammation from studies of specific miRNAs, mRNAs and angiogenic proteins in glioblastoma and neuroblastoma tumor cells that have released EXs and EMVs , Huntington\u2019s disease (HD), stroke and other neuro-inflammatory degenerative conditions. This indicates a definitive role for EX and EMV miRNA cargoes in neurological disease processes with an inflammatory component which may have considerable diagnostic, prognostic and/or therapeutic value brains (Lukiw 2007 ) This inkiw 2007 ; Valadi kiw 2007 ; Hunter kiw 2007 ; Sethi akiw 2007 ; Zhao etkiw 2007 ; Hammondkiw 2007 ). The fil., 2007 ); (ii) fl., 2008 ; Briand l., 2008 ; Skog etl., 2008 ). As disl., 2018 ; Mathieul., 2018 ; Li et al., 2018 ; Serpentl., 2018 ; Upadhyal., 2018 ). Becausl., 2018 ; BarbagaAccumulating evidence continues to implicate secreted miRNAs, including EX and EMV-encapsulated miRNAs, in the pathogenic spreading of progressive, age-related and incapacitating neurodegenerative diseases with an uncontrolled or deregulated inflammatory component and synaptic deficits. These include Alzheimer\u2019s disease (AD), age-related macular degeneration (AMD), Parkinson\u2019s disease (PD), Huntington\u2019s disease (HD), prion disease (PrD), multiple sclerosis (MS), Japanese and viral-induced encephalitis and many related amyloidopathies, tauopathies and synucleinopathies , hypoxil., 2016 ), by coml., 2016 ) and micl., 2016 ; Lukiw el., 2016 ; Alexandl., 2016 ). As obsl., 2016 ; Ludwig l., 2016 ).(i) the signals and pathways essential for stimulation and the origin of their formation, as well as the mechanism of their release from many different cell types in the CNS and their proficiency for modulating functions of target cells; (ii) the molecular-genetic injury and/or environmental factors which stimulate their release for this evolutionarily-conserved type of information communication system amongst astrocytes, microglia and neurons; (iii) their increased production and release during the initiation and spread of progressive age-related inflammatory neurodegeneration; (iv) the actual molecular contents, stoichiometry and packaging of the contents in the vesicles themselves; (v) the magnitude and signaling impact of their plasma membrane-packaged vesicular cargo; (vi) the regulation of their trafficking and targeting to neuronal cells via plasma membrane-mediated cell-surface reception mechanisms; (vii) the lipidomic, proteomic and transcriptomic profiles of these vesicles and what miRNA and/or mRNA encoded information these vesicles may be carrying; (viii) whether or not EXs and/or EMVs can transfer their miRNA-enriched intraluminal cargoes to other cell types and/or to other species; (ix) the role of circular RNA (circRNA) which have been shown in some cases to act as natural \u201canti-miRNA sponges\u201d of specific miRNA activities , microparticles (MP), exosomes (EX) and extracellular microvesicles (EMV), the nature of their vesicular cargoes and miRNA composition. These include: kiw 2013 ; Zhao etkiw 2013 ; Fakhourkiw 2013 ; Pogue akiw 2013 ; Zhao ankiw 2013 ; Avsar ekiw 2013 ; Groot akiw 2013 ; Hou et kiw 2013 ; Li et akiw 2013 ; Ma et akiw 2013 ; Serpentkiw 2013 ). A singThe neurobiology of EX and EMV genesis, release, translocation and uptake by target cells, and their containment of select miRNA populations enriched in the CNS, indicate that they are significant components of a highly dynamic system of intercellular communication via extracellular translocation and targeting in brain cell health, aging and disease. Multiple independent studies indicate that while vesicle-mediated intercellular signaling is important in the homeostatic maintenance of brain cell functions, they have a substantial role in the proliferation of injury, cancer and inflammatory neurodegeneration signaling, as is observed in the AD brain. Many of the details of the mechanisms by which EXs and EMVs and their miRNA cargoes are generated and released by the activation of astrocytes and microglia and their trafficking to target brain cells, primarily neurons, remain to be further clarified. A greater understanding of the mechanisms underlying EX and EMV biogenesis, cargo selection and loading, vesicle release, translocation exterior to the cells of origin and targeting to adjacent or distant neural cells remains critical for unlocking the immense neurobiological and therapeutic potential for these ubiquitous organelles. Not only could an increased understanding of EX and EMV systems and their containment of miRNAs in the brain be useful in the treatment of CNS injuries and progressive age-related inflammatory neurodegeneration, but may also prove useful as delivery vehicles for therapeutic miRNAs, anti-miRNAs and both bioactive and neuroactive pharmaceuticals."} +{"text": "Phase-contrast enhanced micro-computed tomography reveals huge discontinuities at the interfaces between dental fillings and the tooth substrate. Despite the complex micromorphology, gaps in bonding could be visualized and quantified in 3D. ImageJ. PCE-CT at sub-micrometre resolution provided images with an impressive increased contrast and detail when compared with laboratory micro-computed tomography. The interface between the dental adhesive and the tooth was often strongly disrupted by the presence of large debonded gaps . The thickness of the gaps spanned 2\u2005\u00b5m to 16\u2005\u00b5m. There was a large variability in the distribution of gaps within the bonding area in each sample, with some regions around the canal exhibiting up to 100% discontinuity. Although only several micrometres thick, the extensive wide gaps may serve as gateways to biofilm leakage, leading to failure of the restorations. They can also act as stress-raising \u2018cracks\u2019 that are likely to expand over time in response to cyclic mechanical loading as a consequence of mastication. The observations here show how PCE-CT can be used as a non-destructive quantitative tool for understanding and improving the performance of clinically used bonded dental restorations.Bonding of resin composite fillings, for example following root-canal treatment, is a challenge because remaining gaps grow and lead to failure. Here, phase-contrast-enhanced micro-computed tomography (PCE-CT) is used to explore methods of non-destructive quantification of the problem, so that countermeasures can be devised. Five human central incisors with damaged crowns were root-filled followed by restoration with a dental post. Thereafter, the crowns were rebuilt with a resin composite that was bonded conventionally to the tooth with a dental adhesive system (Futurabond U). Each sample was imaged by PCE-CT in a synchrotron facility with a pixel size of 650\u2005nm. The reconstructed datasets from each sample were segmented and analysed in a semi-automated manner using To fix this, the dentist restores the tooth shape and function using a combination of bonded composite materials that need to tightly adhere to the substrate. Biomaterials are typically chosen for mechanical durability and for aesthetic considerations, while matching the mechanical behaviour to the remaining tooth structure. Such dental biomaterials are used to return the tooth into function for patient satisfaction, and specifically to make sure the reconstruction withstands the repeating loads of mastication for many years. In fact, a main objective of contemporary dental treatment is to establish strong and continuous bonds between the filling and the tooth substrate. Current treatment protocols advocate bonded sealing for a variety of reasons. These include increased mechanical stability of the restoration due to an improved distribution and resistance to stresses, as well as prevention of bacterial percolation at the interface of the crown to attributes of the bonding system to increase contrast. 3D measurements of whole, root-treated teeth have previously been demonstrated using PCE-CT, revealing different density dental materials at micrometre resolution accentuates interfaces due to the combined effects of high flux \u2018partial-coherence\u2019 X-rays. This facilitates the differentiation between materials with similar density by the Ethical Review Committee of the Charit\u00e9 Universit\u00e4tsmedizin Berlin, Germany, and were stored in an antiseptic solution prior to the experiment. Each tooth had its root canal treated following a standardized protocol each tooth was restored using a fibreglass dental post. This was cemented into the root canal with a self-adhesive resin cement, following manufacturers recommendations . Such d2.4.Each tooth crown was restored immediately after post cementation. For that, the exposed tooth cervical area was acid etched for 10\u2005s, followed by rinsing and air drying. A dental adhesive system was applied following manufacturers recommendations, including light curing for 10\u2005s Table 1. A stand2.5.After tooth restoration, each sample was mounted in a transparent vial , padded with wet foam to maintain humidity and to avoid dehydration during imaging.ImageJ 1.52d, National Institute of Health, USA; Amira ZIB-Edition, Konrad-Zuse-Zentrum f\u00fcr Informationstechnik Berlin, Germany). The cervical areas, including the rim between root and the crown restoration were\u00a0selected for imaging by PCE-CT in a synchrotron. Each sample was scanned on beamline ID19 of the European Synchrotron Radiation Facility using inline propagation-based contrast microtomography was used to first image each specimen . Following reconstruction the architectures of the restorations were examined in both 2D and 3D ]. The resulting binary images had their different sets of connected pixels and voxels individually numbered and labelled using the \u2018Connected components Labelling\u2019 algorithm. The different components localized to the adhesive layer were then visually selected, discarding the irrelevant (outside of the interface) labels by using the \u2018Select Labels\u2019 function of MorpholibJ. The final Boolean volume comprising only the interfacial gaps between adhesive and tooth was overlaid onto the original volume , is due to the cutting lines, used to remove excess soft cement during restoration construction: a sharp straight scalpel was used to clear overflow prior to crown construction. Thus PCE-CT even reveals laying steps during restoration fabrication. With 2D slices containing the conditioned tooth area and gap, the percentage of gaps at the interface was calculated. Radial, orientation-specific variations in the non-bonded regions were quantified to assess the distributions of gaps at the interface. With the centre of the restoration/post defined as a pivot, the tooth and gap areas were divided in 18 equal segments (sectors) spanning 360\u00b0 around the tooth long axis, each extending 20\u00b0 on the tooth surface. For each sector, the ratio of gaps to the area of bonding was quantified and plotted against the azimuthal angular axis.To isolate debonding and interfacial gaps, the datasets were processed using the free extension package me Fig. 5 to verifme Fig. 5. The ext3.Laboratory \u00b5CT scans generated datasets that reproduced the approximate geometry and the main components of the tooth restoration within the root and the reconstructed crown. In such data, however, there was no difference in contrast between the resin composites used for crown reconstruction and the resin cement Fig. 5, nor wasPCE-CT at sub-micrometre resolution provided 3D datasets with an impressive contrast and a remarkable amount of detail . The micromorphology of the tooth and restoration were fully visible, revealing micrometre-diameter dentin tubules, highlighting the presence of fillers in the different resin composites and well reproducing the layout of single fibres within the fibreglass post. Example overviews and slices in the datasets are shown in a longitudinal slice in Fig. 6e.g. Fig. 6The enhanced contrast brought about with PCE-CT highlights gaps between the adhesive Fig. 6, tooth sThe results from the analysis of 18 sections of each sample, shown in the graphs of Fig. 74.in\u00a0vitro, it is extremely difficult to produce a predictable sealed bonded interface. From a biological standpoint, establishing a continuous interface between tooth and adhesive is a major objective of treatment. It is a clinical objective aimed at preventing damages associated with water sorption, infiltration of bacteria and bacterial by-products, reportedly associated with secondary caries and/or re-infection of the root canal system at the interfaces in adhesively restored human teeth. Of great concern is the observation that in each tooth at least 50% of the visible section of the outer imaged rim exhibited some form of gap, as seen\u00a0in the central column of Fig. 7et al., 2019et al., 2004et al., 2005et al., 2019et al., 2014et al., 2015et al., 2019et al., 2014et al., 2014et al., 2019Due to the lack of contrast and resolution in the laboratory \u00b5CT images, it was not possible to observe the adhesive layer or the interfaces between materials. PCE-CT resolved this problem as it is extremely effective and may be the only possible means to non-destructively image interfacial gaps between the low-density polymer material and other structures within the tooth restorations (Soares et al., 2004et al., 2005et al., 2019et al., 2014et al., 2015et al., 2019Discontinuities at the interface between restorative materials and tooth substrate have been reported using high-contrast dyes such as silver nitrate. This radiopaque liquid has been widely used in dental research due to its high contrast in radiographs (Mollica et al., 2015et al., 2011et al., 2019et al., 2018The use of semi-automated image segmentation presented here will benefit from further improvements but already has many advantages. Since the data are obtained non-destructively, effects of artefacts and filtering can be tested and the 3D data can be quantified in a repeatable, quantitative operator-independent way (Carrera et al., 1996et al., 2007et al., 2004et al., 2005et al., 2011et al., 2014et al., 2016et al., 2016et al.,\u00a02016et al., 2019et al., 2011et al., 2018et al.,\u00a02020Synchrotron \u00b5CT imaging is an effective tool for examining different biological samples. The high photon flux density and almost parallel beams with a wide range of energies is suited for imaging both low- and high-density structures. Phase-contrast imaging (Cloetens et al., 2017in vitro performance record, comparable with other universal adhesive systems (Chen et al., 2015et al., 2015et al., 2010The adhesive used for testing here was Futurabond U, a clinically used universal adhesive system. It can be deployed either with or without prior acid etching of the tooth substrate (Sofan 5.in vitro. It is also a valuable tool for imaging interfacial gaps between thin polymer adhesives and tooth substrate in restored hydrated, treated teeth in 3D. Flaws in the thin polymer bonding layer were quantified non-destructively. Although a possible quantitative data processing pipeline was proposed, much more can be done for improved image segmentation and analysis. The use of PCE-CT will assist efforts to systematically assess the integrity of contact between different clinically used dental materials.The present work provides details about steps needed to measure gaps and the areas that they affect, without the use of any tracer dye and while strictly adhering to clinically used materials and procedures allowing reproducibility and comparability. The observations here clearly establish the use of PCE-CT for advancing our understanding about the ability of clinically used dental adhesive systems to form continuous interfaces with tooth tissues when measured"} +{"text": "Background: CrossFit\u00ae training is a high-intensity functional training program that aims to increase physical functional performance through biochemical responses, i.e., hormonal, metabolic, and inflammatory responses. Most hormonal, metabolic, and inflammatory changes induced by CrossFit\u00ae training have been reported in isolated clinical studies. The purpose of this review was to systematically explore the existing literature on characterization of hormonal, metabolic, and inflammatory responses resulting from CrossFit\u00ae training.Methods: A systematic search of the literature was conducted in PubMed, Web of Science and Scopus from August 2019 to October 2019. Studies were selected through critical review of the content. Using specific keywords, 623 articles were found, of which 597 were excluded for ineligibility, and 25 were eligible. The papers were separated according to subject area: hormonal (n = 8), metabolic (n = 19), and inflammatory (n = 6) changes. All were published between 2015 and 2019.Results: This review reveals potential effects of CrossFit\u00ae training on hormonal, metabolic, and inflammatory responses. However, studies had low levels of evidence and reliability due to methodological limitations.Conclusion: In summary, the results showed a greater volume and intensity of workouts accentuate the responses, that are of paramount importance for improving understanding of the effects of CrossFit\u00ae training and serve as a basis for prescribing future exercise protocols. The training consists of a combination of different exercise elements: cardiovascular (CV), gymnastic and weightlifting exercises , is structured into joint mobility, warm-up, a technical portion, and the main portion. WODs are performed with short or no breaks between exercises, repetitions, and rounds is a training program that emphasizes functional movements. HIFT uses a combination of movements, and self-selected time periods of work and rest , exercise modalities , methods (for time or AMRAP), and intensities (absolute or relative load). In reviewing the literature, it was observed that most of the hormonal, metabolic, and inflammatory changes related to CrossFit\u00ae training have been reported in isolated clinical studies.Studies on the physiological changes resulting from CrossFit\u00ae training.To date, no systematic review of such changes has been performed. The purpose of this review was to systematically review the existing literature on characterization of hormonal , insulin-like growth factor 1 (IGF-1), adrenaline, noradrenaline, metabolic , and inflammatory responses associated with CrossFitA systematic literature search was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines , metabolic and inflammatory (IL-6 and IL-10) parameters. CK, that is biomarkers of muscle damage, was included in inflammatory responses.\u00ae training interventions.Study design: Randomized and non-randomized trials, using either cross-over or parallel groups, comparing different types of CrossFit\u00ae training; (3) a sample of men and women; (4) studies that investigated at least one hormonal, metabolic, or inflammatory/muscle damage variable relevant to the analysis in the present study. Studies were excluded in the following cases: (1) duplicate articles; (2) articles that were not in the English language; (3) articles that presented training protocols not based on CrossFit\u00ae training; (4) articles with special populations; (5) articles that were systematic reviews, conference abstracts, dissertations, theses, and book chapters.The specific inclusion criteria were as follows: (1) articles that were original research; (2) intervention based on CrossFitThe systematic literature search was carried out until October 2019 using the following databases: PubMed, Web of Science and Scopus. The articles were searched using a combination of keywords corresponding to the theme of the review: CrossFit OR \u201chigh-intensity functional training\u201d OR HIFT. Medical Subject Headings (MeSH) was consulted to check possible entry terms related to the keywords. After combining the research results and discarding duplicate studies in the databases, two researchers (NJ and MRD) independently selected titles and abstracts to identify relevant studies. The included articles were retrieved, read in full (full text) and independently assessed for eligibility by the same two researchers (NJ and MRD) according to the criteria described above. A meeting was held, and in the case of disagreement regarding the selection of articles, a third author (JN) was consulted to resolve the disagreement.\u00ae training protocols, and main conclusions was extracted (see Standardized data extraction forms were completed by two researchers (NJ and MRD) and verified by another researcher (JN). Information on the type of study design, characteristics of the participants, sample size, time of experience in the profile, data collection, CrossFitcted see \u20133. The eThe Cochrane Collaboration's risk of bias assessment tool was used to evaluate the internal validity of the studies were selected for a complete full-text review. Finally, 25 articles were selected . All were published between 2015 and 2019, and most were of a cross-sectional design.n = 21; pre-training and post-training or comparison between groups), longitudinal or descriptive of a cross-sectional cohort .The study design was described as acute assessment, in which the HR remained between 85.9 and 97.4% of the maximum HR . There was a consensus that lactate levels are high immediately after a CrossFiti et al. , when evGlycemia was often investigated (8 studies). Similar to lactate, several studies showed an increase after independent WOD (Tibana et al., \u00ae training (Cadegiani et al., Cholesterol, creatinine, GOT and GPT were each investigated in only one study. Cholesterol showed no differences after training (Shaw et al., Six studies (Tibana et al., CK seemed to increase after training (Durkalec-Michalski et al., IL-6 increased after WOD-independent training, while IL-10 increased as a function of WOD characteristics (Tibana et al., \u00ae training. The results of this systematic review showed that there are still few studies for each observed variable. Of the 25 studies analyzed, all had different training protocols regarding the training stimulus administered. This methodological difference was found because CrossFit\u00ae training is constantly varied and consists of a combination of different exercise elements: CV, gymnastic and weightlifting exercises (Glassman, \u00ae training seem to be related to the training variables, and the protocols with more volume and intensity provided greater biochemical responses. In addition, psychological factors, such as pre-competitive anxiety, can alter the physiological status of athletes (Mangine et al., The purpose of this review was to systematically examine the existing literature on characterization of hormonal, metabolic, and inflammatory responses associated with CrossFit\u00ae training practitioners and create an environment conducive to an increase in cortisol (Mangine et al., The studies demonstrated that increases in testosterone and cortisol levels occurred after WODs, with longer recovery intervals (Mangine et al., \u00ae training.Some limitations were observed in the study by Mangine et al. , who eva\u00ae training, while cortisol levels decreased (Poderoso et al., Chronically, testosterone levels in men rose after 6 months of CrossFitThe training dose required to cause hormonal changes is difficult to determine. Notably, training with a greater volume promoted increases in GH concentrations (Kliszczewicz et al., \u00ae training practitioners have higher levels of testosterone and GH and lower levels of noradrenaline than practitioners with overtraining syndrome. The higher levels of noradrenaline in practitioners with overtraining syndrome may be a compensatory attempt to maintain performance during exercise due to reduced conversion of catecholamines to metanephrines (Cadegiani et al., \u00ae training has a high metabolic component may explain the increases in GH levels (Kliszczewicz et al., On the other hand, increases in testosterone levels may be closely related to the health of the participants. In a descriptive cross-sectional study, Cadegiani et al. showed tCatecholamines showed acute elevations after WODs (Kliszczewicz et al., \u00ae practitioners with other sport athletes, the responses may differ. From this perspective, Arruda et al. (\u00ae training (Poderoso et al., When comparing the chronic hormonal responses of CrossFita et al. observed\u00ae training studies was blood lactate. An acute increased lactate concentration response was observed after training sessions (Fernandez-Fernandez et al., \u00ae training, WODs generally do not have a standard break time, i.e., as the training is \u201cfor time\u201d or AMRAP, the intervals are self-selected according to the suitability of the participants. Therefore, this characteristic can keep lactate elevated for a longer duration after the session (Goto et al., The main metabolic marker evaluated in CrossFit\u00ae training practitioners are inconclusive.As for chronic metabolic responses, the lack of change in lactate response may be the result of the intensity utilized for each WOD. It must also be considered that pre-training lactate was not registered (Murawska-Cialowicz et al., \u00ae training session due to increased catecholamines. The increase in the glycemic rate in response to a training session is due to the need for greater utilization of glucose to meet the energy required for the sport, which has the particular characteristic of always being performed at high intensity (Glassman, Blood glucose was another variable observed. According to Tim\u00f3n et al. and Klis\u00ae training session (Shaw et al., Cholesterol response does not appear to be affected by a CrossFit\u00ae training performance and aerobic capacity. Although the primary objective of the study was to examine the effectiveness of a sodium bicarbonate supplementation protocol, the CK response was similar, regardless of the supplement, increasing after the WOD. Another study found an increase in CK level after different WODs, which continued for up to 24 h (Tim\u00f3n et al., Acute muscle damage responses through CK were investigated in three studies (Durkalec-Michalski et al., \u00ae training practitioners to a higher volume and intensity of training for successive days may expose the practitioner to the risk of damage associated with muscle cell necrosis.In addition to being an indicator of muscle damage, CK levels have been shown to be high in people with rhabdomyolysis (Honda et al., \u00ae competition. In contrast, Heavens et al. (IL-6 and IL-10 were evaluated in two studies (Tibana et al., s et al. showed ts et al. used mens et al. studied s et al. .\u00ae training on hormonal, metabolic and inflammatory factors. However, studies evaluating such aspects have a low level of evidence and reliability due to methodological limitations and biases that hinder the convergence of results. Apparently, hormonal, metabolic, and inflammatory stress marker levels increase after CrossFit\u00ae training, regardless of the protocol used. However, a greater volume and intensity of workouts accentuate the responses. Some parameters are inconclusive, such as blood glucose and IL-6 and IL-10 levels, due to different results and the small number of studies. Thus, this review sheds light on specific knowledge gaps that should be further investigated. Nevertheless, the results are of paramount importance for improving understanding of the effects of CrossFit\u00ae training and serve as a basis for prescribing future exercise protocols.The present review demonstrates the potentially significant effect of CrossFitAll relevant data is contained within the article. Further inquiries can be directed to the corresponding author.NJ structured and designed the research. NJ and MD carried out the review of studies. JGV and MD realized risk of bias. NJ, JN, JGV, MD, and JV contributed to the conception and writing of the article, reviewing, and editing the manuscript. DB corrected the final version and English grammar. All authors approved the final version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Various bacteria, archaea, and microbial eukaryotes also evolve H2 as a diffusible end product during fermentative metabolism through the activity of [FeFe]- or [NiFe]-hydrogenases (Horner et al., 2 is a facultative trait that is regulated through the expression and maturation of hydrogenases (Schwartz et al., 2 represents a substrate that organisms utilize to supplement their energy metabolism, thereby allowing for an expansion of their niche space in ecosystems where other sources of reductant are low or variable in supply (e.g., Amenabar et al., Approximately a third of sequenced microorganisms, spanning at least 70 microbial phyla, encode hydrogenases and are thus predicted to be capable of interconverting H2 in ecosystem level processes is increasingly being realized in both environmental and biomedical settings. A wide range of ecosystems have now been described where H2 cycling supports the bulk of primary production and where it forms the basis by which species interact, leading to ecologically structured communities. Much of the research on H2 metabolism to date has focused on ecosystems where H2 is present at elevated concentrations due to biological activity (e.g., anoxic sediments, gastrointestinal tracts; S\u00f8rensen et al., 2 can serve as source of reductant for aerobic soil microorganisms and that this can influence the composition of the atmosphere (Conrad, 2 metabolism is critical for the virulence of numerous pathogens, including Helicobacter, Clostridia, and Enterobacteriaceae (Kaji et al., The implications of H Conrad, . In para2 metabolism from the molecular to the ecosystem scale. In the area of anaerobic metabolism, there are articles exploring the metabolism of H2-metabolizing bacteria capable of sulfate reduction, acetogenesis, halorespiration, and fermentation. Two articles investigate H2 oxidation in sulfate-reducing bacteria using the model system Desulfovibrio vulgaris (Fauque et al., Smith et al. present a mathematical model of the growth and metabolism of this bacterium, whereas L\u00f6ffler et al. investigate the kinetic isotope fractionation associated with its H2 oxidation activity. A comprehensive review led by Schuchmann et al. covers recent advances in understanding clostridial H2 metabolism; it details the discovery and characterization of multimeric electron-bifurcating [FeFe]-hydrogenases, including those associated with formate dehydrogenases (Schut and Adams, Dragomirova et al. focuses on heterologous expression of a [NiFe]-hydrogenase from dehalogenating Chloroflexi (Kublik et al., Pinske explores a third type of formate dehydrogenase-linked hydrogenase, namely the classical formate hydrogenlyase complex of Enterobacteriaceae (McDowall et al., This special issue, featuring 10 articles from 46 different authors, explores microbial H2 metabolism. Islam et al. report two other novel iron-sulfur proteins in mycobacteria, demonstrating that they are essential for the activity of the two high-affinity hydrogenases described in this lineage (Greening et al., Carere et al. meanwhile, build on the recent discovery that verrucomicrobial methanotrophs are facultative mixotrophs (Carere et al., Methylacidiphilum varies depending on H2 availability. Three articles also explore H2 metabolism at the ecosystem level. Adam and Perner explore the diversity of aerobic and anaerobic H2 metabolism in deep-sea hydrothermal vent systems, whereas Meyer-Dombard et al. investigate the influence of H2 on biogeochemical cycling in serpentinizing springs in the Philippines. Teng et al. review the previously underexplored area of H2 metabolism in bioremediation, including in the reduction of organohalides, nitroaromatic compounds, and heavy metals (Chardin et al., Several articles also investigate aerobic H2 in microbial metabolism and uncovers novel enzymes and pathways that mediate this process. This body of work highlights the intricate linkages between H2 cycling and the cycling of various other compounds, including methane, formate, carbon dioxide, sulfate, and organohalides, among others. In turn, these findings pave way for future studies on the biochemistry, physiology, ecology, and industrial applications of microbial H2 metabolism.In summary, this special Research Topic sheds light on the diverse role of HCG and EB drafted this editorial together and approve its submission.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Salmonella enteric serovar Typhimurium (ST), by oral consumption of a genetically modified (GM) probiotic strain in mice , and a significantly improved preventive effect was displayed. However, successful probiotic intervention of ST, one of the most prevalent foodborne pathogens, is challenging, and more work is necessary. We know that many probiotic strains have an inhibitive effect on ST by different mechanisms .ST is a Gram-negative, non-spore-forming, facultative anaerobic bacterium and can infect any warm-blooded animal ST is rapidly expanding (Obaidat and Stringer, L. casei promotes overall bacterial species diversity and increases the abundance of Lactobacillus and Bifidobacterium in the cecum in mice (Peng et al., Lactobacillus spp. is mainly in the small intestine, and Bifidobacterium spp. is mainly in the large intestine (Donaldson et al., In chickens, ST first attaches to the cecal epithelial cells and then spreads to the liver, spleen, and oviduct. In pigs, early ST infection disrupts microbiome composition and functionality principally at the ileum (Arg\u00fcello et al., Probiotics play important roles in human health. As normal commensals, they exert their prophylactic and therapeutic properties against ST in four main ways (Gut et al., L. casei modulates host immunity by regulating the expression of intestinal inflammation-related cytokines [e.g., suppressing pro-inflammatory cytokines and provoking anti-inflammatory cytokines after ST infection (Peng et al., Lactobacillus spp. modulate ST by regulating gene expression related to colonization and virulence (Muyyarikkandy and Amalaradjou, L. pentosus AT6 and its cell-free culture supernatants inhibit ST growth and its adhesion as well as invasion (Liu et al., In fact, probiotics can be modulators, producers, and residents after being administered. As modulators, probiotics effectively modulate either the host or the pathogen. L. casei or the GM counterpart has displayed a significant protective effect on ST infection (Peng et al., Salmonella enteritidis infection is efficacious in broilers, but data on prophylactic treatment timing regarding ST is not available (Higgins et al., Pre-administration of probiotics is an effective method demonstrated in animal studies. For example, 1-week pre-administration of either wild-type Bacillus spp., Bifidobacterium spp., Clostridium spp., Escherichia coli Nissle 1917, and Lactobacillus spp., yeast, and so on (Kanmani et al., Bifidobacterium spp. and Lactobacillus spp., harbor most of the well-characterized probiotic strains and are widely commercialized. These probiotics reduce more than 90% of caecal ST load, prevent invasion of organs, and even completely eradicate ST (Gut et al., Akkermansia muciniphila, Eubacterium hallii, and Faecalibacterium prausnitzii (Almeida et al., Probiotics are any non-pathogenic microorganisms that confer health-promoting properties when administered in adequate amounts (Hill et al., E. coli inhibits ST growth with improvement in fitness (Palmer et al., L. casei improves the protective effect on ST more than the wild-type strain by increasing CLA (Peng et al., Besides the traditional application of wild-type probiotics, GM probiotics are also studied. Although there are safety issues, GM probiotics attracted much interest due to their extra advantages and strengthened effects (Barra et al., Pseudomonas aeruginosa and reducing vancomycin-resistant Enterococcus by GM probiotic E. coli Nissle 1917, similar approaches might be also promising for ST prevention (Hwang et al., Along with the rapid development of synthetic biology, interest has increased on the design and construction of GM probiotics as live biotherapeutics for a range of medical applications (Chua et al., 6 CFU/g or CFU/mL viable cells may be an acceptable way. Considering the GRAS status, consumption of them can be without control. However, to cure ST infection, NG or GM probiotics are more promising. These probiotics can be formulated as concentrated pills or capsules containing more than 109 CFU/g or even more cells. They may be more suitable as over-the-counter drugs. Nevertheless, for any purposes, release of both conventional and GM probiotics as live biotherapeutical products in the market needs full assessment of safety (O'Toole et al., As pointed out, formulation of viable probiotics while enabling cost-effective biomass yield is a critical step toward product development of translational application (Almeida et al., ZS conceived the opinion. YS and JL wrote the draft manuscript. YS collected reference and drawn the figure. ZS finalized the manuscript and acquired funding. All authors discussed the content.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Resveratrol is a natural polyphenol that has anti-aging and anti-inflammatory properties against stress condition. It is reported that resveratrol has beneficial functions in various metabolic and central nervous system (CNS) diseases, such as obesity, diabetes, depression, and dementia. Recently, many researchers have emphasized the connection between the brain and gut, called the gut\u2013brain axis, for treating both CNS neuropathologies and gastrointestinal diseases. Based on previous findings, resveratrol is involved in glucagon-like peptide 1 (GLP-1) secreted by intestine L cells, the patterns of microbiome in the intestine, the 5-hydroxytryptamine (5-HT) level, and CNS inflammation. Here, we review recent evidences concerning the relevance and regulatory function of resveratrol in the gut\u2013brain axis from various perspectives. Here, we highlight the necessity for further study on resveratrol's specific mechanism in the gut\u2013brain axis. We present the potential of resveratrol as a natural therapeutic substance for treating both neuropathology and gastrointestinal dysfunction. Resveratrol is a polyphenol that is secreted by grapes and berries -activated protein kinase (AMPK) pathway and phosphodiesterases (PDEs) Chung, Figure .SIRT1 gene, considered an anti-aging related gene, in the duodenum and also rescues insulin resistance and improves neuronal networks in the brain , an incretin hormone and a major hormone of the gut\u2013brain axis, is linked to the control of energy homeostasis and the development of obesity , and finally lead to improved cognitive function is expressed in both the CNS and gastrointestinal tracts, and currently 5-HT has been considered as an important target in the gut\u2013brain axis.5-HT is a growth factor, a paracrine factor, and an enteric neurotransmitter synthesis through 5-HT receptors (Prasad et al., Considering previous data, 5-HT derived from gut and brain contributes to nervous systems globally, and the circulation of 5-HT in the body mediates the gut\u2013brain axis (Yano et al., 2C receptor-dependent signaling (Peng et al., A current study proved that resveratrol regulates the gut\u2013brain axis by controlling the 5-HT-dependent pathway in an irritable bowel syndrome rat model and specifically that resveratrol influences various organs including brain hippocampus, ileum, and colon through 5-HT axis (Yu et al., Another recent study demonstrated that resveratrol could increase the expression of 5-HT, leading to the improvement of brain function (Nabavi et al., Several studies have mentioned the neurological role of resveratrol in depression and anxiety (Yu et al., One current study reported that the inhibition of 5-HT release attenuates the activation of GLP-1 receptor signaling and highlighted the relationship between GLP-1 and 5-HT serotonin system (Anderberg et al., Another study mentioned that GLP-1 receptor agonist liraglutide could reduce the expression of 5-HT2A receptor and subsequently reduces body weight and inhibits serotonin synthesis in mice model (Nonogaki and Kaji, Ripken et al. suggested that serotonin treatment could boost GLP-1 release, and the blocking of 5-HT receptor could affect the production of GLP-1 (Ripken et al., A recent study proved that 5-HT enterochrnomaffin cells in gut regulates gut microbial metabolism and homeostasis and is affected by the activation of GLP-1 (Lund et al., Further, ghrelin, known as a hormone for regulation of motivation and reward system among brain function, has been interacted with GLP-1 and the monoamine transmitter 5-HT (Currie et al., GLP-1 derived from brain mainly is produced by the nucleus tractus solitarius in brain (Alhadeff et al., Given previous evidences, resveratrol can control 5-HT and its receptor and also modulate release of 5-HT through GLP-1 regulation. Ultimately, resveratrol could control the neuropathology of neurological diseases such as depression and stress-induced anxiety. Also, resveratrol can regulate gut dysfunction in irritable bowel syndrome via 5-HT. Thus, we emphasize the necessity for further study of the specific mechanism and cellular pathways regulated by resveratrol and mediated by 5-HT to fully understand the gut\u2013brain axis.Resveratrol is involved in the gut\u2013brain axis through another mode in addition to the GLP-1 pathway and 5-HT system.Recently, gut microbiota is emerging as an important node in the gut\u2013brain axis (Louwies et al., trans-stilbene have been reported to be the major microbiota-derived metabolites made from resveratrol (Juan et al., Resveratrol administration could be metabolized by the liver, intestinal tract, and gut microbiota Walle, . A recenBifidobacteria infantis and Lactobacillus acidophilus are strongly linked to piceid production from resveratrol (Basholli-Salihu et al., Enterococcus faecalis and increasing the Lactobacillus and Bifidobacterium populations (Qiao et al., Specifically, dihydroresveratrol as a metabolite of resveratrol is produced in the intestines such as the cecum, colon, and rectum through fermentation by the gut microbiota (Amri et al., Recently, resveratrol has been reported to improve gut microbiota in bowel diseases under harsh oxidative stress (Hu et al., A clinical study has reported that resveratrol treatment exerts cardiovascular and anti-obesity effects by ameliorating gut microbiota diversity (Bird et al., These previous findings demonstrate that resveratrol and gut microbiota influence each other. Furthermore, resveratrol could enhance the gut microbiota diversity and the gut barrier's homeostasis. These effects of resveratrol should be investigated further to determine the specific gut bacteria that affect the gut\u2013brain axis.Here, we reviewed previous significant evidence of the effect of resveratrol on the gut\u2013brain axis . We summJC, J-HJ, and JS contributed to the writing of the text. JC and J-HJ made and revised all figures. JS wrote and finalized the revised manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Ten years ago, Archives of Toxicology has issued an Editorial by R.D. Combes entitledIn the following, important research lines of computational toxicology were the development and refinement of computational models for relevant toxicological endpoints, such as liver injury, cardiotoxicity, renal toxicity and genotoxicity Ekins . Best prAs far as regulatory acceptance is concerned, a decisive breakthrough was the integration of (Q)SAR methodologies into the guideline ICH M7 \u201cAssessment and control of DNA-reactive (mutagenic) impurities in pharmaceuticals to limit potential carcinogenic risk\u201d, issued by the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human use (Amberg et al. Advanced computational methodologies (Kusko and Hong Perspectives are emerging for computational approaches to predict the toxicity of nanomaterials Buglak and of cComputational toxicology continues to assist in refining PBPK modelling (Savvateeva et al. A burst in manuscript submissions to \u201cArchives of Toxicology\u201d covering the in silico/computational field is noticed since 2019, signaling both increased scientific importance and regulatory relevance in the twenty-first century of this research area (Krewski et al. A current tendency, both in the United States (Kosnik et al. Given this exciting development, further submissions of manuscripts from these fields to Archives of Toxicology are highly encouraged!"} +{"text": "This is one of the few polymers that the Food and Drug Administration (FDA) has approved for human administration due to its biocompatibility and biodegradability [Poly/poly(lactic acid) (PLGA/PLA) carriers were prepared through spray-drying to incorporate the microcrystals that were previously prepared by ultra-sonication. In vivo testing in rat models was demonstrated to prolong drug retention in joints. The TA remained there for over 28 days, which was more 21 days compared with the TA-free group. Furthermore, these nanocarriers were demonstrated to be stable for one year.In the first research study presented in this Special Issue, Ho et al. developed polymeric microspheres which contain micronized triamcinolone acetonide (TA) in order to increase the drug retention time in joints after intra-articular administration . Poly(laThe group of Peula-Garc\u00eda used PLGA nanoparticles to carry bone morphogenetic protein (BMP-2) . The nanIn another study, Hwang et al. fabricated PLGA carriers combined also with a hydrogel matrix. They produced oxaliplatin-loaded PLGA microparticles using a double emulsion technique and then loaded them into hyaluronic acid and carboxymethyl cellulose sodium-based cross-linked hydrogels . This drKim et al. developed an original system to be used in the topical delivery of trolamine salicylate (TS), a topical anti-inflammatory analgesic used for the treatment of small joint pain . Here, tDuse et al. used PLGA nanoparticles to encapsulate curcumin, a well know natural compound that present anticancer benefits . It was Another interesting study proposed by Varga and colleagues, who contributed with an interesting study of nanoparticle design and optimization where the (\u00b1)-\u03b1-Tocopherol (TP) with vitamin E activity was encapsulated in PLA and PLGA nanoparticles . To stab2) cells.Morelli et al. improved paclitaxel delivery in the gastro-intestinal tract by encapsulating the drug in PLGA nanoparticles coated with PEG . The nanWith the objective to overcome the undesired lag time of the commercially available risperidone, Janich et al. encapsulated this drug in PLGA\u2013lipid microcapsules and PLGA\u2013lipid microgels . The carA research work using PLGA nanoparticles for ocular application was also collected. Ryu et al. produced rapidly dissolving dry tablets containing alginate and dexamethasone-loaded PLGA nanoparticles . These nAn interesting approach based on a combination of cell and drug delivery for the treatment of Huntington\u2019s disease (HD) was proposed by Andr\u00e9 et al. . The autTwo works lead by Roing and Wacker present new theranostic PLGA-based nanoparticles. In the first one, biodegradable and photoluminescent polyester (BPLP) with PLGA polymer was used to fabricate biocompatible photoluminescent nanocapsules . AdditioPLGA toxicity was investigated by Bakhaidar et al. . Here, tAnother relevant study was performed by Operti et al., who used microfluidics technology as a tool to manufacture particles in a highly controllable way . In theiFinally, a research study regarding the importance of new techniques to characterize PLGA nanoparticles was included in this special edition. Shmool et al. investigated the dynamics of PLGA microspheres prepared by freeze-drying . The watThe papers presented in this Special Issue represent a small part of the research that is ongoing in the field of PLGA nanocarriers all over the world. The huge potential of PLGA nanoparticles make them a promising drug delivery system with outstanding properties and with much more potential for exploring in the coming years. With this Special Issue, the editors expect that the readers from the field find it stimulating and contributing more ideas or methodologies for their future work."} +{"text": "Parkinson\u2019s disease (PD) is characterized by the progressive loss of dopaminergic neurons in the substantia nigra. However, other non-dopaminergic neuronal systems such as the serotonergic system are also involved. Serotonergic dysfunction is associated with non-motor symptoms and complications, including anxiety, depression, dementia, and sleep disturbances. This pathology reduces patient quality of life. Interaction between the serotonergic and other neurotransmitters systems such as dopamine, noradrenaline, glutamate, and GABA controls the activity of striatal neurons and are particularly interesting for understanding the pathophysiology of PD. Moreover, serotonergic dysfunction also causes motor symptoms. Interestingly, serotonergic neurons play an important role in the effects of L-DOPA in advanced PD stages. Serotonergic terminals can convert L-DOPA to dopamine, which mediates dopamine release as a \u201cfalse\u201d transmitter. The lack of any autoregulatory feedback control in serotonergic neurons to regulate L-DOPA-derived dopamine release contributes to the appearance of L-DOPA-induced dyskinesia (LID). This mechanism may also be involved in the development of graft-induced dyskinesias (GID), possibly due to the inclusion of serotonin neurons in the grafted tissue. Consistent with this, the administration of serotonergic agonists suppressed LID. In this review article, we summarize the interactions between the serotonergic and other systems. We also discuss the role of the serotonergic system in LID and if therapeutic approaches specifically targeting this system may constitute an effective strategy in PD. Parkinson\u2019s disease (PD) is one of the most common neurodegenerative disorders, which is characterized by the progressive loss of dopaminergic neurons in the substantia nigra compacta (SNc). Dopamine replacement therapy using the precursor L-DOPA is the main treatment for the disease. However, long-term use of L-DOPA leads to the development of dyskinesias and non-motor manifestations contains the largest group of serotonin-producing neurons, and changes in DRN function have been implicated in neuropsychiatric diseases and movement disorders Hornung, . Classic1\u20137) and at least 15 receptor subtypes have been identified may have a constitutive activity, which may be associated with pathophysiological conditions appear particularly interesting for PD , which was reduced by dopaminergic and serotonergic lesions and suppressed by NMDA glutamate receptor antagonists, suggesting that stimulation of glutamate receptors is essential for the observed neuronal response , which is the main brain neurotransmitter mediating inhibitory signals. Deficiency in brain serotonin results in alterations in the GABAergic system in striatal dopaminergic terminals. However, in advanced stages of the disease, the dopaminergic denervation is almost complete and other cell types showing AADC activity convert exogenous L-DOPA into dopamine, including serotonergic terminals (Arai et al., 2A antagonists (Meco et al., 1A agonist have been shown to reduce LID without compromising L-DOPA efficacy (Meadows et al., Compounds acting through the serotonin system such as anpirtoline, (B\u00e9zard et al., 1A agonists such as buspirone (Politis et al., 3 receptors also reduced LID (Kwan et al., 1 agonists with drugs that modulate the glutamatergic function (Tison et al., Clinical trials with serotonergic drugs are ongoing, revealing the promising antidyskinetic effects of 5HT1A receptor agonist buspirone produced significant dampening of GID in grafted patients. However, this effect could also be explained by the dopamine D2 receptor partial antagonistic effects of the drug (Politis et al., 1A and 5-HT1B agonists, suggesting that the effect of these drugs on GID is mediated by the activation of presynaptic host-derived receptors (Shin et al., 6 receptors (Aldrin-Kirk et al., Clinical trials using transplants of fetal dopamine neuroblasts have shown promising results, although many patients have developed GID (Freed et al., Interactions between serotonergic and other neurotransmitter systems reveal that serotonin plays a crucial role in the control of movement by the basal ganglia. These interactions are of great interest for understanding the pathophysiology of PD and to develop novel therapeutic strategies. Manipulation of the serotonergic system represents a valuable target to treat LID and GID in PD patients. However, further investigation is required to clarify mechanisms of neurotransmitter interactions and to determine optimal compounds and doses for effective therapies.All authors have contributed to this work and approved its final version for submission. AM developed the idea for this review and wrote the manuscript. AL-L, CL, and JL-G prepared the figure and were involved in the literature review and preparation and revision of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Preclinical models for the definition of anti-cancer drug safety and efficacy are constantly evolving. Indeed, tumor development in humans is not always fully reproducible and predictable in other animals. In turn, two-dimensional (2D) cultures, used for many years to test drug effects, are limited by the lack of tissue structure and architecture, which can influence both pharmacokinetics and pharmacodynamics, thus impairing the prediction of anticancer drug efficacy. An interesting and reliable alternative is represented by three-dimensional (3D) culture systems, including spheroids and matrix-based collagen or synthetic scaffolds, which have been validated by the EU Reference Laboratories as preclinical models to overcome at least some of the above-mentioned drawbacks.This thematic issue collects studies involving both advances in the techniques used to create and analyze some 3D culture systems and their applications in different cancer models for drug combination testing. The switch from 2D to 3D culture changes several conditions for cell growth, the first being a reduced surface for direct targeting, with the inner cell layers being protected from the external environment. At the same time, inner cells can suffer a reduced nutrient or oxygen supply, drastically changing their metabolism to a hypoxic one, causing the transcription of genes typically involved in drug resistance. This picture is even more complicated when co-cultures of different cell types are generated.A. R. Holub et al. used an The bioprinting approach tested by Seokgyu Han et al. allowed Ezequiel Monferrer et al. comparedAlthough these models of scalable complexity allow us to investigate the interactions between tumor spheroids and the tissue microenvironment, their analysis is usually confined to classic microscopy approaches. Optical microscopy shows intrinsic limits that can be usually outdone only by high cost instruments. However, Eliana Steinberg et al. have demAnother approach to increasing the information derived from 3D models has been described by Tarapong Srisongkram et al. , applyinAlthough new techniques allow researchers to extract an increasing amount of data from 3D cultured cells, the more immediate application of these models is still drug testing. This goal was pursued by Sadaf E. Pustchi et al. , testingA combined therapeutic regimen was also investigated by Layla Mohammad Hadi et al. in 2D anIn summary, 3D cultures have contributed deeply to improving the reliability of in vitro pre-clinical models in anti-cancer drug testing. Moreover, with the establishment of organoids from various types of epithelial cancers, co-cultures of cancer organoids and immune cells have become a highly informative strategy for the development and testing of cancer immunotherapy, representing an additional useful application of 3D culture systems in cancer treatment."} +{"text": "Bacterial proteins exhibiting two or more unrelated functions, referred to as moonlighting proteins, are suggested to contribute to full virulence manifestation in pathogens. An expanding number of published studies have revealed the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to be a multitasking protein with virulence impact in a number of pathogenic bacteria. This protein can be detected on the bacterial surface or outside the bacterial cell, where it interacts with host proteins. In this way, GAPDH is able to modulate various pathogenic processes. Moreover, it has been shown to be involved in non-enzymatic processes inside the bacterial cell. In this mini review, we summarize main findings concerning the multiple localization and protein interactions of GAPDH derived from bacterial pathogens of humans. We also briefly discuss problems associated with using GAPDH as a vaccine antigen and endeavor to inspire further research to fill gaps in the existing knowledge. Although bacterial glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a classic glycolytic enzyme catalyzing the conversion of glyceraldehyde-3-phosphate to 1, 3-bisphosphoglycerate , two distinct pathways participate in GAPDH secretion. In cells grown in Dulbecco's modified Eagle's medium, the GAPDH secretion was mediated by the type III secretion system generally involved in translocation of various effector proteins into the infected cells. Additionally, the interaction of GAPDH with CesT, a specific chaperone for type III effectors, was established. The associated chaperone may stabilize the GAPDH molecules and prevent them from interacting with other cytosolic partners, thus enabling their targeting to the type III secretion apparatus. The other secretory pathway has not been further described and is responsible for GAPDH secretion in EPEC and in probiotic E. coli strains grown in Lysogeny broth medium . Its binding specificity differs among individual pathogens, but plasminogen is the most common target . PathogeMycobacterium tuberculosis secrets iron-binding molecules known as siderophores that compete with the host's iron-transport proteins (De Voss et al., M. tuberculosis utilizes surface-localized GAPDH to capture the human transferrin and then internalizes the transferrin\u2013GAPDH complex. Three years later, the same research group (Malhotra et al., M. tuberculosis has even greater binding affinity to lactoferrin. M. tuberculosis GAPDH thus acts as dual receptor for both transferrin and lactoferrin. The ability of surface-localized GAPDH to bind transferrin has newly been demonstrated for Streptococcus agalactiae (Nagarajan et al., Staphylococcus ssp. Whereas, Modun and Williams (Streptococcus pneumoniae utilizes hemoglobin or heme instead of transferrin or lactoferrin as an iron source. GAPDH of this pathogen can bind both these proteins and has been proposed to participate in iron scavenging for bacterial needs (Yang et al., The ability of an invading bacterial pathogen to survive and proliferate within a host organism also depends on the availability of several trace elements, such as iron, an essential cofactor for diverse biochemical reactions. In a healthy mammalian organism, almost all the iron is bound to the transport proteins transferrin or lactoferrin or is stored in ferritin, because free iron catalyzes the production of toxic free oxygen radicals. The free ionic iron both in extracellular fluids and inside the cells is thus far too low to support bacterial growth. Bacterial pathogens have developed several strategies, however, to exploit iron from those iron-binding proteins (Cornelissen and Sparling, Williams identifiWilliams . StreptoListeria monocytogenes. The surface-localized GAPDH interferes with the host Rab5a protein (Alvarez-Dominguez et al., Listeria monocytogenes escapes rapidly from the phagosomal compartment to the cytosol, where it replicates (V\u00e1zquez-Boland et al., L. monocytogenes GAPDH evidently has the ability to ADP-ribosylate the Rab5a protein, thus blocking its function in phagosome\u2013endosome fusion. As a consequence of this strategy, L. monocytogenes delays the phagosome maturation and gains time for escape from the vacuole prior to its fusion with endolysosome that would result in the pathogen's destruction (Alvarez-Dominguez et al., Extracellular localization of GAPDH has also been demonstrated for bacteria with intracellular life cycles. One can therefore assume that intracellular bacteria might use GAPDH for manipulating some host cellular processes in order to customize the host cell milieu for their successful survival and proliferation. So far, the only study supporting this hypothesis was performed on intracellular Gram-positive S. agalactiae, S. pyogenes, and Staphylococcus aureus was reported to induce apoptosis in murine macrophages (Oliveira et al., GAPDH derived from extracellular pathogens seems to affect host cellular processes, too, as GAPDH secreted by Streptococcus pyogenes is one of few known bacteria able to bind and inhibit the C5a component of the complement system. As an integral part of the innate immunity, the complement system acts in early defense against pathogens prior to the activation of acquired immune response. It promotes cell killing by the formation of a membrane attack complex and production of molecules that stimulate the function of phagocytic cells and contribute to the inflammation manifestation. The C5a component is a potent anaphylatoxin and chemoattractant for neutrophils and macrophages (Kajita and Hugli, 2O2 production in neutrophils (Terao et al., S. agalactiae, the secreted GAPDH exerts a stimulatory effect on B lymphocytes and to a lesser extent also on T cells. GAPDH-induced production of the anti-inflammatory cytokine interleukin-10 suppresses neutrophil recruitment in mice, which finally contributes to successful host colonization by this pathogen (Madureira et al., Immunomodulatory activities of surface proteins represent another strategy for promoting a pathogen's survival in its host organism. S. agalactiae (Madureira et al., S. pneumoniae (Sun et al., Bacillus anthracis (Matta et al., L. monocytogenes (Calderon-Gonzalez et al., L. monocytogenes, the GAPDH peptide was tested as a component of a cell-based vaccine against listeriosis. Dendritic cells were used as adjuvants for immunostimulation. Despite those promising results, the high GAPDH sequence homology between the bacterial strains and humans should be taken into consideration in vaccine development. Homologous sequences can be responsible for inducing cross-immune responses that result in deleterious effects on human health (Perez-Casal and Potter, S. agalactiae or suffering from autoimmune diseases which excludes as potential candidates for a subunit vaccine (Razim et al., Surface localization plus its role in virulence together with the binding to ECM proteins make GAPDH a suitable vaccine candidate for preventing infectious diseases. The host immune responses triggered by bacterial GAPDH have been studied extensively, and as a vaccine component GAPDH was able to induce protection against several pathogenic bacteria (Argiro et al., In summary, it is evident that our current knowledge concerning bacterial GAPDH remains quite limited and provides much space for further research. It is indisputable that this protein has other functions unrelated to its enzymatic role, and it meets the criteria to be included into the family of moonlighting proteins. Data from previously published studies indicate that multiply localized GAPDH participates in other non-glycolytic processes : CytosolAll authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Parkinson\u2019s disease (PD) is the second most common neurodegenerative disorder characterized by age-dependent motor dysfunction and degeneration of the midbrain dopaminergic neurons. The deposition of neuronal inclusion, named Lewy body (LB), in the affected regions is a pathological feature of PD and related disorders such as dementia with LB (DLB). Lewy body formation is thought to begin with \u03b1-synuclein aggregation and fibrillation. Experimental studies based on the knowledge obtained by epidemiological and genetic studies continue challenging researchers to make PD risk predictable and surmountable. In this context, the development of experimental models of PD has contributed to the understanding of PD etiology and the development of therapeutics. The current 11 contributions that comprise this Special Issue highlight the PD-associated phenotypes and their evaluation methods and the development of therapeutic strategies using animal models of PD .+) by glial monoamine oxidase B is transported to dopaminergic neurons probably through the dopamine transporter and inhibits the mitochondrial respiratory complex I subunits [+ and its precursor, MPTP, to make animal or cellular models of PD. Kinoshita et al. [The discovery of the mitochondrial toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) focused the spotlight on the roles of mitochondria in dopaminergic neurons [a et al. . Another neurotoxin, 6-hydroxydopamine (6-OHDA), is a dopamine analogue which produces selective damage to dopaminergic neurons by generation of reactive oxygen species (ROS). Unlike MPTP, 6-OHDA does not cross the blood\u2013brain barrier and is used to induce the degeneration of the nigrostriatal pathway by intracerebral stereotactic injection. 6-Hydroxydopamine-induced rodent models are generally unilateral lesion models and exhibit a rotation response by apomorphine. Rosa et al. reportedThe pesticide rotenone is known to inhibit the mitochondrial complex I, generating ROS. Chronic systemic exposure to rotenone has been reported to reproduce selective nigrostriatal dopaminergic degeneration with LB-like \u03b1-synuclein-positive inclusions in rat . HoweverHyposmia, constipation, and rapid eye movement sleep behavior disorder are considered prodromal symptoms of PD that often precede motor symptoms. These phenotypes are particularly important in developing disease-modifying therapies that prevent the onset or control progression of PD. Taguchi et al. reviewedA synaptic vesicle-binding protein, \u03b1-synuclein, is a key protein to produce PD symptoms, forming LBs in the associated neurons. Recent cellular and animal model studies have revealed that \u03b1-synuclein has a prion-like property, ascending from peripheral to central neural circuits. Mori et al. reviewedPINK1 and Parkin genes cause early-onset familial PD [Mutations of ilial PD ,15. The ilial PD . Torii eilial PD reviewedDrosophila is a powerful tool for genetics and has revealed the molecular relationship between PINK1 and Parkin in mitochondria [Drosophila is now commonly used as PD models to evaluate genetic association. Elvira et al. [Drosophila. Historical perspective of overall PD models was also well summarized by Chia et al [chondria . Drosopha et al. reportedia et al .In summary, all articles appearing in this Special Issue cover the interesting and current topics in PD model studies. Although most PD models do not faithfully reproduce all aspects of this disease, PD model studies would advance our knowledge and promote the development of drugs and therapeutic strategies, receiving new inputs from clinical studies. This Guest Editor would like to thank all of the authors for their contributions to this Special Issue and expects significant advancement to our knowledge of PD in future studies."} +{"text": "Mounting evidence shows genetic overlap between multiple psychiatric disorders. However, the biological underpinnings of shared risk for psychiatric disorders are not yet fully uncovered. The identification of underlying biological mechanisms is crucial for the progress in the treatment of these disorders.We applied gene-set analysis including 7372 gene sets, and 53 tissue-type specific gene-expression profiles to identify sets of genes that are involved in the etiology of multiple psychiatric disorders. We included genome-wide meta-association data of the five psychiatric disorders schizophrenia, bipolar disorder, major depressive disorder, autism spectrum disorder, and attention-deficit/hyperactivity disorder. The total dataset contained 159 219 cases and 262 481 controls.We identified 19 gene sets that were significantly associated with the five psychiatric disorders combined, of which we excluded five sets because their associations were likely driven by schizophrenia only. Conditional analyses showed independent effects of several gene sets that in particular relate to the synapse. In addition, we found independent effects of gene expression levels in the cerebellum and frontal cortex.We obtained novel evidence for shared biological mechanisms that act across psychiatric disorders and we showed that several gene sets that have been related to individual disorders play a role in a broader range of psychiatric disorders. We used precomputed LD scores that were provided by LD score regression, which were calculated using the European panel of the 1000 Genomes Project. No constraining of the intercept was applied.SNP heritability gene sets (n\u00a0=\u00a05917) and canonical pathways (n\u00a0=\u00a01329). The GO gene sets contain genes annotated by the same GO term and cover biological processes, cellular components, and molecular functions. The canonical pathways are representations of biological processes that are compiled by domain experts. Second, we selected 126 expert-curated gene sets that have been tested to date for one of the five disorders in studies based on whole genome approaches . These gene sets cover different types of expert-curated sets: sets of functionally related genes, sets of co-expressed genes, and sets representing protein complexes or networks (online Supplementary Table S1). Third, we obtained gene-expression values of 53 tissues from the GTEx project v7 . The expression values of all genes were Winsorized at 50 reads per kilobase of transcript per million reads mapped (RPKM) and subsequently log2 transformed with pseudocount 1 derived from publicly available databases, and (2) expert-curated, i.e., genes annotated by experts in a specific research field that commonly involves an extensive experimental or methodological design and interpretation. In addition, we investigated tissue-specific gene-expression values as gene properties . First, we selected from the molecular signature database . Considering the significantly associated gene sets, the three calcium channel sets and the gene set involved in membrane depolarization showed equal association strengths, while the other gene sets present small to moderate reduced associations when taking the direction of SNP effects into account. This implies that the effects of SNPs related to calcium channels, or more broadly related to action potentials and depolarization, increase the risk on the different disorders in the same direction, while the effects of genetic variants related to the other identified functions partly act in opposite directions across disorders. In addition to this post hoc analysis, we performed a gene-set and gene-property analysis weighting the disorders according to sample size. These results were highly consistent with the initial unweighted analysis .We compared the results of these analyses \u2013 which are based on meta-analyzing gene-based associations of the individual disorders \u2013 with a gene-set and gene-property analysis including as input the meta-analyzed SNP associations of the five disorders. Hence, the direction of the SNP effects is taken into account. The results of these analyses and the initial analyses showed strong correlations [Pearson's correlation of logTo investigate the contribution of the association signal of the individual disorders to the cross-disorder association, we performed the gene-set and gene-property analyses for the individual disorders as well , indicating that the associations of the identified gene sets are not merely because they comprise genes expressed in the brain.FMRP targets (Darnell et al., GO high voltage gated calcium channel activity, GO postsynapse, MIR137 targets (Lewis et al., GO modulation of synaptic transmission, GO membrane depolarization during cardiac muscle cell action potential, GO gamma aminobutyric acid signaling pathway, and GO transcription factor activity RNA polymerase II core promoter sequence specific showed independent effects. However, the associations with the gene sets GO synapse part, GO synapse, GO neuron spine, GO voltage gated calcium channel complex, and GO excitatory synapse could fully be accounted for by the other identified gene sets. The large overlap of genes in these sets with the other identified gene sets (Third, we applied the forward selection procedure to the gene sets to shed light on the relations between their associations. We excluded the highly-brain-expressed gene set from this step as gene expression levels are already captured by conditioning on the average, cerebellum and frontal cortex tissue expression levels included in the previous step. The eight gene sets ene sets clearly et al., et al., et al., The current gene-set analyses revealed various new sets of genes \u2013 in particular related to the synapse and neuronal functions \u2013 and gene-expression profiles of multiple brain tissues that play a role in shared genetic risk across five psychiatric disorders. The most strongly associated gene set was the highly-brain-expressed genes, which has previously been related to ASD (Pinto et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., de novo mutations (Iossifov et al., et al., et al., et al., et al., et al., et al., In addition, we identified multiple gene sets related to the synapse, which aligns with synaptic functions of several identified genes for multiple individual psychiatric disorders (Schizophrenia Working Group of the Psychiatric Genomics Consortium, Of note, the biological annotations of gene sets comprise a complex and challenging process, e.g., due to the multiple functions of many genes and incomplete knowledge. The construction of gene sets is in general based on different approaches such as shared cellular mechanism, co-expression patterns, protein-protein interaction, or co-localization. Hence, sets of genes may be based upon different inclusion criteria, creating an overlap between gene sets, as also illustrated by the current study. Clearly, it is important to recognize the impact of particular annotations on gene-set analysis results and their biological interpretation.et al., et al., To address this issue of confounding and redundancy in gene sets, we applied conditional analyses. This provided insight in how different gene-set associations relate to each other, and whether identified functions may not be biologically relevant to the disorders but rather induced by confounding factors (de Leeuw et al., Our cross-disorder gene-set and gene-property analyses are built on a meta-analysis of the gene-based associations with the individual disorders, therefore possible opposite effects of genetic variants are not taken into account. To explore if genetic variants are related to multiple disorders but with opposite effects, we performed an SNP-based meta-analysis of the five disorders and conducted a gene-set and gene-property analysis based on those results. In this analysis, genetic variants with opposite effects across disorders are cancelled out. Although these results showed strong correlations with the original analysis, we detected differences in association strength that point to partial differences in direction of SNP effects between the disorders for most identified gene sets. Interestingly, the effects on calcium channel activity are unidirectional across disorders. The outcome of different effects across disorders is supported by the recent finding that the highly correlated disorders SCZ and BD are differentiated by several genetic loci with opposite directions of effects (Bipolar Disorder and Schizophrenia Working Group of the Psychiatric Genomics Consortium, et al., et al., We note that the associations of our identified gene sets were to a large extent driven by SCZ. This is in line with previous studies that reported multiple gene sets associated with SCZ (Ripke In conclusion, the current study provides novel evidence for shared biological mechanisms that act across psychiatric disorders based on gene-set and gene-property analyses. We showed that several gene sets that previously only had been associated with individual disorders also play a role in a broader range of psychiatric disorders, supporting the view of a common pathogenesis across disorders. This indicates that the genetic overlap between disorders is not randomly distributed, but can be explained by specific biological mechanisms. The strongest evidence in our results was for the involvement of synaptic functions, and gene expression profiles of the cerebellum and frontal cortex. The genetic data collection of additional psychiatric disorders is rapidly increasing and will make it possible to extend our analyses to other disorders in the near future. Understanding the shared biological mechanisms between psychiatric disorders may provide a hint towards a general vulnerability for multiple psychiatric disorders, and could result in potential treatment for a broad spectrum of psychiatric disorders."} +{"text": "Annals of Intensive Care about the dose adjustments of vitamin C in critically patients undergoing renal replacement therapy [We have read with interest the manuscript from Honore et al. in therapy . After aMedicine journal in 2019 (online: 2019 November 27) [The authors included in their study the Wu et al.\u2019s case report of a patient with hemolytic jaundice induced by pharmacological dose of ascorbic acid in glucose-6-phosphate dehydrogenase (G6PD) deficiency . This mamber 27) , becauseThe authors searched for studies and used studies published in 2020, like Fujii et al.\u2019s study , so the We conclude that the exclusion of Wu et al.\u2019s study does not probably affect the overall conclusions of Honore et al.\u2019s study, but it affects directly the particular conclusion exposed previously because Wu et al.\u2019s study failed to provide valid scientific evidence.We recommend to exclude it, but a good and accurate literature review is a key element in high-quality original studies."} +{"text": "Mutations in the encoding genes in patients and mouse models underlie severe phenotypes including kidney stones with CLCN5 and osteopetrosis or hypopigmentation with CLCN7. Dysfunction of those intracellular CLCs that are expressed in neurons lead to neuronal defects. Loss of endosomal ClC-3, which heteromerizes with ClC-4, results in neurodegeneration. Mutations in ClC-4 are associated with epileptic encephalopathy and intellectual disability. Mice lacking the late endosomal ClC-6 develop a lysosomal storage disease with reduced pain sensitivity. Human gene variants have been associated with epilepsy, and a gain-of-function mutation causes early-onset neurodegeneration. Dysfunction of the lysosomal ClC-7 leads to a lysosomal storage disease and neurodegeneration in mice and humans. Reduced luminal chloride, as well as altered calcium regulation, has been associated with lysosomal storage diseases in general. This review discusses the properties of endosomal and lysosomal Cl\u2212/H+ exchange by CLCs and how various alterations of ion transport by CLCs impact organellar ion homeostasis and function in neurodegenerative disorders.The regulation of luminal ion concentrations is critical for the function of, and transport between intracellular organelles. The importance of the acidic pH in the compartments of the endosomal-lysosomal pathway has been well-known for decades. Besides the V-ATPase, which pumps protons into their lumen, a variety of ion transporters and channels is involved in the regulation of the organelles' complex ion homeostasis. Amongst these are the intracellular members of the CLC family, ClC-3 through ClC-7. They localize to distinct but overlapping compartments of the endosomal-lysosomal pathway, partially with tissue-specific expression. Functioning as 2Cl Data from patients, mouse models and cell biophysical measurements highlight an important role for Cl\u2212 which is accumulated in a secondary active transport by CLC Cl\u2212/H+ exchangers possess a further glutamate, referred to as \u201cproton glutamate,\u201d whose mutation abolishes or strongly diminishes the transport of both protons and chloride display similar transport properties but differential subcellular localization and the loss of VGLUT1, SV acidification was not impaired in primary neurons from ClC-3-deficient mice was reported to be reduced, suggesting that glutamate toxicity due to excessive glutamate release contributes to the neurodegeneration pointed to the activity of ClC-7 as a ClClcn7\u2212/\u2212 mice , displayed conspicuous loss of hippocampal and cortical neurons. Starting in the CA3 region of hippocampus neuronal loss progressed into the dentate gyrus and by the age of 1.5 years no hippocampal structures in Clcn7lox/lox;EMX1cre mice could be detected revealed slowed lysosomal degradation of endocytosed protein in humans (Kornak et al., CLCN7 mutations (Cleiren et al., CLCN7-related ARO in about 50% of the cases, as well as OSTM1-related ARO, are also neuropathic with primary neurodegeneration manifesting in developmental delay, hypotonia, retinal atrophy and seizures (Steward, CLCN7 mutations, which may lead to subcellular mislocalization of the ClC-7/Ostm1 complex or the impingement of the dysfunctional subunit on the ion transport properties of the unaffected subunit (Schulz et al., Clcn7\u2212/\u2212 mice with death after only a few weeks and neurodegeneration (Alam et al., PM/Ostm1 (Leisle et al., Clcn7+/G213R ADO2 mice also presented fibrosis in non-skeletal tissues such as lung and muscle; their brains exhibited perivascular fibrosis, \u03b2-amyloid accumulation and astrogliosis, and the animals showed behavioral abnormalities (Maurizi et al., Mutations in Steward, , which iSteward, . DominanPM/Ostm1 (Leisle et al., During the first electrophysiological analysis of osteopetrosis-causing ClC-7 missense mutations, it was surprisingly found that, besides loss-of-function mutations due to impaired ER exit or reduced ion transport, several pathogenic ClC-7 mutations accelerated the voltage-dependent activation of ClC-7de novo mutation of ClC-7 was identified in two children with hypopigmentation and delayed development (Nicoli et al., Y715C led to a drastic enlargement of late endosomal/lysosomal compartments (Nicoli et al., Y553C mutant (Polovitskaya et al., Y715C, the enlarged compartments were reported not to be acidified in contrast to surrounding smaller, hyperacidified lysosomes, which was attributed to the hyperactivity of the ClC-7 mutant (Nicoli et al., Recently, a heterozygous \u2212/H+ exchangers on distinct, but overlapping organelles of the endosomal-lysosomal pathway (Stauber et al., Loss-of-function and in some cases also gain-of-function of the intracellular CLCs, ClC-3/ClC-4, ClC-6 and ClC-7/Ostm1, lead to neuropathies, often neurodegeneration. They all function as Cl\u2212 export from endosomes/lysosomes. At membrane voltages in the range of those measured for endosomal/lysosomal compartments, which can be up to inside-positive 100 mV (Koivusalo et al., \u2212 accumulation by CLCs and the necessity of their ion transport. Mutation of the \u201cgating glutamate\u201d does not only convert the exchanger into a pure Cl\u2212 conductor, but also abolishes its voltage dependence. Therefore, such a mutation may indeed represent a gain of function in respect to Cl\u2212 transport. Interestingly, while the uncoupling mutation of ClC-5 or ClC-7 leads to effects that are similar to the respective knock-out in mouse models, the heterozygous mutant of ClC-6 results in a much more severe disorder in patients than in the murine gene knock-out model (Po\u00ebt et al., In some cases, it has been proposed that the CLC serves as a structural protein that functions to recruit other proteins such as components of the transport machinery by direct protein-protein interaction. Amongst others, the existence of different missense mutations which unlikely all prevent the same interactions argues against this. Although a role a role for protein-protein interactions of intracellular CLCs in vesicular trafficking cannot be excluded, the most straight-forward explanation for the importance of CLCs is their role in the regulation of the vesicular ion homeostasis. In this respect, the pronounced outward rectification of the vesicular CLCs remains enigmatic, because it would strongly favor Cl\u2212 by their Cl\u2212/H+ exchange mechanism is pivotal \u2013also for ClC-5, in addition to the acidification- (Novarino et al., \u2212 concentration rises to > 100 mM in the lysosome (Saha et al., \u2212 concentrations, but a normally low pH, have been found in nematode models of various neurodegenerative lysosomal storage diseases including Gaucher and Nieman-Pick A and B (Chakraborty et al., \u2212 concentration has been shown to influence the enzymatic activity of the lysosomal protease cathepsin C (Cigi\u0107 and Pain, \u2212 affects vesicular function is elusive (Stauber and Jentsch, The luminal pH is of undisputed importance for endosomes and lysosomes and impaired acidification is linked to neurodegeneration (Mellman et al., \u2212/H+ exchanger does not only \u2013if at all\u2013 affect the luminal Cl\u2212 concentration and pH, but ion homeostasis in general, including the transmembrane voltage and concentrations of other ion species (Ishida et al., 2+ (Chakraborty et al., The presence of a Cl\u2212/H+ exchangers impinges on the trafficking, function and possibly signaling of endosomal and lysosomal organelles and interconnected pathways such as autophagy, which in turn leads to cell death of vulnerable neurons. Future work is required to uncover in detail the molecular mechanism by which these CLCs contribute to the functioning of the endosomal-lysosomal pathway.In summary, dysfunction of the intracellular CLC ClSB, HH, and TS wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Nursing workforces are strengthened in COVID-19 units to enable closer monitoring of patients\u2019 vitals and personalized care. Yet, the healthcare teams confronted with COVID-19 are particularly strained and require active psychological support from psychologists and unit managers . Although benzodiazepines are often used as first-line treatments in case of acute agitation, after ruling out any somatic cause , randomized control trials don\u2019t support their use (McDermott and Gruenewald, et al., Unfortunately, in case of a drug shortage, alternative treatments must be considered. Midazolam can be used either orally or by subcutaneous injection to treat acute behavioral symptoms (McDermott and Gruenewald, et al., et al., Good clinical practice for the prescription of psychotropic drugs (Livingston et al., If chemical restraint is not possible, physical restraint may exceptionally be prescribed preferably with abdominal and pelvic straps (Livingston The COVID-19 outbreak is overwhelming all healthcare systems and we need to provide new guidelines for better medical management of elderly BPSD patients and their families and preserve hospital staff by streamlining their procedures."} +{"text": "With an enormous prevalence worldwide, diseases of the oral cavity and respective tissues are a highly relevant global health issue . Beside n = 23) with or without depression based on the Research Diagnostic Criteria for Temporomandibular Disorders (RDC/TMDs) Axis II, respectively. All study participants underwent an electromyographical examination to assess their bioelectrical resting activity of temporal and masseter muscles. The resting activity of selected masticatory muscles did not differ between the two groups. The authors concluded that these preliminary findings should lead to further research with larger sample size to gain insight the psychological factors related to bioelectric parameters in masticatory muscles. A Polish study by Gerreth et al. [The high variety and complexity of oral-systemic interactions is displayed by ten excellent articles in this special issue \u201cOral Health and Systemic Diseases\u201d. Thereby, seven studies are reported, complemented by three systematic reviews. A case-control study by Gerreth et al. compreheh et al. examinedh et al. examinedh et al. was perfThree systematic reviews are part of this special issue. The systematic review by P\u00e9rez-Losada et al. also appAltogether, the ten articles in the special issue \u201cOral Health and Systemic Diseases\u201d show different aspects of oral and systemic disease interaction and provide several implications for dental care of these patients. One main conclusion is the necessity of further research, especially large-scaled prospective studies to get a better understanding of the needs of respective patient groups. Furthermore, patient centered dental special care appears recommendable for different patient groups, which were examined in the different studies. The special issue \u201cOral Health and Systemic Diseases\u201d gives the reader an interesting and clinically relevant insight into oral and systemic disease interactions, an emerging field of dental and medical research."} +{"text": "Escherichia coli chromosome by forming a filamentous axial core from which DNA loops emanate, similar to the action of condensin in mitotic chromosome formation. MukBEF action, along with its interaction with the partner protein, MatP, also facilitates chromosome individualization by directing opposite chromosome arms (replichores) to different cell halves. This contrasts with the situation in many other bacteria, where SMC complexes organise chromosomes in a way that the opposite replichores are aligned along the long axis of the cell. We highlight the similarities and differences of SMC complex contributions to chromosome organization in bacteria and eukaryotes, and summarize the current mechanistic understanding of the processes.Structural maintenance of chromosomes (SMC) complexes are ancient and conserved molecular machines that organize chromosomes in all domains of life. We propose that the principles of chromosome folding needed to accommodate DNA inside a cell in an accessible form will follow similar principles in prokaryotes and eukaryotes. However, the exact contributions of SMC complexes to bacterial chromosome organization have been elusive. Recently, it was shown that the SMC homolog, MukBEF, organizes and individualizes the Escherichia coli MukB was the first SMC protein to be identified through its role in chromosome segregation complexes, play multiple important roles in chromosome organization and individualization [reviewed in plays an essential role in determining the axial core shape. Deletion of matP led to formation of uniform circular axial cores, because MatP displaces MukBEF from ter, while MatP+ cells have linear cores as a consequence of MatP-directed displacement of MukBEF complexes from ter in wild-type cells, while MukBEF clusters localize equally with all genetic regions tested in MatP\u2212 cells.By modestly increasing E. coli chromosome placed randomly within a cell? Early imaging studies showed that in new-born E. coli cells that have not initiated replication, the left and right replichores are organized into separate cell halves, while oriC is at midcell . Indeed, MukBEF complexes and MatP-matS are largely confined to \u03b3-proteobacteria (Br\u00e9zellec et al. oriCs located at the old pole in new borne cells and ter at the new pole (Fig. \u2212E.coli exhibit a similar organization (Danilova et al. S systems that are the main driving force behind chromosome segregation in many bacteria and which additionally recruit SMC complexes to the chromosome at specific parS sites near oriC (Fig. Vibrio cholerae encodes for two types of ParABS system, each directed to a specific one of the two separate chromosomes, despite encoding MukBEF and MatP-matS (David et al. The action of MukBEF-MatP in individualization of chromosome arms, by directing left and right arms to opposite cell halves, contrasts with the situation in many bacteria that encode SMC\u2013ScpAB complexes rather than MukBEF (e.g., ole Fig. e. IntrigriC Fig. . IntriguB. subtilis arms could be explained by higher order SMC action. The first in vitro single-molecule studies of loop extrusion by condensin showed asymmetric loop extrusion (Ganji et al. The mechanistic and functional differences between MukBEF and SMC\u2013ScpAB complexes remain elusive, but in our opinion, it is likely that they both act through ATP hydrolysis-driven loop extrusion. The requirement of MukBEF dimers of dimers for function (Badrinarayanan et al. matS, along with other proteins (Br\u00e9zellec et al. \u2212 and Gm+ bacteria (Petrushenko et al. SMC complexes also have co-evolved with other chromosome binding proteins that can cooperate their activity with prospective loop formation; for example, MukBEF and MatP-As in most biological systems, investigation of how SMC complexes function has been limited by the available assays. Early studies primarily exploited classical genetics and biochemistry, while later on new imaging techniques, particularly FISH-painting techniques, along with ensemble techniques like ChIP-seq and chromosome conformation capture techniques began to play important roles; in the latter case these can now be applied to single-cells [reviewed in (McCord et al."} +{"text": "Individuals who exercise regularly are protected from type 2 diabetes and other metabolic syndromes, in part by enhanced gene transcription and induction of many signaling pathways crucial in correcting impaired metabolic pathways associated with a sedentary lifestyle. Exercise activates Calmodulin-dependent protein kinase (CaMK)II, resulting in increased mitochondrial oxidative capacity and glucose transport. CaMKII regulates many health beneficial cellular functions in individuals who exercise compared with those who do not exercise. The role of exercise in the regulation of carbohydrate, lipid metabolism, and insulin signaling pathways are explained at the onset. Followed by the role of exercise in the regulation of glucose transporter (GLUT)4 expression and mitochondrial biogenesis are explained. Next, the main functions of Calmodulin-dependent protein kinase and the mechanism to activate it are illustrated, finally, an overview of the role of CaMKII in regulating GLUT4 expression, mitochondrial biogenesis, and histone modification are discussed. In developing countries, around 50 % of people with diabetes are undiagnosed. As a result, many do not receive adequate treatment and care to manage the disease, putting them at greater risk of serious complications and even death. According to the International Diabetes Federation (IDF) , the number of people with type 2 diabetes is increasing in every country. In 2019, diabetes affected at least 463 million people worldwide and is expected to reach 700 million by 2045 (IDF). The economic impact of diabetes is expected to continue to grow by 2045 as there is no effective cure to prevent or treat diabetes.Type 2 diabetes is one of the fast moving public health problems in both developed and developing countries. According to the World Health Organization (WHO), developing countries will likely suffer from diabetes epidemics in the 21Regulation of carbohydrate and lipid metabolism is very important in patients with insulin resistance and type 2 diabetes. Glucose is most commonly utilized for energy production in mammals. The regulation of glucose metabolism is very important to ensure glucose availability for the central nervous system, which almost entirely depends on glucose for its fuel. Under hyperglycemic conditions, glycogen synthesis is the major pathway in glucose metabolism, and muscle glycogen synthesis rate decreases by 50 % in type 2 diabetes as compared with healthy subjects . SkeletImpairment of insulin-mediated glucose uptake correlates with altered fatty acid metabolism as the biochemical pathways of fatty acid and glucose metabolism are fully integrated . Lipids2+) is an important second messenger involved in the regulation of many cellular events . Raisinal., 2000). It is al., 2000; Raney aal., 2000; Wu et aal., 2000). This pExercise or physical activity is one of the cornerstones for the prevention and management of type 2 diabetes in both men and women . SeveraRegular exercise also reduces the risk of ectopic (non-adipose tissue) lipid accumulation and type 2 diabetes through increased lipid oxidation and lipid oxidative capacity . A studWith regards to insulin sensitivity, exercise also shows a beneficial effect on insulin sensitivity in normal as well as insulin-resistant populations. Individuals with insulin resistance and type 2 diabetes are characterized by impaired insulin-stimulated glucose uptake in skeletal muscle . HoweveReduction in the risk of diabetes by exercise is owing to increased glucose transport capacity . GlucosPlasma membrane GLUT4 content correlated with glucose transport activity in both rat and human skeletal muscle . A studSeveral studies show that exercise increases glucose transport and GLUT4 expression in skeletal muscle. A vigorous 7-day exercise program increased insulin sensitivity and muscle GLUT4 content in younger and older people . Again,A study by McGee et al. (2009) shows tRegular exercise induces mitochondrial biogenesis, resulting in increased lipid oxidation capacity and turnover and improved glucose transport . MitochFewer and smaller-sized mitochondria are found in skeletal muscle of insulin-resistant, obese, or type 2 diabetes subjects, and the size of mitochondria correlates with mitochondrial oxidative capacity as well as in oxidative capacity and increases markers of mitochondrial biogenesis, which include PGC-1, mitochondrial transcriptional factor a (Tfam), and citrate synthase . Increaal., 2010; Ojuka eal., 2010). Activaal., 2010). Increaal., 2010). Increa2+/Calmodulin-dependent serine/threonine-specific protein kinase . It act Figure 2 . A typiman, 2002). When Tal., 1989; Hanson al., 1989).2 max) cycling exercise increases phosphorylation of CaMKII at Thr286. Another study by Serpiello et al. and in al. (2011) also shal. (2011). Besideal. (2011) indicat2+, AMP, and ROS . Increaal., 2003 McConellal., 2003). PGC-1 A study by Mukwevho et al. (2008) reporte3+ groups of lysine amino acid residues. Acetylation is regulated by a factor called Histone acetyltransferases (HATs). HATs help the transfer of an acetyl group from a molecule of acetyl Coenzyme-A to the NH3+ group on lysine. Deacetylation of lysine is facilitated by a factor called Histone deacetylases (HDACs), which catalyzes the removal of the acetyl group with a molecule of H2O . The splis, 1998; Grunstelis, 1998).A study by McGee and Hargreaves (2004) showed"} +{"text": "In this mSphere of Influence article, he reflects on how two papers, \u201cLong-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett\u2019s epithelium\u201d by Sato et al. and \u201cT helper cell cytokines modulate intestinal stem cell renewal and differentiation\u201d by Biton et al. , have influenced his research by describing the development of intestinal organoid cultures and implementation of high-throughput sequencing analysis. The combination of these forefront technologies has expanded opportunities for mechanistic interrogation of host immunity to enteric pathogens.Jose Lemme-Dumit works in the field of mucosal immunology and vaccines. In this mSphere of Influence article, he reflects on how two papers, \u201cLong-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett\u2019s epithelium\u201d by Sato et al. (T. Sato, D. E. Stange, M. Ferrante, R. G. J. Vries, et al., Gastroenterology 141:1762\u20131772, 2011, Although their use is practical, the genetic modifications and aberrant behavior of immortalized cells render results unreliable and nongeneralizable. Other models involve ex vivo primary cultures. The limited viability of human primary cells restricts and limits their use. A major advancement in tissue culture technology was achieved by the identification of the \u201cstemness\u201d marker Lgr5 (leucine-rich-repeat-containing G-protein-coupled receptor 5) at the intestinal crypts human intestinal stem cells and for establishment of long-term organoid cultures. These factors included ligands for Wnt signaling and epidermal growth factor, both of which are necessary for intestinal stem cell proliferation, as well as Noggin (a bone morphogenetic protein inhibitor), an inducer of cell expansion. The authors found that gastrin and nicotinamide improved culture efficiency, with nicotinamide being essential for prolonging culture viability for up to 1\u2009month. Further, inhibition of MAPK (p38) and transforming growth factor \u03b2 (TGF-\u03b2) type 1 (Alk4/5/7) signals increased intestinal stem cell stability and expansion, extending the duration of cultures by at least 6\u2009months. Besides identifying essential factors for intestinal stem cell expansion, Sato and colleagues showed that organoids retain essential aspects of the tissue from which they were derived, including aspects such as architecture, cell type composition, self-renewal dynamics, and normal karyotype. Organoids can be biobanked, which adds practicality. The technology described in that seminal paper has been broadly applied to produce organoids from other polarized epithelial organs such as stomach, lung, bile duct and pancreas, and prostate and mammary gland. Since the original publication of that paper, conditions appropriate for use in establishing 2D organoids have been described. Advantages of working with polarized epithelial cells in a steady monolayer include ease of handling and direct access to apical and basolateral compartments for treatment and analysis.Homeostasis and host defenses in the human gut are finely regulated. The intestinal epithelium provides a physiological and immunological barrier that protects the host from pathogens and harmful agents while maintaining a peaceful coexistence with nutrients and the gut microbiota. A variety of epithelial cell culture models have been used to study biological processes in the human gut. The majority of these l crypts . This brthelium\u201d , CleversThe discoveries by Clevers\u2019 group detailed in their report and follow-up studies have inspired and catalyzed not only my research but an entire field of study. Avoiding the drawbacks represented by cancer cell lines and the difficulty of working with animals and their host restriction, their approach opened a new avenue to the investigation of gut physiology and, in my case, of host-pathogen interactions and mucosal immunity. The organoid system as originally described still lacks the major cells and components that support and interact with the intestinal epithelium and that have important functional roles: innervation, fibroblasts and extracellular matrix components, and immune cells. My work has focused on the development and characterization of human enteroid-immune cell cocultures and the interrogation of immune mechanisms of host defense against enteric pathogens.+ intestinal stem cell subsets with different proliferative capacities capable of interacting with and activating naive Th cells via major histocompatibility complex class II (MHCII) antigen presentation. Treg and interleukin-10 (IL-10) were found to promote organoid expansion, while Th1, Th2, and Th17 cells and derived cytokines depleted intestinal stem cells and induced organoid differentiation. In addition, luminal signals (bacteria or parasite) influenced the intestinal stem cell-Th axis and shaped epithelial cell composition, increasing levels of MHCII-expressing stem cells. While the work was conducted using murine organoid and intestinal mouse crypts, the report highlights an important form of epithelial and immune cell cross talk as a regulated response for maintenance of epithelial self-renewal or for promotion of its differentiation. These results challenge the prevailing paradigm of adaptive immune cell function by showing that they not only contribute to antimicrobial surveillance but also support intestinal tissue growth and differentiation. The role of Th cells activated by intestinal stem cells in host immunity and tolerance is wide open for investigation. Single-cell analysis is the \u201cgold standard\u201d for discerning cell responses and interaction. The approach of Biton and colleagues stimulated ideas that I am applying in my own research to decipher interactions between the human intestinal epithelium and immune cells in the steady state, in response to infection, and during the phase of recovery, facilitating tissue repair.The topic of the complexity of the molecular and cellular processes involved in mucosal immunity to enteric organisms brings me to my second selected paper, which illustrates the unprecedented opportunity offered by recent technological advances to fill important knowledge gaps. Cutting-edge tools, including cell imaging, gene editing, and molecular analyses such as single-cell RNA sequencing (scRNA-seq), are now available for deep mechanistic interrogation of coordinated multicellular host responses. In the research article \u201cT helper cell cytokines modulate intestinal stem cell renewal and differentiation\u201d , the autThe two elegant publications discussed here illustrate the impact of cutting-edge technologies and their potential to advance translational research. Organoids and single-cell analyses could be powerful tools for personalized medicine; they would allow evaluation of drugs or therapies, understanding of genetic risks or disease susceptibility, and even correction of inherited defects."} +{"text": "Scientific evidence shows that food consumption is one of the main causes that increases the risk of developing a non-communicable disease (NCD). One of the mechanisms introduced to ensure more informed food purchases that lead to healthier diets is the introduction in the marketplace of functional food products to provide information on the nutritional and health properties that certain foods possess. This information is transmitted to consumers via different nutritional and health claims. Two studies investigated the prevalence of front-of-package (FoP) claims in Brazilian packaged food. Duran et al. found thTwo studies explored the context of foods and drinks with healthy and nutritious attributes in the United Kingdom (UK). Cesar Revoredo-Giha et al. indicateFour studies focused on consumers\u2019 preferences and willingness to pay (WTP) for functional food products, while one study examined the role of functional food in disease prevention. Vischeccia et al. analyzedTwo studies investigated consumers\u00b4 preferences for food products carrying sustainable and nutritional labels simultaneously. Almli et al. observedTwo lines of investigation analyzed how consumers ranked different nutritional claims. Gracia and Barreiro-Hurl\u00e9 reportedTwo studies explored consumers\u2019 choices for health information. Sogari et al. tested tFour studies focused on psychological factors affecting consumers\u2019 preferences for NCs. Guzek et al. determinThe last two studies investigated new determinants influencing purchase intention for functional foods. Berhaupt-Glickstein et al. , investiThe present Special Issue focused on the role of nutritional properties and/or health-related claims of food products and functional food products on choice preferences, choice behavior, healthy eating/healthy diet and the willingness to pay for certain foods."} +{"text": "Mercury (Hg) is a well-recognized biohazard for the nervous system. Methylmercury (MeHg) is an organic methylated form of Hg, highly toxic to humans, targeting the brain, as MeHg is rapidly absorbed, and easily reaches and crosses the blood-brain barrier , and related intricate mechanisms during homeostasis and disease states. In addition, we discuss possible ways how MeHg may affect hippocampal neurogenesis and the potential lasting consequences for brain neurodegeneration.In mammals, the intestinal microbiota is first acquired either by contact with maternal skin (if a cesarean labor) or directly maternal microbiota transfer immediately after birth intestinal barrier function. Impaired intestinal barrier function has been associated with increased MeHg intestinal absorption in the large intestine ; stage 2, containing transition cells/progenitor cells type 2a and type 2b and stage 3 . After stage 3, NSCs reach neuronal maturity by the extension of dendritic and axon processes and functional synaptic activity into neural circuitry 7 (Falluel-Morel et al., A reduction of 22% of hilus cells and 27% of granular layer cells, without affecting CA1 to CA3 hippocampal fields, after the MeHg challenge, has been found in mice, suggesting that MeHg preferentially affects SGZ's NSC (Sokolowski et al., E. coli, Clostridium, Staphylococcus spp., and hemolytic bacteria, could affect redox-sensitive transcription factor HIF1\u03b1 and ERK1/2 MAP kinase and intestinal barrier function (Holota et al., Falluel-Morel work also documented a decrease in the extracellular signaling of ERK1/2, required for the transition from G1 to S phase and inhibition of Bmi-1 gene expression, responsible for controlling the self-renewal potential of NSC. Genes linked to cell senescence have also been shown to be altered by MeHg, such as increased expression levels of HP1-\u03b3 and HMGA-1 (Falluel-Morel et al., Actinobacteria and decreases in the class Clostridia (Dunphy-Doherty et al., Interestingly, early life stress, such as social isolation, can impact neurogenesis and the intestinal microbiota. Socially isolated rats early in life show significantly fewer BrdU/NeuN positive cells in the dentate gyrus than controls and altered microbiota composition with increases in One possible crosstalk pathway between intestinal microbes and the brain may involve the vagal nerve. Vagotomy has been implicated in impaired hippocampal neurogenesis and BDNF levels (O'Leary et al., The better understanding how the MeHg-altered gut microbiota affects the hippocampal neurogenic niche, by tracking neurogenic biomarkers during NSC maturation in critical time windows of brain development, is in most need for novel therapeutic strategies to ameliorate these deleterious effects that may have lasting consequences for human health.This opinion paper discussed the role of the intestinal microbiota on MeHg neurointoxication with potential consequences for the hippocampal neurogenic niche. Novel breakthrough findings are much supportive of human neurogenesis even in elderly (Boldrini et al., Accumulating evidence implies the gut-brain axis as a pathway for MeHg harmful neurotoxic effects and a potential factor for later neurodegenerative disorders. The MeHg may induce a hormesis-related neuronal toxicity. Hormesis is an important redox dependent aging-associated neurodegenerative/ neuroprotective issue (Calabrese et al., All authors have read and approved the manuscript. All authors have equally contributed to the opinion paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "L. de Swart et al. and \u201cLong-term measles-induced immunomodulation increases overall childhood infectious disease mortality\u201d by M.Rory de Vries works in the field of viral pathogenesis and focuses on interactions between respiratory viruses (or corresponding vaccines) and the host immune system. In this mSphere of Influence article, he reflects on how the articles \u201cPredominant infection of CD150 + lymphocytes and dendritic cells during measles virus infection of macaques\u201d by R. L. de Swart et al. and \u201cLong-term measles-induced immunomodulation increases overall childhood infectious disease mortality\u201d by M. J. Mina et al. made an impact on him. These articles studied interactions between measles virus and the host and influenced him by making two important points. (i) It is crucial to use nonadapted (recombinant) viruses in disease-relevant model systems when studying virus-host interactions. (ii) Studying viral pathogenesis requires a combination of in vitro, ex vivo, and in vivo studies, and a group of researchers with multiple expertises. He learned that only when all these aspects are combined, can one truly answer the question: \u201cHow does a virus cause disease?\u201dRory de Vries works in the field of viral pathogenesis and focuses on interactions between respiratory viruses (or corresponding vaccines) and the host immune system. In this mSphere of Influence article, he reflects on how the articles \u201cPredominant infection of CD150 I have implemented these lessons in our current studies on virus-host interactions of human respiratory syncytial virus (HRSV), where we make use of recombinant viruses directly based on clinical isolates and study their behavior in respiratory organoids or differentiated primary airway epithelial cells cultured at the air-liquid interphase , and scientists should always remain vigilant and \u201ccritical.\u201d If one has a hypothesis that deviates from the current dogma, you should be ambitious and perform the experiments to either prove or disprove that hypothesis with perseverance and indomitable spirit.Both articles described here were game-changers in the field of measles research; combined with follow-up studies, they led to revisions of the textbooks. Shaping me as a researcher, this is one of the \u201clife lessons\u201d I have taken from these articles: what you read in textbooks or articles is not right"} +{"text": "An algorithmic strategy to design stoichiometric quaternary and solid-solution quinary solids is described. The strategy involves recognition of structural inequivalences to generate ternary and quaternary cocrystals which can then be extended to five-component solid solutions through matching of suitable interactions. Ever since Gerhard Schmidt 1971 proposedet al., 2000et al., 2005et al., 2020et al., 2009et al., 2021et al., 2020et al., 2020From early work in the design of molecular crystal structures . Multidrug cocrystals can be expected to produce a synergistic therapeutic effect, while crystal engineering of the molecular packing and dominant non-covalent interactions may allow the modulation of physicochemical properties to improve drug formulation and delivery. To date, the only approaches to succeed in preparing ternary API solids have been the \u2018drug\u2013bridge\u2013drug\u2019 strategy developed by Liu al. 2018 and the IUCrJ , tetramethylpyrazine (TMP), either 2,2\u2032-bipyridine (22BP) or 2,2\u2032-bithiophene (22TBP), and 1,2-di(4-pyridyl)ethane (DPE). A flowchart approach (see Fig. 1Aside from the intrinsic beauty of the resulting structures, this is an elegant example of a retrosynthetic approach being applied to systems built on noncovalent interactions. While hydrogen bonding is the dominant interaction in the examples reported here, the approach lends itself to use with other forms of supramolecular interactions such as halogen bonding, coordination bonding and \u03c0\u2013\u03c0 interactions.An algorithmic approach, such as that described here, is a positive step in the direction of developing an understanding of non-covalent interactions and their influence on synthesis of multicomponent crystals. This is essential for the advancement of crystal engineering into the new era of true design of functional materials."} +{"text": "In addition, microfluidics platforms enable us to measure the mechanical properties of cells by establishing defined flow or confined microstructures through viscoelastic particles/cells focusing and droplet microfluidics. Finally, the flexible microdevices have become widely employed.Microfluidics has proven to be a useful platform to understand the material properties and technical applications of soft matter, including emulsions, polymer solutions, hydrogels, and cellulose papers. The study of the characteristics of soft matter, like viscoelasticity, non-Newtonian fluid mechanics, and deformation, has greatly benefitted from using microfluidics to accurately control conditions in time and space. Microfluidics has also served as a useful platform to study biological cell and tissues systems, including mechanobiology. Using microfluidics, external mechanical stress is regulated in physiologically-relevant systems for studying cells, tissues and organisms to understand how mechanical cues are sensed and transduced into biochemical and electrical signals that influence mechano-transduction. Furthermore, the characteristics of soft matter are exploited when combined with microfluidic platforms to mimic in-vivo microenvironment mimics and (6) mechanobiology research.In this Special Issue, we highlight recent progress in microfluidics with research papers and review articles that focus on novel methodological developments and applications of microfluidics devices for soft matter and mechanobiology. It contains ten research papers and two review articles on the following aspects of microfluidic application regarding soft matter and mechanobiology: (1) droplet generation and its application (2) viscoelasticity-based handling of particles/cells (3) paper-based assays (4) flexible devices (5) Droplet generation and its application: S\u00e1nchez et al. reviewedC. albicans [Viscoelasticity-based handling of particles/cells: Cho et al. found thalbicans in sheatPaper-based assays: Kim et al. reviewedFlexible devices: Lee et al. demonstrin-vivo microenvironments: Yue et al. [Mimic of e et al. engineerMechanobiology research: Feng et al. discusseWe wish to thank all authors who submitted their manuscripts to this Special Issue. We would also like to acknowledge all the reviewers for dedicating their time to provide careful and timely reviews to ensure the quality of this Special Issue."} +{"text": "Dear Editor,Recently, Gao and colleagues published a study about the role of Kupffer cells in hepatotoxicity . The au3 . IntereKupffer cells are known as important modifiers of hepatotoxicity (Kessler et al., 2014; Reif etThe authors declare no conflict of interest."} +{"text": "Although climate change is altering the productivity and distribution of marine fisheries, climate-adaptive fisheries management could mitigate many of the negative impacts on human society. We forecast global fisheries biomass, catch, and profits to 2100 under three climate scenarios and five levels of management reform to (1) determine the impact of climate change on national fisheries and (2) quantify the national-scale benefits of implementing climate-adaptive fisheries reforms. Management reforms accounting for shifting productivity and shifting distributions would yield higher catch and profits in the future relative to today for 60\u201365% of countries under the two least severe climate scenarios but for only 35% of countries under the most severe scenario. Furthermore, these management reforms would yield higher cumulative catch and profits than business-as-usual management for nearly all countries under the two least severe climate scenarios but would yield lower cumulative catch for 40% of countries under the most severe scenario. Fortunately, perfect fisheries management is not necessary to achieve these benefits: transboundary cooperation with 5-year intervals between adaptive interventions would result in comparable outcomes. However, the ability for realistic management reforms to offset the negative impacts of climate change is bounded by changes in underlying biological productivity. Although realistic reforms could generate higher catch and profits for 23\u201350% of countries experiencing reductions in productivity, the remaining countries would need to develop, expand, and reform aquaculture and other food production sectors to offset losses in capture fisheries. Still, climate-adaptive management is more profitable than business-as-usual management in all countries and we provide guidance on implementing\u2013and achieving the benefits of\u2013climate-adaptive fisheries reform along a gradient of scientific, management, and enforcement capacities. Marine fisheries provide a vital source of food for over half the world\u2019s population and support the livelihoods of over 56 million people globally . HoweverThe response of fishers and managers to these changes could either exacerbate or mitigate the impacts of climate change on human society and must be considered in forecasts of climate impacts on marine fisheries ,14. For Gaines et al. providedHere, we use the Gaines et al. climate-MSY) determined by aggregating values from Costello et al. [We used the Gaines et al. climate-o et al. S1 Fig)MSY) deteo et al. . Projecto et al. species o et al. bioeconoo et al. and the The modified Garc\u00eda Molinos et al. species K), intrinsic growth rate (g), and a shape parameter (\u03d5) that determines the proportion of carrying capacity at which production is maximized. Parameters were developed for species-stocks following the procedure detailed in Gaines et al. [The modified Costello et al. bioeconos et al. and are s et al. sourced s et al. and catcs et al. fit to ts et al. . The shas et al. , which ms et al. for a des et al. which pos et al. \u201334 and ts et al. . AlthougThe harvest rate is based on the following five management scenarios: business-as-usual , productivity shift adaptation only, range shift adaptation only, full adaptation, and \u201crealistic\u201d adaptation revenues were calculated as catch multiplied by species-specific ex-vessel prices and (2) We evaluated the impact of climate change and management reform on the fisheries of 156 coastal sovereign countries summing across their domestic and territorial exclusive economic zones (EEZs). We scaled the projections of Gaines et al. from theMSY, the biomass that produces MSY when fished at FMSY, by century\u2019s end (2091\u20132100). This is a common target for fisheries management . This performance metric better reflects the goals of fisheries management than percent change in biomass. For example, decreasing biomass in a previously undeveloped fishery is an expected consequence of economically optimal management and should only be perceived negatively when the decrease reduces biomass below the target.For Approach 1, we compared the percent difference in harvests and profits in 2100 relative to today under each management scenario. While Gaines et al. performeMaximum sustainable yield (MSY) of the evaluated stocks is forecast to decrease by 2.0%, 5.0%, and 18.5% from 2012\u20132021 to 2091\u20132100 under RCPs 4.5, 6.0, and 8.5, respectively . Note thMSY \u2264 1.2 and 0.8 \u2264 F/FMSY \u2264 1.2) in the initial year management results in both lower catches and profits in the future relative to today under all three emissions scenarios . In contial year ; thus, rWhile business-as-usual management results in lower catches and profits relative to today for the majority of countries (82\u201385% of countries), full adaptation yields higher catches and profits for a majority of countries in all but the most severe emission scenario . In thisOverall, our results indicate that climate change will dramatically alter the distribution and productivity of marine fisheries, but plausible climate-adaptive management reforms could minimize or eliminate negative impacts in most countries. This reinforces and expands upon the work of Gaines et al. in two iOur model predicts shifts in productivity that are consistent in both pattern and magnitude with a recent ensemble model that aveImportantly, however, our approach differs from these studies, because, in addition to forecasting the impact of climate change on the biological potential of fisheries, we consider the impact of alternative human responses to these changes, which could either exacerbate or alleviate the impacts of changing biological potential . Indeed,The development and implementation of stock assessment methods and management strategies necessary to achieve benefits in the face of climate change is nascent but rapidly developing. For example, Skern-Mauritzen et al. reviewedFurthermore, achieving the benefits of climate-adaptive fisheries reform will require accounting for shifting productivity and distributions along a gradient of scientific, management, and enforcement capacities. Many countries lack the monitoring programs required to detect and describe shifts in distribution and productivity, the scientific capacity for conducting either climate-agnostic or climate-adaptive stock assessments, and the management capacity for setting and enforcing fisheries regulations ,42,43. TFortunately, a growing body of literature provides guidance on accounting for shifting distributions and productivity in fisheries assessment and management ,17,45,46Historically, well-managed fisheries have been among the most resilient to climate change , and ourAdapting to climate change will require dynamic, flexible, and forward-looking management. This can be achieved by aligning management policies with the spatio-temporal scales of climate change, ecosystem change, and socioeconomic responses . In highShifting distributions are already generating management challenges and the rates of these shifts and associated conflicts are expected to increase with climate change ,18,70. NThe impact of climate change on fishing communities can be reduced through measures that increase socioeconomic resilience and adaptive capacity to environmental variability and changing fisheries ,76,77. AEven the best climate-adaptive management will be unable to maintain current catch and profits in most tropical developing countries. Although these countries should still pursue climate-adaptive reforms to maximize catch and profits from capture fisheries, they will also need to develop, expand, and reform other sectors to compensate for capture fishery losses and meet growing production demands . Marine Although climate change is expected to reduce the productivity of marine fisheries globally , climateS1 Table* See Table S1 in Gaines et al. for eart(DOCX)Click here for additional data file.S1 FigMSY \u2264 1.2 and 0.8 \u2264 F/FMSY \u2264 1.2).The transparent grey box indicates near optimal fisheries management Click here for additional data file.S2 Fig(TIFF)Click here for additional data file.S3 FigThe percentage labels indicate the percentage of countries falling in each quadrant of catch and profit outcomes.(TIFF)Click here for additional data file.S4 FigThe percentage labels indicate the percentage of countries falling in each quadrant of catch and profit outcomes.(TIFF)Click here for additional data file."} +{"text": "Mycobacterium abscessus (Mab) is a fast-growing cousin of the infamous Mycobacterium tuberculosis. Both bacteria cause difficult-to-cure lung disease. In contrast to the slow-growing obligate pathogen M. tuberculosis, Mab is an opportunistic pathogen. Ubiquitously present in soil and water, Mab typically causes disease in vulnerable populations, including immune-compromised patients and people suffering from lung disorders such as cystic fibrosis and chronic obstructive pulmonary disease. Whereas, a drug regime that cures tuberculosis within 6 months is available, cure rates for Mab with the currently recommended combinations are 50% at best (Kwak et al., in vitro potency. Instead, the cornerstone of Mab disease therapy is a macrolide, either azithromycin or clarithromycin. It is plausible that the lack of a rifamycin in Mab regimens contributes to unfavorable outcomes (Ganapathy et al., A key drug in the regimen against tuberculosis is rifampicin. Inclusion of this rifamycin in anti-tuberculosis therapy in the 1960's resulted in dramatic treatment shortening and formed the basis of the 6-month curative regimen still in use today (Ganapathy et al., in vitro (Aziz et al., In a screen of approved drugs against Mab, we were surprised to find that the rifampicin analog rifabutin (Crabol et al., To provide additional preclinical data supporting repurposing of rifabutin, we recently measured its efficacy in a murine model of Mab lung disease. Rifabutin was as efficacious as the first line drug clarithromycin, both administered at the mouse equivalent of their clinically approved doses. As expected, rifampicin lacked efficacy (Dick et al., in vitro results are emerging suggesting that rifabutin is not only active as a single agent, but also appears to suppress inducible macrolide resistance, an intrinsic resistance mechanism frequently encountered in Mab isolates (Nash et al., whiB7-erm41 system (Hurst-Hess et al., in vivo, this finding, would support a one-two punch attack against the infection.Interestingly, Concomitant to these encouraging preclinical data, a first clinical success story was recently reported (Cheng et al., Our preclinical results, together with these early clinical data suggest that rifabutin may improve outcomes of refractory Mab disease. Just as inclusion of a rifamycin was a game changer in the treatment of tuberculosis, rifabutin may both improve cure rates and reduce treatment duration of largely incurable Mab lung disease.The author confirms being the sole contributor of this work and has approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Aspergillus, Cryptococcus, Candida, Histoplasma, Blastomyces, and Coccidioides, and discuss potential strategies to manipulate monocyte responses in order to enhance anti-fungal immunity in susceptible hosts.Monocytes and their derivatives, including macrophages and dendritic cells, play diverse roles in the response to fungal pathogens. Sensing of fungi by monocytes triggers signaling pathways that mediate direct effects like phagocytosis and cytokine production. Monocytes can also present fungal antigens to elicit adaptive immune responses. These monocyte-mediated pathways may be either beneficial or harmful to the host. In some instances, fungi have developed mechanisms to evade the consequences of monocyte activation and subvert these cells to promote disease. Thus, monocytes are critically involved in mediating the outcomes of these often highly fatal infections. This review will highlight the roles of monocytes in the immune response to some of the major fungi that cause invasive human disease, including Lineage tracing studies suggest that non-classical monocytes develop directly from classical monocytes or a monocyte-dendritic cell progenitor (MDP) , including C-type lectin receptors (CLRs), Toll-like receptors (TLRs), and NOD-like receptors (NLRs), can detect pathogen-associated molecular patterns (PAMPs) like \u03b2-glucan, chitin and mannose in the fungal cell wall and trigger downstream signaling pathways to coordinate the innate immune response production , and reactive nitrogen species (RNS) produced by inducible nitric oxide synthase (iNOS or NOS2) in response to pro-inflammatory stimuli macrophages or anti-inflammatory, alternatively-activated (M2) macrophages macrophages and monocytes, activation of these cells with cytokines, including IFN\u03b3 and GM-CSF, restricts the intracellular growth of H. capsulatum, in part by sequestering nutrients like zinc ions that are needed for fungal growth can survive within unstimulated macrophages in vitro, but the addition of IFN\u03b3 or TNF enables fungal killing requires stimulation by pro-inflammatory cytokines cells to secrete GM-CSF that is required for neutrophil fungicidal activity to prime T cells and induce adaptive immune responses that promote fungal clearance (Roy and Klein, C. neoformans or bind cryptococcal antigens, resulting in DC maturation and the subsequent activation and proliferation of T cells (Syme et al., H. capsulatum antigen can dampen harmful Th2 responses by reducing IL-4 production by CD4+ T cells (Szymczak and Deepe, H. capsulatum antigen acquired from apoptotic macrophages to promote CD8+ T cell cytotoxic responses under conditions where CD4+ T cells are absent or low, as might be found in HIV/AIDS patients (Lin et al., B. dermatitidis and H. capsulatum, robust CD4+ T cell priming is dependent on monocyte recruitment to the immunization site (Wuthrich et al., C. immitis antigen can induce T cell proliferation and IFN\u03b3 secretion (Richards et al., A. fumigatus conidia to the draining mediastinal lymph nodes and trigger beneficial CD4+ T cell responses (Bozza et al., C. gattii, but concurrently, the fungus is able to prevent further DC maturation that would lead to a robust adaptive immune response (Huston et al., In a murine model of chronic cryptococcosis, monocytes can differentiate into DCs that mediate the generation of a Th1 adaptive response that aids in clearance of the fungus (Osterholzer et al., The adaptive immune response, which includes the generation of memory T and B cells, is the classic mechanism by which the immune system retains memory of foreign antigens to ensure a rapid and specific response upon re-exposure. However, recent studies indicate that monocytes and other innate immune cells can also contribute to immunological memory through the process of trained immunity (Netea et al., C. albicans can cause histone modifications and metabolic changes in monocytes and macrophages (Quintin et al., C. albicans, these trained monocytes and macrophages had enhanced cytokine production and improved survival of the infected mice (Browder et al., Staphylococcus aureus (Di Luzio and Williams, Saccharomyces cerevisiae can enhance monocyte responses to C. albicans as well as gram-positive and gram-negative bacteria (Rizzetto et al., Fungal antigens have been found to induce trained immunity in monocytes and their derivative cells. Exposure to \u03b2-glucan and to heat-killed or sublethal doses of the commensal fungus C. neoformans indicate that splenic DCs undergo histone modifications that enhance cytokine responses upon rechallenge with a virulent strain of C. neoformans (Hole et al., S. aureus, and C. albicans. The fungal component of C. neoformans that may be involved in stimulating this DC memory also remains to be identified.There is some evidence that DCs may also have memory-like capabilities. Studies using a vaccine strain of C. albicans infection (Saz-Leal et al., Strides are being made to further enhance the effects of trained immunity. For example, deleting SHIP-1 in trained macrophages increases their production of pro-inflammatory cytokines and improves their protection against lethal + macrophages vs. moDCs (Menezes et al., Pneumocystis jirovecii, Fusarium spp., the Zygomycetes like Rhizopus spp. and Mucor spp., and emerging pathogens like Candida auris (Friedman and Schwartz, The multiple roles of monocytes and their derivative cells in the host response to fungal pathogens highlight their importance in mediating the outcomes of infection. Dissecting the specific mechanisms by which monocytes carry out these functions may enable us to develop novel therapeutics that can target these pathways to improve the mortality rates from invasive fungal infections. With the current intense focus on the role of the microbiome in human health, it will be interesting to further uncover the roles that commensal organisms may play in the trained immunity of monocytes as a key defense mechanism against pathogenic fungi. There is ongoing work to determine whether the heterogeneity of monocyte responses may be tied to their origins in the hematopoietic tissues. For instance, there is evidence that the fate of monocytes is predetermined in the bone marrow and may originate from differences in expression of the transcription factor PU.1, which can dictate their eventual differentiation into iNOSLH conceptualized, wrote, and edited the manuscript.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "In the field of Neuroelectronic Interfaces it seems as though the lines between reality and science-fiction/fantasy are often blurred. One of the inspirations for our most recent Gordon Research Conference in March 2018 aimed at \u201cBridging the Gap in Neuroelectronic Interfaces\u201d dates back to 1999 when Chapin et al. , very muUnfortunately, after this very promising start two decades ago, these technologies were \u201clost in translation\u201d useful to further explore the foreign body responses or nano-particle based stimulations. The prospects of bioactive, so called \u201cliving electrodes\u201d were discussed by Adewole et al., who points out several differing approaches to \u201ctrick\u201d the brain into incorporating artificial implants. The biomechanics of neuronal adhesion may play an important role in this context and can be assessed by frequency analysis of quartz crystal oscillators, as is pointed out by Khraiche et al.. As important as microscopic biomechanics is in our field of research, it is equally difficult to quantify in brain tissue. Help is offered from localized probing of biomechanics by Atomic Force Microscopy (AFM) as is briefly reviewed by Viji Babu and Radmacher. Another exciting review by Aplin and Fridman extensively discusses the rarely used constant current (DC) stimulation of neural tissue, a potential new field of neuromodulation enabled by recent developments in microfluidics.Several insightful reviews support our conference's look outside the box and give an overview into microfluidic based model systems . They were able to show optogenetic stimulation of muscles in non-human primates! It goes without saying, flexibility was not a feature expected from silicon probes successfully used in large animals as reported by Ulyanova et al..As one common denominator, several groups reported on recent approaches to a long-standing idea to use of flexible or compliant substrates. For example, Electrode composition and performance was an issue investigated in several articles.Meijs et al. deposited different layers of Boron Doped Diamond (BDD) on TiN electrodes and compared them electrochemically, identifying good candidates for further in vivo testing. The article from Ferlauto et al. demonstrates a reduction of electrical noise by inserting conductive polymers as compliant intermediary on Pt-electrodes. Whereas, the work by Neto et al. informed us to stop worrying about impedances\u2014at least of recording micro-electrodes in the usual range (0.1 to 2 M\u03a9)\u2014as long as they exhibit a low shunting capacity. In contrast, potentiostatic experiments done by Harris et al. concluded that the use of Ohm's law to describe electrical stimulation over Pt-electrodes is an unwarranted oversimplification, ignoring the electrically complex, spatially varying tissue-electrode interface. In order to further the quality control with MEAs Suarez-Perez et al. introduced spectral definitions of SNR based on cortical slow oscillations (SO) providing a less disputable \u201csignal\u201d (LFP UP state) over a \u201cnoise\u201d state.Several articles dealt with improvements for artificial sensing front ends:Losada et al. took the well-known mushroom electrodes . A novel simulation tool was presented by Al Abed et al. to shed light on in vivo electroporation in context of gene therapeutic improvements of the cochlea-electrode-interface.Improvements of optical techniques were presented by several other articles.Nambiar et al. demonstrated an algorithmic pipeline to reconstruct brain tissue surrounding explanted \u201chybrid\u201d array electrodes. Esquibel et al. employed the label free, optical sectioning method of second harmonic generation to examine implanted brain slices and showed unusual collagen fiber patterns not found in normal brains. Quite remarkably, by applying a custom made optoacoustic imaging setup Gottschalk et al. monitored neuronal calcium dynamics under blood-free conditions deep in an ex vivo maintained whole mouse brain. It will not be the last we are going to hear from genetically encoded calcium indicators (GECI). Improvements in 2-photon imaging presented in the review by Dorand et al. show a complicated and dynamic response to BBB-rupturing, substantial immune activation and microglia participation which might warrant a systematic application of different medications. Wellman et al. further supports the quest to widen the circle of usual suspects around brain implanted devices as they reveal an involvement of a wealth of other players like oligodendrocytes or even pericytes. A view shared by Bedell et al. and Hermann et al. poihc who not only vote for minimizing the cross sectional area of implants, but propose benefits from targeting the TLR/CD14 pathway as a therapeutic mechanism\u2014in particular focusing on infiltrating peripheral immune cells, while allowing the resident microglia to facilitate neuroprotection.As hoped for while organizing the conference, several overarching cutting-edge topics were presented to the community for the first time. The conference organizers then crystalized the momentum from the meeting into in the subsequent articles reported in this virtual issue. Unfortunately, the COVID-19 pandemic postponed the most recent installment of our meeting. Please continue to check the Gordon Research Conference website for information about the rescheduled conference.UH and JC both wrote and edited this article.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Dear Editor,st century. Individuals with COVID-19 are at an increased risk of developing lower respiratory tract disorders, such as pneumonia, acute respiratory distress syndrome (ARDS), and even death , a novel coronavirus infection, is fast becoming the epidemic of the 21al., 2020). To stoRecently, the use of vitamin D as a preventive/therapeutic intervention against COVID-19 infection has received much attention . It hasal., 2011). In addal., 2011; Moise aal., 2011; Li, 201al., 2011; Manasekal., 2011; Remmeltal., 2011). For inal., 2011). COVID-al., 2011; Sun et al., 2011). Conseqal., 2011). Vitamial., 2011), which al., 2011; Suh et al., 2011). If theal., 2011), may inin vitro and in vivo vitamin D-mediated ACE2 metabolism and its interactions with other ACE2-inducing drugs. Finally, until further data regarding the efficacy of vitamin D on the COVID-19 infection is available, clinicians should exercise caution in the overuse of vitamin D, especially in the elderly patients who also use other drugs which may alter ACE2 expression. Until further studies can fill these gaps, the single daily dose of 800-1000 IU vitamin D may be sufficient to meet the needs of most of the population. To summarize, first, the review of literature on the beneficial effects of vitamin D against COVID-19 infection leads to some doubts and controversies; therefore, well-designed clinical trials are needed to assess the potential risk-benefit of vitamin D supplementation for the prevention and/or treatment of COVID-19 infection. Second, although appropriate vitamin D doses may show some benefits in reducing the respiratory tract infections, higher doses may be harmful. Third, limited information is available on the None of the authors have any conflicts of interest or financial ties to disclose.Not applicable."} +{"text": "This research provides a guide to sweetpotato breeders for developing and deploying appropriate sweetpotato cultivars capable of meeting targeted consumer needs for sweetpotato fries and other fried sweetpotato products in distinct segments. It will also enable stakeholders in the sweetpotato value chain strategise in order to increase sweetpotato adoption. Prepared foods are increasing in popularity in West Africa alongside rapid urbanisation. Growing demand for fried products calls for targeted breeding efforts to meet consumer needs, but little is known regarding consumer preferences. This research identified the sensory attributes of fried sweetpotato preferred by different consumer groups using a combination of consumer acceptance testing and descriptive sensory analysis. Market and community surveys identified three consumer segments in Ghana and Nigeria with contrasting preferences for fried sweetpotato sensory attributes. One group preferred crispy, crunchy, mealy and sweet fried sweetpotato; another preferred characteristic yam flavour and dry texture; and the third preferred uniform orange colour appearance, ripe plantain flavour and palm nutty flavour. Such consumer segmentation can help emerging West African fried sweetpotato industries identify target markets and provides valuable information to breeders, growers and retailers to prioritise attributes in their breeding, growing or product sourcing decisions. Sweetpotato is a nutritionally important crop and increased consumption has been attributed to several factors. Though primarily a starchy staple, it is also rich in antioxidants, vitamins and minerals in both its roots and leaves. Orange\u2010fleshed, provitamin A\u2010rich types have been well\u2010established for their ability to combat vitamin A deficiency, when combined with a nutrition education component . The market survey set included five contrasting sweetpotato cultivars from breeding trials of the Crop Research Institute and Savanna Research Institute of the Council for Scientific and Industrial Research, Ghana and the International Potato Center. These included both released cultivars, and clones at the advanced stage of the varietal selection process Table\u00a0; Fig.\u00a01.et al. . Oil was heated to a temperature of 180\u00b0C before frying for about 8\u201310\u00a0min. Samples were placed on a white paper towel inside a clean disposable bowl to drain excess oil for about 2\u00a0min before wrapping them with aluminium foil. Sensory evaluation was conducted within 10 mins after frying with initial inner strip temperature of about 80\u00b0C. Samples for the consumer sensory tests in the community survey were prepared by expert fryers at each community using local common practices that varied slightly as described by Ssali et al. . The majet al., Eight panellists were used to profile the two sets of sweetpotato materials Table\u00a0 using a Consumer preference tests of the five varieties in the market survey were carried out in four major Ghanaian regional markets purposively selected. Three of these regions are known to have major sweetpotato producing communities. Bawku in the Upper East Region, Cape Coast in the Central Region and Akatsi in the Volta Region were selected for being the major regional markets and Agbogbloshie, Malata, Kaneshie and Accra Mall markets in the nation\u2019s capital, Accra, were selected due to high levels of economic activities in those markets.Sweetpotato fries were prepared and served to randomly selected consumers at the selected markets. In total, 332 consumers evaluated samples across the four regions. Samples were prepared and coded with three letters generated using XLSTAT software . Consumers were asked to evaluate fries and rate them for overall liking, using a 9\u2010point hedonic scale (1\u00a0=\u00a0Extremely dislike through 9\u00a0=\u00a0Extremely Like). Samples were served on white disposable plates with subjects handling samples with disposable white tissue papers. Consumers were asked to rinse their mouths with water between different samples.et al., Following the gendered mapping for fried sweetpotato product in Kano and Kwara States in Nigeria and Bawku in the Upper East Region of Ghana, consumer preference tests were conducted in the communities with 191 respondents , 101 females (F)) with means separation conducted using Tukey test at 5% significant level for each sensory attribute . Principal component (PCA) analysis using the correlation matrix of significantly different sample attributes was used to visualise how sweetpotato cultivars were differentiated across sensory attributes.Consumer liking scores in both surveys were subjected to one\u2010way ANOVA using JMP Pro 11 and mean separation performed by Tukey test. Cluster analysis was also carried out using Agglomerative Hierarchical Clustering (AHC) to group consumers into different segments based on shared characteristics using XLSTAT . One\u2010way ANOVA was then performed on the overall liking scores for each cluster using JMP Pro 11 and mean separation by Tukey test. An external preference mapping (PREFMAP) was created using a vector model by regressing consumer cluster groups onto the factor scores of the first two principal components from the DSA.et al., et al., et al., The appearance of fried sweetpotato was measured by uniform colour, surface browning and fibrousness Table\u00a0. Most ofet al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., Textural attributes Table\u00a0 are veryet al., et al., et al., et al., et al., Dioscorea rotundata), popularly called Pona, is the most widely consumed and preferred root and tuber variety Table\u00a0 and Kuffet al., et al., et al., Due to the overlapping usage of flavour and taste attributes in sensory descriptions, taste attributes were described as perceived by only the tongue which included the four basic taste sensations and umami while flavour attributes were described to include aroma through the nasal cavity as well as through the mouth. Sweetpotato is generally a sweet crop due to its ability to easily breakdown starch to maltose as a result of amylase activity and to the presence of other sugars and Kuffour (Ghana) used for the community surveys Fig.\u00a0. Obare, et al., et al., et al. across the locations), regardless of the different sensory attributes Table\u00a0. This is, et al. , where n, et al. . In addi et al., . The lea et al., , where y et al., since swet al., et al., et al., et al., et al., Consumers were generally grouped into three segments through agglomerative hierarchical clustering (AHC) of overall liking scores Table\u00a0 in GhanaP\u00a0=\u00a00.035) value indicates that age group distribution is dependent on type of cluster. Older people are more likely to prefer sweetpotatoes with characteristic attributes of this segment. The last group was driven by flavour attributes such as doughnut, ripe plantain and palm nutty flavours and uniform colour, which were all associated with orange\u2010fleshed cultivars. In terms of preference for orange\u2010fleshed cultivars, most consumers were in this group (47.8%). Younger consumers (ages below 25\u00a0years) were highest in this group. This was in line with the findings of Tomlins et al. compared to the less sweet type (PGA14351\u20104) in Ghana. However, consumer segmentation showed that different cultivars were preferred by different consumer groups due to their unique attributes. Three consumer segments with varying attribute preferences were identified. One group preferred sweetpotatoes with sweet taste, crispy, crunchy and mealy textures, while another group was driven by dry texture and yam flavour. The third group was predominantly influenced by attractive colour, flavour and possibly softer textures. The test of independency showed that only age group was dependent on segment groupings. Community surveys in Ghana and Nigeria also indicated a similar trend. Though most currently available orange\u2010fleshed cultivars are generally perceived as poor candidates for fried products by processors due to higher oil consumption than other available cultivars, they command high consumer demand due to their attractive colour and unique flavour attributes. Sweetpotato cultivars of any colour, with dry, crispy, mealy texture and moderately sweet taste could be an ideal sweetpotato for many consumers. There is also a potential market for varieties with low sweetness with the above mentioned attributes. Clearly, these findings will contribute to the development of improved product profiles for sweetpotato breeders in West Africa. These findings could also aid industries to developed appropriate products to reach targeted consumers.Eric Kuuna Dery: Conceptualization ; Data curation (lead); Formal analysis (lead); Investigation (lead); Methodology (lead); Visualization (supporting); Writing\u2010original draft (lead); Writing\u2010review & editing (lead). Edward E. Carey: Conceptualization (supporting); Investigation ; Methodology (supporting); Supervision ; Writing\u2010original draft (supporting); Writing\u2010review & editing . Reuben Ssali: Investigation ; Supervision ; Writing\u2010original draft (supporting); Writing\u2010review & editing . Jan W. Low: Project administration (lead); Resources (lead); Supervision (supporting); Writing\u2010review & editing . Suzanne M Johanningsmeier: Methodology (supporting); Validation (supporting); Writing\u2010original draft ; Writing\u2010review & editing . IBOK NSA ODURO: Supervision (supporting); Writing\u2010original draft (supporting); Writing\u2010review & editing (supporting). Abena Boakye: Data curation (supporting); Formal analysis (supporting); Investigation (supporting); Supervision (supporting); Validation (supporting); Writing\u2010original draft (supporting). Rachel M. Omodamiro: Investigation (supporting). Hauwa Ladi Yusuf: Investigation (supporting); Writing\u2010review & editing (supporting).The authors declare no conflict of interest in this work.Children were not used in the study. Respondents were informed about the study, they could stop the interview at any point, written consent from sensory panellists and from consumers participating in this study were obtained and the research respected the rules of voluntary participation and anonymity. Food samples were prepared according to good hygiene and local practices.A blindfold or red lightening system could have been used during the descriptive evaluation to mask the effect of colour on other attributes. Again, unlike the market survey and descriptive sensory analysis that used French fries sizes, the community survey employed chunk fries which were bigger.Table S1. Average scores and standard deviations of descriptive sensory characteristics for French fried sweetpotato.Click here for additional data file."} +{"text": "Medical Genetics, Genomics and Bioinformatics\u201d, \u201cMedical Genetics, Genomics and Bioinformatics\u20132020\u201d and \u201cMedical Genetics, Genomics and Bioinformatics\u20132021\u201d [Medical Genetics, Genomics and Bioinformatics\u20142022\u201d collected papers on medical genomics, human population genetics and computational biology applications in biomedicine, continuing the topic of medical genetics and genomics. Here, we focused on bioinformatics and systems biology approaches to medical genetics problems, molecular oncology and bioinformatics approaches for medical genomics.The analysis of molecular mechanisms of disease progression challenges the development of bioinformatics tools and omics data integration. We have presented an earlier series of journal special issues: \u201chttps://bgrssb.icgbio.ru/2022/, accessed on 12 May 2023). The current collection continues the series of post-conference journal special issues presenting the highlights from the set of meetings on genetics and systems biology highlighting recent trends in cancer genomics [The papers on bioinformatics applications were originally discussed at the \u201cBioinformatics of Genome Regulation and Structure/Systems Biology\u201d (BGRS/SB) multiconference 2022 and its biomedical symposia in Novosibirsk, Russia and the new IJMS journal issue New Sights into Bioinformatics of Gene Regulations and Structure . The recent special issue on computational genomics in Life and Genes journal also has works on medical bioinformatics [Molecular mechanisms of human disease progression ,2 are beMedical Genetics, Genomics and Bioinformatics\u20142022 presents recent studies on medical genomics. It contains eight research manuscripts and one review, each concerning a bioinformatics solution for the analysis of the molecular mechanisms underlying disease progression.This issue Jeong-An Gim presenteAlmost all the research papers in this special issue deal with NGS data to study disease mechanisms and find candidate genes. Ionut-Florin Iancu analyzedFrontiers in genetics special journal issue . Recent work by the same team on lymphatic dissemination in prostate cancer complemented this study [Elena Pudova et al. analyzedis study . Overallis study have shoYaron Trink and co-authors studied heterogeneity in Wilms\u2019 tumor pediatric malignancy related to faulty kidney development . Wilms\u2019 The topic of cell classification is continued by Olga Krasnova and colleagues presentiIJMS special issue, Bioinformatics of Gene Regulations and Structure\u20132022 the same authors\u2019 group presented a new tool for scRNA-seq imputation via integration with single-cell ATAC-seq that increases the power of the analysis [Mark Melamud et al. studied analysis .https://deafnessvariationdatabase.org/, accessed on 12 May 2023) for the selection of potential diagnostically important parts of this gene. Initial diagnostic testing for hearing loss was suggested. Studying genetic variants in populations is an important approach applied earlier for diabetes [Valeriia Danilchenko and colleagues studied diabetes . Olga Sadiabetes considerdiabetes as a biodiabetes ,26.IJMS special issue New Sights into Bioinformatics of Gene Regulations and Structure topic at to continue the paper selection on bioinformatics and genomics in human diseases, as well as the research topic at Frontiers in genetics .Overall, the current special issue on bioinformatics confirmed research interests in medical genomics and bioinformatics studies ,2. We no"} +{"text": "The ongoing coronavirus disease COVID-19) pandemic caused by SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) is putting our public health services under enormous strain 9 pandemi, MERS-CoEffective disinfection, sterilization, and decontamination procedures are essential for reducing the environmental contamination of pathogens. A reduction of more than a few log units is required for effective treatments. Various innovative technologies for pathogen inactivation have recently been developed. However, some pathogens display intrinsic resistance to both chemical and physical inactivation.There are five generally recognized categories in the hierarchy of resistance as follows: (i) extremely resistant (prions), (ii) significantly resistant , (iii) resistant , (iv) susceptible , and (v) highly susceptible (enveloped viruses) ,17,18. TThis Special Issue comprises two original articles, one communication and one review describing various disinfection or sterilization procedures as well as discussing the underlying mechanisms of inactivation.The article by Takashi Yokoyama et al., titled \u201cVirucidal Effect of the Mesoscopic Structure of CAC-717 on Severe Acute Respiratory Syndrome Coronavirus-2\u201d , describMicroorganisms titled \u201cUniversal Virucidal Activity of Calcium Bicarbonate Mesoscopic Crystals That Provides an Effective and Biosafe Disinfectant\u201d [The inactivation effect of SARS-CoV-2 by CAC-717 revealed by Yokoyama et al. has recently been supported and further expanded by the results of a study by Kirisawa et al. published in an article in fectant\u201d . The stuStaphylococcus aureus and induces growth. Intriguingly, the supernatants of S. aureus cultures incubated with the DACC-coated dressing were found to downregulate inflammation associated with the cytokine overexpression of TNF-\u03b1 and TGF-\u03b21 as well as diminishing gelatinase activity in macrophage cultures and fibroblast/macrophage co-cultures. These findings should help stimulate the application of DACC-coated dressing-based tools in the management of acute or chronic wounds.The article by Silvestre Ortega-Pe\u00f1a et al., titled \u201cDialkyl Carbamoyl Chloride\u2013Coated Dressing Prevents Macrophage and Fibroblast Stimulation via Control of Bacterial Growth: An In Vitro Assay\u201d reportedS. aureus and Enterococcus faecalis) and Gram-negative bacteria were inhibited by treatment with CFEAS at concentrations of >95%. Furthermore, the anti-biofilm activity of CFEAS was effective at similar levels of 1.5% polyhexamethylene biguanide/betaine (PHMB). The study also found that CFEAS can be used as a safe antiseptic. Specifically, fibroblasts were shown to be less sensitive to CFEAS-induced cytotoxicity than macrophages. In addition, the authors showed that the treatment of subacute and chronically infected wounds with CFEAS significantly decreased the viable bacterial number of colony-forming units in the wound biopsies of patients with a venous leg ulcer. These findings indicate that CFEAS can effectively inhibit biofilm formation in a clinical setting. In conclusion, the use of CFEAS for antiseptic and antibiofilm treatment is a promising new approach for wound-healing.The article by Alejandro Cabrera-Wrooman et al., entitled \u201cAntiseptic Effects and Biosafety of a Controlled-Flow Electrolyzed Acid Solution Involve Electrochemical Properties, Rather than Free Radical Presence\u201d , investiA review by Pianpian Yan et al., titled \u201cNew Clinical Applications of Electrolyzed Water: A Review\u201d , summariMicroorganisms, which highlights the research of eminent scientists working on the topics of disinfection, sterilization, and decontamination. The Editor thanks all the authors of this Special Issue for sharing their invaluable experience and for their help in compiling the respective articles. The Editor also wishes to thank Meirong Duan and other members of the editorial staff at the Multidisciplinary Digital Publishing Institute (MDPI) for their unwavering commitment throughout the publication process. I hope that this Special Issue inspires readers and contributes to the development of the research field of the disinfection, sterilization, and decontamination of microorganisms. It has been an honor to organize this Special Issue for"} +{"text": "Nature Plants, a new study reveals how plants assemble translational condensates to balance tissue health and disease resistance.Biomolecular condensates assembled through phase transitions regulate diverse aspects of plant growth, development, and stress responses. How biomolecular condensates control plant immunity is poorly understood. In Nature Plants, Zhou et al. contributes to the formation of biomolecular condensates (Molliex et al. So, what can we learn from this study? First, it reinforces the emerging roles of biomolecular condensates in plant organismal defense (Zavaliev et al. In summary, Zhou and colleagues\u2019 discovery of a phase separation control mechanism of gene translation provides a major conceptual advance in understanding the complex plant immune system. Similar to a circuit rheostat, plants appear to fine-tune the intensity of immune signaling through HEM1 phase separation to balance cell survival and death. Future research using synthetic approaches to harness HEM1\u2019s phase behavior could have practical applications in both agriculture and human health."} +{"text": "Insights in multiple sclerosis and neuroimmunology 2021.\u201d These cover a wide aspect of multiple sclerosis (MS)- related themes as well as themes in autoimmune encephalitis.A total of 19 articles are published in the Frontiers Research Topic \u201cMeca-Lallana et al. show in their article \u201cConsensus on early detection of disease progression in patients with multiple sclerosis\u201d that such consensus statements could help clinicians to find early in the disease course patients with secondary progressive MS (SPMS). Such an early identification is important to perform adequate therapeutic management. Standardized clinical assessments are meaningful in MS care. In the article \u201cMaking every step count: minute-by-minute characterization of step counts augments remote activity monitoring in people with multiple sclerosis,\u201d Block et al. used a model to predict disease progression over the longer term (>2 years) based on obtained measurements. These findings will be used to develop further descriptive metrics for activity. In their article \u201cCurrent status and future opportunities in modeling clinical characteristics of multiple sclerosis,\u201d Liu et al. suggest that there is a strong need to develop validated models of MS clinical outcomes by using cellular or/and molecular biomarkers. In the article titled \u201cModels of care in multiple sclerosis: a survey of Canadian health providers,\u201d Marrie, Donkers et al. claim that the ideal MS service is multidisciplinary in nature, ideally integrated, and with prompt access to care.There is a need for consensus criteria about the identification of certain types of MS. Coerver et al. show in the article \u201cThe association between blood MxA mRNA and long-term disease activity in early multiple sclerosis\u201d that MxA mRNA is expressed in inflammatory pathology in MS that is dependent on the endogenous type-1 interferon system and that this might be a prognostic biomarker for long-term inflammatory disease activity in MS. In the article \u201cGenetic risk variants for multiple sclerosis are linked to differences in alternative pre-mRNA splicing\u201d by Putscher et al., the authors show that genetic variants from MS risk loci affect pre-mRNA splicing. Amoriello et al. investigated soluble HLA-G (sHLA-G) levels in MS in the article \u201cInvestigating serum sHLA-G cooperation with MRI activity and disease-modifying treatment outcome in relapsing-remitting multiple sclerosis.\u201d They found that the HLA-G genotype strongly influences sHLA-G levels. Autoantibodies are of importance in various neuroimmunological disorders, and their role and mechanism of action are partly undefined. In the article \u201cPeptidylarginine deiminase 2 autoantibodies are linked to less severe disease in multiple sclerosis and post-treatment Lyme disease,\u201d Kim et al. make the case that anti-peptidylarginine deiminase 2 (PAD2) antibodies may attenuate inflammation. This effect is observable in tissues with high expression of PAD2. The role of hemolysis was analyzed in the article \u201cPeripheral hemolysis in relation to iron rim presence and brain volume in multiple sclerosis\u201d by Krajnc et al. The authors found an influence of hemolysis on the brain volume but not on the presence of iron rim lesions in progressive MS. Investigations about metabolomics in neuroimmunological disorders is of increasing interest. In their study \u201cMetabolomics of cerebrospinal fluid in multiple sclerosis compared with healthy controls: a pilot study,\u201d \u017did\u00f3 et al. investigated cerebrospinal fluid (CSF) from MS patients compared to controls regarding metabolomic profiles. They found differences in amino and fatty acids in the CSF of newly diagnosed patients with MS in comparison with controls. The most significant changes were seen in levels of arginine, histidine, and palmitic acid. They concluded that such a metabolomic profile may predict inflammatory disease activity in MS. In the article \u201cEffects of vascular comorbidity on cognition in multiple sclerosis are partially mediated by changes in brain structure,\u201d Marrie, Patel et al. showed that vascular comorbidity leads to changes in brain macrostructure and microstructure. In addition, this is associated with lower cognitive function in patients with MS.The myxovirus resistance protein A (MxA) has been long used as a marker for exogenous interferon-beta efficacy in MS treatment. Bridging therapies with injectable immunomodulatory drugs in the management of multiple sclerosis: a Delphi survey of an Italian expert panel of neurologists,\u201d Marfia et al. suggest that the value of bridging therapy with injectable immunomodulatory drugs in MS disease conditions is underscored. The article focuses on patients with MS who plan to become pregnant and patients with MS at risk for cancer recurrence. Ozanimod is a selective sphingosine-1-phosphate (S1P)-receptor 1 (S1P1) and S1P5 modulator used for the treatment of active forms of relapsing-remitting MS (RRMS). Ziemssen et al. present their real-world and long-term study \u201cOzEAN study to collect real-world evidence of persistent use, effectiveness, and safety of ozanimod over 5 years in patients with relapsing-remitting multiple sclerosis in Germany.\u201d The results of this study will add to the safety profile and efficacy profile of ozanimod in the treatment of RRMS. In the study \u201cSafety, adherence and persistence in a real-world cohort of German MS patients newly treated with ocrelizumab: first insights from the CONFIDENCE study,\u201d Weber et al. describe the safety profile of ocrelizumab in the CONFIDENCE real-world MS population study. The findings were consistent with the findings in pivotal clinical trials for the anti-CD20 B cell-depleting antibody ocrelizumab used for the treatment of patients with RRMS and patients with primary progressive MS (PPMS). Importantly, high treatment persistence and adherence were seen in this real-world MS population study. Fathi et al. suggested in their article, \u201cDynamic changes in kynurenine pathway metabolites in multiple sclerosis: a systematic review,\u201d that quinolinic acid is a possible player in the pathogenesis of MS. This conclusion is mainly based on the finding that quinolinic acid levels in CSF were higher in patients with MS than in healthy controls. The value of disease models induced in mice and rats on certain novel MS therapeutic approaches is outlined by Jayaraman and Jayaraman in their article \u201cImpact of histone modifier-induced protection against autoimmune encephalomyelitis on multiple sclerosis treatment\u201d about histone deacetylase (HDAC) inhibitors. HDAC inhibitors such as valproic acid and hydroxamates as well as others are possible candidates for future treatment of MS.In \u201cZhang et al. show in their article \u201cLong-term prognosis of patients with anti-N-methyl-D-aspartate receptor encephalitis who underwent teratoma removal: an observational study\u201d that early detection and removal of teratoma resulted in a favorable long-term prognosis in patients with anti-NMDAR encephalitis. Case studies can be of importance for defining potential new disease entities and for the description of rare disease variants. In the case study \u201cAcute cerebellitis associated with anti-homer 3 antibodies: a rare case report and literature review\u201d by Miao et al., the authors underscore the need for immune-mediated causes to be considered in acute cerebellitis. Importantly, immunotherapy can contribute to the improvement of cerebellar syndrome. Neuropsychological assessment is important in phenotyping and care of patients with neuroimmunological disorders and especially autoimmune encephalitis. In the article by Chan et al., \u201cCognitive and mood profiles among patients with stiff person syndrome spectrum disorders,\u201d it is clarified that neuropsychological testing in stiff person syndrome should include testing of verbal learning and recall, phonemic verbal fluency, attention, and processing speed.It has been shown that in paraneoplastic forms of autoimmune encephalitis, the removal of the associated cancer entity is of primary importance in long-term disease outcomes. For teratoma, Insights in multiple sclerosis and neuroimmunology 2021\u201d gives novel insight into current research themes on MS and autoimmune encephalitis.In conclusion, the Research Topic \u201cRW outlined and wrote the Editorial."} +{"text": "Selenium in soil-plant-animal systems and its essential role for human health published original research reports and critical reviews representing different but interrelated research disciplines involving physiochemical and biological behaviors of Se within the larger foundation topics of agricultural soil, bioavailability, plant uptake, physiological responses, genetics, molecular biology, microbial communities, Se-biofortification strategies, and animal health.With approximate 1 billion people facing some degrees of selenium (Se) deficiency worldwide, it is imperative that the Se community work together and share the latest knowledge on various inter-related aspects of Se in supporting and protecting animal, human, and ecosystem health. In collaboration with Frontiers of Plant Science, Frontiers in Nutrition, and Frontiers in Veterinary Science, this Research Topic entitled Selenium is unevenly distributed in the soil, which has consequently resulted in soil Se deficiency issues and further low Se dietary intake in many parts of the world. To increase Se intake by consumption of Se-enriched plant- and animal-derived food products, we need to better understand and identify those effective strategies for Se delivery though agricultural production systems in different geographical regions. Enhancing bioavailable Se in soil will not only increase Se accumulation in crops but also result in the accumulation of specific bioactive Se compounds in food products. Importantly, the true value of successfully increasing Se concentrations in plant- and animal-based products could be highly determined by the fractionation and the speciation of Se, such as seleno amino acids or selenoproteins in Se-biofortified food products. The different Se compounds accumulated in plant tissues would further determine their bio-accessibility and the absorption of Se through human digestion systems. Similarly, animal health and reproduction are also very much dependent on the bioavailability and the absorption of Se from feeding materials. The adequate intake of Se from the feeding materials or using supplementary Se significantly affect animal\u2019s vital physiological functions that are related to reproduction or pregnancy health, and their auto-immune functions.In this Research Topic, 15 high-quality research papers that addressed various topics or faces of Se research, ranging from the biogeochemical cycling of Se to cellular and molecular processes that elucidate mechanistic functions of Se in human and animal health. Inorganic and organic Se transformations and physio-chemical properties of soils could all ultimately determine soil Se bioavailability for plant uptake and accumulation. Both the concentration and the speciation of Se in soil could affect the Se content and the Se status of crops, particularly in edible plant materials. Recent studies demonstrated that biofortification as an agronomic-based strategy can be utilized to mitigate a low transfer of Se and other nutrients from soil into the food chain and produce Se-enriched food products, which helps increasing dietary Se intake throughout Se-deficient susceptible regions of the world.Se biofortification of soybean genotypes in a tropical soil via Se-enriched phosphate fertilizers\u201d, Silva et\u00a0al. showed that the application of Se-amended phosphate fertilizers could be an effective method to deliver Se to the crop. Adding Se to the commonly used fertilizers could also be challenging due to the soil Se concentration baselines, soil types, redox protentional, pH, and soil microbial or invertebrate communities. Song et\u00a0al. indicated in \u201cSelenium effect threshold for soil nematodes under rice biofortification\u201d that, with the application of selenite for rice biofortification, higher concentrations of soil Se can negatively affect soil nematodes, suggesting that the presence of soil nematodes could be used as an effective bioindicator for the soil environmental changes related to Se content. In addition to the uptake of inorganic Se, plants can also absorb Se via organic Se application, as shown in \u201cUptake and translocation mechanisms of different forms of organic Se in rice\u201d by Wang, Q. et\u00a0al. This rice study provides important insights into the mechanisms underlying the uptake and translocation of organic Se, especially selenomethionine (SeMet), in plants. As an alternative to soil applied Se, foliar application has been used to apply Se to plants. Schiavon et\u00a0al. indicated that \u201cFoliar Se fertilization alters the content of dietary phytochemicals in two rocket species,\u201d while Wang, M. et\u00a0al. further outlined the differences between soil and foliar Se applications in a paper entitled \u201cSoil and foliar Se application: Impact on accumulation, speciation, and bio accessibility of Se in wheat\u201d. In addition to foliar Se application, Malka et\u00a0al. evaluated potential interactions between Se and Zn in foliar application, and indicated that \u201cSeparate foliar sodium selenate and zinc oxide application enhances Se but not Zn accumulation in pea seeds\u201d. Foliar application of Se may additionally influence plant metabolism, as well as increasing Se content in plant tissues, as shown by de Sousa et\u00a0al. in \u201cSelenium enhances chilling stress tolerance in coffee species by modulating nutrient, carbohydrates, and amino acids context\u201d, and demonstrated that foliar Se application improved coffee plants\u2019 ability to tolerate chilling stress.Agronomic Se biofortification has been commonly practiced by adding Se-amended fertilizers to the soil. In Brazil, soybean is a potential major crop for biofortification. In \u201cBanuelos et\u00a0al. in \u201cSalsola soda (agretti) as a Se biofortification crop grown under high saline and boron conditions.\u201d Under field conditions Se-biofortified Salsola soda was produced with poor quality waters containing high levels of Se. Careful attention must, however, be paid in regions where Se-biofortified crops are grown in naturally Se-rich soils or with poor quality waters because of the potential presence of toxic metals in the environment. In \u201cPrediction models for monitoring Se and its associated heavy-metal accumulation in four kinds of acro-foods in seleniferous area\u201d, Jiao et\u00a0al. demonstrated that models can be used to effectively predict toxic metal accumulation in Se-enriched foods in those concerned regions.To produce Se-enriched agricultural products, the biofortification strategy can also be practiced in regions where there are naturally high levels of Se in the soil and/or in irrigation waters, as demonstrated by Hu et\u00a0al. reviewed the importance of \u201cSeleno-amino acids in vegetables\u201d, a review of their forms and metabolism and thereby affect protein structures, functional properties and antioxidant capacity in newly-germinated Se-enriched soybeans. Relatedly, Huang et\u00a0al. also reported in \u201cSelenium biofortification of soybean sprouts: effects of Se enrichment on proteins, protein structure, and functional properties\u201d that Se-biofortified seeds also contain proteins whose quality has also been influenced by Se content. In addition, Li et\u00a0al. evaluated \u201cThe use of selenium for controlling plant fungal diseases and insect pests\u201d, indicating that Se improves the plant resistance to fungal diseases.Se-enriched food products can increase Se intake and promote human health with absorption of plant tissue containing different Se compounds including seleno-amino acids. Earlier studies have clearly demonstrated the important role of Se in plant and animal physiological processes and functions. Hall et\u00a0al. discussed Se biofortification through forages raised for livestock feed in \u201cImpact of selenium biofortification on production characteristics of forages grown following standard management practices in Oregon,\u201d demonstrating that foliar selenate treatment increased forage Se concentrations in a dose-dependent manner, and that coupling Se amendment with standard fertilization practices promoted forage growth and forage Se concentrations. In cases of low soil Se, providing sulfur fertilization could reduce forage Se and potentially alter Se supply to livestock consuming those forages.Excessive low or high Se in soil and consequently Se concentrations in animal feeds can pose health and reproduction risks for animals. Animal-based food products for human consumption are an excellent source of dietary Se intake for the human population. Thus, safely providing Se biofortified feed materials to animals would result in increased Se concentrations in animal-based food products for humans. Dahlen et\u00a0al. reviewed the role of Se in male and female reproductive process and the impacts of maternal dietary Se on offspring outcomes in ruminants in their paper \u201cSelenium supplementation and pregnancy outcomes.\u201d The scientific evidence indicates that Se plays a major role in both male and female reproductive processes and, therefore, as a micronutrient, Se is instrumental to ensure successful animal reproductive efficiency. Increasing the maternal supply of Se alters offspring outcomes in ways that are typical of developmental programming; thereby implying that Se supply to the mother can have significant effects into the next generation of livestock. In animals, mitochondrial function is essential to bioenergetics and consequently life functions. Clearly, the role of Se in antioxidants plays a role in normal cellular metabolism and consequently whole animal health, production, and wellbeing. In addition, Se appears to have a role in mitochondrial function besides through antioxidants. Wesolowski et\u00a0al. reviewed the non-antioxidant the roles of Se in mitochondrial function in \u201cBeyond antioxidants: Selenium and skeletal muscle mitochondria.\u201d The review paper demonstrates our emerging understanding of the role of Se in skeletal muscle mitochondrial function beyond the traditional constructs of antioxidants, and further highlights the importance of a greater understanding of Se in mitochondrial function and energetics.A major determinant of livestock production, health, and well-being is effective and efficient reproductive process that lead to healthy offspring. Selenium is one of the most influential natural-occurring micronutrient elements for living systems. Recognizing selenium\u2019s impact on a multitude of processes in nature requires multi-disciplinary research on Se absorption, chemical transformation, and biochemical and physiological metabolisms in soil-plant-animal systems that can help us develop and implement effective strategies to mitigate public health impacts or concerns of Se deficiencies in the world. In this Research Topic, with different contributions from original research to critical reviews, some of the most influential researchers have provided their latest research findings and demonstrated significant advances in the field concerned with Se in food chains and its effects on human and animal health.All authors contributed to the article through writing, reviewing and editing and have approved the submitted version."} +{"text": "Dear Editor, I agree that accurate representation of research is of great importance, and Iapologize for these mistakes.I am grateful to Dr Anne Witt and Dr Sheila Kredit for drawing attention to errors I havemade in Appendix 1 of \u2018Re-thinking Benign Inflammation of the Lactating Breast:Classification, Prevention, and Management\u2019.Dr Witt and Dr Kredit are correct to state that I have falsely represented: study andThe rates of follow-up in the Witt et al. systematic review.Analysis of Witt et al. in Anderson et al.\u2019sThe Witt et al. study is not a randomized controlled study (RCT), but a nested case-controlstudy. It is referred to as \u2018quasi-experimental\u2019 in the Anderson et al. systematic review, notas an RCT. In Appendix 1 of my article, I wrongly represent the numbers who responded tofollow-up emails in the Witt et al. study, wrongly attributing these inaccurate numbers toAnderson et al. The Witt et al. study demonstrated excellent follow-up in the cohort whoreceived Therapeutic Breast Massage in Lactation (TBML) and also in the control group.Although debate is welcomed, and accurate representation of research essential, Inevertheless contend that TBML should not be recommended to breastfeeding women asevidence-based management of breast inflammation on the basis of Witt et al.\u2019s study, for fourreasons:1.\u2003TBML was delivered as one element in a complex breastfeeding intervention. Itsefficacy was evaluated in small numbers for mastitis and plugged ducts, in the absenceof a control group.TBML was delivered in the context of full breastfeeding support provided by an InternationalBoard Certified Lactation Consultant/registered nurse and/or breastfeeding medicine physician,which included latch correction, feeding patterns, antibiotic prescription, milk removal oranalgesia as clinically indicated. The component of the study which investigates efficacy ofTBML for mastitis and plugged ducts is a small, pre- and post-TBML assessment , which lacks a comparison group. That is,pre- and post-intervention comparisons do not take into account the neurobiological effects ofpatient expectation (placebo effect), as Witt et al. acknowledge in their article.2.\u2003TBML did not show improvements in pain at 2-day and 12-week follow-up when theengorgement group was compared to the control group.Of the 15 participants with engorgement [in the TBML intervention group], measurementswere taken from each breast, giving a total of 30 separate pain scores .\u2004.\u2004. These scoreswere treated independently (n = 30) in the pre-post analysis and combined (n = 15) for thecomparison between the intervention and control groups, making interpretation quitedifficult.Anderson et al. state in their analysis of Witt et al.,In the component of Witt et al. which investigates efficacy of TBML for engorgement, theintervention group (n\u2009=\u200915) was compared to a control group (n\u2009=\u200973); 47% of the interventiongroup had severe engorgement compared to 7% of the control group. Comparison of theengorgement intervention and control groups showed no meaningful difference in pain at day 2nor in pain, exclusive breastfeeding or breastfeeding complications at week 12 in emailfollow-up.3.\u2003Pre- and post-TBML improvements can be explained by the ductal dilations (milkejection) and milk removal components of TBML alone.TBML in the Witt et al. study achieves milk removal by alternating hand expression of milkwith the massage technique, and by allowing direct breastfeeding of the infant during TBML(see Supplement Appendix A). The reduction in breast pain and also in size of plugged ductsobserved immediately after TBML can be explained by the milk removal components of TBML alone,which are associated with milk ejections and ductal dilations.4.\u2003There is no pathophysiological model which explains the proposed efficacy of thegentle areola-to-axilla massage component of TBML.Is increased lymphatic drainage the proposed pathophysiological mechanism of light massagefrom the areola to the axillae? If so, this proposed mechanism isn\u2019t supported by the latestresearch concerning the function of lymphatic vasculature. Interstitial fluid diffuses intothe initial lymphatic capillaries in response to rising pressure gradients between breaststroma and lymphatic capillaries, which mechanically opens these capillaries. Lymphaticcollection vessels contain valves, have smooth muscle in their walls, and are intrinsicallycontractile, actively pumping lymph towards the nodes. Although there is no convincingphysiological rationale to support the belief that application of external pressurefacilitates lymphatic removal of breast stroma interstitial fluid, there is reason to beconcerned that an external pressure application which moves towards the axilla risks increasedintra-alveolar milk pressures.Various breast massage techniques are offered to breastfeeding women around the world, as DrWitt and Dr Kredit note. Anderson et al. analyse the efficacy of a range of massage techniquesin three RCTs and three quasi-experimental studies, including Witt et al.\u2019s study of TBML.Although Anderson et al. conclude \u2018Overall, different types of breast massage were reported aseffective in reducing immediate pain for the participants\u2019, I contend that neither Witt etal.\u2019s data or Anderson et al.\u2019s data support Therapeutic Breast Massage as an evidence-basedintervention for presentations of lactation-related breast inflammation, despite its inclusionin Academy of Breastfeeding Medicine Clinical Protocol #36: The mastitis spectrum.Using the GRADE Working Group grades of evidence in their Summary of Findings, Anderson etal. report low certainty of outcomes for reduction in pain, increase in breast milk supply,and reduction or resolution of symptoms of breast inflammation, noting that \u2018the true effectmay be substantially different from the estimate of the effect\u2019. Anderson et al. observe thatthe ability to replicate or generalize results of the six studies are limited by:Significant heterogeneity of study methods, interventions and outcome measuresLack of detailed explanation of breast massage techniquesUse of invalidated toolsSmall sample sizes and Oketani massage techniques, or requirement for seven consecutive days of massage combined with preparation of freshtopical cactus and aloe leaf lotion and pre- and post-massage application of aloe and cactusflesh, may not be practical in many settings.Anderson et al. also note that requirement for extensive training for traditional Gua Sha breastfeeding women are commonly referred to multiple providers for unproveninterventions when problems emerge. Many popular treatments such as TBML lack both aconvincing evidence-base and a robust underlying pathophysiological model. Such treatments mayincrease the financial burden for families and health systems, and raise the spectre ofdiscriminatory breastfeeding support globally, with ease of access limited to affluentfamilies in advanced economies.Because clinical breastfeeding support remains a research frontier,Thank you for the opportunity to correct my mistaken representation of the Witt et al. andAnderson et al. studies in Appendix 1 of my article, for which I apologize. I welcomerespectful discussion and debate concerning interpretation of existing studies and also theopportunity to amend errors, knowing that as clinicians and researchers we share the samecommitment to improved outcomes for breastfeeding women and their babies.Pamela DouglasKind regards,"} +{"text": "Thermal manipulation has garnered considerable attention for its potential applications in diverse areas, including microelectronics, thermal logic devices, and thermoelectrics. Manipulating heat transfer in nanostructured materials is particularly desirable, especially for structures with high thermal conductivity used in thermal dissipation. However, the demand for high-power micro/nano chips exceeds the capabilities of even cutting-edge thermal management technologies, which currently represent a bottleneck in further development. Conversely, structures with low thermal conductivity are of interest in various areas such as thermal barriers and thermoelectrics. Understanding heat transfer is thus a fundamental problem that requires comprehensive study. However, studying heat transfer in nanostructures presents significant challenges due to the dominance of interfaces, boundaries, and imperfections in its behavior, and the underlying physical mechanisms remain unclear .This Special Issue, entitled \u201cHeat Transfer in Nanostructured Materials\u201d, provides a platform for presenting original and review articles that showcase recent advances in heat transfer within low-dimensional materials and nanofluids. The issue will systematically introduce and discuss the design, construction, characterization, and potential applications of these structures.\u22121 K\u22121. Li et al. [2 nanowires that showed an increase in jump during metal-insulator transition temperature with increasing sample thickness. Phonons were identified as major carriers that dominate thermal transport in the nanowires. Jiang et al. [Low-dimensional structures have demonstrated significant potential for nanoscale dissipation as thermal interface materials (TIMs). Lv et al. developei et al. revealedg et al. investigg et al. examinedg et al. presente3O4 nanoparticles as nanofluids. The connector enhanced the heat transfer coefficient within the second microchannel by increasing the randomness of molecules and particles, refreshing the fluid\u2019s memory before entering the second channel. The effects of Reynolds number and nanoparticles on the connector were also studied, revealing that introducing Fe3O4 nanoparticles increased overall thermal conductivity and the heat transfer coefficient. The connector effectively promoted the random motion of molecules and nanoparticles, with the enhancement being significant at low Reynolds numbers but becoming negligible with increasing Reynolds number. This paper also presented a brief review of current advancements in studying the effects of nanoparticles on fluid thermal conductivity, viscosity, and heat transfer coefficient. Arshad et al. [Heat transfer via nanofluids has emerged as an effective approach for thermal management in complex systems. Optimization of nano additives and microchannel designs can significantly improve heat transfer efficiency. Apmann et al. investigd et al. reportedThis Special Issue is expected to be of interest to readerships in both fields of science and engineering. Understanding heat transfer mechanisms in nanostructured materials is fundamental for further developments of nanoscale devices, yet it remains an extreme challenge. We anticipate more emerging works focusing on depicting the underlying physical pictures of heat transfer and developing strategies for thermal manipulation in nanostructures."} +{"text": "Cancer is still one of the leading causes of death worldwide, despite that tremendous resources are being invested in drug discovery . Recent The majority of cancer medications consist of small molecule inhibitors. These inhibitors are engineered to enhance their efficacy by binding to specific cellular protein targets, which in turn initiate a series of downstream changes in cancer signaling and metabolic pathways . High-thin vivo patient-derived xenograft (PDX) samples proliferation-associated phenotypes, or 2) intermediate phenotypes, such as transcriptomic alterations. Numerous well-established data portals, including The Cancer Genome Atlas (TCGA), DepMap Portal , and The samples . These i samples . These t samples . The selFunctional screening for cancer drug discovery: from experimental approaches to data integration\u201d is to highlight the recent advances in high-throughput functional genetic approaches, especially how results from such new technologies can be applied for future studies in cancer drug mechanisms of action. To achieve this goal, we carefully reviewed every submitted manuscript and screened for highly qualified reviewers. Eventually, we accepted and published nine articles including eight \u201cOriginal Research\u201d articles, and one systematical \u201cReview\u201d article on the mechanism and clinical trials of hepatocellular carcinoma immunotherapy in the onset and progression of gastric cancer (GC). Ren et al. developed a necroptosis-related prognostic signature to reveal immune infiltration, which could predict drug sensitivity and inform personalized drug therapy for hepatocellular carcinoma (HCC) patients. Wang et al. reported a novel hub gene signature closely associated with ferroptosis, serving as a potentially effective biomarker for predicting the prognosis of HCC patients.The research articles presented in this Research Topic encompass efforts in cancer drug discovery from both experimental and computational perspectives. Liu et al. conducted a comprehensive analysis to elucidate the roles of cuproptosis-associated genes in tumor biology and cancer drug sensitivity across various cancers. Chen et al. discovered a novel diagnostic four-gene signature for hepatocellular carcinoma based on an artificial neural network, with applications in drug screening. Li et al. explored the functional effects of FDX1 in tumors, and further validated the inhibitory effect of FDX1 in copper-induced cell death, confirming FDX1\u2019s role as a cuproptosis biomarker. Ruan et al. leveraged pan-cancer analysis to identify DDX56 as a prognostic biomarker associated with immune infiltration and drug sensitivity. Zhang et al. uncovered an LncRNA signature related to cuproptosis, serving as a novel biomarker of prognosis in immunotherapy and drug screening for clear cell renal cell carcinoma.We believe that the articles featured in this Research Topic can offer valuable insights into the application of functional screening methods for cancer drug discovery. The findings presented in these studies are anticipated to enhance our understanding of the molecular mechanisms governing cancer progression and, ultimately, we hope they will positively impact drug and target discovery in the near future."} +{"text": "This method has broad-range applicability to understanding microbial communities and their associations with biosignatures and soil carbon and mineralogic characteristics relevant to climate science and astrobiology.Permafrost is important from an exobiology and climate change perspective. It serves as an analog for extraplanetary exploration, and it threatens to emit globally significant amounts of greenhouse gases as it thaws due to climate change. Viable microbes survive in Earth's permafrost, slowly metabolizing and transforming organic matter through geologic time. Ancient permafrost microbial communities represent a crucial resource for gaining novel insights into survival strategies adopted by extremotolerant organisms in extraplanetary analogs. We present a proof-of-concept study on \u223c22 Kya permafrost to determine the potential for coupling Raman and fluorescence biosignature detection technology from the NASA Mars Perseverance rover with microbial community characterization in frozen soils, which could be expanded to other Earth and off-Earth locations. Besides the well-known utility for biosignature detection and identification, our results indicate that spectral mapping of permafrost could be used to rapidly characterize organic carbon characteristics. Coupled with microbial community analyses, this method has the potential to enhance our understanding of carbon degradation and emissions in thawing permafrost. Further, spectroscopy can be accomplished It is located along the valley floor of Goldstream Creek in a region of discontinuous permafrost. Permafrost in this area is syngenetic ice-rich silt (loess) formed through sediment deposition, causing the permafrost layer to expand upward , a custom-built deep ultraviolet (DUV) Raman and fluorescence spectrometer designed by the NASA Jet Propulsion Laboratory. This same technology is employed on the Mars Perseverance rover. MOBIUS uses a 248.56\u2009nm NeCu pulsed laser . The laser spot is annular in shape with an outer diameter of \u223c44\u2009mm and an effective illuminated area of 3540\u2009\u03bcm2 at a spectral resolution of 3.8\u2009cm\u22121/pixel. For fluorescence measurements, a grating of 300 lines/mm was used with a spectral range of 250\u2013410\u2009nm and a resolution of 0.16\u2009nm/pixel. Spectral positions were calibrated before sample measurements by validating the position of the zero-order reflection, the secondary laser line at 252.93\u2009nm . Local Raman maps covered 0.5\u2009\u00d7\u20090.5\u2009mm at 100 um spacing, resulting in 25 individual point spectra at 1200 laser pulses per point (30-second acquisitions).et al.,Raman scan locations were selected based on the fluorescence intensity distribution obtained during the initial survey to prioritize areas of intense fluorescence that may indicate the presence of concentrated organic/biological material. A Canon camera provided context imaging of the mapped sample with a \u223c20\u2009mm/pixel resolution. Data processing was performed using custom Python programs and visualization tools based on a Loupe software package to process and visualize hyperspectral Raman and fluorescence data sets Uckert, . Spectra2.3.We performed triplicate DNA extractions from each core section directly from 0.5\u2009g of soil using the FastDNA Spin Kit for Soil . An additional cleanup to remove humic material and other inhibitors was performed using the Qiagen DNeasy PowerClean Pro Cleanup Kit . The 16S rRNA gene was amplified with the 515F/926R barcoded primer sets and conditions recommended by the Earth Microbiome Project. Libraries were sequenced on the Illumina MiSeq platform, generating 2\u2009\u00d7\u2009250\u2009bp paired-end reads.et al.,et al.,et al.,et al.,et al.,et al.,et al.,16S rRNA amplicon sequences were processed using the exact sequence variants (ESVs) pipeline in QIIME2, version 2022.2 16S ribosomal RNA sequences database (updated 2022/10/30) Madden, and extr3.3.1.\u22121). The appearance of this Raman peak in a fluorescence spectrum is consistent with the prevalence of water ice in permafrost . The relative intensity of the water band to organic fluorescence varied significantly between and throughout each sample and provides a measure of relative changes in the organic/ice concentration ratio.The three permafrost samples exhibited consistent fluorescence signatures indicative of a similar continuum of organic matter present throughout each sample. This signature comprised broad fluorescence peaks at \u223c320 and \u223c410\u2009nm, typical of biological macromolecules such as nucleic acids and proteins, which are consistent with expectations based on the prevalence of biomass in permafrost , 2B Fri. The twormafrost , 2B. How2O stretching mode. However, when measured with higher spectral resolution and longer exposures, this was better resolved as an asymmetric peak with a narrow component at 3155\u2009cm\u22121 and a broader, weaker component at 3350\u2009cm\u22121 . Proteobacteria and Firmicutes were the most abundant phyla , followed by Acidobacteria (1.1%) and Chloroflexi (0.62%) . We did Clostridium sensu stricto 13, Bacillus, Pseudomonas, Massilia, and Ralstonia), which together account for 78% of all sequences, to known 16S rRNA gene sequences. Close relatives (>97% sequence identity) have been isolated from an assortment of environments and conditions and employ a diversity of life and metabolic strategies, indicating that multiple divergent mechanisms can be employed to survive in ancient cryoenvironments.To better understand the microbial inhabitants of late Pleistocene permafrost, we compared ESV sequences of the five most abundant genera . In contrast to Clostridium, close relatives of Bacillus ESVs are primarily mesophilic (plus a few psychrophiles) and are derived from various environments and conditions. Previous data suggest that Clostridium may persist as vegetative cells in permafrost, while Bacillus form endospores , which can then be subsampled for DNA sequencing. Since there is fine-scale variation in permafrost soils, sequencing the same imaged regions will likely reveal relationships between spectral signatures and community structure.Permafrost cross-sections showed a widespread distribution of partially degraded organic carbon, pockets of water ice, and known microbial constituents. While we found no definitive spectral signatures corresponding to microbial community structure, this may be due to subsectioning methods. Given the high prevalence of organic material in permafrost, signatures from individual microbes may be lost in the background continuum of signals from all other organic matter present when analyzed with spectroscopy alone. To ameliorate this in our first-order analysis, core sections were divided into two pieces: one for spectroscopy and the other for microbial analysis. In the future, fluorescence and Raman spectroscopy can be applied to the core samples et al.,Previous analyses from the tunnel demonstrated that replicate samples from directly adjacent cores had small variations in microbial community structure but highly significant differences when comparing permafrost of different radiocarbon ages located tens of meters apart , and the action of microbial communities as they slowly metabolize and transform organic matter through geologic time and slow thaw. Together with carbon dating of sampled strata, microbial analyses, and greenhouse gas measurements, this has the potential to identify and predict the relationships among many complex factors and reveal how these ultimately drive the contribution of permafrost thaw to the climate change equation.These methods provide a unique means of studying permafrost in a changing climate. The 1700 billion tons of ancient carbon stored in permafrost exists in varying forms, from labile to recalcitrant (Jorgenson 4.3.et al.,et al.,et al.,et al.,Characterizing Earth's ancient permafrost microbial diversity and survival strategies provides a baseline to guide the search for microbial life on other frozen desert worlds. Permafrost is found in the subsurface of Mars and is expected in the mixed crustal and ice silicates of icy planets and moons across the Solar System, including Ceres, Callisto, and possibly Ganymede and Titan Johnson, . Microbiet al.,The ability to rapidly and successfully characterize microbiologic and chemical conditions of ice and carbon-rich environments has important implications for extraplanetary exploration and studying permafrost on Earth. Recent breakthroughs in rapid DNA sequencing (Castro-Wallace et al.,et al.,et al.,et al.,et al.,et al.,et al.,in situ sampling methodologies with established microbial community interrogation techniques will save time in site exploration, retrieval methodology, and post-retrieval analysis while building a library to be used as a proxy for exoplanet biosignatures.Permafrost on Earth is dynamic in structure and formation, providing additional proxies for hypersaline environments such as brine lenses, legacy frozen substrate, and ice-rich sediment or ice wedges (Niederberger 5.et al.,in situ could greatly reduce transport and laboratory cost, shortening the timeline from sampling to results. Creating an index of microbial community characteristics and survival mechanisms for extraplanetary exploration will inform the search for extraterrestrial life. As the search for life continues, any information to constrain search locations saves money and shortens the time to discovery. Proxies for exobiology detection currently exist on Earth, and every effort should be made to systematically characterize their diversity before they are lost to climate change.Up to 40% of northern latitude permafrost may thaw by the end of the century (Chadburn"} +{"text": "Drug reaction with eosinophilia and systemic symptoms (DRESS).Five articles are published in this Frontiers' Research Topic on Chen et al., offering an elegant review of DRESS. This comprehensive review delves into the pathogenesis of DRESS, potential biomarkers, and the relevant therapeutic rationales. With our current understanding of the genetic susceptibility, models of antigen presentation, and T-cell activation by drugs, DRESS along with other severe cutaneous adverse drug reactions can no longer be dismissed as unpredictable, at least for specific drugs. Prevention of DRESS associated with some medications is possible; the most successful example is that of HLA B1502 screening prior to carbamazepine use in the Han Chinese population . PLHIV experience a higher frequency of drug eruptions when compared with the non-HIV population . Immune Kuchinskaya et al.. This report underscores the challenges in recognizing and differentiating DRESS from other systemic inflammatory syndromes with similar presentations and laboratory findings. Lastly, Manieri et al. provided us a review of DRESS in childhood and highlighted that rapid-onset DRESS affects children more often than adults and is usually triggered by antibiotics or iodinated contrast media rather than by anticonvulsants.Furthermore, DRESS do occur in children, as highlighted in the case report by In conclusion, this Frontiers Research Topic provides valuable insights into the complex realm of DRESS, offering a deeper understanding of its mechanisms, treatment options, and challenges in various populations.Enjoy the wealth of knowledge shared in this Research Topic on DRESS!BJ: Writing \u2014 original draft, Writing \u2014 review & editing. YL: Writing \u2014 original draft, Writing \u2014 review & editing. SN: Writing \u2014 original draft, Writing \u2014 review & editing."} +{"text": "Streptococcus pyogenes (Sp) Cas9 revealed through these techniques.Cas9 is an RNA-guided endonuclease from the type II CRISPR-Cas system that employs RNA\u2013DNA base pairing to target and cleave foreign DNA in bacteria. Due to its robust and programmable activity, Cas9 has been repurposed as a revolutionary technology for wide-ranging biological and medical applications. A comprehensive understanding of Cas9 mechanisms at the molecular level would aid in its better usage as a genome tool. Over the past few years, single-molecule techniques, such as fluorescence resonance energy transfer, DNA curtains, magnetic tweezers, and optical tweezers, have been extensively applied to characterize the detailed molecular mechanisms of Cas9 proteins. These techniques allow researchers to monitor molecular dynamics and conformational changes, probe essential DNA\u2013protein interactions, detect intermediate states, and distinguish heterogeneity along the reaction pathway, thus providing enriched functional and mechanistic perspectives. This review outlines the single-molecule techniques that have been utilized for the investigation of Cas9 proteins and discusses insights into the mechanisms of the widely used By measuring the resonance energy transfer efficiency studies the surface structure and properties of samples by detecting the extremely weak interatomic interaction between the sample surface and a miniature force-sensitive sensor , whereas PAM-distal mismatches still allow for the stable binding of the complex to DNA targets. Specifically, 9\u201310 PAM-proximal matches are sufficient for ultrastable SpCas9\u2013gRNA binding. Moreover, as the dwell-time analysis shows two characteristic binding times, a two-step mechanism of Cas9\u2013RNA binding involving PAM surveillance and RNA-DNA heteroduplex formation (see the next section) was proposed . Fully unwound protospacer DNA coupled with full R-loop formation possibly drives the docking of the HNH domain, thus licensing cleavage-competent SpCas9 (see the following section \u201cDNA dissociation after cleavage\u201d). Modifications of gRNA or the engineering of SpCas9 could rebalance the unwinding-rewinding equilibrium and make it stricter to reach the cleavage-competent state, thus minimizing off-target effects. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al.SpCas9 is a multidomain DNA endonuclease. Structures of SpCas9 showed two distinct lobes, the alpha-helical recognition (REC) lobe and the nuclease lobe (NUS), as well as the more variable C-terminal domain (CTD) , the 3\u2019 flap generated by the cleaved NTS is possibly exposed and can be digested by exonucleases ( et al.One distinguished characteristic of SpCas9 is its stable binding to the on-target site after cleavage. Both ucleases (Wang et et al.. Therefo et al.in vitro, could facilitate the dissociation of DNA-cleaved SpCas9 from DNA. Zhang et al. used optical tweezers to examine the consequence of encountering a BLM helicase with a DNA-bound dSpCas9 from both sides ( et al. et al. et al. et al.The long lifetime of the SpCas9\u2013gRNA\u2013DNA complex limits the efficient usage of each SpCas9 protein and impairs the repair of DSBs (Clarketh sides . They prAs evident from this review, single-molecule studies provide not only a fundamental understanding of Cas9 mechanisms but also a framework for rational design aiming at improving Cas9 efficiency and minimizing off-target effects. Based on these studies, a detailed dynamic picture of DNA interrogation and cleavage of SpCas9 has been generated . Upon coQian Zhang, Ziting Chen and Bo Sun declare that they have no conflict of interest."} +{"text": "IARC). Hepatocellular carcinoma is the most common type of liver cancer representing about 90% of all the cases of hepatic malignancy mainly: METTL6, GSTZ1, ADH4, ADH1A, and LCMT1 can be utilized as a predictor of HCC patient\u2019s prognosis. Yang et al., have demonstrated the role of HIF2-alpha, a key metabolic regulator, along with VEGF in cellular proliferation and migration of HCC cells in response to insufficient radiofrequency ablation. Angiogenesis plays a very important role in the pathogenesis of several types of cancer including HCC. Tang et al., have shown that expression of angiogenesis-related genes (ARGs) exhibits predictive value in the prognosis of HCC patients. They identified about differential expression of 97 ARGs and further constructed 9 genes-based models to predict the prognosis of HCC. Tang et al., have demonstrated the importance of senescence-related genes in the prognosis of HCC patients. Their study demonstrates that cellular senescence-related genes can be utilized as a prognostic marker as well as a biomarker of therapeutic response. These studies altogether establish the importance of metabolic alterations related to gene expression and angiogenesis in the prognosis of HCC patients.Metabolic alterations are considered a hallmark of cancer . Tian etWang et al., have identified that genes related to N6-methyladenosine (m6A): B2M and SMOX can serve as prognostic signature and their expression may guide to design of novel therapeutic strategies for HCC patients. In a similar study, Huang et al., have demonstrated a 12 genes-based risk signature model in the prognosis of HCC patients. They have shown that SDC3, NCF2, BTN3A3, and WARS genes can serve as novel prognostic factors for HCC.Epigenetic modifications regulate several aspects of cellular physiology and different disease progression by regulating the gene expression machinery. The role of epigenetic alterations in the pathogenesis of different types of cancers including HCC is well-established but not completely understood. By utilizing the publicly available databases, Wang et al., have established a correlation between mitophagy-related genes and immune infiltration in a subset of HCC patients. They have shown that a subset of patients exhibiting higher mitophagy-related gene expression show poor prognosis and suppressed immune function. In another study, Qu et al., have shown that M2-like macrophage markers like PAM and LGALS3 expression positively correlate with the sensitivity of simvastatin and ARRY-162. Further, they have predicted ten anticancer drugs with higher sensitivity towards the high-mitophagy gene expression group. Further, Cao et al., have shown that March ligases expression regulates immune cell infiltration in HCC tumors. By utilizing the TCGA data set of liver cancer patients, Wang et al., have shown that genes related to copper metabolism correlate with immune infiltration in HCC. Their finding may be very useful in establishing the immunotherapy response biomarker. Liang et al., have shown that ferroptosis regulator membrane protein SLC7A11 exhibits the highest expression correlation with the immune checkpoint gene PD-L1. They have also established that SLC7A11 can serve as an independent prognostic signature itself for HCC patients. Furthermore, another study by Zhang et al., has shown that ferroptosis-related genes were significantly correlated with tumor immune infiltration and immune checkpoint genes expression. Long et al., have also explored the correlation of immune regulatory genes with HCC patients\u2019 survival. Their study has demonstrated that 5 immune regulatory genes expression has significant predictive importance in HCC patients\u2019 survival. In a review article Si et al., have discussed the importance of IL32 and IL34 expression in HCC pathogenesis and therapeutic targeting.Immune therapeutics are revolutionizing cancer treatment, so it\u2019s high time to identify the novel immune modulators of HCC pathogenesis and potential immune therapy targets. in silico analysis of publicly available databases, and very limited validation has been done in laboratory conditions. Most of the studies are correlative, so it will be premature to conclude their direct role of a predicted gene signature in HCC pathogenesis. More preclinical and clinical studies are needed to validate these findings.In general authors of these articles have done molecular characterization of HCC patient samples gene expression, and immune infiltration and studied their impact on the prognosis of HCC patients. Some studies have explored the correlation between gene signatures and therapeutic response along with their prognostic values. The role of metabolic alteration-related genes, angiogenesis-related genes, and genes involved in different types of cell death such as cuproptosis and ferroptosis have been shown to possess prognostic value and correlate well with the different immune phenotypes of HCC tumors. A key limitation of most of the studies is that results are based on purely"} +{"text": "The potential benefits of Reporting and Data Systems (RADS) are well known: improve the communication between the radiologists and physicians; reduce the omission of relevant information in reports; reduce the variability and errors in image interpretation; facilitate the monitoring of results and provide a tool to ensure quality and research. Furthermore, the slightest error in the interpretation of the legend describing the results of an imaging study can modify both the medical decision to start or not a treatment, and to do it with a more or less invasive technique.In this sense, for example, the American College of Radiology (ACR) started the development of the Breast Imaging Reporting and Data System (BI-RADS) in 1993. In recent years, several systems have been developed to diagnose other malignancies based on BI-RADS. However, its utility has yet to be demonstrated which is why scoring systems are continually being revised to apply them to daily clinical practice. The RADS systems developed so far are specially designed for breast, colorectal, gynecological, liver, lung, prostate and thyroid cancers.Incorporation of Reporting and Data Systems into Cancer Radiology\u201d Research Topic. These manuscripts demonstrate how RADS benefit cancer patients, and how improvements to these systems can further benefit patients and optimize disease outcomes. For instance, in Wang et\u00a0al., the authors investigated the value of contrast-enhanced ultrasound in the differential diagnosis and risk stratification of ACR TI-RADS category 4 and 5 thyroid nodules with non-hypovascular, establishing a risk score with ability to improve both aspects.These aspects are clearly reflected in the research papers accepted in the \u201cSun et\u00a0al., it was evaluated the value of multiparametric magnetic resonance imaging (MIR) including synthetic MRI, diffusion-weighted imaging, dynamic contrast enhanced MRI, and clinical features in breast imaging\u2013reporting and data system (BI-RADS) 4 lesions, and develop an efficient method to help patients avoid unnecessary biopsy.In Zhou et\u00a0al., the authors established a predictive model incorporating clinical features and contrast enhanced ultrasound liver imaging- reporting and data system (CEUS LI-RADS) to improve the prediction of microvascular invasion (MVI) in hepatocellular carcinoma (HCC) patients.In Singh et\u00a0al., to minimize inter-radiologist variability, the diagnostic performance of an in-house developed semi-automated model using machine learning methods for prostate imaging-reporting and data system version 2.1 (PI-RADS v2.1) scoring was evaluated in prostate cancer patients. The authors in Meng et\u00a0al., assessed the value of using quantitative parameters from synthetic magnetic resonance imaging (SyMRI) and the Kaiser score (KS) to differentiate benign and malignant breast lesions in patients, identifying that the incorporation of T1 values improves the sensitivity and specificity of the KS protocol alone.In Yang et\u00a0al., a retrospective study was performed to create a predictive machine learning model based on liver imaging and reporting and data system (LI-RADS) features to identify microvascular invasion in hepatocellular carcinoma (HCC) patients, concluding that LI-RADS may help optimize the management of these patients.In Wu et\u00a0al., the authors conducted a retrospective Chinese population-based study of patients screened for lung cancer by computed tomography to assess the correlation between the probability of lung cancer and the number of lung nodules, concluding that this probability does not change with the number but does change with the size of the nodules.Finally, in All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication."} +{"text": "Thus, migratory behaviour neither weakens nor strengthens bird\u2013haemosporidian cophylogenetic congruence, suggesting that other avian host traits are more influential in generating phylogenetic congruence in this host\u2013parasite system.Parasites display various degrees of host specificity, reflecting different coevolutionary histories with their hosts. Avian hosts follow multiple migration patterns representing short but also long distances. As parasites infecting migratory birds are subjected to multiple environmental and biotic changes through their flyways, migration may disrupt or strengthen cophylogenetic congruence between hosts and parasites. On the one hand, parasites might adapt to a single migratory host, evolving to cope with the specific challenges associated with the multiple habitats occupied by the host. On the other, as migrants can introduce parasites into new habitats, higher rates of host switching could also disrupt cophylogenetic patterns. We analysed whether migratory behaviour shapes avian haemosporidian parasite\u2013host cophylogenetic congruence by testing if contributions of host\u2013parasite links to overall congruence differ among resident and short-, variable- and long-distance migrants globally and within South America only. On both scales, we found significant overall cophylogenetic congruence by testing whether overall congruence differed between haemosporidian lineages and bird species. However, we found no difference in contribution towards congruence among links involving resident Using a worldwide avian haemosporidian database collected from 347 different host species and representing 430 localities around the globe , 101 host species and 113 localities.We extracted the dataset on haemosporidian lineages from the MalAvi database each represented ~25% of our global dataset with most tropical species (~67%) sampled in South America. Haemosporidian parasites and their avian hosts showed phylogenetic congruence greater than expected by chance on both global and regional scales .In the Bayesian models evaluating the effect of host migratory distance category on the squared residual values of each individual link , we found that hosts contribute equally to parasite\u2013host cospeciation independently of whether or not they migrate, or their migration distance and 2. Wet al., Parasite\u2013host cospeciation occurs when both clades speciate in tandem, creating congruent phylogenies, which can occur due to neutral processes such as host dispersal and isolation or due to host-driven selection such as host defence (Clayton et al., et al., et al., et al., et al., et al., et al., et al., et al. (Plasmodium juxtanucleare, a haemosporidian parasite previously observed only in Galliformes, infected wild passerines in Brazil. Such events can disrupt cophylogenetic congruence, leading to apparent incongruence between haemosporidians and their hosts (Ricklefs et al., Host defences may favour parasite cospeciation and lead to congruent phylogenies between parasites and their hosts, if they select for specific counter-adaptations that limit parasite success to the original host species (Clayton , et al. recentlyet al., et al., et al., et al., et al., et al., et al., Furthermore, we demonstrated that host migratory behaviour does not affect haemosporidian cophylogenetic congruence, suggesting that other host features can be more important in determining haemosporidian infection of novel hosts, such as cell invasion ability or immunological defence. Similarly, even though migrants can disperse haemosporidians (de Angeli Dutra et al., et al., Culex quinquefasciatus in Hawaii and New Zealand enabled the infection of local endemic and na\u00efve fauna by Plasmodium species, especially the highly generalist Plasmodium relictum (Warner RE, et al., Vectors can also play a fundamental role in determining haemosporidian parasite transmission in nature (Yan et al. (Leucocytozoon, another related genus of avian haemosporidian parasites. As we also observed, host phylogeny influenced parasite\u2013host cophylogeny in all models, indicating that other phylogenetically conserved host traits may contribute towards parasite\u2013host cophylogeny. However, the narrower climatic niche and higher host specificity of Leucocytozoon spp. (Valki\u016bnas, et al., et al., et al., Leucocytozoon parasites, whereas migrants may be exposed to parasites from multiple regions and continents. This pattern could also lead to links involving associations between Leucocytozoon lineages and resident hosts contributing more towards cophylogenetic congruence compared to those involving migrant hosts. It is worth mentioning, however, that our relatively small number of migrants, especially for the South American analyses, might have hidden an association between migratory status and haemosporidian\u2013bird cospeciation.It is important to note, however, that Jenkins et al. identifiPlasmodium and Haemoproteus separately. Nonetheless, we detected an effect of host phylogeny on host\u2013parasite cophylogenetic congruence, suggesting that congruence among parasites and their hosts may be linked to other host traits, and possibly also to vector specificity. It must be noted that, despite the fact haemosporidians and their avian hosts appear to have coevolved, these parasites are still capable of switching to phylogenetically distant hosts (Ferreira-Junior et al., Here, we demonstrate that haemosporidian parasites and their avian hosts generally share a common evolutionary history and show significantly congruent phylogenies. Furthermore, we also found that bird migration is not weakening or strengthening the congruence observed between parasites and their hosts, even when considering different spatial scales or the genera"} +{"text": "Coxiella, Francisella, Rickettsia or Candidatus Midichloria mitochondrii endosymbionts, indicative of their importance to tick physiology. Genomic and experimental data suggest that endosymbionts promote tick development and reproductive success. Here, we review the limited information currently available on the potential roles endosymbionts play in enhancing tick metabolism and fitness. Future studies that expand on these findings are needed to better understand endosymbionts\u2019 contributions to tick biology. This knowledge could potentially be applied to design novel strategies that target endosymbiont function to control the spread of ticks and pathogens they vector.Ticks transmit pathogens and harbour non-pathogenic, vertically transmitted intracellular bacteria termed endosymbionts. Almost all ticks studied to date contain 1 or more of There are 2 main tick families Ixodidae (hard ticks), which possesses a sclerotized hard shield called scutum, and Argasidae (soft ticks), which lacks scutum , Francisella endosymbionts (FEs), Rickettsia endosymbionts (REs) and Candidatus Midichloria mitochondrii (CMM) and hard ticks and the argasid tick O. amblus (CLEOA) revealed that they have complete pathways to produce thiamine (vitamin B1), riboflavin (vitamin B2), niacin (vitamin B3), biotin (vitamin B7) and folate (vitamin B9) and pyridoxine (vitamin B6) and A. sculptum (CeAS-UFV) have complete pathways to synthesize riboflavin, biotin and folate and partial pathways for niacin, pantothenic acid and pyridoxine has complete pathways for the synthesis of riboflavin, pyridoxine, biotin and folate and a partial pathway for the synthesis of thiamine , A. maculatum (FLE-Am) and F. persica, the endosymbiont of the soft tick Argas arboreus, showed that they possess complete pathways for the synthesis of riboflavin, biotin and folate but F. persica seems to only possess a partial pathway for this process and CLEAA, CLEOA and CERM possess complete pathways to produce lipoic acid , NADP+, pyridoxal 5\u2032-phosphate, S-adenosyl-L-methionine and thiamine diphosphate .Many cofactors and coenzymes that are critical to the functioning of essential enzymes are derived from B vitamins Douglas, . Severalin silico flux balance analyses of CRt and CE of R. sanguineus (CRs) identified excessive production of the amino acid proline, which is thought to play a significant role in arthropods due to its involvement in energy production and nitrogen metabolism to O. moubata restored its reproductive fitness, indicating a role for FLE-Om in provisioning B vitamins required for normal tick development and reproduction , which in turn reduced the blood intake of the tick (Zhong et al., et al., H. longicornis, administration of antibiotics to R. sanguineus and R. microplus also reduced the density of CEs and tick blood intake, and transcriptomic analysis of CERM-free R. microplus metanymphs revealed that genes associated with tick blood-feeding capacity such as DAP-36, lipocalin, trypsin inhibitor-like family, Kunitz-type inhibitors, cystatin and evasins were significantly under-expressed (Guizzo et al., et al., et al., et al., Experiments also suggest that endosymbionts may contribute to the blood-feeding capacity of ticks . TreatmeI. ricinus and feeding females with tetracycline-containing bovine blood produced CMM-free ticks within 2 generations. Larvae that hatched from eggs laid by CMM-free females consistently performed poorly during blood feeding, suggesting that CMM is required for the emergence of larvae with intact blood-feeding ability (Guizzo et al., I. ricinus nymphs fed with gentamicin-treated blood had significantly lower engorgement weights, lower moulting proportions and lower weights of moulted unfed adult females in comparison to nymphs fed on antibiotic-free blood (Militzer et al., Francisella symbionts in H. doenitzi significantly increased in number during blood feeding, and Rickettsia sp. phylotype G021 and CMM multiplied massively in I. pacificus and I. ricinus, respectively, following blood meals (Sassera et al., et al., et al., CMM is the most common endosymbiont associated with D. variabilis and A. americanum larvae infected with Rickettsia symbionts displayed increased motility than uninfected larvae [42]. The locomotive ability of newly hatched larvae was determined on flat and inclined surfaces and Rickettsia-containing larvae displayed increased locomotive speed relative to uninfected larvae. Tick motility plays a role in host-questing success; thus, infection with Rickettsia symbionts may impact the disease risk posed by tick-borne pathogens.Finally, et al., et al., et al., et al., Coxiella, Francisella and Candidatus Midichloria branches (Epis et al., et al., et al., et al., In summary, a major function of tick endosymbionts seems to be the provisioning of B vitamins, especially riboflavin, biotin and folate . B vitamet al., et al., C. burnetii and F. tularensis are not vertically transmitted (Genchi et al., et al., Relatedly, another aspect of tick biology that we do not fully understand is how CEs and FEs evolved from pathogenic ancestors (Gerhart C. burnetii and F. tularensis (Zogaj and Klose, et al., In addition to vertical transmission, an essential factor that sustains endosymbiosis is the presumed dependence of ticks on nutrients provided by endosymbionts. Going forward, functional studies to identify specific metabolites that sustain tick\u2013endosymbiont relationships should be prioritized. Developing genetically tractable tick\u2013endosymbiont model systems would accelerate this area of research by facilitating the disruption of endosymbiont genes to assess their impact on tick physiology and fitness. Although methodologies to culture and genetically manipulate CEs or FEs have not yet been developed, genetic tools and culture media are available for related pathogens et al., et al., et al., Future functional studies should also device alternatives to the current practice of using antibiotics to generate endosymbiont-free ticks. This is because antibiotics may eliminate other members of the tick microbiota, thus making it difficult to determine whether any observed effect is solely due to the loss of the endosymbiont. Another key aspect to consider while investigating endosymbiont function is the potential contributions made by the rest of the tick microbiota towards tick physiology. For instance, gut microbiota may modify metabolites present in blood meal to make them amenable for use by tick or endosymbiont. Similarly, antibacterial peptides produced by the tick innate immune system in response to gut bacteria could impact the location and functions of tick endosymbionts (Narasimhan et al., Lastly, targeting keystone taxa among tick microbiota is an innovative approach to inhibit the spread of ticks. Recent studies showed that microbiota were disrupted in ticks fed on blood from mice vaccinated against keystone taxa (Mateos-Hern\u00e1ndez"} +{"text": "Integrative neuroscience involves investigations into the sensory, motor, and cognitive systems. In contrast, translational neuroscience moves fundamental knowledge and interdisciplinary discoveries into clinical practice or applications to benefit human health. Bridging the gap between both fields could profoundly impact our comprehension of brain function and clinical applications . SpecifiFujiki et al., introduced the \u201cquadripulse theta burst transcranial magnetic stimulation\u201d (QTS), a new way to overcome failure rates in producing motor evoked potentials by transcranial magnetic stimulation. These authors employed a similar stimulation pattern as in previous studies (Szel\u00e9nyi et al., Chandrasekaran et al. introduced a novel, highly flexible spinal electrode array for cervical dorsal root stimulation employed in people with motor complete spinal cord injury. This targeted stimulation increased volitionally generated force and tactile sensations within a 6\u20138-week period, but only in particular muscles showing discernable force production during the pre-intervention assessment. These results are comparable to those found by other authors (Freyvert et al., The first two papers in this Research Topic have reported new methods to push the limits of precision in brain and spinal cord neurostimulation. They are relevant because non-invasive neurostimulation of the brain, peripheral nerves, and the spinal cord has unprecedented clinical interest. In this context, Soto et al. is a review covering the development of new active vestibular implantable devices to regain or modulate specific systemic functions. Like cochlear implants, pacemakers, or deep brain stimulators, the vestibular neuroprostheses are a strong example of how bridges between integrative and translational neuroscience are needed to achieve beneficial breakthroughs that will impact the lives of individuals. Vestibular dysfunction affects over 1.8 million people worldwide (Chow et al., The third paper by Ilic et al. deals with visual imagery in dreams of congenitally blind people. This theme is controversial (Andrade, Ilic et al. in this Research Topic analyses studies on the presence and nature of visuospatial imagery in dreams of blind people to elucidate how blind people \u201csee\u201d, whether they may recreate visuospatial imagery via sensory substitution, and whether they can dream in images. At the neurophysiological level, neuroimaging and sensory substitution studies suggest that the \u201cblind\u201d occipital cortex may be able to integrate non-visual sensory inputs, generating visuospatial impressions and enabling the development of a typical spatiotemporal organization of early visual areas even in the life-long absence of vision. This could explain the ability of some congenitally blind individuals to draw symbolic representations of various visual images in striking likeness to those drawn by normally sighted. Therefore, elucidating the mechanistic nature of visual impressions could open new translational possibilities for treating these neuro-disabilities.The fourth article by Andrade, . It has Mesmoudi et al. presented another interesting research covering the gap between integrative neuroscience and translational neuroscience in the context of the recent global health crisis of COVID-19, in which the discovery of ACE2 receptor mechanisms played a fundamental role in understanding this sickness. They employed data on mRNA expression levels of genes provided by the Allen Institute for Brain Science. Moreover, the localization of brain functions was provided by the LinkRbrain platform. These authors investigated which cognitive and sensorimotor functions are associated with the brain regions where ACE2/TMPRSS2 is overexpressed, hypothesizing that the infection might particularly affect them. The results show that central regions specific to ACE2 and MPRSS2 were localized in the brain stem, the subcortical, the orbitofrontal, and some occipital areas (see also Chen et al., Finally, In conclusion, these five papers, taken together, emphasize that we must bridge the gap between knowledge and practice, between theory and therapy. Thus, the synergy between integrative and translational neuroscience involving new neurotechnological developments could serve as the bridge that will lead us to a future where neurological conditions are better comprehended and more effectively treated. As we persist in exploring the frontiers of the brain, let us bear in mind that we could discover the key to unlocking the full potential of neuroscience for the benefit of humanity through the fusion of these two established disciplines.EM: Writing\u2014review and editing. GC: Writing\u2014review and editing. KS: Writing\u2014review and editing. AL: Writing\u2014review and editing."} +{"text": "Wnt signaling in endocrine and metabolic disorders\u201d aims to emphasize the functional role of the Wnt signaling pathway in human endocrinology, focusing on metabolic disease. Endocrine and metabolic disorders encompass a wide range of conditions affecting various organ systems and physiological processes. The Wnt signaling pathway, originally recognized for its role in embryonic development and tissue homeostasis , used for more than 1,300 years in China as a treatment for lumbodynia, may exert its therapeutic effect on steroid-induced ischemic necrosis of the femoral head, is through targeting exosomal microRNAs (miRNAs) to regulate multiple signaling pathways, including Wnt, PI3K-Akt, and MAPK . In another original report investigating miRNAs and Wnt signaling, Tripathi et\u00a0al. demonstrate that miR-539-3p overexpression in osteoblasts downregulates several components of the Wnt signaling pathway and deteriorates trabecular microarchitecture, leading to decreased bone formation in ovariectomized mice. In the third original article in our Research Topic, a group of investigators led by Xiaolin Tu found that the small molecule C91 (CHIR99021) promotes osteogenic differentiation of bone marrow stromal cells via the activation of Wnt signaling .The first study in this Research Topic clarified that one of the mechanisms by which the \u201cVilaseca et\u00a0al. provide an interesting overview of the functional roles of estrogen deficiency in the processes involved in the development of Alzheimer\u2019s disease, including Wnt signaling and glucose transport in the brain, amyloid precursor protein processing to form senile plaques, and Tau phosphorylation forming neurofibrillary tangles.Franco et\u00a0al. elegantly explain the main differences between the physiological roles of canonical Wnt signaling and its pathological involvement in the development of several human diseases, including cancer. Correctly interpreting the molecular bases of Wnt signaling and metabolism, ideally in a cell-type and tissue-specific manner (Franco et\u00a0al.; A very comprehensive review concludes our Research Topic: In conclusion, understanding the intricate interplay between Wnt signaling and endocrine/metabolic disorders holds great promise for the development of targeted therapies and improved patient outcomes.FC: Writing \u2013 review & editing. GS: Writing \u2013 original draft"} +{"text": "However, in vivo validation is currently lacking. A previous metatranscriptomics study into the cause of idiopathic chronic diarrhoea in macaques reported the presence of an unidentified protozoan parasite related to Trichomonas vaginalis. In this work, we performed a reanalysis of the published data in order to identify the parasite species present in the macaque gut. We also leveraged the information-rich metatranscriptomics data to investigate the parasite behaviour in vivo. Our results indicated the presence of at least 3 genera of Trichomonad parasite; Tetratrichomonas, Pentatrichomonas and Trichomitus, 2 of which had not been previously reported in the macaque gut. In addition, we identified common in vivo expression profiles shared amongst the Trichomonads. In agreement with previous findings for other Trichomonads, our results highlighted a relationship between Trichomonads and mucosal bacterial diversity which could be influential in health and disease.Trichomonads, anaerobic microbial eukaryotes members of the phylum Parabasalia, are common obligate extracellular symbionts that can lead to pathological or asymptomatic colonization of various mucosal surfaces in a wide range of animal hosts. Results from previous The vast majority of this work has focused on the human sexually transmitted parasite Trichomonas vaginalis. However, the importance of the proposed mechanisms during colonization of the complex mucosal environment in vivo is unclear. Validation of hypotheses in the natural setting is essential to avoid misinterpretation of results microbiota were used to examine Parabasalia gene expression. For transcript annotation, assembled parasite contigs were aligned to the T. vaginalis G3 annotated proteins and Table S3 .A et al., de novo assembled contigs homologous to Parabasalia genes of interest, with E value, percentage identity and query coverage cut-off values of 1\u00a0\u00d7\u00a010\u221210, 88% and 90%, respectively. To broaden the taxonomic sampling for genes of interest, additional homologues were identified by consulting the literature and through the use of online BLAST . Summary statistics for the dataset and assembly are presented in Table S1. The de novo assembly is available in Data file S2.We aimed to investigate the identity of Trichomonad parasites reportedly present in this dataset (Westreich \u03b1) loci to identify the Parabasalia colonizing the macaque gut. Amongst all the samples, we assembled 58, 10 and 11 18S rRNA, actin and EF-1\u03b1 sequences, respectively, which shared greater than 88% sequence identity with reference Parabasalia sequences for at least 90% of their length (Table S2). We assessed the diversity of sequences present by maximum likelihood analysis and identified 10 well-supported clades for the 18S rRNA locus , 4 for the actin locus , and 1 for the EF-1\u03b1 locus . We generated phylogenies using representative sequences from each clade alongside a range of Parabasalia reference sequences in order to refine the identity of the parasite sequences. Analysis of a single representative sequence from each of the 18S rRNA sequence groups revealed at least 3 major lineages, related to Tetratrichomonas, Pentatrichomonas and Trichomitus spp., with strong bootstrap support only present for the latter parabasalid genera which were identified , followed by Pentatrichomonas (mean abundance 0.025%) and Tetratrichomonas (mean abundance 0.020%). A substantial number of sequences (mean abundance 0.093%) were identified as parabasalid in origin but could not be assigned to a particular genus. Unidentified parabasalid reads appeared more abundant among animals in which Tetratrichomonas, Pentatrichomonas or Trichomitus classified reads were abundant, likely suggesting that they originated from 1 or more of these genera.To taxonomically assign the metatranscriptome reads, we included entified . AccordiTrichomonas (mean abundance 0.019%). This most likely reflects the greater representation of Trichomonas whole genome sequences available in the reference database, including T. vaginalis and T. gallinae, whereas the genera of interest were only represented by in vitro RNA-Seq data, which is likely to have an incomplete gene content. However, while it cannot be ruled out that Trichomonas spp. were present amongst the samples, we have focused our analysis on the most likely genera based on the phylogenetic results. Only 2 control macaques showed a total abundance of Parabasalia greater than 0.125%, limiting the statistical power for tests correlating variables with Parabasalia abundance amongst the control animals.In addition, a notable fraction of reads were classified as et al., We focused on the most abundant putative Parabasalia-derived contigs to explore the most biologically important functions, which are summarized in Trichomitus with strong support, and an additional contig NODE_1833 may have originated from Pentatrichomonas or Tetratrichomonas, although this was poorly supported. In addition, we identified a contig with high similarity to T. vaginalis coronin, an actin-binding protein implicated in phagocytosis . Results suggested that the lysozyme-like contig NODE_2008 originated from T. vaginalis genes previously implicated in pathobiology. Of particular interest for parasite adhesion to host or microbial cells . Cysteine peptidases are also implicated in Trichomonas pathobiology , a secreted cysteine peptidase demonstrated to induce host cell apoptosis based on the microbial profile showed clear separation between the healthy and ICD groups. The macaques with ICD appeared to resolve into 3 subgroups, potentially indicating distinct microbial communities . An obvibundance . Of partP values derived from linear regression ranged from 0.21 to 0.27), although this is likely to have been influenced by the Parabasalia scarcity amongst the control animals , whereas Parabasalia abundance did not . Intriguingly, ICD macaques with greater Parabasalia abundance appeared to more closely resemble control macaques in terms of alpha diversity.Our results suggested a possible relationship between parasite abundance and microbiota diversity. Amongst the macaques with ICD, there was a significant positive relationship between Parabasalia abundance and microbial alpha diversity measures . There was also a significant negative relationship between Parabasalia abundance and Simpson evenness, indicating a more non-uniform distribution of abundance amongst microbial taxa in animals with greater abundance of Parabasalia . However animals . In addivs all correlation analysis at the genus level. We focused on the macaques with ICD and included only the most abundant taxa (greater than 0.005% in at least 1 sample). Amongst 358 taxa, with a total of 64\u00a0261 possible interactions, our results indicated 11\u00a0606 significant abundance correlations between genera. Of the 3 parabasalid genera of interest, Tetratrichomonas showed the greatest number of significant correlations with bacteria (110) followed by Pentatrichomonas (53), and Trichomitus, which showed far fewer significant correlations (17). Tetratrichomonas and Pentatrichomonas substantially overlapped in terms of bacterial genera showing significant positive and negative correlations, possibly indicating shared relationships with bacteria. In contrast, Trichomitus did not share common negative or positive relationships with any bacterial genera with either of the other parabasalid genera , with a moderate network clustering coefficient of 0.431, indicating a greatest potential interdependence with bacteria. Within network 3, the closeness centrality of Tetratrichomonas was 0.465, the 10th highest in the 41-node network, suggesting a relatively central hub-like position in comparison to most bacterial nodes. In contrast, Pentatrichomonas (network 7) and Trichomitus (network 23) inhabited smaller and more sparsely interconnected components, with 7 and 2 genera, respectively.To investigate specific interactions between Parabasalia and bacterial members of the microbiota, we performed an all d genera . Amongstmponents . Of the Tetratrichomonas and Pentatrichomonas were Gram negative, but this pattern did not extend to Trichomitus , 5 out of 53 for Pentatrichomonas and 2 out of 17 for Trichomitus . This may indicate that the microbial community structure and interdependence was dramatically different between the ICD and control conditions.The majority of relationships identified amongst the macaques with ICD were not consistent amongst the control group. Only 1188 out of 11\u00a0606 total significant relationships for the macaques with ICD were homodirectionally concordant amongst the control animals, including 2 out of 110 relationships for Tetratrichomonas and Pentatrichomonas , but not Trichomitus, amongst the macaques with ICD compared with the healthy controls. The original authors ruled out several known common GI pathogens as the cause of ICD by culture and microscopy-based methods. To complement this, we searched the dataset for potentially pathogenic viral lineage amongst the taxonomic profile. We focused on a selection of 29 potential primate-infecting eukaryotic viruses which we identified by the literature search .We performed a differential abundance analysis between the ICD and control groups in order to investigate any potential impact of the parabasalids on disease aetiology. Interestingly, differential abundance analysis suggested a moderate significantly higher abundance of Tetratrichomonas abundance . We identified a significant negative relationship between the abundances of 12 MetaCyc pathways and that of Tetratrichomonas amongst the macaques with ICD and glycogen storage and processing (M\u00fcller, et al., T. vaginalis BspAs have been implicated in host adhesion in vitro (Handrich et al., T. vaginalis BspAs are differentially expressed in response to Mycoplasma symbionts, suggesting a potential role in parasite\u2013bacteria interactions in modulating parasite binding to host cells (Margarita et al., Our results suggested commonality in the expressed functional genes across the Trichomonads. We observed similar energy generation mechanisms for the macaque-infecting parabasalids as have been previously reported for et al., et al., et al., et al., et al., Trichomitus, our results suggested Tetratrichomonas had the greatest abundance correlation with differences in the microbiota. Tetratrichomonas participated in the greatest number of significant abundance correlations, and was a central node within a densely interconnected microbial positive correlation network. Correlation networks have been effectively utilized to identify keystone species within microbial communities with biological significance (Duran-Pinedo et al., Pentatrichomonas and Tetratrichomonas. In addition, of particular interest, a positive abundance correlation with Prevotella, which we observed for Tetratrichomonas in the macaque gut, has been described for T. vaginalis in the human UGT (Martin et al., et al., Tetratrichomonas and Pentatrichomonas were Gram negative. This is notable because other Trichomonads such as Dientamoeba fragilis (Chan et al., in vitro growth. Microbial interactions identified in this study varied hugely between the diseased and healthy conditions, similarly to previous results from the healthy and diseased human oral microbiota (Duran-Pinedo et al., et al., et al., Our results also indicated a potential influential interaction between Trichomonads and microbial diversity in the macaque gut, as has been reported for other hosts and mucosa (El Sayed Zaki et al., et al., et al., T. vaginalis (Pinheiro et al., Trichomitus, whereas Tetratrichomonas was the only Trichomonad genus correlated with bacterial functional expression. Negative correlation of Tetratrichomonas abundance with bacterial degradation pathways for monosaccharides such as GlcNAc and Sia5NAc, potentially derived from mucin glycoproteins (Yurewicz et al., et al., Previous studies have suggested the interaction between Trichomonads and the vaginal microbiota is bidirectional. The microbial profile can influence the ability of Trichomonads to colonize the mucosa (Rathod Campylobacter genus amongst the animals with ICD was originally reported (Westreich et al., Pentatrichomonas and Tetratrichomonas could indicate a causal role in disease. High P. hominis abundance in macaques with ICD was previously reported, but not causally liked to disease (Laing et al., et al., T. gallinae and Tritrichomonas musculis decreases GI microbial diversity (Ji et al., et al., et al., The absence of several known GI bacteria pathogens and microbial parasites was confirmed by culture and microscopy-based methods, and thus may be excluded as causative agents of ICD in the macaques. We performed an additional search for potentially pathogenic viruses amongst the datasets. However, we did not identify any clear differences for any putative host-infecting virus when comparing between diseased and control animals, suggesting viral infection may not be the primary cause of ICD. A greater abundance of reads classified as originating from the in vivo. Reference sequences from closely related parasite strains would also have greatly facilitated analysis (Breitwieser et al., in vivo metatranscriptome (Li et al., Our results revealed a relatively low parasite abundance in the macaque fecal samples, highlighting the need for greater sequencing depth or selective target enrichment (Gaudin and Desnues, in vivo insight into Trichomonad mucosal colonization, which validates numerous in vitro studies (M\u00fcller, et al., et al., et al., et al., et al., In summary, these metatranscriptomics analyses of Trichomonads in the macaque gut have provided the first"} +{"text": "The unprecedented global climate change has severely impacted our environment and engendered severe threats to agricultural productivity . This haThe advancements in genomics and gene editing technologies have offered immense opportunities and potential solutions for the genetic improvement of crops . A plethThe deployment of these technologies has laid down the foundation of modern breeding for effectively channelizing the underutilized diversity hidden in the crop wild relatives into elite gene pools . The advDhakate et al., \u201cAdvances in crop breeding through precision genome editing\u201d by Nerkar et al. and \u201cIntegrating CRISPR-Cas and next-generation sequencing in plant virology\u201d by Mushtaq et al. that have very well built up the narrative of the Research Topic. The other crop-specific comprehensive reviews entitled \u201cCRISPR for accelerating genetic gains in under-utilized crops of the drylands: progress and prospects\u201d by Sharma et al., \u201cRecent advances in date palm genomics-a comprehensive review\u201d by Rahman et al., \u201cUnclasping potentials of genomics and gene editing in chickpea to fight climate change and global hunger threat\u201d by Singh et al., \u201cA CRISPR way for accelerated improvement of cereal crops\u201d by Basu et al., \u201cCRISPR/Cas genome editing system and its application in potato\u201d by Zhang et al., \u201cPhysiological and molecular basis of drought and heat stress tolerance to enhance productivity and nutritional quality of peanuts in harsh environments\u201d by Puppala et al., \u201cA perspective on selectable marker-free genome engineered rice: past, present and future scientific realm\u201d by Singh et al. and \u201cCRISPR/Cas genome editing in potato: current status and future\u201d by Tiwari et al. provide an up-to-date detailed compilation of the published research in date palm, dry-land crops, peanuts, chickpea, potato, and rice.In this Research Topic, we have collated a total of 19 articles, including original research and review articles, to get an essence of the spectrum of current efforts undertaken by applying modern tools for crop trait improvement. To develop a background on the theme, we have included three review articles entitled \u201cComprehending the evolution of gene-editing platforms for crop trait improvement\u201d by Dhakate et al., Tiwari et al. and Basu et al. have comprehensively reviewed the evolution of CRISPR/Cas systems into new-age methods of genome engineering across various plant species and the impact that they have had on tweaking plant genomes and associated outcomes on crop improvement initiatives. Hou et al. reviewed the four types of CRISPR/Cas structures and mechanisms available today and the application of CRISPR/Cas9 systems in overcoming the challenges of self-incompatibility and improving the quality and resistance of potato. The review describes how precise knocking or targeted mutagenesis of S-RNase and Sli genes using CRISPR/Cas9 has helped to create self-compatible and self-incompatible potatoes, respectively. Additionally, the suitability of CRISPR/Cas9 and CRISPR/Cas13a systems in knocking out more than 22 genes has been detailed. Kor et al. have described the role of RNA Pol III promoters in precisely targeting genetic variants in genome editing. Nerkar et al. has focused on the advancements in crop breeding through precision genome editing. This review has included an overview of different breeding approaches for agronomic traits such as disease resistance, abiotic stress tolerance, herbicide tolerance, yield, and quality improvement, reduction of anti-nutrients, improved shelf life; genome editing tools and approaches used for crop improvement and an update on the regulatory approval of the genome-edited crops. Sharma et al. has described the opportunities of implementing genome editing technologies in under-utilized crops to increase genetic gains.There has been continuous evolution in the development of gene editing-based technologies involving CRISPR/Cas platforms. Traditionally used CRISPR/Cas nucleases followed Sequence-Specific Nucleases (SSNs) such as Zinc-Finger Nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs), and led to domains such as epigenome editing, base editing, and prime editing. Zang et al. generated Nud mutants in wheat (TaNud) and barley (HvNud) using CRISPR/Cas9-based gene editing and investigated the heritability of the mutations in wheat. Nud gene is a transcription factor controlling the formation of the caryopsis, and loss-of-function mutation in the gene leads to the naked hull phenotype in barley. With the latest CRISPR/Cas9-based gene editing, combined with PCR-RE (polymerase chain reaction-restriction enzyme) approach, Zang et al. achieved a high editing efficiency (51.7%) of the three Nud alleles/homologs in wheat. This study has proven that with the improved vector system and CRISPR/Cas technology, it is not difficult to achieve precise genetic modification even in complex polyploid crops such as wheat which remained calcitrant to genetic modification technologies for a long time.Ravikiran et al. comprehensively investigated genetic variation in RSHT tolerance with the GWAS approach using the cutting-edge genotyping arrays available in rice. They utilized three GWAS models to identify significant marker-trait associations (MTAs) for spikelet fertility and grain yield. Most significantly, the authors reported a set of stable MTAs for both traits, showing an advantage of 6\u201310\u00a0g for yield and up to 28% for spikelet fertility. Additionally, they identified promising tolerant genotypes that carried favorable alleles of 29, 28, 25, or 24 putative MTAs, which could serve as new donors in nurseries.Heat and drought stresses cause substantial yield losses to crop production. According to the latest estimates, the heatwave episodes will particularly intensify in the Indo-Gangetic plains of India, which supports rice\u2013wheat cropping system . SimulatPuppala et al. focuses on the significant progress that has been made towards the characterization of germplasm for drought and heat stresses tolerance and identifying MTAs and QTLs associated with drought tolerance. Advances in phenomics and artificial intelligence to accelerate the timely and cost-effective collection of phenotyping data in large germplasm/breeding populations are also reviewed and discussed. A holistic breeding approach that considers drought and heat-tolerant traits to simultaneously address both stresses is introduced as a successful strategy to produce climate-resilient peanut genotypes with improved nutritional quality.Peanuts exposed to drought stress at the reproductive stage are prone to aflatoxin contamination, which imposes a restriction on the use of peanuts as health food and also adversely impacts the peanut trade . The revKumar et al. reported development of bread wheat variety HD3411 following marker-assisted backcross breeding for drought tolerance. They reportedly transferred four drought tolerance quantitative trait loci (QTLs) controlling traits, viz. canopy temperature, normalized difference vegetative index, chlorophyll content, and grain yield from the drought-tolerant donor line, C306 into a popular high-yielding, drought-sensitive variety HD2733. Marker-assisted selection coupled with stringent phenotypic screening was used to develop a promising genotype, HD3411. The line HD3411 has shown higher yield over selected cultivars and has been identified for varietal release and testing in the northeastern plain zone of the wheat-growing region in India.Mushtaq et al. have shown that the NGS coupled with CRISPR-based genome editing have enabled rapid engineering of resistance by directly targeting specific genomic sites of plant viruses and viroids. They also discussed the emerging developments in NGS technologies and CRISPR/Cas-based DNA or RNA tests for the characterization of plant viruses along with their potential advantages and limitations. Kaur et al. employed BSA-seq approach in a wild species of rice Oryza glaberrima and identified a novel locus on chromosome 6 for resistance to root-knot nematode (Meloidogyne graminicola). They reported 3 potential candidate genes within QTLs qNR6.1, qNR6.2 and qNR2.1. This research has expanded the breadth of genes available for resistance to root-knot nematode and the possibilities of deploying new genes in rice breeding.Besides abiotic stresses, plant disease outbreaks threaten global food security significantly. The crop pathogens are responsible for a substantial reduction in global crop yield and productivity. The global burden of viral, bacterial and fungal pathogens in farmers\u2019 fields ranges between 20% and 30% . It poseMontesinos-L\u00f3pez et al. in the article \u201cA general-purpose machine learning R library for sparse kernels methods (SKM) with an application for genome-based prediction\u201d demonstrated the usefulness of six machine learning models based on sparse kernel algorithm in two major crops, wheat and maize. Most importantly, this new package with six models allows user to use different formats of data, i.e., binary, categorical, count and continuous response variables. Recently, ML models are also being explored to resolve the off-target problems associated with CRISPR-Cas9. An article on \u2018Machine learning in the estimation of CRISPR-Cas9 cleavage sites for plant system\u2019 by Das et al. developed models on three ML-based techniques to estimate the cleavage sites of a given sgRNA. Out of the 11 trained models, the models based on the random forest technique, Artificial Neural Networks (ANN1-ReLU) and Support Vector Machine (SVM-Linear), performed better in the estimation of CRISPR-Cas9 cleavage sites showing an accuracy of 96.27%.Machine learning (ML) and artificial intelligence (AI) algorithms have been increasingly used nowadays to improvise genomic prediction models to predict the phenotypes of newly developed breeding lines. Punica granatum L.) breeding programs. The genetic architecture of seed-type trait has not been investigated much using modern approaches, and this has hampered the development of farmer-preferred and commercially viable pomegranate varieties. The recent advances in whole genome sequencing and transcriptome profiling in pomegranate have opened vistas for large-scale discovery of markers for trait discovery and improvement. A previous study in pomegranate had identified novel pre-miRNAs for seed-type trait in pomegranate . We believe that the articles compiled in this Research Topic will expand the existing knowledge on the strategies of crop improvement to mitigate climate change and ensure future food security.To summarize, a diverse collection of research and review articles included in this Research Topic has generated valuable information on the development of genetic and genomic resources in a variety of cereals , legumes (chickpea and peanut), fruit and underutilized dryland crops. While, the review articles presented information on the evolution and refinement of new-age genomics, genome editing, and genome prediction models based on ML and AI algorithms for crop improvement, the research articles involved their application culminating into disease resistant, drought and heat resistant, high yielding crop varieties for instance line HD3411 in wheat ("} +{"text": "Trianthema portulacastrum Linn. on dermal wounds via removal of oxidative stress and inflammation\u2019 by Ekta Yadav et al., RSC Adv., 2018, 8, 21621\u201321635, https://doi.org/10.1039/C8RA03500H.Correction and removal of expression of concern for \u2018Ameliorative effect of biofabricated ZnO nanoparticles of The authors regret that there was an error in This correction supersedes the information provided in the Expression of Concern related to this article.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "Objective of the present analysis is to represent the phenomenon of Heat\u2013mass transfer on MHD micro polar fluids caused by permeable and continuously stretching sheet along with slip impacts fostered in a porous medium. Consequently, the equation of energy includes the term of non-uniform heat source/sink. The equation regarding species concentration in cooperates the terms indicating order of chemical reaction to characterize the chemically reactive species. The application software MATLAB with governing syntax of bvp4c technique are employed to reduce equations of momentum, micro-rations, heat, and concentration into suitable required simplifications to derive necessary arithmetic manipulations of available non-linear equations. Various dimensionless parameters are portrayed in the available graphs with essential consequences. Analysis discovered that micro-polar fluid improves velocity and temperature profile while it suppresses micro-rations profile also magnetic parameter ( Investigations of micro polar fluids are of significant recognition because of numerous applications in various industries particularly suspension solutions, solidification of liquid crystals, animal bloods, and exotic lubricants. Bhargava and Takhar2 explored heat transfer of the micro-polar boundary layer (BL) near a stagnation point on a moving wall. Anika et al.3 analyzed consequences of thermal diffusion on the unsteady viscous MHD micro-polar fluid flow past an infinite plate together with hall and ion-slip current. Bhargava et al.4 performed numerical investigations for micro-polar transfer phenomena prompted by non-linear stretching sheet availing two distinct techniques of finite element and finite difference. Takhar et al.5 exercised mixed convection in MHD flow of micro-polar fluids across the stretchy sheet. Bhargava and Rana et al.6 examined nonlinear convective heat and mass transfer in a micro-polar fluid with continuously variable conductivity by employing the objectives of finite element technique.In recent past academic attainment of micro-polar fluid has drawn attracted attention among several engineering community and scientist community as a reason of its limited circumference associated with Newtonian fluids. These fluids are influentially determined by spin inertia and reinforces stress moments and body moments. The theory of microfluids is identified as complex theory against the case of constitutively linear theory and the corresponding underlying mathematical manipulations are not easily amenable to the solution of non-trivial problems in this field. A subclass of these fluids is defined as the micropolar fluids that exhibits micro-rotational effects and micro-rotational inertia. The classical framework of Navier\u2013Stokes model founds certain degree of limitation particularly listing as it cannot describes and elaborates the category of fluids pertaining microstructure characteristics, fluids possessing effective and influential applications. Therefore, analysis of micro polar fluids suggested by Eringen7 has fascinated many researchers to investigate alike problems on the boundary layer (B.L.) flow due to a stretching sheet, as it has numerous applications in industry like the extrusion of polymer sheet by a dye, crystal growing, continuous casting and drawing of plastic films. The pace of cooling and the stretching process are the only factors that directly affect the desired properties of the finished product. The stretching sheet may not be necessarily linear, as we can take in nonlinear fashion also, even though problem may not have noticeable technological relevance. In view of this, Vajravelu8 proposed the flow across a nonlinearly stretching sheet, while Cortell10 studied the flow and heat-transport caused by a stretching sheet for two unalike types of thermal boundary (TB) conditions on the sheet, viz., constant surface temperature (CST) and prescribed surface temperature (PST). Ganji et al.11 reported analytical solution for magneto hydrodynamic flow due to a stretching sheet in nonlinearly manner. Similar work has been studied by Ishak et al.12, Prasad et al.13, Van Gorder et al.14, Raftari et al.15, Abbas and Hayat16, Dadheech et al.17, Olkha et al.18 and Abel et al.19, among others.The flow of fluid across continuously stretching sheet under the influence of available magnetic field has significant emphasis on several domains of engineering particularly plasma investigations, geothermal energy extraction etc. Investigations pertaining to MHD effects on flow of fluid under consideration past a stretching sheet are indexed in an open literature. The first study by Crane20 examined chemically reactive species diffusion due to a plane elastic surface. Abo-Eldahab and Salem21 studied flow and heat transfer of non-Newtonian power law fluid flow with mass diffusion and chemical reaction on a moving cylinder under consideration of magnetic field effect. Chauhan and Jakhar22 reported 2D non-Newtonian flow and heat transport in a channel with suction at the top and a naturally permeable medium at the bottom. Chauhan and Ghiya23 suggested heat-transfer in second order fluid flow in between two stable permeable disks together with the consequences of magnetic field. Kumar24 investigated analysis of finite element combined with heat-mass transfer in hydromagnetic micro-polar flow past a stretching sheet. Emad et al.25 explored the investigations of flowing/suction impacts on the hydromagnetic heat-transfer by the application of mixed convection from continuously stretching surface together with internal heat generation/absorption. Tripathy et al.26 examined the numerical evaluations of hydromagnetic micropolar fluids past the stretching sheet embedded in a porous channel together with non-uniform heat sources and permissible chemical reactions. Chen and Taiwan27 inspected the theory of heat-mass transfer in MHD flow prompted by natural convection from permeable and suitably inclined stretching surface embedded with variable temperature of wall and concentration. Alam et al.28 examined numerical proposals of combined free-forced convection and mass transfer flow past the available vertical, porous plate in the porous channel together with heat generation and thermal diffusions. Aydin and Kaya29 investigated the MHD mixed convective heat transfer flow about the suitably inclined plate. Reddy and Reddy30 suggested investigations of mass transfer and heat generation consequences on MHD free convection flow across the inclined vertical surface in porous medium. Patil et al.31 proposed the influential consequences of Eyring\u2013Powell fluid across the stretching surface in the existence of magnetic field and chemical reactions.The consolidated impacts of heat mass diffusion together with chemical reaction has their dominant significance on several processes emerging in cooling of nuclear reactors, thermal insulation, geothermal reservoirs etc. Andersson et al.32 established melting impacts on the mechanism of heat transfer. Yacob et al.33 examined melting heat transfer in boundary layer stagnation point flow towards a stretching/shrinking sheet in a micropolar fluid. Hayat et al.34 examined Powell-Eyring stagnation point flow towards a surface stretching linearly with melting heat transfer. Melting heat and mass transport effects in non-Newtonian flow over a stretching surface with non-linear radiation and magnetic field effect was discussed by Khan et al.35. Gireesha et al.36 investigated melting heat transfer in MHD flow of dusty Casson fluid over a stretching surface.Fundamental phenomenon of melting heat transfer finds dominant significance in various technological and industrial exercises like comprehending melting of permafrost, magma solidification, metal purification, welding etc. Epstein and cho et al.37 investigated slip effects in viscoelastic fluid flow through porous medium due to a porous oscillatory stretched sheet. Govindarajan et al.38 discussed slip and mass transfer effects in a vertical channel under consideration of heat source and radiation. Olkha and Dadheech40 discussed entropy analysis for MHD flow for different non-Newtonian fluid caused by a stretching sheet with slip effect and heat source. Dadheech et al.41 investigated MHD flow for Casson fluid caused by a stretching sheet with slip effect. Dadheech et al.42 discussed entropy analysis for Williamson fluid caused by a vertical plate with Cattaneo-Christov heat flux and slip effect. The boundary layer flow for different fluids and geometrical configurations has been considered by59 in the presence of magnetic field.A fluid sometimes gets adhered to the solid boundary but in some circumstances, it does not get a hold like as in suspensions, melting of polymers, emulsion processes and several other non-Newtonian fluids often exhibits macroscopic wall slip. Fluids which manifest boundary slip finds applications in various domains such as polishing of heart valves, internal cavities and various other technological procedures. Ali et al.In perspective of given literature review we have observed that there are relatively few studies are performed on MHD Micro-Polar fluid prompted by melting stretching sheet. The main objective of current study is to determine flow behavior and heat transfer of Micro-Polar over a melting stretching sheet. The novelty of the presented work is increased by substantial validating slip effects with chemical reaction and non-uniform heat source/sink. The examinations furnished in the given article can be further utilized to make investigations in fuel industries, flow of crushed water problems, and in the extrusion of polymer sheets. The consequences of the investigations made are employed in various engineering designs, metallurgy industries also for improving the working efficiency of systems for flow of thermos fluids.26 with relevant boundary conditions are given as:In the momentum equation we take micropolar fluid, magnetic field and porous medium term. The magnetic field Bo is applied perpendicular to the stretching sheet and the effect of induced magnetic field is neglected since the magnetic Reynolds number is assumed to be small. We further assume that the impressed electric field is zero and Hall effect is neglected.The thermal contribution of non-uniform heat source and sink is introduced effectively in the energy equation.The mass transfer phenomenon due to diffusion of chemically reactive foreign species has been accounts for by considering the chemical reaction term of first order.Steady two-dimensional incompressible micro-polar fluid flows caused by a stretching sheet are examined. Corresponding velocity components mentclass2pt{minimContinuity equationMomentum equationAngular momentum equationEnergy equationSpecies equationhere 39) for flow, concentration and temperature is21)The appropriate boundary condition \u201352\u20135) reThe local \u201cskin friction coefficient\u201d The \u201ccouple stress\u201d at the surfaceThe \u201clocal surface heat flux 19. Later it has been determined that computed consequences had essential significant influences.The essential objective of given investigation is to demonstrate the influence of several physical parameters on velocity mentclasspt{minimaquations\u00a0 are evalmentclass2pt{minim, in micropolar fluids, the material parameter that can affect the velocity profile is known as the micropolar fluidity parameter (K). When the micropolar fluidity parameter (K) increases, it implies that the microstructure or internal degrees of freedom have a stronger effect on the fluid flow. This can lead to an increase in the complexity of the flow patterns and the velocity profile.Figure\u00a0Figure\u00a0Figure\u00a0Figure\u00a0Figure\u00a0Figure\u00a0Figure\u00a0Figure\u00a0Contours showing the impact of micro-rotation parameter, Moreover, the micro-rotation parameter affecting the velocity and temperature of a micropolar fluid more intensely near the surface slightly varies according to the boundary conditions (i.e. when The velocity The influence of The concentration profile Reduction in velocity In the present analysis, a numerical investigation of micro polar fluid flow due to melting stretchy surface in a porous medium has been carried out. The influence of abundant quantities on velocity, microrotation, temperature and concentration distribution are outlined as follows:"} +{"text": "Similarly to the previous Special Issue entitled \u201cMolecular Mechanisms of Allergy and Asthma\u201d , this SpConsidering their pivotal role in allergies, T cell-related molecular and cellular mechanisms were the focus of four independent investigational works. Perveen et al. showed tSeveral other papers focused on the molecular mechanisms related to mast cells (MCs), the pivotal effector cells behind allergic responses. Ashikari and coworkers identifiFurthermore, while Nguyen and colleagues focused Finally, Zhernov et al. exhaustively described the molecular mechanisms behind eosinophilic esophagitis and scomIt is with great pleasure that I am presenting the articles included in this Special Issue to the asthma and allergy research community."} +{"text": "The circular interactions between type 2 diabetes (TMD2) and major depressive disorder (MDD) are well documented but the understanding of their mechanisms has only recently gained more clarity. Latest research indicates, that the association between TMD2 and MDD is largely mediated by insulin resistance (IR).A metabolic subtype of MDD can be distinguished from other MDD subpopulations, that is characterized by predominantly atypical clinical presentation, IR and different responsiveness to antidepressant interventions. IR is a predictor of nonresponse to some antidepressants. The IR seems to be a state-marker of clinical or subclinical depression and the relationship between IR and MDD varies between sexes and ethnicities. Insulin has a direct impact on the monoaminergic systems known to underlie MDD symptoms: serotoninergic and dopaminergic, which are dysregulated in IR subjects. Several trials assessed the efficacy of insulin-sensitizing drugs in MDD with mixed results for metformin and more consistent evidence for pioglitazone and lifestyle intervention/physical activity.Recently published data suggest a significant role of IR in the clinical presentation, pathophysiology and treatment response in MDD. Further research of IR in MDD and integration of existing data into clinical practice are needed. Their work showed, that IR was elevated in atypical but not typical depression. Some researchers focused on specific symptoms and attempted to disentangle their links to metabolic markers. Chae et al.[\u25aa\u25aa] reported an analysis of German nationwide database which assessed data on the clinical presentation, metabolic syndrome components and inflammation variables in MDD patients. Regarding insulin levels, results showed significant associations between higher insulin and increased appetite, hypersomnia, insomnia and suicidal ideation. In sum their work unraveled several symptom-marker connections between: higher metabolic markers and increased appetite; lower metabolic markers and decreased appetite; lower metabolic markers and insomnia; higher insulin and increased appetite; higher insulin and lower albumin and insomnia. In line with the findings of the Netherlands Study of Depression and Anxiety [\u25aa\u25aa], their work reinforced the links between metabolic markers and atypical presentation of MDD and the observation, that those relationships are mainly noted for insomnia rather than hypersomnia. Similarly, in a cross-sectional analysis of primary care population Shell et al. reported that IR is only elevated during acute MDD episodes, while comparisons of patients with remitted MDD and general population indicated no significant differences. Another work comparing subjects with current MDD, remitted MDD and controls also concluded, that IR was significantly higher in acute MDD vs. controls [et al.[\u25aa\u25aa] noted that IR decreased in patients who experienced symptomatic improvement. They also noted that while baseline C-reactive protein (CRP) levels had a significant effect on the changes in depression severity it was fully mediated by IR. A study comparing IR in normal weight, nondiabetic adolescents included MDD and euthymic participants. It found associations between MDD and IR, which became insignificant after controlling for age, sex and BMI. However, the study sample was small (n\u200a=\u200a196) and less than a fourth of subjects presented IR (defined as HOMA >2.5), therefore the study was potentially underpowered to detect IR related differences [The above-mentioned meta-analysis by Fernandes et al.\u25aa\u25aa reportcontrols . Rashidis [et al.\u25aa\u25aa noted mall n\u200a=\u200a6 and lesferences .et al.[\u25aa\u25aa] published a cross-sectional study exploring the links between IR and depression status in obese individuals without diabetes, not medicated with antidiabetic drugs. They observed, that in female subjects IR (defined as 4th quartile vs. 1st\u20133rd quartiles HOMA that is >5.5) was significantly associated with mild, moderate or severe depression, with higher depression severity being related to higher risk of IR. To the contrary, no significant link between IR and depression was noted in male participants. \u0141api\u0144ska et al.[et al.[\u25aa\u25aa] performed a longitudinal assessment of links between baseline IR and depressive symptoms measured biennially for 8\u200ayears. The results indicated that high IR (4th vs. 1st quartile of studied population) was associated with progression of depressive symptoms. This relationship was significant in subgroups of men, overweight and elderly subjects but not in women, participants with normal BMI, those aged <65\u200ayears.He et al.\u25aa\u25aa publiska et al. studied l.. On the other hand, Gruber et al.[\u25aa\u25aa] summarized data coming from animal and human trials exploring the impact of insulin and IR on dopaminergic transmission. Based on the animal studies they have concluded that (a) insulin differently modulates dopamine release, increasing it if applied in the striatum and inhibiting it if applied in the ventral tegmental area (VTA), (b) insulin deficiency and resistance limits the synthesis and reuptake of dopamine as well as the excitatory stimulation and firing frequency of dopaminergic neurons in VTA, (c) restricted insulin signaling/IR in the nucleus accumbens reduces dopamine release and reuptake, (d) IR is linked to increased food intake, motivation to work for food, dampens reward sensitivity, impairs learning and preference formation as well as promotes behavioral despair resulting in anhedonic/anxious phenotype. Moreover, they summed human studies noting that (i) peripheral IR is a surrogate measure of central IR, (ii) intranasal insulin alters the activity and connectivity of dopaminergic circuitry and modulates reward behavior, (iii) the effects of intranasal insulin on reward processing differs with its dose, with low and high doses increasing the thresholds for reward the most, (iv) IR disrupts the reactivity to food cues, learning and motivation for reward (the last association is highly related to the state of satiety/hunger). While the relationship between high BMI and dysregulation of reward processing of food cues and intake is well established, a study exploring the effects of intranasal insulin on functional connectivity of the dopaminergic midbrain in humans reported, that the, these alterations are better predicted by IR rather than BMI [The alterations of serotonin, dopamine transmission are known to induce depressive symptoms. In an animal study, Martin the drug \u25aa\u25aa. On ther et al.\u25aa\u25aa summarthan BMI .et al.[et al.[\u25aa\u25aa,Surprisingly, IR seems to differently moderate treatment outcomes depending on its modality. In the earlier mentioned work by Brouwer et al., the trel.[et al.\u25aa\u25aa,26 obset al.\u25aa\u25aa,\u25aa. Also, et al.\u25aa\u25aa,. On the et al.\u25aa\u25aa,.et al.[et al.[et al.[et al.[Moulton et al. performel.[et al. publishel.[et al.. Lucianol.[et al.\u25aa reportel.[et al.\u25aa assesseIn sum, latest research suggests that a metabolic subtype of MDD can be distinguished from other MDD populations by the presence of particular pathophysiological mechanisms, symptomatology and responsiveness to treatment. IR seems to play an important role in this metabolic MDD subtype and it is positively linked to atypical depressive symptomatology. Insulin has a direct effect on the serotoninergic and dopaminergic neurotransmission which becomes disrupted by IR. The relationship between IR and MDD seems restricted to current depressive states of either clinical of subclinical severity and it differs between sexes, ethnicities normal/overweight subjects. Moreover, IR limits the efficacy of antidepressant drugs but light therapy might be more effective for subjects with IR than insulin sensitive ones. Insulin-sensitizing treatments might be beneficial for MDD patients, with most robust data supporting the addition of pioglitazone or interventions promoting healthy lifestyle and physical activity. Studies exploring the role of IR in MDD need replication in larger groups/patients of different ethnicities/in various metabolic states and so do trials verifying the potential efficacy of insulin-sensitizing treatments in MDD. Existing data already allow for a step towards personalizing therapeutic approaches and may inform clinical decisions as well as future research for more individualized and efficacious MDD treatment.None.None.There are no conflicts of interest."} +{"text": "Marek\u2019s disease virus (MDV), an Alphaherpesvirus belonging to the genus Mardivirus, causes T cell lymphomas in chickens and remains one of the greatest threats to poultry production worldwide. While losses caused by Marek\u2019s disease have been reduced through live-attenuated vaccines, field strains have increased in virulence over recent decades. MDV research has led to a profound understanding of virus-induced pathogenesis and tumor development ,2,3. OurA timely summary of the major methods of manipulating herpesvirus genomes was provided by Liao et al., with an emphasis on their applications in MDV research. The authors reviewed both the \u201ctraditional\u201d methods, such as site-directed mutagenesis and the use of cosmids, and more recent methods, like bacterial artificial chromosomes (BACs) and CRISPR/Cas9-mediated genome editing . The samYou et al. reported the characterization of a new splice variant of viral interleukin 8 (vIL-8), a chemotactic cytokine, showing that this new vIL-8 isoform is dispensable for MDV replication and pathogenesis .Two papers addressed pressing questions in MDV research using CRISPR/Cas9 approaches: the switch between lytic and latent MDV infections and the use of gene technology to generate MDV-resistant chickens. Roy et al. used CRISPR activation (CRISPRa) of viral genes to investigate the lytic\u2013latent switch. This switch seems to rely on the two MDV phosphoproteins pp24 and pp38 [In an attempt to improve MDV vaccines in the context of immunosuppression commonly caused by MDV and its widely used vaccines, Conrad et al. generated genetically modified vaccine candidates and assessed their vaccine-mediated protection and immunosuppressive effects .Ikaros cancer driver gene can contribute to MDV-induced tumorigenesis. Similar mutations also exist in some human cancers, suggesting that somatic mutations may explain the origin of MDV-induced lymphomas [Three contributions highlight the value of MDV infections in chickens as a model for human diseases. Erf et al. used a Mardivirus in vivo model to gain new insights into vitiligo, a chronic autoimmune disorder . A genomymphomas . Gennartymphomas .Finally, the methods paper on a cell culture system to study MDV integration by You and Vychodil et al. provided a method that allows the investigation of molecular mechanisms of MDV telomere integration, as well as the quantification of virus genome integration, without requiring laboratory animals .Although much progress has been made in MDV research and a growing body of knowledge about this DNA tumor virus exists, a number of challenges need to be overcome. Through this Special Issue, we hope to contribute to a better understanding of MDV as an important poultry pathogen, but also as a valuable animal model for herpesvirus research, virus\u2013host interactions, and virus-induced tumorigenesis."} +{"text": "Glioblastoma (GBM) is the most lethal and prevalent primary brain tumor in adults with a five-year survival rate of only around 5 percent, afflicting about 3.19 people in every 100,000 . It is wEpigenetics and epitranscriptomics\u2014molecular changes of cellular DNA, chromatin, and RNA\u2014can alter the levels of gene transcripts and subsequently change the amount of proteins produced, including those responsible for tumor suppression, progression, or maintenance . Until nIn the early 2000\u2019s, Helen Blau et.al., introduced an evolving concept for adult stem cells suggesting that stem cells is rather a biological function instead of an entity . AccordiYu et\u00a0al., combined analysis of transcriptome and DNA methylation profiles of TCGA to derive a 6-gene model-based risk score. This approach allowed the authors to predict survival and the role of personalized treatment plans for GBM. Wang et\u00a0al., analyzed tumor associated fibroblasts infiltration by Estimating the Proportion of Immune and Cancer cells (EPIC) based on multiple glioma databases. They describe a novel transcript signature that predicts glioma prognosis. Shi et\u00a0al., showed that individual glioblastoma cell lines displayed increased expression of the short splice variant of YKL-40 after low serum treatment. In addition, unlike the full-length (FL) version, which was localized to all cell compartments, the short isoform could not be secreted and was localized only to the cytoplasm. Functionally, FL YKL-40 promoted cell proliferation and migration, whereas SV YKL-40 suppressed them. The authors described pathways that regulate the expression of the SV YKL-40 and discuss the significance for development of new therapies. Majc et\u00a0al., investigated the use of bioactive peptides from venoms as novel therapies targeting cancer specific pathways in glioblastoma. Finally, Basilico et\u00a0al., revealed that changing the rigidity of the mechanical environment tuned the response of glioblastoma cell lines through change in morphological features, epithelial-mesenchymal markers , EGFR and ROS expressions in an interrelated manner. Their work highlights the importance of microenvironment stiffness in the regulation of glioblastoma invasive properties.This Research Topic includes 5 original research papers. NT wrote the initial draft of the editorial. H-WL edited and both agreed on the final version."} +{"text": "In the last decades, complex networks have been used extensively in neuroscience and other fields by employing networks to model interactions among system\u2019s components . MoreoveA common research theme in the study of complex networks is that of the determination of the role and impact of network topology on the emerging properties due to interactions and dynamics. How structural properties in brain regions affect neural dynamics or how do they differ in healthy and diseased subjects? How different types of plasticity affect the dynamics of neural activity in a network?This Research Topic contributes to better understand the impact of dynamics at different spatiotemporal scales, structure and plasticity on brain synchronous activity. To this end, it hosts interdisciplinary analytical and computational works from different fields, such as from complex systems, biophysics, systems biology, and computational neuroscience.Liu et al., aims to better understand the effect of the amyloid \u03b2 peptide (A\u03b2) which is hypothesized to be the major factor driving Alzheimer\u2019s disease (AD) pathogenesis. To this end, the authors derived and investigated a concise mathematical model for A\u03b2-mediated multi-pathway astrocytic intracellular Ca2+ dynamics. They investigated several interventions, such as ion channel blockers or receptor antagonists and demonstrated that a \u201ccombination therapy\u201d targeting multiple pathways simultaneously is more effective towards the treatment of AD.The first paper, by Cambell et al. investigated the fractal dynamics of blood oxygen level-dependent (BOLD) signals during naturalistic conditions. They implemented fractal analysis to quantify scaling behavior using the Hurst exponent (H) in data from the Human Connectome Project to compare H values across movie-watching and rest. Their results showed that movie-watching induces fractal signal dynamics and markedly different than dynamics observed during conventional tasks.van der Vlag et al. introduced a new computational tool called RateML that enables users to generate whole brain network models from a succinct declarative description, in which the mathematics of the model are described without specifying how their simulation should be implemented. With RateML, the end user describes the model\u2019s mathematics once and generates and runs code for different languages/platforms, targeting both CPUs for fast single simulations and GPUs for parallel ensemble simulations .Lei et al. introduced a new type of burst-oscillation mode (BOM), i.e. an alternating transition between two distinct phases . BOM was derived by extensively investigating the response dynamics of a one-dimensional (1D) paced excitable system with unidirectional coupling. These findings may facilitate a deeper understanding of bursts in nature and will have a useful impact in related fields.Shi et al. proposed a deep attributed network representation learning with community awareness (DANRL-CA) framework and two variants, i.e., DANRL-CA-AM and DANRL-CA-CSM, which incorporate the community information and attribute semantics into node neighbours with different methods. They designed a neighbourhood enhancement autoencoder module to capture the 2-step relations between node pairs. They conducted comparisons for node classification and link prediction and found that DANRL-CA-CSM can more flexibly coordinate the role of node attributes and community information in the process of network representation learning, and shows superiority in the networks with sparse topological structure and node attributes.Madadi Asl et al. employed a computational model to show that inhibitory spike-timing-dependent plasticity (iSTDP) at pallido-subthalamic synapses can account for pathological strengthening of pallido-subthalamic synapses in Parkinson\u2019s disease (PD) by further promoting correlated neuronal activity in the globus pallidus (GPe) - subthalamic nucleus (STN) network. Their results may shed light on how abnormal reshaping of GPe-STN connectivity by synaptic plasticity during parkinsonism is related to PD pathophysiology and contribute to the development of therapeutic brain stimulation techniques targeting plasticity-induced rewiring of network connectivity.Zheng et al. studied the emergence of spatiotemporal patterns in a general networked Hindmarsh-Rose (HR) model. Furthermore, they investigated stability properties, namely they obtained conditions leading to Turing instability in networks without delays and showed that there is a difference between the collected current and the outgoing current affecting the neuronal activity, which is relevant in the generation mechanism of short-term memory.Li et al. investigated the dynamics of networks of identical coupled oscillators in a setting where coherent oscillations coexist with incoherent ones, the so-called chimera state. They showed that stable and breathing chimera states in the original two coupled networks typically have very small basins of attraction. Then, they studied the emergence of chimera states by stimulating brain regions and quantified it using the order parameter and chimera index. These two indices were found to be weakly and negatively correlated."} +{"text": "Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmia syndrome characterized by defective cardiac ryanodine receptor (RyR2) calcium release during times of adrenergic stimulation, resulting in bidirectional or polymorphic ventricular tachycardia. Flecainide is a class 1c anti-arrhythmic drug that has demonstrated therapeutic efficacy in treating CPVT. However, its mechanism of action remains disputed. One group proposes a direct effect of flecainide on RyR2-mediated calcium release, while another proposes an indirect effect via sodium channel blockade and modulation of intracellular calcium dynamics. In light of recent studies, this commentary aims to explore and discuss the evidence base for these potential mechanisms. Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmia syndrome characterized by adrenergically mediated polymorphic or bidirectional ventricular tachycardia, leading to syncope and sudden cardiac death. The most common genetic mutations resulting in CPVT are found within genes responsible for coding the cardiac ryanodine receptor (RyR2). These are usually inherited in an autosomal dominant manner and are termed CPVT1. A recessive form (CPVT2) is caused by gene mutations for cardiac calsequestrin. CPVT is linked to seven genes with autosomal dominant or autosomal recessive inheritance [This results in a transient inward current, perpetuating the onset of malignant arrhythmias via delayed afterdepolarizations (DAD) and triggered activity. The first-line drug therapy for CPVT is \u03b2-adrenergic receptor blockers. However, some patients are refractory and experience persistent tachyarrhythmias. The class 1c antiarrhythmic drug flecainide, a known inhibitor of the sodium current (INa), has demonstrated effectiveness in treating CPVT; however, the mechanism responsible for the therapeutic efficacy of flecainide in CPVT remains disputed.Gaining a comprehensive mechanistic understanding of the clinical efficacy of flecainide in CPVT is critical for cases of arrhythmias that may share similar clinical presentation but differ significantly in their underlying mechanisms. Furthermore, abnormalities in calcium handling, including defective RyR2 functioning, are implicated in several other diseases of significant global burden, including muscular dystrophy, malignant hyperthermia, and Alzheimer\u2019s disease. Consequently, RyR2 is a major therapeutic target, and the development of new drugs targeting RyR2 carries significant clinical potential for treating CPVT and other diseases.et al. [Hilliard et al. comparedet al. [In a recent study, Kryshtal et al. investiget al. [Other studies proposed contrasting mechanisms explaining wave suppression. Liu et al. examinedet al. [In adult rat cardiomyocytes, Sikkel et al. conducteet al. [Bannister et al. investiget al. [et al. [Several factors should be considered to account for the varying results of flecainide on SR Ca2+ release via RyR2, which suggest a direct effect in some studies and no eet al. demonstr [et al. only appet al. [et al. [Yang et al. used in [et al. . As estaThe debate regarding the primary mechanism responsible for the therapeutic efficacy of flecainide in CPVT remains highly disputed and unresolved. Numerous studies propose INa-mediated modulation of intracellular calcium dynamics, while several others propose direct RyR2 inhibition as the primary mechanism. Moving forward, the mechanisms of flecainide in CPVT will increase our knowledge of Ca2+ dysregulation in cardiac myocytes and aid in the development of a more specialized therapeutic approach for CPVT."} +{"text": "Exercise and lifestyle modifications have established themselves as the pillars of primary care, proving to be potent weapons in the war against non-communicable diseases. With the World Health Organization (WHO) naming physical inactivity as a leading risk factor for global mortality , it is cThere is extensive research highlighting the wide-ranging benefits of physical activity, spanning from mitigating the risks of cardiovascular diseases, diabetes, and stroke, as well as mental health conditions includinConsidering this, primary care professionals across the world are progressively incorporating exercise-based lifestyle modifications, particulAddressing the evaluation of physical activity within the context of primary care practice presents a noteworthy challenge . The impIn addition, adherence rates continue to fall below satisfactory levels, underscoring the imperative need for the development of strategies aimed at bolstering acceptance and participation . CentralZhang et al.; Hu et al.; Liu Y. et al.; Cheng et al.; Mainous et al.; Lin et al.; Wattanapisit et al.; Liu Z. et al.; Feng et al.; Felemovicius et al.; Heyn et al.).Furthermore, illustrating instances of successful integration of exercise recommendations within real-world settings can serve as practical models for future endeavors . CollaboZhang et al. in their paper \u201cExercise for Neuropathic Pain: A Systematic Review and Expert Consensus\u201d emphasize that a proper exercise program can serve as an effective alternative treatment or complementary therapy for most patients with neuropathic pain (NP). This consensus provides actionable recommendations for clinicians and policymakers alike in formulating exercise prescriptions to treat NP.Hu et al. evaluated all-cause mortality and cardiovascular mortality in the Guangzhou Heart Study (GZHS), an ongoing prospective population-based cohort study in South China. Specifically, they investigated the effect of the Healthy Lifestyle Index and lifestyle patterns on the risk of mortality in a large Chinese population. Their study results suggest that accumulative dimensions of a healthy lifestyle can lower the risk of death, and adherence to the healthy lifestyle pattern was associated with non-smoking and low-level alcohol consumption which reduces the risk of all-cause mortality. These findings highlight the need to consider multi-dimensional lifestyle approaches when developing health promotion strategies.A population-based study by Efficacy of electro-acupuncture in postpartum women with diastasis recti abdominis: A randomized controlled clinical trial\u201d by Liu Y. et al. evaluated the long-term efficacy and safety of electro-acupuncture (EA) in treating diastasis rectus abdominis (DRA) during postpartum. The study results showed that AE treatment improved multiple health parameters and symptoms related to Diastasis Recti Abdominis (DRA), displaying lasting effects up to 26 weeks postpartum.The clinical trial \u201cCheng et al. investigated the effectiveness and response of a multidisciplinary Workplace health promotion (WHP). They used a retrospective cohort sample of healthcare workers participating in a multidisciplinary WHP program in five healthcare facilities. The 20-week intervention included exercise classes, nutrition consultation, and behavioral education followed by anthropometrics, body composition, and physical fitness (PF) measures. Their study results demonstrated that a multidisciplinary WHP could enhance anthropometric and physical fitness profiles among healthcare workers.Furthermore, Mainous et al. evaluated the relationship between depression-high depressive symptomatology and adherence to lifestyle interventions among patients with prediabetes. They analyzed the 2017-20 20 National Health and Nutrition Examination Survey (NHANES), a nationally representative population of U.S. adults. Their findings showed that depression-high depressive symptomatology decreases the likelihood of adherence to exercise-based lifestyle recommendations among patients with a confirmed diagnosis of prediabetes underscoring the interplay between mental health and lifestyle changes.Lin et al. conducted a randomized controlled trial with 66 full-term infants with eczema randomly assigned to an eczema control (EC) group and an eczema with MPIM (EM) group as compared to a healthy control (HC) group. The mothers in the EC group received the instruction of routine care, while the mothers in the EM group applied massage on the infants plus receiving the same instruction of the routine care. HC group received no specific intervention. Compared with the EC group, the EM group showed significantly lower scores on eczema outcomes supporting the reduction of infantile eczema, along with relieving maternal anxiety and depression.A randomized trial by Wattanapisit et al. highlights the importance of training, integrating, and applying knowledge and skills pertaining to PA promotion in clinical settings with the goal of covering several aspects of sport and exercise science. They remind us that PCPs are usually not specialists in these subject areas, and it is essential to supplement and provide knowledge for proper clinical practices. They include the collective perspective of experts in the field.An intriguing opinion paper by Liu Z. et al. conducted a systematic review and meta-analysis to evaluate the effects of a traditional Chinese Qigong therapy called Baduanjin which is characterized by symmetrical body posture and actions, breathing control, meditative state, and concentration. They reviewed randomized controlled trials evaluating Baduanjin therapy's effects on neck pain and functional movement in older individuals. Although the results of the synthesis methods support Baduanjin therapy as a safe and positive treatment for neck pain in older individuals, they called for caution as more studies are needed to validate and support its benefit.Feng et al. through a population-based study in Taiwan, investigated whether physical activity (PA) is associated with a reduced risk of hemorrhagic stroke (HS). Their study supports the beneficial effect of PA on reducing HS risk. However, high-PA did not appear to have a greater protective effect than low-PA in diabetes and hypertension outcomes. Thus, their conclusions support that even <90 min of PA per week might be beneficial in reducing HS risk. They also note that recommendations based on low PA levels are more likely achievable and sustainable across the general population. Additionally, personalized recommendations, based on pre-existing comorbidities, may help optimize the beneficial effects of PA on HS prevention.Felemovicius et al. evaluated a novel composite topical Lidocaine agent treatment for Pruritus ani, or rectal or anal itch, which is a common perianal disorder that affects approximately five percent of the population of the developed world. The SOOTHER Trial showed efficacy in providing rapid and effective relief of pruritus ani in an ambulatory population.The SOOTHER Trail by Heyn et al.) from the University of Colorado School of Medicine and Colorado Children's Hospital underscores the importance of lifelong monitoring of children with disabilities to identify and modify disease-induced risk factors through lifestyle interventions. Their study, \u201cThe association between isometric strength and cognitive function in adults with cerebral palsy,\u201d supports using simple tests like hand grip strength to evaluate early signs of frailty and neurocognitive decline in adults with Cerebral Palsy.Finally, a study by a research team , it becomes evident that they underscore the pivotal significance of exercise and lifestyle modifications within primary care. Through their findings, they shed light on the multifaceted roles of these interventions across various health contexts. As we continue to navigate the intricacies involved in implementing efficacious exercise prescriptions and clinical physical activity recommendations in primary care settings, these studies serve as valuable resources that enrich our comprehension and improve our ability to promote healthier lifestyles effectively for diverse patients within the realm of primary care.In summary, when considering the results of the studies from this Research Topic collectively (PH: Writing\u2014original draft, Writing\u2014review and editing."} +{"text": "HormoneWang et. al.), but its regulatory network in many non-model fruits is not yet fully understood. Li et\u00a0al. investigated the regulation of ethylene in mango fruit ripening, using ethylene and 1-methylcyclopropene (1-MCP), the action inhibitor of ethylene.They found that MiERF2 and MiERF8 may play critical roles in regulating mango fruit softening. Wang et\u00a0al. reported that blueberry had an atypical climacteric ripening process, where a respiratory climacteric character was developmentally regulated by ethylene, but with non-autocatalytic ethylene production during fruit ripening. Except for ethylene, increasing evidence show that other hormones such as auxin, abscisic acid (ABA), brassinosteroids (BRs), gibberellins (GAs), melatonin (MT) and cytokinins (CKs) control fruit ripening . It is generally considered that ethylene is the key actor of climacteric fruit ripening and ABA is the core player of non-climacteric fruit . However, increasing evidences have shown that fruit ripening is regulated by a balance of multiple hormones rather than by a single one, and the crosstalk between ethylene and ABA signaling plays a leading role in regulating fruit ripening. In climacteric fruits, ABA usually accumulates before the initiation of climacteric period that can promote ethylene biosynthesis via upregulating ethylene biosynthesis related genes and accelerating ethylene-triggered ripening plays a negative role in fruit ripening process (Liu et\u00a0al. also found that CK suppressed the loss of total sugar and sucrose and the accumulation of acetic acid and ethanol in the fruit aril, reduced the malondialdehyde accumulation in the pericarp of the overripe litchi fruit. Cytokinin treatment repressed the litchi fruit ripening process and maintained fruit quality in the on-tree overripe fruit. However, more information is needed about the interaction of CKs with other hormones.GA works as a negative regulator of fruit ripening, which inhibits the ethylene biosynthesis and perception during fruit ripening of tomato . SA regu process . The app process . Liu et\u00a0In conclusion, the exciting new findings presented in the present topic collection and other works provide a broad and in-depth understanding of hormone signal interactions in regulating fruit ripening. It is believed that increasing new knowledge will emerge in this exciting and evolving field.XZ and QZ wrote the manuscript, HZ edited and reviewed the manuscript. All authors contributed to the article and approved the submitted version."} +{"text": "Skeletal muscles are an indispensable actor for daily activities, playing an essential role in locomotion through both the control of posture and position and by joint stabilization. In addition, they are involved in body temperature maintenance and constitute nutrient reserves. Skeletal muscle represents approximately 40% of human body weight, but this percentage physiologically decreases when tissue undergoes atrophy. During aging, a decline in muscle strength and mass can be observed and muscle mass decreases by approximately 3\u20138% per decade after 30 years of age and this reduction appears significant after 60 years . Muscle With regard to sarcopenia, Shin et al. (2023) discusseIn their paper, Jeun and Choung (2023) describeAnother high-quality protein that contributes to the maintenance of skeletal muscle mass is egg white protein (EGG), an arginine-rich protein source with a lower amount of leucine. Koshinaka et al. (2023) demonstrated that EGG supplementation in rats had a higher efficiency in muscle gain compared to other common animal-based proteins .Continuing the discussion of dietary protein as an important strategy to manage and prevent sarcopenia, Lees et al. (2023) , in theiThis Special Issue also includes a paper evaluating rosemary leaf extract activity in primary skeletal muscle cells . RosemarAmong the original articles published in this Special Issue, Salucci et al. (2023) describeFinally, this Special Issue contains two reviews that recognize nutritional interventions as an important strategy to manage and prevent skeletal muscle loss. In this regard, Massini and colleagues (2023) discusseNutrients, titled \u201cNutrition and Regulation of Muscle Protein Synthesis\u201d, highlights the strategic role of nutritional approaches, based on supplementation with vitamin (B6), herbal extract (GBE), phenolic compounds , and high-quality dietary proteins , in the prevention of muscle mass loss associated with diseases or aging.In conclusion, this Special Issue in The Guest Editor warmly thanks all of the authors for their contributions to this Special Issue."} +{"text": "Klebsiella variicola strain during a laboratory evolution study, resembling genetic changes observed in clinical isolates.The appearance of colony morphotypes is a signature of genetic diversification in evolving bacterial populations. Colony structure highly depends on the cell\u2013cell interactions and polymer production that are adjusted during evolution in an environment that allows the development of spatial structures. Nucci and colleagues describe the emergence of a rough and dry morphotype of a noncapsulated Klebsiella variicola.Commentary on the paper by Nucci et al that explains the genetic basis and fitness effects of specific mutations leading to rdar-like colony morphotypes in noncapsulated IS elements drive rapid loss of K. pneumoniae capsule production in experimentally evolved population (Nucci et al. Bacillus thuringiensis, where an IS4-like element disrupts a gene encoding a guanylyltransferase, causing increased hydrophobicity and aggregation (Lin et al. B. thuringiensis fuzzy morphotypes, K. variicola rdar-like derivatives display diminished aggregation (Nucci et al. In their recent ria Fig.\u00a0. While tK. variicola display increased growth rate and fitness advantage compared with the ancestor (Nucci et al. The rdar-like clones of K. variicola background was used, while colonies with distinct morphology were less apparent in capsulated strains that followed a different evolutionary path (Nucci et al. mrkH or nac alleles demonstrated that these mutations convey lower influence on cell\u2013to\u2013cell aggregation in the capsulated background and the fitness effects were only marginally larger in capsulated strains compared to noncapsulated strains. Historical contingency, where the existing mutations influence the subsequent adaptation path might explain the larger rdar-like morphotype frequency in noncapsulated K. variicola. This highlights the opportunity to discover novel evolutionary paths in strains lacking the most frequently mutated targets. Deleting the three most frequently mutated pathways in Pseudomonas fluorescens and subsequent experimental evolution in a static microcosm revealed 13 new mutational pathways that all result in wrinkly spreader colony morphotype (Lind et al. B. subtilis permits the evolution of clones with enhanced biofilm formation, explained by the production of novel, cysteine containing amyloid fibre variants (Drago\u0161 et al. P. fluorescens example, the reconstituted B. subtilis strains with cysteine-encompassing amyloid fibres convey a disadvantage in the presence of the exopolysaccharide (Drago\u0161 et al. The rdar-like morphotype was detected after plating by Nucci and colleagues when a nK. pneumoniae genomes revealed comparable IS element insertion in the mrkH gene of numerous clinical isolates. Isolates with interrupted mrkH genes were mostly originated from human host samples, including urine and blood as the main source (Nucci et al. Klebsiella isolates, potentially explained by the negative frequency selection of these mutations. The detection of specific mutations or gene disruptions in natural populations further validate the relevance of experimental evolution studies in the laboratory settings as previously reported in Klebsiella (Nucci et al. Finally, the work by Nucci et al. highlights the relevance of laboratory evolution for real-life scenarios. Detailed analysis of The study of Nucci et al. highligh"} +{"text": "Ralstonia solanacearum is one of the most devastating agents affecting different plants in 45 families. Several management practices have been used effectively for the management of this bacterium in different plants, including the use of biological control agents . However, because of difficulties in handling, culturing, and maintaining biocontrol agents or the issues related to their practical efficacy in soil, the use of their antimicrobial compounds is considered a good alternative. Ye et\u00a0al. investigated an antibacterial furoic acid compound, 5-(hydroxymethyl)-2-furoic acid, produced by the fungus Aspergillus niger. The soil application of this compound effectively controlled the R. solanacearum population in soil by direct killing effect as well as enhancing host resistance in tomato plants. Wang et\u00a0al. reviewed other studies for the management of this bacterium, including biological, organic, breeding, genetic engineering, physical, cultural, chemical and nano-technological approaches.Soil-borne plant pathogens pose a significant threat to plants, and their management is comparatively more difficult than other plant pathogens because of their nature of the hidden enemy, long persistency in soil, vast genetic diversity and host range . HoweverDutta et\u00a0al., in their review, focused on several aspects of the use of nanotechnology in the management of soil-borne plant pathogens highlighting the role of nanoparticles as protectants and inducers of plant defense against soil-borne plant pathogens. Uncovering the mechanism of host resistance and discoveries of host genes/proteins that regulate host resistance against soil-borne plant pathogens will help plant genetic engineers develop plants that can better fight against soil-borne plant pathogens. Tian et\u00a0al. reported that small G-protein StRab5b positively regulates potato resistance against the soil-borne fungus Phytophthora infestans. Pazarlar et\u00a0al. found that Bacillus cereus EC9 protects tomatoes against Fusarium wilt through JA/ET-activated immunity. In another study by Ling et\u00a0al., it was shown that WRKY genes confer resistance to Cucumis metuliferus against root-knot nematodes (RKN). The RKN-resistant variety of C. metuliferus (cm3) was found to specifically recruit beneficial bacterial communities in soil upon infestation with RKN, as reported by Song et\u00a0al. Similarly, Sikandar et\u00a0al. reviewed overall management strategies, including several recent efforts to improve host resistance against Meloidogyne enterolobii.Nanotechnology emerged as one of the most rapidly advancing sciences of the twenty-first century, and plant scientists revealed the effective application of nanotechnology for the management of plant pathogens. Dutta et\u00a0al. unveil the molecular mechanism by which Trichoderma fungus, one of the most used and studied biocontrol fungus, induce host resistance against pathogens including the role of exogenous elicitors in the induction of host resistance. Zhou et\u00a0al. proved that exogenous application of elicitor isotianil could significantly alleviate the symptoms of Fusarium wilt on the banana. They further confirmed that isotianil application might contribute to disease control by inducing host plant defense against Fusarium infection. Investigation into how the pathogens achieve virulence at the molecular level is also essential for understanding the action mechanism of the pathogen. Xu et\u00a0al. revealed the role of cAMP phosphodiesterase in the formation of sclerotia and achieving virulence in Sclerotinia sclerotiorum, an important phytopathogenic fungus that causes stem rot and white mold disease.Enhancing host resistance is also a prominent action mechanism of many biocontrol agents. In their review, Most investigations conducted for the management of soil-borne pathogens are based on greenhouse or controlled laboratory conditions. Although the outcomes of these studies provide useful insights into the action mechanism and control efficacy, field investigations are vital for assessing the practical applications of these strategies. Field assessments consider the complex interplay between plants, pathogens and soil, and environmental factors that can affect disease management and development. This more accurately illustrates how these strategies perform in real agricultural scenarios. Moreover, field assessment can give an actual clue of management strategy on economic feasibility.RK: Writing \u2013 original draft, Writing \u2013 review & editing, Conceptualization."} +{"text": "The 38th issue of Financial Innovation (FIN), Volume 9, No. 2 (2023) presents 23 papers contributed by authors and co-authors from nineteen countries and areas: Canada, Chile, China, France, Germany, Italy, Netherlands, New Zealand, Oman, Portugal, Romania, Russia, Spain, South Africa, Taiwan, T\u00fcrkiye, UK, USA and Vietnam. These papers can mainly be categorized into three sub themes. \u2022 Financial risk management and analysisG\u00f3mez-\u00c1guila et al. (2022) introduce a new procedure to improve the estimation of the Hurst exponent in financial time series and prove that pure price series are not necessarily self-similar. Yeh and Liu (2023) use visualization techniques to help explore the Growth Value Model (GVM) intuitively. De Blasis (2023) addresses the calibration issues of the weighted-indexed semi-Markov chain (WISMC) model applied to high-frequency financial data. Ivanyuk (2023) presents an optimization approach\u2014residual-based bootstrap averaging (RBBA) and shows why and how it works. Monteiro et al. (2023) examine the linear interdependencies between international industries, with a focus on the relationships between the US and other six countries, focusing mainly on international stock indexes or firm-level returns. Mo et al. (2023) present an adaptive measurement model of spatiotemporal correlation coefficients based on the clustering theory and conduct an empirical analysis based on the model. Loughran and McDonald (2023) find a slight positive correlation between industry performance and the use of pandemic-related words, as opposed to the expected negative correlation. Gupta and Pierdzioch (2023) prove that aggregate and state-level economic conditions can predict the subsequent realized volatility of oil price returns.\u2022 Behavior financeCheng et al. (2023) find that rumor propagation outperforms management shocks and other variables in predicting abnormal trading behavior. Wang and Lee (2023) chose three life insurance stock futures in India and one in Taiwan as samples and examine that insurance futures are a safe haven for COVID-19 pandemic risks. Dur\u00e9ndez et al. (2023) find that Management control systems (MCS) and managers\u2019 risk attitudes fully mediate the relationship between financial literacy (FL) and innovation. Alp et al. (2023).examine the presence of investor overconfidence by employing an artificial intelligence technique and a nonlinear approach to impulse responses to analyze the impact of different return regimes on the overconfidence attitude. Madeira (2023) demonstrates how households\u2019 characteristics affect their choice of lenders, consumer debt amounts and default behavior. Cruz et al. (2023) propose an approach to the profile of microcredit holders from the Hispanic minority in the US to identify the factors that explain the punctuality of their microcredit repayments. Zhou et al. (2023) develop a behavioral heterogeneous agent model (HAM) comprising fundamentalists, chartists, and stabilizers to investigate investors\u2019 dynamic switching mechanisms under government intervention.\u2022 Stock marketHtun et al. (2023) find that correlation criteria, random forest, principal component analysis, and autoencoder are the most widely used feature selection and extraction techniques with the best prediction accuracy for various stock market applications. Zhang et al. (2023) propose three novel strategies to address common issues in predicting high-frequency stock prices using machine learning methods. Cepoi et al. (2023) explore the impact of the COVID-19 pandemic on information asymmetry for the case of the Romanian capital market. Meng et al. (2023) examine the effect of stock market liberalization on market efficiency by using a recent stock market liberalization reform policy in China\u2014the Stock Connect\u2014as a quasi-natural experiment. Mensi et al. (2023) provide strong evidence of quantile dependence between gold and stock returns. Li and Chen (2023) conduct a theoretical analysis of low-price collusion in SBIC from the perspective of strategic interaction between institutional investors and the regulator. Mestre (2023) proposes a wavelets approach to estimating time\u2013frequency-varying betas in the capital asset pricing model (CAPM) framework. Carrillo-Hidalgo et al. (2023) develop a dynamic regression model to predict the behavior of shares in the Spanish tourism sector according to the evolution of the COVID-19 pandemic in the medium term. It is confirmed that both the number of deaths and cases are good predictors of abnormal stock prices in the tourism sector."} +{"text": "Neurological disorders are a large and heterogeneous field of research that can be tackled through a variety of approaches, ranging from epidemiology to molecular biology, through clinical, biostatistical, and laboratory experiments. The objective of this article collection was to gather the most recent evidence on signaling and genetic regulation of major neurological disorders . The articles included in the collection offer interesting insights into a range of biological processes involved in neurodegeneration. In addition, one contribution described an in silico approach to develop drugs for Parkinson\u2019s disease (PD). Barrera-Vaquez et al., 2023 sought tAlzheimer\u2019s disease (AD) is certainly the best known neurodegenerative disorder. It is characterized by deficits in multiple cognitive domains always including the memory domain and always involving neurodegeneration in the hippocampus. Several hypotheses have been proposed to explain the complex pathophysiology of AD, including altered neurogenesis and neuroinflammation. Olabiyi et al., 2021 [1\u221242, caspase 3, caspase 9, and cytosolic phospholipase A2. To further test their findings, the authors performed an in vitro experiment by exposing HT22 cells to a 12/15-LOX shRNA and a p38 MAPK inhibitor in high-glucose medium. The finding that the 12/15-LOX pathway plays a role in diabetic brain damage by activating p38 MAPK to promote inflammation and neuronal apoptosis, and that this mechanism could be inactivated by 12/15-LOX inhibitors is worth future research, perhaps not only limited to diabetic cognitive impairment.The role of lipid-mediated inflammation in neurodegeneration was evaluated by Chen et al., 2022 . The autIn their contribution, Zhang et al., 2022 studied Jang et al., 2022 studied Neurodegeneration does not necessarily result in neuronal death. Indeed, brain cancer development can be a consequence of a neurodegenerative process that leads to loss of function, immortalization of cancer cells, and eventually, death of the individual. Oncogenesis shares pathways with neurodegeneration. For example, Chuang et al., 2021 found thNeurodegeneration is highly influenced by lifestyle habits, including alcohol (ab)use. A deeper understanding of the genetic basis of the risk of alcohol use and tendency to abuse is highly sought after. Al-Sabagh et al., 2022 tested tWhich aspect of neurodegeneration should be addressed first?"} +{"text": "Xie et.al identify a homozygous missense variant in DND1 that causes non-obstructive azoospermia in humans . Aprea et.al. identify the pathogenic gene variants in CCDC39, CCDC40, RSPH1, RSPH9, HYDIN, and SPEF2 that cause defects of sperm flagella composition and male infertility. They find that immunofluorescence microscopy in sperm cells is a valuable tool to identify flagellar defects related to the axonemal ruler, radial spoke head, and the central pair apparatus . Yuan et al. finds a Gly684Ala substitution in the androgen receptor that causes azoospermia and Fang et al. reports the phenotypic findings and genetic considerations of congenital absence of the vas deferens with hypospadias or without hypospadias: , which might advance the genetic diagnosis and clinical genetic counseling for male infertility. Zhu et al. also verifies that FISH could analyze numerical chromosomal abnormalities in the sperm of Robertsonian translocation der carriers . Moreover, Wang et.al review various phenotypes resulting from different pathogenic genes, including sperm ultrastructure and encoding proteins with their location and functions as well as assisted reproductive technology outcomes, providing additional clinical views and broadening the understanding of this disease . Finally, Xu et al. reports a case of the birth of a boy after intracytoplasmic sperm injection using ejaculated spermatozoa from a non-mosaic KS man with normal sperm motility, which increases our knowledge of non-mosaic KS . The findings in these studies broaden our understanding of genetic factors in male infertility and were also included in our Research Topic scope.Our Research Topic is \u201cGenetic Factors in Male Infertility\u201d and the goal of this Research Topic is to collect more papers on the aspect of identifying different gene variants and various types of RNAs mainly focused on male infertility including azoospermia, oligozoospermia, teratozoospermia, and asthenozoospermia. In order to achieve this goal, we carefully reviewed every submitted manuscript and screened for highly qualified reviewers. Eventually, we accepted and published seven articles including four \u201cOriginal Research\u201d articles, one \u201cBrief Research Report\u201d article, one \u201cCase Report\u201d article, and one \u201cReview\u201d. These articles covered the genetic factors of non-obstructive azoospermia and teratozoospermia and the clinical outcomes of men with non-mosaic Klinefelter syndrome (KS)."} +{"text": "Despite increasing awareness of the ubiquity of microplastics (MPs) in our environments, little is known about their risk of developmental toxicity. Even less is known about the environmental distribution and associated toxicity of nanoplastics (NPs). Here, we review the current literature on the capacity for MPs and NPs to be transported across the placental barrier and the potential to exert toxicity on the developing fetus.This review includes 11 research articles covering in vitro, in vivo, and ex vivo models, and observational studies. The current literature confirms the placental translocation of MPs and NPs, depending on physicochemical properties such as size, charge, and chemical modification as well as protein corona formation. Specific transport mechanisms for translocation remain unclear. There is emerging evidence of placental and fetal toxicity due to plastic particles based on animal and in vitro studies.Nine out of eleven studies examined in this review found that plastic particles were capable of placental translocation. In the future, more studies are needed to confirm and quantify the existence of MPs and NPs in human placentas. Additionally, translocation of different plastic particle types and heterogenous mixtures across the placenta, exposure at different periods of gestation, and associations with adverse birth and other developmental outcomes should also be investigated.The online version contains supplementary material available at 10.1007/s40572-023-00391-x. Since the production of plastics accelerated after the early 1950s, approximately 6300 million metric tons of plastic waste have been generated worldwide, over three-quarters of which have been discarded in various environments .The most commonly used plastic polymers are high- and low-density polyethylene, polyvinyl chloride, polyethylene terephthalate, polypropylene, and polystyrene . PrimarySince MPs and NPs are known to be widely present in food, water, and air, humans are most commonly exposed through ingestion and inhalation routes . A commoThe toxicity of micro- and nanosized plastic particles likely depends on their physicochemical properties including size, shape, molecular structure, functionalization, surface charge, and material type . There iThe potential for wide-ranging health risks associated with MP and NP exposure combined with the heightened susceptibility to environmental agents during fetal development underscores the importance of investigating the maternal-to-fetal transfer of such particles across the placenta. The role of the placenta is to facilitate essential nutrient, gas, and waste exchange for the fetus, but it is not impervious to environmental toxicants. The placental barrier consists of the syncytiotrophoblast layer, cytotrophoblast cells, and the endothelial cell layer of the fetal capillaries see Fig.\u00a0 21\u2022]. T\u2022. T21\u2022].Existing reviews cover the health risks posed by MPs, NPs, and other nanomaterials including placental transport, but none focus solely on the transport of plastic particles across the placenta , 27,\u00a028.Articles were identified for review through a comprehensive literature search using PubMed and Web of Science databases. The search strategy utilized terms such as \u201cplacenta,\u201d \u201ctransport,\u201d \u201cparticle,\u201d and \u201cplastic\u201d with Boolean operators and Web of Science\u2019s Core Collection (N\u2009=\u2009435). A total of 146 duplicate articles were identified and removed using Endnote. Nineteen additional articles were retrieved from the reference lists of included reviews and other studies. Titles of all papers were screened, abstracts of 51 papers were screened, and 36 full-text papers were assessed for eligibility. Articles were excluded if they did not assess the transport of particles made of plastic across the placental barrier , if they did not explicitly investigate transport across the placental barrier, or if they were not primary research studies . The final selection of 11 primary research articles included 4 studies using in vitro human cell models, 2 in vivo animal studies, 1 study with both in vitro human cells and an in vivo animal model, 3 studies using ex vivo human placental perfusion models, and 1 observational study in humans. The study selection process is outlined in Fig.\u00a0The literature search was conducted in July 2021 and updated in February 2022. A total of 752 articles were identified using PubMed are provided for the translocation studies specifically. The reported \u201cmajor findings\u201d include results from toxicological investigations as well Table .Using an ex vivo human placental perfusion model, Wick et al. (2010) observed size-dependent maternal-to-fetal placental translocation of fluorescent polystyrene particles. After 180\u00a0min of perfusion, fluorescence measurements and transmission electron microscopy micrographs showed that beads sized 50, 80, and 240\u00a0nm were able to cross the placenta to the fetal compartment, while 500-nm beads did not \u2022. LeveraUsing BeWo b30 cells, Kloet et al. (2015) observed that translocation of 50-nm polystyrene nanoparticles was not dependent on the surface charge of the particles. Translocation of three types of 50-nm polystyrene NPs, one positively charged and two negatively charged, was assessed [protein corona. Using the ex vivo human placental perfusion model, Gruber et al. (2020) found that dynamic protein coronas influenced the translocation of plain 80-nm polystyrene nanoparticles across the placental barrier. First, the group showed the addition of plasma to the perfusion medium increased the maternal-to-fetal transfer of NPs [It is plausible that the interaction of plastic particle surfaces with various endogenous biomolecules affects particle transport. In biological fluids, proteins can rapidly cover the surface of nanomaterials forming a r of NPs \u2022. The cor of NPs \u2022. For exr of NPs \u2022. Using r of NPs \u2022. Media r of NPs \u2022. In allKloet et al. (2015) also investigated the role of specific transport mechanisms by assessing whether the distribution of NPs in the compartments changed in the presence of inhibitors of endocytosis and specific ATP-binding cassette transporters. They found that inhibitors of P-glycoprotein and breast cancer resistance protein had no effect on translocation of either NP and that an inhibitor of multidrug resistance protein 1 increased basolateral translocation of the positively charged NP and had no effect on the translocation of the negatively charged NP \u2022. OveralIn contrast, Grafmueller et al. (2015) posit that the translocation of polystyrene particles across the placenta mainly involves an active, energy-dependent transport pathway. The investigators used the ex vivo human placental perfusion model to assess the bidirectional transfer of both plain and carboxylate modified polystyrene particles with fluorescent labels 50\u2013300\u00a0nm in diameter \u2022. After Since there is accumulating evidence that plastic particles can cross the placental barrier, it is important to investigate the toxic potential of these particles. In the studies using ex vivo placental models by Grafmueller et al. (2015) and Wick et al. (2010), general measures of placental viability, functionality, and transfer were observed not to be affected by perfusion of plastic particles \u2022, 35\u2022. HIn different in vitro models, the human 3A-Sub-E trophoblast cell line and villous cytotrophoblasts derived from term placentas of normal pregnancies, Huang et al. (2015) did observe cytotoxicity of carboxylate-modified NPs. Following exposure to 20- and 40-nm particles in both trophoblast models, they observed elevated cleaved caspase 3, a marker of apoptosis induction, based on Western blot analysis \u2022. They aInterestingly, Hesler et al. did not observe transport across a placental BeWo b30/HPEC-A2 co-culture model at either concentration tested (10 and 100\u00a0\u00b5g/mL) after 24\u00a0h of exposure. The researchers used a different detection system than previous studies, asymmetrical flow field-flow fractionation (AF4), and noted the possibility of translocated particles being present below the limit of detection \u2022. Using Given some evidence of the toxicity of plastic particles on trophoblast and embryonic cell lines, the distribution of plastic particles in fetal organs after transport across the placenta warrants consideration. Kenesei et al. (2016) used an in vivo model to investigate the distribution of surface-modified fluorescent polystyrene nanoparticles after intravenous injection of pregnant mice. Using spectral imaging fluorescence microscopy, they found carboxylated NPs in the lacunas of the placenta 5\u00a0min after particle administration \u2022. ParticIn a pilot observational study, Ragusa et al. (2021) were the first to report MPs found in human placenta samples, confirming the placental translocation of plastic particles observed in ex vivo and in vitro models. The researchers obtained placentas from 6 patients in Italy with uneventful pregnancies \u2022\u2022. SamplEleven studies investigating the capacity for MPs and NPs to cross the placental barrier were reviewed. Placental translocation of plastic particles was observed in multiple experimental models including in vitro cell cultures, in vivo animal models, and ex vivo human placental perfusion systems as well as in an observational study of MPs in human placentas after delivery. One study conducted in an in vitro co-culture context and one study conducted in mice did not observe placental translocation of the plastic particles tested. Given the wide variety of systems used, it is important to weigh the strengths and limitations of the different models when comparing study results.While in vitro models are easier to obtain and utilize for high-throughput studies, they can be over-simplified compared to an in vivo environment. Promisingly, translocation of compounds across in vitro models has been shown to correlate well with the ex vivo human placental perfusion model \u2022, 39, 40In vivo studies of placental translocation of plastic particles in a whole-body system are valuable for understanding more realistic and comprehensive exposure conditions. As such, the study Fournier et al. (2020) conducted in rats provides insight into fetal deposition of NPs after maternal inhalation. Additionally, Kenesei et al. (2016) and Huang et al. (2015) investigated placental translocation of NPs in mice. However, extrapolation of findings from animal studies to humans is difficult due to significant interspecies differences in the placenta; rats show differences in placental morphology, yolk sac function, and steroid hormone biosynthesis, for example , 43.The ex vivo human placenta model used by Wick et al. (2010), Grafmeuller et al. (2015), and Gruber et al. (2020) provides advantages over animal models by more accurately representing the structure of the human placental barrier and over cell culture models by allowing for dynamic flow during exposure. The process of tissue degradation, however, limits experiments with these ex vivo models to a few hours \u2022. BecausThe current literature suggests that the placental translocation and toxicity of MPs and NPs depend on physicochemical properties such as size, charge, and chemical modification. Wick et al. (2010), Cartwright et al. (2012), and Aengenheister et al. (2018) showed size-dependent transport of polystyrene particles across the placenta, while Kloet et al. (2015) found that polystyrene particles of the same size had different translocation properties likely dependent on functional groups. Kloet et al. (2015) also showed that functional groups and charge affect the cytotoxicity of polystyrene particles on placental trophoblast cells. This has implications for the risk assessment of the developmental toxicity of MPs and NPs\u2014more characteristics modulating the translocation and toxicity of plastic particles justify the assessment of more variations and types. Furthermore, Gruber et al. (2020) showed dynamic protein coronas affect placental translocation of NPs, posing another variable in experimental risk assessment.Nine out of eleven studies examined in this review found that plastic particles were capable of placental translocation; observations of MPs in human placentas by Ragusa et al. (2021) make this consensus even more convincing. The mechanism that plastic particles use to cross the placenta remains unclear: Kloet et al. (2015) suggested it was passive diffusion and Grafmueller et al. (2015) suggested it was active transport. It is possible that these mechanisms are not mutually exclusive and the process depends on some combination of both.All of the experimental studies utilized mostly uniform, spherical, polystyrene particles. Polystyrene is widely used to package foods and has been found to be one of the most frequently detected plastic polymers in human blood \u2022. MPs anGiven the evidence that MPs and NPs can not only translocate across the placenta but also subsequently deposit in fetal tissues and exert toxicity \u2022, 34\u2022\u2022, Supplementary file1 (DOCX 17 KB)Below is the link to the electronic supplementary material."} +{"text": "Over the last few decades, great efforts have been dedicated to the discovery of various nanomaterials. Due to their unique optical, magnetic, and electrical properties (among others), they have found applications in medicine (drug delivery), agriculture, electronics, catalysis, etc. Thus, due to the increasing possibilities, the need to design and fabricate novel nanoparticles is rapidly increasing. In this Special Issue \u201cAdvanced Nanomaterials in Biomedical Application\u201d, a total of seventeen articles\u2014including five reviews\u2014have been published, addressing the most recent advances in nanomaterials in terms of both synthesis and characterization as well as technological applications. In the following, we provide a brief overview of the key findings presented in this Special Issue.50 values were more than 1000 times lower compared to cisplatin. Furthermore, the derivative with the highest activity cisplatin\u2013diacetyl caffeate conjugate and its MSNs induced apoptotic cell death by causing potent caspase activation. Moreover, in vivo studies conducted using BALB/c mouse models with breast tumors showed that the same compound and its MSNs exhibit tumor growth inhibition with a reduced necrotic area and lowered mitotic rate. The review paper by Spoial\u0103 and containing 72 Mo(VI) and 60 Mo(V), to the existing states of the human papillomavirus (HPV) major capsid protein, L1-pentamer (L1-p), and virus-like particles (VLPs). The color responses result from the different binding modes between [Mo132] and the capsid protein. This straightforward colorimetry approach is of importance to estimating the existing states of the HPV capsid protein and could be used in the future to analyze the quality of the HPV vaccine and the existing states of other viruses.The studies conducted by Xue et al. have shoMany studies have shown that CSC chemokine receptor 4 (CXCR4) is a promising target for cancer therapies, and intracellular siRNA delivery to suppress CXCR4 expression in cancer cells is an effective therapeutic strategy. Thus, Cao et al. synthesiMin and co-authors presenteThe work of Tyubaeva et al. exploresRen and coauthors summarizDing et al. designedIn their review article addressing SARS-CoV-2, a hot topic lately, Kianpout et al. gave an A new nano-emulsion adjuvant based on squalane containing CpG was prepared, and its in vivo properties were examined . The SNAS35A with poly acid (PLGA) nanoparticles, the selective delivery of cytotoxin to macrophages, in comparison to epithelial cells, was suggested. This drug delivery system presents anti-inflammatory effects.Harada et al., reported a useful technique for protein delivery to macrophages . By applIn the study by Wen et al. , osteoim"} +{"text": "They confirmed that [NiFe] large subunits dimerize in the absence of their small subunit (Hartmann et al., Kisgeropoulos et al. investigated the effect of amino acid exchanges of the primary proteinaceous proton acceptor in [FeFe]-hydrogenase. The data expand previous work (Cornish et al., Ka of the proton acceptor is one factor controlling the catalytic bias of the enzyme. Three further articles dealt with functional aspects of hydrogenases. Morra reviewed the identification and characterization of novel [FeFe]-hydrogenases, described their functional roles, and recent findings regarding their O2 tolerance. Kpebe et al. reported on the essential role of a bifurcating [FeFe]-hydrogenase in the sulfate reducer Solidesulfovibrio fructosivorans, showing that during ethanol oxidation, the hydrogenase confurcates electrons from NADH and reduced ferredoxin to produce H2, which is later on used for energy conservation using sulfate as electron acceptor. The function of a bidirectional [NiFe]-hydrogenase in Synechocystis sp. PCC 6803 was investigated by Burgstaller et al.. They showed that this hydrogenase is essential for growth on arginine and glucose in the presence of light and O2, and interestingly this function seems to be unrelated to H2 catalysis. Instead, an H2-independent role for the transfer of electrons into the photosynthetic electron transfer chain is proposed, which is an important hypothesis that may also be relevant for certain multisubunit hydrogenases in other species.This Research Topic comprises 11 articles that aimed to bring together recent advances in hydrogenase research, including structure, function, maturation, and application. In terms of structural investigation, Haase and Sawers investigated residues that contribute to [NiFe]-hydrogenase maturation. They found a histidine residue in a HypC-type chaperone that is important for efficient binding to its maturation partner HypD (Blokesch and B\u00f6ck, Fan et al. significantly improved the [NiFe]-cofactor incorporation in the course of heterologous production of a [NiFe]-hydrogenase from Cupriavidus necator in Escherichia coli. Previously, cofactor insertion was not functional, resulting in production of inactive hydrogenases (Fan et al., On the subtopic of hydrogenase maturation, Hogendoorn et al. isolated the novel strain \u201cCandidatus Hydrogenisulfobacillus filiaventi\u201d R50 gen. nov., sp., nov. which was characterized as a chemolithoautotrophic thermoacidophilic aerobic H2-oxidizing bacterium. This strain excretes about half of the fixed CO2 in the form of amino acids, which makes it a promising candidate for the industrial production of organic compounds from CO2, using H2 as energy source. H2 oxidation in this strain is facilitated by two [NiFe]-hydrogenases from the groups 1b and 1h, with the latter conferring high affinity toward H2 (S\u00f8ndergaard et al., Kobayashi et al. engineered the acetogenic bacterium Moorella thermoacetica for enhanced acetate, ethanol and acetone production. They found that H2 supplementation under mixotrophic conditions increases NADH levels but can inhibit growth due to an unbalanced redox status. This study emphasized the role of a reversible electron-bifurcating group A3 [FeFe]-hydrogenase for balancing the cellular redox state. These observations may also be exploited for biohydrogen production by that strain. Another way to produce H2 was analyzed by Barahona et al.. In order to enhance nitrogenase-driven H2 production in Rhodobacter capsulatus, they used a sensory hydrogenase coupled to the production of a fluorescence signal. By inducing genome-wide mutations and using high-throughput fluorescence-activated cell sorting, they were able to generate mutants with elevated H2 evolution capabilities. Finally, Schumann et al. reviewed current state-of-the-art and future perspectives for efficient light-driven H2 production using phototrophic microorganisms. This technology potentially enables conversion of solar energy into a chemical compound that enables storage and transport. However, as discussed by the authors, extensive engineering is required for this technology to be efficient and scalable.Another four articles explore the application of hydrogenases. 2-based biologically green technologies.Taken together, the Research Topic advances our knowledge especially on the function and application of hydrogenases and provides important perspectives for future HSF: Writing\u2014original draft, Writing\u2014review and editing. CP: Writing\u2014review and editing. FV: Writing\u2014review and editing. CG: Writing\u2014review and editing."} +{"text": "Metastasis and resistance to cancer therapeutics are critical barriers to curing cancer. This special issue entitled \u201cCancer Metastasis and Therapeutic Resistance\u201d contains nine original contributions. The articles span a variety of human cancers, including breast, lung, brain, prostate, and skin and touch upon significant areas of interest such as cancer stem cell function, cancer immunology, and glycosylation. This special issue contains one review article by Kilmister et al., which touches on the important topic of cancer stem cells (CSC) . These sSriratanasak et al. also tackle important aspects of CSC regulation in a research article that examines the impact of cisplatin-induced senescence on lung cancer cells . TypicalThis issue had a significant set of original research, tackling different aspects of breast cancer. The article by Barutello et al. examines a combination of methods to target the cystine-glutamate antiporter xCT and p53 using the drug APR-246, which restores the wild-type function of p53 in mutant p53 tumors . FindingThe research article by Cheng et al. looks more distinctly at guanylate binding protein 5 (GBP5) and its expression in triple-negative breast cancer (TNBC) . This stPrior studies have shown that fucosylation, a type of glycosylation, promotes metastatic phenotypes and signaling in breast cancer cells ,10. DoudRhosin is a RhoGTPase RhoA/C-yes associated protein (YAP) pathway inhibitor . TsubakiPenas-Mart\u00ednez et al. provided an interesting brief study about the effect of antithrombin (AT) on glioblastoma . RecentlSpine magnetic resonance imaging (MRI) can be typically used to guide treatment for vertebral metastasis. Pichon et al. aimed to improve imaging techniques for metastatic breast and medullary thyroid cancers with metastasis of the spine . The stuIuliani et al. contributed a brief report investigating the effect of an agent abiraterone (ABI), which is a selective inhibitor of androgen biosynthesis, on the osteoblast secretome . These sArticles in this special issue span many topics, all aimed to increase knowledge in the area of metastasis and therapeutic resistance. Common themes include areas of significance in understanding the CSC function as well as breast cancer biology. The overall aim of this issue is to provide a broad representation of research areas that examine mechanisms and approaches for confronting metastasis and the failure of cancer treatments."} +{"text": "Perrier et al. conducted NGS in six patients with leukodystrophy and described a range of pathogenic variants of leukodystrophies. Ek et al. emphasized that a genome-wide analysis, with variant calling strategies extended to structural variants (SV) and short tandem repeat expansions (STRs) in addition to single nucleotide variants and small insertions/deletions (SNVs/INDELs), was critical to enhance diagnostic yield for neuromuscular disorders (NMDs). Corroborating literature review and genomic sequencing, Bar et al. identified 22 candidate genes for cyclic vomiting syndromes (CVS), which further suggests a cellular model of the disease mechanism.Next-generation sequencing (NGS) has propelled the diagnosis of neurological disorders, the discovery of new candidate disease loci, and precision therapy. He et al. performed whole-exome sequencing and Sanger sequencing in three patients with adrenomyeloneuropathy (AMN) and identified one known mutation (c.1415_1416delAG) and two novel ABCD1 variants (c.217C>T and c.160_170delACGCAGGAGGC) in the Chinese population, indicating the importance of ABCD1 gene analysis in the diagnosis of patients with spastic paraplegia. Zambrano et al. used NGS to describe two Ecuadorian siblings with muscular dystrophy and deafness who carried EMD and EYA4 mutations associated with phenotypes. Genotypes are also linked to clinical manifestations and laboratory tests. Yang et al. identified differences in serum ceruloplasmin levels correlated to the age of symptom onset and genotypes (ATP7B variant); the authors established the cutoff value (0.13 g/L) of serum ceruloplasmin levels for the diagnosis of Wilson disease (WD) in a Chinese cohort with high sensitivity and specificity. It is hoped that diagnosing and treating neurogenetic illnesses will become easier with the advancement of diagnostic methods.In studying several rare diseases of the nervous system, SM and CL prepared the original draft. BY, LY, and HS critically review and edit the manuscript. All authors have reviewed and approved of the final manuscript."} +{"text": "Jin Despite the implementation of numerous weight loss initiatives by various governments, the global prevalence of obesity continues to rise . This noet al [et al [Since its rediscovery in adult humans, research into thermogenic fat has increased tremendously . This iset al and Yadal [et al now provet al [In the first study by Jin et al VSG and et al . When thet al , again ret al [Firmicutes and decreased the fecal abundance of Bacteriodetes, associated with stabilization of the intestinal epithelial barrier. Mass spectrometry analysis then revealed that 3 metabolites were increased in the feces of VSG-operated mice: licoricidin, muramic acid, and 3-hydroxybutyryl carnitine. Moreover, fecal licoricidin not only negatively correlated with various gut microbiota known to promote metabolic disease, but circulating licoricidin levels were doubled in VSG-operated mice suggesting that it could communicate directly with subcutaneous white fat. To formally test if gut microbiota-derived products contribute to thermogenesis in subcutaneous white fat after VSG, mice were treated with a broad spectrum antibiotic cocktail via their drinking water for 2 weeks. Strikingly, this not only prevented the weight loss and metabolic benefits associated with VSG similar to a previous study in diet-induced obese mice [per se contributes to the activation of thermogenesis in subcutaneous white fat after VSG.Jin et al then reaese mice , but it et al [Next, Jin et al determinet al , 21 and et al , respectet al , are neeet al [et al [et al [et al [18F-fluorodeoxyglucose positron emission tomography (18F-FDG PET) imaging experiments to determine if brown fat contributes to increased energy expenditure in mice receiving post-RYGB fecal microbiota. This revealed that brown fat 18F-FDG uptake in response to overnight cold exposure, the natural stimulus for brown fat, almost doubles in mice receiving post-RYGB feces compared with mice receiving pre-RYGB feces. Accordingly, brown fat UCP1 protein expression was also markedly enhanced.In the other study by Yadav et al stool sal [et al apart frl [et al is that l [et al found th18F-FDG uptake. This could have provided evidence that post-RYGB microbiota enhance blood glucose clearance in response to insulin treatment through using brown fat as a glucose sink. It would have also been interesting to determine if the patients themselves showed increased oxygen consumption and brown fat 18F-FDG uptake after RYGB, which would have provided the key evidence that this beneficial metabolic change in the host is potentially gut microbiota-mediated and is transmissable to germ-free mice.Considering that the post-RYGB feces-treated mice showed enhanced insulin sensitivity in the absence of any differences in body weight, it would have been interesting to determine if insulin increases brown fat et al performed metabolomic analyses of their feces. This revealed that thermogenic molecules like the short chain fatty acid butyrate [et al [Finally, after showing that differences in patient fecal microbiota after RYGB can be transferred to germ-free mice, such as an increase in Akkermansia municiphila, Yadav butyrate , 25 and butyrate were incbutyrate were redbutyrate . Considee [et al . It woulet al [et al [Figure 1). Notably, changes in bile acid metabolism by gut microbiota after VSG leads to the generation of cholic acid-7-sulfate in the liver and its accumulation in the gut [While we are witnessing the dawn of a new era for obesity pharmacotherapy with gut hormone preperations causing unprecendented levels of weight loss, bariatric surgery remains the most effective . The higet al and Yadal [et al are grou the gut , which h the gut . Thus, uet al [et al [The findings of Jin et al and Yadal [et al demonstrl [et al , and actl [et al ."} +{"text": "This is a Research Topic on thoracic oncology. Thoracic malignancy is a term used to describe any cancer presented in organs, glands, or structures within the thoracic cavity. Lung cancer is the second most frequent malignancy after breast cancer, accounting for 2.21 million cases annually, and the leading cause of cancer mortality worldwide (1.8 million deaths) for women and men combined inhibitor and is administered as first-line treatment to ALK-positive metastatic non-small cell lung adenocarcinoma patients. Xing et\u00a0al., systematically reviewed the efficacy and safety of Brigatinib. Tyrosine kinase inhibitors is another type of lung cancer targeted treatment. Currently, mutation status is determined by examining lung tumor tissue biopsies. Hu et\u00a0al., discuss the advances of PET/CT in establishing EGFR mutation status in lung cancer and their clinical significance.Better understanding of the disease has advanced and improved lung cancer treatment and clinical management. Patients may receive chemotherapy, radiotherapy, targeted therapy, immunotherapy, and surgery. Mizuno et\u00a0al., discuss the current status and future perspectives of PD-1/PD-L1 immune checkpoint blockade in lung cancer. ICIs may cause immune-related adverse events. Hao et\u00a0al., present the pathogenesis, risk factors, and clinical presentation of immune checkpoint inhibitor-related pneumonitis. Non-small cell lung cancer patients with mutations on the MET pathway present poor clinical outcomes. The development of targeted tyrosine kinase inhibitors and bispecific antibodies for MET genetic alterations have benefited this cohort of patients. Michaels and Bestvina discuss the evolution and current state of MET selective therapy. Surgery remains the first-line treatment for early stage lung cancer patients with resectable tumors. The surgical methods have developed resulting to smaller surgical traumas, fewer complications, and quicker post-operational recovery periods. Fuzhi et\u00a0al., outline the importance of evaluating pulmonary function in individuals who have undergone surgery for lung cancer, as well as the alterations in pulmonary function that occur after surgery. Additionally, they discuss strategies for effective rehabilitation of pulmonary function and factors that may affect the success of such rehabilitation.The introduction of immune checkpoint inhibitors (ICI) revolutionized cancer treatment and extended survival. However, not all patients respond to ICI therapy and benefit in terms of survival. It is still not clear which is the cohort of patients that would benefit the most. Addala et\u00a0al., 2022; Zhao et\u00a0al., 2022). Parotid and gastric metastases for patients with lung cancer are rare and thus not very well studied. Wang et\u00a0al., and Tang et\u00a0al., present two reviews on primary lung cancer with parotid and gastric metastases.Metastasis is a major factor that leads to mortality, and approximately 90% of cancer deaths are attributed to metastases. Malignant pleural effusion (MPE) is a common clinical problem for patients with lung cancer. The conduction of large-scale randomized clinical trials advanced diagnosis and clinical management. However, treatment focuses on symptom relief and control of fluid accumulation (Boulanger et\u00a0al., reviewed the connection between financial toxicity, quality of life, and survival in high value care.Financial toxicity refers to the adverse impact of cancer treatment expenses on a patient\u2019s quality of life. Lung cancer survivors often experience a rise in unemployment, psychological stress and a decrease in wages, indicating the persistent impact of financial toxicity. Carcinogen derived thoracic cancers including lung and oesophageal are amongst the most frequently diagnosed malignancies worldwide. Despite advances like the introduction of ICIs the clinical management of these patients remains challenging. Endotyping of lung cancer patients in combination with targeted treatments has improved survival. Metastases are common and more studies are necessary to understand the underlying biology. Finally, the effect of cancer on the financial sustainability and stability of patients is an important factor we need to investigate and gather more data.The author confirms being the sole contributor of this work and has approved it for publication."} +{"text": "Wu H. et al., Wu H.-N. et al., Li et al.).Multidrug-resistant (MDR) microbes infection for critically ill patients is a big challenge in clinical practice and is associated with greatly increased mortality . This ReWu H.-N. et al. retrospectively analyzed the distribution and antibiotic resistance of pathogens based on the clinical data of intensive care patients with bloodstream infections presented to a Chinese tertiary hospital and explored the value of procalcitonin (PCT) for the differentiated diagnosis of bloodstream infections caused by various pathogens. Gram-negative bacteria were the most frequently isolated microorganisms and were associated with a higher percentage of complications such as brain dysfunction, acute kidney injury, and thrombocytopenia. It was observed that PCT was a not good biomarker to distinguish bloodstream infections caused by various pathogens or fungi. Given that the mortality for patients with carbapenem-resistant Klebsiella pneumoniae (CRKP) bloodstream infection is reported to be as high as 30%\u221270%, Wu H. et al. used a logistic analysis to assess the association between the neutrophil-to-lymphocyte ratio (NLR) on 4th-day and 28th-day mortality. After balancing the confounders, NLR on the 4th day was associated with the 28th-day mortality, whereas the appropriate initial therapy was an independent protective factor. Moreover, the authors suggested that the trend of the NLR during therapy may help to evaluate the efficacy of different anti-infection therapy strategies at an early stage. In another article, Li et al. described their experience in the management of post-neurosurgical central nervous system infection caused by MDR Gram-negative bacteria with combined intraventricular and intravenous polymyxin B administration. After a mean duration of 14 days of treatment, all six cases caused by CRKP or carbapenem-resistant Acinetobacter baumannii (CRAB) were cured and no obvious kidney injury occurred.Karlsson et al., Yoon et al., Casarotta et al.). Yoon et al. demonstrated that bacterial superinfections were common in a tertiary Korean academic hospital and that more than one-third of the bacterial superinfection cases were caused by multidrug-resistant pathogens. Moreover, bacterial superinfection was associated with significantly fewer ventilator-free days, longer ICU and hospital stays. As many studies reported that a CRAB-associated bloodstream infection was the crucial risk factor for death in patients with COVID-19 . In a prospective longitudinal study in Sweden conducted by Karlsson et al., the authors evaluated the complicated bacteriuria and antibiotic resistance for ICU-admitted COVID-19 patients. They found that the vast majority of patients received antibiotics on ICU admission. Longer stays in ICUs linearly correlated with bacteriuria, and the authors proposed that biofilms in urinary catheters act as a reservoir of pathogenic bacteria with the potential to develop and disseminate antibiotic resistance.This Research Topic also includes three articles on the superinfection of patients with SARS-CoV-2 infection who were admitted to the ICU study, given that CPR scenarios are at high risk for healthcare-associated infections. By studying Advanced Cardiovascular Life Support (ACLS) courses in a manikin simulation, they found more than half of hand-cleaning indications could have been accomplished without delaying patient resuscitation and they concluded that hand disinfection can be implemented without compromising quality in acute care. In patients with severe acute pancreatitis (SAP), secondary MDR pathogen infection plays a vital role in increased mortality and prolonged hospital and ICU stays to enhance the resolution of late-phase organizing pneumonia caused by CRKP. Organizing pneumonia is a pattern of lung-tissue repair after injury and it can be cryptogenic or a response to a specific lung injury in many diverse clinical contexts. Given the therapy for organizing pneumonia is empirical and few therapies have been confirmed besides systemic glucocorticoid therapy (This Research Topic also includes an original study by therapy , they deQL: Writing\u2014original draft. S-JZ: Writing\u2014review and editing. J-CZ: Writing\u2014original draft, Writing\u2014review and editing."} +{"text": "Methodological advancements have played a pivotal role in driving progress within the field of biomedicine. A striking illustration of this trend is the groundbreaking discovery of the CRISPR/Cas9 technology by Emmanuelle Charpentier and Jennifer A. Doudna in 2012, a feat that earned them the Nobel Prize in Chemistry in 2020. This innovation, which facilitates precise gene editing, has revolutionized the capability to incorporate gene sequences into genomes with an unprecedented level of accuracy and efficiency (Jinek et al., Streptococcus pyogenes. Within this context, Charpentier uncovered an entirely novel molecule, tracrRNA, which constitutes a crucial component of the bacterium's innate defense mechanism aimed at neutralizing viruses by cleaving their DNA strands (Deltcheva et al., The genesis of the CRISPR/Cas9 technology emerged unexpectedly from investigations into Consequently, the CRISPR/Cas9 technology has emerged as a cornerstone of modern biotechnology over the past decade. Its global utilization across bioscience domains underscores its impact and significance, and it applications have extended far into the field of clinical neurosciences. So far, CRISPR/Cas9 has inseminated Cellular Neuropathology in the following ways:Treating Genetic Disorders: CRISPR/Cas9 technology holds promise for treating genetic disorders in the nervous system. Researchers have been exploring its potential for correcting mutations associated with neurodegenerative diseases such as Huntington's disease (Morelli et al., Modeling Neurological Disorders: CRISPR/Cas9 has been used to generate cellular and animal models of various neurological disorders. This allows scientists to study disease mechanisms more accurately, potentially leading to insights into the underlying causes of disorders like Alzheimer's disease (Schrauben et al., Understanding Neurodevelopment: By precisely manipulating genes in developing organisms, researchers have been able to uncover the roles of specific genes in brain development (Zhang et al., Functional Genomics: CRISPR/Cas9 technology has enabled scientists to perform large-scale functional genomics studies in neural cells (Sandberg et al., Pain Management: Researchers have explored the use of CRISPR/Cas9 technology to modulate pain perception by targeting specific genes involved in pain pathways (Reese et al., Gene Therapies for Neurological Disorders: CRISPR/Cas9-based gene therapies have shown promise in preclinical studies for various neurological disorders (Ou et al., Regenerative Medicine: CRISPR/Cas9 technology has been investigated for promoting neuronal regeneration after CNS injuries (Keatinge et al., It is important to note that while CRISPR/Cas9 technology holds immense potential, there are also challenges to overcome, including off-target effects, delivery methods, and the need for rigorous safety assessments before any clinical applications can be more widely adopted. The use of CRISPR/Cas9 in clinical neuroscience has also brought about important ethical discussions. Ensuring the safety and specificity of gene editing techniques in the complex and delicate nervous system is a critical consideration.in vitro and in vivo. With this Research Topic, we would like to expand current knowledge in the field functional genomics and gain further insight into neurodevelopmental disorders. This Research Topic is open for all disease areas. Papers outlining limitations and challenges of CRISPR/Cas9 technologies are particularly invited. In this search for the best ideas and concepts, Original research, Reviews, Perspectives and Opinions are envisaged. Papers will be reviewed based on excellence, originality and innovation potential. Outstanding papers will be featured in an editorial. We are looking forward to your contributions to this new Research Topic.In an effort to identify the most promising concepts in translational neurosciences, the Cellular Neuropathology section of Frontiers in Cellular Neurosciences recently launched a platform, the Hot Topics hub Hermann, . Within DH: Conceptualization, Investigation, Writing\u2014original draft, Writing\u2014review and editing."} +{"text": "MWCNTs. The hybrid nanofluid is under the influence of magnetohydrodynamic effects and chemical reaction with activation energy. The governing partial differential equations (PDEs) are transformed into ordinary differential equations (ODEs) using suitable similarity transform. Homotopy analysis method is used to solve the non-linear system of ODEs and MWCNTs while the mass transfer rate depicts contrasting behavior.Hybrid nanofluids are extensively analyzed in recent studies due to their better performance in numerous areas such as heat and mass transfer enhancement, biological fluid movement, medical equipment, heat exchangers, electronic cooling and automotive industry. In current study the nanoparticle concentration utilized is much important in biomedical industry. Major applications include drug delivery, radio-pharmaceuticals, centrifuging blood to obtain red blood cells and plasma, medical implants, onco therapeutics and photo thermal cancer therapy. In this regard, the primary focus of this study is to simulate a blood based unsteady hybrid nanofluid flow between two rotating, stretching disks and convective boundaries. The two nanoparticles in this study are uranium dioxide Jabbaripour et al.2 examined a water based three dimensional hybrid nanofluid flow with aluminum and copper nanoparticles at slip boundary conditions. Subhani and Nadeem3 analyzed water based micro-polar hybrid nanofluid flow with copper and titanium oxide nanoparticles over a stretching surface. Khan et al.4 studied the convective flow of Casson-nanofluid with gold nanoparticles through a rotating disk under impact of non-linear thermal radiation. Izaday et al.5 numerically scrutinized a Ag nanoparticles passing through an artery is analyzed by Chahregh and Dianrvand6 to better understand the blood circulation through the respiratory system. Ghasemian et al.7 presented their work on three-dimensional unsteady Maxwell nanofluid. Alghamdi et al.8 examined the flow of a 9 conducted a study on flow of 10 considered MFD viscosity effects on flow of a magnetic nanofluid. Waqas et al.11 simulated the free convective flow of a water based nanofluid. 12 simulated a stagnation point flow of a hybrid nanofluid with respect to the masses of two types of nanoparticles i.e., magnesium oxide and silver. Mansourian et al.13 analyzed the flow of a ferro-hybrid nanofluid passing over a stretching sheet.The nanofluids are formed by adding particles of nano-meter size into base fluid either by directly mixing (one-step method) or synthesizing nanoparticles first and then mixing (two-step method). The nanofluids consist of two-phases, fluid phase (base-fluid) and solid-phase (nanoparticles). Base fluids usually are water, ethylene glycol, blood and engine oil etc. while nanoparticles are mostly metal oxides, carbides or carbon nanotubes CNTs. When two types of nanoparticles are mixed into the base fluid then hybrid nanofluid is formed. Hybrid nanofluids enhance the efficiency of base fluids in terms of effective thermal conductivity, diffusivity, viscosity and heat transfer rates. These fluids are potentially useful in microelectronics, hybrid-powered engines, solar energy collectors, heat exchangers, drug transport and many medical equipment. Due to vast applicability, many researchers have attempted to analyze and simulate various hybrid nanofluid models in most recent studies. Rashidi et al.14. He developed a differential setup to analyze the hydrodynamical flow over an infinite rotating disk. His work was further extended by Griffiths15 where he analyzed generalized Newtonian fluids which provided a comprehensive description to non-Newtonian boundary layer flows. Rashidi et al.16 investigated the slip flow of a nanofluid on a rotating porous disk. Khan et al.17 numerically analyzed Oldroyd-B nanofluid flowing over an exponentially stretched surface with radiative effects. Hayat et al.18 studied a third grade nanofluid flow over a single rotating and stretching disk with thermophoresis and Brownian motion. Shah et al.19 investigated a nanofluid flow between two rotating and stretching disks with silver based CNTs under MHD effects. Usman et al.20 presented work on enhancement of heat transfer in a blood based nanofluid with power-law model and heat source/sink stimulated by two rotating stretchable disks. Convective flow of a Newtonian fluid between co-rotating stretching disks with Soret and Dufour effects is examined by Sharma et al.21. Rauf et al.22 explored the rate of heat transfer in a hybrid ferrofluid boundary layer flow passing over a rotating and non-linearly stretching disk in presence of an alternating magnetic field. Usman et al.23 presented steady flow model of a power law fluid co-axially rotating between two stretchable disks with heat source/sink.Flow originated by rotating stretching disks have gathered researchers\u2019 interest due to their applications in food processing, medical equipment, industrial and engineering sectors. Fruitful outcomes have been drawn through several researches but the pioneering work on rotating disk was presented by Karman24, Salahuddin et al.25, McCash et al.26, Raza et al.27 and Nisar et al.28 explored effects of chemical reaction with activation energy on various non-Newtonian and nanofluid models. Saleem et al.29 studied the effects of chemical reaction on flow of a second-order viscoelastic fluid with heat generation effect. Gowda et al.30 recently analyzed the heat and mass transfer rate in a non-Newtonian second grade nanofluid model undergoing chemical reaction with activation energy. Zaib et al.31 applied binary chemical reaction with MHD effects on Casson nanofluid flowing over a wedge. Khan et al.32 analyzed a chemically reactive nanofluid flow over a moving needle with viscous dissipation. Flow of cross nanofluid with immersed gyrotatic microorganisms is presented by Azam et al.33 under effects of non-linear thermal radiation.Addition of surfactants, stabilizers and various nanoparticles in blood causes major chemical reactions. Activation energy is the minimum amount of energy required to start off that chemical reaction. Arrhenius equation is utilized to describe the change of rate constant with changing temperature in a chemical reaction. These reactions take place mostly in chemical reactors which are most of the time limited through the rate of mass transfer. In this context, it becomes much important to incorporate chemical reaction effects in order to analyze the flow problem in this study. Other researchers including Hamid et al.34 in 2009 pioneered working with convective boundary conditions while investigating the Blasius flow. Afterwards, various authors analyzed different fluid models in this regard. Yao et al.35 analyzed the heat transfer in viscous fluid flow past a stretching/shrinking wall with a convective boundary condition. Hayat et al.36 investigated the stagnation point flow of a Casson fluid with mixed convection over a linearly stretching surface with thermally convective boundary. Wang et al.37 studied the bio-convective flow of a Maxwell nanofluid with slip effects and passing over an exponentially stretched surface. Haq et al.38 investigated the flow behavior of a Casson nanofluid with convective boundaries. Zaib et al.39 simulated flow of a nanofluid with convective boundaries in a Darcy-Brinkman porous medium. Boundary layer flow of a Casson fluid is examined by Hussain et al.40 with convective boundary conditions and flow over a stretching wedge. Furthermore, recent researchers on convective boundaries are presented by various authors including Akhtar et al.41, Anuar et al.42, Mabood et al.43, Becerro et al.44 and Rasheed et al.45.Convective conditions are characterized by interaction between boundaries of the machinery and the surrounding environment. Heat exchange by convection is the main cause of thermal response in the machine tools which is important in describing the fluctuations due to environmental changes or addition of cooling liquid. The heat transfer coefficients (HTCs) in these conditions are the proportionality constants when convective heat flux and temperature difference (between fluid and structure) are related. AzizMWCNTs. The governing unsteady non-linear PDEs are converted into ODEs by applying suitable similarity transformations. The obtained system of ODEs is then solved by using homotopy analysis method (HAM)46. To provide convergence of the solution In light of the literature review stated above, it is observed that an unsteady flow of a blood base hybrid nanofluid between two rotating and stretching disks with chemical reaction and activation energy along with convective boundaries has not been investigated. Moreover, uniqueness and need of present work is discussed in comparison with existing recent literature on unsteady hybrid nanofluid flow in Table We consider an unsteady blood hybrid nanofluid flow between two rotating and stretching disks in cylindrical coordinate system mentclass2pt{minimu, v and w are radial, tangential and axial velocities in r, z directions, respectively. T represents hybrid nanofluid temperature and C is the concentration. Chemical reaction rate is denoted by s is a constant power. Also, b is a positive constant with dimension of k is thermal conductivity and D is the thermal diffusivity. MWCNTs are presented in Table entclass1pt{minima57Pr is the Prandtl number, M is the magnetic interaction parameter.In order to non-dimensionalize the problem and to convert the system of partial differential equations into ordinary ones , the following similarity transforms are introducedmentclass2pt{minim use Eq. and obtamentclasspt{minimaSkin friction sing Eq. we obtaiIn order to solve system of highly non-linear ordinary differential equations in Eqs. \u201311) we we 11) w The final form of series solution obtained as a result is as followsough Eq. and geneAfter computing the series form solution through homotopic approach the convergence of results is optimized through entclass1pt{minimaThe flow problem is simulated in the fluid domain to depict the behavior of hybrid nanofluid under various effect and physical parameters. In this regard, each fluid phenomena is discussed in detail for hybrid nanofluid in following sections for velocity, temperature and concentration profile.M. In Fig. M. As M increases, the viscous forces in fluid layers decreases causing increase in velocity in all directions. This increase in velocity results in increased temperature of hybrid nanofluid. The temperature profile is analyzed for both fully convective and non-convective boundaries. It is observed that temperature in fully convective boundaries is higher when compared with thermally non-convective (The ratio of electromagnetic force to the viscous forces in a fluid flow is characterized by magnetic interaction parameter, Unsteadiness parameter, The parameters MWCNTs in blood increases the velocity of the hybrid nanofluid in radial, tangential and axial direction. Temperature of the hybrid nanofluid decreases with increased volume fraction of MWCNTs. It is also observed that fully convective boundaries offer higher temperature than non-convective boundaries in case of increasing volume fraction of carbon nanotubes.In Fig. Pr decrease fluid temperature due to reduced thermal conductivity inside the fluid. Fully convective boundaries offer higher temperature as compared to non-convective disks when Pr is increased. Increase in activation energy parameter Convective and non-convective boundary cases for temperature and concentration are shown in Fig. M increases the skin friction due to enhanced Lorentz forces between fluid particles. Increasing volume fraction of CNTs increases the skin friction as nore solid nanoparticles move through the fluid. Unsteady parameter U and stretching parameter Figure Pr, CNTs increases the heat transfer in Fig. Pr, Rate of heat transfer is the ratio of convective heat transfer and conductive heat transfer during a fluid flow. Figure CNTs volume fraction The ratio of mass transfer by convection and diffusion is the Sherwood number which is the mass transfer rate in the fluid. Figure M and Radial, tangential and axial velocity of the hybrid nanofluid increases with increase in Increase in M and Pr.Temperature of the hybrid nanofluid boosts with higher values of Convective boundary conditions result in higher temperature of hybrid nanofluid when compared with non-convective boundary condition case.Increase in Skin friction increases with increase in both volume fractions The rate of heat transfer decreases with increasing volume fraction CNTs decreases the rate of mass transfer in the hybrid nanofluid.Increase in volume fractions of both nanoparticles Current investigation focuses on simulating an unsteady and convective flow of blood based hybrid nanofluid undergoing chemical reaction with activation energy. A novel semi-analytical approach that is homotopy analysis method is utilized to solve the modeled system of non-linear ODEs. Convergence control parameters"} +{"text": "Pharmacoepidemiology, the study of the use and effects of medicines in large human populations, is a bridging science between clinical pharmacology and epidemiology. Pharmacovigilance is \u201cthe science and activities related to the detection, evaluation, understanding, and prevention of adverse effects or other problems associated with medicines.\"This Research Topic contains six manuscripts using pharmacoepidemiological methods, five original research papers, and one perspective paper, including one paper using electronic health records as an information source, two papers using spontaneous reporting systems, and three papers using nationwide medical information based on claims data.Alsowaida et al.). The global epidemic of COVID-19 has made the development of effective drugs for the treatment and prevention of COVID-19 a global priority. Several post-marketing studies have reported significant bradycardia with remdesivir administration, and this article provides validation in an evidence-based source.First, a retrospective cohort study using electronic medical records was reported by to methotrexate (MTX)-based therapy could help reduce the dose-dependent adverse events of MTX, providing clinical evidence to support the beneficial effect of FA.Lin et al. hypothesize that statins inhibit MSU-induced gout flares through their anti-inflammatory properties, and a cohort study using the 2000 Longitudinal Generation Tracking Database (LGTD 2000), a randomly selected dataset of 2 million NHI recipients, found that statins have chemopreventive potential against MSU.Taiwan\u2019s National Health Insurance (NHI) program covered more than 99.9% of the 14 Taiwanese population by 2014 . The NHIYen et al. recruited participants with type 2 diabetes mellitus (T2DM) and cirrhosis from the NHIRD between 1 January 2000 and 31 December 2017 and followed them until 31 December 2018. This report found that using alpha-glucosidase inhibitors was associated with a reduced risk of mortality, hepatocellular carcinoma, compensated cirrhosis, and liver failure in patients with diabetes and compensated cirrhosis.Shida et al. carefully explained the details of these studies.This type of study has not been conducted exclusively in Taiwan. The Pharmaceuticals and Medical Devices Agency (PMDA), the Japanese regulatory authority, conducts various pharmacoepidemiological studies based on actual data from its medical information database for post-marketing drug safety evaluation. Since various medical information is a source of pharmacoepidemiological studies, researchers should properly characterize the sources and consider the study design.The inverse signals detected by pharmacoepidemiological studies are known to be helpful in the search for drug candidates , and man"} +{"text": "Alternative splicing is a major mechanism to increase the number of proteins that can be made from the limited number of genes present in the human genome. During transcription of genes into precursor messenger RNA (pre-mRNA), non-coding introns are spliced out to make a messenger RNA (mRNA) that encodes the functional protein. During the splicing process some exons can be included or excluded and this process is termed alternative splicing. This is a highly regulated process that produces diverse mature mRNA transcripts from a single gene. Alternative splicing is present in almost every gene and is widespread in eukaryotic evolution. Moreover, the majority of genes expressed in the mammalian central nervous system undergo extensive alternative splicing, with some genes capable of contributing to over a thousand isoforms. This results in a variety of proteoforms exhibiting differences in function, binding preferences, catalytic activity, and localization. Disruptions in alternative splicing have been associated with numerous neurological disorders. A comprehensive understanding of its role in healthy and pathological nervous system function is still emerging. It is timely to gather current knowledge, advancement and challenges in this field. With this objective, we brought together several articles that discuss involvement of splicing and associated genetic perturbations in the central nervous system across the evolutionary scale\u2014from fly to human.Feng et al. focuses on the heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), a key RBP associated with neurodegeneration and cancer. The authors discuss hnRNPA1's role in gene transcription, mRNA translation, and stability, highlighting its importance and potential as a therapeutic target. Another study by Titus et al. reveals the functional role of the RBP Caper in Drosophila, emphasizing its significance in sensory and motor neurons development and its regulatory role in Drosophila gravitaxis behavior.Many RNA binding proteins (RBPs) play a crucial role in splicing regulation. The review by Lu Y. Q. et al. reveal the causative role of TANK-binding kinase (TBK1) in Amyotrophic Lateral Sclerosis (ALS) through mutational analysis, emphasizing the importance of intronic sequencing and pre-mRNA splicing analysis in understanding the complex mutational spectrum and pathogenesis of ALS. Reis et al. uncover a severe early onset dementia syndrome caused by an intronic splice donor variant in expanding our understanding of early onset dementia syndromes with a digenic background. Chen et al. identify a de novo splicing variant of the FOXP1 (Forkhead Box P1) gene in a patient with FOXP1 syndrome , providing insights into the genetic basis of global developmental delay, intellectual disability, and language delay. Fan et al. identify a disease-causing and aberrant splicing-inducing variant of TSC complex subunit 2 gene (TSC2) in a Han-Chinese family with Tuberous Sclerosis Complex (TSC), expanding the phenotypic and genetic spectrum of TSC and potentially contributing to its diagnosis and treatment. Wang et al. report a novel heterozygous STXBP1 (Syntaxin Binding Protein 1) splice variant with abnormal intron retention in a patient with Ohtahara syndrome (a rare form of epilepsy), highlighting the significance of splicing defect analysis in understanding the pathophysiology of neurodevelopmental disorders. Levchenko et al. reveal a deep intronic variant in the SNX14 (Sorting Nexin 14) gene in patients with spinocerebellar ataxia type 20, providing insights into the molecular pathogenic mechanism underlying the formation of a novel donor splicing site and potential therapeutic implications.Several studies identify splicing mutations associated with various neurological conditions. Xia et al. assess multiple mutations in three repeat (3R) tau for microtubule binding properties and prion-like aggregation propensity, contributing to the understanding of diverse presentations of tauopathies. Using bioinformatics pipelines, Farhadieh and Ghaedi reveal alternative splicing events (ASEs) in postmortem brain tissue with a cell-specific perspective, providing insights into AD pathology at the cell level. Lu Y. et al. identify several significant AS events in an AD mouse model, offering novel pathological mechanisms mediated by splice changes. Alalwany et al. investigate the neuroprotective effects of VEGF splice isoforms against AD-related neurotoxicity, suggesting potential therapeutic avenues.Tauopathies, including frontotemporal dementia and Alzheimer's disease (AD), are neurodegenerative diseases caused by tau brain aggregates. Tau protein, a microtubule-associated protein, can be disrupted in disease states due to the balance of tau splice isoforms. Winsky-Sommerer et al. analyze the transcriptome and translatome in the female mouse hippocampus at different ages, revealing age-associated splicing changes and their potential role in age-related deficits in hippocampal-dependent behavior. The study provides a comprehensive resource for understanding age-associated splicing changes with implications for neurological diseases.Aging is a major risk factor for neurological disorders including dementia. Baxter et al. explore the correlation between high K+ exposure, delayed-onset NMDA receptor-dependent neuronal death, and exon inclusion levels in neurons and astrocytes in vitro. The study highlights the neurotoxic nature of certain stimulation paradigms and emphasizes the importance of NMDA receptor blockade.Differential splicing of exons in neurons can alter protein properties, including ion channels, neurotransmitter receptors, and synaptic cell adhesion molecules. The Research Topic brings together up-to-date research focused on the biology of splicing and its regulators and associated mutations in neurological diseases. It provides new insights into the pathophysiological role of splicing modulations and offers possible strategies for therapeutic targets.KL-A: Conceptualization, Writing \u2013 original draft, Writing \u2013 review & editing. MS: Conceptualization, Writing \u2013 original draft, Writing \u2013 review & editing."} +{"text": "Epidemiological studies indicate an alarming narrowing in the gender gap of alcohol and tobacco use especially in adolescents, which may reflect changes in sociocultural patterns in women. Moreover, women use more often alcohol to cope with stress and negative feelings. Pharmacokinetics and pharmacodynamics differences, reward process specificities, and female hormones play a major role in gender differences. Women\u2019s consumption of alcohol may be associated with serious birth and developmental consequences in newborns.Thibaut F et al. WFSBP and IAWMH Guidelines for the treatment of alcohol use disorders in pregnant women. The World Journal of Biological Psychiatry 2019 20(1):17-50None Declared"} +{"text": "Vibrio campbellii is described in the paper by Zhou et al. .A new chitin-active AA10 lytic polysaccharide monooxygenase from the marine bacterium The longest linker, connecting the CBM73 to the other three domains, has typical features of an extended linker, with low sequence complexity and abundance of proline (four) and glutamate (four). Indeed, based on various observations, including lacking electron density for the CBM73 in one of the et al. have gone a long way in the further characterization of their LPMO domain, thus providing important information for the LPMO field. In particular, their data on binding of various metal ions are unique and shed light on the binding potential of the unique copper binding histidine motif in LPMOs. As a point of warning, the pH and temperature optima for \u2018LPMO activity\u2019 described in the paper are based on measuring H2O2 production resulting from an off-pathway oxidase reaction of the LPMO with molecular oxygen have a role in the pathogenicity-related physiology of Vibrio species. The novel, complete crystal structure of VhLPMO10A and the adjacent functional data provide an excellent starting point for digging deeper into the true biological role of these enzymes. Importantly, GbpA, VhLPMO10A and several other LPMOs with putative roles beyond biomass processing (e.g. Askarian et al., 2021et al., 2022i.e. a polymer of a sugar, N-acetyl\u00adglucosamine, that is abundant in carbohydrate-containing microbial structures, such as cell walls. It needs to be questioned whether chitin is the true substrate of these LPMOs. In this respect, it is worth noting that the topology of the substrate-binding surfaces of the LPMO domains of GbpA and VhLPMO10A is somewhat different from those of LPMOs known to play a key role in chitin degradation, such as SmLPMO10A (also known as CBP21; see arrows in Fig. 3 in Zhou et al., 2023Zhou et al., the biological function of proteins such as VhLPMO10A and GbpA remains largely enigmatic. From work on GbpA, it seems certain that these proteins are important mediators of the interaction between Vibrio and relevant host surfaces (Kirn et al., 2005et al., 2012Vibrio cells. Interestingly, the GbpA ortholog of the non-pathogenic bacterium Aeromonas veronii Hm21 stimulates epithelial cell proliferation in Zebrafish (Banse et al., 2023VhLPMO10A may have substrates that are yet to be discovered.Despite the structural and functional insights provided by Zhou"} +{"text": "The detrimental effects of APOE4, the strongest genetic risk factor for AD, have been characterized in different brain cell types, including oligodendrocytes isoform encoded by the \u201cC\u201d allele of the rs11136000 polymorphism impairs oligodendrocyte progenitor cell (OPC) proliferation and myelination through paracrine signaling from astrocytes and microtubule-associated protein tau (MAPT) genes , is expressed in microglia and mediates extracellular lipid sensing (Deczkowska et al., TREM2 and other genetic risk variants specific to microglia, neuroimmune myelinoids can be generated by seeding iPSC-derived microglia onto the myelinoid to allow microglia infiltration and interaction with other cell types in the organoid (The roles of genetic risk factors for AD in myelin homeostasis can be defined by deriving genetically engineered myelinoids that express genetic risk variants, such as APOE4 . As a tres APOE4 (Huang ees APOE4 . Anotherorganoid (Cerneckorganoid . Given torganoid .+ T cells in the aging mouse brain, leading to oligodendrocyte activation and death (Kaya et al., Peripheral effectors, such as immune cells and serum proteins have been recently implicated in AD pathogenesis as well (Gate et al., in vivo to promote advanced myelinoid maturation and vascularization (in vitro (Mansour et al., Finally, various myelinoids discussed earlier can be transplanted rization . Indeed,It is also important to consider various limitations of the brain organoid technology for modeling AD, so that appropriate experimental controls can be utilized (Cerneckis et al., Although AD has historically been considered a gray matter disease, emerging evidence indicates concurrent white matter dysfunction during AD progression. The versatile myelinoid platform offers unprecedented access to human brain myelination models that can be tailored to address specific hypotheses pertaining to myelin dysfunction in AD. For example, microglia can be incorporated into myelinoids described in JC: Writing\u2014original draft, Writing\u2014review and editing. YS: Writing\u2014review and editing."} +{"text": "The rapid advancement of biomedical sensor technology has revolutionized the field of functional mapping in medicine, offering novel and powerful tools for diagnosis, clinical assessment, and rehabilitation. The ability to collect and analyze various physiological signals, even in real-time, has provided unprecedented insights into the \u201chidden\u201d functioning of the human body. Biomedical sensors have not only enhanced our understanding of human physiology but have also significantly impacted clinical decision-making, patient management, and the development of personalized medical interventions.This Special Issue presents a collection of 14 papers that showcase the diverse applications of biomedical sensors in the context of functional mapping. The papers can be grouped into three sections, highlighting their contributions to (i) medical diagnosis, detection and prediction; (ii) neurological and rehabilitation assessment; and (iii) medical applications and monitoring. Together, these papers shed light on the transformative role of biomedical sensors in understanding physiological mechanisms and enhancing healthcare practices.This section focuses on the application of biomedical sensors for medical diagnosis, detection and prediction. The papers included in this section have a specific focus on the detection of conditions such as COVID-19 and hand osteoarthritis and the prediction of emotions by biosignals. Furthermore, novel approaches based on artificial intelligence and cutting-edge technologies are described.The paper \u201cCOVID-19 Detection Using Photoplethysmography and Neural Networks\u201d [In the paper \u201cToward Early and Objective Hand Osteoarthritis Detection by Using EMG during Grasps\u201d , researcThe third paper \u201cApplications of Laser-Induced Fluorescence in Medicine\u201d exploresAdditionally, the paper \u201cPredicting Emotion with Biosignals: A Comparison of Classification and Regression Models for Estimating Valence and Arousal Level Using Wearable Sensors\u201d delves iThis section explores the applications of biomedical sensors in neurological assessment and rehabilitation. The papers collected in this section describe interesting advancements in the analysis of electroencephalography (EEG), EMG and Near-Infrared Spectroscopy (NIRS) signals for the extraction of biomarkers to characterize individual status in neuromuscular applications that can have a potential impact, for example, on the assessment of rehabilitation effectiveness.The paper \u201cReliability of Mental Workload Index Assessed by EEG with Different Electrode Configurations and Signal Pre-Processing Pipelines\u201d evaluateIn the paper \u201cA Novel Approach for Segment-Length Selection Based on Stationarity to Perform Effective Connectivity Analysis Applied to Resting-State EEG Signals\u201d , researcThe paper \u201cReliable Fast (20 Hz) Acquisition Rate by a TD fNIRS Device: Brain Resting-State Oscillation Studies\u201d introducThe paper \u201cCombined Use of EMG and EEG Techniques for Neuromotor Assessment in Rehabilitative Applications: A Systematic Review\u201d presentsNext, the paper \u201cWhole-Body Adaptive Functional Electrical Stimulation Kinesitherapy Can Promote the Restoring of Physiological Muscle Synergies for Neurological Patients\u201d introducFinally, the paper, \u201cTechnology Acceptance Model for Exoskeletons for Rehabilitation of the Upper Limbs from Therapists\u2019 Perspectives\u201d addresseThis last section emphasizes the role of biomedical sensors in medical applications and monitoring, describing novel technologies and tools to improve health monitoring in different medical scenarios.The paper \u201cTowards a Practical Implementation of a Single-Beam All-Optical Non-Zero-Field Magnetic Sensor for Magnetoencephalographic Complexes\u201d introducThe paper \u201cExperimental Assessment of Cuff Pressures on the Walls of a Trachea-Like Model Using Force Sensing Resistors: Insights for Patient Management in Intensive Care Unit Settings\u201d investigContinuing in the realm of prosthetic control, the paper \u201cQuestioning Domain Adaptation in Myoelectric Hand Prostheses Control: An Inter- and Intra-Subject Study\u201d delves iThe last paper in this section, \u201cMulti-Scale Evaluation of Sleep Quality Based on Motion Signal from Unobtrusive Device\u201d , introdu\u201cBiomedical Sensors for Functional Mapping: Techniques, Methods, Experimental and Medical Applications\u201d presents a comprehensive collection of cutting-edge research in the field of biomedical sensors. The papers cover a wide range of applications, including medical diagnosis and detection, neurological assessment and rehabilitation, and medical monitoring. These advancements pave the way for improved healthcare practices, patient outcomes, and personalized medicine. As biomedical sensor technology continues to evolve, the findings from these research studies hold significant promise in revolutionizing medical practices and addressing complex health challenges, ultimately leading to better human health and well-being.The Special Issue"} +{"text": "Ocular neurodegeneration including high myopia, glaucoma, macular degeneration, optic nerve atrophy, and retinopathy can lead to blindness without timely and appropriate treatment. As the retina is actually an extension of the brain, studies on the molecular mechanisms by which these eye diseases develop are currently one of the hottest research areas in neuroscience.Novel mechanisms, diagnostic and therapeutic strategies for ocular neurodegeneration\u201d is to extend our knowledge related to retina and vision disorders by bringing together work in ophthalmology, optometry, psychology, neuroscience, and vision science.Investigations on neuronal correction of visual deficits have enriched our knowledge of functional eye diseases including ocular neurodegeneration. Nevertheless, it is urgently needed to further our understanding of how these eye diseases develop. Notably, novel therapies with superstar pharmacological intervention or new methods such as gene therapy or stem cell therapy have been attracted into this research area. The aim of this Research Topic \u201cMolcak et al. have summarized the expression, distribution, functions, and interactions of purinergic receptors in the retina and included potential crosstalk with other systems. Dissection of how these processes are affected will improve our understanding of the mechanisms that drive age-related macular degeneration (AMD).Avrutsky et al. have shown their original research data that caspase-9 inhibition has significant retinal protection from retinal vein occlusion (RVO). To be more specific, they have compared the therapeutic effect of caspase-9 inhibition with VEGF neutralization in an established mouse model of RVO, and they have conducted a series of examinations, including fundus angiography, optical coherence tomography (OCT), and electroretinography (ERG), for analyzing pathological changes.Ye et al. have investigated the long-term safety, efficacy, and binocular balance of monovision surgery using Implantable Collamer Lens (ICL) V4c implantation and Femtosecond Laser-Assisted in situ Keratomileusis (FS-LASIK) for the treatment of myopic patients with presbyopia. The results show that CL V4c implantation and FS-LASIK monovision treatment have long-term safety and binocular visual acuity at various distances.Lan et al. have demonstrated the control ability and characteristics of fixational displacement among healthy adults in a convenient method by using eye-tracking technology. A total of 100 healthy people were recruited for this study, providing an objective view that the fixation stability decreased significantly in the group aged over 50 years old.Chen et al. have reported a non-invasive diagnosis tool to assess blood flow perfusion in a visual pathway for ocular ischemic syndrome (OIS), which is attributable to chronic hypoperfusion caused by marked carotid stenosis. They have detected blood flow perfusion in a visual pathway by 3D pseudocontinuous ASL (3D-pCASL) using 3.0T MRI (magnetic resonance imaging). The results indicate that that there is a lower blood flow perfusion value in the visual pathway in patients with OIS.Velmurugan et al. have summarized different gene therapy methods in Leber hereditary optic neuropathy (LHON), suggesting that a mitochondrially targeted AAV (adeno-associated virus) gene therapy is more efficient than an allotopic AAV gene therapy for rescuing the LHON phenotype.Zheng et al. have presented a surprising treatment for restoring vision in adult amblyopia rats. By using molecular and histological approaches, they have revealed that low-frequency repetitive transcranial magnetic stimulation (rTMS) reinstates the amplitude of visual evoked potentials without influencing the impaired depth perception of amblyopic rats. They conclude that rTMS enhances functional recovery and visual plasticity in an adult amblyopic animal model.Zhang et al. have given a bibliometric analysis of apoptosis in glaucoma. This research will broaden our comprehension about the role of apoptosis in the process of glaucoma and provide guidelines for us in basic research and disease treatment.Zhen et al. have summarized rhodopsin-associated retinal dystrophy and its disease mechanisms and therapeutic strategies. In particular, they emphasize that innovative therapy strategies, such as gene therapy and stem cell therapy, are promising methods for the future treatment of retina pigmentosa. Nevertheless, greater efforts are needed from basic researchers and clinicians to facilitate the translation of recent research findings from the laboratory into clinical practice.Xiang et al. have interrogated the question of whether children with monocular myopia need to wear glasses. They consider the facts that (1) monocular myopia could lead to the accommodative dysfunction and unbalanced input of binocular visual signals, resulting in myopia progression; (2) monocular myopia may also be accompanied by stereopsis dysfunction, and long-term uncorrected monocular myopia may worsen stereopsis acuity in adulthood; (3) patients with monocular myopia could exhibit stereopsis dysfunction at an early stage; thus, they come to a conclusion that children with monocular myopia must wear glasses to restore binocular balance and visual functions, thereby delaying myopia progression.Boal et al. have presented a study showing that retinal ganglion cells adapt to ionic stress in experimental glaucoma. Their data indicate that in response to prolonged IOP (intraocular pressure) elevation, RGCs undergo an adaptive process that reduces sensitivity to changes in K+ while diminishing excitability. These experiments give insight into the RGC response to IOP stress and lay the groundwork for mechanistic investigation into targets for neuroprotective therapy.Li et al. have systemically analyzed independent risk factors for the progression of different degrees of diabetic retinopathy and non-diabetic retinopathy among type 2 diabetic patients. They conclude that young age, short axial length, and higher levels of FBG (fasting blood glucose) and urinary albumin creatinine ratio (UACR) were the independent risk factors for the progression of diabetic retinopathy in type 2 diabetes.Wareham et al. have presented data on collagen mimetic peptide repair of the corneal nerve bed in a mouse model of dry eye disease. Their data suggest that repair of underlying collagen in conditions that damage the ocular surface could represent a novel therapeutic avenue in treating a broad spectrum of diseases or injury.Wang and Wang have shown a preliminary study of spectral-domain optical coherence tomography (OCT) combined with ERG in the assessment of conbercept for neovascular age-related macular degeneration (nAMD). Their data suggest that (1) conbercept is useful for the short-term treatment of nAMD; (2) it can safely improve the visual acuity of affected eyes; and (3) it can restore the structure and function of the retina.Zhou et al. have shown that increased intraocular inflammation in retinal vein occlusion is independent of circulating immune mediators and is involved in retinal oedema. Their results suggest that (1) intraocular inflammation in RVO is driven primarily by local factors but not circulating immune mediators; (2) intraocular inflammation may promote macular oedema through the PI3K-Akt, Ras, MAPK, and Jak/STAT signaling pathways in RVO; and (3) systemic factors, including cytokines and lipid levels, may be involved in retinal microvascular remodeling.Wang et al. have assessed the precision and reliability of a novel computerized heterophoria test (CHT). Their data suggest that (1) the CHT can be used to demonstrate excellent inter- and intra-examiner repeatability and good correlation with the POCT ; (2) the differences between the CHT and POCT are within the permissible range of error; and (3) the CHT could provide a precise and reliable measurement for clinical applications.Altogether, this Research Topic of articles emphasizes novel diagnostic and therapeutic approaches for a variety of ocular neurodegeneration diseases, which are necessary to further explore.WW: Funding acquisition, Writing\u2014original draft. WT: Writing\u2014review and editing. YL: Writing\u2014review and editing. TH: Funding acquisition, Writing\u2014review and editing. DW: Writing\u2014review and editing. HL: Conceptualization, Funding acquisition, Writing\u2014original draft, Writing\u2014review and editing."} +{"text": "Worldwide, one new case of dementia is detected every 3 s , Ramer et al. examined whether childhood 24HAC behaviors were associated with executive function or academic performance in adolescence. Self-reported sleep quality, recreational screen time, and actigraphy-assessed physical activity were collected during grade 5 when the children were ~11 years; executive function and academic performance were collected during grade 9 at age ~15 years. Using structural equation modeling, the authors found: (1) adolescent executive functions were negatively affected by greater childhood recreational screen time and positively affected by better sleep quality; and (2) the negative association between childhood recreational screen time and adolescent executive functions were attenuated by greater childhood physical activity. Interestingly, childhood physical activity was negatively associated with adolescent executive functions; however, this relationship was mitigated by greater sleep.In a longitudinal analysis from the United States of data from the National Institute of Child Health and Human Development (NICHD) Longitudinal Study of Early Child Care and Youth Development to data from the Adult Changes in Thought study and examined the interactive relationships of 24HAC behaviors with global cognitive function. Physical activity and sedentary behavior were assessed by actigraphy, while sleep was estimated as self-reported time in bed. No significant associations were found across all three analytic methods in this cross-sectional study.Mellow et al. cross-sectionally examined the associations between 24HAC composition and cognitive function among 384 Australian healthy adults, aged 60\u201370 years. The 24HAC was assessed by actigraphy and by questionnaires. Cognitive measures included global cognition, memory, executive functions, and processing speed. Using compositional data analysis, no association between 24HAC composition and cognitive function was found.Hicks et al. cross-sectionally explored how chronotype\u2014an individuals' preferred activity and sleep pattern\u2014and physical activity were associated with cognitive performance in 153 American adults aged > 60 years, with and without self-reported sleep disorders. Wrist-worn actigraphy assessed physical activity and chronotype. Using multivariate analysis of covariance, no differences in physical activity or cognitive performance between different chronotypes were found.The results indicate that there is still much we do not understand about the 24HAC and cognitive health relationship. It is difficult to broadly characterize the 24HAC and cognitive health relationship at this time given that: (1) most of the evidence is cross-sectional; and (2) there are few studies on the 24HAC and cognitive health relationship in children, adolescents, younger, or middle-aged adults.Wu et al., Mellow et al., and Hicks et al. all found no association between 24HAC composition and cognitive function in healthy older adults; only Hyodo et al. found an association between 24HAC composition and cognitive function. It is possible that time-use composition fails to capture aspects of the 24HAC that may be more important for cognitive health.Nevertheless, there are several conclusions we can draw from these studies. Notably, time-use composition of the 24HAC does not appear to be strongly associated with cognitive function in healthy older adults. Ramer et al. used a longitudinal design. We strongly urge that future studies use longer follow-up periods and include wider age-ranges in study samples.There is an obvious lack of longitudinal studies in this emerging field of research. Only Our article collection mainly includes studies in older adults, but does not include any research among clinical populations at risk for, or living with, cognitive decline or dementia . People living with clinical conditions which are associated with cognitive decline are often less active, more sedentary, and have poorer sleep (Falck et al., Finally, it is unclear what modifiable and non-modifiable moderators and mediators can impact the 24HAC and cognitive health relationship. None of the studies in this Research Topic explored sex differences. Females have twice the risk of dementia (Ferretti et al., Preserving cognitive abilities across the lifespan is critical for qualify of life and wellbeing. The 24HAC holds promise as a means of maintaining cognitive health from childhood into older adulthood. Future work is needed to identify the temporal 24HAC and cognitive health relationship, as well as understand how different populations, mediators, and moderators such as sex, may impact this relationship across the lifespan.RF wrote the first draft of the manuscript. TL-A, JV, HM, DM, PG, and HS each provided critical edits and wrote portions of the manuscript. All authors contributed to the article and approved the submitted version."} +{"text": "Viruses are medically important obligatory parasites that can impact the health and quality of life of infected patients. Historically, prophylaxis against viral diseases like poliomyelitis and measles became possible by the successful development and roll out of vaccines in the 1950s and 1960s [Virus Microscopy, Coomer and Padilla-Parra discuss the imaging of HIV-1 virus entry and transmission in organoids of the female genital tract and colorectal tract, using techniques such as two-photon confocal microscopy and light sheet microscopy. Hu and Noda describe the structural analyses of Ebola and Marburg virus (Filoviridae) nucleocapsid structures as dissected by single particle analysis using cryo-electron microscopy. Menke et\u00a0al. elaborate on techniques such as super-resolution microscopy and quantitative image analysis for investigating the cell entry and infection biology of Orthohantaviruses. Finally, Petkidis et\u00a0al. highlight the power of label-free microscopy including quantitative phase imaging and atomic force microscopy to investigate virus entry and infection.In this Microscopy special issue"} +{"text": "Primary breast neuroendocrine neoplasms (BNENs) are a rare form of breast cancer, accounting for less than 0.1% of all breast malignancies. These neoplasms have a similar clinical presentation as conventional breast carcinomas, differing mainly in their histopathology and expression of neuroendocrine (NE) markers, chromogranin and synaptophysin. Owing to their rarity, current knowledge of these tumours comes mainly from corroborative case reports or retrospective case series. There is hence a lack of randomised data on the treatment of these entities and current protocol suggests similar treatment as that of conventional breast carcinomas. We report the case of a 48-year-old with a breast mass, which on further work-up was diagnosed as locally advanced carcinoma breast, that required a mastectomy and axillary node dissection on the same side and revealed NE differentiation on histopathological examination. Hence, immunohistochemical staining was indicated which confirmed NE differentiation. We discuss the current knowledge on incidence, demographics, diagnosis, histopathological and staining characteristics, prognostic factors and treatment modalities of BNENs. Primary breast neuroendocrine neoplasms (BNENs) constitute an under-recognised subtype of breast malignancies. This entity accounts for less than 0.1% of all cases of carcinoma breast and under 1% of primary neuroendocrine neoplasms (NENs) .The updated WHO Classification of Tumours of the Breast, in 2019, defined BNEN as a tumour with greater than 90% of cells showing NE differentiation, that is, expressing NE markers \u2014 chromogranin and synaptophysin (SYN) . First dThe present case report outlines a unique case of a primary BNENs.A 48-year-old nulliparous lady without comorbidities presented with a 3-month history of a lump in the lower outer quadrant of her left breast. On examination, a 2 \u00d7 2 cm, irregular, hard lump fixed to the surrounding breast tissue was palpated. Multiple, non-tender enlarged level 1 lymph nodes were also felt in the left axilla.Mammography showed an irregular, infiltrating, Breast Imaging-Reporting and Data System (BIRADS) -V lesion in the posterior aspect of the lower outer quadrant measuring 2 \u00d7 2 \u00d7 1 cm. Significant left axillary lymphadenopathy was also noted.Fine needle aspiration cytology of a level 1 axillary lymph node revealed metastatic breast cancer. Ultrasound-guided tru-cut biopsy of the breast lump yielded nests of cells with nuclear moulding and coarse chromatin. A provisional diagnosis of carcinoma with NE differentiation was made.A chest X-ray showed normal findings and an ultrasound study of the abdomen and pelvis revealed post-hysterectomy status with no other sonological abnormalities.She underwent a left modified radical mastectomy with axillary node clearance, following the workup.On gross examination, a 3 \u00d7 2.5 \u00d7 1 cm, grey-white, well-circumscribed, unifocal, firm mass was noted. Histologically, the tumour was composed of densely cellular nests and trabeculae. The cells were arranged as solid sheets with rounded margins with delicate fibrovascular stroma separating them. Neoplastic cells were larger and polygonal, with eosinophilic, granular cytoplasm and coarse and fine nuclear chromatin .in situ (DCIS) was found. Electron microscopy revealed dense core secretory granules. On Immunohistochemistry (IHC), the tumour was hormone receptor- oestrogen and progesterone positive and negative for HER2/neu (a non-hormone IHC marker for breast cancer that guides treatement options). More than 90% of the cells were strongly positive for neuron-specific enolase (NSE) and SYN and Cyclophosphamide (600 mg/m2) followed by four cycles of single-agent docetaxel (75 mg/m2). The chemotherapy was followed by adjuvant radiation. 50 Gy of three-dimensional conformational radiation therapy with radical intent was given in 25 fractions without concurrent chemotherapy.She received standard-of-care therapy with four cycles of Adriamycin show similar round or spindle-shaped cells with palisading of the nucleus, abundant eosinophilic and fine granular cytoplasm, and nuclei with \u2018salt and pepper chromatin\u2019. Tumour cells form islands, nests and trabeculae which are surrounded by a delicate fibrovascular stroma. According to the WHO classification of tumours 2019, essential criteria for BNENs include histological and immunohistochemical features of NE differentiation and co-existing DCIS is described as a desirable criterion . In the et al [et al [WHO describes BNENs as tumours with >90% of cells showing histological and IHC features of NE differentiation, mainly the expression of NE markers such as SYN and chromogranin A (CGA). CD56 and NSE may also be positive but are not as specific . In addiet al found thet al . Previouet al . In our l [et al noted thAs clinical characteristics and imaging features of BNEN are non-specific, histological features and immunohistochemical staining of tissues remain the cornerstone of diagnosis.As per 2019 WHO classification, well-differentiated BNENs were described as NETs which are considered low to intermediate-grade tumours in the current classification. Poorly differentiated BNENs, earlier described as NECs (neuroendocrine carcinoma) are considered high-grade tumours which include small-cell NEC and large-cell NEC . BNEN paThe differential diagnosis for these primary carcinomas is essentially metastasis from NECs at other sites. Presence of DCIS and the absence of tumours in other organs in radiology help in clinching the diagnosis of primary NEC of the breast . In the In terms of the prognosis of BNENs, the current literature is rather ambiguous with different investigators presenting conflicting reports. This ambiguity can also be attributed to the comparative smaller incidence of these tumours and the lack of larger studies on them.et al [et al [et al [et al [Previously, based on small studies, BNENs were thought to have a prognosis similar or even et al and Yangl [et al suggest l [et al . Cloyd el [et al have shol [et al where thl [et al . In the l [et al .Presently, there is limited information to recommend a specific treatment protocol for invasive breast carcinomas with NE differentiation and current guidelines suggest treatment same as that of conventional breast cancer (IDC-NOS).et al [et al [Surgery remains the mainstay of treatment for early-stage BNEN. The type of surgery is decided upon by criteria similar to that of IDC-NOS. Of these, the location of the tumour and its size determines the surgical method. There is no sufficient evidence to date showing the most efficient chemotherapy regimen for these cancers. Generally, chemotherapy used in patients depends on the histological features of the patient\u2019s tumour. Regimens containing platinum-etoposide, usually indicated for small-cell lung cancer, can be used in poorly differentiated small-cell BNENs , while aet al in 2015 et al . Adjuvanet al . Somatoset al . Studiesl [et al stated tl [et al .Our patient was surgically treated with a modified radical mastectomy of the affected breast with standard adjuvant chemotherapy indicated for Her2/neu negative breast carcinomas. Afterwards, she was started on adjuvant endocrine therapy and has since been in clinical remission. Treatment given was independent of NE features and according to principles of treatment for IDC-NOS, as indicated in the existing literature.There is an unmet need for further studies into these rare tumours for further understanding of these unusual clinical entities and for developing new treatment interventions. This case strengthens the importance of histopathology and early identification of NE features in patients with invasive breast cancer, which will help in early treatment and improved outcomes.The authors have no conflicts of interest to declare.There was no funding received for publishing this case report."} +{"text": "Autophagy in Inflammation Related Diseases, Volume II\u201d to collect related studies for the discussion of such issue.Autophagy is a vital catabolic mechanism to degrade and recycle long-lived proteins and useless organelles relying on lysosomes . Since iWang et al. revealed that aspirin-triggered Resolvin D1 (AT-RvD1) produced an alleviative effect on neuropathic pain through the induction of autophagy-mediated suppression of the NLRP3 inflammasome. In addition, Pei et al. demonstrated that alantolactone attenuated interleukin (IL)-1\u03b2-induced inflammatory responses, relieved cartilage degeneration and promoted impaired autophagy via restraining of signal transducer and activator of transcription 3 (STAT3) and nuclear factor (NF)-\u03baB signaling pathways in osteoarthritis. In a review paper, Feng et al. reported that autophagy was involved in the regulation of heparinase-mediated promotion of coagulation disorder and pulmonary fibrosis in acute respiratory distress syndrome (ARDS). In other two review papers, the role of autophagy in the regulation of fibrosis and immunopathogenesis of inflammatory bowel disease was discussed in detail . In addition, Huang et al. demonstrated the hepato-protective role of autophagy in non-alcoholic fatty liver disease (NAFLD).In our Research Topic, six brilliant studies have been collected and officially published in Frontiers in pharmacology. Among them, Autophagy in Inflammation Related Diseases,\u201d has collected several latest original studies for the exploration of potential targets taking advantage of autophagy in the treatment of several kinds of inflammation-related diseases. Furthermore, several brilliant review papers have discussed the role of autophagy in several inflammation-related disorders through reviewing and summarizing the previous studies. We believe that our Research Topic would bring new insights in the investigation of autophagy in inflammation-related diseases.All in all, our current Research Topic, together with the former one entitled \u201c"} +{"text": "Biosensors, \u201cElectrical/Optical Biosensing and Regulating Technology\u201d, presents a collection of papers that describe the latest advances in electrical/optical biosensors and systems. These papers report on the development and application of electrical/optical biosensing and regulating technologies that are expected to significantly enhance biomedical research. By bringing together these cutting-edge technologies and research findings, this Special Issue facilitates the advancement of biosensing and regulatory technology and its impact on the field of biomedical research.Biosensing has emerged as a powerful tool for exploring biomedical mechanisms. Utilizing highly sensitive electrical and optical sensing technologies, biosensors can detect weak signals and trace biomarkers in a dynamic, real-time, and label-free manner. The current Special Issue of Sun and colleagues have extensively reviewed the application of optical and electrical sensors in flexible wearable wound detection . They emZhan et al. conduct a review of electrochemical sensors and their applications in detecting glycated hemoglobin (HbA1c) . This stMicrofluidic paper-based analytical devices (\u03bcPADs) have greatly promoted the development of in vitro diagnostics due to their low cost, high efficiency, and ease of use. In their review, Zhang et al. provide an overview of the origin, fabrication methods, detection techniques, and innovative applications of \u03bcPADs in in vitro diagnostics . They al2+ is an innovative approach. The detection system has good sensitivity, with a detection limit (LOD) of 1.8 \u03bcM in bovine serum samples, and good selectivity, with a recovery rate of 92.8~110.1% and a relative standard deviation (RSD) of 1.72~4.89%. The platform\u2019s advantages of low cost, easy portability, strong operability, high throughput, and good repeatability make it a promising tool for point-of-care testing (POCT) applications.The smartphone-based ratio fluorescence probe (SRFP) platform developed by Wu et al. is promising for the detection and quantification of calcium ions in serum . The useYang et al. have developed an innovative platform for high-throughput biochemical analysis that integrates a spectrophotometer with a high-precision ball screw-driven two-dimensional motion slider . The steFunctional near-infrared spectroscopy (fNIRS) is a non-invasive method that can be utilized to detect cerebral hemodynamic responses and reflect the pattern of brain activity under different levels of stress. This technique can be used to assess individual cognitive abilities and psychological/physical health. Bak et al. conducted a study that focused on utilizing the laterality index (LIS) values calculated by fNIRS to differentiate between different types of stress . The autLiang et al. have proposed a novel method for quantitatively analyzing global DNA methylation using methylation-specific antibodies (5mC) modified magnetic beads (MB) for immune recognition and affinity enrichment . The metTerahertz radiation is a relatively new and unique radiation source that has been widely applied in various fields. In a recent study by Qi et al., the effects of 0.14 THz terahertz radiation on mouse behavior were investigated . The stuElectrical/optical biosensing and regulating technology can provide accurate and real-time measurements of various biological and environmental parameters, which can help researchers and scientists better understand complex biological systems and environmental processes. This Special Issue showcases the progress and potential applications of different sensor technologies in various fields, opening up new avenues for the future development of biosensing."} +{"text": "Mitochondrial bioenergetics and its function in tissue homeostasis are vital for regulating energy processes. As cells age, mitochondrial functions are often disrupted by both intrinsic and extrinsic threats including oxidative stress . AlthougGr\u00fcn et al.; Zhang et al.) and one on human non-small cell lung cancer along with a review article by Jagtap et al., collects new knowledge on mitochondrial function and shed light on the role of oxidative stress in altered mitochondrial bioenergetics and its relevance with aging-related pathologies. The aging-related decline in proteomic turnover, protein processing/folding, and quality control processes exaggerates the risk of oxidative stress to mitochondrial energetics and reduced GO (rGO) that was found to be mediated by oxidative stress, lipid peroxidation, and mitochondrial dysfunction and altered activities of several enzymes/indicators associated with myocardial activity and lipid peroxidation, respectively. These data revealed evidence of GO and rGO cardiotoxicity to myocardial cells that was mediated by oxidative stress, lipid peroxidation, and mitochondrial dysfunction.function ; 2. UsinLi et al. provided evidence of the anticancer function of ROS, which was essentially promoted by NRF2 inhibition , Li et al. analyzed ethanol fractions of Helicteres angustifolia L. root and showed that 40% ethanol fraction produced selective toxicity to NSCLC cells. Mechanistically, this bioactivity of EF40 was found to be linked with reduced expression of NRF2 that abundantly expresses across several cancers. Of note, EF40-led suppression of NRF2-dependent cellular defense response promoted the intracellular accumulation of ROS in the NSCLC cells. The biochemical analyses further explained the activity of EF40 in cell cycle arrest and apoptosis, promoted through the ROS-led DNA damage response. An evident decrease in matrix metalloproteinases (MMPs) and hnRNP-K expression levels in EF40-treated NSCLC cells further suggested its anti-migratory activity, which was corroborated by significantly reduced tumor growth and lung metastasis in an in vivo xenograft mice model. Conclusively, Li et al. provided direct evidence of the utility of ROS function in promoting anticancer activity that also reaffirmed the significance of NRF2-regulated defensive antioxidant response in cell survival.Although the oxidative stress produced by high ROS levels is central to the cardiotoxicity of myocardial cells, its lethality can be seen in cancer cells. In line with this, an original study by hibition ; 3. UsinJagtap et al. in a review report comprehensively discussed the new knowledge and developments in the stream (Protein synthesis, protein processing and folding, and quality control mechanisms are critical cellular processes that are directly linked with mitochondrial homeostatic function, which progressively decline with aging. Although the role of protein homeostasis or \u2018proteostasis\u2019 in cellular function is understood, its significance in mitochondrial physiology under neurodegenerative conditions needs to be resolved. To this end, e stream ; 4. The Conclusively, the original research and review articles published under the present Research Topic shed light on the role of oxidative stress in altered mitochondrial bioenergetics and its outcome. It also discusses new knowledge on impaired mitochondrial bioenergetics and its relevance with age-related pathologies."} +{"text": "Mood disorders such as major depressive disorder (MDD) and bipolar disorder (BD) are often resistant to current pharmacological treatment. Therefore, various alternative therapeutic approaches including diets are, therefore, under investigation. Ketogenic diet (KD) is effective for treatment-resistant epilepsy and metabolic diseases, however, only a few clinical studies suggest its beneficial effect also for mental disorders. Animal models are a useful tool to uncover the underlying mechanisms of therapeutic effects. Women have a twice-higher prevalence of mood disorders but very little is known about sex differences in nutritional psychiatry. In this review, we aim to summarize current knowledge of the sex-specific effects of KD in mood disorders. Ketone bodies improve mitochondrial functions and suppress oxidative stress, inducing neuroprotective and anti-inflammatory effects which are both beneficial for mental health. Limited data also suggest KD-induced improvement of monoaminergic circuits and hypothalamus\u2013pituitary\u2013adrenal axis\u2014the key pathophysiological pathways of mood disorders. Gut microbiome is an important mediator of the beneficial and detrimental effects of diet on brain functioning and mental health. Gut microbiota composition is affected in mood disorders but its role in the therapeutic effects of different diets, including KD, remains poorly understood. Still little is known about sex differences in the effects of KD on mental health as well as on metabolism and body weight. Some animal studies used both sexes but did not find differences in behavior, body weight loss or gut microbiota composition. More studies, both on a preclinical and clinical level, are needed to better understand sex-specific effects of KD on mental health. High-fat low-carbohydrate ketogenic diet (KD) was originally introduced into clinical practice for the management of drug-resistant epilepsy nearly 100\u00a0years ago Peterman and is sBesides epilepsy, metabolic and cardiovascular diseases growing amount of data suggest a beneficial effect of KD also in neurodegenerative diseases is a leading mental disorder affecting up to 300 million people worldwide, producing a huge burden on society\u2019s wellbeing and the world economy , medium-chain FA (MCFA) and long-chain FA (LCFA) , BD, psychotic disorders and schizophrenia , but have not reported their results yet. No RCTs of KD have been performed or started for MDD, while animal studies, case reports and a few small clinical trials suggest its beneficial effect on the mood. A recent small trial found improved depressive symptoms in 6 MDD patients and 12 BD patients after 3\u00a0weeks on KD have been performed for KD in any mental disorder up to date. Few studies are registered on Disturbed monoamine neurotransmission is a key mechanism of depression, schizophrenia, ADHD, ASD and other mental disorders axis dysregulation is a core symptom of mood and anxiety disorders with elevated cortisol level and disturbed feedback inhibition and Firmicutes metabolism in the motor and somatosensory cortex, but not in the midbrain and basal ganglia. However, the total level of dopamine, serotonin and noradrenaline remained unchanged in all brain regions explored in this study and Firmicutes better in men compared to pre-menopausal, but not post-menopausal women gene expression (Salberg et al. Rats fed with a high-fat high-sugar diet for 12\u00a0weeks showed opposite changes in HPA axis activity with decreased corticosterone level in males but increased level in females (Sahagun et al. Sex-specific effects of KD on mental health were evaluated almost only in animal studies, although one study with obese volunteers did not find any sex differences in KD-induced changes in mood and cognitive function (Halyburton et al. Still very little is known about sex differences in gut microbiota in general (Reviewed in (Jaggar et al. Sex differences in diet-induced changes in gut microbiota were reported only in a few studies with polyphenol (Most et al. Data from experimental animal models and small clinical studies suggest that the ketogenic diet may have beneficial effects both in MDD patients and in the healthy population. Its repeatedly proven neuroprotective and anti-inflammatory effect is thought to be a key mechanism of its potential antidepressant effect. Moreover, key pathways of MDD pathogenesis, such as HPA axis hyperactivity and brain monoamine circuit disturbances have also been shown to be improved by KD in animal stress models. Recent animal studies using depression models showed synergistic effects of antidepressants combined with omega-3-rich fish oil (Paula Farias Waltrick et al."} +{"text": "NAFLD-MAFLD Conundrum\u201d, is to discuss the current debate on renaming nonalcoholic fatty liver disease (NAFLD) to metabolic dysfunction-associated fatty liver disease (MAFLD) and the implications of changing terminology, summarizing the current state of knowledge regarding MAFLD and anticipating the necessity for a new nomenclature which is metabolic dysfunction-associated steatotic liver disease (MASLD) given inOverall, four original articles, a systematic review and two narrative reviews have been published in this Research Topic.Wen et\u00a0al. which included 10 cohort studies and showed that patients diagnosed with MAFLD alone had higher cardiovascular mortality than those diagnosed with NAFLD alone.The importance of a new definition is well established in the systematic review by Liao et\u00a0al., where they observed that the number of publications on MAFLD increased dramatically, especially in the last three years. In particular, gut microbiota became an important research direction for etiological and therapeutic investigations into MAFLD, as well as, insulin resistance, a key factor in studying the development of MAFLD. They concluded that liver fibrosis was an important focus of disease development.Our Research Topic aimed to identify the hot topics and new trends in the gut-liver axis in NAFLD/MAFLD research starting from the bibliometric analysis conducted by Yang et\u00a0al. confirmed the previous analysis revealing that gut microbiota, inflammation, insulin resistance, short-chain fatty acids, and randomized controlled trials will be the new focus of research in the field.Another bibliometric analysis by Li et\u00a0al. in a cross-sectional study among 4,195 participants aimed to evaluate the diagnostic efficacy of different anthropometric indices, including body mass index (BMI), waist circumference (WC), waist-to-height ratio (WHtR), waist-to-hip ratio (WHR), cardiometabolic index (CMI), triglyceride-glucose (TyG) index, hepatic steatosis index (HSI), lipid accumulation product (LAP), body roundness index (BRI), visceral fat index (VAI), abdominal volume index (AVI), cone index (CI), and body fat index (BAI), in adults with MAFLD to identify the best cut-off point for diagnosis of MAFLD in United States adults. In particular, the authors concluded that LAP may be a sensitive marker for diagnosing MAFLD in United States adults.Xiao et\u00a0al. is the skeletal muscle mass (SMM). In the same cohort of Li et\u00a0al., the authors demonstrated that the distribution of SMM differently affected MAFLD and significant fibrosis in the sex groups. Higher appendicular SMM was associated with a lower risk of MAFLD, while the risk of significant fibrosis in females was increased with the trunk SMM. Concluding the two published reviews focused the attention on molecular characterization of NAFLD/MAFLD and on the latest treatments .Another important parameter taken into account by We hope that this Research Topic will help to produce new evidence about NAFLD/MAFLD and contribute to better understand the new definition of MASLD, in order to clear the complexity of this disease.AD: Conceptualization, Writing \u2013 original draft. RK: Writing \u2013 review & editing. SA: Validation, Writing \u2013 review & editing."} +{"text": "Obsessive-compulsive disorder (OCD) is a psychiatric disorder characterized by recurrent unwanted thoughts (obsessions) and associated repetitive behaviors (compulsions), affecting around 1.1\u20131.3% of the global population ; it has been shown to play a key role in neurogenesis and neural development, and possibly in synaptic vesicle transport. In SPRED2-deficient mice, alterations in neural transmission were observed in thalamo-amygdala circuits. Subsequently, it was found that these mice, like those in which SAPAP3 had been deleted, also showed altered ultrasonic vocalizations. These changes were observed in both young and older mice, and appeared to increase with age of SAPAP3 has been specifically associated with the Contamination/Washing dimension of OCD, as well as with a poor response to serotonin reuptake inhibitors , coding for a protein related to SAPAP3 which is also involved in synaptic connectivity, were significantly associated with clinical OCD have also been associated with childhood OCD (Gazzellone et al., SAPAP1 has been knocked out exhibit impaired scaffolding at glutamatergic synapses and altered social behavior (Coba et al., BDNF and NTRK2 genes, which are proximal components of the same cellular cascade as SPRED2, may exert a protective effect against OCD; these effects may be mediated by beneficial effects on this particular signaling pathway (Alonso et al., Copy number variants in Taken together, these findings suggest that genes involved in neurodevelopment and synaptic connectivity, when disrupted, induce not just OCD-like behavior but alterations in brain development, sensory processing, cognitive functioning and social behavior in animals. At least one of these genes is also associated with certain facets of OCD in humans. There is evidence from animal research that alterations in these genes are associated with functional changes involving prefrontal, striatal and limbic brain regions. The consistency of these findings across rodents and humans suggests that at least some of the genetic mechanisms underlying OCD could be evolutionarily conserved. The fundamental phenotype involved in this process may represent lower-order deficits arising from alterations in neural development, which could influence higher-order cognitive processes in a \u201cbottom-up\u201d manner (Benzina et al., The argument presented above would gain support if it were possible to demonstrate neurodevelopmental antecedents of OCD in humans. In this case, too, the available evidence suggests that at least some types of OCD have developmental antecedents. The evidence for these developmental alterations has been summarized in recent reviews (Poletti et al., SAPAP3 or SPRED2 knock-out mice.Specific phenotypes have also been linked to these developmental alterations. For example, patients with OCD show evidence of impaired olfaction, which is a marker of brain dysfunction of developmental origin (Crow et al., An endophenotype is a heritable trait that can be measured in an objective manner, and which is present in individuals with a given psychiatric disorder, as well as their unaffected first-degree relatives, at rates significantly higher than in healthy controls or in the general population (Gottesman and Gould, A tentative integration of the findings described above is presented in During the course of cognitive development in early life, alterations in these processes affects \u201chigher-order\u201d cognitive processes such as flexibility and decision-making capacities (Abramovitch et al., The model outlined above represents a tentative yet coherent approach to understanding the mechanisms through which evolutionarily conserved cellular and neurobiological processes could contribute to the development of OCD in humans. Much remains to be learned about the specific association of each process with OCD, their relationship to the different dimensions of OCD, and the opportunities they offer for early intervention, improved treatment, and the identification of specific endophenotypes such as sensory over-sensitivity (Fontenelle et al., The sole author of this work was responsible for the conceptual framework, literature review, writing, editing, and proofreading of this paper."} +{"text": "Neurodegenerative and neurodevelopmental diseases represent neurological disorders that variably affect individuals and produce negative consequences, such as altered social interaction, restricted/repetitive behavior, and other medical and mental health conditions that result in lifelong functional and social impairments with high economic and social costs and presents a compelling argument in support of salivary biomarkers to diagnose this complex disorder, which currently relies solely on behavioral assessments .The mini review entitled, \u201cSalivary MicroRNA Profiling Dysregulation in Autism Spectrum Disorder: A Pilot Study\u201d, demonstrates how saliva can help clinicians support the ASD diagnostic process through the standardization of conditions used to isolate biomarkers that can robustly detect disease in a more simple way . The second, entitled \u201cSalivary Inflammatory Biomarkers are Predictive of Mild Cognitive Impairment and Alzheimer's Disease in a Feasibility Study,\u201d provides evidence for the feasibility of saliva as a valuable source of biomarkers for the early detection of cognitive impairment in individuals on the AD continuum and potentially other neurodegenerative diseases.This mini review is followed by two original research articles. The first, entitled \u201cNon-Invasive Diagnosis and Monitoring Tool of Children's Mental Health: A Point-of-Care Immunosensor for IL-6 Quantification in Saliva Samples\u201d and \u201cMultisite Dopamine Sensing with Femtomolar Resolution Using a CMOS Enabled Aptasensor Chip\u201d provide examples of how point-of-care (POC) biosensors can help societies fast track patient needs in the early diagnostic process. For instance, preliminary analysis of retrospective PoliBiobank data demonstrates that miRNA and inflammatory molecule biomarkers in saliva samples collected from healthy individuals and those diagnosed with neurodegenerative and neurodevelopmental diseases, using traditional techniques, can be detected via real-time PCR and the MSD platform, respectively (unpublished data).The two remaining studies, \u201cFinally, as we highlighted in Goldoni et al. , our hopAll authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication."} +{"text": "Neurological disorders (ND) are now globally recognized as the primary cause of death and disability. The prevalence, incidence, deaths, and disability-adjusted life years (DALY\u2019s) were estimated from 1990 to 2016 in 195 countries for nearly 15 common neurological disorders . Based oThe rapid development of India resulted in an increased burden of neurological disorders, which form a significant proportion of non-communicable diseases. A survey on the burden of neurological disorders in India revealed that non-communicable neurological disorders doubled, whereas communicable neurological disorders reduced to about one-fourth from 1990 to 2019 . The preIn the present era, a significant increase in neurological disorders has been observed throughout the globe due to the COVID-19 pandemic or as co-morbidities of post COVID-19 complications. The partial or complete lockdown situations in the last 2\u00a0years have confined everyone in their respective territories, leading to changes in neurological complications like anxiety, depression, stress, and psychological disturbances in the general public, especially in children. It has been reported that isolation and post-traumatic stress was the leading cause of depression and altered mood in post COVID-19 patients . A studyIt is the hour of need to deal with the increasing number of neurological disorders in developing and developed countries with safer and therapeutically efficacious medicines. We can achieve this goal with advanced clinical and translational research and bioinformatics tools on traditionally used and medicinally potential natural products . Considering the need to enrich more information about natural products and neurological disorders, researchers and clinicians working in the Natural Products/Pharmacology field were invited to contribute their valuable research to this Research Topic article collection. Research articles on pre-clinical and clinical studies of Natural Products/Traditional Plants in neurological disorders , Case studies/clinical reports emphasizing the role of natural products in neurological disorders, and Review articles focusing on \u201cNeuropharmacological Studies of Natural Products\u201d covering Clinical and translational research with bioinformatics tools, SAR and molecular docking studies and Mechanistic Insights were considered under the scope of this Research Topic.Puri et al., entitled \u201cNatural product-based pharmacological studies for neurological disorders,\u201d focused on targets associated with neurological disorders, traditional holistic approaches for managing neurological disorders and mechanistic approach of phytoconstituents in treating various neurological disorders. A mini-review entitled \u201cUnderstanding the role of \u201csunshine vitamin D\u201d in Parkinson\u2019s disease: A review,\u201d published by Behl et al., explained the role of sunshine vitamin D in Parkinson\u2019s disease. In the systematic review article entitled \u201cThe neuroprotective effects of melatonin against diabetic neuropathy: A systematic review of non-clinical studies,\u201d published by Hosseini et al., the authors have critically analyzed the role of melatonin in diabetic neuropathy and explained its mechanisms taking reference from pre-clinical data. A research paper entitled \u201cEvaluation of Mollugo oppositifolia Linn. as cholinesterase and \u03b2-Secretase enzymes inhibitor,\u201d published by Das et al., emphasized in vitro neuroprotective effect of Mollugo oppositifolia in Alzheimer\u2019s disease. The authors have identified phytochemicals in bioactive plant extract and performed in silico molecular docking studies to identify more potential and stable metabolites with potential for Alzheimer\u2019s disease targeting. Another research paper, \u201cLong-term oral administration of naringenin counteracts aging-related retinal degeneration via regulation of mitochondrial dynamics and autophagy,\u201d stressed the role of a CNS-acting flavonoid, Naringenin, in counteracting aging-related retinal degeneration.We received ten articles for consideration for publication in the thematic issue. After a thorough review, five articles were accepted out of 10 submitted articles. Amongst these, two articles are original research papers, one systematic review, one review, and one mini-review. A review article published by"} +{"text": "The application of glial cell line-derive neurotrophic factor (GDNF) to cell cultures and animal models has demonstrated positive effects upon dopaminergic neuronal survival and development, function, restoration, and protection. On this basis, recombinant GDNF protein has been trialled in the treatment of late-stage human Parkinson\u2019s disease patients with only limited success that is likely due to a lack of viable receptor targets in an advanced state of neurodegeneration. The latest research points to more refined approaches of modulating GDNF signalling and an optimal quantity and spatial regulation of GDNF can be extrapolated using regulation of dopamine as a proxy measure. The basic research literature on dopaminergic effects of GDNF in animal models is reviewed, concluding that a twofold increase in natively expressing cells increases dopamine turnover and maximises neuroprotective and beneficial motor effects whilst minimising hyperdopaminergia and other side-effects. Methodological considerations for measurement of dopamine levels and neuroanatomical distinctions are made between populations of dopamine neurons and their respective effects upon movement and behaviour that will inform future research into this still-relevant growth factor. The discovery of glial cell line-derived neurotrophic factor (GDNF) as a secretion product from the rat B49 glioma cell line in 1993 heralded the arrival of a powerful promotor of dopaminergic cell survival with enormous potential for treating Parkinson\u2019s disease (PD). Lin et al after biSide- and off-target effects are important to consider for future therapies as they affect the tolerability and utility of GDNF. Side-effects following intracerebroventricular (icv) GDNF injection in humans are documented as principally nausea, vomiting, paresthesias, hyponatremia, anorexia, and weight loss channels in addition to striatum (40%). ERK1 and ERK2 phosphorylation were increased in SN and striatum, respectively, and microdialysis showed an increase in DA release in response to K+\u2009and amphetamine was unchanged following nigral GDNF injection in 4\u20136\u00a0month old rats, yet given a twofold increase in K\u2009+\u2009-induced DA release, there was calculated to be a 1.75-fold increase in TC (DA clearance) when measured electrochemically in vivo which fine-tunes behaviour /tTA-responsive promotor system under CAMKIIa to overexpress GDNF at two- to threefold levels in cortex, hippocampus and brainstem in any cells that normally express CAMKIIa showed increased TH activity and DA synthesis yet, unusually, no increase in K+\u2009-stimulated DA release via microdialysis of the mouse Gdnf 3\u2032UTR has been excised via Cre-LoxP. Homozygous removal of the 3\u2032UTR resulted in a more than threefold increase in Gdnf mRNA and affected prepulse inhibition in mice, suggesting schizophrenia-like behaviour (M\u00e4tlik et al. In further support of the endogenous elevation route, the One important methodological consideration arose in measuring extracellular dopamine levels, as reported in Kumar et al. . ElectroThere is evidence for GDNF signalling via multiple pathways and for unexplored roles in maintaining normal striatal function outside of canonical RET signalling. NCAM1, integrins \u03b1v and \u03b21, and N-cadherin induce neurite outgrowth, proliferation, survival, and influence axon guidance (Chao et al. , respectively. This may affect the tolerability of future therapies in humans due to effects upon cognitive and behavioural functions.A clearer framework for working with GDNF in terms of overexpression, localisation, and off-target effects is now evident. This highlights the pitfalls of exogenous application and the benefits of endogenous upregulation as well as the marked effects of GDNF upon DA transport. DA overflow in systems with greatly increased dopamine transporter activity may not be an accurate marker of DA release as accurate measurement may be masked by increased reuptake kinetics see Fig.\u00a0. This is"} +{"text": "RISPR-Cas9, have made zebrafish one of the most powerful model systems for breakthroughs in human diseases and provide the foundation for translational research [Our understanding of fundamental biological mechanisms and the pathogenesis of human diseases has been greatly improved by studying the genetics and genomics of zebrafish . The fieresearch .In this Special Issue of \u201cZebrafish: a powerful model for genetics and genomics\u201d, we present 16 research articles and reviews concerning zebrafish modeling in genetics and genomics that aid in understanding human diseases. Notable publications include three articles that illustrate the generation of novel transgenic zebrafish, which can be used in addressing important biological questions: (1) Mao et al. created a transgenic zebrafish to visualize adipocyte development, facilitating the characterization of white adipose tissues in vivo and the development of therapeutic interventions to treat metabolic diseases in humans; (2) Li et al. generated an animal system to monitor the in vivo dynamics of spatiotemporal calcium signaling useful for normal animal physiology as well as stressful and pathophysiological conditions; (3) Jeong et al. developed an optogenetic manipulation system for dissecting olfaction-related behaviors and their underlying neural circuitry.In addition to transgenesis, two studies have shown advanced applications of CRISPR-Cas9 technology that could assist users in patient-specific gene tailoring as follows: (1) de Vrieze et al. revealed an improved strategy in developing knock-in zebrafish models; (2) Schellens et al. demonstrated the therapeutic potential using the exon skipping approach for modeling retinitis pigmentosa.ift74 gene, developing gradual photoreceptor degeneration that is distinguishable from other previously published IFT-B mutants in zebrafish. (2) Berlingerio et al. demonstrated that cystinosis, a disease characterized by cystine accumulation in the lysosome resulting in nephropathy and vision problems in humans, was recapitulated in ctns gene mutant zebrafish. (3) Alexandro-Moreno et al. showed that primary congenital glaucoma, mainly associated with CYP1B1 gene mutations, was recapitulated in a cyp1b1 KO zebrafish and displayed a novel pathway involving the dysregulated expression of extracellular matrix genes. (4) Kim et al. identified potential regulators responsible for vanishing white matter disease by performing a comparative proteome between eif2b3 KO zebrafish and the WT control. These all show novel molecular processes that underlie disease etiology and, thus, could provide therapeutic clues for corresponding human diseases.What makes this Special Issue more interesting is the addition of articles that each showed a novel gene knockout (KO) zebrafish that recapitulated clinical symptoms associated with specific human diseases. (1) Zhu et al. induced ciliopathy via a mutation in the Additionally, several articles are included in this Special Issue to provide immediate benefit for researchers in regenerative biology, hematopoiesis, brain\u2013skin communications, or environmental health. Ellman et al. described a tutorial paper for setting up an apex resection model to study heart regeneration in lab settings, providing an improved development model and assessing the heart\u2019s capacity for regeneration using zebrafish. Huang et al. discovered that NELF (negative elongation factor), the promoter-proximal pausing controller of RNA polymerase II, is specifically involved in granulocyte development but not erythrocyte development, emphasizing the importance of NELF in regulating hematopoiesis. In addition, Ren et al. identified a biological mechanism that underlies environmental change, particularly acute cold resistance. Furthermore, Hong et al. developed a platform for screening MSH-related activity, identifying an effective chemical treatment for autism spectrum disorder.FOXC1 and PITX2 and discussed their analyses on the utility of zebrafish with the corresponding gene mutations for dissecting potential molecular mechanisms and therapeutic approaches.Finally, three review articles were included in the Special Issue in order to highlight the current status of disease modeling using zebrafish and the underlying molecular pathogenesis. Specific topics include hematopoiesis, rare neurological disorders, and Axenfeld\u2013Rieger syndrome (ARS): (1) Zhang et al. summarized a comprehensive review on erythropoiesis using zebrafish models to explore human anemias; (2) Son et al. described the disease modeling of rare neurological disorders, focusing on zebrafish models; (3) French C.R. wrote a review on the development of ARS induced by mutations in In summary, this Special Issue is a collection of 16 relevant articles that support the use of zebrafish modeling in genetics and genomics for treating human diseases. To further boost the benefits for the communities of researchers who use zebrafish models, we are accepting original research papers or review articles for the 2.0 version with a setup similar to this Special Issue: \u201cZebrafish: A Powerful Model for Genetics and Genomics 2.0\u201d."} +{"text": "Recognizing the future leaders of Hematology is fundamental to safeguarding tomorrow's driving force in innovation. Indeed, early career researchers have faced a significant and potential \u201clong\u201d impact from the pandemic , 2.Therefore, this Research Topic aimed to showcase the high-quality work of internationally recognized researchers in the early stages of their careers. We highlighted research by leading scientists of the future across the entire breadth of Hematology, and present advances in theory, experiment and methodology with applications to compelling problems. Contributions to the Research Topic were by invitation only. All Rising Star researchers were suggested by established Editors within the Frontiers board in recognition of their influence on the future directions in their respective fields.All articles submitted to us for this Research Topic underwent a rigorous peer review process. Ultimately, 11 articles were published.Fouquet et al. highlighted the link between genotype and the unusual clinical phenotype.(i) An interesting case report in hematology was presented by analysis of a heterozygous somatic BLNK mutation associated with T-cell LGL (large granular lymphocyte) leukemia and autoimmune diseases. Liang et al. indicated the feasibility of the NGS approach and the evaluation of its therapeutic effect.(ii) Application of metagenomic next-generation sequencing (NGS) in the diagnosis of visceral leishmaniasis and its treatment evaluation was presented by a case report. Fattizzo and Motta covered a broad spectrum of novel and exciting treatment options in rare congenital and acquired anemias.(iii) A review article presented an update on emerging treatment strategies in rare anemias. Fu et al. presented an artificial intelligence method of excellent performance for the diagnosis and potentially automatic detection of malaria in the future.(iv) An original article reported an intelligent detection method for plasmodium based on self-supervised learning and attention mechanism. Selvakumar et al. focused on epidemiology and potential mechanisms for adverse long-term sequelae of iTTP, best practices in survivorship care, and presented a research agenda for the future.(v) A review article on immune thrombotic thrombocytopenic purpura (iTTP) provided up-to-date knowledge on long-term outcomes and survivorship. Ramadas and Sparkenbaugh reported novel insights into the activation of PAR1 by APC and thrombin, the APC-EPCR-PAR1 axis, and their potential roles in SCD.(vi) A mini review article discussed activation of APC-EPCR-PAR1 axis in sickle cell disease (SCD). Kerbauy et al. reviewed literatures on applying these novel techniques in autologous and allogeneic transplantation across different clinical entities.(vii) A review article focused on total marrow irradiation in hematopoietic stem cell transplantation for hematologic malignancies. Lysandrou et al. described the study rationale and design, including patient screening, product manufacturing, infusion, and participant follow-up to data collection, management, and analysis.(viii) A study protocol article presented the phase I/II trial of induced HLA-G+ regulatory T cells in patients undergoing allogeneic hematopoietic cell transplantation from an HLA-matched sibling donor. Ahmed et al. reported that many patients who were eligible for ide-cel were not able to secure a timely slot, with high mortality rates as a result.(ix) A brief research report article highlighted \u201cwaitlist mortality\u201d for myeloma patients with limited access to BCMA therapy. Whyte has introduced readers into the world of \u201cthromboinflammation\u201d or \u201cimmunothrombosis\u201d.(x) A mini review article explained the interactions of the fibrinolytic and innate immune systems. Shah et al. provided in-depth knowledge of the concept of point-of-care (POC) manufacturing or decentralized in-house production.(xi) A review article delineated promises and challenges of a decentralized CAR T-cell manufacturing model. Considering the multi-thematic character of this Research Topic, our hope is to inspire researchers and physicians to continue their explorations into novel advances in Hematology.EG: Writing \u2013 original draft. ML: Writing \u2013 review & editing. PC: Writing \u2013 review & editing. MA: Writing \u2013 review & editing."} +{"text": "Workplace moral behavior and immoral behavior have received substantial attention over the past decades moral behavior, offering the diverse insights to the individual emotion-centric models and social cognitive process models, exploring the individual dynamic moral emotions and moral cognition before and after the conduct of workplace (im)moral behavior, and clarifying the respective boundary conditions. Totally, we have accepted 28 papers for publication in the Frontiers in Psychology, which deal with different aspects of workplace (im)moral behavior, exploring mechanisms of these behaviors from the perspectives of moral cognition and moral emotion, and investigating the relevant antecedents and outcome variables. Some studies examined positive leadership and its impacts on subordinate performance and behavior . We also included articles that investigate immoral behaviors in the organizations such as abusive supervision and counterproductive behavior. These studies have systematically examined the role of immoral behavior in the organizations from both moral cognition and emotion perspectives, which provide a complete understanding of the mechanisms underlying the immoral behavior. Moreover, we included one systematic review of executives' unethical behavior. Here, we provide an overview of the articles that have been included in this Research Topic.Saeed et al., which examines the impact of ethical leadership on knowledge sharing through the social learning theory. In this study, psychological ownership serves as a mediating variable and professional commitment serves as a moderating variable. The data was collected from employees of 307 Pakistani listed companies. The findings reveal that ethical leadership had a positive effect on knowledge sharing via psychological ownership which was buffered by professional commitment, enhancing the understanding in the field of leadership and knowledge management by identifying the role of ethics.The study \u201cLinking Ethical Leadership to Followers' Knowledge Sharing: Mediating Role of Psychological Ownership and Moderating Role of Professional Commitment\u201d was conducted by Cheng, Guo, et al. employ social information processing theory and social learning theory to build a moderated mediation model. Based on the sample of 557 participants, the results suggest that responsible leadership could inhibit unethical pro-organizational behavior and customer-oriented perspective taking partially may mediate the negative link between responsible leadership and unethical pro-organizational behavior.What about the relationship between responsible leadership and unethical pro-organizational behavior? In their work \u201cStanding in Customers' Shoes: How Responsible Leadership Inhibits Unethical Pro-Organizational Behavior,\u201d Chen J. et al., have used the sample of 287 teachers to examine the relationship between moral leadership and innovation performance.In their work \u201cThe Impact of Moral Leadership on Physical Education Teachers' Innovation Behavior: The Role of Identification with Leader and Psychological Safety,\u201d Zada S. et al. utilized social learning theory and data from 347 employees in 56 teams to investigate the relationship among servant leadership, psychological safety, and knowledge hoarding. The findings suggest that servant leadership negatively affects knowledge hoarding by positively influencing psychological safety, and mastery climate moderated the relationship.The article \u201cServant Leadership Behavior at Workplace and Knowledge Hoarding: A Moderation Mediation Examination,\u201d written by Zada M. et al., examined the impact of servant leadership on the task performance of employees in virtual working environments during the COVID-19 crisis. Drawing on the conservation of resources theory, the findings revealed that servant leadership is positively related to task performance in a virtual environment during crises. The study also found that psychological empowerment partially mediates this relationship, and perceived supervisor support positively moderates the relationship between servant leadership and task performance.The study \u201cHow Classy Servant Leader at Workplace? Linking Servant Leadership and Task Performance During the COVID-19 Crisis: A Moderation and Mediation Approach\u201d by Liu L. et al. have examined one type of destructive leadership, the self-serving leadership. Drawing from the social identity theory and 377 survey data via a three-wave time lagged design, the findings showed that self-serving leadership may induce employees' deviant behavior, organizational identification partially mediates self-serving leadership and employees' deviant behavior, and employees' moral identity negatively moderates the relationship between self-serving leadership and employees' organizational identification.In their work \u201cThe Influence of Self-Serving Leadership on Deviant Behaviors in the Workplace: A Moderated Mediation Model,\u201d Zhu et al.. This bibliometric analysis has reviewed 428 articles published between the years 2000 and 2020 on executives' unethical behavior in the emerging markets.The study \u201cExecutives' unethical behavior with directions for future research,\u201d is written by Xu et al. have used conservation of resources theory to build a moderated mediation model with emotional exhaustion as a mediating variable and supervisor-subordinate guanxi as a moderating variable. The data was collected from 440 tourism employees. The results show that leaders' knowledge hiding is positively linked to employees' withdrawal behavior and emotional exhaustion.In their work \u201cThe influence of leader\u2013signaled knowledge hiding on tourism employees' work withdrawal behavior: A moderated mediating model,\u201d Su et al., employed the social cognitive theory and social exchange theory to explain how, when and why self-sacrificial leaders may trigger knowledge sharing. Totally, 481 pair sample has been used to test the theoretical model. The results showed that good guanxi between employees and their leaders, could lead employees to better understand leaders' self-sacrificial behavior and engage in knowledge sharing.Regarding the relationship between self-sacrificial leadership and employees' knowledge sharing, they study \u201cHow Does Self-Sacrificial Leadership Foster Knowledge Sharing Behavior in Employees? Moral Ownership, Felt Obligation and Supervisor-Subordinate Guanxi,\u201d conducted by Liu X. et al.. Drawing upon purposeful work behavior theory and using two-wave time lagged design, data was collected from 216 employee-supervisor dyads. The findings showed that duty orientation and achievement orientation have opposite relationship to pro-social rule breaking. Furthermore, job autonomy strengthens the positive effect of duty orientation and the negative effect of achievement orientation on pro-social rule breaking.The study entitled \u201cDoes Self-Sacrifice Make Me Great? Research on the Relationship Between Employee Conscientiousness and pro-Social Rule Breaking\u201d is written by Wang and Ma used social comparison theory and a sample of 331 employees, to examine the interaction between perceptions of coworkers' receiving idiosyncratic deals and respective deprivation. The findings showed the moderating role of conscientiousness, which may weaken the relationship between deprivation and social undermining.We have included another contribution related to employee consciousness. In their work \u201cWhen Do Coworkers' Idiosyncratic Deals Trigger Social Undermining? \u2013 The Moderating Roles of Cored Self-Evaluations and Conscientiousness,\u201d Zhang H. et al., examined the impacts of three helping behaviors on helpers themselves. Drawing from the resource conservation theory and using a three-wave time-lagged design, the findings suggested that caring and coaching were more negatively associated with emotional exhaustion and helpers of these two styles were less likely to adopt subsequent deviant behaviors. This study has also identified the role of extrinsic career goals in the direct relationship between the three helping behavior and emotional exhaustion.They study \u201cWhen and How Workplace Helping Promotes Deviance? An Actor-Centric Perspective,\u201d conducted by Cheng, Hu, et al., uses the data collected from 218 salespeople in the internet technology service company and develops a contingent model to show how moral identity and impression management motives could moderate the links between pro-organizational motives, unethical pro-organizational behavior, and organizational behavior.The contribution \u201cA Contingency Perspective of Pro-Organizational Motives, Unethical Pro-Organizational Behavior, and Organizational Citizenship Behavior,\u201d written by Xiao J. et al., has examined the implicit coordination from the perspective of team mentality, and discussed the incentive mechanism of status competition by using knowledge sharing as a mediating variable and psychological safety as a moderating variable. The empirical study has used a sample of 367 employees from 44 companies. The findings revealed that prestige-type status competition was positively related to implicit coordination, while dominant-type status competition was negatively related to implicit coordination. Furthermore, knowledge sharing mediated the relationship between both types of status and implicit coordination, and psychological safety enhanced both relationships.The article \u201cStatus Competition and Implicit Coordination: Based on the Role of Knowledge Sharing and Psychological Safety\u201d written by Zhang M. et al. uncovered the situational factors that influence employees' unethical pro-organizational behavior to repay the organization. Based on the social exchange theory, the authors used multisource data from 139 human resource managers and 966 employees to examine why and how high-commitment work systems affect employees' unethical pro-organizational behavior. The findings revealed that high-commitment work systems promote employees' unethical pro-organizational behavior through relational psychological contract. Besides, the findings showed that balanced reciprocity beliefs strengthen the positive relationship between relational psychological contract and employees' unethical pro-organizational behavior.In the study \u201cResearch on the Relationship Between High-Commitment Work Systems and Employees' Unethical Pro-Organizational Behavior: The Moderating Role of Balanced Reciprocity Beliefs,\u201d Zhang Z. et al. conducted the study \u201cDoes Technostress Increase R&D Employees' Knowledge Hiding in the Digital Era?\u201d and introduced work exhaustion as a mediating variable for exploring how five sub-dimensions of technostress impact R&D employees' knowledge hiding. The authors have used job demand-resource theory to examine technostress\u2014an antecedent of knowledge hiding. The sample of 254 participants was collected through a two-stage survey. The findings showed that techno-invasion, techno-insecurity, and techno-complexity were significantly and positively associated with work exhaustion. Furthermore, work exhaustion mediated the relationship between aforementioned three variables and knowledge hiding, while workplace friendship negatively moderated the association between techno-invasion, techno-insecurity and work exhaustion, reducing the emergence of knowledge hiding, while it also positively moderated the association between techno-complexity and work exhaustion.Wu et al. examined the relationship between workplace suspicion and employees' silence, as well as the mediating role of knowledge hiding from a colleague's perspective. Drawing from resource conservation theory and self-regulation theory, data of 303 pair sample from 23 companies in China was collected through three-waves questionnaire method. The results revealed that workplace suspicion positively influenced employees' silence via knowledge hiding, and knowledge-based psychological ownership strengthened the mediating effect. However, face consciousness weakened the positive impact of workplace suspicion on knowledge hiding.The study \u201cWorkplace Suspicion, Knowledge Hiding, and Silence Behavior: A Double-Moderated Mediation Model of Knowledge-Based Psychological Ownership and Face Consciousness\u201d conducted by Zheng et al. uses 176 valid responses to examine whether employees' personal ethics and perceptions of corporate hypocrisy can be beneficial for reducing employees' mental fatigue. The theories used to develop the hypotheses are stakeholder theories and sociological theories. The findings showed that employees' mental fatigue reduces when internal or external corporate social responsibility (CSR) has a positive impact on employees' altruistic choice and employees' mental fatigue increases when CSR has a negative effect on ethical egoism.The study \u201cImpacts of Corporate Social Responsibility on Employees' Mental Fatigue\u201d written by Jiang et al. conducted the study of \u201cBenefits of Non-Work Interactions with Your Supervisor: Exploring the Bottom-Up Effect of Employee Boundary Blurring Behavior on Abusive Supervision\u201d and explored how employees' boundary blurring behavior can prevent themselves from being abused by supervisors. Drawing from the self-disclosure theory, authors have used a scenario-based experimental study and a multi-wave field study to find out that employees' boundary blurring behaviors inhibit the emergence of abusive supervision through the mediating effect of supervisor liking toward the employee.Existing research seldom employs a bottom-up perspective to examine how employees can reduce abusive supervision. In their work, Fousiani et al. explored the effect of morality and competence in recruiters' hiring decisions. The authors used the Big Two theoretical framework to examine how instrumental or relational goals of organizations might influence the importance of morality or competence of candidates during the hiring process. The authors conducted three studies to test the proposed hypotheses. The findings showed that the primacy effect of morality might hold when organizational goals are relational but might get reversed when organizational goals are instrumental. They also found that perceived appropriateness of a candidate positively affects hiring recommendations.In the article \u201cAppearing competent or moral? The role of organizational goals in the evaluation of candidates,\u201d Carminati and H\u00e9liot, titled \u201cBetween Multiple Identities and Values: Professionals' Identity Conflicts in Ethically Charged Situations,\u201d adopted a qualitative approach. Forty-seven semi-structured interviews had been conducted among doctors and nurses working for the English National Healthcare Service. The findings showed that micro processes such as cognitive and emotional perspective taking, plus identifying with the other may trigger identity conflict.The contribution of Khan et al. examined the relationship between perceived overqualification and counterproductive work behavior (CWB) among textile sector employees, considering job boredom as a mediator and job crafting as a moderator. The findings showed a positive relationship between perceived overqualification and CWB. Furthermore, the study found that job boredom mediated the relationship between perceived overqualification and CWB, and job crafting moderated the positive association between perceived overqualification and job boredom.The study \u201cPerson\u2013Job Misfit: Perceived Overqualification and Counterproductive Work Behavior\u201d by Shen et al. titled \u201cHow I Speak Defines What I Do: Effects of the Functional Language Proficiency of Host Country Employees on Their Unethical Pro-Organizational Behavior\u201d investigates the relationship between functional language proficiency and unethical pro-organizational behavior of host country employees in multinational corporations (MNCs). The authors used data from 309 full-time host country employees to test their predictions guided by social identity theory. The findings suggested that host country employees' functional language proficiency enhances their unethical pro-organizational behavior through their linguistic group identification and moral disengagement.The article by Liu J.-N. et al.. Drawing from planned behavior theory and 200 civil servants' data, the findings showed that public sector leaders' information-sharing behavior is positively related to their subordinates' taking charge behavior, and public service motivation mediates this relationship. The results also found emotional trust strongly moderates the effect of leaders' information-sharing behavior on subordinates' taking charge behavior.The article \u201cHow does leaders' information-sharing behavior affect subordinates' taking charge behavior in public sector? A moderated mediation effect,\u201d is written by Dai et al. have examined the role of positive workplace gossip through a moderated mediation model. Data from 327 employees in the Pearl River and Yangtze River Delta regions of China was collected to test the theory model. The results showed that positive workplace gossip may promote employee innovation.In their work \u201cIs not workplace gossip bad? The effect of positive workplace gossip on employee innovative behavior,\u201d Wang and Tang aimed to investigate the impact of daily supervisor abuse and coworker support on daily work engagement. Drawing from the conservation of resources (COR) theory, the study utilized a daily diary approach and collected data from 73 employees during five consecutive days in China. The results showed that daily abusive supervision had a negative impact on daily work engagement and that daily negative emotions mediated this relationship. Coworker support had a cross-level moderating effect between daily abusive supervision and daily negative emotions.The article \u201cHow Daily Supervisor Abuse and Coworker Support Affect Daily Work Engagement\u201d by Xiao W. et al. investigates the impact of crowdsourcing innovation community reference on creative territory behavior from the perspective of reference group theory. A two-stage survey has been used and 524 valid responses were collected. The results suggested that the crowdsourcing innovation community reference influences members' impression management behavior and then inhibits creative territory behavior. Interestingly, there are different community reference effects among members of different community age groups. These findings contribute to understanding the influence of the crowdsourcing innovation community on crowd participation decision-making and suggest implications for exploring the cooperation mechanism of crowdsourcing innovation.The study \u201cInfluence of Crowd-sourcing Innovation Community Reference on Creative Territory Behavior\u201d by Chen H. et al. examined the relationship between leader's aggressive humor and bystander's workplace withdrawal behavior. The study used the Cognitive-Affective Personality System Theory and collected data from 443 employees and their direct supervisors in the Chinese enterprises. The results showed that leader-aggressive humor positively affected bystander affective rumination and bystander workplace anxiety, which in turn mediated the relationship between leader-aggressive humor and bystander workplace withdrawal behavior. Additionally, organization-based self-esteem moderated the indirect impact of leader-aggressive humor on bystander workplace withdrawal behavior through bystander affective rumination and bystander workplace anxiety.In another perspective, the article \u201cWhy Does Leader Aggressive Humor Lead to Bystander Workplace Withdrawal Behavior?\u2014Based on the Dual Path Perspective of Cognition Affection,\u201d Dai et al. have explored the effect of positive workplace gossip on employee's innovative behavior; Zhang H. et al. have studied when and how workplace helping promotes workplace deviance from an actor-centric perspective; Zhang M. et al. have explained the dark side of high-commitment work systems and linked it to employees' unethical pro-organizational behavior. These findings point out the importance to review and manage workplace (un)ethical/(im)moral behaviors with a balanced but critical perspective.This Research Topic has made the following contributions. First, in this Research Topic, scholars attempted to conduct research from different perspectives and methodologies on the \u201cgood\u201d behaviors and negative behaviors , which have enriched the research on the \u201cdark sides\u201d of workplace positive behavior and the \u201cbright sides\u201d of workplace negative behavior. For example, the study of Cheng, Guo, et al. have examined how positive leadership can prevent employees' workplace unethical/immoral behaviors. This contribution has greatly enriched the current understanding of how responsible leadership is related to employees' unethical pro-organizational behavior. Furthermore, this study has explicitly explained the impacts of positive leadership and the important role that positive leadership can play in inhibiting and governing the negative behaviors of employees.Second, prior research has explored the relationship between various types of destructive leaders' behavior and workplace unethical/immoral behaviors such as counterproductive work behavior, and the relationship between positive leadership and employees' positive workplace behaviors such as innovative behavior and prosocial behavior. However, how positive leadership can help prevent unethical organizational behaviors requires further development. In this Research Topic, Xu et al. have explored the influence of leader-signaled knowledge hiding on tourism employees' withdrawal behavior. Jiang et al. have advocated the benefits of employees' non-work interactions with their supervisors and verified the bottom-up effect of employee boundary blurring behavior on abusive supervision. Liu J.-N. et al. have investigated how leaders' information sharing behavior affect subordinates' taking charge behavior in public sector. Chen H. et al. have further studied the relationship between leader's aggressive humor and employee's withdrawal behavior from a bystander's perspective. Su et al. have demonstrated the impacts of leaders' self-sacrificial behaviors and supervisor-subordinate guanxi on employees' knowledge sharing. Wang and Tang have integrated daily supervisor abuse and coworker support in influencing employees' daily work engagement. These contributions point out that when managing leaders' or employees' workplace (un)ethical/(im)moral behaviors, one should not only look for the reasons and respective governance strategies from the actors' perspectives. In fact, the influence of others is an important source of the formation of individual behaviors in the workplace. Managers should improve the positive interpersonal interactions through building the healthy and meaningful relationship at the workplace, and reduce the contagion effects through lead by example, open communication, promotion of positive corporate culture, rewards on the ethical/moral behaviors, and so on.Third, this Research Topic focuses on the mechanisms by which relationships and interpersonal interactions between supervisors and subordinates and between coworkers influence each other's (un)ethical/(im)moral behaviors. We have included studies of top-down leadership behaviors and their impacts on employees' behaviors, and the studies of the bottom-up employees' behaviors and their impacts on leaders' behaviors. The (un)ethical/(im)moral behaviors in the workplace are examined through multiple directions. For example, Liu J.-N. et al.; Su et al.; Xiao J. et al.; Saeed et al.), knowledge hiding , and knowledge hoarding have attracted increasing attention. The contributions have employed theoretical lens such as the conservation of resources theory, the self-regulation theory, the framework of \u201ccognition-motivation-behavior,\u201d and the psychological safety and psychological ownership perspectives, to examine the formation mechanism between knowledge sharing behavior and counterproductive knowledge behavior. In addition, these contributions have provided a systematic review related to the moral concern of knowledge hiding. Thanks to these studies, we are able to enrich the knowledge hiding research by diversifying the existing theoretical perspectives, realizing the interdisciplinary studies, introducing and integrating the moral ownership, moral self-regulation and other moral psychology, cognitive psychology and other disciplines in the theoretical perspectives, forming new insights to call for research on the novel knowledge hiding phenomenon . Furthermore, the contributions that have been included in this Research Topic have taken the lead in exploring how technostress increase R&D employee knowledge hiding in the digital era. These findings draw practitioners' attention to the ethical nature of knowledge hiding behaviors and the respective moral issues. This Research Topic offers practitioners with knowledge on how to better anticipate the harmfulness of immoral behavior and the dark sides of moral behavior, developing the knowledge governance practices from the perspectives of employees' moral emotion and moral cognition.Last, knowledge-related behaviors such as knowledge sharing (see for example All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication."} +{"text": "Animal welfare and economic implications of infectious diseases in cattle demand an efficient surveillance as the foundation for control and eradication programmes. Bovine respiratory syncytial virus (BRSV), Parainfluenza virus type 3 (PI3V), Bovine herpes virus-1 (BoHV-1), Bovine viral diarrhoea virus (BVDV), and Enzootic bovine leukosis virus (EBLV) cause common and often underdiagnosed diseases in cattle that are endemic in most countries [ Th. Th31]. All assays were highly sensitive and able to discriminate the presence of antibodies from the 1\u200a:\u200a2 dilution. Nonetheless, the dilution that characterized the detection limit varied slightly for each antigen, with the 1\u200a:\u200a1024 dilution successfully discriminating positive samples from non-specific binding values for all five antigens tested. Albeit assays could discriminate positive samples in a wide range of dilutions, the MFI was diminished at low dilutions (1\u200a:\u200a8 or below). This counter-intuitive phenomenon (hook effect) occurs when an excess of antibodies impairs their effectiveness to form immune complexes . With hiet al. [et al. 2007 [et al. [Multiplexing in its various platforms will likely substitute monoplex assays for massive serological surveillance. As with ELISA, xMAP allows for the in-house development of assays, optimizing the antigens of interest and permitting the customization of antigen panels to suit specific needs. The development of an xMAP assay is no more complicated than that of an ELISA that uses the same antigens, in most cases yielding similar or even higher sensitivity. Several multiplex assays have been developed by other research groups to study bovine diseases. Fontana et al. created al. 2007 ). Anders [et al. devised [et al. . NonetheThe viruses chosen for the development of the pentaplex assay in this study , have been targeted in eradication/control programmes in Europe and North America due to increasing disease prevalence and the"} +{"text": "With remarkable progress being witnessed in recent years in the development of sensors, these advances in sensor technology provide unprecedented opportunities for (1) the early diagnosis and prevention of human diseases by detecting critical biomarkers; (2) health assessments by monitoring and analyzing human physiological signals in healthcare and biomedical applications; and (3) the efficient evaluation of human-health-relevant environmental factors by monitoring and measuring environmental determinants.This Special Issue aims to provide an overview of recent advances being made in sensing technology, including sensors and smart devices, and their applications in healthcare, biomedical, and environmental research. The purpose of this Special Issue is to publish a collection of papers that highlight insightful and influential concepts, designs, algorithms, and techniques in this field. We expect these high-quality papers to be widely read and cited for improving the understanding of physiological mechanisms through accurate and sensitive detection, providing new perspectives and technology to solve health-related problems and state-of-the-art designs to integrate multifunctional sensors.Mingyu Sun et al. proposedRamona B. Damalerio et al. fabricatHamed Samimi and Hilmi R. Dajani focused Haoran Liu et al. proposedChuan Dai et al. proposedAli Barzegar Khanghah et al. presentePanayiotis Antoniou et al. investigJie Huang and Daqing Huang designedn = 19) were increased to either 25, 35 or 45 mmHg for 4 h before returning to normal pressure levels at 15 mmHg for the remaining 4 h. Changes in tissue hydration were assessed by differences in spectral slopes between 0.4 and 0.8 THz. They demonstrated that THz can noninvasively assess the corneal endothelium and provide valuable complimentary information for the study and diagnosis of corneal diseases that perturb the hydration of the tissue.Andrew Chen et al. utilizedPengsu Mao divided n = 8), fractal analysis (n = 3), recurrence quantification (n = 2), and the Lyapunov exponent (n = 2). This review demonstrated that, in adolescents assessed in a real context, entropy measures are preferred when studying the complexity of human movement, especially when examining multiscale entropy.Sandra Silva et al. aimed toWe would like to express our profound appreciation for the authors and reviewers who made this Special Issue possible. Our reviewers and authors have dedicated tremendous effort into the preparation of this Special Issue, in order to allow it come to life and flourish."} +{"text": "Pain Medicine community regarding the recent Cochrane review entitled \u201cSpinal cord stimulation for low back pain\u201d by Traeger et al.,We write to the When making broad recommendations about the use of SCS in the real world, the full context and breadth of available literature must be taken into consideration. The authors designed search criteria that included clinical trials comparing SCS to placebo or \u201cno treatment\u201d studies with parallel-group design).While high-quality placebo-controlled studies of SCS for CLBP are indeed needed, there is an abundance of Level 1 comparative effectiveness data that supports the effectiveness of SCS for CLBP. These studies have long-term follow-up and answer key clinical questions, such as defining the optimal SCS waveform for a specific patient phenotype, and whether SCS provides outcome and cost benefits over revision decompression and/or fusion surgery. The summation of these data demonstrates large magnitudes of effect, although with indirectness and potential for risk of bias. As such, GRADE assessment should reveal moderate-certainty evidence of medium to long-term effectiveness of SCS for CLBP.2 statistical analysisTraeger et al.,The authors\u2019 conclusions about SCS\u2019s probable lack of efficacy rested singularly on the Hara et al.Surgery, the ultimate placebo and 2) Hippocrasy: How doctors are betraying their oath. Furthermore, while a pain physician was acknowledged at the end of the publication,Generalizations about CLBP care interventions, drawn from data limited by narrow search criteria, are problematic and misleading. The reader is left questioning whether Traeger et al.We respectfully urge the Cochrane Library to retract and revise the Traeger et al."} +{"text": "Tan et al. and Zhu et al. A diversity of preclinical/translational research studies were also presented in the Research Topic. Lozon et al. presented a novel chemotherapy combination that was validated in vitro in colon cancer and lung cancer cell lines. Abdollah et al., Yang et al., and Lee et al. highlighted a promising anticancer activity for a variety of natural products against hepatocellular carcinoma. On the other hand, the work of Lee et al., Lin et al., Irshad et al., and Kuttikrishnan et al. presented mechanistic insights in pancreatic cancer, breast cancer, non-small-cell lung cancer, and leukemia. The Research Topic also encompasses two literature reviews by Bannoura et al. and Yuan et al.Continuous research efforts have significantly unraveled fundamental molecular pathways that have contributed to paving the way for the development of a multitude of therapeutic strategies, which have and expanded the arsenal of weapons against cancer. This has been tremendously reflected on the uplifting of the cancer survival curve and the improvement of the quality of life of cancer patients. The Research Topic herein was dedicated to highlighting the emerging talents of student researchers pursuing studies in the area of pharmacology of anticancer drugs. The articles in this Research Topic have shed light on the quality and diversity of student researchers across this field and focused on three main themes: 1) Remodeling Cancer Epigenome: Therapeutic Advances; 2) Cancer Immunosurveillance: From Mechanisms to Therapy; 3) Anticancer Drug Discovery and Translational Oncology. The studies herein include two reports of clinical trials by Zhu et al. employed a Markov decision-analytic model to compare the cost-effectiveness of T-DXd with chemotherapy in HER2-low ABC patients. While T-DXd showed improved quality-adjusted life years (QALYs), its incorporation led to increased costs and an unfavorable incremental cost-effectiveness ratio (ICER) compared to chemotherapy, making it devoid of cost-effectiveness in HER2-low ABC patients in the United States. The authors, however, believe that it may still provide health benefits to patients with HR+/HER2-low ABC.Trastuzumab deruxtecan (T-DXd), an antibody\u2013drug conjugate, has shown antitumor activity in patients with HER2-low advanced breast cancer (ABC) in clinical trials. However, the cost-effectiveness of T-DXd was not clearly established. In order to determine whether the benefits of this treatment outweigh its costs, Tan et al. suggested that pembrolizumab plus nab-paclitaxel might be a potential treatment option for patients with cholangiocarcinoma (CCA), which is a highly aggressive malignancy with poor overall survival. In this report, a metastatic extrahepatic CCA patient achieved durable response and good tolerance to a combination treatment of pembrolizumab plus nab-paclitaxel following progression on a chemotherapy of gemcitabine and capecitabine.In their case report, Lozon et al. contributed an original article highlighting the importance of safranal (SAF) as a promising candidate-sensitizing agent of human colon cancer cells (HCT116) and lung cancer cells (A549) to the cytotoxic effect of the topoisomerase inhibitor topotecan (TPT). The combination augmented TPT-induced alterations in DNA repair and boosted the incidence of double-strand breaks with subsequent induction of apoptosis. The sensitization only occurred when the cells were pretreated with SAF 24\u00a0h before TPT treatment. However, simultaneous exposure or adding SAF 24\u00a0h after TPT did not recapitulate the same results. Hence, the effect was sequence dependent.Abdollah et al. aimed to determine whether fucoidan\u2019s chemomodulatory benefits may be enhanced by using it in combination with the FDA-approved antiangiogenic drugs sorafenib and Avastin (bevacizumab) in order to augment their anticancer activity in hepatocellular carcinoma (HCC) cells. The authors reported that the combination treatment inhibited the PI3K/AKT/mTOR and KRAS/BRAF/MAPK pathways in addition to apoptosis induction in HCC both in vitro and in vivo.An article by Yang et al. identified a potential pharmacological mechanism by which Ganji Fang (GJF) can treat HCC at the systemic level. The underlying mechanisms were shown to involve immune control, cell migration, cell proliferation, apoptosis, and inflammation induction. Furthermore, it was demonstrated that the G0/G1 phase cycle arrest subsequent to the apoptosis after GJF treatment in liver cancer cells was triggered by blocking the PI3K/Akt signaling pathway. Pachymic acid has been shown to be the significant active component of GJF that exhibits anticancer action, and EPHA2 may be a potential key target for GJF in HCC.Lee et al. reported that WGM extracts induced cell death by targeting mTOR and MAPK signaling pathways in liver cancer cells, suggesting that they could be used as a pharmacologically safe natural dietary chemopreventive agent for HCC treatment.It was previously found that white genius mushroom (WGM), a popular edible mushroom in Taiwan, mediates strong antiproliferative activities against human Hep3B liver cancer cells. However, the underlying mechanisms have not been fully investigated. Lee et al. investigated the efficacy and molecular mechanisms of ivermectin/gemcitabine combination in pancreatic cancer. They observed that a combination treatment of ivermectin and gemcitabine suppressed pancreatic cancer both in vitro and in vivo more effectively than gemcitabine alone. Mechanistically, this combination was found to exert its effects by inhibiting cell proliferation via G1 cell cycle arrest and augmenting apoptosis by inducing mitochondrial dysfunction. Based on these findings, the study concluded that ivermectin, an antiparasitic drug, can exhibit synergistic effects with gemcitabine and may be repurposed to serve as a promising therapeutic agent for pancreatic cancer therapy.The article by Lin et al. demonstrated the anticancer effects of gallic acid, a phenolic acid known for its antioxidant properties, against triple-negative breast cancer cells in vitro. The study showed that gallic acid was able to inhibit the growth of HCC1806 cells and stimulate their apoptosis by triggering the production of reactive oxygen species, which modulate the PI3K/AKT/EGFR and MAPK signaling pathways.Irshad et al. employed integrative network pharmacology, molecular docking, and in vitro experiments to elucidate the mechanism of action of glycyrrhetinic acid (18\u03b1-GA), a triterpenoid found in licorice against non-small-cell lung cancer (NSCLC). Their network pharmacology study identified EGFR, AKT1, PI3KR1, MAPK1, IGF1, and SRC as crucial hub targets for 18\u03b1-GA against NSCLC. The authors further showed that 18\u03b1-GA augmented G1 cell cycle arrest, triggered apoptosis, reduced the migratory potential, and inhibited the EGFR-PI3K/AKT pathway in NSCLC cell lines.Kuttikrishnan et al. explored the role of neosetophomone-B (NSP-B), a meroterpenoid fungal secondary metabolite, on the FOXM1/BUB1B signaling pathway. The development and progression of various types of cancer, including chronic myelogenous leukemia, have been linked to the abnormal expression of FOXM1 and BUB1B genes. A TCGA data analysis elaborated that BUB1B is overexpressed in most cancers and linked with poor prognosis. Using gene expression profiling, the authors showed the significant downregulation of BUB1B in leukemia cells treated with NSP-B. In addition, they validated their in silico findings in vitro by showing that NSP-B suppresses the expression of FOXM1 and BUB1B in a dose-dependent manner, leading to compromised cell viability and apoptosis induction in leukemia cells.Bannoura et al. summarized the recent data on targeting KRAS G12D, which is among the most common mutations (45%) in pancreatic cancer that are associated with poor prognosis. The article discussed several modalities under development for targeting KRAS G12D, including small molecule inhibitors and immunotherapy.The minireview by Yuan et al. contributed a bibliometric article of the literature from the Web of Science Core Research Topic (2002\u20132021) on the emerging trends and research foci of the natural compound berberine in cancer. Berberine is a multitarget Chinese medicine monomer compound that is extensively studied for its antitumor/antiproliferative effects and its capacity to sensitize cancer cells to chemotherapy. The collected data showed that berberine exhibits anticancer capacity through a diversity of mechanisms, including halting the cell cycle, inhibition of tumor cell invasion and migration, and inducing autophagy and apoptotic cell death."} +{"text": "Natural products have played important roles in drug discovery and development as more than 60% drugs are associated with natural products . Yunnan Chen et al., Lei et al., Yan et al., and Clements-Decker et al.), new synthetic approaches towards the key units of natural products , total synthesis of natural products , and structure-activity-relationship (SAR) studies on bioactive natural-product-like molecules . Taken overall, 10 contributions including 2 reviews and 8 original research articles comprise this Research Topic.This Research Topic presents a Research Topic of reviews and original research articles on isolation and structural characterization of novel natural products . A study on antioxidant and anti-inflammatory activity of constituents isolated from Dendrobium nobile (Lindl.) by Lei et al. shows that nineteen compounds, including two new vitamin E homologues, one new sesquiterpene, and two new dendrobines were isolated. New compound aldehyde-\u03b1-tocopherol demonstrated significant antioxidant activity compared with ascorbic acid (VC), as well as equal cytotoxic effect against Hela cell lines to cisplatin, indicating its potential application in the pharmaceutical and food industries. Yan et al. found fourteen C19-lycaconitine-type diterpenoid alkaloids, including six new alkaloids, grandiflolines A\u2013F, isolated from Delphinium grandiflorum L. New alkaloids grandiflolines A-C and E possess a characteristic \u03942,3 functional group in the A ring, while grandiflolines E and F feature a rare OH-16 substituent. New alkaloids exhibit potential inhibition activities of NO in LPS-activated RAW 264.7 macrophages. A review conducted by Clements-Decker et al. delved into the antimicrobial properties of lipopeptides derived from various bacteria strains. The study sheds light on the structures and recently discovered frameworks of lipopeptide families produced by these bacteria, which possess promising antimicrobial properties. Furthermore, utilizing the genome mining approach, underexplored sources of novel antimicrobial lipopeptides have been uncovered. A detailed understanding of the mode of action and biosynthesis included in the review provides a clear path for the development of potential antimicrobial therapeutics in the future.Rao et al. reported a new transitionmetal-free direct oxidative cyclocondensation reaction. The protocol highlighted the use of readily available o-aminobenzyl alcohols and N,N-dimethyl enaminones as starting materials, thus provided a flexible strategy for the preparation of 3-substituted or 3,4-disubstituted quinolones with broad substrate scope in moderate to excellent yields. The strategy enriched the quinoline synthesis method. Jiang et al. reported an effective palladium-catalyzed method for the synthesis of aryl acrylonitriles. This process uses simple and readily available arylacetonitriles and vinyl halides/triflates as raw materials, and the resultant aryl acrylonitriles can undergo a series of useful conversion reactions, such as reduction, hydrolysis and epoxidation.Wei et al. reported a general strategy for the total synthesis of arylnaphthalene lactone lignans (NALLs) including justicidins B and E and taiwanin C. Key features of this synthesis include an aryl\u2013alkyl Suzuki cross-coupling, a novel intramolecular cation-induced cyclization, and a base-mediated oxidative aromatization. Wei et al. explored the uses of dioxinones in synthesizing macrocyclic natural products and terpenoids. The review highlights the versatility and efficiency of dioxinones as reactive intermediates, making them valuable tools for the synthesis of diverse and complex natural products.Zhou et al. designed and synthesized a series of anti-ZIKV active compounds, acetylarylamine-S-DACOs, and conducted in-depth research on the action mechanism of the representative compound through molecular docking analysis and a series of biological experiments. The results confirmed the anti-ZIKV activity at the molecular and protein levels, and discovered that this selected compound targeted ZIKV RNA-dependent RNA polymerase (RdRp). Yin et al. created a series of podophyllotoxin derivatives containing nitrogen-containing heterocycles, which may have potential as anticancer drugs. After synthesizing several derivatives, imidazolium salts and triazolium salts were found to be the most effective against different types of human tumor cells. Additionally, experiments on cell cycle and apoptosis revealed that these compounds could trigger G2/M cell cycle arrest and apoptosis in HCT-116 cells.We hope that this Research Topic could provide an opportunity to school fellows of YNU to introduce their recent research findings in natural product chemistry to the chemical community, which could also provide an incentive for further scientific collaborations between school fellows of YNU and other researchers in this field."} +{"text": "Mast cells have existed for almost 500 million years in many The international Journal of Molecular Sciences recently published Special Issues dedicated to aspects of the pathophysiology of mast cells, such as \u201cMast cells in human health and diseases\u201d edited by Giovanna Traina , and \u201cMaThe emphasis of this Special Issue entitled \u201cThe Role of Mast Cells and Their Inflammatory Mediators in Immunity\u201d was to address aspects of mast cell involvement in immune and inflammatory processes not covered by previous volumes.A major review communicated via Dr. Domenico Ribatti and authored by Solimando et al. described in detail the production of interleukins by mast cells, as well as their important role in immune processes . Given tFor the longest time, the notion persisted that all mast cells are the same. Then, they were separated into mucosal-type (MMC), containing only tryptase, and those of the connective-tissue-type (CTMC), containing both chymase and tryptase. However, increasing evidence indicates that there are unique subpopulations with distinct histological and secretory characteristics. The submission via Dr. Lars Hellman by Akula et al. using quantitative transcriptome analysis describes a particular subtype of mucosal mast cell in bronchoalveolar lavage fluid (BALF) that contributes to inflammation in the lungs of asthmatic horses that mayThe paper submitted via Dr. Claudia Gonz\u00e1lez-Espinosa by Mart\u00ednez-Aguilar et al. identifies the augmenting effect of lysophosphatidylinositol on mast cell functions promoting inflammation through the differential participation of the GPR55 and cannabinoid 2 (CB2) receptors . PI servThe submission via Dr. Zenke by De Toledo et al. describes the important finding that human-induced pluripotent stem cell (hiPSC)-derived mast cells can mimic mastocytosis-like mast cells , suggestThe article by Drs. Tsilioni and Theoharides reports for the first time that nanomolar amounts of recombinant coronavirus spike protein stimulated human mast cells to secrete proteases via the activation of the angiotensin-converting enzyme 2 (ACE2) receptor, but stimulates the release of the proinflammatory cytokine IL-1b via the activation of TLR4; these processes are augmented by alarmin IL-33 . This noThe contribution communicated via Dr. Tsilioni by Jingshu et al. reported the development of a 3-D hydrogel formed by collagen I and culture human mast cells and showed that different A\u03b2 peptides could stimulate mast cells to secrete inflammatory mediators. This model could incorporate microglia and neurons that could better reflect the mechanisms involved in the pathogenesis of neuroinflammation and Alzheimer\u2019s disease .The submission submitted via Dr. Marcus Maurer by Dr. Buttgereit et al. describes that the mitochondrial Complex I can act as a critical innate inhibitory component of the mast cell secretion of pro-inflammatory mediators . ComplexFinally, the review by Theoharides and Kempuraj discusses the potential role of the ezrin, radixin, moesin (ERMS) family of proteins, especially moesin, in regulating the exocytotic mechanism of mast cell secretion of granule-stored mediators .These papers highlight key aspects of the potential role of mast cells in immunity and inflammation by presenting novel findings on the subtypes, regulatory mechanisms, and unique associations with innate and external triggers. However, there are still gaps in our knowledge, such as the effect of the microenvironment on the tissue mast cell phenotype and ways to regulate mast cell activation. An improved understanding may be obtained through the use of in vitro disease surrogate models using organoids with hiP"} +{"text": "Leishmania parasites and cancer cells. The information regarding common expressed proteins, as possible therapeutic targets, in Leishmania parasites and cancer cells is scarce. Therefore, the current study reviews proteins, and investigates the regulation and functions of several key proteins in Leishmania parasites and cancer cells. The up- and down-regulations of such proteins were mostly related to survival, development, pathogenicity, metabolic pathways and vital signalling in Leishmania parasites and cancer cells. The presence of common expressed proteins in Leishmania parasites and cancer cells reveals valuable information regarding the possible shared mechanisms of pathogenicity and opportunities for therapeutic targeting in leishmaniasis and cancers in the future.The association of leishmaniasis and malignancies in human and animal models has been highlighted in recent years. The misdiagnosis of coexistence of leishmaniasis and cancer and the use of common drugs in the treatment of such diseases prompt us to further survey the molecular biology of Leishmania. Annually, approximately 1.5\u20132 million new cases are reported worldwide being 310 million people at risk. The mortality rate of the disease varies from 40\u00a0000 to 70\u00a0000 cases per year are mostly restricted to the skin lesions with diverse appearances, from localized to body extended wounds or mucosal affectations. However, visceral leishmaniasis (VL) is characterized by severe organic symptoms and might lead to death can also raise the risk of developing some types of cancers and may contribute to the appearance of malignancies which makes them possible models to study host\u2013parasite interactions and mechanisms of cancer Liao, . Howeveret al., et al., et al., et al., et al., et al., et al., Leishmania parasites also modulate and destabilize the host chromatin structure leading to potential changes in relevant immune-related genes and responses pathway and PKC. The metabolic action of miltefosine affects the biosynthesis of glycolipids and membrane glycoproteins of the Leishmania parasite, leading to apoptosis signalling , interleukin-12 (IL-12), IFN-\u03b3, inducible nitric oxide synthase and IL-2, and finally, inducing tumour cell death. Cancer therapy is an essential research area and protein kinases are major targets for drugs. The mTOR is a highly conserved serine/threonine protein kinase that is central regulating essential cellular processes and mTOR inhibitors are used in cancer therapy and parasite survival inside macrophages through down-regulation of the nuclear factor-\u03baB and signal transducer and activator of transcription 3 oncogene factors levels led to the reduction of lesions size and decreased the load of parasites in skin in the L. amazonensis-infected BALB/c mice. However, the use of such an inhibitor also induced hepatotoxicity in BALB/c mice were correlated with the development of oral cancer selectively targeted proliferation and survival of tumour initiating cells in SCC and ovarian cancer cells. This compound prevented cancer cells from entering the S-phase under growth-factor stimulation without cell death induction is an inhibitor of HSP90, which has been investigated in the treatment of cancer such as solid tumours and leukaemia. Alternatively, geldanamycin, and its analogues, 17-dimethylamino ethylamino-17-demethoxygeldanamycin (17-DMAG) and 17-AAG, may show a promising therapeutic activities against leishmaniasis . However, the delivery of 17-AAG is difficult because of its poor aqueous solubility and is overexpressed in the promastigote forms compared to the amastigotes and a combination of gossypol (a pan-ALDH inhibitor) and phenformin leads to cancer cell death and beta (TOP 2B) which exhibit differences in their molecular weight, genetic regulation and the location of the active site. TOP II expression in cancer lines has been largely studied Doyle, . Althoug\u03b4) and epsilon (Pol \u025b). It also interacts with other proteins involved in cell-cycle progression which are not parts of the DNA polymerase complex. PCNA has demonstrated its role in the replication and repair of DNA, chromatin assembly and RNA transcription. Its importance in Leishmania pathology is related to its significant role in drug response in clinical isolates dimers polymerize to form microtubules, which serve as a skeletal system for living cells and participate in several essential functions, such as mitosis or intracellular transport among others has been suggested as potential drug target in leishmaniasis treatment structure. Data produced by genome sequencing in protozoa has indicated the presence of TOM-40 homologues in in vitro\u2019. It seems that TOM-40 increases the replication of cancer cells through regulating the mitochondrial activity and improving cellular energy and redox status , the expression of OAT is up-regulated and the mechanism of this gene up-regulation is related to the activation of \u03b2-catenin signalling (Cadoret et al., et al., \u03b2-catenin signalling and the metabolism of glutamine are important factors in carcinogenesis especially in HCC (Cadoret et al., Leishmania parasites (Zigmond et al., OAT as a Leishmania parasites (Iribar et al., et al., et al., Selenoproteins are a group of enzymes bearing selenocysteine in their catalytic domain. Many of the Se-bearing proteins participate in oxidative stress protection as observed in et al., et al., Leishmania, with promising results. However, this compound seems not to target selenoproteins in these parasites, but rather interact with trypanothione reductase, a key enzyme of Leishmania polyamine-dependent redox metabolism (Ilari et al., et al., et al., et al., The role of selenoproteins and their metabolites including methylselenol, selenodiglutathione, Se-methylselenocysteine and selenomethionine has been underlined in the metabolism of lung cancer cells (Seng and Tiekink, Leishmania parasites has been previously reported (Hart and Opperdoes, et al., et al., Leishmania. Therefore, they might be related to the pathogenicity of these parasites (Blattner et al., et al., Leishmania isolates suggesting resistant strains require higher energy to protect against antimony-induced oxidative stress. Alternatively, the overexpression of such enzyme might lead to enhancing in pyruvate which can remove peroxides and participate to reduce oxidative stress (Biyani et al., Leishmania and mammalian cells, PGKs are encoded by two genes: gene B and gene C, and PGK-1 and PGK-2, respectively (Watson and Littlechild, et al., T. brucei PGK. Of these, 2-amino-N6-substituted analogues represented appropriate activity against the parasite kinase compared with the N6 compounds that lacked the C2 amino group, although activity was still weak (Merritt et al., PGKs are transferases involved in ATP production from ADP and 1,3-diphosphoglycerate. Their involvement in the glycolytic pathway and survival of et al., et al., et al., et al., et al., et al., et al., The importance of PGK-1 in cancer development resides on its involvement in drug-resistance and its dual action depending on the cellular environment. Under intracellular hypoxia conditions, it plays an oncogenic role. However, it decreases tumour growth when it is secreted extracellularly through angiogenesis inhibition (Daly Leishmania parasites and cancer cells. As an interesting issue, the regulation of these markers can be investigated in interactions that can be occurred between Leishmania parasites and cancer cells under \u2018in vivo\u2019 and \u2018in vitro\u2019 conditions. The reliable validation of the expression of such proteins in Leishmania parasites and cancer cells using further experiments is warranted to subsequently confirm their possible functions. Further investigation of the differentially regulation of common expressed proteins between cancer cells, normal human cells, low-pathogenic and high-pathogenic forms of Leishmania parasites might elucidate novel and attractive information concerning such proteins. The existence of common triggering factors reflects mutual features in the etiopathogenetic mechanisms underlying leishmaniasis and cancer. Given these similarities, lessons learned from strategies against cancer may be relevant to design adequate approaches to reduce and eliminate leishmaniasis. Herein, we focused specifically on the shared mechanisms at protein scale. Taken together, the introduction of common expressed proteins in Leishmania parasites and cancer cells might reveal valuable information regarding the possible common mechanisms of pathogenicity and therapeutic targets in leishmaniasis and cancers. Taking into account that current therapies for neglected diseases are based in drugs lacking effectiveness, the lack of new specific anti-Leishmania compounds and of research focused on this group of diseases, drug repurposing constitutes a useful tool to find effective candidates in leishmaniasis control and elimination. This review reinforces the likely functional similarities between many proteins in cancer and parasites, some of them being recognized therapeutic targets and thus the potential use of drugs with proven efficacy in the treatment of cancer for treating parasitic diseases and vice versa, opening new avenues to the one health approach.The up- and down-regulation of the aforementioned proteins were mostly related to the survival, development, pathogenicity, metabolic pathways and vital signalling in"} +{"text": "Iron is essential for life, and the dysregulation of iron homeostasis can lead to severe pathological changes in the neurological system. Iron deficiency slows the development of the neural system and causes mental and emotional disorders ,2,3, whiIn this Special Issue, \u201cIron Metabolism, Redox Balance and Neurological Diseases\u201d, five original research articles and five scientific review papers are published. These papers highlight the most recent advances in different aspects of iron regulation and redox imbalance in various diseases, including the molecular mechanisms of iron-induced oxidative damage in disease pathogenesis, potential therapeutic targets and approaches for the regulation of iron metabolism and related damages, and challenges to current studies attempting to understand an aberrant iron metabolism in the pathology of different diseases and its potential clinical applications.In the original research articles, Han et al. revealed a novel role of iron in the maintenance of cell stemness via the Wnt/GSK-3\u03b2/\u03b2-catenin signaling pathway . The intBao et al. designed and characterized a mitochondrial-targeted pseudo-mitochondrial membrane potential (PMMP) constructed by antioxidant MitoQ , which sLiu et al. demonstrated that IRP2 not only regulated cellular iron homeostasis, but also medicated tissue iron distribution by managing the involvement of hypoxia-inducible factor 2 (HIF2) and nuclear receptor coactivator 4 (Ncoa4) . This stHan et al. investigated the underlying mechanisms of CY-09, a specific inhibitor of the NOD-like receptor protein 3 (NLRP3) inflammasome, on ameliorating AD classical pathology and cognitive impairment in AD mice . Their fChen et al. revealed that the dopaminergic neuronal death and Parkinsonian symptoms in OTU domain-containing protein 3 (OTUD3) knockout mice might be caused by activating inositol-requiring enzyme 1\u03b1 (IRE1\u03b1) signaling, which mediated endoplasmic reticulum (ER) stress . The OTUIn the review articles, Luo et al. summarized the molecular mechanisms of ferroptosis in glioma cell growth, invasion, migration, and resistance, and introduced potential applications and challenges of manipulating ferroptosis in the development and treatment of gliomas . They alJim\u00e9nez-Jim\u00e9nez et al. systematically reviewed the role of antioxidant coenzyme Q10 (CoQ10) in AD and other dementias . CombineHolbein et al. described biological iron requirements, iron regulation, and the nature of iron dysregulation in detail in various disease conditions , includiLee et al. reviewed the interplay between intracellular iron homeostasis and neuroinflammation in neurodegenerative diseases . They inGao et al. summarized and elucidated the interplay between the dysregulation of iron metabolism, redox imbalance, and different neurological diseases such as In conclusion, the original research and review articles in this Special Issue provide an updated overview of the advances on the mechanisms or treatments of neurological diseases related to iron dysregulation and redox imbalance. These papers offer fresh perspectives on the expanding knowledge and research possibilities in the field of iron metabolism, redox balance, and neurological diseases, and may stimulate future studies to better target the regulation of brain iron metabolism for the prevention and treatment of neurological diseases."} +{"text": "Advanced Parkinson\u2019s disease is characterized by periods of poor mobility, dyskinesia and progressive decline in functional independence of the affected person despite the manipulation of levodopa doses and the introduction of supplemental therapies such as catechol-O-methyl transferase inhibitors, monoamine oxidase-B inhibitors and dopamine agonists. The implementation of drug delivery systems allows to bypass problems related to irregular and often unpredictable intestinal absorption of oral levodopa, which significantly affects its bioavailability and contributes to the development and persistence of motor complications. Subcutaneous apomorphine and levodopa/carbidopa jejunal infusion systems have been available for many years and their efficacy is confirmed by randomized studies and long-term experience in many centers worldwide. Recently, a new formulation of levodopa/carbidopa infusion gel that includes the catechol-O-methyl transferase inhibitor Entacapone has been introduced to the market. The use of entacapone allows to reduce total daily dose of administered levodopa. Two different soluble formulations of levodopa/carbidopa (ND0612 and ABBV-951) have completed clinical development, and both can ensure subcutaneous delivery by a portable pump infusion system. ABBV-951 uses a foslevodopa/foscarbidopa formulation, both prodrugs to improve absorption and tolerability. Both systems provide effective improvement of motor complications and are likely to expand the therapeutic options in advanced patients. Future efforts should focus on the earlier detection of patients who are candidates for device-aided therapies, increasing appropriate referral and broadening the availability of these treatments globally. Parkinson\u2019s disease (PD) is a progressive neurodegenerative disorder characterized by loss of nigrostriatal dopamine neurons and its incidence is increasing fast globally is defined as a condition where periods of poor mobility with or without dyskinesia are present and have an impact on functional independence of the affected person in a water solution of carboxymethyl cellulose into the patient\u2019s proximal small intestine through a portable pump and a tube placed via percutaneous endoscopic gastro-jejunostomy (PEG-J) of withdrawal of 1.72\u00a0h in \u201cGood ON\u201d time. The trial also demonstrated positive and clinically meaningful results for the key secondary endpoint of \u201cOFF\u201d time (p\u2009<\u20090.0001) and other secondary endpoints including the MDS-Unified Parkinson's Disease Rating Scale Part II score (MDS-UPDRS motor experiences of daily living sub-score) (p\u2009<\u20090.0001); the Patient Global Impression of Change (PGIC) (p\u2009<\u20090.0001); and the Clinical Global Impression of Improvement (CGI-I) (p\u2009<\u20090.0001).ND0612 is a drug-device combination consisting of a sterile solution of levodopa/carbidopa continuously delivered via a dedicated subcutaneous pump (Olanow et al. ABBV-951 is a new soluble formulation of foslevodopa/foscarbidopa, both levodopa and carbidopa prodrugs, which can be delivered subcutaneously for up to 24\u00a0h/day. After administration, foslevodopa/foscarbidopa is quickly converted to levodopa/carbidopa by alkaline phosphatases, reaching and maintaining therapeutic steady-state plasma levels in a very short time (Rosebraugh et al. Research in PD has increasingly focused on the development of new and effective biological therapies for disease modification (Antonini et al. Limitations will still be related to the lack of direct comparative efficacy and safety studies among advanced treatments and the difficulty of identifying specific patients\u2019 profiles.Infusion therapies might also become more integrated with remote monitoring technologies and telemedicine, allowing healthcare providers to track patient responses, adjust treatments and offer support from a distance (Guerra et al. Along with the progressive increase in the number of options, the selection of more appropriate device-aided therapy for APD will be more complex, it will involve multiple specialties as well as the carers and affected ones. A shared decision approach is, therefore, essential. While treatment decisions need to be individualized, the choice of device-aided therapies can be guided by some general principles based on the patient\u2019s age, cognitive and behavioral status, dyskinesia and frailty.Understanding the comparative benefits of each treatment provides additional information that can help patients, caregivers and providers in the selection of the most appropriate therapy to ensure optimal symptom control and improved QoL (Martinez-Martin et al."} +{"text": "Flow cytometry is a single-cell based technology aimed to quantify the scattering of light and the emission of multiple fluorescence signals by individual cells, biological vesicles, or synthetic microscopical particles when examined one by one at high speed using lasers or other suitable illumination sources ,2. In coThis new Special Issue entitled \u201cFlow Cytometry and Its Applications to Molecular Biology and Diagnosis 2.0\u201d of the International Journal of Molecular Sciences includes a total of five contributions: three original articles and two reviews, providing new information about advanced applications of flow cytometry in the fields of molecular and cell biology for basic and translational research.Rossi et al. extensivDrug development has evolved rapidly, and in the last years, cell and gene therapies are becoming efficient alternatives to small chemical and biomolecule pharmacology. Several cell and gene therapies have reached regulatory approval, specially in the field of tumor immunotherapy, and such a breakthrough demands suitable bioanalytical techniques for fully characterizing the pharmacokinetics of these therapies. However, these different techniques may not always lead to concordant results, and this fact requires a deep understanding of the technical grounds of each technique, which data are generated, and how these data can be compared and interpreted. In their review, Hays et al. critical+ extracellular vesicles (EVs). These results support that the conjugation of antitumor drugs to SiNPs is an efficient way for drug delivery that potentiates drug cytotoxicity, while reducing side effects.Among the new directions for cancer therapy, nanomaterials appear as a promising tool for drug delivery into specific cells or subcellular compartments. In this regard, fluorescent silica nanoparticles (SiNPs) appear to be a promising imaging platform, as they show intramitochondrial colocalization in biological cell models and can be used as efficient drug delivery systems when conjugated to a therapeutic molecule. Sola et al. performeCampylobacter jejuni and other Gram-negative bacteria. In the first part of their in vitro experimental design, Montanari et al. showed that CDT-containing bacterial lysates induce lethal and sublethal alterations in intestinal (CaCo-2) and myeloid (U937) cell lines, including cell cycle arrest, mitochondrial dysfunction, and oxidative stress. These intracellular changes were accompanied by relevant changes in lysosomal exocytosis, secretory autophagy, and EV release. Based on these effect on the secretion processes, Montanari et al. investigated the presence of active CDT in EVs secreted by CDT-treated CaCo-2 cells and found that CDT-like effects were transferred from infected CaCo-2 cells to uninfected heterologous (U937) and homologous (CaCo-2) cells. Their results suggest that EVs from CDT-treated CaCo-2 cells are reliable CDT carriers and could potentially be used in the treatment of colorectal cancer.The paper by Montanari et al. exploredEscherichia coli B strains deficient in key genes of the antioxidant defense, namely oxyR, sodA, and sod B. J\u00e1vega et al. applied this model for systematically assessing issues of ROS specificity of several relevant fluorescent probes and the involvement of different ROS in the oxidative stress induced on these strains by exogenous prooxidants. Their results suggest that the antioxidant-deficient Escherichia coli B strains can be used as ROS biosensors in flow cytometric studies of oxidative stress and confirm the specificity issues of ROS-sensitive fluorescence that have already been detected in eukaryotic models. In addition, J\u00e1vega et al. provided recommendations for the proper design of flow cytometric studies involving ROS detection.Analysis of reactive oxygen species (ROS) and oxidative stress is a very relevant application of flow cytometry. However, to ascertain the specific role of ROS in oxidative stress studies via cytomic methodologies, it is essential to detect and characterize these species accurately. While there is a large availability of fluorescent probes for ROS analysis via flow cytometry, these reagents exhibit important limitations in their specificity and sensitivity that may affect the accuracy of the analysis. J\u00e1vega et al. presente"} +{"text": "Real-world data from wearable devices has the potential to understand mental health status in everyday life. We aimed to investigate the feasibility of estimating mental health status using a wrist-worn wearable device (Fitbit Sense) that measures movement using a 3D accelerometer and optical pulse photoplethysmography (PPG).Participants were 110 patients with mental illnesses from different diagnostic groups. The study was undertaken between 1 October 2020 and 31 March 2021. Participants wore a Fitbit Sense on their wrist and also completed the State\u2013Trait Anxiety Inventory (STAI), Positive and Negative Affect Schedule (PANAS), and EuroQol 5 dimensions 5-level (EQ-5D-5L) during the study period. To determine heart rate (HR) variability (HRV), we calculated the sdnn , coefficient of variation of R-R intervals, and mean HR separately for each sleep stage and the daytime. The association between mental health status and HR and HRV was analyzed.The following significant correlations were found in the wake after sleep onset stage within 3\u2009days of mental health status assessment: sdnn, HR and STAI scores, HR and PANAS scores, HR and EQ-5D-5L scores. The association between mental health status and HR and HRV was stronger the closer the temporal distance between mental health status assessment and HR measurement.A wrist-worn wearable device that measures PPG signals was feasible for use with patients with mental illness. Resting state HR and HRV could be used as an objective assessment of mental health status within a few days of measurement. BecauseHowever, because of concerns about the time and effort involved in self-administered questionnaires, an objective and less burdensome method of monitoring mental health status using wearable devices has attracted attention. One way of estimating mental health status is to use heart rate (HR) variability (HRV), as HR is controlled by the autonomic nervous system (ANS) . The parThere is growing interest in the use of wrist-worn wearable devices that can measure movement using a 3D accelerometer and optical pulse photoplethysmography (PPG) to obtain biometric information in the real world, rather than in a special environment such as a laboratory. However, the validity of such data for the assessment of patients with mental illness has not been adequately demonstrated. HRV measures the variation in time between heartbeats and is usually calculated from electrocardiogram (ECG) R-R intervals; however, wearable devices record an optical PPG signal and therefore the peak of the R waves cannot be detected accurately. The accuracy of PPG-based HR data has been previously reviewed and validated in comparison with ECG data; PPG-based HR data is typically accurate within a 10% error range . As reseIn this study, we examined the feasibility and validity of using wearable devices to estimate mental health status in patients enrolled in the Mental Illness Registry, using standardized tests for anxiety, affect, and QOL as a reference. As a wearable device, Fitbit Sense was chosen because Fitbit device is the most researched , widely 2.2.1.A total of 110 patients who were enrolled in the Mental Illness Registry during the study period between 1 October 2020 and 31 March 2021 and provided their informed consent, participated in the study. Participants were either inpatients or outpatients at six sites: the National Center of Neurology and Psychiatry Hospital, Nara Medical University Hospital, Kansai Medical University Hospital, Nagoya University Hospital, Hokkaido University Hospital, and Akita University Hospital. The sample included patients with a wide range of clinical presentations from different diagnostic groups . Most paThe Ethics Committee of the National Center of Neurology and Psychiatry approved the study protocol and experimental procedures (B2021-086).2.2.This was a multisite observational study. Participants were either inpatients or outpatients at six sites: the National Center of Neurology and Psychiatry Hospital, Nara Medical University Hospital, Kansai Medical University Hospital, Nagoya University Hospital, Hokkaido University Hospital, and Akita University Hospital. The software setup for data acquisition was conducted in advance by the researchers. Participants had only to wear a Fitbit Sense on their left or right wrist for as long as they could tolerate it between the day of consent and their next visit to the hospital (for outpatients), or until their first visit to the hospital after discharge (for inpatients). While wearing the Fitbit Sense, participants were contacted via their smartphones to inquire about their current socioenvironmental situation and to ask them to complete the following self-administered questionnaires: the STAI FORM X-I, II to meas and cvrr , which are modulated by the ANS. We also assessed HR. The sdnn, cvrr, and HR were calculated using a sampling time of 5\u2009s with 30\u2009min as one epoch; if there were multiple epochs appropriately recorded, the average value was used as the representative value. Assuming that during the daytime HR is sensitive to activity, we excluded data in which the HR was greater than 90/min or fell below 50/min, and in which the difference between the maximum and minimum within one epoch was greater than 40/min or less than 5/min. During sleep, only the latter condition was used; data in which the difference between the maximum and minimum HRs within one epoch was greater than 40/min or less than 5/min were excluded because of the suspected possibility of measurement noise. The Fitbit device used in the present study records an optical PPG signal and thus the peak of the R waves cannot be detected accurately. Therefore, the interval between the pulse wave (estimated by 60/mean HR) was used as a surrogate for the R-R intervals obtained from an ECG.Sleep stage information in this study was provided by the Fitbit Research Library, which analyzed the 3D accelerometer and optical PPG data collected in this study. Unfortunately, the algorithm for determining sleep stage from 3D accelerometer and PPG data obtained from Fitbit is not publicly available. However, Beattie et al. provides an overview of the analysis using the generated features on a 30\u2009s time scale based on motion, HRV, and breathing rate parameters were used to develop an automated sleep staging algorithm . Specifi2.4.As reference, we used electronic self-reported assessments of anxiety, affect, and QOL. Patients reported their data using a smartphone. The following self-administered questionnaires were used: the STAI Form X-I and Form X-II to measure state and trait anxiety, the PANAS to measure positive and negative affect, and the EQ-5D-5L visual analog scale (VAS) and utility score to measure QOL.The STAI Form X-I and Form X-II were developed by Spielberger et al. ; they haThe PANAS consists of 20 items that comprise adjectives representing positive and negative affect . The rel5) possible health states, and utility scores are derived from a preference-based algorithm used to calculate quality-adjusted life years . In this study, an algorithm developed using survey results based on the composite time trade-off in Japan was used were performed to examine associations between HR and HRV indices and mental health status variables for each sleep stage and the daytime to estimate which time period HR and HRV indices best reflected mental health status. Then, to explore the effect of focusing on the period showing the strongest association, the effect of temporal distance between the time of HR measurement and mental health status assessment on the association between them was examined using univariate correlation analyses (Pearson\u2019s product moment correlation) between HR and HRV indices and mental health status variables when HR and HRV data were restricted to within 7\u2009days and within 3\u2009days of the mental health status assessment. For the data set that was ultimately estimated to have the strongest association, multivariable regression analysis was performed with each HRV index, age, sex, chlorpromazine-equivalent of antipsychotics, imipramine-equivalent dose of antidepressants, and diazepam-equivalent dose of anxiolytics and hypnotics as independent variables, and each mental health status variable as a dependent variable, to adjust for possible confounding factors , 21.P-value of <0.05 was considered statistically significant for all tests.The statistical analysis was performed using JMP version 13.0.0 ; a 3.Of the 110 patients who consented to participate in the study, available data on HRV indices were obtained from 92 patients . For theThe exploratory analyses of correlations between HR and HRV indices and mental health status variables for each sleep stage and the daytime showed a significant correlation between sdnn and STAI Form X-II scores, between HR and STAI Form X-I and Form-II scores, between HR and EQ-5D-5L VAS and utility scores in the WASO stage; between HR and STAI Form X-I scores and EQ-5D-5L VAS scores in the light stage; between HR and STAI Form X-I scores, PANAS positive affect scores, and EQ-5D-5L VAS scores in the deep stage; and between HR and STAI Form X-I scores in the REM stage . As can Focusing on data obtained in the WASO stage, the correlations between HR and HRV indices and mental health status variables within 14\u2009days, 7\u2009days, and 3\u2009days of the time of mental health status assessment are shown in P\u2009=\u20090.035); HR and STAI Form X-I scores ; HR and PANAS positive affect scores ; HR and EQ-5D-5L VAS scores ; and HR and EQ-5D-5L utility scores .Multivariable regression analyses were conducted on the variables that showed significant intercorrelations for data obtained within 3\u2009days of mental health status assessment . After a4.In this study, we explored the feasibility of using PPG obtained by a wrist-worn wearable device to assess mental health status in patients with mental illness. Of 110 patients, 92 (83.6%) returned usable HR data. HR and HRV measured within 14\u2009days of the mental health status assessment were significantly associated with anxiety and QOL in different sleep stages, and the association was strongest in the WASO stage. Although exploratory, the results also suggest that HR and HRV in the WASO stage may reflect anxiety, positive affect, and QOL within 3\u2009days.ECG data on HR and HRV are one of the most important biomarkers of emotion . When anOur findings suggest that HR and HRV in the WASO stage better reflect current mental health status than data in other sleep stages and in the daytime. This may be because the resting state is maintained during the WASO stage, as it is less affected by daytime activities or ANS activity during REM sleep. In addition, the accuracy of wearable devices in estimating sleep stages in healthy individuals has been reported as reasonable , 14. OneA recent review found thThe present study had several limitations. Although a common technique is to develop a peak detector algorithm to detect the peaks in the PPG signal, and the time between PPG peaks is used as a surrogate for the R-R intervals in the pThis study examined the feasibility and potential of a wrist-worn wearable device to assess the mental health status of mentally ill patients. Although the results were exploratory and cross-sectional, they suggest the potential utility of this method. Additional validation studies, including longitudinal studies, are warranted. If it is possible to objectively monitor mental health status in the home by the wearing of a wrist-worn device for a relatively short period, this could reduce the burden on patients in cohort studies and improve their well-being.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by the Ethics Committee of the National Center of Neurology and Psychiatry. The patients/participants provided their written informed consent to participate in this study.KN contributed toward conceptualization, methodology, investigation, project administration, and writing of the original draft. KMin and TF contributed toward data curation, provision of resources, and formal analysis. MM, MU, MK, NO, SM, KI, NH, AT, MO, MT, and KMis contributed toward conceptualization, methodology, and investigation. MOb, KT, and HO contributed toward conceptualization, data curation, and supervising the statistical analysis. All authors contributed toward acquisition of data, critical revision of the manuscript for important intellectual content, approval of the final version of the manuscript, and agree to be accountable for all aspects of the work and ensure that any questions related to the accuracy or integrity of any part of the work will be appropriately investigated and resolved.This research was supported by AMED under Grant number JP21dk0307103 and an Intramural Research Grant (3-1) for Neurological and Psychiatric Disorders of NCNP. KN confirms that he had full access to all the data in the study and takes final responsibility for the decision to submit for publication.KN reports grants from AMED during the conduct of this study and from Otsuka Pharmaceutical Co., Ltd., Sumitomo Pharma Co., Ltd. and Janssen Pharmaceutical K.K., and personal fees from Otsuka Pharmaceutical Co., Ltd., Sumitomo Pharma Co., Ltd., Meiji-Seika Pharma Co., Ltd., Janssen Pharmaceutical K.K., MOCHIDA PHARMACEUTICAL CO., LTD., Mitsubishi Tanabe Pharma Corp., Viatris Inc., and Eisai Co., Ltd. He has received payment for advisory work from Takeda Pharmaceutical Co., Ltd., Lundbeck Japan, and Nippon Boehringer Ingelheim Co., Ltd., outside the submitted work. MM has received research grants from Sumitomo Pharma Co., Ltd. and Kyowa Kirin Co., Ltd. MK has received grant funding from the Japanese Ministry of Health, Labour and Welfare, the Japan Society for the Promotion of Science, SENSHIN Medical Research Foundation, the Japan Research Foundation for Clinical Pharmacology, and the Japanese Society of Clinical Neuropsychopharmacology, and speaker honoraria from Sumitomo Pharma Co., Ltd., Otsuka Pharmaceutical Co., Ltd., Meiji-Seika Pharma Co., Ltd., Eli Lilly Japan K.K., Merck & Co., Inc., Pfizer Inc., Janssen Pharmaceutical K.K., Shionogi & Co., Ltd., Mitsubishi Tanabe Pharma Corp., Takeda Pharmaceutical Co., Ltd., Lundbeck Japan, Viatris Inc., Eisai Co., Ltd., and ONO PHARMACEUTICAL CO., LTD., and has participated in advisory/review boards for Otsuka Pharmaceutical Co., Ltd., Sumitomo Pharma Co., Ltd., Shionogi & Co., Ltd., and Nippon Boehringer Ingelheim Co., Ltd. NO has received research support or speaker honoraria from, or has served as a joint researcher with, or a consultant to, Sumitomo Pharma Co., Ltd., Eisai Co., Ltd., Otsuka Pharmaceutical Co., Ltd., KAITEKI, Mitsubishi Tanabe Pharma Corp., Eli Lilly Japan K.K., MOCHIDA PHARMACEUTICAL CO., LTD., DAIICHI SANKYO CO., LTD., TSUMURA & CO., Takeda Pharmaceutical Co., Ltd., Meiji-Seika Pharma Co., Ltd., EA Pharma Co., Ltd., Viatris Inc., Ricoh Co., Ltd., Nippon Boehringer Ingelheim Co., Ltd., Lundbeck Japan, Nihon Medi-Physics Co., Ltd., and Nippon Chemiphar Co., Ltd., outside the submitted work. KI has received speaker honoraria from Eisai Co., Ltd., Kyowa Kirin Co., Ltd., Meiji-Seika Pharma Co., Ltd., Merck & Co., Inc., Otsuka Pharmaceutical Co., Ltd., Sumitomo Pharma Co., Ltd., Taisho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Co., Ltd., TOWA PHARMACEUTICAL CO., LTD., and Viatris Inc., outside the submitted work. NH has received personal fees from Janssen Pharmaceutical K.K., Yoshitomiyakuhin Corporation, Otsuka Pharmaceutical Co., Ltd., Sumitomo Pharma Co., Ltd., Novartis Pharma K.K., Meiji-Seika Pharma Co., Ltd., Lundbeck Japan, and Takeda Pharmaceutical Co., Ltd. AT received a research grant from Nippon Boehringer Ingelheim Co., Ltd. and personal fees from MOCHIDA PHARMACEUTICAL CO., LTD., Sumitomo Pharma Co., Ltd., and Otsuka Pharmaceutical Co., Ltd. KMis has received research grants from Eisai Co., Ltd., Sumitomo Pharma Co., Ltd., Takeda Pharmaceutical Co., Ltd., and Sony Corporation and has also received speaker honoraria from Eisai Co., Ltd., Nobelpharma Co., Ltd., Takeda Pharmaceutical Co., Ltd., Merck & Co., Inc., Otsuka Pharmaceutical Co., Ltd., and Viatris Inc. MT has received speaker honoraria from Takeda Pharmaceutical Co., Ltd., Otsuka Pharmaceutical Co., Ltd., DAIICHI SANKYO CO., LTD., Sumitomo Pharma Co., Ltd., Meiji-Seika Pharma Co., Ltd., Viatris Inc., Merck & Co., Inc., Eisai Co., Ltd., and Yoshitomiyakuhin Corporation outside the submitted work. KMin and TF were employed by TechDoctor Inc.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."} +{"text": "International Journal of Molecular Sciences includes original research and reviews on the molecular mechanisms of active, natural products and phytochemicals in vitro and in vivo.The Special Issue \u201cMolecular Mechanisms of Natural Products and Phytochemicals in Immune Cells and Asthma\u201d in the Allergic asthma, an increasingly common immunologic disease in industrialized countries, is a chronic inflammatory condition of the airways that causes airway hyperresponsiveness (AHR). A Th1-Th2 cytokine imbalance has been hypothesized to underlie allergic asthma through a change in immune responses from a Th1 profile to a Th2 profile. Additionally, the recruitment of inflammatory cells such as T cells, mast cells, eosinophils, epithelial cells, macrophages and neutrophils is mediated via a number of chemokines and their receptors. Chemokines and their receptors are important therapeutic targets in asthma and allergic diseases because of their key role in immune cell recruitment and activation during inflammation [Two highly relevant papers have recently been published in the Special Issue of Bioactives and Nutraceuticals entitled \u201cMolecular Mechanisms of Natural Products and Phytochemicals in Immune Cells and Asthma\u201d. These two papers exhibit antiallergic and antiasthmatic effects of the active components in natural products.Lithospermum erythrorhizon on Mast Cells and Ovalbumin-Induced Allergic Rhinitis\u201d by Le et al. [Lithospermum erythrorhizon, N,N\u2019-dicoumaroylspermidine, inhibited the release of \u03b2-hexosaminidase, as well as the production of Th2 cytokines by IgE-sensitized and BSA-stimulated RBL-2H3 cells. Using the allergic rhinitis mouse model, they showed that N,N\u2019-dicoumaroylspermidine reduced the number of inflammatory cells in nasal lavage fluid and the production of serum OVA-specific IgE. N,N\u2019-dicoumaroylspermidine isolated from Lithospermum erythrorhizon exhibits antiallergic properties, and it can be potentially effective for allergic rhinitis.The first article, \u201cAntiallergic Effects of N,N\u2019-dicoumaroylspermidine Isolated from e et al. revealedGynostemma pentaphyllum Attenuates Airway Inflammation and Th2 Cell Activities in a Murine Asthma Model\u201d by Huang et al. [G. pentaphyllum. The study revealed that gypenoside A significantly reduced the expression of inflammatory genes and proteins in the lungs and bronchoalveolar lavage fluid and Th2 cytokine expression. Moreover, gypenoside A significantly inhibited the secretion of chemokines and inflammatory cytokines in tracheal epithelial cells. Gypenoside A is a natural phytochemical that can effectively attenuate airway hyperresponsiveness (AHR) and inflammation in asthma, mainly by blocking Th2 cytokines production. In summary, the two papers successfully demonstrate the antiallergic effects of phytochemicals from Lithospermum erythrorhizon roots and Gynostemma pentaphyllum.The second article, \u201cGypenoside A from g et al. investigRecently, natural products and their phytochemicals play increasingly critical roles, especially multi-ingredient synergistic effects . The theWe hope that this Special Issue can provide a comprehensive overview of the current state of research on molecular mechanisms of natural products and phytochemicals in immune cells and asthma.In conclusion, the Guest Editor would like to thank all authors for their remarkable contributions and the Editorial Board for their support."} +{"text": "The ripening process of fruits is complicated and coordinated and is characterized by obvious changes in pericarp color (green color loss and non-photosynthetic pigment increases), flesh texture , taste (variation of sugars and organic acids), and aroma flavour (volatile productions) of fleshy fruits . SpecifiTeixeira et\u00a0al. showed that the exocarp of green and mature grape berries is rich in chloroplasts, and they applied proteomic analysis of chloroplasts from the two phases. The authors observed that proteins associated with the Calvin cycle were stimulated in green berries, while those related to energy-generating metabolism were enriched in mature berries. Wang et\u00a0al. systematically identified a batch of lignin biosynthesis-related genes and constructed a co-expression network of these genes via weighted gene co-expression network analysis. Specifically, they found the major lignin biosynthesis genes involved in ripening process and stress resistance in banana. Liu et\u00a0al. reviewed NAC transcription factors, which show extensive participation in fruit yield and quality and regulate fruit ripening by directly acting on critical genes related to the biosynthesis and signal transduction of the plant hormones abscisic acid (ABA) and ethylene (ET). These articles provide the basis for the improvement of fruit development and ripening.The material basis and regulator underlying fruit development and ripening still remain challenges. Huang et\u00a0al. and Jiang et\u00a0al. reported that salicylic acid (SA) treatment could maintain the organoleptic quality and postharvest storability of pummelo and blueberry. Li et\u00a0al. recorded that pear had a greasy coating and yellowing process during postharvest storage, while 1-methylcyclopropene (1-MCP) could decrease the wax content of postharvest pear and delay the development of peel greasiness and yellowing by suppressing the transcription of a series of cluster genes associated with ethylene synthesis, ethylene signal transduction, wax accumulation, and chlorophyll degradation. Choi et\u00a0al. investigated the molecular details of kiwifruit ripening using ethylene and its action inhibitor 1-MCP. Through a time-course transcriptomic analysis, they found that the genes related to ET synthesis and signalling suffered from opposite influence from postharvest application of 1-MCP and ET, conversely, in the process of kiwifruit ripening. They identified that the ET transcription factor AcEIL might exhibit an essential function in ET-induced kiwifruit ripening. These articles suggest a practical foundation for improving the protective mechanisms in relation to fruit ripening and senescence.Postharvest ripening and senescence restrict the shelf life of horticultural crops. Both Lin et\u00a0al. compared two cultivars of hardy kiwifruit that have high frost hardiness and show similar trends in antioxidant capacities and nutritional compounds. The authors noted that the antioxidant capacity of the two hardy kiwifruit cultivars decreased but glucose increased progressively during maturation, in which the conversion from starch to total sugar was dominantly due to the expression of sucrose phosphate synthase (SPS) and fructokinase (FK). The predominant acids in the two hardy kiwifruit cultivars were quinic acid and citric acid from the early developmental to late maturity stages, respectively. These findings are conducive to a wider understanding of the physiological and biochemical basis of hardy kiwifruit for the cultivation of chilling-tolerant cultivars.Sensitivity to chilling can influence plant growth in the field and storability and quality during the postharvest storage period . AccordiLin et\u00a0al. assessed the effect of fucoidan application on cold storage quality, reactive oxygen species (ROS) homeostasis, and energy metabolism in cucumber fruit. The authors determined that the optimum concentration of coated fucoidan was 15 g/L, which could increase DPPH and -OH scavenging rates and reduce H2O2 accumulation. The authors suggested that the improved chilling tolerance in cucumbers with fucoidan treatment may be related to the increased antioxidant enzyme activities and ROS scavenging rates, as well as high levels of ATP, ADP, and energy charge. In accordance with the effects of fucoidan in cucumbers, Zhou et\u00a0al. confirmed that \u03b3-aminobutyric acid (GABA) could lighten CI symptoms in peach fruit and that the reduction efficiency of GABA on chilling injury was associated with the accumulation of ascorbic acid (AsA) and glutathione (GSH) contents and the amplified expression profiles of AsA-GSH recycling-related genes. Moreover, the authors indicated that several ERF transcription factors, which are potentiated by GABA treatment in peach fruit, regulate AsA and GSH contents to reduce chilling injury. Overall, the authors proposed potential strategies of fucoidan coating and GABA immersion in alleviating CI symptoms in postharvest horticultural crops.Xing et\u00a0al. investigated the mechanism of action of Sly-miR171d on chilling injury in tomato fruit. They found that down-regulated Sly-miR171d promotes GRAS24 expression, which obviously increased gibberellin production and C-repeat binding factor (CBF) expression and maintained cell membrane stability, therefore enhancing the chilling tolerance of tomato fruit. This study sheds light on the genetic improvement of postharvest tomato to chilling injury.In general, microRNAs (miRNAs) are a class of small, non-protein coding RNA molecules that function as negative regulators of target gene messages , and theIn summary, the articles in this Research Topic provide advanced information on ripening and chilling injury alleviation in horticultural crops. New insights into phytohormones, transcription factors and epigenetic modifications will impel our future applied research in the alleviation of crop ripening and chilling injury.CL: Conceptualization, Writing \u2013 original draft. ZY: Supervision, Validation, Writing \u2013 review & editing. SC: Writing \u2013 review & editing. CW: Supervision, Writing \u2013 review & editing. KW: Conceptualization, Writing \u2013 review & editing."} +{"text": "We as dentists and surgeons would like to bring to the notice of our medical colleagues periodontal disease as yet another co-risk factor of pancreatic cancer.In a recent editorial by Dr Ga\u00ebtan Romain Joliat entitled \u2018Latest Advances and Future Challenges in Pancreatic Surgery\u2019, the authors stress on how development and improvement in the fields of chemotherapy, targeted therapy and immunotherapy will benefit patients with pancreatic cancers.P < .001) as well as in patients between 50 and 70 . Periodontal disease, tooth loss and root canal infections showed a positive association with an increased risk of developing pancreatic cancer. Previously, Heikkil\u00e4 et al. (2018), in their register-based cohort study of 68\u00a0273 adults, reported a higher pancreatic cancer mortality among individuals with periodontitis . An even stronger association was noted after adjustments . Fan et al. (2018) in their population-based nested case-control study have provided supportive evidence towards the role of oral periodontopathogenic microbiota in the aetiology of pancreatic cancer.Yu et al. (2022) also confirm the association between poor oral health and pancreatic cancer risk. They found a statistically significant association between periodontitis and an increased risk of pancreatic cancer in patients under the age of 50 , Fusobacterium nucleatum and Tannerella forsythia have a more potent role in the causation of cancers of the digestive tract than previously thought. They are able to evade the immune mechanisms and colonise the gastrointestinal tract, disrupting the epithelial integrity. Of these, Td is a notoriously invasive motile anaerobe with an equally harmful surface-bound chymotrypsin-like proteinase virulence factor. The possible mechanisms by which it can promote carcinogenesis include (a) hydrolysis of bioactive peptides; (b) inhibition of apoptosis; (c) promotion of cellular invasion; (d) degradation of multiple host proteins; and (e) modulate immunity and inflammation.Evidence shows that periodontopathogens like Females with periodontal disease were reported to be 3 times more likely to develop oesophageal cancer. Hence, periodontal disease could be a significant risk factor for cancer, especially in mature women.Additionally, research has found that certain potent periodontopathobionts are implicated in the tumorigenesis and progression of cancers affecting not only the oral cavity but also oesophageal, breast and gall bladder carcinomas. According to Nwizu et al. (2020), periodontal disease raises the general cancer risk by 14%.Patients with pre-existing gum disease are currently likely to have an increased risk for pancreatic cancer. As periodontal disease is a modifiable risk factor, it is of uppermost importance that regular and valid screening measures are employed for its early detection.9To further add to the knowledge, dentilisin acts against various immunomodulatory proteins critical for the regulation of tumour microenvironment and inflammation, thereby further contributing to tumour progression. It also has the ability to modulate immunomodulatory proteins, including matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs), both of which have been implicated in regulation of tumour microenvironment and metastasis of gastrointestinal cancers.MMPs are derived from host PMNs, endothelial, epithelial and smooth muscle cells in latent pro-MMP form. Of all MMPs, particularly activity of MMP-8 (collagenase) is elevated in patients suffering from gum disease as it causes destruction and digestion of type I collagen which is present in periodontal connective tissue.13 Elevated aMMP-8 levels in mouthrinse and patients\u2019 periodontal disease can be conveniently detected by aMMP-8 POCT kits readily available in the market.P-8 POCT . These aThe authors recommend utilising non-invasive biomarkers like aMMP-8 for early detection of periodontal disease since it is a risk factor for carcinomas."} +{"text": "Bone is a vital tissue as it carries out various metabolic functions: support of the body, protection of the internal organs, mineral deposit and hematopoietic functions. It has recently been observed that bone tissue also has endocrine functions, producing substances with hormonal activity such as osteocalcin and Fibroblast Growth Factor-23 (FGF-23) ,2.As is well known, bone tissue is continuously renewed through the bone remodelling process. The first phase involves bone resorption, mediated by osteoclasts, and the subsequent phase bone formation, mediated by osteoblasts. The continuous succession of these two phases keeps the skeleton young and allows the repair of microcracks .Bone metabolism, which is finely regulated by different biologic signals ,3, can bThe identification of mechanisms underlying the pathophysiology of metabolic bone diseases continues to be an area of significant research efforts, attracting scientists from different fields.For this Special Issue, we received different scientific contributions, spanning in vitro studies to clinical research articles and reviews of the scientific literature.The paper by Jansen et al., entitled \u201cIncreased bone resorption during lactation in pycnodysostosis\u201d , exploreNa et al., in the paper entitled \u201cAesculetin inhibits osteoclastic bone resorption through blocking ruffled border formation and lysosomal trafficking\u201d , showed Osteoporosis is generally considered more prominent among women than men. However, in the review by Rinonapoli et al., entitled \u201cOsteoporosis in men: a review of an underestimated bone condition\u201d , the autIn the review entitled \u201cMechanisms of bone fragility: from osteogenesis imperfecta to secondary osteoporosis\u201d , El-GazzRecent research suggests that the microbiota is involved in the regulation of bone metabolism, and that its alteration may induce osteoporosis. This is the topic of the review by De Martinis et al. entitled \u201cThe osteoporosis/microbiota linkage: the role of miRNA\u201d . In part"} +{"text": "Natural killer (NK) cells are innate immune cells that demonstrate cytolytic activity against tumor cells, virus-infected cells and other physiologically stressed cells, such as senescent cells. The scientific community continues to discover novel biological aspects of NK cells in different organs, tumor microenvironments, and age groups. Recently, cancer immunotherapy has achieved exceptional success. Novel agents and combination therapies that modulate the immune system, including NK cells, provide promising routs for future treatment. International Journal of Molecular Sciences, entitled \u201cNatural Killer Cells and Immunotherapy\u201d, includes eight contributions: five original articles and three reviews that each provide new information on NK cell biology and immunotherapy. This Special Issue of the In their review \u201cThe Biological Role and Therapeutic Potential of NK Cells in Hematological and Solid Tumors\u201d, Velichinskii et al. reviewedIn the review entitled \u201cPostoperative Natural Killer Cell Dysfunction: The Prime Suspect in the Case of Metastasis Following Curative Cancer Surgery\u201d, Market et al. introducSome immune cells exhibit unexplained features and functions in our immune system. Natural killer T (NKT) cells possess the common features of T and NK cells in terms of their immune responses. In particular, invariant NKT (iNKT) cells, expressing an invariant TCR \u03b1-chain, constitute a major population of NKT cells . In the Five original articles introduce the novel findings of NK biology and immunotherapy under different contexts.Cytokines are critical determinants of the phenotypic characteristics and functional activity of NK cells in immune response and development. In the article entitled \u201cPro- and Anti-Inflammatory Cytokines in the Context of NK Cell-Trophoblast Interactions\u201d, Mikhailova et al. introducIn the article \u201cNK Cell-Dependent Antibody-Mediated Immunotherapy Is Improved In Vitro and In Vivo When Combined with Agonists for Toll-like Receptor 2 in Head and Neck Cancer Models\u201d, Gruijs et al. investigIn a short communication article entitled \u201cA Chimeric IL-15/IL-15R\u03b1 Molecule Expressed on NF\u03baB-Activated Dendritic Cells Supports Their Capability to Activate Natural Killer Cells\u201d, Bosch et al. reportedIn the article \u201cEx Vivo Expanded and Activated Natural Killer Cells Prolong the Overall Survival of Mice with Glioblastoma-like Cell-Derived Tumors\u201d, Shida et al. reportedIn the final article presented in this publication, \u201cNLRP3 Deficiency in Hepatocellular Carcinoma Enhances Surveillance of NK-92 through a Modulation of MICA/B\u201d, Lee et al. [As described in this Special Issue, NK cells are representative immune effector cells for the development of immunotherapies for hematological and solid cancer treatments. The possibility of using NK cells is growing rapidly, and will open up new opportunities and prospects for the development of immunotherapy in both hematological and solid cancer treatments; however, many questions remain to be elucidated in clinical trials and basic research. The results and discussions of these articles shed considerable light on NK biology and immunotherapy."} +{"text": "Hepatocellular carcinoma (HCC) and biliary tract cancers (BTC) represent the two major forms of primary liver cancers. Despite the growing efforts to translate the increasing knowledge on molecular alterations of these cancers into treatment options for patients, the actual clinical outcomes remain unsatisfying .BTCs are fatal gastrointestinal cancers with very poor 5-year survival rates . The incHCC is the most common form of liver cancer . This deGuo et al.; Shen et al.; Yang et al.) and seven original research papers .Due to the ineffectiveness and development of resistance to current therapies, the need to identify and provide alternative therapeutic approaches is of paramount importance to alleviate the suffering of BTC and HCC patients. Therefore, the current Research Topic provided a structural platform to identify mechanisms of resistance, novel relevant therapeutic targets and prognostic/predictive markers as well as to demonstrate promising innovative treatment options. The call for papers attracted an astonishing number of 10 highly interesting publications and over 65 authors contributed to this Research Topic in the form of three structured reviews , non-invasive preoperative prediction of angiogenesis related markers in BTC , optimizing strategies for immunotherapy and the current status of adjuvant therapies in HCC are discussed , characteristics of extrahepatic cholangiocarcinoma (eCCA) are analyzed , and novel therapeutic strategies for BTC are demonstrated. .This Research Topic covers a variety of subject areas: promising new survival markers after surgery for BTC patients .High levels of vascular endothelial growth factor (VEGF) expression and microvessel density (MVD) correlate with tumor progression and poor prognosis in eCCA patients . HoweverJansson et al.'s study, three preoperative immunologic plasma markers were identified - CSF1, TIE2, and TRAIL - that predict survival after surgery in a cohort of 102 BTC patients utilizing high-throughput multiplex immunoassay. CSF1 and TIE2 were found to be negative prognostic factors in BTC, while TRAIL was demonstrated to be a positive prognostic factor .BTC patients typically experience cancer recurrence within 5\u00a0years of surgery, but prognostic factors such as lymph node metastasis and tumor grading can only be observed after tumor resection . In JansShen et al.).Immunotherapy is a therapeutic option for HCC patients; however, the immune microenvironment of many tumors suppresses the effectiveness of this treatment .To address this Research Topic, Liang et al. also identified an efficacy enhancement of immunotherapy in HCC by combining anti-PD-1 antibodies with Abrine, an inhibitor of indoleamine 2,3-dioxygenase 1 (IDO1). In their study, Liang et al. showed that IDO1 is upregulated in HCC cells and can lead to tumor immune escape. They found that using Abrine along with anti-PD-1 antibodies can inhibit immune escape and increase CD8+ T cell infiltration, leading to a stronger immune response and anti-tumor effect .Taken together, this Research Topic provides insight into the latest efforts to overcome resistance mechanisms of current therapies, discover novel prognostic and predictive markers, and identify alternative anti-BTC/HCC strategies.We sincerely thank all the authors for their valuable contributions to this Research Topic."} +{"text": "Molecular biomarkers are emerging as the key indices for the management of patients with significant diseases. Motivated by the systematic effort to define the human genome, the creation of rapid analytic technologies for evaluating nucleic acids and proteins has provided the technological \u201cboom\u201d for the development of molecular biomarkers. Collaboration and cooperation between stakeholders involved in biomarker development, application, and regulation may be the most expeditious route toward the translation of laboratory discovery into patient management. In summary, intensive research has originated multiple factors or biomarkers that are likely to be helpful in diagnosis, characterization, and therapy selection of different patients. A thorough understanding of the relevance of each biomarker will be the key to efficiently diagnose a disease and direct the patients towards the drugs more likely to be of benefit based on their particular profile. The papers selected for this special issue represent an excellent panel for addressing the molecular biomarkers as the future tools of medicine. This special issue contains thirteen papers.Circulating microRNAs and kallikreins before and after radical prostatectomy: are they really prostate cancer markers?,\u201d M. G. Egidi et al. presented 38 patients with prostate cancer. They suggested that two miRNAs (miR-21 and miR-141) could be involved in post surgical inflammatory processes. Postoperative serum kallikreins showed a significant decrease, highlighting the potential usefulness of kallikreins apart from PSA as potential prostate cancer markers.In the paper entitled \u201cA novel differential predict model based on matrixx-assisted laser ionization time of flight mass spectrometry and serum ferritin for acute graft-versus-host disease,\u201d. C.-Y. Zhang et al. investigated the possibility of pre warning the risk of an acute graft-versus-host disease (aGVHD) before and after allogeneic hematopoietic stem cell transplantation by serum profiling combining with serum ferritinin. Their joint prewarning model could predict the risk of aGVHD, especially severe aGVHD before transplant which provide a reliable method to continuously monitor the condition of patients.In the paper entitled \u201cThe use of multidimensional data to identify the molecular biomarker for pancreatic ductal adenocarcinoma,\u201d L. Zhuang et al. presented that they have adopted an integrative approach to simultaneously identify biomarker and generate testable hypothesis from multidimensional omics data. They have found that PER2 expression was highly associated with the survival data, thus representing a novel biomarker for earlier detection of pancreatic ductal adenocarcinoma (PDAC).In the paper entitled \u201cClinical evaluation and cost-effectiveness analysis of serum tumor markers in lung cancer,\u201d R. Wang et al. showed that combinations of four tumor markers improved the sensitivity for lung cancer and different combination panels had their own usefulness. NSE, CEA, and CYFRA21-1 were the optimal combination panel with highest Youden's index (0.64), higher sensitivity (75.76%), and specificity (88.57%), which can aid in clinical diagnosis of lung cancer.In the paper entitled \u201cImmune parameters in the prognosis and therapy monitoring of cutaneous melanoma patients: experience, role, and limitations,\u201d M. Neagu et al. reported the follow-up for 36 months of the immune parameters of patients diagnosed in stages I\u2013IV, namely, pre- and postsurgery immune circulating peripheral cells and circulating intercommunicating cytokines.In the paper entitled \u201cComparative gene expression profiling in human cumulus cells according to ovarian gonadotropin treatments,\u201d S. Assou et al. provided an exclusive study characterizing gene expression profiles in cumulus cells (CCs) of periovulatory follicles from patients undergoing HP-hMG and rFSH gonadotropin treatments during in vitro fertilization cycles. This project has characterized the expression of these genes as biomarkers of in vitro embryo quality.In the paper entitled \u201cAptamers: novel molecules as diagnostic markers in bacterial and viral infections?,\u201d F. M. Zimbres et al. urged an urgent need to discover novel diagnostic as well as therapeutic tools against infectious agents. They viewed that the systematic evolution of ligands by exponential enrichment (SELEX) represents a powerful technology to target selective pathogenic factors as well as entire bacteria or viruses. SELEX uses a large combinatorial oligonucleic acid library (DNA or RNA) which is processed by a high-flux in vitro screen of iterative cycles.In the paper entitled \u201cDistribution of ABO blood group and major cardiovascular risk factors with coronary heart disease,\u201d S. Biswas et al. viewed that the AB blood group decreases the risk of CHD in healthy controls; it might be due to the higher concentration of high density lipoprotein cholesterol (HDL-c), while the O blood group increases the risk of CHD due to lower HDL-c levels in Bengali population of eastern part of India.In the paper entitled \u201cImmunomodulatory effect of continuous veno-venous hemofiltration during sepsis: preliminary data,\u201d G. Servillo et al. reported that severe sepsis and septic shock are the primary causes of multiple organ dysfunction syndrome (MODS), which is the most frequent cause of death in intensive care unit patients. Many pro- and anti-inflammatory mediators, such as interleukin-6 (IL-6), play a key role in septic syndrome. Continuous renal replacement therapy (CRRT) removes in a nonselective way pro- and anti-inflammatory mediators. The authors investigate the effects of continuous venovenous hemofiltration (CVVH) as immunomodulatory treatment of sepsis in a prospective clinical study.In the paper entitled \u201cAcetylcholinesterase as biomarker in environmental and occupational medicine: new insights and future perspectives,\u201d M. G. Lionetto et al. viewed that Acetylcholinesterase (AChE) is a key enzyme in the nervous system (NS), since it terminates nerve impulses by catalyzing the hydrolysis of acetylcholine. As a specific molecular target of organophosphate and carbamate pesticides, AChE activity and inhibition are a human biological marker of pesticide poisoning. Thus, it is used to study the effects of the exposure to organophosphate and carbamate pesticides on NS in occupational and environmental medicine. This paper reviews and discusses the recent findings about AChE, including its sensitivity to other pollutants and expression of different splice variants. These insights open new perspectives for the use of this biomarker in environmental and occupational human health monitoring.In the paper entitled \u201cPolyisoprenylated methylated protein methyl esterase is both sensitive to curcumin and overexpressed in colorectal cancer: implications for chemoprevention and treatment,\u201d F. Amissah et al. discussed polyisoprenylated methylated protein methyl esterase (PMPMEase) which cocatalyzes the polyisoprenylation pathway required to process various monomeric G proteins. Mutation of these G proteins is considered to be responsible for 50% of colorectal cancers. This interesting finding suggests that elevated PMPMEase activity and its overexpression can be one of the candidate markers for early diagnosis of colorectal cancer. Susceptibility of this enzyme to curcumin also suggests that PMPMEase can be a potential candidate for targeted anticancer therapy.In the paper entitled \u201cEmerging therapeutic biomarkers in endometrial cancer,\u201d P. Dong et al. reviewed the current status of molecular therapies tested in clinical trials and mainly discussed the potential therapeutic candidates that are possibly used to develop more effective and specific therapies against endometrial cancer progression and metastasis.In the paper, entitled \u201cMouse prostate epithelial luminal cells lineages originate in the basal layer where the primitive stem/early progenitor cells reside: implications for identifying prostate cancer stem cells\u201d J. Zhou et al. have developed an in vivo cell fate tracing mouse model and an in vivo slow-cycling cell label mouse model to provide further insight into this question. Through genetic manipulation in the animals, their findings indicate that the basal cell lineage can produce more differentiated luminal cells; the putative mouse prostate stem cells (which are slow-cycling and responsible for tissue maintenance) likely reside in the basal layer.In the paper entitled \u201c Though the selected topics and papers are not an exhaustive representation of the entire area of molecular biomarkers and tools of medicine, yet they represent the rich and many-faceted knowledge that we have the privilege of sharing with the readers."} +{"text": "The growing concern about cannabis use, the most commonly used illicit drug worldwide, has led to a significant increase in the number of human studies using neuroimaging techniques to determine the effect of cannabis on brain structure and function. We conducted a systematic review to assess the evidence of the impact of chronic cannabis use on brain structure and function in adults and adolescents.Papers published until August 2012 were included from EMBASE, Medline, PubMed and LILACS databases following a comprehensive search strategy and pre-determined set of criteria for article selection. Only neuroimaging studies involving chronic cannabis users with a matched control group were considered.One hundred and forty-two studies were identified, of which 43 met the established criteria. Eight studies were in adolescent population. Neuroimaging studies provide evidence of morphological brain alterations in both population groups, particularly in the medial temporal and frontal cortices, as well as the cerebellum. These effects may be related to the amount of cannabis exposure. Functional neuroimaging studies suggest different patterns of resting global and brain activity during the performance of several cognitive tasks both in adolescents and adults, which may indicate compensatory effects in response to chronic cannabis exposure.However, the results pointed out methodological limitations of the work conducted to date and considerable heterogeneity in the findings.Chronic cannabis use may alter brain structure and function in adult and adolescent population. Further studies should consider the use of convergent methodology, prospective large samples involving adolescent to adulthood subjects, and data-sharing initiatives. Cannabis is the illicit drug most widely available and used worldwide Animal studies have consistently demonstrated that delta-9-tetrahydrocannabinol (THC), the main psychoactive component of cannabis It is remarkable to note that although the onset of cannabis use is typically during adolescence, a few imaging studies have been conducted with adolescent users The growing concern about cannabis use has led to a significant increase in the number of human studies using neuroimaging techniques to determine the effect of the substance on brain structure and function, as well as to several recent reviews examining this topic Data for this systematic review was collected with an advanced document protocol in accordance with the PRISMA guidelines Electronic searches were performed using EMBASE (1980-August 2012), Medline (1966-August 2012), PubMed (1966-August 2012) and LILACS (1982-August 2012) databases. The following key words were used: cannabis; marijuana; marihuana; delta-9-tetrahydrocannabinol; THC; cannabidiol, CBD; neuroimaging; brain imaging; computerized tomography, CT; magnetic resonance, MRI; single photon emission tomography, SPECT; functional magnetic resonance, fMRI; positron emission tomography, PET; diffusion tensor MRI, DTI-MRI; spectroscopy, MRS. All the studies published up to August 2012 were included without language restriction.A general review of all neuroimaging studies investigating brain structure or function was initially performed. We obtained a total of 142 published papers . StudiesWe defined chronic cannabis users as persons who used cannabis several times a week and who had done so for at least two years. Recreational cannabis users were defined as persons who had used cannabis sporadically (less than four times a month), and na\u00efve users or healthy controls were persons who had used cannabis less than 15 times in their lifetime, according to standardized strict criteria Any publication that reported data using two different neuroimaging techniques from the same subjects or a study examining the same subjects with two different cognitive tasks was considered as two studies in this review.Data was independently extracted by two reviewers. In case of disagreement, opinion from a third senior researcher was sought to assess whether study criteria were fulfilled. From the articles included we recorded names of authors, year of publication, socio-demographic and cannabis use characteristics , imaging type and design, exclusion criteria , confirmation of abstinence from other drugs (whether checked by urine test), rest/active condition , type of cognitive task performed during functional imaging and psychopathological variables assessed . With regard to alcohol use, we assessed if subjects met criteria for alcohol abuse or for excessive alcohol consumption based on the reported data. For structural and functional imaging data, the primary measures of interest were global and regional volume, and global and regional activity . The secondary outcome was its correlation with clinical variables. We collected the statistically significant results of each outcome variable, and recorded whether a multiple comparison correction was done to prevent bias towards false positives.a priori selection criteria Of the 142 studies identified, thirty-six did not meet the We identified 11 structural MRI studies that examined adult chronic cannabis users and met our selection criteria . StructuOf the seven studies comparing global brain volume measures between chronic cannabis users and healthy controls, there was only one study reporting significant differences et al. (2005) et al. (2011) et al. (2005) Among the six studies employing a whole-brain analysis approach With regard to the three studies that focused on specific regions of interest, all studies reported bilateral volumetric reductions in the hippocampus et al. (2011) et al. (2005) With respect to other brain regions, Cousijn et al. (2008) et al. (2011) et al. (2011) Four studies have used DTI to examine the integrity of white matter tracts in chronic cannabis users et al. et al. (2011) Three volumetric studies in adolescent chronic cannabis users were included, two of which consist of the same sample et al. (2010) et al. (2011) et al. (2009) In terms of correlations, Medina 15O-PET 133Xe-SPECT 18F-FDG-PET 11C]- raclopride-PET 18F]FMPEP-d2 11C]-raclopride-PET studies et al. (2000) et al. (2001) et al. (2008) 11C]-raclopride-PET binding potential and glucose metabolism 11C]- raclopride-PET studies et al. (2012) et al. (2012) et al. (2011) We included eight case-control studies comparing resting rCBF in adult chronic cannabis users and non cannabis using healthy controls . The imaWe identified 16 studies in adult chronic cannabis users that compared regional activation during the performance of a cognitive task with healthy controls , four wiet al. (2006) et al. (2010) et al. (2012) Chang 15O-PET study, Block et al. (2002) et al. (2007) et al. (2006) In a H2et al. (2004) et al. (2005) et al. (2005) et al. (2004) 15O-PET during the performance of a modified Stroop test. However, they also reported a reduced dorsolateral prefrontal cortex activation and a greater activation in the hippocampus bilaterally et al. (2009) Eldreth et al. (2005) et al. (2011) 15O-PET, and Wesley et al. (2011) et al. (2005) et al. (2011) et al. (2011) et al. (2011) et al. (2010) et al. (2010) et al. (2010) et al. (2010) et al. (2010) Bolla et al. (2011) King et al. (2009) Gruber We included five case-control fMRI studies in adolescent cannabis users comparing brain activity with healthy controls during a cognitive task performance. As an exception, two of them et al. (2007) et al. et al. (2007) et al. (2008) et al. (2010) et al. (2011) Padula et al. (2007) In a group of abstinent cannabis users, Tapert 1-rich areas implicated in several cognitive functions, which may be related to the amount of cannabis use; (2) Altered neural activity during resting state and under several different types of cognitive paradigms, that may reflect a different recruitment of brain areas during the tasks, particularly within the prefrontal cortex; and (3) The few studies conducted in adolescents suggest that both structural and functional alterations may appear soon after starting the drug use and may be related to gender.In this systematic review, we identified 43 studies suitable for inclusion regarding the impact of chronic cannabis use on brain structure and functioning, of which eight (19%) were in the adolescent population. Despite the high degree of heterogeneity among the studies reviewed herein, several relatively consistent findings emerged from this review. These findings, discussed in detail below, include: (1) Structural brain abnormalities, mainly in CBIn terms of structural findings, specific regional brain analyses demonstrated evidence of structural abnormalities when adult chronic cannabis users were compared with healthy controls. The most consistently reported brain alteration was reduced hippocampal volume Functional neuroimaging studies that have evaluated the resting state in active and abstinent adult chronic cannabis users suggest that resting global Functional imaging studies comparing activation in both adult and adolescent chronic cannabis users with healthy controls during the performance of different cognitive tasks indicated that chronic cannabis users would use similar brain areas that engage these cognitive processes but often demonstrating an altered pattern of brain activity A further important issue emerging out of this review is that few studies have investigated the effects of chronic cannabis use on the brain in adolescence subjects. In light of the popularity of cannabis among teenagers Results presented here have pointed out some important methodological differences that limit the generalisation of results and comparison between studies and have doubtless contributed to the slightly disparate array of findings. Despite the use of a strict definition of chronic cannabis user and robust application of inclusion and exclusion criteria in an attempt to avoid excessive heterogeneity between samples, studies often diverged on certain socio-demographic characteristics and cannabis use parameters, such as gender-bias, age of onset, lifetime use and abstinence period before the acquisition of imaging data. Moreover, it is well known that the THC content of smoked cannabis varies markedly between sources and preparations, with potency reported to have increased substantially over the past ten years With regard to other methodological limitations, some studies have reported modest sample sizes, sometimes below the threshold that would be currently regarded as acceptable Table S1Click here for additional data file."} +{"text": "The seventh annual Midsouth Computational Biology and Bioinformatics (MCBIOS) conference took place February 19 and 20, 2010 at Arkansas State University in Jonesboro, AR, presided over by Daniel Berleant, this year\u2019s President of MCBIOS. Keynote speakers were Elaine Ostrander of NIH, renowned for her work on dog genomics, Clayton Naeve, CIO of St. Jude Children's Research Hospital, and Robert Cottingham, Leader of the Computational Biology and Bioinformatics Group, Oak Ridge National Laboratory. The record-breaking attendance exceeded 200 participants which necessitated parallel talk sessions for the first time. Student oral presentation award winners were Heidi Pagan (first place) of Mississippi State University (MSU), Juliet Tang (second place) of MSU, and Aleksandra Markovets (third) of Mississippi Valley State University. In addition, a record number of posters were also presented. Due to the number of posters, student poster awards were given in two categories, Biological Focus and Computational Focus. Winners were N. Platt and V. Chaitankar (1st place), G. Cooper and L. Pillai (2nd), and M. Ammari and C. Gearheart (3rd). Reflecting the integration of these foci in the field, winners in one category frequently scored high in the other category as well. MCBIOS is also pleased to have this year acquired legal status as a non-profit organization.A total of twenty eight papers describing research presented at the 2010 conference were accepted for publication in these proceedings out of a total of forty three papers submitted for consideration (~65%). The number of papers submitted for consideration was the largest submitted since the inception of MCBIOS with a 34% increase over last year\u2019s proceedings , and theet al. describe an expansion of the FDA-developed, ArrayTrackTM genomics tool, into a platform that supports microbial data to enable the rapid detection of food borne pathogenic bacteria during outbreak scenarios [et al. describe new SNP (single nucleotide polymorphism) and QTL (quantitative trait locus) libraries for the ArrayTrack system which provide users with extensive data including links to other resources, and motivated in part by the need to augment gene association study results with biological context [et al. report on the new web-based tool, IView, which graphically displays and makes searchable introgression data by marker name, chromosome number, or map position [Bioinformatics was largely born as a field through the need to analyze and understand sequence data. Partly as a result of ultra-high-throughput sequencing technology, the field has seen a strong resurgence recently with new computational methods being developed and applied to downstream applications such as the identification of human mutations -25 as wecenarios . Xu et context . Christoposition .Quest and colleagues working in the Cottingham laboratory address the problem of brittle genome annotation systems with an OWL-based approach that provides several significant advantages . Work reAn examination of substitution rates over evolutionary time by Ulrich Melcher compared viral and non-viral sequences to reveal that the rate of nucleotide interchanges in plant viruses is not constant, impacting phylogeny studies. The evidence suggests that different evolutionary rules may apply to recent divergences than to those linked to distant speciation events . et al. using a proteomics approach to evaluate the host protein expression differences. Two gene sets were found, each specific to one disease form which showed significant functional differences [et al. address the need for additional genomic information for Northern bobwhite, an avian species critical to ecosystem maintenance. Using next generation sequencing of cDNAs from a multi-tissue library, they generated ESTs which were pipelined into a unigene database and made publicly available at a searchable website [An in depth analysis of the two forms of Bovine Viral Diarrhea, a worldwide cattle disease, was completed by Mais Ammari ferences . Arun R website . et al [et al [Arabidopsis. The conserved gene evolution indicated that the study in the model species can be translational to human disease studies. Rowena Kelly et al [Aspergillus flavus resistance in Maize such as gene expression, proteomic, QTL genetic mapping studies and sequence data from the literature to help identify and select candidate A. flavus resistance genes.Cyriac Kandoth et al mined thl [et al carried ly et al have conBiological information may be encoded in a one-dimensional sequence, but its manifestation takes place in three-dimensional space in the form of proteins, cells and tissues among other structures. Computational analysis and prediction of these structures is challenging, to say the least, and an active area of recent bioinformatics interest -48. Compet al. report on a method for modeling interactions between 8R-lipoxygenase and its substrate [et al., which can serve as an important training and practice tool for surgeons [et al.[To better understand and improve of the creation of anti-inflammatory drugs through inhibition of lipoxygenase, an essential enzyme in the inflammatory pathway, Shuju Bai ubstrate . A mixedsurgeons . The pos [et al.. They ret al [et al [et al compared density based clustering and Fuzzy C-Means clustering algorithms for border detection in dermoscopy images finding that density based clustering performed best with a low border error, high precision and recall; however, the Fuzzy C-Means clustering algorithm had poor performance especially in border detection. Mete et al proposed two new methods, boundary driven density based clustering based algorithms which performed better delineation with noisy images and an active contour model that gave the best detection with optimum parameters. Denise Koessler et al [Sinan Kockara et al and Mutll [et al examineder et al developeThere are many areas within biology that are amenable to computational analysis, each of which is usually approached separately. Somewhat recently, the term \u201csystems biology\u201d has emerged to describe interconnected analyses, particularly those that help reduce complexity in these systems, as defined by the number of interconnections between component parts, into a more functional understanding . The term \u201csystems biology\u201d is often used broadly and not always consistently, but is an emergent area of high interest in bioinformatics -57. et al.[et al. report on the development of a Web-based gene-discovery bioinformatics tool, FAUN (Feature Annotation Using Nonnegative matrix factorization), which identifies implied gene relationships from biomedical literature, enabling hypotheses on functional characterization [A novel method for identifying subnetwork markers in a human protein-protein interaction network is described by Junjie Su et al.. They rrization . et al., explores the important problem of inferring gene regulatory networks from time sliced microarray data. They claim that mutual information (MI) based approaches have inherent limits to their capabilities in this context which they were able to improve upon with algorithms based on modifications to the mutual information metric [ A paper by Vijender Chaitankar, n metric .Kumar and Nanduri present a downloadable database, HPIDB, which serves as a resource of host-pathogen protein-protein interactions integrated non-redundantly from public databases. The authors report on the flexibility of the database for querying and an output format which allows portage to downstream applications .Microarrays have long been a subject of bioinformatics analysis not only for better understanding transcription, a process affected by both genetic and epigenetic factors ,63, but A new non-stationary Dynamic Bayesian Network with a flexible lag choosing mechanism that detects potential genetic regulators and has improved structure prediction is reported by Yi Jia and Jun Huan .et al. address two interesting QC questions of whether expired Affymetrix GeneChip microarrays, an expensive resource, are still useful up to four years after expiration and if RNA stored at -80\u00b0C for the same period was suitable as template source. In spite of a decrease in sensitivity, the authors found that these arrays generated data consistent with that from unexpired arrays and report favorably on the stability of the RNA [Zhining Wen the RNA .There are a few other papers published in the proceedings but, for space, are not summarized here: -69.http://www.ISCB.org). For information regarding MCBIOS and our future meetings, see http://www.MCBIOS.org.MCBIOS 2011 will be held in College Station, Texas. The 2010-2011 President of MCBIOS is Ulisses Braga-Neto of the University of Texas A&M, and Susan Bridges of Mississippi State University is now the President-elect. MCBIOS is a regional affiliate of the International Society for Computational Biology (The authors have no competing interests to declare.All authors served as editors for these proceedings, with JDW serving as Senior Editor. All authors helped write this editorial."} +{"text": "Transcranial direct current stimulation (tDCS) is a noninvasive neuromodulatory technique with putative cognitive enhancing and therapeutic applications. Since the year 2000, almost 1000 papers have been published on tDCS, reflecting the possible significance of a cheap, safe, and easily applied neuromodulatory technology. Whether or not this potential is tapped depends on understanding how tDCS affects brain functioning, a question explored in a recent publication by Stagg et al. ; presenttDCS is thought to decrease neuronal resting membrane potential beneath the anodal electrode by pumping in positive ions; vice versa for cathodal stimulation underwent two tDCS sessions in a counterbalanced order, separated by a week. tDCS electrodes were applied to left DLPFC and the contralateral supraorbit. In each session, participants underwent a 40-min resting-state ASL functional MRI scan in which there was a 10-min baseline, 20-min of concurrent tDCS-ASL and a 10-min post-stimulation period. Separately, but using the same protocol, a second group of participants (N = 12) were scanned once under sham stimulation only. Stagg et al. compared resting-state CBF between these three periods and examined changes in functional connectivity between the regions beneath each electrode and the rest of the brain. The same analyses were conducted using the sham stimulation group to confirm that the changes were not driven by nonspecific temporal drifts.Stagg et al. studied the effect of tDCS on cerebral perfusion using ASL. Subjects to group 2 (stimulation\u2014baseline) should be conducted; finding a statistically significant effect in one sample, but not another is not a valid approach to establish the between-group effect (Nieuwenhuis et al., The electrical current used in tDCS studies is insufficient to generate neuronal action potentials. Instead, spontaneous firing changes resulting from alterations in neuronal resting membrane potential are thought to underlie the neuromodulatory effects of tDCS (Bikson et al., While the clinical potential for tDCS is high, it is obscured by our lack of insight into its neural effects. Studies such as Stagg and colleagues' build on previous findings and pave the way to an improved understanding. Only with this comprehension can the full clinical possibility of tDCS be utilized and directed toward improving treatment.Camilla L. Nord, N\u00edall Lally, and Caroline J. Charpentier wrote the manuscript together."} +{"text": "Frontiers in Fungi and their Interactions details the exciting progress in developing vaccines and immunotherapy for fungi.Invasive fungal diseases have increased many fold over the past 50 years. Current treatment regimens typically require prolonged administration of antifungal medications that can have significant toxicity. Moreover, our present potent antifungal armamentarium fails to eradicate fungal pathogens from certain compromised hosts. Additionally, invasive fungal diseases continue to have unacceptably high mortality rates. A growing body of work has focused on the utility of vaccines and/or immunotherapy as a powerful tool in combating mycoses, either for the active treatment, as an adjuvant, or in the prevention of specific fungal pathogens. This Research Topic \u201cVaccines and Immunotherapy against fungi: a new frontier\u201d in Candida albicans (Vecchiarelli et al., Aspergillus fumigatus (Diaz-Arevalo et al., Cryptococcus neoformans (Hole and Wormley, Paracoccidioides brasiliensis (Travassos and Taborda, P. brasiliensis is also presented (Mayorga et al., Artocarpus heterophyllus, which modulates immunity against P. brasiliensis (Ruas et al., Histoplasma capsulatum (Nosanchuk et al., The critical requirement for understanding the degrees of engagement of host defense pathways in responding to fungal invasion has led to an increased focus on host-pathogen interactions. In this Research Topic, Carvalho et al. review oIn sum, these articles broadly paint the current spectrum of investigations on host-pathogen interactions and provide a review of the state-of-the-art in vaccinology and immunotherapy against fungi. The information presented also underscores the rich areas for future study, all promising improved therapeutics against fungal invaders."} +{"text": "APP are linked to early onset of familial Alzheimer\u2019s disease (FAD); APP processing generates \u03b2-amyloid (A\u03b2) peptides, which are the principal components of the amyloid pathology. Therefore, understanding the role of APP in neuronal function and dysfunction will provide crucial insights to AD pathogenesis.Genetic and biochemical studies establish a central role of the amyloid precursor protein (APP) in Alzheimer\u2019s disease (AD): Genetic mutations and gene amplification of We have a long-standing interest in studying the physiological functions of APP in neurons and synapses. Analysis of various loss-of-function mutants of APP and combining with a mixed-culture system allowed us to identify APP as a synaptic adhesion protein. We recently uncovered a potent role of APP in mediating adult neurogenesis in dentate gyrus: Loss of APP results in an aberrant increase in progenitor proliferation but impaired newborn neuron differentation, maturation and integration. Intriguingly, we found that APP is highly expressed in GABAergic interneurons and that specific deletion of APP in these neurons, but not in excitatory neurons, leads to similar neurogenesis defect. Our mechanistic and functional studies indicate that this activity is mediated by a general role of APP in regulating GABA release through its synaptic adhesion property.AD is an age-related disease. Adult neurogenesis declines sharply with aging. Increasing evidence supports a role of hippocampal adult neurogenesis in brain function and its impairment in AD. Therefore, perturbation of APP-mediated adult neurogenesis may contribute to neuronal dysfunction and AD pathogenesis."} +{"text": "It is unique in its strict selection of a single member for activation, a process termed monoallelic expression. The conceptual advances that have emerged from studying var genes show striking common epigenetic features with many other clonally variant gene families or even single-copy genes that show a variegated expression in parasite populations. However, major mechanistic questions, such as the existence of a potential expression site and the identity of transcription factors or genetic elements driving singular gene choice, are still unanswered. In this review we discuss the recent findings in the molecular processes essential for clonal variation, namely silencing, activation, poising and switching. Integrating findings about all clonally variant gene families and other mutually exclusive expression systems will hopefully drive mechanistic understanding of antigenic variation.Phenotypic variation in genetically identical malaria parasites is an emerging topic. Although antigenic variation is only part of a more global parasite strategy to create adaptation through epigenetically controlled transcriptional variability, it is the central mechanism enabling immune evasion and promoting pathogenesis. The The sites of host\u2013parasite interaction are constantly exposed to recognition by the host immune system. Protozoan pathogens have developed a wide range of sophisticated immune evasion strategies such as antigenic variation . Most var genes are in subtelomeric regions, whereas others are arranged in more chromosome central positions. Var genes consist of an Exon1, coding polymorphic sequences forming the extracellular domain, an Exon2, coding the semi-conserved intracellular domain, connected by a single highly conserved intron.The protozoan pathogen d stream A. Duringvar gene expression studies have wide implications for other plasmodial gene families and single-copy genes controlled by similar epigenetic mechanisms A and B subtypes, as well as some members of the rifins, another clonally variant gene family. Inactivation of a second member of the Sir2 family, called Sir2B, showed some complementary de-repression effect on other, mostly upsC, var gene members , which is enriched in promoter regions of repressed var genes to silent but not active P. falciparum than was anticipated and Histone 3 lysine 9 acetylation (H3K9ac), which renders it permissive for transcription in var gene is active within a single cell. The HB3 P. falciparum strain contains two nearly identical copies of the var2csa gene. Despite the fact that one copy lies on chromosome 12 and the other on chromosome 1, RNA FISH analysis showed that simultaneous expression of both copies occurs at the same site in the nucleus . Interestingly, an episomal promoter of the rifin gene families also colocalizes with the active var gene expression site when activated through drug selection . An alternative view is that an activation complex is recruited to the var gene in situ. This initiates the transcriptional activation cascade including its relocation away from the repressive zone. This scenario predicts that any perinuclear site outside the repressive centres may form an expression site. Depending on which model turns out to be accurate, var gene activation would occur before segregation from the repressive cluster or segregation would be a precondition for activation. In either case, the recent discovery of nuclear actin associated to var gene intron regions opens a new exciting avenue. Nuclear actin could provide a mechanical framework for spatial organization of active and silent genes .A different hypothesis for a mutual exclusive expression mechanism postulates the existence of a unique Var genes are only transcribed during the early stages of the blood stage cycle and intrinsic switching rates determined from in vitro cultured parasites can reach up to 2% per cycle (Roberts et al., var genes will be re-activated after the next round of invasion (var promoter in the late blood cell stages, while H3K9me3 is prevented from spreading into the promoter region (Lopez-Rubio et al., var gene locus in post-ring stages, whereas repressing marks are excluded from this site, presumably contributing to the poised state (Volz et al., P. falciparum awaits further investigation.invasion A. ChIP aSwitching between the clonally variant genes is central to antigenic variation and must be adapted to the host so the variant gene repertoire is not depleted too fast whilst effective immune evasion is still possible. It is important to keep in mind that the parasite needs to control its own proliferation so the host is not severely harmed by high parasitemia and effective transmission is guaranteed.var members (Horrocks et al., var genes during the non-transcribed phase can be experimentally erased by promoter titration (Dzikowski and Deitsch, et al., var genes is independent of previous stages. Another recent study on switch pathways used experimental evidence combined with mathematical modelling to conclude that var switching is non-random and necessitates a balanced process of parasite-intrinsic switching and immune-mediated selection (Recker et al., var gene switching patterns using silencing with drug-selectable markers in multiple isolates suggests that a var gene switching pattern is conserved throughout different genetic backgrounds (Enderes et al., et al., To this day, no genes modifying switch rates have been identified and no functional data is available on the molecular mechanism of switching. Nevertheless, descriptive studies demonstrated that transition rates can vary between var gene silencing may be modulated by external factors. For example, Sir2 depends on NAD levels for its activity. Low nutrition states of patients may influence Sir2 activity and hence alter the switch rate of distinct var subtypes. Experimental data from in vitro cultured parasites using peroxide or starvation stress can lead to a slight up-regulation of central var genes (Rosenberg et al., var gene expression pattern in patients (Merrick et al., Another critical question is whether the observed intrinsic switch rate is influenced by external factors, i.e. does the parasite react to its environment, or is the switching pattern hard-wired into the parasite's genome? Although no longitudinal data from human patients are available to support this concept directly, the epigenetic machinery controlling var expression studies from malaria patients, however, support the idea that host factors contribute to var gene transcriptional control. Unlike in vitro cultured parasites, ups A-type var genes were frequently expressed in P. falciparum-infected patients (Bachmann et al., et al., var genes mostly of the ups B and C-type. It will be crucial to investigate whether host factors modify the intrinsic switch rate of ups A-type var genes.The relative paucity of patient data related to this topic makes it difficult to investigate switching rates in the human host. Two recently published var genes cooperate with chromatin modifying enzymes in a complex interplay. On top of this, spatial regulation creates specific nuclear sub-compartments most likely critical for default silencing and monoallelic expression. Other factors such as non-coding RNA produced in subtelomeric regions adjacent or within var genes may contribute as well to antigenic variation (Epp et al., et al., Mutually exclusive expression relies on distinct layers of genetic and epigenetic control factors. Genetic elements, such as the promoter and the intron of var gene expression site remain to be explored. The biggest puzzle, however, in understanding mutual exclusive expression, is the absence of a putative activation factor. The concept of a limiting factor driving monoallelic var transcription has been postulated on many occasions but remains to be demonstrated. The notion that a unique enhancer element is the activating factor or expression site body would seem plausible, since there is exactly one copy in the nucleus of this haploid parasite. A transcription factor, whose expression is restricted to extremely low levels, may be considered but it seems difficult to really make sure that exactly one transcription factor is present. Considering that under special conditions more than one promoter can be activated we favour a model where potential positive feedback loops within the activating complex could ensure aggregation around the expressed var gene. In the absence of candidate genes for a monoallelic activating factor, identification of mutant parasites that have lost var gene expression remains the biggest challenge in the field. Traditional biochemical and mass spectrometric analysis of factors interacting with key genetic elements (Zhang et al., et al., et al., et al., Properties and composition of the hypothetical It remains enigmatic how one gene is active while others are silenced, when and how a specific gene is poised rather switched, but many exciting research avenues are left to explore in the field of mutually exclusive expression."} +{"text": "Dear Editor,In your June 2011 issue, we found an interesting study by Farzanegan et al. entitled, \u201cEffects of lumbar discectomy on disability and depression in patients with chronic low-back pain.\u201d , 4. DeprThe association between depression and pain after low-back surgery is not consistent across studies. Depression and disability have been found to be highly influential in patients undergoing lumbar spine surgery. The psychological profile of depression has predicted poorer outcomes in patients with chronic sciatica pain and disability . The stu"} +{"text": "In contrast to the current therapy, which antagonises the formation of proinflammatory mediators, the \u201cproresolving\u201d therapy promotes natural antiinflammatory processes. It is likely that several drugs and phytochemicals would act in this way, but this point has not been investigated and thus might be totally overlooked. In this paper, effects of curcumin (diferuloylmethane) were analysed, considering the ability of this natural compound to affect resolution of inflammation through modulation of its important inputs \u2013 activity and apoptosis of neutrophils. The presented data indicate that, besides its well-known ability to suppress mechanisms engaged at the onset and progression of inflammation, curcumin could support resolution of inflammation through decreased activity and enhanced apoptosis of neutrophils. This substance decreased the formation of oxidants in neutrophils, both under in vitro conditions and after oral administration to arthritic rats. Moreover, curcumin accelerated spontaneous apoptosis of neutrophils, as indicated by increased externalisation of phosphatidylserine, by intercalation of propidium iodide and by enhanced activity of the executioner caspase-3.Novel strategies of antiinflammatory therapy are based upon pharmacological agents capable to enhance the resolution \u2013 During the inflammatory process, different cytokines, growth, transforming and chemotactic factors are synthesised and several types of cells become activated. Neutrophils (polymorphonuclear leukocytes) are considered to be central cells of acute inflammation. These cells most rapidly reach the site of injury or infection and liberate antimicrobial proteins, proteases and produce reactive oxygen species. Prolonged or excessive liberation of these very effective and toxic substances could intensify the inflammatory process and enhance tissue damage. Therefore activity of neutrophils declines consecutively by the effect of antiinflammatory mediators and programmed death of these cells (apoptosis) is initiated. These processes are essential for the termination (resolution) of the beneficial inflammation , luminol, isoluminol and PMA (phorbol-myristate-acetate) were obtained from Sigma and methotrexate was from Pliva-Lachema . Human Annexin V/FITC Kit was purchased from Bender MedSystems , Caspase-Glo 3/7 Assay from Promega and human purified caspase-3 was from Enzo Life Sciences .in vitro conditions, isolated human neutrophils were stimulated with 0.05 \u00b5M PMA and chemiluminescence was enhanced either with isoluminol (extracellular) or with luminol in the presence of extracellular scavengers \u2013 superoxide dismutase and catalase and propidium iodide. Only Annexin positive cells were considered pre-apoptotic cells and double positive cells (Annexin+/propidium+) were considered late-apoptotic or dead cells.et al., Effect of curcumin on caspase-3 activity was evaluated in cell-free system by the luminescence method, using Caspase-Glo 3/7 Assay kit and human purified caspase-3 elevated the percentage of preapoptotic and apoptotic neutrophils A and incet al., 2, myeloperoxidase, collagenase, as well as to its ability to modulate activities of T lymphocytes and macrophages were reduced more effectively than oxidants formed within neutrophils (involved in elimination of phagocytosed pathogens and fulfilling a regulatory role). Compared to previous studies (Limasset in vivo conditions \u2013 in arthritic rats. Neutrophils modified by inflammation produced several times more oxidants than did healthy controls and this hyperreactivity was suppressed by orally administered curcumin. Since the excessive formation of radicals was ascribed to the enhanced activity of NADPH oxidase in the presence of cytokines (Fairhurst et al., et al., et al., et al., et al., The decreasing effect of curcumin on neutrophil activity was further observed under et al., Curcumin-related inhibition of neutrophil oxidative burst was comparable with the activity of methotrexate \u2013 a drug widely used in the therapy of arthritic patients, which triggers synthesis of the endogenous anti-inflammatory mediator adenosine (Cronstein, The presented data indicate that, besides its ability to suppress mechanisms engaged at the onset and progression of inflammation, curcumin could support resolution of inflammation through decreased activity and enhanced apoptosis of neutrophils."} +{"text": "Induction of premature (stress induced) cellular senescence of normal cells provides the mechanism eliminating proliferative capability of potentially carcinogenic cells and thereby is considered to be a barrier preventing neoplastic tranformation . On the Additional evidence on the role of perturbed dNTP pools in induction of DNA damage and replication stress was provided by Bester et al.,. Their sG12V, a classical model of oncogene-induced senescence. Among the enzymes down-regulated by activated HRAS were TS, ribonucleotide reductase subunits M1 and M2 (RRM1 and RRM2). Ectopic coexpression of TS, RRM1 and RRM2 or addition of deoxyribonucleosides to a large extent prevented DNA damage, decreased expression of senescence-associated phenotypes as well as attenuated the extent of proliferation arrest. Interestingly, overexpression of TS, RRM1 and RRM2 in quiescent NHFs had no effect on their proliferation arrest suggesting that unlike the OIS, quiescence is not induced by perturbation of dNTP pools. Moreover, in a separate study, Mannava et al.,[In the subsequent studies Mannava et al., provideda et al.,demonstraMost of the above findings were reproduced in a study of Aird et al., on a rolThe seminal findings on different cellular and oncogene models that perturbation of dNTP pools leads to DNA damage, replication stress, irreversible senescence -8,may ha"} +{"text": "Cardiac hypertrophy is a distinguished feature of several physiological and pathological remodeling (Frey et al., Distinct underlying signaling pathways for physiological and pathological hypertrophy have been identified (Maillet et al., Unlike pathological hypertrophy, only a little studies described how miRNAs response to physiological hypertrophy (Soci et al., Traditionally, the adult mammalian heart is recognized as a post-mitotic organ with no regenerative capacity for cardiomyogenesis (Rosenzweig, With the strategies for checking myocyte hypertrophy and new cardiomyocyte formation, the miRNA basis of physiological hypertrophy will be revealed, which will help develop a miRNA-based therapy for heart failure."} +{"text": "MAOA, COMT, and 5HTTLPR each have predictable effects on the availability of the monoamine neurotransmitters dopamine, noradrenaline, and serotonin. We hypothesized that 5HTTLPR genotype would show little association with prefrontal cognitive performance, but that COMT and MAOA would have interacting effects on cognition through their shared influence on prefrontal catecholamine availability. We assessed the individual and epistatic effects of functional polymorphisms in COMT, MAOA, and 5HTTLPR on children's prefrontal cognitive function in nearly 6,000 children from the population-based Avon Longitudinal Study of Parents and Children (ALSPAC). Neither MAOA nor 5HTTLPR polymorphisms showed significant effects on cognitive function. In boys but not girls, there was a modest but statistically significant interaction between MAOA and COMT genotypes such that increased prefrontal catecholamine availability was associated with better working memory. These results suggest that assessment of multiple genes within functionally related systems may improve our understanding of the genetic basis of cognition. \u00a9 2010 Wiley-Liss, Inc.Individual differences in cognitive function are highly heritable and most likely driven by multiple genes of small effect. Well-characterized common functional polymorphisms in the genes Experimental manipulation of human and primate neurotransmission has provided insight into the distinct roles that the monoamine neurotransmitters serotonin (5-HT), dopamine (DA), and noradrenaline (NA) play in prefrontal cognition [Clarke et al., O-methyltransferase (COMT), one such enzyme [Karoum et al., COMT knockout mice show normal prefrontal NA levels and administration of the COMT-inhibitor tolcapone increases the release of DA, but not NA, in rat PFC [Gogos et al., Two major routes for the deactivation of monoamine neurotransmitters are reuptake by transporter molecules in the presynaptic cell membrane, and enzymatic degradation within the neuron or synapse. Both serotonin and NA transporters are abundant in PFC [Mantere et al., COMT, MAOA, and the serotonin transporter-linked polymorphic region (5HTTLPR) affect the level and/or activity of their products in defined manners, summarized in COMT Val allele was associated with worse performance on a measure of executive function, the Wisconsin Card Sort Test (WCST). While meta-analysis of similar studies shows little consistent cognitive effect [Barnett et al., MAOA and 5HTTLPR polymorphisms on cognition. MAOA genotype has been reported to significantly affect IQ among healthy Chinese women [Yu et al., 5HTTLPR polymorphism have been predominantly negative for standard neuropsychological measures such as IQ, response inhibition, and sustained attention [Fallgatter et al., Individual differences in cognitive function are highly heritable [Devlin et al., 158Met on cognitive performance at ages 8 and 10 in a population-based sample of more than 5,000 children from the Avon Longitudinal Study of Parents and Children (ALSPAC) [Barnett et al., MAOA and 5HTTLPR. We also assess evidence for epistasis between the MAOA, 5HTTLPR, and COMT polymorphisms, in an attempt to broaden the candidate gene approach to include assessment of functionally interactive gene systems, here represented by the overlapping effects of these SNPs on synaptic neurotransmitter availability. We hypothesized that genes that exclusively affect serotonergic function (5HTTLPR) would show little effect on these prefrontally driven cognitive tasks, but that main genetic effects, and potential epistasis, would be demonstrated for variants affecting dopaminergic or noradrenergic availability , with 5 \u00b5M of each primer and 25 mM dNTPs in a total reaction volume of 25 \u00b5l. Amplifications were performed on a Perkin Elmer 9700 thermocycler with one cycle at 96\u00b0C for 10 min followed by 30 cycles of 94\u00b0C for 15 sec, 60\u00b0C for 15 sec, 72\u00b0C for 30 sec, and a final 3-min extension at 72\u00b0C. The forward primer was labeled with the fluorescent dye 6-FAM, and amplicons were visualized on an ABI 3100 capillary sequencer. Allele sizes were determined using Genotyper 2.5 (Applied Biosystems). MAOA 3, 3.5, and 4-repeat allele frequencies in girls were consistent with Hardy\u2013Weinberg equilibrium .DNA, obtained from blood and mouthwash samples, was extracted and processed [Jones et al., 5HTTLPR, low activity individuals were those with SS, SLG, or LGLG genotypes, medium activity were SLA or LGLA genotypes, and LALA genotypes were denoted high activity [Hu et al., COMT, Met/Met individuals were considered low, Val/Met individuals medium, and Val/Val individuals high activity, because Val allele carriers show higher COMT enzyme activity, and hence faster degradation of catecholamines at body temperature [Lachman et al., MAOA we excluded genotypes involving the rare 2- or 5-repeat alleles, whose functional effects are not yet clear [Sabol et al., MAOA is located on the X chromosome, boys carry only one allele. Boys with the 3-repeat allele were denoted low MAOA activity and those with 3.5- or 4-repeat alleles as high activity, since they show higher transcriptional efficiency and hence result in higher levels of synaptic MAOA [Sabol et al., Individuals were classified into high, medium, or low activity groups with respect to the functional effects of the polymorphism in each of the three genes. In each case, \u201chigh activity\u201d groups comprised the alleles leading to fastest clearance of the synaptic neurotransmitter and \u201clow activity\u201d the alleles leading to slowest clearance. For 5HTTLPR, and for MAOA in girls only. For boys, we compared cognitive scores in high versus low activity groups using t-tests. Main effects of COMT in this sample have been previously reported [Barnett et al., Main effects of genes on cognition were assessed by comparing cognitive scores between high, medium, and low activity groups using one-way ANOVA for the 5HTTLPR and MAOA genotypes, and a catecholaminergic one between COMT and MAOA genotypes. We assessed evidence for these interactions using general linear models with each cognitive measure as an outcome variables predicted by two genetic main effects and a gene\u2013gene interaction term. As before, all genotypes were coded as low, medium, or high activity. These analyses were conducted separately in boys and girls because MAOA is located on the X chromosome, and because COMT is known to have sexually dimorphic effects on a range of functional and neuropsychiatric phenotypes [Harrison and Tunbridge, Two possible sources of epistatic effects on cognition were considered: a serotonergic interaction between 5HTTLPR genotype. There were no cognitive differences between children for whom 5HTTLPR, MAOA, or COMT genotypes were or were not available . Allele frequencies were, for 5HTTLPR alleles S = 41.4%, LA = 51.5%, LG = 7.1%, and for MAOA, 2-repeat = 0.2%, 3-repeat = 34.0%, 3.5-repeat = 2.0%, 4-repeat = 62.4%, 5-repeat = 1.5%. COMT allele frequencies, as previously reported, were Val = 48.6%, Met = 51.4%. The number of children considered as low, medium, and high activity genotypes for whom at least one cognitive measure was available are shown in The number of children included in each analysis varied by the availability of cognitive data and genotypes: cognitive data were available on around two-thirds of those for whom DNA is available. As previously reported [Araya et al., MAOA on cognitive measures were assessed separately in boys and girls. t-Tests between high and low (minimum n = 854) activity MAOA groups in boys showed no effects on any cognitive measure. Similarly in girls, one-way ANOVA between low, medium, and high activity MAOA groups revealed no cognitive differences such that better performance was seen in the high than medium activity group (Tukey's post hoc P = 0.03). One-way ANOVA revealed no cognitive differences between the three genotypes within the low activity group.Groups defined by low, medium, and high serotonin transporter promoter activity were compared on all cognitive measures using one-way ANOVA. There were no differences between groups on measures of IQ or verbal inhibition see . NominalCOMT genotype and MAOA genotype, and a COMT \u00d7 MAOA interaction term, were fitted separately for girls and boys. In boys, there was evidence of interaction between COMT and MAOA only for working memory score . Nonetheless, this sample remains the largest so far examined with respect to MAOA and cognition, and the complete lack of difference in cognitive score between genotypes in either sex is again a decisive negative result.Although the lack of cognitive effects of MAOA, there was a modest but significant interactive effect of MAOA genotype and COMT genotype on working memory performance in boys but not girls. With few exceptions, cognitive genetic studies have thus far had inadequately sized samples to realistically assess possible gene\u2013gene (epistatic) interactions. The COMT \u00d7 MAOA interaction observed here for working memory is plausible given the dependence of working memory on prefrontal dopaminergic availability. That no similar effect was found for IQ is surprising: COMT genotype has a strong, male-specific effect on verbal IQ in this sample [Barnett et al., MAOA on IQ but not measures of executive function [Yu et al., In contrast to the absence of main effects of Separate analyses were carried out for boys and girls and for multiple cognitive phenotypes. Since scores on the four cognitive measures were correlated, strict correction for multiple comparisons using, for example, a Bonferroni correction, was not suitable. Nonetheless, while the large sample used in the study reduces the chances of Type II error, the possibility of Type I error remains present.MAOA has a widespread distribution in the brain [Grimsby et al., MAOA expression may be predominantly determined by genetic and epigenetic factors other than this VNTR, including a currently unknown locus with which it shows strong linkage disequilibrium [Balciuniene et al., COMT expression appears greater in PFC than other regions [Matsumoto et al., COMT is complex, with tissue-specific monoallelic expression [Gimelbrant et al., COMT SNPs that act in concert with the Val158Met polymorphism in influencing cognitive function in this sample [Barnett et al., COMT haplotype groups produced no meaningful change in results from those seen using just the Val158Met genotype .While both tasks that have a high working memory content [Owen, MAOA genotype affects working memory only in the context of high prefrontal catecholamine availability. There is, of course, a distinction between demonstrating a statistical interaction, and understanding the biological mechanism of interaction between two genes [Clayton and McKeigue, COMT and/or MAOA expression affect cognitive function. One such possibility is NA, which, like DA, is catabolized to some extent by both COMT and MAOA. At present, dopaminergic pathways remain the more obvious candidate, because COMT appears to play a relatively minor role in regulating NA in the PFC [Gogos et al., The gene\u2013gene interaction found here suggests that COMT \u00d7 MAOA effect on working memory was in accordance with our previous study [Barnett et al., COMT genotype affected a wide range of cognitive functions in boys but had no effect in girls. There is additional wide-ranging evidence to suggest that the effects of COMT may be different between the sexes [Harrison and Tunbridge, COMT knockout mice, which show sex-specific behavioral phenotypes [Gogos et al., COMT in human PFC [Chen et al., COMT are the bilateral relationships between COMT and estrogen-related compounds: estrogens mediate COMT expression [Xie et al., COMT metabolizes catechol estrogens, a process regulated by Val158Met variation [Worda et al., COMT, the location of the MAOA gene on the X chromosome complicates association studies, with the result that little is known about the relative expression and effects of MAOA in the two sexes.The sex-specific nature of COMT and MAOA, two genes involved in the functional deactivation of DA and other neurotransmitters, show interactive effects on working memory performance in normal children from a large, homogeneous, and representative sample. While neither gene has been unquestionably linked to psychiatric disorder, normal variation in brain function is likely to be partly determined by genetic variation in neurotransmitter pathways. Understanding genetic effects on normal brain function is a necessary stage in understanding the basis of abnormal function, but large samples are required to tease out relatively subtle effects on cognitive development at the population level.In conclusion, we present evidence that functional polymorphisms in"} +{"text": "Impulsivity can be defined as a predisposition toward rapid, unplanned reactions to internal or external stimuli regardless of negative consequences of these reactions for the impulsive individual or for others , lack of premeditation (difficulty in thinking and reflecting on consequences of an act), lack of perseverance (inability to remain focused on a task), and sensation seeking (tendency and openness to try and enjoy exciting or dangerous activities). The BIS-11 assesses impulsivity on the subscales attentional impulsivity (inability to focus attention or concentrate), motor impulsivity (acting without thinking), and non-planning impulsivity (lack of future orientation or forethought). Both questionnaires are highly correlated with each other (r = 0.67), but correlations between their subscales are only weak and inconsistent, supporting the notion that both measures cover different aspects of impulsivity (Meule et al., Beyond the fact that self-report and behavioral measures seem to capture different aspects of impulsivity, conceptualizations also vary between the different self-report instruments. For instance, two of the most widely used impulsivity questionnaires are the Only a few studies have done this as yet. For instance, differential relationships between subscales of the BIS-11 and eating disorder symptomatology have been found in clinical samples. Patients with binge eating disorder had higher scores on the motor impulsivity subscale compared to healthy controls, but did not differ on the other two subscales (Nasser et al., Eating Inventory (formerly Three-Factor Eating Questionnaire, Stunkard and Messick, hunger subscale and attentional impulsivity. Furthermore, both attentional and motor impulsivity were correlated with disinhibition (Lyke and Spinella, Studies investigating non-clinical samples also revealed differential associations between BIS-11 subscales and various measures of eating behavior. For example, Lyke and Spinella examinedconcern for dieting subscale of the Restraint Scale (Herman and Mack, eating concern subscale of the Eating Disorder Examination\u2014Questionnaire (Fairburn and Beglin, In a recent study by van Koningsbruggen and colleagues , only thr = 0.94, Spinella, While the BIS-11 contains 30 items, Spinella developeFood Cravings Questionnaire\u2014Trait, Cepeda-Benito et al., Mood Eating Scale, Jackson and Hawkins, Night Eating Questionnaire, Allison et al., Perceived Self-Regulatory Success in Dieting Scale, Meule et al., Restraint Scale, Herman and Mack, Yale Food Addiction Scale, Gearhardt et al., In a range of studies, positive correlations could be found between the BIS-15 and various constructs that are related to overeating (Table external eating subscale of the Dutch Eating Behavior Questionnaire (van Strien et al., Up to now, only one other research group has examined relations between eating behavior measures and BIS-15 subscales. In this study (Hou et al., In sum, it appears that attentional impulsivity is most consistently related to various measures that are associated with overeating. Positive, but less consistent, relationships can also be found with motor impulsivity, particularly in patients with binge eating behavior. Non-planning impulsivity seems to be only weakly related to overeating. Neither subscale seems to be consistently correlated with BMI, which may be due to the fact that BMI is influenced by many factors other than eating behavior. Future research needs to address the question why attentional impulsivity in particular has such a prominent role in overeating. First evidence suggests that attentional impulsivity may increase the susceptibility that highly palatable food-cues attract attention and trigger eating behavior (Hou et al.,"} +{"text": "Evidence-based medicine is supposed to be a conclusive summary of available empirical knowledge on certain medical issues and as such serving as the basis of guidelines and treatment recommendations. Meant to be self-explaining, the term actually appears to be crucial. It seems inconsistent and inconclusive since recommendations of single countries/institutions are based on different evidence measures\u2014some derive evidence from meta-analyses, whereas others exclusively follow randomised controlled studies. In a philosophical side trip, M\u00f6ller criticalIn mild major, minor or subsyndromal depression, the total score of the Hamilton Depression Rating Scale (HAMD) so far is regarded as \u2018gold standard\u2019 in evaluating efficacy of antidepressant treatment. Nevertheless, in comparing the full scales HAMD17 and the Inventory of Depressive Symptomatology IDS-C28) plus diverse unidimensional subcales in a randomised, placebo-controlled trial in a representative patient sample, Helmreich et al. [8 plus diGRIA1, GRIA2 and GRIA4, Chiesa et al. [In the pathophysiology of depression, glutamate plays an important role. Exploring the association of major depressive disorder with some single nucleotide polymorphisms within the glutamatergic AMPA receptor subunits a et al. in a relAlthough an association between affective disorders and the metabolic syndrome has been suspected, past studies did not bear clear results in this respect. Hence, Kahl et al. have exaCognitive deficits play a major role in psychiatric disorders like depression and schizophrenia, but also in neurodegenerative diseases. In an fMRI study in healthy probands, Voss et al. investigDTI also was used by Konrad et al. to invesIn all, cognitive deficits are core symptoms of psychiatric disorders and responsible for an unfavourable outcome. The investigation of the neurobiological basis of these symptoms is needed to develop new regenerative treatment strategies in psychiatric and neurodegenerative diseases."} +{"text": "A commentary onNeural correlates of moral sensitivity in obsessive compulsive disorderby Harrison, B. J., Pujol, J., Soriano-Mas, C., Hern\u00e1ndez-Ribas, R., L\u00f3pez-Sol\u00e0, M., Ortiz, H., et al. (2012). Arch. Gen. Psychiatry 69, 741\u2013749.Archives of General Psychiatry, Harrison et al. (In a recent issue of n et al. providedThese brain areas were found to be active on healthy humans experiencing moral emotions such as indignation or moral disgust (see Moll et al., The results are striking in so far as they show that patients with OCD demonstrated significantly increased activation of the ventral frontal cortex, particularly of the medial orbitofrontal cortex. Moreover, significant positive associations were documented between the patients' DY-BOCS ratings of total symptom severity and the activation of the anterior insula (Harrison et al., The evidence from this study offers the opportunity to discuss two emerging related issues.First, it adds further support on a clinical model to the view that visceral and moral disgust share, at least in part, common neural and cognitive mechanisms Jones, . In factThus, their heightened moral sensitivity might reflect an enhanced disgust for immoral outcomes.This hypothesis is supported by previous evidence coming from neuroimaging studies. For instance, an overlapped recruitment of ventromedial prefrontal and orbitofrontal cortices have already been reported during the processing of both sensorial and moral disgust (Moll et al., A similar result was announced for the insula. For example, Wicker et al. have disAll these findings suggest that, the heightened moral sensitivity in OCD can be grounded on the same neural mechanisms responsible for their altered sensitivity to outcomes which can induce sensory and emotional disgust (Shapira et al., Another remarkable aspect deserving some discussion is that OCD participants were not different from healthy control subjects in the subjective rating of the moral dilemmas, despite the difference reported in neural activation. This result, which probably argues some distinction between the patients' increased neural responses and their perceived emotional experience during a moral dilemma (Harrison et al., This suggestion is supported by the recent evidence of a negative correlation between interoception and anxiety (Pollatos et al.,"} +{"text": "To the Editor:In our manuscript entitled \u201cSingle-neuron RNA-Seq: technical feasibility and reproducibility,\u201d published online on July 6th, 2012, we have made a few serious mistakes which need to be corrected:J. Neurosci. 31, 6939\u20136943.\u201dWe referenced several points and quoted statements from a recent study by Okaty et al., but we neither cited the paper nor placed quotation marks on the statements. The full citation should be \u201cOkaty, B. W., Sugino, K., and Nelson, S. B. (2011). Cell type-specific transcriptomics in the brain. We request to add a citation after \u201cThe analysis of each single-cell transcriptome consists of several independent steps\u201d in the first paragraph of Results, as \u201cThe analysis of each single-cell transcriptome consists of several independent steps ,\u201d given that these points were previously raised by Okaty et al. We request to add a citation to the first paragraph of Introduction, as \u201cstudies of gene expression and function in the brain were restricted to a relatively small number of genes (Luo and Geschwind, Moreover, gene expression may be regulated in opposing directions in different cell types, thereby appearing static in composite data.ToMoreover, as previously discussed by Okaty et al., \u201cgene expression may be regulated in opposing directions in different cell types, thereby appearing static in composite data (Okaty et al., We apologize to the readers of Frontiers in Genetics and to the authors of the Okaty et al., manuscript, for our failure to correct these mistakes during the review process of our manuscript."} +{"text": "Frontiers in Pharmacology brings together a group of review and original articles focusing on IBS.Irritable bowel syndrome (IBS) is a very common functional disorder of the digestive tract. Despite the prevalence and the social impact of IBS, the exact etiopathogenesis is incomplete and the pharmacological treatment is unsatisfactory. In this research topic, Fabrizio De Ponti brilliantly outlined the drugs currently used in\u2014or in development for\u2014IBS, within a scenario in which the specific armamentarium of medications is unsatisfactory activation on the production of kynurenine in IBS and selective 5-HT4 receptor agonist in vitro with strong in vivo gastrointestinal activity in guinea pigs, rats, dogs, and in healthy humans. TD-8954 may have value in the treatment in C-IBS sufferers.Beattie et al. investigated the Interleukin-6 (IL-6) is elevated in the plasma of D-IBS patients (Rana et al., 1 and/or CB2 receptor activation\u2014exert pharmacological actions which are potentially beneficial in IBS (Izzo and Coutts, 1 and CB2 receptor agonists in normalizing the accelerated transit (Kimball et al., 1 and CB2 responsiveness is maintained long after an initial inflammatory period and suggest a role of cannabinoid receptors in the underlying pathophysiology of PI-IBS.Cannabinoids\u2014via CBThe differentiated Caco-2 cells intestinal cell line, derived from a human colon carcinoma has been used for drug absorption studies as well as to evaluate the epithelial barrier integrity in relation to IBS as well as to investigate the intestinal permeability of IBS drugs (Catalioto et al., In summary, this research topic should provide a useful resource for IBS researchers, both basic and clinical. The pharmacological studies, together with new strategies for drug discovery, highlight that more research is urgently required to translate novel and innovative basic concepts into prescribing options for healthcare professionals. It is hoped that this collection of articles will inspire further research into IBS."} +{"text": "The fundamental role of D-serine as a co-agonist at the N-methyl-D-aspartate receptor (NMDAR), mediating both physiological actions of glutamate in long term potentiation and nociception and also pathological effects mediated by excitotoxicty, are well-established. More recently, a direct link to a chronic neurodegenerative disease, amyotrophic lateral sclerosis/motor neuron disease (ALS) has been suggested by findings that D-serine levels are elevated in sporadic ALS and the G93A SOD1 model of ALS (Sasabe et al., It has only recently been recognized that D-amino acids play an important physiological role in the central nervous system. The evidence is most convincingly demonstrated for D-serine through its action as a co-agonist at the N-methyl-D-aspartate receptor (NMDAR), a major glutamate receptor subtype, involved in synaptic plasticity , TAR DNA binding protein 43 (TARDBP) and fused in sarcoma (FUS). Arginine199 is highly conserved across species from bacteria to man and lies within the active site close to the FAD and substrate binding sites of the enzyme. The effect of the R199W-DAO mutation is very profound abolishing enzyme activity , the major biosynthetic for D-serine, in mammalian brain has been characterized extensively was backcrossed with C57BL/6J mice and maintained as homozygotes (DAO\u2212/\u2212) in order to study the motor phenotype in more detail has been identified in a mouse line, which shows abnormal locomotor activity and in two overlapping ALS-like conditions, progressive bulbar palsy and primary lateral sclerosis , compared to normal controls and a control neurological group consisting of conditions associated with similar muscle loss compared to controls, the greatest change being an increase of 57% in glycine which suggested a potential impairment in glycine transport (de Belleroche et al., An impaired clearance of both glycine, as indicated from these results (Lane et al., \u2212/\u2212, where D-serine synthesis is reduced. On the other hand, glycine facilitates NMDAR-mediated currents after afferent stimulation, diminished by GO but not by DAO (Li et al., \u2212/\u2212 knockdown or depletion of glycine by GO reduce LTP (Li et al., In hippocampus and amygdala (Li et al., The interplay between glycine site co-agonists has not been extensively characterized in brain stem and spinal cord. However, studies in rat hypoglossal motor neurons suggest that an increase in synaptically released glycine in response to metabolic stress may play a role in exacerbating NMDA receptor-mediated excitotoxicity in motor neurons (Kono et al., Experimental evidence obtained to date highlights the selective localization of DAO in spinal cord motor neurons, brain stem motor nuclei, cerebellum and other CNS centers involved in motor control. DAO is also found in glial cells in spinal cord and cerebellum. This distribution of enzyme activity is consistent with the lower levels of D-serine found in posterior brain compared to forebrain. The primary findings that implicate D-serine in ALS and potentially in neurodegeneration in general are that elevated levels of D-serine in spinal cord are found in ALS and the SOD1 mouse model of ALS (Sasabe et al., In order to further substantiate this concept, we have explored the potential toxic properties of the FALS-associated R199W-DAO mutation in cell culture and have shown that whether expressed in astrocytes or motor neurons the mutation promotes apoptosis but most interesting when expressed in glial cells the mutant protein is able to initiate apoptosis in neighboring neurons in co-culture that lack the mutation. The way in which this effect is transmitted between cells appears to be crucially dependent on the presence of an NMDAR co-agonist as the effect is blocked by the selective antagonist DCKA. In the situation where DAO is inhibited due to the presence of a mutation, D-serine levels are elevated (Sasabe et al., It is of considerable importance to note that increased synthesis of D-serine by neuronal and glial SR is known to be activated by cellular stressors such as AMPA, amyloid-beta and lipopolysaccharide (Wu and Barger, The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Dear Sir,Upsala Journal of Medical Sciences. This article provides an interesting analysis of estimating renal function in critically ill patients, but I believe there are several aspects that may warrant further discussion:I read with great interest the article entitled \u2018Significant differences when using creatinine, modification of diet in renal disease, or cystatin C for estimating glomerular filtration rate in ICU patients\u2019 by Lipcsey et al. in the MFirst of all, the equations estimating glomerular filtration rate (GFR) are all notoriously flawed in the setting of rapidly changing renal function, that is, acute kidney injury. The creatinine-based formula for creatinine clearance is shown to over-estimate renal function when actual GFR is low, due to its multiple interfering factors and tubular secretion nature, but the cystatin C-based formula is also affected by muscle mass and adiposity . To compSecond, the duration of illnesses and timing of tests are also not taken into consideration in Lipcsey's article. Nejat et al. in their multicenter observation study have indicated that cystatin C rises earlier than creatinine in patients with acute kidney injury by 4\u20135 hours from a general ICU population .Finally, is there any better way of determining renal function in the critically ill with convenient point-of-care feasibility? Tidman et al. in their study of GFR determination in chronic kidney disease patients have provided a new thought: combining creatinine-based and cystatin C-based results for estimation . Though"} +{"text": "Bevacizumab (Avastin) has rapidly gained status as a broadly active agent for malignancies of several different histologies in adults. This activity has spawned a range of uses in pediatrics for both oncologic and non-oncologic indications. Early analyses indicate that pediatric cancers exhibit a spectrum of responses to bevacizumab that suggest its activity may be more limited than in adult oncology. Most exciting, is that for low-grade tumors that threaten vision and hearing, there is not only evidence for objective tumor response but for recovery of lost function as well. In addition to oncological indications, there is a range of uses for non-oncologic disease for which bevacizumab has clear activity. Finally, a number of mechanisms have been identified as contributing to bevacizumab resistance in cancer. Elucidating these mechanisms will guide the development of future clinical trials of bevacizumab in pediatric oncology. Angiogenesis, or the formation of new blood vessels from pre-existing vessels is essential for normal organogenesis and tumorigenesis. The wide range of requirements for new blood vessel growth during development and in the setting of pathology is served by numerous angiogenic factors that recruit endothelial progenitors and other pro-angiogenic and vascular supporting cells, and promote endothelial cell proliferation and survival. Vascular endothelial growth factor (VEGF) is essential to both normal and pathology-associated angiogenesis and consequently, VEGF antagonism is the primary focus of anti-angiogenic therapies. Multiple isoforms of VEGF are generated through alternate splicing and post-translational modifications of the products of a multi-gene family comprised of VEGF-A, -B, -C, -D and placenta growth factor Ferrara, . Most of121, VEGF165, VEGF189, VEGF206) as well as two co-receptor, Neurophilin 1 and 2. While binding to VEGFR-1 occurs with higher affinity than binding to VEGFR-2, it is VEGFR-2 that mediates the pro-angiogenic effects of VEGF on endothelial cells in patients who had progressed after prior treatment every 2\u2009weeks. Fifteen of the patients had no objective sustained response. Eight of the patients with GBM had stable disease for \u226512\u2009weeks with a median of 176\u2009days and median PFS of 4.2\u2009months. Median PFS for patients with DIPG was 2.3\u2009months. This study concluded that there is a lack of efficacy of combined bevacizumab and irinotecan in children with recurrent GBM and DIPG.Outcomes for children with high-grade gliomas (HGG) remain dismal with 5-year-survival rates of 5\u201315% for grade IV gliomas and 20\u201340% for grade III gliomas every 2\u2009weeks. Seven had a radiographic response and seven had clinical improvement . At the conclusion of the study, eight of the nine patients had not progressed . The 14th patient was progression-free at 45\u2009months. Significant responses to bevacizumab in children with LGG is consistent with reported activity for other anti-angiogenic therapies such as thalidomide-based regimens are the most common childhood brain tumor. Treatment varies as a function of age and tumor location between surgical resection alone, or combined surgery and chemo or radiation therapy. Common indications for adjuvant therapy include recurrence in surgically inaccessible locations and threats to function particularly for deep-seated midline tumors growth of which can result in vision loss, or hypothalamic or brainstem dysfunction. While LGG are slow growing, biologically benign tumors, they possess increased vasculature . Although benign, these tumors tend to inexorably progress and often result in loss of all functional hearing by middle age. Standard treatment includes surgery and radiation however, these approaches are frequently ineffective at preserving hearing . Importantly, four of the seven evaluable patients had 57% improvement in word recognition beginning approximately 8\u2009weeks after initiation of therapy and four of those five also reported improvement in tinnitus. Mautner et al. treated with bevacizumab (5\u2009mg/kg every 2\u2009weeks for 12\u2009months) had a significant reduction in both MRI fluid-attenuated inversion-recovery (FLAIR) and T1-post-Gd contrast abnormalities. The average daily dexamethasone dose was reduced by 8.6\u2009mg. A retrospective review at Children\u2019s Hospital of Denver evaluated the response to bevacizumab of patients with DIPG (n\u2009=\u20094) , a severe complication of allogeneic stem cell transplantation. Yabe et al. reportedVascular endothelial growth factor is a potent inducer of vascular permeability. Blocking it in patients with severe edema such as radiation necrosis and CLS may benefit some patients. Further studies are needed to further evaluate these findings.Bevacizumab has been used in several case reports of patients with angiomatosis, a neoplasm-like entity. Smith et al. reportedBevacizumab has also been reported to benefit a patient with multifocal lymphangiomatosis or Gorham\u2013Stout disease (GSD), presenting as idiopathic osteolysis of the bones. GSD is possibly triggered by occult neoplastic cells that produce large amounts of IL-6 and VEGF. Grunewald et al. reportedp\u2009=\u20090.002). Treatment of ROP with bevacizumab is now the standard of care at many centers. Albini and Murry reported a retrospective, non-comparative, open-label, interventional case series in 35 eyes of 33 patients with diseases such as CVN, Coat\u2019s disease, familial exudative vitreoretinopathy and sickle cell retinopathy who were treated with bevacizumab . One patient with sickle cell retinopathy and central retinal vein occlusion had significant improvement in central macular thickness over 6\u2009months after one injection (811\u201356\u2009\u03bcm). Similar effectiveness was also reported for treatment of CNV after Stevens\u2013Johnson syndrome , choroidal neovascularization (CNV), Coat\u2019s disease, and familial exudative vitreoretinopathy can lead to severe vision loss. Vision loss can occur for many reasons, including retinal ischemia and VEGF-induced neovascularization and vascular leakage of fluid, blood, and exudates. Mintz-Hittner et al. conducteMaturo and Hartnick reportedn\u2009=\u20091) including anaphylaxis, cerebral ischemia, posterior reversible encephalopathic syndrome, proteinuria, lymphopenia, and pneumonia.In adult studies, serious adverse events have been reported with the use of bevacizumab including CNS hemorrhage (1\u20135%), hypertensive crisis (<1%), and blood clots (6\u20139%) (Scappaticci et al., In the cases reported by Hwang et al. who wereIn patients with NF2 treated with bevacizumab at 5\u2009mg/kg/dose, no thromboembolic events, hemorrhage, congestive heart failure, gastrointestinal perforation, reversible posterior leukoencephalopathy, or Grade III/IV events have been reported (Plotkin et al., Secondary amenorrhea was reported in a few patients. In the Phase I COG study, two of three post-menarchal females had a rise is LH and FSH, one subsequently missed her periods (Glade Bender et al., The patient treated for CAT syndrome was noted to have multiple punched out boney lesions after four doses of bevacizumab (Smith et al., Together, these studies indicate that bevacizumab is generally well tolerated in the pediatric population at doses up to 15\u2009mg/kg/dose. Serious side effects seen in adults have not been reported in the pediatric population.Targeting VEGF pathways clearly has a role in the treatment of a broad range of human disease. Some applications, like the treatment of post-irradiation edema or CLS, relate more to VEGF-mediated regulation of vascular permeability rather than to VEGF-stimulated angiogenesis. In these circumstances bevacizumab has demonstrated significant effect and it is likely that its use as a single agent for these indications will increase. In contrast, as detailed above, in cancer treatment when the angiogenic functions are the critical target for VEGF directed therapies, bevacizumab fails in most cases to produce a lasting clinical response. Identifying the mechanisms of resistance and appropriately modifying treatment approaches will be essential for the full realization of the potential of anti-angiogenic therapies. Here we will briefly review proposed mechanisms by which cancers adapt and evade VEGF antagonism.In discussion of and investigation into mechanisms of bevacizumab resistance, much has been made of an apparent change in the pattern of recurrence in bevacizumab-treated, versus conventionally treated, GBM. Some retrospective and prospective studies have reported an increase in the frequency of diffuse and distant recurrence after bevacizumab therapy from approximately 10\u201350% (Norden et al., The mechanisms that drive resistance to bevacizumab therapy are not yet fully defined. However, preclinical and clinical studies have revealed multiple intratumoral and systemic pathways that can contribute to tumor progression on bevacizumab therapy and these have been well reviewed by Bergers and Hanahan . In respIn addition to developing therapeutics that can target the multiple mechanisms of angiogenesis and tumor recurrence, a great clinical challenge as we advance anti-angiogenic therapies for cancer will be the application of laboratory and imaging techniques that can better define response early in therapy. This will be especially critical in the application of anti-angiogenic therapies to brain tumors where serial biopsy is not currently available. In this regard, serum evaluation for the up-regulation of pro-angiogenic factors, especially those associated with resistance like VEGF, PIGF, PDGF, FGF, IL8, CXCL12 may provide insight into shifts in response (Murukesh et al., In addition, imaging, particularly MRI-based spectroscopy, perfusion, diffusion, and oxygen consumption sequences may be able to detect functional changes in the vasculature relevant to anti-angiogenic responses and recurrent angiogenesis (Pope et al., Finally, PET imaging for therapy-induced changes in expression of specific components of angiogenic pathways as well as for changes in vascular function is an additional promising approach for detecting response to anti-angiogenic therapies that is just entering clinical evaluation (Chen and Chen, Together, sophisticated and combinatorial monitoring of response to anti-angiogenic therapies may support the timing of adjunctive interventions to circumvent resistance mechanisms and enhance efficacy.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "The MUS-81, SLX-1, and XPF-1 structure-selective endonucleases have been implicated in meiotic crossover (CO) formation in a variety of organisms, but their contributions to C. elegans CO formation have been unclear. In this issue of PLOS Genetics, Agostinho et al., Saito et al., and O'Neil et al. demonstrate that MUS-81 and XPF-1 function in two parallel pathways during the formation of meiotic crossovers in C. elegans and provide important insights into the interplay between endonucleases and the Bloom helicase ortholog during crossover formation.Saccharomyces cerevisiae have provided extensive genetic and biochemical support for this model. Although initiation by DSBs appears to be universal and a progression of joint molecule (JM) intermediates that link DNA duplexes on homologous chromosomes and S. cerevisiae Sgs1. Sgs1 disassembles early meiotic JMs, both to generate noncrossovers and to prevent formation of aberrant multi-chromatid JMs et al. report that him-6 mutants have defects in processing early intermediates, leading to chromosome fragmentation et al. propose that HIM-6 also has a late function in generating COs in conjunction with XPF-1 Drosophila BLM Additional potential similarities arise from studies of C. elegans meiosis might work together to resolve HJs. However, it is possible that the proteins act on a different structure, such as nicked HJs (e.g., see reference Another important question concerns the exact nature of the pre-crossover JM. The authors suggest that these resolvases are acting on HJs, and resolution of chromatin bridges by GEN1 supports this suggestion. Agostinho"} +{"text": "Kenneth Hugdahl's second affiliation is Department of Radiology, Haukeland University Hospital, Bergen, Norway.lower degrees of language lateralization as determined with fMRI (van Ettinger-Veenstra et al., higher degrees of lateralization.\u201dOn page 1 the final sentence in the second column should read: \u201cMoreover, individuals with n = 16 to find correlations between ear asymmetry and behavioral language tests in the non-forced condition of the Bergen DL task.\u201dOn page 7 the final sentence of the first column should read: \u201cFor the same reason van Ettinger-Veenstra et al. might habut see Catani et al., On page 8 the final paragraph of the discussion should read: \u201cAs far as language is concerned, however, stronger lateralization seems to be associated with better performance in verbal abilities (Boles et al.,"} +{"text": "Was\u2212/\u2212 mouse. Both cellular and humoral immune defects in WAS patients contribute to the onset of severe clinical manifestations, in particular microthrombocytopenia, eczema, recurrent infections, and a high susceptibility to develop autoimmunity and malignancies. Autoimmune diseases affect from 22 to 72% of WAS patients and the most common manifestation is autoimmune hemolytic anemia, followed by vasculitis, arthritis, neutropenia, inflammatory bowel disease, and IgA nephropathy. Many groups have widely explored immune cell functionality in WAS partially explaining how cellular defects may lead to pathology. However, the mechanisms underlying the occurrence of autoimmune manifestations have not been clearly described yet. In the present review, we report the most recent progresses in the study of immune cell function in WAS that have started to unveil the mechanisms contributing to autoimmune complications in WAS patients.Wiskott\u2013Aldrich Syndrome (WAS) is a severe X-linked Primary Immunodeficiency that affects 1\u201310 out of 1 million male individuals. WAS is caused by mutations in the WAS Protein (WASP) expressing gene that leads to the absent or reduced expression of the protein. WASP is a cytoplasmic protein that regulates the formation of actin filaments in hematopoietic cells. WASP deficiency causes many immune cell defects both in humans and in the WAS murine model, the WAS gene is located on the X chromosome and encodes a 502 amino acid protein is also an important regulator of WASP activation by inducing a stable acting form is a rare X-linked Primary Immunodeficiency (PID) that affects 1\u201310 out of a million male individuals is observed in some patients with substantial protein expression cells in T cells and T Cell Receptor (TCR)-dependent activation and fungal infections as compared to WASP-positive patients , followed by vasculitis , arthritis (29%), neutropenia (25%), inflammatory bowel disease (9%), and IgA nephropathy (3%). Henoch\u2013Sch\u00f6nlein-like purpura, dermatomyositis, recurrent angioedema, and uveitis have also been reported in some patients , most available therapies are not curative. Intravenous immunoglobulins (IVIG) and antibiotic prophylaxis are often used to reduce the risk of infections in WAS patients, but it is not clear whether these treatments effectively reduce the incidence of life-threatening infections is available, BMT leads to 73\u2013100% survival . This adverse event gives rise to some concerns on the safety of RV-mediated GT for WAS. A multicenter clinical trial using a third generation LV carrying WAS gene driven by the endogenous promoter is on going in Milan, Paris, and London. Preclinical data in the murine model indicate that the LV-mediated GT approach is effective in restoring immune cell functionality . Two different Gene Therapy (GT) clinical trials have been approved: a Retroviral Vector (RV)-mediated gene transfer family member Fas ligand (FasL) that is released and binds its receptor Fas in an autocrine fashion , Multiple Sclerosis (MS), and Systemic Lupus Erythematosus . In the last decade, it has been clearly defined that the lack of WASP causes defects in the cytoskeletal functions of B cells, including adhesion, migration, and homing and a reduced or absent antibody production to polysaccharides and other T cell-independent antigens co-engagement in Was\u2212/\u2212 B cells from chimeras could mediate tolerance breakdown, since the loss of Myeloid Differentiation primary response gene 88 (MyD88) signaling abolished the production of anti-dsDNA antibodies, germinal center formation, and development of systemic autoimmune disease , Recher et al. which in turn activate pDCs to produce high levels of type I IFN , in the pathogenesis of systemic autoimmune diseases. In particular, IFN-\u03b1 produced by pDCs upon recognition of foreign nucleic acids via TLR7 and TLR9 contributes to tolerance breakdown in several autoimmune diseases, such as SLE, SS, and psoriasis (Ronnblom, Was\u2212/\u2212 mice abolish the production of anti-dsDNA antibodies (Becker-Herman et al., \u2212/low B cells (Ng et al., \u2212 B cells in WAS patients (Park et al., Was\u2212/\u2212 mice (Westerberg et al., Triggering of autoreactive B cells by self-nucleic acid-containing ICs can be another possible mechanism underlying the production of autoantibodies in WAS. In fact, it is known that self-nucleic acid-containing ICs can activate B cells through synergistic engagement of BCR and TLR7 or TLR9 (Leadbetter et al., In conclusion, together with the defects already described in the literature, these future lines of enquiry underline the greater than expected extent to which the WASP deficiency affects the immune system. Further research is necessary to define the underlying molecular and cellular mechanisms leading to autoimmunity, which represents the main collateral damage caused by WASP deficiency.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "While Frye and Jackson do a good job describing the common mechanisms of resistance found in food animals in the U.S., they err in stating that in the U.S. extended spectrum \u03b2-lactamases (ESBLs) \u201cthus far have only been found in human and not food animal isolates.\u201dEscherichia coli expressing CTX-M-type ESBLs in fecal isolates from healthy cattle at a livestock market and from a diagnostic isolate submitted to the Ohio Animal Disease Diagnostic Laboratory. Since then, CTX-M-type ESBLs have been found in E. coli collected from healthy dairy calves in the western U.S. (Davis et al., Salmonella enterica isolates from swine in Minnesota and from turkeys in 4 states (Wittum et al., E. coli from swine finishing barns in Michigan and Ohio and in Klebsiella pneumoniae from swine in Illinois (Mollenkopf et al., In fact, Wittum et al., in 2010 first reported the collection of E. coli isolate expressing CTX-M-type ESBLs from Pennsylvania has been reported (Doi et al., E. coli expressing OXA-type ESBLs (Davis et al., We are not aware of studies finding CTX-M-type ESBLs in isolates from broiler chickens, but a retail chicken meat"} +{"text": "MTHFR gene includes a common polymorphism (rs1801133 or C677T), which is associated with enzyme activity. The T-allele of the C677T polymorphism has been associated with earlier age at onset of schizophrenia in a Scandinavian population, although no association was found in replication attempts in other populations. Extending the study to five Nordic samples consisting of 2,198 patients with schizophrenia, including the original Scandinavian samples, there was no significant association between MTHFR C677T polymorphism and age at onset in schizophrenia. The present results do not suggest that the investigated MTHFR polymorphism has any significant influence on age at onset of schizophrenia in the Nordic population. \u00a9 2012 Wiley Periodicals, Inc.Methylenetetrahydrofolate reductase (MTHFR) is an enzyme involved in metabolic pathways of importance for nucleotide synthesis and methylation of DNA, membranes, proteins and lipids. The Methylenetetrahydrofolate reductase (MTHFR) is an enzyme involved in metabolic pathways of importance for nucleotide synthesis and methylation of DNA, membranes, proteins and lipids [Frankenburg, MTHFR gene includes two common polymorphisms which both alter enzyme activity and homocysteine concentrations [Frosst et al., MTHFR C677T among 40 polymorphisms associated with schizophrenia susceptibility in the Han Chinese population [Yue et al., The MTHFR C677T functional polymorphism, and the metabolism of folate, methionine, and homocysteine have been extensively studied in relation to etiologically complex chronic diseases, and a relationship between MTHFR C677T and age at onset have been observed in for example, coronary artery disease, breast cancer, and Parkinson disease [Mager et al., MTHFR 677 T-allele predispose to an earlier age of onset in unrelated patients with schizophrenia of Scandinavian origin and found similar results in a family-based sample from western China [Vares et al., MTHFR polymorphisms in unrelated patients with schizophrenia to a combined analysis on age at onset. However, we were unable to replicate the findings outside the initial Scandinavian sample [Vares et al., The MTHFR C677T polymorphism and age at onset in two additional samples of Icelandic and Danish patients with schizophrenia, and combine the results with the previous Scandinavian results in a meta analysis.To determine whether the initial finding in unrelated individuals was restricted to populations of Scandinavian origin, or more likely due to chance alone, we here analyse the relationship between The Icelandic sample consisted of patients with schizophrenia, who were recruited all over Iceland as previously described [Stefansson et al., The sample from Denmark/Aarhus has also been previously described [Demontis et al., The samples from the initial report [Vares et al., P < 1 \u00d7 10\u22125, or frequency difference between chip types or typing centers with P < 1 \u00d7 10\u22126 were also excluded. Only samples with yield >90% were included, and the lower yield of each pair of duplicates was removed. Markers had control HW equilibrium P values > 0.001, and, in each group, yield in cases and controls was >95%.Genotyping of the Icelandic group using Illumina Human Hap300Beadchip genome-wide arrays was carried out as previously described [Stefansson et al., In Denmark/Aarhus, DNA was extracted from dried blood spots provided by the Danish Newborn Screening Biobank using the Extract-N-Amp Blood PCR kit . DNA was subsequently whole-genome-amplified using the RepliG mini kit and genotyped on the Illumina Infinium HD Human610-Quad BeadChip as previously described [Hollegaard et al., MTHFR C677T SNP was genotyped at the SNP Technology Platform in Uppsala, Sweden (http://www.genotyping.se), using the Illumina BeadStation 500GX and the 1536-plex Illumina Golden Gate assay (Illumina Inc.) as previously described [Vares et al., In the initial Scandinavian sample [Vares et al., For each study, we estimated the allele association between the C677T polymorphism and age at onset of schizophrenia with a general linear model. In this primary analysis, age at onset was treated as a quantitative trait, and modeled as a function of gender and the number of T alleles . The analyses were conducted in parallel, with R for the Icelandic and Danish/Aarhus sample and with SAS/STAT\u00ae software (version 9.3) for the previously investigated Scandinavian samples [Vares et al., Meta analysis of the five studies was done with a simple random-effects model with Proc Mixed in the SAS/STAT\u00ae software . In the analysis, the allele association was modeled as a function of the fixed effect of the intercept and the random effect of study. The beta coefficients (regression slopes) for C677T from the primary analyses were used as the observed effect size, and the corresponding squared standard error (SE) was treated as known variance in the diagonal of the variance\u2013covariance matrix R. HW equilibrium was tested using Fisher's exact test as implemented in PEDSTATS [Wigginton and Abecasis, MTHFR 677T allele frequency varied from 27% (Sweden) to 36% (Iceland) in the samples . A negative association between 677T and earlier age at onset was also apparent in the Norwegian sample, although in this sample the association strength was weaker . We noted that this sample consisted of patients with an earlier age of onset than the other Nordic samples, and most patients became ill before the effect of the MTHFR C677T polymorphism was apparent in the original Scandinavian samples [Vares et al., MTHFR C677T polymorphism and age at onset of schizophrenia in the Icelandic sample (P = 0.86). Thus the initial finding of an age dependent association between the MTHFR C677T polymorphism and age at onset of schizophrenia in the Scandinavian population could not be replicated in two large and independent samples of similar origin. A meta-analysis of all five samples resulted in a pooled association estimate of 0.42 \u00b1 0.26 years earlier age at onset of schizophrenia per T-allele (mean \u00b1 SE), which did not reach statistical significance (P = 0.17).In the independent Danish sample (Aarhus), the age at onset decreased with on average 0.25 years per MTHFR 677T-allele and age at onset of schizophrenia in two independent samples of northern European heritage (Denmark and Iceland), or in meta-analysis including the initial Scandinavian samples. These results are in line with earlier analyses outside Scandinavia [Peerbooms et al., In the present study, we were unable to replicate our original finding of a negative association between the MTHFR 677T-allele and earlier age at onset in our original Scandinavian sample was primarily due to the Danish (Copenhagen) sample, and a similar tendency was present in the Danish (Aarhus) replication sample. Age at onset varied among the cohorts included in the study, and part of this variation may have been related to the different definitions of age at onset used . The heterogeneity in the definitions of age at onset is likely to have increased phenotype variation (and thus decreased the power to detect a true but weak association), but we observed no relationship between the definition of age at onset and association strength, neither in this nor in our previous analysis of multiple samples [Saetre et al., MTHFR polymorphism with respect to varying environmental conditions, such as availability of folic acid in food and/or practices of folic acid supplementation during pregnancy. Moreover, the power to detect an effect of the MTHFR 677T allele on late onset schizophrenia would be limited in the Danish (Aarhus) sample simply because of its younger age structure. Thus we cannot exclude the possibility of a weak negative association in populations of North European origin.The association between MTHFR 677T-allele has no significant effect on the age at onset of schizophrenia in the general population, irrespective of the geographical location, ethnicity or genetic background. Nevertheless, given the important role of MTHFR in the metabolism on folic acid, methionine, and homocysteine, it is possible that MTHFR gene variation not only affects schizophrenia susceptibility but may also modify symptom severity [Herran et al., However, given the data at hand, and previous analysis of samples of different geographical and ethnical origins [Peerbooms et al.,"} +{"text": "Phosphodiesterase-5 (PDE5) inhibitors have shown a beneficial effect in a variety of clinical conditions, such as benign prostate hyperplasia, pulmonary arterial hypertension, female sexual arousal disorder, overactive bladder, and incontinence, Raynaud's disease, heart failure and stroke among others pools (see Figure in vivo models of prostate cancer (Das et al., The accumulating body of evidence suggests that PDE5 inhibitors could interfere with the efflux functions of the ABC transporters, thus sensitizing cancer cells toward cytotoxic agents that are substrates of ABC transporters (Ding et al., In summary, there is evidence that suggests that PDE5 inhibitors may have an anticancer action either by increasing cGMP-PKG and coupled downstream events or by their ability to inhibit ABC transporter\u2014mediated s efflux of anticancer drugs. The safety, high tolerability, and wide availability of PDE5 inhibitors have made this class of drug an attractive tool to investigate their role in cancer chemotherapy. Since a detailed understanding of PDE5 inhibitors as anticancer adjuvants is limited, further studies are warranted to characterize their mechanisms and establish their role."} +{"text": "DNA methylation is one of the essential epigenetic mechanisms that are closely correlated with the mechanisms underlying cell growth, differentiation and transformation in eukaryotes. Global changes in the epigenetic landscape are considered to be a hallmark of cancer. The initiation and progression of cancer are mediated through epigenetic modifications along with genetic alterations. Aberrant methylation of promoter regions is an epigenetic abnormality of the human genome that is highly characteristic of cancer. In this review, we aimed to summarize our current understanding of the alterations in the epigenetic landscape and investigate the potential use of DNA and RNA methylation in effective molecular treatment strategies. Complex diseases, such as malignant tumors and diabetes, are a common occurrence and represent a major public health concern. Despite the significant advances in cancer treatment, the overall cancer-related mortality is ~90%, due to late-stage diagnosis and failure to optimize treatment. Therefore, effective biomarkers for cancer diagnosis are urgently needed.Cancers such as lung, colon and breast cancer are frequently diagnosed at a late stage. Despite intensified efforts focused on improving the survival of cancer patients, only a moderate improvement is generally achieved. Failure of early diagnosis often leads to low treatment efficiency and poor prognosis; thus, the identification of a signal characterizing the early stages of formation and progression of cancer may reduce the incidence of this disease . Early fThe mechanisms underlying cancer development are complicated and cancer was originally perceived as a genetic disease. However, it was previously demonstrated that the initiation and progression of cancer involve epigenetic abnormalities, such as DNA methylation, histone modifications, nucleosome positioning and aberrant expression of non-coding RNAs, specifically microRNAs \u20137. The tcis-acting elements with trans-acting factors and gene expression. Various epigenetic experiments, involving genomic imprinting . Chromosome remodeling or reactivation of transposable elements was generated via demethylation of exons and introns or repetitive DNA sequences. An increased number of DNA hypomethylation loci in CpGs was shown to increase chromosomal instability and oncogene activation. For example, the hypomethylation of LINE-1 and Alu retrotransposons is frequently associated with lung cancer . The distribution of m5C within DNA is unique and may be used for genome-scale methylation analysis . For exaAs a specific pattern of gene expression in mammals, DNA methylation is essential for tissue development. Abnormal DNA methylation commonly disrupts molecular signaling mechanisms and leads to the development of various diseases, such as cancer. DNA methylation was the first epigenetic alteration to be identified in cancer . Therefoet al were found to harbour methylated sites (ESR1 and 14-3-3-\u03c3 promoters were significantly different compared to healthy controls (et al (et al (The DNA methylation status is correlated with cancer and the methylation level is inversely correlated with mRNA expression levels. Jin et al reporteded sites . In breacontrols . Glocknes (et al reportedl (et al comparedCDH1, APC, MGMT, RASSF1A, GSTP1, p16 and RAR-\u03b22, affect the activity of tumor suppressor genes, typically leading to transcriptional silencing. A number of important genes that undergo silencing interfere with important cancer-related cell pathways. Thus, the aberrant methylation of the promoters of cancer-related genes may be deemed as a potential biomarker contributing to early cancer detection and prediction of prognosis.The identification of patients with organ-confined carcinoma is key to early-stage diagnosis of cancer. Common organ-confined cancers, such as lung, hepatic, breast, cervical, colorectal and genitourinary tract cancers, result in patient death due to the lack of effective clinical diagnosis. In these cancers, the methylation of the promoter regions of tumor suppressor genes, such as SEPT9, is commonly detected in the plasma of patients with primary CRC and was submitted to the FDA for marketing application in 2010 as a molecular marker for early clinical stage CRC. SEPT9 as a DNA methylation biomarker was also associated with breast cancer; Gonzalez et al (SEPT9 expression may contribute to the pathogenesis of certain types of breast cancer. Another potential biomarker for breast cancer is the methylation of PITX2. The evaluation of the PITX2 methylation status among different breast cancer patient populations successfully increased the outcome prediction performance. Harbeck et al (PITX2 methylation in 399 breast cancer specimens and identified low-risk patients with hormone receptor-positive and node-negative disease. Hartmann et al (PITX2 in 241 breast cancer specimens and concluded that methylation of PITX2 was correlated with clinical outcome. Hrasovec et al (TMEM25 were correlated with CRC, with TMEM25 hypermethylation possibly playing a significant role in altering the expression of this gene in CRC.A series of novel DNA methylation biomarkers in the plasma and stool were developed for various detection purposes. Vimentin is transcriptionally silent in normal epithelia and aberrant vimentin expression has been used as a cancer marker in fecal DNA testing . In 2005ez et al indicateck et al investignn et al also anaec et al reportedThe elucidation of the mechanisms that underlie DNA methylation changes in cancer cells may help identify a number of cancer-specific methylation markers, assisted by promising detection methods, ultimately resulting in optimized clinical applications. The realization that DNA methylation may be involved in human malignancies and is ubiquitous in human diseases is likely to promote the development of novel diagnostic, preventive and therapeutic strategies.et al (et al (et al (RASSF1A to be an epigenetic marker for the detection of fetal DNA in maternal plasma. Epigenetic information passed from parent to offspring and DNA methylation from gametes was shown to be predominantly maternal (et al (The association between methylation and heredity is currently an emerging research topic. The presence of modified m5C affecting the phenotype of the offspring may be parentally inherited. Carone et al indicatel (et al reportedl (et al considermaternal . Similarl (et al recentlyThe modified DNA m5C that extensively occurs within the genome was extensively investigated over the last few decades; however, numerous putative methylated RNAs have been identified and characterized, regarding location, mechanism of formation and cellular function . Additio"} +{"text": "Almost 40 years ago, Takatsuki et al. recognized the existence of a peculiar T cell leukemia in Kyoto, Japan that they named Adult T Leukemia (ATL). They reported a series of 13 patients in 1976 (Uchiyama et al., Almost 10 years ago, ours colleagues Kuan Teh Jeang and Mitsuaki Yoshida organized a special issue on HTLV infection in Oncogene. In setting up this issue, we cannot forget the memory our friend Teh.We called upon the expertise of different research groups from Europe, Japan, and USA. However, we regret that the format of this issue prevented us from soliciting many other colleagues. The following reviews will deal with many fascinating aspects of viral cycle, but summarizes also new approaches that should allow a better integrated research.A first group of articles provides information about HTLV-1 epidemiology and associated-pathogenesis. The article from Gessain and Cassar provides an updated view on HTLV-1 distribution, based on data obtained from 1.5 billion individuals originating from endemic areas (Gessain and Cassar, A second group of articles summarizes the interaction between the virus and the host's cells. Before causing diseases, HTLV-1 has to enter the cell. However, the mechanisms of HTLV-1 transmission and cell entry have remained elusive for a long period of time. Pique and Jones have summarized recent insights about those mechanisms both at the cell level but also between individuals (Pique and Jones, in vivo (Bai and Nicot, A third group of articles reports data on individual viral proteins that play important roles in the viral cycle and/or in pathogenesis. Nakano and Watanabe remind us the important role played by Rex, which uses cellular pathways to export unspliced or singly spliced viral mRNAs in the cell cytoplasm, therefore allowing expressing of structural proteins (Nakano and Watanabe, Finally, Duc Dodon and colleagues remind us that studying HTLV-1 pathogenesis requires animal models (Dodon et al.,"} +{"text": "Frontiers in Neural Circuits in response to the \u201cSpecial topic\u201d entitled \u201cCortical NO interneurons: from embryogenesis to functions.\u201d Although it is practically impossible to address all aspects of NO interneurons, this special issue organized in four sections covers different facets of neocortical and hippocampal NO interneurons: from their diversity and embryonic origins to their functions in the cortical circuits and in neurovascular coupling.Neuronal processing and physiology of cortical circuits rely on a delicate interplay between glutamatergic excitatory neurons and GABAergic inhibitory interneurons in a spatially, temporally, and cell-type specific manner. Understanding these processes is further complicated by the large diversity characterizing the cerebral cortex (Ascoli et al., Studying the functional roles of NO-producing cortical interneurons has been challenging. NO being a highly diffusible gas (Wood and Garthwaite, stratum radiatum of the CA1 area exhibit characteristic properties of ivy cells and express NPY. In contrast to their counterparts in the stratum pyramidale (Fuentealba et al., stratum radiatum ivy cells display a different pattern of axonal and dendritic arborizations suggesting that these two subpopulations of hippocampal NO interneurons are involved in different microcircuits.This issue was revaluated in the whisker-to-barrel cortex by using NADPH-diaphorase histochemistry (Nogueira-Campos et al., Concomitantly with improvements in nNOS detection and the advent of genetic tools allowing lineage analysis of interneuron precursors, the developmental temporal and spatial origins of hippocampal and cortical nNOS neurons have been clarified with three studies showing that hippocampal and cortical type I NO interneurons originate from the medial ganglionic eminence (Jaglin et al., Another important question addressed in this special topic, is how NO interneurons are modulated by afferent signals and what their main neuronal targets are. Type I NO interneurons were found to express selectively the NK1 receptor whose activation by its natural agonist substance P induced their depolarization (Dittrich et al., Although NO is a well-established vasodilator, the relevance of NO interneurons in neurovascular coupling, the tight relationship between neuronal activity and local blood perfusion, has been questioned for decades (Cauli and Hamel,"} +{"text": "Brain-derived neurotrophic factor (BDNF) is a potent regulator of neuronal development and synaptic plasticity that is fundamental to neural circuit formation and cognition. It is also involved in the control of appetite and body weight, with mutations in the genes for BDNF and its receptor, TrkB, resulting in remarkable hyperphagia and severe obesity in humans and mice. Recent studies have made significant progress in elucidating the source, action sites, and regulatory pathways of BDNF with regard to its role in the control of energy homeostasis, and have shed light on the relationships between BDNF and other molecules involved in the control of body weight. Here we provide a comprehensive review of evidence from pharmacological, genetic, and mechanistic studies, linking BDNF to the control of body weight. This review also aims to organize the main findings on this subject into a more refined framework and to discuss the future research directions necessary to advance the field. NTR). The Trk receptors have different neurotrophins as their preferred ligands, with NGF binding to TrkA, BDNF and NT4/5 binding to TrkB and NT3 binding to TrkC, while all of the neurotrophins can bind to p75NTR is a member of the neurotrophin family of secreted signaling molecules that includes nerve growth factor (NGF), neurotrophin-3 (NT3), and neurotrophin-4/5 (NT4/5). Signal transduction can occur through the binding of two distinct classes of receptor proteins: the tropomyosin receptor kinase (Trk) family of receptor tyrosine kinases, which includes TrkA, TrkB, and TrkC, or the p75 neurotrophin receptor , dorsomedial hypothalamus (DMH), ventromedial hypothalamus (VMH), paraventricular hypothalamus (PVH), and brainstem Flier, . The ARCBdnf genes have nine exons, the first eight of which contain 5\u2032 untranslated regions (5\u2032 UTRs), with exon IX containing the entire coding sequence as well as the 3\u2032 untranslated region (3\u2032 UTR) , ventral tegmental area (VTA), substantia nigra and nucleus of the solitary tract (NTS) , after which the signal peptide is cleaved, leaving proBDNF mice, which had increased risk of obesity resulting from increased food intake, concomitant with elevated serum leptin and insulin levels infusion of neurotrophins on presynaptic cholinergic function is the major site of thermogenesis. Sympathetic inputs from the stellate ganglion increase the expression of uncoupling protein 1 (UCP1) in IBAT, which allows the energy generated from \u03b2 oxidation of fatty acids to dissipate as heat in response to physiological stimuli such as cold exposure and overeating expressing Cre recombinase indeed caused modest hyperphagia and obesity was used to delete the Bdnf gene in SF1-expressing VMH neurons, the resulting mutant mice did not display any body weight phenotype (Dhillon et al., Cre transgenic mice will greatly facilitate elucidation of the role of VMH BDNF in the control of feeding behavior and body weight.The VMH is likely a site for the synthesis of BDNF protein that is critical for energy homeostasis. The Bdnf gene expression.Food deprivation also decreased levels of BDNF protein in the rat DVC, whereas refeeding or peripheral injection of the anorexigenic hormones leptin or CCK increased BDNF protein levels in this area (Bariohay et al., TrkB gene expression was reduced to a quarter of the normal amount, or the Bdnf gene was deleted in CaMKII\u03b1-expressing neurons, displayed markedly increased food intake on high fat diet compared to low fat diet (Xu et al., Bdnf mRNAs in the VTA and deletion of the Bdnf gene in the VTA induced hyperphagia on high fat diet but not low fat diet (Cordeira et al., Mice in which Bdnf mRNA encoding the same protein due to the presence of two alternative polyadenylation sites: short 3\u2032 UTR Bdnf mRNA and long 3\u2032 UTR Bdnf mRNA. One study indicates that BDNF protein regulating food intake is translated from long 3\u2032 UTR Bdnf mRNA, likely in dendrites (Liao et al., Bdnf polyadenylation site, such that the long Bdnf 3\u2032 UTR is truncated (Gorski et al., Bdnf mRNA (An et al., Bdnf mRNA in the VMH and nearby regions completely prevented these animals from developing hyperphagia and obesity (Liao et al., Bdnf mRNA to regulate appetite.Neurons produce two forms of NTR (Reichardt, NTR, lead to obesity in humans and mice (Lee et al., Not much is known about the action sites of BDNF with regard to its role in the control of energy homeostasis. BDNF exerts its biological effects through two receptors, TrkB and p75TrkB mouse knockouts. Once the neuronal populations are identified, researchers can focus on the mechanisms by which BDNF modulates the function of these neurons.Pharmacological studies have suggested several putative sites where BDNF acts to control energy homeostasis. Direct injection of BDNF into either the PVH or the VMH of adult rats was found to decrease food intake and increase energy expenditure (Wang et al., TrkB mRNA in the VTA (Cordeira et al., 1, such that peripheral injection of a D1 receptor agonist normalized the hyperphagia on high-fat diet (Cordeira et al., 1 is likely indirect, as the vast majority of TrkB-expressing neurons are striatal medium-sized spiny neurons that express the dopamine receptor D2 (Baydyuk et al., In addition to the hypothalamus and the brainstem, the mesolimbic dopamine system may be another target for BDNF to affect food intake, especially hedonic eating. Consumption of high fat diet was found to increase It is worth noting that one study reported that while central administration of TrkB agonists was anorexigenic in non-human primates, similar to what has been observed for rodents, peripheral administration was orexigenic, opposite to what is observed for rodents (Lin et al., db/db mice, diet-induced obesity mice and lethal yellow agouti mice, peripheral administration of BDNF has been shown to prevent or ameliorate the diabetic and obese phenotypes (Nakagawa et al., db/db mice, BDNF could potentiate the action of insulin in peripheral tissues, reducing serum glucose in obese, diabetic db/db mice (Ono et al., db/db mice by affecting blood glucose control, rather than food intake. BDNF administration also normalized fasted blood glucose levels, possibly through reduced hepatomegaly (Tonra et al., db/db mice, suggesting a long-lasting effect of BDNF on the control of glucose metabolism (Ono et al., db/db mice were restored to normal levels by BDNF treatment, with increased islet beta-cell area in BDNF-treated mice and a reversal of the decrease in pancreatic secretory granules, indicating a role for peripheral BDNF in restoring impaired pancreatic insulin production and secretion in db/db mice (Nakagawa et al., Obesity in mice with BDNF deficiency is associated with hyperglycemia and impaired glucose tolerance (Kernie et al., db/db mice led to increased insulin receptor activation in the liver and insulin-stimulated PI3K activity in the liver, skeletal muscle and IBAT. However, no direct effect of BDNF was found on cultured hepatocytes, L6 muscle cells or 3T3-L1 adipocytes, suggesting an indirect route of action. This may possibly be through regulation of central mechanisms leading to peripheral signaling, as suggested by the observation that centrally administered BDNF also had hypoglycemic effects, and led to similar increases in liver insulin receptor activation and insulin-stimulated PI3K activity (Nakagawa et al., db/db mice, indicating the activation of sympathetic pathways to regulate glucose metabolism (Yamanaka et al., CNS neurons may mediate some effects of peripheral BDNF administration on glucose homeostasis. Chronic subcutaneous BDNF administration in Bdnf mRNA levels in the VMH (Komori et al., Bdnf mRNA in dendrites, as leptin increased dendritic Bdnf mRNA translation in cultured hypothalamic neurons (Liao et al., Bdnf mRNA and BDNF protein, likely by activating BDNF-expressing neurons through a polysynaptic neural circuit.Several studies indicate that BDNF acts downstream of anorexigenic factors such as leptin and CCK to regulate food intake and body weight. Peripheral leptin administration was found to increase Bdnf mRNA (Liao et al., Bdnf mRNA, leptin injection activated LepRb normally in the ARC, DMH, and VMH, as indicated by phosphorylation of STAT3; however, leptin-induced c-Fos expression was impaired in the ARC and VMH and abolished in the DMH (Liao et al., BDNF signaling through the TrkB receptor is necessary for the anorexigenic action of leptin. Peripheral leptin injections reduced food intake in wild-type mice, but failed to do so in mice lacking long 3\u2032 UTR TrkB mutant mice and Mc4r\u2212/\u2212 knockout mice (Xu et al., TrkBF616A mice in which the phenylalanine residue at position 616 of the TrkB receptor is changed to an alanine residue, which makes the TrkB kinase sensitive to inhibition by the small molecule 1NaPP1 (Chen et al., Bdnf mRNA levels in the VMH (Xu et al., BDNF also interacts with the melanocortin system to regulate energy homeostasis. The initial consideration of this interaction arose from the appreciation of similar obesity phenotypes between Bdnf gene is a common allele (Shimizu et al., Cre transgenic mice and circuitry-mapping tools, it is becoming feasible to identify the neural circuits mediating the effects of BDNF on energy homeostasis. Identification of these circuits and elucidation of the mechanisms by which BDNF acts on them will greatly enhance our understanding of human obesity and hopefully bring us closer to a strategy for designing effective drug treatments for this disease.Strong evidence of a crucial role for BDNF in the control of energy homeostasis has been accumulated from genetic and pharmacological studies during the last decade or so. The Val66Met polymorphism in the The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "The mechanisms of eNOS-uncoupling seem multiple and complex. Recent research provides emerging evidence supporting an essential role of increased activity of arginases including arginase-I and arginase-II in causing eNOS-uncoupling, which results in vascular oxidative stress and inflammatory responses, and ultimately leading to vascular diseases. This review article will summarize the most recent findings on the functional roles of arginases in vascular diseases and/or dysfunctions and the underlying mechanisms in relation to oxidative stress and inflammations. Moreover, regulatory mechanisms of arginases in the vasculature are reviewed and the future perspectives of targeting arginases as therapeutic options in vascular diseases are discussed.Oxidative stress and inflammation in the vascular wall are essential mechanisms of atherosclerosis and vascular dysfunctions associated with risk factors such as metabolic diseases, aging, hypertension, etc. Evidence has been provided that activation of the vascular endothelial cells in the presence of the risk factors promotes oxidative stress and vascular inflammatory responses, leading to acceleration of atherosclerotic vascular disease. Increasing number of studies from recent years demonstrates that uncoupling of endothelial nitric oxide synthase (eNOS), whereby the enzyme eNOS produces detrimental amount of superoxide anion Atherosclerotic cardiovascular disease and vascular complications associated with risk factors such as diabetes mellitus, hypercholesterolemia, hypertension, aging, etc., remain the most important challenge for our society and nitric oxide (NO\u22c5) are important signaling molecules involved in the regulation of vascular functions, including vascular relaxations, inflammatory responses, and cell proliferation . 2O2 which is either detoxified to H2O by peroxiredoxins, glutathione peroxidases, and catalase or metabolized to the powerful oxidant molecules such as hydroxyl radical (OH\u22c5), peroxynitrite , flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), and tetrahydrobiopterin (BH4). Electrons from NADPH are transferred in trans from the carboxyl terminal reductase domain of one eNOS monomer, via the flavins FAD and FMN, to the heme in the amino-terminal oxygenase domain of the other monomer, where BH4, oxygen, and l-arginine are bound that competes with l-arginine for eNOS binding and arginase-II (Arg-II), which is encoded by two separate genes. The human Arg-I gene which maps to chromosome 6q23, encodes a 322 amino acid protein as well as intracellular l-arginine concentration (0.05\u20130.2\u2009mmol/L) far exceed the Km of eNOS (2\u201320\u2009\u03bcmol/L) even in the presence of high extracellular concentration of l-arginine (0.4\u2009mmol/L) does not have any benefits on vascular stiffness and left ventricular ejection fraction, but increases mortality .The underlying mechanisms of the detrimental effects of chronic \u22c5 production is well documented and peroxynitrite increase Arg-I gene expression in porcine coronary arterioles is also implicated in regulation of arginase expression and activity in endothelial cells. p38MAPK is a member of the superfamily of MAPKs which serves as cellular a stress sensor for a variety of cellular stresses including hyperglycemia, oxidative stress, and inflammatory cytokines and Arg-II in endothelial cells (Yepuri et al., /\u2212\u2212 background show accelerated atherosclerosis (Vaisman et al., Arginase-II as therapeutic target in cardiovascular diseases has shown promising beneficial effects in genetic modified mouse models. Systemic deficiency of Arg-II reduces systemic and vascular inflammations in mice fed high cholesterol diet and high fat diet, and improves endothelial function in aging, reduces atherosclerosis, and improves insulin sensitivity and glucose homeostasis (Ming et al., It is however, interesting to notice that pharmacological agents that target arginase indirectly through blockade of signaling transduction pathways that regulate arginase gene expression or activity show beneficial effect on vascular functions. Early studies demonstrate that statins which inhibit arginase activity through inhibition of the small G protein or GTPase RhoA improves endothelial function (Ming et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "To compare the efficacy of fine-needle non-aspiration cytology (FNNAC) with that of fine-needle aspiration cytology (FNAC) of thyroid lesions.FNAC and FNNAC techniques were studied in 50 cases of thyroid lesions. All the needle-sampling procedures were done by a single operator. The samples were assessed cytologically and evaluated using five parameters, that is, background blood or clot, amount of cellular material, degree of cellular degeneration, and degree of cellular trauma and retention of appropriate architecture.Wilcoxon signed rank test was performed using SPSS14 software. Differences between all the individual parameters as observed in FNAC and FNNAC smears were insignificant.After evaluation of FNAC and FNNAC on the basis of these scores, greater numbers of diagnostically superior samples were obtained by FNNAC; however, by FNAC more number of diagnostically adequate smears were observed. The numbers of unsuitable smears were also more by FNNAC technique. In vascular organs, an alternative method of fine-needle non-aspiration cytology (FNNAC) also known as cytopuncture was developed in France in 1982 by Brifford et al.[The study population comprised all patients who presented with thyroid swellings at the Department of Pathology (cytology section) from May 2005 to April 2007. After a thorough clinical examination, all the patients were subjected to both FNAC and FNNAC. A total of 50 cases of thyroid lesions were included in this study. The procedure was explained to the patient and verbal consent was obtained prior to performing the procedure. The patients were subjected to FNAC and FNNAC using 23-gauge needles and 10-cc plastic syringes. All the procedures were performed by a single operator. FNAC or FNNAC sampling was carried out randomly with lesions, irrespective of consistency and size of lesions. Every slide was assessed without the prior knowledge of techniques utilized. The study was thus single blind and also prevented the observer bias. The smears were scored according to criteria using a predetermined scoring developed by Mair et al..A cumulative score between 0 and 10 points was obtained for each specimen which was then categorized into one of the following three categories:Category 1\u2014(Score 0\u20132) Unsuitable for diagnosis.Category 2\u2014(Score 3\u20136) Adequate for cytological diagnosis.Category 3\u2014(Score 7\u201310) Diagnosis superior.P=0.05.The difference in the score for the individual parameter was assessed by Wilcoxon signed rank test using SPSS14 software. All the results were analyzed considering the statistical significance at a level of The non-aspiration technique yielded less diagnostically adequate but more diagnostically superior smears when compared with aspiration technique. A total of 19 cases were unsuitable for cytodiagnosis by non-aspiration as compared with 17 cases by aspiration technique .P value obtained by Wilcoxon signed rank test was not statistically significant in favour of non-aspiration sampling for any parameter. However, the average scores for each parameter favoured non-aspiration sampling than aspiration sampling [sampling .Non-aspiration sampling displayed more cellular material, less cellular trauma and degenerative changes, better retention of architecture and less likelihood of obscuring by blood. The average score per case was 6.04 by non-aspiration technique and was 5.90 by aspiration technique. FNAC, since its inception in 1847, has passed through two phases of initial scepticism and interim enthusiasm and has successfully reached the final stage of acceptance as identified by Orell in his aThe important advantage of FNNAC sampling is easy operation and absolute control over operating hand, especially for neck, breast, cutaneous or subcutaneous tissue. The FNNAet al.,[et al.[et al,[et al.[Results when compared for background blood contamination supported the non-aspiration technique Figures and 2 buet al., the amouet al., and 4, b.,[et al. observed.,[et al. was greal.[et al, but the l.[et al, and 4 sil,[et al. but the l,[et al. and 4 wiet al.[For the five parameters studied objectively, there was no statistically significant difference observed between the two techniques, and a similar result has been found by Haddadi-Nezhad et al. Whereas et al.11et al.[et al.[In the present study, more diagnostically superior and less diagnostically adequate samples were obtained more by non-aspiration technique in comparison to aspiration technique. It was observed that the percentage of inadequate sampling was more with non-aspiration (38%) than with aspiration 34%) technique in contrast to the observations of Santos and Leiman[% techniqet al. With thel.[et al.Both the techniques have their own merits and demerits and neither is superior to the other. By combining both the techniques, better diagnostic accuracy can be achieved. However, FNNAC technique is easier to perform with better patient compliance."} +{"text": "Dear Editor in Chief,With a great interest, we read a recently published paper in Frontier in Endocrinology journal by Vuolo et al., entitled \u201cVitamin D and Cancer\u201d by b-galactosidase and sialidase of B and T cells, respectively, which can activate macrophage to phagocyte the neoplastic cells. Group-specific component protein (Gc) as DBP preserve vitamin D in body fluids and put it available for tissues. But an enzyme produced by neoplastic cells deactivate and denaturize this factor and provide neoplastic cells to spread (Yamamoto and Naraparaju,"} +{"text": "Arabidopsis and maize have identified a number of genes that are required for epidermal cell differentiation. However, the mechanism that specifies shoot epidermal cell fate during plant organogenesis remains largely unknown. Particularly, little is known regarding positional information that should restrict epidermal cell fate to the outermost cell layer of the developing organs. Recent studies suggested that certain members of the HD-ZIP class IV homeobox genes are possible master regulators of shoot epidermal cell fate. Here, we summarize the roles of the regulatory genes that are involved in epidermal cell fate specification and discuss the possible mechanisms that limit the expression and/or activity of the master transcriptional regulators to the outermost cell layer in plant shoots.Land plants have evolved a single layer of epidermal cells, which are characterized by mostly anticlinal cell division patterns, formation of a waterproof coat called cuticle, and unique cell types such as stomatal guard cells and trichomes. The shoot epidermis plays important roles not only to protect plants from dehydration and pathogens but also to ensure their proper organogenesis and growth control. Extensive molecular genetic studies in The shoot epidermis is a single layer of surface cells that are morphologically characterized by anticlinal cell division patterns. The outer surface of the shoot epidermis is covered with a hydrophobic structure called a cuticle, which prevents water loss, pathogen attacks, and post-genital fusion of organs was identified as an epidermis-specific homeobox gene that belongs to the HD-ZIP class IV family after the embryos have undergone tangential cell divisions to generate outer protodermal cells and inner cells caused severe phenotypes associated with defects in epidermal cell specification , a rice HD-ZIP class IV gene, was induced on the cut surface during callus regeneration mutant, which develops multilayered epidermis by amplification of differentiated epidermal cells, expression of an HD-ZIP class IV gene was detected only in the outermost epidermal layer , which serve as precursor of cuticular wax and zhoupi (zou) mutants are defective in cuticle formation in organs generated during embryogenesis . Plant embryos may require ALE1 and GSO1/GSO2-mediated signaling for efficient deposition of cuticle on the surface of the protodermal cells that develop in close physical contact with surrounding endosperm cells.crinkly4 (cr4) is a maize mutant with defects in the development of leaf epidermis and aleurone layer ] cause phenotypes defective in epidermal cell differentiation, lateral root initiation, and root initial cell maintenance and TOADSTOOL2 (TOAD2) are leucine-rich repeat receptor-like kinases redundantly required for epidermal cell differentiation in the embryo in the surface cells is a calpain like cysteine protease that is conserved among land plants (Lid et al., DEK1 mRNA is expressed ubiquitously, suggesting that its activity is regulated post-translationally (Wang et al., Arabidopsis was not sufficient to upregulate the expression of ATML1 (Johnson et al., DEK1 activity affected cell division and growth also in internal tissues, suggesting that the action of DEK1 is not epidermis-specific (Johnson et al., dek1 may cause discontinuity and abortion of the epidermis (Johnson et al., Localization of some HD-ZIP class IV transcription factors was not limited to nuclei of heterologous cells (Zhang et al., in planta and it is possible that dimerization with other HD-ZIP class IV proteins changes the activity of ATML1 in a cell-type dependent manner, although ectopic expression of ATML1 alone was sufficient to induce epidermal cell fate in inner tissues (Takada et al., ATML1 was shown to heterodimerize with PDF2 atml1;pdf2 and DEK1 knockdown lines showed ectopic differentiation of mesophyll cells on the surfaces of leaves and cotyledons, respectively, (Abe et al., rpk1;toad2 embryos exhibited ectopic subepidermal marker expression in the outermost cell layer (Nodine et al., ATML1 decreased differentiation of green mesophyll cells in leaves (Takada et al., cr4 and dek1, which are defective in \u201csurface\u201d aleurone layer differentiation in the maize endosperm, cause ectopic differentiation of \u201cinner\u201d starchy endosperm cells on the surface of the endosperm (Becraft et al., Several lines of evidence show that acquisition of epidermal cell fate is associated with a loss of mesophyll or internal cell fate. First, epidermis-deficient Mesophyll cells possibly represent a primitive state of leaf cells, considering that ancestral aquatic algae are composed mainly of mesophyll-like cells. Land plants may have repressed mesophyll cell differentiation to evolve an epidermis on the surface. Evolutionary studies, including comparative genomics, may be useful for identifying molecular components that promote epidermal cell formation (Zalewski et al., Despite the extensive molecular genetic studies in model plants, positional signals that specify shoot epidermal cell fate remain unknown Figure . Most ofThe cuticle-bearing outermost cells should have distinct mechanical properties compared with inner cells. Moreover, cells located at the surface are unique, in that they are in constant contact with the environment. These unique properties could influence the differentiation of epidermal cells. Attempts to directly isolate epidermis-promoting biomolecules and to identify physical/environmental constraints influencing epidermal cell fate may shed new light on the issue.Shinobu Takada wrote the main manuscript text and Hiroyuki Iida prepared Figure The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "With as many as 300,000 United States troops in Iraq and Afghanistan having suffered head injuries Miller, , traumat It is estimated that as many as 300,000 U.S. troops in Iraq and Afghanistan have suffered head injuries Miller, . In the Largely due to population-wide increases in life-span, AD is rapidly becoming the public health crisis of our time. There are currently over three million Americans afflicted with the disease, a figure that is projected to increase to nearly nine million Americans and over 100 million world-wide by 2050 peptides], neurofibrillary tangles (NFTs), and widespread loss of cortical neurons Selkoe, . AlthougThe term \u201cTBI\u201d encompasses a wide variety of traumas. In fact, any form of brain injury is broadly classified as a TBI. Nevertheless, brain traumas can grossly be divided into two categories: (1) closed head injuries (where a rapid deceleration or blow to the head causes brain damage) or (2) penetrating head injuries (caused by a foreign object piercing the skull). Closed head injuries can come in the form of skull fractures, brain contusions caused by brain-skull impact, hematomas, and diffuse axonal injuries brought on by shearing forces. Notably, closed head injuries associated with concussions from contact sports and shockwave blasts from improvised explosive devices have garnered much recent attention. TBIs may range from mild to severe, with about 75% of injuries coming in the form of concussions or other mild TBIs has been demonstrated in the acute response to brain injury (Roberts et al., A very recent study examined survivors of a single TBI 1\u201347 years after the trauma, and reported that NFTs and A\u03b2 pathology were present in approximately one-third of these patients. Such findings demonstrate the long-term consequences of a single TBI event (Johnson et al., As mentioned above, TBI is a strong epigenetic risk factor for development of AD later in life. Strikingly, several defining AD pathological hallmarks have been observed following TBI in patient brains and in numerous TBI animal models. In addition to neuronal and synaptic loss (Kotapka et al., A\u03b2 deposits and widespread axonal A\u03b2 accumulation have been found in patients' brains shortly after TBI (Roberts et al., de novo following brain trauma (Cribbs et al., In an attempt to clarify mechanisms of plaque appearance after brain trauma, several non-transgenic rodent and rabbit models have been utilized. While these animal models have proved useful to characterize axonal A\u03b2 accumulation after TBI, wild-type rodents and rabbits did not manifest cerebral \u03b2-amyloid plaques. This is likely owed to the fact that these TBI animal models have relatively low abundance of brain endogenous A\u03b2 species that do not reach a critical threshold for aggregation (Lewen et al., An important related concept is the idea of A\u03b2 pathology \u201cspreading.\u201d Interestingly, studies from Mathias J\u00fccker's laboratory and others have shown that intracerebral infusion of brain extracts containing aggregated A\u03b2 can initiate A\u03b2 deposition in brains of APP transgenic mice (Kane et al., TBI has also been shown to induce tauopathy. In that regard, it is important to note that a similar process has been described for spreading of NFTs by axonal transport after injection of abnormally folded tau filaments into a mouse model of cerebral amyloidosis (Clavaguera et al., In patients' brains as well as in experimental animal models, TBI has been associated with microglial activation (Carbonell and Grady, Depending on their activation state, microglia can be deleterious or beneficial in the context of cerebral amyloid deposition (Town et al., The development of clinically-relevant animal models is critically important to enable future study at the intersection of TBI and AD research. Animal models of AD fail to exhibit some of the key pathological earmarks of the human syndrome, even after significant brain injury (Uryu et al., By contrast, we have recently published a novel rat transgenic model of AD, line TgF344-AD. This transgenic line expresses mutant human APP and presenilin-1, which are each independent genetic causes of early-onset familial AD. Notably, this rat displays the full spectrum of human AD hallmarks, including cerebral amyloidosis, tauopathy, gliosis, and most importantly, large-scale apoptotic loss of neurons in cortical and hippocampal regions. Moreover, these animals display significant age-dependent cognitive disturbance (Cohen et al., The correlation between brain injury and neurodegenerative disease is now well-established (Szczygielski et al., These mechanistic investigations and the development of pre-clinical therapeutics will rely critically on a clearer understanding of both human pathologies. A key limiting factor is the large gap in our knowledge of the link between post-mortem observations in humans after TBI with animal model systems. Part of the uncertainty can be attributed to limitations inherent to experimental models of TBI. Therefore, it is expected that more precise modeling of pathological hallmarks in animal models will allow us to fill the knowledge gap. Specifically, it will be critical to develop models that accurately mimic the forces impacting the human brain under a variety of circumstances (Morales et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Computed tomography revealed a marked decrease in the size of the metastases following three therapeutic courses, and no lung metastases or new lesions were detected following nine therapeutic courses. The response was declared clinically complete. The patient refused additional treatment following nine therapeutic courses, and there was no recurrence 36 months after the final course of therapy. This case demonstrates the efficacy of IRIS plus bevacizumab as a first-line combination therapy against lung metastases of rectal cancer.This report presents the case of a 72-year-old male who had undergone abdominoperineal resection following a diagnosis of lower rectal cancer with multiple lung metastases. Pathologically, the resected specimen exhibited advanced rectal cancer with regional lymphoid metastases and was classified as stage IV disease. S-1 and irinotecan (IRIS) plus bevacizumab combination therapy was used to treat the lung metastases following the surgery. S-1 (100 mg/body) was administered orally on days 1\u201314 of a 28-day cycle, and irinotecan (125 mg/m As a result of marked improvement in systemic chemotherapy against unresectable and/or recurrent colorectal cancer, the median survival time of patients with metastatic colorectal cancer has improved to >20 months with administration of fluorouracil (5-FU), irinotecan and oxaliplatin . Accordi2) and bevacizumab (7.5 mg/kg) were administered by intravenous infusion on days one and 15. Following three courses of therapy, the metastatic right lung tumor decreased in size to ~10 mm in diameter, and the left lung tumor had decreased in size to ~3 mm in diameter with a diagnosis of rectal cancer. Patient medical history was otherwise unremarkable. On physical examination, all findings were unremarkable with the exception of slight pallor in the palpebral conjunctiva. Hematological investigations revealed anemia . Other laboratory tests and serum levels of carcinoembryonic antigen and carbohydrate antigen 19-9 were all within normal limits. Colonoscopy revealed the entire circumference of an elevated tumor with central depression and erosion at the lower rectum. A biopsy specimen from the tumor was indicative of a moderately differentiated adenocarcinoma. Abdominal computed tomography (CT) revealed thickening of the rectal wall with regional lymph node swelling but no liver metastasis. Chest CT revealed two metastatic lung tumors measuring 25 mm (in the middle lobe of the right lung) and 10 mm in diameter (in the lower lobe of the left lung) . A diagndiameter . Followidiameter . Followidiameter . The reset al (et al (et al (Multidisciplinary therapies, including systemic chemotherapy against unresectable and/or recurrent colorectal cancer, have improved, and FOLFOX, CapeOX and FOLFIRI combination chemotherapies plus bevacizumab or anti-EGFR monoclonal antibody can improve the survival times of patients. In the present case, the patient received IRIS plus bevacizumab combination therapy against lung metastases of rectal cancer. Regarding IRIS chemotherapy, Muro et al reportedl (et al and Tourl (et al identifil (et al , IRIS iset al (et al (et al (The effectiveness of IRIS plus bevacizumab combination therapy as a first-line therapy for metastatic colorectal cancer has been reported. In a phase II study, Komatsu et al reportedl (et al reportedl (et al reportedTreatment of lung metastases from colorectal cancer is a debated issue. The overall survival of patients with completely resectable lung metastases is better than that of patients with unresectable lung metastases \u201317. The In conclusion, IRIS plus bevacizumab combination therapy is well tolerated and efficacious as first-line chemotherapy for metastatic colorectal cancer. As a CV port is not required and patients can be released from the infusion pump while at home, IRIS plus bevacizumab combination therapy could contribute to improved quality of life in patients with metastatic colorectal cancer."} +{"text": "Normal aging is generally characterized by a slow decline of cognitive abilities albeit with marked individual differences. Several animal models have been studied to explore the molecular and cellular mechanisms underlying this phenomenon. The excitatory neurotransmitter glutamate and its receptors have been closely linked to spatial learning and hippocampus-dependent memory processes. For decades, ionotropic glutamate receptors have been known to play a critical role in synaptic plasticity, a form of adaptation regulating memory formation. Over the past 10\u2009years, several groups have shown the importance of group 1 metabotropic glutamate receptor (mGluR) in successful cognitive aging. These G-protein-coupled receptors are enriched in the hippocampal formation and interact physically with other proteins in the membrane including glutamate ionotropic receptors. Synaptic plasticity is crucial to maintain cognitive abilities and long-term depression (LTD) induced by group 1 mGluR activation, which has been linked to memory in the aging brain. The translation and synthesis of proteins by mGluR-LTD modulate ionotropic receptor trafficking and expression of immediate early genes related to cognition. Fragile X syndrome, a genetic form of autism characterized by memory deficits, has been associated to mGluR receptor malfunction and aberrant activation of its downstream signaling pathways. Dysfunction of mGluR could also be involved in neurodegenerative disorders like Alzheimer\u2019s disease (AD). Indeed, beta-amyloid, the main component of insoluble senile plaques and one of the hallmarks of AD, occludes mGluR-dependent LTD leading to diminished functional synapses. This review highlights recent findings regarding mGluR signaling, related synaptic plasticity, and their potential involvement in normal aging and neurological disorders. Over the next decades, a majority of developing countries will face one of the biggest challenges in history: accelerated aging of its populations. In human, aging is characterized by physical, psychological, and social changes. Interestingly, major individual differences are known to exist and low probability of disease and disability, high cognitive and physical capacity, and active engagement in life in general are associated to successful aging. Several animal models have been developed to investigate the processes behind this phenomenon test was introduced in 1984, to study hippocampus-dependent memory in rats Morris, . CognitiEarly on, alterations in cholinergic neuronal activity were linked to memory impairments in the Long-Evans rat model receptor subtypes , another form of synaptic plasticity has been closely related to memory formation -terminal domain of group 1 mGluR isoform and Alzheimer\u2019s disease (AD). Results published on animal models of those diseases and clinical trials conducted with patients will thus help to understand the processes involved in the aging of the brain.FMR1 gene transcription which encodes the Fragile X mental retardation protein (FMRP; Pieretti et al., FMR1 KO mice and FXS patients could dysregulate synaptic plasticity and lead to memory deficits (Luscher and Huber, Group 1 mGluRs have been implicated in the development of the FXS (Dolen et al., FMR1 KO mice, the lack of this protein synthesis suppressor may unbalance cellular mechanisms and exaggerate mGluR-dependent signaling (Dolen and Bear, FMR1 KO mice brain, mGluR5 is associated more with Homer 1a stimulating agonist-independent signaling pathways than long Homer isoforms (Ronesi et al., FMR1 KO mice, mGluR-LTD formation does not require the acute stimulation of protein synthesis which is probably related to the constitutive overexpression of proteins implicated in LTD formation and maintenance to compensate for the lack of FMR1 (Hou et al., FMR1 KO mice, LTP priming is reduced by mGluR decoupling from protein synthesis (Auerbach and Bear, The stimulation of group 1 mGluR with the specific agonist DHPG normally induces FMR protein translation in dendrites (Weiler et al., Drosophila fly model (McBride et al., in vivo treatment with group 2 mGluR antagonists in these mice (Choi et al., FMR1 KO mice by reducing mGluR-LTD with a 50% reduction in mGluR5 expression (Dolen et al., The first demonstration of mGluR modulation rescuing phenotypes in FXS animal models was attained by pharmacologically rescuing social interaction and memory impairments in the FMR1 KO mice brain (D\u2019Agata et al., Drosophila FMRP gene and presenilin, a protein mutated in the familial form of AD (McBride et al., 2+ from intracellular stores, it can also facilitate the hyperphosphorylation of tau by up-regulating protein kinase activities (Tsai et al., 2+ signaling, synaptotoxicity, and eventually synapse deterioration (Renner et al., A possible link between FXS and AD has been proposed since FMR protein binds to the amyloid precursor protein (APP; Sokol et al., After several decades of intense studies on processes involving ionotropic glutamate receptors in the aging brain, their metabotropic counterparts, and particularly post-synaptic group 1 mGluRs are becoming key targets to unravel synaptic plasticity processes underlying learning and memory. mGlu receptors, Homer proteins, and downstream signaling pathways likely all play critical roles in the maintenance of high cognitive abilities in old age. Further studies are thus highly warranted especially considering their apparent role in FXS and possibly AD.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "The Athenian statesman and poet Solon (638\u2013558 BC) believed that \u201cno man can be happy,\u201d being \u201cwretched all those who contemplate the sun.\u201d One century later, the lyric poet Pindar (c. 522\u2013443 BC) wrote that there has never been a man \u201cdevoid of trouble\u201d on earth, and there will never be one. In a recent Frontiers article, inspired by up-to date neuroscience evidence on the perception of pain, Lelard and colleagues investigated the interrelations between behavior and emotion in painful situations (Lelard et al., Unfortunately, we all know the unpleasant sensory and emotional experience of pain. What we can now explore scientifically in regards to pain has been complexly expressed through language and philosophy. The ancient Greeks used the word To determine how imagining oneself in painful situations might influence muscular and physiological responses, posturographic and physiological data were collected from participants viewing pictures of both painful and non-painful situations, while instructed to imagine themselves experiencing each situation. A displacement of center-of-pressure measure collected from a posturographic platform indicated that movement in the anteroposterior direction was shorter when subjects were presented with painful images. Responses of the tibialis anterior and soleus muscles, which have been associated with freezing responses in previous reports (Carpenter et al., Previous investigations have differentiated the \u201crepresentational characters\u201d that emerge while imagining a situation from the \u201cinstinctual characters\u201d that arise in more automatic bodily responses, and have suggested that these may emerge from cortical vs. subcortical activity, respectively (Giummarra et al., The article by Lelard and colleagues suggests that emotional content is capable of changing muscular responses in specific configurations with ecological significance (Lelard et al., Additional questions may inform future research. For example, in what situations might emotionally-laden stimuli evoke responses different from those observed in this study\u2014a muscular response indicative of fleeing rather than freezing? Would active suppression of empathy or perspective-taking for painful stimuli alter muscular responses? In addition, would suppression effectiveness differ for tasks with more visceral-response vs. representational-response evoking images? Possible experimental avenues for theoretical exploration that might support the implications of this study may exist in research examining individuals with unusual perspective-taking in healthy and pathological conditions\u2014i.e., those with mirror-touch synesthesia (Banissy and Ward,"} +{"text": "They have important microbicidal activities, however in inflammatory conditions they may secondarily attack surrounding tissues. Overproduction of reactive oxygen species, prolonged or excessive liberation of MPO and other effective yet also toxic substances from neutrophils may participate in disturbed apoptosis, intensify the inflammatory processes and result in serious human diseases. The inhibitory effect of quercetin on PMA stimulated SO generation in isolated human neutrophils was found to be dose-dependent, without affecting the activity of intact isolated neutrophils. At comparable conditions, quercetin was more potent in inhibiting MPO release than SO generation. Our results indicate that quercetin could support resolution of inflammation through decreased activity of neutrophils, The concentration of 10 \u00b5mol/l significantly inhibited only MPO release by 36%. Our results are in agreement with findings of Nosal et al., and Pere (et al. where quet al., et al., et al., 2+-ATPase (Horakova, et al., et al.,Quercetin is believed to protect human organism against several degenerative diseases (Hollman & Katan, Our study provided evidence supporting the potential beneficial effect of quercetin in diminishing tissue damage at the site of inflammation by inhibiting MPO release and by decreasing the generation of superoxide and the subsequently derived ROS."} +{"text": "The phenotype of DBA/2-mdx mice also seems to depend on the function of satellite cells. In this review, we summarize the methodology of direct isolation, characterization, and molecular regulation of satellite cells based on our results. The relationship between the regenerative capacity of satellite cells and progression of muscular disorders is also summarized. In the last part, we discuss application of the accumulating scientific information on satellite cells to treatment of patients with muscular disorders.Skeletal muscle has great regenerative capacity which is dependent on muscle stem cells, also known as satellite cells. A loss of satellite cells and/or their function impairs skeletal muscle regeneration and leads to a loss of skeletal muscle power; therefore, the molecular mechanisms for maintaining satellite cells in a quiescent and undifferentiated state are of great interest in skeletal muscle biology. Many studies have demonstrated proteins expressed by satellite cells, including Pax7, M-cadherin, Cxcr4, syndecan3/4, and c-met. To further characterize satellite cells, we established a method to directly isolate satellite cells using a monoclonal antibody, SM/C-2.6. Using SM/C-2.6 and microarrays, we measured the genes expressed in quiescent satellite cells and demonstrated that Hesr3 may complement Hesr1 in generating quiescent satellite cells. Although Hesr1- or Hesr3-single knockout mice show a normal skeletal muscle phenotype, including satellite cells, Hesr1/Hesr3-double knockout mice show a gradual decrease in the number of satellite cells and increase in regenerative defects dependent on satellite cell numbers. We also observed that a mouse's genetic background affects the regenerative capacity of its skeletal muscle and have established a line of DBA/2-background One of the best-known examples of regeneration is the ability of newts to regenerate limbs and tails. This process was believed to utilize specialized multipotent cells that have the potential to produce all types of cells. The existence of this type of multipotent stem cells seemed to be the reason newts, but not humans, can regenerate limbs. However, a recent study of the axotl has suggested a different model cells, and muscle-resident interstitial cells, that seemed to function as stem cells for muscle regeneration integrin \u03b21(+)Cxcr4(+)CD34(+)CD45(\u2212)Sca-1(\u2212)Mac-1(\u2212) fraction contained only myogenic cells constitute a subgroup of the transforming growth factor (TGF)-\u03b2 superfamily, and are known as myogenic differentiation regulators. Gamell et al. reported that Bmp2 induces phosphorylation of Akt and migration of C2C12 were upregulated (>5-fold) in the activated state in our microarray results. The most highly upregulated gene (334-fold) was As mentioned above, satellite cells occupy a unique location and do not express the myogenic determination gene MyoD. Therefore, we hypothesized that the genes responsible for maintaining satellite cells are specifically expressed in quiescent satellite cells in skeletal muscle. To isolate such genes, we also prepared non-myogenic cells from skeletal muscle for comparison with quiescent and activated satellite cells, and 63 genes were finally identified as \u201cquiescence genes,\u201d which are highly expressed in quiescent satellite cells but not in cultured myoblasts and non-myogenic cells in skeletal muscle , but these genes are also expressed in non-myogenic cells in skeletal muscle. Another discrepancy is the expression of Notch-related genes in quiescent satellite cells. We identified Notch signaling-related genes (Notch3 and HeyL/Hesr3) as the most highly expressed genes in quiescent satellite cells, although Pallafacchina's results did not show the importance of Notch signaling in quiescent satellite cells. However, recent studies have clearly demonstrated the essential roles of Notch signaling for maintaining quiescent satellite cells in vivo as well as developmental stages (see below). On the other hand, quiescent satellite cells share common features in our and Pallafacchina's results, for example, some cell adhesion molecules and transcriptional factors. Although our reports had not mentioned it, Pallafacchina et al. intriguingly found up-regulation of anti-oxidative genes in quiescent satellite cells. In fact, our original data also included ant-oxidative genes in quiescent-stage specific manner. These results imply the importance of oxidative stress in quiescent satellite cells. Farina et al. analyzed gene expressions of quiescent satellite cells, activated satellite cells , and proliferating myoblasts and Hesr are known as primary targets of Notch signaling , which leads to a decrease in BrdU uptake leads to the generation of mononuclear cells that proliferate is a well-known inherited muscular disorder, and the causative gene, mdx mouse (the correct nomenclature is C57BL/10-DMDmdx) is the most widely used model animal of DMD show similar phenotypes to humans; loss of muscle weight, increased fibrosis, accumulation of adipocytes, and decreased muscle force and demonstrated that mdx/mTR mice exhibit severe muscular dystrophy and a decrease in the loss of satellite cell proliferation . Recently, Cappellari et al. reported that Dll4 and PDGF\u03b2 signals convert myogenic cells to pericyte-like cells without erasing their myogenic memory (Cappellari et al., Satellite cells undoubtedly have the best potential to produce new myofibers in vitro reduces their regenerative activity, as described above (Montarras et al., in vitro (Gilbert et al., in vivo regenerative potential (Parker et al., Another problem is the difficulty of obtaining large numbers of satellite cells from a donor. In addition, expansion of satellite cells Another source of cells for therapy for muscle disorders is iPS cell-derived myogenic cells (Mizuno et al., mdx phenotype (Le Grand et al., The physiological self-renewal mechanism of satellite cells during regeneration must also be revealed. Some evidences have indicated an asymmetrical model of self-renewal of satellite cells (Conboy and Rando, In addition, another mechanism must be understood for the successful cell transplantation for muscle disorders. To date, we have not paid attention to the fusion process between transplanted cells and myofibers. When donor cells are transplanted, they have to cross the basal lamina and fuse with myofibers. Horsley et al. indicated the myotube-myoblast fusion process requires IL4/IL-4R signaling mediated by NFATc2 (Horsley et al., For 10 years, the isolation, characterization, and molecular regulation of satellite cells have been studied, and accumulating evidence is starting to reveal the molecular mechanisms for maintaining the satellite cell pool. In addition, recent studies are beginning to identify the roles of satellite cells in several different disorders. For instance, McCarthy et al. indicated that acute hypertrophy is not dependent on satellite cells (McCarthy et al., The generation of iPS cells has assisted the cell therapy approach to many disorders including muscular dystrophy (Takahashi and Yamanaka, Skeletal muscle is indispensable for motility. In addition, skeletal muscle has the potential to control other tissues. For instance, Bostrom et al. showed that a skeletal muscle-derived cytokine, irisin, converts white fat cells to brown fat-like cells (Bostrom et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "A decline in skeletal muscle mass and function with aging is well recognized, but remains poorly characterized at the molecular level. Here, we report for the first time a genome-wide study of DNA methylation dynamics in skeletal muscle of healthy male individuals during normal human aging. We predominantly observed hypermethylation throughout the genome within the aged group as compared to the young subjects. Differentially methylated CpG (dmCpG) nucleotides tend to arise intragenically and are underrepresented in promoters and are overrepresented in the middle and 3\u2032 end of genes. The intragenic methylation changes are overrepresented in genes that guide the formation of the junction of the motor neuron and myofibers. We report a low level of correlation of gene expression from previous studies of aged muscle with our current analysis of DNA methylation status. For those genes that had both changes in methylation and gene expression with age, we observed a reverse correlation, with the exception of intragenic hypermethylated genes that were correlated with an increased gene expression. We suggest that a minimal number of dmCpG sites or select sites are required to be altered in order to correlate with gene expression changes. Finally, we identified 500 dmCpG sites that perform well in discriminating young from old samples. Our findings highlight epigenetic links between aging postmitotic skeletal muscle and DNA methylation. We found an overall global trend of genomic hypermethylation in human skeletal muscle between the young and old groups Fig. . In ordeet al., et al., et al., CpG islands (CGI) are present in 60% of gene promoters in the 450 K chip, and methylation deregulation in CGI overlapping the promoter has often been linked with cancer negatively correlates with the corresponding promoter methylation level was the transcription factor CTCF . This suggests that the dmCpG sites we identified within the aged group are relevant to muscle tissue. There were 54 enriched terms with a P < 0.01 (Table P = 2.05E-07) and axon guidance (P = 2.11E-07), which may be related to muscle innervation and aging. A decline of function in the neuromuscular junction has long been thought to contribute to the decline of muscle mass with age , cytoskeleton (P = 0.0003), and cytoskeleton organization (P = 0.001) are all linked to cytoskeleton function, and the latter plays an important role in proper muscle contractile function , homophilic cell adhesion (P = 8.43E-08), and cell adhesion (P = 1.70E-05), with cell adhesion being associated with fiber degeneration (Table P = 6.16E-10) with 50 of 216 pathway members (23.1%) differentially methylated in at least one intragenic site .A recent report utilized genes identified as having dmCpGs with age to examine stem cell differentiation pathways occurs with aging and is a major contributory factor in sarcopenia (Doherty et al., et al., et al., et al., et al., In aging human skeletal muscle, we identified a strong preference for the dmCpG sites to localize within a gene, and in the central and 3\u2032 end regions of genes, but surprisingly no preference for the promoter. Some studies have suggested that intragenic DNA methylation could regulate alternative splicing (Sati et al., A potential issue with the biopsy procedure used in our study is tissue heterogeneity between biopsies, which may impact the resultant molecular signatures. There are other cell types in muscle that might contaminate muscle-specific methylation signatures. Such sample variation may influence the results. However, it was previously demonstrated that there is remarkably little technical variation between discrete biopsies sampled from the right and left leg of the same individual with regard to fiber type differences, gene expression as assessed by microarray, fiber size, or even strength (Tarnopolsky et al., et al., In summary, we describe for the first time a genome-wide DNA methylation survey of a postmitotic tissue between young and old healthy human skeletal muscle. We also determined that there were several hundred robust dmCpGs that discriminated younger males from older males. Ontology analysis of the terms associated with dmCpGs provided several interesting hints of a potential role for epigenetic changes in the neuromuscular junction during aging. Resistance exercise can reverse specific aspects of the gene expression changes in aging to more youthful levels (Melov All methods and experimental procedures are available in online supplement."} +{"text": "There is not too much success in the antioxidant treatment of heart deceases in humans. However a new approach is now developed that suggests that depending on their structures and concentrations antioxidants can exhibit much more complicated functions in many pathological disorders. It is now well established that physiological free radicals superoxide and nitric oxide together with their derivatives hydrogen peroxide and peroxynitrite and reactive nitrogen species (RNS)) play a more important role in heart diseases through their signaling functions. Correspondingly this work is dedicated to the consideration of damaging signaling by ROS and RNS in various heart and vascular disorders: heart failure (congestive heart failure or CHF), left ventricular hypertrophy (LVH), coronary heart disease, cardiac arrhythmias, and so forth. It will be demonstrated that ROS overproduction (oxidative stress) is a main origin of the transformation of normal physiological signaling processes into the damaging ones. Furthermore the favorable effects of low/moderate oxidative stress through preconditioning mechanisms in ischemia/reperfusion will be considered. And in the last part we will discuss the possibility of efficient application of antioxidants and enzyme/gene inhibitors for the regulation of damaging ROS signaling in heart disorders. Heart disease (cardiopathy) and cardiovascular diseases are a group of numerous pathological disorders such as heart failure (congestive heart failure or CHF), left ventricular hypertrophy (LVH), coronary heart disease, cardiac arrhythmias, and so forth, in which signaling processes of reactive oxygen and reactive nitrogen species (ROS and RNS) play an important role. Contemporary studies identified major sources of ROS and RNS productions: NADPH oxidases (Nox), xanthine oxidase, mitochondria, and nitric oxide synthases (NOS). As a rule, heart and cardiovascular diseases are characterized by ROS overproduction whereas the formation of major RNSs nitric oxide and peroxynitrite (diamagnetic molecule) can decrease or increase depending on the nature of heart injury. Free radicals are usually considered to be the damaging factors in various pathologies, but on the other hand ROS and RNS are important signaling species in many physiological and pathophysiological processes. For example the critical role of these species has been shown in preconditioning and other survival processes (see below). A major aim of this work is to consider the role of ROS and RNS signaling in various heart and cardiovascular diseases.NADPH oxidases generate superoxide by the one-electron reduction of dioxygen:2O2+NADPHphox) was an important factor of the development of Ang II-induced cardiac hypertrophy independently of the change in blood pressure in mice. Similar effect of NADPH oxidase-derived superoxide was demonstrated by Nakagami et al. [Phagocyte NADPH oxidase Nox2 plays important role in heart injury. Bendall et al. found thi et al. Li et ali et al. showed ti et al. . Similari et al. demonstri et al. . \u03b2) induced NADPH oxidase activation and ROS overproduction that accelerated atherosclerosis, hypertension, and myocardial remodeling in apolipoprotein E-deficient (apoE(\u2212/\u2212) mice. It was found that Rac1 initiated hypertrophic response in the heart dependent on NADPH oxidase-generated ROS . Hingtgephox\u2009\u2009on internal membranes in epithelial cells. It was also found that in contrast to the other NADPH oxidase isoforms Nox4 produced mainly hydrogen peroxide and the very small amounts of superoxide. Cytosolic oxidase proteins or the GTPase Rac are not required for the activity of this enzyme. Serrander et al. [NADPH oxidase Nox4 is a NADPH oxidase isoenzyme which plays an important role in heart and vascular diseases. Martyn et al. suggester et al. also fou There is a lot of uncertainty in the studies of Nox4-dependent ROS production. Conclusion that Nox4 produces mainly hydrogen peroxide and not superoxide contradicts a majority of the other experimental data. It is possible that unreliable methods such as nitroblue tetrazolium (NBT) reduction were applied for superoxide detection in the above works \u201316. On t Nox4 is apparently located in different way in cardiac cells comparing to other NADPH oxidases. Thus Kuroda et al. found th Surprisingly, Zhang et al. found th Obviously these findings contradict those obtained by Kuroda et al. . In bothZhang et al. suggestePrincipal reaction catalyzed by xanthine oxidase (XO) is the oxidation of xanthine into uric acid: ablished and Chamablished suggesteablished . Althougablished , 27. However, it is now generally agreed that there is a significant increase in cellular XO level and activity in the cardiovascular system under pathological conditions even though these changes may not be easily detected under physiological conditions. For example Thompson-Gorman and Zweier measured xanthine oxidase-mediated free radical generation in isolated rat heart . They fo Similarly to NADPH oxidases xanthine oxidase stimulated many ROS-dependent heart disorders. Landmesser et al. showed t\u03b1) and induced the activation of xanthine oxidase and superoxide generation leading to coronary endothelial dysfunction in a murine model. Yamamoto et al. [ Duncan et al. found tho et al. showed to et al. demonstrMitochondria are an essential ROS producer in heart and vascular diseases although its significance as a ROS source comparing to NADPH oxidases and xanthine oxidase remains a subject for discussion. It is now well established that superoxide is generated by mitochondria due to electron leak from the two electron carriers of respiratory chain; these sources of superoxide are Complex I and Complex III . Many au\u03b1-induced mitochondrial superoxide production impaired respiratory complex I activity and led to mitochondrial damage in the left ventricle (LV) in rats. Mitochondrial depolarization and enhanced ROS production mediated by lipoxygenase and arachidonic acid were probably responsible for arrhythmias following ischemia-reperfusion injury [ Redout et al. have stun injury . Howevern injury found thNitric oxide synthases (NOS) catalyze conversion of L-arginine to L-citrulline and nitric oxide but under uncoupling conditions these enzymes also produce superoxide:oxidants . Thus Lioxidants showed toxidants found th As NOSs are able to produce both RNS and ROS, the effects of these enzymes on cardiovascular system can be very complicated\u2014they can enhance or diminish heart damage. Nitric oxide is the endothelium-derived relaxing factor (EDRF); therefore its function must be mainly favorable at the heart. However, the diffusion-controlled reaction of nitric oxide with superoxide produces the highly reactive peroxynitrite, a really harmful agent. Khadour et al. have stuRegulation of superoxide/nitric oxide balance is important for the prevention of heart damage. This balance is partly achieved by the interaction of principal enzymatic ROS producers during pathological changes in heart. Thus Saavedra et al. showed t Saraiva et al. suggesteA brief episode of myocardial ischemia makes the heart remarkably resistant to a subsequent ischemia, the phenomenon named ischemic preconditioning. It has now been shown that ROS and RNS signaling play an important role in ischemic preconditioning and cardioprotection. For example it was found that 30\u2009min of ischemia triggered by acetylcholine and an opioid receptor in isolated rabbit hearts stimulated preconditioning which included the activation of ROS- and RNS-dependent cascade of the epidermal growth factor (EGF) receptor, phosphatidylinositol 3-kinase (PI3-K), protein kinase B (Akt), nitric oxide synthase (NOS), and ROS-dependent opening of mitochondrial (mito)K(ATP) channels . This ca Kimura et al. found th It is usually proposed that both K(ATP) channel opening and ischemic preconditioning protect the ischemic heart by acting at K(ATP) channels in the inner mitochondrial membrane. However Brennan et al. found th Van-Cuong et al. studied \u03b4, PI3K, and ERK kinases. It is thought that these processes stimulate the inhibition of mitochondrial permeability transition pore formation and trigger the entrance into the preconditioned state. In recent work Vigneron et al. [\u03b2 (GSK-3\u03b2), the opening of mitoK(ATP), and ROS generation activating the target of rapamycin (mTOR) pathway and induced cardioprotection. These findings suggest that cardioprotection involved a prosurvival mTOR pathway. Cohen et al. have revn et al. showed t Overproduction of ROS and deregulation of RNS production are important factors of the development of heart and cardiovascular diseases. Mechanisms of ROS and RNS generation by major producers, NADPH oxidases, xanthine oxidase, mitochondria, and nitric oxide synthases in these diseases as well as in preconditioning were discussed above and presented in \u03baB and cardiomyocyte hypertrophy in mice. As it has been already noted, Akt also participates in preconditioning [Among various enzymes, protein kinases B and C and mitogen-activated protein kinases (MAPK) play a very important role in ROS and RNS-dependent enzymatic cascades responsible for heart damage. One of these kinases is the serine/threonine protein kinase B (Akt). It has already been noted that Akt participates in Nox2-initiated Ang II-dependent cardiomyocyte hypertrophy . Recentlitioning , 65. Theitioning found thitioning proposed\u03b4, and PKC\u03b5 in heart damaging processes has been also demonstrated [\u03b4 or selective activation of PKC\u03b5 reduced oxidative damage in the heart following myocardial infarction. cGMP-dependent protein kinase (PKG) showed protective activity in the heart [ Participation of protein kinases PKC, PKCnstrated , 71, 87.he heart , 89.Gaitanaki et al. [\u03b2-Estradiol (E2) improved congestive heart failure (CHF) in rats by antioxidative mechanism that involved thioredoxin (Trx) upregulation, the inhibition of Rac1 mediated NADPH oxidase activity, and the apoptosis signal-regulating kinase 1(ASK-1)/JNK/p38-mediated apoptosis. Cai et al. [\u03b1 inhibited endothelium-dependent NO-mediated dilation of coronary arterioles from porcine heart by the ceramide-induced activation of JNK and subsequent production of superoxide by xanthine oxidase. Widder et al. found thi et al. showed ti et al. demonstri et al. showed ti et al. studied i et al. showed tvia a novel zinc-finger protein, MCPIP (MCP-1-induced protein). MCPIP stimulated enzymatic cascade through the activation of MAP kinases JNK and p38. These findings suggested that MCPIP induced ROS/RNS production that stimulated ER stress, autophagy and apoptosis. Hikoso et al. [\u03baB in cardiomyocytes in response to pressure overload. They demonstrated that I\u03baB kinase (IKK)-(NF-\u03baB) signaling cascade was protective in cardiomyocytes due to the attenuation of oxidative stress and JNK activation. Younce and Kolattukudy studied o et al. investig\u03b1) is upregulated in a number of cardiomyopathies inducing adverse cardiac remodeling and dilation due to the degradation of the extracellular matrix by matrix metalloproteinases (MMPs). Awad et al. [\u03b1 (rTNF) induced stronger superoxide production and increased expression of several MMPs in mouse neonatal cardiomyocytes comparing to cardiofibroblasts. Phosphatidylinositol 3-kinase (PI3K-\u03b3) mediated TNF-dependent superoxide production and MMP expression. Lu et al. [ It has been shown that tumor necrosis factor proteins depend on ROS levels and can stimulate or decrease longevity of experimental animals. Klotho gene regulates cell senescence and aging. Mechanisms of ROS regulation in such gene/enzymatic processes were recently discussed [ It is known that several genes regulate ROS formation in pathological states. Thus it has been shown that the suppression of iscussed . These genes can also participate in ROS-dependent heart damage. Alcendor et al. showed t\u03b11-adrenergtic receptor (\u03b11-AR) pathway together with protein kinases PKC\u03b5 and PKC\u03b4 and induced Akt-FOXO3a phosphorylation in rat cardiomyocytes. Sengupta et al. [ Guo et al. showed ta et al. showed ta et al. .Heart Diseases and Antioxidants), therefore we need to choose some principal studies. Antioxidants and free radical scavengers can suppress free radical-dependent heart disorders by direct reactions with reactive hydroxyl and peroxy free radicals or through the regulation of ROS signaling in gene- and enzyme-catalytic cascades. We will consider some important examples. For a long time antioxidants were considered important pharmacological agents for the treatment of heart diseases. It is impossible to discuss even a small part of works published on this problem against ischemia-reperfusion injury in the heart. It has been proposed that Sildenafil, a selective inhibitor of phosphodiesterase type 5 induced powerful protection against myocardial I/R injury through the activation of cGMP-dependent protein kinase G (PKG). Das et al. suggeste Thus antioxidants of various classes might be considered to be of potential use for the prevention and maybe treatment of heart and cardiovascular diseases.The data discussed in this work show that practically all pathological disorders in heart and cardiovascular system are associated with damaging ROS signaling. These damaging processes can be initiated by ROS overproduced by various sources such as NADPH oxidases, mitochondria, xanthine oxidase, and NO synthases , or norm\u03b1, or monocyte chemotactic protein-1 (MCP-1) are responsible for ROS overproduction. ROS overproduction might be also initiated by cellular disorders for example initiated by hyperthermia increased the expression of thioredoxin (Trx) and the suppression of NADPH oxidase/ASK-1/c-Jun/p38 cascade causing a decrease in apoptosis and the stimulation of CHF recovery [ On the other hand ROS signaling in enzymatic/gene cascades might also decrease disorders in heart and cardiovascular system. As is seen from farction . 17\u03b2-estrecovery . Decreasrecovery , 78. It is of interest that the participation of certain genes in signaling pathways frequently led to diminishing heart disorders. As is seen from As ROS overproduction is one of the most important stimulators of heart disorders, it is logically to suggest that the application of antioxidants should be useful for fighting the heart and cardiovascular diseases. It has been shown above that the effects of antioxidants are very complicated and changed from direct interactions of antioxidants with reactive free radicals to the influence of enzymatic/gene pathways. For example protein kinases and the antioxidant/prooxidant genes can enhance or diminish ROS formation stimulating surviving and death of cardiomyocytes. Another source of difficulty is dependence of the effects of antioxidants or prooxidants and various ROS inhibitors dependent on the levels of oxidative stress. As it was pointed out, low levels of ROS (low oxidative stress) might have prosurvival effects while high ROS levels (severe oxidative stress) will damage biomolecules. These effects can in turn be complicated by the survival effects of prooxidants-induced preconditioning and the induction of antioxidant enzymes (MnSOD and CuZnSOD). Therefore a major question is how we might take into account all miscellaneous effects of antioxidants, prooxidants, and gene/enzymatic inhibitors for the selection of the right compounds and methods in the antioxidant (or prooxidant) treatment of heart diseases? Of course there is no simple answer. However, it is probably useful before beginning the treatment of patients to consider possible effects of the selected compound in major ROS and RNS signaling processes."} +{"text": "Vitamin C and the Common Cold\u201d Linus Pauling included an interesting chapter on \u201chuman biochemical individuality\u201d that defined some important parameters on individual human genotypic versus phenotypic variation, based in part on studies from hemoglobin genetics . If we assume that there are about 26,600 human genes available to be expressed in each cell and that each gene is responsible for at least one inherited trait or genetic function (a number that is probably vastly underestimated), in a global human population exceeding 7 billion it then becomes exceedingly difficult to define \u201chuman genetic normalcy.\u201d These ideas form the basis for the evolving concept of \u201chuman genetic individuality\u201d and our ongoing efforts to better understand the genotypic basis of human phenotypic diversity in both health and disease and miRNA abundance, speciation and complexity in defined brain anatomical regions from different human samples. These studies have been very valuable since the profiling of mRNA and/or miRNA can provide a powerful \u201csnapshot\u201d into the physiological status of a human cell or tissue in health and disease, and may even be predictive for the prognosis and/or diagnosis for the future outcomes of other AD patients. Steady-state mRNA and miRNA levels from different individuals clearly indicates that the abundance and speciation of these RNAs within clearly defined anatomical regions can significantly differ between samples analyzed, suggesting that genetic variation and extraneous effects, including age, gender, body mass index (BMI), apolipoprotein E (ApoE), beta-amyloid cleavage enzyme (BACE) and other AD-relevant allele status, life-style and intrinsic population effects can influence the profile of mRNA or miRNA abundance and complexity (Colangelo et al., APOE genotype (Tang et al., To illustrate one important example is the miRNA abundance and speciation of a small family of inducible, NF-kB-sensitive miRNAs in two different American populations\u2014Caucasian Americans and African Americans afflicted with AD Figure . A pathoVariation in miRNA patterns lends further strength to the idea that AD is not a single, definable neurological disease entity such as sickle-cell anemia Pauling, , but rat\u201chuman genetic individuality.\u201d If molecular-genetic and epigenetic profiles of AD brain samples are any indication of AD phenotypic variation then there may be real and significant inter-ethnic differences in AD epidemiology, incidence, disease course and progression. This further suggests that an equally wide variety of diagnostic and individualistic prevention and treatment strategies will be required to more effectively address such progressive, age-related neurological disorders of the human CNS, including the implementation of novel combinatorial therapeutic strategies such as anti-NF-kB and anti-miRNA approaches that have not yet been considered (Lukiw, Lastly, much independently derived data comparable to that shown in Figure d Lukiw, ."} +{"text": "Dear Editor,I read with great interest the work published by Mogadam et al., entitled \"Comparison of Analgesic Effect between Gabapentin and Diclofenac on Post-Operative Pain in Patients Undergoing Tonsillectomy\" . I found"} +{"text": "Synovial sarcomas commonly occur in the soft tissue of the extremities, while a primary occurrence in the mediastinum is quite rare. The current study reports the case of an 11-year-old male who presented with a neck mass, which computed tomography showed was due to a giant mediastinal mass involving the thyroid gland. The tumor was resected by thoracotomy and diagnosed as monophasic synovial sarcoma by histopathology. The patient received adjuvant combination chemotherapy and radiation therapy following surgery. At the 3-month follow-up, no local tumor recurrence was found. The present case report highlights the significance of recognizing the unusual presentation and clinical manifestation of synovial sarcoma to aid clinical management. Written informed consent was obtained from the patient\u2019s family. Synovial sarcoma is a type of mesenchymal tissue cell tumor that exhibits epithelial differentiation, which most frequently arises in the extremities, while a primary occurrence in the mediastinum is quite rare ,2. Prima2 in the left thyroid gland, however nothing of significance was revealed in the chest or abdominal regions. Computed tomography (CT) scans scans and 2 shmination showed mmination \u20137, confimination showed amination was perfet al in 2002 as a type of mesenchymal tissue cell tumor that exhibits epithelial differentiation , which met al, of the Primary mediastinal synovial sarcoma is a type of malignant tumor with no specific differences from other mediastinal tumors with regard to clinical manifestation, imaging or histology, therefore, it is difficult to diagnose. With regard to clinical manifestations, mediastinal synovial sarcomas reveal various initial symptoms due to their different scopes of infringement. The common symptoms include chest pain \u20137, shortet al Although mediastinal synovial sarcomas have limited clinical data and no standardized treatments, complete surgical excision remains the cornerstone of therapy ,6,8,9. Aet al retrospe al(et al, adjuvan al(et al also fou al(et al. In the al(et al, the 5-y al(et al recently al(et al,9,17. Th al(et al and 9 sh al(et al, it was al(et al must be A previous study showed tMediastinal synovial sarcomas are malignant tumors with a low incidence, no specific clinical manifestations and a lack of unified and effective treatments. These factors highlight the challenges preventing its diagnosis and treatment in clinics. The existing data supports surgery as the preferred treatment for mediastinal synovial sarcoma, but appropriate auxiliary treatments, including radiotherapy and chemotherapy, must be taken into consideration according to the factors affecting prognosis. In addition, the SYT-SSX fusion gene in synovial sarcoma must be further investigated in hope of identifying novel targeted therapies."} +{"text": "Clustering and classification of large-scale chemical data are essential for navigation, analysis and knowledge discovery in a wide variety of chemical application domains. The maximum common structure (MCS) for a group of compounds is an important element of such classification, providing insight into activity patterns and enabling scaffold alignment for a more consistent 2D depiction. Most modern, exact MCS implementations use back-tracking or cliqu"} +{"text": "A goal of the COG Ewing Sarcoma (ES) Biology Committee is enabling identification of reliable biomarkers that can predict treatment response and outcome through the use of prospectively collected tissues and correlative studies in concert with COG therapeutic studies. In this report, we aim to provide a concise review of the most well-characterized prognostic biomarkers in ES, and to provide recommendations concerning design and implementation of future biomarker studies. Of particular interest and potentially high clinical relevance are studies of cell-cycle proteins, sub-clinical disease, and copy number alterations. We discuss findings of particular interest from recent biomarker studies and examine factors important to the success of identifying and validating clinically relevant biomarkers in ES. A number of promising biomarkers have demonstrated prognostic significance in numerous retrospective studies and now need to be validated prospectively in larger cohorts of equivalently treated patients. The eventual goal of refining the discovery and use of clinically relevant biomarkers is the development of patient specific ES therapeutic modalities. First described by James Ewing as an endothelioma of bone Ewing, , Ewing sIt was the advent of consistency in diagnosis that enabled cooperative groups worldwide to develop multi-center ES clinical trials. Over the past three decades these trials have systematically evaluated and optimized local and systemic treatment protocols for patients with ES , and increasing patient age have all been implicated as negative prognostic features. None of these are as significant as the presence of metastatic disease and studies have demonstrated variability in these individual features , and sub-clinical disease measurement. Fusion type will be discussed to demonstrate the importance of prospective evaluation and validation of biomarkers in the context of evolving therapy. The remaining categories were selected for in depth discussion after consideration of REMARK criteria. In the following sections we will highlight the features of each of these putative biomarkers that lead us to propose that their parallel evaluation and validation in the next series of prospective therapeutic trials is warranted. Several of these, including CNAs and cell-cycle proteins were recently discussed at a European Network for Cancer Research in Children and Adolescents (ENCCA) summit of 35 international experts and RB1. Kovar et al. (CDKN2A deletions in 30% of tumors (N\u2009=\u20098/27) and 52% of ES cell lines (N\u2009=\u200912/23) and several retrospective studies have demonstrated an association between CDKN2A alterations and clinical outcome in ES patients. Wei et al. , while Tsuchiya et al. (CDKN2A deletions in 17% of tumor samples (N\u2009=\u20094/24). Patients in both studies were found to have worse disease-specific survival in univariate and multivariate analyses. Maitra et al. (CDKN2A downregulation by immunohistochemistry in 20% of patients (N\u2009=\u20094/20), and this correlated with metastatic disease at presentation and trended toward shortened survival. A meta-analysis examining the prognostic significance of CDKN2A alterations in ES based on six separate studies (N\u2009=\u2009188) concluded that the estimated pooled risk ratio (RR) for worse outcome with CDKN2A alterations was 2.17 and the estimated pooled RR of metastasis at diagnosis was 2.60 and 10% of primary tumors (N\u2009=\u20094/42) and this over-expression was associated with advanced disease at diagnosis, poorer treatment response, and a worse overall survival. Significantly, this effect was independent of site, local treatment, or tumor necrosis. Similarly, a study by de Alava et al. (N\u2009=\u20096/55) and increased p53 protein expression was found to be the strongest prognostic factor that was associated with worse overall survival. Huang et al. (TP53 mutations in 13.3% of patient samples (N\u2009=\u20098/60), as well as CDKN2A homozygous deletions in another 13.3% of samples (N\u2009=\u20098/60). TP53 mutations and/or CDKN2A deletions were significantly associated with a poor response to chemotherapy (P\u2009<\u20090.0001) and, in a multivariate analysis, TP53 and/or CDKN2A alteration status as a single combined variable was identified as the most significant prognostic factor (P\u2009<\u20090.001). Finally, using immunohistochemistry and fluorescent in situ hybridization (FISH), Lopez-Guerrero et al. (P\u2009=\u20090.025), and worse progression-free survival (P\u2009=\u20090.012) and disease-specific survival (P\u2009=\u20090.006) in patients with localized disease.The potential of u et al. detecteda et al. identifig et al. reportedo et al. analyzedTP53 and CDKN2A as negative prognostic biomarkers in ES. Currently, COG and the ES Biology Committee are performing a large-scale analysis of TP53 and CDKN2A status in over 150 prospectively collected tumors from patients treated on the most recent AEWS0031 therapeutic study. Should this study confirm prior observations, analysis of these cell-cycle regulatory proteins will become a strong candidate for inclusion as a prognostic biomarker that can inform treatment decisions in future clinical trials.In summary, compelling data from several retrospective studies implicates alterations of Genomic instability with subsequent CNAs have been well-documented in ES and these alterations have been recently reviewed by Jahromi et al. . The recIn summary, independent studies of both small and large tumor cohorts have identified individual and global patterns of CNAs as putative prognostic biomarkers in ES. We anticipate that the continued improvement in next generation sequencing platforms will allow for greater characterization of structural variations in tumors, and will generate even more data to test associations between CNAs and clinical outcome. It is the recommendation of this committee that tumor and germline DNA be collected from all patients registered on future therapeutic studies of ES in order that CNAs and other genetic mutations can be evaluated as prognostic and predictive biomarkers in homogeneously treated patients. To that end, COG has discussed the prospective incorporation of CNA and genomic analysis in their upcoming ES trial for relapsed/refractory patients.Assessment of minimal residual disease (MRD) has been established as a critical part of therapeutic decision making in childhood acute lymphoblastic leukemia protocols and therefore received similar therapy , and 19% of patients (N\u2009=\u200918/92) with non-metastatic disease at presentation. Circulating transcripts were identified in 20% of patients (N\u2009=\u200929/144) at diagnosis, and were more frequently observed in patients with large tumor burdens. In patients with localized disease, RT-PCR positivity in bone marrow and peripheral blood correlated with significantly poorer outcomes. In contrast, a study of peripheral blood samples from 26 children was unable to identify a significant progression-free survival difference in patients with detectable fusion transcript at diagnosis with localized disease, 50% of patients (N\u2009=\u20093/6) with isolated pulmonary metastases, and 100% of patients (N\u2009=\u20096/6) with bone metastases. However, the study did not establish a correlation between marrow positivity for ES transcript and progression-free disease. Results of other smaller studies have been recently summarized by Wagner et al. for molecular diagnosis, isolation of quality RNA has become less practical. Feasibility will only diminish as additional rare non-EWSR1 translocations are identified.To rigorously address whether the detection of circulating tumor transcript is of prognostic significance, the multi-center European EURO-E.W.I.N.G. 99 trial prospectively collected bone marrow samples for over 10\u2009years. As the first large prospective trial examining sub-clinical disease via RT-PCR in ES patients, the findings of this study will be critical to evaluate the feasibility and usefulness of this modality as a biomarker for ES. Based on our own experience with a much smaller cohort of patients in COG we, as a committee, are skeptical that RT-PCR-based assays will be clinically optimal for prognostication and treatment stratification. We base this assertion on our combined observations regarding issues of technical reproducibility of the assay between individual laboratories, and the technical expertise required to consistently obtain sufficient quality RNA for valid and reliable RT-PCR analysis. Although these issues could be addressed with the establishment of a central College of American Pathologists (CAP)-Clinical Laboratory Improvement Act (CLIA)-certified reference laboratory, the issue of RNA degradation in sample shipments would remain. In addition, RT-PCR-based analysis requires knowledge of the precise breakpoint. With the increasing use at many COG institutions of closed needle biopsy for diagnostic tissue collection and fluorescence In summary, although of potential prognostic significance, technical and logistic realities regarding tissue collection and RNA-based studies of blood and bone marrow specimens significantly diminish this committee\u2019s enthusiasm for RT-PCR analysis of sub-clinical disease in routine clinical practice. Should the aforementioned Euro-Ewing study validate RT-PCR of bone marrow as a significant prognostic variable, this issue will need to be re-addressed. At such time, consideration would need to be given to optimizing collection and submission of quality RNA and to creation of a CAP-CLIA certified COG reference laboratory.5 peripheral blood mononuclear cells. Ash et al. -directed therapy. Activating mutations in the EGFR gene are detectable in only a small minority of NSCLC patients but it is these patients who selectively respond to EGFR-directed therapy patients who will respond to Epidermal Growth Factor Receptor (in vitro and in vivo models of ES are highly sensitive to the PARP1 inhibitor Olaparib alone and in combination with the drug temozolomide. Moreover, in a drug screening of several hundred cancer cell lines a marked and selective susceptibility of ES cell lines to Olaparib was also discovered (Garnett et al., The findings that only a small subset of patients with relapsed ES respond to IGF-1R targeted monotherapy serve as a sobering example of the critical need for predictive biomarkers in this disease. As trials investigating novel agents move forward, it is paramount that strategies that will permit evaluation of predictive biomarkers be simultaneously implemented. This will enable identification of patients who may preferentially benefit from such interventions in the future and allow for more selective inclusion and exclusion of patients in a manner that will lead to improved response rates. One potential treatment modality to emerge from recent pre-clinical investigations is PARP1 inhibition. PARP1 is a key enzyme involved in single-strand repair of DNA (Wang et al., Numerous prognostic biomarker studies for ES have been published in recent years. Of particular interest and potentially high clinical relevance are studies of cell-cycle proteins, sub-clinical disease, and CNAs. All of these have demonstrated prognostic significance in numerous retrospective studies and now need to be validated prospectively in larger cohorts of equivalently treated patients. The challenges in identifying and validating clinically relevant biomarkers in ES highlight a significant hurdle for the individualization of therapy in any rare cancer. Prospective therapeutic trials with standardized treatments remain the optimum source of biologic material and clinical correlative information to drive successful biomarker identification. Since these trials can take years to complete it is essential that biomarker studies be meticulously designed and incorporated up front in therapeutic studies. It is imperative that these studies are designed vigilantly to maximize levels of evidence and ensure adherence to REMARK guidelines. In addition, biomarkers that can be tested and validated on blood or fixed tumor specimens will have the best chance of translation into routine clinical practice. As new agents are developed, predictive biomarkers will need to be developed to assess the benefit of these therapies and rationally design treatment stratification based on likelihood of response. The choice of technical platforms must also be carefully considered in trials involving rare diseases. Although characteristics such as sensitivity are important when choosing a methodology, issues such as availability, cost-effectiveness, and sample requirements are equally important. Rare cancers require the participation of multiple institutions, and it is imperative that samples from each site are similarly collected and processed. Cooperative groups can play a critical role to ensure that biomarker studies are carefully selected, rigorously designed and, whenever possible, incorporated into therapeutic studies.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Graft versus host disease (GVHD) is the major complication of allogeneic hematopoietic stem cell transplantation. GVHD is characterized by an imbalance between the effector and regulatory arms of the immune system which results in the over production of inflammatory cytokines. Moreover, there is a persistent reduction in the number of regulatory T (Treg) cells which limits the ability of the immune system to re-calibrate this proinflammatory environment. Treg cells are comprised of both natural and induced populations which have unique ontological and developmental characteristics that impact how they function within the context of immune regulation. In this review, we summarize pre-clinical data derived from experimental murine models that have examined the role of both natural and induced Treg cells in the biology of GVHD. We also review the clinical studies which have begun to employ Treg cells as a form of adoptive cellular therapy for the prevention of GVHD in human transplant recipients. Although hematopoietic stem cell transplantation (HSCT) has been a successful therapeutic strategy for treating hematological malignancies for several decades, its broad application is limited by the high incidence of graft versus host disease (GVHD). GVHD is primarily a donor T cell-mediated syndrome whereby T cells in the graft elicit an immune response, resulting in host tissue damage . These cells, termed Treg cells, express the forkhead box transcription factor Foxp3, which is both necessary and sufficient for the suppressive ability of Treg cells cells comprise 5\u201310% of the CD4+ T cell compartment and develop in the thymus which are the sites of GVHD-associated tissue damage which is a ligand for the CD1d molecule has been shown to expand donor-derived Treg cells in a dose-dependent manner and reduce GVHD-associated mortality (Duramad et al., in vivo expansion of nTreg cells and in vivo conversion and/or expansion of iTreg cells. Thus, it is difficult to exclude that these approaches may also result in the expansion of iTreg cell populations as well.Pharmacological strategies have also been tested in murine GVHD models to determine whether Treg cell numbers can be augmented after allogeneic HSCT. To that end, Shin et al. demonstrde novo iTreg cell generation in recipient mice is negligible during GVHD (Chen et al., in vitro induction/expansion of this population followed by adoptive transfer into recipient animals. In initial studies, iTreg cells were stimulated with allogeneic dendritic cells or treatment with anti-CD3/anti-CD28 antibodies in the presence of TGF-\u03b2 and IL-2 to induce Foxp3 expression. Administration of in vitro-differentiated iTreg cells along with BM grafts containing alloreactive donor T cells did not result in any significant protection from lethal aGVHD (Koenecke et al., in vivo survival of these cells which was accompanied by instability of Foxp3 expression, resulting in a loss of suppressive function early post transplantation (Koenecke et al., While the majority of rodent models of GVHD have focused on the biology of nTreg cells, there has been much less attention devoted to the role of iTreg cells in GVHD biology. This has been due, in part, to the fact that there are no proven cell surface markers that distinguish nTreg cells from iTreg cells. Consequently, isolation of a pure iTreg cell population from donor animals for selective adoptive transfer studies is not currently feasible. Furthermore, in vivo is not clear, but one potential explanation is that the proinflammatory cytokine milieu that occurs during GVHD may also render iTreg cells more unstable. Supporting this premise are data demonstrating that in vivo-derived iTreg cell conversion is significantly enhanced when mice are treated with monoclonal antibodies that block signaling through IL-6 or IL-21 which serves to reduce inflammatory cytokine production (Bucher et al., in vivo, an alternative approach has involved the culture of CD4+CD25\u2212 T cells with the hypomethylating agent 5-azacytidine. Choi et al. (+ T cells both in vitro and in vivo, and that transplantation of these cells ameliorated GVHD severity.The reason that iTreg cells are unstable i et al. reported+ Treg cell populations.We would note that instability of Foxp3 expression has also been noted to occur in nTreg cells in non-transplant models (Zhou et al., + Treg cells are classically defined as being a subset of the CD4+ T cell compartment. However, a CD8+ Foxp3+ Treg population has been described and found to be capable of suppressing T cell responses in animal models of autoimmunity and allergen exposure (Hahn et al., + Foxp3+ T cells have also been documented in the tumor microenvironment of patients with colon and prostate cancer, suggesting that they may be a mechanism by which tumors escape immune surveillance (Kiniwa et al., + Foxp3+ iTreg cells are induced early during GVHD (Beres et al., + counterparts, these cells were found to be dependent on TGF-\u03b2 and IL-2 for induction (Sawamukai et al., + or CD8+ iTreg cell population was required to prevent increased GVHD-associated mortality (Beres et al., + iTreg cells could be expanded in GVHD recipients using IL-2 antibody complexes in conjunction with Rapamycin as has been previously described with CD4+ Treg cells (Shin et al., + Foxp3+ T cells have been induced in vitro and found to suppress GVHD in a humanized mouse model (Zheng et al., in vitro or in vivo methodological approaches for translational application.Foxp3+ Treg cells to CD8+ T cells was significantly decreased at the mucosal interface of GVHD patients as compared to patients with intestinal inflammation unrelated to GVHD (Rieger et al., The approach that has been employed to address whether Treg cells may serve to modulate the severity of GVHD in man has been to correlate the absolute number and/or frequency of Tregs with the subsequent incidence and severity of aGVHD and cGVHD. Several reports have demonstrated a decreased frequency of Treg cells in the peripheral blood of patients with high clinical grades of aGVHD as compared to patients with lower grade aGVHD or no GVHD (Li et al., +CD25hi Treg cells as compared to individuals without GVHD. This was supported by a more recent study that reported increased peripheral Treg cell numbers in transplant recipients that developed cGVHD with no prior aGVHD diagnosis (Ukena et al., It is important to note, however, that not all studies have demonstrated a correlation between reduced Treg frequency and GVHD severity. Clark et al. observedThe reason for the differences observed in these studies is not entirely clear. For the most part, however, studies that have failed to demonstrate that a reduction in Treg cell frequency and/or absolute numbers is associated with increased GVHD severity have relied on CD25 expression to delineate Treg cell populations, whereas those that have reported a positive correlation have tended to employ Foxp3 expression as a readout for this Treg cell population. Thus, it is possible that the reliance on different phenotypic markers may result in somewhat different populations being examined and be a potential explanation for these discordant results.+Foxp3+ Treg cells in the peripheral blood of the donor negatively correlated with the incidence of GVHD in the graft recipient. Several subsequent studies confirmed this correlation in recipients of HLA-identical sibling and unrelated donor stem cell grafts (Pabst et al., +CD25+CD127lo Treg cells as compared to bone marrow grafts, the frequency of peripheral blood Treg cells is reversed post transplantation. This was presumed to be due to the fact that PBSC Treg cells tended to be CD62Llo as a consequence of both granulocyte colony stimulating factor treatment for mobilization and the subsequent leukapheresis process (Blache et al., + Treg cells in the graft have been found to correlate with reduced GVHD incidence (Lu et al., + Treg cell population is more potent at suppressing GVHD than the corresponding CD62Llo population (Taylor et al., An alternative approach to examine the effect of Treg cells on GVHD severity in human allogeneic HSCT has been to assess the number of donor-derived Treg cells within the graft prior to transplantation. In this regard, Rezvani et al. determin+CD25hi CD127\u2212 or CD4+CD25hiICOS+ Treg populations were likely to be most suitable for human adoptive transfer studies. Many groups have also identified in vitro expansion protocols that yield high number of Treg cells for adoptive transfer (Karakhanova et al., ex vivo-expanded cells also ameliorated disease in a xenograft model of GVHD (Chakraborty et al., Less than two decades after their discovery, Treg cells are now entering into clinical trials in allogeneic HSCT recipients. Pre-clinical murine models of GVHD have provided much insight into Treg cell-based therapy, but most mouse studies have been performed using Foxp3-GFP reporter mice where Foxp3-expressing Treg cells can be definitively isolated for adoptive transfer studies. This is not a luxury that is available in human studies where CD25 expression necessarily serves as a surrogate for Foxp3. However, since CD25 is upregulated on all activated T cells, further phenotypic characterization of these cells has been generally thought to be necessary for their use in man. To that end, Ukena et al. comparedOne of the first reported clinical studies was conducted by Brunstein et al. who performed a phase I clinical prophylaxis trial with cord blood-derived Treg cells. The rationale for the use of cord blood-derived Treg cells was based, in part, on earlier studies, that had shown that they express similar levels of CTLA-4, Foxp3, GITR, and CD25 as adult peripheral blood Treg cells, and when stimulated by alloantigen, were potent suppressors of T cell expansion (Chang et al., A second clinical trial was performed by Di Ianni et al. , in whicThere has been one small study involving two patients in which expanded Treg cells were administered to patients with documented GVHD (Trzonkowski et al., + donor leukocyte infusions, used to treat relapsed hematologic malignancies post HSCT, resulted in Treg cell expansion in vivo. This same group then utilized this strategy to treat glucocorticoid refractory cGVHD patients. The administration of low dose IL-2 was associated with an amelioration of disease severity and this correlated with an increase in the number of Treg cells (Koreth et al., in vivo expansion of Treg cells may be a more clinically feasible strategy to enhance Treg reconstitution post transplantation, as compared to more costly expansion strategies.An alternative approach to harness the potential for Treg cell therapy in humans is based on the requirement of these cells for IL-2. Specifically, Zorn et al. found thCollectively, these studies are exciting evidence that Treg cell therapy has now entered into the clinic. Going forward, well-designed trials will be necessary to determine whether these cells are indeed capable of preventing and/or treating patients with established GVHD.+ nTreg cells, CD4+ iTreg cells, and CD8+ iTreg cells in GVHD biology remain unclear. Elucidating the mechanisms by which the respective cell subsets function may provide insight for developing better therapeutic strategies. Furthermore, additional studies are required to ascertain whether CD4+ and CD8+ Treg cell populations function cooperatively or whether they have overlapping redundant roles in GVHD biology.Despite the significant progress that has been made in understanding the role of Treg cells in GVHD biology, a number of questions remain. First of all, the relative roles of CD4in vitro, have unstable Foxp3 expression. Since Foxp3 expression is necessary for suppressive function, further inquiry is needed to determine whether Foxp3 expression can be stabilized especially under pro inflammatory conditions which characterizes the GVHD milieu (Koenecke et al., Secondly, accumulating evidence indicates that Treg populations, particularly those that are expanded ex vivo expansion remain unresolved. Moreover, it is possible that alloantigen-specific Treg cells may be more potent in suppressing GVHD, and should be studied further (Albert et al., Finally, current Treg cell-based immunotherapy approaches rely on the expansion of polyclonal populations of Treg cells. The best source of Treg cells and the optimal culture conditions for The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Neuropeptide W (NPW), which was first isolated from the porcine hypothalamus, exists in two forms, consisting of 23 (NPW23) or 30 (NPW30) amino acids. These neuropeptides bind to one of two NPW receptors, either NPBWR1 (otherwise known as GPR7) or NPBWR2 (GPR8), which belong to the G protein-coupled receptor family. GPR7 is expressed in the brain and peripheral organs of both humans and rodents, whereas GPR8 is not found in rodents. GPR7 mRNA in rodents is widely expressed in several hypothalamic regions, including the paraventricular, supraoptic, ventromedial, dorsomedial, suprachiasmatic, and arcuate nuclei. These observations suggest that GPR7 plays a crucial role in the modulation of neuroendocrine function. The intracerebroventricular infusion of NPW has been shown to suppress food intake and body weight and to increase both heat production and body temperature, suggesting that NPW functions as an endogenous catabolic signaling molecule. Here we summarize our current understanding of the distribution and function of NPW in the brain. In 1995, O'Dowd et al. . In 2002in situ hybridization histochemistry has revealed that NPW mRNA is expressed in a few restricted brain regions, including the rat periaqueductal gray (PAG), Edinger-Westphal nucleus (EW), and dorsal raphe nucleus , PAG, superficial gray layer of the superior colliculus, and subfornical organ. In general, NPBWR1 is most commonly expressed at high levels in the amygdala (Singh et al., In humans, RT-PCR analysis has demonstrated that NPBWR1 mRNA is highly expressed in the amygdala, hippocampus, neocortex, and hypothalamus (Lee et al., i et al. reportedi et al. used [12NPBWR1 knockout mice are hyperphagic and show decreased energy expenditure, suggesting that NPW may act as a modulator of feeding. icv infusion of NPW in male rats has been shown to increase food intake during the first 2 h in the light phase (Shimomura et al., ob/ob and db/db mice. Therefore, NPW may play important roles in feeding and energy metabolism, functioning as a substitute for leptin (Date et al., We have carried out a series of neuroanatomical studies to examine the neural relationship between NPW and other neuropeptides involved in the regulation of feeding. Very close neuronal interactions were observed between NPW-containing nerve fibers and orexin- or melanin-concentrating hormone-containing neuronal cell bodies and nerve fibers in the rat brain (Takenoya et al., 2 consumption and increased CO2 production, as well as an increase in body temperature (Mondal et al., Very recently, Skrzypski et al. have demImmunohistochemical studies have reported that NPBWR1 is expressed in the PVN, pituitary gland, and adrenal medulla in the human, mouse and rat (O'Dowd et al., in vitro study has reported that log molar NPW23 concentrations, significantly alter prolactin, growth hormone and ACTH release from dispersed rat anterior pituitary cells (Baker et al., in vivo studies have revealed that icv infusion of NPW23 stimulates an increase in plasma prolactin levels (Baker et al., On the other hand, icv infusion of NPW23 stimulates prolactin release in the rat (Shimomura et al., Niimi and Murao have repThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "In western industrialized nations approximately 25% of all deaths are caused directly or indirectly by the consumption of psychotropic substances. Substance-related addictions therefore constitute the most frequently occurring psychiatric disease category (McGinnis and Foege, In their recently published manuscript Pascoli et al. elucidatSince the first description of a patient, whose alcohol addictive behavior was positively influenced by high-frequency accumbal DBS (Kuhn et al., Although the mechanisms of deep brain stimulation are, despite its decade-long application, not yet fully understood it has been argued that the modulation of dopaminergic transmission \u2013 especially in the context of addictive disorders \u2013 underlies the therapeutic effect. While this assumption could only be demonstrated for the rat nucleus accumbens (Sesia et al., The influence of impaired dopaminergic functioning on cognition and neuronal plasticity has been much debated. It is assumed that an activation of accumbal D1 receptors by phasic dopamine release facilitates limbic afferents and promotes cortical synaptic plasticity. At the same time, D2 receptor activation might promote the reinforcement of prefrontal influences, which is especially relevant in the context of addiction given the reduced density of both striatal and accumbal D2 receptors in addicted patients (Lee et al., The results of Pascoli et al. help expThe short-term application of psychotropic substances, in this case cocaine, apparently triggers a fast modification of D1-receptor expression in terms of focal synaptic plasticity and could, hence, bring forth a lasting shift in the D1/D2 ratio, which eventually might lead to a fortification of limbic and drug-associated afferents.Current pharmacologic approaches to ameliorate the dysregulated dopaminergic system are not sufficiently effective (Spanagel and Vengeline,"} +{"text": "Cells with damaged and unrepaired genomes represent a potential threat for the organism and therefore their elimination is beneficial. The biological safeguard barriers responsible for elimination of such hazardous cells rely on two principles: intrinsic - achieved through cellular senescence/apoptosis, and extrinsic - performed by the immune system. The concept of immune system-mediated clearance of senescent cells is well established , howeverResearch performed in the last two decades shows that cellular senescence as an essentially irreversible block of cell proliferation can be triggered or bypassed via manipulation of expression levels of several dozens of genes indicating the complexity and redundancy of regulatory machineries controlling this antitumor barrier. While the regulatory circuits are not understood in much detail, the unifying feature of cellular senescence is the activation of cell cycle checkpoints that block cell-cycle progression at G1/S or G2/M boundary. Checkpoint activation reflects suprathreshold long-term expression, commonly triggered by persistent DNA damage response (DDR) signaling, of protein inhibitors of cyclin-dependent kinases (CDKs), the key drivers of cell-cycle progression. The multiple pathways leading to the induction of individual inhibitors of CDKs (CDKi) form the basis for redundancy of mechanisms for induction and maintenance of senescence. The robustness of response, manifested as the possibility to impose senescence even in tumor cells lacking two pivotal senescence mediators p53 and Rb, stems from the fact that the regulatory circuits are interconnected by numerous cross-talks of signaling pathways and several feedback mechanisms [et al. reported recently [per se . Production of ROS in cells during inflammation contributes to aging and development of age-related diseases. Nox4 is a member of the NADPH oxidase family known to regulate production of ROS, especially superoxide forms to induce DNA damage and premature senescence. Weyemi et al. showed that knock-down of NOX4 decreased RAS-induced DDR [et al. reported that Ras-induced senescence is mediated via Nox1 and Nox4, and overexpression of both genes is sufficient to induce senescence via activated DDR [Cytokines secreted by senescent cells play here the important role not only in shaping the senescent phenotype by autocrine and paracrine signaling and reinforcing the cell-cycle block by secondary induction of diverse CDKi, but also, as Hubackova recently , by caus se Fig. . In thisated DDR .et al. did not explore the presence of cytokine-induced secondary bystander senescence in vivo, this can be anticipated based on the recent study of Braumuller et al. [et al. show that T-helper-1 cell cytokines TNF\u03b1 and IFN\u03b3 cooperatively induce senescence in mouse beta-cell tumors, both in vivo and in vitro, indicating a reciprocal relationship between the immune system and cellular senescence. Importantly, the concerted action of these two T-cell produced cytokines induced senescence in cancer cells indicating the role of immune surveillance in control of tumor cell proliferation by cytokine-induced senescence. Although not assessed in their study, it can be predicted based on previous reports that the observed senescence-inducing effect in response to both IFN\u03b3 and TNF\u03b1 is also triggered by DNA damage. TNF\u03b1 and IFN\u03b3 were also found to increase both intracellular and extracellular ROS production. Importantly, binding of NF\u03baB, a crucial mediator of cytokine effects, on NOX4 promoter was observed [Even though Hubackova r et al. . The cytobserved . Thus, NIt is becoming widely accepted that secretome of senescent cells can also modulate the microenvironment, in both normal or tumor tissues. However, due to the overall complexity and variability of the secreted cytokine species, dependent on specific cell types, (patho)physiological context and senescence-inducing stimulus, it is currently hard to predict all outcomes of senescence-associated cytokine signals on tissue homeostasis. Nevertheless, what can be conceived now, is that the genotoxic effects of several cytokine species produced by senescent cells can spread damage in tissues manifested as secondary (and tertiary) senescence and thus to contribute to aging and pathogenesis of aging-associated diseases. Therefore, the elimination of senescent cells from the organism may provide a rejuvenation effect, as has already been documented in progeroid mice ."} +{"text": "IFT122, and demonstrated impaired ciliogenesis in patient fibroblasts. This report on IFT122 broadens the phenotype of CED and expands its allelic heterogeneity.Cranioectodermal dysplasia (CED) is a very rare autosomal recessive disorder characterized by a recognizable craniofacial profile in addition to ectodermal manifestations involving the skin, hair, and teeth. Four genes are known to be mutated in this disorder, all involved in the ciliary intraflagellar transport confirming that CED is a ciliopathy. In a multiplex consanguineous family with typical CED features in addition to intellectual disability and severe cutis laxa, we used autozygosity-guided candidate gene analysis to identify a novel homozygous mutation in IFT122,WDR35,C14ORF179, and WDR19) is a skeletal dysplasia characterized by typical craniofacial features in the form of dolichocephaly, sagittal craniosynostosis, and facial dysmorphism , and skeletal anomalies in the form of narrow thorax and short extremities, in addition to ectodermal dysplastic features in the form of thin sparse scalp hair and micro/hypodontia and was absent from 374 Saudi control chromosomes.In this report, we describe a multiplex family containing three affected siblings born to healthy first cousin Saudi parents Fig.\u2003A. In addIFT122 as a novel CED disease gene (Walczak-Sztulpa et\u2003al. IFT122 in CED (Tsurusaki et\u2003al. In order to confirm the pathogenicity of this mutation, we cultured fibroblasts from the foreskin of the younger brother following circumcision and proceeded with stress-induced ciliogenesis assay, essentially as described before (Shaheen et\u2003al."} +{"text": "Recent progress in the field of epigenetics has provided a new study angle for our research efforts on reproductive medicine and gynecologic malignancies. We have acquired valuable insight into the regulatory mechanism and biological effects of DNA methylation and histone modification, the two major epigenetic pathways. The newly acquired knowledge effectively complements that gained from the genetic standpoint and holds great potential for the prevention, diagnosis, risk assessment, and treatment of these diseases. Specifically, the DNA methylation and imprinting mechanisms are implicated in fertilization, early embryonic development, placental function, and pathogenesis of preeclampsia and intrauterine growth retardation. Aberrant DNA methylation and chromatin modification lead to gene-specific silencing of numerous tumor suppressor genes, DNA repair genes, and steroid hormone receptors. This special issue presents a collection of peer-reviewed papers focusing on these areas. While the issue is not intended as an exhaustive representation of all of the potential topics, they nevertheless provide insightful and multifaceted information that we consider a pleasure to share with the readers.This special issue includes 9 articles: three of which are related to the IGF-II imprinting in placenta and the effects of reproduction procedures on imprinting; two describe epigenetic mechanisms and genetic test for infertility; another paper documents the effects of in vitro maturation on histone acetylation in oocytes and early cleavage embryos; two address DNA methylation changes in cancers; one paper discusses rational design of primer for methylation assays.Effects of in vitro maturation on histone acetylation in metaphase II oocytes and early cleavage embryos,\u201d Wang et al. document a reduced expression of histone acetyltransferase GCN5 (GCN5) and histone deacetylase 1 (HDAC1) in two-cell embryos but a normal level of these enzymes after the two-cell stage. The results indicate that in vitro maturation could affect protein and gene expression related to histone acetylation in oocytes and early cleavage embryos. However, by function of selection, parts of the changes could be recovered in late embryo development. In the first paper entitled \u201cImprinting and promoter usage of insulin-like growth factor II in twin discordant placenta,\u201d Luo et al. analyze the imprinting and promoter usage of IGF-II in placenta of normal twins and twins with weight or phenotype discordance and conclude that promoter 3 specific LOI of the IGF-II gene may be closely related to phenotype discordance, but not to weight discordance. In the second paper entitled \u201cOxidative stress and DNA methylation in prostate cancer,\u201d Donkena et al. present a comprehensive review on the effects of oxidative stress on DNA methylation and cancer progression, life style and diet as factors involved in ontogenesis and epigenetic interference for cancer prevention, and DNA methylation as a biomarker for cancer detection. Updates on the application of DNMT inhibitors to chemotherapy are also provided. In the third paper entitled \u201cPreimplantation genetic screening: an effective testing for infertile and repeated miscarriage patients?,\u201d Wang et al. compare results from different laboratories on preimplantation screening of aneuploidy and assess the efficacy, risks, and benefits of the procedure. They conclude that the use of preimplantation genetic screening should be reconsidered.In the fourth paper entitled \u201cStudy on the imprinting status of insulin-like growth factor II (IGF-II) gene in villus during 6\u201310 gestational weeks,\u201d Chen et al. compared the rate of loss of GF-II imprinting in the placental villous tissues between normal and abnormal embryo development and observed a significantly increased loss of imprinting in the abnormal group, suggesting that the imprinting status of IGF-II may be functionally related to embryo development.In the fifth paper entitled \u201cEffects of assisted reproduction technology on placental imprinted gene expression,\u201d Katagiri et al. investigate the impact of assisted reproduction techniques (ART) on imprinted gene expression in human placenta. Different changes in the mRNA levels of imprinted genes are observed in the ART group compared with the spontaneous conception group, suggesting that ART may modify epigenetic status.In the sixth paper entitled \u201cSpecificity of methylation assays in cancer research: a guideline for designing primers and probes,\u201d Barekati et al. discuss the critical parameters to be considered for a rational design of PCR primers used for the detection of methylated DNA. The authors also provided applicable tools/algorithms and useful websites.In the seventh paper entitled \u201cEpigenetic regulatory mechanisms associated with infertility,\u201d Minocherhomji et al. review the epigenetic mechanisms involved in spermatogenesis and infertility. Topics discussed in detail include the regulation and potential role of epigenetics in infertility by high-order chromatin organization, epigenetic control of genes associated with pericentromeric regions of chromosome 9 and Y, and noncoding RNAs. In the eighth paper entitled \u201cIn the ninth paper entitled \u201cHypermethylation of SOX2 promoter in endometrial carcinogenesis\u201d, Wong et al. report their studies on the methylation profiles of SOX2, a gene encoding the stem cell-related transcription factor SOX2 in endometrial carcinomas. Compared to normal control tissues, cancer tissues show hypermethylation and decreased expression of SOX2. The authors conclude that epigenetic silencing mechanisms may play a crucial role in transcriptional regulation of SOX2 and loss of SOX2 expression.Shi-Wen JiangShi-Wen JiangBrian BrostBrian BrostSean DowdySean DowdyXing XieXing XieFan JinFan Jin"} +{"text": "Physiotherapists play an inherent role in the multidisciplinary palliative care team. Existing knowledge, attitudes, beliefs and experiences influence their team participation in palliative care.The objective of this study was to assess the changes in knowledge, attitudes, beliefs and experiences among student physiotherapists who attended a palliative care training program.Preliminary quasi-experimental study design, conducted at an academic institution.Fifty-two student physiotherapists of either gender of age (20.51\u00b11.78 years) who attended a palliative care training program which comprised lectures and case examples of six-hours duration participated in this study. The study was performed after getting institutional approval and obtaining participants\u2019 written informed consent. The lecture content comprised WHO definition of palliative care, spiritual aspects of life, death and healing, principles, levels and models of palliative care, and role of physiotherapists in a palliative care team. The physical therapy in palliative care-knowledge, attitudes, beliefs and experiences scale - modified from palliative care attitudes scale were used for assessing the participants before and after the program.t-test and Wilcoxon signed rank test at 95% confidence interval using SPSS 11.5 for Windows.Paired P<0.05) were noted for all four subscales- knowledge (7.84\u00b14.61 points), attitudes (9.46\u00b18.06 points), beliefs (4.88\u00b13.29 points) and experiences (15.8\u00b111.28 points) out of a total score of 104 points.Statistically significant differences (The focus-group training program produced a significant positive change about palliative care in knowledge, attitudes, beliefs and experiences among student physiotherapists. However, communication and coordination within the palliative care team has to be improved to minimize the negative impact of symptom distress on patient well-being and quality of life. Also, physical therapists must develop strategies for patient empowerment and methods for assessing and evaluating qualitative aspects of physical therapy in palliative cancer care.et al.,[According to Glazer-Waldman et al., the attiet al., if patieet al.[et al.[Previous studies that evaluated effects of training in palliative care utilized the educational program as part of a curriculum development among other healthcare professionals like critical care medicine trainees, medical et al. which wal.[et al.et al.[Effects of education and training programs on knowledge, attitudes, beliefs and practice were studied by other authors in the fields of nursing and child health.14 In phyet al. studied et al. who founExisting knowledge, attitudes, beliefs and experiences influence their team participation in palliative care. The objective of this study has been to assess if any change that occurred in knowledge, attitudes, beliefs and experiences among physical therapy students who attended a 6-hour educational training program on palliative care.The study ethical conduct was approved by the Institution and written informed consent was obtained from all participants prior to commencement of the study.Study design: This study used a pre-post quasi-experimental design, in which data were collected from a convenience sample of physical therapy students who attended a formal training program on palliative care and role of physical therapy.Participants: Final year physical therapy students of the institution attended upon free registration. The physical therapy curriculum had four years of academic training followed by six months of internship in undergraduate degree program. The students had previous experience of evaluating and treating pain in their third and early fourth year of academics during their clinical postings in multispeciality government hospital focused in treatment of cancer patients. Participants were given an initial description and instructions on filling the questionnaires by a qualified physical therapist.Training program: The training program was an elective educational intervention which comprised a lecture, case examples and active demonstration which consisted of WHO definition of palliative care, spiritual aspects of life, death and healing, principles, levels and models of palliative care, and role of physiotherapists in a palliative care team- for a total duration of six hours, where direct contact training of physiotherapists in palliative care emphasizing on introduction to palliative care, principles and models of assessment and care of persons at hospice, supportive, palliative, end-of-life and bereavement perspectives, goal planning and strategic implementation of physical therapy treatment methods in palliative care was done. Interaction duration of 15 min was allowed both between participants and also with the invited speaker (first author), who was a qualified physical therapist, experienced in pain relief methods for seven years.The training program required free registration of the participants and was conducted annually on a not-for-profit basis by the institution. The training program is one of its kinds listed in International Association for Hospice and Palliative Care website.et al.[Outcome measurement: The data was collected pre and post-training through a self-administered questionnaire- physical therapy in palliative care-knowledge, attitudes, beliefs and experiences scale . The scale is a 37-item self-report measure consisting of both qualitative and quantitative data. The full version of the questionnaire is given in et al. on critiThe PTiPC-KABE scale was was pilot-tested for test-retest reliability and was then used for assessing the participants before and after the program as a primary outcome measure. To avoid bias and to maintain participant anonymity, coding and decoding during data mining and analysis was done by another independent blinded physical therapist.Evaluation of test-retest reliability of PTiPC-KABE scale: For this purpose, we randomly selected 24 physical therapy students by Lots method from the same sample and we administered the questionnaire twice with a time-gap of 1 h, 2 h before the commencement of the actual study.Participants who could not understand English, who refused consent or who returned incomplete questionnaires were excluded from analysis. Completed questionnaires were collected by another qualified physical therapist, who was blinded to the study. The data were initially mined and entered for analysis by another blinded physical therapist.t-test and Wilcoxon signed rank test. Test for normality was done using Kolmogorov-Smirnof test. All analyses were done using SPSS 11.5 for windows at 95% confidence interval.Evaluation of test-retest reliability was done using intra-class correlation coefficient (ICC) for the total scores of two trials of the PTiPC-KABE scale. A mixed-methods model of primary analysis was done to compare pre-post scores using paired Of the total 82 physical therapy students- who attended the training program, 76 provided consent and were willing to participate in this study. 24 filled for the initial test-retest reliability of the scale (ICC=.80-.90) for the total score and the rest 52 were considered for this pre-post study. A total of 52 participants- 12 male (23.1%) and 40 female (76.9%) with age 20.51\u00b11.78 years- filled the questionnaire twice- before the commencement of training program and after the completion of the training program.P<.05) changes post-program was observed for all items of the PTiPC-KABE scale after we adjusted for acquiescence bias of Likert scaling. We combined the moderately agree and strongly agree into \u201cagree\u201d and same for disagree option. No response was given a score of \u201czero\u201d, disagree was given \u201cone\u201d, neutral/ unsure was given \u201ctwo\u201d and agree was given \u201cthree\u201d. Such a scoring helped us to have a continuous data for computation purposes. We also inverted the scores for the items with negative impact on palliative care such as items 10, 17, 24, 27-33. Thus a total score can thus be derived after adding all the raw scores.Statistically significant , attitudes (9.46\u00b18.06 points), beliefs (4.88\u00b13.29 points) and experiences (15.8\u00b111.28 points) subscales of PTiPC-KABE scale when analyzed using paired t-test .t-test [There was an overall statistically significant change in total score of 36.61\u00b114.35 points out of a total of 104 points (35.2% change) when analyzed using paired t-test .Our study is the first of its kind reported on physiotherapist population and effects of a palliative care training program. One of the positive consequences of such training programs is the development of curricular content to suit student needs. Such educational interventions pave way to develop better curricular models and to integrate into regular professional and post-professional education.et al.[Marion et al. describeGoals of care should guide use of technology; and decision-making should be patient- centered.Know how to use prognostic scoring systems and recognize their limitations; be familiar with the ethical principles and guidelines for forgoing life-sustaining treatments; know how to withdraw life-sustaining treatments in ways that avoid distress; know how to monitor, identify and treat symptoms of distress, discomfort, anxiety or pain; and, know how to use opiates and sedatives to titrate to effect.Respect patients as individuals with diverse preferences; value the role of families in shared decision-making; respect ethnic and religious differences; and, be aware of their own values regarding death.Listen and communicate effectively and empathically; form trusting relationships with patients and families; explain details of illness, treatment strategy and goals of care simply; collaborate with the patient and family in balancing benefits and burdens; make recommendations and decisions in the face of uncertainty; give advice based on the patient\u2019s preferences; talk comfortably about death, dying and loss; support families or other intimates during their bereavement; and, work effectively in collaboration with multidisciplinary teams.et al,[We see these- knowledge and attitudes among physical therapists largely shown to influence their practice patterns and levels of care. Earlier reports by Weed and Zimny, Zimny anet al, commenteet al.[et al.[Population-based interventions are much better as educational interventions aimed at team training in palliative care. Sato et al. found pol.[et al. showed pet al.[The study had few limitations- the lack of established validity of the questionnaire used and lack of long-term follow-up of participants to observe knowledge translation into action process. We understand that measurement error of the said questionnaire would be nullified in pre-post designs if the scale has appreciable test-retest reliability. Accordingly the test-retest reliability was excellent. Further research on validation is in progress. Long-term carry-over effect of gained knowledge and its implication into actual real-life situation in palliative care is also under scrutiny currently. Long-lasting improvements in knowledge was found to be absent among medical and nursing students education previously by Velayuthan et al. who alsoet al.[The implementation of the above suggested findings, however, face their own barriers. Lai et al. listed tet al.,[There are already well-established multidisciplinary palliative care training centers for healthcare professionals in operation in Kerala as described by Seamark et al., to name According to Fins and Nilson, as acadeet al.[et al,[et al.[Considering that physical therapists have largely favorable attitudes toward evidence-based practice as reported by Jette et al. and a tret al. and a wil.[et al, there isl,[et al. This knoPhysical therapists, let\u2019s now move on, from ignorance to knowledge.The focused training program on palliative care had positively influenced a group of physiotherapy students in this study by bringing about a significant change in their knowledge, attitudes, beliefs and experiences."} +{"text": "Further expanding the major findings of the landmark study by Harrison and colleagues showing that late-life pharmacological inhibition of the enzyme mammalian Target Of Rapamycin (mTOR) -a conserved integrator of nutrient and growth factor signaling- is sufficient to significantly extend lifespan in mice on rapamycin feeding , Selman If activation of AMPK (and/or inhibition of mTOR/S6K1) constitutes the best metabolic response to avoid that a normal growth rate would compromise somatic integrity in progeroid animals , 5, it iEven if findings from Colman and colleagues demonstrating significant lifespan extension in rhesus monkeys can definitely substantiate CR as an effective pro-longevity strategy for humans , many peMetformin treatment has been recently found to selectively target tumor-initiating cancer stem cells (CSCs) in breast cancer cell cultures -31, likeTranslation of in vitro results into in vivo applications may significantly alter, however, the assumed CRM nature of the biguanide metformin. A recent study by Anisimov and colleagues published in a previous issue of Aging (Albany NY) may certainly establish that metformin should be defined as geroprotective or gerosuppressant rather than bona fide CRM. Long-living female mice from the outbred SHR strain were fed metformin in drinking water beginning at 3, 9 or 15 months of age and they were then analyzed for reproductive aging, mean & maximal lifespan and incidence of malignant tumors . MetformEarlier studies from the same group showed that metformin treatment increased mean lifespan of SHR mice by >37% and increased maximum lifespan by >10 months . MetformIf metformin both prevents cancer and extends lifespan in cancer-prone (e.g. neu-N transgenic mouse model of mammary cancer) strains of rodents, metformin's ability to prolong lifespan without affecting cancer in non-cancer-prone strain of rodents (e. g. SHR) may suggest that metformin can prolong life (and delay aging) by mechanisms unrelated to its ability to suppress cancer. It may not, however, if this discrepancy simply relies with the cellular response that oncogenes and tumor suppressors have on the phenotype of energetic metabolism. Derived from the FVB/N strain, neu-N transgenic mice express the nontransforming rat neu cDNA under the control of a mammary-specific promoter . As a coWhen considering recent studies describing that pre-malignant intraepithelial neoplasias such as ductal carcinoma in situ (DCIS) of the breast do contain pre-existing carcinoma precursor cells , 64, it It remains to be elucidated how metformin treatment, if started early and/or late in life, might significantly impact on late-onset diseases, like Alzheimer dementia, coronary artery diseases or, perhaps more importantly, certain familial forms of cancer. In this regard, an ideal scenario relates to genetic susceptibility to breast and ovarian cancer in women arising from a mutation in the BRCA1 gene, which is one of the most widespread genetic diseases , 77. WomAnisimov and colleagues studied markers of cellular senescence in fibroblasts from skin of SHR mice treated with metformin since the 3rd and the 9th months of life . AlthougAs mentioned above, one of the hallmarks of senescent cells is that the senescence growth arrest is essentially permanent and cannot be reversed by known physiological stimuli . TherefoPioneeringly revealed by current Anisimov's studies, a previously unrecognized ability of metformin to regulate the senescent phenotype in vivo may provide crucial insights to definitely distinguish a/the molecular mechanisms underlying metformin-promoted anti-aging and/or anti-cancer effects. On the one hand, studies of human tissues and cancer-prone mice argue strongly that cellular senescence suppresses cancer in vivo -114. SenAn unexplored scenario relates to metformin's ability to induce and/or enhance senescence responses in tumor cells themselves. Senescence-inducing stressors such as oxidative damage, DNA damage and/or oncogenes normally reach sufficient intensity to trigger senescence at the pre-malignant tumor stage. In agreement with a role of senescence in cancer prevention, the subsequent invasive progression of pre-malignant lesions almost inevitably involves one or more events that inhibit or impair the senescence pathway . Pret al [et al [et al [Landmark studies of Lin et al and Campl [et al have recl [et al . Senescel [et al ? Althougl [et al ; b.) phal [et al to promol [et al , 129. Inl [et al , Skp2 inl [et al -132 gettl [et al , 134, itl [et al . Becausel [et al ), a molel [et al -139. Finl [et al -142, it l [et al and to pl [et al .Cellular senescence can drive are-related pathologies other than cancer. If metformin reduced abundance of senescent cells is central to metformin's ability to actively regulate lifespan extension, it would be interesting to test if metformin treatment may decelerate rapid accumulation of senescent cells and/or the rapid development of age-related phenotypes including growth retardation, loss of weight, lipodystrophy (i.e. loss of adiposity), hair loss and bone density defects. A mouse model of Hutchinson-Gilford progeria syndrome can be extremely informative because it develops phenotypes that overlap with those of HGPS children and do not include cancer even when causing a significant alteration in the number and proliferative capacity of epidermal stem cells , 146. AdThe key question that remains to be answered in Anisimov's studies relates to the molecular mechanism(s) through which metformin treatment increased lifespan and delayed tumor formation in female SHR mice. An ever-growing experimental body of evidence strongly suggests that metformin, at the cellular level, works as an efficient deactivator of the mTOR/S6K1gerogenetic pathway owing its ability to activate the metabolic rheostat AMPK . Althoug"} +{"text": "This strategy might yield a novel class of more efficacious anti-epileptics with fewer side effects by specifically addressing disease pathophysiology. Moreover, this strategy may have ramifications for other adult seizure syndromes in which GABA receptor-mediated depolarizations play a pathogenic role, such as temporal lobe epilepsy.Neonatal intensive care has advanced rapidly in the last 40\u2009years, with dramatic decreases in mortality and morbidity; however, for neonatal seizures, neither therapies nor outcomes have changed significantly. Basic and clinical studies indicate that seizures in neonates have long-term neurodevelopmental and psychiatric consequences, highlighting the need for novel pharmacotherapeutics. First-line treatments targeting GABAA receptors, like barbiturates and benzodiazepines, are limited in their efficacy and carry significant risks to the developing brain. Here, we review the use of current GABA agonist therapies for neonatal seizures and suggest other treatment strategies given recent developments in the understanding of disease pathogenesis. One promising avenue is the indirect manipulation of the GABAergic system, via the modulation of neuronal Cl Seizures are the most common neurological emergency in the neonate, with an estimated prevalence of 1.8\u20135 seizures/1000 live births in the US Jensen, . In factA receptor, or by antagonism of NMDA receptors. Although these drugs are effective in adults, they do not control neonatal seizures well. When given as monotherapy, phenobarbital controls seizures in less than half of newborns often secondary to an increased neuronal accumulation of Cl\u2212, though \u2212 homeostasis. Among these are the bumetanide-sensitive Na+-K+-Cl\u2212co-transporter NKCC1, which transports Cl\u2212into the cell, and KCC2, which normally extrudes it is actively enrolling patients in a randomized, double-blind, controlled dose-escalation study of bumetanide as an add-on therapy to treat refractory seizures caused by hypoxic-ischemic encephalopathy. The estimated study completion date is December 2015. A second trial (NCT01434225) is being performed by a large, multi-center European group in an \u201copen-label,\u201d dose-escalation fashion to assess the effect of bumetanide in addition to phenobarbital for the treatment of neonatal seizures caused by hypoxic-ischemic encephalopathy. Data from these pilot studies will be utilized to guide the design of larger Phase III trials that will determine the efficacy of bumetanide in the treatment of neonatal seizures.GABA via NKCC1-knockdown or KCC2-overexpression may result in cortical neurons with fewer and shorter dendrites (Cancedda et al., \u2212 gradients in GABA transmission. Nevertheless, in the context of our enhanced understanding of the mechanisms of GABA signaling in the neonatal brain, novel therapies that target Cl\u2212 homeostasis are promising new targets for the treatment of neonatal seizures (Loscher et al., \u2212 homeostasis should also be considered. One unexplored mechanism of Cl\u2212 modulation is the manipulation of neuronal Cl\u2212 gradients via targeting of the serine-threonine regulatory kinases of NKCC1 and KCC2 (Kahle et al., in vitro, and does so via changes in transporter phosphorylation at critical regulatory residues (Figure \u2212 homeostasis, such as temporal lobe epilepsy, in which abnormal expression of Cl\u2212 channels also renders GABA excitatory (Palma et al., There are some concerns about the modulation of depolarizing GABA responses based on its importance during neurodevelopment. Studies suggest the \u201cpremature\u201d shift of Es Figure (Kahle es Figure . Given tThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Dear Editor:i.e.: \u2018every cell has a sex\u2019 In 2001, The United States Institute of Medicine (IOM) Committee on Understanding the Biology of Sex and Gender Differences concluded that \u2018Sex\u2026should be considered when designing and analysing studies in all areas and at all levels of biomedical and health-related research\u2026\u2019 and stated an apparent paradox It is unchallenged that there are health differences between males and females and that social and cultural factors could contribute to the observed differences. Anyway, the sex-dependent differences also have a biological base which sometimes has not been deeply investigated. Scientists studying health differences between male and female aim to both considering social/cultural environment and investigating biological/molecular mechanisms different between sexes. Some experimental studies have elucidated important differences in cell death control et al., via an apoptosis-inducing factor-dependent pathway (a caspase-independent pathway) in male neurons while proceeds via a cytochrome C-dependent pathway (a process mediated by caspase activation) in female neurons in vivo studies. In particular, it has been demonstrated that microRNA-23a, by binding the mRNA of the caspase inhibitor named XIAP, induces its translational repression in females, leading to enhanced caspase signalling in the ischaemic female brain. This effect has been shown to be independent of circulating oestrogen levels , contributing to female cellular higher resistance after ischaemic brain injury. Gupta NC et al. in vitro study. In addition, at least to some extent, sex-matched or sex-unmatched cell controls may be necessary in many experimental settings. In conclusion, sex-related differences in cell death mechanism may have strong implications for experimental studies and sexual dimorphism dependent on chromosomal rather than hormonal differences have important implications for planning preclinical studies and clinical interventions.However, it is currently emerging that some cell death programs are differentially controlled by sex-related hormone-independent cellular genetics. Differences in cell death sensitivity in male and female may then occur in the absence of an hormonal context. This is not an immediately obvious finding; Penaloza C"} +{"text": "Frontiers in Neurology, on the treatment of West syndrome (WS) by using valproate as monotherapy, prompted us to rethink about the past and present treatment strategies and the outcome in this severe epileptic syndrome.The recent article by Chandra et al. , publishEven though the study by Chandra et al. leaves sHistorically, some previous studies in children with WS, which employed valproate as monotherapy, proved effective in controlling either hypsarrhythmia and/or the epileptic spasms \u20136. BesidOverall, the treatment strategies in WS [either the first-line treatments or the more classical non-golden treatments ] are based on the assumption that an early initiation of therapy coupled with a rapid control of seizures in these patients may prevent the arrest or the decline in cognitive development.clinical spasms with onset in infancy is wider than previously thought and is currently comprised under the umbrella term of Infantile Spasms syndrome (ISs), which defines an epileptic syndrome (occurring in children younger than 1\u2009year\u2009\u2013 rarely older than 2\u2009years), with clinical spasms usually occurring in clusters whose most characteristic EEG finding is hypsarrhythmia . WS refers to a form (a subset) of ISs, characterized by the combination of clustered spasms and hypsarrhythmia on an EEG. Additional (less common) phenotypes falling within the ISs include the so-called infantile spasms single-spasm variant (ISSV: in which spasms may occur singly rather than in clusters), hypsarrhythmia without infantile spasms [HWIS: in which hypsarrhythmia can be recorded without any evidence of clinical spasms], and infantile spasms without hypsarrhythmia . There Osborne . Notably Osborne . In thisFor all the above reasons, the results of Chandra et al. highligh"} +{"text": "Atrial fibrillation (AF), as a sustained arrhythmia, is featured by uncoordinated atrial activation with the consequent deterioration of mechanical function in the atrium (Mestroni, Three classical models have been proposed for the genesis of AF, including focal activity, single-circuit reentry, and multiple-circuit reentry model (Fatkin et al., Although AF is a most prevalent type of arrhythmia, its genetic etiology remains unclear (Franco et al., Pitx2a, Pitx2b, and Pitx2c (Schweickert et al., Pitx2d is also expressed in human, functioning as a dominant negative protein (Cox et al., The Pitx2 gene mainly encodes three distinct isoforms including The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Rhizobia are also controlled by systemically regulated or autoregulated processes on top of the basic innate immunity response. The induction of systemic immunity responses like ISR (induced systemic resistance) by some beneficial rhizosphere bacteria or the SAR (systemic acquired resistance) response provoked by pathogens are results of multiple response cascades employed by the plant host to respond to microbial and other environmental interactions. However, the entire response network is by far not yet revealed. For example, bacteria-induced plant responses resulting in improved resistance towards pathogens can also be due to the perception of secondary metabolites, like the surfactin lipopeptide, produced by certain biocontrol Bacilli such as chitin or lipochitooligosaccharides, peptidoglycan, lipopolysaccharides or flagellum structures, and the initiation of efficient plant defence reactions type is tightly regulated in response to cell density or the cell \u201cquorum\u201d or pathogenic (Pseudomonas aeruginosa) bacteria provoked at concentrations as low as nano- to micromolar significant changes in the accumulation of over 150 proteins. Auxin-responsive and flavonoid synthesis proteins were induced and also a secretion of plant metabolites that mimic QS compounds were found, which may have the potential to disrupt QS signaling by associated bacteria. In tomato plants, a specific induction of systemic resistance proteins after inoculation of the roots with C4- and C6-side chain AHL-producing Serratia liquefaciens MG1 was discovered independently in MG1-inoculated plants. Furthermore, Serratia plymuthica HRO-C48, producing C4-/C6- and OHC4-/OHC6-homoserine lactones, is able to induce ISR-like systemic protection of bean and tomato plants against the fungal leaf pathogen Botrytis cinnera; this response was greatly reduced with mutants impaired in AHL-production N-acyl AHL-compounds in a different manner: C4- and C6- homoserine lactones alter the expression of selected hormonal regulated genes which results in changes of the plant's hormone content, in particular an increased auxin/cytokinin ratio Urban) (G\u00f6tz et al., Lotus corniculatus produce lactonases which degrade AHLs and prevent their uptake and transport. In barley, it could further be demonstrated that C8- and C10-AHLs are taken up in a cell energy dependent manner by ABC-transporters into the root and transported via the central cylinder into the shoot (Sieper et al., The first demonstration of specific responses of a plant to bacterial AHLs was demonstrated for the legumes o et al. found thu et al. and Jin u et al. demonstrRhizobia (P\u00e9rez-Montano et al., Gypsophila was shown to influence QS, type III secretion system and gall formation activity by Pantoea plantarum (Chalupowicz et al., Serratia marcescens 90\u2013166 (Ryu et al., Interestingly, several plants have been demonstrated to produce AHL-mimic substances or to develop other activities influencing QS of plant associated bacteria (Gao et al., Pseudomonas aeruginosa, was shown to selectively impair the regulation of the nuclear transcription factor NF-\u03ba B which controls innate immune responses in mammalian cells (Kravchenko et al., N-acylhomoserine lactones as modifying bacterial effector molecules.Uptake of AHL-compounds and specific perception of AHLs in animal cells were also studied intensively in recent years (Teplitski et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "We discuss the cellular machinery required for CIL and the molecular signals that regulate it. We focus on our recent finding that in prostate cancer cells, Contact inhibition of locomotion is regulated by a balance between EphA and EphB receptor signalling. We show that, as recently described for chick heart fibroblasts, microtubule dynamics are required for Contact inhibition of locomotion in prostate cancer cells and we propose that stabilization of microtubules could account for defective Contact inhibition of locomotion between cancer cells and noncancer cells.Contact inhibition of locomotion (CIL) occurs when a cell stops migrating in a particular direction upon contact with another cell. Many cancer cells show Contact inhibition of locomotion when contacting one another but display contact-unimpeded migration following collision with noncancer cells. Here we review current understanding of Contact inhibition of locomotion, from Abercrombie's historical studies of cells in tissue culture to more recent analyses of Contact inhibition of locomotion This indicates that CIL has occurred. However, when PC-3 cells are treated with EphA2/EphA4 siRNA, the difference between free migration and migration following contact was significantly reduced, indicating that these cells do not display CIL . There is no significant difference between the free and contact Cx values of taxol-treated cells indicating that taxol treatment leads to failure of CIL and suggests that defective CIL in heterotypic collisions between PC-3 cells and fibroblasts is mediated by EphB3 and EphB4 signalling. DU-145 cells may not display defective CIL because they do not have increased expression of EphB receptors and so EphA signalling predominates and CIL occurs in heterotypic collisions between DU-145 cells and fibroblasts (Astin et al., Reverse-transcription PCR profiling of the Eph receptor and ephrin expression in PC-3 and DU-145 cells indicated that PC-3 cells have increased expression of EphB3 and EphB4 compared to DU-145 cells (Astin Here we show that CIL between prostate cancer cells is regulated by EphA receptors, specifically EphA2 and EphA4. These receptors appear to act together to coordinate CIL. By contrast, PC-3 cells display contact-unimpeded migration following collisions with fibroblasts. We find that this is due to their increased expression levels of EphB3 and EphB4, which engage ephrin-B2 expressed on fibroblasts. Knockdown of these two EphB receptors can restore CIL between PC-3 cells and fibroblasts. We propose that during cell\u2013cell collisions, cell migratory responses are regulated by a balance between repulsive EphA versus attractive EphB signalling.et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., Recently, we have shown that Cdc42 is activated downstream of EphB receptors while RhoA is activated following EphA receptor activation (Astin et al., et al., et al., et al., et al., Our finding that EphB4 is increased in the more metastatic cell line, PC-3, compared to the less metastatic prostate cancer cell line, DU-145, is consistent with previous reports (Xia in vitro and in vivo (Abercrombie & Heaysman, et al., et al., et al., Since Abercrombie's early observations on the social behaviour of migrating cells in tissue culture, several studies have shown that CIL is an important process in defining the migratory behaviour of cells"} +{"text": "Endothelial dysfunction is the hallmark of hypertension, which is a multifactorial disorder. In the cardiovascular system reactive oxygen species play a pivotal role in controlling the endothelial function and vascular tone. Physiologically, the endothelium-derived relaxing factors (EDRFs) and endothelium-derived contractile factors (EDCFs) that have functions on the vascular smooth muscle cells. The relaxation induced by the EDRFs nitric oxide (NO), prostacyclin, and the endothelium-derived hyperpolarization factor (EDHF) could be impaired in hypertension. The impaired ability of endothelial cells to release NO along with enhanced EDCFs production has been described to contribute to the endothelium dysfunction, which appears to lead to several cardiovascular diseases. The present review discusses the role of oxidative stress, vascular endothelium, and vascular tone control by EDRFs, mainly NO, and EDCFs in different models of experimental hypertension. Hypertension is a multifactorial disorder that involves many mechanisms leading to risk factors for cardiovascular diseases. Endothelial dysfunction is defined as the imbalance between the production and bioavailability of endothelium-derived relaxing factors (EDRFs) and endothelium-derived contractile factors (EDCFs), associated with increased bioavailability of oxygen reactive species (ROS) and decreased antioxidant capacity characterized as oxidative stress. In this review we will discuss the involvement of oxidative stress and vascular endothelium as well as the importance of vascular tone control, relaxation, and contraction in hypertension.4), flavin-adenine-dinucleotide, flavin-mononucleotide, and nicotinamide-adenine-dinucleotide-phosphate and hydrogen peroxide (H2O2) increase arginase activity/expression in the endothelial cells. This should lead to NOS uncoupling with reduced NO production and augmented superoxide anion (O\u22122) production. As shown by Romero et al. , which convert L-arginine and molecular oxygen to L-citrulline and NO, using such co-factors as tetrahydrobiopterin or Ser1179 (bovine) activates eNOS or Thr497 (bovine) decreases its activation revealed higher baseline NO and H2O2 concentrations than normotensive rats -mediated activation of NADPH oxidase (Heitzer et al., 495 residue in the Ca2+/CaM binding domain by PKC (Mount et al., 495 dephosphorylation results in eNOS uncoupling and O\u22122 production rather than NO generation (Lin et al., 495 eNOS phosphorylation site is more phosphorylated in hypertension or contains uncoupled eNOS remains unknown.Increased ROS bioavailability, decreased antioxidant capacity, or both occur in many models of hypertension such as SHR (Suzuki et al., 4 supplementation reverses the effect of uncoupled NOS induced by TERPY (Bonaventura et al., We have investigated the vascular mechanisms involved in the vasorelaxation induced by NO donors that present potential capacity to replenish vascular NO upon reduced NO bioavailability. Most of the studies using NO donors are performed on endothelium-denuded arteries to avoid interference of endogenously produced NO (Bonaventura et al., A and ETB receptors. ETA receptors are expressed on smooth muscle cells and promote contraction. ETB receptors are located on endothelial and smooth muscle cells, with opposite effects. Smooth muscle ETB activation evokes contraction, whereas endothelial ETB activation induces relaxation (Taddei et al., The altered function of endothelial cells leads to enhanced contraction (Endemann and Schiffrin, The SHR aorta exhibits a characteristic endothelial dysfunction that is not due to decreased EDRF release, but it is the result of simultaneous EDCF release. Indomethacin, a non-selective COX inhibitor, restores the blunted relaxation in SHR aorta to the level of normotensive (L\u00fcscher and Vanhoutte, 2, TXA2, and ROS are proposed as COX-derived EDCFs. Increased endothelial [Ca2+]i is required to evoke EDCF-mediated responses. Dysfunction in Ca2+ handling within the endothelium is important for the exacerbation of endothelium-dependent contractions in SHR aorta (Tang et al., Endoperoxides, PGI2O2 modify the vascular activity of NOS and COX in concentration-dependent way (Cai et al., 2 evokes vasorelaxation, whereas in aging animals or SHR it induces contraction (Vanhoutte, Independent of the genesis of hypertension, specific ROS such as HInhibitors of COX (Taddei et al., In conclusion, the data presented in this work suggest that decreased NO availability along with enhanced EDCFs production contribute to the endothelium dysfunction and impaired vascular relaxation in hypertension Figure . ConsideThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Our article reports a meta-analysis of available evidence on high-dose chemotherapy followed by autologous stem cell transplantation for primary breast cancer. A previous publication by Berry et al. reported a closely related meta-analysis and the authors regret not citing this article:Berry DA, Ueno NT, Johnson MM, et al. High-dose chemotherapy with autologous stem cell support as adjuvant therapy in breast cancer: Overview of 15 randomized trials. J Clin Oncol 2011;29:3214-3223Berry et al. provide evidence that high-dose chemotherapy followed by autologous stem cell transplantation (HDCT) support prolongs relapse-free survival but does not improve overall survival in patients with high-risk primary breast cancer. In our subgroup analysis, age and hormone receptor status had a significant interaction with OS. In the study by Berry et al., OS was statistically different by treatment arm in the subgroup for women with both hormone receptor-negative and HER2-negative tumor, for whom there was a 33% reduction in the risk of death. The hypothesis suggest that this breast cancer category presents increased sensitivity to dose intensification of alkylating agents and should remain the subject of clinical HDCT studies.While our meta-analysis evaluated publication bias and heterogeneity in the included studies, our analysis did not include an analysis of individual patient data, which Berry et al. did carry out. The lack of an analysis on individual patient data can be considered as a limitation of our meta-analysis."} +{"text": "Physiotherapy is concerned with identifying and maximizing movement potential, within the spheres of promotion, prevention, treatment and rehabilitation. Physical therapists practice in a broad range of inpatient, outpatient, and community-based settings such as hospice and palliative care centers where as part of a multidisciplinary team of care, they address the physical and functional dimensions of the patients\u2019 suffering. Physiotherapy treatment methods like therapeutic exercise, electrical modalities, thermal modalities, actinotherapy, mechanical modalities, manual physical therapy and assistive devices are useful for a range of life-threatening and life-limiting conditions like cancer and cancer-associated conditions; HIV; neurodegenerative disorders like amyotrophic lateral sclerosis, multiple sclerosis; respiratory disorders like idiopathic pulmonary fibrosis; and altered mental states. The professional armamentarium is still expanding with inclusion of other miscellaneous techniques which were also proven to be effective in improving quality of life in these patients. Considering the scope of physiotherapy in India, and in palliative care, professionals in a multidisciplinary palliative care team need to understand and mutually involve toward policy changes to successfully implement physical therapeutic palliative care delivery. World Confederation for Physical Therapy (WCPT) defines Physical Therapy as; \u201c\u2026 providing services to people and populations to develop, maintain and restore maximum movement and functional ability throughout the life-span. Physiotherapy includes the provision of services in circumstances where movement and function are threatened by the process of ageing or that of injury or disease. Full and functional movement are at the heart of what it means to be healthy. Physiotherapy is concerned with identifying and maximizing movement potential, within the spheres of promotion, prevention, treatment and rehabilitation. Physiotherapy involves the interaction between physiotherapist, patients or clients, families and care givers, in a process of assessing movement potential and in establishing agreed upon goals and objectives using knowledge and skills unique to physiotherapists\u201d.Physical therapists practice in a broad range of inpatient, outpatient, and community-based settings, including the following:Hospitals Outpatient clinics or officesRehabilitation facilitiesSkilled nursing, extended care, or subacute facilitiesHomesEducation or research centersSchools and playgrounds Hospices or palliative care centersCorporate or industrial health centersIndustrial, workplace, or other occupational environmentsAthletic facilities Fitness centers and sports training facilities.Physiotherapists play an inherent role in the multidisciplinary palliative care team emphasizing on improving function and quality of life in patients who are deemed to require physical and functional dimensions of care. PhysicalFunctional dimension is defined as one\u2019s perceived ability to perform accustomed functions and activities of daily living, experienced in relation to expectations and adaptations to declining functionality. Functionet al.[Inspite of most of population being rural, the current status of medical personnel and facilities in our country is scarce; with only 34% of physicians and 25% of all hospital beds are available in rural areas. Nursing is considered as a low-status job and is not a much-sought-after profession for young people. Hence the need for an efficient allied health professional to fill in the current needs of palliative care team sees the emergence of physical therapists, with their thorough professional background and clinical expertise, as invaluable members in the team of care.Physicians address the physical dimension to their extent and nurses in functional dimension. Addressing both the aspects simultaneously can be more beneficial for the patient. Considering the current healthcare scenario in India as reported by Seamark et al. Inspite The objective of this paper is to update the palliative care clinicians, physical therapists and other team members on the role of a physical therapist in a palliative care team by detailed view of physical therapy treatment methods and their evidence for application into conditions requiring palliative care.It comprises passive movement, assisted active movement, active movement, assisted-resisted active movement, and resisted movement techniques. The techniques can be applied in anatomical planes or as functional movement direction. The techniques can be performed on land or in water. The latter is termed as \u201chydrotherapy\u201d. Best examples of therapeutic exercise techniques are relaxation, massage, suspension therapy, muscle- re-education, progressive resisted exercise, floor aerobics, active mobility exercises, mobilization and stabilization exercise, proprioceptive neuromuscular facilitation (facilitation and inhibition techniques); breathing exercise; postural training; work simulation, work conditioning and work hardening; graded activity program and cognitive-behavioral training. ExerciseLow-frequency modalities like neuromuscular electrical stimulation and functional electrical stimulation, iontophoresis, high-voltage pulsed galvanic current, transcutaneous electrical nerve stimulation (TENS) and diadynamic currents; medium frequency modalities like Russian currents and Interferential therapy. High frequency modalities are usually grouped under deep-heating modalities under thermal modalities. Electrical modalities are very useful adjuncts in pain management.Cryotherapy ; superficial heating agents ; hydrotherapy (whirlpool and contrast bath). Thermal modalities are effective adjuncts to exercise and/or electrical modalities.Traction therapy, compression therapy, therapeutic taping and continuous passive motion. Compression therapy can be very useful in managing lymphedema.Ultraviolet, LASER, Extracorporeal Shock Wave therapy are useful in selected situations.Biofeedback is useful in patients with limited cognitive abilities and neuromuscular dyscontrol.Massage, deep transverse frictions, myofascial release, trigger point therapy, muscle energy techniques, motor control retraining.Joint mobilization using passive physiological and passive accessory (joint play) movements, combined movements, mobilization with movements, manipulation (high-velocity low amplitude thrust techniques).Neurodynamic techniques of neural tissue loading and nerve massage.Manual physical therapy techniques are used in a variety of settings ranging from hospital-based to home-based. The effects of the techniques depend upon the skills of the handling therapist.Orthosis- splints/ braces: supportive devices for the body parts.Prosthesis: artificial limbs.Mobility aids: locomotor training devices like wheelchair, prone crawlers.Walking aids: canes, crutches and walkers.Assistive devices are useful for training functional activities for patients with limited function.et al,[et al,[Pate et al, and Bryal,[et al, reportedl,[et al,Toot in additet al,[Laakso emphasizet al, said, inOxford Textbook of Palliative Medicine, as follows;The importance of physical therapy is widely stated in the most-read textbook- Physiotherapy aims to \u201coptimise the patient\u2019s level of physical function and takes into consideration the interplay between the physical, psychological, social and vocational domains of function\u2026\u2026 The physiotherapist understands the patients underlying pathological condition, but this is not the focus of treatment. The focus of physiotherapy intervention is, instead, the physical and functional sequelae of the disease and/or its treatment, on the patient.\u201d[patient.\u201dPhysiotherapy aims to: maintain optimum respiratory function; maintain optimum circulatory function; prevent muscle atrophy; prevent muscle shortening; prevent joint contractures; influence pain control; optimize independence and function; and, education and participation of the carer.The following section describes the role of physical therapy in common conditions that require palliative care.Physical therapists have a very important role to play in holistic care of patients diagnosed with cancer as stated by Flomenhoft and RashPhysical therapy treatment techniques have also been reported in cancer-related fatigue by Watson and Mock, breast c2023et al,[et al,[Narayanan and Koshy emphasizet al, said thal,[et al, performeThe Chartered Society of Physiotherapy (CSP)\u2019s evidence summary emphasizes the effectiveness of physical therapy in the palliative care of older people. Physicalet al,[Multiple sclerosis (MS) is the met al, also fouet al,et al,[Weih et al, performeet al,[et al.[et al,[et al,[The use of motor imagery (MI)\u2014presumed to be a visual and kinesthetic neural representation of the overt behavior\u2014has relied on two major assumptions. The first assumption is that the internally generated movement patterns involve the same neuronal correlates as the overt behaviors . Second, it assumes that using MI will lead to cortical and subcortical neuronal modification that is of benefit to a person who has experienced a stroke. Mental practice as an effective technique for locomotor training and rehabilitation was also employed in most neurological conditions as found in their comprehensive review by Malouin and Richards.[Ginis et al, reportedl,[et al. Katz et l.[et al, earlier l,[et al, in theirl,[et al, mentioneRichards.The role of physical therapy in palliative care of patients with respiratory disorders ranges from home-based care such as training symptom control for cough and breathlessness to providing interventions such as airway clearance techniques in the intensive and critical care units in hospital-based rehabilitation. The renowned professional body for respiratory and cardiac conditions, the American Thoracic Society emphasisPulmonary rehabilitation is a multidisciplinary program of care for patients with chronic respiratory impairment that is individually tailored and designed to optimize physical and social performance and autonomy. ATS has listed four essential components of pulmonary rehabilitation as; (1) exercise training , (2) education , (3) psychosocial and behavioral intervention , and (4) outcome assessment. This was also mentioned by Lanken et al,[en et al, that pulThe therapeutic efficacy of pulmonary rehabilitation was demonstrated convincingly in many systematic reviews and randomized controlled trials and hence physical therapy in the form of exercise training in globally accepted and widely practised position statements and treatment guidelines.et al,[Nici et al, mentioneet al,[Of the respiratory disorders requiring palliative care, the most life-limiting condition is idiopathic pulmonary fibrosis. Raghu et al, stated tet al, reportedet al, physicalKeenleyside and Vora recommenLeGrand stated tSachs and Weinberg explaineet al,[Emery et al, found exCiesla elaboratThe reported benefits of formalized exercise training to an informal recreational physical activity in chest physical therapy also extended to include systemic conditions like chronic renal failure to have positive effects on quality of life by Eng and Ginis. Also selet al,[et al,[et al,[et al,[Palliative care improved outcomes in patients living with HIV or AIDS. Home palliative care and in-patient hospice care improved a number of patient outcomes, particularly in terms of pain and symptom control, anxiety, insight and spiritual well-being. Harding et al, also stal,[et al, and hencl,[et al, 2005, O\u2019l,[et al, found aeet al,[et al,[et al,[et al,[Earlier proponents like O\u2019Brien et al, and O\u2019Brl,[et al, advocatel,[et al, in anothl,[et al, found cosound mind and a sound body\u201d and authors like Wilfey and Kunce[et al,[Exercises as a treatment for altered psychological states have been through over the years grounded on the principle, \u201cand Kunce and Tuckand Kunce found eace[et al, also fouThough exercise had been a part of behavioral medicine for treating altered physiological states like obesity, diabetes, cardiovascular risk modification and smoking according to Martin and Dubbert, through et al,[et al,[et al,[et al,[Exercise therapy programs such as aerobic exercise training was shown by McCann and Holmes to positet al, confirmeet al, for anxiet al, who founl,[et al, found chl,[et al, found exl,[et al, found adSteptoe and Cox found thTkachuk and Martin in their84Though theoretically not part of physical therapy, physical therapists are trained in the following techniques and they do perform these in their regular practice.Tarler-Benlolo emphasizet al,[EMG Biofeedback, exercise91et al, and exeret al, was showet al, and, Qiget al,Other complementary therapies which are often included in physical therapist\u2019s treatment armamentarium are acupuncture, aromatherapy, reflexology, relaxation and massage.Massage therapy as a complementary therapy technique for feet and handPhysical therapy was shown to have positive influence on quality of life and perceived well-being in a wide range of patient populations requiring palliative care such as cancer, HIV, neurological disorders, cardiopulmonary conditions and mental illnesses.The scope of physiotherapy practice is influenced by the ratio of qualified physiotherapists to the population. The number of physiotherapists per head of population in India is 1:212,000. This oftet al,[Meier et al, suggesteFuture studies are warranted on the following aspects;Assessment of knowledge, attitudes, beliefs and experiences toward palliative care among physical therapistsEvolution of a palliative care training program for physical therapists.Qualitative research on experiences of palliative care team members with physical therapistsInfluence of physical therapy on patient and caregiver perceptions and quality of life in other palliative care conditions.The authors wish to recommend three approaches to improve physical therapy in palliative care.Improving professional knowledge and skillsChanging professional attitudes toward care at end-of-lifeRecognizing the palliative care healthcare system in India.Coming together is the beginning, keeping together is progress, working together is success\u201d\u201c- Henry Ford."} +{"text": "Mitochondrial outer membrane permeabilization (MOMP) is the ultimate step in dozens of lethal apoptotic signal transduction pathways which converge on mitochondria. One of the representative systems proposed to be responsible for the MOMP is the mitochondrial permeability transition pore (MPTP). Although the molecular composition of the MPTP is not clearly understood, the MPTP attracts much interest as a promising target for resolving two conundrums regarding cancer treatment: tumor selectivity and resistance to treatment. The regulation of the MPTP is closely related to metabolic reprogramming in cancer cells including mitochondrial alterations. Restoration of deregulated apoptotic machinery in cancer cells by tumor-specific modulation of the MPTP could therefore be a promising anti-cancer strategy. Currently, a number of MPTP-targeting agents are under pre-clinical and clinical studies. Here, we reviewed the structure and regulation of the MPTP as well as the current status of the development of promising MPTP-targeting drugs. Traditionally, MPTP has been regarded as a multimeric complex, which putatively consists of the voltage dependent anion channel (VDAC) in the OMM, the adenine nucleotide translocase (ANT) in the IMM, cyclophilin D (CyP-D)\u2014a mitochondrial matrix protein that exhibits peptidylprolyl cis-trans-isomerase activity, and some other proteins such as hexokinase (HK) ; pro-apoptotic multidomain proteins (Bax and Bak); pro-apoptotic BH3-only proteins , promotes binding of Bcl-XL to VDAC, leaving Bax free from Bcl-XL. Free Bax interacts with Bak and/or Bax to form pore structures for the release of cytochrome c -4-hydroxyphenyl]-2-naphthalene carboxylic acid) are well known to induce clinical remission in patients with APL is an expanded porphyrin which displays an elevated oxidizing potential, thereby triggering excess generation of ROS and dithiodipyridine (DTDP) can cause thiol oxidation of a critical cysteine residue (Cys 56) of ANT, through which BMH and DTDP may induce MOMP and cell death irrespective of the expression level of Bcl-2 (Costantini et al., Resveratrol, a polyphenolic compound from grapes and wine, can inhibit mitochondrial ATP synthesis and trigger MOMP (Fulda et al., Curcumin is a major constituent of turmeric powder from the plant Curcuma longa. Among several anti-cancer mechanisms of curcumin are the modulation of Bcl-2 family proteins and cellular ROS, inhibition of the NF-\u03ba B survival pathway, and inhibition of cyclooxygenase-2 (Divya and Pillai, Betulinic acid, a natural pentacyclic triterpenoid of the lupane class, is known to trigger mitochondrial apoptosis in cancer cells (Fulda et al., Berberine, an alkaloid derived from plants of the Berberidaceae family, has been shown to exert direct effects on mitochondria, including the interaction with ANT (Pereira et al., L on the OMM to prevent them trapping Bax, allowing free Bax to form OMM channels (Neuzil et al., in vitro data, which warrant further clinical trials with \u03b1-TOS (Neuzil et al., \u03b1-TOS, a vitamin E analogue, competes with ubiquinone in binding to Q sites of respiratory complex II, which results in the displacement of ubiquinone from complex II, disrupts the electron flux, and consequently generating ROS (Dong et al., Honokiol is a small molecule polyphenol isolated from the genus Magnolia (Fried and Arbiser, Again, GSK3 plays a role in the opening of the MPTP through the phosphorylation of CyP-D. In 206 osteosarcoma \u03c1\u00b0 cells with an extreme Warburg phenotype, constitutively active ERK was shown to oppose this signaling by phosphorylating and inhibiting GSK3 (Masgras et al., Mitochondria have pivotal opposing roles in energy generation for cell survival and cytochrome c release for apoptotic cell death. Although the concept of the MPTP is still evolving, mounting evidence indicates that the MPTP is directly responsible for cytochrome c release, leading to apoptotic cell death. Targeting the MPTP for cancer treatment has two advantages: tumor specificity and bypass of the resistance mechanisms. Metabolic reprogramming and mitochondrial alterations in cancer cells could provide important clues for developing tumor-specific anti-cancer agents. MOMP finally occurs as a consequence of upstream pro-apoptotic signaling events, which are frequently deregulated in many cancers and which become resistant to most classical anti-cancer agents that target upstream regulators of MOMP (Fulda et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Understanding the role of different proteins in controlling synapses formation and stabilization may help elucidating, at the network level, the mechanisms by which inhibitory transmission controls network excitability and oscillatory behavior, crucial for information processing in the brain.Aim of this e-book is to highlight recent advances in these processes, bringing together leading experts in the field, who have made major contributions to our understanding of the cellular and molecular mechanisms regulating the appropriate assembly, location, and function of pre and postsynaptic specializations at inhibitory synapses.This e-book comprises nine reviews, one perspective and three research articles organized in a logic way following the information flow from the pre to the postsynaptic site.via protein\u2013protein interactions across the synaptic cleft.In the first article, Jovanovic and Thomson (School via postsynaptic calcium signals, triggered by the depolarizing action of GABA and activation of voltage-dependent calcium channels.Grantyn et al. (Instituvia transcriptional and post-translational modifications.Early in postnatal life, the depolarizing action of GABA is crucial for the construction of neuronal circuits. This process depends on intracellular chloride homeostasis which is under control of the cation-chloride co-transporter KCC2. In addition, by interacting with actin cytoskeleton, KCC2 exerts, independently of its role on chloride homeostasis, a crucial role on spines morphogenesis. Chamma et al. (INSERM In the following research paper, Bhumbra et al. (DepartmA receptor antagonists or gating modifiers. GABA transient may limit the activation of postsynaptic receptors and their binding reaction to mono-ligand state, promoting low probability channel opening and fast deactivation kinetics.Once released from presynaptic terminals, GABA diffuses in the synaptic cleft and binds to postsynaptic receptors. This process is very fast and occurs in non-equilibrium conditions. Barberis et al. (Departmtransporters may exert a homeostatic control on GABA release is put forward in a perspective article by Conti et al. (The intriguing hypothesis that GABA i et al. (DepartmA receptor isoforms, physiologically relevant to phasic and tonic inhibition. The highest potency would be compatible with extrasynaptic receptors, containing the \u03b1 6 subunit and exposed, during spillover of GABA from synapses, to low agonist concentrations, while the lowest potency would be compatible with synaptic GABAA receptors containing the \u03b12/\u03b13\u03b2\u03b3 subunits, exposed, during vesicular release, to high agonist concentrations.In the following review, Mortensen et al. (DepartmA-mediated conductance. They suggest that the \u03b2 3 subunit may be an important pharmacological target for the treatment of striatal disorders.Using a genetic approach, Janssen et al. (DepartmA receptors and other ligand-gated ionic channels or G-protein coupled receptors co-localized on the postsynaptic membrane. The interaction may occur via a direct coupling between two receptors or via the activation of intracellular signaling pathways. Usually the interaction results in a reduced GABAergic inhibition with consequent disinhibition. A similar receptor\u2013receptor interaction may occur also at extrasynaptic sites to regulate tonic GABAA-mediated inhibition.Next, Shrivastava et al. (DepartmSasso\u00e8-Pognetto et al. (DepartmA receptors in front of presynaptic releasing sites, by directly interacting with several types of \u03b1-containing subunits. Its heterogeneity, obtained through alternative splicing and post-translational modifications, associated with complex biophysical properties of its domains leads to the construction of diverse structural scaffolds that can accommodate different GABAA receptor subtypes. Gephyrin is also recognized as a crucial hub for multiple signal transduction pathways and trans-synaptic signaling that ultimately impact on synaptic dynamics and synaptic plasticity.The complex and still \u201cenigmatic\u201d role of the scaffolding molecule gephyrin at GABAergic synapses is exquisitely reviewed by Tretter et al. (DepartmPapadopoulos and Soykan (Max-PlaA receptors subunits mRNA in the hippocampus and cortex of post mortem brains of individual suffering from alcohol dependence. Interestingly, the observed changes refer mainly to extrasynaptic GABAA receptors, which mediate tonic inhibition responsible for regulating basic neuronal excitability.Finally, Jin et al. (Departm"} +{"text": "Actin is a protein abundant in many cell types. Decades of investigations have provided evidence that it has many functions in living cells. The diverse morphology and dynamics of actin structures adapted to versatile cellular functions is established by a large repertoire of actin-binding proteins. The proper interactions with these proteins assume effective molecular adaptations from actin, in which its conformational transitions play essential role. This review attempts to summarise our current knowledge regarding the coupling between the conformational states of actin and its biological function. Proteins are essential building blocks of the living systems. They are involved in all the biological functions playing structural and/or regulatory roles. In most of the cases proteins are required to be adaptive, i.e., to change their structure and function under certain cellular conditions. A frequent example of this adaptation is the up and down regulation of their activity, which occurs in many cases through interactions with other proteins and/or small regulatory molecules. The conformational elasticity of the proteins is essential for their adaptive properties. In many cases the details of the relationship between the various conformational states and the functions of proteins are well established. In some cases the characterisation of the coupling between the structural transitions and the modifications in the biological function is not complete. As proteins are often central components of cellular machineries, the detailed description of their conformational dynamics is desirable.The interest in actin has extensively increased since its discovery by Straub . In theiAfter some debate since the first report on actin as an intranuclear entity dated back to the 60s Lane, actin alActin is not restricted to metazoans but can also be found in plants. Since the discovery of the actomyosin complex in plants [Vorobeva and Poglazov, Actin has a large repertoire of interacting partners including metal ions, nucleotides and actin-binding proteins, which attribute versatile functions to actin [Sheterline et al., It is essential to understand how actin can adapt and modify its conformation for the various biological processes, what cellular components are effective to regulate these conformational transitions and how these different structural states are coupled to the complex and tightly regulated mechanisms through which actin fulfils its manifold biological activity. Here we review the present knowledge on the conformational dynamics of actin from structural, spectroscopic and cellular biology studies of complexes that actin forms with its interacting partners. We will also attempt to discuss how these conformational differences contribute to the functional segregation of actin structures in living cells.Since its discovery in 1942 Straub, from aceActin exists in both monomeric (globular or G-actin) and polymeric (filamentous or F-actin) form .. The fiUnder physiological ionic conditions actin monomers assemble into filaments. Actin polymerisation proceeds through kinetically distinct steps. It is initiated by the slow formation of actin nuclei (dimers/trimers), which serve as seeds for the subsequent filament elongation. During elongation more actin monomers associate to than dissociate from either of the two ends, which results in the net growth of both filament ends. The steady-state phase is characterised by a dynamic equilibrium where the length of the actin filaments remains constant, while actin monomers continually associate to and dissociate from the ends. In this dynamic equilibrium a stationery population of free actin monomers is established, called critical concentration, whose value lies between the critical concentration of the barbed and pointed end. Besides structural polarity, determined by the arrangement of actin protomers within the filament, actin filaments also exhibit a kinetic polarity, which is defined by the different monomer association and dissociation rates at the two ends. The barbed or plus end binds actin monomers faster than the pointed or minus end. Although its ATPase activity is not crucial for actin polymerisation, actin self-assembly is associated with the ATPase cycle, which powers treadmilling process Wegner, .The first high-resolution structural model of the actin filament at a resolution of 8 \u00c5 was proposed by [Holmes et al., fs range reflect the rearrangements of atoms/molecules, and these structural changes can be resolved by X-ray crystallography, electron microscopy (EM), cryo-EM and femtobiological approaches [Egelman, ns correlation times are related to the change in the restricted segmental motion of a monomer/protomer or a few neighbouring protomers and can be determined by time-dependent fluorescence anisotropy [Ikkai et al., Rather than being rigid structures, both monomeric and filamentous actin have remarkable conformational flexibility and adopt many various structural states in response to interaction with their partner molecules [Orlova and Egelman, Egelman, . The ns KD = 10\u221210 M) than ADP (KD = 10\u22128 M) [Neidl and Engel, KD \u223c10\u22129 M) is thought to be occupied by magnesium in cells [Estes et al., \u22121) [Schuler, Pi). This results in the appearance of an ATP/ADP-Pi cap at the barbed end, while the rest of the filament contains ADP-bound actin protomers [Brenner and Korn, Pi almost simultaneously, which results in homogeneous ADP-bound actin protomers along the whole filament [Yao et al., The main ligands that bind to the central cleft of the actin monomers are an adenosine nucleotide and a divalent cation inset a Schuler, , which iSchuler, . In the The Holmes model postulated the importance of an interstrand hydrophobic plug-pocket interaction in filament integrity [Holmes et al., The importance of this cross-strand hydrophobic interaction and loop mobility in actin filament integrity was supported by disulfide cross-linking studies. These experiments showed that mutant G-actin\u2014in which the loop is locked to the protein backbone\u2014could not polymerise [Shvetsov et al., 2+-ADP actin monomers had lower steady-state phosphorescence emission anisotropy, and thus greater torsional flexibility than filaments polymerised from Mg2+-ATP actin monomers [Rebello and Ludescher, Cys374 in S1 and substantially greater intermonomer flexibility of filaments that were assembled from ADP-actin monomers than those assembled from ATP-actin monomers. These data suggest that more tenuous interprotomer connections are formed when the filament is polymerized from ADP-bound monomers [Nyitrai et al., The influence of the different nucleotides on the conformational dynamics of actin filaments was first demonstrated by the analysis of EM images of negatively stained actin filaments, and by dynamic elasticity and viscosity measurements of solutions of F-actin. These measurements suggested that actin filaments formed from ATP-actin monomers were more rigid than actin filaments assembled from ADP-actin monomers [Janmey et al., Lp = 9 \u03bcm), which suggests similar bending flexibilities [Isambert et al., 3\u2014which mimics the \u03b3-phosphate [Combeau and Carlier, Pi-state resulted in higher flexural rigidity of F-ADP-Pi-actin (Lp = 13.5 \u03bcm) compared to F-ATP, or -ADP-actin [Isambert et al., A detailed light microscopy analysis of the thermal fluctuations of fluorescently labelled F-actin showed that there was no difference in the persistence length of actin filaments assembled from either ATP-actin or ADP-actin monomers [Hild et al., Gln137 close to the \u03b3-phosphate of the bound ATP. As Gln137 is implicated in the ATPase mechanism these structural rearrangements may be related to the regulation of ATPase activity of actin [Oda et al., Consistently, FRET measurements showed that the replacement of ATP by ADP in the nucleotide-binding cleft resulted in conformational changes that bring 3-F-actin and ADP-F-actin showed that the structure of S2 is more disordered in the ADP-state which results in breaking one of the longitudinal bonds and destabilization of the filament [Orlova and Egelman, Actin self-assembly and ATPase activity were also shown to alter the conformation of the DNase I binding loop (or D-loop) inset c 2+-F-actin were shown to be higher compared to Ca2+-actin filaments using spectroscopic and EM approaches [Orlova and Egelman, 2+-F-actin than for Mg2+-F-actin [Nyitrai et al., Tightly bound cations can modify the conformational state of actin as well. Both the torsional and bending flexibilities of Mg2+-G-actin and Mg2+-G-actin in the range of 6\u201326 \u00b0C, while above 26 \u00b0C a conformational transition was detected in Ca2+-actin monomers [Nyitrai et al., 2+-F-actin was found to be lower than that of Ca2+-F-actin in the range between pH 6.5 and pH 7.4 [Hild et al., 2+-F-actin. The interprotomer connections were more rigid at both pH 6.5 and 7.4 in Mg2+-F-actin than in Ca2+-F-actin [Hild et al., Temperature-dependent FRET measurements revealed no difference between the flexibility of the outer domain (S1 and S2) of Ca2+-actin filaments was proved to be unaffected (Lp \u223c8 \u03bcm) when the filaments were polymerized at different pH values (between pH 5 and 9) [Arii and Hatori, 2+-F-actin increased from \u223c4.5 \u03bcm to \u223c9 \u03bcm as the pH increased from 5 to 9, suggesting higher mobility of actin protomers within Ca2+-F-actin at lower pH values [Arii and Hatori, 2+-actin and Mg2+-actin filaments as the pH decreased [Kron and Spudich, The persistence length of MgThe polymerisation of actin filaments was accelerated and the helical pitch between the protomers was increased by lowering the pH [Zimmerle and Frieden, The observations described above demonstrate that small ligands\u2014such as nucleotides and cations\u2014and also external conditions\u2014such as the pH\u2014modify the conformation of actin. Despite the large amount of accumulated data the biological function of these structural modifications remained somewhat ambiguous. Some observations indicated that ATP hydrolysis altered the thermodynamic and mechanical properties of actin filaments and their interactions with actin-binding proteins. These results led to the hypothesis that ATP hydrolysis may serve as a biological clock in living cells. The maturation of the filaments is sensed and reflected by the change in the nucleotide state, and thus in the conformation of actin filaments [Allen et al., In many cases, when the conformation of actin filaments is changed upon ligand-binding, the effects propagate along the filaments through the interaction of neighbouring actin protomers, i.e., through long-range allosteric interactions. Such allosteric interactions were reported for many actin-binding compounds Oosawa, and are Amanita phalloides, is the best characterised so far. Phalloidin binds tightly to actin filaments, reduces the rate of Pi release [Dancker and Hess, Pi-actin , which suggests different interprotomer interactions in F-actin in the ADP-Pi state compared to the ATP-, or ADP state [Orban et al., Several actin-binding natural products that have cytotoxic activity exhibits cooperativity [Wieland and Faulstich, Jaspis johnstoni)\u2014also binds to actin filaments competitively with phalloidin [Bubb et al., Another cyclic peptide, jasplakinolide\u2014that can be found in a marine sponge can alter the regulation of the cytoskeleton and/or bind directly to actin [Richard et al., Apart from these examples many other poisonous chemicals (such as for example jararhagin) [Costa and Santos, D. discoideum has 17 [Romans and Firtel, D. melanogaster and mammals have 6 genes [Fyrberg et al., The tuning of the conformation and thus cellular function of actin can be achieved by the various actin isoforms which coexist in living cells. Actin is expressed as a variety of isoforms generated by gene duplications. Yeasts have 1 actin gene [Gallwitz and Seidel, S. cerevisiae and D. discoideum actin filaments based on 3D helical reconstruction from EM images show that these actin filaments are similar in terms of the overall three-dimensional morphology, which confirms that these parameters were conserved through the evolution [Orlova et al., S. cerevisiae actin filaments than in muscle actin [Orlova and Egelman, S. cerevisiae actin protomers than in muscle actin [Orlova et al., S. cerevisiae actin filaments by time-dependent phosphorescence anisotropy decay measurements indicated higher torsional flexibility for yeast F-actin than for muscle F-actin [Prochniewicz and Thomas, S. cerevisiae G-actin compared to muscle actin in the barbed end, in the region of S1 and S2 and in protomer-protomer contact areas within the actin filaments as well [Stokasimov and Rubenstein, S. cerevisiae F-actin [Orlova et al., D. discoideum F-actin in the Ca2+-bound form displays less extensive interstrand contacts between the two long pitch helix than muscle actin, while these contacts are more massive in D. discoideum F-actin in its physiologically relevant Mg2+-bound form [Steinmetz et al., D. discoideum Mg2+-F-actin is longer (Lp = 4.2 \u03bcm) than that of the D. discoideum Ca2+-F-actin (Lp = 1.6 \u03bcm) or the muscle Mg2+-F-actin (Lp = 2.3 \u03bcm). Therefore, the enhanced interstrand connectivity seems to provide the structural basis for the altered bending flexibilities of the actin filaments.Available structural analyses of rabbit muscle, 2+-actin filaments were thermodynamically more stable than the \u03b1-skeletal Mg2+actin filaments. On the other hand, \u03b1-cardiac Mg2+-actin filaments are more stable than the \u03b1-cardiac Ca2+-actin filaments [Orban et al., The muscle specific \u03b1-skeletal and \u03b1-cardiac actin isoforms differ only in four amino acids [Vandekerckhove and Weber, Although the experiments of recent years have shed light on many aspects of the isoform specific functional variations of actin, the understanding of how these conformational dynamics differences of polymers assembled from different actin isoforms contribute to the isoform specific functions demands further investigations.Apart from small ligands and peptides described above actin interacts with a large number of partner proteins. These proteins can change the conformation of actin when regulating its biological functions. Although the coupling between these structural-functional changes is not completely understood yet, there are indications that the actin-binding protein induced structural modifications are established for certain biological functions. In the next session we attempt to provide examples for these conformational changes.2+-F-actin [Orlova and Egelman, Pi is bound to myosin) the binding of myosin subframent-1 was not cooperative and was accompanied by a smaller change in the microsecond rotational dynamics of F-actin than in the rigor state [Prochniewicz et al., Myosins interact with actin in all of their known biological activities. As a classic example, myosins play special and central role in the manifestation of muscle contraction [Geeves et al., A recent study has found evidence that the effect of the myosin on the conformation of actin depends on the myosin isoform as well [Prochniewicz et al., The field focusing on the functions of various myosins is large and growing. It is well established that the specialised forms of myosins play essential roles in many biological functions in synergic interactions with actin. Despite the decades of investigations the information regarding the conformational effects of myosins on the structural and dynamic properties of actin filaments is limited, indicating that further investigations will be needed to properly describe and understand the biological functions of these interactions.2+-ATP actin monomers in a nucleotide dependent manner (KD(Mg-ATP) = 2 \u03bcM, KD(Mg-ADP) = 80 \u03bcM) [ is a small (5kDa) WH2-domain (Wiskott-Aldrich syndrome protein-homology 2) containing protein. It sequesters actin monomers by forming a 1:1 nonpolymerisable complex with them, which does not participate in actin assembly at either end of the filament [Cassimeris et al., 2+-ATP-G-actin at intermediate rates (100\u2013400 s), which suggests that the conformational dynamics of G-actin is restricted upon T\u03b24-binding [De La Cruz et al., Trp and the Tyr band of the near-UV CD spectrum of Mg2+-ATP-G-actin [De La Cruz et al., Tyr residues around the nucleotide binding cleft led to the proposal that the nucleotide binding cleft is narrower in the T\u03b24-G-actin complex. In support of this, FRET measurements showed that T\u03b24-binding decreased the distance between probes attached to Lys61 (S2) and Cys374 (S1) and to Lys61 (S2) and \u025b-ATP, while increased the distance between Gln41 (S2) and Cys374 (S1). This indicates that T\u03b24 rotates the D-loop towards the bound nucleotide away from S1 enclosing the nucleotide binding cleft [Dedova et al., Extensive spectroscopic and biochemical analysis revealed that T\u03b24-binding was accompanied by significant changes in the conformation of actin monomers [De La Cruz et al., KD = 0.1 \u2212 1 \u03bcM [Pantaloni and Carlier, Profilin is an essential actin binding protein that plays important role in controlling actin dynamics and actin-based motile processes. The structure of different profilin isoforms in complex with actin isoforms were solved by X-ray crystallography [Schutt et al., The high-resolution crystal structure of the profilin-actin complex shows that binding of profilin to G-actin results in the rotation of the two major domains by 4.7\u00b0 relative to each other in a \u2018clamp\u2019-like fashion, closing around the profilin and opening up the nucleotide-binding cleft [Schutt et al., ADF/cofilin (actin-depolymerising-factor) (AC) family of proteins can be grouped into five functionally distinct classes of actin-binding proteins which are characterized by the presence of the ADF homology (ADF-H) actin-binding module [Lappalainen et al., An extensively studied member of the AC protein family is ADF/cofilin that contains a single ADF-H domain. ADF/cofilin binds both G-, and F-actin, preferentially their ADP-bound form in a 1:1 and 2:1 stoichiometry, respectively [Hayden et al., Gln41 (S2) and Cys374 (S1) [Dedova et al., Lys61 (S2) and Cys374 (S1) [Blondin et al., The recently solved crystal structure of twinfilin's ADF-H domain in complex with a G-actin molecule reveals that the binding interfaces are located in the groove between S1 and S3 of actin [Paavilainen et al., Pi release from ADP-Pi-F-actin [Blanchoin and Pollard, The binding of ADF/cofilin to actin filaments exhibits a high degree of kinetic cooperativity [Hawkins et al., Gln41 (S2) and Cys374 (S1) in neighbouring protomers and an increase in their mobility, which suggests a more flexible protein matrix around the probes and loosening of the intermonomer contacts within the long-pitch strand of F-actin [Scoville et al., The atomic model of ADF/cofilin-decorated F-actin revealed extensive contacts of the bound ADF/cofilin molecule with the outer domain of actin protomer [McGough et al., The analysis of the thermally driven fluctuations of fluorescently labelled actin filaments revealed that ADF/cofilin increased the bending flexibility and decreased the persistence length of actin filaments by 5-fold [McCullough et al., 2+-dependent severing activity, with gelsolin remaining bound to the barbed end of the severed filament [Hesterkamp et al., The gelsolin family consists of seven different proteins characterized by repeats of gelsolin-like (G) domains, including gelsolin, adseverin, villin, capG, advillin, supervillin and flightless I, which are involved in the regulation of actin dynamics [Silacci et al., Cys374. Further cross-linking and fluorescence measurements showed that the nucleation by gelsolin was promoted by conformational changes between the D-loop and the C-terminal of protomers, which propagate along the filament from the gelsolin capped barbed ends [Khaitlina and Hinssen, Helical reconstruction of cryo-EM images showed that by binding to the side of F-actin gelsolin bridges two neighbouring actin protomers within the short pitch helix, and induces distortions within the actin filament which may sufficiently weaken the noncovalent interactions to break and sever the filaments Bearer, . EM recoBoth in vitro and in vivo, the rate of the spontaneous polymerisation of actin is limited by the instability of the initial actin dimers/trimers. In cells, to overcome this kinetic barrier and regulate precisely the spatiotemporal initiation of actin structures, membrane-associated stimuli-dependent nucleation factors catalyze the de novo formation of actin filaments by unique mechanisms. The first identified nucleation machinery includes the WASP/WAVE/Scar proteins (Wiskott-Aldrich syndrome protein/WASP family verprolin homologous/suppressor of c-AMP response) which activate the Arp2/3 complex to generate a branched daughter filament from a pre-existing mother filament Pollard, . RecentlS. cerevisiae was also proposed to induce conformational changes within yeast actin filaments, which was indicated by the increase of the pyrene excimer fluorescence in formin-nucleated actin filaments [Wen and Rubenstein, The mechanisms by which nucleation factors catalyze filament assembly and regulate barbed end dynamics has been extensively studied over the past few years. For detailed information we direct the readers to recent reviews [Kerkhoff, Minor changes in the highly conservative amino-acid composition of actin can have deep impact on the structure of actin, its function and interactions with its partner molecules [Bookwalter and Trybus, Mutations in actin are frequently manifested in the form of skeletal [Sparrow et al., Under in vitro conditions, the M132V nemaline actin mutant obtained from human biopsies was demonstrated to have lower polymerization capability and increased velocity in the actomyosin motility assay [Marston et al., The T278I mutation of the ACTG1 gene coded cytoskeletal \u03b3-actin can also cause autosomal dominant hearing loss [van Wijk et al., In the case of the familial hypertrophic cardiomyopathy some mutant actin presented slower folding in vitro. The incorporation of the monomers into the filamental structure affected adversely as well [Vang et al., Besides mutations in actin itself, mutations in genes encoding actin-associated proteins also lead to genetic conditions. Dystrophin and its homologous utrophin are members of the spectrin-superfamily. These proteins are associated with the costameric cytoskeleton in striated muscle that circumferentially locates around the myofibrils in register with the Z-disk and physically couples the force-generating myofibrils to the sarcolemma Ervasti, . The mutThe effects of dystrophin and utrophin on the structural dynamics of actin filaments were studied with transient phosphorescence anisotropy. The results showed that both of these proteins altered the rotational dynamics of actin filaments [Prochniewicz et al., It is difficult to pinpoint in a simple model the biological function related to the conformational changes in actin monomers or filaments. The difficulty comes from two sources. Actin has various and complex biological functions and apparently it can effectively and relatively quickly adapt to many intracellular situations and binding partners. On the other hand there are a large number of molecules from small cations to proteins which contribute to the broad conformational landscape of actin. While these conformational changes are extensively studied in vitro, very little is known about the conformational dynamics of actin in the cellular environment. One of the aims of the previous sections of this review was to provide examples of these specific interactions and environments, and to give an overview regarding the role of the conformational state of actin.Although the described interactions were specific some of them could provide bases for a more general model regarding the regulatory function of actin conformation. Recent observations suggested that formins, a group of actin nucleation factors, could substantially change the conformation of actin filaments by making them more flexible [Bugyi et al., So far, the conformational dynamics of actin, and the effects of different factors were investigated in depth under in vitro conditions. Considering the rich variety of actin functions and the large amount of data accumulated in these studies some of the couplings between the structural changes and biological functions were revealed. In many other cases the complete understanding of the roles of intramolecular mechanisms in actin demands further studies. Cellular actin networks interact simultaneously with more proteins that can induce different changes in the conformational dynamics of individual actin monomers or filaments. How are these changes superimposed\u2014enhanced or dampened\u2014and determine the overall conformational dynamics of actin networks? How does the conformational dynamics of actin filaments play a role in the establishment of the functional properties of relevant actin networks? Recent advances in the development of novel technologies (such as fluorescence lifetime imaging microscopy (FLIM) or fluorescence anisotropy decay imaging microscopy (FADIM) [Suhling et al., fs to ms and can be measured by different approaches of the rotation. The rotational correlation times describing the rotational motion of G-, or F-actin are distributed to a broad time scale; from ches see .mechanical property of actin filaments, it measures the resistance of the filament to an external twisting torque.mechanical property of actin filaments, it measures the resistance of the filament to bending forces.K) of F-actin by the following equation:describes the flexural rigidity of actin filaments. It equals the arc length of the filament over which the tangent angle at every point along the arc length correlates in three-dimensional motion. The persistence length is the distance over which the filament bends due to thermal fluctuations. The persistence length is related to the flexural rigidity (kB is the Boltzmann-constant and T is the absolute temperature. Actin filaments belong to the semiflexible polymers with typical persistence length of 0.1 \u2013 20 \u03bcm.where"} +{"text": "The human genome harbors an impressive number of genes encoding enzymes that primarily metabolize or transport drugs or other xenobiotics (XMEs). Genetic and functional variation in these genes is tremendous and has complex consequences, depending, for example, on whether enzyme structure or expression is affected, or whether the produced metabolite is pharmacologically or toxicologically active or not. Despite numerous impressive examples of the impact of genetic variation on pharmacokinetics and drug response, today's knowledge is incomplete regarding most XME genes and fragmentary even for many well-investigated XMEs. This is one of the reasons why clinical pharmacogenetic studies are often controversial and clinical application in personalized medicine is presently limited. Advanced technology and ongoing large-scale projects are rapidly uncovering the existing genetic variation in all populations on earth, ultimately enabling the personal genome in the very near future. A wealth of mostly rare novel variants is awaiting functional characterization either by high-throughput expression/phenotyping techniques or by prediction using improved algorithms to estimate functional relevance.With this Research Topic we would like to give an up-to-date overview about the current knowledge in this field by covering both, known hard facts as well as cutting-edge advancement in novel genetic and genomic variation of XMEs and their functional consequences. Five major subtopics which include 20 research or review papers are included in this E-book. These are the following:Clinical application of CYP2C19 pharmacogenetics toward more personalized medicine Lee, , review.Pharmacogenetics of cytochrome P450 2B6 (CYP2B6): advances on polymorphisms, mechanisms, and clinical relevance : resolving the puzzle (Stephens et al., MDMA, methamphetamine, and CYP2D6 pharmacogenetics: what is clinically relevant? (de la Torre et al., Molecular interactions between NAFLD and xenobiotic metabolism (Naik et al., Toward a clinical practice guide in pharmacogenomics testing for functional polymorphisms of drug-metabolizing enzymes. Gene/drug pairs and barriers perceived in Spain (Ag\u00fandez et al., Clinical implications of XME gene variantsCYP3A4 in three South African populations (Dr\u00f6gem\u00f6ller et al., Characterization of the genetic variation present in Frequencies of 23 functionally significant variant alleles related with metabolism of antineoplastic drugs in the Chilean population: comparison with Caucasian and Asian populations (Roco et al., Pharmacogenomic diversity among Brazilians: influence of ancestry, self-reported color, and geographical origin (Suarez-Kurtz et al., Inter/intraethnic variability of XME gene variantsImpact of the interaction between 3\u2032-UTR SNPs and microRNA on the expression of human xenobiotic metabolism enzyme and transporter genes (Wei et al., CYP2C19 (Helsby and Burns, Molecular mechanisms of genetic variation and transcriptional regulation of Regulation of XME gene expressionImpact of genetic polymorphisms on chemotherapy toxicity in childhood acute lymphoblastic leukemia (Gervasini and Vagace, Multilocus genotypes of relevance for drug metabolizing enzymes and therapy with thiopurines in patients with acute lymphoblastic leukemia (Stocco et al., Functional polymorphisms in xenobiotic metabolizing enzymes and their impact on the therapy of breast cancer (Vianna-Jorge et al., High-resolution melting analysis of the common c.1905+1G>A mutation causing dihydropyrimidine dehydrogenase deficiency and lethal 5-fluorouracil toxicity (Borr\u00e0s et al., Polymorphisms of phase I and phase II enzymes and breast cancer risk (Justenhoven, CYP2C8 gene rs11572080 with regard to colorectal cancer risk (Ladero et al., Analysis of the functional polymorphism in the cytochrome P450 Pharmacogenetics in cancer therapy"} +{"text": "Easton (In speech-related fields, researchers had begun formulating ideas for modularizing speech movements even prior to Bernstein's influence. Cooper et al. , for ins. Easton had firs. Easton shifted . Easton , with liMeanwhile, researchers in other areas have built a substantial volume of experimental and modeling research around the neuromuscular organization and biomechanics of non-speech movement, including work on complex fine motor systems such as the fingers (e.g., Overduin et al., The great majority of evidence for modularization derives from experiments on non-human spinal structures (see Tresch et al., In our view, neuromuscular modules are built specifically to drive body structures that are biomechanically efficacious, enabling them to operate feed-forward, i.e., with little or no central feedback control. This has often been assumed as a premise underlying modularization (e.g., Loeb et al., While there remains some controversy around whether these modules are best defined in terms of their neural (e.g., d'Avella and Bizzi, Developing a theory of speech production that accords with current work on neuromuscular modularization, we believe, has the potential to link a number of fields and methodologies surrounding a central question in cognitive science, with implications for all aspects of speech research, from phonetics and phonology to the phylogenetic and ontogenetic development of speech. In addition to bringing another complex motor system into the broader discussion of neural modules, modularizing speech at the neuromuscular level promises a major advance for speech models, constituting a \u201cmissing link\u201d between speech movement primitives (Ramanarayanan et al.,"} +{"text": "The arbitrariness of the linguistic sign is a fundamental assumption in modern linguistic theory. In recent years, however, a growing amount of research has investigated the nature of non-arbitrary relations between linguistic sounds and semantics. This review aims at illustrating the amount of findings obtained so far and to organize and evaluate different lines of research dedicated to the issue of phonological iconicity. In particular, we summarize findings on the processing of onomatopoetic expressions, ideophones, and phonaesthemes, relations between syntactic classes and phonology, as well as sound-shape and sound-affect correspondences at the level of phonemic contrasts. Many of these findings have been obtained across a range of different languages suggesting an internal relation between sublexical units and attributes as a potentially universal pattern. Linguistic theory widely adopts Saussure's essentiaEmpirical evidence for such phenomena primarily comes from signed languages sometimes further imitating the emotional impression they have on us, e.g., the German \u201cUff\u201d which transposes the ejected breath (ff) with which we instinctively express a reaction of relief into written German .According to Wundt , some onOrganon model, they also fulfill the conative/appealing function as in the calling (German \u201che\u201d) or the request to keep silent (German \u201cssst\u201d).Following Schrott and Jacobs , in inteTesting cross-cultural agreement in the understanding of phonological iconicity of interjections, Sauter et al. asked nakyoro kyoro for \u201clooking around\u201d or \u201cspinning\u201d; Tamil thuru thuru for \u201ceager\u201d or \u201cactive\u201d). Following Dingemanse, sensory imagery is perceptual knowledge that derives from sensory perception of the environment and the body. Although scarcely represented in Indo-European languages, Atoda and Hoshino but lack the central feature of compositionality to qualify as morphemes. They even appear across language borders in non-cognate-words of remote languages also seem to depict sensory imagery and therefore might qualify as iconic mappings.According to Bergen , availabSapir initiateMore recently, Pe\u00f1a et al. reportedShrum et al. extendedmaluma with a curvy round shape and takete with a spiky angular shape. The effect was subsequently labeled as \u201ckiki/bouba effect\u201d and replicated across a wide range of unrelated languages such as Himba (Bremner et al., Substantial evidence for phonological iconicity as a cross-linguistic phenomenon was derived from a seminal experiment of K\u00f6hler . Within Maurer et al. found thDevelopmental and cross-linguistic studies strongly suggest an innate origin of iconic mappings. However, dependent variables used are offline measures and especially adults' judgments might reflect metacognitive strategies.To overcome this problem, Westbury implemenUsing an implicit learning categorization task combined with EEG, Kovic et al. presenteLikewise, Ramachandran points to possible synkinetic mappings of hand and jaw movements, controlled in two adjacent areas in the Penfield motor homunculus (Ramachandran and Hubbard, Building on their research on sound-size correspondences, Taylor and Taylor asked moFocusing on real text instead of artificial word material, F\u00f3nagy contrastIn a more general approach, Heise extendedAryani et al.' softwareAnother account of systematic mappings of phonology to affective dimensions was proposed by Zajonc et al. , who conSystematic form-meaning mappings are abundant in many languages, although not always necessarily iconic in nature. Yet, these latter ones hold strong implications for the essence of human language and its origin.Given the relatively small inventory of phonemes and the potentially infinite number of concepts to be expressed, the Saussureian principle of arbitrariness certainly remains a general key feature of human language Gasser, , allowinFay et al. point inStrictly arbitrary relations between levels of phonology and semantics as assumed by psycholinguistic models (e.g., Levelt et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Schizosaccharomyces pombe. This simple model has contributed significantly to our understanding of how the cell cycle is regulated, and serves as an excellent model for studying aspects of cytokinesis. Here we will discuss the state of our knowledge of how the contractile ring is assembled and disassembled, how it contracts, and what we know of the regulatory mechanisms that control these events and assure their coordination with chromosome segregation. \u00a9 2011 Wiley-Liss, Inc.Cytokinesis is the final stage of the cell cycle, and ensures completion of both genome segregation and organelle distribution to the daughter cells. Cytokinesis requires the cell to solve a spatial problem and a temporal problem (to coordinate cytokinesis with mitosis). Defects in the spatiotemporal control of cytokinesis may cause cell death, or increase the risk of tumor formation [ S. pombe model, giving references to reviews addressing aspects of fission yeast biology that are beyond the scope of this overview. As their common name implies, cells of the fission yeast Schizosaccharomyces pombe divide by medial fission, reminiscent of cell division of animal cells. The cells take the form of a cylinder capped by hemispherical ends. During interphase, cells grow mainly at their tips, with cell length being a measure of cell cycle progression. Upon commitment to mitosis, cells stop elongating and reorganize the actin and tubulin cytoskeletons in preparation for nuclear and cell division [McCully and Robinow,S. pombe. This has led to the use of this simple model system for studies addressing the regulatory and mechanistic aspects of cytokinesis, which have provided many insights into the regulation of this critical stage of the cell cycle. Cytokinesis is regulated by a group of protein kinases known as the septation initiation network (SIN). The SIN is essential for cytokinesis, and is coregulated with mitotic events, to ensure the coordination of mitosis and cytokinesis (see below). S. pombe. We will briefly introduce the S. pombe has a single, essential mitotic cyclin-dependent kinase (CDK), named cdc2p: its function is conserved in distantly related yeasts such as S. cerevisiae where it is named CDC28, and in human cells, where it is known as CDK1 [Beach et al.,Reorganization of the actin and tubulin cytoskeletons for mitosis and cytokinesis depends upon commitment mitosis and CDK activity [reviewed by Marks et al.,S. pombe [He et al.,S. pombe [reviewed by Martin,Unlike mammalian cells, fission yeast undergo a \u201cclosed\u201d mitosis, in which the nuclear envelope does not break down, and the spindle forms inside the nucleus [Hagan and Hyamsmid1 and pom1 [Huang et al.,The PH-domain protein mid1p, is a key factor of positioning CR at the medial cell cortex [Chang et al.,The second is nuclear export of mid1p at the G2/M transition, which targets mid1p to the cortex proximal to the nucleus [Sohrmann et al.,An elegant study from the Pollard lab [Wu et al.,When cells enter metaphase, F-actin and myosin II (myo2p) independently appear on the medial cortex and then interact with each other to form the CR [Naqvi et al.,for3-null mutant [Nakano et al.,de novo polymerization of F-actin is the dominant pathways for CR assembly in S. pombe. Actin subunits depolymerized from actin patches in cell tips are bound to profilin cdc3p [Balasubramanian et al.,S. pombe differs from animal cells, where assembly of the CR is induced after inactivation of CDK1 and anaphase onset. Formation of the S. pombe CR early in mitosis is mediated by mid1p, which acts as an organizing scaffold for the actin polymerization machinery and myo2p to the future division site. Mid1p has a C-terminal PH domain with low similarity to the metazoan cytokinesis proteins of the anillin family, which directly bind to both F-actin and myosin II [reviewed by D'Avino,In addition to myo2p, an IQGAP-like actin-crosslinking protein rng2p, the formin cdc12p, and the F-BAR protein cdc15p are also incorporated into cortical mid1p nodes in early metaphase, independently of F-actin [Wu et al.,mid1 mutant cells, although CR formation is not initiated at metaphase, myo2p associates with a cable-like F-actin structure elongating from the cell cortex during anaphase, resulting in formation of an abnormally shaped CR, which is frequently displaced from the cell middle [Huang et al.,S. pombe division is symmetrical while another system, probably SIN-mediated, supports CR-assembly. Interestingly, at least two other species of the Schizosaccharomyces genus, S. japonicus and S. octosporus do not have a clear ortholog of mid1p and in these organisms CR assembly is initiated during anaphase [Alfa and HyamsS. pombe, which is required for formation of the septin ring and cell separation in S. pombe [Berlin et al.,In S. pombe ortholog of the conserved CDC14-family of phosphoprotein phosphatases; it is implicated in many cellular processes, and is regulated by phosphorylation and localization [Cueille et al.,S. pombe. The CR is assembled stepwise during mitosis [Wu et al.,Flp1p/clp1p is the Drosophila POLO [Ohkura et al.,plo1, cdc7, or spg1 promotes CR and septum formation from any stage of the cell cycle, uncoupling the usual dependency of cytokinesis upon entry into mitosis [Fankhauser and Simanis,The SIN is a group of protein kinases which are essential for cytokinesis. Signaling requires the activity of three protein kinases, each of which has a regulatory subunit (kinase-regulator); cdc7p-spg1p [Fankhauser and Simanis,The SIN has been implicated in assembly of contractile actin ring (CR) [Hachet and Simanis,S. pombe SPB is composed of cytoplasmic and nuclear components which are separated by the nuclear envelope and connected by fine striations [Ding et al.,Laser ablation of SPBs in fission yeast during mitosis suggests that at least one SPB must be intact during anaphase B for cytokinesis to occur [Magidson et al.,sid4 or cdc11 block association of SIN proteins with the SPB, and prevent SIN signaling. Ectopic activation of the SIN in a sid4 mutant fails to promote septum formation, indicating that SPB association of SIN proteins is important for signaling [Balasubramanian et al.,The SIN proteins associate with the SPB via a tripartite scaffold comprised of ppc89p, sid4p and cdc11p [Chang and Gould,in vitro [Furge et al.,During interphase, spg1p, byr4p, and cdc16p are all observed at the SPB; cdc16p and byr4p are interdependent for localization [Sohrmann et al.,Spg1p in its GTP-bound form interacts with cdc7p during mitosis [Sohrmann et al.,spg1 and cdc7 [Guertin et al.,cdc7 mutants and some alleles of spg1 [Sparks et al.,The protein kinase sid1p and its regulatory subunit cdc14p appear at the new SPB only after the inactivation of CDK and onset of anaphase B [Guertin et al.,Biochemical analysis has shown that cdc16p, spg1p, mob1p, and cdc13p (the mitotic cyclin for cdc2p) all bind to the N-terminal domain of cdc11p [Morrell et al.,par1, the B\u2032 regulatory subunit of PP2A [Jiang and Hallberg,fin1 [Grallert et al.,The transition from the symmetric to the asymmetric configuration of the SIN and the initiation of septation requires inactivation of mitotic CDK [Yamano et al.,zfs1 and scw1 are RNA binding proteins [Beltraminelli et al.,cdc7 mutant at the permissive temperature [Mulvihill et al.,A number of putative regulators of the SIN have been identified genetically; S. pombe SIN in another yeast, Saccharomyces cerevisiae, is called the mitotic exit network (MEN). In addition to a role in cytokinesis, the MEN is required for the inactivation of CDK, and exit from the mitotic state into G1; for reviews, see [Burke,The biological counterpart of the ee Burke,. Many coee Burke,. Some coee Burke,. The kinee Burke,.As mentioned above, the SIN seems to be important for stabilizing the CR after onset of anaphase and to sustain assembly of CR components until cytokinesis is finished. The SIN and flp1p/clp1p, are both implicated in a cytokinesis checkpoint, which blocks the next round of mitosis in response to perturbed assembly of the CR [Le Goff et al.,Overexpression of C-terminal truncated cdc12p, probably corresponding to a dominant active form lacking the auto-inhibitory domain, induces cdc15p- and CR-dependent cytokinesis even in interphase cells [Yonetani and Chang,S. pombe, only a small subset of these can trigger reorganization of F-actin, CR formation and septation when their activity is altered [Matsuyama et al.,Though whole genome-based screening shows that more than 200 proteins are localized to the division site in After the completion of nuclear division, the CR constricts, which is followed by the primary septum synthesis. At present, it is unclear whether CR constriction is an active, motor-driven process, or whether it occurs passively, as the septum is deposited behind it. Previous studies have shown the following: Mutant cells that are unable to assemble a coherent CR deposit septum materials at the cell cortex [Streiblova et al.,S. pombe, the Arp2/3-complex is not required for CR assembly [Wu et al.,Araidopsis thaliana [Boutte et al.,Drosophila early embryo [Cao et al.,S. pombe cells the Rab11-homolog ypt3p localizes to the medial region during cytokinesis, dependent on an intact actin cytoskeleton [Cheng et al.,S. pombe.As the primary septum is synthesized, actin patches accumulate around the region of septation. In ace2 produces multicompartmented cells [Alonso-Nunez et al.,The primary septum is composed of linear chains of 1,3-\u03b2-glucan [Humbel et al.,S. pombe CR with those of mammalian cells, and speculate upon possible mechanisms for its assembly and constriction. We will begin with a brief review of the mechanics of cytokinesis in mammalian cells.In this section, we will discuss the assembly, constriction and disassembly of the CR, with particular emphasis on the mechanical and structural aspects of these processes. We will also compare the properties of the In animal cells, cytokinesis is brought about by membrane ingression, which has been named the cleavage furrow (CF). The CF is thought to be induced by geometrical asymmetry of the cortical tension in the cell. Several mechanisms for CF formation have been proposed [reviewed by Wang,Caenorhabditis elegans oocyte Centralspindlin also reduces Rac GTPase-activity, which inhibits the formation of an Arp2/3-complex-dependent branched actin network and thereby ensures formation of the formin-dependent CR, which is composed of straight F-actin [Canman et al.,C. elegans oocyte [reviewed by Werner and Glotzer,In animal cells, the central spindle and overlap region of astral microtubules (MTs), which extend from opposite spindle poles, promote CR assembly at telophase. Centralspindlin, a protein complex consisting of MKLP1, a kinesin-6 dimer, and Rho-family GTPase-activating protein (RhoGAP) subunits, plays the central role in this system [Mishima et al.,Sulfolobus acidocaldarius [Lindas et al.,Cytokinesis is accomplished by the scission of a \u201cbridge\u201d connecting the daughter cells. Dynamic reorganization of membranes including scrambling of outer and inner sides of plasma membrane, SNARE\u2013mediated vesicle fusions, and membrane scission by ESCRT complex is required for this step [reviewed by Prekeris and Gould,de novo at mitosis (and does not arise from conversion of interphase actin cables or patches), but they are differ significantly with regard to how CR F-actin is formed: from dozens of cdc12p nodes or from a small number of cdc12p spots. Recent studies have shown that the cdc12p spot formation depends on its FH3 domain and the actin-bundling protein ain1p, and is dispensable for CR assembly [Yonetani et al.,In this section, we will address two questions: first, what is the origin of CR F-actin? Second, is the CR formed by a spot/leading cable mechanism, or from a band of nodes? Considerable effort has been devoted to analysis of CR assembly [Vavylonis et al.,de novo assembly. The cortical flow of preformed F-actin is supported by direct observations of flux of fluorescently labeled actin [Cao and Wang,de novo assembly of CR F-actin was observed as accumulation of actin at the equator of blebbistatin-treated mitotic cells where the cortical flow is abolished [Guha et al.,At least two nonexclusive mechanisms seem to be involved in recruitment of actin to the equatorial region during cytokinesis in animal cells: cortical flow and S. pombe cells. The CR actin filaments have mixed polarities, and interdigitating myosin-like thick filaments between them were also observed in animal cells [Sanger and Sanger,S. pombe is discussed below.It is generally accepted that the CR is comprised mainly of actin and myosin II. However, their precise configuration in the ring remains undetermined. EM observations in several dividing cells have revealed that CR F-actin generally aligns parallel with the equator beneath the plasma membrane at the division site [Schroeder,As mentioned above, cortical F-actin filaments oriented perpendicular to CR, which would not be involved in CR constriction according to the purse-string model, are often detectable. Filaments with this orientation have escaped detection by EM observation. Given the high turnover rates of actin and myosin II in CR, this perpendicular F-actin may also be dynamic, probably more than CR F-actin, and hence may be difficult to preserve during severe fixation. Functional dissection of these filaments might substantiate alternative mechanisms for cortical ingression during cytokinesis Wang,. Along sDrosophila and HeLa cells [Field and Alberts,Some actin-binding proteins (ABPs) localized selectively at CR are thought to characterize the special properties of CR F-actin bundles. The actin-bundling protein \u03b1-actinin localizes to the CF in dividing NRK cells and is involved in accumulation of CR F-actin [Mukhina et al.,It is noteworthy that the equatorial accumulation of F-actin is not always evident in dividing cells, whereas ABPs or myosin II are clearly detectable in most cases [e.g., Neujahr et al.,It also remains possible that myosin II contributes to organization of CR F-actin bundles. Myosin II is implicated in rearrangement of the actin cytoskeleton in protruding lamellae of fibroblast cells [Verkhovsky et al.,In the purse-string hypothesis, myosin II motors are presumed to generate a force driving CR constriction via interaction with CR F-actin. Inhibition of the ATPase activity of myosin II using blebbistatin blocks cytokinesis in mammalian cells without affecting CR F-actin assembly [Straight et al.,S. pombe has two myosin II isoforms, myo2p and myo3p , both of which localize to the CR [Bezanilla and Pollard,S. pombe cytokinesis remains unclear. F-actin accumulates at the division site in myo2-null cells but fails to form a ring [Kitayama et al.,myo2-E1 allele), significantly reduces the actin-binding and motor activity of myo2p [Lord and Pollard,myo2-E1 mutant cells, node condensation into a ring abolished at restrictive temperature and is delayed at the permissive temperature [Coffman et al.,myo2-E1 cells constrict twofold slower than those in wild-type cells [Stark et al.,myo3 exacerbates the phenotype of myo2-E1 cells, the two myosin-IIs seem to share overlapping functions in cytokinesis [Motegi et al.,C. elegans, UNC-45 colocalizes with myosin II during cytokinesis in the early embryos [Barral et al.,S. pombe rng3p belongs to the same UCS -family and associates with myo2 and other four myosin proteins cotranslationally [Amorim and Mata,myo2-E1 cells [Lord et al.,Folding of the myosin head domain requires a co-chaperone of the (UNC-45/CRO1/She4p) family of proteins. In Dictyostelium or Drosophila S2 cells, accumulation of individual myosin-II filaments at the equatorial region has been visualized as small rods less than 1 \u03bcm in length by fluorescence microscopy [Yumura and Fukui,One of the observable characteristics of myosin II molecules is to assemble into bipolar thick filaments through their tails [Kaminer and Bell,FRAP analysis has shown that CR F-actin and cortical F-actin turn over rapidly both in fission yeast and mammalian cells [Pelham and Chang,S. pombe cells, the sole ADF, named as adf1p, localizes to the CR and is essential for accumulation of F-actin at the division site during cytokinesis [Nakano and Mabuchi,Xenopus ADF/cofilin, XAC, localizes to the leading edge of the CF in a fertilized egg and also participates in its formation [Abe et al.,In S. cerevisiae, the CR is not essential for cytokinesis, and septation alone manages to carry out the closure of bud neck [Bi et al.,It is unequivocal that in dividing animal cells net inward forces are generated along the equator, whether by the CR or by other mechanisms, and they drive the constriction of the medial cortex [Rappaport,in vitro. Fine and time-resolved EM observation of CR and the medial region of dividing fission yeast cells may also help to address this issue. An attractive model for generation of the contractile force has been proposed, which posits that polymerization and depolymerization of CR F-actin can produce mechanical force for the ring contraction in the presence of end-tracking crosslinkers even without myosin II motors [Zumdieck et al.,Whatever the actual function of CR constriction is, its mechanism(s) is still an open question. As discussed above, the purse-string model should be challenged by examining whether myo2p assembles into minifilaments Schizosaccharomyces pombe has proved to be an informative model for the study of cytokinesis and its coordination with mitosis. Many of the structural components required for cytokinesis are well conserved through evolution, so the fission yeast provides an excellent background in which to test the in vivo effects of defined mutations; when proteins are involved in multiple processes, hypomorphic alleles may permit separation of these functions. Though we have learnt a great deal about how cytokinesis occurs in S. pombe through a judicious mixture of cell biology, genetics and biochemical analysis, it is clear from the foregoing discussion that much remains to be done. Scientifically speaking, the next few years should be very exciting!The fission yeast"} +{"text": "Microorganisms transform inexpensive carbon sources into highly functionalized compounds without toxic by-product generation or significant energy consumption. By redesigning the natural biosynthetic pathways in an industrially suited host, microbial cell factories can produce complex compounds for a variety of industries. Isoprenoids include many medically important compounds such as antioxidants and anticancer and antimalarial drugs, all of which have been produced microbially. While a biosynthetic pathway could be simply transferred to the production host, the titers would become economically feasible when it is rationally designed, built, and optimized through synthetic biology tools. These tools have been implemented by a number of research groups, with new tools pledging further improvements in yields and expansion to new medically relevant compounds. This review focuses on the microbial production of isoprenoids for the health industry and the advancements though synthetic biology. Escherichia coli or Saccharomyces cerevisiae, which serves as a microbial cell factory and recombined control elements were screened to select the E. coli strain that produced sevenfold more mevalonate , human 5-phosphomevalonate kinase (hPMK), yeast 5-diphosphomevalonate decarboxylase (yPMD), and E. coli IPP/DMAPP isomerase , by introducing heterologous genes for IPP isomerase from Nostoc punctiforme, which were inserted into the chromosome of E. coli. This plasmid-free strain created astaxanthin as its only carotenoid at 1.4\u2009mg/g dcw method\u201d to put together multiple genes in a single step, Nishizaki et al. improved lycopene production. \u201cMultiplex automated genome engineering\u201d (MAGE) was proposed by Wang et al. They modified 24 genetic components at once from a degenerate pool of synthetic DNA, achieving a fivefold increase in lycopene production in just 3\u2009days , was achieved was overexpressed to produce \u03b1-santalene, a skin cancer chemopreventative, at 0.21\u2009mg/g dcw and kaurene synthase-like (SmKSL) as well as GGPP synthase (BTS1) and FPP synthase (ERG20) in S. cerevisiae (Zhou et al., Biosynthetic pathways for various diterpenes and sesquiterpenes have also been engineered for improved production through synthetic biology. To maximize production of several sesquiterpenes, Asadollahi et al. replaced the nativeS. cerevisiae, but production levels of the Taxol intermediate, taxadiene, were low. Several changes to taxadiene synthesis in yeast were introduced, including an alternate geranylgeranyl diphosphate synthase from S. acidocaldarius and a codon-optimized taxadiene synthase from Taxus chinensis, ultimately resulting in a 40-fold titer increase to 8.7\u2009mg/l (Engels et al., E. coli as a host, Ajikumar et al. (Application of synthetic biology tools to microbial production of the cancer chemotherapy drug paclitaxel will decrease its cost and increase its availability. Paclitaxel, known as Taxol, is a potent chemotherapy drug, which is very difficult to chemically synthesize (Chandran et al., r et al. divided de novo design of biosynthetic pathways (Ajikumar et al., The past decade has witnessed the potential of synthetic biology to make the microbial isoprenoid production become industrially relevant. However, further improvements in yield and expansion to new medically important compounds can be attained through the development of additional tools. An incomplete understanding of the complexity of biosynthetic pathways limits the ability to fully forward engineer microbial production (Nielsen and Keasling, The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Lower limb injuries in sport are increasingly prevalent and responsible for large economic as well as personal burdens. In this review we seek to determine which easily implemented functional neuromuscular warm-up strategies are effective in preventing lower limb injuries during sports participation and in which sporting groups they are effective.Seven electronic databases were searched from inception to January 2012 for studies investigating neuromuscular warm-up strategies and injury prevention. The quality of each included study was evaluated using a modified version of the van Tulder scale. Data were extracted from each study and used to calculate the risk of injury following application of each evaluated strategy.Nine studies were identified including six randomized controlled trials (RCT) and three controlled clinical trials (CCT). Heterogeneity in study design and warm-up strategies prevented pooling of results. Two studies investigated male and female participants, while the remaining seven investigated women only. Risk Ratio (RR) statistics indicated 'The 11+' prevention strategy significantly reduces overall 0.54 to 0.84) and overuse lower limb injuries as well as knee injuries among young amateur female footballers. The 'Knee Injury Prevention Program' (KIPP) significantly reduced the risk of noncontact lower limb and overuse injuries in young amateur female football and basketball players. The 'Prevent Injury and Enhance Performance' (PEP) strategy reduces the incidence of anterior cruciate ligament (ACL) injuries . The 'HarmoKnee' programme reduces the risk of knee injuries in teenage female footballers. The 'Anterior Knee Pain Prevention Training Programme' (AKP PTP) significantly reduces the incidence of anterior knee pain in military recruits.Effective implementation of practical neuromuscular warm-up strategies can reduce lower extremity injury incidence in young, amateur, female athletes and male and female military recruits. This is typically a warm-up strategy that includes stretching, strengthening, balance exercises, sports-specific agility drills and landing techniques applied consistently for longer than three consecutive months. In order to optimize these strategies, the mechanisms for their effectiveness require further evaluation. Historically, stretching as part of a warm-up strategy before exercise has been strongly advocated to prevent injury . Howeveret al. [et al. [Neuromuscular training programmes are hypothesized to improve joint position sense, enhance joint stability and develop protective joint reflexes, ultimately preventing lower limb injuries. H\u00fcbscher et al. recentlyet al. . However [et al. systematThe aims of this systematic review were: (1) to evaluate the efficacy of functional neuromuscular warm-up strategies which do not require additional equipment in preventing lower limb injury in order to guide clinical and sporting practice; and (2) to identify the common elements of successful strategies in order to guide future research.Embase, SPORTDiscus, Google Scholar, PubMed, ISI Web of Knowledge, Scirus and PEDro were searched for articles from inception to June 2011 and updated in January 2012. Search terms included (movement training OR neuromuscular OR proprioceptive OR proprioception OR plyometric) AND (training OR program OR programme) AND prevent* AND (injury OR injuries). Limits included English language (due to the cost of translation) and human studies. The reference list of retrieved articles was manually checked for potentially relevant studies.Inclusion/exclusion criteria are shown in Table et al. [et al. [A modified version of the nine item van Tulder et al. was used [et al. criteria [et al. . Two indDetails of study design, participant characteristics, interventions, statistical analysis, results and study limitations were extracted and tabulated from each included study by one reviewer (KH). Additionally, two reviewers (KH and CB) extracted data related to participant numbers and injury incidence for the various types of lower limb injuries reported. Review Manager version 5.0 was used to calculate risk ratios (RR) and their 95% Confidence Intervals (CI) for all comparisons as well as to produce forest plots to represent this data visually. The number needed to treat (NNT) was calculated only for variables producing a statistically significant RR . Sensitivity analysis was completed to identify if the use of equipment improved injury prevention. To complete this, the effectiveness of a selection of eight studies, five randomized controlled trials (RCTs) -16 and tThe initial search identified 766 articles Figure . DuplicaTable Details of each study are summarized in Table RRs for the effectiveness of neuromuscular warm-up strategies in preventing undefined lower limb injuries are shown in Figure None of the strategies evaluated were able to produce significant reductions in hip or thigh injuries, with calculated risk ratios shown in Figure P = 0.046). The AKP PTP [RRs for the effectiveness of neuromuscular warm-up strategies in preventing knee injuries are shown in Figure AKP PTP was ableRRs for the effectiveness of neuromuscular warm-up strategies in preventing lower leg and ankle injuries are shown in Figure This systematic review investigated the effectiveness of neuromuscular warm-up strategies for injury prevention. Based on available data a number of strategies appear to be effective in preventing lower limb injuries. Specifically, 'The 11+' strategyThe quality assessment criteria revealed that the studies had various methodological weaknesses affecting their internal validity. Firstly, sample sizes were often too low to evaluate specific injuries . If evaluating the effectiveness of neuromuscular warm-up programmes on more specific injuries, sample size calculations prior to commencement and recruitment of larger samples are recommended. Additionally, future studies should ensure blinding of assessors, concealment of treatment allocation, intention to treat analysis and more adequate randomization procedures to reduce the impact of issues relating to internal validity. External validity was also limited, in particular the applicability of the findings to age groups other than between 13 and 26 years.There is also a need to determine the mechanism of effectiveness of neuromuscular warm-up strategies and determine whether injury reduction is the result of each individual component or due to a combination of exercises. No studies were identified which compared two different components or combinations of neuromuscular warm-up strategies and, in general, programmes targeted varying risk factors associated with a variety of specific injuries. Addressing this through further research will enable more emphasis on effective components of injury-specific interventions and facilitate the development of more successful neuromuscular warm-up strategies for injury prevention, specifically in reference to specific lower limb injuries.et al. [et al. [et al. [et al. [et al. [There is limited homogeneity between the prevention strategies and the methods of recording injury incidence, making data pooling for meta-analysis inappropriate. Injury incidence was reported by a certified athletic trainer , a coachet al. , Gilchri [et al. , LaBella [et al. , Kiani e [et al. and Brus [et al. . This poAdverse effects were only noted in four studies ,28,31,33The effectiveness of three neuromuscular warm-up strategies in preventing the total number of lower limb injuries was evaluated in studies included in this review. Of these, only 'The 11+' and KIPPThe PTP may haveHip and thigh injuries were recorded during the evaluation of two neuromuscular warm-up strategies, 'The 11+' and 'TheKnee injury rates were recorded in all of the nine studies, with six of these recording ACL injuries. Based on available data, four neuromuscular warm-up strategies were found to be effective in preventing knee injuries. These included individual studies showing 'The 11+' and 'HarSuccess of the AKP PTP may relaet al. [et al. [et al. [et al. [et al. [et al. [et al. [Despite investigating the same warm-up strategy , Mandelbaum et al. demonstr [et al. showed o [et al. was a CC [et al. performe [et al. there wa [et al. informed [et al. while th [et al. ,28-34.The PTP , and KLIThe 'HarmoKnee' programmet al. [The effectiveness of four neuromuscular warm-up strategies which did not require additional equipment in preventing lower leg and ankle injuries were evaluated in studies from this review. Based on the results, no neuromuscular warm-up programme was able to reduce lower limb injury risk significantly. However, it should be considered that the KIPP indicateet al. which evet al. results A previous systematic review comparing balance work and neuromuscular exercises revealed that ankle sprains were reduced by 36% and 50%, respectively . AdditioAccording to the present systematic review, several practical neuromuscular warm-up strategies which do not require additional equipment that is not readily available at the usual amateur competition or training venues are effective to varying degrees at preventing lower limb injuries. However, in some instances a large number of participants would need to undertake a strategy before one injury is prevented. This is the case with the PEP strategyImportantly, this systematic review highlights several areas that may account for significantly better injury prevention when incorporating neuromuscular warm-up strategies. These include: (1) incorporation of stretching, strengthening and balance exercises, sports-specific agility drills and landing techniques; (2) completing the strategy for longer than three consecutive months; and (3) completing of the strategy at all training sessions. In addition to these programme specifics, further evaluation of the '11+' programmFurther studies need to determine whether 'The 11+' , KIPP 3, 'HarmoKThe current systematic review identified five practical neuromuscular warm-up strategies which do not require additional equipment and which may effectively reduce the risk of lower limb injuries. Specifically 'The 11+' reduced overall and overuse lower limb injuries and knee injuries in young amateur female football players, the 'KIPP' reduced non-contact overall and overuse lower limb injuries in young amateur female football and basketball players, the 'HarmoKnee' programmThe authors declare that they have no competing interests.KH and CB were the primary initiators of the study while all authors made substantial contributions to data analysis and interpretation. All authors were involved in drafting and revising the manuscript and approved the penultimate version. The final version was approved and submitted by DM.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1741-7015/10/75/prepub"} +{"text": "Cannabis sativa (cannabis) should be considered doping in sports. Results from a 2010 report in the United States the main psychoactive constituent and responsible for the observed toxic effects after smoking, while other cannabinoids are responsible for minor effects, such as cannabinol (CBN), which is 10% as psychoactive as THC Huestis, . THC is The non-psychoactive cannabidiol (CBD) is anxiolytic in humans following a single dose (Zuardi et al.,"} +{"text": "Research in the last two decades has made clear that astrocytes play a crucial role in the brain beyond their functions in energy metabolism and homeostasis. Many studies have shown that astrocytes can dynamically modulate neuronal excitability and synaptic plasticity, and might participate in higher brain functions like learning and memory. With the plethora of astrocyte mediated signaling processes described in the literature today, the current challenge is to identify, which of these processes happen under what physiological condition, and how this shapes information processing and, ultimately, behavior. To answer these questions will require a combination of advanced physiological, genetical, and behavioral experiments. Additionally, mathematical modeling will prove crucial for testing predictions on the possible functions of astrocytes in neuronal networks, and to generate novel ideas as to how astrocytes can contribute to the complexity of the brain. Here, we aim to provide an outline of how astrocytes can interact with neurons. We do this by reviewing recent experimental literature on astrocyte-neuron interactions, discussing the dynamic effects of astrocytes on neuronal excitability and short- and long-term synaptic plasticity. Finally, we will outline the potential computational functions that astrocyte-neuron interactions can serve in the brain. We will discuss how astrocytes could govern metaplasticity in the brain, how they might organize the clustering of synaptic inputs, and how they could function as memory elements for neuronal activity. We conclude that astrocytes can enhance the computational power of neuronal networks in previously unexpected ways. Thanks to those receptors astrocytes are not only capable to respond to the classical neurotransmitters glutamate and \u03b3-aminobutyric acid (GABA) released from synapses [through metabotropic glutamate receptors (mGluRs) mGluR1-5 and GABAB receptors respectively; Pasti et al., 2+ signals through the astrocytic syncytium . Their activation leads to the intracellular activation of phospholipase C (PLC), followed by the production of the messenger molecule inositol trisphosphate transients mediated by channels, exchangers, and transporters whole-cell Ca2+ transients have led to the idea that unlike neurons, astrocytes display exclusively particularly slow responses, and that their signals are not suited to be restricted to small cellular compartments, as happens for example, in dendritic spines. However, in vivo experiments have shown that faster local responses of astrocytes in the somatosensory cortex can occur upon hind limb stimulation channel. TRPA1 mediated spotty Ca2+ signals seem to play an important role in setting the basal intracellular Ca2+ concentration of hippocampal astrocytes . But what is the impact of this on neuronal functioning? Numerous studies in different brain regions have uncovered a multitude of ways in which astrocytes can modulate neuronal excitability and synaptic transmission. In the following section we will review several of these different pathways.The excitability of neurons is one of their most fundamental properties. Dynamic modulation of excitability is a powerful way of implementing state-dependent changes in neuronal computation. Several studies have shown that astrocytes can regulate neuronal excitability. Astrocytes can achieve this through several mechanisms: by regulation of the extracellular ionic composition, by maintaining a tonic extracellular transmitter concentration, by regulation of basal synaptic transmission, and by the induction of phasic events in neighboring neurons. We will shortly discuss these different mechanisms.+. Both hippocampal astrocytes receptor mediated current, and a decrease in neuronal excitability , or long-lasting (long-term plasticity). Short-term plasticity (a change in synaptic strength lasting up to 10s of seconds) is thought to underlie critical computational functions of neuronal networks ions (Dingledine et al., 2+ into the postsynaptic neuron, which can activate second messenger cascades leading to either LTP or LTD (Malenka and Bear, d-serine is a more likely co-agonist for the NMDAR then glycine (Mothet et al., d-serine in the brain seems to be the astrocyte. Astrocytes release d-serine into the extracellular space through vesicular fusion, and this astrocytic d-serine release is necessary to obtain sufficient NMDAR activation for induction of LTP in hippocampus (Yang et al., d-serine release (Zhang et al., Most forms of long-term synaptic plasticity depend on activation of postsynaptic NMDARs. The NMDAR classically is described as a coincidence detector of pre- and post synaptic neuronal activity. This is because for activation it needs both binding of presynaptically released glutamate as well as a postsynaptic depolarization to relieve it from block by magnesium (Mgd-serine as shown in hippocampus was described in the hypothalamic supraoptic nucleus (SON; Panatier et al., d-serine for postsynaptic NMDARs is greatly reduced, leading to a shift in the activity dependence of LTP and LTD (Figure A similar dependence of NMDAR mediated long-term plasticity on astrocyte-derived D Figure .d-serine can actively gate LTP in the rat barrel cortex in vivo (Takata et al., d-serine release from the astrocytes upon activation. These findings highlight the importance of d-serine as a modulator of plasticity both in vitro and in vivo. Furthermore, since cholinergic input into the cortex has been suggested to gate cortical plasticity (Bakin and Weinberger, Interestingly, a recent study showed that astrocytic release of d-serine can also induce synaptic plasticity independent of its role at the NMDAR (Kakegawa et al., d-serine from Bergmann glia. This d-serine subsequently activated postsynaptic \u03b42 glutamate receptors, which in turn caused internalization of postsynaptic AMPARs. Interestingly, interfering with this form of LTD disrupted motor coordination in vivo, showing its relevance for cerebellar development. From the study by Kakegawa et al. (Apart from acting as a NMDAR co-agonist, a recent study has shown that astrocytic release of a et al. it is noa et al. . Furthera et al. . Therefoa et al. and hippin vivo (Navarrete et al., Another gliotransmitter that can mediate long-term plasticity is glutamate. Previously, we mentioned that astrocyte stimulation at hippocampal Schaffer collateral synapses can induce short-term potentiation through astrocytic glutamate release followed by activation of presynaptic mGluRs (Fiacco and McCarthy, in vivo, by application of the cannabinoid receptor agonist \u03949-THC, leads to LTD of Schaffer collateral synapses instead of LTP. This LTD also requires astrocytic glutamate release, but is mediated by activation of postsynaptic NMDARs, followed by endocytosis of postsynaptic AMPARs (Han et al., Additionally, a recent study showed that pharmacological activation of astrocytes 1Rs (Sj\u00f6str\u00f6m et al., 1Rs together governed the induction of t-LTD. We showed that activation of presynaptic NMDARs during induction of t-LTD is mediated by astrocytic glutamate release (Min and Nevian, 2+ signaling. This in turn leads to glutamate release from the astrocyte, which activates presynaptic NMDARs. Activation of the presynaptic NMDARs induces a long-lasting decrease in synaptic release probability, although the signaling cascade downstream from presynaptic NMDAR activation is still unclear (Figure In the neocortex, we have recently shown that astrocytic release of glutamate is necessary for the induction of spike-timing-dependent depression (t-LTD; Min and Nevian, r Figure .2+ signals, and therefore presumably the astrocytic glutamate release, is not correlated with respect to the pre- and post-synaptic action potentials (Min and Nevian, 2+ block of the presynaptic NMDARs to hamper their efficient recruitment. However, it was recently shown that presynaptic NMDARs in the developing sensory cortex incorporate the NR3A subunit, which renders the receptor insensitive to Mg2+ block (Larsen et al., One interesting question rising from these results is how the presynaptic NMDARs are efficiently activated. Because the timing of the astrocytic CaThese results show that astrocytes form a crucial part of the retrograde signaling cascade for induction of t-LTD. Since endocannabinoid mediated forms of LTD occur in numerous brain regions and serve important functions (Heifets and Castillo, 7 receptors. Activation of these postsynaptic receptors leads to insertion of AMPARs, and thereby to a long-lasting postsynaptic increase in the efficacy of excitatory synapses (Gordon et al., 2+ signaling and subsequent release of ATP through mGluR activation. This protocol can lead to a similar long-lasting postsynaptic upscaling of the synaptic efficacy (Gordon et al., Astrocytes also play a role in homeostatic control of synaptic transmission. This was first shown in cultured neurons, where glial release of the cytokine TNF\u03b1 increases the synaptic expression of AMPARs, thereby controlling the weight of all excitatory synapses (Beattie et al., Finally, long-term synaptic plasticity is costly in terms of energy. As mentioned before, astrocytic supply of energy to neurons is essential for maintaining synaptic transmission (Rouach et al., d-serine, they can directly induce synaptic plasticity by releasing glutamate or other gliotransmitters, and they can mediate homeostatic plasticity. In addition, dynamic regulation of metabolic support by astrocytes is crucial for synaptic plasticity.In conclusion, astrocytes play a role in several forms of long-term plasticity. Astrocytes can either set the threshold for LTP/LTD induction by controlling the extracellular concentration of In the previous section, we have discussed how astrocytes can modulate neuronal functioning. Based on the above described astrocyte-neuron interactions, we will summarize some possible roles that astrocytes could play in neuronal computation. We suggest that the functional role of astrocytes adds to the computational power of neuronal networks.d-serine, astrocytes are ideally positioned to mediate metaplasticity. The level of astrocytic d-serine release will determine the possible amount of NMDAR activation and therefore of NMDAR mediated postsynaptic Ca2+ influx upon synaptic activity. Since many forms of LTP and LTD require NMDAR mediated Ca2+ influx for their induction, astrocytes can shift the threshold for LTP and LTD induction (Figure d-serine and therefore, the induction threshold for LTP and LTD (Panatier et al., d-serine release upon astrocyte activation by cholinergic inputs opens the window for subsequent LTP induction in the in vivo neocortex (Figure d-serine release is a physiologically important metaplasticity signal in the brain. It can translate the activity state of the neuronal network into a modulatory signal that determines the corresponding learning rule: during a high activity state due to arousal or heightened attention the neuronal and astrocyte networks are highly active supporting the induction of LTP. Future behavioral studies should determine what the importance of this astrocyte mediated metaplasticity is for learning and memory formation.Metaplasticity is a higher-order form of synaptic plasticity, defined as a change in the ability to induce synaptic plasticity (Abraham and Bear, n Figure . A clearx Figure . Therefo2+ signaling spreads along an astrocytic process and results in the release of gliotransmitters at neighboring synapses. Heterosynaptic short-term plasticity could play a role in switching between synaptic ensembles during information processing. As described earlier, astrocytes mediate heterosynaptic short-term depression at excitatory synapses onto CA1 pyramidal neurons (Zhang et al., Single astrocytes form non-overlapping domains (Bushong et al., Heterosynaptic long-term plasticity has been implicated in the homeostatic control of synaptic inputs to a neuron (Chistiakova and Volgushev, 2+ signaling, directly translates into synaptic plasticity. For example, we found that astrocyte activation with a voltage-clamp depolarization protocol results in increased Ca2+ signaling in the astrocyte, but that this activity alone does not change synaptic transmission strength at excitatory cortical synapses. An additional activation of the presynaptic axon was necessary in order to induce LTD (Min and Nevian, It is important to note that not every activation of an astrocyte, manifested by increases in Ca2+ transients gradually increases during the induction of t-LTD (Min and Nevian, The induction of many forms of synaptic plasticity requires a repeated presentation of a certain stimulus pattern (Petersen et al., 2+ signals in the processes of astrocytes can be used to perform chemical computations similar to linear and non-linear Ca2+ signaling in neurons. Efficient Ca2+ buffering (Neher, 2+ microdomains that limit the release of gliotransmitters in space and time. Only certain patterns of Ca2+ signals might cause a high enough concentration of Ca2+ in the astrocyte processes to trigger vesicular release. This could be accomplished by local Ca2+ buffer saturation and a specific spatial relationship between Ca2+ release sites in the ER and the vesicular Ca2+ sensor triggering exocytosis (Marchaland et al., 2+ signaling in astrocytes is needed to understand their chemical computation.How can an astrocyte act as a thresholding unit? The sophisticated Cag Neher, , receptog Neher, and extrin vivo. However, recent advances in genetics have made it possible to specifically alter astrocyte signaling in vivo while keeping neuronal signaling intact. This has led to some important insights into astrocyte involvement in information processing. For example, it is now clear that astrocyte derived adenosine modulates the build-up of sleep pressure as well as the cognitive effects of sleep deprivation (Halassa et al., 1 receptor, Han et al. (in vivo (Saab et al., in vivo experiments with the subcellular astrocyte-neuron signaling mechanisms as described in this review.As described above, there is ample experimental evidence for an active role of astrocytes in neuronal plasticity. The most important question remaining is the functional relevance of the above described astrocyte-neuron interactions. This has proven very hard to address, since experimental approaches until recently were unable to specifically modify and study the role of astrocytes in neuronal computation and behavior n et al. showed tn et al. . Finallyin vitro experiments and behavioral readouts in vivo is the use of mathematical models. As mentioned earlier, progress has been made with mathematical models of astrocyte Ca2+ signaling. In addition, several models have included bidirectional signaling between astrocytes and neurons on the synaptic level, leading to new insights into the function of this bidirectional signaling. For example, a study by Nadkarni et al. (One possible way to bridge the gap between cellular mechanisms observed with high-resolution i et al. suggestsi et al. . In anoti et al. . Such moAn example of a recently developed neuron-glia network model comes from a recent study by Wade et al. . Here, i2+ dynamics, thereby potentially enhancing the computational power of neuronal networks. The challenge for the future is to understand how and when astrocyte mediated signaling processes are involved in computation in the brain. To answer this fascinating question will require a combination of state-of-the-art experimental techniques with advanced computational modeling.In conclusion, research on the involvement of astrocytes in neuronal signaling in the last years has resulted in a rich array of possible astrocyte-neuron interactions. It is now evident that beyond their permissive role in synaptic and network function astrocytes can perform integration of neuronal signals by means of their CaThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "To the Editor: Kaba et al. (We conducted a retrospective subanalysis of results from a multicenter therapeutic trial assessing HEV seroprevalence among HIV/hepatitis C co-infected patients in France (The difference between our study, which demonstrated low HEV IgG prevalence in IDU patients, even in southern France, and the results from Kaba et al. ( In Response: The letter of Larrat et al. (,\u2013Seroprevalence studies of different populations, especially those with differing immune responses, cannot lead except by chance to the same result. Of note, we recently reported that HEV seroprevalence was 2.3% among injection drug users infected with HIV ("} +{"text": "Dear Editor:et al. about the relationship between down-regulation of osteopontin and breast tumour progression in vivois highly interesting [The article by Chakraborty eresting . Recent et al. in a recent study have reported that nearly 67% of oral squamous cell carcinomas and nearly 54% of all oral carcinomas in situ lesions are immunoreactive for osteopontin [et al. suggests that osteo-pontin may be an important marker of early invasion in lingular squamous cell carcinomas [For instance, Devoll eopontin . Similarrcinomas . In factBesides oral malignancies, osteopontin has also been implicated in the etio-pathogenesis of numerous other systemic malignancies such as non-small cell lung cancers and breast cancers . Clearly"} +{"text": "Dictyostelium discoideum) and plants gene family, which is 70% identical to glycogen synthase kinase-3 from mammals, and rice (LOC_Os05G33050) also possess homologous proteins comprising ARM repeats and a BTB/POZ domain signaling (Samuel et al., AtPUB19 showed that it is upregulated in response to drought, salt, cold and ABA (Liu et al., ATPUB18 as a negative regulator has been put forward in ABA-mediated stomatal closure and drought responses (Seo et al., ATPUB22/23 in Capsicum annum known as CaPUB1 was found to be highly inducible in response to various abiotic stresses such as drought, cold and salt (Cho et al., A biological role for the U-box/ARM protein Nicotiana, two U-box/ARM proteins NtCMPG1 and tobacco ACRE276 and their functional homolog in Arabidopsis, AtPUB17 has been implicated as positive mediators of plant defense and stress signaling (Gonzalez-Lamothe et al., Another report suggested the role of AtCHIP, an Arabidopsis U-box/ARM protein in response to extreme temperature conditions. Subsequently, AtCHIP was reported to be involved in the ABA stress signaling pathway by mediating interaction with protein phosphatase 2A (Yan et al., On the basis of facts described above, it can be concluded that animal and plant ARM repeat proteins share many resemblances. Therefore, it is possible that at least some transcription effectors involved in Wnt signaling are evolutionary conserved. These elements include nuclear accumulation in response to extracellular signal, phosphorylation and degradation. Apart from the common response, plants possess specific signaling pathways mediated by ARM proteins. In plants, ubiquitination is critically involved in the function of ARM proteins. The proliferation of \u03b2-catenin-like ARM proteins in plants suggest their significance in the regulation of diverse biological fuctions in them. Further study of these proteins in plants would contribute to our understanding of the molecular factors involved in response to abiotic stress.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Obesity is a major public health concern and there are increasing calls for policy intervention. As obesity and the related health conditions develop during childhood, schools are being seen as important locations for obesity prevention, including multifaceted interventions incorporating policy elements. The objective of this systematic review was to evaluate the effects of policies related to diet and physical activity in schools, either alone, or as part of an intervention programme on the weight status of children aged 4 to 11\u00a0years. A comprehensive and systematic search of medical, education, exercise science, and social science databases identified 21 studies which met the inclusion criteria. There were no date, location or language restrictions. The identified studies evaluated a range of either, or both, diet and physical activity related policies, or intervention programmes including such policies, using a variety of observational and experimental designs. The policies were clustered into those which sought to affect diet, those which sought to affect physical activity and those which sought to affect both diet and physical activity to undertake random effects meta-analysis. Within the diet cluster, studies of the United States of America National School Lunch and School Breakfast Programs were analysed separately; however there was significant heterogeneity in the pooled results. The pooled effects of the physical activity, and other diet related policies on BMI-SDS were non-significant. The multifaceted interventions tended to include policy elements related to both diet and physical activity (combined cluster), and although these interventions were too varied to pool their results, significant reductions in weight-related outcomes were demonstrated. The evidence from this review suggests that, when implemented alone, school diet and physical activity related policies appear insufficient to prevent or treat overweight or obesity in children, however, they do appear to have an effect when developed and implemented as part of a more extensive intervention programme. Additional evidence is required before recommendations regarding the focus of policies can be made and therefore, increased effort should be made to evaluate the effect of policies and policy containing intervention programmes upon weight status. Obesity among children is associated with significant psychological, social and health consequences including insulin resistance, cardiovascular disease, low self-esteem and poorer education and employment outcomes ,2. The rTo date the systematic reviews which have examined the effect of obesity related school policies have evaluated diet and physical activity outcomes rather than weight status -16. JaimGuidance from The Cochrane Collaboration and the National Health Service Centre for Reviews and Dissemination informed the development of the review protocol, which is available upon request ,18.Two search strategies were developed for this systematic review, one for diet related and one for physical activity related policies [Dialog Datastar], British Education Index [Dialog Datastar], Australian Education Index [Dialog Datastar], Cumulative Index to Nursing and Allied Health Library (CINAHL Plus) [Ebscohost], and The Cochrane Library [Wiley Online]. The search strategy was developed in Medline ; source of funding; ethics approval; recruitment; summary characteristics of the study population; details of the intervention ; treatment of any control group; definition of obesity; duration of follow-up/exposure; and results. Standard tools were used to assess the quality of the studies -24. The d) in body mass index standard deviation score (BMI-SDS), were calculated for each study using standard calculations and the R package MAd ), where \u2018m\u2019 is the average number of participants in each school and \u2018ICC\u2019 is the intra-cluster correlation . An ICC et al.,31. Furt2 statistic [Random effects meta-analysis of each cluster was undertaken in Stata to obtaitatistic .The study identification process and reasons for exclusion are illustrated in Figure\u00a0Ten studies examined diet related policies, five physical activity related policies, and six examined policies with both diet and physical activity related components (henceforth known as combined policies) Table\u00a0. Despiteet al.[All the included studies examined BMI as an outcome categorised as overweight or obese, or adjusted to standard deviation scores (BMI-SDS), percentiles (BMI%), growth rates or Healthy Fitness Zone (BMIHFZ) . The Heaet al.. One stuet al..Study quality is summarised in Table\u00a0Due to the nature of policy interventions, the randomised controlled trials and controlled before and after studies could not meet some of the quality criteria generally applied to these study designs. Blinding of the outcome assessment may not always have been possible and, for some studies, loss to follow up was greater than 20%. However, each study employed a valid design, in terms of the use of second sites as controls, random allocation and protection from contamination.The demographics and baseline weight status of the participants of each study are listed in Table\u00a0Fifteen studies utilised the Center for Disease Control and Prevention (CDC) 2000 BMI reference categories and an additional paper appears to have used this categorisation but did not report it . Two stuet al.[Key results from each study are presented in Table\u00a0et al. and Thomet al.. Table\u00a04Thirteen studies evaluated diet related policies, including five evaluating the NSLP ,44,47,50The pooled result of participation in the NSLP was a small non-significant rise in BMI-SDS -0.193 to 0.269) and the effects of these policies have not been combined. However, the individual results and effect sizes are detailed in Table\u00a0et al.[et al.[et al.[Six studies evaluated policies with both diet and physical activity related components, one of which did not report the quantitative results just the direction and significance and therefore effect sizes could not be calculated -59,63,64le\u00a0et al. reportedl.[et al.,64. The l.[et al., was siget al.[The aim of this systematic review was to examine the effect of school diet and physical activity related policies upon anthropometric outcomes among children aged 4\u201311\u00a0years. Twenty-one studies were identified which examined a range of policies which were clustered as either diet related or physical activity related or both (combined policies) for analysis. Within the diet related policies cluster, eight studies evaluated the NSLP and SBP and as these policies target a subset of the population they were analysed separately from the other diet related policies. The NSLP was associated with a non-significant rise in BMI-SDS results, whereas the SBP was associated with a significant decrease in BMI-SDS and the other diet related policies were associated with a non-significant decrease in BMI-SDS, however, significant heterogeneity remained in the NSLP and SBP sub-clusters reducing the validity of these results Figures\u00a0, 3 and 4et al. as imporet al.,26,31. Get al.. Five ofet al.,47,55,632\u2009=\u200965.6%. Millimet and Tchernis [A strength of this review was the broad search strategy. School policy evaluation may be reported by a variety of disciplines and inside and outside of peer-reviewed journals and therefore through the variety of databases searched, the grey literature search and the inclusion of literature such as dissertations all the relevant studies were sought. Primarily, this demonstrated that there is a paucity of scientific evaluations of school policies as only 21 eligible studies were identified from the 6,894 retrieved, yet among the eligible studies where were a variety of designs, quality and policies which impinge upon the review. Among the 21 studies reviewed only five utilised experimental study designs which prevented the consideration of causal pathways in this review Table\u00a0. Loss toTchernis and MillTchernis used comet al.[et al.[et al.[et al.[et al.[et al.[et al.[et al.[In order to calculate effect sizes, assumptions about the outcome correlations in studies using independent and non-independent samples were made; these assumptions were relaxed in a sensitivity analysis, reported in Additional file et al. and Choml.[et al. both foul.[et al. found a l.[et al. found thl.[et al. argue thl.[et al. argue thl.[et al. recentlyl.[et al. using rol.[et al. with thol.[et al. did not l.[et al.,67.et al.[This review evaluated the effect of school policies upon an objective measure of weight status (BMI-SDS) unlike previous reviews which have evaluated physical activity and diet outcomes, which may be more subjective -16. Theret al. found siet al.. SubsequThe evidence from this systematic review suggests that diet and physical activity related policies need to be located within more complex approaches to preventing childhood obesity which focus on multiple factors and at multiple levels of influence as advocated by the Centers for Disease Control and Prevention guidelines . No poliThe complex web of factors which influence weight have been illustrated in the obesity systems map which also highlights the range of levels of influence from micro to macro . Within 95% CI: 95% confidence interval; AVHPSP: Annapolis valley health promoting schools program; BMI: Body mass index; BMI%: Body mass index percentile; BMIHFZ: Body mass index healthy fitness zone; BMI-SDS: Body mass index standard deviation score; CDC: Centers for disease control and prevention; ECLS-K: Early childhood longitudinal study \u2013 kindergarten cohort; FMI: Fat mass index; HAES: Health at every size; ICC: Intra-cluster correlation; IOTF: International obesity task force; MeSH: Medical subject headings; NSLP: National school lunch program; PE: Physical education; SBP: School breakfast program; UK: United Kingdom; USA: United States of America; USDA: United States Department of Agriculture.The authors declare that they have no competing interests.AJW was involved with the conception and design of the review, undertook the searches and participated in the study identification, data extraction and quality assessment, he then undertook the analysis and drafted the manuscript. WEH was involved with the conception and design of the review, advised on and supervised the analysis and assisted with drafting the manuscript. CAW was involved with the conception of the study and had input into the final manuscript. AJH participated in the study identification, data extraction and quality assessment and proofread the final manuscript. SL contributed to the drafting of the final manuscript and interpretation of the results. KMW was involved with the conception and design of the review, participated in the data extraction and quality assessment, assisted with the interpretation of results and drafting of the final manuscript. All authors read and approved the final manuscript.Search strategy.Click here for fileSensitivity analysis.Click here for fileEffect size calculations.Click here for fileDiet related policies meta-analysis with Rappaport, Daskalakis and Sendacki [ replacing Foster, et al. [.Sendacki replacin, et al. .Click here for file"} +{"text": "Exome sequencing now allows us to reliably identify these mutations using a single genomic test, and we have recently implemented exome sequencing in the diagnostic follow-up of these patients.Germline coding de novo mutations in genetic disease and the associated risk factors such as local genomic structure and paternal age. Next, I will describe our recent work using a diagnostic family-based exome sequencing approach to test this de novo mutation hypothesis in 100 patients with unexplained ID, as well as targeted follow-up studies of several candidate ID genes in 750 additional patients. A total of 79 unique coding de novo mutations were identified and validated in 52 patients. Damaging de novo (n = 10) as well as X-linked maternally-inherited (n = 3) mutations were detected in known ID genes, resulting in a minimal diagnostic yield of 13% in this cohort. In addition, potentially causative de novo mutations in novel candidate ID genes were detected in 22 patients. For three of these candidate genes, recurrent de novo mutations were identified in patients with similar phenotypes, confirming that they are true ID genes. To further expand the possibilities of exome sequencing for mutation detection, we have recently implemented automatic CNV detection on exome data, and compared its performance to that of high-resolution genomic microarrays. This analysis shows that exome sequencing can reliably detect the large majority of pathogenic de novo CNVs, responsible for approximately 15% of ID.In this presentation, I will first discuss the role of de novo mutations therefore represent an important cause of ID, and exome sequencing is an effective diagnostic strategy for their detection.In conclusion,"} +{"text": "PLoS Genetics, Joyce et al. report the first comprehensive RNAi screen of genes regulating somatic chromosome pairing in Drosophila Many generations of biologists have been intrigued by the myriad structures that eukaryotic chromosomes can adopt and have questioned how their form relates to function Drosophila presents a unique opportunity for identifying molecular regulators that establish, maintain, and antagonize homolog pairing because its homologous chromosomes are almost always paired in somatic cells. Metz described somatic cell homolog pairing in 1916 Suppressor of Hairy Wing (Su(Hw)) and Topoisomerase II as pairing promoting factors Cap-H2, was shown to be necessary and sufficient to antagonize pairing of homologs in the context of polytene chromosomes Cap-H2 mutant Drosophila males have chromosome unpairing defects in meiosis I, also providing evidence for a Cap-H2 anti-pairing activity Recent evidence has raised the exciting possibility that both pairing and anti-pairing forces may act on chromosomes to regulate the spatial juxtaposition of homologous sequences . Two preThe ability to perturb homolog pairing by RNAi depletion The pairing and anti-pairing genes code for cell cycle, protein turn-over machinery, and chromatin proteins, among others. Previous studies suggested that cell cycle regulation and chromosome pairing are related by showing that entry into S-phase and G2/M disrupt pairing Perhaps the most exciting broad conclusions from this study are that chromosome pairing is much more complicated and dynamic than anyone had anticipated, and that an abundance of \u201cpairing promoting\u201d and \u201canti-pairing\u201d factors provide opposing forces. The authors suggest that the degree of homolog pairing in somatic cells, at the gene level and at the whole chromosome level, is likely determined by the relative activities of pairing and anti-pairing factors . It is n"} +{"text": "Surgery, radiotherapy and chemotherapy are universally recognized as the most effective anti-cancer therapies. Despite significant advances directed towards elucidating molecular mechanisms and developing clinical trials, cancer still remains a major public health issue. Recent studies have showed that cancer stem cells (CSCs), a small subpopulation of tumor cells, can generate bulk populations of nontumorigenic cancer cell progeny through the self-renewal and differentiation processes. As CSCs are proposed to persist in tumors as a distinct population and cause relapse and metastasis by giving rise to new tumors, development of CSC-targeted therapeutic strategies holds new hope for improving survival and quality of life in patients with cancer. Therapeutic innovations will emerge from a better understanding of the biology and environment of CSCs, which, however, are largely unexplored. This review summarizes the characteristics, evidences and development of CSCs, as well as implications and challenges for cancer treatment. The former can be divided into two progeny stem cells or differentiated progenitors and thus controlling the self-amplification of stem cells, whereas the latter produces one differentiated progenitor and another daughter [in vivo, and the microenvironment plays an important role in the function preservation of stem cells [Stem cells, which are rare in most tissues, are defined as cells with the ability to perpetuate themselves by self-renewing and to differentiate into a variety of specialized cells in a tissue or organ ,2. Both daughter . Generaldaughter . Stem ceem cells .CSCs are a small subpopulation of tumor cells with an infinitely proliferative potential existing in tumor tissues . They plCSCs and stem cells have a host of similar characteristics, such as self-renewal, indefinite self-replication, asymmetric cell division, generating a large number of differentiated cells and expressing specific molecules ,18. Addiet al.[+/CD38\u2212) [+/CD38\u2212 cells can form tumors that phenotypically resemble the patient\u2019s original tumor [Growing evidence has shown that tumors are derived from and maintained by a rare population of dysregulated stem cells. The CSC hypothesis was first raised by Mackillop et al. in 1983.+/CD38\u2212) . After tal tumor ,24, indial tumor . This noal tumor , lung [2al tumor ,28, liveal tumor , ovary [al tumor , colon [al tumor , pancreaal tumor [Further evidence of CSCs comes from histology and immunocytochemistry studies. For example, many tumors are very heterogeneous and contain multiple cell types native to the host organ. Heterogeneity is commonly retained by tumor metastases, which implies that the cell that produced them had the capacity to generate multiple cell types. Ginestier et al. showed tl.[et al. confirmel.[et al., brain [l.[et al., and livl.[et al. were alsl.[et al., are botl.[et al.,48. In 2ma (NPC) . Furtheret al.[et al.[According to label-retaining cell (LRC) trial, adult stem cells can be identified based on their ability to retain nucleoside analog, such as bromodeoxyuridine. In accordance with this principle, Zhang et al. found thl.[et al. confirmeet al.[Growing evidences suggests the existence of a dynamic equilibrium and bidirectional conversion between CSCs and cancer progenitors . On the et al. demonstrin vivo, whereas most daughter cells exited from the cell cycle are not able to proliferate in vitro; (2) Differentiation to a limited extent in vitro, and the capacity can be analyzed by special surface markers at the different differentiation stages. During this process, the cellular morphology was often bizarre and maturation is incomplete. Another study [Cancer progenitor cells display low a self-renewal capacity and a higher probability of terminal differentiation compared with CSCs . Variouser study showed t+ stem cells from CML patients were insensitive to STI571 in vitro, thereby these immature Ph+ progenitor cells can survive, while the overall sensitivity of CML CD34+ progenitor cells to STI571 is mainly determined by cell cycle status [Growing evidences indicated that drug-targeted therapies to control tumors either at CSCs or cancer progenitor cells level exhibited different sensitivity. Chronic myeloid leukemia (CML) stem cells were insensitive to tyrosine kinase inhibitors like imatinib, dasatinib and nilotinib, while sensitive to leukemia progenitor cells \u201362. The e status .high/+ CD24low/\u2212 expression was found in breast tumor-initiating stem-like cells. However, it was not clear whether CD44 and CD24 consistently distinguished tumorigenic from non-tumorigenic cells. Subsequently, these CSC-like cells were verified intrinsically resistant to conventional chemotherapy [et al.[et al.[+ stem and early progenitor cells lost their CD133 expression, giving rise to late progenitors and finally differentiated progeny. These lineage programs for cell fate determination can be restricted by PcG proteins, such as Bmi1, which regulates tumor initiation in CD133+ stem and early progenitor cells, while regulates tumor maintenance of proliferation, differentiation and cell fate determination in CD133\u2212 proliferative progenitors. Likewise, Stewart et al.[Increasing evidences showed that the surface protein markers expressed by CSCs and cancer progenitor cells were somewhat dissimilar. In 2003 , CD44higotherapy and ioniotherapy . Jiang ey [et al. suggestel.[et al. showed trt et al. found th+/CD38\u2212 cells, in leukemia is similar to normal hematopoietic progenitor cells [To date, the cell of origin of CSCs remains to be a pendent and troubled problem around the world. There are two hypotheses for the origin of CSCs . One staor cells . The eviIt has been proposed that CSCs and normal stem cells can interconvert into each other. The more important consequence of this event is that normal stem cells can generate CSCs that ultimately induce a new tumor. Emerging evidence has supported this notion, as CSCs share many properties of normal stem cells. For examples, both have the capacity of self-renewal and non-directional differentiation potential, and many classic cancer related signal transduction pathways also regulate the development of normal stem cells. In this scenario, cancer cells could simply utilize the existing stem cell regulatory pathways to stimulate their self-renewal. In addition, both stem cells and CSCs have telomerase activity and amplified telomere repeats, while most adult human somatic cells lack detectable telomerase. Another theory associates stem cells with the formation of tumors, which is most often related with tissues with a high rate of cell turnover. In these tissues, it has long been expected that stem cells are responsible for tumor formation. Tissue with fast renewal, such as epithelial tissue and those of the hematopoietic system, are sites with high incidence of cancer. The faster tissues renew, the higher the rate of mutation that will occur during replication and transcription. Although it is not clear which target cells mutate and transform to tumors, experimental data obtained from a variety of tumors show that certain colon cancers and leukemia result from an accumulation of multiple mutations of stem cells . Due to Some researchers presume that CSCs may be obtained by the mutation of committed progenitor cells with an ability of self-renewal. For example, leukemia stem cells can be transformed from granulocyte-macrophage progenitors with the assistance of MLL-AF9 fusion protein . Anotheret al.[+ cells could generate additional lgr5+ cells as well as all other adenoma cell types, thus exhibiting activity of CSCs in mouse intestinal adenomas [et al. found that the yellow fluorescent protein (YFP) could been expressed in around 1% of basal papilloma epithelial cells in mice, and these YFP-labeled tumor cells were capable of generating all cell types that comprised the tumor [Despite the lack of direct experimental evidence, some studies show that CSCs may be the fusion of stem cells and other cells . These net al.. The CSCet al., which het al., A549 luet al., colon cet al. and glioet al.. Terminaet al.\u201383. Howeadenomas ; (2) theadenomas ; (3) usihe tumor .At present, the molecular mechanisms underlying regulating the development of CSCs remain to be unexplored. Various signaling pathways have been suggested, and some of them are reviewed as follows.The Notch signaling pathway is a highly conserved cell signaling system present in most multicellular organisms, which regulates widely the development and homeostasis of vertebrate and invertebrate embryos and adult individuals through the local interaction between cells, and controls how cells respond to intrinsic or extrinsic developmental cues that are necessary to unfold specific developmental programs . Notch aStudies indicate that Notch signaling is likely to be implicated in the pathogenesis of many human tumors, including leukemia and pancWnt proteins are secreted signaling molecules of Wnt signaling, and nuclear \u03b2-catenin function as a key mediator. One indicator of Wnt pathway activation is the nuclear accumulation of its main effector \u03b2-catenin, which is one component of a transcriptional activation complex that includes members of the T-cell factor/lymphoid enhancer factor (TCF/LEF) family of DNA binding proteins . In normet al.[et al.[Wnt signaling pathway regulates many developmental processes through transcriptional regulation and its et al. demonstrl.[et al. documentl.[et al.,112.in vivo[+ glioma CSCs [et al.[in vitro (7). The signal transducer and activator of transcription 3 (STAT3) is a crucial transcriptional regulator involved in tumorigenesis. Inhibition of STAT3 with specific inhibitors or targeting STAT3 with specific shRNAs disrupts proliferation and maintenance of GSCs [Other molecular pathways or factors that play a critical role in the development of CSCs include following: (1) mTOR signaling pathway, which is frequently aberrantly activated in human cancers, and significantly correlated with biological cell behaviors . Recent in vivo. In addiin vivo; (2) Fibin vivo. In addiin vivo and gastin vivo. Studiesin vivo. Using toma CSCs . In addioma CSCs ; (4) Recoma CSCs suggest oma CSCs ; (5) Epioma CSCs . This nooma CSCs ; (6) Baos [et al. in 2008 of GSCs ,125.Once a cancer has been diagnosed, treatments vary according to cancer type and severity. Surgery, radiation therapy, chemotherapy or hormonal therapy represents traditional approaches designed to remove or kill rapidly-dividing cancer cells. However, there has been hardly any substantial progress with new therapies regarding clinical endpoints, despite significant advances in molecular mechanisms of cancer. Cancer remains a major public health issue. Conventional anti-cancer treatments target the more mature cancer cells that form the bulk of the tumor, but do not target the CSCs, which are relatively quiescent and intrinsically resistant, thus possibly accounting for treatment failures . To target al.[Tumor metastasis is a complex process, and is also the main cause of the death of cancer patients in clinic. It is the key to improve the prognosis of patients by removing CSCs selectively with no significant toxicity . Severalet al. confirm et al.; (3) Somet al.. In breaet al.. Subsequet al.. Cell diet al., especiaet al.,140; (4)et al..et al.[Another way to control the tumor progression is to induce differentiation of CSCs. Study by Piccirillo et al. showed aet al.. The CSCet al.. The bioet al.[et al.[et al.[Recent studies have found that CSCs are the main reason for tumor growth, recurrence and metastasis . Dingli et al. in 2010 et al.. (3) Manet al.. (4) Meal.[et al. and Lianl.[et al. have obsl.[et al.,152,153,l.[et al..Despite recent clinical studies have begun to monitor the behavior of CSCs during chemotherapy, it is still an urgent requirement of more clinical studies to assess how responses to therapy correlate with CSC biomarkers. Development of new CSC-targeted strategies is currently hindered by the lack of reliable markers for the identification of CSCs and the poor understanding of their behavior and fate. Although cancers represent a major therapeutic challenge, with better understanding in the CSCs, the more specific markers to look for this lethal disease. There is no doubt that the application of CSC theory to study the tumorigenesis mechanisms will lead a paradigm shift in the cancer research and the understanding of the essence of cancer, supplying a new way to effectively diagnose tumor sites and find functional proteins as potential therapy targets."} +{"text": "VSG by RNA polymerase I (Pol-I), with silencing of other VSGs, and periodic switching of the expressed gene, typically via DNA recombination with duplicative translocation of a new VSG to the active site. Thus, telomeric location, epigenetic controls and monoallelic transcription by Pol-I at an extranucleolar site are prominent features of VSGs and their expression, with telomeres, chromatin structure and nuclear organization all making vitally important contributions to monoallelic VSG expression control and switching. We discuss VSG transcription, recombination and replication control within this chromosomal and sub-nuclear context.African trypanosomes are lethal human and animal parasites that use antigenic variation for evasion of host adaptive immunity. To facilitate antigenic variation, trypanosomes dedicate approximately one third of their nuclear genome, including many minichromosomes, and possibly all sub-telomeres, to variant surface glycoprotein (VSG) genes and associated sequences. Antigenic variation requires transcription of a single Given the important role of tracts of imperfect 70\u2009bp repeats in VSG recombination reactions (Fig.\u2009VSG switching (Glover et\u2009al., Although RAD51-dependent mechanisms of T.\u2009brucei nuclear DNA replication and suggest a link with antigenic variation. The first link between the DNA replication machinery and VSG transcriptional control was based on studies of T.\u2009brucei ORC1/CDC6 (Godoy et\u2009al., VSGs in insect-stage cells (Tiengwe et\u2009al., et\u2009al., et\u2009al., procyclin loci in bloodstream-form cells following knockdown of MCM-binding protein (Kim et\u2009al., T.\u2009brucei replication has yet to be demonstrated. These findings implicate DNA replication in VSG transcriptional control, but the basis of this, and in particular whether there is a common mechanistic action of ORC1/CDC6 and MCM-binding protein, remains unclear. Association of ORC1/CDC6 with telomeres (Benmerzouga et\u2009al., P.\u2009falciparum and other organisms, both at telomeres and elsewhere (Sasaki and Gilbert, T.\u2009brucei ORC1/CDC6 is remarkably small relative to Orc1 orthologues and lacks the N-terminal bromo-adjacent homology domain involved in binding HP1, which acts in heterochromatin-mediated silencing in other organisms (Flueck et\u2009al., et\u2009al., A number of studies are providing insights into the machinery, co-ordination and regulation of VSG expression are also possible. Loss of ORC1/CDC6 is likely to reduce the number of replication origins and hence replication efficiency, which in turn is likely signalled by the cell-cycle checkpoint machinery; in other eukaryotes ORC mutations trigger a Rad9-dependent checkpoint, arresting cells in S-phase (Ide et\u2009al., T.\u2009brucei, the equivalent histone residue, H3K76, is di-or tri-methylated by two enzymes, DOT1A and DOT1B, respectively (Janzen et\u2009al., et\u2009al., VSG transcriptional control and VSG switching dynamics present in dot1b mutants, may be due to a link between replication and checkpoint signalling (Stockdale et\u2009al., VSG expression and switching may relate to chromosome dynamics after replication. ORC in other eukaryotes is implicated in sister chromatid cohesion; pre-replication complexes can direct loading of cohesin, and ORC provides a cohesin-independent route for sister chromatid association in budding yeast (Diaz-Martinez et\u2009al., T.\u2009brucei cohesin knockdown, which also causes elevated BES switching (Landeira et\u2009al., Two less direct explanations for the roles of ORC1/CDC6 and perhaps MCM-binding protein in VSG BESs (Boothroyd et\u2009al., et\u2009al., VSGs in their previously transcribed or silent states clearly depends upon the replication process. However, mechanistic data are currently lacking here, and we do not yet know the timing, rate or direction of replication through BESs or other VSG loci.Other studies also suggest that DNA replication acts in antigenic variation. The DNA DSBs found within the VSG allelic exclusion and recombination, both essential aspects of antigenic variation in T.\u2009brucei, are critically dependent upon the telomeric environment. Emerging evidence also reinforces the importance of distinct chromatin territories within the nuclear space, although cause or consequence is less certain here. The sub-telomeric context likely provides an environment that experiences more frequent breaks, which allowed T.\u2009brucei to effectively co-opt and potentially modify a natural response to DNA breaks to achieve efficient antigenic variation. These typically heterochromatic loci also facilitated the massive expansion of the VSG gene family without multi-gene expression. Modifications have been achieved through exploiting minichromosomes to increase the maximum telomeric VSG-count by approximately 10-fold, by expansion of recombinogenic 70\u2009bp repeats flanking VSG genes and, potentially, through BRC-repeat expansions within BRCA2. Clusters of large numbers of silent telomeric VSGs likely now facilitate the homology search and improve access to templates for repair. T.\u2009brucei has also co-opted the Pol-I machinery for VSG expression, leading to the formation of a novel extranucleolar, telomere-associated Pol-I compartment.The available evidence indicates that VSG loci, and it will be important to determine the cis-regulatory sequences, the trans-acting factors and how they interact to drive VSG exclusion and switching. An approach focussed on T.\u2009brucei homologues of DNA repair, transcription regulatory and chromatin-associated factors identified in other systems has been fruitful. However, an important goal for the future is to seek factors that play more direct and specific roles in VSG expression control, some of which may represent exploitable drug-targets. Such factors should further illuminate the mechanisms underlying monotelomeric VSG expression and recombination, the processes that make T.\u2009brucei such a persistent parasite.There are a complex variety of chromatin states that could impact transcription, recombination and replication at"} +{"text": "Digestive malignancies remain one of the leading causes of cancer-related death worldwide despite the fact that increasing clinical and biological knowledge has emerged. Among all the newly discovered cancer cases, colorectal cancer, stomach cancer, liver cancer, and esophagus cancer rank in the front and the mortality rate for them also tops the list. The poor prognosis of digestive tumors is partially due to late diagnosis, delayed initiation of treatment, and unsatisfactory reaction to cancer therapies. Many molecular markers have been discovered for early diagnosis and better therapeutic outcomes from which we do benefit a lot. However, diagnosis and treatment of alimentary cancers require further optimization. We believe further study of novel molecular targets for early detection and better treatment would be helpful to reduce mortality rate and to improve the prognosis of malignant diseases of digestive system.In this current issue, we focus on recent advances in the research of novel molecular targets which would help reveal the possible mechanism of tumorigenesis, progression, metastasis, and recurrence of digestive malignancies. The potential value of these molecular targets in cancer therapy is also highlighted. We present 10 articles on novel molecular targets in digestive system of which six articles discuss the molecular markers in hepatocellular carcinoma, three articles discuss the molecular makers in gastric carcinoma, and one makes a comprehensive review on one anticancer target in digestive system cancer therapy.In the paper entitled \u201cH-Ras oncogene expression and angiogenesis in experimental liver cirrhosis,\u201d by G. \u00d6. Elpek et al. evaluated the relation between H-Ras expression and angiogenesis in liver cirrhosis which can progress to liver carcinoma. The oncogene H-Ras is elevated in liver cirrhosis and correlates significantly with angiogenesis.\u201cLEPREL1 expression in human hepatocellular carcinoma and its suppressor role on cell proliferation\u201d by J. Wang et al. found that LEPREL1 was downregulated in hepatocellular carcinoma (HCC) tissues both in mRNA and protein levels, and the down-regulation was not associated with conventional clinical parameters of HCC. LEPREL1 could serve as a potential tumor suppressor gene by inhibiting HCC cell proliferation.The research paper \u201cExpression of potential cancer stem cell marker ABCG2 is associated with malignant behaviors of hepatocellular carcinoma\u201d by G. Zhang et al. found that ABCG2 could probably function as a liver cancer stem cell marker because of its close relationship with tumorigenesis and also because it could promote cell proliferation, drug resistance, and metastasis. This molecule may represent an attractive target for the innovation of cancer stem cell-directed therapy for HCC.in vitro and in vivo,\u201d S. He et al. found that the interference of NET-1 could enhance the anticancer effect of sorafenib. The interference of NET-1 could lead to impaired ability of proliferation and migration and could induce apoptosis in HCC cell line. NET-1 may be a promising molecular target to develop adjuvant therapy in combination with the only effective targeted drug, sorafenib for HCC.In \u201cStudy of RNA interference targeting NET-1 combination with sorafenib for hepatocellular carcinoma therapy Another two articles talked about the function of microRNA in HCC. X.-Y. Huang et al. showed that miR-29 was upregulated in HCC and correlated with poor outcomes of HCC. It might function by promoting cell proliferation and inhibiting cell apoptosis. The work by Z. Wang et al. clarified the association of miR-499 and miR-34b/c polymorphisms with susceptibility to HCC and got to the final conclusion that rs3746444 was not associated with susceptibility to HCC while rs4938723 was associated with increased HCC risk.\u201cMast cells positive to tryptase and c-kit receptor expressing cells correlates with angiogenesis in gastric cancer patients surgically treated\u201d by M. Ammendola et al. studied the angiogenesis in gastric cancer and found that MCTP and c-kitR-EC correlated positively with microvascular density. Drugs against c-kitR and tryptase could be promising agents in antiangiogenic therapy.\u201cSignificance of glutathione peroxidase 1 and caudal-related homeodomain transcription factor in human gastric adenocarcinoma,\u201d J. J. Han et al. demonstrated GPX1 and CDX2 might participate in the carcinogenesis, differentiation, and progression of gastric adenocarcinoma, and CDX2 might be an independent prognostic factor.\u201cIndirect comparison showed survival benefit from adjuvant chemoradiotherapy in completely resected gastric cancer with D2 lymphadenectomy\u201d by Q. Yang et al. confirmed the role of adjuvant chemoradiotherapy in D2-resected gastric cancer patients with discussion of underlying molecular mechanism of this benefit.The review article \u201cPP2A-mediated anticancer therapy\u201d by W. Chen et al. made a general review of the tumor suppressor PP2A by focusing on PP2A structure and the possible mechanism of its participation in anticancer therapy.In summary, this special issue presents several intriguing achievements in the field of novel molecular targets in digestive malignancies which we hope could be utilized in the future for early diagnosis and treatment."} +{"text": "Pichia anomala, Acinetobacter spp. and Pseudomonas putida; specialists such as Dunaliella salina, Saccharomyces cerevisiae, Lactobacillus spp. and other lactic acid bacteria; freshwater autotrophs Gonyostomum semen and Microcystis aeruginosa; obligate anaerobes such as Clostridium acetobutylicum; facultative pathogens such as Rhodotorula mucilaginosa, Pantoea ananatis and Pseudomonas aeruginosa; and other extremotolerant and extremophilic microbes such as Aspergillus spp., Salinibacter ruber and Haloquadratum walsbyi. Some microbes, such as Escherichia coli, Mycobacterium smegmatis and Pseudoxylaria spp., exhibit characteristics of both weed and non-weed species. We propose that the concept of nonweeds represents a \u2018dustbin\u2019 group that includes species such as Synodropsis spp., Polypaecilum pisce, Metschnikowia orientalis, Salmonella spp., and Caulobacter crescentus. We show that microbial weeds are conceptually distinct from plant weeds, microbial copiotrophs, r-strategists, and other ecophysiological groups of microorganism. Microbial weed species are unlikely to emerge from stationary-phase or other types of closed communities; it is open habitats that select for weed phenotypes. Specific characteristics that are common to diverse types of open habitat are identified, and implications of weed biology and open-habitat ecology are discussed in the context of further studies needed in the fields of environmental and applied microbiology.Competition between microbial species is a product of, yet can lead to a reduction in, the microbial diversity of specific habitats. Microbial habitats can resemble ecological battlefields where microbial cells struggle to dominate and/or annihilate each other and we explore the hypothesis that (like plant weeds) some microbes are genetically hard-wired to behave in a vigorous and ecologically aggressive manner. These \u2018microbial weeds\u2019 are able to dominate the communities that develop in fertile but uncolonized \u2013 or at least partially vacant \u2013 habitats via traits enabling them to out-grow competitors; robust tolerances to habitat-relevant stress parameters and highly efficient energy-generation systems; avoidance of or resistance to viral infection, predation and grazers; potent antimicrobial systems; and exceptional abilities to sequester and store resources. In addition, those associated with nutritionally complex habitats are extraordinarily versatile in their utilization of diverse substrates. Weed species typically deploy multiple types of antimicrobial including toxins; volatile organic compounds that act as either hydrophobic or highly chaotropic stressors; biosurfactants; organic acids; and moderately chaotropic solutes that are produced in bulk quantities . Whereas ability to dominate communities is habitat-specific we suggest that some microbial species are archetypal weeds including generalists such as: As the collective metabolism and ecological activities of microorganisms determine the health and sustainability of life on Earth it is essential to understand the function of both individual microbes and in their communities. The cellular systems of an insignificant portion of microbial species have been intensively characterized at the levels of biochemistry, cell biology, genomics and systems biology; and a substantial body of (top-down) studies has been published in the field of microbial ecology in relation to species interactions, community succession and environmental metagenomics. There are, however, some fundamental questions that remain unanswered in relation to the behaviour of microbial species within communities: what type of biology, for instance, enables microbes to dominate entire communities and their habitats and thereby determine levels of biodiversity in specific environments?et\u2009al., et\u2009al., et\u2009al., et\u2009al., dominate their respective communities. In the field of plant ecology it is open habitats (such as freshly exposed fertile soil) that facilitate the emergence of weed species both within specific ecosystems and across evolutionary timescales. We propose that weed behaviour is equally prevalent in some microbial habitats, that open microbial habitats promote the emergence of microbial weed species, and that microbial weed biology represents a potent ecological and evolutionary mechanism of change for some microbial species and their communities.Plant species that primarily inhabit freshly disturbed habitats \u2013 known as weeds \u2013 are characterized by vigorous growth; tolerance to multiple stresses; exceptional reproduction, dispersal and survival mechanisms; a lack of specific environmental requirements; production of phytotoxic chemicals; and/or other competitive strategies . The bioThis article focuses on the following questions: (i) what types of substrate or environment facilitate the emergence of dominant microbial species, (ii) are there archetypal weed species that can consistently dominate microbial communities, (iii) what are the stress biology, nutritional strategies, energy-generating capabilities, antimicrobial activities and other competitive strategies of microbial weeds, (iv) which components and characteristics of microbial cells form the mechanics of weed biology, (v) what are the properties of open habitats that promote the emergence of weed species, and (vi) how are microbial weeds conceptually distinct from plant weeds, and other ecophysiological groups of microorganism?et\u2009al., et\u2009al., et\u2009al., et\u2009al., Aureobasidium pullulans, Candida spp., Debaryomyces hansenii, Metschnikowia pulcherrima, Pichia guilliermondii, Pichia manshurica, Rhodotorula mucilaginosa and Zygowilliopsis californica and generalists such as Aspergillus, Pichia and Pseudomonas species , Haloquadratum walsbyi , and Gonyostomum semen and Microcystis aeruginosa (eutrophic freshwater) ability to dominate can be restricted to a specific type of habitat. Most of these microbes do not populate their respective habitat as a one-off or chance event; they can consistently dominate their microbial communities and do so regardless of their initial cell number . Furthermore dominating species span the Kingdoms of life and, collectively, can be observed in most segments of the biosphere; from bioaerosols to solar salterns, oceans and freshwater to sediments and soils, the phyllosphere and rhizosphere, to those with molar concentrations of salts or sugars (see Table\u2009S1). The phenomenon can also be observed during food-production, food-spoilage and waste-decomposition processes where resource-rich habitats are available for colonization (see Table\u2009S1). The materials present in and the microbes living on these substrates are ultimately derived from the natural environment so community successions resulting in single-species dominance are likely to be commonplace in comparable open habitats within natural ecosystems . Environments in which P.\u2009putida is a major ecological player (Table\u2009S1) typically contain an array of chemically diverse aromatics and hydrocarbons that induce chaotropicity-mediated stresses environments including soils, plant necromass, saline and very high-sugar habitats (Table\u2009S1). At least 20 species of Aspergillus and the closely related genus Eurotium are xerotolerant strains are capable of reasonable growth rates close to 0\u00b0C may be attributed, in part, from enhanced energy-generating capability. Aspergillus species (like many fungi) can utilize a range of polyols \u2013 as well as trehalose \u2013 as compatible solutes based on their individual strengths as protectants against osmotic stress, desiccation rehydration, chaotropes etc. accumulates carotenoids in response to light which can protect against oxidative stress that is a potential hazard in salterns exposed to high levels of ultraviolet , also uses the \u2018salt-in\u2019 compatible-solute strategy, and \u2013 along with S.\u2009ruber \u2013 has light-activated protein pumps that generate proton-motive force and thereby boost energy generation can successfully compete with the multitude of halophilic Archaea found in 5\u2009M NaCl environments . This alga preferentially utilizes glycerol as a compatible solute, is able to accumulate glycerol to molar concentrations either primarily use glycerol as a compatible solute or preferentially accumulate this chaotrope at low temperature , the acidic surface-film of sphagnum moss (as well as other plant tissues) and various surfaces within the human body and other animals a polyextremophile that is psychrophilic, highly salt-tolerant, and can growth over the majority of the pH range known to support microbial life ohmeri and Pichia sydowiorum that exhibited the greatest tolerance to the widest range of solute-imposed stresses (Table\u2009D.\u2009hansenii and Hortaea werneckii), and/or otherwise lacked the capacity for vigorous growth on the high-sugar substrate at 30\u00b0C of the parent plant have a favourable water activity and high nutrient availability and are therefore potentially habitable by a wide range of microbes , accumulate a compatible solute (glycerol) that affords protection against this chaotrope (see below), and at the same time generate energy without any loss of ATP-generation efficiency \u2013 which is activated under cellular stress. This regulatory system enables the coordination of an array of stress responses in an energy-efficient manner (Table\u2009S.\u2009cerevisiae responses to osmotic stress [via the high-osmolarity glycerol-response (HOG) pathway], oxidative stress, and challenges to protein and membrane stability are exceptionally efficient which can be prevalent in relatively low-nutrient aquatic and non-aquatic habitats . Despite their copiotrophic phenotype, pseudomonads utilize a number of strategies to maintain growth and metabolism under phosphate-, iron- or nitrogen-depleted conditions metabolic versatility in nutrient utilization can enhance the competitive ability of microbes , and polysaccharides . In order to out-compete other species in such habitats, microbes must utilize these macromolecular substrates that typically have a low bioavailability, require specialist enzymes to degrade, can be toxic, and/or are energy-expensive to catabolize. Facultatively saprotrophic microbes such as Aspergillus, Pichia, Rhodotorula and Acinetobacter species are able to access and catabolize recalcitrant polysaccharides; some weed species can produce exceptional amounts, or high-affinity versions, of inducible saprotrophic enzymes, such as xylanases can retain activity over a wide range of environmental stresses high chaotropicity weeds are able to dominate their habitats due largely to the production of phytotoxic inhibitors that can be exuded from leaves, roots and plant necromass and which inhibit the germination and/or growth of other plant species is unique to microbial weed species Tables\u2009 and 2. Wet\u2009al., et\u2009al., et\u2009al., et\u2009al., et\u2009al., octanol-water <\u20091.95 typically partition into the aqueous phase of cellular macromolecules and can act as potent chaotropic stressors also inhibits the growth of competing species by acidifying the environment which it achieves via the production of a number of organic acids , including efflux pumps is conferred by an array of mechanisms : killer toxins, hydrolytic enzymes and volatile substances have an unusually high resistance to the net chaotropicity of the ammonia, ethanol, butanol, acetone and other chaotropic substances in their habitat that are highly effective at expulsion of hydrocarbons such as toluene as well as many other stressors and toxins are inhibitory to competing species gives an insight into the complexity of the microbial warefare that takes place in open habitats: 57 compounds were detected including terpenes, alcohols, ketones and esters . This is a product of its metabolic versatility, exceptional tolerance to hydrocarbons and other solutes, and antimicrobial activities that emerges as a dominant genus; and in the green layer at lower pH (2\u20132.5), R.\u2009mucilaginosa has the competitive advantage (Table\u2009S1). Phyllosphere, rhizosphere and other habitats can also remain continuously open if they are replenished with organic substances from ions released from soil particles, extracellular polymeric substances and compatible solutes from microbial cells, root diffusates, animal wastes, diverse sources of necromass, etc.The concept of an open habitat is a fundamental, well-established principle in the fields of plant and animal ecology. For microorganisms, open habitats can be defined according to the diversity, interactions and community succession of their inhabitants in contrast to other types of microbial habitat that are defined by physicochemical characteristics, substrate type or nutrient concentration Table\u2009. Hydrocas Tables\u2009 and 6. O\u22121 mole\u22121 chaotropic activity, <\u2009pH\u200911 or\u2009>\u2009\u221215\u00b0C or Aspergillus species . The stress biology, energy-generating capabilities and antimicrobial activities of these species undoubtedly contribute to their success . Several ecophysiological classification systems that are based on microbial growth-phenotype and/or substrate utilization (Table\u2009S2). Microbial generalists can occupy a wide range of ecological niches, and typically achieve this via the ability to utilize a wide range of nutrients and substrates (Table\u2009S2). Conversely microbial specialists, such as S.\u2009cerevisiae, H.\u2009werneckii and many basidiomycetes have specific nutritional and/or physicochemical requirements . By contrast P.\u2009putida appears to be an exceptional example of a microbial weed both in terms of its cellular biology and its ability to dominate diverse types of open habitat.Microorganisms have been previously categorized based on the way in which their phenotype impacts ecological behaviour: as autotrophs and heterotrophs; generalists and specialists; Saccharomyces cerevisiae supports global industries in fermented products, biofuel, biocides (ethanol is also an important disinfectant; McDonnell and Russell, P.\u2009anomala produces a substantial catalogue of biotechnologically useful substances and is employed in production of foodstuffs (see Walker, et\u2009al., et\u2009al., et\u2009al., et\u2009al., et\u2009al., P.\u2009putida is used for the commercial production of bulk and fine chemicals, industrial biocatalysis, and as the basis of many other industrial and environmental biotechnologies (Kruijt et\u2009al., et\u2009al., et\u2009al., et\u2009al., Aspergillus spp., P.\u2009putida and Clostridium spp. play primary roles in the treatment or decomposition of plant matter, oil and hydrocarbons, xenobiotics in polluted soils, sludge and slurry treatments, and other organic wastes (Tables\u2009S2 andAspergillus species are used for production of foods (Rose, et\u2009al., S.\u2009cerevisiae, Rhodotorula spp., P.\u2009anomala, P.\u2009putida, lactic acid bacteria and other weed species invaluable as agents of biological control, for preventing spoilage of drinks, foodstuffs and animal feeds (see Calvente et\u2009al., et\u2009al., et\u2009al., It is irrefutable that a small minority of microbial species recurrently appear as the dominant members of microbial communities; and we suggest here that these microbes have shared phenotypic traits. This implies that open-habitat ecology has impacted the evolutionary trajectories of these microbes, and that the activities of weeds determine microbial diversity in many localities within the biosphere. Whereas microbial weed species are not always useful to humankind see , they mads Rose, , fine chMicrobial weeds are also used as model systems for research, yet key aspects of their biology remain enigmatic. Many plant weeds have exceptional reproduction and dispersal systems and it rCrop plants such as sunflowers, potatoes, wheat and barley are known to have weedy ancestors (Anderson,"} +{"text": "However, in humans there is limited evidence of endocrine disruption caused by EDCs. EDCs are a large group of persistent organic pollutants (POPs), such as polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs), polychlorinated biphenyls (PCBs), and polybrominated ethers (PBDEs), chloronaphtalenes (PCNs), and bisphenol A (BPA), stable, lipophilic pollutants that affect fertility and cause serious reproductive problems. Xenoestrogens are synthetic compounds, but there are also numerous natural molecules in food that exhibit estrogen-mimetic activities. These natural molecules are mainly phytoestrogens isoflavones, and the most consumed are genistein and daidzein. Additionally, certain mushrooms or fungi can contain estrogen-like compounds called mycoestrogens.The potential effect of the so-called endocrine disruptors (EDCs) or xenoestrogens on human health and the proven effect on wildlife have got considerable attention in the scientific community. Endocrine disruption represents one of the most controversial environmental issues despite the fact that many substances, both natural and artificial, have been recognized to interfere with endocrine signaling pathways. Such interactions have been documented both in laboratory animal studies as well as Endocrine-disrupting chemicals: some actions of POPs on female reproduction.\u201dXenoestrogens activities in oocyte, ovary, placenta, and mammary gland and in the consequent serious reproductive problems, including ototoxic action, lack of ovulation, premature ovarian failure (POF), or polycystic ovarian syndrome (PCOS) are discussed in the review E. Gregoraszczuk and A. Ptak \u201cAssessment and molecular actions of endocrine-disrupting chemicals that interfere with estrogen receptor pathways\u201d discuss different molecular actions of some of the major xenoestrogens found in food or the environment and summarize the current models used to evaluate environmental estrogens. This paper is accompanied by clinical study of Caserta et al. \u201cCorrelation of endocrine disrupting chemicals serum levels and white blood cells gene expression of nuclear receptors in a population of infertile women\u201d compares the internal exposure to bisphenol A (BPA), perfluorooctane sulphonate (PFOS), perfluorooctanoic acid (PFOA), monoethylhexyl phthalate (MEHP), and di(2-ethylhexyl) phthalate (DEHP) in serum samples of 111 infertile women and 44 fertile women and analyses levels of gene expression of nuclear receptors as biomarkers of effective dose.G. Kerdivel et al., in their paper \u201cTwo of the papers deal with aspects of alkylphenols action as endocrine disruptors. Association between endocrine disrupting phenols in colostrums and maternal and infant health\u201d showed that most neonates who are exposed to BPA rather than NP or OP via colostrum are recommended continuous biomonitoring of the phenols to clarify their suspected health risk on neonates and pregnant or gestation mothers. Furthermore, A. Hejmej in their paper \u201cPhotoperiod-dependent effects of 4-tert-octylphenol on adherens and gap junction proteins in bank vole seminiferous tubules\u201d evaluating in vivo and in vitro effects of 4-tert-octylphenol (OP) on the expression and distribution of adherens and gap junction proteins, N-cadherin, \u03b2-catenin, and connexin 43 (Cx43), in testes showed that long-term treatment with OP resulted in the reduction of junction proteins expressions independent of FSH indicating that OP acts directly on adherens and gap junction proteins in the testes.The paper by B. Yi et al. \u201cDiverse effects of phytoestrogens on the reproductive performance: cow as a model\u201d review how exposure of soybean-derived phytoestrogens can have adverse effects on reproductive performance in female adults. Authors suggest that these findings should be specially taken into consideration when recommendations are made regarding dietary or therapeutic phytoestrogen intake in humans. Particularly that they are commonly recognized as therapeutic compounds.I. Woc\u0142awek-Potocka et al. in their paper \u201cAction of halowax 1051 on enzymes of phase I (CYP1A1) and phase II (SULT1A and COMT) metabolism in the pig ovary\u201d describe local ovarian metabolism of PCNs in ovarian tissue and suggest that fast activation of phase I enzymes with simultaneous inhibition of phase II enzymes indicates that androgenic action of PCNs on follicular steroidogenesis may partially result from metabolite action occurring locally in ovarian follicles.Polychlorinated naphthalenes (PCNs) are new player as endocrine disruptors. Data concerning their potency and action on ovarian function are scarce. Dr J. Barc et al. in their paper \u201cTo complete the issue, B. Ayala-Garc\u00eda et al. revise current knowledge about the epigenetic mechanisms that underlie the effects of EDs on phenotypic variability and plasticity to stress the value of using the information derived from experiments with EDs to unveil the mechanisms that underlie phenotypic variability and speciation through epigenetic phenotypic plasticity.Taking into account that in this special issue have been published review articles, research articles and clinical studies, we hope that the information published in this special issue enriches the knowledge of our readers and scholars interested in the influence of xenobiotics on human health.Ewa L. GregoraszczukEwa L. GregoraszczukRadmila KovacevicRadmila Kovacevic"} +{"text": "In heart failure (HF), exercise has been shown to modulate cardiac sympathetic hyperactivation which is one of the earliest features of neurohormonal derangement in this syndrome and correlates with adverse outcome. An important molecular alteration related to chronic sympathetic overstimulation in HF is represented by cardiac \u03b2-adrenergic receptor (\u03b2-AR) dysfunction. It has been demonstrated that exercise reverses \u03b2-AR dysfunction by restoring cardiac receptor membrane density and G-protein-dependent adenylyl cyclase activation. In particular, several evidence indicate that exercise reduces levels of cardiac G-protein coupled receptor kinase-2 (GRK2) which is known to be involved in both \u03b21-AR and \u03b22-AR dysregulation in HF. Similar alterations of \u03b2-AR system have been described also in the senescent heart. It has also been demonstrated that exercise training restores adrenal GRK2/\u03b1-2AR/catecholamine (CA) production axis. At vascular level, exercise shows a therapeutic effect on age-related impairment of vascular reactivity to adrenergic stimulation and restores \u03b2-AR-dependent vasodilatation by increasing vascular \u03b2-AR responsiveness and reducing endothelial GRK2 activity. Sympathetic nervous system overdrive is thought to account for >50% of all cases of hypertension and a lack of balance between parasympathetic and sympathetic modulation has been observed in hypertensive subjects. Non-pharmacological, lifestyle interventions have been associated with reductions in SNS overactivity and blood pressure in hypertension. Several evidence have highlighted the blood pressure lowering effects of aerobic endurance exercise in patients with hypertension and the significant reduction in sympathetic neural activity has been reported as one of the main mechanisms explaining the favorable effects of exercise on blood pressure control. It has been generally accepted that regular physical activity is associated with beneficial effects on the cardiovascular system are associated with adverse outcome (Kaye et al., Since it has been demonstrated that myocardial GRK2 levels and activity mirror those measured in peripheral lymphocytes in HF patients (Iaccarino et al., Exercise training in patients with stable HF, can relieve symptoms, improve exercise capacity and quality of life, and reduce disability, hospitalization, and mortality (Piepoli et al., The crucial role of \u03b2-AR dysregulation in the pathophysiology of HF is well established. GRK2, which plays a key role in the regulation of \u03b2-AR, is significantly elevated in human and experimental HF (Ungerer et al., As mentioned above, exercise training appears to reduce autonomic derangement and neurohumoral excitation at rest in HF. The effects of exercise training on adrenergic hyperactivation in HF patients have not been completely clarified. Recently, an important molecular mechanism has been identified that contributes to the sympathetic overdrive of the failing heart. This mechanism involves the upregulation of GRK2 in adrenal medulla of HF animals, which leads to downregulation and G protein uncoupling of the \u03b12-ARs present in the chromaffin cell membranes of the adrenal gland that normally exert negative feedback control on CA turnover (Lymperopoulos et al., Noteworthy, alterations of \u03b2-AR system, similar to those observed in HF, have been described also in the senescent heart (Davies et al., Exercise has been shown to modulate GRK2 levels/activity by reducing levels of this kinase in the heart and, consequently, inducing \u03b2-AR \u201cresensitization.\u201d It has been previously demonstrated in rats that both exercise and \u03b2-blockers reverse \u03b2-AR dysfunction by restoring cardiac receptor membrane density and G-protein-dependent adenylyl cyclase activation (Leosco et al., At vascular level, studies conducted in the aorta and carotid arteries of old rats have shown a reduced \u03b2-AR-dependent vasorelaxation (Chapman et al., In old healthy subjects, it has been demonstrated that physical training ameliorates age-related deterioration of cardiac function in terms of enhanced LV inotropic response to CA (Ehsani et al., The most common form of hypertension is neurogenic hypertension that is associated with sympathetic overdrive, loss of parasympathetically mediated cardiac variability, and excessive angiotensin II activity Esler, . EvidencClinical interventions showing impressive blood pressure lowering effects by targeting reductions in SNS activation (Krum et al., Exercise training could elicit adaptations in the adrenergic system, since SNS is activated during each bout of exercise and repeated activation of SNS may result in an attenuation of sympathetic activity (Grassi et al., SNS overactivity is common in many cardiovascular disease states and is related to a higher incidence of morbidity and mortality. It is widely accepted that exercise training is associated with reductions in SNS activity, whether at rest or during conditions that produced sympathoexcitation, and this effect may represent an important mechanism by which exercise may contribute to long term cardiovascular health. Future studies are needed to further identify the molecular mechanisms that are involved in physical activity dependent changes in the control of SNS activity.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "The objective of this article was to review the effects of xenobiotics on total antioxidant capacity (TAC). Measurement of TAC is appropriate for evaluation of the total antioxidant defenses of blood, cells, and different kinds of tissues and organs. TAC is reduced by alcoholism, smoking, and exposure to radiation, herbicides, carbon monoxide, carbon tetrachloride, lead, arsenic, mercury, cadmium, aluminum, and other toxic elements. The test is also an important tool in evaluating environmental and occupational exposure. The involvement of unstable free radicals and reactive species from oxygen (ROS), nitrogen (RNS), and chlorine in cell and tissue damage is well established , hydroxyl radical (\u2022OH), nitric oxide (NO\u2022), peroxynitrite (ONOO\u2013), though others are not is any molecule that has one or more incomplete orbitals. A FR can gain electrons, oxidizing another atom/molecule or lose them, thus reducing an element. Some reactive oxygen species (ROS) are FR [superoxide anion , proteins (protein peroxidation), nucleic acids (DNA or RNA oxidation), and carbohydrates (glycosilation) (Vladimirov & Proskurnina 2\u2022\u2013) by mitochondria. As a further step, SOD and GPx convert O2\u2022\u2013 into hydrogen peroxide (H2O2). However, as H2O2 is also a toxic reactive oxygen species, it should also be modified to inocuous water and atomic oxygen by the enzyme CAT.Since the 1960's, many research groups have been evaluating oxidative and nitrosative stress and the antioxidant defenses by measuring the activity of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione redutase (GSH) and glutathione peroxidase (GPx). As a consequence of oxidative phosphorylation, 3\u20135% of oxygen in converted to the free radical superoxide anion of a biological sample , food or vegetable extract or of living tissues and organs . Many xenobiotics can be toxic to cells, tissues and organs, constituting environemntal and occupational health problems.et al., et al., et al., et al., et al., In order to know if a therapeutic drug can preserve or consume the antioxidants of a patient, TAC determination is done in medicinal drugs. Thus the perioperative anesthetics dopamine (1080\u00b1162 mmolET/L), propofol (100\u00b118 mmolET/L), dobutamine (80\u00b116 mmol ET/L), and noradrenaline (62\u00b116 mmolET/L) were found to present higher TAC values , one of the most potent liver toxins, presents a toxicological dose-response effect characterized by oxidative stress and lipid peroxidation events that induce reduction of liver TAC, liver degeneration, necrosis and fibrosis are important metal-scavenging proteins protecting cells against cadmium, mercury, zinc and copper cytotoxicity can also reduce plasma TAC by 20% and activating the nuclear antioxidant response element which is further responsible for the increase in TAC levels. This could explain why exposure to magnetic fields has been correlated to increased TAC levels in male workers (Sirmatel et al., et al., Exposure to eletromagnetic radiation of 900MHz from mobile phones induced lipid peroxidation in the hippocampus and brain cortex of rats as well as oxidative stress and histopathological changes in the rats\u2019 endometrium, effects which were reversed by antioxidants (K\u00f6yl\u00fc et al., et al., et al., (It should be noted that the tecniques for determination of TAC in blood and biological fluids are still very recent. Thus potential clinical correlations between TAC and disease are yet in progress. Similarly, in many pathophysiological conditions the relationship with TAC levels has yet to be established. In a study with different degrees of injury, there was no correlation between plasma TAC levels and severity of lesions in patients with burns (Farriol et al., revealedAlcohol, smoking, heavy metals and toxic elements, some pesticides, some occupational exposures, and eletromagnetic and nuclear radiations can decrease TAC, rendering subjects less resistant to oxidative and nitrosative injuries and subsequent diseases. More research is needed to address the role of antioxidant supplementation in xenobiotic exposure and disease prevention."} +{"text": "To the Editor: Anthrax is a global zoonotic disease, but human infections are rare in countries of Western Europe. During 2009\u20132010, a total of 119 (47 laboratory-confirmed) drug-abuse\u2013related cases of anthrax were reported in the United Kingdom and Germany user; the disease was described in 1 person who died in Norway in 2000 plnthracis (7). TheB. anthracis into Western Europe (This reemergence of drug-related anthrax in Europe strengthens the view that heroin may provide a continuing route of entry for"} +{"text": "Since time immemorial, in search for rescue for their disease, people looked for drugs in nature. The traditional use of medicinal plants can lead to the discovery of new potent botanical agents in the treatment of several diseases. Some 7000 natural compounds are currently used in modern medicine; most of these had been used for centuries by traditional healers and the global market value of medicinal plant products exceeds $100 billion per annum. In spite of the development of pharmacological agents for the treatment of chronic diseases, the use of medicinal plants continues to flourish. Over the last century, the drastic changes of human life style and eating habits lead to the emergence of various chronic diseases. The decreasing efficacy of some synthetic drugs and the increasing contraindications of their usage make the usage of natural drugs topical again. Thus, the study of phytotherapy for chronic diseases treatment might yield an excellent return in potential sources of medicinal plants which play vital roles in disease prevention and their promotion and use fit into all existing prevention strategies. In this issue, we aim to present some recent advances in the use of medicinal plants for treating the chronic diseases such as diabetes, cancer, cardiovascular diseases, inflammation, and neurologic disorders. Hydrangea paniculata, slows down the progression of membranous glomerulonephritis by anti-inflammatory effects and inhibiting immune complex deposition,\u201d investigated the renoprotective activity of skimmin, one of the major pharmacologically active molecules present in Hydrangea paniculata. They studied also the underlying mechanisms of the observed renoprotective effects of skimmin in a rat model of membranous glomerulonephritis induced by cationic bovine serum albumin which may be the inhibition of IL1\u03b2 and IL-6 expression. In another study \u201cTraditional Chinese medicine Tang-Luo-Ning ameliorates sciatic nerve injuries in streptozotocin-induced diabetic rats,\u201d D.-W. Zou et al. describe the beneficial effect of Tang-Luo-Ning (TLN), an effective traditional Chinese medicine for the treatment of diabetic peripheral neuropathy (DPN). To illustrate the underlying neural protection mechanisms of TLN, the effect of TLN on electrophysiology and sciatic nerve morphology was investigated in a model of streptozotocin-induced DPN. G. Belcaro et al., \u201cGrape seed procyanidins in pre- and mild hypertension: a registry study,\u201d studied the efficacy of a standardized grape seed procyanidins extract in decreasing blood pressure when associated with nondrug intervention (diet and lifestyle modification) in a controlled registry study involving healthy prehypertensive and mildly hypertensive subjects. The authors supported that the effect on blood pressure adds to the beneficial effects of grape seed procyanidins on the cardiovascular disease phenotype associated with the oxidation of membrane lipids . A study titled by C. C. Velusami et al. demonstrates that both methanol and successive water extracts of Nelumbo nucifera petals had an effect on inhibition of lipid storage in adipocytes and increasing lipolysis. In addition, methanol extract exhibited the concentration dependent inhibitory effect on lipase activity. Furthermore, Nelumbo nucifera petal extracts showed evident agonist and antagonist activity towards serotonin and cannabinoid receptors, respectively, while it showed no effects towards melanocyte concentrating hormone and melanocortin receptors. Another clinical study performed by G. Belcaro et al. \u201cGreenselect Phytosome for borderline metabolic syndrome\u201d demonstrates that Greenselect Phytosome, a proprietary lecithin formulation of a caffeine-free green tea catechin extract, was especially effective for weight/waist changes in a controlled registry study on asymptomatic subjects borderline for metabolic syndrome factors and with increased plasma oxidative stress. A. Hunyadi et al., \u201cMetabolic effects of mulberry leaves: exploring potential benefits in type 2 diabetes and hyperuricemia,\u201d report a series of relevant in vitro and in vivo studies on the bioactivity of an extract of mulberry leaves and its fractions. In vivo antihyperglycemic and antihyperuricemic activity, plasma antioxidant status, in vitro glucose consumption by adipocytes in the presence or absence of insulin, xanthine oxidase inhibition, free radical scavenging activity, and inhibition of lipid peroxidation were analyzed.In a clinical study, \u201cEffect of eucalyptus oil inhalation on pain and inflammatory responses after total knee replacement: a randomized clinical trial,\u201d Y. S. Jun et al. demonstrate the beneficial effects of eucalyptus oil inhalation on pain and inflammatory responses after total knee replacement surgery. S. Zhang et al., \u201cSkimmin, a coumarin from Andrographis paniculata nees for treating lung adenocarcinomas,\u201d Y.-T. Tung et al. have studied the antipulmonary cancer effects of andrographolide in a lung tumor mouse model induced by human vascular endothelial growth factor A165 (hVEGF-A165). The antiangiogenesis and chemotherapeutic potential of andrographolide may provide a cure for pulmonary tumors in the future. Z.-Z. Meng et al., \u201cEffect of Xiaoyaosan decoction on learning and memory deficit in rats induced by chronic immobilization stress,\u201d observed the effect of Xiaoyaosan (XYS) decoction on chronic immobilization stress- (CIS-) induced learning and memory deficit in rats from behaviors and changes of proteins in hippocampus. The findings suggested that XYS decoction may be helpful in reversing CIS-induced learning and memory deficit by increasing the levels of postsynaptic density protein 95 and synaptophysin on the hippocampal nerve synapses and improving synaptic plasticity.In addition, known bioactive constituents of mulberry were identified and quantified. A study performed by H.-M. Zhao et al., \u201cSi Shen Wan inhibits mRNA expression of apoptosis-related molecules in p38 MAPK signal pathway in mice with colitis,\u201d demonstrated that Si Shen Wan, a formula of traditional Chinese medicine used to treat ulcerative colitis, allergic colitis, and chronic colitis, effectively inhibited mRNA expression of apoptosis-related molecules in p38 MAPK signal pathway to downregulate colonic epithelial cells apoptosis in colonic mucosa from mice with colitis. In another study \u201cTherapeutic potential of andrographolide isolated from the leaves of Garcinia origin (\u2013)-HCA,\u201d summarizes the update of chemical constituents, significance of in vivo/clinical antiobesity effects, and the importance of the current market potential of Garcinia/hydroxycitric acid especially as a potential supplement for weight management and as antiobesity agent. A study realized by Y. Liang et al., \u201cEffects of the Chinese traditional prescription Xiaoyaosan decoction on chronic immobilization stress-induced changes in behavior and ultrastructure in rat hippocampus,\u201d demonstrated the potential mechanism of Xiaoyaosan (XYS) decoction's antidepressant-like effect in \u03b1-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors related to synaptic plasticity in the hippocampus rats induced by chronic immobilization stress. The study demonstrates that XYS decoction may produce an antidepressant-like effect, which appears to be involved with AMPA receptors related synaptic plasticity of hippocampus. In another study \u201cCitrus Bergamia Risso elevates intracellular Ca2+ intracellular stores in human umbilical vein endothelial cells,\u201d G. H. Seol et al. demonstrate that Citrus Bergamia Risso mobilizes Ca2+ from primary intracellular stores via Ca2+-induced and IP3-mediated Ca2+ release and affect promotion of Ca2+ influx, likely via an store-operated Ca2+ mechanism. This finding supports the potential roles of bergamottin in cardiovascular function. J. Hoscheid et al., \u201cInhibitory effect of the hexane fraction of the ethanolic extract of the fruits of Pterodon pubescens Benth in acute and chronic inflammation,\u201d confirm the anti-inflammatory activity of the hexane fraction of an ethanolic extract of Pterodon pubescens Benth. The anti-inflammatory activity was measured with increasing doses of the hexane fraction by using a carrageenan-induced rat model of pleurisy and a rat model of complete Freund's adjuvant-induced arthritis. The results of biochemical, hematological, and histological analyses indicated a significant decrease in glucose, cholesterol, and triglycerides levels and reduction in the number of total leukocytes and mononuclear cells. The study demonstrates also the absence of toxicity for the doses used. H. Park et al., \u201cAmpelopsis Radix protects dopaminergic neurons against 1-methyl-4-phenylpyridinium/1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced toxicity in Parkinson's disease models in vitro and in vivo,\u201d have demonstrated that Ampelopsis Radix has neuroprotective effects with antioxidant mechanisms in Parkinson's disease models. The standardized extract of Ampelopsis Radix protected dopaminergic neurons by inhibiting reactive oxygen species generation in vitro, showed potent radical scavenging activities in vitro, and protected the dopaminergic neurons in the brain in the mouse Parkinson's disease model leading to motor improvements. Another paper by H. Ha et al., \u201cAntiatopic dermatitis effect of Artemisia iwayomogi in dust mice extract-sensitized Nc/Nga mice,\u201d demonstrates the anti-inflammatory and antiatopic dermatitis effects of Artemisia iwayomogi both in vitro and in vivo. Artemisia iwayomogi inhibited the nitric oxide and histamine productions in RAW264.7 and MC/9 cells. Furthermore, isochlorogenic acid A, chlorogenic acid, and scopoletin were demonstrated to be the major components of this plant. Additionally, in the mice, the topical application of Artemisia iwayomogi reduced the dermatitis scores in the dorsal skin and ears and reduced the plasma levels of IgE. E. Panzarini et al., \u201cAdministration dependent antioxidant effect of Carica papaya seeds water extract,\u201d demonstrate that Carica papaya seeds water extract is potentially useful for protection against oxidative stress. The authors have assessed the antioxidant activities of the Carica papaya seeds water extract against hydrogen peroxide oxidative stress in human skin Detroit 550 fibroblasts. An in vivo study by M.-H. Chen et al., \u201cAntidiabetic and antihyperlipidemic effects of Clitocybe nuda on glucose transporter 4 and AMP-activated protein kinase phosphorylation in high-fat-fed mice,\u201d demonstrates that amelioration of diabetic and dyslipidemic state by Clitocybe nuda extract in high-fat-fed mice occurred by regulation of GLUT4, glucose-6-phosphatase, and AMP-activated protein kinase phosphorylation. The plant extract decreased hepatic glucose production in the liver and enhanced glucose uptake in skeletal muscle. This study presents a deep analysis of mechanisms of action involved in the antidiabetic and antihyperlipidemic activities of Clitocybe nuda.A review conducted by L. O. Chuah et al., \u201cUpdates on antiobesity effect ofAfter the first volume of this special issue published in 2012, we hope that this issue will present valuable information for scientists and clinicians."} +{"text": "These genes are thought to be functionally important for optimal viral replication and persistence in infected individuals. Primate lentiviruses can be classified by the composition of these accessory genes. While viruses of the HIV type1 (HIV-1) group have vpx gene derived from HIV-2 GL-AN clone (Kawamura et al., Although PPM consisting of seven consecutive prolines has been demonstrated to be required for efficient HIV-2 Vpx translation, thereby acquiring viral infectivity in macrophages, the effects of PPM mutations on the degradation of Vpx in cells was not formally analyzed as yet (Fujita et al., Various expression plasmids were transfected into human 293T cells (Lebkowski et al., Proteasomal degradation is generally triggered by the polyubiquitin modification of lysine residues in a protein. There are three lysines in the Vpx of HIV-2 GL-AN clone (Khamsri et al., In total, proteasomal or lysosomal degradation does not account for the extremely low expression level of Vpx exhibited by the PPM mutants. This is consistent with our previous conclusion that PPM is critical for efficient translation of Vpx (Miyake et al.,"} +{"text": "Assessment of lymph node status is a critical issue in the surgical management of gallbladder cancer. The aim of this study was to compare the anatomical location of positive nodes, number of positive nodes, and lymph node ratio (LNR) as prognostic predictors in gallbladder cancer.\u03c72 scores calculated with the Cox proportional hazards regression model.We conducted a retrospective analysis of 135 patients with gallbladder cancer who underwent a radical resection with regional lymphadenectomy. A total of 2,245 regional lymph nodes were retrieved . The location of positive nodes was classified according to the AJCC staging manual (7th edition). \u2018Optimal\u2019 cutoff values were determined for the number of positive nodes and LNR based on maximal P\u2009<\u20090.001), number of positive nodes , and LNR as significant prognostic factors. Multivariate analysis identified number of positive nodes as an independent prognostic factor ( P\u2009=\u20090.004); however, location of positive nodes and LNR failed to remain as an independent variable.Lymph node metastasis was found histologically in 59 (44%) patients. The \u2018optimal\u2019 cutoff values for the number of positive nodes and LNR were determined to be three nodes and 10%, respectively. Univariate analysis identified location of positive nodes is valid for stratifying patients based on the prognosis after resection. Lymph node status is an established prognostic factor in various gastrointestinal malignancies-7. Thereet al.[et al.[In the cases of gallbladder cancer, both the American Joint Committee on Cancer and the et al. first reet al.) in patiet al.. Howeverl.[et al. were the\u03c72 scores calculated by the Cox proportional hazards regression model.The current study compared the prognostic power of positive node location , numberFrom May 1982 to January 2009, 148 consecutive patients underwent a radical resection for gallbladder cancer in the study department, defined as a resection of both the primary tumor and regional lymph nodes. Thirteen patients with an invasive primary malignant tumor in other organs were excluded, leaving 135 patients for this retrospective study. They included 94 women and 41 men with ages ranging from 37 to 85 years.en bloc regional lymph node dissection. Late-stage diseases often required more extensive resections such as major hepatectomy , pancreaticoduodenectomy (the Whipple procedure or pylorus-preserving procedure), or major hepatectomy combined with pancreaticoduodenectomy , duodenum (n\u2009=\u20094), portal vein (n\u2009=\u20093), stomach (n\u2009=\u20091), and inferior vena cava (n\u2009=\u20091). Among the total of 135 patients, 111 underwent an initial radical resection and 24 underwent a radical second resection after a prior simple cholecystectomy for presumed benign disease.en bloc. In the patients who underwent a pancreaticoduodenectomy, the right portion of the superior mesenteric node group was also dissected together with the above node groups. In some patients with early-stage disease, advanced age, or comorbid diseases, a less aggressive regional lymphadenectomy was performed at the discretion of the individual surgeons. In this series, 48 patients with suspected (or confirmed) regional nodal disease also underwent a dissection of the paraaortic lymph nodes [The regional lymph nodes of the gallbladder included the cystic duct, pericholedochal, posterior superior (posterosuperior) pancreaticoduodenal, retroportal, right celiac, and hepatic artery node groups,14,17. I artery),14,17.All pathological findings were documented by using the AJCC cancer staging manual (7th edition). The priImmediately after resection, the surgeon(s) retrieved lymph nodes from the node-bearing adipose tissues of the fresh surgical specimen, and grouped them according to location. A total of 2,829 lymph nodes were retrieved from the 135 patients. A representative section, 3-\u03bcm thick, was cut from each lymph node retrieved, and the nodes examined for metastases on routine histological examination using hematoxylin and eosin.The number of positive lymph nodes as well as the total lymph node count (TLNC) was recorded for each patient. Paraaortic lymph nodes (if any) were not included in the TLNC, and any metastases detected in these lymph nodes were categorized as distant metastases and designated as pM1. Thus, iThree parameters were used to assess the nodal status in individual patients: the location of positive lymph nodes, the number of positive lymph nodes, and LNR. The location of positive nodes was classified into three categories: pN0, pN1, pN2, according to the AJCC cancer staging manual (7th edition). LNR wasThree patients died post-resection during a hospital stay, giving an in-hospital mortality rate of 2%. Adjuvant treatment after resection was administered at the discretion of the individual surgeons. Thirty-six patients received oral administration of 5-fluorouracil or its derivatives. Eight patients received intravenous administration of 5-fluorouracil alone or in combination with other agents. Six patients received intravenous administration of gemcitabine. No patients received adjuvant radiotherapy.Patients discharged home were followed regularly in outpatient clinics every one to six months for at least five years, with a median follow-up period of 146 months. At the time of disease status assessment, 53 patients had died of tumor recurrence and 19 patients had died of other causes with no evidence of tumor recurrence. One patient was alive with recurrent disease, and the remaining 62 patients were alive without the disease.Medical records and survival data were obtained for all patients. The survival time in each patient was defined as the interval between the date of the definitive resection and the date of the last follow-up or death. Only deaths from tumor recurrence were treated as failure cases in the analysis of disease-specific survival (DSS). The Kaplan-Meier method was used to estimate cumulative DSS rates, and the log rank test was used to evaluate differences between groups.\u03c72 scores, which were calculated using the Cox proportional hazards regression model. Eleven conventional variables were found to be significant by univariate analysis was used for all statistical evaluations. All tests were two-tailed, and A total of 2,245 regional lymph nodes were retrieved from the 135 patients, with TLNC per patient ranging from 3 to 55 . Of the study patients, 59 (44%) had a total of 252 positive lymph nodes; the number of positive nodes ranged from 1 to 26 per patient, and LNR ranged from 2.6% to 93% .\u03c72 score, the \u2018optimal\u2019 cutoff value was three nodes for the number of positive nodes , number of positive lymph nodes , LNR , pM classification, histological type, histological grade, lymphatic vessel invasion, venous invasion, perineural invasion, and residual tumor status as significant prognostic factors and the 59 patients with TLNC\u2009>\u200916 P\u2009=\u20090.10; Table2P\u2009=\u20090.707).We then focused on a subgroup of 76 patients without nodal disease (pN0) for further survival analysis. Even in this subgroup of patients, no significant difference in DSS was noted between 51 with TLNC\u2009\u2264\u200916 and 25 with TLNC\u2009>\u200916 recommended \u2018analysis of a minimum of three lymph nodes\u2019 for accurate staging of gallbladder cancer. Howeveret al. and Mayo[et al. disclose[et al.. The abo[et al.-5,12,18.et al.[et al.[et al.[et al.[In contrast, Japanese hepatobiliary surgeons, including us, maintain an aggressive attitude toward regional lymphadenectomy for gallbladder cancer,13,14,23et al. and Mayo[et al. independ[et al. and Negi[et al. independ[et al.,18) is wet al.[et al.[et al.[et al.[In various gastrointestinal malignancies, evaluating a limited number of lymph nodes may result in an underestimated number of positive nodes, leading to \u2018stage migration\u2019 -5,12,18.et al., Lee et [et al., Sakata [et al., and Sie[et al. for pancet al.[\u03c72 scores calculated by the Cox proportional hazards regression model and the et al. suggesteet al.), of poset al.). In addThe current study has several limitations: the retrospective nature of the analysis, the relatively small number of patients spanning a long period of time, some variability in the degree of nodal dissection, and the short follow-up time for some patients. We believe, however, that these limitations did not greatly affect the results of the study as the differences between groups were too marked to have resulted from bias. In addition, the role of the number of positive nodes in assessing the nodal status for gallbladder cancer is now more clearly defined than previously based on the current study. Our results thus provide useful information for accurately staging nodal disease, predicting prognosis after resection, and selecting candidates for adjuvant chemotherapy after resection. The current study also emphasizes the need to retrieve a larger number of lymph nodes than ever in rThe number of positive lymph nodes better predicts the outcome after resection than either location of positive lymph nodes or LNR in gallbladder cancer, provided that lymph node evaluation is sufficient. Dividing the number of positive lymph nodes into three categories is valid for stratifying patients based on the prognosis after resection.Written informed consent was obtained from the patients for publication of this report.AJCC, American Joint Committee on Cancer; DSS, Disease-specific survival; LNR, Lymph node ratio; TLNC, Total lymph node count.The authors declare that they have no competing interests.YS conceived the study and drafted the manuscript. JS helped to draft the manuscript and performed statistical analyses. TW performed chart review and follow-up of the study cohort. TO helped with chart review and patient follow-up. YA provided histological data. KH was responsible for the whole study and participated in its coordination. All authors read and approved the final manuscript."} +{"text": "Synovial fluid is a viscous solution found in the cavities of synovial joints. The principal role of synovial fluid is to reduce friction between the articular cartilages of synovial joints during movement. The presence of high molar mass hyaluronan (HA) in this fluid gives it the required viscosity for its function as lubricant solution. Inflammation oxidation stress enhances normal degradation of hyaluronan causing several diseases related to joints.This review describes hyaluronan properties and distribution, applications and its function in synovial joints, with short review for using thiol compounds as antioxidants preventing HA degradations under inflammation conditions. The human skeleton consists of both fused and individual bones supported and supplemented by ligaments, tendons, and skeletal muscles. Articular ligaments and tendons are the main parts holding together the joint(s). In respect of movement, there are freely moveable, partially moveable, and immovable joints. Synovial joints , the free.g. those covering the bone ends in the knee joint \u2013 belong to mechanically highly stressed tissues in the human body. At walking, running, or sprinting the strokes frequency attain approximately 0.5, 2.5 or up to 10 Hz.In a healthy synovial joint, heads of the bones are encased in a smooth cartilage layer. These tough slippery layers \u2013 et al.,in vivo.Cartilage functions also as a shock absorber. This property is derived from its high water entrapping capacity as well as from the structure and intermolecular interactions among polymeric components that constitute the cartilage tissue are localized/embedded. Their primary function is to continuously extrude high-molar-mass hyaluronans (HAs) into synovial fluid.The synovial fluid (SF) of natural joints normally functions as a biological lubricant as well as a biochemical pool through which nutrients and regulatory cytokines traverse. SF contains molecules that provide low-friction and low-wear properties to articulating cartilage surfaces.et al.,et al., 2001), hyaluronan (HA) comprised of tissue macrophage A cells, fibroblast-like B cells , it has b2\u2022\u2013, H2O2, \u2022OH) are generated in abundance by synovial neutrophils from RA patients, as compared with synovial neutrophils of osteoarthritis (OA) patients and peripheral neutrophils of both RA and OA patients and/or catalase, which suggests the possibility that there is pathologic oxidative damage to synovial fluid components in RA patients. Dahl et al. to be in sterically favorable equatorial positions while all of the small hydrogen atoms occupy the less sterically favorable axial positions. Thus, the structure of the disaccharide is energetically very stable. HA is also unique in its size, reaching up to several million Daltons and is synthesized at the plasma membrane rather than in the Golgi, where sulfated glycosaminoglycans are added to protein cores in cosmetic surgery. It is reported that injection of such products into the dermis, can reduce facial lines and wrinkles in the long term with fewer side-effects and better tolerability compared with the use of collagen initiated by the HO2.Propagation phase: formation of a peroxy-type C-macroradical of hyaluronan in a process of oxygenation after entrapping a molecule of OFormation of a hyaluronan-derived hydroperoxide via the reaction with another hyaluronan macromolecule.Formation of highly unstable alkoxy-type C-macroradical of hyaluronan on undergoing a redox reaction with a transition metal ion in a reduced state.et al.,et al.,et al.,et al.,et al.,Termination phase: quick formation of alkoxy-type C-fragments and the fragments with a terminal C=O group due to the glycosidic bond scission of hyaluronan. Alkoxy-type C fragments may continue the propagation phase of the free-radical hyaluronan degradation reaction. Both fragments are represented by reduced molar masses preserving the redox status of cells and protecting tissues against damage caused by the elevated reactive oxygen/nitrogen species (ROS/RNS) levels, by which oxidative stress might be indicated.In vitro. High molecular weight hyaluronan samples were exposed to free-radical chain degradation reactions induced by ascorbate in the presence of Cu(II) ions, the so called Weissberger's oxidative system. The concentrations of both reactants were comparable to those that may occur during an early stage of the acute phase of joint inflammation balance and regulates signaling pathways during oxidative stress/conditions. GSH is mainly cytosolic in the concentration range of ca. 1\u201310 mM; however, in the plasma as well as in SF, the range is only 1\u20133 \u00b5M , another significant precursor of the GSH biosynthesis, has broadly been used as effective antioxidant in a form of nutritional supplement ions to Cu(I), forming N-acetyl-l-cysteinyl radical that may subsequently react with molecular O2 to give O2\u2022\u2013 , when added at the beginning of the reaction, exhibited a clear antioxidative effect within ca. 60 and 80 min, respectively is a cystine-depleting compound with antioxidative and anti-inflammatory properties; it is used for treatment of cystinosis \u2013 a metabolic disorder caused by deficiency of the lysosomal cystine carrier. CAM is widely distributed in organisms and considered to be a key regulator of essential metabolic pathways , when added before the onset of the reaction, exhibited an antioxidative effect very similar to that of GSH . Moreove"} +{"text": "Recently, the Notch system has been shown to be an important bridge between APCs and T cell communication circuits. In the present review, we discuss recent findings that explore the mechanisms in the linkage of innate and adaptive immunity, including granulomatous formation though TLRs and Notch activation.Granulomas represent a spectrum of inflammatory sequestration responses that may be initiated by a variety of agents, including non-infectious environmental factors and infectious microbial pathogens. Although this reaction is designed to be protective, the associated tissue injury is often responsible for a profound degree of pathology. While many of the mechanisms that sustain the development of the granuloma are enigmatic, it is accepted that the maintenance of this inflammatory process is dependent upon dynamic interactions between an inciting agent, inflammatory mediators, various immune and inflammatory cells, and structural cells of the involved tissue. The best studied of the host-dependent processes during granuloma development is the innate and adaptive immune response. The innate immune response by antigen-presenting cells is initiated quickly to protect from overwhelming pathogens, but with time, can also activate the adaptive immune response. APCs, essential regulators of the innate immune response, can respond to microbial ligands through Toll-like receptors (TLRs), which function in the recognition of microbial components and play an important role to link the innate and adaptive immune responses. CD4 The granulomatous response is a complex host defense mechanism that has evolved to provide containment of infectious and/or environmental agents Warren, . The maiThe initial response to infection is the engagement of the host's innate immune system that triggers a rapid, multifaceted anti-infectious response involving the release of proinflammatory cytokines and eventually leads to the activation of the adaptive immune response -\u03b3, Interleukin (IL)-12, and tumor necrosis factor (TNF) were necessary for lung lesion progression , were shown to be maintained by IL-4, IL-5, and IL-13, hallmarks of a type 2 response or Schistosoma mansoni antigen followed by a pulmonary challenge with sized Sepharose beads coated with a known amount of tuberculin purified protein derivative (PPD) , that promote a dominant Th2 response (Herbert et al., +, 15\u201330% are CD8+ T cells, and there are also about 2%\u03b3\u03b4 T cells (Tsai et al., + T cells in the control of mycobacterial infection has been highlighted by many studies in knockout mice, and also in HIV patients (Ladel et al., The typical granuloma contains mostly macrophages and DCs, surrounded by T lymphocytes, while myeloid DCs also have been found in the granulomas of tuberculosis patients and mouse tuberculin models (Ulrichs and Kaufmann, Innate immunity is the first line of host defense directed against invading pathogens and is designed to maintain host integrity (Si-Tahar et al., M. tuberculosis infection in vivo (Bafica et al., Mycobacterium tuberculosis (Bafica et al., Microbial products, including mycobacterium antigen, activate specific TLRs, which in turn induce specific gene transcription resulting in the up-regulation and secretion of select chemokines and cytokines (Krutzik and Modlin, + Th cells (Kumar et al., Pathogens such as bacteria, helminths, fungi, and viruses are recognized by and activate APCs, which in turn activate CD4in vitro data will require a number of in vivo studies using diverse granulomatous models, including bacterial, parasitic, and fungal, as well as autoimmune models.In addition, it has been shown that Notch proteins are also important in the induction of Th responses (Amsen et al., \u2212/\u2212 mice when compared to wild-type mice, Th2 cytokine levels of IL-4, IL-5, and IL-13 were increased in these knockout mice (Ito et al., \u2212/\u2212 mice and defined a role for TLR9 in the induction of both Notch ligand Dll4 and Th17 expression using both in vivo and in vitro approaches (Ito et al., \u2212/\u2212 mice exhibited significantly larger granuloma formation, following an impaired Th17-like response with decreased expression of Dll4 on DCs from TLR9\u2212/\u2212 mice compared with WT mice. Dll4 was the primary Notch ligand upregulated by mycobacterial infection of DCs in WT mice. When Dll4 was specifically blocked in vivo using anti-Dll4 Ab during mycobacteria-induced pulmonary granuloma formation, Th17 cellular responses were significantly inhibited and larger granulomas were observed. Moreover, in in vitro experiments, anti-dll4 antibody specifically blocked IL-17 production by CD4+ T cells, while overexpression of Dll4 augmented IL-17 production by CD4+ T cells, suggesting that Dll4 plays an important role in promoting Th17 activity during a mycobacterial challenge. In contrast, the observed histologic alterations in lung granuloma development in TLR9\u2212/\u2212 mice also coincided with a significant decrease in lung myeloid DCs, which are crucial to the differentiation of Th17 cells, as well as decreased levels of dll4 on DCs when compared with wild-type mice. This impaired migration of lung myeloid DCs in granuloma was attributed to decreased production of chemokine CCL20. Chemokines constitute a family of structurally related chemotactic cytokines that direct the migration of leukocytes throughout the body under both physiological and inflammatory conditions (Matsushima, in vivo in normal mice via homeostatic proliferation express CCR6 as well as CCL20 (Hirota et al., \u2212/\u2212 mice and in lungs with anti-dll4 Ab is correlated with not only impaired DC migration but also reduced numbers of Th17 cells in lungs during mycobacteria induced pulmonary granuloma formation (Ito et al., While the mechanism of granuloma formation is unclear, this distinct cellular response is considered a histologic hallmark for a protective immune response, involving both innate and adaptive immunity. TLR9 is known to play a role in the regulation of Th1 responses (Bafica et al., Schistosoma mansoni eggs to the lungs (Schaller et al., Our data suggest that an understanding of Dll4 regulation of Th17 responses through Notch may provide mechanistic approaches for modifying and controlling the immune response induced by the Th17 phenotype. Moreover, a number of studies have demonstrated that Notch proteins are important in the induction of Th1 responses. In the presence of functional MyD88, PAMP binding to TLR up-regulates Dll4, which causes the differentiation of na\u00efve Th cells to a Th1 phenotype. In addition, when Dll ligands are overexpressed on APCs or are cross-linked as fusion proteins, they also promote Th1 cell differentiation (Maekawa et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "DING proteins constitute a recently discovered protein family that is ubiquitous in eukaryotes. The structural insights and the physiological involvements of these intriguing proteins are hereby deciphered. ab initio the complete and exact sequence of this 38\u2005kDa protein by utilizing mass spectrometry and X-ray data in tandem. Taking advantage of this first complete eukaryotic DING sequence, a immunohistochemistry study was undertaken to check the presence of DING proteins in various mice tissues, revealing that these proteins are widely expressed. Finally, the structure of a bacterial representative from Pseudomonas fluorescens was solved at sub-angstrom resolution, allowing the molecular mechanism of the phosphate binding in these high-affinity proteins to be elucidated.DING proteins constitute an intriguing family of phosphate-binding proteins that was identified in a wide range of organisms, from prokaryotes and archae to eukaryotes. Despite their seemingly ubiquitous occurrence in eukaryotes, their encoding genes are missing from sequenced genomes. Such a lack has considerably hampered functional studies. In humans, these proteins have been related to several diseases, like atherosclerosis, kidney stones, inflammation processes and HIV inhibition. The human phosphate binding protein is a human representative of the DING family that was serendipitously discovered from human plasma. An original approach was developed to determine MS experiments, including ESI-MS on intact HPBP, were used to correct errors from crystallographic sequencing, including those for the few peptides that could not be sequenced. Finally, this technique allowed, ab\u00a0initio and without ambiguities, the 38\u2005kDa HPBP to be sequenced fragmentation in LC-MS/MS and MALDI-MS/MS experiments to be obtained. The primary sequence obtained by X-ray crystallography was used like an \u2018Ariane wire\u2019, useful to align peptide sequences subsequently obtained by mass spectrometry, without the need of having overlapping peptides. It can be noted that X-ray crystallography techniques provided important information that can barely be obtained using MS, such as the exact number of amino acids and the presence of the disulfide bridges, and the discrimination of residues that possess the same mass (Leu, Ile, 3.et al., 2008Taking advantage of obtaining the HPBP sequence (Diemer et al., 2010Leishmania major genome and western blot studies on plant tissues (Perera et al., 2008et al., 2010et al., 2007DING proteins were observed in all tested tissues, namely brain, skin, heart, aorta, lung and liver, suggesting that these proteins are widely expressed within the organism (Collombet SJ suppresses expression of HIV-1 genome (Darbinian et al., 2008SJ with human C/EBP\u03b2 transcription factor and viral Tat transactivator. Moreover, p27SJ possesses a phosphatase activity inducing a dysregulation at S and G2/M phases in cell cycles related to alteration of the Erk1/2 phosphorylation state (Darbinian et al., 2009et al., 2005The immunohistochemistry study also reveals that the DING protein cellular localization is tissue-dependent, being exclusively nuclear in neurons, and nuclear and cytoplasmic in the heart muscle. The nuclear localization of DING proteins fits well with previous observations concerning biological activities of DING proteins, showing a clear involvement of these proteins in complex processes within the nucleus. For example, p274.et al., 2006Pseudomonas fluorescens called PfluDING (Ahn et al., 2007et al., 2007a et al., 1999Escherichia coli phosphate-binding protein. Interestingly, the unique feature of DING proteins compared with pstS is the presence of four protruding loops at the protein surface (Fig. 2b Two structures of DING representatives are available: the structure of HPBP (Morales 5.K D of approximately 1\u2005\u00b5M (Ahn et al., 2007et al., 2009Although their phosphate-binding ability has not been clearly related to their biological functions until now, DING proteins are able to bind phosphate with high affinity. Indeed, it has been shown that HPBP and PfluDING bind phosphate with a a K a values of the phosphate moiety, PfluDING binds only dibasic phosphate both at acidic and basic pH. The structures show that the phosphate ion is bound via 11 normal hydrogen bonds plus a highly energetic hydrogen bond, between a phosphate oxygen and the carboxylate side chain of Asp62 (Fig. 3b K a of atoms in the binding site. Indeed, the fact that PfluDING binds only dibasic phosphate both at acidic and basic pH can be explained by the finding of a very positively charged binding site, capable of altering dramatically the phosphate pK a.The quality of the obtained data allows most of the H atoms in the protein structure to be located precisely (Fig. 36.ab initio HPBP using mass spectrometry and X-ray data in tandem. Taking advantage of the very high diffracting power of DING protein crystals, we elucidated the molecular mechanism of phosphate binding in high-affinity proteins. These studies illustrate that DING proteins are widely expressed in eukaryotic tissues, and their cellular localization is tissue-dependent, albeit being mostly nuclear. This nuclear localization partly explains some observed biological activities, such as the role in the cell cycle and the inhibition of the HIV replication by interacting with the viral protein Tat and the human transcription factor CEBP/\u03b2. The involvement of DING proteins in several important human diseases, together with their genetic mystery and our findings of unknown HMW-DING in eukaryotes, enhance the emerging scientific interest on this protein family.The DING proteins family is an intriguing protein family that seems ubiquitous in eukaryotes, albeit their coding genes are missing. This unconventional protein family requires, for its investigation, some methodological developments. For example, an original approach was developed in order to sequence"} +{"text": "Cytokinins (CKs) are a large group of plant hormones which play a crucial role in many physiological processes in plants. One of the interesting functions of CKs is the control of programmed cell death (PCD). It seems that all CKs-dependent phenomena including PCD are accompanied by special multi-step phosphorelay signaling pathway. This pathway consists of three elements: histidine kinase receptors (HKs), histidine phosphotransfer proteins (HPs) and response regulators (RRs). This review shows the r\u00e9sum\u00e9 of the latest knowledge about CKs signaling pathways in many physiological processes in plants with special attention paid to PCD process. Programmed cell death (PCD) is a process that normally occurs during seed germination, development and senescence. This process is crucial for proper functioning of all multicellular organisms, both plants and animals \u2014kinetin and benzylaminopurine \u2014at high concentrations are able to induce PCD in plants of roots which seems to be crucial for this synthesis. In cells, CKs are present in chloroplasts or as complexes bound with tRNA or aromatic compounds and their derivates. Derivates of zeatin and iP as well as their sugar conjugates are most popular, but their occurrence depends on plant species, stage of development and tissue , a purine-derived CK, discovered as a degradation product of DNA, plays a crucial role in plant cell division. It was isolated by Professor Scoog in 1955 and now it seems that it is the best-known CK Fig.\u00a0. Active A. thaliana and organismal response to stress, pathogens, senescence which are responsible for reception of the CK signals that mediate phosphoryl group transfer between AHKs and ARRs were also characterized were observed. In rice, only two of HPs (OHP3 and OHP4) are involved in the CK signaling multi-step phosphorelay system. The RRs present in rice are also divided into two groups: OsRRs and ORRs. The former is similar to type-A ARRs were described as negative regulators of CK signaling, whereas type-B seems also to play a positive role in transcription factors engaged in CK signaling . They were closely related to AHK4, AHK3, and AHK2 receptors, respectively and A. thaliana (L.) Heynh. In both carrot and Arabidopsis, PCD was induced by accelerating senescence or senescence-like process both in vitro (in cell cultures) and in vivo is engaged in CK-stimulated induction of PCD (Vescovi et al. Zea mays and Arabidopsis (Caesar et al. The question arises V. faba ssp. minor after kinetin treatment (Kunikowska et al. Subcellular AHK localization sheds a new light on hormone functioning (Caesar et al."} +{"text": "Bian Zheng) is a traditional diagnostic method to categorize patients' syndromes based on their presented conditions. Currently, combination of Zheng classification and biomedical diagnosis becomes a common model in TCM diagnostics in clinical practice. Clinical treatments of a patient rely on the classification of a specific TCM Zheng. This special issue collects articles describing the clinical trials with TCM Zheng classification, Zheng classification methodology from clinical data, systematic reviews on clinical studies using Zheng classification, and the clinical pharmacological evaluation on TCM interventions using the omics platforms as well as bioinformatics.According to the traditional Chinese medicine (TCM) theory, TCM Zheng is an analysis on disease presentation by TCM practitioners. Zheng classification for mild-to-moderate systematic lupus erythematosus: a pilot prospective, single-blinded, randomized controlled study\u201d by Zhong et al. showed that Zi Shen Qing is safe and effective for decreasing severity of systemic lupus erythematosus activity and withdrawal dosage of corticosteroids in the mild-to-moderate patients with \u201cDeficiency of Qi and Yin\u201d Pattern. \u201cTherapeutic efficacy of Fuzheng-Huayu tablet based traditional Chinese medicine syndrome differentiation on hepatitis-B-caused cirrhosis: a multicenter double-blind randomized controlled trail\u201d by Song et al. revealed that Fuzheng-Huayu decreases TCM syndrome scores and improves the quality of life of hepatitis-B-caused cirrhosis patients. The only longitudinal study included in this issue, \u201cIncreases in Xu Zheng and Yu Zheng among patients with breast cancer receiving different anticancer drug therapies\u201d by Huang et al. assessed Zheng using the TCM Constitutional Scale. It is significant that more and more clinical trials pay attention to the Zheng classification and put it as a criterion for participants selection and outcome assessment, and such changes not only provide the change to demonstrate the characteristics of TCM therapy, but also provide the more comprehensive assessment of an intervention compared with conventional biomarker-oriented assessment.Among included randomized controlled trials, \u201cPrescription of Chinese herbal medicine and selection of acupoints in pattern-based traditional Chinese medicine treatment for insomnia: a systematic review\u201d by Yeung et al. included 227 studies, 17,916 subjects, and 87 TCM patterns. They highlighted high-quality studies to examine the additional benefits of pattern differentiation and pattern-based TCM treatment. \u201cModified Dachengqi decoction combined with conventional treatment for treating acute exacerbation of chronic obstructive pulmonary disease: a systematic review based on randomized controlled trials\u201d by Wu et al. analyzed 16 studies involving 1,112 patients and showed that Dachengqi decoction combined with routine treatment enhances the effectiveness. \u201cTianma Gouteng Yin as adjunctive treatment for essential hypertension: a systematic review of randomized controlled trials\u201d by Wang et al. reveals no confirmed conclusion about the effectiveness and safety of Tianma Gouteng Yin for essential hypertension. \u201cZHENG-omics application in ZHENG classification and treatment: Chinese personalized medicine\u201d by Dai et al. gives the brief introduction and preliminary validation and discusses strategies and system-oriented technology. \u201cClinical applications of omics technologies on ZHENG differentiation research in traditional Chinese medicine\u201d by Song summarized the typical omics application in common studied diseases and TCM Zhengs and discussed the main problems and countermeasure of Zheng classification researches. \u201cSyndrome differentiation in Chinese herbal medicine for irritable bowel syndrome: a literature review of randomized trials\u201d by Li et al. involved 735 RCTs and 67,784 patients and indicated that 30.5% studies applied Zheng classification. Reviews and evidences, \u201cMetabonomics-based study of clinical urine samples in suboptimal health with different syndromes\u201d by Cui et al. identified the specific metabolic products of the syndrome of stagnation of liver Qi. \u201cSimilar connotation in chronic hepatitis B and nonalcoholic fatty liver patients with dampness-heat syndrome\u201d by Dai et al. discussed the molecular mechanism of similar connotation that exists in chronic hepatitis B and nonalcoholic fatty liver by metabonomics to give the modern understanding of dampness-heat syndrome. \u201cDifferences of excess and deficiency Zheng in patients with chronic hepatitis B by urinary metabonomics\u201d by Sun et al. suggested that chronic hepatitis B patients could be categorized into two groups with diverse pathogenesis. \u201cDifferences in metabolites of different tongue coatings in patients with chronic hepatitis B\u201d by Zhao demonstrated that tongue coatings have objective material basis. \u201cMetabonomics study of essential hypertension and its Chinese medicine subtypes by using gas chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy\u201d by Li et al. distinctly classified Yin deficiency, Yang-hyperactivity syndrome, and Yin-Yang deficiency syndrome by principal components analysis and partial least squares discriminant analysis. \u201cVisual agreement analyses of traditional Chinese medicine: a multiple-dimensional scaling approach\u201d by Lo et al. developed a novel visual agreement analysis for TCM via multiple-dimensional scaling. \u201cThe pattern element scale: a brief tool of traditional medical subtyping for dementia\u201d by Shi et al. evaluated the utility of the new pattern element scale in dementia patients. \u201cValidity of a diagnostic scale for acupuncture: application of the item response theory to the five viscera score\u201d by Tomura first applied the item response theory model to the five viscera score to test its validity by evaluating the ability of the questionnaire items to identify an individual's latent traits. \u201cInterrater reliability of Chinese medicine diagnosis in people with prediabetes\u201d by Grant et al. provides initial evidence of variation in the biomarkers of people with prediabetes according to the different TCM Zhengs. \u201cValidation of the constitution in Chinese medicine questionnaire: does the traditional Chinese medicine concept of body constitution exist?\u201d by Wong et al. adapted and validated the constitution in Chinese medicine questionnaire in Hong Kong Chinese people. \u201cClassification and progression based on CFS-GA and C5.0 boost decision tree of TCM Zheng in chronic hepatitis B\u201d by Chen et al. applied correlation-based feature selection, genetic algorithm, and decision tree for Zheng classification and progression in the TCM treatment of chronic hepatitis B (CHB). \u201cUnderstanding the molecular mechanism of interventions in treating rheumatoid arthritis patients with corresponding traditional Chinese medicine patterns based on bioinformatics approach\u201d by Jiang et al. indicates that molecular network analysis could provide insight into the full understanding of the underlying mechanism of how TCM pattern impacts the efficacy. \u201cThe correlation between high-sensitivity C-reactive protein, matrix metallopeptidase 9, and traditional Chinese medicine syndrome in patients with hypertension\u201d by Wu et al. investigated the relationships between Zheng and the two inflammatory biomarkers in patients with essential hypertension. \u201cResearch on Zheng classification fusing pulse parameters in coronary heart disease\u201d by Guo et al. illustrated that the nonlinear dynamic variables of TCM pulse can improve the performances of Zheng classification models. \u201cA two-level model for the analysis of syndrome of acute ischemic stroke: from diagnostic model to molecular mechanism\u201d by Dai et al. analyzed that collateral obstruction syndrome, the integration of diagnostic model and molecular mechanism analysis creates an interesting perspective to better understand Zheng. \u201cProgression from excessive to deficient syndromes in chronic hepatitis B: a dynamical network analysis of miRNA array data\u201d by Chen et al. suggested that miRNAs are important mediators for TCM Zheng classification and could be potential diagnosis and therapeutic molecular markers. \u201cCirculating miR-583 and miR-663 refer to ZHENG differentiation in chronic hepatitis B\u201d by Zhang et al. indicated that 354 putative targets for miR-583 and 68 putative targets for miR-663 were mainly involved in Axon guidance, Neurotrophin, and MAPK signaling pathway; miR-583 and miR-663 may be potential markers for Zheng differentiation in CHB. Besides, \u201cAutophagy inhibition enhances apoptosis induced by dioscin in Huh7 cells\u201d by Hsieh et al. showed that dioscin, a new water-soluble steroidal saponin from Dioscorea nipponica Makino, causes autophagy in Huh7 cells and suggested that dioscin has a cytoprotective effect.Zheng diagnosis for patients is sometimes inconsistent among different practitioners. This is in part due to the paucity of evidence-based diagnostic methods in TCM. To solve this problem, establishment of validated diagnostic tool is inevitable. \u201cClinical trial on the characteristics of Zheng classification of pulmonary diseases based on infrared thermal imaging technology\u201d by Ni et al. showed that the infrared thermal images characteristics of a different Zheng of pulmonary disease were distinctly different. These studies provide the potential of how to improve the quality of Zheng diagnosis. Further studiese are needed to systematically improve the quality of Zheng diagnosis and the integrate this method with modern medicine.Furthermore, Recently, a great progress has been made in the clinical studies focusing on the exploration of the effect of TCM interventions in the patients with clarified TCM Zheng in some diseases. The randomized controlled trials and systematic reviews provide strong evidence for Zheng classification. This special issue presents, even though partially, the efficacy of TCM practice with integration of TCM Zheng classification, biomedical disease diagnosis, and the biological basis of Zheng. We hope that our readers could share these works and further contribute to this important field."} +{"text": "Although research in mice has elucidated a number of fundamental features of NK cell biology, mouse, and hNK cells significantly differ in their subpopulations, functions, and receptor repertoires. Thus, there is a need for a model that is more closely related to humans and yet allows experimental manipulations. Non-human primate models offer numerous opportunities for the study of NK cells, including the study of the role of NK cells after solid organ and stem cell transplantation, as well as in acute viral infection. Macaque NK cells can be depleted in vivo or adoptively transferred in an autologous system. All of these studies are either difficult or unethical to carry out in humans. Here we highlight recent advances in rhesus NK cell research and their parallels in humans. Using high-throughput transcriptional profiling, we demonstrate that the human CD56bright and CD56dim NK cell subsets have phenotypically and functionally analogous counterparts in rhesus macaques. Thus, the use of non-human primate models offers the potential to substantially advance hNK cell research.Human NK (hNK) cells play a key role in mediating host immune responses against various infectious diseases. For practical reasons, the majority of the data on hNK cells has been generated using peripheral blood lymphocytes. In contrast, our knowledge of NK cells in human tissues is limited, and not much is known about developmental pathways of hNK cell subpopulations NK cells are lymphocytes that have evolved to provide a first line of immune protection against viruses and malignancies before adaptive immune responses emerge Lanier, . IncreasThe study of murine NK (mNK) cells has clearly elucidated a number of fundamental principles, some of which seem to universally apply to all NK cells, such as the activation of NK cells by absent or altered MHC class I molecule expression, also known as the \u201cmissing self hypothesis\u201d , and CD16+ NK cells on highly purified CD56+, CD16+, and DN rmNK cells. We either utilized rhesus-specific ABI TaqMan assays, when available, (Life Technologies), or custom-designed primers and probes based on rhesus mRNA sequences. A complete list of real-time PCR assays employed in our study is available upon request. All assays were subjected to a rigorous selection process to ensure that targeted rhesus macaque genes were orthologous to human genes.A previous transcriptome analysis study revealed a number of differentially expressed genes in CD56+, CD16+, and DN rmNK cell subsets revealed a segregation of NK cells into groups corresponding to the CD56s Figure . Similars Figure . Converss Figure as expecs Figure . In addis Figure . FinallyF Figure .+ and CD16+ NK cells, both in the PCA analysis as well as for most of the gene expression data presented in Figure bright NK cells represent a less mature developmental stage of NK differentiation, whereas CD56dim cells exhibit a more differentiated effector profile in rhesus macaques with an acquired and transmissible immunodeficiency (Daniel et al., \u2212CD8+CD16+ cells (De Boer et al., + and DN rmNK cell subpopulations, and may have limited the authors' ability to accurately assess the effects of SIV infection on the full repertoire of rmNK cells.Multiple phenotypic and functional changes within the NK cell compartment have been documented in HIV infection (Fauci et al., dim NK cells and an increase of the CD56\u2212CD16+ NK cell subset (Alter et al., + NK cells (Reeves et al., bright and CD56+ cells, respectively, without affecting the frequencies of cells expressing CD62L (Reeves et al., Although NK cells from chronically HIV- or SIV-infected donors display a hyper-activated state, their capacity to respond to PMA and ionomycin is diminished (Azzoni et al., Whereas most hNK studies have focused on NK cell subsets from peripheral blood, much less is known about NK cells in tissues. The complexity and heterogeneity of hNK cells residing in various organs, such as uterus, brain, spleen, liver, pancreas, and skin, as well as various mucosal tissues, including the gut, is an area of research that is garnering increasing interest (Shi et al., + hNK cell subset was recently discovered in human and mouse mucosal-associated lymphoid tissues (Cella et al., + NK cells (Reeves et al., + NK cells displayed an altered functional profile with increasing resemblance to conventional NK cells. These alterations were linked to gut inflammation and the up-regulation of indoleamine 2,3-dioxygenase 1 (Reeves et al., A novel IL-22- and IL-17-producing NKp44+ NK cell subsets in treated HIV-patients with incomplete or suboptimal CD4+ T cell recovery (Sips et al., + NK cells could play a compensatory role in patients with ongoing compromised immunity. However, to our knowledge, the impact of HIV infection on IL-22-producing NKp44+ hNK cells has not yet been addressed.A recent study described an expansion of intraepithelial and lamina propria NKp46in vivo effects of cytokines on lymphocyte homeostasis and disease can be addressed in rhesus macaques (Kuramoto et al., + T cells (Mueller et al., In addition to improved access to tissue samples, a number of experimental procedures that have the potential to significantly advance our understanding of NK cells can be carried out in non-human primates. For instance, the in vivo setting.To evaluate the antiviral contributions of NK cells in control of SIV infection, mouse monoclonal antibodies against CD16 have been administered to rhesus macaques (Choi et al., ex vivo expanded cells. This approach could generate novel insights into NK cell turnover, differentiation, migratory behavior, in vivo killing of target cells, and other areas. Moreover, induced pluripotent stem cells have been used to generate hNK cells with antiviral activity against HIV (Knorr and Kaufman, in vivo NK cell development and differentiation. These and many other experimental manipulations of the immune system in non-human primates open a number of new avenues to address basic immunological questions and highlight the potential of these animal models.Furthermore, non-human primates could also be used in autologous transfer studies using fluorescently labeled cells and Here we present two arguments as to why NK cell research in non-human primate models has the potential to yield significant insights into hNK biology. First, as a consequence of the phylogenetic relatedness of humans and non-human primates, there are many shared properties between hNK and non-human primate NK cells. Second, as an animal model, non-human primates offer access to tissues and allow a variety of manipulations, which the \u201chuman model\u201d cannot offer due to ethical constraints. The work in non-human primates is challenging, as animals and their housing are relatively cost-intensive and require dedicated facilities and staff. Nonetheless, we believe that their significance as a disease model and the potential clinical applicability of immunological findings derived from these animals continue to make research in non-human primates highly rewarding.Non-human primate models not only bear relevance for humans as outstanding disease models but also as a valuable resource to address basic immunological questions. The exciting answers to these questions and the lessons non-human primates can teach us certainly deserve a greater consideration by the scientific community.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Cryptococcus neoformans, chitin-like molecules associate with capsular glucuronoxylomannan (GXM) to form functionally distinct glycan complexes. Such interactions between glycans that result in the formation of structures with different functions strongly suggest that additional molecular complexes with unknown properties may exist in fungal pathogens. Moreover, the identification of these novel complexes has stimulated the search of new immunogens with potential to protect human and animal hosts against systemic mycoses.The biological properties of fungal immunogens have historically utilized testing of isolated molecules. Recent findings, however, indicate that fungal glycans differing in structure and function can interact to form hybrid complexes with unique properties. In the pathogenic yeast The surface of fungal cells is rich in polysaccharides and protein- or lipid-bound oligosaccharides chitooligomers promoted enlargement of GXM fibrils, and (3) exposure of C. neoformans cells to an inhibitor of N-acetylglucosamine synthesis caused a decrease in capsular dimensions (Fonseca et al., C. neoformans to form glycan complexes composed of chitin-derived structures and GXM, their production during infection, impact on the host's immune system, and structural determinants regulating this glycan-glycan interaction were unknown until very recently.The supposition that GXM and chitin-derived structures interact has been confirmed by a number of approaches (Rodrigues et al., N-acetyl amino group of chitin, but not on carboxyl and O-acetyl groups of GXM. Importantly, this study shows that glycan complexes formed by GXM and chitin-derived molecules also arise during macrophage infection. Injection of either isolated molecules or the glycan complexes into mice induced distinctly different cytokine responses. In fact, the glycan complexes were efficient in inducing the production of lung IL-10, IL-17, and TNF\u03b1, while the cytokine profiles of mice challenged with either GXM or chitin oligomers alone were similar to cytokine levels in control animals. The fact that glycan complex structures produce enhanced immunosuppressive and pro-inflammatory cytokine responses while chitin oligomers and GXM alone did not suggested that cell-associated C. neoformans glycans form hybrid structures with unique functions.A recent study (Ramos et al., C. neoformans model (Ramos et al., The discovery of the formation of functionally distinct glycan complexes raises a number of puzzling questions. For instance, the surface of fungal pathogens is decorated with many different glycans that coexist in several microenvironments (Nimrichter et al., C. neoformans is in fact increased in the lungs of infected rats (Fonseca et al., in vivo observations demonstrating that chitooligomer detection and capsule enlargement are more evident in host tissues manifesting higher activity of this enzyme (Fonseca et al., Chitooligomers are the products of enzymatic hydrolysis of chitin. Chitinase expression is induced during pulmonary cryptococcosis in rodents (Vicencio et al., C. neoformans. Microscopic examinations of C. neoformans yeast cells, in fact, support this possibility. Co-staining of cryptococci with antibodies raised to GXM and to \u03b11,3 glucan reveal that \u03b11,3 glucan is widely distributed in the capsule (Cordero et al., C. neoformans GXM (Reese and Doering, C. neoformans, the presence of the capsule is well-known to slow down the molecular traffic across the cell surface (Nosanchuk et al., GXM has the potential to associate with a number of hydrophilic components, mainly because of its high efficiency in the formation of hydrogen bonds. Thus, GXM-chitin interactions probably have other counterparts in Surface molecules do not exist in their isolated form in cellular systems and approaches investigating interacting molecules can provide a deeper understanding of complex biological processes than the study of individual purified molecules. The discovery of hybrid glycans with previously unknown functions suggests new venues of investigation on the roles of polysaccharides and glycoconjugates in fungal infections. In addition, the connections between glycan association and functional variation strongly indicate that molecular complexes with still unknown properties may exist in fungal pathogens. This conclusion encourages new perspectives on models aiming at the discovery of protective immunogens.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Distal expression quantitative trait loci are genetic mutations that affect the expression of genes genomically far away. However, the mechanisms that cause a distal eQTL to modulate gene expression are not yet clear. Recent high-resolution chromosome conformation capture experiments along with a growing database of eQTLs provide an opportunity to understand the spatial mechanisms influencing distal eQTL associations on a genome-wide scale. We test the hypothesis that spatial proximity contributes to eQTL-gene regulation in the context of the higher-order domain structure of chromatin as determined from recent Hi-C chromosome conformation experiments. This analysis suggests that the large-scale topology of chromatin is coupled with eQTL associations by providing evidence that eQTLs are in general spatially close to their target genes, occur often around topological domain boundaries and preferentially associate with genes across domains. We also find that within-domain eQTLs that overlap with regulatory elements such as promoters and enhancers are spatially more close than the overall set of within-domain eQTLs, suggesting that spatial proximity derived from the domain structure in chromatin plays an important role in the regulation of gene expression. Expression quantitative trait loci (eQTL) experiments map mutations in a genome to variation in gene expression . They haWe determine whether spatial proximity plays a role in eQTLs regulating their target genes as illustrated in et al. submitted for publication) spanning 10 publications and six cell types (L. Mangravite ication) . Of theset al. observed for an edge E with read pairs mapping within the fragments u and v. The total frequency of a fragment v is then defined as et al. afford the use of an error-correction method by Yaffe and Tanay .For each chromosome, Hi-C data in its raw form can be represented as a weighted graph nd Tanay , and we et al. Domain boundaries for the Hi-C assays described earlier in the text were identified using a hidden Markov model and published in the same location as the Hi-C data. Each domain in this sequence of topological domains n is the number of fragments in et al., yet still enriched for similar chromatin marks. The domain-finding method of Dixon et al. uses a Hidden Markov Model with a local \u2018directionality index\u2019 statistic, whereas the method for the alternative definition explicitly encodes the quality score of a domain in a dynamic program. Dixon et al.\u2019s method results in domains at a particular scale of genomic length , whereas the alternative definition identifies domains that persist across multiple length scales.We obtained topological domains for each chromosome from Dixon domains that havet al. submitted for publication) (Q of eQTLs.eQTLs were obtained from the eQTL browser (eqtl.uchicago.edu), which compiles genome-wide eQTL data from 10 publications ; National Institutes of Health (NIH) [1R21AI085376 and 1R21HG006913]; Alfred P. Sloan Research Fellow (to C.K.). Funding for open access charge: NIH [IR21HG006913].Conflict of interest statement. None declared."} +{"text": "In this issue Mandl and colleagues replicated the findings of a previous study , observed as an increase in the gray matter (GM) magnetic resonance signal , White matter fraction (wmf = 1 \u2212 fgm), TE (78 ms) and b-value (1000 s/mm2) (Mandl et al., To test this hypothesis we simulated the effect which a partial-volume of gray matter would have on parallel and transverse diffusivity using published parameters. Relaxation rates Figure 3 resolution, it is likely that several WM voxels could be contaminated with volumes of GM, even after using standardized white matter templates. Noise in MRI acquisitions is thought to cause an overestimation of FA in both isotropic and anisotropic structures (Pierpaoli and Basser, A more technical consideration is the possible effect of image noise and partial volumes on FA quantification (Basser and Jones, +, K\u2212-ATPase utilization to recover post-activation transmembrane ion gradients, which in turn might translate into changes in vascular oxygenation levels. However, the extant evidence from BOLD fMRI and PET studies does not support a metabolic explanation for the observed effects. In vitro studies in the rat brain\u2014which are free from confounding vascular effects - show that massive depolarization and increases in metabolism have a minimal effect upon WM ADC quantification (Anderson et al., A final possibility is that FA increases may reflect activity-evoked glial swelling associated with increases in extracellular potassium levels (Ransom et al., R2 are excluded, requires 18 separate parameters (Basser and Jones, In order to advance the use of functional DTI, a more detailed exploration of the origin of the observed changes is vital. To describe the basic WM, GM, and CSF model, even when contributions from blood and The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Paxillin encodes a focal adhesion-associated protein and is involved in the progression and aggressive phenotypes of malignancies through its interactions with the actin cytoskeleton and key signal transduction oncogenes. The present study aimed to investigate the clinicopathological and prognostic significance of paxillin in gastric cancer. The expression of paxillin was evaluated using tissue microarrays of gastric adjacent non-cancerous mucosa, adenoma and carcinoma specimens by immunohistochemistry. Paxillin expression was compared against clinicopathological parameters and the survival time of the patients. Paxillin was highly expressed in gastric adenoma compared with that in non-neoplastic mucosa and carcinoma (P<0.05). Paxillin expression was lower in the younger carcinoma patients compared with that in the elder carcinoma patients (P<0.05). Paxillin expression was negatively correlated with tumor size, depth of invasion and lymph node metastasis, but not with patient gender, lymphatic or venous invasion, or TNM staging (P>0.05). Higher paxillin expression was observed in intestinal-type compared with diffuse-type carcinoma (P<0.05). Kaplan-Meier analysis indicated a positive association between paxillin expression and cumulative survival rate in all, advanced and intestinal-type carcinoma patients (P<0.05). Multivariate analysis using the Cox proportional hazards model indicated that patient age, depth of invasion, lymphatic invasion, lymph node metastasis, TNM staging and Lauren classification were independent prognostic factors for all gastric carcinomas (P<0.05). Aberrant paxillin expression may be involved in the growth, invasion, metastasis and differentiation of gastric carcinoma. Altered paxillin expression may, therefore, be employed as an indicator of pathobiological behaviors and prognosis of gastric carcinomas. Despite a global decline in the incidence and mortality of gastric cancer in the last 60 years, it remains the fourth most common and second most frequent cause of cancer-related mortality. Gastric cancer continues to be a major health concern due to the slow decrease in incidence in Asia and high mortality from diagnosed gastric carcinomas in the West, despite the advanced diagnostic and operative techniques that are commonly used in clinical practice ,2. An inet al and adjacent non-neoplastic mucosa (n=197) collected at Toyama University Hospital from 1993 to 2006. The adenoma samples were resected from endoscopic biopsy at Toyama University Hospital from 1997 to 2008. The patients with gastric carcinomas were 120 males and 272 females . Archival materials were obtained from the Department of Pathology of Toyama University Hospital. In 151 cases, tumor development was accompanied by lymph node metastasis. None of the patients underwent chemotherapy, radiotherapy and adjuvant treatment prior to surgery. All patients were followed up by consulting their case documents and by telephone.All tissues were fixed in 10% neutralized formalin, embedded in paraffin, cut into 4-\u03bcm sections and stained with hematoxylin and eosin (H&E) in order to confirm the histological diagnosis and microscopic characteristics of the specimens. The staging for each gastric carcinoma was evaluated according to the Union for International Cancer Control system, which indicates the extent of tumor spread . HistoloFrom H&E-stained sections of the tumor cases, representative areas of solid tumor were selected for sampling and 2-mm diameter tissue cores per donor block were punched out and transferred to a recipient block with a maximum of 48 cores using a tissue microarrayer . Sections (4-\u03bcm) were consecutively cut from the microarrays and transferred to poly-lysine-coated glass slides.Serial sections of TMA were deparaffinized with xylene, rehydrated with alcohol, and subjected to immunohistochemical staining with intermittent microwave radiation, as previously described . Rabbit Immunoreactivity for paxillin showed a cytoplasmic pattern . One hunStatistical evaluation was performed using Spearman\u2019s rank correlation test. Kaplan-Meier survival plots were generated and comparisons between survival curves were made with the log-rank test. Cox proportional hazards model was employed for multivariate analysis. SPSS 17.0 software was applied to analyze all data, and P<0.05 was considered to indicate a statistically significant difference.As indicated in Follow-up information was available on 392 of the gastric carcinoma patients for periods ranging from 0.2 months to 121 months . de novo carcinogenesis is well understood, particularly in diffuse-type gastric carcinomas (Paxillin is a cytoskeletal protein that was recently identified as a component of focal adhesions and links between F-actin and integrin . In the rcinomas . These fet al(et al(et al(Cai et al found thet al. Li et a al(et al document al(et al found th al(et al. HoweverAlthough all types of gastric cancer are malignant tumors that originate from the same gastric epithelium, the morphological features of the cancers vary substantially in individual patients. According to Lauren classification, gastric intestinal-type carcinoma is characterized by cohesive carcinoma cells that form gland-like tubular structures, such as well- and moderately differentiated carcinoma; while cell cohesion is less apparent or absent in diffuse-type carcinoma, such as poorly differentiated or signet ring cell carcinoma ,17. Our et al(et al(To date, there have been several studies describing the prognostic significance of paxillin expression in malignancies ,12,14,26et al found thIn conclusion, aberrant paxillin expression may be important in the malignant transformation of gastric epithelial cells. Its reduced expression was closely correlated with growth, invasion, metastasis and a worse prognosis of gastric carcinomas. Its expression may be employed to differentiate between the intestinal- and diffuse-type carcinomas. It was considered as a promising marker to indicate the pathobiological behaviors and prognosis of gastric carcinomas."} +{"text": "Historically there has been a distinction between basal ganglia dependent and hippocampus dependent memory systems (e.g., Squire, A newly published study by Foerde et al. also supThe failure to learn from delayed feedback in patients with hippocampal lesions could be related to the loss of function of area CA1, since CA1 receives afferents from areas known to respond phasically to reinforcing stimulation (Gasbarri et al., Overall, the results described by Foerde et al. provide"} +{"text": "To the Editor: We challenge the conclusions of Feingold et al. that \u201cregional density of livestock is a notable risk factor for nasal carriage of LA-MRSA for persons with and without direct contact with livestock\u201d (Staphylococcus aureus (MRSA), but they retrospectively analyzed 87 culture-confirmed MRSA cases reported to a reference laboratory. These were a mixture of clinical disease isolates and screening isolates that were unevenly distributed between the groups was >180 times higher in 352 occupationally exposed persons than in 2,094 rural residents without farm exposure (0.24%) (Finally, the contention of Feingold et al. that pig production in the Netherlands is \u201cgreatly overshadowed by the density of pig-farming operations in the United States\u201d is mistaken . Pig den"} +{"text": "An elevated resting heart rate is one of the strongest predictors of cardiovascular mortality and is independently associated with sudden cardiac death (SCD). Agents capable of reducing heart rate without significant side effects are therefore of particular interest for the prevention of SCD. Recent human and animal studies have shown that omega-3 fatty acids can reduce heart rate. Our work has shown that omega-3 fatty acids significantly reduce membrane electrical excitability of the cardiac myocyte by lowering its resting membrane potential and the duration of the refractory period through inhibition of ion channels. We propose that these actions may be the underlying mechanisms for the omega-3 fatty acid-induced reduction of heart rate observed in both humans and animals. The heart rate-lowering capability of omega-3 fatty acids may contribute to their preventive effect against SCD. The cardioprotective effects of omega-3 fatty acids have become widely recognized. One of the most significant effects is the prevention of sudden cardiac death (SCD) is independently associated with SCD. Multiple prospective studies have shown that even after adjusting for common cardiovascular health-related variables, such as age, weight, smoking, alcohol consumption, diabetes, blood pressure, physical activity, blood cholesterol, medications, and socioeconomic status, elevated heart rate remains a risk factor for SCD and a predictor of time to cardiac death (Shaper et al., Given these consistent and robust findings, drugs or supplements that reduce heart rate are of particular interest for preventing SCD. In fact, studies that have examined outcomes for patients with chronic heart failure have revealed that pharmacotherapy to reduce heart rate is associated with better outcomes for at least 5 years (Franke et al., in vitro work provide a likely mechanism by which omega-3 fatty acids act on cardiac myocytes to reduce heart rate.The relationship between omega-3 fatty acids and cardiovascular disease is well studied, and has appeared inconsistent at times (Harris et al., The effect of omega-3 fatty acids on heart rate has been observed in many different populations, both with and without cardiovascular disease. A meta-analysis of 30 randomized, double-blind, placebo-controlled trials concluded that fish oil consumption can significantly reduce heart rate (Mozaffarian et al., In addition, several large-scale, population-based studies showed that increased dietary fish and omega-3 fatty acid intake was associated with a significant reduction in heart rate. Dallongeville et al. analyzedFish oil also effectively reduces heart rate during times of increased cardiac demand such as exercise. A study of 25 Australian football players revealed that 6g/day of fish oil reduced heart rate during submaximal exercise over a period of 5 weeks (Buckley et al., Another set of interesting findings comes from a population perhaps the least likely to experience cardiovascular illness: infants. Term infants treated with varying amounts of DHA in their formulas for 12 months show that DHA supplementation reduces heart rate compared to infants whose formula does not contain DHA, with no evidence of a dose response (Pivik et al., Finally, Harris and colleagues performed a small prospective study that provides a valuable indication of which mechanisms are likely to underlie the omega-3 fatty acid-driven reduction in heart rate. The group enrolled heart transplant patients, ensuring that their transplants had occurred more than three months prior and there had been no transplant-related hospitalizations (Harris et al., Similar reductions in heart rate due to omega-3 fatty acids have been observed in animals. In a rat model, animals fed a DHA-enriched diet had lower heart rates than animals fed a control diet, a pattern that was achieved by 2 months and maintained until the end of the 32-week study (Ayalew-Pervanchon et al., Overall, it is apparent that a wide range of human and animal subjects, with or without cardiac disease, all respond to omega-3 fatty acid supplementation with reductions in resting and stress-induced heart rates. These findings suggest a highly consistent and robust effect of omega-3 fatty acids on heart rate.Although omega-3 fatty acids may reduce heart rate through several different mechanisms, our early studies demonstrated a direct effect of omega-3 fatty acids on cardiac cell membrane electrical excitability that contributes to reduced heart rate. We used isolated, neonatal rat cardiac myocytes that retain spontaneous beating behavior, allowing us to assess the effect of EPA and DHA on contraction as well as electrophysiological activity without neural or hormonal input. We found that EPA and DHA promptly reduced the contraction rate of the cardiac myocytes by 50\u201380% without a significant change in the amplitude of the contractions. This effect of omega-3 fatty acids on the excitability of the cells was similar to that produced by the class I antiarrhythmic drug lidocaine Figure , top Ka. In addiThe reduction of electrical excitability of cardiac myocytes by omega-3 fatty acids can be demonstrated directly by their response to electrical pacing (Kang and Leaf, To better elucidate the mechanism of action of omega-3 fatty acids on heart rate, we employed a patch-clamp technique to examine the electrophysiological activity in isolated neonatal rat cardiac myocytes. First, we induced the action potential in the myocytes exposed to EPA or DHA and measured the strength of the current required to elicit an action potential (Kang et al., At this point in our research, the manner by which omega-3 fatty acids reduce membrane excitability was still unclear. Therefore, we tested the effects of omega-3 fatty acids on single ion channel activity in neonatal rat cardiac myocytes. The results demonstrated a prompt inhibitory action of omega-3 fatty acids on the Na+ currents through fast sodium channels responsible for the phase 0 of the action potential in isolated neonatal rat cardiac myocytes. The inhibition of this ion channel was dose, time, and voltage dependent, but not use dependent Figure Xiao et. SubsequRegulation of heart rate in humans is highly complex. Sympathetic output, vagal tone, and systolic and diastolic left ventricular function are only a few of the factors that contribute to the regulation of heart rate. While omega-3 fatty acids could potentially affect any or all of these factors, our studies strongly suggest a direct impact of omega-3 fatty acids (Leaf et al., The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Post-translational modification of lysine residues of specific proteins by ubiquitin modulates the degradation, localization, and activity of these target proteins. Here, we identified gains of ubiquitylation sites in highly conserved regions of human proteins that occurred during human evolution.We analyzed human ubiquitylation site data and multiple alignments of orthologous mammalian proteins including those from humans, primates, other placental mammals, opossum, and platypus. In our analysis, we identified 281 ubiquitylation sites in 252 proteins that first appeared along the human lineage during primate evolution: one protein had four novel sites; four proteins had three sites each; 18 proteins had two sites each; and the remaining 229 proteins had one site each. PML, which is involved in neurodevelopment and neurodegeneration, acquired three sites, two of which have been reported to be involved in the degradation of PML. Thirteen human proteins, including ERCC2 and NBR1, gained human-specific ubiquitylated lysines after the human-chimpanzee divergence. ERCC2 has a Lys/Gln polymorphism, the derived (major) allele of which confers enhanced DNA repair capacity and reduced cancer risk compared with the ancestral (minor) allele. NBR1 and eight other proteins that are involved in the human autophagy protein interaction network gained a novel ubiquitylation site.The gain of novel ubiquitylation sites could be involved in the evolution of protein degradation and other regulatory networks. Although gains of ubiquitylation sites do not necessarily equate to adaptive evolution, they are useful candidates for molecular functional analyses to identify novel advantageous genetic modifications and innovative phenotypes acquired during human evolution. Ubiquitin is a 76-residue polypeptide that is highly conserved among eukaryotes. Ubiquitylation of the lysine residues of substrate proteins targets the ubiquitylated proteins for degradation by the proteasome . The ubiA large number of genetic modifications have occurred in the human lineage during primate evolution that might be responsible for the emergence of human phenotypes ,7. TheseN-acetylglucosamine modification sites [To assess the impact of PTMs on human proteome evolution and to identify candidates for evolutionarily innovative PTM sites, a large amount of PTM data from human cells is needed. Recent progress in high-throughput screening by mass spectrometric analysis has enabled the large-scale characterization of PTM sites in the human proteome, including phosphorylation sites ,18, O-lion sites , lysine on sites , and ubion sites -25.et al.[et al.[et al. and Wagner et al.\u2019s datasets include lysines modified not only by ubiquitin, but also by ubiquitin-like proteins such as SUMO, ISG15, and NEDD8. In this report, we therefore use the term \u201cubiquitylation\u201d to indicate both ubiquitin and ubiquitin-like protein modifications.We hypothesize that appearance of novel ubiquitylation sites in proteins along the human lineage during primate evolution may have modified protein regulatory networks, potentially resulting in the acquisition of novel phenotypic traits. To address this possibility, we developed a bioinformatics method to systematically identify gains of novel ubiquitylation sites in the human lineage during primate evolution. As a pilot study, we used ubiquitylation data for human proteins reported by Kim et al. and Wagnl.[et al. as inputhttp://www.uniprot.org) and PhosphoSitePlus (http://www.phosphosite.org) [et al.[et al.[We aimed to identify human ubiquitylated lysines located in highly conserved regions of mammalian proteins that first appeared along the human lineage during primate evolution. To do this, a large amount of ubiquitylation site data and multiple sequence alignments of orthologous mammalian proteins are required. To assess ubiquitylation sites, one can use databases containing PTM data, such as UniProt (ite.org) , or largite.org) -23,25. I) [et al. and Wagnl.[et al., as welll.[et al.. The oveThe timing of the gain of a ubiquitylated lysine was determined by finding the branch that enclosed the earliest shared lysine between humans and other primates on the mammalian phylogenetic tree. For example, the human PML residue Lys 394 protein, which is also known as XPD, is involved in transcription-coupled nucleotide excision repair and is implicated in cancer-prone xeroderma pigmentosum, trichothiodystrophy, and Cockayne syndrome . In the The neighbor of BRCA1 gene 1 (NBR1) protein has been identified as one of the principle cargo receptors for selective autophagy of ubiquitylated targets ,32. AbnoPML gene is often fused with the retinoic acid receptor \u03b1 (RARA) gene, which is associated with acute promyelocytic leukemia [Of the 281 ubiquitylation sites, 269 sites in 243 human proteins were acquired along the human lineage during primate evolution, and are shared with chimpanzees and other primates has a ubiquitylated Lys 33 that is shared with chimpanzees and gorillas, while other early-diverged primates (including orangutans) and all other mammals examined have a glutamine (Q) residue at this position Figure . NGDN fuThe scavenger receptor class B member 1 (SCARB1) protein is a plasma membrane receptor for high-density lipoprotein cholesterol (HDL). It mediates cholesterol transfer to and from HDL and is iWe found that 56 novel ubiquitylation sites in 54 proteins first appeared in the common ancestor of catarrhine primates. One representative case is WD repeat-containing protein 35 (WDR35) Lys 684, at which position most other mammals have a glutamic acid (E) Figure . WDR35 hATXN2 gene causes spinocerebellar ataxia type 2 [Of the 281 novel human ubiquitylated lysines, 116 in 107 proteins are shared with simians. One example is ataxin 2 (ATXN2) Lys 349, at which position all the other mammals examined have an arginine (R) Lys 211 first appeared in primates after their divergence from the common ancestor of Euarchontoglires and is shared in all primates examined glutamine allele is designated as the mutant, which shows reduced DNA repair capacity; carriers of this minor allele therefore have an increased cancer risk . The gaiInterestingly, among the 252 proteins, nine proteins have been found in human autophagy protein interaction networks . NBR1 haet al.[Hagai et al. showed tet al.. To explWe developed a bioinformatics method to identify novel ubiquitylation sites that evolved along the human lineage, resulting in the identification of 281 novel ubiquitylation sites. The gain of novel ubiquitylation sites could result in novel ubiquitin-associated protein regulatory interactions. Proteins with a novel ubiquitylation site are useful candidates in the search for genetic modifications implicated in the emergence of novel phenotypes during human evolution.et al.[et al.[To identify ubiquitylation sites in human proteins, we used the large-scale analysis datasets of Kim et al. and Wagnl.[et al.. These rl.[et al.. Peptidehttp://genome.ucsc.edu). The \u2018CDS FASTA alignment from multiple alignment\u2019 data, which are derived from the \u2018multiz46way\u2019 alignment data [Multiple sequence alignments of the human proteins and orthologous proteins from other mammalian species were obtained from the University of California Santa Cruz (UCSC) Genome Browser Database was used to collect protein sequences for some species. The multiple sequence alignments were generated using MUSCLE (http://www.drive5.com/muscle).The National Center for Biotechnology Information (NCBI) Protein database and may differ from those of the UniProt or NCBI Protein databases.Finally, 281 sites in 252 proteins were collected. We examined the multiple alignments to estimate the timing of the gain of the ubiquitylated lysine residue. Possible functional consequences of the gain of the ubiquitylation site were assessed by a literature survey. The positions of the residues noted in this manuscript are derived from the datasets of Kim et al. and Wagnl.[et al., which aThe authors declare that they have no competing interests.YH conceived of this study, conducted the programming work, and prepared the manuscript. DSK participated in the sequence analysis. Both authors read and approved the final manuscript.List of proteins with novel ubiquitylation sites.Click here for fileDetailed alignments of surrounding regions of novel ubiquitylation sites.Click here for filePhylogenetic tree of the 37 mammals used in this study.Click here for file"} +{"text": "Our laboratory has been evaluating the potential contribution of environmentally bioavailable neurotoxic metals to the onset, development and progression of AD for about 30 years is perhaps in vitro and in vivo aluminum promotes inflammatory signaling via the pro-inflammatory transcription factor NF-kB, another prominent feature characteristic of AD brain 2\u00b712H2O, or alum, as a clarification and \u201cfinishing\u201d agent that at physiologically realistic concentrations, aluminum strongly promotes amyloid aggregation and accumulation, a key feature of AD neuropathology Exley, ; (ii) thn Bondy, ; (iii) tn Bondy, ; (v) than Bondy, ; (vi) th Flaten, ], and . A commonly used Tg2576 mouse model overexpresses a mutant form of beta amyloid precursor protein (\u03b2 APP), APPK670/671L, linked to early-onset familial AD, and develops amyloid plaques and progressive cognitive deficits as the mice age. Tg2576 mice exposed to dietary aluminum have been shown to develop oxidative stress and robust amyloidogenesis, key features of AD neuropathology (Pratic\u00f2 et al., in vitro and in vivo models for AD with AD itself. Indeed, the abundance of specific miRNAs are highly selective, and potential indicators and predictors of human health and disease, including progressive neurological disorders such as AD (Alexandrov et al., We would like here to briefly include some recent genetic data on aluminum and its effects on miRNA abundance in a highly relevant transgenic animal model for AD that shows strong parallels to miRNA profiles which are found in AD brain Figure . There ain vitro studies (Kruck et al., Lastly, more research into the potential contribution of aluminum to the AD process is clearly warranted. There are currently no treatments for AD that effectively prevent or cure AD's insidious onset or propagation. We think it important to emphasize that the most effective clinical treatment yet devised for moderate- to late-stage AD patients was the implementation of the first generation anti-oxidant and trivalent iron/aluminum chelator desferrioxamine to attempt to remove aluminum from the brains of AD patients (Crapper McLachlan et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "The tumor necrosis factor superfamily (TNFSF) members play pivotal roles in embryonic development of lymphoid tissue and their homeostasis. RANKL is recognized as an important player in bone homeostasis and lymphoid tissue formation. In its absence bone mass control is deregulated and lymph nodes fail to develop. While its function in bone is well described, there is still little functional insight into the action of RANKL in lymphoid tissue development and homeostasis. Here we provide an overview of the known functions of RANKL, its signaling receptor RANK and its decoy receptor OPG from the perspective of lymphoid tissue development and immune activation in the mouse. Expressed by the hematopoietic lymphoid tissue inducing (LTi) cells and the mesenchymal lymphoid tissue organizer (LTo) cells, RANKL was shown to stimulate Lymphotoxin (LT) expression and to be implicated in LTi cell accumulation. Our recent finding that RANKL also triggers proliferation of adult lymph node stroma suggests that RANKL may furthermore directly activate LTo cells. Beyond bone, the RANKL-RANK-OPG triad plays important roles in immunobiology that are waiting to be unraveled. There is now an emerging consensus to refer to the receptor as RANK and, as a consequence and for simplicity, its ligand is called RANKL. The acronym OPG has remained in use.Tumor necrosis factor (TNF) and Lymphotoxin (LT) were identified as the first members of a large family, now called the TNF-superfamily (SF). Not surprisingly, the receptors for these proteins also constitute a SF with sequence homology, named TNF Receptor (TNFR) SF. A hallmark of these ligand-receptor pairs lies in a threefold symmetry, where by the oligomeric binding arrangement amplifies their avidity and introduces flexibility. Further complexity arises through different partner affinities and generation of soluble ligand and receptor forms cells and V\u03b35+ thymocytes as well as later arising CD4+CD8\u2212 single positive thymocytes and \u0393\u03b4T cells are equipped with RANKL and cryptopatches (CPs) and abnormalities of the spleen (Dougall et al., Rankl\u2212/\u2212 and Lt\u03b1\u2212/\u2212 mice have lower number of LTi cells in mesenteric LNs of newborn mice (Kim et al., \u2212/\u2212 mice display fewer LTi cells in mesenteric LNs at E 17.5 but not at E15.5 (Yoshida et al., In view of the finding that LTi cell recruitment is LT independent (Eberl et al., Rankl\u2212/\u2212 mice. Second, mucosal LNs develop in LT\u03b2\u2212/\u2212 mice but not in Rankl/\u2212\u2212 mice (Alimzhanov et al., Rankl\u2212/\u2212 embryos cannot rescue LN genesis (Kim et al., In the current model of LN development a positive feedback loop takes place between LTi and LTo cells Figure . RANK siLT\u03b2R signaling in mTECs induces RANK expression (Mouri et al., Rankl\u2212/\u2212 mice (Knoop et al., A number of observations support a role of RANKL in SLO growth. The LNs that developed after neutralization of RANK signaling in embryos were smaller (Eberl et al., \u2212/\u2212 mice: it was noted that in these LNs B cells and FDCs were absent (Yoshida et al., \u2212/\u2212Rankl\u2009 mice and observed that most stromal cells in the B cell compartment lacked VCAM-1 and CXCL13 expression. Finally, postnatal RANKL overexpression resulted in an increase in small but clearly defined B cell follicles, which all comprised FDCs (Hess et al., B cell recruitment and organization into follicles occur in a CXCL13-dependent manner at later stages of SLO formation (Ansel et al., in vitro generated DCs and in combination with other TNFSF members (Wong et al., in vivo (Dougall et al., Activated CD4 and CD8 T cells express surface and soluble RANKL (Josien et al., Porphyromonas gingivalis (Kajiya et al., P. gingivalis-induced periodontitis and therefore reduce alveolar bone loss (Arizon et al., Th17 T cells represent an important osteoclastogenic T cell type by robust RANKL production and activation of RANKL release by mesenchymal cells (Sato et al., The RANK-RANKL-OPG axis plays a recognized role in bone homeostasis through the regulation of osteoclastogenesis. It is also implicated in SLO development and regulation of the immune response. There are many incentives to answer remaining questions. In addition to a restless curiosity of the researcher, tertiary lymphoid tissues that arise in inflamed tissue rely on similar if not identical cellular dialogs as those found in SLO development. Defining RANKL function in lymphoid tissue development will open new therapeutic avenues to treat inflammatory diseases and provide new strategies for vaccine development.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Burkholderia cepacia, Pseudomonas aeruginosa or Stenotrophomonas maltophilia, which cause diseases only in patients with predisposition or in hospital (Berg et al., Just like humans, plants have recently been recognized as meta-organisms, possessing a distinct microbiome and revealing close symbiotic relationships with their associated microorganisms (Berg et al., Microbiomes of humans and plants are currently intensively studied using the same methods and addressing similar scientific questions (Ram\u00edrez-Puebla et al., Despite the fact that the majority of our lifetime is spent in indoor environments such as home, work place, or public buildings, our knowledge of microbial diversity inside buildings is limited. We are not alone in these indoor environments: they provide new habitats and residence to numerous microbial communities comprising possibly hundreds of individual bacterial, archaeal and fungal species including diverse viruses. Recent studies analyzed potentially pathogenic and allergenic indoor microorganisms with mainly cultivation-based methods (T\u00e4ubel et al., 6 bacteria per person-hour as measured via 16S rRNA gene quantification from aerosols (Qian et al., Indoor microbial communities are an important component of everyday human health (Arundel et al., Indoor microbiomes originate primarily from human skin, pets, or the outside air (Flores et al., Empirically the positive effects of houseplants and flowers are well-known, but there is also evidence for psychological effects such as stress reduction and creative task performance (Fjeld et al., Plant DNA as frequently detected as chloroplast 16S rRNA gene sequences in amplicon surveys is a substantial part of all indoor microbiomes, but mainly filtered out for the presentation of data (Oberauner et al., Typical and often dominant plant-associated bacteria are members of the indoor microbiome. A relationship of bacteria genera occurring on plants and indoors is given in Figure Burkholderia cepacia, Pseudomonas aeruginosa and Stenotrophomonas maltophilia (Ryan et al., At species level, no differentiation was possible for clinical and plant-associated isolates. This was studied for Interestingly, Thaumarchaeota, originally described to be associated with ammonia-oxidation in soil and the rhizosphere of plants, have been found on human skin (Probst et al., Comparing indoor with plant microbiomes, it is our opinion that both outside and inside plants are of importance for our indoor microbiome. Plants provide beneficial bacteria for indoor rooms and therefore can positively influence human health. The following facts support our opinion about the importance of plants as source for a beneficial microbial biodiversity:Based on these facts, we speculate the following:Enclosed environments and their microbiomes\u2014like private/public buildings, hospitals, and clean rooms, which are more or less separated from outside, are especially shaped by human influence and human associated microbes (Hospodsky et al., Microbes, which live in close vicinity to human beings, are adapted to us as symbionts, commensals, or pathogens, whereas these life-styles are changeable dependent on the host-microbe balance. Indoors we share these microbes, which might get deposited on various surfaces by one person and afterwards get collected by another. Human-associated microbes e.g., skin associated, are confronted with totally new biotic and abiotic factors in the built environment. Here they have to adapt to new surface materials, compete with others for scarce nutrients and withstand stresses associated to cleaning reagents etc. However, in the case of houseplants we allow them to proliferate in a protected environment. Plant associated microbes stay on the leave or stem surface, where they have adapted to and are sheltered from cleaning procedures. Although these phyllosphere communities are confronted with an absence of direct sun light and rain as well as other changed meteorological parameters like air/dust turbulences, their rhizosphere and surrounding soil communities stay in their natural habitat. Hence, these well balanced plant communities, which we bring inside, have the potential to balance an indoor microbiome, by increasing its diversity and filter airborne microbes.Members of the plant microbiome are an important source for indoor microbiomes. Both, plants from inside and outside can contribute to the micro-flora. Plant-associated bacteria could act as counterparts against pathogens within the microbial ecosystems. They stabilize the ecosystem, enhance biodiversity and avoid outbreaks of pathogens. However, more research is necessary to understand the microbiology of indoor environments. Currently used cleaning and hygiene strategies in built environments especially in hospitals and ICUs often promote multi-resistant pathogens instead of supporting beneficials. In future, it is important to re-think our understanding of necessary sterility and our relationship to our surrounding microbiomes. This \u201cparadigm shift in ecology\u201d is not only required for plants, humans Jones, but also"} +{"text": "Spruyt et al. report aHow can an alcohol-avoidance bias be a predictor of relapse, while alcohol-avoidance training has positive effects in alcohol-dependent patients? Consider two recent clinical alcohol-avoidance training studies. In our first study in 214 alcohol-dependent patients interventions, such as pharmacological treatment for alcohol craving: perhaps those patients with higher risk relapse had undergone different treatments that affected their approach/avoidance associations with alcohol. These particular third-variable explanations may or may not be true for the results of Spruyt et al.; they only serve to illustrate that a correlation between avoidance associations and relapse does not imply that the relapse risk was caused by the avoidance associations; and even less that inducing an avoidance association via training would cause an increased relapse risk.r\u2009=\u2009\u22120.05, p\u2009=\u20090.37, Field, personal communication). In our own first re-training study, we also used the relevant-feature approach-avoid Implicit Association Test . Hence, measuring an approach-bias with a relevant-feature task appears to be unrelated to measuring, and perhaps changing, an approach-bias with an irrelevant-feature task. It could at least theoretically be the case that the kind of avoidance associations as measured by a relevant-feature SRC cause an increased chance of relapse, while alcohol-avoidance associations retrained and assessed by an irrelevant-feature AAT decrease the chance of relapse.Second, different tasks were used in our studies and the study by Spruyt et al. In their prediction study, Spruyt et al. used the relevant-feature R-SRC, while we used an irrelevant-feature alcohol Approach Avoidance Task to measure the approach bias (alcohol AAT; Wiers et al., In conclusion, the evidence does not support the idea that the induction of an avoidance bias is likely to be harmful in alcoholic patients: the study of Spruyt et al. does not allow conclusions regarding causality, and a recent training study in fact showed that a relative increase in avoidance mediated the beneficial effects of avoidance training. We do, however, concur with Spruyt et al. that more research is needed regarding the assessment and modification of biases in action tendencies (Watson et al.,"} +{"text": "Cachexia is a complex metabolic syndrome associated with many chronic or end-stage diseases, especially cancer, and is characterized by loss of muscle with or without loss of fat mass. The management of cachexia is a complex challenge that should address the different causes underlying this clinical event with an integrated or multimodal treatment approach targeting the different factors involved in its pathophysiology. The purpose of this article was to review the current medical treatment of cancer-related cachexia, in particular focusing on combination therapy and ongoing research. Among the treatments proposed in the literature for cancer-related cachexia, some proved to be ineffective, namely, cyproheptadine, hydrazine, metoclopramide, and pentoxifylline. Among effective treatments, progestagens are currently considered the best available treatment option for cancer-related cachexia, and they are the only drugs approved in Europe. Drugs with a strong rationale that have failed or have not shown univocal results in clinical trials so far include eicosapentaenoic acid, cannabinoids, bortezomib, and anti-TNF-alpha MoAb. Several emerging drugs have shown promising results but are still under clinical investigation . To date, despite several years of coordinated efforts in basic and clinical research, practice guidelines for the prevention and treatment of cancer-related muscle wasting are lacking, mainly because of the multifactorial pathogenesis of the syndrome. From all the data presented, one can speculate that one single therapy may not be completely successful in the treatment of cachexia. From this point of view, treatments involving different combinations are more likely to be successful. Cachexia is a complex metabolic syndrome associated with underlying illness and characterized by loss of muscle with or without loss of fat mass. Cachexia can occur as part of many chronic or end-stage diseases such as infections, cancer, AIDS, congestive heart failure, chronic renal failure, rheumatoid arthritis, tuberculosis, and chronic obstructive pulmonary disease. The prominent clinical feature of cachexia is weight loss in adults (corrected for fluid retention). Anorexia, inflammation, insulin resistance, and increased muscle protein breakdown are frequently associated with cachexia. Cachexia is distinct from starvation, age-related loss of muscle mass, primary depression, malabsorption, and hyperthyroidism, and is associated with increased morbidity. CachexiaThe best management of cancer cachexia is to cure the cancer as this will completely reverse the cachexia syndrome. Unfortunately, this remains an infrequent achievement in adults with advanced solid tumors. A second option could be to counteract weight loss by increasing nutritional intake, but since in the majority of cachectic patients, anorexia in only a part of the problem, nutrition as a unimodal therapy was not completely able to reverse the wasting associated to cachexia. Indeed, a large number of randomized controlled trials of nutritional intervention did not show a significant benefit with regard to weight change or quality of life. These results have led to attempts to manipulate the process of cachexia with a variety of pharmacological agents, with the main purpose of providing symptomatic improvement. To date, however, despite several years of coordinated efforts in basic and clinical research, practice guidelines for the prevention and treatment of cancer-related muscle wasting are lacking, mainly because of the multifactorial pathogenesis of the syndrome.Progestagens, that is, Medroxyprogesterone Acetate (MPA) and Megestrol Acetate (MA) are currently considered the best available treatment option for CACS, and they are approved in Europe for treatment of cancer- and AIDS-related cachexia. However, progestational agents are nonetheless limited in their ability to treat cancer cachexia. Fewer than 30% of patients treated with MA experience short-term appetite stimulation, and alth5in vitro study,[et al., carried out a randomized placebo-controlled study[et al.,[In a subsequent ro study, they shoro study, Simons eled study to invesled study undertooy[et al., carried Among orexigenic agents, corticosteroids are widely used. In randomized controlled studies, they have been shown to improve appetite and quality of life compared with placebo.14 MA andet al.,[et al.,[et al.,[et al.,[Drugs able to inhibit the synthesis and/or release of cytokines (i.e. eicosapentaenoic acid (EPA), melatonin, etc.), the cytokine action -12, and IL- 15), and drugs able to inhibit the proteasome activity (i.e. bortezomib) have been tested in experimental models of cachexia, with some positive results. Unfortunately, most clinical trials in humans have provided limited or disappointing results. N-3 fatty acids, especially EPA, may have anticachectic properties. The first study by Fearon[et al., comparinet al., comparedet al., conclude,[et al., and Stra,[et al., have fai,[et al., showed n,[et al., showed tet al.,[Thalidomide has multiple immunomodulatory and antiinflammatory properties; its inhibitory effect on TNF-\u03b1 and IL-6 production may be responsible for its apparent anticachectic activity. Thus, thalidomide has been used for treatment of cachexia associated with acquired immunodeficiency syndrome, tuberculosis, and cancer. In the current literature, there are few studies that have assessed the anabolic effects of thalidomide in gastrointestinal cancer cachexia. Gordon et al., undertooet al.,[Research on experimental animal models has shown that non-steroidal anti-inflammatory drugs, including cyclooxygenase-2 (COX-2) inhibitors, may palliate cachexia through the suppression of systemic inflammation. Lai et al., carried Recently, much research interest has focused on ghrelin, a 28 amino acid peptide produced by the P/D1 cells of the stomach. Not only does ghrelin stimulate GH secretion (via the GH secretagogue-1a (GHS-1a) receptor) but it also promotes food intake (via the orexigenic NPY system) and decreases sympathetic nerve activity. Synthetic human ghrelin has been shown to improve muscle wasting and functional capacity in patients with cardiopulmonary-associated cachexia and to improve energy intake in anorexic cancer patients. Based on the animal studies and short-term human trials, there appears to be much promise for further studies to investigate the use of ghrelin and GHS-R agonists for the treatment of cachexia caused by multiple underlying conditions. Significant questions remain to be answered, however, before its widespread use, most prominently whether the gains produced by GHS R agonists maintain safety and efficacy with long-term use in human diseases. Clearly, more long-term research is needed.et al.,[of ghrelin to humans with cachexia has shown no univocal efficacy in increasing food intake with single dose intravenous administration.26 In a set al., 21 adultet al.,[et al.,[Neary et al., carried ,[et al., carried et al.,[Lundholm et al., carried Branched-chain amino acids are neutral amino acids with interesting and clinically relevant metabolic effects: They interfere with brain serotonergic activity and inhibit the overexpression of critical muscular proteolytic pathways. The potential role of branched-chain amino acids as antianorexia and anticachexia agents was proposed many years ago, but only recent experimental studies and clinical trials have tested their ability to stimulate food intake and counteract muscle wasting in anorectic, weight-losing patients. In experRecently, a prospective, randomized, phase III trial comparing the effects of oxandrolone (10 mg bid) and MA (800 mg q.d.) on weight, body composition, and quality of life (QOL) in 155 adult patients with solid tumors and weight loss receiving chemotherapy demonstrated that patients treated with oxandrolone experienced an increase in LBM, a reduction in fat mass, and reduced self-reported anorectic symptoms.et al.,[Olanzapine, an atypical neuroleptic with safe therapeutic window for several psychotic diseases, induces significant weight gain and positive metabolic effects. Preliminary data from a phase I pilot study by Braiteh et al., suggest et al.,[et al.,[n=12) or fish oil, 2 g tid, plus celecoxib 200 mg bid (n=10). All patients in both groups received oral food supplementation. After 6 weeks of treatment, patients receiving fish oil+placebo or fish oil + celecoxib showed significantly more appetite, less fatigue, and lower C reactive protein (C-RP) values than their respective baselines values . Additionally, patients in the fish oil+celecoxib group also improved their body weight and muscle strength compared to baseline values . Comparing both groups, patients receiving fish oil + celecoxib showed significantly lower C-RP levels and higher muscle strength , and body weight than patients receiving fish oil+placebo. The addition of celecoxib improved the control of the acute-phase protein response, total body weight, and muscle strength. In the context of combined approaches, one of the most intriguing ones was our open phase II trial.[L-carnitine 6 (4 g/day), (4) thalidomide (200 mg/day), or (5) medroxyprogesterone acetate/megestrol acetate plus pharmacologic nutritional support plus L-carnitine plus thalidomide. Treatment duration was 4 months. The sample size was 475 patients. The different single agents were selected on the basis of the following rationale. The antioxidant agents were shown to be effective in our previous studies.[4et al.,[From all the data presented, one can speculate that one single therapy may not be completely successful in the treatment of cachexia. From this point of view, treatments involving different combinations are more likely to be successful. Cerchietet al., carried ,[et al., aimed toII trial.38 The ai studies.\u201344 The p studies. Synthetistudies.4\u201349 The \u03c9studies.4\u201352 Barbe[4et al., demonstr[4et al., in 200 p[4et al.,54 Patien[4et al., and the in vitro and slow muscle wasting in rats with cancer cachexia.[Current new trends include anti-IL-6 humanized monoclonal antibody which in murine models appear to inhibit cancer cachexia; IL-15, acachexia. Formotercachexia. Recentlyet al.,[A new class of nonsteroidal SARMs is being developed for use in cancer cachexia. SARMs are designed to have predominantly anabolic activity in muscle and bone with minimal androgenic effects in most other tissues. Evans et al., carried et al., Recent eet al., Predictiet al., Key defiet al.,In summary, based on current views on the cachexia syndrome in cancer patients, we put forward the following recommendations:Wasting is a predictable event in many cancer patients, readily diagnosed by assessment of weight, change in appetite, and food intake. Often these patients will also have anemia and low albumin, with a concomitant increase in C-reactive protein. The above simple assessments should form a consistent part of the record of all advanced cancer patients.Use a systematic formal guide to rule out treatable secondary causes of wasting.At the onset and throughout the course of illness, offer patients nutritional counseling , encourage them to take part in a rehabilitation program tailor made for their needs and abilities, and consider the use of specific nutraceutical and pharmacologic interventions. Follow-up visits should not only note careful evaluation of antitumor therapy and tumor volume, but also regular assessment of symptom control, weight, appetite, and function.Take careful note of the full medication profile of patients who are wasting. These might include drugs that could have a favorable effect on cachexia and other agents that may be deleterious .Testosterone status should be established in cancer patients with the cachexia syndrome. If clearly reduced, physiologic testosterone supplementation should be considered after discussion with the patient.Patients must be assured of a reasonable intake of amino acids. Protein-containing foods are indicated and rich sources of both essential and nonessential amino acids will support any anabolic potential.Clinical researchers should be more cognizant of the work of their colleagues in sports medicine, AIDS, and geriatrics. Learning from their enterprises, further studies on creatine and supraphysiologic amounts of amino acids with a particular role in protein synthesis should be conducted. Similarly, the role of supraphysiologic doses of anabolic agents, in combination with nutrients and compounds that control muscle proteolysis, should receive high priority.There are few, if any, negative exercise trials. Patients should be encouraged to keep active or take part in tailored exercise programs, and studies on nutritional and pharmacologic agents should incorporate the potential additive effects of exercise."} +{"text": "Studies over the past decade have helped to decipher molecular networks dependent on Toll-like receptor (TLR) signaling, in mycobacteria-infected macrophages. Stimulation of TLRs by mycobacteria and their antigenic components rapidly induces intracellular signaling cascades involved in the activation of nuclear factor-\u03baB and mitogen-activated protein kinase pathways, which play important roles in orchestrating proinflammatory responses and innate defense through generation of a variety of antimicrobial effector molecules. Recent studies have provided evidence that mycobacterial TLR-signaling cross talks with other intracellular antimicrobial innate pathways, the autophagy process and functional vitamin D receptor (VDR) signaling. In this article we describe recent advances in the recognition, responses, and regulation of mycobacterial signaling through TLRs. Mycobacterium tuberculosis (Mtb). In this state, healthy immune-competent individuals are able to combat infection by mounting of an effective immune response for recognition of various molecular patterns of mycobacteria occurs as early as 24 h post-infection. It is obvious that Mtb elicits inflammation-dampening signals in macrophages which would favor its survival.Numerous studies have revealed that TLR2 is involved in the innate recognition and responses in innate immune cells such as macrophages and dendritic cells of a variety of mycobacterial cell wall antigens including 19-kDa mycobacterial lipoprotein, glycolipids like lipoarabinomannan (LAM), LM, 38-kDa antigen, LprG lipoprotein, phosphatidylinositol mannoside (PIM), triacylated (TLR2/TLR1), or diacylated (TLR2/TLR6) lipoproteins [reviewed in Jo et al. ; KleinniMany previous studies have reported that mycobacterial components are involved in innate recognition and responses through TLR signaling , Rv0315, and Rv0577 induce dendritic cell maturation and activation leading to increased expression of costimulatory molecules and proinflammatory cytokines , and potentiate the Th1 immune response . TDM has been reported to be tethered to several receptors, including TLR2, the class A scavenger receptor MARCO, Fc receptor-\u03b3 (FcR\u03b3), and macrophage-inducible C-type lectin (Mincle) , TLR2 signaling activates Wnt-\u03b2-catenin signaling. The authors suggest that TLR2 integrates Wnt-\u03b2-catenin signaling to modulate a battery of genes associated with T(Reg) cell lineage commitment. At low MOIs, Mtb-triggered macrophage apoptosis depends on a TLR2 signaling cascade associated with ASK1/p38 MAPK- and c-Abl-dependent phosphorylation of c-FLIP and its degradation, leading to caspase 8 activation (Kundu et al., Recent work by Bansal et al. suggestsTLR-dependent NF-\u03baB signaling and MAPK pathways contribute to antimycobacterial innate immunity through secretion of antibacterial effector molecules, cytokines, and chemokines, thus recruiting various immune cells to the site of infection (Jo et al., M. bovis BCG, are linked with the increased production of Th1-type T cell cytokines through regulation of monocyte IL-10 production (Randhawa et al., M. bovis BCG infection (Davila et al., Human genetic polymorphisms of TLRs have a role in regulating innate recognition of microbes and in determining susceptibility to mycobacterial infection. The relationship between TLR polymorphisms, disease susceptibility, and innate immune activities against mycobacteria has been described in several reviews (Texereau et al., Previously, it was shown that human TLR1 deficiency is linked with impaired mycobacterial innate immune signaling and susceptibility to leprosy (Misch et al., Several other innate immune pathways are closely linked with the TLR signaling pathway, thus cooperatively defining the innate immune response which enables clearance of mycobacteria inside cells. Activation of autophagy has a critical effector role in innate immune responses such as enhancement of phagosomal maturation and coordination of the innate and adaptive immune systems Deretic, , 2012. IRecent studies have uncovered the interplay of vitamin D-dependent antimicrobial responses and autophagy pathways in the activation of host defense against mycobacterial infection (Yuk et al., in vivo and in vitro (Kim et al., The impact of host autophagy on phagosome maturation and Mtb killing gives rise to the idea that compounds that activate or modulate the autophagy pathway, could potentially enhance antimicrobial activities against Mtb infection (Gutierrez et al., Developing vaccines and chemotherapeutic agents is the cornerstone of the successful management of tuberculosis. Understanding the molecular mechanisms of specific interactions of Mtb with its host should augment efforts in both these areas. Immune modulation as a strategy to combat mycobacterial infection remains underexplored. This review brings to light how mycobacterial modulins signaling through TLRs, contribute either to an effective host immune response or to immune evasion by the pathogen. Targeting those modulins that facilitate immune evasion, or exploiting those that facilitate a robust innate and adaptive immune response, could offer new avenues for controlling infection. The review also brings forth the attractive possibility of developing therapeutics designed to augment autophagy as a means of restricting mycobacterial survival and combating infection.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Spatial and bodily representations are multisensory processes that imply the integration of several afferent signals into a coherent internal model of our egocentric space. Crucially, this model involves also the vestibular information from the balance organs in the inner ear (Ventre et al., CVS has been used to modulate a wide range of cognitive and sensory functions in brain-damaged patients and in healthy participants (Utz et al., specificity of these effects. Does CVS directly affect the somatosensory processing? Are the observed effects mediated by non-specific factors, such as ocular movements, spatial attention or general arousal?Various hypotheses have been suggested to explain the CVS-induced modulation on tactile perception. In particular, one of the most controversial issues in the classical and current literature concerns the Since Rubens , most ofConversely, the role of non-specific effects such as spatial attention is still a matter of debate. This hypothesis argues that CVS may induce a reorientation of spatial attention toward the hemispace ipsilateral to the stimulated ear. Strong evidence against this hypothesis derives from a recent study on brain-damaged patients (Bottini et al., More recent behavioral and electrophysiological studies, in healthy participants, have strengthened this suggestion. There are at least three main crucial observations ruling out interpretation in terms of non-specific attentional effects. First, left-cold CVS affects the perception of distinct somatosensory sub-modalities, i.e., touch and pain, for both the ipsilateral and contralateral hand (Ferr\u00e8 et al., Previous studies exploring more widely CVS effects also support the idea that spatial attention does not have a pivotal role. Rorden et al. did not specific and related to the activation of cortico-subcortical networks (Lopez et al., To conclude, this evidence suggests that in healthy volunteers the effects of CVS are"} +{"text": "Biological Psychiatry, Levy-Gigi et al. found that a 12 weekly 90-mins cognitive behavioral therapy (CBT) treatment in individuals with posttraumatic stress disorder (PTSD) is associated with an increase in hippocampal volume and expression of glucocorticoid receptor genes, known as FKBP5 (Levy-Gigi et al., In a recent study in Interestingly, Levy-Gigi et al. found that CBT ameliorates all aspects of PTSD, including avoidance, re-experiencing, and hyperarousal symptoms. However, it is not clear from the Levy-Gigi et al. study whether changes to the hippocampal volume are associated with amelioration to which PTSD symptoms. Prior studies show that there is a trend for a negative correlation between higher re-experiencing symptoms and hippocampal volume (Shucard et al., At the neural level, several studies show that PTSD is associated with abnormalities to various brain areas, including reduced activity and volume of the hippocampus (Gilbertson et al., How can hippocampal dysfunction be related to PTSD symptoms? As noted by Levy-Gigi et al., the ventral hippocampus-amygdala pathway has been shown to be related to increased stress and anxiety (Fanselow and Dong, The Levy-Gigi et al. study also showed that CBT alters the expression of genes, including the FKBP5 gene. FKBP5 regulates glucocorticoid receptor sensitivity, and reduce the efficacy of cortisol in the brain (Mahon et al., Along these lines, Felmingham et al. found thFuture computational modeling work is needed to explain how changes to the hippocampal volume and gene expression can ameliorate PTSD symptoms [see for example Krasne et al. ; Li et aIn summary, Levy-Gigi et al. have ext"} +{"text": "Recent advances in sequencing technologies have revealed extensive intratumour heterogeneity (ITH) both within individual tumours and between primary and metastatic tumours for different cancer types. Such genetic diversity may have clinical implications for both cancer diagnosis and treatment with increasing evidence linking ITH and therapeutic resistance. Nonetheless, whilst limiting the activity of targeted agents, tumour genetic heterogeneity may provide a new therapeutic opportunity through generation of neo-antigens that could be recognised and targeted by the patient's own immune system in response to immune-modulatory therapies. Longitudinal genomic studies assessing tumour clonal architecture and its correlation with the underlying immune response to cancer in each particular patient are needed to follow tumour evolutionary dynamics over time and through therapy, in order to further understand the mechanisms behind drug resistance and to inform the development of new combinatorial therapeutic strategies. Current Opinion in Pharmacology 2013, 13:497\u2013503CancerThis review comes from a themed issue on Massimo Santoro and Francesca CarlomagnoEdited by Issue and the EditorialFor a complete overview see the Available online 7th May 20131471-4892/$ \u2013 see front matter, \u00a9 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.coph.2013.04.006The existence of distinct subpopulations of cancer cells within a tumour harbouring different behavioural phenotypes, including tumourigenicity, ability to metastasise and evolve resistance to treatment, has been recognised for many years . Recent In this article we review the clinical implications of ITH for the genetic stratification of tumours, the emerging evidence that suggests the need to investigate the changing nature of tumour subclonal architecture through therapy and the potential impact of such diversity on anti-tumour immunity. We argue that an in-depth understanding of tumour evolution over time, the mechanisms driving tumour diversity and its impact on immunity may lead to the improved management of cancer patients .Phenotypic heterogeneity observed in tumours results from both genetic and non-genetic causes of heterogeneity. Spontaneous tumours are known to arise through Darwinian-like somatic clonal evolution involving the acquisition of \u2018driver\u2019 events, such as genetic mutations or copy number variations, believed to affect cancer cell proliferation or survival, along with \u2018passenger\u2019 events, assumed to be phenotypically silent without a selective fitness advantage . Non-genRecent advances in massively parallel sequencing technologies have enabled the analysis of the complex clonal architecture of both primary and metastatic tumours . PatternHER2 predicts response to trastuzumab but its distribution can be heterogeneous in primary tumours and associated with shorter disease-free survival times compared to patients with homogenous HER2 amplification [et al. [HER2 amplification in oesophageal adenocarcinoma independently predicted worse disease-specific survival and overall survival compared to non-heterogeneous HER2 amplified tumours.The validation of predictive biomarkers may be simpler and less subject to tumour sampling bias when present in all regions of a tumour and sustained during disease progression. However, ITH for the expression of genetic and phenotypic biomarkers has been shown in several tumour types. In breast cancer, the amplification of fication . Yoon et [et al. showed tEGFR predict response to gefitinib, but discordance for the EGFR mutation has been shown between primary and metastatic tumours [HER2 amplification and HER2 protein overexpression has been shown within the same tumour, and between diagnostic biopsies and resected tumours [HER2 amplification between primary and metastatic tumours has also been shown in breast cancer [et al. [KRAS, NRAS, BRAF, PIK3CA and TP53 genes. However, in patients with a history of more than one colorectal primary tumour and interval treatment, there was evidence for discordance in TP53. These examples demonstrate that relying on a single tumour biopsy may lead to sampling bias in some cases and risk missing potentially therapeutically relevant lesions or contribute to the allocation of a mutation as actionable without establishing clonal dominance [Primary and metastatic tumours can evolve independently and acquire different phenotypes leading to significant genetic divergence, and therefore discordance, between primary and metastatic tumours in terms of biomarkers detected in the diagnostic biopsy . In non- tumours . In prim tumours . Discordt cancer . In colo [et al. found muominance . Furtherominance . This maT790M mutation known to confer insensitivity to gefitinib [et al. [EGFR mutations treated with EGFR TKIs, the presence of low frequency subclones harbouring T790M mutations before the onset of treatment was associated with shorter progression-free survival, and Turke et al. [MET amplification was associated with EGFR TKI resistance. In colorectal cancer, wild-type KRAS predicts sensitivity to anti-EGFR antibody therapies such as panitumumab. Diaz et al. [KRAS wild-type tumours, the emergence of mutations in KRAS could be detected during the course of therapy resulting in acquired resistance. They concluded that subclonal populations harbouring KRAS mutations existed before commencing treatment, and that under the selective pressure of anti-EGFR blockade, resistant subclones rapidly expand and repopulate the tumour. In chronic myeloid leukaemia and gastrointestinal tumours, resistance to imatinib due to mutations in the BCR-ABL fusion protein [Most advanced cancers still remain incurable despite significant progress in the fields of cancer research and therapy. Response to therapy is generally of limited duration. This may be due to the inevitable evolution and proliferation of resistant subclonal populations, which may exist before the onset of treatment, under the selective pressure of therapies \u2022\u2022. In NSefitinib \u2022\u2022. Su et [et al. demonstre et al. \u2022 showed z et al. \u2022\u2022 showed protein and KIT protein respecti protein . These eet al. [EGFR mutant NSCLC with an EGFR TKI and EGFR-specific antibody could prevent resistance associated with the expansion of a subclone harbouring a T790M mutation. Approaches like this would require the development of biomarkers predicting likely resistance mechanisms in different patients, and such mechanisms could be targeted either in combination, or alternating, with standard treatment regimens [In light of increasing evidence in support of ITH and its role in treatment resistance, there is a need for alternative therapeutic approaches. Gillies et al. argue thet al. \u2022\u2022, and tregimens . Treatmeregimens and melaregimens . Other aregimens .post hoc analyses of data originally generated by Sjoblom et al. [in vivo \u2018vaccine\u2019 or priming effect which could be further enhanced by interference with immune-modulatory pathways. Whilst the neo-antigenic repertoire generated by ITH could be seen as non-self by the immune system, the type of tumour cell death and inflammatory environment within the tumour will define their immunogenicity and the final outcome of the immune response (i.e. tumour progression versus regression). Importantly, immunity to tumour-associated antigens can be potentiated given proper identification and manipulation of immune-regulatory checkpoints restricting T cell function [Whilst emerging evidence supports the notion that ITH limits the efficacy of conventional and targeted therapeutics, its overall effect on the immune response to cancer may still be of potential benefit for the patient since intratumoural mutational diversity can provide neo-antigens that may be perceived by the immune system as non-self, producing unique opportunities for the generation of anti-tumour immunity. The wealth of data now being generated through whole genome sequencing of tumour samples provides further support for this concept. In silico-based computer algorithms combined with high-throughput m et al. revealedm et al. . Furtherm et al. . MSI is m et al. . One potm et al. . Based ofunction . This hafunction . In addifunction \u2022).Although ITH may complicate diagnostic and treatment decisions, it can be clinically useful in predicting clinical outcome. In Barrett's oesophagus \u2022 and breWith the development of improved technologies allowing the interrogation of ITH, our understanding of tumours and their evolutionary trajectories may lead to better design of clinical trials in search of improved therapeutic interventions to anticipate the emergence of drug resistance mechanisms and generate improved predictive and prognostic biomarkers . Whilst None declared.None.\u2022 of special interest\u2022\u2022 of outstanding interestPapers of particular interest, published within the period of review, have been highlighted as:"} +{"text": "A recent study found that mosquito-transmitted (MT) lines of rodent malaria parasites elicit a more effective immune response than non-transmitted lines maintained by serial blood passage (non-MT), thereby causing lower parasite densities in the blood and less pathology to the host. The authors attribute these changes to higher diversity in expression of antigen-encoding genes in MT cf. non-MT lines. Alternative explanations that are equally parsimonious with these new data, and results from previous studies, suggest that this conclusion may be premature. Plasmodium chabaudi or other malaria species are transmitted through mosquitoes, their virulence and asexual replication rate in the blood is reduced [et al.[Many studies have shown that when parasite lines of the rodent malaria reduced -6. This reduced ,6-13. Spet al. [et al.\u2019s [The family of genes that Spence et al. found toet al.\u2019s observatet al. (Plasmodium chabaudi in C57/Bl6 laboratory mice) parasite lines were evolved by SBP through either immunized or na\u00efve mice and, at the end, virulence of the evolved lines was measured, both in immunized and na\u00efve mice, and both before and after mosquito transmission [Does this resetting render MT parasites more controllable by the immune system, or is it just that MT parasites are less intrinsically virulent? In previous experiments studying the evolution of parasite virulence in the same experimental model used by Spence smission . Before et al.[et al.[et al.\u2019s [However, when the I-lines were compared to the N-lines in pre-immunized mice, the differences disappeared. In the light of new data from Spence et al., it is hl.[et al., these ret al.\u2019s interpreet al.[However, several alternative explanations that invoke changes to the parasite\u2019s intrinsic virulence, as opposed to the immunity it provokes, are similarly parsimonious with the data of Spence et al. and fromet al.,2. Severet al. and expeet al., bottlenet al. virulencet al.[Plasmodium falciparum, the most deadly of the human malaria parasites, that certain subsets of the variable surface antigens are highly pathogenic [Although virulence attenuation in laboratory models of malaria -6 is higthogenic -20 thus The author declares that she has no competing interests."} +{"text": "Plants imperatively have to cope with adverse conditions owing to their lack of mobility and to the high amounts of reactive oxygen species (ROS) generated from both respiration and photosynthetic metabolism. Although thiol redox homeostasis in plants is mainly preserved by the cellular glutathione pool, specific strategies have been adopted by the plant kingdom during evolution to manage these \u201cextra\u201d pro-oxidative conditions. Unlike human or yeast, plants generally possess a higher number of genes coding for antioxidant proteins, including protein families responsible of dithiol/disulfide exchange reactions. During the last decades, redox-dependent post-translational modifications of proteins proved to be pivotal to many cellular functions. In particular, this is critically important under some situations of environmental constraints taking into account the alterations and fine adjustment of the cellular redox status occurring during and after any biotic or abiotic stresses.Indeed, thiol groups of cysteinyl residues are highly sensitive to oxidation which might critically perturb cellular homeostasis. Members of the thioredoxin superfamily are key proteins involved in the regulation of cysteine/protein redox state. They share two common and well-known features: (i) the presence of an active center containing at least one catalytic cysteine residue, and (ii) a highly conserved 3D-structure, the so-called thioredoxin fold, which consists of a four-stranded anti-parallel \u03b2-sheet surrounded by three \u03b1-helices. Key members of this super family are thioredoxins (TRX) and glutaredoxins (GRX). Representatives of both subgroups are distributed in most cellular compartments and contain at least one TRX motif in their structures. While TRXs are generally reduced by thioredoxin reductases (TR), the reduction of GRXs depends on reduced glutathione (GSH).The 19 reports of this Research Topic provide timely overviews and new insights into redox regulation, focusing on both TR/TRX and GSH/GRX reduction systems in plants. The biochemical characteristics of these systems as well as their target proteins and functions in metabolic and signaling pathways are discussed. Several contributions to this Research Topic deal with the role of TRX systems in plastid metabolism. Michelet et al. summarizArabidopsis, and on the discovery of trans-thylakoid redox pathways controlling disulfide bond formation and reduction.Within the photosynthetic context, Karamoko et al. propose in vivo approach showing the positive effect of NTRC overexpression in plant performance.In addition, the importance of NADPH thioredoxin reductase C (NTRC) in plastid redox regulation is also reported in four articles. The paper by Puerto-Gal\u00e1n et al. discusseThe importance of redox regulation in plant mitochondria is also highlighted in this Research Topic by L\u00e1zaro et al. . These ah with selected target proteins using surface plasmon resonance. The presented data reveal a stronger preference of TRX h for an oxidized target, thus explaining the selective association of TRX with oxidized proteins. Zaffagnini et al. (Some aspects of the cytosolic redox regulation pathways have also been developed in this Research Topic. Hara and Hisabori analyzedi et al. review hgrx mutants, that GRXs are essential for stress adaptation in cyanobacteria. Finally, Knaff and Sutton (GRXs are oxidoreductases of the TRX family which display a particularly rich diversity in higher plants. Beyond classification in three main subgroups based on sequence and structural features, Couturier et al. indicated Sutton describeArabidopsis. The data demonstrate that cellular glutathione homeostasis influences the root architecture and the leaf area under optimal and stress conditions. Another aspect where glutathione and homoglutathione are crucial molecules is nodule development and in the context of legume-rhizobium mutualistic interactions. Indeed, these organs are peculiar due to the formation of a bacteroid in which the oxygen-sensitive nitrogenase reduces di-nitrogen to ammonia. However, the importance of other redox systems in this unique organ has been poorly documented. Frendo et al. (Glutathione is a key component in regulation and maintenance of cellular thiol redox homeostasis. It also plays key roles in different aspects of plant development. Here, Rahantaniaina et al. provide o et al. review to et al. summarizThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "N-acetyl-beta-d-glucosaminidase), markers of inflammation and markers of oxidative stress. Despite the promise of some of these new biomarkers, further large, multicenter prospective studies are still needed before they can be used in everyday clinical practice.Diabetes prevalence is increasing worldwide, mainly due to the increase in type 2 diabetes. Diabetic nephropathy occurs in up to 40% of people with type 1 or type 2 diabetes. It is important to identify patients at risk of diabetic nephropathy and those who will progress to end stage renal disease. In clinical practice, most commonly used markers of renal disease and progression are serum creatinine, estimated glomerular filtration rate and proteinuria or albuminuria. Unfortunately, they are all insensitive. This review summarizes the evidence regarding the prognostic value and benefits of targeting some novel risk markers for development of diabetic nephropathy and its progression. It is focused mainly on tubular biomarkers (neutrophil-gelatinase associated lipocalin, kidney injury molecule 1, liver-fatty acid-binding protein, Chronic kidney disease (CKD) is an important public health problem and the prevalence is estimated to be 8%\u201316% worldwide . DiabeteAccording to the above mentioned, it is clear that new biomarkers are needed to predict patients who will develop diabetic nephropathy or are at risk of progressing to ESRD. Some of the promising new biomarkers in serum and urine will be presented in this paper.Tubulointerstitial damage is an important feature of diabetic nephropathy and is, in addition to glomerular damage, an important predictor of renal dysfunction ,5,6,7. INeutrophil gelatinase-associated lipocalin (NGAL) is small (25-kDa) protein that is released from injured renal tubular cells in acute kidney injury into the blood and urine, long before a decrease in the glomerular filtration rate can be detected ,9,10. Seet al., included 96 patients with CKD stages 2\u20134, among them 20% with diabetic nephropathy [et al., 74 type 2 diabetic patients were included [et al., 158 patients with CKD stage 3 and 4 were included, and 6% were patients with diabetic nephropathy [2) over one year. Adding urine NGAL-to-creatinine ratio demonstrated the greatest benefit in identifying patients with CKD progression only in group of patients with relatively low proteinuria. In the study by Nauta et al., 94 diabetic and 45 non-diabetic control subjects were included [et al., in 63 type 1 diabetic patients with diabetic nephropathy who were followed for three years [et al., serum and urine NGAL were used in addition to albuminuria to predict the GFR decline rate in type 2 diabetic patients, but only urine albumin excretion rate was significantly associated with eGFR and the eGFR decline rate [In one of the first large studies, Bolignano hropathy . Patientincluded . They wehropathy . The basee years . Accordiine rate .According to conflicting results of previously mentioned studies, and due to the strong link with proteinuria in most of the studies, serum and urine NGAL do not offer additional prognostic information compared to already established biomarkers.Kidney injury molecule 1 (KIM-1) is a transmembrane tubular protein with unknown function, not detectable in the normal kidney, but elevated in experimental and clinical kidney damage . It is aet al., in biopsies from various renal diseases and controls, renal KIM-1 was significantly increased in all diseases versus controls, except minimal change [versus controls and correlated positively with tissue KIM-1 and negatively with renal function, but again not with proteinuria [et al., 145 renal transplant recipients were included and were followed for four years for graft loss [In the study by von Timmeren l change . KIM-1 wl change . Renal Kl change . At the l change . Urinaryteinuria . In the aft loss . At baseaft loss . In multaft loss . Urinaryaft loss ,27,28,29Liver-fatty acid-binding protein (L-FABP) is an intracellular carrier protein that is expressed in proximal tubules of the kidney and liver. Although its precise function is unknown, it is believed to be endowed with protective functions . L-FABP et al., including 165 normoalbuminuric type 1 diabetic patients urinary L-FABP predicted the development of micro- and macro-albuminuria independent of recognized biomarkers [et al., including over 100 type 2 diabetic patients, high urinary L-FABP levels were associated with the increase in albuminuria, progression of diabetic nephropathy to ESRD or induction of haemodialysis [et al., only urine albumin excretion rate was significantly associated with eGFR and eGFR decline rate in type 2 diabetic patients and not L-FABP [In the large prospective study by Nielsen omarkers . In anotdialysis . In the t L-FABP .et al., including type 2 diabetic patients, heart fatty acid-binding protein (H-FABP), a marker of distal tubular damage, was the only tubular marker associated with eGFR after adjustment for other risk markers of progressive diabetic nephropathy [In the study by Nauta hropathy .According to published studies, members of the FABP family are emerging as the tubular markers with the greatest chance of offering added predictive value for progressive diabetic nephropathy over and above that offered by established risk markers .N-acetyl-beta-d-glucosaminidase (NAG) is a lysosomal enzyme that is constitutively expressed by the proximal tubule and a well-studied urinary marker of established proximal tubule cell injury [Urinary l injury . NAG is l injury .et al., lower urinary NAG levels were associated with the regression of microalbuminuria in type 1 diabetes mellitus [et al., including type 1 diabetic patients who participated in the Diabetes Control and Complications Trial (DCCT), baseline levels of urinary NAG independently predicted the development of micro- and macro-albuminuria during the follow up period of nine years [In the study by Vaidya mellitus . In the ne years . The lacne years . FurtherSeveral factors are involved in the development and progression of diabetic nephropathy. Growing evidence indicates that pathogenesis and progression of diabetic nephropathy is associated with the presence of a chronic subclinical low-grade inflammatory state and oxidative stress ,43. IncrCytokines are low molecular weight polypeptides and their most important function is regulation of the inflammatory process. They contribute in accelerating and maintaining chronic inflammation. The first studies suggesting the role of inflammatory cytokines in the development of diabetic nephropathy were published more than 20 years ago ,45.et al., 194 type 1 diabetic patients from multi-centre Finnish Diabetic Nephropathy Study (FinnDiane) were included [et al., including 29% of patients with diabetic nephropathy baseline IL-6 levels gradual increase from control subjects to CKD stage 3 and 4 and to CKD stage 5 patients [et al., five inflammatory markers\u2014IL-6, IL-8, monocyte chemoattractant protein 1 (MCP-1), interferon \u03b3-inducible protein (IP-10), and macrophage inflammatory protein 1d\u2014were measured in urine samples collected from individuals with type 1 diabetes [Interleukin-6 (IL-6) is a proinflammatory cytokine, which is produced by many cells and is also associated with visceral obesity and insulin resistance . In the included . They weincluded . In thisincluded . In the patients . In the diabetes . Baselindiabetes . By multdiabetes .et al., 151 type 2 diabetic patients with various degrees of nephropathy, as well as 80 healthy volunteers, were included [In the study by Moriwaki included . Signifiincluded . In addiincluded .et al., serum and urinary IL-18 and serum IL-6 levels were also significantly elevated in patients with type 2 diabetes compared to control subjects [In the another study by Nakamura subjects . In uni-subjects .et al., reported in two studies direct and significant association between serum TNF-\u03b1 and urinary protein excretion in diabetic patients with normal renal function and microalbuminuria, as well as in subjects with overt nephropathy and renal insufficiency [et al., where significant differences in serum levels of TNF-\u03b1 were observed between the type 2 diabetic patients and control subjects [The primary source of tumour necrosis factor-\u03b1 (TNF-\u03b1) are monocytes and macrophages, although intrinsic renal cells are also able to synthesize this cytokine . It was ficiency ,56. Urinficiency ,56. Multficiency ,56. Simisubjects . In patisubjects .et al., only levels of soluble forms of receptors 1 and 2 for TNF-\u03b1, and not TNF-\u03b1, were found to be associated with GFR in a multivariate analysis [et al., 410 patients with type 2 diabetes were included and were followed for 12 years [et al., 628 patients with type 1 diabetes, normal renal function and no proteinuria were included and followed for 12 years; in the follow up period, 69 patients developed eGFR less than 60 mL/min per 1.73 m2 [Interestingly, in the study by Niewczas analysis . In anot12 years . In foll12 years . In the 1.73 m2 . In this 1.73 m2 . SimilarInfiltration of leukocytes into inflammatory lesions is mediated by adhesion to endothelial cells and transmigration from vascular lumen to inflammatory sites . Adhesioet al., type 1 diabetic patients and healthy subjects were included [et al., baseline levels of ICAM-1 and VCAM-1 were measured from stored blood samples from the 1441 participants of the DCCT [et al., 30 hypertensive type 2 diabetic patients and 30 non-diabetic normotensive subjects were included; their VCAM-1, ICAM-1 and selectin levels were measured [In the study by Clausen included . In thisincluded . In the the DCCT . Only anthe DCCT . In the measured . The diameasured . Due to Growing evidence suggests that recruitment of inflammatory cells from the circulation into renal tissue plays a pivotal role in the progression of various renal diseases, including diabetic nephropathy. Infiltration of activated T cells and monocytes initiate renal damage and lead to a progressive loss of renal function ,67. Chemet al., 45 type 2 diabetic patients and 20 healthy subjects were included [et al., levels of urinary MCP-1 in type 2 diabetic patients with normal renal function were significantly higher than those in healthy adults, and urinary MCP-1 levels increased gradually from normo-, micro- to macro-albuminuria [et al., urinary levels of MCP-1 in patients with macroalbuminuria were significantly elevated compared to the levels in patients with normo- and micro-albuminuria [et al., in type 1 diabetic patients, baseline urinary MCP-1 was significantly higher in those with an early progressive kidney function decline compared with those who displayed stable kidney function [In the study by Wada included . Urinaryuminuria . Similaruminuria . In thisuminuria . In prevfunction .et al., showed that blockade of the renin\u2013angiotensin system in patients with type 2 diabetes was associated with a reduction in urinary MCP-1 levels as well as an improvement in renal function [et al., found that aldosterone blockade by spironolactone may offer beneficial renoprotective effects through anti-inflammatory mechanisms via the modulation of MCP-1 [Two interesting studies demonstrating the effect of treatment with ACE inhibitors and spironolactone on MCP-1 in type 2 diabetic patients have been published. In the first, Amann function . In the of MCP-1 .etc., and reduced levels of antioxidant agents and enzymes, such as bilirubin, superoxide dismutase and antioxidant vitamins, thus promoting oxidative stress [Under normal physiological conditions, there is a balance in the generation of oxygen-free radicals and the antioxidant defence mechanisms used to deactivate free radical toxicity . Experime stress ,76. Mease stress ,78. 8-OHe stress ,78. Urine stress ,78.et al., 396 Japanese type 2 diabetic patients with normoalbuminuria or microalbuminuria were included and followed for five years [In the large study by Hinokio ve years . Authorset al., 52 patients with type 2 diabetes mellitus (32 with nephropathy and 20 without) and 20 healthy control subjects were included [The multivariate logistic regression analysis suggested that the urinary 8-OHdG was the strongest predictor of nephropathy among several known risk factors. In the another study by Serdar included . The conAccording to conflicting results of clinical studies, it is questionable if markers of oxidative stress add additional prognostic information in relation to the development or progression of diabetic nephropathy compared to established risk markers.etc. [Today, in clinical practice, most commonly used markers of renal disease and progression of CKD and also diabetic nephropathy are serum creatinine, eGFR and proteinuria/albuminuria but unfortunately they are all insensitive. Some new biomarkers reviewed in this paper are promising but further large, multicenter prospective studies are needed before they can be used in everyday clinical practice. The main problem is that most of the biomarkers are still at an intermediate phenotype level, which is too distant from the gene level and most of these biomarkers are deeply influenced by environment, genetics, sex differences, etc. . It is aDespite that the purpose of this paper is to present the biomarkers of renal disease and progression in patients with diabetes, it is also important and should be mentioned that, in patients with diabetic nephropathy, there is also a concomitant increase in cardiovascular morbidity and mortality . Some of"} +{"text": "Recently, Christoph Thiel and colleagues from Aachen University published an improved physiologically based pharmacokinetik modeling (PBPK) technique for mouse to human extrapolation . This pThe translation of preclinical knowledge often generated in mice to first-in-human studies represents a critical step . More tspecies-specific physiology, such as differences in organ size, perfusion, etc. the species-specific non-protein bound fraction of the test compound, max and KM for the primary route of excretion, and kinetic parameters, such as Vtissue-specific gene expression of the metabolizing key enzymes and transporters.The authors start with a na\u00efve extrapolation where humans are considered as 'large mice' where the same dose per body weight was administered . This nInterspecies differences represent a major problem in toxicology (Dohnal et al., 2014; BernauePBPK modeling has been used since long to predict absorption, distribution, metabolism and excretion (Sterner et al., 2013; Lee et"} +{"text": "G-nitro-L-arginine methyl ester was performed throughout fructose intake. L-NAME treatment itself induces increase in blood pressure and relative heart weight as well as impairment in arterial relaxation and contractility. However, in these rats, fructose administration did not cause further elevation of blood pressure and other abnormalities observed in rats receiving fructose without L-NAME. Our results showed that in the state of NO deficiency (induced by L-NAME administration) fructose does not induce cardiovascular and metabolic alterations which develop in rats with a functional NO system. This indicates that impairment of the NO system may participate in many of the adverse effects induced by high-fructose intake.The aim of this study was to evaluate the involvement of nitric oxide (NO) system damage in the deleterious effects of high-fructose intake in rats. Fructose was administered as 10% solution in drinking water to twelve-week-old male Wistar rats for the period of 8 weeks. Blood pressure was measured by tail-cuff plethysmography. After sacrificing the rats at the end of the treatment, relative weights of heart and liver and biochemical parameters in blood plasma were determined. Reactivity of isolated conduit arteries was measured using a force-displacement transducer for recording isometric tension. Fructose drinking rats had increased blood pressure and impaired acetylcholine-induced relaxation of the thoracic aorta in comparison with control rats drinking just tap water. Relative liver weight and plasma concentrations of glucose and triglycerides were also elevated after fructose administration. In a further group of Wistar rats, inhibition of NO production by administration of N NG-nitro-L-arginine methyl ester \u2013 L-NAME) causes NO deficiency and impaired vascular relaxation leading to sustained blood pressure increase plays a critical role as a molecular mediator in a variety of biological processes, including vasodilatation, neurotransmission and macrophage-mediated immunity. In spite of its pleiotropic effects in organisms, its role has been most extensively studied in physiology and pathology of the cardiovascular system. It is its prominent role in endothelium-dependent relaxation of vessels thanks to which the function of NO in the cardiovascular system was discovered and identified , fructose drinking rats (receiving 10% solution of fructose for 8 weeks), L-NAME treated rats (receiving 40 mg/kg/day of L-NAME in drinking water for 8 weeks), L-NAME treated fructose drinking rats (receiving 40 mg/kg/day of L-NAME in 10% solution of fructose for 8 weeks). The animal protocols used in this study were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health, and approved by the Animal Health and Welfare Division of the State Veterinary and Food Administration of the Slovak Republic.Twelve-week-old male Wistar rats were housed at 22\u201324\u00b0C on a 12:12-h dark-light cycle (06.00\u201318.00 h lights on) and maintained on a standard laboratory rat chow th week of life), all rats were fasted overnight and then they were sacrificed under CO2 anesthesia. Samples of their blood were collected immediately and used for measurement of plasma glucose, cholesterol and triglyceride concentration. Wet weight of the left heart ventricle and of the liver and the length of the tibia were determined for calculation of the ratio of left heart ventricle weight to tibia length and of liver weight to tibia length. The thoracic aorta and superior mesenteric artery were carefully removed and prepared for functional studies performed by isometric tension recording.Systolic blood pressure and heart rate were measured in conscious rats by the non-invasive tail-cuff method. At the end of the experiment (in the 202 + 5% CO2) modified Krebs solution maintained at 37\u00b0C. The Krebs solution had the following composition (in mmol/l): NaCl 118, KCl 5, CaCl2 2.5, MgSO4 1.2, NaHCO3 25, KH2PO4 1.2, glucose 11, CaNa2.EDTA 0.03. The arterial rings were set up for isometric tension recording using a force-displacement transducer Sanborn FT 10 . The preparations were equilibrated under a resting tension of 10mN for 60\u201390 min and the solution was changed every 15 min.The arteries were cut into rings (3.0\u20133.5 mm in width) and suspended in 20 ml organ baths filled with oxygenated (95% O\u20136 mol/l). When the contraction reached a plateau, increasing concentrations of acetylcholine were applied in a cumulative manner (10\u20139\u201310\u20135 mol/l).To examine the endothelium-dependent vasorelaxation, the preparations of the aorta were first precontracted by phenylephrine (10\u201310\u20133\u00d710\u20135 mol/l).Adrenergic contractions were determined in endothelium-intact thoracic aortas and mesenteric arteries as the responses to cumulatively applied exogenous noradrenaline . Frequency-response curves to electrical stimuli were obtained using square pulses of 0.5 ms in duration, at supramaximal voltage (>30 V), applied at 1\u201332Hz, for a period of 20 s. In our preliminary observations we found that the contractions of rat mesenteric arteries elicited by electrical stimulation (using the described parameters of stimulation) were blocked by phentolamine or tetrodotoxin, indicating that they were induced by nerve-released (endogenous) noradrenaline.N\u03c9-Nitro-L-arginine methyl ester hydrochloride (L-NAME) were purchased from Sigma-Aldrich (Germany); other chemicals were purchased from local commercial sources.Acetylcholine chloride, L-Noradrenaline hydrochloride and The results are presented as means \u00b1 S.E.M. The arterial responses to particular pharmacological and electrical stimuli are expressed as absolute values in mN and normalized to the cross sectional area of the respective ring preparation.p<0.05.Statistical evaluation was carried out by using one-way analysis of variance (ANOVA). The results were considered to be significant when After eight weeks of treatment with fructose, the Wistar rats had significantly elevated blood pressure without change in heart rate, when compared to control untreated rats. Body weight and relative weight of the left heart ventricle did not differ between control and fructose drinking rats. However, the relative liver weight, as well as plasma glucose and triglyceride concentrations were increased due to fructose treatment .In the thoracic aorta, endothelium-dependent relaxations in response to acetylcholine were significantly decreased after fructose administration . ContracEight-week L-NAME administration to control Wistar rats caused significant increase in blood pressure, relative weight of left heart ventricle, and in plasma cholesterol level . It alsoIn this study we found that eight-week-lasting fructose administration to adult Wistar rats induced metabolic changes associated with increase in plasma glucose and lipids, enlargement of liver, mild but significant blood pressure elevation, and reduction in acetylcholine-induced arterial relaxation. We also showed that in rats made NO deficient by L-NAME treatment, the high-fructose intake did not evoke such alterations. These observations indicate that impairment of the NO system might represent an important mechanism of high-fructose induced damage.et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., It was shown by other investigators that production of NO decreased after feeding normal rats with excess of fructose and that this effect may induce hypertension development in such treated individuals which exhibit similar severity of hypertension as L-NAME-treated rats and their arteries have also diminished endothelium-dependent relaxation. However, in conduit arteries of adult SHR the NO synthase activity is markedly elevated and their resting relaxant capacity is still sufficiently NO dependent (Puzserova et al., et al., 1996; Yoneyama et al., The presented observations show that fructose administration had no effect on the magnitude of arterial contractions induced by exogenous or endogenous noradrenaline. On the other hand, treatment of rats with L-NAME alone led to diminution of arterial contractile responses. This paradoxical effect of inhibiting NO synthase was demonstrated by many authors (Dowell et al. (et al., Inhibition of NO production eliminated also the high-fructose evoked increase in liver mass and in plasma glucose and lipids. It is known that NO is implicated in a myriad of mechanisms regulating liver metabolism and its higher amount may have the potential for both protecting the liver as well as exacerbating its injury. Spruss et al. demonstr et al., or in ra et al., , variousIn conclusion, in this study we demonstrated that NO deficiency abolished the abnormalities observed during fructose administration in normal rats. It may thus be supposed that impairment of the NO system may participate in many of the adverse effects induced by high-fructose intake since a functional NO system is necessary for their manifestation."} +{"text": "The endothelium is the orchestral conductor of blood vessel function. Pathological blood vessel formation or the inability of endothelial cells (ECs) to perform their physiological function (a condition known as EC dysfunction) are defining features of various diseases. Therapeutic intervention to inhibit aberrant angiogenesis or ameliorate EC dysfunction could be beneficial in diseases such as cancer and cardiovascular disease, respectively, but current strategies have limited efficacy. Based on recent findings that pathological angiogenesis and EC dysfunction are accompanied by EC-specific metabolic alterations, targeting EC metabolism is emerging as a novel therapeutic strategy. Here, we review recent progress in our understanding of how EC metabolism is altered in disease and discuss potential metabolic targets and strategies to reverse EC dysfunction and inhibit pathological angiogenesis. Partialet al, et al, et al, et al, The hexosamine biosynthesis pathway starts with the conversion of the glycolytic intermediate fructose-6-phosphate (F6P) into glucosamine-6-phosphate (GlucN6P) Fig . GlucN6Pet al, et al, et al, et al, Other metabolic pathways are less well characterized in ECs. Fatty acid (FA) oxidation (FAO) and glutamine oxidation have been implicated in replenishing the TCA cycle to produce ATP via oxidative phosphorylation Fig in ECs. Arginine is a metabolite in the ornithine cycle and converted into citruline and nitric oxide (NO) by endothelial nitric oxide synthase (eNOS) Fig , which is an allosteric activator of 6-phosphofructo-1-kinase (PFK-1) in the rate-limiting step of glycolysis. EC-specific PFKFB3 deletion diminishes retinal and hindbrain vascularization in mice, showing that increased glycolytic flux is required for growth factor-induced angiogenesis is metabolically demanding and mediated by adaptations in EC metabolism Fig A. While in vivo -1-(4-pyridinyl)-2-propen-1-one (3PO) or EC-specific genetic silencing of PFKFB3 inhibits tumor growth et al, et al, et al, et al, et al, in vivo and that pharmacological blockade of MCT1 inhibits angiogenesis and reduces tumor growth in mice Fig A is characterized by heightened pressure in pulmonary arteries caused by excessive EC proliferation and vascular dysfunction levels are another hallmark of PAH ECs are partly reminiscent of the metabolic profile of angiogenic ECs. It would be thus interesting to determine if reducing glycolysis by pharmacological blockade of PFKFB3 can reduce the hyperproliferative rate in PAH ECs. Alternatively, the beneficial effects of PDK inhibition in PAH to induce oxidative metabolism could also be beneficial to block angiogenesis by preventing the glycolytic switch in ECs. Indeed, PDK blockade with dichloroacetate inhibits angiogenesis in glioblastoma patients and reactive nitrogen species (RNS), which might be mediators of EC dysfunction Fig Blake &. High glet al, et al, et al, et al, et al, et al, et al, et al, et al, et al, Accumulation of F6P increases the flux through the hexosamine biosynthesis pathway (HBP), which produces UDP-GlcNac, an important precursor of glycosylation reactions Fig A Brownl. While get al, et al, et al, et al, et al, et al, et al, et al, in vitro and transgenic overexpression of glyoxalase-I in rats reduces vascular AGE formation and improves vasoreactivity at the expense of NADPH, increasing ROS. Sorbitol is subsequently converted into fructose and the highly reactive 3-deoxyglucosone (3DG), which promotes the formation of AGEs and by a salvage pathway from dihydrobiopterin (BH2) via dihydrofolate reductase (DHFR) Fig B metabolism centers around the ability of folate-derived co-enzymes to carry activated 1C units Fig Tibbett. DHFR caEC metabolism is best characterized in the diseases discussed above. However, these represent only a minor fraction of the disorders in which pathological EC responses are presumably involved. Indeed, it is highly likely that EC metabolic alterations are also involved in the pathogenesis of other diseases such as ischemia, pre-eclampsia, vasculitis, vascular neoplasms and others although this has hardly been studied.et al, et al, et al, et al, in vitro, and in vivo levels of serum nitrites (an indicator of NO production) are inversely proportional to serum uric acid concentrations is common in patients with hypertension and may even be a root cause of EC dysfunction leading to cardiovascular disease (Feig et al, et al, A broader characterization of EC metabolism in the future might reveal novel therapeutic targets in metabolic pathways that are generally not considered to be important in pathological EC function. Recent findings that endothelial cholesterol efflux to high-density lipoprotein regulates angiogenesis (Fang The findings in this review suggest that blood vessel pathology is mediated, or at least characterized, by disease-specific alterations. However, at present, there are no studies that incorporate state-of-the-art metabolomics tools to characterize EC metabolism in disease. Metabolic profiling using isotope incorporation studies and metabolic flux analysis could greatly increase our understanding of the metabolic alterations that underlie EC pathology.In vivo studies to characterize EC metabolism in animal models of human disease could provide highly relevant insight in disease-specific metabolic alterations. However, this requires isolation of ECs from diseased tissue, which at present poses technical and interpretational challenges for proper analysis of metabolism using advanced metabolomics methods.in or ex vivo models. The recent development of new protocols to isolate ECs from patient tissue offers the possibility to study metabolism in clinically relevant systems. Accordingly, such studies could greatly advance the identification of novel biomarkers and therapeutic targets in EC metabolism.Another pressing issue is the lack of studies characterizing metabolism in patient-derived tissue using either et al, et al, et al, et al, Overall, it is clear that pathological blood vessel responses are associated with metabolic alterations in ECs. These metabolic adaptations are not just innocent bystanders, but in many cases mediate important aspects of disease. Increased EC glucose metabolism is emerging as a key feature of angiogenic and hyper-proliferative ECs. Targeting EC glucose metabolism has recently been shown as a viable strategy to curb pathological angiogenesis, but is still in its infancy (Schoors"} +{"text": "Cryptococcus species, with neoformans strains mostly isolated from individuals with impaired immunity strain that lacks the sterylglucosidase enzyme and administered to mice preceding fungal infection confers complete protection in animals challenged with lethal doses of either C. neoformans or C. gattii , a class of immunomodulatory glycolipids, in a genetically engineered non-pathogenic C. neoformans pulmonary infection and reduced phagocytosis, which can be restored by administration of IgM (Subramaniam et al., C. neoformans infected rodents produce fungal binding IgM and depletion of these cells resulted in impaired macrophage function facilitating cryptococcal dissemination to the brain (Rohatgi and Pirofski, B cells play a critical role in protection against experimental cryptococcosis (Subramaniam et al., C. neoformans. For instance, NKT cells are increased in the lungs of mice infected with C. neoformans, associating the chemokine MCP-1 in their recruitment and accumulation (Kawakami et al., sgl1 strain (Kawakami et al., C. neoformans in the lungs correlates with macrophage polarization (Davis et al., C. neoformans.SGs have been shown to modulate cytokine production (Lee et al., sgl1 strain might provide a potential vaccination strategy against cryptococcosis. Rella et al., provide a proof of concept study that opens a novel area of research and a potential therapeutic strategy to prevent and reduce the devastating consequences of cryptococcosis.Further studies focusing on the immune responses generated by SGs and safety issues associated with delivering live-attenuated cryptococci, heat-killed fungi, or vesicle formulations containing SGs preparations are necessary. However, these findings are significant in the setting of HIV/AIDS immune deficiency suggesting that the \u0394The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Ischemia-reperfusion injury represents a pathological condition characterized by an initial undersupply of blood to an area or organ followed by a restoration of perfusion and concomitant reoxygenation (= reperfusion). Ischemia typically occurs in the presence of embolism or thrombosis but can also be triggered by surgery and transplantation. Anyway, the disturbance in perfusion results in a severe imbalance between metabolic supply and demand, subsequently causing tissue hypoxia . NotablyInterestingly, restoration of blood flow and reoxygenation is commonly associated with an exacerbation of tissue injury and a profound inflammatory response (\u201creperfusion injury\u201d) , 2. IschFor example, myocardial ischemia followed by reperfusion typically manifests in microvascular dysfunction, death of myocytes, and myocardial stunning or dysfunction.Ischemia-reperfusion injury (IRI) of the lung, for example, following transplantation, is characterized by nonspecific alveolar damage, edema formation, and hypoxemia. The clinical spectrum of pulmonary IRI may range from mild hypoxemia to acute respiratory distress syndrome.In contrast to other organs, the brain is particularly susceptible to ischemia and irreversible neuronal damage already occurs after only 5 minutes of complete ischemia . For braIRI of the kidney may occur in the setting of transplantation and cardiac arrest and during cardiac surgery. Here it is important to note that renal injury is usually associated with a high morbidity and mortality. The cortical-medullary region is the most susceptible region to tubular injury, inflammation, and vascular alterations.Generally, IRI of a single organ causes the release of different proinflammatory mediators, which may subsequently induce inflammation in other organs, thereby potentially contributing to multiple organ dysfunction or even failure .\u03baB (NF-\u03baB) [Different pathological processes contribute to tissue injury secondary to ischemia-reperfusion. During ischemia, limited oxygen availability leads to an impaired endothelial cell barrier function with a concomitant increase in vascular permeability and leakage due to decreases of intracellular cAMP levels caused by a reduced adenylate cyclase activity . Further (NF-\u03baB) .Reperfusion of ischemic tissue activates a complex inflammatory response without the involvement of pathogenic triggers, a phenomenon also referred to as sterile inflammation. During the initiation of this inflammatory response, endogenous molecules act as alarmins or danger-associated molecular patterns (DAMPs) . The inf \u201cThe effects of remote ischemic preconditioning and N-acetylcysteine with remote ischemic preconditioning in rat hepatic ischemia-reperfusion injury model\u201d by B. U. Togrul et al., \u201cThe effects of spinal, inhalation, and total intravenous anesthetic techniques on ischemia-reperfusion injury in arthroscopic knee surgery\u201d by S. C. Karahan et al., \u201cEfficacy and safety of hepatectomy performed with intermittent portal triad clamping with low central venous pressure\" by D. Dohman et al., \u201cAdalimumab ameliorates abdominal aorta cross clamping induced liver injury in rats\u201d by Y. Demirci et al., \u201cEvidence for the use of isoflurane as a replacement for chloral hydrate anesthesia in experimental stroke: an ethical issue\u201d by B. Mich\u00e8le et al., \u201cThe effect of dexmedetomidine on oxidative stress during pneumoperitoneum\u201d by S. C. Karahan et al., \u201cThe comparison of the effects of sevoflurane inhalation anesthesia and intravenous propofol anesthesia on oxidative stress in one lung ventilation\u201d by D. Dohman et al., and \u201cRole of ethyl pyruvate on systemic inflammatory response and lung injury in an experimental model of ruptured abdominal aortic aneurysm\u201d by G. Altun et al.This special issue is devoted to the modulation of ischemia-reperfusion injury by different measures and contains eight original papers addressing this clinically relevant topic. These papers are accompanied by two review articles dealing with the effects of anesthetics on ischemia-reperfusion injury. Papers from B. U. Togrul et al., D. Dohman et al., and Y. Demirci et al. are focusing on ischemia-reperfusion injury of the liver. In two of these three papers, different therapeutic interventions on hepatic ischemia-reperfusion injury are evaluated, whereas the third paper is a retrospective study in which the authors investigated the efficacy and safety of intermittent portal triad clamping with low central venous pressure during liver resection. In this context, it has been reported that remote ischemic preconditioning and therapeutic interventions can reduce liver damage after inducing ischemia-reperfusion injury. The studies by S. C. Karahan et al., B. Mich\u00e8le et al., S. C. Karahan et al., D. Dohman et al., and G. Altun et al. elucidate the effects of different anesthetic techniques and drugs on ischemia-reperfusion injury. These eight papers are entitled as follows:Alexander ZarbockAhmet ErogluEngin ErturkCan InceMartin Westphal"} +{"text": "Advances in medical science and technology allow people live longer lives, which results in age-related problems. Humans cannot avoid the various aged-related alterations of aging; in other words, humans cannot remain young at molecular and cellular levels. In 1956, Harman proposed the \u201cfree radical theory of aging\u201d to explain the molecular mechanisms of aging. Telomere length, and accumulation of DNA or mitochondrial damage are also considered to be mechanisms of aging. On the other hand, stem cells are essential for maintaining tissue homeostasis by replacing parenchymal cells; therefore, the stem cell theory of aging is also used to explain the progress of aging. Importantly, the stem cell theory of aging is likely related to other theories. In addition, recent studies have started to reveal the essential roles of tissue-resident mesenchymal progenitors/stem cells/stromal cells in maintaining tissue homeostasis, and some evidence of their fundamental roles in the progression of aging has been presented. In this review, we discuss how stem cell and other theories connect to explain the progress of aging. In addition, we consider the mesenchymal progenitor theory of aging to describing the process of aging. Several theories to explain the aging-related alterations in our bodies have been proposed and accepted. The free radical theory of aging was first proposed by Harman in 1956 as one factor of aging Harman, . The numAll adult stem cells exist in a unique microenvironment, which is known as a niche. The niche is consistent with heterogeneous types of cells and extracellular matrix proteins. The blood vessel has been proposed as a niche in common. Using expression patterns of cell surface receptors of the SLAM family in hematopoietic stem cells, Kiel et al. showed that many hematopoietic stem cells are associated with sinusoidal endothelium in the spleen and bone marrow differentiation and self-renewal potentials. They are indispensable for renewal and regeneration of parenchymal cells after damage. In adult mammals, many but not all tissues have functional resident tissue-specific stem cells that satisfy the criteria, including hematopoietic, skeletal muscle, pigment, epithelial, sperm, adipose, intestinal, and neural stem cells. Although a subset of adult stem cells is maintained in a quiescent state in the homeostasis of skeletal muscle and hematopoiesis , some of the reactive oxygen species (ROS), and their reactive products. ROS consist of superoxide anions (O\u22122), hydrogen peroxide (H202), and hydroxyl radicals (OH.), which are mainly generated in cells by the mitochondrial respiratory chain is another molecule that regulates the ROS pathway in hematopoietic stem cells. The FOXO subfamily is known as the downstream target of the PI3K-AKT signaling pathway. Mice conditionally depleted of C. elegans. Two genes were identified as responsible for longevity in C. elegans. One is the daf-2 gene, an equal homolog to both the mammalian insulin and IGF-1 receptors, and the other is age-1, a homolog to the worm PI3K-kinase catalytic subunit and SKN-1 (homolog of NRF2) , which is a truncated and farnesylated form of LAMIN A. HGPS affects mesenchymal linages. Zhang et al. produced iPS cells from HGPS dermal fibroblasts and differentiated them into neural progenitors, endothelial cells, fibroblasts, vascular smooth muscle, and mesenchymal stem cells and an increase of mitochondrial MnSOD-dependent ROS is an inherited disorder that causes premature aging and shortens the life span. The causative gene of HGPS is s Figure . Lentiviin vivo importance of telomere length, mice with the telomerase RNA component Terc knocked out (\u2212/\u2212Terc) were generated by Blasco et al. \u2212/\u2212Terc mice exhibited failures in highly proliferative organs including the hematopoietic and reproductive systems knockout (\u2212/\u2212Atm) mice, and the offspring showed increased telomere erosion and genomic instability . Compared with mdx mice, mdx/mTR mice showed severe dystrophic phenotypes and decreased proliferative potential of muscle stem cells is a well-known inherited X-linked disorder occurring in one in 3500 boys. The causative gene is \u2212/\u2212Terc and +/\u2212Terc littermates (CD45.2). First, they observed impaired B cell lymphopoiesis and increased myeloid proliferation in \u2212/\u2212Terc recipient mice. Twelve-month-old \u2212/\u2212Terc mice showed a more severe defect of B cell lymphogenesis and accelerated myelopoiesis compared with 2-month-old \u2212/\u2212Terc mice. The environment of \u2212/\u2212Terc mice limited the engraftment of even wild-type hematopoietic stem cells. They also showed a shortened telomere length in mesenchymal progenitors in \u2212/\u2212Terc mice and a decrease in their number. Taken together, these results suggest that the shortening of telomere length in both stem cells and mesenchymal progenitors are factors of the telomere theory aging in various tissues is known to cause a photosensitive form of the brittle hair disorder trichothiodystrophy. A study of de Boer at al. showed that mice with mutated Xpd genes (TTDXpd) exhibit many aged-related symptoms including osteoporosis, kyphosis, osteosclerosis, early graying, cachexia, infertility, and a reduced life span although the mice are born with developmentally normal phenotypes , Nijnik found that impairment of the NHEJ pathway causes a progressive loss of hematopoietic stem cells during aging -deficient mice pathway is a well known mechanism to repair DNA double-strand breaks. One of its components is DNA ligase IV. Using ice Lig4Y88C mice,Accumulating studies also demonstrated that a component of serum is altered during aging. By utilizing parabiotic pairings, Conboy et al. showed that the age-related decline of muscle stem cell activity can be modulated by systemic factors that change with age (Janzen et al., p16INK4a expression induced senescence, and a knockdown of p16INK4a prevented the senescence of human mesenchymal stem cells (Shibata et al., Cellular senescence is a unique state generally defined as irreversible cell cycle arrest. Recent studies have shown that senescent cells exhibit a robust increase in mRNA expression and secretion of numerous proinflammatory cytokines, which work in a paracrine manner (Campisi et al., Fibroblasts can be observed in all tissues. Fibroblasts and mesenchymal progenitors show similar morphologies, but their differentiative potentials distinguish them. Sudo et al. investigated the differentiation potential of human fibroblasts derived from various tissues and found that cells originally considered fibroblasts have potential to differentiate into the mesenchymal lineage, which includes osteoblasts, chondrocytes, and adipocytes (Sudo et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "To better understand the type and range of health issues initiated by patients and providers in \u2018high-quality\u2019 primary-care for adults with diabetes and low socio-economic status (SES).Although quality of care guidelines are straightforward, diabetes visits in primary care are often more complex than adhering to guidelines, especially in adults with low SES who experience many financial and environmental barriers to good care.We conducted a qualitative study using direct observation of primary-care diabetes visits at an exemplar safety net practice in 2009\u20132010.In a mainly African American (93%) low-income population with fair cardiovascular control , visits addressed a variety of bio-psychosocial health issues [median: 25 problems/visit (range 13\u201332)]. Physicians most frequently initiated discussions about chronic diseases, prevention, and health behavior. Patients most frequently initiated discussions about social environment and acute symptoms followed by prevention and health behavior.Primary-care visits by diabetes patients with low SES address a surprising number and diversity of problems. Emerging new models of primary-care delivery and quality measurement should allow adequate time and resources to address the range of tasks necessary for integrating biomedical and psychosocial concerns to improve the health of socio-economically disadvantaged patients. Africanet al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., Previous research has shown patient, provider, or system factors associated with improved diabetes outcomes, such as better medication adherence, greater medication intensification, fewer competing demands at the office visit, greater access to care, greater patient motivation, and better continuity of care from a county hospital in Northeast Ohio, performing in the top quartile on their biomedical quality of care scores for their patients with diabetes in 2009\u20132010 after obtaining institutional review board approval. The biomedical quality of care scores are locally adapted National Council of Quality Assurance measures for adults with diabetes, and included the percent of diabetes patients with blood pressure (BP) <140/90 mmHg, hemoglobin A1c<7%, and low-density lipoprotein cholesterol (LDL-c) <100 mg/dL. We purposively selected adult patients with diabetes who were scheduled for routine follow-up visits with their primary-care provider at the practice site.We used participant/direct observation for data collection, as we were interested in the observed number, range, and types of health issues brought up at the primary-care visit for adults with diabetes and as the primary author was a provider at the practice site. For each of the practices\u2019 four primary-care physicians, we audio taped, observed, and transcribed three to four routine follow-up visits with diabetes patients after obtaining informed consent from patients and physicians. We excluded the primary-care provider who authored this paper due to potential biases; however, all the other providers participated in the study. Providers were told that we were observing the visit to evaluate current primary-care practice. We continued the enrollment of patients until we reached saturation of themes . The research assistant and internal medicine physician developed and piloted an observation form, environmental checklist, performed key informant interviews of providers and patients, wrote daily field notes, and composed reflective memos. For this analysis, we focused on analyzing the transcribed office visits, as these were felt to be most pertinent to our objective of evaluating the range and types of health issues that arose at the visit. The research assistant (a PhD sociology candidate) audio-recorded and observed most of the patient visits, whereas a primary-care provider/health services researcher audio-recorded and observed the remaining patient visits.et al., et al., A multidisciplinary team used a purposeful, constant comparative approach to count and categorize health issues, raised by the patient or the physician at the primary-care visit (Bradley n=5) were more likely to be men but were otherwise similar to participants in terms of age and race. The providers were all family practice physicians, Caucasian, and married. Three providers were women and one provider was a man.The safety net clinic serves a mainly low-income African American population . Out of the 20 patients asked to enroll, 15 patients consented to participate. The 15 patients were mainly African American (93%) with fair cardiovascular risk factor control . The patients had a mixture of insurance categories: private (33%), private plus medicare (20%), medicare alone (7%), medicaid (7%), and uninsured (33%). These numbers were similar to the numbers of the overall clinic population outlined above. Patients who refused to participate , prevention , changes in sexual partners, her spiritual beliefs as they relate to her health, and two acute issues.In-depth study reveals a rich subtext of the attitudes, beliefs, knowledge, and behaviors, as well as the role of popular culture underpinning patients\u2019 attempts to manage their diabetes. It is instructive for \u2018Dr Smith\u2019 to know that \u2018Ms Jones\u2019 \u2018ate at Subway for a whole month\u2019 (because Jared lost weight doing so) and decided that because the contestants on the Biggest Losers maintained their weight loss \u2018at home, after the show,\u2019 she could do so as well. Ms Jones acknowledges that she allows herself \u2018wiggle room\u2019 in trying to manage her diabetes: \u2018I ate only half the foot long for lunch and the other half for dinner.\u2019 Ms Jones\u2019 expression of the belief that \u2018my body feels better when my sugar is higher\u2019 initiated a lengthy discussion about how to manage low as well as high blood sugar.Her physician resourcefully integrates both the physician-directed health priorities such as chronic disease management of her diabetes and the patient-directed priorities such as her social environment into the overall plan of care. For instance, the physician incorporates the patient\u2019s concerns about potential job loss and concern for medication costs when writing prescriptions, and the provider and patient work together with the social worker to derive the most appropriate regimen that will minimize costs while maximizing adherence. In the end, the provider prescribes some expensive medications such as insulin, which will be obtained through the help of the social worker via a free pharmaceutical company program that will mail the patient her medications. Other generic medications were prescribed that cost four dollars a month, such as her cholesterol medicine. Furthermore, the physician incorporates the patient-directed comments about her social environment regarding diet into the plan for weight loss by encouraging recent weight loss while modifying the \u2018subway\u2019 diet to have less bread.Patients and providers discussed a wide range of issues at these primary-care visits . Importaet al., et al., Although we expected the primary-care visit for adults with diabetes and low SES to be diverse in the range of health issues, the surprisingly large number of health issues (median 25) and the inter-relationship with the social environment of patients\u2019 lives was remarkable. Similar to our study, one previous observational study reported that primary-care physicians deal with an average of 17 topics, questions, and symptoms at the primary-care diabetic visit (Parchman et al., Quantitative studies often fail to capture other issues such as the social environment (Samuels et al., et al., et al., These findings show what is possible in dedicated primary care \u2013 possibilities that provide hope for overcoming the chasms of fragmentation and poor quality that characterize much of the US healthcare system IOM, . Study fSeveral limitations to our study deserve mention. As this was a qualitative study that was carried out at one clinic, the conclusions may not be generalizable to the overall population. However, we were not trying to generalize findings but explore the range of health issues discussed in a primary-care visit to inform future potential interventions to improve care. Second, although we tried to combat bias in our analysis by including sociologists and primary-care physicians along with third-party physician review to confirm or refute findings, we recognize that some biases are hard to fully address. For instance, providers are more likely to conclude that they need more time with patients as we have suggested in this study. However, we also note that patients are more likely to bring up psychosocial issues than providers during an office visit; therefore, not all of our findings are biased toward positive provider performance.et al., et al., et al., et al., This study also has several important implications. First, the study raises questions about how \u2018productivity\u2019 is assessed in primary care. From bio-psychosocial and patient-centered points of view, the observed visits were highly productive. However, most of the present productivity measures and financial incentives emphasize \u2018throughput\u2019 (Morrison and Smith, Systems that incentivize quality and care integration over quantity may have the benefit of allowing scheduling of complex patients at more appropriate intervals and allow for more adequate resources for caring for biomedical and psychosocial aspects of patient health. In this time of tumultuous change in care organization and payment, we should support models of primary-care delivery and quality measurement, which allow adequate time and resources to address the range and complexity of tasks that are necessary for improving the health of socio-economically disadvantaged patients."} +{"text": "In 2014, Schlichting and co-workers have opened up new opportunities to collect damage-free diffraction data from randomly oriented sub-micron-sized crystals using serial femtosecond crystallography (SFX) (Chapman al. 2015 demonstrIUCrJ, Schlichting and co-workers (Nass et al., 2016et al., 2014In this issue of They then applied the same methodology to a thaumatin data set collected at the LCLS using the native sulfur as a source of anomalous scatterering Fig. 1. A photoet al., 2015de novo phasing of SFX data, particularly with only native crystals, is still far from trivial. Millions of crystals and many hours of beamtime are usually required to collect a sufficient amount of data for structure determination. Nass et al.\u2019s study has demonstrated that with optimized data processing technology, single-wavelength anomalous diffraction data from the native sulfur atoms can allow protein structures to be solved ab initio. Improvements in XFEL instrumentation and processing software should further reduce the number of images required to phase SFX data.X-ray free-electron lasers have provided a new way to obtain SFX data that is advantageous over traditional synchrotron sources in terms of better data resolution, smaller sample size and minimal radiation damage (Kang"} +{"text": "Pseudomonas aeruginosa biofilm (Costerton et al., Bacillus subtilis can choose between a variety of lifestyles such as sporulation, motility as an explorative lifestyle, biofilm formation, and the acquisition of genetic competence for the uptake of foreign DNA (L\u00f3pez and Kolter, In their endless struggle to survive in harsh and rapidly changing environments, many bacteria depend on their ability to live together as multicellular communities, also known as biofilms. In these communities cells are embedded within a self-produced slimy matrix that is mainly composed out of extracellular polysaccharides and proteins (Hall-Stoodley et al., B. subtilis biofilm communities, different groups of cells fulfill distinct functions, which are important for the well-being of the whole community of clonal identical bacteria. Some bacteria produce extracellular polysaccharides and proteins and thereby provide the matrix for the community. Other cells secrete exoproteases for degradation of protein as an alternative energy source (Marlow et al., In the B. subtilis suggest that tyrosine phosphorylation plays an important role in the regulation of biofilm formation and cell differentiation, in addition to the known mechanisms of transcriptional regulation and protein-protein interactions (for review see Vlamakis et al., B. subtilis encodes two protein tyrosine kinase/ modulator couples, PtkA/ TkmA, and EpsB/ EpsA. Interestingly, the simultaneous deletion of either both kinase or modulator genes totally abolished extracellular polysaccharide production causing a biofilm defect. The single mutants did not phenocopy the kinase or modulator double mutant and were still able to produce exopolysaccharides. However, colony structure and pellicle formation was affected in the single mutants (Gerwig et al., B. subtilis at different levels: EpsB acts downstream of the central regulator of cell differentiation, Spo0A, whereas PtkA is likely to act upstream of Spo0A (see Figure Recent studies in In principle, the stochastic phosphorylation state of the Spo0A protein determines to which promoters the protein binds and consequently if cells differentiate into a spore or become a matrix producer. High levels of phosphorylated Spo0A induce spore development, whereas medium levels lead to matrix production (Fujita and Losick, In order to influence sporulation efficiency as shown by Kiley and Stanley-Wall , PtkA moptkA mutant, Kiley and Stanley-Wall (In order to explain altered biofilm formation and the sporulation defect of the ley-Wall conducteley-Wall were notley-Wall , except ley-Wall . ClearlyA more obvious problem for the identification of tyrosine phosphorylated proteins with the potential to control biofilm formation and sporulation is that most published data relates to cells harvested from exponentially growing cultures rather than from biofilms. Moreover, the studies were performed with strains derived from a domesticated strain that does not produce robust bofilms. Thus, it is reasonable to assume that not all of the proteins that might be relevant for biofilm formation and sporulation are expressed under these conditions. Furthermore, regulatory phosphorylation is a rapid method for adapting cellular processes to environmental changes. Thus, it seems safe to assume that not all phosphorylations are present permanently. Interestingly, all currently identified tyrosine phosphorylation reactions with functional relevance have been found by attempts other than large-scale phosphoproteomics analyses. Examples include the regulator of unsaturated fatty acid synthesis FatR (Derouiche et al., eps operon for exopolysaccharide production. Hence, the regulation of the two corresponding genes is similar. The eps operon is only strongly expressed if the SinR anti-activator protein is inhibited by either of its antagonists SinI and SlrR under biofilm forming conditions (Kearns et al., epsB gene only affects exopolysaccharide production but leaves sporulation unaffected (Gerwig et al., The second level of regulatory tyrosine phosphorylation is provided by the EpsB kinase that phosphorylates the glycosyltransferase EpsE (Elsholz et al., Straight signal transduction is an important issue for many conserved multi-component signal transduction system families and has been extensively studied for two-component regulatory systems and phosphotransferase system-controlled RNA-binding antitermination proteins. These systems have evolved to avoid non-cognate interactions either by restricting the interactions with non-cognate proteins partners, ligands, and target molecules. Moreover, differential gene expression of the non-cognate components has been observed to prevent non-productive cross-talk (Schilling et al., Staphylococcus aureus that also contains two similar tyrosine kinase/ modulator couples. In this case, the Cap5A1 modulator protein of one couple and the Cap5B2 protein tyrosine kinase of the other couple show functional cross-talk suggesting that interplay between different tyrosine/ modulator couples might not be limited to B. subtilis (Soulat et al., However, this might be different for regulatory tyrosine phosphorylation, as suggested for the interplay between EpsB and PtkA. In yeast (Shi et al., B. subtilis is one of the most exciting results of recent studies. This is underlined by the observation of extensive links between the different signal transduction systems that involve post-translational modifications (van Noort et al., ptkA deletion mutant. Furthermore, large-scale phosphoproteomics under biofilm-promoting conditions could help to identify potential tyrosine phosphorylated targets. Additional tasks are the identification of substances that can be sensed by the PtkA modulator protein TkmA and to further dissect the potential cross-talk between the two known tyrosine kinase/ modulator couples EpsB/ EpsA and PtkA/ TkmA in B. subtilis.The detection of a regulatory interplay between protein tyrosine phosphorylation and classical sensing via the phosphorelay in the control of cell differentiation in The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Microarray technologies facilitate high-throughput gene expression analysis. However, the diversity of platforms for rice gene expression analysis hinders efficient analysis. Tools to broadly integrate microarray data from different platforms are needed.http://www.ricearray.org) to explore gene expression across 1,867 publicly available rice microarray hybridizations. The ROAD\u2019s user-friendly web interface and variety of visualization tools facilitate the extraction of gene expression profiles using gene and microarray element identifications. The ROAD supports meta-analysis of genes expressed in different tissues and at developmental stages. Co-expression analysis tool provides information on co-regulation between genes under general, abiotic and biotic stress conditions. Additionally, functional analysis tools, such as Gene Ontology and KEGG (Kyoto Encyclopedia of Genes and Genomes) Orthology, are embedded in the ROAD. These tools facilitate the identification of meaningful biological patterns in a list of query genes.In this study, we developed the Rice Oligonucleotide Array Database contains supplementary material, which is available to authorized users. Oryza sativa) is a staple food for more than 50% of the human population. Because of the high level of genomic colinearity and conservation of gene function among grass species, rice serves as a useful research model in other grass studies . The co and Gale), has br and Gale; Itoh et and Gale; Tanaka and Gale). Despit and Gale).japonica and indica) have been reported and their characteristics are summarized in Table\u2009japonica and indica cultivars, respectively. Agilent has constructed a 22K Rice Oligo Microarray Kit based on rice FLcDNAs and recently announced a 44K version was designed by the Beijing Genomics Institute and Yale University and based on the draft indica and japonica sequences. The University of California, Davis, led a National Science Foundation (NSF) supported effort to design, print and validate 22K and 45K oligonucleotide arrays based on gene model predictions from TIGR\u2019s osa1 version 3.0 release.Microarray technologies are an important strategy for genome-wide expression pattern analysis and is becoming increasingly important for gene functional analysis . Severano et al.). The Orhttp://ricexpro.dna.affrc.go.jp/), which is based on the Agilent 44K microarray, provides an overview of the spatiotemporal gene expression profiles of various organs and tissues provides a meta-analysis toolbox to explore gene expressions across a wide variety of biological contexts for rice and other species, but it is commercial and not completely publicly available , which integrates the most comprehensive public microarray datasets and provides several functional analysis tools. With a user-friendly web interface, the ROAD is a useful reference for elucidating rice gene expression and function.These rice microarray platforms have been successfully used in characterizing gene expression profiles from different tissues and organs , differto et al.). Geneveuz et al.). Other uz et al.), RicePLuz et al.), Bio-Aruz et al.) and Yaluz et al.) are usend Fobert). This sWe collected information from six rice microarray platforms, including the Affymetrix, Agilent 22K and 44K, BGI/Yale, and the NSF 20K and 45K, to construct the ROAD. Probe sequences from each platform were extracted and mapped onto cDNAs to match the probes to the expressed genes; the cDNAs were drawn from the Rice Genome Annotation Project V6 , Rice A2(Cy3/Cy5)) were used and the color-swap hybridizations were manually corrected to make them comparable among other samples. Normalized data were integrated into the ROAD, thus simplifying the retrieval process of their gene expression profiles. After entering a list of genes or microarray element IDs into the ROAD, the user can then select the microarray platform and specific experiment to search against to assemble microarray data from different experiments into context-related profiles (meta-profiles). This large-scale combination and analysis of expression data from a single organism using a single platform allows the identification of biologically meaningful expression patterns of individual genes . One drSimilarity of gene expression profiles (co-expression) can provide powerful information to identify new genes functionally related. The rapid accumulation of microarray data in past decade allows the creation of co-expression networks by examining the co-expression patterns of genes over a large number of experimental conditions. Several online co-expression analysis tools have been developed for rice, including OryzaExpress , RiceArp value is calculated for each GO/KO term, whose value is based on comparisons of the observed number from the queried gene list and the expected number from the genome scale.Gene Ontologies (GO) provide controlled vocabulary to describe the biological process, molecular function, and component of the cell to which a gene product putatively contributes , R package marray in Bioconductor was used to do the normalization with within-array Lowess and between-array MAD scale normalization methods were collected from NCBI GEO , EBI ArCleveland; Wang etCleveland). The coad et al.). The sead et al.). Regardhttp://www.ricearray.org. Heatmap and classic line plots were generated by the PHP library JpGraph (http://jpgraph.net/).The ROAD database was constructed with PHP (Hypertext Preprocessor) and MySQL, run on a Windows 2003 server. The http address isAfter MAS normalization of all Affymetrix microarray samples, outliers were detected using the arrayQualityMetricsBioconductor package, which uses three different statistical tests to identify outliers and KO sa et al.). The RAka et al.). Then hTable S1. Detailed information of rice microarray experiments available in ROAD. (XLS 204 KB)Additional file 1: Figure S1. Screenshots of meta-analysis in ROAD queried with 19 root-preferential genes for anatomy (a) and developmental stages (b) of Affymetrix array platform, and anatomy (c) and developmental stages of Agilent 44K array platform. (JPEG 5 MB)Additional file 2: Authors\u2019 original file for figure 1Authors\u2019 original file for figure 2Below are the links to the authors\u2019 original submitted files for images."} +{"text": "This Research Topic aims to cover recent progress in research studying how genetic make-up and environmental factors can contribute to the development of mental disorders such as anxiety, depression, schizophrenia, and psychoactive substances abuse. It has brought together leading experts in the field to address these questions from different angles in eleven reviews, seven original research articles and two theoretic/opinion papers.1A receptor KO mice compared to WT animals. Then, by performing loose-seal cell-attached electrophysiological recordings in 5-HT transporter knockout (Sert\u2212/\u2212) and tryptophan hydroxylase-2 knockout (Tph2\u2212/\u2212) mice, Araragi et al. in the pathophysiology and the treatment of psychiatric disorders. First, Prof. Gardier nicely si et al. demonstret al.'s results The following eleven articles provide excellent insights into the interaction between gene and environment in mental disorders as well as the role of several transmitters/neuropeptides and the different therapeutic strategies. El-Hage et al. elegantlin vivo. They present novel methods using [11C]-(+)-PHNO and PET which could provide insights into the function of D3 receptors in addiction.The last five articles cover neuroscience research on drug of abuse. In order to better understand the processes by which peer influences take effect in prairie voles, Anacker and Ryabinin measure In summary, these studies illustrate how mental disorders can arise from multiple sources. It even seems that the entire body can impact on mental state and psychiatric health (Renoir et al., The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Most individuals experience these threats at least once during their lives, and some individuals experience them daily and other forms of interpersonal rejection threaten individuals' physical and psychological well-being because these behaviors are more likely to achieve re-inclusion; anti-social behaviors should be more likely to fortify power/provocation needs (control and meaningful existence) because these behaviors will likely provoke acknowledgement from the ostracizers . Thus, participants who give a small amount could be interpreted as either being less aggressive or potentially more pro-social because they are obeying the experimenter nominally but also not subjecting the target to unnecessary discomfort. Further, how do researchers categorize participants who actively choose not to allocate any hot sauce at all? Is this simply lack of aggression or also an independent pro-social behavior toward the target?Future research needs to address directly these two conflicting behavioral responses. Often, researchers only give participants one behavioral option. Because of this, it is hard to rule out the possibility that ostracized participants are simply responding more extremely than included participants using whatever option they are given because it is the only option they have to fortify some of their basic needs. Some behavioral measures can be interpreted as pro- or anti-social depending on how participants respond and devaluate the same target simultaneously (Sommer and Bernieri, Other threat-focused models [e.g., rejection-based threats, Smart Richman and Leary , or threResearchers could also measure theoretically meaningful individual difference variables to test potential moderation of the anti-social/pro-social paradox. For example, ostracized participants who have a higher dispositional need to belong (Leary et al., future expectations argument. Wesselmann et al. (Some individual differences should moderate anti-social responses. Individuals higher in rejection sensitivity (i.e., the tendency to expect and easily perceive rejection; Ayduk et al., n et al. offer ann et al. , but theflight response (compared with fight responses, i.e., pro- and anti-social behaviors; Williams, freeze response (Williams, flight and freeze responses.Williams developemeaning/significance (also Jonas et al., control (Kruglanski, Chronically ostracized individuals may also be susceptible to recruitment by predatory/extreme groups because these groups may offer a last bastion of inclusion (Wesselmann and Williams, The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Epichlo\u00eb, Bacon et al., Balansia, Porter et al., Claviceps purpurea where the alkaloids, typically ergotamine and ergocristine, are responsible for the resultant ergotism associated with C. purpurea. The presence of these toxins can compound livestock issues with the concomitant consumption of ergovaline produced by the endophyte. In terms of livestock production systems, associated ergot alkaloid toxicities are not limited to pasture or feeding pasture products. While Claviceps Africana is widespread throughout Africa and Asia, the first reported case of toxicity was in Australian sorghum in 1996 (Ryley et al., C. Africana-infested sorghum has been demonstrated to be detrimental to steer performance in Australian feedlots that utilize this feedstuff (Blaney et al., Ergot alkaloids have been associated with endophyte-infected grasses (e.g., the While ergot alkaloid incidences are rare in humans resulting from increased regulation of grain processing (Flieger et al., If fungi that synthesize ergot alkaloids pre-date the human race, and knowledge of ergot properties has been recorded as far back as 1100 BC Schiff, , why havA multi-disciplinary approach will be needed to solve most ergot alkaloid related issues. This research topic, Recent Investigations of Ergot Alkaloids Incorporated into Plant and/or Animal Systems, epitomizes that reality through diverse scientific approaches addressing the core issue of ergot alkaloids in agriculture. Innovative research articles highlight the numerous effects that ergot alkaloids can have on livestock (Aiken and Flythe, The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Chandelier cells are a specialized GABAergic interneuron subtype that selectively innervates pyramidal neurons at the axon initial segment (AIS), the site of action potential generation. ChC connectivity allows for powerful yet precise modulation of large populations of pyramidal cells, suggesting ChCs have a critical role in brain functions. Dysfunctions in ChC connectivity are associated with brain disorders such as epilepsy and schizophrenia; however, whether this is causative, contributory or compensatory is not known. A likely stumbling block toward mechanistic discoveries and uncovering potential therapeutic targets is the apparent lack of rudimentary understanding of ChCs. For example, whether cortical ChCs are inhibitory or excitatory remains unresolved, and thus whether altered ChC activity results in altered inhibition or excitation is not clear. Recent studies have shed some light onto this excitation-inhibition controversy. In addition, new findings have identified preferential cell-type connectivities established by cortical ChCs, greatly expanding our understanding of the role of ChCs in the cortical microcircuit. Here we aim to bring more attention to ChC connectivity to better understand its role in neural circuits, address whether ChCs are inhibitory or excitatory in light of recent findings and discuss ChC dysfunctions in brain disorders. Discovered in the 1970s, ChCs quickly gained intrigue as a unique and potentially powerful subtype of GABAergic interneurons that selectively innervates pyramidal neurons at the axon initial segment (AIS), directly regulating the site of action potential generation , L3, L5a and L5b , ChC axons can vary in their complexity and localization in different cortical areas and layers, and depends on the type and age of the animal mice (Kirmse et al., Whether ChCs are inhibitory or excitatory is not currently agreed upon. ChCs activate GABAd Kaila, . However\u2212 regulation at the postsynaptic AIS (Szabadics et al., \u2212 concentrations, Szabadics et al. used the gramicidin perforated patch technique (Szabadics et al., in vivo. Nevertheless, a possibility for ChC excitation is hypothesized to occur during \u201cdown\u201d states, when pyramidal neurons are hyperpolarized and sodium channels are deinactivated (Szabadics et al., ChCs in L2/3, on the other hand, have been shown to be capable of mediating excitatory activity in brain slices from animals well past the developmental period when GABA-mediated excitation is thought to occur (Szabadics et al., in vivo conditions indicated that L2/3 ChCs were predominately inhibitory (Woodruff et al., In vivo studies that clearly demonstrate whether ChCs are inhibitory or excitatory are lacking, but some results suggest that ChCs are not excitatory (Klausberger et al., Because the perforated patch technique may still alter GABAergic activity, concerns with this technique and the results obtained were raised (Glickfeld et al., in vivo data, suggest that ChCs are inhibitory, as originally assumed (Somogyi, ChC-mediated excitation is an enigmatic issue. This is largely due to the complications when recording GABAergic responses. Nevertheless, currently those experiments using the least invasive techniques, along with Somogyi, and demoSomogyi, .A receptor \u03b12 subunit is increased in pyramidal neurons in schizophrenia (Volk et al., ChC dysfunctions are well associated with schizophrenia (Lewis et al., Schizophrenia is associated with significant changes in neural activity. These changes are shown with both structural and functional alterations and result in abnormal neural network oscillations and synchrony (Meyer-Lindenberg et al., in vivo recordings show that ChCs fire more robustly than other types of cortical neurons when overall cortical excitation increases (Zhu et al., ChC dysfunction is also implicated in epilepsy (DeFelipe, A significant impediment that prevents elucidating the link between ChC dysfunctions and brain disorders is the uncertainties in the role of ChCs in the cortical microcircuit, including whether ChCs are inhibitory or excitatory. Ideally, the ability to specifically target ChCs with the identification of specific markers will greatly aid in resolving the role of ChCs in brain functions. One such undertaking is the creation of the Nkx2.1CreERT2 mouse line that can label a portion of interneurons, the majority of which are ChCs (Taniguchi et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer GGB and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review."} +{"text": "Autism spectrum disorder (ASD) is an umbrella term for a heterogeneous group of developmental disorders that present with persistent deficits in social communication/interaction and repetitive/restricted patterns of behavior, interests, or activities that cannot be better explained by intellectual disability or global developmental delay could directly affect the enteric nervous system (ENS), a nervous tissue within the gut wall often called \u201cthe second brain\u201d due to its size, structure, complexity, and autonomic regulation of gut functions, such as bowel motility, secretion/absorption and local blood flow Furness, , 2012. FS-nitrosoglutathione (GSNO) , traditionally considered as only supportive cells of the ENS, are emerging as local GI regulators. They are strategically located at the interface between the neurons and other non-neuronal cells in the gut, such as enterocytes or immune cells, and participate in the regulation of gut motility, intestinal epithelial barrier, and inflammatory processes in Rett Syndrome (Yasui et al., Recent study showed the role of EGCs' Cx43 hemichannels in the regulation of the GI motility (McClain et al., Concurrently, Cx43 on EGCs is taking part in the inflammatory process. Neuronal loss is one of the characteristics of intestinal inflammation and is driven by the activation of neuronal purinergic receptor (Gulbransen et al., 2+oscillations that can consequently regulate gene transcription (Dolmetsch et al., Animals with ablated Cx43 in EGCs also exhibited an increased fluid content in stools, indicating disabled water reabsorption/secretion (McClain et al., EGCs are also in proximity of capillaries within the gut wall (Fu et al., In summary, we presented a hypothetical link between the Cx43 of EGCs and GI related symptoms in patients with autism Figure . EGCs inThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Literature indicates that racism, sexism, homophobia and HIV-related stigma have adverse impacts on health, well-being, and quality of life among HIV-positive women of African descent (African/Black diaspora). However, limited evidence exists on the effectiveness of interventions aimed at reducing stigma tailored for these women. This study systematically reviewed randomized controlled trials (RCTs), non-randomized observational and quasi-experimental studies evaluating the effectiveness of interventions aimed at reducing stigma experienced by this population.The Cochrane methodology was used to develop a search strategy in consultation with a librarian scientist. Databases searched included the Cochrane Library, Ovid EMBASE, PsycInfo, and 10 others. Two reviewers independently assessed the studies for potential relevance and conducted the Cochrane grading of RCTs to assess risk of bias and the Newcastle\u2013Ottawa scale to assess the quality of non-randomized studies. Eligible papers were selected if they employed an intervention design with African/Black diasporic women living with HIV as the target population and had a primary outcome of stigma reduction.Of the five studies that met all of the eligibility criteria, four demonstrated the effectiveness of interventions in reducing HIV-related stigma. Only two of the five studies were designed specifically for HIV-positive African/Black diasporic women. Limitations included the absence of interventions addressing other forms of stigma and discrimination .Our findings suggest that there are limited interventions designed to address multiple forms of stigma, including gender and racial discrimination, experienced by HIV-positive African/Black diasporic women. Both in the global South and the global North, the HIV epidemic has disproportionately impacted people of African descent. Disproportionate rates of HIV in women of African descent have been reported in Western countries including Canada, the United States and the United Kingdom ,2. For tThe demographic profile in Canada signifies that a high proportion of HIV-positive women were born outside of Canada, particularly in Africa and the Caribbean . CurrentA preponderance of research has documented negative associations between singular forms of marginalization \u2013 HIV-related stigma, racism, sexism \u2013 and the health and well-being of people with HIV . For HIVThe term intersectional stigma was coined to describe the co-occurrence of stigma based on multiple identities of women living with HIV, including ethnoracial identity, gender and gender identity, national identity, sexual orientation and HIV serostatus . InterseAs recent HIV research recommends interventions that address stigma/discrimination at the micro, mesa and macro levels, there is limited evidence of interventions that address intersecting forms of marginalization ,43. Neveet al. [et al. [While evidence syntheses have been conducted evaluating the effectiveness of interventions designed to reduce HIV-related stigma ,54, theret al. evaluate [et al. examinedTo identify interventions designed for African/Black diasporic women with HIV and to examine their effectiveness at reducing stigma, we reviewed randomized controlled trials (RCTs), non-randomized quasi-experimental and observational studies that reported on the efficacy of stigma reducing interventions compared to treatment as usual in reducing the multiple forms of stigma and discrimination experienced by HIV-positive African/Black diasporic women. For the purposes of this review, we define African/Black diaspora as people of African ancestry who are living outside of the African continent with a sFor our systematic review of stigma reducing interventions for African/Black diasporic women, we identified intervention research that targeted any level or form of stigma and discrimination experienced by this population. As applicable, we also evaluated the effectiveness of stigma reducing interventions at addressing well-being and self-efficacy among African/Black diasporic women living with HIV.This project was not prospectively registered. A protocol was developed during the planning process (Grant# CIHR No. 97106 U of T Fund No. 487453 sub-grant 11).This systematic review adheres to guidelines outlined by the Cochrane Collaboration . The PRIIn June 2013 (current end date of electronic search), we conducted a search of 13 electronic databases that covered national and international literature in medical/health sciences, psychology, and social sciences. The search strategy was applied to Ovid MEDLINE (1946\u2013current) and Ovid EMBASE (1980\u2013current), then adapted for AgeLine Database (1978 \u2013 current), ASSIA: Applied Social Sciences Index and Abstract database (1987 \u2013 current), CINAHL (1980 \u2013 current), Clinicaltrials.gov (1999 \u2013 current), the Cochrane Library, a collection of six databases including the Cochrane Database of Systematic Reviews (CDSR), Cochrane Central Register of Controlled Trials (CENTRAL), Cochrane Methodology Register (CMR), Database of Abstracts of Reviews of Effects (DARE), Health Technology Assessment (HTA) Database, and NHS Economic Evaluation Database (NHS EED) (1898 \u2013 current), Dissertation Abstract International (1637 \u2013 Current), PsycINFO (1806 \u2013 present), Social Services Abstracts database (1979 \u2013 current), Social Science Abstracts (1972 \u2013 current), Sociological Abstracts (1952 \u2013 current), Social Sciences Citation Index (1900 \u2013 current). Keywords searched included: (1) HIV/AIDS keywords ; (2) stigma/discrimination keywords intervention search terms ). We also undertook a hand search of 19 journals (Supplementary Appendix) from 1995 to August 2013 to identify articles missed by our search, and conducted a citation search of the reference list of included studies. As only published manuscripts were included, authors were contacted by email to ascertain if a complete manuscript or publication was available. If any additional information or clarification were required, authors were also contacted.Two reviewers divided and screened all titles from the literature search for obvious exclusions. The remaining abstracts were independently assessed by two reviewers , with disagreements resolved by a third reviewer (LAC). We included published intervention studies if: (1) study population included HIV-positive women of African ancestry ; (2) the intervention aimed to reduce stigma and discrimination; (3) the study provided quantitative outcome data for: (a) stigma and discrimination , and (b) well-being , or (4) self-efficacy . Only RCTs and observational study designs were included.Three reviewers designed and independently piloted a data extraction form. The following items were extracted: (1) study information ; (2) participant information ; (3) intervention information ; (4) outcomes and measures used. Two investigators independently extracted data from relevant research. The definition of stigma reduction intervention is any intervention for which the reduction of stigma or discrimination was considered an outcome of the intervention. Our definition of stigma included HIV-related stigma as well as other forms of stigmatization or discrimination identified . The datasets were compared for each study, and a third party settled disagreements (LAC). Our review did not have any jurisdiction restrictions; though we were only able to extract data from papers published in English.The risk of bias of each eligible study was assessed by two investigators using the eight-item Newcastle\u2013Ottawa Scale for non-random and observational studies and the An a priori decision was made to conduct a meta-analysis; however, due to the heterogeneity of the study findings, including type and measurement of health outcomes, it prevented us from pooling the results using meta-analysis. Instead, we reported on study findings and conducted a descriptive analysis based on reported outcomes.Our study selection process is described in Supplementary Fig. 2 using an adapted version of the PRISMA flow diagram . After rThe details of the study characteristics of the included studies are reported in Supplementary Table 1. Of the five studies that met criteria for inclusion, three reported findings from RCTs ,62,64, aThe review sample totalled 238 participants of which 188 participants (79%) identified as African\u2013American women with HIV. Sample size ranged from 11 to 109 per study. Although all of the included studies focused on HIV-positive populations and considered cultural differences such as gender and ethnoracial identity in their analysis, only two of the five studies designed their interventions specifically for African\u2013American women ,65. The et al. [et al. [et al. [et al. [All five of the included studies reported on the effectiveness of interventions where stigma reduction was an expected outcome. Abel et al. and Abelet al. evaluate [et al. evaluate [et al. evaluate [et al. evaluateet al. [et al. [The results of the risk of bias assessments are reported in Supplementary Table 2 for the included RCTs and Supplementary Table 3 for observational studies. Overall, the three RCTs had a moderate risk of bias. Of the two prospective cohort studies Hosek et al. had a lo [et al. had an uWe conducted a descriptive synthesis of findings based on stigma reduction outcomes and reported on the effectiveness of the interventions for reducing stigma and discrimination. We also summarized findings on physical and mental well-being as reported in each study. The resulting data from each included study are presented in Supplementary Table 4.All five included studies reported stigma outcomes. Four studies measured perceived HIV stigma \u201364. One et al.\u2019s [et al. [Findings from four of the included studies indicated that the specified interventions demonstrated promise in reducing stigma. Both of the studies with an exclusively African/Black diasporic female sample reported reductions in stigma post-intervention. Rao et al.\u2019s evaluati [et al. reportedet al. [The two studies with a mixed ethnoracial female sample also demonstrated reductions in stigma post-intervention. Findings from Abel et al. and Abelet al. indicateet al. , and 12 et al. .et al. [The one study with a gender and ethnoracially diverse sample demonstrated mixed results in regards to stigma reduction. The Project ACCEPT intervention evaluated by Hosek et al. reportedet al. .et al. [et al. [et al. [Findings on physical and mental well-being post-intervention were mixed. Abel et al. reported [et al. reported [et al. noted siWe performed a comprehensive systematic review of the literature to evaluate the efficacy of current HIV-interventions in reducing stigma and discrimination experienced by African/Black diasporic women with HIV. We identified five studies that met our inclusion criteria, of which four studies demonstrated that their interventions had a positive effect on reducing HIV-related stigma in women living with HIV. These findings indicate that stigma reducing interventions can be of benefit for African/Black diasporic women with HIV. These findings could be useful for practitioners wishing to identify interventions that are effective in addressing HIV-related stigma in this population and the subsequent health benefits of such interventions.et al. [et al. [Of these studies, two exclusively sampled African/Black diasporic women with HIV and developed interventions that were culturally appropriate for this population. Rao et al. adapted [et al. develope [et al. ; oral an [et al. ), peer fet al. [et al. [The included studies identified interventions that showed promise in reducing the effects of stigma on African/Black diasporic women with HIV; however, only two studies were designed specifically for this population. While Abel et al. and Abelet al. conducteet al. . Althoug [et al. revealedet al.\u2019s [et al. [et al. [Furthermore, only one of these interventions was specifically designed to address both internal and perceived experiences of stigma. Rao et al.\u2019s interven [et al. , the aim [et al. addresse [et al. ,61. Over [et al. integratThis review also shows limited evidence of the long-term effectiveness of stigma reducing interventions for African/Black diasporic women with HIV. The follow-up duration ranged from four weeks to six months, with only two studies having a follow-up period of three months or longer ,64. WhilAdditionally, while all of these studies reported HIV-related stigma outcomes using similar measures, there is a lack of data demonstrating the effectiveness of stigma reducing interventions addressing other forms of stigma experienced by HIV-positive African/Black diasporic women including sexism and racism. These interventions were also focused on addressing stigma at the interpersonal or intrapersonal level; there is little evidence of interventions addressing stigma and discrimination at community, institutional or structural levels, which has been identified in the literature as essential to combating stigma . MoreoveSeveral limitations in our review merit discussion. First, although our study did not exclude based on language, in our analysis we were unable to include studies published in languages other than English. Our search strategy did not include an electronic database search for grey literature, though we did conduct manual searches that could capture non-peer reviewed or unpublished literature. Our study definitions of stigma/discrimination and health derived from Westernized concepts and may not have captured non-Westernized conceptualizations of these terms. Lastly, we were unable to pool the data for meta-analysis because of the significant heterogeneity in many study features.In summary, our systematic review contributes to the emerging body of literature on stigma reducing interventions for people with HIV ,53,54. NClick here for additional data file.Click here for additional data file."} +{"text": "Increasing distress about climate change consequences is noticeable in daily press releases and science news articles. In the last 5 years more than 3 hundred thousand scientific articles included the terms \u201cclimate change,\u201d \u201cdrought stress\u201d and/or \u201cclimate adaptation\u201d as a main topic. This build-up of energy devoted to understand climate change significance parallels the fact that any living organism must be able to cope with environmental changes to survive. Plant's sessile condition reinforces even more the need of an efficient adaptive response to counteract a suboptimal environment. Such adaptive strategies synchronize growth and development adjustments, as well as cellular and molecular activities, aimed at an efficient use of scarce resources, e.g., water.Arabidopsis HD-Zip I transcription factors, named ATHB7 and ATHB12, down-regulating a number of genes encoding ABA-receptor proteins, in addition to up-regulating protein phosphatases type 2C. Both ABA receptors and protein phosphatases 2C are well established components of the ABA signaling pathway accumulation upon drought perception serves as an initial signal for long-term acclimation reactions, which eventually involve the differential expression of genes leading to changes in transcript and protein patterns (Vald\u00e9s et al., As previously mentioned, developmental changes and morphological alterations are part of the plant adaptation and, besides controlling stress responses, HD-Zip I genes have additional roles in controlling development (Ariel et al., in silico interactions between genes early regulated in the shade-avoidance response and, ATHB7 and ATHB12 that highlight their potential participation also in light signaling pathways (Ciolfi et al., Besides water availability, the plant environmental context is defined by additional, simultaneous external factors, e.g., light and temperature, and should these factors influence the transcript levels of HD-Zip proteins the dynamic behavior of the ABA-driven stress response becomes automatically dependent on such factors. This implies that cross-communication between different signaling systems should be mediated by the same HD-Zip proteins. In this sense, available genomic and proteomic data have predicted The intricate network established within this superfamily of transcription factors suggest that the plant-specific and evolutionary highly conserved HD-Zip proteins are crucial players modulating stress responses and may be linking patterning and adaptation by acting to adjust developmental programs to specific environmental situations.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "The exponential growth in generation of large amounts of genomic data from biological samples has driven the emerging field of systems medicine. This field is promising because it improves our understanding of disease processes at the systems level. However, the field is still in its young stage. There exists a great need for novel computational methods and approaches to effectively utilize and integrate various -omics data.Systems medicine has been growing rapidly in part due to the emerging technologies to gather high-volume measurements from biological samples. One of the first such technologies, the mRNA microarray, is being replaced by next generation sequencing (NGS), which provides a much higher resolution of genetic information . Array and NGS-based methods to characterize genetic variation , DNA methylation changes, microRNAs (miRNAs) differential expression, and other types of biological information have dramatically expanded the generation of biological data. Other sources of data from mass spectrometry-based proteomics and metabolomics to high-throughput determination of protein-protein interactions and regulatory relationships provide further information for a systems-level understanding of disease. Finally, collection of clinical data and electronic medical records (EMRs) has made modern biomedical research possible on the full scale of data integration, that is, an integration scenario of using genomic, transcriptomic, proteomic, metabolomic, and phenotypic data.The rationale of discovery-based high-throughput investigation of disease is that there are molecular signatures that can be identified for better diagnosis, prognosis, and/or treatment of disease. However, challenges arise in the analysis of high-throughput data because of the large number of possible variables raising the very real potential for false-positive predictions and overfitting of data, as well as many other potential problems . To ameliorate these problems, computational approaches have been developed that utilize existing knowledge, such as overlaying high-throughput observations on regulatory or protein-protein interaction networks or canonical biological pathways.For this special issue we solicited manuscripts in several different subject areas including data integration from multiple high-throughput sources, NGS data analysis and applications, personalized medicine and translational bioinformatics, modeling of pathways and networks, and data mining and pattern recognition in biomedical applications. We briefly describe the accepted papers in this special issue in the remainder of this editorial.MultiRankSeq: multi-perspective approach for RNAseq differential expression analysis and quality control\u201d by Y. Guo et al. and \u201cQPLOT: a quality assessment tool for next generation sequencing data\u201d by B. Li et al., describe algorithms for analysis of NGS data. Y. Guo et al. introduce a novel tool, namely, MultiRankSeq, which combines the output of three independent programs to determine differential expression from RNAseq data to provide a single improved output. QPLOT is a tool for assessing the quality of NGS runs by providing both summary quality metrics and graphical representations of these metrics. In another paper, entitled \u201cComparative study of exome copy number variation estimation tools using array comparative genomic hybridization as control,\u201d Y. Guo et al. systematically compare four different tools for detecting copy number variations (CNVs) from whole exome sequencing (WES) against a standard array-based method for CNV evaluation.Two papers, \u201cComputational analysis of transcriptional circuitries in human embryonic stem cells reveals multiple and independent networks,\u201d X. Wang and C. Guda assess the role of core transcription factors in the pluripotency of embryonic stem (ES) cells. Their computational analyses identified several additional transcriptional regulatory networks that might be involved in this complex regulatory process, providing interesting hypotheses about mechanisms of fate determination in ES cells. The paper \u201cNetwork-assisted prediction of potential drugs for addiction\u201d by J. Sun et al. describes computational analyses of drug-target networks for addictive and nonaddictive drugs. The authors analyzed the topology of these networks and found that drugs with similar effects could cluster together and identified a set of nonaddictive drugs that might have therapeutic benefits for treatment of addiction. This paper was called out in the recent \u201cTranslational Bioinformatics Year-in-Review\u201d in 2014 Joint Summits on Translational Science (http://www.amia.org/jointsummits2014). In \u201cDeGNServer: deciphering genome-scale gene networks through high performance reverse engineering analysis,\u201d J. Li et al. describe their webserver to infer transcriptional regulatory networks from large-scale datasets. The server makes use of a computer cluster to run a number of network inference algorithms and return the results to the user very quickly, thus facilitating genome-scale network reconstruction.In \u201cDevelopment of dual inhibitors against Alzheimer's disease using fragment-based QSAR and molecular docking\u201d and \u201cNovel natural structure corrector of ApoE4 for checking Alzheimer's disease: benefits from high throughput screening and molecular dynamics simulations,\u201d deal with molecular docking simulations to determine small-molecule inhibitors targeting Alzheimer's disease. In the first paper, M. Goyal et al. used a fragment-based quantitative structure activity relationship (QSAR) analysis to identify lead compounds that might inhibit interaction of proteins that drive Alzheimer's disease pathogenesis. In the second paper, the authors describe large-scale docking simulations to screen for inhibitors of the conformational change of apolipoprotein E4 (ApoE4) that is thought to drive Alzheimer's pathogenesis. They further show the value of molecular dynamics simulations to screen candidates to eliminate molecules that do not have stable binding properties with targets. In \u201cHGF accelerates wound healing by promoting the dedifferentiation of epidermal cells through \u03b21-integrin/ILK pathway,\u201d J.-F. Li et al. experimentally investigate the contribution of hepatocyte growth factor (HGF) to wound healing. They showed that treatment of diabetic mice promoted proliferation and migration of epithelial cells and that this effect could be blocked by silencing the \u03b21-integrin signaling pathway.Two papers from M. Goyal et al., \u201cIntegrative analysis of miRNA-mRNA and miRNA-miRNA interactions,\u201d the authors first generated RNAseq data for normal and tumor cell lines and then identified aberrantly expressed mRNAs and miRNAs. Groups of similarly expressed miRNAs and mRNAs were analyzed to highlight examples of flexible and selective regulatory networks underlying these interactions. In \u201cA diverse stochastic search algorithm for combination therapeutics,\u201d M. U. Caglar and R. Pal show how the use of a stochastic search algorithm can be useful in identification of optimal combinations of drugs for therapy, the so-called drug cocktails. Their novel method greatly reduces the number of experimental steps needed to assess the optimal combination of drugs for a particular therapy. In \u201cEvaluating word representation features in biomedical named entity recognition tasks\u201d by B. Tang et al., the authors present a comparative analysis of three different methods for word representation in recognition of named entities from biomedical literature. Their findings indicate that a combination of the complementary approaches can improve results on benchmark recognition tasks.In the paper \u201cMultiple biomarker panels for early detection of breast cancer in peripheral blood,\u201d the authors describe the use of machine-learning approaches to identify a five-gene panel that can identify breast cancer from peripheral blood samples. In the paper by Jiang et al., \u201cNew aQTL SNPs for the CYP2D6 identified by a novel mediation analysis of genome-wide SNP arrays, gene expression arrays, and CYP2D6 activity,\u201d the authors develop a novel approach for the detection of transexpression quantitative trait loci (eQTLs) from genome-wide association studies by considering indirect effects introduced by a mediator gene. They apply their method to analyze indirect regulatory effects on the important liver enzyme, CYP2D6. Finally, in \u201cExpression sensitivity analysis of human disease related genes,\u201d L.-X. Ma et al. examine the expression of genes implicated in a range of diseases. They report that genes that are robustly expressed under different perturbations are more likely to be associated with lethal diseases, whereas less robustly expressed genes are associated with nonlethal diseases.In the paper by F. Zhang et al., \u201c"} +{"text": "Although 3D cell cultures better mimic in vivo microenvironments of human tissues and provide an in-depth understanding of the morphological and functional features of tissues, they are also limited by having relatively low throughput and thus are not amenable to high-throughput screening (HTS). One attempt of making 3D cell culture amenable for HTS is to utilize miniaturized cell culture platforms. This review aims to highlight miniaturized 3D cell culture platforms compatible with current HCI technology.High content imaging (HCI) is a multiplexed cell staining assay developed for better understanding of complex biological functions and mechanisms of drug action, and it has become an important tool for toxicity and efficacy screening of drug candidates. Conventional HCI assays have been carried out on two-dimensional (2D) cell monolayer cultures, which in turn limit predictability of drug toxicity/efficacy To address this issue, high content imaging (HCI) or high content screening (HCS) technology\u2014which refers to a high-throughput, automated microscope-based assay that provides information on multiple properties or features of individual cells simultaneously with several fluorescent dyes\u2014has been adapted to a more systematic and accurate evaluation of drug candidates ,9998,99]G2 cells . Similarll lines . Apart fll lines . Cheong [et al. . Additio [et al. ,105,106 [et al. . One of [et al. ,109. High-throughput HCI on miniaturized 3D cell culture has a potential to decipher toxicodynamic and toxicokinetic traits of drugs and helps us to understand complicated toxicology pathways and related adverse responses in early stages of drug discovery. Simplifying steps for miniaturized 3D cell culture and integrating the cell culture platforms with automation systems for liquid handling and image acquisition/analysis are critical to implement 3D HCI. Microwell and cellular microarray platforms are more compatible with automated liquid dispensing robots such as microarray spotters than microfluidic devices, resulting in increased throughput of HCI, whereas microfluidic devices are inherently low throughput in HCI due to the sample loading processes and large pumping units . In addi"} +{"text": "Studies over the last decade have provided insights on the molecular and structural determinants of Gag-membrane binding. The human immunodeficiency virus type-1 (HIV-1) Gag polyprotein adopts a compact \u201cfolded over\u201d conformation and exists in the monomeric or low-order oligomeric states prior to targeting to the PM in the pericentriolar region of the infected cell and therefore. Structural details of M-PMV MA binding to single phospholipids are discussed. Dick et al. describe the principles that govern Gag interactions with membranes, focusing on RSV and HIV-1 Gag , a channel mediating release of Ca2+ from ER stores, as a cellular factor differentially associated with HIV-1 Gag that might facilitate ESCRT function in virus budding. In a research article, Ehrlich et al. show that Gag modulates ER store gating and refilling and how various membrane components facilitate these events. It is well established that assembly of most of retroviral Gag proteins occurs on the plasma membrane (PM) (Ono et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Streptomyces, which includes several hundred species, many of which produce biotechnologically useful secondary metabolites. The last 2 years have seen numerous publications describing Streptomyces genome sequences . Our blastn searches (using these two cluster sequences as queries) failed to detect a complete streptomycin gene cluster in the S. gancidicus genome, but there were some regions of sequence similarity on a 111 kb contig (GenBank: AOHP01000057). An antiSMASH 2.0 search failed to find any aminoglycoside biosynthetic cluster in this genome. We are not aware of any experimental evidence that this strain produces the aminoglycoside streptomycin and conclude that these seven genes highlighted by the authors and relatively high error rates associated with these platforms can lead to rather fragmented and/or incomplete genome assemblies. The situation is not helped by the biased sequence composition (approximately 70% G + C) of Streptomyces DNA. Furthermore, non-ribosomal peptide synthases (NRPS) and polyketide synthetases (PK) are long, modular proteins made up of many repeated domain units. This means that the genes encoding these key enzymes can be particularly difficult to assemble accurately from short sequence reads. To overcome this issue, the authors of the Mg1 genome project and which is notable for its production of the antibiotic moenomycin A , which has an 11.14 Mbp genome. Rather, strains SPC6 and DSM 40736 are closely related and fall within a clade with several other strains for which draft genomes are available and with Streptomyces venezuelae for which a complete finished genome sequence is available (Pullan et al., S. venezuelae reference sequence. Evidently, genome reduction has also occurred in S. albus strain J1074 (Zaburannyi et al., S. somaliensis (Kirby et al., Among bacteria, streptomycetes have some of the largest genomes, typically within the range of 8.7 Mbp to 11.9 Mbp (Zhou Streptomyces and related species [e.g. (Liu et al., Streptomyces sp. strain OH-4156, revealing an unusual class of post-translationally modified ribosomally synthesized peptides (Claesen and Bibb, et al., Streptomyces genome comes from strain PRh5, an endophyte of wild rice that produces nigericin, an antibiotic effective against mycobacteria (Yang et al., The availability of cheap sequencing has led to the generation of numerous genome sequences for"} +{"text": "Escherichia coli is a heterogeneous species that can be part of the normal flora of humans but also include strains of medical importance. Among pathogenic members, Shiga-toxin producing E. coli (STEC) are some of the more prominent pathogenic E. coli within the public sphere. STEC disease outbreaks are typically associated with contaminated beef, contaminated drinking water, and contaminated fresh produce. These water- and food-borne pathogens usually colonize cattle asymptomatically; cows will shed STEC in their feces and the subsequent fecal contamination of the environment and processing plants is a major concern for food and public safety. This is especially important because STEC can survive for prolonged periods of time outside its host in environments such as water, produce, and farm soil. Biofilms are hypothesized to be important for survival in the environment especially on produce, in rivers, and in processing plants. Several factors involved in biofilm formation such as curli, cellulose, poly-N-acetyl glucosamine, and colanic acid are involved in plant colonization and adherence to different surfaces often found in meat processing plants. In food processing plants, contamination of beef carcasses occurs at different stages of processing and this is often caused by the formation of STEC biofilms on the surface of several pieces of equipment associated with slaughtering and processing. Biofilms protect bacteria against several challenges, including biocides used in industrial processes. STEC biofilms are less sensitive than planktonic cells to several chemical sanitizers such as quaternary ammonium compounds, peroxyacetic acid, and chlorine compounds. Increased resistance to sanitizers by STEC growing in a biofilm is likely to be a source of contamination in the processing plant. This review focuses on the role of biofilm formation by STEC as a means of persistence outside their animal host and factors associated with biofilm formation. Escherichia coli is a diverse species of bacterium that includes members of the normal commensal flora of humans and animals but also pathogenic strains of veterinary and medical importance. Pathogenic members are usually classified in two major groups: intestinal E. coli (InPEC) and extraintestinal E. coli (ExPEC). The latter group is typically responsible for urinary tract infections [uropathogenic E. coli (UPEC)], neonatal sepsis, and meningitis in humans and various infectious diseases in animals including mastitis associated with Crohn's disease, diffusely adherent E. coli (DAEC), enteroaggregative E. coli (EAEC), enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), Shiga-toxin producing E. coli (STEC) that includes enterohemorrhagic E. coli (EHEC), and enteroinvasive E. coli (including Shigella) (EIEC) strain will not be covered in this review because it does not fit within the classic STEC pathotype.The predominant STEC serotype associated with outbreaks is O157:H7. Since it was one of the first serotypes identified as causing hemolytic uremic syndrome (HUS) and the most severe illness, EHEC O157:H7 is the most commonly reported STEC serotype in the media , but in complex communities called biofilms. Biofilms are aggregates of microorganisms enclosed in a self-produced extracellular polymeric matrix that are attached to a biotic or abiotic surface , several of which encode fimbrial adhesins for the matrix architecture among 51 STEC strains found that the presence of autotransporter genes within the genome was variable among STEC serotypes , colanic acid, and/or cellulose. The genes encoding proteins that are involved in the synthesis of these polysaccharides are present in the genomes of STEC strains EDL933 and Sakai and capsules, which are surface structures, have been implicated in biofilm formation by E. coli group 4 capsule, which is composed of the same sugar repeats as the LPS O-antigen and acetamido sugars in their repeat-unit structures and the export machinery (CsgE-G) , the signal molecule for AI-1, secreted by others bacterial species. In Sharma et al. systems in EPEC and aggregative adherence fimbriae (AAFs) in EAEC, results in the detachment of bacteria from the biofilm and surface are also significantly up-regulated when E. coli O157:H7 interacts with lettuce roots , expel vesicles containing viable E. coli O157:H7, whereas Colpoda steinii and Acanthamoeba palestinensis (flagellates) do not . The persistence of STEC in the presence of disinfectants gives rise to the probability that STEC survive and grow within a biofilm in processing plants and 4\u00b0C (production hours temperature) (Dourou et al., E. coli O157:H7 attachment increased at 4\u00b0C over time in the presence of a fat-lean tissue homogenate (Dourou et al., E. coli O157:H7 EDL933 is able to adhere and produce a dense biofilm on surfaces that are not favorable for its attachment when collagen I is present, which is a muscle fibrous extracellular matrix protein (Chagnot et al., E. coli O157:H7 is also able to form biofilms on stainless steel when grown in spinach leaf lysates (Carter et al., E. coli O157:H7 biofilms produce large amounts of AI-2 when cultured in pork, beef or spinach broth (Silagyi et al., In the processing plant environment, temperatures are normally controlled and maintained between 4 and 15\u00b0C. Many studies have shown that STEC are able to grow in a biofilm within this temperature range (Dourou et al., E. coli O157:H7 biofilms were more resistant to sanitizers than at lower RH (Bae et al., E. coli O157:H7 that were able to regrow as a biofilm on polyurethane (Marouani-Gadri et al., Fouladkhah et al. showed that the use of quaternary ammonium compound-based and peroxyacetic-based chemical sanitizers on biofilms that had matured for 1 week were more effective at 4\u00b0C than 25\u00b0C. However, these commercial sanitizers used at concentrations recommended to kill planktonic STEC were not able to kill or remove STEC biofilms from stainless steel surfaces (Fouladkhah et al., Comamonas testosterone, Acinetobacter calcoaceticus, Burkholderia caryphylli, and Ralstonia insidiosa, can initiate biofilm formation and may allow E. coli O157:H7 to integrate within a pre-formed biofilm, resulting in a mixed biofilm (Marouani-Gadri et al., C. testosteroni can enhance the ability of E. coli O157:H7 to form biofilms (Marouani-Gadri et al., C. testosteroni within the biofilm did, however, decrease the number of colony forming units of E. coli O157:H7 following chemical treatment when compared to chemical treatment of a single species E. coli O157:H7 biofilm (Marouani-Gadri et al., Interestingly, non-pathogenic bacteria isolated from processing plants, such as E. coli O157:H7 (Ryu et al., E. coli O157:H7 from sanitizer treatments (Ryu et al., E. coli O157:H7 could colonize a mature biofilm formed by EPS-producing bacteria (Castonguay et al., The ability to secrete EPS is related to biofilm formation on stainless steel surfaces, but it was shown that overproduction of the EPS inhibits the initial attachment of E. coli in general, a slow-growing and dormant subpopulations are highly tolerant to antibacterial treatments (Lewis, In addition to the protection offered by the biofilm matrix against sanitizers, it is well established that for s Lewis, . Cells fs Lewis, . The emes Lewis, . This noIt is known that STEC biofilms are more resistant to sanitizers than their planktonic counterparts (Wang et al., E. coli O157:H7 inside a biofilm. However, the biofilm matrix remains attached to the surface (Perez-Conesa et al., E. coli O157:H7 biofilms from processing plants. Others strategies such as bacteriophage treatments of E. coli O157:H7 biofilms have also been investigated. The KH1 bacteriophage reduces the population of O157:H7 cells attached to stainless steel, but not those incased within a biofilm matrix (Sharma et al., E. coli O157:H7 within the biofilm and remove the biofilm matrix from the contaminated surface. For example, a combination of steam and lactic acid were able to kill E. coli O157:H7 and remove the biofilm matrix from stainless steel surfaces (Ban et al., Many essential oils have been shown to have good antibiofilm activity against food-borne pathogens (Giaouris et al., Contamination of the environment and processing plants with cow feces containing STEC is a major concern for food and public safety, especially since STEC can survive for prolonged periods of time outside its host. Biofilm formation appears to contribute significantly to STEC survival on produce, in rivers, and in processing plants. Several factors involved in biofilm formation such as curli, cellulose, PGA, and colanic acid are involved in plant colonization and attachment to different surfaces often found in meat processing plants. However, the factors involved in STEC survival within biofilms in rivers remain unknown. Furthermore, STEC biofilm formation on farms, in manure, and in soil has not been thoroughly explored despite the presence and persistence of STEC in these environments. The Stx toxin, which is a key factor in human host pathology, also appears to be an important factor for STEC survival against protozoan predation. In the food industry, resistance to sanitizers improves the ability of STEC to persist in the processing plant. Despite the development of new strategies to eradicate biofilms formed by food-borne pathogens, no effective solutions to remove STEC biofilms from surfaces have been identified. Therefore, future research should focus on the identification of factors promoting STEC survival, especially non-O157 STEC, and the persistence of STEC in environmental biofilms on the farm.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "N = 106) , a recently developed therapy for resistant hypertension, is generally regarded as a safe procedure renal artery stenosis causes recurrent hypertension in denervated patients (Kaltenbach et al., Long-term randomized trials are needed. Two major randomized trials on RDN, i.e., the Symplicity HTN-2 and HTN-3 trials (Esler et al., Imaging methods monitoring renal artery stenosis need to be standardized. Symplicity HTN trials did not standardize renal artery imaging methods during follow ups. Ultrasonography, magnetic resonance angiography, and computerized tomographic angiography were used (Krum et al., It is likely that improved catheters for RDN using lower power radiofrequency over a shorter time will reduce the local tissue injury at the ablation site compared with that caused by the first generation RDN systems (Versaci et al., The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "We appreciate Lioy et al.\u2019s interest in our recent article documentn-butyl phthalate (DnBP) and di(2-ethylhexyl) phthalate (DEHP) is encouraging, but the rising trend in other phthalates, such as diisobutyl phthalate (DiBP) and diisononyl phthalate (DiNP), is worrisome. Lioy et al. note that DiNP is less potent as an antiandrogen than the other phthalates. However, its increasing presence in the U.S. general population warrants public health concern: The U.S. Environmental Protection Agency (EPA) has expressed concern about DiNP\u2019s use [Phthalates Action Plan and other large environmental health studies to evaluate the presence of potential \u201csubstitute\u201d chemicals not only of phthalates but also of other environmental chemicals to provide the best human exposure information. In fact, for the most recent NHANES survey (2011\u20132012), scientists at the Centers for Disease Control and Prevention are beginning to release population-level data for the urinary metabolite of 1,2-cyclohexane dicarboxylic acid diisononyl ester (DINCH), a nonphthalate plasticizer that is used as a replacement for some of the high-molecular-weight phthalates . Indeed,"} +{"text": "Growth factors such as transforming growth factor-\u03b2 (TGF-\u03b2), vascular endothelial growth factor (VEGF), insulin-like growth factor-1 (IGF-1), platelet-derived growth factor (PDGF), and basic fibroblast growth factor (bFGF) have been associated with the tendon healing process as a novel method for treating tendon and ligament injuries is also discussed.Human tendon healing process is classified into five phases as follows: immediate post injury phase, inflammatory phase, proliferation phase, reparative phase, and remodeling phase consist of MSCs and HSCs, which can further differentiate into various cell types that secrete growth factors is impeded by the lack of soft tissue around it. However, partially ruptured ACL can regenerate with growth factor administration and after cell transplantation. Intra-articular transplantation therapies using MSCs have been used for treating torn ACLs (Agung et al., Recently, we showed that partially transected rodent ACL can be repaired by injection of BMCs or cultured MSCs into the articular cavity at 1 week after transection (Oe et al., Stem cell therapies use several stem cell sources such as human embryonic stem cells, MSCs, BMCs, and bone marrow mononucleated cells have been reported (Chen et al., Few clinical trials have been performed using stem cells transplantations for treating tendon and ligament injuries in humans. Ahmad et al have reviewed 5 clinical studies that used stem cell therapy for treating tendon injuries and concluded that stem cells can have positive effect on tendon healing (Ahmad et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Our earlier studies showed that lactational exposure to lead (Pb) caused irreversible neurochemical alterations in rats. The present study was carried out to examine whether gestational exposure to Pb can cause long-term changes in the brain cholinergic system and behavior of rats. The protective effect of calcium (Ca) supplementation against Pb toxicity was also examined. Pregnant rats were exposed to 0.2% Pb (Pb acetate in drinking water) from gestational day (GD) 6 to GD 21. The results showed decrease in body weight gain (GD 6\u201321) of dams, whereas no changes were observed in offspring body weight at different postnatal days following Pb exposure. Male offspring treated with Pb showed marginal alterations in developmental landmarks such as unfolding of pinnae, lower and upper incisor eruption, fur development, eye slit formation and eye opening on postnatal day (PND) 1, whereas significant alterations were found in the righting reflex (PNDs 4\u20137), slant board behavior (PNDs 8\u201310) and forelimb hang performance (PNDs 12\u201316). Biochemical analysis showed decrease in synaptosomal acetylcholinesterase (AChE) activity and an increase in acetylcholine (ACh) levels in the cortex, cerebellum and hippocampus on PND 14, PND 21, PND 28 and in the four-month age group of rats following Pb exposure. Significant deficits were also observed in total locomotor activity, exploratory behavior and open field behavior in selected age groups of Pb-exposed rats. These alterations were found to be maximal on PND 28, corresponding with the greater blood lead levels observed on PND 28. Addition of 0.02% Ca to Pb reversed the Pb-induced impairments in the cholinergic system as well as in behavioral parameters of rats. In conclusion, these data suggest that gestational exposure to Pb is able to induce long-term changes in neurological functions of offspring. Maternal Ca administration reversed these neurological effects of Pb later in life, suggesting a protective effect of calcium in Pb-exposed animals. Developmental exposure to Pb has become an important public health concern because of the possible toxic impact on sensitive development and programing of organ functions is an essential mineral for the newborn infant and alteration in Ca signaling may have life-long consequences in brain functions (Bass Lead acetate (99.99% purity) was purchased from Sigma Chemicals and all other chemicals from Merck, India.et al.,et al.,et al.,et al.,Female and male albino rats (Wistar) were acclimatized for one week before mating. Two females and one male were placed overnight in a cage and the presence of a vaginal sperm plug was recorded. Pregnancies determined by the presence of sperm in the vaginal smear were considered gestational day (GD) 1. On GD 6, the dams were randomly assigned to three experimental groups exposed rats and Group III \u2013 animals exposed to 0.02% calcium supplement together with Pb. The animals were housed individually. Pregnant animals were exposed to low level Pb (0.2%) by intake of Pb-acetate dissolved in deionized water from gestational day 6 (GD 6) until the birth of pups (GD 21) . Calciumet al.,et al.,Starting with PND 1, the number of pups was counted and gender differences were recorded. The male pups were weighed and observed for developmental signs, such as fur development (the appearance of fur sufficient to cover skin), incisor teeth eruption (the first appearance of the upper and lower incisors), pinnae detachment (both ears completely unfolded from the head), eye slit formation, eye opening (both eyes fully open) and crown rump length stretched between two wooden blocks was then permitted to grasp the string with its forepaws, where the point was released, so that it was suspended from the string by its forepaws. The latency to fall from the string (maximum 60 s) was measured and each pup was tested for a single trial on each of the five test days at the designated time periods (PNDs) for a period of two hours in the morning (08.00\u201310.00AM). The activity was presented as counts/min.Open field behavior: The open field behavior was measured in a chamber measuring 90\u00d790\u00d790 cm. The floor of the arena was divided into 36 equal squares. The number of squares crossed with all paws (crossing), standing on the hind legs (rearing), standing on the hind legs and placing the forelimbs on the wall , placing the nose against the wall or floor (sniffing), and wiping, licking or combing of any part of the body (grooming) were recorded. The sum of all tasks in open field was considered the total behavior containing three equally spaced holes (3 cm in diameter) in the floor. Each rat was placed in the center of the arena for 5 min., the time during which the number of head dips and head-dipping duration (in seconds) were recorded. A head dip was scored if both eyes disappeared into the hole.et al.,For the determination of Pb levels, 5.0 ml of whole blood was taken by heart puncture. After digestion with concentrated nitric acid using a microwave digestion system followed by addition of 30% hydrogen peroxide, the samples were brought to a constant volume. Blood Pb levels were determined with an atomic absorption spectrophotometer (AAS-Shimadzu-AA 6300) with graphite furnace (GFA-EX7i) followed by Student-Newman-Keuls (SNK) post hoc test using statistical package for social sciences (SPSS 16) to compare the effects among various groups. The 0.05 level of probability was used as the criterion for significance. All the data in this study were analyzed using the dam or litter as the experimental unit. For some behavioral measures, two pups from each litter were tested, but the values were averaged and only one value from each litter was considered for statistical analysis. The variation within the litter was less than 5%.p<0.05), while the body weight of male offspring from Pb-exposed mothers showed no significant changes on PND 7, PND 14, PND 21, PND 28 and in 4-month-old rats compared to controls (p<0.01) was observed in the latency to turn in the righting reflex and slant board behavior of Pb-exposed offspring (p<0.01) recorded from PND 12 to PND 16 and levels of acetylcholine (ACh) were determined in the cortex, cerebellum and hippocampus on PND 14, PND 21, PND 28 and in the 4-month age groups of control and Pb exposed rats and 7. Ap<0.001) increased the Pb levels in all selected age groups of rats levels at any age induce neuro-degeneration that leads to impairment of neurobehavioral functions (Schwartz et al., observed et al., also obset al.,et al., (et al.,et al.,et al.,et al.,et al.,et al.,et al.,Dietary Ca intake can support normal fetal growth and development during pregnancy in animals and humans (Han et al., have sugConsistent with the published work and results from our laboratory, it is clear that gestational exposure to Pb is sufficient to induce late life alterations in neurobehavioral functions in rats. These neurobehavioral changes continue even long after the Pb exposure was stopped. Although Ca supplement reversed the Pb-induced neurotoxicity, longterm protection by Ca is partial, suggesting that adequate calcium intake would be beneficial in treating Pb-induced toxicity during pregnancy in rats."} +{"text": "Recent advances in modern molecular technology and next-generation sequencing platform have brought in enormous insights into evolution. Although challenges remain, evolutionary research now enters a new era and becomes a hybrid discipline encompassing the fields of molecular biology, genetics and genomics, metabolomics, neuroscience, structural and chemical biology, bioinformatics, proteomics, pharmacology, statistics, and computational biology. The common bond or theme that unifies the various fields is the need to reveal complex genotype-phenotype relationships and their mechanistic regulations, attempting to understand the diversifying functionality in life and ultimately find the underlying causes and effective means of treating disease.Articles in this special issue cover a broad range of topics related to the evolution of genomes, transcriptomes, proteomes, and interactomes in diverse organisms, including humans.Signal transduction networks are dynamic and constantly under environmental selection in evolution. This topic is covered in the long review article \u201cMulti-OMICs and Genome Editing Perspectives on Liver Cancer Signaling Networks\u201d by S. Lin et al. This review covers multiple advances in the multi-OMICs and genome editing technologies, now available for use in biomedical and cancer research, and provides new perspectives in targeted therapy and precision medicine. Proteome and interactome networks are also investigated in an accompanying research paper \u201cPositive Selection and Centrality in the Yeast and Fly Protein-Protein Interaction Networks.\u201d S. Chakraborty and D. Alvarez-Ponce study the selective pressures of yeast and fly proteome and show that long-term positive selection has preferentially targeted the periphery of the yeast interactome, while interestingly in flies genes under positive selection encode significantly more connected and central proteins.Transcriptional and functional regulation and conservation during evolution are touched upon in two other reviews in this special issue. In \u201cCircadian Control of Global Transcription,\u201d S. Li and L. Zhang discuss circadian rhythms through investigating expression of clock-controlled genes (CCGs). They summarize the circadian control of CCG transcription in five model organisms, including cyanobacterium, fungus, plant, fruit fly, and mouse, providing an overview to the function of the circadian clock, as well as its output mechanisms adapted to meet the demands of diverse environmental conditions. In \u201cRoles of Hsp70s in Stress Responses of Microorganisms, Plants, and Animals,\u201d A. Yu et al. review the heat shock protein 70s family members, a class of molecular chaperones that are highly conserved and ubiquitous in organisms ranging from microorganisms to plants and humans. This review provides an overview of the specific roles of Hsp70s in response to stress, particularly abiotic stress, in diverse taxa. Mycobacterium tuberculosis and Lung Cancer: Implications into Host-Pathogen Interaction and Coevolution,\u201d Y. Tian et al. study the cross-link between pathogens and cancer. They examined the association between the Mycobacterium tuberculosis (MTB) and lung cancer and found that 62% of the lung cancer samples are MTB-L positive, suggesting a link between MTB infection and lung cancer development. In another paper \u201cLost Polarization of Aquaporin4 and Dystroglycan in the Core Lesion after Traumatic Brain Injury Suggests Functional Divergence in Evolution,\u201d H. Liu et al. study the roles of aquaporin4 and dystroglycan in brain edema formation after traumatic brain injury. They found that at an early stage of traumatic brain injury aquaporin4 and dystroglycan maintained the polarized distribution, which were subsequently lost from perivascular end-feet and induced cytotoxic brain edema. In addition, in the study entitled \u201cIdentification and Evolutionary Analysis of Potential Candidate Genes in a Human Eating Disorder,\u201d U. Sabbagh et al. set out to find genes linked with eating disorders and associated with both metabolic and neural systems . Through a text-based analysis, they identified a number of potential candidate genes including VGF. VGF human polymorphism studies contribute to eating disorders and obesity, suggesting a new approach to connect eGWAS and GWAS in NES patients.Evolutionary principles and adaptation lessons learned from research have been successfully applied to human disease studies, revealing important insights into potential targets for therapy. In the paper \u201cClinical End-Points Associated with flfl Triggers JNK-Dependent Cell Death in Drosophila,\u201d J. Huang and L. Xue study a gene, falafel (flfl), that encodes a fruit fly Drosophila homolog of human SMEK. They performed both gain-of-function and loss-of-function analysis in Drosophila and identified flfl as a negative regulator of JNK pathway-mediated cell death. In another study \u201cNeurotrophin, p75, and Trk Signaling Module in the Developing Nervous System of the Marine Annelid Platynereis dumerilii,\u201d A. Lauri et al. study the evolution of neurotrophic signaling in neuronal development, neural circuit formation, and neuronal plasticity using the worm Platynereis system. They discovered and validated nucleotide sequences encoding putative neurotrophic ligands and receptors in two annelids, suggesting roles of these molecules in nervous system and circuit development, as well as their involvement in shaping neural networks during protostome-deuterostome evolution.Model systems have been instrumental in elucidating signaling mechanisms underlying biological function, providing interesting cues in evolutionary research areas. In the article \u201cLoss ofThe great leap in evolutionary research would not be possible without the development of modern molecular technologies. CRISPR/Cas9 system is a powerful technology to perform genome editing in a variety of cell types. In the paper \u201cA CRISPR-Based Toolbox for Studying T Cell Signal Transduction,\u201d S. Chi et al. adapted CRISPR/Cas9-based tools to study human Jurkat cells. They showed that distinct Cas9 variants, including wild-type Cas9, dCas9-KRAB, and sunCas9 function as expected in different Jurkat cell lines.With the advent of next-generation sequencing platforms, a large number of genomewide sequencing data become available, which greatly facilitate the evolution research field to promote to a new height. In the paper \u201cComputational Analysis of the Binding Specificities of PH Domains,\u201d Z. Jiang et al. study Pleckstrin homology domain proteins with significant insights into binding specificity and abnormal regulation in disease. In addition, in another report \u201cSimFuse: A Novel Fusion Simulator for RNA Sequencing (RNA-Seq) Data,\u201d Y. Tan et al. developed the SimFuse pipeline to accelerate fusion discovery from RNAseq data. SimFuse utilizes real sequencing data as the fusions' background to closely approximate the distribution of reads from a real sequencing library and uses a reference genome as the template from which to simulate fusions' supporting reads, improving the performance metrics, rigor, and control.The future evolution researchers will need to be well versed in interdisciplinary fields and have the appropriate background to leverage the biological, clinical, and computational resources necessary to understand their data and track dynamic evolution. The communication among these specialty disciplines, acting in unison, will be critical as we strive to uncover answers underlying the complex and often puzzling molecular events in evolution.We hope that this special issue would shed light on major advances in the broad area of signal transduction with evolutionary insights and engender novel hypotheses and research innovations to investigate deeper mechanisms and further to engineer signaling circuits for personalized therapeutics in human disease."} +{"text": "Low let-7 levels resulted in up-regulation of oncogenes including MYCN, AURKB and LIN28 itself, the latter through a direct feedback mechanism. Targeting LIN28, or restoring let-7 levels, both led to effective inhibition of this pathway. In summary, paediatric malignant GCTs show biological differences from their adult counterparts at a genomic and protein-coding transcriptome level, whereas they both display very similar microRNA expression profiles. These similarities and differences may be exploited for diagnostic and/or therapeutic purposes.Genomic and protein-coding transcriptomic data have suggested that germ cell tumours (GCTs) of childhood are biologically distinct from those of adulthood. Global messenger RNA profiles segregate malignant GCTs primarily by histology, but then also by age, with numerous transcripts showing age-related differential expression. Such differences are likely to account for the heterogeneous clinico-pathological behaviour of paediatric and adult malignant GCTs. In contrast, as global microRNA signatures of human tumours reflect their developmental lineage, we hypothesized that microRNA profiles would identify common biological abnormalities in all malignant GCTs owing to their presumed shared origin from primordial germ cells. MicroRNAs are short, non-protein-coding RNAs that regulate gene expression via translational repression and/or mRNA degradation. We showed that all malignant GCTs over-express the miR-371\u2013373 and miR-302/367 clusters, regardless of patient age, histological subtype or anatomical tumour site. Furthermore, bioinformatic approaches and subsequent Gene Ontology analysis revealed that these two over-expressed microRNAs clusters co-ordinately down-regulated genes involved in biologically significant pathways in malignant GCTs. The translational potential of this finding has been demonstrated with the detection of elevated serum levels of miR-371\u2013373 and miR-302/367 microRNAs at the time of malignant GCT diagnosis, with levels falling after treatment. The tumour-suppressor In addition to considering genomic and protein-coding transcriptomic changes, we will emphasize findings from expression profiling studies of short non-protein-coding RNAs, termed microRNAs, in GCTs. Our improving knowledge of the molecular mechanisms underlying the pathogenesis of GCTs is contributing to the identification of new biomarkers and therapeutic targets, and the development of clinico-biological algorithms for disease segmentation and risk stratification. Combined with the developing collaborations between international clinical trial groups, we can be cautiously optimistic that these approaches will, in the foreseeable future, improve the clinical management of the children, adolescents and young adults affected by this disease , regardless of patient age, tumour site and histological subtype Teilum, . Despiteet\u00a0al., et\u00a0al., et\u00a0al., et\u00a0al., et\u00a0al., et\u00a0al., et\u00a0al., et\u00a0al., et\u00a0al., et\u00a0al., 1996bet\u00a0al., et\u00a0al., Malignant GCTs rapidly became a highly curable disease, even when diagnosed at advanced clinical stages, with the advent of cisplatin-based chemotherapy in the 1970s located at the common region of 12p gain (12p13.31), suggesting that they are likely to be important in tumorigenesis. However, other non-seminomatous tumours, for example YSTs, which also have 12p gain, do not over-express these genes, suggesting other mechanisms must be important.As the only consistent structural chromosomal abnormality in malignant GCTs of adult patients, 12p gain is implicated in invasive malignant GCT development. The observation that the pre-invasive testicular lesion intratubular germ cell neoplasia unclassified demonstrates a similar pattern of overall genomic changes to those seen in invasive TGCTs, except for 12p gain, supports this theory and novel cancer genes in TGCT pathogenesis. Gene expression patterns in malignant GCTs characteristic of embryonic stem cells (ESCs) were confirmed and a distinctive transcriptomic programme was identified for individual histological subtypes , TFAP2C and UTF] and paediatric YSTs were associated with genes such as AFP, those involved in differentiation , lipid metabolism and proliferation pathways are not only involved in normal PGC development, resulting in oogenesis and spermatogenesis, but are also implicated in GCT development are associated with the development of bilateral GCTs in seminomas, resulting in phosphorylation of KIT and PI3K and therefore constitutive activation of the PI3K pathway, even in the absence of KITLG . Although MAPK1 is globally expressed in adult GCTs, mutations of KRAS and BRAF are rare events, pointing to the involvement of KIT as an upstream protein in activation of RAS/RAF signalling of target mRNAs, and whose expression profiles are dysregulated in cancer reported data from 48 paediatric samples, including gonadal and extragonadal malignant GCTs . Consistent with these observations, the miR-29 family is under-expressed in YSTs, when compared with seminomas and human choriogonadotrophin (HCG) assist malignant GCT diagnosis, they have limitations in sensitivity and specificity family of microRNAs, which regulate cell proliferation is not a feature of malignant GCTs (Palmer et\u00a0al., LIN28 over-expression (Murray et\u00a0al., LIN28/let-7 axis represent promising targets for novel therapies in malignant GCTs. As well as depleting LIN28, an alternative strategy is direct replacement of let-7 family members (Murray et\u00a0al., et\u00a0al., In addition, low LIN28/let-7 axis in all malignant GCTs also suggests a pathway that may be a target for the development of novel therapeutic agents.Our increasing knowledge of the biology of malignant GCTs needs to be harnessed to improve outcomes for both adult and paediatric patients. In particular, genomic and protein-coding transcriptomic data confirm that malignant GCTs of childhood are biologically distinct from those of adulthood and provide evidence supporting the different management approaches employed in patients of different ages. In contrast, all malignant GCTs over-express the miR-371\u2013373 and miR-302/367 clusters regardless of patient age, histological subtype or anatomical tumour site. The detection of elevated serum levels of these microRNAs at the time of malignant GCT diagnosis, with levels falling after treatment, highlights the universal diagnostic potential of this finding. In addition, the dysregulation of the Further studies are now required, integrating genomic changes using high-resolution methods with combined analysis of mRNA and microRNA expression profiles in malignant GCTs from both adult and paediatric patients with good and adverse clinical outcomes. These approaches are likely to identify regulatory pathways and networks associated with treatment sensitivity and resistance. In particular, such studies should aim to define further the clinical relevance of age-specific biological characteristics of malignant GCTs. These insights may assist the optimal management of affected patients and identify biological targets suitable for the development of novel therapeutic agents. The ultimate aim of this approach will be to improve clinical outcomes for this under-investigated patient group, both through improved survival of patients with poor-risk disease and reduced late-effects of treatment for those with low-risk disease."} +{"text": "Diabetes is a progressive metabolic disorder that can ultimately lead to serious chronic vascular complications including renal failure, vision loss, and cardiac dysfunction , a novel class of non-coding RNAs of 22~24 nucleotides in length, act as post-transcriptional regulators of gene expression by binding to the 3\u2032-untranslated region (3\u2032-UTR) of target mRNA that induces mRNA degradation and/or translational repression (Lim et al., Cardiomyocyte hypertrophy, myocardial fibrosis, and cardiomyocyte apoptosis are important features of diabetic cardiomyopathy (Ruiz and Chakrabarti, in vitro, indicating that overexpression of miR-223 might be a compensatory response to restore glucose metabolism in diabetic heart (Lu et al., Hyperglycemia, oxidative stress and mitochondrial damage are involved in the etiology of diabetes-induced cardiac dysfunction and diabetic cardiomyopathy (Shantikumar et al., Accumulating evidence has indicated that circulating miRNAs can be used as sensitive biomarkers for certain diseases such as cardiovascular diseases and cancers Fabbri, . DespiteTaken together, desregulated miRNAs are potentially involved in the etiology and pathogenetic processes of diabetic cardiomyopathy. An in-depth understanding of their functional roles and molecular mechanisms in the development of diabetic cardiomyopathy will provide better prospects to identify sensitive clinical biomarkers and novel therapeutic targets for diabetic cardiomyopathy.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Production of fuels and chemicals through a fermentation-based manufacturing process that uses renewable feedstock such as lignocellulosic biomass is a desirable alternative to petrochemicals. Although it is still in its infancy, synthetic biology offers great potential to overcome the challenges associated with lignocellulose conversion. In this review, we will summarize the identification and optimization of synthetic biological parts used to enhance the utilization of lignocellulose-derived sugars and to increase the biocatalyst tolerance for lignocellulose-derived fermentation inhibitors. We will also discuss the ongoing efforts and future applications of synthetic integrated biological systems used to improve lignocellulose conversion. One of the daunting challenges faced by the modern world is our unsustainable dependence on petroleum as the primary source for transportation fuels and many chemical products including solvents, fertilizers, pesticides, and plastics Service, . To fulfd-glucose linked by \u03b2-1,4 glycosidic bonds while a mixture of pentoses, especially d-xylose, and hexoses comprises the main component of hemicellulose (20\u201340% of biomass dry weight) represents arguably the most important renewable feedstock on the planet. Lignocellulose is a complex matrix of various polysaccharides, phenolic polymers, and proteins that are present in the cell walls of woody plants Saha, . Converst) Saha, . Lignin t) Saha, . Chemicat) Saha, . After pt) Saha, and therse Saha, . Althougse Saha, . Third, se Saha, . For exase Saha, , 2011. Ise Saha, . The conse Saha, . OverlimDespite government incentives and mandates, these grand challenges have prohibited the commercialization of lignocellulose conversion into fuels and chemicals at low cost , yield , and productivity (4\u2009g/L/h) in a complex medium and then converts pyruvate into ethanol and CO2 after extensive metabolic engineering and adaptive laboratory evolution and yield (0.48\u2009g/g xylose) using mineral salts medium in 16\u2009h to release catabolite repression; (2) overexpression of a glucose transporter from Z. mobilis to restore glucose transport and metabolism; (3) overexpression of genes rpiA, tktA, rpe, and talB to increase pentose phosphate pathway. Recently, a completely different approach to decrease glucose repression has been developed , organic acids, and phenolic compounds Saha, . FurfuraE. coli and S. cerevisiae conferred the tolerance to furan aldehydes including furfural and 5-HMF in E. coli (Miller et al., E. coli biocatalysts (Wang et al., A significant amount of effort has been contributed to the identification and optimization of biological components to increase the resistance to furan aldehydes, especially furfural Table . The toxS. cerevisiae gene disruption library was screened for mutants with growth deficiencies in the presence of furfural and ZWF1 was found to relate to furfural tolerance (Gorsich et al., ZWF1 increased furfural tolerance (Gorsich et al., ZWF1 encodes glucose-6-phosphate dehydrogenase, which catalyzes the first step of the pentose phosphate pathway, the major pathway providing NADPH when utilizing glucose as the carbon source. A similar approach using genome-wide RNAi screen showed that inactivation of the SIZ1 gene increased furfural tolerance (Xiao and Zhao, SIZ1 encodes E3 SUMO-protein ligase and inactivation of SIZ1 increases the tolerance to oxidative stress besides furfural (Xiao and Zhao, S. cerevisiae cells and to cause damage to mitochondria, vacuole membranes, and cytoskeletons (Allen et al., YAP1 or its target genes CTA1 and CTT1encoding catalases increased tolerance to furan aldehydes (Kim and Hahn, E. coli. The expression of the genes in major oxidative regulons such as OxyR and SoxRS regulons is not activated by the presence of furfural (Miller et al., E. coli, an oxidoreductase UcpA with an undefined function was found to be associated with furfural tolerance by a transcriptomic analysis and its overexpression increased furan aldehyde tolerance (Wang et al., E. coli on plates. Beneficial plasmids containing the thyA gene were recovered from all three genomic libraries. The thyA gene encodes thymidylate synthase, important for dTMP biosynthesis, suggesting furfural toxicity is possibly related to DNA damage (Zheng et al., E. coli mutants led to the discovery of some polyamine transporters including PotE, PuuP, PlaP, and PotABCD with a beneficial role for furfural tolerance (Geddes et al., E. coli (Glebes et al., lpcA, groESL, ahpC, yhiH, rna, and dicA genes are associated with furfural tolerance although the overexpression of these genes individually only showed limited positive effect (Glebes et al., E. coli the tolerance to furan aldehydes (Wang et al., A variety of genomic and transcriptomic approaches have yielded many beneficial genetic traits related to furan aldehydes tolerance Table . S. cerepntAB and the deletion of the yqhD gene together, made cells less tolerance to furfural than the cells with either one of these two beneficial genetic traits alone (Wang et al., There are at least two major challenges for designing such an integrated detoxification system. First, most epistatic interactions between beneficial genetic traits are not predictable and the experimental search for the optimal combination of multiple effector genes is time-consuming and labor-intensive (Sandoval et al., Efficient xylose metabolism and tolerance to furan aldehydes are desired features of microbial catalysts used in lignocellulose conversion. Past efforts of synthetic biology focused on identification and optimization of individual biological parts needed for a successful lignocellulose conversion. We have gradually accumulated much knowledge about xylose metabolism and transport, glucose repression, and furan aldehyde toxicity. Limited success of lignocellulose conversion has been achieved using these individual optimized parts (Sandoval et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Diabetes is a risk factor for Alzheimer disease (AD). Apolipoprotein E (ApoE) and several genes related to AD have recently been identified by genome-wide association studies (GWAS) as being closely linked to lipid metabolism. Lipid metabolism and glucose-energy metabolism are closely related. Here, we review the emerging evidence regarding the roles of lipid and glucose metabolism in the modulation of \u03b2-amyloid, tau, and neurodegeneration during the pathogenesis of AD. Disruption of homeostasis of lipid and glucose metabolism affects production and clearance of \u03b2-amyloid and tau phosphorylation, and induces neurodegeneration. A more integrated understanding of the interactions among lipid, glucose, and protein metabolism is required to elucidate the pathogenesis of AD and to develop next-generation therapeutic options. Familial AD is caused by mutations in the amyloid precursor protein (Goate et al., ApoE is an essential regulator of cholesterol metabolism and is the strongest genetic risk factor for AD Ashford, . The Apoin vitro experiment in which transient membrane cholesterol loading increased A\u03b242 secretion (Marquer et al., Moreover, independently of ApoE\u03b54, high levels of low-density lipoprotein cholesterol and low levels of high-density lipoprotein cholesterol are associated with higher amyloid-PET indices (Reed et al., In addition to the ApoE gene, recent GWAS studies have identified novel risk genes for AD (Hollingworth et al., in vivo and in vitro, statins reduced the A\u03b2 level in the brain (Fassbender et al., Although clinical studies have indicated that statins have no beneficial effect on cognitive function (McGuinness et al., Normal tau promotes the assembly and stabilization of microtubules. However, hyperphosphorylated tau sequesters normal tau and disrupts microtubules, forming NFT (Iqbal et al., As the largest pool of cholesterol resides in neuronal myelin membranes, disorders that impair sterol synthesis or intracellular trafficking of lipids in neurons cause hypomyelination and neurodegeneration (Saher and Stumpf, +-ob/ob mice, generated by crossing diabetic ob/ob mice, display increased A\u03b2 deposition in the cerebral vasculature (Takeda et al., Diabetes in midlife is associated with mild cognitive impairment (MCI; Roberts et al., db/db mice (Kim et al., in vitro and in animal models (Kickstein et al., Several neuropathological studies suggest that the magnitude of NFTs in the brain at autopsy is not different between AD patients with and without diabetes Kalaria, . HoweverDiabetes causes structural deficits in the brain (Sato and Morishita, Recent large, long-term, randomized controlled trials suggest that a multidisciplinary intervention, including exercise and diet, could improve or maintain cognitive function in at-risk elderly people (Ngandu et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Stem cell (SC) research holds the promise of controlling and/or curing a variety of diseases. SC applications include cell therapy, disease modeling, and developmental biology. This commentary focuses on an important study that examined Notch signaling in murine satellite muscle SCs in which Notch signaling is specifically blocked in the satellite SC using the dominant negative pan-Notch inhibitor, dnMaml1. The Pax7-Cre+/dnMaml1+ mutant mice showed myopathy manifested as muscle damage and weakness as early as 6 months of age and a shortened life span of about 8 months. The satellite SC pool was totally depleted in the mutant mice due to skewing of the balance between self-renewal and differentiation to differentiation rather than self-renewal. Further, the progeny of myoblasts showed reduced proliferation compared to controls. These experiments and others post cardiotoxin (CTX)-induced injury provided evidence that Notch pathway blockade leads to loss of quiescence and depletion of the satellite SC pool. These results are concordant with reports of other studies that blocked murine Notch pathway signaling by selectively inhibiting the recombining binding protein-Jj (RBP-Jj), a nuclear factor involved in the canonical Notch signaling pathway (Bjornson et al., To add further complexity, NOTCH family members have been shown to carry out different and sometimes opposing functions in the same tissue and/or cell type (Yin et al., NOTCH gene mutations have been found in approximately 15% of HNSCC (Gaykalova et al., NOTCH1 may act as a tumor suppressor gene in HNSCC (Pickering et al., In addition to its important role in muscle development, regeneration and repair, the NOTCH pathway is dysfunctional commonly in several developmental, vascular and cardiac diseases, including leukoencephalopathy and pulmonary arterial hypertension (Fouillade et al., Notch1 knockout mice (Nicolas et al., The Lin et al. paper isTargeted therapies against stemness regulatory pathways like NOTCH are promising in a variety of disorders. Owing to the high possibility of muscle side effects after long-term administration, we recommend following patients by physical exam for muscle wasting and avoiding concomitant use of steroids, which can lead to myopathies. The study by Lin and colleagues highlighThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Selective motor neuron degeneration is a hallmark of amyotrophic lateral sclerosis (ALS). Around 10% of all cases present as familial ALS (FALS), while sporadic ALS (SALS) accounts for the remaining 90%. Diverse genetic mutations leading to FALS have been identified, but the underlying causes of SALS remain largely unknown. Despite the heterogeneous and incompletely understood etiology, different types of ALS exhibit overlapping pathology and common phenotypes, including protein aggregation and mitochondrial deficiencies. Here, we review the current understanding of mechanisms leading to motor neuron degeneration in ALS as they pertain to disrupted cellular clearance pathways, ATP biogenesis, calcium buffering and mitochondrial dynamics. Through focusing on impaired autophagic and mitochondrial functions, we highlight how the convergence of diverse cellular processes and pathways contributes to common pathology in motor neuron degeneration. SOD1; Rosen, Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease characterized by loss of large motor neurons in the brain and spinal cord, resulting in progressive voluntary muscle wasting and respiratory failure. Patient death typically ensues 3\u20135 years following symptom onset . AMPK, activated by a drop in cellular energy, phosphorylates ULK1 on Serine 317 and Serine 777 , a well-established autophagosomal marker have emerged as the most common cause of both sporadic and familial ALS and Parkin. Mutations in The downstream events that link the targeting of depolarized mitochondria by PINK1-Parkin to the canonical autophagy core machinery are not yet entirely clear. Direct interaction of Parkin with Beclin1 represents one possible explanation to glycolysis and induction of mitophagy without compromising membrane polarization. Importantly, PINK1 stabilization is not required for this process; iron chelation-induced mitophagy is efficiently activated in fibroblasts deficient in Parkin mutations have been linked with ALS and other degenerative diseases, including dementia . In this process, electrons pass along a sequence of protein complexes (I-IV) located in the inner mitochondrial membrane Polymerase 1 (PARP1), thereby increasing its activity and potentiating PARP1-induced cell death (Joshi et al., Further evidence concerning additional roles for constituents of the autophagy machinery in traditional caspase-dependent pathways implicates even broader contributions to cell death. For example, cleavage of the autophagy-related gene Motor neuron cell death in both familial and sporadic forms of ALS is attributable to necroptosis (Re et al., SOD1 or VCP-associated inclusion body myopathy mutant models did not suggest a similar beneficial effect (Zhang et al., SOD1 mutant mice lacking mature lymphocytes, a modest survival increase was noted following rapamycin treatment (Staats et al., SOD1 mutant model (Castillo et al., ATG genes through an mTOR-independent pathway.That protein aggregates are characteristic of ALS and other neurodegenerative diseases suggests that the autophagy pathway may be an attractive therapeutic target for the prevention and treatment of neurodegeneration (Vidal et al., G2019SLRRK2 mutation implicates autophagic induction as an early event. Accordingly, RNAi-mediated knockdown of autophagy factors LC3 or ATG7 is sufficient to rescue neurite length (Plowey et al., Atg7-deficient mice that negatively regulating autophagy is sufficient to promote cell survival of hippocampal pyramidal neurons following ischemic insult (Koike et al., Although direct defects in autophagy genes may not be the causal link in all cases of ALS, dysfunction of autophagy exacerbates disease phenotypes (Bruijn et al., As increasing evidence suggests a prominent role for mitochondrial dysfunction and oxidative stress in motor neuron degeneration, enhancing mitochondrial health represents a promising strategy for treating ALS and related diseases. In line with this, long-term users of the antioxidant vitamin E show a decreased risk for ALS (Wang et al., Atg genes is sufficient to cause neurodegeneration in mice, suggesting a prominent role for autophagic clearance pathways in neurodegenerative disease (Hara et al., Protein aggregates are chief characteristics of many neurodegenerative diseases, including ALS (Blokhuis et al., BME, NM, and YCM conceived of, drafted, edited, and approved the corresponding review.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "The consequence is impaired utilisation of glucose in brain cells. Where are published papers denying the results of Edge et al. and Nyenwe et al.?The authors write \u201cEvidence that significant harmful effects are derived from metabolic acidosis by itself has not been provided in human beings and therefore the successful management of metabolic acidosis require the therapy of the underlying causative disorder.\u201d According to Edge et al. and NyenThe authors write \u201cBoth retrospective and prospective studies have consistently documented that sodium bicarbonate therapy does not improve metabolic responses, biochemical parameters, acid-base balance normalisation, or clinical outcomes among patients with DKA, either children or adults.\u201d In diabetic ketoacidosis, life-threatening is only its most severe stage, coma. If the facts presented in paragraph 1 are correct, thus increase of the very low blood-pH in the comatose patient after infusion of alkalising solution (such as sodium bicarbonate) should result in recovery to normal state of consciousness. And this has been reported in reality: lethality of coma in diabetic ketoacidosis is zero with treatment which includes infusions of alkalising solutions, for example, Umpierrez et al. . WithoutIn the paper of Adeva-Andany et al. there ar"} +{"text": "RNA-induced silencing complex (RISC), whose core component is one of the Argonaute family proteins. MiRNAs then direct RISCs to target mRNAs, which are recognized through partial sequence complementarity. Bioinformatic prediction and experimental target gene identification have shown that a miRNA binds mRNAs of hundreds of protein coding genes, which often span a broad spectrum of functional categories are endogenously encoded single-stranded RNAs of about 22 nucleotides (nts) in length that play essential roles in a large variety of physiological processes in animals and plants Ambros, . Mature Bartel, . The fun Bartel, . Subsequ5 copies of mature miRNAs in a cell (Calabrese et al., 4 copies per cell (Neilson et al., 4 copies per cell in hepatocytes (Chang et al., 4\u20131.7 \u00d7 105; Janas et al., in vivo mechanism of action of mammalian miRNAs remains to be a central question in the field of miRNA research.However, the model miRNA used in the aforementioned zebrafish study, miR-430, is unique in that its expression is rapidly induced and reaches millions of copies per cell in a few hours after fertilization. This expression level of miR-430 is at least 10 times more than all mature miRNAs combined in a mammalian cell, and serves the single purpose of degrading its target genes, maternal mRNAs, at the maternal-zygotic transition (Giraldez et al., In contrast to these desperate efforts to search for a unified model of miRNA mechanism of action, studies of individual functional targets in primary cells or tissues from miRNA mutant mice are painting a rather different picture. Depending on miRNAs, target genes, and cellular contexts, the outcome of miRNA-target mRNA interactions could be predominantly translation repression or mRNA degradation, or a mixture of both. This heterogeneity in miRNA mechanisms of action has been increasingly recognized as more and more miRNA mutant mice are generated and analyzed (Olive et al., cis-elements in mature miRNAs and target mRNAs determine the mechanism of miRNA action. Future investigation is warranted to identify these cis-elements, if they exist at all.Here we sought to summarize the relative contribution of translation repression and mRNA degradation to miRNA regulation of functional targets in miRNA mutant mice. We focused on miRNA target genes whose protein and mRNA levels were measured concurrently in primary cells or tissues from mutant mice with genetic ablation or transgenic expression of individual miRNA genes. This includes a total of 159 target genes from 77 miRNA mutant mice Table . Our anaFrom a practical standpoint, measuring target gene protein levels is preferred to mRNA levels for the purpose of studying the effect of a miRNA on its target genes. Even for target genes predominantly regulated by mRNA degradation, the miRNA effect can still be captured by measuring their protein abundance. In the same vein, translatome analysis is more appropriate for measuring the global effect of a miRNA on its target genes, while transcriptome analysis often failed to identify any significant effect of miRNA deletion on its target genes, despite the obvious functional consequences in mutant mice (Matkovich et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Citation: Cowie, S. M., P. Knippertz, and J. H. Marsham (2013), Are vegetation-related roughness changes the cause of the recent decrease in dust emission from the Sahel?, Geophys. Res. Lett., 40, 1868\u20131872, doi:10.1002/grl.50273[1] Since the 1980s, a dramatic downward trend in North African dustiness and transport to the tropical Atlantic Ocean has been observed by different data sets and methods. The precise causes of this trend have previously been difficult to understand, partly due to the sparse observational record. Here we show that a decrease in surface wind speeds associated with increased roughness due to more vegetation in the Sahel is the most likely cause of the observed drop in dust emission. Associated changes in turbulence and evapotranspiration, and changes in large-scale circulation, are secondary contributors. Past work has tried to explain negative correlations between North African dust and precipitation through impacts on emission thresholds due to changes in soil moisture and vegetation cover. The use of novel diagnostic tools applied here to long-term surface observations suggests that this is not the dominating effect. Our results are consistent with a recently observed global decrease in surface wind speed, known as \u201cstilling\u201d, and demonstrate the importance of representing vegetation-related roughness changes in models. They also offer a new mechanism of how land-use change and agriculture can impact the Sahelian climate. Goudie and Middleton, Engelstaedter and Washington, Zender and Kwon, Moulin and Chiapello, Prospero and Lamb, Evan et al., Olsson et al., Fensholt et al., Mahowald et al., Evan and Mukhopadhyay, Chiapello et al., Mahowald et al., [2] North Africa is the world's largest dust source A general challenge in investigating the causes of this dust trend is the sparse observational record from source regions. Satellite-based data sets are short and mainly provide aerosol optical thickness but not emission directly [Ackerman and Cox [FDE) as the fraction of all reports containing these ww codes. We purposely omitted the frequently reported ww code 6 to exclude transport events. The seven stations were selected on the basis of a minimum of 1000 dust observations overall during the time period 1984\u20132010 and at least 500 observations per year for each of the 27 years.[4] We use the seven Sahelian stations of Nouakchott , Nema (61497), Tombouctou (61223), Gao (61226), Niamey (61052), Agadez (61024), and Gour\u00e9 (61045) see . Wind sp and Cox ). We defu and v wind vectors from the European Centre for Medium-Range Weather Forecasts ERA-Interim reanalysis at a horizontal resolution of 80 km were used for the area inside the blue box shown in DUP) diagnostic parameter In addition, 6-hourly 10 m Klink [[6] An analysis of the possibility of artificial trends due to instrument changes (as in Klink ) suggestV) decrease from 4.6 ms\u20131 in the mid-1980s to 3.3 ms\u20131 in recent years , and FDE and DUP (0.93) reflect the strong control of wind speed on the occurrence of dust emission. Surprisingly, corresponding trends in regionally averaged mean wind and DUP computed from ERA-Interim reanalysis are substantially smaller (dashed lines in \u20131) and \u221214%, respectively.[7] e et al. , shows a[8] We have also analyzed the trends for: (a) day/night-time time data, (b) data at each major SYNOP hour , and (c) data in each season. In each case the results are robust , supporting the hypothesis that the observed trends are real and not an artefact of a change in sampling through the period.Helgren and Prospero, Foltz and McPhaden, Fensholt et al., v25 and v75, at which the probability of dust emission is 25% and 75%, respectively are in boreal winter , while no clear trends are found at higher latitudes (15\u00b0N\u201330\u00b0N), which are probably more strongly influenced by the very sparsely vegetated Sahara [Decadal Sahel dust trends analyzed with surface observations and new diagnosticsWind-speed changes dominate over soil changes in recent dust emission decreaseVegetation-induced roughness changes are the main control on wind-speed trends"} +{"text": "The disrupted in schizophrenia 1 (DISC1) gene is found at the breakpoint of an inherited chromosomal translocation, and segregates with major mental illnesses. Its potential role in central nervous system (CNS) malfunction has triggered intensive investigation of the biological roles played by DISC1, with the hope that this may shed new light on the pathobiology of psychiatric disease. Such work has ranged from investigations of animal behavior to detailed molecular-level analysis of the assemblies that DISC1 forms with other proteins. Here, we discuss the evidence for a role of DISC1 in synaptic function in the mammalian CNS. Within this family, segregation is observed between the translocation event and a spectrum of psychiatric disorders, including major depression, schizophrenia, and bipolar disease development in both humans and rodents, and gradually decreases during life fractions, and has been also shown to be present in dendritic spines by the use of ultrastructural methods reagents. A recent methodological approach that has been exploited successfully in studies of DISC1 biology is et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., DISC1 knockdown by in utero delivery of short hairpin RNA . The most prevalent observations involve modifications to neurogenesis, neuronal migration, and integration of neurons into the neuronal parenchyma of their final destination. The last of these results in subpopulations of slightly misplaced neurons and alterations in mature neuronal morphology, such as changes in process branching increases both the number of spines and their size, whereas continuing inhibition of DISC1 expression up to 6 days results in fewer and smaller spines is injected into the medial prefrontal cortex of young rats , and LTP was also small in both genotypes, probably because the experiments were not performed with GABA receptor antagonists, which is the normal methodology employed for studying perforant pathway LTP in brain slices.Comparatively high levels of DISC1 are expressed both in the dentate gyrus and in its major output region, the hippocampal CA3 subfield. Manipulation of DISC1 is known to produce effects on adult neurogenesis and wiring in this brain area enhancement of mossy fiber paired-pulse facilitation at all interstimulus intervals examined, but no change in 1-Hz frequency facilitation studied with 20 consecutive stimuli . Kvajo et al. , is probably the most widely studied single pathway in the mammalian brain. Synaptic transmission in this pathway was investigated in mice engineered to express a C-terminal fragment of DISC1 in the forebrain in an inducible and reversible fashion. These animals, which showed a number of behavioral changes when expression of the DISC1 fragment was induced, also had a depressed input\u2013output relationship in the SCCP at the age of 3\u20134 months. However, no changes in either short-term plasticity or LTP were found . In the DISC1tr cohort, neither pathway showed changes in input\u2013output relationships or short-term synaptic dynamics. Also, the ratio of N-methyl-d-aspartate (NMDA) receptor-mediated and AMPA receptor-mediated components of the synaptic response was not genotype-dependent. Interestingly, however, we found a clear enhancement of theta burst-induced LTP in the SCCP of DISC1tr mice, but a complete loss of LTP in the temporoamonic pathway. The mechanisms underpinning these pathway-specific effects of the truncated form of DISC1 on long-term synaptic plasticity require further investigation. Our article also describes our single-cell recordings used to compare a broad range of intrinsic excitability properties in CA1 pyramidal cells from 4-month-old DISC1tr mice and wild-type littermates. These identified no substantial dependence on genotype.The SCCP has also been studied in DISC1et al., DISC1. These experiments suggested that reducing the level of DISC1 by \u02dc60% may serve to increase the duration of action potential bursts, although whether this reflects an intrinsic or synaptic change is not apparent from this type of data.One report concerning DISC1 function in hippocampal neurons describes the use of mixed hippocampal cultures prepared from embryonic day 17.5 mouse embryos and grown on multielectrode arrays by \u02dc30%, probably reflecting the increased number of spines present under these conditions. Cortical cultures have also been employed to demonstrate that knockdown of DISC1 with siRNA increases NMDA receptor-mediated currents in response to exogenous agonist application .Other neurophysiological studies in the cortex have also employed brain slices. One study examined slices from two different mouse models \u2013 the first was the DISC1, et al. . In this et al., , spontanin utero knockdown of DISC1 (Niwa et al., in utero electroporation, have unaltered membrane resistance or membrane potential. However, dopamine D2ergic modulation of electrically evoked excitatory postsynaptic potentials in these deep layer neurons is strongly attenuated when DISC1 is knocked down. This observation is thought to be related to the disturbed dopaminergic innervation of this cortical area produced by the knockdown of DISC1 (Niwa et al., Acutely prepared slices of mouse prefrontal cortex have also been used to study the neurophysiological consequences of et al., et al., et al., et al., et al., et al., et al., et al., DISC1 levels are unquestionably high in postsynaptic elements. Additionally, a number of DISC1 binding partners also show prominent postsynaptic localisation and possess known postsynaptic roles. Consequently, investigations of DISC1 neurophysiology at synapses have tended to concentrate on postsynaptic functionality. However, DISC1 expression appears also to be a feature of some axons and presynaptic terminals (Kirkpatrick in utero electroporation to introduce Cre-dependent inducible expression vectors for full-length DISC1 or a C-terminal truncation of DISC1 or DISC1 RNAi reagents, in addition to channelrhodopsin2. This permitted both expression of both DISC1-related contructs and light-mediated activation to be confined entirely to presynaptic elements. Recording from layer 2/3 cells, the authors found that spontaneous EPSC frequencies were enhanced by a truncated form of DISC1 (Maher & LoTurco, et al. (DISC1 knockdown lowers the probability of release. In future, it will be interesting to determine which proteins DISC1 is interacting with to produce these actions in presynaptic elements. Possibly related to these functional observations of altered presynaptic function is the observation that full-length DISC1 promotes, and C-terminally truncated DISC1 retards, transport along neuronal processes of vesicles containing a synaptic vesicle protein target (Flores et al., A recent elegant study using optogenetic methodologies has provided more direct evidence for presynaptic functions of DISC1 in the neocortex of Wistar rats (Maher & LoTurco, , et al. in transin vivo studies in appropriate models. Such work is currently ongoing in our group and presumably elsewhere as well, so we should expect to see data on in vivo DISC1-related neurophysiology in the near future.In conclusion, neurophysiological investigation of the neurobiological functions of DISC1 is still in its infancy. We believe that further analyses using neurophysiological measures will provide important insights into the biology of this protein and, consequently, pathways with important roles in psychiatric disease. The data produced to date indicate that alterations to DISC1, such as those that elicit psychiatric disease, can affect multiple processes. As described above, there is evidence for activities on both the presynaptic and postsynaptic sides of the fast chemical synapse, and for interactions with processes controlling long-term synaptic plasticity. The first hints of how these might impact on the behavior of intact complex networks are appearing, although there is a clear need for suitable"} +{"text": "Drosophila and vertebrate systems has implicated a family of proteins, the Teneurins, as a new transsynaptic signal in both the peripheral and central nervous systems. The Teneurins have established roles in neuronal wiring, but studies now show their involvement in regulating synaptic connections between neurons and bridging the synaptic membrane and the cytoskeleton. This review will examine the Teneurins as synaptic cell adhesion molecules, explore how they regulate synaptic organization, and consider how some consequences of human Teneurin mutations may have synaptopathic origins.To achieve proper synaptic development and function, coordinated signals must pass between the pre- and postsynaptic membranes. Such transsynaptic signals can be comprised of receptors and secreted ligands, membrane associated receptors, and also pairs of synaptic cell adhesion molecules. A critical open question bridging neuroscience, developmental biology, and cell biology involves identifying those signals and elucidating how they function. Recent work in The developing neuron has a myriad of tasks to complete along its path to become part of a functioning brain network. The final goal is to form a reliable synaptic connection with its defined partner. While synapse formation has been intensively studied because of its expression in specific stripes of the fly embryo has been the most frequently studied synapse in Drosophila, Teneurins have two distinct expression levels. They are highly expressed at connections between select pairs of pre- and postsynaptic partners (Hong et al., all neuromuscular and olfactory connections, a lower, basal level of expression exists, suggesting a more general role. Here, the interaction is heterophilic between presynaptic Ten-a and postsynaptic Ten-m. Perturbation of either component of this basal level at the NMJ causes a myriad of phenotypes including fewer synaptic boutons, failed active zone apposition, disorganization of synaptic proteins, failed pre- and postsynaptic differentiation, and reduced function (Mosca et al., In Drosophila use Teneurins to ensure proper partner matching (Hong et al., Both the olfactory system and the NMJ in These findings revealed a number of facets about Teneurin biology, synaptic regulation, and the logic underlying neuronal development. First, it identified the Teneurins as novel, critical components of the transsynaptic cadre of signals. Ten-a and Ten-m regulate cytoskeletal organization and cooperate with known transsynaptic signals like Neurexin/Neuroligin (Mosca et al., 2+ signaling (Silva et al., Recent evidence further suggests synaptic roles for Teneurins in diverse vertebrate systems. An unbiased proteomics screen (Silva et al., in vivo (O\u2019Sullivan et al., teneurin-3 knockdown impairs the morphology and connectivity of retinal ganglion cells (Antinucci et al., teneurin-3 knockdown, the role of Teneurins at vertebrate synapses remains an exciting question of great importance for future work.Latrophilins regulate excitatory synapse development and strength The Teneurins are one of a critical suite of cell surface molecules for synapse formation, organization, and function. These include cell adhesion molecules, ligand-receptor complexes, and secreted factors (Johnson-Venkatesh and Umemori, The Teneurins are synaptic cell surface molecules with such diverse functions. They are synaptic organizers (Scheiffele et al., Drosophila has compared Teneurin and Neuroligin1 perturbations (Mosca et al., Beyond synaptic induction, the Teneurins also ensure an ordered cytoskeleton, a role which may be more unique to this family. While Nectins can organize actin (Mori et al., Drosophila, only two Teneurins exist, but regulate all of these events (Baumgartner et al., Drosophila NMJ, where Teneurins have been most mechanistically studied, but further consider translation. While the more complex vertebrate nervous systems may have evolved additional mechanisms for controlling synaptic organization absent in the fly, it is important to note the CNS and PNS conservation of Teneurins in the Drosophila (Mosca et al., A critical question is how the Teneurins regulate such diverse processes as neuronal wiring, synapse organization, morphogenesis, and patterning. In At the NMJ, the Teneurins function to organize the cytoskeleton and ensure properly apposed active zones. Muscle Ten-m interacts in a complex with \u03b1-spectrin (Mosca et al., While spectrin is also presynaptic, the microtubule cytoskeleton instead is the predominant player in ensuring proper NMJ morphology and function (Hummel et al., neuroligin and ten-a mutants synergize, suggesting an alternative mechanism. Indeed, ultrastructural defects like detached and misshapen active zones following Teneurin perturbation (Mosca et al., Beyond the cytoskeleton, the Teneurins have an independent role in regulating active zone apposition and structure. Teneurin perturbation causes failures (Mosca et al., C. elegans, the Teneurin ten-1 interacts with the Integrin and Dystroglycan homologs ina-1 and dgn-1 and the prolyl 4-hydroxylase phy-1 to regulate collagen IV and maintain basement membranes during embryonic development (Trzebiatowska et al., ten-1 phy-1 double mutants, embryos display gross defects in epidermal development, body wall musculature, and enhanced lethality. These phenotypes also synergize with mutations in collagen IV, leading to a model whereby epidermal TEN-1 binds collagen IV in the basement membrane. In the absence of phy-1, collagen IV is improperly processed and fails to be secreted into the basement membrane to bind TEN-1, weakening the muscle structure. Similar defects and interactions also occur with Integrin and Dystroglycan mutations (Trzebiatowska et al., Teneurins can interact heterophilically with other Teneurins (Oohashi et al., Drosophila, synaptic Integrins and Dystroglycan regulate NMJ development (Hoang and Chiba, pgant3 and pgant35A, two protein alpha-N-acetylgalactosaminyltransferases that regulate integrins. Mutations in these genes control the levels of the synaptic integrin receptor \u03b1PS2 and Ten-m (Dani et al., pgant. Further supporting this interplay is evidence that Ten-m and \u03b1PS2 directly interact (Graner et al., trans (Graner et al., In Drosophila, Teneurins mediate partner matching and synaptic organization at the same synapses (Hong et al., In in vivo will be able to offer important clarity on these differences are achieved.An intriguing possibility may lie in the Teneurins themselves. If their intrinsic structural properties could distinguish homo- and heterophilic interactions, this would enable both signaling modes with the fewest restrictions Figure . BiophysIn recent years, the synaptic basis of neurological disorders has been more concretely appreciated (Thompson and Luscher, How may the Teneurins be synaptopathic? Teneurin mutations have been implicated in a number of intellectual disabilities. Large regions of the human X and 5th chromosomes containing Teneurins-1 and -2, respectively, are linked to mental retardation (Tucker and Chiquet-Ehrismann, A tantalizing link between Teneurins and synaptopathies rests with bipolar disorder. Genome-wide association studies linked Teneurin-4 mutations to enhanced susceptibility to bipolar disorder (Psychiatric GWAS Consortium Bipolar Disorder Working Group, Drosophila, they order the underlying synaptic cytoskeleton and ensure proper synaptic function, differentiation, and morphology. Understanding their complete synaptic role, however, is in its infancy, and remains an exciting area for future study. Further work is needed to decipher the mechanisms of synaptic Teneurin function, leading to how they affect human brain function. Indeed, while human nervous systems have more complex circuit regulatory requirements than invertebrates, the marked conservation in worm, fly, and mouse models for Teneurin function suggests that their diverse roles may rely on similar core mechanisms. It will be critical to determine how these synaptic roles differ from those of partner matching. Understanding their downstream effectors and how homo- and heterophilic Teneurin signals are distinguished in partner matching vs. synaptic organization will achieve this goal. Linking these mechanistic studies to those of patients with Teneurin mutations will further enhance our understanding of how these proteins function. As future work addresses these questions, the Teneurins may take their place alongside known synaptopathic and ASD genes like Neurexin and Neuroligin as critical synaptic determinants, highlighting their importance in producing a functioning, organized brain.The Teneurins have emerged as transsynaptic, cell surface molecules essential for synaptic organization. In The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "The effective management of cardiac arrhythmias, either of atrial or of ventricular origin, remains a major challenge. Sudden cardiac death due to ventricular tachyarrhythmias remains the leading cause of death in industrialized countries and a large capacity (mc) per unit area.\u201d This \u201ccable model\u201d considers that the intracellular and extracellular potentials vary along the longitudinal axis only, and that both the cytoplasm and the extracellular spaces can be approximated as ideal ohmic conductors (with ir and er respective resistances per unit length). Hence, propagating cardiac action potentials along a fiber can be described by the following second-order partial differential equation (PDE):ionicI is the nonlinear membrane ionic current density (\u03bcA/cm2), defined by the active/stochastic electrical properties of the cell. Alternatively, multiplying by mr, Equation (1) can be re-written asmcmr is the trans-membrane time-constant (e.g., Plonsey, Engelmann was perhUsing this theoretical framework, Weidmann demonstrMyocardial infarction and/or acute ischemia provoke profound changes in the passive electrical properties of cardiac muscle (De Groot and Coronel, in vivo. Therefore, del Rio et al. (The role that changes in intrinsic properties of pacemaker cells (Yaniv et al., o et al. studied The authors hope that this monograph will provide a better appreciation of the crucial role that myocardial passive electrical properties play in not only the maintenance of a normal cardiac rhythm but also how changes in these parameters can trigger atrial and ventricular arrhythmias. The application of this knowledge should facilitate the development of more effective anti-arrhythmic therapies.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Tests al., 2015). This tal., 2015). In thial., 2015). An intin vitro systems for hepatotoxicity, because they may improve the throughput and avoid difficulties due to interspecies extrapolation, because human cells can be used . Howeveeif, 2014; Ghallabeif, 2014, 2014; Cam; Camin val., 2015; Driesseal., 2015; Tolosa al., 2015; Grinberal., 2015). Other"} +{"text": "The 13th International Conference on Bioinformatics (InCoB2014) was held for the first time in Australia, at Sydney, July 31-2 August, 2014. InCoB is the annual scientific gathering of the Asia-Pacific Bioinformatics Network (APBioNet), hosted since 2002 in the Asia-Pacific region. Of 106 full papers submitted to the BMC track of InCoB2014, 50 (47.2%) were accepted in BMC Bioinformatics, BMC Genomics and BMC Systems Biology supplements, with three papers in a new BMC Medical Genomics supplement. While the majority of presenters and authors were from Asia and Australia, the increasing number of US and European conference attendees augurs well for the international flavour of InCoB. Next year's InCoB will be held jointly with the Genome Informatics Workshop (GIW), September 9-11, 2015 in Tokyo, Japan, with a view to integrate bioinformatics communities in the region. The 13th InCoB , an official conference of the Asia-Pacific Bioinformatics Network (APBioNet) [We offered authors four tracks to submit manuscripts for potential publication in the supplement issues of BMC Bioinformatics, BMC Systems Biology or BMC Genomics (BMC track) and PeerJ . Of the BMC Medical Genomics [BMC Bioinformatics supplement [BMC Systems Biology supplement [The 20 articles in this supplement cover mainly \"genomic\" topics, with three medically-oriented papers going into Genomics for the pplement comprisepplement . Five mopplement .et al.[de novo assembly software for fungal genome data. Proteogenomics is increasingly used for the accurate annotation of protein coding regions using proteomic data. As currently available proteogenomic tools are tailored specifically for human and eukaryotic data, Uszkoreit et al.[With genome sequencing technologies becoming more and more accessible and affordable, the genetic origin of the Marwari horse was established by whole genome sequencing while thet al. have assit et al. have devet al.[et al.[et al.[et al.[et al.[Seven papers are devoted to transcriptome analysis addressing a range of challenges from epigenetics ,14 to unet al. have idel.[et al. have pinl.[et al. have idel.[et al. have idel.[et al. have appet al.[Guanine-rich nucleotide sequences form four-stranded G-quadruplex structures. Yano and Kato have useet al. have devet al. have devet al.[In the era of genomic medicine, it is possible that some approved drug molecules can be used for diseases other than those they were originally approved for. Yang and co-workers propose et al. have optet al.[et al.[Taguchi and co-workers have ideet al.. Xu et al.[et al. have devet al.[Papers specifically relating on genome-scale analysis with a disease focus are presented in a new supplement in BMC Medical Genomics and a brief overview of these articles is presented here. With pandemic viral infections spreading rapidly by jet travel, understanding cross-species transmissibility of vectors is addressed by Tan and co-workers for inflet al. have anaWith the growth in regional bioinformatics meetings, including ISCB-Asia meetings and the 2013 IEEE International Conference on Bioinformatics and Biomedicine (BIBM) in China , there iThe authors declare that they have no competing interests.SR wrote the introduction. CS and SR (Program Committee Co-chairs) managed the review and editorial processes, respectively. TWT supported the post-acceptance manuscript processing.List of Program Committee Members and Additional Reviewers in Alphabetical Order.Click here for file"} +{"text": "Picornaviridae and genus Hepatovirus, which is a sole member of this family. HAV has radically different properties from those of other picornaviruses. First, HAV is extremely resistant to degradation caused by environmental conditions, which include thermal denaturation , acid treatment , 20% ether and chloroform, and detergent inactivation . Second, the HAV has a highly de-optimized codon usage and grows slowly in a tissue culture. Third, VP1-2A or PX, which is a 67-residue C-terminal extension of VP1, has been implicated in the particle assembly (Graff et al., Hepatitis A, which is a major global health problem (Nainan et al., Picornavirus family. VP4, which is a small viral protein, was first detected in HAV mature virions. The external surface of HAV exhibits fewer features than those observed in enteroviruses and cardioviruses (Wang et al., Recently, structures of a mature virion and an empty capsid assembly intermediate of HAV were determined by X-ray crystallography (Wang et al., Although the structure of HAV presents significantly different properties compared with other previously characterized viruses, reveals the phylogenic relationship between typical picornaviruses and insect viruses and also indicates a novel uncoating mechanism, several important biological parameters, such as the entry of HAV and release of an RNA genome, still need to be clarified."} +{"text": "International Journal of Molecular Sciences [The author would like to insert the citation after the following sentence, \u201cThese RBPs are detected on laser trackswithin one minute after laser irradiation, and are excluded from the laser tracks shortly [Sciences .et al. [R-loops), they presented several data supporting that the exclusion of these factors is part of a DDR Pi3K-like kinases-dependent mechanism operating at DNA damage sites to prevent R-loops accumulation.The author wants to stress that an important reference has been inadvertently omitted in the appended review. Britton et al. recently"} +{"text": "In a recent review paper on theoretical explanations of affective stimulus-response compatibility (aSRC) effects between positive/negative stimuli and approach/avoidance-related movements, Kozlik et al. ; KNL argKrieglmeyer et al. report a2. We agree that smiling and frowning are linked to positive and negative affects, respectively, more rigidly than non-facial actions commonly are. We also agree that aSRC effects for facial responses are less affected by instructions of arbitrary action goals result in greater left frontal activation, while other smiles do not to AE.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "The use of synthetic non-coding RNAs for post-transcriptional regulation of gene expression has not only become a standard laboratory tool for gene functional studies but it has also opened up new perspectives in the design of new and potentially promising therapeutic strategies. Bioinformatics has provided researchers with a variety of tools for the design, the analysis, and the evaluation of RNAi agents such as small-interfering RNA (siRNA), short-hairpin RNA (shRNA), artificial microRNA (a-miR), and microRNA sponges. More recently, a new system for genome engineering based on the bacterial CRISPR-Cas9 system , was shown to have the potential to also regulate gene expression at both transcriptional and post-transcriptional level in a more specific way. In this mini review, we present RNAi and CRISPRi design principles and discuss the advantages and limitations of the current design approaches. Natural regulatory RNAs are a heterogenous group of endogenous non-coding RNAs that modulate biological processes at many levels through different mechanisms. They have inspired the design of synthetic RNA molecules, such as riboswitches, sensors, and controllers, as key elements for programing cellular behaviors, as well as antisense-based approaches for specific gene expression regulation, which is the focus of this mini-review was discovered in 1998, when Andrew Fire and Craig C. Mello reported the capability of exogenous double-stranded RNAs (dsRNA) to silence genes in a specific manner in miRNAs are small endogenous non-coding RNAs, typically 18\u201322\u2009bp long, which derive from longer hairpin-shaped precursors called pre-miRNA Bartel, . A pre-msiRNAs are mostly exogenous dsRNA molecules derived from viral RNAs or artificially introduced into the cell has been recently proposed . Sequence rules concern the position of the binding site in the target transcript and its nucleotide composition. The target region should be chosen preferably 50-100\u2009nt downstream of the start codon and should avoid the middle of the coding sequence , which possesses the highest affinity toward complementary RNA, Bifunctional oligodeoxynucleotide/antagomiR constructs, which ensure high transfection efficiency and prevention of unintended immune stimulation, and morpholino oligomers, which have been shown to be efficient inhibitors of both pri-miRNA and mature miRNA activity in zebrafish and Xenopus laevis are efficient sponges with structurally accessible and indigestible miRNA binding sites Figure H Haragu, 2012. TAn exciting and promising advance in the field of artificial gene regulation comes from Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR). CRISPR is a natural adaptive immune system used by archaea and bacteria against phage and plasmids in order to perform genome editing in eukaryotes mechanisms, thus this system can be used to either disrupt or edit a gene . Many tools are currently available online for the design of sgRNAs Table .Besides genome-editing applications, the CRISPR/Cas9 system can be employed for gene expression regulation. The system, known as CRISPR interference (CRISPRi), is based on a catalytically dead Cas9 (dCas9) lacking endonuclease activity co-expressed with a sgRNA (Gilbert et al., A \u201cseed\u201d region has been identified as the 12nt region adjacent to the PAM site. Mismatches in the seed region can dramatically reduce the repression, while mismatches in the non-seed area can cause a mild effect. Design guidelines recommend using a length of 20\u201325\u2009nt as the base-pairing region of the sgRNA (Larson et al., Gilbert et al. also introduced the sunCas9 CRISPRa system, in which expression of a single sgRNA with one binding site is sufficient to turn on genes that are poorly expressed or that increase the expression of well-expressed genes (Gilbert et al., in vivo by site-specific genome engineering (Bassett et al., CRISPRi can also be successfully employed to knock out miRNAs, by using a sgRNA/Cas9 complex targeting the pre-miRNA sequence (Zhao et al., Finally, although current tools for CRISPRi are based on the DNA targeting approach described above, the discovery of other Cas proteins targeting RNA molecules, such as Cmr, suggests an alternative post-transcriptional methodology similar to RNAi (Hale et al., Both RNAi and CRISPRi represent valid approaches for artificial gene regulation and both can suffer from significant side effects which may result from factors beyond sequence match. One clear advantage of CRISPRi over RNAi is that being an exogenous system it does not compete with the endogenous machinery of miRNA processing. Nevertheless, both techniques require more work in terms of enhancing targeting efficiency and reducing side effects.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.http://www.frontiersin.org/Journal/10.3389/fbioe.2014.00065/abstractThe Supplementary Material for this article can be found online at Click here for additional data file."} +{"text": "PLOS Genetics, Head et al. with caffeine; e.g., increases in citrate synthase and cytochrome c oxidase mRNA were observed 24 hours after caffeine exposure during contractions and increased fatigue resistance [2+] and muscle weakness [From an evolutionary perspective, the SR Caweakness , in muscweakness , and in weakness . In thissistance in non-sweakness .ACTN3 577xx allele promote increased energy expenditure and improved mitochondrial function without requiring an increase in physical activity? Perhaps treatments to induce a controlled SR Ca2+ leak provide such an opportunity, but then the risk of causing impaired muscle function due to excessive Ca2+ leakage has to be overcome.Human evolution and athletic performance are fascinating, but the findings of Head et al. provide additional avenues for future studies with important implications for human health, since the benefits of improved mitochondrial function span far beyond increased exercise capacity. Obesity and the metabolic syndrome are associated with impaired mitochondrial function, and of course, constitute a widespread and rapidly increasing health problem. Could strategies that phenocopy the effects of the"} +{"text": "In response: We agree with Berry et al. (Bulinus snails, which can serve as intermediate hosts for Schistosoma haematobium, were found during a malacological survey in 2014 (As stated in our manuscript, we cannot exclude the possibility that our case definition generated false-positives; the potential limitations of our findings have already been discussed ("} +{"text": "We discuss Wittmann et al. \u201cEffectsThe brain integrates partial sensory input with internal representations to construct the elaborate story we know as time Hammond, . Our ord2A receptors in internal clock models (ICMs) in duration discrimination and temporal control of motor performance. The study revealed a decreased ability to accurately produce intervals longer than 3 s and synchronize finger-tapping to auditory beats separated by more than 2 s. This suggests that effects of psilocybin on temporal processing are specific to relatively long durations, attributable to memory, and decision-making components of the ICM (Gibbon et al., Serotonergic hallucinogens generally slow the perceived flow of time Shanon, . PharmacComparable results are observed in Wackermann et al. , assessiTemporal processing of longer durations is impaired in people with schizophrenia Fuchs, . However2A receptor agonists may induce clinical symptoms of schizophrenia such as hallucinations, delusion, psychomotor poverty, and distorted perception (Teixeira et al., 2AR activation have shown to assist NMDAR-dependent memory mechanisms (Zhang and Stackman, 2A R occupancy in the prefrontal cortex of schizophrenic patients (Zhang and Stackman, Psychopharmacological research suggests that drugs such as psilocybin may serve as useful tools for understanding temporal serotonergic signaling mechanisms underlying psychosis, due to their capacity to cause distorted perception in normal subjects (Rammsayer, 2A receptor antagonist ketanserin.Impairments in working memory, selective attention, and executive control, as seen in schizophrenia, lead to distorted sequencing and integration of past, present, and future into a personal narrative. Carter et al. demonstr2A receptor activity is associated with time distortion in both psychiatric disorders and hallucinogenic experiences. Manipulating antagonists/agonists provides an approach to utilizing psychoactive drugs as tools in research for understanding time perception in the ordinary brain. It would be fruitful to compare healthy subjects under the influence of psilocybin with patients with acute schizophrenia, utilizing a common paradigm as in Wittmann et al. (5-HTn et al. . Howevern et al. excludesn et al. . An fMRIn et al. . Negativn et al. .Neuroimaging techniques combined with psychophysical tests of time perception (for a review see Grondin, A2 receptor activities. Commonalities across pharmacological treatments and neurological disorders should be explored within a common experimental paradigm to better understand neurochemical processes mediating temporal processing in ordinary states.Slowing of perceived time is induced by psilocybin and schizophrenia; having a common basis in 5-HTKS obtained comprehensive research and drafted original article, with collaboration of JB's efforts. JB provided substantial advisory of research materials and writing processes. Both authors edited and revised the article, insured the integrity of the product, and agreed on the final version for submission.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "The design of biomaterials and the sourcing for appropriate cells are two integrated aspects of tissue engineering to construct a tissue implant for clinical applications. During the past decades, many innovative biomaterials with desirable biological and mechanical properties have emerged, while stem cells have been shown to be a promising cell source to differentiate into many cell types. However, the testing of these bioartificial tissue constructs in the clinical trials is far from satisfactory. How microenvironments in the biomaterials regulate cellular signaling pathways and functions, how stem cell-derived target cells respond to extracellular cues presented by the biomaterials, and how implanted tissue constructs interact with host tissues remain to be investigated. The information of the interplay between cell and biomaterials would be helpful to guide us in improving our current strategy to refine the tissue constructs for effective tissue repair in regenerative medicine. in vivo and in vitro studies. They suggest that well-designed randomized animal trials are needed before moving into clinical trials. Then we introduce three types of innovative biomaterials: (1) bionanocomposites based on bacterial cellulose and magnetic nanoparticles (magnetite) for efficient chronic wounds healing reported by B. Galateanu et al.; (2) the elastomeric poly(\u03b5-caprolatone urethane) (PCLU) scaffolds using a high internal phase emulsion for bone tissue regeneration developed by S. Changotade et al.; and (3) a novel gelatin-alginate-polyacrylamide 3D interpenetrating network with superior performance in promoting chondrogenesis using human adipose-derived stem cells designed by S. Dinescu et al. Most importantly, our special focus will be given to the insight studies on the cell-biomaterial interactions. C.-H. Wang et al. demonstrate that the migration ability of bone marrow stem cells can be regulated by varying the porous structure of the artificial ligaments and this regulation is related to the RhoA/ROCK signaling pathway. M. Deng et al. demonstrate the endothelial differentiation of human adipose-derived stem cells on the polyglycolic acid/polylactic acid (PGA/PLA) mesh and propose the synergistic effect of 3D environments and biochemical signals such as growth factors on the acquisition of mature characteristic endothelial phenotype. S. Fu et al. have investigated the protective effect of neuropeptide Substance P on bone marrow stromal cells against apoptosis induced by serum deprivation through Wnt signaling. J. Michel et al. have reviewed a wide range of hydroxyapatite containing scaffolds and their interactions with mesenchymal stem cells in in vitro and in vivo contexts.In this special issue, we first present a thorough review by M. Ramamoorthi et al. on the efficacy of dental stem cell therapy in bone regeneration in preclinical"} +{"text": "Neovascular ocular disorders such as diabetic retinopathy and neovascular age-related macular degeneration (neovascular AMD) are an important cause of blindness in the world . In thesA flow cytometric analysis of vitreous inflammatory cells in patients with proliferative diabetic retinopathy.\u201d Histological and flow cytometric analyses have recently demonstrated the importance of histiocytes/macrophages and T lymphocytes in the development and activation of this disease, but no prediction on the visual prognosis was made. Moreover, higher CD4/CD8 ratio in the vitreous of patients with PDR compared to that in their blood was consistent with local inflammatory response in the disease.Inflammation has an important role in the development of proliferative diabetic retinopathy (PDR) as described in the paper by M. Urban\u010di\u010d et al., 2013, \u201c ex vivo cultured cells growing out of human fibrovascular epiretinal membranes (fvERMs) from PDR can give insight into their function in immunity, angiogenesis, and retinal detachment, as described in the paper by Z. Ver\u00e9b et al., 2013, \u201cFunctional and molecular characterization of ex vivo cultured epiretinal membrane cells from human proliferative diabetic retinopathy.\u201d Several surface markers such as hematopoietic and mesenchymal stem cell markers have recently been reported in fvERMs from patients with PDR. Additionally, secretion of different angiogenesis-related factors was demonstrated in cells growing out of the fvERMs.Characterization of the cell surface marker phenotype of \u201cCandidate genes for proliferative diabetic retinopathy.\u201d Several pathogenetic mechanisms have been implicated in the development of PDR such as alteration in retinal blood flow, hemostatic abnormalities, metabolic changes, increased oxidative stress, increased polyol and hexosamine pathway flux, activation of protein kinase C isoforms, and increased advanced glycation end-product formation, growth factors, and so forth . The role of epigenetics in diabetic retinopathy is now an emerging area as described in \u201cEpigenetic modifications and diabetic retinopathy\u201d by R. A. Kowluru et al., 2013. It is well known that diabetic environment facilitates epigenetics modifications, which can alter the gene expression without permanent changes in DNA sequence. It has been shown recently that genes encoding mitochondrial superoxide dismutase and matrix metalloproteinase-9 are epigenetically modified, and, by activation of epigenetically modified enzymes, DNA methyltransferases are increased and micro-RNAs responsible for regulating nuclear transcriptional factor and growth factors are upregulated.Epigenetic mechanisms are expected to be involved in the pathogenesis of PDR as well. Gene polymorphisms and epigenetic mechanisms responsible for PDR are reviewed in this paper in the angiogenic cascades to developing new therapies for retinal vascular diseases. Anti-VEGF agents such as ranibizumab and aflibercept have become increasingly well-established therapies as shown in the paper by M. Hanout et al., 2013,Does the adult human ciliary body epithelium contain \u201ctrue\u201d retinal stem cells?\u201d). However, until fully accepted as an important treatment modality in different eye disorders, their usefulness must be confirmed in well-designed clinical trials.Stem cell therapy is a promising approach in different diseases, including those causing blindness. Recent reports of retinal stem cells being present in several locations of the adult eye have sparked great hopes that they may be used to treat the millions of people worldwide who suffer from blindness as a result of retinal disease or injury . A populDaniel Petrovi\u010dQuan Dong NguyenBorut PeterlinGoran Petrovski"} +{"text": "Cryptococcus neoformans (Cn) has become one of the leading causes of mortality in AIDS patients. Understanding the interactions between Cn and phagocytes is fundamental in exploring the pathogenicity of cryptococcal meningoencephalitis. Cn may be extracellular or contained in the monocytes, macrophages, neutrophils, dendritic cells and even endothelial cells. The internalized Cn may proliferate inside the host cells, or cause the lysis of host cells, or leave the host cells via non-lytic exocytosis, or even hijack the host cells (Trojan horse) for the brain dissemination, which are regulated by microbe factors and also immune molecules. Coexistence of protective and deleterious roles of phagocytes in the progression of cryptococcosis warrant further investigation.Meningoencephalitis caused by Cn has been co-evolved with the phagocyte predators, e.g., amoebas and proliferates in the brain parenchyma. As Cn is a facultative intracellular pathogen, it is speculated that the transmigrating Cn might be extracellular or within some phagocytes, thereby invading the central nervous system (CNS) via a trans-cellular pathway or Trojan horse pathway promotes the inflammatory cytokines (Retini et al. The recruited neutrophils in the lung internalize Cn after intratracheal inoculation (Feldmesser et al. Candida albicans infection (Romani et al. Neutrophils are not only phagocytes but also the modulators of immune responses. Depletion of neutrophils results in a Th2 response and renders mice susceptible to high monocytes (Osterholzer et al. Upon Cn airway infection, CCR2 mediates the recruitment of Ly6G+ T cell responses against Cn (Bauman et al. In the lymphnodes, Langerhans cells and myeloid DC induce protective CD4Candida albicans also invades brain endothelial cells via endocytosis (Filler and Sheppard Different from monocytes/macrophages, neutrophils and dendritic cells, endothelial cells are not professional phagocytes. Yet, Cn is observed in the brain endothelial cells of infected mice (Chretien et al. Serological evidences suggest that people may have been infected with environmental Cn in early childhood (Goldman et al."} +{"text": "This special issue on photorespiration focuses on recent advances in this topic. The majority of the papers summarizes and extends contributions given at the 2nd workshop, \u2018Photorespiration\u2013key to better crops\u2019, held in Warnemuende, Germany in June 2015. This was organized by the DFG (German Research Foundation)-supported research network, \u2018Photorespiration: origins and metabolic integration in interacting compartments\u2019 (FOR 1186\u2013Promics).2 uptake and CO2 release. It is closely associated with photosynthetic CO2 assimilation and represents one of the major highways of carbon metabolism in most plants. By mass flow, surpassed only by photosynthesis, PR actually constitutes the second most important process in the land-based biosphere. Plants using the most widespread C3 type of photosynthesis for CO2 assimilation display particularly massive photorespiratory CO2 production. PR is initiated by competition of O2 with CO2 at the active site of the universal carboxylating enzyme Ribulose 1,5-bisphosphate Carboxylase/Oxygenase (Rubisco) (2-containing atmosphere (The term photorespiration (PR) describes a light-induced biochemical process that converts 2-phosphoglycolate (2PG) into 3-phosphoglycerate (3PGA) and is accompanied by ORubisco) , which pmosphere . To conv2 and NH3. Quantitatively, PR can decrease photosynthesis by up to 30% under current atmospheric concentrations of CO2 and O2 and even more at elevated temperature , chlorophytes, C3 and C4 plants were not viable in ambient air and could only be rescued under artificially enhanced CO2/O2 ratios highlight the important role of PR in the evolution of C4 photosynthesis via intermediary stages, in which the capacity for PR is lost from leaf mesophyll cells and relocated to the bundle sheath cells. As shown by D\u00f6ring et al. (this issue), in fully evolved C4 plants such as sorghum, the majority of genes encoding components of PR are also expressed preferentially in bundle sheath cells. Khoshravesh et al. (this issue) highlight the importance of organelle positioning in bundle sheath cells and the relocation of photorespiratory activity to this tissue during the evolution of C4 photosynthesis in grasses. Hagemann et al. (this issue) review the current position on the continuous coevolution of photosynthesis and PR. The evolution of all photorespiratory enzymes was elucidated and it was revealed that the present-day plant photorespiratory enzymes originated from archaeal, bacterial, and cyanobacterial sources, which served as eukaryotic host cell (Archaea), and mitochondrial (proteobacteria) or plastdial (cyanobacteria) endosymbionts, respectively. Moreover, calculating in terms of the geological era, ancient CO2/O2 ratios indicated that photorespiratory metabolism existed from the invention of oxygenic photosynthesis and remained necessary in cells evolving different types of carbon-concentrating mechanisms (CCM).The insight paper and three reviews discuss the current view on PR and its evolution. Sage (this issue) summarized the stepwise development of Cet al. (this issue), by using mutants that lack the activity of mitochondrial NADH dehydrogenase, analysed the role of the mitochondrial electron transport chain in photosynthesis. Using transcriptomic analysis of Lotus japonicus wild type and GS2 mutant plants on a range of different nitrogen concentrations and at ambient and elevated CO2, P\u00e9rez-Delgado et al. (this issue) show that primary nitrogen assimilation and PR are transcriptionally co-regulated. They also identify candidate transcription factors mediating this co-ordinated response. Betti et al. (this issue) summarize recent advances obtained in photorespiratory engineering and discuss the potential of realizing gains in crop productivity through manipulation of the photorespiratory pathway. Nunes-Nesi et al. (this issue) explore natural genetic variation in yield and photosynthetic capacity and point to the fact that photorespiratory capacity, in part, is genetically determined. Obata et al. (this issue) review the tight integration of PR with other metabolic pathways which reach beyond the recycling of carbon from the Calvin\u2013Benson cycle, a topic that is further extended by Hodges et al. (this issue). Timm et al. (this issue) focus on the still limited understanding of regulatory interactions between plant PR and photosynthesis and discuss its critical impact for successfully engineering photosynthesis. Montgomery et al. (this issue) also explore the regulatory effect of light reactions on PR in their opinion paper. They employ the framework of modularity in the cyanobacterium Fremyella diplosiphon and suggest a highly controlled interplay among light reactions, PR, and CCM. The opinion paper by Orf et al. (this issue) also centres on cyanobacteria. According to their comparative meta-analysis of cyanobacterial and plant metabolite profiles, the authors propose that cyanobacteria can serve as a much simpler surrogate to study the complex, highly compartmentalized, plant PR metabolism.Another set of contributions deals with the intertwining of the photorespiratory pathway with the central metabolism and its significance for engineering plant productivity. For example, Fromm et al. (this issue). Alonso-Cantabrana and von Caemmerer (this issue) report on using carbon isotope discrimination as a tool to quantify C4-like photosynthesis in C3\u2013C4 intermediate species. Labelling with the stable carbon isotope 13C also revealed a strong effect of reduced mitochondrial malate dehydrogenase activity on PR . Sharwood et al. (this issue) emphasize the importance of standardized and validated protocols for quantifying carbon fixation capacity in plants with differing carbon assimilation strategies, with particular emphasis on quantifying Rubisco activity.A deeper understanding of PR requires technology to determine rates of PR and photosynthesis accurately and this is reviewed by Hanson et al. (this issue). Dellero et al. report on the impact of reduced photorespiratory glycolate oxidase activity on leaf metabolism in Arabidopsis and review recent advances in the understanding of glyoxylate metabolism in different plant organs . Knocking out glycolate oxidase of Cyanidioschyzon merolae resulted in the first mutant with a photorespiratory phenotype among red algae . This finding revealed that the plant-type photorespiratory cycle using a peroxisomal glycolate oxidase evolved before the split of red and green algae, and it represents a further example that organisms, though carrying a CCM, also depend on functional PR.Four papers deal with the specific role of the central enzyme, glycolate oxidase. The biochemistry of this peroxisomal enzyme converting glycolate into glyoxylate is reviewed by Hodges 4 plants. Not only is it essential to support photosynthetic carbon assimilation, it is also heavily intertwined with other metabolic pathways and is the driving force for the evolution of C4 photosynthesis would be an important future aim. However, in the long-term, it is also envisaged that synthetic pathways for carbon assimilation (i.e. pathways not existing in nature), that are not affected by oxygen, might become a reality (The past decades of research on the process of PR have revealed that this pathway is an indispensable companion to oxygenic photosynthesis and includes photosynthetic organisms that feature highly efficient CCMs such as cyanobacteria, many algae, and C"} +{"text": "Autism spectrum disorder (ASD) is a set of complex neurodevelopmental disorders. ASD is characterized by early-onset difficulties in social interaction, repetitive behavior, and verbal and non-verbal communication. Worldwide prevalence of ASD is about 1% .There is both direct and indirect evidence for the role of prenatal testosterone and related androgens in the etiology of autistic traits and ASD. Recently, Baron-Cohen et al. found dip\u2009<\u20090.05) in mothers of autistic children compared to control subjects.Autistic-like traits are the first signs of ASD is a disease that leading to overproduction of adrenal androgens due to an enzyme deficiency [usually of 21 hydroxylase (21-OH)] and the fourth digit (the \u201cring\u201d finger) is an indicator of prenatal sex steroid levels Manning, . HoweverSeveral studies have investigated 2D:4D ratios among individuals with ASD, and the majorities of them reported lower 2D:4D ratios in autistic probands [reviewed in Teatero and Netley ]. For exAttention deficit hyperactivity disorder is a group of behavioral symptoms including hyperactivity, inattention, and impulsiveness. ADHD symptoms are often observed in individuals with ASD. Both disorders are more frequent in males than females ; in contrast, the sex ratio was significantly decreased in pregnant women with low levels of anti-Toxoplasma IgG antibodies . Moreover, infected women had significantly more facial hair (p\u2009=\u20090.001) and hair reduction (p\u2009=\u20090.002) than the control group.Ingudomnukul et al. reportedSeveral complex neurodevelopmental disorders may be associated with higher levels of testosterone, including antisocial and aggressive behavior that involved in the etiology of various neuropsychiatric disorders (Vyas et al., T. gondii infection (Miller et al., Different mechanisms have been shown to involve in the etiology of neuropsychiatric disorders and behavioral alterations during Several direct and indirect evidences support the role of increased prenatal testosterone in ASD and latent toxoplasmosis. Latent toxoplasmosis may also be involved in the etiology of various testosterone-related medical disorders and plays different roles in the etiology of neuropsychiatric disorders. Taken together with the high prevalence of latent toxoplasmosis in different nations (approximately 25\u201330% of the world\u2019s human population (Robert-Gangneux and Dard\u00e9, The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Journal of the American Academy of Child and Adolescent Psychiatry . On page 169, two of the 95% CIs reported in An error appeared in the article \u201cCognitive Training for Attention-Deficit/Hyperactivity Disorder: Meta-Analysis of Clinical and Neuropsychological Outcomes From Randomized Controlled Trials\u201d by Samuele Cortese, Maite Ferrin, Daniel Brandeis, et\u00a0al., published in the March 2015 issue of the"} +{"text": "Cell fate conversion is considered as the changing of one type of cells to another type including somatic cell reprogramming (de-differentiation), differentiation, and trans-differentiation. Epithelial and mesenchymal cells are two major types of cells and the transitions between these two cell states as epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) have been observed during multiple cell fate conversions including embryonic development, tumor progression and somatic cell reprogramming. In addition, MET and sequential EMT-MET during the generation of induced pluripotent stem cells (iPSC) from fibroblasts have been reported recently. Such observation is consistent with multiple rounds of sequential EMT-MET during embryonic development which could be considered as a reversed process of reprogramming at least partially. Therefore in current review, we briefly discussed the potential roles played by EMT, MET, or even sequential EMT-MET during different kinds of cell fate conversions. We also provided some preliminary hypotheses on the mechanisms that connect cell state transitions and cell fate conversions based on results collected from cell cycle, epigenetic regulation, and stemness acquisition. As two major types of cells in most animals, epithelial cells are known for their basement membrane, apical-basal axis of polarity, gap junction, immobility, and so on, while the characteristics of mesenchymal cells are almost just opposite, loosely associated, no polarity, and high mobility. Although the two types of cells are so different from each other, the transitions between epithelial and mesenchymal states, epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET), have been observed clearly and studied extensively during a variety of biological processes including embryonic development, cancer progression, and somatic cell reprogramming.The first observation of EMT can be dated back to as early as 1890 when some ductal epithelial cells were described to acquire mesenchymal characteristics in breast tumors progression as reviewed previously Nieto, . It is sEMT has been observed in multiple biological processes, especially embryonic development. The generation of adult tissues and organs requires multiple rounds of sequential EMT-MET, which is used to refer an EMT followed with its reversed process, MET into embryonic stem cells (ESC)-like cells with Oct4, Klf4, c-Myc, and Sox2. The generated cells were named induced pluripotent stem cells (iPSC). iPSC are able to generate tumors containing a variety of tissues from all three germ layers cells after being transplanted into nude mice and viable, fertile live-born progeny by tetraploid complementation (Takahashi and Yamanaka, During the generation of iPSC, multiple changes have been observed with the MEF, including those in gene expression profiles (Brambrink et al., Although MET is necessary for fibroblasts reprogramming, there are still several questions remaining unclear. Firstly, since the process from MEF to iPSC can be considered as a reversed process of embryonic development at least partially, the reversed process of sequential EMT-MET, which is also a sequential EMT-MET, might be observed during iPSC generation. Secondly, the different or even opposite regulatory roles of the four transcriptional factors, like on TGF\u03b21 and TGF\u03b2 receptor 2, make MET induction more complex (Li et al., Briefly, we determined reprogramming efficiencies with totally 74 different infection sequences. One particular infection sequence, Oct4 and Klf4 first, c-Myc next and Sox2 last, generated iPSC with the highest efficiency, about 600% of basal level (Liu et al., The reports on MET during iPSC generation in 2010 suggest this temporary EMT should generate more difficulties for later MET and decrease the reprogramming efficiency at the first glance (Li et al., To explain how short EMT was induced on MEF which are already in mesenchymal state and why sequential EMT-MET promotes iPSC generation, we proposed a new model in previous report (Liu et al., Another available hypothesis is that the optimal mesenchymal state may be more suitable for the cell fate conversions, which is partially supported by the connection between EMT and stem cell characteristics (Hayashida et al., in vitro differentiation of ESC or iPSC is also useful for EMT/MET research because of their similarity to embryonic development and relative simplicity of the system. As a membrane marker for epithelial cells, E-cadherin has also been used as one of the markers for undifferentiated ESC (Li et al., Multiple rounds of sequential EMT-MET make embryonic development an excellent model and hot topic for EMT/MET research Nieto, . HoweverTake the differentiation from iPSC to NSC as an example, immediate up-regulation of N-cadherin, a marker for mesenchymal cells, is essential for the efficient differentiation. However, E-cadherin expression is required to support the self-renewal of NSC (Karpowicz et al., The successful trans-differentiation of somatic cells into functional neurons (Sheng et al., The observations of EMT/MET during different kinds of cell fate conversions do not enable us to answer the question that EMT/EMT is a by-product or a significant cause for cell fate conversions. Take MET during iPSC generation from MEF for example, MEF and iPSC definitely have the characteristics of mesenchymal and epithelial cells respectively. Thus the successful conversion from MEF to iPSC must be accompanied by a MET process. MET is demonstrated to be required for MEF reprogramming, because reprogramming was greatly impaired when EMT was induced or MET was inhibited (Li et al., One way to answer the question above is to study the reprogramming of epithelial cells. Ciliary body epithelial cells have been reported to have higher reprogramming efficiency to iPSC than fibroblasts (Ni et al., Unfortunately, the studies on MET are fewer than those on EMT not only because MET is regarded as a reversed and following process of EMT during embryonic development but also because both MET and EMT employ same regulatory system and most method to inhibit EMT can induce MET (Li et al., 1 phase similar to ESC (Neganova and Lako, Cell cycle regulation is an old topic in cell biology and is observed during almost all kinds of cell fate conversions. iPSC have a high proliferation rate and short G1/S phase, where lies the major difference on cell cycle signatures between MEF and iPSC (Yang et al., On the other hand, EMT and cell cycle regulation are also inter-connected. TGF\u03b2 can lead to proliferation arrest and has extensive interaction with p53 (Adorno et al., 0 phase.To explain the positive correlation between proliferation rate and reprogramming efficiency, Dr. Jaenisch\u2019s laboratory has provided a model which suggested the early phase of MEF reprogramming was a stochastic process (Buganim et al., 1 phase, whereas cells are focused to DNA replication in S phase. Thus genes that specify the original cell fate would be silenced in S phase, as partially confirmed by the relative higher DNA methylation in S phase than G1 phase (Brown et al., 1 phase. Since genes specify original cell fate is in a hypo-methylated state in contrast to the hyper-methylated state of genes specify other cell fates, the cells would keep their original fate without outside stimulants. However, when the expression of genes specifying for other cell fates were increased significantly like during iPSC generation and trans-differentiation, the possibility for cells to switch cell fate will increase significantly. In addition, it is also reasonable to propose that decreasing the overall DNA methylation might facilitate cell fate conversion by reducing the methylation levels on the genes specifying for other cell fates. This hypothesis is supported by the study on Tet1 and lysine-specific demethylase 1 (Chen et al., The following question is what kind of events or markers during cell cycle promote reprogramming. One possibility is the DNA methylation regulation during cell cycle progression (Bou Kheir and Lund, As just discussed on DNA methylation, histone modifications are also required to be duplicated and inherited during cell cycle progression (Probst et al., The histone modifications affect cell fate conversions from a variety of aspects (Papp and Plath, +/CD24\u2212/low on cell surface (Al-Hajj et al., +/CD24\u2212/low population with properties associated with mammary epithelial stem cells like abilities to form mammosphere and to differentiate into myoepithelial or luminal epithelial cells (Mani et al., As mentioned above, EMT was first observed in breast cancer and plays critical roles during cancer progression (Thiery et al., However, the studies on ESC and iPSC are not fully consistent with the hypothesis above. ESC as isolated from inner cell mass are considered to be typical epithelial cells. The epithelial marker, E-cadherin, together with proteins like SSEA1, alkaline phosphatase, Oct4, Nanog, and others have been used to determine the undifferentiated state of ESC (Horie et al., We have provided a brief review on the connection between EMT/MET and cell cycle, epigenetic regulation, and stemness to provide possible mechanisms underlying the contributions of EMT/MET to cell fate conversions. Of course, EMT/MET also twists with other biological processes or factors including cell senescence with telomerase reverse transcriptase (Qiao et al., In our opinion, the well established connections between EMT/MET and large amount of biological processes is the major problem faced by researchers who intend to study the contributions of EMT/MET to cell fate conversions. Firstly, the complex interactions among different biological processes make it difficult to study one aspect at one time. For example, cell cycle, DNA methylation, and tumor progresses have been suggested to function together under certain circumstances (Robertson et al., To solve the problem above, one possibility is to put EMT and MET together and study sequential EMT-MET during cell fate conversions. Sequential EMT-MET has been observed during embryonic development and iPSC generation (Liu et al., To explain why temporary EMT before EMT promotes iPSC generation from MEF, we proposed a hypothesized model with optimal mesenchymal and optimal epithelial states as above. Considering the sequential EMT-MET, the preliminary hypothesis that an optimal mesenchymal may function as intermediate for efficient cell fate conversions might be more reasonable. This hypothesis is able to explain the beneficial effects of temporary EMT during iPSC generation and the opposite roles of EMT on stemness regulation. The hypothesis is supported by the chromosomal instability during EMT and in mesenchymal stem cells (Miura et al.,"} +{"text": "Modifications aiming at improving their efficiency and their delivery to their target cells are studied. However, their use as drugs is not straightforward. The biological activities of these fragments are mediated by several receptor families. Several matricryptins may bind to the same matricellular receptor, and a single matricryptin may bind to two different receptors belonging or not to the same family such as integrins and growth factor receptors. Furthermore, some matricryptins interact with each other, integrins and growth factor receptors crosstalk and a signaling pathway may be regulated by several matricryptins. This forms an intricate 3D interaction network at the surface of tumor and endothelial cells, which is tightly associated with other cell-surface associated molecules such as heparan sulfate, caveolin, and nucleolin. Deciphering the molecular mechanisms underlying the behavior of this network is required in order to optimize the development of matricryptins as anti-cancer agents.The extracellular matrix (ECM) is a source of bioactive fragments called matricryptins or matrikines resulting from the proteolytic cleavage of extracellular proteins and proteoglycans . Matrix metalloproteinases (MMPs), cathepsins, and bone-morphogenetic protein-1 release fragments, which regulate physiopathological processes including tumor growth, metastasis, and angiogenesis, a pre-requisite for tumor growth. A number of matricryptins, and/or synthetic peptides derived from them, are currently investigated as potential anti-cancer drugs both Matricryptins are biologically active fragments released from extracellular matrix (ECM) proteins and glycosaminoglycans by proteases collagen prolylyl hydroxylase is composed of two membrane associated proteins (the protective protein/cathepsin A and neuraminidase-1) and of the elastin-binding protein, an inactive spliced variant of lysosomal \u03b2-galactosidase . These trials include phase I (Lin et al., Clinical trials of endostatin mostly focus on solid tumors in association with cytotoxic drugs (fundus oculi angiogenesis diseases (Zhang et al., in vivo and exerts anti-fibrotic activity (Nishimoto et al., Several matricryptins such as the propeptide of lysyl oxidase, which is a tumor suppressor (Min et al., SV drafted the Section Receptors and Co-receptors of Matricryptins and Table The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Recently, two groups have reported that FMRP is important in the replication stress (RS) response and may play a role in meiosis in response to RS, rather than double strand break (DSB), is suppressed when endogenous FMRP is down-regulated. This suppression can be rescued by exogenous expression of wild-type FMRP but not by its nucleosome-binding-deficient mutant T102A or Y103L. Similar phenomena were also observed by Zhang et al. in a Drosophila S2 cells, while Alpatov et al. demonstrated that Fmrp levels in total lysate of mouse embryonic fibroblasts (MEFs) reduces slightly upon aphidicolin (APH) treatment. Using fractionation and immunofluorescence (IF) data, they both conclude that FMRP is recruited to chromatin upon RS.Zhang et al. found that dFmr1 increases at both the mRNA and protein levels in replication-stressed Due to the nature of FMRP, such a fractionation strategy may not be suitable for researching the intracellular localization of the protein. It has been well-established that FMRP is tightly associated with the ribosome and the rough endoplasmic reticulum (RER) to facilitate IF detection of nuclear FMRP. Zhang et al. demonstrated that dFmr1 accumulate in an S2 nucleus treated with combination of hydroxyurea (HU) and LPB, but not with HU or LPB alone, and that the dFmr1 signal concentrates in the Hoechst dull staining area. In MEFs, Fmrp staining is proximal to DAPI-condensed chromocenters, reminiscent of the centromere localization of PARP-1, which has been reported to interact with FMRP , indicating that FMRP chromatin function is independent of its canonical mGluR function. However, as reported by Reeve et al. is necessary to clarify its locus on chromatin (if ChIP-grade anti-FMRP is available). The data will provide deeper insight into the chromatin function of FMRP and also provide supporting information about whether or what histone modification recruits FMRP st year since the property of FMRP protein was initially characterized. As a cytoplasmic protein also functioning in chromatin, FMRP opens a new chapter of its story (Fig.\u00a0This year marks the 21"} +{"text": "Together these diverse perspectives spin an intricate web of ion channel regulation in the nervous system.Ion channels are complex hetero-oligomeric structures characterized by large, dynamic interaction networks, or \u201cinteractomes.\u201d In addition to directing channel localization, density and ion fluxes, these complexes facilitate downstream signaling events. Moreover, pathological modulation of these networks contributes to neurological dysfunction. Our contributors to this Research Topic, Described by Fan et al. as a \u201cmuSeveral other contributions shed light on the new insights into the function and composition of interactomes of various voltage-gated channels, regulated leak channels, and so called large pore channels.+ channel regulatory proteins are growing in complexity in terms of function and type. Known to regulate activation and trafficking of muscarinic receptor-activated Kir3 channels, Zylbergold et al. , by the so-called auxiliary subunits, dipeptidyl peptidase-like proteins (DPLPs) and Kv4 channel interacting proteins (KChIPs). While these were amongst the first identified interactors and low voltage-activated Ca2+ channels (Cav3) functionally interact with other conductances to regulate signal processing in the cerebellum.Traditionally viewed as auxiliary subunits, Kd et al. provide d et al. focus spd et al. describes et al. review hElusive until recently, understanding of this regulated leak channel whose loss in mice is lethal (Lu et al., Permeable to ions and small metabolites like ATP, Panx1 channels gained early notoriety as \u201cdeath pores\u201d in ischemic stroke and seizure (Thompson et al., 2+ entry. They describe foundational work implicating STIM1 as the Ca2+ sensor in this process critical for maintaining neurotransmission. Further they outline key physical interactions between STIM1 with Ca2+-release activated channels and voltage-gated Ca2+ channels that coordinate the activation and inhibition of these types of channels, respectively. Finally, two papers by Wilson et al. (2+ channels.A number of contributions underscore the capacity of \u201cpromiscuous\u201d intracellular proteins to modulate a variety of ion channels and receptors through physical interaction. Reviewed by Donnelier and Braun , cysteinn et al. ,b focus Adding further complexity to ion channel networks is consideration of lipid membrane composition and lipid second messengers. In the sole lipidome-oriented contribution, Raboune et al. identifyWhile daunting, elucidating these macromolecular intricacies has a translational silver lining: while difficult to identify and unravel, the myriad interaction loci revealed by studying these interactions present unique opportunities for discrete, and potentially safer therapeutic intervention. For example, selective blockade at key interaction loci with cell-permeable peptides now provides an infinite number of ways in which interactomes can be discretely modulated to improve health outcomes.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Depression is a heterogeneous mood disorder that has been classified and treated in a variety of ways. Although, a number of synthetic drugs are being used as standard treatment for clinically depressed patients, but they have adverse effects that can compromise the therapeutic treatments and patient's compliance. Unlike, synthetic medications, herbal medicines are widely used across the globe due to their wide applicability and therapeutic efficacy associated with least side effects, which in turn has initiated the scientific research regarding the antidepressant activity. This review is mostly based on the literature of the last decade, aimed at exploring the preclinical profile of plant-based alkaloids (the abundant secondary metabolite) as an emerging therapy for depression. Depression is a state of low mood and aversion to activity that can affect a person's thoughts, behavior, feelings and sense of well-being Sandra, . People The current antidepressant drugs are facing many challenges as mentioned by Alexander and Preskorn , in his Alkaloids are a group of naturally occurring chemical compounds that contain mostly basic nitrogen atoms. This group also includes some related compounds with neutral (McNaught and Wilkinson, Psychotria myriantha Mull. which exhibited antidepressant-like effect when studied on a 5-HT system in rat hippocampus (Farias et al., Annona cherimolia, including 1,2-dimethoxy-5,6,6a,7-tetrahydro-4H-dibenzoquinoline-3,8,9,10-tetraol, anonaine, liriodenine, and nornuciferine. The results showed that repeated treatment with this plant produced an antidepressant-like action in mice (Mart\u00ednez-V\u00e1zqueza et al., Rhazya stricta. Acute administration of the lyophilized extract of R. stricta resulted in a significant antidepressant-like effect in experimental animals (Ali et al., Mitagyna spicosa. Mitagynine i.p injection significantly reduced the immobility time of mice in both forced swim test and tail suspension test without any significant effect on locomotor activity (Idayu et al., Ziziphus apetala which showed strong activity against 11-\u03b2-hydroxysteroid dehydrogenase inhibition in vitro (Han et al., Aconitum baicalens exhibited an antidepressant-like effect in an animal model of depression (Nesterova et al., Boerhaavia diffusa Linn. It showed significant antidepressant activity in unstressed and stressed mice in different models (Dhingra and Valecha, Evodia fructus and found that it could reverse the decreases of sucrose preference, a number of crossing, 5-HT, and Na level and also increase immobility time (Jiang et al., Sceletium tortuosum have antidepressant property in animal studies (Loria et al., Piper nigrum. Its antidepressant activity has been observed in mice exposed to both chronic and acute stress. It caused a significant change in both immobility and swimming times (Wattanathorn et al., Piper laetispicum. When tested in the forced swim test, it caused a significant dose-dependent decrease in mobility at various test doses and thus possessed antidepressant activity (Yao et al., Dactylicapnos scandens Hutch, had an antidepressant effect in mice. It dose-dependently reduced the immobility time in the tail suspension test and thus could be effective for the moderate state of depression (Xu et al., The antidepressant effect of various plant alkaloids has been reported in the literature Table . Braziliin vitro and, therefore, could be the possible mechanism for its antidepressant effect (Han et al., Aconitum baicalense, improved serotonergic system activity in an animal model of depression (Nesterova et al., in vitro assays (Xu et al., Strictosidinic acid probably exhibited antidepressant effect due to an inhibition of monoamine oxidase activity (Farias et al., Our review on the basis of available literature suggested that alkaloids could play a potential role as natural antidepressants. Keeping in mind their abundance in nature, the alkaloids could be an economical source of healing the depressive disorder. The available therapeutic agents fail to produced effect in all patients; approximately 30\u201340% failure has been reported to first-line antidepressant drugs accompanied by the very slow onset of action. Several alkaloids are in clinical practice and producing outstanding results in different therapeutic classes. These reported alkaloids though evoked antidepressant effects in various animal studies, but still deficient in clinical evidence. In conclusion, enough scientific evidence gathered in our review supported that the plant-based alkaloids can serve as leads for antidepressant drug discovery. It is key to subject these alkaloids to further clinical studies for efficacy, potency, and safety to ensure their clinical status.SP carried out all the literature survey and written an initial draft of the review and AK draw the structures of compounds and help in review-draft. HK supervised the overall project and finalized the final version of the review.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Brueelia-complex (Bush et al., 2015Brueelia-complex and 30 outgroup taxa selected from across the order Phthiraptera. Also included are phylogenetic reconstructions based on Bayesian inference analyses of combined COI and EF-1\u03b1 sequences for Brueelia-complex species and outgroup taxa.Data is presented in support of a phylogenetic reconstruction of one of the largest, and most poorly understood, groups of lice: the"} +{"text": "International experience and evidence-based practices have shown that reduction in variability through use of protocolized perioperative care improves surgical outcomes and reduces costs to patients and healthcare systems. In this series of Expert Opinions, we provide consensus recommendations for the various components of perioperative care to aid with the development of enhanced recovery after surgery protocols. Early ambulation, early feeding, and early return of bowel function are linked with quicker recovery, earlier discharge, and improved satisfaction. Postoperative nausea and vomiting (PONV) is a common complication following abdominal surgical procedures with patient , anesthetic (use of opioids and volatile anesthetics), and surgical factors contributing to its incidence and severity. Nausea and vomiting should be viewed as existing on a continuum, and sequelae and patient discomfort vary based on patient experience, surgical procedure, and recovery profile. Guidelines for its avoidance and treatment are based on evidence-based literature, expert opinion, and professional association recommendations [ASPAN\u2019S evidence-based clinical practice opinion for the prevention and/or management of PONV/PDNV. J Perianesth Nurs 2006 [Gan et al. 2007] Gan et al. 2014. Gan et al. 2003. Use of volatile anestheticsIntraoperative use of opioidsSurgical/anesthetic timeSmoking (tobacco use decreases PONV risk)Modifiable risk factorsFemale sexYoung age (<50)History of PONVSurgical location Surgical technique (laparoscopic)Non-modifiable risk factors Apfel et al. 2012a Prophylaxis of PONV is based on a number of risk factors\u00a0. Prophylaxis and treatment should rely on modification of anesthetic technique, if possible, and the use of medications that work at a variety of receptor sites allowing for full multimodal benefit. Non-pharmacologic techniques include ensuring appropriate analgesia and hydration, acupuncture/acupressure, and aromatherapy. Pharmacologic techniques include medications that work at different receptor sites:All patients should receive PONV prophylaxis during the perioperative period. The numbers of medications used for treatment and prophylaxis should be determined by the number of modifiable and non-modifiable risk factors. Medications used should represent different mechanisms of action in an attempt to achieve multimodal benefit Tram\u00e8r 2001."} +{"text": "During the Oligo\u2010Miocene, major phases of phosphogenesis occurred in the Earth's oceans. However, most phosphate deposits represent condensed or allochthonous hemipelagic deposits, formed by complex physical and chemical enrichment processes, limiting their applicability for the study regarding the temporal pacing of Miocene phosphogenesis. The Oligo\u2010Miocene Decontra section located on the Maiella Platform is a widely continuous carbonate succession deposited in a mostly middle to outer neritic setting. Of particular interest are the well\u2010winnowed grain to packstones of the middle Miocene Bryozoan Limestone, where occurrences of authigenic phosphate grains coincide with the prominent carbon isotope excursion of the Monterey event. This unique setting allows the analysis of orbital forcing on phosphogenesis, within a bio, chemo, and cyclostratigraphically constrained age\u2010model. LA\u2010ICP\u2010MS analyses revealed a significant enrichment of uranium in the studied authigenic phosphates compared to the surrounding carbonates, allowing natural gamma\u2010radiation (GR) to be used as a qualitative proxy for autochthonous phosphate content. Time series analyses indicate a strong 405 kyr eccentricity forcing of GR in the Bryozoan Limestone. These results link maxima in the GR record and thus phosphate content to orbitally paced increases in the burial of organic carbon, particularly during the carbon isotope maxima of the Monterey event. Thus, phosphogenesis during the middle Miocene in the Mediterranean was controlled by the 405 kyr eccentricity and its influence on large\u2010scale paleoproductivity patterns. Rare earth element data were used as a tool to reconstruct the formation conditions of the investigated phosphates, indicating generally oxic formation conditions, which are consistent with microbially mediated phosphogenesis. Phosphogenesis during the Monterey event forced by 405 kyr eccentricity controlled paleoproductivityGamma radiation is directly linked to phosphogenesis via U enrichment of authigenic phosphateTrace elements show the phosphates formed under microbially mediated and well\u2010oxygenated conditions This also presupposes the need for sections with well\u2010established correlations to both global chronostratigraphy as well as global climatic records. Furthermore, phosphogenesis needs to be studied with sufficiently high temporal resolution to estimate changes in the amount of phosphate formed over time, in order to accurately correlate them with established palaeoecological and paleoclimatological records. To that end the Decontra section, which is a well\u2010established shallow\u2010marine reference section, with a robust orbitally tuned stratigraphic framework [F\u00f6llmi, Mutti and Bernoulli, Hubert et al., Crosby and Bailey, F\u00f6llmi, Filippelli, Delaney, Schenau et al., Slomp et al., van der Zee et al., Paytan and McLaughlin, Slomp and Van Cappellen, Recent studies found that most occurrences of phosphogenesis in the present day and ancient oceans were directly mediated by microbial activity, irrespective of the marine setting in which they formed [The present work deals with three questions regarding phosphogenesis in the Decontra section during the MMCO and Monterey event: (1) Determine the phosphate contents on a sufficiently high\u2010resolution to correlate them with global climatic records; (2) Apply selected rare earth element (REE) proxies to reconstruct the formation history of the phosphates, in order to put them into relation to the already established paleoenvironmental history of the section; (3) Find the dominant uranium\u2010bearing phase using electron microprobe and LA\u2010ICP\u2010MS analyses and understand the proxy\u2010relationship between these phases and existing gamma log data.2Vecsei and Sanders, Crescenti et al., Mutti et al., Vecsei et al., Vecsei and Sanders, Carnevale et al., The late Oligocene to late Miocene Decontra section lies on the northern slope of the Orfento river valley southeast of the village Decontra on the Maiella mountains in Central Italy The Cerratina cherty Limestone\u2014a 35 m thick succession of hemipelagic wackestones to packstones dominated by planktic foraminifers containing chert nodules, phosphatized foraminiferal tests, and sponge spicules. The first occurrence of the planktic foraminifer Praeorbulina sp. in the upper part of this unit indicates an age of <16.2 Ma; (3) The 32 m thick Bryozoan Limestone dominated by winnowed bryozoan grainstones with abundant planktonic and benthic foraminifers; (4) The 3 m thick Orbulina Limestone with abundant Orbulina sp.; (5) The Lithothamnium Limestone characterized by abundant red algal fragments overlying the Orbulina Limestone with a sharp contact. The base of the Lithothamnium Limestone is a 1.5 m thick horizon containing abundant Heterostegina fragments [see Reuter et al., Vecsei and Sanders, Carnevale et al., Reuter et al., Auer et al., Vecsei and Sanders, Cornacchia et al., Reuter et al. [Within the section five lithostratigraphic units are described Figure : (1) Ther et al. and the inifera; The CerrMutti and Bernoulli, Reuter et al., Mutti and Bernoulli, Reuter et al., Reuter et al., Auer et al., Orbulina Limestone, is interpreted as the Ser4/Tor1 sequence boundary [Reuter et al., Orbulina Limestone to \u223c15.24\u2013\u223c11.92 Ma, indicating generally low but continuous sedimentation within this part of the section [Auer et al., The Decontra section is also well known for occurrences of authigenic phosphate, particularly within the middle Miocene Bryozoan Limestone, where a prominent phosphatic hardground occurs [3Reuter et al., Auer et al., 2O5 content and check for impurities in the limestone as well as JLS\u20101 were measured as unknowns and reproduced within the reported errors for major components including P2O5.The gamma\u2010ray measurements were carried out in the field in 2012 using a portable \u201cGS\u2010512\u201d gamma\u2010ray spectrometer , and are reported in total counts (TC) [2O5 was subsequently correlated with the reported gamma\u2010ray values from the field measurements. Comparison was done by averaging the gamma\u2010ray measurements in the vicinity of the sampling spot. The results of this comparison together with the correlation coefficient were plotted in a standard cross plot of the samples were prepared and documentation of phosphatic grains was performed by backscattered electron (BSE) imaging, using a Jeol Superprobe JXA\u20108200 electron microprobe (EMP) at the Eugen F. Stumpfl Electron Microprobe Laboratory, UZAG . Figure .Williams et al., [Fryer et al., Using the BSE images, suitable phosphates were selected for LA\u2010ICP\u2010MS analyses. LA\u2010ICP\u2010MS analyses were performed at the NAWI Graz Central Lab \u201cWater, Minerals and Rocks\u201d (University of Graz and Graz University of Technology), in order to obtain concentrations for U, Th, and selected rare earth element 610 of the National Institute of Standards and Technology, Gaithersburg, MD, USA. Values for the SRMs reported by m et al. were appJochum et al., Evans and M\u00fcller, Caragnano et al., Williams et al., http://georem.mpch-mainz.gwdg.de) [Jochum et al., While the NIST SRM glasses are not matrix\u2010matched to the analyzed carbonate and phosphate material, they offer distinct benefits compared to various matrix\u2010matched standards . Several analytical studies using biogenic carbonates and authigenic apatite found that the analytical advantages offered by the NIST SRM glasses outweigh the disadvantages of standardization using an unmatched matrix, which is largely based on the much better known trace element concentration of the NIST reference glasses compared to other standards [Koenig et al., Electron microprobe (EMP) analyses were subsequently carried out in close proximity to the LA\u2010ICP\u2010MS craters, after repolishing the samples. Since the grains are complex and often highly porous mineral aggregates of calcium fluor apatite and calcium carbonate, EMP analysis did not yield results close to the theoretical value of fluor apatite and calcite. Thus, all calculations of analyzed phosphates as well as calcium carbonates utilize a consistent CaO concentration of 55 wt. % as the internal standard value [e.g., 3.1Prior to interpretation, the measurements were evaluated for mixed signals of phosphate grains and surrounding carbonate rock based on their major elements . This preevaluation excluded several spots from the subsequent data analyses .McLennan, Alibo and Nozaki, Shields and Stille, Haley et al., Garnit et al., REE concentrations for each spot were transformed into shale\u2010normalized REE concentrations using the Post Archean Australian Sedimentary Rocks (PAAS) standard [German and Elderfield, Bau and Dulski, Morad and Felitsyn, Shields and Stille, Garnit et al., German and Elderfield [De Baar et al. [PAAS normalized REE concentrations were then used to calculate the Ce anomaly, a useful tool for the characterization of paleoredox conditions [derfield defined r et al. . [Hammer et al., The REDFIT power spectrum for the Bryozoan Limestone shown in Figure r et al. and used44.1Amphestigina, Elphidium, and miliolids) and corallinaceans are common [Reuter et al., Mutti and Bernoulli, Reuter et al., Thin section analysis of the 27 samples taken within the Bryozoan Limestone reveals well\u2010winnowed carbonatic grainstones, predominantly composed of bryozoan and echinoid derived skeletal fragments containing a variable amount of planktonic and benthic foraminifera. In the lowermost 2 m of the unit below, the microbially formed phosphatic hardground, notable occurrences of benthic foraminifera , showing that potassium is not a major gamma\u2010ray source for the sediment .Results of the XRF analysis show that all samples are very pure carbonates, with only race amounts of SiO4.2LA\u2010ICP\u2010MS and EMP analyses reveal that most investigated phosphatic grains likely represent complex microcrystalline aggregates of phosphate and calcium carbonate, making accurate analysis of the mineral composition difficult. This was further corroborated by the performed electron microprobe analysis of the samples. Nevertheless, matrix standardized results reveal a significant U enrichment of phosphate containing grains in gamma\u2010ray sources compared to the surrounding purely carbonatic host material. Results of the LA\u2010ICP\u2010MS analyses show that the uranium content in the investigated phosphate grains is consistently elevated ranging between 13.5 and 131 ppm, with an average of 48.4 ppm and a median of 43.5 ppm. By comparison, the uranium concentration in the carbonate matrix ranges between 0.17 and 1 ppm, with an average of 0.44 ppm and a median of 0.39 ppm. The standard deviation of the U concentration in the biogenic carbonates is 0.25 ppm. These values result in an average enrichment of roughly 1:111 of uranium in the phosphatic grains compared to the carbonate matrix. Thorium content is comparably low in most analyzed grains, with only three grains showing significantly elevated Th content, in the range of \u223c30 ppm , indicating overall weak contribution of 40K to the total gamma ray counts. K2O is furthermore only weakly correlated with gamma\u2010ray intensity and both large and small\u2010scale patterns occur within the gamma\u2010ray (GR) record of the Decontra section [2O5 and total gamma radiation counts . TOC is generally lost in the sedimentary record, as it is quickly dissolved and refracted by biological processes in most settings. This also explains the comparably low organic carbon content of the investigated grainstones Figure . TOC val5.2Auer et al., 2O5 content of the XRF measurements of discrete rock samples is assumed to be the predominant U bearing mineral phase within the sediment [s Figure a.Compton et al., The fact that no other significant U sources occur within the sediment, and U concentrations of the carbonates remained relatively constant in the marine realm [\u22121) [Auer et al., O'Brien et al., Mutti and Bernoulli, Reuter et al., Auer et al., The comparably low sedimentation rates for the Bryozoan Limestone , the main constituent of marine snow, acts as a transport mechanism for organically bound phosphate to the sediment.F\u00f6llmi, Mutti and Bernoulli, Arning et al., Filippelli, Crosby and Bailey, This setting led to a complex interplay of organic matter transport , authigenic/microbial phosphate formation, and uranium incorporation. Release of OM\u2010derived P in the sediment pore water occurred through degradation of both microbial mats and marine snow through microbial activity within the sediment. Microbes at the sediment water interface used POM as nutrient source, which in turn liberated P and U into a closed biochemically controlled micromilieu at the sediment water interface [Davey and O'toole, F\u00f6llmi, Schenau et al., van der Zee et al., Paytan and McLaughlin, Filippelli, Crosby and Bailey, Reuter et al., Auer et al., Thin section and geochemical evidence within in the sediment, without significant mixing or enrichment from the overlying ocean water and in conjunction with the activity of magnetotactic bacteria [Bau and Dulski, German and Elderfield, German and Elderfield, Bau and Dulski, Alibo and Nozaki, Morad and Felitsyn, Haley et al., Shields and Stille, Garnit et al., Emsbo et al., REE signatures show a negative Ce anomaly in both the carbonate matrix and all investigated phosphatic grains using the Ce/Ce* versus Pr/Pr* plot [Reynard et al., Shields and Stille, Garnit et al., Reynard et al., A slight trend toward more negative La/Sm values as well as enriched La/Yb values occurs in our data. This suggests that both phosphates and carbonates in the Decontra section were affected by substitution processes as well as adsorption processes during early diagenesis [Auer et al., This creates a good explanation for the largely in\u2010phase variability of both the magnetic susceptibility and GR records of the Decontra section [5.4Filippelli, Mutti and Bernoulli, Reuter et al., Vincent and Berger, Jacobs et al., Abels et al., Mourik et al., Holbourn et al., J. C. Zachos et al., J. Zachos et al. Harzhauser et al., Economically viable phosphorites are generally deposited as lag deposits or hardgrounds formed during episodes of nondeposition or even active sediment removal. Thus, virtually all global phosphorite deposits are generally unrelated to primary P accumulation within the sediment and subsequent phosphogenesis [F\u00f6llmi, Filippelli, van der Zee et al., Filippelli, Holbourn et al., Our results indicate that the well\u2010known link between the carbon and phosphorous cycle [Compton et al., F\u00f6llmi, Jacobs et al., John et al., F\u00f6llmi et al., Holbourn et al., Diester\u2010Haass et al., Holbourn et al., Our results provide insights into the pacing and formation requirements of the widespread middle Miocene shallow marine phosphorite deposits [13C record during the Monterey event [Holbourn et al., Diester\u2010Haass et al., Holbourn et al., Diester\u2010Haass et al., Holbourn et al., Our model is thus in accordance with the hypothesis that variations in ocean circulation are the cause of the 405 kyr eccentricity\u2010forcing observed in the \u03b46Mutti and Bernoulli, Reuter et al., Auer et al., The present study uses LA\u2010ICP\u2010MS data of carbonate grainstone as well as authigenic phosphate to characterize microbially mediated phosphogenesis in the middle Miocene Bryozoan Limestone of the Decontra section in the Maiella mountain range . Our results show that: (1) primary microbially controlled phosphogenesis persisted throughout the Bryozoan Limestone in accordance with previous studies [Supporting Information S1Click here for additional data file.Table S1Click here for additional data file."} +{"text": "Matricaria chamomilla L. essential oils and a potent pro-apoptotic molecule and enhanced the production of tumor growth factor-beta 1 (TGF-\u03b21) on NCTC 2544 keratinocytes, although it did not change the activity of monocytes and dendritic cells , as the major mechanism of tumor immune escape, a crucial hurdle for tumor immunotherapy (Jacobs et al., in vitro cultured keratinocytes (Frikeche et al., +CD25+FoxP3+ regulatory T cells, through TGF-\u03b2 expression (Baumgartner et al., in situ.Darra et al., reported that the anti-neoplastic action exerted by \u03b1-bisabolol, derives fundamentally by its ability in inducing mitochondria-mediated apoptosis in cancer cells (Darra et al., In vitro research usually neglected this issue, as most of investigations based on cell lines obviously never consider the immune microenvironment existing in the in vivo situation. In this perspective, the recent article by Frikeche et al., raises some criticism about the actual role of \u03b1-bisabolol as a real, promising chemopreventive molecule.Despite its promising activity as an anti-tumor molecule, \u03b1-bisabolol does not possess so different features respect to the widest family of plant-derived anti-inflammatory and chemopreventive polyphenols (Chirumbolo, Alpha bisabolol might affect mitochondrial permeability transition also in non cancer cells (Leanza et al., in vitro cancer lines, such as MiaPaCa, should be considered with caution.As with other phytochemicals, the role of \u03b1-bisabolol on cancer therapy should be expanded in future debates, while any further proposals to investigate this organic compound on The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "The innate immune system plays a dualistic role in the evolution of ischemic brain damage and has also been implicated in ischemic tolerance produced by different conditioning stimuli. Early after ischemia, perivascular astrocytes release cytokines and activate metalloproteases (MMPs) that contribute to blood\u2013brain barrier (BBB) disruption and vasogenic oedema; whereas at later stages, they provide extracellular glutamate uptake, BBB regeneration and neurotrophic factors release. Similarly, early activation of microglia contributes to ischemic brain injury via the production of inflammatory cytokines, including tumor necrosis factor (TNF) and interleukin (IL)-1, reactive oxygen and nitrogen species and proteases. Nevertheless, microglia also contributes to the resolution of inflammation, by releasing IL-10 and tumor growth factor (TGF)-\u03b2, and to the late reparative processes by phagocytic activity and growth factors production. Indeed, after ischemia, microglia/macrophages differentiate toward several phenotypes: the M1 pro-inflammatory phenotype is classically activated via toll-like receptors or interferon-\u03b3, whereas M2 phenotypes are alternatively activated by regulatory mediators, such as ILs 4, 10, 13, or TGF-\u03b2. Thus, immune cells exert a dualistic role on the evolution of ischemic brain damage, since the classic phenotypes promote injury, whereas alternatively activated M2 macrophages or N2 neutrophils prompt tissue remodeling and repair. Moreover, a subdued activation of the immune system has been involved in ischemic tolerance, since different preconditioning stimuli act via modulation of inflammatory mediators, including toll-like receptors and cytokine signaling pathways. This further underscores that the immuno-modulatory approach for the treatment of ischemic stroke should be aimed at blocking the detrimental effects, while promoting the beneficial responses of the immune reaction. As highlighted by recent expression profiling studies, the majority of the genes acutely modulated in the blood of stroke patients are implicated in the regulation of the innate immune system and specialized cells, activated in the brain or recruited from the periphery, actively participate to the detrimental processes implicated in tissue damage, as well as to the repair and regeneration phases and cytokine signaling pathways -\u03b3 proteins and other DAMPS, plays an important role in ischemic brain injury , represent promising therapeutic options, effective in reducing the inflammatory response to stroke injury is a member of the immunoglobulin family of cell surface receptors and has been implicated in the development and progression of stroke. The full-length, membrane-bound RAGE isoform (fl-RAGE) is mainly expressed in neurons and in microglia/macrophages. Up-regulation of this receptor has been documented after both permanent and transient focal cerebral ischemia in rodents has been reported in rats subjected to either transient or permanent focal brain ischemia -1\u03b1 have been correlated with favorable long-term outcome and MIP-1\u03b1 have been shown to play an important role in promoting tissue damage via recruitment of inflammatory cells deficiency exacerbates ischemic brain damage by upregulation of proinflammatory cytokines and this Zn finger-containing immunoregulatory protein also participates in LPS- and electroacupuncture-induced ischemic stroke tolerance occurring in peri-ischemic regions 2\u20137 days after focal ischemia in mice is coincident with the delayed increase of MMP-9, suggesting its involvement in neurovascular remodeling (Zhu et al., 2+ homeostasis in neurons through activation of EP1 receptors (Kawano et al., After an ischemic insult, the expression of COX-2 is elevated in neurons, vascular cells and neutrophils, as demonstrated both in stroke patients and in animal models (Nogawa et al., in vitro (Kim et al., COX-2 has been implicated in hyperbaric oxygen preconditioning, since pharmacological inhibition of this enzyme abolishes the beneficial effects of the conditioning stimulus in a rat model of transient global cerebral ischemia (Cheng et al., de novo expression of inducible NOS contribute to ischemic tissue damage (Iadecola et al., While elevated expression of inducible NOS is associated with ischemic (Cho et al., NO plays a crucial role in cortical spreading depression-induced tolerance to transient focal cerebral ischemia in rats (Horiguchi et al., Although ischemic stroke is a major cause of mortality and long-term disability worldwide (Go et al., All the authors participated in the collection, review, and analysis of the relevant literature, as well as to drafting and revising of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Sepsis is an infection-initiated systemic inflammatory syndrome with an estimated incidence of 18 million cases annually worldwide. Despite advances in intensive care and supportive technology, the mortality rate of sepsis still ranges from 15% to 80%, reminding scientists and clinicians that it remains to be a major clinical challenge. The key to winning the \u201ccampaign\u201d to combat sepsis is improved understanding of the epidemiology, pathogenesis, and biomarkers of sepsis and discovery of novel therapies. The present special issue shows several encouraging results and provides comprehensive reviews of the latest advances in this field. in vitro, in vivo, and genetic studies on the effects of defensins as well as the corresponding mechanisms within sepsis. Their review, \u201cDefensins and Sepsis,\u201d also points out that the function of defensins reflects both their immunomodulatory and broad-spectrum antimicrobial effects.The effector cells from the innate and adaptive immune systems play a crucial role in sepsis. Dendritic cells, in particular, serve as professional antigen presenting cells and are involved in the aberrant immune response to sepsis. In this special issue, X. Fan et al. discuss the effects of sepsis on the amount, surface molecule expression, cytokine secretion, and T-cell activating function of dendritic cells and the underlying mechanisms in their review \u201cAlterations of Dendritic Cells in Sepsis: Featured Role in Immunoparalysis.\u201d Recent postmortem studies of patients who died of sepsis showed that depletion of CD4 and CD8 lymphocytes is an important characteristic. Thus, knowledge of these circulating lymphocyte abnormalities is relevant for the understanding of sepsis pathophysiology. R. de Pablo et al., who have previously reported on the alteration of B cells, natural killer cells, and T-cell function in septic patients, summarize their latest findings on the role of blood lymphocytes in sepsis and discuss the different kinetic patterns of lymphocyte subsets and their relationship to outcome in their review \u201cRole of Circulating Lymphocytes in Patients with Sepsis.\u201d Both the clinical and basic researches have shown that sepsis-associated immunosuppression is associated with adverse outcomes. A novel heterogeneous population of immature myeloid cells that possess immunosuppressive activities, termed myeloid-derivedsuppressorcells(MDSCs), has gainedmuchattention in recent sepsis studies. D. Lai et al. discuss the complex functions of MDSCs in the pathogenesis of sepsis. Their review \u201cMyeloid-Derived Suppressor Cells in Sepsis\u201d also proposes that the overall role of MDSCs involves much more than simply being an immunosuppressive cell population. These 3 review articles provide a comprehensive analysis of the major important immune cells in sepsis and highlight potential therapeutic targets. As a group who have investigated the function of the family of defensins in sepsis for nearly 10 years, G.-H. Xie et al. summarize the\u03baB-Mediated Inflammatory Response.\u201dAlthough the international Surviving Sepsis Campaign guidelines have been released for 10 years, sepsis remains a fatal syndrome due to the lack of efficient biomarkers and novel treatments. D. N. Nguyen et al. investigated plasma cortisol levels in septic patients with delirium and coma and found that cortisol is a potential biomarker of brain dysfunction in their article \u201cCortisol Is an Associated-Risk Factor of Brain Dysfunction in Patients with Severe Sepsis and Septic Shock.\u201d F. Song et al. and P. Madhusudan et al. discuss two important but controversial issues related to the Surviving Sepsis Campaign Guidelines. In a meta-analysis of 12 randomized trials involving 4100 septic patients, \u201cIntensive Insulin Therapy for Septic Patients: A Meta-Analysis of Randomized Controlled Trials,\u201d F. Song et al. reported no benefit and a higher incidence of hypoglycemia with intensive insulin therapy compared with conservative glucose management. P. Madhusudan et al. discuss the current debate on the choice, amount, and end points for fluid resuscitation in sepsis in their review \u201cFluid Resuscitation in Sepsis: Reexamining the Paradigm.\u201d K. Xie et al. investigated the therapeutic function of hydrogen gas in a septic animal model for several years, and, in their present review, \u201cHydrogen Gas Presents a Promising Therapeutic Strategy for Sepsis,\u201d they summarize the progress of hydrogen treatment in sepsis. J. Zhou et al. and X. Li et al. explore novel drugs for sepsis from the perspective of the neuroendocrine network in sepsis in their two studies, \u201cEpinephrine Enhances the Response of Macrophages under LPS Stimulation\u201d and \u201cAgmatine Protects against Zymosan-Induced Acute Lung Injury in Mice by Inhibiting NF-In this present special issue about the pathogenesis, biomarkers, and treatment of sepsis, the authors provide comprehensive reviews and attractive research perspectives on the mechanisms of sepsis which we hope will inspire researchers investigating novel biomarkers and therapeutic sepsis targets."} +{"text": "Escherichia coli (STEC) are a group of diarrheagenic bacteria associated with foodborne outbreaks. Infection with these agents may result in grave sequelae that include fatality. A large number of STEC serotypes has been identified to date. E. coli serotype O104:H4 is an emerging pathogen responsible for a 2011 outbreak in Europe that resulted in over 4000 infections and 50 deaths. STEC pathogenicity is highly reliant on the production of one or more Shiga toxins that can inhibit protein synthesis in host cells resulting in a cytotoxicity that may affect various organ systems. Antimicrobials are usually avoided in the treatment of STEC infections since they are believed to induce bacterial cell lysis and the release of stored toxins. Some antimicrobials have also been reported to enhance toxin synthesis and production from these organisms. Various groups have attempted alternative treatment approaches including the administration of toxin-directed antibodies, toxin-adsorbing polymers, probiotic agents and natural remedies. The utility of antibiotics in treating STEC infections has also been reconsidered in recent years with certain modalities showing promise.Shiga toxin-producing Escherichia coli (STEC) are a group of bacterial organisms that are capable of producing one or more types of Shiga toxin (Stx). STEC are associated with a disease spectrum ranging from diarrhea and hemorrhagic colitis (HC) to the potentially fatal hemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP). STEC infections are typically food-borne that has acquired the ability to produce Stx2, typically produced by enterohemorrhagic E. coli (EHEC) rather than EAEC group members. This may have occurred via horizontal gene transfer resulting in a new E. coli virotype dubbed the Enteroaggregative Hemorrhagic E. coli or EAHEC (Bloch et al., E. coli O104:H4 and humans are believed to be the major reservoir for this organism (Wieler et al., E. coli O104:H4 was relatively similar to that caused by other STEC infections some pertinent differences existed. For instance, about a quarter of subjects affected developed HUS during the 2011 outbreak, which is 2\u20135 fold higher than the rate usually observed for an STEC infection (WHO, Perhaps highlighting the relevance of monitoring these non-O157 serotypes was the emergence of the rather notorious The lack of an effective treatment strategy for an STEC infection has made these agents a prominent public health threat and a burden to the medical community at large. The currently recommended management of an STEC infection mainly relies on supportive therapy and hydration Thorpe, . The useThe debatable use of antimicrobial agents for the treatment of an STEC infection has led to the rise of various alternative treatment approaches Table . These hin vitro and were demonstrated to protect challenged mice when administered intravenously (Nishikawa et al., in vitro and in animals (Kitov et al., in vitro and upon testing in animals (Paton et al., in vitro. This agent, however, was not effective in clinical trials (Trachtman et al., Various agents that mimic Stx receptors and bind them thus reducing their availability to cellular receptors have been developed. Carbosilane dendrimers harboring Gb3 at their termini neutralize Shiga toxins cycl, was shown to protect cells in culture against Stx (Noel et al., Cell permeable agents that can bind Stx2 and potentially interfere with its intracellular trafficking have been reported. These include Ac-PPP-tet (Watanabe-Takahashi et al., E. coli O104:H4 outbreak (Lapeyraque et al., E. coli O104:H4-induced HUS results in no additional benefits (Kielstein et al., Preparations of antibodies that can bind Shiga toxins and neutralize their effects have been reported. Anti-lipopolysaccharide antibodies have shown protective abilities upon laboratory assessment (Paton et al., in vitro or in experimental animal models; however, they have not been evaluated in clinical studies. Worth noting is a study that showed a synergistic effect between green tea extract and an antibiotic, levofloxacin, in the treatment of an STEC-infected mouse model (Isogai et al., Various natural products have been considered as potential therapeutic agents for STEC infections. These have included lactic acid (Pittman et al., The use of antimicrobial agents in treating STEC infections has been controversial and the subject of an ongoing debate. While some studies indicated that the use of particular agents may increase the risk of HUS, others have reported a decrease of this risk upon implementation of antimicrobials. While these observations may be particular to certain agents at some doses, the potential risk of antimicrobial treatment inducing HUS has led to a general contraindication of such agents (Qadri and Kayali, in vitro; these include the quinolones, trimethoprim, and furazolidone (Kimmitt et al., E. coli O104:H4 from the 2011 outbreak in Europe do not display an increase in toxin production upon treatment with meropenem, ciprofloxacin, chloramphenicol, or fosfomycin, unlike E. coli O157:H7 (Corogeanu et al., E. coli O157:H7 and in E. coli O104:H4. A sub-MIC concentration may, after all, be the concentration available locally at the site of infection. We noted that the response is variable depending on the isolate used and the concentration of antimicrobial implemented (Nassar et al., Several antimicrobial agents have been shown to enhance the release or the production of Shiga toxins from STEC cells E. coli O104:H4 during the 2011 outbreak (Geerdes-Fenge et al., E. coli O157:H7 cells remain viable, followed by treatment with gentamicin at a bactericidal concentration. This strategy was effective in decreasing toxin release compared to solely treating the cells with a bactericidal gentamicin concentration (Kanbar et al., E. coli O157:H7 infection mouse model resulted in an improved animal survival rate (Rahal et al., E. coli O104:H4 infected mice similarly resulted in an improved survival rate compared to untreated control mice that were infected with the organism; however, the highest survival rate observed was with mice treated with gentamicin alone, unlike our observations with E. coli O157:H7 (Fadlallah et al., Reconsideration of treating STEC infections with antimicrobial agents has nevertheless gained ground in recent years. Ciprofloxacin was recently reported to decrease the risk of HUS in subjects infected with in vitro beneficial effects of probiotics and that inoculation of animal models with a probiotic prior to an experimental STEC infection has preventative capabilities (Asahara et al., in vitro study showed that cultivation of STEC organisms in the presence of various Bifidobacterium, Pediococcus, and Lactobacillus strains results in a decreased production of Stx2. This was attributed to a decrease in pH due to the acids produced by these agents (Carey et al., Although probiotics may not have a therapeutic benefit in the management of an STEC infection, they may have a relevant preventative utility. Probiotics are probably capable of disrupting host-infectious agent/toxin interactions by occupying cellular receptors themselves, by producing decoy receptors that take up the toxins or by modifying the local milieu, hence making these interactions unfavorable (Corr et al., in vitro (Niu et al., Another preventative measure proposed as a means of controlling STEC is the application of lytic phages. Lytic phages have been shown to reduce STEC numbers Various vaccine approaches have also been attempted including the development of preparations that contain bacterial peptides and virulence factors (Wen et al., In conclusion, despite the passage of more than three decades since STEC organisms were first associated with human clinical illness CDC, , a generThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Schizosaccharomyces pombe undergoes a closed mitosis, it exhibits a short period during meiosis when nuclear and cytoplasmic proteins are mixed in the presence of intact NE and nuclear pore complexes (NPC). This \u201cvirtual\u201d nuclear envelope breakdown (vNEBD) involves changes in the localization of RanGAP1, an activator of Ran-GTP hydrolysis. Recently, Nup132, a component of the structural core Nup107-160 subcomplex of the NPC, has been shown to be involved in the maintenance of the nuclear cytoplasmic barrier in yeast meiosis. In this review, we highlight the possible roles of RanGAP1 and Nup132 in vNEBD and discuss the biological significance of vNEBD in S. pombe meiosis.Ran, a small GTPase, is required for the spindle formation and nuclear envelope (NE) formation. After NE breakdown (NEBD) during mitosis in metazoan cells, the Ran-GTP gradient across the NE is lost and Ran-GTP becomes concentrated around chromatin, thus affecting the stability of microtubules and promoting the assembly of spindle microtubules and segregation of chromosomes. Mitosis in which chromosomes are segregated subsequent to NEBD is called \u201copen mitosis.\u201d In contrast, many fungi undergo a process termed \u201cclosed mitosis\u201d in which chromosome segregation and spindle formation occur without NEBD. Although the fission yeast In eukaryotic cells, the nucleus is enclosed by a nuclear envelope (NE) in interphase. The NE is a double membrane structure which separates the nucleoplasm from the cytoplasm. Macromolecules are transported between the nucleus and the cytoplasm across the NE through nuclear pores which are formed by large protein complexes called nuclear pore complexes occurs (open mitosis) , nuclear and cytoplasmic molecules are mixed similar to open mitosis. Interestingly, both the NE and NPCs are maintained in this phase and thus this phenomenon is called \u201cvirtual\u201d NEBD that regulate its localization unts Figure . NotablyS. pombe. Interestingly, the metazoan Nup107-160 subcomplex is known to re-localize to the kinetochores upon NEBD and functions in kinetochore-microtubule attachment (Lo\u00efodice et al., S. pombe meiosis I. In addition, Nup133, a human homolog of S. pombe Nup132, is required for proper localization of RanGAP1 at the kinetochore in human cells (Zuccolo et al., Our discovery raises the question of how Nup132 regulates both kinetochore assembly and NE permeability during meiosis I in nup132 mutant activates the spindle assembly checkpoint and delays meiosis I progression (Figure Inadequate kinetochore-microtubule attachment in the n Figure . The durn Figure . Alternan Figure . These rS. cerevisiae Nup133 results in Ran being uniformly localized in the cell rather than being enriched in the nucleus (Gao et al., S. pombe and S. cerevisiae, Nup132/Nup133 is required for the uniform distribution of NPCs along the NE (Doye et al., nup132 mutant will help determine whether changes in nuclear permeability during anaphase I involve NPC disassembly or NE rupture.As mentioned above, Nup132 depletion results in the loss of barrier function of the NE during anaphase I (Yang et al., S. pombe may be regulated by phosphorylation or other post-translational modifications of nucleoporins. Further studies are required to elucidate the role of post-translational modifications of Nup132 or other nucleoporins in the regulation of barrier function of NPCs and vNEBD during meiosis in S. pombe.Functional alterations in Nup132 or other nucleoporins may change the barrier function of the NE without inducing NPC disassembly. Post-translational modifications in nucleoporins may alter the barrier function of NPCs. In mammals, nucleoporin Nup50 is phosphorylated by ERK kinase, which in turn changes the affinity of importin \u03b2 to NPCs (Kosako et al., S. pombe cells produce spores, and a correlation of vNEBD with spore formation has been established (Arai et al., mes1 mutant, which is deficient in blocking the degradation of cyclin Cdc13 (Izawa et al., mes1 mutant shows no vNEBD (Arai et al., tws1 mutant, a meiosis-specific allelic mutant of Cdc2 (MacNeill et al., tws1 mutant, nuclear proteins diffuse to the cytoplasm and RanGAP1 localizes to the nucleus during meiosis I (Arai et al., S. japonicus (Aoki et al., The timing of vNEBD corresponds to meiosis II during which S. pombe produces spores under nutritionally starved conditions. These spores are metabolically inactive until they experience growth-favorable conditions (Shimoda and Nakamura, S. pombe (Nishijima et al., vNEBD occurs specifically during anaphase II and results in the diffusion of nuclear proteins into the cytoplasm and cytoplasmic proteins into the nucleus, which is similar to that observed during open mitosis. vNEBD may allow cytoplasmic proteins to function in the nucleus during or after anaphase II. Occurrence of vNEBD is correlated with spore formation (Arai et al., S. pombe. This provides an example in which the regulated barrier function of the NE plays an important role for regulating meiotic processes in eukaryotes.The phenomenon of vNEBD suggests the importance of \u201copening\u201d the gate between the nucleus and cytoplasm during meiosis. Nucleoporins and RanGAP1 may play key roles during open meiosis without physically breaking down the NE in The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Gan-Cao, coming from extracts of dried roots of Glycyrrhiza uralensis, Glycyrrhiza glabra, and Glycyrrhiza inflata, of which chemical analyses were performed (Zhang and Ye, A recent paper by Glycyrrhiza species (Fiore et al., According to these authors, 18-\u03b2-glycyrrhetinic acid and glycyrrhizin possess an anti-viral potential, assessing previous evidence about the antiviral properties of Glycyrrhiza extracts contributes in hampering a full comprehension of their beneficial effects. Licocalchone A downregulates the inflammation-induced P450 1B1 while isoliquiritigenin exerts an opposite effect, then differentially influencing the role of estrogens in chronic inflammation and in carcinogenesis (Dunlap et al., Molecular interactions between 18-\u03b2-glycyrrhetinic acid and glycyrrhizin modulates the inhibition of several drug-metabolizing enzymes and efflux transporters (Feng et al., The authors did not address either clinical trial about licorice activity against microbes.Glycyrrhiza-derived purified substances, such as glabridin, are recently addressed as promising tools to prevent bacterial infections and against parasites (Cheema et al., Moreover, the role exerted by licorice in blood pressure may influence immunity and DCs functionality, as the equilibrium between the inflammatory response induced by T cell and T cell suppressor responses is critical for the regulation of blood pressure levels (Rodr\u00edguez-Iturbe et al., in vitro, yet surely clinical evidence is needed.This evidence supports the idea that the introduction of a whatsoever can be considered as a beneficial phytochemical from raw plants in the highly complex network of signaling interactions between cells and organs, even considering the different individual ability coming from genetics and phenotypic polymorphism or from epigenetics and lifestyle, cannot allow physicians, as well as researchers, to foresee the \u201creal\u201d effect of that substance in the whole organism. An interesting hypothesis is that phytochemicals, as toxic substances, produced by plants to protect themselves are beneficial only in a restricted range of doses. In this perspective, there is interesting evidence about the role of low doses of phytochemicals, particularly flavonoids, on stress response and immune reactions The author confirms being the sole contributor of this work and approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "CCDC151), which has been shown to be essential in motile cilia of many animals and other vertebrates but its effects in humans was not observed until currently. We observed a novel nonsense mutation in a homozygous state in the CCDC151 gene (NM_145045.4:c.925G>T:p.[E309*]) in a clinically diagnosed PCD patient from a consanguineous family of Arabic ancestry. The variant was absent in 238 randomly selected individuals indicating that the variant is rare and likely not to be a founder mutation. Our finding also shows that given prior knowledge from model organisms, even a single whole-exome sequence can be sufficient to discover a novel causal gene.Primary ciliary dyskinesia (PCD) is an autosomal-recessive disorder characterized by impaired ciliary function that leads to subsequent clinical phenotypes such as chronic sinopulmonary disease. PCD is also a genetically heterogeneous disorder with many single gene mutations leading to similar clinical phenotypes. Here, we present a novel PCD causal gene, coiled-coil domain containing 151 ( Primary ciliary dyskinesia is a genetically heterogeneous ciliopathy inherited in an autosomal-recessive fashion [Bush et al., DNAI1, DNAH5, DNAH11, RPGR, and OFD1) had been identified and these accounted for just 40% of the cases [Zariwala et al., CCDC151 deficiency as a further cause of PCD.Understanding the genetic aetiology of PCD has been challenging. In 2007, only five genes , and a long history of wet cough with light-green colored sputum and his unaffected brother were studied . The parents\u2019 blood samples were also collected for further analysis. Details on all methods used in this study can be found in the Supp. Materials and Methods. Summary statistics from WES of proband can be found in Supp. Results.CCDC151 gene and the other was a frameshifting insertion in the ZNF595 gene (p.N201fs). The stop gain was absent in dbSNP, Exome Variant Server (EVS), our internal database (which includes his unaffected sibling), and 1000 Genomes Project databases, whereas the insertion was present (in a homozygous state) in many of our previously whole-exome sequenced controls (of Arabic ancestry) and also had a total MAF of 8.6% in EVS. The filtering process is depicted in Figurehttp://databases.lovd.nl/shared/variants/0000040146#04563).Initially, all LRoH regions identified by Plink [Purcell et al., PCR was used to amplify a region 221 bp long (harboring the stop gain) in the proband, the unaffected brother, and the parents, which was then subsequently sequenced (using Sanger sequencing method) and digested with AvrII enzyme to confirm the variant status is in the local population, a buccal swab sample from 238 randomly selected individuals of Saudi Arabian ancestry were collected . The PCR amplicons produced (using primers in Supp. To deduce how common the c.925G>T:p.(E309*) variant in CCDC151 gene was first identified as a potential human ciliome gene by Ostrowski et al. (CCDC151 was among the 110 proteins (with Accession no: BAB01602) identified by one-dimensional polyacrylamide gel electrophoresis analysis of human ciliary axonemes . More recently, Jerber et al. -dependent cilia in Drosophila. In the same analysis, they reported that Ccdc151 was expressed in tissues with motile cilia in zebrafish, and morpholino-induced depletion of the gene product lead to, similar to human PCD phenotypes, left\u2013right asymmetry defects [Jerber et al., Ccdc151 is strongly expressed in (and was restricted to) motile-ciliated tissues, where it is required for dynein arm assembly and for the transport of the docking complex Ccdc114 [Jerber et al., Ccdc151 is important in the control of IFT-dependent dynein arm assembly in many animals [Jerber et al., Ccdc151 in IMCD3 mouse cells resulted in a deregulated ciliary length [Jerber et al., Chlamydomonas ODA10 gene that is the homolog of the mouse Ccdc151 gene was calculated to be approximately 0.0009 . Additional evidence from the absence of mutation in EVS and internal database , GERP scores [Davydov et al., CCDC151 carried out by Jerber et al. \u03a6 mutations in autosomes were quantified in three healthy individuals (two of Arabic and one of European ancestry); and this figure was found to be approximately 60 on average. However, a very large proportion of these were in a heterozygous state and in olfactory, taste- and hearing-related genes. The amount of \u03a6 mutations that fell in the entire ciliome database (i.e. list 1 and 2) was on average one. The ciliome database contains within it 2,004 genes related to all types of cilia , but a crude estimate on the number of \u201crespiratory cilia\u201d-related genes (not including the known PCD genes) was estimated to be approximately 100 . As aforementioned, known PCD genes explain around 70% of PCD, which leaves the other 30% to these 100 genes. Thus, the probability of one of these \u03a6 mutations falling in one of the second-tier candidate PCD genes in an autozygous region , certain combinations of ultrastructural defects and/or sterility (or subfertility) are far from being completely understood [Vincensini et al., CCDC151 to cilia [Jerber et al., CCDC151 were not observed (until now) to associate these findings to the human model. Here, we report a homozygous nonsense mutation in CCDC151 in a patient who has been clinically diagnosed with PCD. The variant was screened in 238 unrelated individuals, and it was found to be absent in all of them indicating that the mutation is not a founder mutation and may have occurred relatively recently and/or is tribal specific. Nevertheless, CCDC151 adds to the already identified 25 genes in which mutations are known to cause PCD and indicates the important role CCDC151 plays in human respiratory ciliary function. Our findings also show that given prior knowledge from an animal model, even a single WES (with high read depth) can be adequate when pinpointing a novel causal gene.To conclude, although animal models have strongly linked"} +{"text": "C) to trigger intracellular signals appears central to neurodegeneration pathways, yet the physiological significance of such signals is rather puzzling. For instance, PrPC deregulation disrupts phenomena as diverse as synaptic transmission in mammals and cell adhesion in zebrafish. Although unrelated, the key proteins in these events -the NMDA receptor (NMDAR) and E-cadherin, respectively- are similarly modulated by the Src family kinase (SFK) Fyn. These observations highlight the importance of PrPC-mediated Fyn activation, a finding reported nearly two decades ago. Given their complex functions and regulation, SFKs may hold the key to intriguing aspects of PrP biology such as its seemingly promiscuous functions and the lack of strong phenotypes in knockout mice. Here we provide a mechanistic perspective on how SFKs might contribute to the uncertain molecular basis of neuronal PrP phenotypes affecting ion channel activity, axon myelination and olfactory function. In particular, we discuss SFK target proteins involved in these processes and the role of tyrosine phosphorylation in the regulation of their activity and cell surface expression.The ability of the cellular prion protein (PrP Yes, there are two paths you can go by but in the long run There's still time to change the road you're on(R. Plant and J. Page)C on neuronal cell surfaces is required for pathogenic prions and a\u03b2 oligomers to trigger cellular damage within lipid rafts, thereby stimulating neurite outgrowth anchor, PrP is unlikely to physically associate with cytosolic SFKs. Nevertheless, co-immunoprecipitation data indicate that, in epithelial cells, PrP and SFKs interact at least indirectly within a larger protein complex (Morel et al., C triggered Fyn activation and hyperphosphorylation of the NR2B subunit of the NMDAR at tyrosine residue 1472 (Um et al., C promotes embryonic cell adhesion by stabilizing E-cadherin at the cell-surface (M\u00e1laga-Trillo et al., C as a modulator of cell-cell adhesion in zebrafish and synaptic transmission in mammals converge at a common mechanism, namely the ability of SFKs to control the internalization of trans-membrane proteins by phosphorylating their endocytic signals and/or the corresponding binding molecules. Given the functional diversity of SFK target proteins, we reasoned that altered SFK activity may account for at least some of the distinct neuronal phenotypes observed in PrP knockout and transgenic mice (Figure At first glance, some of these findings have no apparent connection with each other, aside from the common involvement of SFKs. However, closer scrutiny reveals mechanistic similarities pertinent to our understanding of PrP function. This is particularly manifest in two of the aforementioned models, where the downstream targets of PrP turned out to be transmembrane proteins modulated by tyrosine phosphorylation. In the study by the Strittmatter lab, binding of a\u03b2 oligomers to PrPC clearly shows that the latter can influence the activity of ion channels (Um et al., C suppresses NMDAR activity and glutamate excitotoxicity via the inhibition of NR2D subunits (Khosravani et al., 2+-mediated excitotoxicity (Solomon et al., 2\u03b4-1 subunit and retain it at the ER, thus disrupting its delivery to the plasma membrane. Notably, while the molecular mechanisms of these two phenotypes seem unrelated, they are both consistent with the ability of SFKs to regulate the activity of ligand- and voltage-gated ion channels (Salter and Kalia, 2\u03b4-1 during biosynthesis is the most likely explanation for its reduced levels at the plasma membrane (Senatore et al., The NMDAR-mediated glutamate excitotoxicity induced by a\u03b2 oligomers and PrPv1.3 voltage-gated potassium channels are expressed along the dendrodendritic synapse and are negatively regulated by Src phosphorylation (Fadool and Levitan, v1.3 null mice have modified action potentials and deregulated potassium currents, resulting in structurally altered olfactory bulbs and a heightened sense of smell (Fadool et al., 2+ influx that triggers inhibitory GABA release (Chen et al., v1.3 channels, and NMDARs provides testable explanations for the olfactory defects of PrP knockout mice.Unique among mouse PrP knockout phenotypes was the finding of impaired olfactory behavior (Le Pichon et al., Re-examination of a mild, late-onset phenotype in PrP knockout mice led to the discovery of defects in the maintenance of peripheral myelin (Bremer et al., As with the olfactory phenotype of Le Pichon et al., copious evidence suggests a potential involvement of SFKs in the PrP-CDP phenotype. Comprehensive reviews on the role of Fyn during myelination are available elsewhere (Kramer-Albers and White, v1.5 and Kv2.1 potassium channels in Schwann cells is controlled by the interplay between Src, Fyn, PTP\u03b1, and PTP\u03b5 (Sobko et al., in vivo, this regulation might be relevant for re-myelination but not for OPC development (De Biase et al., Finally, voltage- and ligand-gated ion channels constitute further regulatory targets of SFKs during myelination. For instance, the phosphorylation state of KC in the proliferation and differentiation of various stem cell lineages (reviewed in Martin-Lanner\u00e9e et al., Altogether, the common regulation of ion channels, olfaction and myelination by PrP and SFKs suggests that this phenomenon may extend to some of the other subtle PrP phenotypes mentioned above. For instance, it would be interesting to see if the role of PrPC to elicit intracellular signals at the cell surface is central to its multiple roles in health and disease. Second, the specificity of these signals is largely determined by extrinsic factors such as the plasma membrane microenvironment and the availability of cell- and tissue-specific protein partners. Third, the ubiquitous expression and functional redundancy of SFKs, along with the diversity of their downstream targets offer plausible explanations for some of the striking facts of PrP biology, such as its functional promiscuity, the viability of PrP knockout mice, and the diversity of PrP transgenic/mutant phenotypes. Based on these considerations, we propose that PrPC acts as a gatekeeper between neuronal survival and toxicity by controlling the activity and/or endocytic trafficking of ion channels, synaptic proteins and cell adhesion molecules via SFKs. Testing these complex scenarios should prove a formidable but rewarding endeavor.Several key observations emerge from the present analysis. First, the ability of PrPThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Recent advances in brain connectivity research suggest that the human brain operates as a complex but economic global network. Novel approaches from graph theory have been applied to a variety of neuroimaging studies and have achieved great success. However, the brain network architecture and plasticity patterns vary across different brain developmental periods, learning activities, and disease effects. Translation of descriptive imaging levels into clinical and other exploitation crucially depends on the potential to induce and monitor plastic changes in the network of interest. This special issue is intended to stimulate the continuing efforts in understanding the architecture and plasticity patterns of brain networks. It also aims to broaden the attention not only to the researchers who are currently working on these areas but to the general scientific audience who are interested in the brain structures and functions as well.This special issue includes eleven papers that presented an up-to-date progress of MR neuroimaging methods on brain network architecture and plasticity investigation employing various neuroimaging modalities: structural MRI, diffusion MRI, and functional MRI. In these papers, there are five studies focused on brain diseases, including Alzheimer's disease, mild cognitive impairment, mild traumatic brain injury, and major depressive disorder. Two studies investigated the brain plasticity changes after visual or hearing deprivations. Also, there are two studies that assessed volunteers after mediation or cognitive training, and, finally, the remaining two studies are on the methodological development for brain network construction.Brain Diseases. In the paper entitled \u201cAbnormal Resting-State Functional Connectivity Strength in Mild Cognitive Impairment and Its Conversion to Alzheimer's Disease\u201d by Y. Li et al., an investigation was performed for individuals diagnosed with mild cognitive impairment. With a 2-year follow-up, those individuals converted to Alzheimer's disease were compared with nonconverters for their alterations on functional connectivity strength and seed-based functional connections.The paper entitled \u201cTopological Properties of Large-Scale Cortical Networks Based on Multiple Morphological Features in Amnestic Mild Cognitive Impairment\u201d by Q. Li et al. investigated the topological properties of cortical networks based on geometric measures change in aMCI patients compared with normal controls.The paper entitled \u201cMultilevel Deficiency of White Matter Connectivity Networks in Alzheimer's Disease: A Diffusion MRI Study with DTI and HARDI Models\u201d by T. Wang et al. conducted an evaluation of how the fiber tractography method could influence the construction of brain white matter network and eventually affect the extraction of brain network properties, as well as group comparison using Alzheimer's disease patients as examples.In the paper entitled \u201cCompensation through Functional Hyperconnectivity: A Longitudinal Connectome Assessment of Mild Traumatic Brain Injury\u201d by A. Iraji et al., patients with mild traumatic brain injury were studied for possible brain functional alterations. Brain functional network was constructed through predefined seeds in a 358-landmark mask and a groupwise clustering algorithm was employed to identify general patterns of functional hyperconnectivity.The paper entitled \u201cReorganization of Anatomical Connectome following Electroconvulsive Therapy in Major Depressive Disorder\u201d by J. Zeng et al. presented a study for patients with major depressive disorder that were treated by electroconvulsive therapy. The plasticity of white matter pathway was evaluated.Sensory Deprivation. In the paper entitled \u201cAlterations of Regional Spontaneous Brain Activity and Gray Matter Volume in the Blind\u201d by A. Jiang et al., authors investigated how the early blindness shapes the regional spontaneous brain activity and gray matter volume in visual areas compared to sighted controls. A correlation analysis is also performed between the onset age of blindness and the brain structure and functions.In the paper entitled \u201cFunctional Reorganizations of Brain Network in Prelingually Deaf Adolescents\u201d by W. Li et al., authors used resting-state fMRI to study prelingually deaf adolescents to access the possible brain network reorganizations due to their experience.Training Influence. The paper entitled \u201cState and Training Effects of Mindfulness Meditation on Brain Networks Reflect Neuronal Mechanisms of Its Antidepressant Effect\u201d by C.-C. Yang et al. designed a longitudinal analysis investigating resting-state fMRI both before and after 40 days of meditation training. Differences in functional connectivity both between states (rest versus meditation) and between timepoints (before versus after training) were assessed and reveal functional specificity of plastic reconfiguration of key networks implied in MDD.The paper entitled \u201cThe Exercising Brain: Changes in Functional Connectivity Induced by an Integrated Multimodal Cognitive and Whole-Body Coordination Training\u201d by T. Demirakca et al. investigated the impact of \u201clife kinetik\u201d training on brain plasticity in terms of an increased functional connectivity during resting-state functional magnetic resonance imaging (rs-fMRI). The training is an integrated multimodal training that combines motor and cognitive aspects and challenges the brain by introducing new and unfamiliar coordinative tasks.Methodology Development. The paper entitled \u201cClosely Spaced MEG Source Localization and Functional Connectivity Analysis Using a New Prewhitening Invariance of Noise Space Algorithm\u201d by J. Zhang et al. proposed a prewhitening invariance of noise space as a new method for localizing closely spaced and highly correlated cortical sources under real magnetoencephalography (MEG) noise, to facilitate the source-level functional connectivity analysis.The paper entitled \u201cNode Detection Using High-Dimensional Fuzzy Parcellation Applied to the Insular Cortex\u201d by U. Vercelli et al. investigated a fuzzy parcellation scheme that partitions insular cortex into a number of regions based on the variances of their functional signals. Furthermore, the identified 12 clusters located in the insular cortex are found to best correlate with distinct brain areas that subserve different brain circuits/functions."} +{"text": "Functionally matured microRNAs (miRNAs) are small single-stranded non-coding RNA molecules which are emerging as important post-transcriptional regulators of gene expression and consequently are central players in many physiological and pathological processes. Since the biological roles of individual miRNAs will be dictated by the mRNAs that they regulate, the identification and validation of miRNA/mRNA target interactions is critical for our understanding of the regulatory networks governing biological processes. We promulgate the combined use of prediction algorithms, the examination of curated databases of experimentally supported miRNA/mRNA interactions, manual sequence inspection of cataloged miRNA binding sites in specific target mRNAs, and review of the published literature as a reliable practice for identifying and prioritizing biologically important miRNA/mRNA target pairs. Once a preferred miRNA/mRNA target pair has been selected, we propose that the authenticity of a functional miRNA/mRNA target pair be validated by fulfilling four well-defined experimental criteria. This review summarizes our current knowledge of miRNA biology, miRNA/mRNA target prediction algorithms, validated miRNA/mRNA target data bases, and outlines several experimental methods by which miRNA/mRNA targets can be authenticated. In addition, a case study of human endoglin is presented as an example of the utilization of these methodologies. MicroRNAs (miRNAs) are an endogenous family of single-stranded 20-25 nucleotide non-coding RNAs that play a critical role in posttranscriptional gene regulation by acting as guide molecules for the miRNA-induced silencing complex (miRISC) to inhibit gene expression by targeting specific mRNAs for translational inhibition and/or degradation . It is now clear that the expression levels of miRNAs vary widely; some are ubiquitously expressed, while others are expressed in a tissue- and/or cell-specific manner, and many show spatiotemporal expression patterns . Importal., 2009; Dombkowal., 2009; Friedmaal., 2009; Gurtan al., 2009). Computal., 2009). Therefal., 2009; Fabian al., 2009). Althoual., 2009; Bartel,al., 2009; Selbachal., 2009), recental., 2009; Mendellal., 2009).The vast majority of functional miRNAs are produced by a canonical multistep biogenic process which is initiated in the nucleus and is completed in the cytoplasm Figure 1 and is retained within the miRISC Figure 1 is found . ImportWith few exceptions, MREs are primarily located in the 3\u2032-untranslated region (3\u2032-UTR) of mRNAs and once recognized, mature miRNAs imperfectly base pair with MREs following a set of rules which have been experimentally and computationally identified and/or deadenylation, decapping, and subsequent decay by a number of silencing factors that are scaffolded to this complex by TNRC6A/GW182 Figure 1 . There http://microrna.gr/microT) even these two algorithms would benefit from more current data input. To begin to address this critical question, several recent review articles have compared and contrasted many of the miRNA/mRNA target algorithms currently available , Targetal., 2009), Pictaral., 2006), and Elal., 2007). The laal., 2007) provideal., 2011; Grimsonal., 2011), DIANA-al., 2013; Reczko al., 2013), and thal., 2010) recentlal., 2010) and Taral., 2010; Grimsonal., 2010) have beIt is also significant to note that updated algorithms identify up to 60 % of all available miRNA/mRNA targets and provide only one valid target in approximately every three predicted targets . It is Given the above review of miRNA biology and miRNA/mRNA target prediction algorithms, we propose the following \u201cwork flow\u201d scheme Figure 2 for the For a case study we have chosen to analyze the human endoglin gene (ENG) for potential MREs. Endoglin is a homodimeric co-receptor for transforming growth factor beta (TGF\u03b2) and is known to play a regulatory role in TGF\u03b2 signaling or miRNA of interest has been chosen, it must then be analyzed by miRNA/mRNA target prediction algorithms Figure 2. Both thwww.ensembl.org) and in the 3\u2032-UTR, the seed sequence binding type, whether or not the predicted MRE is conserved , what sal., 2013). This ial., 2013; Wang etal., 2013) and \u223c70al., 2013). Importal., 2013; Park etal., 2013; Sandberal., 2013; Tan et al., 2013). TherefInterestingly, the human ENG gene generates two distinct mRNAs through alternative splicing, which results in isoforms that differ in a portion of their CDS and 3\u2032-UTR . As a result, Table 1Since the mRNA isoform which encodes S-endoglin harbors the longest 3\u2032-UTR, the Diana-microT-CDS algorithm will only utilize this sequence for computing miRNA/endoglin mRNA target interactions. When this analysis is performed, a total of 259 (threshold set to 0.4) miRNAs are predicted to interact with the human S-endoglin mRNA isoform at 797 individual MREs, with 186 target sites located in the CDS and 611 sites in the 3\u2032-UTR (data not shown). Table 1In contrast to Diana-microT-CDS, the TargetScan algorithm results include the identified miRNAs and the predicted location of MREs in the 3\u2032-UTR but not in the CDS. However, this tool does allow the user to analyze any annotated splice variant for a given gene. For example, TargetScan will analyze both L-endoglin and S-endoglin 3\u2032-UTRs. The TargetScan results also include the number and type of seed match of conserved and poorly conserved miRNA binding sites, and a total context score (predicted efficacy of targeting) . Interehttp://www.microrna.gr/tarbase) . DIANA-al., 2006). http://mirtarbase.mbc.nctu.edu.tw/) and > 500,000 interactions derived from high-throughput experiments . At leaal., 2014) and miRal., 2009), are al-throughput sequencing of RNA isolated by crosslinking immunoprecipitation) experimental approach involves the transfection of a given miRNA mimic into a cell line of choice followed by ultraviolet (UV) cross-linking to generate AGO/miRNA/RNA cross-linked regions Table 7, human eUCUCUAAGGAAGCGCAUUUC 3\u2032, the partially complementary motifs are underlined) was identified 40 nts downstream from the transcription initiation start site. Given that this predicted MRE is harbored in the 5\u2032-UTR region of the human endoglin mRNA isoforms, miR-522-3p/endoglin mRNA interactions would not be identified by the target algorithms discussed above since they are not programmed to analyze this region. Furthermore, with the tendency of miR-522-3p to interact with noncanonical MRE sequences, Tan et al. Table . These ial. (2014) demonstIn conclusion, it is important to note that although only two of the ten DIANA-TarBase v7.0 cataloged experimentally identified miRNA/human endoglin mRNA interactions Table 7 Selbach, there iFor this review article we subjected the human endoglin mRNA to Diana-microT-CDS and TargetScan miRNA target analyses and examined DIANA-TarBase v7.0 data sets of cataloged and published experimentally supported miRNA/human endoglin mRNA interactions (see above). Given that the majority of the identification and cataloging of miRNA/mRNA target interactions by DIANA-TarBase v7.0 result from high-throughput techniques withouthttp://www.ncbi.nlm.nih.gov/pubmed). Six publications were identified demonstal., 2014) . Shyu eal., 2014) speculaal., 2014). Therefal., 2014; Vasudeval., 2014), it is miRNA profiling expression experiments utilizing ovarian cancer cells and ovarian cancer clinical samples demonstrated that a number of miRNAs were aberrantly expressed, including miR-370-3p, which was down-regulated in these studies . ImportTijsen et al. (2014) Table focused Again, it is important to note that the miR-15/107 family members, miR-16-5p, miR-103a-3p, and miR-107 were identified to interact with human endoglin mRNAs by the HITS-CLIP technique Table 7 demonstGiven that endoglin has been established to play a regulatory role in TGF\u03b2 signaling . This fOnce the plethora of information from prediction algorithms, published validations of miRNA/mRNA interactions, and manual sequence inspections of miRNA binding sites has been assembled, prioritization of specific miRNA/mRNA target interactions to investigate can more effectively proceed. Among the number of putative miRNA/human endoglin mRNA interactions documented above, the remainder of this review article will focus on miR-370 Table 8 since thClearly a given miRNA and its target mRNA must be co-expressed in order for the miRNA to regulate the expression of a given biological target. Therefore, miRNA and target mRNA co-expression experimental studies should be performed first Figure 2, since tCo-expression is typically demonstrated by simply performing Northern blot analysis or quantitative real-time PCR (qPCR) using total RNA isolated from a specific cell type or tissue, and probes or primers specific for a given miRNA and mRNA target . We recin situ hybridization and immunohistochemical experiments utilizing paraffin-embedded, formalin-fixed tissues to address the question of co-expression . Additiovo, 2010; Sansom ovo, 2010).As described in the \u201cAnalysis of the Published Literature\u201d section above Table 8, recent Importantly, Chen et al. (2014) initiatAfter the demonstration of co-expression of the miRNA and target mRNA of interest, the physical interaction of a specific miRNA with a candidate MRE harbored in a target mRNA needs to be confirmed Figure 2. The majFor construction of chimeric luciferase reporter constructs, the predicted MRE sequence from the target gene, most often located in the 3'-UTR but also in the 5'-UTR and CDS see , is clohttp://www.mirbase.org) and a passenger strand that is split in two separate antisense chemically synthesized LNA modified RNA oligonucleotide strands . After transfection into cells, the segmented nature of the passenger strand ensures that only the mature miRNA (guide strand) is loaded into the RISC with no resulting miRNA activity from the passenger strand. Regardless of the chemical makeup of the mimic utilized, transfection of a miRNA mimic into cells will increase the proportion of RISC containing this particular miRNA and therefore, gain-of-function studies can assess the biological consequences (i.e. repression of luciferase reporter levels/activity) resulting from an increase in the activity of the mimicked miRNA . In contrast, miRNA inhibitors, which are utilized for loss-of-function experiments, are chemically synthesized, single-stranded, modified antisense RNA oligonucleotides which are designed to bind with and form highly stable heteroduplexes with the complementary endogenous miRNAs when introduced into cells engineeAlthough the ability of miRNAs to repress the activity of a chimeric luciferase reporter gene is a useful screening device, it remains a surrogate assay for testing the effects of miRNAs on their putative mRNA targets. Therefore, after confirming the physical interaction of a miRNA with a candidate MRE harbored in target mRNAs by reporter assays, we recommend that miRNA gain- and loss-of-function transfection experiments also be performed to validate miRNA-mediated post-transcriptional regulation of target genes of interest Figure 2. Experimental manipulation of endogenous miRNA activity by miRNA mimic and miRNA inhibitor transfection should correspond to predictable changes in target protein levels . TherefAlthough gain- and loss-of-function experiments are powerful, it is important to remember that results can be confounded by side effects of transfection . Under in vivo . Recallal., 2014). These al., 2014). Alternal., 2014). Therefal., 2014; Helwak al., 2014; Martin al., 2014; Tan et al., 2014). Chen et al., (2014) demonstAfter miRNA gain- and loss-of-function transfection experiments have confirmed that a given miRNA mimic and inhibitor mediate the inverse protein expression of a target gene of interest, it is finally necessary to demonstrate that this regulation equates to changes in biological function Figure 2. Dependiin vivo gain- and loss-of-function experiments in mice or rats . For ex al, 2014; Montgom al, 2014), by inf al, 2014; Hsu et al, 2014; Huang e al, 2014), or by al, 2014; Kota et al, 2014; Miyazak al, 2014). In con al, 2014; McClure al, 2014), by inf al, 2014; Seeger al, 2014; Tijsen al, 2014), or by al, 2014; Baigude al, 2014).Chen et al. (2014) demonstmiRNAs are emerging as important post-transcriptional regulators of gene expression and consequently are central players in many physiological and pathological processes should equate to altered biological function. To date only a small proportion of miRNA/mRNA target interactions have been functionally validated. The unique experimental outline described here can be applied to the validation of any miRNA/mRNA interaction. As relevant targets are identified, the biological functions of a specific miRNA can be unraveled and assist in development of miRNA therapeutics.The authors declare that they have no conflict of interest.We express our appreciation to Ms. Emily Keeler for generating the figures and tables."} +{"text": "In March 2014, over 400 individuals from 35 countries in sub-Saharan Africa and 59 international partner organizations gathered in Accra, Ghana for an integrated Community Case Management (iCCM) Evidence Review Symposium. The objective was 2-fold: first, to review the current state of the art of iCCM implementation and second, to assist African countries to integrate lessons learned and best practices presented during the symposium into their programmes. Based on the findings from the symposium this supplement includes a comprehensive set of articles that provide the latest evidence for improving iCCM programs and ways to better monitor and evaluate such programs. Since early 2000 the use of integrated community case management (iCCM) strategy to deliver pneumonia, malaria and diarrhea treatments to children under 5 has dramatically increased. In 2005 there were only 10 countries in sub-Saharan Africa with policies supporting implementation of iCCM of which 7 included pneumonia treatment [iCCM, in the hands of well trained, supplied and supervised community health workers can reduce child mortality ,4. RecogSince the joint statement was released an increasing amount of evidence has been generated on the strengths and limitations of iCCM, as well as the outcome and impact of iCCM within a variety of different country contexts . A numbeBetween 3 and 5 March 2014, over 400 individuals from 35 countries in sub-Saharan Africa and 59 international partner organizations gathered in Accra, Ghana for an iCCM Evidence Review Symposium. The objective of the symposium was 2-fold: first, to review the current state of the art of iCCM implementation by bringing together researchers, donors, governments, implementers and partners to examine the current iCCM implementation landscape and status of evidence in key programme areas, in order to summarize lessons learned and best practices, and identify priorities and gaps in knowledge for improving maternal-newborn and child health. Second, to assist African countries to integrate lessons learned and best practices presented during the evidence symposium into their programmes and identify key actions to include in their national plans.We conceptualized a theory of change model as to what factors may increase utilization of quality iCCM services . In addition lessons learned were documented in each area. This supplement includes articles, based in part on these experiences and lessons learned. The thematic and additional areas, their relationship to the model, and their associated articles are shown in Box 1Coordination, Policy Setting and Scale-up: the current state of iCCM policies in Africa and challenges in development of policy and scale up\u2013 RELATES TO POLICY1. Rasanathan et al. Community case management of childhood illness in Sub-Saharan Africa: Findings from a cross-sectional survey on policy and implementation (article # 020401)Rasanathan et al. Where to from here? Policy and financing of integrated community case management of childhood illness (iCCM) in sub-Saharan Africa (article # 020304)Human Resources and Deployment: community health worker (CHW) selection, geographic disbursement, motivation and retention \u2013 RELATES TO DEPLOYMENT2. Pratt et al. Spatial distribution and deployment of community-based distributors implementing integrated community case management (iCCM): GIS mapping study in three South Sudan states (article # 020402)Supervision & Performance Quality Assurance: strategies to ensure high quality care including strategies for effective training, use of alternative models for supervision, and the role of mHealth to support and motivate CHWs to provide quality care \u2013 RELATES TO QUALITY3. Bosh-Capblanch and Marceau. Training, supervision and quality of care in integrated community case management (iCCM) programmes in sub-Saharan Africa (article #020403)Strachan et al. The scale up of integrated community case management of malaria, pneumonia and diarrhoea across three African countries: A qualitative study exploring lessons learnt and implications for implementation (article # 020404)Supply Chain Management: which systems ensure continuous supply, how best to forecast needs \u2013 RELATES TO SUPPLY4. Chandani et al. Evidence for improving community health supply chains from Ethiopia, Malawi, and Rwanda (article # 0204005)Sheishia et al. Strengthening community health supply chain performance through an integrated approach: Using mHealth technology and multilevel teams in Malawi (article # 020406)Costs, and cost-effectiveness and financing: identifying cost drivers, improving cost-effectiveness and the importance of minimizing patient costs \u2013 RELATES TO POLICY AND DEMAND5. Jarrah et al. The costs of integrated community case management (iCCM) programs: A multi-country analysis (article # 020407)Monitoring, Evaluation and Health Information Systems: innovations in monitoring, integrating with health management information systems, using results to drive programmatic decision-making and improvements, evaluation design and methods \u2013 RELATES TO ALL AREAS6. Guenther et al. Routine monitoring systems for integrated community case management programs: Lessons from 18 countries in sub\u2013Saharan Africa (article # 020301)Oliphant et al. Multi-country analysis of routine data from integrated community case management programs in sub-Saharan Africa (article # 020408)Diaz et al. A proposed model to conduct process and outcome evaluations and implementation research of child health programs in Africa using integrated community case management as an example (article # 020409)Demand generation and social mobilisation: the relationship between iCCM and care-seeking, treatment utilisation and treatment adherence, effective strategies to generate demand \u2013 RELATES TO DEMAND7. Sharkey et al. Demand generation and social mobilisation for iCCM and child health: Lessons learned from successful programmes in Niger and Mozambique (article # 020410)Impact and outcome evaluations: Issues with measuring mortality and using coverage to model mortality\u2013 RELATES TO COVERAGE AND MORTALITY8. Amouzou et al. Assessing the impact of the integrated community case management (iCCM) programmes on child mortality: Review of early results and lessons learned in sub-Saharan Africa (article # 020411)Friberg et al. Using the Lives Saved Tool as part of evaluations of community case management programs (article # 020412)Additional topic areas:Newborns \u2013 Aboubaker et al. Community health workers: a crucial role in newborn health and survival (article # 020303)Research \u2013 Wazny et al. Setting global research priorities for integrated community case management (iCCM): Results from a CHNRI exercise (article # 020413)Private sector \u2013 Awor et al. Integrated community case management and the private sector in africa \u2013 a relevant experiences and potential next steps (article # 020414)Conclusions \u2013 Young et al. The way forward for integrated community case management programmes (article # 020304)In regards to policy and scale up Rasanathan et al report on the results of survey on iCCM in sub-Saharan Africa and in a viewpoint summarizing policy issues that impact scale up and sustainability. In the area of human resources and deployment Pratt et al detail the use of GIS mapping in three states in Southern Sudan to improve deployment of CHWs. For training and quality Bosh-Capblanch et al provides a systematic review of training supervision and quality of care, while Stratchan et al present a qualitative assessment of implementation practices in 3 African countries. In regards to maintaining supply chains Chandani et al describe how three countries were able to ensure commodities and supplies reached community health workers, while Sheishia et al describe an innovative technology to track supplies in Malawi. To examine costs of iCCM, Jarrah et al describe the cost drivers of iCCM in multiple countries using standardized methods. In the area of monitoring and evaluation Guenther et al put forth components and attributes of a comprehensive monitoring system for iCCM, while Oliphant et al use routine monitoring from multiple country programs to demonstrate the utility of these data to examine key factors of program success or failure. Diaz et al review the study design and data elements collected for multiple recent evaluations of iCCM to suggest how such evaluations can be done in the future. In regards to demand, Sharkey et al present experience in two countries and the lessons learned on demand generation and social mobilization for iCCM. Finally, in the area of impact and outcome evaluation Agbessi et al report on the limitations of measuring and using mortality to assess iCCM as an end point in several countries in Sub-Sharan Africa and Friberg et al demonstrate how the Lives Saved Tool could be used to model the mortality impact of iCCM.In addition to the articles covering the key thematic areas of the symposium we also have three additional articles. Awor et al describe how the private sector can contribute to iCCM. Aboubaker et al also present the role of Community Health Workers in newborn survival. Based on a systematic process to prioritize research areas, known as CHNRI Wazny et al report on priority iCCM research areas that are still needed. Finally, using the evidence available, Young et al suggest a way forward to improve and sustain iCCM where it is needed. This comprehensive set of articles provides the latest evidence for improving iCCM programs and ways to better monitor and evaluate such programs."} +{"text": "Among the tools proposed to assess the athlete's \u201cfatigue,\u201d the analysis of heart rate variability (HRV) provides an indirect evaluation of the settings of autonomic control of heart activity. HRV analysis is performed through assessment of time-domain indices, the square root of the mean of the sum of the squares of differences between adjacent normal R-R intervals (RMSSD) measured during short (5 min) recordings in supine position upon awakening in the morning and particularly the logarithm of RMSSD (LnRMSSD) has been proposed as the most useful resting HRV indicator. However, if RMSSD can help the practitioner to identify a global \u201cfatigue\u201d level, it does not allow discriminating different types of fatigue. Recent results using spectral HRV analysis highlighted firstly that HRV profiles assessed in supine and standing positions are independent and complementary; and secondly that using these postural profiles allows the clustering of distinct sub-categories of \u201cfatigue.\u201d Since, cardiovascular control settings are different in standing and lying posture, using the HRV figures of both postures to cluster fatigue state embeds information on the dynamics of control responses. Such, HRV spectral analysis appears more sensitive and enlightening than time-domain HRV indices. The wealthier information provided by this spectral analysis should improve the monitoring of the adaptive training-recovery process in athletes. The optimization of the training process in elite athletes requires the quantification of the training loads (Borresen and Lambert, most reliable and practically applicable measure for day-to-day monitoring\u201d (Plews et al., In a recent review (Buchheit, However, despite its accessibility/simplicity, even with all the above-mentioned methodological improvements (see Plews et al., In our view, recording HRV clues in both supine and standing positions is also convenient and provides more information about the actual autonomic settings, their interplay and how they are resorted (Schmitt et al., A recently published study accurately displayed how individual patterns of spectral analysis of HRV divert in \u201cfatigue\u201d states from \u201cno fatigue\u201d condition (Schmitt et al., In summary, RMSSD measures and their derived variables have an effective practical usefulness, which can help the practitioner to identify a global \u201cfatigue\u201d level. However these variables do not allow the clustering of different sub-categories of \u201cfatigue,\u201d at variance with the spectral HRV analysis in both supine and standing positions, which likely consider the current ability to control in a dynamic setting.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Chronic tendinopathy is a painful common condition affecting athletes as well as the general population undergoing to tendon overuse. Although its huge prevalence, little is known about tendinopathy pathogenesis, and even cloudier is its treatment. Traditionally, tendinopathy has been defined as a lack of tendon ability to overcome stressing stimuli with appropriate adaptive changes. Histologic studies have demonstrated the absence of inflammatory infiltrates, as a consequence conventional antinflammatory drugs have shown little or no effectiveness in treating tendinopathies. New strategies should be therefore identified to address chronic tendon disorders. Angiofibroblastic changes have been highlighted as the main feature of tendinopathy, and vascular endothelial growth factor (VEGF) has been demonstrated as one of the key molecules involved in vascular hyperplasia. More recently, attention has been focused on new peptides such as Substance P, nitric oxide, and calcitonin gene-related peptide (CGRP). Those new findings support the idea of a nerve-mediated disregulation of tendon metabolism. Each of those molecules could be a target for new treatment options. This study aimed to systematically review the current available clinical and basic science in order to summarize the latest evidences on the pathophysiology and its effect on treatment of chronic tendinopathy, and to spread suggestions for future research on its treatment. With the increasing number of amateur sport practitioners, a growing prevalence of tendinopathy has been recorded in the last few years in Europe and United States , between June and August 2015. The combinations of key-words used were the following: \u201c(\u201ctendinopathy\u201d[MeSH Terms] OR \u201ctendinopathy\u201d[All Fields]) AND ,\u201d \u201c(\u201ctendinopathy\u201d[MeSH Terms] OR \u201ctendinopathy\u201d[All Fields]) AND (\u201csubstance p\u201d[MeSH Terms] OR \u201csubstance p\u201d[All Fields] OR \u201cp substance\u201d[All Fields]),\u201d \u201c(\u201ctendinopathy\u201d[MeSH Terms] OR \u201ctendinopathy\u201d[All Fields]) AND OR \u201cneurotransmitter agents\u201d[All Fields] OR \u201cneurotransmitter\u201d[All Fields]).\u201d The search was aimed to retrieve any level of evidence studies concerning molecular pathways involved in pathogenesis of tendinopathy, clinical associated features and therapeutic implications. Both clinical and experimental in vivo and in vitro studies were included. No study types were excluded except for literature reviews and case reports. No time interval for publication was set. Of each of the retrieved articles, the whole bibliography was carefully checked to enrich the research with possible studies relevant for the present work. Results of the studies were read, analyzed, and tabulated. The included studies have been divided into three categories: vascular function, nervous function, and therapeutic studies. The study selection process was carried out as shown in Figure Articles research has been carried out using PubMed online database , fibroblast growth factor-2 (FGF-2), cyclooxygenase-2 (COX-2), sphingosine kinase-1 (SPHK1), (transforming growth factor) TGF-\u03b1, VEGF-A, and VEGF-C. Comparable results were obtained by Petersen et al. (p = 0.0001 and p = 0.046 respectively). An in vivo study on rabbits, by means of specific exercise protocols, Andersson et al. ; group 2: knock-out (iNOS\u2212/\u2212); and group 3: knockout (iNOS\u2212/\u2212) + systemic NO synthase inhibition through aminoguanidine (AG) administration. When systematically inhibiting the NO synthase in iNOS\u2212/\u2212 mice (group 3), the cross-sectional area of the healing Achilles tendon was significantly reduced. However, no significant differences were found between the wild-type (group 1) and the knock-out mice (group 2) concerning both the cross-sectional area and biomechanical features of the healing Achilles tendon. Moreover, the same authors and nitric oxide synthase (NOS) expression have been carried out, in order to evaluate endothelial activation during tendinopathy and its effects on tendon tissue. An overuse protocol applied to supraspinatus tendons was evaluated by Szomor et al. , that fo2, in patellar tendons with tendinopathy in respect to normal tendons.It has been recently considered a role for neurotransmitters in the evolution of tendinopathy. The main molecule that has been investigated is Substance P (SP), which is known to play several roles in proliferation of fibroblasts, angiogenesis, and pain transmission patches on elbow extensors tendon (McCallum et al., The SP administration in tendinopathy has been extensively studied by Burssens et al. who studThe increasing knowledge concerning tendon dysfunction, clinically expressed as tendinopathy, leads to the individuation of a huge array of factors implied in the pathogenesis and repair mechanisms of this disease. Among single molecules and pathways involved in pathogenesis and healing process of tendinopathy, vascular, and neuronal factors play a major role (Papalia et al., Some studies have investigated the role that VEGF has in both the pathogenesis and to the healing response of tendinopathy, also using VEGF and its splice variants as an efficient treatment (Zhang et al., SP administration in surgically repaired rat tendons (Burssens et al., The local vascular activation is actually considered a still unclear chapter of the wide topic of tendinopathy. Studies have demonstrated an increased expression of NOS isoforms produced by endothelium in diseased tendons (Szomor et al., However, since VEGF and its pathways are the broadly known factors among a so complex pathogenesis, it should probably be the main factor to pay efforts on. An interesting research line is the regulation of VEGF action during the pathogenesis of tendinopathy (Lu et al., Vascularization and neuronal transmission play a key role in determining the pathogenesis of the tendinopathy. The mainly known factors implied in the process are VEGF, Substance P, and Nitric Oxide, although their exact role in the mechanism of tendinopathy is not well determined. More research should be carried out, especially studies involving human subjects, in order to assess the timing of action of those factors, to find out how therapies targeted to the phase of the disease process may fasten the healing process and the clinical recovery.AD, RP, and VD supervised the articles selection process and reviewed the final manuscript. SV, BZ and GT provided articles selection, manuscript writing, and table filling up.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "A selection of papers prepared by participants of 7th Young Scientists School SBB\u201915 is presented in three Supplementary Issues to the journals of BMC series, namely, BMC Genomics, BMC Genetics and BMC Microbiology.http://conf.nsc.ru/sbb2015). The series of Young Scientists Schools in Systems Biology and Bioinformatics (SBB) started several years ago, in 2008, as educational workshop associated with the large international conference series on bioinformatics known as BGRS\\SB (Bioinformatics of Genome Regulation and Structure\\Systems Biology) (http://conf.bionet.nsc.ru/bgrssb2016). After its inauguration year of 1998, BGRS/SBB held biannually at the Institute of Cytology and Genetics SB RAS in Novosibirsk. In a recent decade, it became a prime meeting venue for biologists, computer scientists, physicists, mathematicians and biochemists working in an interdisciplinary field of systems biology and computer genomics. Being the largest system biology meeting series in Russia, BGRS\\SB-14 attracted participants from 27 countries. In the past BioMedCentral had published series of special issues based on best materials presented at the conference in BMC Genomics , BMC Evolutionary Biology , BMC Genetics and BMC Systems biology . In addition, the protein structure related studies presented by BGRS/SBB participants were collated as special issues in Journal of Bioinformatics and Computational Biology . [SBB\u201915 took place June 22nd-25th in Novosibirsk, Russia and data analysisEvolutionary bioinformaticsSystems biology and gene network modelingThe scientific topics discussed in 2015 were presented at the following sections:In SBB\u201915 Supplements to BMC journals Genomics, we collected the best studies presented at the conference. Due to the general breadth of the field of Systems Biology and Bioinformatic, the manuscripts were accepted into three separate tracks, thus forming three Supplementary Issues, BMC Genomics, BMC Genetics and BMC Microbiology. Below we will describe contents of these issues.The paper by Fedoseeva et al. titled \u201cComparative transcriptional profiling of renal cortex in rats with Inherited Stress-Induced Arterial Hypertension and normotensive Wistar Albino Glaxo rats\u201d dissect genetic underpinnings of arterial hypertension, a disease which cannot be explained as progression along a straightforward route . Hence, The paper of Klimov et al. \u201cGenome-wide transcriptome analysis of hypothalamus in rats with inherited stress-induced arterial hypertension\u201d continues in the wake of previous paper with a systems biology analysis of expression changes in hypothalamus of the same two strains of rats, and pinpointed the changes within three categories of genes: \u2018the regulation of hormone level\u2019, \u2018the regulation of the blood pressure level\u2019, and \u2018the immune system processes\u2019 .The paper of Lavrov and co-authors titled \u201cFrequent variations in cancer-related genes may play prognostic role in treatment of patients with chronic myeloid leukemia\u201d convincingly demonstrated that the gene variants present or absent in the genome of patient influence relative efficiency of the therapy for chronic myeloid leukemia, thus, paving a way to personalized approach to the treatment of this disease .Finally the paper \u201cGenomic Determinants of Birth Weight Variability in the Pig\u201d by Wang and co-author explored the polimorphisms influencing the weight of newborn piglets, and highlighted a number of genes involved in glucose and lipid metabolism as well as in maternal-fetal lipid transport as important for this agriculturally important trait. Additionally, there a basic science related vibe in this study as it may provide some mechanistic explanation to relative success of intrauterine competition of the fetuses in species with larger size of litter .http://www.biomedcentral.com/1471-2164/16/S13/S1).The paper by Ivanov et al.\u201cNon-random fragmentation patterns in circulating cell-free DNA reflect epigenetic regulation\u201d convincihttp://www.biomedcentral.com/1471-2164/16/S13/S2) deals with the peculiarities of the exon-intron structure, which affect the functionality of the encoded protein. The paper shows that protein functional sites predominantly encoded by relatively long exons.The paper by Medvedeva et al. \u201cComputer analysis of protein functional sites projection on exon structure of genes in Metazoa\u201d describes a theoretical model that assesses tissue-specific gene knockout effect on gene expression in various structures of the brain, and predicts that the most pronounced tissue-specific effects are detected for genes that participate in the apoptosis/survival network [The paper by Petrovskiy et al. \u201cPrediction of tissue-specific effects of gene knockout on apoptosis in different anatomical structures of human brain\u201d .Gunbin and colleagues continued the evolutionary analysis of miRNA sequences in brain using unique paleogenetics data described in the manuscript titled \u201cThe evolution of l brain\u201d presents an in silico analysis of 22 known nucleotide polymorphisms and arrived at functionally important conclusion about the mechanism of action for at least one of these DNA variants.The paper by Arkova et al. \u201cObesity-related known and candidate SNP markers can significantly change affinity of TATA-binding protein for human gene promoters\u201d , the functional transcription factor binding sites ranking in the fruitflies , and comparative mining of stress- related expression profiles .Finally, the works by Menzorov et al. , Kozlov The paper by Klimenko et al. titled \u201cBacteriophages affect evolution of bacterial communities in spatially distributed habitats: a simulation study\u201d explored evolutionary scenarios that may be influenced by the spatial location of initial phage invasion and showed that the speciation rate is lower when invasion penetrated the fully formed community of sedentary cells .In their paper \u201cOn the control mechanisms of the nitrite levels in Escherichia coli cells\u201d Khlebodarova, Ree and Likhoshvai describe the mathematical model of nitrite utilization in E.coli cells cultured in the flow chemostat, the process relevant to many biotechnological and clinical applications .The paper of Bryanskaya et al. titled \u201cThe role of environmental factors for the composition of microbial communities of saline lakes in the Novosibirsk region (Russia)\u201d for the first time described microbial composition of these saline lakes, their dependence on physical-chemical parameters of waters, as well as evaluated possible avenues for their bioprospecting .http://conf.bionet.nsc.ru/bgrssb2016/).We wish you an interesting reading and sincerely await you at our next BGRS conference that will take place in Novosibirsk 29 August \u2013 2 September 2016 ("} +{"text": "Salmonella enterica adjusts and adapts to different environments while attempting colonization. In the course of infection nutrient availabilities change drastically. New techniques, \u201c-omics\u201d data and subsequent integration by systems biology improve our understanding of these changes. We review changes in metabolism focusing on amino acid and carbohydrate metabolism. Furthermore, the adaptation process is associated with the activation of genes of the Salmonella pathogenicity islands (SPIs). Anti-infective strategies have to take these insights into account and include metabolic and other strategies. Salmonella infections will remain a challenge for infection biology.The human-pathogenic bacterium Salmonella enterica is a Gram-negative enterobacterium closely related to Escherichia coli strike with endotoxins, typhoid fever, and severe systemic illness. The millions of infections and thousands of fatal cases every year are an important reason for a better understanding and control of Salmonella infection . Adaptations include multiple abilities for oxygen and nitrate respiration deficient strain to utilize gluconate, but not other sources such as glucose encoding a Fe2+/Mn2+/Zn2+transporter. Thus, to study the mechanisms of systemic disease caused by Salmonella, infection models using Salmonella-susceptible inbred mouse strains such as BALB/c or C57BL/6 with defective Slc11a1 allele are frequently used facilitate studies on virulence mechanisms and metabolic activities on a molecular level and allow a detailed picture of host-pathogen interactions. Comparative genomics was used for example to identify the presence of different metabolic pathways for non-typhoidal and typhoidal pathovars of Salmonella for additional high resolution typing of Salmonella isolates by phage types and different PFGE patterns. This allows investigation in unprecedented detail of virulent strains as well as their correlation with metabolic resistance features such as pathways for degradation of antibiotics.Methods useful in analyzing the global impact of gene deletions on Salmonella regulates its metabolic pathways in response to changing nutritional environments. A study performed by Blair et al. focused on changes in transcriptomic profiles when using LB or various minimal media for growth. Transcription profiles were established and the article instructively starts from microarray experiments and verifies putative differences by quantitative real-time PCR , oxidative , and iron-limiting metabolic protection. In the four ferritins, bacterioferritin (Bfr) was found to be down-regulated in LP strains.The second \u201c-omics\u201d level, namely transcriptomics including microarrays and high throughput sequencing approaches, gives insights into how Salmonella protein is a challenge for MS analyses. Nevertheless, with more effort even quantification of proteins is possible applying different labeling techniques and standards. A good example for the application of the technique to Salmonella is the enzyme quantifications of ex vivo purified Salmonella performed by Steeb et al. (Mass spectroscopy (MS)-based proteomics is a method of choice when analyzing gene products: with this approach protein expression is directly measured. Typically only several matching peptides from a protein are identified applying the knowledge of the genome sequence and identified reading frames. This only partial peptide coverage for a given b et al. , also ilSalmonella metabolism. In particular, isotopolog profiling (IP) allows analysis of current metabolic fluxes under defined conditions. For a detailed method explanation see the study by H\u00e4rtel et al. coupled to different cell culture techniques (including establishing tissue infection models) offer here a wealth of information. A nice example including bioluminescent Salmonella, the Streptomycin mouse model and bioimaging is Pontier-Bres et al. with proteins mediating only this function in the complete network. Furthermore, hubs, central nodes in the network receiving many connections and indicating strongly connected genes or proteins, are of interest. For instance, interactome networks describing protein-protein interactions are built up and serve as scaffolds for further analysis for each condition. This was compared to a genome-scale network connecting genes with metabolic pathways and cellular functions. Looking at the top five connecting hub proteins from the transcriptional network as well as the hubs in the genome scale metabolic pathway and cellular function network , all these hubs were found to be dispensable for virulence in mutation studies. However, double mutants of these two sets of regulatory proteins showed clear effects on virulence in mouse infection experiments produced by the NO synthase of several immune cells of the host has a severe impact on central carbon metabolism of Salmonella. NO targets the pyruvate and \u03b1-ketoglutarate dehydrogenase complexes (Richardson et al., ch Table . FurtherSalmonella making it more stress resistant (Figure Citrate is a TCA cycle intermediate Figure and is at Figure .The broad influence of amino acids on metabolic adaptation during infection. The work on acetylation regulation in Salmonella by Wang et al. (g et al. also undg et al. .ArgT is an essential virulence determinant which decreases the host's cellular arginine content and reduces by this way the NO production of the host (Das et al., Salmonella appears to be without influence on NO production. Although arginine degradation pathways are up-regulated in Salmonella during infection of macrophage and essential for virulence, this is due to other mechanisms but not related to substrate degradation of iNOS (Choi et al., In particular, the bacterial arginine permease Salmonella. In a study on cysteine biosynthesis during oxidative stress, cysteine biosynthesis regulation was blocked in \u0394cysB and \u0394cysE mutants and oxidative defense pathways encoded by katG and soxS were up-regulated compared to the wild-type strain (Turnbull and Surette, Salmonella survival and replication (Bjur et al., Cysteine is a key amino acid during oxidative stress response in SsrAB virulon controlling SPI2 gene expression is induced under nutrient-poor conditions (e.g., presence in the phagosome, Kuhle and Hensel, The Salmonella enterica (Table Various metabolic pathways which have an impact on the SPI1 activity of ca Table . One exaca Table and secrca Table .Salmonella invasiveness and to induce strong inflammatory host responses (Humphreys et al., There are also indications for the influence of SPI1 functions on the host's metabolism in order to facilitate survival in the intestine and subsequently intracellular to promote the infection process. Thus, the SPI1-T3SS effector protein SopE is known to increase Salmonella virulence (Table Although SPI1 and SPI2 are induced under very distinct nutritional environments (SPI1 in a nutrient rich environment, SPI2 by nutrient starvation, Kuhle and Hensel, ce Table . One exace Table .Intracellular adaptation and metabolism of Salmonella. While conditions in the intestinal lumen are nutrient rich, the situation changes after Salmonella invades into the epithelial cells and is phagocytosed at the basolateral cell side by macrophages or dendritic cells. Staying inside the SCV, the pathogen has to deal with nutrient limitations. To investigate which metabolites could interact with expression of genes in SPI1 or mainly SPI2, one issue is to define the nutritional situation of Salmonella gain inside the SCV and to figure out which metabolites Salmonella has access to. Mouse infection experiments showed on the one hand that intracellular Salmonella get access to a wide range of nutrients, including nearly all amino acids except proline. On the other hand, it was shown that the ability to manifest a full systemic infection is dependent on the utilization of \u201cglycerol, fatty acids, N-acetylglucosamine, gluconate, glucose, lactate, and arginine\u201d (Steeb et al., Salmonella is able to counteract various defense mechanisms in order to facilitate growth or reduce immune responses (Table es Table . Invasioes Table .Shigella-infected cells, amino acid levels of epithelial cells invaded by Salmonella normalized 3 h after infection, which leads to relocalization of mTor invasion sustaining pathway to the SCV, phosphorylation of ATG protein 13, leading to a low ATG protein 1 activity and thus reduced autophagy (Ganley et al., Salmonella is able to avoid autophagy in epithelial cells. Further investigations are required to clarify if normalization of amino acid levels is directly induced by Salmonella. At least the invasion-induced membrane disturbance is only severe in the first hour of infection and somehow repaired faster than in cases of invasion by other intracellular pathogens (Tattoli et al., However, in contrast to Salmonella adapts rapidly and successfully to changing conditions including intracellular survival in macrophages, in epithelia and in the gut, we will now examine which antibiotic strategies are nevertheless available for Salmonella infections. A seminal work by Becker and co-workers showed that the robust metabolism of Salmonella limits possibilities for new antibiotics (Becker et al., Salmonella virulence (Steeb et al., Salmonella are highly resilient (Barat et al., Salmonella in particular, as well as by exploring novel ways of anti-infectives. One inspiring example is metabolic engineering of Salmonella vaccine bacteria in the mevalonate pathway to boost human V\u03b32V\u03b42 T cell immunity (Workalemahu et al., Salmonella also vulnerable also in conserved and well investigated pathways, such as TCA cycle and its anaplerotic reactions. Thus, Salmonella Typhimurium is controlled by host NO production as shown in mice experiments in vivo. Methionine or lysine auxotrophy results from reduced succinyl-CoA availability as the lipoamide dehydrogenase activity is targeted by NO while compensatory Salmonella pathways to achieve more succinyl-CoA are again blocked by NO (Richardson et al., As Salmonella to facilitate intracellular survival within the SCV in host cells and its nutrient supply.We saw that multiple \u201c-omics\u201d and especially metabolomic data are currently used to determine the needs for Salmonella's generalist metabolic lifestyle meets all types of environmental challenges, be it ROS or nutrient limitation by its broad metabolic capabilities. The broad metabolism suggests nevertheless potential for novel anti-infective strategies. However, under severe conditions Salmonella regulation and metabolism are spiked up by input from SPI1, SPI2, T3SS and T6SS, modified invasion abilities, redox protection and central metabolism to turn the neutral environmental lifestyle of Salmonella into a pathogenic lifestyle for its host. On top of this such genetic modules catalyze rapid genetic exchange between Salmonella strains showing that only an integrated picture will help to sustain antibiotic efficiency against Salmonella infections.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Fusarium graminearum is a phytopathogenic Ascomycota that can cause Fusarium head blight in wheat and other cereals worldwide, leading to important yield losses as well as reduced grain quality. The analysis of the F. graminearum genome sequence revealed the presence of 20 non-ribosomal peptide synthases, 15 polyketide synthases, and 17 terpenoid synthases, all potentially involved in the production of a panel of secondary metabolites, including yet unknown ones, such as the mycotoxins deoxynivalenol (DON) and other type B trichothecenes , and both types can lead to either adaptation and survival, or cell death. Here, cell death does not necessary mean a failure to implement an effective stress response but can be the result of the response pathway itself when a destructive response is implemented. The effects of various stresses that phytopathogenic fungi are likely to encounter in the context of host invasion have been the subjects of numerous works. In particular, oxidative stress, as the result of the early defensive \u201coxidative burst\u201d triggered in the host plant upon infection, has been intensively examined.Stresses caused by variations of the environment can be biotic (the surrounding microbiome) or abiotic as signals that initiate/modulate biosynthesis (reviewed in Hong et al., Aspergillus nidulans, the Yap-like bZIP factor NapA, was shown to be involved in tolerance to oxidative stress (Asano et al., NapA (Yin et al., Aspergillus ochraceus and the NapA orthologue AoYap1 (Reverberi et al., AoYap1 but toxin accumulation is also enhanced in untreated conditions (Reverberi et al., In Aspergillus parasiticus have binding sites for the same bZIP transcription factor AtfB that is activated upon oxidative stress via MAPK signaling (Hong et al., A. parasiticus is grown in toxin-inducing medium (Roze et al., Aspergillus species (see Reverberi et al., Botrytis cinerea, the BcAtf1 factor was also shown to positively regulate the production of secondary metabolites (Temme et al., On the mechanistic side, mobility shift assays provided experimental evidence that antioxidant and aflatoxin biosynthetic genes in F. graminearum, previous results indicate that oxidative stress with H2O2 could be a pre-requisite for the biosynthesis of the mycotoxin DON, which may suggest that DON production and endogenous oxidative stress could be connected (Ponts et al., FgAP1 that activates the transcription of antioxidant enzymes (Montibus et al., F. graminearum to its own benefit. Recent data may bring clues as of the mechanism of activation of DON biosynthesis upon oxidative stress. A glycogen synthase kinase GSK3 was shown to be essential for both virulence and DON production F. graminearum, and to be up regulated upon oxidative stress by H2O2 (Qin et al., In F. graminearum. An acidic pH is a prerequisite for DON production (Merhej et al., FgPac1, homologous to the member of the pH regulator system PacC in A. nidulans (Merhej et al., The pH of the environment was shown to be particularly critical for the initiation of trichothecene B biosynthesis in F. graminearum, the cell wall integrity pathway involves the MAP kinase FgMgv1 (Hou et al., F. graminearum, FgHOG1 of the osmoregulation MAP kinase pathway, mediated by the FgOS-2 kinase, is involved in hyperosmotic as well as cell membrane stress responses. FgHOG1 also plays a role in ROS-mediated signaling. Its deletion causes a drastic reduction of DON accumulation, also caused by hyperosmotic conditions, as well as other developmental defects (Van Thuat et al., in planta (Van Thuat et al., Different response pathways can be triggered to counteract stresses that modify the organization and stability of the fungal cell wall. For example, the cell wall integrity pathway is responsive to changes in osmotic pressure and oxidative stress (see Hayes et al., F. graminearum, transcription factor Tri6 has been evoked above (Seong et al., Aspergilli, LaeA has been described as a secondary metabolism-specific regulator involved in switching from inactive heterochromatin to the transcriptionally permissive euchromatic state. In F. graminearum, FgLae1 is a regulator of secondary metabolism that activates the production of DON (Kim et al., FgLae1 has pleiotropic effects in F. graminearum, also affecting sexual development for example (Kim et al., Regulators of fungal secondary metabolite biosynthesis that play a role in regulating other aspects of fungal life, including response to stress, have been described. The example of the F. graminearum's response to oxidative stress, i.e., defensive plant-produced H2O2 may serve as a signal to produce DON (Ponts et al., F. graminearum for its own development and secondary metabolism (Audenaert et al., e.g., caspofungin, nikkomycin Z, tunicamycin, fluconazole; see Hayes et al., Previous observations made about A. parasiticus, co-localize with stress response proteins to the endosome/transport vesicles/vacuoles fraction of a fungal cell extract (Linz et al., F. graminearum, previous work showed that the endoplasmic reticulum stress response and oxidative stress are tightly linked (Malhotra et al., F. graminearum secondary metabolism and stress response pathways are indisputably very closely interconnected. Although further investigation is required, an attractive hypothesis is that secondary metabolism pathways could be part of the fungus' stress-response system. Under this scenario, more than a coupling of pathways, the production of DON and other secondary metabolites would be integral part of the fungus' arsenal to cope and adapt to its always-changing environment, including in the context of host-pathogen exchanges.The hypothesis of a coupling between stress response and mycotoxin production is reinforced by recent evidence that proteins involved in secondary metabolite pathways, including the aflatoxin one in The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Dear Editor,et al. reported that abdominal compression reduced the movement of lung tumors (thereby possibly reducing treatment uncertainty), with portal fluoroscopy being used to measure the tumor movement [et al. [et al. [et al. also reconfirmed the validity of abdominal compression using 4D CT and reported that the internal target volume was significantly reduced for lower lobe tumors [et al. reported that abdominal compression increased the variation of tumor motion by referring to their 4D cone-beam CT (CBCT) data, contending that longer treatment time to include the abdominal compression procedure may reduce the reproducibility of tumor motion [et al. [et al. [Several different approaches have been applied to stereotactic hypofractionated radiotherapy for lung tumors, including free breathing, breath-hold, gating, and tracking. Negoro movement . Heinzer [et al. and Han [et al. confirmer motion . Richmon [et al. and Mamp [et al. also repUsing a 4D planning CT imager, Aquilion LB, we calculated 3D lung tumor motion trajectories with an Anzai belt and stereotactic body frame with an abdominal compression plate for five patients who received four-fraction VMAT stereotactic ablative body radiotherapy (SABR). In addition, the motion trajectories of lung tumors were calculated using 4D CBCT imaging functionality provided by an X-ray Volume Imaging (XVI) system version 4.5 both immediately before and during treatment. The pre-treatment 4D CBCT data were acquired by the built-in XVI software, Symmetry, whereas in-treatment 4D CBCT was obtained by in-house software using projection images acquired during VMAT delivery . The brex, y and z axes correspond to the lateral, anteroposterior and craniocaudal directions, respectively. A large interpatient variability was observed: Fig. Figure Figure In conclusion, we confirmed the reproducibility of lung tumor movement using 4D planning CT and 4D CBCT for five patients who received four-fraction VMAT SABR under constrained breathing conditions. The results appear to be clinically acceptable, but further study is needed because of the small data size of this preliminary study. It is anticipated that the flattening-filter-free technique may increase breathing trajectory reproducibility due to its faster dose delivery . In addi"} +{"text": "Allium cepa, Nicotiana tabacum, Vicia faba, or Arabidopsis thaliana but this number largely increased in the last years. Plant comet assay has been used to study the genotoxic impact of radiation, chemicals including pesticides, phytocompounds, heavy metals, nanoparticles or contaminated complex matrices. Here we will review the most recent data on the use of this technique as a standard approach for studying the genotoxic effects of different stress conditions on plants. Also, we will discuss the integration of information provided by the comet assay with other DNA-damage indicators, and with cellular responses including oxidative stress, cell division or cell death. Finally, we will focus on putative relations between transcripts related with DNA damage pathways, DNA replication and repair, oxidative stress and cell cycle progression that have been identified in plant cells with comet assays demonstrating DNA damage.The systematic study of genotoxicity in plants induced by contaminants and other stress agents has been hindered to date by the lack of reliable and robust biomarkers. The comet assay is a versatile and sensitive method for the evaluation of DNA damages and DNA repair capacity at single-cell level. Due to its simplicity and sensitivity, and the small number of cells required to obtain robust results, the use of plant comet assay has drastically increased in the last decade. For years its use was restricted to a few model species, e.g., The first reports on the use of comet assay in plants date from the 1990's and the fact that root is usually the organ directly in contact with contaminated soil and water, have also influenced the establishment of plant comet assays in ecotoxicological approaches. Technical details concerning plant comet assays in different organs and species have been thoroughly reviewed by Gichner et al. .Allium cepa, Nicotiana tabacum, Vicia faba, and Arabidopsis thaliana that allows the quantification of several comet parameters, including the tail DNA %, tail length, tail extension moment or Olive tail movement in light excess-induced DNA damages in Ipomoea aquatica root protoplasts, and correlated DNA damages observed by comet assay with chlorophyll degradation. However, these two studies did not take into consideration the potential role of UV in light-induced DNA damages. In a study designed to investigate UV-A and UV-B effects, Jiang et al. , compared to DNA polymerase \u03bb UV-B sensitive mutants. UV-C was also shown to induce both SSBs and DSBs in Arabidopsis plumbaginifolia protoplasts -rays are used to increase seed vigor and/or enhance plant tolerance to environmental stresses. Navarrete et al. pioneerea et al. , Koppen a et al. and Verba et al. optimizea et al. , 2008a uArabidopsis homologous recombination deficient mutants subjected to \u03b3-rays. On the other hand, Vandenhove et al. seedlings after exposure to \u03b3-rays concomitant with a difference in expression profiles of three miRNAs, and an increase of reactive oxygen species (ROS) levels. Combining the use of the comet assay, and the expression of genes encoding DNA repair-related proteins, Nishiguchi et al. . Don\u00e0 et al. presented by soils were significantly genotoxic to A. cepa roots, with DNA damages measured by comet assay and compared to the effects of increasing \u03b3-ray doses.Concerning radioactive contaminations, Saghirzadeh et al. successfMost of the contaminated sites worldwide are contaminated with heavy metals. In Europe, heavy metals contaminated almost 50% of the investigated sites developed adaptive responses and protection mechanisms against genotoxic effects of the mutagenic agents methylmercuric chloride (MMCl) and ethyl methanesulfonate (EMS) genotoxicity has been the most studied during the last years. Achary et al. , 2012a aN. tabacum in hydroponic and soil experiments. These results were confirmed on Talinum triangulare roots and correlated with Pb-induced oxidative stress including genotoxic aspects was reviewed by Pourrut et al. . Using cA. cepa roots was shown to induce significant DNA damages in V. faba leaves and roots, in a dose-dependent manner and that these effects were associated with oxidative stress. Sturchio et al. toxicity mechanism in plants involves DSBs and possibly replication blocks, with plant condensin II playing a critical role in DNA damages repair was shown to induce significant DNA damages induces higher DNA damages in roots compared to leaves. This differential effect was possibly attributable to the higher accumulation of Zn (II) in roots, compared to shoots. Tkalec et al. were demonstrated to be relevant genotoxicity biomarkers. Despite still restricted to a few number, some studies have already used plant comet in field ecotoxicology assays of soils contaminated with metals (see Section \u201cContaminated Matrices\u201d below).It is worth noting that the interest of using the comet assay as a reliable biomarker on ecotoxicological assays is increasing, and Bandyopadhyay and Mukherjee applied o et al. used a bA. cepa, supporting the genotoxic potential of this type of nanomaterials.Plant comet assays are also increasingly used to assess the phytotoxicity of small-scale materials Table , e.g., n2 NPs in A. cepa were shown to induce DNA damages in Brassica rapa ssp. rapa, and this result was confirmed by DNA laddering and TUNEL assays.Recently, using higher NPs concentrations, Thiruvengadam et al. also demA. cepa plants. These data supported the concomitant observation of chromosomal aberrations and mitotic aberrations in the same tissues oxide NPs increased the nuclear DNA damages in s Liman, .The alkaline comet assay showed an increase of DNA damages in tomato seedlings exposed to NiO-NPs up to 2 mg/ml oxide and tin (IV) oxide is a mixture widely used in industrial coating. A significant increase in DNA damages was recently observed of Medicago sativa cells. In this and other pioneer studies, the comet assay can play a pivotal role as a tool to assess environmental impacts of suspected emerging nanocontaminants.Besides metal oxide NPs, quantum dots form another type of nanomaterials increasingly prevalent in the environment. Quantum dots are nanomaterials used in electronics which possess semiconducting properties, composed for example of arsenic (As), selenium (Se) and tellurium (Te) in various proportions. Despite their increasing prevalence in the environment, the toxicity of quantum dots in plants is largely unknown. In a pioneer study, Santos et al. used a bSeveral researchers have used the comet assay to monitor DNA damages induced in plants by numerous organic pollutants Table . The mos2O2 induce DNA damages and/or HPetunia grandiflora and Gaillardia grandiflora by comet assay, in a pioneer study of plant\u2013plant association for phytoremediation involving the treatment of textile dyes roots after exposure to doses up to 10 mg/L roots used treated by two herbicides 2,4-D and Dicamba . These results were confirmed in the same study by RAPD analysis. Recently, Liman et al. were demonstrated in plants, e.g., barley radicles treated with Juglans regia husk water extracts. It should be noted that the authors stressed the need of performing accurate and appropriate statistical evaluations of comet results, an emerging topic of discussion. Ci\u011ferci et al. may have cytotoxic and genotoxic effects or have protective roles against stressing conditions in a wide number of species, including humans. The way phytocompounds influence oxidative stress balances, and regulate programmed cell death pathways and cell cycle chekpoints, support their wide therapeutic use was recently shown to inhibit plant growth of Oryza sativa, A. thaliana, Brassica rapa or Lactuca sativa and AtRad21.1 (SYN2) as important effectors in early repair of DSBs, after treatment with bleomycin might occur during S phase and stimulate HR in fas mutants. Also, levels of formed DSBs were compared in rice wild type plants vs. an aphidicolin-sensitive phenotype. Without aphidicolin treatment, both WT and osrecql4-2 mutants produced very low levels of DSBs, but these increased in the mutants after treatment hypersensitive to excess of boron (B). Excess of B induced DNA damages and affected the expression of HEB1 and HEB2, which encode respectively the CAP-G2 and CAP-H2 subunits of the condensin II protein complex, important in maintenance of chromosome structure. These results suggested that DSBs are a cause of B toxicity and that condensin II reduces the incidence of DSBs and PhAPX (encoding for a cytosolic isoform of ascorbate peroxidase).Roy et al. , 2013 reV. faba and telomeric regions in Arabidopsis mutants by Roy et al. , top1 (DNA topoisomerase I), MtTFIIS (transcription elongation factor II-S) and MtTFIIS-like. So, despite comet assay has not been consistently applied to these environmental stresses in plants, the available data of their interference with DNA integrity, opens a perspective of their use in the near future. Also, Confalonieri et al. (Medicago truncatula the MtTdp2\u03b1-gene overexpression prevented the accumulation of DSBs in absence or presence of osmotic stress, and that the MtMRE11, MtRAD50 and MtNBS1 genes that are involved in DSB sensing/repair, being up-regulated in the MtTdp2\u03b1-overexpressing plants grown under physiological conditions, were no further up-regulated under osmotic stress (Confalonieri et al., Salt, drought and osmotic stress are ever more emerging as abiotic defies intimately related with soil overuse and climate changes (e.g., Santos et al., y et al. who suppi et al. demonstrIn this review we have highlighted most relevant studies that used comet assay in plants to study the impact of stress conditions on plant DNA damages. This work was mostly focused on the most recent major advances in the last five, regarding conventional and emerging contaminants and complex matrices. The recent advances in the use of the plant comet assay to both a larger number of plant species, and a larger number of conditions, support the use of this technique as a robust and sensitive technique to assess DNA damages induced by stress conditions. Data also support that this simple and robust technique may be a powerful tool to complement conventional and -omics tools in situ environmental pollution monitoring. Moreover, new fields of research using plant comet assay are open, not only in environmental studies, but also in plant physiology, as this technique may help elucidating pathways involved in plant development, cell cycle/programmed cell death, or even plant disease resistance. Also, it remains an important field of research deciphering genetic mechanisms underlying processes related with DNA damage/repair, in which comet assay will have undoubtedly a crucial role.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Yet the creatinine concentration decreased significantly (p<0.05) when the cadmium animals treated with 200 and 400 mg/kg body weight of the extract were compared with the cadmium control. Furthermore, histological alterations in the kidney were observed in cadmium untreated rats and these were ameliorated in cadmium treated rats by co-administration of IG extract. IG showed apparent protective and curative effect on Cd-induced nephrotoxicity.Cadmium has been considered a risk factor for humans as it accumulates in body tissues, such as the liver, lungs, kidneys, bones, and reproductive organs. The aim of the present study was to evaluate the effect of Irvingia gabonensis (IG) against cadmium (Cd)-induced nephrotoxicity. The study was performed on twenty (20) male rats divided into four groups: control group, cadmium group , cadmium + extract and cadmium + extract . Changes in the kidney biochemical markers, namely glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), aminotransferase (ALT), aspartate aminotransferase (AST) activities and levels of malondialdehyde (MDA), urea, and creatinine were determined in serum. Histological examinations were monitored. Exposure to Cd lowered the activities of kidney antioxidants, while it increased LPO levels. Levels of all disrupted parameters were alleviated by co-administration of IG extract. The malondialdehyde concentration of the rats treated with 200 and 400 mg/kg body weight of the extract significantly decreased ( It is a highly accumulative toxicant with very long biological half-life . Cd nephrotoxicity was reported to result from generating free radicals and thus inducing cell necrosis and apoptosis in membranes of erythrocytes and tissues, such as kidney, liver, brain, and testes, with thiobarbituric acid reactive substances (TBARS) and hydroperoxides used as indicators of oxidative damage (IG) is a commercial and indigenous fruit tree of West and Central Africa and identified as the most important tree for domestication were purchased from Aldrich Chemical Co. . Glutathione, hydrogen peroxide, 5, 5\u2019-dithios-bis-2-nitrobenzoic acid (DNTB) and epinephrine were from Sigma Chemical Co., Saint Louis, MO USA. Trichloroacetic acid (TCA) and Thiobarbituric acid (TBA) were purchased from British Drug House (BDH) Chemical Ltd., Poole, UK. Other reagents were of analytical grade and the purest quality available.Irvingia gabonensis was collected on 16th January, 2014 in Ado-Ekiti (Ekiti State) and authenticated at the Department of Plant Science, Ekiti State University. The stem bark of Irvingia gabonensis was air-dried and crushed into fine powder. The powdered part extracted with ethanol using maceration and the extract was concentrated in vacuum at 40 \u00b0C with a rotary evaporator and water bath to dryness. The yield of the extraction was 5.01%.The stem bark of Irvingia gabonensis stem bark for the detection of various phytochemicals. Tests for common phytochemicals were carried out by standard methods weighing between 80\u2013120 g were bought from the animal house of the Department of Chemical Sciences, Biochemistry Unit, Afe Babalola University, Nigeria. The animals were kept in aired cages at room temperature (28\u201330 \u00b0C) and received normal laboratory chow and water ad libitum.Male Wistar rats for laboratory animal care and use. The ethical committee of the Afe Babalola University approved this study. The use of all animals in this study followed the guidelines of the institutional Animal Ethical Committee given by the Committee for Control and Supervision of Experiments on Animals (CPCSEA).Cadmium was induced in groups II, III and IV. Briefly, Cadmium dissolved in distilled water was given by intravenous injection (through tail vein) at a dose of 4 mg/kg body weight.Irvingia gabonensis (200 mg/kg b.w.) (14 days) + cadmium chloride (4 mg/kg b.w.); Group IV \u2013 Irvingia gabonensis (400 mg/kg b.w.) (14 days) + cadmium chloride (4 mg/kg b.w.) according to the method of Ojo et al. ; Group II\u2013cadmium chloride (4 mg/kg b.w.); Group III \u2013 et al. .Kidney tissues were quickly removed, washed in ice-cold isotonic saline and blotted individually on ash-free filter paper. The tissues were then homogenized in 0.1 M 2-amino-2-(hydroxymethyl)-1,3-propanediol hydrochloride buffer, pH 7.4, using a Potter-Elvehjem homogenizer at 4 \u00b0C. The crude tissue homogenate was centrifuged at a speed of 9 000 rpm for 15 min in a cold centrifuge and the supernatant was kept at \u201320 \u00b0C for estimation of GSH, SOD and CAT activities.Blood collected from the heart of the animals into plain centrifuge tubes was allowed to stand for 1 h. Serum was prepared by centrifugation at 3 000 g for 15 min in a Beckman bench centrifuge. The clear supernatant was used for assessing the serum lipid profile and enzymes.et al. followed by the Duncan multiple range test for analysis of biochemical data using SPSS (20.0). Values were considered statistically significant at The ethanolic extract was found to contain compounds known to have antioxidant activity, like tannins, phlobatannins, flavonoids, anthocyanin, cardiac glycosides and alkaloids .p<0.05) in the relative weight of the kidney of cadmium untreated rats when compared with the control, while treatment with Irvingia gabonensis stem bark (100 and 200 mg/kg) significantly decreased the relative weight of the kidney of cadmium-induced rats to values statistically comparable to the control (p>0.05). All these changes induced by cadmium intoxication restored significantly (p<0.05) to near normal levels on administration of Irvingia gabonensis stem bark.p<0.05) serum and kidney lipid peroxidation (LPO) products measured as thiobarbituric acid reactive substances confirmed the nephro-protective activity as a significant recovery of nephron damage and decreased necrosis was evident against cadmium-induced nephrotoxicity in the kidney of the rats, comparable to their control. The histological results further corroborated the biochemical findings suggesting the useful effects of Irvingia gabonensis stem bark in cadmium-induced toxicity in rats.Histology of the kidney slide of cadmium untreated rats showed tubular degeneration, necrosis and severe renal cortical congestion . TreatmeIrvingia gabonensis stem bark extracts revealed the presence of polyphenol-rich compounds. Polyphenols have been suggested to decrease oxidative stress in humans. Flavonoids found in the extract may inhibit oxidative stress by scavenging free radicals, acting as reducing agents, hydrogen atom donating molecules or singlet oxygen quenchers, chelating metal ions and sparing other antioxidants such as alkaloids, glycosides, saponins, tannins, flavonoids and phenolic compounds, which are responsible for the antioxidant activity.Cadmium is a well-known human carcinogen and a potent nephrotoxin. It is a potent inducer of oxidative stress and affects the cellular antioxidant defense potential bi-phasically by reserve and improvement of several antioxidant enzymatic and non-enzymatic molecules. The phytochemical study of Irvingia gabonensis stem bark examined against cadmium-induced nephrotoxicity in rats showed that the mean body weight of the cadmium-exposed group decreased with the increase in relative liver weight, which agrees with the findings of other authors (EI-demerdash et al., et al., et al. (Irvingia gabonensis, the changed body weight and liver weight parameters recovered to near normal levels due to the antioxidant effects of Irvingia gabonensis stem bark.In the present study, the potent chelation therapy with , et al. reportedet al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., Many studies have shown that cadmium induces oxidative damage by producing ROS (Liu et al., et al., et al., Irvingia gabonensis in the treated groups, indicating a prominent nephroprotective effect of Irvingia gabonensis against cadmium nephrotoxicity. The increased levels of serum AST and ALT in cadmium exposed rats indicate an increased permeability and damage and/or necrosis of the kidney. In our study, we found that the extract of Irvingia gabonensis at a dose of 400 mg/kg caused a significant decrease in the activities of serum AST, ALT, which further supports the beneficial effects of the extract of Irvingia gabonensis in cadmium-induced rats.Moreover, the protective effects of this extract may be related to its ability to chelate or sequester cadmium via formation of cadmium-flavonoid complexes (el-Ashmawy Serum total protein represents a complex mixture containing a number of components which differ in properties and function. Hypo-proteinemia, protein deficiency in plasma, may be partly due to dietary insufficiency with subsequent impairment of the protein synthetic machinery, or to excessive excretion Chawla, . In the et al., et al., et al., et al., et al., Irvingia gabonensis had a protective effect against cadmium-induced kidney damage, as established by the significant decrease in urea and creatinine levels in the Irvingia gabonensis treated group compared with the cadmium group.It has been postulated that increased levels of serum urea and creatinine are linked to kidney disease Chawla, . Urea isIrvingia gabonensis treatment of cadmium-induced nephrotoxicity in rats revealed that the observed pathological impairments caused by cadmium recovered significantly, indicating that Irvinga gabonensis is capable of preventing the nephron damage induced by cadmium. It is thus suggested that Irvingia gabonensis may inhibit Cd-induced kidney damage. However, further studies are necessary to find out the actual mechanism of action of phytochemicals and their doses in the presence of oxidative stress due to Cd intoxication.Histological examination revealed that cadmium intoxication caused abnormal ultra-structural changes in kidney tissue, including tubular degeneration, necrosis and severe renal cortical congestion. Regarding the histopathological observation, Irvingia gabonensis exhibited protective effects against Cd-induced nephrotoxicity. For populations exposed to Cd, the use of the ethanolic extract of Irvingia gabonensis could thus be recommended.In conclusion, the ethanolic extracts of"} +{"text": "Dear Editor,Morbilivirus which belongs to family Paramyxoviridae . The dial., 2011; Yanagi al., 2011; Fazlalial., 2011). Measleal., 2011). Howeveal., 2011; Muscat al., 2011). Historal., 2011). Recently, increased number of measles outbreaks with high morbidity and mortality has been observed in various regions of Pakistan during recent years as outlined in supplemental material . A total of 871/1053 (82.71 %) children from Faisalabad and 647/813 (79.58 %) children from Jhang were found vaccinated either with single or dual dose of measles vaccination Table 1. Out of Samples from non-vaccinated children showed high prevalence (63 %) which was an indication of previous measles infection in these particular children. These findings were suggestive and may be correlated with confirmatory sero-diagnosis of recent outbreaks in these areas. The non-vaccination status against measles was considered as one of the major risk factor in children . The prevalence of anti-measles IgG antibodies from samples collected from Faisalabad was found 79.54 % whereas in Jhang 67.42 % of samples were observed as positive. There was no significant difference of sero-prevalence between Faisalabad and Jhang. The possible explanation for non-significant prevalence could be that these two areas are closely related geographically, traditionally and are closely situated to each other. These factors may be considered for a similar trend towards vaccination coverage and sero-conversion against measles as previously described by Hussain et al. (2008[The prevalence of anti-measles IgG antibodies from samples collected from male children was higher as compared to female children with non-significant difference. However, few of the available literature has reported that risk of measles and level of anti-measles IgG antibodies was observed higher in females as compared to male children (Rahim et al., 2011; FowotadBased on various age groups, prevalence of anti-measles IgG antibodies were observed as 75.35 % in children from 1-6 years of age, whereas 67.56 % in children from 6-10 years of age were positive with no significant difference. These findings were not in accordance with some of the previous studies which showed the highest incidence of measles during the age of 6 months to 3 years (Matsumura et al., 2005; Rahim eIt was concluded from the overall results of present study that vaccination coverage against measles was below the standards of WHO and Health Department, Govt. of Punjab, Pakistan. Further, vaccine efficacy and development of humoral immune response in children was not optimum according to WHO guidelines and higher risk of measles incidence was found in non-vaccinated children. Based on these findings, it is recommended that routine immunization against all vaccine preventable diseases in general and against measles in particular should be carried out as per guidelines of WHO to completely control and eradicate measles from Pakistan.The authors declare that they have no conflict of interest."} +{"text": "Neural stimulation is a critical technique in treating neurological diseases and investigating brain functions. Traditional electrical stimulation uses electrodes to directly create intervening electric fields in the immediate vicinity of neural tissues. Second-generation stimulation techniques directly use light, magnetic fields or ultrasound in a non-contact manner. An emerging generation of non- or minimally invasive neural stimulation techniques is enabled by nanotechnology to achieve a high spatial resolution and cell-type specificity. In these techniques, a nanomaterial converts a remotely transmitted primary stimulus such as a light, magnetic or ultrasonic signal to a localized secondary stimulus such as an electric field or heat to stimulate neurons. The ease of surface modification and bio-conjugation of nanomaterials facilitates cell-type-specific targeting, designated placement and highly localized membrane activation. This review focuses on nanomaterial-enabled neural stimulation techniques primarily involving opto-electric, opto-thermal, magneto-electric, magneto-thermal and acousto-electric transduction mechanisms. Stimulation techniques based on other possible transduction schemes and general consideration for these emerging neurotechnologies are also discussed. Neural stimulation is an essential technique for restoring lost neural functions and correcting disordered neural circuits in neurological diseases the strong cytotoxicity of QDs is a concern, particularly when a thin coating is used to achieve an active QD-neuron interface nanoparticles were targeted to the biotinylated peptide of a genetically engineered anchor protein in the membrane of neurons expressing the temperature-gated TRPV1 ion channels (Placement II; Huang et al., 2+ influx through the TRPV1 ion channels, depolarized the neurons to fire action potentials in vitro, and triggered thermal avoidance in worms.Widely used superparamagnetic nanoparticles can convert alternating magnetic fields to localized heat via magneto-thermal transduction (Laurent et al., 2+ influx into the cells faster than in the above work (Huang et al., A more specific ion-channel targeting strategy was also implemented in a mouse xenograft model by tethering nanoparticles directly to the TRPV1 ion channels (Placement III; Stanley et al., in vivo stimulation feasibility, untargeted superparamagnetic iron oxide nanoparticles were dispersed in the vicinity of TRPV1-expressing human embryonic kidney HEK-293FT cells, dissociated hippocampal neurons and neurons at the ventral tegmental area of mice (Placement I; Chen et al., 2+ influx in the HEK-293FT cells, stimulated hippocampal neurons to fire action potentials, and activated the neurons at the ventral tegmental area of mice to have an enhanced expression of c-fos, achieving a stimulation response with a latency of 5 s after the onset of the magnetic field. And the stimulation in the mouse model remained effective for at least 1 month thanks to the good biocompatibility, stability and decreased endocytosis of extracellularly dispersed nanoparticles.To improve the temporal resolution for neuronal activation and realize a long-term 2+ permeability, and thus temperature-gated Na+ ion channels are desired as the target (Kn\u00f6pfel and Akemann, Magneto-thermal neural stimulation enabled by superparamagnetic nanoparticles can achieve a uniform stimulation of the target cell population due to the uniform expression of TRPV1 ion channels in these cells across the tissue (Huang et al., As a wirelessly transmitted primary stimulus, ultrasound interacts with tissues weakly and can penetrate deep into soft tissues with minimal energy absorption Tyler, . It can 2+ and Na+ influxes in an ultrasonic field (Placement II; Marino et al., Piezoelectric nanomaterials can convert ultrasound waves to electric fields via acousto-electric transduction due to their piezoelectricity (Wang and Song, Nanomaterial-enabled neural stimulation is an emerging class of neurotechnologies, with numerous exciting breakthroughs in the past decade. As a powerful enabling tool, nanomaterials can be either applied alone or combined with other approaches including synthetic biology to facilitate innovative neural stimulation schemes. These new techniques not only allow non- or minimally invasive neural stimulation of a high spatial resolution and cell specificity, but also improve the safety by significantly reducing the required power of the primary stimulus (Huang et al., Nanomaterials of other transduction mechanisms, such as magneto-mechanical, acousto-mechanical, and opto-optical transductions, are also worth considering for potential development of additional neural stimulation schemes. Magneto-mechanical transduction via magnetic nanoparticles can convert magnetic fields to localized mechanical forces to activate mechanosensitive ion channels such as the TREK-1 channels (Hughes et al., To select the primary and secondary stimuli, several factors are considered. The primary stimulus needs to penetrate tissues deeply, be easy to focus at an appropriate spatial resolution and be safe for long-term exposure. The secondary stimulus needs to be selected according to an adequate expression of the target ion channels in the neuron's membrane. For example, it is not necessary to genetically modify the target neurons with voltage-gated ion channels to use electric fields as the secondary stimulus, whereas, to use heat, the TRPV1 ion channels may need to be genetically inserted into the membrane of target neurons (Huang et al., in vitro. To move forward, many issues including biocompatibility, stability, consistency, efficiency and reliability will need to be addressed (Gomez et al., in vitro and/or in vivo studies. Clinical application is promising, but remains very challenging due to concerns on the safety of nanomaterials, viral vectors for gene delivery, and genetic modification to the target neurons (Manilla et al., This diverse class of nanomaterial-enabled neurotechnologies is still in their early stages of development, with many having only been validated YW and LG analyzed the relevant published work, designed the perspective and structure, and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Preliminary follow-up studies seem to support the efficacy and safety of SVF/ASCs enrichment and the additional benefit from the combined use of autologous platelet-derived growth factors and hormones during breast reconstruction procedures. In the present review we highlighted the complex interplay between resident or grafted ASCs, mature adipocytes, dormant or active breast cancer cells and tumor microenvironment. Actually, data concerning the permissive role of ASCs on breast cancer progression are contrasting, although no clear evidence speaking against their use exists.Breast cancer is the most common cancer in women and autologous fat grafting is an important clinical application in treatment of post-surgical deformities. The simplicity of fat grafting procedures and the absence of subsequent visible scar prompted an increasing interest for this technique. The plasticity of adipose-derived stem cells (ASCs) obtained from stromal vascular fraction (SVF) of adult adipose tissue provided exciting perspectives for regenerative medicine and surgery. The recent discovery that SVF/ASC enrichment further ameliorates clinical efficacy of grafting ASCs suggest as ASC-mediated new adipogenesis and vasculogenesis. ASC adipogenic differentiation involves Akt activity and EGFRs, FGFRs, ERbB2 receptor-mediated pathways that also play a pivotal role in the regulation of breast cancer growth. Moreover, the finding that platelet-derived growth factors and hormones improved long-term maintenance of fat grafting raises new concerns for their use during breast reconstruction after cancer surgery. However, it remains unclear whether grafted or resident ASCs may increase the risk of However, the term \u201cadipose-derived stem cells\u201d (ASCs) has been successively recommended for the consistency between research groups (Zhao et al. in situ lesions. This finding induced caution and suggested some concerns about the use of fat grafting with SVF/ASC enrichment in breast reconstruction following cancer surgery. In the present review, we tried to describe the biomolecular pathways regulating proliferation and differentiation of ASCs, in order to define potential implications of breast cancer cell biology and risks for their use in post-surgery breast cancer reconstruction.Adult adipose tissue is a multifunctional organ that contains various cellular types, including mature adipocytes, macrophages and stromal cells, supported by connective tissue surrounding fine capillaries (Zuk et al. in vitro as a function of time and/or passage in culture (Mitchell et al. in vitro, ASC surface immunophenotype resembles that of MSCs, with a similarity greater than 90% (Gimble et al. + cells and to differentiate them from circulating precursors (Pittenger et al. + cells and the perivascular origin of ASCs (Figure\u00a0in vitro (Figure\u00a0ASCs share with MSCs the differentiation potential along several mesenchymal lineages (Gimble et al. s Figure\u00a0C-E. Cytoo Figure\u00a0. Besideso Figure\u00a0.Table 1The fascinating differentiative pluripotency of ASCs and their ability to enhance vascularization (Bertolini et al. \u03b1, heparin-binding epidermal growth factor, insulin-like growth factor-II and adipsin (Manabe et al. in vivo and in vitro studies demonstrated relevant phenotypic changes in adipocytes surrounding breast cancer (Dirat et al. Many studies focused the relationship between mature adipocytes and breast cancer cells. Rat mature adipocytes affect the biological behavior of epithelial cells through the production of leptin, adiponectin, tumor necrosis factor-in vivo and in vitro reported that ASCs favor tumor growth, increasing extracellular matrix deposition and vascularization, suggesting that ASCs may directly contribute to the dense network of fibroblasts and desmoplastic reaction surrounding breast cancer (Wang et al. in vitro documented that co-culture of human ASCs and breast cancer cells induce high levels of metalloproteinases (Pinilla et al. Differently from mature adipocytes, the interplay between resident mesenchymal cells, including ASCs, and breast epithelial cells is still partially unknown. In this respect, it is still unclear whether preadipocytes act differently from mature adipocytes. ASCs are located in perivascular niches and contribute to cell turn-over, vascular network for the maintenance of adipose tissue tropism (Strawford et al. in vivo and in vitro have been performed to verify ASCs influence on dormant tumor cells and on their growth and invasiveness. In literature is not yet clear whether dormant tumor cells are out of cell cycle, or persist in a dynamic state of proliferation and death. The transition between dormant and active states requires the presence of various signals, such as cytokines, hormones and growth factors (Donnenberg et al. To better clarify their role in cancer progression, studies in vivo interaction and define how selectively stimulate ASCs regenerative function without influencing tumorigenesis.Altogether, these studies highlight the concept that resident ASCs and cancer cells may interact in a complex and dynamic fashion influencing the tumor behavior. Further studies are needed to better clarify this in vitro (Cervelli et al. In vitro data alone seem to suggest the caution in the local use of growth factors in addition to fat graft and further investigation of the interplay between ASCs and breast cancer cells should be performed also in vivo.The proliferative arrest and/or cell loss are potential limitations in regenerative surgery strategies. So, exogenous growth factors should provide the necessary microenvironmental signals to accelerate cell proliferation, extracellular matrix synthesis and tissue deposition (Chen et al. Autologous fat grafting is a procedure widely used in breast reconstruction after cancer surgical treatment (Gentile et al. in vivo. MSCs can be readily transduced via adenoviral, retroviral or lentiviral vectors without compromising the capability for differentiation or the expression of surface markers. Consequently, MSCs are potentially suitable for a gene approach in cancer therapy through the induction of a more chronic and slow release of drugs that are often limited by their toxicity or short life (Kucerova et al. In vitro, MSCs transduced with adenoviral vector carrying human Interferon-\u03b2 and co-cultured with breast cancer cells induced the reduction of cancer cells growth (Studeny et al. in vivo, when Interferon-\u03b2\u2009\u2212\u2009transfected MSC cells are injected intravenously in a xenograft breast cancer mouse model (Studeny et al. In vitro studies documented that cancer stem cell exert antiapoptotic effect on breast cancer cells and counteract cell-cycle changes caused by tamoxifen, so promoting tumor growth and invasiveness (Wang et al. Conventional cancer therapies include surgery, chemotherapy and radiotherapy. A certain number of preclinical studies recently proposed the use of MSCs as candidates to deliver anti-cancer drugs. Chemokines secreted by breast tumor cells are capable of stimulating MSCs migration and recruitment, suggesting a potential role for MSCs as delivery agents for chemotherapeutic purposes in breast tumours Aromatase inhibitors are used as second-line therapy or as first-line adjuvant therapy, but they have the disadvantage to inhibit indiscriminately aromatases, including those in bone and brain tissues, with adverse effects in terms of bone mineralization and cognitive function, respectively (Rubin et al. in vivo seem to confirm the efficacy of SVF/ASCs enrichment and the beneficial additional use of autologous platelet-derived growth factors and hormones in breast reconstruction. The improvement in long-term maintenance strongly supports the additional combined use of fat grafts with autologous platelet-derived growth factors and hormones, such as insulin. However, additional translational research studies are needed to better clarify the possible impact of these procedures on tumor microenvironment, in particular their potential effect on cancer cells. Different studies confirmed the complex and dynamic interplay between cancer cells and resident ASCs. Latters, in the tumor microenvironment, seem to affect only active cancer cells, so promoting neoangiogenesis, matrix remodeling and intercellular communication via gap-junction. In addiction, it has been hypothesized the presence of cancer stem cells, from resident stem cell or dedifferentiated tumor cells, that may favour the epithelial-mesenchymal transition, supporting tumor growth and invasiveness. In addition, the interaction between grafted ASCs and resting cancer cells doesn\u2019t seem to be responsible for cancer recurrence because resting cancer cells are more resistant to apoptosis and they don\u2019t require stroma or vascular structure for their survival. Preliminary data describe that SVF/ASCs enrichment did not show increased risk of new cancer or relapse compared with control group.Current evidence sustains that ASCs represent a promising tool for innovative therapies in regenerative surgery and play a significant role in lipofilling-mediated breast reconstruction after breast cancer surgery. SVF/ASCs enrichment seems to favor long-term fat graft maintenance in reconstruction of tissue defects, likely promoting vascularization and collagen synthesis. Preliminary studies Finally, ASCs characteristics appear promising for their engineered use as \u201ccarrier\u201d of adjuvant chemotherapeutic agents against residual breast cancer cells. So, the growth of malignant cells may be counteracted by local release of drugs in tumor microenvironment while systemic plasma concentration remain low, avoiding the problems related to toxicity and short life."} +{"text": "Carragher et al.In an attempt to analyse the cross-sectional data correctly, I obtained information on alcohol consumption for eight of the study regions from published WHO data for 2010,I did a multiple regression analysis using methods described by KronmalIn my regression model, I considered total alcohol consumption in each area as the response, with GDP, population and TEASE-16 score the predictors. Only population size was significantly associated with total alcohol consumption , and exaThe relationship between TEASE-16 and GDP per capita is shown in Neither TEASE-16 score nor income, (measured by GDP), were significantly correlated with alcohol consumption across the eight areas in the year 2010. It seems unlikely that analysis of the original data used by Carragher et al."} +{"text": "Machine learning (ML) has been well recognized as an effective tool for researchers to handle the problems in signal and image processing. Machine learning is capable of offering automatic learning techniques to excerpt common patterns from empirical data and then make sophisticated decisions, based on the learned behaviors. Medicine has a large dimensionality of data and the medical application problems frequently make the human-generated, rule-based heuristics intractable. In this special issue, we provide a forum to present the cutting-edge machine learning techniques in medical applications, including the learning of similarities across different image modalities, organ localization, learning of anatomical changes, tissue classification, and computer-aided diagnosis. \u201cAdaptive neuro-fuzzy inference system for classification of background EEG signals from ESES patients and controls\u201d introduced an adaptive neurofuzzy inference system for classification of background EEG signals from the patients of slow-wave sleep syndrome and control subjects. Their study showed that the entropy measures of EEG were significantly different between the patients and normal subjects. Therefore, a classification framework based on entropy measures was proposed. S. Jirayucharoensak et al. in \u201cEEG-based emotion recognition using deep learning network with principal component based covariate shift adaptation\u201d proposed the utilization of a deep learning network (DLN) to discover unknown feature correlation between input signals. The DLN was implemented with a stacked autoencoder (SAE) using hierarchical feature learning approach. D. Al-Jumeily et al. in \u201cA novel method of early diagnosis of Alzheimer's disease based on EEG signals\u201d introduced three neural synchrony measurement techniques: phase synchrony, magnitude squared coherence, and cross correlation for classification of mild Alzheimer's disease patients and healthy subjects. K. Zhang et al. in \u201cAdaptive bacteria colony picking in unstructured environments using intensity histogram and unascertained LS-SVM classifier\u201d presented a novel approach for adaptive colony segmentation in unstructured environments by treating the detected peaks of intensity histograms as a morphological feature of images. In order to avoid disturbing peaks, an entropy based mean shift filter was introduced to smooth images as a preprocessing step. The relevance and importance of these features can be determined in an improved support vector machine classifier using unascertained least square estimation. M. Cabrerizo et al. in \u201cInduced effects of transcranial magnetic stimulation on the autonomic nervous system and the cardiac rhythm\u201d demonstrated that repetitive transcranial magnetic stimulation (rTMS) could induce changes in the heart rhythm.The topics of the accepted papers in this Special Issue spread from electroencephalography (EEG) signal processing to image segmentation. Z. Yang et al. in"} +{"text": "Major histocompatibility complex I may be important for both normal development and pathogenesis of some CNS diseases including Parkinson\u2019s.Neuronal expression of major histocompatibility complex I (MHC-I) has been implicated in developmental synaptic plasticity and axonal regeneration in the central nervous system (CNS), but recent findings demonstrate that constitutive neuronal MHC-I can also be involved in neurodegenerative diseases by playing a neuroinflammtory role. Recent reports demonstrate its expression The major histocompatibility complex (MHC) gene family encodes molecules on the surface of cells that enable the immune system to recognize presented self- and foreign-derived peptides and beta 2 microglobulin (\u03b22m) chains is immune-privileged and that MHC-I is not expressed by neurons Lampson, . HoweverThis review summarizes the pattern of expression and the different implications of neuronal MHC-I in the brain, and focuses in particular in the potential role of constitutive MHC-I expression by specific subsets of neurons in neurodegenerative diseases such as PD.in vitro, usually triggered by exposure to interferon gamma (IFN-\u03b3), and in vivo. The initial such study showed that MHC-I genes expression were induced by IFN-\u03b3 in cultured rat hippocampal neurons , and are far more susceptible to MHC-I induction than other neuronal populations tested, including cortical, striatal and thalamic neurons subunit, CD3zeta , exhibit reduced retinal synaptic activity, incomplete developmental refinement of connections between retina and its central targets, and reduced retinal ganglion cell dendritic motility with increased dendritic density has been linked to MHC-I and CTLs in the CNS (Friese and Fugger, Our findings in human postmortem samples of adult control individuals and PD patients show that MHC-I is expressed by SN DA and LC NE neurons (Cebri\u00e1n et al., Neuronal MHC-I expression plays multiple roles. First, it regulates synaptic plasticity during brain development. Second, it regulates axonal regeneration and the appropriate specification of synaptic inputs following injury. Third, in neuronal diseases including neurotropic viral infections, neuronal MHC-I expression is upregulated and may initiate T cell mediated responses. While current research in each of these areas is ongoing, the suggestion of a role in neurodegenerative disease is the most recent and the least understood: while there is a consensus that many neurodegenerative diseases feature a robust inflammatory response, it remains unclear how this is related to chronic disease processes.In vitro experiments indicate that DA primary human neurons derived from human embryonic stem cells and primary catecholamine murine neurons are more susceptible to MHC-I induction by IFN-\u03b3 than other neuronal populations (Cebri\u00e1n et al., Our recent study demonstrates neuronal MHC-I expression in both normal and PD adult brain; such expression to date appears to be particular for catecholaminergic/monoaminergic neurons. These possibilities are consistent with recent demonstrations that microglia can be activated by substances released from degenerating neurons in PD, such as \u03b1-syn (Zhang et al., Neuronal display of antigenic MHC-I could participate in a range of additional neurological disorders. For example, Japanese encephalitis virus can induce MHC-I expression in non-neuronal cells by interferon type 1 (Abraham et al., Together, these data indicate that in human brain, neuronal MHC-I expression, antigen presentation and the presence of T cells could occur simultaneously under certain circumstances, leading to the death of targeted neurons. In some neurodegenerative diseases, and in particular for PD, we propose that activation of lymphocytes in the periphery may occur in response to self or non-self proteins, or as an initial insult and disruption of the blood brain barrier, with a subsequent penetration of lymphocytes in the brain. When catecholamine neurons die with subsequent release of \u03b1-syn and NM to the extracellular space, the activation of microglial cells will release proinflammatory substances such as IFN-\u03b3, leading to an upregulation of MHC-I in the membrane of catecholamine neurons that could present neuronally derived antigens. If lymphocytes are close, they could recognize these antigens and target and kill the cell, which would again release \u03b1-syn and NM Figure , leadingFuture studies are necessary to identify which antigens are presented by MHC-I expressing catecholamine neurons and how T cells might interact with them. If these interactions occur, immune therapies used in other diseases including classical autoimmune disorders such as Type 1 diabetes or MS may be adapted to provide future treatments for PD.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Heme oxygenase (HO) is a ubiquitous enzyme with various properties, but its main function is catalyzing the rate-limiting step in heme degradation to produce equimolar quantities of biliverdin, iron, and carbon monoxide (CO) and Hmox1\u2212\u2215\u2212 mice, Soares et al. cells, restores placentation, and fetal growth, while normalizing blood pressure (Linzke et al., Zenclussen et al. review hThe relevance of HO-1 in the regulation of immune responses during pregnancy is further highlighted in the article by George et al. . They inIn summary, the HO-1/CO axis may play a pivotal role in sustaining pregnancy, and thus understanding its biology during pregnancy may reveal promising therapeutic approaches for pregnancy complications.Both the authors contributed in writing the editorial.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "In a recent study, Cid-Fernandez et al. tested aOther prior studies have investigated changes in motor changes in aging Light, . In one What is important about this study is Cid-Fernandez and colleagues (a) have tested both motor and cognitive processes in (b) three age groups. Most existing studies on aging often compare younger (aged 18\u201330 or 50 years old) with older (aged 60 or 65 years and over) adults Scott, , and thuImportantly, there are studies that test behavioral performance in more age groups than those recruited in the Cid-Fernandez et al. study s. One sucOther studies have recruited participants across various age groups and correlated behavioral performance with age (Davis et al., Cid-Fernandez et al. suggest The Cid-Fernandez et al. findingsThe author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Chorioamnionitis (CA) describes an intrauterine status of inflammation and/or infection of placental membranes, refering to both histological and clinical CA . It is c"} +{"text": "Recent objectification research found results consistent with the sexualized body-inversion hypothesis (SBIH): People relied on analytic, \u201cobject-like\u201d processing when recognizing sexualized female bodies and on configural processing when recognizing sexualized male bodies processing becomes more important . Because Bernard et al. presenteIn their first study, Schmidt and Kistemaker examined symmetry in Bernard's stimuli and found that female bodies were more asymmetrical than male bodies. Strikingly, neither the interaction between stimulus gender and stimulus orientation nor the three-way interaction involving stimulus set-up emerged. These results suggest that differences in symmetry between inverted (vs. upright) female bodies are not more pronounced in the Bernard set-up than in the counterbalanced set-up.In their second study, these authors replicated Bernard et al. 's findindirectly address this putative symmetry confound as they allow comparison within symmetry-matched stimuli . A visual inspection of Figure 2 and asymmetrical female bodies (seventh vs. fourth bar); (ii) asymmetrical inverted female bodies (fourth bar) were not recognized better than symmetrical inverted female bodies (eighth bar).Schmidt and Kistemaker identified a potential stimulus set-up confound in their second study (contrary to Bernard et al., In sum, contrary to Bernard et al. , SchmidtSchmidt and Kistemaker also found an inversion effect for male and female bodies and this pattern occurred for naked bodies with or without masked sexual body parts. They concluded that Bernard et al.'s findings are not driven by target sexualization (Schmidt and Kistemaker, conceptual replication perspective, Schmidt and Kistemaker provide evidence in favor of restricted generalizability of Bernard et al.'s findings but from a direct replication perspective Schmidt and Kistemaker's paper cannot address the role of target sexualization in Bernard et al.'s stimuli because they did not manipulate sexualization of these stimuli. Bernard et al. (First, although informative with regard to the role of target sexualization and inversion, the SBIH was posited to explain differences in recognition of sexualized male vs. sexualized female bodies (i.e., stimulus gender effect: Bernard et al., d et al. , howeverIn sum, contrary to Bernard et al. , SchmidtThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Hexokinase-2-mediated aerobic glycolysis is integral to cerebellar neurogenesis and pathogenesis of meduloblastoma. Cancer and Metabolism 2013, 1(2):17A response to Gershon et al. : Gershon et al. have invfl/fl conditional knockout that they have used to study the metabolism and migratory behavior of CGNPs during their development not only targets those neurons[Gershon et al. interpretation of the results of their Fig. 5 may also be discussed as the hGFAP-cre;Hk2 neurons. Thus anOne might thus feel that in their work, Gershon et al. may have overlooked an important aspect of the cerebellar developmental process and one could suggest that they give more attention to the possible involvement of Bergmann glia in modulating the role of Hexokinase-2-mediated aerobic glycolysis during cerebellar neurogenesis."} +{"text": "Reelin, a multifunctional extracellular protein that is important for mammalian brain development and function, is secreted by different cell types in the prenatal or postnatal brain. The spatiotemporal regulation of Reelin expression and distribution during development relates to its multifaceted function in the brain. Prenatally Reelin controls neuronal radial migration and proper positioning in cortical layers, whereas postnatally Reelin promotes neuronal maturation, synaptic formation and plasticity. The molecular mechanisms underlying the distinct biological functions of Reelin during and after brain development involve unique and overlapping signaling pathways that are activated following Reelin binding to its cell surface receptors. Distinct Reelin ligand isoforms, such as the full-length protein or fragments generated by proteolytic cleavage differentially affect the activity of downstream signaling pathways. In this review, we discuss recent advances in our understanding of the signaling transduction pathways activated by Reelin that regulate different aspects of brain development and function. A core signaling machinery, including ApoER2/VLDLR receptors, Src/Fyn kinases, and the adaptor protein Dab1, participates in all known aspects of Reelin biology. However, distinct downstream mechanisms, such as the Crk/Rap1 pathway and cell adhesion molecules, play crucial roles in the control of neuronal migration, whereas the PI3K/Akt/mTOR pathway appears to be more important for dendrite and spine development. Finally, the NMDA receptor (NMDAR) and an unidentified receptor contribute to the activation of the MEK/Erk1/2 pathway leading to the upregulation of genes involved in synaptic plasticity and learning. This knowledge may provide new insight into neurodevelopmental or neurodegenerative disorders that are associated with Reelin dysfunction. Cleavage at this site releases a six amino acid carboxy-terminal peptide, reducing signaling activity and hindering dendrite development in the postnatal neocortex and the very low-density lipoprotein receptor (VLDLR). These proteins belong to the low-density lipoprotein receptor (LDLR) family. They have partial functional redundancy and play an essential role in Reelin-mediated neuronal migration based on the observation that double knockout mice display a in vitro is an intracellular adaptor protein that is essential for Reelin signal transduction. This protein binds the cytoplasmic tail of lipoprotein receptors, including ApoER2 and VLDLR (Trommsdorff et al., Crk/CrkL mutant mice display a reeler-like cortical phenotype (Park and Curran, Genetic studies demonstrated that Dab1, and thus Reelin signaling, is specifically required for a specific mode of radial migration termed somal or terminal translocation, but not for glial-guided locomotion (Franco et al., PAFAH1b1 gene that is responsible for human lissencephaly type I, may be particularly relevant to cortical development. Lis1 binding to phospho-Dab1 is Reelin-dependent, and genetic interaction between Dab1 and PAFAH1b1 demonstrates a functional relationship between these proteins (Assadi et al., PAFAH1b alpha subunits bind specifically to VLDLR, potentially promoting the interaction between Lis1 and Dab1 downstream of this receptor (Zhang et al., In addition to Crks and Rap1, biochemical studies identified several molecules that may be involved in Reelin-dependent neuronal migration. These include proteins that regulate cytoskeletal dynamics and cell motility, such as Lis1, Nck\u03b2 and N-WASP (Assadi et al., reeler mice. Dendritic defects are also apparent in immature hippocampal or cortical cultures isolated from mutant mice, but not in mature cultures (Niu et al., . Since Reelin treatment rescued these defects, these in vitro studies first demonstrated that Reelin directly promotes dendrite development. Following studies further demonstrated that Reelin enables initial dendritic outgrowth by promoting the extension of the Golgi apparatus into apical dendrites (Matsuki et al., in vitro in a manner that is dependent on SFK activity and Dab1 phosphorylation (Beffert et al., in vivo studies demonstrated that the Crk adaptor proteins are required for Reelin-induced Akt phosphorylation, placing the kinase functionally downstream of these adaptors (Park and Curran, Dendrite outgrowth is disrupted in homozygous Other molecules that have been implicated in Reelin-dependent dendrite outgrowth include the amyloid precursor protein (APP; Hoe et al., reeler mice exhibit altered hippocampal synaptic plasticity and multiple behavioral abnormalities, such as defects in executive function and contextual fear conditioning learning (Brigman et al., in vivo (Beffert et al., ++ influx through the NMDAR (Beffert et al., ++ influx, Erk1/2 signaling and CREB-dependent IEGs transcription (Telese et al., Heterozygous ++ signaling (Hellwig et al., Reelin gene in adult mice does not result in impaired synaptic plasticity. However, it renders the adult brain strikingly sensitive to amyloid-induced synaptic suppression, leading to profound learning disabilities (Lane-Donovan et al., In addition to its well-documented postsynaptic effects, Reelin also acts presynaptically, causing a rapid enhancement of spontaneous neurotransmitter release. This effect is due to the mobilization of VAMP7-containing synaptic vesicles, and requires canonical ApoER2/VLDLR receptors, PI3K and CaGHL wrote the first draft of the manuscript and made the figures. GD wrote and revised the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "The grant number is GR09/0021.In the Funding section, the second grant number from the funder Instituto de Salud Carlos III (Madrid, Spain;"} +{"text": "Morbilivirus . The clal., 2011; Fazlalial., 2011). Expandal., 2011; Liu et al., 2011). Furtheal., 2011; Leuridaal., 2011). Vaccinal., 2011; Sheikh al., 2011). Many ial., 2011). Among al., 2011). Furtheal., 2011). Therefore, keeping in view the importance and significance of measles, this preliminary study was designed to develop an ELISA for detection of humoral immunity. For this purpose ELISA plates were coated with measles virus CAM-70 strain which contain not less than 1000 CCID50 in 0.5 ml. Coating of plates was performed using carbonate buffer as described by van der Werff (2008). BrieflThe authors declare that they have no conflict of interest."} +{"text": "The fossil record of crocodylians and their relatives (pseudosuchians) reveals a rich evolutionary history, prompting questions about causes of long-term decline to their present-day low biodiversity. We analyse climatic drivers of subsampled pseudosuchian biodiversity over their 250 million year history, using a comprehensive new data set. Biodiversity and environmental changes correlate strongly, with long-term decline of terrestrial taxa driven by decreasing temperatures in northern temperate regions, and biodiversity decreases at lower latitudes matching patterns of increasing aridification. However, there is no relationship between temperature and biodiversity for marine pseudosuchians, with sea-level change and post-extinction opportunism demonstrated to be more important drivers. A \u2018modern-type' latitudinal biodiversity gradient might have existed throughout pseudosuchian history, and range expansion towards the poles occurred during warm intervals. Although their fossil record suggests that current global warming might promote long-term increases in crocodylian biodiversity and geographic range, the 'balancing forces' of anthropogenic environmental degradation complicate future predictions. Crocodylians and their relatives have a rich evolutionary history. Here the authors show long-term decline of terrestrial crocodylians driven by decreasing temperatures but no relationship between temperature and biodiversity for marine crocodylians over their 250 million year history. Ecological models can predict the responses of extant species distributions to rising temperatures. However, only the fossil record provides empirical evidence of the long-term interactions between climate and biodiversityOngoing climate change, with projected global warming of 2.0\u20134.8\u00b0C over the next centurySarcosuchus, which reached around 12\u2009m in length and weighed up to 8 metric tons68Pseudosuchia is a major reptile clade that includes all archosaurs closer to crocodylians than birds, and made its first fossil appearance nearly 250 million years ago, at the time of the crocodile\u2013bird split8101112Here we examine the effect of climate on spatiotemporal patterns in pseudosuchian biodiversity over their 250 million year history, using a comprehensive fossil occurrence data set. Our study is the first to analyse climatic drivers of pseudosuchian biodiversity through the group's entire evolutionary history, applying rigorous quantitative approaches to ameliorate for uneven sampling across both time and space. Furthermore, this is the only comprehensive, temporally continuous fossil occurrence dataset for a major extant vertebrate group: equivalent datasets are not currently available for mammals, birds, squamates, teleosts, or other groups with evolutionary histories of similar durations.An uncorrected global census of pseudosuchian genera shows anN=38, R2=0.221; Subsampled pseudosuchian biodiversity reached a peak during the earliest intervals of the group's history and shows a long-term pattern of gradual decline towards the present day . SubstanOnly the crocodylomorph clade survived the Triassic/Jurassic mass extinction (201 Myr ago)10162416Sampling of palaeotropical non-marine crocodylomorphs is limited throughout the Jurassic\u2013Cretaceous and 4a. 88The Cretaceous witnessed the non-marine radiations of notosuchians819931The effect of the K/Pg mass extinction on crocodylomorphs has previously been perceived as minor or non-existent192819283436353636\u03b418O palaeotemperature proxy17Relative changes in subsampled non-marine biodiversity in both North America and Europe track each other and the 7 during the early Paleogene greenhouse world 66\u201341 Myr ago). There is no evidence for transient biodiversity increases driven by the short-term Paleocene\u2013Eocene Thermal Maximum (56 Myr ago), possibly because the timescale of species origination and phenotypic divergence that would allow speciation to be recognisable in the fossil record is longer than that of this rapid climatic event includes a near-comprehensive dataset of all published pseudosuchian occurrences spanning the Middle Triassic through to the Pleistocene, a period of nearly 250 million years. Pseudosuchian body fossil occurrences that could be assigned to genera were downloaded from this database , accessed via Fossilworks (http://fossilworks.org), on 23 February 2015.Following extensive work to ensure that occurrences and taxonomic opinions were consistent and up-to-date with the literatureGenera were used so as to incorporate specifically indeterminate material, enabling us to include more data points in our analyses, and also to avoid problems with using species as a unit for estimating palaeobiodiversityAlligator and Crocodylus, have been used as \u2018wastebasket taxa' for indeterminate or non-referable fossil species. Therefore, we modified occurrences of Crocodylus and Alligator before analysis, as explained in the Extant genera, especially et al. (in review). Environmental assignment (marine versus non-marine) for post-Ypresian taxa was based primarily on facies data recorded in the PaleoDB and information presented in refs et al.Aktiogavialis, Piscogavialis and Siquisiquesuchus; these were omitted from the marine crocodylomorph data set of Martin et al.Tomistoma were probably marineT. schlegelii) are non-marine, or their environments are unknown; as such, here we treat Tomistoma as non-marine.The resultant pseudosuchian data set was subdivided into non-marine stratigraphic time bins . Althougu, singleton taxa were defined based on occurrences within collections58In determining Good's Patterns in non-marine and marine taxa were analysed separately, to avoid problems with sampling from heterogeneous environments58http://www.scotese.com) to obtain reconstructed palaeogeographic positions for each occurrence.In addition to producing sub-sampled terrestrial and marine biodiversity curves through time, we also analysed the palaeolatitudinal distribution of terrestrial biodiversity. This was implemented through plots of subsampled regional biodiversity against the regional palaeolatitudinal centroid, as well as plots of subsampling curves within time bins . Plots o\u03b418O palaeotemperature proxies and sequence stratigraphic eustatic sea level estimates in two sets of analyses: (1) terrestrial pseudosuchian biodiversity in North America and Europe were each compared to the Zachos et al.et al.\u03b418O from the Prokoph et al.We compared subsampled genus biodiversities at a quorum of 0.4 to \u03b418O and sea level, with and without the application of first differencing.Although a contentious issue in crocodylomorph phylogeny, we follow the most recent placement of Thalattosuchia as a basal clade outside of Crocodyliformeset al.\u03b418O values of fish teeth from the Western Tethys. One potential problem with this method is that the fish teeth are from a variety of different species and genera, with L\u00e9cuyer et al.\u03b418O can occur. In addition, there might be differences between the isotopic fractionation that occurs between phosphate and water, and that which takes place in the fish teeth\u03b418O curves of Prokoph et al.et al.\u03b418O dataset for deep sea palaeotemperatures of Zachos et al.et al.1716The similarity of these independent isotopic databases17Statistical comparison was made using time series approaches, specifically generalised least squares (GLS) regression incorporating a first-order autoregressive model .Supplementary Figures 1-7, Supplementary Tables 1-2, Supplementary Methods and Supplementary ReferencesNon-marine pseudosuchian dataMarine pseudosuchian dataTime intervals tablePERL script for SQS (provided by John Alroy)et al. (2008) palaeotemperature proxy isotope dataZachos et al. (2005) sea level dataMiller et al. (2008) palaeotemperature proxy isotope dataProkoph"} +{"text": "Key components in this response include enzymes involved in the biosynthesis of deoxymugineic acid , the deoxymugineic acid efflux transporter (TOM1), the Fe(III)-deoxymugineic acid transporter (OsYSL15), and Fe2+ transporters . In whole roots, these proteins are expressed in a coordinated manner with strong transcriptional induction in response to Fe deficiency. Radial transport of Fe to xylem and phloem is also mediated by the mugineic acid family phytosiderophores, as well as other chelators and their transporters, including Fe(II)-nicotianamine transporter (OsYSL2), phenolics efflux transporters (PEZ1 and PEZ2), and citrate efflux transporter (OsFRDL1). Among these, OsYSL2 is strongly induced under conditions of Fe deficiency. Both transcriptional induction and potential feedback repression mediate the expressional regulation of the genes involved in Fe uptake and translocation in response to Fe deficiency. The transcription factors IDEF1, IDEF2, and OsIRO2 are responsible for transcriptional induction, whereas the ubiquitin ligases OsHRZ1 and OsHRZ2, as well as the transcription factors OsIRO3 and OsbHLH133, are thought to mediate negative regulation. Furthermore, IDEF1 and OsHRZs bind Fe and other metals, and are therefore candidate Fe sensors. The interacting functions of these regulators are thought to fine tune the expression of proteins involved in Fe uptake and translocation.Iron (Fe) is an essential element for most living organisms. To acquire sparingly soluble Fe from the rhizosphere, rice roots rely on two Fe acquisition pathways. The first of these pathways involves Fe(III) chelators specific to graminaceous plants, the mugineic acid family phytosiderophores, and the second involves absorption of FeThe online version of this article (doi:10.1186/s12284-014-0027-0) contains supplementary material, which is available to authorized users. Among u et al. ). The tr2+ uptake is coupled to ferric-chelate reductase activity on the root surface, which is strongly induced under conditions of Fe deficiency . Howevearschner ), thus i2+ uptake, although this effect may be limited as the PEZ2 transcript shows little induction under conditions of Fe deficiency . To avoUnder Fe-deficient conditions, the enzymes and transporters responsible for Fe uptake are induced not only in the epidermis/exodermis, but also in the cortex and vascular bundle Table . The Ose et al. ; Ishimare et al. ). The NAe et al. ) are thoOsFRDL1 is specifically expressed in root pericycle cells, with no apparent induction under conditions of Fe deficiency (Table Citrate is an Fe(III) chelator and is thought to play a dominant role in xylem Fe transport in non-graminaceous plants . In ricPEZ1 is expressed specifically in the vascular bundle, while PEZ2 is expressed in the epidermis/exodermis, cortex, and vascular bundle . Both PArabidopsis thaliana (AtFPN1/AtIREG1) is a likely candidate for theOsVIT1, OsVIT2, and MIT expression are repressed under conditions of Fe deficiency . The miAs reviewed above, the levels of expression of numerous rice genes involved in Fe uptake and translocation are strongly induced under conditions of Fe deficiency at the transcriptional level. Highly conserved temporal and spatial patterns of expression are observed, especially with regard to genes involved in DMA-based Fe uptake . IDE1 ai et al. , 2004]))cis-actii et al. , 2009];;cis-actii et al. ). IDEF1 on Table . At latei et al. , [2009],i et al. ). IDEF2 o et al. ), whereai et al. , 2013]))cis-actii et al. , [2009],i et al. ; Ogo et i et al. ; Itai eti et al. ). Both Ig et al. ).OsIRO2 itself is positively regulated by IDEF1, forming a transcriptional cascade enhancing the expression of genes involved in Fe(III)-DMA uptake and translocation . Howeveg et al. ).in silico prediction of cis-sequences over-represented in Fe deficiency-induced gene promoters revealed that the IDEF1-, IDEF2-, and OsIRO2-binding sequences are enriched within the 500-bp promoter regions of Fe deficiency-induced genes and AGCTAGCT (designated DCEp1 for putative downstream core element 1), as well as common regulatory sequences, such as TATA-box and upstream TFIIB-recognition elements near the transcription start sites of Fe deficiency-inducible genes . The eni et al. ). Regulavs. other metals.IDEF1 binds ferrous Fe and other divalent metals, such as zinc (Zn) and nichel, via its histidine-asparagine repeats and proline-rich regions . In ricOsHRZ1 and OsHRZ2 knockdown show substantial tolerance to low Fe availability, and accumulate large amounts of Fe in shoots and seeds irrespective of Fe nutritional conditions. These knockdown lines also show enhanced expression of Fe deficiency-inducible genes involved in Fe uptake and/or utilization even under Fe-sufficient conditions, but this enhancement is much less obvious under prolonged Fe deficiency interacts indirectly with the OsIRO3 homolog POPEYE (PYE) by binding another subset of basic helix-loop-helix transcription factors from thcy Table . OsHRZ1 y Figure . In Arabg et al. ). BTS ang et al. ). BTS kng et al. ).OsHORZ1 knockdown show increased susceptibility to Fe deficiency and repression of Fe deficiency-inducible genes . OsHORZi et al. ). Thus, OsNAS1, OsNAS2, OsYSL15, and OsIRT1 under both Fe sufficiency and deficiency, and this enhancement may be mediated by the OsIRO2 pathway . Knockoi et al. ). GibberTakasaki ; Yoshii Takasaki ). A jasmTakasaki ) has recg et al. ). OsRMC In Strategy-I plants, systemic Fe signal derived from shoots is thought to determine whether Fe deficiency response in roots takes place (Giehl et al. ; Garc\u00eda 2+ uptake. Research has also elucidated the function of DMA in internal Fe translocation, in addition to its known role in Fe uptake. The differential spatial expression of genes responsible for Fe uptake and translocation in response to Fe deficiency is highly coordinated and is well correlated with their physiological functions. In addition to positive regulation of these genes at the transcriptional level, negative regulation at both the transcriptional and translational levels may also function in fine tuning the Fe deficiency response. Although the Fe-sensing mechanism remains unclear, further characterization of IDEF1 and OsHRZs may in fact consolidate their respective roles as Fe sensors. Despite these advances in our understanding, a potentially large proportion of the molecules involved in intercellular and subcellular Fe movement have not yet been identified. Further investigation into these issues will help to develop novel tools for producing Fe-efficient and Fe-fortified crops with increased versatility.Rice responds to low Fe availability by inducing enzymes, transporters, and regulators that participate in Fe uptake from the rhizosphere and Fe translocation within the plant body. Recent studies demonstrated that rice utilizes both classical DMA-based Fe uptake and direct FeTK and RNI analyzed the microarray data. TK wrote the manuscript with critical revision by RNI and NKN. All of the authors read and approved the manuscript."} +{"text": "Ependymal cells (ECs) form a barrier responsible for selective movement of fluids and molecules between the cerebrospinal fluid and the central nervous system. Here, we demonstrate that metabolic and barrier functions in ECs decline significantly during aging in mice. The longevity of these functions in part requires the expression of the myristoylated alanine-rich protein kinase C substrate (MARCKS). Both the expression levels and subcellular localization of MARCKS in ECs are markedly transformed during aging. Conditional deletion of MARCKS in ECs induces intracellular accumulation of mucins, elevated oxidative stress, and lipid droplet buildup. These alterations are concomitant with precocious disruption of ependymal barrier function, which results in the elevation of reactive astrocytes, microglia, and macrophages in the interstitial brain tissue of young mutant mice. Interestingly, similar alterations are observed during normal aging in ECs and the forebrain interstitium. Our findings constitute a conceptually new paradigm in the potential role of ECs in the initiation of various conditions and diseases in the aging brain. They are highly polarized with adherens junctions at their apical interface allowing them to form a highly selective barrier between the interstitial tissue and cerebrospinal fluid (CSF) in the ventricles is critical for previously unappreciated biological functions in ECs related to their aging. Selective deletion of MARCKS in ependyma results in precocious emergence of biomarkers for aging, both in ECs and in the brain interstitium. MARCKS is a known regulator of cell polarity, cytoskeletal signaling, cell migration, and embryonic brain development was confirmed using antibody labeling, which additionally revealed that its subcellular localization mirrored the distribution of MARCKS in 2M ECs, enriched at the apical surface mouse brains revealed MARCKS loses its apical clustering and becomes less polarized in the cytosol, whereas p-MARCKS is sparsely distributed Fig. and S5. ines Fig.. Thus, M\u03b6 in ECs, we focused on determining how the phosphorylation state of MARCKS may influence its localization and function. In lung epithelial cells, phosphorylation of MARCKS is known to regulate its association with filamentous actin (F-actin) near the plasma membrane. MARCKS dissociates from actin networks and the plasma membrane upon phosphorylation by PKC -bisphosphate P\u2082) in neurons and by performing a FITC\u2013dextran (FD4) assay in wholemounts using Ussing chambers Fig.. TER, a Finally, the impact of precocious aging of MARCKS-cKO ECs on potential gliosis and neuroimmune responses in interstitial brain tissue was assessed. First, effects of normal aging on the integrity of the ependymal layer in the forebrain were documented in wholemount preparations of the ventricular walls in young and old brains. Imaging clearly illuminated scarring of ECs accompanied by astrocyte infiltration into the scarred regions . These pet\u00a0al., et\u00a0al., et\u00a0al., et\u00a0al., MARCKS-dependent mechanisms used as a model in our study likely represent one of many that can influence aging and maintenance of ECs. ECs have been credited with multiple functional attributes which are possibly influenced by MARCKS-dependent cellular and molecular mechanisms. For example, a unique population of ependyma in the choroid plexus is responsible for the production of CSF and various transport proteins may be indicative of a potential role for MARCKS on biomechanical properties of stroke in EC cilia. Indeed, the apical clustering of MARCKS near the base of EC cilia is suggestive of potential interaction with molecular motors that facilitate the beating of motile cilia. Ependyma in the young adult brain have been described to be heterogeneous based on their multiciliated or biciliated morphology was originally identified in intestinal goblet cells and gained attention for its selective expression in the lung epithelium and its role in mucus secretion during airway hyper-responsiveness (Nakanishi et\u00a0al., et\u00a0al., et\u00a0al., et\u00a0al., et\u00a0al., et\u00a0al., Ependyma have been shown to accumulate lipids in the aged mouse brain (Bouab et\u00a0al., We also found elevation of 4-HNE and nitrotyrosine in ECs of MARCKS-cKO mice similar to aged wild-type brains. 4-HNE is an \u03b1,\u03b2-unsaturated hydroxyalkenal that is produced by lipid peroxidation and tyrosine nitration. It is upregulated in instances of elevated reactive nitrogen species and, together with nitrotyrosine, is routinely used as a biomarker for oxidative stress. Interestingly, elevated intracellular oxidative stress has been demonstrated to lead to induction of lipid droplet formation and fatty acid accumulation in several cell types (Kuramoto et\u00a0al., et\u00a0al., et\u00a0al., Our findings have revealed that selective disruption of barrier functions in ECs can age the interstitial brain tissue Fig.. This paet\u00a0al., et\u00a0al., However, ependyma-infiltrated astrocytes may contribute toward elevated oxidative damage in periventricular tissues. For example, it was recently demonstrated that age-associated increase in mitochondrial metabolism in astrocytes produces detrimental reactive oxygen and nitrogen species (Jiang & Cadenas, in\u00a0vivo model for assaying the role of ECs in maintenance of brain homeostasis under various disease states and environmental stimuli with potential relevance to human aging.In summary, our study demonstrates for the first time that MARCKS is required for maintaining the ependymal barrier integrity and sustaining forebrain homeostasis during normal aging. In addition, we have identified that mucins are expressed by, and may be secreted from, ependymal cells into the brain interstitium in an age- and MARCKS-dependent manner. These functions are correlated with lipid droplet formation in ependyma raising the possibility that MARCKS-dependent mucin expression and distribution in the brain parenchyma may be related to lipid metabolism in aging ECs. Thus, conditional MARCKS deletion in ependyma together with the apparent precocious aging of ECs provides a highly suitable Experimental Procedures are available in Supporting information."} +{"text": "Escherichia coli and related enteric species is cytoplasmic accumulation of phosphorylated sugars such as glucose-6-phosphate or the non-metabolizable analog \u03b1-methylglucoside-6-phosphate. This \u201cglucose-phosphate stress\u201d triggers a dedicated stress response in \u03b3-proteobacteria including several enteric pathogens. The major effector of this stress response is a small RNA (sRNA), SgrS. In E. coli and Salmonella, SgrS regulates numerous mRNA targets via base pairing interactions that result in alterations in mRNA translation and stability. Regulation of target mRNAs allows cells to reduce import of additional sugars and increase sugar efflux. SgrS is an unusual sRNA in that it also encodes a small protein, SgrT, which inhibits activity of the major glucose transporter. The two functions of SgrS, base pairing and production of SgrT, reduce accumulation of phosphorylated sugars and thereby relieve stress and promote growth. Examination of SgrS homologs in many enteric species suggests that SgrS has evolved to regulate distinct targets in different organisms. For example, in Salmonella, SgrS base pairs with sopD mRNA and represses production of the encoded effector protein, suggesting that SgrS may have a specific role in the pathogenesis of some \u03b3-proteobacteria. In this review, we outline molecular mechanisms involved in SgrS regulation of its target mRNAs. We also discuss the response to glucose-phosphate stress in terms of its impact on metabolism, growth physiology, and pathogenesis.Bacteria adapt to ever-changing habitats through specific responses to internal and external stimuli that result in changes in gene regulation and metabolism. One internal metabolic cue affecting such changes in Over the last decade, small RNAs have emerged from relative obscurity to take their places as central regulators of gene expression in organisms from all three domains of life. While hundreds of small RNAs in dozens of bacterial genomes have been identified by computational or experimental methods, the functions of the vast majority of these remain a mystery. Nevertheless, we have learned a great deal about a small number of \u201cmodel\u201d bacterial sRNAs, including how their production is regulated, what targets they in turn regulate and the physiological outcomes of sRNA-mediated regulation. In this review, we first provide a brief overview of features of regulatory sRNAs that act on mRNAs through base pairing interactions. We then focus on one well-characterized sRNA, SgrS (sugar transport related sRNA) and describe its activities on target mRNAs and how these activities regulate bacterial metabolism and pathogenesis.Several mechanistic classes of sRNAs have been identified in diverse bacterial species. Many characterized sRNAs act by base pairing with mRNA targets to control mRNA stability and translation. Such sRNAs are often between 50 and 300 nucleotides in length and require an RNA chaperone, Hfq, for their stability and regulatory effects on target mRNAs Hfq-binding sRNA and this is independent of manX regulation , a glutamate-pyruvate aminotransferase is highly conserved, suggesting that SgrR regulates sgrS expression in all these organisms. All identified SgrS homologs contain a Rho-independent terminator and most possess an additional stem-loop structure upstream of the terminator; these two structures are important for Hfq binding to SgrS was not sufficient to allow growth rescue during glucose-phosphate stress conditions. This is in part due to very low levels of SgrT produced from the native sgrS allele in E. coli -1 and SPI-2 (Brumell et al., sopD by SgrS involves base pairing interactions between the conserved region of SgrS and the early coding sequence of sopD mRNA (Figure sopD mRNA degradation (Papenfort et al., Salmonella encodes a second SopD protein, SopD2, which shares 42% identity with SopD and likely arose from a duplication (Jiang et al., sopD2 base pairing interaction differs from SgrS-sopD at only a single position, a wobble G:U base pair instead of the G:C base pair. Remarkably, this interaction that differs by only a single hydrogen bond prevents regulation of sopD2 by SgrS (Papenfort et al., Although SgrS is conserved among enteric bacteria, divergence in primary sequence has resulted in species-specific target regulons, exemplified by the finding that A Figure ; the intsopD regulation by SgrS is not yet clear, the inclusion of sopD in the Salmonella SgrS regulon illustrates plasticity in the evolution of sRNA regulons. The presence of sgrR-sgrS-sgrT in the same genomic context in pathogenic and non-pathogenic \u03b3-proteobacteria (Horler and Vanderpool, Salmonella. Studies of other SgrS homologs in pathogenic and non-pathogenic enteric bacteria will surely shed light on the breadth of regulatory activities of this fascinating dual-function sRNA.While the biological significance of The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Moreover, growing evidence suggests a key role of intracellular CuAOs and PAOs in several facets of plant development. Here, we discuss recent advances in understanding the contribution of different CuAOs/PAOs, as well as their cross-talk with different intracellular and apoplastic metabolic pathways, in tissue differentiation and organ development.Plant polyamines are catabolized by two classes of amine oxidases, the copper amine oxidases (CuAOs) and the flavin adenine dinucleotide (FAD)-dependent polyamine oxidases (PAOs). These enzymes differ to each other in substrate specificity, catalytic mechanism and subcellular localization. CuAOs and PAOs contribute to several physiological processes both through the control of polyamine homeostasis and as sources of biologically-active reaction products. CuAOs and PAOs have been found at high level in the cell-wall of several species belonging to Fabaceae and Poaceae families, respectively, especially in tissues fated to undertake extensive wall loosening/stiffening events and/or in cells undergoing programmed cell death (PCD). Apoplastic CuAOs and PAOs have been shown to play a key role as a source of H In Arabidopsis thaliana 10 CuAO genes are present, among which only eight encode for putative functional CuAOs AtCuAO\u03b51 and AtCuAO\u03b52 are consecutive fragments of a copy of AtCuAO\u03b4 gene. Phylogenetic analysis evidenced that plant CuAOs form three clades (I-III), clade I being composed of three subgroups (Ia-Ic) and clade II of two putrescine (Put), cadaverine (Cad), spermidine (Spd), spermine (Spm), and thermospermine (Therm-Spm) are involved in several physiological processes, such as cell proliferation, differentiation and defense responses are localized in the apoplast and the other members of clade III in peroxisomes , AtCuAO\u03b31 (clade IIa) as well as Nicotiana tabacum CuAO1 (NtDAO1), oxidize mainly Put, Cad, and Spd , which shows preference for N-methyl-Put and is involved in nicotine biosynthesis, although all three proteins are clustered together in clade III was shown to produce Nor-Spd from Therm-Spm , which are localized intracellularly and show an -AtPAO5, 2O2; hydroxyl radical, \u2022OH; singlet oxygen, 1O2) and nitric oxide (NO) in orchestrating developmental processes, as well as in being involved in signaling of both local and systemic defense responses in plants. The apoplast is a major \u201chub\u201d for these chemical species. Their accumulation in large amounts and the complexity of the regulatory mechanisms involved in their biosynthesis reflect the peculiar role of this compartment in physiological events that depend on temporarily regulated and spatially restricted ROS and NO signatures , transition metal ions and H2O2 -mediated NO production distribution in plants , which is clustered together with AtCuAO\u03b4 in clade IIb, was attributed to the implication of PA metabolism in physiological processes taking place during fruit ripening , are controlled by Therm-Spm and Spm and an AtPAO5-like (GhPAO4) PAO may play a crucial role in the generation and differentiation of embryogenic callus during somatic embryogenesis have a role in shoot regeneration from root cultures (Lim et al., Some studies suggest that in CuAO and PAO gene families regarding catalytic activity, subcellular localization, expression pattern and physiological roles of the encoded proteins. Indeed, important links to developmental and stress-related events are emerging for CuAOs and PAOs through ROS/NO production and regulation of specific PA levels.Numerous recent studies have evidenced an extraordinary complexity in All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Although their causative mechanisms are quite diverse, these diseases share the common feature of an uncontrolled and progressive accumulation of fibrous tissue macromolecules in affected organs leading to their dysfunction and ultimate failure. The pathogenesis of fibrotic diseases is complex and despite extensive investigation has remained elusive. Numerous studies have identified myofibroblasts as the cells responsible for the establishment and progression of the fibrotic process. Tissue myofibroblasts in fibrotic diseases originate from several sources including quiescent tissue fibroblasts, circulating CD34+ fibrocytes, and the phenotypic conversion of various cell types including epithelial and endothelial cells into activated myofibroblasts. However, the role of the phenotypic transition of endothelial cells into mesenchymal cells in the pathogenesis of fibrotic disorders has not been fully elucidated. Here, we review the evidence supporting EndoMT\u2019s contribution to human fibrotic disease pathogenesis.Fibrotic diseases encompass a wide spectrum of clinical entities including systemic fibrotic diseases such as systemic sclerosis, sclerodermatous graft The resulting fibrosis disrupts the normal architecture of the affected organs, leading to their progressive dysfunction and eventually to their functional failure ,2,3. Thee (GVHD) , and IgG disease ,11, as w disease ,13, and disease , pulmona disease , liver [ disease , and kid disease . Further disease , asbesto disease , or aris disease , and oth disease and Chag disease . The mosFibrotic disorders collectively affect a very large number of individuals, and owing to the current lack of effective therapeutic approaches, result in very high morbidity and mortality rates. Indeed, it has been estimated that the mortality caused by fibrotic diseases in the Western developed countries is as high as 45% of the total mortality . AlthougGiven the wide variety of affected organs, the chronic nature of the fibrotic processes, the lack of effective antifibrotic therapies, and the large number of individuals suffering their devastating effects, fibrotic diseases pose a serious public health problem representing an enormous challenge to health services worldwide and causing a serious economic burden to society. Despite the remarkable detrimental consequences of the fibrotic disorders for human health, there are very few approved therapeutic agents for these conditions. However, the recent elucidation of important alterations in crucial regulatory pathways involved in the fibrotic process and the identification of the central role of activated myofibroblasts in the production and abnormal deposition of ECM in this process provide a sound basis for the search and eventual identification of novel and effective means of therapy for these devastating diseases ,28,29.in vivo, myofibroblasts induce changes in the biomechanical properties of the affected tissues causing a progressive increase in tissue stiffness, a newly recognized extremely potent profibrotic stimulus [Numerous recent studies have demonstrated that myofibroblasts are the cells ultimately responsible for the severe fibrotic process in the fibrotic disorders ,31,32,33stimulus ,37.et al. [Given the crucial role of myofibroblasts in the pathogenesis of systemic and organ-specific fibrotic disorders including SSc and Idiopathic Pulmonary Fibrosis (IPF), there has been extensive investigation aimed at the precise identification of their cellular origin ,39,40,41et al. . Indeed,et al. ,70. HoweIn the following sections, we will review the available evidence that supports a role for EndoMT in the pathogenesis of various human fibrotic disorders. First, we will review studies of the molecular mechanisms of EndoMT emphasizing various pathways known to participate in the pathogenesis of tissue fibrosis, then, we will briefly discuss the occurrence of EndoMT in various experimentally-induced animal models of tissue fibrosis, and finally, we will review the occurrence of EndoMT in cultured human EC and in tissues from patients with various human fibrotic disorders.In contrast with the extensive studies on the molecular events and pathways involved in EMT , the mecThe TGF-\u03b2 family of proteins comprises several pleiotropic growth factors that play crucial roles in numerous physiological processes including embryogenesis, cellular development and differentiation, immunologic system development, inflammatory response functions, and wound repair ,72. Owinin vitro; (2) TGF-\u03b2 induction of EndoMT was associated with a strong upregulation in the expression of the transcriptional repressor Snail1 indicating that Snail1 is directly involved in TGF-\u03b2-induced \u03b1-SMA expression; and (3) induction of \u03b1-SMA expression involved the c-Abl kinase and protein kinase C-\u03b4 (PKC-\u03b4), as specific inhibition of their kinase activities with imatinib mesylate and rottlerin, respectively, or by knockdown of their corresponding transcripts with specific siRNA abrogated the marked increase in TGF-\u03b2 induced \u03b1-SMA and Snail1 expression and protein levels. These observations confirmed the potent induction of EndoMT by TGF-\u03b21 in vitro [in vivo studies. The study of Cooley et al. demonstrated that a TGF-\u03b2-neutralizing antibody decreased EndoMT and resulted in a profound reduction in neo-intima formation in a mouse model of interpositional vein grafts [et al. generated heterozygous mice with endothelium-specific knock-out of the TGF-receptor II (T\u03b2RII) gene and demonstrated that the resulting partial T\u03b2RII deletion abrogated EndoMT and reduced tubulointerstitial kidney fibrosis in two murine models of renal fibrosis [Given the crucial role that the TGF-\u03b2 family of profibrotic growth factors plays in the development of tissue fibrosis and in the pathogenesis of numerous fibrotic diseases, several studies have demonstrated that TGF-\u03b2 is involved in the generation of myofibroblasts through EndoMT ,82,83,84n grafts . In anotfibrosis . The rolfibrosis .The morphogens Notch and Hedgehog play crucial roles during embryonic development. Although in the adult these pathways are very highly regulated, their aberrant activation may lead to various pathological consequences, including the development of fibrotic diseases ,88,89. TRecently, the Sonic Hedgehog (SHh) pathway, another important morphogen, has also been shown to participate in the pathogenesis of various fibrotic disorders ,96,97,98The Wnt proteins comprise a multigene family of secreted glycoproteins that play crucial roles during embryogenesis signaling through canonical and non-canonical pathways ,100. NumCav1 knock out mice (Cav1\u2212/\u2212) showed that these mice exhibited extensive skin and lung fibrosis [Cav1\u2212/\u2212 mice [Cav1\u2212/\u2212mice in vivo and suggested that CAV1 deficiency participates in the development and progression of tissue fibrosis and fibrotic diseases through the establishment of EndoMT.CAV1, the main protein component of caveolae plays an important role in the internalization, trafficking and degradation of TGF-\u03b2 receptors and, therefore, is involved in the regulation of TGF-\u03b2 signaling and TGF-\u03b2-mediated fibrotic responses ,109,110.fibrosis . Subsequfibrosis ,112,113,fibrosis . These s\u2212/\u2212 mice . The resThe important role of ET-1, the most potent endogenous vasoconstrictor polypeptide identified to date, in the regulation of multiple vascular functions and its participation in various human diseases including Primary and Secondary Pulmonary Arterial Hypertension (PAH) has been well recognized. However, numerous recent studies have shown that ET-1 also displays strong profibrotic effects and it is involved in various aspects of the fibrotic process ,117. Recin vitro and in vivo [Numerous recent studies have demonstrated an important role of hypoxia in the development or progression of various fibrotic diseases including SSc, kidney, and cardiac fibrosis ,123,124. in vivo . However in vivo and anot in vivo .One novel pathway that has been recently recognized as a potentially important participant in the pathogenesis of fibrotic processes through reciprocal interactions with TGF-\u03b2 involves reactive oxygen species (ROS) ,131. AltAlthough there are multiple sources of intracellular ROS, extensive studies have shown that most ROS production derives from the activation of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase system. NOX4 is one of seven NADPH isoforms, however, unlike other members of the NOX family, it is constitutively active and the regulation of its effects occurs mainly at the expression level. The role of NOX4 as an important downstream mediator of TGF-\u03b2-induced myofibroblast generation, its contribution to the potent TGF-\u03b2 profibrotic effects, and its participation in the pathogenesis of tissue fibrosis in various fibrotic disorders such as IPF and liver fibrosis have been recently demonstrated ,143,144.MicroRNAs (miRNAs) are small non-coding RNA that modulate the expression of a large number of protein coding genes at the post-transcriptional level . Recent et al. [The pioneering studies of Zeisberg et al. clearly et al. . Anotheret al. . Other set al. ,118. Howet al. .et al. [et al. [et al. [Li and Bertram recentlyet al. examinedet al. . In anot [et al. employed [et al. . These s [et al. employin [et al. ,167.et al. [in vivo in this animal model.The role of EndoMT in experimentally induced pulmonary fibrosis and pulmonary arterial hypertension was examined in several recent studies. The pioneering studies of Hashimoto et al. evaluateet al. . The resin vitro with EC isolated from affected tissues. The study of Zeisberg et al. [in vitro studies with isolated human coronary artery EC. These studies showed that, following TGF-\u03b21 treatment, the cells became spindly shaped and lost their EC molecular markers and acquired the ability to produce various fibroblast-specific macromolecules including vimentin and type I procollagen. Several other studies have examined the occurrence of EndoMT in cultured human EC of various types. A study by Kitao et al. [in vitro in cultured human dermal microvascular EC and demonstrated the induction of a mesenchymal cell phenotype by TGF-\u03b21 with a change to a spindle cell morphology in monolayer culture and initiation of expression of \u03b1-SMA and COL1A. In another study Rieder et al. described the phenotypic conversion of human microvascular intestinal EC into mesenchymal cells following exposure to a combination of proinflammatory cytokines in vitro and suggested that intestinal EC exposed to an inflammatory environment may participate in the intestinal fibrotic process which accompanies intestinal inflammatory diseases in vitro [et al. examined the effects of interferons \u03b1 and \u03b3 on human dermal microvascular EC and demonstrated that interferon \u03b3 induced morphological and molecular/gene expression changes in these cells consistent with their transdifferentiation into profibrotic myofibroblasts [in vitro [In contrast with the extensive evidence from animal models demonstrating the occurrence of EndoMT in the development of experimentally-induced tissue fibrosis there are only few studies that have provided evidence indicative of the occurrence of EndoMT in human fibrotic disorders. Most studies have been performed g et al. describio et al. examinedin vitro . Chrobakroblasts . Recentlin vitro . The resin vitro . The samin vitro .in vitro following exposure to TGF-\u03b21 or to other cytokines or inducing agents. In contrast, there are few studies that have demonstrated the occurrence of EndoMT directly in vivo, although some recent publications have described results demonstrating the presence of EC expressing simultaneously EC molecular markers such as, for example, CD31/PECAM-1 or von Willebrand Factor and proteins such as \u03b1-SMA, or type I and III interstitial collagens that are specific for myofibroblastic mesenchymal cells. Most of these studies identified cells co-expressing both types of molecular markers in the affected tissues employing immunofluorescence microscopy studies with confocal microscopy. These studies are summarized in All the studies described in the previous section demonstrated that EC isolated from fibrotic tissues from various human fibrotic disorders were capable of undergoing EndoMT et al. [et al. [et al. [Despite the clinical importance and the demonstration of morphological and molecular alterations indicative of EndoMT during experimentally-induced pulmonary fibrosis , there aet al. were amoet al. and the et al. . In the [et al. employed [et al. , assesseWe recently performed a study to examine the possibility that EndoMT is involved in the fibrotic process of SSc-associated pulmonary fibrosis . ImmunohTissue fibrotic reactions represent the most serious complications of radiation therapy and intense investigation is being conducted to understand the intimate mechanisms involved in the devastating organ fibrosis and dysfunction induced by radiation therapy. Recent studies have examined the potential role of EndoMT in the severe radiation-induced fibrotic process. One study examined the role of EndoMT in the development of radiation-induced pulmonary fibrosis . Using iet al. [et al. [in vivo during the development of diabetic kidney disease-associated kidney fibrosis [Several studies have described evidence supporting the occurrence of EndoMT in human tissues from patients with cardiac fibrosis ,171 and et al. examinedfibrosis .The possibility that EndoMT of portal vein endothelium via TGF-\u03b2/Smad activation may be involved in portal venopathy was examined recently . The resin vitro studies with human EC of various tissue origins, experimental animal models of tissue fibrosis, and analysis of tissues from patients with various fibrotic diseases certainly indicate that EndoMT plays an important role in the pathogenesis of these common and often fatal disorders. The extensive literature published about the participation of EndoMT in the fibrotic process and the demonstration of its occurrence in affected tissues from patients with SSc-associated pulmonary fibrosis and PAH, vein graft fibrosis, intestinal fibrosis, and radiation-induced organ fibrosis indicate that such a role should no longer be considered controversial but it is a reality, as discussed recently [The results of the numerous studies reviewed here including recently ,176. A c"} +{"text": "For entry and infection viruses have developed numerous strategies to subjugate indispensable cellular factors and functions. Host cell lipids and cellular lipid synthesis machinery are no exception. Not only do viruses exploit existing lipid signalling and modifications for virus entry and trafficking, they also reprogram lipid synthesis, metabolism, and compartmentalization for assembly and egress. Here we review these various concepts and highlight recent progress in understanding viral interactions with host cell lipids during entry and assembly. The biological functions of lipids range from membrane formation to energy storage and signalling , glycophosphatidylinositol (GPI)-anchored proteins, and transmembrane proteins can cluster into discrete domains, called lipid rafts or glycosphingolipids . Upon specific binding to ganglioside GM1, SV40 reduces GM1\u2019s diffusion rate. This stabilized SV40-GM1 complex then recruits cholesterol to generate a lipid raft. The interaction induces actin-dependent immobilization of the virus-ganglioside complex, followed by virus-induced invagination of the plasma membrane. An elegant study by Ewers et\u2009al., et\u2009al., et\u2009al., Low-density lipoprotein receptors, also known as cholesterol receptors, are used by several members of the Flaviviridae family, including hepatitis C virus (HCV) and Niemann-Pick C1-like 1 (NPC1L1) are important entry factors for filovirus . Entry of VSV has been suggested to require the interaction between the viral glycoprotein G and the negatively charged phospholipid phosphatidylserine (PS) on the cell surface is the least abundant phospholipid in the cell membrane, but it is also one of the most versatile signalling molecules in cells and plays a central role in endosome trafficking and maturation. Differential phosphorylation of PI, regulated by specific PI kinases and phosphatases, results in the formation of different PI phosphate (PIP) species signalling pathway is one of the most important PI-mediated signalling cascades activated during virus entry. Activation of PI3K and subsequent generation of PIP3 serves as docking platform for proteins carrying lipid-binding domains, including Akt, the main effector of PI3K signalling provide a scaffold for the viral replication machinery, serve to concentrate the viral and cellular factors needed for assembly of new virions, and provide a protective environment for avoidance of cellular innate immune responses. Examples include the large dsDNA poxviruses which transiently recruit endoplasmic reticulum (ER)-derived cisternae around viral RCs , for instance, recruits PI4K-III\u03b1 to virus replication sites to increase local levels of PI(4)P for assembly of infectious viral particles cycle or by manipulating the carbon source used by the cells to generate energy and macromolecules, several viruses take control of central energy metabolism to promote synthesis of cholesterol and fatty acids. This phenomenon has been described for two large enveloped viruses: HCMV or endosomes (Ichihashi and Oie, et\u2009al., et\u2009al., et\u2009al., et\u2009al., et\u2009al., et\u2009al., et\u2009al., et\u2009al., et\u2009al., et\u2009al., et\u2009al., et\u2009al., et\u2009al., et\u2009al., et\u2009al., et\u2009al., Since its inception, viral apoptotic mimicry has proven to be a widespread lipid-mediated entry mechanism used by several enveloped viruses including: pichinde, cytomegalo, lassa fever, lenti, dengue, ebola and Marburg viruses (Callahan The fundamental role of lipids in virus biology and infection is becoming increasingly clear. New technologies, such metabolomics, and advances in mass spectrometry-based lipidomics are allowing for systematic characterization of the alterations in host lipid metabolism, as well as cellular and viral lipid profiles induced by viral infection.As highlighted in this review, lipids serve to orchestrate different stages of viral replication, ranging from entry to spread. Viruses take control of lipid-mediated signalling to co-ordinate viral entry and intracellular trafficking. Later during infection, they actively modify intracellular membrane for replication, re-direct lipid metabolism to produce sufficient membrane for the assembly of new particles, and modify cell membrane lipid content to ensure infectivity of those virions.Systematic characterization of how viruses take control of and alter lipid metabolism is now needed to unravel the common strategies used by these different viruses. Such studies may serve for the identification of infection biomarkers; and together with the development of therapeutics targeting subsets of lipid synthesis enzymes it may be possible to identify novel broad-spectrum therapeutic agents that target virus modified lipid metabolism. Overall, a deeper understanding of how viruses manipulate the host cells lipid program will serve to further our understanding of the cellular mechanisms that govern lipid modification, compartmentalization and metabolism."} +{"text": "The Editorial on the Research TopicImmunogenic Cell Death in Cancer: From Benchside Research to Bedside RealityGarg et al.; .Immunogenic cell death (ICD) has emerged as a cornerstone of therapy-induced antitumor immunity \u20133. ICD i et al.; ]. The imDerer et al. (Udo S. Gaipl lab). It is noteworthy that ICD can also be achieved by various \u201csmart\u201d combinatorial strategies\u2009\u2013\u2009an important point for clinically applied non-ICD inducers, discussed in details by Bezu et al. (Guido Kroemer lab).Most potent ICD inducers, characterized so far, elicit danger signaling through oxidative-endoplasmic reticulum stress . SeveralFucikova et al. (Radek Spisek lab). The promising results generated by systemically administered ICD inducers have also paved way for application of ICD-based dendritic cell (DC) vaccines and lymphoma by Zappasodi et al. (Massimo Di Nicola lab). In the latter case, it is clear that the field is moving toward chimeric antigen receptor (CAR)-T cell\u2019s application, and it will be interesting to see its combination with ICD in near future.Several lines of experimental evidence have established the validity of ICD. However, the overreliance on usage of prophylactic vaccination in transplantable (heterotopic) tumor models has attracted some criticism . While tvaccines . This imKersten et al. (Karin E. de Visser lab). The strategies for targeting epigenetic processes to improve immunotherapy are further discussed by Wachowska et al. (Jakub Golab lab).Nevertheless, the insurmountable complexity of cancer makes it inevitable that in certain contexts, ICD may fail. This failure may stem from various factors, e.g., tumor heterogeneity , MHC-levGarg et al.). This classification paper brings together >50 authors from the fields of ICD and DAMPs, and tries to reach a comprehensive accord on various terminologies related to DAMPs/ICD, the historical background of these concepts, ICD classification system (Type I vs. Type II inducers), and the relevant preclinical/clinical criteria crucial for the field(s) . We hope that this consensus paper will be a useful literature resource for various researchers/clinicians. These contributions, while summarizing the status quo, have also exposed a set of major questions and challenges that still need to be addressed.We believe that the valuable contributions of key researchers/clinicians toward this research topic/special edition have largely fulfilled its primary aim, i.e., to foster a critical discussion on experimental and clinical relevance of ICD. In fact, to further summarize and organize the fields of ICD and DAMPs, we have produced a multi-author consensus paper within this research topic that attempts to classify DAMPs and ICD inducers with an eye on translational potential of ICD apoptosis-associated immunosuppressive processes?Does the role of ROS in ICD extend beyond a proximal stressor? e.g., ROS-elicited oxidation-associated molecular patterns/OAMPs have been shown to mediate immunogenic potential cancer mice models clinical trial.Can ICD withstand the operational/regulatory (GLP/GMP/GCP) hurdles associated with anticancer vaccines-production? [indications for which are emerging (emerging ]Characterizing ICD-resistance mechanisms in the clinic.Characterizing reliable ICD-biomarker(s) detectable in patient tumor/sera-samples.Investigating ICD as a source of robust prognostic/predictive/mechanistic biomarkers [a point investigated recently in some studies is incontrovertibly valid; but, owing to the incomprehensible complexity of cancer, the \u201cspecifics of ICD\u201d will always remain open to amenability and variations. We envisage that overtime various \u201cvariants\u201d of ICD may emerge that differ from each other in a manner dependent upon, the type of anticancer therapy, cancer cell death pathways, cancer-types, tumor antigen make-up, the ADG wrote the manuscript. PA provided senior supervision and critically revised the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Huntington's disease (HD) is a hereditary neurodegenerative disorder caused by the expansion of a polyglutamine stretch within the huntingtin protein (HTT). The neurological symptoms, that involve motor, cognitive and psychiatric disturbances, are caused by neurodegeneration that is particularly widespread in the basal ganglia and cereberal cortex. HTT is ubiquitously expressed and in recent years it has become apparent that HD patients experience a wide array of peripheral organ dysfunction including severe metabolic phenotype, weight loss, HD-related cardiomyopathy and skeletal muscle wasting. Although skeletal muscles pathology became a hallmark of HD, the mechanisms underlying muscular atrophy in this disorder are unknown. Skeletal muscles account for approximately 40% of body mass and are highly adaptive to physiological and pathological conditions that may result in muscle hypertrophy or atrophy . The atrophy is caused by degeneration of myofibers and their replacement by fibrotic tissue is the major pathological feature in many genetic muscle disorders. Under normal physiological conditions the muscle function is orchestrated by a network of intrinsic hypertrophic and atrophic signals linked to the functional properties of the motor units that are likely to be imbalanced in HD. In this article, we highlight the emerging field of research with particular focus on the recent studies of the skeletal muscle pathology and the identification of new disease-modifying treatments. HTT) gene which are normally observed in healthy objects. Neurodegeneration, particularly widespread in the striatal nuclei, basal ganglia and cereberal cortex, is a source of neurological symptoms that involve motor, cognitive and psychiatric disturbances is neurodegenerative disorder caused by the expansion of polyglutamine stretch within the huntingtin protein (HTT) developed signs of a slowly progressing myopathy with elevated creatine kinase levels many years before first signs of chorea were detected. Muscle biopsy revealed a mild myopathy with mitochondrial pathology including a complex IV deficiency could be considered as an additional mechanism of HD pathology transcripts were significantly reduced and defects in mRNA processing were detected (Waters et al., Transcriptional deregulation is a typical feature of HD pathology in the brain (Luthi-Carter et al., To better understand a mechanism underlying muscle wasting in the R6/2 mouse model, key pathways governing protein metabolism, apoptosis and autophagy were examined. R6/2 mice exhibited increased adiposity and elevated energy expenditure without altered food intake. A total protein synthesis was unexpectedly increased in the gastrocnemius muscle by 19%, which was associated with over-activation of rapamycin mTOR signaling (She et al., HTT resulted in increased PGC-1\u03b1 expression in HD myoblast, while PGC-1\u03b1 rescue led to increased expression of markers for oxidative muscle fibers and reversal of blunted response for GPA in HD mice (Chaturvedi et al., c oxidase by 15%. Complex I\u2013dependent respiration of HD mitochondria showed more sensitivity to inhibition by Ca2+ than in wild-type mitochondria (Gizatullina et al., Since mitochondrial dysfunction might play a crucial role in HD pathology (Quintanilla and Johnson, As it has been mentioned in the previous paragraph, enhancing PGC-1\u03b1 activity might be a good strategy to improve skeletal muscles function in HD. Indeed, pharmacologic treatment with the pan-PPAR agonist bezafibrate restored the PGC-1\u03b1, PPARs and downstream genes to wild type levels. It also prevented conversion of type I oxidative to type II glycolytic muscle fibers as well as increased muscle mitochondria numbers. Finally, bezafibrate rescued lipid accumulation and apparent vacuolization of brown adipose tissue in the HD mice (Johri et al., in vivo (Fujimoto et al., The other strategy to improve muscle function in HD is based on the heat shock machinery modulation that could suppress mHTT aggregation (Labbadia and Morimoto, Recent studies also showed that HDAC4 function in the cytoplasm (Mielcarek et al., HD is a complex disease that has a peripheral component to its pathophysiology. Transcriptional changes in the HD skeletal muscles were comparable to those observed in the different brain regions and skeletal muscle wasting/atrophy is likely to be an important portion of HD pathogenesis. Some of the molecular and physiological changes in HD muscles can be detected, even in the pre-symptomatic HD individuals. On the molecular level, mitochondrial dysfunctions, PPAR alpha signaling and HSF1 activation were identified as major players in the muscle HD-related pathology. The major pathological pathways identified in skeletal muscles have been summarized in Figure Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Adeno associated vectors (AAV) have shown considerable promise to treat various genetic disorders in both preclinical and clinical settings mainly because of its safety profile. However, efficient use of AAV to deliver genes in immune-competent sites like muscles and liver requires very high doses which are associated with concomitant cellular immune response against the viral capsids leading to destruction of the transduced cells. Coupled with that, there are enough evidences that at high doses, AAV particles are subjected to increased cellular phosphorylation/uniquitination leading to proteasome mediated degradation and loss of the viral particles. The presence of preexisting immunity against AAV further adds on to the problem which is acting as a major roadblock to efficiently use it as a gene therapy vector in the clinics. To overcome this, rational bioengineering of AAV capsid becomes a prime tool by which specific amino acid residue(s) can be suitably modified/replaced by compatible residue(s) to create vectors having lower host immune response and higher intracellular trafficking rate. This article reviews the various aspects of rationally designing AAV capsids like by site-directed mutagenesis, directed evolution and combinatorial libraries which can create vectors having not only immune evasive property but also enhanced gene expression and transduction capability. One or more combinations of these strategies have strong potential to create novel vectors which will have suitable clinical efficiency even at a low dose. Despite the reported success it is becoming increasingly clear that humoral and cell mediated immune response against the vector is a major impending factor towards the efficacy of gene therapy [Adeno-associated virus (AAV) is a non-pathogenic parvovirus that has been widely used as a vector of choice for gene therapy. Although the virus has been detected in many different tissues of several animal species it has n therapy . Preexis therapy ,11.AAV has been used in several clinical trials for both inherited and non-inherited diseases with considerable success Table\u00a0. In the In contrast to these clinical studies, which targeted immune privileged sites, AAV has limited success when it came to treat monogenic diseases like haemophila B and lipoprotein lipase (LPL) deficiency following intravenous, intrahepatic or intramuscular administration Table\u00a0. For exaet al., tried to transiently suppress the immune system to inhibit AAV capsid specific T cell response against transduced hepatocytes expressing F.IX transgene in rhesus macaques [One of the major barriers to successful gene delivery with AAV vectors is the humoral immunity to wild type vectors. Humans are natural carriers of AAV genome. Neutralizing antibodies (NAb) to AAV AAV1 and 2) in humans was first reported in the early 60s and 70s ,19. Rece and 2 inmacaques . But no To summarize, immune-suppression can be advantageous as it represses the body\u2019s immune response long enough for the AAV capsid proteins to be not recognized by our defense mechanism thereby preventing NAb formation and allowing readministration of the vector. However this strategy will not be useful to circumvent preexisting NAb against AAV capsid. Thus, alternate strategies like rational capsid modifications must be looked into to evade these neutralizing antibodies.invitro. Extending this finding invivo the authors were able to elucidate a negative effect of tyrosine phosphorylation on viral intracellular trafficking and transgene expression [invitro (~10 fold) and invivo transduction efficiency of the novel vectors.The ubiquitin\u2013proteasome pathway has been shown to play an essential role in AAV intracellular trafficking ,27 and tpression . Thus baFollowing this finding, tyrosine mutant AAV vectors were used in other target sites like retina , skeletaApart from tyrosines, serines (S), threonines(T) and lysines(K) are also potential sites for phosphorylation and or ubiquitination on the AAV capsids and traditionally mutating them could augment AAV transduction efficiency. It has been shown earlier that targeted inhibition of the serine/threonine kinase phosphorylation of a cellular protein FK506-binding protein (FKBP52), improved AAV mediated gene transfer by 30-fold compared to ~5 fold increase seen with inhibition of tyrosine kinases alone . It is aGoing by this logic, several studies by our laboratory at Christian Medical College, Vellore, India, had generated S/T/K mutants on AAV1, 2, 5 and 8 which has proven to be more efficient in hepatic gene delivery as compared to their wild type counterparts -40. S/T Tenney et al., [In summary, rational point mutations on AAV capsids have shown considerable promise and this field is still wide open to explore especially since we have access to the 3 dimensional (3D) structures of several clinically important AAV serotypes -49. Usin et al., could deet al., [et al., [et al., in 1999 [et al., [et al., described a rationally designed chimeric AAV2.5 [et al., [Another approach to create novel AAV variant is to insert known ligands into the AAV capsid thereby allowing retargeting to specific cell types to which the WT vectors have a low affinity . By thiset al., where th[et al., demonstr in 1999 . In this in 1999 . However[et al., generate[et al., . Two new[et al., -59. Bowl[et al., describe[et al., as well [et al., .in vitro evolution strategy was first described by Grimm et al.,, in 2008 [et al., in 2008 [et al., [in vivo at serum levels which were much higher than what is required to neutralize the wild type vector. Additionally, both the mutants elucidated increased gene expression when compared to the wild type. Using DNA shuffling in an in vitro model of human ciliated airway epithelium, Li et al., [et al., [invivo. In this study they enriched for an AAV variant which showed widespread delivery to the outer retina and reverses the disease phenotypes of X-linked retinoschisis and Leber\u2019s congenital amaurosis in murine models. This vector also efficiently transduced primate photoreceptors from the vitreous.Chimeric capsids based on in 2008 . In this in 2008 created [et al., utilized et al., were abl[et al., successfet al., [et al., [et al., [et al., [We have seen in clinical trials using AAV that in targeting immune competent sites like liver and muscles, preexisting humoral immunity acts as an impediment to long term gene expression. Thus it necessitates basic knowledge of immunogenic epitopes on AAV capsids to rationally design AAV variants that can evade this immune response. Peptide scanning to map neutralizing epitopes for antibodies against AAV capsid opens up another rational approach to bioengineer AAV capsids. In one such early study Moskalenko et al., identifi[et al., where th[et al., . These A[et al., . In late[et al., which le[et al., created [et al., and Hutt[et al., demonstrAlthough AAV has gained immense popularity as a gene therapy vehicle to treat several genetic disorders, there is still a persistent need to further improve on the vector capsid design and engineering which can bypass the problem of neutralization by preexisting antibodies as well as T-cell mediated immune clearance. Over the past decade, many technologies have been used to make the AAV capsids less immunogenic and more efficient. For example, coating of AAV particles by inert polymers like polyethylene glycol (PEG) has been shown to modestly decrease its immunological properties. Site directed mutagenesis of amino acid residues (S/T/K/Y) on AAV capsids based on their phosphorylation status and presence on B- cell epitope has created novel vectors with reduced antibody response as well as high transgene expression. Rationally creating point mutations does not seem to interfere with their overall safety profile or packaging efficiency when compared to wild type vectors. Thus, it enables us to achieve high gene expression at a low vector dose which will further reduce the chance of eliciting immune response against the viral particles. Also, inserting known peptides at specific sites on AAV capsids can alter the natural tropism of AAV which is extremely helpful for targeted gene delivery at specific organs. Finally directed evolution of AAV can create novel chimeric vectors which can also have reduced neutralizing antibody response along with high target site specificity.An alternate strategy that can be employed in quest of a \u2018stealth\u2019 vector is to isolate/screen novel AAV\u2019s from human sources . This st"} +{"text": "Listeria monocytogenes and highlights recent discoveries on the molecular mechanisms evolved by this intracellular bacterium to subvert IFN responses.Interferons (IFNs) are secreted proteins of the cytokine family that regulate innate and adaptive immune responses to infection. Although the importance of IFNs in the antiviral response has long been appreciated, their role in bacterial infections is more complex and is currently a major focus of investigation. This review summarizes our current knowledge of the role of these cytokines in host defense against the bacterial pathogen Listeria monocytogenes is a pathogenic Gram-positive bacillus responsible for a foodborne disease in humans and animals called listeriosis , activation of pathogen-recognition receptors (PRRs) and subsequent production of pro-inflammatory cytokines production during infection. Type I IFN production by infected cells can be controlled by Listeria multidrug efflux pumps MdrM and MdrT, via the secretion of the second messenger cyclic-di-AMP , Salmonella typhimurium and some viruses is a paradigm for this, being an important mediator of innate and adaptive immune responses with a key role in clearance of viral and bacterial pathogens and in tumor control. IFN-\u03b3 was first described as an antiviral protein , Brucella abortus, Chlamydia muridarum, Francisella novicida, Salmonella enterica, Staphylococcus aureus, and Yersinia pestis , which encode proteins involved in a broad range of cellular functions (reviewed in MacMicking, Listeria infection has mostly been studied in the context of the IFN-\u03b3 pathway.The beneficial or detrimental effects of IFNs on Listeria cytoinvasion (Myers et al., Listeria infection by coordinating a potent oxidative and vesicular trafficking program (Kim et al., L. monocytogenes (Sonoda et al., L. monocytogenes (Cole et al., Listeria within granulomatous structures, thus avoiding massive T cell activation (Popov et al., The antilisterial activity of IFN-\u03b3 in phagocytic cells involves induction of oxidative and nitrosative defences, via increased expression of enzymes that control production of reactive oxygen and nitrogen species, such as NOX2/CYBB, DUOX2, and iNOS/NOS2 (MacMicking, Listeria infection is less documented. Zwaferink et al. have observed that upregulation of iNOS/NOS2 by IFN-\u03b2 promotes necrotic death of macrophages (Zwaferink et al., Listeria infection of epithelia is not understood.The function of type I IFN-associated ISGs in L. monocytogenes (Archambaud et al., Of interest, a subset of ISGs is amongst the most induced genes in the intestinal tissue of gnotobiotic humanized mice infected orally with Listeria has evolved several mechanisms to avoid immune detection and evade IFN responses. It has been demonstrated that deacetylation of Listeria peptidoglycan by the deacetylase PgdA confers resistance to host lysozyme, thus preventing release of MAMPs, such as DNA, RNA and lipopeptides, that trigger IFN-\u03b2 production (Boneca et al., Listeria pgdA mutants are rapidly killed in murine macrophages, which produce lysozyme, and induce a strong secretion of IFN-\u03b2 compared to wild-type Listeria. The role of PgdA is not limited to the control of type I-IFN production as a pgdA mutant hyperinduces pro-inflammatory cytokines as well. Modification of peptidoglycan by PgdA is an extremely efficient mechanism of immune escape used by Listeria, which correlates with its critical role in virulence.Listeria has evolved a sophisticated strategy to modulate, either negatively or positively, the expression of ISGs in epithelial cells, by targeting a chromatin-repressive complex, BAHD1 (Bierne et al., Listeria infection promotes, albeit via an unknown mechanism, the targeting of BAHD1 at the promoter of a set of ISGs, thereby downregulating type I- and type III-IFN responses. On the other hand, Listeria can produce a nucleomodulin, LntA, which when secreted by intracellular bacteria, enters the nucleus of infected cells, binds BAHD1 and inhibits its function (Lebreton et al., Remarkably, Listeria-host interactions. Yet, our understanding of the immune response to Listeria, and more specifically the role IFNs and of their downstream effectors, is far from complete and often relies on studies performed in cultured cells or in mice. However, murine and human listeriosis differ in many aspects (Lecuit, Listeria in epithelial cells, is not functional for Listeria uptake in the mouse. Thus, the route of entry of Listeria is not strictly the same in mice and humans. Moreover, ISGs induced in response to infection are not identical in mice and humans. Additionally, murine hepatocytes do not respond to type III-IFNs (Hermant et al., L. monocytogenes. Altogether, species-specific differences provide limits to the use of mouse models in characterizing IFN pathways engaged during Listeria infection in humans, especially in key epithelial organs such as the gut, liver and placenta. It will be important to perform future studies using adapted animal models, such as humanized mice permissive to oral infection or transgenic mice with human xenografts (Walters et al., Listeria infection depends on the route and time of infection (Pontiroli et al., Listeria infection in vitro, but the relevant ISGs and their cellular functions remain to be identified. Validation of ISGs identified in cultured cells in adequate in vivo models or deduced from analyses of patient samples, will be required to address the complex role of IFNs and bacterial subversion strategies and provide new insights into Listeria pathogenesis.We have an extensive knowledge of the molecular and cellular mechanisms involved in Lecuit, . For insThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "S-nitrosoglutathione (GSNO) indicates the involvement of these organelles in the sulfur metabolism. This could suggest the participation of a new family of molecules designated as reactive sulfur species (RSS) which will possibly provide new functions for peroxisomes.Peroxisomes are ubiquitous organelles with a notable oxidative metabolism. In plants, these subcellular compartments have been shown to be involved in the metabolism of reactive oxygen and nitrogen species (ROS and RNS), whose components, hydrogen peroxide and nitric oxide (NO), are important molecules involved in signaling processes. The presence of new elements in plant peroxisomes such as glutathione reductase (GR), sulfite oxidase (SO), glutathione (GSH), and \u00b7\u22122), hydrogen peroxide (H2O2), and RNS and GSNO which are characterized by a broad spectrum of physiological/pathological activities. Both these molecular families (ROS and RNS) include radical molecules containing an unpaired electron as well as non-radical molecules and can also have dual effects depending on their cellular concentration. Thus, H2O2 and NO at low concentrations can function as signal molecules in the cell or may cause damage to cell components when overproduced as a consequence of adverse conditions which does not reflect the complexity of their enzymatic composition has been shown to be generated from L-cysteine by the pyridoxal-5\u2032-phosphate-dependent enzyme. Thus, endogenous H2S can act as a neuromodulator in rat brain . Thus, high cellular GSH concentrations in an oxidative environment and the decomposition of S-nitrosothiols generate disulfide-S-oxides is an essential mineral for plant growth and development . This RNS is a highly oxidant compound capable of catalyzing the conversion of xanthine dehydrogenase to xanthine oxidase . SO catalyzes the conversion of sulfite to sulfate with the concomitant generation of H2O2 (Jim\u00e9nez et al., In this context, the interactions of ROS, RNS and possibly RSS components in plant peroxisomes open up new challenges and a new area of research to determine the biochemical interactions and potential functions of these reactive species of oxygen, nitrogen and sulfur in peroxisomes, some of which play a very important role as signaling molecules in physiological and phyto-pathological processes (Yamasaki, The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Within this physiologic range of RBC turnover, a small fraction of hemoglobin (Hb) is released into plasma as free extracellular Hb. In humans, there is an efficient multicomponent system of Hb sequestration, oxidative neutralization and clearance. Haptoglobin (Hp) is the primary Hb-binding protein in human plasma, which attenuates the adverse biochemical and physiologic effects of extracellular Hb. The cellular receptor target of Hp is the monocyte/macrophage scavenger receptor, CD163. Following Hb-Hp binding to CD163, cellular internalization of the complex leads to globin and heme metabolism, which is followed by adaptive changes in antioxidant and iron metabolism pathways and macrophage phenotype polarization. When Hb is released from RBCs within the physiologic range of Hp, the potential deleterious effects of Hb are prevented. However, during hyper-hemolytic conditions or with chronic hemolysis, Hp is depleted and Hb readily distributes to tissues where it might be exposed to oxidative conditions. In such conditions, heme can be released from ferric Hb. The free heme can then accelerate tissue damage by promoting peroxidative reactions and activation of inflammatory cascades. Hemopexin (Hx) is another plasma glycoprotein able to bind heme with high affinity. Hx sequesters heme in an inert, non-toxic form and transports it to the liver for catabolism and excretion. In the present review we discuss the components of physiologic Hb/heme detoxification and their potential therapeutic application in a wide range of hemolytic conditions.Hemolysis, which occurs in many disease states, can trigger a diverse pathophysiologic cascade that is related to the specific biochemical activities of free Hb and its porphyrin component heme. Normal erythropoiesis and concomitant removal of senescent red blood cells (RBC) from the circulation occurs at rates of approximately 2 \u00d7 10 Hemolysis with release of free hemoglobin (Hb) and heme occurs in a wide range of disease states and clinical interventions, including genetic and acquired anemias such as sickle cell disease, burns, extracorporeal circulation and massive blood transfusion state. The 32-kD Hb dimer readily passes through the glomerulus\u2014if not bound by Hp\u2014and is rapidly cleared by the kidneys , Hb exists as a tetrameric heme containing protein with a molecular weight of 64 kD. Following release from RBCs during intravascular hemolysis, extracellular Hb is in a dynamic tetramer \u2194 dimer equilibrium 16-fold faster than tetrameric Hb (kox = 0.015 h\u22121) Hb. While oxidized ferric (Fe3+) Hb usually remains below the level of detection in plasma, a large fraction of excreted Hb ultimately appears in the urine in the oxidized ferric state may accumulate. The large Hb-Hp complex cannot be filtered by the kidney and in vitro translocation across endothelial monolayers is almost completely blocked stabilizes Hb in a reduced state oxidation state -6 effector pathway, increases expression of Hp in the liver and many parenchymal and non-parenchymal cells. In many species, Hp is one of the most extensively responding acute-phase plasma proteins. IL-6 has also been reported to enhance expression of CD163 on macrophages, suggesting that enhanced Hb sequestration and clearance capacity are general adaptive responses to infection and tissue injury . Additionally, NO synthase (NOS), may be uncoupled due to oxidation of the essential cofactor BH4, thus leading to the generation of O\u00b7\u22122 in place of NO. Accumulation of ONOO- may further contribute to the reduction of NOS activity and the disruption of the NOS dimer.Hx also appears to support vascular NO homeostasis as demonstrated by studies in heme-overloaded Hx-null mice and mouse models of hemolytic disorders or lipid peroxides] occur in many disease states, particularly within the context of inflammatory reactions and during cycles of ischemia followed by reperfusion oxo-ferryl Hb [Hb(Fe4+ = O)] generation, (2) ferric Hb [Hb(Fe3+)] generation, and (3) protein radical generation [\u00b7Hb(Fe4+ = O)] during the peroxidative reaction sequence. However, Hp binding limits secondary reactions of these compounds with internal and/or external substrates or it can transfer to susceptible external molecules such as lipoproteins (\u201cextrinsic reactions\u201d). During the \u201cintrinsic\u201d free radical reactions within Hb, a defined peptide hotspot that is located at the \u03b1-globin/\u03b2-globin interface becomes the primary site where amino acid oxidation occurs. Among these amino acids is the highly susceptible \u03b2-chain Cys93, which can be oxidized to cysteic acid, as well as the \u03b1-chain Tyr42, which appears to be an important radical hub within Hb Hb to low-affinity heme acceptors Hb \u2014the substrate for heme transfer\u2014does not usually accumulate in plasma, even under severe hemolytic conditions Hb-Hp to accumulate and to reach significant plasma concentrations. In a hemolysis model in guinea pigs, we detected up to 50% of ferric Hb-Hp 24 h after infusion of an Hp bolus for treatment of severe blood transfusion-associated hemolysis. Conceptually, the Hb-Hp complex in plasma may therefore be better compared with a polymerized Hb such as a hemoglobin-based oxygen carrier (HBOC). The intentionally slow clearance rates of HBOCs also allow these substances for significant accumulation of ferric species in vivo. Following infusion of most HBOCs there is no barrier against heme release and the oxidative damage that is associated with heme loss may be a significant component of vascular toxicity that accompanies administration of these therapeutic candidates . Hx sequesters heme in an oxidatively inert conformation in a complex with a 1:1 stoichiometry, until it is cleared by the liver. Heme transfer to more reactive biomolecules is therefore suppressed by Hx. Accordingly, heme-overloaded Hx-null mice show increased oxidative stress in blood vessels, endothelial activation, enhanced inflammation and decreased NO bioavailability are therefore likely to modulate pathologies of the cardiovascular system such as atherosclerosis or typical vascular complications of hemolytic anemias. Oxidized lipoproteins are an established promoter of atherosclerosis and both Hb and heme can promote LDL oxidation (Balla et al., In humans, an Hp gene polymorphism exists that determines the three major phenotypes: 1-1, 2-1, and 2-2 (Levy et al., in vivo administration studies. These experimental Hp products resulted from industrial efforts to develop phenotype-specific therapeutic Hp products from pooled human plasma. Comparative investigations of two Hp products with predominant Hp 1-1 or Hp 2-2 composition found no significant differences in the intravascular sequestration or renal and vascular short term protection provided by dimeric and multimeric Hp phenotypes in a therapeutic setting of acute intravascular Hb exposure in guinea pigs and dogs (Lipiski et al., In the past, multiple functions of Hp, such as its anti-oxidative and CD163 adaptor functions, have been reported to be phenotype dependent (Asleh et al., Epidemiologic studies have also found controversial associations of Hp genotypes with the prevalence and clinical sequelae of atherosclerosis in the general population and in specific patient populations, particularly those with diabetes mellitus. However, more stratified analysis of several large observational studies now convincingly revealed that the Hp 2-2 genotype/phenotype is associated with increased relative risk of coronary artery disease of up to 10-fold in diabetic patients with a glycosylated HbA1c > 6.5 (Cahill et al., Thus, far, the pathophysiology underlying these associations is unknown. According to studies discussed above, the primary Hb detoxifying functions of Hp 1-1 and Hp 2-2 are comparable and can therefore not easily explain the epidemiologic differences. However, the biologic functions of Hp that have been investigated so far are more representative of the short term protective functions of Hp during acute hemolysis and may not fully reflect longer term antioxidant or immune modulatory functions of the protein that might be more relevant for chronic vascular disease development.Most epidemiologic studies of phenotype association with cardiovascular disease have been controlled for overall population heterogeneity and for established cardiovascular risk factors; however, these studies were not systematically controlled for differences in Hp plasma concentrations (Cahill et al., The controversial findings related to Hp phenotype-specific functions and associated disease state risks should certainly stimulate more extensive investigations. However, it now appears that, for therapeutic considerations, the Hp phenotype selection of a product will likely not be a primary determinant of clinical success (Lipiski et al., A genetic Hx polymorphism has been reported in populations of African ancestry (Kamboh et al., The only available data suggesting a strong association between plasma Hx level and disease risk come from studies evaluating the mouse model of Hx deficiency (Tolosano et al., Mouse models of sickle cell disease have a phenotype of chronic heme-driven endothelial activation and dysfunction that could be recovered by repeated Hx administration (Vinchi et al., Hemolysis has also been recognized to exacerbate some renal and vascular complications related to the transfusion of stored red blood cells. Retrospective clinical observation studies suggested a link between the storage duration of red blood cells and the incidence of cardiovascular complications such as myocardial infarction, stroke and death. One component of the so-called red blood cell storage lesion is an increased RBC fragility, which may lead to hemolysis post-transfusion. In a guinea pig transfusion model, increased fragility of older RBC could be linked to increased hemolysis in the post-transfusion period, appearance of free Hb in the circulation and Hb-triggered injury in the kidney and vasculature. All these pathologic changes could be prevented by Hp treatment at the time of old blood transfusion (Baek et al., Hemolysis with increased free Hb/heme levels and accompanying Hp/Hx depletion can be observed in some patients with severe sepsis. In a mouse sepsis model, Hx administration significantly reduced organ injury and mortality (Larsen et al., The rationale for the use of Hp and Hx as therapeutic agents is based on the idea that they act by scavenging circulating Hb and heme, with particular relevance to pathological conditions associated with hemolysis. Central to this concept is the notion that acute and chronic hemolytic conditions are characterized by depletion of the Hb and heme scavengers. Pre-clinical proof-of-concept animal models have demonstrated that Hp and Hx effectively attenuate Hb- and heme-induced vascular and renal pathologies. Based on these preliminary observations, it would appear rational to develop Hp and Hx for therapeutic use (Boretti et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Blood pressure homeostasis is maintained by several mechanisms regulating cardiac output, vascular resistances, and blood volume. At cellular levels, reactive oxygen species (ROS) signaling is involved in multiple molecular mechanisms controlling blood pressure. Among ROS producing systems, NADPH oxidases (NOXs), expressed in different cells of the cardiovascular system, are the most important enzymes clearly linked to the development of hypertension. NOXs exert a central role in cardiac mechanosensing, endothelium-dependent relaxation, and Angiotensin-II (Ang-II) redox signaling regulating vascular tone. The central role of NOXs in redox-dependent cardiovascular cell functions renders these enzymes a promising pharmacological target for the treatment of cardiovascular diseases, including hypertension. The aim of the present review is to focus on the physiological role of the cardiovascular NOX-generating ROS in the molecular and cellular mechanisms affecting blood pressure. Blood pressure is regulated by a dynamic equilibrium of different complex mechanisms Opie, . The maiNOX enzymes are membrane NADPH oxidases with the unique role of producing superoxide anions by one electron reduction of oxygen using NAD(P)H as electron donor have been identified Lambeth, Figure and togeROS have been for long time considered as toxic byproducts of the chemical utilization of oxygen within the cells and oxidative stress has been linked to the pathogenesis of many disorders Sies, . It was Multiple physiological functions have been so far attributed to NOX enzymes. DUOX1 and 2 enzymes, first discovered in thyroid with a role in thyroid hormones synthesis , vascular smooth muscle cells (VSMCs), and adventitial fibroblasts.ECs express NOX1, NOX2, NOX4, and NOX5 , is involved in many vascular processes including vasoconstriction, fibrosis, hypertrophy, inflammation, and aging . For the role of NOX-dependent Ang II signaling in the endothelium see NOXs in the endothelium-dependent relaxation.Ang II induces activation of the enzymatic activity and increases expression of NOXs both in cultured VSMCs and intact arteries generated by endothelial nitric oxide synthase (eNOS). The liposolubile NO diffuses across the membranes reaching VSMCs, where it increases cGMP levels by activating the soluble guanylate cyclase; the subsequent activation of cGMP-dependent kinases leads to a decrease of intracellular calcium levels and relaxation. Superoxide anions produced by NOXs react with NO to produce peroxynitrite close to NOX2. Then, ryanodine receptors-2 activation leads to an increase of local cytosolic Ca2+ concentration and force development (Prosser et al., in vitro results in an increase of ROS levels correlated with the amplitude and the frequency of stretch (Prosser et al., in vivo during the normal cyclic stretching and shortening of cardiomyocytes at each heartbeat, where the Ca2+ spark can be dynamically modulated by ROS in dependence of pre-load and heart frequency.NOX-generating ROS contribute to the positive inotropic response to mechanical stretch in cardiomyocytes. Physiological stretch triggers a microtubule-mediated activation of NOX2 localized at t-tubule membranes. This mechanism referred by Prosser et al. as X-ROS2+ concentration and myocardial contractility due to mechanical stretch, known as Anrep effect. This slow response follows within 1\u20132 min an increase of the afterload reaching a maximum after 10\u201315 min. In this case, NOX2-derived ROS mediates Ang II dependent ET-1 release. In cardiomyocytes, Ang II released by mechanical stretch (Sadoshima et al., +/H+ exchanger-1 (Akram et al., +, inhibition of Na+/Ca2+ exchanger, increase of cytosolic calcium concentration and contraction.NOX2 is also involved in the slow enhanced increase in intracellular Ca2+-ATPase, plasma membrane Ca2+ ATPase, L-Type Ca2+ channels and Nav are some examples of molecular target of ROS leading to modulation of intracellular Ca2+ levels linked to myocyte contractility (Sag et al., The role of NOXs in cardiomyocytes are not limited to mechanosensing. ROS produced by NOXs and by other sources such as mitochondria, are able to modulate different kinases phosphorylating proteins involved in calcium signaling; sarco/endoplasmic reticulum CaThe role of ROS in hypertension has been well documented (Lee and Griendling, Numerous studies using ROS scavengers or more specific NOX inhibitors, were aimed at evaluating the role of NOXs in the elevation of blood pressure in hypertensive animals (Lass\u00e8gue et al., 2O2 could mediate compensatory relaxation acting as an endothelium-derived hyperpolarizing factor (Yada et al., NOX2 elevation is correlated with hypertension. Indeed, in transgenic mice with endothelial-specific overexpression of NOX2, Ang II causes a greater increase in ROS production and attenuated acetylcholine-induced vasorelaxation compared to wild-type (Murdoch et al., Human studies aimed at linking NOX dysfunction with hypertension, have shown that some polymorphisms in the gene encoding p22 phox and affecting enzymatic activity, are associated with hypertension (Zalba et al., Finally, another interesting aspect of NOX involvement in blood pressure homeostasis impairment is related to cigarette smoke, a risk factor of hypertension. Cigarette smoke condensate exposures have been correlated with ROS production, downregulation of enzymatic antioxidant cellular systems and cell toxicity (Russo et al., in vitro experiments. However, due to the low bio-availability, peptide inhibitors are not promising therapeutic tools.NOXs are now being considered as target of pharmacological intervention in patients with hypertension (Cai et al., A number of small molecule NOXs selective inhibitors have been developed. Among them there are NOX1 and NOX 4 selective inhibitors like GKT137831, GKT136901, and GKT901 (Takac et al., Multiple molecular mechanisms regulating blood pressure involve NOX signaling. Of great importance is the central role of NOXs in angiotensin signaling, in the availability of NO and in the cardiac mechanosensing. A general scheme of the main overall effects of NOX-mediated signaling in the cells of the cardiovascular system leading to blood pressure modulation are shown in Figure The current challenge in the NOX biology research field is represented by the better understanding of the mechanisms by which NOX isoforms exert their differential biological effects leading to the development of substances able to modulate specific redox-dependent cell functions.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Many neurological disorders, including neurodevelopmental disorders, report hypersynchrony of neuronal networks. These alterations in neuronal synchronization suggest a link to the function of inhibitory interneurons. In Fragile X Syndrome (FXS), it has been reported that altered synchronization may underlie hyperexcitability, cognitive dysfunction and provide a link to the increased incidence of epileptic seizures. Therefore, understanding the roles of inhibitory interneurons and how they control neuronal networks is of great importance in studying neurodevelopmental disorders such as FXS. Here, we present a review of how interneuron populations and inhibition are important contributors to the loss of excitatory/inhibitory balance seen in hypersynchronous and hyperexcitable networks from neurodevelopmental disorders, and specifically in FXS. FMR1) gene and the consequent loss of the gene product of FMR1\u2014Fragile X Mental Retardation Protein repeat located in the 5\u2019 untranslated region of the gene expands to a length of more than 200 repeats. The loss of this protein is far reaching because FMRP interacts with approximately 4\u20138% of all synaptic mRNAs and regulates the translation of numerous synaptic proteins and receptor systems is one of several disorders associated with autism spectrum disorders (ASDs)\u2014a heterogeneous group of behaviorally identified neurodevelopmental disabilities. The prevalence rate of autism in FXS reportedly ranges from 25% to 52% , but not in the cornu ammonis area 1 (CA1) subregion played by this large and heterogeneous cell population (Buzs\u00e1ki et al., 2+-dependent spike initiation and/or propagation (Miles et al., As earlier stated, cortical networks in FXS are hyperexcitable and highly synchronous (Gon\u00e7alves et al., Fmr1 KO mouse model (Gibson et al., Fmr1 KO mice (Selby et al., Fmr1 KO mice compared to wild type animals (Paluszkiewicz et al., Fmr1 KO mice reduces inhibitory output which in turn alters the synchronization and spike output of excitatory neuronal networks in layer II/III (Paluszkiewicz et al., Fmr1 KO mice (Gibson et al., In FXS, EEG recordings show elevated relative theta power and reduced relative upper-alpha power (Van der Molen and Van der Molen, Fmr1 KO mouse model (Xu et al., There is additional evidence that suggest a role for interneurons in FXS with respect to specific activation via neuromodulators. Inhibitory interneurons have differential response to neuromodulators, among them, acetylcholine muscarinic receptors (Cea-del Rio et al., Interestingly, inhibitory neurotransmission dysfunction appears to be region selective. As stated above, studies in the cerebral cortex reveal interneuron specific problems. There is a clear lack of excitatory drive to FS interneurons in layer IV (Gibson et al., In summary, while enhanced excitatory neurotransmission leads to hyperexcitable phenotypes, inhibitory interneurons are not just contributing factors but are likely playing a major role in hyperexcitable, hyperresponsiveness and hypersynchronicity of neuronal networks in FXS (Gibson et al., Since many FXS patients also present with one or more features of ASDs, insights gained from studying the monogenic basis of FXS could pave the way to a greater understanding of the role of inhibitory interneurons in autism. At this point most of the evidence for interneuron participation is indirect in terms of neuromodulatory activation and downstream excitatory network activation, but very promising in terms of the relevance of their contribution. Thus, understanding how interneurons participate in neuronal network abnormalities seen in FXS lends to a greater understanding for neurodevelopmental disorders that fall in the autism spectrum.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Plants form the foundation for our global ecosystem and are essential for environmental and human health. With an increasing number of available plant genomes and tractable experimental systems, comparative and functional plant genomics research is greatly expanding our knowledge of the molecular basis of economically and nutritionally important traits in crop plants. Inferences drawn from comparative genomics are motivating experimental investigations of gene function and gene interactions. This special issue aims to highlight recent advances made in comparative and functional genomics research in plants. Nine original research articles in this special issue cover five important topics: (1) transcription factor gene families relevant to abiotic stress tolerance; (2) plant secondary metabolism; (3) transcriptome-based markers for quantitative trait locus; (4) epigenetic modifications in plant-microbe interactions; and (5) computational prediction of protein-protein interactions. The plant species studied in these articles include model species as well as nonmodel plant species of economic importance .Evolution of Transcription Factor Gene Families Relevant to Abiotic Stress Tolerance. The extant flowering plants have experienced multiple rounds of genome duplication and gene duplication is the primary source of gene family evolution. X.-L. Wang et al. in \u201cDivergence of the bZIP Gene Family in Strawberry, Peach, and Apple Suggests Multiple Modes of Gene Evolution after Duplication\u201d explored the evolutionary dynamics of the bZIP family, which contains transcription factors associated with plant tolerance to abiotic stress. They performed evolutionary analysis of the bZIP family in three rosaceous species in multiple aspects such as selection pressure on protein-coding sequences and genomic synteny. Another transcription factor gene family relevant to abiotic stress, the CCCH zinc finger family, was studied by W.-J. Chen et al. in \u201cSignificant Microsynteny with New Evolutionary Highlights Is Detected through Comparative Genomic Sequence Analysis of Maize CCCH IX Gene Subfamily.\u201d They performed comparative analysis of the CCCH IX subfamily in three cereal grain species and found that segmental duplication has played an important role in the expansion of this gene family. Their analysis also indicates that deletions, multiplications, inversions, and purifying selection have contributed to the evolution of the CCCH IX subfamily.Plant Secondary Metabolism. Plants produce a wide range of secondary metabolites that underpin functional diversity in plants. Gene expression profiling through transcriptome sequencing is a powerful approach for understanding the molecular basis of plant secondary metabolism. H. Tian et al. in \u201cAnalysis of Polygala tenuifolia Transcriptome and Description of Secondary Metabolite Biosynthetic Pathways by Illumina Sequencing\u201d analyzed expression of secondary metabolite biosynthetic genes in P. tenuifolia, a well-known medicinal plant, using RNA-seq approach. Their analysis revealed candidate genes that are potentially involved in biosynthesis of several important secondary metabolites such as triterpene saponins and phenylpropanoid. Similarly, R. Li et al. in \u201cDe Novo Transcriptome Sequencing of the Orange-Fleshed Sweet Potato and Analysis of Differentially Expressed Genes Related to Carotenoid Biosynthesis\u201d performed RNA-seq analysis of secondary metabolism in Ipomoea batatas, an important food crop. Through comparing the global gene expression profile in relation to the differences in the carotenoid content of two I. batatas cultivars, they identified more than 50 genes potentially involved in carotenoid biosynthesis. Also, Y. Wei et al. in \u201cGenome-Wide Identification of Genes Probably Relevant to the Uniqueness of Tea Plant (Camellia sinensis) and Its Cultivars\u201d performed comparative analysis of RNA-seq data in several species of the genus Camellia, an important source for tea production. They identified differentially expressed genes relevant to the biosynthesis of flavonoid, theanine, and caffeine. Furthermore, their sequence comparison revealed nonsynonymous mutations that are potentially related to the diversity between the two cultivars of C. sinensis.Transcriptome-Based Markers for Quantitative Trait Locus. Quantitative trait locus (QTL) analysis has been widely used for elucidating the genetic basis of complex traits in plants. Molecular makers are prerequisites for QTL analysis. QTL makers can be developed from either genome sequences or transcriptome sequences. Development of genome-based markers requires genome-sequencing data, which are available only in model plant species or major crop species. For nonmodel plant (crop) systems, transcriptome-based markers can be a better choice for QTL analysis with a limited budget. E. S. Seong et al. in \u201cExpressed Sequence Tags Analysis and Design of Simple Sequence Repeats Markers from a Full-Length cDNA Library in Perilla frutescens (L.)\u201d developed simple sequence repeats (SSR) markers based on approximately 1,000 expressed sequence tags (ESTs) derived from cDNA libraries for this member of the mint family used in traditional Asian medicine. They identified 18 SSR makers that could be very useful for understanding of genomic basis of medicinal function in P. frutescens. Recent advance in next-generation sequencing technology greatly enhances the capability for molecular marker development. Q. Ding et al. in \u201cCharacterization and Development of EST-SSRs by Deep Transcriptome Sequencing in Chinese Cabbage (Brassica rapa L. ssp. pekinensis)\u201d identified 10,420 SSR markers from 51,694 nonredundant unigenes assembled from RNA-seq data. This large set of SSR makers could facilitate genome-wide discovery of QTLs in Chinese cabbage. Also, R. Li et al. in \u201cDe Novo Transcriptome Sequencing of the Orange-Fleshed Sweet Potato and Analysis of Differentially Expressed Genes Related to Carotenoid Biosynthesis\u201d identified 1,725 SSR markers in the transcriptome data for sweet potato.Epigenetic Modifications in Plant-Microbe Interactions. While it is widely accepted that genetics governs plant growth, development, and response to environment, an increasing number of studies have showed that epigenetics also plays an important regulatory role in plants. The paper by K. Melmaiee et al. entitled \u201cQuantification and Gene Expression Analysis of Histone Deacetylases in Common Bean during Rust Fungal Inoculation\u201d revealed that epigenetic modification via histone deacetylases is involved in the response of common bean to rust fungal inoculation. The results from this paper provide new insight into the molecular mechanism underlying plant-microbe interactions.Computational Prediction of Protein-Protein Interactions. Protein-protein interaction (PPI) is an important molecular mechanism underlying various biological processes. Computational prediction of protein-protein interactions based on protein sequences is a straightforward approach to the utilization of whole-genome gene annotation for the global view of protein-protein interaction network in an organism. Various algorithms have been developed for protein sequence-based PPI prediction, though with limited success. J. Yao et al. in \u201cPPCM: Combing Multiple Classifiers to Improve Protein-Protein Interaction Prediction\u201d present a machine learning approach for PPI prediction based on various features derived from protein sequences. Their results demonstrated that integration of multiple features could significantly improve the PPI prediction accuracy as compared with prediction classifiers based on individual features. This novel approach has a great potential for PPI prediction in nonmodel organisms, including plant species."} +{"text": "Valero-Mu\u00f1oz et al. showed that heart failure with preserved ejection fraction (HFpEF) induced beiging in adipose tissue. They reported that in HFpEF brown adipose tissue (BAT) reduced the expression of some browning markers, such as uncoupling protein 1 (ucp-1), cell death-inducing DFFA-like effector a (cidea), and epithelial V-like antigen (eva), which were yet expressed by white adipose tissue (WAT) during beiging and further deepen the role of immune cells, particularly M1 and M2 macrophages (Liu et al., A complex machinery of interactions between bone marrow precursors, pre-adipocytes, mature adipocytes and de-differentiated/mesenchymal progenitors, strongly suggests that the \u201cadipose\u201d compartment is a complex dynamic system that acts on several physiological districts to regulate energy balance, survival and tissue renewal (Hausman and Hausman, Further markers and investigations are needed to elucidate the role of adipose tissue and its beiging in heart function and heart failure.The author confirms being the sole contributor of this work and approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Assessments of climate change impacts on forests and their vitality are essential for semi-arid environments such as Central Asia, where the mountain regions belong to the globally important biodiversity hotspots. Alterations in species distribution or drought-induced tree mortality might not only result in a loss of biodiversity but also in a loss of other ecosystem services. Here, we evaluate spatial trends and patterns of the growth-climate relationship in a tree-ring network comprising 33 juniper sites from the northern Pamir-Alay and Tien Shan mountain ranges in eastern Uzbekistan and across Kyrgyzstan for the common period 1935\u20132011. Junipers growing at lower elevations are sensitive to summer drought, which has increased in intensity during the studied period. At higher elevations, juniper growth, previously favored by warm summer temperatures, has in the recent few decades become negatively affected by increasing summer aridity. Moreover, response shifts are observed during all seasons. Rising temperatures and alterations in precipitation patterns during the past eight decades can account for the observed increase in drought stress of junipers at all altitudes. The implications of our findings are vital for the application of adequate long-term measures of ecosystem conservation, but also for paleo-climatic approaches and coupled climate-vegetation model simulations for Central Asia. Uzbekistan (UZ) and Kyrgyzstan (KG) belong to a region commonly referred to as Central Asia. This largely semi-arid to arid mountainous region is particularly vulnerable to ongoing and predicted climate change , 2. TempObserved impacts of anthropogenic climate change include an increase in health risks , decreasJuniperus spp.) dominates with around 80% of the forested areas at mid-to high elevations in Central Asia ).et al [et al [2 concentrations have been related to increased intrinsic water-use efficiency, where increased drought stress can lead to reduced stomatal conductance to compensate water loss at the expense of secondary growth [Although changes in climate during the past eight decades were remarkable and comparatively rapid, trends in juniper growth are less obvious and uniform. Combining the obtained results, we can summarize that juniper trees at the sites in Karakol, KG, with humid growing season climate seem to et al found sil [et al , reviewiy growth \u201354. Howey growth and 2) ty growth .Over the full 1935\u20132011 period, the growth-climate relationships of the juniper sites generally showed benefiting effects on tree growth from warm temperatures at high altitudes and abundant moisture supply at low elevation sites , agreeinJ. seravschanica in the Zaamin National Park, UZ [et al [et al [Conversely, junipers at lower altitudes showed distinct and persistent drought responses, which has previously been shown for Park, UZ . Liu et Z [et al or Liangl [et al noted all [et al , are likl [et al . Moreovel [et al .The identified growth-climate response shifts in our study, which occurred only within the past 40 to 50 years, most likely indicate impacts of the ongoing climate change on juniper growth in Central Asia. We therefore stress the importance of long-term conservation measures to counteract possible losses or reductions of the mountain juniper forest ecosystems in UZ and KG. Our findings also highlight the need of time-dependency analyses in paleo-climatological studies when using high elevation juniper trees, as well as the integration of growth-climate response shifts when assessing and predicting vegetation dynamics in Central Asia.Juniperus spp. and local drought-induced juniper die-backs in this biodiversity hotspot. Consequently, alterations in the floral composition of the temperate coniferous forest biome in Central Asia can be expected. We therefore stress the need to apply adequate long-term measures for ecosystem conservation and to consider this ongoing response shift in paleo-climate and model simulation approaches for this region.Using a unique and extensive juniper network established for the Tien Shan and northern Pamir-Alay mountain ranges, we detected changes in growth-climate responses during the past eight decades. Junipers at low elevation sites mainly increased their climate sensitivity to drought during summer and the entire growing season. At the highest elevation sites, however, juniper growth was favored by high summer temperatures during 1935\u20131964, but was limited by increasing drought conditions during the past ~30 years. This change in climate sensitivity of the junipers likely demonstrates the effect of the ongoing climate change, and also explains the low reconstruction skills of high elevation juniper sites in Central Asia. Across the study area, changes in climate response were detected at all regions during summer, but also during winter, spring and autumn. This may be the cause of the spatially non-uniform changes in growth trends across the study region. Finally, our results indicate that a further rise in temperature together with decreasing rainfall amounts, as predicted for the coming decades, will most likely increase the risk of altitudinal range shifts of S1 TableJuniperus sp. (JUSP), J. seravschanica (JUSE), J. semiglobosa (JUSM), and J. turkistanica (JUTU)), the number of trees (n) and TRW series (n), start and end year over the full and truncated (n(i) < 5 series) period, interseries correlation (Rbar), mean segment length (MSL), mean sensitivity (MS), Expressed Population Signal (EPS), and highest correlations with climate (rMAX) for the analyzed 1935\u20132011 period.Information include country code (CC), latitude , longitude , elevation , exposition (Exp), species ((EPS)Click here for additional data file.S2 TablePearson correlation results for all juniper sites and sites closest grid point data for monthly (current year January to December) and seasonal a) temperature means and b) precipitation sums for the full 1935\u20132011 period. Sites within the region are sorted based on increasing longitude and within the region from high to low elevation sites . Seasons include previous year December to current year February (pDJF), March\u2013May (MAM), June\u2013July (JJ), June\u2013August (JJA), September\u2013November (SON), May\u2013September (M\u2013S), April\u2013October (A\u2013O), and the entire year.(EPS)Click here for additional data file.S3 TableDifferences in climate response from 1935\u20131964 to 1982\u20132011 of all juniper sites for monthly (current year January to December) and seasonal a) temperature means and b) precipitation sums. Sites within the region are sorted based on increasing longitude and within the region from high to low elevation . Seasons include previous year December to current year February (pDJF), March\u2013May (MAM), June\u2013July (JJ), June\u2013August (JJA), September\u2013November (SON), May\u2013September (M\u2013S), April\u2013October (A\u2013O), and the entire year.(EPS)Click here for additional data file.S1 FigSee (EPS)Click here for additional data file.S1 File(XLSX)Click here for additional data file."} +{"text": "Loss of auditory sensory hair cells (HCs) is the most common cause of hearing loss. This review addresses the signaling pathways that are involved in the programmed and necrotic cell death of auditory HCs that occur in response to ototoxic and traumatic stressor events. The roles of inflammatory processes, oxidative stress, mitochondrial damage, cell death receptors, members of the mitogen-activated protein kinase (MAPK) signal pathway and pro- and anti-cell death members of the Bcl-2 family are explored. The molecular interaction of these signal pathways that initiates the loss of auditory HCs following acoustic trauma is covered and possible therapeutic interventions that may protect these sensory HCs from loss via apoptotic or non-apoptotic cell death are explored. Auditory hair cells (HCs) are important for the conversion of acoustic sound energy into electrical impulses that travel to the auditory centers of the brain for hearing. Sensorineural hearing loss (SNHL) is a form of hearing impairment that occurs most commonly from damagedHCs within the cochlea; it is a prevalent disability, affecting one in five people and more than 48 million Americans , endonuclease G (EndoG), apoptosis inducing factor (AIF), second mitochondria-derived activator of caspases/direct inhibitor of apoptosis protein binding protein with low pI (Smac/DIABLO), and mammalian homolog of bacterial high temperature requirement protein A2 and recruits pro-caspase-9 to form an apoptosome . XIAP is a cytosolic protein that has three baculoviral inhibitory repeat (BIR) domains\u2014BIR1 and BIR2 specifically bind and inhibit caspase-3 and -7, while BIR3 is a specific inhibitor of caspase-9 binding of a death ligand to its complementary receptor; 2) recruitment of adaptor molecules such as FAS-associated death domain protein (FADD) and tumor necrosis factor receptor type 1-associated death domain protein (TRADD); (3) binding, dimerizing, and activation of initiator caspase-8 and -10; and (4) formation of a death-inducing signaling complex , respectively are important regulators of cell survival and cell death. Necroptosis refers to a RIPK3-dependent molecular cascade of events that promotes regulated necrotic cell death polymerase 1 (PARP1) dependent form of non-apoptotic death. PARP1 is recruited to sites of DNA damage where it is able to catalyze ADP ribosylation of various proteins and histones and generate negative charges by overconsumption of nicotinamide adenine dinucleotide (NAD) during the ADP ribosylation process. Ultimately, other factors important for DNA repair are recruited. However, when PARP1 becomes hyper-activated in situations of persistent DNA damage, there is a depletion of NAD and inhibition of mitochondrial ATP synthesis, which is essential in ATP-dependent apoptosis pathways , caspase-3 activation, and intrinsic cell death , and hydroxyl expression of extracellular pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF\u03b1) and recruited of neutrophils and macrophages to the cochlea; and 2) generation of oxidative stress in the form of ROS and reactive nitrogen species (RNS) such as superoxide on the cell surface of auditory HCs and initiate a signaling cascade that can lead to cell death that promotes mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NFkB) signaling have also been detected in OC and cochlea following various inner ear challenges such as exposure to gentamicin, electrode insertion trauma, and autoimmune responses mediated by inner ear tissues 1 and 2 (ERK1/2), c-Jun N-terminal kinases , and p38 kinases and blocking transcription of anti-apoptotic genes (such as Bcl-2) have demonstrated protection against HC and hearing losses following aminoglycoside, acoustic, and electrode insertion injury to the cochlea contains two unpaired electrons. Superoxide anion -mediated apoptosis; however there is some recent evidence that oxidative stress can turn on extrinsic cell death signaling. This again demonstrates the many levels of communication that occur both upstream and downstream in events of programmed cell death. ROS can activate apoptosis signal-regulating kinase-1 (ASK-1), which is a MAPKKK that can phosphorylate and activate mediators of the JNK and p38 pathways of extrinsic programmed cell death that can trigger up regulation of genes important for cell death such as Bim and Bcl-6 . Acoustic trauma can also initiate inflammation and edema of the stria vascularis and compromise the blood supply to the cochlea (Smith et al., Acoustic trauma can lead to ROS release by the marginal cells of the stria vascularis as a result of reductions in cochlear blood flow and cochlear hypoxia (Yamane et al., c (Wang et al., When TNF\u03b1 binds to its complementary receptor TNFR1, TRADD and FADD are recruited along with caspase-8. Caspase-8 can activate executioner caspase-3 to promote extrinsic cell death through DNA fragmentation and chromatin condensation via CAD, ACINUS, and HELI-CARD or it can cleave BID into tBID, which can activate Bax-mediated intrinsic, mitochondrial cell death (Liu et al., c into the cytoplasm, generation of apoptosomes, and activation of caspase-3 dependent cell death. AIF and EndoG are also released into the cytosol, translocate into the nucleus, and can initiate chromatin condensation and DNA fragmentation of auditory HCs (Nicotera et al., Oxidative stress from noise exposure can also initiate intrinsic apoptotic cell death in auditory HCs, resulting in mitochondrial release of cyt In addition to apoptosis, acoustic trauma can result in regulated necrosis through RIPK3/RIPK1 activation in rats, that was reversed with necrosis inhibitor necrostatin-1 (Zheng et al., Acoustic trauma can also promote swelling and rupture of dendritic terminals of cochlear nerve afferent fibers (Spoendlin, Furthermore, intense noise exposure can increase intracellular calcium in auditory HCs and activate calcium-dependent calpains\u2014cysteine proteases that promote proteolysis and breakdown of cytoskeletal and membrane proteins, kinases, phosphatases, and transcription factors (Wang et al., Otoprotective drugs can target different levels in apoptosis and necrosis signaling pathways following acoustic trauma Figure . GlucocoNumerous diverse insults to the inner ear can cause auditory HC damage and hearing loss. The evolutionarily conserved apoptotic and necrotic cell death signaling that occurs in auditory HCs is shared among many ototoxic and traumatic stressor events. The most well studied molecular mechanisms behind cell death in auditory HCs involve TNF\u03b1 signaling, JNK and p38 activation and the effect of high levels of oxidative stress. Although their effects on intrinsic and extrinsic pathways of apoptosis have been studied extensively, there are likely many levels of cross communication between signaling cascades that are still undiscovered. Research in this area is becoming more prevalent, as well as research into mechanisms of regulated necrosis in auditory HCs. A number of otoprotective drug therapies target different levels along this pro-inflammatory pro-death signaling cascade to block downstream events that lead to cell death and promote auditory HC viability and hearing protection.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "CYP3A2) mRNA expression and DNA fragmentation, as well as on contents of different cholesterol fractions and protein thiol groups in rat testes. Wistar albino male rats were divided into two groups: I \u2013 control , II \u2013 chronic alcoholism (15% ethanol self-administration during 150 days). Following 150 days of alcohol consumption, testicular free amino acid content was found to be significantly changed as compared with control. The most profound changes were registered for contents of lysine (\u201353%) and methionine (+133%). The intensity of DNA fragmentation in alcohol-treated rat testes was considerably increased, on the contrary CYP3A2 mRNA expression in testis cells was inhibited, testicular contents of total and etherified cholesterol increased by 25% and 45% respectively, and protein SH-groups decreased by 13%. Multidirectional changes of the activities of testicular dehydrogenases were detected. We thus obtained complex assessment of chronic alcoholism effects in male gonads, affecting especially amino acid, protein, ATP and NADPH metabolism. Our results demonstrated profound changes in testes on the level of proteome and genome. We suggest that the revealed metabolic disorders can have negative implication on cellular regulation of spermatogenesis under long-term ethanol exposure.There is good evidence for impairment of spermatogenesis and reductions in sperm counts and testosterone levels in chronic alcoholics. The mechanisms for these effects have not yet been studied in detail. The consequences of chronic alcohol consumption on the structure and/or metabolism of testis cell macromolecules require to be intensively investigated. The present work reports the effects of chronic alcoholism on contents of free amino acids, levels of cytochrome P450 3A2 ( Spermatet al., et al., et al., Ethanol can cause disturbance in the main metabolic pathways Zakhari, . AlcoholCYP3A2) mRNA expression and DNA fragmentation, as well as on contents of different cholesterol fractions and proteins thiol groups in rat testes.Based on these facts and considering that alcoholism is a chronic disease highly prevalent in the world population, the present work reports the effects of chronic ethanol consumption on free amino acids, levels of cytochrome P450 3A2 , relative humidity of 40% to 70%, lighting (12 h light-dark cycle), and on a standard pellet feed diet . The study was performed in accordance with the recommendations of the European Convention for the Protection of Vertebrate Animals Used for Experimental and other Scientific Purposes and approved by the Institutional Animal Care and Use Committee. For the experimental model, reproducing male rats were selected according to the method for measuring voluntary alcohol self-administration in rats, which provides a continuous choice between an alcohol solution and water (two-bottle preference test) were used as controls. From the beginning of the experiment, they were kept in the same conditions as the experimental animals, but were given only water After 150 days, both the experimental and control rats were sacrificed under a mild diethyl ether anesthesia by decapitation. The right testis was used for histochemical analysis and the left testis for other investigations.et al. activity in the testes was demonstrated by the method of Nachlas et al. was histochemically detected according to the technique of Hess g, 10 min, 4 \u00b0C) equal volumes of 3% sulfa-salicylic acid were added and the obtained mixtures were left for 10 minutes in the refrigerator at 4 \u00b0C. The formed sediments were removed by centrifugation and then fractionated through 2% agarose gels . After electrophoresis, the gels were stained with ethidium bromide and visualized under a UV transilluminator . Analysis of electrophoresis data was carried out with Quantity One Software (USA).The DNA from the testes was isolated by a modified method from Current Protocols in Toxicology . Data were compared using Student's t-test. Differences were considered to be statistically significant at Investigation of chronic alcoholism effects on rat testis pools of free amino acids showed tInvestigation of rat testes DNA fragmentation demonstrated its essential intensification following 150 days of ethanol administration in comparison with control . In the In testes of alcohol-treated rats 8 fractions of DNA fragments with weights over 1000 (4 different fractions), 700, 550, 30 and 20 b.p. were present. Main of high-weighted DNA fragments were fractions with weights over 1000 b.p., while among low-weighted DNA fragments fractions were approximately equal (by peak intensity).CYP3A2 mRNA expression was indicated in testes of rats with chronic alcoholism and reduced form of nicotinamide adenine dinucleotide phosphate (NADPH), both at the stage of glycolysis (changes of glycine and serine) and in the citric acid cycle (changes of methionine) , with superoxide anions, oxygen molecules, hydroperoxide, organic peroxides and peroxide radicals Stepuro, . Thus, cet al., et al., et al., et al., Changes of methionine in testicular pools deserve special attention. This amino acid can act as antioxidant and immunomodulator Pavlov, . Methionet al., et al., et al., et al., It is reasonable to propose that biosynthesis of S-adenosylhomocysteine and homocysteine could be also broken due to excessive alcohol intake. Changes in contents of methionine, glycine and serine \u2013 amino acids involved in the synthesis of these compounds , changes in its contents can cause changes in free amino acid contents .Changes of such amino acids as glycine in the testis pool are of special importance because of the involvement in glutathione biosynthesis Marks, . Such chet al., et al., et al., DNA is an important molecular target for toxicants by mitochondria, driven by acetaldehyde metabolism, is a common trigger of both mechanisms . In our previous study, we showed CYP2E1 mRNA expression and protein content elevation in alcohol-treated rat testes with simultaneous spermatogenesis violations , which are involved in testosterone biosynthesis , a member of the nuclear receptor superfamily, regulating gene transcription in a ligand-dependent manner and much of this function is attributed to the thiol groups present on them. One or more reduced thiol (-SH) groups are essential for the function of many proteins (Top\u00e7uoglu et al., Our results on the decrease of protein SH-groups in testes of alcohol-treated rats are in good accordance with data of clinical studies on the decrease of total thiol in alcohol abusers (Prakash Our results suggest that the self-administration of 15% alcohol in rats during 150 days led to multidirectional changes of the activity of testicular dehydrogenases, which play a crucial role in supplying energy needed for various metabolic functions in germ cells Mathur, . Dehydroet al., Forming part of complex II of the respiratory chain, SDH, is situated at the intersection of the citric acid cycle and oxidative phosphorylation. This combination of functions places SDH at the center of two essential energy-producing metabolic processes of the cell (Cervera et al., It is well known that spermatogonia may utilize glucose as the major energy substrate, but spermatocytes and spermatids suffer a rapid decline in their ATP content in glucose-supplemented media and require lactate/pyruvate for the maintenance of their ATP concentrations (Jutte et al., et al., Glucose transport into the cell and the lactate LDH isoenzyme system, which reversibly catalyzes the inter-conversion of pyruvate and lactate, are biochemical steps which participate in the regulation of lactate production (Riera Chemically induced stress causes elevated LDH activity, which can be used as a good diagnostic tool in toxicology (Ksheerasagar & Kaliwal, CYP3A2 mRNA expression and DNA fragmentation processes, as well as changes in cholesterol and protein thiol group contents allowed us to obtain complex estimation of this pathologic influence in male gonads, especially on the metabolism of amino acids, proteins, ATP and NADPH. Our results demonstrated profound changes in testes on the level of proteome and genome. We suggest that the revealed testicular metabolic disorders could have negative implications on cellular regulation of spermatogenesis under long-term ethanol exposure.Thus investigation of chronic alcoholism effects on testicular levels of free amino acids, rates of"} +{"text": "Cilia are whip-like projections that are widely conserved in eukaryotes and function as a motile propeller and/or sensory platform to detect various extracellular stimuli. In vertebrates, cilia are ubiquitously found in most cells, showing structural and functional diversities depending on the cell type. In this review, we focus on the structure and function of cilia in choroid plexus epithelial cells (CPECs). CPECs form one or two dozen non-motile 9+0 cilia, which display transient acquisition of motility during development. Genetic malfunction of cilia can lead to failure of multiple organs including the brain. Especially, several groups have demonstrated that the defects in CPEC cilia cause the communicating form of hydrocephalus. In order to elucidate the molecular mechanisms underlying the hydrocephalus, we have previously demonstrated that the cilia possess an NPFF receptor for autocrine signaling to regulate transepithelial fluid transport. In this perspective, we also discuss the potential involvement of cilia in the other aspects of choroid plexus functions, such as the regulation of brain development and neuroinflammation. Cilia are hair-like projections on the cell surface with a diameter of ~250 nm and various lengths of typically 5\u201310 \u03bcm Figure . Their sFor example, ependyma (ependymocytes) lining brain ventricles form hundreds of motile cilia to circulate the cerebrospinal fluid (CSF). The axoneme of this ciliary subtype has a central pair of singlet microtubules (termed \u201c9+2\u201d), and is heavily equipped with axonemal dyneins and their regulatory complexes, which collectively drive the back-and-forth movement of cilia Figure . In contGenetic defects leading to ciliary malfunctions cause disorders with clinically variable phenotypes. Such disorders are called ciliopathies and include primary ciliary dyskinesia, polycystic kidney disease, Leber congenital amaurosis, nephronophthisis, Senior-L\u00f8ken syndrome, Joubert syndrome, Bardet-Biedl syndrome, and Meckel Gruber syndrome carries tubulin and other materials along the axoneme that produce CSF with high efficiency (Damkier et al., As described above, mature ependyma form hundreds of motile 9+2 cilia that beat in a concerted manner to circulate CSF. In mouse, the multiciliogenesis initiates after birth and requires about 2 weeks for full maturation Figure . In contCelsr2, an ortholog of the planar cell polarity gene Flamingo, an impairment of ciliogenesis is observed in ependyma but not in CPECs (Tissir et al., Genetically modified mouse models have also shown differences in the mechanism of ciliary formation and/or the maintenance of cilia in CPECs and ependyma. In a knockout mouse for Tg737RpwIft88 mouse that has defects in IFT88 expression and function, Banizs et al. observed a communicating form of the hydrocephalus at neonatal periods, when most ependyma lack mature motile cilia. During these stages, CPEC cilia show an accumulation of polycystin-1, the defects of which cause autosomal dominant polycystic kidney disease, in a bulb-like structure at the tip. This abnormal ciliary structure and protein localization coincide with an increase in cellular cAMP levels and aberrant regulation of intracellular pH and ion transport activities in CPECs (Banizs et al., Pkd1 knockout mice, which encodes polycystin-1, and observed hydrocephalus at perinatal periods in both mouse lines.Regarding the unique function of CPEC cilia, several groups including ours have reported the potential involvement of CPEC cilia in the regulation of CSF production. Analysis of CPEC cilia in relation to the hydrocephalus was first described by Yoder et al. (Banizs et al., We used a primary culture system for swine CPECs to analyze ciliary function and showed that deciliation by chloral hydrate increases both intracellular cAMP levels and basolateral-to-apical transepithelial fluid transcytosis, which is consistent with the above observations by Banizs et al. (Narita et al., Bbs1, Bbs2, Bbs4, and Bbs6 mutant mice (Swiderski et al., Swiderski et al. investigated the mechanism of ventriculomegaly that is common in ciliopathy models of Kif3a in cranial neural crest cells, using a Wnt1 promoter-driven Cre recombinase (Liu et al., cre expression in E16.5 choroid plexuses, they concluded that the hydrocephalus is due to overproduction of CSF (Liu et al., Recently, Liu et al. generated a conditional knockout of Rfx3 knockout mouse also exhibits corpus callosum agenesis (Benadiba et al., The above studies implicate defects in CPEC cilia as a cause of the communicating form of hydrocephalus. However, reports by Durand et al. suggest additional mechanisms. They generated mice deficient for Rfx3, a transcription factor that regulates ciliogenesis, and demonstrated marked inhibition of ciliogenesis in both CPECs and ependyma, which is associated with the communicating form of hydrocephalus (Baas et al., A growing body of evidence suggests that the choroid plexus functions as a selective and educative gate for circulating immune cells in the immune surveillance of the CNS to resolve neuroinflammation under pathological conditions (Schwartz and Baruch, Recently, we performed proteomic analysis of CPEC cilia from swine and identified >800 proteins (Narita et al., According to the traditional view, CPECs have been regarded as solely responsible for the production of CSF. However, based on our current understanding of CSF production, we should re-interpret or re-evaluate the traditional views of CSF homeostasis (Iliff et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "The most common diseases resulting in irreversible blindness or vision impairment include age-related macular degeneration (AMD), glaucoma, diabetic retinopathy, cataract, and dry eye. These diseases seriously affect the quality of life in elderly people worldwide. Therefore, understanding their pathogenesis and the development of strategies allowing earlier detection and treatment demands more effort and attention from both basic and clinical fields. This issue focuses on different aspects of these diseases in elderly population.\u03b2, IL-18, or IL-17\u201d by S. Chen et al., the authors investigated the responses of multipotent retinal stem cells (RSCs), isolated from mice, to the proinflammatory signaling molecules including IL-1\u03b2, IL-18, and IL-17A. They found that the addition of IL-1\u03b2, IL-18, or IL-17A in the cultured cell decreased RSC viability but increased pyroptotic and/or necroptotic cells. The study is innovative and unique, because, instead of RPE19, a new cell type was used as the model system. Additionally, the results fill gaps in understanding immunological mechanism of AMD pathogenesis.AMD is one of the most common diseases resulting in irreversible blindness worldwide in the elderly population. Among multifactorial pathogenesis, immune dysregulations are leading theories of AMD pathogenesis. Recently IL-17 pathway was reported to be involved in AMD pathogenesis. In the original article \u201cResponses of Multipotent Retinal Stem Cells to IL-1Like AMD, glaucoma and diabetic retinopathy are common causes of blindness in older adults. Glaucoma is often caused by damage to the optic nerve due to an abnormally high pressure in your eye, while diabetic retinopathy is a diabetes complication, caused by damage to the retinal blood vessels. However, both of them have no symptoms or warning signs at early stage. Thus, it is important to have regular eye exams to measure intraocular pressure and ocular blood flow. In the review paper \u201cOcular Blood Flow Autoregulation Mechanisms and Methods\u201d by X. Luo et al., the authors summarized the methods for ocular blood flow evaluating and discussed mechanism and treatment of ocular blood flow regulation, particular in glaucoma and diabetic retinopathy.Cataract is one of the major causes of visual impairment of elderly people. Although recent bioinformatics studies revealed susceptibility genes, such as EPHA2, for age-related cataract, the mechanism underlying its pathogenesis remains elusive. In the original paper \u201cThe Polymorphisms with Cataract Susceptibility Impair the EPHA2 Receptor Stability and Its Cytoprotective Function\u201d by J. Yang et al., the authors found that EPHA2 signaling can protect the lens epithelial cells from oxidative stress-induced cell death. In the original paper \u201cEpigenetic Regulation of Werner Syndrome Gene in Age-Related Cataract\u201d by X. Zhu et al., the authors investigated the promotor methylation and histone medication of Werner syndrome gene (WRN). They found that both mRNA and protein levels of WRN were significantly decreased only in anterior lens capsules in age-related cataract, suggesting that the strategies to intervene epigenetic alteration in this disease should aim to anterior lens capsules. By investigating very large cataract patient population in rural China, X. Cao et al. presented a normative ocular biometry of adult cataract patients in rural China. They found that the axial length is normally distributed with a positive skew and a big kurtosis and corneal astigmatism may affect rural Chinese vision acuity.Dry eye is a multifactorial disorder of the tears and ocular surface and is a common and often unrecognized disease affecting tens of millions of individuals worldwide. Q. Long et al. evaluated the biomechanical behavior of the cornea in dry eye using, for the first time, Corneal Visualization Scheimpflug Technology (CorVis ST), a new noncontact tonometry system. Their results provide insight into its full usefulness for dry eye patients. B. Wang et al. compared dry eye disease that resulted from two refractive surgeries [small-incision lenticule extraction (SMILE) versus femtosecond laser in situ keratomileusis (FS-LASIK)] in high myopia. They found that SMILE is a safe and successful alternative for the correction of refractive error and may provide a more superior and safer refractive outcome than FS-LASIK in the first six months following surgery. CorVis ST, the very latest technology, has been used by J. Wang et al. to assess the biomechanical parameters of the cornea in myopic and emmetropic eyes.Vitreous hemorrhage (VH) is one of the ophthalmologic emergency situations. In the paper contributed by D. Y. Kim and colleagues, the authors analyzed causes and prognosis of acute-onset preoperatively unknown origin VH in 169 eyes and found that retinal vein occlusion, retinal break, and AMD are the most common causes. In addition, aging may be an important factor for influencing visual prognosis following vitrectomy.Optic neuritis is one of the common optic neuropathies and is highly associated with multiple sclerosis. In the original paper \u201cEvaluation of Retinal Nerve Fiber Layer and Ganglion Cell Complex in Patients with Optic Neuritis or Neuromyelitis Optica Spectrum Disorders Using Optical Coherence Tomography in a Chinese Cohort\u201d by G. Tian et al., the authors reported that spectral-domain optical coherence tomography, SD-OCT, is a very useful and objective method to evaluate the thickness of the peripapillary retinal nerve fiber layer and macular ganglion cell complex in optic neuritis and neuromyelitis optica.Common age-related ocular diseases demand attention as a global health problem. This special issue covered pathogenesis, diagnosis, and treatment of most of these diseases.Jun ZhangJun ZhangJingsheng TuoJingsheng TuoZhongfeng WangZhongfeng WangAiqin ZhuAiqin ZhuAnna Machali\u0144skaAnna Machali\u0144skaQin LongQin Long"} +{"text": "Hypoxia is an important micro-environmental characteristic of rheumatoid arthritis (RA). Hypoxia-inducible factors (HIF) are key transcriptional factors that are highly expressed in RA synovium to regulate the adaptive responses to this hypoxic milieu. Accumulating evidence supports hypoxia and HIFs in regulating a number of important pathophysiological characteristics of RA, including synovial inflammation, angiogenesis, and cartilage destruction. Experimental and clinical data have confirmed the upregulation of both HIF-1\u03b1 and HIF-2\u03b1 in RA. This review will focus on the differential expression of HIFs within the synovial joint and its functional behavior in different cell types to regulate RA progression. Potential development of new therapeutic strategies targeting HIF-regulated pathways at sites of disease in RA will also be addressed. It is an autoimmune and polyarthritic condition, characterized by inflammation of the synovium (synovitis), progressive cartilage destruction, and bone erosion, which ultimately results in loss of integrity of the affected joints are activated as an adaptive mechanism. HIFs are considered the \u201cmaster regulators\u201d and macrophage-like synoviocytes and an underlying loose connective tissue called the sub-lining (sub-intima) layer Smith, . The arcInflammatory factors are known to upregulate the expression HIF-1\u03b1 and HIF-2\u03b1 in RA. Pro-inflammatory cytokines such as IL-1, TNF-\u03b1, and IL-33 have previously been reported to increase the expression of HIF isoforms in synovial fibroblasts are pattern recognition receptors that are mainly expressed in immune cells and RASF cells in RA to regulate inflammatory responses (Brentano et al., Immune cells also play an important role in synovial inflammation. HIF-1\u03b1 and HIF-2\u03b1 are highly expressed in immune cells, in particular macrophages, in the RA synovium (Hollander et al., Synovial angiogenesis is likely a consequence of synovial hypoxia in RA (Konisti et al., The articular cartilage is an avascular, aneural, and alymphatic tissue, which is composed primarily of chondrocytes. The role of HIFs in cartilage destruction in RA is not yet fully characterized, with the majority of the work conducted in healthy articular cartilage. Cells in the articular cartilage normally reside in a hypoxic environment, with oxygen tension varying from 6\u201310% at the joint surface to 1% in the deeper layers (Gibson et al., in vitro on cells derived from RA synovium, further ex vivo and in vivo studies are warranted to assist in strengthening our understanding of the complex role of HIF in cartilage degradation in RA.Hypoxia has been demonstrated to upregulate the levels of MMPs in cells derived from RA synovium, although additional pathways independent of HIF may also be involved (Canning et al., Studies to date suggest that HIFs are promising targets for novel RA treatments. Approaches that may be considered for targeting hypoxia in RA cells include the use of hypoxia-activated prodrugs, specific HIF inhibitors, gene therapy, or targeting indirect pathways important in hypoxic cells. The majority of these targeted therapies have come from research on the effects of hypoxia on the growth of tumors (Phillips, A number of HIF inhibitors have been developed that possess inhibitory activity against cancer and HIF-related diseases (Ban et al., in vivo model of RA (del Rey et al., The use of delivery carriers may improve the efficacy of HIF-related therapeutic agents following systemic administration, by overcoming the pharmacokinetic and stability issues. The best example of this is in the delivery of gene therapy targeting HIFs, which have previously been shown to modulate cellular responses in hypoxia-related diseases (Post et al., Indirect strategies to target downstream HIF signaling pathways have also been investigated in RA. For example, many therapeutic approaches have successfully resolved angiogenesis in preclinical animal models of RA by administering antibodies targeting VEGF or small molecule inhibitors targeting the VEGF receptor (Maruotti et al., Accumulating evidence supports hypoxia and HIFs in regulating a number of important pathophysiological characteristics of RA, including synovial inflammation, angiogenesis, and cartilage destruction. Therefore, HIF inhibitors are likely to target multiple important RA processes. Experimental and clinical data have confirmed the upregulation of both HIF-1\u03b1 and HIF-2\u03b1 in RA. At this time, the relative importance of each isoform in RA pathology and disease severity is still unclear. These two isoforms show different sensitivity to oxygen tension and display distinct, and sometimes opposing, cellular activities. In addition, further studies are required to clarify the interrelationship between HIFs and other simultaneous pathways in perpetuating RA disease. This will allow us to determine whether specific HIF-1\u03b1 or HIF-2\u03b1 inhibition is likely to be required for successful clinical outcomes in RA. To optimize their effects, HIF inhibitors may require encapsulation within delivery vehicles directed toward affected RA tissue to improve therapeutic accumulation and stability following systemic administration, as well as reduce off-target effects.SH was responsible for assembling, drafting and revising the manuscript, and preparing the associated figure and table. TD contributed to the drafting of this manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "The liver is famous for its strong regenerative capacity, employing different modes of regeneration according to type and extent of injury. Mature liver cells are able to proliferate in order to replace the damaged tissue allowing the recovery of the parenchymal function. In more severe scenarios hepatocytes are believed to arise also from a facultative liver progenitor cell compartment. In human, severe acute liver failure and liver cirrhosis are also both important clinical targets in which regeneration is impaired, where the role of this stem cell compartment seems more convincing. In animal models, the current state of ambiguity regarding the identity and role of liver progenitor cells in liver physiology dampens the enthusiasm for the potential use of these cells in regenerative medicine. The aim of this review is to give the basics of liver progenitor cell biology and discuss recent results vis-\u00e0-vis their identity and contribution to liver regeneration. The liver has the amazing potential to regenerate when mild liver damage occurs. During this process, remnant resting hepatocytes will re-enter the cell cycle and efficiently replenish the liver through proliferation. A good example of the capacity of adult hepatocytes and bile epithelial cells to proliferate is seen during recovery from partial hepatectomy in rats and mice, when two-third of the liver is removed , liver stem cells (LSCs), atypical ductal cells (ADCs), or intermediate hepatobiliary cells, it is sometimes difficult to deducewhether researchers are studying the same cell. While it is desirable to come to a nomenclature and classification of these -maybe different- cells, in this review we will use the term HSPCs to encompass the various liver stem/progenitor cell populations irrespective of species or injury model.Liver regeneration can be broadly characterized into hepatocellular or biliary regeneration, which is dependent on the type of injury. Adaptive, but flexible crosstalk between the microenvironment (i.e. extracellular matrix (ECM) and neighboring cells, like Kupffer cells, myofibroblasts and hepatic stellate cells) and the stem-cells themselves are required to allow the activation of HSPCs . Differin vitro for their cell renewal and differentiation capacity or in vivo for their ability to repopulate an injured liver with HSPC-derived hepatocytes and cholangiocytes ), Prom1 Figure 2.The problem with the aforementioned markers is that they are also expressed on regular biliary epithelial cells. The current view is that, once awakened by signals from the surrounding liver tissue, HSPCs become transit amplifying cells expressing some proteins not expressed by regular biliary epithelial cells such as Foxl1 (Forkhead Box l1), TACSTD2/Trop2 or LGR5 ((leucine-rich-repeat-containing G protein-coupled receptor 5) -like weak inducer of apoptosis (TWEAK), which is produced by monocytes, T lymphocytes and macrophages whose expression increases in contexts of acute injury, inflammatory disease and cancer. The expression of its receptor Fn14, normally expressed by epithelial and mesenchymal cells, is also relatively low in healthy tissue but is dramatically induced in injured and diseased tissue, such as in livers following a DDC treatment. HSPC activation by DDC was significantly reduced in Fn14-null mice and by the use of an anti-TWEAK antibody while overexpression of TWEAK in hepatocytes or exogal., 2005). Indeedal., 2005; Murakamal., 2005; Steilinal., 2005). FGF7-dal., 2013). Obvioual., 2013). Contraal., 2013). This sin vivo /c-MET and Epidermal growth factor (EGF)/EGF receptor (EGFR) are key regulatory elements to determine HSPC activation and differentiation. In c-MET knockout mice, HSPC response was significantly less upon DDC injury al., 2012) suggestal., 2012). Wnt3a al., 2012, 20137]in vivo ).+/EpCAM+ cells are defined as hepatoblasts expressing albumin and they are capable of differentiating into hepatocytes (EpCAM-/Dlk1-/Alb+/CK19-) and cholangiocytes (EpCAM+/Dlk1-/Alb-/CK19+) . These ductal plate derived YFP positive cells co-expressed SOX9, OPN and other oval cell markers , the caal., 2012; Tanaka al., 2012). Only o4. This was in contrast to a later study that showed that lineage tracing of LGR5-CRE positive cells yielded hepatocyte-like cells after one single injection of CCl4 or the DDC and MCDE diets Figure. Until ral., 2013). None oal., 2013; Schaub al., 2013) or regual., 2013) and appal., 2013; Feil etal., 2013). Severaal., 1989) and traal., 1989). If theal., 2011). Carpenal., 2011) showed al., 2011) is likeal., 2011; Yanger al., 2011). The real., 2011), ~ 28.7al., 2011), ~ 10.5al., 2011) to 7 % al., 2011); the fiAn alternate hypothesis that might at least partially explain the obtained results from these studies, is that the low hepatocyte repopulation from HSPCs is a matter of hepatobiliary linkage but not of massive hepatocyte production . In thide novo formation of mature hepatobiliary cells An overarching mechanism by which an aberrant microbiota negatively impacts health is by driving chronic inflammation Gut-derived endotoxins play a central role in the initiation of acute liver injury and progression to chronic liver disease ; (iii) An imbalanced intestinal homeostasis results in a breach of the gut barrier and subsequent microbial translocation ; (iv) Selective intestinal decontamination with antibiotics is beneficial for patients and prevents experimental liver injury ; (v) Mice with genetic deletions in the lipopolysaccharide (LPS) signaling pathway are resistant to experimental liver injury and fibrosis Transplantation of microbiota from diseased mice to germfree mice transfers some aspects of diseased phenotypes, indicating that altered microbiota plays a role in disease establishment and manifestation . Thus, \u03b1,\u03b2) into hepatocytes. Many research efforts are focused on ways to expand hepatocytes or have perfect differentiation conditions of stem cells towards functional hepatocytes. Both are not current practice; hepatocytes in culture lose their functionality very quickly while stem cell differentiation towards hepatocytes often only reaches the hepatocyte-like cell level . Currenal., 2012; Snykersal., 2012; Takase al., 2012) and manal., 2012) ). The seid, 2000). CurrenIn vivo, HSPCs are not only influenced by growth factors and cytokines, changes in the extracellular matrix (ECM) seem to effect the proliferation and differentiation as well. The ECM is a complex structure made up of protein fibers that serve as a dynamic substrate that supports tissue repair and regeneration . Changeal., 2011; Kallis al., 2011; Van Hulal., 2011) and Lamal., 2011; Espanolal., 2011) deposital., 2011; Shupe eal., 2011). Uygun al., 2011) successal., 2011).Similar decellularized matrixes also increased hepatocyte differentiation of human fetal HSPCs when used as a coating substrate for regular tissue culture plates . ClearlIn these last couple of years we have learned much about the pathways and conditions involved in HSPC activation thanks to sophisticated genetically modified mouse models. These same models are currently in conflict about the existence and function of HSPCs during liver injury and regeneration. Perhaps novel mouse liver injury models, more representative of human disease, need to be developed to fully unravel the existence, identity and function of HSPCs.LD & LvG: Interuniversity Attraction Poles (IAP) - phase VII - contract P7/47 (10/2012-09/2017); SV: Flemish Government Agency for Innovation by Science and Technology, IWT/SB/121548 (2013-2016).Leo A. van Grunsven and Laurent Doll\u00e9 contributed equally to this publication."} +{"text": "In continuation of the last year's special issue \u201cTCM Zheng Classification and Clinical Trials,\u201d the time for 2014 issue has come. Now we are on the way from TCM Zheng classification in clinical trial to development of clinical practice guideline. Clinical effectiveness is what matters most in any treatment, including traditional Chinese medicine (TCM). Many clinical trials have been conducted in China to evaluate the effectiveness of TCM, but many of the studies are with poor quality and may be inaccessible to non-Chinese scientists. As shown in This 2014 special issue contains 9 articles accepted from a total of 19 submissions consisting of 6 research articles, 1 systematic review (SR) article, and 2 narrative review articles.Two cohort studies were gathered, \u201cChinese Herbal Decoction Based on Syndrome Differentiation as Maintenance Therapy in Patients with Extensive-Stage Small-Cell Lung Cancer: An Exploratory and Small Prospective Cohort Study\u201d; 357 herbal decoctions were prescribed for 29 patients based on Zheng classification to observe the treatment effect and treatment length of herbal decoctions. Another cohort study \u201cThe Functional Difference of Dendritic Cells in HBeAg Negative Chronic Hepatitis B Patients with Three Different Spleen Deficiency Syndromes and the Therapeutic Evaluation of Chinese Medicine Intervention Based on Syndrome Differentiation\u201d by H. Song et al. suggested that Chinese medicine intervention according to Zheng classification could advance the maturity and function of dendritic cells and improve the therapeutic effect.One case analysis study, \u201cStudy on TCM Syndrome Differentiation of Primary Liver Cancer Based on the Analysis of Latent Structural Model\u201d by X. Yue et al., analyzed 559 inpatients records to provide evidences for TCM Zheng classification of primary liver cancer.Another paper focuses on clinical practice guideline; \u201cEnhanced Evidence-Based Chinese Medicine Clinical Practice Guidelines in Hong Kong: A Study Protocol for Three Common Diseases\u201d by A. Lu et al. presented a novel of three-phases clinical practice guidelines research protocol for insomnia, chronic gastritis, and cerebral infarction. This study may serve as model for future guidelines development in the area.To answer the question of what are the differences between different types of Zheng? \u201cThe Th17/Treg Immune Balance in Ulcerative Colitis Patients with Two Different Chinese Syndromes: Dampness-Heat in Large Intestine and Spleen and Kidney Yang Deficiency Syndrome\u201d by M. Jiang et al. identified different mechanism on immune imbalance of ulcerative colitis patients with two types of TCM Zheng. D. Wang et al. identified that 26 potential biomarkers in the plasma of coronary heart disease (CHD) patients and 19 differential metabolites contributed to the classification of two main types of TCM Zheng \u201cphlegm Zheng\u201d and \u201cblood stasis Zheng\u201d in \u201cA Metabonomics Profiling Study on Phlegm Syndrome and Blood-Stasis Syndrome in Coronary Heart Disease Patients Using Liquid Chromatography/Quadrupole Time-of-Flight Mass Spectrometry.\u201dWeijing Decoction) Combined with Pharmacotherapy for the Treatment of Acute Exacerbations of Chronic Obstructive Pulmonary Disease\u201d by C. C. Xue's research group included 15 RCTs, 986 subjects. They concluded that Weijing decoction, a universally used herbal formula for treating chronic obstructive pulmonary disease (AECOPD), appeared to be beneficial for acute exacerbations of AECOPD and well-tolerated when taken concurrently with routine pharmacotherapy (RP).Systematic review paper \u201cChinese Herbal Medicine , would be valuable for the researchers for conducting TCM clinical trials. We, the editors, appeal that more proper designed clinical trials on long term major outcomes should be funded. Hopefully, this special issue will give helpful insights to scientists, physicians, and patients and will also be able to make a progress in the field of TCM clinical studies."} +{"text": "Cognitive impairment is the final outcome of a complex network of molecular mechanisms ultimately leading to dementia. Despite major efforts aimed at unraveling the molecular determinants of dementia of Alzheimer type (DAT), effective disease-modifying approaches are still missing. An interesting and still largely unexplored avenue is offered by nutraceutical intervention. For instance, robust epidemiological data have suggested that moderate intake of red wine may protect against several age-related pathological conditions as well as DAT-related cognitive decline. Wine is highly enriched in many polyphenols, including resveratrol. Resveratrol is a well recognized antioxidant which may modulate metal ion deregulation outcomes as well as main features of the Alzheimer\u2019s disease (AD) brain. The review will discuss the potentiality of resveratrol as a neuroprotectant in dementia in relation to the oxidative stress produced by amyloid and metal dysmetabolism. French paradox arises from the epidemiological fact that French people, despite their indulgence to a high fat diet, show a relative low incidence of cardiovascular diseases extracellular deposition of misfolded \u03b2-amyloid (A\u03b2) in senile plaques (SPs); (2) intracellular accumulation of hyperphosphorylated tau in neurofibrillary tangles (NFTs); (3) severe brain atrophy; and (4) the presence of areas of chronic inflammation has been considered the main trigger for AD-related synaptic dysfunction. Amyloid has been therefore the major target for therapeutic intervention can lead to direct deacetylation of acetylated tau, thereby promoting its proteasomal degradation dependent deacetylase) which in turn sets in motion a cascade of PGC-1\u03b1-dependent events that ultimately lead to improved mitochondrial functioning and biogenesis and boost cellular ROS scavenging by efficiently scavenging hydroxyl, superoxide, and metal-induced radicals , Amyotrophic Lateral Sclerosis (ALS), Prion Protein disease, Huntington\u2019s disease (HD), and AD. All these neurodegenerative conditions share common pathological features that include deposition of misfolded proteins, metal ion deregulation and exposure to oxidative stress (Shin et al., Iron deregulation has been linked to AD (Weinreb et al., Zinc dyshomeostasis has been proposed as a risk factor for AD. Accumulation of excessive zinc, or its deficiency, are both involved in the neuronal loss which leads to AD and aging related cognitive decline Brewer, . While zin vitro (Granzotto and Zatta, in vivo the downstream events of aluminum overload, namely the aluminum-related ROS production and neuroinflammatory response activation (Zaky et al., Aluminum lacks modulatory functions in biological processes; however, its accumulation in the brain has been demonstrated to be linked to several neuropathological conditions (Zatta et al., Resveratrol is a multi target compound and may represent an effective therapeutic tool in aging-related neurodegenerative processes. Consistently, several clinical trials are ongoing to test its effectiveness as dietary supplement to slow dementia progression (ClinicalTrials.gov, in vivo may act on other uninvestigated biological targets (Herskovits and Guarente, In summary, major effects are associated with its scavenging activity as well as in the activation of SIRT1 (see Bordone and Guarente, Resveratrol is a multi-target, simple, safe, and cost-effective dietary supplement. Nevertheless, it should be reminded that its role as therapeutic agent is not devoid of potential problems. The pro-oxidant activity in presence of labile copper, the poor bioavailability and ease degradation all represent major issue that require new sophisticated efforts (Goldberg et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "This volume features state-of-the-art approaches to determining the effects of ageing on visual perception, visual cognition, and visually guided behavior. They incorporate psychophysics, eye movements, electrophysiology, and neuroimaging to determine how ageing affects vision in health and pathology.Brockmole and Logie present 2 ingestion, they observed age-equivalent fractional cerebral blood flow (\u0394CBF) in the presence of age-related increases in fractional cerebral metabolic rate of oxygen (\u0394CMRO2). Reductions in \u0394CBF responsiveness to increased \u0394CMRO2 in elderly participants led to paradoxical age-related BOLD decreases. Age-related \u0394CBF/\u0394CMRO2 ratio decreases were associated with increases in behavioral reaction times, suggesting that age-related slowing resulted from less efficient neural activity. Alichniewicz et al. (Hutchison et al. investigz et al. used funz et al. examinedz et al. present Bieniek et al. investigIt is our hope that these studies contribute to a better understanding of the effects of ageing on visual perception and visual cognition.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "There are errors in the Funding section. The correct funding information is as follows: This study was funded by the FIS co-financed by the European Union through the Fondo Europeo de Desarrollo Regional , and by the Grupo de Excelencia Investigadora URJC-Banco Santander No. 30VCPIGI03: Investigaci\u00f3n traslacional en el proceso de salud\u2014enfermedad (ITPSE). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."} +{"text": "Plasmopara viticola, the causal agent of grapevine downy mildew, is one of the most devastating grape pathogen in Europe and North America. Although phytochemicals are used to control pathogen infections, the appearance of resistant strains and the concern for possible adverse effects on environment and human health are increasing the search for alternative strategies. In the present investigation, we successfully tested two protein hydrolysates from soybean (soy) and casein (cas) to trigger grapevine resistance against P. viticola. On Vitis vinifera cv. Marselan plants, the application of soy and cas reduced the infected leaf surface by 76 and 63%, as compared to the control, respectively. Since both hydrolysates might trigger the plant immunity, we investigated their ability to elicit grapevine defense responses. On grapevine cell suspensions, a different free cytosolic calcium signature was recorded for each hydrolysate, whereas a similar transient phosphorylation of two MAP kinases of 45 and 49 kDa was observed. These signaling events were followed by transcriptome reprogramming, including the up-regulation of defense genes encoding pathogenesis-related (PR) proteins and the stilbene synthase enzyme responsible for the biosynthesis of resveratrol, the main grapevine phytoalexin. Liquid chromatography analyses confirmed the production of resveratrol and its dimer metabolites, \u03b4- and \u03b5-viniferins. Overall, soy effects were more pronounced as compared to the cas ones. Both hydrolysates proved to act as elicitors to enhance grapevine immunity against pathogen attack. Vitis vinifera) has a surveillance system considered as the first defense line against invader pathogens, activated after signal molecule perception. These molecules may come from infectious agents or non-pathogenic microorganisms and therefore be designated as pathogen- or microbe-associated molecular patterns (PAMPs or MAMPs), respectively; they may correspond also to secondary products released during pathogen invasion, called damage-associated molecular patterns (DAMPs) into elicited cells, considered cardinal for defense response activation , proved to enhance efficiently grapevine defense responses , dwarf pea (Pisum sativum), and tomato (Solanum lycopersicum) susceptible to P. viticola, were grown in glasshouse conditions, as described by Gauthier et al. (V. vinifera cv. Gamay) were cultivated as described by Vandelle et al. and casein (cas), produced for the pharmaceutical and food industry were supplied by \u201cA. Costantino & C. S.p.A.\u201d . Since the two products are not yet on the market and protected by a trademark, their physicochemical characteristics cannot be reported. Solutions at 1.6 mg/ml in sterile ultra-pure or distilled water were prepared 24 h before use, 0.20 \u03bcm filtered and stored at 4\u00b0C. This concentration was selected based on previous trials . Plants were placed for 5 h in a humid chamber , and then transferred back to the greenhouse. Five days post inoculation (dpi), plants were placed again in the humid chamber overnight to stimulate sporulation. Disease was assessed estimating the leaf surface area (%) covered by sporulation. Five plants were used per condition, evaluating three leaves per plant that were marked before treatment.Both faces of leaves of 8-weeks old grapevine plantlets were sprayed by 2+ and MAPK), cells were transferred in the M10 buffer , re-suspended at 0.1 g of fresh weight of cells (FWC) per ml and equilibrated for 1 h in light (130 rpm at 24\u00b0C) before treatments as described in Dubreuil-Maurizi et al. (In vivo reconstitution of aequorin was performed adding 5\u03bcl of coelenterazine (5 mM stock solution in DMSO) to 5 ml of aequorin-transformed cell suspension for at least 3 h in the dark. Aequorin was quantified adding 300 \u03bcl of lysis buffer . Finally, variations in [Ca2+]cyt were calculated following the calibration equation developed by Rentel and Knight and 50 \u03bcl of 0.3 mM luminol. Relative luminescence was recorded within a 10-s period using a luminometer and expressed as nmol H2O2 per gram of FWC.Hr et al. . BrieflyWestern blot analyses were performed as described by Trd\u00e1 et al. . AliquotDead cells were quantified according to Binet et al. . Briefly\u2212\u0394\u0394Ct method and reaction efficiencies were calculated.Aliquots (2 ml) of treated cells were harvested by filtration on GF/A filters following a time course , frozen and ground in liquid nitrogen. Total RNA isolation was obtained by Trizol . The RNA yield and quality were determined by NanoDrop 2000 . cDNAs were synthesized by reverse-transcribing 400 ng of total RNA using Superscript III reverse transcriptase kit . Amplifications were run in a 96 well-plates iCycler iQ thermal cycler and quantification was performed with the iCycler iQTM associated software . Differential expression was determined according to the 22) ranging from 0.94 to 0.99 separated by filtration on GF/A filters following the same time course of qPCR analyses. The culture medium samples were directly analyzed, whereas cell samples were stirred vigorously and incubated in 2 ml absolute ethanol for 24 h at 4\u00b0C to extract stilbenes. Samples were analyzed by an Acquity UPLC system equipped with a model 2996 photo-diode array detector, as described by Boutegrabet et al. . Each sasoy and cas), data were subjected to ANOVA . Significant differences (P \u2264 0.05) were identified by the General Linear Model (GLM) procedure with the Duncan's Multiple Range Test (DMRT). Percentage data of incidence of decay underwent arcsine-square-root transformation before ANOVA analysis. For gene expression data, since just two conditions were compared (relative expression by soy and cas), Student's t-test was applied. Data were processed using the software package Statistics for Windows .When more than two conditions were present . Thereafter, plants were sprayed by soy and cas at 1.6 mg/ml, inoculated by P. viticola sporangia 24 h later and evaluated for the leaf sporulating area at 5 dpi. A significant reduction of disease severity by 76% for soy and 63% for cas was observed as compared to the control. However, no difference was found between the two hydrolysates .Generation of Hsoy and cas hydrolysates was also evaluated by cell viability assays, in which the number of red colored grapevine cells was assessed at 24 hpt. We did not recorded any toxic effect of the two hydrolysates compared to the water control (data not shown).The toxicity of PR1), a \u00df-1,3-glucanase (PR2), a chitinase 4c (PR3), a protease inhibitor (PR6), a polygalacturonase-inhibiting protein (PGIP) and a stilbene synthase (STS). Overall, soy and cas induced a rapid and high transcript accumulation of most of the tested genes with different kinetics and intensities; a more pronounced effect of soy was generally observed , showed a biphasic expression profile. It peaked at 4 or 8 hpt for soy- and cas-treated cells, respectively.In this study we evaluated by qPCR in time-course experiments the expression in grapevine cell suspensions of six selected defense genes known to be activated in response to various elicitors: those encoding a pathogenesis-related protein 1 .soy and cas hydrolysates to trigger resistance against gray and green mold (Lachhab et al., in vitro of the different pathogens (Lachhab et al., 2+]cyt variations, H2O2 production, MAPK activation, defense genes expression, and phytoalexin production. Both soy and cas induced specific changes of [Ca2+]cyt, considered essential for defense response activation (Lecourieux et al., 2+]cyt, as compared to water control, similarly to other elicitors as OG, cryptogein, BcPG1, and Flg 22 (Lecourieux et al., soy and cas showed two specific signatures, characterized by different kinetics and duration, suggesting different active molecules in each hydrolysate (Blume et al., soy-treated cells, the increase in [Ca2+]cyt was more rapid than that triggered by cas, and the level of free cytosolic calcium remained higher as compared to water and cas treatments. A similar behavior was observed in [Ca2+]cyt signatures induced by the polypeptide cryptogein in tobacco cells (Lecourieux et al., soy is likely not an oligosaccharide as assumed elsewhere (Lachhab et al., soy and cas chemical composition by the manufacturer is in progress.On the bases of the effectiveness of soy-treated cells a fast, strong, and lasting MAPK activation, which is consistent with the high level of free cytosolic calcium concentration. These results are similar to those of Lecourieux et al. (2+]cyt and MAPK stimulation is more evident considering the cas- effect. Indeed, the rapid decrease of the [Ca2+]cyt level is correlated with a very low activation of MAPKs after 60 min, as previously demonstrated in cryptogein-treated tobacco cells (Lecourieux et al., Calcium signaling is known to act upstream of the MAPK pathway in some plant defense responses (Link et al., x et al. who reco2+]cyt variations and MAPKs activation, many authors described the rapid release of H2O2 in cell suspensions as a response to various elicitors (Poinssot et al., soy- and cas-elicited cells did not show any variation in H2O2 production. Furthermore, similarly to the \u03b2\u2013glucan derivative PS3, the two hydrolysates did not have any toxic effect on grapevine cells, supporting the hypothesis that the resistance to pathogen invasion may depend on other defense responses rapidly activated during pathogen infection (Aziz et al., 2O2 generation is not necessary for the induction of defense reactions. As shown by Galletti et al. (Arabidopsis, without affecting the expression of genes involved in defense responses effective against B. cinerea. In addition, Pauw et al. (Catharanthus roseus.In addition to [Cai et al. , a null w et al. showed tP. viticola of some Vitis species as V. riparia, was associated to a more rapid and stronger induction of defense gene expression, as compared to susceptible cultivars, e.g., V. vinifera (Polesani et al., PR1 and PR6 (Lu et al., PR6 is in agreement with the protective effect provided by the two hydrolysates to grapevine against Botrytis attack (Lachhab et al., STS the key gene of the biosynthesis of resveratrol, known to increase resistance against pathogens (Coutos-Th\u00e9venot et al., Phomopsis viticola and B. cinerea and affecting P. viticola zoospore mobility (Adrian et al., Consistently with previous reports, the two tested hydrolysates caused an up-regulation of 6 known defense-related genes (B\u00e9zier et al., soy, induced a high and rapid accumulation of resveratrol at 4 hpt, the first time-assessment in our trials, meaning that STS gene was induced even before. This finding is in agreement with the concept that stilbene is the primary inducible response of grapevine against a number of biotic and abiotic stresses (Adrian et al., As expected, the two protein hydrolysates, in particular Interestingly, resveratrol was present at high amounts in culture medium, but showed low accumulation in cells. As suggested by Adrian et al. , part ofP. viticola (Pezet et al., soy showed pronounced efficiency in inducing defense responses, as compared to cas treatment, in particular concerning stilbene accumulation. The different results may be ascribed to their different proteic origins. Indeed, soybean contains different proteins as lipid transfer proteins (Kido et al., P. viticola.The \u03b4- and \u03b5-viniferin dimers, considered as important markers for resistance of grapevine to pathogens (Malacarne et al., cas and particularly soy hydrolysates as enhancers of grapevine innate immunity and thus good candidates to replace or reduce fungicide applications in modern sustainable viticulture, as they are cheap, easily available and safe to humans and the environment.In conclusion, the presented data support the use of Nihed Lachhab: conducted the experiments, analyzed data and prepared the manuscript; Simona M. Sanzani: designed the experiments, analyzed the data and prepared the manuscript; Marielle Adrian: supervised phytoalexin quantification, analyzed the data and edited the manuscript; Annick Chiltz and Suzanne Balacey: helped in conducting the experiments; Maurizio Boselli: helped to design the experiments and edited the manuscript; Antonio Ippolito: helped to design the experiments and edited the manuscript; Benoit Poinssot: designed the experiments, analyzed the data and prepared the manuscript. All authors have read the manuscript and agree with its content.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Recently, Bin Zhao and colleagues from the Chinese Academy of Science in Beijing and University of California, Sacramento, have published a review about identification of cardiotoxic compounds by rapid screening methods derived cardiomyocytes have been introduced and several gene and microRNA cardiotoxicity markers have been identified . Meanwhal., 2015). Currenal., 2015, 20139]in vitro al., 2015; Grinberal., 2015), kidneyal., 2015; Bulacioal., 2015) and neual., 2015; Shinde al., 2015), which al., 2015; Vartak al., 2015; Ghallabal., 2015; Schliesal., 2015; Hoehme"} +{"text": "Renal fibrosis represents a common pathway leading to progression of chronic kidney disease. Renal interstitial fibrosis is characterized by extensive fibroblast activation and excessive production and deposition of extracellular matrix (ECM), which leads to progressive loss of kidney function. There is no effective therapy available clinically to halt or even reverse renal fibrosis. Although activated fibroblasts/myofibroblasts are responsible for the excessive production and deposition of ECM, their origin remains controversial. Recent evidence suggests that bone marrow-derived fibroblast precursors contribute significantly to the pathogenesis of renal fibrosis. Understanding the molecular signaling mechanisms underlying the recruitment and activation of the bone marrow-derived fibroblast precursors will lead to novel therapy for the treatment of chronic kidney disease. In this review, we summarize recent advances in our understanding of the recruitment and activation of bone marrow-derived fibroblast precursors in the kidney and the development of renal fibrosis and highlights new insights that may lead to novel therapies to prevent or reverse the development of renal fibrosis. Chronic kidney disease (CKD) is a global public health problem. Worldwide, over 1 million people die from CKD yearly. It is estimated that more than 20 million Americans have CKD and more than 450,000 Americans suffer from end-stage renal disease (ESRD) requiring renal replacement therapy. CKD has become the eighth leading cause of death in the United States. Regardless of various etiologies, a common pathological feature of CKD is renal fibrosis, which is characterized by accumulation of fibroblasts/myofibroblasts with increased production and deposition of extracellular matrix (ECM) including collagen type I, III, IV, fibronectin, vimentin, and proteoglycans , macrophages, and immune cells, which suggest that interaction and communication among these cell types regulates the development of fibrotic disorders (Kisseleva and Brenner, The recruitment of circulating cells into sites of injury is mediated by locally produced chemokines. Chemokines are classified based on the relative position of cysteine residues near the NH2 terminus into four major families: CC, CXC, C, and CX3C Rollins, . Recent in vivo in a murine model of renal fibrosis induced by obstructive injury (Okamura et al., in vitro (Xia et al., in vitro and in vivo (Izquierdo et al., The chemokine CXCL16 is a recently discovered cytokine belonging to the CXC chemokine family (Matloubian et al., CXCR6 is the receptor for CXCL16. CXCR6 was first cloned as an orphan receptor by three independent groups and was termed STRL33, BONZO, or TYMSTR (Alkhatib et al., In these studies, we have observed that genetic deficiency of CXCL16 or CXCR6 does not completely block bone marrow-derived fibroblast precursor infiltration into the kidney and renal fibrosis development, suggesting that other chemokine/receptor pairs may be involved in the process of recruiting bone marrow-derived fibroblast precursors into the kidney. Consistent with this notion, CCR2 and CCL21/CCR7 have been reported to play a role in the recruitment of bone marrow-derived fibroblast precursors into the kidney and the development of renal fibrosis (Sakai et al., in vitro and in vivo (Chen et al., in vitro. Smad3-knockout mice accumulate significantly fewer bone marrow-derived fibroblasts in the kidney after obstructive injury, exhibit less myofibroblast activation, and express less \u03b1-SMA in the obstructed kidney. Furthermore, genetic deletion of Smad3 reduces total collagen deposition and suppresses expression of extracellular matrix proteins. Additionally, wild-type mice engrafted with Smad3\u2212\u2215\u2212 bone marrow cells displayed fewer bone marrow-derived fibroblasts in the kidney with obstructive injury and showed less severe renal fibrosis compared with wild-type mice engrafted with Smad3+\u2215+ bone marrow cells. These results indicate that Smad3 of bone marrow-derived cells plays an important role in bone marrow-derived fibroblast activation. However, Samd3 deficiency does not completely abolish bone marrow-derived fibroblast activation, collagen deposition, and ECM protein expression in vivo. These results suggest that other factors may be involved in bone marrow-derived fibroblast activation.The activation of bone marrow derived fibroblast precursors plays a crucial role in the pathogenesis of renal fibrosis (Yang et al., + T cells can differentiate into two major distinct phenotypes, Th1 and Th2 cells, which are characterized by specific cytokine expression patterns (Wynn, in vitro. Furthermore, CP690550 treatment or STAT6 deficiency inhibits bone marrow-derived fibroblast activation and ECM protein production in the kidney in response to obstructive nephropathy. To further confirm the role of bone marrow STAT6 signaling in myeloid fibroblast activation, we performed bone marrow chimeric experiments. Wild-type mice transplanted with STAT6 null bone marrow cells exhibit fewer bone marrow-derived fibroblasts and develop a lesser degree of renal fibrosis. These results suggest that inhibition of JAK3/STAT6 signaling may serve as a novel therapeutic target for fibrotic kidney disease.The activation of bone marrow-derived fibroblasts is modulated by inflammatory cells in the microenvironment. T cells plays an important role in the pathogenesis of renal fibrosis (Tapmeier et al., ns Wynn, . Th2 celns Wynn, . Howeverns Wynn, . SpecifiAdiponectin is a multifunctional cytokine and an important regulator of lipid and carbohydrate metabolism. Emerging evidence suggests that circulating adiponectin levels are elevated in patients with CKD, and a high level of adiponectin is linked to increased cardiovascular mortality (Zoccali et al., It is generally thought that macrophages do not produce ECM proteins. These cells promote fibrosis indirectly by producing profibrotic cytokines that activate fibroblasts (Wynn and Barron, Recent studies have shown that renin angiotensin system plays an important role in the development of fibrotic kidney disease (Mezzano et al., Recent studies have demonstrated that bone marrow derived fibroblast precursors contribute significantly to the pathogenesis of renal fibrosis. Recruitment and activation of bone marrow-derived fibroblasts are mediated through the interaction between chemokines/cytokines and their receptors Figure . TherefoAlthough chemokines are involved in recruiting bone marrow-derived fibroblasts into the kidney in response to injury, the molecular signaling mechanisms underlying chemokines-induced bone marrow-derived fibroblast recruitment remains to be defined. Ras proteins are members of a family of small GTPase that control signaling pathways involved in cell migration, proliferation, differentiation, and survival (Rodr\u00edguez-Pena et al., JY drafted the manuscript. ZZ, LJ, and YW reviewed and edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Ginsenoside-Rb1 is a chemically pure steroid glycoside from the root of Panax ginseng, which has a 3000-year history of medicinal use in the Orient.Betts et al 2011) reported that Rb1 inhibits vascular endothelial growth factor (VEGF) release by retinal pigment epithelial cells, which is a major source of VEGF in the eye of ginsenosides and their derivatives. By simple acylation of the panaxadiol, an analog of ginsenoside, its anti-tumor activity was significantly enhanced, compared with the non-acylated parent (Using a different cancer cell line (lung fibroblast), Dong et al. (2011) noted that the removal of the sugar moiety (by hydrolysis) significantly increased its cytotoxic potency, based on MTT assays, to measure viable cell numbers . Du et aSuch SAR reports are not limited to chemical modification of ginsenosides leading to inhibition of cancer cell proliferation only. Wei et al (2009) reported that oxidation of panxadiol leads to triperpene derivatives with a significant increase in the ability to inhibit the HIV-1 protease enzyme, which is required for maturation and infectivity of the HIV virus . FinallyRecently, there has been an increased interest in SAR in medicinal chemistry. These reports provide further evidence for the need of exploring how chemically modified ginsenosides may enhance inhibition of VEGF in ocular and other cell types. Such discoveries will provide new knowledge for novel intervention methods to treat angiogenic diseases such as diabetic retinopathy, AMD and cancer."} +{"text": "Stem cell research is of high interest, because precursor cells can be directed to differentiate into practically all mature cell types. This represents a potential perspective for therapeutics of severely damaged organs and also an alternative to animal drug and toxicity testing. Nonetheless, an essential question still remains: How similar are differentiated stem cells to the desired mature cells, e.g. liver cells? This highlight report focusses on a study recently published in the Journal of Hepatology where tThe liver has a spectacular ability to regenerate developThe authors applied biostatistical techniques to compare gene clusters using real liver cells (primary human hepatocytes), stem cells and six different stem cell-derived hepatocyte-like cells . The coin vitro systems play a major role in testing of neurotoxicity represe represe5al., 2014; Krug etal., 2014), nephroal., 2014; Yang etal., 2014) and hepal., 2014; Godoy eal., 2014; Godoy 2al., 2014; Godoy aal., 2014; Grinberal., 2014; HengstlThis is the first time that such a systematic genome wide comparison of stem cell derived and genuine hepatocytes has been performed. The recent published study in the Journal of Hepatology offers"} +{"text": "Progression of solid tumors is characterized by an evolutive growing cellular mass containing variable proportions of a complex network of tumor stroma cell populations surrounding cancer cell (CC) foci. This cellular intricacy determines mechanical, molecular and functional interactions involved in multiple mechanisms driving reciprocal aspects of paracrine and justacrine crosstalks between TSC and adjacent CC. There is increasing evidence that tumor stroma is a multicellular organ containing vascular structures and a large variety of cellular populations, including resident carcinoma-associated fibroblasts (CAF), myofibroblasts (MF), leucocytes and macrophages, bone marrow-derived mesenchymal stem cells, and in breast cancer, adipose progenitor cells. This cellular heterogeneity sustains tumor growth and angiogenesis, deregulated cancer cell proliferation, survival, chemoresistance, epithelial-to-mesenchymal transitions (EMT), invasion and tumor metastasis.In primary tumors and their metastases, these intercommunications between cancer cells and their surrounding TSC are organized in part by the convergent secretion of various ECM components building the architectural formation of ECM interfaces between these two critical cellular populations. ECM remodeling during cancer progression is regulated by the concomitant secretion of CC and TSC soluble factors and cytokines as well as subcellular exosomes involved in genetic tranfer between cancer cells and their stromal microenvironment (reviewed in ref. ). PlastiSeveral studies pointed the critical roles played by the ECM, CAF and MF in tumor progression. Decorin is a member of the Small Leucine-rich Repeat Proteoglycan (SLRP) family expressed and secreted in the interstitial ECM in breast stroma (reviewed in ref. ). In ECMIn this context, decorin was shown to sequester latent form of TGF\u03b21 (L-TGF\u03b21) in the ECM and to interact with the active TGF\u03b21 ligand, thus preventing its binding to TGF\u03b21 receptors . Most imIn the background of ductal carcinoma in situ (DCIS) of the breast, Van Bockstal et al reveal that the function of decorin is involved in breast cancer cell spreading and that both TGF\u03b21 and bFGF down-regulated the ECM protein decorin in CAF-associated breast tumors . In turnin vitro and in vivo will provide a rationale for new therapeutic options aimed to neutralize the oncogenic role of TGF\u03b21 as a repressor of the decorin tumor suppressive functions. As reported by Van Bockstal et al [These findings collected in breast cancer-associated fibroblasts are of pal et al this ass"} +{"text": "The integration of information from genome to metabolome allows the establishment of associations between genetic potential and final phenotype, a feature not realizable by only considering single \u2018omes\u2019. Therefore, in our opinion, integrated omics will become the future standard for large-scale characterization of microbial consortia including those underpinning biological wastewater treatment processes. Systematically obtained time and space-resolved omic datasets will allow deconvolution of structure\u2013function relationships by identifying key members and functions. Such knowledge will form the foundation for discovering novel genes on a much larger scale compared with previous efforts. In general, these insights will allow us to optimize microbial biotechnological processes either through better control of mixed culture processes or by use of more efficient enzymes in bioengineering applications.Biological wastewater treatment plants harbour diverse and complex microbial communities which prominently serve as models for microbial ecology and mixed culture biotechnological processes. Integrated omic analyses are currently gaining momentum towards providing enhanced understanding of community structure, function and dynamics Biological wastewater treatment plants host diverse and dynamic microbial communities possessing varied metabolic capabilities over changing environmental conditions, e.g. microorganisms accumulating various storage compounds of biotechnological importance. Given their structural and functional diversity, BWWT processes hold great potential for future sustainable production of various commodities from wastewater as well as from other mixed substrates with well-defined physico-chemical boundaries and are widespread in developed and developing countries biomolecular extractions protocols and computational analyses ineffective yet highly active populations minimizing errors by cancelling out noise and biases stemming from single omic analyses and (ii) optimizing/maximizing overall data usage.Following standardized and systematized biomolecular isolations, multi-omic datasets are generated in addition to the physico-chemical parameters recorded at the time of sampling Fig.\u2009; step 2.et\u2009al., et\u2009al., et\u2009al., in silico data processing and analysis methods, which in turn will allow integrated omics to provide comprehensive multi-level snapshots of microbial population structures and functions in situ , fully integrated omic analyses should be applied routinely in the study of microbial consortia for greater effectiveness. For instance, despite this wealth of information, current metagenomic assemblies and analysis schemes, metagenomic (and metatranscriptomic) data resulting from the use of current short-read sequencing and assembly approaches do not allow the comprehensive resolution of microdiversity, e.g. genetic heterogeneity of microbial populations , coupled to carefully controlled laboratory experiments, will allow the effective elucidation of novel functions within BWWT plant microbial communities with potential biotechnological applications.Although integrated omics-based approaches are highly effective for large-scale analysis and formulation of hypotheses (including within the context of BWWT plant communities), these efforts are limited due to current high-throughput measurement methods (see previous section) and the reliance on et\u2009al., et\u2009al., et\u2009al., et\u2009al., Knowledge of gene function, regulation and physiological potential derived from integrated omic data over different spatial and temporal scales holds great promise in harnessing the biotechnological potential of microbial consortia. In particular, advancements in integrated omics followed by hypothesis testing may generate new knowledge (Muller None declared."} +{"text": "Phenotypic integration among different anatomical parts of the head is a common phenomenon across vertebrates. Interestingly, despite centuries of research into the factors that contribute to the existing variation in brain size among vertebrates, little is known about the role of phenotypic integration in brain size diversification. Here we used geometric morphometrics on the morphologically diverse Tanganyikan cichlids to investigate phenotypic integration across key morphological aspects of the head. Then, while taking the effect of shared ancestry into account, we tested if head shape was associated with brain size while controlling for the potentially confounding effect of feeding strategy.The shapes of the anterior and posterior parts of the head were strongly correlated, indicating that the head represents an integrated morphological unit in Lake Tanganyika cichlids. After controlling for phylogenetic non-independence, we also found evolutionary associations between head shape, brain size and feeding ecology.Geometric morphometrics and phylogenetic comparative analyses revealed that the anterior and posterior parts of the head are integrated, and that head morphology is associated with brain size and feeding ecology in Tanganyikan cichlid fishes. In light of previous results on mammals, our results suggest that the influence of phenotypic integration on brain diversification is a general process. Brain size is highly variable among vertebrates,2. This The cichlids of Lake Tanganyika are an interesting model group to investigate the integration between skull and brain because of their remarkable diversity in both brain size and head morphology. The relative brain size of Tanganyikan cichlids is correlated with several ecological and social factors such as diet,24, habiAnolis lizards[The high degree of integration between the different parts of the head is widely reported across vertebrates . Intere lizards. These a lizards. Hence, lizards which wo lizards,15. AlteIn this study, we use landmark-based geometric morphometric phylogenetic analyses to test for the existence of phenotypic integration between various aspects of head morphology and brain size in Lake Tanganyika cichlids. According to our hypothesis, we test two aspects of integration. First, we test if the head is composed of morphologically independent modules or if it represents an integrated morphological unit. We then investigate whether head morphology is integrated with brain size or brain region volumes while considering the potentially confounding effect of prey utilization patterns that may affect head morphology.Benthochromis tricoti) for which we had two specimens was much higher than within-species between-sex variation and the interaction between species and sex . Our data should thus be robust against within-species sex differences. Specimens were sacrificed using an overdose of benzocaine. After measuring standard length and head width , the head was severed and preserved in 4% paraformaldehyde in a phosphate buffer for tissue fixation and preservation. Whole brains were obtained from dissected heads following fixation and weighed using a Precisa 125A electronic scale . All cranial nerves, optic nerves and meningeal membranes were removed and the brain was severed from the spinal cord just posterior of the dorsal medulla. Since brain volume and brain weight are highly correlated in our dataset , we use\u2009=\u20090.96,,36. Feed\u2009=\u20090.96,,47. To a\u2009=\u20090.96,. Therefo,[et al., Konings,[et al.,50, Yama,[et al.-53, Yuma,[et al., Takeuchl.[et al., Ochi[5l.[et al..p\u2009>\u20090.62 in all cases). We measured the head length as the distance between the foremost point of the snout and the rearmost point along the operculum using a scale photographed in the background of the images. Using TpsDig version 2.16, we digitized six homologous landmarks and seven semi-landmarks along the edge of the forehead to capture the variation in forehead shape where the largest proportion of variation is concentrated[F550, 2112\u2009=\u20093.58, p\u2009<\u20090.001) than intraspecific between-sex differences . Generalized Procrustes Analysis (GPA) was performed to mathematically remove the variation of position, size, and rotation of the landmark configurations[Images of the lateral sides of 156 individuals were taken with a reflex digital camera (Nikon D 70 with an AF Micro Nikkor 60\u00a0mm 1:2.8 D macro lens). Assuming symmetry, we used the best quality images either from the right or left side. A permutation test using Procrustes distance confirmed that the shape was not different between the photos of the separate sides , 0.08 (PC2), 0.005 (PC3), 0.003 (PC4)), suggesting the use of residuals would not introduce bias in the analysis[\u03bb\u2009=\u20090.95) and PSC (\u03bb\u2009=\u20091), indicating the necessity for phylogenetic correction. PPCA and PSC were performed using the phytools package[In order to control for the allometric effect of body size on brain weight, we used phylogenetic principal component analyses (PPCA). Prior tanalysis. We perf package in R ver package.\u03bb\u2009<\u20090.001). Following Horn\u2019s parallel analysis[\u03bb\u2009=\u20091 for the residuals of the model. Then, we performed mPGLS with \u03bb\u2009=\u20091, i.e. a Brownian motion model, to test the integration between head shape and brain size. We also tested the robustness of our test using overall brain size against structural heterogeneity of the brain by performing mPGLS on size of the six major brain regions as a predictor variable and head shape (PC1-4) as a response matrix, using overall brain weight to control for the effect of size and brain size were correlated with head shape. The direction of the shape change associated with brain size is presented in Figure\u00a0The associations between head shape and the three independent variables are summarized in Table\u00a0Our study represents one of the first macro-evolutionary studies of phenotypic integration between brain size and head morphology in non-mammalian vertebrates and several insights can be gained from our results. First, we found support for phenotypic integration rather than modularity between the pre-orbital and post-orbital parts of the head. Second, head morphology was linked to prey utilization. Third, when controlling for the association between food utilization and head morphology, we found that brain size in Lake Tanganyika cichlids was closely associated with variation in head morphology. Together, these findings indicate that brain evolution and trophic adaptations may interact through phenotypic integration of brain size and head shape.et al.[et al.[Perissodini) in their dataset while we did not. Second, our choice of landmarks and semi-landmarks are slightly different from Parsons et al.[et al.[et al.[et al.[Our test for modularity did not support the existence of independent modules within the head. Our result therefore disagrees with a previous comparative analysis which suggested the existence of independent modules within the head of East African cichlids. We propet al.. First, l.[et al. includedns et al.. Howeverns et al.,31, diffns et al., while Pl.[et al. investigl.[et al.. It is tl.[et al.,23,60. Hl.[et al. did not l.[et al., it is pInstead, our results indicated phenotypic integration between the pre-orbital parts (which included the skull) and the post-orbital parts (which included the upper and lower jaws) of the head. Given the close association between forehead shape and skull shape this intper se, a round forehead with a downward-pointing mouth are thought to be adaptations for grazing algae from the substrate[Our results confirmed the widely reported evolutionary link between head morphology and feeding ecology in Lake Tanganyika cichlids-37. The ubstrate. Howeverubstrate,24-26.After controlling for the interaction between feeding mode and head morphology, our results confirmed a strong association between head morphology and brain size. We showed that species with a high head profile have larger brains while species with a more elongated head profile have smaller brains. Given that an elongated body shape is associated with a smaller head volume in cichlid fishes, our resOur multivariate phylogenetic comparative study found support for integration of the head morphology of Lake Tanganyika cichlids, and that evolutionary associations exist among head morphology, feeding mode, and brain size. Our study therefore indicates that head-brain phenotypic integration might have played an important role in forming the macro-evolutionary variation of brain size in cichlid fishes, a group which has been an important model system for the study of phenotypic diversification. Given the strong and general association of ecology and head shape in teleost fishes-36, our We declare that we have no competing interests.All authors have read and approved the final manuscript. MT participated in the design of the study, carried out the analyses, and drafted the manuscript. AG-V conducted brain dissections and data collection, assisted in the analyses, and critically revised the manuscript. NK conceived the study, obtained funds, participated in the design of the study and helped to draft the manuscript.Heterogeneity of the brain.Click here for file"} +{"text": "Zhao and Devine exhaustiShevchuk and co-workers describeOstermeier and collaborators review eCasagrande and co-workers focus onAlthough a large body of evidence points out to a negative role of platelet activity during pathological conditions, a protective role is also well-established. Accordingly, due to their enriched cargo in bioactive compounds, including growth factors and anti-inflammatory molecules, platelet preparation lysates represent a successful strategy for treatment of inflammatory conditions and regenerative medicine. By investigating the in vitro anti-inflammatory activity of various platelet-rich fibrin preparations (PRFs), Kargarpour and colleagues highlighEvodia rutaecarpa), as therapeutic agents against thromboembolic disorders is the focus of two Chinese studies [The potential of two phytochemicals, i.e., pterostilbene and rutaecarpine (a bioactive component of the well-known Chinese medicine studies ,9. Lin a studies find tha2+ channel ORAI and its activating Ca2+ sensor STIM that are associated with platelet activation. These two proteins are upregulated by hyperphosphatemia occurring in chronic kidney disease and the authors reported the beneficial action of MgCl2 and the CaSR agonist GdCl3 in counteracting this detrimental phenomenon, thus suggesting that pharmacological modulation of CaSR by both Mg2+ and Gd3+ might restore calcium homeostasis in blood.Zhou\u2019s group investigFinally, a rapid and reproducible test for routine laboratory identification of platelet activation is described by Watanabe\u2019s group , who set"} +{"text": "Why does Frontiers in Multiple Sclerosis and Neuroimmunology publish Case Reports? There are several reasons. First, we can learn from case reports regarding novel disease entities and rare diseases. Second, case reports can give insight into better pathophysiological understanding of rare diseases. Third, case reports can be of interest regarding translational aspects and modeling in animal models. Fourth, case reports can be important regarding exploration of novel treatment regimen. Possibly, more reasons in favor of case reports can be found, the above indicated are most relevant for the Speciality Chief Editors of Frontiers in Multiple Sclerosis and Neuroimmunology. Of course, it is important that the presented cases bring additional value to the field. The ideal type of case report would contain data regarding the clinical manifestation, immunological, genetic, and pathological aspects of the disease and a novel treatment approach would be presented. Also, the findings should be discussed in the context of the relevant literature. Ideally, there should be two or more similar patients per case report. This is not trivial, especially if the case reports are coming from an individual center. Sometimes, case reports are the entrance into research for the involved physicians especially early in career, since the case has initiated a scientific argument. Therefore, the value of case reports also in gaining scientific thinking should not be underestimated. Frontiers of Multiple Sclerosis and Neuroimmunology is proud to present the \u201cMultiple Sclerosis and Neuroimmunology\u2014Case Report Collection I\u201d. This collection contains 27 case studies that were published between 2021 and 2022. We would like to thank the Associate and Review Editors who have evaluated the manuscripts and significantly contributed with their constructive criticism.The 27 case reports of \u201cMultiple Sclerosis and Neuroimmunology\u2014Case Report Collection I\u201d can be ordered as follows: (1) Reports regarding neurological manifestations of coronavirus disease 2019 (COVID-19). (2) Case reports regarding vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) and nervous-system related side effects. (3) Case reports with novel aspects of the emerging field of autoimmune encephalitis (AE). (4) Case reports of viral encephalitis. (5) Case reports regarding autoimmune peripheral nervous system disease. (6) Reports of cases of neuroimmunological side effects by pharmacological treatment. (7) Case reports regarding rare disease variants leading to demyelination within the central nervous system (CNS). (8) Case reports of rare disease variants successfully treated by immune therapy.Ishaq et al. present a case of a patient with opsoclonus myoclonus syndrome a rare neurological disease entity that was successfully treated with intravenous immunoglobulins (i.v. Ig). Gilio et al. present findings regarding the overlap of functional neurological disorders with long COVID. They indicate that stress and inflammation might drive disease precipitation of functional neurological disorders. Kimura et al. reports a patient with Bickerstaff brainstem encephalitis possibly triggered by COVID-19 and emerging Takotsubo cardiomyopathy (TC). They indicate that TC should be considered early when hemodynamic status remains unstable in patients with Bickerstaff brainstem encephalitis.There are several neurological manifestations that can be associated with COVID-19 and post- and long COVID. Maniscalco et al. present a patient with multiple sclerosis (MS) with a relapse shortly after vaccination with BNT162b2 from Pfizer-BioNTech. Nistri et al. present 16 cases regarding relapse manifestation of MS triggered by vaccination against SARS-COV-2 with 10 patients vaccinated with BNT162b2 from Pfizer-BioNTech, two patients vaccinated with mRNA-1273 from Moderna and four patients vaccinated with ChAdOx1 from AstraZeneca. In a case series by Ancau et al. three patients are reported with acute hemorrhagic encephalomyelitis (AHE) after SARS-COV-2 vaccination with ChAdOx1 from AstraZeneca. The authors indicated that these vaccination-related neurological diseases are rare events. They argue for the need of a robust post-vaccination surveillance.Song et al. report a patient with coexistence of anti-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor encephalitis and biomarkers of Alzheimer\u2018s disease. There are AE that are antibody-negative. Possibly, the relevant antigen has not been defined for these disease entities so far. Park and Kim present a patient of antibody negative AE that was successfully treated with B cell depletion by rituximab arguing for an underlying immunological disease-driving pathophysiology. Beside autoimmunity as driver for AE, there is paraneoplasia as disease initiator of AE, mediated by the anti-neoplasia-directed immune response. Gogia et al. report a case with amphiphysin antibody-associated stiff limp syndrome and myelopathy in a patient with breast cancer. Fang, Pan, et al. report a patient with relapses of anti-AMPA encephalitis with progressive brain atrophy and speculate regarding the underlying mechanisms. They observe partial functional recovery even in presence of severe brain atrophy after treatment with immunotherapy. Vaux et al. present a patient that was diagnosed with schizophrenia and was subsequently identified as a patient with anti-N-methyl-D-aspartate (NMDA) receptor encephalitis. Treatment with immune therapy led to improvement of symptoms in this patient. This case argues that patients with psychiatric diagnosis should be routinely explored for AE as a potential cause of their disease. This is important since immunotherapy can lead to improvement of symptoms and can in some patients even result in cure. Hashimoto\u2018s encephalopathy is a highly debated and questioned disease entity. Amano et al. present a patient with Hashimoto\u2018s encephalopathy associated with lymphomatosis cerebri and periodic synchronous discharges resembling prion, namely Creutzfeld-Jacob disease.Different types of autoimmune encephalitis (AE) are rare diseases with varying clinical presentations. Much can be learned regarding neurobiology from these diseases. Kolesnik et al. report a patient with herpes simplex virus 2 (HSV-2) encephalitis presenting with the clinical manifestation of chorea. This is a very rare clinical manifestation in HSV-2 encephalitis.Huang et al. report a patient with Guillain-Barr\u00e9 syndrome (GBS) and unilateral facial palsy. They summarize the current literature regarding such clinical presentations and report 28 cases. They speculate regarding the underlying immune response. Belgrado et al. had two patients with GBS and posterior reversible encephalopathy (PRES). They speculate that autoimmune dysregulation associated with GBS may be a trigger factor for co-emergence of PRES. A patient with reported combined central and peripheral demyelination (CCPD) was presented by Alshamrani et al. This patient had the coexistence of radiologically isolated syndrome (RIS) and Miller-Fisher-syndrome (MFS). Most so far reported cases of CCPD were diagnosed as having co-existence of MS and chronic inflammatory demyelinating polyradiculoneuropathy (CIDP).Koska et al. (a) report a patient with MS with severe lymphopenia due to treatment with fingolimod. The discontinuation of fingolimod treatment led to clinical deterioration with presence of neuropsychological symptoms that was difficult to overcome. Progressive multifocal leukoencephalopathy (PML) should be also taken early into consideration regarding differential diagnosis in such patients. Tang et al. present a patient with recurrent encephalopathy after treatment with ornidazole. Ornidazole is an antibiotic used for the treatment of protozoan infections. The patient recovered after discontinuation of the treatment with ornidazole. Longitudinal extensive transverse myelitis (LEMS) evolved and novel autoantibodies emerged in a patient treated with pemprolizumab reported by Charabi et al. The humanized antibody pemprolizumab targets programmed cell death protein 1 (PD1) on lymphocytes. Pemprolizumab is used in the treatment of various types of cancers. The emergence of LEMS was B cell mediated. A putative novel autoantigen was hypothesized in this disease condition that has not been defined so far.Baert et al. APRIL provides a favorable environment for plasmocytes in the NMO lesion. In addition, APRIL induces an anti-inflammatory response in the NMO lesion. This indicates that targeting of APRIL by novel immunological therapies should only be executed with extreme caution since there is a dichotomy of APRIL-related functions in the NMO lesion. Differential diagnosis can be challenging in patients with atypical clinical presentations. Fang, Tong, et al. report a patient with primary CNS lymphoma that was initially misdiagnosed with glial fibrillary acidic protein (GFAP) astrocytopathy. Early diagnosis to differentiate these diseases is important, due to grossly different treatment approaches. Gao et al. report a patient with GFAP astrocytopathy associated with an area postrema syndrome. Ma et al. report a patient with bilateral meningo-cortical involvement of anti-myelin-oligodendrocyte-glycoprotein (MOG) IgG associated disease. \u0160toura\u010d et al. present the difficulties in the diagnosis and treatment of progressive tumefactive demyelination. The presented patient had an unfavorable outcome. Neurosarcoidosis can be difficult to diagnose. Braun et al. present a patient with myelopathy that was finally diagnosed with neurosarcoidosis.The expression of a proliferation inducing ligand (APRIL), belonging to the TNF superfamily, in the CNS was investigated in a patient with neuromyelitis optica (NMO) reported by Koska et al. (b) report a patient with Marburg variant of MS who was successfully treated with cyclophosphamide and ocrelizumab. Eculizumab was successfully used to treat a patient with seronegative NMO by Digala et al. This argues for a role of complement in seronegative NMO.The case series \u201cMultiple Sclerosis and Neuroimmunology\u2014Case Report Collection I\u201d demonstrates the power of case studies as well as their limitations. Possibly, the cases will help to gain progress regarding clinical, pathophysiological, immunological, and treatment-related understanding in clinical and research environments dealing with related patient groups and evolving scientific topics in neuroimmunology. Interested physicians, physician-scientist and researchers will possibly have a benefit that will help to transform scientific thinking in medicine to a patient-focused research approach regarding disease biology and rationally well-founded medical and rehabilitative treatments.RW outlined and wrote the editorial.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."} +{"text": "Neurodegenerative diseases are a heterogeneous group of disorders characterized by gradual progressive neuronal loss in the central nervous system . These iThis Special Issue of Medicina, entitled \u201cBiomarkers in Alzheimer Disease and Other Dementias: What\u2019s Next into Pathophysiology to Support Clinical Practice and Drug Development\u201d, includes seven articles dealing with the role of CSF plasma/serum biomarkers in the understanding the biochemical mechanisms and diagnosis of dementing disorders, and their drug development.The CSF AD profile is highly predictive of the progression of cognitively unimpaired subjects to mild cognitive impairment (MCI) and dementia. In their interesting study, Scarmeas et al. [Using ultrasensitive single-molecule array assays (Simoa), it is possible to measure low concentrations of NfL, not only in the CSF but also in blood samples, with very high sensitivity. In this prospective study of patients with AD conducted by Kern et al. , higher In the future, biomarkers should be available not only for early diagnosis, but also for indicating responses to specific therapeutic interventions that help to guide treatment decisionsfor AD. Alharbi et al. demonstrPrion diseases are rare, irreversible neurodegenerative disorders with rapidly progressive dementia, cerebellar ataxia and myoclonus. Altuna et al. reviewed\u03b1-syn species measured for further validation of these biomarkers in clinical practice.Continuous efforts will be also necessary to establish useful CSF biomarkers for the early diagnosis of dementia with Lewy bodies (DLB). In their review, Foska et al. highlighCompared to protein-based biomarkers, miRNAs are more stable inbodily fluids; moreover, their easy measurement usingvarious commonly used laboratory methods make them potential biomarkers for atypical dementing disorders such asmultiple system atrophy (MSA) and progressive supranuclear palsy (PSP). In our systematic review , three sPotagas et al. suggesteOverall, the papers published in this Special Issue contribute to a deeper understanding of biochemical markers in dementing disorders, including neurodegenerative proteinopathies with novel biomarkers. Thereby, this Special Issue might initiate hypothesis-driven refinement of current and novel biomarkers in AD and other dementias."} +{"text": "It is unclear whether seven interventions recommended by Public Health England for preventing and managing common musculoskeletal conditions reduce or widen health inequalities in adults with musculoskeletal conditions.We used citation searches of Web of Science (date of \u2018parent publication\u2019 for each intervention to April 2021) to identify original research articles reporting subgroup or moderator analyses of intervention effects by social stratifiers defined using the PROGRESS-Plus frameworks. Randomized controlled trials, controlled before-after studies, interrupted time series, systematic reviews presenting subgroup/stratified analyses or meta-regressions, individual participant data meta-analyses and modelling studies were eligible. Two reviewers independently assessed the credibility of effect moderation claims using Instrument to assess the Credibility of Effect Moderation Analyses. A narrative approach to synthesis was used (PROSPERO registration number: CRD42019140018).Of 1480 potentially relevant studies, seven eligible analyses of single trials and five meta-analyses were included. Among these, we found eight claims of potential differential effectiveness according to social characteristics, but none that were judged to have high credibility.In the absence of highly credible evidence of differential effectiveness in different social groups, and given ongoing national implementation, equity concerns may be best served by investing in monitoring and action aimed at ensuring fair access to these interventions. Studies had to be of adults aged 18\u00a0years and over with a relevant musculoskeletal condition and not restricted only to patients with inflammatory disease or trauma/injury. Protocol papers, editorials, correspondence, conference abstracts and non-English language articles were excluded.Screening of titles and abstracts and review of full-text articles for inclusion were conducted by two independent reviewers with disagreement resolved by consensus, using RayyanData extraction was conducted by one reviewer using a form that we designed before data extraction began. Data extraction was checked by a second reviewer resolving discrepancies through discussion. In addition to standard descriptive fields, we extracted information on (i) baseline distribution of PROGRESS-Plus characteristics; (ii) methods, results and author conclusions on moderator analyses by each PROGRESS-Plus variable; (iii) selective participation or attrition by PROGRESS-Plus characteristics.We assessed the credibility of potentially relevant treatment effect moderation reported in analyses of a single RCT or in meta-analysis of multiple RCTs using the Instrument to assess the Credibility of Effect Moderation Analyses (ICEMAN).Given heterogeneity in populations, interventions and outcomes, we anticipated a narrative synthesis approach rather than formal meta-analysis. Where available, we sought to summarize both relative and absolute differences in effectiveness of interventions by social stratifier.The citation search yielded 1480 potentially relevant articles after removal of duplicates, of which 12 were included .,,,Six of the 12 included studies were moderator (subgroup) analyses of data from single RCTs, typically the original trial, of the BeST,Within the economic analysis of the BeST trial data,et al.In addition to pre-specified moderator analyses of the BeST trial, Underwood et al.Using latent class analysis of baseline psychological and symptom severity variables in the BeST trial, Barons et al.In a complier average causal effects analysis of the BeST trial data, Knox et al.et al.Bernard et al.Martinez-Calderon et al.P\u00a0=\u00a00.028) with the beneficial effect of the STarT Back intervention greatest among higher SES participants and least in lower SES participants. This is suspected to be due to a lack of effect of promoting self-management without further face-to-face physiotherapist support in lower SES patients with low health literacy . The interaction term for educational level was in the same direction, suggesting greater benefit among more educated participants, but was statistically non-significant (P\u00a0=\u00a00.109). Interaction terms for age, sex and employment status were non-significant (P\u00a0>\u00a00.20).In a secondary analysis of STarT Back trial data, Beneciuk et al.et al.,et al.\u2019s review included Yoga for Healthy Lower Backs trial findings alongside other forms of mindful exercise for chronic low back pain on the outcomes of pain intensity and disability. Martinez-Calderon et al.\u2019s included various exercise intervention trials for low back pain with a focus on self-efficacy outcomes up to 12\u00a0months after intervention.Meta-regression analyses by Zou et al.,In pre-specified moderator analyses Salisbury et al.Meta-regression analyses by Niknejad et al.In contrast, Kroon et al.,et al.,et al.,et al.,We applied ICEMANTheoretically informed, pre-specified subgroup analyses of RCT data, adequately powered and appropriately conducted and reported, provide the best available evidence of differential effectiveness of interventions. We found no claims of differential effectiveness of interventions recommended in the PHE ROI tool that met this high bar of evidence. Some evidence that low back pain (LBP) patients of lower occupational class might benefit less from the STarT Back stratified care intervention was judged as moderately credible. However, this was not a pre-specified subgroup analysis in the original trial and, unlike secondary analyses of the BeST intervention,,et al.,,,,The absence of highly credible, \u2018confirmatory\u2019 evidence of differential effectiveness of interventions is not unexpected: a similar conclusion was reached in previous reviews in the general medical literature,,,,,The most comprehensive body of research to date has been on therapist-delivered interventions for low back pain, spanning subgroup analysis of the BeST trial,Our review extends previous research to specifically consider equity issues and provide a critical synthesis of currently available evidence on differential effectiveness for the range of interventions recommended in the ROI tool. Despite the high burden of musculoskeletal conditions there has been surprisingly little focus on equity when investigating the effects of interventions. Given the well-recognized challenges in obtaining rigorous evidence on equity effects from quantitative analysis of single and multiple trials, our review underscores the importance of embedding equity considerations across the research cycle including intervention development and process evaluation. While none of the interventions in our review was designed deliberately to be \u2018equity focussed\u2019, they have demonstrated overall effectiveness in populations with varying degrees of social diversity and many are now already at fairly advanced stages of implementation. At the time of this review almost 1300 health professionals had been trained to deliver ESCAPE-pain. Prior to COVID-19, it was offered at 295 NHS facilities and leisure/community centres across UK, including some in more deprived neighbourhoods. In total, 19\u00a0300 people have taken the programme and an online version has been released .We used a citation search strategy that will tend to miss relevant studies of other similar interventions. We restricted our attention to studies with an appropriate comparison of effect between social groups. There may be other studies, including those excluded at title/abstract stage, which include useful information or discussion on equity-related matters in relation to these or similar interventions, but we think it unlikely that we missed additional rigorous evidence of effect moderation. For example, one systematic reviewWe found no highly credible evidence against the assumption that seven interventions recommended for the prevention and management of musculoskeletal health are equally effective in different social groups. A policy of restricting or targeting these interventions to subpopulations is not supported. Most of these interventions are already being actively implemented, many achieving substantial reach nationally. Equity concerns may be best served by investing in equity-focussed action aimed at ensuring fair access to, and participation in, these interventions. Routine collection of key social variables during implementation should underpin this.Effects_on_health_inequalities_PHE_systematic_review_SUPPLEMENT_fdac014Click here for additional data file."} +{"text": "Direct care workers play a key role in supporting the health and wellbeing of older adults and people with disabilities across care settings\u2014yet their own health risks are largely overlooked. The four papers in this symposium address this critical knowledge gap. First, McCall and colleagues will present a comparative analysis of the health status, health insurance coverage, and healthcare experiences of direct care workers across long-term care using National Health Interview Survey data. Next, Lee et al. will present the trends and characteristics of occupational injuries and illnesses among California\u2019s long-term care workers from 2019 to 2020 using California Workers\u2019 Compensation data, assessing the impact of COVID-19 on their occupational health. Sterling will characterize the physical and mental health of the direct care workforce before and during COVID-19 using data from the CDC's Behavioral Risk Factor Surveillance Survey, as well as drawing on qualitative and survey-based studies of unionized, agency-employed home care workers in New York. Sterling will also present findings from a pilot feasibility study of an intervention aimed at improving home care workers\u2019 well-being. Finally, Quinn et al. will synthesize findings on home care workers\u2019 occupational hazards\u2014including needlesticks, musculoskeletal strain, violence and infections\u2014and examine how preventing these risks can improve safety for both workers and clients. The discussant will draw out themes and implications from across these complementary studies, highlighting the importance of safeguarding direct care workers\u2019 health as a key step toward improving care quality and outcomes for older adults and people with disabilities."} +{"text": "The prevalence of aging-related neuronal diseases is increasing. However, there is still a huge unmet need for diagnosis, treatments, and drug discovery of these diseases. With the development of modern high-throughput omic measurement platforms, vast amounts of biological data have been generated which can be integrated in multi-omics studies to examine the complex molecular underpinnings of diseases, thus impacting the development of aging-related neuronal diseases' therapy.In this editorial, we presented an account of how integrative multi-omics studies have greatly facilitated the diagnosis, treatments, and drug discovery of aging-related neuronal diseases. This editorial is based on 13 research articles and 3 regular reviews which shed light on the power of integrative multi-omics studies to improve aging-related neuronal diseases' therapy including but not limited to Alzheimer's disease (AD), ischemic stroke (IS), and Parkinson's disease (PD).Han et al.'s cross-sectional study, chi square test, correlation analysis, and regression analysis were employed to analyze the influencing factors of cognitive impairment. Consequently, gender, age, education level, hypertension, and LDL-C were found to have significant differences in the incidence of cognitive impairment, providing a basis for the early screening and intervention in the elderly . Two research articles create prediction models based on variables of interest. Kang et al. developed an accurate, efficient nomogram with a model created by Cox regression method and further built a corresponding website to help clinicians improve their assessment of patient outcomes. In Li J. et al.'s study, they applied machine learning classification model light gradient boosting machine (LightGBM) to analyze presurgical variables of endovascular treatment (EVT) for acute ischemic stroke (AIS) induced by large-vessel occlusion (LVO) and construct a unique prediction model which was used to establish feasible and accurate presurgical prediction scale in identifying unfavorable outcomes of AIS after EVT.Several research articles analyze variables for prediction. In Zhang C. et al. first employed the limma R package to the got the significant differentially expressed genes (DGEs) in Depression. These DEGS were then put into weighted gene co-expression network analysis (WGCNA) and protein\u2013protein interaction (PPI) analysis and at last the common gene general transcription factor IIF polypeptide 2 (GTF2F2) which may serve as a promising diagnostic biomarker and treatment target of depression was thus identified . Most of the bioinformatic approaches in this study were implemented in the article of Gu et al., where Stepwise regression and logistic regression analyses were employed to get hub genes and diagnostic model related to Iron Metabolism in AD. They also retrieved eight drugs targeting hub genes from the DrugBank database . A similar study in IS was completed by Wang X. et al. where Boruta algorithm was used for genes' further screening. Importantly, they validated the gene signature with many methods, such as enrichment analyses through GO, KEGG, and GSEA pathways, ROC curves, and immune cell infiltration. Moreover, Li D. et al. demonstrated that gene methylation can also be utilized as signatures. In their research, least absolute shrinkage and selection operator cox regression analysis were carried out to construct a diagnostic signature related to PD . In Zhang W. et al.'s study, the molecular mechanism of Xingnao Kaiqiao Pill in the treatment of perioperative neurocognitive disorder (PND) was investigated from the perspective of network pharmacology and molecular docking technology. They constructed the network of \u201cXingnao Kaiqiao Pill\u2013traditional Chinese medicine\u2013compound\u2013common target\u201d by Cytoscape software. Molecular docking stimulation was used to further verify the interaction between the active components and key targets . Huang et al. and Chen Y. et al. both performed multi-omics integration analysis based on single cell technology. In their study, scRNA analysis, differential expression analysis, cell-cell communication analyses, and cell trajectory inference analyses were performed to identify candidate ligands or receptors, as well as the corresponding cell types. Combined with molecular docking, Huang et al. found that Quercetin targets VCAM1 to prevent diabetic cerebrovascular endothelial cell injury , while Chen Y. et al. further identified differentially expressed transcription factors in AD associated with exercise using a modified SCENIC method. Chen Y. et al. finally constructed a network of exercise-regulating TFs in monocytes, revealing the mechanism by which exercise regulated monocytes to confer therapeutic benefits against AD and its complications. Furthermore, through target gene's knockdown and bioassays, Xia et al. found that GDF15 effectively alleviated neuronal ferroptosis post Spinal cord injury (SCI) via the p62-Keap1-Nrf2 signaling pathway and promoted locomotor recovery of SCI mice, which is suggested as a potential target on SCI pathogenesis and treatment.Eight research articles capture gene signature (models) by integrating multi-omics information through different bioinformatics analysis and machine learning approaches. Zhu et al. exploited a novel iterative method called linear neighborhood similarity-based protein multifeatures fusion (LNSPF) to predict potential key proteins. The gene expression data downloaded from the benchmark database are used for further optimization through linear neighborhood similarity . To find biologically important imaging genetic relations more powerfully, Wang et al. imposed the GraphNet regularization penalty on the existing model and presented an improved fusion paired group lasso structured sparse canonical correlation analysis algorithm (FGLGNSCCA). Experiment results shown that the new FGLGNSCCA model proposed in their manuscript is superior or equivalent to traditional methods. With FGLGNSCCA algorithm, more AD-related biomarkers can been found .Notably, two research articles focus on the development of computational approaches which can integrate multi-omics information. Based on topological features extracted from a protein-protein interaction (PPI) network and functional features extracted by integrating subcellular localization and homologous information of proteins, Chen W. et al.'s review focuses on optogenetics in neurobiology, including how to use optogenetics to control nerve cells, study neural circuits, and treat diseases by changing the state of neurons. Wang Y. et al. revealed potential protective role of polysaccharides of Neurodegenerative Diseases (NDs), highlighting the contributions of polysaccharides and the prospects of their mechanism studies for the treatment of NDs. Ji et al. concentrated on body fluids biomarkers of early neurological deterioration (END) that have shown potential to be transferred into clinical practice.Finally, three reviews give a comprehensive understanding of the progress in the field of aging-related neuronal diseases from different aspects. In conclusion, the research articles and reviews in this Research Topic show how integrative multi-omics are applied to better understand and treat aging-related neuronal disease. For fully utilizing data from various omics sources to gain insights into disease, multi-omics approaches are becoming more relevant every day.This editorial was designed by MT and XG and written by CS and XL. YL, HZ, and YG had revised it. All authors have made a direct and intellectual contribution to this topic and approved the article for publication.This work was supported by grants from Jiangsu University (19JDG039) and the National Natural Science Foundation of China (32002235).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."} +{"text": "APOE \u03b54 allele is the greatest genetic determinant for AD and is widely reported to mediate detrimental effects on mitochondria function and mitophagic process. Given the continuity of the physiological process, this review takes the mitochondrial dynamic and mitophagic core events into consideration, which highlights the current knowledge about the molecular alterations from an APOE-genotype perspective, synthesizes ApoE4-associated regulations, and the cross-talk between these signaling, along with the focuses on general autophagic process and several pivotal processes of mitophagy, including mitochondrial dynamic , mitophagic induction . These may shed new light on the link between ApoE4 and AD and provide novel insights for promising mitophagy-targeted therapeutic strategies for AD.Alzheimer's disease (AD) is one of the major worldwide causes of dementia that is characterized by irreversible decline in learning, memory loss, and behavioral impairments. Mitophagy is selective autophagy through the clearance of aberrant mitochondria, specifically for degradation to maintain energy generation and neuronal and synaptic function in the brain. Accumulating evidence shows that defective mitophagy is believed to be as one of the early and prominent features in AD pathogenesis and has drawn attention in the recent few years. Alzheimer's disease (AD) is the most common and well-known form of dementia and features age-related and progressive recognition impairment, memory loss, and learning failure, accompanied by encephalatrophy, and extracellular amyloid-\u03b2 (A\u03b2) plaques, intracellular tangles of hyperphosphorylated Tau allele remains the strongest genetic risk for sporadic AD , inner mitochondrial membrane (IMM), intermembrane space, and mitochondrial matrix performs diverse essential roles in multiple intracellular homeostasis events, including tricarboxylic acid cycle, \u03b2-oxidation of fatty acids, genetic information storage, CaHere, we have reviewed emerging findings for the central and multi-faceted roles that mitophagy plays in the pathogenesis of AD, described physiological changes from the pre-mitophagy events to mitophagy proceeding considering the continuity of the physiological processes, integrated the signaling pathways suggesting the relationships between ApoE4 and mitophagy deficit, and discussed the underlying mechanisms that how ApoE4 triggers these abnormalities and ultimately contributes to AD pathogenesis, while highlighting potential therapeutic targets that may correct these dysfunctions .Autophagy is a ubiquitous event highly conserved from yeast to mammals and functions as a recycling system for maintaining metabolic homeostasis and cellular self-renewal , and mitophagy-specific induction.Mitochondria is a highly dynamic organelle that can form long tubular networks for highly interconnected networks, and continually undergoes the cycle of fusion and fission in response to environmental changes and higher phosphorylation level of dynamin-related protein1 (DRP1), leading to reductive mitochondria fragmentation and neuronal energy dysfunction possess pro-fission activation and fusion inhibition are the most broadly investigated fission regulators network in recent years Chan, . Mitochoin vivo and in vitro, ApoE4 interferes with SIRT1 transcription, expression, and enzyme activity more than ApoE3 in the frontal cortex tissue from AD patients are growing and the polyubiquitinated level of MFN-2 is reduced, while the fission proteins (FIS1 and GLP1) are not distinctively altered in HEK293 cells overexpressing human Tau, and rat primary hippocampal neurons machinery, then is cleaved by PARL in a mitochondrial membrane potential (MMP)-dependent manner followed by degradation and mitophagic genes (BNIP3 and PINK1) in APOE \u03b54 carriers. Collectively, ApoE4 mediated full-scale inhibition of mitophagy via FOXO3a signaling, including its expression, activity-dependent on chemical modification, and the induction of its downstream effects.FOXO3a served as a transcription factor highly represented in the brain and a new target of AD diagnosis is a deacetylase, highly expressed in high-metabolic tissues, and strongly associated with mitochondrial quality (Meng et al., via direct interaction with TOMM40 (Bertolin et al., 2a cells by proteomic profiling (Mise et al., APOE (Simonovitch et al., TOMM40 expression is closely associated with APOE expression (Mise et al., APOE-TOMM40-APOC1 variants are strongly associated with a high instance of AD (Gottschalk et al., APOE \u03b54 has closely linked to various TOMM40 polymorphisms carried adverse impacts (Roses et al., APOE \u03b54, and its degradation is blocked under an ApoE4-induced context, which may be helpful to explore the underlying mechanisms of AD.TOMM40, a channel-forming subunit at OMM constituting the TOMM40 complex for protein import into mitochondria (Humphries et al., 2a cells performed by proteomic profiling assays (Orr et al., via double-labeling analysis, and reconfirmed the interaction without isoform-dependent difference via co-IP in H9c2 cells, a type of embryonic rat heart-derived cells (Chen et al., APOE-TR mice or AD model mice, even the APOE \u03b54 carriers and AD subjects, in order to explore whether the interaction exists in the neuronal system, whether there is a differential interaction between ApoE3 and ApoE4, and whether it is associated with AD pathogenesis. Incidentally, the VDAC1-A\u03b2 and VDAC1-Tau interaction is confirmed by double-labeling analysis and co-IP in the brains of AD subjects and several AD model mice (Manczak and Reddy, VDAC1, a porin protein abundantly located at OMM that mediates ATP production and the trafficking of nucleotide and metabolites, functions as a target for Parkin-directed poly-ubiquitin chains, and regulates Parkin recruitment (Sun et al., A\u03b2 aggregation causes reductive cytosolic Parkin and PINK1 levels in the cytoplasm (Kerr et al., via multi-faced downstream regulators (Sohn et al., Collectively, ApoE4 and PINK1-Parkin comprise a potential mitochondrial signaling cascade response pathway, and these observations indicate ApoE4-mediated inhibition on PINK1-Parkin mitophagy Deficient mitochondrial quality control is established to interfere with cellular bioenergetic metabolisms and neuronal survival, leading to the occurrence and development of neurodegenerative disorders (Chinnery, 2+ (Cereghetti et al., APOE genotype. Third, the neuro-degeneration processes may share a common mechanism with AD and other neurodegenerative diseases. ApoE has been extensively studied in multiple fields and displays new findings, such as the interaction of ApoE4-TFEB promoter (Parcon et al., SIRT1 promoter (Theendakara et al., APOE-genotype perspective, hence, completing a related investigation may be helpful to better understand the complicated alterations in AD (Sun et al., in vivo and in vitro studies and further develop specific therapies.Despite current studies about mitophagic control, some important issues are required to be addressed. First, ApoE4 participates in diverse mitophagic signaling pathways at various processes in direct or indirect manners (Horner et al., In general, mitophagy is a multifunctional event preventing AD pathogenesis along with multiple challenges to its beneficial effects. Current findings of the ApoE4 role of mitophagy in AD seem to be limited to some extent, and remain controversial still need further investigation in detail. Discovering the gaps in the underlying mechanisms are greatly enlightened to screen for possible and predominant mechanisms and open a new avenue of research to facilitate earlier diagnosis or potential neuroprotective therapies for AD.GC had the idea for the article and made the final approval of version to be submitted. HC wrote and critically revised the manuscript the work. YiJ and FC supervised the overall writing and edited the manuscript. GH and LZ performed the literature search, sorting, and integration. FS and MZ provided guidance, helpful discussion, and careful revision. YC and YaJ helped the schematic diagrams. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."} +{"text": "The Women in Avian Physiology Research Topic is part of an inclusive Frontiers series across all sections focused on Women in Physiology. The purpose of the series is to showcase physiological research of women and to highlight their achievements. Submissions were welcomed covering all areas of Avian Physiology. Submissions were encouraged from early career researchers and/or where the lead/last author were females. A total of 12 submissions were accepted for publication in this Research Topic covering the areas of: 1. Skeletal Muscle Physiology and Meat Quality; 2. Female Reproduction; 3. Pathobiology; 4. Genetic Selection; and 5. Neurobiology.Swanson et al., Bordini et al., and Xu et al.Swanson et al. reviewed phenotypic plasticity to environmental variation focusing on metabolic rates and skeletal muscle physiology in wild birds. The ultrastructural plasticity of skeletal muscle with regard to thermal variation and increased workload was reviewed and correlated with myostatin, Insulin-like growth factor-1, and satellite cell proliferation. Xu et al. examined the effects of temperature and selection for growth on the proliferation, differentiation, adipogenic potential of turkey myogenic satellite cells through frizzled-7-mediated Wnt planar cell polarity (Wnt/PCP) pathway. It was found that thermal stress altered frizzled-7 regulation of the Wnt/PCP pathway in a growth-dependent manner affecting the growth potential of the breast muscle and protein to fat ratio. Bordini et al. contributed an original Research Topic studying molecular pathways and key genes associated with White Striping and Wooden Breast in chickens. Using Weighted Gene Co-expression Network Analysis to identify clusters of co-expressed genes associated with White Striping and Wooden Breast, they found that endoplasmic reticulum stress may underly the inflammatory condition in affected breast muscles and Collagen type IV may have significant role in the events leading to White Striping and Wooden Breast.Three contributions addressed skeletal muscle physiology and/or meat quality by Hanlon et al. provided a comprehensive review on the roles of 17\u03b2-estradiol in non-gonadal tissues and its impact on reproduction in both laying and broiler breeder hens. Estradiol-17\u03b2 are involved in reproduction, liver metabolism, and medullary bone formation. Thus, this hormone may regulate all aspects of egg formation and hence the timing of estradiol-17\u03b2 is critical to reproduction which has been altered by genetic selection for intense growth. Mehlhorn et al. investigated how hen line, age and housing affect estradiol-17\u03b2 on egg laying performance. High performance hen lines had higher estradiol-17\u00df concentrations compared to low performing hens. Regardless of line, maximal estradiol-17\u00df concentration was measured at their 49th to their 51st week of age. Furthermore, cages hens had highest estradiol-17\u03b2 levels compared to floor housed hens. We could show that laying performance is strongly linked with estradiol -17\u00df concentration. This concentration changes during laying period and is also influenced by the housing system.Salmonella vaccine was shown to decrease Salmonella enterica serovar enteritidis load in immunized broiler chickens by Acevedo-Villanueva et al.Shanmugasundaram et al. contributed an original Research Topic demonstrating that subclinical doses of combined fumonisins and deoxynivalenol (mycotoxins contaminating poultry diets) predispose Clostridum perfringens inoculated broilers to necrotic enteritis, an economically important disease negatively impacting digestion and absorption of nutrients in broilers.Use of an oral-killed chitosan nanoparticle Bernardi et al. reported on chemerin as a possible genetic selection tool for embryo survivability pending larger scale testing. Research on how the somatotrophic axis has changed with commercial growth selection and its relationship to increased growth was reported by Vaccaro et al. They found the expression of insulin-like growth factors 1 and 2 was greater in the modern commercial chicken and maybe linked with increased breast muscle growth and overall muscle accretion. In broilers divergently selected for ultimate pH, it was found by Beauclercq et al. that there is an association between serum lipid profile, ultimate pH and meat quality. Furthermore, since ultimate pH can be obtained on live birds, it may be a useful marker in genetic selection.Two contributions addressed the effects of genetic selection. Loveland et al. contributed a perspective paper on how inversion variants can affect neural circuitry. This review covered how behavior polymorphisms can evolve from genetic inversions, especially when inversions are associated with sets of genes involved with hormonal regulation. Primary focus was on the three-morph system of the ruff , two alternative morphs (Satellites and Faeders) each with distinct behaviors and low circulating testosterone that is genetically determined by an inverted region on an autosomal chromosome. Franco et al. examined the bird sense of sight and found that broilers raised under blue light were more hyperopic than those raised with white light. The blue light reared broilers had better spatial vision and higher success in selecting the right feeder."} +{"text": "Advances made in the Internet of Things (IoT) and other disruptive technological trends, including big data analytics and edge computing methods, have contributed enabling solutions to the numerous challenges affecting modern communities. With Gartner reporting 14.2 billion IoT devices in 2019 , smart aHowever, there remain practical challenges in large-scale and rapid deployment of sensors for diverse applications, such as problems affecting siting optimization methods and participant recruitment and incentive mechanisms. On a higher level, the deluge of data sources that drive the IoT phenomenon grows every day. With the rise of smartphone-enabled citizen sensing data via social networks or personal health devices, as well as with increasing connectedness in transport, logistics, utilities, or manufacturing domains, this range and complexity of available data calls for even more advanced data processing, mining and fusion methods than those already applied.The goal of this Special Issue was to solicit high-quality original papers aimed at demonstrating effective and efficient deployment of sensor networks in the IoT, encompassing both physical and virtual sensor networks (through modelling) as well as social networks. Related issues of data processing, in addition to challenges of the fusion and visualisation of the resultant IoT big data, are also reflected in the published papers. This Special Issue consists of 11 papers. All submissions were strictly and thoroughly peer-reviewed by experts. These submissions cover many of the relevant research issues. In the following sections, we summarize these articles and highlight their major contributions.In the article entitled \u201cegoDetect: Visual Detection and Exploration of Anomaly in Social Communication Network\u201d, Pu et al . presentThe article entitled \u201cRecognizing Context-Aware Human Sociability Patterns Using Pervasive Monitoring for Supporting Mental Health Professionals\u201d, contributed by Moura et al. , presentZurbuchen et al. , in theiThe article entitled \u201cIoTCrawler: Challenges and Solutions for Searching the Internet of Things\u201d by Iggena et al. addresseThe paper entitled \u201cEnergy Management Expert Assistant, a New Concept\u201d by Linan-Reyes et al. presentsThe topical theme of user device location privacy is explored in the paper \u201cPerturbed-Location Mechanism for Increased User-Location Privacy in Proximity Detection and Digital Contact-Tracing Applications\u201d by Lohan et al. , which pThe predictive maintenance of sensors is addressed in the article entitled \u201cProviding Fault Detection from Sensor Data in Complex Machines That Build the Smart City\u201d by Gasc\u00f3n et al. through The application of IoT-enabled services to smart tourism scenarios is explored in the article \u201cAn Architecture for Service Integration to Fully Support Novel Personalized Smart Tourism Offerings\u201d by Sabbioni et al. . The artThe use of short texts from social networks as a data source in the IoT is the focus of the article entitled \u201cInvestigating the Efficient Use of Word Embedding with Neural-Topic Models for Interpretable Topics from Short Texts\u201d, submitted by Murakami and Chakraborty . The aut\u201cA Novel Privacy Preserving Scheme for Smart Grid-Based Home Area Networks\u201d, submitted by Ali et al. , presentA review article, entitled \u201cApplications of Wireless Sensor Networks and Internet of Things Frameworks in the Industry Revolution 4.0: A Systematic Literature Review\u201d by Majid et al. , complet"} +{"text": "Determination of antisuicidal factors (AF) in balance with risk factors for suicidal behavior (SB) is essential for treatment and prophylactic measures.Study AF in a sample of schizophrenic recovered patients according to operational criteria R.P. Liberman et al. (2002).The content analysis of published self-reports of a sample (n = 13) of Russian and foreign psychiatrists and clinical psychologists with psychotic experience was used as a part of a more extensive qualitative analysis of \u00abwounded healers\u00bb.In the history of > \u00bd ex-patients, repeated SPs (aborted suicides), as well as non-suicidal self-harm , were noted during the active period of the disease, and in four of them \u2013 during untreated psychosis. Following AFs can be distinguished in recovery state: clinical social , and existential .AF is an important integral component of recovery in schizophrenia as a process of personality development despite a burden of severe mental disorders.No significant relationships."} +{"text": "Enterobacter faecalis MSI12 and its effect on the disruption of Candida albicans biofilm\u2019 by G. Seghal Kiran et al., RSC Adv., 2015, 5, 71573\u201371585, DOI: 10.1039/C5RA10302A.Correction for \u2018Characterization of an exopolysaccharide from probiont The authors regret that incorrect versions of Accordingly, the experimental methods followed in the phase contrast microscopy and large-size images of The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "The second volume of the Special Issue on \u201cMicro-Electro Discharge Machining: Principles, Recent Advancements and Applications\u201d confirms the growing interest in the micro-EDM technology as a suitable and efficient technology for machining novel, multi-material components, with demanding requirements in terms of precision, accuracy and productivity.This volume consists of 10 original research papers which involve several approaches to micro-EDM and cover the enhancement of the process performance, such as the material removal rate, surface roughness, or accuracy of the machining, using advanced optimization methods. Some studies also consider several dielectric fluid additives and investigate the processability of new materials. Others investigate the combination of Reverse-micro-EDM with laser beam micromachining or explore new applications for the micro-EDM for fabricating antimicrobial nanosilver colloid.In more detail, in order to improve the machining accuracy of detail features in micro-EDM milling, a theoretical model is developed by Jing et al. to simulThe influence of the near-dry WEDM technique to reduce the environmental impact of wet WEDM was investigated by Chaudhari et al. . The stuA mathematical\u2013statistical computational (MSC) model for predicting high productivity and quality of the machined area is formulated by Straka et al. by the aWire-cut electro-discharge machining (Wire-EDM) of polymer composite material (PCM) was studied by Ablyaz et al. . Tests wSidhu et al. used theAblyaz et al. investig2O3). The study evaluated effects on material removal rate (MRR), relative electrode wear rate (REWR), and surface roughness on machining (EDM) of ultrafine particle type tungsten carbide and observing single discharge crater and hole machining tests. The results showed that adding CnF enhanced the material removal rate under all conditions. In contrast, Si and Al2O3 powders only improved the machining performance at a high discharge energy of 110 V. Improvement in surface roughness was observed prominently at high voltages for all the powders. However, alumina improved the surface roughness the most among the three powders.Gattu et al. mixed thEsser et al. observedReverse-\u00b5EDM was considered by Kishore et al. with theFinally, an alternative study has been performed by Tseng et al. with theWe thank all the authors who submitted their papers to this Special Issue, \u201cMicro-Electro Discharge Machining: Principles, Recent Advancements and Applications\u201d. We would also like to acknowledge all the reviewers for dedicating their time to providing careful and timely reviews to ensure the quality of this Special Issue."} +{"text": "Research focusing on innovative nanomaterials for applications in biomedicine and bioengineering has steadily gained attention over the last 20 years. This is due to the unique physical and chemical characteristics that can be provided by nanomaterials compared to their bulk counterparts, including augmented surface reactivity; improved mechanical, electrical, magnetic, optical and thermal properties; and their enhanced bioactivity, drug loading capacity or permeability through biological barriers. Advanced nano-biomaterials with tailored surface topography and chemistry can be designed to create environmental conditions favorable for proper protein and consequently for accelerated cell adhesion, proliferation and differentiation ,2. In thNanostructured modifications of metal scaffolds represent a promising approach to accelerate implant osseointegration through the increasing in endothelial commitment of mesenchymal stem cells (MSC). Gardin et al. explored the possibility that exosomes secreted by hMSCs grown on the different nanotubular Ti surfaces could act as biomimetic tools to modulate the biological properties of human umbilical vein endothelial cells (HUVECs) in vitro . The resNanoscale surface modifications may also have an impact on peri-implant cell fate and implant loading, both of which have an impact on early bone healing. This issue was addressed by De Barros E Lima Bueno et al., who studied how mechanical loading affects healing around implants with nanotopography . At 7 daIn addition to nanotopography, the composition of the composition certainly plays a key role in determining the final performance of biomedical devices. Poly:polystyrene sulfonate (PEDOT:PSS) is a widely used conductive polymers for a plethora of applications, including electroactive scaffolds for bone tissue regeneration. As a viable alternative, PEDOT:Nafion is emerging due to its better electrochemical properties than PEDOT:PSS. However, its biocompatibility has not yet been studied. Guzzo et al. investigated the in vitro cytotoxicity of nanostructured PEDOT:Nafion coatings obtained from a water-based formulation of PEDOT:Nafion, using a primary cell culture of rat fibroblasts . The resMagnetic iron oxide nanoparticles are widely used in applications for targeted anti-cancer radiotherapy and imaging techniques using radioisotopes emitting \u03b2+, \u03b2\u2212, \u03b1, and \u03b3 radiation. In this context, Nieciecka et al. demonstrated that the conjugation of iron oxide nanoparticles with terbium ions and guanosine-5\u2032-monophosphate were more promising for hypothermia-based treatments than standard unconjugated nanoparticles .4:Yb3+/Er3+ UCNPs coated with SiO2 using the hydrothermal method [Up-conversion rare-earth nanoparticles (UCNPs) are an extremely interesting class of materials due to their high fluorescence intensity as well as the possibility to absorb near-infrared radiation and converting into visible light through non-linear optical processes. Zhang et al. reported a new method for synthesizing Ag-NaYFl method . These nA new and promising strategy for cancer therapy is sonodynamic therapy (SDT). In SDT, effective sonosensitizers are of paramount importance. Ma et al. showed that urchin-shaped copper-based metalloporphyrin liposome nanosystem can be considered highly effective sonosensitizer, as it was found to massively generate reactive oxygen species capable to kill 4T1 tumor cells under ultrasound irradiation . In the This Special Issue also contains four review articles on key topics in the field, which can be very interesting and useful, especially for young researchers involved in this multidisciplinary and highly fascinating field of research. The first review by Duta et al. covers the production of biphasic calcium phosphate materials derived from fish wastes , known as fish discards, in the context of material recycling and the circular economy . This woNanomaterials covers a wide range of different nanomaterials, including nanostructured coatings, nanoparticles, nanosystems, biomimetic devices and drug carriers that are expected to be the building blocks of next-generation of biomaterials. Although not fully representative of the huge number of different nanomaterials and solutions available for biomedical applications, this Special Issue manages to provide a quick overview of some of the most promising solutions in this rapidly evolving and interdisciplinary field.In summary, this Special Issue of"} +{"text": "F. Duffy et al. Nature Communications 10.1038/s41467-022-32996-5 (2022)P. falciparum transcriptomic datasets with approaches centred on the tight transcriptional pattern governing P. falciparum along its ~48\u2009h intraerythrocytic asexual cycle, and we showed a relation between circulation of more developed parasites within each ~48\u2009h asexual cycle and lower parasitaemias or milder malaria symptoms1. Previously unpublished data from Duffy and colleagues is not fully aligned with our published conclusions. Here we discuss their comments on our recent study.We have recently reanalysed several 1 we included among others, the 2018 publication by Tonkin-Hill et al.2 which at the time did not report individual patients\u2019 parasitaemias, total parasite biomass estimates in the form of Ptot or the levels of HRP2, haematocrit, and weight for its calculation, or that parasitaemias of the subset of the patients whose samples they used for differentially expressed gene (DEG) analysis were significantly different between samples of severe and uncomplicated malaria cases, contrary to their report for the comparison including the entire group of samples2, hence we could not have included these data in our publication.In our manuscript2, we included their reported DEG list in the analyses of our manuscript similarly to what we did for other studies1. We concluded that the comparison between non-severe and severe cases without reported strong parasitaemia differences in Tonkin-Hill et al. resulted in less pronounced differences in peak of expression of DEGs and in proportions of predicted parasite stages. We agree with Duffy et al. that we could have more clearly transmitted to the reader that the normalization efforts originally applied to their dataset would prevent showing such clear trends as the other studies, and accept their criticism. Nevertheless, despite the normalizations implemented, using the reported DEG list by Tonkin-Hill et al. we still estimate higher ring-stage proportion in severe versus non-severe malaria cases ; and we observed gene expression patterns associating with number of housekeeping-gene reads, which we used instead of individual parasite levels, given that those were not originally reported 2. Duffy et al. now report that RNAseq read counts of this housekeeping gene and individual parasitaemias do not correlate, which we had no possible way of knowing at the time of publication of Thomson-Luque et al.1. We do not fully agree with the additional arguments based on correlations proposed by Duffy et al. The proportion of ring- and non-ring- asexual stages is expected to sum up to 1 (or very close to 1 whenever gametocytes are present). Positive correlations to one of the groups imply negative correlations to the other, as in fact Duffy et al. observed. That the correlation found by Duffy et al. is positive with the proportion of rings, supports our rational that severe cases have higher proportion of rings and higher parasitaemias . The variation of the glycine tRNA ligase shown on PlasmoDB highlights slightly lower expression of the gene at very young ring stages or schizonts, which would lead to proportionally lower normalised glycine tRNA ligase reads in parasites of younger developmental stage, and not reflect youth of parasites as proposed by Duffy and colleagues.Although we were aware of the different efforts Duffy and colleagues performed in Tonkin-Hill et al. to statistically normalize by developmental stage the differential expression between parasites causing severe and non-severe malaria1 as only Lee et al. reported individual values of HRP23 of the ten studies included.In Fig. 5 of Thomson-Luque et al. we have not investigated transcriptome correlates with circulating parasite density within each individual study, as we believe these would be difficult to detect due to small sample size, and short ranges in parasite densities in the vast majority of the included studies. We also did not report nor inferred any correlates between total parasite biomass and circulating parasite age in Thomson-Luque et al.2. At the time of publishing Thomson-Luque et al. we referred to the parasite density comparison reported in Tonkin-Hill et al.2, showing a non-significant difference between severe and uncomplicated malaria cases n\u2009=\u200944 p\u2009=\u20090.87262, and we could not foresee that the authors would now present an analysis of the same cohort with a lower sample number showing a significant difference .Duffy et al. now report that parasitaemias of the subset of the patients whose samples they analysed for differentially expressed gene (DEG) analysis were significantly different between samples of severe and uncomplicated malaria cases, however, this data is novel and different from the one they published in Tonkin-Hill et al.2. And in Thomson-Luque et al. we wrote: \u201cTonkin-Hill et al. detected no differences between severe and uncomplicated malaria cases in total number of var gene reads, but identified segregation at the multidomain and individual domain level between severe and uncomplicated disease. However, Tonkin-Hill et al. also found genes involved in PfEMP1 transport and regulation to be downregulated in severe malaria\u201d. We believe we did not interpret Tonkin-Hill data or statement wrongly.Regarding a possible misinterpretation of Tonkin-Hill var gene expression data, objectively, in Tonkin-Hill et al. can be read \u201cgenes involved in PfEMP1 transport and a gene involved in regulation of var genes were down-regulated in severe malaria. This suggested that var gene expression was modulated but PfEMP1 surface presentation was reduced.\u201d4, in line with the with the conclusions in Thomson-Luque et al. Nevertheless, we acknowledge that our combination of very different datasets may be a limitation, and we believe that future studies, including parameters like the ones now presented by Duffy and colleagues, alongside novel approaches like single-cell analysis should provide greater resolution of the parasite life-cycle stages. This may help improve interpretations and correct any unintended imperfect conclusion in Thomson-Luque et al. and the other bulk RNAseq-studies.We published a series of observations obtained through multiple approaches and applied to several studies, which we interpreted considering the data included in the original reports, to allow the detection of broad patterns that could otherwise be overseen. Since the publication of Thomson-Luque et al., a study investigating expression profiles of cerebral malaria and uncomplicated malaria isolates from Benin found that the mean parasite age expressed in hour post invasion was statistically lower in cerebral malaria cases, and confirmed the difference by microscopic reading of thin blood smearsFurther information on research design is available in the\u00a0Reporting Summary"} +{"text": "Non-coding RNAs including long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) are implicated in the regulation of gene expression at transcriptional, posttranscriptional, and epigenetic levels. Several studies in cell lines, animal models, and humans, have revealed that non-coding RNAs play crucial roles in the pathogenesis of Alzheimer's disease (AD). Detailed knowledge on their mechanism of implication in the AD pathogenesis can help to develop novel therapeutic and disease management strategies. The two main pathological hallmarks of AD are amyloid plaques resulting from the \u03b2-amyloid accumulation, and neurofibrillary tangles (NFT) due to the phosphorylated tau accumulation. Several lncRNAs and miRNAs play crucial roles in both these hallmarks of the AD pathogenesis and other AD-related pathological procedures such as neuronal and synaptic plasticity, neuroinflammation, neuronal differentiation and neuronal apoptosis. In this review, we outlined the non-coding RNAs and further discussed how they are implicated in these AD-related pathological procedures. Alzheimer's disease (AD) is a devastating neurodegenerative disorder and the most common cause of dementia, accounting for approximately 60 % to 80 % of cases among the elderly worldwide comprise a class of endogenous regulatory RNA molecules that are over 200 nucleotides (nt) in length, without protein-coding capacity, and make up the largest proportions of the mammalian non-coding transcriptome , and th2O2 increase the expression of lncRNA BACE1-AS, which in turn makes BACE1 mRNA more stable, leading to enhanced APP processing and toxic A\u03b2 production by a post-transcriptional feed-forward mechanism is an enzyme central to the pathology of AD that contributed to the formation of A\u03b2 peptides and extracellular plaque deposition . BACE1 al., 2008). Producal., 2008) . Besideal., 2008). BACE1-al., 2008; Fotuhi al., 2008).The neuronal sortilin-related receptor 1(SORL1) gene encodes SORLA protein, which is involved in the processing of amyloid-\u03b2 protein precursor (APP) and decrease A\u03b2 peptides production in neurons . StudieLow density lipoprotein receptor-related protein 1(LRP1), as a receptor is involved in a variety of cellular processes including cell signaling, lipoprotein metabolism, and APP trafficking and processing . LRP1 iOne of the neuropathological features of AD is synaptic dysfunction that occurs in the first stages of the disorder , is widely synthesized in the central nervous system, has a physiological role in synaptic plasticity and survival of neurons . BDNF gal., 2016, 2009[98al., 2012). Guo etal., 2012). It wasal., 2012; Ding etal., 2012), and thal., 2012). More rGlial cell line-derived neurotrophic factor (GDNF) is an important biologically active trophic factor for development and survival of the midbrain dopaminergic neurons . It hasNeuroblastoma differentiation marker 29 (NDM29), a RNA pol II-transcribed lncRNA, mapped to a genomic region of chromosome 11, and its synthesis is regulated by an extragenic type 3 promoter . NDM29 Sox2 is a component of the core transcriptional regulatory network that is essential for maintaining pluripotency of stem cells and neurogenesis . Sox2 oRecently, Magistri et al. used RNA sequencing in order to discover significant alterations in expression profile of the lncRNAs from AD brains and found that lncRNA EBF3-AS is abundantly expressed in the brain of late-onset AD cases compared to control individuals . FurtheLee and colleagues identified 47 upregulated and 158 downregulated lncRNAs in rat model of AD compared with healthy controls using microarray analysis . MoreovMicroRNAs (miRNAs) are endogenous ~22 nucleotide non-coding RNAs, responsible for direct posttranscriptional regulation of gene expression, either by targeting mRNA degradation or by inhibition of protein translation at both mRNA and protein levels . After \u2032-UTR region of the BACE1 to reduce A\u03b2 accumulation, which makes it as a novel drug target , a rate-limiting enzyme involved in the formation of A\u03b2, plays a crucial role in AD progression . Similaal., 2014; Zhu et al., 2014). Other al., 2014). Furtheal., 2014). In anoal., 2014). Du et al., 2014). In sum\u2032-UTR of the APP mRNA resulted in decreased level of APP in patients with AD . Intereal., 2020).in vitro and in vivo . Severaal., 2016). Smith al., 2016). It hasal., 2016). Studieal., 2016). Moreoval., 2016).In this review, we summarized the relationship between ncRNAs and AD pathogenesis based on recent evidence. Multiple studies have shown the critical role of ncRNAs in various biological processes, including cell growth, apoptosis, and differentiation, indicating that they might be involved in the occurrence and progress of neurodegenerative diseases including AD can be used as potential therapeutic targets and biomarkers.Reliable biomarkers for early diagnosis are required to halt the disease development and improve the effects of therapeutic strategies.Authors appreciate the Cognitive Sciences and Technologies Council of Iran for supporting this work.This work was supported by the Cognitive Sciences and Technologies Council of Iran, grant number 6300.The authors have no relevant financial or non-financial interests to disclose.Majid Khodayi-Shahrak performed research and wrote the manuscript; Mohammad Khalaj-Kondori supervised research, reviewed and edited the manuscript; Mohammad Ali Hosseinpour Feizi reviewed and edited the manuscript; Mahnaz Talebi reviewed and edited the manuscript."} +{"text": "Snowdon et al., RSC Adv., 2019, 9, 6752\u20136761, DOI: 10.1039/C9RA00034H.Correction for \u2018Comparative study of the extrinsic properties of poly(lactic acid)-based biocomposites filled with talc The authors regret that the values given for oxygen and water vapor permeability in The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "The intensive genetic selection of broiler (meat-type) chickens over the past 80\u00a0years has focused narrowly on economically important traits, namely growth rate, feed efficiency, and breast yield . AlthougGrowth rates have dramatically increased by over four times between 1957 and 2005 and modede novo lipogenesis and/or endogenous (adipogenesis/lipogenesis) sources. As commercial broilers are fed lipid-poor diets (<10%), the majority of the accumulated fat is derived from the liver, which is the main site for ogenesis . Fat metKato et al. showed that two-week intracerebroventricular infusion of neurosecretory protein GM (NPGM) increased body mass, and the mass of abdominal and gizzard fat in chickens, without effects on feed intake. At molecular levels, they showed that NPGM might induce hepatic fat deposition via down regulation of the hepatic PPAR\u03b1 gene expression.At the central level, Massimino et al., on the other hand, used high throughput real-time quantitative PCR to determine the effect of embryonic thermal manipulation on the hepatic lipid and carbohydrate metabolism, stress, cell proliferation, and thyroid hormone pathways in mule ducks. They identified several key genes that might be involved in thermal long-term programming of hepatic metabolism. Surugihalli et al. used mass spectrometry-based metabolomics to evaluate the rapid remodeling of hepatic mitochondrial and cytoplasmic networks in chicken E18 embryo and d3 post-hatch chicks. Several metabolites were profiled in both plasma and liver showing a transition from lipid oxidation in embryonic liver to de novo lipogenesis in neonatal liver. Using forced molting laying hens, cecal metabolomics, and liver transcriptomics, Ruirui (abstract only) identified regulatory intestinal-liver lipid metabolism factors affecting reproductive performance in laying hens.At the peripheral level, the Bonnefont group has used a high throughput metabolomics approach to identify hepatic biomarkers for Foie Gras quality in duck. A total of eighteen quality-associated metabolite signatures were determined, with five specific to liver weight and four specific for technological yield at cooking. Zhao et al. determined the developmental changes of adipocyte differentiation, lipid synthesis, lipolysis, fatty acid \u03b2-oxidation, and lipid content from chicken embryo day 12 to day-9 post hatch. They showed that the mitochondrial copy number and fatty acid \u03b2-oxidation increased during the post hatch period, indicating that subcutaneous adipose tissue plays an important role in energy supply. Sun et al. used the Iso-Seq technology and identified several long non-coding RNA and alternative splicing in abdominal and subcutaneous adipose tissues of pekin ducks. Na et al. reported several reference genes for real-time quantitative PCR in chicken adipose tissue and adipocytes. Kim et al. determined the effect of in ovo injection of different doses of all-trans retinoic acid on quail embryonic adipogenesis. They found that all-trans retinoic acid promoted hypertrophic fat accretion in quail embryos via upregulation of PPAR\u03b3 and FABP4 and down regulation of Dlk1. The Gilbert group studied the effect of dietary baicalein supplementation on adipose tissue gene expression profiles during the first week post hatch in chickens. They showed a reduction of growth performance , breast muscle weight, and subcutaneous and abdominal fat weights along with a modulation of several genes involved in adipogenesis and fat storage. In a separate paper, the Cline group reviewed the effects of dietary flavonoids on lipid metabolism in liver, skeletal muscle, and adipose tissue of poultry species. Finally, Kim and Voy provided a thorough review associated with the beneficial effects of n-3 polyunsaturated fatty acids in reducing fat accretion in poultry.In adipose tissue, In summary, the papers within the current Research Topic provide new insights and discoveries related to lipid metabolism in various avian species and suggest some solutions and perspectives for future investigations aiming to reduce fat accretion in poultry. It is my fervent hope that this ebook and the Research Topic is a great resource for the readers, and we look forward to a second volume to expand more research and knowledge associated with other molecular pathways of lipid metabolism in various other avian species."} +{"text": "Protein kinases regulate nearly every aspect of cell life, and changes in their expression or gene mutations cause cancer and other diseases. As many human diseases result from mutations and overexpression of kinases, this enzyme class symbolizes an important targeted strategy for drug development. Kinases are also essential in signaling pathways that regulate tumor cell functions. Kinase dysregulation causes a number of pathophysiological changes that promote cancer cell proliferation and metastasis. This targeted, structure-based drug design is gaining traction; several anticancer drugs targeting specific kinases are in various stages of clinical trials. When compared to a decade ago, the new opportunities, developments, and results in this field are almost unbelievable.The development of superior drugs that target specific signaling molecules has had a significant impact on the pipelines of pharmaceutical companies, leading to the development of multiple marketed kinase inhibitors. The development of selective small molecules against kinases has the potential to provide greater potency and selectivity than was previously possible.The authors included in this Research Topic discuss how various classes of kinases are used as potential drug targets, with a particular emphasis on anticancer therapy. The content covers both the challenges and opportunities for future kinase inhibitor development for anticancer therapy. Contributions also discuss how the problem of drug resistance to kinase inhibitors is being addressed, as well as the future of kinase drug discovery. Kinases are currently being studied extensively for the treatment of a variety of life-threatening diseases. This Research Topic brings together several reviews and research articles, contributed by scientists and researchers in the fields of medicinal chemistry and molecular biology, as well as new emerging multi-drug targets.Hepatic Rupture as the Initial Presentation of an EGFR-Mutated Lung Adenocarcinoma: A Case Report\u201d by Mirallas et al., which describes a female patient who presented with a metastatic hepatic rupture and was later diagnosed with EGFR-mutated lung adenocarcinoma. Hepatic rupture is a rare complication in patients with solid tumor malignancies, particularly lung adenocarcinomas, and it is associated with a very poor prognosis.This Research Topic starts with a research article on \u201cAlam et al. examining \u201cBax/Bcl-2\u2019s Cascade Is Regulated by the EGFR Pathway: Therapeutic Targeting of Non-Small Cell Lung Cancer.\u201d The review summarizes recent information on the molecular mechanisms of the Bax/Bcl-2 cascade and the EGFR pathway in NSCLC and targets them for therapeutic implications. This study looked at the therapeutic potential of Bax/Bcl-2/EGFR SMIs, particularly those with higher potency and selectivity, such as gefitinib, EGCG, ABT-737, thymoquinone, quercetin, and venetoclax. Furthermore, they also discuss the EGFR pathway and ongoing in vitro, in vivo, and clinical studies in NSCLC. Exploration of such inhibitors facilitates future NSCLC treatment and management.The next article in this Research Topic is a review by Wu et al. in their contribution, \u201cLestaurtinib Has the Potential to Inhibit the Proliferation of Hepatocellular Carcinoma Uncovered by Bioinformatics Analysis and Pharmacological Experiments,\u201d identifies a new candidate STAT family. This factor is involved in the development of HCC and new treatment Candidate STAT family genes for HCC were found using bioinformatics web resources such as Oncomine, Gene Expression Profiling Interactive Analysis (GEPIA), The Human Protein Atlas (HPA), Tumor Immune Estimation Resource (TIMER), and GSCALite. In this study, the author performed in vitro experiments using lestaurtinib as a treatment for patients with liver cancer and verified that lestaurtinib, a tyrosine kinase inhibitor, can inhibit the growth of liver cancer cells. The author explores the effects of lestaurtinib in hepatocellular carcinoma through in vitro and in vivo systemic studies to provide a new strategy for the treatment of hepatocellular carcinoma. In vivo studies focus on addressing how to limit toxicity at optimal biological doses.Research by Therapeutic Implications of Caffeic Acid in Cancer and Neurological Diseases,\u201d is a review by Alam et al. that focuses on the chemical, physical, and pharmacological properties of caffeine, including its antioxidant, anti-inflammatory, anticancer, and neuroprotective effects. The features, therapeutic potential, and future possibilities of CA are also discussed by the author.The fourth article in this Research Topic, \u201cChen et al. identified a novel LDLR-ROS1 fusion in patients with resectable stage IIIA NSCLC, entitled \u201cCase Report: Adjuvant Crizotinib Therapy Exerted Favorable Survival Benefit in a Resectable Stage IIIA NSCLC Patient With Novel LDLR-ROS1 Fusion.\u201d After receiving crizotinib as adjuvant therapy, the patient experienced no substantial harmful side effects and maintained recurrence-free survival (RFS) for 29\u00a0months. This example supports the use of adjuvant treatment with the ROS1 inhibitor exhibiting clinical survival advantage in ROS1 fusion-positive resected NSCLC, according to the authors\u2019 report of a novel LDLR-ROS1 fusion responding to crizotinib in a patient with lung adenocarcinoma.The fifth article in this Research Topic by Avery et al. on \u201cOnco-immunomodulatory properties of pharmacological interference with RAS-RAF-MEK-ERK pathway hyperactivation.\u201d This article outlines the rationale and concepts for utilizing the immunomodulatory properties of RAS-RAF-MEK-ERK inhibition while emphasizing the role of MEK inhibition in combinatorial and intermittent anemia treatment. The considerable scientific efforts made to address the difficulties encountered during the clinical transition of different therapeutic drugs were also highlighted in the hunt for the most efficient and secure patient- and tumor-tailored treatment strategy.The sixth article in this Research Topic is a review by Case Report: A Lung Adenocarcinoma With Brain Metastasis Harbored Novel MET 14 Skipping Alteration Sensitive to Savolitinib,\u201d was written by Li et al. and describes a 61-year-old woman who had lung adenocarcinoma and was treated with Savolitinib after having a novel MET exon 14 skipping (c.3004). These two MET changes were also validated by the CytoTest MET/CCP7 FISH Probe (c-MET/CCP7 Ratio:1.41 and mean gene copy number:6) and qPCR, which used the ABI 7500. Savolitinib treatment lasted for 2\u00a0months, and the clinical assessment showed a partial response (PR). In conclusion, this discovery not only broadened the range of the MET exon14 variation (METex14). Targeted NGS analysis may enhance the ability to detect MET changes in everyday practice.The seventh article in this Research Topic, \u201cRET Signaling Pathway and RET Inhibitors in Human Cancer,\u201d by Regua et al. reviews biological understanding of RET signaling, the effects of RET hyperactivity on tumor progression in a variety of tumor types, and the efficacy of RET inhibitors in preclinical and clinical settings.The final article in this Research Topic, \u201cKinase Inhibitors in Cancer Therapy.\u201dI would like to thank all authors who contributed to this Research Topic on \u201c"} +{"text": "After tBritish Medical Journal, Wallis et al. [Annals of Thoracic Surgery, Rong et al. [Addressing the issue of disparities between men and women can seem redundant, since this dichotomy is well recognized in the professional world. It is nevertheless essential in our surgical field since it is still a male-dominated world: most recent data from the public report of the American Association of Medical Colleges (AAMC) in 2019 on active physicians sex and specialtyindicates that 92% of practicing cardio-thoracic surgeons are men . Despites et al. reportedg et al. reported\u2018I do not wish to give (women) a first place, still less a second one\u2014but the complete freedom to take their true place, whatever it may be\u2019.\u2014Elizabeth BlackwellSadly, despite this documented equality in operative results, the academic career ladder still seems steeper for female surgeons: women with an MD are less likely to author a paper than men with the same degree and instead, a PhD becomes much more essential for women . They alThe Women in Cardio-Thoracic Surgery Committee currently consists of seven female surgeons from various career levels and countries: Prof. Jolanda Kluin (The Netherlands); Dr Indu Deglurkar (UK); Dr Lena Emrich (Germany); Dr Francesca D\u2019Auria ; Dr Julie Cleuziou (Germany); Dr Miia Lehtinen (Finland); and Dr Maroua Eid (France). This heterogeneity seems essential, allowing all female surgeons to relate to the committee.female surgeons [The committee holds an important mission: establishing a network for surgeons . Such coet al. \u2018attention should be given to the potential unconscious bias in leadership in cardiothoracic surgery\u2019 [Gender should not intervene in one\u2019s career choices; only abilities and perseverance should be considered. A shift towards an increased representation of female surgeons is to be expected in our field, and as suggested by Shemanski surgery\u2019 . For our"} +{"text": "Artificial intelligence (AI) has emerged as a key player in modern healthcare, especially in the pharmaceutical industry for the development of new drugs and vaccine candidates. AI technology has shown promising results in target identification, protein\u2013protein interactions, protein\u2013drug interactions, metabolic pathway identification, drug repurposing, facilitating de novo drug design to combat diseases, and increasing short-term research productivity. This Special Issue highlights recent advances in computational modeling and dynamics simulations for elucidating phosphodiesterase 9 (PDE9) inhibitors with different metal systems; the identification of potential inhibitors of coronavirus disease 2019 (COVID-19); the prediction of convolutional network-based drug\u2013target interaction modules; the building of transformer-based deep neural networks for metabolomics; and the development of autoencoder-based deep learning models for drug repurposing for Alzheimer\u2019s disease. Signal transduction systems such as cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) regulate cellular functions in organisms, and the dysregulation of these signaling pathways leads to various diseases. To minimize the dysregulation of signaling pathways and the disruption of cellular function, Sivakumar et al. targetedThe repurposing of \u2018existing\u2019 drugs to treat common and emerging diseases increasingly involves the targeting of lower-risk compounds and results in reduced development costs and times. The COVID-19 pandemic has disrupted healthcare systems globally since 2019, calling for new therapeutics that target severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is responsible for the disease. Antiviral treatments have been known to reduce disease spread, infection, and severity for over 30 years. Mohammad et al. [The prediction and identification of compound\u2013protein interactions (CPIs) play an important role in the recent discovery and development of safe drug candidates. Many studies point to the advantages of deep learning models for learning task-related functions from large datasets on compound\u2013protein interactions. Wang et al. developeIn the past decades, high-throughput screening (HTS) experiments have greatly accelerated the identification of drug\u2013target interactions (DTIs). However, HTS experiments are costly and cumbersome, and cannot meet the need to reveal DTIs for millions of existing drug and target combinations. Jin et al. developeMass spectrometry (MS) fingerprinting is key to metabolomics . The identification of metabolite molecules is currently performed primarily by comparing MS data with those of a limited number of authentic chemical standard libraries. Only 10% of molecules can typically be identified experimentally from reproducible spectral features of complex matrices , despite many heuristics . To address the above issues, Shrivastava et al. developeFinally, Chyr et al. proposedBiomolecules for the opportunity to work with them on this enlightening editorial journey.We hope that the interdisciplinary topics covered in this collection will prompt further discussion among researchers and promote innovation in the field of artificial intelligence for drug design and development. Above all, we would like to thank the authors for their excellent contributions. We also thank the scientific experts in AI, computational modeling, and drug design who offered invaluable comments and suggestions to improve the quality of this Special Issue. Finally, we would like to thank the Editor-in-Chief, Section Managing Editor, and all the editorial staff of MDPI\u2014"} +{"text": "Matson et al., RSC Adv., 2021, 11, 32269\u201332274. DOI: 10.1039/D1RA06151HCorrection for \u2018X-ray absorption spectroscopy of exemplary platinum porphyrin and corrole derivatives: metal- The authors regret that the one of the author affiliations was incorrectly shown in the original manuscript. The corrected list of affiliations is as shown above.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "The COVID-19 pandemic dramatically changed the nature of social interaction, creating negative impacts and challenges, but also opportunities for progressing how we communicate, as humans. Social distancing policies, lockdown measures, and mandatory quarantines accelerated technological communication. For example, Artificial Intelligence (AI) mediated communication grew at an unprecedented rate, willingly or otherwise. As part of pandemic control efforts, many activities, such as workplace meetings, education, and conferences, moved online, using social media platforms, the metaverse, and specialized programs accessed through mobile devices or laptops. As a result, digitally mediated channels became critical for information acquisition and communication across a wide spectrum of human activities in our personal and professional lives. As the scientific understanding of COVID-19 improved, pandemic restrictions loosened. However, it remains to be seen whether the pandemic communication paradigm, characterized by heavy technological mediation and reliance on non-human agents, will gradually decline, or whether the paradigm shift will become deeply entrenched with further acceleration of the dependency on technological mediation and non-human agents.Such unprecedented reliance on technological mediation and non-human agents for information and communication is akin to a social-psychological experiment on a global scale. Much remains unknown, however, since this communication paradigm shift began almost 3 years ago. There are two main problems that require attention. First, the psychological impact of and underlying mechanisms behind the extended and extensive reliance on technological mediation and non-human agents is not yet well understood, especially regarding the influence of AI technologies. Second, the extent to which the impact of this reliance is likely to persist and influence future communication is also not well understood, especially in different national and cultural contexts. Research involving new approaches to data collection, such as big data techniques, together with more traditional data techniques, can help in providing greater insights into these two problems.In this Research Topic, we sought to extend current understanding about how the COVID-19 pandemic has reshaped human communication, especially cognitive, psychological, and behavioral shifts in social interactions. We focused on research that explored new possibilities for interpersonal communication practices in the post-pandemic era. Eighteen manuscripts were selected for inclusion and draw intensive attention with more than 26,000 views.Information technologies, such as social media platforms, offer individuals various functionality to support the maintenance, development, and sustainability of individuals and organizations. For instance, when COVID-19 emerged, the provision of resources and social support became a major concern for many.The Importance of Project Description to Charitable Crowdfunding Success: The Mediating Role of Forwarding Times,\u201d Lu et al. considered 205 COVID-19 charitable projects and 11,177,249 donors to assess the process by which nonprofit organizations raise funds through information descriptions about project descriptions. The results of the study indicated that understandability and a negative tone for descriptions help to improve the amounts raised. A question remains: might quarantines and economic disadvantages exacerbate social anxiety among impoverished individuals?In the article titled \u201cSocial Media as Online Shelter: Psychological Relief in COVID-19 Pandemic Diaries,\u201d Feng et al. explored how individual narratives on social media affect people's psychological health during a state of emergency using data collected from 19 interviews with Chinese diary writers. The study found that a pandemic diary could promote self-relief, public communication, emotional drive, meaning connection, and identity construction in public spaces, thus helping shape a sense of unity and belonging and facilitating the psychological reconstruction of people who are vulnerable to potential mental health crises.In the article titled \u201cOnline Collaborative Documents as Media Logic: The Mediatization of Risk Response in the Post-pandemic Era,\u201d Jiang et al. used a mixed-method design approach to examine treating online collaborative documents as a special approach to risk response in public health and natural disaster situations. The study found that the editability, accessibility, activability, and normality of technological affordances connected the functional features of a digital platform with users' potential actions.In the article titled \u201cIn this Research Topic, the authors also explored the drawbacks of information technology adoption. As the COVID-19 pandemic progressed, human reliance on information technology increased. For example, social media and the metaverse are now routinely being used for entertainment, learning, daily communication, and work.Personal network protects, social media harms: Evidence from two surveys during the COVID-19 pandemic,\u201d Ren and Yan demonstrated the increasingly critical and multifaceted role of communication technologies in affecting mental health conditions, indicating the destructive outcomes of the overuse of social media.In the article titled \u201cHow do Internet moms raise children? The reshaping of Chinese urban women's parenting psychology by COVID-19 online practices,\u201d Zhao and Ju, focusing on a special group of internet mothers, examined how they raised their children using the internet during the pandemic. The study found that social media created a new platform for information empowerment, mutual action, and ideation of motherhood for urban women formerly bound to family and parenting matters.In the article titled \u201cMetaverse as a possible tool for reshaping schema modes in treating personality disorders,\u201d Yin et al. showed the potential role of the metaverse and virtual worlds in reshaping schema modes for treating personality disorders by reconstructing a new object world for a patient with the prescription of a psychotherapist.In the article titled \u201cPsychological distance and user engagement in online exhibitions: Visualization of moir\u00e9 patterns based on electroencephalography signals,\u201d Li J. et al. followed an Electroencephalography (EEG) signaling approach to highlight the possibility of EEG-visualization media devices in reducing the psychological distance and promotion of interpersonal communication between two participants experiencing an online exhibition.In the article titled \u201cSocial media also provides new opportunities for many as we enter a post-pandemic era, including the public, institutions, media, and governments.The application of network agenda setting model during the COVID-19 pandemic based on latent Dirichlet allocation topic modeling\u201d and \u201cEvent history analysis of the duration of online public opinions regarding major health emergencies,\u201d Liu K. et al. and Liu X. et al. explored the public opinion landscape of the pandemic, as well as the dynamics of public opinion evolution.In the articles titled \u201cSocial media interactions between government and the public: A Chinese case study of government WeChat official accounts on information related to COVID-19\u201d and \u201cGovernment crisis communication innovation and its psychological intervention coupling: Based on an analysis of China's provincial COVID-19 outbreak updates,\u201d Shao et al. and Zhou et al. drew on the perspective of government-public relationships to focus on issues pertaining to government-public interactions and government crisis communication in an attempt to provide practical implications for crisis communication systems and public administrations during a public health crisis.In the articles titled \u201cRelationship Between Hardiness and Social Anxiety in Chinese Impoverished College Students During the COVID-19 Pandemic: Moderation by Perceived Social Support and Gender,\u201d Cheng et al. studied 673 impoverished Chinese college students and found that perceived social support moderated the effect of hardiness on social anxiety.In the article titled \u201cSelf-Efficacy, Proxy Efficacy, Media Literacy, and Official Media Use in COVID-19 Pandemic in China: A Moderated Mediation Model,\u201d Li Q. et al. explored the determinants of self-efficacy for fighting against the COVID-19 pandemic under social cognitive theory. The authors found that official media use was a negative moderator of the association between media literacy and proxy efficacy.In the article titled \u201cUnpacking the effects of personality traits on algorithmic awareness: The mediating role of previous knowledge and moderating role of internet use,\u201d Fang and Jin revealed that previous knowledge of algorithms and internet use are mediators and moderators between personality and algorithm awareness.In the article titled \u201cThe mediating role of personal values between COVID-19-related posttraumatic growth and life satisfaction among Chinese college students: A two-wave longitudinal study,\u201d Xie et al. established that COVID-19-related posttraumatic growth is positively associated with life satisfaction, while self-transcendence and self-enhancement values partially mediate this association.In the article titled \u201cPersonal space increases during the COVID-19 pandemic in response to real and virtual humans,\u201d Holt et al. suggested that personal space boundaries were expanded during the pandemic. The authors provided an index of recovery from the psychological effects of the crisis.In the article titled \u201cExploring factors that influence COVID-19 vaccination intention in China: Media use preference, knowledge level and risk perception,\u201d Chen et al. examined the role of media use preference, knowledge level, and risk perception in predicting people's intentions to take a COVID-19 vaccine in the Chinese context.In the article titled \u201cInfluence of personality traits on online self-disclosure: Considering perceived value and degree of authenticity separately as mediator and moderator,\u201d Lv et al. revealed the role of personality traits in online selfdisclosure, while separately assessing the perceived value and degree of authenticity as mediator and moderator.Finally, in the article titled \u201cIn summary, this Research Topic aimed to unite efforts to explore various aspects of communicative practices during and after a major crisis, although most of the studies were situated in the Chinese context. While we cannot say that this Research Topic provides a comprehensive knowledge map of post-pandemic communication practices, we hope that it will contribute to broadening the scope of conventional theoretical versions of information, media technology, and psychology.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication."} +{"text": "In the published article, there was an error. We mistakenly described the trend of changes in the levels of some metabolites after treatment of SpA patients.Results, \u201cDynamic alterations in the metabolic profile before and after treatment of spondyloarthritis,\u201d Paragraphs 2 and 4. These sentences previously stated:A correction has been made to \u201cKapoor et al. and BoguAnd\u201cIn addition, in the serum of JIA patients, MTX therapy increased the level of omega-3 unsaturated fatty acids (docosahexanoic acid and linoleic acid), which are anti-inflammatory mediators.\u201dThe corrected sentence appears below:\u201cKapoor et al. and BoguAnd\u201cIn addition, in the serum of JIA patients, MTX therapy reduced the level of omega-3 unsaturated fatty acids (docosahexanoic acid and linoleic acid), which are anti-inflammatory mediators.\u201dThe authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated."} +{"text": "Cells, several up-to-date reviews and original studies have provided new insights into the basic and translational aspects of AMPK signaling.5\u2032-adenosine monophosphate (AMP)-activated protein kinase (AMPK) is an enzyme regulating numerous cellular processes involved in cell survival as well as health- and lifespan. AMPK has emerged not only as a key cellular energy sensor but also as an important integrator of signals that manages cellular energy balance. Due to this property and being ubiquitously expressed in all mammalian cell types, AMPK has attracted interest in virtually all areas of biomedical research. In this Special Issue of Visnjic et al. considerIn the other review paper, Aslam and Ladilov describeObesity has been categorized by the American Medical Association as a chronic progressive metabolic disorder and is a growing public health concern worldwide. In the paper by van der Vaart et al. , an impoFinally, an original study by Li et al. providedIn a sophisticated study, Olivier et al. investigNon-alcoholic fatty liver disease (NAFLD) is emerging as a leading cause of chronic liver illness affecting > 30% of the European population, and even higher rates can be seen in the US population ,12. The Both transcriptional and genetic alterations in AMPK pathways may impact tumor microenvironments in a tissue-dependent manner either positively or negatively . This asLastly, Bhutta et al. reviewed"} +{"text": "Many researchers rely on animal studies to elucidate the mechanisms underlying diverse disease processes and to test the safety of emerging medical interventions. Animal research serves as a bridge between in vitro experiments and human trials in the preclinical stage. The majority of animal research employs almost genetically identical laboratory-bred mice or rats. Testing genetically similar individuals in a laboratory environment provides a level of control not available in clinical trials and is crucial for all phases of biomedical research. Numerous animal models have been developed to imitate pathophysiological abnormalities observed in humans. For an animal model of a human disease to be optimal, it must have similar pathophysiology, phenotypic, and histopathological traits; predictive biomarkers for disease progression or prognosis; therapeutic responsiveness; and levels of pharmacological safety or toxicity responses ,2. Thus,Various animal models have been critical in basic scientific and preclinical research on human neurological disorders, including traumatic or non-traumatic, neuroinflammatory, neuropsychological, and neurodegenerative diseases. The animal models are designed to mimic several facets of the disorders, including their genetic basis, neuropathological lesions, and clinical symptoms . These aHence, the Special Issue \u201cModeling Neurological Disorders in Experimental Animals: New Insights and Emerging Roles \u201d aimed tThe research articles in this Special Issue have mostly focused on the alterations of behavioral and histopathological characteristics and signal transduction, as well as the testing of potential therapeutic compounds in genetically manipulated and/or exogenous factor-induced animal models for neurological disorders. To begin, the articles by Ang et al., entitled \u201cTranscriptome Profiling Reveals Novel Candidate Genes Related to Hippocampal Dysfunction in SREBP-1c Knockout Mice\u201d and \u201cSREThis Special Issue contains two additional research articles on animal models of schizophrenia. Chang et al. reported in their article \u201cInteraction of Prenatal and Postnatal Risk Factors in the Behavioral and Histological Features of a \u201cTwo-Hit\u201d Non-Genetic Mouse Model of Schizophrenia\u201d that disAnother article in this Special Issue demonstrated a non-genetic and exogenous factor-induced animal model of neurological disorders. Although exposure to radiofrequency electromagnetic fields (RF-EMFs) has increased in the pediatric population, data on the consequences of the exposure to the central nervous system to RF-EMFs in them are scarce. The article by Kim et al. entitled \u201cExposure to RF-EMF Alters Postsynaptic Structure and Hinders Neurite Outgrowth in Developing Hippocampal Neurons of Early Postnatal Mice\u201d reveled This Special Issue featured a research article testing a prospective therapeutic drug in a genetically manipulated mouse model of neurological disorder. Ding et al., in their article \u201cCarbamazepine Restores Neuronal Signaling, Protein Synthesis, and Cognitive Function in a Mouse Model of Fragile X Syndrome\u201d , uncoverAdditionally, in a research paper on the hormonal effect on maternal behavior, the article by Leko et al. entitled \u201cTranscriptome Sequencing in the Preoptic Region of Rat Dams Reveals a Role of Androgen Receptor in the Control of Maternal Behavior\u201d revealedsgsh Mutant Zebrafish Recapitulates Molecular and Behavioural Pathobiology of Sanfilippo Syndrome A/MPS IIIA\u201d by Douek et al. [sgsh (Deltaex5-6) zebrafish mutant could be a valuable resource for gaining a better understanding of MPS IIIA pathobiology and developing timely and effective therapeutic interventions.Apart from the rodent models mentioned above, this Special Issue included an article describing the development of a zebrafish model that mimicked a human disease. Mucopolysaccharidosis IIIA , a pediatric neurological lysosomal storage disease, is caused by a deficiency of the enzyme N-sulfoglucosamine sulfohydrolase (sgsh), resulting in an impairment in heparan sulfate-glycosaminoglycan catabolism and its accumulation in tissues. The article \u201cAn Engineered k et al. suggesteThe review articles presented in this Special Issue demonstrated the ingenuity of researchers in addressing the critical task of modeling human neurological disorders in animals in meaningful ways. The articles mainly reviewed various animal models for neurological disorders, including amyotrophic lateral sclerosis (ALS), Alzheimer\u2019s disease (AD), epilepsy, Parkinson disease (PD), peripheral nerve injury, and post-traumatic stress disorder (PTSD). These animal models will be useful to gain better understanding of the mechanisms leading to signs of neurological disorders, validating drug targets, and providing assurance that therapeutic approaches will ultimately benefit patients. ALS is a fatal, multigenic, multifactorial, and non-cell autonomous neurodegenerative disease characterized by upper and lower motor neuronal loss. Bonifacino et al. reviewedAD is the most common cause of dementia, and its pathogenesis is complex, including A\u03b2 deposits, tau aggregates, excitotoxicity, and neuroinflammation. Robert et al. reviewedPD is the second most prevalent neurodegenerative disorder after AD. No cure for PD has been discovered, and treatment strategies are aimed at relieving symptoms through the restoration of dopaminergic functions. Liu and Cheung reviewedNeurons are structurally distinct and have injury-prone dendrites and axons. Peripheral nerves that have been injured, but not central nerves, have the ability to recover and reinnervate their target organs. Numerous animal models have been used to elucidate the mechanisms of axon regeneration. Lee and Cho reviewedKetamine has evolved into a versatile drug, which is used for a number of indications ranging from trauma analgesia to depression and PTSD therapy. Due to the inherent ethical problems associated with performing prospective clinical studies on traumatic events and stress-related diseases in humans, researchers frequently use preclinical rat models to investigate the effects of ketamine on fear memory and PTSD-like behaviors. A review by Choi et al. summarizConsequently, this Special Issue provides an update on current knowledge of the importance of animal models in mechanistic and therapeutic research on neurological disorders. I hope that readers will gain an understanding of current research trends and insights on future research directions through this Special Issue. However, I recognize that this Special Issue is insufficient to address the diverse animal models for a variety of neurologic disorders and that the articles included in this issue cannot possibly cover all on this topic. Thus, to complement any latest research data or research trends, I have re-launched the Special Issue \u201cModeling Neurologic Disorders in Experimental Animals: New Insights and Emerging Roles 2.0 \u201d to soli"} +{"text": "Pseudomonas aeruginosa via LasIR/RhlIR circuitry\u2019 by Mayank D. Shah et al., RSC Adv., 2019, 9, 40228\u201340239.Correction for \u2018Potassium 2-methoxy-4-vinylphenolate: a novel hit exhibiting quorum-sensing inhibition in The authors would like to add affiliation d as the current affiliation for Prashant S. Kharkar. The corrected list of affiliations is as shown above.The authors regret that one of the affiliations (affiliation The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "Countrywide multi-serotype outbreak of Salmonella Bovismorbificans ST142 and monophasic Salmonella Typhimurium ST34 associated with dried pork sausages in France, September 2020 to January 2021\u2019 by Pardos de la Gandara et al., upon publication on 12 January 2023, the title was missing the year of 2020 after September. This was corrected on 13 January 2023 at the request of the authors.In the article \u2018"} +{"text": "Nonalcoholic fatty liver disease (NAFLD), which is characterized by excessive triglyceride (TG) accumulation in hepatocytes, have become a serious health problem worldwide . Due to Decades of studies have demonstrated that cytokines derived from metabolic organs, including liver, adipose tissues and skeletal muscles, play crucial role in the regulation of hepatic and systemic lipid homeostasis . These oGuo et\u00a0al., identified dysregulated myokines in the development of NAFLD and NASH through comprehensive transcriptome profiling. Mao et\u00a0al., identified adipokines and hepatokines associated with high-salt-diet in mice. Wang et\u00a0al., analyzed the relationship between circulating Ism1 and diabetes and diabetes-associated NAFLD. Gao et\u00a0al., provided a novel view on endoplasmic reticulum-related and secretome gene in NAFLD progression.Therefore, at this stage, we set up a Research Topic entitled \u201cThe Roles and Mechanisms of Hepatokines, Adipokines and Myokines in the Development of Non-Alcoholic Fatty Liver Disease (NAFLD)\u201d in the Frontiers in Endocrinology. Through this Research Topic, we aimed to further establish the roles of hepatokines, adipokines and myokines in NAFLD and NASH. In this collection, Overall, these studies together identified some new cytokines associated with NAFLD pathogenesis, which further strengthened our understanding of metabolic liver disease. However, intensive work is still required, such as investigations into the roles and mechanisms of these cytokines through genetic models, translational studies in human subjects, and screening potential therapeutic target for treatment. We hope that more and more studies on this Research Topic would help us better understand the molecular mechanisms of NAFLD development and identify more therapeutic targets for its treatment.The author confirms being the sole contributor of this work and has approved it for publication."} +{"text": "Berrones-Reyes et al., RSC Adv., 2019, 9, 30778\u201330789.Correction for \u2018Quantum chemical elucidation of the turn-on luminescence mechanism in two new Schiff bases as selective chemosensors of Zn The correct email address is The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "As a result of their rapid development, polymer composites are seeing wider use in transportation infrastructure in China and worldwide. Styrene\u2013butadiene\u2013styrene (SBS), epoxy resin (ER), polyethylene (PE), expanded polyethylene (EP), polyurethane (PU), crumb rubber and other polymer materials can be used to modify asphalt and improve the performance of asphalt mix. Meanwhile, the application of polymer admixtures such as PE fiber, polyvinyl alcohol (PVA) fiber, polypropylene (PP) fiber, etc., in bituminous, cementitious or soil materials also improves their strength and durability. Polymer materials are also widely used in pavement maintenance and treatment; they can quickly restore road function and extend the road\u2019s service life.This Special Issue contains sixteen research papers and one systematic review. The low-temperature properties of SBS-modified asphalt are not satisfactory; thus, Chen et al. analyzed the main factors for improving the low-temperature performance of SBS-modified asphalt base on the orthogonal tests . To solvSome research studies are devoted directly to the polymer materials applied in bituminous, cementitious or soil materials. Ren et al. studied the fatigue life and the material design of polymer concrete including ER and PU . Ji et aThe Special Issue concludes with a review on fiber-reinforced geopolymers and highlights the difficult aspects of current research, in addition to assessing the literature records ."} +{"text": "Humulus lupulus L.) as a potent monoacylglycerol lipase inhibitor for treatments of neuroinflammation and Alzheimer\u2019s disease\u2019 by Min-Che Tung et al., RSC Adv., 2021, 11, 31062\u201331072, https://doi.org/10.1039/D1RA05311F.Correction for \u2018Discovery of 8-prenylnaringenin from hop ( The authors regret that the name of one of the authors (Hsing-Mien Hsu) was shown incorrectly in the original article. The corrected author list is as shown above.The authors also regret an incorrect version of The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "The brain weighs approximately 2% of the body, but utilizes about 20% of the total energy and oxygen supply, thus energy supply is critically important for the brain. In addition, the brain has no energy reserve, except some glycogens in the astrocytes, and the neurons can only use ATP, which comes from glucose degradation in the essential structure mitochondria. Mitochondria impairment, such as electron transport chain damage, can induce decreased ATP levels, decreased antioxidant defense, which is prevalent in many neurodegenerative disorders. For example, ATP dysfunction has been suggested to be the major impaired in depression . NumerouIt is critically important to understand the central facets of physiology and pathophysiology of energy supply in the brain, however, the energy supply regulation in the brain is not clear, for example how emotion affects energy supply, and does it activated by monoamine neurotransmitters. Emotion plays an important role in the regulation of the energy supply for the brain. Emotion parallels with behavior all the time, from the initiation of a behavior, the process of behavior to the end of the behavior. Emotion is the tendency for behavior, is also the motivation of a behavior. Emotion is also important for appraisal of behavioral consequences, marking the behavior as reward or punishment . Emotionvia autonomous nervous system, but also affect the energy supply in the brain. For example, the monoamine transmitters stimulate Na+, K+-ATPase activity in astrocytes and facilitate their role in ion concentrations , and suggested the berberine might be used as encouraging antidepressants to modulate depressed emotion.In another paper titled \u201cCurrent evidence and future directions of berberine intervention in depression\u201d, via TRPV1\u201d, the authors Meng et al. studied the effects of berberine on transient receptor potential vanilloid (TRPV1) in dorsal root ganglia inflammation via inhibiting NF-\u03baB and activating the JNK/p38 MAPK pathways in early injury, which inhibited the overexpression of TRPV1. The study suggested that berberine can reverse neuropathic pain response via inhibiting TRPV1 expression.In the experimental study about berberine titled \u201cBerberine Alleviate Cisplatin-induced Peripheral Neuropathy by Modulating Inflammation Signal Li et al. introduced another kind of natural product, echinacoside, which is a kind of phenylethanoid glycoside (PhG) in Cistanche tubulosa. In the paper \u201cTherapeutic potential and molecular mechanisms of Echinacoside in neurodegenerative diseases\u201d, they reviewed many recent studies about its mechanisms in neuroprotective efficacy in the prevention and treatment of neurodegenerative diseases, and proposed that the major effect of echinacoside is improving mitochondrial function and reducing anti-oxidative stress.In another review, the authors Tao et al. summarized recent in vitro and in vivo studies about Chinese herbs in treating dementia, and found that the extracts of these Chinese herbs work on reducing generation of reactive oxygen species and inhibiting inflammation and neurotoxicity.In the review paper titled \u201cProgress in the mechanism of autophagy and traditional Chinese medicine herb involved in dementia\u201d, Lyu et al. studied a kind of Chinese herb Yi-zhi-fang-dai formula, and its effects on pyroptosis and glymphatic dysfunctions. The study suggested that the Chinese herb Yi-zhi-fang-dai formula could inactivate pyroptosis via inhibiting caspase-1/11 activation and gasdermin D cleavage, and induce AQP-4 depolarization thus increase glymphatic function to reduce neuronal damage.In the experimental paper titled \u201cYi-zhi-fang-dai formula exerts neuroprotective effects against pyroptosis and blood-brain barrier-glymphatic dysfunctions to prevent amyloid-beta ccute accumulation after cerebral ischemia and reperfusion in rats\u201d, the authors Yin et al. studied the antioxidant activity of natural product rhein, and also its effects in clearance of \u03b2-amyloid (A\u03b2) in Alzheimer\u2019s disease. The study found that rhein could significantly reduce reactive oxygen species, and activate mitochondrial biogenesis by increased cytochrome C oxidase and superoxide dismutase activities.In the experimental paper titled \u201cRhein relieves oxidative stress in an A\u03b21-42 oligomer-burdened neuron model by activating the SIRT1/PGC-1\u03b1-regulated mitochondrial biogenesis\u201d, the authors Liu et al. introduced a study about a kind of Chinese herb named danzhi xiaoyao powder and its effects on the symptoms of generalized anxiety disorder. The results suggested that the power could improve the weight growth and improve appetite and reduce the anxious mood via notch signaling pathway in the hippocampus. The study suggested that danzhi xiaoyao powder is a good therapy method for mood disorders.In the experimental paper \u201cNeuronal Regeneration by Downregulating Notch Signaling Pathway in the Treatment of Generalized Anxiety Disorder\u201d, the authors He et al. introduced a kind of classic Chinese herb formula, qiangji decoction in the paper \u201cQiangji Decoction alleviates neurodegenerative changes and hippocampal neuron apoptosis induced by d-galactose via regulating AMPK/SIRT1/NF-\u03baB signaling pathway\u201d. The herbs have been widely used in the traditional Chinese medicine, but the mechanisms are not clear. This paper found the major effect of qiangji decoction is reducing inflammation via reducing TNF-\u03b1, IL-1\u03b2 and IL-6 in the hippocampus via regulating AMPK/SIRT1/NF-\u03baB signaling pathway in hippocampal neuron apoptosis.In the experimental study, Zheng et al. introduced a kind of Chinese herb ginkgo biloba extract and donepezil on Alzheimer\u2019s disease. In the paper titled \u201cEffects of ginkgo biloba extract and donepezil on functional recovery in Alzheimer\u2019s disease: a multi-level characterized study based on clinical features and resting-state functional magnetic resonance imaging\u201d, they used neuroimage study and found that the herbs could change the ALFF values in right gyrus rectus and decreased PerAF values in left fusiform gyrus. And the authors concluded that the imaging metrics in specific brain regions may serve as biomarkers for therapeutic efficacy of medicines.In the experimental study, Zheng et al. introduced a study on the mechanism of sytisine on temporal lobe epilepsy, in the paper titiled \u201cCytisine exerts an anti-epileptic effect via \u03b17nAChRs in a rat model of temporal lobe epilepsy\u201d. Cytisine is an agonist of \u03b17 nicotinic acetylcholine receptors (\u03b17nAChRs) and has shown neuroprotection in many neurological diseases. This study found that sytisine could increase hippocampal function via enhancing ACh levels and \u03b17nAChR expression, and decrease glutamate level to reduce seizures. The authors Arrodi et al. introduced one paper titled \u201cModulatory effects of alpha- and gamma-tocopherol on the mitochondrial respiratory capacity and membrane potential in Alzheimer\u2019s disease an in vitro model of Alzheimer\u2019s disease\u201d, which suggested that mitochondrial abnormalities are an early feature in the pathogenesis of AD. They found that alpha-tocopherol or gamma-tocopherol could modulate mitochondrial function by increasing the production of ATP and reducing mitochondrial reactive oxygen species via altering mitochondrial metabolic pathways such as oxidative phosphorylation.The authors in vitro: implication for a potential therapy of Alzheimer\u2019s disease\u201d, Cheng et al. investigated the role of mitochondrial dysfunction in the pathogenesis of Alzheimer\u2019s disease. In addition, they investigated the effects of tortoise plastron gelatin and dear antler gelatin in preventing neuronal mitochondria function. The found that these two natural products could increase cell viability by modulating intracellular ATP and calcium level, and also regulate mitochondrial membrane potential (MMP) and ultrastructure, and finally inducing anti-dementia effects.In the paper titled \u201cTortoise plastron and dear antler gelatin prevents against neuronal mitochondrial dysfunction In all, these studies demonstrate that astrocytic function and neuronal mitochondria function play important roles in many neurodegenerative disorders. In addition, this topic introduced many natural drugs in treating these diseases. We expected that this Research Topic will stimulate interest in the study of the mechanisms of modulating the energy supply."} +{"text": "Current studies in the domain of cognitive neuroscience are devised to ensure a high signal-to-noise ratio. Measures from numerous participants are averaged to allow for commonalities to emerge, washing out possible differences. Albeit generally a good thing, a \u201cone-size-fits-all\u201d approach has a drawback: it fails to take inter-individual variability into account, a limitation that is no longer tenable given the increasing attention to so-called precision medicine , to relatively innocuous, though possibly intrusive, routines (as in superstitions), to frankly pathological states (as in compulsions). The common thread is the reliance on the mechanisms supporting habit formation. Neurofunctional models of OCD describe an imbalance between the goal-directed and the habit system of action control, which would lead to over-reliance on the latter (Gillan and Robbins, disturbance in the way in which one's body weight or shape is experienced,\u201d the other criteria being significantly low weight and intense and persistent fear of becoming fat. In AN body size is generally overestimated (Schneider et al., Perceiving our body in space is instrumental to all approach/avoidance interactions with the environment (Sirigu et al., Overestimation of bodily space is not exclusive to AN. Young and perfectly healthy individuals misjudge their body size, particularly along the width dimension (Casper et al., So far, we provided two among many possible examples (e.g., agoraphobia, Indovina et al., For example, detecting and learning associations, a relevant step in habit formation, varies considerably based on personality traits and cognitive style (Kaufman et al., In sum, susceptibility factors for pathology may be nested within cognitive variability and\u2014though still poorly explored\u2014should be considered alongside psychosocial and biological markers (Jacobi et al., variability into the newly developing models of disease.Recent neuropsychological approaches to psychiatry have underlined the multifactorial origin of mental illnesses, drawing attention to cognitive variables (Wood et al., The study of cognitive variability is notoriously laden by methodological and reliability issues (Hedge et al., and applying the visuospatial skills required to view oneself \u201cfrom the outside\u201d (deVignemont, and constitute a vulnerability trait for disease. Detecting this trait within the healthy population (whereby variability exists; Samuel et al., Consider the example of body-size delusions. Describing one's body implies reporting beliefs and/or attitudes related to it Thus, while not implying a causal link between one cognitive style and development of a psychiatric condition, investigating cognitive variability could prove fruitful in characterizing disease and informing on the most probable direction malfunctioning could take, should other factors co-occur.ED drafted the paper. All authors discussed the ideas presented in the paper. All authors reviewed and approved the submitted version."} +{"text": "Xu et al.) respectively.Our interest in Non-coding RNA and Wnt/\u03b2-catenin Signaling Pathway in Human Cancer were initiated from our earlier studies: one was that the long non-coding RNA (LncRNA-NEF) antagonized epithelial to mesenchymal transition and cancer metastasis via cis-regulating FOXA2 and inactivating Wnt/\u03b2-catenin signaling and anotOut of the many manuscripts that we have received, seven were published in this research topic, including 2 reviews and 5 original research articles.Mu et al. summarized recent studies on the function and mechanisms of tumor resistance to cisplatin mediated by circular RNAs (circRNAs). The authors described various types of mechanisms in detail and the role of circRNAs in regulation of tumor proliferation, invasion, chemosensitivity, and other biological behaviors in the tumor microenvironment (TME). The authors emphasized that circRNA can be used as a promising target gene to reverse drug resistance and improve therapeutic efficacy.One review contributed by Li et al. summarized studies on the regulatory mechanisms of lncRNAs and their target gene signaling pathways in laryngeal squamous cell carcinoma, which is the second most frequent tumor of the respiratory system. This has significance in that by summarizing ncRNAs biological functions and important regulatory mechanisms in laryngeal squamous cell carcinoma the authors provide ideas for the improvement of diagnosis, prognostic evaluation, and development of pre-clinical targeted drugs.A second review contributed by Zhu et al. reported that the lncRNA LINC-PINT suppresses cell proliferation, invasion and Epithelial\u2013Mesenchymal Transition (EMT) by blocking Wnt/\u03b2-catenin signaling in glioblastoma. They started with bioinformatics prediction of the role of LINC-PINT, followed by in vitro RT-PCR, clonal assays, and wound healing experiments to study the mechanism, and then they used in vivo tumor grating experiments to confirm the role of this lncRNA.The article contributed by Liu et al. and colleagues reported that microRNA (miRNA)-142-3p inhibits tumorigenesis of colorectal cancer (CRC) via suppressing the activation of Wnt signaling by directly targeting \u03b2-catenin. They used clinical samples and compared miRNA expression profiles between healthy donors and CRC patients. Colony formation and MTT assays were used to test cell proliferation. Luciferase assay, immunohistochemistry, and Western blotting were employed to explore the molecular mechanisms.Chen et al. reported that the lncRNA SNHG1 regulates the proliferation, apoptosis and autophagy of prostate cancer cells (PCa) via the Wnt/\u03b2-catenin and PI3K/AKT/mTOR signaling pathways. The PCa cells were transfected with a small interfering RNA plasmid (si-SNHG1) and si-SNHG1+multicellular protein EZH2 small interfering RNA plasmid (si-EZH2) to study the molecular mechanisms. Another article related to PCa was contributed by Jia et al. who reported that a traditional Chinese medicine Astragalus polysaccharides (APS) inhibits tumorigenesis and lipid metabolism through the miR-138-5p/SIRT1/SREBP1 pathway in prostate cancer. The approach used was microarray studies upon drug (APS) exposure, and they have successfully shown that ecoptic expression of SIRT1 inhibits the expression and nuclear translocation of SREBP1 via activating AMPK phosphorylation.The article contributed by Shao et al. studied the anti-tumor mechanisms of curcumin in hepatocellular carcinoma (HCC). The authors reported a novel role for curcumin in inducing cell cycle arrest and apoptosis by downregulating the lncRNA LincROR, in turn reducing \u03b2-catenin and inactivating Wnt signaling. They approached the study by choosing lncRNAs that were previously reported to be related to tumorigenesis, and LincROR was the most down-regulated in the curcumin-treated HCC cells by examination of their expression levels.Altogether, this research topic on non-coding RNA and Wnt/\u03b2-catenin Signaling Pathway in Human Cancer provided new ideas and mechanistic data on the role of several ncRNAs in cancers, shedding new light on the current state of the field and, more importantly, providing new avenues for future diagnostic and therapeutic avenues."} +{"text": "Chronic Inflammation and Neurodegeneration in Retinal Disease, Volume II\u201d presents eight original research articles and one mini review from seven different countries with important contributions in the field of retinal inflammation. Most of the contributions are related to diabetic retinopathy (DR), the others cover age-related macular degeneration (AMD), retinitis pigmentosa, and the spontaneous polygenic model of inherited retinal dystrophy.Inflammation and neurodegeneration have a widely recognized role in the pathogenesis of the main retinal conditions. However, the exact mechanism through which inflammation causes alteration of the retinal structure\u2014with consequent dysfunction of the retinal pigment epithelium, of neurons, and ultimately of photoreceptors\u2014is not entirely known. This Research Topic \u201cStravalaci et al. focused on long pentraxin 3 (PTX3), an emerging new player in ocular homeostasis and a potential pharmacological target in neurodegenerative disorders of the retina. Physiologically present in the human eye and induced in inflammatory conditions, this protein is strategically positioned at the blood retinal barrier interface, where it acts as a \u201cmolecular trap\u201d for complement and modulates inflammation both in homeostatic and pathological conditions such as AMD and DR. Gesualdo et al. presented an interesting study on fingolimod and DR, investigating the interactions between fingolimod, a sphingosine 1-phosphate receptor (S1PR) agonist, and melanocortin receptors 1 and 5 . This Research Topic is a typical example of repurposing since fingolimod is a drug approved to treat relapsing-remitting multiple sclerosis. The authors demonstrated, in an in vivo model of DR, that fingolimod has anti-angiogenic activity mediated not only through S1P1R, but also by melanocortin receptors. Another interesting pre-clinical study on DR was provided by Canovai et al. The authors showed the efficacy of a novel substance containing cyanidin-3-glucoside (C3G), verbascoside, and zinc to maintain the integrity of the blood retinal barrier and retinal function in streptozotocin-induced diabetic rats.The review by in vitro study by Schmalen et al. underlined the importance of M\u00fcller cell signaling in the inflamed retina, indicating an active role in chronic retinal inflammation. These authors demonstrated an intense signaling capacity of M\u00fcller cells, which reacted in a highly discriminating manner upon treatment with different cytokines, showing several characteristics of atypical antigen-presenting cells.Accumulating data provide evidence for a pivotal role of M\u00fcller cells in the pathogenesis of DR. In this regard the Parravano et al., where the authors carried out a randomized clinical trial on patients with diabetic macular edema (DME) treated with a special oral curcumin formulation with a polyvinylpyrrolidone-hydrophilic carrier and intravitreal injections of dexamethasone. They demonstrated a significant reduction in central retinal and inner retinal layer thickness with the combined therapy in patients affected by DME. This study also showed that the pharmacological combination therapy (curcumin and dexamethasone) was well-tolerated. On this regard, a good long-term safety profile of intravitreal dexamethasone has been demonstrated on real-world studies when used to manage DME -induced macular edema, steroid implant is a valid option for DME patients not responding to anti-VEGF therapy and non-DME patients with macular edema. The final contribution on the topic of DME focused on the anti-inflammatory effects of subthreshold micropulse yellow laser (SMYL). The authors, Bonfiglio et al., demonstrated that SMYL may reduce macular thickening and improve best-corrected visual acuity in eyes with persistent macular edema after pars plana vitrectomy and membrane peeling for tractional DME.An important clinical contribution on the Research Topic of DR has been provided by nage DME . Furthernage DME . AnotherHollingsworth et al.) have used systems genetics to identify possible models of spontaneous polygenic AMD by mining the BXD family of mice using single nucleotide polymorphism analyses of known genes associated with the human retinal disease. The goal of these scientists was to propose a pre-clinical mouse model (BXD32) to better understand the pathophysiology of progressive retinal dystrophies and discover efficacious treatments. Their study demonstrated that the BXD32 mouse strain exhibits a severe neurodegenerative phenotype accompanied by adverse effects on the retinal vasculature. Finally, Canto et al. showed that sulforaphane, a natural compound, modulates the inflammation and delays neurodegeneration in a retinitis pigmentosa (RP) mouse model. Specifically, they assessed the modulation of glial cells in the RP rd10 mouse model showing that sulforaphane treatment regulated the microglial activation state.A group of scientists from University of Tennessee (Overall, the contributions of the present Topic Research focused on inflammation and retinal degeneration, highlighting new insights in retinal diseases mechanisms and novel pharmacological approaches."} +{"text": "Inflammation is a well-known consequence of many traditional cancer treatments that can serve to enhance antitumor immunity and promote unwanted side effects. These side effects are often sustained long after cancer treatment has ended and may coincide with sustained chronic low-grade systemic inflammation that compromises the health of organs in the body and central nervous system (CNS). Assessment of circulating cytokines and their downstream products are considered to serve as potential biomarkers for systemic inflammation, identifying cancer survivors at risk of inflammation-associated disorders. In relation to the CNS, inflammatory markers are associated with cancer-related fatigue ; anxietyWe congratulate Bower et al. for condThe longitudinal nature of the study is a majBower et al.\u2019s findings\u039aB) transcription pathway. Studies in patients with schizophrenia who, apart from psychosis, share several anxiety, depression, and cognitive symptoms with cancer patients, have identified a subgroup of patients that exhibit elevated brain and blood cell NF-\u039aB\u2013mediated cytokines. These low-grade cytokine elevations are explained by dysregulation of the NF-\u039aB, including reduced expression of NF-\u039aB pathway inhibitors (Although CRP and cytokine levels in Bower et al.\u2019s study arhibitors . Althoughibitors compelliIn addition to providing a platform for new approaches and discoveries regarding the measurement of inflammatory biomarkers in cancer patients, Bower et al.\u2019s work claAnother direction for investigation is related to the evolving treatment landscape. For example, an increasing number of women are receiving T-DM-1. Given there are more prevalent and severe toxicities associated with T-DM1 compared with Trastuzumab , future As the RISE study focuses on fatigue and longitudinal design , we awaiNo funding was used for the writing of this editorial.Role of the funder: Not applicable.Disclosures: The authors have no disclosures to declare.Author contributions: AKW, RJC, JLV: writing\u2014original draft, writing\u2014review & editing."} +{"text": "Renal fibrosis is the progressive and complicated process manifested by histological aberrance and functional decline in the kidney. The pathogenesis involves multiple molecular pathways and cellular targets leading to myofibroblast activation and accumulation of extracellular matrix, which is in response to excessive epithelial injury and inflammation. Formation and exacerbation of fibrosis during the development of chronic kidney disease (CKD) is the common pathway to end-stage renal failure. However, few interventions are available that specifically target the pathogenesis of renal fibrosis. Emerging evidences prove that natural product therapy directly targets the pathogenesis of renal fibrosis, and exhibits beneficial effects in clinical. Further research (such as multi-omics studies and network pharmacology) is urgently needed to investigate the puzzle from the active compounds to underlying mechanisms and therapeutic targets of natural product against renal fibrosis.The Research Topic intends to highlight the latest advances from the active compounds to underlying mechanisms and therapeutic targets molecular mechanism of natural product against renal fibrosis. The issue includes 25 articles that is contributed by more than 200 authors in the fields of renal pharmacology. We have generated a collaborative discussion that facilitated the development of new mechanisms, new therapeutic targets, and candidate drugs from natural product against renal fibrosis.Zheng et al. illustrate that astragalus polysaccharide extract plays a beneficial role in reducing renal inflammation and fibrosis and in improving renal function by regulating the TGF-\u03b2/ILK pathway in hypertensive mice. Ren et al. confirm that natural flavonoid pectolinarigenin alleviates renal fibrosis to delay hyperuricemic nephropathy via suppressing TGF\u03b2/SMAD3 and JAK2/STAT3 signaling pathways. By targeting non-TGF-\u03b2 pathway, Yu et al. report the medical leech saliva extract, hirudin, protects the kidney from fibrotic injury by ameliorating renal autophagy impairment via PI3K/Akt pathway. Liu et al. demonstrate that quercetin alleviates podocytes apoptosis in vitro and in vivo by regulating the EGFR pathway, providing a novel approach to reveal the therapeutic mechanisms of quercetin against DN. Gong et al. identify proanthocyanidins (OPC) as active compounds of grape seeds. OPC exhibits beneficial protection against cadmium-induced DN in multidimensional aspects by the regulation of oxidative-antioxidative status, metal-binding ability, mediation of the levels of essential elements, and activation of the p38 MAPK and Keap1/Nrf2 signaling pathways.New mechanisms of renal fibrosis and the protective effects of natural product have been investigated and uncovered. Fibrosis-related signaling pathways are the common therapeutic targets, especially TGF-\u03b2 pathway. Ma et al. prove that farrerol reverses oxidative stress, inflammation, and fibrosis in renal tubular epithelial cells by activating Nrf2 and subsequently increasing PINK/Parkin-mediated mitophagy and eliminating damaged mitochondria. Xiang et al. show that the restoration of PPAR\u03b1 activity delays diabetic nephropathy progression and attenuates lipid metabolism disorders by downregulating miR-21 expression to improve mitochondrial function. Li et al. report that maintaining the balance of mitochondrial dynamics exhibits renoprotection in kidney by inhibiting mitochondrial fission and promoting mitochondrial fusion via the downregulation of the primary mediator proteins of mitochondrial fission (Drp1 and Fis1) and the upregulation of fusion proteins (Opa1 and Mfn1). Zhang et al. carries out the serum metabonomics analysis of patients with diabetic kidney disease and shows the potential role of specific gut microbiota in the progression of renal fibrosis and diabetic kidney disease that involves the dysfunction of phenylalanine and tryptophan metabolisms. This study suggests the kidney-gut axis functions as a potential therapeutic target of renal fibrosis.Additionally, metabolic regulation and kidney-gut axis function as a promising therapeutic target against renal fibrosis. Shao et al. summarized five Chinese herbal medicines with sufficient clinical efficacy, high frequency of use, and well-studied mechanism, including Abelmoschus manihot and Huangkui capsule, Salvia miltiorrhiza and its components ; Rhizoma coptidis and its monomer berberine; Tripterygium wilfordii and its components ; Kudzu root Pueraria and its monomer Puerarin. The researches of these five Chinese herbal medicines set the study pattern for natural product against renal fibrosis, which are the promising candidate for widespread and standardized application.Since Chinese herbal medicine exhibits beneficial effects against renal fibrosis, the lack of randomized controlled trial and clear mechanism hinder natural product to pass modern assessments. Here, Dong et al. designed a multicenter, nonrandomized, single-arm clinical trial to explore the clinical effects of MFSD on idiopathic membranous nephropathy (MN), presenting an inspiring result that MFSD has significantly beneficial effects on idiopathic MN treatment, and has the same beneficial effects on patients with MN who are newly treated and who accepted with immunosuppressive therapy without remission. Further study from the same group by Gao et al. investigates the main active compounds of MFSD by high-performance liquid chromatography-mass spectrometry (HPLC-MS) and more than 30 active compounds are identified. These active compounds alleviate podocyte injury by modulating autophagy-related protein and Wnt/\u03b2-catenin pathway, indicating autophagy and Wnt/\u03b2-catenin pathway as potential targets of MFSD for MN treatment. Zhang et al. demonstrate the anti-fibrotic and anti-inflammatory effects of Bupi Yishen Formula in vivo and in vitro by suppressing TLR4-mediated NF-\u03baB signaling.The study of Luo et al. reveals the anti-oxidant and anti-inflammatory ability of Shenkang injection (SKI) in kidney, and identifies chrysophanol, emodin, and rhein as active compounds against renal fibrosis via simultaneously targeting I\u03baB/NF-\u03baB and Keap1/Nrf2 signaling pathways. Jia et al. report TLYS decoction improves mitochondrial dynamics to attenuates renal fibrosis and renal function decline by suppressing oxidative stress and mitophagy. The comparative network pharmacology analysis is the alternative approach to explore the mechanisms of natural products with limited experiments. Chan et al. performed comparative network pharmacology to figure out the mechanism of Liu-wei-di-huang-wan. Liu-wei-di-huang-wan may ameliorate fibrosis, angiogenesis, inflammation, disease susceptibility, and oxidative stress via modulating TNF signaling pathway, which could be validated through clinical trials.Beyond to five Chinese herbal medicines, studies from the present issue provide the clinical evidences of natural products against renal fibrosis and explore underlying mechanisms. Mahuang Fuzi and Shenzhuo Decoction (MFSD), a Chinese herbal formula, is a promising candidate for renal fibrosis and CKD treatment in clinial. In conclusion, the collection of 25 articles in the Research Topic contributes to better understanding of natural products against renal fibrosis from the active compounds to underlying mechanisms and therapeutic targets. These studies provide promising and potential candidates against renal fibrosis by modulating novel pathways in clinical trial and animal experiment. Accompanying with the evolution of high throughput screening method and model, novel candidates and therapeutic targets will emerge to facilitate the drug discovery, which hold great potential for treatment with renal fibrosis."} +{"text": "The implementation of sustainable food packaging solutions within future circular food supply chains is essential to protect customers and ensure food quality, safety, and optimal shelf-life. This will be improved by new innovative packaging materials and will contribute to reducing food waste. In this direction, it is important to employ lifecycle assessment (LCA) to define food supply chain impacts, taking into consideration food waste, global food industry environmental impacts, and shipping distances, with the aim of achieving consumer satisfaction. It is important to share data on (i) the consequences of specific food product\u2013package interactions, (ii) the consideration of the utilization of novel packaging biomaterials, and (iii) overall consumer behavior and satisfaction as a critical focus. The aim of this Special Issue was to bring the most updated information in the new era of sustainability and food packaging.D\u00f6rnyei et al. proposedThe work of Wang et al. presenteThe study of Shin et al. showed tPleva et al. investigChen et al. developeSalmonella enterica via different contact materials (polypropylene from reusable plastic crates (RPCs), corrugated cardboard, and medium-density fiberboard (MDF) from wooden boxes). The survival of the pathogenic microorganism was studied in cauliflowers and the contact materials during storage. The LCA approach was used to evaluate the environmental impact of produce-handling containers fabricated from the different food-contact materials tested. The results showed a higher risk of cross-contamination via polypropylene compared with cardboard and MDF. Another outcome of the study was the potential of Salmonella surviving both in cross-contaminated produce and in contact materials under supply chain conditions. Regarding environmental sustainability, RPCs showed a lower environmental impact than single-use containers (cardboard and wooden boxes).L\u00f3pez-G\u00e1lvez et al. assessedCruz et al. presenteIn the article of Miller et al. various The review from Krauter et al. contextuBauer et al.\u2019s study aiIn another study, Bauer et al. presenteJunior et al. presente"} +{"text": "At present, cancers are described in both biological and clinical settings with static models that characterize tumors by the phenotypic and genotypic features observed at a given time point. However, cancers are highly dynamic processes that evolve based on specific genomic and epigenomic changes. Such dissonance between the model used to describe tumors and their true nature reverberates all the way from basic biological research to clinical practice. For instance, it is well established that anti-cancer treatments impose selective pressures leading to the emergence of resistant clones . This evHistorically, the first barrier to the characterization of the evolutionary properties of cancer was the limited amount of available data to accurately reflect tumor heterogeneity, which was traditionally based on a single tissue biopsy . Severala priori expectation when we proposed this research topic was to be confronted by a barrage of works focusing mainly on the genetic aspects of cancer evolution. Looking retrospectively at the collection of accepted manuscripts, however, we were surprised to observe some specific yet unexpected topics recurrently emerging.The articles in this research topic address important aspects of cancer evolution, encompassing models associated with a diversity of tumor types including renal cell carcinoma, glioblastoma, breast, colorectal, lung, ovarian, pancreatic and prostate cancer. Our Duic\u0103 et al.; Gao et al.; Qi et al.; Li et al.; Zheng et al.; Li et al.; Chen et al.; Zhao et al.). Duic\u0103 et al. presented a review of the role of miRNAs in cancer-relevant processes, focusing on gynecological malignancies . Another study evaluated exosomal miRNAs as a mechanism of resistance in small cell lung cancer with particular elevation of exosomal miR-92b-3p that was associated with the PTEN/AKT pathway based on preclinical models . Importantly, this study also included a clinical cohort of 50 patients to help validate the authors\u2019 hypothesis that downregulation of this biomarker was associated with better clinical outcomes. Junchen Li et al., instead, focused on the anti-cancer mechanism of andrographolide in patients with luminal-like breast cancer through the inhibition of miR-21-5p . Similarly, Chen et al. investigated the role of circular RNA (circRNAs) in prostate cancer. The authors focused on the regulation, expression, and functional effects of circNOLC1 for in vitro and in vivo models, proposing circNOLC1 as a potential biomarker and target for prostate cancer treatment.Multiple articles within this collection focused on the role of non-coding RNA in cancer, including in particular microRNA (miRNA), circular RNA (circRNA) and long non-coding RNA (lncRNA) . Qi et al. evaluated lncRNAs as potential biomarkers to assess heterogeneity in metastasis for colon and rectal cancers. The analysis identified two biomarker lncRNAs, KCNQ1OT1 and SNHG1, associating with cancer initiation and metastatic potential. Interestingly, the authors proposed different mechanisms of actions of these two lncRNAs in colon and rectal cancers . Finally, Zheng et al. identified a panel of prognosis-associated lncRNAs that were significantly associated with survival in ovarian cancer across multiple independent cohorts, including The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO). The authors further selected and validated the expression of four lncRNAs in vitro on multiple ovarian cancer cell lines .Multiple studies focused on lncRNAs as well. Zheng et al. published a review on the communication between EMT and cancer stem cells (CSC). While this was once thought to be an unidirectional evolution, more recent studies suggest that this transition is bi-directional, stochastic, and mediated by the tumor microenvironment, ultimately showing how such \u201chybrid state\u201d or \u201cplasticity\u201d is linked to poor prognosis and resistance. Cui et al. studied the role of ENC1 in accelerating EMT processes in colorectal cancer, whereas the study by Shou et al. reported an inverse relation between tissue inhibitor matrix metalloproteinases 1 (TIMP1) and prognosis in samples from patients with renal cell carcinoma. The study evaluated TIMP1 as a biomarker to enhance metastasis via the EMT signaling pathway. Another study explored EMT-related genes using TCGA and local samples for pancreatic ductal adenocarcinoma . This study identified a 8-gene signature that impoved prediction compared to clinical variables alone, particularly to assess adjuvant treatment response, such as to immune checkpoint inhibitor therapy. In the future, evaluating therapies that target cells in this \u201chybrid\u201d EMT state may have specific applications to prevent seeding of metastatic sites.A second theme that was recurrent in our collection is the plasticity of the epithelial-mesenchymal transition (EMT). Within this collection, Lu et al. produced a review of Liquid\u2013liquid Phase Separation (LLPS) and protein/nucleic acid condensates. LLPS are a new paradigm in the study of cellular activities recently coming under more intense research focus. Recent progress has been made to understand the roles of LLPS in cancer. Additionally, Dai et al. published a review on RNA modifications and their role in cancer with a deep discussion on the YTH protein family of m6A readers, summarizing the recent advances in structure and biological function of YTH family proteins, and their roles in human cancer and therapy applications.Finally, it is worth mentioning two reviews on protein condensates and RNA modifications. This collection of articles highlights promises and challenges of characterizing the dynamic aspects of cancer. They nicely expose the critical need for multi-omics biomarkers to track cancer evolution in both research and clinical settings. Interestingly, we observed an emerging interest towards previously less studied molecular players, such as non-coding RNAs, condensates, and RNA modifications, both in the context of cancer cells and tumor microenvironment. We anticipate that the use of integrated models based on multiple biomarkers will be necessary to capture the complexity of cancer evolution, especially in today\u2019s clinical settings dominated by a paradigm shift from monotherapy approaches towards combinations of chemotherapy, immune checkpoint inhibitors and targeted therapies."} +{"text": "Cassia occidentalis Linn. stem using liquid chromatography tandem mass spectrometry: application to its pharmacokinetic studies\u2019 by Mohammed Riyazuddin et al., RSC Adv., 2020, 10, 4579\u20134588. DOI: 10.1039/C9RA07482ACorrection for \u2018Simultaneous quantification of five biomarkers in ethanolic extract of The authors regret that the one of the affiliations (affiliation d) was incorrectly shown in the original manuscript. The corrected list of affiliations is as shown above.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "Considered a simple and versatile technique, electrospinning has emerged as a technology for developing 3D materials for a wide range of applications. Electrospun fibers ranging from nanometers to micrometers in diameter have been applied in many research areas including chemistry, materials science, engineering, and chemical engineering, as well as medicine, pharmacology, and pharmaceutics. Based on a large variety of polymers , types of collectors, and nozzle configurations, a vast and complex number of electrospun materials can be obtained, allowing those electrospun fibers to be applied for catalysis , separatBased on In their review entitled \u201cTunable Spun Fiber Constructs in Biomedicine: Influence of Processing Parameters in the Fibers\u2019 Architecture\u201d, Felgueiras et al. described how processing parameters can affect the architecture of electrospun fibers . In addiSt. John et al., in their review entitled \u201cAdvances in Electrospun Nerve Guidance Conduits for Engineering Neural Regeneration\u201d, reported the capability of the electrospun approach to fabricate fibers of various scales and which have a large surface area with a three-dimensional porous structure resembling the native extracellular matrix for regeneration of the nervous system . ConsideIn the third review published in this Special Issue\u2014\u201cOsteochondral Tissue Engineering: The Potential of Electrospinning and Additive Manufacturing\u201d by Costa et al.\u2014the most recent advances in osteochondral tissue engineering are summarized . The autIn two different research articles, Tawfik et al. reported the uses of coaxial electrospun fibers for as fast dissolving matrixes. In the paper entitled \u201cFabrication and Characterization of Fast-Dissolving Films Containing Escitalopram/Quetiapine for the Treatment of Major Depressive Disorder\u201d , authorsChen et al. demonstrated uses of aligned core-shell fiber for the delivery of docosahexaenoic acid and brain-derived neurotropic factor . The cord,l)-lactide-co-glycolide (PLGA) nanofibers were loaded and spun on the metal stent. Electrospun fibers were characterized and the presence of drug on the material\u2019s surface was suggested based on water contact angle. The drug release was evaluated, in which the telmisartan was released over 30 days. In vitro and in vivo results of hybrid stent/PLGA nanofibers loaded with telmisartan indicated that this material can controllably release disease-relevant therapeutics, enhance the migration of endothelial progenitor cells, providing complete endothelial coverage and recovery, and reducing intimal hyperplasia.The research article \u201cTelmisartan Loaded Nanofibers Enhance Re-Endothelialization and Inhibit Neointimal Hyperplasia\u201d by Lee, Kuo, Liu and co-authors demonstrated the electrospun nanofibers loaded with telmisartan for sustained and local delivery of drug to injured arterial vessels . Poly evaluated the antibacterial activity of the fibers loaded with curcumin against polyresistant bacteria, using hyaluronic acid sodium as a polymer basis . Based ol-lactide-co-poly-\u03b5-caprolactone (PLA-PCL) electrospun tubular scaffold engineered with mesenchymal stem cells (MSCs), in order to promote and speed up the regeneration process, ensuring an adequate support to esophageal tissue reconstruction, avoiding the use of autologous conduits. They observed an interesting correlation between the asymmetrical shape of the scaffold and its p-MSCs cellularization process, which helped in keeping the scaffold\u2019s mechanical properties suitable for esophageal application. The authors also observed that the advantage of their pro-posed scaffold with hybrid composition (polymer combined to p-MSCs). Preliminary results collected after in vivo implantation in a porcine model were promising; but further studies are required to statistically validate the data.In terms of biologic applications, Pisani et al., in their research entitled \u201cEngineered Full Thickness Electrospun Scaffold for Esophageal Tissue Regeneration: From In Vitro to In Vivo Approach\u201d , developSteinberg et al., , developKim, D., Kim, S., and co-authors detailed in their article entitled Enhanced Differentiation Capacity and Transplantation Efficacy of Insulin-Producing Cell Clusters from Human iPSCs Using Permeable Nanofibrous Microwell-Arrayed Membrane for Diabetes Treatment a biodegDean, Thomas, and co-authors detailed, in \u201cUni-Directionally Oriented Fibro-Porous PLLA/Fibrin Bio-Hybrid Scaffold: Mechano-Morphological and Cell Studies\u201d , a biohyCurrently, numerous polymers, blends and hybrid systems have been electrospun in order to improve these materials in biomedical applications. In addition to the several approaches in order to obtain electrospun fibers with a vast range of morphologies, including coaxial and side-by-side nozzles, and different collectors, combining this strategy with others microfabrication techniques has increased the applicability of these materials for different biological systems and chemical sciences."} +{"text": "RSC Adv., 2018, 8, 18396\u201318399.Correction for \u2018Synthesis and optical properties of lead-free cesium germanium halide perovskite quantum rods\u2019 by Lin-Jer Chen, The affiliation in the original article was incorrect. The correct affiliation is listed herein.RSC Advances article and their previous related paper published in Journal of Physical Chemical Letters, cited as ref. 21 in the original article.The author apologises that there are portions of text overlap between this In addition, there is an error in the \u2018Characterization and measurements\u2019 section of the ESI. The first sentence should be changed from \u201cThe as-prepared samples were characterized by X-ray powder diffraction (XRD), and transmission electron microscopy (TEM)\u201d to \u201cThe as-prepared samples were characterized by X-ray photoelectron spectroscopy (XPS), and transmission electron microscopy (TEM)\u201d. There was no XRD data reported, therefore the ESI has been updated online to reflect this change.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "The skin is comprised of multiple types of cells that serve as a protective barrier. Mutations in the genes that are responsible for protecting the functional integrity of the skin are often found in many inherited skin diseases, more commonly known as the Mendelian human skin disorders. Advances in molecular techniques and sequencing technologies have enabled identification of novel pathogenic variants, which helps to provide insight into genotype\u2013phenotype correlations and to define the genetic basis of these skin disorders. In this Research Topic, a total of ten articles are published, including those describing findings from case studies and original research, as well as a mini review of current genetic diagnosis strategies, novel gene variants, and genotype\u2013phenotype correlations in human Mendelian skin disorders.Xu et al. report on the use of WES in identifying a de novo pathogenic variant c.2T>C (p.M1T) in KLHL24 in Chinese twin boys with epidermolysis bullosa simplex. Similarly, Wang et al. demonstrate the detection using WES of a missense mutation, c.1156G > A (p.Ala386Thr) in DKC1, which leads to a benign form of dyskeratosis congenita syndrome with the mucocutaneous triad. In another article appearing in the Research Topic, WES analysis also reveals a heterozygous missense mutation c.293G>A in GJB3, which is associated with erythrokeratodermia variabilis, ichthyosis, and non-syndromic hearing loss Gao et al.Recent developments in genome-wide association studies (GWAS) and next-generation sequencing (NGS) techniques have resulted in an integrative approach to the use of functional genomics and expression data in deciphering the causative genetic variants of inherited skin diseases. Because most of the mutations identified in human Mendelian skin disorders reside in protein-coding genes, whole exome sequencing (WES) has been widely used in the identification of such pathogenic variants, and in the genetic diagnosis of Mendelian skin conditions with atypical or unique phenotypes. Wang et al. the genetic profiling of epidermolysis bullosa cases Alharthi et al. and the discovery of a novel CREBBP variant for genetic diagnosis of Rubinstein\u2013Taybi Syndrome Lee et al. Recent evidence from large-scale expression studies (using microarrays and RNA sequencing) has also revealed the roles played by non-coding RNA molecules, such as small non-coding RNAs or miRNAs, in the pathogenesis of several complex skin diseases with a significant pathogenicity score. In addition, a novel homozygous missense mutation (p.L154R) in gene ABHD5 has been detected in a patient with Chanarin\u2013Dorfman syndrome, a rare autosomal recessively inherited genetic disease Liang et al. Finally, Wu et al. also report in this Research Topic on a new case of congenital poikiloderma with a novel missense mutation in the FAM111B gene c.1883G>A (rs587777238).Molecular genetic studies based on family case reports and large-scale regional profiling analyses often provide significant insight into novel pathogenic variants, thereby helping to extend the spectrum of the genetic profile, improve diagnosis, and establish an improved understanding of human Mendelian skin disorders. In this Research Topic, Xu et al. provide an initial description of their discovery of a c.2T>C pathogenic variant in KLHL24, affecting twins in China, and its correlation with epidermolysis bullosa simplex. Their research has identified correlations between phenotypes and genotypes in epidermolysis bullosa, in which KLHL24 pathogenic variants are associated with the mild phenotype. In contrast, the study by Liang et al. on genotype\u2013phenotype analysis in patients with reported Chanarin\u2013Dorfman syndrome reveals no correlation. Meanwhile, as indicated in a review by How et al. there is no significant genotype\u2013phenotype relation in incontinentia pigmenti, a rare type of X-linked dominant genetic disease characterized by ectodermal dysplastic disorder. However, the literature does suggest that variation in a combination of the types of mutations, functional domains affected, X-inactivation, and genomic background may lead to the variability observed in incontinentia pigmenti phenotypes How et al. The authors propose that a detailed understanding of the genotype\u2013phenotype correlation in incontinentia pigmenti will support further investigations concerning prognosis and future reproductive options. Meanwhile, a recent large cohort study involving investigation of genotype\u2013phenotype correlations in patients with autosomal recessive ichthyosis has provided new insights on and definitions of specific phenotypic clues for corresponding genetic mutations (Certain phenotypic features of inherited skin disorders may be associated with particular gene mutations, but paradigms for clinical genotype\u2013phenotype correlation remain unclear in many instances due to the highly variable phenotypic expressivity of the relevant mutations; these paradigms require further refinement. In this Research Topic, utations . In addiutations .In summary, this Research Topic enhances our knowledge of recent exciting progress in the field of genodermatosis, including molecular diagnostics protocols, novel pathogenetic variants, and genotype\u2013phenotype correlations. Together, these studies provide value in the form of greater diagnostic precision, a source of information for clinical assessments, and ways to improve clinical care and management."} +{"text": "Cardiovascular and metabolic bone diseases are demanding health problems with high morbidity and mortality ,2. AlthoIn this context, the Special Issue (SI) \u201cDietary Bioactives: Their Role in the Prevention and Treatment of Cardiovascular and Metabolic Bone Diseases\u201d has published nine novel papers on this topic ,13,14,15The narrative review was published by Mandatori et al. (2021). The authors summarized the most relevant recent knowledge concerning the role of Vitamin K2, a bioactive compound with a key role in the \u201ccalcium paradox\u201d phenomenon, which involves both vascular and bone tissue . The chaLycii radicis cortex in a paper published by Park et al. (2021). Using an animal model of ovariectomized-induced osteoporotic mice, the authors identified scopolin as the candidate bioactive compound extracted from Lycii radicis cortex capable of preventing and treating osteoporosis [Cucurbita moschata leaves, a pumpkin cultivar in Western countries, were published by Lambertini et al. [Subsequently, the specific mechanism of Vitamin K2 in bone health was also addressed in an in vitro study . Specifioporosis . Finallyi et al. .Allium sativum extract in an ex vivo study on mouse heart samples exposed to E. coli lipopolysaccharide inflammatory stimulus [i) the capability of Sasa quelpaertensis to ameliorate metabolic dysfunction conditions including dyslipidemia, insulin resistance, and hepatic lipid accumulation, induce in rats by a high-fructose-diet [ii) the protective effects of Vitis labrusca on cardiovascular dysfunction due to hypertensive conditions\u2014employed the model of Spontaneously Hypertensive Rats [Regarding cardiovascular health, in this Special Issue, one in vitro, one ex vivo and two pre-clinical animal studies were published. Baldassare et al. (2021) reported the anti-inflammatory and anti-oxidative role of myo-inositol using a model of cultured human endothelial cells isolated from the umbilical cord vein of women affected by gestational diabetes . Indeed,stimulus . Additioose-diet and (ii)ive Rats .Finally, in this SI, Esposito et al. (2021) published a cross-sectional analysis performed on a sub-cohort of 4592 subjects from the Moli-sani Study (2005\u20132010) which suggested that intake polyphenols, which contribute to slowing down the biological aging process, may exert protective effects on the long-term risk of cardiovascular and metabolic bone disease development .In conclusion, this SI allowed us to publish a number of encouraging scientific studies based on in vitro, ex vivo and in vivo approaches confirming the increasing interest of researchers in the discovery of new potential bioactive compounds for human health. However, future research must better understand the mechanisms of action of natural molecules and nutritional supplements for the management of cardiovascular and metabolic bone diseases."} +{"text": "The article of Yamashita et al. illustrates the ability of in situ crystal growth to bypass kinetic barriers between phases, demonstrating the importance of this approach in phase discovery (Oswald et al., 2008Although chemically simple, the salt hydrates are very sensitive to external conditions, with complex energy landscapes containing multiple viable phases. However, transformations between solid phases are frequently kinetically hindered, and KCl\u00b7H"} +{"text": "The innate immune system is the first line of defense against bacterial and viral infections and sterile inflammation through the recognition of pathogen-associated molecular patterns (PAMPs) as well as danger-associated molecular patterns (DAMPs) by pathogen-recognition receptors (PRRs), and produces proinflammatory and antiviral cytokines and chemokines . Cells is devoted to many aspects of innate immunity and gives an overview of different DAMPs, immune cells, special mechanisms, and therapeutic options for treating diseases related to chronic inflammation or infections.This Special Issue of One of the well-known DAMPs is the high-mobility group box 1 protein (HMGB1), which is either passively released by dying cells or actively secreted by immune and other cells and was described as implicated in both stimulating and inhibiting innate immunity. Andersson et al. reported that the pro- and anti-inflammatory activities of HMGB1 depend on post-translational modification of its disulfide bonds by binding to different extracellular cell surface receptors either directly or as a cofactor of PAMPs . Another DAMP, extracellular ribosomal RNA, which is released under pathological conditions from damaged tissue, acts synergistically with Toll-like receptor 2 ligands, inducing the release of cytokines in a nuclear factor kappa B-dependent manner in vitro as well as in vivo. Grote et al. suggest that extracellular RNA might sensitize Toll-like receptor 2 to enhance the immune response under pathological conditions and therefore might serve as a new target for the treatment of bacterial or viral infections .Arnholdt et al. demonstrate that cells related to innate immunity and influencing immunoregulatory and inflammatory processes, such as gamma delta T cells, play an important role in angiogenesis and tissue generation. By using a femoral artery ligation model in mice, depletion of this subset of T cells was demonstrated to impair angiogenesis, increase the number of leukocytes and inflammatory M1-like macrophages, and promote the formation of neutrophil extracellular traps (NETs) . The topic of autoinflammation is also covered in this Special Issue. The review of P. Georgel provides some examples of autoimmune/autoinflammatory diseases caused by the deregulated expression of type I interferons and interleukin-1\u03b2. The role of interleukin-1 and type I interferons and their crosstalk in autoinflammatory diseases such as rheumatic diseases are analyzed to reveal novel therapeutic opportunities .Gullet et al. discuss the key components of programmed cell death pathways and highlight the plasticity of pyroptosis, apoptosis, and necroptosis as well as significant crosstalk among these pathways. The concept of PANoptosis, an inflammatory cell death pathway that integrates components of different cell death pathways and is implicated in driving innate immune responses and inflammation, is explained . A review by Papendorf et al. provides a comprehensive overview of molecular pathogenesis disorders caused by proteostasis perturbations, and current knowledge of the various mechanisms by which impaired proteostasis promotes autoinflammation is summarized .2+-binding protein [To investigate the crosstalk between coagulation and innate immunity, the effect of thrombin on macrophage polarization is investigated by Ukan et al. Results demonstrate that thrombin induces an anti-inflammatory phenotype in macrophages, which shows similarities to as well as differences from the classical M2 polarization states regarding the expression of secreted modular Ca protein .D. Suzuki third-instar larvae is described by Carrau et al. as a valuable tool for investigating hemocyte-derived effector mechanisms against pathogens, particularly for the formation of extracellular traps [To investigate insect innate immunity, the in vitro cultivation of primary hemocytes from ar traps . Drugs such as ganciclovir and its pro-drug valganciclovir are often used to treat viremic patients transfected with, e.g., human cytomegalovirus (HCMV). Results from Land\u00e1zuri now suggest that binding and signaling through endothelin receptor B (ETBR) is crucial for viral replication and that selected ETBR blockers inhibit HCMV infections .Lin et al. report that albumin attenuates chronic liver diseases (CLDs) via alleviating inflammation of Kupffer cells caused by bacterial products, which might provide a compelling rationale for albumin therapy in patients with CLDs ."} +{"text": "Wang et al. develop a new iron cathode electro-Fenton process coupled with a pH-regulation divided electrolysis cell for p-nitrophenol degradation. In the electrochemical Fenton system, an iron plate was used as the cathode to inhibit the release of iron ions and promote the reduction of Fe3+ to Fe2+. Therefore, excellent electrocatalytic degradation performance towards organic pollutants was achieved. Wang and Wang synthesized a NiO modified BiVO4 nanocomposite by a hydrothermal and calcination method. The as-prepared nanocomposite showed enhanced photoelectrochemical performance due to the unique NiO lamellar structure that provided a large number of active sites.Numerous metals and metal oxides have been employed as electro- or photo-catalysts for energy conversion and pollutant degradation. Wang et al. used a sol-gel self-combustion method to prepare carboxylate-rich carbon-modified Fe3O4 magnetic catalysts for heterogeneous Fenton degradation of organic pollutants. The prepared Fe3O4-based catalysts displayed improved heterogeneous Fenton degradation performance due to the enhanced pollutant adsorption. Zhu et al. synthesized a CdS/microcrystalline cellulose nanocomposite photocatalyst using an ultrasonic-assisted method. The prepared nanocomposite photocatalyst displayed enhanced pollutant degradation performance under visible light due to the heterojunction formation that efficiently separates the photogenerated electrons and holes of the photocatalyst. Wang et al. prepared a Co3O4/Ti cathode by electrodeposition for electrocatalytic reduction of nitrate, in which the NO3\u2212 was reduced to N2 and NH4+ by the catalysis of Co3O4/Ti, and then NH4+ was selectively oxidized into N2 assisted by chloride ions and using IrO2-RuO2/Ti as the anode. Qiu et al. prepared Pt-modified TiO2 nanotubes as catalysts for photocatalytic degradation of Rhodamine B (RhB) under UV light. It was reported that the superoxide radical anions (O2\u2212\u2219), photogenerated hole (h+) and hydroxyl radical (OH\u2219) were the main active species contributing for RhB degradation.Bai et al. reported the Fischer\u2013Tropsch synthesis performance of Co-based catalysts supported on graphitized ordered mesoporous carbon. The high catalytic performance resulted from the highly crystallized graphitic structure of the mesoporous carbon and the uniform dispersion of CoO on the support. Dai et al. used ion-exchange, in situ modification and complexation-excessive impregnation modification methods to modify SAPO-11 molecular sieves with Ni. The Ni-modified SAPO-11 molecular sieves were supported by NiWS catalysts for hydroisomerization of n-Hexadecane. The complexation-excessive impregnation modification method led to the best hydroisomerization performance. Huang et al. studied the effect of Ga2O3 on the hydrodesulfurization performance of 4,6-dimethyldibenzothiophene catalyzed by the stepwise impregnation method. Ga2O3 promoted Ni and Mo species to disperse uniformly and doping of more Ni atoms into the MoS2 crystals, increasing the average stacking number and the length of MoS2. As a result, enhanced hydrodesulfurization performance was achieved due to the formation of more NiMoS active phases in the system.In addition, metal and metal oxide based or modified materials have also been used for other catalytic applications. Zhang et al. prepared a series of nanostructured Fe-Cu binary oxides for arsenic removal. The crystallinity and structure of the Fe-Cu binary oxides had a significant impact on the arsenic adsorption performance. The oxides with lower crystallinity showed higher surface hydroxyl density and better adsorption performance. Li et al. reviewed the preparation, classification and applications of templated materials, particularly adsorbents in wastewater treatment. The templating method can endow materials with high specific area and unique porous structures, thereby enhancing the material sorption performance towards aqueous pollutants. Wei et al. reviewed the composite adsorbents for fluoride removal, including the adsorbent types , preparation and sorption performance. The adsorption mechanisms for fluoride removal involving electrostatic attraction, ion exchange, complexation, and hydrogen bonding were also discussed.Adsorption is a simple but effective way for environmental decontamination . VariousYu et al. prepared new biochar from excess sludge, followed by acetic acid modification. The modified sludge-derived biochar displayed improved porosity and enriched\u2013COOH functional groups, thereby enhancing its adsorption performance to uranium. However, the catalytic performance of the sorbent was not discussed. Zeng et al. fabricated porous glass-ceramics based on coal fly ash without using pore forming agents by direct overfiring, in which borax was used to destroy the structure of quartz and amorphous vitreous body in coal fly ash and thus reduce the sintering temperature by the B-O bond. Chen et al. fabricated a non-sintered ceramsite from pyrite tailings for phosphorus removal. Both Plackett-Burman Design (PBD) and Box-Behnken Design (BBD) based response surface methodology were used to optimize the fabrication parameters.Recently, with the promotion of the circular economy, waste based materials have attracted growing interest for various applications, such as fertilizers , carbon Wang et al. reviewed the significant roles of surfactants in oriented immobilization of cellulase on nanocarriers as well as a surfactant reversed micelle system.Cellulase plays a key role in the production of fuel ethanol by enzymatic hydrolysis of lignocellulose, and immobilization of cellulase on the nanocarriers is an effective way to improve the hydrolysis efficiency. In summary, this Research Topic discussed various inorganic materials as catalysts or adsorbents with unique nanostructures and functionalities for energy conversion and environmental decontamination. In the future, inorganic materials will continue to play a vital role in addressing global energy and environmental challenges, such as climate change, energy shortages and environmental pollution. Engineering new high performance heterogeneous catalysts and understanding the limiting factors and their mechanisms in the catalytic reaction are two key research directions that should be paid more attention to."} +{"text": "The rapidly growing industry of crop biostimulants leverages the application of plant growth promoting rhizobacteria (PGPR) to promote plant growth and health. However, introducing nonnative rhizobacteria may impact other aspects of ecosystem functioning and have legacy effects; these potential consequences are largely unexplored. Nontarget consequences of PGPR may include changes in resident microbiomes, nutrient cycling, pollinator services, functioning of other herbivores, disease suppression, and organic matter persistence. Importantly, we lack knowledge of whether these ecosystem effects may manifest in adjacent ecosystems. The introduced PGPR can leave a functional legacy whether they persist in the community or not. Legacy effects include shifts in resident microbiomes and their temporal dynamics, horizontal transfer of genes from the PGPR to resident taxa, and changes in resident functional groups and interaction networks. Ecosystem functions may be affected by legacies PGPR leave following niche construction, such as when PGPR alter soil pH that in turn alters biogeochemical cycling rates. Here, we highlight new research directions to elucidate how introduced PGPR impact resident microbiomes and ecosystem functions and their capacity for legacy effects. Like other introduced species, plant growth promoting rhizobacteria (PGPR) have unpredictable ecosystem consequences and legacy effects; these outcomes are a current knowledge gap. Commercially available crop biostimulants sometimes contain PGPR that directly or indirectly increase plant productivity or crop yield, stimulate nutrient uptake, improve nutrient cycling efficiency, reduce pathogen loads, and increase plant tolerance to abiotic stress . Interest in this research topic has expanded in recent years. Before 2015, fewer than 10 articles per year were published on this topic; the count jumped to 50+ per year since 2019. Despite increased interest in PGPR, only 12 of the 313 articles included the search term \u2018ecosystem\u2019 and one article included \u2018legacy\u2019. Here, we highlight potential effects of PGPR species introductions that may extend beyond the host plant to ecosystem functions that may persist longer than the PGPR populations , Box 1How do PGPR affect macronutrient and micronutrient cycling beyond host\u2013plant acquisition? PGPR may have traits to solubilize and mobilize macronutrients and micronutrients. Some research into nitrogen and potassium root acquisition and soil leaching currently exists. More research is needed into micronutrients important to plant productivity and organic matter persistence, such as iron and manganese.How do PGPR affect organic matter persistence? PGPR may shift relative abundance or activity of key players in the resident microbiome. These changes in microbe\u2013microbe interactions could cascade to changes in soil properties mediated by microbes such as pH, erodibility, porosity, and water holding capacity of soil. Experimental applications of PGPR under field conditions that quantify soil parameters are needed.What are the multi\u2010trophic consequences of introduced PGPR? PGPR are known to release volatile organic compounds (VOCs) that can deter herbivores. Some VOCs attract pollinators, but whether PGRP VOCs attract pollinators is unexplored. Indirect multitrophic interactions, mediated by changes in plant phenology like budbreak or flowering, may be spurred by PGPR and this remains a current knowledge gap. Additionally, PGPR may indirectly affect resident microbial abundances through antagonisms with bacterivores or fungivores.Do PGPR spread to other plant hosts in the ecosystem? Host specificity is low among biostimulant microbes due to their application across various crops. Thus, escape from crops to adjacent ecosystems has a high potential, although persistence may be negligible. Mitigating effects caused by the escape of biostimulant microbes is an area of active research contains Bacillus velezensis GB03. Application is intended to reduce pathogens such as those causing blight , root rot (Pythium), and wilt (Fusarium). The hypothesized mechanism is that B.\u2009velezensis induces a systemic immune response across a wide range of plant hosts. Another potential mechanism is that the PGPR increases anti\u2010fungal indigenous taxa (Xiong et\u2009al., Another ecosystem consequence that affects crop yield is plant disease suppression. Many PGPR in biostimulants are added to crops for the purposes of reducing pathogen loads (Pieterse et\u2009al., et\u2009al., et\u2009al., et\u2009al., Microbial interactions affect organic matter persistence. Biostimulant PGPR may have indirect effects on ecosystem functions such as the accumulation and decomposition of organic matter through their interactions with the resident microbiome (Hellequin et\u2009al., et\u2009al., et\u2009al., et\u2009al., et\u2009al., Introduction of new microbes could lead to genetic changes in the inoculant or community, yet the genetic impact of PGPR introductions remains largely unexplored. Any location with a high microbial density, such as the rhizosphere, can support rapid and pervasive horizontal gene transfer (Kent et\u2009al., et\u2009al., et\u2009al., et\u2009al., Legacy effects on resident microbial community structure can arise as PGPR establish and persist following introduction. Establishment and persistence can occur through at least two mechanisms: augmentation and displacement (Kurkjian et\u2009al., et\u2009al., Plant growth promoting rhizobacteria that persist following introduction can leave a legacy by altering their microenvironment to promote their own fitness, through a process known as \u2018niche construction\u2019 (Callahan et\u2009al., et\u2009al., et\u2009al., et\u2009al., et\u2009al., et\u2009al., et\u2009al., Plant growth promoting rhizobacteria introductions that do not persist in the community may still leave legacy effects (Mallon et\u2009al., Introducing PGPR may have unintended effects that cascade throughout the resident microbiome, adjacent plant communities, and the whole ecosystem Box\u2009. We urgeJAMM wrote the initial draft of this manuscript and conceived original ideas. JAMM, PEA, JKM, WM and MAC contributed to idea refinement, writing, and revising the manuscript."} +{"text": "Stroke stands as a major cause of death and disability with increasing prevalence. The absence of clinical improvement after either intravenous thrombolysis (IVT) or mechanical thrombectomy (MT) represents a frequent concern in the setting of acute ischemic stroke (AIS). In an attempt to optimize overall stroke management, it is clinically valuable to provide important insight into functional outcomes after reperfusion therapy among patients presenting with AIS. The aim of the present review is to explore the predictive value of leukoaraiosis (LA) in terms of clinical response to revascularization poststroke. A literature research of two databases (MEDLINE and Scopus) was conducted in order to trace all relevant studies published between 1 January 2012 and 1 November 2022 that focused on the potential utility of LA severity regarding reperfusion status and clinical outcome after revascularization. A total of 37 articles have been traced and included in this review. LA burden assessment is indicative of functional outcome post-intervention and may be associated with hemorrhagic events\u2019 incidence among stroke individuals. Nevertheless, LA may not solely guide decision-making about treatment strategy poststroke. Overall, the evaluation of LA upon admission seems to have interesting prognostic potential and may substantially enhance individualized stroke care. Stroke represents not only the second leading cause of death but also the major cause of acquired disability among adult individuals, mostly accompanied by a considerable unfavorable effect on the long-term functional independence of stroke survivors ,2,3. TakThe beneficial effects of reperfusion therapy following stroke, either with intravenous thrombolysis (IVT) or mechanical thrombectomy (MT), have been well documented. IVT with recombinant tissue plasminogen activator (rt-PA) has emerged as a first-line treatment strategy for patients with acute ischemic stroke (AIS) within 4.5 h of symptom onset . IncreasGiven the fact that prompt forecasting of each patient\u2019s propensity for recovery substantially contributes to the decision-making in terms of poststroke treatment strategy, it becomes essential to be provided with an accurate and timely outcome prognosis . SeveralIntroduced by Hanchski in 1987 , the terConsidering the expected substantial increase in stroke patients with pre-existing LA undergoing reperfusion therapy, as stroke burden rises among the elderly and life expectancy continues to significantly expand in developed countries, it is of key importance to elucidate the potential contribution of LA on forecasting clinical response to IVT or MT poststroke. Thus, the objective of our study was to review all available literature published within the last decade dealing with baseline LA as a prognostic tool following stroke recanalization.The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA registration number: CRD42022369840) were used to guide this study. Our study\u2019s methods were a priori designed. Two investigators conducted literature research on two databases (MEDLINE and Scopus) (SK and AS) to trace all relevant studies published between 1 January 2012, and 1 November 2022. Search terms were as follows: (\u201cleukoaraiosis\u201d OR \u201cwhite matter hyperintensities\u201d OR \u201cWMHs\u201d) AND (\u201cthrombolysis\u201d OR \u201cthrombectomy\u201d OR \u201cbrain revascularization\u201d OR \u201creperfusion\u201d). The retrieved articles were also hand-searched for any further potential eligible articles. Any disagreement regarding the screening, or selection process, was solved by a third investigator (KV) until a consensus was reached. Only full-text original articles published in the English language were included. Secondary analyses, reviews, guidelines, meeting summaries, comments, unpublished abstracts, or studies conducted in animals were excluded. There was no restriction on study design or sample characteristics.Data extraction was performed using a predefined data form created in Excel. We recorded the authors, year of publication, number of participants, follow-up time, method of leukoaraiosis assessment, time of computed tomography execution, time from symptom onset to recanalization, the scale used to examine stroke severity and clinical outcomes, and the main results of each study.No statistical analysis or meta-analysis was performed due to the high heterogeneity among studies. Thus, the data were only descriptively analyzed. Overall, 262 records were retrieved from the database search. Duplicates and irrelevant studies were excluded; hence, a total of 90 articles were selected. After screening the full text of the articles, 37 studies were eligible for inclusion .A total of 37 publications fulfilled our inclusion criteria, as shown in In total, 20 studies utilized the Fazekas score, 11 the van Swieten scale, three the Age-Related White Matter Change Scale, two differentiated between the presence or absence of LA, one used the CREDOS WMH visual rating scale, and four estimated WMH volume on imaging. In total, all the studies included in this review were longitudinal. They were either retrospective or prospective cohorts.The total number of stroke patients included in all studies ranges from n = 56 to n = 3In none of the 37 included studies, stroke patients are contrasted to demographically-matched healthy individuals and none of the studies include a disease-control group other than stroke patients.National Institutes of Health Stroke Scale (NIHSS) and modified Rankin Scale (mRS) have been simultaneously used in 30 studies. NIHSS was the only scale in three studies and mRS exclusively in one study. In two studies NIHSS was combined with the Fugl-Meyer rating scale (FMS) and in one study with the Oxford Handicap Scale (OHS).A literature review over the last decade was conducted in order to elucidate baseline LA prognostic value in AIS patients undergoing reperfusion therapy. A total of 37 full-text original articles dealing with the potential utility of LA evaluation in forecasting stroke survivors\u2019 clinical response to revascularization were identified and classified into three groups based on the implemented revascularization technique.With respect to poststroke outcome after EST, Zhang et al. , having Additionally, Liu et al. further As far as the role of brain atrophy and LA in forecasting futile recanalization poststroke is concerned, Kaginele et al. , having With regard to clinical and radiographic variables able to act as predictors of futile recanalization among AIS patients undergoing MT, Gilberti et al. , having Regarding the relationship between both LA severity and time to successful reperfusion with 3-month functional outcome in an acute stroke setting, Milkati et al. exploredWith respect to the occurrence of hemorrhagic transformation (HT), Shi et al. investigApart from that, Lee et al. reportedOn the contrary, Atchaneeyasakul et al. , having As for the prognostic utility of LA burden in patients presented with AIS due to large cerebral artery occlusion undergoing intra-arterial therapy (IAT), Giurgiutiu et al. , having With respect to the clinical response to IV rt-PA utilization in ischemic stroke patients in the setting of detectable LA on baseline CT scan, Huang et al. examinedAdditionally, Zhong et al. , having As far as the role both early ischemic and pre-existing brain imaging signs are to play in terms of predicting clinical response to IVT in ischemic stroke patients, Delcourt et al. observedConsidering that the assessment of the global burden of small vessel disease (SVD), as reflected by both white matter changes and pre-existing lacunar infarcts, may constitute a more precise marker with valuable prognostic potential in terms of clinical outcome and hemorrhagic complications following IVT among stroke patients, Arba et al. investigConsidering the influence of pre-existing LA on CT scans of stroke patients admitted for IVT on 3-month functional outcome and risk of symptomatic ICH, Yang et al. examinedDespite being associated with ICH occurrence and 3-month unfavorable outcome following IVT, LA severity was not reported as an independent prognostic marker of symptomatic ICH, mortality, or clinical outcome in a study conducted by Capuana et al. within aWith respect to the prognostic significance of LA in terms of tPA-related bleeding events, Willer et al. , having Regarding the occurrence of remote post-thrombolytic hemorrhagic complications of IVT, Chen et al. exploredAs far as the prognostic potential of LA burden among elderly stroke patients is concerned, Nighoghossian et al. , having Regarding the prognostic potential of endothelial dysfunction markers as the soluble tumor necrosis factor-like inducer of apoptosis (sTWEAK) in an acute ischemic stroke setting, da Silva-Candal et al. exploredTaking everything into consideration, the present review provides evidence regarding the prognostic significance of LA severity, as evaluated on baseline head imaging within the early phase following stroke. Our findings suggest the significant role LA assessment may play in forecasting clinical response to reperfusion treatment strategy. LA burden, serving as a surrogate marker of biological age and consequently \u201cbrain frailty\u201d among stroke patients, appears to be able to yield additional information in terms of cerebral reserve and brain parenchyma resilience to emerging ischemia, thus reliably predicting both reperfusion status and clinical outcome after either IVT or MT. Determining the extent of pre-existing white matter abnormalities may properly guide the decision-making in a setting of acute stroke, as a greater degree of these lesions is usually coupled with unfavorable prognosis and poorer outcomes after the selected intervention is implemented. Additional data support the prognostic value of baseline LA on the potential development of adverse hemorrhagic events after IVT or MT, with a higher LA burden identified on admission head imaging being accompanied by an increased incidence of intervention-related ICH. Nevertheless, LA assessment should be interpreted in a clinical context and may constitute a more powerful tool when being paralleled with other clinical and neuroimaging biomarkers in an attempt to facilitate individualized stroke care. The aforementioned results highlight the need to further investigate LA as a promising imaging marker for clinical outcomes after IVT or MT to enhance patients\u2019 risk stratification and promote overall stroke management. Additional studies among stroke individuals on the linkage between LA burden and prognosis after stroke intervention are recommended in order to provide further insight into this clinically important relationship."} +{"text": "Epigenetic mechanisms and post-translational modifications as novel therapeutic targets in cancer\u201d collected 11 articles on epigenetic mechanisms, post-translational modifications, cell cycle and signaling pathways related to tumorigenesis and development. Our aim is to provide new research directions for targeted cancer therapies and drug development.This Research Topic \u201cZhou et al. summarized the role and mechanism of the modification related enzymes \u201cWriters\u201d and \u201cErasers\u201d in cancer, providing a basis for the development of anti-tumor epigenetic drugs. Tang et al. assessed the causal relationship between smoking-related DNA methylation and breast cancer risk by Mendelian randomization. The results suggest that DNA methylation plays an important role in linking smoking to breast cancer, especially the subtype of ER+ breast cancer. Zeng et al. evaluated pyroptosis-related genes in esophageal adenocarcinoma and found that the expressions of GSDMB and ZBP1 were influenced by DNA methylation levels. Huo et al. focused on the role of RNA modification in cancer. They summarized the molecular mechanism of RNA modification in the occurrence and development of head and neck squamous cell carcinoma and discussed the related treatment options. Jing et al. demonstrated that hypoxia induced upregulation of ELFN1-AS1 expression in colorectal cancer cells. As a potential target of competing endogenous RNA, ELFN1-AS1 relieved the inhibition of TRIM14 by sponging miR-191-5p, thus promoting the proliferation and invasion of colorectal cancer cells. Lu et al. elucidated the role of acetylation modification in tumor immunity by focusing on histone acetyltransferases and deacetylases, and discussed the clinical application of acetylation-modified drugs in tumor therapy.Epigenetic modifications affect genetic expression without the sequence change of DNA, and its disorders are closely related to the occurrence and progression of cancer. Methylation is a widely studied form of epigenetic modification. Hou et al. provided evidence that low expression of USP47 in the primary colorectal cancer was associated with disease-free survival. USP47 deubiquitinated and stabilized the expression of transcription elongation factor A3 (TCEA3). Knockdown of UPS47 or TCEA3 can enhance doxorubicin-induced apoptosis and pyrotosis of colorectal cancer cells, which provides a potential target for the treatment of colorectal cancer.Post-translational modifications increase the protein diversity and play critical roles in regulating the protein activity, localization, and interaction with other molecules. Xia et al. identified aminoquinoline as a novel multi-kinase inhibitor of CDK4/6 and PI3K/AKT for the treatment of hepatocellular carcinoma. Zhao et al. revealed that lysophosphatidic acid induced transactivation of EGFR through MMP-dependent pathway, which upregulated geminin expression and promoted DNA replication in gastric cancer cells. Guo et al. found that a natural microbial product, Ilicicolin A, as a novel EZH2 antagonist, inhibited the EZH2-mediated signaling pathway to enhance the sensitivity of castration-resistant prostate cancer cells to enzalutamide. Yang et al. investigated the role of kinesin family member 2C (KIF2C) in cervical cancer. KIF2C expression was significantly upregulated in cervical cancer, promoted cervical cancer cells proliferation, invasion, and migration. Knockdown of KIF2C inhibited the development of cervical cancer by activating p53 signaling pathway, providing a new target for the treatment of cervical cancer.In normal cells, cell cycle and signal transduction are strictly regulated, and their dysregulation and abnormal activation lead to various diseases including cancer. Epigenetic mechanisms and post-translational modifications as novel therapeutic targets in cancer\u201d Research Topic emphasizes that epigenetic and post-translational modification provide new targets for the development of anti-cancer drugs and bring new strategies for treatment of cancer.In conclusion, the \u201c"} +{"text": "It is challenging to conduct in-depth research of the molecular mechanisms associated specifically with human age-related diseases, and how internal and external factors are regulating these processes. For these reasons, research has focused on finding innovative therapies that increase the specificity of the treatment and reduce their drawbacks. This Research Topic on \u201cNLiu et al. offered insights into the heterogeneity of osteoarthritis, which provided in-depth understanding of the transcriptomic diversities within synovial tissue. This transcriptional heterogeneity may improve an understanding on osteoarthritis pathogenesis and provide potential molecular therapeutic targets for osteoarthritis. Zhao et al. investigated the aging-based diagnostic gene signature and molecular subtypes with diverse immune infiltrations in atherosclerosis. Huang et al. did comprehensive characterization of ageing-relevant subtypes associated with different tumorigenesis and tumor microenvironment in prostate cancer. The authors proposed that ageing-relevant molecular subtypes and gene signature might be of great significance to determine clinical outcomes and tumor microenvironment features as well as immunotherapeutic responses in prostate cancer. Song et al. identified reliable gene signatures through combination strategies of diverse feature selection methods, which facilitated the early detection of ischemic cardiomyopathy and revealed the underlying mechanisms. Liu et al. developed of a toll-like receptor (TLR)-based gene signature that can predict prognosis, tumor microenvironment, and chemotherapy response for hepatocellular carcinoma. They also proposed that this TLR-based gene signature might assist clinicians to select personalized therapy programs for HCC patients.This Research Topic described some high-throughput methods for examining age-related molecular signatures. Lin et al. summarized the role of macrophage phenotypic diversity in the progression of the dynamic atherosclerotic plaque, and the possibility of treating atherosclerosis by targeting macrophage microenvironment. Wang et al. reviewed and concluded the AMPK as a potential therapeutic target for intervertebral disc degeneration. The role of sonic hedgehog pathway in the development of the central nervous system and aging-related neurodegenerative diseases was discussed by Yang et al.You at al. investigated the influence of anemia on postoperative cognitive function in patients undergo hysteromyoma surgery. The results from He et al. showed that secoisolariciresinol diglucoside (SDG) can significantly increase mitochondrial DNA copy number and slow down the process of telomere shortening. The authors also indicated that SDG could improve ovarian reserve by inhibiting oxidative stress. Qin et al. discovered that rational design and synthesis of 3-morpholine linked aromatic-imino-1H-indoles as novel Kv1.5 channel inhibitors sharing vasodilation effects. Du et al. found that the 4-methoxydalbergione could inhibit bladder cancer cell growth through inducing autophagy and inhibiting Akt/ERK signaling pathway. Wu et al. suggested that the sevoflurane could alleviate myocardial ischemia reperfusion injury via inhibiting P2X7-NLRP3 mediated pyroptosis. Jia et al. demonstrated that circulating LBX2-AS1 could be an underlying diagnostic marker in multiple myeloma (MM). Targeting LBX2-AS1 suppressed tumor progression by affecting mRNA stability of LBX2 in MM. Hence, LBX2-AS1 could be a novel therapeutic marker against MM. Lan et al. uncovered that the olfactory impairment could be an early indicator to guide early intervention for postoperative cognitive dysfunction.This Research Topic also discussed novel mechanistic insights and targeted therapies for a personalized treatment against various age-related diseases. Xing et al.Lu et al. reviewed the vitamin D and lipid profiles in postmenopausal women and concluded that the vitamin D administration in postmenopausal women could decrease the concentrations of triacylglycerol, and HDL-cholesterol, but have no effects on LDL-cholesterol and total cholesterol. Xu et al. summarized the recent progress in the functions and mechanisms of newly discovered circular RNAs in intervertebral disc degeneration. Zheng et al. stated that ginkgo biloba extract 80 could have cardioprotective properties through the activation of AKT/GSK3\u03b2/\u03b2-Catenin signaling pathway. Yin et al. investigated the cordyceps militaris-derived polysaccharide CM1 and demonstrated that it could alleviate atherosclerosis in LDLR (\u2212/\u2212) mice by improving hyperlipidemia. Zhou et al. revealed that propofol could ameliorate ox-LDL-induced endothelial damage by enhancing autophagy through PI3K/Akt/m-TOR pathway, which might offer a novel therapeutic strategy in atherosclerosis. A type I collagen-targeted MR imaging probe for staging fibrosis in Crohn\u2019s disease was conducted by Li et al. Their results demonstrates that targeted MRI probe (EP-3533) supplies a better enhanced effect compared to Gd-DTPA and could be a promising method to evaluate the progression and monitor the therapeutic response of bowel fibrosis. Yang et al. found that the sinomenine could suppress development of hepatocellular carcinoma cells through inhibiting MARCH1 and AMPK/STAT3 signaling pathways. This study provides a new support for SIN as a clinical anticancer drug and illustrates that targeting MARCH1 could be a novel treatment strategy in developing anticancer therapeutics.New formulations or new therapeutic molecules useful for increasing anti-aging effectiveness and reducing toxicity are illustrated here. The atheroprotective effects and molecular mechanism of berberine were discussed by Wang et al. found that the aging-related gene signature was in relation to tumor immunity and stromal activation in rectal cancer, which might predict survival outcomes and immuno- and chemotherapy benefits. Li et al. stated that the miR-330\u20135p in small extracellular vesicles derived from plastrum testudinis-preconditioned bone mesenchymal stem cells could attenuate osteogenesis by modulating Wnt/\u03b2-Catenin signaling. Zeng et al. observed that knockdown of miR-615-5p reversed the suppression of circRNA_100146 silence on the proliferation and invasion of prostate cancer cells. The authors also stated that the tumor growth was also suppressed by silencing circRNA_100146 in vivo. Lu et al. identified two osteoarthritis-specific markers HTR2B and SLC5A3 and their knockdown ameliorated apoptosis and inflammation of Osteoarthritis synovial cells. Zhang et al. also discovered that mitomycin C could inhibit esophageal fibrosis by regulating cell apoptosis and autophagy via lncRNA-ATB and miR-200b.Finally, some genes and proteins involved in aging or anti-aging as well as substances that inhibit or rejuvenate aging are also presented in this Research Topic. We believe that researchers could find this Research Topic to be a useful Research Topic of articles on novel molecular mechanisms and innovative therapeutic approaches for age-associated diseases."} +{"text": "Myeloid neoplasms (MN), namely myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN), and acute myeloid leukemias (AML) are characterized by disrupted myelopoiesis encompassing increased apoptosis of bone marrow (BM) progenitors, differentiation arrest and increased proliferation . This reBarcellini and Fattizzo asked themselves the \u201cegg or chicken\u201d question as to whether immune phenomena comes before or after MN. They examined their epidemiological association, and discussed that autoimmunity and immunodeficiency are the two faces of a dysregulated immune tolerance and surveillance possibly resulting in tumor escape and infections. Alterations of the microbiota and of mesenchymal stem cells in MN are also discussed to highlight the importance of a permissive microenvironment for tumor growth. Finally, the authors highlight how novel therapies for MN (including checkpoint inhibitors and chimeric antigen receptor T-cells) may increase autoimmune phenomena.Cominal et al., focused on Philadelphia chromosome-negative MPN that display inflammatory alterations of BM niche. They studied BM soluble mediator signatures using a multiplex assay and found a distinctive profile in polycythemia vera with increased levels of chemokines, and growth factors compared to essential thrombocytopenia and primary myelofibrosis. Deregulation of soluble mediators was associated with abnormal blood counts, thrombosis, treatment status and risk stratification and this might represent a therapeutic target. Additionally, JAK inhibitors also affect the levels of inflammatory cytokines in MPN patients, as described by Cattaneo and Iurlo. They also discussed how these drugs affect several components of the innate and adaptive immune systems such as dendritic cells, natural killer cells, T helper cells, and regulatory T cells, resulting in a level of immune deficiency with increased infectious risk.Scium\u00e8 et al., focused on another rare \u201cproliferating\u201d condition: systemic mastocytosis. They described a KIT D816V mutated patient who evolved into MN with PDGFRA rearrangement and responded to imatinib therapy; they discuss how immunological mechanisms may play a role in promoting clonal prevalence of one entity (mastocytosis) over the other (MN).Bucelli et al., who described a large series of patients with co-occurrence of myeloid and lymphoid neoplasms. Patients mainly suffered from MPN with associated non-Hodgkin lymphomas; nearly a half required anti-lymphoma therapy and 1/3 experienced a high-grade infection that was significantly associated with mortality.The clinical and prognostic aspects of the concomitant presence of distinct hematological clonal entities was further addressed by Whether the myeloid and lymphoid clones share a common origin or develop autonomously is still debated, and another interesting example is the association of large granular lymphocyte (LGL) expansion with MN and BM failure syndromes. Our group performed a literature review and discussed how LGL clones, found in up to 1/3 of MN, are associated with deeper cytopenia (likely through immune mediated apoptosis) and good response to immunosuppression. Far from being innocent bystander, LGL clones may contribute to immunosurveillance, as their depletion after immunosuppression may favor leukemic escape.Li et al., developed and validated an innovative prognostic model based on a novel immune-17 signature derived from transcriptome data from The Cancer Genome Atlas (TCGA) and The Genotype-Tissue Expression (GTEx) databases. They confirmed that immune biology processes and transcriptional dysregulations are critical factors in the development of AML. Interestingly, the incorporation of the immune-17 signature to the ELN2017 risk score improved patient stratification. This immune signature may be therapeutically exploited, as described by Sun Yao et al., that treated an AML patient with PD-1 blockade in combination with azacytidine after allogeneic hematopoietic stem cell transplantation; these strategies that reactivate anti-leukemic immune surveillance may in turn result in devastating autoimmune/autoinflammatory responses, as in the case described who developed fatal graft versus host disease.Focusing on AML, Razanamahery et al., described a case of Erdheim\u2013Chester disease (ECD), a rare histiocytosis, characterized by somatic mutations of MAP-kinase pathway in CD14+ monocytes. They found a correlation between disease activity and increased CD14++CD16\u2212 \u201cclassical monocyte\u201d and decreased CD14lowCD16++ \u201cnon-classical monocyte\u201d highlighting the contribution of a phenotype switch of innate immunity in this rare disease.Moving to innate immunity effectors, Giannotta et al., reported a patient with MPN who developed clinically overt PNH requiring anti-complement therapy. They discuss that the selection and expansion of PNH clones in MPN is likely to be ascribed to the same immunological bottlenecks described in BMF: autoimmunity against BM precursors, toxicity of therapies, and acquirement of cooperative somatic mutations.Another very rare condition associated with autoimmunity and MN is paroxysmal nocturnal hemoglobinuria (PNH). Caprioli et al., described how the use of single-cell technologies represent powerful tools to assess the cellular composition of the complex tumour ecosystem and its immune environment (Finally, ironment Figure\u00a01In conclusion, this Research Topic highlights the multifaceted immunologic aspects of pathogenesis, clinical course, and treatment of MN. This expanding field will increasingly benefit from sophisticated molecular tools to further identify druggable pathways/targets and optimize management of MN and other rare entities.All\u00a0authors\u00a0listed have made a substantial, direct, and intellectual\u00a0contribution\u00a0to the work and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."} +{"text": "Aminul Islam et al., RSC Adv., 2022, 12, 7835\u20137849, DOI: 10.1039/D2RA00768A.Correction for \u2018Efficacy of surface-functionalized Mg The authors regret that the name of one of the authors (Md. Mahbubul Haque) was shown incorrectly in the original article. The corrected author list is as shown here.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "Due to demographic trends, the importance of optimal treatment of proximal femur fractures in elderly patients will undoubtedly continue to increase. The clinical outcome for this vulnerable patient group depends on many factors, some of which we as surgeons may influence . However, patient-specific factors are also of upmost importance and may raise our suspicion for a complicative course.In this issue, several authors highlight different surgery-related and patient-specific factors that may influence the perioperative course of geriatric patients with proximal femur fractures.Kristen et al. retrospeIn the context of surgical timing, Forssten et al. investigIn addition to the above reported impact of timing, Weing\u00e4rtner et al. highlighIn their randomized double-blinded trial, Zhang et al. evaluateA retrospective, observational study evaluated whether beta-blockers therapy provided a survival benefit following hip fracture surgery . Ahl et Finally, Becker et al. reviewedWe hope that this issue will provide you with useful information regarding your daily practice with this vulnerable patient group."} +{"text": "Management of hemodialysis patients.\u201dChronic kidney disease (CKD) is a highly prevalent condition affecting >10% of the general population . Though Assessing the efficacy and safety of Juan Bi Tang for dialysis-related myofascial pain in the fistula arm: Study protocol for a randomized cross-over trial,\u201d Hsu et al. outline a protocol for randomizing HD patients to use of the Chinese herbal medicine Juan Bi Tang, to determine if this therapeutic strategy could alleviate dialysis-related myofascial pain. The authors note wide use of Juan Bi Tang in treatment of other musculoskeletal disorders, though efficacy in treatment of fistula-related pain has not been explored. The results of this study could provide a more effective strategy for managing access arm pain in HD patients.Patients who receive HD through an arteriovenous fistula may experience pain during or after dialysis treatments, which can be related to alterations in extremity perfusion . These sFlash glucose monitoring to assess glycemic control and variability in hemodialysis patients: The GIOTTO study\u201d, Mambelli et al. demonstrate the acceptability of flash glucose monitoring in patients on HD. Though further studies are warranted to validate its accuracy and precision in the setting of other comorbidities, the use of flash glucose monitoring in HD patients could provide an additional layer of safety in managing these vulnerable patients.Additionally, HD treatments can induce hyperglycemia and hypoglycemia in ESKD patients . In factKang et al., \u201cAssociation between alkaline phosphatase and muscle mass, strength, or physical performance in patients on maintenance hemodialysis,\u201d the authors found that high alkaline phosphatase levels were associated with poor physical performance in ESKD patients on HD, as assessed by several tests of muscle mass and function including handgrip strength and a 6-min walk test. High alkaline phosphatase levels in HD patients typically represent high-turnover bone disease, and have been shown to associate with increased risk of hospitalization and death values are evaluated during HD treatments to predict short-term and long-term changes in PTH levels. This model would use blood and dialysate samples to guide management of mineral bone disease in HD patients, which is highly prevalent and contributes to significant morbidity. We anticipate that big data and mathematical modeling will continue to enable development of prediction models useful in management of HD patients.Given the massive amount of data collected and available from cohort studies such as the Dialysis Outcomes and Practice Patterns Study , advanceDialyzer classification and mortality in hemodialysis patients: A 3-year nationwide study cohort,\u201d Abe et al. report that protein-leaking dialyzers may be associated with a reduction in mortality. Compared to low-flux and high-flux dialyzers, protein-leaking dialyzers in a group of >250,000 patients on HD in Japan were associated with lower mortality , though the exact contributions of \u03b22-microglobulin removal were not evaluated. Further information is needed to clarify relationships between removal of variable dialyzable proteins and outcomes such as mortality (There are also several studies focusing on clinical endpoints in HD patients, such as mortality. Some risk of mortality may be related to HD treatments in themselves. In their study, \u201cortality .Tylicki et al., \u201cCOVID-19 vaccination reduces mortality in patients on maintenance hemodialysis\u201d, provides even more data to support the use of COVID-19 vaccination in patients on HD to reduce mortality.Finally, in the era of an ongoing pandemic, we must also acknowledge that non-dialysis related factors can significantly influence the outcomes of patients with ESKD. A study by Management of hemodialysis patients\u201d, contribute valuable information on assessing and treating some of the variable aspects of increased morbidity and mortality in ESKD patients on hemodialysis.In sum, the seven articles presented in this Research Topic, \u201cDP drafted the editorial. All authors contributed to editing and finalization of the manuscript."} +{"text": "As the first evolutionary group that comprises of innate immunity and adaptive immunity, fish is considered as a supreme model for clarifying the evolutionary and regulatory mechanisms of vertebrate immunity. However, the types and characteristics of fish immune cells are still not quite clear. In this Research Topic, eight articles including six original research articles and two review articles highlight the advance of novel techniques to identify immune cell population in fish.Fei et\u00a0al. reviewed the reagents (including mAbs of surface markers and immune cells) available for the research of fish immunity. The authors further discussed the potential applications of fluorescence-activated cell sorting and droplet-based microfluidics in screening and identifying antigen-specific B lymphocytes with a high-throughput manner and suggested to incorporate the alternative technologies to promote the production of specific antibodies in a high-throughput and cost-effective way. Similarly, the review article by Chan et\u00a0al. summarized the protein marker and partial corresponding mAbs of teleost fish immune cells, and presented the interaction of fish T cells, B cells and dendritic cells via surface molecules for modulating adaptive immune response. More importantly, they further reviewed the advance for application of single-cell RNA sequencing (scRNA-seq) in teleost immunology and explored future directions of the methods developed for studying fish immunity at the cellular level. Mart\u00edn et\u00a0al. isolated two homologs of mammalian CD38 (CD38A and CD38B) from Rainbow trout (Oncorhynchus mykiss). By using the mAb against CD38A, CD38A+ populations among IgM+ B cells and IgM- leukocytes of head kidney (HK) were screened via flow cytometry. The IgM+ CD38A+ B cells increased post-inactivated Aeromonas salmonicida stimulation in vitro, which produced higher levels of IgM and enhanced B cell differentiation gene transcription than the cells lacking CD38A.The monoclonal antibody (mAb) specific for leukocyte surface markers is the classical approach to identify fish immune cells. Chan et\u00a0al., already applied to identify previously unknown cell markers of teleost immune cell populations. In anterior kidney (AK) of Nile tilapia (Oreochromis niloticus), Wu et\u00a0al. identified five distinct immune cell subsets including B cells, T cells, granulocytes, macrophages, and dendritic cells (DCs) and further uncovered different subsets of B-cell and T cells based on distinct transcriptional level of the transcription factors (TFs) and cytokines. Additionally, Huang et\u00a0al. analyzed scRNA-seq data of Orange-spotted grouper (Epinephelus coioides) with a full-length transcriptome as a reference, which was aimed to develop alternative approach for the fish samples without any published genome. In their study, four cell types including T cells, B cells, monocytes/macrophages (Mo/M\u03c6) and NCC (non-specific cytotoxic cells) were identified and two subsets of Mo/M\u03c6 (M1 and M2 type), as well as four subsets in B cells . Moreover, the finding of syngnathid fish, Syngnathus typhle (a fish species lacking the spleen and major histocompatibility class II (MHC II) pathway) by Parker et\u00a0al. indicated that the loss of CD4+ T cells accompanied the loss of the MHC II pathwapresencehe present of regulatory T cells and cytotoxic T cells.The scRNA-seq is a newly developed technique that, also reviewed by Smith et\u00a0al. examined the mRNA transcriptome profiles at different stage of Atlantic salmon adherent head kidney leukocytes (HKLs) using microarray, and revealed that the adherent HKL cell population differentiates in vitro to become macrophage-like without exogenous stimulation, which might be regulated by miRNA and targeted differentially expressed genes (DEGs) associated with macrophage differentiation and function. Using transcriptome analysis, Park et\u00a0al. revealed that the macrophage heterogeneity in adherent intestinal cells (AIC) and adherent head kidney cells (AKC), as well as the functional characteristics of mucosal and systemic macrophages in Atlantic salmon. Their data also suggested that interaction of miRNA and mRNAs related to macrophages and epithelial cells were involved in macrophage activation and differentiation.Although the scRNA-seq technique develops rapidly and contributes to the research field of fish immune cell, the analysis of transcriptome profile is still a reliable method to clarify the activity and development of specific immune cell type in fish. In summary, all collected articles in this Research Topic exhibited recent novel works regarding fish immune cell identification and provided new strategy to better uncover the composition and function of teleost immune system, which can also help to expand our understanding for the evolution of the vertebrate immune system.All authors contributed to this editorial insight and approved the submitted version.This work was supported by National Natural Science Foundation of China , Natural Science Foundation of Guangxi (2020GXNSFAA297243), Technology Planning Project of Guangdong Province of China (grant no. 2015A020209181), National Key R&D Program of China (2018YFD0900501), Independent Project of Southern Marine Science and Engineering Guangdong Laboratory (Zhanjiang) (No. ZJW-2019-06), Special Foundation for \u201cAchieving the First Class\u201d of Guangdong Province , The Project of Guangxi Mangrove Coastal Wetland Ecological Protection and Sustainable Utilization Talent Highland (BGMRC202101), Guangdong South China Sea Key Laboratory of Aquaculture for Aquatic Economic Animals, Guangdong Ocean University (No. KFKT2019YB11), the Japan Society for the Promotion of Science KAKENHI Grants .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."} +{"text": "K-edge HERFD-XANES is evaluated in mine waste samples and synthetically generated mixtures of reference compounds.The improvement in As speciation resulting from As 2O3), orpiment (As2S3), getchellite (AsSbS3), arsenopyrite (FeAsS), ka\u0148kite (FeAsO\u00ad4\u00b73.5H2O), scorodite (FeAsO4\u00b72H2O), sodium arsenate (Na3AsO4), and realgar (As4S4) were selected for their importance in mine waste systems. Statistical methods of principal component analysis and target transformation were employed to determine whether HERFD improves identification of the components in a dataset of mixtures of reference compounds. LCF was performed on HERFD- and total fluorescence yield (TFY)-XANES spectra collected from mine waste samples. Arsenopyrite, arsenolite, orpiment, and sodium arsenate were more accurately identified in the synthetic HERFD-XANES spectra compared with the transmission-XANES spectra. In mine waste samples containing arsenopyrite and either scorodite or ka\u0148kite, LCF with HERFD-XANES measurements resulted in fits with smaller R-factors than concurrently collected TFY measurements. The improved accuracy of HERFD-XANES analysis may provide enhanced delineation of As phases controlling biogeochemical reactions in mine wastes, contaminated soils, and remediation systems.High-energy-resolution fluorescence-detected (HERFD) X-ray absorption near-edge spectroscopy (XANES) is a spectroscopic method that allows for increased spectral feature resolution, and greater selectivity to decrease complex matrix effects compared with conventional XANES. XANES is an ideal tool for speciation of elements in solid-phase environmental samples. Accurate speciation of As in mine waste materials is important for understanding the mobility and toxicity of As in near-surface environments. In this study, linear combination fitting (LCF) was performed on synthetic spectra generated from mixtures of eight measured reference compounds for both HERFD-XANES and transmission-detected XANES to evaluate the improvement in quantitative speciation with HERFD-XANES spectra. The reference compounds arsenolite (As The oxidation of residual As-sulfide minerals or dissolution of As-bearing minerals in mine wastes releases dissolved As. Predicting the geochemical fate of As is important when designing remediation or prevention measures in mine wastes and other contaminated systems. Precipitation of stable and sparingly or insoluble minerals that incorporate or contain As promotes the attenuation of As. Understanding the mechanisms of As adsorption and mineralization processes, as well as the geochemical conditions that favour As attenuation, provides a foundation for the design of As remediation systems.et al., 1998et al., 2009et al., 2013et al., 2005et al., 2005et al., 2009et al., 2009et al., 2016et al., 2004et al., 2006et al., 2018et al., 2019et al., 2020et al., 2021et al., 2022et al., 2010et al., 2009et al., 2017et al., 2018et al., 2019Arsenic mobility in environmental systems has been extensively studied ; (2) low concentrations of elements of interest wet chemical methods that rely on pre-treatments that convert the solid phase to a liquid- or gas-phase sample, et al., 2009et al., 2002et al., 2002et al., 2006et al., 2007et al., 2009et al., 2009et al., 2011et al., 2014et al., 2017et al., 2018et al., 2014et al., 2021K-edge was developed to improve the detection limit, increase the resolution of the features in XANES spectra and principal component analysis (PCA) techniques were employed to evaluate improved spectral-feature resolution in HERFD-XANES compared with transmission-detected XANES for standard compounds. Arsenic speciation analyses, HERFD-XANES, and total fluorescence yield (TFY)-XANES were conducted to determine the impact of detection mode on the quantification of As species in mine waste samples. The findings of this study will allow us to evaluate the benefits of HERFD-XANES over conventional detection modes for As speciation as applied to both standard compounds and environmental samples.2.2.1.2O\u00ad3) and sodium arsenate (Na3AsO4) and mineral specimens of orpiment (As2S3), getchellite (AsSbS3), arsenopyrite (FeAsS), ka\u0148kite (FeAsO\u00ad4\u00b73.5H2O), scorodite (FeAsO4\u00b72H2O), and realgar (As4S4) . The mineral specimens contained a combination of host rock and the mineral of interest, which was visually identified and removed from the host rock with a fine-tipped diamond Dremel to avoid contamination. Environmental samples were collected from Long Lake Mine, an abandoned gold mine in Sudbury, ON, Canada, that operated intermittently from 1909 to 1939. Samples were collected in aluminium tubing using a piston-coring technique . Solid samples were then freeze-dried and stored at 4\u00b0C to preserve their mineralogical composition. All samples and reference compounds were finely ground with an agate mortar and pestle in an anaerobic chamber in preparation for analysis. Samples and reference compounds were transported in an anerobic canister and removed immediately prior to analysis.Arsenic reference compounds included reagent-grade arsenic trioxide was performed to confirm the identities of the arsenic reference compounds. Ground reference compounds were loaded into Kapton capillaries and sealed at both ends with Locktite 454 ep\u00adoxy for PXRD analysis. Analyses were performed at CMCF-BM at the Canadian Light Source in Saskatoon, SK. Specifications of the beamline and endstation are fully described elsewhere was used for further data processing, including diffractogram background subtraction and phase identification with the peak search and match algorithm using the COD-Inorg REV218120 2019.09.10 database .Diffraction images were background-subtracted, calibrated, and integrated using 2.3.Hephaetus . The energy resolution of the crystal analyzer is approximately 1\u2005eV. Normally, filters can be applied upstream of the sample to attenuate and lower the intensity of the incident beam to optimize the count rate for fluorescence yield (FY) detection; however, retaining the maximum signal intensity is critical for HERFD. When FY and HERFD were measured simultaneously, the FY count rate was optimized using aluminium foil to directly filter the signal before it entered the detector.All XAS measurements were performed at 20-ID-C at the Advanced Photon Source at the Argonne National Laboratory in Lemont, IL, USA, where monochromatic X-rays in the hard X-ray regime are produced by an insertion device and a monochromator (Si 311). Ground reference compounds were spread thinly on Kapton tape and layered to achieve the appropriate thickness for collection of transmission-detected XAS. The thickness, calculated for each reference mineral using 2.4.LARCH , orpiment (As2S3), getchellite (AsSbS3), arsenopyrite (FeAsS), ka\u0148kite (FeAsO4\u00b73.5H2O), scorodite (FeAsO4\u00b72H2O), sodium arsenate (Na3AsO4), and realgar (As4S4). Computer-generated spectra of mixtures were created with the spectra from each of the reference compounds, measured in both HERFD and transmission. All computer-generated mixtures were a combination of four reference compounds, each with a minimum contribution of 5% of the total mixture; these limitations were selected to represent speciation with a commonly acceptable number of fit components and detection limit. The computer-generated mixtures were analyzed with LCF and PCA. The datasets of mixtures were created with both HERFD and transmission reference compounds and each unique reference compound was included in 3500 synthetic spectra. A dataset was generated for each of the 70 combinations of four reference compounds to decrease sampling bias. A set of 100 random mixture ratios was generated using the NumPy module . LCF analysis on the mine waste samples was completed in Athena . A small shift of \u22120.5 to \u22121\u2005eV is observed in white-line energy in HERFD measurements compared with transmission measurements ; variations in set-up angles were minimized by the magnetically positioned sample holder and consistent sample mounting technique ensuring the sample surface position was unchanged between samples and references. This configuration allows comparison of measurements across samples and reference compounds.The resolution increases observed in the HERFD-XANES spectra are similar to other studies , scorodite , and sodium arsenate reference spectra for both the HERFD and transmission data, but additional components did not further decrease the residual standard deviation. The synthetic mixtures of arsenopyrite, arsenolite, sodium arsenate, and scorodite were selected for PCA and target transformation because each had unique spectral features . The longer air-path to the area detector used for HERFD measurements could attenuate up to 56% of the As K\u03b1 fluorescence emission which would decrease the signal-to-noise ratio. A helium bag placed in the air-path would decrease the attenuation and improve the signal-to-noise ratio. However, improved speciation using HERFD-XANES was observed in samples of As-bearing mill tailings collected from the Long Lake mine, which were identified to have a binary mixture of arsenopyrite and scorodite or kankite and arsenopyrite, scorodite and kankite are likely chemical components of the dataset whereas getchellite, orpiment, realgar, arsenolite and sodium arsenate are unlikely to be chemical components of the dataset . On average, the LCF on HERFD-XANES spectra obtained from Long Lake samples with binary mixtures resulted in fits with significantly lower R2 than fits on FY XANES spectra obtained from the same samples could be impacted by over-absorption (self-absorption) that dampens the amplitude of the XAFS oscillations were not measured on the same samples used for XAS measurements. If over-absorption is present in the samples, the quality of the fits in both HERFD and TFY would be impacted.Sample spectra collected using FY detection were fit with reference compounds measured in transmission detection, which could be a source of misfit between the sample and standard spectra. Spectra collected in HERFD or TFY from thick samples containing high As concentrations (Table S3) showed no improvement in R2 with HERFD-XANES spectra. The lack of improved fit quality may be attributed to reference materials that do not exactly represent, but have similar molecular bonding environments to, the species present in the sample. Getchellite, orpiment and realgar are rare As-sulfide minerals that are not often identified as secondary minerals in oxidized mine waste environments; however, the spectra for these minerals are similar to arsenical pyrite (arsenopyrite), As(III)\u2013S (getchellite), As(III)\u2013O (arsenolite) and As(V) (sodium arsenate) . Arsenopyrite was identified in some of the tailings samples with conventional XRD measurements, and Fe\u2013As\u2013O-containing phases were previously identified with SEM-EDS and As(V) sorbed to various relevant iron oxides and iron hy\u00addroxy-sulfates, such as goethite, ferrihydrite, jarosite, and hematite. XANES and EXAFS studies show that As sorbs to and is incorporated into secondary precipitates .HERFD-XANES provides resolution of unique spectral features that may not be detectable with transmission-detection XAS analyses, and this additional resolution enhances LCF fitting when the appropriate standards are available. Slight improvements in spectral resolution are also expected when As is dilute or in a complex matrix, which is one of the main benefits of HERFD for environmental samples. Developing a database of environmentally relevant As reference compounds is vital to the application of LCF with HERFD-XANES to quantitatively determine the speciation of As in mine wastes and other haza\u00adrdous materials. Use of standard spectra from a HERFD-XANES database is critical with respect to the application of this technique (10.1107/S1600577522007068/ye5018sup1.pdfDetails on Datasets 1 and 2. Figures S1 to S15. Tables S1 to S3. DOI:"} +{"text": "Vascular disease is now the leading cause of death worldwide, with cardiovascular and cerebrovascular diseases being the main contributors . HoweverYu et al. attempted to illustrate the association between plasma metabolic profiles and cerebral collateral circulation in patients with acute ischemic stroke (AIS). They found that the sphingosine-1-phosphate (S1P) level in plasma showed significant positive correlation with good collateral circulation and which might be a potential diagnostic biomarker for predicting collateral circulation status in patients with AIS. The paper by Xue et al. focused on excavating the potential biomarkers in lacrimal diabetic angiopathy and its potential mechanism. Hub genes App, F5, Fgg, and Gas6 related to the regulation of circulation and coagulation were identified. Meanwhile, certain small molecular compounds were considered that might reverse the altered differentially expressed genes. This study might empower the novel potential targets to treat lacrimal angiopathy and other diabetes-related diseases. The paper by Fan et al. found that myricanol could inhibit proliferation and migration of vascular smooth muscle cells (VSMCs) induced by platelet-derived growth factor-BB by suppressing the platelet-derived growth factor receptor-\u03b2 and NF-kB p65 translocation. Furthermore, myricanol was found suppressing the intimal hyperplasia in mice with carotid stenosis. The paper by da Costa et al. found the events that HFD-fed leading decreased Nrf2 nuclear accumulation, decreased mRNA expression and activity of Nrf2-regulated enzymes were prevented in castrated mice. The study indicate that testosterone would downregulate Nrf2, leading to oxidative stress and vascular dysfunction in HFD-fed obese mice.As the dominating part of this Topic, papers of basic research that we primarily collected have revealed some interesting relationships between metabolic abnormalities and vascular diseases. The paper by Hu et al. showed that there was no significant correlation between increased serum uric acid levels and the risk of first stroke in the Chinese adults with hypertension. Meanwhile, risk of the first stroke in patients with hyperuricemia less than 60 years old was significantly higher than control. The paper by Yu et al. was based on bioinformatic analysis of metabolomic and proteomic to reveal that the dysregulation of glutamate and glycine metabolism, upregulated glycolysis and fatty acid synthesis in the endothelial progenitor cells that treated with the oscillatory shear stress.However, papers of clinical research presented in this Topic have significant discoveries as well. The paper by Ning et al. with creative perspective summarized the potential mechanism underlying metabolic perturbation that type 2 diabetes mellitus (DM) affect the hypertension (HTN), what may be involved in the metabolism of insulin and angiotensin II, sympathetic nervous system as well as the energy reprogramming.Meanwhile, the Review paper by"} +{"text": "In this Research Topic several studies have analysed the clinical and molecular profile related to cardiovascular disease (CVD) development in a range of inflammatory and systemic autoimmune diseases, including systemic lupus erythematosus (SLE), psoriatic arthritis, rheumatoid arthritis and spondylarthritis among others. Novel molecular mechanisms and biomarkers have been characterized along with clinical tools and approaches to manage this prevalent comorbidity.Quevedo-Abeledo et\u00a0al., studied the levels of three CVD molecules involved in the metabolism of triglycerides, such as lipoprotein lipase (LPL), angiopoietin-like protein 4 (ANGPLT4), and apolipoprotein C-III (ApoC3), in 185 patients with SLE and 162 healthy donors. Performing a multivariable analysis, they identified that the LPL, ANGPLT4 and ApoC3, axis is altered in SLE and associated with the disease damage score. Wang et\u00a0al., used public transcriptomic data to gain insight into the involvement of new players and pathways related to the development of atherosclerosis in SLE patients. They identified 5 hub genes which might promote the monocytes differentiation into macrophages and the pathway of IL-17 signalling as potential mechanisms involved in the atherosclerotic process of SLE patients. Guzman-Martinez et\u00a0al., reviewed the association of the immune system with the pathogenesis of endothelial damage and atherosclerosis in SLE patients, including inflammatory mediators, specific circulating cell populations and autoantibodies. Additionally, they discussed the utility of the immune system as early CVD biomarkers and therapeutic targets in SLE.In the case of SLE, three studies have explored at the molecular level the underlaying mechanisms associated with CVD. Remuzgo-Mart\u00ednez et\u00a0al., analysed the role of the protein irisin as a serological and genetic biomarker of CV risk, disease severity and subclinical atherosclerosis in a cohort of 725 patients with axial spondylarthritis (axSpA). Their results suggested that low levels of irisin in the serum could be considered a marker of high CV risk, more severe disease and the presence of subclinical atherosclerosis, in axSpA patients. Furthermore, they found that irisin may also constitute a biomarker of disease activity in axSpA at the genetic level.On the other hand, Barbarroja et\u00a0al., reviewed the interplay of hepatic alterations and CVD, analysing different mechanisms where autoimmunity, chronic inflammation, metabolic deregulation and treatments seem to have an key role. They also discussed the latest controversies regarding the effects of anti-inflammatory therapies used in PsA and RA in the liver damage, such as biologics and DMARDs such as, leflunomide or methotrexate. Schwartz et\u00a0al., also analyzed in a cohort of PsA patients, the associations of metabolic dysregulation and systemic inflammation with coronary artery disease (CAD) measuring traditional CVD risk factors, serum markers of metabolic dysfunction, inflammatory cytokines and inflammation in specific tissues by using positron emission tomography-computed tomography (PET-CT). They identified metabolic and inflammatory players associated with subclinical CAD in PsA, including inflammation in adipose tissue which might be considered as novel target for CVD prevention and treatment in PsA.In the case of inflammatory arthritis like rheumatoid arthritis (RA) and psoriatic arthritis (PsA), Gao et al., using mendelian randomization and genome-wide association study (GWAS). The analysis suggested a potential causal link between CVD and psoriasis.The association between Psoriasis and the risk of CVD was also explored by Lastly, two studies in this issue evaluated the CV involvement in rare systemic autoimmune disorders.Zhou et\u00a0al., analyzed in anti-melanoma differentiation-associated gene 5 (anti-MDA5) antibody-positive dermatomyositis (DM)//clinically amyopathic dermatomyositis (anti-MDA5 Ab+ DM/CADM) the myocardial involvement. Anti-MDA5 dermatomyositis is a rare autoimmune disease, with high prevalence in Japanese patients with clinically amyopathic dermatomyositis and progressive interstitial lung disease. It also shows systemic manifestations affecting skeletal muscle, skin, and other organs.In a cohort of seventy-six hospitalized patients suffering this disorder, this study identified twelve patients with MI (16%), associated with severe cardiovascular complications and adverse evolution of the disease. They concluded that myocardial involvement is an independent poor prognostic factor of patients with anti-MDA5 Ab+ DM/CADM.Huang et\u00a0al., investigated the efficacy of percutaneous transluminal pulmonary angioplasty (PTPA) in patients with Takayasu arteritis (TA) and pulmonary hypertension (PH) and pulmonary artery stenosis.Takayasu\u2019s arteritis is a rare chronic inflammatory autoimmune condition that impact the largest blood vessels in the body (aorta) and its branches, the supra-aortic trunks.Results of this study, analyzing 183 lesions from 79 surgeries carried out on 32 patients with TA and PH, indicated that PTPA improved clinical symptoms, hemodynamic parameters, and exercise tolerance, in patients with TA pulmonary artery stenosis and PH. Furthermore, reperfusion pulmonary edema was significantly reduced, and no patient died of PTPA-related complications with guidance from the pressure wire.Overall, these findings might be important for healthcare resource planning and preventive approaches. These 9 articles substantial CV-risk observed in autoimmune diseases patients, suggests that strategies to reduce CV-risk should become a routine part of the management of autoimmune disease patients. However, the causes of cardiovascular involvement associated with systemic autoimmune diseases, and their potential therapy, require further research.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."} +{"text": "Organisms respond and adapt to the internal and external environmental changes through various cell signaling. The abnormal alteration of cell-signaling pathways could disrupt cell homeostasis and cause diseases . For insCell Signaling Status Alteration in Development and Disease, we collected studies providing new insights into the roles of cell signaling in development and disease occurrence and progression. A total of 5 articles, including two reviews, two original researches and one method article, were published in this Research Topic. We summarize and discuss the main findings of these studies in this editorial.Although cell signaling can be measured through development of sequencing technologies and the application of effective bioinformatic tools , many quAbreu de Oliveira et al.). Yang et al. comprehensively reviewed the role of cyclic GMP-AMP synthase (cGAS)\u2014a stimulator of the interferon gene (STING) signaling pathway in various diseases, such as acute injury, pneumonopathy and kidney diseases, providing a theoretical basis for immunotherapy targeting the STING signal pathway . Ma et al. found that miR-654-5p overexpresses in activated human hepatic stellate cells and TGF\u03b2-treated human hepatic LX-2 cells augmented liver fibrosis in mice that were intraperitoneally injected with CCl4 by targeting the RXR\u03b1 receptor . Zhang et al. proposed a robust method to acquire finely resolved transcriptional programs with few cells from snap-frozen or RNAlater-treated clinical tissues that was sufficient enough to resolve even isoforms based on immunofluorescence-guided laser capture microdissection (immuno-LCM-RNAseq). With this method, the researchers were able to analyze transcriptional networks and signaling pathways during development, pathogenesis, and aging of specific cell types within native tissues . Lou et al. integrated both immune and hypoxia signaling to establish reliable prognostic signatures for lung adenocarcinoma (LUAD) across different omics data, and provided a robust prognosis predictor for the LUAD patients .Wnt signaling plays an important role in the mammary gland development and adult homeostasis in virtually all animal species. Willy et al. provided a systematic review on Wnt signaling involved in breast cancer and explored the impact of Wnt signaling alteration on the tumorigenesis and disease occurrence (The studies published in this Research Topic presented a diversity of intriguing and meaningful results covering a range of cell signals, which could facilitate our understanding of development and disease. We hope that this Research Topic will inspire researchers to systematically investigate development and disease progression from the perspective of cell signal in a systematic way."} +{"text": "Insights in Cardiovascular Therapeutics: 2021\u201d has achieved great success and is attracting interest from the cardiovascular community. Here, we spotlight 12 studies published in our section that related to cell death and cardiovascular injuries, as well as some recent advances in the field that have tremendous potential in cardiovascular therapy. In addition, these highlights may serve as the foundation for some new developments in our Cardiovascular Therapeutics areas. In 2022, we will keep working to create a fantastic platform for cardiologists, translational cardiovascular scientists, and cardiovascular pharmacological scientists to share new results and data in clinical cardiology and translational cardiovascular therapeutics.With the effort and support of the authors, editorial office, and editorial team, the Frontiers in Cardiovascular Medicine, Cardiovascular Therapeutics Section-Research Topic \u201cWu et al., Liao et al., and Dash et al., atrial fibrillation reported by Lee et al. and Zheng Wang et al., refractory angina reported by Ambari et al., In-stent restenosis reported by Zhu et al., critical limb ischemia reported by Quiroz et al., protein conformational diseases reported by Zheng Song et al., mitochondrial dysfunction reported by Chen et al., and myocardial injury reported by Barbieri et al. and Cao et al.Cardiovascular diseases (CVDs) are the leading cause of morbidity and mortality worldwide. An estimated 17.9 million people live with CVDs each year with no effective cures \u20134. In thIn recent decades, new mechanisms that orchestrate various cell death pathways have been discovered, and this field continues to expand. The current well-established forms of cell death pathways include intrinsic or extrinsic apoptosis, necroptosis, pyroptosis, ferroptosis, mitochondrial permeability transition (MPT)-driven necrosis, autophagic cell death (autosis), lysosome-dependent cell death, immunogenic cell death , cellula5 myocytes production pathway and has initially discovered in response to viral infections. Following infection with a virus such as influenza A virus (IAV), a master regulator of PANoptosis, Z-DNA-binding protein 1 (ZBP1) , 42, int1 (ZBP1) . It is b1 (ZBP1) , resulti1 (ZBP1) . PANopto1 (ZBP1) . AlthougWu et al., Zheng et al., Wang et al., Liao et al., Dash et al., Lee et al., Ambari et al., Zhu et al., Quiroz et al., Zheng Song et al., Chen et al., Barbieri et al., and Cao et al. on our Research Topic to illustrate the cutting-edge treatments for different cardiovascular diseases. Readers could use Medical experts and scientists have long searched for potential cardiac disease treatments and surviving and improving patients' lives. The Frontiers in Cardiovascular Medicine -Cardiovascular Therapeutics section has provided a platform for distinguished scientists to communicate, inspire, and seek more potential therapeutic solutions , 47. In KX carried out literature collections, research analyses, and drafted the manuscript. MK, JY, NS, SW, RV-P, and HW provided editing input. XY supervised and edited the manuscript. All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.This work was supported by the National Institutes of Health Grants to XY , HW , JY (HL153599), MK (HL135177), and America Heart Association Award to KX (916828).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."} +{"text": "Pogostemon cablin ameliorates ethanol-induced acute liver injury in rats via inhibition of oxidative stress and lipid accumulation\u2019 by Qiong-Hui Huang et al., RSC Adv., 2018, 8, 24399\u201324410.Correction for \u2018Patchouli oil isolated from the leaves of The authors regret that Corrected Fig. 1 caption: GC-MS analysis of patchouli oil. Numbers indicated on peaks correspond to those in The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "Mesenchymal stromal/stem cell (MSC) therapies are increasingly explored as novel regenerative and immunomodulatory approaches to treat or prevent diseases is the only curative option available today for several hematological and non-hematological diseases. However, HSCT is associated with several early and late complications, such as low HSPC engraftment and impaired hematopoietic recovery, immune-mediated graft rejection, and GvHD in the case of allotransplantation : a potential game changer for the COVID-19 crisis,\u201d to employ their immunomodulatory and regenerative properties for lung repair. In turn, Yang et al. from the \u201cCenter for Respiratory Medicine\u201d at the China-Japan Friendship Hospital in Beijing, China, review \u201cTherapeutic applications of MSCs in IPF.\u201d The authors present an in-depth analysis of relevant factors for the optimization of MSC treatment, including the use of MSCs from different sources for lung repair, different administration routes, timing, dosing, frequency, and potential pretreatment of the clinical MSC products, to guide the design of future clinical research and to identify novel therapeutic options for these complex diseases.Due to the perivascular nature of MSCs surrounding and maintaining the integrity and function of blood vessels : a liver-derived MSC-like population with pro-regenerative properties.\u201d These HLSCs were first described in 2006 as a new stem cell population obtained from the highly vascularized healthy human livers . The most studied MSC types were isolated from umbilical cord (UC-MSCs), adipose tissue (AT-MSCs) and bone marrow (BM-MSCs). The most studied diseases were wounds, ulcers, burns and psoriasis. Montero-Vilchez et al. report in their article \u201cMSC-conditioned medium for skin diseases: a systematic review\u201d that MSC-CCM improved wound healing, hair restoration, skin rejuvenation, atopic dermatitis, and psoriasis, in both animal models and humans, and decreased hypertrophic scars and flap ischemia in animal models, with further studies needed to standardize CCM manufacturing. In addition, Qui\u00f1ones-Vico et al. reviewed \u201cThe role of exosomes derived from MSCs in dermatology\u201d for both in vitro and in vivo models of different skin conditions.The skin is the largest organ of the human body and skin injuries can damage this important barrier. Three review articles were contributed to our topic from researchers at the \u201cAndalusian Network of Design and Translation of Advanced Therapies\u201d at \u201cVirgen de las Nieves University Hospital\u201d in Granada, Spain. These reviews give an excellent overview on the treatment of skin disease with \u201ccell products\u201d and \u201ccell-conditioned\u201d media (CCM), as well as \u201cMSC-derived exosomes\u201d in dermatology. Khan et al. from \u201cThe Interdisciplinary Stem Cell Institute\u201d at the University of Miami share their experiences in their article: \u201cThe Interdisciplinary Stem Cell Institute's Use of Food and Drug Administration-Expanded Access Guidelines to Provide Experimental Cell Therapy to Patients With Rare Serious Diseases.\u201dIn some cases, patients present a life-threatening condition or serious disease that requires the urgent access to investigational medical product . For those cases, the U.S. Food and Drug Administration (FDA) may authorize expanded access, to gain access to an investigational medical product for treatment outside of clinical trials. An in-depth understanding of the molecular mechanisms underlying MSCs' beneficial therapeutic effects in different clinical indications is essential to manufacture safe and effective therapies tailored to the individual patient need and endothelial progenitor cells (EPCs) -MSC-derived EVs, thus suggesting that the EVs confer neuroprotection to the damaged hippocampus. De Luna et al. reviewed the role of \u201cMSC-derived EVs as silver linings for cartilage regeneration.\u201d Ghafouri-Fard et al. summarize \u201cThe emerging role of exosomes in the treatment of human disorders with a special focus on MSC-derived Exosomes.\u201d Zhong et al. review \u201cThe emerging potential of exosomes on adipogenic differentiation MSCs\u201d. Zhang J. et al. review the \u201cPotential networks regulated by MSCs in acute-on-chronic liver failure (ACLF): exosomal miRNAs and intracellular target genes,\u201d identifying beneficial effects of MSC-derived exosomes. Zhang Y. et al. investigated the therapeutic potential of exosomes derived from amniotic fluid stem cells in a rat model of skin wound repair. They found accelerated wound healing and improved regeneration of hair follicles, nerves, vessels, and increased proliferation of cutaneous cells, and more natural distribution of collagen during wound healing, while preventing excessive aggregation of myofibroblasts and the extracellular matrix. Lai et al. found that AT-MSC-derived exosomes decreased cardiomyocyte apoptosis and hypertrophy in the heart of mice with myocardial ischemia/reperfusion injury, concluding that they act as potent cardioprotectors.The production and release of EVs, including exosomes, from MSCs is perceived to be a major mediator of their therapeutic effects for several pathological conditions influence the secretome of MSCs and their interaction with monocytes from stroke patients to promote the beneficial antiinflammatory effect of MSCs. In turn, Maisonneuve et al. shed light on how human dental pulp derived stromal cells (DPSCs) react toward interaction with Streptococcus mutans, which is involved in dental pulp necrosis and cardiovascular tissue infections. In response to infection, DPSCs adopted a proinflammatory profile, strengthening the establishment of the dental pulp inflammation.in vivo characterization and cell sorting, preconditioning of MSCs, cellular engineering and the use of structural supports/3D systems.Recent research efforts focus on strategies for further improving the therapeutic efficacy and safety of MSCs and their products, thus helping to achieve better treatment outcomes. These entail amongst others improved Valiuliene et al. from the \u201cLife Sciences Center\u201d at Vilnius University in Lithuania studied the \u201cMetabolic profile and neurogenic potential of human amniotic fluid MSC-like stem cells from normal versus fetus-affected gestations.\u201d The authors found some characteristic differences in surface markers, phosphorylation rate, ATP, ROS, and cytokine secretion between the two groups, which may indicate metabolic and phenotypic differences that need to be considered in their clinical use. Xie et al. from Sun Yat-sen University in Guangzhou, China, studied how \u201cCardiac-derived CD51-positive MSCs enhance cardiac repair through SCF-mediated angiogenesis in mice with myocardial infarction.\u201d Most vascularized tissues contain resident MSCs, but there is no typical marker to identify resident cardiac MSCs, and the authors here identify CD51 as a novel marker for cardiac resident MSCs.Zhao et al. demonstrate that hypoxic culture preconditioning may constitute a promising strategy to enhance cellular viability and angiogenesis of transplanted AT-MSCs. Lucciola et al. have studied the \u201cImpact of sustained TGF-\u03b2-receptor inhibition on chromatin accessibility and gene expression in culture human endometrial EM-MSCs.\u201d The authors found that A83-01, a selective TGF-\u03b2-receptor inhibitor, promotes the expansion of EM-MSCs in culture by blocking differentiation and senescence, with in-depth analysis of the underlying gene networks and genome-wide chromatin changes. Bai et al. report how \u201cGlycyrrhizic acid promotes osteogenic differentiation of human BM-MSCs by activating the Wnt/\u03b2-catenin signaling pathway,\u201d thus indicating the potential of pharmacological pretreatment for improvements in bone repair. Preconditioning of MSCs also modifies the content and secretion of EVs. Peltzer et al. present that \u201cIFN-\u03b3 and hypoxia priming have limited effect on the miRNA landscape of human MSC-derived EVs.\u201d Li et al. found that \u201cmiRNA-27a-5p is abundant in small EVs derived from epimedium (EPI)-pre-conditioned BM-MSCs stimulate osteogenesis by targeting Atg4B-mediated autophagy.\u201d Feng et al. reviewed the \u201cEffect of melatonin for regulating MSCs and their EVs\u201d in preclinical and clinical studies.An injured tissue constitutes an ischemic and hypoxic environment that damages the implanted therapeutic cells, leading to apoptosis and thus compromising their capacity in the early stages of cell transplantation. Garcia-Bernal et al. from Murcia in Spain studied how \u201cExofucosylation of AT-MSCs alters their secretome profile.\u201d The authors found that the exofucosylation modification that improves MSC in vivo trafficking also improved their secretome to promote their antiinflammatory properties, which could be beneficial in treatment of autoimmune, inflammatory, and degenerative diseases. Pawitan et al. reviewed the \u201cEnhancement of the therapeutic capacity of MSCs by genetic modification.\u201d Of the 85 articles reviewed, 51 studies focused tumor/cancer/metastasis, while 34 studies focused on non-cancerous pathologies. In line, Han et al. studied how \u201cKnockdown of POSTN inhibits osteogenic differentiation of MSCs from patients with steroid-induced osteonecrosis,\u201d while Zhang W. et al. studied how \u201cUpregulation of PARKIN accelerates osteoblastic differentiation of BM-MSCs and bone regeneration by enhancing autophagy and \u03b2-catenin signaling.\u201dLin et al. from \u201cTsing Hua University\u201d in Taiwan demonstrate how \u201c3D-spheroids of UC-MSC-derived Schwann Cells (SCs) promote peripheral nerve regeneration.\u201d The authors used a well-defined non-genetic approach to phenotypically, epigenetically, and functionally convert UC-MSCs into SC-like cells that simulated sprouting of neurites from neuronal cells. To enhance their therapeutic effectiveness, the authors assembled the cells in 3D spheroids with a marked increase in their neurotropic, proangiogenic and anti-apoptotic profile. Marinaro et al. from C\u00e1ceres in Spain tested \u201cLaparoscopy for the treatment of congenital hernia: Use of surgical meshes and MSCs in a clinically relevant animal model.\u201d The authors combined laparoscopy and stem cells incorporated into a mesh by using a fibrin-sealant solution for the treatment of hernia in a swine model, with improved in vivo performance in the MSC containing group compared to cell-free mesh.Among the strategies to preserve MSC biological functions are those mimicking the natural habitat of MSCs during cell therapy. This multidisciplinary Research Topic has brought together specialists in Stem Cells, Immunology, Bioengineering, and Cellular Neuroscience, to share their knowledge on current therapeutic strategies and the mechanisms underlying the immune modulation and tissue regeneration that are orchestrated by MSCs. The different articles contained in this Research Topic reflect the great interest, knowledge and innovation in these fields. Here, we have given a summary on the latest clinical developments, efforts to refine the understanding on the MoA of MSC therapeutics, and introduced novel strategies to enhance the efficacy of MSCs and their derived products. Importantly, inflammation and the regulation thereof is an essential part of the healing and regenerative cascades in the human body. The modulation of inflammation is an important aspect of MSCs beneficial properties in the healing of tissue injury or suppression of tissue damage. The many studies contained in this Research Topic indicate that MSCs employ a host of mediators and mechanisms to achieve this goal, with great promise for clinical use.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.VC-G receives support of the Consejer\u00eda de Transformaci\u00f3n Econ\u00f3mica, Industria, Conocimiento y Universidades co-funded by Fondos FEDER (PY20/00481), the Institute of Health Carlos III co-funded by Fondos FEDER (CP19/00046 and PI20/00341), the Fundaci\u00f3n Cient\u00edfica de la Asociaci\u00f3n Espa\u00f1ola Contra el C\u00e1ncer (IDEAS20051CAPI), and the Asociaci\u00f3n Pablo Ugarte (+VIDA project). VH-P receives funding from the Spanish Ministry of Science, Innovation and Universities (PCI2018-093062) and the Valencian Council for Innovation, Universities Science and Digital Society (PROMETEO/2019/075). Contributions from GM were made possible by the German Research Foundation (DFG) and the German Federal Ministry of Education and Research (BMBF) through the BSRT (GSC203) and BCRT, respectively, and in part supported by the European Union's Horizon 2020 Research and Innovation Program (Horizon 2020 Framework Program) under the Grant Agreements No. 733006 (PACE) and No. 779293 (HIPGEN).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."} +{"text": "The literature demonstrates that infections like Borrelia and Lyme, as well as other infections like group A beta-hemolytic streptococcal (GABHS), can cause an autoimmune reaction and associated antibodies target dopaminergic loci in the mesolimbic region of the brain, which interferes with brain function and potentially causes RDS-like symptoms/behaviors. The treatment of PANDAS remains controversial, especially since there have been limited efficacy studies to date. We propose an innovative potential treatment for PANDAS based on previous clinical trials using a pro-dopamine regulator known as KB220 variants. Our ongoing research suggests that achieving \u201cdopamine homeostasis\u201d by precision-guided DNA testing and pro-dopamine modulation could result in improved therapeutic outcomes.Pediatric autoimmune neuropsychiatric disorders associated with group A streptococcal infections (PANDAS) is a concept that is used to characterize a subset of children with neuropsychiatric symptoms, tic disorders, or obsessive-compulsive disorder (OCD), whose symptoms are exacerbated by group A streptococcal (GAS) infection. PANDAS has been known to cause a sudden onset of reward deficiency syndrome (RDS). RDS includes multiple disorders that are characterized by dopaminergic signaling dysfunction in the brain reward cascade (BRC), which may result in addiction, depression, avoidant behaviors, anxiety, tic disorders, and/or OCD. According to research by Blum Th. Thi.e.,s of RDS .Group A beta-hemolytic streptococcal (GABHS) infections are thought to be the root cause of RDS and a variety of aggressive and depressive behaviors . CumulatThe original concept of PANDAS can be traced back to a clinical trial that was conducted at the National Institutes of Health (NIH) in the US . The nos2.It is currently believed that PANDAS is an autoimmune reaction that, in part, mimics rheumatic fever and differs from the etiological links associated with spectrum disorders or RDS. As a result, rheumatic fever can be thought of as an autoimmune disorder caused by streptococcal infections in which antibodies affect the brain and induce neuropsychiatric conditions. In their article on Tourette\u2019s Syndrome and OCD, Lombroso & Scahill introduced the idea of \u201cmolecular mimicry\u201d as a potential explanation for PANDAS . Accordiet al. in 1998 ..et al. iet al. conducted a study involving 311 subjects who self-reported neuropsychiatric symptoms and also had concurrent group A streptococcal infection status ..et al. a3.et al. came to the conclusion in 2018 that rigorously conducted research regarding treatments for PANDAS is scarce, and published studies have a significant risk of bias and other anti-dopaminergic reward gene risk polymorphisms as measured by genetic risk assessment at the DNA level in compromised pre- and post-adolescence children with PANDAS/CANS. Our ongoing work posits a potential positive clinical outcome as a function of the induction of \u201cdopamine homeostasis\u201d with precision-guided DNA testing and pro-dopamine regulation ."} +{"text": "SARS-CoV-2 spike protein comprises a conserved hydrophobic pocket that binds linoleic acid (LA). LA supplementation interferes with viral entry into the cell and replication, and thus could be used as an antiviral. The COVID-19 pandemic and concomitant lockdowns presented a global health challenge and triggered unprecedented research efforts to elucidate the molecular mechanisms and pathogenicity of SARS-CoV-2. The spike glyco\u00adprotein decorating the surface of SARS-CoV-2 virions is a prime target for vaccine development, antibody therapy and serology as it binds the host cell receptor and is central for viral cell entry. The electron cryo-microscopy structure of the spike protein revealed a hydrophobic pocket in the receptor-binding domain that is occupied by an essential fatty acid, linoleic acid (LA). The LA-bound spike protein adopts a non-infectious locked conformation which is more stable than the infectious form and shields important immunogenic epitopes. Here, the impact of LA binding on viral infectivity and replication, and the evolutionary conservation of the pocket in other highly pathogenic coronaviruses, including SARS-CoV-2 variants of concern (VOCs), are reviewed. The importance of LA metabolic products, the eicosanoids, in regulating the human immune response and inflammation is highlighted. Lipid and fatty-acid binding to a hydrophobic pocket in proteins on the virion surface appears to be a broader strategy employed by viruses, including picornaviruses and Zika virus. Ligand binding stabilizes their protein structure and assembly, and downregulates infectivity. In the case of rhinoviruses, this has been exploited to develop small-molecule antiviral drugs that bind to the hydrophobic pocket. The results suggest a COVID-19 antiviral treatment based on the LA-binding pocket. Spike makes the first contact with the receptor on the host cell, human angiotensin-converting enzyme 2 (ACE2), and mediates infection by virus\u2013host cell membrane fusion . The furin-cleavage site in spike is key to the increased pathogenicity of SARS-CoV-2 . S2 comprises the membrane-fusion machinery, including the fusion peptide (FP), the fusion peptide-proximal region, heptad repeat (HP) 1, a central helix region (CH), HP2, a transmembrane anchor and a cytoplasmic C-terminal tail . The central helices of spike form a coiled-coil trimeric core. When spike interacts with ACE2, the S2 core and a second proteolytic site (S2\u2032) become exposed leading to an elongated coiled-coil structure formed by HP1, the central helix and HP2 which positions the N-terminal fusion peptide close to the host cell membrane . In the closed conformation of spike the RBDs are all \u2018down\u2019, resulting in threefold symmetry. Importantly, the closed conformation of spike is considered to be a non-infectious form because the RBM is inaccessible. ACE2 interacts with spike in open states with one, two or all three RBDs pointing \u2018up\u2019 , and it has been suggested that ACE2 interaction enhances the opening of the RBDs of the RBD is accessible Fig. 1b. In thp\u2019 Fig. 1b, and i2.et al., 2016et al., 2016et al., 2020et al., 2006et al., 2020et al., 2021et al., 2021et al., 2020et al., 2020C3-symmetrized structure reached a resolution of 2.85\u2005\u00c5. Surprisingly, the RBDs moved closer to each other compared with available atomic models. In a pocket of the RBD, additional tube-shaped density was identified which could not be attributed to protein and LC-MS-MS, the ligand in the RBD was confirmed to be LA in mammalian cells and stabilization of the spike trimer by a trimerization domain . Notably, the RBM is completely structured, which is not the case in the LA-free closed conformation, where regions including the RBM were disordered. In the presence of LA, the RBM is tucked away between the RBDs and is inaccessible for ACE2 binding , resulting in increased release of LA from the membranes and increased intracellular LA concentration , including LA. The media used for insect-cell culture contain FFAs as additives, thus mimicking the host environment. Secondly, the transfection of a plasmid encoding spike protein into mammalian cells does not faithfully recapitulate SARS-CoV-2 viral infection. The latter causes extensive membrane and lipidome remodelling and cPLA2.1.et al., 2020et al., 2022et al., 2022et al., 2017et al., 2022et al., 2019et al., 2022et al., 2021Sequence alignments of spike proteins of the other human betacoronaviruses SARS-CoV, MERS-CoV, HKU1-hCoV and OC43-hCoV indicated that the hydrophobic pocket in the RBD (named the B domain in HKU1-hCoV and OC43-hCoV) is conserved application of the force resulted in an opening of the closed spike in the absence of LA. In contrast, only 14% of the LA-bound spikes opened on the application of the same force. This demonstrates that LA binding stabilizes the locked state of spike, increasing the energy required to raise a single RBD and reach an open conformation simulations were performed to assess the relative stability of the closed and LA-bound locked spike proteins which coordinates the carboxy headgroup of LA spike was found to contain a lysine-to-asparagine mutation (K417N) leading to loss of a positive charge at the RBD\u2013RBD interface. This may affect the intersubunit LA\u2013RBD interaction and weaken the stabilization effect of LA in this VOC spike , significantly reduced attachment to human cells was observed compared with mini-virions with apo spike devoid of LA. Importantly, saturated fatty acids such as palmitic acid (PA) did not show this inhibitory effect, and the mini-virions bound with similar efficiency as mini-virions with apo spike , an enzyme target for antiviral drug development in a negative-feedback mechanism of SARS-CoV-2 spike (Wuhan) has been shown to bind the heme metabolites bilirubin and biliverdin . Receptor binding to the canyon displaces a hydrophobic \u2018pocket factor\u2019 from a pocket in VP1 below the canyon . This \u2018pocket factor\u2019 in the VP1 pocket has been suggested to stabilize picornavirus assembly. Receptor recognition and concomitant loss of the \u2018pocket factor\u2019 destabilizes the capsid and helps to release the picornavirus RNA genome during infection and vitamin A were shown to have antiviral activity against SARS-CoV-2.\u2005A recent cryo-EM structure reveals that ATRA indeed binds to the hydrophobic pocket of SARS-CoV-2 spike protein . Importantly, ATRA inhibits the entry of SARS-CoV and MERS-CoV pseudoviruses into human cells, confirming the conservation of the hydrophobic pocket by desaturation and elongation (Hanna & Hafez, 2018M (Richieri & Kleinfeld, 1995M in a so-called fatty acid\u2013lipid storm (Yan et al., 2019et al., 2021et al., 2022et al., 2022et al., 2020et al., 2021et al., 20202s releasing long-chain PUFAs from host cell membranes has been implicated in severe COVID-19 (Barberis et al., 2020et al., 2021et al., 2020et al., 2020Under physiological conditions, the levels of unsaturated FFA are maintained below 0.1\u2005\u00b52 activation and lipid metabolome remodelling, which are both common elements of viral infection (Goodwin et al., 2015et al., 2019et al., 2019et al., 2015et al., 2015et al., 2020et al., 2020et al., 2001The dysregulation of FFA levels during COVID-19 is suggested to result from cPLA4.2 and lipid remodelling, allowing the formation of viral replication compartments. LA release from the membranes contributes to the later-stage fatty acid\u2013lipid storm caused by pro-inflammatory eicosanoids. Release of PUFAs, including LA, from the membrane by cPLA2 changes the fluidity of the cellular membranes and contributes to pulmonary and cardiovascular disease. Taken together, this suggests that early-stage supplementation of LA, i.e. while the viral infection is localized in the upper airways, may be beneficial to interfere early with SARS-CoV-2 infection, replication and viral transmission. Current insights into the mechanism of SARS-CoV-2 infection further provide a rationale for the development of intranasal, pulmonary or oropharyngeal drug-delivery systems for self-administration, targeting mucosal epithelial cells with high ACE2 concentration. Given worldwide persisting SARS-CoV-2 infections and the conservation of the pocket in spike protein, LA delivery could present a novel and sustainable strategy for early COVID-19 treatment and prophylaxis.LA binding to an evolutionarily conserved hydrophobic pocket of the RBD of SARS-CoV-2 spike protein was discovered in the spike cryo-EM structure and substantiated by LC-MS-MS. LA impacts viral infection and disease progression by several mechanisms. LA stabilizes spike protein in a non-infectious locked conformation, avoiding premature S2\u2032 cleavage and dissociation of the S1 subunit of spike. Important immunogenic epitopes in the RBD of spike, including the RBM, evade immune recognition in the LA-stabilized locked conformation. In the human host cell, LA binding to spike and viral membranes contributes to hyperactivation of cPLA"} +{"text": "Prediction and Explanation in Biomedicine using Network-Based Approaches\u201d putting together \u201cprediction\u201d and \u201cexplanation\u201d . Scientific methodology is aware of the different, albeit related, status of the two perspectives since long time , gene expression correlation and protein-protein interaction networks . In particular, Uversky and Giuliani review the most recent results in terms of hierarchical organization of complex biological systems, remarking the benefits of analyzing such systems in a multi-level fashion, hence going beyond the standard causative model where events originate at molecular level and then show up at the \u2018top\u2019 of the hierarchy . Causality is also the key in , where the authors review how causality can help in shaping disease networks, shedding light on using also functional information alongside physical proximity for a thoughtful modelling. In Tran et al., the authors question the suitability of MCF-7 cell line for in vitro breast cancer research. They use a network-based approach to compare two MCF-7 datasets against a human breast invasive ductal carcinoma dataset taken from The Cancer Genome Atlas (TGCA), showing how they have only minimal similarity in biological processes, hence concluding that using MCF-7 to study breast cancer can hide important gene targets. Finally, TGCA plays an important role also in , where the authors use a network-based approach to find hub genes related to acute lymphoblastic leukemia.Biological systems are the most evident paradigm of complexity, and this is why it is much more productive to focus on the dynamics of their correlation structure with respect to an in-depth analysis of isolated features. In this Research Topic, this point is made evident by papers exploring correlation structures located at different organization layers: contacts between amino-acid residues of a protein molecule motivated by the solution of relevant biomedical problems. Specifically, Kuznetsov et al. use a variational autoencoder to generate a synthetic 1-cycle ECG which not only looks quite natural, but can also be generated starting from just 25 features automatically learned by the autoencoder. As instead, Nazarenko et al. show an interesting network-based approach based on parenclitic and synolytic networks to describe multidimensional data via a suitable graph that makes the data easier to inspect, visualize and analyze. Tests on synthetic and benchmark data corroborate the competitiveness of using parenclitic and synolytic networks against common machine learning approaches.It is important not to confuse the integration of data analysis and explanatory perspectives with the too-often repeated statement of the substantial irrelevance of the hypothesis-driven approach when in presence of massive amount of data ; the sitChen et al.; Jung et al.; Luo et al.; Thomas et al.), that give us the strong impression that network-based approaches are here to stay. In detail, Thomas et al. review how network biology can help in understanding inflammatory bowel disease by discussing different network modelling , with some examples also on multi-layered networks. Chen et al. build a predictive model based on network analysis and circular miRNA to address recurrent implantation failure (RIF). Luo et al. also aim at characterizing RIF, but the authors exploit network-based approaches (protein-protein interaction and circRNA\u2013miRNA\u2013mRNA networks) to highlight four hub genes that may be involved in the development of RIF. Finally, Jung et al. briefly review computational models based on machine learning, network modelling and genome-scale metabolic models to characterize drug-resistant cancer cells.In this Research Topic, the application potential to biomedical practice of network-based approaches is explored in (After all, this is not surprising at all, \u201cnetwork biology\u201d is nothing else than \u201cbiology as such\u201d given any biological system derives its peculiar behaviour from the interaction of many different element players at different organization layers with no privileged causal layer of explanation . This is"} +{"text": "More than 50 million individuals worldwide suffer from dementia with ~60\u201370% affected by Alzheimer's disease (AD). As a progressive and neurodegenerative disorder and the leading cause of dementia in the elderly, AD lacks effective treatments to modify or stop disease progression. Over the past years, studies have identified new molecular mechanisms and modifiable risk factors of AD. Accumulating data suggest that advancing the understanding of AD mechanisms and risk factors may allow the development of targeted interventions to potentially prevent or cure the disease. There are an increasing number of preclinical studies focusing on mechanism-driven therapies, which need to be further characterized before introducing into clinical trials to determine their efficacy.Progress of Translational Medicine in Alzheimer's Disease\u201d in Frontiers in Aging Neuroscience includes a series of 13 articles that discuss recent advances in the field of translational studies in AD and highlight current challenges and outstanding questions requiring further study in order to advance research in this domain. These articles are discussed based on two main specific themes, noted below.Here, the Research Topic \u201cXing et al. have completed a meta-analysis, combing through discoveries which describe the diagnostic values of exosome-derived biomarkers in AD and MCI (Mild Cognitive Impairment). The meta-analysis includes 19 eligible studies involving 3,742 subjects and supports the potential diagnostic value of exosome-derived biomarkers in AD and MCI, and lays a foundation for future research to further confirm this finding.Emerging evidence has shown that exosome-derived biomarkers are potentially related to early occurrence and development of AD, which requires a consolidated analysis. Vogrinc et al. summarize more than 100 AD risk loci, with many of them potentially serving as biomarkers of AD progression, even in the preclinical stage of the disease. Furthermore, the analysis of GWAS data has led to identification of key pathways underlying AD pathogenesis, including cellular and metabolic processes, biological regulation, localization, transport, regulation of cellular activities, and neurological system processes. Thus, gene clustering into molecular pathways may help identify novel molecular targets and support the development of more tailored and personalized intervention of AD.Over the past decade, numerous genome-wide association studies (GWAS) have identified significant genes associated with increased risk of AD, for which a systemic review to identify potential molecular mechanisms was overdue. In a review article, Homann et al. conduct an analysis of GWAS on AD brain imaging biomarkers and neuropsychological phenotypes using the European Medical Information Framework for Alzheimer's Disease Multimodal Biomarker Discovery Dataset (EMIF-AD MBD). Their results highlight the power of using quantitative end phenotypes as outcome traits in AD-related GWAS analyses and nominates new loci underlying cognitive decline.Next, Shi et al. investigate plasma proteomic biomarkers associated with AD through a meta-analysis. The study assesses the diagnostic performance of their own group's previously identified blood biomarkers, and identifies proteins significantly associated with AD, including alpha-2-macroglobulin (A2M), ficolin-2 (FCN2) and fibrinogen gamma chain (FGG).Although plasma biomarkers for the diagnosis and stratification of AD have been intensively analyzed, no plasma markers have so far been well-established and confirmed for AD diagnosis. To this end, Logan et al. explore convolutional neural networks (CNN), a class of deep learning algorithms, for classifying multi-modal neuroimaging data such as magnetic resonance imaging (MRI) and positron emission tomography (PET). These networks may successfully enable early detection of AD and provide insight into AD classification using 3D architectures for multi-modal PET/MRI data.Presently, another challenging barrier to understanding AD and related dementias is availbility of suitable artificial intelligence (AI) methodologies for analyzing massive AD datasets. In a comprehensive review, Wu et al. summarize recent findings involving altered gut microbiota of patients with AD, and discuss pathogenetic mechanisms of gut microbiota in AD, suggesting gut microbiota\u2013targeted therapies for AD.Recently, increasing evidence has suggested the involvement of gut microbiota dysbiosis in association with AD. In a through review, Mee Hayes et al. discusses recent progress and potential mechanisms for targeting aggregates with proteasomes and disaggregases in liquid droplets.Despite the long-standing and established findings supporting insoluble protein deposition as pathological hallmarks of AD and other neurodegenerative disorders, studying the reverse process of aggregate disassembly and degradation has only recently gained momentum, following reports of enzymes with distinct aggregate-disassembly activities. A timely review by Stonebarger et al. present an article challenging a major obstacle in understanding the etiology of normative and pathological aged brains in AD\u2014the availability of suitable animal models. This article highlights our current knowledge in the area and examines the use of the rhesus macaque monkey as a pragmatic translational animal model to progress future research in this area.Furthermore, Morgan et al. have performed text-mining and generated an exhaustive, systematic assessment of the breadth and diversity of biological pathways within a corpus of 206,324 dementia publication abstracts. The results of this research may be applicable to the context of the broader AD literature corpus.The issues surrounding the large number of poorly understood underlying functional mechanisms is a major stumbling block in AD research today. Blevins et al. present a review article looking into the AD-related NLRP3 inflammasome, a multiprotein complex that plays a common and pivotal role in regulating innate immunity and inflammation underlying human pathophysiology.Finally, Zhong et al. focusing on D-penicillamine (D-Pen), a water-soluble metal chelator that can reduce A\u03b2 aggregation. The authors report that D-Pen significantly improves the cognitive functions of APP/PS1 mice and reduces A\u03b2 generation with an associated increase in ADAM10 via the non-amyloidogenic processing pathway.The first article that addresses potential therapies for AD is a study by Ma et al. present the results of their study describing the effects and molecular mechanisms of cornel iridoid glycoside (CIG), an active ingredient of the traditional Chinese herb Cornus officinalis, in pathological tau P301S transgenic mice. The data support CIG as a promising molecule for potentially treating AD-related tauopathy.Next, Carranza-Naval et al. analyze liraglutide (LRGT), a glucagon-like peptide-1 agonist, for its effects on reducing pathological changes in the brain of a mixed murine model of AD and T2D (APP/PS1 x db/db mice). These results support the potential for LRGT therapy to reduce AD-associated brain complications.Because type 2 diabetes (T2D) is a major contributor to the development of AD and AD patients with co-occurrence of T2D are regularly observed in the clinical arena, In closing, we would like to thank all the contributing authors and reviewers for their valuable expertise and effort toward successfully compiling this series of research focusing on translational medicine for AD. We realize that although these selected articles cover a good number of studies reporting up to date research findings, future studies will be necessary to better understand the \u201celephant in the room:\u201d the etiology of AD, which is our primary goal in developing this Research Topic. We sincerely hope that this Research Topic of articles will provide a useful point of reference and stimulate future studies committed to ultimately understanding the complex pathogenesis of AD and advancing the development of useful therapeutics for AD.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.EF has CRADA arrangement with ChromaDex and is consultant to Aladdin Healthcare Technologies, Vancouver Dementia Prevention Center, and Intellectual Labs. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."} +{"text": "Formulations from nanotechnology platform promote therapeutic drug delivery and offer various advantages such as biocompatibility, non-inflammatory effects, high therapeutic output, biodegradability, non-toxicity, and biocompatibility in comparison with free drug delivery. Due to inherent shortcomings of conventional drug delivery to cancerous tissues, alternative nanotechnological-based approaches have been developed for such ailments. Ovarian cancer is the leading gynecological cancer with higher mortality rates due to its reoccurrence and late diagnosis. In recent years, the field of medical nanotechnology has witnessed significant progress in addressing existing problems and improving the diagnosis and therapy of various diseases including cancer. Nevertheless, the literature and current reviews on nanotechnology are mainly focused on its applications in other cancers or diseases. In this review, we focused on the nanoscale drug delivery systems for ovarian cancer targeted therapy and diagnosis, and different nanocarriers systems including dendrimers, nanoparticles, liposomes, nanocapsules, and nanomicelles for ovarian cancer have been discussed. In comparison to non-functionalized counterparts of nanoformulations, the therapeutic potential and preferential targeting of ovarian cancer through ligand functionalized nanoformulations\u2019 development has been reviewed. Furthermore, numerous biomarkers such as prostatic, mucin 1, CA-125, apoptosis repeat baculoviral inhibitor-5, human epididymis protein-4, and e-cadherin have been identified and elucidated in this review for the assessment of ovarian cancer. Nanomaterial biosensor-based tumor markers and their various types for ovarian cancer diagnosis are explained in this article. In association, different nanocarrier approaches for the ovarian cancer therapy have also been underpinned. To ensure ovarian cancer control and efficient detection, there is an urgent need for faster and less costly medical tools in the arena of oncology. Then extracellular matrix gets degrade and the dissociation of cells takes place from primary cancer. The dissociated cells become abdominal free-floating cells or clumps together that forms multicellular aggregates.In the context of patient survival, chemotherapy and radiation therapy is under clinical trials to evaluate the ovarian cancer initial stage and approach the benefits of survival (Boev\u00e9 et al., In the early phase ovarian cancer detection, the current treatment and diagnostic methods are not enough efficient and sensitive as well. Furthermore, delayed diagnosis has been observed as a virtue of non-specified detection and high costs of the applied methods. Recently, nanotechnology is emerged as a growing field in the treatment and diagnosis of ovarian cancer that offers the development of novel and potential strategies to overcome ovarian cancer (Zhou et al., Liposomes exhibit a lipid bilayer that holding hydrophobic interface that enables the attachment of therapeutic and diagnostic agents of both hydrophilic and hydrophobic nature to the geometry of liposomes. The core region of dendrimers is closer to the hydrophobic component and hydrophilic termini that helps in the delivery of hydrophobic payloads within water insoluble domain. On another hand chemical conjugation of moieties with the exterior is take place for its delivery. Adsorption of carbon nanotubes to various agents is take place for consecutive delivery. Micelle provides delivery platform for hydrophobic anticancer drugs with prolong circulation.2.In recent years, ovarian cancer is globally classified as deadly gynecological cancer (Boitano et al., Biomarkers manifest versatile class of biomolecules that indicates a specific disorder or biological function Ueland, . In the 2.1.Optical biosensor utilizes an optical transducer system with sensing features that are used for analysis purposes. It senses and transmits those signals that are directly proportional to the biomarker . These bTo improve the surface glycosylation visibility of sample cells an accelerating technique was used that showed efficient results. During the technique the MUC1 precise glycosylation on the cancer cell surface was pictured through a quantum dots label using the strategy of RMC magnification. This study offers a sort of suggested scheme for protein-specific glycosylation and thus indicates the mentioned technique as a feasible strategy (Yang et al., 2.2.Electrochemical nanosensors have shown potential methodology and role for the precise detection of biomarkers in ovarian cancer and usually have the detection power in minute quantities of biomarkers and other analytes (Parmin et al., Another research group worked on the CA-125, biomarker identification through the development of immuno-based electrochemical nanosensor. The developed nanosensor was composed of multiwall carbon nanotubes, gold nanoparticles and 3D reduced graphene oxide composite. The developed system displayed an excellent detection limit i.e. 6 \u00b5U/mL .2.3.Micro fluidic laboratory-on-chip nanosensor integrates easily the various features of multiple sensing systems with less sampling rate and thus pose it a versatile miniaturized system (Jamshaid et al., 2.4.Within a portable system, it is the unique feature of giant magnetoresistive biosensors that it measures multiple biomarkers all together. In addition, other advantages include integrated circuit connectivity, high accuracy and easy biomolecular detection (Xuan et al., 3.In the recent years, many nanoconjugates and nanoformulations as a drug delivery system have been developed and they have improved the therapeutic drug delivery to the target side due to unique features such as biocompatibility, high therapeutic efficacy as compared to free drug, non-toxicity, biodegradability, off-target side effects and ease of manufacturing scale-up (Larra\u00f1eta et al., 3.1.Liposomes are nontoxic phospholipids-based sphere-shaped small vesicle ranges in size from 400\u2009nm to 2.5\u2009mm. Liposomes are extensively employed renowned clinically delivered nanosystems with biodegradable nature and have the capability to entrap hydrophobic and hydrophilic biomacromolecules, i.e. RNAs, peptides and proteins without modification in their inherent properties (Sun et al., The co-loading of two chemotherapy agents into a single liposome in clinical trials has shown many advantages but the loading of two chemotherapeutic drugs into a single liposome is quite challenging. In this context, irinotecan and doxorubicin were encapsulated and loaded into liposomal nanoformulations to treat ovarian cancer xenograft. Various ratios between drugs to the drug were used during loading into the liposome. The encapsulation efficiency was 80%, and it was found that after storage for 6 months the liposomal stability attributes significantly influenced the drug encapsulation. After the administration of developed nanosystem via intraperitoneal route considerably enhanced the survival of the animals with tumor, which was attributed to increased exposure of liposomal nanoformulations to the systemic circulation (Shaikh et al., 3.2.Several nano-sized drug delivery systems have been tested in cancer treatment to reduce the side effects of traditional anticancer drugs while increasing the antitumor efficacy of target therapy (Mohammadzadeh et al., In addition to metal nanoparticles, biomacromolecules such as vitamins, nutrients, hydrophobic drugs and phenolic compounds were immensely delivered to multifaceted biological systems using chitosan nanoparticles. Chitosan-a chitin-deacylated biopolymer of natural origin that exhibit useful features such as inertness, biodegradability, biocompatibility and the presence of hydroxyl and amino group that make it a suitable candidate for the drug delivery (Muddineti et al., In another research study, cannabidiol-loaded polymeric nanoparticles were developed and evaluated in ovarian cancer cell for growth inhibition potential. The encapsulation efficiency found was 95% with 240\u2009nm size nanoparticles and displayed controlled release kinetics for cannabidiol after internalization to SKOV-3 ovarian cancer cells for an extended period of 96\u2009hours. After encapsulation, the antiproliferative effect of cannabidiol was found excellent against the SKOV-3 cells. Apoptosis was also induced by the applied drug-loaded nanosystem that was confirmed when PARP protein was found provoked. Importantly, the ovarian tumor growth inhibition was slightly higher for cannabidiol-loaded nanoparticle as compared to free cannabidiol (Fraguas-S\u00e1nchez et al., In the arena of ovarian cancer targeted drug delivery epigallocatechin gallate-loaded chitosan base mesoporous silica nanoparticles were developed and evaluated. Furthermore, the fabricated system was attached with AS1411 aptamer through conjugation with the amine group present in the chitosan using electrostatic attraction. The aptamer functionalized nanosystem followed macro-pinocytosis and displayed efficient internalization potential in SKOV-3 cell lines. Consequently, it resulted in an augmented cytotoxic effect in 93% of ovarian cancer cells along with G1 cell cycle arrest and down-regulation of ERK2 expression level (Alizadeh et al., 3.3.Solid-lipid nanoparticles are made up of surfactants, lipids and therapeutic drugs enclosed in spherical shape colloidal nanocarriers exhibiting 50\u20131000\u2009nm diameters. The lipid core and nano size range of solid-lipid nanoparticles make them versatile drug delivery carriers from nanotechnology platforms. Such nanocarriers have attained greater interest also due to their considerable biocompatibility, excellent circulation time and marked accumulation at the target site (Radhakrishnan et al., 3.4.l-buthionine sulfoximine\u2014a \u03b3-glutamylcysteine ligase inhibitor, the glutathione synthesis was hampered significantly. Systemic toxicity consideration in this context helped in overcoming the issue of carboplatin resistance by using polyurea-based dendrimer formulations (Cruz et al., Dendrimers are spherical nano size, regularly branched, three-dimensional tree-like architectures of high molecular weight in which the branch lengths are stearic limited exhibiting an inner core. The inner microenvironment and surface of dendrimers are composed of functional moieties that help in the attachment of delivering cargoes (Mishra et al., 3.5.Among nanostructured materials micelles represent an intriguing class of nanoarchitecture in which amphiphilic molecules above critical micelle concentration aggregates to form such a versatile class of nanocarriers (Karayianni & Pispas, 3.6.Nanocapsules manifest a nanoscale vesicular system that is composed of central cavity that gives space to the drugs of interest. In addition, an outer polymeric shell is also there surrounding inner core and it helps in the attachment of various targeting ligands and moieties during surface functionalization (Li et al., A summary of various nanosystems is provided in 4.The knowledge and research on nanotechnology-based formulations has bloomed in recent years, however, only few formulations have made their way successfully to clinics. Most of such formulations fail to demonstrate similar results when tested in vivo and halt their progress to clinical trials. For clinical translation, every nanoformulation has particular challenges, however, most of them confront biological, technological, and study-design-related challenges.Biological challenges include lack of routes of administration, tempering biodistribution, hannelling nanosystems across biological barriers, toxicity and degradation (Lv et al., Controlling nanoparticle\u2019s biological fate is difficult and requires focus. There is a possibility of liver, lung, and kidney damage, despite the fact that nanoparticles are made of biosafety materials. Surface area, shape, solubility, particle size and agglomeration are the factors that are reported to cause toxicity (Jia et al., Technological challenges of nanosystems include equal optimization, performance predictions and scale-up synthesis. Most nanoparticles used are produced in small batches, and scaling up is not always possible due to instrumentation and other factors. In animal models, the best lead clinical candidates are not always systematically designed and optimized. To circumvent this, we can employ specific methods that permit the testing of numerous nanoformulation and the selection of a single optimized formulation via selective iterations (Dobrovolskaia et al., N\u2009=\u20091 clinical studies will be needed for personalized medicine. This includes genetic, environmental, and medical history factors (Love et al., Study-design challenges such as study size, intent, and timing of nanoparticle therapies during therapy have a significant impact on clinical studies. The majority of research is based on \u201ccell and animal models,\u201d which may not translate to human trials. Therefore, it is challenging to mimic natural human body reactions using a single model. Metastasis is an important attribute of cancer, so \u201cmodels of cancer metastasis\u201d should be researched. Nanotechnology enables personalized oncology, in which cancer therapy and diagnosis are tailored to each patient\u2019s tumor molecular profile, and predictive oncology, in which genetic and/or molecular markers predict development and progression of disease and clinical outcomes. The National Cancer Institute in the US has recently allocated funds to eight national Centers of Cancer Nanotechnology Excellence due to its potential impact on cancer research (Misra et al., 5.The precise delivery of therapeutic cargoes into ovarian tumor cells through novel engineered carriers from nano scaffolds has been widely explored in recent years. In the diagnosis and treatment of ovarian cancer, the chemotherapeutic drugs\u2019 adverse effects were significantly curtailed, the site-specific delivery was augmented and solubility issue of hydrophobic cargoes was resolved using numerous nanotechnology-based carrier systems. In ovarian cancer, nanotechnology from nanoscience domain addresses the site-specific delivery and provides new-enhanced and targeted therapy possibilities. Several formulations show interesting results but only a few will make their way to clinical trials and clinics because the progress of most of them is halted in pre-clinical stages due to a number of factors. Regarding clinical translation, each nanoformulation has particular challenges related to either biological, study-design, or technology related. In addition, the toxicity, biotransformation and excretion of nanocarriers-based systems for ovarian cancer will also be a challenging aspect. Therefore, in the design stages, such considerations should be kept in mind for developing biocompatible and nontoxic carrier systems. Numerous nanotechnology-based biosensors have been proposed and tested in recent years for the diagnosis of ovarian cancer. The associated concerns with ovarian cancer diagnosis and treatment could be resolved through these nanomaterials based biosensors due to their high sensitivity and selectivity. However, the development of nanosensors is a time-consuming and complex process and thus often fails in the critical evaluation of biomarkers. Importantly, in the identification of ovarian cancer biomarkers, the demand of portable nanosensors that could be used to rescue subjects outside the clinical settings is of much value. In the scenario of ovarian cancer diagnosis, microfluidic and paper-based nanosensors fabrication offers feasible prospect for biosensors\u2019 commercialization. In a nutshell, in the near future thrilling developments are required for sensing procedure scale-down using nanotechnology platform that hopefully would enable the patients to check their health easily and indeed would open new avenues in the diagnosis and treatment of ovarian cancer."} +{"text": "Dementia-related continuing education opportunities are important for rural primary health care (PHC) professionals given scarce specialized resources. This report explores the initial perceptions and continuing education needs of rural interprofessional memory clinic team members and other PHC professionals related to a short series of dementia-related education webinars. Three webinars on separate topics were delivered over an 8-month period in 2020 in Saskatchewan, Canada. The research design involved analysis of webinar comments and post-webinar survey data. Sixty-eight individuals participated in at least one webinar, and 46 surveys were completed. Rural memory clinic team members accounted for a minority of webinar participants and a majority of survey respondents. Initial perceptions were positive, with webinar topics and interactivity identified as the most effective aspects. Continuing education needs were mainly aligned with professional roles; however, some overlap of interests occurred. Future webinars will further explore learning needs within an interprofessional environment. Meant tet al., et al., et al., et al., et al., IPE in primary care that promotes interaction and learning between different professional groups is important for strengthening collaboration skills and behaviours and fostering collaboration within teams team members and the developer (DS) of the Primary Care Dementia Assessment and Treatment Algorithm (PC-DATA\u2122), which has been adapted and integrated into the clinic model. Prior to 2020, continuing education was offered periodically to one team at a time, delivered by clinical specialists and other experts via telehealth videoconference. Teams suggested topics in which they were keen to receive further training, which included differential diagnosis, capacity and competency, medication for dementia, and driving assessment. More teams were invited to take part in the sessions as the model spread to other communities. In a recent process evaluation, teams indicated participation in training and continuing education improved self-confidence in abilities and supported implementation of memory clinics (Morgan 1). Each webinar began with a 30\u201345 minute presentation followed by 30\u201345 minutes of discussion. Team members had the flexibility to join remotely with their chosen device since they often travel between settings and communities. During the webinars, the presenter\u2019s camera and audio were live and participants could offer written or oral comments at any time; however, participant\u2019s cameras were turned off.In February 2020, the researchers introduced a cross-team dementia-related continuing education series delivered via WebEx. Three webinars were delivered in 2020 on topics suggested by the teams and are included in this analysis .2). Six Likert items and two open-ended questions were adapted from previous evaluations of dementia-related continuing education sessions for health professionals . Regarding webinar content, the majority agreed the sessions were appropriate for their professional needs and new information was learned (96%). Most found the interactive format and webinar environment effective for learning (96%). A majority also intended to apply the information in their practice and appreciated the participation of other PHC teams and professionals; however, these particular items were endorsed by fewer participants (<90%).Among survey respondents across the webinars, overall satisfaction was high (94%) (data not shown in table). Regarding the first two webinars which were more medication-focused than the third session, some AHPs found themselves engaged although they were not in a prescribing role and did not feel highly knowledgeable about the topic . Illustrative quotations of themes are provided in Supplemental Table\u00a0This study explored the perceptions and continuing education needs of rural memory clinic team members and other PHC professionals participating in interprofessional dementia-focused webinars. Findings from post-webinar survey evaluations indicated positive initial perceptions of webinar content and format, and education suggestions that mainly corresponded to professional roles.et al., et al., et al., et al., et al., Our findings are consistent with research showing rural health professionals react positively to dementia care learning opportunities delivered remotely . To help improve management capacity, the rural memory clinic model and other task-sharing approaches collaborate with highly qualified and experienced dementia specialists to further train and support PHC professionals (Canadian Academy of Health Sciences, et al., et al., et al., Suggestions by survey respondents for future education topics generally aligned with professional roles. The mutual interest of AHPs and FP/NPs in pharmacological and non-pharmacological management, and emphasis by FP/NPs on management interventions, underscores the challenge of these issues in primary care. Interest in medication-related topics is consistent with recent research showing potentially inappropriate prescribing is associated with higher levels of comorbidity among people living with dementia (Delgado Limitations of this study should be considered. Survey items focused only on a few selected perceptions of webinar content and format. Other measures of content and format, and other domains, may have been pertinent to participant experience but were not included. Survey participation was voluntary; therefore, the results are subject to non-response bias. Respondents may also have tended to agree rather than disagree with statements, reflecting acquiescence bias. Furthermore, the initial findings from a small sample of professionals from one geographic area limit the generalizability of the findings.There have been few published studies of initiatives that offer dementia-related continuing education in an interprofessional setting to PHC professionals. Initial results from this study indicate favourable feedback from rural participants of a short series of interprofessional dementia-related education webinars. The findings also revealed opportunities to seek further input on varying education needs within teams, to inform future webinars and research."} +{"text": "For patients with a variety of severe diseases, including primarily hematopoietic malignancies, immunodeficiency syndromes, and genetic disorders, allogeneic hematopoietic stem cell transplantation (aHSCT) represents a potentially curative therapeutic approach. In this context, despite significant progress in the optimization of aHSCT, the development of graft-versus-host disease (GVHD) remains a challenge to long-term transplant success after aHSCT. It is associated with significant morbidity and mortality and is the major cause of non-relapse mortality.GVHD occurs when donor T cells are primed by recipient antigens subsequently eliciting an inflammatory response against the host. Clinically, two types of GVHD are distinguishable: an acute form (aGVHD), and a chronic form (cGVHD). In brief, the main characteristics: aGVHD occurs in 30-50% of aHSCTs and is a multi-organ disorder resulting from inflammatory cytokines and donor T cells which primarily damage skin, liver, gastrointestinal tract, and eye. cGVHD, with a prevalence of 30-70% of aHSCTs, is induced by T and B cells resulting in a heterogeneous immunological complication affecting virtually every organ.Traditionally, broad immune-suppressive drugs (with considerable toxicities) including calcineurin inhibitors (CNI) (cyclosporin or tacrolimus), together with methotrexate or mycophenolate mofetil (MMF), and mTOR inhibitors (Sirolimus/Rapamycin) are used as GVHD prophylaxis. But despite first success reports, significant GVHD still occurs with these drugs. Other prophylaxis strategies like pre-transplant anti-thymocyte globulin (ATG) are effective in reducing severe GVHD but have no survival benefits and steroids have serious side effects.One of the most critical challenges in aHSCT is the development of less toxic and more targeted therapies that maintain the graft-versus-leukemia/tumor (GVL/T) effect but suppress GVHD while facilitating enhanced immune reconstitution relative to existing strategies. Recently, several prophylaxis strategies for GVHD have been developed and others are currently in development, including, for example in the case of haploidentical HSCT, post-transplant cyclophosphamide (PTCy), which seem to be very promising.In the frame of this specific Research Topic, we aimed to collect recent developments of innovative methods for both prevention and treatment of GVHD, without impairment of GVL. In 8 original research articles and 4 reviews, this edition provides a deep insight into the role of the microbiome and metabolism as well as recent advances of small molecule and cell therapy development.Mohamed et\u00a0al., summarize metabolic pathways contributing to GVHD and discuss metabolic targets for acute and chronic GVHD in immune and non-immune cell as well as the immunomodulatory function of microbial metabolites. Furthermore, they examine the metabolic effects of co-inhibitory pathway blockade (PD-1) and cellular therapies (Tregs/MSCs/Bregs) in aHSCT. The mini review by Karl et\u00a0al., provides an overview of metabolic T cell alterations in GVHD and illustrates the impact of conventional GVHD therapy on T cell metabolism.We are glad, that experts in the field highlight the recent progress in the broad field of immune cell metabolism with two comprehensive reviews. Ghimire et\u00a0al., investigates the role of G-protein coupled receptors (GPR43 and GPR109A) which engage microbial derived metabolites, like short chain fatty acids, in the mitigation of GVHD in intestinal biopsies from patients after allo-HSCT. A second study by Heidrich et\u00a0al., describes an association of dental biofilm microbiota dysbiosis with the risk of aGVHD.Recent studies have shown the association of microbiome dysbiosis and aGVHD. Here, primary research by Campe and Ullrich. Moreover, Agbogan et\u00a0al., explore the immunomodulatory effect of adoptively transferred CpG-activated B cell progenitors to alleviate GVHD symptoms. In addition, Scheurer et\u00a0al., describe an in vitro generated sub-population of CD11b+CD11c+ myeloid-derived suppressor cells (MDSCs) as potent immune modulators leading to the prevention of GVHD without negatively affecting tumor cytotoxicity. Another innovative and attractive strategy using CRISPR/Cas9 has been described by Majumder et\u00a0al., for genetical engineering of na\u00efve T cells pre transplant as a method for GVHD prevention in a major murine mismatch model.In the context of cell therapy development, the biological relevance of T helper cell lineage defining transcription factors as potential targets for GVHD therapy has been delineated in a review article by Braun and Zeiser thoroughly review the role of kinase inhibition as novel treatment strategies for acute and chronic GvHD after allo-HCT. Thangavelu et\u00a0al., evaluate the efficacy of a novel agonist of the retinoic X receptor (RXR), IRX4204, to treat cGVHD in two complementary murine models with bronchiolitis obliterans or sclerodermatous manifestations. Primary research by Matos et\u00a0al., analyzes a possible association of anti-thymocyte globulin (ATG) treatment and serum levels of 25-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 in 4 HSCT cohorts with different vitamin D3 supplementation. Lastly, Hadjis et\u00a0al., characterize post-transplant cyclophosphamide as superior in ameliorating pre-clinical GVHD compared to five other optimally dosed chemotherapeutics that vary in mechanisms of action and drug resistance.In addition, the recent therapeutic advances in the area of drug development, e.g. small molecules and antibodies, are also addressed. Finally, this Research Topic makes us again aware of how complex the regulation of GVDH is and in which fragile balance between GVHD and GVL patients after aHSCT find themselves. We are aware that this issue can only compile a first selection of innovative findings and treatment strategies that are currently being developed for the prevention and treatment of GVHD.GVHD biology and treatment remains a field that is always influenced by current research developments and new advances can be expected in a short time. Therefore, we will continue to monitor the field and provide updates to the authorship of Frontiers in Immunology.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.EU is a member of Phialogics and receives research support from BMS and Gilead. AB is a scientific cofounder of Aamuthera Biotech GmBH and Dualyx NV.The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."} +{"text": "Fossil-based heteroatom-containing compounds are crucial core scaffolds or key intermediates in a wide range of pharmaceutical molecules, fiber dyes and printing ink , which cLiu et al., Zhu et al., Liu et al., Zhang et al., Sun et al., Yao et al., Zhou et al., Zhao et al., Yang et al., and Zhou et al.), biodiesel production , and green synthesis of heteroatom-containing bioactive compounds and functional materials . Also, the Research Topic provides interesting insights into the green photocatalysis of organic pollutants .This Research Topic is Volume II of a series, and here we present a collection of original research and review articles with topics on green and sustainable chemistry, including catalytic conversion of biomass feedstocks . Zhang et al. use glycerol waste to comparatively evaluate the ameliorative effect on lignocellulose under microwave or conventional heating method. During fast pyrolysis, levoglucosan produced from microwave-treated samples (32.9%) was far more selective than the conventional heating group (18.8%), and the content of aldehydes (high toxicity to the downstream fermentation) after glycerol waste and microwave pretreatment was decreased by 2.5 times compared with the untreated counterpart. In addition to directly using raw biomass resources, simple sugars like fructose can be efficiently converted to 5-hydroxymethylfurfural (up to 82% yield) by dehydration over a stable Ti-doped SBA-15 catalyst in DMSO at 140\u00b0C for 1\u00a0h , or to 5-ethoxymethylfurfural (80.4% yield) by cascade dehydration-etherification using a UIO-66-SO3H catalyst in ethanol under the same thermal conditions . Zhang et al. manufacture a biomass-based solid acid catalyst (SiO2@Cs-SO3H) with a large specific surface area (21.82\u00a0m2/g) and acidity (3.47\u00a0mmol/g) using renewable chitosan as raw material through sulfonation procedure under relatively mild conditions, which is active for esterification of oleic acid and methanol to produce biodiesel (98.2% yield).Original research paper of Chen et al. reports an unprecedented inactivation process of the indanol-derived NHC catalysts bearing N-C6F5 groups, giving an unexpected multi-cyclic complex product from the 3-component reaction with 1-methylcyclopropyl-carbaldehyde, 2,2,2-trifluoroacetophenone and the NHC catalyst. Pan et al. develop an acid-catalyzed 2-alkylation of indole molecules catalyzed by traceless HI, and 2,3-disubstituted indole molecules bearing congested tertiary carbon centers are obtained in moderate to good yields. Some functional catalytic materials such as hierarchical porous SAPO-34 , bimetallic Zn-Zr metal-organic framework , and graphene oxide-silver nanoparticles composite are prepared in sustainable ways, and found to be efficient for the synthesis of value-added chemicals or degradation of organic pollutants.The work of Liu et al., Sun et al., Zhou et al., Yang et al., Yao et al., Liu et al., Wu et al., Liu et al., Pan et al., and Zhang et al.). Liu et al. review the application of recyclable heterogeneous non-noble Zr/Hf-containing catalysts with acid-base bifunctionality for catalytic transfer hydrogenation using the safe liquid hydrogen donor, with emphasis on evaluating the reaction mechanisms and conversion pathways. In a more detailed manner, the research progress of catalytic synthesis of \u03b3-valerolactone from furfural by Zr/Hf-based catalysts is reviewed by Sun et al., and the effects and regulation approaches of Lewis acid-base and Br\u00f8nsted acid sites in the catalysts on each steps in the reaction process are discussed. Zhou et al. reveal the significance and potential of using titanate nanotubes-based materials as sustainable and environmentally benign solid catalysts/supports for synthesis of various bio-based chemicals, such as glycerol-derived solketal, jet fuel range alkanes precursors, biomass-derived esters, aldehydes, and aromatic compounds. Yang et al. propose the research development trend for improving the institutional mechanism of the utilization of crop straw resources, strengthening technology research and development, exploring the economic model of green cycle agriculture, accelerating the construction of the industrial system, and designing new paths of resource utilization in multiple ways. Yao et al. mainly review some latest studies about the conversion of cellulose to 5-hydroxymethylfurfural catalyzed by solid acids with Br\u00f8nsted and/or Lewis acidic sites, such as sulfonated solid acids, carbon-based acids, and zeolites. Liu et al. summarize the mechanisms of several important processes of producing 5-ethoxymethylfurfural from lignocellulosic biomass-derived sugars and the research progress of the developed acid catalysts. In addition, advancements in tobacco (Nicotiana tabacum L.) seed oils and lipid extraction from microalgae using green solvents for biodiesel production are also collected. For some structurally somplex natural products such as sex pheromones , and momilactones and related 9\u03b2-H pimarane skeleton , the recent advances in their synthetic strategies with the involved challenges are overviewed.This Research Topic features several review articles with distinct scopes (We wish this Research Topic attracts interested colleagues, enlightening more eco-friendly and sustainable synthetic procedures, shedding light on renewed catalytic strategies and routes developed for the production of bio-based heteroatom-containing compounds, and providing enthusiasm in research and studies. Enjoy its reading!"} +{"text": "Back in 2010, when we first published data on the in vivo nutrigenomic effects of virgin olive oil polyphenols within the frame of the Mediterranean diet [Research is growing at a fast pace, acknowledging that gene\u2013diet interactions play an enormous role in human health and are typically only partially assessed. Healthcare providers, on the other hand, receive scarce knowledge of these interactions, thus jeopardizing the application of precision nutrition ,3. At thThis Special Issue on the effects of gene\u2013diet interactions in human health includes six original research articles showing data that will bring readers closer to the state-of-the-art developments in the field of nutritional genomics. From human intervention studies in Spain and Latin America to large-scale genome-wide association studies (GWAS), such as the UK Biobank, and the study of molecular mechanisms in Drosophila melanogaster, this issue includes the latest advances driving our understanding of gene\u2013diet interactions.A genetic correlation and two-sample mendelian randomization study was performed by Xu et al. assessinGranado-Serrano et al. studied Rivera-I\u00f1iqguez et al. studied A high polygenic risk score for Type 2 diabetes mellitus was used by Lopez-Portillo et al. to assesDu et al. used DroGene\u2013diet interactions, once fully incorporated into clinical practice, hold great promise as a precise tool to improve quality of life, health span, and reduce healthcare costs. This special issue added a piece of knowledge in that direction with more precise data and analysis."} +{"text": "Polasa et al. use an extensive set of equilibrium and non-equilibrium molecular dynamics simulations to describe the conformational changes in the YidC during membrane insertion . Similarly, Li et al. consider the effect of cholesterol and membrane composition on C99 dimerization with ramifications in Alzheimer\u2019s disease . Be it atomistic or coarse-grained, molecular dynamics uses a physics-based approach to explicitly describe the interaction between molecular moieties of interest with an outstanding resolution. Aslam and Alvi present a different computational approach based on systems biology. Their mathematical modeling allowed the complete exploration of a novel postsynaptic channel for glutamatergic synaptic transmission and its effector molecules composed of ions, diacylglycerides, and protein kinases (Aslam and Alvi). Unlike molecular dynamics, systems biology works at much larger scales and aims to characterize emergent phenomena from complex and interdependent processes and interactions in biological systems. Advancing our knowledge of membrane-related processes depends on experimental methods as well. The Research Topic includes the purely experimental work by Kurakin et al., who studied lipid vesicle and divalent ion interactions . This process is of paramount importance in signaling and membrane structure. Overall, we show that advancement in membrane research requires synergies from widespread techniques.Our knowledge of cellular membranes and their components\u2019 critical role is recent and so far, unfinished. Bilayers of lipids were proposed less than 100\u00a0years ago . \u201cFluid"} +{"text": "Resco de Dios et al. Nature Communications 10.1038/s41467-022-32013-9 (2022)In their letter, Resco de Dios et al. discuss fire regimes in China using a dataset of annual area burned from a satellite product, i.e., the Moderate Resolution Imaging Spectroradiometer (MODIS) Collection 6 (C6) MCD 64 A1. They suggest that: (1) the fraction of burned area in China\u2019s subtropics is low, (2) no dipole pattern exists between the burned area in western and eastern subtropical China, (3) the El Ni\u00f1o-Southern Oscillation (ENSO) has only weak effects on area burned in subtropical China, and (4) the fire suppression policy has little effect on fire activity in subtropical China. Their analysis of satellite-derived annual area burned raises some interesting issues that deserve further discussion.1. Even though MODIS-derived fire products, such as active fires and area burned, have much improved in recent years2, biases still remain in these products because of surface topography, fast vegetation growth rates, and clouds2. In addition to these biases, crop data gaps due to clouds occur in these fire products2 and this is especially the case in cloudy subtropical China. As a result, fire patterns in southwestern China have been found to be inconsistent between MODIS fire products and ground observations3. To address these biases, we calculated fire occurrences from MODIS fire points4 and further distinguished between wildfire and crop fire in the fire occurrence dataset. We find a high fraction of crop fires in the MODIS fire data in northern China, particularly in the North and Northeast China Plains and Resco de Dios et al. may be the primary reason for the discrepancies between the two studies. We developed the Wildfire Atlas of China (WFAC), a robust ground-truthed fire occurrence dataset, in which fire occurrences were detected by multiple satellites (including MODIS) and carefully validated through surface observations by local forestry departments and Resc1. To support our previous findings, we have now implemented empirical orthogonal function (EOF) analyses on the WFAC field only (Fig.\u00a0Based on their analysis of the annual area burned, Resco de Dios et al. argue the lack of a dipole pattern in fire activity between western and eastern subtropical China suggested in our study. We detected this pattern based on the co-varying patterns between the coupled fields of fires in WFAC and sea surface temperaturenly Fig.\u00a0. The firResco de Dios et al. claim that the modulation of ENSO on fire in China is weak. They base their claim on the insignificant correlations they find between gridded area and ENSO indices on individual grid points in China. Unlike their analysis of individual grid points, our analyses were based on the covariance of data on these grid points. Combining all grid points, our correlation analysis increases the degree of freedom, raises the likelihood of a significance test, and therefore is reliable and robust. Fire in individual grid points can be noisy on a local scale, while climate plays a more critical role in modulating large-scale fires.8. Resco de Dios et al. stated that the ENSO could only influence the ignitions and thus has little effect on fire activity. In fact, fuel availability and flammability are also key factors in fire occurrence, particularly for large-scale fires9. This is evidenced by the strong correlations between fire occurrence and interannual climate variability.Many previous studies revealed the dominant impacts of ENSO in different regions of China10.China\u2019s fire policy not only suppresses existing fires but also prevents human-ignited fire occurrences. As revealed in previous studies, the fire suppression policy since 1987 decreased not only burnt areas but also fire occurrencesThe study by Resco de Dios et al. was based on MODIS-derived annual area burned, which differs from our ground-truthed WFAC fire occurrence dataset. The MODIS cannot sufficiently distinguish the wildfire from the frequent crop fires and thus vastly misinterrupt the crop fires as wildfire, especially over the northern plains where forests are rare. Here, we show that the EOF analyses of the WFAC can also reveal the dipole fire pattern between southwestern and southeastern China. We highlight that the dipole fire pattern and ENSO modulation are on large scales. The fire control policy not only suppresses existing fires but also prevents human-ignited fire occurrences, and thus plays an effective role in reducing five activities in China."} +{"text": "Aboo Shuhailath et al., RSC Adv., 2016, 6, 54357\u201354370, DOI: 10.1039/C6RA07836B.Correction for \u2018Photoactive, antimicrobial CeO The corrected list of affiliations is as shown above.The authors regret that the one of the affiliations (affiliation The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "Epithelial-mesenchymal transition (EMT) is a core cellular program in vertebrate embryonic development where the expression of key junctional and cytoskeletal proteins such as E-Cadherin and Vimentin orchestrate stages of embryogenesis such as gastrulation or morphogenesis . In 2003When epithelial tumors progress, they lose cell\u2013cell contacts and apico\u2013basal polarity, transforming into spindle-shaped mesenchymal structures. Such cells also gain capabilities of motility and invasiveness for metastasis . At metaVuletic et al.). EMT-associated gain of NK cell ligands in cancer cells may enhance NK cell mediated cytotoxicity, whereas an immune suppressive tumor microenvironment (TME) may activate enzymes that lead to a loss of ligands. He et al. show that the expression of PSMC5 altered the type of immune cells in the TME of CRC by suppressing the infiltration of CD8+ T cells and enhancing the infiltration of innate immune cells such as macrophages and neutrophils and was associated with EMT .EMT proteins can have various non-EMT related functions and act in a tissue-specific manner such as acquisition of immunosuppression and cancer stem cell (CSC)-like features . The tumYu et al.). Song et al. show that expression of \u03b2-arrestin1 enhanced the motility of CRC cells via the activation of Wingless/integration-1 (Wnt)/\u03b2-catenin signaling .EMT may enhance chemoresistance with evidence for enhanced drug efflux, slower cell proliferation and avoiding apoptosis signaling pathways. Slug was implicated in the development of partial EMT and resistance to doxorubicin via upregulation of drug efflux transporters and stemness in liver cancer cell lines . Yu et aReliance on one or two genes to evaluate a process as complex as EMT as well as the use of acute tumor models may not be sufficient to recapitulate the heterogeneity, metabolic idiosyncrasies, and growth pattern of a slow growing tumor . Over thJia et al.). Moreover, modeling experiments suggest that partial EMT can facilitate hybrid metabolic states with combination of glycolysis and oxidative phosphorylation, contrary to primary tumors that mostly rely on glycolysis . The results reported provide new insights into EMT plasticity and its implications in CRC."} +{"text": "For a comprehensive understanding of protein function and dynamics, it is crucial to study their mechanical properties ,9,10,11.Recent progress regarding theoretical AI-based approaches for the prediction of 3D structures has creaVan der Sleen and Tych reviewedGala and \u017dold\u00e1k applied Single-molecule force experiments can explore protein folding and unfolding under mechanical force. However, such experiments are challenging, cost-demanding and require many years of highly specialized expertise. Bauer and \u017dold\u00e1k describeAtomic force spectroscopy was used to examine the mechanical properties of two peptides on the surfaces of gold nanoclusters (Au NCs) as small as 2 nm . Such smNon-equilibrium pulling data and derived force-dependent kinetic rates measurements show a systematic discrepancy between the total distance between the native (N) and the unfolded state (U) from elastic models and the sum of the measured distances for folding and unfolding kinetics. Rico-Pasto et al. performeSziklai et al. applied Microtubule disassembly and protein degradation are essential processes in the cell, which are mediated by highly specialized hexameric nanomachines. Varikoti et al. conducteTo summarize, as shown by the research articles in this Special Issue, protein nanomechanics is a fruitful emerging concept which describes the internal dynamics of highly mechanically anisotropic proteins. The further development of experimental methods and bioconjugation strategies will strongly contribute to developing our fundamental understanding of complex protein nanomachines and protein behavior at solid interfaces. Based on our knowledge of protein mechanics, we should be able to design and develop nanomachines and proteinaceous surfaces with tailored-made functional and elastic properties."} +{"text": "Studies have identified high rates of mental disorders in refugees, but most used self-report measures of psychiatric symptoms. In this study, we examined the percentages of adult refugees and asylum seekers meeting diagnostic criteria for major depressive disorder (MDD), post-traumatic stress disorder, bipolar disorder (BPD), and psychosis.A systematic literature search in three databases was conducted. We included studies examining the prevalence of MDD, post-traumatic stress disorder, BPD, and psychosis in adult refugees according to a clinical diagnosis. To estimate the pooled prevalence rates, we performed a meta-analysis using the Meta-prop package in Stata (PROSPERO: CRD42018111778).I2= 99%) for MDD, 31% for post-traumatic stress disorder, 5% for BPD, and 1% for psychosis. Subgroup analyses showed significantly higher prevalence rates of MDD in studies conducted in low-middle income countries than high-income countries studies , and in studies which used the Mini-International Neuropsychiatric Interview compared to other diagnostic interviews . Studies among convenience samples reported significant (p = 0.001) higher prevalence rates of MDD and PTSD than studies among probability-based samples .We identified 7048 records and 40 studies (11 053 participants) were included. The estimated pooled prevalence rates were 32% (95% CI 26\u201339%; This meta-analysis has shown a markedly high prevalence of mental disorders among refugees. Our results underline the devastating effects of war and violence, and the necessity to provide mental health intervention to address mental disorders among refugees. The results should be cautiously interpreted due to the high heterogeneity. In 2020et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., During the last years, several studies have examined the prevalence of mental disorders in refugees and asylum seekers. However, the resulting prevalence rates in this population present great variability , and psychosis] in refugees and asylum seekers from conflict-affected areas. Serious mental disorders are conditions resulting in serious functional impairment, which interferes with major life activities NIH, . Other det al., et al., v. high-income countries, and among different kind of population sample (convenience samples v. probability-based samples).Similar to previous studies (Fazel www.prisma-statement.org) statement from inception to June 4, 2020, by a medical librarian. Search terms included controlled terms as well as free text terms. The following terms were used (including synonyms and closely related words) as index terms or free-text words: \u2018refugees\u2019 and \u2018mental illnesses\u2019. A search filter was used to limit the results to adults. The search was performed without date or language restrictions. Duplicate articles were excluded. The full search strategies for all databases can be found in the online Supplementary materials.et al., et al., Studies were considered eligible for this meta-analysis if they examined (a) the prevalence of serious mental disorders , (b) in an adult (\u2a7e18 years) population of refugees and asylum seekers who had to cross their country borders, (c) according to the criteria of Diagnostic and Statistical Manual of Mental Disorders or International Classification of Diseases (ICD 9 or10) (d) assessed with a structured or semi-structured clinical interview, such as the Structured Clinical Interview \u2013 SCID, Mini-International Neuropsychiatric Interview \u2013 MINI, or Composite International Diagnostic Interview \u2013 CIDI. No cut-off has been considered to evaluate the diagnosis' severity. Disorders have been evaluated serious according with the National Institute of Mental Health (NIH) and GBD considerations and asylum seekers (a person who seeks protection from persecution or serious harm in a country other than their own) were in agreement with the UNHCR Master Glossary of Terms UNHCR, and AsylAll title and abstracts were screened by two researchers independently (MP/MS or SG). We retrieved the full texts of all abstracts that seemed eligible for inclusion. Subsequently, we performed a full-text selection according to our pre-specified eligibility criteria. Disagreements between the reviewers were resolved by discussion.For every eligible study, two reviewers independently (MP/MS or SG) extracted data related to the year of publication, the country of data collection, the country of the refugees' origins, the sample size of the population, the age, the gender, the time elapsed as refugees or asylum seekers, the diagnostic tool used. Finally, we extracted the prevalence rates of MDD (current episode and recurrent), PTSD, BPD and psychotic disorder. In the absence of absolute numbers and if allowed by the data reported, the percentages of prevalence rates were converted into numbers. The time since displacement was reported in three different categories: (1) people who have spent more than five years as refugees or asylum seekers (\u2a7e5), (2) people who have spent between 5 years and 1 year as refugees or asylum seekers (>1\u00a0<\u00a05) and (3) people who have spent less then 1 year as refugees or asylum seekers. Countries were categorised as low-, middle- (LMIC) and in high-income (HIC) based on the World Bank list of economies 2020 evaluated the assessment of the methodological quality of individual studies using a modified version of the Joanna Briggs tool \u2013 JBI Critical Appraisal Checklist for Studies Reporting Prevalence Data . Prevalence rates were calculated per each disorder separately. Pooled rates for subgroups were indicated, when at least three studies were present for each disorder. All statistical analyses were conducted using Meta-prop package in STATA/SE 16.1 for Mac of MDD between studies conducted in refugees resettled in LMICs higher prevalence rate of MDD has been reported in studies higher prevalence of MDD and PTSD than studies . The other analyses of the MDD, reMDD and PTSD subgroups, showed no significant differences among prevalence rates and high heterogeneity , followed by PTSD (31%), recurrent episode of MDD (16%), and BPD (5%). The prevalence of psychotic disorders was 1%. Subgroup analyses showed that MDD appeared to be more prevalent (47%) among studies conducted in LMICs than in HICs (28%), and when the MINI (37%) has been used, compared to other diagnostic instruments (26%). PTSD and MDD showed higher prevalence rates (34% and 35% respectively) in studies where participants were from convenience samples in comparison to studies that used probability-based samples. PTSD and MDD were the most frequently evaluated disorders with 36 studies and 32 studies respectively, as opposed to BPD and psychosis of which we had only a few studies.et al., et al., et al., et al., et al., et al., et al., According to previous studies, the world-wide prevalence rate reported for MDD is 4.4% WHO, , the lifet al., et al., et al., et al., Our results are in line with the prevalence rates of the Blackmore and colleagues , and that its administration is shorter and its outcomes therefore potentially less precise.Despite the high heterogeneity and methodological limitations of included studies, a high prevalence of serious mental disorders was found in refugees and asylum seekers diagnosed through structured clinical interviews. However, specific instruments culturally adapted for use across different local cultures and contexts for refugees would be advisable, since no one of the diagnostic instruments currently in use has been developed for the non-western populations, who represent the highest sample of refugees. For this reason, to reduce the high heterogeneity, more rigorous studies, using representative samples and culturally adapted instruments, to measure cultural concepts of distress would be recommended. Moreover, to strengthen the evidence base concerning the more serious mental disorders, further research on the prevalence of psychosis and BPD is imperative. To follow this goal, international and government investments in mental health research, population screening, and specific interventions on asylum seekers and refugees are warranted. However, until we have more rigorous studies and adequate diagnostic tools, due to the high heterogeneity between these studies, our results should be considered with caution.In sum, our systematic review and meta-analysis show that it is imperative that governments and actors across the world acknowledge the devastating effect of war and prosecution on individuals' mental health. In order to prevent cycles of violence and victimisation, effective public mental health responses may be put in place to address worldwide suffering.All the data involved have been included in Tables and Figures of this paper, including online Supplementary materials."} +{"text": "Image and video processing operatons are significant in our life as most electronic devices, such as PCs and mobiles, are all developed by signal processing. Therefore, signal processing can be considered as a part of computer science. Information entropy is very important in signal processing, and it was developed by Claude Shannon and Harry Nyquist who defined data representations of the physical world. Nowadays, deep learning is widely used in signal processing due to its good performance. This Special Issue calls for current studies on several signal (including image and video) processing approaches that are based on information entropy and machine learning. Papers that were both applicative and theoretical in nature were welcomed (both research papers and reviews), and contributions regarding new signal processing tools for the entropy research community were also welcome. Finally, six papers were selected for this Special Issue.In the contribution by Jia et al., \u201cChaotic Mapping-Based Anti-Sorting Radio Frequency Stealth Signals and Compressed Sensing-Based Echo Signal Processing Technology,\u201d the signal-generating design principle of anti-sequential difference histogram (SDIF) arrangements was studied for the principal arrangement approach of the SDIF . In addiIn the contribution by Bao et al., \u201cPCQNet: A Trainable Feedback Scheme of Precoder for the Uplink Multi-User MIMO Systems,\u201d the authors presented a CNN-based compression network titled PCQNet to minimalize the feedback\u2019s overhead . The autThe contribution by Wang et al., \u201cEfficient Entropic Security with Joint Compression and Encryption Approach Based on Compressed Sensing with Multiple Chaotic Systems\u201d, focused on a novel approach that uses compressed sensing for image compression and encryption concurrently . In thisIn the contribution by Zhao et al., \u201cA Complex-Valued Self-Supervised Learning-Based Method for Specific Emitter Identification,\u201d the authors present a complex self-supervised learning approach to fully examine the unlabeled examples . In the In the contribution by Liu et al., \u201cMulti-Scale Mixed Attention Network for CT and MRI Image Fusion,\u201d the authors present a CNN-based CT and MRI image fusion approach, which uses a pictorial saliency-based plan to reserve more valuable information . In thisIn the contribution by Sun et al., \u201cOn Architecture Selection for Linear Inverse Problems with Untrained Neural Networks,\u201d the authors attempted to broaden the application and understanding of untrained neural network priors by exploring the interaction between the choice of architecture and measurement models . The aut"} +{"text": "To the Editor: We read with great interest the article by Lima et al. (Melioidosis is not a notifiable disease in India. Even so, from a single tertiary-care teaching hospital at Odisha, we have reported >100 cases of culture-confirmed cases during 2016\u20132021 (Clinical severity of melioidosis is predominantly a function of host immunity ("} +{"text": "Li et al.; Sequencing technologies including bulk and single-cell approaches have been employed broadly to investigate molecular changes and the underlying mechanisms of cancer development, progression, and metastasis . For insThe Application of Sequencing Technologies and Bioinformatics Methods in Cancer Biology, we aimed to collect novel findings and methods in the field of cancer biology related to mining bulk and single-cell sequencing data with bioinformatics approaches for illuminating different types of tumors and immune checkpoint expression or tumor mutation burden was an effective prognostic biomarker for immunotherapy. Their findings highlighted that SRS could regulate the immune microenvironment of LUAD.Zhou et al. explored the immune-oncology profile of LUAD and identified a set of immune genes significantly correlated with progression-free survival. They also found that the risk signature based on several of these immune genes was associated with neutrophil infiltration. Therefore, these three studies screened out different types of prognostic biomarkers for LUAD patients, which could be useful and promising for survival analysis.Three studies on this Research Topic dissected lung adenocarcinoma (LUAD) from different perspectives and identified a number of potential prognostic biomarkers. For example, Liu et al. identified five CRC survival-related genes based on hub gene analysis of co-expression and protein-protein interaction networks. They also constructed a promising risk model using three of them for prognostic prediction. Their results indicated that these hub genes could be correlated with CRC development and the survival of patients. Wang et al. constructed a scoring model based on four necroptosis-related genes , which can effectively predict the prognosis and response of colon cancer patients to chemotherapy and immunotherapy. They also found that the activation of necroptosis-related genes could promote the metastasis of colon cancer. Unlike these two studies, Bartlett et al. investigated the association between miRNAs and immune checkpoint blockade for CRC. They detected a number of miRNAs that were correlated with mutation burden, microsatellite instability, or PD-L1 expression. Among them, three miRNAs were related to the M1 macrophage polarization state and their targets had the potential to impact the TGF-\u03b2 pathway.For colorectal cancer (CRC), three studies discovered potential prognostic biomarkers or treatment preditors by analyzing large-scale datasets. For instance, Zhao et al. discovered that loss of ARID1A was strongly correlated with the high expression of CD47 in gastric cancer. They identified CD47 as a potential direct downstream target of ARID1A, while the combination of ARID1A and CD47 would be a promising prognostic biomarker. Pan et al. examined the landscape of papillary thyroid cancer (PTC) at single-cell resolution and detected molecular signatures correlated with the disease-free survival of patients. They also found that dendritic cells and B cells could play critical functions in preventing PTC progression. identified GPX1 as a promising prognostic biomarker for breast cancer based on whole-exome sequencing and single-cell RNA-seq data. They found that RUNX3 could be a potential driver and therapeutic target for RRMM. Wu et al. detected KIF23 as an effective prognostic biomarker for clear cell renal cell carcinoma (ccRCC). They also revealed that KIF23 could promote the nuclear translocation of \u03b2-catenin, while the knockdown of KIF23 would reduce the proliferation, migration, and invasion of ccRCC. For liver cancer, Wu et al. showed that up-regulated expression of POSTN, LAYN, and HTRA3, and down-regulated expression of AANAT and AFM were associated with poorer overall survival of patients. They also found that the gut microbial metabolites of trimethylamine N-oxide TMAO and POSTN were potential targets for liver cancer treatment. Jiang et al. revealed that TMEM176B could be a diagnostic and prognostic biomarker for skin cutaneous melanoma. The expression of TMEM176B was shown to be correlated with tumor-infiltrating lymphocytes, pathological stage, therapy sensitivity to radiation, as well as tumor ulceration.In addition to biomarker exploration articles in lung and colorectal cancer, we received articles that explored biomarkers in gastric, thyroid, breast, and other tumor types. For example, t cancer . They obWu et al. systematically analyzed AP3S1 in diverse tumor types and found that its expression was widely associated with the immunosuppressive microenvironment. They also demonstrated that AP3S1 could be a pan-cancer biomarker for prognosis and immunotherapy. The other study explored the gene expression profiles of 988 cell lines from 20 distinct cancers by employing several computational methods Ding et al. They identified robust pan-cancer biomarkers for differentiating a variety of cancer types. Overall, the aforementioned studies discovered different types of potential biomarkers for diverse tumors, which could be useful for the diagnosis, prognosis, or treatment of corresponding cancers.Another two studies conducted pan-cancer analyses and detected potential biomarkers for the clinical management of cancers. For example, Dai et al. developed a novel model based on the score of cell type compositions (CTCs) for improving the prognostic analysis of acute myeloid leukemia patients (AML). They further showed that the CTC score could potentially benefit the individualized treatment of AML patients. Chen et al. systematically investigated the chromosome instability (CIN) profile of 280 patients with bone metastatic cancer based on the copy number variations inferred from cell-free DNA (cfDNA) sequencing data. They revealed that CIN quantification with cfDNA provided an effective and non-invasive method for predicting the survival of spine metastasis patients. Yuan et al. developed a scoring approach based on 15-DNA repair gene signatures for effectively predicting the prognosis of gastric cancer patients who received immunotherapies. The scoring system developed by them may benefit the tailored immunotherapy of gastric cancers. Chen et al. proposed an efficient strategy for identifying the circular RNAs (circRNAs) with protein-coding potential in CRC. They also suggested that those circRNAs might be functional in promoting proliferation and invasion ability, while the peptides derived from circRNAs could be potential targets for CRC therapy or diagnosis. Hu et al. designed a panel based on 28 breast cancer-related genes for long-read sequencing . They demonstrated that this approach can effectively detect structural variations in breast cancer patients, which could be used in related clinical investigations. Shi et al. systematically evaluated a gene panel containing \u223c1,300 key immuno-oncology genes designed for characterizing tumor microenvironments. Based on the analysis of >1,200 formalin-fixed paraffin-embedded tumor samples, they showed that this panel was comparable with orthogonal platforms . The computational strategies developed in these studies were useful and promising for exploring and characterizing various aspects of different cancers.Besides biomarker investigation, several studies on this Research Topic developed new computational strategies for profiling tumors from different aspects. For instance, Taken together, the studies published in this Research Topic presented a diversity of interesting and meaningful results for a range of different cancers, which could facilitate our understanding of cancer biology. It is well known that bulk and single-cell sequencing technologies can respectively obtain whole-system and cellular views of tumors. Multi-omics and multimodal strategies are superior to single-omics methods for dissecting cancers since different types of data could be complementary . Third-g"} +{"text": "Micro and nano molding technologies are continuously being developed due to enduring trends such as increasing miniaturization and the higher functional integration of products, devices and systems. Furthermore, with the introduction of high-engineering-performance polymers, feedstocks and composites, new opportunities in terms of materials properties can be exploited, and consequently, more micro product and micro/nano structured surfaces are currently being designed and manufactured.Innovations in micro and nano molding techniques are seen in different processes employed in production ; in the use of new and functional materials including, e.g., nanocomposites; for an ever-increasing number of applications ; in several key enabling technologies that support the successful realization of micro and nano molding processes and their integration into new manufacturing process chains.Accordingly, this Special Issue seeks to showcase research papers focusing on the latest developments in micro and nano scale manufacturing using molding techniques as well as their related key enabling technologies to produce both micro products and micro/nano structured surfaces.The Special Issues consists of 10 original research papers and 2 review papers, which cover fundamental molding process technology development, key enabling technologies, as well as the design and application of these technologies for the fabrication of micro/nano devices and micro structured components.The papers included in the Special Issue address research, development and recent advancements in four main domains of micro/nano molding: (1) process technology developments and characterization; (2) modeling and simulation; (3) tooling technologies and micro tool design; (4) applications.(1)Process technology developments and characterization. Calaon et al. analyzed(2)Modeling and simulation. Weng et al. modeled (3)Tooling technologies and micro tool design. Wang et al. develope(4)Applications. Kim et al. presenteWe wish to thank all of the authors who submitted their papers to this Special Issue, entitled \u201cLatest Advancements in Micro Nano Molding Technologies\u2014Process Developments and Optimization, Materials, Applications, Key Enabling Technologies\u201d. We would also like to acknowledge all of the reviewers whose careful and timely reviews ensured the high quality of this Special Issue.http://www.prosurf-project.eu/, accessed on 5 April 2022, 2018\u20132021, Project ID: 767589), as well as the Marie Sk\u0142odowska-Curie Action Innovative Training Networks MICROMAN and DIGIMAN4.0 , is acknowledged. The support and funding from the Danish Innovation Fund , through the research projects MADE DIGITAL, Manufacturing Academy of Denmark , Work Package WP3 \u201cDigital manufacturing processes\u201d and QRprod is acknowledged.The support and funding from the European Commission Horizon2020 Framework Programme for Research and Innovation through the ProSurf project (\u201cHigh Precision Process Chains for the Mass Production of Functional Structured Surfaces\u201d,"} +{"text": "Development of a risk assessment profile tool to determine appropriate use of SARS-CoV-2 rapid antigen detection tests for different activities and events in Ireland, since October 2021\u2019 by Mallon et al., published on 20 January 2022, the name of author Aileen Conway was corrected . This change was made on request of the authors on 4 February 2022.In the article \u2018"} +{"text": "Lane CA, Barnes J, Nicholas JM, et al. Associations between blood pressure across adulthood and late-life brain structure and pathology in the neuroscience substudy of the 1946 British birth cohort (Insight 46): an epidemiological study. Lancet Neurol 18: 942\u2013522019; \u2014The appendix of this Article has been corrected as of June 17, 2020."} +{"text": "Aberrations in epigenetic regulation at various levels, including DNA methylation, chromatin architecture, and regulatory RNAs, are often associated with, and significantly contribute in most carcinogenesis . TranscrParrello et al. discussed the possibility of targeting factors that control global transcriptional regulation. Li et al. discussed the dual roles of CBX7, a component of polycomb repressive complex, in cancer where it can either help in cancer progression by downregulating tumor suppressor genes or help cancer suppression by modulating cell cycle related proteins. CBX7 interacts with various regulatory RNAs, including micro RNAs, long non-coding RNAs, circular RNAs. Regulatory RNAs play a significant role in carcinogenesis, including chemo-resistance . Sun et al. showed that plasma-derived exosomal micro RNA, miR-2276-5p in glioma patients could serve as a potential diagnostic and prognostic marker. Transcriptomics based gene signatures are emerging as promising biomarkers in cancer .Genome-wide transcriptional control is often dysregulated in cancer. In this research topic, Fatima et al. and Raina et al.). Abbas et al. reported that maternal diet rich in omega-3 fatty acid can reprogram epigenetic and transcriptomic landscapes in F1 generation mice and provide resistance to breast cancer development. Pu et al. reported that methylation profiles of zinc finger genes, specially ESR1 and ZNF132, could be potential biomarkers for the early diagnosis of colorectal cancer patients carrying KRAS mutations. Another study by Gua et al. showed that APOA1 gene is downregulated by DNA methylation in hepatocellular carcinoma that could be a potential biomarker to predict survival. Role of NTPCR in epithelial ovarian cancer and FGFR1\u2013GLI1 axis as a potential therapeutic target in breast cancer were also reported.Epigenetic landscape is altered in cancer cells that results in transcriptional dysregulation. Various dietary components have ability to modulate epigenetic aberration (Gazova et al. used CRISPR-Cas9 to generate homozygous inactivating mutation in USP16 gene using leukemia cell line and studied how these cells adapt to the extreme selection pressure through compensatory pathways. Authors also cautioned targeting USP16 in leukemia as cancer could develop resistant to USP16 inhibitors. A timely review by Amir et al. discussed the usefulness of combination therapy of tyrosine kinase inhibitors with epigenetic drugs in chronic myeloid leukemia. Leszczynska et al. reviewed the emerging therapeutic approaches against pediatric high-grade gliomas, particularly those having mutations in genes coding for histone 3 variants that result in substitution of lysine at 27 to methionine.In conclusion, epigenetic aberration and transcriptional homeostasis disruptions are associated with cancer. In-depth understanding of these processes and their interdependencies is needed to better understand carcinogenesis and to develop novel and effective therapeutic approaches."} +{"text": "L\u00fcke et al.).The aim of anakoinosis is \u201ctissue editing,\u201d meaning that bioactive, regulatory acting principles are combined to re-establish tissue homeostasis at primary and metastatic tumor sites by communicatively reprogramming tumor tissue and cell recruitment . Pro-anaL\u00fcke et al.; L\u00fcke et al.; Heudobler et al.). The clinical responses outline the possibility to specifically activate unique tumor tissue dynamics in response to pro-anakoinotic effectors, either by combining differently acting biomodulatory drugs, or associating DNA damage with fungal infection.This Research Topic highlights differential clinical outcome characteristics in three histologically completely different neoplasia as a response to \u201ctissue editing\u201d when treated either with pro-anakoinotic approaches or accidentally initiated by severe fungal infection and reduced intensity induction chemotherapy in acute lymphoblastic leukemia (ALL) .DNA damage response following \u201cone shot\u201d chemotherapy in ALL seems to serve as a homeostatic trigger in the case of parallel fungal infection, presumably supervising and amplifying immunological response by the innate immune system, thereby preventing both early relapse and persistence of minimal residual disease, as indicated by long-term continuous complete remission in both described ALL cases .In r/r NSCLC, explorative studies of the randomized trial provide strong hints that pre-treatment with combined biomodulatory therapy may be the basis for successful consecutive immune checkpoint inhibitor (ICPi) therapy: even though the progression-free survival rate of the biomodulatory treatment arm is significantly inferior to that of the ICPi arm, it, however, exerts tissue modifications that render successive ICPi more efficacious. These results stimulate the hypothesis that the addition of ICPi to the biomodulation could be beneficial .The immune-modulatory acting MEPED schedule for r/r cHL resulted in pivotal outcomes in six cases. Whether a patient is frail and does not respond to reduced standard first-line therapy, or patients are r/r after autologous hematopoietic-stem-cell transplant (autoHSCT), or even allogeneic hematopoietic-stem-cell transplant , MEPED may induce complete PET negative remission. The remissions could be maintained by alloHSCT. But even after discontinuation of MEPED in a frail patient, CR continued without any HSCT. Like in NSCLC, a possible beneficial supplementation of the immune-modulatory schedule could be ICPi therapy has been given by the group of dulators .Favero et al.; L\u00fcke et al.; L\u00fcke et al.; Heudobler et al.). In vitro studies by Del Favero identify novel biological paths induced by bio-physical circumstances, such as shear stress, and give a first hint on how to target respective biologic structures for reprogramming tumor functions. Drugs used in pro-anakoinotic schedules, like mTOR inhibitors, e.g., in cHL, may reprogram a response to shear stress, as experimentally shown .Besides therapeutic modulation of cell metabolism, immune response, or tumor-associated inflammation in neoplasia, as exemplified in cHL, NSCLC, and ALL or GVHD, reprogramming the mechanical properties of tumor and adjacent stromal cells may be an important new therapeutical direction . The ALL studies highlight that clinical courses of malignant diseases may cause tissue alterations that render therapeutically accessible homeostatic disbalances previously hidden .For successful development of combination therapies in the field of anakoinosis, the detailed description of complex tissue functions supporting disease-related, pathophysiologically dysbalanced homeostasis is necessary. This message clearly emerges from the contributions on GVHD, and from the mechanistic analysis of how T24 bladder cancer cells cope with fluid shear stress by modifying their shape .Development and evaluation of biomodulatory drugs is already ongoing .A novel technology for the evaluation of systems biological drug interactions, relevant for anakoinosis research, has been presented by Liao et al.).Gold nanorods, currently studied as potential therapeutics in photothermal therapies, may also contribute to evaluating pathophysiology in tumor tissues, and as such the impact on tissue homeostasis, by identifying specific binding sites of gold nanorods in single-cell compartments (in vitro test systems, as indicated by the Research Topic, may promote the exploration of biomodulatory drugs derived from different pharmacological tools for designing pro-anakoinotically acting combination therapies, particularly for overcoming therapeutic problems with refractory neoplasia and complex non-malignant diseases associated with tissue dysregulated homeostasis (Kumari et al.; L\u00fcke et al.; L\u00fcke et al.; Heudobler et al.; In summary, this research topic provides a novel clinical collection of pro-anakoinotic approaches modifying dysregulated homeostasis in quite different ways for long-term tumor control or even healing of refractory tumor disease. Hopefully, pre-clinical"} +{"text": "With interest, we read the recent article by Wang et al. in which their logistic regression analysis identified the use of vasopressor agents, the presence of a neurological disease, high APACHE II and SOFA scores, an acute gastrointestinal injury (AGI)\u2009\u2265\u2009grade II, and the use of mechanical ventilation or continuous renal replacement therapy (CRRT) as independent risk factors influencing the success rate of placement of a naso-jejunal tube (NJT) . The imp"} +{"text": "Cancer is a genetic disease associated with the rapid growth of abnormal cells, which provoked the death of almost 10 million people worldwide during 2020 , placingSanit\u00e0 et al. reviewed the most recent techniques for surface modification and functionalization of nanoparticles in order to improve their biocompatibility and cellular uptake behavior. Similarly, Cheng et al. summarized and analyzed the current research progresses and challenges in tumor microenvironment-responsive shrink-sized drug delivery nanosystems. Cheng et al. also discussed the current implications and knowledge for promoting deep penetration into tumors using nanoparticles. Meanwhile, Chen et al. used two novel HLA-A2-restricted cytotoxic T lymphocyte epitopes (SV95\u20136 and SV95\u20137 peptides) derived from survivin , and were then loaded into human dendritic cell/poly(lactic-co-glycolic) acid-based nanoparticles, thus obtaining advanced materials specific against cancer cells. It should also be noted that Chen et al. carried out major histocompatibility complex peptide binding algorithms to predict a range of modified SV95 decamers (from SV95\u20132 to SV95\u201310) based on the natural SV95\u2013104 peptide sequence of ELTLGEFLKL. On the other hand, Sharifiaghdam et al. designed and synthesized new layer-by-layer selenium-based nanocomplexes as carriers of small interfering RNA with improved stability and a dual mode of action against tumors: gene silencing and apoptosis induction in cancer cells. To close this research topic, Shah et al. reported theranostic optical imaging probes based on shortwave infrared (SWIR)-emitting rare earth-doped nanoparticles encapsulated with human serum albumin (ReANCs), which demonstrated superior surveillance ability for detecting micro-lesions at depths of 1\u00a0cm in an animal models of breast cancer metastasis, thereby promising an ability for follow-up therapy based on SWIR fluorescence measurements from tumor-targeted ReANCs.Traditional cancer treatments have shown several limitations. Nonetheless, diverse technologies based on nanotechnology have shown significant advances with the aim of obtaining a more efficient and safe cancer therapy. Despite this, several key obstacles related to the use of nanoparticles for cancer therapy such as the complexity and heterogeneity of tumor biology, a lack of understanding of nano-bio interactions, as well as chemical, manufacturing and control challenges must be further studied for clinical success. This research topic addresses some novel aspects of engineering that take advantage of our growing understanding of bionano behaviors and interactions to develop more efficient nanotherapies for cancer patients. Keeping this in mind, in this research topic, The role of nanotechnology in cancer research has grown dramatically in recent years. However, only a few dozen nano-based technologies have reached the market so far, primarily cell-scale targeted bionanosystems, and controlled and sustained carries of desired biomolecules. To change this, we must reconsider traditional views and rethink how we conduct translational cancer nanomedicine research."} +{"text": "Editorial on the Research TopicHearing Loss: Mechanisms and PreventionHearing Loss: Mechanisms and Prevention has encompassed 34 contributions from experts who are dedicated to recent advances in the mechanisms and prevention of hearing loss in ways of hair cell damage and prevention, spiral ganglion cell development and inherited hearing loss in animal models and patients, as well as a range of novel treatment approaches for hearing loss.Hearing loss, which is often referred as an \u201cinvisible disability,\u201d is one of the most common sensory impairments worldwide. The prevalence of hearing loss is high and it is estimated that 20 percent of the world\u2019s population is affected by hearing loss to some degree in 2021 according to the WHO report. The resulting consequences are significant burdens to the economy and society. There are numerous contributing factors to hearing loss, such as noise exposure, congenital, infectious, traumatic, and immune-mediated causes and the severity of hearing loss is always related with age. To develop preventative and treatment strategies specific to the underlying causes, it is crucial to understand the pathophysiology of these contributing factors. This Frontiers Research Topic entitled Sun et al. identified that the G protein-coupled receptor 125 is expressed in multiple cell types dynamically in the developing and mature cochlea in mice. Stereocilia play an important role in hearing and balancing sensation. Du et al. investigated the role of the Rho GTPase cell division cycle 42 in mice and reported it as a vital regulator in stereocilia development of cochlear hair cells. In recent decades, new technologies have emerged in inner ear research which help researchers reveal the development of hair cells. As such, single-cell sequencing technology is a powerful tool for analyzing gene expression variations across different cell types, and it has also been proven to be useful in inner ear research. Wu et al. reviewed recent applications of single-cell sequencing in inner ear research, covering from identifying unknown cell subtypes, discovering novel cell markers, to revealing dynamic signaling pathways during development. Meanwhile, by using single-cell RNA sequencing analysis, Chen et al. Identified different cell subtypes in the greater epithelial ridge cells in the cochlear duct related to their degeneration during postnatal development in rats. During P1 and P7 rats, five cell clusters reduced significantly while four clusters, enriched with genes associated with the degeneration of the greater epithelial ridge cells, had high similarity in gene expression patterns and biological properties. Besides utilizing single-cell sequencing technique, Wu et al. from a different group developed a 3D imaging technique for the three-dimensional examination of the microstructure of the full thickness of the tympanic membranes in mice. With this imaging technique, they discovered the 3D form of the elastic and collagen network, as well as the close spatial relationships between the elastic fibers and the elongated fibroblasts in the tympanic membranes, which provides important information for hair cell development.Hair cells are most important part in sound conduction and balancing sensation. Their development is tightly correlated with the function of cochlea. To reveal cochlear development and hearing, Liu et al. reported excessive accumulation of calcium due to acoustic overexpression and slow clearance around the presynaptic ribbon might lead to disruption of calcium homeostasis by compared the consequences of noise-induced cochlea synaptopathy of C57BL/6J and CBA/CaJ mice. The susceptibility of noise-induced cochlear synaptopathy in CBA mice is caused by mitochondrial dysfunction of inner hair cells. Xiao et al. investigated the molecular behavior of high-mobility group box 1 (HMGB1) in the cochlea following noise exposure both in mice and in vitro and reported that HMGB1 has a possible negative effect on cellular lifespan indicated by the higher cell viability observed in the HMGB1 knocked-down mice after stimulation with H2O2. In addition to resolving the intrinsic cause of noise-induced hearing loss, researchers are also working on the approaches to protecting noise-induced damge. The use of FK506 (tacrolimus) to treat noise-induced hair cell loss and noise-induced hearing loss (NIHL) has been applied clinically and He et al. identified the downstream mechanisms of FK506-attenuated NIHL. They found that FK506 treatment not only inhibits calcineurin activity to attenuate moderate-noise-induced outer hair cell loss and hearing loss, but also inhibits reactive oxygen species and activates autophagy. Badash et al. demonstrated that endolympahtic hydrops are correlated with noise-induced cochlear synaptopathy by exposing live CBA/CaJ mice to various noise intensities and using optical coherence tomography to measure endolymph volume. Liang et al. also found a positive role sirtuin-3 in protecting cochlear hair cells against noise-induced damage via the superoxide dismutase 2/reactive oxygen species signaling pathway. Many different factors are also involved in the development of hair cells and contribute to hearing loss. Ding et al. identified that the ototoxicity of 2-hydoxypropyl-beta-cyclodextrin (HP\u03b2CD) spread from the high-frequency base towords the low-frequency apex of the cochlea from P3 to P28 and the HP\u03b2CD-induced outer hair cell (OHC) death is correlated with the upregulation of prestin in OHCs. In addition, 4\u20136\u00a0weeks post-HP\u03b2CD treatment, there is a second, massive wave of degeneration involving inner hair cells, pillar cells, auditory nerve fibers and spiral ganglion neurons. An interesting effect of caffeine in cochlear hair cells was identified by Tang et al. They showed that caffeine induces autophagy and apoptosis in auditory hair cells via the SGK1/HIF-1\u0251 pathway, which suggests overdoes of caffeine may lead to hearing impairment. Gong et al. described the importance of claudin h in morphogenesis and auditory function of the hair cells. Zebrafish with deficiency of claudin h have significant reduction of otic vesicle size and loss of utricle otolith and loss of hair cells in neuromasts caused by the deficiency of claudin h can be rescued by claudin h mRNA in zebrafish. Tu et al. demonstrated that the deficiency of small muscle protein, x-linked (SMPX) causes stereocilia degeneration in cochlea and progressive hearing loss using an Smpx null mouse model by CRISPR-Cas9 technique. Kwesi et al. presented a study of effect of high jugular bulb (HJB) on the hearing loss in patients with large vestibular aqueduct syndrome (LVAS). LVAS patients with concurrent HJB show higher air conduction thresholds.A great effort has been made to study the cause of hair cell damage and the approaches to protecting them. Noise can induce cochlear hair cell damage and it is the most common cause of hearing impairment. Noise-induced hearing loss involves different mechanisms and pathways. Li et al. Although hearing loss can be caused by various factors, new approaches to treating hearing loss are emerging. Dong et al. revealed the positive function of optic atrophy1 (OPA1) in hearing by examining the ability of OPA1 to protect against cisplatin-induced cochlear cell death both in vitro and in vivo. They showed overexpression of OPA1 prevented cisplatin-induced ototxicity, which suggests a possible role of OPA in ototoxicity and/or mitochondria-associated cochlear damage. It has been demonstrated before that neither N-acetylcysteine nor dexamethasone can protect hair cells from oxidative stress when at ineffective concentrations, but Bai et al. reported when these two drugs combine together, they show a better therapeutic effect both ex vivo and in clinical patients. Chen et al. developed a stable and effective to deliver dexamethasone (DEX) via an electrode coated with polycaprolactone. This device maintains stabilityof DEX concentration for more than 9\u00a0months and shows promising application in cochlear implantation. New technologies are also making contributions to treatment of hearing loss, such as stem cell-based therapies as reviewed in He et al. and nanoparticle treatment as reviewed by Huang et al. In the prior review, they fully described the ways of inducing the differentiation of stem cells, the implantation operation and regulation of exogenous stem cells after implanted into the inner ear, and elaborated the relevant inner ear signal pathways and the clinical applications of new materials. In the latter review, they summarized recent developments challenges of nanoparticles in diagnostics and treatment of hearing loss.Apart from cochlear hair cell damage, hearing loss can also relate several neurological disorders, such as Alzheimer\u2019s disease, Parkinson\u2019s disease, Huntington\u2019s disease and autism spectrum disorder, as thoroughly reviewed in Sun et al. explored the regulatory mechanisms of atrial natriuretic peptide (ANP) underlying functional properties of auditory neurons in vitro and reported that ANP could support and attract neurite outgrowth of sprial ganglion neurons (SGN) and possesses a high capacity to improve neuronal survival of SGNs against glutamate-induced excitotoxicity via triggering the natrieretic peptide receptors-A/cGMP/PKG pathway. Ma et al. indicated that the expression levels of vesicle transporter protein 3, glutamate/aspartate transporter protein, and Na+/K+-ATPase \u02511 are disrupted in spiral ganglion cells in mice after noise exposure, suggesting that disruption of glutamate release and uptake-related protein expression may exacerbate the occurrence of synaptopathy. Some gene mutations are also participated in the development of spiral ganglion neurons. Qiu et al. investigated the pathological role of mutant ATP6V1B2 in the auditory system with transgenic mice carrying c.1516 C > T (p.Arg506\u2217) in Atp6v1b2, Atp6v1b2Arg506\u2217/Arg506\u2217. They showed the transgenic mice have hidden hearing loss at early stages and developed late-onset hearing loss. The degeneration of spiral ganglion neurons are induced by apoptosis activated by lysosomal dysfunction and the subsequent blockade of autophagic flux, which then further impairs the hearing. Meanwhile, scientists are searching for potential approaches to protecting spiral ganglion neurons. Wang et al. described a transgenic mice with tumor necrosis factor 2/4 double knockout show the attenuation of spiral ganglion neuron degeneration by the differential regulation of some core molecules. Chen et al. explored a way to reprogram cochlear Sox2+ glial cells into functional spiral ganglion neurons by induction of small molecules. In the field of regeneration research of spiral ganglion neurons in the inner ear, utilization of specific genetic tool of animal models is a common research approach. For example, a specific spiral ganglion neuron damage approach is described by Hu et al. They generated a strain of transgenic mice exhibiting inducible SGN-specific Cre activity in the inner ear which may serve as a valuable SGN damage model.Spiral ganglion neurons are bipolar neurons connecting the primary auditory receptor cells, the hair cells, with the auditory brain stem. Their development and protection are highly linked with the auditory system. Xu et al. identified a spontaneous mutation of coiled-coil domain-containing 154 gene as a new osteopetrosis-related gene can induce congenital deafness. In porcrine model, Ren et al. studied the population statistics, hearing phenotype, and pathological changes of congenital single-sided deafness (CSSD) which is highly resembled with human non-syndromic CSSD disease. The deaf cochlear of this strain show cochlear-saccular degeneration. In vitro, Wen et al. investigated the mechanisms of Waardenburg syndrome (WS) by inducing an iPSC line derived from a WS patient with SOX10 mutation. The induced cells differentiated into neural crest cells (NCCs) and SOX10 deficiency had a significant impact on the gene expression patterns throughout NCC development in the iPSC model. In children, Liang et al. identified 18 new potential genes associated with congenital deafness and 87 potential new genes associated with otitis media by using a network-based method incorporating a random walk with restart algorithm, as well as a protein-protein interaction framework. In patients, Wang et al. reported a phenotypic heterogeneity of post-lingual and/or milder hearing loss with the GJB2 c.235delC homozygous mutation. Zhu et al. reported a compound heterozygous variant of the OTOF gene in familial temperature-sensitive auditory neuropathy and the auditory neuropathy can be diagnosed by the presence of cochlear microphonics with absent or markedly abnormal auditory brainstem responses (ABRs). Wang et al. further demonstrated the significance of genetic testing for auditory neuropathy patients with p.E818K in the ATP1A3 gene. All these findings have made contributions to a genetic understanding of inherited deafness and provide novel biomarkers for clinical screening.Although various environmental insults can cause damage to hair cells and spiral ganglion neurons, inherited factors will also lead to hearing loss verified both in animal models and clinical patients. In mice, In conclusion, the collection of research articles and reviews presented in this Research Topic provides a comprehensive set of information on the factors attributing to hair cell development, the mechanisms of hair cell damage and the approaches to protect them, as well as spiral ganglion neuron development and protection and genes involved in the inherited hearing loss. Most importantly, there are various new potential treatment approaches to hearing loss. Together, the achievements included in this Research Topic make huge contributions to further understand the underlying causes of hearing loss and may facilitate the development of novel therapies to treat hearing loss in the near future."} +{"text": "Following publication of the original article phenotype which means the mutant was not able to grow at ambient CO2 level to LC condition , an efficient inhibitor for both CCMs and the GlyDH (glycolate dehydrogenase) in C2 cycle (Taubert et al. Synechocystis, nor in mutant with HCR phenotype (Eisenhut et al. 2 level, without displaying the HCR phenotype. It is worthy to further investigate the underlying mechanism of glycolate excretion of strain WT-\u0394glcD.Additionally, inactivation of glycolate metabolism was reported to render a high-CO"} +{"text": "Escherichia coli and Saccharomyces cerevisiae, over the past few years, oleaginous yeasts that naturally accumulate high-content lipids have been directly used or genetically modified for producing diverse bioproducts, although early trials on the commercial production of microbial oil date back to World War I. This Research Topic concentrates on the advancement of bioengineering of oleaginous yeasts, including Yarrowia lipolytica and Rhodosporidium (Rhodotorula) toruloides, for producing biofuels and bioproducts, with particular emphasis on the establishment of synthetic biology tools and novel engineering strategies.The current Research Topic provides an effective communication platform, collecting both original research articles and review papers examining explorations of the mechanism for lipid accumulation, biotechnological applications, and metabolic engineering efforts related to oleaginous fungi including the non-conventional yeasts. Microbes have been harnessed for the production of hydrocarbon with a high-energy density as \u201cdrop-in\u201d fuels, renewable chemicals, and value-added compounds. In addition to the commonly used model organisms such as Y. lipolytica biological engineering cycle for strains development and improvement. The sets of molecular biology toolbox have been established for the genetic manipulation of non-conventional yeasts Y. lipolytica has been engineered for producing microbial lipid with a titer of 99 g L\u22121 and a rate of 1.2 g/L/ h , the oleaginous yeast Y. lipolytica, NADPH to support lipid biosynthesis was primarily generated from the oxidative pentose phosphate pathway (PPP) when glucose was used as a carbon source, this resulted in carbon loss as released CO2 for the biosynthesis of the end product (Wasylenko et al., Y. lipolytica, different synthetic pathways were engineered in yeast cytosol to convert glycolytic NADH into NADPH (Qiao et al., Y. lipolytica (Markham et al., Y. lipolytica (Xu et al., Aspergillus terreus in Y. lipolytica (Zhao et al., Y. lipolytica (Larroude et al., To construct the productive cell factories of oleaginous microorganisms, some novel metabolic engineering strategies including engineering central carbon metabolism and pathway compartmentalization have been employed. In the oleaginous yeast XX wrote the manuscript. YX and JQ provided comments and helped with the revision of the manuscript. All the authors approved the submission of this manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "The interest in utilizing food-derived compounds therapeutically has been rising. With the growing prevalence of systematic chronic inflammation (SCI), efforts to find treatments that do not result in the side effects of current anti-inflammatory drugs are underway. Bioactive peptides (BAPs) are a particularly promising class of compounds for the treatment of SCI, and the abundance of high-quality seafood processing byproducts (SPB) makes it a favorable material to derive anti-inflammatory BAPs. Recent research into the structural properties of anti-inflammatory BAPs has found a few key tendencies including they tend to be short and of low molecular weight (LMW), have an overall positive charge, contain hydrophobic amino acids (AAs), and be rich in radical scavenging AAs. SPB-derived anti-inflammatory BAPs have been observed to work via inhibition of the NF-\u03baB and MAPK pathways by disrupting the phosphorylation of I\u03baB\u03b1 and one or more kinases , respectively. Radical scavenging capacity has also been shown to play a significant role in the efficacy of SPB-derived anti-inflammatory BAPs. To determine if SPB-derived BAPs can serve as an effective treatment for SCI it will be important to understand their properties and mechanisms of action, and this review highlights such findings in recent research. Food has been used as a therapeutic treatment of diseases for millennia, however the discourse surrounding the role that food and food-derived compounds play in human health has been reinvigorated of late . Recent Systemic chronic inflammation (SCI) is one of the greatest challenges to human health and wellbeing in the 21st century . SCI proSeafood processing byproducts (SPB) are an ideal candidate to derive useful biomolecules and have already garnered attention in the fields of nutrition, medicine, and cosmetics ,11. A laBAPs are small, liberated protein fragments, around 2-20 amino acid (AA) bases long. The small size and exposed AA side chains of BAPs enable ready interaction with other molecules ,21,22. BThe properties of anti-inflammatory protein products and the mechanisms by which they act are intrinsically linked. A better understanding of how BAPs structural properties and mechanisms are connected will be required to harness their potential in the coming years. This review provides a resource in which properties and mechanisms of fish- and shellfish-derived anti-inflammatory protein products are discussed based on the recent evidence. Previous work by Chakrabarti et al., Bechaux et al., Venkatraman et al., Cal et al., Chalamaiah et al., Olsen et al., Daliri et al., La Manna et al., Zamora-Sillero et al., Nongonierma et al., Urakova et al., Guha et al., and Zhu et al. have expounded on aspects of marine-derived bioactive compounds ,32,33,34Inflammation is an important biological mechanism that promotes healing and resolution in cases of infection and injury . FurtherPancreatic cancer, for instance, can be initiated by DNA damage resulting from inflammation and the associated increase in reactive oxygen and nitrogen species . SCI proNSAIDs are a common treatment for inflammation, both chronic and acute. While NSAIDs are an invaluable tool in combating inflammation, problems arise when used to treat chronic inflammation over long periods of time, the risk of associated gastric bleeding, ulceration, renal failure, and a myriad of other side effects can occur ,25,40. AA number of broad trends have been observed in the structure of anti-inflammatory peptides. Anti-inflammatory BAPs tend to be positively charged, have hydrophobic AA residues, be rich in antioxidative AAs, and be short, of low molecular weight (LMW) peptides 34]. N-. N-34]. Anti-inflammatory peptides tend to have an overall positive charge, and contain an abundance of positively charged AAs such as arginine, lysine, and histidine, particularly in the N- and/or C-terminal positions ,44,45,462+ signaling, which plays a central role in NF-\u03baB signaling and cytokine production . NS. NS43]. NF-\u03baB is a family of inducible transcription factors that contribute to an immune and inflammatory response. NF-\u03baB is a large biochemical pathway that stimulates the production of numerous cytokines and chemokines that can be easily measured via a multitude of methods, both in vivo and in vitro . NF-\u03baB iThe canonical pathway for NF-\u03baB activation relies upon the phosphorylation and subsequent degradation of the subunit I\u03baB\u03b1 by I\u03baB, which then allows for nuclear translocation of NF-\u03baB ,61,63. ACOX-2 stimulates the conversion of arachidonic acid into prostaglandins, and is the target of most NSAIDs . ProstagIL-1\u03b2 and IL-6 are cytokines that are upregulated during inflammation. IL-1\u03b2 deregulation is implicated in inflammation related pain as well as many of the numerous chronic inflammation-related diseases previously mentioned . IL-6 upActivation of MAPK is another notable cellular process under inflammation. MAPK is a highly conserved pathway integral to numerous cellular functions including cell proliferation, differentiation, and inflammatory response ,79. MAPKTNF-\u03b1 is cytokine produced by macrophage cells during inflammation and is both an activator of inflammatory pathways and a product of inflammation. TNF-\u03b1 expression is regulated by both MAPK and NF-\u03baB pathways. Research by Gao et al. indicateOxidative stress and inflammation are intimately connected. Reactive oxygen species and reactive nitrogen species (RNS) are ubiquitous in the body and regulate cellular function when at healthy levels ,70. HoweChronic inflammation is one of the preeminent threats to the human health in the 21st century, in fact over 50% of deaths worldwide are attributable to diseases associated with chronic inflammation . It is i"} +{"text": "Circular RNA (circRNA) is a novel class of single-stranded RNAs with a closed loop structure. The majority of circRNAs are formed by a back-splicing process in pre-mRNA splicing. Their expression is dynamically regulated and shows spatiotemporal patterns among cell types, tissues and developmental stages. CircRNAs have important biological functions in many physiological processes, and their aberrant expression is implicated in many human diseases. Due to their high stability, circRNAs are becoming promising biomarkers in many human diseases, such as cardiovascular diseases, autoimmune diseases and human cancers. In this review, we focus on the translational potential of using human blood circRNAs as liquid biopsy biomarkers for human diseases. We highlight their abundant expression, essential biological functions and significant correlations to human diseases in various components of peripheral blood, including whole blood, blood cells and extracellular vesicles. In addition, we summarize the current knowledge of blood circRNA biomarkers for disease diagnosis or prognosis. EGFR) gene was used to guide the response of EGFR tyrosine kinase inhibitors in non-small cell lung cancer (NSCLC) patients, which was approved by the FDA in clinical practice are a group of endogenous noncoding RNA molecules Chen, . They weDue to the importance of liquid biopsy biomarkers in precision medicine is covalently joined with an upstream 3-prime ss Fig. Li et a. There aWith the rapid application of high-throughput RNA-sequencing (RNA-seq) technology , is the first circRNA that was discovered to function as an RBP sponge at the promoter region of their parental genes and enhances their transcription and cyclin-dependent kinase inhibitor 1 (p21), which facilitates the inhibition of CDK2 by p21, thus blocking cell cycle progression modification of circRNAs , such as BodyMap dataset, we observed tissue-specific circRNA expression in all 11 rat tissues, which may be closely related to the physiological functions of those tissues patients after surgery in adipose-secreted exosomes was found to regulate deubiquitination via the suppression of miR-34a and the activation of deubiquitination-related USP7 in plasma samples of hepatocellular carcinoma (HCC) patients, which could reduce DNA damage and promote HCC cell growth patients and 55 normal subjects. They found that serum exosomal circFLI1 levels were significantly higher in SCLC patients, especially in SCLC patients with distant metastasis under normal conditions and identified circRasGEF1B as a conserved positive regulator of the LPS response and lung cancer patients (Huang et al., hsa_circ_001937 had good discriminative power with an AUC of 0.873. After anti-TB treatment, the expression level of hsa_circ_001937 was significantly decreased compared to that of healthy controls. These results suggest that PBMC hsa_circ_001937 may be a TB diagnostic biomarker (Huang et al., circ_0000798 expression in PBMCs of HCC patients, which was associated with poor overall survival (Lei et al., circ_0000798 expression could distinguish HCC patients from healthy controls with an AUC of up to 0.703. The authors suggested that PBMC circ_0000798 has potential as a blood biomarker for HCC diagnosis and prognosis (Lei et al., , hsa_circ_0057762 and hsa_circ_0003090, that can differentiate children with SLE from healthy controls (Li et al., hsa_circ_0054633 in peripheral whole blood as a sensitive and specific diagnostic biomarker for prediabetes and T2DM (Zhao et al., With increasing knowledge of blood cell circRNAs and their function, many circRNAs in blood cells or whole blood have been proposed as liquid biopsy biomarkers for human diseases Fig. C Aufier. For insThe high stability, abundance and spatiotemporal specific expression of blood circRNAs make them ideal biomarkers for liquid biopsy. In the past several years, many studies have shown that blood circRNAs, both cell-free blood circRNAs and circRNAs in blood cells, have great potential as biomarkers of many human diseases in liquid biopsy. A biomarker with broad clinical application must have demonstrated analytical validity, clinical validity and clinical utility (Byron et al.,"} +{"text": "N- and N,N\u2032-alkyl indigo derivatives\u2019 by Daniela Pinheiro et al., Chem. Sci., 2020, DOI: 10.1039/d0sc04958a.Correction for \u2018Deep in blue with green chemistry: influence of solvent and chain length on the behaviour of The authors regret that incorrect compound names and values are reported in The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "We have read the study by Jerjes-S\u00e1nchez et al with interestCould you detail how many of your PA-VTE developed left-sided extensive iliofemoral DVTs?Chan et al performed a systematic review in 124 patients with PA-DVT in which 84 (88%) of the known 96 patients, for which the side was known to be affected, were reported to have left-sided DVT; 87 (71%) of the 122 DVTs were restricted to the proximal veins, without distal calf vein involvement, and 64% 56/87) were iliofemoral DVTs, concluding that if larger studies confirmed such findings, these could positively impact our understanding of pathophysiology and derivation of diagnostic algorithms in PA-VTE.6/87 wereWax et al performed a retrospective care series in four women with MTS, describing diverse clinical scenarios; previous ischemic stroke from presumed paradoxical emboli, chronic LE venous congestion treated prepregnancy with stenting, previous history of iliofemoral DVT treated with catheter-directed thrombolysis, anticoagulation and stenting, and active third trimester acute iliofemoral DVT. The first three patients received thromboprophylaxis doses of anticoagulation, while the fourth one was fully anticoagulated.DeStephano et al described successful pharmacomechanical percutaneous thrombectomies in three patients with extensive iliac DVTs in pregnancy from MTS, concluding that endovascular interventions may play an important role in carefully selected patients, reducing thrombotic burden, and decreasing long-term complications like postthrombotic syndrome.Regarding MTS and pregnancy, evidence suggests that MTS confers an anatomic predisposition to DVTs as vein compression leads to endothelial damage and subsequent buildup of elastin and collagen and stagnant blood flow.Should societal clinical practice guidelines consider including MTS as a strong preexisting risk factor for the development of PA-VTE?Given the paucity of data regarding MTS during pregnancy and postpartum in the form of case reports and case series,"} +{"text": "Imaging technology is being recognized as an important tool for breaking through the bottleneck of drug development as it is able to provide great insights into the morphology or functionality of pharmacological models, including cell, tissue, and animal. Imaging has several advantages compared to traditional evaluation methods, which include high spatiotemporal resolution, imaging sensitivity, and tissue specificity. In addition, imaging can be utilized to determine the gene expression, metabolism of various substances, cancer detection, drug development, as well as other fields.Imaging technology includes both microscopic and macroscopic imaging scales, and encompasses fluorescence-based microscopy , Raman-bFang et al. systemically summarized the recent advances that have been made in the development of noninvasive imaging and radiotherapy agents for the different molecular subtypes of breast cancer in preclinical studies in their study, \u201cTheranostics for the different molecular subtypes of breast cancer.\u201d The researchers provided a conceptual examination of recent or current original articles that were published within the last decade in the field of preclinical breast cancer nuclear imaging. Data were extracted from the PubMed database and filtered according to the key words \u201cbreast cancer,\u201d \u201cpreclinical,\u201d \u201cPET/SPECT,\u201d and \u201ctargeted imaging.\u201d In order to help guide future investigations of novel theranostic agents, they listed different imaging agents and cell lines that were tested in preclinical studies. They think molecular imaging can help with diagnosis, staging, guiding treatment, and predicting response to corresponding targeted therapy. The review of Zeng et al. which was titled \u201cTreatment coherent Raman scattering microscopy in oncology pharmacokinetic research\u201d highlighted coherent Raman scattering (CRS) microscopy as a novel emerging platform to facilitate oncology pharmacokinetic research. It would be of great importance to develop label-free optical microscopy that is able to assess stability and dissolution of drugs in the solid state, and uptake, distribution, interaction, and excretion of anticancer drug nanocarriers in a biological environment. Therefore, they summarized the recent technical advances and applications of CRS microscopy in the field of anticancer drug pharmacokinetics at the single cell level, drug stability and dissolution in the solid state, as well as the activities of anticancer drug nanocarriers in single cells. According to their review, there are several reasons to believe that CRS microscopy with label-free, chemically selective, high temporospatial resolution, and highly sensitive imaging can offer novel opportunities for investigation of anticancer drugs.Two review articles demonstrated the role of one imaging technology in the treatment of cancer. in vitro studies. Zhang et al. in the article \u201cMetabolic reprogramming of sulfur in hepatocellular carcinoma and sulfane sulfur-triggered anti-cancer strategy\u201d uncovered reprogramming of sulfur metabolism in hepatocellular carcinoma (HCC) and were able to provide a potential therapeutic strategy for HCC by donating sulfane sulfurs. Herein, the cell imaging assay was carried out to support their hypothesis. Their findings suggest that application of sulfane sulfurs may be an effective therapeutic strategy, particularly for HCC tumor cells that have reprogrammed the sulfur metabolism. Jin et al. in their article \u201cHirsutella sinensis fungus regulates CD8 + T cell exhaustion through involvement of T-Bet/Eomes in the tumor microenvironment\u201d have provided insights into the application of Hirsutella sinensis fungus (HSF) in a tumor immune treatment. Their study demonstrated that HSF exerts antitumor effects mainly by reducing the expression of immune checkpoints by inhibiting T-bet in T cells, which lowers Tex cell production in the tumor microenvironment. Additionally, HSF could promote the Eomes expression in order to enhance T cell function. In vivo imaging technology was utilized to evaluate the effects of HSF on various tumor mouse models. Their findings were able to provide novel insights into the effect of HSF on tumor immune responses.In order to explore a novel strategy of cancer treatment, imaging technology can be applied across several in vivo evaluation of drugs. Xu et al. in their article \u201cBioluminescence imaging-based assessment of the anti-triple-negative breast cancer and NF-Kappa B pathway inhibition activity of britanin\u201d were able to evaluate the anti\u2013breast cancer activity of britanin. Their results demonstrated that britanin induces apoptosis via inhibition of the NF-\u03baB pathways. The bioluminescence imaging screening system is useful for accelerating application of britanin in the antitumor field, which provides a useful tool for evaluating the efficacy of phytochemicals in inhibiting cancer cell proliferation in animal models. Zhan et al. in their article \u201cin vivo improvement of synergistic chemotherapy in esophageal cancerConstruction of biocompatible dual-drug loaded complicated nanoparticles for \u201d developed a doxorubicin and \u03b2-elemene\u2013loaded mesoporous silica nanoparticle system to exert inhibitory effects in esophageal cancer treatment. Fluorescence images were applied in order to validate efficacy of the combination therapy in vivo. Zhang et al. in their article \u201cTerphenyllin suppresses orthotopic pancreatic tumor growth and prevents metastasis in mice\u201d reported a novel marine-derived natural product terphenyllin with potent anti-pancreatic cancer (PC) activity. Herein, terphenyllin was found to significantly suppress PC cell growth and metastasis in vitro and in vivo. Terphenyllin induced PC cell apoptosis by increasing the expression of proapoptotic proteins and decreasing the expression of antiapoptotic proteins. The Panc1-Luc cell lines were utilized to develop an orthotopic mouse model, which may be able to closely mimic the original situation in human PC patients and may be better able to predict the therapeutic efficacy of the test compound. The in vivo imaging technique demonstrated significant inhibitory effects of terphenyllin on tumor growth. Their results reveal the therapeutic potential of terphenyllin in PC, which can help provide a basis for further developing this natural compound as an anticancer therapeutic agent.Imaging technology was a powerful tool for the Li et al.\u2019s \u201cManganese-based targeted nanoparticles for postoperative gastric cancer monitoring via magnetic resonance imaging,\u201d an Mn-based contrast agent for MRI was synthesized to provide accurate evaluation of therapeutic effects and guide treatment strategy adjustment over time. A series of in vitro and in vivo imaging experiments were employed to assess the characters of Mn3O4@PEG-RGD NPs. Their results indicated that Mn3O4@PEG-RGD NPs likely have a great potential for the MRI postoperative monitoring of gastric cancer and give an appropriate strategy for following chemotherapy. Xu et al. in their research \u201cin vitro anticancer activity of fullerenol-foxorubicin conjugates alpha 3 function by colchicineSynthesis, characterization, cellular uptake, and s\u201d designed and synthesized the fuller enol (FU)-DOX conjugates and folic acid (FA)-grafted FU-DOX conjugates in order to improve the selectivity and activity of DOX in cancer cells. They synthesized DOX and FU conjugates (FU-DOX) through the use of the acid-sensitive hydrazone bond and were further modified by FA in order to obtain FA-FU-DOX conjugates for improving tumor-targeting effects. In their study, fluorescent microscopy was utilized to monitor cellular uptake. Indeed, FA-FU-DOX conjugates may optimize the safety and efficacy profile of DOX. Zhou et al. also wrote another review article \u201cpH-Sensitive and long-circulation nanoparticles for near-infrared fluorescence imaging-monitored and chemo-photothermal synergistic treatment against gastric cancer\u201d which reported photothermal\u2013chemotherapy combined nanoparticles (PCC NPs) that have functions of chemo-photothermal synergistic therapy and continuous imaging for gastric cancer. The PCC NPs consisted of dopamine, poloxamer, DOX, and IR-820 via \u03c0\u2013\u03c0 stacking and demonstrated good biocompatibility both in vitro and in vivo. Their study can offer a novel postoperative treatment for gastric cancer.The integrated diagnosis and treatment of nanoparticles will provide precise information for a cancer treatment strategy. In Zhao et al. in their article \u201cAccuracy of endoscopic diagnosis of helicobacter pylori based on the Kyoto classification of gastritis: a multicenter study\u201d provided evidence of clinical accuracy and robustness of the Kyoto classification of gastritis in the Chinese population. Furthermore, they discovered that the reappearance of two indicators (unclear atrophy boundary and unclear atrophy boundary) in atrophic mucosa could help sufficiently determine the presence of Helicobacter pylori (H. pylori) infection on an endoscopic basis. Their prospective and multicenter study was based on 650 Chinese patients. In order to prevent the occurrence and development of gastric cancer (GC) early on, their study offered an important novel finding for screening of early GC based on the close relationship between H. pylori and GC.Moreover, imaging technology also plays a significant role in a clinical anticancer medication strategy. in vitro, evaluating in vivo anticancer effects, and benefiting the clinical diagnosis. These articles provide deep insights into methodology and applications of imaging technology. We believe that imaging technology would be increasingly welcome in oncology pharmacological research.In conclusion, a collection of 11 articles contributed to this research topic, which covers two reviews and nine research articles. It is important to note that these published articles cover a wide spectrum of applications of imaging technology in oncology pharmacological research, which includes exploring a novel anticancer chemotherapy strategy"} +{"text": "Matricaria chamomilla, commonly known as chamomile, and one of the most popular medicinal plants in the world. Thanks to a high content of phenolic compounds and essential oils, preparations from chamomile flowers demonstrate a number of pharmacological effects, including antioxidant, anti-inflammatory, antimicrobial and sedative actions as well as improving gastrointestinal function. Several recent studies have shown certain positive effects of chamomile preparations in the prevention of obesity and complications of diabetes. These effects were associated with modulation of signaling pathways involving the AMP-activated protein kinase, NF-\u03baB, Nrf2 and PPAR\u03b3 transcription factors. However, the potential of chamomile in the management of obesity seems to be underestimated. This review summarizes current data on the use of chamomile and its individual components to treat obesity and related metabolic disorders in cell and animal models and in human studies. Special attention is paid to molecular mechanisms that can be involved in the anti-obesity effects of chamomile preparations. Limitation of chamomile usage is also analyzed. Obesity is an increasing health concern related to many metabolic disorders, including metabolic syndrome, diabetes type 2 and cardiovascular diseases. Many studies suggest that herbal products can be useful dietary supplements for weight management due to the presence of numerous biologically active compounds, including antioxidant polyphenols that can counteract obesity-related oxidative stress. In this review we focus on AMPK 5' adenosine monophosphate-activated protein kinaseAP-1 activator protein-1FOXO1 forkhead box O protein 1GLUT-4 glucose transporter type 4HFD high fat dietFFAs free fatty acidsLDL low density lipoproteinsNF-\u03baB nuclear factor-\u03baBNrf2 nuclear factor erythroid 2 related factor 2PPAR\u03b3 peroxisome proliferator-activated receptor gammaROS reactive oxygen speciesSTZ streptozotocinTAG triacylglyceridesTNF\u03b1 tumor necrosis factor alphast century . Intensal., 2017; Kanda eal., 2017; Matsudaal., 2017; Catryssal., 2017; Bayliakal., 2017). Metaboal., 2017; Laclausal., 2017; Saklayeal., 2017). Global anti-obesity strategies are focused on dietary and lifestyle changes, i.e., change the energy balance so that calories spent prevail over calories consumed along with an increase in physical activity . NaturaMatricaria chamomilla (synonym: Matricaria recutita), commonly known as chamomile, is one of the most popular medicinal plants in the world and phenolic compounds - phenolic acids, coumarins and flavonoids . Preparal., 2011; Europeaal., 2011), and coal., 2000; McKay aal., 2000; Haghi eal., 2000). In vitro and in vivo studies have shown that polyphenols have protective effects against oxidative stress-related diseases, including metabolic syndrome and obesity . In linal., 2006, 2020) an) an(7) Anta, 2012). It seeSeveral human clinical trials have confirmed the effectiveness of chamomile tea in the improvement of inflammatory markers, lipid profile and insulin resistance in patients with type 2 diabetes , and thgenerally recognized as safe) list of the United States Food and Drug Administration (FDA), which means a substance added to food is considered safe by experts . Howeveal., 2010; Benito al., 2010; Europeaal., 2010), and thal., 2001; Paulsenal., 2001). Chamomal., 2001), rhinital., 2001) and eyeal., 2001). In addal., 2001; Europeaal., 2001; Nakagawal., 2001). Peopleal., 2001). Sesquial., 2001) and coual., 2001) are conal., 2001). Due to chamomile mild sedative effects , long-tSince chamomile essential oils and phenolic compounds possess a strong antibacterial activity, the oral consumption of chamomile preparations at high concentrations can affect gut microbiota composition. In this, context, both beneficial and undesirable gut bacteria can undergo qualitative and quantitative changes resulting in health adverse effects such as gastrointestinal problems. However, future clinical studies on this issue are needed. At high concentrations, plant phenols exert pro-oxidant activity ; therefChamomile flowers contain a wide range of polyphenolic compounds and essential oils that possess various biological activities including antioxidant, anti-inflammatory and energy metabolism modulating effects. Due to these properties chamomile preparations can be effectively used for obesity prevention and treatment. Whole chamomile extract seems to be more effective than isolated individual components since the latter show some differences in cellular and protein targets and together may demonstrate synergetic effects. More research is needed to establish the molecular mechanisms of action of chamomile preparations. In particular, there is a need to further explore the involvement of PPAR\u03b3 and Nrf2 signaling pathways because these were found to play dual roles in obesity demonstrating both anti-obesity and obesity-promoting effects to VIL and a grant from National Research Foundation of Ukraine (#2020.02/0118) to MMB.The authors declare that they have no conflict of interest.Maria M. Bayliak provided idea and design of the article, prepared figures and tables, performed review and editing; provided funding acquisition; Tetiana R. Dmytriv collected literature, wrote the original draft and prepared figures; Antonina V. Melnychuk and Nadia V. Strilets collected literature and wrote the original draft; Kenneth B. Storey performed review and editing and provided valuable discussion; Volodymyr I. Lushchak performed analysis, review and editing the manuscript, provided funding acquisition. All authors read and approved the final manuscript."} +{"text": "Endogen (DAMPs) . Upon PA et al.) .Nucleic Acid-Associated Inflammation\u201d by Roca Suarez et al., as well as Xu et al.Given the central role of PRRs in the control of invading pathogens and endogenous threats, it is not surprising that genetic or etiological alterations of inflammation foster a wide range of human pathologies. Underscoring this concept, the persistent dysregulation of nucleic acid-associated inflammatory pathways has been associated with the development of chronic liver diseases. The two main etiology agents that are linked with these liver pathologies are hepatitis B virus (HBV) and hepatitis virus C (HPC). These viruses have distinct genomes and viral life cycles but can both repress innate anti-viral defenses through common mechanisms. These strategies are being discussed in our Research Topic \u201cvia comparative analyses of innate immune responses, from biological models (zebrafish and mouse) to human. This underscores the existence of tissue- and species- specificities, and is discussed in six reviews of the Research Topic which cover various aspects, ranging from the role of transposable elements to the limitations of in vivo models and provides cues towards the development of high content therapeutic strategies in relevant physiological models .Innate immunity largely relies on the recognition of evolutionarily conserved structures that can be identified Santa et al. and by Kumar give a comprehensive overview of the regulatory circuitries of nucleic acid-sensing pathways.However, despite major advances in the field of innate immunity to identify the pathways involved in the onset of cytokine production in response to immune-stimulatory nucleic acids, there are still many open questions. Specifically, how these signalling pathways are regulated in respect to various nucleic acid substrates and tissue insults. In this special Research Topic, key opinion leaders in the field offer an overview of (1) the major molecular and cellular aspects of nucleic acid sensing across species; (2) the complexity of innate and adaptive immune responses and their key role in the maintenance of tissue homeostasis; and (3) the intricate connections between deregulated nucleic acid sensing machinery and human disease. For instance, reviews by Constanzo et al. and by Taffoni et al.Even though nucleic acid-associated inflammation is the first line of defense of the host, activation of innate immunity is not always guaranteed. Indeed, microbes and malignant cells have developed a variety of strategies to prevent inflammation, in order to counteract the host response or escape the induction of anti-tumor immunity . SupportHemphill et al. from this Research Topic is discussing the therapeutic potential of the three-prime repair exonuclease 1 (TREX1) targeting as a novel immunotherapy strategy against cancer.Regulating abnormal nucleic acid sensing is emerging as a potent strategy against inflammatory diseases. Thus, PRRs and their downstream effectors have become attractive targets for the identification of biomarkers and the development of therapeutic agents with broad-range efficacy against inflammatory disorders. Review by Nucleic Acid-Associated Inflammation focuses on one specific aspect of nucleic acid sensing, encompassing signaling, regulation, interspecies specificities, and pathology relevance. The Research Topic is equally addressed to expert investigators who may wish to extend their knowledge on inflammation, innate and nucleic acid immunity, and to newcomers to this exciting and quickly progressing field of investigation.In conclusion, each one of the reviews and articles presented in All authors wrote the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.In Vivo Investigation (EMERG\u2019IN\u00a0DOI: 10.15454/90CK-Y371). DO is supported by the Lundbeck Foundation (R335-2019-2138), a young talented cancer researcher grant from Kr\u00e6ftens Bek\u00e6mpelse (R279-A16218), the Br\u00f8drene Hartman Fond, the H\u00f8rslev Fond, the fabrikant Einer Willumsens mindelegat, and the Eva og Henry Fraenkels Mindefond. VT is supported by an LSHTM/Wellcome Institutional Strategic Support Fund (ISSF) Fellowship (204928/Z/16/Z). CV-B is supported by a startup grant from the Dept. of Radiation Oncology at Weill Cornell Medicine and by WCM Prostate Cancer SPORE Development Research Program Award. EV is supported by ANRS (ECTZ104527), LabEx HepSYS (ANR-10-LAB-28), and the French National Research Agency (ANR-21-CE15-0035-01).NL is supported by the European Research Council , \u201cLA LIGUE pour la recherche contre le cancer\u201d and the \u201cAgence Nationale de Recherche sur le SIDA et les H\u00e9patites virales\u201d (ANRS: ECTZ117448). CL is supported by the EU INFRAIA project VetBioNet (EU H2020 project 731014) and received institutional support from INRAE. The INRAE Infectiology of Fishes and Rodents Facility belongs to the National Distributed Research Infrastructure for the Control of Animal and Zoonotic Emerging Infectious Diseases through The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."} +{"text": "Moreover, since 2013, 12 different in vivo inhibition of TASK-1 in rats with A293 at 10 mg/kg/day induced the development of significant early signs of PAH, with abnormal elevation of right ventricular systolic pressure (RVSP), abnormal pulmonary vascular cell proliferation, pulmonary vessel remodeling, and lung inflammation in a porcine model of persistent AF was associated with an increase in pulmonary arterial pressure (Wiedmann et al., + currents, and the in vitro and in vivo selective TASK1 inhibition reduces the T cell proliferation and cytokine production (Meuth et al., In line with these results, Wiedmann et al., found that chronic in vivo pharmacological inhibition of TASK-1 should be extremely managed in treated-patients with AF.For all these physiological role played by TASK-1 in several tissues, the Based on these results, one can hypothesize that A293 may induce PAH and right ventricular dysfunction in humans. Given the long history of drug-induced PAH [anorexigens (Montani et al., HL, DM, and FA drafted the manuscript. All authors reviewed and revised the final version and approved manuscript submission.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Biomedicines journal. The mechanisms of HDL modifications and their functional implications was exhaustively reviewed by M\u00e1rquez et al. [Epidemiological studies have shown that low levels of plasma high-density lipoprotein cholesterol (HDL-C) are associated with increased atherosclerotic cardiovascular disease (CVD). However, accumulating experimental evidence has challenged this epidemiologic notion in the last decade, since HDL-C-raising strategies failed to confer increased cardioprotection in subjects at high risk for cardiovascular disease ,2. Thus,z et al. . Other sz et al. and subjz et al. , which iz et al. .Beyond the predictive value of compositional and functional properties of HDL, a retrospective study also revealed the role of low HDL-C in predicting the risk of developing coronary artery ectasia in healthy subjects . In lineThe clinical significance of HDL changes in composition or function do not only relate to atherosclerosis. Dysfunctional HDL has also been associated with other diseases, including cognitive impairment ,11 and aRecent findings from epidemiological studies suggest an inverse relationship between HDL-cholesterol (HDL-C) and Alzheimer\u2019s disease (AD). In the context of neurological disabilities, accumulating experimental evidence supports an HDL-mediated protection against memory deficits, neuroinflammation, and endothelial dysfunction . HoweverApart from traditionally assigned cardioprotective favorable functions of HDL , accumulating data suggest that HDL is also involved in host defence as part of immune system (reviewed in ). In linAn impaired insulin signaling is frequently associated with NAFLD and increased risk of cardiovascular disease . Plasma Finally, the work reported by Ced\u00f3 et al. was a clIn summary, studies published in this Special Issue provided clinical evidence of the usefulness of alterations in HDL composition and function in a wide spectra of diseases and helped define novel targets for the diagnosis/prognosis/therapeutics of cardiovascular/neurological/autoimmune diseases."} +{"text": "There has been recent debate regarding the efficacy of electroconvulsive therapy in the treatment of depression. This has been based on narrative reviews that contradict existing systematic reviews and meta-analyses. In this special article, we highlight the mistakes that occur when interpreting evidence using narrative reviews, as opposed to conventional systematic reviews and meta-analyses. For example, a group of mental health professionals, patients and relatives recently wrote to the Chair of the Care Quality Commission (CQC) in the UK, stating: \u2018Given the high risk of permanent memory loss and the small mortality risk, the longstanding failure to determine whether or not ECT works means that its use should be immediately suspended until a series of well designed, randomised, placebo controlled studies have investigated whether there really are any significant benefits against which the proven significant risks can be weighed\u2019 Read, . Calls hv. simulated ECT (sECT) for treatment of depression , and RCTs comparing different forms of ECT and pairwise and network meta-analyses of these trials.Briefly, clinical trial evidence for ECT in depression can be divided into original placebo-controlled (sECT) RCTs, further RCTs assessing ECT Ten ECT-sECT RCTs for depression have been published (summarised in n\u00a0=\u00a0294), remission rates were significantly higher for ECT v. rTMS with a significantly more pronounced reduction of depressive symptoms in the ECT group and remission . Neither review found a significant difference in discontinuation between groups. Whilst this illustrates improved trial design (contemporary ECT modes and active comparators), these trials usually lack blinding of participants.The original ECT-sECT RCTs, conducted between the 1950s and mid-1980s, would not meet contemporary standards of evidence-based medicine (EBM) . Numerous additional modern RCTs have assessed the efficacy of ECT against non-pharmacological interventions such as rTMS. In a systematic review and meta-analysis comparing ECT with rTMS of persistent and, for some, permanent brain dysfunction, primarily evidenced in the form of retrograde and anterograde amnesia, and the evidence of a slight but significant increased risk of death, the cost-benefit analysis for ECT is so poor that its use cannot be scientifically justified.\u2019et al., et al., These bold claims directly contradict evidence available at the time, as well as recently published reviews states only five of ten studies were statistically significant. Such vote-counting is a flawed method of research synthesis, and acknowledged as misleading (see I2\u00a0=\u00a00) and non-significant . This effect size is comparable to that reported by the UK ECT group, \u22120.91 (95% CI \u22121.27 to \u22120.54) derived from six trials.Given the lack of reporting of an effect size across all included trials, we conducted our own meta-analysis of trials included in the narrative review. Read and Bentall suggested Ulett et al. be exclu63]) see . Heteroget al. , and cognitive impairment in depression is recognised in MDD ; the study was powered based on this measure, which showed a statistically significant improved outcome with real ECT is discouraged\u2019. The failure to employ any standardised assessment of quality critically undermines both any objectivity and ability to draw conclusions about quality \u2013 especially as many such scales exist that are standardised, reliable and widely employed. As noted decades ago, Greenland and Begg and Mazumdar Kendall's \u03c4 were non-significant. The funnel plot however showed some visual asymmetry and a trim and fill analysis suggested three potentially missing studies \u2013 adjusting the effect size downward from \u22120.85 to \u22120.68.Meta-analyses of small underpowered trials can exaggerate effect sizes . This has implications for their general criticism of past meta-analyses, where they argue, \u2018All five of the meta-analyses claim that ECT is effective for depression but, as we have seen, they are all of a poor standard, not least because none of them pay sufficient attention to the quality of the papers on which they base this claim.\u2019 The current analysis suggests at least two possibilities: either study quality is not a key driver of effect size in sECT trials or the Read et al. assessment of study quality fails to capture relevant quality differences.Importantly, meta-regression showed the Read et al. quality et al. in trials where previous ECT was unknown or unreported (\u22121.13 [95% CI \u22121.50 to \u22120.76]) rather than those reporting previous treatment with ECT (\u22120.67 [95% CI \u22120.97 to \u22120.37]) . Hence, trials where patients previously received ECT did not bias in favour of the ECT arm through patients being \u2018unblinded\u2019. It is also worth noting that significant differences demonstrated when comparing different modes of ECT in contemporary trials would likely not be consistent with contemporary standards yet are still of high enough standard to contribute to the evidence base. Second, it represents a misunderstanding of principles of EBM that both Read and Bentall and Readet al. focus exet al., (et al., et al., et al., et al., Read et al., fail to et al., and non-v. sECT and has significant effects on patient safety that merit a public enquiry. Examining their evidence, we have identified numerous substantial problems that stem from these narrative reviews having inherent biases and major methodological shortcomings. We would suggest those concerned with interpreting evidence continue to use conventional standardised methods of systematic review and meta-analysis where possible and that policy decisions must continue to be based on this level of evidence.Recent calls for banning ECT are based on selective narrative reviews written by authors who suggest ECT does not have efficacy et al.'s empirical arguments, moving beyond disagreement is crucial. We therefore advocate for modern trials to optimise ECT side-effect monitoring, and studies to elucidate the mechanism of action of one of the most effective treatments we have in Psychiatry.Whilst we cannot agree with most of Read"} +{"text": "Editorial on the Research TopicAdvances in Molecular Docking and Structure-Based ModellingThe three-dimensional (3D) structures of proteins form the structural framework of their functions. Having access to the structure allows scientists to better apprehend molecular details of protein functions; it is also crucial for protein engineering, e.g., to modify and optimize an enzyme for a certain biochemical reaction; or for designing new and improved drug molecules based on the structure of the target protein. Structures are also needed to investigate how proteins interact; a vast majority of the protein-protein interface residues are involved in extensive intra-protein interactions apart from inter-protein interactions .https://www.rcsb.org) , and thephaFold2 , it is ein silico methodologies are often complex, have limitations, and the results must be associated with appropriate statistical and quality measures. The objective of this Research Topic was to bring together various contributions based on cutting-edge computational methodologies; these include computational analysis of structures and complexes with developments and applications that integrate docking and molecular dynamics approaches, and complex experimental data such as cryogenic electron microscopy (cryo-EM).These ab-initio. The first work by Olek and Joseph dealt with the quality of models obtained by cryo-EM. In fact, the final atomic model is often incomplete or contains regions where atomic positions are less reliable or incorrectly built. They presented a software tool for the validation of the backbone trace of atomic models built in the cryo-EM maps. They use the false discovery rate analysis to segregate molecular signals from the background and show how this approach can properly complement current measures Olek and Joseph. Launay et al. tackled the challenging question of scoring in protein\u2013protein docking. They explored several ways to perform consensus-based rescoring. They showed that rescoring performs worse than the traditional physics-based evaluation but the two complement each other and can be used in combination .Two articles underline the importance of new computational approaches for evaluating atomic models derived from experimental data or built Pitard et al. studied the interaction of calmodumin (CaM) with the bacterial virulence factor, Edema Factor (EF). The system is of great interest as orthosteric and allosteric ligands have been proposed to inhibit EF activity. Using state-of-the art MD simulations, they underlined the presence of cavities at the interface between EF and CaM that could be linked to allosteric events Pitard et al.; Tang et al. combined molecular modelling and MDs to apprehend new mechanistic insights into the exciting CRISPR-Cas9 system in the DNA cleavage state. Their results provide useful guidance for engineering new CRISPR-Cas9 editing systems with improved specificity Tang et al.; Kumari et al. look at the Farnesoid X receptor (FXR) that is essential in regulating the network of genes involved in maintaining bile acid and lipid homeostasis. MDs of FXR with or without cofactors allowed a precise description of critical residue positioning during conformational changes that explain FXR activation state underlying main differences between bound and unbound forms Kumari et al.; Ghoula et al. analysed the multi domain ceramide transfer protein (CERT) implicated in the transport of ceramide from the endoplasmic reticulum to the Golgi and plays a major role in sphingolipid metabolism. Combining docking and MD simulations, the binding affinity of 14 ligands was tested. This study suggests a novel inhibitory mechanism of CERT for limonoid compounds involving the stabilization of the sub-domain interface and could help in developing new and potentially more selective inhibitors of this transporter Ghoula et al.Classical approaches such as molecular dynamics (MD) are useful to apprehend new biological systems. In this field, Gheyouche et al. applied different approaches to model the structure of RHOA-ARHGEF1 complex and they further analysed the protein-protein interface. They refined the models using MD simulations and highlighted the importance of data-driven human inspection. The modelled RHOA-ARHGEF1 interface will be extremely useful for the design of inhibitors targeting this protein-protein interaction (PPI). Gheyouche et al. Similarly, Pal et al. look at systems of economic interest. They have characterized, using molecular docking, immune response molecules of duck protein TLR3, TLR7, and RIGI and predicted to have potent antiviral activities against different identified strains of Avian influenza Pal et al.; Gobinath et al. combined experiments and docking approaches in COVID research. They have screened and proposed new indole derivatives on the famous spike glycoprotein of SARS-CoV-2 Gobinath et al.As previously mentioned, experimental 3D structures or structural models are crucial for the design of new drug molecules. Chakraborti et al. looked at the infectious pathogen with a serious global impact: Mycobacterium tuberculosis. There is a constant need to search for novel therapeutic strategies because of the emergence of multidrug-resistant tuberculosis (MDR-TB). Universal stress protein is a perfect target in this field. A library of 1.9 million compounds was subjected to virtual screening, which led to the selection of 2,000 hits through an enrichment process, then 22 potential binders of Rv1636 were prioritized for experimental validations where two compounds of natural origin showed promising binding affinities Chakraborti et al.; Vedithi et al. looked at the proteome of Mycobacterium leprae. They presented a large set of computational approaches to unravel new potential druggable targets Vedithi et al.At a larger-scale, Souza et al. presented innovative approaches to perform high-throughput ligand-protein docking calculations by using coarse-grained molecular dynamics simulations, based on the most recent version of the Martini force field. Their approach, characterized by excellent computational efficiency, offers a level of accuracy comparable to all-atom simulations Souza et al.; Jiang et al. looked at the Interaction of leukocyte integrin macrophage-1 antigen (Mac-1) to platelet glycoprotein Ib\u03b1 (GPIb\u03b1) implicated in haemostasis and inflammatory responses. They performed a series of \u201cramp-clamp\u201d steered molecular dynamics (SMD) simulations and compared the results with single molecular AFM measures. The concordance in the results underlined the importance of such approach to understand the platelet\u2013leukocyte interaction in haemostasis and inflammatory responses under mechano- and microenvironments Jiang et al.Finally, This special issue is dedicated to the loving memory of Prof. Narayanaswamy Srinivasan who left us too soon on the third of September 2021 . As a pa"} +{"text": "The maintenance of genomic stability in multicellular organisms relies on the DNA damage response (DDR). The DDR encompasses several interconnected pathways that cooperate to ensure the repair of genomic lesions. Besides their repair functions, several DDR proteins have emerged as involved in the onset of inflammatory responses. In particular, several actors of the DDR have been reported to elicit innate immune activation upon detection of cytosolic pathological nucleic acids. Conversely, pattern recognition receptors (PRRs), initially described as dedicated to the detection of cytosolic immune-stimulatory nucleic acids, have been found to regulate DDR. Thus, although initially described as operating in specific subcellular localizations, actors of the DDR and nucleic acid immune sensors may be involved in interconnected pathways, likely influencing the efficiency of one another. Within this mini review, we discuss evidences for the crosstalk between PRRs and actors of the DDR. For this purpose, we mainly focus on cyclic GMP-AMP (cGAMP) synthetase (cGAS) and Interferon Gamma Inducible Protein 16 (IFI16), as major PRRs involved in the detection of aberrant nucleic acid species, and components of the DNA-dependent protein kinase (DNA-PK) complex, involved in the repair of double strand breaks that were recently described to qualify as potential PRRs. Finally, we discuss how the crosstalk between DDR and nucleic acid-associated Interferon responses cooperate for the fine-tuning of innate immune activation, and therefore dictate pathological outcomes. Understanding the molecular determinants of such cooperation will be paramount to the design of future therapeutic approaches. Innate immunity, the first line of host defense, is classically triggered in response to pathogen infection or local lesions to promote infection clearance or wound-healing processes. The activation of innate immune responses vastly relies on pattern-recognition receptors (PRRs) that detect danger associated molecular patterns (DAMPs) or pathogen-associated molecular patterns (PAMPs). Upon recognition of PAMPs or DAMPs, PRRs trigger signaling cascades leading to the production of soluble mediators, such as type I Interferons, cytokines and chemokines. Pathogen-derived nucleic acids constitute major PAMPs that are detected by a vast array of PRRs that operate in specific subcellular localizations. In recent years, self-nucleic acids, originating from replication stress , DNA or A plethora of cytosolic nucleic acid sensors have been described to participate in triggering Interferon responses. Such receptors notably include the ubiquitous DNA-dependent activator of Interferon regulatory factors (DAI) , AIM2 77, 8, IntHowever, there is emerging evidence for an intricate signaling network beyond the cGAS-STING cascade, which cannot be overlooked in therapeutic strategies aiming to boost STING activation. Of particular importance is the fact that cGAS and STING have been both described as involved in genotoxic stress response and to participate to the maintenance of genomic integrity. Furthermore, the DNA-PK complex, which is best known for its function in non-homologous end-joining (NHEJ)-mediated repair of dsDNA breaks (DSB), has been shown to serve as an alternative route to stimulate type I Interferon production \u201321. In pmini review, we discuss this interconnection between DNA repair mechanisms and nucleic acid immunity, by focusing on the cGAS and IFI16 receptors and the way in which they control STING activation. While several DNA repair proteins have been involved in the fine tuning of inflammatory responses , \u201cLA LIGUE pour la recherche contre le cancer\u201d and the \u201cAgence Nationale de Recherche sur le SIDA et les H\u00e9patites virales\u201d (ANRS). CT was supported by Merck Sharp and Dohme Avenir (MSD-Avenir \u2013 GnoSTic) program, followed by an ANRS fellowship (ECTZ119088). JM was supported by a \u201cConventions Industrielles de Formation par la Recherche\u201d (CIFRE) fellowship from the \u201cAgence Nationale de Recherche Technologie\u201d (ANRT). AS is supported by the ERC-PoC DIM-CrIC (893772). IV was supported by the European Research Council (637763) followed by the Prix Roger PROPICE pour la recherche sur le cancer du pancr\u00e9as of the Fondation pour la Recherche M\u00e9dicale . HC is supported by a PhD Fellowship from \u201cLa Ligue contre le cancer\u201d (TAGQ21108). We acknowledge the SIRIC Montpellier Cancer Grant INCa_Inserm_DGOS_12553 for support.JM was employed by Azelead.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Vaccines entitled \u201cInfluenza Virus and Vaccine Development\u201d. This Special Issue collects one review and seven research papers that cover multiple aspects of influenza vaccine development, including the development of candidate vaccine virus (CVV) and master donor viruses for generating high growth reassortants, new vaccines and adjuvants, assay evaluation and vaccine safety. These investigations provide a wealth of experience and lessons from recent influenza pandemics, which can also be applied to current COVID-19 vaccine development.While the scientific community has been focusing on combating novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that is responsible for the current COVID-19 pandemic, we also want to draw your attention to this Special Issue of The review article by Rockman, Laurie and Barr , highligFour other manuscripts in this Special Issue report how novel vaccines can increase the strength and breadth of immune responses beyond the traditional HA response in animal or clinical studies. Bazhan et al. describeIn this Special Issue, Carnell et al. focused The paper by Ambati et al. investigWe sincerely hope these lessons learned from past and current influenza research will benefit the ongoing battle against the COVID-19 pandemic."} +{"text": "Matrix nanocomposites are high performance materials possessing unusual features along with unique design possibilities. Due to extraordinary thermophysical characteristic contained by these matrix nanocomposites materials they are useful in several areas ranging from packaging to biomedical applications. Being an environment friendly, utilization of nanocomposites offer new technological opportunities for several sectors of aerospace, automotive, electronics and biotechnology. In this regards, current pagination is devoted to analyze thermal features of viscous fluid flow between orthogonally rotating disks with inclusion of metallic matrix nanocomposite (MMNC) and ceramic matrix nanocomposites (CMNC) materials. Morphological aspects of these nanomaterials on flow and heat transfer characteristics has been investigated on hybrid viscous fluid flow. Mathematical structuring of problem along with empirical relations for nanocomposites materials are formulated in the form of partial differential equations and later on converted into ordinary differential expressions by using suitable variables. Solution of constructed coupled differential system is found by collaboration of Runge\u2013Kutta and shooting methods. Variation in skin friction coefficient at lower and upper walls of disks along with measurement about heat transfer rate are calculated against governing physical parameters. Impact of flow concerning variables on axial, radial components of velocity and temperature distribution are also evaluated. Contour plots are also drawn to explore heat and thermal profiles. Comparison and critical analysis of MMNc and CMNc have been presented at lower and upper porous disks. Our computed analysis indicates that hybrid nanofluids show significant influence as compared to simple nanofluids with the permutation of the different shape factors. Initially, nanofluids are considered as single phase flows but it is experimentally proved that consideration of nanoliquid as two phase compositions is much more beneficial. Yamada et al.2 discussed the combined two or more than two organic and inorganic constituents at nanoscale which are obliged in polymers. This idea executed hybridization of nanoparticles with different organic/inorganic compositions. The hybridization of base fluid with multiply structured nanoparticles change dynamics of industrial world3.With advancement in technology modern thermal management systems like central processing units in computers, light emitting diodes, transistor, solar collectors, audio amplifiers and so forth requires liquids with improved thermal conductivity. Since, ordinary fluids are unable to meet desired requirements for advanced industrial and technological procedures so insertion of nanoparticles in poorly conducting ordinary liquids are managed. The appropriate mixture of nanoparticles in specified ratios has generated exceptional thermal performance of weak thermalized systems. The concept of addition of nanoparticles was inaugurated by Choi and Eastman2, Aluminum Oxide (Al2O3) because of fact that they provide strong wear resistance and compressive power. In addition, majority volume of matrix metal composites are filled by a ceramic such as nitrides, oxides, silicides and borides. To gain optimized usage and evoking of precise nanoscopic features the ceramics as well as metallic elements are uniformly distributed. Zeeshan et al.4 investigated influence of morphological aspects of nanoparticles by varying shape on features of fluid floating across a spinning disk. Ahmad et al.5 depicted magnetized nano squeezed flow between two parallel disks along with consideration of viscous dissipation and multi natured hybrid particles. Haq et al.6 inserted Manganese-Zinc ferrite and Cobalt ferrite in viscous fluid flow between parallel disks and adumbrated enhancement in thermal conductance. Heat transfer features by inducting platelet, cylinder, and brick-shaped copper nanoparticles in water was studied by Khan et al.7. Hajabdollahi et al.8 examined morphometric aspects of (Al2O3) nanoparticles by considering platelets, spherical, cylindrical shapes, and bricks shapes on performance of tube heat exchangers. Kucharik et al.9 discussed absorption of metallic particles in water for formation of colloidal substances used in heat transfer diffusion processes in lasers. Ghozatloo et al.10 analyzed experimentally about dynamics of insertion of gold nanoparticles in water and ethylene glycol and build comparative framework regarding affectivity of particles against viscosity variant base liquids.From advent of twenty-first century a new findings in nanotechnology is discovered which is the concept of matrix nanocomposites materials. It is a two phase material with one phase consisting of solid material and other phase is nanometer sized particles. Nanomaterials are lightweight, highly strength full, sustainable, elevated temperature strengthened, corrosion resisted can be used in a wide variety of applications, e.g. aerospace, automotive, biomedical engineering etc. On basis of such mechanical, electrical, thermal, optical, electrochemical and catalytic features nanocomposites are characterized in to ceramic/metal matrix nanocomposites. MMNC is the material which composes of metal matrix distributed in concrete, ceramic, or organic substances. The reinforcement and distribution of matrix material into nano-scale particles which generates remarkable improvement in mechanical properties of formed composition. In metal matrix composites metals like copper, magnesium, and aluminum are capitalized as base metal. Since, base materials are metal which have stiffness and strength so MMNC is obliged in aviation, aerospace, defensive weapons and other areas. The most widely used reinforcements in matrix nanocomposites are Silicon Carbide (SiC), TiO11 investigated unsteady squeezing flow and upsurge in convective motion of fluid molecules bounded between two parallel disks. Mochizuki et al.12 conducted experimental analysis the mount in thermal conductivity of viscous fluid flowing between two parallel heated disks rotated radially. Characteristics of hall current on hybridized nanoliquid flow across spinning disk was demonstrated by Acharya et al.13. Bhattacharyya et al.14 examined enhancement in heat transfer properties of viscous liquid between two coaxial disks by adding carbon nanotubes. Bhat et al.15 analyzed heat transfer in flow through addition of nanostructures in a porous disk by employing tangential slip level constraint at boundary. Maskeen et al.16 established hybridized nano fluid flow with combination of alumina and copper particles under impact of Lorentz magnetic field. Ghaffar et al.17 numerically studied heat transfer in incompressible flow of fluid between two orthogonally passing coaxial porous disks with interaction of ferromagnetic nanoparticles.Heat is form of energy that arises due to temperature difference. Heat transfer with in flow procedures has received several applications like in electric power production, vehicle propulsion and climate control devices, home heating, and cooling appliances and so many. Heat transmits during the flow by the way of conduction, convection, and radiation. Generally, heat transfer in fluid arises due to convective motion of liquid molecules and enhanced is done by uplifting thermal conductivity of liquid. Researchers have experimentally discovered that this is executed by adding nanoparticles. This experimentation is proved to be successful when cooling and heating of domestic appliance like refrigerator, coils and electrical device is raised by nanoparticles inclusion. Chamkha et al.18 used hybridization of Titanium dioxide with Silicon Carbide in heavy water limited between moveable disks under effect of vertical magnetic field. Use of hybrid nanofluid during treatment of cancer patients was disclosed by Hayat et al.19. Enhancement in thermal transfer by using hybrid nanoliquid during industrial procedures was explained by Hussein et al.20. Devi et al.21 examined hydromagnetic hybrid nano-liquid (Cu-Al2O3/water) for raise in temperature and heat transfer up gradation in liquid flowing in permeable flow domain. Sarkar et al.22 evaluated optimistic impact of hybridization of nanoparticles for enhancing temperature profile. Sundar et al.23 provided comparative analysis regarding heat transfer features attained with addition of ordinary and hybridized nanoparticles.In recent years some inconsistencies in reported results regarding heat transfer rate with addition of nanoparticles is found. So, researchers have tried to use hybridized nanofluid which is engineered suspension of dissimilar nanoparticles in mixture or in composite form. This idea has further improved heat transfer and pressure drop characteristics and removed disadvantages of individual suspension. With the formation of hybridized nanofluids a better thermal network containing synergistic effect of nanomaterials is attained. In this regard several studies is available in literature like Behnam et al.24 analyzed MHD flow and its practical appliance in multiple sectors. The field of magneto hydrodynamics was introduced by Alfven et al.25. Emad et al.26 demonstrated analytical and numerical treatment about hybrid magnetic nano material in porous stretching/shrinking medium. Ghadikolaei et al.27 analyzed heat transfer features of electrical conducting hybridized nano liquid (TiO2-Cu) in water by varying shape of nanoparticles. Hayat et al.28 explored magnetically effected fluid flowing between two parallel spinning disks by using analytical approach. Ahmad et al.29 explored impact of magnetic field on asymmetric flow of fluid and found decrease in momentum profile. Rashidi et al.30 magnetic flow of viscous fluid by interpreting impact of it on thermal conductivity. Raddya et al.31 explored impact of magnetic field on nano-liquid flow in rotating frame and found reduction in velocity against Hartmann number. Mliki et al.32 demonstrated convective flow of nano fluid under appliance of MHD in an enclosure by examining thermo physical features. Turkyilmazoglu et al.33 explicated heat transfer features of magnetized viscous fluid flow generated by spinning disks. Some recent literature about flow characteristics of fluid under appliance of magnetic field in different physical configurations is enclosed in38. Sheikholeslami et al.39 aimed to investigate hot gas flow inside inner pipe and operating fluid in the annulus region filled with nanoparticles composed of CuO. Wang et al.40 analyzed utilization of hybrid nanofluid in solar photovoltaic system using the spectral beam splitting technology. Chuanpan et al.41 described a high-efficiency sensing system working with carbon nanofibers and discussed it efficiency by varying shape and amount of fibers. Guo et al.42 founded application hybridized nano based materials in environmental protection. Yun et al.43 determined degradation performance of nano-TiO2\u00a0as a coating material used in roads and bridge construction. Sheikholeslami & Farshad44 discussed the solar collector system with turbulater effect for hybrid nanoparticles. Hybrid nanofluid is useful to increase the exergy efficiency of solar system and produces higher overall efficiency45.Magneto hydrodynamics describe flow behavior of moving conducting liquid which in turns polarized it. Impact of magnetic field is evaluated in industrial processes like fuel industry, electric generators, nuclear plants, aerodynamics, crystal production and so forth. Tamim et al.To the best of the author\u2019s knowledge there has been a scarcity in research regarding analysis of morphology effect of hybrid nanofluids coupled with matrix metal and ceramic matrix nanocomposites on thermo physical features of fluid flow between two orthogonally placed porous disks. Mathematical modeling in the form of partial differential equations is developed by considering shape and size of different (MMNC) and (CMNC). Later on, attained PDE\u2019s are converted into ODE\u2019s by using appropriate variables. Solution of intricate coupled differential system is attained by implementing Runge\u2013Kutta and shooting method jointly. Impact of involved dimensionless parameters on associated profiles is adorned.2-Cu/water nanoparticles between two orthogonally moving porous coaxial disk in the presence of the external magnetic field applied in z-direction. The diameter of boundary disks is 2r. The physical model takes in a cylindrical coordinate system Let us consider an incompressible, viscous, laminar, unsteady, 2D flow of hybrid nanofluid containing TiOmentclass2pt{minim46HNfd).Law of conservation of mass, momentum, and energy are as follows referred to Kashif et al.Thermophysical properties of is defined in Hybrid nanofluid effective heat capacitance is denoted byHNfd), and3O4), second (Al2O3), third (TiO2) and fourth (Cu) NP\u2019s (n1 and n2) based on thermal conductivity of (HNfd). The thermal conductivity of copper nanoparticles is high than titanium oxide.HNfd) The thermal conductivity of hybrid nano fluid of specific size and shape factor of NP\u2019s is described as underFor boundary situations the flow state is:Here, Following similarity variables are usedAssociated boundary conditionsR\u2009=\u2009Here, \u03b1\u2009=\u2009R, and consider the case following Majdalani et al.50 when \u03b1 is a constant, Finally, we set F\u2009=\u2009Boundary conditions at lower and upper wall of channelSkin friction and Nusselt number at both porous walls are computed coefficients which are of engineering interest are computed in this section.The The calculation at the lower and upper disk for heat transfer rate (Nusselt numbers) Since attained couple system of ODEs manipulated in Eqs. and 29)29) are iIn a shooting method, the missing initial condition at the initial point of the Interval is assumed, and the DE is then integrated numerically as an initial value problem. The accuracy of the assumed missing initial condition is then checked by comparing the calculated value of the dependent variable at the terminal point with its given value here. If a difference exist, another value of the missing initial condition must be assumed and the process is repeated. This process is continued until the agreement between the calculated and the given condition. For this purpose, given below Table An extensive representation of non-linear coupled system of ordinary differential equations along with coefficients possessing features of matrix composite materials and hybrid nano fluidPutting values of Eq. , Eq. 2323, Eq. , Eq. 2525, Eq. and Eq. in Eqs. We followed the RK methodology with the inclusion of shooting methods for the solution purpose of the existing flow model. The following replacement remain ingredient to start the process:First, transform the model system of ODEs Eq. and Eq. in the fBy interchanging, embedded in Eq.\u00a0, the folConsequently, the initial condition are:46. Table This section is presented to elaborate the impact of flow concerning equations like expansion/contraction ratio parameter entclass1pt{minimamentclasspt{minimaentclass1pt{minimaR was seen in Fig.\u00a0R.Figure\u00a0entclass1pt{minimamentclasspt{minimamentclasspt{minimaIn this study numerical analysis of MHD hybrid nanofluids flows through porous surfaces is adumbrated. Thermophysical features of metallic and ceramic-metallic nanocomposites are combined with hybrid nanofluid. Evaluation about shape, size factors and particle volume fraction on velocity, temperature profile is executed. Numerical and graphical consequences are gained regarding skin friction coefficient and Nusselt number. n\u2009=\u20095.7 shows good results in MMNC than shape factors at both porous walls.Nusselt number atMagnetic parameters have a significant effect on skin friction and Nusselt number for hybrid flow.In absence of injection/suction all governing parameters at lower porous surface show good performance as compared to upper surface.R indicate better results as compared to nanofluids.Hybrid nanofluids under the effect of M and For the contracting case Nusselt and skin friction coefficients for hybrid nanofluid show significant results.Increase in volume fraction thermal boundary layer thickness increases but permeable Reynold number reduces thermal boundary layer thickness.Some key findings are itemized as below"} +{"text": "Chemical signals are routinely propagated through biomacromolecules to modulate the structure of active sites and protein-protein interfaces. However, our understanding of the fundamental mechanisms coupling disparate regions of proteins is limited by the lack of accurate, atomic resolution information on their intrinsic dynamics. Indeed, the pathways most critical to chemical information flow are often intimately linked with the conformational ensembles populated by biomolecules, and spatially distant from traditional catalytic or ligand binding sites. A \u201choly grail\u201d of biophysical chemistry has been to understand, at the molecular level, how ligand binding information is transmitted through a protein matrix to induce a functional response. Though a majority of information about dynamically-driven biological function comes from lower molecular weight proteins, an exploding number of studies have taken advantage of advances in spectroscopy, electron microscopy, and molecular simulations to synergistically map dynamic pathways that underlie long-range communication in multidomain systems.de novo spatial and temporal regulation of protein function. The articles in this Research Topic tackle this knowledge gap by reporting on the structural and dynamic components that govern intra- and inter-domain crosstalk.The advantage of visualizing the solution ensembles of large biomolecules lies in the potential to leverage flexible hotspots for drug discovery or Redzic et al. demonstrated an intricate link between micro-millisecond protein dynamics and allostery in biliverdin reductase \u03b2 (BLVRB). Strikingly, evolutionary differences in amino acid sequence distal to the catalytic site induce a substantial variability in molecular motions that work in concert to regulate BLVRB function. A thermodynamic and kinetic investigation by Dubrow et al. offers insight into the role of protein motions in organizing biomolecular interfaces. Through site-directed mutagenesis, this work captures the per-residue impact on conformational selection during the binding transition state of influenza A nonstructural protein 1 and human p85\u03b2. The allosteric interactions of cholesterol with the chemokine receptor CCR3, critical to immune cell trafficking, is characterized by van Aalst and Wylie with circular dichroism and fluorescence polarization. The authors identified cholesterol as a critical mediator of substrate binding and receptor activation that drives signal transduction in GTPase assays. Cole and Igumenova used NMR spectroscopy to explore the interplay between Cd2+, Zn2+ and the conserved homology 1 (C1) zinc finger domain of protein kinase C, revealing the atomistic structural and thermodynamic properties of the C1 coordination sphere that facilitate competition between divalent metals.Purslow et al. highlighted the influence of protein dynamics on the activity of bacterial phosphotransferase Enzyme I by dissecting the contributions of active site flexibility to enzymatic turnover, most notably through the rotameric equilibrium of the catalytic His. Another elegant NMR relaxation study, performed by Zeng et al., revealed an RNA-driven disorder-to-order transition in the N-terminus of a bacterial RNase P, which serves as a dynamic checkpoint to ensure substrate alignment and enzyme activation. Baudin et al. reported an NMR structural study of the SERPINE1 mRNA binding protein (SERBP1), revealing SERBP1 to be intrinsically disordered but capable of sampling several compact conformations. The authors further define its RNA binding preferences and propensity for liquid-liquid phase separation, providing seminal molecular details of its mechanism.In another study, Le et al. carried out a novel structural study of the SpeG N-acetyltransferase, defining the structural basis for an allosteric mechanism that is unique within this enzyme family. This work implicates a dynamic loop, along with several \u03b2-strands, as mediators of enzyme activity and lays the foundation for expanded structure-function studies of SpeG. Long-range allostery in \u03b1-tryptophan synthase (\u03b1TS) is shown by D\u2019Amico et al. to involve networks of flexible residues that propagate \u223c25\u00a0\u00c5 chemical signals. Here, a novel role for surface-exposed residues in modulating dynamic crosstalk in \u03b1TS is revealed by NMR and molecular simulations. Skeens et al. and Cui and Lisi explored the intrinsic dynamics of cytokines as a driver for promiscuous and non-overlapping functions. Site-directed mutagenesis, novel structural engineering, and receptor binding demonstrated an intimate link between multi-timescale conformational dynamics and several biological activities.in silico predictions. For instance, Raniolo and Limongelli combined quantum-mechanics and free-energy calculations to improve standard ligand parametrization, allowing enhanced sampling (Funnel-Metadynamics) simulations of the paradigmatic benzamidine/trypsin molecular binding system that elegantly reproduced the high-resolution crystallographic ligand binding mode, providing a very accurate description of the binding mechanism. Hajrediniand and Ghose, instead, showed how enhanced sampling MD simulations could provide insight into the structural mediating role of a conserved \u201ccatalytic\u201d residue that inactivates two distantly related kinase families, i.e., bacterial tyrosine and shikimate kinases.The computational works contributing to this Research Topic covered various flavors and challenges of modern atomistic simulations, demonstrating the benefits of obtaining information with atomistic resolution that can be directly compared with experimental evidence and highlighting the potential of Estarellas et al. computationally assessed changes in the structural and dynamical properties of distinct isoforms of the adenosine monophosphate-activated protein kinase complexes. The comprehensive analysis of molecular dynamics simulations, also involving network theory tools, enabled characterization of key molecular factors that mediated activation of pan-activator PF-739, identifying distinctive features that correlate with the affinities of different isoforms. Massi and Morgan also combined molecular dynamics simulations with network analysis to show that substrate specificity in the enzymatic activity of the oligosaccharyltransferase of Campylobacter lari is regulated by modulation of dynamic allosteric pathways. Finally, Pacini et al. provided a perspective on the future challenges of network theory calculations aimed at elucidating the link between the information encoded in protein primary sequences, their dynamics and functions. Jernigan and Kumar proposed, instead, the use of elastic network models to infer the dynamics of a variety of proteins (with known bound and unbound structures) by observing the transfer of fluctuations among distant regions upon binding of an allosteric ligand.Frontiers in Molecular Biosciences highlights once more the power of combining computational and experimental approaches to characterize protein allostery with atomic resolution. We expect that further strengthening and exploiting such synergy will be instrumental toward a complete dissection of the structure/dynamics/function relationship in proteins and the development of predictive tools to identify allosteric networks and hot spots for drug design.Overall, the collection of manuscripts selected for this special issue of"} +{"text": "However, few studies integrate genetic, epigenetic, social, and environmental determinants of early life phenotypes to understand their links with diseases in later life. In this Mancilla et al. reviewed the literature to examine health inequality within the context of social epigenomics. Sasaki et al. investigated the effect of sample handling on DNA methylation profiles. Candelo et al. investigated a possible association between Zika virus infection and cyclin-dependent kinase 5 regulatory subunit-associated protein 2 (CDK5RAP2) mutation. Le et al. investigated the mechanisms linking assisted reproductive technology (ART) to cholesterol metabolic and respiratory disorders later in life. Xu et al. investigated the use of maternal serum human leukocyte antigen-G (sHLA-G) to detect prenatal chromosomal abnormalities. Ferreira and Dantas Junior reported a case study of a neonate with Beare-Stevenson Syndrome whose father had Congenital Bilateral Absence of the Vas Deferens (CBAVD). Luo et al. presented a whole exome sequencing study of Joubert Syndrome (JBTS), a type of ciliopathies.This topical collection presents original research, review articles, and case studies on a scope of exposures and health outcomes spanning the pre-natal period through adulthood. Future studies integrating a spectrum of genetic and epigenetic studies along with relevant exposures have a potential to inform mechanisms that underlie the associations between maternal phenotypes, birth outcomes, and offspring adult diseases."} +{"text": "Thyroid cancers (TCs) are the most prevalent malignancy of the endocrine system and the seventh most common cancer in women. According to estimates from the Global Cancer Observatory (GCO) in 2020, the incidence of thyroid cancer globally was 586,000 cases. As thyroid cancer incidences have dramatically increased, identifying the most important metabolic pathways and biochemical markers involved in thyroid tumorigenesis can be critical strategies for controlling the prevalence and ultimately treatment of this disease. Cancer cells undergo cellular metabolism and energy alteration in order to promote cell proliferation and invasion. Glutamine is one of the most abundant free amino acids in the human body that contributes to cancer metabolic remodeling as a carbon and nitrogen source to sustain cell growth and proliferation. In the present review, glutamine metabolism and its regulation in cancer cells are highlighted. Thereafter, emphasis is given to the perturbation of glutamine metabolism in thyroid cancer, focusing on metabolomics studies. KG \u03b1-ketoglutarateTCA tricarboxylic acidOAA oxaloacetateGSH GlutathioneGLUD glutamate dehydrogenaseGOT glutamate oxaloacetate transaminaseGPT glutamate pyruvate transaminaseATC anaplastic thyroid cancerFNAB fine-needle aspiration biopsyCITED1 Cbp/p300-interacting transactivator1TTF-1 thyroid transcription factor 1CEA carcinoembryonic antigenHBME-1 hector battifora mesothelial-1ATP adenosine triphosphateGln glutamineNEAA non-essential amino acidASCT2 amino acid transporter-2GLS glutaminasemTORC1 mammalian targets rapamycin complex 13 TriiodothyronineTGSSG oxidized glutathione Globally, thyroid cancers (TCs) are the most common head and neck malignancy and the seventh most prevalent cancer in women . HistolThe complexity of cancer in terms of diverse processes that drive the formation, growth, development, and progression of tumor cells continues to present a great challenge to the medical field. Over the past decade, many theories of the molecular mechanisms involved in cancer with a focus on individual factors and processes have emerged. Presently, several defined hallmarks for the cancer process are co-opted together to provide a conceptual framework for the complexity inherent in cancer . MetaboGlutaminolysis is another considerable metabolic reprogramming of cancer cells that mediates anaplerotic reactions to supply tricarboxylic acid (TCA) cycle intermediates . GlutamThis paper will review the experiments conducted on the role of Gln metabolism in TCs with additional consideration on possible mechanisms linking perturbation of Gln metabolism to pathological conditions.4+ and can occur in several tissues such as the brain, liver, lungs, adipose and skeletal muscles . Furtheal., 2007; Roth, 2al., 2007). This ral., 2007). 4+ into Gln and ATP products and glutaminase. GS in the cytosol is subject to transform glutamate plus NHal., 2017; Krebs, al., 2017). In facal., 2017; Neu et al., 2017). It is al., 2017). GLS inal., 2016; Curthoyal., 2016).via SLC7A5/ SLC3A2, and leucine enters the cell; thereby, adapting a rate-limiting step for mTORC1 ac-tivation . Among Figure 1 . Moreoval., 2017). Furtheal., 2017; Yang etal., 2017). It shoal., 2017). In thial., 2009). Besideal., 2009). Additial., 2009), c-Jun al., 2009), p53 , K-Ras al., 2009), Rb , and Rhal., 2009) have be3) hormone produced by the thyroid gland is essential for numerous functions in the body. T3 can modulate Gln release rate from several tissues, including the liver, kidney, intestine, and immune system cells . Althoual., 1990). Nevertin vivo/in vitro metabolites . Over t1H HRMAS NMR spectroscopy to find metabolic changes in benign (nodular goiter (NG)), malignant , and normal thyroid tissue lesions , no neoplastic nodules (NN), follicular adenomas (FA), and malignant thyroid cancer [65]. In that study, glutamate was one of the remarkable elevated metabolites in all samples . The real., 2013). Metaboal., 2013). D\u2010glutal., 2013, Seo et al., 2013, Tian etal., 2013). Table al., 2013; Gu et aal., 2013; Lu et aal., 2013; Ryoo etd cancer . In thatal., 2013; Skorupaal., 2013; Tian etal., 2013; Wang etal., 2013; Wojtowial., 2013; Xu et aal., 2013; Zhou etal., 2013) shows a1H-NMR spectroscopy combined with GC/flame ionization detector (FID)/MS techniques in comparison with healthy thyroid tissues (n = 10). Their work led to the observation that the total GSH was increased in cancer lesions compared to the healthy control group , MNG (n = 16), and healthy volunteers (n = 20), in a GC-MS-based metabolomics approach. It was observed that glutamic acid level was substantially increased in the PTC group compared to healthy subjects. Moreover, pathway analysis showed perturbations in D-Gln and D-glutamate and GSH metabolism with common impacts in both PTC and MNG tumorigenesis . While,al., 2020). Our teBRAF V600E mutation, and the expression of ASCT2 expression was raised in MTC , FTC: 112), MTC: (70), poorly differentiated carcinoma PDC: (23), and ATC: (8), and follicular adenomas, FA (152), a distinct expression pattern in Gln metabolism-related protein among the histopathological subtypes of thyroid cancer was identified . In thaal., 2016). Microa2, MTC: and tissue samples using several molecular and biochemical approaches revealing that the GLS, a key enzyme in glutaminolysis, was overexpressed in cancer specimens and played a prominent role in the development and progression of PTC . In 201At a glance, increasing evidence suggests that Gln, as a NEAA, plays an important role directly or indirectly in tumorigenesis and cancer cell survival. However, despite the emerging understanding of the biological relevance of Gln in cancer, the research underlying perturbation of Gln metabolism on TCs is restricted. From the studies mentioned above, it is evident that most of the metabolomics studies that reported changes of Gln/glutamate and related metabolic pathways were those that performed NMR approaches with untargeted strategies. Hence, it will be essential to perform quantitative and targeted assays in cohort projects to unravel the biological mechanism underlying Gln metabolism and then find the effect of other factors on it in TCs. Thyroid gland plays pivotal roles in the growth and development of the human body and keeps metabolism under control, and by releasing certain hormones, regulates Gln metabolism. Therefore, significant efforts must be made to untangle the role of Gln metabolism in thyroid tumorigenesis, which can result in a comprehensive understanding of disease progression and ultimately lead to the discovery of novel therapeutic strategies. Crispin R. Dass and Mehdi Hedayati 22432500, Fax: +98(21) 22416264, E-mail: hedayati47@gmail.com, hedayati@endocrine.ac.ir) contributed equally as corresponding author.The authors declare that they have no conflict of interest.All authors contributed to the study conception and design. The first draft of the manuscript was written by Raziyeh Abooshahab. Kourosh Hooshmand and Fatemeh Razavi edited the manuscript. Mehdi Hedayati and Crispin R. Dass approved the final version. All authors participated in the critical revision of the manuscript for important intellectual content."} +{"text": "Culinary skills are important objects of study in the field of Public Health. Studies that propose to develop instruments for assessing such construct show lack of methodological uniformity to report validity and reliability of their instruments.To identify studies that have developed instruments to measure culinary skills in adult population, and critically assess their psychometric properties.We conducted a systematic review according to the PRISMA statement. We searched literature PubMed/Medline, Scopus, LILACS, and Web of Science databases until January 2021, and consulted Google Scholar for relevant grey literature. Two reviewers independently selected the studies, conducted data extraction, and assessed the psychometric quality of the instruments. A third reviewer resolved any doubts or disagreements in all steps of the systematic review.The search identified 1148 potentially relevant studies, out of which 9 met the inclusion criteria. In addition, we included 3 studies by searching the related articles and the reference lists of these studies, totaling 12 included studies in this review. Ten studies reported the development of tools measuring culinary skills in adults and 2 studies performed cross-cultural adaptations of original instruments. We considered adequate quality of internal consistency reliability in four studies. One study received adequate rating for test-retest reliability. No studies presented adequate rating for content validity and four studies showed satisfactory results for at least one type of construct validity. One study reported criterion validity and the quality of this psychometric property was inadequate.We identified many studies that surveyed culinary skills. Although the isolated measures appraised in this review show good promise in terms of quality of psychometric properties, no studies presented adequate measures for each aspect of reliability and validity. A more consistent and consensual definition of culinary skills is recommended. The flaws observed in these studies show that there is a need for ongoing research in the area of the psychometric properties of instruments assessing culinary skills. The discussion about the improvement of culinary skills and food practices has proven to be an important object of study in the field of Public Health; these skills are key factors associated with eating behaviors and with several complexities that represent social determinants of health .Several authors define the term culinary skills in their publications \u20136, howevCulinary skills are associated with other concepts that involve the practice of proper and healthy eating, such as food literacy, which takes into account the broader social and environmental dimensions of eating together, associated with an individual\u2019s abilities . Those cTime devoted to cooking has decreased and has been viewed as a global trend: food industry investments in advertising and marketing to \u201csolve the everyday food problem\u201d devalue cooking as an emancipatory competence associated with a healthy food routine . Such deet al. (2001) [The main source of cooking knowledge and skills is through parents \u201317. This. (2001) found frIn this scenario, culinary skills among adults, especially those responsible for preparing household meals, have been an important focus of research , 17, 19.Before being considered suitable, the instruments must offer accurate, valid, and interpretable data for the population\u2019s assessment. Moreover, the measures are supposed to provide scientifically robust results. These results are established based on measures of reliability and validity of the instruments \u201322. ReliThere are public health policies focused on cooking in several parts of the world . Despiteet al. (2014) [McGowan . (2014) conducteAdditionally, previous reviews have not proposed to appraise the quality of psychometric properties of instruments measuring culinary skills, which justifies the importance of this study, given the fact that the diagnosis of one\u2019s skills entrusted to the application of these instruments may be flawed. This could result in planning inappropriate food and nutrition educational actions for providing emancipatory and self-care practices.Therefore, this systematic review aimed to identify studies that have developed instruments to measure culinary skills in adult population, and critically appraise the quality of their psychometric properties.We hope that this study can provide evidence-based guidance on the psychometric properties of instruments measuring culinary skills, to subsidize the selection of valid and reliable instruments by healthcare professionals to assess these subjects in clinical and public health settings and avoid unrealistic expectations about the information that such measures may provide.http://www.crd.york.ac.uk/PROSPERO/; registration number CRD42019130836). The protocol is available in the We registered the protocol of this systematic review on the International Prospective Register of Systematic Reviews . A third reviewer (T.M.L.) resolved any doubts or disagreements between the reviewers regarding the inclusion or exclusion of articles. The third reviewer compared the results of the independent selection of articles carried out by the two reviewers. If the third reviewer identified any differences, he would ask the two authors to discuss their opinions. If the two reviewers did not reach an agreement, the third reviewer would present his opinion.This review, included articles meeting the following criteria: 1) address culinary skills in adults; 2) describe the instrument\u2019s validation and reliability process, which can be original or adapted instruments. No filters for year of publication, country or language were employed. Articles that developed original instruments or reporting cross cultural adaptation of instruments addressed to measure culinary skills in children and adolescents or those whose instruments were not available (in the article or upon request to the authors) were excluded. For initial screening of abstracts and titles, we used the Reviews . Two autTwo authors (A.R.T. and D.B.) independently performed data extraction using a preformatted spreadsheet in Microsoft Excel. A third reviewer (T.M.L.) resolved any disagreements or doubts resolved any disagreements or doubts occurred in this step, by comparing the data extraction carried out by the two reviewers. If the third reviewer identified any differences, he would ask the two authors to discuss their interpretations. If the two reviewers did not reach an agreement, the third reviewer would present his opinion. We also consulted the third reviewer in case of any doubts regarding the inclusion of potentially relevant articles identified during this step of the systematic review.The information extracted consisted of descriptive data of the study .et al. [et al. (2007) [We determined the psychometric quality according to the rating system adapted from Hair Jr, Black, Babin et al. ; Pedrosaet al. ; and Ter. (2007) . The criet al., 2011 [et al, 2017 [et al, 2011 [et al, 2018 [et al, 2017 [et al, 2019 [et al, 2019 [et al, 2013 [et al, 2013 [The electronic search (including gray literature databases) identified 1148 potentially relevant studies. After reviewing the titles and abstracts, we selected 16 articles for full-text examination. Of these, nine studies met theal, 2013 ). We ideal, 2013 ; Condraset al, 2011 [et al, 2013 [et al, 2017 [et al, 2019 [et al, 2019 [et al, 2013 [et al, 2018 [et al, 2011 [et al, 2017 [et al, 2011 [Studies were carried out in the United States of America , Brazilal, 2017 ; Martinsal, 2019 ), Canadaal, 2019 ; Kennedyal, 2019 ), Switzeal, 2013 ), Portugal, 2018 ), Scotlaal, 2011 ) and Noral, 2017 ). All ofal, 2011 ).et al, 2013 [et al, 2017 [et al l, 2019 [et al, 2019 [et al, 2017 [et al, 2018 [et al, 2011 [et al, 2013 [et al, 2011 [et al, 2011 [et al, 2011 [et al, 2019 [et al, 2019 [et al, 2018 [Included papers had distinct purposes: those reporting the development of an original instrument, or cross-cultural adaptation of a tool to explicitly measure cooking/food skills or a part thereof (n = 7) and original tools developed to evaluate a cooking and food skills intervention (n = 5). Most tools assessed cooking skills in adults from a particular country , parental, 2019 ), univeral, 2019 ; Jomori al, 2017 ; Kowalkoal, 2018 ) and adual, 2018 ; Condrasal, 2011 ; Condrasal, 2013 ; Barton al, 2011 ). Study al, 2011 , Condrasal, 2011 ; Martinsal, 2019 ; Kennedyal, 2019 ; Kowalkoal, 2018 ; Michaudal, 2018 ; Warmin,al, 2018 ; Vrhovnial, 2018 ). The paAll studies provided description of the construct, with conceptual framework or clear rationale to define their instruments\u2019 construct.et al, 2011 [et al, 2013 [et al, 2011 [et al., 2017 [Six studies reported the development or crosl., 2017 ) aiming et al. (2011) [et al. (2013) [et al. (2017) [Michaud (2007) , develop. (2011) reported. (2013) then ada. (2017) describeet al. (2011) [et al., 2011 [et al., 2013 [et al., 2017 [Barton . (2011) also des. (2011) ; Warmin,. (2011) ; Condrasl., 2011 ; Condrasl., 2013 ; Jomori l., 2017 ).et al., 2013 [et al., 2018 [et al., 2017 [et al., 2019 [et al., 2019 [The remaining studies describet al., 2013 [et al., 2018 [The Cooking Skills Scale focusedet al. (2017) [rate how good they are at: Microwave food, including heating ready-meals).Lavelle . (2017) developeet al.\u2019s cooking scale (2013) [et al.\u2019s cooking confidence measure (2017) [et al., (2019) [Unlike the items shown in Hartmann e (2013) and Lavee (2017) , the Coo, (2019) focused et al.\u2019s food skills confidence measure (2017) [et al.\u2019s food skills questionnaire (2019) [et al.\u2019s [rate your confidence in boiling, steaming or stewing) and using basic ingredients and seasoning . Vrhovnik\u2019s Food skills survey tool (2012) [Lavelle e (2017) consistee (2019) focused et al.\u2019s instrumel (2012) also conet al., 2019 [et al., 2013 [et al., 2013 [et al., 2011 [et al., 2019 [et al., 2019 [et al., 2018 [et al., 2017 [et al., 2011 [et al., 2017 [et al., 2018 [et al., 2011 [et al., 2017 [et al., 2019 [et al., 2019 [et al., 2011 [et al., 2011 [et al., 2018 [et al., 2017 [et al., 2019 [et al., 2019 [et al., 2013 [The studies reported analysis of the psychometric properties of their instruments: Only two studies presented statistical methods derived from the experts\u2019 judgment for content validity . Six oul., 2019 ; Condrasl., 2013 , Hartmanl., 2013 ; Barton l., 2011 ; Kennedyl., 2019 ; Martinsl., 2019 ). Two stl., 2018 , Jomori l., 2017 ). Only ol., 2017 ) reportel., 2017 ; Condrasl., 2011 ; Jomori l., 2017 ; Kowalkol., 2018 ; Barton l., 2011 ; Vrhovnil., 2011 ; Lavellel., 2017 , Kennedyl., 2019 ; Martinsl., 2019 ) and/or l., 2019 ; Warmin,l., 2019 ; Condrasl., 2011 ; Barton l., 2011 ; Kowalkol., 2018 ; Lavellel., 2017 , Kennedyl., 2019 ; Martinsl., 2019 ; Hartmanl., 2013 ). Table We describe the quality of the psychometric properties of the instruments in et al., 2011 [et al., 2018 [et al., 2017 [et al., 2019 [et al., 2017 [et al., 2019 [et al., 2011 [et al., 2013 [et al., 2013 [We considered adequate quality of internal consistency reliability in four studies Three sl., 2019 ; Jomori l., 2017 ; Kennedyl., 2019 ) showed l., 2011 did not l., 2011 did not l., 2011 ; Condrasl., 2013 ; and Harl., 2013 ) did notet al., 2011 [et al., 2013 [et al., 2018 [et al., 2011 [et al., 2017 [et al., 2019 [et al., 2019 [et al., 2018 [et al., 2019 [Kappa, despite adequate design and method. Five studies showed inadequate time interval . Howevel., 2018 ; Martinsl., 2019 ), since l., 2011 ; Hartmanl., 2013 ) or inadl., 2013 ; Warmin,l., 2013 ; Condrasl., 2011 ; Lavellel., 2017 ); therefet al., 2019 [et al., 2017 [et al., 2011 [et al., 2011 [et al., 2013 [et al., 2019 [et al., 2013 [No studies reporting the development or small changes of an original instrument provided adequate measures to show content validity. The authors did not calculate any index of agreement for content validity , or stal., 2013 ; Kennedyl., 2019 ). Moreovl., 2013 ).Regarding construct validity, six studies reported at least one kind of analysis .et al., 2011 [et al., 2018 [et al., 2017 [et al., 2017 [et al., 2017 [et al., 2017 [Self-Efficacy for Using Basic Cooking Techniques (SECT)). Two studies performed cross-cultural adaptations of original instruments . We clal., 2017 ) and retl., 2017 ). In addl., 2017 ), hence,l., 2017 ). Jomoril., 2017 performel., 2017 and Kowal., 2018 ). One ofl., 2018 ). Six stl., 2018 ; Condrasl., 2013 ; Hartmanl., 2013 ; Barton l., 2011 ; Kennedyl., 2019 ; Martinsl., 2019 ).Most studies did not provide information on criterion validity. Only one study performTo our knowledge, this is the first systematic review to identify and appraise quality of psychometric properties of instruments for assessing culinary skills in adults. This article has provided a comprehensive critical analysis of the studies\u2019 characteristics and their psychometric properties. We found twelve studies developing original instruments to measure culinary skills in adults, or performing cross-cultural adaptations.This systematic review has highlighted gaps in these instruments, suggesting the need to develop new studies with robust and standardized psychometric methodology that shows validity and reliability of culinary skills measurements. Although we considered adequate quality of internal consistency reliability in four studies, only one study received adequate rating for stability (test-retest reliability). No studies developing original instruments presented satisfactory measurement for content validity since the authors did not calculate any index of agreement. Only four studies showed satisfactory results for at least one type of construct validity and only one study reported criterion validity, however, we considered inadequate quality of this measurement property. These results indicate that although there are isolated measures appraised in this review that show good promise in terms of quality of psychometric properties, no studies presented satisfactory results for each aspects of reliability and validity.et al., 2019 [Most studies are originally from countries whose native language is English. One Brazilian study original., 2019 .et al., 2011 [Regarding submission of psychometric studies for ethical approval, one study justifil., 2011 , 50.et al., 2019 [Most studies reported the development of scales, indexes, and questionnaires. One study classified their instrument as an index ; howevel., 2019 .A scale, on the other hand, measures levels of intensity at the variable level, like to what extent a person agrees or disagrees with a particular statement. A scale is a type of measure composed of several items that have a logical or empirical structure among them. The most commonly used scale is the Likert scale. The sum of scores for each of the statements creates an overall score of the intensity related to the assessed latent phenomenon .et al., 2013 [et al., 2018 [et al., 2019 [The majority of the included studies presented instruments with items assessing cooking self-efficacy (regarding food preparation techniques), meal planning and food selection and purchase. The main difference between the instruments referred to the conceptualization of culinary skills: some authors comprehend that such skills comprise the ability to prepare certain dishes, including those based on pre-prepared products and convenience foods . Howevel., 2019 . Thus, ul., 2019 . Hence, et al. (2017) [et al. (2019) [Some authors identify cooking skills as a distinct construct from food skills. Lavelle . 2017) define c. (2017) however,. (2019) seem to . (2019) conceptu define . (2019) .Although all instruments reported some psychometric information, the evaluation of the psychometric quality using the criteria adopted in this systematic review exhibited some missing data.et al., 2011 [et al., 2017 [et al., 2018 [et al., 2011 [et al., 2017 [et al., 2019 [et al., 2019 [Regarding the reliability of the instruments, most studies reported internal consistency reliability . Internl., 2019 .et al., 2019 [et al., 2017 [Cooking Behavior scale . Similar results were observed in Jomori et al.\u2019s (2017) [Cooking Behavior (CB) and Cooking Attitude (CA) scales showed low internal consistency reliability. The later authors argued that problems in the process of cross-cultural adaptations concerning translation of the original instrument into Brazilian Portuguese might have occurred. The items corresponding to these scales might not represent the constructs the authors intended to measure [Three studies showed insufficient results for Cronbach\u2019s alpha . Two ofl., 2017 showed is (2017) study: T measure . Thus, i measure .et al. (2011) [Barton . (2011) did not . (2011) tested oet al., 2011 [et al., 2011 [et al., 2013 [et al., 2017 [et al. (2013) [Kappa values (<0.7) in two studies that reported test-retest reliability. Therefore, we rated inadequate quality of this attribute.We considered indeterminate quality of stability reported in six studies, due to insufficient sample size or inadequate time interval to perform test- retest reliability . Hartma. (2013) performe. (2013) . Particiet al., 2013 [et al., 2013 [et al., 2011 [et al., 2019 [et al., 2019 [Studies that relied exclusively on internal consistency reliability and stability analysis, without performing other psychometric measurements to validate their instruments, may not provide trustworthy results because these instruments reproduce only a consistent result in time and space from different observer (reliability), without measuring exactly what they propose , 53. Sixl., 2013 ; Hartmanl., 2013 ; Barton l., 2011 ; Kennedyl., 2019 ; Martinsl., 2019 ). The auAll studies aiming to develop and validate an original instrument failed to show proper content validity: most studies relied on face validity, literature research, and experts\u2019 judgment; however, the authors did not calculate any index to confirm experts\u2019 agreement. Content validity based on the use of statistical methods derived from the experts\u2019 judgment, proves itself to be essential. Otherwise, the mere fact that the experts report on the lack or excess of items representative of the construct, or that they simply determine to what extent each element corresponds to the latent phenomena, does not itself provide relevant information for the validation process , 39, 54.et al., 2011 [et al., 2017 [et al., 2018 [et al., 2017 [et al., 2017 [et al., 2017 [et al., 2018 [We evaluated the quality of construct validity measures of studies reporting structural validity , hypothl., 2017 ; Jomori l., 2017 ) or crosl., 2017 ; Kowalkol., 2018 ). We obsa priori ideas about the latent variables researchers intend to measure [Regarding structural validity, two studies performed principal component analysis and three studies performed exploratory factor analysis. No studies performed Confirmatory Factor Analysis (CFA). According to Gruijters, 2019 , explora measure , 31.et al., 2017 [et al., 2018 [Only two out of five studies reporting structural validity describl., 2018 .EFA is a \u201clarge-sample\u201d procedure and that generalizable or replicable results are unlikely if the sample is too small.Michaud (2007) performeet al. (2017) [Self-Efficacy for Using Basic Cooking Techniques (SECT)). This type of validity evaluates the presence of differences in the measurements obtained between the groups, not whether the measure actually measures the intended construct [The cross-cultural adaptation of Michaud\u2019s (2007) instrume. (2017) was adeqonstruct , hence, Vrhovnik (2012) did not provide statistical results for items factor loadings, which may imply inadequate decisions regarding retention or exclusion of an item .et al.\u2019s study (2017) [Buying in Season; Using leftovers to create another meal; Keeping Basics in the cupboard and Reading the best before date), however they were retained in the \u2018Food Skills\u2019 factor. When a variable is found to have more than one significant loading, it is hard to make those factors be distinct and represent separate concepts [a priori factor structure could be flawed [Microwave food (not drinks/liquid) including heating ready-meals\u2019. The Brazilian Food Guide (2014) states that although microwaving may be used in meal preparation it is not seen as a cooking skill.Despite satisfactory results for convergent validity in Lavelle y (2017) , the expconcepts . If an ie flawed . Moreoveet al., 2018 [et al.\u2019s cooking skills instrument (2013) [Kowalkowska l., 2018 performet (2013) . Howevert (2013) recommenRegarding criterion validity, little information was available in the included studies. Only one study presented criterion validity . However, we considered inadequate quality of this attribute. These findings were expected since most of the time, the criterion validity is a challenge for the researcher, because it demands a \u201cgold standard\u201d measure to be compared with the chosen instrument, which cannot be easily found in all knowledge areas , 59.This review has some limitations. It is possible that some studies were missed out because they were not indexed in the databases searched, or were published for institutions, foundations, or societies. In addition, although the criteria were adapted from previous studies, the difficulty of interpreting the studies may have under- or overestimated the quality of the instruments\u2019 psychometric properties.This review identified many studies surveying culinary skills; we considered most instruments insufficient, according to the quality of their psychometric properties. Thus, the flaws observed in these studies show that there is a need for ongoing research in the area of the psychometric properties of instruments assessing culinary skills. Moreover, our findings contribute to supporting the selection of valid and reliable instruments by healthcare professionals in clinical and Public Health settings.Measuring culinary skills involves several separate but related domains, which integrate other constructs related to the culinary practices. Therefore, it is recommended that a more consistent and consensual definition of culinary skills as a construct be generated. Instruments should cover items and domains without overestimating one\u2019s skills, based on his/hers ability of heating convenience food. Considering items measuring culinary skills related to the use of using basic ingredients and seasoning proves itself essential for greater understanding of barriers and facilitators related to healthy culinary practices.S1 Appendixhttps://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42019130836.From: Aline Rissatto Teixeira, Daniela Bicalho, Tacio de Mendon\u00e7a Lima. Evidence for the validation quality of culinary skills instruments: a systematic review. PROSPERO 2019 CRD42019130836. Available from: (PDF)Click here for additional data file.S2 Appendix(PDF)Click here for additional data file.S1 Table10.1371/journal.pmed1000097.From: Moher D, Liberati A, Tetzlaff J, Altman DG. The PRISMA Group (2009). Preferred Reporting Items for Systematic Reviews and Meta-Analyses: The PRISMA Statement. PLoS Med 6(7): e1000097. doi:(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file."} +{"text": "The importance of prenatal maternal somatic diseases for offspring mood and anxiety disorders may be overlooked or undervalued. We conducted the first systematic review and meta-analysis assessing the risk of offspring mood and anxiety disorders in the context of prenatal maternal somatic diseases.We screened articles indexed in Embase , PsycARTICLES and PsycINFO databases up to August 2021. 21 studies were included. We examined the overall associations between prenatal maternal somatic diseases and offspring mood/anxiety disorders. Analyses were stratified according to maternal somatic diseases and follow-up duration.We observed an increased risk of mood and anxiety disorders in the context of prenatal maternal somatic diseases ; maternal obesity, hypertensive disorders and infertility were risk factors for mood disorders; maternal polycystic ovary syndrome , severe obesity and moderate obesity were risk factors for anxiety disorders. Prenatal maternal somatic diseases increased the risk of mood disorders in childhood and adulthood , as well as the risk of anxiety disorders in adulthood .The results indicate that prenatal maternal somatic diseases are associated with offspring mood and anxiety disorders, and that the associations may be long-lasting. Mood disorders (bipolar and depressive disorders) and anxiety disorders are complex mental disorders resulting from multiple factors such as genetic predisposition, parenting style, family environment, socioeconomic status. Fetal origins of mental disorders have been attracting increasing attention. Awareness of prenatal risk factors is crucial for prevention strategies.et al., et al., et al., et al., et al., et al., Prenatal maternal health plays an important role in the subsequent mental health of offspring , PsycARTICLES (via EBSCO) and PsycINFO (via EBSCO). The search strategy consisted of relevant Medical Subject Heading (MeSH) terms, Emtree term-exploded, keywords and word variants. The detailed search strategies for each database were fully described in online Supplementary Table S1. In addition, we searched the reference lists of all included studies, related reviews for further potential studies.in utero , (2) control group(s): had a comparison group(s) without the exposure(s), (3) outcomes: included mood or anxiety disorders diagnosed according to any recognised diagnostic criteria or self-report , (4) statistical indicators were provided to examine the effect of prenatal maternal somatic diseases on mood or anxiety disorders in offspring, (5) study design: cohort studies, including population-based cohort studies and registry-based studies.Articles were eligible if they met the following criteria: (1) exposures: offspring were exposed to maternal somatic diseases Articles were excluded if they met the following criteria: (1) exposed group was mixed with maternal somatic diseases diagnosed postpartum; (2) reviews, meta-analyses, abstracts or conference proceedings.When there were multiple groups of useful data in one article, only the data derived from the group with the largest sample size or the most severe exposure were selected for the meta-analysis. In addition, we did not delete articles that presented overlapping samples as they included different maternal somatic diseases or outcomes, but only the data from the largest sample size was used in the meta-analysis.After removing duplications, titles and abstracts were reviewed independently by two researchers for initial screening, and then full text. Any disagreement was resolved through group discussions. Endnote was used as the bibliographic software.Two researchers independently extracted data. Any disagreement was resolved through group discussions. The following data were extracted: author, year of publication, the country where the study was conducted, sample and source, exposure and measure, outcome and measure, adjusted confounders, and measure of association. All included articles were assessed in terms of methodological quality according to the Newcastle\u2013Ottawa Quality Assessment Scale (NOS) for cohort studies. NOS consists of eight items across three domains: selection , comparability and outcome . Studies were graded as good quality (7\u20139 stars), fair (4\u20136 stars) and poor (0\u20134 stars).Q test and I2 statistics were used to evaluate the heterogeneity. A fixed-effects model was adopted when I2\u00a0<\u00a050%, otherwise, a random-effects model was used. Funnel plot, trim and fill method and Egger test were used to detect potential publication bias. To assess the stability of the meta-analysis, sensitivity analysis was performed. Sensitivity analysis was performed by excluding studies one by one to explore the impact of each study on the overall results. Furthermore, analyses were stratified according to maternal somatic diseases and follow-up duration (or offspring's age at diagnosis). Quantitative meta-analysis was conducted for an outcome when more than one study presented relevant data. The reason why we adopted stratified analysis rather than traditional subgroup analysis was that the former can make better use of the data in our study. Analyses were performed with Stata 16.0.The method was based on the relative risk (RR) with 95% confidence intervals (CIs) obtained in each study. If the RR was not reported, the hazard ratio (HR)/odds ratio (OR) is considered to be approximately equal to RR. RRs fully adjusted were preferentially pooled in our analyses. Cochrane et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., In total, 21 articles were eligible for inclusion .Fig. 2.Analyses were stratified according to maternal somatic diseases. Pooled effect of two studies showed that maternal obesity was significantly associated with offspring mood disorders . In the et al., et al., et al., et al., et al., et al., et al., In the stratified analyses, we did not perform meta-analyses for maternal PCOS, cancer, asthma, diabetes, thyroid diseases and rheumatoid arthritis due to the limited number of eligible studies. These studies reported that prenatal exposure to maternal PCOS , including childhood (aged\u00a0<\u00a018 years) and adulthood (aged\u00a0\u2a7e\u00a018 years). In the meta-analysis of five studies, prenatal maternal somatic diseases were associated with the increased risk for mood disorders in childhood . The metet al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., I2\u00a0=\u00a033%, p\u00a0>\u00a00.05) .Analyses were stratified according to maternal somatic diseases. Pooled effect of two studies showed that the risk of offspring anxiety disorders significantly increased in the context of maternal PCOS . In the et al., et al., In the stratified analyses, we did not perform a meta-analysis for maternal infertility and hyperemesis gravidarum due to the limited number of eligible studies. One study reported a significant association between maternal infertility and anxiety disorders of offspring . In the meta-analysis of five studies, prenatal maternal somatic diseases were not associated with offspring anxiety disorders in childhood . The metp\u00a0>\u00a00.05), the asymmetric funnel plot suggested the possibility of publication bias . The \u2018trim and fill\u2019 method was then used to recalculate the pooled results. The adjusted pooled effect of mood disorders exhibited a similar trend with two potential studies filled . The result for anxiety disorders was not altered with two potential studies filled .Fig. 4.Sensitivity analysis did not significantly alter these findings, indicating that our results were relatively stable .To our knowledge, this is the first systematic review and meta-analysis assessing the risk of offspring mood and anxiety disorders in the context of prenatal maternal somatic diseases. The overall meta-analyses confirmed that prenatal maternal somatic diseases were associated with an increased risk of mood and anxiety disorders in offspring. For mood disorders, we identified that maternal obesity, hypertensive disorders, and infertility were risk factors. For anxiety disorders, risk factors were maternal PCOS and obesity. Furthermore, our study emphasised the impact of prenatal maternal somatic diseases on mood and anxiety disorders may be long-lasting.et al., et al., The overarching finding of the meta-analysis showed that prenatal maternal somatic diseases were related to offspring mood/anxiety disorders. Mood and anxiety disorders are complex mental disorders, the RRs generally <2 indicated that they each modulated risk by a relatively small. Prenatal exposure to both maternal somatic diseases and psychiatric factors implicates an adverse intrauterine environment, but they might point toward differing underlying mechanisms of an increased risk of mood/anxiety disorders. Although most of the included studies (16 of 21) adjusted maternal psychiatric disorders or distress, the effect of maternal psychosocial factors cannot be excluded, as a relationship has been reported early between somatic diseases and mental health were associated with an increased risk of mood disorders in offspring, but the association with anxiety disorders was not statistically significant. Although a previous meta-analysis found that preeclampsia was associated with an elevated risk of offspring schizophrenia, the association with other psychiatric disorders was inconclusive was significantly stronger than those postnatally diagnosed in one of our included studies (Momen In the context of maternal somatic diseases, the risk of mood disorders was increased in both childhood and adulthood, as was the risk of anxiety disorders in adulthood. The findings suggested that the effects of prenatal maternal somatic diseases were long-lasting and even persist into old age. However, the results should be interpreted with caution because the two ages were not evaluated in the same study.There are some limitations as well. First, although a total of 21 studies were included, the majority of the studies were from northern European populations, and there was a lack of data from Asian populations. Second, when it came to specific exposures, the number of studies was small, and for some exposures, there were not enough studies to allow quantitative analysis. Third, a pooled risk consistently adjusted for the same variables could not be reached because different variables are adjusted in each study. Forth, different diagnostic criteria and follow-up periods were adopted for the same outcome, which may be the reason for the significant heterogeneity of some results. Fifth, in the context of maternal somatic disease, the effects of the disease itself and treatment measures are included, yet we cannot distinguish their effects in the associations. Sixth, it may be difficult to diagnose children, though mood and anxiety disorders occur at any time throughout the lifespan. And both mood and anxiety disorders may not be diagnosed for a long time after the onset. Therefore, the real risk may be underestimated.In conclusion, the results of our study indicate that prenatal maternal somatic diseases can be associated with offspring mood and anxiety disorders, and that the associations may be long-lasting. These findings advance the understanding of the prenatal origins of risk for mood and anxiety disorders. More high-quality prospective studies are needed to resolve the limitations mentioned above."} +{"text": "The ancient membrane protein TSPO is phylogenetically widespread from archaea and bacteria to insects, vertebrates, plants, and fungi. TSPO\u2019s primary amino acid sequence is only modestly conserved between diverse species, although its five transmembrane helical structure appears mainly conserved. Its cellular location and orientation in membranes have been reported to vary between species and tissues, with implications for potential diverse binding partners and function. Most TSPO functions relate to stress-induced changes in metabolism, but in many cases it is unclear how TSPO itself functions\u2014whether as a receptor, a sensor, a transporter, or a translocator. Much evidence suggests that TSPO acts indirectly by association with various protein binding partners or with endogenous or exogenous ligands. In this review, we focus on proteins that have most commonly been invoked as TSPO binding partners. We suggest that TSPO was originally a bacterial receptor/stress sensor associated with porphyrin binding as its most ancestral function and that it later developed additional stress-related roles in eukaryotes as its ability to bind new partners evolved. E. coli and Saccharomyces cerevisiae was found to be a monomer was conclusively shown to be located in the ER/Golgi by fluorescent tagging of N-terminal 6-histidine-tagged AtTSPO in organisms of all kingdoms, questions that may only be answered by identifying specifically associated proteins.On the surface this ancient protein appears to have very different functions in different organisms, yet most of these functions relate to stress-induced changes in metabolism.2+ signaling may alter TSPO\u2019s binding to its partners, thereby affecting its role in various metabolic processes.It has been suggested that TSPO was originally a bacterial receptor/stress sensor that developed additional roles in eukaryotes (Gatliff and Campanella Perhaps the most widely researched interaction of TSPO is that with the voltage-dependent anion channel (VDAC), a channel in the mammalian OMM that controls a wide range of processes involving transport of molecules into and out of mitochondria . This finding led the authors to suggest that TSPO could be directly or indirectly involved in regulation of the UPR (Rolland et al. Another potential TSPO interacting partner was proposed by Jurkiewicz et al. : the NACPseudomonas strains suggested several proteins for further study (Leneveu-Jenvrin et al. Pseudomonas homolog of VDAC, was predicted to functionally interact with TSPO (Leneveu-Jenvrin et al. RsTSPO and Sinorhizobium meliloti TSPO (Leneveu-Jenvrin et al. RsTSPO; the Kat B catalase, also predicted to interact with S. meliloti TSPO; hemolysin II/III, virulence factors that remove iron from heme; the adenine phosphoribosyltransferase Apt; a putative nucleoside-diphosphate-sugar epimerase Pfl01_0720 and the UDP-N-acetylmuramate-alanine ligase MurC, both involved in cell wall synthesis; orthologs of the thiol oxidoreductase PSPTO4367, involved in oxidative stress protection; and finally a putative hybrid histidine kinase Pfl01_2810, the interaction with which was suggested to be specific to P. fluorescens Pf0-1 (Leneveu-Jenvrin et al. RsTSPO (Li et al. A STRING database search for functionally-interacting partners for TSPO from RsTSPO (Yeliseev et al. AtTSPO (Jurkiewicz et al. Amid its many proposed binding partners and functions, can an ancestral function and partner for TSPO be discerned? It seems reasonable to conclude that some part of TSPO function, along with aspects of its structure, must be conserved throughout evolution, as mammalian TSPO has been shown to functionally substitute for the bacterial F. diplosiphon (Busch et al. Drosophila had reduced mitochondrial respiration and increased mitochondrial oxidative stress (Lin et al. The most widely observed function in all organisms is the binding of porphyrins, with affinities ranging from low micromolar in bacteria (e.g., 5\u00a0\u00b5M in the cyanobacterium In higher plants, the role of porphyrins and oxidative stress are strongly connected (Frank et al. R. sphaeroides (Yeliseev and Kaplan The handing of porphyrins to porin for export in bacteria, similar to what has been suggested for TSPO is an ancient protein whose varied and complex functions appear to be dictated by its location and its orientation in the membrane, as well as its binding partners. In many aspects of its behavior TSPO resembles a receptor or sensor; indeed, it shows some structural similarity to GPCRs (Li et al."} +{"text": "Mining genetic variation including structural variation from mammalian genomes is a crucial step towards investigating the relationship between genotype and phenotype. However, compared to the detection of single nucleotide variants and small indels, characterizing large and particularly complex structural variation is much more difficult and less intuitive . In the et\u00a0al. presented high-quality draft genomes of the grey wolf (Canis lupus) and dhole , and then identified a large number of dog-specific structural variants [In the paper, Wang variants . Functio"} +{"text": "Among the multiple reasons why plasticity rules in vivo might differ significantly from in vitro studies is that extracellular calcium concentration use in most studies is higher than concentrations estimated in vivo. STDP, like many forms of long-term synaptic plasticity, strongly depends on intracellular calcium influx for its induction. Here, we discuss the importance of considering physiological levels of extracellular calcium concentration to study functional plasticity.Since its discovery, spike timing-dependent synaptic plasticity (STDP) has been thought to be a primary mechanism underlying the brain\u2019s ability to learn and to form new memories. However, despite the enormous interest in both the experimental and theoretical neuroscience communities in activity-dependent plasticity, it is still unclear whether plasticity rules inferred from Spike timing-dependent plasticity (STDP) is a form of long-term synaptic modification thought to constitute a mechanism underlying formation of new memories. The polarity of synaptic modifications is controlled by the relative timing between pre- and post-synaptic action potentials , commonly between 2 and 3 mM and [Ca2+]e (S\u00fcdhof, 2+]e is associated with decreased synaptic transmission (Borst and Sakmann, 2+]e and is not triggered by Ca2+ entry via VDCC (Scanziani et al., Neocortical STDP relies heavily on presynaptic glutamate release and presynaptic firing rate (Markram et al., S\u00fcdhof, . Reducedin vitro under physiological conditions will allow a more robust application in vivo.All studies use different protocols . The numin vitro studies, and by definition, in vivo studies are inherently in a physiological calcium concentration. First demonstration of STDP in vivo was performed in the retinotectal pathway of Xenopus (Zhang et al., in vitro studies are performed in juvenile rodent while in vivo studies are performed in older animals. This may constitute an additional limitation to the transposition of the results observed in vitro. Several studies suggest that the capacity to induce t-LTD decreases with age (Banerjee et al., in vivo are obtained in the anesthetized animal. Although there are variations in extracellular calcium concentration of about 0.2 mM between awake and anesthetized animals (Ding et al., In opposition to As demonstrated by Inglebert et al. fine tuning of pre- and postsynaptic activity can restore t-LTD and t-LTP in physiological extracellular calcium condition. But would it be possible to restore classic plasticity rules under regular patterns of activity? The key component could be neuromodulation. Interestingly, replay of activity from place-cells with overlapping firing field in hippocampal slices induced t-LTP only in the presence of Carbachol, a cholinergic agonist (Isaac et al., The use of physiological external calcium concentration not only modulates the learning rules for long-term synaptic plasticity but it also enhances context-dependent synaptic plasticity. Analog-digital modulation of action potential-evoked synaptic transmission lies on modification of spike shape, by either broadening the axonal spike (Shu et al., H or IA (Daoudal and Debanne, Hebbian plasticity and Intrinsic plasticity are closely linked and are synergistically modified (Debanne et al., YI and DD wrote the article and YI built the figures. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."} +{"text": "Dear Editor,Rosmarinus officinalis) extracts have been extensively studied for their ability to ameliorate traits of metabolic dyshomeostasis . Carnosal., 2015). Carnosal., 2015; Lee et al., 2015; Lipina al., 2015; Naimi eal., 2015; Ou et aal., 2015, 2018[13al., 2015; Park anal., 2015; Song etal., 2015; Tsai etal., 2015; Wang etal., 2015, 2019[24al., 2015; Xie et al., 2015, 2018[29al., 2015), antiobal., 2015), and neal., 2015) propertal., 2015).Rosemary or rosemary-derived preparations have been demonstrated to modulate glycemic parameters in human subjects. Consumption of rosemary tea for 90 days has been reported to reduce glycated hemoglobin levels in addition to alleviating insulin resistance in type 2 diabetes subjects . ReductAuthors are thankful to Jain (Deemed to be University), Bangalore for the support.None."} +{"text": "Micronutrient deficiencies are known to affect more than two billion people globally . The magn = 32) of the studies in this systemic review measured child health outcomes in early infancy while the remainder measured outcomes in children whose ages ranged from 24 h after birth until school age. Quezada-Pinedo et al. concluded that there was a need for more high-quality studies examining the long-term effects of maternal iron status during pregnancy on child health outcomes.Several articles in this issue focus on anaemia and iron deficiency. In a systematic review including 44 studies published from 1975 to January 2021, Quezada-Pinedo et al. [In a narrative review including 14 articles published in the 10 years up until 2021, Wawer et al. reportedThe World Health Organization recommends iron and folic acid (IFA) supplementation for all pregnant women to prevent anaemia and adverse foetal outcomes . In a stThe original research by Ahmed et al. found a n = 71,728), Sol\u00e9-Navais et al. [n = 2628), the authors failed to observe any association between whole blood selenium concentration and birth weight or SGA. Sol\u00e9-Navais et al. pointed out that this lack of association could be due to the homogenous group of subsamples with fairly good selenium status. The authors concluded that, while a maternal diet rich in selenium during pregnancy may be beneficial for foetal growth, further interventional studies were needed to confirm the causal relationship.Using data from a large Norwegian pregnancy cohort published prior to October 2020, Zgliczynska and Kosinska-Kaczynska [In another review including 85 studies published between 1981 and 2020, Jouanne et al. reportedThe collection of articles in this issue brings together the latest research on a broad range of topics on micronutrients and pregnancy, including the prevalence of various micronutrient deficiencies and their risk factors, as well as highlights the potential benefits of adequate micronutrient and/or risk of deficiencies on children\u2019s birth outcomes. I am sure that the findings of these studies will contribute to enhancing our current knowledge of the importance of micronutrients during pregnancy and will encourage readers to conduct further studies in this important area of research."} +{"text": "Clostridium clariflavum for utilization in biofuel industry\u2019 by Asma Zafar et al., RSC Adv., 2021, 11, 9246\u20139261, DOI: 10.1039/D1RA00545F.Correction for \u2018Efficient biomass saccharification using a novel cellobiohydrolase from The authors regret that in the original article, the name and affiliation for one of the co-authors (Hasan Ufak Celebioglu) were incorrectly given. The correct name and affiliation are shown here.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "Retirement is a major life transition that may improve or worsen mental health, including depression. Existing studies provide contradictory results. We conducted a systematic review with meta-analysis to quantitatively pool available evidence on the association of retirement and depressive symptoms.v. cross-sectional studies), study quality score (QS) and considering studies using validated scales to diagnose depression. Heterogeneity between studies was evaluated with I2 statistics.We applied PRISMA guidelines to conduct a systematic review and meta-analysis to retrieve, quantitatively pool and critically evaluate the association between retirement and both incident and prevalent depression and to understand better the potential role of individual and contextual-level determinants. Relevant original studies were identified by searching PubMed, Embase, PsycINFO and the Cochrane Library, through 4 March 2021. Subgroup and sensitivity meta-analyses were conducted by gender, study design from 60 datasets suggested a protective effect of retirement on the risk of depression , although with high statistical heterogeneity between risk estimates . Funnel plot asymmetry and trim and fill method suggested a minor potential publication bias. Results were consistent, confirm their robustness and suggest stronger protective effects when progressively restricting the included studies based on quality criteria: (i) studies with the highest QS , (ii) studies with a high QS and using validated assessment tools to diagnose depression and (iii) studies of high quality, using a validated tool and with a longitudinal design . We observed a progressive reduction in funnel plot asymmetry. About gender, no statistically significant difference was found .Forty-one original studies met our a priori defined inclusion criteria. Meta-analysis on more than half a million subjects (Pooled data suggested that retirement reduces by nearly 20% the risk of depression; such estimates got stronger when limiting the analysis to longitudinal and high-quality studies, even if results are affected by high heterogeneity.As retirement seems to have an independent and protective effect on mental health and depressive symptoms, greater flexibility in retirement timing should be granted to older workers to reduce their mental burden and avoid the development of severe depression. Retirement may also be identified as a target moment for preventive interventions, particularly primary and secondary prevention, to promote health and wellbeing in older ages, boosting the observed impact. Briefly, we used a combination of free text and exploded MeSH headings, identifying: (i) the concept of \u2018retirement/transition to retirement\u2019 and (ii) \u2018depression/depressive symptoms\u2019. Further studies were retrieved from manual reference listing of relevant articles and consultation with experts in the field. Details on inclusion and exclusion criteria are reported in ad-hoc developed data extraction spreadsheet. The data extraction spreadsheet was piloted on ten randomly selected papers and modified accordingly. Data extraction included: full reference details, country of study conduction, study design, study setting, study population details, sample size, exposure details, outcomes of interest, including validated assessment tools for depression, and quantitative results, including ESs and corresponding confidence intervals (CIs). Corresponding authors were contacted by e-mail in case of incomplete data. Quality appraisal of included studies was carried out applying the 14-item scoring system developed by Shim et al. for population-based studies on retirement as a risk factor in a two-step process; a first screening was performed based on title and abstract. Then, full texts were retrieved for a second screening. At both stages, disagreements among reviewers were resolved by consensus and by consulting a third senior author (A.O.) when disagreement persisted. Data were independently extracted by two authors (V.G. and G.P.V.), supervised by a third author (A.O.), using an et al., s.e.s), we mathematically converted them into ORs with corresponding CIs and visual inspection of funnel plots. We performed sensitivity analyses progressively limiting meta-analysis to: (i) high-quality studies; (ii) high-quality studies using validated scales to diagnose depression; (iii) high-quality longitudinal studies using validated scales to diagnose depression. Moreover, we conducted a subgroup meta-analysis by gender strata and study design.We performed descriptive analysis to report and pool the characteristics of included studies using ranges and average values. With regard to the pre-specified outcomes of interest, we would expect variability between studies, e.g. by study design and population. We, therefore, applied random-effects meta-analyses to acquire estimates of the association between retiring and risk of depression/depressive symptoms, rather than to assume a single true value in a fixed-effects approach software.We assessed publication bias with funnel plot visual inspection published in the last 5 years. The majority of the studies were conducted in Europe and in the USA . Four studies were conducted in Asia, one in Brazil, one in Australia; four were multi-centre studies conducted at the global and European level.We identified 2470 studies by searching the selected databases and listing references of relevant articles. After removing duplicates, 1619 records were retrieved. Papers were screened and selected, as illustrated in n\u00a0=\u00a012, 60.0%) having less than 10 years of follow-up. Most of the longitudinal analyses were derived from the Survey on Health and Ageing and Retirement in Europe , followed by the Health and Retirement Study and the Gaz et Electricit\u00e9 cohort study . Twenty-one studies (51.2%) had a cross-sectional study design, while only one study reported both cross-sectional and longitudinal data . One study included only males studies were longitudinal studies; their follow-up time ranged from 2 to 25 years, with most of them and Question 13 [Was the loss to follow-up appropriately addressed and/or adequately described in the study?] (n\u00a0=\u00a011) reported the lowest scores .More than ninety per cent of included studies used validated tools to diagnose depression-related outcomes, including the Center for Epidemiologic Studies Depression scale (CES-D) in 17 studies (41.5%), the Euro Depression-scale (EURO-D) in eight studies (19.5%), the Geriatric Depression Scale (GDS) in three studies (7.1%) and the Zung Self-rating Depression Scale (ZSDS) in two studies (4.9%). The International Classification of Diseases-10 (ICD-10) was used to identify depression-related conditions in three studies (7.1%). The Patient Health Questionnaire-8 (PHQ-8), the Hospital Anxiety and Depression Scale (HADS), the Composite International Diagnostic Interview (CIDI), the Depression Adjective Check List (DACL) and the Clinical Interview Schedule-Revised (CIS-R) were used in only one study each . Three sn\u00a0=\u00a015, 36.6%) of included studies reported a statistically significant negative association between retirement and depression (i.e. retirement decreased the risk of depression) reported a positive association did not report statistically significant associations between retirement and depression (n\u00a0=\u00a019), ORs (n\u00a0=\u00a011), \u03c72 (n\u00a0=\u00a03), relative risks (RRs) (n\u00a0=\u00a01), hazard ratios (HRs) (n\u00a0=\u00a01) and mean differences (n\u00a0=\u00a02). Almost all included studies reported adjusted effect estimates . The funnel plot resulted slightly asymmetrical at visual inspection, showing a low potential for publication bias, not confirmed by Egger's linear regression test . Moreover, the ES change after the trim and fill method was minor [0.84 (95% CI\u00a0=\u00a00.75\u20130.94)], and two studies were trimmed in the lower right quarter of the funnel plot . Then, we limited the analysis to studies with high QS and using validated assessment tools to diagnose depression. In this analysis, 44 datasets were included, for a total of 239\u00a0453 subjects, strengthening the significant association between retirement and decreased risk of depression . Finally, only studies (i) with a QS equal or higher than 15, (ii) using validated assessment tools to diagnose depression and (iii) with a longitudinal study design were included. We report a statistically significant association between retirement and depression , with high statistical heterogeneity , but no publication bias, as confirmed by funnel visual inspection and Egger's test , a statistically significant association between retirement and depression was equally found and high heterogeneity and high heterogeneity between studies (Gender-strata meta-analyses are reported in online Supplementary Fig. 2b and 2c. When only considering women, the analysis included 21 datasets and a total of 219\u00a0655 subjects, reporting no statistically significant association between retirement and depression (pooled ES\u00a0=\u00a00.79, 95% CI\u00a0=\u00a00.61\u20131.02, ogeneity . About m studies . In bothPooled data from 41 original studies and more than half a million subjects suggested that retirement or transition to retirement reduce by nearly 20% the risk of depression or depressive symptoms; such estimates remain consistent when limiting the analysis to longitudinal and high-quality studies.et al., Before interpreting our findings further, we must account for the considerable heterogeneity among the included studies, which might limit the generalisability of pooled effect estimates. To overcome this and test the results level of strength, we first applied a random-effect model. Secondly, we conducted sensitivity and stratified meta-analyses by study design and QS. The reasons behind the high level of heterogeneity among the included studies are to be explored in light of, on one side, the wide variety of studies\u2019 designs, settings and populations, definitions and methodological quality and, on the other side, of the complex, multi-determinant and multi-mediator relationship between the process of retirement and mental health and wellbeing (Pesaran et al., Despite half of the retrieved studies being cross-sectional, which did not allow us to explore causality, they accounted for less than one-third of included subjects. Another limitation to consider is that duration of retirement was not reported in most studies, so we could not differentiate among the potential risk of depression for short- or long-term exposure to retirement. A subgroup analysis considering the work before retiring was not possible since only two included studies stratified results for this variable (Belloni To the best of our knowledge, this is the first systematic review and meta-analysis pooling all original studies investigating the association of retirement with prevalent and incident depression. We used a comprehensive range of databases and search terms to maximise the number of studies retrieved and minimise the chance of publication bias. Besides, further studies were retrieved from the reference listing of relevant articles. Such a comprehensive and rigorous summary of the available evidence offers several meaningful insights, valuable to plan, implement and evaluate public health and preventive strategies, public policies, as well as future avenues of research.Despite the well-known assumption that considers retirement as a potentially stressful life event Kremer, , one of Characteristics of the transition refer to the type and conditions of retirement, which were available in 76% of included studies. For instance, we report different impacts on depression between voluntary and involuntary retirement, with the more considerable impact of the latter (Mosca and Barrett, et al., et al., et al., et al., et al., There is extensive literature on how employment characteristics influence health after retirement (Hernberg, et al., et al., et al., With reference to resources, access to social and financial resources around retirement might compensate and mitigate the impact of lifestyle changes and the psychological consequences of retiring. We reviewed data where the risk of depression at retirement is differentially distributed by household socioeconomic status (Arias-De La Torre Concerning individual appraisal, personality characteristics influence the meaning assigned to retirement and the ability to cope with this change. Negative expectations and fears about retirement are more likely related to adverse repercussions on individuals\u2019 wellbeing (Barnes-Farrell, Regarding gender, differences in primary role between women and men, at home and work, respectively, could explain differences in adapting to the event and in health outcomes by gender Moen, , but neeet al. (et al. (et al. (Overall and sensitivity analyses results are consistent with other reviews on the topic. Van Der Heide et al. focused (et al. analysed (et al. ; they suet al., et al., et al., et al., et al., et al., et al., Regarding public health and preventive strategies, we demonstrated that, besides other factors influencing the risk of late-life depression, transition to retirement, as a life event that almost the entire population experience at some point (Clark and Oswald, et al., et al., et al., et al., About public policies, our data complement the accumulating evidence on the impact of pension reforms on health and mental health (Eibich, et al., et al., Concerning research, it clearly emerges from our analysis that, in order to reduce heterogeneity and accumulate solid evidence, shared methodological standards and definitions should be followed in the future. More extended longitudinal studies should be preferred so as to reduce inverse causality issues and might help disentangle and quantify the different components that mediate the effects of retirement on the risk of depression and its determinants and monitor such association's temporal evolution. It would also be necessary to further differentiate between contextual and individual characteristics to adapt coping strategies at the public health and clinical levels. Special attention should be paid to health inequalities to investigate better socioeconomic status indicators role in the relationship between retirement and health (Adler As a matter of fact, despite current trends in extending working lives, life expectancy after regular retirement is projected to grow faster than increases in the pension age, reaching 20.3 years for men and 24.6 years for women in 2050 OECD, . Therefo"} +{"text": "This review outlines how CRISPR/Cas9 has transformed T cell research allowing immunologists to rapidly probe the roles of genes in T cells thus paving the way for novel therapeutics. Furthermore, this review describes how these tools reduce the requirement for genetic mouse models, while increasing the translational potential of T cell research.Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 allows for precise gene targeting in mammalian cells, including T cells, allowing scientists to disrupt or edit specific genes of interest. This has enabled immunologists to investigate T cell functions as well as opened the path for novel therapeutics involving gene editing of T cells By enabling easy, swift, and precise manipulation of the genetic code, scientists can probe the roles of novel genes with an unprecedented speed and precision. The Nobel prize awarded to Jennifer Doudna and Emmanuelle Charpentier in 2020 for their discoveries of CRISPR/Cas9 celebrates the beginning of a biological revolution. CRISPR was discovered in bacteria as a defense mechanism used to disrupt invading foreign DNA from invading bacterial viruses (i.e. bacteriophages) by guiding a DNA cleaving nuclease to the foreign genomic DNA composed of a trans-activating CRISPR RNA (tracrRNA), which facilitates binding of the Cas9 enzyme, and a CRISPR RNA (crRNA), which engages the target DNA and the tracrRNA bound in the Cas9 enzyme, thus guiding the enzyme specifically to its target site. The gRNA-bound Cas9 enzyme binds gRNA-complementary genomic DNA sequences upstream of protospacer adjacent motifs (PAM) with high specificity and efficacy . These et al., et al., in vivo after Cas9-mediated editing. To overcome this challenge, scientists could combine Cas9-modified human T cells with organoid systems, where organized three-dimensional tissue cultures are grown in culture , and are limited to studying certain tissue types. Cas9-edited human T cells can also be studied in vivo in so-called humanized mice, which are immunodeficient mice that are reconstituted with human immune cells. These have shown some potential in increasing translational potential of therapies from preclinical models (Tao and Reese, et al., in vivo setting. Unfortunately, these models are not trivial to set up and are generally not broadly available to the research community (et al., CRISPR/Cas9 has already aided in discovery of multiple gene programs critical for human T cell activation, proliferation, and signaling (Shifrut ommunity (Allen e et al., . NonetheIn summary, these described model systems have the advantage of being high throughput compared with conventional genetically modified mouse models. Furthermore, as Cas9-edited human T cells are somewhat easier to generate, it is now possible to conduct studies in a model with greater translational potential, while accommodating the principles of the 3Rs. These benefits of using CRISPR/Cas9 in T cell research are also applicable to many other cell types. Although it is still a challenge to effectively implement these tools with many other primary human cell types, future innovation will likely enable this.et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., CRISPR/Cas9 has further allowed for generation of genetically modified T cells that can be used in patients for therapies. CRISPR/Cas9 has been adapted for generating efficacious CAR-T cells for therapy, which have an artificially introduced antigen receptor (Eyquem CRISPR/Cas9 has changed the way scientists conduct research in multiple fields, including the field of immunology. In T cell research CRISPR/Cas9 mutagenesis has already been used for multiple important discoveries, and has paved the way for novel, faster, and more translational techniques that in coming years have the potential to disrupt the way T cell studies are performed. Future developments will likely further enable CRISPR/Cas9-mediated development of highly efficacious T cells for cancer therapies, including CAR-T cells and adoptive T cell transfer."} +{"text": "Prevalence of SARS-CoV-2 specific neutralising antibodies in blood donors from the Lodi Red Zone in Lombardy, Italy, as at 06 April 2020\u2019 by Percivalle et al., published on 18 June 2020, incorrect protocol numbers were mistakenly quoted in the Ethical statement. The text was corrected on 20 January 2022 at the request of the authors. In the article \u2018"} +{"text": "More than 85% of pre-clinically tested drugs fail during clinical trials, which results in a long, inefficient and costly process, suggesting that animal models are often poor predictors of human biology . The abiThe research topic of this issue is aimed at providing further context to the use of iPSC-derived cells as disease models (\u201cdisease in a dish\u201d models) for screening leads for drugs.In this context, Trudler et al. , Lu QianWang et al. describeAccording to Vokner et al.\u2019s review, Interestingly, iPSC-based models can also serve for screening potential drugs against complex diseases such as Parkinson\u2019s disease , AlzheimThe model of iPSC-derived cardiomyocytes with very long-chain acyl-CoA dehydrogenase deficiency (VLCADD), studied by Knottnerus et al. , impliesiPSC-derived cardiomyocytes also serve as attractive models for dilated cardiomyopathy, such as propionic acidemia (PA), caused by mutations in either the PCCA or PCCB genes encoding both subunits of the mitochondrial propionyl-CoA carboxylase (PCC) enzyme and in CUrea cycle disorders are enzymopathies resulting from inherited deficiencies in any genes of the cycle. Zabulica et al. demonstrTo study the rare disease riboflavin transporter deficiency (RTD), Marioli et al. used iPSAmong the applicative future goals in studying iPSCs is the potential to generate patient specific organs such as liver, hearth patch, etc. Olgasi et al. describePregnancy miscarriages have many unknown causes and are complex processes that require solution. Bohnke et al. were abl"} +{"text": "Homocysteine levels can be increased by different conditions including genetic factors, diet, life style, several medications, etc. Elevated levels of homocysteine, called hyperhomocysteinemia (hHcy), are associated with a higher risk of neurovascular diseases, dementia, migraine, developmental impairments or epilepsy. The mechanisms underlying the neurotoxicity of homocysteine include oxidative stress, DNA damage, protein thiolation or protein homocysteinylation, triggering apoptosis and excitotoxicity. Recent data indicate that inflammation during hHcy comes along with increased levels of several cytokines and changes in DNA methylation.Homocysteine is a non-proteinogenic sulfhydryl-containing amino acid derived from methionine and is a homologue of cysteine. The concentration of homocysteine is regulated by two key pathways: remethylation back to methionine or transsulfuration to cysteine with the simultaneous production of hydrogen sulfide , which is a major regulator of cellular methylation reactions that occur in eukaryotic and prokaryotic organisms. SAHase activity is a significant source of homocysteine and adenosine. The author presents structural characteristics of the two principal domains of the SAHase subunit, which are based on the Rossmann fold. The study highlights similarities and differences in the spatial arrangements of both major domains. In his review, Brzezinski summarizIn their review Rizzo and Lagane summarizhHcy which has been linked to different systemic and neurological diseases, is well known as a risk factor for systemic atherosclerosis and cardiovascular disease (CVD), and has been identified as a risk factor for several ocular disorders, such as diabetic retinopathy (DR) and age-related macular degeneration (AMD). Oxidative stress, endoplasmatic reticulum (ER) stress, inflammation, and epigenetic modifications have been suggested as possible mechanisms of hHcy-induced blood retinal barrier (BRB) dysfunction. More recently hHcy-induced brain inflammation was reported as a mechanism of blood-brain barrier (BBB) dysfunction and pathogenesis of Alzheimer\u2019s disease (AD). The contribution by Tawfik et al. focuses The original study by Elsherbiny et al. demonstrIn the paper by Kovalska et al. , a methiN-methyl-d-aspartate receptors (NMDARs) and a reducing agent. In the paper by Sibarov et al. [Homocysteine (HCY) combines distinct pharmacological properties as an agonist of v et al. , the rolIvanova et al. provide 2S in kidney ischemia-reperfusion injury and oxidative stress in the heart.Hydrogen sulfide has been implicated in cardiovascular protection through redox balance and vessel relaxation. The paper by Wijerathne et al. emphasiz2S. Prenatal hyperhomocysteinemia (hHCy) was found to induce behavioral impairments and oxidative stress in brain tissue of rats, with a decreased expression of the H2S generating enzyme-cystathionine-beta synthase and concentrations of H2S in the brain. The administration of the H2S donor to females with hHcy during pregnancy prevented behavioral alterations and oxidative stress of their offspring. The acquisition of behavioral studies together with biochemical studies will improve our knowledge about homocysteine neurotoxicity and proposes H2S as a potential agent for the therapy of hHcy-associated disorders.Finally, Yakovleva et al. accentuaThis Special Issue describes several pathological conditions associated with elevated levels of homocysteine, some further mechanisms of homocysteine action, and opens new possibilities for treatments."} +{"text": "Cell membranes develop extraordinarily complex lipids and proteins geared to perform functions required by cells. The collective processes occurring within membranes strongly impact their cellular behavior and biochemistry; thus, understanding these processes poses unique challenges due to the often complex and multiple interactions among membrane components. The plasma membrane surface accommodates different types of lipid and protein clusters; however, the functional role of membrane surface clustering has not been fully understood yet. Super-resolution microscopy (SRM) has been extremely helpful in expanding our knowledge and changing the paradigm on both the structure and function of these clusters. These SRM techniques have been used to examine the organization and dynamics of plasma membrane components, thus giving insight into the fundamental interactions determining membrane functions.Membranes Special Issue, entitled \u201cDynamics and Nano-Organization in Plasma Membranes\u201d, presents both an update and a comprehensive overview of recent developments in plasma membrane nano-organization. It features eight contributions, namely six research papers and two reviews. A brief descriptive summary of the scientific contributions is reported here. This Kure et al.\u2019s review p2/s, which is comparable to similar studies of other aquaporins. From the kICS analysis, it can be concluded that the addition of actin polymerization-inhibiting compounds, cytochalasin D and latrunculin, did not significantly affect the diffusion coefficient of AQP9 in these cells, indicating that there was no correlation between the actin cytoskeleton network and AQP9; in addition, decreases in the diffusion coefficients were observed when adding Methyl-\u03b2-Cyclodextrin (M\u03b2CD). These observations, when combined, suggest that, unlike other AQPs , AQP9 is not a nano-domain membrane protein.Kure et al. also repIn the paper by Jaszul et al. , the molSezgin et al. reportedYang et al. reviewedIn their paper, Allender et al. discusseLast, Tintino et al. studied In conclusion, the papers in this Special Issue provide a significant contribution to increasing our understanding of the complexity in cell membrane organization. Super-resolution microscopy techniques are highlighted as powerful tools for obtaining new information about plasma membrane nano-organization, nanodomains, lipid as well as protein diffusion, along with membrane proteins and their interaction with plasma membranes. Moreover, important contributions related to the development of new methodologies to generate model lipid membranes as well as an alternative theory were described in order to explain the physical origin of membrane domains."} +{"text": "Dvorak describes that VEGF is widely believed to induce angiogenesis by its direct mitogenic and motogenic actions on vascular ECs. However, he emphasizes the role of VEGF in vascular permeability, the role of delayed hypersensitivity in pathological stroma generation, particularly extravascular fibrin deposition and clotting within the tumor microenvironment (TME). Based upon his prior work where he concludes that tumors are \u201cwounds that do not heal,\u201d he now proposes that tumors are wounds that continually exhibit elements of local healing but do damage the host (patients) whereas the healing process actually facilitates tumor survival and growth. Moreover, solid tumors, and healing wounds feature enlarged feeder arteries, draining veins, and several other abnormal vessel types in addition to new angiogenic blood vessels. He also proposes that that VPF/VEGF induces stroma formation primarily by way of its potent \u201cVPF\u201d function. Much remains to be explored about the signaling and functions of VEGF in wound healing, tumor progression and chronic inflammatory diseases.We begin with vascular endothelial growth factor (VEGF), also known as vascular permeability factor (VPF), one of the most important growth factor in EC and vascular biology. This important proangiogenic molecule was discovered over 3\u00a0decades ago by the Dvorak Laboratory at Harvard Medical School and was Wang et al. describe the basics of VEGF/VEGFR-2 signaling and provide an update on VEGFR-2 signaling-mediated physiological and pathological functions as well as potential treatment strategies. Potent VEGF signaling also promotes angiogenesis and dictates arterial fate but inhibits venous specification, and this may depend on Notch signaling status and thus conferring therapeutic resistance. They also discuss the Notch signaling crosstalk between CSCs and vascular ECs within the TME. They believe that therapeutic approaches could be developed by targeting the essential role of Notch pathway in CSCs and development of the arteriolar niche that promotes the self-renewal of CSCs , a predog status . Akil et of CSCs .Alfaidi et al. emphasize the role of Nck1/2 adaptor proteins in vascular biology, vascular permeability and angiogenesis, and discuss their therapeutic potential. A study on an adaptor protein widely expressed in vascular ECs by Liu et al. demonstrates that the mitogen-inducible gene 6 acts as a potent anti-angiogenic factor in the regulation of physiological and pathological angiogenesis. In terms of anti-angiogenesis, thrombospondin-1 (TSP-1) is one of the first discovered endogenous angiogenesis inhibitor, and negatively regulates EC functions . Although VEGF-activation of Notch signaling is known as the key to arterial specification, they have performed carefully analysis in the settings of de novo vasculogenesis of the dorsal aorta during early embryogenesis and vasculature development in the neonatal mouse retina and described novel signaling mechanisms and a new understanding of this subject. They also emphasize the role of shear stress in the maintenance of arterial identity after blood circulation is established, as shear-induced Notch signaling activation and cell cycle arrest may contribute to A/V specification process mainly originates from the mitochondria in diabetes, a study from Zhao et al. describe the paradox of adenosine monophosphate-activated protein kinase (AMPK), a heterotrimeric serine-threonine kinase, in the involvement of vascular remodeling and the development of pulmonary hypertension. They emphasize the differential effects of AMPK on pulmonary vasoconstriction and pulmonary vascular remodeling, which may also be involved in the regulation of pathological microvascular remodeling and angiogenesis under obese conditions . They functionally classify ECs as quiescent ECs involved in maintaining homeostasis, proliferative ECs, inflammatory ECs, remodeling ECs, ECs involved in EndMT, and ECs involved in angiogenesis. This classification may well explain two new connected processes such as inflammatory angiogenesis and non-inflammatory regenerative angiogenesis dissected in hind-limb ischemia-triggered angiogenesis shows that lipopolysaccharide induces transcriptional activation of forkhead box protein C2 and promotes itself expression in a histone acetylation manner using lung ECs and a sterile sepsis model in neonatal mice.EC heterogeneity is emerging as an important area in vascular biology. It is well known that ECs show tissue- and organ-specificity, and significantly contribute to the formation of different types of blood and lymphatic vessels. They serve as an important cell type in the development of vasculogenesis, angiogenesis, venogenesis and arteriogenesis under physiological and pathological conditions. As newly classified innate immune cells , ECs areogenesis . Dawson Fang et al. propose that an EndoMT program is partially and reversibly activated event in angiogenic ECs to support acquisition of the subset of mesenchymal characteristics, which is necessary to develop sprouting angiogenesis. They also discuss the potential signaling and regulatory mechanisms that may control the EndoMT program as well as potential therapeutic approaches in cancers. EndoMT is known to be regulated by transforming growth factor-\u03b2 (TGF-\u03b2) family. Ma et al. report that TGF-\u03b22 may be essential for this process by regulation of the balance between transcription factor SNAIL and ID factors and this may also regulate angiogenesis . Additionally, a study by Hunyenyiwa et al. shows that obesity inhibits angiogenesis via TWIST1-SLIT2 signaling, similar to an angiogenic phenotype occurred in tumor angiogenesis under diet-induced obesity conditions and partial epithelial-to-mesenchymal transition (EMT)/EndoMT have been extensively reported. EndoMT is a process whereby an EC undergoes a series of molecular events that lead to a change in phenotype toward a mesenchymal cell. In a hypothesis and theory article, nditions . It shouPaulson et al. show that primary cilia exists in lymphatic vasculature, and surprisingly is expressed on the abluminal surface of the lymphatic vessel. They also present evidence that lymphatic vessel patterning is regulated by a primary cilium protein IFT20 in lymphatic ECs during development and inflammation, which could lead to a new paradigm in the field of lymphangiogenesis. Last but not the least, Norden and Kume provide an elegant review about current status and the mechanisms by which lymphatic permeability and function are regulated in a tissue- and organ-specific manner, including lacteals of the small intestine. Disrupted lymphatic EC junctions are associated with various diseases, including lymphatic leakage present in chylothorax and lymphedema, metabolic syndrome, and impaired immune surveillance. They also describe key signaling pathways and factors that control lymphatic EC junctional integrity including VEGF, S1P, LPA signaling molecules and FOXC1 and FOXC2 transcription factors as well as RhoA/ROCK and Ephrinb2-Ephb4 pathways.In addition to new sprouting from blood vessels, the sprouting of lymphatic vessels, lymphangiogenesis, is actively involved in many pathological processes including tissue inflammation and tumor dissemination but is insufficient in patients suffering from lymphedema. Cilia, a microtubule-based organelle expressed on the apical surface of blood ECs is thought to function as a blood flow sensor. via toll-like receptor 2 signaling (de novo arteriogenesis, particularly under pathological conditions (Ren et al.; Heterogeneous endothelium and vasculature play a pivotal role in the regulation of tissue and organ homeostasis in human health and disease progression. This topic only covers the tip of iceberg in the field of EC and vascular biology, and significant studies are urgently needed to move the field forward. One emerging area is the immunological properties of ECs and their role in angiogenesis and lymphangiogenesis. ECs show distinguished immunological functions and have innate and trained immunity . McCoy eignaling . Secondlignaling . The resignaling . Thirdly"} +{"text": "Mast et al. analyzed transcriptome data derived from RNA-sequencing (RNA-seq) of COVID-19 patient bronchoalveolar lavage fluid (BALF) samples, as compared to BALF RNA-seq samples from a study investigating microbiome and inflammatory interactions in obese and asthmatic adults . Based on their analysis of these data, Mast et al. concluded that mRNA expression of key regulators of the extrinsic coagulation cascade and fibrinolysis were significantly reduced in COVID-19 patients. Notably, they reported that the expression of the extrinsic coagulation cascade master regulator Tissue Factor (F3) remained unchanged, while there was an 8-fold upregulation of its cognate inhibitor Tissue Factor Pathway Inhibitor (TFPI). From this they conclude that \u201cpulmonary fibrin deposition does not stem from enhanced local [tissue factor] production and that counterintuitively, COVID-19 may dampen [tissue factor]-dependent mechanisms in the lungs\u201d. They also reported decreased Activated Protein C (aPC) mediated anticoagulant activity and major increases in fibrinogen expression and other key regulators of clot formation. Many of these results are contradictory to findings in most of the field, particularly the findings regarding extrinsic coagulation cascade mediated coagulopathies. Here, we present a complete re-analysis of the data sets analyzed by Mast et al. This re-analysis demonstrates that the two data sets utilized were not comparable between one another, and that the COVID-19 sample set was not suitable for the transcriptomic analysis Mast et al. performed. We also identified other significant flaws in the design of their retrospective analysis, such as poor-quality control and filtering standards. Given the issues with the datasets and analysis, their conclusions are not supported. Since the emergence of SARS-CoV-2 in December of 2019, there have been over 230 million reported cases and more than 4.7 million deaths . The sciIn Mast et al. the authors concluded that the extrinsic coagulation cascade regulation in the lung was not majorly impacted by SARS-CoV-2. They proposed that the bradykinin storm mediated pathogenesis originally proposed in The first issue we identified was related to the designation of the BALF bulk RNA-sequencing samples from Michalovich et al (GEO data set - PRJNA434133) as \u201cHealthy Controls\u201d by Mast et al. Analysis of the meta-data associated with the described \u201cHealthy Control\u201d subjects published in Michalovich et al. demonstrates that their samples were overall not healthy and also not representative of the average American population in terms of obesity (42.4%),The issues with using the samples from Michalovich et al. as the healthy control samples is made clear in the findings of the original manuscript. The study found significant changes in systemic and pulmonary inflammation when comparing individuals with obesity, asthma, or smoking history to their healthy subjects. Specifically, they found elevated levels of circulating inflammatory mediators and proteins regulating coagulation . Additionally, they reported significant changes in BALF concentrations of C reactive protein, Serum Amyloid Alpha, IL-5, and other proteins that would impact coagulation and inflammation. Gene ontology analysis of transcriptional differences in the BALF that were published in Michalovich et al. identified significant enrichments in tissue remodeling and inflammation ontologies amongst obese and asthmatic groups relative to the three healthy controls. These significant inflammatory, pro-coagulant, and transcriptional changes within the samples that Mast et al. designated as \u201chealthy controls\u201d have many overlapping similarities with the phenotypic changes that are associated with SARS-CoV-2. Such changes would significantly disrupt the ability to accurately characterize SARS-CoV-2 differential expression as these comorbidities are not controlled for in Mast et al. The presence of such disparate sample types within the \u201chealthy control\u201d group does not yield an averaged and more representative control group as was implied in Mast et al., instead the pro-coagulant transcriptional changes associated with the co-morbidities observed in much of the control group would likely mask relevant COVID-19 induced transcriptional changes. Additionally, averaging of highly disparate samples within the control group during differential expression analysis would not yield a more representative data set, but rather would generate a noisy control group with averages significantly weighted towards more abundant sample types.Based on the description of the RNA sequencing library preparation methods described in Zhou et al. and Michalovich et al., very different approaches were used to prepare sequencing libraries. The type of library preparation can significantly modify the RNA content of sequencing libraries via polyA enrichment, rRNA depletion, and other major differences in molecular processes underlying library preparation. Dissimilar libraries, particularly those with non-similar polyA enrichment and ribosomal RNA (rRNA) depletion, cannot reliably be used for differential expression analysis with transcripts per million (TPM) based normalization, which Mast et al. utilized in their analysis .Michalovich et al. uses libraries that are enriched for mRNA via polyA enrichment, while Zhou et al. does not. Michalovich et al. also uses a TruSeq Stranded RNA library Prep kit with RiboZeroTMGold ribodepletion probes. This library preparation approach yields libraries that are selectively depleted of ribosomal reads (which are the predominant RNA species in cells), while enriching for mature mRNA transcripts . By contTo confirm the functional differences in library preparation methods, we analyzed the proportion of reads aligning to rRNA transcripts using the same CLC genomics alignment settings and reference transcriptomes described in Mast et al. This confirmed that the total rRNA content was at a significantly greater proportion in SARS-CoV-2+ patient samples relative to control samples. The amouRNA-seq approaches for differential expression analysis require that enough sequencing reads be collected to accurately quantify the total expression of transcripts across the genome. In order to have statistical meaningful numbers of reads mapping to each gene for differential expression analysis minimum read depth requirements must be met. If a particular transcript is lowly expressed relative to other transcripts, then a low number of reads may be stochastically detected during sequencing. Such a dynamic could artificially inflate or deflate the relative expression of a particular transcript, especially when normalization approaches are applied to compare libraries sequenced at different depths or with radically different RNA compositions. It is generally accepted in the field that experiments investigating eukaryotic global gene expression typically require at least 30 million poly-A and ribo-depleted reads per sample . In humaIn Mast et al., there are major discrepancies in the relative depths of the sequencing libraries used for the \u201chealthy control\u201d samples and the SARS-CoV-2+ patient samples . Of the To illustrate a specific instance, Mast et al. report that thrombomodulin (THBD) expression in the BALF was decreased by 2200% during SARS-CoV-2 infection. They reported the expression level to be approximately 9.6 counts per million reads in COVID-19 infection and 224 counts per million reads in the control sample set. However, at the level of raw counts, control samples averaged 8,377.68 counts while COVID-19 positive samples averaged 59.88 counts. The normalization process for the counts per million based normalization was further biased by the inclusion of between 16%\u201380% of the total rRNA in only COVID-19 samples. These rRNA reads would be included in the total number of mapped reads used to calculate the CPM normalization factor in a manner that was not consistent with the normalization of control samples. Additionally, such a bias would significantly decrease the likelihood of detecting mRNA transcripts in the COVID + genes, including thrombomodulin transcripts. These confounding factors could bring into question the accuracy of the reported magnitude of the differential expression, the reported directionality of the differential expression, and the subsequent pathway analysis performed.The significant issues we have identified regarding the heterogeneity of control samples, dissimilar library preparation methods, and insufficient sequencing depth collectively bring into doubt the validity of many of the conclusions drawn in Mast et al. The normalized manner in which the gene expression data were reported in the supplement and manuscript of Mast et al. made it difficult for reviewers and readers to identify these issues when analyzing the manuscript. Mast et al. additionally did not provide supplemental data regarding the raw reads that were processed during alignment, the raw counts that were normalized and processed during differential expression analysis, or any NGS quality control standards that should have been conducted by the authors before analyzing the data set. From our analysis of their raw data, we conclude that the sample set and experimental design implemented in Mast et al. are fundamentally flawed. The concerns are significantly magnified knowing that others researching COVID-19 are citing these poorly substantiated results in publications or integUpon processing the raw data as described in our results section, serious issues with relative sequencing depth quickly became apparent. Review of the count data which we have summarized in Table 2 and the differential expression results for genes of interest reported in Table 1 of Mast et al. demonstrate the flawed nature of this analysis. Overall, 23 out of 35 genes of interest reported in Mast et al. average less than 10 mapped reads per gene but were still included in the analysis. 8 of thoBy far the most notable result reported in Mast et al. is the reported observation that tissue factor, the key initiator of the extrinsic coagulation cascade, is not significantly impacted by SARS-CoV-2 infection. They reported no significant difference in expression levels and concluded that tissue factor biology was not a significant factor in the thrombotic complications of SARS-CoV-2 in the lung. They postulated that COVID-19 may dampen tissue factor dependent mechanisms in the lung. This analysis was confounded by the above-described issues including relative depth, rRNA differences between control and COVID sample sets, and normalization This statement is important as the field has also begun converging on tissue factor as a key player in the pathogenesis and coagulopathy complications of SARS-CoV-2 infection. For instance, patients with COVID-19 have been shown to have elevated levels of tissue factor laden microvesicles circulating in their blood, along with other markers of the extrinsic coagulation cascade . FurtherAt the time the manuscript was submitted, several higher quality data sets were available and the authors of Mast et al. should have redone their analysis on sample sets that were collected with the intent of resolving transcriptomic signatures to accurately characterize the host response to SARS-CoV-2 . Additio"} +{"text": "In the search for effective therapeutic strategies for depression, repetitive transcranial magnetic stimulation (rTMS) emerged as a non-invasive, promising treatment. This is because the antidepressant effect of rTMS might be related to neuronal plasticity mechanisms possibly reverting connectivity alterations often observed in depression. Therefore, in this review, we aimed at providing an overview of the findings reported by studies investigating functional and structural connectivity changes after rTMS in depression.A bibliographic search was conducted on PubMed, including studies that used unilateral, excitatory (\u2a7e10 Hz) rTMS treatment targeted on the left dorsolateral prefrontal cortex (DLPFC) in unipolar depressed patients.The majority of the results showed significant TMS-induced changes in functional connectivity (FC) between areas important for emotion regulation, including the DLPFC and the subgenual anterior cingulate cortex, and among regions that are part of the major resting-state networks, such as the Default Mode Network, the Salience Networks and the Central Executive Network. Finally, in diffusion tensor imaging studies, it has been reported that rTMS appeared to increase fractional anisotropy in the frontal lobe.The small sample size, the heterogeneity of the rTMS stimulation parameters, the concomitant use of psychotropic drugs might have limited the generalisability of the results.Overall, rTMS treatment induces structural and FC changes in brain regions and networks implicated in the pathogenesis of unipolar depression. However, whether these changes underlie the antidepressant effect of rTMS still needs to be clarified. No limitation was posed regarding publication date. We included studies using unilateral, excitatory, high-frequency (\u2a7e10\u00a0Hz) TMS treatment targeting the left DLPFC. Only studies that investigated brain structural and FC using whole-brain neuroimaging techniques before and after rTMS protocols in depressed patients were included. Studies (a) employing other techniques , (b) based on bilateral or inhibitory rTMS protocols, or (c) targeting areas other than left DLPFC were excluded. The reference list of the selected articles was checked in order to find relevant references not emerged from the main query. Only 13 studies were selected as eligible. Of these, two studies included, in addition to unipolar depressed patients, a small cohort of depressed bipolar disorder type II patients. Finally, nine studies used resting-state functional magnetic resonance imaging (rs-fMRI), three studies employed diffusion tensor imaging (DTI) techniques, and one study employed single-photon emission computed tomography (SPECT).Connectivity changes induced by rTMS treatment are briefly discussed below, divided according to the investigated connectivity domain. FC results focused on two brain areas, the DLPFC and the subgenual anterior cingulate cortex (sgACC), whose connectivity emerged to be affected by the selected rTMS protocol. Although our focus was on connectivity changes from before to after rTMS, we also listed, when reported, the baseline features predictive of treatment response.et al. , a decrease in FC between sgACC and DMN, consistently with the result reported by Liston et al. (v. sham) on the Montgomery-\u00c5sberg Depression Rating Scale (MADRS) and FC was demonstrated, suggesting that reduction in FC between sgACC and DMN may parallel the reduction in depressive symptoms, with no specific effect of active v. sham rTMS. Moreover, with regards to the CEN, the authors found a decrease in FC between sgACC and CEN. This last result is in contrast with the one reported by Liston et al. , a marker of white matter microstructure, measured through DTI and major resting-state networks ) were found to be associated with rTMS treatment, possibly mediating rTMS therapeutic efficacy.et al., et al., et al., et al., et al., et al., et al., In particular, in the reviewed studies, a decrease in FC between DLPFC is part of the SN, a key circuit directing attention and cognitive control Menon, , and comet al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., The three DTI studies here reviewed (Kozel et al., et al., The reviewed studies suffer from some limitations. First, the sample size was often modest and some studies (Liston In conclusion, the abovementioned results support the hypothesis that rTMS induces neuronal plasticity and reorganisation of key networks in the pathogenesis of unipolar depression. However, whether these changes underlie the antidepressant effect of rTMS is not defined yet. Further studies including larger and more homogeneous samples are needed to better clarify the effect of rTMS on brain connectivity and the relationship with its therapeutic effect in unipolar depression.All data described in this review have been included in"} +{"text": "Dear Editor,The diverse microbial community present in the human intestine plays a vital role in translating the food to nutrients and metabolites essential for maintaining host physiology, including digestion, lipid and glucose metabolism, immune homeostasis, and proper development of the brain and cognitive functions . Consumal., 2018). Modulaal., 2018). The poal., 2018; \u00c1ngel Gal., 2018). A deepal., 2018; Conternal., 2018; de Olival., 2018; Guevaraal., 2018; Istas eal., 2018; Lima etal., 2018; Medina-al., 2018; Ntemirial., 2018; Park etal., 2018; Vilela al., 2018) summariThe authors thank the American University of Ras Al Khaimah for the support and facilities provided.The authors declare no conflict of interest."} +{"text": "Khatouri et al., RSC Adv., 2021, 11, 7059\u20137069, DOI: 10.1039/D0RA09804C.Correction for \u2018Effect of hydrophobically modified PEO polymers (PEO-dodecyl) on oil/water microemulsion properties: The authors regret that the name of one of the authors was shown incorrectly in the original article. The corrected author list is as shown above.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "Dear editor,We are grateful for the comments of \u201cEarliest details of Dermatology by Ayurveda\u201dSimilarly, we are grateful for the comments by Mirzaei MR et al.None declared.Iago Gon\u00e7alves Ferreira: Conception and/or design of the study; literature review and article selection; content analysis; results analysis; preliminary review and final drafting.Magda Blessmann Weber: Conception and/or design of the study; literature review and article selection; content analysis; preliminary review and final drafting.Renan Rangel Bonamigo: Conception and/or design of the study; literature review and article selection; content analysis; preliminary review and final drafting.None declared."} +{"text": "Childhood trauma is associated with an elevated risk for psychosis, but the psychological mechanisms involved remain largely unclear. This study aimed to investigate emotional and psychotic stress reactivity in daily life as a putative mechanism linking childhood trauma and clinical outcomes in individuals at ultra-high-risk (UHR) for psychosis.N = 79 UHR individuals in the EU-GEI High Risk Study. The Childhood Trauma Questionnaire was used to assess self-reported childhood trauma. Clinical outcomes were assessed at baseline, 1- and 2-year follow-up.Experience sampling methodology was used to measure momentary stress, affect and psychotic experiences in the daily life of \u03b2 = \u22120.14, p = 0.010) and negative affect was modified by transition status such that stress reactivity was greater in individuals who transitioned to psychosis. Moreover, the association of stress with negative affect and psychotic experiences was greater in individuals exposed to high v. low levels of childhood trauma. We also found evidence that decreased positive affect in response to stress was associated with reduced functioning at 1-year follow-up . In addition, there was evidence that the association of childhood trauma with poor functional outcomes was mediated by stress reactivity , but no evidence that stress reactivity mediated the association between childhood trauma and transition .The association of stress with positive (Emotional and psychotic stress reactivity may be potential mechanisms linking childhood trauma with clinical outcomes in UHR individuals. The effects of childhood trauma will be mediated via pathways through stress reactivity (i.e. a mediation model).Furthermore, other possibilities of how childhood trauma and stress reactivity may combine with each other may be relevant the magnitude of associations between momentary stress and negative affect, positive affect and psychotic experiences is modified by transition status, and (ii) the effect of childhood trauma on transition status will be mediated via pathways through stress reactivity (i.e. a mediation model).et al., et al., The sample comprises UHR individuals from London (UK), Melbourne and Amsterdam/The Hague (the Netherlands) recruited as part of the EU-GEI High Risk Study . The target constructs show high and continuous variation over time. To obtain a representative sample of participants\u2019 experiences in daily life and to capture relevant variation in these target constructs with high resolution, a time-contingent sampling design with a blocked random schedule and a high-sampling frequency was used for ESM data collection, i.e. ten times a day for six consecutive days at random moments within set blocks of time , an established 25-item self-report measure enquiring about traumatic experiences during childhood . Second, we added two-way interaction terms for stress\u00a0\u00d7\u00a0childhood trauma to examine whether the associations between momentary stress, negative affect, positive affect and psychotic experiences were modified by childhood trauma (H2). The hypothesis that the associations of momentary stress with affect and psychotic experiences were greater in individuals exposed to high v. low levels of childhood trauma was tested by using the \u2018testparm\u2019 command for computing Wald tests to assess statistical significance of two-way interaction terms and the \u2018lincom\u2019 command to compute linear combinations of coefficients nested within participants (level-2), the \u2018mixed\u2019 command in Stata 15 was used to fit two-level, linear mixed models . To control for confounding of findings by comorbid disorders, all analyses were controlled for comorbid major depressive and anxiety disorders. In addition, analyses for testing H3 and H4 were controlled for time to follow-up to account for variation in time to follow-up. Unadjusted analyses and sensitivity analyses in a restricted sample assessed in a \u00b16 month time interval around the expected follow-up time points are displayed in online Supplementary materials 4\u20136.Restricted maximum-likelihood (H1 and H2) or maximum-likelihood estimation (H3 and H4) were applied, allowing for the use of all available data under the relatively unrestrictive assumption that data are missing at random and if all variables associated with missing values are included in the model . Assessment of clinical outcomes was completed for 48 participants at 1-year follow-up and 36 participants at 2-year follow-up . Nine individuals (11%) transitioned to psychosis by the final follow-up time point. Participants were on average 23 years old (s.d.\u00a0=\u00a04.93) and 56% were women. The majority (67%) of the sample was white, followed by 15% with black ethnicity. Seventy-six percent of the participants were diagnosed with a comorbid axis I disorder. Comparing the current study's participants to individuals included in the EU GEI High-Risk study, for whom ESM data were not collected (N\u00a0=\u00a0266), there were no differences in demographics or overall prevalence of comorbid disorders . However, the current sample showed higher levels of childhood trauma , a higher prevalence of specific phobias and a lower prevalence of major depressive disorder compared to participants, for whom ESM data were not collected 0.27 to 0.36, p\u00a0<\u00a00.001) and psychotic experiences as well as with a moderate decrease in positive affect .Momentary stress was associated with small to moderate increases in negative affect and psychotic experiences . Furthermore, we found a non-significant indication that childhood trauma modified the association between momentary stress and positive affect .Childhood trauma modified the associations of momentary stress with negative affect and lower level of functioning at 1-year follow-up .B\u00a0=\u00a01.74, 95% CI 0.36 to 3.11, p\u00a0=\u00a00.016). Moreover, perceptual abnormalities at 1-year follow-up were predicted by emotional and psychotic stress reactivity . There was no evidence that emotional or psychotic stress reactivity predicted anxiety or tolerance to normal stress.Decreased positive affect in response to stress was associated with higher illness severity . We found no evidence that emotional and psychotic stress reactivity mediated the association of childhood trauma and level of functioning. The association of childhood trauma and unusual thought content at 2-year follow-up was mediated by increased negative affect in response to stress . In addition, the association of childhood trauma and perceptual abnormalities at 1-year follow-up was mediated by increased negative affect and psychotic experiences in response to stress . High levels of childhood trauma were associated with more intense reactivity in the form of a stronger increase of negative affect and psychotic experiences in response to stress, which, in turn, was associated with higher illness severity, unusual thought content and perceptual abnormalities at follow-up. We found no evidence for direct effects of childhood trauma on anxiety and tolerance to normal stress and no mediation via stress reactivity.In exploratory analyses, there was no evidence for a direct effect of childhood trauma on transition status and no mediation via stress reactivity .Using an experience sampling design, we found strong evidence that minor daily stressors were associated with emotional and psychotic stress reactivity in UHR individuals (H1). Childhood trauma modified the effect of daily stressors on negative affect and psychotic experiences, with more intense psychotic experiences and stronger increases in negative affect for individuals exposed to high levels of childhood trauma (H2). In addition, we found some evidence to suggest stress reactivity predicts clinical outcomes at follow-up (H3). Finally, there was partial evidence that stress reactivity mediates the association of childhood trauma and clinical outcomes (H4).et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., The reported findings should be interpreted in light of several methodological considerations. First, childhood trauma was measured with a retrospective self-report questionnaire. A common concern about retrospective self-report is that recall bias and cognitive distortions might lead to invalid ratings (Dill et al., et al., In accordance with previous ESM studies, we found that momentary stress was associated with small to moderate increases in negative affect and psychotic experiences and moderate decreases in positive affect in UHR individuals (Reininghaus et al., et al. (et al., et al., et al., et al., et al., et al., et al., et al., When considering the role of childhood trauma and stress reactivity in clinical trajectories, several possibilities of how these may combine with each other may be relevant (Schwartz and Susser, , et al. , we inveet al., et al., et al., et al., Taken together, our findings underscore the relevance of reactivity to daily stressors as a putative mechanism linking childhood trauma with clinical outcomes in UHR individuals. Adding evidence to the mediated synergy model, the study suggests early adversity in childhood links to more severe clinical trajectories via, and in interaction with, subsequently elevated stress reactivity in adulthood. Therefore, the findings underline the relevance of ecological momentary interventions targeting stress reactivity in daily life (e.g. EMIcompass, a transdiagnostic ecological momentary intervention for improving resilience in youth; Schick"} +{"text": "Prostate cancer (PCa) is one of the most frequently diagnosed malignancies of men in the world. Due to a variety of treatment options in different risk groups, proper diagnostic and risk stratification is pivotal in treatment of PCa. The development of precise medical imaging procedures simultaneously to improvements in big data analysis has led to the establishment of radiomics - a computer-based method of extracting and analyzing image features quantitatively. This approach bears the potential to assess and improve PCa detection, tissue characterization and clinical outcome prediction. This article gives an overview on the current aspects of methodology and systematically reviews available literature on radiomics in PCa patients, showing its potential for personalized therapy approaches. The qualitative synthesis includes all imaging modalities and focuses on validated studies, putting forward future directions. In global cancer statistics of men prostate cancer (PCa) is the most frequently diagnosed malignancies in the world and the fifth leading cause of death worldwide The Radiomics Pipeline Figure is the e(1) Image acquisition and preprocessing(2) High-throughput feature extraction(3) Data integration and data analysisThe Image Biomarker Standardization Initiative (IBSI) published a reference manual to harmonize the feature extraction by providing (i) definitions, (ii) a standardization of the radiomics pipeline, (iii) reference datasets and (iv) a reporting scheme e.g., by intensity inhomogeneity correction or noise filtering for MR-images All imaging modalities mentioned in the introduction can be utilized for PCa radiomics: TRUS, MRI, PSMA-PET, CT and bone scan. It is important to mention that heterogeneities in image acquisition und image reconstruction algorithms due to different local standards are culpable for missing repeatability and reproducibility of RF The spatial and gray level information of the segmented voxels is used in numerous mathematical calculations to extract pre-defined \u201chand-crafted\u201d RF. They can be computed with various open-source packages like PyRadiomics e.g., the depth of a tree or a deep-learning architecture and (iii) one test set for the final assessment. The latter might be used independently for validation. During cross-validation (CV) usually small datasets are divided accordingly in an iterative process. K-fold CV partitions the dataset in k subsets using one as a validation and the rest for training. This process is repeated for each subset. Leave-one-out CV operates similarly but leaves one patient for validation while using the rest for training CV should be used with caution especially with leave-one-out CV tending to be overly optimistic Often a vast number of RF are computed, and the abundance of RF demands feature selection and/or reduction to avoid overfitting and to exclude not relevant or redundant features. Many features are correlated with each other; these redundant features might be depicted with heatmaps and should be omitted st order features and texture features. Shape features describe the morphology of the VOI for instance the size, volume or diameter. 1st Order features are based on an intensity histogram derived of the segmented voxels et al. the gray level co-occurrence matrix (GLCM) assesses the gray levels of pairs of neighboring voxels \u201cHand-crafted\u201d RF st of January 2014 st of January 2021. 251 articles were located. Additionally, 22 manuscripts were identified through other sources . 35 duplicates were removed. Only articles that met inclusion criteria were included. Finally, 77 studies were included in the qualitative synthesis. Please see Figure Studies eligible for inclusion complied with the following criteria: articles had to be on PCa radiomics with predefined \u201chand-crafted\u201d features derived from MRI, TRUS, CT, Choline- or PSMA-PET and needed to apply internal or external validation. Excluded were papers not written in English and non-original articles. Two of the co-authors (SKBS and ASB) performed independently a PubMed/Medline, EMBASE and Cochrane Library database search for the terms: (cancer of prostate[MeSH Terms]) AND ((texture features) OR (radiomics)). If the two independent readers included or excluded studies differently a third reader (CZ) decided on eligibility. This was performed in 11 cases. The time period considered in this literature review was from 1Furthermore, ongoing clinical trials were screened on \u201cclinicaltrials.gov\u201d. Studies eligible for inclusion fulfilled the following criteria: ongoing trials on PCa RFs with \u201chand-crafted\u201d features derived of MRI, TRUS, CT or PSMA-PET. Trials with unknown status were excluded. CZ performed the search for the terms (\u201cCondition or disease: prostate cancer\u201d AND \u201cradiomicsl\u201d OR \u201ctexture features\u201d). Six trials were located, and one trial (NCT03294122) was excluded due to unknown trial status.Literature research revealed 57 original papers computing RF on multiparametric magnetic resonance tomography (mpMRI) imaging et al. proposed a RF based framework for PCa detection and localization et al. (ROC-AUC 0.683-0.768 across 3 sites), showed good area under the receiver operating characteristics curve (ROC-AUC) for PCa detection et al. developed a framework of combined T2w, DWI and DTI features for differentiation of PCa and non-PCa voxels et al. demonstrated that the addition of DCE-RFs does not improve performance of T2w and DWI-RF based models to detect clinically significant PCa in the PZ et al. showed lower results for a PZ specific classifier for PCa detection with ROC-AUC of 0.6-0.71 In two preliminary studies, Cameron et al. introduced novel RF based on Joint Intensity Matrix to predict GS (ROC-AUC 0.64-0.82 depending on GS groups) et al. however, could not show that ADC features were predictive for GS upgrading in intermediate-risk prostate cancer et al. demonstrated that RF and quantitative histomorphometry correlate and are predictive for GS et al. investigated the prediction of clinically significant PCa (GS\u22657) in PIRADS 3 lesions, which could be a useful tool for biopsy guidance et al. reported that RFs from T2w and DWI sequences are associated with clinically significant PCa, being even more relevant than PIRADSv2 evaluation in some patients GS prediction and discrimination were assessed in 22 studies et al. and Zhang et al. showed that mpMRI derived RFs show good performance for bone metastasis prediction in untreated PCa with an ROC-AUC up to 0.92 et al. externally validated an ADC based RF (SZEGLSZM), which was identified in a previous study et al. demonstrated a ML classifier derived from T2w and ADC RF with good prediction of BCR after surgery or RT. which was externally validated with a AUC of 0.73 et al. showed good performance for BCR prediction after RT of localized PCa et al. elaborated a quality system to asses automated prostate segmentations with external validation et al. and Giannini et al. addressed RF-based PCa segmentation Five studies investigated RF for the prediction of extracapsular extension and reported high AUC values between 0.80-0.90 for radiomic signatures based on T2w and ADC sequences Literature research revealed five original papers et al. reported two distinct RFs with good performance to detect significant PCa lesions not visible in PSMA-PET/CT. This result was externally validated by an independent cohort et al. demonstrated a RF based machine learning model to predict lymph node involvement, presence of metastases, GS prediction (\u22658) and presence of extracapsular extension Three studies aimed for GS discrimination Literature research revealed six original papers using CT scans et al. implemented RF for automatic prostate gland delineation in TRUS images et al. suggested that RF derived from CT images might enhance interpretation of treatment response of bone metastases et al. demonstrated that RFs derived from CT images of PSMA-PET/CT scans could accurately distinguish between metastatic lesions and sclerotic area et al. outperformed conventional measures for detection of lymph nodes metastases GS discrimination by RF was the aim of four studies using TRUS In total, 5 studies were identified using mpMR imaging n = 4), PET (n = 1), CT (n = 1) and bone scans (n = 1) to extract RF may outperform biopsy mapping for GS 7 vs \u22658 discrimination et al. examined the PSMA expression in a combined cohort of more than 18 000 radical prostatectomy specimens and observed a correlation between PSMA expression and the GS The other focus is GS discrimination, reflecting the need for improvements in risk stratification. It is not surprising that most of the studies chose GS discrimination, since GS is the most established histologic biomarker. In clinical routine, the GS before primary PCa therapy is evaluated in tissue cores obtained by biopsy. However, due to intratumoral heterogeneity the GS in biopsy cores and prostatectomy specimen is discordant in 20-60% of the patients e.g. by genomic analyses) might outperform GS for risk prediction et al. showed weak correlations between RF and hypoxia gene expressions, providing an opportunity to assess the hypoxia status in PCa et al. and Switlyk et al. demonstrated an association between RF and the genetic marker phosphatase and tensin homolog et al. identified radiomic signatures which reflected genes that are over- and underexpressed in aggressive prostate cancer et al. suggests that RF signatures could distinguish between lesions of different aggressiveness However, several studies proposed that a thorough analysis of PCa tissue characteristics address extraprostatic extension (n = 6), BCR (n = 6), segmentation (n = 4), bone metastasis (n = 3), lymph node detection (n = 3) and radiotherapy toxicity (n = 2). Considering that most PCa patients are long-term survivors after treatment a reliable prediction of toxicity is warranted. Due to the lack of predictive models for toxicity prediction, we consider this field of major interest for future studies. Some of the excluded studies featured interesting concepts for the use of radiomics and treatment associated toxicity in PCa patients. Radiotherapy toxicity prediction was investigated for femoral head fractures Overall, most of the included studies presented good to high AUC values. However, these findings need to be considered diligently regarding publication bias and the variability observed in RF. As illustrated above the radiomic pipeline is a sequence of operations and each operation can be modified et al. developed a 3D printable phantom to measure repeatability and reproducibility of MRI-based radiomic features which could facilitate multi-center studies to harmonize image protocols and thereby tackling some of these challenges Texture features are increasingly sensitive to acquisition parameters with growing spatial resolution Multiple segmentations can reduce variability and bias in RF extraction of manually, semiautomatically or automatically segmented VOIs et al. investigated different generally adopted image intensity normalization techniques for T2w-MRI images and demonstrated a relevant impact on reproducibility of RFs Isaakson et al investigated normalization techniques to enhance comparability across different subjects and visits et al. investigated the variability of RF in MRI by using different filters, normalization, and image discretization techniques and observed that RF were sensitive to these pre-processing procedures. Hence, they recommended detailed reporting of the pre-processing steps and the use of open-source software et al. reported that ComBat harmonization is efficient and enables MRI data pooling from different scanners and centers Schwier et al. investigated repeatability of RF derived from CBCTs and reported that only five radiomic features were repeatable in < 97% of the reconstruction and preprocessing methods et al. proposed an approach to assess RF stability without multiple acquisitions and segmentations that could be used for preliminary RF selection. In addition, the authors advocated that RF derived of ADC maps behave differently based on the region extracted e.g. RF derived from head and neck tumors are less stable than those derived of sarcomas et al. recommends to investigate the repeatability of RF for every tumor type as well and for every PET-Tracer Two studies investigated repeatability of MRI-derived RFs and concluded that repeatability of many RFs is moderate and that a set of reproducible image features is desirable These papers demonstrate the fragility of RFs and the need of reproducible RF sets in order to enable a broad clinical application.et al. pitfalls could be uncovered and described Consequentially, more research on prostate MRI and PSMA-PET RF robustness should be performed. Other approaches to tackle RF variability is the standardization of RF definitions and calculations which IBSI tries to promote Nevertheless, validation is pivotal considering the variability of RF. 35 of 238 articles were excluded due to missing validation. In internal validation different types might be utilized like the aforementioned ML algorithms, k-fold CV or leave-one-out CV, as well as independent datasets for model development and validation. A proper methodology and the separation of training and validation dataset is demanded at all times et al. used deep learning in combination with \u201chand-crafted\u201d features and has successfully applied it in differentiating unilateral breast cancer from low-risk patients Many studies used ML for model building and verification. ML and deep learning as a subfield are emerging and harbor great potential This review focusses on the clinical aspects of RF demonstrating its great potential to affect management of PCa. However, some technical aspects have not been further investigated: information on the used algorithms for RF extraction or ML approaches were not provided. Additionally, we did not state whether the published models or the parameters are publicly available.In conclusion, most research in PCa radiomics focuses on PCa detection and GS discrimination. MRI as SoC is the most used imaging modality for RF computation for now, but PSMA-PET is gaining evidence in a wide variety of clinical settings. Most of the results suggest good to high performance of radiomics models but should be considered carefully due to RF variability. Further research is demanded on RF sensitivity and robustness especially on RF extracted of prostate MRI and PSMA-PET."} +{"text": "Inflammation is divided into acute inflammation and chronic inflammation. Chronic inflammation has been proved to be one of the major culprits of tumor occurrence and development . Once acLiu et al. described the role of IL-17A in Chronic Obstructive Pulmonary Disease (COPD), and discussed that IL-17A and its downstream regulators are potential therapeutic targets for COPD and subsequently COPD-derived lung cancer. Zhang et al. explored the role of FDX1 in the prognosis and metabolism of lung adenocarcinoma. They showed that FDX1 was closely related to glucose metabolism, fatty acid oxidation and amino acid metabolism. Another interesting study performed by Zeng et al. found chemokine ligand 14 (CXCL14) is involved in the proliferation and migration of ROS-induced colorectal cancer (CRC) cells, suggesting that aberrant ROS may promote colorectal cancer cell proliferation and migration through an oncogenic CXCL14 signaling pathway. Also, Wang et al., from another aspect, discussed the carnitine palmitoyltransferase system as a new target for anti-inflammatory and anti-cancer therapy, which may work as a promising anti-inflammatory/anti-tumor therapeutic strategy for numerous disorders. In tumor microenvironment field, Du et al. studied the tumor microenvironmental m6A as a prognostic biomarker associated with the hepatocellular carcinoma (HCC). And Wei et al. found that inhibition of BCL9 modulates the cellular landscape of tumor-associated macrophages in colorectal cancer.Here, we are committed to developing new strategies and treatments to activate the immune system of patients with chronic inflammation and cancer, in order to discover new drugs that can be used to combat chronic inflammation and induce cancer immune activation. In our research topic, Wu et al. explored T cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT), which is highly expressed in a subset of Treg cells, as a novel target for bladder cancer immunotherapy. Li et al. discussed the cGAS-STING pathway to explore the DNA immune recognition regulation in autoimmune disease and cancer. They summarized the current progress on cGAS-STING pathway modulators and laid the foundation for further investigating therapeutic development in autoimmune diseases and tumors. Wang et al. discussed the inhibitory effect of methylseleninic acid on esophageal squamous cell carcinoma through EGFR-IL-6 signaling axis. For metastatic cancer, Guo et al. used a xenograft model in zebra fish, and found lncRNA OR3A4 acts as the inflammatory cytokine to promote the proliferation and metastasis of ovarian cancer through KLF6 pathway.Today, in cancer prevention and treatment, many clinical trials aiming at evaluating the efficacy of inflammatory regulators are underway. Though the single use of anti-inflammatory drugs has shown limited efficacy in cancer treatment, inflammation modulators can synergistically increase the anti-cancer drug\u2019s efficacy in cancer therapies . For example, in some patients with advanced cancer, after conventional treatment, immunotherapy can still alleviate or even prevent the further development of disease . It is wIn summary, the collection of aforementioned articles in this research topic provide either overview of novel targets in immunotherapy, or new fundamental findings and summaries related to chronic inflammation and cancer. Additional researches, for example, drug delivery systems and synthetic antibody engineering, are needed to gain further insights as we move toward to improve the safety and efficacy of novel immunotherapy applications in chronic inflammation and cancer."} +{"text": "Stroke is a multifactorial disease and an extremely serious and socially important medical condition ,2. A recThis Special Issue (SI) entitled \u201cGenomics of Stroke\u201d provides a platform for a wide range of papers related to genetic studies of the pathogenesis, progression, diagnosis, and treatment of stroke. This SI includes six research articles and four reviews.The research articles in our SI are focused on association and functional studies of stroke-related genes or noncoding RNAs, and model systems, as well as pharmacogenomics and bioinformatics studies of stroke.First, we consider a paper entitled \u201cMicroRNA Analysis of Human Stroke Brain Tissue Resected during Decompressive Craniectomy/Stroke-Ectomy Surgery\u201d by Carlson et al. It should be noted that the authors conducted a comprehensive study. Specifically, human brain samples were obtained during craniectomy and brain tissue resection in patients with severe stroke who had life-threatening brain swelling. The tissue samples were subjected to histopathological and immunofluorescence microscopy evaluation, next-generation miRNA sequencing (NGS), and bioinformatic analysis . As a reThe role of another type of regulatory RNA\u2014namely, circular RNAs (circRNAs), in cerebral ischemia conditions was studied by Filippenkov et al., as reported in their paper entitled \u201cGenome-Wide RNA-Sequencing Reveals Massive Circular RNA Expression Changes of the Neurotransmission Genes in the Rat Brain after Ischemia\u2013Reperfusion\u201d. This type of RNA remains a highly promising target for research. Filippenkov et al. conducted a genome-wide RNA-Seq analysis of the subcortical structures of the rat brain containing an ischemic damage focus and penumbra . They fop = 0.048). SNPs rs62278647 and rs2316710 (PTX3) were associated significantly with IS . These correlations for rs62278647 and rs2316710 were found only in women, which suggests a sex-specific association of the PTX3 polymorphism [Interesting results were also obtained by Khrunin et al. in their paper entitled \u201cExamination of Genetic Variants Revealed from a Rat Model of Brain Ischemia in Patients with Ischemic Stroke: A Pilot Study\u201d. The authors were able to correlate the results of gene expression obtained in a rat model of tMCAO with the genomic characteristics of patients with ischemic stroke (IS). Previously, the authors had developed a bioinformatic approach to exploring single-nucleotide polymorphisms (SNPs) in human orthologues of rat genes expressed differentially under conditions of induced brain ischemia . Using tLee et al. present their paper entitled \u201cAssociation of CYP26C1 Promoter Hypomethylation with Small Vessel Occlusion in Korean Subjects\u201d related to epigenetic studies of stroke. In their study, the authors measured the level of DNA methylation in the CYP26C1 promoter and the 5\u2032 untranslated region of 115 normal subjects and 56 patients in Korea with small-vessel occlusion (SVO) . They foTanaka et al. present their paper entitled \u201cInfluence of Renal Impairment and Genetic Subtypes on Warfarin Control in Japanese Patients\u201d, which refers to pharmacogenomic research. It is known that warfarin is an effective means of anticoagulant therapy. However, warfarin has a narrow therapeutic range and shows a wide variation in doses between patients because of numerous environmental and genetic factors that influence warfarin pharmacokinetics and pharmacodynamics. Moreover, the genotypes of vitamin K epoxide reductase complex 1 (VKORC1) and cytochrome P450 2C9 (CYP2C9) can influence therapeutic warfarin doses ,11,12. TFinally, our SI includes an interesting research article by Leira et al. based on the application of bioinformatics methods entitled \u201cNetwork Protein Interaction in the Link between Stroke and Periodontitis Interplay: A Pilot Bioinformatic Analysis\u201d. In this exploratory study, the authors conducted a protein\u2013protein network interaction (PPI) search with documented encoded proteins for both stroke and periodontitis . Genes oAdditionally, results and prospects for stroke genomics were systematized and discussed in substantial reviews.The first review in our SI entitled \u201cMonogenic Causes of Strokes\u201d by Chojdak-\u0141ukasiewicz et al. reports that monogenic disorders are rare but play significant roles in the causes of strokes, especially in young people. The monogenic causes of stroke are recognizable by key clinical features and radiographic pictures. Genetic tests are expensive but should be part of routine diagnostic procedures in younger patients with cerebrovascular events, especially in the absence of typical vascular risk factors . The mosThe SI also includes a review characterizing one of the examples of ethnic features of stroke genomics. In their review entitled \u201cGenetic and Genomic Epidemiology of Stroke in People of African Ancestry\u201d, Prapiadou et al. describe significant racial/ethnic differences in the incidence, subtype, and prognosis of stroke between people of European and African ancestry, of which only about 50% can be explained by traditional stroke risk factors . HoweverAnother review entitled \u201cThe Genetic Landscape of Patent Foramen Ovale: A Systematic Review\u201d by Paolucci et al. will undoubtedly attract attention to our SI. It should be noted that paradoxical embolism through patent foramen ovale (PFO) is an important cause of cryptogenic IS, especially in younger patients. Consistent with this observation, the rate of PFO in siblings of young patients with IS and PFO is three times higher than in siblings of patients without PFO ,17,18. IFinally, the review entitled \u201cInfluence of Haptoglobin Polymorphism on Stroke in Sickle Cell Disease Patients\u201d by Edwards et al. is of substantial interest for our SI. This review outlines the current clinical research investigating how the haptoglobin (Hp) genetic polymorphism and stroke occurrence are implicated in sickle cell disease (SCD) pathophysiology . Hp is aIn conclusion, the diversity and quality of the studies presented in this SI indicate the constant advancement of knowledge in the field of genomics of stroke.We hope that our SI will be useful and interesting for readers. Further development of the field of stroke genomics will bring society closer to improving diagnostic, prognostic, and therapeutic measures to combat stroke and related pathologies."} +{"text": "Mental health (MH) service users have increased prevalence of chronic physical conditions such as cardio-respiratory diseases and diabetes. Potentially Preventable Hospitalisations (PPH) for physical health conditions are an indicator of health service access, integration and effectiveness, and are elevated in long term studies of people with MH conditions. We aimed to examine whether PPH rates were elevated in MH service users over a 12-month follow-up period more suitable for routine health indicator reporting. We also examined whether MH service users had increased PPH rates at a younger age, potentially reflecting the younger onset of chronic physical conditions.A population-wide data linkage in New South Wales (NSW), Australia, population 7.8 million. PPH rates in 178 009 people using community MH services in 2016\u20132017 were compared to population rates. Primary outcomes were crude and age- and disadvantage-standardised annual PPH episode rate (episodes per 100 000 population), PPH day rate and adjusted incidence rate ratios (AIRR).MH service users had higher rates of PPH admission and a larger number of hospital days than other NSW residents due to increased likelihood of admission, more admissions per person and longer length of stay. Increases were greatest for vaccine-preventable conditions , and chronic conditions . The highest number of admissions and relative risks were for respiratory and metabolic conditions, including chronic obstructive airways disease and diabetic complications . One-quarter of excess potentially preventable bed days in MH service users were due to vaccine-related conditions, including vaccine-preventable respiratory illness. Age-related increases in risk occurred earlier in MH service users, particularly for chronic and vaccine-preventable conditions. PPH rates in MH service users aged 20\u201329 were similar to population rates of people aged 60 and over. These substantial differences were not explained by socio-economic disadvantage.PPHs for physical health conditions are substantially increased in people with MH conditions. Short term (12-month) PPH rates may be a useful lead indicator of increased physical morbidity and less accessible, integrated or effective health care. High hospitalisation rates for vaccine-preventable respiratory infections and hepatitis underline the importance of vaccination in MH service users and suggests potential benefits of prioritising this group for COVID-19 vaccination. Disadvantage, lifestyle, increased prevalence of chronic illness, medication side effects, reduced help-seeking and the accessibility and quality of health care all contribute to this mortality gap rates of PPH in MH service users, examining whether increased rates observed in longer-term follow-up studies are also observed over shorter time periods more suitable for routine indicator reporting. Second, to examine the relationship between age and the risk of PPH admission. People with MH conditions have a younger age at onset of risk factors such as obesity and conditions such as cardiovascular disease state government hospital and community services are provided through 15 geographically organised Local Health Districts and three Speciality Health Networks, which are responsible for physical care as well as MH care. State government-operated community MH services are organised around geographical catchment areas, are free of charge and can be accessed without referral by a General Practitioner (primary care physician). Private office-based primary and specialist care, private hospital care, and pharmaceuticals are funded or subsidised by the Australian Federal Government with a varying degree of consumer co-payment. Referral by a General Practitioner is required to access subsidised private specialist care. Private hospitals mainly provide non-emergency care (including voluntary MH care) for individuals opting in to private health insurance: in the study period, private hospitals provided 23% of total acute overnight hospital episodes in NSW and 27% of acute overnight hospital days MH service user status, defined as any contact with state specialist MH services in the preceding 2 years, (ii) age and (iii) socio-economic status of person's area of residence. MH service user status was defined by linkage to state-wide community MH datasets.The study group (MH group) were NSW residents aged 0\u2013100 years who had clinical contact with any NSW government ambulatory MH service between 1 July 2015 and 30 June 2017. These included face-to-face, telephone or videoconference contacts in community MH centres, home visits, hospital outpatient clinics or emergency departments. Services to non-NSW residents, administrative contacts, case conferences and in-reach services to hospital inpatients were excluded. Age, sex and area of residence were defined at the index contact in the observation period. Primary care, private specialist care and non-government organisation contacts are not available in the dataset.The main outcome was admission to any NSW public or private hospital in 1-year observation period between 1 July 2016 and 30 June 2017 with a primary or secondary diagnosis of a PPH, as defined using Australia's national specification program, a data linkage examining premature mortality in MH service users. Data were linked by the NSW Centre for Health Record Linkage . Data sets and linkage methods are described elsewhere any PPH, (ii) three PPH groups and (iii) 21 individual PPH conditions. For ease of interpretation, chronic conditions were further sub-grouped into cardiovascular, metabolic and respiratory conditions, and acute conditions into acute infections, seizures and other acute conditions. Episodes with multiple PPH diagnoses were counted separately for condition-specific rates but treated as a single episode when calculating overall PPH rates. Age-specific rates were calculated using 5-year bands defined by age on 30 June 2016. Differences in the proportion of people with any PPH were tested using a two-sample test of proportions. Differences in the number of PPH episodes per person and the average length of stay of PPH episodes were compared using Wilcoxon's rank-sum test.Adjusted Incidence Rate Ratios (AIRRs) were calculated by direct standardisation to the NSW non-MH population distributions for age group and for the socioeconomic disadvantage index of the person's area of residence (expressed as a quintile). Disadvantage was measured using the Australian Bureau of Statistics Index of Relative Socioeconomic Disadvantage (IRSD) PPHs occurring prior to first MH contact in the observation period, (ii) admissions to private hospitals and (iii) PPH with associated MH care.The study was approved by the NSW Population and Health Service Research Ethics Committee and the Aboriginal Health and Medical Research Council of NSW (Ref 1564/19).The study is overseen by (i) a Steering Committee which includes representation from peak organisations representing NSW health consumers, MH service users and MH carers, and (ii) an Aboriginal Advisory Committee which includes Aboriginal people with lived experience, community organisation, policy and research expertise.et al., A total of 178\u00a0009 people had contact with NSW ambulatory MH services in the observation period. Half (50.1%) were male. MH service users were younger than the NSW population: most were aged between 15 and 44 , see Supplementary Table s2. Diagnostic characteristics of our community cohort are described elsewhere: the most common specific diagnoses are affective disorders, psychotic disorders and other MH conditions including anxiety and adjustment disorders , particularly chronic obstructive airways disease . Rates were also significantly increased for metabolic conditions including diabetic complications and for cardiovascular conditions . Amongst acute conditions, the highest rate ratios were for seizures and epilepsy , which comprised 12% of total PPH admissions in the MH group but only 5% in the non-MH group.After adjustment for age and disadvantage, MH service users experienced 3.6 (95% CI 3.5\u20133.6) times the rate of PPH compared to other NSW residents . AIRR weother vaccine-preventable conditions , which includes hepatitis B and C, mumps, herpes zoster, diphtheria and pertussis, as well as for vaccine-preventable pneumonia and influenza which includes influenza and haemophilus or streptococcal pneumonia.PPH risk was increased for both categories of vaccine-preventable conditions. Risk was elevated for The risk of PPH admission varied with age . Preventable hospital days were increased in most conditions, particularly chronic respiratory conditions , diabetes complications , vaccine-preventable conditions , acute pneumonia and seizures and epilepsy . The average length of stay was increased for almost all PPH conditions.MH service users had more than five times the number of hospital days per 100\u00a0000 population compared with other NSW residents . Chronicchronic obstructive pulmonary disease, diabetic complications, congestive cardiac failure, cellulitis, seizures and urinary tract infection. Vaccine-preventable conditions contributed nearly one-quarter (24.5%) of excess bed days because of their greater incidence and substantially increased length of stay.MH service users experienced 63\u00a0134 days in the hospital due to PPH conditions in 2016\u20132017; 80% more than the expected number among other NSW residents , excluding PPH admissions to private hospitals significantly increased the gap between MH service users and other NSW residents, because MH service users had fewer PPH admissions in private hospitals. Excluding PPH episodes occurring in specialised MH units, or PPH episodes occurring prior to a person's first community MH contact did not significantly alter the AIRR for PPH episode rates and caused a small but statistically significant reduction in AIRR for PPH days.et al., et al., et al., et al., et al., In the NSW population of nearly 8 million, MH service users had 3\u20134 times more preventable hospital admissions for physical health care, were admitted at a much younger age and spent substantially longer in hospital for equivalent conditions when compared to people without MH service contact. These findings are likely to reflect several interacting factors. First, rates of medical morbidity are increased in people with MH conditions. Our finding of more than five-fold increases in admission rates for chronic respiratory disease and diabetes complications is consistent with the increased prevalence of these conditions in people with MH conditions (Firth et al., et al., et al., et al., et al., Second, increased preventable admission is likely to reflect health system factors. PPHs are an effective measure of access to primary and specialist care (Parchman and Culler, et al., We found that MH service users had five-fold increases in per capita rates of preventable hospital bed days. Of the total, 80% of bed days for MH service users were excess to those which would be expected if PPH incidence and the average length of stay were the same as for other NSW residents. MH service users are around 2% of the NSW population, but account for around 10% of preventable bed days for chronic respiratory and metabolic conditions and up to 20% of bed days for vaccine-preventable conditions. These findings highlight the significant burden of these preventable hospitalisations for individuals and for the health system. The finding of longer average stays for equivalent conditions underlines the importance of proactive consultation-liaison psychiatry, and integrated physical and MH care in general hospital settings (Oldham higher than in earlier cohorts with longer follow-up periods. Kisely et al. (et al. (The current study examined whether increased PPH rates reported in previous longer-term studies were also observed over a 12-month time period more suitable for routine indicator reporting. Relative rates in the current study were y et al. examined (et al. followedet al., The second aim of the current study was to examine the interaction between age and PPH in MH service users. One study has reported a U-shaped relationship between age and PPH rates, finding higher rates of PPH among children, youth and older people (Guo This study uses linked data from routine health collections and therefore shares the limitations and biases of such data sets. In particular, the current dataset is biased towards the identification of people with more acute or severe MH conditions. Each year more than 10% of Australians access a MH service (Australian Institute of Health and Welfare, et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., relative risk. However, our findings may not generalise directly to other health systems which use different definitions.Medical comorbidity and premature mortality in MH care is a global problem (Liu et al., et al., et al., et al., Socioeconomic disadvantage influences population PPH rates (Pappas et al., Twelve-month PPH rates appear to be a potentially useful indicator for measuring the personal and health system impacts of increased medical morbidity in MH service users. Collaborative efforts such as the \u2018Equally Well\u2019 initiatives in the UK, Australia and New Zealand (National Mental Health Commission,"} +{"text": "Alzheimer's disease (AD) is a neurodegenerative disorder and the most common cause of dementia. There is currently no effective treatment, which makes preclinical prediction for AD particularly important applications.Wang X. et al., focus on subjects with low and high plasma A\u03b2 levels among individual with SCD. They investigate the microstructural changes in white matter (WM) based on diffusion tensor imaging from dataset of Sino Longitudinal Study on Cognitive Decline (SILCODE). Result shows a correlation between WM integrity and plasma \u03b2-amyloid (A\u03b2) 40 levels rather than A\u03b242 in individuals with SCD. This indicates plasma A\u03b240 levels may represent a useful biomarker to predict different trajectories of aging in individuals with SCD.The author Qiao et al., analyses the associations between WM disruptions and cognitive declines at the early stage of subcortical vascular cognitive impairment (SVCI). This study concludes the damage of long WM in right hemisphere in the pre-SVCI patients and correlated with declines in executive functions and spatial processing.Another case-control study by Huang et al., uses multi-kernel support vector machine (SVM) to examine whether WM structural networks can be used for screening SCD and aMCI. Their findings promote the development of potential brain imaging markers for the early detection of AD.The study by Yang et al., explore microstructural and cerebral blood flow (CBF) abnormalities in individuals with SCD plus and aMCI. They point out the mean kurtosis of DKI may be used as an early potential neuroimaging biomarker and may be more sensitive than CBF at the very early stage of AD.Based on diffusional kurtosis imaging (DKI) and three-dimensional (3D) arterial spin labeling (ASL), Wu Z. et al., examines group differences in gray matter surface morphometry, including cortical thickness, the gyrification index (GI), and the sulcus depth. The authors aim to track the progression of the disease in different stages of AD, including health controls, early MCIs, late MCIs, and ADs. Based on region-of-interest (ROI) analysis, their study shows that cortical thickness and sulcus depth indices are predominant during AD progression while GI is insensitive. The findings highlight the relevance between gray matter surface morphometry and the stages of AD, laying the foundation for in vivo tracking of AD progression.The paper by Fu et al., extracts gray matter volumes to predict the regional densities in the whole brain in normal control (NC), SCD, Amnestic mild cognitive impairment (aMCI) and AD. In this study, decreased structural covariance and weakened connectivity strength are observed in SCD compared with NC. In addition, increased structural covariance in aMCI and decreased structural covariance in AD are also found. These results provide evidence to the structural disconnection hypothesis in individuals with SCD.The study by Li et al., points out the impairment in spatial navigation (SN) in patients with MCI. They demonstrate that structural connectivity network abnormalities, especially in the frontal and parietal gyri, are associated with a lower SN accuracy, independently of white matter hyper intensities, which providing a new insight into the brain mechanisms associated with SN impairment in MCI.The study by Cui et al., points out different functional activity of the SCD patients with aMCI patients, which suggest SCD may be a separate stage of cognitive decline before aMCI and is helpful to the study of preclinical cognitive decline.The study by Tao et al., further identify individuals with SCD or aMCI from healthy control (HC) and to describe the relationship of pathological changes in these two stages. They conclude that the neural degeneration from SCD to aMCI follows a gradual process, from abnormalities at the nodal level to those at both nodal and network levels.Based on the topological characteristics of the WM network, Chen Q. et al., identifies distinct functional states and explore the reconfiguration functional connectivity (FC) in individuals with SCD. Results indicate that the alterations of dynamic FC may underlie the early cognitive decline in SCD patients and could be served as sensitive neuroimaging biomarkers.The study by Wei et al., discover four interactions among self-reference network (SRN), dorsal attention network (DAN), and salience network (SN) using resting-state fMRI. These results point out that the influence of the SRN in the ultra-early stages of AD is non-negligible.Taking the important role of self-reference processing into account, Xu et al., explores the specific characteristic based on the multimodal brain networks, including individual morphological, structural and functional brain networks. Results highlight the role of cortical-subcortical circuit in individuals with SCD, providing potential biomarkers for the diagnosis and prediction of the preclinical stage of AD.The study by Wu L. et al., investigates the cognitive impairment in individuals with chronic pontine stroke based on voxel-mirrored homotopic connectivity. Results indicate the important role of lingual gyrus and precuneus as ROIs in the early diagnosis of cognitive impairment individuals with chronic pontine stroke.The study by Wang Y. et al., demonstrates that the carotid calcifications are associated with post-stroke cognitive impairment (PSCI). They conclude that the significant role of large vessel atherosclerosis in PSCI should be concerned in future study.The study by Chen Y. et al., concludes that the methylation of peripheral NCAPH2 could be used as a useful peripheral biomarker in the early stage of AD screening. Low levels of NCAPH2 methylation are observed in SCD, and which is independent of the APOE \u03b54 status. In addition, there is a positive correlation between NCAPH2 methylation levels and the hippocampal volumes in SCD APOE \u03b54 non-carriers.The study by Dakterzada et al., compares the results of Innotest enzyme-linked immunoassay (ELISA) with two automated methods (Lumipulse and Elecsys). Both Lumipulse and Elecsys methods are highly concordant with clinical diagnoses, and the combination of Lumipulse Ab42 and P-tau has the highest discriminating power. They recommend both automated methods for the measurement of CSF biomarkers.The study by Shi et al., explores whether adenosine receptor 1 (A1 R) is involved in electroacupuncture (EA) pretreatment induced cognitive impairment after focal cerebral ischemia in rats. The results showed that EA pretreatment revered cognitive impairment, improved neurological outcome, and inhibited apoptosis at 24 h after reperfusion. Pretreatment with CCPA (a selective A1 receptor agonist) could imitate the beneficial effects.The study by Lin et al., examines the relationship between spinal cord injury (SCI) and olfactory dysfunction. They point out that the SCI initiates pathological processes, including inflammatory response and impaired neurogenesis. These results provide a basis for pathological mechanisms of early neurodegenerative diseases involving the olfactory bulb and enable early clinical drug intervention.The study by Wu P. et al., proposes the sustained clinical efficacy of unilateral magnetic resonance-guided focused ultrasound (MRgFUS) thalamotomy in Chinese patients with ET.Essential tremor (ET) is occasionally associated with a high risk for MCI and dementia. The retrospective study by JJ, YH, and FJ have written this editorial for the Research Topic they have edited. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."} +{"text": "The study of child and adolescent learning has generally focused on aspects specifically tied to individual academic performance. However, a new emerging perspective is that any \u201cdeficit\u201d and/or disability and conversely any achievement is not the result of a single event, such as an isolated reaction, but it is formed, through numerous biosocial contributing variables, during a child's attempt to adapt to learning conditions and settings. The fit between such adaptations and normative criteria is often associated with labels such as \u201cfulfillment,\u201d \u201cstrengths\u201d \u201cresilience\u201d or \u201cweaknesses,\u201d \u201crisk,\u201d \u201cvulnerability\u201d and \u201cdisability.\u201dThis Research Topic explored the overlapping challenges and themes related to developmental adaptations (as defined above) in the context of formal and informal settings for learning primarily within childhood and adolescence.Jefferies et al. investigated the relationships between the multidimensional constructs of physical literacy and resilience in 227 school Canadian children aged 9\u201312 years old. They found that resilience was predicted by movement capacity, confidence, and competence, environmental engagement, and overall perceptions of physical literacy. Their research highlights the importance of introducing physical literacy in schools.To start off in this path, Ato et al. examined the impact of temperament on academic achievement and sociometric status in a sample of 295 6\u20137-year-old Spanish children. Parents completed the Temperament in Middle Childhood Questionnaire, while sociometric status and academic achievement were derived from teachers' reports. Latent profile analysis showed that Children in the \u201cNegative/Undercontrolled\u201d profile were at higher risk for academic failure and peer rejection, while \u201cSociable/High regulated\u201d showed the reverse pattern. The findings have very important (so far underexplored) implications for ways in which schools could integrate \u201cdifficult children.\u201dIn a similar vein, Yao et al. examined the association of dopamine-related genes with mental and motor development and gene-environment interaction in 201 preterm and 111 term Taiwanese children, who were followed from 6 to 36 months and were genotyped for 15 single-nucleotide polymorphisms (SNPs) in dopamine-related genes . MAOA SNPs were robustly associated with the mental (but not motor) development scores throughout early childhood in the premature children but not in the term counterparts. This warrants further investigations on whether the MAOA variants could help develop personalized interventions for preterm children.Two contributions focused on early developmental determinants. Firstly, Li et al. examined whether executive function training (EFT) could improve children's emotional competence (EC). Fifty-five 4-year-old Chinese children were assigned to either EFT or no-EFT group. Pre-test vs. post-test training between-and- within-subjects effects were analyzed to quantify improvement. EFT was associated with significantly higher scores on EC and changes in inhibition control and working memory abilities significantly predicted variation in EC. The findings suggest that intervening on inhibition control and working memory abilities via training may improve preschool children's emotional abilities.Considering another early developmental period, Caramia et al. recruited 29 young Italian adolescents to test whether walking with a smartphone increased fall and injuries risk, and to quantify these possible outcomes, participants were asked to walk along a walkway, with and without a concurrent writing task on a smartphone. Concurrency of walking and smartphone use resulted in reduced step length, gait speed and general aspects of gait stability, regardless of experience or frequency of use, suggesting that using the smartphone while walking may determine an increased risk of injury or falls also for young digital natives.At the other end of the spectrum, adult studies have shown that the concurrency of a smartphone-related task and walking can increase instability and risk of injuries. Zhao et al. developed the Brief Haze Weather Health Protection Behavior Assessment Scale-Adolescent Version (BHWHPBAS-AV), and tested its validity and reliability in two randomly selected districts of Baoding, China, and involving 22 middle-school classrooms and 1,025 valid questionnaires. The BHWHPBAS-AV scale showed promising reliability and validity suggesting it may be applied to assess adolescent haze weather health protection behavior, and help school and medical staff administer targeted behavioral and preventative interventions or health education programs.Adopting an approach to prevent air pollution, Grant et al. assessed the cognitive profiles of 360 Canadian adults and children ranging from 7 to 80 years of age with disability in reading alone, mathematics alone and both (comorbidity), with tests widely used in both psychoeducational and neuropsychological batteries. Through a systematic exhaustive review of clinical neuroimaging literature, they mapped the complex set of domain-specific and domain-general impairments shown in the comorbidity of reading and mathematical disabilities to correspondingly plausible neuroanatomical substrates of dyslexia and dyscalculia. According to their hypothetical model, reading-math comorbidity seems due to atypical development of the left angular gyrus. This neuroeducational framework may assist to improve both early prediction and intervention across developmental periods.In a second prevention study, Malboeuf-Hurtubise et al. reported a pilot study based on a new intervention, which combines mindfulness meditation and Philosophy for Children (P4C) activities, with the goal of improving mental health in pre-kindergarten children. Thirty-eight pre-kindergarten Canadian children took part in this study and were randomly allocated to the experimental or wait-list control conditions. Teachers completed pre- and post-intervention questionnaires. Although there were no significant effects, some improvement trends were found for internalized symptoms and hyperactivity. The results partly contradict previous research and suggest that mindfulness and P4C may not be effective intervention for mental health in children. However, the study also suggests a host of other confounding variables that might be responsible for the null findings and should be addressed in future research.Finally, In summary, this Research Topic collection explored how children's preferences, profiles and predispositions are shaped by the social and biological activities that form the background of their everyday living. These papers addressed integrated multidisciplinary issues of education, development and public health, contributing practical examples of viable and sustainable local targeted programs, which, hopefully, may stimulate future research.AD'A wrote the first draft. All authors contributed equally to edit to final version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "GBA1, the gene encoding the enzyme glucocerebrosidase. Deficiency of glucocerebrosidase leads to the accumulation of the substrates glucocerebroside and glucosylsphingosine in macrophages and neuronal cells. Variants in GBA1 are a significant genetic risk factor for Define- Parkinson disease (PD) methods is a rare autosomal recessive lysosomal storage disorder caused by pathologic variants in GBA1 is located in a gene-rich region on chromosome 1q21 that encompasses seven genes and two pseudogenes within an 85-kb region , non-processed pseudogene, GBAP1 , encoding for part of the mitochondrial outer membrane import complex protein 1, which is transcribed convergently to GBA1 , which now includes over 600 different GBA1 variants with differing predicted clinical significance (https://www.centogene.com/centolsd.html). Many NGS protocols have utilized short-read sequencing, which can provide data at a relatively low cost and high accuracy but may be less reliable in cases where there are structural variants and repetitive or highly homologous regions (Mandelker et al., GBA1, Zampieri et al. initially aligned reads to the whole genome, but missed almost all recombinant alleles, which had aligned preferentially to GBAP1 (Zampieri et al., GBA1 rather than the whole genome, they were able to identify recombinant variants that had initially been false negatives, except for the 55-bp deletion mutation. Alignment against the whole genome did not significantly affect the mapping quality and coverage of missense mutations unrelated to recombination. Care should therefore be taken not only in the library preparation, but also in data analysis steps of NGS pipelines to sequence GBA1.Next-generation sequencing is able to generate vast amounts of sequence data and has been applied to identify variants in GBA1 variants using the Oxford Nanopore MinION (Leija-Salazar et al., NciI. Another study aimed to expand upon this protocol by refining and applying it to the discovery of GBA1 variants in a PD longitudinal cohort from New Zealand (Graham et al., GBA1 and updated the software pipeline, improving accuracy and reducing the computational workload, but were unable to detect any recombinant gene conversion alleles or deletions of >50 bp.The introduction of long-read sequencing technology in recent years addressed some limitations of short-read sequencing (Goodwin et al., GBA1 is complicated by complex gene-pseudogene rearrangements and recombination. Recombinant alleles make up a significant proportion of GBA1 mutations and sites of recombination events have been identified between intron 2 to exon 11 (Hruska et al., GBA1 and its pseudogene, there is an increased likelihood of both reciprocal and non-reciprocal recombination (Tayebi et al., NciI. In this allele, the site of crossover of chromosomal mispairing between gene and pseudogene occurs within intron 9 and continues to the 3'-UTR of the gene, introducing three exon 10 nucleotide mismatches (Latham et al., RecTL, covers the pseudogene sequence from intron 8 or the beginning of exon 9 [GBAP1 (Martinez-Arias et al., MTX and its pseudogene as well (Tayebi et al., GBA1 alleles (Velayati et al., Accurate and comprehensive NGS analysis of GBA1 gene rearrangements cannot be sufficiently captured using most current NGS methods. Several recent studies using NGS technology without Sanger sequencing validation have not reported the presence of recombinant alleles, including a recent study performed on more than 3,000 PD cases (den Heijer et al., The complexity of de novo or germline mutations on the maternal allele (Saranjam et al., GBA1 and S78L in MPZ (Benko et al., GBA1 mutation on the same allele (Hassan et al., GBA1 screening, and for now, Sanger sequencing should be used for the most accurate genotyping.Gaucher disease is an autosomal recessive disorder that usually results from the inheritance of a mutant allele from each parent. Increasing evidence suggests this may not always be the case, and that there are important exceptions to the traditional Mendelian pattern of inheritance, such as new mutations or uniparental disomy (Nakka et al., GBA1 in many Parkinson disease NGS analyses, it is important to consider the effects of the nearby homologous pseudogene. Recombinant alleles in GBA1 have been identified in patients with GD and with PD that might be missed when relying on NGS analysis alone without Sanger sequencing validation.Next-generation sequencing allows for unbiased simultaneous analysis of many genes. In addition, it makes it more feasible to analyze specific genes in larger cohorts for the study of common diseases like Parkinson disease. With the inclusion of GBA1 confirmed by Sanger sequencing. In addition, there is evidence to suggest that the choice of polymerase used could be a factor in the accuracy of NGS variant identification. A recent study performed a large-scale screening of GBA1 based on an NGS protocol and found a significant number of false negatives due to a polymerase-dependent allelic imbalance (den Heijer et al., Challenges exist in short-read NGS methods for sequencing highly homologous regions. The shorter reads cannot be mapped uniquely to the reference genome, especially in cases where there are recombinant alleles aligning to the homologous region. A computational custom scaffold-based approach was recently introduced to improve the detection and phasing of targeted complex variants using short reads (Zeng et al., GBA1 from their pseudogenes. An additional advantage is the ability to phase mutations and assign haplotypes. It can also detect intronic SNPs. There are still some limitations, including high costs, low throughput, and high per-base error rates. Long-read NGS also has a limited ability to accurately resolve homopolymers and to detect small insertions and deletions. Importantly, it is still unable to consistently detect recombinant alleles.Long-read NGS has many advantages, one of which is an improved ability to discriminate functional genes such as GBA1 variants and recombination remains Sanger sequencing. Without validation by Sanger sequencing, the frequencies of GBA1 variants based on NGS analysis may be underestimated, particularly for complex recombinant alleles. Real-time PCR can also be used to identify recombinant alleles (Velayati et al., GBA1 NGS analysis, it is still currently limited in its capacity to recognize recombinant alleles. The shortcomings identified will likely also be pertinent for the analysis of other genes with highly homologous pseudogenes. As sequencing technology continues to rapidly progress, we will likely continue to see improved detection of GBA1 variants. This has exciting potential for clinical diagnostics and studies of large patient cohorts.The most accurate method for detecting all EW: drafted the manuscript and figure. NT: organized the topic, reviewed the literature, and edited the manuscript. ES: conceived of the topic and edited the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Motoric Cognitive Risk (MCR) syndrome is a clinical syndrome combining slow walking speed and subjective cognitive complaints (SCC) that was first reported by Verghese et al. in 2013 [Recently, we reported that MCR is associated with anxio-depressive disorders and depression (ADDD) in a cross-section of a large population-based cohort known as \u201cThe Canadian Longitudinal Study on Aging\u201d . We demoThe risk of major neurocognitive disorders in individuals with MCR is more than twice that of those without MCR . FurtherIn addition to MCR, depression has been independently associated with an increased risk of major neurocognitive disorders . In indi"} +{"text": "Cancer research in the field of Computational Systems Biology attempts to address questions that will advance current knowledge in the mechanisms of cancer progression or treatment resistance. By analyzing multi-omics data and developing a predictive mathematical and/or computational model of an unknown biological system, we can systematically understand 1) the mechanisms that tie altered gene expression and downstream molecular mechanisms to functional cancer phenotypes ; 2) and/Artificial Intelligence (AI) Optimized Systems Modeling for the Deeper Understanding of Human Cancers\u201d in Frontiers in Bioengineering and Biotechnology, and Frontiers in Genetics aims to provide an international forum for:1) bringing together the greatest research efforts in cancer-specific molecular/network signature identification by integrating multi-omics/multi-level data;etc., to provide novel ideas and solutions in mathematical modeling for tumor growth, drug resistance, and targeting effect prediction;2) exploring future-generation interesting and practical biomedical applications in AI, machine learning, big data sciences, knowledge-based system, 3) addressing the real-world challenges in the fields of AI-based patient diagnosis or disease progression prediction by utilizing modern machine learning or statistical strategies, and produce a more reliable and promising application environment to develop those technologies.This special issue entitled \u201cetc. The final acceptance rate is 50% with 18 accepted papers in this special issue. The summaries of these papers are outlined below.1) New bioinformatic approaches to Identify key molecular/network signatures for precision diagnosis or treatment of cancersSubmission for this special issue started from May 2020 and closed in Oct 2020. In nearly one and half years, we received in total 36 paper submissions. All submitted manuscripts had gone through at least two rounds of revision with reviewers in the related fields, including bioinformatics, computational biology, machine learning, and clinical study, Identification of signatures of prognosis prediction for melanoma using a hypoxia score\u201d by Shou et al. The authors developed a computational method to identify the gene signatures of melanoma in hypoxic condition for prognosis prediction.In the article entitled \u201cIdentifying hypoxia characteristics to stratify prognosis and assess the tumor immune microenvironment in renal cell carcinoma\u201d by Zhang et al. The authors established a hypoxia-related risk model to predict the prognosis of patients.In the article entitled \u201cPrediction of Radiosensitivity in Head and Neck Squamous Cell Carcinoma Based on Multiple Omics Data\u201d by Liu et al. The authors identified 12-gene signature based on multiple omics data achieved the best ability for predicting radiosensitivity in Head and Neck Squamous Cell.In the article entitled \u201cAn Effective Graph Clustering Method to Identify Cancer Driver Modules\u201d by Zhang et al. The authors proposed a graph clustering method, called \u201cMCLCluster\u201d, to identity cancer driver modules that drive cancer progression.In the article entitled \u201cExploring the differential expression and prognostic significance of the COL11A1 gene in human colorectal carcinoma: an integrated bioinformatics approach\u201d by Patra et al. The authors developed an integrated bioinformatics approach to reveal the COL11A1 gene as a prognostic biomarker in colorectal carcinoma.In the article entitled \u201cMicroRNA-126 Modulates Palmitate-induced Migration in HUVECs by Downregulating Myosin Light Chain Kinase via the ERK/MAPK Pathway\u201d by Zhu et al. The authors evaluated the effects of miR-126 on the cell migration and uncovered the underlying mechanism in HUVECs treated with palmitate.In the article entitled \u201cIntegrated analysis of DEAD\u2010box helicase 56: a potential oncogene in osteosarcoma\u201d by Zhu et al. The authors set up a novel integrated analysis protocol and found that DDX56 is a potential therapeutic target for the treatment of osteosarcoma.In the article entitled \u201cA machine learning approach for tracing tumor original sites with gene expression profiles\u201d by Liang et al. The authors developed a machine learning approach by integrating random forest and Naive Bayesian, to predict the primary origin sites of tumors.In the article entitled \u201cA deep learning framework to predict tumor tissue-of-origin based on copy number variation\u201d by Liang et al. The authors proposed a deep learning framework composed of an autoencoder (AE) and a convolution neural network (CNN) to predict the primary origin sites of tumors.In the article entitled \u201cTOOme: a novel computational framework to infer cancer tissue-of-origin by integrating both gene mutation and expression\u201d by He et al. The authors integrated somatic mutation and gene expressions t infer the primary original sites of tumor and obtained a great accuracy.2) New studies of clinical informatics for speeding up the development of cancer diagnosisIn the article entitled \u201cDiagnosis of cervical cancer with parametrial invasion on whole-tumor dynamic contrast-enhanced magnetic resonance imaging combined with whole-lesion texture analysis based on T2-weighted images\u201d by Li et al. The authors integrated DCE-MRI images and texture analysis for diagnosis cervical cancer.In the article entitled \u201cPredictive value of the texture analysis of enhanced computed tomographic images for preoperative pancreatic carcinoma differentiation\u201d by Zhang et al. The authors extracted 396 features from patient CT images and selected the optimal feature subset to predict the pathological degree of differentiation of pancreatic carcinoma.In the article entitled \u201cRA-UNet: A hybrid deep attention-aware network to extract liver and tumor in CT scans\u201d by Jin et al. The authors developed a 3D network model, RA-UNet, to precisely extract the liver region and segment tumors from the liver. Testing on public datasets show that the proposed architecture obtains competitive results.In the article entitled \u201cClassification of Infected Necrotizing Pancreatitis for Surgery within or beyond Four Weeks Using Machine Learning\u201d by Lan et al. The authors applied machine learning models to predict the optimal timing of surgical intervention and identified the key factors associated with surgical timing for infected necrotizing pancreatitis.In the article entitled \u201cPrediction of Proximal Junctional Kyphosis after Posterior Scoliosis Surgery with Machine Learning in the Lenke 5 Adolescent Idiopathic Scoliosis Patient\u201d by Peng et al. The authors developed a machine learning model for proximal junctional kyphosis (PJK) prognostication in Lenke 5 adolescent idiopathic scoliosis (AIS) patients undergoing long posterior instrumentation and fusion surgery.In the article entitled \u201cA New Method Based on CEEMD Combined with Iterative Feature Reduction for Aided Diagnosis of Epileptic EEG\u201d by Peng et al. The authors proposed a computational method based on complementary ensemble empirical mode decomposition (CEEMD) combined with iterative feature reduction for aided diagnosis of epileptic EEG.3) New strategies for optimizing the data preprocessing and quality controlIn the article entitled \u201cAssessing the impact of data preprocessing on analyzing next generation sequencing data\u201d by He et al. The authors compared commonly used data preprocessing software and found differences in the detection of hotspot mutations and HLA typing. They also explained the impact of data preprocessing steps on downstream data analysis results.In the article entitled \u201cRF-PCA: A New Solution for Rapid Identification of Breast Cancer Categorical Data Based on Attribute Selection and Feature Extraction\u201d by Bian et al. The authors developed a hybrid model RF-PCA, which significantly reduce the time required for the classification, but also improved the accuracy.In the article entitled \u201cThe guest editors would like to thank all authors submitting their valuable works to this special section of Frontiers in Bioengineering and Biotechnology, Frontiers in Genetics, as well as all peer-reviewers for their great effort reviewing the submitted articles, providing constructive comments and suggestions and assisting the editors reaching the final decision. Special thanks will be sent to the editor-in-chief (EIC), Ranieri Cancedda and Jos\u00e9 AG Ag\u00fandez, for their precious time and valuable instructions that help us prepare and finalize this special issue."} +{"text": "They constitute important components in the human diet, acting as nutraceutical compounds with antioxidant, chemopreventive, antimitotic, neuroprotective, cardioprotective, and anti-inflammatory activities. Several phenylpropanoids are considered high-value biochemicals employed in the production of fragrances, pharmaceuticals and biopolymers functional characterization of genes involved in the phenylpropanoid metabolism and its coordination with physiological processes; and (4) biotechnological approaches to exploit the economic, medicinal, and nutraceutical potential of phenylpropanoids.This Research Topic aimed to gather recent findings in all aspects of phenylpropanoid metabolism gained by means of systems biology approaches and the utilization of biotechnology to exploit the economic, medicinal and nutraceutical potential of phenylpropanoids. The topic is organized into four sections: (1) structural, molecular and computational approaches toward unraveling the biosynthetic pathways involved in synthesis of diverse phenylpropanoid-derived metabolites; (2) discovery of genes and/or gene networks involved in distinct aspects of phenylpropanoid metabolism Delli-Ponti et al. (in this volume) have reviewed how gene expression and co-expression networks can be used as tools to uncover specialized metabolism biosynthetic pathways. Also using a computational approach, Elder et al. (in this volume) have applied density functional theory (DFT) calculations to evaluate the thermodynamics of coupling modes and subsequent rearomatization reactions between coniferyl alcohol and hydroxystilbene glucosides, which has been detected as a natural monomer in the bark lignin of Norway spruce.The chemical diversity of phenylpropanoids results from the modification and amplification of a set of core structures derived from the shikimate pathway. A vast array of regulatory proteins, biosynthetic enzymes, oxidases and other genes are recruited to produce the various classes of phenolic metabolites. Additionally, many phenylpropanoids are specific to just one or a few plant species, underscoring the complexity of phenylpropanoid biosynthesis and the need for comprehensive characterization studies in diverse species that expand our knowledge base beyond traditional model plant and crop species. Structural, molecular and computational approaches have been applied to identify genes, enzymes and metabolites involved in the biosynthesis of phenylpropanoids in different plants. Du et al. (in this volume). Lignin is a phenolic polymer important for plant growth and development but it is also considered a major bottleneck to the efficient conversion of plant biomass into downstream products. Rosado et al. (in this volume) have reported an in-depth characterization of the structural characteristics of lignins present in rice husks and straw, which are agricultural by-products that can be used to produce chemicals and materials in biorefineries. To identify the timing and key parameters of cell wall recalcitrance across different switchgrass genotypes, Saha et al. (in this volume) measured cell wall composition and phenylpropanoid/lignin biosynthesis gene expression in three switchgrass genotypes representing lowland and upland ecotypes. Yao et al. (in this volume) reviewed recent progress in defining the lignin biosynthetic pathway in lycophytes, monilophytes, gymnosperms, and angiosperms, and integrated new insights on major transcriptional regulators. In another study with evolutionary implications, Rencoret et al. (in this volume) structurally characterized the lignin-like fractions isolated from several ancestral plants, including those from moss, lycophyte, horsetail, fern, cycad, and gnetophyte species. Blaschek and Pesquet (in this volume) provided an overview of the differences and similarities in the structures, reaction mechanisms, substrate specificities, and functional roles between phenoloxidases. Because grasses are able to synthesize phenylpropanoids from either phenylalanine (Phe) or tyrosine (Tyr), Simpson et al. (in this volume) employed 13C isotopic-labeled precursors and mass spectrometry-based metabolomics to determine the downstream metabolites derived exclusively from Phe and Tyr in sorghum. Several phenylpropanoids show bioactivity that might influence plant growth and development or might be beneficial for human health. El Houari et al. (in this volume) reviewed reports describing altered accumulation of bioactive phenylpropanoids (or phenylpropanoid-derived metabolites) as the causal factor for observed phenotypes of lignin mutants in Arabidopsis. Cappellini et al. (in this volume) reviewed the recent progress in understanding the anthocyanin biosynthetic pathway in plants, with special emphasis on the differences in molecular mechanisms between monocot and dicot plants, and discuss the biological activities of anthocyanins as beneficial components of the human diet. Similar to anthocyanins, tannins form another group of phenolic compounds with beneficial effects on human health. Wang et al. (in this volume) performed a genome-wide analysis of the tannase gene family to identify candidate genes responsible for tannin metabolism in three nut tree species in the Juglandaceae family: walnut, pecan, and Chinese hickory.The biosynthesis of phenylpropanoids is often triggered by environmental stimuli. To this end, the effect of chilling treatment on the accumulation of phenylpropanoids and on antioxidant activity in seedlings of two rice varieties (contrasting for chilling tolerance) was studied by Tang et al. (in this volume) leveraged single-molecule real-time sequencing technology to elucidate flavonoid synthetic pathways in blueberries. Their transcriptome analyses led to the discovery of a R2R3 MYB transcription factor that can positively regulate anthocyanin synthesis in fruits. 5-aminolevulinic acid (ALA) is a plant growth regulator that induces fruit coloration and thereby finds potential applications in modern fruit production. A transcriptome study by Zheng et al. (this volume) identified the differentially expressed genes associated with ALA-induced anthocyanin accumulation in apple, including two R2R3-MYB transcription factors involved in flavonoid accumulation. A study by Ani\u010di\u0107 et al. (this volume) investigated flavonoid metabolism during fruit development in rockrose, a traditional medicinal plant rich in bioactive phenylpropanoids, using comparative metabolomic and transcriptomic approaches. This work highlights correlations between expression patterns of biosynthetic genes and the content of proanthocyanidins. Phenolic compounds are modulated by biotic and abiotic stresses, and a study by Laou\u00e9 et al. (in this volume) used quantitative trait locus (QTL) mapping and RNA-Seq to explore the complex polygenic network underlying the constitutive production of specific stilbenoids, flavonoids, and lignans in white spruce. Understanding the formation of secondary cell walls (SCWs) and their lignification has important agro-industrial applications. Hixson et al. (this volume), by undertaking an integrated analysis of the metabolome, transcriptome, and proteome of Arabidopsis lines mutated in arogenate dehydratase genes, exposed the involvement of novel proteins and additional post-transcriptional and translational processes that govern phenylpropanoid/lignin biosynthesis. As a proxy to study SCW formation in the bioenergy crop sugarcane, Sim\u00f5es et al. (this volume) established a lignifying cell culture system that they probed with transcriptomic and metabolomic analyses to illuminate the molecular mechanisms involved in this differentiation process, leading to the discovery of regulatory modules that control SCW deposition.The identification of genes and transcriptional networks responsible for specific accumulation patterns of phenylpropanoids during a physiological development process or a stress response is essential to elucidate and harness the fine regulatory mechanisms involved in these patterns. Recent advancements in omics technologies enable integrated approaches to unravel these mechanisms at the transcriptomic, proteomic, and metabolomic levels. These studies provide platforms to guide future research on improving crops for human health and wellness. Chen et al. (in this issue) identified two non-selective uridine diphosphate (UDP) glycosyltransferases (UGTs) from Isatis indigotica Fort. that catalyze the addition of a sugar molecule onto several structurally diverse lignin acceptor substrates. Shi et al. (in this issue) sought to explore the transcriptional regulatory mechanisms of anthocyanin and proanthocyanidin biosynthesis in Chinese bayberry, of which the fruit is considered an important dietary source of natural antioxidants. They identified a MrMYB6 gene that is highly upregulated during the latter stages of fruit development and determined it is a negative regulator of anthocyanin and proanthocyanidins through formation of a complex with two transcription factors, bHLH and WD40. Busche et al. (in this issue) carried out a study five 2-oxoglutarate-dependent dioxygenases involved in the formation of the flavonoid aglycon in banana (Musa): flavanone 3-hydroxylase, flavonol synthase and anthocyanidin synthase. Biochemical analysis of several recombinant candidate proteins showed that MusaF3H1 and MusaF3H2 act as flavanone 3-hydroxylases, MusaFLS1 and MusaFLS3 both function as flavonol synthases, and MusaANS has anthocyanidin synthase activity. Elucidating the activity of these genes will facilitate the development of bananas with higher nutritional value. Hydroxycinnamoyl acid amides, such as clovamide, are phenylpropanoid metabolites that play roles in protecting plants from biotic and abiotic stresses. Sullivan and Knollenberg (this issue) identified, cloned and biochemically characterized a hydroxycinnamoyl-CoA:L-DOPA hydroxycinnamoyl transferase (HDT) from red clover that is capable of synthesizing clovamide and related hydroxycinnamoyl amides in vitro. Characterization of this enzyme activity expands our knowledge of the poorly characterized family of BAHD hydroxycinnamoyl-CoA transferase enzymes and will aid in future studies aimed at understanding the molecular basis of substrate specificity within this important family.The phenylpropanoid pathway in plants plays a major role in the synthesis of a wide variety of secondary metabolites. Metabolites originating from this pathway are frequently involved in plant structure or chemical signaling and defense, including flavonoids, lignins, hydroxycinnamic esters, flavonoids, anthocyanins and tannins. Dietary flavonoids, anthocyanins, proanthocyanidins, hydroxycinnamoyl acid amides and lignans are bioactive compounds that have been shown to exhibit multiple health promoting and antioxidant activities. Lignans are plant secondary metabolites composed of a core scaffold that is formed by two or more phenylpropanoid units that can adopt a spectrum of different structural forms. Lin et al. (in this issue) provide direct evidence that two key enzymes involved in monolignol biosynthesis, 4-Coumaric acid:CoA ligase (4CL) and 4-hydroxycinnamoylCoA:shikimic acid hydroxycinnamoyl transferase, form a Ptr4CL-PtrHCT complex in Populus trichocarpa and its formation is a potential mechanism to modulate metabolic flux during secondary cell wall synthesis. The brown midrib (bmr) phenotype found across several C4 grasses has been critical for identifying mutants compromised in lignin synthesis. Tetreault et al. (in this issue) used a combined bulk segregant analysis (BSA) and next-generation sequencing (NGA) approach to show that bmr30 encodes a chalcone isomerase (CHI) and is involved in synthesis of the flavonoid tricin and not a monolignol. In Populus species, lignin can also be further modified by acylation with p-hydroxybenzoate. Zhao et al. (in this issue) used wild type (WT), lignin p-hydroxybenzoate deficient, and p-hydroxybenzoate overproduction plants to investigate the role of this modification in the response of plants to gravitropic/mechanical stress. They showed that lignin-bound p-hydroxybenzoate content increased during tension wood formation. This increase is correlated with a significant induction of expression of a gene encoding a BAHD family acyltransferase, namely, p-hydroxybenzoyl CoA: monolignol p-hydroxybenzoyltransferase 1 (PtrPHBMT1) whose gene product preferentially conjugates p-hydroxybenzoate to S-lignin monomer sinapyl alcohol.Lignin is a heterogeneous phenolic polymer that is highly abundant in the secondary cell walls of all land plants and is composed of three major monolignol subunits: 4-hydroxyphenyl (H), guaiacyl (G), and syringyl (S). The monolignol building blocks of lignin are synthesized by enzymes acting in concert that catalyze sequential reactions. Ferreira and Antunes (this volume) reviewed current progress on synthetic biology and highlighted the application of biosensors for re-engineering and autonomously controlling plant phenylpropanoid metabolism. Lam et al. (this volume) reviewed the understanding and bioengineering of the biosynthesis of tricin, a type of plant flavonoid that is an essential plant defense chemical and a promising nutraceutical. Sullivan et al. (this volume) established a de novo hydroxycinnamoyl-malate ester biosynthetic pathway in alfalfa via heterologous expression of a red clover gene and enhanced alfalfa post-harvest protein protection. A transcriptomic study of transgenic tomato plants by Zhao et al. (this volume) defined a GATA transcription factor mediating the co-regulation of drought stress response and phenylpropanoid biosynthesis. Genetic, biochemical and physiological studies from Lee et al. (this volume) found that Arabidopsis needs optimal anthocyanin content for better growth under high nitrate and high salt conditions. A study by Roldan et al. (this volume) using transgenic white clover with high levels of foliar condensed tannins discovered that condensed tannins bind to forage proteins to reduce anthropogenic greenhouse gas emission. Huber et al. (this volume) chemoenzymatically synthesized a series of new phenylpropanoid derivatives and studied their structures and biological effects. Using qualitative and quantitative phytochemical analyses, Gampe et al. (this volume) demonstrated that Ononis hairy root cultures produce isoflavonoids with less chemical divergence and in higher quantity, suggesting a promising system for large-scale isoflavonoid production.Plant phenylpropanoids and their derivatives are essential for plant growth, stress responses, and health benefits for humans. A comprehensive understanding of the biosynthetic mechanisms and transcriptional regulatory network(s) of phenylpropanoid metabolism in various plant species is central for developing biotechnological approaches to produce economically desirable traits and products. Additionally, advancements in synthetic biology and biosensor technology illuminate the potential of real-time control of phenylpropanoid metabolism in the future. Systems biology and biotechnology have largely contributed to enhance our understanding on the molecular mechanisms underlying the biosynthesis of phenylpropanoids in plants, as well as to manipulate the phenylpropanoid metabolism to exploit its economic, medicinal and nutraceutical potential. Articles in this volume further contribute to these goals, covering different aspects and branches of the pathway. Novel insights and exciting biotechnological strategies involving the phenylpropanoid pathway are expected in the years to come.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.IC is indebted to Funda\u00e7\u00e3o de Amparo \u00e0 Pesquisa do Estado de S\u00e3o Paulo (FAPESP) for the Research Grant No. 2019/25587-0 and to Conselho Nacional de Desenvolvimento Cient\u00edfico e Tecnol\u00f3gico (CNPq) for the research fellowship 302927/2018-2. MX is supported by the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research, as part of the Quantitative Plant Science Initiative at Brookhaven National Laboratory. BU is supported by the Center for Bioenergy Innovation (CBI), a U.S. Department of Energy Bioenergy Research Center supported by the Office of Biological, Environmental Research in the DOE Office of Science.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."} +{"text": "The encapsulation of seeds in hard coats and fruit walls (pericarp layers) fulfils protective and dispersal functions in many plant families. In angiosperms, packaging structures possess a remarkable range of different morphologies and functionalities, as illustrated by thermo and hygro\u2010responsive seed pods and appendages, as well as mechanically strong and water\u2010impermeable shells. Key to these different functionalities are characteristic structural arrangements and chemical modifications of the underlying sclerenchymatous tissues. Although many ecological aspects of hard seed encapsulation have been well documented, a detailed understanding of the relationship between tissue structure and function only recently started to emerge, especially in the context of environmentally driven fruit opening and seed dispersal (responsive encapsulations) and the outstanding durability of some seed coats and indehiscent fruits (static encapsulations). In this review, we focus on the tissue properties of these two systems, with particular consideration of water interactions, mechanical resistance, and force generation. Common principles, as well as unique adaptations, are discussed in different plant species. Understanding how plants integrate a broad range of functions and properties for seed protection during storage and dispersal plays a central role for seed conservation, population dynamics, and plant\u2010based material developments. In fruits and seeds, tissue hardening is often a result of lignification, but hard secondary cell walls may also consist mainly of polysaccharides. A good example are the hard, nonlignified endosperm cell walls in many palm seeds \u2013 for example, date palm (Phoenix dactylifera) or the tagua nut (Phytelephas macrocarpa) \u2013 which serve simultaneously as storage and protective tissues against physical, chemical, and biological damage Fig.\u00a0. In hardBertholletia excelsa; Sonego et al., Ibicella lutea; Horbens et al., Hamamelis mollis; Poppinga et al., Based on their performance, we classify sclerenchymatous encapsulations as either static, meaning dimensionally stable and indehiscent , Proteaceae and woody cones in the Pinaceae (e.g. Pinus spp.) and Cupressaceae (e.g. Sequoiadendron giganteum), which may all reduce seed loss or damage from granivores, desiccation, and fire die and typically dry together with the embyro(s) inside. After this point, seed viability depends strongly on the hydration level. Therefore, a major function of many hard encapsulation tissues is to retain a low moisture content inside the seed. A low seed moisture content may increase the desiccation tolerance of the embryo and induces dormancy in a large number of species Werker, . In addiet al., et al., et al., et al., et al., et al., et al., et al., et al., In static encapsulations, waterproofing may only be beneficial until conditions are suitable for germination. As germination requires water uptake by the embryo (imbibition), all covering layers play a central regulatory role in this step Fig.\u00a0. In addiet al. represents a unique adaptation for epizoochorous dispersal via trample\u2010burrs. The tissue is entirely fibrous for separation and are often simply realized by means of a locally reduced tissue and cell wall thickness, and/or lignification gaps . This i, et al. , known a, et al. and dry et al., . These p et al., , for exawns Fig.\u00a0 that alset al., et al., Aethionema arabicum), which consequently show different modes of dispersal and different levels of physical dormancy , plants develop follicles (responsive systems) with different valve curvatures along a climatic gradient, which results in higher levels of serotiny and higher opening temperatures in drier regions , four genes related to the flavonoid metabolism and seven peroxidases and thio/peroxiredoxins have been associated with differential dormancy along an aridity gradient have far\u2010reaching consequences for dispersal, protection, and germination. They may occur within the lifetime of an individual and eastern black walnut (J. nigra). In both species, the seed is surrounded by an ellipsoidal hard shell and partly separated by a septum. However, the septum in J.\u00a0regia is thin and mechanically insignificant, whereas in J.\u00a0nigra it is extremely thick and reinforces the shell internally . Interestingly, they also occur in many species of the palm family (Arecaceae); for example, in the Borneo giant fan palm , the Bismarck palm (Bismarckia nobilis), and in Eugeissona palms (Eugeissona spp.) . Shell geometry is particularly important to resist shell cracking by granivores via compression loading, for example. Owing to a higher rigidity, small spherical shells are more advantageous than elongated shells of similar size and thickness (Huss et al., et al., et al., et al., et al., Hard encapsulations for seed protection and dispersal are morphologically extremely diverse. On a general basis, the functionality of packaging structures arises from characteristic modifications of the tissues surrounding the embryo. Despite large genetic and ecological differences, many encapsulations share rather common features, such as predetermined breaking points, employment of fibres for force generation, and the incorporation of flavonoids for long\u2010term storage and protection. However, many questions still remain open. In the context of cell shapes, for example, it is unclear why 3D puzzle sclereids are rather rare in static structures despite their high strength (Huss"} +{"text": "Oxytocin is widely used for induction and augmentation of labour, particularly in low- and middle-income countries (LMICs). In this systematic review and meta-analysis, we examined the effect of intra-partum Oxytocin use on neonatal encephalopathy.The protocol for this study was registered with PROSPERO (ID: CRD42020165049). We searched Medline, Embase and Web of Science Core Collection databases for papers published between January 1970 and May 2021. We considered all studies involving term and near-term (\u226536\u2009weeks\u2019 gestation) primigravidae and multiparous women. We included all randomised, quasi-randomised clinical trials, retrospective studies and non-randomised prospective studies reporting intra-partum Oxytocin administration for induction and/or augmentation of labour. Our primary outcome was neonatal encephalopathy. Risk of bias was assessed in non-randomised studies using the Risk Of Bias In Non-randomised Studies of Interventions (ROBINS-I) tool. The RoB 2.0 tool was used for randomised studies. A Mantel-Haenszel statistical method and random effects analysis model were used for meta-analysis. Odds ratios were used to determine effect measure and reported with 95% confidence intervals.p\u00a0<\u20090.00001).We included data from seven studies of which 3 took place in high-income countries (HICs) and 4 in LMICs. The pooled data included a total of 24,208 women giving birth at or after 36\u2009weeks; 7642 had intra-partum Oxytocin for induction and/or augmentation of labour, and 16,566 did not receive intra-partum Oxytocin. Oxytocin use was associated with an increased prevalence of neonatal encephalopathy (Odds Ratio 2.19, 95% CI 1.58 to 3.04; Intra-partum Oxytocin may increase the risk of neonatal encephalopathy. Future clinical trials of uterotonics should include neonatal encephalopathy as a key outcome.The online version contains supplementary material available at 10.1186/s12884-021-04216-3. Clinical administration of exogenous Oxytocin is the most common way to induce and augment labour as it increases the frequency, duration and strength of uterine contractions . Recent Intra-partum Oxytocin administration has been shown to reduce the mean duration in labour in several clinical trials . This isImproper use and administration of high doses of Oxytocin has been found to precede perinatal sentinel events such as uterine rupture, cord prolapse or placental abruption as a result of uterine hyperstimulation, leading to fetal asphyxia . InapproThe protocol for this study was registered with PROSPERO (ID: CRD42020165049). Any amendment or modification to the original protocol during the course of the review was submitted to PROSPERO. The Cochrane handbook for systematic reviews of interventions was used to frame this review. One investigator (CB) searched the published literature between January 1970 to May 2021 on Medline (Ovid), Embase (Ovid) and Web of Science Core Collection databases to access relevant studies. The years were limited to 1970 to reflect current clinical practice as much as possible to allow the translation of findings. We searched for the concepts (1) encephalopathy/HIE, (2) oxytocin/uterotonic, expanded with risk factors and intrapartum injections/treatment/intervention/management/surveillance to find papers not explicitly mentioning oxytocin and (3) newborn infants/newborn infant diseases and obstetric, using controlled terms (i.e. MeSH-terms in MEDLINE) and words in title and abstract. , quasi RCTs, non-randomised prospective cohort studies and retrospective studies reporting intrapartum use of Oxytocin.Primigravidae and multiparous women giving birth at or after 36\u2009weeks\u2019 gestation.Intra-partum Oxytocin for induction (continuous and discontinuous) and augmentation of labour through intravenous and intramuscular routes at all doses. We excluded studies where Oxytocin was used after the delivery of the baby for prevention of post-partum bleed.Women who did not receive Oxytocin or any other uterotonic drug during labour.Babies with signs of neonatal encephalopathy following Oxytocin administration during labour. Neonatal encephalopathy was defined as the need for prolonged resuscitation at 5\u2009min of age and/or a 5-min Apgar score\u2009<\u20097 or lack of crying by 5\u2009min of age for babies born at home.2 index. An I2 value of size 0\u201340% was considered a low, 30\u201360% as moderate, 50\u201390% as substantial and 75\u2013100% as considerable heterogeneity according to the Cochrane Handbook [2 index. A Chi2 test was conducted to determine subgroup differences. Lastly, the overall effect was presented using a Z-test. All statistical analyses were performed on the RevMan V.5.1.4 software.Raw data was extracted from the selected studies on Excel spreadsheets by two independent investigators (CB/MM) and cross-checked by a third investigator (PM). Any disagreement during study selection or data extraction and analysis were resolved by consensus or by involving a senior reviewer when no consensus was reached. Risk of bias was assessed for each study by one investigator (CB) using the Risk Of Bias In Non-randomised Studies of Interventions (ROBINS-I) tool, which evaluates risk of bias in estimates of the effectiveness or safety (benefit or harm) of an intervention. The RoB 2.0 tool was used to assess the risk of bias of randomised studies. Grading of Recommendations, Assessment, Development and Evaluations (GRADE) was used to assess certainty in the body of evidence. A Mantel-Haenszel statistical method and random effects analysis model were used for meta-analysis. Odds ratios were used to determine effect measure and reported with 95% confidence intervals. Heterogeneity between studies was assessed to determine variability in the data using the IWe identified seven studies following full text screening, of which one had its data collected during a previous community-based cluster randomised controlled trial. These seven studies included neonatal encephalopathy as an outcome, of which 6 were case controls and 1 was a cluster randomised trial \u201317 , asphyxiated infants (Apgar score of <\u20096 at 1\u2009min) with no sign of encephalopathy (n\u00a0=\u20091 study) or term infants with a Thompson score\u2009<\u20093 (n\u00a0=\u20091 study). Data from the remaining study compared women with no injections during labour and women with injections during labour. Three of the included studies were undertaken in HICs, 4 in LMIC settings and all reported Oxytocin use and the prevalence of neonatal encephalopathy in newborns (Supplementary Table\u00a0Inclusion criteria for participants in selected studies were comparable: (1) a gestational age of at least 36\u2009weeks with 2) a classification of the baby into mild, moderate or severe neonatal encephalopathy according to Sarnat scoring and (3) administration of Oxytocin for induction and/or augmentation of labour. Studies compared neonatal encephalopathy infants with term infants with no sign of encephalopathy at birth . The Committee defines neonatal encephalopathy as a clinically defined syndrome of disturbed neurological function within the first week of life in term infants , manifested by difficulty initiating and maintaining respiration, depression of tone and reflexes, subnormal level of consciousness, and often, seizures. Upon further reading, we established that the PMMRC defines moderate and severe neonatal encephalopathy as Sarnat stages 2 and 3 [Two of the studies, however, did not use Sarnat scoring to determine neonatal encephalopathy severityis et al assessednn et al employedny et al considerFarquhar et al de 2 and 3 .Risk of bias was evaluated for 6 studies using the ROBINS-I tool, which is used in observational studies comparing health effects of interventions. The RoB 2.0 tool was used to assess for of bias of the cluster-randomised study. Selected studies ranged from low to severe risk of bias due to confounding domain and co-interventions, as these were not assessing specifically the effect of Oxytocin on neonatal outcomes [Ellis et al, Futrakul et al, Mullany et al, Tann et al) [Farquhar et al [, Hayes et al [Milsom et al [, Ellis et al [, Futrakul et al [, Mullany et al [Tann et al [Pooled data from HIC studies \u201313 revean et al) \u201317 reporar et al , Hayes ees et al and Milsis et al , Futrakuul et al , Mullanyny et al and Tannp\u00a0=\u20090.02). Furthermore, the subtotal difference in the onset of neonatal encephalopathy between the Oxytocin and the Oxytocin-free groups was significantly different in studies from LMICs . The overall effect combining results from both HICs and LMICs demonstrates a significant difference between the Oxytocin and Oxytocin-free group in the onset of neonatal encephalopathy . Subgroup differences analysis (I2) revealed that 72.2% of the variability in effect estimates from both groups is due to subgroup differences rather than sampling error was available for 158 infants in the Oxytocin group and 175 infants in the Oxytocin-free group in high-income settings. Pooled data from HIC studies showed a significant difference in the subtotal for the onset of neonatal encephalopathy between the Oxytocin and Oxytocin-free 175/600; 29%) groups . The heterogeneity amongst subgroups was of 44% for HIC studies and 51% for LMIC studies.The included studies had an overall substantial heterogeneity and LMICs that included intra-partum Oxytocin use in mothers of term infants with neonatal encephalopathy. Our main finding was an association between Oxytocin use and the onset of neonatal encephalopathy, which was significantly higher in term babies of women induced/augmented with Oxytocin compared to Oxytocin-free women . Moreover, this was found to be even higher in LMICs compared to HICs . A higher percentage of heterogeneity across LMIC studies may be linked to variability in social environment as well as settings where studies were taking place such as home births in rural areas of southern Nepal [We assessed a total of seven studies from HICs summary. Supplementary Table\u00a04. Risk of bias for Mullany et al., 2013 (RoB 2.0). Supplementary Table\u00a05. Summary of Findings table (GRADE)."} +{"text": "Fueled by advances in computing power, algorithms, and big data, the last decade has witnessed widespread applications of artificial intelligence (AI) in every major field, including medicine and healthcare. Generally speaking, AI is expected to help realize the promise of precision medicine in three major areas: (1) disease prevention, (2) personalized diagnosis, and (3) personalized treatment. In this Research Topic, \u201cArtificial Intelligence for Precision Medicine,\u201d we aim to set up an open stage in the community where breakthrough application examples of AI for precision medicine are presented. We envisage that AI technologies, if applied openly, fairly, robustly, and in close collaboration with human intelligence, will open new doors for effective and personalized healthcare worldwide.Hart et al.- AI-aided diagnosis and early detection of diseases: Chen et al.; Jensen et al.; Mistro et al.; Wang et al.- AI-enhanced treatment and delivery: Barua et al.- Clinical decision support with AI techniques: Luo- Enhancing patient care via AI applications: Zhang et al.- Radiomics and quantitative imaging: Kapelner et al.; Namdar et al.- Bioinformatics for more effective healthcare: Chan et al.- Innovative AI applications for patient safety: Hart et al. developed seven machine learning algorithms based solely on personal health data from the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial (PLCO), and compared them with 15 practicing physicians in stratifying endometrial cancer risk for 100 women. The results indicate that their random forest model achieves a testing AUC of 0.96, 2.5 times better at identifying above-average risk women with a 2-fold reduction in the false-positive rate. A novel concept named \u201cStatistical Biopsy\u201d was proposed for the first time.In their work, Chen et al. reported their development of a deep-learning convolutional neural network (DCNN) for enhanced organ-at-risk (OAR) segmentation on cone beam computed tomography (CBCT), trained with forty post-operative head and neck cancer patients. The developed DCNN improved CBCT in terms of Hounsfield unit (HU) accuracy, image contrast, and OAR delineation accuracy.Jensen et al. demonstrated that their novel machine learning model can be used to quickly estimate the Pareto set of feasible dose objectives in cancer radiotherapy, which may directly accelerate the treatment planning process and indirectly improve final plan quality by allowing more time for plan refinement. Their model outperforms the existing machine learning techniques by utilizing optimization priorities and output initialization.Using a cohort of 100 prostate cancer patients, Mistro et al. have demonstrated that knowledge models can be effectively used as teaching aid to bring inexperienced planners to a level close to experienced planners in fewer than 2 days. The proposed tutoring system can serve as an essential component in an AI ecosystem that will enable clinical practitioners to use knowledge-based planning effectively and confidently for personalized radiation treatment.As a first attempt, Wang et al. have demonstrated a novel deep learning framework for pancreas stereotactic body radiation therapy (SBRT) planning, which can predict a fluence map for each beam, hence bypassing the lengthy inverse optimization process.Based on 85 training cases and 15 test cases, Barua et al. demonstrated that a Multivariate Functional Principal Component Analysis (MFPCA) approach can be used to characterize the temporal trajectories of mandibular subvolumes receiving radiation. Their work suggests that temporal trajectories of radiomics features derived from sequential pre- and post-RT CT scans correlate with radiotherapy-induced mandibular injury, which may be used to aid in earlier management of osteoradionecrosis, a major side-effect in radiation therapy of oropharyngeal cancer patients.In their work, Luo summarized three major approaches currently employed in predicting cervical cancer outcomes: statistical models, medical images, and machine learning, and discussed some of the challenges in making clinical outcome prediction more accurate, reliable, and practical.In a mini-review, Zhang et al. proposed a transfer learning-based prognostication model for overall survival in pancreatic ductal adenocarcinoma patients. The model achieved the area under the receiver operating characteristic curve (AUC) of 0.81, significantly higher than that of the traditional radiomics model of 0.54. Their result suggests that transfer learning-based models may significantly improve prognostic performance in typical small sample size medical imaging studies.Kapelner et al. introduced and discussed a novel R package called \u201cPersonalized Treatment Evaluator (PTE)\u201d developed by them. They combined randomized comparative/controlled trial (RCT) data with a statistical model of the response to estimate outcomes under different treatment allocation protocols. Their PTE package can be used to evaluate personalization models in medicine as well as fields outside of medicine.To evaluate the overall effectiveness of personalized medicine, Namdar et al. presented first a comprehensive review of AUC metric, and then proposed a modified version of AUC that takes confidence of the model into account and incorporated AUC into Binary Cross Entropy (BCE) loss function. They demonstrated the validity of the new concept on MNIST, prostate MRI, and brain MRI datasets.In their paper, Chan et al. discussed and summarized the various applications of machine learning approaches in machine-specific and patient-specific quality assurance (QA), a key component in safeguarding patient safety during the radiation treatment of cancer patients.In a review paper, Precision medicine is an evolving healthcare approach focused on tailoring medical decisions, treatments, practices, and products to individual patients based on their genetic, environmental, lifestyle, and other factors. In this Research Topic, eleven teams reported promising results from their experience in applying AI for precision medicine. Moving forward, we anticipate that more work needs to be done to eliminate biases in the AI models and make these models interpretable, therefore ultimately achieving the promise of precision medicine, i.e., delivering the right treatment to the right patient at the right time.JD drafted the editorial. JD, TH, EC, JC, and FE-S revised and approved the final version. All authors contributed to the article and approved the submitted version.Research reported in this publication was supported by the National Institute of Biomedical Imaging and Bioengineering of the National Institutes of Health under Award Number R01EB022589, by the National Science Foundation under Award Number DMS 1918925, by the National Cancer Institute under Award Number 21X130F, and by the Department of Energy under Award Number DE-SC0021655 to JD. EC was supported by the grant NSF 19-500 under Award Number DMS 1918925 and 1922843 in years 2019-2022.The content is solely the responsibility of the authors and does not necessarily represent the official views of those institutions.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."} +{"text": "A study of natively iodinated bovine thyroglobulin demonstrates that structural details of biologically important chemical reactions can now be visualized by electron cryo-microscopy. Its circulation regulates metabolism and many other fundamental processes, including neuronal tissue growth (Caravalho & Dupuy, 2017Acta Cryst. D (Kim et al., 2021et al., 1989et al., 2020in vivo conditions and those made available under the in vitro conditions of iodination assays (Coscia et al., 2020et al. did not observe the presence of mono-iodinated tyrosine residues in the bovine TG structure. Similarly, biochemical assays performed by Coscia et al. did not reveal the presence of T3, the active form of thyroxine hormone. Hence, direct synthesis of T3 still needs to be demonstrated. The bovine TG structure exposes another interesting issue potentially related to the regulation of the redox potential of thyroid follicles. TG structures contain numerous cysteine residues, many of which are present in the cysteine-rich thyroglobulin type domains. In the human TG structure, all cysteine residues are linked by disulfide bonds. Due to the limiting resolution of density maps, this conclusion was often only supported by their proximity. In contrast, the bovine TG structure contains cysteine residues not involved in disulfide bridges despite their proximity, as described by Kim et al.In the cryo-EM structure of bovine TG from natural sources presented in this issue of et al., 1985et al., 1999et al., 1996et al., 2020et al. stated, the bovine TG material at first also appeared unstable on the grids. They hypothesized that the instability was caused by the inter\u00adactions at the solvent\u2013air interface and that a small addition of detergent already successfully applied in other studies (Noble et al., 2018et al., 2019et al. is a triumph of sample preparation and the maturation of electron cryo-microscopy. The structure demonstrates that structural details of bio\u00adlogically important chemical reactions can now be visualized by electron cryo-microscopy.Although TG has been extensively studied, its three-dimensional structure could not be determined for decades. Isolation of purified TG (Heidelberger & Palmer, 1933"} +{"text": "Inspired by how the human brain works, neurocomputing algorithms, including deep learning, reinforcement learning, and neurodynamic optimization, have achieved tremendous success in various applications across many domains, e.g., visual object tracking, speech recognition, human-level control, text understanding, and real-time optimization.Various types of intelligent equipment and hardware devices have been developed to implement neurocomputing models for engineering systems. Deep learning has been employed for industrial robotic applications, including stereo reconstruction, object pose recognition, and product quality check. With the advent of the Internet of Things and edge computing devices, deep reinforcement learning has become popular in the predictive maintenance of engineering equipment. Embedded convolutional neural networks are widely utilized for autonomous vehicle control. The success of applying neurocomputing approaches and related hardware implementations in different engineering domains, such as intelligent manufacturing, energy internet, and smart healthcare, has proven the potential of employing neurocomputing for solving real problems in various engineering fields.In recent years advances in sensor and data storage technologies have enabled the accumulation of a large amount of data from engineering systems. Driven by big data generated from engineering systems, neurocomputing, and its hardware implementation will continually transform engineering systems into more intelligent forms.This Research Topic aims to provide a forum for researchers to present the latest research on applications of neurocomputing algorithms and neurocomputing-based hardware in engineering systems. It brings together 14 high quality papers reporting on the latest applications of neurocomputing in transportation, manufacturing, biomedical engineering, electrical engineering, and knowledge management.Self-Triggered Consensus of Vehicle Platoon System With Time-Varying Topology,\u201d Wang et al. designed a secure event-triggered controller considering the safe distance for the vehicle platoon system. Based on the new event-triggered function, a more energy efficient self-triggered control strategy was developed by using the Taylor formula. The new self-triggered control strategy could avoid continuous calculation and measurement of vehicles.In the paper entitled \u201cIndustrial Control Malicious Traffic Anomaly Detection System Based on Deep Autoencoder\u201d by Wang et al. proposes a method of detecting abnormal traffic in industrial control networks based on autoencoder technology. The Kullback\u2013Leibler divergence was added to the loss function of the proposed model to improve the ability of feature extraction and the ability to recover raw data. The gas pipeline dataset was used to verify the performance of the proposed method.The paper on \u201cData-Driven Hybrid Equivalent Dynamic Modeling of Multiple Photovoltaic Power Stations Based on Ensemble Gated Recurrent Unit\u201d Long et al. report on a data-driven hybrid equivalent model for the dynamic response process of the multiple PV power stations. The data-driven hybrid equivalent model contained the simple equivalent model and data-driven error correction model. The simulation results validated the super-performance of the proposed model both in response speed and accuracy.In a contribution on \u201cSun et al. contribute a paper on \u201cAutoPath: Image-Specific Inference for 3D Segmentation,\u201d which introduces AutoPath, an image-specific inference approach for more efficient 3D segmentations. The proposed AutoPath dynamically selected enabled residual blocks regarding different input images during inference, thus effectively reducing total computation without degrading segmentation performance. Experimental results on a liver CT dataset showed that the proposed approach not only provided an efficient inference procedure but also attained satisfactory segmentation performance.Boosting Knowledge Base Automatically via Few-Shot Relation Classification\u201d by Pang et al. investigated a fully automatic method to train a relation classification model which facilitates to boost the knowledge base. In the proposed method, various multiple instance learning methods were incorporated into the classic prototypical networks, reducing sentence-level noises.A paper on \u201cGu et al.'s article \u201cInvestigating the Impact of the Missing Significant Objects in Scene Recognition Using Multivariate Pattern Analysis\u201d adopted multivariate pattern analysis to explore the object-scene association in scene recognition when different amounts of significant objects were masked. The analysis results suggested that the lateral occipital complex was sensitive to the loss of significant objects and mainly involved in scene recognition by the object-scene semantic association.Machine Learning Models to Predict Primary Sites of Metastatic Cervical Carcinoma From Unknown Primary,\u201d Lu et al. conducted a series of bioinformatics analyses based on a dataset from The Cancer Genome Atlas (TCGA) RNA-Seq data of squamous cancer and TCGA Pan-Cancer data. Three machine learning models, random forest, neural networks, and support vector machine, were developed to explore potentially effective tools to distinguish these squamous cancers.In the paper \u201cA Manufacturing-Oriented Intelligent Vision System Based on Deep Neural Network for Object Recognition and 6D Pose Estimation\u201d Liang et al. present a new two-stage intelligent vision system based on a deep neural network with RGB-D image inputs for object recognition and 6D pose estimation. A dense-connected network fusing multi-scale features was first built to segment the objects from the background. The 2D pixels and 3D points in cropped object regions were then fed into a pose estimation network to make object pose predictions based on the fusion of color and geometry features.In their contribution examining \u201cArtificial Intelligence-Based Application to Explore Inhibitors of Neurodegenerative Diseases\u201d by Deng et al. explores an integrated new approach for finding lead compounds that inhibit galectin-3, by combining universal artificial intelligence algorithms with traditional drug screening methods. Manifold artificial intelligence algorithms were performed to validate the docking results and further screen compounds.The paper titled \u201cEnergy Investment Risk Assessment for Nations Via Seq2seq Model\u201d Liang et al. propose a sequence to sequence model to evaluate the energy investment risk of 50 countries. Bi-long-short term memory was used as an encoder to process the historical statistics in the proposed method.In their \u201cA Relational Adaptive Neural Model for Joint Entity and Relation Extraction\u201d by Duan et al. describes a relational-adaptive entity relation joint extraction model based on multi-head self-attention and densely connected graph convolution network. In the model, the multi-head attention mechanism was specifically used to assign weights to multiple relation types among entities to ensure that the probability space of multiple relations was not mutually exclusive.The paper on \u201cClassification of Metastatic and Non-Metastatic Thoracic Lymph Nodes in Lung Cancer Patients Based on Dielectric Properties Using Adaptive Probabilistic Neural Networks\u201d by Lu et al. proposes a classifier to identify metastatic and non-metastatic thoracic lymph nodes in lung cancer patients based on dielectric properties. Compared with the other methods, the proposed classifier achieved a higher classification accuracy based on dielectric property data collected from lung cancer patients.An article on the \u201cXiang et al.'s contribution on the \u201cPrediction of Dangerous Driving Behavior Based on Vehicle Motion State and Passenger Feeling Using Cloud Model and Elman Neural Network\u201d presentes a new method for dangerous driving behavior prediction using a hybrid model consisting of cloud model and Elman neural network based on vehicle motion state estimation and passenger's subjective feeling scores.A New Way of Airline Traffic Prediction Based on GCN-LSTM\u201d by Yu employed graph convolutional neural network and long short memory network to construct an airline traffic prediction system with short-term prediction ability.The paper titled \u201cThese successful applications of neurocomputing in various domains demonstrate the significant potential of applying neurocomputing approaches to solving complex engineering problems. With the help of big data and increasing computing power, neurocomputing will play a vital role in future engineering systems.LW wrote the manuscript. ZZ, ZS, and CH edited the manuscript. All authors contributed to the article and approved the submitted version.This work was supported in part by the National Natural Science Foundation of China under Grant 62002016, in part by the Guangdong Basic and Applied Basic Research Foundation under Grants 2020A1515110431 and 2019A1515111165, and in part by the Fundamental Research Funds for the Central Universities and the Youth Teacher International Exchange & Growth Program under Grant QNXM20210037.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."} +{"text": "The 2021 Nobel Prize in Physiology or Medicine honors the discoveries of the temperature or mechanically activated channels, whose structural studies provided insights of channel gating at atomic level. Using capsaicin, a natural pungent agent that induces pain in our skin similar to that induced by heat, David Julius identified the TRPV1 ion channel as a heat-activated nociceptor in the peripheral nervous terminus Unexpectedly, the atomic structures of these channels were all determined by single-particle cryo-electron microscopy (cryo-EM), a structure determination technique that does not require crystallization but, instead, images individual biological molecules embedded in a thin layer of vitreous ice using an electron microscope followed by extensive computational image analysis (Cheng, 2018et al., 2013et al., 2013et al., 2016Interestingly, progress in structural studies of TRPV1 and the technological developments and advancements of single-particle cryo-EM were tightly entwined. With the methodological breakthroughs in single-particle cryo-EM, the first structure of TRPV1 was determined in 2013 (Cao etc. These exogenous stimuli generate pain that serve as a warning signal for avoidance. TRPV1 activation can also be modulated by endogenous stimuli, such as extracellular protons generated by tissue acidosis associated with inflammation. Potentiation of TRPV1 by extracellular protons causes heat hypersensitivity. Without the restriction of crystallization, single-particle cryo-EM not only enabled atomic structure determination of TRPV1 (see Fig. 1et al., 2013et al., 2016et al., 2021As a polymodal signal detector, TRPV1 can be activated by exogenous stimuli, such as heat, capsaicin, plant toxins and peptide toxins from spider et al., 2015et al., 2019et al., 2018As for Piezo1/2, soon after its initial discovery, structural biology had entered this new era of single-particle cryo-EM. As such, structural studies of Piezo channels were carried out exclusively using this method and comprehensive structural studies have progressed rather rapidly (Ge The major goals of structural biology go beyond knowing the atomic structures of these channels and what they look like, but address the harder questions of how they work to generate physiological responses. In this regard, there are still many unanswered questions concerning these channels. Among them, the mechanisms of temperature activation, such as TRPV1 by heat or TRPM8 by cold, and the mechanism of mechanosensing by Piezos, remain among the most pressing unanswered questions. Recognition of the Nobel Prize not only honors the seminal discoveries of these channels, but also stimulates excitement among structural biologists to continue pursuing these burning questions."} +{"text": "Sci., 2020, DOI: 10.1039/d0sc04044d.Correction for \u2018Direct synthesis of the organic and Ge free Al containing BOG zeolite (ITQ-47) and its application for transformation of biomass derived molecules\u2019 by Qintong Huang In the original manuscript, the affiliations of authors Anmin Zheng and Avelino Corma were displayed incorrectly. The corrected affiliations are as displayed above.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "Alzheimer's disease (AD) is a complex neurodegenerative disease, affecting a significant part of the population. The majority of AD cases occur in the elderly with a typical age of onset of the disease above 65 years. AD presents a major burden for the healthcare system and since population is rapidly aging, the burden of the disease will increase in the future. However, no effective drug treatment for a full-blown disease has been developed to date. The genetic background of AD is extensively studied; numerous genome-wide association studies (GWAS) identified significant genes associated with increased risk of AD development. This review summarizes more than 100 risk loci. Many of them may serve as biomarkers of AD progression, even in the preclinical stage of the disease. Furthermore, we used GWAS data to identify key pathways of AD pathogenesis: cellular processes, metabolic processes, biological regulation, localization, transport, regulation of cellular processes, and neurological system processes. Gene clustering into molecular pathways can provide background for identification of novel molecular targets and may support the development of tailored and personalized treatment of AD. Alzheimer's disease (AD) is a progressive neurodegenerative disorder, affecting the cerebral cortex and hippocampus in human brain , presenilin-1 (PSEN1), and presenilin-2 (PSEN2) are associated with early-onset AD (EOAD), developing in fourth or fifth decade of life can be observed already 15\u201320 years prior to the onset of the clinical symptoms and meta-analyses combining multiple GWAS dataset (n = 21) in AD risk evaluation were included (n = 16) were analyzed separately (n = 13) combined identified genotype alterations in GWAS and meta-analyses with changes in AD-related biomarkers enrichment analysis was performed, using Cytoscape plug-in ClueGO . This toIn our dataset of genes related to AD risk, we observed significant enrichment for four major GO biological process categories: cellular process, metabolic process, biological regulation, and localization .APP, PSEN1, and PSEN2 genes result in overproduction of the hydrophobic A\u03b240 and A\u03b242 peptides, leading to aggregation and formation of insoluble plaques family that includes PS1 and PS2 have increased risk for early development of memory impairment E4 allele (APOE4), which is associated with more extensive A\u03b2 deposition is considered a major risk factor for LOAD and rs7412 (p.Arg158Cys) define polymorphic alleles APOE2, APOE3, and APOE4 that encode three respective protein variants: apoE2 , apoE3 , and apoE4 , followed by APOE4 (5\u201335%) and APOE2 allele (1\u20135%) gene were associated with AD. PICALM rs3851179 was associated with decreased AD risk rs744373 SNP was associated with risk for LOAD was associated with AD family were linked to AD. Rs11136000 was associated with decreased risk for AD or increased (rs9331888) risk for AD were associated with AD and type 2 diabetes (T2D), indicating potential shared molecular pathways between the diseases /proton (H+) exchanger in the Golgi, important in maintenance of homeostasis cluster encodes a family of cell surface proteins that participate in the regulation of calcium signaling and was shown to considerably increase AD risk , important for tau phosphorylation and NFT formation in CNS . Rs1466662 within this DCHS2 was associated with AD is a gene, encoding for poliovirus receptor 2, immunoglobulin expressed in neuronal cell tissues, that is important in T-cell activation were identified as novel AD related risk alleles forms one of the primary pores via which proteins can readily enter the mitochondria. The TOMM40 gene is the only gene identified in genetic studies to date that presumably contributes to LOAD-related mitochondria dysfunction locus, contributing to the diverse and specific Ig forming in the adaptive immunity and blood plasma biomarker levels can predict neurodegenerative changes in AD progression and memory decline and are often used in clinical diagnostics. Except for their diagnostic potential, biomarkers can be applied in studies of AD molecular mechanisms and could be used to monitor the biochemical effects of potential disease intervention linked to molecular pathways identified in this review were associated with CSF A\u03b2 and tau levels , important mediator of immune system, were associated with CSF A\u03b2 and tau levels , were associated with LOAD protein expression in CSF [271]. Furthermore, association of rs2228145 with CSF and serum IL6R levels revealed the effect on age of onset in AD [171]. GLIS3 rs514716 association with CSF A\u03b2 and tau levels was observed were associated with CSF levels of ACE gene product and APOE-containing lipoproteins that were associated with CSF A\u03b2 levels in AD subjects gene, is important enhancer of immune response encodes ATIP3, inhibitor of extracellular signal-regulated kinase 2 (ERK2) and cell proliferation. Similarly, rs3092960 within CCR2 encoding for CCL2 receptor was also associated with significant levels of CCR2 protein in CSF were associated with CCRL2 protein expression in CSF as well were associated with biomarker levels, with rs573521 being the best predictor of MMP3 protein expression in CSF in LOAD as a novel AD-related locus (Zhong et al., BCAM was associated with CSF levels of phosphorylated tau181 and A\u03b242 (Huang et al., BCAM is a gene encoding Lutheran blood group glycoprotein, an immunoglobulin important in laminin recognition (Parsons et al., ARHGAP24 rs111882035 was associated with memory tests outcomes in MCI individuals (Chung et al., ARHGAP24 is important in actin cytoskeleton remodeling and specifically suppresses Rac1 and Cdc42 activity (Lavelin and Geiger, Although not enriched in GO analysis, many other genes were manually annotated cellular processes and are also summarized in Genes and key SNPs, involved in biological regulation, associated with biomarkers in GWAS and their meta-analyses, are summarized in CD1A polymorphisms: rs16840041, rs2269714, and rs2269715 were associated with increased plasma neurofilament light level, a potential protein biomarker for AD (Wang et al., Three ATP6V1H rs1481950 was associated with higher CSF BACE activity (Hu et al., Additionally, one biomarker associated gene was manually annotated to biological regulation . A polymGenes and key SNPs, involved in localization, associated with biomarkers in GWAS and their meta-analyses, are summarized in GRIN2B rs10845840 was reported as a risk loci for AD, associated with temporal lobe atrophy (Stein et al., GRIN2B encodes the NR2B subunit of NMDA receptor that mediates a Ca2+ dependent synaptic transmission in the CNS (Hu et al., MAPT rs242557 was associated with plasma tau levels (Chen et al., MAPT encodes for tau, the prominent component of NFTs. H2 haplotype is associated with MAPT expression and LOAD risk (Allen et al., BCHE rs509208 with PET imaging of cortical A\u03b2 in AD subjects was revealed (Ramanan et al., Genes and key SNPs, involved in neurological system process, GO term specific for biomarker gene set, are summarized in Among all AD related biomarker loci, obtained from GWAS and meta-analyses that were not enriched in GO analysis, additional 18 were manually annotated.The remaining seven genes could not be associated with any of the seven main enriched pathways, even though they were linked biomarker changes in AD GWAS and meta-analysis. Although some of them were linked to a specific function in the literature, they have not been annotated with any of the GO terms. Genes and key SNPs with no known function associated with AD biomarkers in GWAS and meta-analyses are summarized in CCDC134 rs7364180 was associated with CSF A\u03b2 levels in AD subjects (Kim et al., CCDC134 is a proliferation promoting molecule, driving cytokine-like activation of CD8+ T-cells (Huang et al., ECRG4 rs34487851 was observed (Chung et al., ECRG4 encodes a peptide hormone that is involved in NFT formation, age-related senescence of precursor cells in the CNS and activation of microglia and peripheral mononuclear leukocytes (Kujuro et al., FRA10AC1 as a novel risk locus (Li et al., FRA10AC1 gene are potential cause of folate-sensitive fragile site FRA10A expression (Sarafidou et al., LUZP2 was also proposed as a novel AD risk locus as rs7943454 was associated with higher plasma neurofilament light levels (Li et al., LUZP2 gene (Stepanov et al., ZNF804B, is not known yet, a ZNF804B rs73705514 was associated with memory tests outcomes in MCI individuals (Chung et al., Alzheimer's disease is the most prevalent neurodegenerative disorder worldwide. A lot of research focuses on the identification of genetic factors that may contribute to the development and progression of the disease. Numerous GWASs and meta-analyses reported different genetic factors associated with AD risk or biomarker levels. A cumulative effect of small but significant contributions of numerous genetic factors can at least in part elucidate the LOAD progression. The pathogenic processes in AD may be influenced on a personalized basis by a combination of variants in key genes and pathways. Apart from serving as a hallmark of the disease, polymorphisms in various genes might help in early diagnostics and prediction of disease progression. Integration of genetic factors and biomarker status may increase the predictive value of diagnostic or prognostic models.Through the GO analysis we compiled a list of the most enriched pathways, associated with AD pathology. Among four GO parental categories in AD risk gene set, immune response, APP metabolism, cholesterol metabolism, endocytosis and biological regulation on different levels can be exposed as important AD related biological processes. Furthermore, enrichment analysis on smaller AD biomarker gene set pinpointed three additional parental categories. Besides neurodegeneration, numerous research evidence link AD with neuroinflammation, lipid metabolism as well as receptor mediated endocytosis, supporting scientific background of our analysis. Several identified genes were associated with more than one biologic process, represented in various GO categories. The intersection of different biological processes creates a complex interconnected network, suggesting multi-pathway approach in AD genetic background evaluation is needed. Additionally, manual annotation of genes that were not associated with the most significant pathways in GO analysis, could help to elucidate their function in AD pathogenesis.This comprehensive summary of genetic variants identified by GWAS studies and their meta-analyses can also provide background for identification of novel molecular targets, and the results may be important for development of personalized medicine. However, GWAS and meta-analyses cannot explain the molecular mechanisms of the contribution of a novel susceptibility locus to the overall genetic risk. Therefore, our compiled and annotated results may serve as a basis for the functional studies of pathophysiological mechanisms of risk genes, identified on a genome-wide scale. Furthermore, better characterization of risk genes functions could enable the stratification of AD patients according to the main molecular mechanisms of pathogenesis, supporting development of tailored and personalized treatment of the disease.The original contributions presented in the study are included in the article/DV performed literature search and gene enrichment analysis. DV, KG, and VD participated in writing and editing of manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "The development of bioactive biomaterials is an important component in the general field of tissue engineering, specifically for the success of tissue repair and regeneration approaches . BioactiEngineered bioactive materials involve conventional biomaterials, such as bioactive glasses and ceramics, as well as biopolymers based on proteins. Polysaccharides and biomoleculecontaining biomaterials . In receSprio et al. reviewed the application of biomorphic transformations to obtain nanostructured 3-D bioceramics. Yanmei Tang et al. reviewed the advances of polydopamine nanoparticles in tissue engineering applications, including the repair of bone, cartilage, skin, heart, and nerve. Fujian Zhao et al. reported tantalum-gelatin methacryloyl-bioactive glass (Ta-GelMA-BG) scaffolds which could enhance osteointegration at the early stage of implantation. Haiping Lu et al. developed Ag and MSCs-derived exosomes-contained PCL scaffold for regulating immune cells and MSCs proliferation and differentiation. Haiping Lu et al. introduced the broad application of \u03b2-TCP in tissue engineering and discussed different approaches to enhance and customize \u03b2-TCP scaffolds, including physical modification.This Research Topic is the second part on the \u201cMultifunctional Bioactive Nanomaterials for Tissue Regeneration\u201d series, which includes several papers demonstrating the main advances of multifunctional nanomaterials in tissue engineering. In this topic, Multifunctional Bioactive Nanomaterials for Tissue Regeneration Part 2\u201d will contribute to inspire future developments of advanced bioactive nanomaterials for regenerative medicine to close the gap between research and clinical applications.The editors hope that the current topic \u201c"} +{"text": "In last few decades, organic materials including polymers have received much attention for their potential applications in electronics, because they have outstanding advantages such as high processibility, mechanical flexibility, and low weight. Extensive research efforts have thus been devoted to the development and advancement of organic materials for various applications, covering a wide range from molecular design to device-fabrication methods. In addition, it has been recognized that surfaces and interfaces play a crucial role in the operation and performance of the devices. For instance, various interactions at organic\u2013metal interfaces are of great importance in organic epitaxy, and also have a strong correlation with intermolecular structures and their electronic properties.In this context, the main focus of this Special Issue was collecting scientific contributions addressing surface and interface engineering with organic materials, and related applications. The diversity of contributions presented in this Special Issue exhibits the potential of organic materials in a variety of applications that are not limited to the fabrication of organic devices. This Special Issue contains eight featured original research papers as regards nanoarchitecture based on organic elements , physicaIn the contributed article entitled \u201cInvestigation on the Adsorption-Interaction Mechanism of Pb(II) at Surface of Silk Fibroin Protein-Derived Hybrid Nanoflower Adsorbent\u201d , Xiang LByung Soo Hwang et al. provided an outlook for the development of stimuli-responsive functional materials through their study of gelation behaviors of hydrogels in the contributed article entitled \u201cThermogelling Behaviors of Aqueous Poly(N-Isopropylacrylamide-co-2-Hydroxyethyl Methacrylate) Microgel\u2013Silica Nano-particle Composite Dispersions\u201d [Peng Xiao et al. presented a facile method for the fabrication of liquid metal electrodes using polydimethylsiloxane, and also demonstrated solar-blind photodetection via surface exfoliation in a series of contributed articles entitled \u201cFabrication of a Flexible Photodetector Based on a Liquid Eutectic Gallium Indium\u201d , and \u201cFlIn the contributed article entitled \u201cCrack-Assisted Charge Injection into Solvent-Free Liquid Organic Semiconductors via Local Electric Field Enhancement\u201d , Ju-HyunIn the contributed article entitled \u201cCesium Doping for Performance Improvement of Lead(II)-acetate-Based Perovskite Solar Cells\u201d , Min-SeoHyeok Jo Jeong et al. reported the sigmoidal concentration dependence of electrical conductivity of poly:poly(styrene sulfonate) processed with linear glycol-based additives in the contributed article entitled \u201cSigmoidal Dependence of Electrical Conductivity of Thin PEDOT:PSS Films on Concentration of Linear Glycols as a Processing Additive\u201d . It was In the contributed article entitled \u201cThe Methods and Experiments of Shape Measurement for Off-Axis Conic Aspheric Surface\u201d , Shijie Materials. I am personally grateful to Ms. Freda Zhang, Managing Editor of this Special Issue.In conclusion, the contributed articles in this Special Issue demonstrate relevant progress and the potential of organic materials in a variety of applications. I wish to thank and acknowledge all the authors for their priceless contributions and the editorial members of"} +{"text": "The bladder exstrophy\u2013epispadias complex (BEEC) is an abdominal midline malformation comprising a spectrum of congenital genitourinary abnormalities of the abdominal wall, pelvis, urinary tract, genitalia, anus, and spine. The vast majority of BEEC cases are classified as non-syndromic and the etiology of this malformation is still unknown. This review presents the current knowledge on this multifactorial disorder, including phenotypic and anatomical characterization, epidemiology, proposed developmental mechanisms, existing animal models, and implicated genetic and environmental components. Congenital anomalies of the lower urinary tract (CALUT) are a group of birth defects of the ureter, bladder, and urethra, which includes bladder exstrophy\u2013epispadias complex . BEEC is an abdominal midline malformation comprising a spectrum of congenital genitourinary abnormalities of the abdominal wall, pelvis, urinary tract, genitalia, anus, and spine . The sevEpispadias is rare, with incidences of one in 101,000 live births in males and one in 1,300,000 in females ,8. CBE hEpispadias is generally diagnosed at birth, although its presentation is dependent on severity and sex. It consists of a dorsal located ectopic urethral meatus as a result of non-closure of the urethral plate during embryological development . In bothCBE presents as a protrusion of the urinary bladder through a defect on the infraumbilical abdomen, in association with a diastasis of the pubic symphysis with distally divergent rectus abdominis muscles A 3]. Pu. Pu3]. PCE is a major birth defect in which the bladder is widely open on the infraumbilical abdominal wall and is separated into two distinct halves. It is often associated with omphalocele, separated pubic bones, short-gut syndrome, and other malformations, including talipes and spina bifida B 1,11].,11.1,11]The vast majority of BEEC cases are non-syndromic, however, a number of cases have been reported whereby BEEC has also been associated with various other syndromes, malformations, and congenital diseases . There aThe majority of individuals affected by BEEC have no positive family history of BEEC. However, even though familial occurrence is rare, 30 multiplex families have been described ,19,20,21n = 28) and triplets (n = 2), including monozygotic (n = 20), dizygotic (n = 3), trizygotic (n = 2), and unknown zygosity (n = 5). Of the CE anomalies within the 20 monozygotic twins, 9 were concordant and 11 were discordant. The higher incidence of CE in monozygotic twins compared to dizygotic twins could suggest a possible genetic contribution to the occurrence of these anomalies. Fullerton et al., 2017 [Reutter et al., 2007 reportedl., 2017 reportedThere is an extensive history of successful disease gene discovery made through the characterization of individuals with chromosomal abnormalities. These chromosomal changes provided a shortcut to identify relevant chromosomal loci for positional cloning approaches before next generation sequencing techniques transformed disease gene discovery over the past decade. Translocations disrupting disease-associated genes and deletions harboring the causative gene led to some of the earliest disease gene identifications in the last century.Cytogenetic analyses have identified a number of chromosomal anomalies in individuals with BEEC, summarized in Multiple autosomal chromosome anomalies have been reported in association with BEEC. Zaki et al., 2012 identifiBoyadjiev et al., 2005 identifiEl-Hattab et al., 2010 reportedThauvin-Robinet et al., 2004 reportedA study undertaken by Harrison et al., 2014 of 17 feDraaken et al., 2010 performeFrom a cohort of 244 BEEC cases, Lundin et al., 2010 reportedA comparison of eight previously reported 22q11.21 duplications in individuals with CBE revealed a 414 kb \u201cphenocritical\u201d region, encompassing 10 candidate protein coding genes . Within In contrast to the potential high penetrance effect of chromosomal changes, candidate and genome wide association studies (GWAS) investigate the contribution of more common variants, conferring a smaller effect size. Both association study approaches seek to genotype large cohorts of individuals with BEEC and compare allele frequencies of SNPs in these against ethnically matched controls.p = 0.026, odds ratio (OR) = 18.33) whereas a four-base-pair insertion was associated with an increased risk in non-Caucasian patients OR = 4.58. Using luciferase assays, they showed a consistent statistically significant reduction in transcriptional efficiencies of the promotor sequences containing indel polymorphisms, suggesting that indel polymorphism of the deltaNp63 promoter leads to a reduction in p63 expression which could potentially lead to BEEC.To date, only a small number of candidate gene association studies have been undertaken, the first of those by Wilkins et al., 2012 to invesReutter et al., 2014 conductep = 2.22 \u00d7 10\u22128). Meta-analysis of rs6874700 from this study and the previous study by Draaken et al., 2015 [p = 9.2 \u00d7 10\u221219). Analysis of ISL1-expressing cells by a lineage tracer mouse model showed ISL1-expressing cells in mouse model in the urinary tract E10.5 and distributed in the bladder E15.5. In zebrafish larvae, staged 48 HPF ISL1 expression was detected in a small region of the developing pronephros. This association together with functional studies in mouse embryos and zebrafish larvae suggest ISL1 as an important susceptibility gene for CBE and as a regulator of urinary tract development.Draaken et al., 2015 performeThe lack of large multiplex families has made traditional positional cloning approaches to disease gene discovery incredibly challenging in BEEC. Ludwig et al., 2009 conducteThe introduction of exome and genome sequencing technologies and large sequence variant databases in healthy controls have opened opportunities to determine the effects of rare variants that may be enriched in individuals with BEEC. Reutter et al., 2016 were theThe application of array-based, GWAS, and next generation sequencing techniques in large BEEC cohorts has helped to identify putative disease-causing genes and chromosomal regions in the human genome for both Mendelian and multifactorial BEEC. Functional analysis of embryonic pathways provides a better understanding of the molecular biological mechanisms underlying normal, urorectal, and genitourinary malformations within the embryology of the human urogenital system.It is reasonable to propose that both inherited and de novo highly penetrant variants could be relevant to the etiology of BEEC as they have been shown for many genetically heterogeneous congenital birth defects such as congenital heart disease.New approaches such as gene and pathway enrichment analyses of high-impact de novo variants from whole exome or whole genome data in parent-offspring trios will likely aid in the identification of novel genes and/or pathways to better understand the underlying genetic mechanisms of BEEC, and the potential to use these data to develop therapeutic approaches to help children affected by this devastating congenital disorder."} +{"text": "Mental health problems early in life can negatively impact educational attainment, which in turn have negative long-term effects on health, social and economic opportunities. Our aims were to: (i) estimate the impacts of different types of psychiatric conditions on educational outcomes and (ii) to estimate the proportion of adverse educational outcomes which can be attributed to psychiatric conditions.N = 2511) were from a school-based community cohort of Brazilian children and adolescents aged 6\u201314 years enriched for high family risk of psychiatric conditions. We examined the impact of fear- , distress- and externalising-related conditions on grade repetition, dropout, age-grade distortion, literacy performance and bullying perpetration, 3 years later. Psychiatric conditions were ascertained by psychiatrists, using the Development and Well-Being Behaviour Assessment. Propensity score and inverse probability weighting were used to adjust for potential confounders, including comorbidity, and sample attrition. We calculated the population attributable risk percentages to estimate the proportion of adverse educational outcomes in the population which could be attributed to psychiatric conditions. Analyses were conducted separately for males and females.Participants (p < 0.05) and grade repetition , respectively. Externalising conditions were associated with grade repetition in males and females , as well as age-grade distortion in males and females . Externalising conditions were also associated with lower literacy levels and bullying perpetration in females. If all externalising conditions were prevented or treated, we estimate that 5.0 and 4.8% of grade repetition would not have occurred in females and males, respectively, as well as 10.2 and 5.3% of age-grade distortion cases and 11.4% of female bullying perpetration.Fear and distress conditions in males were associated with school dropout (odds ratio (OR) = 2.76; 95% confidence interval (CI) = 1.06, 7.22; The study provides evidence of the negative impact of psychiatric conditions on educational outcomes in a large Brazilian cohort. Externalising conditions had the broadest and most robust negative impacts on education and these were particularly harmful to females which are likely to limit future socio-economic opportunities. The global prevalence of these conditions in young people is around 13.4% on a range of educational indicators beyond attainment in young males and females separately, while implementing robust adjustment for confounders and a random subsample based on all eligible children (n\u00a0=\u00a0958). These families (n\u00a0=\u00a02511) were selected for full household assessment by lay interviewers (parent interview) and trained psychologists (child interview) at baseline (6\u201314 years) and at follow-up . Participation was associated with higher maternal education, socioeconomic group (SEG), living in Porto Alegre, and anxiety-related conditions at baseline and 3-year follow-up (2013\u20132014) data from the Brazilian High-Risk Cohort Study for Psychiatric Conditions (BHRCS), a large school-based community cohort enriched for high family risk for psychiatric conditions , University of S\u00e3o Paulo and Federal University of Rio Grande do Sul ethics committees. Written informed consent was obtained from parents and participants that were able to read, write and clearly understand the written consent.et al., et al., Current psychiatric conditions were assessed at baseline using parent-report on the Brazilian\u2013Portuguese version of the Development and Well-being Assessment (DAWBA) were excluded due to divergent literature as to which broad group they might belong.Based on previous literature . Age-grade distortion was calculated for participants reporting grade repetition, dropout or expulsion since baseline, but still enrolled in education. For these participants, age-grade distortion was measured in years and calculated by subtracting the participant's current age from the expected age range for the current school grade. For example, if a child was 12\u201313 years and enrolled in 5th grade (expected age 10\u201311 years), we estimated an age-grade distortion of 2 years. For subjects not currently enrolled in school but reporting grade repetition or dropout since baseline, to be conservative, we attributed the value of 1 year.et al., Reading and writing literacy was measured via the School Performance Test (\u2018Teste de Desempenho Escolar\u2019 \u2013 TDE) ; intelligence (IQ); study site and the dummy comorbidity variable described above.SEG was assessed using the classification from the \u2018Associa\u00e7\u00e3o Brasileira de Empresas de Pesquisa\u2019 (Brazilian Association of Research Companies) ABEP, . ClassifIQ was assessed by trained psychologists with the vocabulary and block design subtests of the Wechsler Intelligence Scale for Children, 3rd edition \u2013 WISC-III .We estimated the impact of baseline psychiatric conditions on educational outcomes at 3-year follow-up using unadjusted and adjusted regression models and using schools as the primary sampling units. Maternal education, any anxiety condition and study site predicted response at follow-up in the present sample , based on weighted predicted probabilities from regression models. \u2018Punaf\u2019 generates estimates based on two scenarios: first, using predicted probabilities from the model and second, if all participants did not have a psychiatric condition. The ratio of predicted prevalence estimates from the two scenarios was then used to calculate PARPs. As we aimed to generalise to the Brazilian population, we included an additional sampling weight for PARP calculations. This sampling weight fully accounted for the family liability oversampling (see sample description). The methods and results can be found in the Supplemental material in Martel et al. due to psychiatric condition category. Estimated PARPs can be interpreted as a percentage of adverse educational outcomes in the total population attributable to psychiatric conditions . The PARP was computed using l et al. . This alFor educational outcomes, we used data from the National Institute of Educational Studies and Research database to estimate the number of Brazilians to which PARPs would translate. Details of estimates are described in online Supplementary material.Potential confounders were adequately balanced (standardised mean difference <0.2) between weighted groups following application of PSWs .Among males, fear-related conditions were associated with higher odds of school dropout, and distress-related conditions were associated with higher odds of grade repetition . ExternaComorbidity between fear-, distress- and externalising-related condition categories was included as a covariate in regression models and associated estimates are presented in online Supplementary Tables S6a to S8b. Females with a comorbid condition in addition to fear-related or distress-related condition had higher odds for school dropout and age-grade distortion, respectively (online Supplementary Tables S5a and S6a). Males with fear-related or distress-related conditions who met criteria for at least one other condition category had higher odds of bullying perpetration and low literacy levels respectively (online Supplementary Tables S5b and S6b).Applying school participation estimates for 2014 in Brazil, described in online Supplementary material (p. 3), we estimate that for the Brazilian population, 154\u00a0000 cases of grade repetition , 591\u00a0000 cases of age-grade distortions and 236\u00a0000 cases of bullying perpetration would have been avoided if externalising conditions were prevented or treated. As hypothesised, some differences according to gender were found, et al., et al., et al., Psychiatric conditions can have multiple short- and long-term impacts on individuals and society were more likely to experience school dropout and grade repetition, respectively. This further increases the gender disparity already experienced by males who have lower educational attainment than females OECD, . Previouet al., et al., et al., et al., To contextualise the population impact of psychiatric conditions, we calculated PARPs for each educational indicator. Previous research has estimated that up to 10.2% of early school termination could be attributed to psychiatric conditions (Vander Stoep et al., et al., et al., As a practical application of our estimates, we consider the case of ADHD in Brazil. Among those with ADHD, it is estimated that only 20% receive medication treatment (Mattos et al., et al., This study has some limitations. First, cohort participants came from two large urban areas and thus findings are more generalisable to urban areas, where more than 80% of the Brazilian population lives OECD, . TherefoIn conclusion, the current study estimated the impact of psychiatric conditions on educational outcomes, including examining different condition categories with several widely used educational indicators. The United Nation Sustainable Development Goals recognise education as \u2018one of the most powerful vehicles for sustainable development\u2019. Our findings provide support that treatment and prevention of psychiatric conditions could prevent around 4.9% of grade repetition and 7.7% of age-grade distortion in males and females, and 11.4% of bullying perpetration behaviour and low literacy performance in females with prior externalising-related conditions. Thus, policies which encourage early intervention and collaboration between education and health sectors to support the mental health of schoolchildren can have profound consequences for lifetime opportunities and socioeconomic well-being."} +{"text": "Retinal vein occlusion (RVO) is a differential diagnosis for Coats\u2019 disease due to retinal arterial Leber\u2019s aneurysms. Occasionally, RVO shows a Coats-like appearance. The differential diagnosis between Coats\u2019 disease and RVO is essential for clinical therapy, especially for those obsolete RVOs with collateral vessels and without retinal hemorrhage. In this case report, we describe and discuss the imaging characteristics of bilateral RVO-simulated Coats\u2019 disease with tortuous retinal arterioles and its prognosis after anti-vascular endothelial growth factor therapy, which will be beneficial for its definite diagnosis and aid further investigation. Coats\u2019 disease was first reported in 1908 as an ocular disorder with retinal telangiectasis and massive intraretinal and subretinal exudation . RetinalA 52-year-old woman was presented to the ophthalmology department of First Hospital of China Medical University complaining of blurred vision in her left eye for two months. She had amedical history of hypertension and no diabetes. The best-corrected visual acuity was 60/60 in her right eye and 15/60 in her left, and the intraocular pressure was 17mmHg in both eyes. Slight cataracts were found in both eyes, and other anterior segment examinations were normal. Wide-field fundus imaging showed bilateral tortuous retinal arterioles with retinal hemorrhage, as well as tortuous and dilated retinal veins in the supratemporal quadrant. Heavy hard exudates were found in the macula of the left eye. The middle phase of fluorescein angiography revealed numerous miliary aneurysms in both eyes and patchy, blocked fluorescence in the left eye. Peripheral fluorescein angiography demonstrated the \u201cfishing net\u201d appearance of retinal capillaries and non-perfusion regions in both eyes . Multico4 and Schatz et al [Herein, we described a bilateral branch RVO with a Coats-like appearance and tortuous retinal arterioles. Hypertension may be a risk factorfor RVO. In contrast to reports by Scimecaetaltz et al , no secotz et al , which iFamilial retinal arteriolar tortuosity is characterized by tortuosity of the second- and third-order retinal arterioles . Khan etIn conclusion, this was the first report of bilateral RVO-simulated Coats\u2019 disease with tortuous retinal arterioles. A definitive diagnosis is crucial for further investigation and treatment."} +{"text": "Proteins as molecular machines have dynamic structures sampling various conformational states, which determine their functionality, ligand binding, and allosteric properties. Allosteric communication as an intrinsic property of proteins simulations, elastic network models (ENM), hybrid and integrated methods, and protein structure networks (PSN).Khan et al., performed an integrated computational study on the estrogen receptor alfa (ER\u03b1), which have been observed to be recurrent in metastatic breast cancer patients. The impact of experimentally-reported ER\u03b1 polymorphisms was studied using techniques such as mCSM stability and binding affinity analysis has become a promising therapeutic approach in cancer immunotherapy. In Liu et al.'s work, the binding features of PD-1 with Nivolumab, a humanized IgG4 antibody approved by the US FDA, were investigated using MD simulations. The computational analysis suggested that the N-terminal loop of PD-1 serves as an important gatekeeper for the anti-PD-1 antibody binding, which might be a potential target for anti-PD-1 antibody design.MD simulation is the most popular computational method used in complementing experimental techniques as it captures the behavior of proteins in full atomistic detail for understanding binding and allosteric events complex was generated as a potential therapeutic target for inflammatory bowel disease. Eight \u201chot spots\u201d residues and six potential binding sites at the OSM-OSMR interface were predicted using computational alanine scanning and FTMap , and identified potential key binding sites. Besides, some possible tunnel pathways of the inhibitors in these CYP51-inhibitor complexes were proposed. Using MD simulations, Du et al. gave a detailed analysis of thermostability factors of barley limit dextrinase, including eight salt-bridges.In the brief research report by Dudas et al. used the hybrid MDeNM enzyme and its allosteric properties. In particular, large fluctuations of the N-terminal domain and its allosteric role were discussed in the context of infectious disease treatment.Normal mode analysis based on ENM facilitates the study of protein dynamics and allosteric effects in a high-throughput manner as the case study. Based on available experimental structures conformationally most variable region of HA was identified as a potential target for diverse ligands. Furthermore, the empirical contact potentials including an ENM-based entropy term were found successful in ranking the free energies of peptide/proteins designed against HA.Halder et al., gave a thorough presentation on the network-dynamical systemic approach to protein function. In addition, their work highlighted the advantages of the side-chain network analysis in studying subtle conformational changes with an emphasis related to allostery. Gosu et al., used PSN to study the MD ensemble of Myeloid differentiating factor 88 (Myd88) and AlloSigMA that could serve as a potential binding site. Yan et al. designed the ANCA webserver for constructing and analyzing PSN for interpretation of functional residues and allosteric regulation.Topological description of protein structures has become a popular tool to quantify protein structures and dynamics is a technique that measures the thermodynamics of binding reactions and reaction kinetics. The review by All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "While vaccines traditionally have been designed and used for protection against infection or disease caused by one specific pathogen, there are known off-target effects from vaccines that can impact infection from unrelated pathogens. The best-known non-specific effects from an unrelated or heterologous vaccine are from the use of the Bacillus Calmette-Gu\u00e9rin (BCG) vaccine, mediated partly through trained immunity. Other vaccines have similar heterologous effects. This review covers molecular mechanisms behind the heterologous effects, and the potential use of heterologous vaccination in the current COVID-19 pandemic. We then discuss novel pandemic response strategies based on rapidly deployed, widespread heterologous vaccination to boost population-level immunity for initial, partial protection against infection and/or clinical disease, while specific vaccines are developed. Since the introduction of the smallpox vaccine in 1796, and the Bacillus Calmette-Gu\u00e9rin (BCG) vaccine in 1921, vaccination-related reductions in non-specific morbidity and mortality have been described. Carl N\u00e4slund was the first to establish the concept of non-specific immunological vaccine effects, also known as heterologous effects of vaccination (HEV), when BCG vaccination started in Sweden (Aaby and Benn Measles vaccination (MV) can also have non-specific effects. After the introduction of MV in Guinea-Bissau, there was a significant decrease in childhood mortality . Since its introduction in South America, reports revealed an interference with the replication of other enteroviruses, resulting in fewer diarrheal deaths , but the primary data in fact only supports the conclusion of increasing risk with decreasing number of DPT innoculations.Until recently, the lack of studies evaluating the underlying mechanisms of HEV has been a major obstacle in recognizing and taking advantage of these effects during a pandemic. In this mini-review, we review the immunological mechanisms and the development of the idea of Heterologous Vaccine Intervention (HVI), referring to the exploitation of heterologous or non-specific effects of current approved vaccines against one or more unrelated pathogens. This novel pandemic \u201cearly response\u201d intervention, that could be rolled out in parallel with non-pharmaceutical interventions (NPIs) could be used for the current COVID-19 pandemic and for future pandemics to come.+ monocytes and imprints a persistent transcriptomic myeloid bias on human hematopoietic stem and progenitor cell compartment in the bone marrow after a primary exposure, such as an infection or vaccination, that leads to an altered response towards a second exposure after the return to a non-activated immune state, is termed \u2018trained immunity\u2019(Netea et al. Other innate immune cells that can contribute to protective HEVs include NK cells by increased cytokine production (Rozot et al. Less well studied mechanisms in BCG and other live vaccines are from viral interference (Sepp\u00e4l\u00e4 et al. In addition to the effect of a heterologous vaccine on innate immune mechanisms, vaccines can also alter adaptive immune responses to unrelated pathogens and antigens. Two different mechanisms have been described: cross-reactivity and bystander activation. The classical adaptive immune response corresponds to T cells that respond to antigen presentation, but these T cells may cross-react with a different antigen with amino acid similarity (Frankild et al. Toxoplasma gondii in adults are stimulated through bystander activation, upon immunization with tetanus toxoid (Goodridge et al. An illustrative example of cross-reactivity resulting from molecular mimicry is the existence of memory T cells specific to viral antigens in unexposed adults, such as herpes simplex virus, cytomegalovirus and HIV-1, possibly as a consequence of cross-reactivity with antigens in the environment. Furthermore, the seasonal influenza vaccine can stimulate T cells specific for a cross-reactive bacterial homologue (Su et al. The durability of the heterologous effects after vaccination is unknown, although vaccinia and BCG vaccinations are associated with better long-term survival during a 40-year follow up (Rieckmannet al. Several ecological studies have suggested a negative association between different vaccines and the prevalence or mortality of COVID-19. Escobar et al. attempted to mitigate potential confounding factors and reported a strong correlation between their BCG index and COVID-19 mortality in different socially similar European countries, specifying that every 10% increase in the BCG index was associated with a 10.4% reduction in COVID-19 mortality (Escobar et al. An inverse association between influenza vaccination coverage rate in the elderly, at a county or region level in the US (Zanettini et al. Using the quadrivalent inactivated influenza vaccine applied in the Netherlands in the 2019\u20132020 influenza season, Debisarun et al. demonstrated the induction of trained immunity response in an in vitro model, including improvement of cytokine responses, after stimulation of human immune cells with SARS-CoV-2 (Debisarun et al. The concept of Heterologous Vaccine Intervention is being developed with BCG vaccination, and several randomized trials are underway to study if BCG vaccination reduces the incidence or severity of COVID-19 in different countries (NIH U.S. National Library of Medicine Although specific COVID-19 vaccines have been released and given emergency use approval, they are still in limited supply and the slow pace of COVID-19 vaccination is a global challenge. Public health policies against pandemics may derive benefit from the concept of HVI, authorizing the early use of non-specific vaccines either generally as a boost, second dose after the COVID-19 vaccine prime (Hupert et al."} +{"text": "Focused electron beam (FEB) and focused ion beam (FIB) technologies have opened novel paths for material science research and technology at the micro and nano scales in recent decades. These technologies are highly adaptable, and flexible tools are used successfully in fundamental and applied research projects allowing visualization, elemental and structural analysis, and additive and subtractive manufacturing, as well as efficient modification of materials. All of this can be done on scales ranging from hundreds of \u03bcm to one nanometer. The unique flexibility of focused beams enables them to be used for rapid prototyping as well as nanostructure editing and analysis. Today, these technologies play an important role in the development of new integrated circuits based on miniaturized electronic devices.Concerning the additive nanofabrication, the use of gas injection systems with advanced programs allow to grow in single-step nanostructures in three dimensions by the partial decomposition of a precursor material produced by the effect of the beam scanning. This technique is called focused ion/electron beam-induced deposition (FIBID/FEBID). Nanostructures grown using this additive manufacturing technique can have arbitrary shapes, both in-plane and out-of-plane , and also can host insulating, metallic, ferromagnetic, or superconducting properties.+ FIBID deposits with in situ heating , nan nan+ FIBs [+ FIB ,8.1) Purification of FEBID nanostructures: Markus Rohdenburg et al. [ Purifica2) High-resolution nanostructures using FEBID: Sangeetha Hari et al. [ High-res3) A new approach for the nanofabrication of metallic nanostructures based on FEB: Luisa Berger et al. [ A new ap4) Tuning of diameters in 3D FEBID nanostructures: Lukas Seewald et al. [ Tuning o5) Evolution of the microstructure and resistivity in Pt FIBID deposits with in situ heating: Chaorong Zhong et al. [ Evolutio+ FIB-induced processing for photonic applications: Mariachiara Manoccio et al. [6) Nanofabrication using Ga Nanofabr+ FIB: Alex Belianinov et al. [7) Fabrication of 3D FIBID nanostructures using He Fabricatv et al. have repI would like to thank all authors who submitted their papers to this Special Issue. I would also like to thank all the reviewers for dedicating their time to provide careful and timely reviews to ensure the quality of this Special Issue."} +{"text": "Tinel et\u00a0al.; Aschauer et\u00a0al.; Wu et\u00a0al.; Okamura et\u00a0al.) and three comprehensive Review articles on immunogenomics of SOT and HSCT that include both animal and human studies.Alloreactivity is caused by the extensive difference in polymorphic genes between allogeneic donor and recipient, primarily in the major histocompatibility complex (MHC). As key mediators of alloresponses, alloreactive T cells are educated by self MHC and thus acquire the ability to recognize non-self MHC, leading to graft rejection and graft-versus-host-disease (GVHD), in solid organ transplantation (SOT) and hematopoietic stem cell transplantation (HSCT), respectively . ThroughShi et\u00a0al. provides an insightful review of genomic-based approaches to the study of transplant rejection, including microarray, RNA sequencing (RNA-seq), and spatial transcriptomic techniques. This review traces the developmental history of these approaches and extends it to the current fast-paced field of emerging technologies, which includes the integration of single cell RNA-seq with T cell receptor (TCR) and B cell receptor (BCR) sequencing to profile immune repertoires, with mass cytometry and featured barcode antibodies to measure protein expression, and with chromatin sequencing to explore gene regulatory networks. The authors discuss the advantages and limitations of each approach. Application of these analysis tools will contribute to a fundamental understanding of the alloresponse and likely promote novel therapeutic options to overcome rejection and GVHD.Tinel et\u00a0al. performed miRNA and mRNA profiling of kidney allograft biopsies to reveal new pathways involved in microvascular Inflammation (MVI), the main histological injury associated with AMR. This study identifies six differentially-expressed miRNAs that are correlated with the intensity of MVI. mRNAs/miRNAs interplay analysis further elucidates the crosstalk between renal-resident and allograft-infiltrating cell subsets and suggests that epithelial, rather than endothelial, metabolism modifications occur during AMR. This study illustrates the great potential of multi-omics to decipher rejection mechanisms.To investigate the role of microRNAs (miRNAs) in antibody-mediated rejection (AMR) after SOT, Tian et\u00a0al. reviews recent advances in TCR-seq and computational tools and discusses the potential of using TCR-seq to profile alloreactive T cell repertoires. Defining and tracing donor- and recipient-reactive TCRs may reveal the fingerprint of alloreactive T cells, providing valuable indication of graft rejection, acceptance and treatment response after SOT.TCR repertoire diversity and turnover dynamics are related to rejection and tolerance in SOT . The preAschauer et\u00a0al. analyzes donor-reactive TCRs in pre-transplant blood and post-transplant kidney biopsies in the presence and absence of TCMR to find little repertoire overlap in these two sites. While circulating donor-reactive repertoire is increased in both groups, donor-reactive T cells in kidney are only enriched during a TCMR episode with substantially diverse TCRs, indicating the capability to respond against a variety of epitopes in the allograft.Frequencies of circulating donor-reactive T cells were elevated in kidney transplant patients receiving conventional immunosuppression , 7. HoweFu et\u00a0al. further discusses the use of TCR-seq to discern the factors behind human T cell repertoire development and how this approach can be used in combination with human immune system mouse models to understand human repertoire selection. The article explains current understanding of the propensity of alloreactive TCRs as a consequence of thymic selection. It also notes the limitations of techniques historically used to study human TCR repertoires and provides descriptions of innovative tools the authors are utilizing TCR diversity is narrowed in allogeneic T cells; 2) top dominant T cell clones are highly shared across circulation and GVHD target organs in allogeneic recipients; 3) clonal expansion of rare rearrangements from pre-transplant donor T cells may account for the sharing of a few clones among allogeneic recipients. Their findings illustrate immune repertoire sequencing-based methods as a novel personalized strategy to guide diagnosis and therapy in GVHD.T cells are considered as not only the main culprit behind TCMR after SOT, but also the driving force of GVHD after allogeneic HSCT . RecoverOkamura et\u00a0al. reports that high levels of complement factor Ba a week after HSCT predict the occurrence of TA-TMA and related non-relapse mortality. This finding, once confirmed in a broader cohort, will lay the groundwork for highly-tailored and complement-targeted therapeutics. C5 blockade\u2014which already revolutionized outcomes of atypical hemolytic uremic syndrome is also a life-threatening medical condition. Although pathogenic variants in complement regulatory genes have been reported as genetic susceptibility to TA-TMA in only a minority of patients , 20, grosyndrome , anothersyndrome .This Research Topic of articles provides an in-depth review of current understanding of alloresponses after transplantation\u00a0in preclinical and clinical settings from the immunogenomics perspective and encourages future investigations in this field.YW, JZ, and JF collected articles and wrote the editorial review. All authors contributed to the review and approved the submitted version.YW was supported by R01 CA258440 from NIH/NCI (PI: Xue-Zhong Yu). JZ was supported by the Fondation Emmanuel Boussard, IMAGINE Institute, DIM-Th\u00e9rapie G\u00e9nique/R\u00e9gion Ile-de-France, Agence de la Biom\u00e9decine, and Agence Nationale de la Recherche . JF was supported by a Congressionally Directed Medical Research Program (CDMRP) Discovery Award W81XWH-20-1-0159 funded by the Department of Defense (DoD), a R21 grant AI166069 supported by NIH/NIAID and Nelson Faculty Development Awards UR011630-01 and UR011630-02 from the Nelson Family Transplantation Innovation Award Program at Columbia University Irving Medical Center.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."} +{"text": "Bipolar disorder is associated with premature mortality, but evidence is mostly derived from Western countries. There has been no research evaluating shortened lifespan in bipolar disorder using life-years lost (LYLs), which is a recently developed mortality metric taking into account illness onset for life expectancy estimation. The current study aimed to examine the extent of premature mortality in bipolar disorder patients relative to the general population in Hong Kong (HK) in terms of standardised mortality ratio (SMR) and excess LYLs, and changes of mortality rate over time.This population-based cohort study investigated excess mortality in 12 556 bipolar disorder patients between 2008 and 2018, by estimating all-cause and cause-specific SMRs, and LYLs. Trends in annual SMRs over the 11-year study period were assessed. Study data were retrieved from a territory-wide medical-record database of HK public healthcare services.Patients had higher all-cause [SMR: 2.60 (95% CI: 2.45\u20132.76)], natural-cause [SMR: 1.90 (95% CI: 1.76\u20132.05)] and unnatural-cause [SMR: 8.63 (95% CI: 7.34\u201310.03)] mortality rates than the general population. Respiratory diseases, cardiovascular diseases and cancers accounted for the majority of deaths. Men and women with bipolar disorder had 6.78 (95% CI: 6.00\u20137.84) years and 7.35 (95% CI: 6.75\u20138.06) years of excess LYLs, respectively. The overall mortality gap remained similar over time, albeit slightly improved in men with bipolar disorder.Bipolar disorder is associated with increased premature mortality and substantially reduced lifespan in a predominantly Chinese population, with excess deaths mainly attributed to natural causes. Persistent mortality gap underscores an urgent need for targeted interventions to improve physical health of patients with bipolar disorder. Specifically, we adopted two mortality metrics, namely SMR and LYLs to quantify the magnitude of excess mortality among patients with bipolar disorder compared with the general population. Changes in SMRs across the study period were also assessed to clarify whether the mortality gap improved or worsened over time.et al., et al., et al., et al., Population statistics and information on all registered deaths in HK between 2008 and 2018 were obtained from the Census and Statistics Department. Data of the patient cohort were extracted from the Clinical Data Analysis and Reporting System , and aged \u2a7e15 years during the study period as the study population. Diagnosis of bipolar disorder was recorded and verified by the International Classification of Diseases, 10th revision (ICD10 codes: F30 and F31). Final diagnostic ascertainment took into consideration the longitudinal illness course (Chang Causes of death were classified according to ICD10 codes (online Supplementary Table S1), and were divided into natural and unnatural causes. Natural causes were categorised into infectious and parasitic diseases (A00\u2013B99), neoplasms (C00\u2013D48), cardiovascular diseases (I00\u2013I99), respiratory diseases (J00\u2013J99), digestive diseases (K00\u2013K93) and genitourinary diseases (N00\u2013N99). An array of specific natural causes was also identified for analyses. As data on specific unnatural causes (V01\u2013Y98) were not available, we treated unnatural deaths as a single category for analyses.P exact tests. To assess trends in all-cause, natural-cause and unnatural-cause SMRs over time, joinpoint regression analysis was performed which estimated the optimal number of linear slopes and joinpoints with modified Bayesian information criteria for all-cause and cause-specific deaths were calculated as the primary mortality measures to quantify the relative mortality rate between patients with bipolar disorder and the general population. First, number of observed deaths and person-years of follow-up were computed for each calendar year (2008\u20132018), sex and age category for the patient group. The number of person-years for each stratum was multiplied by the corresponding mortality rate in the general population to produce expected number of deaths, indirectly standardising overall mortality ratio by age, sex and calendar year. SMRs were estimated by dividing the observed number of deaths by the expected number of deaths. Crude mortality rates (CMRs) per 100\u00a0000 person-years as well as SMRs for all-cause and cause-specific deaths of the patient group over the whole study period, stratified by sex and four broader age groups , were then calculated, with 95% confidence intervals (CIs) of SMRs being derived by mid-et al., et al., et al., et al., et al., et al., lillies for all-cause mortality were also calculated as a complementary mortality measure. Following the method adopted in previous research for LYL estimation with 106\u00a0147 persons-years of follow-up , and a total of 1042 deaths, of which 821 (78.8%) had a known cause. The numbers of person-years and cause-specific death counts for each demographic subgroup of patients are listed in online Supplementary Table S2.All-cause SMR for bipolar disorder was significantly increased in the total sample and in each demographic subgroup relative to the general population and 2. ASMRs for natural and unnatural causes were significantly increased in the total sample and in each demographic subgroup of patients with bipolar disorder, except the youngest age-group who showed no significant increase in natural-cause SMR and 2. BNatural causes accounted for most of the known-cause deaths in patients. Approximately two-thirds of all known-cause deaths were attributed to respiratory diseases, cancers and cardiovascular diseases (online Supplementary Table S2). Respiratory diseases represented the leading cause of death and accounted for around one-thirds of all known-cause deaths. Cancers and cardiovascular diseases contributed to about 1 in 5 and 1 in 7 known-cause deaths, respectively. Unnatural causes accounted for approximately one-fifths of known-cause deaths. Men had generally higher CMRs for most listed causes of death than women . MortaliAs shown in Joinpoint regression fitted a linear model of all-cause, natural-cause and unnatural-cause SMRs with no joinpoints over time for the total sample, men and women subgroups. As shown in To our knowledge, this is the first study using LYLs complementary with relative mortality risk measure (SMR) to evaluate the excess mortality associated with bipolar disorder. Our results showed that patients with bipolar disorder had 2.6-fold increased mortality risk compared with the general population. Both men and women with bipolar disorder exhibited a substantially shorter lifespan than the general population, with approximately 7 years of excess LYLs. Natural causes, specifically respiratory diseases, cardiovascular diseases and cancers, accounted for the majority of known-cause deaths, while unnatural cause had markedly elevated SMR, particularly among patients aged <35 years. Generally, the mortality gap persisted over 11 years, albeit slightly improved in men with bipolar disorder.et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., Our finding on all-cause SMR for bipolar disorder is broadly consistent with the literature which reported 2\u20133 times greater overall mortality risk than the general population (Chang et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., Until now, very few studies have comprehensively examined cause-specific SMRs for natural deaths in bipolar disorder. Consistent with the literature (Crump et al., et al., et al., et al., Patients with bipolar disorder had approximately 8 times greater risk of dying from unnatural causes than the general population. This finding is concordant with the unnatural-cause summary SMR of 7.42 reported by a recent meta-analysis (Hayes et al., et al., et al., et al., et al., Although our analysis suggested small, albeit statistically significant, reduction in annual all-cause mortality rate over time in men with bipolar disorder, the overall mortality gap remained largely unchanged over 11 years. Both natural-cause and unnatural-cause mortality differences also showed no significant improvement across the study period in either sex or in the overall sample. Our results thus agree with most previous studies (Medici et al., et al., et al., Several study limitations should be noted. First, several demographic variables including educational level, socioeconomic or employment status and a number of premature mortality risk factors such as unhealthy lifestyles and obesity were not adequately recorded in medical database and thus were not included in the analysis. Second, missing data on patients\u2019 death causes may compromise the accuracy in evaluating cause-specific SMRs. Third, as information on specific unnatural causes was not available, we were not able to investigate mortality risk for individual categories of unnatural deaths including suicide and accidents. Fourth, the study data did not contain information denoting the disorder subtypes, precluding us from examining potential differences between bipolar I and II disorders in mortality rate and life expectancy. Fifth, patients\u2019 age of illness onset was defined by age of first-recorded diagnosis of bipolar disorder. However, previous studies showed that there could be significant delays between illness onset and ascertainment of bipolar disorder diagnosis (Dagani In conclusion, this large population-based study indicates that bipolar disorder patients have increased mortality risk and markedly reduced life expectancy in a predominantly Chinese population, with excess mortality being primarily attributable to natural causes. Our findings of persistent mortality gap across the study period highlights an urgent need for further research to identify its underlying causes and to implement multi-level, targeted interventions to promote physical health of bipolar disorder patients so as to significantly reduce their risk of preventable physical morbidity and premature mortality."} +{"text": "The increase in number of people suffering from neurodegenerative dementia (ND), such as Alzheimer's disease (AD) and Parkinson's disease (PD), has placed a huge burden on society. Precise detection of ND in an early phase is still challenging in daily clinical practice , 4-[(E)-2-(6-{2-[2-(2-(18F)fluoroethoxy)ethoxy]ethoxy}pyridin-3-yl)ethen-1-yl]-N-methylaniline (18F-AV45), and 7-(6-(18F)fluoropyridin-3-yl)-5H-pyridoindole (18F-AV1451) have been widely used in research and diagnostic imaging of ND. Development of bioinformatics analysis and artificial intelligence methods has significantly facilitated advances in research of ND. Nowadays, researchers are facing difficulties in choosing the best method in order to conduct research in a comprehensible fashion with estimates of accuracy and reproducibility.In addition to metabolic changes such as altered glucose metabolism, alterations of amyloid-beta and tau protein are biomarkers associated with pathology of AD, whereas alpha-synuclein is proven to be related to PD and other NDs e4 allele might affect the relationship between serum lipid levels and cognitive impairment. In addition, Hu, Li, Zhao, et al. provided evidence that the combination of telmisartan and rosuvastatin might be an effective prevention and/or treatment strategy for cognitive impairment and dementia, especially in hypertensive patients with the APOE e4 allele. Kuang et al. demonstrated that the e4 genotype leads to distinct default mode network (DMN) functional alterations in early phases of AD using persistent homology approach. Lin investigated whether CDGSH iron-sulfur domain 2 (CISD2) gene attenuation had an influence on anti-inflammatory effects and M1-M2 polarization in microglia. This study promised a potential therapeutic target for ND.Based on univariate and multivariate analyses, Jiao et al. focused on plasma biomarkers which are less expensive and invasive than those necessitating a spinal fluid sample. The results of their study showed that the multifactor model of plasma amyloid-beta 42 and total-tau in combination with Montreal Cognitive Assessment (MoCA) could be a viable model separate health and AD subjects in clinical practice. Gao et al. characterized the relationship between plasma amyloid-beta levels and cognitive decline in 1,240 cognitively normal participants. The relationship between plasma amyloid-beta 40 and cognitive decline was an inverted-U shape in a cognitively normal population. None of relationship between plasma amyloid-beta 42, amyloid-beta 42/40, and cognitive decline was found during a 2-year follow-up. Lin et al. enrolled cognitively normal amyloid-beta-positive participants from 2 cohort studies, all types of resilience to cerebrospinal fluid (CSF) amyloid-beta could predict longitudinal cognitive decline.For clinical diagnosis of AD, Yu et al. identified 16 hub genes correlated to the neuropathological stage and 35 potential biomarkers for the diagnosis of AD. Yuen et al. demonstrated a systematic workflow for evidence synthesis of transcriptomic studies using both meta-analysis and bioinformatics methods to identify potential pathogenic factors. The results showed that reduced amyloid-beta clearance in AD pathogenesis was associated with genes encoding Fyn and EGFR, which were key receptors in amyloid-beta downstream signaling. Robin et al. comprehensively profiled phenotypic features over time in one commercially-available induced Pluripotent Stem Cell (iPSC)-derived human neuron cell line. This study provided a tool to investigate neurodegenerative and other central nervous system (CNS) diseases. Deng et al. applied multivariate model to estimate the association between leukocyte telomere length (LTL) and cognitive performance. Their results suggested that LTL might be a biomarker of cognitive aging.Identification of novel molecular biomarkers for diagnosis and treatment of AD is urgently demanded. Based on bioinformatics analysis, Kong et al. explored the links between diabetes and AD by studying the advanced glycation end products (AGEs) and the receptors for AGEs (RAGE). The results of their study suggested that patients with diabetes were at a higher risk of developing AD. They further reviewed the interaction between RAGE and amyloid-beta as well as tau, which highlighted the potential of RAGE to be used as an effective target for AD diagnosis and treatment. Ho et al. explored type 2 diabetes mellitus (T2DM) pathogenesis in the amyloidogenic evolvability. A better understanding of the role of T2DM in amyloidogenic evolvability might reveal new targets for therapeutic intervention in AD patients who are comorbid with T2DM.Li et al. found that T2DM could give rise to the white matter atrophy of several brain regions, including left posterior cingulate, precuneus, insula, and right rostral middle frontal gyrus. In addition, they investigated the white matter structural network disruption in T2DM patients with MCI. Chen et al. developed MR glucose chemical exchange saturation transfer (glucoCEST) imaging in a rat model of AD. The findings from their study showed that this method could explore the occurrence and progress of diabetes-related AD or dementia.Using magnetic resonance (MR) imaging, Feng et al. explored the role and underlying mechanism of calcium-sensing receptor (CaSR) in cognitive deficits in AD mice. Their study might provide novel insights on the potential of CaSR as a therapeutic target for AD. Wu et al. examined whether steroid receptor coactivator 1 (SRC-1) is involved in pathogenesis of AD. Tyagi et al. reviewed the role of cyclooxygenases (COX) and mammalian/mechanistic target of rapamycin (mTOR) and potential therapeutic approaches targeting COX-2 and mTOR in AD and cancer.Based on live-cell imaging combined with behavioral tests, Bjorkli et al. reviewed the preclinical and clinical investigations of commonly used biomarkers in animal models of AD and AD patients respectively. They also provided recommendations for standardization of procedures in sample collection to enhance the translational validity of preclinical study using AD animal models.Zhang, Chen, et al. reviewed the researches about TDP-43 and its relationship with limbic-predominant age-related TDP-43 encephalopathy (LATE).TDP-43 is a protein related to amyotrophic lateral sclerosis (ALS) and many cases of tau-negative frontotemporal lobar degeneration. Zhang, Xie, et al. reported prospects of 2 kinds of reprogramming technologies for neurodegenerative diseases: (1) convert adult somatic cells to iPSCs and (2) directly reprogramming adult somatic cells to induced Neurons (iN).In an effort to develop neurodegenerative disease model at cellular level, Jang et al. provided updates on current progress in stem cell-derived dopaminergic neuron transplantation as a therapeutic alternative for PD. Yang, Zhang, et al. aimed to uncover the metabolic pathways across anatomical regions in the brain of PD and levodopa-induced dyskinesia (LID). Based on principal component analysis (PCA) and multivariate general linear model, the midbrain and right cortex were identified as the primary regions of metabolic abnormalities in PD and LID rats. In addition, PD and LID rats exhibited lower levels of synaptophysin (SYP). All results provided key insights for developing targeted therapies in PD. Harsanyiova et al. discussed the relationship between gastrointestinal tract and the pathology or treatment of PD symptoms.Bao et al. surveyed the various positron emission tomography (PET) radiotracers available for AD imaging and discussed their clinical applications especially in terms of early detection and cognitive relevance. Based on [18F]-APN-1607 PET tracer, Lu et al. detected tau deposition in AD and reported that individual tauopathy is correlated with impaired cerebral glucose metabolism and cognitive function. Using combined PET and MR imaging, Kim et al. investigated the effect of conductive hearing loss in an AD mouse model. The findings from their study indicated that even partial hearing loss could aggravate memory impairment in AD.11C-PE2I is a PET radiotracer targeting neuronal dopamine transporters (DaT). Ivanidze et al. investigated neurovascular unit (NVU) integrity by using arterial spin labeling (ASL) MR imaging and correlated the findings of NVU integrity with striatal DaT density from 11C-PE2I PET imaging. This exploratory research could serve as a foundation for further development of combined NVU and striatal DaT density as early disease biomarkers and potential new therapeutic targets. Based on 11C-CFT and 18F-FDG PET imaging data, Fei et al. studied the relevancy between UPDRS motor scores and PDQ39 mobility sub-scores.31P MR spectroscopy of parieto-occipital lobes with 7-Tesla MR imaging, Das et al. accurately quantified high-energy phosphate and membrane phospholipid metabolites in amnestic MCI (aMCI). Furthermore, they have also found that brain energy metabolism and membrane phospholipid indexes were related to cognitive performance in domains of executive function (EF), memory, attention, and visuospatial skills using aMCI.Based on partial volume-coil Liu, Jiang, et al. explored functional and structural properties of abnormal brain networks associated with PD. The authors showed that both the expressions of metabolic and structural patterns in PD patients were significantly higher than healthy controls, and verified their results in connectome analysis, which provided new information for elucidating the neuropathological mechanisms of PD. Shu et al. developed an integrative nomogram based on white matter radiomics biomarkers and nonmotor symptoms for the identification of early-stage PD.Tsai et al. explored the skull score (SS) distribution of tremor patients, and correlated the SS with image feature from customized skull density ratio (cSDR). This study provided useful information for clinical study of MR-guided focused ultrasound thalamotomy. Chiu, Tzeng, et al. found that the patients with tremor and vascular cognitive impairment (VCI) had high possibility of mixed pathology of PD and Lewy body disease (LBD).Ye et al. investigated the volumetric changes in thalamus and hypothalamus in ALS. The results from their study revealed no significant difference of the volume in thalamus and hypothalamus between ALS patients of normal frontotemporal function and healthy controls.Yu et al. identified the brain function activity differences between MCI patients with depression and MCI patients without depression using resting state MR imaging measurements. This study provided useful information for a better understanding of the relationship between depressive symptoms and memory deficits. Xing et al. explored the alterations in intra- and inter-network functional connectivity of multiple networks in presbycusis patients, suggesting that functional network connectivity can be used to predict potential cognitive impairment in their early stage.Wan et al. compared the changes in subcortical nuclei in older adults with cognitive frailty (CF) and studied their relationship with cognitive decline and physical frailty. Their results showed significant volume reductions in five subcortical nuclei, including the bilateral thalami, left caudate, right pallidum, and accumbens area in older adults with CF. Lee et al. used surface-based analysis to evaluate subcortical structural characteristics and its relationship with early onset Alzheimer's disease (EOAD) and late-onset Alzheimer's disease (LOAD). The results from their study demonstrated that EOAD and LOAD might have different courses of pathomechanism.Qiao et al. examined the neural substrates and mechanisms that generate memory deficits, seizures, and neuropsychiatric abnormalities in encephalitis with leucine-rich, glioma-inactivated 1 (LGI1) antibodies. The results showed that neural disorder and behavioral deficits of anti-LGI1 encephalitis might be associated with extensive changes in brain connectivity and microstructure.Bruchhage et al. performed machine learning to assess the contribution of volume fraction myelin and gray matter volume. They proposed a refined model of cerebellar contribution to early AD development. The results from their study showed higher anterior cerebellar contribution to MCI and higher posterior cerebellar contribution to mild/moderate stages of AD for each tissue property. Ma et al. proposed a generative adversarial network (GAN) framework to distinguish brain images of human subjects of normal brain aging from those of human subjects with AD and frontotemporal dementia (FTD). The results from their study showed an accuracy of 88.28% in distinguishing human subjects of normal brain aging from those of human subjects with AD and FTD based on GAN framework derived from brain image.Hu, Li, Zhang, et al. found that periodontitis was associated with learning and memory impairment, probably induced by neuroinflammation via activating the toll-like receptor 4/Nuclear factor kappa B signaling pathway. Ding et al. aimed to evaluate the value of odors in olfactory identification (OI) test and other known risk factors for predicting incident dementia. The results from their study suggested that peppermint smell capability might be one of the useful indicators for predicting dementia. Nie et al. assessed the differences of eye movement parameters between healthy elderly individuals and patients with MCI. They found that cognitive deficits and eye movement indexes were correlated, which could be further explored as early markers for MCI.Yang, Huang, et al. proposed a neuroimaging approach to identify MCI using a deep learning method and functional near-infrared spectroscopy (fNIRS). Their results indicated that fNIRS imaging approach based on temporal feature maps as a promising diagnostic method for early detection of MCI and clinicians might use it as a tool for evaluation of MCI.The paper by Tseng et al. used multivariate time-series electrocardiogram (ECG) analysis to diagnose cardiovascular diseases. They investigated various ECG features and found some associations between features of ECG and medical records .Chiu, Hung, et al. studied the relationship between freezing of speech (FOS) and dementia with Lewy bodies (DLB). They designed a freezing of speech single questionnaire (FOSSQ) and compared the association factors of FOS in non-demented participants, patients with AD, vascular dementia (VaD), and DLB. The results of their study supported validity of the FOSSQ for discriminating DLB from individuals with non-demented or other forms of dementia. Similarly, Wang, Hung, et al. designed a novel questionnaire for visuospatial dysfunction (VSD) in DLB.Gupta et al. proposed different neuroimaging modalities combined with APOE genotype to form a multimodal system for discrimination of AD to increase the classification accuracy.To distinguish stable MCI (MCIs) from converting MCI (MCIc), Lin et al. developed an extreme learning machine (ELM)-based grading method to predict MCI-to-AD conversion. The method was validated by Alzheimer's Disease Neuroimaging Initiative (ADNI) cohort, with an accuracy of 84.7% in prediction of MCI progression to AD within 3 years.In order to determine whether a MCI patient is at high risk of progressing to AD, Wang, Wei, et al. conducted a systematic review and meta-analysis to assess the available preclinical evidence and possible mechanisms of baicalein for animal models of PD. Li et al. investigated the efficacy and safety of 3 Erinacine A-enriched Hericium erinaceus mycelia (EAHE) capsules for the treatment of patients with mild AD. The study lasted for 49 weeks and the results showed that EAHE was well-tolerated without significant side effects. Flavonoid containing natural products, Myricetin (MYR) and Dihydromyricetin (DMY) are abundant in fruits and vegetables. Liu, Guo, et al. reviewed the benefits of MYR and DMY in AD patients at molecular level, including effects on amyloid-beta protein imbalance, neuroinflammation, dyshomeostasis of metal ions, autophagy disorder, and oxidative stress.Lai et al. compared and ranked 9 treatment methods for MCI in AD based on meta-analysis. They pointed out that music therapy might be the best treatment for MCI followed by acupuncture, among the nine treatment methods included for their meta-analysis. Similar to those non-pharmacological interventions, Pei et al. proposed a neurofeedback training based on mismatch negativity to regulate sensory ability and memory.The early intervention for MCI could decrease the rate of conversion from MCI to AD. Stuckenschneider et al. investigated the effects of a 12-month structured exercise program on the progression of 183 amnestic MCI patients. No significant improvement of cognitive performance was found based on the results from their study. In another study based on event-related measurement of auditory memory, Laptinskaya et al. found no improvements of cognitive performance in a 10-week unimodal cognitive or physical training and an active lifestyle for older adults at risk for dementia. In the narrative review by Meng et al., patients with AD who presented with long-term exercise interventions appeared to have improved blood flow, increased hippocampal volume, and improved neurogenesis. These results indicated that exercise intervention might be an important moderator to prevent long term disease progression.Kumar et al. reviewed and discussed nano-enabled drug delivery systems and their current and potential applications for the treatment of various NDs, including AD and PD, in studies to overcome the limits of blood-brain barrier (BBB). Huang et al. developed a medical red light treatment (RLT) device to treat older adults with mild to moderate AD. They planned a study protocol to verify the safety and efficacy for a 24 weeks period. On the other hand, Zhu et al. found that there was more efficacy via tele-health interventions in lowering depression for careers of dementia patients based on meta-analysis.Luo et al. reviewed the therapeutic effect of DBS in AD, and analyzed its stimulation parameters and potential mechanism of action. Tan et al. used a convolutional neural network (CNN) to classify handwritten digits and letters and applied dropout at different stages to simulate DBS effects on engrams. The results of their study showed that dropout of engram nodes might be a possible mechanism by which neuromodulation techniques could disrupt or enhance memory. Hwang et al. pointed out the applications of phase amplitude coupling-based phase-dependent DBS technique in PD, which aimed to deliver timed stimulation pulses to a specific phase precisely to modulate pathological network activities and behavior in real time.Deep brain stimulation (DBS) is widely used in the field of mental and neurological diseases. Tang et al. investigated the effect of electroacupuncture (EA) on cognitive impairment and the role of c-Jun N-terminal kinase (JNK) signaling pathway in AD model mice. The results of their study showed that EA could reverse cognitive deficits and substantially lower the burden of amyloid precursor protein. In another study, Hongna et al. demonstrated the improvements of locomotor function by promoting autophagy flux and inhibiting necroptosis in rats with spinal cord injury treated with Jia-Ji electro-acupuncture.In summary, large number of articles collected in this Research Topic reflected recent advances in mechanistic study of pathophysiology of AD and PD, development of biomarkers and molecular imaging techniques for diagnosis and treatment of AD and PD. We hope that publications of this Research Topic will not only report recent advances in ND research, but also facilitate translation of new discovery to development of new diagnostic tests and therapeutic agents for early diagnosis and treatment of ND, including AD and PD.JJ and KS wrote the draft. FP copyedited for the language. C-YH and W-MK reviewed and revised the manuscript. All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."} +{"text": "Neurodegenerative diseases, including Alzheimer\u2019s disease (AD), Parkinson\u2019s disease (PD) and amyotrophic lateral sclerosis (ALS), are typically characterized by progressive neuronal loss and neurological dysfunctions in the nervous system, affecting both memory and motor functions. Neuregulins (NRGs) belong to the epidermal growth factor (EGF)-like family of extracellular ligands and they play an important role in the development, maintenance, and repair of both the central nervous system (CNS) and peripheral nervous system (PNS) through the ErbB signaling pathway. They also regulate multiple intercellular signal transduction and participate in a wide range of biological processes, such as differentiation, migration, and myelination. In this review article, we summarized research on the changes and roles of NRGs in neurodegenerative diseases, especially in AD. We elaborated on the structural features of each NRG subtype and roles of NRG/ErbB signaling networks in neurodegenerative diseases. We also discussed the therapeutic potential of NRGs in the symptom remission of neurodegenerative diseases, which may offer hope for advancing related treatment. Neurodegeneration is characterized by the progressive loss of neuronal structure and function, including the death of neurons, which deteriorates over time and eventually leads to dysfunction of the nervous system. Neurodegenerative diseases, including Alzheimer\u2019s disease (AD), Parkinson\u2019s disease (PD), Huntington\u2019s disease (HD), amyotrophic Ilateral sclerosis (ALS) and spinocerebellar ataxia belong to the epidermal growth factor family of extracellular ligands, participating in the regulation of normal cells and tumor cell growth and survival through the ErbB family receptor tyrosine kinases is a family of growth factors that plays multiple roles in many neurological disorders, including ALS has been differently named as neu differentiation factor , heregulin , acetylcholine receptor-inducing activity , glial growth factor , and sensory and motor neuron-derived factor was originally found in DNA sequence databases and DNA libraries when screening for genes with sequence similarities to members of the NRG1 family was originally detected in adult pancreas and muscle and was initially identified as a necessary factor for tissue growth is a neurodegenerative disease with insidious and progressive onset, mainly in the elderly, and its incidence increases with age is a continuously progressive disease clinically characterized by changes in cognition or behavior is pathologically characterized mainly by: (1) senile plaque formed by abnormal deposition of \u03b2-amyloid protein outside neurons; and (2) NFTs formed by abnormal phosphorylation of tau protein such as the C-terminal fragments of APP (APP-CTs) have been reported to exert cytotoxic effects in neuronal cells represents a potential treatment for AD, and lots of BACE inhibitors are progressing through clinical trials at present increase the levels of pErbB4 receptor and pAkt, and increase the level of Bcl-2 both in in vitro studies and APP/PS1 transgenic mice family, NRG2 can bind directly to ErbB3 and ErbB4, thus transactivating ErbB2 , a specific ligand for ErbB4 and a neuronal-enriched neurotrophin, has been identified as a protein structurally related to the NRG1. It is involved in the genetic predisposition to a broad spectrum of neurodevelopmental, neurocognitive and neuropsychiatric disorders, including AD, autism and schizophrenia and the risk of AD development and in the family sample (p = 0.0166), indicating that genetic variants in the NRG3 gene play a role in AD and SNPs in the NRG3 genes and were more strongly associated with AAO of AD follows AD as the second severe neurodegenerative disease toxin-based mouse model of PD, researchers also found that systematic administration of NRG1\u03b21-ECD can rescue nigral dopaminergic neurons via the ErbB4 receptor tyrosine kinase , also known as motor neuron disease (MND) or Lou Gehrig\u2019s disease, is a disease that causes the death of neurons controlling voluntary muscles of spinal motor neurons (Lasiene et al., In a recent study on the relationship between ErbB4 and ALS/FTD, ErbB4 mutation was found for the first time in ALS/FTD and that its mutation reduced auto-phosphorylation of upon NRG1 stimulation (Sun et al., Schizophrenia is one of the serious psychiatric diseases whose etiology is not fully elucidated. The main clinical characteristics of schizophrenia are perception, thinking, emotion and behavioral disorientations (Owen et al., NRG1 (Harrison and Weinberger, NRGs and the increased risk of schizophrenia development may be determined by the wide-ranging effects of NRGs on brain functions (Wang et al., Studies suggest that NRG1/ErbB4 interactions play a vital role in the pathological mechanism of schizophrenia (Li et al., NRG1 or ErbB4 gene in some schizophrenia patients (Walss-Bass et al., NRG1 or ErbB4 may only account for a small part of schizophrenia cases (Harrison and Weinberger, The direct and chronic disturbance of NRG1/ErbB4 signal transmission, such as the mutation of NRG3 are associated with cognitive and psychotic symptom severity, which is accompanied by increased expression of prefrontal cortical NRG3 (Meier et al., In order to reveal the role of NRG2 in the modulation of behaviors with relevance to psychiatric disorders, Vullhorst et al. found a Although scientists are looking forward to finding cures for neurodegenerative diseases with complex and unclear pathogenesis, no comprehensive and effective treatments and drugs have been developed. The results of a growing number of studies suggest that NRG1 regulates cell maintenance, differentiation, proliferation, migration, and survival or apoptosis in both neuronal and nonneuronal cell types (Mei and Xiong, Current evidence has indicated that NRGs play important roles in neurological disorders such as brain trauma (Fricker et al., In order to strengthen the understanding of NRGs in neurodegenerative disease prevention and treatment, further relevant mechanisms need to be clarified in the future. More in-depth future research on NRGs is necessary to improve diagnostic solutions for neurodegenerative diseases, thereby reducing the burden on both patients and families.W-jZ: conceptualization. W-jZ, G-yO, and W-wL: writing\u2014original draft preparation. W-jZ and G-yO: writing\u2014review and editing.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Sci., 2021, 12, 14432\u201314440, DOI: 10.1039/d1sc04138j.Correction for \u2018Plasticizer and catalyst co-functionalized PEDOT:PSS enables stretchable electrochemical sensing of living cells\u2019 by Jing Yan The authors regret that there was an error in the equation of the calibration curve of PPL/PDMS in The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "Helicobacter pylori) has been classified by the International Agency for Cancer Research as carcinogenic in humans but many other are the subject of intense research. Most studies on the role of microbiota in GI tract oncogenesis focus on pancreatic and colorectal cancers with the following three species: Helicobacter pylori, Escherichia coli, and Porphyromonas gingivalis as likely causative factors. This review summarizes the role of bacteria in GI tract oncogenesis.Disturbances in gastrointestinal (GI) microbiota could play a significant role in the development of GI cancers, but the underlying mechanisms remain largely unclear. While some bacteria seem to facilitate carcinogenesis, others appear to be protective. So far only one bacterium ( Cancer is currently a major world-wide health problem: it is estimated that approximately 18.1 million new cancer cases and 9.6 million cancer-related deaths occurred in 2018 alone, and there is a 20% risk of developing cancer before turning 75\u00a0years old, and 10% risk of dying from it correlated positively with tumor development, while members of the Clostridiales, especially Clostridium Group XIVa, were associated with decreased cancer risk, probably due to the production of butyrate, which has anti-inflammatory and anti-tumorigenic properties mouse model in which gut microbiota changes, which occurred during tumorigenesis, supported increased tumorigenic process in the later stages , which develop spontaneous colitis due to microbial-induced activation of effector T cells , Firmicutes (mainly Streptococcus), and Actinobacteria (mainly Rothia) , while Firmicutes predominated in saliva and mouthwash (predominant taxa Streptococcus and Prevotella). Interestingly, Acinetobacter and Fusobacterium, which were enriched in the neoplastic tissue, remained increased in the late stage of OSCC facilitating cancer progression by their ability to cause local inflammation. Zhang et al. (F. nucleatum infections may cause cancer through their effect on MMP9 pathways and upregulation of cytokines such as tumor necrosis factor (TNF)-\u03b1, IL-1\u03b2, and IL-6 (Whitmore and Lamont 2014).Zhang et al. examinedg et al. performeg et al. . These ag et al. . It was Streptococcus has become the focus of extensive microbiota analysis , but infection alone is not sufficient dying within 5\u00a0years of initial diagnosis 2. Furthermore, the authors found that hypoxia, a dominant feature of the PDAC microenvironment, greatly enhances P. gingivalis intracellular survival , complement activity and TLR4 activation. These effects facilitate local inflammatory responses that contribute to progression of periodontitis is currently the third leading cause of cancer-related death worldwide . Although HCC is closely related to chronic infection with hepatitis B virus and hepatitis C virus as well as to chronic liver damage increased intestinal permeability caused by alterations in the tight junctions between enterocytes allowing for the inflow of such harmful substances as LPS into portal blood. (2) Modification of specific receptor activity which consequently allows for passage of microbial metabolites into circulation. (3) Increased secretion of biochemically active factors was proposed by Ni et al and 13 potentially harmful bacteria, which are often increased in these patients is the second leading cause of cancer death in the USA showed that B. fragilis was proposed as a likely carcinogen because of its ability to produce metalloprotease in CRC patients are also likely to be important for colonic tumorigenesis through TLR4 expression on CRC cells F. nucleaKanazawa et al. studied Bacteroides fragilis toxin which decreases E-cadherin on the surfaces of epithelial cells loosening intercellular junctions and thus allowing for an increased inflow of harmful substances and antigens from the gut (Sears et al. Several mechanisms have been proposed to explain the effect of bacteria on CRC development. The \u201calpha-bug\u201d hypothesis assumes that bacteria induce CRC by a specific action such as the one described for E. coli and B. fragilis already at an early noncancerous stage (Dejea et al. Interestingly, the colonic mucosa biofilm in patients with familial adenomatous polyposis was reported to be composed mainly of F. nucleatum has been confirmed in studies involving global cohorts. However, this biomarker is not sufficiently specific and sensitive to allow for non-invasive CRC diagnosis (Chang et al. Streptococcus bovis, which has been associated with colon cancer (Tjalsma et al. S. bovis antigen profiles could distinguish 11 out of 12 colon cancer patients from 8 control subjects, whereas E. coli antigen profiles were not useful (Tjalsma et al. S. bovis antigen profiles were also detected in patients with polyps, suggesting that this infection occurs in the early stage of carcinogenesis. This could be a promising diagnostic tool for the early detection of human colon cancer (Tjalsma et al. Based on metagenomic analyses a number of microbes have been proposed as biomarkers of CRC, but only Probiotics show promise as agents of host\u2013microbiome modulation therapies for several diseases, including CRC (Torres-Maravilla et al. Although the overwhelming majority of studies support the role of the microbiome in cancer, some authors did not find significant associations. For example, in the study by Olson et al. there weA large number of studies have been devoted in recent years to the analysis of the role of microbiota in GI oncogenesis. New treatments such as fecal transplantation, phage-based therapy, antibiotics, and probiotics therapies are currently tested in clinical settings to detect, prevent or improve the clinical course of various cancers. There is also a concerted effort to develop a fast, non-invasive, sensitive, and specific cancer detection test. However, these novel treatment interventions and diagnostic tests require validation in rigorous clinical trials before they could enter clinical practice."} +{"text": "Dauncey et al., Chem. Sci., 2019, 10, 7728\u20137733.Correction for \u2018A dual photoredox-nickel strategy for remote functionalization The authors regret that The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "Obesity is the result of an energy imbalance caused by an increased ratio of caloric intake to energy expenditure. In conjunction with obesity, related metabolic disorders, such as type 2 diabetes mellitus, dyslipidemia and hypertension, have become global health problems . ReducinGanoderma lucidum inhibits differentiation and lipid accumulation of 3T3-L1 adipocytes via reducing the expression of genes responsible for lipogenesis and the phosphorylation of mitogen-activated protein involved in cell proliferation and differentiation and is, thus, a promising natural agent for obesity and related metabolic diseases. Lee et al. [Schisandra chinensis ameliorates lipid accumulation and induces a brown fat-like phenotype through AMP-activated protein kinase in 3T3-L1 adipocytes. The authors also described that gomisin N inhibits adipogenesis and lipogenesis by enhancing fatty acid oxidation and thermogenesis and may have a potential preventive and therapeutic agent to combat obesity. Lee et al. [In studies using cell and animal models, Jeong and Park providede et al. showed te et al. demonstrIn a human study, Auguet et al. reportedIn a recent review, Kury\u0142owicz and Puzianowska-Ku\u017anicka summarizThis Special Issue offers interesting findings from cell, animal and human studies on medicines for the treatment of obesity and suggests strategies for the development of future antiobesity therapies."} +{"text": "Chinese university students are at high risk for depressive symptoms and the ongoing coronavirus disease 2019 (COVID-19) pandemic may have exacerbated the mental health of university students. However, existing studies on depressive symptoms in Chinese university students during the COVID-19 pandemic reported a wide range of prevalence estimates, making mental health planning for this population difficult. The objective of this study was to conduct a systematic review and meta-analysis of surveys that assessed the prevalence of depressive symptoms in Chinese university students amid the COVID-19 pandemic.Major Chinese and English databases and preprint platforms were searched to identify cross-sectional studies containing data on the prevalence of depressive symptoms in Chinese university students during the pandemic. Two authors independently retrieved the literature, evaluated the eligibility of potential studies, assessed the risk of bias (RoB) of included studies, and extracted data. RoB was assessed with the Joanna Briggs Institute Critical Appraisal Checklist for Studies Reporting Prevalence Data.I2 = 99.9%, p < 0.001). The pooled prevalence of depressive symptoms was 26.0% (95%CI: 23.3\u201328.9%), which was significantly higher in female than in male students , in postgraduates than in undergraduates , in students living inside than in those living outside the COVID-19 epicentre , in students from universities at the epicentre than in those from universities outside the epicentre , in students who had close contact with COVID-19 than in those who did not , and in students who had acquaintances or relatives infected with COVID-19 than in those who did not. Five sources of heterogeneity were identified from the subgroup analysis: survey period, % of males among the survey sample, scale of depressive symptoms, cutoff score of the scale and level of RoB.In total, 1177 records were retrieved, and 84 studies involving 1 292 811 Chinese university students during the pandemic were included. None of the included studies were rated as completely low RoB. Statistically significant heterogeneity in the prevalence estimates of included studies was detected (Over one-fourth of Chinese university students experienced depressive symptoms during the COVID-19 pandemic. Mental health services for this population should include periodic evaluation of depressive symptoms, expanded social support and psychiatric assessment and treatment when necessary. It is also necessary to design depression prevention programmes that target higher-risk cohorts of university students. The transition is challenging because of the high level of academic and employment stress and the prevalent interpersonal, romantic and emotional problems in this particular stage for university students pandemic has caused a global mental health crisis. Lessons learned from the 2003 severe acute respiratory syndrome (SARS) epidemic in China suggest that depressive symptoms are one of the most common mental health problems among university students; for example, during the SARS epidemic, 25.4\u201329.6% of the Chinese university students had depressive symptoms guidelines, and the protocol was registered in the International Prospective Register of Systematic Reviews (PROSPERO) with the registration number CRD 42020206666.The inclusion criteria for eligible studies were (a) cross-sectional surveys or baseline surveys of cohort studies with meta-analysable data ; (b) study subjects were Chinese university students, including overseas students and postgraduates; (c) the presence of depressive symptoms was assessed with standardised instruments and (d) the study was conducted during the COVID-19 pandemic (since 1 January 2020). We excluded studies with mixed samples that did not present results separately for university students and studies that assessed depressive symptoms with unstandardised instruments .We searched potential studies published between 1 January 2020 and 10 February 2021 in both Chinese and English bibliographic databases: China National Knowledge Infrastructure, Wanfang data, VIP Information, PubMed, Embase and PsycInfo. Key terms used were: (adolescen* OR teenager* OR youth* OR student* OR young adult* OR undergraduate* OR universit* OR college*), (coronavirus disease 2019 or severe acute respiratory syndrome coronavirus 2 or COVID-19 or COVID) and (depress*). To avoid missing relevant studies, reference lists of the retrieved reviews and included studies were also hand-searched. Preprint servers were also searched to retrieve grey literature: medRxiv, bioRxiv, PsyArXiv, ChinaXiv and Research Square. The literature search was ended on 12 February 2021. Detailed search strategies are provided in online Supplementary Table 1.By using a predesigned electronic form, the following variables were extracted from included studies: first author, study site, study period, characteristics of the study sample, sampling method, sample size, survey method, assessment of depressive symptoms and rates of depressive symptoms. According to the State Council Information Office of the People's Republic of China Critical Appraisal Checklist for Studies Reporting Prevalence Data (abbreviated as \u2018JBI checklist\u2019 hereafter) to assess the RoB of included studies , a fixed-effect model was used to generate the pooled estimates; otherwise, the random-effect model was used. The pooled rates of various cohorts were compared by using the Z test. We used subgroup analysis to explore the source of heterogeneity in the prevalence estimate of depressive symptoms. The Q-value test was used to test the significance of differences in prevalence rates between subgroups. Publication bias was assessed with funnel plots and Begg's test, since Begg's test is fairly powerful for large meta-analyses that include 75 or more original studies for the prevalence of depressive symptoms in the whole sample and in various cohorts of the sample. Forest plots were adopted to display the prevalence rates and pooled estimates. We used the et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., n\u00a0=\u00a037), followed by Zung's Self-rating Depression Scale (SDS) (n\u00a0=\u00a022), the depression subscale of the Symptom Checklist-90-Revised (SCL-90-R) (n\u00a0=\u00a08), the depression subscale of the Depression, Anxiety and Stress Scale \u2013 21 Items (DASS-21) (n\u00a0=\u00a07) and the Center for Epidemiologic Studies \u2013 Depression Scale (CES-D) (n\u00a0=\u00a07). The average and median reported prevalence rates of depressive symptoms were 27.3% and 25.8%, respectively. Other detailed characteristics of the included studies are shown in The process of study inclusion is shown in n\u00a0=\u00a062) and problematic sampling method (n\u00a0=\u00a058) (online Supplementary Table 2).In total, 31 studies had a RoB score of \u20180\u20133\u2019, 42 had a RoB score of \u20184\u20136\u2019 and 11 had a RoB score of \u20187\u20138\u2019. No study was scored nine. The two most common methodological issues were inappropriate sample frame . Pooled v. 28.6%, p\u00a0<\u00a00.001), in students with siblings than in only child students , in overseas than in domestic students , in postgraduates than in undergraduates , in students living in Hubei than in those living in provinces other than Hubei , in students from universities of Hubei than in those from universities of other provinces , in students who were in close contact with COVID-19 than in those who had no history of COVID-19 contact , and in students who had friends, classmates or relatives infected with COVID-19 than in those who did not .Table 2p value of the Begg's test was 0.169. No statistically significant publication bias was detected across the 84 included studies.As shown in v. 21.8%, p\u00a0=\u00a00.015), in studies with a percentage of males <50% than in those with a percentage of males \u2a7e50% , in studies assessing depressive symptoms with CES-D than in those using SCL-90-R , in studies defining the presence of depressive symptoms as \u2018PHQ-9\u00a0\u2a7e\u00a05\u2019 than in those defining it as \u2018PHQ-9\u00a0\u2a7e\u00a010\u2019 , and in studies with a high RoB than in those with a low RoB .Five factors were identified as sources of heterogeneity across included studies : survey v. males), in students with siblings (v. only children), in overseas students (v. domestic), in postgraduates (v. undergraduates), in students living within the COVID-19 epicentre (v. those living outside), in students from universities at the epicentre (v. those from universities of provinces other than Hubei), in close contacts of COVID-19-infected persons (v. those without a history of COVID-19 contact) and in students who had COVID-19-infected friends, classmates or relatives (v. those who did not). In addition, 1.69% of Chinese university students had severe depressive symptoms.This systematic review and meta-analysis summarised studies estimating the prevalence of depressive symptoms among Chinese university students amid the COVID-19 pandemic. We found an overall prevalence rate of 26.0% of depressive symptoms in Chinese university students and significantly higher rates in female students .et al., et al., et al., One possible explanation for the higher risk of depressive symptoms in overseas than in domestic students is the status of ethnic minority groups in foreign countries , the sample representativeness of overseas students may be limited in our study. Finally, patterns of utilisation of mental health services among depressed students are very important for mental health planning and policy-making in the context of the COVID-19 pandemic, but the included studies provided little information on service use.This study has some limitations. First, none of the included studies were rated as completely low RoB. Subgroup analysis according to RoB level found a significantly higher prevalence of depressive symptoms in studies with a high level of RoB, so it is possible that the reported overall pooled estimate overestimates the true prevalence. Second, because several included studies used strict criteria to define the presence of depressive symptoms (i.e. PHQ-9\u00a0\u2a7e\u00a010), we may have underestimated the prevalence of depressive symptoms. Given the above two limitations, it is difficult to assess the magnitude and direction of bias in the prevalence estimate. Cautions are needed when generalising our findings. Third, even after stratifying the studies, high levels of heterogeneity were still kept within each strata of study in the subgroup analysis, so there remained other factors associated with the risk of depressive symptoms that were not identified. The heterogeneity of the results suggests that further rigorously designed studies using widely accepted assessments of depressive symptoms and representative samples of Chinese university students amid the COVID-19 pandemic are warranted to arrive at accurate estimates. Fourth, because of the small number of studies during the postoutbreak period, longitudinal data are needed to examine the trajectory of depressive symptoms in Chinese university students in the postpandemic era. Fifth, since the sample size of overseas students was relatively small indicate that cohort-specific prevention programmes, which are probably cost-effective, need to be designed.China is a mental health services resource-poor country, so university managers and staff, including campus psychological counselors, should have a critical role in depression prevention; for example, they could provide expanded social support to students at risk, engage in follow-up care, mental health education and periodic screening of depressed students and promote social connectedness between students. Although the pandemic increases physical distances between staff and students, support services can be easily provided to students via smartphones.In addition, the 28.9% prevalence of depressive symptoms during the postoutbreak era in this study and some"} +{"text": "There is growing evidence that damage to spermatozoa by reactive oxygen species (ROS) play a key role in male infertility. The aim of the present study was to assess seminal plasma levels of total antioxidant capacity (TAC), free 8-Isoprostane and activities of catalase and superoxide dismutase (SOD) in men with asthenozoospermia, asthenoteratozoospermia and oligoasthenoteratozoospermia compared with normozoospermic males.The patients consisted of 46 men with seminal parameters abnormalities. The patients were grouped into asthenozoospermic (n = 15), asthenoteratozoospermic (n = 16) and oligoasthenoteratozoospermic (n = 15). The control group consisted of 16 healthy males with normozoospermia. Catalase activity was measured by Aebi spectrophotometeric method. Levels of TAC and SOD were measured by commercially available colorimetric assays. Level of free 8-Isoprostane was assessed by commercially available enzyme immunoassay (EIA) method. Differences between groups were assessed using Mann-Whitney U test and Kruskal-Wallis test. Coefficients of correlation were calculated using Spearman's correlation analysis. All hypothesis tests were two-tailed with statistical significance assessed at the p value < 0.05 level with 95% confidence intervalsLevels of catalase and TAC were significantly lower in patients than the control group. No significant changes were seen in SOD activities. Levels of free 8-Isoprostane were significantly higher in patients than the control group. Furthermore, asthenozoospermic, asthenoteratozoospermic and oligoasthenoteratozoospermic groups had significantly lower values of catalase activity and TAC when compared to normozoospermic males. Levels of free 8-Isoprostane were significantly higher in all patients subgroups than the control group. Levels of catalase and TAC were positively correlated with sperm motility and morphology. Free 8-Isoprostane levels showed an inverse correlation with sperm motility and morphology.Decreasing seminal plasma antioxidants levels, especially catalase and TAC, could have significant role in etiology of impaired sperm function. Measurement of 8-Isoprostane may be used as a specific biomarker for assessing oxidative stress on sperm. In the etiology of male infertility, there is growing evidence that damage to spermatozoa by reactive oxygen species (ROS) play a key role ,2. Spermet al study showed statistically significant change in activity of SOD in infertile men compared to normozoospermic samples. They also observed that the SOD activity exceeds values obtained for normozoospermic samples only in oligozoospermic males [et al investigated activities of SOD and catalase in men with asthenozoospermia, teratozoospermia and oligozoospermia compared to normozoospermic males. Their study showed a significant elevation in intracellular activity of SOD and decreasing in catalase activity in infertile samples [et al study showed that seminal plasma activity of SOD in infertile men is significantly grater than in fertile men while catalase activity is not different between these groups [et al showed seminal plasma enzymatic and nonenzymatic (TAC) antioxidant capacities do not alter in the asthenozoospermic specimens, whereas SOD activity is lower in oligoasthenozoospermic samples than normozoospermic males [et al investigation showed that there is not a significant difference in seminal plasma or sperm SOD activity between normozoospermic and oligo- or asthenozoospermic males [et al observed that whole semen SOD activity is higher in men with oligoszoospermia than those with normozoospermia [et al study showed that seminal plasma TAC in infertile asthenozoospermic and asthenoteratozoospermic males is lower than fertile men [The findings on the seminal plasma catalase and SOD activities and total antioxidant capacity (TAC) are controversial. Sanocka ic males . In anot samples . Zini ete groups . The stuic males . Hsieh eic males . This grospermia . Koca ettile men . They alAvailable data on the impact of oxidative stress on sperm are based on the measurement of seminal plasma and sperm levels of malondialdehyde (MDA) by the thiobarbituric acid-reacting substance (TBARS) assay -25. ReceThe aim of the present study was to assess seminal plasma levels of TAC and free 8-Isoprostane and activities of catalase and SOD in men with asthenozoospermia, asthenoteratozoospermia and oligoasthenoteratozoospermia compared to normozoospermic males.6/ml of ejaculate and specimens with hyperviscosity were excluded from this study. The criteria for sperm normality were as follows: sperm concentration \u2265 20 \u00d7 106/ml of ejaculate, sperm motility \u2265 50% and normal sperm morphology \u2265 30% [A case-control study was designed. Following Institutional Review Board approval, the semen samples were collected from the case and the control groups. All specimens were collected into sterile plastic containers by masturbation after an abstinence period of 3\u20135 days, and were analyzed within 1 h of collection. After allowing at least 30 min for liquefaction to occur, semen analysis was performed to measure sperm concentration, sperm motility and sperm morphology using Sperm Quality Analyzer IIC ,30. Sampgy \u2265 30% ,29,30. Tgy \u2265 30% ,31. The 2O2 (250 \u03bcmol/L) was then added to all tubes, and absorbance (A2) was read exactly after 3 minutes. The difference between A2 and A1 (\u0394A) was calculated. The TAC of the sample was then calculated by the following formula: TAC = Concentration of the Standard \u00d7 (\u0394A Blank - \u0394A Sample)/(\u0394A Blank - \u0394A Standard). The results were expressed as mM.TAC was measured by colorimetric assay ,33. We u10 of standards and SOD activity was expressed as U/ml.SOD activity was measured by colorimetric assay ,31. We uCatalase activity was estimated by the method of Aebi . CatalasWe assessed free form of 8-Isoprostane and only the fraction shedded to seminal plasma from cell membranes.Free 8-Isoprostane was purified by affinity chromatography method . We usedAt first, the elution solution was evaporated to dryness using a vacuum centrifugation. Then, the concentration of free 8-Isoprostane was measured by enzyme immunoassay (EIA) method . We usedDifferences between groups were assessed using Mann-Whitney U test and Kruskal-Wallis test. Coefficients of correlation were calculated using Spearman's correlation analysis. All hypothesis tests were two-tailed with statistical significance assessed at the p value < 0.05 level with 95% confidence intervals. The data were expressed as the mean \u00b1 SEM. Statistical computations were calculated using SPSS 11.5 for windows software .Seminal parameters of the subjects are reported in Table Then, we examined the correlation between seminal parameters and seminal plasma levels of 8-Isoprostane, TAC, SOD and catalase in total case group. Levels of TAC showed a positive correlation with sperm motility and morphology . We also observed a direct correlation between catalase activity and sperm concentration , sperm motility , and sperm morphology . Seminal plasma levels of free 8-Isoprostane showed an inverse correlation with sperm motility and sperm morphology . Levels of SOD did not show any correlation with seminal parameters.The most relevant findings of this study were (i) significant elevation of seminal plasma levels of free 8-Isoprostane and decreasing of catalase and TAC levels in asthenozoospermic, asthenoteratozoospermic, oligoasthenoteratozoospermic samples compared with normozoospermic men (ii) both catalase and TAC showed a positive correlation with sperm motility and morphology while an inverse correlation was seen with free 8-Isoprostane levels.et al [et al results about catalase activity in infertile men [et al. evaluated antioxidant capacity of seminal plasma in asthenozoospermic and oligoasthenozoospermic specimens with normal viscosity and hyperviscosity [et al., we observed a significant decrease in catalase activity and TAC in men with asthenozoospermia compared to normozoospermic men. Our finding about SOD activity in asthenozoospermic men was similar to Siciliano et al study. In another study, Hsieh et al evaluated SOD activities in seminal plasma and spermatozoa in infertile men with normozoospermia and oligoasthenozoospermia [et al also observed that SOD activities of seminal plasma and sperm are positively but nonsignificantly correlated with sperm motility and concentration. Our results about the SOD activity of the seminal plasma in oligoasthenozoospermic men were similar to Hsieh et al study. Koca et al evaluated TAC in infertile asthenozoospermic and asthenoteratozoospermic men compared to normozoosperic fertile men [et al study also showed that TAC correlates positively to sperm motility. We also observed these findings. Tkaczuk-Wlach et al evaluated activity of SOD in the whole semen of patients with oligozoospermia compared to patients with normozoospermia [et al study was limited by the fact that they used whole semen sample, because the membrane-bound oxidases or antioxidants associated with cellular debris and/or organelles can influence activities of antioxidant enzymes such as SOD and catalase [Our findings about activities of SOD were contradicted the results of Sanocka et al ,11. Our iscosity . Their sospermia . They obtile men . Their sospermia . Their scatalase .et al study [et al also observed that there is an inverse significant correlation between seminal plasma TAC and ROS levels. They suggested that the inverse correlation between TAC and ROS might be associated with an increase in the consumption of soluble, non-enzymatic antioxidants in seminal plasma which is resulted from over production of ROS. In our study the correlation between TAC and sperm morphology was positive. According to Gil-Guzman et al study, this finding could be interpreted that in semen with high rate of abnormal forms, because of high levels of ROS production, consumption of non-enzymatic antioxidants will be higher.Immature spermatozoa with abnormal morphology and cytoplasmic retention are the most sources of ROS production in semen. This has been confirmed by Gil-Guzman al study . Their set al [et al and our study might suggest that the higher level of antioxidant status prevents lipid peroxidation in spermatozoa and therefore results in higher sperm motility. Hsieh et al observed a slightly positive correlation between seminal plasma SOD activity and sperm concentration [et al. Immature spermatozoa generate primary superoxide anion. This anion is dismuted to hydrogen peroxide by SOD activity. Detoxification of hydrogen peroxide is carried out by catalase activity. Hydrogen peroxide is the primary toxic ROS for human spermatozoa that its high concentration induces lipid peroxidation and results in cell death. Therefore, the balance of the SOD and catalase activities in semen is important for maintaining sperm motility [The inverse correlation between lipid peroxidation and sperm motility has been shown by Keskes-Ammar et al . In our ntration . Their imotility .Our results are agreed with some previous studies that show increasing of lipid peroxidation by measuring MDA in sperm and seminal plasma in males with asthenozoospermia, asthenoteratozoospermia and oligoasthenoteratozoospemia ,23. SimiMDA is widely used index of lipid peroxidation due to its simplicity. The TBARS test application to body fluids and tissue samples is unreliable. Application of a gas chromatography/mass spectrometry (GC/MS) assay for MDA has indicated that the commonly used TBARS assay overestimates the actual MDA levels by more than 10-fold, possibly resulting from cross reactivity with other aldehydes and the harsh conditions used in sample preparation .sn-2 position of phospholipids by phospholipase A2, isoprostanes are formed initially in situ, where they may contribute to the effects of oxidative stress on membrane biophysics. Measurement of 8-Isoprostane may provide a reliable marker of lipid peroxidation in vivo, because, it is a stable compound. In addition, 8-Isoprostane is specific product of free radical-induced lipid peroxidation. 8-Isoprostane has also been found to be present in detectable quantities in all normal biological tissues and in free form in all normal biological fluids. This is important because it allows the definition of a normal range such that small increases in its formation can be detected in situations of mild oxidant stress. Finally, the levels of 8-Isoprostane is unaffected by lipid content of the diet [Recent studies have focused on 8-Isoprostane, as an index of lipid peroxidation. Isoprostanes are formed in situ in cell membranes; following free radical attack on the arachidonic acid. Unlike prostaglandins, which are formed from arachidonic acid following its release from the the diet ,28.Evidence is beginning to emerge suggesting that isoprostanes are not only markers of oxidative injury, but active participants in the pathophysiology of some disorders. The capacity of isoprostanes to readily esterify to cell lipid membranes, and the resulting marked distortion of membrane structure and function, undoubtedly contribute to their pathophysiologic potential. As well, the existence of specific receptor for isoprostanes has been proven . So, becIt is concluded that decreasing seminal plasma antioxidant status, especially catalase activity and TAC, may have significant role in the etiology of impaired sperm function. Measurement of 8-Isoprostane may be used as a specific biomarker for assessing oxidative stress on sperm. However, further studies with a larger sample size are required to confirm these findings.The author(s) declare that they have no competing interests.Ali Khosrowbeygi carried out all of the experiment and participated in data analyzing and writing the manuscript.Nosratollah Zarghami designed the study and participated in data analyzing and writing the manuscript.The pre-publication history for this paper can be accessed here:"} +{"text": "Several authors advocate spleen preserving distal pancreatectomy, because of the increased complication rate after splenectomy.Postoperative complications and survival after distal and total pancreatectomy, were recorded and retrospectively analyzed according to spleen preservation. Patients, who underwent distal and total pancreatectomy without histologically proven adenocarcinoma, or extrapancreatic disease, were included in the cohort which was divided into splenectomy and no splenectomy groups. Statistical analysis was performed using Fisher's test.The study group consisted of 62 patients who underwent distal and total pancreatectomy between 26/11/1987 to 6/1/2006. Splenectomy was performed in 35 out of 62 patients (56.5%), distal pancreatectomy was performed in 49 out of 62 patients (79%). Morbidity rate was 28.6% in splenectomy group and 14.8% in the no splenectomy group (p = 0.235), while 30 days mortality rate was 2.9%; one patient died in the splenectomy group (p = 1).Spleen-preservation did not influence the outcomes after distal and total pancreatectomy in our series. Pancreatectomy may be accompanied with splenectomy in distal and total pancreatic resections. Elective peripheral pancreatectomy is safer than pancreaticoduodenectomy, but carries a high morbidity rate -4; intraIn our study, postoperative complications after distal and total pancreatectomy, were recorded and analyzed according to spleen preservation, in patients with pancreatitis (chronic and acute), benign neoplasms and other benign diseases.th of November 1987 and 6th of January 2006. Patients with histologically proven adenocarcinoma, patients with cystadenocarcinoma, patients who underwent completion pancreatectomy after postoperative complication of pancreaticoduodenectomy, patients who underwent pancreatectomy because of abdominal trauma, patients who had hepatic metastases in laparotomy, patients who had cancer in the pancreatic head or lower common bile duct and patients who had additional procedures such as gastrectomy and colectomy were excluded from the study. The patients were divided into splenectomy and no splenectomy group. The following parameters were recorded and analyzed for each of the above mentioned groups: sepsis (SIRS and MODS), acute renal failure, pulmonary complications , ARDS , cardiac complications , central nervous system complications , intra abdominal abscess (defined as an infected fluid collection identified by CT or ultrasound scan-guided needle aspiration and microbiologic culture), postoperative primary intra abdominal hemorrhage , postoperative primary gastrointestinal hemorrhage (1ry GI), delayed gastric emptying, wound infection, wound dehiscence, first 30 postoperative days mortality.Prospective collected data were retrospectively analyzed for patients who underwent distal or total pancreatectomy with or without splenectomy between 28\u00ae, Chicago, IL, USA). A p value less than 0.05 was considered significant.Statistical analysis was performed using Fisher's two-tailed test, in the \"Statistical Package for the Social Sciences\" version 12 for Windows . After fulfilling the exclusion criteria, our study group consisted of the rest 62 patients who underwent total and distal pancreatectomy with or without splenectomy. The demography, types of operations and final diagnoses are shown in Tables Hospital data included 160 patients who underwent distal and total pancreatectomy between 28Using the Fisher's test no studied factor was correlated with splenectomy vs no splenectomy groups Table . InteresThe two cases of intra abdominal access were treated by the radiologist with a CT guided drainage. The only case of primary postoperative hemorrhage needed a reintervention after a failed embolization.The 30 days mortality rate in the study group of patients was 2.9%. One patient died (stroke) in the splenectomy group (p = 1). There was not recorded any postoperative death due to postsplenectomy sepsis.et al., [et al., [et al., [et al., [et al., [Spleen preserving distal pancreatectomy has been advocated by many authors, because of splenectomy associated immunologic alterations and septic postoperative complications ,16,24,25et al., and Beno[et al., studied [et al., studied [et al., ,27. Aldr[et al., in a gro[et al., demonstr[et al., studied [et al., , in a co[et al., ,4,28 aftThe authors conclude that spleen preservation does not influence the outcome after distal or total pancreatectomy, in benign diseases and selected benign neoplasms.The author(s) declare that they have no competing interests.IK: Designed the study, performed bibliographic research, drafted and revised the manuscript.AT: Carried out the data and participated in the writing process.RB: Participated in the design of the study, helped to draft the manuscriptand and performed the statistical analysis.SB, JB, DM: Participated in manuscript revision process.All authors read and approved the final manuscript."} +{"text": "The importance of prophylactic aspirin use in both developed and developing countries can hardly be overemphasized. I am troubled, however, by Stafford and colleagues' failure to cite and discuss United States studies that show much higher rates of antithrombotic use than they report . Using aTwo factors probably account for these differences. First, Stafford et al.'s federal surveys of ambulatory care encounters miss a significant proportion of aspirin use. Studies in a variety of populations that used direct patient surveys and other methods, some of which are cited in Brown et al. , have foThe experience of the nonprofit-integrated systems is also important because it calls into question Stafford et al.'s suggestion, emphasized in PLoS's accompanying synopsis, that direct-to-consumer advertising of statins explains the 1997\u20132000 dip in aspirin use in their data. Kaiser Permanente and HealthPartners members were equally exposed to direct advertising, but maintained high aspirin use during this period\u2014despite probably also using statins (and angiotensin converting enzyme [ACE] inhibitors) at higher rates than individuals in other US care-delivery settings.The US nonprofit HMO experience nevertheless reinforces the authors' main conclusion that \u201caggressive and targeted interventions are needed to enhance provider and patient adherence to consensus guidelines for CVD risk reduction\u201d . Aggress"} +{"text": "The condition appears to be inherited in an autosomal dominant mode, although sporadic cases have been reported. It is a rare disease, a few families have been described in the literature. In the British family, the locus for oculo-oto-dental syndrome was mapped to 20q13.1 within a 12-cM critical chromosomal region. Dental management is complex, interdisciplinary and will include regular follow up, scheduled teeth extraction and orthodontic treatment. Hearing checks and, if necessary, hearing aids are mandatory, as well as eye examination and ad hoc treatment if necessary.The otodental syndrome also named otodental dysplasia, is characterised by a striking dental phenotype known as globodontia, associated with sensorineural high frequency hearing loss and eye coloboma. Globodontia occurs in both primary and permanent dentition, affecting canine and molar teeth ( The Otodental syndrome has been described under various names:\u2022 Otodental dysplasia ,2;\u2022 Familial otodentodysplasia ;\u2022 Globodontia; the term globodontia refers to the enlarged bulbous fused malformed posterior teeth with almost no discernable cusps or grooves -6;\u2022 In some families, an associated ocular phenotype was recognized and it wThe condition has been described in at least 9 families. The first case of otodental syndrome was described in Hungary in a mother and her son by Denes and Csiba, 1969 ,8, a girThe otodental syndrome, also named otodental dysplasia, is characterised by a striking dental phenotype known as globodontia, associated with sensorineural high frequency hearing loss and eye coloboma.per se diagnostic. It consists mainly of enlarged canine and molar fused teeth both in the primary and in the permanent dentition. The incisors are not affected. In a few cases described in the literature the condition was discovered in young children (around 3 years of age) [The dental phenotype is of age) ,11 consuSensorineural high frequency hearing loss and coloboma was reported in the British family only ,8.et al., 1976 [et al., 2002 [et al., 1975 [Some authors have reported dysmorphic facial features. Witkop l., 2002 had alsol., 1975 had symmet al., 1975 [Constitutional short stature was diagnosed in a patient followed by Levin l., 1975 . HoweverThe association of sensorineural hearing loss and dental anomalies can be found in other syndromes:\u2022 autosomal recessive sensorineural hearing impairment, dizziness and hypodontia ;\u2022 bilateral sensorineural hearing loss and multiple anterior dens invaginatus ;\u2022 Kantaputra and Gorlin, 1992 [et al., 2002 [OOD was mapped to 20q13.1 within a 12-cM critical chromosomal region.The condition appears to be inherited on an autosomal dominant basis. In the British family described by Vieira l., 2002 , the locet al., 2002 [Sensorineural hearing loss of about 65 dB is found at all frequencies but is more pronounced at about 1000 Hz. It usually plateaus by the fourth decade . The agel., 2002 . Speech Frequent ear abscesses were noted in one patient .Differential diagnostic audiometric test suggested a cochlear site of lesion .et al., 2002 [Eye phenotype was described by Vieira l., 2002 . AbnormaThe gingiva was described as inflamed and enlarged in a patient . GingivaThe oral/dental anomalies affect both the primary and the permanent dentition and could be classified as anomalies of eruption, number, size, shape and structure.There was a significant delay in eruption of the primary and permanent dentition ,16, espeet al., 1988 [Missing teeth, especially premolars, were reported . The prel., 1988 .Large bulbous canines and molars crowns can summarize the clinical intraoral findings.globodontia [et al., 1981 [et al., 1975 [Permanent molars are malformed with fusion of cusps. The crowns of the canines and posterior teeth are enlarged, bulbous and malformed with multiple prominent lobules. The deciduous dentition is more severely involved. The relation between cusps and the major groove is eliminated hence the use of the term bodontia . Cook etl., 1981 describel., 1975 noted laThe canines have been described as large with a marked bulbous cingulum . Based oet al., 1971 [et al., 2001 [The average size premolars had convex occlusal surfaces with no developmental grooves or fossae . In the l., 1971 premolarAn enamel defect (hypoplasia) was frequently found on the buccal surface of canines .et al., 2002 insisted that the incisors were not normal, demonstrating also the yellowing and pitting enamel defects [The teeth might be prone to decay . The ena defects .The molars displayed taurodontism (an inverted crown-body/root ratio). The roots were short and tapered and some were taurodont in configuration . The decet al., 2002 described also the enlarged pulp chambers, the pulp stones and the abnormal root defects [Vieira defects .et al. [Various malocclusions were described in the family members examined by Vieira et al. . Case 1 et al., 1988 [The boy presented by Chen et al., 1984 [Odontomas, the most common type of odontogenic tumors, were reported by Beck-Mannagetta l., 1984 in the pet al., 1984 and Toledo et al., 1971 [The histological findings described by Beck-Mannagetta l., 1971 ,20 showeIn the area of hypoplastic enamel, slightly reduced enamel thickness and alterations existed with prominent enamel rods, irregular incremental lines and rod sheath area containing voids, defects very similar to those observed in hypomaturation enamel defects. The amelodentinal junction in these areas was displaced towards the surface of the tooth and the subjacent underlying dentin had scanty irregular tubules .Genetic counselling is important. Inheritance is clearly autosomal dominant with complete to variaad hoc treatment if necessary.Dental management is complex, interdisciplinary and will include regular follow up, scheduled tooth extraction and eventually orthodontic treatment. Hearing check and, if necessary, hearing aids are mandatory; as well as eye examination and The identification of the gene involved in this disease is of importance in our understanding of the development of various tissues and organs ."} +{"text": "Editorial on the Research TopicYing and Yang Members of the Tumor Necrosis Factor Superfamily: Friends or Foes in Immune-Mediated Diseases and CancerBalomenos et al.; Chakrabandhu and Hueber; Roberts et al.; Saxena et al.; Volpe et al.; Yamada et al.; Yolcu et al.; O\u2019Reilly et al.). Unfortunately, other critical members of the TNF superfamily, including CD40L/CD40, GITR, BAFF, APRIL, TACI, and GITR were beyond the scope of this research topic. However, we refer the reader to scholarly reviews about other members of TNF/TNFR superfamilies that have not been covered here, including their roles in rheumatic diseases , and general aspects of their signaling pathways (Tumor necrosis factor (TNF) and TNF-receptors (TNFR) superfamilies represent one of the earliest immunological systems to be targeted for immunotherapy as typified by the use of anti-TNF\u03b1 agents for treating Crohn\u2019s disease in patients refractory to corticosteroids and conventional immunosuppressives . It is adiseases , neuroinpathways .The articles published under this research topic help to provide a better understanding of the roles of some of the major members of the TNF/TNFR superfamily in the pathogenesis of several diseases and generate considerable hope that their manipulation could lead to therapeutic interventions:Hamad\u2019s paper on the \u201cExpansion of FasL-Expressing CD5+ B Cells in Type 1 Diabetes Patients\u201d discusses a newly identified subpopulation of FasL+ CD5+ CD19+ B cells that was significantly elevated in Type 1 Diabetes (T1D) subjects. In contrast, to IL-10+ CD5+ B cells that do not express FasL but produce IL-10, FasL+ CD5+ cells do not produce cytokines and are more resistant to activation-induced cell death (AICD). These intriguing findings could lead to better understanding of the mechanisms underlying the pathogenic role of FasL in T1D and cell types expressing it , 5.Yamada et al.; Hamad et al.).Yamada\u2019s group\u2019s paper on the \u201cDual Role of Fas/FasL-Mediated Signal in Peripheral Immune Tolerance\u201d uses data from research studies on Fas/FasL gene mutations in mice and humans to discuss the dual function of the Fas pathway signaling leading to apoptosis or cell proliferation of immune cells depending on local environmental circumstances, and how mutations and disruptions of this process leads to the pathogenesis of autoimmunity .Yolcu\u2019s paper on \u201cFas/Fas-Ligand Interaction As a Mechanism of Immune Homeostasis and \u03b2-Cell Cytotoxicity: Enforcement Rather Than Neutralization for Treatment of Type 1 Diabetes\u201d focuses on the immunogenic activities of Fas/Fas-L interactions, as well as how TNF functions in both apoptosis and growth signaling, thereby regulating both immune reactions and injury repair. The paper further details how these processes may be involved in the pathogenesis of T1D and presents arguments for and against therapeutic neutralization of Fas and/or FasL to potentially abrogate T1D .Balomenos\u2019s paper, \u201cOn How Fas Apoptosis-Independent Pathways Drive T Cell Hyperproliferation and Lymphadenopathy in Volpe et al.).Volpe\u2019s paper, \u201cFas-Fas Ligand: Checkpoint of T Cell Functions in Multiple Sclerosis,\u201d discusses Fas\u2013Fas ligand interactions, and their involvement in Activation Induced Cell Death (AICD) and negative selection to destroy autoreactive lymphocytes in the context of autoimmune disease and cancer. In particular, this article focuses on the Fas\u2013FasL pathway and how defects in its interaction involving Th17 and Treg can contribute to the pathogenesis of multiple sclerosis (Chakrabandhu and Hueber).Chakrabandhu\u2019s paper, \u201cFas Versatile Signaling and Beyond: Pivotal Role of Tyrosine Phosphorylation in Context-Dependent Signaling and Diseases,\u201d discusses the role of the Fas/FasL system as an apoptosis activator as well as a source of non-death signals that can promote survival, proliferation, migrations, and cell invasion. This article focuses on the recent identification of tyrosine phosphorylation in the death domain as a regulator of the reversible signaling switch between the two seemingly opposite roles of Fas/FasL, the factors and context that contribute to the switch, and how pathologies can develop in the signaling pathways . Interestingly, upregulation of IL-10 as a result of FasL blockade plays a critical role in preventing islet insulitis in NOD mice bearing a loss-of-function gld mutation of FasL .O\u2019Reilly\u2019s paper \u201cThe Janus Face of Death Receptor Signaling during Tumor Immunoediting\u201d addresses the issue of tumor cells\u2019 ability to proliferate and evade immune-mediated elimination by resisting the death ligand-induced apoptotic pathway through cleavage and mutations in death ligand receptors, or overexpression of decoy receptors and pro-survival proteins. The article discusses the role of death receptor signaling, and the early elimination and escape phase of tumor immunoediting as it relates to the tumor cells\u2019 ability to resist cell death and metastasize. The article also discusses the opportunities and challenges of developing death receptor agonists for cancer therapeutics (In summary, diversity of these studies reveal the complex tasks carried out by TNF superfamily members, which provides a great opportunity for identifying therapeutic targets and at the same time, underlines the challenges.AG and RA read and summarized manuscripts published under the Research topic. AH, HY served as associate editors for the Research Topic and TD read, commented, and edited the editorial.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Inflammation has been widely demonstrated to be involved in various stimuli, such as oxidative stress, bacterial and virus infection, and some physiological process, while a chronic and excessive inflammatory response is a significant risk factor for developing various human diseases .An increasing number of compelling reports are recently published suggesting that some nutrients, like amino acids, oligosaccharides, and short-chain fatty acids, exhibit anti-inflammatory effect , 3, whicThe articles contained in this special issue include 4 review papers and 10 original research papers that are focused on characterizing the contribution and molecular mechanisms associated with nutrients and inflammation. A brief description of these 14 works is detailed below.Amino acids and their metabolites have been widely demonstrated to exhibit anti-inflammatory effect on various inflammatory models, such as inflammatory bowel disease. X. Bao et al. provide a detailed review of the literature on the relationship between amino acids and inflammatory bowel disease in their paper titled \u201cRoles of Dietary Amino Acids and Their Metabolites in Pathogenesis of Inflammatory Bowel Disease.\u201dIn the paper titled \u201cRole of Uric Acid Metabolism-Related Inflammation in the Pathogenesis of Metabolic Syndrome Components Such as Atherosclerosis and Nonalcoholic Steatohepatitis,\u201d A. Kushiyama et al. outline the molecular mechanisms underlying inflammation occurrence in relation to uric acid metabolism.Inflammation contributes to the development of various metabolic diseases, such as obesity and diabetes. S. Chen et al. review the anti-inflammatory properties of quercetin in relation to obesity and type 2 diabetes in the paper titled \u201cTherapeutic Effects of Quercetin on Inflammation, Obesity, and Type 2 Diabetes.\u201dIntestinal microbiota is highly involved in host physiology and pathology through activity of the microbiome and its metabolic products. \u201cDiet-Intestinal Microbiota Axis in Osteoarthritis: A Possible Role\u201d by Y. Li et al. concludes that intestinal microbiota is a major hidden risk factor for osteoarthritis and an important explanation for person-level risk factors.Four groups from R. Garib et al., H. Xiao et al., H. Ni et al., and M. Li et al. investigated the anti-inflammatory effects of amino acids on different inflammatory models. The papers include \u201cEffect of Previous High Glutamine Infusion on Inflammatory Mediators and Mortality in an Acute Pancreatitis Model,\u201d \u201cN-Acetyl-L-cysteine Protects the Enterocyte against Oxidative Damage by Modulation of Mitochondrial Function,\u201d \u201cEffects of Glutamate and Aspartate on Serum Antioxidative Enzyme, Sex Hormones, and Genital Inflammation in Boars Challenged with Hydrogen Peroxide,\u201d and \u201cHigh-Methionine Diet Attenuates Severity of Arthritis and Modulates IGF-I Related Gene Expressions in an Adjuvant Arthritis Rats Model.\u201d\u03baB signaling pathways involve in the mechanism of anti-inflammatory effects of fatty acids from royal jelly.In the article \u201cIn Vitro Anti-Inflammatory Effects of Three Fatty Acids from Royal Jelly,\u201d Y.-F. Chen et al. evaluate and compare the in vitro anti-inflammatory effects of three fatty acids on lipopolysaccharide-stimulated RAW 264.7 macrophages and find that MAPK and NF-Escherichia coli, G. Liu et al. find that dietary chitosan markedly alleviated intestinal inflammation. The paper is titled \u201cChitosan Modulates Inflammatory Responses in Rats Infected with Enterotoxigenic Escherichia coli.\u201dIn rats infected with enterotoxigenic Veronicastrum axillare were collected: \u201cCurcumin Alters Neural Plasticity and Viability of Intact Hippocampal Circuits and Attenuates Behavioral Despair and COX-2 Expression in Chronically Stressed Rats\u201d by G.-Y. Choi et al. and \u201cVeronicastrum axillare Alleviates Lipopolysaccharide-Induced Acute Lung Injury via Suppression of Proinflammatory Mediators and Downregulation of the NF-\u03baB Signaling Pathway\u201d by Q. Ma et al.Traditional medical plants and plant extracts have been widely explored to treat inflammatory diseases. In this issue, two articles about curcumin and A clinical trial titled \u201cThe Effect of Immunonutrition on the Postoperative Complications in Thymoma with Myasthenia Gravis\u201d by Y. Xin et al. concludes that preoperative immunonutrition support is effective in reducing postoperative complications in patients of thymoma with myasthenia gravis.High-mobility group box 1 protein (HMGB1) and autophagy are vital to maintain cellular homeostasis and protect against inflammatory response. In the paper titled \u201cRegulation of Autophagy-Related Protein and Cell Differentiation by High Mobility Group Box 1 Protein in Adipocytes\u201d by H. Feng et al., it focuses on the relationship between HMGB1 and autophagy in adipocytes."} +{"text": "As members of the Ca2+ permeable transient receptor potential (TRP), five of the channels (TRPV1-4 and TRPM2) are activated by different heat temperatures, and two of the channels (TRPA1 and TRPM8) are activated by cold temperature. Accumulating evidences indicates that antagonists of TRPA1 and TRPM8 may protect against cisplatin, oxaliplatin, and paclitaxel-induced mitochondrial oxidative stress, inflammation, cold allodynia, and hyperalgesia. TRPV1 was responsible from the cisplatin-induced heat hyperalgesia and mechanical allodynia in the sensory neurons. TRPA1, TRPM8, and TRPV2 protein expression levels were mostly increased in the dorsal root (DRG) and trigeminal ganglia by these treatments. There is a debate on direct or oxaliplatin-induced oxidative cold stress dependent TRPA1 and TRPV4 activation in the DRG. Involvement of molecular pathways such as cysteine groups, glutathione metabolism, anandamide, cAMP, lipopolysaccharide, proteinase-activated receptor 2, and mitogen-activated protein kinase were also indicated in the oxaliplatin and paclitaxel-induced cold allodynia. In this review, we summarized results of five temperature-regulated TRP channels as novel targets for treating chemotherapy-induced peripheral painAbnormal Ca Chemotherapeutic agents such as taxanes and platinum analogs are used in treatment of several cancer types. Taxanes inhibit progression of mitosis through stabilization of tubulin in the treatment of solid tumors overload plays an important role. Ca2+ enters into cells by different ways including cation channels. Voltage gated calcium channels (VGCC) and chemical channels are well-known calcium channels neurons have important roles in the pathobiology of neuropathic pain. There is no barrier between the DRG and blood, and compounds with a high molecular weight can easily diffuse into the DRG channels .Excessive reactive oxygen species (ROS) and low levels of antioxidants play a pivotal role in the pathobiology of cancers has a significant role in the development and maintenance of neuropathic pain. The involvement of TRPA1 through activation of p38 MAPK in oxaliplatin-induced acute cold hypersensitivity in mice DRG neuron was recently reported is a member of PAR subfamily of G protein-coupled receptors and activation of these receptors regulates several pathophysiological processes including inflammation and pain , menthol, icilin and low-voltage-activated (LVA) channels are very selective to CaTRPV1 is a member of vanilloid subfamily of the TRP superfamily. The channel was firstly expressed in rats through activation of high temperature and pungent hot chili pepper component (capsaicin) in mice DRG (Caterina et al., ROS are produced in physiological levels as part of normal mitochondrial function and phagocytic activity. During the removal of bacteria and viruses, ROS are produced by anti-inflammatory cells such as macrophages microphages and microglia. Therefore, there is direct relationship between increased levels of ROS and inflammatory hyperalgesia (Oehler et al., Cisplatin-induced TRPV1 channel expressions were investigated in DRG neuron by Hori et al. and theyReports on cisplatin-induced thermal sensitivity in rodents are conflicting. For example, cutaneous mechanical allodynia and hyperalgesia but not noxious thermal sensitivity was reported by cisplatin treatment (Hori et al., In a study, diameters of TRPV1 remained unchanged in mice DRG neurons following cisplatin treatment, although the occurrence of TRPV1 in the neurons was increased by cisplatin treatment (Khasabova et al., 2+ concentrations induced release of excessive substance P from the central and peripheral nerve terminals of DRG neurons in response to noxious stimuli (Sacerdote and Levrini, Increased intracellular CaThe involvement of oxaliplatin on the release of calcitonin gene-related peptide from rat sensory neurons in culture was recently reported (Pittman et al., Another member of TRP superfamily is the TRPV2 and the channel is also a member of thermosensitive TRP channels and it is activated by a very high-threshold heat temperature (>52\u00b0C; Ahluwalia et al., As a member of TRP superfamily, TRPV4 was firstly described with mammalian osmotransducer property (Liedtke et al., Induction of hyperalgesia through activation of \u03b12\u03b21 integrin and Src tyrosine kinase pathways in rat DRG neuron was reported in the TRPV4 knockout mice by paclitaxel treatment (Alessandri-Haber et al., Accumulating evidence suggests that neuropathic pain and painful neurotoxicity in the rodents are increased by selected chemotherapeutic agent through increased sensitization of TRPA1, TRPM8, and TRPV1. In addition, antagonists of TRPA1 and TRPM8 were able to attenuate cisplatin, oxaliplatin, and paclitaxel-induced mitochondrial oxidative stress, inflammation, cold allodynia, and hyperalgesia, although TRPV1 was responsible for cisplatin-induced heat hyperalgesia and mechanical allodynia in sensory neurons. TRPA1, TRPM8, and TRPV2 protein expression levels were mostly increased in the DRG and trigeminal ganglia neurons by chemotherapeutic agents. There is a debate on direct or oxaliplatin-induced oxidative cold stress dependent TRPA1 and TRPV4 activation in the DRG. Involvement of molecular pathways such as cysteine group, GSH, anandamide, cAMP, lipopolysaccharide, proteinase-activated receptor 2, and mitogen-activated protein kinase were also indicated in oxaliplatin and paclitaxel-induced cold allodynia. Therefore, there is growing evidence for the potential role of TRP channel inhibitors as modulators of chemotherapy-induced neuropathic pain in the clinic.A new member of the TRP superfamily is TRPM2. The enzyme ADPR pyrophosphatase in the C-terminal domain of TRPM2 is sensitive to ROS and RNS (Wehage et al., MN formulated the present hypothesis and he was responsible for writing the report. NB made critical revision for the manuscript. The original figures were produced by MN.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Editorial on the Research TopicSelf-Eating on Demand: Autophagy in Cancer and Cancer TherapyThe field of autophagy has grown enormously over the past 10\u201315\u2009years, with rapid advances in our understanding of the regulatory mechanisms that control autophagy pathways in mammalian systems, and an improved understanding of the physiological influences of autophagy in health and disease. Supporting such progress, there has been substantial diversification in assessment and modulation tools . SponsorTransAutophagy comprises five different thematic Working Groups with activities designed to synergize and support translation of our ever-advancing basic autophagy knowledge into biomedical and biotechnological applications . TargetiMathiassen et al. (Cecconi\u2019s lab) discusses mounting evidence for new regulatory intersections between autophagy and the cell cycle, which need to be urgently validated in vivo. At the mechanistic level, the tumor suppressor role of autophagy has been ascribed to its vital cell-autonomous functions in mitigating damage and maintaining cellular integrity during metabolic stress. An emerging and intriguing link, which is discussed in the review of Kania et al. (Bultynck\u2019s/Parys\u2019s labs), is the regulation of autophagy in cancer cells through Ca2+ transfer from the ER to mitochondria via the inositol 1,4,5-trisphosphate receptor (IP3R) at ER\u2013mitochondria contact sites. In a developing research area with enormous potential, the impact of miRNA-mediated autophagy regulation on the tumor microenvironment and cancer growth, and their potential as cancer biomarkers and therapeutic targets, is discussed in the review of Gozuacik et al. (Gozuacik\u2019s lab).The review of Viry et al. (Janji\u2019s lab), cancer cell-associated autophagy in hypoxic tumors plays a crucial role in modulating immunosurveillance and in fostering the immunosuppressive tumor microenvironment, by suppressing key mechanisms of innate and adaptive antitumor immunity, thus favoring tumor outgrow and dissemination. Consistent with this pro-tumorigenic role, advanced tumors often display an \u201cautophagy-lysosomal addiction,\u201d which appears to be required to maintain their energy balance through the recycling of intracellular components into biosynthetic pathways or ATP synthesis and to regulate secretion of pro-tumorigenic factors. In the review of New et al. (Tooze\u2019s lab), the idea that advanced and aggressive mutant KRAS-driven tumors exploit a heightened autophagy\u2013lysosomal pathway under the transcriptional control of the MiF/TFE factors to support energy metabolism and to allow growth under conditions of energy deficit and metabolic stress is discussed (Iovanna (Iovanna\u2019s lab) highlights the key role played by the pancreatitis-associated vacuolar protein 1 (VMP1) in pancreatic acinar cells and how its elevated expression drives early autophagy and cooperates with the KRAS oncogene to promote carcinogenesis in the pancreas.In established tumors, elevated levels of autophagy are often associated with poorly oxygenated regions where the demand for nutrients and the need to withstand diverse metabolic stresses are increased. As further discussed in the review of iscussed . FurtherKeulers et al. (Rouschop\u2019s lab), is the ability of advanced cancer cells to use autophagy as a trafficking and export mechanism of pro-tumorigenic factors, such as pro-inflammatory/pro-angiogenic cytokines or chemotactic/pro-invasive molecules. This cancer cell-autonomous trait further illustrates the plasticity of tumor-associated autophagy, which can enable and modulate the crosstalk between cancer and stromal cells thereby affecting the tumor microenvironment, a property that needs to be taken into consideration when considering therapeutic approaches. Based on the growing relevance of tumor-associated autophagy, many labs are developing and testing the effects of autophagy modulators in cancer therapy. The recognition of the prevalent\u2014albeit not unique\u2014cytoprotective and stress adaptation roles of autophagy in advanced cancers has led to the assumption\u2014as supported by in vitro and preclinical data\u2014that blocking cancer cell-intrinsic autophagy may curtail cancer cell resistance to chemotherapy, thereby improving therapy outcome. Thus, the first-generation autophagy blockers, e.g., chloroquine and its derivative hydroxychloroquine (Fulda (Fulda\u2019s lab). Finally, although autophagy is a highly dynamic process, the expression of certain autophagy genes in aggressive tumors like melanoma, may provide novel independent prognostic biomarkers for early stage neoplasms, as discussed in the review of Tang et al. (Lovat\u2019s lab). This may help to identify patients at risk of disease progression, thus facilitating earlier patient therapeutic intervention and stratification for personalized therapeutic approaches.Another emerging aspect linking autophagy to tumor progression, discussed in the review of oroquine , 7 are cAuthors contributed equally.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "International Journal of Molecular Sciences on 11 May 2017 [We read with great interest the article \u201cCirculating Cell-Free DNA and Circulating Tumor Cells as Prognostic and Predictive Biomarkers in Advanced Non-Small Cell Lung Cancer Patients Treated with First-Line Chemotherapy\u201d published by Coco, S. et al. in The authors chose a device which is simple, inexpensive and allows to study the biological characteristics of CTCs isolated from 3 ml of blood. Nevertheless, Haematoxylin\u2013Eosin (H&E) staining alone does not allow a correct enumeration of CTCs, which would necessitate further molecular characterization for cancer-specific biomarkers, such as, in this case, thyroid transcription factor 1 (TTF-1) for adenocarcinomas and high-molecular weight (HMW) keratins (CK5/6) for squamous-cell carcinomas . Indeed, as early as 2013, El-Heliebi et al. reported that reliable detection of CTCs should be confirmed by immunocytochemical and/or molecular biological methods [Furthermore, the association between a higher number of CTCs and better prognosis is definitely uncommon and would deserve further discussion. Our feeling is that, similarly to what is reported by Juan et al. , the autAs a suggestion, the authors could perform a leukocytes depletion before detecting CTCs by using a ScreenCell Cyto kit and subsWe hope that our observation and advice will be accepted as a productive analysis."} +{"text": "Editorial on the Research TopicCD4 T Follicular Helper Cells in HIVFH cells could also hold clues to a functional cure for HIV; as reservoirs of latent HIV during antiretroviral therapy (ART), TFH cells facilitate HIV persistence. This special topic \u201cCD4 TFH cells in HIV\u201d synthesizes reviews from experts in the field to explore, discuss, and share recent discoveries on the intricate relationship between TFH cells and HIV. This special topic also seeks to offer perspectives on areas needing further inquiry in this rapidly evolving field.CD4 T follicular helper cells are vital for induction of long-lived humoral immunity; this specialized function makes them highly desirable immunological targets for designing an effective HIV vaccine. Intriguingly, TFH cells during HIV infection. Leong et al. delineate potential cell intrinsic and extrinsic factors driving viral replication within TFH cells during HIV infection. These include lack of cellular host restriction factors within TFH cells, exclusion of cytolytic CD8 T cells from B cell follicles within germinal centers (GCs), and trapped virus on GC follicular dendritic cells; a constellation of factors that create a perfect storm to promote unfettered viral replication within GC TFH cells.Despite the success of ART, achieving a functional cure for HIV, i.e., sustained viral suppression after ART interruption, remains a challenge due to persistence of viral sanctuaries under ART. As a result, there is a critical need to understand the reservoir to devise effective strategies to purge the latent virus and eliminate life-long dependence on ART. In this special topic, experts in the HIV field synthesize and evaluate recent discoveries in humans and non-human primate model systems to understand the complex dynamics of TFH cells are not depleted as dramatically as other CD4 T cell subsets. Zaunders et al. report a clear increase in proportion of TFH cells compared to other CD4 T cell subsets in serial fine-needle aspirates from lymph nodes of SIV-infected macaques. Miles et al. reveal that an increase in interleukin 6 and interferon gamma and a corresponding reduction in IL-2 could promote TFH proliferation. Hong et al. and Wang et al. argue that a relative increase in TFH cell proportions occurs in the face of net decrease in TFH cells numbers. They attribute the decrease in TFH numbers to increased expression of programmed death-ligand 1 on dendritic cells following infection. Hong et al. also state that an increase in transforming growth factor-b-mediated collagen deposition and fibrosis and loss of fibroblastic reticular cells drives disruption of the lymphoid architecture impairing TFH cell numbers and function. Miles et al. state that an increase in T follicular regulatory cells impairs TFH function in HIV infection. While these observations may seem at odds with each other, given the complex immunopathology of HIV infection and progression to AIDS, it is highly likely that TFH cell numbers and function vary widely over the course of infection and between infected animals and humans. Ultimately, B cell responses are impaired with aberrant B cell activation and hypogammaglobulinemia in the face of poor HIV-specific antibody responses. Chiodi et al. raise the possibility that IL-7, a cytokine whose level is elevated during HIV-1 infection, may have a role in increased expression of B cell costimulatory molecules on TFH cells leading to abnormal B cell differentiation and apoptosis.Surprisingly, despite being hotbeds for viral replication, TZaunders et al. contend that not all immune responses are impotent but that effective immune responses drive virus evolution resulting in Envelope divergence and diversification. The generation of neoantigens in the infected host could recruit naive CD4 T cells, which upon activation drive viral replication leading to eventual depletion of CD4 T cells.However, antibody responses are not compromised in all HIV-infected individuals. In some infected individuals, broadly neutralizing antibodies that have acquired extensive hypermutation and even deletion/insertion mutations appear several years after initial infection. Graff-Dubois et al. assert that TFH cell frequency varies widely according to (i) the stage of HIV/SIV infection, (ii) the severity of the disease, and (iii) the ability to develop broadly neutralizing antibodies highlighting that a blanket increase or decrease in TFH cells is not a unifying characteristic of all HIV/SIV infections. Given the intricate link between virus and lymph node cells, blocking HIV replication in lymphoid tissues might be a prerequisite for induction of effective TFH responses and anti-HIV antibodies and could have the benefit of dramatically decreasing seeding of the latent reservoir. While the lymph node has received a great deal of attention, other compartments are also emerging as critical parameters deserving of attention.In their review, Thornhill et al. review what is known about peripheral(p) TFH cells, which are now emerging as valuable surrogates for GC TFH cells and indicate that the preservation of pTFH cells during HIV infection by early ART initiation is associated with better viral suppression and lack of B cell dysfunction. Moukambi et al. highlight the importance of TFH cells in the spleen, the primary organ for B cell activation and differentiation. Their recent observations indicate early and profound loss of splenic TFH cells. There is also emerging interest in studying TFH cells in mucosal compartments\u2014this understanding of the TFH cell response in distinct tissue compartments with differences in viral dynamics will undoubtedly shed further insights in the relationship of CD4 TFH cells and HIV. Thus, there remains a great deal to learn about TFH cells, and as with HIV, these cells are likely to attract the attention of immunologists and virologists alike.The author confirms being a contributor of this work and approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Molecular Psychiatry, Huang et al. heroically investigated the cortical functional changes following methamphetamine abuse both in animal model and human addicts -induced motor evoked potential (MEPs) (Huang et al., Motor cortex is commonly a neglected region in addiction field. However, neuroimaging findings demonstrated that craving evoked by drug-associated cues involved motor and sensory regions (Yalachkov et al., Cortical plasticity is affected by a number of factors, such as genetic susceptibility to activity-dependent plasticity, trophic factor expression, neurotransmitters (Li Voti et al., Cortical plasticity impairment, however, is not limited to addiction. Previous studies reported that schizophrenia (Fitzgerald et al., All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Brain is the most energetically demanding organ of the body, and is thus vulnerable to even modest decline in ATP generation. Multiple neurodegenerative diseases are associated with decline in mitochondrial function, e.g., Alzheimer\u2019s, Parkinson\u2019s, multiple sclerosis and multiple neuropathies. Genetic variances in the mitochondrial genome can modify bioenergetic and respiratory phenotypes, at both the cellular and system biology levels. Mitochondrial haplotype can be a key driver of mitochondrial efficiency. Herein, we focus on the association between mitochondrial haplotype and risk of late onset Alzheimer\u2019s disease (LOAD). Evidence for the association of mitochondrial genetic variances/haplotypes and the risk of developing LOAD are explored and discussed. Further, we provide a conceptual framework that suggests an interaction between mitochondrial haplotypes and two demonstrated risk factors for Alzheimer\u2019s disease (AD), apolipoprotein E (APOE) genotype and chromosomal sex. We posit herein that mitochondrial haplotype, and hence respiratory capacity, plays a key role in determining risk of LOAD and other age-associated neurodegenerative diseases. Further, therapeutic design and targeting that involve mitochondrial haplotype would advance precision medicine for AD and other age related neurodegenerative diseases. A central role of mitochondria in age-related metabolic and neurodegenerative diseases has long been proposed, and an association between mitochondrial dysfunction and Alzheimer\u2019s disease has been long proposed across multiple investigative strategies from analysis of human tissue to cell model systems Beal, . The \u201cmiHere we review the link between mitochondrial dysfunction and the risk of AD, the contribution of mitochondrial genetic variances to the risk, and how the risk is modulated by other factors, such as apolipoprotein E (APOE) genotype and sex differences.Unlike many other organelles, mitochondria have their own genome. The human mitochondrial genome is a circular set of 16,569 base pairs encoding 37 genes. Thirteen of these genes encode protein subunits required for four of the five electron transport chain complexes: complex I (NADH ubiquinone oxidoreductase), complex III (cytochrome bc1 complex), complex IV (cytochrome c oxidase), and complex V (ATP synthase); two encode rRNAs for mitochondrial ribosomes 12S and 16S), and 22 encode tRNAs activity was observed in both platelets and postmortem AD brain cycle enzymes was observed in AD brain. Autopsy-confirmed AD brain had significantly reduced activity in pyruvate dehydrogenase complex, isocitrate dehydrogenase, and \u03b1-ketoglutarate dehydrogenase complex, whereas the activity of succinate dehydrogenase and malate dehydrogenase were increased than non-J participants and total energy expenditure (TEE) were measured in 294 participants in the health, aging and body composition study . Different sets of haplogroups are identified in different studies and different studies could identify opposite effects of the same mitochondrial haplotype on risk of LOAD. Moreover, multiple studies involving Caucasians of European descendant did not detect associations between any haplogroup and AD (Chinnery et al., APOE\u03b54 genotype is a widely recognized risk factor for AD, and has been repeatedly confirmed in the studies reviewed herein (Corder et al., In longitudinal studies, APOE\u03b54 carriers had significantly greater rCMRglc decline in the vicinity of temporal, posterior cingulate, and prefrontal cortex, basal forebrain, parahippocampal gyrus, and thalamus (Reiman et al., In vitro studies also suggested that truncated APOE\u03b54 fragment can interact directly with mitochondria and cause mitochondrial dysfunction and neurotoxicity (Chang et al., At the cellular level, APOE\u03b54 gene expression in human was associated with down-regulation of genes involved in mitochondrial oxidative phosphorylation and energy metabolism (Xu et al., After stratifying by APOE\u03b54 status, three interesting modes of interactions between APOE\u03b54 status and mitochondrial haplogroups in modulating the risk of AD were apparent (see Table The first mode is a neutralizing effect of mitochondrial haplogroup on the effect of APOE\u03b54 on risk of AD (see Table The second mode is an enabling effect of APOE\u03b54 on mitochondrial genetic variances as risk factors for AD (see Table A synergistic effect was also observed between APOE\u03b54 and mitochondrial haplotypes (see Table As with the association between mitochondrial haplotype and the risk of late onset AD, the interaction between APOE\u03b54 and mitochondrial genetic variances in modulating the risk of late onset AD remains debatable. Multiple studies failed to identify any correlation between mitochondrial haplogroup and APOE\u03b54 status or failed to identify an interaction between the two on the risk of developing AD (Zsurka et al., Females are at greater lifetime risk for LOAD, and also have higher prevalence and incidence rate than all age-matched males (Brookmeyer et al., While almost all the reviewed studies recognized sex differences on risk of LOAD, only a few studied the effects of mitochondrial genetic variances in each sex (van der Walt et al., In contrast, some variances affected only males (see Table These results indicate that previously observed associations between mitochondrial haplogroups and AD could be driven by sex. Conversely, the effects observed in one sex could be diluted in the whole population, or be neutralized by the other sex, resulting in non-significant associations.Mounting evidence suggested a central role of mitochondrial dysfunction in the etiology of late onset AD. As reviewed above, disruption of mitochondrial bioenergetics, structure and dynamics have all been indicated in AD patients. Given the maternal pattern of inheritance of late onset AD and mitochondrial genome, herein we reviewed the association of mitochondrial genetic variances on bioenergetics, respiratory phenotypes and risk of developing LOAD. While the outcomes remain debatable, a large body of science supports an association between mitochondrial genetic variances and differences in bioenergetics and AD susceptibility. Several factors can help explain the disagreement. First, although most studies were conducted in descendants of European origin, the geographic distribution of participants were drastically different, which could result in diverse nuclear genetic background. Since mitochondria communicate extensively with the nucleus, uncontrolled nuclear background can potentially mask effects of mitochondrial genetic variances. Second, as we have reviewed, observations could be driven by certain subhaplogroups, thus results obtained from different populations may be biased by their dominant subhaplogroup. Third, given the heteroplasmic nature of the mitochondrial DNA, accumulated mutations throughout aging may be haplotype and tissue specific Wallace, , which cThe data also indicate that mitochondrial haplotype is one factor among several that impacts risk of AD. This is consistent with the multifactorial nature of aging trajectories and risk for LOAD. A systems biology approach that integrates mitochondrial genetic variances and risk factors such as APOE\u03b54 genotype and sex is a step towards resolving disparities across studies of mitochondrial haplotype and risk of neurodegenerative diseases associated with mitochondrial dysfunction (see Figure YW and RDB wrote and reviewed the manuscript together.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Saccharomyces cerevisiae biosynthesis in heterologous robust hosts to produce a wide array of compounds use of fluorescent compounds as acceptor substrates whose fluorescence signal decreases upon sugar conjugation (Gantt et al., E. coli, S. cerevisiae, and Streptomyces. These biosynthesis approaches include de novo pathway engineering, central carbon flux redirection for co-factor or precursor synthesis, the selection of alternative enzymes, protein engineering/mutagenesis, precursor directed mutasynthesis, modular metabolic engineering, system and synthetic biology tools, such as codon optimization, vector, promoter, ribosome binding site engineering, and the engineering and manipulation of other non-protein-coding sequences (Pandey et al., E. coli. This hurdle can be addressed by regulating intermediate consuming pathway genes in the chromosome of host organisms. Several studies have been reported by our group (Malla et al., E. coli by constructing background E. coli mutants (Kim et al., Recently, advances have been made in the construction of robust microbial cells for the synthesis of several biomolecules in in vivo fermentation approach for producing NP glycosides is inadequate for producing a significant amount of glycosides by simple fermentation even though the process can be easily subjected to industrial scale up. To produce future NP glycosides by fermentation, a robust system should be developed that is capable of producing diverse NDP-sugars in the cell cytosol while engineering promiscuous GTs for sugar transfer to acceptor molecules. High level production of target non-natural NP glycosides can be achieved by simple microbial fermentation upon proper implementation of recently developed system/synthetic biology tools for the engineering of microbial host cells. Cumulatively, this approach of GT-mediated exchange of microbial non-natural glycones with exogenous aglycones offers huge combinatorial potential for the biosynthesis of novel future molecules for human use.The RP and JS wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Innate lymphoid cells (ILCs) are a newly classified family of immune cells of the lymphoid lineage. While they could be found in both lymphoid organs and non-lymphoid tissues, ILCs are preferentially enriched in barrier tissues such as the skin, intestine, and lung where they could play important roles in maintenance of tissue integrity and function and protection against assaults of foreign agents. On the other hand, dysregulated activation of ILCs could contribute to tissue inflammatory diseases. In spite of recent progress towards understanding roles of ILCs in the health and disease, mechanisms regulating specific establishment, activation, and function of ILCs in barrier tissues are still poorly understood. We herein review the up-to-date understanding of tissue-specific relevance of ILCs. Particularly we will focus on resident ILCs of the skin, the outmost barrier tissue critical in protection against various foreign hazardous agents and maintenance of thermal and water balance. In addition, we will discuss remaining outstanding questions yet to be addressed. ILCs have the property of classic lymphoid cells but lack rearranged antigen-specific receptors NKp46 cells, ILCs are conventionally divided into three groups based on their differential expression of effector cytokines and developmental requirements for transcription factors cells were also described and Peyer\u2019s patches has long been recognized (De Togni et al., + ILC3s, can induce inflammation in the intestines of both mice and humans (Bernink et al., Although ILCs normally play host-protective roles, dysregulated activation of ILCs could lead to inflammatory diseases (Artis and Spits, Chronic ILC2 activation was reported to contribute to a large variety of tissue inflammatory disorders such as asthma in the lung and atopic dermatitis in the skin, which are generally associated with over-production of the type 2 inflammatory cytokines such as IL-4, IL-5, and IL-13. ILC2s expand and produce the type 2 cytokines in mice with allergic lung inflammation (Chang et al., \u2212 cells within human colorectal carcinoma tumors (Kirchberger et al., ILC3s could contribute to inflammatory diseases and tumorigenesis in various tissues. IL-17- and IL-22-producing ILC3s have been linked with the inflammatory skin disease psoriasis, inflammatory bowl disease, obesity-associated asthma, and multiple sclerosis (Buonocore et al., in vitro development analysis (Scoville et al., Specific functions of ILCs in homeostasis and inflammation are associated with their unique localization in peripheral tissues. Recently, several groups reported that ILCs are predominantly tissue-resident cells that do not circulate in the body. In parabiotic studies, helper-like ILCs in both non-lymphoid tissues and lymphoid organs such as the small intestine, salivary gland, lung, mensenteric lymph node, and adipose tissue of two joined adult mice with shared blood circulation systems remain locally and do not move from one mouse to another (Gasteiger et al., + ILC1-type cells although their origin and lineage association are not known (Almeida et al., + and NCR\u2212 subsets, account for about 50% of total skin ILCs in humans (Dyring-Andersen et al., Skin is the outmost barrier tissue constantly exposed to assaults of various foreign agents. Skin is enriched with ILCs and ILCs of all the three groups could be found in the skin (Yang et al., + dendritic cells are involved in the programming of skin-homing CCR10+ ILCs in skin-draining lymph nodes (Yang et al., Since ILCs share many common regulatory pathways with helper T cells for their development and function, we investigated whether skin-specific ILCs are programmed in skin-draining lymph nodes to acquire their skin-homing property for establishment of their skin residency, paralleling the process by which the skin-homing property of conventional \u03b1\u03b2 T cells is imprinted in skin-draining lymph nodes (Yang et al., + ILCs in skin-draining lymph nodes, local inflammatory conditions suppress programming of skin-homing CCR10+ ILCs (Yang et al., Dependent on environmental cues of lymphoid organs in which T cells and ILCs are activated, they could be also imprinted to acquire homing properties to other distinct peripheral tissues (Campbell and Butcher, Compared to the healthy skin, ILC2s are increased in the skin of patients with atopic dermatitis (Kim et al., + ILC3s are increased in both lesional and non-lesional skin and NKp44\u2212 ILC3s have the potential to differentiate to NKp44+ ILC3s in response to stimulation of IL-1\u03b2 and IL-23 (Teunissen et al., + ROR\u03b3t+CD56+ ILC3s were found in the psoriatic skin in a separate study (Dyring-Andersen et al., Increasing evidence implicates involvement of ILC3s in psoriasis, a skin inflammatory disease largely resulting from overproduction of Th17-associated cytokines such as IL-17A (Dyring-Andersen et al., While ILCs are relatively well studied for their participation in development of skin inflammatory diseases, their physiological role in maintenance of the skin homeostasis and host protection is still poorly understood. Since ILC2s are capable of producing amphiregulin, a member of epithelial growth factor family, they could potentially contribute to wound healing (Salimi et al., + T cells and dampen their responses to microbiota (Hepworth et al., + ILCs and T cells plays a critical role in maintaining the immune homeostasis of the skin (Yang et al., Rag1\u2212/\u2212 mice that lack T and B cells, there are significantly reduced CCR10+ ILCs in the skin, while transfer of CD4+ T cells could partially restore the homeostatic presence of CCR10+ ILCs (Yang et al., Il2rg\u2212/\u2212Rag2\u2212/\u2212 mice, which lack all ILCs as well as T/B cells, donor CD4+ T cells in skin of hosts contain significantly higher percentages of IL-17A-producing Th17 and IL-5-producing Th2 cells but much lower percentages of IFN\u03b3-producing Th1 cells than those transferred into Rag1\u2212/\u2212 recipients that have ILCs (Yang et al., Skin ILCs interact with other skin-resident immune cells to maintain the immune homeostatic condition. In the steady-state skin, the preferential interaction of ILC2s and mast cells has been detected (Barnig et al., The research over past several years has started to reveal roles of ILCs in the skin and unravel mechanisms regulating establishment and function of the skin-resident ILCs. However, several major questions remain to be addressed. First, physiological functions of skin-resident ILCs in host protection and immune homeostatic maintenance are still poorly understood. While ILCs have been generally implicated in protection against infection, control of commensal bacteria and regulation of immune activation in various barrier tissues, the function of resident ILCs specifically in the skin is not well studied. Considering that ILCs are maintained locally in different barrier tissues, functions and regulation of ILCs in the skin and other barrier tissues are likely regulated differently with local environmental cues. For example, the CCR10/CCL27 axis is involved in the regulation of resident ILCs in the skin but not other sites (Yang et al., + ILCs are dominant in the healthy skin where they promote balanced maintenance of resident T cells (Yang et al., \u2212/\u2212, Foxp3\u2212/\u2212 or topical Calcipotriol-treated wild-type mice, CCR10+ ILCs are decreased while CCR10\u2212 ILCs are increased in the skin (Yang et al., + and CCR10\u2212 ILCs might be differently involved in skin immune homeostatic regulation and activation (Fig.\u00a0+ and CCR10\u2212 skin ILCs are regulated and function should help understand basic biology of the skin-specific ILCs in the health and disease.Second, mechanisms regulating differential involvement of ILCs in the skin homeostasis and inflammation are largely unknown. Studies of ILCs of the skin and other barrier tissues have showed that ILCs could be involved in both homeostatic regulation and tissue inflammatory processes. However, it is currently unknown whether and how ILCs involved in homeostatic regulation are different from ILCs involved in immune inflammatory processes in the skin. Our studies have found that CCR10+ ILC1s to ILC3s (Bernink et al., Third, the involvement of ILCs in promoting diseases has raised interest in modulating ILC functions for the treatment of diseases. In other tissues, antibodies against ILC2-activating cytokines, such as IL-25 and IL-33, as well as those targeting ILC2-produced cytokines, including IL-5 and IL-13, have been found to suppress ILC2 proliferation and function in tissue inflammatory diseases in the lung (Gauvreau et al.,"} +{"text": "Phospholipids represent the highly conserved structural basis of biological membranes from bacteria to humans. However, plants and other photoautotrophic organisms are unique in using non-phosphorus galactolipids as primary components of their photosynthetic membranes. In light of the biomass of green tissues as compared with that of the overall plant body and the highly stacked thylakoid membrane structures in chloroplasts, galactolipids are the most abundant membrane lipids on the earth. Historically, the roles of galactolipids have been studied mainly in relation to photosynthesis, and recent advances in molecular biology with Arabidopsis and other model organisms have revealed an essential role of galactolipids in photosynthesis. However, these galactolipids are also abundant in non-photosynthetic organs, especially flowers, which suggests their distinct role apart from photosynthesis. The aim of this mini-review is to describe distinct biochemical properties of flower galactolipids and possible new roles, with a summary of the current understanding of galactolipid biosynthesis in Arabidopsis.The online version of this article (doi:10.1186/1999-3110-54-29) contains supplementary material, which is available to authorized users. Its activity was initially observed as a major DGDG synthetic activity in isolated chloroplasts or envelope preparations (van Besouw and Wintermans, SENSITIVE TO FREEZING 2, a gene essential for freezing tolerance in Arabidopsis, encodes a processive galactosyltransferase that produces oligogalactolipids by transferring galactose groups from MGDG (Moellering et al., Because of the odd enzymatic property of GGGT, its identity has long been an issue. Recently, Moellering and the Benning group found that Petunia hybrid to explore yet-unknown roles of galactolipids in flowers (Figure\u00a0Given that chloroplasts are differentiated as a form of plastids, an important question was whether galactolipids are unique in chloroplasts or are widely found in different plastids. Biochemical studies found significant levels of MGDG and DGDG in non-photosynthetic organs (Kleinig and Liedvogel, s Figure\u00a0 (Nakamurs Figure\u00a0; (Nakamus Figure\u00a0; (Nakamus Figure\u00a0.Figure 3The level of MGDG in petals is about one third of that in leaves (Nakamura et al., 2003dad1, which is deficient in anther dehiscence, is mutated in a gene encoding lipase that liberates 18:3 fatty acid from the glycerlipid backbone for jasmonic acid synthesis (Ishiguro et al., Among the 3 floral organs, stamens show the highest MGDG levels and the level is about half that in leaves (Nakamura et al., Pistils are the most unique among the floral organs we discuss in terms of predominant DGDG levels relative to that of MGDG (Nakamura et al., Galactolipids can be utilized even during pollen tube growth. Glycerolipid profiling of lily pollen tubes before and after elongation revealed a 5.7-fold increase in DGDG level and 2.8-fold increase in MGDG level (Nakamura et al., mgd1 mutant is challenging because it has a lethal phenotype (Kobayashi et al., Galactolipid biosynthesis is well understood in the model system Arabidopsis. Meanwhile, ample biochemical data from different botanical flowers show intriguing features of galactolipid biosynthesis in floral organs. Although the functional difference between type A and B MGD is clear and type B is more involved in MGDG synthesis in flowers, double knockout of MGD2 and MGD3 does not result in defective flower development nor MGDG level (Kobayashi et al.,"} +{"text": "Neuroinflammation plays a critical role in the onset and progression of many neurological disorders, including Multiple Sclerosis, Alzheimer's and Parkinson's diseases. In these clinical conditions the underlying neuroinflammatory processes are significantly heterogeneous. Nevertheless, a common link is the chronic activation of innate immune responses and imbalanced secretion of pro and anti-inflammatory mediators. In light of this, the discovery of robust biomarkers is crucial for screening, early diagnosis, and monitoring of neurological diseases. However, the difficulty to investigate biochemical processes directly in the central nervous system (CNS) is challenging. In recent years, biomarkers of CNS inflammatory responses have been identified in different body fluids, such as blood, cerebrospinal fluid, and tears. In addition, progress in micro and nanotechnology has enabled the development of biosensing platforms capable of detecting in real-time, multiple biomarkers in clinically relevant samples. Biosensing technologies are approaching maturity where they will become deployed in community settings, at which point screening programs and personalized medicine will become a reality. In this multidisciplinary review, our goal is to highlight both clinical and recent technological advances toward the development of multiplex-based solutions for effective neuroinflammatory and neurodegenerative disease diagnostics and monitoring. Neurological disorders account for an increasing number of disability-adjusted life-years worldwide, especially in high-income countries. Alzheimer's disease, Parkinson's disease and Multiple Sclerosis are the most prevalent causes of neurological disability cleavage of amyloid precursor protein] and its clearance, leading to the formation of toxic oligomers (A\u03b2O), subsequent synaptic dysfunction and neuronal cell death the formation of amyloid plaques is promoted by an increased production of A\u03b21\u221242, while in sporadic AD it is mainly due to impaired A\u03b2 clearance is a neurodegenerative disorder primarily affecting neocortical regions and characterized by progressive episodic memory loss leading to significant behavioral changes deposition imaging. Biomarker analysis, including \u03b1-syn, in serum or CSF, is not performed in standard clinical practice , GBA (glucocerebrosidase), PRKN (parkin), and LRRK2 (leucine-rich repeat kinase 2) which are expressed in microglia , the second most common neurodegenerative disorder, is characterized by the early and progressive loss of dopaminergic neurons in the Multiple Sclerosis (MS) is a chronic inflammatory demyelinating disorder of the CNS of unknown etiology but with a genetic predisposition and environmental influence or \u201cinside-out\u201d (CNS-intrinsic initiation of the inflammatory cascade) models which specifically recognizes the biomarker of interest and a transducer which converts biochemical interactions into a quantifiable electrical signal proportional to biomarker concentrations. Biosensors are commonly classified in electrochemical, optical, piezoelectric, or magnetic, based on the signal transduction mechanism. These technologies have broad applications in health provided by the use of gold nanoparticles (NPs) , which mediates the neuronal binding and toxicity of A\u03b2O, was used as a recognition element for the specific detection of the oligomers. The biosensor presented a LOD of 0.5 pM and successfully detected cell-derived A\u03b2O from conditioned media of 7PA2 Chinese Hamster Ovary (CHO) cells that naturally secrete A\u03b2O based on its oxidation and subsequent aggregation into NPs (polydopamine) (Yildirim and Bayindir, 2 nanotube arrays for sensitive \u03b1-syn quantification with a detection limit of 34 pg/mL (An et al., substancia nigra (Choi et al., 2 NPs (Heydari-Bafrooei et al., Although \u03b1-syn has been the most intensely studied and recognized biomarker for PD, its application in biosensing is very limited. A photoelectrochemical biosensor was developed by An et al. based on Au-doped TiOThe heterogeneous nature of MS, characterized by distinct patterns associated with the demyelination process, makes it highly improbable that a single diagnostic marker is capable of covering the full spectrum of MS subtypes (Lucchinetti et al., in situ early diagnostics of neuroinflammatory and neurodegenerative diseases. Altogether, these advantages will surely bring great benefits for both academic and medical fields.An increasing number of studies are uncovering the beneficial and detrimental roles of microglia in neurodegenerative disease onset and progression. Pro-inflammatory cytokines can be used in combination with classical biomarkers for neurodegenerative and neuroinflammatory disease diagnostics and monitoring of disease progression. Technologies for simultaneous detection and quantification of different biomarkers are rapidly developing and future devices are aimed at bringing valuable advantages, specifically related to lower sample volumes, detection time and limits, higher specificity and sensitivity. Decreasing the need for biological samples processing, while integrating biosensing platforms in portable lab-on-a-chip systems would, in turn, allow point-of-care use by semi-skilled operators toward real-time and All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "The explosive growth of health-related data presented unprecedented opportunities for improving health of a patient. Machine learning plays an essential role in healthcare field and is being increasingly applied to healthcare, including medical image segmentation, image registration, multimodal image fusion, computer-aided diagnosis, image-guided therapy, image annotation, and image database retrieval, where failure could be fatal.The purpose of this special issue is to advance scientific research in the broad field of machine learning in healthcare, with focuses on theory, applications, recent challenges, and cutting-edge techniques.The quality level of the submissions for this special issue was very high. A total of 24 manuscripts were submitted to this issue in response to the call for papers. Based on a rigorous review process, 8 papers (33%) were accepted for the publication in the special issue. Below, we briefly summarize the highlights of each paper.One of the papers of this special issue, \u201cDiagnosis of Alzheimer's Disease Based on Structural MRI Images Using a Regularized Extreme Learning Machine and PCA Features,\u201d R. K. Lama et al. proposes a method and compared Alzheimer disease (AD) diagnosis approaches using structural magnetic resonance (sMR) images to discriminate AD, mild cognitive impairment, and healthy control subjects using a support vector machine (SVM), an import vector machine (IVM), and a regularized extreme learning machine (RELM). By means of experiments on the ADNI datasets, it has been concluded that RELM with the feature selection approach can significantly improve classification accuracy of AD from mild cognitive impairment and healthy control subjects.S. Alam et al. presented a method for distinguishing AD from healthy control using combination of dual-tree complex wavelet transforms, principal coefficients from the transaxial slices of MRI images, linear discriminant analysis, and twin support vector machine in their article \u201cTwin SVM-Based Classification of Alzheimer's Disease Using Complex Dual-Tree Wavelet Principal Coefficients and LDA.\u201dA semisupervised learning approach for cell detection is presented in the third article of this special issue by N. Ramesh et al. in their paper entitled \u201cCell Detection Using Extremal Regions in a Semisupervised Learning Framework.\u201d The method requires very few examples of cells with simple dot annotations for training.In the paper entitled \u201cPatient-Specific Deep Architectural Model for ECG Classification\u201d by K. Luo et al., a method for ECG classification is proposed. The method is based on time-frequency representation and patient-specific deep learning architectural model, and it uses deep neural network classifier.An automatic method for segmentation of 3D magnetic resonance imaging (MRI) data, useful in the clinical diagnosis of brain tumor, named as Glioma is presented by Z. Li et al. in the article \u201cLow-Grade Glioma Segmentation Based on CNN with Fully Connected CRF.\u201d The method combined a multipathway convolutional neural network (CNN) and fully connected conditional random field (CRF). Experimental results have shown that the method is useful for low-grade glioma.A general system for hybrid disease diagnosis adopting classifier optimization procedure using evolutionary algorithms is presented by M. R. Nalluri et al. in their article \u201cHybrid Disease Diagnosis Using Multiobjective Optimization with Evolutionary Parameter Optimization.\u201dY. Chou et al., in their paper \u201cA Real-Time Analysis Method for Pulse Rate Variability Based on Improved Basic Scale Entropy,\u201d proposed a method named sliding window iterative base scale entropy analysis by combining base scale entropy analysis and sliding window iterative theory for analyzing heart rate variability signal.Another paper of this special issue by J.-S. Park et al. titled \u201cR Peak Detection Method Using Wavelet Transform and Modified Shannon Energy Envelope\u201d presents an R peak detection method using the wavelet transform and a modified Shannon energy envelope for rapid ECG analysis.These 8 selected contributions basically can reflect the new achievements in the machine learning applications in healthcare, and we hope they can provide a solid foundation for future new approaches and applications."} +{"text": "Iron (Fe) is an essential plant micronutrient but is toxic in excess. Fe deficiency chlorosis is a major constraint for plant growth and causes severe losses of crop yields and quality. Under Fe deficiency conditions, plants have developed sophisticated mechanisms to keep cellular Fe homeostasis via various physiological, morphological, metabolic, and gene expression changes to facilitate the availability of Fe. Ethylene has been found to be involved in the Fe deficiency responses of plants through pharmacological studies or by the use of ethylene mutants. However, how ethylene is involved in the regulations of Fe starvation responses remains not fully understood. Over the past decade, omics approaches, mainly focusing on the RNA and protein levels, have been used extensively to investigate global gene expression changes under Fe-limiting conditions, and thousands of genes have been found to be regulated by Fe status. Similarly, proteome profiles have uncovered several hallmark processes that help plants adapt to Fe shortage. To find out how ethylene participates in the Fe deficiency response and explore putatively novel regulators for further investigation, this review emphasizes the integration of those genes and proteins, derived from omics approaches, regulated both by Fe deficiency, and ethylene into a systemic network by gene co-expression analysis. Iron (Fe) is an essential element for living organisms including plants. Fe-containing proteins play a variety of vital roles in cellular respiration, intermediary metabolism, oxygen transport, and DNA stability and repair, as well as photosynthesis in plants. In human beings, Fe deficiency causes severe healthy problems, including anemia, which affects billions of people worldwide . The first step in this strategy is the acidification of the rhizosphere mediated by the H+-translocating P-type ATPase AHA2 transcription factor FER-LIKE Fe DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (FIT) , have been carried out in a broad range of plant species, including model strategy I plant Arabidopsis, Medicago, soybean, as well as Strategy II plant rice and others from the four data sets, herein named Fe deficiency-response core genes, with 61 being up-regulated and 10 down-regulated from S-adenosylmethionine (SAM), underlying the importance of controlling the expression of NAS genes including NAS1 gene recycling for ethylene synthesis, is encoded by single gene in Arabidopsis genome, indicating the importance to tightly control its expression oxygenase family; proteins in this family is generally considered to possess oxidoreductase activity catalysing the 2-oxoglutarate- and Fe(II)-dependent oxidation of an organic substrate using a dioxygen molecule. Previous study showed that the ethylene synthesis protein ACC oxidase also belongs to 2OG-Fe(II) oxygenase superfamily , functioning as ferric iron binding and participating in the cellular iron ion homeostasis, are essential to protect cells against oxidative damage and flowering was the gene AT3G13610 encoding a Fe (II)- and 2-oxoglutarate-dependent dioxygenase family gene F6'H1 and Fe (II)-dependent oxygenase superfamily protein putatively assumed to be involved in hormone metabolism by MapMan analysis. The gene co-expressed with AT3G12900 is AT5G38820, whose expression is also regulated by ethylene , and this study does have uncovered new layer of regulation in gene activity in response to Fe deficiency that were induced in Brassica napus phloem sap upon Fe deficiency, which is consistent with the decrease of ACC content (Gutierrez-Carbonell et al., Medicago truncatula under Fe limitation (Rodriguez-Celma et al., Although nearly no protein with changes in abundance upon Fe deficiency has been uncovered across a wide range of plant species, a comprehensive comparison of these studies indeed has revealed some common elements in proteome under Fe limitation. In brief, proteins associated with \u201coxidative stress and defense,\u201d \u201cC metabolism,\u201d \u201cN metabolism,\u201d \u201ccell wall,\u201d \u201csecondary metabolism, particularly the phenylpropanoid metabolism,\u201d \u201cenergy and ATP-coupled transport processes,\u201d and \u201cprotein metabolism\u201d have been identified as differentially accumulated proteins among plant species, which is well-summarized in an excellent review published in 2013 (Lopez-Millan et al., Omic approaches have been widely employed to explore the responses of plant to Fe deficiency, and have uncovered diverse metabolic adaptations upon Fe starvation. A subset of conserved Fe-responsive genes and some common metabolic pathways have been revealed by transcriptome and proteome across a range of plant species. It has been clear that the concordance between the abundance of mRNA and their related proteins is not strong correlated (Lan et al., All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Stroke is an important cause of morbidity and mortality as well as functional impairment particularly in the elderly people . PredictSeveral previously published studies found an association between high serum CRP level and development of stroke , 8-10. RSimilarly, Ridker et al., found a positive correlation between serum hsCRP and severity of stroke . In a pr"} +{"text": "Over the last decade, an increasing number of reports underscored the importance of epigenetic regulations in brain plasticity. Epigenetic elements such as readers, writers and erasers recognize, establish, and remove the epigenetic tags in nucleosomes, respectively. One such regulation concerns DNA-methylation and demethylation, which are highly dynamic and activity-dependent processes even in the adult neurons. It is nowadays widely believed that external stimuli control the methylation marks on the DNA and that such processes serve transcriptional regulation in neurons. In this mini-review, we cover the current knowledge on the regulatory mechanisms controlling in particular DNA demethylation as well as the possible functional consequences in health and disease. Among several other epigenetic tags, methyl tags on the DNA were generally considered as repressive marks. However, an increasing number of studies showed that the DNA methylation at intergenic regions as well as gene regulatory regions might enhance gene expression in vivo 45 proteins (GADD45A and GADD45B) that in neurons take part in active DNA demethylation processes was first described in the 1972 or apolipoprotein B mRNA editing complex (APOBEC) is an alternative path to successive oxidation reactions by TET enzymes for the initiation of DNA demethylation Figure . The modBdnf gene that is quite well investigated in the context of synaptic plasticity and learning , methyl CpG binding protein 2 (MeCP2), histone deacetylases (HDAC) 1 and 2. The repressor complex dissociates following phosphorylation of MeCP2 and nitrosylation of HDAC2 in response to Ca+2 influx (Chen et al., Bdnf IV in a stimulus-dependent manner (Ratnu et al., In the long-lived nature of postmitotic neurons, genomic stability needs to be maintained for decades while at the same time their remarkable plasticity has to be kept at a poised state ready to respond (Baker-Andresen et al., Gadd45b gene expression (Keeley et al., Unfortunately, conflicting reports have been published on the role of GADD45B in learning and memory processes. Fear conditioning induces Interestingly, 5hmC is not only an intermediate DNA demethylation form but also an epigenetic mark on its own, which is enriched within promoters and gene bodies (Kaas et al., Another critical issue is cell-type specificity of DNA demethylation. In most studies brain tissue that contains different neuronal and glial cell types was used. The current knowledge on how the DNA demethylation machinery functions in different cell-types and responds to neuronal activity is therefore very limited. DNA methylation patterns vary between neurons and non-neuronal cells. Ventromedial prefrontal cortex neurons have higher global DNA methylation levels compared to non-neuronal cells (Li et al., Bdnf IX promoter was found, which is in line with reduced Bdnf IX expression (Gavin et al., Arc was also associated with reduced levels of GADD45B and DNA demethylation in this stress model (Grassi et al., Bdnf IX promoter methylation level is abolished and mRNA expression is perturbed (Ma et al., Bdnf gene expression (Dong et al., Given the principal functions of chromatin modifications in regulating gene transcription programs, it\u2019s not surprising that the number of studies, which report the involvement of DNA demethylation machinery in neurological disorders, is steadily increasing. Enhanced GADD45B levels were reported in two different cohorts of major psychotic patients (Gavin et al., Based on the initial GADD45B-dependent demethylation hypothesis (Gavin et al., GB and MK are invited to contribute to the article collection for the special issue of the \u201cEpigenetic Mechanisms Regulating Neural Plasticity\u201d. GB outlined the mini review and GB and MK wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "However, this notion contrasts with the mild phenotype associated with tau deletion. Instead, an analysis of cellular hallmarks common to different tauopathies, including aberrant patterns of protein phosphorylation and early degeneration of axons, suggests that alterations in kinase-based signaling pathways and deficits in axonal transport (AT) associated with such alterations contribute to the loss of neuronal connectivity triggered by pathogenic forms of tau. Here, we review a body of literature providing evidence that axonal pathology represents an early and common pathogenic event among human tauopathies. Observations of axonal degeneration in animal models of specific tauopathies are discussed and similarities to human disease highlighted. Finally, we discuss potential mechanistic pathways other than microtubule destabilization by which disease-related forms of tau may promote axonopathy.Tauopathies are a diverse group of diseases featuring progressive dying-back neurodegeneration of specific neuronal populations in association with accumulation of abnormal forms of the microtubule-associated protein tau. It is well-established that the clinical symptoms characteristic of tauopathies correlate with deficits in synaptic function and neuritic connectivity early in the course of disease, but mechanisms underlying these critical pathogenic events are not fully understood. Biochemical Proper brain function relies on appropriate connectivity between specific neuronal populations. An essential cellular process underlying such connectivity involves the generation and continued maintenance of molecular constituents within axons and dendrites , frontal temporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), chronic traumatic encephalopathy (CTE), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), and Pick's disease (PiD) and glial cells (tufted astrocytes and coiled bodies) is characterized by severe atrophy of the frontal, temporal, and parietal lobes. Cytoplasmic neuronal tau inclusions known as Pick bodies represent the major neuropathological hallmark of PiD, and these are typically found in the dentate gyrus of the hippocampus and frontal and temporal cortices promoter, which provides relatively selective expression of tau in forebrain neurons vectors encoding P301L tau into the EC (Siman et al., Collectively, data from various animal models of tauopathy reveal axonal degeneration as a prominent pathological feature, and further suggest there is a contribution to this phenotype from glial cells. Based on their resemblance to this specific aspect of human tauopathies, these animal models may help define mechanisms and specific molecular components mediating the toxic effect of pathogenic tau on axonal maintenance and function.in vitro (Weingarten et al., in vivo has yet to be provided.The initial discovery of tau protein was based on biochemical procedures that revealed its ability to associate with purified microtubules and modulate their dynamic growth behavior in vivo. Furthermore, axonal development is normal in cultured primary neurons obtained from some tau knockout mice (Harada et al., Collectively, data from the available four transgenic tau knockout mouse lines (reviewed in Ke et al., Available experimental evidence suggests that tau performs functions other than microtubule stabilization in neurons. Numerous studies suggest a role for tau in multiple cellular processes and compartments, some of which may not include microtubule interactions. The functional repertoire of tau includes microtubule-actin cytoskeleton interactions (Selden and Pollard, In vitro studies using purified components of the AT system further supported a model where tau would elicit this effect by competing with conventional kinesin heavy chain subunits for microtubule binding (Seitz et al., in vivo extend the conclusions from squid axoplasm studies to mammalian neurons, showing normal AT rates in the optic nerve of tau-overexpressing mice (Yuan et al., The dependence of axons on proper anterograde and retrograde AT is evident because mutations in selected conventional kinesins or cytoplasmic dynein subunits suffice to promote dying-back degeneration of neurons (Morfini et al., in vitro and in situ also appears inconsistent with the notion that tau physically blocks motor proteins (Samsonov et al., The highly dynamic nature of the interaction between tau and microtubules The major motor proteins responsible for AT, conventional kinesin and cytoplasmic dynein, are regulated by specific protein kinases providing a novel mechanism linking pathological forms of tau to deficits in AT (Kanaan et al., 2+ dysregulation may negatively impact axonal connectivity (Figure 2+ from the endoplasmic reticulum, leading to inhibition of synaptic vesicle exocytosis and synaptic transmission through a mechanism involving PAD exposure and GSK3 activation (Figure 2+ imbalance and cell death in induced pluripotent stem cells (Imamura et al., 2+ levels by directly inhibiting plasma membrane Ca2+ ATPase (Berrocal et al., 2+ buffering in cells (Werth and Thayer, 2+ levels (reviewed in Eckert et al., 2+ levels may promote abnormal activation of calcium-activated proteases and proteolysis of critical cytoskeletal protein components (Figure In addition to the mechanisms above, mechanisms linking pathological tau to Cay Figure . For exan Figure . Oligomen Figure and longn Figure in cultun Figure . Studiess Figure . Consists Figure , and inhs Figure . Interess Figure . Collect2+ homeostasis, altered glial function, and others (Figure The landscape of tauopathies is marked by significant heterogeneity in clinical presentation, cellular topography, and neuropathological features. Despite this diversity, an examinations of human brains and animal models of tauopathies reveal axonopathy as a common feature of these diverse diseases (Hyman et al., s Figure . DevelopAK, BC, KC, GM, and NK all contributed equally with writing, editing and figure preparation for the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Malaria has remained a greatest health and socioeconomic burden in the tropical and subtropical regions of the world. According to World Health Organization, approximately, 429,000 deaths with 212 million cases were reported from malaria in the world , China , and Pakistan and it shares the shortest land border with Afghanistan (106 Km) among all the bordering countries of India , China (79%), Nepal (78%), and Bhutan (60%) while its prevalence in Myanmar and Bangladesh is 34 and 7%, respectively followed by Myanmar (66%), Bhutan (40%), Nepal (22%), Pakistan (19%), China (11%), and Afghanistan (5%) in the northeast region of India of hanistan 06 Km gene in this fatal human malaria parasite gene gene amplification were characterized by increased susceptibility to chloroquine but decreased susceptibility to mefloquine (Imwong et al., pvmdr1 in conferring resistance to chloroquine is still elusive and controversial (Faway et al., pvmdr1 gene copies correlated with mefloquine use history, in vivo data showing a direct relationship between mefloquine resistance and pvmdr1 gene amplification form mefloquine-treated patients are needed (Khim et al., P. vivax.i et al. . Howeveri et al. . AccordiKinley Wangdi and colleagues have highlighted the malaria elimination efforts carried out by the neighboring countries, however, they did not mention the Malaria Control Programme of Pakistan bordering Gujarat and Rajasthan, the high malaria transmission western states of India (Wangdi et al., Wangdi and colleagues have overlooked the malaria deaths and prevalence in children. According to WHO, \u201cA child dies of malaria every 2 minutes\u201d (World Health Organization, All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Shen Yuan et al. highlighIn the discussion, the authors state: \u201cOur study is the first meta-analysis investigating the protective role of chocolate consumption against diabetes.\u201dHowever, this represents a slight imprecision; in 2011, Buitrago-Lopez et al. [Another interesting difference between the two studies is that Shen Yang et al. found a chocolate protective effect occurred in the category of moderate chocolate consumption (<6 serving/week), while Buitrago-Lopez et al. found that a higher chocolate consumption (definitions of which vary from the number of servings per week or grams per day) confers the lowest incidence of diabetic and cardiometabolic events. Such differences deserve a detailed exploration in order to delimitate the ideal chocolate recommendation which provides optimal flavonoid amounts for a cardiometabolic protective effect without"} +{"text": "Tsianos et al. demonstrThe possibility of combining neuroimaging and/or eye-tracking with visual search paradigms offers a promising avenue for cognitive style research. Visual search tasks can investigate the allocation of attention during task completion (i.e., Galpin and Underwood, We also argue that future cognitive styles research would benefit from not only adopting a mixed method experimental approach, but also from investigating other dimensions of cognitive style beyond the visualizer\u2013verbalizer dimension. For instance, it has been shown that individual differences in brain structure and function are related to preferences in field-dependence/field-independence (Hao et al., Whilst some authors argue that cognitive styles are more dynamic than static, so can change or alter Zang, , others A decade has passed since Coffield et al.'s heavy crRB, opinion concept, main conclusions, article drafting; AG, LM, and SC verification of opinion concept, main conclusions, article drafting.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "IUCrJ (2017), 4, 108\u2013118] discuss progress on using in-cell NMR in prokaryotic and eukaryotic cell preparations.High-resolution NMR provides increasing possibilities to probe biomolecular structure in the native state. In this issue, Luchinat and Banci [ One of the unique properties of magnetic resonance (MR) has been its ability to non-invasively yield molecular information from the test tube to applications in cells, tissue and even entire organisms. In the context of MR imaging (MRI) or spectroscopy (MRS), water or other small molecules including metabolites can be used as MR-active probes to infer localized chemical information at the micrometre scale.in vitro solution-state or, more recently, solid-state NMR conditions. Applying the full and growing arsenal of such NMR methodology for studying larger molecules in a native setting has, however, required the development of novel and tailored NMR-based cellular structural biology concepts. Compared to the in vitro case, NMR studies under native conditions have to simultaneously address several challenges. Firstly, adequate spectroscopic sensitivity is of utmost importance as the native cellular environment is usually far more complex than what can be mimicked under well defined test-tube conditions. Another challenge relates to the fact that NMR signals of interest must be detected in a complex, usually dominating, molecular background. In practice, concepts suppressing signals of the surrounding molecular background so far have employed a combination of tailored biochemical, cell biology and spectroscopic methods. Thirdly, studying \u2018live\u2019 cell preparations furthermore calls for NMR approaches that allow NMR data to be rapidly acquired or involve instrumental setups that keep cells viable by allowing exchange of nutrients and removal of metabolic byproducts.In parallel, nuclear magnetic resonance (NMR) spectroscopy has become a well established structural biology approach to comprehensively study (at the atomic level) three-dimensional molecular structure and dynamics under IUCrJ, E. Luchinat and L. Banci and in human cells. In the latter case, Luchinat and Banci discuss various protein delivery methods that allow proteins or other biomolecules to be incorporated into cells as well as intracellular protein expression approaches. With these developments, solution-state NMR has become a powerful tool for studying three-dimensional molecular structure and the effect of molecular crowding at the atomic level. In addition, their review highlights elegant studies probing post-translational modifications and (mis)folding of proteins in living cells.In their review in this issue of et al., 2015et al., 2015et al., 2014et al., 2015et al. native conditions. Again, promising applications have been seen in recent years. Next to the work discussed by Luchinat and Banci, such studies have targeted large protein complexes embedded in bacterial cell envelopes (Kaplan in vitro and in vivo NMR can be of great value in dissecting molecular structure and interactions in the native state. For example, successful demonstrations included the use of cell lysates (Frederick et al., 2015et al., 2015in vivo extracts (Qiang et al., 2017Finally, hybrid approaches that bridge the gap between"} +{"text": "Recen in vivo . Further in vivo has, howTanda and his colleagues further assessed specificity of the blocking effects ofthe TLR4 antagonists on self-administration of remifentanil or cocaine . ConsistThe more recent results by Tanda et al. indicate"} +{"text": "Sorop Figure 1). However, not all patients have all comorbidities and the unpredictable interplay between different comorbidities is likely to result in multiple HFpEF phenotypes. Indeed, unbiased cluster analysis of densely phenotyped HFpEF patients suggests the presence of distinct \u2018phenogroups\u2019 with different clinical characteristics and outcomes.A substantial proportion of patients with heart failure have a left ventricular (LV) ejection fraction (EF) in the \u2018normal\u2019 range, a form of the syndrome that is termed heart failure with preserved ejection fraction (HFpEF). Patients with HFpEF have significant morbidity and mortality but unlike heart failure with reduced EF, there are currently no effective validated therapies. HFpEF is therefore an important area for further research. Patients with HFpEF have cardiac and extra-cardiac manifestations, including LV diastolic dysfunction, abnormal heart rate and rhythm, microvascular dysfunction, increased aortic stiffness, and abnormal ventriculo-vascular coupling, which impair systolic and diastolic reserve capacity upon exercise.Clinical studies to investigate the pathomechanisms involved in HFpEF have typically involved small numbers of highly selected patients subjected to invasive physiological assessment and cardiac biopsy-based analyses. A recent extensively-promoted model based on such studies posits that comorbidities induce systemic inflammation and vascular endothelial dysfunction as a consequence of an abnormal balance between reactive oxygen species (ROS) production and nitric oxide (NO) bioavailability.et al.Experimental animal models that allow deeper analysis of the pathogenesis of HFpEF would clearly be valuable. Rodent models that have been employed include the DOCA-salt model of hypertension and models of obesity and/or diabetes.et al.,,et al.et al.\u2019s findings that cardiomyocyte size and body weight were both reduced in their model rather than being increased as might be expected. With regard to inflammation, the authors measured systemic TNF\u03b1 levels but it would have been informative to also quantify myocardial cytokines and inflammatory cell infiltration.Sorop et al.et al.Figure 1).A more general critique of the model reported by Sorop The work was supported by the British Heart Foundation (BHF CH/1999001/11735) and the Department of Health via a National Institute for Health Research (NIHR) Biomedical Research Centre award to Guy\u2019s & St Thomas\u2019 NHS Foundation Trust in partnership with King\u2019s College London and King\u2019s College Hospital NHS Foundation Trust.Conflict of interest: none declared."} +{"text": "Hair cells in the inner ear convert mechanical stimuli provided by sound waves and head movements into electrical signal. Several mechanically evoked ionic currents with different properties have been recorded in hair cells. The search for the proteins that form the underlying ion channels is still in progress. The mechanoelectrical transduction (MET) channel near the tips of stereociliary in hair cells, which is responsible for sensory transduction, has been studied most extensively. Several components of the sensory mechanotransduction machinery in stereocilia have been identified, including the multi-transmembrane proteins tetraspan membrane protein in hair cell stereocilia (TMHS)/LHFPL5, transmembrane inner ear (TMIE) and transmembrane channel-like proteins 1 and 2 (TMC1/2). However, there remains considerable uncertainty regarding the molecules that form the channel pore. In addition to the sensory MET channel, hair cells express the mechanically gated ion channel PIEZO2, which is localized near the base of stereocilia and not essential for sensory transduction. The function of PIEZO2 in hair cells is not entirely clear but it might have a role in damage sensing and repair processes. Additional stretch-activated channels of unknown molecular identity and function have been found to localize at the basolateral membrane of hair cells. Here, we review current knowledge regarding the different mechanically gated ion channels in hair cells and discuss open questions concerning their molecular composition and function. Hair cells of the inner ear are specialized mechanosensory cells, which convert mechanical stimuli provided by sound waves (cochlea) or head movement (vestibular system) into electrical signals. Hair cells are highly polarized cells with extraordinary morphological specialization for sensing mechanical stimuli. The most prominent morphological specialization of a hair cell is the hair bundle. It protrudes from the apical surface of a hair cell and is formed by an array of F-actin based stereocilia that are arranged in a staircase of decreasing heights Figure . The senBesides the sensory MET channels at tip links, a second mechanically activated channel was recently identified in hair cells that is located at their apical cell surface where stereocilia emanate from the cell body Figures . This MEHair cells in the mammalian cochlea come in two flavors, outer hair cells (OHCs) and inner hair cells (IHCs). OHCs have an important function in amplifying input sound signals while IHCs transmit sound information to the CNS and low in Ca2+ (0.03 mM; Bosher and Warren, +. However, Ca2+ profoundly affects channel function where channel activity is increased when the external Ca2+ is decreased from a mM to a \u03bcM concentration (Corey and Hudspeth, The MET channel is non-selective for cations (Corey and Hudspeth, 2+ selectivity and single-channel conductance also show tonotopic characteristics in OHCs but not in IHCs. In 20 \u03bcM external Ca2+, single-channel conductance varies from 145 to 210 pS for OHCs along the tonotopic axis but is about 260 pS for IHCs along the entire length of the cochlea (Beurg et al., The organ of Corti in mammals has the ability to separate sound frequencies along its length\u2014high-frequency tones at the proximal end and low-frequency at the distal end of the organ. The Ca2+ either to the MET channel itself or to a binding side near the channel. Slow adaptation is thought to be regulated by a myosin motor complex at the upper insertion site of tip links (Crawford et al., 2+ (Peng et al., 2+ entry and voltage, while channel open probability is modulated by divalent ions interacting with the local lipid environment (Peng et al., 2+ influx (Corns et al., 2+ selectivity (Effertz et al., Sensory MET channels in hair cells adapt to mechanical stimuli, which leads to a decrease in current during a constant stimulus but additional stimulation again increases current. Adaptation is thought to set the resting tension of the transduction channel to position the channel near the most sensitive point of activation, and is important for providing amplification for mechanical signals (reviewed in LeMasurier and Gillespie, 2+, and sensitivity to channel blockers are similar but not identical between the two channels (Beurg et al., 2+ and ~90 pS at 0.07 mM Ca2+ (Beurg et al., During the early development of hair cells, their hair bundles are less directionally sensitive. Transducer currents can be observed by deflection of the hair bundle both towards the shortest and longest stereocilia (Waguespack et al., + and Na+ (Iwasa et al., \u2212, which is thought to regulate prestin function (Oliver et al., Several studies identified stretch activated MET currents in the basolateral membrane of hair cells, but the properties of these currents differed between reports. At least three different currents that are affected by mechanical force have been reported in OHCs. One type of current was activated by stretch and a single-channel conductance of 38\u201350 pS was determined for the underlying channel. This ion channel was non-selective to cations and had a reversal potential ~ \u221212 mV (Ding et al., 2+ imaging, it was demonstrated that the sensory MET channel is localized near the lower end of tip links (Beurg et al., The search for the molecular constituents of the MET channel in stereocilia has been in progress for decades. Using high speed CaTmc1 and Tmc2 are members of a gene family consisting in mammals of eight genes (Keresztes et al., Tmc1 and Tmc4 are the main family members that are expressed in adult cochlear hair cells, while Tmc2 is only transiently expressed in the cochlea during early postnatal development but can be detected in vestibular hair cells into adulthood (Kawashima et al., Tmc3 belongs to the same gene subfamily as Tmc1 and Tmc2, it does not appear to be essential for MET by hair cells (Beurg et al., Tmc1/2 deficient hair cells (Kawashima et al., 2+ selectivity and adaptation time constant in developing hair cells lacking either TMC1 alone or TMC2 alone differ (Kim and Fettiplace, 2+ selectivity of IHCs and OHCs lacking TMC2 but not TMC1 is significantly reduced (Kim and Fettiplace, Tmc1 has been reported to reduce Ca2+ permeability and single-channel conductance in IHCs (Pan et al., 2+ selectivity of the MET channel can be explained by variations in the stoichiometry of TMC1/2 (Pan et al., Tmc1 reduces single-channel conductance (Beurg et al., Tmc1 and Tmc2 can be caused by modulating the concentration of PIP2 in hair bundles (Effertz et al., tmc ortholog in the worm has been reported to relate to sodium-sensitive channel for salt sensation (Chatzigeorgiou et al., C. elegans (Zhang et al., Drosophila, TMC was found to play a function in providing sensory feedback for laval locomotion (Guo et al., Drosophila TMC (Zhang et al., However, whether TMC1 and TMC2 form the channel pore is still under debate. It was proposed that the tonotopic gradient in the conductance and CaTmhs/Lhfpl5 mutations cause deafness and lead to a dramatic reduction in mechanotransduction currents in cochlear hair cells of mice (Xiong et al., Tmhs/Lhfpl5-deficient hair cells. Single channel recordings demonstrated that in the absence of TMHS/LHFPL5 the conductance of the MET channel is affected as well as its activation and adaptation kinetics (Table TMHS/LHFPL5 is a second protein that has been implicated to be an integral component of the mechanotransduction channel in hair cells. TMHS/LHFPL5 is a member of a small subfamily within the large superfamily of proteins with four transmembrane domains (Petit et al., cs Table . The toncs Table . Taken tcs Table . Since scs Table . HoweverTmie-deficient cochlear hair cells, no MET currents can be detected, even though tip links remain intact and all known components of the MET machinery including TMC1/2 can travel into stereocilia (Zhao et al., TMIE is a protein with two transmembrane domains and linked to deafness in both human and mice (Mitchem et al., 2+ concentration (Wu et al., The similarities in single-channel conductance and pharmacological properties of the normal and reverse-polarity current in hair cells initially suggest that these two mechanically gated currents are carried by the same channel pore (Kim et al., \u2212 influx through a stretch-sensitive channel in the basolateral membrane of OHCs was reported to allosteric modulate prestin, thus potentially functioning in OHC amplification (Oliver et al., Piezo2-deficient hair cells demonstrated that PIEZO2 is not essential for electromotility (Wu et al., We still know next to nothing about the molecular composition of ion channels that carry the stretch activated currents in the basolateral membrane of OHCs (Ding et al., Recent studies have provided compelling evidence that hair cells express several molecularly distinct ion channels with different function. The best studied of these is the sensory MET channel at tips of stereocilia. Substantial evidence suggests that TMC1/2, TMHS and TMIE are integral components of the sensory MET channel Figure but whicXQ and UM wrote the manuscript. Figures were designed by XQ.UM is a co-founder of Decibel Therapeutics. The other author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Drosophila melanogaster have uncovered that DANs in the fly brain are also spontaneously active, and that this activity reflects the behavioral/internal states of the animal. Strikingly, genetic manipulation of basal DAN activity resulted in behavioral alterations in the fly, providing critical evidence that links spontaneous DAN activity to behavioral states. Furthermore, circuit-level analyses have started to reveal cellular and molecular mechanisms that mediate or regulate spontaneous DAN activity. Through reviewing recent findings in different animals with the major focus on flies, we will discuss potential roles of this physiological phenomenon in directing animal behaviors.Dopamine modulates a variety of animal behaviors that range from sleep and learning to courtship and aggression. Besides its well-known phasic firing to natural reward, a substantial number of dopamine neurons (DANs) are known to exhibit ongoing intrinsic activity in the absence of an external stimulus. While accumulating evidence points at functional implications for these intrinsic \u201cspontaneous activities\u201d of DANs in cognitive processes, a causal link to behavior and its underlying mechanisms has yet to be elucidated. Recent physiological studies in the model organism Animals need to modify behaviors according not only to the external world but also to their internal states, such as sleep need, hunger, or sexual motivation was observed when animals are in REM sleep has a central role in the regulation of the sleep-wake balance. A single pair of DANs projecting to the dFB promotes wakefulness, and dopamine receptors are necessary in the dFB neurons to process the waking signal strongly correlates with the behavioral activity of rats without external stimuli. In flies, repetitive training of paired presentations of odor cues and electric shocks with intervals (spaced training) is commonly used for the induction of aversive LTM , a gustatory center in the insect brain, is reported to control taste sensitivity to sucrose cluster encode protein hunger : D1- to D5-type receptors (D1R\u2013D5R). It is commonly accepted that D1R and D5R mainly recruit the G\u03b1Drosophila, four dopamine receptors, DopR1 , Dop2R, DopR2 and DopEcR exist in the genome (Adams et al., Xenopus oocyte suggested it to be excitatory (Reale et al., in vitro but with different cell lines, and never been directly compared. It is thus important to measure the threshold of these receptors for correct interpretation of functional results.In Nonetheless, accumulating behavioral and physiological evidence suggests the critical role of DopR2 in the detection of spontaneous activity of DANs. DopR2 was shown to be critical in regulation of sleep in the dFB (Pimentel et al., in-vivo electrophysiology experiments demonstrated that DopR2 in the dFB neurons mediates the wake-promoting dopamine signaling (Pimentel et al., o and thereby hyperpolarizes the membrane potential through modulating specific K+ channels (Pimentel et al., in vitro (Han et al., Exquisite DopR2 in the MB is also responsible for detecting the spontaneous activity of MV1 or MP1 DANs during memory maintenance (Berry et al., Drosophila addressed intracellular signaling molecules that mediate the effect of spontaneous DAN activity (Cervantes-Sandoval et al., o family (Thambi et al., Xenopus oocyte system (Reale et al., Two of the recent studies in Drosophila studies. Importantly, different internal animal states, e.g., hunger, sleep need, or sexual drive, are represented by different yet partially overlapping DAN cell types. This combinatorial state coding is reminiscent of various reinforcement signals conveyed by different combinations of DANs (Aso et al., In this article, we have reviewed diverse functions of spontaneous activity of DANs, paying special attention to recent In some cases, spontaneous DAN activity functions as an information filter to enable animals to respond differently to the same sensory input, such as food, food-associated cues or potential mating partners, depending on the physiological state (Inagaki et al., TI, HT, and NY wrote the manuscript and designed the figures.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Musculoskeletal pain due to ischemia is present in a variety of clinical conditions including peripheral vascular disease (PVD), sickle cell disease (SCD), complex regional pain syndrome (CRPS), and even fibromyalgia (FM). The clinical features associated with deep tissue ischemia are unique because although the subjective description of pain is common to other forms of myalgia, patients with ischemic muscle pain often respond poorly to conventional analgesic therapies. Moreover, these patients also display increased cardiovascular responses to muscle contraction, which often leads to exercise intolerance or exacerbation of underlying cardiovascular conditions. This suggests that the mechanisms of myalgia development and the role of altered cardiovascular function under conditions of ischemia may be distinct compared to other injuries/diseases of the muscles. It is widely accepted that group III and IV muscle afferents play an important role in the development of pain due to ischemia. These same muscle afferents also form the sensory component of the exercise pressor reflex (EPR), which is the increase in heart rate and blood pressure (BP) experienced after muscle contraction. Studies suggest that afferent sensitization after ischemia depends on interactions between purinergic (P2X and P2Y) receptors, transient receptor potential (TRP) channels, and acid sensing ion channels (ASICs) in individual populations of peripheral sensory neurons. Specific alterations in primary afferent function through these receptor mechanisms correlate with increased pain related behaviors and altered EPRs. Recent evidence suggests that factors within the muscles during ischemic conditions including upregulation of growth factors and cytokines, and microvascular changes may be linked to the overexpression of these different receptor molecules in the dorsal root ganglia (DRG) that in turn modulate pain and sympathetic reflexes. In this review article, we will discuss the peripheral mechanisms involved in the development of ischemic myalgia and the role that primary sensory neurons play in EPR modulation. Pain is a common clinical complaint resulting in a significant financial burden to both patients and society. In the U.S. alone, studies have estimated the mean cost of pain per patient at about $9K in adults and $12K in adolescents. The annual cost to society is over $635 billion injury that is characterized by the generation of free radicals Debold, and reacex vivo , a well-studied cardiovascular reflex arc that causes an increase in heart rate and blood pressure (BP) during exercise and unmyelinated (C) neurons, whose cell bodies rest in the dorsal root ganglia (DRG), consist primarily of a long axon that gives rise to free nerve endings in the muscle tissue Stacey, . Some stInitially, group III and IV muscle afferents were studied to determine their roles in the generation of the EPR were chemosensitive. In line with the work of Light et al. , as well as polymodal nociceptors have been extensively characterized electrophysiologically both t et al. , two dist et al. .The two previously mentioned sub-populations of metaboreceptors (\u201clow metabolite\u201d responders) and metabo-nociceptors (\u201chigh metabolite\u201d responders) along with their response properties are extensively altered following ischemic injury. Transient or prolonged ischemic insult to the muscles decreased mechanical thresholds and increased firing to mechanical stimulation in group III and IV muscle afferents (Ross et al., The increased mechanical sensitivity in primary afferents as well as the enhanced response to \u201clow metabolites\u201d, combined with the greater number of afferents responding to both noxious and non-noxious metabolite stimuli, correlate with increased behavioral responses after ischemic injury. In rats, models that cause ischemia-reperfusion via a hind limb tourniquet induced mechanical hyperalgesia and allodynia in the treated animals, accompanied by cold hyperalgesia (Coderre et al., After ischemic injury to the periphery, a diversity of channels and membrane receptors are upregulated in the DRGs Figure . Many ofin vitro from these animals showed increased responses to capsaicin, a TRPV1 agonist, compared to neurons from sham animals. Also in this model, there was an increase in the sympathetic response to arterial injection of capsaicin in the animals exposed to femoral artery occlusion compared to controls. In another femoral occlusion model, the pressor response evoked by intra-arterial injection of capsaicin into the injured hind limb more than doubled the response elicited by the same injection in the contralateral, uninjured limb (Tsuchimochi et al., TRPV1 has been associated with the development of ischemic pain in different models. Studies in humanized hemoglobin transgenic SCD mice have shown that TRPV1 plays a role in cutaneous afferent sensitization (Hillery et al., However, these findings are in contrast with other reports in which gene expression analysis in male mouse DRGs that innervate muscle tissue exposed to I/R or prolonged BAO injury did not show changes in TRPV1 mRNA expression 24 h after injury (Ross et al., There is ample evidence for the role of P2 receptors and how they affect afferent response to ischemia. As an example, the non-selective P2 receptor inhibitor PPADS, attenuates the EPR elicited by static contraction of the muscle (Kindig et al., ASIC and P2X receptors may also be key players in the afferent sensitization that is observed after ischemic injury (Dunn et al., The role of other P2X receptors in the development of ischemic myalgia is less clear. P2X3 is upregulated after I/R injuries and the total number of positive neurons in the DRG innervating ischemia-affected muscle tissue are also increased (Cairns et al., in vitro study showing that ATP enhances the response of ASIC3 to low pH. In this report, only the interaction between P2X5 and ASIC3 activation mimics the enhanced response to low pH and ATP that is observed in sensory neurons. Furthermore, about 25% of DRG neurons express P2X5 and of these neurons, about half co-express ASIC3 (Birdsong et al., in vivo or ex vivo is still required.P2X receptors also modulate the function of ASIC3, another key mediator of pain generation. Targeting this acid-sensing ion channel can effectively reduce muscle pain in different animal models (Sluka et al., In the specific context of ischemia, total DRG ASIC3 mRNA expression is increased in different injury models, and the total number of ASIC3 positive cells in the DRG increases (Queme et al., P2X receptors are not the only purinergic receptors that are relevant after ischemic injuries. In a prolonged ischemia model, expression of the ADP sensitive, P2Y1 receptor, was found to be upregulated in the DRGs (Ross et al., Ischemic injury alone does not likely drive all of the aforementioned changes in primary muscle afferents. Increased gene expression and concomitant afferent sensitization can also be linked to increased signaling from the damaged muscle tissue. Current evidence points at two important sources: cytokines and growth factors. These molecules are released into the intramuscular environment in response to the tissue damage caused by ischemia (Ascer et al., NGF has been frequently linked with the development of pain and hyperalgesia in various animal models and clinical conditions (Amaya et al., GDNF, another growth factor frequently tied to pain perception, is highly expressed in the muscles after ischemic injuries (Ross et al., One of the better characterized pro-nociceptive signals that is increased in injured muscles after ischemic injury is IL1\u03b2. This cytokine has been associated with pain development in multiple models ranging from muscle overuse (Noma et al., Another possible site of action for these various cytokines and growth factors is the DRG itself. Reports have highlighted the contributions of glial cells through cytokine release in models of neuropathic pain (Mika et al., Chronic pain conditions are more prevalent in women (Wijnhoven et al., in vivo behavioral results suggesting lower mechanical thresholds in females, mechanical thresholds were found to be significantly higher in females during patch clamp recordings of retrogradely labeled afferents (Hendrich et al., Recent studies have provided evidence for sex-dependent immune reactions that lead to differential brain and spinal cord sensitization mechanisms in a variety of rodent injury models (Sorge et al., In our own investigation of female muscle afferents, we have found distinct changes in gene expression within the affected DRGs following I/R. Whereas males show a robust upregulation of ASIC3 after I/R, which corresponded with alterations in behavior and afferent sensitivity (Ross et al., Interestingly, human and animal studies have shown that females also have decreased EPRs compared to males (Ettinger et al., Adequate management of the multiple complications in patients with ischemic injuries presents a variety of challenges. While patients with conditions like PVD and FM experience great benefits from an active lifestyle and physical therapy (Busch et al., SCD presents different challenges. Many patients with this condition are children and teenagers (Wilson and Nelson, Skeletal muscle ischemia is a strong driver of peripheral afferent sensitization, exerting robust effects through complex signaling cascades, resulting in the development of deep tissue pain and altered EPRs Figure . MultiplLFQ and MPJ planned the manuscript. LFQ and JLR analyzed the literature and wrote the manuscript with guidance from MPJ. All authors edited, read and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Although MCS between endoplasmic reticulum and mitochondria have been well studied and characterized in different contexts, emerging evidence indicates that lysosomes also exhibit close proximity with mitochondria, which translates in their mutual functional regulation. Indeed, as best illustrated in NPC disease, alterations in the lysosomal-mitochondrial liaisons underlie the secondary accumulation of specific lipids, such as cholesterol in mitochondria, resulting in mitochondrial dysfunction and defective antioxidant defense, which contribute to disease progression. Thus, a better understanding of the lysosomal and mitochondrial interactions and trafficking may identify novel targets for the treatment of Niemann-Pick disease.Lysosomal storage disorders (LSD) are characterized by the accumulation of diverse lipid species in lysosomes. Niemann-Pick type A/B (NPA/B) and type C diseases Niemann-Pick type C (NPC) are progressive LSD caused by loss of function of distinct lysosomal-residing proteins, acid sphingomyelinase and NPC1, respectively. While the primary cause of these diseases differs, both share common biochemical features, including the accumulation of sphingolipids and cholesterol, predominantly in endolysosomes. Besides these alterations in lysosomal homeostasis and function due to accumulation of specific lipid species, the lysosomal functional defects can have far-reaching consequences, disrupting NPC1 and NPC2 genes, resulting in functional defects in the lysosomal proteins NPC1 and NPC2, involved in cholesterol efflux from lysosomes.Niemann-Pick (NP) diseases encompass a group of autosomal recessive lysosomal storage disorders (LSD), characterized by the accumulation of diverse lipid species in lysosomes. While these diseases were initially considered a single entity with overlapping biochemical, pathological and clinical features, developing evidence demonstrated differential etiological causes phosphate, glucocerebroside, GM2 and GM3 gangliosides and sphingosine also accumulate in lysosomes and NPC diseases are caused by defects in lysosomal homeostasis and function, there are significant differences between these diseases in relation to the degree of cholesterol trafficking to mitochondria, with the consequent impact in mitochondrial dysfunction and impairment in antioxidant defense strategies. In this review, we summarize the biochemical and genetic features of both diseases, highlighting their commonalities and differences regarding lysosomal-mitochondrial cholesterol trafficking and communication as the molecular basis to understand the differential involvement of mitochondrial dysfunction in NPC disease. Further understanding the lysosomal and mitochondrial liaisons in NP diseases may thus provide the opportunity to improve and expand the current armamentarium for the treatment of these lysosomal disorders.The clinical diagnosis of NPA and NPB diseases is mainly based on the presence or absence of neurological symptons , which is located on chromosome 11 locus 11p15.4-p15.1. More than 180 pathogenic mutations in the SMPD1 gene in patients with NPA and NPB have been identified, which are concentrated in exon two and represent a valid model to examine lysosomal-mitochondrial communications that underlie the widespread defects in intracellular lipid transport.NPC1 is a multi transmembrane protein located in late endosomes and lysosomes constitutes a highly dynamic membrane structure that plays a key role in the maintenance of cellular homeostasis, as well as in the digestion and recycling of cellular components and in lipid metabolism and trafficking to the inner mitochondrial membrane (IMM), where it is converted to pregnenolone by P450scc Stocco, . MutatioThe trafficking of free cholesterol to OMM is mediated by several steps that involve various intracellular organelles, including lysosomes and lipid droplets (LD) and specific proteins, such as the translocator protein (TSPO) and voltage-dependent anion channel (VDAC) (Elustondo et al., Interestingly, 15 genes identified by sequence homology with the StAR hydrophobic lipid-binding pocket domain of approximately 210 amino acids have been described in human and mouse (Ponting and Aravind, The extramitochondrial source of the cholesterol pool that reaches mitochondria is not fully understood and could originate from LD, ER, the endosomal pathway or the plasma membrane (Rone et al., Another source of mitochondrial cholesterol is the ER. To reach mitochondria, cholesterol from the ER is transported by cytosolic proteins, such as the PAP7 protein, which interacts with TSPO and StARD proteins (Liu et al., Due to the relevance of lysosomal-mitochondrial liaisons in NPC, understanding the trafficking of lysosomal cholesterol to mitochondria may be essential for the pathophysiology of the disease. Interestingly, although astrocytes from NPC1 deficient mice exhibit decreased expression of StARD1 protein and mRNA levels (Chen et al., Mln64 mutant allele exhibited minor perturbations in the metabolism and in the intracellular distribution of cholesterol, questioning its contribution in the intramitochondrial trafficking of cholesterol (Kishida et al., critical role of this protein in steroidogenesis and hence in the trafficking of cholesterol to IMM for processing. Thus, although the understanding of the pathways of mitochondrial cholesterol trafficking and accumulation in NPC disease still remains elusive, this process is important for the progression of the disease and its further characterization may be key for the design of future therapies. Whether StARD1 in partnership with StARD3 are critical in this process remains to be fully established.Despite this evidence for a putative role of StARD3 in mitochondrial cholesterol trafficking, targeted mutation of the StARD3 StART domain has been shown to cause only modest alterations in cellular sterol metabolism and mice homozygous for the Mitochondria are double-membrane organelles that are essential for energy supply, metabolism, production of reactive oxygen species and apoptosis signaling Hatefi, . MitochoBesides Parkin and PINK1, which play a key role in mitophagy (McLelland et al., \u2212/\u2212 mice than in NPC fibroblasts (Ordonez, A considerable body of evidence suggests that impaired autophagy contributes to lysosomal lipid storage in LSDs through the accumulation of ubiquitinated proteins and dysfunctional organelles, including mitochondria (Platt et al., Ordonez, . MoreoveOrdonez, .Another factor that can contribute to impaired autophagy and defective clearance of dysfunctional mitochondria is the accumulation of sphingosine in lysosomes, which has been shown to disrupt calcium homeostasis (Lloyd-Evans et al., \u2212/\u2212 mice exhibit impaired autophagy flux determined by the combination of rapamycin with or without chloroquine, an effect that was accompanied by increased LC3BII and p62 levels. In addition, ASMase\u2212/\u2212 hepatocytes displayed impaired fusion of autophagosome-containing mitochondria with lysosomes in response to acetaminophen, which translated in increased susceptibility to acetaminophen-mediated liver injury by sustaining mitochondrial damage (Baulies et al., \u2212/\u2212 mice from acetaminophen-mediated liver injury. Moreover, human B lymphocytes from patients with NPB disease exhibit alterations in the rate of autophagic vacuole accumulation, mitochondrial fragmentation and mitophagy induction, indicating impaired clearance of damaged mitochondria (Canonico et al., In parallel with these findings in NPC, there is also evidence of autophagy defects in NPA/B diseases (Fucho et al., \u2212/\u2212 mice (Figure In contrast to defective autophagy, which is common to NPA and NPC, intracellular cholesterol trafficking and accumulation in mitochondria is a differential feature between NPA/B and NPC diseases Figure . Althouge Figure , and thie Figure . Moreovee Figure . The mece Figure . As acide Figure , it remae Figure , which ie Figure . As the e Figure .\u2212/\u2212 mice and in fibroblasts from NPC patients, leading to increased median survival and maximal life span of Npc1\u2212/\u2212 mice, protection against oxidative stress and oxidant-induced cell death and restoration of calbindin levels in cerebellar Purkinje cells, which improved locomotor impairment in Npc1\u2212/\u2212 mice. In addition, high-resolution respirometry analyses showed that GSH-EE treatment improved oxidative phosphorylation, coupled respiration and maximal electron transfer in cerebellum of Npc1\u2212/\u2212 mice (Torres et al., \u2212/\u2212 mice (Fu et al., \u2212/\u2212 mice (Mar\u00edn et al., Increased mitochondrial cholesterol content in NPC cells can lead to important functional consequences, such as decreased mitochondrial membrane fluidity (Colell et al., 2+ buffering capacity of mitochondria (Kiselyov and Muallem, 2+ homeostasis (Jennings et al., 2+ homeostasis preceded by the accumulation of sphingosine that impairs the endocytic pathway (Lloyd-Evans et al., 2+ content and function remains to be established. In addition, further work is needed to demonstrate a causal role for the disruption of Ca2+ homeostasis in the death of the Purkinje neurons in NPC disease, as shown in other related diseases (Girard et al., In addition to these events, mitochondrial cholesterol loading may impair mitochondrial dynamics reflected in the balance between fusion and fission events. Disruption of appropriate mitochondrial fluidity following cholesterol accumulation can prevent the fusion of mitochondria with adjacent healthy mitochondria (Baker et al., Although the genetic causes of NPA and NPC disease are well understood, the consequences at the level of disruption of intracelular trafficking and interorganelle communication is still incomplete, thus limiting the availability of effective therapy. Enzyme replacement therapy for ASMase and NPC1 deficiency is expected to successfully treat peripheral non-neuronal symptoms of both diseases (Schuchman and Desnick, ST, EB, SZ, CE, CG-R, and JF-C discussed findings, analyzed literature and wrote the manuscript. ST, CG-R, and JF-C designed the schematic Figures.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "An overview of the most relevant papers in the International Journal of Cardiovascular Imaging over the year 2016 for the different modalities. Relatively few manuscripts in the field of X-ray imaging were published in 2016. The far majorities were in MSCT, MRI and echocardiography, followed by nuclear cardiology and intravascular imaging, of which the last one is not included in this overview.Author He et al. described the use of the Myocardial Perfusion Frame Count (TMPFC) technique as an indicator to predict left ventricular systolic dysfunction in the sub-acute phase of STEMI . They coThe team of Starek et al. tested various acquisition protocols for the right atrium and the left atrium to create 3D models of the atria and esophagus using 3D rotational angiography . They usAnother topic that received attention was longitudinal stent deformation and the longer term clinical outcomes . Guler eTimmins et al. compared in a very small cohort of patients (n\u2009=\u20095) with non-obstructive coronary artery disease biplane angiographic versus intravascular ultrasound derived reconstructed coronary geometries, assessed wall shear stress and the association of wall shear stress and CAD progression after 6\u00a0months [Donmet et al. studied in a population of 81 patients the changes over time in non-culprit lesions in patients wih ST segment elevated myocardial infarction using QCA . They foIn 2016 different excellent paper in the field of nuclear cardiology were published in the journal. In this review we selected a few papers on technical advances, atherosclerosis imaging, imaging in heart failure and FDG PET/CT imaging in infective endocarditis.Perfusion SPECT imaging may be technically challenging in obese patients and data on its prognostic value in the obese are rather scarce. De Lorenzo et al. studied High efficiency CZT cameras provide also an opportunity to lower the injected activities of radiopharmaceuticals for SPECT myocardial imaging. Kincl et al. evaluateSeveral excellent reviews on the topics of atherosclerosis and multi-modality imaging were published in 2016. FDG-PET has shown promise in detecting metabolically active inflammatory cell infiltrates associated with vulnerable atherosclerotic plaque. In contrast to previous ex vivo studies incuatong plaque specimens with FDG after excision, Liu et al. investigLeft ventricular dyssynchrony (LVD) is an independent predictor of adverse cardiovascular events and progression to heart failure. It can also be potentially corrected with cardiac resynchronization therapy. LVD can be diagnosed using phase analysis on myocardial perfusion imaging with ECG-gated SPECT. Tavares et al. evaluateRecent studies have shown promising results using (18)F-FDG PET/CT for the diagnosis of prosthetic valve endocarditis (PVE) and the use of this nuclear imaging technique has been recently advocated in American and European guidelines on the management of endocarditis. However, negative controls were usually lacking in these studies. Fagman et al. comparedIn the 2016 edition of the International Journal of Cardiovascular Imaging several interesting studies were published in the field of cardiac ultrasound. Some of them are discussed in this overview.The American and European scientific echo community have published an algorithm for the grading of diastolic function. However, the ability to use this algorithm effectively in daily clinical practice has not been investigated. Van Dalen et al. hypothesized that in some patients it may be difficult to grade diastolic dysfunction with this scheme, since there may be discrepancies in the assessed parameters . The ASE2 of 44.9% was obtained. When excluding either of LAVImin or E/e\u2032 the model fit was significantly reduced. In contrast, when LAVImax was excluded the model fit was preserved. To detect an NT-proBNP level of >125\u00a0ng/L, LAVImin yielded a significantly larger area under the receiver operating characteristic curve than LAVImax and E/e\u2032. In this community-based sample, LAVImin was more strongly associated with NT-proBNP than LAVImax. Moreover, the discriminatory power to detect an elevated NT-proBNP level was stronger in LAVImin than in LAVImax and E/e\u2032. These findings support previous data that LAVImin may be more closely related to left ventricular filling function than LAVImax.Previous data have demonstrated that left atrial (LA) minimum volume indexed for body surface area (LAVImin) is more strongly associated with the Doppler echocardiographic E/e\u2032 ratio than LA maximum volume index (LAVImax). Hedberg et al. sought to explore if LAVImin was more closely related to serum levels of NT-proBNP than LAVImax and E/e\u2032 in the community . A commuDe Groot-de Laat et al. assessed the incremental value of two-dimensional and three-dimensional transoesophageal echocardiography over two-dimensional transthoracic echocardiography in three reader groups with different expertise in a total of twenty patients and five healthy persons . OverallParavalvular leak after transcatheter aortic valve implantation (TAVI) is challenging to quantitate. Transthoracic echocardiography is the main tool used for the assessment of paravalvular leak but is modestly reproducible. Abdelghani et al. sought to develop a reproducible echocardiographic approach to assess paravalvular leak in the post-TAVI setting . Four obDespite successful aortic coarctation (CoA) repair, systemic hypertension often recurs which may influence left ventricular function. Menting et al. aimed to detect early left ventricular dysfunction using left ventricular global longitudinal strain (GLS) in adults with repaired CoA, and to identify associations with patient and echocardiographic characteristics . In thisOxborough et al. simultaneously assessed longitudinal strain and left ventricular volume/ right ventricular area in 92 male athletes subdivided according to varying sporting demographics . AthleteSchinkel et al. wrote an interesting overview on the role of contrast-enhanced ultrasound (CEUS) in the evaluation of patients with known or suspected atherosclerosis . CEUS isThere were a number of interesting advancements in cardiovascular MRI (CMR) in 2016.Reval et al. studied Improved border sharpness of post infarct scar was demonstrated by Rutz et al. with a sThe prognostic value of stenosis class as measured by magnetic resonance angiography over traditional risk factors in patients with peripheral arterial disease was demonstrated by van den Bosch et al. . ImproveFaletti et al. found th1-regularization (IS SENSE) was demonstrated [The performance of an accelerated CMR protocol using iterative SENSE reconstruction and spatio-temporal Lnstrated . In relanstrated used thinstrated .The feasibility of heart deformation analysis for measuring regional myocardial velocity with CMR was tested in normal volunteers . CardiacMarkl et al. performeA dose correction for post-contrast T1 mapping of the heart was proposed by Gai et al. and applA semi-automated cardiac segmentation tool was found to improve the reproducibility and speed of right heart MRA .KnowledgIn 2016 a large number of original articles, covering a variety of topics related to CT, were submitted to the journal and underwent a rigorous review/selection process. While the submission, review, and editorial selection process has well-known biases, the articles published reflect current trends in computed tomography.Coronary artery disease is a major focus of cardiac CT. While clinical prevention guidelines suggest a limited role, coronary calcium scoring (CAC) remains a topic of ongoing research. Published papers describe the effective radiation exposure among participants from the MESA cohort , and disIn contrast to CAC, which identifies only calcified plaque, contrast-enhanced CTA can assess overall plaque burden and plaque characteristics. While it is not an indication for CTA itself, plaque burden is evaluated by experienced clinical readers in coronary CTA indicated for suspected obstructive CAD and is a topic of significant interest in research. Published papers describe a correlation between elevated HBA1c and higher frequency of obstructive CAD and vulnerable atherosclerotic coronary plaque characteristics (positive vessel remodeling and low-attenuation plaques) in patients with type 2 diabetes , 57 An oCT) has gained significant interest. One published study examined the feasibility of FFRCT in a small \u2018unselected\u2019 cohort of patients with suspected significant CAD [CTcould be measured in the majority of consecutive, patients who had suspected significant CAD by CCTA and demonstrated good diagnostic performance for detecting hemodynamically significant CAD even in patients with calcified vessels. Another important study demonstrated the impact of image resolution on geometrical reconstruction and subsequent FFR calculation with invasive and CT FFR [Based on the experience with invasive coronary angiography as well as CTA, the limitations of luminal stenosis assessment for the prediction of hemodynamic significance of CAD are well known. CT techniques to evaluate lesion functional significance are therefore a major focus of research. Among them, non-invasive fractional flow reserve measured by coronary computed tomography angiography by micro-computed tomography (mCT) in a coronary bifurcation model . The traAn important focus of CT are indications in the context of structural heart disease intervention. For TAVR planning, one study evaluated an automatic aortic root landmarks detection method with automated determination of annulus radius, annulus orientation, and distance from annulus plane to right and left coronary ostia . Other sExciting progress is described in analysis of CT data. Published studies examined quantitative semi-automated methods for assessment of coronary luminal stenosis severity and fullThe above selection of published articles about cardiovascular CT reflects current clinical use and research interests. Use of CT, like any other diagnostic test, has to balance anticipated benefit against potential risk, specifically for CT radiation exposure and contrast administration. This risk assessment takes into account the susceptibility of specific patient populations , 79. The"} +{"text": "Despite the characterization of ribosomal S6 kinase 2 (RSK2) as a protein kinase acting as a downstream effector of the well characterized ERK MAP-kinase signaling pathway, it turns out to be a challenging task to link RSK2 to specific neuronal processes dysregulated in case of mutation. Animal models such as mouse and Drosophila combine advanced genetic manipulation tools with in vivo imaging techniques, high-resolution connectome analysis and a variety of behavioral assays, thereby allowing for an in-depth analysis for gene functions in the nervous system. Although modeling mental disability in animal systems has limitations because of the complexity of phenotypes, the influence of genetic variation and species-specific characteristics at the neural circuit and behavioral level, some common aspects of RSK2 function in the nervous system have emerged, which will be presented. Only with this knowledge our understanding of the pathophysiology of CLS can be improved, which might open the door for development of potential intervention strategies.Loss of function mutations in the RSK2 knock-out mice (RSK2\u2212) and mutants of the single RSK ortholog in Drosophila (D-RSK) were analyzed at the behavioral and neurophysiological level. We summarize findings with both animal models and their implications to better understand the neuropathophysiology of CLS.Coffin-Lowry syndrome is a rare X-chromosome linked disorder with an incidence of 1:50,000\u2013100,000. Clinical characteristics are heterogeneous and variable in expressivity. They include facial dysmorphism, digit and skeletal abnormalities, and growth delay. Prominently, CLS patients suffer from severe mental disabilities (IQ: 15\u201360). Less frequently, stimulus-induced drop attacks, epileptic seizures and hearing loss are manifested. The risk to develop psychiatric diseases like depression and psychosis might be increased. No treatment exists for this disease family. With the exception of a 140 amino acid N-terminal extension, D-RSK shows equal sequence similarities of about 70% to RSK1\u20134. Notably, all sequence motifs required for RSK activation are also present in D-RSK belongs to the AGC kinase family and the C-terminal kinase domain (CTKD) is related to the CaK Figure . From biK Figure . HoweverDrosophila eye development, D-RSK acts as a cytoplasmic anchor for ERK, thereby inhibiting ERK nuclear translocation and phosphorylation of nuclear targets carried a mutation in RSK2 and later on developed clearer characteristics of CLS, which generally resemble those of RASopathies induces features of depression, expressed as general inactivity. Furthermore, activity recordings allow correlating disease associated genes with circadian and sleep disturbances as one characteristic of some mental illnesses.The similarities of molecular pathways controlling the generation and differentiation of neurons, neural circuit wiring and synaptic communication make animal models a powerful tool to characterize cellular and physiological processes and link them to human disorders. A more challenging task is to model mental disabilities or mental health disorders in animal systems because of the complexity of symptoms, the influence of genetic variation on disease outcome and species-specific characteristics at the molecular, neural circuit and behavioral level and the habenula, playing a role in motivational and rewarding aspects of behavior from CLS patients uncovered a reduction in total brain volume with a particular impact on temporal lobe, cerebellar and hippocampal volumes but more pre-existing neurons (Castillon et al., RSK2\u2212 mice in a context-dependent manner.Do the behavioral phenotypes seen in mice and RSK2\u2212 mice (Mehmood et al., RSK2\u2212 animals showed a growth and developmental delay (Ammar et al., in vivo (Fischer et al., RSK2\u2212 behavioral deficits.Loss of RSK2 interferes at several levels with neuronal properties (Figure de novo synthesis, trafficking, variations in heterotetrameric subunit composition, posttranslational modifications and interaction with scaffold proteins at postsynaptic densities (Huganir and Nicoll, RSK2\u2212 mice, upregulated levels of the GluA2 subunit of AMPA-receptors at synaptic sites were observed (Mehmood et al., RSK2\u2212 mice or from hippocampal slices demonstrated decreased synaptic transmission, altered AMPA and NMDA receptor properties, but normal paired-pulse facilitation and long-term potentiation. Altogether, these findings argued for a postsynaptic function of RSK2 in neurotransmission (Morice et al., Accumulating data support the view that RSK2 influences ionotropic glutamatergic synapses. A focus of RSK2 research is the AMPA-subfamily of glutamate receptors because of their fundamental role in synaptic plasticity as a crucial step for learning and memory formation. The number of AMPA-receptors and their channel properties are influenced by RSK2\u2212 mice. Thus, RSK2 seems to mediate at least some of its effects by modulation of scaffold proteins of the postsynaptic density in an activity-dependent manner.Another function of RSK2 at the postsynapse is interaction with proteins of the Shank family, which act as major scaffolds for AMPA-, NMDR- and metabotropic glutamate receptors (Sala et al., RSK2\u2212 animals are increased with an accompanying up-regulation of dopamine receptor2 expression (Pereira et al., 2A receptor, thereby linking growth factor induced MAP-kinase signaling with regulation of a G-protein coupled receptor (Sheffler et al., RSK2\u2212 brains were detected (Pereira et al., Emotional behaviors like anxiety, depression or hyperactivity are regulated by the neurotransmitters dopamine and serotonin. Dopamine levels in the mouse cortex of Drosophila, analysis has focused on the larval neuromuscular system as a well-established model for synapse formation, neurotransmission and synaptic plasticity (Harris and Littleton, D-RSK mutants, overall bouton number is increased but this phenotype is counteracted by a much stronger decrease in synapse number per bouton (Fischer et al., Drosophila D-RSK is a postsynaptic requirement for efficient synaptic transmission.In Drosophila gave first insights into the impact of RSK2 on neuronal functions. In CLS patients it is still unknown, if similar neurite growth and synaptic defects exist like in animal models. Patient derived iPSCs could be differentiated into distinct neuron subtypes and analyzed at the molecular, biochemical and physiological level. Besides analysis of molecular interactions, a particular emphasis for further studies should be the analysis of RSK2 in experience-dependent synaptic changes. A good example for future research about CLS is the fragile-X mental retardation syndrome, where parallel experiments in animal models have paved the way to bridge the gap to better understand the human disease phenotypes. For example, metabotropic glutamate receptor and ERK-pathway dependent protein synthesis were affected, which led to clinical trials in patients with a mGluR5 antagonist, unfortunately turning out to be not effective (Berry-Kravis et al., RSK2/D-RSK phenotypes, our understanding of neuronal dysfunction is far from complete at all levels of analysis.While no information exists about the neuronal basis of the clinical phenotype in CLS patients, recent findings in mouse and MF and TR wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "The earlier identification of selective inhibitors of fungal SL biosynthesis promised potent broad-spectrum anti-fungal agents, which later encouraged testing some of those agents against protozoan parasites. In this review we focus on the key enzymes of the SL de novo biosynthetic pathway in protozoan parasites of the Apicomplexa and Kinetoplastidae, outlining the divergence and interconnection between host and pathogen metabolism. The druggability of the SL biosynthesis is considered, alongside recent technology advances that will enable the dissection and analyses of this pathway in the parasitic protozoa. The future impact of these advances for the development of new therapeutics for both globally threatening and neglected infectious diseases is potentially profound.Sphingolipids (SLs) are an integral part of all eukaryotic cellular membranes. In addition, they have indispensable functions as signalling molecules controlling a myriad of cellular events. Disruption of either the Protozoa (kingdom Protista) are single-cell organisms that can be free-living or parasitic in nature Baron, . Out of Trypanosoma brucei , Leishmania spp. and Trypanosoma cruzi ; Sporozoa \u2013 the apicomplexan Toxoplasma gondii (toxoplasmosis), Cryptosporidium spp. (cryptosporidiosis) and Eimeria spp. (coccidiosis in poultry and cattle), Theileria spp. (East Coast Fever in cattle) and Plasmodium spp., including Plasmodium falciparum the causative agent of severe malaria and one of the \u2018Big Three\u2019 global infectious diseases alongside HIV and tuberculosis or Neglected Zoonotic Diseases King, and wereThis review presents sphingolipid (SL) biosynthesis and ceramide (CER) homoeostasis as a potential gold mine of tractable drug targets for these protozoan parasites.In general, available treatments for the diseases caused by the Kinetoplastidae and Apicomplexa are outdated (if not historic), with relatively few examples that were introduced recently, toxic and require a long treatment regimen, and therefore close monitoring of patients.The kinetoplastid pathogens in focus here all cause NTDs and as such there are significant problems with the available drug regimens:et al.et al.et al.et al.et al.et al.et al.et al.The treatment of leishmaniasis often requires a long course of intravenous pentavalent antimony drugs (e.g. Glucantime and Pentostam), aminosidine (paromomycin) or liposomal amphotericin B , and 660\u00a0000 (429\u00a0000 in 2015) associated deaths; although the actual numbers might be even higher , as the primary complex mammalian SL; and inositol phosphorylceramide (IPC) in fungi, plants and numerous protozoa and ceramide-1-phosphate (C1P) that can be protozoa . These ma et al.. Furthertrans double bond) affect their biophysical properties rendering these molecules different from their glycerolipid counterparts, i.e. SM vs phosphatidylcholine (PC) to L\u03b1 (lamellar phase) transition near the physiological temperature of 37 \u00b0C, in contrast, this transition for naturally occurring glycerolipids is near or below 0\u00a0\u00b0C. Additionally, the long saturated alkyl chains of SLs allow them to pack tightly with sterols, stabilized by hydrogen bonding of SPH allows it to remain partially uncharged at physiological pH retaining the ability to move across membranes for new drug targets or regimens via serine palmitoyltransferase (SPT), to produce dihydrosphingosine. The latter comprises first the formation of CER in the ER by the action of ceramide synthase (CerS), and then the formation of complex SLs in the Golgi. These products vary depending on the species, and are formed under the catalysis of what could be generically termed SL synthases: SM synthase in mammals and IPC synthase in fungi, plants and protozoa. It is worth mentioning that another Golgi localized metabolic pathway results in the formation of glycosylated CER species, and also contributes to the regulation CER levels . However, clear divergence is observed in the second and the third steps, both of which represent a cell-fate modulator process. CerSs exhibit differential preferences for the chain length of the acyl-CoA substrate around the PLP-binding lysine (in bold) -dependent . Subsequent minor metabolic differences are encountered across different species; mainly concerning the order of the hydroxylation (in fungi and higher plants) and acylation to produce CERs has previously indicated the presence of CerS activity in transfer protein CERT in mammals to produce IPC via IPC synthase with respect to the animal SM synthases and SLs ([phyto]ceramide and IPC/SM/EPC). Accordingly, these enzymes act as regulators of a delicate balance between pro-apoptotic CER and pro-mitogenic DAG , a depsipeptide, was first reported by Ikai et al. and soon. et al.. The tarLeishmania major and T. gondii are not susceptible to AbA inhibition compared with the single copy found, for example, in L. major and T. gondii , chain length variation, the hydrophobic nature of the involved enzymes and the presence of multiple pathways that can operate in parallel subcellular localisation, (b) regulation (c) chain length specificity, (d) kinetics of trafficking and (e) mechanism of action. For example, phosphorylation of 1\u20133% cytosolic SPH may double the levels of S1P that acts on G protein-coupled receptor (GPCR) to elicit a specific response in a particular cellular locality for certain period of time , in the pursuit of identifying new compound scaffolds active against the Leishmania spp IPC synthase utilising yeast and function makes SL biosynthesis highly alluring for drug intervention, after all, everybody needs SLs, right?"} +{"text": "Atrial fibrillation (AF) is maintained by reentrant excitation forming stable or meandering rotors, leading circle reentry, or multiple circulating wavelets repolarization in antiarrhythmic drug therapy. AF circuits were found to be less stable and more likely to self-terminate when APDINa blockade using Class I drugs has been extensively tested in clinical, experimental, and in silico studies. While these drugs are effective in treating paroxysmal AF, their efficacy may be related to suppression of triggered activity via non-canonical effects on RYR2 rather than INa (Salvage et al., INaK blockade impacts nodal cell firing via regulation of the so-called calcium clock (Sirenko et al., INaK blockade than a whole-heart virtual model that incorporates neural feedback and the conduction system, simulates the ventricular response rate to AF, and tests the potential risk of proarrhythmia by digitalis toxicity. Finally, the importance of IK1 in fibrillatory dynamics is well-established. Noujaim and colleagues (Noujaim et al., via its inhibitory effects on IK1. However, this strategy must be approached with caution since IK1 density is greater in ventricular compared to atrial myocardium. A notable concern is the potential for unmasking ventricular ectopy (Miake et al., This line of inquiry has clear implications for ion channel pharmacotherapy, an area of major challenge considering the suboptimal efficacy of many ion channel drugs against AF as well as their risk of inducing ventricular pro-arrhythmia. The focus on the aforementioned targets in an atria-only model is interesting from a theoretical perspective but less so from a pragmatic one. For example, in silico studies are always constrained by strong experimental and clinical measurements to guarantee their relevance for human AF.Nonetheless, the present work by Sanchez et al. establisFA and CC drafted, revised, and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Neurodegenerative diseases affect millions of individuals in North America and cost the health-care industry billions of dollars for treatment. Current treatment options for degenerative diseases focus on physical rehabilitation or drug therapies, which temporarily mask the effects of cell damage, but quickly lose their efficacy. Cell therapies for the central nervous system remain an untapped market due to the complexity involved in growing neural tissues, controlling their differentiation, and protecting them from the hostile environment they meet upon implantation. Designing tissue constructs for the discovery of better drug treatments are also limited due to the resolution needed for an accurate cellular representation of the brain, in addition to being expensive and difficult to translate to biocompatible materials. 3-D printing offers a streamlined solution for engineering brain tissue for drug discovery or, in the future, for implantation. New microfluidic and bioplotting devices offer increased resolution, little impact on cell viability and have been tested with several bioink materials including fibrin, collagen, hyaluronic acid, poly(caprolactone), and poly(ethylene glycol). This review details current efforts at bioprinting neural tissue and highlights promising avenues for future work. Neurodegenerative diseases affect over 55 million individuals annually in North America, creating a multi-billion dollar burden on the health-care industry due to the costs associated with treatment, and rehabilitation therapy , which are pluripotent stem cells derived from a human embryo; mesenchymal stem cells (MSCs), which are multipotent stromal cells that can differentiate into osteoblasts, chondrocytes, myocytes, and adipocytes; neural stem/progenitor stem cells, which are multipotent and can differentiate into neurons, astrocytes, and oligodendrocytes; and human induced pluripotent stem cells (hiPSCs), which are adult cells taken back to a pluripotent state methacrylamide, and poly(a-hydroxy-acids) from hESCs and hiPSCs, but they impose unnatural geometric constraints on the cells uses a melted thermoplastic which is deposited layer-by-layer onto a flat substrate to build a 3-D construct (Tan et al., Selective laser sintering uses a similar process as FDM, but SLS has a higher resolution (O\u2019Brien et al., Stereolithography is the highest resolution option for bioprinting (O\u2019Brien et al., Inkjet bioprinting uses a modified inkjet printer to deposit cells encapsulated in a bioink onto a chosen substrate (O\u2019Brien et al., Bioplotting using syringes to print tubes or spheroids layered on top of each other (O\u2019Brien et al., Microfluidic extrusion represents an extension of bioplotting (Pfister et al., Several groups have bioprinted neural tissue using various cell types with varying levels of success Table . In 2006In 2014, Lorber et al. inkjet printed retinal glial cells and disassociated retinal cells, resulting in 57% cell death in glial cells and 33% cell death in retinal cells compared with controls of unprinted cells grown on tissue culture plates (Lorber et al., Suri et al. photopatCurley et al. also useLee et al. combinedGu et al. extrudedin vivo study implanted 3-D printed constructs into cerebellum-lesioned zebrafish. Treated fish showed increased spontaneous coiling contraction and increased hatching rate compared with lesioned untreated fish, indicating cellular restoration.Similarly, Lozano et al. extrudedLee et al. used micThese studies differ greatly in the number of cells lost due to the stress of the printing process. Cell viability allows the user to seed at the correct cell density. However, some studies do not report cell death while others report up to 57% cell death during the printing (Lorber et al., Current work indicates that a wide variety of bioink materials may be suitable for 3-D printing neural tissue. However, more research needs to be done comparing the printability of each of these materials in terms of efficiency and ease-of-use, both which become important when scaling up production. This review has covered multiple methods of 3-D printing neural constructs. Inkjet bioprinting is the most well documented but is limited in both bioink material and geometries. Microfluidic extrusion has recently seen success in printing complex shapes with various neural cell types and remains an option of interest that needs further research in creating ideal bioink compositions. Other possibilities, such as stereolithography and SLS, remain underused for neural tissue applications.What remains to be done is finding a cohesive unit of bioink and bioprinting method which results in a high cell viability post-printing and is adaptable enough to print multiple different neural cell types with a bioink which has controllable elastic properties and porosity and can be loaded with factors to further control differentiation.In addition, most studies lack a hands-off manner of controlling bioprinting. Incorporating CAD and microtechnology into printing projects would help fully realize the high-throughput nature of 3-D bioprinting tissue, as the field is still largely limited by human-controlled systems. The use of CAD would further assist in increasing cell resolution within printed constructs. Advancing the resolution of bioprinting could also allow the printing of vascular networks within a designed tissue, something which would allow neural models to be scaled-up beyond a maximum achieved size of mm. This development would allow more physiologically relevant constructs to be printed for disease modeling and drug discovery.Bioprinting can change how neural tissue are engineered, moving it from a time consuming, hands-on process that can vary from lab-to-lab to a sterile, high-throughput process that can rapidly produce physiologically accurate brain constructs for applications in cell therapy and drug screening. The low throughout methods for engineering brain tissue limit their applicability for drug screening. Cell therapy has had limited success for the same reason: the number of cells required for injection requires lengthy culture time in addition to the difficulty controlling cell diffusion and differentiation. For bioprinting to succeed as the new standard for engineering neural tissue more bioinks must be done to accurately control brain region development, and the issue of vascularization must be solved to print accurate constructs suitable for long-term culture. However, such bioprinted neural tissues hold great promise for applications in both cell therapy and for drug screening.MT and SW both contributed to the authorship. SW proposed the topic and provided feedback through the writing process. MT wrote the initial draft and completed revisions based on feedback provided.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Protein kinases are major regulators of mitosis, with over 30% of the mitotic proteome phosphorylated on serines, threonines and tyrosines. The human genome encodes for 518 kinases that have a structurally conserved catalytic domain and includes about a dozen of cell division specific ones. Yet each kinase has unique structural features that allow their distinct substrate recognition and modes of regulation. These unique regulatory features determine their accurate spatio-temporal activation critical for correct progression through mitosis and are exploited for therapeutic purposes. In this review, we will discuss the principles of mitotic kinase activation and the structural determinants that underlie functional specificity. Protein phosphorylation is a key regulatory mechanism influencing various cellular processes such as cell growth, cell motility, cell differentiation and cell division. Most notably, protein phosphorylation peaks during mitosis and the timing coincides with the cell division-related chromosomal and cytoskeletal reorganization and an \u03b1-helical C-terminal lobe at each end family . Though the activation loop auto-phosphorylation is sufficient for basal activity, facilitating the right conformation of the loop is required for full activation, and is achieved by the binding of TPX2 and INCENP to Aurora A and Aurora B kinases, respectively and kinetochore-microtubule attachment Elowe, . Cdc20, CDK Activating Kinase belonging to the family of CDKs) which is essential for their increased kinase activity and the budding yeast Hrr25 (Casein kinase) critical for meiosis co-orientation of kinetochore do not require the activation loop phosphorylation and their kinase domains are constitutively active (Eswaran et al., Kinases achieve substrate specificity through multiple mechanisms in mitosis (Ubersax and Ferrell, in vitro and in vivo thus far has helped define kinase substrate specificity. Typically about 4-6 amino acids flanking the phospho-acceptor residue P can contribute to the selectivity of kinases for their substrate. The molecular basis for substrate recognition mainly comes from the structural work on CDK2/cyclin A bound to its substrate (Brown et al., Kinase substrate specificity is generally determined by the architecture of the substrate binding site, which might select negatively against certain residues flanking the phosphorylation site. The identification of substrates in vitro phosphorylation of kinetochore proteins by Bub1 followed by phosphorylation-directed staining and mass spectrometric analyses identified many prospective Bub1 substrates with a putative consensus motif \u03d5-X5-S/T (Breit et al., The identification of multiple budding yeast kinetochore Aurora B kinase substrates, allowed to define an Aurora consensus phosphorylation sequences (Cheeseman et al., in vivo (Dephoure et al., The use of peptide libraries against kinases has greatly contributed to the identification of optimal peptide sequence motifs and \u201canti-motifs\u201d for kinases (Hutti et al., Kinases may use docking sites or sites that are primed by another kinase to enhance their substrate selectivity. For example, to bind and to be phosphorylated by Plk1, a substrate generally needs to be primed by another kinase (Lee et al., CDK-cyclins also use docking sites to recognize and phosphorylate temporally their substrates. Certain cyclin partners have a hydrophobic docking patch that recognizes an \u201cRXL\u201d motif on substrates 40\u00c5 away from the catalytic site of the CDK active site (Schulman et al., Many mitotic kinases rely on spatial targeting to phosphorylate their specific substrates. This restricts the activity of the kinase to generate gradients of kinase activity. The most well characterized spatially-targeted kinases are Aurora A and B kinases and they appear to share the same substrate specificity (Fu et al., The molecular basis for the recruitment of Mps1 kinase to the outer kinetochore is also well established. Multiple kinases including CDK1, Aurora B, Plk1, and Mps1 itself are implicated in Mps1 kinetochore targeting (Morin et al., High resolution mechanistic understanding of kinase regulation is essential not only to define how kinases achieve error-free cell division, but also to exploit the differences in their regulatory mechanisms to specifically target them in mitosis-related human disorders. Structural studies of kinases thus far have provided key insights into the similarities and differences in the modes of activation and regulation of many mitotic kinases. Although kinases possess a broadly conserved catalytic core, their level of kinase activity and substrate specificity are determined by specific inter/intra molecular interactions and phosphorylation. In this review, we summarize how key structural regulatory elements such as the relative orientation of the C-helix, activation segment conformation and spatial regulatory elements responsible for correct kinase sub-cellular localization achieve accurate kinase function. However, there are still many open questions, particularly on factors determining the graded level of kinase activation and its implications on their mitotic role. More structural analyses of kinases in complex with their regulatory binding partners with and without bound substrates, and their functional implications in cells will undoubtedly further advance our mechanistic understanding of this essential class of mitotic regulators.JW and AJ have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "A small proportion of patients with Parkinson\u2019s disease (PD), chronically under dopamine replacement therapy, may undergo an addiction-like behavioral disturbance, named dopamine dysregulation syndrome (DDS). This behavioral disorder is characterized by the increase of doses beyond those required for motor control, and its management remains difficult; thus, early recognition and careful monitoring of at-risk individuals are crucial. We report the cases of two PD patients with a previous unsatisfactory switching from an immediate release (IR) to an extended release (ER) pramipexole formulation who developed DDS. PD patients unsatisfactorily switched from an IR to an ER formulation of dopamine agonists should be considered as at-risk individuals for DDS development."} +{"text": "The increasing use of nipple-sparing mastectomy (NSM) and skin-sparing mastectomy (SSM) in the treatment of nonmetastatic breast cancer is justified by considerations linked to their therapeutic index. In selected patients, efficacy results tend to be similar to those observed after radical modified mastectomy and at the same time, subcutaneous mastectomies preserve the patient\u2019s body image. Yet the oncologic safety of the two former surgical approaches is still a matter of debate, also in consideration of the almost complete absence of clinical studies directed to prospective, controlled comparisons between subcutaneous and radical modified mastectomies. In addition, no clear statement\u2014and consequently no consensus\u2014emerges from the rather rare reports addressing the issue of whether or not there exist robust algorithms for guiding decision-making in delivering postoperative radiotherapy after NSM or SSM. The objective of the present review article is to revisit the dataset recently provided by the literature, which might help oncology teams optimise local treatment in this patient population. Throughout the past two decades, both nipple-sparing mastectomy (NSM) and skin-sparing mastectomy (SSM) have been gaining ground in the surgical management of nonmetastatic breast cancer patients. The main reason for this trend relates to the therapeutic index elicited for NSM and SSM, which was shown to compare favourably with that observed for modified radical mastectomy (MRM). Indeed, the literature suggests that treatment outcomes of NSM, SSM and MRM tend to be similar as regards efficacy endpoints, and at the same time, patients draw a number of advantages from the two former surgical modalities, especially in terms of organ and body image preservation , 2\u20139.Whether to deliver postoperative radiotherapy (PORT) after NSM or SSM or not is nowadays an issue essentially addressed by multi-disciplinary tumour boards involving not only surgeons and radio-oncologists but also diagnosticians, pathologists and medical oncologists. Often, such a decision-making process turns out to be a challenging task, for no straightforward, prospective comparison between subcutaneous radical mastectomies and MRMs has emerged so far from reports analysing the exact relevance of PORT after NSM and SSM , 11.The objective of this review article is to revisit the messages recently provided by the literature which might help oncology teams optimise local treatment in this patient population.http://www.ncbi.nlm.nih.gov/pubmed).We used search strings on studies addressing the issue of interaction between NSM and SSM and adjuvant radiotherapy, via the following search terms: breast cancer, SSM, NSM, MRM, PORT, treatment outcome and late complications. This review article is essentially based on full articles published since 2000 and retrieved from the PubMed search engine by tumour microfoci was demonstrated by Cense et al in nearlet al . Neverthet al .et al [The persistence of mammary tissue after SSM or NSM obviously increases the risk of local failure if tumour cells are present in this residual tissue \u201319. In tet al , the locAfter SSM, the most significant risk factors for local recurrence identified in two studies are nodal involvement, tumour size, poorly differentiated carcinomas, and lympho-vascular invasion (LVI) , 21. In In 2007, Meretoja published a report on a cohort of 207 consecutive women who underwent, from 1992 to 2001, SSM and immediate breast reconstruction immediate breast reconstruction (IBR) without any adjuvant radiotherapy [et al [It has been repeatedly substantiated that PORT delivered to reconstructed breast carries a higher risk of complications compared to that of no PORT \u201326. Befoet al showed tet al .et al [P = 0.03), nipple loss , and rates of reconstruction failure . On multivariate regression analysis, PORT , age >55 years , breast volume \u2265800 cm3 , smoking , and peri-areolar incision were independent risk factors for complications. The authors concluded that despite higher complication rates after PORT, reconstruction failure and nipple/areola necrosis remained infrequent. Moreover, capsule formation can readily be treated with capsulotomy or capsulectomy [In a recent report published in 2015, Tang et al analysedet al . Compareulectomy .et al [et al [et al [et al [et al [Out of 17 studies analysed by Janssen et al in 2015,l [et al and Burdl [et al deliverel [et al deliverel [et al , PORT wal [et al . The uneet al [n = 800) and those receiving delayed PORT (n = 201), on 5 days per week [At the European Institute of Oncology in Milan, Petit et al performeper week , 32, 34.et al [Finally, an international survey, conducted by Marta et al , collect p < 0.001 for each of them).Answers to the questionnaire resulted in the identification of nine risk factors, practitioners include into the decision to deliver PORT or not after NSM/SSM and IBR. These factors were: 1) nodal involvement; 2) LVI; 3) grade III carcinomas; 4) triple negative histo-type; 5) young age; 6) positive surgical margins; 7) tumour size; 8) extracapsular extension (ECE) and 9) multi-centric presentation. As expected, radio-oncologists and surgeons did not fully agree on the magnitude of the risk level affecting each of these factors. For instance, the answers from the two professional disciplines most significantly diverged for the first five factors reported here above than mastectomy patients.In a recent analysis of the current role of PORT after NSM, a total of 112,817 patients were isolated from the SEER database . Over thet al [Up until recently, the European Institute of Oncology in Milan recommended the use of intraoperative RT to the NAC in this latter patient population . In patiet al indeed sRetrospective studies on NAC indicate that 10% to 30% of cases receive PORT , 39\u201346. There is also a grey zone in the literature as regards the specific impact of nodal status on treatment outcome after NSM or SSM. It is therefore legitimate to take into consideration the data retrieved from the Early Breast Cancer Trialists\u2019 Collaborative Group\u2019s meta-analysis . Compareet al [In the present state of knowledge, the current review strongly suggests that the recommendations by Marta et al are in aFinally, this review confirms that, despite the fact that an increase in complications is documented in patients receiving PORT, immediate breast reconstruction using tissue expansion and implant is an acceptable option for women undergoing NSM and SSM for breast cancer .Whether SSM or NSM is safe in patients to whom no PORT is delivered remains a matter of controversy, especially in the absence of direct, controlled comparisons between patients with or without adjuvant radiotherapy. The literature on PORT after NSM and SSM continues to elicit rather large inconsistencies when treatment efficacy is the main endpoint. In particular, NSM, which spares a small amount of glandular tissue to protect the areola blood supply, raises concerns about the oncologic safety of this type of surgery, especially in high-risk patients. PORT is likely to reduce the risk of local failure in patients treated with subcutaneous mastectomy. Although some reasonable recommendations emerge from large-scale pooled analyses or surveys, the absence of prospective studies still prevents surgeons and radio-oncologists to identify those cases that are bound to draw a clear benefit from PORT after SSM or NSM. This definitely warrants the activation of randomised trials in these clinical settings.The author has no conflicts of interest and did not receive any funding to publish this review article."} +{"text": "The induction of brown-like adipocyte development in white adipose tissue (WAT) confers numerous metabolic benefits by decreasing adiposity and increasing energy expenditure. Therefore, WAT browning has gained considerable attention for its potential to reverse obesity and its associated co-morbidities. However, this perspective has been tainted by recent studies identifying the detrimental effects of inducing WAT browning. This review aims to highlight the adverse outcomes of both overactive and underactive browning activity, the harmful side effects of browning agents, as well as the molecular brake-switch system that has been proposed to regulate this process. Developing novel strategies that both sustain the metabolic improvements of WAT browning and attenuate the related adverse side effects is therefore essential for unlocking the therapeutic potential of browning agents in the treatment of metabolic diseases. Adipose tissue is sensitive to changes in nutrient supply and ambient temperature: an evolutionary development that has allowed animal species to adapt to food shortage and cold temperatures. In higher vertebrates, white adipose tissue (WAT) primarily stores energy in the form of triglycerides in unilocular white adipocytes, which can then be released as fatty acids when food is scarce , as well as various peptides and hormones adipocytes, the activation of which upregulates Ucp1 and other genes involved in energy expenditure in WAT. Browning of WAT is an adaptive and reversible response to environmental stimuli, including cold exposure, pharmacological agents such as \u03b2de novo differentiation centers the browning transcriptional network. It has been proven to be necessary and sufficient for adipocyte differentiation and function Farmer . ChronicModulations of PPAR\u03b3 through ligands, posttranslational modifications, isoform distinction . Adipoq knock-out mice show increased thermogenic response , and fibroblast growth factor 21 (FGF21)\u2014are produced in response to \u03b2Besides the mainstreams of transcriptional and hormonal regulations, various mechanisms have been identified to regulate browning that include cytoskeleton remodeling genetically obese mice resulted in reductions in total body weight and subcutaneous fat stores , an enzyme involved in prostaglandin synthesis, also induced browning of WAT and consequently increased energy expenditure and reduced adiposity are PPAR\u03b3 agonists that were widely used as insulin sensitizers in the treatment of type 2 diabetes. In addition to their insulin sensitizing function, TZDs are well known to induce thermogenic gene expression in both white and brown adipocytes , whereas Fgf21-deficient mice show an impaired response to cold stress due to diminished thermogenic activity emerges as an insulin-mimetic hormone that regulates systemic energy balance and has beneficial effects on body weight, insulin sensitivity, dyslipidemia, and pancreatic \u03b2-cell growth mediate thermogenesis in BAT and lipolysis in WAT; thus, activating these receptors with selective pharmacological agonists is an attractive strategy for stimulating the browning of WAT. A number of \u03b23-AR agonists have been developed as anti-obesity agents. However, their harmful side effects have called into question whether the long-term stimulation of \u03b23-ARs is safe and beneficial. Himms-Hagen et al. demonstrated that chronic treatment with a \u03b23-AR agonist, CL 316,243, led to the appearance of multilocular brown adipocytes in WAT, promoted thermogenesis, and delayed the development of obesity in rats fed a high-fat diet and T3 (triiodothyronine) are key regulators of metabolism and energy homeostasis, and have been shown to induce WAT browning , induced ectopic expression of UCP1 in rat abdominal WAT T Moolman . THs hav Moolman , which d Moolman . This fuUcp1 and the appearance of brown adipocyte clusters is a member of the superfamily of transforming growth factor-\u03b2. It has been shown to singularly promote the differentiation of mesenchymal progenitor C3H10T1/2 cells to a brown adipocyte lineage , and chronic diseases and Parathyroid-hormone-related protein (PTHrP) have been implicated in the browning of WAT in cachexia. PTHrP was originally recognized for its beneficial effects on skin, cartilage, placenta, and bone development (Maioli et al. Burn trauma causes hypermetabolism due to marked increases in catecholamines, which have been reported years after the initial injury (Kulp et al. Foxa3 resulted in a lean, energy inefficient and more insulin sensitive phenotype in mice older than one year old (Ma et al. Aging is arguably a major risk factor for metabolic syndrome (Tchkonia et al. In recently years, significant progress has been made in identifying stimuli and signaling pathways that can induce browning of WAT and trigger adaptive thermogenesis. These advancements in knowledge have garnered great support in exploiting adipose tissue plasticity together with browning agents as therapeutic tools for obesity, albeit with side effects as discussed above. The revelation of the two sides of the coin regarding the browning of WAT\u2014namely, that it mitigates the metabolic consequences of obesity but propagates a hypermetabolic state in other pathologic conditions\u2014suggests that browning \u201cwastes energy\u201d and thus is not a favorable physiological state. Therefore, we hypothesized that the body needs to tightly regulate this browning process via a \u201cbrake-switch\u201d system to prevent the negative outcomes of both hyper- and hypo-activation of browning activity (Ferrannini et al. Prmd16 gene expression (Ng et al. Zfp423 in white adipocytes led to the accumulation of beige adipocytes in WAT in adult mice, while its gain-of-function converted brown adipocytes into a more white-like phenotype (Shao et al. This \u201cbrake-switch\u201d hypothesis is supported by the recent identification of HOXC10, a homeobox domain-containing transcription factor, as a negative regulator of browning of WAT (Ferrannini et al. The browning of WAT has become an increasingly favorable strategy for ameliorating the effects of obesity and subsequent metabolic dysfunction. However, it is energetically inefficient and thus is physiologically unfavorable. Furthermore, recent evidence has implicated browning in the development of cachexia, lipotoxicity, and other detrimental outcomes under acute and chronic hypermetabolic conditions (as summarized in Fig."} +{"text": "Alcohol is one of the most commonly abused substances in the United States. Chronic consumption of ethanol has been responsible for numerous chronic diseases and conditions globally. The underlying mechanism of liver injury has been studied in depth, however, far fewer studies have examined other organs especially the heart and the central nervous system (CNS). The authors conducted a narrative review on the relationship of alcohol with heart disease and dementia. With that in mind, a complex relationship between inflammation and cardiovascular disease and dementia has been long proposed but inflammatory biomarkers have gained more attention lately. In this review we examine some of the consequences of the altered cytokine regulation that occurs in alcoholics in organs other than the liver. The article reviews the potential role of inflammatory markers such as TNF-\u03b1 in predicting dementia and/or cardiovascular disease. It was found that TNF-\u03b1 could promote and accelerate local inflammation and damage through autocrine/paracrine mechanisms. Unraveling the mechanisms linking chronic alcohol consumption with proinflammatory cytokine production and subsequent inflammatory signaling pathways activation in the heart and CNS, is essential to improve our understanding of the disease and hopefully facilitate the development of new remedies. Alcohol is one of the most commonly abused substances in the United States. The effect of alcohol on organ systems of the body extends beyond the liver, where it is metabolized, to include the central nervous system, cardiovascular system, kidneys, lung, gastrointestinal tract, pancreas, and the immune system is another recognizable complication of chronic excessive alcohol consumption deficits. The severity of this damage depends on the duration, and frequency of exposure to ethanol during gestation. There is strong evidence that during prenatal development alcohol exposure has negative consequences, however, the causes ethanol-induced neurodegeneration are poorly understood. Alcohol has been linked to hyper-inflammation, reactive oxygen species (ROS) generation and ultimately neuronal death , which can increase the influx of proinflammatory mediators and leukocytes into the brain. The BBB consists of the mainly of specialized endothelial cells lining the cerebral blood vessels, surrounded by pericytes, astrocytical processes, and neurons. It is known that alcohol can directly pass through the BBB, ultimately reaching the brain cells and causing subsequent neuronal toxicity, however the underlying mechanism is not fully understood. It was indeed suggested that chronic ethanol exposure induces oxidative stress and neuroinflammation in part by affecting platelet endothelial cell adhesion molecule-1 (PECAM-1) expression.Endothelial PECAM-1 is a cell adhesion molecule that allows for the interaction of immune cells and the endothelium which facilitates the transmigration of leukocytes and thus contributes to the endothelial cell permeability barrier , not only is there abnormal production of TNF-\u03b1, but TNF-\u03b1 has also been linked to the pathological neuromodulation of the disease (McAlpine et al., Alcohol is well-known for its dysregulation of cytokines levels in several body organs such as the liver, brain, lung, and plasma. It has been postulated that such changes are responsible for undesirables CNS alterations, which results in long term effects in behavior and permanent neurodegenerative effect. Hence, understanding the role of such cytokines is essential to determine the exact pathogenesis of alcohol-associated neurological disorders (Achur et al., Along with other cytokines, TNF-\u03b1 was found to be dysregulated by alcohol-related tissue destruction as is evident by the abnormal levels of circulating cytokines in alcoholic patients (Achur et al., Alcohol can be beneficial or harmful and may affect the heart and whole cardiovascular system in many ways. Some of the cardiovascular diseases that are associated with heavy drinking are: cardiomyopathy, cardiac arrhythmias, hypertension, atherosclerosis and heart failure Table . In thisAccording to Djousse and Gaziano, approximately half a million Americans are diagnosed with heart failure each year and some of those cases are associated with alcohol consumption (Djousse and Gaziano, Cardiac disease remains an essential burden that causes death in chronic alcoholic population Kannel, . ExcessiDespite the lack of clear understanding of the exact mechanism by which chronic alcohol consumption causes heart failure, stroke volume reduction and low ejections fraction was found in alcoholic population (Patel et al., Recent evidence shows that cardiomyocyte apoptosis appears to play a major role in many alcoholic cardiomyopathies leading to heart failure (Fernandez-Sola et al., K, by IGF-1 mainly through PI-3K/Akt activation (Teos et al., K reported earlier maybe mediated by IGF-1-dependent signaling pathway.Sparagna et al. has shown that low-dose alcohol attenuates apoptosis in neonatal rat cardiocytes through Akt and AMP-activated kinase (Sparagna et al., Even though long-term alcohol abuse has been associated with defect in cardiac contractility and eventual development of dilated cardiomyopathy and low-output heart failure Table , interes2+ (2+ channels nor their inactivation parameters. Interestingly, no data is available on the electrophysiological effects of low alcohol exposure or on the exposure frequency. Clinically relevant concentrations of ethanol induced elevation of Ca,L (Brown et al., + currents of rats (Nakamura et al., 2+i in a concentration dependent manner (Ren, Most of the experimental evidence indirectly relates chronic alcohol-dependent changes in intracellular Caner Ren, . Interes Lieber, . Interes Lieber, .Although once thought of as part of inflammatory cells only, cytokines and chemokines are now recognized to play pivotal role in cardiac homeostasis and repair (Tarzami et al., in vivo study on pregnant Wistar Rats, the authors extrapolated that the inflammation and oxidative stress are the mechanisms of the destructive effect of ethanol on their hearts (Shirpoor et al., The relationship between inflammation and inflammatory markers with alcoholic heart disease has gained much attention lately. In an Among these inflammatory markers, TNF-\u03b1 was particularly interesting for its role in cardiovascular diseases Ferrari, . In addiThe locally produced autocrine TNF-\u03b1 was found to have a noticeable role in alcohol-related heart failure (Meldrum et al., Alcoholism is a multifactorial disorder that requires a multidisciplinary approach to treat depending on the organs affected. Heavy drinkers suffer from many organ damages; among the most effected organs are liver and kidney. As we mentioned before, heart and brain can be affected either directly or indirectly by alcohol and or its breakdown metabolites. So far, conventional treatment strategies have used a combination of a reduction of ethanol-dependent inflicted damage by control drinking and increasing local and systemic protective mechanisms of the body by using antioxidant supplementation (Mailloux, Other non-conventional suggested approaches included the use of microRNAs since they are shown to play an important role in multi-organ alcohol-induced damages including brain and heart (Natarajan et al., Alcohol consumption effects cardiovascular system and predisposes to the development of cardiac abnormalities such as cardiac remodeling, cardiac arrhythmia, cardiomyopathy myocardia infarction, and even SCD Table . ConsequHeavy drinking has been shown to increase the risk of heart failure, thus in addition to the conventional approach to control alcoholism, it is critical to focus on preventive measures that could reduce the risk to heart failure in these patients. It was reported that normal heart does not express TNF but failing heart procures robust amount of TNF-\u03b1, Hence, more studies are dedicated to understanding the role of TNF-\u03b1 in heart failure patients. It is known that TNF-\u03b1 is elevated in chronic heart failure patient in accordance with their functional class (Heberto Herrera Garza et al., Attempts to integrate TNF-\u03b1 antagonist into the realms of therapeutic medicine has been suggested, with possible effectiveness for symptomatic heart failure patients (Heberto Herrera Garza et al., Interestingly, in brain injury, TNF-\u03b1 has been postulated to potentiate glutamate-mediated cytotoxicity in astrocyte leading to neuronal degeneration (Pickering et al., SDF-1, otherwise known as CXCL12, plays a major role in regulating stem cell recruitments, inflammation and inflammation mediated injury (Doring et al., In the heart, various cells including cardiomyocytes, stromal cells, endothelial cells, fibroblasts, and dendritic cells express SDF-1 (Wei et al., Helicobacter Pylori induced peptic ulcer, rotator cuff disease, skin inflammation squamous cell carcinoma of the lung, renal ischemia-reperfusion injury, glioblastoma tumor, and AIDS-associated neurologic disorders (Han et al., in vitro, which indicates the pathological roles of SDF-1 in increasing the severity of neurological impairment due to increased astrocyte cell death (Table SDF-1 alpha has also been shown to play roles in other disease conditions that affect other systems in the body commonly affected by heavy alcohol exposure. SDF-1 has been shown to increase in toxic liver damage, neonatal sepsis, autoimmune, inflammatory diseases, th Table . Moreoveth Table . TogetheActivation of TNF-\u03b1 by SDF-1 has also been postulated to promote the reactivation of latent HIV in macrophages and microglial cells Table . Other pCollectively, these data suggest that the alcohol induced alteration in circulating chemokines is a major contributor in alcoholic mediated organ damages via both direct and indirect mechanisms Figure . The preIn conclusion, alcohol's action are complex, chronic alcohol consumption can increase the risk of heart disease, brain damage, dementia, neuropathy, metabolic disturbances, nutritional deficiencies, certain cancers, liver, and other faces of morbidity and mortality. In this review we have highlighted the contribution of inflammatory process in alcohol-mediated tissue damage and organ dysfunction. What is apparent from literature review is that the full understanding of the related cytokine signaling pathways in alcoholic injuries will be essential for further understanding of their potential contribution and their complex biological effects on alcohol-induced organ damage. Investigation of agents that interfere with inflammatory cytokine production is needed in order to enhance our understanding on their potential contributions into pathogenesis of diseases that are associated with excessive alcohol consumption.AO and AP: performed the literature search and designed the tables/figures and edited the manuscript; ST: wrote the manuscript; JL and GH: performed the literature search and discussed the content of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Cancer is a leading cause of death worldwide, and its incidence is rising with numbers expected to increase 70% in the next two decades. The fact that current mainline treatments for cancer patients are accompanied by debilitating side effects prompts a growing demand for new therapies that not only inhibit growth and proliferation of cancer cells, but also control invasion and metastasis. One class of targets gaining international attention is the aquaporins, a family of membrane-spanning water channels with diverse physiological functions and extensive tissue-specific distributions in humans. Aquaporins\u22121,\u22122,\u22123,\u22124,\u22125,\u22128, and\u22129 have been linked to roles in cancer invasion, and metastasis, but their mechanisms of action remain to be fully defined. Aquaporins are implicated in the metastatic cascade in processes of angiogenesis, cellular dissociation, migration, and invasion. Cancer invasion and metastasis are proposed to be potentiated by aquaporins in boosting tumor angiogenesis, enhancing cell volume regulation, regulating cell-cell and cell-matrix adhesions, interacting with actin cytoskeleton, regulating proteases and extracellular-matrix degrading molecules, contributing to the regulation of epithelial-mesenchymal transitions, and interacting with signaling pathways enabling motility and invasion. Pharmacological modulators of aquaporin channels are being identified and tested for therapeutic potential, including compounds derived from loop diuretics, metal-containing organic compounds, plant natural products, and other small molecules. Further studies on aquaporin-dependent functions in cancer metastasis are needed to define the differential contributions of different classes of aquaporin channels to regulation of fluid balance, cell volume, small solute transport, signal transduction, their possible relevance as rate limiting steps, and potential values as therapeutic targets for invasion and metastasis. Xenopus laevis expression system, introduced AQP1 channels enabled high osmotic water flux across the plasma membrane as compared to non-AQP control oocytes are a family of water channels that also include a subset of classes shown to mediate transport of glycerol, ions, and other molecules , which stimulates endothelial cell proliferation and angiogenesis in response to hypoxia , induce AQP1 expression in low oxygen conditions occurs in normal physiological conditions such as implantation, embryogenesis, and organ development, as well as pathological processes such as cancer invasion and metastasis degradation and retraction Figure . Cell poA migrating cell extends its leading edge into the ECM by assembling a branched network of intracellular actin filaments, predicted to yield a physical force that dynamically pushes the membrane out, alternating with relaxation and actin depolymerization Wang, . Membranin vitro, and augmented metastasis in a mouse model -mediated phosphorylation of AQP4 at serine 180 correlated with a decreased glioma cell invasion have been shown to interact with adhesion molecules and to influence adhesive properties of migrating cells. Increased AQP1 in mesenchymal stem cells enhances migration by a mechanism involving \u03b2-catenin and the focal adhesion kinase (FAK) chloride HgCl cells (Gao et al., + is an inhibitor of voltage-gated potassium channels, calcium-dependent potassium channels, the nicotinic acetylcholine receptor, and it has also been shown to block AQP-1,\u22122, and\u22124 water permeability in Xenopus laevis oocytes and kidney derived cell lines (Brooks et al., + is variable, having been confirmed by some groups (Detmers et al., + in erythrocytes with native AQP1, or in epithelial cells transfected with AQP1, and suggested previous positive results might have been due to inhibition of K+ channels and altered baseline cell volume; however, the observation that site-directed mutation of AQP1 altered TEA sensitivity (Brooks et al., + block of AQP1 water permeability reduced cell migration and invasion in in vitro models of osteosarcoma and hepatocellular carcinoma (Pelagalli et al., + as a possible AQP1 inhibitor. However, given the variability in efficacy and cross-talk with other channels, TEA+ is not an ideal candidate for clinical development, although the targets causing the observed block of cancer cell migration and invasion might merit further investigation.TEAXenopus laevis oocytes (Migliati et al., in vitro (Dorward et al., 2, and NO (Nakhoul et al., 2 and cation transport properties (Yang et al., Xenopus oocytes stimulated with forskolin was first reported in 1996 (Yool et al., in vitro, but not in vivo (Klebe et al., in vitro remains to be determined.Bumetanide is a sulfamoylanthranilic acid derivative used clinically to increase diuresis by blocking sodium cotransporter activity at the loop of Henle in the nephron. Molecular derivatives of bumetanide have been synthesized and found to exhibit inhibitory effects on classes of AQP channels. For example, the bumetanide derivative AqB013 blocks osmotic water fluxes mediated by mammalian AQP1 and AQP4 channels expressed in Plant-based derivatives that reduce cancer cell migration and invasion include agents that have also have been found to inhibit AQPs. Bacopa monnieri is a perennial herb native to the wetlands of India that is used in alternative medicinal therapies. Chemical constituents bacopaside-I and bacopaside-II, were shown to block AQP1 but not AQP4 water channels (Pei et al., 2 (Zelenina et al., 4 (Zelenina et al., Mercury has classically been used as an AQP1 inhibitor. In the human AQP1 monomer, the NPA motif in loop E is near cysteine 189, which is the site at which mercury inhibits osmotic water permeability (Preston et al., Aquaporin-dependent mechanisms serve as key steps throughout the process of metastasis, in angiogenesis, cellular dissociation, cell migration and invasion. AQPs\u22121,\u22122,\u22123,\u22124,\u22125,\u22128, and\u22129 contribute to one or more processes, generally potentiating cancer invasion and metastasis by boosting tumor angiogenesis, enhancing cell volume regulation, regulating cell-cell and cell-matrix adhesions, interacting with the actin cytoskeleton, regulating proteases and ECM degrading molecules, contributing to the regulation of epithelial-mesenchymal transition in cancer cells, and interacting with specific signaling pathways important in cancer cell motility and invasions. Pharmacological agents for aquaporin channels have therapeutic promise for improving cancer treatment, and include derivatives of bumetanide, organic metal compounds, plant medicinal agents, and other small molecule compounds. Although conflicting evidence has been raised for some compounds, there is nevertheless a compelling need to continue identifying novel candidates for AQP-specific modulators relevant not only for the treatment of cancer, but other pathological conditions. In conclusion, although much remains to be defined for molecular mechanisms in cancer invasion and metastasis, the roles of AQP channel function in cancer progression will inspire new therapeutic targets for improving treatment of malignant and invasive carcinomas.MD: wrote the manuscript; AY: reviewed and edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "AD is a multifactorial disease that occurs in familial and sporadic forms, but is always accompanied by neurotoxic accumulations of amyloid and tau proteins. The deposition of amyloid was postulated to be central for AD pathogenesis in the \u201camyloid cascade hypothesis\u201d , which belongs to a small family of transmembrane proteins together with amyloid precursor-like proteins (APLP) 1 and 2. APP can undergo distinct secretase-mediated cleavages, following either the \u201cnon-amyloidogenic\u201d or \u201camyloidogenic\u201d pathway. The first serves a range of important physiological functions, including regulation of transcription and synaptic plasticity (reviewed by M\u00fcller et al., In this commentary, we want to highlight two recent papers that addressed this question using overlapping and complementary methods: Puzzo et al. and WangOn the other hand, Puzzo et al. investigTaken together, the results from Wang et al. and PuzzAnother important open question is whether similar results would have been obtained using mice with a more acute, conditional APP reduction, such as a tamoxifen-inducible APP-KO line (Callahan et al., The new pathological role for APP has implications for studies using transgenic AD mouse models, which typically overexpress human APP (recent overview in Jankowsky and Zheng, In conclusion, the studies by Wang et al. and PuzzAS conceived the study and wrote the original draft with critical and substantial input from AU. AL-H provided valuable ideas and revisions. All authors reviewed relevant literature, participated in discussions, and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Our understanding of reactive oxygen species (ROS)\u2014a group of highly reactive chemicals containing oxygen\u2014has changed in the last few years from ROS as just harmful substances to crucial intra- and extracellular messengers as well as important regulators controlling a wide spectrum of signaling pathways. Nevertheless, there are still many uninvestigated points and open questions regarding ROS, especially in pathophysiology. Delicately controlled ROS homeostasis is critical for maintaining normal cell functions and any disruption in the oxidation-antioxidation balance leads to oxidative stress associated with a wide spectrum of human disorders such as chronic inflammation, age-related diseases, and cancers. In health, the intracellular ROS level is tightly controlled by various antioxidants. In contrast, cancer cells have an abnormally high level of ROS due to an increased ROS production and/or impaired ROS detoxification that can damage intracellular macromolecules such as nucleic acids, proteins, and lipids. Elevated ROS production in cancer cells may result from an aberrant metabolic activity, mitochondrial dysfunction, disturbed cellular signaling, oncogene activity, and interaction with tumor infiltrating immune cells.2O2 has the opposite effects on cancer cell proliferation depending on its concentration and cancer type. G. Vilema-Enriquez et al. in their article \u201cMolecular and Cellular Effects of Hydrogen Peroxide on Human Lung Cancer Cells: Potential Therapeutic Implications\u201d review effects of hydrogen peroxide on human lung cancer. The authors discussed effects of H2O2 on migration and invasion, calcium release, and other molecular features of cancer cells. Furthermore, they describe the link between hydrogen peroxide and inflammation. Finally, the authors hypothesize that novel therapeutic approaches against lung cancer may be based on the use of H2O2. Y.-C. Hung et al. in their review \u201cRoles of Reactive Oxygen Species in Anticancer Therapy with Salvia miltiorrhiza Bunge\u201d deal with Danshen as a drug of the traditional Chinese medicine and provide a systematic review of its antioxidant capacity and potential anticancer effects. Moreover, they conclude that based on the existed preclinical data this drug may be pipelined in clinical trials. A research paper by W. Li et al. deals with hyperglycemia in pancreatic cancer cells. The authors succeeded in finding the link between hyperglycemia and epithelial-mesenchymal transition through the production of hydrogen peroxide. Another research report on breast cancer of D. M. Badr et al. (\u201cThe Combination of \u03b1-Tocopheryl Succinate and Sodium Selenite on Breast Cancer: A Merit or a Demerit?\u201d) shows in vitro and in vivo that sodium selenite antagonizes effects of \u03b1-tocopheryl succinate on apoptosis induction in cancer cells via inhibition of oxidative stress. An intriguing review came from France, authored by M. Assi and A. R\u00e9billard, and was devoted to the problem of cachexia in cancer patients (\u201cThe Janus-Faced Role of Antioxidants in Cancer Cachexia: New Insights on the Established Concepts\u201d). As regulators of catabolic pathways ROS are involved in muscle atrophy in cachectic cancer patients, the authors summarize and discuss contradictory data on the effects of antioxidants in such patients.The ultimate purpose of this special issue is to publish high-quality research communications as well as review articles dedicated to the role of ROS in cancer biology, anticancer therapy, and related topics. Five articles published in this special issue are devoted to reactive oxygen species in cancer biology. Presently, it is rather well known that HThe next topic highlighted in this issue is devoted to ROS in tumor immunology. A review by X. Chen et al. (\u201cReactive Oxygen Species Regulate T Cell Immune Response in the Tumor Microenvironment\u201d) gives readers an overview of ROS in the tumor microenvironment and especially in the tumor-induced immunosuppression. The authors, based on improvement of anticancer T cell response, consider an antioxidant treatment as a promising option for cancer therapy. A. Scala et al. in their research article \u201cAlterations in Red Blood Cell Functionality Induced by an Indole Scaffold Containing a Y-Iminodiketo Moiety: Potential Antiproliferative Conditions\u201d deal with a prediction of the antiproliferative effects of heterocyclic scaffolds, which could be important for development of new therapeutic approaches against cancer.A research article by M. Weniger et al. considers pancreatitis as a main risk factor for pancreatic cancer. The authors show an unexpected analgesic effect of the new antioxidant SkQ1 during pancreatic inflammation. The last article from this issue deals with the oxidative stress in cancer-prone diseases in pediatric age. S. Perrone et al. in \u201cOxidative Stress in Cancer-Prone Genetic Diseases in Pediatric Age: The Role of Mitochondrial Dysfunction\u201d review recent literature on such diseases and discuss molecular mechanisms of oxidative stress associated with mitochondrial dysfunction. They conclude that mitochondria-targeted medicines could be applied into the clinics to improve the quality of life of patients with cancer-prone genetic diseases.Summarizing, the wide spectrum of review and research articles presented in this issue provides recent interesting data on ROS in the context of cancer biology and anticancer therapy.Alexandr V. BazhinAlexandr V. BazhinPavel P. PhilippovPavel P. PhilippovSvetlana KarakhanovaSvetlana Karakhanova"} +{"text": "Mandibular advancement surgery may positively affect pharyngeal airways and therefore potentially beneficial to obstructive sleep apnea (OSA).To collect evidence from published systematic reviews that have evaluated pharyngeal airway changes related to mandibular advancement with or without maxillary procedures.PubMed, EMBASE, Web of Science, and Cochrane Library were searched without limiting language or timeline. Eligible systematic reviews evaluating changes in pharyngeal airway dimensions and respiratory parameters after mandibular advancement with or without maxillary surgery were identified and included.This overview has included eleven systematic reviews. Maxillomandibular advancement (MMA) increases linear, cross-sectional plane and volumetric measurements of pharyngeal airways significantly (p<0.0001), while reducing the apnea-hypopnea index (AHI) and the respiratory disturbance index (RDI) significantly (p<0.0001). Two systematic reviews included primary studies that have evaluated single-jaw mandibular advancement, but did not discuss their effect onto pharyngeal airways. Based on the included primary studies of those systematic reviews, single-jaw mandibular advancement was reported to significantly increase pharyngeal airway dimensions (p<0.05); however, conclusive long-term results were lacking.MMA increases pharyngeal airway dimensions and is beneficial to patients suffering from OSA. However, more evidence is still needed to draw definite conclusion related to the effect of single-jaw mandibular advancement osteotomies on pharyngeal airways. Pharyngeal airway dimensions are inevitably affected by skeletal jaw movements during orthognathic surgery. Both one-jaw mandibular advancement, 2 and tSurgeons and orthodontists have gained increasing interest in pharyngeal airway evaluation, as it affects patients\u2019 health and quality of life. The effThe aims of this overview were to examine systematic reviews for changes in pharyngeal airway dimensions and/or respiratory parameters related to mandibular advancement osteotomies with or without concomitant maxillary osteotomies, and to critically appraise the quality of the reported systematic reviews.https://www.crd.york.ac.uk/prospero/display_record.asp?ID=CRD42016046489).The reporting of this systematic review adheres to the Cochrane\u2019s recommendation on overview of systematic reviews and the rd April 2017. There was no search limitation set for publication language or dates. The search results were exported into Endnote X7 and duplicate articles were removed. Next, the title and abstract of all articles were screened for potential eligibility, and the full text of relevant articles was retrieved. Lastly, the reference lists of those relevant articles were manually searched to screen for further relevant articles. Both electronic and manual searches were performed independently by two authors (TSK and RAZ). Disagreement was resolved by discussion with the other authors.The electronic databases PubMed, EMBASE, Web of Science, Scopus and Cochrane Library were searched using the search strategy outlined in This overview has included systematic reviews that have assessed changes of pharyngeal airways related to mandibular advancement osteotomies with or without concomitant maxillary osteotomies. Eligible systematic reviews had to report outcome measures of pharyngeal airway dimensions and their post-surgical changes, i.e. linear, cross-sectional plane or volumetric measurements. Furthermore, data from reviews reporting on respiratory parameter changes have also been evaluated and included.Systematic reviews that have studied specific target group (i.e. edentulous patients and morbidly obese OSA patients), or pharyngeal airways in cleft lip and palate, syndromic or distraction osteogenesis patients have been excluded from this overview.Data from included systematic reviews was extracted independently by two authors and inserted in pre-tabulated data sheets . Any disagreement related to data extraction was resolved by consensus in discussion with the other authors to ensure consistency and reliability of extracted data. The data extraction included authors, publication year and title, methods of analyses, number and study design of included studies, sample population ; type of interventions, outcome measures and main findings, follow up period and meta-analyses\u2019 result when available.The methodological quality of the included reviews was assessed independently by TSK and RAZ using the Assessment of Multiple Systematic Reviews (AMSTAR) tool. On the 2 test for heterogeneity (p = 0.1) and the \u03992 measure of inconsistency. A significant heterogeneity was considered when p< 0.1 for \u03c72 test or when \u03992> 50%. Treatment effects across the studies were combined using the fixed effect model when there was no heterogeneity observed (p> 0.1); in case of heterogeneity observed, the random effect model was applied. Funnel plot was used to assess publication bias, while Egger test for funnel plot asymmetry will be used when more than ten primary studies were included in an analysis[A narrative overview is provided summarizing the data gathered from included systematic reviews. Meta-analyses have been performed whenever possible by pooling the data across different reviews using the software \u201cReview Manager\u201d . The heterogeneity of trial results was assessed with the \u03c7 analysis, 24.An electronic search of the databases has generated an overall of 1642 articles. Titles and abstracts of 1211 articles were screened after removing the duplicates. The full texts of 23 relevant articles were retrieved and assessed for their eligibility of inclusion. No other relevant article was found while manually searching the reference lists of those 23 articles. Ultimately, 11 systematic reviews \u201318 have Two systematic reviews, 16 haveThe AMSTAR tool analysis revealed one systematic reviewwith a hiAlthough a self-declared no conflict of interest was found in eight systematic reviews, 16, 18,Eight out of eleven systematic reviews have analyzed the quality of evidence of their included primary studies. Three reviews, 9, 11 aet al[For the 38 primary studies that have been assessed based on risk of bias or methodology quality, only two were reported as low risk of bias or high methodological quality. Besides, half of them showed a moderate risk of bias or methodological quality. Zaghi et al did not The narrative information from the included systematic reviews is elaborated below. The results of the meta-analyses of included systematic reviews are shown in Although two systematic reviews, 16 inclMeta-analyses of three systematic reviews showed a significant increase of minimum cross-sectional area (CSA), pharynget al[et al[I2 = 0%), the fixed effect model was used. The meta-analysis indicated that MMA with or without genioplasty or genial tubercle advancement (GTA) lead to a significantly increased total pharyngeal volume after the surgery (p<0.00001). Although there was no statistically significant different (p = 0.62) between the subgroups, MMA with genioplasty or GTA has higher increased total pharyngeal volume (mean = 8.73ml) in comparison with MMA alone after the surgery. A symmetry funnel plot was noted suggesting a low risk of publication bias ..et al[etion bias et al[et al[et al[et al[Two primary studies reported by Butterfield et al, 43 were al[et al of the a al[et al meta-ana al[et al, 46 incl al[et al were fou al[et alwas eventMeta-analyses of post-MMA data reported by included systematic reviews in this overview revealed a significant reduction of the AHI, 18, Reset al[et al[Two systematic reviews, 18 haveet al reportedHsieh and Liao did not et al[et al[2 nadir (p = 0.04) to be associated with a higher post-MAA OSA cure rate (AHI<5/h).Univariate analysis of Holty et al suggesteet al. These r al[et al who haveThe here presented overview detected significantly reduced AHI after MMA with a relatively high treatment success rate (>85%) in OSA patients. This is comprehensible and in line with consistently increased post-MMA linear, cross-sectional area and volumetric pharyngeal airway measurements. The minimum CSA is one of the most commonly used airway measurements, and hasMandibular advancement with bilateral sagittal split osteotomies (BSSO) is a well-established procedure in the treatment for patients with retrognathic mandible, with concomitant beneficial effect on pharyngeal airways. However,et al[Based on CBCT analysis, Hernandez-Alfaro et alhave repoet al of MMA cet al, 18 withet al, 18 and et alare assocet al and OSA The maximum follow-up period varies across primary studies between 5 weeks to 12 years, with a vast majority of less than 5 years. Therefore, some of these follow-up periods were definitely too short since recurrence of OSA has been reported as late as 10 to 15 years after MMA. A standAround one third of the included systematic reviews have performed electronic search in only one database and therefore posed a significant threat to selection bias. Moreover, none of the included systematic reviews has disclosed the \u2018conflict of interest\u2019 status of their included primary studies. Besides, four systematic reviews did not describe the characteristics of their included primary studies. As the quality of systematic reviews is affected directly by the quality of its included primary studies, a thorough investigation and reporting of each included study are mandatory.et al[Publication bias is another critical aspect to be investigated in the systematic reviews. Only two, 18 out et al suspecteThe following shortcomings of this overview have to be highlighted. Most primary studies of the included systematic reviews were of moderate and only a few of high quality, which might have affected the quality of those systematic reviews. Besides, seven of the included systematic reviews\u201315, 17 hMaxillomandibular advancement (MMA) increases pharyngeal airway dimensions, providing positive post-surgical effects in patients suffering from OSA. However, still more evidence is needed to draw conclusions related to effect of single-jaw mandibular advancement osteotomies on pharyngeal airways.S1 Text(PDF)Click here for additional data file.S2 Text(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file."} +{"text": "Alternative splicing of precursor mRNA is an important mechanism that increases transcriptomic and proteomic diversity and also post-transcriptionally regulates mRNA levels. Alternative splicing occurs at high frequency in brain tissues and contributes to every step of nervous system development, including cell-fate decisions, neuronal migration, axon guidance, and synaptogenesis. Genetic manipulation and RNA sequencing have provided insights into the molecular mechanisms underlying the effects of alternative splicing in stem cell self-renewal and neuronal fate specification. Timely expression and perhaps post-translational modification of neuron-specific splicing regulators play important roles in neuronal development. Alternative splicing of many key transcription regulators or epigenetic factors reprograms the transcriptome and hence contributes to stem cell fate determination. During neuronal differentiation, alternative splicing also modulates signaling activity, centriolar dynamics, and metabolic pathways. Moreover, alternative splicing impacts cortical lamination and neuronal development and function. In this review, we focus on recent progress toward understanding the contributions of alternative splicing to neurogenesis and brain development, which has shed light on how splicing defects may cause brain disorders and diseases. Alternative splicing is a crucial step of post-transcriptional gene expression that substantially increases transcriptome diversity and is critical for diverse cellular processes, including cell differentiation and development as well as cell reprogramming and tissue remodeling. Our understanding of the physiological significance and disease implications of alternative splicing has been greatly improved by genetic approaches and RNA deep sequencing. In this review, we focus on alternative splicing in neuronal differentiation from stem/progenitor cells, neuronal migration and functional development of neurons Figure .trans-acting splicing regulators and cis-elements of pre-mRNAs and muscleblind-like 1 (MBNL1) exhibit switched expression during heart development to regulate splicing of cardiac mRNAs can be distinguished from neurons in the developing brain promotes exon 10 inclusion of PTBP2 and thus maintains PTBP2 level in neurons in NPCs and hence maintains apical progenitors. Genetic mutations that generate aberrant Flna splice isoforms in NPCs are linked to periventricular nodular heterotopia, a neuronal migration disorder. Thus, a better understanding of the mechanisms of neuronal alternative splicing may provide plausible treatment strategies for neuronal disorders.Among neuronal splicing regulators, PTBP1 is exclusively expressed in embryonic stem cells and NPCs, whereas PTBP2 and Rbfox proteins are mainly expressed in neurons. A recent report showed that PTBP1 and Rbfox antagonistically modulate neuronal fate via their roles in regulating alternative exon selection splice isoforms, i.e., PKM1 and PKM2 in neurons and glial cells, respectively (Zhang et al., PKM1 and PKM2 isoforms result from mutually exclusive exon selection. Selective expression of PKM isoforms is also critical for regulating glucose metabolism in muscle and cancer (Christofk et al., Brain tissues comprise a variety of cell types including neural precursor cells, neurons, and various subtypes of neuroglia. Tantalizing issues remain as to whether and how alternative splicing influences neural fate determination and which splicing regulators are involved (Raj and Blencowe, Reeler mutant mice and mice with spontaneous or targeted mutations of Dab1 or either of the receptors exhibit similar phenotypes characterized by ataxia, tremors, and a reeling gait (D'Arcangelo et al., Dab1 occurs during brain development, resulting in multiple splice isoforms (Gao et al., Dab1 exon 9b/c (Yano et al., Dab1. Differential selection of exons 7 and 8 of Dab1 is also intriguing because these two exons encode a domain containing critical tyrosines that are targets of Reelin-mediated phosphorylation. Moreover, ApoER2 also undergoes alternative splicing. The exon 19-containing domain of ApoER2 is important for synapse formation and function via its interaction with PSD-95 (Beffert et al., ApoER2 and that blocking SRSF1-binding sites using an antisense oligonucleotide has therapeutic potential (Hinrich et al., The mammalian cerebral cortex has a highly organized six-layered structure consisting of a variety of neuron subtypes (Molyneaux et al., Scn9a mRNA increases the SCN9A level (Eom et al., Alternative splicing also regulates neurologic functions such as axon guidance and synaptogenesis. A number of neuronal mRNAs undergo alternative exon selection to generate isoforms in response to neuronal stimulation. Synaptic activity promotes exon 19 inclusion of ApoER2, which then binds Reelin and enhances long-term potentiation (Beffert et al., The combination of various genetic tools and RNA-seq has advanced our knowledge of the impact of alternative splicing on neural development and function. Recently, the use of cell-surface or genetically engineered fluorescent protein markers and fluorescence-activated cell sorting has enabled the isolation of stem/progenitor cells and specific neuronal types (Zhang et al., C-HS, DD, and W-YT: Jointly wrote this review; W-YT: Defined the scope of the review and edited the draft. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "At the cellular and subcellular levels, acid-base balance is achieved by ion exchange.Most physiological processes are sensitive to pH and regulation of pH within tissues and body fluids represents a fundamental homeostatic process in living organisms. At the organismal level, acid-base balance is achieved by fast respiratory modulations of the partial pressure of CORecently in Current Biology, Jalalvand et al. describein vitro spinal cord was studied. While mammals regulate arterial blood pH (pHa) at 7.4 at their normal body temperature of 37\u00b0C, Jalalvand et al. (a and CSF pH (pHCSF) increase by ~0.015 unit per \u00b0C when body temperature decreases in ectothermic vertebrates upon intense and exhaustive exercise that interfaces with neuronal membranes. Chesler , the Danish Natural Sciences Research Council (FNU) (TW), the National Institutes of Health- NS091836 to Sherif Elbasiouny (SD), and the National Science Foundation IOS-1257338 (LH) for funding.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Anterior shoulder instability has been successfully managed arthroscopically over the past two decades with refined \u201canatomic\u201d reconstruction procedures involving the use of anchors for the repositioning and re-tensioning of the antero-inferior capsuloligamentous complex, in an effort to recreate its \u201cbumper effect\u201d.Research and online content related to arthroscopic treatment of shoulder instability was reviewed and their results compared.The short- and mid-term results of this technique have been very satisfactory. The greatest number of recent reports suggests that long-term results (>5 years follow-up) remain rather satisfactory, especially in the absence of significant glenoid bone loss (>20-25%). In these studies recurrent instability, in the form of either dislocation or subluxation, ranges from 5.1 to over 20%, clinical scores, more than 5 years after the index procedure, remain good or excellent in >80% of patient population as do patient satisfaction and return to previous level of activities.As regards arthroscopic non-anatomic bony procedures (Latarjet or Bristow procedures) performed in revision cases or in the presence of >20-25% bone loss of the anteroinferior aspect of the glenoid, recent reports suggest that their long-term results are very satisfactory both in terms of re-dislocation rates and patient satisfaction.It appears that even \u201clege artis\u201d performance of arthroscopic reconstruction decelerates but does not obliterate the degenerative procedure of dislocation arthropathy. The presence and grade of arthritic changes correlate with the number of dislocations sustained prior to the arthroscopic intervention, the number of anchors used and the age at initial dislocation and surgery. However, the clinical significance of radiologically evident dislocation arthropathy is debatable. Despite prompt reduction and immobilisation for a reasonable but not excessive period of time before further rehabilitation, it can often lead to recurrent shoulder instability -3. It apet al. , Porcell [et al. and Yian [et al. have sug [et al. , 8.A Bankart lesion or various Bankart-variants appear to be the \u201cessential lesion\u201d in most instability cases and is most usually amenable to \u201canatomic\u201d procedures that aim to reconstruct and recreate the anatomy of the antero-inferior capsule-ligamentous complex. Over the past 10 years refined arthroscopic reconstruction techniques involving the use of suture anchors have been developed and offer very satisfactory short- and mid-term outcomes that are comparable to classic open techniques. On the other hand, \u201cnon-anatomic\u201d procedures such as the Latarjet or Bristow coracoid transfer or other \u201cbone-block\u201d procedures are being used all the more often to address patients with a combination of osseous and ligamentous injuries. These procedures have recently been performed arthroscopically by a number of experienced surgeons, with a very promising outcome -12. In t1 & 2). Therefore, only studies reporting on the long-term outcome following arthroscopic stabilisation with the use of anchors will be analysed in this review. Both metal and bio-absorbable anchors have been used very successfully in the treatment of shoulder instability, neither one providing superior clinical outcomes. However, the visibility of the drill holes was significantly greater after using poly-L-lactic acid polymer implants [Initial clinical studies on the long-term clinical outcome (>5-year mean patient follow-up time) following arthroscopic Bankart procedures describe the clinical outcome in patient series where techniques involving the use of tacks and trans-glenoid sutures were employed and report relatively high re-dislocation rates and haveimplants .et al. [Kim et al. report oRhee and colleagues on the oUnlike Rhee and colleagues Larain aCastagna and colleagues in 2010 analysedet al. in 2007 [Porcellini in 2007 reportedPlath and colleagues in a veret al. [Van der Linde et al. report aet al. for failet al. . It alsoArthroscopic or open techniques involving bone block procedures or coracoid transfer techniques are used more and more often in both revision and primary instability surgery, especially in the presence of >20-25% bone loss of the anteroinferior aspect of the glenoid , 22. Recet al. [et al. have also reported a low recurrence of instability (4 out of 68 shoulders- 5.9%) in their long term results study of open Latarjet procedure [Schmid et al. have reprocedure .et al. [Dumont et al. have repet al. in 1983, who introduced the term \u201cdislocation arthropathy\u201d for this pathology and classified it radiologically [et al. [Arthritic changes in the shoulder joint following shoulder dislocation were first described by Samilson ogically . The cla [et al. and cons [et al. .No such evidence was available concerning the long term effect of arthroscopic anatomic procedures on the prevalence and evolution of dislocation arthropathy until recently, when a number of publications have demonstrated that the long-term incidence of moderate and severe dislocation arthropathy is similar to that reported following open anatomic procedures , 19, 28.et al. reviewed the long term results of 68 shoulders treated with the Latarjet procedure. One fourth of the patients showed progression of arthritis [3).Mizuno rthritis . This perthritis and the It appears that initial joint trauma sustained during anterior shoulder dislocation has long-term biologic effects on the shoulder joint cartilage and the joint physiology in general. Avoiding numerous pre-operative dislocations appears to prevent to some extent the occurrence and severity of post-operative dislocation arthropathy . One shoAnterior first time shoulder dislocation has significant short and long term effects on the shoulder joint. It can lead to anterior instability, loss of function and dislocation arthropathy. Open or arthroscopic anatomic procedures in the absence of severe glenoid bone loss provide good to excellent long term results even for the high demand athlete. Long term results of non-anatomic procedures in the presence of significant glenoid bone loss have been reported as satisfactory. Shoulders with a history of anterior dislocation are very likely to eventually develop some grade of arthritis even when they are treated surgically, however the presence of arthritis does not correlate with functional scores."} +{"text": "Stem Cells International set out to publish a special issue focused on the \u201cEx Vivo and In Vivo Stem Cell-Based Tissue Engineering Strategies for Their Use in Regenerative Medicine.\u201d This special issue resulted in a collection of nine outstanding articles which include two reviews and seven original studies made by recognized researchers from seven countries across Europe, Asia, South America, and North America.Tissue engineering is an emergent discipline focused on the generation of bioartificial tissue-like substitutes through the combination of biomaterials, cells, and growth factors. In this context, stem cell-based strategies have made substantial contributions in the field. Researchers worldwide have demonstrated that stem cells have an extraordinary capability to differentiate into different cell lineages, promote tissue regeneration, release a wide range of growth factors, and modulate the host immune response. Based on the current impact of tissue engineering and stem cells in medicine, Concerning the review articles, M. Cimino et al. contributed with a complete review entitled \u201cXeno-Free Strategies for Safe Human Mesenchymal Stem/Stromal Cell Expansion: Supplements and Coatings,\u201d where authors suggest that culture/expansion of human mesenchymal stem cells under xeno-free conditions is still needed to improve their clinical translation. The review made by A. Owczarczyk-Saczonek et al. discussed \u201cThe Use of Adipose-Derived Stem Cells in Selected Skin Diseases ,\u201d highlighting that these stem cells are promising alternatives to generate new engineered or stem cell-based strategies to treat skin diseases.in vivo studies were published in this special issue. On the one hand, M. Mata et al. published the article entitled \u201cIn Vivo Articular Cartilage Regeneration Using Human Dental Pulp Stem Cells Cultured in an Alginate Scaffold: A Preliminary Study\u201d in which they demonstrated, through different histological approaches, that human dental pulp stem cells have a positive impact on the regeneration of articular cartilage in rabbits. On the other hand, V. Chapman et al. demonstrated that late passage marrow-derived mesenchymal stem cells were more efficient than early passage cells in the treatment of osteoarthritis on their article entitled \u201cTherapeutic Benefit for Late, but Not Early, Passage Mesenchymal Stem Cells on Pain Behaviour in an Animal Model of Osteoarthritis.\u201d These articles provide new evidence related to the usefulness of stem cells in cartilage tissue engineering.In the field of cartilage tissue engineering, two interesting Cryptomphalus aspersa's eggs induced the differentiation of adipose stem cells to myofibroblast on their study entitled \u201cHigh Sensitivity of Human Adipose Stem Cells to Differentiate into Myofibroblasts in the Presence of C. aspersa Egg Extract.\u201d In another interesting ex vivo approach, E. Oliveira et al. on their article \u201cInfluence of Different ECM-Like Hydrogels on Neurite Outgrowth Induced by Adipose Tissue-Derived Stem Cells\u201d demonstrated the synergetic effect of the correct combination of biomaterials and adipose stem cells to increase the neurite outgrowth from DRG explants. In these articles, the usefulness and versatility of adipose stem cells in tissue engineering were well demonstrated.Currently, adipose-derived mesenchymal stem cells are considered one of the most promising mesenchymal stem cells by several and well-supported reasons. Within this special issue, N. Garcia-Honduvilla et al. demonstrated that a natural extract purified from in vivo approach, M. Garrido et al. demonstrate the potential clinical application of amniotic membrane in digestive surgery on their article \u201cTransplantation of Human Amniotic Membrane over the Liver Surface Reduces Hepatic Fibrosis in a Cholestatic Model in Young Rats.\u201d These three articles highlight the potential clinical usefulness of extraembryonic-derived cells and scaffolds in tissue engineering.Extraembryonic tissues are an important source of stem cells and natural scaffolds. In this regard, G. P. Liao et al. successfully repaired diaphragmatic defects in rats by using decellularized rat diaphragm containing human amniotic fluid-derived stem cells on their article entitled \u201cTissue Engineering to Repair Diaphragmatic Defect in a Rat Model.\u201d In addition, in order to solve the problem associated to the expansion of epithelial cells, S. M. Nam et al. investigated the use two stem cells sources as feeder cells of corneal epithelial cells on their article entitled \u201cEx Vivo Expansion of Human Limbal Epithelial Cells Using Human Placenta-Derived and Umbilical Cord-Derived Mesenchymal Stem Cells.\u201d In an Finally, in these nine articles, authors rigorously discussed and demonstrated the high versatility of different kinds of stem cells to differentiate, promote tissue healing, modulate host immune response, and serve as feeder platform for the ex vivo expansion of differentiated cells. In conclusion, the articles published in this special issue provide new tissue engineered-based strategies and scientific evidence that support the potential clinical usefulness of stem cells in regenerative medicine.V\u00edctor CarrielStefano GeunaMiguel Alaminos"} +{"text": "ICC) is the second most common primary hepatic malignancy with poor prognosis. Despite improvements in its diagnosis and therapy, the prognosis for ICC patients remains poor. An improved understanding of ICC pathogenesis and consequential identification of novel therapeutic targets would improve the prognosis of ICC patients. MicroRNAs (miRNAs) are a class of highly conserved, endogenous, small non\u2010coding RNA molecules of 18\u201323 nucleotides in length, which regulate gene expression through complementary base\u2010pairing with target messenger RNAs and subsequent gene silencing. Several studies have shown deregulated expression of miRNAs in ICC cell lines and tissues, in which these miRNAs play important roles in ICC apoptosis, cell proliferation, invasion, migration and metastasis. In this review, we illustrate the potential role of miRNA in the pathogenesis of ICC and explore the possibilities of using miRNAs as prognostic and diagnostic markers, as well as therapeutic targets in ICC.Intrahepatic cholangiocarcinoma ( Intrahepatic cholangiocarcinoma (ICC) originating from cholangiocytes is the second most common primary tumour of the liver MicroRNAs (miRNAs) belong to a new class of small non\u2010coding, endogenous RNAs comprising 18\u201323 nucleotides that negatively regulate gene expression through inducing mRNA degradation or repressing translation by annealing with the complementary sites in 3\u2032\u2010untranslated regions (3\u2032\u2010UTRs) of target mRNAs via exportin\u20105 Previous data showed that miRNA synthesis and maturation consists of a stepwise process that is compartmentalized between the nucleus and the cytoplasm et al. using 27 ICC tissues, 10 normal cholangiocyte samples and 8 normal liver tissues Deregulated miRNAs in ICC were listed in Table Further study on miRNAs expression profiling identified a number of significantly deregulated miRNAs in two ICC cell lines (HuCCT1 and MEC) as compared with a normal intrahepatic biliary epithelial cell line (HIBEpiC) et al. Oishi et al. Plieskatt et al. Wang et al. Karakatsanis et al. Recently, Zhang et al. Plieskatt via derepressing or suppressing important signalling mediators along specific signalling pathways pertinent to cancer development.A number of aberrantly expressed miRNAs have been reported to function as tumour suppressors or oncogenes in ICC Table via dereet al. Hu et al. Wang et al. in vitro and impaired tumour growth in vivo. Furthermore, PTPN14 and PTEN were identified as direct and functional targets of miR\u201021. High expression levels of miR\u201021 were closely related to adverse clinical features, diminished survival and poor prognosis in ICC patients.Wang The biological functions and/or prognostic significance of two down\u2010regulated miRNAs, namely miR\u2010204 and miR\u2010320, have been studied in details et al. Twist, and decreased E\u2010cadherin levels by directly targeting the Twist gene. Oishi et al. Li et al. in vitro and reduced the protein levels of SMYD3 and downstream target genes . Knockdown of SMYD3 inhibited cell migration and invasion resembling that of miR\u2010124 overexpression.Hepatitis C virus core protein (HCVc) plays an important role in the development of ICC. Zeng et al. Iwaki et al. Qiu et al. Li et al.Bernuzzi The dismal prognosis and aggressive progression associated with ICC have led researchers and clinicians to explore new avenues of potential treatment for ICC patients The authors declare no conflict of interest."} +{"text": "The present study seeks to determine potential associations between viral infections and neuropsychiatric diseases. To address this issue, we investigated the peptide commonalities between viruses that have been related to psychiatric and neurological disorders\u2014such as rubella, human immunodeficiency virus, and herpesviruses\u2014and human distal-less homeobox (DLX) proteins expressed in developing brain\u2014namely, DLX1, DLX2, DLX5, and DLX6. Peptide matching analyses revealed a high degree of pentapeptide sharing. From an immunological perspective, this overlap is relevant because pentapeptides are endowed with immunogenicity and antigenicity\u2014that is, they are immune determinants. Moreover, infection-induced immune cross-reactions might have functional, spatial, and temporal implications related to the functions and expression patterns of DLX1 and DLX5 in the fetal and adult human brain. In sum, our data support the hypothesis that viral infections may be linked to neuropsychiatric diseases through autoimmune cross-reactions caused by molecular mimicry between viral proteins and brain-specific DLX self-antigens. Infections, neuropsychiatric diseases, and language disorders are often concomitant pathological events that can have early etiological roots in fetal life and then become apparent in any stage across the life span of the individual, from the postnatal period to the late age family\u2014namely DLX1, DLX2, DLX5, and DLX6\u2014that have been thoroughly investigated in numerous studies on neurodevelopment. Indeed, the four TFs are expressed during early fetal neurodevelopment , where DLX proteins may be expressed , DLX2 , DLX5 , and DLX6 were dissected into pentapeptides offset by one aa residue: MTMTT, TMTTM, MTTMP, and so forth. Then, each of the resulting pentapeptides was analyzed for matches to a sample library of 25 viral proteomes using the Protein Information Resource (PIR) match program (https://research.bioinformatics.udel.edu/peptidematch/batchpeptidematch.jsp) was used as a control neural protein. Glutamate decarboxylases 1 and 2 were additionally investigated as DLX-related proteins.The primary amino acid (aa) sequences of human DXL1 : Borna disease virus ; Epstein-Barr virus ; Hendra virus ; Hepatitis B virus genotype C subtype ayr ; Hepatitis C virus genotype 1a ; Human cytomegalovirus ; Human herpesvirus 1 ; Human herpesvirus 2 ; Human herpesvirus 6A ; Human herpesvirus 6B ; Human immunodeficiency virus type 1 group M subtype A ; Human parvovirus B19 ; Influenza A virus, H5N5 ; Influenza A virus, H1N1 ; Influenza A virus, H7N7 ; Influenza B virus ; Influenza C virus ; Measles virus ; Rotavirus A ; Rotavirus C ; Rotavirus X ; Rubella virus ; Vaccinia virus ; Varicella-zoster virus ; and Zika virus .www.iedb.org) resource and 4.4% of the DLX2 peptide sharing ;the extent of the peptide sharing is independent of the virus protein length. For example, Rubella virus and the eleven times longer HHV3 share exactly the same number of pentapeptides\u2014namely, three\u2014with DLX1 protein;quantitatively, the extent of the peptide sharing is unexpectedly high compared to the mathematical expected value of the pentapeptide sharing between the five neural proteins and the 25 viral proteomes. The expected value can be calculated as follows: given two protein entities and assuming that all aa occur with the same frequency, the expected probability for the two entities to share a pentapeptide is expressed by the formulam is the number of pentapeptides present in the DLX1 protein ; n is the number of pentapeptides present in the set formed by the 25 viral proteomes , and N is the number of all possible pentapeptides. Since each residue can be any of 20 aa, then N is 205 . Developing the equation, the expected pentapeptide sharing between DLX1 and the 25 viral proteomes is equal to 1,02668314453125e-5 or, practically, zero.where: www.immuneepitope.org; Vita et al., n < 12.The pentapeptide matching between viral and neural proteins illustrated in Table A comparative analysis of DLX expression in fetal and adult neurogenic areas of the human brain was conducted using the online database and resources of the Allen Institute for Brain Science (Lein et al., On the whole, Figure Actually, few data are available on DLX protein expression in humans. Rakic and Zecevic studied https://www.proteinatlas.org/on DLX1 and DLX5 protein expression in the adult human brain. The data are shown in Figure Such experimental results obtained in human fetal developing brain are flanked by data collected from the Human Protein Atlas (www.proteinatlas.org; Uhl\u00e9n et al., In essence, we found a vast and unexpected peptide sharing between DLX proteins and numerous infectious agents that constellate human life, from prenatal time to adulthood. The peptide platform defined in Table In this context, different immunological pathogenic mechanisms might be theoretically account for the neuronal damage according to the type of immune response, i.e., humoral vs. cell-mediated, and the timing of infection-induce maternal immune activation in relationship to the expression patterns of DLX proteins in the fetus (see Figure On the other hand, a cell-mediated mechanism could also theoretically be implied in the cross-reactive immune-mediated subclinical damage of the fetal nervous systems, since memory T-cell trafficking between mother and fetus is also a well-known phenomenon (Jeanty et al., Based on data from Figure Functionally, infection-induced immune cross-reactions would affect two TFs that, according to numerous studies, are implicated in crucial functions and fundamental processes during neurodevelopment and adult neurogenesis, and are potentially relevant to language competence and other higher cognitive functions see Box ;Spatially, infection-induced immune cross-reactions would damage brain structures where adult neurogenesis occurs and that are involved in the neural circuitry of language and memory, and in cognitive and emotional functions (Ming and Song, Temporally, infection-induced immune cross-reactions suggest a two-hit model that, depending on the DLX protein expression profiles, comprehends targets allocated in two time-windows in the life of an individual with a subclinical damage in fetal life and clinical onset in adulthood.Box 1DLX1:regulates the development of the ventral forebrain, craniofacial patterning, and the early diencephalic subdivision (Eisenstat et al., regulates neurite maturation and migration (Cobos et al., regulates the fate switch between cortical and striatal interneurons: cells that ordinarily would become cortical interneurons transform toward a subtype of GABAergic striatal interneurons, thus reducing glutamatergic input to the hippocampus (McKinsey et al., its loss leads to subtype-specific loss of inhibitory interneurons with a reduction in inhibitory currents and generalized seizures in mice (Cobos et al., contributes to promote cortical interneuron migration from the basal forebrain by direct repression of the semaphorin receptor neuropilin-2 (Le et al., when altered, might be related to Mowat-Wilson syndrome (McKinsey et al., is downregulated or altered in autism spectrum disorders (ASD; Liu et al., has low thalamic expression in psychosis (Kromkamp et al., has been proposed as a language-associated protein (Boeckx and Ben\u00edtez-Burraco, DLX5:promotes neuronal differentiation in SVZ (Shu et al., its loss leads to defective neuronogenesis (Perera et al., contributes to convert fibroblasts into induced GABAergic interneurons (Colasante et al., regulates development of peripheral and central components of the olfactory system (Long et al., is a candidate genes for autism (Nakashima et al., is involved in Rett syndrome (Itaba-Matsumoto et al., participates to the regulation of the expression of the glutamic acid decarboxylases (St\u00fchmer et al., its loss preferentially reduces the number of mature parvalbumin- interneurons (Wang et al., in vivo, the possibility of obtaining animal models of neuropsychiatric disorders by immunizing pregnant animals with DLX proteins could be examined. Moreover, analyses of sera from human patients with neuropsychiatric diseases, for example schizophrenia, are warranted to measure immunoreactivity against the peptides shared between viruses and DLX proteins. Possibly, the results of these joint basic and clinical in vivo approaches might also help design new therapeutic approaches in neuropsychiatry.In synthesis, the present study confirms previous reports (Kanduc et al., All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "We are aware of concepts of osmotic and oncotic pressure in clinical physiology. Per Starling's law, the freely movable ions do not significantly contribute to the retention of vascular volume, or the maintenance of interstitial fluid pressure was predicted for a long time (Stiernet et al., In the beta cells of the pancreas, the insulin granules are not freely soluble molecules in the cytosol but rather exists as packaged vesicles. Electron micrographs of beta cells and physiological experiments have revealed that these insulin granules undergo regulated exocytosis to a glucose load, rather than random secretion Dean, . PotassiRecently, it has been suggested that the exocytosis involving insulin granules resembles remarkable similarity to the pattern of inhibitory enteric neuromuscular neurotransmission (Chaudhury, \u2212/\u2212 mice, the number and appearances of secretory granules in islet \u03b2-cells of knockout mice were normal (Sakamoto et al., The SLC17A9 channel transports ATP within the insulin containing dense core vesicles (Sawada et al., Type II diabetes mellitus is characterized by progressive exhaustion of the beta cells of the pancreas (Butler et al., In this commentary, we advance the hypothesis that SLC17A9 dysfunction may contribute to the ongoing inefficacy of insulin exocytosis, leading to progressive diabetes. The lack of entry of ATP through a dysfunctional SLC17A9 (Sakamoto et al., What causes SLC17A9 dysfunction is not clear. Interestingly, SLC17A9 is gated by ATP itself (Sawada et al., There have been recent advances of novel markers that can tag synaptic proteins, especially those acting on the membranes. PET imaging may detect these global aspects of neurotransmission (Finnema et al., ArC, conceptualized and development of manuscript, drafted manuscript. CD, major intellectual contribution. VD, major intellectual contribution. CM, important discussion. NS, important discussion. WM, important discussion and conceptualizations. RR, important discussion. AM, important discussion, manuscript recheck. AdR, important discussion and prospects. All authors read and approved final version of manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Helicobacter pylori-mediated inflammation has been implicated in gastric cancer in many previously established studies, and Fusobacterium nucleatum presence has been observed with greater intensity in colorectal cancer patients. Despite ambiguity in the exact mechanism, infection-mediated inflammation may have a link to cancer development through an accumulation of potentially mutagenic DNA damage in surrounding cells. The multiple DNA repair pathways such as base excision, nucleotide excision, and mismatch repair that are employed by cells are vital in the abatement of accumulated mutations that can lead to carcinogenesis. For this reason, understanding the role of DNA repair as an important cellular mechanism in combatting the development of cancer will be essential to characterizing the effect of infection on DNA repair proteins and to identifying early cancer biomarkers that may be targeted for cancer therapies and treatments.Pathogenic and commensal microbes induce various levels of inflammation and metabolic disease in the host. Inflammation caused by infection leads to increased production of reactive oxygen species (ROS) and subsequent oxidative DNA damage. These in turn cause further inflammation and exacerbation of DNA damage, and pose a risk for cancer development. The significance of cancer as a disease that affects a large percentage of the world population is undeniable. It is one of the leading causes of death worldwide and according to World Health Organization, it causes 8\u20139 million deaths/year. In the United States alone, it is projected that 39.6% of the population will have some type of cancer during their life. Consequently, there are enormous expenditures in the field of cancer care. The expenditures for cancer care in the United States were nearly $125 billion in 2010 and could reach $156 billion in 2020; as mentioned in the website of National Cancer Institute. Infection and infection-associated inflammation is the major threat of cancer. Chronic inflammation from infection causes abnormal immune response, obesity, DNA damage and cancer. The best example is the inflammatory bowel disease where Ulcerative colitis and Crohn's disease develop colon cancer.Helicobacter pylori (gastric cancers), hepatitis B and C viruses (hepatic cancers), and Fusobacterium (Colon cancer). The detailed list of microbes that have been researched in relation to their effect on certain DNA repair proteins are added in the Table In 1863, Rudolf Virchow was the first scientist to link inflammation with cancer. He mentioned that the origin of cancer was at sites of chronic inflammation and a group of irritants with tissue injury causes cell proliferation of innate immune system . The inflammatory cytokines have been reported to generate ROS and reactive nitrogen intermediates (RNI) using NADPH oxidase in phagocytic cells and epithelial cells directly induces DNA damage. The versatility of different ROS is reflected in the wide array of DNA damage that they can cause. In general, the hydroxyl molecule (OH\u2212) is the most damaging form of ROS, but other forms like the oxygen molecule (O2), RO2, and RO are also capable of different types of damage. For instance, while OH\u2212 can react with all of the bases and the deoxyribose backbone of DNA, O2 preferentially targets guanine residues that inflict damage on the host DNA such as DNA strand breaks that may affect tumor suppressors or oncogenes . The MMR, BER, and NER pathways respond to specific lesions in DNA residues. DSBs are particularly dangerous lesion and are repaired by two principal pathways: NHEJ pathway functions in all phases of the cell cycle, while the high-fidelity HR pathway requires a template for repair and utilizes available sister chromatids during the S and G2 phases of the cell cycle.Nucleotide excision repair, or NER, is a versatile repair pathway that recognizes bulky adducts and general base lesions that cause a distortion of the double-helix structure of DNA. The major sources of these types of DNA damage are ultraviolet radiation and various types of genotoxic chemicals and transcription-coupled repair (TC-NER) pathways. In GG-NER, DNA damage is removed from the whole genome while TC-NER is primarily involved in repairing the damage on the coding strand of actively transcribed genes is now considered a specialized sub-pathway of BER. They share several common proteins including APE1, Pol\u03b2, LIGIII\u03b1, along with the nick sensor poly (ADP-ribose) polymerase 1 (PARP1) and the scaffold protein X-ray cross-complementation group 1 (XRCC1) . On the other hand, the mammalian bifunctional DNA glycosylases have an associated AP lyase activity generating 3\u2032dRP or 3\u2032-P (NEIL1 and 2) and 5\u2032-P. The DNA polymerase then fills in the gap using the template DNA strand and finally, DNA ligase seals the nick by completing the repair of the DNA duplex , RAD50, and NBS1 recruits ATM (ataxia telangiectasia mutated), generates DNA breaks followed by phosphorylation of histone H2AX (generating \u03b3H2AX) that amplifies the damage signal and generate DSBs that ultimately progress to gastro-intestinal cancer and Colibactin. Cdt is present in There are multiple mechanisms for bacterial infections to induce cancer, and we focused gastric cancer and colon cancer here. Further studies in this arena will give more in depth insight to the mechanisms of association between specific bacterial infections and cancers, but in the next section we will discuss the well-known and characterized bacterial-infection associated cancers especially through the lens of ROS induced DNA damage and alteration of DNA repair pathways.Chlamydia pneumonia and Mycobacterium tuberculosis and failure in these bring DNA damage/mutation and genomic instability. Microbial infection is one of the major reasons of the failure of DDRs.Microbes such as bacteria, virus and parasites are able to activate or alter various signaling pathways that may lead to either activation of oncogenes or down-regulation of tumor suppressor genes in a contribution to cancer progression is able to bind E-cadherin to induce greater \u03b2-catenin release and ultimately activate WNT signaling, which is oncogenic and outer inflammatory protein A (OipA) are involved in epidermal growth factor receptor activation which leads to PI3K-AKT signaling and ultimately activates \u03b2-catenin , the major pathway that repairs small-scale mutations such as single base pair mismatches, is inhibited (Kim et al., H. pylori infection. Additional MMR proteins MLH1, MSH3, MSH6, PMS1, and PMS2 have also been shown to be down-regulated upon H. pylori infection of gastric cell lines AGS and BG (Machado et al., H. pylori infection are supposed to be repaired by the BER pathway in a normally functioning cell. In a cell infected with H. pylori, there is decreased expression of vital BER proteins. APE-1, an AP endonuclease, and YB-1, an early-stage repair protein of BER, are down-regulated upon H. pylori infection (Machado et al., H. pylori infection. Reduced expression of OGG1 allows for accumulation of abasic sites that would not be repaired by the other DNA repair pathways and lead to carcinogenic mutation-build up and cellular transformation (Kidane et al., An accumulation of DNA damage caused by chronic inflammation and resulting ROS has the potential to induce cellular transformation. In a normally functioning cell, the multiple DNA repair pathways are able to curb the accumulation of DNA mutations by addressing the damage as it occurs. In a cell infected by H. pylori induced gastric cancer has been. Certain pathogenic species such as enterotoxigenic Bacteroides fragilis and Escherichia coli strain NC101 have been linked to colitis-associated colon cancer (Wu et al., The association between bacterial infection and colon cancer has not been elucidated to the extent that Fusobacterium nucleatum in abundance in colorectal tumor tissues (Kostic et al., F. nucleatum is a gram-negative microbe more often associated with the oral cavity. However, the bacterial species have been identified in the early stages of cancer in colorectal adenomas as well as carcinoma samples (McCoy et al., F. nucleatum to mice was shown to speed up colonic tumorigenesis and induce a pro-inflammatory state through NF-\u03baB signaling (Kostic et al., F. nucleatum has also been positively correlated with mortality linked to colorectal cancer, meaning that greater abundance of the bacteria more likely resulted in mortality due to the cancer (Mima et al., F. nucleatum is a causative agent in colorectal carcinogenesis, but the preliminary studies have indicated the involvement of bacteria in the induction of cancer (Ray, One area of interest in this field is the presence of cer Ray, .F. nucleatum is a very invasive bacterial species due to its ability to adhere well to mucous surfaces (McCoy et al., F. nucleatum can therefore act similar to H. pylori and adhere to host cell surfaces to alter cellular pathways with its bacterial proteins. Once F. nucleatum adheres to host cells, bacterial FadA adhesin binds E-cadherin to activate \u03b2-catenin signaling to increase cell growth and proliferation as well as to regulate inflammatory response of the cell (Rubinstein et al., F. nucleatum has also been found to activate TLR4 and ultimately lead to NF-\u03baB activation (Yang et al., F. nucleatum to a pro-inflammatory state (Kostic et al., F. nucleatum in the colon (Rubinstein et al., F. nucleatum contributing to carcinogenesis and tumorigenesis by inducing genetic mutations as a result of prolonged inflammation and potentially the down-regulation of various DNA repair pathways, similar to what occurs in H. pylori-mediated inflammation leading to gastric cancer.In the oral cavity, H. pylori and Fusobacterium, Salmonella typhi infection has been associated with the development of gallbladder cancer (Mager, Other bacteria associated cancers: Other that r Mager, . All othHelicobacter pylori infection correlated to a lower risk for esophageal cancer development (de Martel et al., Streptococcus pyogenes and Serratia marcescens in the late 1800's to induce an initial fever followed by treatment for many different types of cancers (de Martel et al., Salmonella enterica serovar Typhi reduced tumor growth and enhanced survival in mice (Vendrell et al., Mycobacterium bovis, is used clinically for the treatment of high-risk urinary bladder cancer (Kucerova and Cervinkova, While a number of bacterial infections have been shown to increase the potential for carcinogenesis, recent developments in the field have provided evidence for a positive role of certain bacteria and toxins in cancer prevention and therapy Mager, . For exaH. pylori, Hepatitis B and C virus, and Human papilloma virus (Mantovani et al., F. nucleatum and colorectal cancer, on the functionality of the various DNA repair pathways, can lead to novel identification of various markers for early cancer detection as well as more effective therapies and treatments that can combat the loss in DNA repair function in host cells.About 20% of the global cancer burden is linked to infectious agents including, but not limited to, All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "In this mini-review we discuss the biological characteristics of studied material and its handling which influence results of histochemical staining with \u201cclassical\u201d fluorochromes. Future perspectives of ROS and RNS imaging with newly designed probes are briefly outlined.Developmental transitions and stress reactions in both eukaryotes and prokaryotes are tightly linked with fast and localized modifications in concentrations of reactive oxygen and nitrogen species (ROS and RNS). Fluorescent microscopic analyses are widely applied to detect localized production of ROS and RNS Quite recently, peroxynitrite (formed upon NO reaction with superoxide anion) was shown as a positive regulator of plant cell signaling by tyrosine nitration in proteins are generated and scavenged over the whole life span of all known types of aerobic organisms. In plants and fungi production of ROS, together with reactive nitrogen species (RNS), has been linked with almost all developmental processes from germination through reproduction until cell death Asada, . ROS andArabidopsis research. Cell-permeable fluorescence-based probes were subsequently introduced to detect tiny real-time changes in ROS and RNS levels within relevant cellular compartments, e.g., DCF DA and DHDCF DA for detection of ROS , proper sample washing, keeping constant time of staining/scanning within a set of experiments, using optimal pH and turgor pressure can contribute to obtaining of correct results. Still it should be emphasized that histochemical staining and subsequent microscopic detection cannot be used for accurate ROS/RNS quantification but the combinations of several different analytical methods can give more reliable estimation of their intracellular levels for hydrogen peroxide , hydatodes (at leaf edge), or nectaria (in flowers) can enhance the introduction of fluorochromes into the living tissues of above-ground plant organs. The fluorochromes uptake in multicellular organs can thus be enhanced by increased external or decreased internal pressure, e.g., by syringe or vacuum infiltration, respectively (Figure Optimization of staining procedures for different photosynthetic and fungal organisms in our laboratory showed that results of ROS/RNS imaging in multicellular biological matrices are significantly influenced by the feasibility of material infiltration with the applied probes Figure . Currenty Figure . Moreovey Figure but sevey Figure .in vivo patterns of ROS and RNS abundance within plant organs and meristems. Newly synthesized probes with increased specificity and improved photostability have been reported, such as Aarhus Sensor Green preferable to SOSG for singlet oxygen (Pedersen et al., Arabidopsis, both on cultured cells and on leaves (Ledoux et al., 2O2 endogenously produced in living macrophages (Yuan et al., Recognized drawbacks of commercially available fluorescence probes for ROS and RNS detection initiated a quest for improved tools to measure more accurately the differential 2O2 with good selectivity based on intramolecular charge transfer (Chen et al., in vitro. However, the \u201cclassical fluorescent probes\u201d based mainly on diaminofluorescein derivatives, still represent important tools to study ROS and RNS in plant science (Nie et al., 2O2, peroxalate esters, and fluorescent dyes as published for in vivo imaging of H2O2 in mouse model (Lee et al., Fluorescein derivatives have become replaced in animal ROS and RNS research by more specific molecular probes based on nanoparticles or redox-sensitive fluorescent proteins (for review see Guo et al., in vivo monitoring. The situation resembles a \u201cpopulation bottle-neck\u201d; only a reduced number of protocols are applicable to ROS and RNS microscopy in plant and fungal models and thus these few remain fixed in routine practice for a substantial period. With increasing knowledge on the importance of localized and tiny intracellular redox fluctuations the quantitative and spatio-temporal analysis of ROS and RNS levels in plant and fungal cells is still highly challenging.Although, a plethora of ROS and RNS sensing molecules have been designed, just a part of them has been confirmed experimentally to be suitable for ROS and RNS MS prepared manuscript based on long-lasting discussions and joint experiments with LL, it was approved and widely discussed by both authors.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "One rationale for the frequent use of MgSO4 in critical care is the high incidence of hypomagnesaemia in intensive care unit (ICU) patients. Correction of hypomagnesaemia along with the neuroprotective properties of MgSO4 has generated a wide application for MgSO4 in ICU.Magnesium (Mg) has been developed as a drug with various clinical uses. Mg is a key cation in physiological processes, and the homeostasis of this cation is crucial for the normal function of body organs. Magnesium sulfate (MgSO Magnesium (Mg) is one of the most abundant cations in the body, and is also a drug with numerous clinical applications. The body usually contains up to 28 g of Mg . Most ovia regulating intracellular calcium availability . Magnesani, 2011). Calciuani, 2011; Noronhaani, 2011). Influx and efflux of Mg plays an important role in different transcellular transports . MagnesOne of the key reasons for the wide use of Mg in critical care is the high prevalence of hypomagnesaemia in ICU patients reduction of intestinal absorption of Mg, (2) increased loss of Mgbyrenal route, and (3) compartmental redistribution . The moIntravenous Mg supplementation rapidly increases serum Mg level following long-term use of PPIs and subsequent hypomagnesaemia. PPIs affect intestinal epithelial cell locally. Oral Mg is not effective in PPI's induced hypomagnesaemia. Discontinuation of PPI use will resultsin quick normalization of serum Mg levels , post-renal transplantation and drug-induced Mg wasting are reasons for renal Mg loss . These Numerous roles for magnesium in critical care medicine have been suggested . Defici4) was used to prevent eclamptic seizures in Germany . Magnesal., 1985). MgSO4 hak, 2002; Ignatavhak, 2002). A numbhak, 2002; Gonzalehak, 2002). Hypomahak, 2002). MgSO4 ard, 1925; Pritchaard, 1925), and coNumerous mechanisms of action have been suggested for magnesium including 1) vasodilatory action, (2) blood-brain barrier (BBB) protection, (3) reduction of cerebral edema, and (4) central anticonvulsant action .4 as a treatment option for patients who are resistant to standard therapy , agonists of \u03b22-receptors, inhaled ipratropium bromide, corticosteroids, anticholinergic drugs and general managements . Researal., 2008; Gontijoal., 2008; Jones aal., 2008; Kew et al., 2008). Life-tal., 2000; Daengsual., 2000; Griffital., 2000; Kew et al., 2000; Kokturkal., 2000; Rowe, 2al., 2000; Rowe anal., 2000; Rower eal., 2000; Singh eal., 2000).+-Ca2+ pumps), (2) muscle relaxation , (3) reduction of inflammatory mediators (inhibition of degranulation of mast cells and T-cells stabilization), (4) depression of the irritability of muscle fibers, and (5) inhibition of prostacyclin and nitric oxide synthesis. These mechanisms lead to a reduction in the severity of asthma reduction of intracellular calcium level .4 is used via intravenous and inhalation routes for the management of acute asthma and peak expiratory flow rate (PEFR) were assessed in different clinical trials . Use ofal., 2000; Daengsual., 2000; Griffital., 2000; Kew et al., 2000; Kokturkal., 2000; Rowe, 2al., 2000; Rowe anal., 2000; Rower eal., 2000; Singh eal., 2000). In somnes, 2008). Unlikeal., 2000, 1996[29al., 2000; Porter al., 2000; Scarfonal., 2000). The imal., 2002; Boonyaval., 2002; Devi etal., 2002; Gallegoal., 2002; Green aal., 2002; Hughes al., 2002; Mahajanal., 2002; Silvermal., 2002; Tiffanyal., 2002). In chial., 2013; Liu et al., 2013). Up to al., 2013; Devi etal., 2013; Green aal., 2013; Singh eal., 2013; Tiffanyal., 2013). Howeveal., 2006; Gallegoal., 2006; Gandia al., 2006; Hill etal., 2006; Hughes al., 2006; Kokturkal., 2006; Mangat al., 2006; Nanninial., 2006; Nanninial., 2006; Rolla eal., 2006; Zandsteal., 2006). Respiral., 2006; Gandia al., 2006; Hill etal., 2006; Mangat al., 2006; Nanninial., 2006; Nanninial., 2006; Rolla eal., 2006; Zandsteal., 2006). In oneal., 2013). In 201al., 2016). In chial., 2016). Adversott, 2008). 4 has been well documented to be beneficial in the management of nervous system injuries especially in the ICU. These injuries include stroke, aneurysmal subarachnoid hemorrhage (ASAH), and traumatic brain injuries . 4 in ASAH in the last two decades velocity and Glasgow Outcome Scale (GOS). The findings showed better outcome in patients with ASAH 90 days post-hemorrhage, but they did not find a significant difference in GOS between the control and treatment groups . Differal., 2002). Also, al., 2002) showed ues (2005) revealeal., 2006). High dal., 2008). MgSO4 al., 2013; Chen anal., 2013; Westermal., 2013). Despital., 2009; Wong etal., 2009). Friedlal., 2009) reporte4 24 hours post-stroke shows a significant decrease in the infarct volume based on the findings of MRI . Saver al., 2009). In theal., 2002). Singh al., 2002) showed al., 2013). It wasal., 2006). Concural., 2012; Zhu et al., 2012).4 on biomarkers in different neuropathies has been assessed in several studies . MgSO4 al., 2012). The inal., 2012; Mirrahial., 2012). The deal., 2012). S100B al., 2012; Lamers al., 2012). Increaal., 2012; Mizukosal., 2012). Increaal., 2012). 4 decreases inflammatory biomarkers such as nitric oxide, prostaglandin E2, interleukin 1\u03b2 and tumor necrosis factor-\u03b1 . Concural., 2013; Ovbiageal., 2013).TBI is an important health problem with high a mortality and morbidity rate . StudieNumerous studies have reported that Mg plays an important role in the prevention and treatment of central nervous system (CNS) injuries. Magnesium protects neurons from ischemic injuries and supports neuronal survival following TBI with different mechanisms such as: (1) blocking NMDA channels, (2) inhibition of presynaptic excitatory neurotransmitters, (3) inhibition of voltage-gated calcium channels, and (4) potentiation of presynaptic adenosine. Moreover, Mg can relax vascular smooth muscles and enhance cerebral blood flow. Serum total and ionized Mg levels are reduced after head injuries .4 in diffuse axonal injury has been shown by Zhao et al. . The inal. (2016). The pral. (2016). The moal. (2016). 4 cannot improve the strength of respiratory muscles in the critically ill patients under mechanical ventilation . Serum al., 1993). The noal., 1993).4 is an important drug in the ICU. MgSO4 can increase brain tissue oxygenation index by 34 % after cerebral artery occlusion . As staal., 2005). Electrolyte imbalance following hypomagnesaemia has been reported by researchers in the ICU , coronary artery bypass surgery and heart valve surgeries . Kaplanal., 2003). Concural., 2012). In subal., 2012). Adminial., 2013).4 as a neuroprotective agent, current evidence suggests that MgSO4 is an important part of the management of ICU patients (Table 14 therapy in the ICU, serum Mg levels should be checked on a daily basis.Despite the controversial views on the effects of MgSOs Table 1. (RefereThe authors have no competing interests to declare."} +{"text": "Editorial on the Research TopicHomeostasis and Allostasis of Thyroid FunctionA basic understanding of thyroid control involving pituitary thyrotropin (TSH) has become a cornerstone for the contemporary diagnosis of thyroid disorders. However, long-held simplistic interpretations of the classical feedback concept fall short of the elusive goal of a universally applicable and reliable diagnostic test. Diagnostic ambiguities may arise from the dynamic nature of thyroid homeostasis. Concentrations of TSH and T3 are governed by circadian and, addConsequently, the clinical care of thyroid patients faces major challenges, foremost ill-defined reference ranges for TSH and thyroid hormones (THs), and persistently poor quality of life in a substantial subset of treated hypothyroid patients . Divergestability through change by modifying setpoints and parameters of feedback control provides an overview of homeostatic mechanisms in the light of recent research. The classical \u201cshort feedback\u201d structure (Astwood-Hoskins loop) (A review article by the editors (ns loop) is now cns loop) , 18, thens loop) \u201323.Hoermann et al.; .Newly identified non-classical processing structures add to the complexity of the control system. They explain both pulsatile thyrotropin release and significant deviations from a log-linear relationship between FT4 and TSH concentrations . In et al.; , therebyrelational stability [Hoermann et al.; .The novel clinical concepts feed back to theory. \u201d models \u201335, demoLeow, extending implications to antithyroid treatment and LT4 substitution.Interpretation of thyroid function tests can be severely affected by homeostatic time constants resulting in hysteresis effects , as reviAlthough sensitive for primary hypothyroidism, TSH measurement has low specificity and is unable to detect dysfunctions of central origin. Isolated TSH measurements may be misleading in certain physiological and alloDietrich et al.) for novel diagnostic approaches based on mathematical modeling, such as functional thyroid reserve capacity and step-up deiodinase activity. These calculated parameters deliver estimates for \u201chidden\u201d structure parameters of thyroid homeostasis and provide early indicators of thyroid failure. Reconstructing the individual equilibrium point of thyroid homeostasis is facilitated by new tools and may prove useful as a personal target for L-T4 dosage titration , e.g., for therapy of stroke or in long-lasting space flights. Based on its pleiotropic effects they question if 3-T1AM can be a safe cryogenic drug. Some of the inconsistencies in reported serum concentrations may result from plasma protein binding, potential role of gut microbiota in the formation of thyronamines from iodothyronines or conversion of 3-T1AM to 3-iodothyroacetic acid (3-TA1), a possible major mediator of thyronaminergic signaling .The traditional view of pituitary\u2013thyroid feedback control holding T4 plasma concentration constant close to a fixed set point has beenHoermann et al.).Deeper insights in the physiology of thyroid function and its homeostatic control warrant a rethinking of diagnostic practice. The old paradigm employing TSH in the center of diagnostic work-up has to be replaced by a relational concept, where TSH is interlocked with FT4 and FT3, and multivariable distributions represent homeostatic equilibria , 30. ThiJD, JM, and RH wrote some of the papers in this Research Topic and participated as guest editors for manuscripts, where they were not coauthors themselves. All authors listed have made a substantial, direct, and intellectual contribution to this editorial and approved it for publication.JD received funding and personal fees by Sanofi-Henning, Hexal AG, Bristol-Myers Squibb, and Pfizer and is co-owner of the intellectual property rights for the patent \u201cSystem and Method for Deriving Parameters for Homeostatic Feedback Control of an Individual\u201d . All other authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported."} +{"text": "We recently elucidated several misperceptions in a field related to toxin/antitoxin (TA) systems, persister cell biology field, born of controversy, is plagued by several prevailing misperceptions. For example, some TA systems stabilize plasmids and other genomic regions; however, the evidence of post-segregational killing by toxins of TA systems is weak. In addition, the evidence is weak for cell death via TA systems like MazF/MazE and weak for TA systems mediating death in phage exclusion systems. Although many aspects of the biological roles of TA systems remain enigmatic, there are now some clear, confirmed TA functions: (i) phage inhibition, (ii) plasmid maintenance, (iii) stress response (including regulation of loci distinct from the TA pair itself), (iv) biofilm formation, and (v) persistence. Therefore, this opinion piece aims to challenge the oft-repeated claims of cell killing related to TA systems with the goal of emphasizing their primary biological role: constraining metabolism in a reversible manner. Hence, their roles in their five confirmed functions all stem from their ability to rapidly and reversibly reduce metabolic activity.Historically, TA systems were deemed cytosolic (not secreted); hence, TA system toxins were considered primarily to affect the metabolism of the host that produced the TA system rather than as serving as a weapon against other cells, such as colicins , regardless of the gene order. In addition, we prefer naming the antitoxin with four letters ending with an \u201cA\u201d , where possible, to more readily identify the antitoxin.How TA systems are classified is also evolving and is based on the mechanisms by with antitoxins mask toxin activity. There are six well-established systems , was discovered in 1983, and was shown to maintain a mini-F plasmid , that also stabilized a plasmid (R1) , and R1 plasmids have a copy number of 6 are also suspect. For example, although roughly 80% of vegetative Myxococcus xanthus cells undergo PCD during nutrient-limiting development , it was claimed that MazF mediated this killing has been proposed as a quorum-sensing element for E. coli type I TA system Hok/Sok inhibits T4 phage propagation was an \u201cantitoxin\u201d .Even this year it has been written that \u201cthe biological roles of many TA modules still remain elusive\u201d eliminating nearly all mRMA via toxin MqsR cleaving 5\u2032-GCU sites and (ii) reducing ATP via toxin GhoT and thereby reduces the translation of the RpoS transcript (Hu et al., S. aureus, the SavS/SavR TA system silences the virulence genes hla and efb by binding their promoters (Wen et al., This role of antitoxins controlling more than their own promoter was confirmed by antitoxin DinJ of the In addition, the TisB/IstR-1 type I TA system has been shown to have the physiological role of producing persister cells when the cell is stressed by ciprofloxacin (D\u00f6rr et al., mqsRA is not governed by conditional cooperativity (Brown et al., E. coli YafQ/DinJ TA system (Ruangprasert et al., Yersinia pestis HicA3/HicB3 TA system (Bibi-Triki et al., Proteus vulgaris HigB-HigA TA system (Schureck et al., It is frequently indicated that conditional cooperativity is the prevalent form of type II TA system regulation (Harms et al., Charles Darwin emphasized the importance of natural selection for beneficial mutations; i.e., changes that lead to improved reproduction will become dominant Darwin, . For bacTW conceived and wrote the manuscript. SS helped write the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Evidence from experimental and clinical studies have shown that oxidative/nitrosative stress and inflammation associated with metabolic disorders such as obesity, hypertension, and diabetes conduce to left ventricular hypertrophy, fibrosis, diastolic dysfunction, heart failure, and ischemia/reperfusion damage , 2.\u03baB, are shown. Information in this issue also includes findings on the relationship between ROS and the activation/maturation of dendritic cells (DCs) that might trigger cardiovascular and metabolic pathologies.In this special issue, original researches on the causative effect of oxidative stress and inflammation on pathologies such as mitral valve prolapse (MVP), arteriosclerosis, hypertension, and ischemia/reperfusion (IR) injury are presented. Also, the evidence of the effect of NLRP3-inflammasome and reactive oxygen species (ROS) production by Ca2+ overload on mitochondrial function, along with the demonstration of the regulatory properties of MG132, demetoxicurcumin, and lycopene on signaling pathways that activate either nuclear factor E2-related factor 2 (Nrf2) or NFThe strong association between osteoprotegerin (OPG) levels and oxidative stress status in patients affected by MVP with severe regurgitation opens its possible use as a serum marker of this pathology according to P. Songia et al. On the other hand, R. Mastrocola et al. demonstrate that the inhibition of NLP3-inflammasome reduces IR injury by activating prosurvival RISK pathway and preserving mitochondrial function. This is interesting and is related to the article presented by Y. Oropeza-Alamaz\u00e1n et al., which demonstrates that silencing of MCU decreases ROS production and mitochondrial dysfunction induced by Ca2+ overload in reperfusion injury.Pharmacological regulation of oxidative stress and inflammation is also included in this special issue. Y. Li et al. demonstrated that the downregulation of cyclooxygenase (COX-2) by demethoxycurcumin (DMC) favors nitric oxide (NO) production via e-NOS activation, preventing endothelial dysfunction, whereas L. Kong et al. demonstrated that MG132 administration inhibits Nrf2 and IkB proteolysis via the proteasome, preventing endothelial dysfunction associated with cardiovascular disease in animals with diabetic nephropathy. The antioxidant and anti-inflammatory properties of lycopene are shown to be related with the downregulation of Rho-associated kinases and the activation of key factor expression in the study described by Y. He et al., who propose that this compound might by a potentially effective method for transplant arteriosclerosis in clinical organ transplantation. Finally, J. Stein et al. show that Nox2-derivated oxidative stress and PKC activation play relevant roles in DCs activation and the atherothrombotic processes that might impinge on cardiovascular and metabolic pathologies.We hope that the research articles included in this issue contribute to the understanding of mechanisms related to the development of cardiovascular diseases and increase the possibility to find novel specific markers and the proposal of new potential therapeutic treatment with the use of new drugs that regulated pathway risk related with the trigger of cardiovascular disease."} +{"text": "Recently, Mikheil Gogiashvili and colleagues from TU-Dortmund have published a study about the metabolomics heterogeneity of breast cancer . The ba1H NMR are still relatively rare in breast cancer. Therefore, the present study of Gogiashvili and colleagues represents an important milestone in this field of research. Currently, the majority of prognostic studies with cancer patients has been performed based on mRNA (Grinberg et al., 2017; 20159]9]; March"} +{"text": "Studies in behavioral economics have shown that social punishment can explain why genetically unrelated individuals are often able to maintain high levels of socially beneficial cooperation and parties that are financially unaffected (\u201cthird parties\u201d \u2014TPP) and mentalizing brain regions in TPP (Baumgartner et al., Pioneering behavioral studies have showed that strong emotions trigger the willingness to punish norm violators (Hirshleifer, Recently, Krueger and Hoffman reviewedInterestingly, these three networks partially overlap with those underlying the detection of norm violations in other social contexts. There is a growing cognitive neuroscience literature on a neural mechanism that detects when individual behavior or beliefs differ from those of others (for reviews, see Izuma, However, amygdala activity was reported only in SPP and TPP studies (Buckholtz et al., Interestingly, TPJ activity during TPP is paralleled by an initial deactivation of the DLPFC (Buckholtz et al., These recent findings raise intriguing and testable questions for future research, e.g., in the use non-invasive brain stimulation to further verify fMRI findings. There is evidence suggesting that transcranial current stimulation could effectively modulate within- and between-network interactions. For example, transcranial alternating current stimulation induced oscillatory desynchronization between the medial frontal and parietal cortices and, therefore, affected value-based decisions but not closely matched perceptual decisions (Polan\u00eda et al., All authors listed have made substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "The IL-23/IL-23R is critical for pathogenic inflammatory Th17 cell differentiation. Experimental data in murine models of arthritis, colitis and encephalomyelitis corroborate these findings. This manuscript reviews the current knowledge on the effects of sodium chloride on innate and adaptive immunity. We also performed a systematic literature review for clinical studies examining the relationships between salt consumption and the development or the activity/severity of the most common IMDs mediated by the IL-23/Th17 pathway, i.e., rheumatoid arthritis (RA), multiple sclerosis (MS), and Crohn's disease (CD). Nine studies were found, 4 in RA, 4 in MS and 1 in CD. An association was found between developments of anti-citrullinated protein antibody (ACPA) positive RA in smokers and salt intake, but these results were not confirmed in another study. For MS, no association was observed in pediatric subjects while in adult patients, a link was found between salt intake and disease activity. However, this result was not confirmed in another study. These conflicting results highlight the fact that further evaluation in human IMDs is required. Moreover, physicians need to develop clinical trials with diet interventions to evaluate the impact of low salt intake on disease activity/severity of IMDs.Immune mediated diseases (IMDs) are complex chronic inflammatory diseases involving genetic and environmental factors. Salt intake has been proposed as a diet factor that can influence the immune response. Indeed, experimental data report the influence of sodium chloride on the differentiation of naive CD4 Immune-mediated diseases (IMDs) are complex chronic inflammatory disorders involving the contribution of different predisposing factors. Indeed, both specific genetic backgrounds and environmental factors participate in their pathogenesis. Environmental factors have been implicated according to the prevalence of these diseases in certain countries or geographical areas, and/or through exposure of the population to local factors, but lifestyle habits as well as diet are also involved. The most common IMDs are type I diabetes, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), psoriasis, multiple sclerosis (MS), and inflammatory bowel diseases, such as Crohn's disease (CD). Contributing environmental factors are specific for each IMD, and for instance, smoking is a well identified factor for the development of RA or CD, while ultraviolet exposure is involved in SLE. The influence of diet has long been suspected as a contributing pathogenic factor for IMD. For instance, a Mediterranean diet is thought to influence the occurrence of RA immune cells. For instance, sodium chloride favored the polarization of pathogenic CD4+ T cells toward a T helper 17 (Th17) phenotype , and especially ILC3 cells, and mucosal-associated invariant T (MAIT) cells transcription factor . IL-23 has the capacity to induce and maintain the expression of SGK1 in Th17 cells. Using protein-protein interaction analysis from a large database, FOXO1 was identified as a transcriptional factor that had a downstream role in the expression of SGK1. SGK1 promoted phosphorylation of FOXO1, which led to reduced FOXO1 activity and increased IL23r mRNA expression and induction of ROR\u03b3t, a major regulator of Th17 differentiation when cultured in a hypertonic milieu has also been reported in normal subjects who have a high salt diet . Finally in a case-control study, sodium intake as evaluated by 24 h urinary sodium excretion was found to be increased in a small cohort of patients with early RA in the colon (Aguiar et al., IL-10 deficient mice that were exposed to a high-salt diet developed also a more severe spontaneous colitis (Tubbs et al., Normal intestinal lamina propria mononuclear cells\u2014extracted from macroscopically and microscopically unaffected colonic samples of 9 patients undergoing resection for cancer colon- express high quantities of IL-17A, IL-23R, TNF-\u03b1, and ROR\u03b3t when exposed to increasing concentrations of sodium chloride (Monteleone et al., One study examined the relationship between salt consumption and CD (Khalili et al., + T cells markedly infiltrated the central nervous system (Kleinewietfeld et al., In EAE, mice that were fed with a high salt diet developed more severe disease. High salt consumption also accelerated the onset of disease. In this setting, IL-17A expressing CD4Our literature search found 4 papers analysing the effect of dietary salt in MS, namely 2 in adult patients and 2 others in pediatric subjects (Farez et al., We found neither experimental nor clinical data examining the relationship between salt consumption and psoriasis.via IL-23R promotion. Indeed, the studies by Wu et al. and Kleinewietfeld et al. both reported that an increased salt concentration promotes Th17 differentiation by SGK1 involvement (Capon et al., Sodium is an essential nutrient for the organism and is a major physiologic player. The description of its role as an environmental factor in inflammation, and especially in IMDs comes from several reports Arora, . Indeed,Lactobacillus murinus. Treatment of mice with L. murinus prevented salt-induced aggravation of actively-induced EAE by modulating Th17 cells. A moderate high-salt intervention in a pilot study in humans reduced intestinal survival of Lactobacillus spp. and increased Th17 cells. These results indicate that high salt intake is connected to the gut-immune axis (Wilck et al., One question is the consequence of a high salt diet on the blood or lymph node compartments. Indeed, besides the hypertonic and vasopressive effects on blood pressure, a high salt diet does not induce a high concentration in the blood or lymph nodes (van der Meer and Netea, Besides sodium chloride, are other salt components involved in the induction of the Th17 lymphocyte subset? For instance, potassium has been reported to be a protective cation for CD (Khalili et al., SGK1 gene polymorphism is another genetic background that merits investigation, despite negative findings in RA (Jiang et al., Despite strong experimental reports, clinical studies did not report consensual results. Indeed, clinical evidence for the relationships between sodium consumption and development and/or activity/severity of IMDs is limited. Three studies reported such a link in the setting of RA (Sundstr\u00f6m et al., The influence of the environment on the development and clinical expression of IMDs is an exciting and challenging issue. The role of sodium chloride in IMDs such as RA, CD, psoriasis and MS is a relevant question for the pathogenesis of these diseases but also for the management of patients. Current experimental data strongly support a link between salt intake and Th17 differentiation. However, the specific influence of dietary salt intake on the expression of IMDs in humans is still not demonstrated, and requires further studies, especially clinical trials with nutritional interventions aimed at comparing low vs. high salt diets. The interaction between salt and other environmental factors, as well as the genetic background, is a promising avenue for further research, in order to improve our understanding of the specific role of sodium chloride in the pathogenesis of IMDs.ET, MB, CV, and PS analyzed and discussed the literature and conceived the outline of the manuscript. ET and PS wrote the manuscript. All authors reviewed the manuscript and provided critical discussion and input.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Moreover, we measured the concentration of reduced and oxidized ascorbate and glutathione that are involved in ROS detoxification together with phenolics, anthocyanins and tocopherols. Among antioxidant enzymes, we analyzed the activities of ascorbate peroxidase , and the soluble and bound forms of polyphenol oxidase and guaiacol peroxidase . The data reported in this paper are related to the research article \u201cMetabolic characterization and antioxidant activity in sweet cherry (Prunus avium L.) Campania accessions\u201d, authored by Mirto et al. (2018) In this article, we reported the original data obtained by the study of metabolites and enzymes involved in sweet cherry antioxidant system. We measured hydrogen peroxide (H Specifications TableValue of the data\u2022The data show the antioxidant metabolites and enzyme activities measured in sweet cherries fruits.\u2022The data highlight the differences among the different sweet cherries Campania accessions.\u2022The data are useful to identify the accessions more suitable for long-term storage.1Selecting fruit accessions rich in antioxidant can help delaying fruit senescence and better preserving its characteristics during postharvest. To this aim, we investigated antioxidant metabolites level and antioxidant enzyme activities in forty-three accessions of sweet cherry fruits from Campania region 2O2), as well as malondialdehyde (MDA) of sweet cherry fruit accessions. MDA is considered a useful index of general lipid peroxidation and a biomarker for oxidative stress In 2 (PPO) or H2O2 (POD), and their subsequent, polymerization to melanin. This phenomenon known as enzymatic browning, that occurs during fruit storage In 2To identify the accessions which better resist to oxidative stress and, therefore, are more suitable for the long-term storage, sweet cherry fruits of forty-three accessions from Campania region were harvested and collected at commercial maturity, in the regional experimental farm \u201cImprosta\u201d, Eboli Campania, Italy . One additional commercial accession (Bigarreaux) and two commercial cultivars (Del Monte and Ferrovia) were used as reference. The data were obtained by biological triplicates, each one constituted by twenty fruits from five plants, analyzed separately. The fleshy part of fruits was cut, frozen in liquid nitrogen, and powdered -1. Hydrogen peroxide (H2O2) malondialdehyde (MDA) were assayed according to Woodrow et al. Reduced and oxidized ascorbate (AsA+DHA) and reduced and total (reduced plus oxidized) glutathione (GSH) were extracted and evaluated as described in Annunziata et al. o-tolidine, used as substrate. PPO analysis was performed using catechol as substrate. Ascorbate peroxidase was extracted and assayed according to Hediye Sekmen et al. Enzyme activity analyses were performed by spectrophotometer assays. Polyphenol oxidase and guaiacol peroxidase were simultaneously extracted by a two-step extraction protocol, that allowed the separation of both soluble and membrane bound isoforms"} +{"text": "Alzheimer\u2019s disease (AD) is ultimately linked to the amyloid precursor protein (APP). However, current research reveals an important synaptic function of APP and APP-like proteins (APLP1 and 2). In this context various neurotrophic and neuroprotective functions have been reported for the APP proteolytic fragments sAPP\u03b1, sAPP\u03b2 and the monomeric amyloid-beta peptide (A\u03b2). APP is a metalloprotein and binds copper and zinc ions. Synaptic activity correlates with a release of these ions into the synaptic cleft and dysregulation of their homeostasis is linked to different neurodegenerative diseases. Metal binding to APP or its fragments affects its structure and its proteolytic cleavage and therefore its physiological function at the synapse. Here, we summarize the current data supporting this hypothesis and provide a model of how these different mechanisms might be intertwined with each other. Alzheimer\u2019s disease (AD) is a fatal neurodegenerative disorder and a severe burden of our aging societies and several unstructured regions in a slightly distorted square pyramidal geometry (a type 2 non-blue site) to three protein ligands and two water molecules almost as high as to CuBD. Copper-binding to the GFLD again correlates with the reduction of a neighboring disulfide bridge (Cys98-Cys105) pointing to redox-activity also of this site. Furthermore, the match of geometry and functionality is suggestive for a complementation of the two copper-binding sites by either a conformational change within the E1 domain into a \u201cclosed\u201d conformation and with low affinity to zinc harbors two or three different metal binding sites, here designated as M1\u20133 spread on three helices to regulate homo- and hetero-dimerization with APP and APLP2 become progressively more acidic down to pH 6 in the trans-Golgi and <5 in lysosomes in the amyloid plaques , ceruloplasmin, tyrosinase and dopamine \u03b2 hydroxylase , or WD. Loss of ATP7A function causes growth failure, brittle hair, hypopigmentation, arterial tortuosity and neuronal loss most prominent in the hippocampus and cerebellum discussed involve S-nitrosylation, oxidation and allosteric modulation, increased anchorage of the neurotransmitter receptors to the membrane, and modulation of neurotransmitter receptor function receptors), Glycine and other surface receptors, such as TrkB. Furthermore, zinc can bind to and regulate ProSAPs/Shanks scaffolding proteins of the postsynaptic density (PSD), involved in synaptic signaling. Indeed, in some cerebral areas nearly 50% of the glutamatergic synapses are actually \u201cglu-zinc-ergic\u201d (Watt et al., In contrast to copper ions there is a substantial amount of zinc loosely bound to biomolecules, designated as reactive or chelatable zinc, which is implicated in neuronal signaling. Reactive zinc is largely distributed within presynaptic vesicles in some axon terminals throughout the telencephalon and co-localizes with a subset of glutamatergic neurons (Frederickson et al., Synaptic function of APP is discussed in detail in other chapters of this special issue. Briefly, loss of APP function leads to a reduced number of dendritic spines (Watt et al., in vitro interaction of APP, and mutations, abolishing copper-binding to the GFLD, reduce APP synaptogenic activity in a cellular assay system (Baumk\u00f6tter et al., As pointed out before, copper binds APP at different sites within the E1 and E2 domain, causing structural changes and altered dimerization properties and heparin binding characteristics (Baumk\u00f6tter et al., in vivo conditions at the synapse. Consistently, addition of 50 \u03bcM zinc to cells expressing APLP1 caused a lateral concentration at cell-cell contact sites (Mayer et al., Different reports suggest binding of APP, APLP1 and to a minor extent also APLP2 to zinc with affinities in the low micromolar range (Bush et al., Despite some major gaps in our understanding of APP/APLPs synaptic function, the current data as presented in this review article strongly suggest that activity-dependent changes in zinc and copper concentrations in the synaptic cleft can be sensed by the APP/APLP family. In turn, they seem to modulate neurotransmission by different pathways including neurotrophic activity of sAPP\u03b1 or trans-cellular dimerization/signaling. However, one major gap in our current understanding, especially in respect to the function of copper, is the limitation of available sensitive sensors, allowing determination of local transient changes in copper concentration. In this regard, live-cell optical imaging with fluorescent sensors offers a potentially powerful approach for interrogating aspects of labile copper accumulation, speciation, trafficking, and redox function in living systems at the molecular level. Such reagents have greatly facilitated the study of calcium and zinc in cell biology, but analogs tools for cellular copper remain underdeveloped (Zeng et al., KW, AA, CUP and SK wrote this review article.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Lin et al. reported that young, asymptomatic APOE4 targeted-replacement mice treated with rapamycin had restored CBF and BBB integrity to the level of wild-type controls [Alzheimer's disease (AD) is the most common form of dementia and the 6th leading cause of death in the US. Inheritance of the Apolipoprotein \u03b54 (APOE4) allele is the strongest genetic risk factor for late-onset AD - APOE4 carriers accumulate beta-amyloid (A\u03b2) and neurofibrillary tau tangles earlier and with more extensive pathology compared to non-carriers. However, decades before the aggregation of A\u03b2 and tau, cognitively normal APOE4 carriers have developed neurovascular deficits, including reduced cerebral blood flow (CBF) and impaired bloodbrain barrier (BBB) integrity . The abocontrols . The preRapamycin is a drug approved by US Food and Drug Administration (FDA). It has been widely used in clinical settings and was originally used as an immunosuppressive agent to prevent rejection of organs in transplant patients. By 1991, Heitman and co-workers discovered a kinase, Target of Rapamyin (TOR) . In mammLin et al. demonstrated that mTOR inhibition reduced proinflammatory pathways in brain vasculature that otherwise impair BBB integrity in the APOE4 transgenic mice [Lin et al. previously reported that inhibition of mTOR activates endothelial nitric oxide synthase and causes the release of nitric oxide, a vasodilator, which in turn increases CBF [Lin et al. found that rapamycin restored their CBF and vascular density, which were associated with reduced accumulation of A\u03b2 and cerebral amyloid angiopathy, and improved memory [nic mice . Loss ofnic mice . Restorinic mice . In addiases CBF . In miced memory . CollectLin et al. further demonstrated that rapamycin restores and preserves neurovascular functions in the APOE4 transgenic mice using MRI. Their results may provide the basis for future AD prevention trials in human APOE4 carriers.In conclusion, mTOR inhibition has been shown to increase lifespan and healthspan in various species. Rapamycin has been approved by the FDA since 1999 for various uses in humans, and these compounds have been given to cancer patients for relatively long periods of time with little change in the quality of life."} +{"text": "Irradiated ice-covered ocean worlds with rocky mafic mantles may provide the conditions needed to drive the emergence and maintenance of life. Alkaline hydrothermal springs\u2014relieving the geophysical, thermal, and chemical disequilibria between oceans and tidally stressed crusts\u2014could generate inorganic barriers to the otherwise uncontrolled and kinetically disfavored oxidation of hydrothermal hydrogen and methane. Ionic gradients imposed across these inorganic barriers, comprising iron oxyhydroxides and sulfides, could drive the hydrogenation of carbon dioxide and the oxidation of methane through thermodynamically favorable metabolic pathways leading to early life-forms. In such chemostatic environments, fuels may eventually outweigh oxidants. Ice-covered oceans are primarily heated from below, creating convection that could transport putative microbial cells and cellular cooperatives upward to congregate beneath an ice shell, potentially giving rise to a highly focused shallow biosphere. It is here where electron acceptors, ultimately derived from the irradiated surface, could be delivered to such life-forms through exchange with the icy surface. Such zones would act as \u201celectron disposal units\u201d for the biosphere, and occupants might be transferred toward the surface by buoyant diapirs and even entrained into plumes. Key Words: Biofilms\u2014Europa\u2014Extraterrestrial life\u2014Hydrothermal systems. Astrobiology 17, 1265\u20131273. Such hydrogenations yield an ever-renewed stock of highly specified organic molecules\u2014the so-called CHNOPS with a typical bonding motif -C\u2013C(H2)\u2013N(H)\u2013C\u2013O reactions as salty ocean waters are reduced to hydrogen and formate on gravitation into the primitive ultramafic crust that are potentially more amenable as acceptors along metabolic pathways than just CO2 or carbonate is a lower potential acceptor but has the advantage of also providing further carbon through a variant of the acetyl coenzyme-A pathway from hydrothermal activity, and an ocean with dissolved oxidants providing a geochemical gradient, the first life-forms could potentially follow some of the same metabolic pathways known to be at, or near, the root of the earliest life on Earth, that is, acetogenesis and methanotrophy , leading to effective delivery through the ocean to the ice-water interface along the ice-water interface to reach sites of down-welling convection in the ice that might bring radiolytically produced oxidants to the ocean at the same time (Vargas et al.,et al.,et al.,et al.,et al.,et al.et al.,et al.,et al.,et al.,The dynamic communities comprising any sub-ice-shell biosphere could constitute a habitable zone, manifested as microbial mats concentrated around oxidant-rich ice-shell sites at the down-welling regions of thermally unstable regions at the ice-water interface (Goodman et al.,et al.,et al.,et al.,et al.,et al.,Future missions to search for life on Europa (Hand"} +{"text": "Recently, Birte Hellwig and colleagues from the Department of Statistics, TU Dortmund University have published a study to predict late metastasis in breast cancer . In breIn the present study Hellwig et al. (2016) used a In recent years numerous studies have been performed to predict prognosis of cancer (Hammad, 2013; Hammad"} +{"text": "Neutrophils are well known for their role in infection and inflammatory disease and are first responders at sites of infection or injury. Platelets have an established role in hemostasis and thrombosis and are first responders at sites of vascular damage. However, neutrophils are increasingly recognized for their role in thrombosis, while the immunemodulatory properties of platelets are being increasingly studied. Platelets and neutrophils interact during infection, inflammation and thrombosis and modulate each other\u2019s functions. This review will discuss the consequences of platelet\u2013neutrophil interactions in infection, thrombosis, atherosclerosis and tissue injury and repair. Platelets are anuclear cell fragments that circulate in healthy humans at about 150,000\u2013400,000 per microliter blood. Platelets are well known for their role in thrombosis and hemostasis. Inherited or acquired defects in platelet count and/or function can be associated with bleeding complications. Conversely, platelets are key players in thrombotic disease and drugs inhibiting platelet function are vital in treatment and prevention of arterial thrombotic diseases such as myocardial infarction and stroke. Recent studies show that platelets have many functions beyond physiological or pathological thrombus formation. They play a role in inflammation, are effectors of injury in a variety of pulmonary disorders and syndromes, facilitate tissue repair and act in the growth and development of metastases of various cancers have been shown to crawl along and adhere to the surface of (activated) endothelial cells, eventually forming an almost continuous layer on the vascular endothelium (von Bruhl et al. At least three distinct mechanisms may be involved in platelet-dependent, neutrophil-mediated induction of thrombosis. First, neutrophils have been demonstrated to transfer tissue factor, the physiological initiator of coagulation, to platelets during thrombus formation in vitro (Giesen et al. The \u2018blood-borne\u2019 TF concept challenges the dogma that tissue factor is normally not located on cells in contact with blood and the source and quantity of blood borne TF is subject to ongoing debate (Butenas et al. A second mechanism by which the platelet\u2013neutrophil axis contributes to initiation and propagation of thrombosis is through generation of NETs (Fuchs et al. A third mechanism linking the platelet\u2013neutrophil axis to thrombosis relates to neutrophil constituents modulating hemostasis. For instance, neutrophil cathepsin G and elastase inactivate natural anticoagulant systems including tissue factor pathway inhibitor, thrombomodulin and antithrombin (von Bruhl et al. Platelets have long been implicated in acute coronary events. Their key role is evidenced by the success of platelet inhibitory drug in treatment and prevention of acute arterial thrombotic events (Jamasbi et al. Sterile tissue injury as for example encountered in injury by toxins (Miyakawa et al. For example, acetaminophen-induced liver injury results in an influx of platelets and neutrophils in the liver microcirculation (Miyakawa et al. Platelets and neutrophils have been implicated as drivers of ischemia/reperfusion injury in liver (Yadav et al. Platelets trigger the release of NETs in a model of experimental transfusion-associated acute lung injury and thereby aggravate disease (Caudrillier et al. Platelet\u2013neutrophil interactions have been shown to facilitate repair in a model of heat-induced liver injury (Slaba et al. Platelets are well known to be required for liver regeneration following partial hepatectomy and it has been suggested that growth factors stored within platelet granules drive platelet-mediated liver regeneration (Lesurtel et al. Finally, NETs have also been demonstrated to contribute to organ injury in sepsis (Czaikoski et al. Experimental and clinical evidence for a role of platelet\u2013neutrophil crosstalk in a variety of clinical conditions including inflammation, thrombosis, atherosclerosis and tissue injury is rapidly emerging. Agents blocking PSGL-1, P-selectin, or MAC-1 that have pleiotropic effects that include inhibition of platelet\u2013neutrophil interactions have been trialed in humans (Mertens et al. Inhibitors of PAD4 may have merit given the key role of PAD4 in NET formation and the intact NET-independent anti-inflammatory properties of PAD4-deficient neutrophils (Li et al. A clear advantage of targeting NETs in thrombotic syndromes is the lack of effect of the intervention on physiological hemostasis. As bleeding is an important side effect of current therapies for venous and arterial thrombosis and disseminated intravascular coagulation, the development of agents with a better risk/benefit ratio in terms of thrombotic potency versus bleeding risk would be a significant improvement.Although platelets are traditionally seen as key players in thrombosis and hemostasis and neutrophils as prime inflammatory cells, recent data demonstrate a complex interplay between these cells with important immune functions for platelets and a clear role of neutrophils in thrombotic diseases. The prime mode of interaction between platelets and neutrophils and the downstream effects of this interaction are summarized in Fig."} +{"text": "HTT is ubiquitously expressed and acts systemically, meaning blood, which is readily available and contains cells that are dysfunctional in HD, could act as a surrogate for brain tissue. We conducted an RNA-Seq transcriptomic analysis using whole blood from two HD cohorts, and performed gene set enrichment analysis using public databases and weighted correlation network analysis modules from HD and control brain datasets. We identified dysregulated gene sets in blood that replicated in the independent cohorts, correlated with disease severity, corresponded to the most significantly dysregulated modules in the HD caudate, the most prominently affected brain region, and significantly overlapped with the transcriptional signature of HD myeloid cells. High-throughput sequencing technologies and use of gene sets likely surmounted the limitations of previously inconsistent HD blood expression studies. Our results suggest transcription is disrupted in peripheral cells in HD through mechanisms that parallel those in brain. Immune upregulation in HD overlapped with Alzheimer\u2019s disease, suggesting a common pathogenic mechanism involving macrophage phagocytosis and microglial synaptic pruning, and raises the potential for shared therapeutic approaches.There is widespread transcriptional dysregulation in Huntington\u2019s disease (HD) brain, but analysis is inevitably limited by advanced disease and postmortem changes. However, mutant HTT gene and is characterised by motor, cognitive and psychiatric features. Onset occurs around 45 years on average and inversely correlates with CAG repeat lengthHuntington\u2019s disease (HD), the most common monogenic neurodegenerative disorder in the developed worldHTT is ubiquitously expressed589101415519HD research has traditionally focused on the brain due to the presence of characteristic mutant huntingtin protein aggregateset al.2324et al.Transcriptional dysregulation is a central feature of HD pathogenesisIn the current study we present a transcriptomic analysis of whole blood in human HD using RNA sequencing (RNA-Seq). We studied differential expression of individual gene transcripts and enrichment of differential expression in gene sets in two independent cohorts from Track-HDPremanifest gene carriers had a mean total motor score (TMS) of 2 and total functional capacity (TFC) of 13 between premanifest and manifest HD in either the Track-HDHTT gene carriers were combined to increase the analytical power in a comparison of HD and controls. Once again there were no individually significant transcripts in independent or combined cohorts, but the differential expression analysis in the combined cohort is given in Attempting to identify both HD specific and stage-specific changes in gene expression (mRNA) level, we compared premanifest, manifest and control subjects, whilst controlling for age and gender. C) of 13 , indicatWe next asked whether networks of genes with similar functional annotation were dysregulated in HD relative to controls. Pathway annotations were collated from publicly available gene ontology databases to form a set of generic pathways using the same method as the recent HD genome-wide association study (GWAS) of modifiers of age at onsetGene set enrichment analysis (GSEA), with a false discovery rate threshold of q\u2009<\u20090.05 to correct for multiple testing, identified 53 upregulated and 14 det al.et al.Through RNA-Seq, Miller, et al.et al.A limitation of using curated pathways from databases is the incomplete or incorrect annotation. One way to overcome this is to use gene co-expression, because genes that are co-expressed often have related functions. WGCNA identifies clusters (modules) of genes with highly correlated expression, constructing original, unbiased gene co-expression networks based on observed dataGSEA for brain co-expression modules was applied to our combined Track-HD and Leiden blood expression dataset. Immune- and inflammatory-related brain modules were upregulated in HD blood, and notable downregulated modules included synaptic function, proteasomal degradation, mitochondrial function and transcription. The 10 most significantly up and downregulated modules in the combined dataset that were also nominally significant (p\u2009<\u20090.05) in both independent cohorts are given in \u20134) correlation between dysregulation p-value in the direction of interest (positive) in HD blood and degree of module membership (kME)The module membership (kME) of a gene is measured by the correlation of its expression with the eigengene, which is representative of all gene expression profiles in the module et al.et al.\u221216) excess , suggesting some concordance in signal at the individual gene level. Furthermore, a significant excess of generic pathways was found to be significantly (p\u2009<\u20090.05) dysregulated in both datasets, most markedly in the positive (p\u2009<\u20090.001) direction, but also negative (p\u2009=\u20090.028), thus showing an overlap in biological signal. Pathways significantly upregulated in both datasets are mainly related to immune response in the 112 gene positive Track-HD subjects . After cWe then tested whether generic pathways that were significantly enriched for upregulated or downr\u22127) and 66 , which were also dysregulated in the HD caudateSimilarly, we tested whether modules dysregulated in HD blood relative to controls also coret al.\u22123). Using the same method as for concordance with Labadorf, et al.et al.\u22127). Thus, we conclude that analysis of TMS in the Track-HD cohort broadly supported the associations reported in Mastrokolias et al.Mastrokolias et al.\u22124).In Alzheimer\u2019s disease, an early inflammatory response involving microglia contributes to pathogenesis3637et al.yellow, being particularly highly enriched (combined Track-HD and Leiden p\u2009<\u20091\u2009\u00d7\u200910\u221216). Notably, this module has immune and microglia-specific functionset al.Zhang, HD research has focused on the brain as the most conspicuous clinical features can be clearly linked to progressive degeneration of specific brain regions45We conducted RNA-Seq of whole blood in two independent cohorts of HD patients. Using gene set enrichment analysis (GSEA) with publicly-available pathway databases and WGCNA modules from HD and control brain datasets, we identified dysregulated genes and gene sets in blood that replicated in both independent cohorts and correlated with clinical motor signs (TMS). These correspond to the most significantly dysregulated modules in caudate nucleus, the most prominently affected region in HD brain. This suggests mutant huntingtin drives a common pathogenic signature in both blood and brain.et al.944RNA-Seq more comprehensively and accurately quantifies mRNA than hybridisation-based microarrays or tag-based methodsDespite these limitations, gene set enrichment analysis identified significantly overlapping dysregulated pathways in the Track-HD and Leiden HD blood datasets, even though they differed in age and disease severity. Thus, through grouping transcripts into biologically relevant pathways and co-expressed transcripts, we could highlight areas of dysfunctional biology in HD. The observed upregulation of immune-related pathways is consistent with that previously identified in transcriptional and functional studies57910PGC-1\u03b1, a member of the dysregulated ATP metabolic process pathway and 66 (CNneg1), were also significantly dysregulated in the same direction in both independent blood datasets. Compared with other brain regions, the caudate has the largest number of expression changes and the highest correlation with HDet al.et al.Our demonstration of a transcriptional signature common to both HD blood and brain supports the use of blood cells to study aspects of HD biology. HD model systems, such as mice, only recapitulate aspects of disease and must be compared to the relevant data in human tissue5519et al.et al.et al.In AD, amyloid plaques are surrounded by chronically activated microglia36All experiments we performed in accordance with the Declaration of Helsinki and approved by the University College London (UCL)/UCL Hospitals Joint Research Ethics Committee and the LUMC IRB. Peripheral blood samples were donated by genetically-diagnosed HD patients and controls, and all subjects provided informed written consent.Manifest subjects demonstrated motor abnormalities that were unequivocal signs of HD, as evidenced by total motor scores (TMS) over 5 on the Unified Huntington\u2019s Disease Rating Scale (UHDRS). Premanifest gene carriers had a burden of pathology score (age x [CAG \u2013 36.5))The Track-HD cohort consisted of 54 premanifest gene carriers, 63 manifest HD subjects and 23 controls. These were a representative sample from the Track-HD study , preseleThe Leiden cohortWhole blood was collected in two PAXGene Blood RNA tubes per subject, and immediately placed upright at room temperature. They were checked at 5\u2009hours for incomplete mixing or separation, and any showing separation were remixed with a further 10 inversions. Tubes were stored overnight at \u221220\u2009\u00b0C and transferred to \u221280\u2009\u00b0C the following morning. They were sent on dry ice to Biorep within 30 days.TM method . Quality control measures were made on globin-reduced samples on the Bioanalyzer RNA 6000 Nano kit .Total RNA extraction was performed using the PAXGene Blood RNA kit , following the supplier\u2019s instructions. Each solution in the kit was divided into aliquots to process batches of 12 samples. Replicate tubes for each subject were processed on different days. RNA was stored at \u221280\u2009\u00b0C before proceeding with the quality measurements and further use. RNA was collected by centrifugation, washing with 70% ethanol, and resuspended in buffer. Quality measurements of total RNA were made using spectrophotometric analysis (Nanodrop), 260/280 ratio denaturing agarose gel, and the RNA 6000 Nano kit for the Agilent Bioanalyzer . Samples were globin reduced using the GLOBINclearTM Poly-A mRNA method (Illumina). In short, poly-A mRNA transcripts were captured from total RNA using poly-T beads and cDNA generated using random hexamer primingIndexed cDNA sequencing libraries were prepared using the TruSeqSequencing failed for six Track-HD samples, including four premanifest, one manifest and one control subject. Quality control analysis was performed using the RNA-SeQC packagehttp://www.ensembl.org/info/data/ftp/index.html, obtained in gtf format, genome build GRCh38.3, gene build updated in June 2015). To remove residual batch effects the R package svaseq was usedRNA-Seq data were aligned to the human reference genome hg19 using TopHat2Enrichment of differential expression among gene sets corresponding to biological hypotheses (pathways) was tested using the Gene Set Enrichment Analysis (GSEA) methodet al.The set of 124 HD brain expression modules derived by Neueder and Bateset al.A set of 117 co-expression modules derived from the Gibbs, We generated a set of 213 co-expression modules from Braineacet al.The set of 111 co-expression modules from Zhang, Weaknesses of relying on public databases to provide pathways for analysis include their restriction to prior biological knowledge and the poor annotation of many genes. To overcome this annotation gap, we also tested the following sets of gene co-expression modules for enrichment of dysregulation:et al.et al.Labadorf, et al. datasets.We used a similar method to test for concordance of fold change in genes between the Track-HD and Mastrokolias All data is deposited at the European Genome-phenome Archive (EGA) and accessible through the authors or the NeurOmics consortium.How to cite this article: Hensman Moss, D. J. et al. Huntington\u2019s disease blood and brain show a common gene expression pattern and share an immune signature with Alzheimer\u2019s disease. Sci. Rep.7, 44849; doi: 10.1038/srep44849 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations."} +{"text": "The Mediator complex was originally discovered in yeast, but it is conserved in all eukaryotes. Its best-known function is to regulate RNA polymerase II-dependent gene transcription. Although the mechanisms by which the Mediator complex regulates transcription are often complicated by the context-dependent regulation, this transcription cofactor complex plays a pivotal role in numerous biological pathways. Biochemical, molecular, and physiological studies using cancer cell lines or model organisms have established the current paradigm of the Mediator functions. However, the physiological roles of the mammalian Mediator complex remain poorly defined, but have attracted a great interest in recent years. In this short review, we will summarize some of the reported functions of selective Mediator subunits in the regulation of metabolism. These intriguing findings suggest that the Mediator complex may be an important player in nutrient sensing and energy balance in mammals. Gene transcription in eukaryotic cells is orchestrated through extremely complicated processes and multiple steps, including initiation, elongation, and termination, with the initiation being the most studied regulation step of gene expression. Disturbance of transcription initiation often results in serious diseases, such as cancer, in humans has sharply increased in the past decades. Dysregulation of genes in the pathways controlling glucose and/or lipid metabolism is common in states of insulin resistance and T2D, especially when patients are also diagnosed with non-alcoholic fatty liver disease (NAFLD) or cardiovascular disease and PGC1 families of transcription cofactors, MED1 contains two LXXLL motifs that provide the binding surfaces for various nuclear receptors, and either one of the LXXLL motifs is sufficient for protein\u2013protein interactions -selective genes and HMG-CoA synthase (Hmgcr) (Amemiya-Kudo et al. The functions of MED15 and other mediator subunits in worms have been recently reviewed (Grants et al. RhoA/MAL or Ras/ELK1, by directly interacting with them in response to upstream signals (Stevens et al. MED23 has been well studied in mammalian model systems. The importance of MED23 in viability has been shown with the embryonic lethality when MED23 was knocked out in mice (Balamotis et al. It is known that HNF4\u03b1 regulates a various set of genes that are not only involved in early development but also in liver and pancreatic cell differentiation, glucose metabolism, and lipid homeostasis (Odom et al. i.e., CDK19, MED12L, and MED13L (Daniels et al. Originally considered as a part of a transcriptional repressor in the large Mediator complex, the kinase module can repress or activate gene expression through kinase-dependent or kinase-independent mechanisms (Malik and Roeder The CDK8\u2013CycC dimer negatively regulates de novo lipogenesis by reducing nuclear SREBP-1a or SREBP-1c protein stability (Zhao et al. In addition to CDK8 function in the negative regulation of de novo lipogenesis through its kinase activity, it has been shown that CDK8 has a positive role in response to serum, likely at the elongation step instead of the initiation step where CDK8 is a negative regulator (Donner et al. Through MED13, the MED12\u2013MED13 dimer links the small mediator as well as the CDK8\u2013CycC dimer (Tsai et al. To date, not much information is available on the functions of MED12L and MED13L. Expressed in both heart and brain, MED13L is associated with early development of both heart and brain since its missense mutations and gene interruption are found in patients with congenital heart defect, learning disabilities, and facial anomalies (Muncke et al. Since the discovery of the Mediator complex as a transcription cofactor of eukaryotes, its subunits have been associated with various biological processes and several diseases ranging from developmental defects to cancer in animal models and humans. The metabolic functions of the Mediator complex have become increasingly significant. In most cases, the Mediator complex functions as a bridge to connect and integrate specific transcription factors to the basal transcription machinery, resulting in expression changes of a selective set of genes. In addition to the presence of many subunits, tissue-specific expression and possible diverse assembly states at different physiological conditions further complicate the biological functions of the Mediator complex. Future studies on the role of the Mediator complex in metabolism may include identification of how the metabolic or nutrient signals may regulate the assembly states and subunit compositions and investigation of tissue-specific functions of each subunit in regulating nutrient and energy metabolism in vivo."} +{"text": "Erythrocytes regulate vascular function through the modulation of oxygen delivery and the scavenging and generation of nitric oxide (NO). First, hemoglobin inside the red blood cell binds oxygen in the lungs and delivers it to tissues throughout the body in an allosterically regulated process, modulated by oxygen, carbon dioxide and proton concentrations. The vasculature responds to low oxygen tensions through vasodilation, further recruiting blood flow and oxygen carrying erythrocytes. Research has shown multiple mechanisms are at play in this classical hypoxic vasodilatory response, with a potential role of red cell derived vasodilatory molecules, such as nitrite derived nitric oxide and red blood cell ATP, considered in the last 20 years. According to these hypotheses, red blood cells release vasodilatory molecules under low oxygen pressures. Candidate molecules released by erythrocytes and responsible for hypoxic vasodilation are nitric oxide, adenosine triphosphate and S-nitrosothiols. Our research group has characterized the biochemistry and physiological effects of the electron and proton transfer reactions from hemoglobin and other ferrous heme globins with nitrite to form NO. In addition to NO generation from nitrite during deoxygenation, hemoglobin has a high affinity for NO. Scavenging of NO by hemoglobin can cause vasoconstriction, which is greatly enhanced by cell free hemoglobin outside of the red cell. Therefore, compartmentalization of hemoglobin inside red blood cells and localization of red blood cells in the blood stream are important for healthy vascular function. Conditions where erythrocyte lysis leads to cell free hemoglobin or where erythrocytes adhere to the endothelium can result in hypertension and vaso constriction. These studies support a model where hemoglobin serves as an oxido-reductase, inhibiting NO and promoting higher vessel tone when oxygenated and reducing nitrite to form NO and vasodilate when deoxygenated. How erythrocytes modulate vascular tone has been widely studied over the last two decades. The vasodilation of the vasculature under hypoxic conditions has inspired much research ranging from the effect of oxygen partial pressure on smooth muscle cell contractility and endothelial nitric oxide synthase (eNOS) activity to nitrite reduction by hemoglobin (Hb) inside erythrocytes and subsequent production of nitric oxide Figure . Here we2 molecule. As hemoglobin binds oxygen, conformational changes occur to the protein and the binding affinity of the protein for molecular oxygen increases. Thus, binding is cooperative so that binding subsequent molecules of oxygen becomes easier after initial binding. Cooperative binding can be understood by a two-state model: The high affinity R-state and the low affinity T-state by eNOS, which requires oxygen as a substrate along with L-arginine extremely fast (6\u20138 \u00d7 10However, as demonstrated above in the work by Chu et al. endothelial NO does play a physiological role in vascular function (Chu et al., One can explain this paradox of NO bioavailability and Hb concentration in the blood by considering Hb encapsulation by RBCs. Experiments where RBCs are mixed with NO in suspension or in a stopped flow apparatus show that RBCs scavenge NO 1,000 times slower than cell-free Hb (Liu et al., In addition to these changes under static conditions, a cell free zone created by the fluid dynamics of blood flow further reduces NO scavenging. The velocity of blood near the vessel wall is slower than in the center of the vessel due to friction. This creates a pressure gradient that pushes RBCs toward the center of the vessel away from the NO producing endothelium. Bugliarello and Sevilla, Cokelet and Liao et al. experimentally showed the presence of a cell free zone and computational modeling by Butler et al. supported its ability to reduce NO scavenging (Bugliarello and Sevilla, In disease, RBC membrane abnormalities promote thrombosis Ataga, . Setty eIn addition to adhesion, polymerization of hemoglobin in sickle cell disease leads to rigid, inflexible red blood cells, which effect blood rheology. Moreover, oxygenated sickle RBC also displays changes in cell mechanics and therefore blood rheology, attributed to altered hemoglobin concentration in the cell and changes to the cell membrane (Nash et al., Work by Bor-Kucukatay et al. suggest vascular function, specifically nitric oxide production could play a role in RBC deformability and rheological alterations to RBCs during hypertension (Bor-Kucukatay et al., As reviewed above, encapsulation of Hb inside RBCs greatly reduces its ability to scavenge NO produced by the endothelium. However, in multiple diseases RBC hemolysis occurs introducing cell-free Hb into the vasculature (Reiter et al., In vitro findings suggest cell-free hemoglobin has the potential to release hemin and take part in oxidative reactions resulting in oxidative stress and inflammation (Miller et al., Cell-free hemoglobin reactions in the vasculature and surrounding tissues through extravasation may add to disease conditions (Schaer et al., In addition to releasing Hb, hemolysis also releases arginase, an enzyme that converts L-arginine to ornithine, into the blood stream. L-arginine is the substrate for nitric oxide synthesis by eNOS and therefore, the release of arginase further diminishes NO production and vascular function (Morris et al., Red blood cell breakdown not only decreases NO bioavailability through NO scavenging by cell-free Hb, but also by production of red blood cell microparticles (Donadee et al., RBC hemolysis contributes to the vascular pathology of diseases and disorders such as thalassemia, hereditary spherocytosis, Glucose-6-phosphate dehydrogenase deficiency, paroxysmal nocturnal, hemoglobinuria, and autoimmune hemolytic anemia (Johnson et al., In contrast to the role played by RBCs in diminishing NO. Research shows deoxy-RBCs promote vasodilation in the presence of nitrite (Cosby et al., 2 caused vessel dilation and an increased concentration of ATP in the effluent (Dietrich et al., ATP activates purinoceptors on endothelial cells leading to the production of NO and alteration of vascular tone (Ralevic and Burnstock, Addition of nitrite to deoxyRBCs showed an enhanced effect on vasodilation when infused (Cosby et al., in vivo SNO-Hb must form during the oxygenation/deoxygenation cycle of RBCs. This leads to the question of how SNO-Hb forms. Huang et al. and Xu et al. ruled out allosterically-controlled transfer of NO from HbNO to the beta-93 cysteine of Hb (Xu et al., The second proposed mechanism for the effect deoxyRBC and nitrite on vascular function is the formation of SNO-Hb in the presence of nitrite and subsequent release of NO from deoxygenated SNO-Hb. Research has demonstrated the hypoxic release of NO from SNO-Hb and the ability of the released NO to relax vessels (Stamler et al., In vitro studies have rejected a significant role in the reduction of nitrite by other RBC molecules such as xanthine oxidoreductase and carbonic anhydrase supporting the hypothesis that hemoglobin plays a dominant role (Liu et al., The last proposed mechanism of RBC vasodilation in the presence of nitrite is the reduction of nitrite to NO by Hb. Brooks first studied the nitrite Hb interaction in 1937 and this work was later extended by Doyle in 1981 Brooks, ,b, to fo2O3 formation (Basu et al., + for the RBC membrane, which in turn could also lead to NO escape through nitrosylation of AE1 (Salgado et al., However, once NO is formed inside a RBC, it must somehow escape rapid scavenging by the intraerythrocytic Hb. Proposed mechanisms of escape of NO bioactivity from the RBC have included S-nitrosothiol formation and the role of the RBC membrane. Research by Basu et al and Roche et al suggested S-nitrosothiol formation could occur through a metHb-nitrite mediated NOther heme-globin nitrite reducers may play a role in modulating vascular function. Deoxymyoglobin reduces nitrite faster than Hb (Shiva et al., The production of NO by deoxyRBCs in the presence of nitrite and other NO donors provide a potential therapeutic role for plasma nitrite in reducing vasoconstriction, RBC adhesion, and potentially improving RBC deformability (Space et al., Red blood cells play an important role in vascular function. Through the compartmentalization of Hb, RBCs deliver oxygen, minimize NO scavenging, sense oxygen tension, and deliver NO bioactivity in hypoxia. In healthy conditions, RBCs promote hemostasis through the well-regulated delivery of oxygen and the balance of NO scavenging and production. As data has shown, the RBC, once only viewed as a sink for NO, has the ability to produce NO bioactivity under hypoxia. The important role of the RBC in vascular function becomes more evident in diseases that alter or compromise the RBC membrane leading to conditions of oxidative stress, hypertension, thrombosis, and vaso-occlusion.CH, MG, and DK-S together conceptualized the article and its content. CH wrote the initial draft of the manuscript and further edited the manuscript. MG and DK-S heavily edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Alternatively, resigned individuals may simply be numb to the negative effects of exclusion regardless of their momentary expectations (Bernstein and Claypool, Future research should examine if and how expectations matter for chronically excluded individuals. Williams refers tExclusion paradigms often blindside participants with exclusion (Williams and Wesselmann, whether participants will choose to respond pro- or anti-socially to exclusion, as well as the degree of their response, which is a current paradox in the literature (Wesselmann et al., get-acquainted paradigms (Wesselmann et al., life alone paradigm (Twenge et al., in vivo paradigms.Participants' expectations of inclusion also likely influence Situational factors that may influence expectations of relational evaluation could be the psychological closeness of the sources of inclusion (e.g., a romantic partner; Arriaga et al., role-based exclusion, Nezlek et al., Individual factors may also influence one's expected relational evaluation levels. For example, narcissistic individuals may expect high relational evaluation and thus respond with more aggression than non-narcissists when their expectations are violated (Bushman and Baumeister, Researchers should investigate the specific role that relational evaluation plays in the consequences of exclusion, and how situation-level and individual-level characteristics influence this construct. EW, JW, and MB each contributed to the theoretical arguments in this article.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Myocardial infarction afflicts close to three quarters of a million Americans annually, resulting in reduced heart function, arrhythmia, and frequently death. Cardiomyocyte death reduces the heart\u2019s pump capacity while the deposition of a non-conductive scar incurs the risk of arrhythmia. Direct cardiac reprogramming emerged as a novel technology to simultaneously reduce scar tissue and generate new cardiomyocytes to restore cardiac function. This technology converts endogenous cardiac fibroblasts directly into induced cardiomyocyte-like cells using a variety of cocktails including transcription factors, microRNAs, and small molecules. Although promising, direct cardiac reprogramming is still in its fledging phase, and numerous barriers have to be overcome prior to its clinical application. This review discusses current findings to optimize reprogramming efficiency, including reprogramming factor cocktails and stoichiometry, epigenetic barriers to cell fate reprogramming, incomplete conversion and residual fibroblast identity, requisite growth factors, and environmental cues. Finally, we address the current challenges and future directions for the field. As the leading cause of death in the United States, heart disease accounts for one out of every four mortalities \u2014that are necessary and sufficient to convert fibroblasts to induced cardiomyocyte-like cells (iCMs) both in vitro inhibition directs cells toward a cardiac lineage fate (Karamboulas et al., Widespread epigenetic repatterning occurs during direct cardiac reprogramming. Zhao et al. performed chromatin immunoprecipitation sequencing of H3K4me2, which marks the promoters and enhancers of transcriptionally active genes, and demonstrated that one week after GHMT cocktail reprogramming 47% of the H3K4me2 peaks had shifted to align with those of primary cardiomyocytes (Zhao et al., Given that extensive repatterning of histone modifications occurs during the reprogramming process, modulation of the deposition and removal of histone modifications may promote greater reprogramming efficiency. Hirai et al. used a small molecule inhibitor of Ezh2, the catalytic component of the PRC2 complex catalyzing H3K27me2/3, to demonstrate that Ezh2 inhibition early in reprogramming increases reprogramming efficiency (Hirai and Kikyo, To identify key epigenetic barriers to the reprogramming process, we conducted a comprehensive loss-of-function screen of epigenetic modifying factors and identified Bmi1 as a critical epigenetic inhibitor of reprogramming (Zhou et al., Ebf1 is a key target of Mll1, and the reduction in Ebf1 expression caused by Mll1 inhibition mediates the observed increase in reprogramming efficiency (Liu et al., In a similar approach, Liu et al. conducted a gain-of-function screen of cardiac development epigenetic modifiers and transcription factors and identified the H3K4 methyltransferase Mll1 as a barrier to direct cardiac reprogramming (Liu et al., Direct cardiac reprogramming is accomplished through the activation of cardiac gene expression but must also be accompanied by complete silencing of the original fibroblast signature. Characterization of epigenetic repatterning during reprogramming by Liu et al. demonstrates a gradual repression of fibroblast loci (Liu et al., Maintenance of residual fibroblast gene expression presents a roadblock to the successful reprogramming of functionally mature cardiomyocytes. MicroRNAs miR-1 and miR-133 enhance direct cardiac reprogramming by suppressing fibrotic gene expression (Muraoka et al., in vitro.In another approach to erasing fibroblast identity, studies have used inhibition of pro-fibrotic signaling pathways to enhance reprogramming efficiency (Ifkovits et al., in vivo (Mohamed et al. Mohamed et al. demonstrate that repression of residual fibroblast signature using TGF\u03b2\u00a0and Wnt inhibition also enhances reprogramming and cardiac function Recent studies have examined the effect of intracellular signaling pathways on direct cardiac reprogramming. Zhou et al. modulated intracellular signaling pathways by screening a library of 192 protein kinases to assess the effect on GHMT transcription factor reprogramming (Zhou et al., Abad et al. screened seven small molecule compounds with a demonstrated role in iPSC reprogramming and found that the Notch inhibitor DAPT enhances GHMT transcription factor cocktail reprogramming (Abad et al., in vitro and in vivo (Mohamed et al., in vitro (Mohamed et al., in vivo (Mohamed et al., Mohamed et al. screened a library of 5,500 small molecule compounds in an unbiased, high throughput approach to determine cell signaling pathways that modulate reprogramming and identified the WNT and TGF\u03b2 signaling pathways as barriers to reprogramming (Mohamed et al., in vivo over in vitro (Qian et al., In vivo reprogrammed iCMs are more similar to endogenous cardiomyocytes than in vitro reprogrammed iCMs. This suggests that unidentified extrinsic factors in the in vivo microenvironment such as topographic cues, mechanical forces, growth factors, cytokines, or paracrine signaling play an important role in promoting iCM maturation.Multiple studies have noted greater conversion efficiency or more complete functional maturation of iCMs reprogrammed in vitro reprogramming generated incompletely converted, immature iCMs (Yamakawa et al., in vitro generation of functionally mature iCMs that contract spontaneously and exhibit calcium oscillations (Yamakawa et al., Lack of requisite growth factors constitutes a barrier to reprogramming. Yamakawa et al. noted that under serum-based culture conditions, in vitro substrate (Chopra et al., In addition to growth factors, environmental cues are important in developing the functional maturity of iCMs. Cultured cardiomyocytes respond differently to the stiffness of the in vitro substrate stiffness or mechanical stretch, reprogramming does respond to other topographical cues. Morez et al. demonstrated that the forward programming of cardiac progenitor cells using the cardiac lineage transcription factor cocktail Myocardin, Tbx5, and Mef2c is enhanced by topographical cues which modulate histone acetylation (Morez et al., + adult progenitor cells were reprogrammed on flat or microgrooved collagen I coated polydimethylsiloxane substrates. Reprogramming efficiency and sarcomere assembly are enhanced on microgrooved substrates compared to flat substrates (Morez et al., While direct cardiac reprogramming is unaffected by in vitro cell culture substrates directly alters reprogramming efficiency and maturity.The identity and composition of the extracellular matrix also impacts cardiomyocyte phenotype and reprogramming. Substrate adhesive ligands alter cardiomyocyte sarcomere organization and maturation through integrin signaling. Sarcomeres are well organized and polarized when cardiomyocytes are cultured on fibronectin coated polyacrylamide substrates but not on collagen I coated polyacrylamide (Chopra et al., in vivo, Li et al. cultured reprogramming iCMs in a 3D fibrin hydrogel and demonstrated that 3D culture enhances direct cardiac reprogramming (Li et al., in vivo compared to in vitro (Li et al., To more accurately mimic environmental stimuli in vivo conditions also has the potential to improve reprogramming. Promoting angiogenesis through preconditioning with VEGF increases in vivo reprogramming efficiency and improves therapeutic restoration of cardiac function (Mathison et al., Myh7 positive cardiomyocytes in the infarct zone of GMT treated hearts and increases ejection fraction by four-fold (Mathison et al., in vivo reprogramming efficiency (Qian et al., Manipulation of Additional research is required to translate direct cardiac reprogramming into a clinical therapy. Necessary steps include continued basic research, research in large animal models, improvement in human reprogramming, and bioengineering of delivery mechanisms.A better understanding is needed of the mechanism of late stage reprogramming events and iCM maturation. Research in this area is currently hindered by inefficiency in the reprogramming process. Asynchronous, heterogeneous cell populations produce low rates of fully reprogrammed cells making it difficult to acquire sufficient cell numbers to study late stage reprogramming events. Early stage studies are aided by the comparative synchrony of cells early in the reprogramming process that provides a large sample population.in vivo monitoring systems, and genetic manipulation, making the mouse a particularly productive model. However, the mouse exhibits significant cardiovascular differences compared to humans. In addition to obvious differences such as small size and short lifespan, mice differ from humans in a range of anatomical, physiological, energetic, electrophysical, and mechanical properties that include heart rate, coronary artery structure, and contraction/relaxation kinetics. Large animal models such as the dog, sheep, or pig have greater physiologic resemblance to humans with similar body size, heart size, and heart rate. In fact, physiological similarities between pigs and humans are close enough to make the pig an ideal xenotransplant donor. Recent progress in genetic manipulation of pigs will contribute to the use of the pig as a cardiovascular disease model.Research in large animal models is also required to move direct cardiac reprogramming toward clinical application. The efficiency of housing, breeding, and handling rodents has made them the most widely used animal models in biomedical research. Additionally, a wealth of tools has been developed specifically for murine research, including imaging techniques, Although significant progress has been achieved in uncovering the molecular barriers to direct reprogramming in mice, research in reprogramming human cells lags far behind. Reprogramming human fibroblasts requires the addition of extra factors but yields far lower conversion efficiency. Spontaneously beating cells are rare, indicating that more work is required to translate findings from the mouse to human and uncover undiscovered molecular barriers in human reprogramming.in vivo research uses direct injection of retroviral vectors into the infarct zone. While conceivably, direct cardiac injection could be achieved in some myocardial infarct patients during coronary bypass surgery, a non-invasive delivery method is preferable. Additionally, retroviral vectors integrate into the host cell genome, incurring the risk of gene disruption and cellular transformation. The ideal delivery vector would be non-integrating with high transfection efficiency, specificity for the target cell type, and adequate capacity to accommodate multiple reprogramming factors. Adenoviruses are promising viral vectors for the delivery of reprogramming factors. The most commonly employed vector in clinical trials, adenoviruses are non-integrating, with large capacity and high transduction efficiency. Additionally, recent research using small molecules to achieve reprogramming and developments in nanoparticle delivery systems offer potential alternatives to viral vector reprogramming factor delivery.Finally, a safe and efficient delivery system is required for the translation of direct cardiac reprogramming to clinical use. Current"} +{"text": "The International Conference on Bioinformatics (InCoB) has been publishing peer-reviewed conference papers in BMC Bioinformatics since 2006. Of the 44 articles accepted for publication in supplement issues of BMC Bioinformatics, BMC Genomics, BMC Medical Genomics and BMC Systems Biology, 24 articles with a bioinformatics or systems biology focus are reviewed in this editorial. InCoB2017 is scheduled to be held in Shenzen, China, September 20\u201322, 2017. Abbas and Bahig investigMycobacetrium tuberculosis could explain how azathioprine could suppress the pathogenicity of this organism in transplant patients. Le and Ou [Molecular recognition factors (MoRFs) located within longer intrinsically disordered proteins perform important cellular functions such as signalling and regulation, but are very difficult to accurately detect. Sharma et al. have adde and Ou have devGutierrez and Nakai have buiCastiglione et al. have devOne of the fundamental assumptions of structural bioinformatics is that hydrophobic residues prefer to be buried in protein structures. By analysing the standard Barton502 dataset using support vector regression on informative physicochemical properties, Liou et al. show thaSmall organic molecules as drug lead compounds is an important area of structural bioinformatics research, where knowledge of the target\u2019s 3D structure enables rational ligand design, for therapeutic applications. Using a series of derivatives from the influenza drug zanamivir, Dholakia et al. have genGiven the spiralling cost of drug development, a growing number of approved drug molecules are being considered for medical conditions different from their original purpose, known as repurposing or repositioning. As the number of known drug-target interactions far exceeds the potential interactions of these drugs with other potential targets, predicting the repurposing of a drug appears to be extremely challenging. Ezzat et al. have addComputer-based morphological analysis present a rapid and efficient way to analyse biological samples. Skull analysis can be used for clinical applications as well as for species classification. Based on human orthodontic parameters used in clinical analysis, Mosleh et al. have devIn pathology applications, cell-based microscopic analysis suffer from out-of-focus images due to depth effects. Intarapanich et al. have devThe quality of the articles reviewed in this introduction are comparable with earlier InCoB conferences while the subject coverage has extended substantially into bioimaging. Next year's conference, InCoB2017 will be held in Shenzhen from Sept. 20\u201322, 2017 , with pa"} +{"text": "Bladder-related pain is one of the most common forms of visceral pain, and visceral pain is among the most common complaints for which patients seek physician consultation. Despite extensive studies of visceral innervation and treatment of visceral pain, opioids remain a mainstay for management of bladder pain. Side effects associated with opioid therapy can profoundly diminish quality of life, and improved options for treatment of bladder pain remain a high priority. Endocannabinoids, primarily anandamide (AEA) and 2-arachidonoylglycerol (2-AG), are endogenously-produced fatty acid ethanolamides with that induce analgesia. Animal experiments have demonstrated that inhibition of enzymes that degrade AEA or 2-AG have the potential to prevent development of visceral and somatic pain. Although experimental results in animal models have been promising, clinical application of this approach has proven difficult. In addition to fatty acid amide hydrolase and monacylglycerol lipase , cyclooxygenase (COX) acts to metabolize endocannabinoids. Another potential limitation of this strategy is that AEA activates pro-nociceptive transient receptor potential vanilloid 1 (TRPV1) channels. Dual inhibitors of FAAH and TRPV1 or FAAH and COX have been synthesized and are currently undergoing preclinical testing for efficacy in providing analgesia. Local inhibition of FAAH or MAGL within the bladder may be viable options to reduce pain associated with cystitis with fewer systemic side effects, but this has not been explored. Further investigation is required before manipulation of the endocannabinoid system can be proven as an efficacious alternative for management of bladder pain. Intravenous treatment of rats with URB937, a FAAH inhibitor with minimal penetration of the CNS decreased activity of bladder-specific afferent nerve in response to controlled filling of the bladder (Aizawa et al., AEA has been shown to be an agonist of the TRPV1 pro-nociceptive channel (Tognetto et al., Clinical trials of FAAH inhibitors failed to alleviate pain associated with osteoarthritis (Huggins et al., Although 2-AG is far more abundant than AEA, less work has been done investigating the therapeutic potential of inhibition of MAGL to ameliorate visceral pain. In a mouse chronic neurogenic pain model, MAGL inhibition alleviated neuropathic pain (Kinsey et al., A major obstacle to management of pain by inhibiting MAGL is the observation that increased systemic 2-AG actually results in enhanced response to painful stimuli due to desensitization of CB1 (Schlosburg et al., Endocannabinoids have been demonstrated to suppress inflammation in bladder (Wang et al., Mechanisms of suppression of inflammation by endocannabinoids are not completely understood, but AEA has been shown to inhibit mitogen-induced T- and B-cell lymphocyte proliferation by increasing apoptosis (Schwarz et al., in vitro damage to hippocampal slices exposed to \u03b2-amyloid by a process mediated by CB1 binding that resulted in diminished ERK 1/2 phosphorylation, decreased NF\u03baB activation, and reduced COX-2 expression (Chen et al., Binding of 2-AG to CB1 inhibits cyclooxygenase-2 in nerves resulting in suppression of MAPK/NF\u03baB signaling (Zhang and Chen, Inflammation plays a key role in release of substances that modulate nociception. Thus, it is highly likely that the analgesic effects of endocannabinoids may in part be due to their anti-inflammatory effects.Evaluation of bladder pain in rodent models can be difficult, and the presence of bladder pain is most often inferred by evaluating referred mechanical sensitivity of the hind paws or abdominal wall or activity of bladder afferent nerves (Sadler et al., in vivo with cyclophosphamide to induce inflammatory cystitis (Walczak et al., ex vivo in the presence or absence of intravesical cannabinoid agonists, and it was determined that intravesical cannabinoids suppressed afferent nerve fiber firing in inflamed bladders via CB1 activation (Walczak et al., Bladders and associated afferent nerves were isolated from mice treated Manipulation of the endocannabinoid system has emerged as an appealing alternative to opioids for management of severe bladder pain. However, the potential for undesirable side effects or lack of efficacy remain significant obstacles to advancement of this therapy. Emergence of dual inhibitors of endocannabinoid degradation and either activation of TRPV1 or COX may address many of the limitations of this approach. Information on the activity of endocannabinoids synthesized in peripheral tissues remains extremely limited. It remains unclear whether or not strategies to address organ-specific pain by manipulation of endocannabinoids is a viable option, but this is an intriguing alternative that has the potential to provide effective analgesia with minimal systemic side effects.All studies were approved by the University of Wisconsin Animal Care and Use Committee prior to performance. Procedures were consistent with guidelines provided by the Guide for the Care and Use of Laboratory Animals, 8th Edition, The National Academies Press, Washington, DC, USA.DB and ZW participated in the concept experimental design, analysis and writing. ZW was directly involved in performing experiments.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "However, remyelination often fails in chronic neurological diseases, such as progressive multiple sclerosis. The lack of currently approved pro-remyelination therapies highlights the need to elucidate the cellular and molecular mechanisms underpinning this regenerative process. Whereas some T lymphocyte subsets such as Th1 and Th17 are implicated in inducing myelin injury, a recent study by Dombrowski et al. reveals a novel role for regulatory T cells (T Regeneration can occur efficiently in the central nervous system (CNS) in the form of remyelination, whereby new myelin is ensheathed around axons to reinstate trophic/ metabolic support and insulation for electrical impulse conduction. This process requires oligodendrocyte progenitor cells (OPCs) to migrate to areas of injury, proliferate, and differentiate into myelin-producing oligodendrocytes. Remyelination is limited or fails altogether in various neurological disorders, most prominently in progressive multiple sclerosis (MS), leading to axon dysfunction or loss. Although it is recognized that this failure largely reflects impaired oligodendrocyte differentiation , demonstrating that these cells directly stimulate remyelination independent of immunomodulation.While it is now recognized that the innate immune system (i.e. macrophages) supports remyelination were detected in lesions at the time of oligodendrocyte differentiation and remyelination initiation (Dombrowski et al., regs. The effects of Treg depletion were mirrored in a distinct demyelination model (Dombrowski et al., regs in remyelination is not dependent on the mode of demyelination nor is it restricted to the spinal cord.Using a focal model of toxin-induced demyelination in the mouse spinal cord, where the temporal distinction between myelin damage and remyelination allows investigations of the regenerative process in isolation, Treg depletion in both models did not alter numbers of total oligodendrocyte lineage cells nor proliferating OPCs, suggesting effects of Tregs on the differentiation of OPCs into mature myelinating oligodendrocytes. Indeed, exposing brain explants to Tregs or their conditioned media enhanced oligodendrocyte differentiation, myelination, and remyelination, in comparison to non-polarized CD4+ T cells (Dombrowski et al., regs on OPCs was confirmed in vitro, where Treg conditioned media enhanced the differentiation of isolated OPCs and accelerated myelination in OPC-neuronal co-cultures (Dombrowski et al., reg conditioned media was carried out to identify pro-remyelination factors, and identified high expression of the growth regulator CCN3 (Dombrowski et al., reg conditioned media, as use of a blocking antibody or specific depletion from the conditioned media abolished the pro-differentiation and pro-myelination effects (Dombrowski et al., reg-derived CCN3 was sufficient to support these responses (Dombrowski et al., Tregs are direct drivers of oligodendrocyte differentiation and remyelination, thereby revealing a novel regenerative function for Tregs beyond immunomodulation. Considering this study, one must now acknowledge that the adaptive immune system is not solely involved in damage induction and modulation of inflammation, but is also a critical component of the regenerative process that follows. A recent study demonstrated that MS patient-derived CD4+ T cells injected into demyelinated mouse CNS show high variability in their ability to support remyelination (El Behi et al., regs to stimulate remyelination. Overall, these studies support that further investigations into the pro-remyelination function of Tregs and CCN3 in MS are warranted for development of novel regenerative therapies.Altogether, these data demonstrate that T"} +{"text": "Machine learning techniques have been increasingly applied in the medical imaging field for developing computer-aided diagnosis and prognosis models. Multimodal medical imaging can provide us with separate yet complementary structure and function information of a patient study and hence has transformed the way we study living bodies. Therefore, using machine learning techniques to deal with multimodal medical images is much more challenging due to the diversity of biophysical-biochemical mechanisms. In these years, researchers mainly adapt modern machine learning and pattern recognition techniques such as supervised, unsupervised, semisupervised, and deep learning to solve multimodal medical imaging related problems.To record the ideas of talents and gather more contributions to these fields, this special issue was launched and supported by this journal. This special issue focuses on the new imaging modalities/methodologies and new machine learning algorithms/applications for the further development in the multimodal medical imaging field, which will provide opportunities for academics and industrial professionals to discuss the latest issues and progresses in the area of multimodal medical imaging. The papers contained in this special issue address the development and application of medical image segmentation, registration, fusion, classification, image restoration, image retrieval, and computer-aided diagnosis.In \u201cEstimation of Response Functions Based on Variational Bayes Algorithm in Dynamic Images Sequences,\u201d B. Shan proposes a nonparametric Bayesian model to estimate the response functions in dynamic medical imaging, in which the nonparametric Bayesian priors are designed to favor desirable properties of the functions and used to improve the estimation of response functions.In \u201cTwo-Layer Tight Frame Sparsifying Model for Compressed Sensing Magnetic Resonance Imaging,\u201d S. Wang et al. propose a two-layer tight frame sparsifying model for compressed sensing magnetic resonance imaging (MRI) by sparsifying the image with a product of a fixed tight frame and an adaptively learned tight frame, which is solved by a three-level Bregman numerical algorithm and enables accurate MRI reconstruction from highly undersampled data with efficiency.In \u201cMany is Better than One: An Integration of Multiple Simple Strategies for Accurate Lung Segmentation in CT Images,\u201d Z. Shi et al. present a novel computerized tomography (CT) lung image segmentation method by integrating multiple strategies, including the guided filter to smooth the image, the optimized threshold to get binary image, region-growing strategy to extract thorax regions, and random walk algorithm to segment lung regions and to get the state-of-the-art segmentation accuracy.In \u201cPulmonary Nodule Detection Model Based on SVM and CT Image Feature-Level Fusion with Rough Sets,\u201d T. Zhou et al. present a pulmonary nodules detection algorithm based on support vector machine (SVM) and CT image feature-level fusion with rough sets to improve the detection accuracy of pulmonary nodules in CT image. Both the unreasonable feature structure and the nontightness of feature representation are taken into consideration in this pulmonary nodules detection algorithm.In \u201cMultigrid Nonlocal Gaussian Mixture Model for Segmentation of Brain Tissues in Magnetic Resonance Images,\u201d Y. Chen et al. propose a novel segmentation method based on the regional and nonlocal information to overcome the impact of image intensity inhomogeneities and noise in human brain magnetic resonance images.In \u201cDTI Image Registration under Probabilistic Fiber Bundles Tractography Learning,\u201d Z. Guo et al. propose a diffusion tensor imaging (DTI) image registration method under probabilistic fiber bundles tractography learning, where the residual error model is modified with finite sample set and the calculated deformation field is then registered on the DTI images.In \u201cAutomated Segmentation of Coronary Arteries based on Statistical Region Growing and Heuristic Decision Method,\u201d Y. Tian et al. propose a fully automated coronary artery segmentation from cardiac data volume based on a statistics region growing together with a heuristic decision to further help cardiovascular radiologists detect and quantify stenosis.In \u201cRapid Retrieval of Lung Nodule CT Images Based on Hashing and Pruning Methods,\u201d L. Pan et al. propose a new retrieval framework based on a hashing method for lung nodule CT images, which can translate high-dimensional image features into a compact hash code to greatly reduce the retrieval time and memory space. Moreover, a pruning-based decision rule is utilized in this algorithm to improve its retrieval precision.In \u201cThe Classification of Tongue Colors with Standardized Acquisition and ICC Profile Correction in Traditional Chinese Medicine,\u201d Z. Qi et al. design a tongue color classification approach using a standardized tongue image acquisition process, color correction, and several machine learning techniques for tongue inspection-based diagnosis in traditional Chinese medicine.In \u201cDiagnostic Method of Diabetes Based on Support Vector Machine and Tongue Images,\u201d J. Zhang et al. develop a SVM-based diagnostic method for diabetes using standardized tongue images. This work shows the potential of applying digitalized tongue images, which are usually used in traditional Chinese medicine, to the diagnosis of diabetes.In \u201cA Computer-Aided Analysis Method of SPECT Brain Images for Quantitative Treatment Monitoring: Performance Evaluations and Clinical Applications,\u201d X. Zheng et al. introduce and validate a computer-aided analysis method to achieve the quantitative treatment monitoring based on single-photon emission computed tomography (SPECT) images, which can provide a convenient solution to generate a parametric image and derive the quantitative indexes from the longitudinal SPECT brain images for treatment monitoring.The papers in this special issue provide a useful message of machine learning techniques in dealing with multimodal medical images. This unique and informative collection of papers highlights the direction of related studies. This special issue illustrates the important role that machine learning techniques play in the multimodal medical imaging fields."} +{"text": "Escherichia coli Bacteriophages Differ in Their Efficacy and Ability to Stimulate Cytokine Release In vitro,\u201d Dufour et al. suggest that the level of cytokine response generated in our paper is due to remaining bacterial debris rather than true differences between individual phages , which to our understanding is achieved through lipopolysaccharide specific binding to a phage derived protein. However, as Dufour et al. rightly point out, \u201cbacterial lysates may also contain several pathogen-associated molecular patterns (PAMPs) able to elicit a pro-inflammatory response, such as flagellin (sensed by TLR-5), unmethylated CpG Oligodeoxynucleotide DNA (TLR-9), lipoteichoic acid from Gram-positive bacteria (TLR-2) and triacyl lipopeptides (TLR-1 with TLR-2) . The additional purification step (the EndoTrap Blue system) binds endotoxins based on the conserved core of lipopolysaccharide (With hindsight and a thorough data audit, we would certainly concur that at least some component of the observed cytokine responses are due to remaining contaminants and that additional purification steps such as chromatography will further reduce possible contaminants. However, such additional steps could come at a cost, reduce overall phage concentration and potentially require additional concentration steps (Boraty\u0144ski et al., However, while it is likely that some component of the cytokine response generated is due to remaining contaminants, additional variation could also be introduced through factors which are uncontrollable, such as genetic variation in Toll receptors between different donors (Netea et al., Despite the suggestion to the contrary provided by the Dufour et al. commentary, the reduction and quantification of endotoxin in phage preparations remains an active area of research (Branston et al., MKM, YH, MN, CC, ES-E, and AN: Writing of the reply and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "The author reports that Newcastle disease virus (NDV)-infected umbilical cord-derived mesenchymal stem cells (MSCs) may provide a novel effective therapeutic approach for targeting glioma stem cells (GSCs) and glioblastoma multiforme (GBM), and for sensitizing these tumors to \u03b3-radiation [I read with great interest the article by Kazimirsky et al. entitledInternational Journal of Cancer [Stem Cell Research & Therapy.A couple of additional points can be made on this topic. The findings of Kazimirsky et al. regardinf Cancer and an af Cancer \u20136. In adf Cancer stated tf Cancer . In partf Cancer . From thContinuing with this important issue, in an article with 70 references none of the previously published work is discussed or cited by Kazimirsky et al. . As we k"} +{"text": "Traditionally, the role of \u201cemotion\u201d has received little attention in research studies of decision-making that can be used to simulate dynamic real-life decision-making behavior as well as test the SMH in each trial and large losses in some trials. The selection of decks C and D results in a small gain (US$50) in each trial and small losses in some trials. On average, selections from decks A and B over 10 trials will cause decision-makers lose US$250, and as such, these are defined as disadvantageous decks. Conversely, selections from decks C and D over 10 trials will cause decision-makers gain US$250, so these are defined as advantageous decks. The advantageous decks provide small immediate gains in each trial, but the long-term outcome is positive; by contrast, the disadvantageous decks provide large immediate gains in each trial, but the long-term outcome is negative . Consequently, findings related to the PDB phenomenon and gain/loss frequency have clearly echoed the main points reported in previous literature concerning behavioral decision-making Must et al. review IGT and depression-related issues; (2) Brevers et al. review studies on IGT and gambling disorders; (3) Linnet provide a review of IGT in the context of dopamine and gambling disorders; (4) Cassotti et al. review IGT in relation to developmental studies; (5) Turnbull et al. consider IGT performance as the processing of emotion-based learning; (6) Overman and Pierce examine the effects of real plus virtual cards and additional trials; and (7) van den Bos et al. provide a global overview of rodent version of the IGT.Category II: Clinical examinations: (1) Sallum et al. discuss the IGT and attention deficit hyperactivity disorder; (2) Xiao et al. combine the IGT and functional magnetic resonance imaging (fMRI) in order to investigate adolescent smoking behavior; (3) Singh describe the connection between sleep deprivation and IGT performance; and (4) de Oliveira Cardoso et al. provide a behavior-image study that investigates the correlation between frontal and cerebellar lesions and IGT performance.Category III: Model construction: (1) Worthy et al. compare predictability between win-stay/lose-shift and Value-Plus-Preservation (VPP) models in the IGT; (2) Steingroever et al. validate the predictive power of the Prospect Valence Learning\u2013Delta model; (3) Dai et al. provide an improved cognitive model for predicting IGT choice behavior; (4) Lin et al. refine a simplified model for estimating IGT performance; and (5) Ahn et al. compare three advanced IGT-related computational models.Category IV: Theoretical integration: (1) Okdie et al. provide a statement on construal level theory for IGT-related performance; (2) Bull et al. consider sensitivity toward reward and punishment in healthy IGT participants; (3) Singh suggest a potential role for reward and punishment during the IGT; and (4) Singh consider the influence of sex-differences, handedness, and lateralization on IGT performance.Category V: Brain imaging technology: (1) He et al. combine IGT and fMRI to investigate decisions involving unhealthy food; (2) Mapelli et al. utilize the IGT and event-related potentials (ERPs) to depict the behavioral performance and brain activation of patients with Parkinson's disease; (3) Tamburin et al. combine the IGT and ERPs to detect choice behavior and brain activation in patients with chronic lower-back pain; and (4) Fernie and Tunney describe a study on the correlation between SCRs and knowledge effects in the IGT.The articles selected for inclusion in this special issue provide good coverage of neuroimaging modalities used in previous IGT experiments. However, there might still be some room for a data-driven data analysis method What types of brain lesions does the IGT truly measure? (2) Can SCRs be combined with the IGT to form a critical index of somatic markers? (3) Does the IGT measure ability for implicit or explicit learning? (4) Does EV or gain/loss frequency primarily guide decision-making behavior in the IGT? (5) Is it possible to devise a more sensitive data analysis method that can allocate more specific brain responses to the precise behaviors of IGT performance, such as the events of win, loss, and the switching of card decks? We recommend that future studies of IGT consider these questions seriously and provide in-depth investigations and discussions.Y-CC, C-HL, and J-TH discussed the main structure of this article. Y-CC and C-HL drafted the preliminary title, literature review, and chapter categorization, as well as the initial draft. J-RD and C-HL provided additional viewpoints for future development in the use of brain imaging for studying the IGT. J-TH and J-RD provided final refinements to this article.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "The term \u201cnutraceutical\u201d, derived from the words \u201cnutrition\u201d and \u201cpharmaceutical\u201d was coined in 1989 to describe substances that could be used as foods that possess health benefits. The range of nutraceutical products is widely diverse and may be divided into three main categories: (i) natural products, comprising, among others, herbs and spices; (ii) dietary supplements; and (iii) functional foods. Based on food sources, nutraceuticals can be classified as: (i) dietary fibres of plant origin; (ii) probiotics, which are live microbial feed supplements; (iii) prebiotics, dietary ingredients that selectively alter the composition or metabolism of gut microbiota; (iv) polyunsaturated fatty acids, omega-3 or omega-6 fatty-acids; (v) antioxidant vitamins, including vitamin C, vitamin E and carotenoids; (vi) biologically active phytochemicals like polyphenols; and (vii) spices, whose main components are terpenes and other constituents of essential oils.In the recent years, the field of nutraceuticals has progressed from being a mere conceptual area within biomedical research to a value-added industry with a promising future ahead. Indeed, lately, both the academic community and the general population have become increasingly interested in the health benefits and potential risks pertaining to beverages and food products beyond their basic nutritional assets. In addition, continuing scientific research has provided evidence regarding the biologically active compounds and underlying physiological mechanisms, highlighting the importance of nutraceuticals as complements to the conventional therapies and medications allowing dose reduction and decreasing the occurrence of adverse effects. Despite the undeniable progress in the field of nutraceuticals, there are still several issues that remain to be addressed. These include clinical evidence supporting in vitro claims, regulatory aspects and assurance of nutraceuticals\u2019 identity, quality and safety.International Journal of Molecular Sciences comprises one review article and 16 original research papers that provide important insights into the state of the nutraceuticals field. Nosrati et al. [Fucus spiralis in a human cell in vitro model (MCF-7 cells). Han et al. [The special issue \u201cNutraceuticals in Human Health and Disease\u201d in the i et al. use mixei et al. charactei et al. investign et al. report tFicus umbellata constituents that appears to be due to apoptosis induction through the ROS-dependent mitochondrial pathway. The work conducted by Dai et al. [Sophora flavescens, inhibits influenza A virus replication and possesses anti-inflammatory activities and that the underlying mechanism may be linked to its ability to inhibit IAV-induced activations of the TLR4, p38 MAPK, and NF-\u03baB pathways.The studies conducted by Forney et al. provide i et al. reveals Acanthopanax senticosus in repressing radiation-induced protein expression changes on prefrontal cortex, and suggest that this compound may be a promising alternative medicine for the treatment of radiation injury in the brain yet further testing are needed to understand whether the bioactive ingredients could be effective through the blood\u2013brain barrier. Klimaszewska-Wi\u015bniewska et al. [Studies developed by Liu and Dey suggest a et al. provide Collectively, these studies support a role for nutraceuticals in the prevention and amelioration of several diseases. Their innovation in the field and their successful future clinical application will be governed by evidence regarding safety, purity and efficacy. The use of these compounds in clinical practice is emerging, yet important pharmaceutical and clinical issues remain to be answered."} +{"text": "To lessen the \u201cwear and tear\u201d of existence, cells have evolved mechanisms that continuously sense DNA lesions, repair DNA damage and restore the compromised genome back to its native form. Besides genome maintenance pathways, multicellular organisms may also employ adaptive and innate immune mechanisms to guard themselves against bacteria or viruses. Recent evidence points to reciprocal interactions between DNA repair, DNA damage responses and aspects of immunity; both self-maintenance and defense responses share a battery of common players and signaling pathways aimed at safeguarding our bodily functions over time. In the short-term, this functional interplay would allow injured cells to restore damaged DNA templates or communicate their compromised state to the microenvironment. In the long-term, however, it may result in the (premature) onset of age-related degeneration, including cancer. Here, we discuss the beneficial and unrewarding outcomes of DNA damage-driven inflammation in the context of tissue-specific pathology and disease progression. To withstand the hazards of existence, multicellular organisms need to preserve their bodily functions for long periods of time and protect themselves against pathogens. Taking the cell as a point of reference, the maintenance is directed inwards to counteract macromolecular damage. This often involves restoring injured nucleic acids back to their native form and interleukin-8 (IL-8) , we recently showed that persistent DDR triggers a chronic auto-inflammatory response leading to severe fat depletion in mice (Karakasilioti et al., Ercc1F/\u2212 fat depots showed hallmarks of persistent DDR together with the marked up-regulation of pro-inflammatory factors, the infiltration of activated macrophages as well as the release of DAMPs known to initiate and perpetuate immune responses (Karakasilioti et al., Ercc1F/\u2212 fat depots in vivo and in adipocytes ex vivo showed that persistent DNA damage signaling triggers the induction of IL-6, IL-8, and TNF\u03b1 by promoting transcriptionally active histone marks and the dissociation of nuclear receptor co-repressor complexes from promoters; the response required ATM and it was instigated in a DNA lesion- and cell type-specific manner. In support of these findings, NF-\u03baB is stochastically activated in tissues of naturally-aged and Ercc1\u2212/\u0394 mice (unlike Ercc1\u2212/\u2212 mice, the Ercc1\u2212/\u0394 animals maintain about 10% of the wild-type ERCC1 protein levels and develop progressive, degenerative changes that markedly resemble those seen in natural aging (Tilstra et al., Ercc1\u2212/\u0394 mice. Moreover, inhibition of IKK/NF-\u03baB activity reduced cellular senescence and oxidative damage in DNA and proteins (Tilstra et al., Zmpste24\u2212/\u2212 and LmnaG609G/G609G progeroid animals. As in Ercc1\u2212/\u0394 animals, genetic and pharmacological inhibition of NF-\u03baB signaling can ameliorate the age-associated features and extend the lifespan of these animal models (Osorio et al., Atm\u2212/\u2212 animals present with infiltration of neutrophils and lymphocytes in the lungs and increased mRNA levels of pro-inflammatory e.g., IL-6, TNF cytokines (Eickmeier et al., Cellular senescence is a term used to describe cells that cease to divide in culture and has been one of the first paradigms to link DNA damage and immunity to disease (Campisi and d'Adda di Fagagna, per se but persistent DDR that triggers the repertoire of innate immune responses (Fumagalli and d'Adda di Fagagna, ssDNA intermediates generated during e.g., transcription or DNA replication may also activate DDR and trigger a pro-inflammatory response (Abe et al., DNA damage-driven inflammation can be both beneficial and detrimental for organismal survival Figure . To undeFuture strategies aimed at identifying new players or delineate key pathways may shed light on the biochemical crosstalk DNA repair and immune factors allowing us to gain insights onto how both systems contribute to disease origin and progression at old age. In this regard, the use of e.g., tissue-specific or tagged knockin animals and high-throughput proteomics and genomics approaches will likely prove valuable toward the development of rationalized interventions (Tilstra et al., AI and EG researched the literature, prepared a schematic first draft, and Figure The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Coffee is the most consumed beverage worldwide. Epidemiological studies with prospective cohorts showed that coffee intake is associated with reduced cardiovascular and all-cause mortality independently of caffeine content. Cohort and case-control studies reported an inverse association between coffee consumption and the degree of liver fibrosis as well as the development of liver cancer. Furthermore, the beneficial effects of coffee have been recently confirmed by large meta-analyses. In the last two decades, various in vitro and in vivo studies evaluated the molecular determinants for the hepatoprotective effects of coffee. In the present article, we aimed to critically review experimental evidence regarding the active components and the molecular bases underlying the beneficial role of coffee against chronic liver diseases. Almost all studies highlighted the beneficial effects of this beverage against liver fibrosis with the most solid results indicating a pivot role for both caffeine and chlorogenic acids. In particular, in experimental models of fibrosis, caffeine was shown to inhibit hepatic stellate cell activation by blocking adenosine receptors, and emerging evidence indicated that caffeine may also favorably impact angiogenesis and hepatic hemodynamics. On the other side, chlorogenic acids, potent phenolic antioxidants, suppress liver fibrogenesis and carcinogenesis by reducing oxidative stress and counteract steatogenesis through the modulation of glucose and lipid homeostasis in the liver. Overall, these molecular insights may have translational significance and suggest that coffee components need clinical evaluation. Coffea, a member of the Rubiaceae family, which includes hundreds of different species. Commercial production mainly exploits the seeds of Coffea arabica (arabica coffees), which represent about 70% of the world market, while Coffea canephora (commonly known as robusta coffees), which has a more bitter taste than arabica, is used principally for instant and espresso coffees (www.ico.org).Coffee is the most consumed beverage worldwide with an impressive impact on the economy of producing countries. Coffee is prepared from the seeds of the coffee plant, genus The association between coffee consumption and all-cause and cause-specific mortality, especially cardiovascular mortality, has been investigated in independent studies evaluating prospective cohorts, with most solid studies finding an inverse association with all-cause and cardiovascular mortality for both caffeinated and decaffeinated blends ,2. RecenIn this review, we aimed to summarize experimental evidence regarding molecular mechanisms underlying the hepatoprotective effects of coffee by reviewing in vitro and in vivo studies. A search of the electronic databases Medline and Embase was performed to retrieve relevant articles published up to May 2016. A combination of the keywords \u201ccoffee\u201d, \u201ccaffeine\u201d, \u201ccoffee polyphenols\u201c, \u201cchlorogenic acid\u201d, \u201ccafestol\u201d, \u201ckahweol\u201d, \u201cmelanoidins\u201d, \u201cliver steatosis\u201d, \u201cliver damage\u201d, \u201cliver injury\u201d, \u201cliver inflammation\u201d, \u201cliver fibrosis\u201d, \u201cliver cirrhosis\u201d, \u201cliver cancer\u201d was searched. All published studies in the English language were considered. Additional papers were retrieved by references of epidemiological studies on coffee and liver diseases. After careful evaluation, the following studies were excluded from the review: (i) studies reporting in vivo effects on acute models of liver damage or failure ; (ii) studies reporting in vitro effects on metabolic processes without assessing effects on cell steatosis or apoptosis (pure metabolic studies).Convincing results from a meta-analysis of 28 prospective studies have indicated that both caffeinated and decaffeinated coffee consumption is associated with lower risk of diabetes in a dose-response manner . ConsistPpar\u03b3), cluster of differentiation (Cd36), fatty acid binding protein 4 (Fabp4) and Mgat1 genes; more interestingly, CGA treatment also attenuated inflammation in the liver and white adipose tissue accompanied by a decrease in the mRNA levels of macrophage marker genes including EGF-like module-containing mucin-like hormone receptor-like 1 (F4/80), cluster of differentiation (Cd68), integrin Cd11b, integrin Cd11c and tumor necrosis factor alpha (Tnf\u03b1), monocyte chemotactic protein 1 (Mcp-1) and C-c chemokine receptor type 2(Ccr2) encoding inflammatory proteins.To our best knowledge, the first experimental study showing a protective effect of a coffee component against liver steatosis was from Rodriguez de Sotillo and Hadley , who shoDue to the tight connection of NAFLD with cardiovascular mortality, it is important to highlight the results from Panchal et al. , who evaRecently, further studies suggested the impact of coffee components not only on metabolic pathways in the liver but also on endoplasmic reticulum stress and autophagy, which are pivot processes in NAFLD . Our groThe association of coffee consumption with fibrosis and cirrhosis development has been investigated in several studies . In a pr4) or thioacetamide and attributed this effect to its nonselective adenosine receptor antagonist activity [Coffee has been consumed for several centuries worldwide for its flavor and taste but also because of the psychoactive effects of caffeine, an alkaloid which is contained in different quantities according to coffee species, techniques of roasting and brewing ,23. To tactivity Table 24 or thioactivity showed tactivity and to ractivity .Arauz et al. and Furtado et al. ,36 indepThe anti-fibrotic effects of caffeine have been also evaluated in in vitro cultures of human and rodent hepatic stellate cells. Shim et al. assessed the effect of caffeine on human hepatic stellate cells (HSC) proliferation and migration and found that caffeine attenuates the progression of liver fibrosis by inhibiting HSC adhesion and activation . Wang etBeside morphological evidence for the anti-fibrotic activity of caffeine coming from the mentioned studies, recently it was elegantly shown by Hsu et al. that caffeine may also favorably impact hemodynamic changes in a rat model of portal hypertension . In this4-induced cirrhosis; the authors found that CGA reduces liver fibrosis and the expression of collagen I and collagen III. Consistently, rats treated with CGA displayed reduced levels of VEGF, TGF-beta and alpha-smooth muscle actin, thus indicating that CGA is able to counteract liver fibrogenesis in rats [Although the anti-fibrotic effects of caffeine are well characterized, few experimental studies have been conducted so far to investigate the effects of polyphenols, such as CGA, in in vitro and in vivo models of liver fibrosis. Shi et al. first explored the effect of the oral administration of CGA in rats with CCl in rats . Success in rats . These f in rats .The epidemiological association between coffee intake and liver cancer prevalence or incidence has been extensively studied throughout the two last decades Table 3Table 3. At the experimental level, different studies have been conducted so far to identify the molecular determinants of such effects. To our best knowledge, the first experiment that showed the beneficial effects of a coffee component on liver carcinogenesis was conducted by Mori H et al. , who rep4. One week after DEN injection, the groups started receiving coffee or 0.1% caffeine ad libitum for 24 weeks. The groups receiving coffee or caffeine alone not only had a reduction in collagen content but also displayed a significant reduction in the size and area of pre-neoplastic lesions and in the mean number of neoplastic lesions [Katayama et al. assessed the effects of coffee in a liver cancer\u2013prone Long Evans Cinnamon rat and showed that coffee administration for 25 weeks delayed the occurrence of hepatitis, significantly improved survival, reduced the expression of inflammatory cytokines, and reduced the incidence of small pre-neoplastic liver foci in Long-Evans Cinnamon (LEC) rats . Similar lesions .As concerns the molecular targets involved in the chemopreventive effects of coffee, evidence points out the importance of Nrf-2. Cavin et al. showed aSeveral epidemiological studies have associated the consumption of coffee with a decreased risk of developing chronic liver diseases. Consistently, experimental data suggest that specific coffee components, in particular caffeine and CCA, favorably impact liver steatogenesis, fibrogenesis and carcinogenesis by acting on different molecular and cellular targets . Therefo"} +{"text": "When facing various extrinsic or intrinsic stimuli, BBB is damaged which is an early event in pathogenesis of a variety of neurological diseases in old patients including acute and chronic cerebral ischemia, Alzheimer\u2019s disease and etc. Treatments that could maintain the integrity of BBB may prevent neurological diseases following various stimuli. Old people often face a common stress of sepsis, during which lipopolysaccharide (LPS) is released into circulation and the integrity of BBB is damaged. Of note, there is a significant decrease of melatonin level in old people and animal. Melatonin has been shown to preserves BBB integrity and permeability via a variety of pathways: inhibition of matrix metalloproteinase-9 (MMP-9), inhibition of NADPH oxidase-2, and impact on silent information regulator 1 (SIRT1) and nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome. More important, a recent study showed that melatonin supplementation alleviates LPS-induced BBB damage in old mice through activating AMP-activated protein kinase (AMPK) and inhibiting gp91 The blood brain barrier (BBB) is a regulated interface between the peripheral circulation and the central nervous system , composed of occludin, claudin and zo-1, are key components of the BBB could produce neuroinflammation Shi, , promotiper se but also on the incidence or severity of age-related diseases /NF-\u03baB signaling pathway in neonatal rats (Hu et al., in vitro cell model (Zhao et al., in vivo mice model (Zhou X. et al., phox up-regulation in brain capillary endothelial cells (Figure AMPK activation has been shown to play important role in maintaining the integrity of BBB (Liu et al., s Figure . AMPK acMMP-9 has been shown to play important role in BBB damage (Jin et al., SIRT1 was reported to be beneficial in sepsis. Using EX527, a SIRT1 inhibitor, the authors figured out that melatonin alleviated BBB damage in mice which subjected to cecal ligation and puncture via SIRT1 to inhibit inflammation, apoptosis and oxidative stress (Zhao et al., Aging and sepsis triggered NLRP3 inflammasome activation (Volt et al., Although acute toxicity of melatonin is extremely low in both animal and human studies, melatonin may still cause minor adverse effects, such as headache, insomnia and nightmares (Malhotra et al., In conclusion, decreased melatonin levels may account for the BBB damage in old people who often face the common stress of sepsis and neuroinflammation. Melation supplementation treatment significantly inhibits such events. Therefore, continuous daily melatonin supplementation may help prevent sepsis and neuroinflammation-related neurological diseases through maintaining the integrity of BBB in old people. Since melatonin has low toxicity profile and high efficacy in many pathophysiological states, it should be more commonly tested/used in the medical and veterinary arenas. Further studies are needed to verify the important significance of daily melatonin supplementation in old people.W-CL, XW, XZ, XC and XJ wrote the manuscript and XC, XJ obtained the funding. XW drew the figures. All authors have approved the final version of this review article.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Recently, a genome-wide study about gene copy number gains and corresponding expression levels has been analyzed in a cohort of 190 non-small cell lung cancer (NSCLC) patients . The auSince decades, much effort has been invested to better understand the relationship between gene expression and prognosis of tumors (Hellwig et al., 2016; Stock e"} +{"text": "During 2014\u20132016, we conducted mosquito-based Zika virus surveillance in Rio de Janeiro, Brazil. Results suggest that Zika virus was probably introduced into the area during May\u2013November 2013 via multiple in-country sources. Furthermore, our results strengthen the hypothesis that Zika virus in the Americas originated in Brazil during October 2012\u2013May 2013. Aedes aegypti mosquito is considered the main vector of Zika virus in urban and suburban areas throughout the world, including Brazil, where the mosquito has been confirmed, together with Ae. albopictus mosquitoes, as a vector for the virus , which we pooled (n = 178) and subjected to Zika virus detection using real-time RT-PCR clustered within the same strongly supported lineage, which included strains detected in Rio de Janeiro and other parts of Brazil in late 2015 and in 2016 Figure. Ae. aegypti mosquitoes that were collected during a mosquitoborne virus surveillance program in Rio de Janeiro. Information regarding Zika virus infection rates is lacking for female and male mosquitoes trapped in the field. However, experiments performed in the laboratory demonstrated transovarial transmission of Zika virus among Ae. aegypti mosquitoes and revealed a minimal filial infection rate of 1:290 (In this study, we detected Zika virus RNA in 2 pools of engorged Ae. aegypti mosquitoes trapped in Rio de Janeiro before the first case of autochthonous Zika virus disease was diagnosed in the city (In conclusion, we showed the presence of Zika virus in engorged Bayesian maximum clade credibility tree representing the time-scale phylogeny of the Zika virus outbreaks in the Americas."} +{"text": "A characteristic of post-surgery patients, particularly the more elderly, can be a persistent self-propagating cerebral inflammatory syndrome referred to as post-operative cognitive dysfunction (POCD). Changes can be analogous to those seen in Alzheimer's disease , a synthetic sedative with analgesic and anxiolytic properties, is widely used in surgery. It is a selective \u03b1Physical trauma, including that caused by surgery, induces an innate immune response that includes release of pro-inflammatory cytokines such as TNF and interleukins (Arvin et al., Since systemic TNF has long been known to cross the blood-brain barrier (Gutierrez et al., ++ homeostasis, and thus the ionic signaling cascades on which normal function of these cells depends (Park et al., Dexmedetomidine has an extensive history of improving neurological function, for example when given preemptively in animal models tibial fracture (Zhu et al., 2-adrenoceptor agonist appears implicated, in that yohimbine, an \u03b12-adrenoceptor antagonist, enhanced TNF levels when the two were compared in a lipopolysaccharide-induced liver damage model (Chen et al., Various pathways of TNF inhibition by dexmedetomidine have been explored. Its action as a \u03b1Using the same mouse tibial fracture model as did others with dexmedetomidine six years later (Zhu et al., Given the pleiotropic nature of TNF, reducing its excess production with dexmedetomidine may also cast light on the mechanisms of other useful outcomes of therapy with this agent that are presently little understood. For instance dexmedetomidine is an acknowledged analgesic, particularly in surgical settings (Vaughns et al., Escherichia coli protecting them completely from harm (Tracey et al., The background information required to rationalize the contrasting outcomes reported in the two trials (Su et al., Thus it seems logical that, in the context of post-surgical delirium (Su et al., A useful step in understanding its mechanism further would be to experimentally compare preemptive use of dexmedetomidine and one of the specific anti-TNF biologicals reported to minimize POCD delirium, pain and anxiety, and to induce morphine tolerance. Because of their molecular size, these biologicals would require administering intracerebroventricularly or perispinally (Tobinick, IC proposed the scope of the review. Both authors were involved in planning and editing the manuscript, blending their complementary expertises. Both authors read, altered and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Serial crystallography was developed for the use at free-electron lasers but the approach has recently also been adapted to synchrotron sources. Here we discuss how the synergy between the two X-ray sources will facilitate a wide application of the technique in microcrystallography, room-temperature structure determination and time-resolved studies. For time-resolved studies, the small crystal size allows for rapid diffusive saturation in mix-and-inject analysis of biochemical reactions, and full optical saturation of the sample by a pump laser in studies of light-driven proteins. The ability to outrun most radiation damage avoids the need for sample cooling and its artifacts, allowing studies of molecular machines at work in their correct room-temperature thermal bath or a controlled chemical environment.Recently we have seen rapid progress in the serial crystallography (SC) method at X-ray free-electron lasers (XFELs). Injection of thousands of protein microcrystals into the \u223c10Despite these achievements, the XFEL community remains relatively small, due to the limited availability of XFEL beamtime which, at present, allows only two simultaneous data collections worldwide (in Japan and USA). In addition, serial femtosecond crystallography (SFX) at XFELs is experimentally demanding and complex, currently requiring sizable interdisciplinary teams of collaborating scientists and engineers. The transfer of the SC approach to next-generation synchrotrons upgraded for higher flux density and with beamlines using sophisticated focusing optics, submicron beam diameters and fast low-noise photon-counting detectors offers a way out of this dilemma. In applications such as room-temperature data collection or phasing from radiation-sensitive microcrystals, serial millisecond crystallography (SMX) at synchrotrons has developed into a viable alternative. In the near future, it may even be possible to extend the method to time-resolved studies. Already it frees beamtime at XFELs to exploit their unique capabilities, such as outrunning damage in structure analysis from nanocrystals or ultrafast time-resolved crystallography.et al., 2008et al., 2011et al. shows many papers on SC, with some of the most cited examples devoted to the interplay between synchrotrons and XFELs.The experimental origins of fast SC can be traced to an early proposal to fire bioparticles in a continuous single-file stream across a beam for diffraction analysis using a Rayleigh jet (Spence & Doak, 2004 al. 1998. Rossmanet al., 2013The term \u2018serial\u2019 in synchrotron studies has recently become popular in a wider sense, and may also refer to taking a series of diffraction patterns along a few larger crystals, or the scanning of fixed target devices. Such serial approaches share the idea of distributing the radiation dose, to get the maximum signal from all of the available crystal volume. Short millisecond exposures may outrun slower secondary radiation damage from diffusing radicals or relaxation of individual damaged molecules (Warkentin et al., 2015et al., 2016cis\u2013trans reaction. Yet both proteins are available in large amounts and can be grown into big crystals suitable for time-resolved Laue diffraction experiments at a synchrotron. A recent study of the light-driven proton pump bacteriorhodopsin, for which only a few micrometre thick crystals were available, resulted in a series of 13 snapshots at logarithmically spaced time points after activation (Nango et al., 2016An important application where injector-based SC has proven advantageous is fast time-resolved pump\u2013probe crystallography. Myoglobin (Barends et al., 2014et al., 2015et al., 2015et al., 2016The work was only feasible using sample-efficient high-viscosity injectors (Weierstall"} +{"text": "Reactive oxygen and nitrogen species are generated during normal cellular metabolic activities but can also play an etiopathogenetic role in a variety of conditions, including cardiovascular diseases, neurodegenerative diseases, and cancer.Although the role of oxidative stress in human physiology and pathology has been intensely studied for several decades, it is still far from being understood. Certainly, the roles of free radicals and antioxidants have been significantly redefined. Some \u201cnegative\u201d actions of free radicals in human biology and pathology are now known to be \u201cpositive,\u201d and the hypothesis that \u201cclassical antioxidants\u201d could be always beneficial for the human health was not confirmed by several epidemiological and clinical studies. The possible reasons of the failures of the current antioxidant therapies, including methodological pitfalls in the drug development and delivery and the lack of good biological markers to select the patients, have been reviewed elsewhere , 2.We currently believe that instead of \u201cantioxidants,\u201d it is more appropriate to develop \u201cmodulators of oxidative stress\u201d because, depending on the condition, it could be more beneficial to reduce or increase the oxidative stress.\u03b2-Synthase: Increased CO Reactivity as a Novel Molecular Mechanism of Pathogenicity?\u201d by J. B. Vicente et al., \u201cProtective Mechanisms of the Mitochondrial-Derived Peptide Humanin in Oxidative and Endoplasmic Reticulum Stress in RPE Cells\u201d by L. Minasyan et al., \u201cEffect of Emodin on Preventing Postoperative Intra-Abdominal Adhesion Formation\u201d by G. Wei et al., and \u201cOpuntia spp.: Characterization and Benefits in Chronic Diseases\u201d by M. del Socorro Santos D\u00edaz et al.), some cardiovascular effects of modulators of oxidative stress , some neurological effects of modulators of oxidative stress , and some effects of modulators of oxidative stress related to cell growth and cancer development Administration Delays Hepatic Cell Proliferation by Altering Oxidative State in the Regenerating Rat Liver\u201d by A. Butanda-Ochoa et al., \u201cPreclinical Antileukemia Activity Of Tramesan: A Newly Identified Bioactive Fungal Metabolite\u201d by M. R. Ricciardi et al., and \u201cMarkers of Oxidative Stress and Inflammation in Ascites and Plasma in Patients with Platinum-Sensitive, Platinum-Resistant, and Platinum-Refractory Epithelial Ovarian Cancer\u201d by J. C. Cant\u00f3n-Romero et al.).In this special issue, several aspects of the modulation of oxidative stress were examined including the role of polyphenols and other natural substances Opuntia spp., stressing that all Opuntia components used as nutritional and pharmaceutical agents exhibit beneficial properties mainly resulting from their high content in antioxidants, while several other phytochemical components , which have been characterized, also contribute to the medicinal properties of Opuntia spp. that might have a great economic potential because these plants grow in arid and desert regions.In particular, this special issue contains two interesting contributions coming from INSERM Toulouse. The first one is an original research paper prepared in collaboration with CNRS and Palau Sabatier University in which C. Camare et al. investigated whether 4-hydroxynonenal (4-HNE), an aldehydic lipid oxidation product abundantly present in oxidized LDL, contributes to its proangiogenic properties. Using the immunofluorescence analysis of human atherosclerotic lesions, they found colocalization of HNE adducts with CD31 (marker of the endothelium), indicating a close relationship between 4-HNE and neovascularization. Moreover, they revealed that physiological concentrations of 4-HNE also enhance the formation of tubes by human microvascular endothelial cells (HMEC-1), through mechanisms involving reactive oxygen species (ROS) and activation of the neutral type 2 sphingomyelinase and sphingosine kinase-1 (nSMase2/SK-1) pathway. Eventually, they found that carbonyl scavengers hydralazine and bisvanillyl-hydralazone inhibited such proangiogenic effects of 4-HNE. In their second paper prepared jointly with partners from Mexico and from INRA in Toulouse, they described in detail features of the well-known plants of In the original research paper prepared by partners from Riga and from Zagreb, we found novel information about growth-modulating effects of the dihydropyridine derivatives (DHPs) with potential antioxidant capacities. Thus, I. Bruvere et al. revealed that cell-type-specific differences in the growth-modifying effects of the DHPs in vitro can be attributed only to the novel types of the DHPs, which differentiate these substances from their well-known predecessor diludine. Since the growth-modifying effects of the novel DHPs indicate possible differential effects on cancer and on nonmalignant cells, which might be also different from their antioxidant effects, these substances deserve particular attention and further studies.The original research paper prepared by three teams from Karlsruhe, Germany, reveals the possible beneficial effects of pentaerythritol tetranitrate (PETN), which were not described before. Namely, pulmonary arterial hypertension was induced by the ranging dosage of the i.v. applied monocrotaline, which induced endothelial dysfunction, pulmonary vascular wall thickening, and fibrosis, as well as protein tyrosine nitration, thus causing an increase in pulmonary arterial pressure, followed by the increase in heart/body and lung/body weight ratios. However, in this study, S. Steven et al. found also that PETN therapy could act beneficially upon these pathophysiological processes, most likely by upregulation of heme oxygenase-1 (HO-1)."} +{"text": "There is a natural decline in cognitive function as we age, particularly in processing speed and working memory. A range of modifiable factors can increase the risk of accelerated cognitive decline including hypertension, chronic inflammation, atherosclerosis, diabetes, atrial fibrillation, stroke, and impaired central nervous system glucose regulation. Given the lack of adequate interventions for cognitive decline and dementia, it is essential that treatments with the potential to reduce the risk of cognitive impairment are thoroughly explored.Nutraceutical and lifestyle medicines, including vitamins, herbs, supplements, physical activity, and diet, have been shown to possess anti-inflammatory, antihyperglycaemic, and antihypertensive properties, suggesting their utility in targeting the pathophysiology associated with risk for cognitive decline. The purpose of this special issue was to explore the role of such complementary medicines in the modification of risk factors for cognitive decline.Clinical trials involving nutraceutical and herbal medicine interventions for people with mild cognitive impairment and dementia are reviewed in G. Z. Steiner et al. The manuscript titled \u201cA Systematic Review of Intervention Studies Examining Nutritional and Herbal Therapies for Mild Cognitive Impairment and Dementia Using Neuroimaging Methods: Study Characteristics and Intervention Efficacy\u201d focuses on papers that feature neuroimaging outcome measures.D. Chang et al. provide a detailed review of the literature on herbal medicine for vascular dementia in their paper titled \u201cHerbal Medicine for the Treatment of Vascular Dementia: An Overview of Scientific Evidence.\u201dVascular disease, such as cerebrovascular disease and atherosclerosis are risk factors for cognitive impairment. X. Zhou et al. review the evidence on this link and promising Traditional Chinese Medicines in their paper titled \u201cVascular Contributions to Cognitive Impairment and Treatments with Traditional Chinese Medicine.\u201dThe fourth review featured in this special issue summarises the evidence on the efficacy of physical activity in reducing depression. D. C. Mathersul and S. Rosenbaum's paper is titled \u201cThe Roles of Exercise and Yoga in Ameliorating Depression as a Risk Factor for Cognitive Decline.\u201dH. Macpherson et al. outline the findings of a clinical trial on older adults in their paper titled \u201cThe Effects of Four-Week Multivitamin Supplementation on Mood in Healthy Older Women: A Randomized Controlled Trial.\u201d\u03b21\u201342.\u201dThe first preclinical paper in this special issue explores the effectiveness of Huannao Yicong extract, a Traditional Chinese Medicine, in reducing tau hyperphosphorylation in Alzheimer's disease model rats. Y. Cao et al.'s paper is titled \u201cTraditional Chinese Medicine Huannao Yicong Decoction Extract Decreases Tau Hyperphosphorylation in the Brain of Alzheimer's Disease Model Rats Induced by A\u03b2 and LRP1 in Hippocampus.\u201dX. Wang et al. report their findings from an electroacupuncture treatment on Alzheimer's disease model mice in their paper titled \u201cImprovement of Electroacupuncture on APP/PS1 Transgenic Mice in Spatial Learning and Memory probably due to Expression of AThe second electroacupuncture paper in this special issue explores the effects of this treatment on cerebral hypoperfusion model rats. C.-X. Zheng et al.'s paper is titled \u201cElectroacupuncture Ameliorates Learning and Memory and Improves Synaptic Plasticity via Activation of the PKA/CREB Signaling Pathway in Cerebral Hypoperfusion.\u201dIn the paper titled \u201cPreservation of Cognitive Function by Lepidium meyenii (Maca) Is Associated with Improvement of Mitochondrial Activity and Upregulation of Autophagy-Related Proteins in Middle-Aged Mouse Cortex\u201d by S.-S. Guo et al., maca was found to improve cognition and behavior, and mitochondrial dysfunction in middle-aged mice.\u03b2-induced neurotoxicity on SH-SY5Y cells are modulated by a the Traditional Chinese Medicine, Yi-Zhi-Fang-Dai formula. The paper is titled \u201cYi-Zhi-Fang-Dai Formula Protects against A\u03b21\u201342 Oligomer Induced Cell Damage via Increasing Hsp70 and Grp78 Expression in SH-SY5Y Cells.\u201dIn an in vitro study by L. Liu et al., the effects of AThe final paper in this special issue by M. A. Akhtar et a. titled \u201cMedicinal Plants of the Australian Aboriginal Dharawal People Exhibiting Anti-Inflammatory Activity\u201d explores the medicinal effects of a range of Eucalyptus plants used in traditional Australian Aboriginal medicine by the Dharawal people.Authors contributed with a range of papers including original research and review articles spanning in vitro, in vivo, and human studies that improve the understanding of the pathology involved in cognitive impairment in older age and the development of evidence-based complementary treatment strategies for cognitive decline. This collection of works provides a snapshot (that is by no means exhaustive) of current research and some of the emerging trends in this field. This special issue features 11 papers including 4 reviews and 7 original research articles. The complementary therapies explored include nutritional supplements (2 papers), herbal and traditional medicines (6 papers), physical activity (1 paper), and electroacupuncture (2 papers). A brief description of these 11 works is detailed below."} +{"text": "In 1920, German neuropathologist Alfons Maria Jakob describeEtymologia series is dedicated to the memory of Richard T. Johnson, MD (1931\u20132015), the leading prion disease authority in the United States for many years and great friend of CDC\u2019s infectious disease programs, so many of which involve central nervous system disorders.This issue of Emerging Infectious Diseases\u2019 long-running"} +{"text": "Neuroinflammation is an important component of many neuroinflammatory and neurodegenerative diseases. Microglia and astrocytes play a key role in neuroinflammation. Activated glial cells under neuroinflammatory condition produce proinflammatory cytokines, chemokines, nitric oxide (NO), and other neurotoxic mediators. Thus, it has been conventionally thought that the inhibition of such glial activation might be an effective neuroprotective strategy under inflammatory or degenerative disease conditions in the central nervous system (CNS). However, recent studies indicated that activated glia could adopt either neuroprotective or neurotoxic phenotype depending on instigating stimuli present in the given microenvironment . LipopolIn a recent study by Song et al. , a phenoIn summary, based on a high-throughput screen of an approximately 3,500-member in-house library, Song et al. identified a novel small-molecule compound that has an anti-neuroinflammatory effect via the phenotypic switch towards the M2-like anti-inflammatory state in glia. The GPM is a specific PPAR-\u03b3 agonist that can be used to fine-tune glial phenotypes and functions. The GPM-mediated anti-inflammatory state of brain glia may lead to beneficial neuroprotective effects and has therapeutic potential in neuroinflammatory and neurodegenerative diseases. Recent studies suggested that functional phenotypes of macrophages and glia are closely associated with their glucose metabolism. A more recent study by Van den Bossche et al. ["} +{"text": "Coxiella burnetii, Brucella abortus, Salmonella enterica serovar Typhimurium, Legionella pneumophila, Chlamydia trachomatis, and Orientia tsutsugamushi manipulate the endocytic and secretory pathways. Understanding how bacterial effector proteins manipulate host processes not only gives us keen insight into bacterial pathogenesis, but also enhances our understanding of how eukaryotic membrane trafficking is regulated.Intracellular bacteria have developed numerous strategies to hijack host vesicular trafficking pathways to form their unique replicative niches. To promote intracellular replication, the bacteria must interact with host organelles and modulate host signaling pathways to acquire nutrients and membrane for the growing parasitophorous vacuole all while suppressing activation of the immune response. To facilitate host cell subversion, bacterial pathogens use specialized secretion systems to deliver bacterial virulence factors, termed effectors, into the host cell that mimic, agonize, and/or antagonize the function of host proteins. In this review we will discuss how bacterial effector proteins from Obligate and facultative intracellular bacteria have developed numerous methods to hijack host membranes to promote uptake, survival, and intracellular replication. Following uptake, intracellular pathogens must engage host organelles and subvert host defense mechanisms to establish their unique intracellular niches. To facilitate interactions with the host, many pathogenic bacteria deliver bacterial virulence proteins, termed effectors, into the host cell using specialized secretion systems. These proteins traffic to distinct subcellular locations within the host cell Rabex-5 are synthesized in the rough endoplasmic reticulum (ER), delivered to the ER-Golgi intermediate compartment (ERGIC), modified as they move through the Golgi, and are ultimately packaged into transport vesicles for delivery to their final destination associates with early and late endosomal compartments , encoded on two Salmonella pathogenicity islands that matures by trafficking through the endocytic pathway and interacts with the secretory pathway in the SCV, altering the membrane surface charge that project from the SCV 2 and 3 play an integral role in maintaining SCV positioning and depletion of SCAMP2 or 3 results in dispersion of the SCV within the host cell GTPases or de-PCylation, respectively . Interestingly, ubiquitination of these proteins does not require host E1 or E2 enzymes, representing a method of ubiquitination that is unique to trans-Golgi network and is recruited to target membranes by GTP-bound Rab7 P and potentially modulates the activity of the retromer complex. Mutation of ridL results in decreased L. pneumophila replication in macrophages, LAMP1 accumulation on the LCV, as well as recruitment of retrograde cargo receptors (Vps10 and CIMPR) and SNX1 and 2 , ectopic pregnancy, and infertility ; IncA (CT119), InaC (CT813), and IPAM (CT223) where it undergoes extensive interactions with the Golgi (Grieshaber et al., C. trachomatis causes relocalization of SNXs from endosomes to the inclusion membrane and induces inclusion tubulation (Aeberhard et al., L. pneumophila, knockdown of retromer components enhances C. trachomatis replication, suggesting the retromer complex controls infection (Mirrashidi et al., trans-Golgi network (Mirrashidi et al., In a study to map the host-Inc interactome, an interaction between the inclusion membrane protein IncE and SNX5/6, a component of the retromer complex, was identified (Mirrashidi et al., C. trachomatis hijacks non-vesicular ER-TGN transport to acquire ceramide, a sphingomyelin precursor. Host lipids including sphingomyelin, cholesterol, phosphatidylcholine, and phosphatidylinositol are incorporated into the bacterial cell (Hackstadt et al., In addition to acquiring nutrients by exploiting host vesicular trafficking pathways, C. trachomatis T3SS substrates localize to the inclusion membrane, an additional subset of T3SS proteins are predicted to be secreted into the host cell cytosol (Subtil et al., While a large number of Orientia tsutsugamushi is an obligate intracellular bacterium that is the causative agent of scrub typhus, a potentially fatal disease that is endemic to the Asia-Pacific region. O. tsutsugamushi is transmitted to humans via the bite of an infected trombiculid mite (Valbuena and Walker, O. tsutsugamushi is internalized by clathrin-dependent endocytosis and associates with early and late endosomes as evident by co-localization with EEA1 and LAMP2, respectively (Chu et al., O. tsutsugamushi is released into the cytoplasm where it moves along microtubules to the MTOC (Kim et al., O. tsutsugamushi escapes the phagosome is unknown, however it encodes a hemolysin, tlyC and a phospholipase D (Ge and Rikihisa, C. burnetii (Pan et al., L. pneumophila (Pan et al., Anaplasma phagocytophilum (Caturegli et al., O. tsutsugamushi Ikeda possesses 47 Ank open reading frames (ORFs; Nakayama et al., Orientia infection, the ER is distended and the Golgi is perturbed. While the exact mechanism of how this occurs and the benefit to Orientia is unknown, it is possible that at least part of this is dependent on Ank9.The ankyrin repeat domain is a 33-residue eukaryotic motif involved in mediating protein-protein interactions for numerous host cell processes including transcription, cell cycle regulation, signal transduction, and cytoskeletal rearrangements (Voth et al., Obligate and facultative intracellular bacteria have developed sophisticated strategies to modulate host endocytic and secretory trafficking to promote formation of their unique replicative niches. The adaption of genetic tools for manipulation of obligate and facultative intracellular pathogens has substantially enhanced our understanding of host-pathogen interactions and effector function. While great strides have been made toward understanding how effector proteins manipulate host processes to redirect membrane and nutrients to the parasitophorous vacuoles, the function of most effector proteins still remains ill-defined and genetic manipulation of some of these organism presents specific challenges. Large-scale screens to identify putative binding partners of ectopically produced type III secreted effectors (Mirrashidi et al., All authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "X-ray free-electron lasers (XFELs) provide ultra-bright femtosecond X-ray pulses that are intense enough to enable data collection of small weakly scattering objects and short enough to outrun most radiation damage effects. These beam features allow not only to study highly radiation-sensitive systems such as metalloproteins and tiny crystals but also to capture fleeting reaction intermediates in time-resolved studies. They therefore expand the structural biologist\u2019s toolbox by a great deal . Second, there is the serial femtosecond crystallography (SFX) data collection scheme that only once probes randomly oriented microcrystals intersecting the XFEL beam and gives a still image. Thus, it had long been doubted that the resulting integrated Bragg intensities are accurate enough for de novo phasing to be successful. A number of XFEL-specific phasing approaches have been suggested using in vivo grown nanocrystals diffracting to better than 2.5\u2005\u00c5 resolution becomes meaningful and fewer images (and thus less sample and beam time) are needed to obtain accurate Bragg intensities.Knowing whether one can solve a structure by SAD phasing and when to stop collecting more data because the anomalous signal in SAD phasing is high enough is still an active area of research [see, for example, Terwilliger al. 2016 and refe al. 2017 in this de\u00a0novo phasing required 60000 indexed patterns for fully automatic model building (Barends et al., 2014et al., 2016et al. (2015et al., 2017In fact, data processing programs have improved significantly in recent years. This is reflected in the finding that fewer and fewer diffraction patterns of a given dataset are needed for phasing. For example, the very first demonstration of al. 2015 could no"} +{"text": "Clinical observations that hyperlipidemia and diabetes are both reversed in mice and in patients taking Gleevec support the drugs' primary metabolic targets by biguanides and statins. This is evident by structural data demonstrating that Gleevec shows pyridine- and phenyl-guanidine homology with Phenformin and identical phenylcarbamoyl structural and ligand binding homology with Lipitor. The misunderstood mechanism of action of Gleevec is emblematic of the pervasive flawed reasoning that genomic analysis will lead to targeted, personalized diagnosis and therapy. The alternative perspective for Gleevec's mode of action may turn oncotargets towards metabolic channel reaction architectures in leukemia and melanoma, as well as in other cancers.Phenformin's recently demonstrated efficacy in melanoma and Gleevec's demonstrated anti-proliferative action in chronic myeloid leukemia may lie within these drugs' significant pharmacokinetics, pharmacodynamics and structural homologies, which are reviewed herein. Gleevec's success in turning a fatal leukemia into a manageable chronic disease has been trumpeted in medical, economic, political and social circles because it is considered the first successful targeted therapy. Investments have been immense in omics analyses and while in some cases they greatly helped the management of patients, in others targeted therapies failed to achieve clinically stable recurrence-free disease course or to substantially extend survival. Nevertheless protein kinase controlling approaches have persisted despite early warnings that the targeted genomics narrative is overblown. Experimental and clinical observations with Phenformin suggest an alternative explanation for Gleevec's mode of action. Using Gleevec's demonstrated anti-proliferative action in chronic myeloid leukemia ushered in the era of targeted therapies with claims that the drug blocks the constitutively active BCR-ABL tyrosine kinase thereby inhibiting phosphorylation of multiple downstream proteins of the mitogenic signaling pathways . Partly et al. [in vitro studies have thrown into similar question whether the single-target kinase signal blocking mechanism is indeed the primary mechanism for STI-571's mode of action [13C-guided precise flux measurements, in a comparative multiple cell line study. Targeted 13C-glucose tracer fate association studies demonstrated the drugs' downstream impact on submolecular fatty acid processing and deuterium depleting metabolic events that occurred independent of Gleevec's molecular target. Clinical observations that hyperlipidemia and diabetes are both reversed in mice and in patients taking Phenformin and Gleevec support the drugs' primary metabolic targets [In their excellent paper Petrachi et al. reiteratf action using 13 targets \u201310.et al.'s work in melanoma [The structural homology between Phenformin and Gleevec certainly links Petrachi melanoma to Gleevmelanoma , the guamelanoma and Gleemelanoma , which hmelanoma , 14 detemelanoma . Strikinmelanoma so that et al.'s work [via six hydrogen bond interactions [via distinct amino acid residues that are also known to bind Lipitor, Metformin and Phenformin in their own protein targets. These low affinity and high capacity drug binding architectures are evidently shared by Lipitor, Phenformin and Gleevec, which include histidine, leucine, iso-leucine, as well as serine, as specific amino acid ligands in two dimensional visual structures [13C-glucose guided metabolomics studies [It is worth knowing that all analogues designed to treat Gleevec resistance share guanide structural similarities and target the same ATP pocket for binding, all of which result in remissions in chronic myeloid leukemia. Petrachi .'s work may exteractions . Such hyractions . Figure ructures . Gleevec studies , 12. The studies to Metfo studies , a struc studies \u201324. Thes studies , 23. MorIn summary, the excellent paper by Petrachi et al. [et al. [i et al. that demi et al. as well i et al. \u201329. The [et al. opens di [et al. , 14, bas [et al. ."} +{"text": "Control of cell-cell coordination and communication is regulated by several factors, including paracrine and autocrine release of biomolecules, and direct exchange of soluble factors between cells through gap junction channels. Additionally, hemichannels also participate in cell-cell coordination through the release of signaling molecules, such as ATP and glutamate. A family of transmembrane proteins named connexins forms both gap junction channels and hemichannels. Because of their importance in cell and tissue coordination, connexins are controlled both by post-translational and post-transcriptional modifications. In recent years, non-coding RNAs have garnered research interest due to their ability to exert post-transcriptional regulation of gene expression. One of the most recent, well-documented control mechanisms of protein synthesis is found through the action of small, single-stranded RNA, called micro RNAs (miRNAs or miRs). Put simply, miRNAs are negative regulators of the expression of a myriad proteins involved in many physiological and pathological processes. This mini review will briefly summarize what is currently known about the action of miRNAs over Cxs expression/function in different organs under some relevant physiological and pathological conditions. Cell-cell communication and signaling is regulated by exchange of soluble factors between cells through gap junction channels (GJC) , the level of miR-1 is downregulated with a concomitant upregulation of Cx43 within the endoneurium of the sciatic nerve the presence of Cx43 GJCs. However, after fusion, Cx43 is downregulated by both miR-206 and miR-1 in myocytes in vitro (Anderson et al., In the cell line C2C12, which is a mouse myoblast cell line, it has been shown that miR-206 promotes muscle differentiation (Kim et al., As in skeletal muscle cells, miR-1 also controls Cx43 levels in smooth muscle cells. Thus, in overactive bladder, it was shown that MYOCD downregulates Cx43 expression by controlling miR-1 levels, showing that reduction of Cx43 could be a key factor in this pathology (Imamura et al., in vivo\u2014was observed (Inose et al., Cx43 is the main Cx expressed in osteocytes, and its presence is fundamental for their differentiation (Civitelli, GJs play a key function in propagating action potentials, and the heart is no exception to this principle. Both Cx40 and Cx43 localize along the axis of atrioventricular conduction, including atrioventricular node, atrioventricular bundle and Purkinje fibers suggesting an important role in conducting the impulse (Gourdie et al., As mentioned, miR-1 is involved in downregulation of Cx43 in skeletal muscle development (Anderson et al., miR-1 is not the sole master switch, controlling Cx43 levels in the heart. On one hand, it has been shown that miR-19 a/b decrease Cx43 levels, and that this change is associated with cardiac arrhythmia observed in a mouse constitutively overexpressing the miR-17-92 cluster in smooth muscle and cardiomyocytes (Danielson et al., in vivo and in vitro, and inhibits tumor growth in vivo (Li et al., Significant changes in gene expression patterns that promote rapid cell division are the unifying hallmark of tumorigenesis. Each different type of cancer has a distinctive signature of \u201cdriver\u201d mutations, which are recurrent across patients and affect genes that encode key components of the cell cycle machinery (Vogelstein et al., In nasopharyngeal carcinoma associated with the Epstein-Barr virus, downregulation of miR-218 has been consistently observed (Alajez et al., In breast cancer cell line MDA-MB-231, transfection of hsa-miR-206 decrease Cx43 levels, which was correlated with a decrease of proliferation rate and cell migration (Fu et al., An interesting potential avenue of research is to better understand whether changes in expression levels of Cxs in different types of cancer are partially or fully mediated by miRNAs. Therefore, biologically accurate models are required in order to dissect the mechanism that underlies promotion of invasiveness and worsens the clinical course of different forms of cancer.This review revisits insurmountable evidence of the relevant role of Cxs in health and disease. In addition to this, we have discussed the most recent findings in microRNA-mediated regulation of Cxs for several muscle and skeletal disorders as well as rhythm-associated and structural heart defects, and several types of cancer. Table JFC and MAR wrote and edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Finally, we demonstrate that vector-mediated expression of alpha-synuclein (SNCA), a transcript decreased in selectively vulnerable motor neurons in all four screens, can extend life span, increase weight and decrease neuromuscular junction pathology in a mouse model of SMA. In summary, we have combined multiple data sets to identify transcripts, which are strong candidates for being phenotypic modifiers, and demonstrated SNCA is a modifier of pathology in motor neuron disease.The term \u201cmotor neuron disease\u201d encompasses a spectrum of disorders in which motor neurons are the primary pathological target. However, in both patients and animal models of these diseases, not all motor neurons are equally vulnerable, in that while some motor neurons are lost very early in disease, others remain comparatively intact, even at late stages. This creates a valuable system to investigate the factors that regulate motor neuron vulnerability. In this study, we aim to use this experimental paradigm to identify potential transcriptional modifiers. We have compared the transcriptome of motor neurons from healthy wild-type mice, which are differentially vulnerable in the childhood motor neuron disease Spinal Muscular Atrophy (SMA), and have identified 910 transcriptional changes. We have compared this data set with published microarray data sets on other differentially vulnerable motor neurons. These neurons were differentially vulnerable in the adult onset motor neuron disease Amyotrophic Lateral Sclerosis (ALS), but the screen was performed on the equivalent population of neurons from neurologically normal human, rat and mouse. This cross species comparison has generated a refined list of differentially expressed genes, including CELF5, Col5a2, PGEMN1, SNCA, Stmn1 and HOXa5, alongside a further enrichment for synaptic and axonal transcripts. As an The term \u201cmotor neuron disease\u201d refers to a group of disorders, causing progressive paralysis of affected patients due to the degeneration of motor neurons cells which control voluntary movements. Importantly, not all motor neurons appear to be affected in the same way, with those that control the face being affected less that those that control the abdomen. The reason why some motor neurons are more vulnerable is unknown; however, understanding this may provide new targets for therapeutics to slow motor neuron degeneration either as stand-alone therapeutics or in combination with SMN-inducing compounds. In this study, we analysed gene expression in different groups of motor neurons and compared this to previously published expression data to identify commonalities. One of the common transcripts was alpha-synuclein (SNCA), which was consistently expressed at lower levels in vulnerable motor neurons. Importantly, when SNCA levels were increased in a mouse model of motor neuron disease, the disease phenotype was significantly reduced, including an extension in survival and reduction in motor neuron pathology. Collectively, these results demonstrate that this approach can identify disease modifiers that can reduce disease severity in models of motor neuron disease and potentially identify new therapeutic targets. SMN1 gene and visualised with Cy3-conjugated secondary antibodies . Muscles were then whole-mounted in Dako Fluorescent mounting media. Confocal microscopy was performed using a Nikon A1RThe percentage of fully occupied endplates was determined by classifying each endplate in a given field of view either fully occupied completely overlies endplate (BTX)), partially occupied ), or vacant (no pre-synaptic label overlies endplate). At least 4 fields of view were analysed per muscle totalling >100 endplates per muscle.All data was assembled and analysed using Microsoft Excel and GraphPad Prism.S1 TableTable shows the ensembl ID , the official gene symbol (gene), the chromosomal location (locus), the average normalised read count from an N of 2 samples for resistant (BS-WT) or vulnerable (SC_WT) samples, the log2 fold change, and the relevant statistics .(XLSX)Click here for additional data file.S2 TableTable includes the mouse official gene symbol and the log2 fold change identified in Brockington et al., Kaplan et al., Murray et al., and Hedlund et al.(XLSX)Click here for additional data file.S3 TableTable includes the mouse official gene symbol and the log2 fold change identified in Brockington et al., Kaplan et al., Murray et al., and Hedlund et al.(XLSX)Click here for additional data file."} +{"text": "I am concerned by an article \u201cDetecting Key Genes Regulated by miRNAs in Dysfunctional Crosstalk Pathway of Myasthenia Gravis\u201d by Cao et al., 2015 [In this study, the authors used data from public databases to compare microarray data from the thymus and miRNome data from peripheral blood mononuclear cells (PBMCs). They analyzed the regulation of mRNAs from the thymus by miRNAs from peripheral blood mononuclear cells: two different \u201ctissues.\u201d Of course, they ended up with results but one can really wonder about the meaning of these results.Moreover, in their manuscript, Cao et al. compiled thymic microarray data altogether while data are from different categories of patients (seropositive (for anti-AChR antibodies) and seronegative patients) ["} +{"text": "Tenascins represent key constituents of the extracellular matrix (ECM) with major impact on central nervous system (CNS) development. In this regard, several studies indicate that they play a crucial role in axonal growth and guidance, synaptogenesis and boundary formation. These functions are not only important during development, but also for regeneration under several pathological conditions. Additionally, tenascin-C (Tnc) represents a key modulator of the immune system and inflammatory processes. In the present review article, we focus on the function of Tnc and tenascin-R (Tnr) in the diseased CNS, specifically after retinal and optic nerve damage and degeneration. We summarize the current view on both tenascins in diseases such as glaucoma, retinal ischemia, age-related macular degeneration (AMD) or diabetic retinopathy. In this context, we discuss their expression profile, possible functional relevance, remodeling of the interacting matrisome and tenascin receptors, especially under pathological conditions. Numerous studies demonstrate that retina and optic nerve degeneration is highly associated with remodeling of various extracellular matrix (ECM) components. Glycoproteins and proteoglycans that surround retinal cells and optic nerve fibers represent major constituents of the ECM meshwork, known as the matrisome , Tnc exhibits high expression during early development. With ongoing maturation, it is progressively downregulated, but re-expressed under pathological conditions , diabetic retinopathy, glaucoma and retinal vascular occlusion (Mizener et al., Several studies reported on a dysregulation of Tnc following cerebral, hepatic as well as myocardial ischemia (Lu et al., In the CNS, tenascins represent main structural and functional constituents of synaptic sites (Dityatev et al., AMD is defined by a deterioration of the macula and represents a major cause of vision impairment worldwide (Jager et al., The tenascin family member Tnx was identified in AMD patients in a genome-wide association study (Cipriani et al., Additionally, high levels of Tnc were observed in choroidal neovascular membranes from AMD patients (Nicol\u00f2 et al., Diabetic retinopathy is also highly associated with retinal vascular dysfunction. Tnc was found in intravitreal membranes of patients with traumatic and idiopathic proliferative vitreoretinopathy as well as in diabetic retinopathy (Hagedorn et al., RGC nerve fibers exhibit a poor regeneration capacity after injury, which often leads to irreversible vision loss. Therefore, multiple studies focused on the improvement of RGC survival as well as axonal regrowth, guidance and pathfinding (Fischer and Leibinger, After optic nerve damage, Wallerian degeneration, demyelination, immune activation and glial scar formation can be observed. In this context, it has become evident that next to the intrinsic cellular repertoire, an inhibitory environment prevents regrowth of optic nerve fibers Fischer, . ECM proin vivo. Tnr was also described as a repulsive guidance molecule of newly growing as well as regenerating optic nerve fibers in the zebrafish (Becker and Becker, in vitro. In contrast to the reduced Tnr expression levels observed in the optic nerve of the salamander (Becker et al., in vivo. In addition, Tnr and axon growth-promoting molecules were found upregulated in the regenerating visual pathway of the lizard Gallotia galloti (Lang et al., Compared to mammals, the CNS of the zebrafish displays a robust axonal regeneration capacity and allows visualization of axonal regeneration and re-myelination Since Tnr is highly associated with myelinated optic nerve fibers and nodes of Ranvier, it was proposed that it might have a functional relevance in myelination processes. Recordings of action potentials from Tnr knock-out mice revealed reduced axonal conduction velocities compared to control mice. In contrast, no significant differences in the number of myelinated optic nerve fibers or in the myelin ultrastructure were observed in Tnr knock-out compared to wild-type mice (Weber et al., A potential role of Tnc in neural repair of the injured rat optic nerve was initially reported by Ajemian et al. . Here, aA huge diversity of interacting molecules can be observed for the tenascin proteins. For Tnc this includes the cell adhesion molecules contactin-1 (Rigato et al., The signaling of integrins in RGC-glia interactions is crucial for RGC survival and process extension (Vecino et al., in vitro. Furthermore, Tnc knock-out mice show an impaired de-differentiation capacity (Besser et al., As mentioned above, CSPGs are major interaction partners of Tnc. In the CNS, CSPGs are widely recognized as major inhibitory constituents of the glial scar (Silver and Silver, in vitro (Tom et al., Likewise, Tnc displays a complex interactome with other ECM glycoproteins. For instance, its interaction with fibronectin and Tnr was reported (Chiquet-Ehrismann et al., The adhesion molecule contactin-1 was identified as an important neuronal receptor for Tnr. Interaction of these two molecules was reported to mediate the repulsion and defasciculation of neurites (Pesheva et al., In the CNS, tenascin glycoproteins are important constituents of a highly regulated and dynamic matrisome. In sum, the current literature supports the notion that Tnc and Tnr are implicated in various pathological processes following retinal and optic nerve degeneration as well as various eye diseases Table . Under pJR wrote the manuscript. LR designed the figures. LR and AF revised the manuscript. All authors have approved the final article.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Inflammatory response, oxidative stress, and endoplasmic reticulum (ER) stress are important pathophysiological bases of the occurrence and development of diabetes mellitus (DM) and macroangiopathy complications. Selenoprotein S (SELENOS) is involved in the regulation of these mechanisms; therefore, its association with DM and macroangiopathy has gradually received attention from scholars worldwide. SELENOS has different biological functions in different tissues and organs: it exerts antioxidant protection and has anti-ER stress effects in the pancreas and blood vessels, while it promotes the occurrence and development of insulin resistance in the liver, adipose tissue, and skeletal muscle. In addition, studies have confirmed that some SELENOS gene polymorphisms can influence the inflammatory response and are closely associated with the risk for developing DM and macroangiopathy. Therefore, comprehensive understanding of the association between SELENOS and inflammation, oxidative stress, and ER stress may better elucidate and supplement the pathogenic mechanisms of DM and macroangiopathy complications. Furthermore, in-depth investigation of the association of SELENOS function in different tissues and organs with DM and macroangiopathy may facilitate the development of new strategies for the prevention and treatment of DM and macrovascular complications. Here, we summarize the consensus and controversy regarding functions of SELENOS on currently available evidence. In 2016, the World Health Organization (WHO) declared that diabetes mellitus (DM) had become the eighth most prevalent cause of disease-related mortality. Its onset is no longer mainly observed in economically developed areas; in recent decades, the number of patients has increased significantly in middle- and low-income countries. Therefore, DM has been confirmed to be a \u201cglobal\u201d public health issue [Selenium (Se), a trace element, is a key component of selenoproteins involved in a wide range of functions, including redox homeostasis, inflammatory regulation, thyroid hormone metabolism, immunity, myocardial and tumoral diseases, and reproduction \u20136. MoreoPsammomys obesus (P. obesus) by Walder et al. [Selenoprotein S (SELENOS), as a member of the selenoprotein family, was first discovered in the liver of r et al. and was r et al. , 14\u201316. r et al. \u201321. The r et al. \u201325. Ther188UGA codon in SELENOS variant 2 CDS can be normally encoded as Sec to produce the protein SELENOS isoform 2, with 189 amino acid residues. Because the SECIS in SELENOS variant 1 is spliced, the 188UGA in the mRNA CDS cannot be encoded as Sec and is recognized as the translation termination signal. Therefore, a short protein, SELENOS isoform 1, with 187 amino acid residues, is produced [The human SELENOS gene (GenBank: NG_013322.1) is located on 15q26.3. Its primary transcript produces two types of transcription variants after selective splicing: SELENOS variant 1 (GenBank: NM_203472.2) and SELENOS variant 2 (GenBank: NM_018445.5). The main difference between these two transcripts is that the selenocysteine insertion sequence (SECIS) in the 3\u2032untranslated region (3\u2032UTR) of SELENOS variant 1 is spliced \u201330. Therproduced , suggest174Cys and 188Sec that has reductase activity [SELENOS is a single-pass transmembrane protein with an ER region (1st\u201325th amino acid residues), a transmembrane region (26th\u201351st amino acid residues), and a cytoplasmic region (52nd\u2013189th amino acid residues). The cytoplasmic region contains the valosin-containing protein (VCP)-interacting motif composed of the 78th\u201388th amino acid residues, and there is a selenosulfide bond between In addition to localizing in the ER membrane , 18, SELWhen Walder et al. first diZhang et al. found thFurthermore, studies in recent years on the association between SELENOS gene polymorphisms and inflammation have also become a hot research topic. Previously, Curran et al. showed t188Sec and was maintained through the restoration of the selenosulfide bond between 174Cys and 188Sec. In other words, in the reduction process, 188Sec of SELENOS and substrates formed a mixed selenosulfide bond to cause substrate reduction. Next, through the restoration of the selenosulfide bond between 174Cys and 188Sec, SELENOS broke the mixed selenosulfide bond between SELENOS and substrates to maintain the activity of the SELENOS reductase. Liu et al. [174Cys and 188Sec depended on the function of thioredoxin (Trx), indicating that SELENOS was a Trx-dependent reductase. In addition, they also showed that SELENOS had peroxidase activity and could break down its substrate, hydrogen peroxide (H2O2), into H2O [2O2 could up-regulate SELENOS expression in HEK293 human embryonic kidney cells, while inhibition of SELENOS expression could further aggravate the LPS-induced increase of reactive oxygen species levels in HepG2 human liver cancer cells, down-regulation of GPx expression, and inhibition of cell viability. These results suggested that SELENOS may have the function of protecting tissues and cells from oxidative stress-induced damage. Previously, Gao et al. [2O2 damage and enhance cell viability. Our study group induced SELENOS overexpression in human umbilical vein endothelial cells (HUVECs) and showed that SELENOS overexpression could significantly increase HUVEC viability after H2O2 stimulation, enhance the activity of superoxide dismutase (SOD), and reduce the production of methane dicarboxylic aldehyde (MDA) [2O2, which was accompanied by the further increase of ROS and MDA levels and further reduction of the GPx activity. They also confirmed that the antioxidant protection function of SELENOS was associated with mitogen-activated protein kinase (MAPK) and c-JUN N-terminal kinase (JNK).Christensen et al. showed tu et al. showed tinto H2O . The abointo H2O and Zenginto H2O . They foo et al. showed tde (MDA) . Gan et de (MDA) showed tde (MDA) indicateAs an ER membrane protein, SELENOS plays an important role in the maintenance of the morphology and distribution of ER in cells . In addiIn addition to investigating the relationship between SELENOS and inflammation, Fradejas et al. also anaMart\u00ednez et al. comparedP. obesus. In addition, the inhibition of SELENOS expression by glucose in a concentration-dependent manner was also confirmed in in vitro cultured HepG2 liver cancer cells. Moreover, Gao et al. [Walder et al. showed to et al. confirmeo et al. induced The expression of SELENOS in 3T3-L1 adipocytes was inhibited by glucose or insulin in a concentration-dependent manner . KarlssoThe contradiction of the association of adipose tissue SELENOS with glucose metabolism in the aforementioned studies was also present in related studies on the association of skeletal muscle SELENOS with glucose metabolism. Walder et al. confirmeSAA can interact with SELENOS, which was confirmed by both a yeast-two hybrid experiment and surface plasmon resonance analysis , and the2O2 damage and enhance cell viability, suggesting that SELENOS was a protective factor in pancreatic islet and could protect pancreatic islet \u03b2 cells from oxidative stress damage. The study by Christensen et al. [174Cys and 188Sec in the molecular structure of SELENOS rendered SELENOS with reductase activity; in addition, the study by Liu et al. [The progressive apoptosis of pancreatic islet \u03b2 cells is an important feature of the occurrence and development of DM, and oxidative stress damage induced by high glucose is one of the reasons for pancreatic islet \u03b2 cell apoptosis. The study of Gao et al. showed tn et al. showed tu et al. confirmeIn addition to the localization of SELENOS in the plasma membrane, secreted SELENOS was also detected in the culture medium of HepG2 liver cells and human serum samples. However, secreted SELENOS was not detected in the culture medium of 3T3-L1 adipocytes, L6 skeletal muscle cells, Min6 pancreatic islet \u03b2 cells, HEK293 human embryonic kidney cells, HUVECs, or human aortic vascular smooth muscle cells (HA/VSMCs), suggesting that serum SELENOS was mainly secreted by hepatocytes , 33. In Therefore, SELENOS in different tissues and organs has different functions. The results are summarized in Fig.\u00a0Hyrenbach et al. used the2O2; the changes after inhibition of SELENOS expression had the opposite results [Vascular endothelial cells play an important role in the maintenance of cardiovascular homeostasis, while endothelial dysfunction is the initiating step in the development of atherosclerosis \u201378. Our results . These r2O2 or TM and increase VSMC apoptosis. Therefore, it was speculated that SELENOS could increase the resistance of VSMCs to oxidative stress and ER stress [VSMCs are important components of the tunica media of vascular walls. Their abnormal proliferation is involved in the formation of early atherosclerosis plagues, and the apoptosis of VSMCs in advanced atherosclerosis plaques will induce the reduction of collagen production to cause fibrous cap thinning and reduce plague stability , which iR stress .2O2. When SELENOS expression was inhibited, Cav-1 and protein kinase C\u03b1 (PKC\u03b1) expression levels increased [2O2, indicating that SELENOS increased the resistance of VSMCs to oxidative stress damage through the MAPK/JNK pathway. In addition, the antioxidant function of SELENOS in blood vessels may also be associated with the aforementioned 188Sec in its molecular structure. Furthermore, the study of Liu et al. [2O2 into H2O, which could partially explain the study results of our group [Our study group further showed that high SELENOS expression could inhibit the increase of caveolin-1 (Cav-1) induced by Hncreased , suggestncreased confirmeu et al. also shour group and thosur group . These rShibata et al. confirmeIt has been confirmed that various SELENOS SNPs are associated with the risk of the development of autoimmune inflammatory diseases. Marinou et al. showed tSELENOS is closely associated with inflammation, oxidative stress, and ER stress. In-depth and comprehensive understanding of its association with the above reactions may not only elucidate or supplement pathogenic mechanisms of relevant diseases but also provide new evidence for clinical physicians to develop new strategies for disease prevention and treatment. However, the current understanding of this aspect is limited; there are still some scientific questions that need to be further confirmed or studied in-depth, such as whether SELENOS can inhibit the expression of other inflammatory factors and whether the regulation of ER stress by SELENOS is different in different cell types. Furthermore, whether the human SELENOS gene promoter region has negative regulatory elements that can regulate SELENOS gene expression and whether there are transcription factors that can interact with these elements should also be further explored. These studies will help to provide new targets for drug intervention and to discover more effective disease treatment methods.The functions of SELENOS in inflammatory reactions, oxidative stress, and ER stress point to its great potential in DM and macroangiopathy. It has been shown that SELENOS expressed in different tissues and organs has different effects on the occurrence and development of DM and its macroangiopathy. The high expression of SELENOS in pancreatic islets and blood vessels can exert an antioxidant protection function and can increase the defense capacity of VSMCs to ER stress, while SELENOS expressed in liver, adipose tissue, and skeletal muscle can promote the occurrence and development of DM and insulin resistance. Based on the above functional characteristics of SELENOS, the method by which tissues and organs specifically regulate its expression to fully utilize the advantages of SELENOS and weaken its adverse effects still require further in-depth studies. SELENOS gene polymorphism is associated with DM, macroangiopathy, tumors, and autoimmune inflammatory diseases; therefore, it is expected to become one of the indicators for the prediction of the risk of the above diseases and one of the theoretical bases for the adoption of primary preventive measures for the population carrying relevant SNPs. However, SELENOS SNPs that have been discovered to be associated with disease development must still be confirmed in different populations using larger sample sizes."} +{"text": "Scientific Reports6: Article number: 3482410.1038/srep34824; published online: 10062016; updated: 12092016The Acknowledgments section in this Article is incomplete:\u201cWe are grateful to Gerrit Ansmann and Christian Geier for interesting discussions and for critical comments on earlier versions of the manuscript and by the Verein zur Foerderung der Epilepsieforschung e.V. (Bonn)\u201d.should read:\u201cWe are grateful to Gerrit Ansmann and Christian Geier for interesting discussions and for critical comments on earlier versions of the manuscript. This study was supported by the Deutsche Forschungsgemeinschaft DFG (Grant No.: LE 660/5-2) and by the Verein zur Foerderung der Epilepsieforschung e.V. (Bonn)\u201d."} +{"text": "The majority of the reported adverse events seen in these studies are mild or moderate in severity and tend to affect the gastrointestinal or nervous systems. These adverse events, which are common in both adults and children, are also typical of symptoms of malaria or concomitant infections present in these patients. The wealth of safety data on artemether/lumefantrine has not identified any neurological, cardiac or haematological safety concerns. In addition, repeated administration is not associated with an increased risk of adverse drug reactions including neurological adverse events. This finding is especially relevant for children from regions with high malaria transmission rates who often receive many courses of anti-malarial medications during their lifetime. Data are also available to show that there were no clinically relevant differences in pregnancy outcomes in women exposed to artemether/lumefantrine compared with sulphadoxine-pyrimethamine during pregnancy. The six-dose regimen of artemether/lumefantrine is therefore well tolerated in a wide range of patient populations. In addition, post-marketing experience, based on the delivery of 250 million treatments as of July 2009, has not identified any new safety concerns for artemether/lumefantrine apart from hypersensitivity and allergies, known class effects of artemisinin derivatives.This article reviews the comprehensive data on the safety and tolerability from over 6,300 patients who have taken artemether/lumefantrine (Coartem Plasmodium falciparum resistance to monotherapy , G\u00fcrkov et al [et al [et al [et al [et al [et al [There have been case reports of neurological problems occurring after administration of herbal artemisinin or artesov et al , and Adjl [et al , as showet al 1, G\u00fcrkov l [et al , and Mayl [et al , as showl [et al or Makanl [et al , which aLumefantrine is chemically related to halofantrine, an anti-malarial known to be associated with significant prolongation of QTc interval. Indeed, QTc prolongation is a known class effect of many anti-malarial drugs. As such, cardiac safety has been thoroughly investigated during the preclinical and clinical development of AL.+ channels have been investigated in stably transfected human embryonic kidney cells (HEK293) using a whole cell patch-clamp technique [in vitro hERG channel assay showed that lumefantrine and desbutyl-lumefantrine have higher IC50 values (approximately 200-fold) than halofantrine have been delivered to malaria-endemic countries. Post-marketing experience has not identified any new specific safety concerns apart from hypersensitivity and skin reactions , a recognized class effect for artemisinin derivatives [\u00ae) from published Novartis-sponsored and independently-sponsored clinical trials. Data are available to support the use of a six-dose regimen of AL as a safe and well-tolerated treatment for P. falciparum malaria or malaria due to mixed infection including P. falciparum in adults, adolescents, children and infants. Indeed, the safety profile of this drug is similar in both adults and children, and many of the reported adverse events are typical of symptoms of malaria or concomitant infections that are commonly seen in these patient populations. Reported adverse events are mainly of mild or moderate severity, with very few serious adverse events reported and even fewer serious adverse events considered related to treatment. In addition, no notable neurological adverse events or cardiac safety concerns were identified in the many studies that examined the safety of AL. As patients often take repeated courses of treatment for malaria, it is reassuring to note that data are available that show repeated administration of AL is not associated with an increased risk of adverse drug reactions, in particular with regard to neurological adverse events. In addition, a thorough review of the clinical data for AL does not identify any potential cardiac safety issues. Data are also available to show that there were no clinically relevant differences in perinatal mortality, neonatal mortality, still birth, pre-term delivery, low birth weight and spontaneous abortion in women exposed to AL compared with SP during pregnancy.This review summarizes some of the safety and tolerability data on AL treatments have been delivered and post-marketing experience confirms the favourable safety and tolerability profile for this drug. In addition, this wealth of safety evidence has not identified any safety concerns for AL apart from rare type 1 hypersensitivity reactions, a recognized class effect of artemisinin derivatives [In conclusion, AL is a safe and well-tolerated treatment for 50: Inhibitory concentration 50 (the concentration that gives 50% inhibition); ICH: International Conference on Harmonization; IKr: Rectifying K+ current; MAS: Mefloquine plus artesunate; MedDRA: Medical Dictionary for Regulatory Activities; SAE: Serious adverse event; SP: Sulphadoxine-pyrimethamine; vs.: Versus; WHO: World Health Organization.ACT: Artemisinin-based combination therapy; AE: Adverse event; AL: Artemether/lumefantrine; AQ: Amodiaquine; AQSP: Amodiaquine plus sulphadoxine-pyrimethamine; AS: Artesunate: ASAQ: Artesunate plus amodiaquine; ASTMH: American Society of Tropical Medicine and Hygiene; CQ: Chloroquine; CQSP: Chloroquine plus sulphadoxine-pyrimethamine; DNP: Dihydroartemisinin plus napthoquine plus trimethoprim; DP: Dihydroartemisinin-piperaquine; ECG: Electrocardiogram ; hERG: Human ether-a-go-go related gene; ICThe authors would like to acknowledge that Novartis Pharma AG sponsored this supplement. However, none of the authors works for, or represents in any way, Novartis Pharma AG.All authors met International Committee of Medical Journal Editors criteria for authorship."} +{"text": "The author conducted a review of studies that compared the efficacy, tolerability and indication for the use of clozapine in current perspectives for the treatment of resistant schizophrenia/ partial responders. In the early 1960s, German psychiatrists working with G. Stille at Wander Pharmaceuticals in Bern, Switzerland, worked to refute the concept that EPS and antipsychotic efficacy were linked.3 ClinicaHowever, enthusiasm for the drug was maintained by a small cadre of clinical investigators including G. Honigfeld at Sandoz, who observed that clozapine, was remarkably effective in treatment-resistant patients. This led to a landmark double-blind study of clozapine in a well-defined group of treatment-resistant patients whose blood cell counts were closely monitored during treatment and ulti1The mechanisms of action which account for the effectiveness of clozapine as a pharmacotherapy for the treatment of neuroleptic non-responders and neuroleptic intolerant schizophrenic subjects remain elusive. It is characterized by generally lower affinities for D2 receptors and relatively greater affinities for serotonin (5-hydroxytryptamine) 5-HT2A receptors in particular, but also for noradrenergic receptors (\u03b11 and \u03b12), muscarinic acetylcholine receptors, histamine and other dopamine (DA) subtype receptors.et al,[11C] SCH23390 and [11C]raclopride to investigate D1 and D2 receptor occupancy in vivo in 25 schizophrenic patients receiving atypical antipsychotic treatment with clozapine, olanzapine, quetiapine or risperidone. They concluded that among the atypical antipsychotics, clozapine appeared to have a simultaneous and equivalent occupancy of dopamine D1 and D2 receptors. Whether its effect on D1 receptors represents agonism or antagonism is not yet clear. This issue is still unresolved in the preclinical arena. The distinctive effect on D1/D2 receptors may be responsible for clozapine's unique effectiveness in patients with schizophrenia refractory to other typical and atypical antipsychotics.Tauscher et al, used PosMeta analysis of controlled trials involving patients who had treatment resistant schizophrenia\u201312 and aet al.,[In an open trial by Agarwal et al., drug-resMoncrieff comparedSeveral authors have questioned whether clozapine should be indicated as a first-line treatment for early psychosis.\u201317 The ret al., reported the interesting observation that clozapine, in comparison with fluphenazine, was similarly efficacious for new-onset patients with schizophrenia or schizoaffective disorder.[2 receptors in their brains. Typical antipsychotics may cause the upregulation because they dissociate slowly from the D2 receptor[Woerner disorder. It is frdisorder. that pat receptor and the receptor may allo receptor Because receptoret al.,[Pragmatic trials, supposed to provide more complete information for physician in clinical practice, support et al., in a phaet al., studied et al., Patientset al,[In another pragmatic trial Lewis et al, studied a second generation antipsychotic, Tuunainen et al,[In a Meta analysis comparing clozapine with en et al, found a et al., found that the magnitude of improvement in mean BPRS and CGI scores from baseline to end of the study was significantly greater in the clozapine group than in the risperidone group. Statistically significant differences in favor of clozapine were also seen for most of the secondary efficacy measures . The adverse event profile was similar for both treatment groups, with a lower risk of extrapyramidal symptoms in the clozapine group.In a Double-Blind Comparative Study, includinClozapine has shown consistent clinical benefit in schizophrenic patient with persistent aggressive and violent behavior.30 Whetheet al.,[McGurk et al., investiget al.,[Bilder et al., examinedet al.,[Purdon et al., examinedIt is estimated that approximately 30% of patients treated with clozapine do not respond adequately, remaining with persistent psychotic symptoms despite taking adequate treatment for sufficient period. Such patients are called \u201cpartial responders to clozapine,\u201d \u201cclozapine resistant,\u201d or even \u201csuper refractory\u201d. Despite 353745In a study conducted at psychiatry center, S.M.S. medical college, Jaipur, by Dr. R.K. Solanki and Dr. Paramjeet Singh, 100 diagnosed cases of schizophrenia on clozapine maintenance (not showing adequate response) were augmented with risperidone (2-4 mg/d). Patients showed significant improvement after 4 week on CGI, BPRS and PANSS scales.et al.,[Lieberman et al., reviewedIn international suicide prevention trial includinet al.,[Turetz et al., treated et al.,[et al.[Green et al., examined.,[et al.It is als.,[et al.Clozapine is least likely to worsen the neurological disorder so it isA number of authors have investigated the factors associated with response to clozapine but findings are contradictory. Reviewing the evidence Chung and Remington suggesteP<0.001), in 12 (60%) it lasted longer (P=0.0368) and in 17 (85%) it occurred more quickly on rechallenge (P<0.001).they concluded that after risks and benefits have been considered, rechallenge may well be justified in some patients.Apart from common side effects like sedation, dizziness, syncope, tachycardia, hypotension, ECG changes, nausea and vomiting, fatigue, weight gain, constipation, anticholinergic effects and subjective muscle weakness, several rare side effects has been described during clozapine treatment. One most important is neutropenia which may lead to fatal agranulocytosis, occurs in about 1% patient. Its mechanism is unclear but may be the result of direct toxicity or an immune response. Annan LJIn a review Wetterling suggested that Average weight gain with clozapine is 1.72 kg /month, occurring most frequently during first 6 to 12 month of treatment. There haet al.,[2. Thirty patients (36.6%) were diagnosed with diabetes during the 5-year follow-up. Weight gain, use of Valproate and total daily dose of clozapine were not significant risk factors for developing diabetes mellitus. Patients experienced significant weight gain that continued until approximately month 46 from initiation of clozapine. There was a nonsignificant increase in total serum cholesterol and a significant increase in serum triglycerides level.In a 5 year naturalistic study Henderson et al., examinedet al.,[Howes et al.,examined et al., Similarlet al.,et al.,[Akathisia occurs even occasionally with clozapine. Very raret al., reviewedet al.,et al.,[Cohen et al., conducteet al.,69 Myocaret al., There iset al.,Clozapine is known to increase seizure frequency. In a review seizure 74Clozapine specifically has two important risks of intestinal dysfunction: potentially severe ileus and sialDe novo emergence or exacerbation of obsessive-compulsive (OC) symptoms during treatment with clozapine has been described in the literature. Lykouras et al.,[s et al., revieweds et al.,Toxic delirium can occur in about 3% of patient.ClozapineData regarding the potential teratogenacity of clozapine are not unanimous. There are some reports of congenital malformations and other complications during pregnancy. But there is report of succeThe ten years of clinical experience of clozapine use in the dose range 100-400 mg/day as monotherapy or augmentation has been significantly safe and acceptable at large by patient subgroups. The side effect profile remained comparable to other SGA's, as evident from the unpublished data of the ongoing study at psychiatry center, SMS medical college, jaipur.The result of the different studies indicated that clozapine exhibit superiority over typical antipsychotics in terms of both efficacy and safety. However the magnitude of its effect is not consistent. Efficacy data for other SGA's in the treatment of refractory schizophrenics were inconclusive.So there is growing need to consider different treatment strategies. Whether monotherapy or adjuvant therapy for non responsive or partially responsive schizophrenics."} +{"text": "Sir,et al for their interest in my letter published in 2003 on traditional behavioural practices and exchange of saliva among sub-Saharan African populations. The comments by M Coluzzi et al on my letter highlight a potentially important behavioural practice associated with human herpesvirus 8 (HHV-8) transmission; the use of saliva to soothe blood-sucking arthropod bites. As mentioned in my letter, I found some evidence of this practice in reviewing ethnographic material from the Human Relations Area Files (HRAF). However, neither the extent nor frequency of saliva-associated behavioural practices, including those highlighted by Coluzzi et al, has been investigated among sub-Saharan African populations. The HRAF files present only descriptive, ethnographic data. Scientifically oriented epidemio-logical studies need to be developed to better characterise the frequency and distribution of saliva-associated behavioural practices and evaluate the potential association between these practices and risk of infection with HHV-8.I thank M Coluzzi et al discuss ecological data from a previous study The \u2018promoter arthropod\u2019 hypothesis raised by"} +{"text": "An outbreak of acute hemorrhagic conjunctivitis occurred in Delhi, India, during August and September 1996. The etiologic agent was confirmed as enterovirus type 70 by a modified centrifugation-enhanced culture method followed by immunofluorescence and neutralization tests. After nearly a decade, this virus is reemerging as a cause of acute hemorrhagic conjunctivitis in India."} +{"text": "Newer techniques of Laser Trabeculoplasty have revived the procedure and gained widespread acceptance by the ophthalmic community. This review was undertaken to address the evolution of different laser trabeculoplaty techniques, proposed mechanisms of action as well as review current studies of the therapeutic effects of these interventions. Laser trabeculoplasty (LT), first described in 1974 by Worthen and Whickham,Several other lasers including krypton (647.1 or 568.2 nm) diode (810 nm), DLT and continuous wave, frequency-doubled Nd: YAG 532 nm), the work by Latina and colleagues (1998)2 nm, the4Most glaucoma specialists regard LT as an adjunct to maximum tolerated medical therapy. Alternatively, it can be used as an initial glaucoma therapy. LT provides successful results in POAG, pseudo exfoliation and pigmentary glaucoma and in eyes with combined mechanism glaucoma. It does not appear to be effective in glaucoma secondary to uveitis, congenital glaucoma, juvenile open angle glaucoma, iridocorneal endothelial syndrome, iridocorneal mesodennal dysgenesis, steroid induced glaucoma, and glaucoma secondary to elevated episcleral venous pressure.SLT results in selective absorption of energy by trabecular pigmented cells sparing adjacent cells and tissues from thermal damage. It delivers a 400 um diameter treatment spot in three nanoseconds, by using short bursts at low power settings . SLT delivers energy fluence levels to the TM cells that are several thousand times lower than with ALT. One consequence of this energy reduction is that SLT seems to produce less thermal tissue damage to TM than ALT.5MLTMLT utilizes an 810-nm diode laser emitting a train of repetitive short pulses to allow the surgeon to control and spatially confine the laser-induced thermal elevation to produce the intended sub lethal photo thermal effects to elicit a thermal stress response in the TM cells. It is typically performed to deliver 60 to 65 (or 120-130) confluent 300-um diameter invisible laser applications covering the whole height of the TM over 180- (or 360-) angle. Due to the combination of (a) low absorption of the 810-nm laser wavelength by the TM and (b) low irradiance of 2W over the relatively large 300 um spot, MLT seems to interact and thermally affect all superficial and deep TM-pigmented cells without producing visible photo thermal changes, tissue blanching, or bubble formation. The treatment is invisible to the surgeon and uneventful for the patient with no pain and no dazzling laser flashes (810 nm is invisible).Many prospective and retrospective studies compare SLT with ALT indicating that a similar short- and long-term efficacy of ALT and SLT can be expected in patients with OAG with a similar and a mild risk profile.The GLT results demonstrate that ALT could be considered a primary therapy for OAG. Several other studies have also demonstrated that ALT is effective at lowering IOP.18Studies investigating the use of SLT as initial therapy using a 180-treatment report a mean reduction of 7.7 plus 3.5 mmHg (30%)19et al.et al.et al.McIlraith et al.et al.et al.Other variables such as age, sex, type of OAG and degree of trabecular meshwork pigmentation are not associated with improved outcomes in the group of participants receiving SLT.et al.et al.et al.et al.Repeat ALT effectiveness was assessed in a few studies. Feldman et al.et alet al.8Since SLT does not induce trabecular meshwork scarring, Latina et al.et al.Repeat SLT was assessed by Shah The findings are encouraging and suggest that 360-degree SLT may be repeated after an initially successful 360-degree SLT fails and with IOP reduction that suggests only slightly diminishing returns. Findings also suggest that these results may be achieved as early as six months after the first treatment.In a randomized pilot study conducted in the University of Missouri Kansas city,Along with medications, LT effectively and safely lowers elevated IOP. SLT and MLT can be performed with minimum iatrogenic damage to the TM- lowest intraoperative and postoperative complications and side-effects. This will eventually gain widespread acceptance by the ophthalmic community. Newer techniques may assume an increasingly important role of LT in our management of glaucoma."} +{"text": "To the Editor: We thank Su and Chee (The range of dengue-related ophthalmic complication is still being investigated, and we agree with Su and Chee that other ophthalmic manifestations may occur in patients with dengue fever. In a retrospective observational case series involving 22 eyes of 13 patients with visual impairment from dengue infection, carried out in our hospital, Chan et al. ("} +{"text": "Environmental Health Perspectives, La Pensee et al. (2009) postulated that bisphenol A (BPA), at nanomolecular doses, confers chemo resistance in estrogen receptor (ER)-\u03b1\u2013positive and \u2013negative breast cancer cells. Certainly, drug resistance is well-known to be an important complication in a variety of cancer chemotherapy options. Several molecular mechanisms have been suggested to explain the onset of drug resistance. Determining the exact mechanism in a particular case is challenging both at the clinical and preclinical research levels because the genome-wide and proteomic approaches to mechanistic studies are still at a developing stage , suggesting that direct or rapid response to E2 is widespread at the mRNA levels in these genes. 2 and environmental endocrine-disrupting chemicals in the uterus of women. Involvement of these gene transcripts, which are present in breast, uterine, and ovarian tissues, in the environment\u2013endocrine inter action suggests the possibility of a utero-ovarian feedback control of breast cancer chemo sensitivity effected by npcRNAs.Recent reports from other laboratories have tended to support a role of npcRNAs in BPA-mediated mechanisms involved in breast cancer and a possible physiochemical interaction of BPA with estrogen and non-estrogen-mediated chemosensitivity-inducing pathway elements. For example, These observations suggest that there may be a utero-ovarian feedback control mediated by ERs on the uterus that cross-talk with vasoneural pathways; the feedback control may mediate estrogen involved in chemoresponsive pathways of breast cancer. Furthermore, these pathways may be regulated by noncoding npcRNAs whose functions may be physiochemically modified by environmental toxicants such as BPA and other related chemials.in Vitro and Laboratory Animal Studies for Assessing Risks to Human Health\u201d in Chapel Hill, North Carolina, on 28\u201330 November 2006 (Since the forum titled \u201cBisphenol A: An Expert Panel Examination of the Relevance of Ecological, ber 2006 , there h"} +{"text": "Time-resolved structural studies of proteins have undergone several significant developments during the last decade. Recent developments using time-resolved X-ray methods, such as time-resolved Laue diffraction, low-temperature intermediate trapping, time-resolved wide-angle X-ray scattering and time-resolved X-ray absorption spectroscopy, are reviewed. Proteins undergo conformational changes during their biological function. As such, a high-resolution structure of a protein\u2019s resting conformation provides a starting point for elucidating its reaction mechanism, but provides no direct information concerning the protein\u2019s conformational dynamics. Several X-ray methods have been developed to elucidate those conformational changes that occur during a protein\u2019s reaction, including time-resolved Laue diffraction and intermediate trapping studies on three-dimensional protein crystals, and time-resolved wide-angle X-ray scattering and X-ray absorption studies on proteins in the solution phase. This review emphasizes the scope and limitations of these complementary experimental approaches when seeking to understand protein conformational dynamics. These methods are illustrated using a limited set of examples including myoglobin and haemoglobin in complex with carbon monoxide, the simple light-driven proton pump bacteriorhodopsin, and the superoxide scavenger superoxide reductase. In conclusion, likely future developments of these methods at synchrotron X-ray sources and the potential impact of emerging X-ray free-electron laser facilities are speculated upon. Currently approximately 20 new structures are deposited daily within the Protein Data Bank, adding to an accumulated total of more than 60\u2005000 structural entries. This mass of structural knowledge has provided detailed biological insight into protein structure and function relationships and uncountable details into the specific biochemistry of cellular reactions. Despite this impressive history of success, understanding the details of protein conformational changes presents a major challenge for structural biology. This is because X-ray diffraction, by far the most successful technique for elucidating new protein structures at high resolution, intrinsically relies upon the presence of well ordered arrangements of identical copies of the same protein. Flexible regions of a protein are frequently not visible within a crystal structure and proteins known to display large levels of flexibility, such as membrane protein transporters and receptors : the first is that the maximum available X-ray flux from the synchrotron insertion device may be utilized; the second is that the broad spectrum of the X-ray beam enables a large number of full X-ray diffraction reflections to be collected without the need to rotate the crystal, which is essential when one wishes to record snapshots on sub-millisecond timescales. Nevertheless, this approach encompasses a large number of technical challenges which have been widely discussed at a given time delay following reaction triggering within three-dimensional crystals. An alternative approach to real-time room-temperature studies is to control the kinetics of the reaction by using temperature or the chemical environment, and thereby find conditions for which a large population of a desired intermediate becomes trapped within three-dimensional crystals. Once conditions have been identified under which the population of a specific intermediate builds up, standard monochromatic X-ray diffraction data can be collected. While conceptually less elegant than time-resolved Laue diffraction, intermediate trapping methods have proven very popular in the search to understand the structural pathways of protein-mediated reactions F obs \u2212 F calc electron density map) is visible close to the iron atom, indicating a reactive hydrogen peroxide species within the active site, but the resolution of the electron density map does not allow an atomic model to be built without additional chemical restraints. To address this issue, off-resonance Raman spectra were recorded from both crystals and solutions of superoxide reductase treated with hydrogen peroxide show very good agreement between the low-temperature structure -induced conformational changes in bacterio\u00adrhodopsin [see Hirai & Subramaniam 2009 and Ande al. 2009 for over4.a).Although time-resolved Laue diffraction and intermediate trapping studies have provided several detailed insights into protein-mediated reactions, these methods necessarily probe protein conformational dynamics within the crystalline state. Structural probes applicable to liquid phases would explicitly side-step this fundamental limitation of time-resolved crystallographic studies of proteins. Time-resolved wide-angle X-ray scattering is an emerging technique that addresses this shortcoming, whereby X-ray scattering data are recorded as a function of time from an ensemble of samples in a liquid environment. Thus the breakage and formation of chemical bonds within small molecules, or the rearrangement of secondary structural elements within proteins, can be visualized since the internal distances between the atoms of the sample change with time. On the other hand, all structural information accessible using wide-angle X-ray scattering is averaged over all orientations of a randomly ordered ensemble of molecules within the sample. As such, the level of structural detail that can be envisioned, even in principle, is significantly less than what can be gleaned using X-ray diffraction. The experimental set-up at the ESRF is shown in Fig. 4et al., 2001et al., 2004et al., 2005 et al., 2005et al., 2006et al., 2006et al., 2004et al., 2005et al., 2005et al., 2009et al., 2006et al., 2008et al., 2008Time-resolved wide-angle X-ray scattering was first successfully applied to probe the structural dynamics of a number of photosensitive small molecules in solution et al., 1998Time-resolved wide-angle X-ray scattering has also recently been extended to probe the conformational dynamics of proteins (Cammarata b)et al. (2008b b c)Difference time-resolved wide-angle X-ray scattering data recorded from solubilized haemoglobin are reproduced in Fig. 4t, following photoactivation. These data were fitted to three basis spectra and their characteristic time constants determined. Structural refinement against the intermediate and late state basis spectra records the modulation of an X-ray absorption spectrum in the energy region from 50\u2005eV to approximately 1000\u2005eV above the absorption edge, whereas X-ray absorption near-edge structure (XANES) focuses upon the smaller energy region up to approximately 50\u2005eV above the edge et al., 2005Myoglobin in complex with carbon monoxide again provided a convenient system for proof-of-principle X-ray spectroscopy studies of protein conformational dynamics about a metal centre. It is more than two decades since the first time-resolved X-ray absorption near-edge spectra of the photodissociation of carbon monoxide from the haem iron of myoglobin were described and Japan (http://www-xfel.spring8.or.jp/) and the first American XFEL facility has recently reported lasing at 8\u2005keV (http://lcls.slac.stanford.edu/). These fourth-generation X-ray sources promise extremely intense hard X-ray bursts of approximately 100\u2005fs in duration, and will thereby create new opportunities for imaging of biological molecules from extremely small samples are currently under construction in Europe (et al., 2005et al., 2008et al., 2007Within the realm of time-resolved pump\u2013probe structural studies, the most obvious benefit of these emerging sources will be their remarkable capability to probe the structural dynamics of light-driven reactions with a temporal resolution of 100\u2005fs. Several landmark measurements have already been reported on this timescale at linear-accelerator-based X-ray sources, including key technical innovations for synchronizing the femtosecond laser and X-ray pulses (Cavalieri et al., 2003et al., 2003et al., 2004et al., 2005et al., 2005et al., 2004et al., 2000et al., 2000While taking a very optimistic viewpoint of these emerging opportunities, it seems likely that several technical challenges will be encountered along this path. For example, when using pump\u2013probe methods to study protein reaction dynamics in the crystalline phase, it may well prove advantageous to use the full width of the background undulator spectrum of the XFEL to perform \u2018pink\u2019 time-resolved Laue-diffraction (Bourgeois et al., 2008et al., 2009et al., 2005et al., 2006et al., 2006Time-resolved wide-angle X-ray scattering (Cammarata In closing, the last decade has witnessed several significant technical improvements to existing methods, as well as the development of novel approaches, for elucidating the structural details of protein catalyzed biochemical reactions as a function of time. Even a conservative extrapolation of these recent developments can foresee intermediate trapping and time-resolved wide-angle X-ray scattering studies becoming increasingly widely applied approaches within structural biology. We also foresee that that domain of applications of time-resolved Laue diffraction and X-ray absorption spectroscopy will grow, albeit more slowly. Finally, the imminent application of ultra-fast extremely intense XFEL-generated X-ray pulses to probe the reaction pathways of light-sensitive proteins will offer unique opportunities, delivering many surprises as it opens new structural windows upon fundamental photochemical events on the timescale at which they\u00a0occur."} +{"text": "Indirect immunofluorescence with serum is used in the diagnosis of pemphigus. We report a case in whom blister fluid was used as the specimen for indirect immunofluorecscence. Immunofluorescence is one of the diagnostic tests done in cases of pemphigus. In 1941, Coons et al. developed the technique of immunofluorescence. In 1964, Beutner and Jordon used the indirect immunofluorescence (IIF) technique to demonstrate antibodies in the sera of pemphigus patients.[Pemphigus vulgaris is an autoimmune blistering disease characterized histologically by suprabasal blister and immunopathologically by A 30-year-old female a known case of pemphigus was biopsied for histopathology and direct immunofluorescence (DIF). Blister fluid along with serum was taken for indirect immunofluorescence (IIF). DIF showed strongly positive IgG deposits. Serial dilutions of blister fluid using phosphate buffered saline (PBS) were incubated with cryostat sections of normal skin (substrate). Subsequently sections were incubated with FITC-labeled anti-human IgG for 30 min at 37\u00b0C. Slides were washed with PBS and mounted in buffered glycerol. Fluorescent microscopy revealed moderately strong ICS deposits of IgG and focal weak ICS deposits of C3 Figures . IIF witet al.[et al.[et al.[Immunofluorescence is nowadays commonly used in the diagnosis of vesicobullous disorders. Direct immunofluorescence requires perilesional skin, whereas serum is used for indirect immunofluorescence. Blisters are formed as a result of a breakdown of tissue integrity and fluid accumulation in a specific compartment of the skin. In pemphigus, simple binding of the antibody to the ectodomain of the antigen (desmoglein 3) triggers blister formation, perhaps by impairing the function of the molecule. Blister l.[et al. comparedl.[et al. had desc"} +{"text": "The resulting cascade of reactions greatly magnifies melatonin's efficacy in reducing oxidative stress and apoptosis even in the presence of mitochondrial electron transport inhibitors. The actions of melatonin at the mitochondrial level are a consequence of melatonin and/or any of its metabolites. Thus, the molecular terrorism meted out by reactive oxygen and nitrogen species is held in check by melatonin and its derivatives.The intracellular environmental is a hostile one. Free radicals and related oxygen and nitrogen-based oxidizing agents persistently pulverize and damage molecules in the vicinity of where they are formed. The mitochondria especially are subjected to frequent and abundant oxidative abuse. The carnage that is left in the wake of these oxygen and nitrogen-related reactants is referred to as oxidative damage or oxidative stress. When mitochondrial electron transport complex inhibitors are used, e.g., rotenone, 1-methyl-1-phenyl-1,2,3,6-tetrahydropyridine, 3-nitropropionic acid or cyanide, pandemonium breaks loose within mitochondria as electron leakage leads to the generation of massive amounts of free radicals and related toxicants. The resulting oxidative stress initiates a series of events that leads to cellular apoptosis. To alleviate mitochondrial destruction and the associated cellular implosion, the cell has at its disposal a variety of free radical scavengers and antioxidants. Among these are melatonin and its metabolites. While melatonin stimulates several antioxidative enzymes it, as well as its metabolites (cyclic 3-hydroxymelatonin, N N-acetyl-5-methoxytryptamine) is an endogenously-produced molecule found throughout the animal kingdom from unicells to humans which is coupled to oxidative phosphorylation provides cells with their major means of generation of its energy requirements inhaled and eventually taken up by cells is processed in the mitochondrial ETC where it is converted to water following its four electron reduction. However, during this reductive process, partially reduced species of O2 are also produced including reactants that are reduced by one, two or three electrons, i.e., the superoxide anion (O2\u00b7\u2013) and hydroxyl radical (\u00b7OH) and one non-radical product, hydrogen peroxide (H2O2). Collectively, these agents are referred to as reactive oxygen species (ROS).The majority of molecular oxygen , respectively. Since H2O2 is the immediate precursor of the highly damaging \u00b7OH, it is imperative that H2O2 be removed from the intramitochondrial environment as quickly as possible. The major enzyme that accomplishes this is glutathione peroxidase (GPx), which metabolizes H2O2 to water and O2; in this process GPx also converts reduced glutathione (GSH) to its oxidized metabolite (GSSG). Given the major importance of GSH in mitochondrial physiology, it is essential that GSSG be reduced back to GSH; this is accomplished by glutathione reductase (GRd) . O2O2 from the mitochondrial environment is never complete and, via the Fenton reaction, some damaging \u00b7OH are always formed. Cellular organelles have no enzymatic means to remove \u00b7OH so it must either be neutralized by a free radical scavenger or it mutilates a bystander molecule. This carnage occurs in the immediate vicinity of where the \u00b7OH is formed because of its extremely rapid reaction rate; the damage is referred to as being site specific.The removal of H\u2013). Mitochondrial NO functions as a reversible antagonist of complex IV of the ETC by competing with O2 for its binding site. Usually tissue concentrations of NO and O2 are, respectively, in the ranges of 100\u2013500 nM and 10\u201330 \u00b5M. In these concentration ranges, NO causes roughly half maximal inhibition of mitochondrial respiration. Thus, NO is a physiological regulator of respiration and also of the rate of ATP synthesis ; two of these are nitric oxide (NO) and the peroxynitrite anion has been proposed to exist in mitochondria (mtNOS) . This latter product was identified by mass spectral analysis and carbon and proton-nuclear magnetic resonance (Tan 1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) , GPx and GRd. The SOD isoforms dismutate Oet al., et al., et al., et al., It has been known for more than a decade that the activities of the antioxidative enzymes mentioned above are heightened in the presence of exogenously administered, pharmacological doses of melatonin , in the production of this tripeptide antioxidant. Often, under elevated oxidative stress conditions, melatonin preserves intracellular GSH levels. This is not necessarily related to the ability of melatonin to stimulate \u03b3-GCS since the indoleamine could preferentially scavenger free radicals and thereby preserve basal intracellular GSH concentrations. The stimulation of \u03b3-GCS, as originally described by Urata et al., .One final aspect should be considered when melatonin's ability to attenuate molecular impairment due to ROS/RNS is discussed. It is what Hardeland refers tThis section briefly summarizes the multiple processes by which melatonin may restrict the destruction of molecules and organelles normally inflicted by ROS/RNS. Whereas these processes have all been documented in vivo, the significance of each in forestalling oxidative damage may be cell specific.Several drugs have been identified which are classified as mitochondrial poisons, i.e., they interfere with the transfer of electrons through the ETC. These drugs greatly exaggerate the escape of electrons into the mitochondrial intramembraneous space leading to a reduction of molecular oxygen and formation of radicals. Additionally, reduced oxidative phosphorylation precipitates a depletion of ATP Beal, . These cet al., et al., Rotenone, a specific inhibitor of complex I of the mitochondrial ETC, causes the generation of an excessive number of free radicals. The damage inflicted by this drug is believed to be a consequence of the \u00b7OH , strongly potentiated rotenone-mediated death of pheochromocytoma (PC12) cells, a response attenuated by melatonin. Furthermore, in the presence of melatonin, free radicals were not detected to be released from PC12 cells co-exposed to rotenone plus the Ca2+ ionophore. Since melatonin did not change the concentration of Ca2+ nor did it prevent the inhibitory effect of rotenone on mitochondrial complex I, the authors concluded that the beneficial effects of melatonin on the mitochondria were primarily related to the antioxidative and free radical scavenging capacity of the indoleamine . It was immediately suspected that the drug caused damage to the dopaminergic cells of the substantia nigra which are the major neurons lost in individuals with idiopathic PD. When these individuals died and the brain was examined there was, in fact, a selective destruction of the mesencephalic dopamine-containing neurons. As tragic as these instances were, the identification of this destructive drug provided experimentalists with an agent that causes parkinson-like signs in animals. Use of MPTP has now become a model of examine the processes of PD as well as to investigate drugs that may modify the course of the disease.+). The latter molecule is then released from the glial cells and is taken into dopaminergic nerves via the dopamine transporter. MPP+ interferes with complex I of the mitochondrial ETC; this leads to cellular energy depletion and eventually to the death of dopaminergic cells.When administered to animals, MPTP is taken up by astrocytes surrounding dopaminergic neurons and terminals where it is metabolized to 1-methyl-4-phenylpyridinium was increased by melatonin treatment. The reduction in cyanide-mediated neural toxicity by melatonin was assumed to be related to the free radical scavenging activity of the indoleamine.Yamamoto and Tang performeWhen cultured rat cortical neurons were exposed to potassium cyanide over a range of concentrations (0.01\u20131.0 mM) lactate dehydrogenase efflux, indicative of cellular damage, into the culture medium was observed. Melatonin significantly reduced escape of the enzyme from the neurons and preserved their morphology opening, cytochrome c release, positive YOPRO-1 staining of the early apoptotic nuclei and DNA laddering. Besides inhibiting the apoptotic events initiated in astrocytes by H2O2, melatonin also reduced free radical formation and other degenerative cellular processes that resulted from the exposure of cells to either tert-butyl hydroperoxide or cumene hydroperoxide. Additionally, the protective effect of melatonin against these damaging agents was better than that provided by vitamin E (Jou et al., In a second, more complete study, this group examined in detail many aspects of apoptosis and showed that melatonin also prevented death of cells caused by the oxidizing agent, Het al., et al., et al., The greater efficacy of melatonin in reducing observable free radical generation and apoptotic processes than vitamin E is a common observation. A number of studies have compared melatonin with classic antioxidants, e.g., glutathione, mannitol, vitamin C, vitamin E, etc., and invariably melatonin performs in a superior manner (Sofic et al., According to Jou and colleagues the openet al. (Besides reducing free radical-mediated, mitochondrial-dependent apoptosis, a process that does not require an interaction of melatonin with a receptor, melatonin may also have a receptor-mediated means of reducing the likelihood of apoptosis. Kilic et al. examinedet al. concludeet al., et al., et al., et al., et al. (L and the reduction in cytochrome c release from mitochondria by melatonin in the damaged newborn rat brain.A follow-up study by the same group that melatonin acts to preserve the function of the PI-3 K/Akt pathway (Kilic , et al. who repoMitochondria are the site of a large percentage of free radicals and related toxicants that are generated in cells. These reactive products cause damage to essential mitochondrial molecules which result in opening of the mitochondrial transition pore, release of cytochrome c and activation of the down stream events that culminate in free radical-mediated, mitochondrial-dependent apoptosis.Since melatonin was discovered to be an indirect antioxidant and direct efficient free radical scavenger, its ability to reduce oxidative stress and to curtail cellular apoptosis has been repeatedly documented. It has also been shown that part of melatonin's ability to quell the oxidation of key molecules stems from its conversion to metabolites, i.e., cyclic 3-OHMEL, AFMK and AMK when it incapacitates free radicals and their related products.The ability of melatonin to protect against oxygen- and nitrogen-based reactants is obvious in situations where toxins that inhibit the mitochondrial electron transport complexes are used. The mitochondrial poisons cause electron leakage with the resultant formation of large numbers of free radicals and consequentially molecular damage. This mutilation is inhibited by melatonin and its metabolites.et al., et al., et al., et al., et al., et al., et al., et al., et al., While melatonin readily resists mitochondrial oxidative damage and cellular apoptosis, there are many unanswered questions remaining. Some of the most noteworthy relate to the intramitochondrial concentrations of melatonin, the precise location of the indoleamine in relation to the complexes of the ETC, is it melatonin or a melatonin derivative that is the active agent in mitochondria and a definitive explanation for its high efficacy in preventing mitochondrial and cellular free radical-mediated destruction. Melatonin's very low toxicity combined with its high efficacy, however, portends its use in clinical medicine to treat conditions that are associated with elevated free radical damage, e.g., septic shock (Gitto"} +{"text": "Human Mutation by Gottlieb et al. [BAK1 genotype among abdominal aortic anerurysm (AAA) patients and some controls. In particular, sequencing revealed a statistically significant number of instances in which single nucleotide polymorphisms (SNPs) present in tissue samples from patient abdominal aorta (AA) were absent from matching blood samples. The authors proposed a novel hypothesis \u201cpostulating that multiple variants of genes may preexist in \u2018minority\u2019 forms within specific non-diseased tissues and be selected for, when intra- and/or extra-cellular conditions change\u201d [Gottlieb et al., An article published in b et al. receivedThe implications of this unusual finding are of potential significance as regards the role somatic mutation and intraorganismal selection might play in genetic disease, not to mention the suitability of using blood-derived genomic DNA to evaluate patient mutational status of genetic diseases that affect various tissues. The work was challenged in a Letter to the Editors by Hatchwell , who argIn the current issue, K\u00fcry et al. discuss Human Mutation, we feel that discussion of the original article by Gottlieb et al. [These four letters were all carefully peer reviewed and evaluated editorially, but within the limitations of the rather constrained \u201cLetter to the Editors\u201d format. As the Managing Editor and Co-Editors of b et al. and its"} +{"text": "Phantom breast syndrome is a type of condition in which patients have a sensation of residual breast tissue and can include both non-painful sensations as well as phantom breast pain. The incidence varies in different studies, ranging from approximately 30% to as high as 80% of patients after mastectomy. It seriously affects quality of life through the combined impact of physical disability and emotional distress. The breast cancer incidence rate in India as well as Western countries has risen in recent years while survival rates have improved; this has effectively increased the number of women for whom post-treatment quality of life is important. In this context, chronic pain following treatment for breast cancer surgery is a significantly under-recognized and under-treated problem. Various types of chronic neuropathic pain may arise following breast cancer surgery due to surgical trauma. The cause of these syndromes is damage to various nerves during surgery. There are a number of assumed factors causing or perpetuating persistent neuropathic pain after breast cancer surgery. Most well-established risk factors for developing phantom breast pain and other related neuropathic pain syndromes are severe acute postoperative pain and greater postoperative use of analgesics. Based upon current evidence, the goals of prophylactic strategies could first target optimal peri-operative pain control and minimizing damage to nerves during surgery. There is some evidence that chronic pain and sensory abnormalities do decrease over time. The main group of oral medications studied includes anti-depressants, anticonvulsants, opioids, N-methyl-D-asparate receptor antagonists, mexilitine, topical lidocaine, cannabinoids, topical capsaicin and glysine antagonists. Neuromodulation techniques such as motor cortex stimulation, spinal cord stimulation, and intrathecal drug therapies have been used to treat various neuropathic pain syndromes. One in eight women will develop breast cancer of which approximately 60% are treated surgically for axillary node staging and primary breast tumor resection. It is estimated that over 50% of women suffer chronic pain following treatment for breast cancer surgery. \u201cPhantom Breast Syndrome\u201d (PBS) is a type of condition in which patients have a sensation of residual breast tissue and can include both non-painful sensations as well as phantom breast pain. Patient may have pain and discomfort, itching, pins and needles sensations, tingling, pressure, burning, and throbbing. The syndrome can start even after more than one year of surgery. The incidence varies across different studies, ranging from approximately 30% to as high as 80% of patients after mastectomy. PBS can et al.,[et al.,[et al.,[et al.,[et al.'s review, there were 21 studies with follow-up periods from 1-96 months (one study of 210 months), which revealed the following widely varying ranges of prevalence estimates: Phantom breast pain 3-44%, intercostobrachial neuralgia (ICN) 16-39% for all breast cancer surgery, ICN in breast-conserving surgery 14-61% and neuroma pain 23-49%.[et al.,[et al.,[In a recent review, Jung et al., found th,[et al., suggeste,[et al., distingu,[et al., revealed,[et al.,6 Inciden,[et al., Neuroma ,[et al., Other ne,[et al., found thThere are a number of assumed factors causing or perpetuating persistent neuropathic pain after breast cancer surgery. There is, however, a lack of large-scale multiple risk factor studies identifying the variables as independent risk factors or evaluating their relationships with other variables, which are known to affect the development of chronic pain. From the literature currently available, the most well-established risk factors for developing phantom breast pain and other related neuropathic pain syndromes are severe acute postoperative pain and greater postoperative use of analgesics.9 These aUnderlying each of the four classifications of pain after breast cancer surgery is damage to various nerves during surgery. Nerve preservation approaches have shown reduced incidence of sensory deficits (53% vs 84% of women) but nerve-sparing is only successful in 65% of the cases where it was attempted. EvidencePhantom breast pain and other pain syndromes severely affect the quality of life of patients. The negative impact on a patient's physical and psychosocial functioning is consistent with many chronic and cancer pain syndromes. It has been reported that up to half of patients report negative impact of pain on their activities and up to one-quarter report moderate to high impact on their daily activities at home and work. Not surprisingly, studies have also found that breast cancer surgery patients with chronic pain have a greater psychological stress and psychiatric morbidity than the general population.12Based upon current incomplete evidence, the goals of prophylactic strategies could first target optimal perioperative pain control and minimizing damage to nerves during surgery. Peri-operative Pain Control: Medications traditionally used for persistent neuropathic pain such as topical EMLA, gabapent16et al.,[There is some evidence that chronic pain and sensory abnormalities do decrease over time. Unfortunet al., found thet al.,[Well-established PBS needs treatment. In 2005, Finnerup et al., reviewedFor persistent post-breast cancer surgery neuropathic pain, there exist very few randomized, double-blind, placebo-controlled trials. The use of topical capsaicin23 or amiHowever, many unanswered questions remain about the optimal doses, timing and coordination of therapy with ongoing adjuvant treatment for breast cancer. There are also a number of medications and multidisciplinary approaches showing benefit for other types of neuropathic pain that have yet to be trialed. In women with early-onset or established post-breast cancer surgery neuropathic pain, neuromodulation techniques such as motor cortex stimulation,26 spinalThe small scale of existing studies in persistent pain after breast cancer surgery creates considerable uncertainty regarding the generalizability of their findings, and also regarding the identification of potentially modifiable risk factors. Recent improvements in neuropathic pain screening tools now make early identification and syndrome classification of neuropathic pain more achievable. Many imp"} +{"text": "Recent evidences prove that, release of potent lysosomal enzymes e.g. \u03b2-Glucuronidase by degranulation of polymorponuclear leukocytes in host gingiva may contribute significantly to tissue destruction and the pathogenesis of periodontal disease. The purpose of the present study was to compare and correlate GCF \u03b2-Glucuronidase with periodontal status among diabetic and non-diabetic patients with chronic periodontitis. A total number of 75 patients were equally divided into Group I (control group), Group II (non diabetic with chronic periodontitis) and Group III (diabetic with chronic periodontitis). Clinical parameters like Plaque index, Gingival index, Probing Pocket Depth and RBS were recorded. The \u03b2-Glucuronidase level in GCF of all three groups was determined by spectrophotometric analysis. It was observed that the periodontitis patients irrespective of their diabetic status, showed increased periodontal destruction with elevated level of \u03b2-Glucuronidase than the controls. Also, the diabetic patients showed increased severity of periodontal destruction and the elevated level of \u03b2-Glucuronidase, thus indicating diabetics at a higher risk for progressive periodontal destruction. Much effort has been made in recent years to identify risk factors responsible for initiation and progression of periodontal diseases. Mounting evidences indicate that, gingivitis and periodontitis are caused by various host responses which are associated with the continuous presence of microorganisms in the gingival crevice. They mayGingival Crevicular Fluid (GCF) provides a non invasive means of studying the host response factor by change of constituents in the fluid. The inflammatory exudates from gingival microcirculation crosses inflamed periodontal tissue and en route collects molecules of potential interest from the local inflammatory reaction. Therefore, the fluid offers a great potential source of factors like enzymes that may be associated with tissue destruction.\u03b2-Glucuronidase is a neutrophil derived lysosomal acid hydrolase enzyme which is stored in the neutrophil primary (azurophil) granules and is released in response to inflammation during periodontal destruction. This enzyme is active in degradation of proteoglycans and the ground substance and is an indicator of neutrophil (PMN) influx into the crevicular environment. Lamster in a recPeriodontal disease has been shown to be more severe in diabetics as compared to non-diabetics. SignificAlthough, a positive relationship of \u03b2-Glucuronidase level and traditional clinical parameters for diagnosis of periodontal disease has been widely studied, but there are inadequate number of studies for comparing \u03b2-Glucuronidase level as a potential marker for disease activity in diabetic and non diabetic patients suffering form chronic periodontitis. Hence, the present study was conducted with the following aims and objectives:To compare GCF \u03b2-glucuronidase activity with periodontal status among diabetic and non-diabetic patients.To correlate GCF \u03b2-glucuronidase activity with periodontal status among diabetic and non-diabetic patients.The patients for this study were selected from the Department of Periodontology and Implantology, Bapuji Dental College and Hospital, Davangere, Karnataka State.Patients with good oral hygiene and clinically healthy periodontium (Group I).Non-diabetic patients having probing pocket depth of 5-9 mm with a radiographic evidence of bone loss on at least 2 teeth, in minimum of two quadrants (Group II).Diabetic patients having probing pocket depth of 5-9 mm with a radiographic evidence of bone loss (Group III) on at least 2 teeth, in minimum of two quadrants.Patients with history of intake of any antibiotics and regular use of anti-inflammatory medication or the use of other drugs known to affect the periodontium, in past 6 months.Patients who had professional teeth cleaning or any other periodontal treatment within last 6 months.Patients who were unable to perform routine oral hygiene procedures.A total number of 75 patients, in the age group of 30-60 years were selected for this study. The selected patients were divided into three groups as follows:Group I: (Control Group) consists of 25 patients with clinically healthy periodontium and good gingival status .Group II: Consists of 25 non-diabetic patients with chronic periodontitis having 5-9 mm probing pocket depth and radiographic evidence of bone loss.Group III: Consists of 25 diabetic patients with chronic periodontitis having 5-9 mm probing pocket depth and radiographic evidence of bone loss.The following clinical parameters were recorded after the selection of test sites.Plaque Index (PI) (Silness and Loe 1964)Gingival Index (GI) (Loe and Silness 1963)Probing Pocket Depth (PD) was measured by using William's graduated periodontal probe, held parallel to the long axis of tooth from free gingival margin to base of pocket.Random blood sugar (RBS) level was recorded for Group II and Group III patients.The rate of hydrolysis of phenolphthalein glucuronidase serves to assay the activity of \u03b2-Glucuronidase.The phenolphthalein liberated is estimated by the red colour which it gives, at alkaline pH. Phenolphthalein glucuronide has hardly any absorption at the same pH.A single test site was selected from each patient of Group I, II and III. After light supragingival scaling, a standard volume of 0.5 \u00b5l GCF was collected extrasulcularly from isolated test site . Collectet al.[The \u03b2-Glucuronidase level in GCF was estimated by using the reagent kit supplied by Sigma Diagnostics and specet al.Between the study groups, a significant increase in PI, GI, PD, RBS and \u03b2-Glucuronidase level was observed . On compPeriodontal disease process is site specific and has multifactorial origin where periodontal pathogens, host response, genetic, systemic and behavioral risk factors interplay to develop the disease process. Hence, various measures have been taken to include microbial, immunologic, systemic, genetic and behavioral factors, in addition to traditional clinical and radiographic parameters, for assessing patient's periodontal status.The activity of \u03b2-Glucuronidase is associated with severity of inflammation and also with the presence of putative pathogenic flora. This enzyme has been detected in GCF of patients with active site of periodontal disease, thus making \u03b2-Glucuronidase as an important biochemical marker for disease activity and for predicting future attachment loss.Diabetes mellitus encompasses a heterogenous group of disorders with common characteristics of altered glucose tolerance and impaired lipid and carbohydrate metabolism. Diabetes develops from either deficiency in insulin production or an impaired utilization of insulin.Diabetes is linked to severe periodontal destruction. Uncontrolled diabetic frequently show a combination of inflammatory and degenerative changes ranging from a mild gingivitis to advanced chronic periodontitis with a widened periodontal ligament, exudation from periodontal pockets, and/or multiple lateral periodontal abscesses. Although it may be more severe in nature, the periodontal disease of the diabetic seems to be clinically similar to that found in non-diabetic individuals. This suggests that the diabetic state serves as a predisposing factor which can accelerate periodontal destruction originated by microbial agents.In the present study, we have attempted to test the hypothesis that \u2018diabetics with chronic periodontitis have higher levels of disease activity markers in GCF as compared to non-diabetics with chronic periodontitis\u2019. The probing pocket depth in chronic periodontitis patients with or without diabetes was standardized, so that these parameters did not have any bearing on the level of activity of GCF \u03b2-glucuronidase, thus allowing comparisons to be made between \u03b2-glucuronidase activity of diabetic and non-diabetic patients. Also, this study was aimed to determine \u03b2-glucuronidase activity in host tissues and not from the bacterial origin hence acidic pH was used to detect activity of enzyme.et al.[In this study, plaque index, gingival index and probing pocket depth values were significantly lower in Group I, as compared to Group II and Group III patients. Though there was statistically non-significant difference in plaque index and gingival index in between Group II and Group III patients, the average probing pocket depth was significantly higher in Group III as compared to Group II. These findings are consistent with that of Oliver and Tervonen and Lamset al.The relationship between oral hygiene to periodontal disease has long been well established in the dental literature. In this study, with respect to oral hygiene (Plaque Index) and gingival status , the values in diabetic and non-diabetic patients with chronic periodontitis (Group III and II) were statistically not significant. However, when compared with Group I patients, these values were significantly higher.et al.[In Group I , low levels of \u03b2-glucuronidase activity could be demonstrated, similar to the findings of Lamster et al. In theirP < 0.001), as compared to healthy individuals, showing highly significant correlation between GCF \u03b2-glucuronidase level and probing pocket depth. These results are consistent with the findings of Bang et al.[et al.[This study demonstrates that there is an increase in GCF \u03b2-glucuronidase level in Group II patients i.e. non diabetic with chronic periodontitis . This finding is in accordance with study by Bacic et al.,[The present study also demonstrates that there is higher level of GCF \u03b2-glucuronidase activity in patients of diabetes with chronic periodontitis as compared to non-diabetic with chronic periodontitis (t=2.09, c et al., who showc et al., demonstret al,[et al,[Shlossman et al, stated tl,[et al, claimed et al,[et al,[It is also observed that there was a significant positive correlation between GCF \u03b2-Glucuronidase and RBS levels, in diabetics with chronic periodontitis. This observation is comparable to the findings of Oliver et al, who demol,[et al, did not Another important observation made in the present study was that even with difference in clinical parameters between study groups, periodontitis patients irrespective of their diabetic status, showed an increased periodontal destruction with elevated \u03b2-Glucurondiase level than control. This suggests that \u03b2-Glucuronidase level can be used as a biochemical marker for chairside diagnostic kit, which can diagnose early phases of disease with reasonable confidence.Following conclusions were drawn from the present study:Periodontitis patients irrespective of their diabetic status, showed increased periodontal destruction with elevated levels of \u03b2-Glucuronidase, than in the control group.Diabetic patients had highest level of \u03b2-Glucuronidase level and increased severity of periodontal destruction. This confirms the fact that diabetes is a risk factor for periodontal disease.The presence of \u03b2-Glucuronidase level in GCF can be used as a biochemical marker for diagnosing the chronic periodontitis cases."} +{"text": "Sir,et al based upon our recent report as the fourth-line therapy. After 8 weeks, a 24% reduction in tumour size per RECIST criteria was observed. The pre-treatment (A) and post-treatment (B) computed tomography images are shown in accompanying figure. The subsequent treatment course was complicated by cholangitis and radiographic progression occurred after 20 weeks.In the commentary by Santini et al that further prospective studies are needed to determine the role of anti-EGFR therapy in SBA. In an attempt to build upon our previous work with the combination of capecitabine and oxaliplatin, CAPOX (We agree with Santini"} +{"text": "In striated muscle, the actin cytoskeleton is differentiated into myofibrils. Actin and myosin filaments are organized in sarcomeres and specialized for producing contractile forces. Regular arrangement of actin filaments with uniform length and polarity is critical for the contractile function. However, the mechanisms of assembly and maintenance of sarcomeric actin filaments in striated muscle are not completely understood. Live imaging of actin in striated muscle has revealed that actin subunits within sarcomeric actin filaments are dynamically exchanged without altering overall sarcomeric structures. A number of regulators for actin dynamics have been identified, and malfunction of these regulators often result in disorganization of myofibril structures or muscle diseases. Therefore, proper regulation of actin dynamics in striated muscle is critical for assembly and maintenance of functional myofibrils. Recent studies have suggested that both enhancers of actin dynamics and stabilizers of actin filaments are important for sarcomeric actin organization. Further investigation of the regulatory mechanism of actin dynamics in striated muscle should be a key to understanding how myofibrils develop and operate. \u00a9 2010 Wiley-Liss, Inc. Actin is one of the major cytoskeletal proteins in eukaryotic cells and plays essential roles in a number of cellular processes including cell migration, cytokinesis, vesicle transport, and contractile force generation . However, the function of CAS-1 is currently unknown.Proteins that bind to G-actin influence actin dynamics by altering polymerization/depolymerization kinetics and exchange rates of actin-bound ATP or ADP [Paavilainen et al., C. elegans striated muscle, tropomyosin functions antagonistically to ADF/cofilin and AIP1 to stabilize sarcomeric actin organization [Ono and Ono, TPM1 cause hypertrophic and dilated cardiomyopathies [Thierfelder et al., TPM2 cause nemaline myopathy [Donner et al., TPM3 cause nemaline myopathy [Laing et al., TPM3 causes nemaline myopathy and reduces affinity of tropomyosin with tropomodulin, a pointed-end capping protein, thereby potentially influencing the actin dynamics at the pointed ends [Akkari et al., Proteins that bind to the side of actin filaments are integral components of sarcomeric thin filaments and commonly stabilize actin filaments. Tropomyosin is perhaps the best characterized side-binding protein that regulates muscle contraction as well as actin filament stability [Gunning et al., NEB) cause nemaline myopathy [Pelin et al., Nebulin is a large protein (600\u2013800 kDa) that has been proposed to be a ruler that determines lengths of sarcomeric thin filaments [McElhinny et al., MYOT) cause myotilinopathy that includes three known types of skeletal muscle diseases: limb girdle muscular dystrophy type 1A, myofibrillar myopathy, and spheroid body myopathy. Myotilinopathy patients commonly exhibit progressive disorganization of sarcomeres with disarrayed Z-bands [Olive et al., Several actin-binding proteins with multiple immunoglobulin-like (Ig) repeats are expressed in striated muscle and associated with myofibrillar actin filaments. The palladin/myopalladin/myotilin family of Ig-repeat proteins localize to the Z-band and play important roles in sarcomeric organization [Otey et al., Drosophila and C. elegans. Kettin is associated with actin filaments during early myofibrillogenesis in Drosophila muscle [Ayme-Southgate et al., sallimus (sls) gene that also encodes zormin and connectin/titin by complex alternative splicing [Machado and Andrew, sls mutant phenotypes is complicated due to defects in multiple sarcomeric proteins. By contrast, C. elegans kettin is encoded by ketn-1 that is a separate gene from other connectin/titin-related genes in C. elegans [Ono et al., C. elegans kettin by RNAi causes disorganization of sarcomeric actin filaments when muscle is subjected to chemically induced hypercontraction, suggesting that kettin is important for stability of sarcomeric actin during contraction. C. elegans kettin cooperates functionally with \u03b1-actinin and ALP-enigma [Ono et al., Kettin is a large protein of 500\u2013700 kDa with 30-35 Ig-repeats that is specifically present in invertebrate striated muscle. Kettin directly binds to actin filaments and localizes to the I-band and Z-band [Lakey et al., Xin is a muscle-specific protein in vertebrates that stabilizes actin filaments. Xin contains 15\u201328 repeats of 16-amino-acid sequence, called Xin repeats. Xin repeats directly bind to actin filaments in vitro and stabilize actin filaments in stress fibers when Xin repeats are expressed in cultured smooth muscle cells [Pacholsky et al., C. elegans cause disorganization of sarcomeric actin filaments in body wall muscle, but this phenotype is suppressed when muscle contraction is reduced, suggesting that UNC-87 stabilizes actin filaments during actomyosin contraction [Goetinck and Waterston, C. elegans does not have nebulin or Xin, UNC-87 might be a functional homologue of these actin stabilizing proteins in striated muscle.UNC-87 is a calponin-like protein that stabilizes actin filaments and is found only in nematode muscle [Goetinck and Waterston, Both pointed and barbed ends of sarcomeric actin filaments are capped by capping proteins. However, as described above, sarcomeric actin filaments are capable of exchanging actin monomers at both ends, indicating that the filaments have dynamic caps rather than permanent caps. The barbed ends of sarcomeric actin filaments are capped by CapZ and aligned in register at the Z-band [Casella et al., Drosophila [Bai et al., C. elegans [Stevenson et al., Tmod1 null cardiac myocytes develop fewer and thinner myofibrils than wild-type cells but with sarcomeric organization of uniformly aligned actin filaments [Ono et al., The pointed ends of sarcomeric actin filaments are capped by tropomodulin (Tmod) [Fowler et al., TPM3 causes nemaline myopathy and reduces the affinity of tropomyosin for Tmod [Akkari et al., C. elegans muscle, TMD-1 (C. elegans Tmod) appears to cooperate with ADF/cofilin and AIP1 in sarcomeric organization of actin filaments [Yamashiro et al., Drosophila sarcomere length short (SALS) functions to enhance elongation of thin filaments by antagonizing the activity of Tmod [Bai et al., Drosophila striated muscle, knockdown of SALS shortens the length of thin filaments, while overexpression of SALS elongates thin filaments from their pointed ends. The sals gene encodes two SALS isoforms that have one or two WASP homology 2 (WH2) domains. However, in vitro, a SALS fragment containing two WH2 domains binds to the actin pointed ends and inhibits elongation rather than promoting elongation as expected from the in vivo observations. Therefore, the precise mechanism of SALS-mediated thin filament elongation remains unclear. An ortholog of SALS appears to be absent in vertebrates and nematodes, and it remains to be determined whether a functional homolog of SALS exists in other organisms.Tmod cooperates with other actin-regulatory proteins and regulates length or stability of thin filaments. Tropomyosin binds to Tmod and enhances its capping activity [Fischer and Fowler, Drosophila SALS.Leiomodin (Lmod) is a Tmod-related protein with a unique C-terminal extension containing a poly-proline sequence and a WH2 domain [Conley et al., C. elegans, FHOD-1 is the only formin of the FHOD subfamily, and it is predominantly expressed in several types of muscle cells. FHOD-1-null mutant worms exhibit some thinner striated muscle cells than wild-type worms but show no major abnormalities in sarcomeric actin organization [Pruyne et al., As mentioned above, an actin nucleator for initial assembly of thin filaments in muscle cells has not been identified. The Arp2/3 complex nucleates branched actin networks [Goley and Welch, Drosophila embryonic muscle, nonmuscle myosin II (zipper) is required for formation of striated sarcomeres [Bloor and Kiehart, Myosin II is the force-generating enzyme in sarcomeres. It is also an important regulator of myofibril assembly. Pharmacological perturbation of myosin motor activity disrupts organized assembly of sarcomeric actin filaments [Soeno et al., Studies in live muscle cells have demonstrated that actin in sarcomeres is dynamic during assembly and even in mature myofibrils. A number of regulators of sarcomeric actin dynamics have been identified, and functional studies have revealed their important roles in myofibril assembly, sarcomere organization, and maintenance of myofibrils . Regulat"} +{"text": "Sir,et al. on the usage of silicone gel for the treatment of hypertrophic scars.[et al. mentioned that \u201cThe results of the self-drying silicone gel have been satisfactory.[et al. reported that \u201csilicone gel is able to reduce the formation of keloid and hypertrophic scars and the signs/symptoms associated with the healing process (paraesthesia, pulling sensation, alterations in colour.[et al. recently performed a comparative study among silicone gel, silicone gel sheeting and topical onion extract, including heparin and allantoin, for the treatment of postburn hypertrophic scars and concluded that \u201cSilicone products, either in gel or sheet, are superior to Contractubex in the treatment of the hypertrophic scar.[et al. also mentioned that \u201cThe therapist should select the most appropriate agent according to the patient\u2019s need and guidelines of these signs.[I read with great interest the report by Puri ic scars. Puri et sfactory.\u201d Indeed,n colour.\u201d Howeverhic scar.\u201d Karagozse signs.\u201d Further"} +{"text": "A century after Robert Koch linkedindividual cultured microbes to specific diseases (Koch's postulates), it hasbecome increasingly apparent that the complex community of microorganisms associated with the human body plays a key role in health and disease. The NationalInstitutes of Health recently announced the Human Microbiome Project as part ofthe NIH Roadmap for medical research, with a primary goal of advancing ourunderstanding of the relationships among host-associated microbial communities,health, and disease. Many physicians and researchers, however, have onlypassing familiarity with the concepts involved in the study and therapeuticmanipulation of complex microbial communities.This special issue was conceived toaccomplish several goals. We wanted to provide readily accessible overviews ofthe concepts and methods used in the study of complex microbial communities,and demonstrate how changes in indigenous microbial communities can play a rolein diseases such as antibiotic-associated diarrhea and bacterial vaginosis. Wealso set out to find examples of how probiotics can be used for the therapeuticmanipulation of the indigenous microbiota.Clostridium difficile infection through the lens of microbial ecology,\u201d by S. Walk and V. Young, discusses the role of thegut microbiota in antibiotic-associated diarrhea and Clostridium difficile infection while Y. Sanz et al., in the seventh article \u201cInsightsinto the roles of gut microbes in obesity,\u201d review the evidence for therole of gut microbes in obesity. In the eighth article \u201cThe human vaginal bacterial biota and bacterialvaginosis,\u201d by S. Srinivasan and D. Fredericks review the human vaginalbacterial microbiota and theninth article \u201cTemporal shifts in microbial communities in non-pregnantafrican-american women with and without bacterial vaginosis,\u201d by J. Wertzet al., examines this microbial community in the setting of bacterialvaginosis. The tenth article \u201cVaginal microbiota and the use of probiotics,\u201d by S. Cribby et al. discusses the use of probiotics toalter the vaginal microbiota. Finally, the eleventh article \u201cProbiotics and gastrointestinalinfections,\u201d by R. Britton and J. Versalovic, and the twelveth article\u201cProbiotic bacteria influence the composition and function of the intestinal microbiota,\u201d byP. O\u2019Toole and J. Cooney, summarize the potential role of probiotics toinfluence gastrointestinal health.We were fortunate to receive a strong collection of reviewarticles and primary research manuscripts to meet the goals of this specialissue. In the firstarticle \u201cConceptualizing the human microbiota: from multicelled organ toecological community,\u201d B. Foxman et al. present a novelconceptualization of the human microbiome that blends perspectives of epidemiologists,classical ecologists and infectious diseases physicians. The second article \u201cApplication ofecological network theory to the human microbiome,\u201d by J. Foster et al.,outlines how ecological network theory can be applied to studies of the humanmicrobiome, while the third article \u201cInteractions of the intestinal epithelium with the pathogen andthe indigenous microbiota: a three way crosstalk,\u201d by C. V. Srikanth and B. McCormick, review the interactions between epithelial pathogens and theindigenous microbiota in the mammalian gut. In the fourth article \u201cApplication of sequence-dependentelectrophoresis fingerprinting in exploring biodiversity and populationdynamics of human intestinal microbiota: what can be revealed?\u201d G. Huyset al. review the use of sequence-dependent fingerprinting methods for studyingthe structure and dynamics of complex microbial systems using the humanintestinal microbiota as an example. The fifth article \u201cEcological characterization of thecolonic microbiota of normal and diarrheic dogs,\u201d by J. Bell, employssuch a fingerprinting method to study the canine colonic microbiota. The sixth article \u201cEmerginginsights into antibiotic-associated diarrhea and"} +{"text": "Helicobacter pylori (HP)-independent growing tumors. However a few cases of regression of high-grade gastric lymphomas after the cure of Helicobacter pylori infection had been described.Treatment of primary gastric diffuse large B-cell lymphoma is still controversial. The treatment of localized disease was based on surgery alone, or followed by chemotherapy and/or radiotherapy. High-grade gastric lymphomas are generally believed to be We report here a case with primary diffuse large B-cell lymphoma that showed a complete pathologic remission after HP eradication and we reviewed the literature. A computerized literature reach through Medline, Cancerlit and Embase were performed, applying the words: high grade gastric lymphoma, or diffuse large B cell, MALT gastric lymphoma, DLBCLL (MALT) lymphoma and Helicobacter. Articles and abstracts were also identified by back-referencing from original and relevant papers. Selected for the present review were papers published in English before January 2007.Helicobacter pylori as first line therapy in high grade gastric lymphoma: 22 of a total of 38 enrolled patients obtained complete remission. Depth of gastric wall infiltration and clinical stage were important factors to predict the response to antibiotic therapy. Our case and the review of the literature show that high-grade transformation is not necessarily associated with the loss HP dependence. In early stage, for high-grade B-cell HP-positive gastric lymphomas, given the limited toxicity of anti-HP therapy, this treatment may be considered as one of the first line treatment options.Forty two cases of primary high grade gastric lymphoma that regressed with anti HP treatment were found. There were anedoctal cases reported and patients belonging to prospective studies; four trials studied the effect of eradication of Helicobacter pylori (HP) infection plays an important role in the development and growth of gastric mucosa-associated lymphoid tissue (MALT) lymphomas [ymphomas ,2. Eradiymphomas .in vitro study by Hussell et al [It has been demonstrated by laboratory and clinical studies that primary gastric large B cell MALT lymphomas are transformed, antigen independent, autonomously growing tumors that are unlikely to respond to eradication therapy of the HP infection. An ll et al showed tll et al ,6.et al [However anedoctal cases of primary gastric large B-cell lymphoma that responded to antibiotic therapy had been described and, more recently, Chen et al reportedWe report here a patient with diffuse large B cell lymphoma of the stomach, that achieved a complete pathologic remission after anti HP therapy and a detailed review of literature is also presented.In May 2003, a 43-year-old man was admitted for epigastric pain of two months duration and weight loss (more than 10% of the body weight). Clinical examination was unremarkable and laboratory data were within normal values; only a mild hypochromic anemia was disclosed (Hb 12.4 g/dl).Helicobacter pylori was identified in the mucosa. The previously reported diagnostic criteria for gastric diffuse large B-cell lymphoma were used [A gastroscopy was performed and revealed an ulcerative lesion in the gastric antrum ranging 3 cm in diameter. Biopsies established the diagnosis of diffuse large B cell lymphoma (DLBCL) of the stomach and ere used ,8 showed a hemicircumferencial thickness of the anterior gastric wall, which was infiltrated until to the serosa. Staging was completed with neck, chest and abdominal computed tomography and with bone marrow biopsy. There were not other lymphoma-deposits outside the stomach, and a clinical stage E IThe patient refused chemotherapy and a surgical treatment was then planned. Waiting this treatment, the patient underwent an HP eradication therapy. He received a triple therapy with omeprazole (20 mg twice a day), amoxicillin (1 g twice a day) and clarithromycin (500 mg twice a day) for seven days, and after that omeprazole (20 mg every day) for other 21 days.Prior to surgery, the patient underwent repeat gastroscopy (a month later) that showed a clear improvement of the ulcerative lesion of the gastric antrum and biopsies showed a complete disappearance of the lymphoma Figure .The patient was informed of the good results from anti HP therapy but he preferred to undergo to subtotal gastric resection. The histological examination revealed complete remission of the lymphoma and absence of Helicobacter pylori. He did not receive additional treatment and is in continuous complete remission after 42 months.We selected all cases reported with primary gastric large B-cell lymphoma treated with anti HP treatment and all cases of primary gastric large B-cell lymphoma treated in prospective studies with anti HP-therapy. According to the WHO classification, low grade MALT lymphoma with focal high grade component constituted by \"solid or sheet-like proliferations of transformed cells\" were included as diffuse large B-cell lymphoma .Tumors were staged clinically according to the modified by Musshoff and Schmidt-Vollmer, Ann Arbor Classification for extrHelicobacter pylori as first line therapy in gastric high grade gastric lymphoma: 22 of a total of 38 (57.9%) enrolled patients obtained complete remission. Data of the 42 responsive patients are reported in Table A total of 61 patients, including the present case, with primary gastric large B-cell lymphoma were treated with anti HP treatment ,10-25 anHelicobacter pylori in one patient) with remaining nodal disease. In one patient, large B cells disappeared, but areas of MALT lymphoma and nodal disease persisted. The patient with Burkitt-like lymphoma, obtained a complete remission.Different schedules of eradication treatment were used and were based on a proton pump inhibitor together with a combination of antibiotics . Forty-two of 61 patients obtained a complete remission of the lymphoma. In two patients there was gastric complete remission in the majority of patients; only in one patient, there was a progression of disease after an initial partial response .Four patients including present case underwent subtotal or total gastrectomy, after endoscopic confirmation that Helicobacter pylori infection was cured and lymphoma regressed ,22.Other patients in complete remission didn't undergo further treatment, except one patient with AIDS who relapsed after 6 months and needed chemotherapy. This is the only one relapse described. Two patients, in partial remission after eradication treatment, gained complete regression of lymphoma after chemotherapy ,22. Becaet al., [The median period of follow-up was 22 months. The longer period of follow-up was reported in the series of Chen et al., : all theInformation about genetics of large B cells didn't express bcl-6 and p53; in the patient with Burkitt-like lymphoma, malignant cells expressed bcl-6 and p53; in the with Burkitt-like lymphoma, malignant cells expressed bcl-6 and not bcl-2; in two patients there were not alterations of p53 and k-ras genes and microsatellite instability .et al., [et al., [et al., [et al., [In 20 patients, tumor response was unexpected, but in 22 cases it was obtained in prospective trials. Chen et al., reported[et al., 5 cases,[et al., 2 cases In the present case, eradication of HP infection obtained with a short course of antibiotic therapy resulted in a complete pathologic remission of a diffuse large B cell lymphoma of the stomach. This complete regression of the disease was confirmed not only by gastroscopy and biopsies but also by gastrectomy.Helicobacter pylori stimulation. This assertion was supported by in vitro and in vivo results.This finding confirms one more time that large B cell HP-positive gastric lymphomas are not necessarily associated with loss of HP dependence. Until few years ago, large B cell gastric lymphoma was considered independent of et al., [in vitro in response to Helicobacter pylori, as MALT lymphoma cells did.A study by Hussell et al., showed tIn vivo confirmation came from the fact that a number of cases of antibiotic-resistant MALT lymphoma contained large B cells in deep layers of the stomach and these cells were thought responsible for absent response of these tumors [et al., [Helicobacter pylori eradication could play a part in optimum treatment of an accompanying low grade component.e tumors ,6. Boot [et al., concludeet al., [et al., [Helicobacter pylori associated chronic active gastritis; she refused chemotherapy and received only eradication treatment with an unexpected tumor remission. These two cases were the first published cases of regression of large B cell lymphoma after eradication therapy. Afterwards analogous surprising situations were reported.In 1997, Rudolph et al., describe[et al., reportedet al., [et al., [et al., [et al., [et al., [et al., [et al., [et al., [et al., [Morgner et al., , collect[et al., , in 50% [et al., and in 5[et al., . Alpen e[et al., started [et al., and Hiya[et al., are a we[et al., . Alpen e[et al., in theiret al., [et al., [et al., [These authors paid attention to different prognostic factors. Hiyama et al., focused [et al., ; for Nak[et al., , 93% of In this review of the literature, age, sex, location of tumor and the presence or absence of areas of MALT lymphoma don't seem to influence the response of anti Helicobacter therapy. Clinical stage and depth of tumor invasion are the most important predictive factors of complete remission . HoweverHelicobacter pylori infection, to MALT lymphoma and large B cell lymphoma. There is consistent evidence for the clonal link between the small cell tumor and the large cell tumor [de novo [de novo. The absence of the low grade component could be due to sampling bias or to overgrowth by large cells [Helicobacter pylori eradication have a common genetic pattern and if cases without areas of MALT lymphoma are transformed lymphomas or de novo lymphomas.Very little is reported about genetics of these tumors ,29. Accoll tumor . This evll tumor . t disease was based on surgery alone, or followed by chemotherapy and/or radiotherapy, however recent studies showed that clinical outcome of localized gastric lymphoma treated by systemic chemotherapy alone was similar to that treated by surgery followed by chemotherapy in terms of tumor response, disease-free survival and overall survival suggesting that surgery be reserved for those with residual lymphoma after chemotherapy -38.According to this review, among patients with complete remission obtained after eradication therapy, only one patient, who was affected by HIV infection, relapsed. These data suggest that after a complete remission, no other treatment including gastrectomy might be necessary, even if full thickness of gastric wall is infiltrated at presentation.Helicobacter pylori eradication, but a late response of up to 45 months has been described [et al., [et al., [In most cases of gastric MALT lymphoma remission is achieved within 12 months after escribed . Among tescribed ,10,22. A[et al., extendedIn all cases that responded to eradication therapy, initial or complete regression of lymphoma was evident at the first endoscopic and histologic examination. Only in one case, there was a disease progression, after an initial response, at the second examination .Our case reported here and the review of the literature allow us to conclude that:1. Complete remission was obtained after HP eradication treatment in 42 of 61 patients with primary gastric HP related DLBCL.2. There is no marker that can predict if the tumor will regress after antimicrobial therapy. However, depth of gastric wall infiltration and clinical stage can strongly predict the probability of a complete remission, it must be emphasized that complete remission was reached in anecdotal cases independently of these factors after anti HP eradication.Helicobacter pylori infection a complete staging with endoscopic ultrasonography, computed tomographic imaging and bone marrow biopsy should be carried out and the patients should first be treated by anti HP treatment. An endoscopic revaluation 4\u20136 weeks after the eradication treatment should be performed. These evolutions of lymphoma can happen:From a practical point of view we suggest that all patients with primary gastric DLBCL associated with - No response or progression: patient must undergo to surgery or other conventional treatment.- Partial remission: lymphoma is probably responsive and could obtain a complete remission; patient must be strictly monitored to detect signs of progression or a complete remission.- Complete remission: patient must be strictly monitored but may not require further treatments.The author(s) declare that they have no competing interests.LC diagnosed and treated the patient, revised and finally approved the manuscript for been published, RP performed bibliographic research and participated in manuscript revision process, PS performed bibliographic research and participated in manuscript revision process, AZ and CP performed pathological diagnosis and histological pictures. All authors read and approved the final manuscript."} +{"text": "Recent advances in understanding the characteristics of renal cell carcinoma (RCC) have brought to our attention many prognostic markers that affect and predict the survival outcome of patients with the disease. For the moment, however, patients with RCC have not received any benefit from such markers. If a patient is diagnosed as \u201chigh risk\u201d by using such prognostic markers, there is no promising systemic therapy available. In this review we mainly focus on biomarkers of RCC that can be applied for therapeutic use reported in recent publications. Several issues and limitations in the reported studies are also highlighted and discussed. Developing biomarkers from the viewpoint of therapeutic application will lead to improvement of the prognosis of RCC patients. Renal cell carcinoma (RCC) is the most common malignant tumor of the kidney. It accounts for 3% of all adult malignancies and for approximately 95,000 deaths per year worldwide. More tha2Somatic and epigenetic mutations of the von Hippel-Lindau (VHL) disease tumor suppressor gene are observed in 42-57% and 5-19% of sporadic clear cell RCCs, respectively. Under hyet al.,[et al.,[Lidgren et al., demonstret al., Therefor,[et al., who demoet al.,[et al., showed that CAIX expression was a significant predictor in univariate and multivariate analyses and proposed accurate systems for predicting survival for patients with localized or metastatic RCC.[et al.,[et al.,[Carbonic anhydrase IX is one of the most validated prognostic biomarkers of RCC. Bui et al., performeatic RCC.13 They c.[et al., reported,[et al., detected,[et al., Carbonicet al.,[et al.,[et al.,[+ target cells and production of IFN-\u03b3 on stimulation with such cells in all patients. However, liver enzyme disturbances reached National Cancer Institute Common Toxicity Criteria Grades 2 to 4 in all three patients. They performed liver biopsy, which suggested that the liver toxicity was caused by a specific attack of the scFv(G250)+ T-cells against CAIX+ bile duct epithelial cells. Therefore, CAIX may not be an appropriate target for specific therapy.Recently CAIX-targeted therapies were reported in several studies. Uemura et al., discusse,[et al., investig,[et al., transducUpregulation of VEGF, the most potent growth factor for tumor vasculature, is significantly associated with upregulation of HIF-\u03b1. Renal cec in the cytoplasm.[c, released by tBID (truncated BH3-interacting-domain death agonist) from the mitochondria, binds and activates apoptotic protease activating factor-1 (Apaf-1). It forms a multiprotein caspase-activating complex (apoptosome) and leads to activation of caspase-9, undergoing autoactivation to promote recruitment and cleavage of caspase 3. Caspase 3 cleaves its target substrates to affect the changes associated with apoptosis.[Apoptosis is essential to sculpt the developing organism by removing outdated or unneeded structures and also central to the homeostasis of adult tissues by maintaining the balance between cell production and cell elimination. Since caytoplasm. Cytochropoptosis.c release by inhibiting the terminal effector caspase-3 and caspase-7 and interfering with caspase-9 activity and processing. It has also been reported to affect cell division, cell cycle progression, signal transduction pathways and protein degradation.[Inhibitor of apoptosis protein can inhibit apoptosis by binding to prevent a common step downstream of mitochondrial cytochrome radation. Eight huin vitro.[et al.,[et al.,[Although the expression of c-IAP1 and c-IAP2 is not tumor-specific, their overexpression can suppress chemotherapy-induced apoptosis in vitro. Kempkens.[et al., performe,[et al., reportedet al.,[et al.,[Survivin is overexpressed in various human malignancies and its expression is associated with features of biologically aggressive disease, resistance to therapy and poor clinical outcome in patients with various malignancies. Parker et al., demonstr,[et al., evaluate,[et al., However,et al.,[Smac (second mitochondria-derived activator of caspase)/DIABLO (direct inhibitor of apoptosis-binding protein with low PI) is a proapoptotic protein that in healthy cells resides in the intermembrane space in the mitochondria, but is released into the cytosol during apoptosis, where it interacts with IAPs and disrupts their ability to bind caspases . The balet al., evaluateIt is well known that RCC frequently harbors numerous tumor-infiltrating lymphocytes (TILs), suggesting that a host antitumoral immune response is stimulated by the malignant transformation of cells. However, there is a paradoxical relation between increased levels of TILs and diminished cancer-specific survival. Tumor-inet al.,[+ cells. Moreover, significantly fewer NK cells in peripheral blood, a lower proportion of CCR5/CXCR3/CD4+ cells and a higher proportion of CCR4/CD4+ cells were observed in patients with metastatic RCC in the study. These results indicate a change in helper T responses during the progression of RCCs. Donskov and von der Maase[+ cells were significant prognostic factors of poor survival both in univariate and multivariate analyses, whereas intratumoral macrophages, CD4+, CD8+, CD20+ and CD56+ cells were not significant ones. However, it is not yet clear why \u201clow\u201d CD57+ NK cells can be a prognostic factor for IL-2-based immunotherapy. Furthermore, there seems to be a discrepancy between the results of the two studies. Thus, the question remains: \u201cIs infiltration of NK cells into tumors a positive prognostic factor or a negative one?\u201dC\u00f3zar et al., evaluateder Maase analyzedNatural killer cells mainly kill tumor cells that have reduced major histocompatibility complex (MHC) Class I expression and can escape killing by cytotoxic T lymphocytes (CTLs). If the tumor cells have acquired escape mechanisms from CTLs, NK cell-infiltration can be a positive finding for suppressing the tumor. Therefore, it is important to evaluate the expression of MHC Class I molecules on RCC cells. We recently demonstrated that MHC Class I was down-regulated in 38% of conventional RCCs and the down-regulation was an independent prognostic factor. Unfortun+ T cells that coexpress CD25, the IL-2 receptor \u03b1-chain. In search of more specific markers for Treg, the transcription factor forkhead box P3 (FOXP3) has been identified. Forkhead box P3 is not only a key intracellular marker but also a crucial developmental and functional factor for Tregs. Siddiqui et al.,[+ CD25+ FOXP3\u2212 (but not CD4+ CD25+ FOXP3+) T cells was significantly associated with higher TNM stage, larger tumor size, the presence of coagulative tumor necrosis and poorer cancer-specific survival. Interestingly, they also showed that CD4+ CD25+ FOXP3\u2212 TILs expressed more IL-10 (cytokine synthesis inhibitory factor) than CD4+ CD25+ FOXP3+ cells, suggesting that FOXP3\u2212 Tregs have a powerful inhibitory function. Moreover, Dannuli et al.,[389 IL-2, acting like a CD25-specific antibody, reduced the number of Tregs present in the peripheral blood of metastatic RCC patients without severe side-effects and abrogated Treg-mediated immunosuppressive activity in vivo. They also demonstrated that the antitumor effects of DAB389 IL-2 followed by vaccination with RNA-transfected DCs significantly improved the tumor-specific T cell responses. Thus, depletion of Tregs is one of the strategies to suppress the progression of RCCs.Another topic of TIL is the regulatory T cell (Treg) that regulates the activation of other T cells and may be necessary to maintain peripheral tolerance to self antigens. One mechanism by which cancers evade immune destruction is by recruiting regulatory cells into the tumor microenvironment. Treg is i et al., demonstri et al., demonstret al.,[et al.,[The B7 family of molecules on antigen-presenting cells (APCs) is one of the best-defined costimulators for T cells. These molecules bind to the CD28 molecule on T cells and provide signals required for the activation of naive T cells. B7-H1, a member of the B7 family, can be induced on T lymphocytes, but aberrant expression on tumor cells has been described in various human malignancies. The expression of B7-H1 on tumor cells is considered to enhance apoptosis of activated tumor-specific T cells. Thompson et al., demonstret al., Furtherm,[et al., indicateet al.,[Here we will introduce two papers with high impacts on biomarker studies. Jiang et al., assessedet al.,[During the past decade, a large number of markers have been studied for their prognostic value in RCC. For example, molecular tumor proliferation markers, including Ki-67 (MIB-1), proliferation cell nuclear antigen (PCNA), topoisomerases and p100, have been investigated in many studies, but their value as prognostic markers is still controversial. Furthermore, urologists cannot apply most such results to improve the survival of RCC patients. Therefore, it is important to identify novel RCC-specific antigens. Recently, bioinformatical approaches have been used to search for such antigens. Yao et al., examinedDuring the past decade, a large number of proteins that are putatively important in carcinogenesis and cancer biology have been studied for their prognostic value in RCC, but their clinical use remains controversial. Recently, however, novel biomarkers have been identified by various methods and some of them have been verified as clinical predictors of prognosis of RCC patients . It is i"} +{"text": "Dear Editor,et al.,[I read the article by Dhingra et al., which deAutofluorescence in Inflammatory CNVMMany reports showed the utility of AF as a marker for early disease and in predicting the outcome of treatment in CNVM due to age-related macular degeneration. There are very few reports on AF findings in inflammatory CNVM.Inflammatory CNVM, which is usually of the classic type (type 2), is seen on AF photography as a precise hyper-autofluorescent area due to the hyperplastic retinal pigment epithelium (RPE). Following treatment, CNVM may contract and leave a zone of absent RPE causing a hypo-autofluorescence.Secondary choroidal neovascularization in multifocal choroiditis and panuveitis (MCP) is readily visible as a hyper-autofluorescencent area originating from a hypo-autofluorescent spot (scar). With increasing follow-up time, the hyper-autofluorescence associated with CNV decreases. AF imaging provides a method to image the appearance of new or enlarging spots that appear, which is more sensitive than using ophthalmoscopically visible signs.More studies are required to understand the role of AF in inflammatory diseases. AF imaging could be an important noninvasive tool to reduce the need for angiography and to help in early diagnosis, as also in the follow-up of inflammatory CNVM patients.Use of Anti Vegf0 in Inflammatory CNVMThere are potential disadvantages of treating inflammatory CNV with photodynamic therapy (PDT). PDT can cause localized inflammation and increase VEGF production. Local release of VEGF, after PDT, may be associated with a higher incidence of recurrent choroidal neovascularization (CNV).et al, studied 10 patients with CNV, who were refractory to previous immunosuppression and PDT or intravitreal triamcinolone (IVTA). They reported that intravitreal bevacizumab improved the best corrected visual acuity (BCVA) and reduced the central macular thickness in the eyes with 2.5 injections (mean), and this was seen at 7.5 months of follow up. In this study they included a patient who recurred three months after pegaptanib injection and responded well.[et al reported successful treatment of inflammatory CNVM with ranibizumab in a patient with multifocal choroiditis and pan uveitis (MCP), who did not responded to bevacizumab or the PDT and IVTA therapy.[Tran ded well. Fine et therapy.et al reported significant improvement of 2.2 lines in BCVA at 24 months, with a significant decrease in foveal thickness (265 microns), with only 1.3 injections (mean). This study confers the long-term benefits of intravitreal bevacizumab.[The recent and largest case series by Monsour ckness 26 microns,Serious side effects and chances of recurrence make PDT an obsolete treatment option. Recent promising results favor intravitreal bevacizumab as a primary / mono therapy in inflammatory CNVM."} +{"text": "The conference venue included two locations: the Lindy Boggs Conference facility at the University of New Orleans (UNO); and Dinner/Speaker venues in the French Quarter \u2013 Broussard's of New Orleans and The House of Blues. The conference featured three days of technical presentations, with the third day partly devoted to a satellite Conference on Nanopore Cheminformatics with an on-site demo presentation of a nanopore detector (from the Research Institute for Children and UNO Nanopore Cheminformatics/Biophysics Labs). The theme of this year's conference was \"Computational Frontiers in Biomedicine\".The Fourth Annual MidSouth Computational Biology and Bioinformatics Society (MCBIOS-IV) conference was held in New Orleans, Louisiana on February 1st place went to 1st: Mutlu Mete of UALR, 2nd place to William Sanders of MSU and 3rd place to Stephanie Byrum of the UALR. In Poster Session II, 1st place went to Matthew Landry of UNO, 2nd place to Terra Colvin, Jr. of UALR, and 3rd place to Iftekhar Amin of UNO. 1st place for outstanding oral presentation went to Vijayaraj Nagarajan of USM.At MCBIOS 2007, awards for outstanding poster presentations were given to the following students: In Poster Session I, 1This year, 24 out of 31 submitted papers were accepted for inclusion in the proceedings (77%), similar to the number published in last year's Proceedings . Each ofet al [One of the most important challenges of current miRNA research is to decipher the \"code\" of miRNA regulation \u2013 to find the connection between miRNA expression and phenotypic changes. Gusev et al report tet al study is that there is a large body of microarray data that is becoming available for analysis. As such, methods to begin inferring regulatory networks from this data are important. In another paper, Peng Li et al compare probabilistic Boolean Network (PBNs) and Dynamic Bayesian Network (DBNs) approaches to correctly inferring regulatory networks [One of the things evident from the Gusev networks . They fiet al [Enterococcus bacteria. A complex response was mapped out involving cytoskeletal rearrangement, immune response, modulation of growth and cellular metabolism, and Wnt signaling, as well as responses heretofore unrecognized because they involve poorly annotated genes.While microarray technology is steadily improving, it still suffers from noise; hence experiments are repeated several times to reduce error. To reduce the amount of replication necessary, Dozmorov et al used F-tet al [Biological analysis spans several different areas from the genome/proteome to the metabolome, collectively referred to as \"omics\" for the study of different biological bodies. In one study, Schnackenberg et al study agS. aureus. Their combination of methods suggests that Msa is membrane-bound with sites for phosphorylation and protein-binding, suggesting it plays a role in signal transduction, which is consistent with its known activity as a modulator of the protein SarA.Nagarajan and Elasri use bioiet al. [Not all peptide fragments are represented equally in mass spectrometry (MS) experiments. To help predict which peptides might be lost or underrepresented, Sanders et al. use artiBridges et al develop DNA-protein binding interactions: (1) TBP \u2013 TATA receptor binding, and (2) HIV Integrase \u2013 HIV DNA Terminus binding. The method is also effective at detecting DNA-DNA binding interactions that occur with annealing of DNA single-strand overhangs [protein-protein binding interactions for the medically important case of antibody-antigen interactions [Four papers explore a new transduction-based nanopore detector mechanism. The first introducverhangs as well ractions .clustering (unsupervised learning) \u2013 a marked departure from the standard supervised-learning approach to SVMs. The author's objective was to have a powerful, non-parametric, method for phase tracking on nanopore transduction signals, a key requirement for extracting binding kinetics from channel current signals. They also describe a new form of Hidden Markov Model (HMM) that has the strengths of the much more complex HMM-with-Duration (HMMwD) models, but at a computational cost approximating the simpler HMM [Pattern recognition is a critical part of making sense of the high-throughput data gathered in modern biomedical experiments. Four papers explore the development of machine learning based pattern recognition methods and their application to resolving complex nanopore-transduction detector signals. The first describepler HMM . The goapler HMM , examinepler HMM , exploreet al [Rather than focus on refinement of methods, several authors report the effectiveness and utility of existing bioinformatics approaches to better understand a biological system. For example, Nan Mei et al studied et al. [Guo et al. used micCircadian rhythms are generally associated with the sleep/wake cycle, but also regulate many activities and affect the expression pattern of practically all genes. Time-course microarrays can potentially detect this baseline oscillation, which Ptitsyn and Gimble use in aIdentification of DNA binding sites for transcription factors (motifs) is important for a complete understanding of co-regulation of gene expression, but has proven to be quite challenging. Das and Dai review pet al [Loganantharaj et al have proet al [Eisenia fetida, which is often used in toxicology studies. They describe the sequencing and analysis of 3,144 new ESTs.Pirooznia et al report tet al [The insertion of new or altered genes into genomes is a key step in many functional analysis studies, and it is important to determine how many copies of each of these transgenes are present. Yuan et al report tet al [k-median, but which uses statistical spatial depth to identify the \"center\" of a cluster. A new subcluster selection rule, Relative Average Depth, is also introduced. In data sets that are noisy or have high dimension and low sample size, which is common in gene expression data sets, the bisecting k-spatial median algorithm does well compared to the component-wise bisecting k-median algorithm.Ding et al propose et al [Cancer diagnosis usually begins with histopathological examinations of tissue biopsies. These evaluations are usually somewhat subjective and the growing number of such images has provides an opportunity to test automated approaches to tissue sample categorization. Mete et al report ard and 24th, 2008. Our web site, , contains further information on the society and future meetings. MCBIOS is a regional affiliate of the International Society for Computational Biology .The fifth annual MCBIOS Conference will be held in Oklahoma City, Oklahoma in the Cox Convention Center in downtown Oklahoma City on February 23The authors declare that they have no competing interests.All authors served as co-editors for these proceedings, with JDW serving as Senior Editor. All authors helped write this editorial."} +{"text": "In May 1998, three large outbreaks of salmonellosis, affecting 91 persons, were identified in the Republic of Georgia. Eighteen Salmonella Typhimurium strains were characterized by arbitrary primed polymerase chain reaction and pulsed-field gel electrophoresis; the results suggested that all cases were part of a single outbreak caused by a distinct clonal strain."} +{"text": "Molecular Neurodegeneration and Institute for Biomedical Research, Xiamen University co-organized the 2009 International Conference on Molecular Neurodegeneration in Xiamen, China on May 18-20, 2009. The objectives of this meeting were to (1) promote cutting-edge neurodegeneration research in China and in neighboring Asian countries; (2) facilitate the exchange of information relevant to neurodegenerative research; (3) provide education opportunity for students, postdocs and physicians; and (4) provide a platform for investigators at different career levels to interact and network, and to foster collaborations at the international levels. About 100 investigators presented their recent discoveries with a wide range of scopes of neurodegeneration research, including new genes, molecular pathways, animal models, and potential therapeutics.Age-related neurodegenerative diseases are great challenges as the aging population grows. To promote neurodegeneration research and to share recent progress in understanding molecular mechanisms underlying these devastating diseases, the journal As life expectancy continues to increase and a growing number of people enter the aged population, neurodegenerative diseases have increasingly become a major problem for which treatments are critically needed. Although much progress has been made in the past 20 years on understanding the mechanisms of neurodegenerative diseases and on developing novel therapeutic strategies, most of these achievements are from the United Stated and European countries. Neurodegeneration research in Asian countries, most of which are very populated and also suffered severely with the spread of neurodegenerative diseases, is lagged far behind. The 2009 International Conference on Molecular Neurodegeneration is designed to bridge this gap. The major aim of this meeting is to provide a unique platform for Asian researchers and scientists from Western countries to exchange information and ideas, to share their most recent research advances, and to establish future collaborations in neurodegeneration studies.Molecular Neurodegeneration , an open access, peer-reviewed online journal that publishes all aspects of neurodegeneration research, and Institute for Biomedical Research, Xiamen University , which is dedicated to revealing the fundamental molecular causes of diseases and devising the innovative therapies of tomorrow. Finacial supports of this meeting come from both foundations and pharmaceutical companies including Alzheimer's Association, Ellison Medical Foundation, National Natural Science Foundation of China, Science and Technology Bureau of Xiamen City, Raptor Pharmaceutical, GlaxoSmithKline, Beckman Coulter, Zeiss, Perkin Elmer, Millipore, Applied Biosystems, and Genetimes Techonolgoy, Inc. Drs. Guojun Bu from Washington University School of Medicine and Huaxi Xu from Burnham Institute for Medical Research, Editors-in-Chief of Molecular Neurodegeneration, are co-chairs of this conference.This conference is jointly sponsored by More than 20 world-renowned scientists, including Dr. Aaron Ciechanover, a 2004 Nobel Laureate in Chemistry for his discovery of the ubiquitin system, and Dr. Steven Heinemann, a member of the US National Academy of Sciences, presented their research progress in the meeting. In addition, researchers from Asian countries, including China , Japan, and South Korea, also presented their work in short talk and poster sessions.See figure \u2022 Keynote Speaker: Aaron Ciechanover : \"The ubiquitin proteolytic system: from basic mechansisms through human diseases and onto drug targeting\"Session I: ApoE and apoE receptors in AD\u2022 Guojun Bu (Washington University School of Medicine): \"ApoE receptors in apoE and A\u03b2 metabolism\"\u2022 Mary Jo LaDu (University of Illinois at Chicago): \"A\u03b242 and apoE structure/function interactions: implications for Alzheimer's disease\"Session II: APP processing: \u03b2- and \u03b3-secretases\u2022 Robert Vassar (Northwestern University): \"Regulation of BACE1 in Alzheimer's disease\"\u2022 Takeshi Iwatsubo (University of Tokyo): \"Alzheimer's disease: unraveling the structure-function relationship of gamma-secretase\"\u2022 Gopal Thinakaran (University of Chicago): \"Function and dysfunction of presenilins\"Session III: Presenilin and amyloid beta\u2022 Sam Sisodia (University of Chicago): \"Presenilin function in adult neurogenesis\"Short Talk Session I\u2022 Taisuke Tomita (The University of Tokyo): \"Identification and analysis of a substrate-specific genetic modulator for the \u03b3-secretase activity\"\u2022 Yingying Le : \"Activation of \u03b2-adrenergic receptor on microglia promotes uptake and clearance of Alzheimer's disease-associated amyloid \u03b2 peptide\"\u2022 Wandong Zhang : \"ABCG2 is up-regulated in Alzheimer's brain with cerebral amyloid angiopathy and acts as a gatekeeper at the blood-brain barrier for A\u03b2 peptides\"Keynote Address- Steve Heinemann : \"New approaches to understanding Alzheimer's disease\"Session IV: Functions of APP and its processing products\u2022 Hui Zheng (Baylor College of Medicine): \"APP function in neurons and synapses\"\u2022 Frederic Checler : \"Regulatory functions of APP intracellular domain\"Session V: Parkinson and trinucleotide repeat disorders\u2022 Jie Shen : \"Impaired neurotransmitter release in Alzheimer's and Parkinson's diseases\"\u2022 Xiaojiang Li (Emory University): \"Synaptic toxicity of mutant huntingtin\"Short Talk Session II\u2022 Hongyu Hu : \"Mechanism of \u03b1-synulcein aggregation associated with Parkinson's disease\"\u2022 Hua Gao (Key Laboratory for Neurodegenerative Disease of the Ministry of Education): \"DJ-1 decrease rotenone-mediated dopaminergic cell death via ERK signaling\"\u2022 Cristine Alves da Costa (Institut de Pharmacologie Moleculaire et Cellulaire): \"DJ-1-mediated cell death control by p53 is regulated by caspase proteolysis\"\u2022 Lingqiang Zhu : \"Activation of glycogen synthase kinase-3 inhibits long term potentiation with synapse-associated impariments\"Session VI: BDNF and novel genes in neurodegeneration\u2022 Eric Tzeng (Beckman Coulter): \"The latest total solution characterizes neurodisorder diseases\"Yunwu Zhang (Xiamen University): \"A novel gene that inhibits Alzheimer's \u03b2-amyloid generation and tau phosphorylation\"Short Talk Session III\u2022 Jin Tae Hong : \"Mutant presenilin 2 (N141I) increases beta-secretase activity through increase of ERK pathway dependent hydrogen peroxide generation\"\u2022 Ling Li (University of Alabama at Birmingham):\"Simvastatin prevents cognitive and hippocampal synaptic deficits in APP/PS1 double transgenic mice\"\u2022 Wangxia Wang (University of Kentucky): \"MicroRNA miR-107 targets genes related to neurodegenerative disease\"\u2022 Qingyan Liu : \"A novel transmembrane protein down regulated in Alzheimer's brains and interacts with a DnaJ-like heat shock protein (DNAJB4)\"\u2022 Xiuqi Bao : \"Establishment of gonadectomy-accelerated brain aging model in mice\"Session VII: Metal, cell cycle and signaling in neurodegeneration\u2022 Ashley Bush (University of Melbourne): \"The metal theory of Alzheimer's disease: from bench to clinic\"\u2022 Karl Herrup (Rutgers University): \"Cell cycle events as risks for neurodegeneration: a look into mechanism\"\u2022 Yong Shen : \"Tumor necrosis factor and Alzheimer's disease\"Session VIII: AD genetics and therapy\u2022 George Martin (University of Washington): \"Classes of gene action with the potential to modulate the times of onset and the rates of progression of neurodegenerative disorders\"\u2022 Edward Koo : \"Gamma secretase modulators, nonsteroidal anti-inflammatory drugs, and Alzheimer's disease\"\u2022 Colin Masters : \"A\u03b2 oligomers as tractable targets for Alzheimer's disease: diagnostics and therapeutics\"The authors declare that they have no competing interests.GB supervised plans for the meeting and made final decisions regarding meeting and planning.YZ helped prepare and set up for the meeting and wrote this article and cover letter.LO helped prepare program and agenda for meeting, cooresponded with all involved and served as administrative contact."} +{"text": "To the Editor: Gupta et al. raise important issues regarding molecular profiling as an epidemiologic tool (Gupta et al. interpret their experience as indicating that, with geographically dispersed isolates, a higher degree of genomic similarity than is reliably provided by single-enzyme PFGE is necessary to improve specificity, thereby avoiding fruitless investigative efforts ("} +{"text": "Sir,et al for their interest in our recent study and for their remarks concerning the safety of using involved-field irradiation in treating limited-stage small-cell lung cancer (SCLC). We have updated our analysis of failure patterns in patients with limited-stage SCLC who were treated in our phase II study of involved-field irradiation during the second and third cycles of carboplatin, paclitaxel and etoposide (We thank Dr Chun-Ru Chien Recently, A phase III trial of concurrent chemoradiotherapy without elective nodal irradiation in limited-stage SCLC, where patients will be randomised to either once daily irradiation to a dose of 66\u2009Gy or twice daily irradiation to a dose of 45\u2009Gy will shortly commence . Such a"} +{"text": "Sulfur mustard (SM), also known as mustard gas, has been the most widely used chemical weapon. The toxicity of SM as an incapacitating agent is of much greater importance than its ability to cause lethality. Acute toxicity of SM is related to reactive oxygen and nitrogen species, DNA damage, poly(ADP-ribose) polymerase activation and energy depletion within the affected cell. Therefore melatonin shows beneficial effects against acute SM toxicity in a variety of manner. It scavenges most of the oxygen- and nitrogen-based reactants, inhibits inducible nitric oxide synthase, repairs DNA damage and restores cellular energy depletion. The delayed toxicity of SM however, currently has no mechanistic explanation. We propose that epigenetic aberrations may be responsible for delayed detrimental effects of mustard poisoning. Epigenetic refers to the study of changes that influence the phenotype without causing alteration of the genotype. It involves changes in the properties of a cell that are inherited but do not involve a change in DNA sequence. It is now known that in addition to genetic mutations, epimutations can also involve in the pathogenesis of a variety of human diseases. Several actions of melatonin are now delineated by epigenetic actions including modulation of histone acetylation and DNA methylation. Future studies are warranted to clarify whether epigenetic mechanisms are involved in pathogenesis of delayed sulfur mustard toxicity and melatonin alleviates delayed toxicity of this warfare agent. Among the available chemical warfare agents, sulfur mustard (SM), also known as mustard gas, has been a widely used chemical weapon. Because of its devastating toxicity, its use during the World War I earned it the sobriquet \u201cking of the battle gases\u201d. Other compounds such as nitrogen mustard (HN2) were developed during World War II, but found to be unsuitable as a munition. Soon after discovering HN2, it became the first non-hormonal agent used in cancer chemotherapy. A number of HN2 derivatives including cyclophosphamide (CP), ifosfamide, mechlorethamine, melphalan and chlorambucil are valuable cytotoxic and radiomimetic agents for the treatment of cancer and triggers the expression of selectins and cellular adhesion molecules, via enhancing nuclear factor (NF)-\u03baB and activator protein (AP)-1 activation, thereby promoting pro-inflammatory responses including most importantly tumor necrosis factor (TNF)-\u03b1 and interleukin (IL)-1\u03b2.ONOO\u2013 also induces apoptosis and necrosis in cells depending on the exposure concentration. In case of higher concentration, a DNA repair enzyme poly (ADP ribose) polymerase-1 (PARP-1), mediates ONOO\u2013-induced necrosis to various nuclear proteins. In case of severe DNA injury, overactivation of PARP-1 depletes the cellular stores of NAD+, an essential cofactor in the glycolytic pathway, the tricarboxylic acid cycle, and the mitochondrial electron transport chain. As a result, the loss of NAD+ leads to a marked reduction in the cellular pools of ATP, resulting in cellular dysfunction and cell death via the necrotic pathway. Experimental evidence has established that the PARP-1 pathway of cell death plays a pivotal role in tissue injury and organ dysfunction in mustard-induced acute toxicity , histone acetyl transferases (HATs) and DNA methyltransferases (DNMTs) is glucocorticoid resistance. Although inhaled glucocorticoids are highly effective in asthma, they provide relatively little therapeutic benefit in COPD, despite the fact that active airway and lung inflammation is present. This may reflect that the inflammation in COPD is not suppressed by glucocorticoids, with no reduction in inflammatory cells, cytokines or proteases in induced sputum even with high doses of inhaled and oral glucocorticoids that have been activated during the chronic inflammatory process. The increased expression of most of these inflammatory proteins is regulated at the level of gene transcription through the activation of pro-inflammatory transcription factors, such as nuclear NF-\u03baB and AP-1. The molecular pathways involved in regulating inflammatory gene expression are now being delineated and it is now clear that chromatin remodeling and a variety of epigenetic mechanisms play a critical role in the transcriptional control of genes. Stimuli that switch on inflammatory genes do so by changing the chromatin structure of the inflammatory gene, whereas glucocorticoids reverse this process.et al., et al., et al., Glucocorticoids produce their effect on responsive cells by activating the glucocorticoid receptor (GR) to directly or indirectly regulate the transcription of target genes. Most of the anti-inflammatory actions of glucocorticoids are due to suppression of the actions of AP-1 and NF-\u03baB Barnes, . The actet al., et al., et al., et al., et al., \u2013 are similar to those that have been observed previously in patients with asthma and chronic bronchitis from other common causes (Emad and Rezaian, \u2013 scavenging agent in both in vivo and in vitro (Sourdeval et al., et al., et al., et al., et al., et al., et al., et al., et al., Melatonin shows beneficial effects against SM-induced acute toxicity as a multifunctional antioxidant and ONOOet al., Despite 75 years of research, there is still no antidote for mustard. This fact is especially crucial when we consider that probably at least a dozen countries have mustard in their arsenals today. Melatonin has been administered in both physiological and pharmacological amounts to humans and animals, and there is widespread agreement that it is a non-toxic molecule. In pregnant rats, maternal lowest no observed effect level has been found to be 200mg/kg/day and developmental no observed adverse effect level is \u2265 200 mg/kg/day (Jahnke"} +{"text": "Frequent ventricular premature complexes (VPC) can result in left ventricular dysfunction. Several case reports have found this association and reversal with radiofrequency ablation -4. OtherThe relation between the burden of ventricular ectopy and left ventricular function was evaluated by Baman TS et al . VPC burImprovement in left ventricular end systolic and end diastolic dimensions as well as ejection fraction has been documented after VPC ablation . In spitDarrieux FC, et al noted anLelakowski J et al reportedTwo articles in this issue of the journal focus on the ablation of VPCs. While Sheldon SH et al discusse"} +{"text": "GABAergic inhibitory neurotransmission contributes to diverse aspects of brain development and adult plasticity, including the expression of complex cognitive processes. This is afforded for in part by the dynamic adaptations occurring at inhibitory synapses, which show great heterogeneity both in terms of upstream signaling and downstream effector mechanisms. Single-particle tracking and live imaging have revealed that complex receptor-scaffold interactions critically determine adaptations at GABAergic synapses. Super-resolution imaging studies have shown that protein interactions at synaptic sites contribute to nano-scale scaffold re-arrangements through post-translational modifications (PTMs), facilitating receptor and scaffold recruitment to synaptic sites. Additionally, plasticity mechanisms may be affected by the protein composition at individual synapses and the type of pre-synaptic input. This mini-review article examines recent discoveries of plasticity mechanisms that are operational within GABAergic synapses and discusses their contribution towards functional heterogeneity in inhibitory neurotransmission. The plasticity of individual synapses occurs downstream of activity or neuro-modulatory signaling and must be reconciled with homeostatic mechanisms to maintain overall network function receptors. These CB1 receptors are pre-synaptically enriched at CCK+ synapses and participate in the depolarization-induced suppression of inhibition , post-synaptic scaffolding and signaling proteins, and trans-synaptic adhesion molecules which facilitate effective communication between the pre- and post-synapse for efficient neurotransmission. GABAARs are composed of pentamers from a family of subunits encoded by 19 distinct genes . Although it has been recently shown that many receptor subunits can access the synaptic space (Hannan et al., ARs composed of the combination of \u03b11\u20133 subunits along with \u03b21\u20133 and \u03b32 subunits, whereas those containing the subunits \u03b14\u20136 and \u03b4 tend to be extra-synaptic (Fritschy and Panzanelli, ARs are trafficked to the plasma membrane from cytoplasmic pools, or diffuse laterally within the membrane in and out of synapses to alter the local concentration of receptors and therefore synaptic strength (Flores and M\u00e9ndez, ARs is an important mechanism by which inhibitory plasticity is achieved (Petrini and Barberis, ARs scales with the size of gephyrin clusters (Specht et al., via decreased confinement of GABAARs (Jacob, AR clustering. For example, activity induction in hippocampal slices leads to inhibitory potentiation that is correlated to increases in gephyrin cluster size concordant with mIPSC amplitude (Flores et al., AR synaptic organization can be used to understand mechanistic bases for synapse alterations.The GABAergic post-synapse contains GABAs Jacob, . SimilarAR synaptic accumulation through CaMKII signaling (Flores et al., ARs at synapses, large increases in calcium leads to reduced retention of GABAARs (Petrini and Barberis, While plasticity occurs at all synapses, basal synapse characteristics such as size, strength, and composition are variable, and therefore the extent of induced synaptic plasticity is also variable. For example, spinal cord synapses contain over four times as many gephyrin molecules per synapse and at a higher density than cortical synapses (Specht et al., ARs, the interaction between GABAARs and post-synaptic scaffolds, or the dynamics of the post-synaptic scaffolds themselves could all contribute to modulating synaptic receptor retention and therefore the function of inhibitory synapses (Choquet and Triller, via altered receptor-scaffold interactions. Of these, modification of GABAARs (Comenencia-Ortiz et al., ARs and gephyrin result in enhanced surface localization (Matt et al., ARs controls both surface trafficking and removal (Comenencia-Ortiz et al., via gephyrin-dependent (Mukherjee et al., Direct modification of GABAARs as well as those of pre-synaptic vesicle release sites clearly demonstrating that synaptic gephyrin nano-domains represent functional organizational units (Crosby et al., ARs has been shown by perturbing gephyrin clustering via overexpression of dominant-negative gephyrin, which causes a reduction in the number and size of GABAAR nano-domains (Crosby et al., ARs at synaptic sites (Battaglia et al., Recent efforts towards describing post-synaptic dynamics have employed live-imaging and super-resolution microscopy to determine real-time and nano-scale re-organization of the post-synapse (Specht et al., AR retention at synapses. A recent study has found that phosphorylation of gephyrin at serine 268 (regulated by ERK1/2; Tyagarajan et al., AR synaptic dwell time (Battaglia et al., AR dynamics outside of synaptic sites, suggesting that gephyrin is involved in extra-synaptic receptor scaffolding regulated by phosphorylation of distinct serine residues (Battaglia et al., via receptor-scaffold interactions. Phospho-proteomic analyses of synaptic proteins indicate that more than just gephyrin and GABAARs are dynamically phosphorylated, and that altered brain states such as sleep deprivation (Wang et al., ARs and gephyrin (\u0160mid\u00e1k et al., PTMs have now been shown to control gephyrin nano-domain structure and GABAARs dynamics and inhibitory synapse function have been identified (Fritschy et al., Models for receptor-scaffold interactions propose that modifying the number of scaffolds or the affinity of receptor-scaffold binding will define the equilibrium governing immobilization of receptors at the synapse (Choquet and Triller, ARs and adaptor proteins (Cajigas et al., AR subunit is disrupted in a loss-of-function mouse model null for the RNA binding protein NONO, leading to a reduction in synaptic GABAARs and gephyrin clustering (Mircsof et al., Recent data suggests that local translation of mRNA coding for synaptic proteins could offer a way to acutely modify synaptic composition in a synapse-specific manner (Rangaraju et al., The findings highlighted in this mini-review article (summarized in BC and ST contributed to the organization and writing of this manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Glycogen storage disease type I (GSD I) is a relatively rare metabolic disease with variable clinical intensity. It is caused by deficient activity of the glucose 6-phosphatase enzyme (GSD Ia) or a deficiency in the microsomal transport proteins for glucose 6-phosphate (GSD Ib). We searched the most recent English literature (1997-2017) regarding any article with the key word of \u201cglycogen storage disease type I\u201d in PubMed, Science Direct, Scopus, EMBASE, and Google Scholar. We will present all of the published articles about the molecular genetic characteristics and old-to-new diagnostic methods used to identify GSD I in regard of methodology, advantages and disadvantages. Diagnosis of GSD type I and its variants is challenging because it is a genetically heterogeneous disorder. Many molecular methods have been used to diagnose GSD I most of which have been based on mutation detection. Therefore, we discuss complete aspects of all of the molecular diagnostic tests, which have been used in GSD type I so far. With the advent of high throughput advanced molecular tests, molecular diagnosis is going to be an important platform for the diagnosis of storage and metabolic diseases such as GSD type I. Next-generation sequencing, in combination with the biochemical tests and clinical signs and symptoms create an accurate, reliable and feasible method. It can overcome the difficulties by the diagnosis of diseases with broad clinical and genetic heterogeneity. Glycogen storage diseases (GSDs) are a group of metabolic disorders determined by the accumulation of glycogen in different tissues. GSDs are caused by the enzyme deficiencies effect on glycogen synthesis, glycogen breakdown or glycolysis (glucose breakdown), typically within muscles and/or liver cells. The disorders were numbered as they were discovered which classified chronologically by GSD type I (von Gierke disease) to GSD type XI . MajoriGSD (Glycogen storage disease) type I or von Gierke disease is the second most common type of GSDs. GSD type I typically presents in early childhood. Von Gierke disease is an autosomal recessive disorder. There is no report regarding ethnic difference in the incidence of GSD type I, however there are different types of mutations in Caucasian, Hispanic, Asian and Jewish populations . The ovHistorically, in 1929, Edgar von Gierke was the first to describe a glycogen storage disease (GSD), which initially was named in his honor regarding any article with the key word of \u201cglycogen storage disease type I\u201d in PubMed, Science Direct, Scopus, EMBASE, and Google Scholar.GSD I is characterized by accumulation of glycogen and fat in the liver and kidneys, leading to hepatomegaly and renomegaly. The clinical manifestations of von Gierke disease include growth retardation, hepatomegaly, hypoglycemia, lactic acidemia, hyperuricemia, osteoporosis and hyperlipidemia . The paThe clinical presentations of GSD Ib is quite similar to that of GSD Ia, however the symptoms of the patients with GSD Ia, such as hepatomegaly, a characteristic ''doll-like'' face, short stature, and chronic fatigue are more severe. Unlike patients with GSD-Ia, most patients with GSD-Ib also suffer from neutrophil dysfunction and neutropenia, lead to frequent bacterial infections. Clinical differential diagnosis of GSD-Ia from Ib is difficult because neutropenia is sometimes periodic or never develops in GSD-Ib was codified the enzyme translocase and mutations within this gene in GSD Ib patients were described to translocate glucose-6-phosphate (G6P) from the cytosol to the lumen of the endoplasmic reticulum (ER) and two other transport systems to transport the reaction products phosphate (Pi) and glucose (G6PT2 and G6PT3 respectively) to the cytosol , which al., 1997). G6PT1 al., 2002). It hasal., 2002). G6PC and most patients with GSD Ic and Id have mutations in the G6PT1 gene SLC37A4, therefore GSD I is now divided into two general subtypes GSD Ia and GSD I non-a, respectively and GSD-Ib, a deficiency in the glucose-6-phosphate (G6P) transporter (G6PT) . But wial., 1995; Veiga-Dal., 1995). Howeveal., 1995; Kishnanal., 1995; Janeckeal., 1995). Among al., 2001).The final and conclusive diagnosis of GSD needed pathologic diagnosis in combination with biochemical and clinical findings; however, liver biopsy is an invasive procedure . PractiPrompt and accurate diagnosis is the most important point for the proper treatment of metabolic diseases. Diagnosis of GSD I is sometimes complicated because there are common features between GSD I and III, including hepatomegaly, hypoglycemia, and hyperlipidemia. Validated and clinically useful tools with a positive predictive value > 90 % are necessary for the diagnosis of GSD I. The diagnosis of GSD I currently relies on clinical features, pathologic diagnosis of liver biopsy, biochemical, and molecular genetic tests and pathologic findings of the liver biopsy in favor of GSD .G6PC or G6PT1 gene unique to Caucasian, Hispanic, Chinese/Japanese/ Korean, and Jewish GSD I patients have been described, suggesting separate ethnic founder effects for some mutations . Transfal. (1999) has conal. (1999) analyzeal. (1999) showed al., 2000; Nakamural., 2000).Eventually, even a single base alteration can be detected by the altered mobility of the single-stranded DNA molecule in SSCP. In 2004, in one study the frequency of two prevalent mutations of GSD Ia patient in Caucasian (the Q347X and R83C mutation) was reported to screen the Ashkenazi Jewish population by SSCP method as an accurate and easy technique which leads to a predicted prevalence five times higher than in the general Caucasian population .According to the above literature, disadvantages of SSCP method include the requirement of highly standardized electrophoretic conditions in order to get constant results. Furthermore, some mutations may remain undetected, and accordingly definite absence of mutation cannot be proven. In HD method single-base substitutions are less stable and excessively sensitive to environmental changes. This fact reduces the sensitivity of this method for this type of mutations, which is frequently found in GSD I , two deletions (540del5 and 158delC) and one nonsense mutation (Q347X). Three of them have not been described previously and the R83C was the most common mutation among the Czech and Slovak patients has been applied for screening of unknown point mutations. Identification of mutations in the al., 2000). This tal., 2000).G6PC (GSD Ia) and SLC37A4 (GSD Ib) full genes can be used for confirming the diagnosis of these diseases precisely have launched that are referred to as massively parallel or next-generation sequencing (NGS). Direct sequencing has proven extremely successful because of its accuracy and affordability, but it is inappropriate for large-scale screening projects because it has been developed to sequence only one amplified DNA molecule at one time in a given sample on both strands . Moreoval., 2014; Janeckeal., 2014). Directal., 2014; Gerin eal., 2014). Directal., 2017). Since al., 2017; Lam et al., 2017; Ihara eal., 2017; Triocheal., 2017; Chiang al., 2017; Koz\u00e1k eal., 2017; Reis etal., 2017; Yuen etal., 2017; Qiu et al., 2017; Ki et aal., 2017; Miltenbal., 2017; Qiu et al., 2017; Tamhankal., 2017; Liang eal., 2017; Gu et aal., 2017; Lu et aal., 2017; Mahmoudal., 2017, Choi etal., 2017), Koreanal., 2017), Brazilal., 2017), Chinesal., 2017; Chiang al., 2017; Qiu et al., 2017; Gu et aal., 2017; Lu et aal., 2017), Indianal., 2017; Karthi al., 2017), Japaneal., 2017), Czech al., 2017), Hungaral., 2017) and Freal., 2017) patiental., 2017), Chinesal., 2017; Qiu et al., 2017; Liang eal., 2017) Japanesal., 2017) populatiga, 2018). Converiga, 2018; Wang etiga, 2018). Briefliga, 2018). SuccesWang et al. (2013) have deSeveral approaches have been used for detection of known mutations. These methods usually started with the polymerase chain reaction (PCR) and additional assay steps are performed based on the type of mutation such as RFLP-PCR . Ihara non-A patients which is advantageous for a primary molecular genetic diagnostic approach. They concluded that the principal advantages of DHPLC are its semi-automated nature, with rapid results (a few minutes per sample), and the feasibility to collect eluted DNA for next analyses, but DHPLC alone cannot provide the details about the nature of mutations. Also, the sensitivity of the method is dependent on the temperature of analysis, the selection of which is dependent on operator experience technique after PCR . Thoughal., 2000). Lam etal., 2000) have real., 2000).G6PC gene because it is a major cause of GSD-Ia among Japanese patients. The high reproducibility and sensitivity of this technique indicates that the method may be safely applied to clinical diagnosis. Afterward, Kojima et al. combined with a TaqMan fluorogenic probe (TaqMan-ASA) used a al. (2004) performal. .Lately, high resolution melting analysis (HRMA), a simple real-time PCR-based method for detecting sequence variations for GSD Ia was developed. The basis of this method is that changes in amplicons of HRM are dependent on their DNA melting curves in the presence of saturating DNA binding dyes . TherefIn the last decade, the use of DNA microarray technique for diagnosis of genetic diseases has been reported , the cost, speed and accuracy of detection increase. The authors declare that they have no conflicts of interest."} +{"text": "The prevalence and social cost of clinical depression are rapidly increasing, and the field continues to attract considerable research interest and a major downstream target of the ERK signaling pathway, is upregulated both in depressive suicides and in animal models of depression induced by unpredictable chronic mild stress (UCMS) and social defeat is associated with the diverse signs and symptoms of depression (see for example, Dwivedi et al., However, the authors did not mention other ways of regulating ELK-1 activity, which have been demonstrated in previous studies. For example, ELK-1 can be degraded by FBXO25, a ubiquitin ligase (Teixeira et al., There are also other issues to be resolved around ELK-1 regulation. SIRT6, a histone deacetylase, interacts with ELK-1 to suppress ELK-1-dependent transcription (Cea et al., Furthermore, ELK-1 has isoforms and splice variants (Rao and Reddy, Besides the issues raised above, the work presented by Apazoglou et al. provides clear evidence that ELK-1 is a promising drug target for clinical depression. Interestingly, ELK-1 has been implicated in diseases comorbid with depression (Mayeux et al., Although the signaling pathways and molecules involved in depression have been studied extensively over the decades (Duman and Voleti, The author confirms being the sole contributor of this work and has approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer HS and handling editor declared their shared affiliation at time of review."} +{"text": "The spatiotemporal control of DSB formation occurs within a specialized chromosomal structure characterized by sister chromatids organized into linear arrays of chromatin loops that are anchored to a proteinaceous axis. Although SPO11 and its partner proteins required for DSB formation are bound to the axis, DSBs occur preferentially within the chromatin loops, which supports the \u201ctethered-loop/axis model\u201d for meiotic recombination. In this mini review, we discuss insights gained from recent efforts to define and profile DSB hotspots at high resolution in eukaryotic genomes. These advances are deepening our understanding of how meiotic recombination shapes genetic diversity and genome evolution in diverse species.Homologous chromosomes must pair and recombine to ensure faithful chromosome segregation during meiosis, a specialized type of cell division that occurs in sexually reproducing eukaryotes. Meiotic recombination initiates by programmed induction of DNA double-strand breaks (DSBs) by the conserved type II topoisomerase-like enzyme SPO11. A subset of meiotic DSBs are resolved as crossovers, whereby reciprocal exchange of DNA occurs between homologous chromosomes. Importantly, DSBs are non-randomly distributed along eukaryotic chromosomes, forming preferentially in permissive regions known as hotspots. In many species, including plants, DSB hotspots are located within nucleosome-depleted regions. DSB localization is governed by interconnected factors, including Meiosis is a specialized cell division program that is essential for sexual reproduction in eukaryotes. During this program, replication of chromosomal DNA to form sister chromatids is followed by two rounds of cell division. Maternal and paternal chromosomes (homologs) segregate at the first division and sister chromatids segregate at the second division. Chromosome number is thereby halved and, in diploid organisms, meiosis culminates in the production of haploid progeny cells (gametes). Chromosome segregation during meiosis is imperative for the continuation of the species, as it enables the formation of a zygote that inherits the full chromosome complement in the next generation by fusion of a male and a female gamete and mouse, DSB hotspots have been defined as loci meeting or exceeding given thresholds for Spo11-oligo density and physical size -based peak-calling approach in gene promoters than in wild-type budding yeast cells, indicative of a negative feedback loop in which homolog engagement following DSB formation suppresses Spo11 activity and prevents further breaks group participate in the assembly of the synaptonemal complex and promote crossing over targeted to meiotic hotspots suppresses crossover recombination , Copia LTR and LINE-1 classes less than expected (Choi et al., Arabidopsis and mouse, however, fewer-than-expected maize DSB hotspots occur in Copia LTR and LINE retrotransposons (He et al., Despite SPO11-1-oligo depletion in Arabidopsis, significant overlap occurs between comparable classes of DNA elements and DSB hotspots, many of which are located within functionally conserved sequences (Choi et al., Arabidopsis hotspots may be more evolutionarily stable. Nonetheless, comparisons between eukaryotes indicate that repeated sequences may influence meiotic recombination initiation landscapes in related ways.Citing the hotspot paradox hypothesis, Yamada et al. speculatATM in budding yeast, suppresses the formation of clustered DSBs in cis over distances of ~70\u2013100 kb (Garcia et al., ATM activity allows DSBs to form independently of one another over \u00b120\u2013100-kb distances, giving rise to DSB formation in neighboring regions at frequencies comparable to those expected by chance. Over distances of \u00b1 ~7.5 kb, by contrast, Tel1ATM inactivation permits the formation of adjacent DSBs significantly more frequently than expected, generating localized regions of \u201cnegative DSB interference\u201d (Garcia et al., cis-interference suggests that hotspots within the same loop domain are \u201cprimed\u201d for cleavage. Cooper et al. (ATM-dependent cis-interference, which restricts DSB formation to only one of the primed intra-loop hotspots. Spatial regulation of meiotic DSB formation also occurs in trans via a mechanism involving Tel1ATM and Mec1ATR, another DDR signal transduction kinase (Zhang et al., trans-interference inhibits DSB formation at the corresponding locus on its sister, its homolog or frequently both. This mechanism is thought to ensure that an intact template is available for DSB repair, and to prevent DSB formation at allelic loci on both homologs (Zhang et al., Meiotic DSB hotspots are identified by mapping the DSB landscape in a population of cells. This landscape reveals a continuum of variation within which loci with high probabilities of DSB formation may be detected (Pan et al., r et al. speculatGenome-wide DSB mapping in different eukaryotes has revealed diversity with regard to the hierarchical combinations of factors that shape meiotic recombination landscapes and hotspots. These distinctions highlight the importance of studying DSB landscapes in diverse eukaryotes and beyond model organisms. Efforts to elucidate the mechanisms that determine DSB hotspot designation may inform genetic or epigenetic manipulations intended to reshape naturally constrained meiotic recombination landscapes. For example, the presence of hotspots in conserved genomic elements, such as nucleosome-depleted promoters, has relevance for targeting crossover recombination to specific loci in plants (Sarno et al., AT and IH wrote and edited the manuscript. AT created Table The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "DNA methylation is an epigenetic marker that has been shown to vary significantly across different tissues. Taking advantage of the methylation differences between placenta-derived cell-free DNA and maternal blood, several groups employed different approaches for the discovery of fetal-specific biomarkers. The aim of this study was to analyse whole-genome fetal and maternal methylomes in order to identify and confirm the presence of differentially methylated regions (DMRs). We have initially utilized methylated DNA immunoprecipitation (MeDIP) and next-generation sequencing (NGS) to identify genome-wide DMRs between chorionic villus sampling (CVS) and female non-pregnant plasma (PL) and peripheral blood (WBF) samples. Next, using specific criteria, 331 fetal-specific DMRs were selected and confirmed in eight CVS, eight WBF and eight PL samples by combining MeDIP and in-solution targeted enrichment followed by NGS. Results showed higher enrichment in CVS samples as compared to both WBF and PL samples, confirming the distinct methylation levels between fetal and maternal DNA for the selected DMRs. We have successfully implemented a novel approach for the discovery and confirmation of a significant number of fetal-specific DMRs by combining for the first time MeDIP and in-solution targeted enrichment followed by NGS. The implementation of this double-enrichment approach is highly efficient and enables the detailed analysis of multiple DMRs by targeted NGS. Also, this is, to our knowledge, the first reported application of MeDIP on plasma samples, which leverages the implementation of our enrichment methodology in the detection of fetal abnormalities in maternal plasma. Using a novel doubleenrichment approach (MeDIP in combination with insolution hybridization enrichment followed by NGS), we have confirmed the presence of a set of 331 DMRs in multiple CVS, WBF and PL samples. The results of this study demonstrate that there is a clear distinction between the methylation levels of fetal and maternal DNA for the selected DMRs. The utilization of a novel doubleenrichment approach in this study provides a significant expansion in the number of fetalspecific biomarkers. This increase in fetal biomarkers sets the foreground for the implementation of our approach in the detection of the most common fetal aneuploidies.2.2.1.In total, 11 WBF, 10 PL and 11 firsttrimester CVS (11\u201314 weeks of gestation) were used in this study . The stuet al., Peripheral blood was collected from women donors into two 8-mL EDTAcontaining tubes. An average of 8 mL of plasma was isolated using a doublecentrifugation protocol as previously described and the QIAamp DNA Mini kit (Qiagen), respectively, according to the manufacturer's instructions. DNA from PL samples was extracted using the QIAsymphony DSP Virus/Pathogen Mini Kit (Qiagen).2.2.DMR identification was initially performed on three WBF, three CVS and two PL samples using wholegenome MeDIPNGS. Based on specific criteria (see Section 3.2) we selected 331 DMRs that were found to be hypermethylated in the fetal tissues and hypomethylated in maternal whole blood and plasma. Confirmation of the methylation status of the 331 DMRs was performed on eight CVS, eight WBF and eight PL samples using MeDIP followed by targeted insolution enrichment and NGS.2.3.\u03b2globin loci, as described previously , according to the manufacturer's protocol. The remaining MeDIP experiments were performed using mouse anti5\u2032methylcytosine monoclonal antibody , as described previously using 18\u201330\u00a0ng of genomic DNA were used in order to confirm primer specificity. PCR reactions were performed using MyTaq HS DNA Polymerase , as described elsewhere , 10\u00d7 blocking agent (Agilent), blocking oligonucleotides the regions selected exhibited consistent DNA hypermethylation profiles in all CVS and hypomethylation in all the female nonpregnant tissues; (b) selected regions had preferentially more than two CpG dinucleotides in the DNA sequence; (c) potential DMRs that were in copy number variable regions or in repetitive element regions were excluded; (d) selected DMRs should be located 200\u00a0bp away from repetitive elements; and (e) the adjusted (Bonferroni correction) et al., P value <0\u00b71 were considered for subsequent analysis. These bins were merged into consecutive regions (DMRs) after specific criteria selection and filtering.Pairwise genomewide methylation comparisons between CVS, WBF and PL were performed using the MEDIPS package . The response variable in this model is the read depth, which in our experiments acts as a proxy for the methylation level of each region, while the categorical variable is the sample type and consists of three levels . The additional random effect allows for different methylation variability between the different DMRs. Subsequent od Holm, .3.3.1.Three WBF, three CVS and two PL samples were subjected to wholegenome MeDIPNGS analysis to enable genomewide identification of fetalspecific DMRs. The resulting alignment files were used as input for the R package MEDIPS, where the differential coverage between two groups of samples (i.e. CVS vs. WBF and CVS vs. PL) was calculated. Using MEDIPS criteria (see Section 2.7.1), 3574 DMRs were identified in the CVS vs. WBF comparisons, of which 1888 regions showed hypermethylation in CVS and hypomethylation in WBF samples, while 1686 regions showed hypomethylation in CVS and hypermethylation in WBF samples . Similar3.1.1.P\u00a0<\u00a02\u00a0\u00d7\u00a010\u221216 for all three pairwise posthoc tests (The overlap of the DMRs obtained from the two comparisons (WBF vs. CVS and PL vs. CVS) provided 1453 common fetalspecific DMRs that showed hypermethylation in CVS and hypomethylation in maternal samples. Those ranged from 100 to 2300\u00a0bp in length. Comparison of the overall methylation status of the aforementioned DMRs showed a clear distinction of the methylation status between CVS and maternal tissues (WBF and PL), with adjusted oc tests a). Overa3.2.To further ascertain and characterize the identified biomarkers, a subset of 331 potential fetalspecific DMRs was selected based on specific criteria (see Section 2.7.1). In addition, selection was focused on autosomal chromosomes and on regions located in significant regulatory regions such as potential promoters, CpG islands (CGIs) and exonic (coding) regions. Specifically, 294, 64 and 73 DMRs were located within coding regions (67\u00b77% in gene bodies and 32\u00b73% in exons), potential promoters and CGIs, respectively . Resulet al., et al., et al., et al., et al., et al., et al., et al., et al., et al., The methylation status of the selected DMRs was also compared with previous studies that utilized methylation differences between fetal and maternal tissue for the identification of fetalspecific biomarkers. Common DMRs were found between our approach and DMRs identified using bisulphite conversion, methylationsensitive restriction digestion and microarrays (Old 4.In this study, we undertook the genomewide biomarker discovery of DMRs between fetal and maternal DNA and confirmed the presence of a subset of these DMRs by combining for the first time MeDIP with insolution targeted enrichment and NGS.et al., Also, this is, to our knowledge, the first reported application of MeDIP in plasma samples. Previous studies have employed MeDIP in order to characterize the methylation status of different tissues using large amounts of input DNA. We were able to overcome this limitation and successfully enrich and characterize the methylome of multiple plasma samples using modifications of an existing MeDIP protocol (Borgel et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., Previous studies have employed different methods for the discovery of fetalspecific biomarkers using methylation differences between fetal and maternal DNA, including sodium bisulphite conversion, methylationsensitive restriction digestion or affinitybased techniques (Gitan et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., Comparison of the selected DMRs with previous reports showed that the methylation patterns of several DMRs that were confirmed in our study are consistent with other methylationbased approaches (Old Based on the characteristics of the validated DMRs and due to the great potential of this approach to be utilized in the clinical setting for the detection of the most common aneuploidies, future work will focus on the identification and characterization of additional DMRs on chromosomes 13, 18 and 21. In addition, the discovery of DMRs across all autosomes using our approach opens the way for identifying and validating markers associated with subchromosomal copy number changes, such us clinically relevant microdeletions and microduplications."} +{"text": "Sci., 2018, 9, 4071\u20134082.Correction for \u2018Thermoreversible crystallization-driven aggregation of diblock copolymer nanoparticles in mineral oil\u2019 by Matthew J. Derry The authors regret that in The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "The FruM proteins have a 101 amino acids (a.a.)-long extension at the N-terminus which is absent from FruCOM. We suggest that this N-terminal extension might confer male-specific roles on FruM interaction partner proteins such as Lola, which otherwise operates as a transcription factor common to both sexes. FruM-expressing neurons are known to connect with other neurons to form a sexually dimorphic circuit for male mating behavior. We propose that FruM proteins expressed in two synaptic partners specify, at the transcriptional level, signaling pathways through which select pre- and post-synaptic partners communicate, and thereby pleiotropic ligand-receptor pairs for cell-cell interactions acquire the high specificity for mutual connections between two FruM-positive cells. We further discuss the possibility that synaptic connections made by FruM-positive neurons are regulated by neural activities, which in turn upregulate Fru expression in active cells, resulting in feedforward enhancement of courtship activities of the male fly.The The genfru gene spans over 150 kb of the genome, and harbors at least four promoters, P1\u2013P4 in females of the number of X-chromosomes over the number of autosome pairs is 1.0 or larger, the cell adopts the female fate, whereas, when the X/A value is 0.5 or smaller, the cell adopts the male fate. Counting of the relative numbers of X-chromosomes is performed by a transcriptional two-directional switch at the Sex-lethal (Sxl) gene, which is transcribed only when X/A exceeds 1.0. Thus the Sxl gene typically produces the Sxl protein only in XX individuals. The female-specific Sxl protein functions as a splicing regulator that induces female-specific splicing of its target, the transformer (tra) gene primary transcript. Only a transcript spliced in the female pattern can encode a functional Tra protein, which in turn induces female-specific splicing distinct from a default splicing that occurs in males in its targets, e.g., the primary transcript from the P1 promoter of the fru gene (fru-P1). Upon binding to the Tra target motif in the fru-P1 primary transcript in neurons and neuron-restricted FruM (P1 products) expression do not necessarily mean that FruCOM is \u201cnon-neural.\u201d Lee et al. (P1-dependent fru-positive neurons (hereinafter fru[+]-neurons) that are sexually dimorphic derive from multiple neuroblasts rather than a few dedicated neuroblasts: in fact, all type II neuroblast lineages bring about sexually dimorphic fru[+]-neurons leads to an increase in the proportion of female-typical neurons at the expense of the male-type neurons in the mAL cluster acts on Or47b olfactory neurons in mature adult males to boost their ligand sensitivity, making these elder males more successful in copulation than younger males (Lin et al., fru[+] neurons called P1 neurons (Chen et al., IR52a-expressing fru[+]-chemosensory neurons on the wing margin mediate input to stimulate male-male courtship (He et al., fru expression in the IR52a-sensory neurons as positive regulators for male-male courtship is also modulated by neural activities during the adult stage.The nervous system of holometabolous insects such as Jallon, . Notably Jallon, . These uon Hall, . Recent fru gene produces two major protein groups: FruM and FruCOM. The FruM proteins have an N-terminal extension that FruCOM proteins lack, but we do not know how important this structural difference is in terms of the protein functions. The expressions of the FruM and FruCOM proteins are mutually exclusive both spatially and temporally (e.g., neuroblasts vs. neurons in the postembryonic nervous system; Sato et al., The robo1 gene, a direct target of FruM (Ito et al., robo1 in males, whereas truncated Lola inhibits full-length Lola\u2019s action to repress robo1, with the result that the ipsilateral neurite forms in males but not females (Sato et al., fru mutations that lost FruCOM while retaining FruM proteins (Song et al., fru mutants (Song et al., Molecular studies on the actions of FruBM protein have revealed that this protein forms a transcriptional complex with an isoform of Lola, a pleiotropic transcription factor, to transcriptionally repress the robo1 gene is the sole established target of FruM (more specifically, FruBM; Ito et al., The The loss of FruM expression by the olfactory receptor mutations observed in adult pheromone neurons (Hueston et al., fru gene became potentiated to achieve a specialist role\u2014i.e., a neural masculinizer role\u2014by creating structurally distinct FruM proteins in addition to FruCOM proteins. We assume that FruM proteins specify coherent signaling pathways in the pre- and postsynaptic neuron pair to form a Fru-labeled neural circuit. This circuit is probably consolidated by the fly\u2019s experience via use-dependent synaptic enhancement. However, this model describing how the actions of fru could induce adaptive changes in the nervous system of a fly during its individual lifetime remains to be tested in future experiments.These considerations prompt us to speculate that the DY: conceptualization, review and editing. KS and DY: funding acquisition and writing the original draft. JG: experimental work. KS: result analysis and visualization.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "The electrophysiology of the paralimbic network (\u201cdefault mode\u201d) for self-awareness has drawn much attention in the past couple of decades. In contrast, knowledge of the molecular organization of conscious experience has only lately come into focus. We here review newer data on dopaminergic control of awareness in humans, particularly in self-awareness. These results implicate mainly dopaminergic neurotransmission and the control of GABAergic function directly in the paralimbic network. The findings are important for understanding addiction, developmental disorders, and dysfunctional consciousness. Self-awareness is a conscious experience with the self as an object. It is an essential part of conscious experience of the world, and is a tool for conscious self-monitoring and for controlling behavior. Its default may have grave consequences. The \u201cneural correlates\u201d of self-awareness have been studied by several investigators, including Devue and Br\u00e9dart and D\u2019Aractive during self-awareness , we have realized that a paralimbic network is indeed instrumental in self-awareness: the network is not only 1 and D2 receptor agonist pergolide is effective in increasing confidence in seeing words and in improving performance in a forced-choice word recognition task. This demonstrates neurotransmitter regulation of subjective conscious experience of perception and provides the first direct evidence that dopamine is instrumental in conscious experience (Lou et al., Functional brain imaging has indicated that abnormal conscious experiences in schizophrenia, like hallucinations and delusions, are associated with abnormal dopaminergic neurotransmission (Changeux and Lou, The site of dopamine-GABA interaction for self-awareness in the human brain was unknown until recently. To clarify this issue, we have used a PET ligand for GABA receptor binding. With this ligand, we were able to detect changes in dopamine-induced GABA binding under different physiological conditions at well-defined brain sites (Lou et al., The GABA receptors are constructed as ligand ion channels. According to Stephens et al. , they arIn premature infants, functional MR imaging together with diffusion tensor imaging-based tractography has been used to study the relationship between performance on the Bayley Scales of Infant Development and early myelination (Cui et al., Newborn infants already show a form of \u201cbasic consciousness\u201d by establishing rudimentary eye contact with their mother (Lagercrantz and Changeux, A recent review showed that the default mode network follows an inverse U-shape, where it is weaker in children and elderly and stronger in adults. Cognitive function is positively correlated with default mode network functional connectivity (Mak et al., In later childhood and adulthood, disturbance of the paralimbic network is linked to severe pathology. Thus, deficient GABA neurotransmission is prominent in disorders with poor self-awareness and self-monitoring such as addiction (Lingford-Hughes et al., To determine if deficient GABA neurotransmission in pathology could be a primary event or secondary to toxic or pathological effects in more complex disorders, we examined if deficient dopamine-GABA neurotransmission was present in a relatively mono-symptomatic disorder such as gambling disorder (M\u00f8ller et al., dopamine. It is also a prominent site of cholinergic activity. Interaction between the cholinergic and dopaminergic system via GABA receptors has been well described (Changeux and Lou, The basal forebrain part of the system is not only regulated by The widespread dysfunction of self-awareness in disease is likely to be a consequence of the exceedingly high oxygen demand of the paralimbic network. The high oxygen requirement is considered to be the result of dense concentrations of parvalbumin GABAergic interneurons in the richly connected hubs of the paralimbic network. In particular, the fast gamma oscillations are susceptible to metabolic disruptions because of their high energy-demand (Kann et al., via GABA receptors in the paralimbic network. This finding has already resulted in a large and promising study of disabled persons with faltering self-awareness and consciousness (Sanz et al., Until recently, conscious experience and self-awareness were considered off-limits for the natural sciences. Neurobiological research shunned the \u201chard question\u201d of how conscious experience and self-awareness arise from a physical basis. Hence, it has been fashionable to limit neuroscience to try to identify neural \u201ccorrelates\u201d of conscious experience and self-awareness. The risk is evident for arriving at two parallel worlds: a mental and a physical, without understanding how they interact. This limitation has impeded our understanding of the biological function of self-awareness, and how it may account for disease. We have here reported data showing that self-awareness and conscious experience can be disturbed by electrophysiological manipulation of the paralimbic network (Lou et al., HL conceptualized the review and wrote the initial draft. KR and J-PC participated in writing the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "We assessed whether the risk of various psychotic disorders and non-psychotic bipolar disorder (including mania) varied by migrant status, a region of origin, or age-at-migration, hypothesizing that risk would only be elevated for psychotic disorders.We established a prospective cohort of 1 796 257 Swedish residents born between 1982 and 1996, followed from their 15th birthday, or immigration to Sweden after age 15, until diagnosis, emigration, death, or end of 2011. Cox proportional hazards models were used to model hazard ratios by migration-related factors, adjusted for covariates.migrants: 2.20, 95% CI 1.96\u20132.47; aHRchildren : 2.00, 95% CI 1.79\u20132.25), affective psychotic disorders , and other non-affective psychotic disorders . For all psychotic disorders, risks were generally highest in migrants from Africa and elevated at most ages-of-migration. By contrast, risk of non-psychotic bipolar disorders was lower for migrants overall, and across all ages-of-migration except infancy , while risk for their children was similar to the Swedish-born population .All psychotic disorders were elevated among migrants and their children compared with Swedish-born individuals, including schizophrenia and schizoaffective disorder (adjusted hazard ratio [aHR]Increased risk of psychiatric disorders associated with migration and minority status may be specific to psychotic disorders, with exact risk dependent on the region of origin. The Cause of Death register was used to obtain the date of death and the Migration Register was used to record date of migration. Individuals who died emigrated for the final time, or who were diagnosed with a psychiatric outcome before age 15 were excluded.et al., We linked participants to the National Patient Register to determine a psychiatric diagnosis in either inpatient or outpatient settings according to the International Classification of Diseases, 10th revision [ICD-10]. We studied four psychiatric outcomes: schizophrenia or schizoaffective disorder ; affective psychotic disorders , other non-affective psychotic disorders and; bipolar disorder or manic symptoms without confirmed psychotic symptoms . For individuals who received diagnoses on multiple visits to in- or outpatient services, we adopted a hierarchal classification system, informed by clinical expertise and consistent with earlier research children of migrants if they were born in Sweden with one or both parents born outside of Sweden, and; (iii) Swedish-born if they were born in Sweden to two Swedish-born parents. The region of origin was defined by Statistics Sweden as Sweden, Finland, other Nordic countries, other European countries, Asia and Oceania, the Middle East, Africa, North America, South America, and unknown, based on country of birth. For migrants, we categorized age-at-first-migration into five groups: infancy (0\u20132 years), early childhood (3\u20136 years), middle childhood (7\u201312 years), adolescence (13\u201318 years) and early adulthood (19\u201329 years). We considered sex, current age, and follow-up period (see below) as a priori confounders. We also controlled for income in supplemental analyses, obtained from the Longitudinal Integration Database for Health Insurance and Labour Market Studies [LISA]. Since 1990, LISA has estimated total disposable family income from all sources each year , weighted for family size. As age 16 is the earliest an individual is included in the LISA, for most participants, we utilized information on their family income at age 15 via linkage to their parents, including adoptive parents where relevant. For migrants arriving in Sweden after age 15, we included their family income as first recorded in the LISA database. Where no income could be estimated, participants were excluded from these analyses (see below). We calculated income quintiles in each year for the entire population and assigned this value to participants in the year of their cohort entry. This method implicitly takes income inflation into account.We defined migrant status according to information in the Total Population and Multi-Generational registers. Participants were classified as (i) et al., et al., We first generated descriptive characteristics of the sample. Next, for each outcome, we investigated whether incidence varied by migrant status, region of origin, and age-at-migration using Cox proportional hazard regression. We estimated unadjusted and adjusted hazard ratios [aHR] and 95% confidence intervals [95% CI] for each exposure. To account for possible period effects , we split the data into 5-year bands of calendar time , modelled as a time-varying covariate. We also modelled age as a time-varying covariate , given risk of psychiatric disorders varies substantially by age , being highest amongst the Swedish-born population (59.2%) and lowest amongst migrants (45.0%). As expected, migrants with psychiatric diagnoses were more likely to be in the lowest income quintile (30.2%) compared to 5.3% of Swedish-born, and 8.6% of children of migrants . Additional cohort characteristics are presented in We identified 1\u00a0796\u00a0257 individuals who contributed over 12.79 million person-years of follow-up . Of thesRisk of all psychotic disorders was elevated among migrants and their children compared with Swedish-born individuals, after adjustment for confounding. For example, risk of schizophrenia and schizoaffective disorder was approximately doubled in migrants and their children , with similar results obtained for other non-affective psychotic disorders . Risk ofThe excess risk of psychotic disorders in migrants persisted for participants from all regions of origin . Thus, fadolescence: 2.38, 95% CI 1.79\u20133.16; aHRearly adulthood: 2.77, 95% CI 1.64\u20134.69). For other non-affective psychotic disorders risk was elevated at all ages-of-migration, except early adulthood . Risk of affective psychosis was elevated among those who migrated in infancy , adolescence and early adulthood , but not childhood. In contrast, bipolar disorder without psychosis was associated with lower risks at all ages-of-migration, except during infancy .Risks of all psychotic disorders were elevated across most ages-of-migration compared with the Swedish-born population . For schThese associations were partially attenuated following adjustment for income, but this did not substantially alter our findings (online Supplementary Tables S1\u2013S3). A sensitivity analysis excluding possible prevalent cases in migrants did not change the pattern of our results (online Supplementary Tables S4\u2013S6). Schoenfeld tests and examination of log-log residual plots revealed no evidence of departure from proportionality across our main exposures for schizophrenia or affective psychosis (online Supplementary Table S7). These tests did suggest a departure from proportionality for our other two outcomes, but log-log residual plots revealed these effects were small and the departure from a zero-slope was negligible .This is the first longitudinal study to investigate how migrant status, region of origin, and age-at-migration affect the risk of schizophrenia, schizoaffective disorder, affective psychotic disorders, other non-affective psychotic disorders, and non-psychotic bipolar disorder. We discovered distinct signatures of risk, which varied according to the presence or absence of psychosis. Thus, migration-related exposures substantially increased the risk of psychotic disorders, albeit with more attenuated effect sizes for affective psychoses. In contrast, non-psychotic bipolar disorder showed a markedly different pattern, with generally lower risks across our three migration-related exposures compared with the Swedish-born population. Our results were impervious to adjustment for income and were unlikely to be explained by prevalent cases amongst migrants, or by age, sex, or period effects.et al., et al., We used to register data from a nationwide cohort of nearly 1.8 million people, with nearly complete coverage, and virtually no loss-to-follow-up. Clinical recording of schizophrenia spectrum disorders is known to be highly complete in the registers with good validity , which may have introduced bias vis-\u00e0-vis residual confounding; from the available data, however, any confounding effect appeared modest, and income may lie on the causal pathway between migration and mental health, making adjustment inappropriate.et al., et al., et al., et al., et al., Further sensitivity analyses suggested our results were unlikely to be attributable to prevalent cases amongst migrants, arguing against selective migration, consistent with previous observations (Selten et al., et al., et al., et al., et al., et al., et al., et al., Our results are consistent with previous research demonstrating an increased risk of schizophrenia-spectrum disorders among migrants and their children (Lloyd et al., et al., et al., Comparisons with previous findings with respect to age-at-migration and psychotic disorders require careful attention. In one study (Veling et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., The different signatures of risk we observed between psychotic and non-psychotic disorders with respect to migration suggest that exposure to adversities related to migration and minority status could act specifically on psychotic rather than affective pathways. Emerging research supports the hypothesis that psychotic and non-psychotic bipolar disorder may have distinct neurodevelopmental origins (Murray et al., et al., et al., Repeated exposure to factors associated with migration \u2013 including social disadvantage, trauma or discrimination experienced prior to, during, or following migration \u2013 may also prime individuals to expect a greater level of threat from the environment (Reininghaus et al., et al., et al., et al., et al., We should also give credence to another possible explanation of our results: misdiagnosis. It has been suggested that higher rates of psychotic disorders in some ethnic minority groups are due to diagnostic bias, with people from the majority group less likely to receive a stigmatizing diagnosis such as schizophrenia compared with bipolar disorder (Schwartz and Blankenship, The shared patterns of risk across three categories of psychotic disorders with respect to various migration-related exposures suggest that migration may act specifically on psychotic rather than affective pathways to disorder. This provides potentially important clues to the aetiology of serious mental illnesses and should galvanise efforts to identify the exact social, environmental, and biological determinants of this preventable, gross inequality experienced by migrant and minority populations (Kirkbride,"} +{"text": "Microtubules (MTs) play a fundamental role in many vital processes such as cell division and neuronal activity. They are key structural and functional elements in axons, supporting neurite differentiation and growth, as well as transporting motor proteins along the axons, which use MTs as support tracks. Tau is a stabilizing MT associated protein, whose functions are mainly regulated by phosphorylation. A disruption of the MT network, which might be caused by Tau loss of function, is observed in a group of related diseases called tauopathies, which includes Alzheimer\u2019s disease (AD). Tau is found hyperphosphorylated in AD, which might account for its loss of MT stabilizing capacity. Since destabilization of MTs after dissociation of Tau could contribute to toxicity in neurodegenerative diseases, a molecular understanding of this interaction and its regulation is essential. Tau is a microtubule-associated protein assembled head-to-tail, which form a pseudo helical lattice , 269\u2013284 (in R2), and 300\u2013313 (in R3) is proposed, based on the attenuation of Tau NMR signal upon addition of paclitaxel-stabilized MTs was used to compare Tau binding mode to Taxol-stabilized MTs and to tubulin when Tau is used as the sole inducer of the polymerization of RB3 . Change in the isoform ratio has an indirect impact on MT assembly and the dynamics of the MT networks because the 3R Tau is known to have a lower capacity of MT stabilization and tubulin polymerization than the 4R Tau (Scott et al., in vitro assays. Surprisingly, R406W Tau is reported to be the most affected, despite the fact that the mutation is not near the MTBR (Hong et al., in vitro assays. However, there is no agreement on the extent of the specific effect of each of these mutations (Barghorn et al., Tau protein is encoded by the in vivo using Paclitaxel treatment in AD mouse models (Cash et al., One of the proposed strategies in seeking AD treatment consists of compensating the loss of the MT-stabilizing Tau function (Cash et al., Drosophila model of tauopathy (Quraishe et al., MT stabilizing peptides are another option chosen to restore MT stability (Quraishe et al., Drosophila, HDAC6 null mutation rescues MT defects through increased tubulin acetylation (Xiong et al., Finally, MTs could be stabilized not by mimicking MAP function, but by modulating MT PTMs, which have a crucial role in MT dynamics. Levels of total \u03b1-tubulin are reduced by approximately 65% in AD-patient brains compared to age-matched control brains but the relative ratio of acetylated tubulin is increased by approximately 31% compared to the controls (Zhang F. et al., Overall, Tau implication in neurodegenerative diseases, and other diseases where MTs play an important role, clearly shows the interest of the Tau/MTs interaction as a potential target for intervention (Pachima et al., The content of the manuscript was drafted and edited by all authors.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "N- and O-sulfation and N-acetylation patterns. Its low concentration in crude extracts, containing other heterogeneous GAGs, leads to a purification process that is very complex, and which is well-guarded by manufacturing companies. Van der Meer et al. [The seven reviews and the eleven articles in this special issue provide an updated survey of recent research and developments in the ever-growing field of heparin, along with low molecular weight heparins (LMWHs) and glycosaminoglycans (GAGs). The complex biosynthetic process, and the variability of tissues and animal species, has led to heparin chains heterogeneous in size and both r et al. , throughAs a consequence of the \u201cheparin crisis\u201d in late 2007, an updating of heparin pharmacopeia monographies in the USA and the EU with new NMR and HPLC tests increased the quality control capabilities for crude and API porcine heparins, with some limitations in detecting the addition of non-porcine crude heparins or other GAG-like contaminants. An improvement to this process [Lima M. et al. reviewedTwo Italian teams review tThe interactions and binding sites of heparin/HS with BMPs and cytokines of the TGF-\u03b2 superfamily are reviewed by Rider C. and Mulloy B. . The actIn a collaborative study of 6 laboratories in the USA, Europe and India [An article reports 2 cells.Three Italian groups exploredA collaborative study by an Israeli and Italian team led to tThe review by authors from the Departments of Neurosurgery at two US Universities suggestsA Belgian team (Minet V.) reviewedCompositional analysis of both LMWH Dalteparin and Danacis-Pt, have been studied \u201cin vitro\u201d and in xenograft models [cis-Pt resistance in a cancer-resistant cell line. In vitro preliminary studies show that Tinzaparin has no effect on cis-Pt accumulation in cis-Pt-resistant xenografts but strongly increases the Pt content in non-cis-Pt-resistant ones.The interactions of Tinzaparin, a LMWH used as antithrombotic prophylaxis in clinical oncology with et al.) . In vitrComponent fractionation of Semuloparin, an ultra LMWH obtained by a depolymerization process preserving the AT binding region, has allowed a team at Sanofi to isola\u00ae, have also been defined on the basis of a determination of single-crystal X-ray conformation. Quantitative NMR were also used, confirming that this method shows intrinsic robustness for content determination [An extended physico-chemical characterization of Fondaparinux, the synthetic \u03b1-methyl glycoside of the AT binding pentasaccharide, and the active ingredient of the anticoagulant drug Arixtra et al.) .A team from Heidelberg University has syntAuthors from the Trondheim University (Norway) (Arlov \u00d8. and Skj\u00e5k-Br\u00e6k G.) review t"} +{"text": "Plants are the primary source of human food and animal feed and also form the basis of numerous industrial and pharmaceutical products. This special issue reflects the diversity of modern research in the field of plant molecular genetics. It covers a wide range of modern technologies and scientific approaches that aim to achieve a better understanding of the various aspects of molecular mechanisms underpinning the key traits in major crops and other commercially important plant species. Phytophthora capsici in Chili Pepper\u201d reported on 11 novel molecular markers targeting resistance to the soil-borne pathogen Phytophthora capsici in chili pepper. The markers developed through high-resolution melting analysis, represent an excellent tool for marker-assisted selection. Another type of molecular markers, SSR, was used for the study of genetic diversity in cultivated and wild melon . The authors found enormous genetic variability within the collection, and deployment of the well-known SSR markers enabled the generation of a phylogenetic tree showing the relationships between melon accessions. An investigation presented by R. Ben Ayed and A. Rebai revealed a significant link between the five SNP markers analysed and the biochemical composition and quality of olive fruits.Three papers in this issue deal with the development, study, and application of molecular markers in plant breeding. N. Kim et al. in \u201cDevelopment of Clustered Resistance Gene Analogs-Based Markers of Resistance toLinum usitatissimum L.)\u201d presented by G. S. Krasnov et al. reported valuable results from RNAseq analysis and candidate gene identification for flax genotypes tolerant or sensitive to a high concentration of Al. The authors' conclusions on glutathione metabolism, oxidoreductase, and transmembrane transporters can be further applied both in academic study and in practical breeding. Very few investigations have been conducted on plant growth in the absence of gravity during space-shuttle orbit around our planet. The paper presented by O. Yu. Yurkevich et al. (\u201cMolecular Cytogenetics of Pisum sativum L. Grown under Spaceflight-Related Stress\u201d) describes a chromosome analysis of pea progenies derived from plants grown in space using a novel FISH approach. Minor chromosome rearrangements were observed in response to \u201cspaceflight-related stress,\u201d which could lead to better guidelines for biological experiments with plants during future space-shuttle missions.Several of the presented reports are related to the genetic control of plant response to abiotic stresses. Drought is one of the major challenges being faced in agriculture. The paper presented by N. M. Kamal et al. (\u201cStay-Green QTLs Response in Adaptation to Post-Flowering Drought Depends on the Drought Severity\u201d) provides important findings on QTLs identified in sorghum grown in Sudan (Africa) under drought conditions. The presented data, generated in cooperation with the International Atomic Energy Agency (IAEA), Austria, can be used for the improvement of grain yield in drought-prone environments using a \u201cstay-green\u201d approach through the application of QTL analysis. Tolerance to aluminum toxicity represents an entirely different type of abiotic stress, but this too is an important agricultural problem, especially in acidic soils. The paper \u201cAluminum Responsive Genes in Flax .\u201d This is an extremely intricate cytogenetic study of the centromeric nucleosomes in dividing cells. The authors presented some critical results from CENH3 gene analysis, a central player in this very complex cytological trait, offering promising potential outcomes.IAEA also supported a study on the generation of mutant bread wheat with improved micronutrient content in the grain. S. Kenzhebayeva et al. presented their paper \u201cMutant Lines of Spring Wheat with Increased Iron, Zinc, and Micronutrients in Grains and Enhanced Bioavailability for Human Health,\u201d where stable breeding lines of wheat were produced from the M Phaseolus beans using diverse characteristics derived from the seed morphology of these species. L. Sinkovi\u010d et al. presented their paper \u201cMorphological Seed Characterization of Common (Phaseolus vulgaris L.) and Runner (Phaseolus coccineus L.) Bean Germplasm: A Slovenian Gene Bank Example,\u201d which contained an interesting comparison of seeds, accompanied by color images. The presented findings are essential for a better understanding of the wide diversity of bean species, which can be directly used for genetics and breeding of these nutritionally important crop varieties. In contrast, another paper \u201cIn Silico Genome-Wide Analysis of the ATP-Binding Cassette Transporter Gene Family in Soybean (Glycine max L.) and Their Expression Profiling,\u201d presented by A. K. Mishra et al., reported on computer analyses of available databases concerning the important gene family of the ATP-binding cassette transporter, in soybean. The authors provided a very accurate and detailed analysis of all identified genes and protein isoforms, arranging these new findings within the context of other pertinent information and comparisons.A remarkable level of genetic polymorphism was found in a germplasm collection ofP. Soundararajan et al. presented a detailed review entitled \u201cInsight on Rosaceae Family with Genome Sequencing and Functional Genomics Perspective.\u201d The Rosaceae family is one of the most significant plant families, and the fruits, berries, flowers, and many other important parts of these plants are familiar to each of us from early childhood. The authors have provided a comprehensive overview on the modern level of genetic knowledge and technology employed in comparative genomics and functional genome analysis for many Rosaceae species. Whole genome sequences in this plant family are so important for genetics, breeding, and agriculture that the presented review will surely be of broad general interest to future investigations in this area.In conclusion, the review presented by N. Borisjuk et al., entitled \u201cGenetic Modification for Wheat Improvement: From Transgenesis to Genome Editing,\u201d summarizes the attempts and results of wheat improvement using various genetic engineering approaches. The authors reflect the progress achieved since the dawn of genetic transformation up until the recent emergence of precise modern genome editing using CRISPR/Cas9 and related systems in wheat. This comprehensive review can provide a strong stimulus for both current and future researchers to pursue work on wheat improvement, and deploy all available modern technologies to secure a sustainable supply of quality agricultural produce for our growing global population.Yuri ShavrukovNikolai BorisjukNarendra K. Gupta"} +{"text": "Several physiological processes such as fertilization, apoptosis, muscle contraction, neuronal activity, and sensory perception are based on the spatial and temporal variation in intracellular Ca2+ and rely on a class of proteins that specifically respond to these highly dynamic stimuli. Neuronal calcium sensor (NCS) proteins are exclusively expressed or enriched in neurons, and their structural and biochemical diversity reflects the multiplicity of their biological roles, which include control of gene transcription, neuronal growth and survival, channel and receptor regulation, neurotransmitter release, synaptic plasticity, and regulation of enzymatic activity. Neurological disorders and neurodegenerative diseases are increasingly associated with altered functions of specific NCS proteins. This Research Topic includes original articles and reviews that provide an interdisciplinary collection of up-to-date biochemical and biophysical research on NCS proteins and their established and novel biological roles in normal and altered conditions.The precise detection and regulation of free intracellular CaRatai et al. show that NCS-1 plays an important role in adipocyte function and its deficiency gives rise to obesity and diabetes type 2 in adult mice, thus suggesting a potential genetic link between psychiatric disorders and the risk of being obese. The progressive degeneration of dopaminergic neurons within the Substantia nigra is the hallmark of Parkinson's disease and causes its motor symptoms. The link between dopamine release and NCS-1 and its possible implications in Parkinson's disease has been reviewed by Catoni et al., who summarize the role of the interplay between Ca2+ and dopamine signaling in neuronal activity and cell death. Simons et al. provide novel data at the transcript level that link NCS-1 deficiency to impaired ATP-production and elevated metabolic stress in Substantia nigra dopaminergic neurons in mice. An overview of other novel roles of NCS-1 is provided by the review of Nakamura et al., which focuses on both the neuronal system and the heart, presenting NCS-1 as a regulator of voltage-gated Ca2+ channels, ionotropic dopamine receptors, and inositol 1,4,5-trisphosphate receptors. A complex interplay between Mg2+, Ca2+ and Zn2+ binding to NCS-1 leads to the appearance of multiple protein conformations and modulate its functional status as suggested by Tsvetkov et al., who demonstrate that NCS-1 binds Zn2+ with differential affinities favoring either the interaction with targets or protein aggregation. Choudhary et al., who focus on optical tweezers investigations to reveal a complex folding mechanism underlaid by a rugged and multidimensional energy landscape, provide insight into the structural and mechanistic details of the folding and misfolding processes of NCS-1 at the single molecule level.Neuronal calcium sensor-1 (NCS-1) is highly conserved from yeast to human, and it has been implicated in a number of psychiatric conditions including autism, bipolar disorder, schizophrenia, and X-linked mental retardation. At odds with other members of the NCS family, NCS-1 interacts with several cellular targets, which is reflected by a variety of roles. 2+-binding motifs and amino acids involved in target recognition has been investigated by Marino and Dell'Orco, who suggest for NCS-1, recoverin and GCAP1 an evolution-driven correlation between the amino acids mediating many persistent interactions and their conservation. An inter-domain interaction triggered by Mg2+-binding is essential for the ubiquitous Ca2+ sensor CIB2 to reach a fully functional conformation, as shown by Vallone et al., who found that the apparently conservative E64D mutation associated with Usher Syndrome 1J and non-syndromic hearing loss prevents this long-range allosteric mechanism. Mutations in calmodulin, another ubiquitous Ca2+ sensor, were long thought to be incompatible with life due to the completely conserved amino acid sequence across all vertebrates. The review by Jensen et al. provides an overview of the human missense mutations found in patients with severe cardiac arrhythmias.The importance of allosteric interactions between Ca2+ dependent. The review by Ames summarizes the results of recent studies on GCAPs, VILIP1 and recoverin dimerization. A paradigmatic example of the myristoyl-switch protein, recoverin is involved in the regulation of the phototransduction cascade in rods and cones as reviewed by Zang and Neuhauss. Four different recoverin isoforms exist in zebrafish photoreceptors. Their specific Ca2+-sensing properties and conformational changes have been investigated by Elbers et al., who found that binding of Ca2+ leads to less pronounced structural rearrangements compared to the bovine ortholog indicating either a modified Ca2+-myristoyl switch or no switch at all. Novel roles for recoverin in health and disease-associated conditions have been found by Zernii et al. who provide in vitro and in vivo evidence that illumination of the mammalian retina leads to the accumulation of disulfide dimers of recoverin, which are thought to favor light-induced oxidative stress and photoreceptor apoptosis.Many NCS proteins form dimers, a process that is often Ca2+. Rehkamp et al. present a chemical cross-link/mass spectrometry investigation on the interaction between GCAP2 and GC-E, while Wimberg et al. investigate five recently identified GC-E mutants associated with Leber Congenital Amaurosis, a cone-rod dystrophy, finding severe alteration of the cGMP synthesis. Another prototypical guanylate cyclase, the atrial natriuretic factor receptor guanylate cyclase is found to be regulated by the Ca2+ sensor neurocalcin \u03b4 and hormone ANF via two distinct and non-overlapping transduction modes, as elucidated by Duda et al.The complex between GCAPs and the retinal guanlylate cyclases is a crucial component of the vertebrate phototranduction machinery as it regulates the interplay between the second messengers cGMP and Ca2+-binding proteins have been found to be involved in the complex etiology of psychiatric disorders. By combining a stereology-based approach and molecular analyses Lauber et al. investigated the involvement of parvalbumin in autism spectrum disorder, finding a dysregulation of its expression in Cntnap2 knockout mouse. The review by Mundhenk et al. presents a detailed structural and biophysical characterization of the Ca2+-sensor proteins caldendrin and calneuron-1 and\u22122, focusing also on their cellular function and their role in neuropsychiatric disorders.Ca2-domain protein otoferlin in modulating the Ca2+-triggered exocytosis at the ribbon synapse in mouse inner hair cells is proposed by Takago et al. by a combination of electrophysiology and biochemical analyses. The importance of Ca2+-binding proteins in regulating the fundamental process of exocytosis and synaptic coupling is emphasized in the review by Maj et al., focusing on secretagogin and its novel roles in developing and adult neuronal cells and Bornschein and Schmidt, focused on synaptotagmin 1 and 2 and their role in presynaptic voltage-gated Ca2+ channel regulation.A role for the multi CPeraza et al. identifies by biochemical and biophysical investigations a novel ligand of DREAM that modulates Kv4 potassium channels currents, with consequences that are relevant for physiology and disease. The role of DREAM/KChIP3 in pain transmission and its possible involvement in emotional processing was studied by Guo et al., who assess the pain sensitivity and negative emotional behaviors of Kcnip3\u2212/\u2212 rats and find a possible role for the protein in central nociceptive processing. Novel tools to regulate the role of DREAM in the endoproteolysis of endogenous presenilin-2 in mouse brain are presented by Naranjo et al., who suggest that the interaction between the two proteins may have a therapeutic potential in Alzheimer's disease. Finally, the review by N\u00e9ant et al. summarizes the current knowledge regarding Ca2+ signaling in quiescent glioblastoma stem-like cells and discussed how Ca2+ via KCNIP proteins may affect gene expression in glioblastoma.Downstream Regulatory Element Antagonist Modulator (DREAM)/KChIP3 exerts multiple functions, including the regulation of A-type outward potassium currents. The contribution by Taken together, this Research Topic delivers new visions to our knowledge on NCS proteins and will stimulate future research. We wish to thank all the authors for having submitted papers of high quality and all the reviewers that contributed with constructive and fruitful suggestions.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Mops condylurus) trapped in the city of N\u2019Zerekore and in a nearby village.In 2018, a previously unknown Ebola virus, Bombali virus, was discovered in Sierra Leone. We describe detection of Bombali virus in Guinea. We found viral RNA in internal organs of 3 Angolan free-tailed bats ( Ebolavirus (family Filoviridae), Bombali virus (BOMV), was discovered in Sierra Leone (Chaerephon pumilus (little free-tailed bat) and Mops condylurus (Angolan free-tailed bat). Both bat species are widespread in Africa, and their ranges include countries where human Ebola virus disease (EVD) outbreaks have occurred. Forbes et al. and in a pool of liver and spleen tissues (Ct 28.2) of an M. condylurus bat from a school in the city of N\u2019Zerekore in March 2019 in China, and another study showed that 3 distinct groups of unclassified filoviruses are circulating in Eonycteris spelaea and Rousettus spp. fruit bats in China (Mar\u00ed Sa\u00e9z et al. (EBOV IgG was detected in the human population of Sierra Leone in 2006, 8 years before the EVD outbreak began in that country (M. condylurus bats provides additional confirmation that BOMV could amplify in these bats and that this species is a reservoir host of this virus.Although BOMV had been detected in the northern part of Sierra Leone (Mops condylurus bats, Guinea.Additional information on Bombali virus in"} +{"text": "Dear Editor,in vitro techniques of liver . In recal., 2013; Grinberal., 2013; Leist eal., 2013; Ghallabal., 2013), kidneyal., 2013; Jiang eal., 2013; Su et aal., 2013; Valenteal., 2013; Lee et al., 2013), neuronal., 2013; Yang etal., 2013; Colaianal., 2013; Sisnaisal., 2013) and deval., 2013, 2016[22al., 2013; Waldmanal., 2013). Stem cIndices have been developed to identify a possible hazard of developmental toxicity based on genome-wide expression data. A precondition is the definition of so-called developmental genes of a test system. Developmental genes are up- or down-regulated during the differentiation process in the absence of test compounds. Developmental potency describes the fraction of all developmental genes, whose expression is altered by test compounds (Shinde et al., 2017; WaldmanMuch progress has been achieved in analyzing disturbed developmental processes. However, it still remains challenging to differentiate stem cells to adult cell types that closely resemble the corresponding mature cell type in vivo (Godoy et al., 2015; Cameronin vitro, but there is still a long way to go until the techniques are ready for routine use and to replace animal studies in pharmacology and toxicology. Stem cell-based alternative test methods offer powerful tools to analyze developmental toxicity The author declares no conflict of interest."} +{"text": "Candida albicans and Staphylococcus aureus interactions, one from the Candida perspective [Staphylococcus perspective (Todd and Peters) [Aspergillus fumigatus and Pseudomonas aeruginosa [Candida albicans and Pseudomonas aeruginosa (Fourie and Pohl) [Cryptococcus neoformans (Mayer and Kronstad) [Candida albicans and the oral bacterial microbiome that contribute to the pathogenesis of oral candidiasis (Bertolini and Dongari-Bagtzoglou) [We would like to thank all the contributors to the Special Issue on Fungal\u2013Bacterial Interactions\u2014Current Knowledge and Future Perspectives. In total, seven 7) reviews/perspective papers were published in the issue. A wide range of various fungal\u2013bacterial interactions were covered. Two papers covered et al.) , and the Peters) . Another et al.) . Other snd Pohl) , bacterironstad) , and dystzoglou) . Lastly, reviews/"} +{"text": "Gracilibacteria (formerly GN02) and Saccharibacteria (formerly TM7) of the recently described Candidate Phyla Radiation and not to Gracilibacteria as reported by Espinoza et al. (Saccharibacteria genomes showed a 7-fold increase in the number of single-copy core genes (To briefly demonstrate their composite nature, we refined some of the key Espinoza et al. MAGs through a previously described approach and the a et al. . A pangere genes . These fre genes . Composire genes recruiteCo-assembly of a large number of metagenomes that contain very closely related populations often hinders confident assignments of shared contigs into individual bins. Nevertheless, even when proper refinement is not possible, reporting composite MAGs as single genomes should be avoided. As of today, highly composite Espinoza et al. MAGs (The rapidly increasing number of MAGs in public databases already competes with the total number of microbial isolate genomes , and inc7\u2013"} +{"text": "Ad-HBV is a product T101) aiming to treat chronic HBV firstly developed by Transgene S.A., France then by Transgene Tasly (Tianjin) Biopharmaceutical Co., Ltd., Tianjin, China in China. So, in the originally published article [01 aimingAdditionally, in the \u201cAcknowledgements\u201d section in the originally published article, \u201cThe Ad-HBV were a kind gift from Xia Meng (Transgene Tasly (Tianjin) Biopharmaceutical Co., Ltd., Tianjin, China). We want to thank Zhiming Wang and Xiaomin Wang (Transgene Tasly (Tianjin) Biopharmaceutical Co., Ltd., Tianjin, China) for teaching neutralization activity detection of anti-Ad antibodies\u201d should be replaced by \u201cWe want to thank Transgene Tasly (Tianjin) Biopharmaceutical Co., Ltd., to provide us the opportunity to participate in the non-clinical safety assessment of T101, and most of the research work appeared in this paper came from the non-clinical safety assessment of T101\u201d."} +{"text": "Protein kinases represent a large and diverse multi-gene family of enzymes, which catalyze the transfer of the \u03b3-phosphate group from its natural co-substrate adenosine triphosphate (ATP) to a free hydroxyl group of an amino acid side chain. They are involved in numerous cell signaling pathways. Diseases might arise when deregulation or mutation of a kinase takes place. Therefore, kinases are promising drug targets for the treatment of several disorders ranging from cancer, autoimmune pathologies, inflammation, or neurodegenerative diseases. After the approval of imatinib in 2001, 38 kinase inhibitors were introduced to the market by the beginning of 2018. Numerous kinase inhibitors are currently in clinical trials.Structural data\u2014e.g., from the structural genomics consortium (SGC)\u2014as well as the high-quality kinase probe programs from the Chemical Probe Portal and the SGC kept pushing forward both the identification of new kinase targets and the design of novel kinase inhibitors within the last years. The numbers of reported type I\u00bd, type III, and type V kinase inhibitors as well as covalent kinase inhibitors have continuously increased in the last few years.This Special Issue deals with the design and development of novel inhibitors addressing different kinases as well as with strategies to inhibit kinases. The Special Issue consists of eight original research articles and four reviews, as briefed below.Brenner et al. described the efficacy of six inhibitors of CDC25, dual-specificity phosphatases that activate cyclin-dependent kinases, on acute myeloid leukemia cells isolated from patients . The resIn their study, Halekotte et al. designed and synthesized optimized 4,5-diarylimidazoles as ATP-competitive inhibitors of CK1\u03b4 and discussed their structural relation to the p38\u03b1 mitogen-activated protein (MAP) kinase . The resb]pyrazine-based FGFR inhibitors starting from a c-Met inhibitor [Zhang et al. described the ligand-based design of pyrrolo2,3--bpyrazinAn analysis of kinase inhibitors and their specificity with respect to their scaffold diversity is described by Dimova and Bajorath . Yang et al. reported the synthesis of novel pyrrolidone-fused methylpyrrole derivatives and their biological evaluation as potential multi-target tyrosine kinase receptor inhibitors . An exteIn their study, Tegethoff et al. showed that 1-methylisoindigo is a Stat3-related inhibitor of various tyrosine kinases . The autThe metabolic stability of two potent pyridinylimidazole-based p38\u03b1 mitogen-activated protein (MAP) kinase inhibitors ML3403 and LN950 was improved by Heider et al. . The novThe number of both approved kinase inhibitors and kinase inhibitors in clinical trials increases continuously. In their research article, Carles et al. presented an extensive analysis of their monthly-updated Protein Kinase Inhibitor Database (PKID) . The PKIIn their review, Lee at al. summarized recent advances in the targeting of the p38\u03b1 MAP kinase as a potential strategy for the treatment of Alzheimer\u2019s disease . InhibitLee et al. discuss the pro- and anti-tumorigenesis functions of senescence as well as the roles of kinase modulators in these processes . An overIn their comprehensive review, Cheng et al. summarized the recent progress of MEK inhibitors and focused on marketed MEK inhibitors and inhibitors in clinical trials . ClinicaSchmidt et al. presented a timely review on the two members of the WEE kinase family, WEE1 and PKMYT1 . Both ki"} +{"text": "This research describes how Department of Veterans Affairs (VA) and non-VA home-based long-term care (LTC) environments prepared for and secured the safety and wellbeing of elderly and disabled persons in the wake of Hurricane Maria, which struck Puerto Rico on September 20, 2017. In-person interviews, home visits, and field observations were conducted in Puerto Rico from January-March 2019. Staff from three of the VA\u2019s Caribbean Healthcare System\u2019s home-based LTC programs were interviewed, as well as caregivers in non-VA LTC environments. Veterans, family members of Veterans, family members of VA caregivers, and community members with expertise in disaster recovery were also interviewed for a total of N = 58 interviews and N = 12 home visits. Preliminary results of qualitative content analysis show VA and non-VA LTC environments prepared residents and caregivers for Hurricane Maria through providing education and recommendations in advance, including having enough medications and food on hand. Participants described Hurricane Maria not simply as a disaster but a \u201ccrisis\u201d and a storm unlike any they had ever experienced. The interconnected nature of the VA seemed to provide a stronger support network compared to non-VA environments that were often independently run. Health of Veterans and non-Veterans was reported to be mostly stable during recovery. Perspectives from VA and non-VA entities allowed for a fuller picture to emerge around how Hurricane Maria impacted home-based LTC environments in Puerto Rico. This research can inform policies and procedures for such environments caring for elderly and disabled populations in areas prone to disasters."} +{"text": "The tumor microenvironment (TME) constitutes an important component of any cancer. The histology of TME consists of a series of normal resident cells and a variety of recruited cells, which are involved in complex and dynamic interactions with cancer cells . These iSachs et al., describe a novel method for overcoming this problem by demonstrating, in different cancer models, the ability of new small molecules to reverse chemotherapy resistance. Munoz et al., address the problem of chemoresistance of glioblastoma multiforme (GBM), a fatal malignancy of the central nervous system. They show that MiR-93 and\u2212193 are specifically expressed in temozolomide (TMZ) resistant glioblastoma cells (GBM), including resistant neurospheres from a patient with TMZ resistance. Part of the resistance occurs by miR-93 and\u2212193 decreasing Cyclin D1 to reduce GBM cycling. This may open up avenues for new therapeutic approaches to reverse such chemoresistance . Besides chemoresistance in GBM, expansion of residual glioma cells is also a challenge in the context of chemotherapy. Work by Tsidulko et al., indicates that conventional anti-glioblastoma therapy such as TMZ affects proteoglycan structure and composition of normal brain tissue. Proteoglycan alteration may be involved in brain extracellular matrix (ECM) deterioration and the development of residual glioma cells .In this Research Topic, diverse facets of the TME are addressed, including different tumor types and tissue specificity of TME . RecentlLadomersky et al., demonstrate a significant association between advanced age of patients and immunosuppression in the circulation and central nervous system. Based on their findings, the authors propose that normal human aging suppresses immune surveillance and immunotherapeutic efficacy against glioma, and thus, contributes to GBM initiation and progression .Although immune checkpoint blockade therapy has afforded new hope for the durable treatment of many cancers, it has not provided yet substantial benefit to patients with glioblastoma. Utilizing a series of epidemiological, genome expression, and immunome databases, Yang et al., focus their study on bronchoalveolar lavage fluid-derived exosomes, released in the TME and demonstrate their role in fueling inflammation and tumor invasiveness via mast cells/neutrophils activation and cytokines release in TME. In a gastric cancer study, Zhao et al., analyze the role of NEDD9 in cancer cell migration under hypoxia. They demonstrate that NEDD9 regulates MICAL1, which facilitates hypoxia-induced gastric cancer cell migration in a Rac1-dependent manner .Using a lung cancer model, Tuo et al., address the role of the cytoplasmic isoform of phosphoenolpyruvate carboxykinase (PCK1) in the progression of hepatocellular carcinoma (HCC). PCK1 is a rate-limiting enzyme in gluconeogenesis which occurs mainly in the liver. The authors find that the expression of PCK1 is down regulated in HCC and associated with poor outcome. In hepatoma cells, the authors demonstrate that reactive oxygen species (ROS) production and nuclear translocation of Nrf2 and thioredoxin reductase 1 (TXNRD1) are suppressed during PCK1 overexpression. Furthermore, the authors show that targeting the TXNRD1 antioxidant pathway sensitizes PCK1-knockout hepatoma cells to sorafenib treatment in vitro .Hu et al., investigate the effect of berberine (BBR) in gastric cancer (GC). BBR is a natural isoquinoline alkaloid, presumably involved in lipid metabolism and glucose homeostasis by regulating the expression of HNF4\u03b1. The authors show that BBR inhibits the proliferation, invasion, and migration of GC cell lines, and thus reduces GC tumor growth in vivo. The antitumor effect of BBR shown appears to involve the AMPK-HNF4\u03b1-WNT5A signaling pathway . Using scutellarin, an active flavone extracted from Erigeron breviscapus Hand-Mazz (EBHM), Sun et al., investigate cisplatin resistance in non-small cell lung cancer (NSCLC). The authors find that scutellarin sensitizes tumor cells to cisplatin by enhancing apoptosis and autophagy via downregulation of p-AKT and c-Met in autophagy and caspase-3-dependent apoptosis. They suggest that the combination of cisplatin and scutellarin may be a novel therapeutic strategy for patients with NSCLC .The role of natural products in the modulation of the TME is likewise addressed in this Research Topic. Tirado-Hurtado et al., highlight the role of DNA Damage Inducible Transcript 4 (DDIT4) in cancer. The DDIT4 gene is expressed under stress situations, turning off the metabolic activity triggered by the mammalian target of rapamycin (mTOR). The authors propose targeting DDIT4 for the development of novel drugs that could be more specific and efficient than current mTOR inhibitors .The impact of DNA damage on TME is also investigated. Chulpanova et al., address the use of MCSs for drug delivery in oncology. The authors focus on MSC and tumor interactions, which are crucial for cancer control. They also describe novel therapeutic strategies using MSCs and MSC-derived membrane microvesicles for cancer therapy .Mesenchymal stem cells (MCSs) play important role in the TME . ChulpanIn conclusion, investigation of TME continues to be a vital focus toward the elaboration of novel strategies that produce more effective treatments for localized cancers and metastases. Findings presented in this Research Topic have the potential to make a major impact on this field and to inspire further discoveries.AL drafted the manuscript. AL, MB, KC-S, and AZ critically reviewed the manuscript for important intellectual content and approved it for publication.AL and KC-S are Founders and Shareholders of Lambda Pharmaceuticals. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "To identify which patient-related effect modifiers influence the outcomes of integrated care programs for type 2 diabetes in primary care.Integrated care is a widespread management strategy for the treatment of type 2 diabetes. However, most integrated care programs are not tailored to patients\u2019 needs, preferences and abilities. There is increasing consensus that such a patient-centered approach could improve the management of type 2 diabetes. Thus far, it remains unclear which patient-related effect modifiers should guide such an approach.PubMed, CINAHL and EMBASE were searched for empirical studies published after 1998. A systematic literature review was conducted according to the PRISMA guidelines.In total, 23 out of 1015 studies were included. A total of 21 studies measured the effects of integrated diabetes care programs on hemoglobin A1c (HbA1c) and three on low-density lipoprotein cholesterol, systolic blood pressure and health-care utilization. In total, 49 patient characteristics were assessed as potential effect modifiers with HbA1c as an outcome, of which 46 were person or health-related and only three were context-related. Younger age, insulin therapy and longer disease duration were associated with higher HbA1c levels in cross-sectional and longitudinal studies. Higher baseline HbA1c was associated with higher HbA1c at follow-up in longitudinal studies. Information on context- and person-related characteristics was limited, but is necessary to help identify the care needs of individual patients and implement an effective integrated type 2 diabetes tailored care program. More than 90% agreement was reached. Therefore, the remainder of the titles and abstracts were screened by 1 reviewer (D.H.). Second, the first 20 full texts were screened independently by two reviewers (D.H. and A.E.). Again, more than 90% agreement was reached and therefore, each reviewer independently screened half of the full texts. Third, the reference lists of the included studies were screened to obtain additional studies. Steps 1 and 2 of the study selection process were then repeated.Descriptive data on studies were extracted by 1 reviewer (D.H.) between August and October 2015. Studies were coded for author names, year of publication, country, study design, length of follow-up, population size, age, percentage of males and CCM components. In case of uncertainties, a group discussion was held with two other authors (A.E. and M.B.).et al., The Effective Public Health Practice Project Quality Assessment Tool (EPHPP) was used to assess the quality of the included studies (Armijo-Olivo Two reviewers (D.H. and M.B.) independently performed the quality assessment for each study. Disagreements were resolved via discussion conform EPHPP guidelines.The included studies were categorized according to: (1) the reported outcome(s) of interest ; and (2) the type of patient characteristic(s) investigated in subgroup analyses. Characteristics were classified as person-related (predisposing), context-related (enabling) or health-related (illness level) characteristics according to Andersen and Newman\u2019s Behaviorn=453), and/or type of care was not integrated (n=257). After the title, abstract and full text screening, 27 studies were included , blinding (n=9) and selection bias (n=9). Almost all studies (n=25) scored high on the domain data collection. The overall study quality was strong for four studies, moderate for 11 studies and low for 12 studies. Most studies with low quality had a cross-sectional study design and did not report on or adjust for possible confounders.The methodological quality of the included studies can be found in Supplementary Table 1. The domains with the most \u2018weak\u2019 ratings were confounders (n=20) was 15 months (range 6\u2013112). The median sample size consisted of 376 individuals (range 80\u2013105 056) with an average age of 60.0 years (range 50.5\u201370.9); the percentage of male subjects ranged from 31.3 to 68.0.Of the included studies, nine (33.3%) were retrospective cohort studies, seven (25.9%) cross-sectional studies, seven (25.9%) (randomized) controlled studies and four (14.8%) prospective cohort studies. n=25), followed by self-management support (n=20). Of the studies that included the components delivery system design, most introduced a care team (n=13), followed by regular follow-up visits (n=8). Self-management support was mostly realized through individual educational sessions on diabetes, health and nutrition (n=14).In total, 18 uncontrolled studies \u2013 including prospective, retrospective and cross-sectional cohort designs \u2013 measured the effects of integrated care programs on HbA1c. In addition, seven studies compared the influence of patient characteristics on the effectiveness of integrated diabetes care programs between intervention and control groups. In total, 51 patient characteristics were assessed as potential effect modifiers of the relationship between integrated care and HbA1c. The results will be presented according to study design. For RCTs all characteristics assessed by this study design will be discussed. Due to the high number of characteristics assessed by the cross-sectional, retrospective and prospective cohort studies, only characteristics assessed by three or more studies will be presented.(Randomized) controlled trials: Five RCTs and two controlled trials (CTs) compared the influence of patient characteristics on the effectiveness of integrated diabetes care programs on the HbA1c level between intervention and control groups . In totaet al., et al., et al., et al., et al., Sex and age were the person-related characteristics evaluated as potential effect modifiers. Three studies assessed sex as a potential modifier, of which two found that women in the intervention group had statistically significant lower HbA1c values at follow-up compared to women in the control group , depression and diabetes mellitus (DM) duration. Each characteristic was assessed by one study. Patients with high FBG (>10 mmol/L) at baseline receiving integrated diabetes care had significantly lower HbA1c levels at follow-up compared to patients receiving usual care and sex (n=9). In seven studies the effect of integrated diabetes care programs on HbA1c differed significantly across ranges of age: younger patients had higher HbA1c levels at follow-up compared to older patients (n=5) and experienced greater change from baseline in HbA1c (n=2) , baseline HbA1c (n=7) and duration of type 2 diabetes (n=6). The effect of integrated diabetes care programs on HbA1c was different for people on insulin therapy. These patients had higher HbA1c levels at follow-up compared with patients on diet and/or oral therapy in five studies , body mass index (BMI) (n=6) and sex (n=5). Four studies of integrated care programs found non-significant associations between age and HbA1c and medication use (n=4). The effect of integrated care programs on HbA1c differed significantly across ranges of diabetes duration in four studies (De Alba Garcia et al., et al., et al., et al., et al., et al., et al., et al., Most examined health-related characteristics were duration of type 2 diabetes (No context-related characteristics were assessed by three or more studies.Three prospective and retrospective cohort studies measured the effect of integrated diabetes care programs on LDL-c. The RCTs and cross-sectional studies included in this review did not measure this effect. In total, 11 patient characteristics were assessed by the studies. Only those results that were assessed by at least two studies will be discussed.et al., et al., Prospective and retrospective cohort studies: The person-related characteristic age was examined by three studies (Sperl-Hillen and O\u2019Connor, et al., The modifying effect of baseline LDL-c on the relationship between integrated diabetes care programs and changes in LDL-c over baseline was assessed by two studies (Sperl-Hillen and O\u2019Connor, No context-related characteristics were assessed by the included studies.Four retrospective and prospective cohort studies measured the effect of integrated diabetes care programs on SBP. In total, nine patient characteristics were assessed by the studies. Only those results that were assessed by at least two studies will be discussed.et al., et al., et al., et al., et al., et al., et al., et al., Retrospective cohort and prospective cohort studies: Age was measured by three studies (Mold et al., et al., et al., et al., et al., et al. (et al., et al. found that the effect of integrated diabetes care programs on health-care utilization was different between males and females (Liu et al., Health-care utilization was assessed by three studies: one RCT (Nielsen , et al. found th et al., . For malThis paper presents a literature review on relevant patient characteristics for guiding tailored integrated type 2 diabetes care in primary care. HbA1c was considered an outcome in 93% of the 27 studies identified. Many different patient characteristics were investigated by these studies. Findings indicate that the effect of integrated primary care programs on HbA1c differs significantly according to a number of person and health-related characteristics. Younger age, longer disease duration, higher baseline HbA1c and insulin therapy were associated with higher HbA1c levels. Health insurance status, living situation and income were the only context-related characteristics in the included studies and were not frequently assessed.Compared to HbA1c, LDL-c, SBP and health-care utilization were included far less. It was found that higher baseline LDL-c lead to greater LDL-c improvement. Patients with higher age had higher SBP levels at follow-up as well as greater improvements in SBP compared to younger patients. The relationship between integrated care and health-care utilization seemed to be modified by sex: women had more consultations per year compared to men.et al., et al., \u03b2 cell function and pancreatic insulin secretion, resulting in the need for a more complex and intensive drug therapy (Al Omari et al., et al., et al., et al., et al., et al., et al., Several factors might explain the elevated HbA1c levels in a subset of patients with type 2 diabetes. Younger patients tend be more non-adherent to oral medication therapy and experience less profound diabetes-related health problems than older patients (Pyatak et al., \u03b2-cell dysfunction (Heianza et al., et al., et al., High HbA1c at baseline also seemed to be predictive of later HbA1c. First, type 2 diabetes is a heterogeneous disease in both pathogenesis and clinical manifestation (Inzucchi et al., et al., et al., Several factors could explain the differences in levels of LDL-c, SBP and health-care utilization between levels of patient characteristics. Prescription of statins usually follows when LDL-c level is 2.5 mmol/L or higher, possibly leading to greater improvements in LDL-c for those patients with high baseline LDL-c levels (The Dutch college of general practitioners, et al., et al., et al., et al., Overall, our results indicate the need to implement integrated diabetes care programs specifically tailored to the needs, values and preferences of younger patients and to those on insulin therapy, with longer disease duration and/or higher HbA1c levels and older patients with high SBP levels. These effect modifiers can help to provide the right care to the right person at the right time. At this moment, not every patient with these characteristics receives such care. Current practice might therefore not be suitable for all patients. Lack of motivation, family support and feeling burned-out from managing diabetes are reported barriers to optimal self-management (Browne et al., et al., et al., et al., This review has several limitations that should be taken into account. First, given the scarceness of studies assessing the differences in the effect of integrated diabetes care programs on diabetes control measures by levels of patient characteristics, it was decided to include RCTs, prospective and retrospective cohort studies. However, this introduced significant heterogeneity and made it impossible to conduct a meta-analysis. Second, quality of the studies was weak for most studies. This was mainly due to the cross-sectional study design of more than one-third of the studies and the use of less robust statistical methods. Fortunately, it is unlikely that these studies altered the results, as their findings were similar to those of the other, more robust studies. Third, very few context- and person-related characteristics were analyzed. Studies performed in a non-integrated diabetes care setting, found that context-related characteristics, such as socio-economic status and social network, are associated with measures of diabetes control and are likely to be strong predictors of diabetes control (Jotkowitz et al., The current review provides a good understanding of which characteristics can help to identify patients with different health-care needs and preferences. However, to implement an effective integrated type 2 diabetes tailored care program, it is necessary to know which context- and person-related characteristics are important to identify patients. Furthermore, implementation of an effective tailored diabetes care program is only possible by taking into account the care preferences of patients and caregivers. In the next phase of the PROFILe project (Elissen"} +{"text": "Dear Editor,n\u2009=\u200943) or omnivorous diet (n\u2009=\u200946). The research compared biochemical parameters and erythrocyte parameters, as well as estimated energy and nutrient intakes, between the two groups. The vegetarian children had a two-fold decrease in serum hepcidin level accompanied by decreased ferritin level and slight but statistically significant increase in concentration of soluble transferrin receptor (sTfR), but no differences in concentration of hemoglobin, mean corpuscular volume, iron, and transferrin compared to the omnivorous group. Moreover, vegetarian children had comparable total iron intake but consumed more (approx. by 30%) ascorbic acid in food [We read with great interest the paper by Ambroszkiewicz et al. entitled in food . The papOwing to the increasing popularity of vegetarian diets, their clinical consequences are becoming clearer and include such potential health benefits as decreased all-cause mortality and decreased risks of obesity, type 2 diabetes, and coronary heart disease . HoweverMost data on the health effects of vegetarian diets were collected from adults, so the study by Ambroszkiewicz et al. , despiteThe study by Ambroszkiewicz et al. reportedp\u2009<\u20090.01) and decreased hepcidin level in lacto-ovo-vegetarian children. Based on these findings, the authors suggest that the vegetarian children may suffer from subclinical iron deficiency. Their view is supported by two references in which similar trends in hepcidin and sTfR levels were found in children diagnosed with iron deficiency [Ambroszkiewicz et al. noted thficiency , 15. Howficiency . Using tficiency , only foWe would like to suggest that instead of diagnosing a potential disease condition in the lacto-ovo-vegetarian children, the biochemical changes could be considered a form of adaptation wherein a slight increase in sTfR and two-fold decrease in hepcidin as observed by Ambroszkiewicz et al. are evidFinally, simple measures can be taken to enhance the availability of dietary iron in children or adults who do develop clinical iron deficiency on vegetarian diets. The consumption of diets containing ferritin-rich seeds provides iron in a more bioavailable form than other vegetables . In someIn summary, there is no evidence that the adaptive changes described by Ambroszkiewicz et al. in veget"} +{"text": "Obesity and metabolic syndrome are considered major public health problems, and their negative impact on cardiovascular disease (CVD) and diabetes mellitus type 2 (DM2) is profound. Targeting modifiable risk factors such as dietary habits is therefore of great importance. Many of today\u2019s health challenges with overweight and obesity may have behavioral roots, and traditional methods such as regulations and campaigns are often insufficient to improve dietary choices. Nudging or choice architecture might be a viable tool to influence people\u2019s everyday choices and behaviors to better outcomes. This paper reviews the current state of the rapidly expanding number of experimental field studies that investigate the effects/associations of nudging on healthy food choices. A systematic literature search was conducted in PubMed, where 142 citations were identified. Based on selection criteria, six randomized controlled trials and 15 non-randomized controlled trials were ultimately included. The results of this systematic review show that many of the studies included traffic-light labeling, which may be a promising strategy. The reviewed findings, however, also highlight the challenges that confront experimental studies examining the impact of nudging on diet. Metabolic syndrome (MetS) affects public health, and has been associated with a doubling of cardiovascular disease (CVD) risk as well as a five-fold increased risk of diabetes mellitus type 2 (DM2) . To be dThroughout the past decades research in behavioral sciences has revealed that human behavior and decision-making is boundedly rational, and as a result, people make suboptimal, often self-destructive decisions . TherefoTo improve health outcomes nudging or choice architecture (a related term) might beSeveral systematic reviews have suggested that nudging may be effective in increasing the consumption of healthy food and decreasing the consumption of unhealthy food: Broers et al., 2017 choice architecture and food, and (3) nudging and healthy food. In order to identify published studies examining nudging and/or the related term choice architecture , versus According to Hollands et al., 2013 . The lasThe majority of studies regarding nudging and/or choice architecture consist of multiple nudges and/or interventions. This makes it a challenge to map the different nudges and to evaluate their effects. However, Hollands et al., 2013 [nudge and food choice), 79 in the second search (choice architecture and food), and 32 in the third search . After screening 74 studies , 62 full-text articles were retrieved and assessed for eligibility. After removing duplicates and those not making it through quality assessment, 21 studies were included in this review. The included studies comprise of six RCTs and 15 non RCTs. A matrix was designed to get an overview over all the included articles. The articles were structured according to reference, participants/site, and results. The study characteristics are provided in The literature search identified 142 citations, of which 31 were found in the first search . The participants were blinded to group assignment. The intervention group received placemats featuring two healthy \u201cKids\u2019 Meals of the Day\u201d upon the restaurant entry, to nudge children toward healthy options. Forty-eight families had looked at the placemat before ordering . After the families finished dining, researchers recorded children\u2019s orders and collected leftovers for quantifying dietary intake via weighed plate waste. Families who were exposed to the study placemats ordered more healthy food compared to controls. However, there were no significant differences in dietary intake when comparing the intervention versus control groups overall. Nevertheless, children who ordered one of the promoted healthy entr\u00e9es consumed less saturated fat across the total meal compared to those who did not (p = 0.04).Anzman-Frasca et al., 2018 investign = 4) or control (n = 10) for five months. Then, the chef schools were further randomized to chef (n = 2) or chef + smart caf\u00e9 (n = 2), and the control schools were further randomized to smart cafe (n = 4) or control (n = 6). In the smart caf\u00e9, vegetables were offered at the beginning of the lunch line. Fruits were placed in attractive containers, or next to the cash registers. Signage and images promoting fruits and vegetables were prominently displayed. White milk selection was placed in front of sugar-sweetened milk . All the modifications were simultaneously present and applied daily by existing food service staff. School food selection was recorded, and food consumption was measured using plate waste methods. The study revealed no association between the smart caf\u00e9 intervention alone and food consumption. Fruit selection increased in the chef , smart caf\u00e9 , and chef plus smart caf\u00e9 schools compared with the control schools. Vegetable selection increased in the chef , smart caf\u00e9 , and chef plus smart caf\u00e9 schools compared with the control schools.Cohen et al., 2015 investign = 4), vegetable (n = 3) or control group (n = 3). However, the paper only focuses on the fruit intervention and control groups. The fruit intervention group made changes to the convenience, visibility, and attractiveness of fruit in their lunchrooms for a period of 6 weeks. The control group made no changes. The selection and plate waste data were assessed. Fruit selection increased overall by 36% (p < 0.001), and fruit consumption increased overall by 23% (p < 0.017) in the fruit intervention group, compared to controls.Greene et al., 2017 conducteHollands et al., 2018 examinedp = 0.044) in energy purchased in the day following the introduction of calorie labeling was found in one site. However, the effect diminished over time. No changes in energy purchased were revealed in the remaining five sites.Vasiljevic et al., 2018 investigVelema et al., 2018 examinedCole et al., 2018 investign = 43, 11\u201322 years) with intellectual and developmental disabilities were included. The intervention included: (i) prompting by \u2018celebrity servers\u2019, (ii) the creation of fruit and vegetable-inspired artwork for the dining hall, (iii) classroom-based taste-testing activities, and (iv) logo naming and branding activities. Selection and plate waste of foods at lunch were assessed. Smarter lunchroom increased selection (whole grains) and consumption of healthy food, and decreased selection and consumption of unhealthy food (refined grains).Hubbard et al., 2015 investigKroese et al., 2016 investign = 4642) of a large hospital in Boston, MA, US, and regular cafeteria patrons. In the first phase, a traffic-light labeling system was introduced to encourage the patrons to purchase healthy items (labeled green) and avoid unhealthy items (labeled red). In the second phase, certain cafeteria items were rearranged, making green-labeled items more accessible and the red-labeled items less accessible. The main outcome measures were proportion of green or red labeled items purchased. Labeling decreased the red item purchases and increased green purchases. The intervention effects were similar across all race/ethnicity and job types.In a 9 months longitudinal study, Levy et al., 2012 investign = 96, BMI = 29.7 \u00b1 6.0 kg/m2) who regularly ate lunch at their workplace cafeteria, were randomly assigned into one of two intervention groups: (1) Environmental change (low-energy-dens foods and food content labeling) or (2) Environmental change, education and pricing incentives. Food intake and energy intake was assessed with scan card technology coupled with computerized cafeteria cash registers. No difference in total energy intake were revealed between the groups over the study period. However, significant changes in energy intake were observed across the groups from baseline to the intervention period, with an increase in the percentage of energy from carbohydrates and a decrease of energy from fat. Lowe et al., 2010 investigNikolaou et al., 2014 investigp < 0.01).Olstad et al., 2014 investigSeward et al., 2016 investigThorndike et al., 2014 investigThorndike et al., 2012 investign = 291) examined the strength of combinations of toppings and bread type, and the main study consisting of a two (bread type) by two (topping type) between-subjects design. The included participants (n = 226) were given a free sandwich at a university stand with an unhealthy deep-fried snack (croquette) or a healthy topping. About half of the participants were offered a whole wheat bun unless they asked for white bun, and the other half were offered a white bun unless they asked for a whole wheat bun. Regardless of the topping, the whole wheat bun was the default option in 94% of the participants. When the default of bread offered was white, 80% of the participants chose the default option. The study revealed a strong default effect of bread type.Van Kleef et al., 2018 investigVan Kleef et al., 2015 investign = 1113) responded to a survey including questions about the breakfast. Results showed that consumption of fun-shaped whole wheat bread rolls almost doubled consumption of whole wheat bread (p = 0.001). However, consumption of white bread rolls did not differ according to shape.Van Kleef et al., 2014 investigVan Kleef et al., 2012 investign = 24, 86%) of french fries consumers noticed the reduction in portion size during the intervention.Vermote et al., 2018 investigAl-Khudairy et al., 2019 have modNudging or choice architecture interventions aim to improve dietary choices, but empirical evidence to support the effectiveness has been scarce. An important criterion for an intervention to qualify as a nudge is that the targeted audience/participants/customers retain their freedom to make a choice . In thisLabeling is the most frequent used nudge in this review and nineteen of the studies include labeling. Previous studies have shown mixed results and labeling has been debated . DespiteAvailability refers to how accessible an object or stimuli are, and rearranging the positions of food is a common nudge, for instance, in food stores. Seven studies used an availability nudge. Placing healthy food near the cash register increase the sale of these food because people are prone to pick up something in the \u201clast-minute\u201d as done in the study by Van Kleef et al., 2012 . HoweverPriming is, according to Marteau et al. (2012) , a promiThe results of this systematic review show that the majority of the studies include traffic-light labeling, and that may be a promising strategy. The results suggest that the majority of the interventions were effective. Thirteen studies measured effects on purchasing healthier food, and nine of them, plus one site in the study of Vasiljevic et al., 2018 , showed In most of the field experiments, the participants were not aware that they were part of an experiment. This limited social influence regarding desirable behavior and observer reactivity . The fieDespite the tremendous interest and studies on nudging and choice architecture regarding public health, it is still a relatively new and under-explored field. Many of the studies reviewed during this systematic review lacked definitional and conceptual clarity that might lead to poor methodology and unclear effects. A benefit of nudging is that it does not requires any additional actions for the individual or the targeted audience. Furthermore, the interventions are usually no cost or low cost, and that makes it easier to implement. Another important issue is that our environment, whether we like it or not, influences our behavior. Then it makes sense to alter the environment in a way that we make better choices that will be beneficial in the long run . Many ofThis systematic review was executed independently by both authors to minimize errors and bias. When there was discrepancy regarding a study, the study was thoroughly discussed based on the inclusion criteria and the quality check list. We prepared a checklist for measuring study quality based on the Downs and Black validated checklist from 1998 , but resNudging and/or choice architecture alone will not solve the worldwide health challenges caused by poor health choices. Although behavioral economics can provide great benefits if appropriately used, it is still necessary with an underlying political fundament . In otheMoving forward, nudging and choice architecture would benefit from a better conceptual clarity and a more systematic approach. There are no \u201cquick fix\u201d or \u201cmagic bullets\u201d when it comes to changing people\u2019s behavior. Additionally, even though it might be challenging, nudging interventions should be compared to more traditional interventions such as information campaigns and educational strategies. Future research should have longer study durations such as Thorndike et al., 2014 , and incThe results of this systematic review show that the effect sizes are very diverse and also low. Many of the studies included traffic-light labeling that might be a promising strategy. Moreover, this study also highlights the challenges that must be addressed when experimental studies concerning nudging are conducted. According to Marteau et al. (2012) , we live"} +{"text": "From their discovery in biological systems, reactive oxygen species (ROS) have been considered key players in tissue injury for their capacity to oxidize biological macromolecules. Aerobic organisms possess a system of biochemical defenses to neutralize the oxidative effects of ROS, but the balance between ROS generation and the antioxidant system is slightly in favor of the ROS so that a continuous low level of oxidative damage exists . When thThe papers reported in this Special Issue deal with different aspects of reactive oxygen species (ROS) actions in living organisms. Some papers consider the role of ROS in inducing cellular dysfunction.2O2 to obtain a cell model of oxidative stress to study the role of sphingomyelin synthase 2 (SMS2) in endothelial disease (ED). They found that SMS2 induces the stress of the endoplasmic reticulum (ER) that leads to ED both activating the Wnt/\u03b2-catenin pathway and promoting intracellular cholesterol accumulation, both of which contribute to the induction of ER stress and finally lead to ED.Thus, Hua et al. treated 2O2 or doxorubicin to verify if trimethylamine N-oxide (TMAO), an organic compound derived from dietary choline and L-carnitine, is a factor involved in the progression of atherosclerosis and other cardiovascular diseases. They show that TMAO does not affect the treatment\u2019s effect on cell viability, sarcomere length, intracellular ROS, and mitochondrial membrane potential. Therefore, they conclude that TMAO cannot be considered a direct cause or an exacerbating risk factor of cardiac damage at the cellular level in acute conditions.Querio et al. used aduAnother work evaluates the role of ROS as agents able to induce cellular protection. Lin et al. demonstrROS can also be involved in the therapeutic action of some antitumoral drugs. Soltan et al. evaluate2O2). Artemisinin prevents cell death at clinically relevant doses in a concentration-dependent manner. Artemisinin restored the nuclear morphology, prevented the increased intracellular ROS, and attenuated apoptosis. These data suggested that artemisinin protected neuronal cells. Similar results were obtained in primary cultured hippocampal neurons. Cumulatively, these results indicated that artemisinin protected neuronal cells from oxidative damage, at least in part through the activation of AMPK. These findings support the role of artemisinin as a potential therapeutic agent for neurodegenerative diseases.The antioxidant capacity to protect against oxidative stress-linked disease has been evaluated by Zhao et al. , who stuMoreover, some reviews are presented in this Special Issue. L\u00e9vy et al. reviewedDamiano et al. examinedXiao and Meierhofer reviewedSiauciunaite et al. summarizThe review of Di Meo et al. analyzedIsmail et al. collecteIt is our opinion that the articles included in this Special Issue, despite dealing with such different topics, represent an important contribution to the knowledge of the physiological and pathological role of ROS, and give some information on the benefits and limitations of antioxidant treatment."} +{"text": "Growing evidence suggests that the mechanical interaction between the cells and their microenvironment is of critical importance to their behaviors under both normal and diseased conditions, such as migration, differentiation, and proliferation. The study of tissue mechanics in the past two decades, including the assessment of both mechanical properties and mechanical stresses of the extracellular microenvironment, has greatly enriched our knowledge about how cells interact with their mechanical environment. Tissue mechanical properties are often heterogeneous and sometimes anisotropic, which makes them difficult to obtain from macroscale bulk measurements. Mechanical stresses were first measured for cells cultured on two-dimensional (2D) surfaces with well-defined mechanical properties. While 2D measurements are relatively straightforward and efficient, and they have provided us with valuable knowledge on cell-ECM interactions, that knowledge may not be directly applicable to in vivo systems. Hence, the measurement of tissue stresses in a more physiologically relevant three-dimensional (3D) environment is required. In this mini review, we will summarize and discuss recent developments in using optical, magnetic, genetic, and mechanical approaches to interrogate 3D tissue stresses and mechanical properties at the microscale.Cells Tissues are composed of a large collection of ECM macromolecules (Frantz et al., Tissues and native ECMs are heterogeneous, anisotropic (Jones et al., The microscale tissue or cellular mechanical properties and constitutive relationships can be probed by active microrheology (Wilson and Poon, ex-vivo tissues (Iwashita et al., By laser tracking the deflection of the probing cantilever tip during indentation and retraction , AFM indin vitro and in vivo.Optical tweezer has been used to probe the viscoelasticity of living cells and tissues (Staunton et al., Micro-injected cell-sized ferrofluid microdroplets can be uECM or tissue deformations provide useful information on the stress states of cells and tissues (Nam and Chaudhuri, TFM is one of the most widely adopted approaches using matrix deformations to compute cell generated traction forces (Dembo and Wang, In principal, TFM can be adapted to 3D (Hall et al., 3D TFM relies mostly on randomly distributed fluorescent beads to compute matrix deformations, which is generally infeasible for tissue deformations. This obstacle can be overcome by monitoring tissue deformations directly after releasing the stored solid stresses (Stylianopoulos et al., in vitro or in vivo (Camp\u00e0s et al., To overcome the complexities associated with the development of 3D TFM, the dependence of the stress calculation on the local tissue mechanical properties must be eliminated. Hence, several recent studies introduced microbead/droplet-based stress sensors of well-controlled mechanical properties to 3D systems. These types of stress sensors can be introduced to virtually any system, in vitro cell aggregates or living embryonic tissues (Camp\u00e0s et al., Cell-sized fluorescent oil microdroplets, with defined mechanical properties and coated with adhesion ligands, can be introduced to in vivo (Mohagheghian et al., In addition to the wide application as a compliant substrate in 2D TFM, elastic polyacrylamide hydrogels have also been used to quantify 3D stresses by creating microbeads that are embedded into tissues to act as cell-like pressure sensors (Dolega et al., Unlike the incompressible microdroplet method which measures anisotropic stresses, the elastic microbead method measures the isotropic stresses using volume strain. With more information about bead deformations, either through surface tracking (Lee et al., Stress sensors can also be engineered to be of the molecular size to detect sub-cellular forces with piconewton sensitivity. Unlike the cell-sized sensor described above, the force-induced displacement in the molecular sensor is readily converted to a shift in emitted fluorescence, such as that observed in FRET . Howeverin vivo applications (Cai et al., Cells can be engineered to directly express FRET-based sensors (Cost et al., Molecular tension sensors can also be functionalized to the substrate to detect cell-matrix forces (Liu et al., Tensions within the plasma membrane can be measured by the tether-pulling method, where controlled forces are applied through functionalized AFM cantilevers or optical/magnetic tweezer beads that are tethered to the membrane (Lieber et al., Unlike the stress probing methods discussed earlier, which are based on either the stress-strain constitutive relationship or the displacement-fluorescence relationship, the last collection of approaches discussed here do not rely directly on strain/displacement information.Fluctuation of a freely diffusing Brownian particle has long been used in passive rheology to measure the particle diffusion coefficient and fluid viscosity (Wilson and Poon, in vitro to in vivo (Scarcelli et al., Mechanical properties and states can also directly affect a material's optical properties, which can therefore be used to probe tissue mechanics. One of such approaches with increasing application in mapping tissue mechanical properties is Brillouin light scattering microscopy based on acousto-optic interaction (Scarcelli et al., With the growing interest in exploring the roles of tissue mechanics in physiology and pathology, there are significant recent advancements in developing tools that can map 3D tissue mechanical properties and stresses at the microscale. Future work may be directed at increasing the throughput and/or accuracy of the 3D mapping of tissue mechanics using existing methods such as active microrheology and stress sensors. Opto-mechanical approaches will provide new insights into tissue mechanics due to their non-invasive, label-free and high-throughput nature. Furthermore, simultaneous mapping of local mechanical properties and measurement of 3D deformations will enable accurate 3D TFM. With our developing knowledge of cellular mechanotransduction, it will be possible to utilize and/or engineer some of the cell's intrinsic behaviors as a type of new mechanical probe, as evidenced by the fluctuation-based approaches. However, caution should also be paid to the discrepancies when interrogating tissue mechanics using different approaches (Wu et al., JZ contributed to the conception of the work and wrote the article. NC wrote the article. CR-K supervised the work and wrote the article.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "In the original article, there was an error. We mistakenly stated that 5-MeO-DMT is part of the ayashuasca brew.Introduction, paragraph one:A correction has been made to the 2A >5-HT2C >5-HT1A receptors (Szabo et al., Ayahuasca, a millenarian decoction used as a sacrament by south American indigenous tribes, known to induce powerful hallucinogenic states when administered with monoamine oxidase inhibitors (MAOI; Ara\u00fajo et al., Ayahuasca is used by many syncretic churches ritualistically, as a way to heal many physical and mental illnesses with or without scientific knowledge about the effects (Frecska et al., Ayahuasca can potentially treat recurrent depression (Os\u00f3rio Fde et al., \u201cPsychoactive tryptamines are a class of molecules that act as a neurotransmitter in the vertebrate brain (Jacob and Presti, Discussion, paragraph three:Additionally, a correction has been made to the Ayahuasca acute administration to depression diagnosed patients (dos Santos et al., Ayahuasca tea, are composed of several psychoactive substances including DMT analogs and MAOi (Frecska et al., Ayahuasca tea the DMT is administrated with MAOi, in order to avoid tryptamines degradation. Using oral or intraperitoneal administration without MAOi may reduce the availability of 5-MeO-DMT to the central nervous system, since the monoamine oxidase will readily destroy any tryptamine, in the bloodstream, guts and also in the brain (Halberstadt et al., per se can increase neurogenesis, at least in vitro cultured hippocampal cells (Morales-Garc\u00eda et al., \u201cThe choice of a single dose treatment, was made to address the gap between the molecular mechanisms, subjective and hormonal effects underlying The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated."} +{"text": "Dear Mr Editor,Herpesviridae reactivation in patients under veno-venous extracorporeal membrane oxygenation (VV ECMO) for severe acute respiratory distress syndrome (ARDS) and raised some important questions. Data available do not allow to conclude once for all, however, we might provide some pieces of answers.Huang et al. recently commented on our publication concerning Herpesviridae reactivation is so common in ARDS patients under ECMO who often combine sepsis and prolonged mechanical ventilation (MV). It would be of high interest to perform a matched controlled study in order to determine if ECMO occurring during ARDS enhances the frequency of Herpesviridae reactivation.First, the \u201cwho?\u201d. In a general population of ARDS patients (without ECMO), Ong et al. showed tHerpesviridae reactivation or active infection in ICU patients is rather touchy. The positivity of biological samples signs the evidence of viral transition from latency to replication, but end-organ disease symptoms can be difficult to identify in such complex patients. One key question would be the comparison of performance diagnosis of blood and airway CMV PCR with antigenemia, which is considered as the reference test in end-organ disease diagnosis. Concerning the timing of HSV and CMV reactivation, Heininger et al. [Second, the \u201chow?\u201d. The precise definition of r et al. , compariHerpesviridae reactivation during ARDS [Herpesviridae reactivation with specific markers such as monocytes HLA-DR expression [Finally, the \u201cwhy?\u201d. Several hypotheses have been stated to explain the poor outcomes of patients exhibiting ing ARDS . Viral ppression .Herpesviridae in ARDS patients, and even more in those under ECMO.To summarize, despite several decades of research, much remains to be done to lighten the role of"} +{"text": "Camellia senenisis plant, is the second most consumed beverage worldwide after water, and is consumed by more than two-thirds of the world population inhibits neuroblastoma cell growth and neurosphere formation in vitro ; the autZhao et al., reported that Fuzhuan brick-tea protects against UVB irradiation-induced photo-aging via MAPKs/Nrf2-mediated down-regulation of MMP-1, and suggested that this tea could be used as not only a functional food but also a good candidate in the development of cosmetic products and medicines for the remedy of UVB-induced skin photo-aging .Annunziata et al., evaluated colon bioaccessibility and antioxidant activity of tea polyphenolic extract by using an in vitro simulated gastrointestinal digestion assay . They foPan et al., report that polyphenols in Liubao tea prevent carbon tetrachloride-induced hepatic damage in mice through their antioxidant function . MoleculShen et al., examined the association between tea consumption and risk of hospitalized fracture in 453,625 Chinese adults. Their study concluded that habitual tea consumption was associated with moderately decreased risk of any fracture hospitalizations, and the participants with decades of tea consumption and those who preferred green tea were also associated with lower risk of hip fracture .Unno et al., determined the stress-reducing function of matcha green tea in both animal experiments and clinical trials . They foRode et al., determined, in a cross-sectional observational study among a population of 273 hypercalciuric stone-formers, whether daily green tea drinkers experienced increased stone risk factors compared to non-drinkers, and found no evidence for increased stone risk factors or oxalate-dependent stones in daily green tea drinkers .Furthermore, Khan and Mukhtar extensively reviewed the health-promoting effects of tea polyphenols , by summIlex, including the large-leaved Kudingcha (Ilex latifolia Thunb and Ilex kudingcha C.J. Tseng), Yerba Mate (Ilex paraguariensis A. St.-Hil), Yaupon Holly (Ilex vomitoria), and Guayusa (Ilex guayusa Loes), and suggested their potential applications in the pharmaceutical or nutraceutical industries [In another review article, Gan et al., summarized the distribution, composition, and health benefits of several caffeinated beverages from the genus dustries .Tea consumption is also considered a natural complementary therapy for neurodegenerative diseases such as Alzheimer\u2019s disease that affects an increasing patient population among the elderly. Polito et al., reviewed epidemiological studies on the association between tea consumption and the reduced risk of Alzheimer\u2019s disease, along with the anti-amyloid effects and the role of tea in preventing this neurodegenerative disease .While beneficial effects by tea consumption have been documented in various human disease models as mentioned above, there are major challenges in developing some tea components (such as green tea polyphenols) as therapeutic agents, including how to improve their bioavailabilities, stability, efficacies, and specificity . FurtherI would like to thank all the authors for their exceptional contributions."} +{"text": "Decades ago, Oriental Medicine doctors could use just traditional tools including acupuncture, moxibustion, and herbal medicine despite its long history. However, technology improves and modern medical devices are invented to assist the Oriental Medicine doctors in performing diagnosis and treatment. Now, they can use exclusive medical devices such as pulse meter, tongue diagnosis, and face system with their own sensors and diagnostic skills to get information of body and disease from patients. They also can use modern therapeutic tools including electroacupuncture, pharmacopuncture, and electric-moxibustion as well as traditional ones. Experimental and translational studies on these new tools are underway.For this special issue, we invited manuscripts with the following topics: improvement of tools in oriental medicine, pharmacopuncture (Chinese medicine injection), electroacupuncture and electric-moxibustion, validation of treatment efficacy, development of pulse meter and tongue diagnosis system, and experimental and translational studies on modern device of Oriental Traditional Medicine. We received 105 manuscripts from various labs for six months and 22 manuscripts were accepted for publication. Here we highlight some of the key ongoing challenges published in this special issue.The efficacy of medical devices using radiofrequency is demonstrated in the papers \u201cMoxibustion-Simulating Bipolar Radiofrequency Suppresses Weight Gain and Induces Adipose Tissue Browning via Activation of UCP1 and FGF21 in a Mouse Model of Diet-Induced Obesity\u201d and \u201cShort-Term Efficacy of Pulsed Radiofrequency Thermal Stimulation on Acupoints for Chronic Low Back Pain: A Preliminary Study of a Randomized, Single-Blinded, Placebo-Controlled Trial\u201d.Granule, one of modern forms of herbal medicine, is used in the studies \u201cStudy of the Treatment Effects of Compound Tufuling Granules in Hyperuricemic Rats Using Serum Metabolomics\u201d and \u201cRandomized, Double-Blind, Placebo-Controlled Study of Modified Erzhi Granules in the Treatment of Menopause-Related Vulvovaginal Atrophy\u201d.The article \u201cCinobufacini Injection Improves the Efficacy of Chemotherapy on Advanced Stage Gastric Cancer: A Systemic Review and Meta-Analysis\u201d reviews the literature on the efficacy comparison between Cinobufacini injection (one of Chinese medicine injections) combined with chemotherapy and chemotherapy solely used in advanced gastric cancer treatment.The protective effect of patches on acupoints against electromagnetic fields is shown in \u201cEvaluation of the Effectiveness of Protective Patches on Acupoints to Preserve the Bioenergetic Status against Magnetic Fields\u201d.The clinical application of the low voltage Meridian Energy Detection System is evaluated in assessing the electrodermal activity in the \u201cThe Development and Application Evaluation of Meridian Energy Detection System in Traditional Oriental Medicine: A Preliminary Study\u201d and the safety of auto manipulation device for acupuncture which can help Oriental Medicine doctors is shown in the \u201cSafety Assessment of the Auto Manipulation Device for Acupuncture in Sprague-Dawley Rats: Preclinical Evaluation of the Prototype\u201d.Different face diagnosis system is used in \u201cAn Herbal Medicine, Yukgunja-Tang is more Effective in a Type of Functional Dyspepsia Categorized by Facial Shape Diagnosis: A Placebo-Controlled, Double-Blind, Randomized Trial\u201d and \u201cDifference between Right and Left Facial Surface Electromyography in Healthy People\u201d.In this special issue, there are more valuable manuscripts besides above. We hope the readers will be interested in improvement of diagnostic and remedial tools of Oriental Medicine."} +{"text": "Vibrio cholerae and downregulate the expression of virulence genes\u2019 by Nicolas Perez-Soto et al., Chem. Sci., 2017, 8, 5291\u20135298.Correction for \u2018Engineering microbial physiology with synthetic polymers: cationic polymers induce biofilm formation in Vibrio cholerae strain used in this work is A1552The The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "There is a significant gap in our understanding of the molecular and cellular biology of cerebrovascular and neurodegenerative diseases to identify new therapeutic targets and develop diagnostic tools to better understand the disease progression and treatment. The cellular and molecular events linking cerebrovascular pathology and neurodegeneration are also not fully understood. The research articles and review papers published in this topic aim at a multifaceted approach to evaluating recent progress in our understanding some of the underlying molecular mechanisms of disease process and potential therapeutics targeting these diseases. Here we summarize the contributing articles to our topic conveying the aim of the pertaining research. The articles in this Research Topic highlight the challenges inspiring future research to address some of the questions and to exploit new opportunities for development of novel therapeutics for cerebrovascular and neurodegenerative diseases.Smith-Dijak et al. studied the effects of pridopidine, a drug that enhances brain derived neurotrophic factor (BDNF) signaling through stimulation of the sigma-1 receptor (S1R) and S1R agonist, in cortical neurons obtained from a mouse model of Huntington disease (HD). Several pathways implicated in synaptic functions are dysregulated in HD, including BDNF and calcium signaling. The data provide evidence for restoration of synaptic plasticity that maintain the stability of neuronal and synaptic function required for new learning and cognitive function. The results suggest a potential new direction for developing therapy to mitigate cognitive deficits in HD and may provide new avenues for neuroinflammation-related disorders treatment.Agouni et al. provided a comprehensive analysis of circulating extracellular vesicles (EVs) from vascular wall, blood, and immune cells in transient ischemic attacks (TIA) and acute ischemic stroke (AIS) patients from Southeast Asia and the Middle East. This study showed that EVs of various origins, especially those associated with endothelial cell injury and platelet activation, are increased in TIA and AIS patients. The levels of EV continue to be high for up to 30-days post-attacks indicating a sustained cellular activation, which may be associated with an increased risk of recurrence of acute events in this population.D'Angelo et al. carried out a review on antiphospholipid syndrome (APS) and multiple sclerosis (MS). APS and MS are both considered as anti-lipid autoimmune diseases with specific pathophysiological mechanisms and events. Isolated neurological APS represents a significant diagnostic challenge, as epidemiological, clinical, and neuroimaging features may overlap with those of MS. The review draws attention to the clinical relevance of diagnosing isolated neurological APS and suggests that prompt and accurate diagnosis and treatment of APS with anti-aggregant and anticoagulant could be vital to prevent or reduce APS-related morbidity and mortality.Wei et al. examined the cellular mechanisms mediating the neuroprotective effects of Homer1a, a short form of a scaffold protein, which is upregulated in rat cortical neurones following oxygen and glucose deprivation (OGD) mimicking ischemia-reperfusion (I/R) injury. The results showed that overexpression of Homer1a reduced OGD-induced lactate dehydrogenase (LDH) release, cell death, and mitochondrial dysfunctions in cultured cortical neurons. Homer1a also protects against OGD-induced injury by preserving mitochondrial function through inhibiting the protein kinase R-like endoplasmic reticulum kinase (PERK) pathway. In addition, mitochondrial protection of Homer1a was blocked by the ER stress activator tunicamycin (TM) suggesting that Homer1a may be a promising target of protecting neurons from cerebral injury.Yang et al. examined the effect of cyclooxygenase (COX2)/prostaglandin D2 (PGD2)-related autophagy on brain injury in diabetic rats suggesting that the COX2-PGD2 pathway is a potential therapeutic target for diabetic brain injury.Xu et al., reported that low-moderate ethanol consumption may prevent ischemic stroke and reduce brain cerebral ischemia/reperfusion injury (I/R) by suppressing inflammation, whereas heavy alcohol consumption may induce ischemic stroke and worsen brain I/R injury by aggravating inflammation.He et al. found that smilagenin, a steroidal sapogenin from traditional Chinese medicinal herbs, can have neuroprotective effect on dopaminergic neurons in a chronic mouse model of Parkinson's disease (PD) suggesting that this drug could prevent the impairment of dopaminergic neurons in PD.Zhang et al. demonstrated that Naringenin (NAR), a grapefruit flavonoid promoted microglia M1/M2 polarization, thus conferring anti-neuroinflammatory effects via the inhibition of mitogen-activated protein kinase (MAPK) signaling activation. These findings provide new alternative avenues for neuroinflammation-related disorders treatment.Hao et al. showed that heterozygous loss of activin receptor-like kinase 1 (Alk1) can lead to hereditary hemorrhagic telangiectasia, which is a vascular disease characterized by direct connections between arteries and veins leading to arteriovenous malformations (AVMs). The results of the study suggest that Alk1 induces the formation of sporadic human cerebral AVMs through affecting migration and proliferation of endothelial cells combined with vascular endothelial growth factor A.Li et al. developed a flow cytometry protocol to identify microglia and monocyte-derived macrophages from mouse intracerebral hemorrhagic (ICH) stroke model induced by collagenase or blood injection. The authors also combined magnetic-activated cell separation system that allows eight tissue samples to be assessed together. This protocol represents a very important tool for biological functions of microglial and monocyte-derived macrophage in ICH stroke and related brain diseases.Jin et al., showed that glucose-regulated protein (GRP78) a chaperone protein located in the endoplasmic reticulum (ER) is involved in the neuroglial response to neurotoxic insult in rats induced by mitochondrial toxin 3-nitropropionic acid (3-NP), which selectively damages striatal neurons. These data provide novel insights into the phenotypic and functional heterogeneity of GRP78-positive cells within the lesion core and the involvement of GRP78 in the activation/recruitment of activated microglia/macrophages and blood-brain-barrier impairment in response neurotoxic insult.Song et al. reported that the knock down of myosin light chain kinase, a key enzyme in smooth muscle cell contraction, in human brain smooth muscle cells (SMCs) caused effects similar to those observed in cultured SMCs from intracranial aneurysm patients. These results indicate that myosin light chain kinase plays an important role in maintaining smooth muscle contractility, cell survival and inflammation tolerance and is crucial to the normal function of intracranial arteries.Hoyk et al. showed elevated serum triglyceride levels, changes in functional and morphological gene expressions and blood brain barrier dysfunction in transgenic mice overexpressing the human APOB-100 protein, a mouse model of human atherosclerosis suggesting that these transgenic mice could be a useful model to study the link between cerebrovascular pathology and neurodegeneration.Yu et al. reported that injection of hydroxysafflor yellow A (HSYA), a major active chemical component of the safflower via carotid artery improves cognitive impairment and synaptic plasticity in a rat model of stroke induced by middle cerebral artery occlusion.Chen et al. showed that Gap19, a selective Connexin 43 (Cx43) -hemi -channel inhibitor, produces neuroprotective effects in cerebral ischemia/reperfusion injury induced by middle cerebral artery occlusion in mice via suppression of Cx43 and Toll-like receptor 4 (TLR4) mediated signaling pathways.Chang et al. developed a new in vitro cerebral micro-bleed model to study the interactions between brain endothelial cells and red blood cells exposed to oxidative stress. Their findings demonstrate that erythrophagocytosis mediated by the brain endothelial monolayer and the passage of iron-rich hemoglobin and RBC, may be involved in the development of cerebral microbleeds that are not dependent on disruption of the microvasculature.Faustino-Mendes et al. reviewed the current experimental models of immature ischemic brain and highlighted the need for new multifactorial experimental models to attain more efficient therapies to treat this complex vascular condition and related long-term conditions.Chen et al. showed that the compound 22a, a promising neuroprotective compound derived from tetramethylpyrazine and widely used as active ingredient of traditional Chinese medicine, effectively prevented glutamate-induced excitotoxicity in cerebellar granule cells (CGNs) via involvement of the PI3K/Akt and PGC1\u03b1/Nrf2 pathways suggesting that this compound might be useful in preventing neuronal death from ischemic stroke.Du et al. tested a hypothesis that chemokine interleukin (IL) eight released by astrocytes and C-X-C motif chemokine receptor 1 (CXCR1) in neurons are involved in neuronal apoptosis induced by methamphetamine (METH), a widely abused illicit drug, which can cause dopaminergic neuron apoptosis and astrocyte-related neuroinflammation. The results suggest that CXCR1 may be a potential target for METH-induced neurotoxicity therapy.Lu et al., aimed to explore the protective effects of rosuvastatin, a 3-hydroxymethyl-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, against haemorrhagic transformation (HT) after recombinant tissue plasminogen activator (rt-PA) treatment in a mouse model of experimental stroke. The beneficial effects are related to inhibition of the inflammation-related nuclear factor kappa B (NF-\u03baB) and mitogen-activated protein kinase (MAPK) pathways.Wang et al. showed that mild ER stress (\u201cpreconditioning\u201d) induced by tunicamycin (TM), can alleviate LPS-induced astrocytic activation and BBB disruption. Their findings provide a better understanding for the regulatory role of ER stress in neuroinflammation and indicate that mild ER stress might have therapeutic value for the treatment of neurodegenerative diseases.Yang et al., found that adapentpronitrile, a new adamantane-based dipeptidyl peptidase-IV (DPP-IV) inhibitor significantly ameliorated neuronal injury and decreased amyloid precursor protein (APP) and amyloid beta (A\u03b2) expression in the hippocampus and cortex of rat model of diabetes fed with high fat diet. These authors showed that adapentpronitrile protected against diabetic neuronal injury by inhibiting mitochondrial oxidative stress and the apoptotic pathway.Jang et al. reported intrastriatal injection of adeno-associated viral vector serotype DJ containing N171-82Q mutant huntingtin (HTT) gene to juvenile mice produced Huntington's disease (HD)-like symptoms including mutant HTT aggregation, neurodegeneration, and Neuroinflammation. The authors suggested that this model will a useful tool to better understand neuropathological mechanisms of HD and develop new therapeutics for this disease.Hao et al. established a stable method for the isolation of endothelial cells (ECs) from human cerebral arteriovenous malformation tissues, which play an important role in the manifestation and development of cerebral vascular malformation as well as haemorrhagic stroke and thrombogenesis. The protocol can also be adapted for other vascular diseases.He et al. demonstrated that rosuvastatin treatment significantly increased neurite outgrowth in cortical neurons after oxygen-glucose deprivation (OGD)-induced damage, reduced the generation of reactive oxygen species, protected mitochondrial function and elevated the ATP levels via Notch1 pathway. These findings highlight Notch1 signaling as important player and novel therapeutic target in promoting brain plasticity.In summary, the present Research Topic encompassed several cutting-edge techniques and models for investigating the cellular and molecular mechanisms of cerebrovascular dysfunction and neurodegeneration. Together, they provide a framework for understanding the way some of the diseases progress and potential pathways for therapeutic interventions. We acknowledged that collection of articles in a single topic cannot deal with this extremely vast subject characterized by complex conditions such as cerebrovascular dysfunction and neurodegeneration. The topics addressed, however, help developing clear ideas, not only in terms of recent studies but also the unmet needs for future research in these areas. We trust that the papers assembled in this Research Topic will prove useful in encouraging and stimulating future progress in research related to cerebrovascular and neurodegenerative diseases.SS prepared the editorial and both SK and SS edited the final version. SS and SK made substantial contributions to the review and approved the manuscripts accepted on this topic.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Progression from latency to active Tuberculosis (TB) disease is mediated by incompletely understood host immune factors. The definitive characteristic of progressive human immunodeficiency virus (HIV) disease is a severe loss in number and function of T lymphocytes. Among the many possible mediators of T lymphocyte loss and ineffective function is the activity of the immune-modulatory enzyme indoleamine 2,3-dioxygenase (IDO). IDO is the rate-limiting enzyme converting tryptophan to kynurenine. IDO activity was initially recognized to mediate tolerance at the foeto-maternal interface. Recently, IDO activity has also been noted to play a critical role in immune tolerance to pathogens. Studies of host immune and metabolic mediators have found IDO activity significantly elevated in HIV and TB disease. In this review, we explore the link between IDO-mediated tryptophan catabolism and the presence of active TB disease in HIV-infected patients. We draw attention to increased IDO activity as a key factor marking the progression from latent to active TB disease in HIV-infected patients. Recently, the effect of indoleamine 2,3-dioxygenase-1 (IDO) activity in various diseases, including tuberculosis (TB) and human immunodeficiency virus (HIV) disease has generated considerable interest. Tryptophan, an essential amino acid in the body, can either be converted to serotonin or oxidized to kynurenines. IDO is the rate-limiting enzyme in the conversion of tryptophan to kynurenines (Higuchi and Hayaishi, Essential micronutrients are those which cannot be produced endogenously and must be acquired through dietary sources. Humans, like other mammals, cannot synthesize tryptophan, and therefore, must obtain it from their diet and internal protein turn-over Brown, . Foods sIDO1 and IDO2 (Yamazaki et al., de novo pathway for NAD+ synthesis when niacin intake is limited in the diet (Higuchi and Hayaishi, In certain cell types, tryptophan can be converted to serotonin or melatonin by tryptophan hydroxylase (Ruddick et al., Munn and colleagues observed IDO expression and activity at the foeto-maternal interface in pregnant mice (Munn et al., Tryptophan depletion and subsequent generation of kynurenine metabolites have considerable effects on the host's immunity. The IDO pathway plays a significant role in T cell hypofunctionality, a critical concept in immune tolerance to pathogens (Mellor et al., Immune cells, in particular macrophages and dendritic cells, are sites of IDO production. IDO expression and activation creates a local immunosuppressive micro-environment, which allows pathogens to escape sterilizing immunity or containment (Nagamatsu and Schust, Downstream metabolites of the IDO pathway such as quinolinic acid, are neurotoxic and have been implicated in HIV-associated dementia (Gostner et al., in vitro (Byrne et al., in vitro studies have reported that measles, influenza, cytomegalovirus, and herpes simplex viral infections are also susceptible to tryptophan depletion (Obojes et al., Amino acids are critical micronutrients for all pathogens. Microbes either synthesize amino acids or depend on their hosts for amino acids for growing and multiplying. The ability of the host to deprive pathogens of essential micronutrients is a crucial feature of innate defense. Tryptophan, just like carbon, iron, and nitrogen are critical for microbial survival (Rohmer et al., In animal models, increased tryptophan metabolism via the IDO pathway has been associated with poor immune responses to M. TB (O'Connor et al., When co-infecting a host, HIV and M. TB influence each other, accelerating deterioration of immune function. HIV infection causes severe loss of T cells, both in number and function (Douek et al., Werner et al. reportedin vitro (Blumenthal et al., in vivo (Gautam et al., Despite extensive work in murine and macaque models of TB, only a few studies have investigated IDO-mediated tryptophan catabolism and its metabolites in human TB . M. TB hIDO can be studied quantitatively by measuring mRNA expression or protein levels or enzymatic activity (product-to-substrate ratio; Li et al., A biomarker is defined as any characteristic that can be objectively measured as an indicator of health or disease process or as a response to a therapeutic intervention (Group et al., ex vivo M. TB challenge in cells or tissue from active or latently infected patients to understand TB pathology. Some studies have examined metabolic profiles or genes from active TB patients to identify metabolites or genes that are switched on or off, which may be implicated in the transition from latency to active disease in both HIV-infected and uninfected individuals (Weiner 3rd et al., in vitro (Zhang et al., Most TB studies have relied on animal models or It remains to be determined whether elevated IDO activity is causative of, or only associated with, progressive HIV and TB disease. Some researchers postulate that human co-existence with latent TB infection may be a human evolutionary adaptation to increase host tryptophan in times of low dietary tryptophan availability (Williams and Dunbar, Chlamydia trachomatis, an intra-macrophage pathogen, which can synthesize tryptophan, blocking IDO activity with levo-1-methyl-tryptophan led to improved susceptibility to doxycycline (Ibana et al., in vivo that M. TB induces IDO expression in the lungs of macaques and mice with active TB disease. In the macaque, inhibition of IDO activity led to a reduced M. TB burden, pathology and improved animal survival (Gautam et al., Currently, several clinical trials are exploring blocking IDO activity for therapeutic use. During infection with In summary, IDO-mediated tryptophan catabolism via the kynurenine-nicotinamide pathway represents a common axis upregulated in HIV infection and TB disease. Rather than being uniquely elevated in TB, cumulative elevation of IDO in HIV and TB may explain the extraordinarily high rate of progression to active TB in HIV-infected patients. It is unclear whether elevated IDO is causative of progression to active disease or a compensatory response to the microbe. There is much promise in using metabolites from the IDO-mediated tryptophan pathway to diagnose active TB and monitor anti-TB treatment. IDO holds promise as a TB biomarker in both HIV-positive and negative patients, but further confirmatory studies are urgently needed. Regarding new TB drug targets, it remains speculative whether IDO inhibition would be beneficial. Further exploration of this pathway is necessary to better understand TB pathogenesis and discover novel host biomarkers and potential drug targets for either TB or HIV disease.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Escherichia coli populations. Toxin\u2013antitoxin systems hold a crucial but still elusive part in bacterial response to stress. This perspective highlights how these modules can also serve as a great model system for investigating basic concepts in gene regulation. However, as the genomic background and environmental conditions substantially influence toxin activation, it is important to study (auto)regulation of toxin\u2013antitoxin systems in well-defined setups as well as in conditions that resemble the environmental niche.Autoregulation is the direct modulation of gene expression by the product of the corresponding gene. Autoregulation of bacterial gene expression has been mostly studied at the transcriptional level, when a protein acts as the cognate transcriptional repressor. A recent study investigating dynamics of the bacterial toxin\u2013antitoxin MazEF system has shown how autoregulation at both the transcriptional and post-transcriptional levels affects the heterogeneity of Escherichia coli K-12 MG1655 strain has at least 37 TA loci in total, with at least 10 toxins characterized as type II toxins with RNA-degrading activity, i.e., endoribonucleases. Type II endoribonucleases can be active independent of ribosomes or in a ribosome-dependent manner, and few of them cleave RNA sequence specifically (Masuda and Inouye Toxin\u2013antitoxin (TA) modules are highly versatile systems, generally widespread in prokaryotic genomes . Overall, the majority of the known type II TA systems are RNA-degrading enzymes, degrading RNA while bound to the ribosome, or in a ribosome-independent manner, such as the MazF toxin regulation in commonly used Measuring toxin and antitoxin levels in single cells in vivo is a challenging task. As toxin and antitoxin proteins form complexes, tagging them with fluorescent reporters would most certainly decrease stability of the complexes and prevent efficient toxin neutralization by the antitoxin. In addition, tagging toxins with fluorescent proteins could interfere with the toxin activity , or as variability in phenotypic traits of the individual cells in time (Ackermann mazEF transcript induced pulse-like behavior and increased temporal variability in MazF levels during stress (Nikolic et al. Several previous studies indicate that the majority of chromosomally encoded type II TA systems elicit phenotypic heterogeneity in stressed bacterial populations by promoting variation in gene expression, cell size, and growth rate (Klumpp et al. mazEF autoregulation is a complex mechanism attained by several protein\u2013protein, protein\u2013mRNA and protein\u2013DNA interactions. Autoregulation of mazEF expression at the transcriptional and post-transcriptional level is responsible for fluctuations in MazF levels during ectopic stress (Nikolic et al. mazG and relA transcripts are parts of the polycistronic mRNA together with mazE and mazF (Gama-Castro et al. mazEFG transcriptional unit (Goormaghtigh et al. relA-mazEF unit (Gama-Castro et al. mazEF expression, will provide necessary details to obtain a full picture of autoregulatory processes. Stress response mechanisms might additionally regulate mazEF expression (Battesti et al. mazEF expression, and to what extent (Yamaguchi and Inouye mazEF expression during physiological induction of the toxin will further determine the importance and impact on this TA system on the behavior of bacterial cells.The"} +{"text": "Sci., 2015, 6, 3242\u20133247.Correction for \u2018Humidity-dependent surface tension measurements of individual inorganic and organic submicrometre liquid particles\u2019 by Holly S. Morris The corrected figure is shown below.An error in The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "The presence of tumor cells in blood vessels, particularly extramural venous invasion (EMVI), is an independent poor prognostic factor in colorectal cancer (CRC).EMVI was present in one-quarter of EC patients after surgery alone, and in 21.6% of patients after nCRT, as confirmed by additional EVG staining.7In future, EMVI should be investigated in a larger group of EC patients undergoing nCRT followed by surgery. Our results in the nCRT group are probably influenced by case mix and less power in a relatively small group with a potential selection bias of non-responders to nCRT."} +{"text": "Background: Space Agencies are planning human missions beyond Low Earth Orbit. Consideration of how physiological system adaptation with microgravity (\u03bcG) will be managed during these mission scenarios is required. Exercise countermeasures (CM) could be used more sparingly to decrease limited resource costs, including periods of no exercise. This study provides a complete overview of the current evidence, making recommendations on the length of time humans exposed to simulated \u03bcG might safely perform no exercise considering muscles only.Methods: Electronic databases were searched for astronaut or space simulation bed rest studies, as the most valid terrestrial simulation, from start of records to July 2017. Studies were assessed with the Quality in Prognostic Studies and bed rest analog studies assessed for transferability to astronauts using the Aerospace Medicine Systematic Review Group Tool for Assessing Bed Rest Methods. Effect sizes, based on no CM groups, were used to assess muscle outcomes over time. Outcomes included were contractile work capacity, muscle cross sectional area, muscle activity, muscle thickness, muscle volume, maximal voluntary contraction force during one repetition maximum, peak power, performance based outcomes, power, and torque/strength.Results: Seventy-five bed rest \u03bcG simulation studies were included, many with high risk of confounding factors and participation bias. Most muscle outcomes deteriorated over time with no countermeasures. Moderate effects were apparent by 7\u201315 days and large by 28\u201356 days. Moderate effects (>0.6) became apparent in the following order, power and MVC during one repetition maximum (7 days), followed by volume, cross sectional area, torques and strengths, contractile work capacity, thickness and endurance (14 days), then muscle activity (15 days). Large effects (>1.2) became apparent in the following order, volume, cross sectional area (28 days) torques and strengths, thickness (35 days) and peak power (56 days).Conclusions: Moderate effects on a range of muscle parameters may occur within 7\u201314 days of unloading, with large effects within 35 days. Combined with muscle performance requirements for mission tasks, these data, may support the design of CM programmes to maximize efficiency without compromising crew safety and mission success when incorporated with data from additional physiological systems that also need consideration. Space Agencies are planning to transition from International Space Station (ISS) missions to Lunar missions including a crewed base from which to test and develop hardware and procedures required for the longer term goal of human Mars missions Foing, . It is wBased on bed rest research and previous spaceflight experience, the ISS provides astronauts with 2.5 h per day for exercise using a treadmill, cycle ergometer and resistance exercise device designed and adapted for \u03bcG to search the following databases up to July 2017: Pubmed, CINAHL, Web of Science, NASA Technical Reports Server and The Cochrane Collaboration Library. No restrictions on type of bed rest or publication dates were applied, and due to the inability to use \u201cBoolean logic\u201d on the NASA Technical Reports Server, the strategy was adapted to keyword searches. The full search strategy is available in A range of relevant terms grouped by main search terms were constructed using Boolean logic (astronauthttps://rayyan.qcri.org/) , blinded to each other's decisions, using Rayyan tool was used to assess risk of bias of all the included studies, with \u201cH,\u201d \u201cM,\u201d and \u201cL\u201d showing high, moderate and low risk, respectively, using pre-defined published definitions for each level were calculated between pre and post-bed rest values for each outcome individually without an overall pooled effect. Hedges' g was used to bias correct for the typically small sample sizes, as only control group data from \u03bcG simulation studies were eventually included. The reported data set that was as close to immediate pre and the end of bed rest was used for the analysis. No exclusion or analysis variation was made based on the individual study analysis methods. The pooled standard deviation for Hedges' g was calculated using the root mean square of the pre and post-group standard deviations. This version does not specifically include the sample size (n), preventing any complications that could arise from inflating n when both group means are from the same sample. Results were first sub-grouped by outcome measure type and then by muscle group before being listed in order of ascending days spent in simulated \u03bcG. Individual effects sizes were calculated and plotted in figures for each outcome at every time point where data were available. To enable a brief overview of the large data set to also be provided, an unweighted mean effect at each common time point within each muscle group was used to provide a summary result. This was only done when more than one study assessed the same outcome at the same time point. These statistics were chosen due to data being from the same sample rather than a separate intervention and control group, thus making a traditional weighted effects meta-analysis pooling inappropriate. Traditional meta-analysis assumes two different sets of individuals in each group or 1.2 (large) was highlighted as a time point when a worthwhile mechanistic change had occurred , (3) muscle activity , (4) muscle thickness , (5) muscle volume (cm3), (6) maximal voluntary contraction force during one repetition maximum (7), peak power (W), (8) performance based outcomes , (9) power , and (10) torques and strength . Within each subgroup data were further sub-grouped for analysis by major muscle groups. For completeness of reporting, any measures that did not fit within major muscle groupings were grouped for analysis and reported as either \u201cother lower limb,\u201d \u201cother trunk,\u201d or \u201cother upper limb\u201d outcomes, to enable every outcome measure extracted from included studies to be reported in the results. The outcomes included in the \u201cother\u201d groupings are listed in the text.Ten sub groups were created based on the measurement methods units used for each for analysis as follows : (1) contractile work capacity (J), (2) cross sectional area generally decreased over time, however a transient increase was seen in Plantar Flexor, Dorsi Flexor and Quadriceps muscles and only at 20 days. In Plantar Flexor and Quadriceps muscles, muscle activity decreased again after 20 days, there were no data for Dorsi Flexor muscles beyond 20 days to establish a post 20 day trend. Moderate effects were apparent in upper limb muscle groups by 15 days but not until 90 days for Dorsi and Plantar Flexor muscles which were the only muscles with data at the 90 day point. The breakdown of individual activity effects per muscle is available in Maximal voluntary contraction during one repetition maximum decreased over time except for other upper limb outcomes that remained mostly unchanged as far as data were available up to 45 days. Moderate effects became apparent by 7 days and large effects by 35 days. Other lower limb outcomes that included maximal isometric force during supine squat, hip extensor force and legs total work never reached a large effect, but had no data available beyond 35 days. The breakdown of individual MVC during one repetition maximum effects per muscle is available in Power decreased over time. Moderate effects became apparent by 7 days and large effects by 20 days, although these were only seen in the Quadriceps muscle data. Hamstring, Hip Flexor, and upper limb muscles that included elbow flexors and extensors reached moderate effects by 20 days. Plantar Flexors never reached a moderate effect but data were only available at 14 days. Other trunk muscles included trunk flexors and extensors tested in combination within functional movements. The breakdown of individual power effects per muscle is available in Performance based outcomes all worsened over time, as although sit to stand, balance, and sprint time outcomes all had positive effects, this was considered a worsening effect within these measures. Endurance reached a large effect by 14 days, jumping a moderate effect at 42 days and large by 44 days, sit to stand and balance reached large effects by 60 days and sprint time by 62 days. Data for most outcomes were only available for one time point and so trends over time for individual outcomes are not able to be determined. The breakdown of individual performance based effects per outcome is available in The main finding of the review was that muscle cross-sectional area, volume, shape, size, activity, power, performance, torque, and force-based outcomes, at either regional or global level, all decline over time, based on the current evidence base. Moderate effects became apparent in the following order: power and MVC during one repetition maximum (7 days), followed by volume, cross sectional area, torques and strengths, contractile work capacity, thickness and endurance (14 days), then muscle activity (15 days). Large effects became apparent in the following order: volume, cross sectional area (28 days) torques and strengths, thickness (35 days), and peak power (56 days). No large effects were found for muscle activity. There were limited data for contractile work capacity and no large effects were apparent. In general, lower limb and trunk muscles appeared to decline more rapidly than upper limb muscles. Locomotion muscles such as Plantar Flexor and Quadriceps muscles also generally appeared to decline more rapidly than other muscles groups and with larger effect sizes.Human spaceflight missions differ in duration, so results have to be placed into the context of mission profiles and operationally important considerations. Operationally, performance-related measures such as power, MVC, torques and strengths are considered most critical. In terms of mission profiles, typical ISS missions involve approximately 180 days in \u03bcG level of strength to achieve mission critical tasks such as donning/doffing and standing up/moving whilst wearing a space suit in low gravity, hatch opening, and pulling/dragging a fellow crew member wearing a space suit during an emergency. The absolute level is defined as the precise required strength outcome in raw units to achieve a task, as opposed to considering relative changes with effect size or percentage changes. A relative (%) reduction in strength will make all tasks with an absolute strength requirement more challenging for all individuals, but the biggest impact will be felt by those who have a lower initial level of absolute strength. For example, a strong individual might be able to lose 30% of their pre-flight strength and still comfortably achieve a mission critical task , whereas a weaker individual might already be close to their physical limit during this task without any deconditioning. Operationally, having an estimate of the most rapid possible rate of change in muscle outcomes may be useful in the case of a crew member with low pre-flight absolute strength, or an individual highly susceptible to \u03bcG adaptation. In the present study, the most extreme negative value within the confidence interval for each outcome provides an estimate of the most extreme worst likely true value that might be encountered with exposure to \u03bcG. Based on this estimation, the results of this analysis suggest that the change experienced by an individual astronaut might reach a large effect size in some muscles within a 7 day lunar transit period for volume, cross sectional area, contractive work capacity, thickness, power, and MVC. However, the confidence intervals are wide due to the small sample sizes across the current evidence base, so this estimate should be treated with caution as it may be exaggerated. Individual effects are difficult to determine in a transferable way to the true population from the data currently available or from individual case studies. Ideally, a population selected for their increased susceptible to unloading/\u03bcG-induced muscular adaptation should be studied in a long-duration \u03bcG analog to produce a representable average effect that could be transferred to the true population with more reasonable confidence. Until such data are available, estimating the maximum rate of decline in an individual in response to \u03bcG exposure of such a duration will remain difficult. In addition, consideration would also be needed should an individual be selected to perform some tasks in a mission that are not considered mission critical, but are essential to other mission goals. It may be that checking for susceptibility to outcomes that are linked more strongly to mission success is checked and made part of astronaut eligibility screening, it could also be any more susceptible mission critical individuals undergo more rigorous preflight and inflight training protocols or consider use of other more removed countermeasures beyond the scope of this review.Exercise countermeasure development may want to consider focusing on those which might best address the more susceptible outcome changes in this review, volume, cross sectional area, contractive work capacity, thickness, power, and MVC while also ensuring any proposed exercises are tailored to tasks considered critical, such as donning/doffing and standing up/moving whilst wearing a space suit in low gravity, hatch opening, and pulling/dragging a fellow crew member wearing a space suit. The impact of any chosen exercise types on future spacecraft exercise hardware would also need further consideration. Future research should consider identifying exercise countermeasures that would best address the more susceptible outcomes and be feasible with any technical constraints of new space vehicles planned for use within Moon and Mars missions.As CM are likely to be needed on the return trip from both Moon and on the journeys to and from Mars, such CM will need developing. Countermeasure devices should support lower limb and trunk muscle exercise as these decline earlier than other body regions and are essential for locomotion and for spinal function on return to G loading Bamman, . Based omuscle changes, additional holistic consideration of other physiological systems will likely be required. Finally, any CM development for exploration missions will also have to consider constraints of space vehicles that will be used, such as available physical space, limited number of devices that can be included in the space craft, consumables, generation of heat, carbon dioxide, and vibration, which are likely to be more restricted than the ISS ISS Long Duration Mission (LDM) exercise prescriptions (Dawson et al., Most of the studies scored four on the bed rest tool, with no studies scoring a full seven points, although 13 studies scored six. The reasons for marking studies down was mostly due it being unclear if criteria had been met rather than clearly failing a point. The most common unclear criteria was related to restricted sunlight exposure followed by ensuring a fixed daily routine. The high risk of bias results were most commonly caused by not clearly showing how confounding factors were managed and providing adequate description of participation. The participation domain considers participant eligibility criteria, source of participants, baseline descriptions, description of sampling frame and recruitment, description of period and place of recruitment, and inclusion/exclusion criteria (Hayden et al., There was some asymmetry in the funnel plot showing potential publication bias toward studies reporting a decrease in muscle outcomes. However, there were studies present on the increasing side of the plot, so the risk is not likely to be high. In addition, it is expected that many of the muscle outcomes would decrease during a period of inactivity such as bed rest, therefore, it not surprising most studies reported decreases. Therefore, while it appears a risk of reporting bias may exist, the presence of some studies reporting increases and the expected pattern of more decreases being reporting suggest this finding should be treated with caution and the potential risk is likely to be low.This review only considered muscle outcomes. Spaceflight is known to affect many more human physiological systems including bone, cardiovascular and vestibular (Pavy-Le Traon et al., The results of this review suggest that moderate effects on a range of muscle function parameters may occur within 7\u201314 days of unloading, with large effects within 35 days. Combined with identification of muscle performance requirements for future exploration mission tasks, these data, may support the design of CM programmes to optimize their efficient use without compromising crew safety and mission success. However, the data suggests CM are likely to still be needed for longer transit/orbital periods of 14\u201328+ days, such as a prolonged Lunar orbit, deep space exploration, or a Mars mission, as moderate effects occur between 7\u201314 days and large effects by 28 days for most muscle outcomes. However, if large effect sizes occur only after 28\u201335 days, to save resources, space agencies might consider short missions without exercise CM, or fixed periods of abstinence during longer \u03bcG exposures, if they could be confident that moderate changes in muscle performance could be reversed in-flight. Finally, several research gaps are highlighted for future bed rest studies in which standardized time points for measurements should be used and clear information provided on sunlight exposure control, fixed daily routine, and control of any confounding factors.1Akima et al., 2Akima et al., 3Akima et al., 4Akima et al., 5Alkner and Tesch, 6Alkner et al., 7Arbeille et al., 8Bamman et al., 9Belavy et al., 10Belavy et al., 11Belavy et al., 12Belavy et al., 13Belavy et al., 14Belavy et al., 15Belavy et al., 16Belavy et al., 17Belavy et al., 18Belavy et al., 19Berg et al., 20Berg et al., 21Berry et al., 22Buehring et al., 23Caiozzo et al., 24Cescon and Gazzoni, 25Convertino et al., 26de Boer et al., 27Dudley et al., 28Duvoisin et al., 29Ellis et al., 30English et al., 31English et al., 32Ferrando et al., 33Ferretti et al., 34Fu et al., 35Funato et al., 36Gast et al., 37Germain et al., 38Greenleaf et al., 39Greenleaf et al., 40Greenleaf et al., 41Holguin et al., 42Holt et al., 43Kawashima et al., 44Koryak, 45Koryak, 46Koryak, 47Koryak, 48Koryak, 49Koryak, 50Koryak, 51Koryak, 52Kouzaki et al., 53Krainski et al., 54LeBlanc et al., 55Lee et al., 56Macias et al., 57Miokovic et al., 58Miokovic et al., 59Miokovic et al., 60Muir et al., 61Mulder et al., 62Mulder et al., 63Mulder et al., 64Mulder et al., 65Mulder et al., 66Narici et al., 67Pisot et al., 68Portero et al., 69Reeves et al., 70Rittweger et al., 71Rittweger et al., 72Schneider et al., 73Shinohara et al., 74Trappe et al., 75Trappe et al., 1Belavy et al., 2Belavy et al., 3Belavy et al., 4Biolo et al., 5Cavanagh et al., 6Shenkman et al., 7Amorim et al., 8Rittweger and Felsenberg, 9Koriak Iu, 10Koriak Iu, 11Koriak Iu, 12Koryak, 13Bamman and Caruso, 14Bamman, 15Felsenberg et al., 16Ferretti, 17Greenleaf et al., 18Grogor'eva and Kozlovskaia, 19Guo et al., 20Hargens et al., 21Judith Hayes et al., 22Ito et al., 23Jaweed et al., 24Koryak, 25Kozlovskaia et al., 26LeBlanc et al., 27LeBlanc et al., 28Liu et al., 29Macias et al., 30Meuche et al., 31Meuche et al., 32Milesi et al., 33Miyoshi et al., 34Moriggi et al., 35Mulder et al., 36Netreba et al., 37Scott et al., AW: initial concept ideas, protocol planning and drafting, search screening, analyzing, and drafting all manuscript versions. JS: methods advice, protocol drafting, and approving final draft. NW: data extraction, analysis, and drafting final version. MV: protocol planning, search screening, data analysis, drafting text, and checking final version. NC: protocol planning, search screening, methods advice, manuscript drafting, and approving final version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Fusarium mycotoxins. Further, we suggest that understanding how mycotoxin levels are regulated by microbial encounters can offer novel insights for mycotoxin control in food and feed. Microbial degradation of mycotoxins provides a wealth of chemical information that can be harnessed for large-scale mycotoxin detoxification efforts.An important goal of the mycotoxin research community is to develop comprehensive strategies for mycotoxin control and detoxification. Although significant progress has been made in devising such strategies, yet, there are barriers to overcome and gaps to fill in order to design effective mycotoxin management techniques. This is in part due to a lack of understanding of why fungi produce these toxic metabolites. Here we present cumulative evidence from the literature that indicates an important ecological role for mycotoxins, with particular focus on Aspergillus fumigatus with the potential to produce a myriad of secondary metabolites. One of the most powerful ways to activate BGCs is by co-cultivation or mixed fermentation of two or more microbes. Microbial interactions that have led to up-regulation of known metabolites or to discovery of novel natural products are numerous benzoic acid and citreoisocoumarinol [ in Trichoderma atroviride and zearalenone produced by Ch\u00e9rif, . TrichodF. oxypsorum in both plant and animal hosts (L\u00f3pez-D\u00edaz et al., F. avenaceum that lacked the ability to synthesize enniatin showed decreased virulence when infected on potato tubers (Herrmann et al., Fusarium oxysporum f. sp. melonis isolates to synthesize beauvericin or enniatin B does not contribute to virulence in melons (Moretti et al., in vitro cultures in F. graminearum and F. pseudograminearum (Mudge et al., F. culmorum, F. graminearum, and F. pseudograminearum cause fusarium head blight as well as fusarium crown rot. However, the former two pathogens are more aggressive pathogens in head blight while F. pseudograminearum shows enhanced fitness as the pathogen of crown rot. This has been attributed to the differential production of DON in the different tissues (stem base vs. wheat heads; Tunali et al., F. graminearum, DON has been demonstrated to inhibit apoptosis-like programmed cell death in Arabidospis thaliana (Diamond et al., F. graminearum (Voigt et al., F. graminearum has been proposed (Audenaert et al., Burkholderia glumae, a seed-borne bacterium and F. graminearum has been recently identified. Co-cultivation of the two microbes resulted in an increase in sporulation and DON production in F. graminearum, which is at least partially in response to a toxic bacterial metabolite. An overall increase in disease severity was observed upon co-infection of rice with the two pathogens. The two microbes were also found to be physically attached upon microscopic observations after co-cultivation (Jung et al., Fusarium spp. It is necessary to note here that microbial interactions can also reduce the virulence of Fusarium species on their plant hosts, as discussed in section Microbial Interactions in Detoxification and Degradation of Mycotoxins.Although secondary metabolites are not \u201crequired\u201d for the growth and development of fungi, they function as fitness factors. Mycotoxins have been shown to contribute to the pathogenicity, aggressiveness and/or virulence of fungi. Fusaric acid has been reported to enhance the virulence of Pseudomonas piscium, from the wheat head microbiome has shown that the bacterium secretes an antifungal agent, phenazine, against F. graminearum. Phenazine, upon entering the fungal cell, inhibits the histone acetyl transferase module of the SAGA complex which subsequently leads to an inhibition of fungal growth and pathogenicity in addition to a complete suppression of DON biosynthesis (Chen et al., Aspergillus nidulans\u2014Streptomyces rapamycinicus association where the bacterium induces histone modification mediated by the SAGA complex which results in production of orsellinic acid and its derivatives by the fungus (Nutzmann et al., Epigenetics has been steadily gaining momentum in the last few decades in the world of transcriptional regulation. There is now growing evidence that microbial communication regulates epigenetic modifiers that in turn control mycotoxin biosynthesis. The SAGA complex, conserved across eukaryotes, induces transcription of genes by mediating histone acetylation of the corresponding promoters. A study that isolated the bacterium, Fusarium mycotoxins mediated by microbes, plants and insects (The literature supports the idea that mycotoxins can be important players in shaping microbial communities and their interaction with hosts. Signaling molecules are regulated \u201ccoinage\u201d and need to be recycled\u2014through various chemical transformation processes\u2014to maintain homeostasis in the community. Thus, it is not surprising that there are several examples of degradation of insects .Fusarium mycotoxin accumulation in grains. Preventative application of Psuedomonas fluorescens strain before inoculation with F. culmorum resulted in a significant reduction in Fusarium head blight as well as DON levels in infected wheat grains (Khan and Doohan, Paenibacillus polymyxa, isolated from wild teosinte, have been shown to produce fusaridins which contribute to the antifungal activity against F. graminearum. Co-existence of these bacteria with F. graminearum in grains during storage at room temperature resulted in a significant decrease in DON accumulation (Mousa et al., Bacteria have been shown to contribute to reduction of Aspergillus tubingenesis NJA-1, a soil isolate, has been shown to convert DON into a less-toxic product that has been speculated to be the result of hydrolysis, based on differences in the mass of the metabolites (He et al., Agrobacterium\u2013Rhizobium strain E3-39 converts DON into 3-keto DON (Shima et al., Nocardioides WSN05-2 forms the non-toxic epimer, 3-epi-DON (Ikunaga et al., Deviosa insulae forms 3-keto-DON (Wang et al., Devosia mutans 17-2-E-8 forms both 3-keto-DON and 3-epi-DON (He et al., Eggerthella spp., isolated from chicken intestine, has been reported to de-epoxify DON over a wide range of temperatures and pH (Gao et al., Tri101, encoding 3-O-acetyltransferase for 3-O-acetylation of the trichothecene ring has been characterized in F. graminearum and has been further identified in other Fusarium species (Kimura et al., Arabidopsis, it has been reported that the UDP-glycosyltransferase catalyzes transfer of glucose from UDP-glucose to the hydroxyl group at the 3-C position in DON (Poppenberger et al., Understanding the mechanisms underlying chemical transformation of mycotoxins could pave the way toward evolving novel techniques for mycotoxin decontamination in food and feed. Several mechanisms of detoxification of DON have been studied as summarized in Clonostachys rosea has been reported to produce a zearalenone-specific lactonase that catalyzes the hydrolysis of the lactone ring, which is followed by spontaneous decarboxylation (Utermark and Karlovsky, C. rosea to zearalenone. Trichosporon mycotoxinivorans has been shown to convert zearalenone into ZOM-1 which is characterized by the opening of the ring structure at the ketone group positioned at C6' (Vekiru et al., Rhizopus arrhizus catalyzes sulfation of the hydroxyl group at the C4 position resulting in the formation of zearalenone-4-O-sulfate conjugate (el-Sharkaway et al., Zearalenone mimics estrogen upon ingestion in animals and humans resulting in sexual and reproductive abnormalities. Microbes possess the ability to degrade and inactivate zearalenone, as reviewed in Ji et al. . ClonostPseudomonas (Altalhi, Bacillus (Cho et al., Rhodoccous, and Streptomyces (De Mets et al., Acinetobacter has been shown to secrete extracellular enzymes that oxidize zearalenone into smaller estrogenic products (Yu et al., Lactobacillus strains with zearalenone although the bacteria removed the mycotoxin from the cultures. The authors were able to recover zearalenone from the bacterial cultures and suggest that the bacteria bind zearalenone in a density-dependent manner (El-Nezami et al., Lysinibacillus sp. isolated from chicken intestine can remove zearalenone from cultures and the process has been shown to be significantly reduced upon heat treatment. The authors suggest a potential enzymatic process that may be involved in the interaction between the bacterium and zearalenone (Wang et al., Pseudomonas putida ZEA-1 utilizes zearalenone as a carbon source (Altalhi, Species of Altalhi, , BacilluAltalhi, .Burkholderia ambifaria, a novel bacterium isolated from barley rhizosphere has been reported to be able to utilize fusaric acid as a sole carbon source (Simonetti et al., Aspergillus tubingensis (Crutcher et al., Colletotrichum sp (Fakhouri et al., Mucor rouxii (Crutcher et al., A significant understanding of the biochemical pathways involved in detoxification processes (Carere et al., The mycotoxigenic fungal species live in complex and nutrient-deficient environments\u2014be it in soil, plant or animal hosts. The soil micro-environment often fluctuates with variations in water availability, air, light, and temperature, among other abiotic factors. Now add to this, a complex cocktail of microbes and hosts that are integral to the environment where survival of microbes heavily depends on active community participation. Mycotoxins play a significant role in the defensive strategies of mycotoxigenic fungi against the resident microbes. Interactions between microbes in such environments may involve competition or compromise where mycotoxins may serve as essential chemical language mediating communication. The host environments are usually unfriendly, thus requiring special adaptations in order for the fungi to thrive in such conditions. Several studies support a view that mycotoxins may act as signaling molecules that modulate host responses and promote successful colonization. Reports of microbes that can metabolize and detoxify mycotoxins are aplenty, highlighting the importance of examining microbial interactions to uncover strategies for mycotoxin detoxification.Fusarium spp. The evolving knowledge on molecular and genetic mechanisms that govern mycotoxin production provides us with valuable tools to study the ecological roles of mycotoxins. This is not only an achievable goal but also has the potential to be highly rewarding. Such knowledge can facilitate development of novel strategies to control infections of mycotoxigenic fungi as well as mycotoxin contamination in food and feed.In this review chapter, we have summarized existing literature that accentuate the ecological significance of mycotoxins with focus on NV along with NK conceptualized and drafted the theme of the review. NV wrote the article and NK reviewed, edited, and refined the manuscript along with NV.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Mycobacterium tuberculosis (Mtb) is the bacterial pathogen that causes the majority of human tuberculosis (TB), the leading infectious disease in the world , a well-documented Mtb complex and in a related species Mycobacterium marinum, but absent from the Mycobacterium bovis Bacille Calmette\u2013Gu\u00e9rin (BCG) genome, encodes an ESX-1 type VII secretion system that has been extensively investigated as a major virulence factor among mycobacterial species have been identified by comparative genomic studies , but their role in Mtb virulence has not been explored. Arora et al. biochemically and functionally characterized the CitE enzymatic subunits. The purified CitE1 and CitE2 proteins degraded acetyl-CoA and propionyl-CoA in vitro and the genes encoding both enzymes were up-regulated when Mtb was exposed to oxidative stress. Moreover, deletion of the citE genes from the Mtb genome reduced the resistance to oxidative stress, intracellular replication in macrophages, and growth in a guinea pig infection model. This study suggests that CitE may be a potential target for TB drug development.Bacterial citrate lyase, which is important for both metabolism and virulence, is composed of three subunits, CitD (\u03b3), CitF (\u03b1), and CitE (\u03b2) with a global collection of Mtb genomes and identified a novel phylogenetic clade that share single-nucleotide polymorphisms (SNPs) in key virulence-associated loci, including the mce1 locus and the phoP gene. This clade includes four hypervirulent strains isolated from geographically diverse regions. The common SNPs and structural variations within the clade may be considered as potential genetic determinants of hypervirulence for future studies.Mtb is the most common cause of human TB, M. bovis can cause TB in both humans and cattle, making it a zoonotic threat to both food safety and public health and folC (encoding FolC-dihydrofolate synthase) genes have been associated with resistance to para-aminosalicylic acid (PAS; Rengarajan et al., Howe et al. found that MetM, a putative amino acid transporter, plays a crucial role in the synthesis of folate precursors, which antagonizes PAS activity.The mutations in the Mtb. In the report done by Ilin et al., FS-1 was used in combination with standard anti-TB antibiotics on guinea pigs infected with an XDR-Mtb strain. The genetic changes in Mtb genomes following infection were analyzed and FS-1 was found to cause a counter-selection of drug-resistant variants that sped up the recovery of the infected animals from XDR-TB. While the drug resistance mutations remained intact in more sensitive isolates, reversion of drug resistance was associated with a general increase in genetic heterogeneity of the Mtb population.Drug induced reversion of antibiotic resistance has drawn recent attention as a prospective approach to combat drug resistance (Baym et al., Mtb virulence, including characterization of new roles for known virulence factors, identification of new virulence factors, and the elucidation of drug-resistance mechanisms and reversion. This Research Topic, together with many recent publications, enhances our understanding of the mechanism of Mtb virulence and pathogenesis.The articles in this Research Topic present new findings regarding the cellular and molecular mechanisms of JS, PC, and FB have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Multimorbidity is widespread, costly, and associated with a range of deleterious outcomes; it affects an estimated 67-80% of older adults. This study tests the validity of a multimorbidity resilience index developed in a Canadian sample of older adults by Wister et al., (2018), with a U.S.-based sample, using National Social Life, Health, and Aging Project (NSHAP) data, and draws upon the index to investigate the effects of resilience on outcomes over time. We mapped Wister et al.\u2019s (2018) index to NSHAP measures, and assessed cross-sectional associations with health outcomes, using logistic regression. To assess the effects of resilience on health outcomes over time, we estimated mixed models of the relationships between resilience on outcomes over a 5-year interval. Total resilience was consistently associated with improved outcomes, including pain level ; reduced utilization ; improved mental health ; self-rated physical health ; and sleep quality . Longitudinal model results indicate change in multimorbidity resilience and number of chronic diseases predict (\u03b1=.001) pain level and self-rated physical health. Effects were moderated by socio-demographic factors. Our findings validate Wister et al.\u2019s (2018) resilience index in a U.S. sample, supporting the importance of this measure to capture core components of older adults\u2019 capacity to sustain well-being in the context of living with multiple, chronic conditions. Results from the longitudinal models provide beginning insights into the effects of resilience on symptom experience and perceived health over time, highlighting potential levers for change."} +{"text": "The value of the nosological distinction between non-affective and affective psychosis has frequently been challenged. We aimed to investigate the transdiagnostic dimensional structure and associated characteristics of psychopathology at First Episode Psychosis (FEP). Regardless of diagnostic categories, we expected that positive symptoms occurred more frequently in ethnic minority groups and in more densely populated environments, and that negative symptoms were associated with indices of neurodevelopmental impairment.plus to estimate five theory-based models of psychosis. We used multiple regression models to examine demographic and context factors associated with symptom dimensions.This study included 2182 FEP individuals recruited across six countries, as part of the EUropean network of national schizophrenia networks studying Gene\u2013Environment Interactions (EU-GEI) study. Symptom ratings were analysed using multidimensional item response modelling in MA bifactor model, composed of one general factor and five specific dimensions of positive, negative, disorganization, manic and depressive symptoms, best-represented associations among ratings of psychotic symptoms. Positive symptoms were more common in ethnic minority groups. Urbanicity was associated with a higher score on the general factor. Men presented with more negative and less depressive symptoms than women. Early age-at-first-contact with psychiatric services was associated with higher scores on negative, disorganized, and manic symptom dimensions.Our results suggest that the bifactor model of psychopathology holds across diagnostic categories of non-affective and affective psychosis at FEP, and demographic and context determinants map onto general and specific symptom dimensions. These findings have implications for tailoring symptom-specific treatments and inform research into the mood-psychosis spectrum. Specifically, FEP individuals were recruited as part of the \u2018Functional Enviromics\u2019 work package, which consisted of an incidence and a case-sibling-control study conducted across six countries with the aim to investigate clinical, genetic, and environmental interaction in the development of psychotic disorders.Individuals suffering from their FEP were recruited between 2010 and 2015 as part of the large EUropean network of national schizophrenia networks studying Gene\u2013Environment Interactions (EU-GEI) study ; central Amsterdam, Gouda and Voorhout (the Netherlands); part of the Veneto region, Bologna municipality, city of Palermo ; 20th arrondissement of Paris, Val-de-Marne, Puy-de-D\u00f4me (France); Madrid , Barcelona, Valencia, Oviedo, Santiago, Cuenca (Spain); and Ribeir\u00e3o Preto (Brazil).et al., We screened all subjects who were referred to mental healthcare services with a suspicion of psychosis. The ascertainment period of cases ranged from 12 months in London to 48 months in Val-de-Marne and Bologna, with a median of 25 months. In each site, a psychiatrist experienced in epidemiology research oversaw the local team, which was centrally trained to minimize non-differential recruitment bias in the different healthcare systems. Written consent was obtained from the subjects who agreed to take part of the case-sibling-control study. For incidence-only cases, local research ethics committees approved the extraction of demographics and clinical information from patient records. More detailed information is available on the EU-GEI core paper on the incidence rates of schizophrenia and other psychotic disorders (Jongsma x.5).Patients were included in the current study if they met the following criteria during the recruitment period: (a) aged between 18 and 64 years; (b) presentation with a clinical diagnosis for an untreated FEP, even if longstanding ; (c) resident within the catchment area at FEP. Exclusion criteria were: (a) previous contact with psychiatric services for psychosis; (b) psychotic symptoms with any evidence of organic causation; and (c) transient psychotic symptoms resulting from acute intoxication . The OPCRIT system allows to: (1) assess the pre-morbid history and current mental state; and (2) establish the diagnosis of psychotic disorders based on algorithms for several diagnostic classification systems. It consists of a checklist which can be filled using different sources, e.g. case records or clinical interviews. Fifty-nine items relate to the mental state examination. We used diagnoses based on Research Diagnostic Criteria (RDC) , omega hierarchical (\u03c9H), and index H. Coefficient \u03c9 is an estimate of the proportion of common variance accounted by general and specific symptom dimensions. Coefficient \u03c9H is an estimate of the proportion of reliable variance accounted by the general dimension, treating variability in scores due to specific dimensions as measurement error the general factor independently from the specific symptom dimensions, and (2) each specific symptom dimension independently from the general factor. To estimate the extent to which symptom dimensions were represented by their own set of OPCRIT items and their replicability across studies, we computed the construct reliability index H , Akaike Information Criterion (AIC), Bayesian Information Criterion (BIC), and Sample-size Adjusted BIC (SABIC) as model fit statistics. For the model showing the best fit, we calculated reliability and strength indices, such as McDonald's omega analysis in STATA 14 and based on clinical records for 49% (n\u00a0=\u00a01070) of the sample. The sample prevalence of psychotic symptoms is presented in Supplementary Table S1.We identified 2774 treated incidence cases of psychosis (Jongsma M\u00a0=\u00a030.1) compared with women . Age-at-first-contact differed across ethnic groups, with individuals of Black ethnicity (M\u00a0=\u00a029) being younger than individuals of White ethnicity . The most common RDC-based diagnosis was broad or narrow schizophrenia (38.6%), followed by schizoaffective disorders (35%), unspecified non-organic psychotic disorder (16.3%), bipolar disorder (5.9%), and psychotic depression (4.2%).Fifty-seven per cent of FEP were men. Subjects were mostly people of a White ethnicity. Other main ethnic groups included Black African and Black Caribbean, North African, Mixed, and Asian. Mean age-at-first-contact with psychiatric services was 32.1 years; this was lower in men or case note review (95% CI 0.56\u20130.65), compared with a classification by chance (95% 0.32\u20130.41). Moreover, symptom dimensions showed similar diagnostic classification accuracy across countries .Findings on factor scores by gender, age-at-first-contact, and ethnicity, are shown in v. Cambridge), whereas a negative association was observed in Spain (online Supplementary Table S5).A moderate positive association was observed for more densely populated environments and the general dimension score. This is the first study on general and specific symptom dimensions in an incidence sample of psychosis. First, we found in our FEP sample that manic and delusional symptoms primarily underlie the identified general psychosis factor across diagnostic categories of non-affective and affective psychosis. Second, findings showed that specific dimensions of positive, negative, disorganized, manic and depressive symptoms are complementary to the general dimension. Third, general and specific symptom dimensions discriminated well between diagnoses of psychotic disorders. Forth, positive symptoms were more common among individuals of Black and North African ethnicity. Fifth, there was some evidence that early age-at-first-contact was associated with higher scores for several dimensions, such as of negative, disorganised and manic symptoms. Sixth, men presented with more negative and less depressive symptoms than women. Finally, higher scores for the general dimension were observed for individuals living in urban neighbourhoods.et al., et al., et al., et al., et al., et al., v. a multidimensional construct of psychosis. Hence, it was a suitable model for addressing dimensionality issues for psychosis and generating reliable phenotypes.Before interpreting our findings, we must consider potential limitations. Symptoms were rated with a semi-structured face-to-face interview or from case note review. Still, study investigators underwent a specific and centrally organized training for OPCRIT and demonstrated good inter-rater reliability for individual item ratings; moreover, OPCRIT is a tool specifically designed to allow use with different sources (McGuffin et al., et al., et al., et al., et al., In our study, the bifactor model of psychopathology best explained the observed symptoms at FEP compared with unidimensional and multidimensional models. Our findings are consistent with, and extend, previous research on psychotic symptoms in people with enduring psychotic disorders (Reininghaus et al., et al., et al., et al., et al., We found some evidence of gender differences in symptom dimension scores. Men showed less depressive symptoms and more negative symptoms compared with women. This finding is consistent with other studies in stable schizophrenia (Shtasel et al., et al., et al., et al., et al., et al., et al., We further found that symptom dimensions vary in terms of ethnicity. Consistent with a previous report (Kirkbride et al., et al., et al., et al., et al., et al., et al., We showed that high population density is positively associated with the general and specific disorganized, negative and manic dimensions. In our multinational sample, we were not able to replicate previous findings on the relationship between urbanicity and the positive dimension (Kirkbride et al., et al., et al., et al., In the context of a general effort to move away from DSM and ICD categories (Demjaha et al., From a research perspective, our findings suggest that the general dimension may reflect a phenotype for the study of general risk factors. For example, urbanicity may impact on the risk and profile of psychosis through the combination of other, more specific socio- or bio-environmental factors. In addition, we showed a substantial variation of sociodemographic determinants at the specific dimension level, which may support an integrated socio-developmental model of psychosis (Morgan et al., et al., et al., et al., We may further suggest using the general dimension as a quantitative measure of psychopathology for research into the genetic component shared across psychotic disorders. The evidence is required to establish the extent to which pathophysiology of schizophrenia, bipolar disorder, and psychotic depression is shared at the level of pathways and neuronal cell mechanisms (Forstner et al., et al., et al., et al., et al., From a clinical perspective, although each patient presents with a specific pattern of psychopathology and response to treatment at FEP, attention has been traditionally focused on the positive dimension management. Mental health professionals may integrate observations of the whole range of symptoms and signs with a consideration of neurodevelopmental and socio-environmental risk factors. Such an approach should aim to plan and optimize pharmacological and non-pharmacological treatments (Murray We may further suggest promoting mental health professionals to adopt treatment plans guided by dimensions, and increasing their confidence in dimensional classifications. Reconciling contradictory concerns of clinicians and researchers (Kendell and Jablensky,"} +{"text": "The incidence of neurological disorders such as multiple sclerosis (MS), Alzheimer\u2019s disease (AD) and Parkinson\u2019s disease (PD) is increasing throughout the world, but their pathogenesis remains unclear and successful treatment remains elusive. Bidirectional communications between the central nervous system and gut microbiota may play some role in the pathogenesis of the above disorders. Up to a thousand bacterial species reside in human intestine; they colonize the gut shortly after birth and remain for life. Numerous studies point to the role of microbiota composition in the development, course and treatment of MS, AD and PD. The purpose of this review is to summarize the current knowledge about the role of gut microbiota composition in the development, course, and prognosis of selected neurological diseases. Global incidence of a number of common neurological disorders is rapidly increasing Bengmark and consThe central nervous system (CNS) and the gut microbiota interact in a bidirectional manner, e.g. the CNS modulates gut functioning in response to psychological and physical stressors affecting motility, secretion and immune reactivity Mayer , whereas11 bacteria cells can be found in 1\u00a0g of colon contents cells target brain and spinal cord cells leading to demyelination and axonal damage is the most common neurological disease of young adults in Europe and North America , which is the most widely used animal model of MS, showed that oral treatment with ampicillin, vancomycin, neomycin, sulfate, and metronidazole can delay the onset and reduce the severity of the disease, decrease levels of pro-inflammatory cytokines and increase levels of interleukin (IL)-10 and IL-13 before the induction of EAE resulted in its delayed onset and milder course is a common neurodegenerative disease of the elderly accounting for 60\u201380% of all dementia cases , A\u03b2 accumulation in brain and inflammatory response , which are not synthetized by humans were found in cerebrospinal fluid and the cerebral cortex of patients with general paresis suggesting that these bacteria might play a role in slowly progressive dementia, cortical atrophy, and local amyloidosis , the second most common neurodegenerative disorder of elderly, is 15\u00a0years consisting of aggregated and phosphorylated \u03b1-synuclein (Braak and Del Tredici Non-motor symptoms, including mood and cognitive impairment, sleep disorders, sensory disruption and GI dysfunction (Postuma et al. Prevotellaceae, Coprococcus, Firmicutes, Clostridium coccoides, Clostridium leptum, B. fragilis and increased representation of Lactobacillaceae, Verrucomicrobiaceae, Ruminococcaceae, Lactobacuillus, Bacteroidetes, Proteobacteria, Clostridiaceae and Akkermansia (Hasegawa et al. Lactobacillaceae could affect the ENS as these bacteria were found to increase the excitability of colonic after-hyperpolarization neurons by inhibiting calcium-dependent potassium channel opening (Kunze et al. Lactobacillus spp. quantity and serum leptin concentrations were found to be positively correlated (Queipo-Ortuno et al. Prevotellaceae and increased abundance of Lactobacillaceae have been correlated with decreased levels of gut ghrelin. The latter hormone may regulate nigrostriatal dopamine function and restrict neurodegeneration in PD (Queipo-Ortuno et al. Prevotellaceae levels are not specific for PD and were reported in other diseases such as autistic spectrum disorders (Kang et al. Prevotellaceae levels have been proposed as an excluding biomarker of PD (Scheperjans et al. Prevotellaceae below 6.5% was found to be diagnostic of PD with 86.1% sensitivity and 38.9% specificity (Dinan and Cryan Enterobacteriaceae positively correlate with the severity of postural instability and gait difficulty (Scheperjans et al. Gut microbiota abnormalities are common in PD patients and their relation to brain dysfunction is being intensively investigated (Scheperjans B. fragilis, C. perfringens, Pseudomonas spp., most strains in Enterobacteriaceae family, 12 species in Clostridium, C. coccoides group and C. leptum subgroup (Hasegawa et al. Initial studies employing per os administration of hydrogen water in rats (Fu et al. Akkermansia and Ralstonia were found to be increased in affected patients (Scheperjans Interestingly, small intestinal bacterial overgrowth was found to contribute to the pathophysiology of motor fluctuation in PD patients and eradication of bacterial overgrowth resulted in clinical improvement (Fasano et al. Some authors also found an increase in gut permeability in patients with PD when using the urinary sucralose test (Julio-Pieper et al. There was a 90% decrease of plant fiber intake between 1800 and 1970, while the consumption of calories from animal fat and refined sugar increased four times in this time period (Burkitt et al. There is ample evidence for the existence of communication between CNS, gut and intestinal microbiome (Rhee et al."} +{"text": "Recent efforts in synthetic biology have shown the possibility of engineering distributed functions in populations of living cells, which requires the development of highly orthogonal, genetically encoded communication pathways. Cell-free transcription-translation (TXTL) reactions encapsulated in microcompartments enable prototyping of molecular communication channels and their integration into engineered genetic circuits by mimicking critical cell features, such as gene expression, cell size, and cell individuality within a community. In this review, we discuss the uses of cell-free transcription\u2013translation reactions for the development of synthetic genetic circuits, with a special focus on the use of microcompartments supporting this reaction. We highlight several studies where molecular communication between non-living microcompartments and living cells have been successfully engineered. Current Opinion in Biotechnology 2019, 58:72\u201380NanobiotechnologyThis review comes from a themed issue on Giovanni Maglia and Wesley R BrowneEdited by Issue and the EditorialFor a complete overview see the Available online 26th December 2018https://doi.org/10.1016/j.copbio.2018.10.006http://creativecommons.org/licenses/by/4.0/).0958-1669/\u00a9 2018 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license reactions constitute ideal model systems for deducing the rules of network composition, developing new communication pathways between cellular mimics and living cells [Simplified models of biological systems could help identify key molecular parameters and as such have the potential to uncover generalisable concepts. Proteins and other components of the transcription and translation machinery can be extracted from cells as a lysate or purified and reconstituted (PURE) in order to perform gene expression ng cells , and creng cells .In this review, we intend to first summarise the use of TXTL reactions for prototyping and implementation of novel genetic networks. We will discuss how microfluidic technologies and semipermeable microcapsules provide essential tools for cell-free synthetic biology in a context where size, composition, and individuality of the reaction are of particular importance. Finally, we will highlight important contributions of encapsulated TXTL to the development of synthetic communication paths.As the complexity of synthetic gene networks increases, more resources are shared between synthetic circuits and their host. As a result, cells experience a decrease in fitness, which limits their growth and the efficiency of synthetic circuits \u2022\u2022. Furthin vivo applications, leveraging the strong potential of riboregulation [Development of complex genetic circuits requires the use of well-characterised regulatory modules. Cell-free TXTL allows the rapid and thorough testing of new genetic parts thus enabling such characterisation .et al. proposed a method for performing TXTL reaction on a chip in which the gene template DNA was attached to a functionalised surface, first under batch conditions using wheat-germ extract [E. coli cell extract [et al. [et al. are a major technological breakthrough that will facilitate the development of numerous synthetic gene circuits in the future.In contrast with methods using free-floating DNA gene templates, Bar-Ziv extract , and sub extract \u2022\u2022. The l [et al. \u2022\u2022 or double (water-in-oil-in-water) emulsions in order to study large number of these reactions at a cellular scale.et al., who optimised a cell-free genetic circuit based on an incoherent feedforward topology using fluorescently barcoded droplets in which lysate-based TXTL reactions can take place [Large numbers of TXTL microdroplets in oil with a controlled dispersity are easy to generate, store, and remain stable over hours, which makes them particularly interesting for screening large numbers of parameters influencing TXTL reactions c. This fke place .et al. encapsulated Xenopus egg extract in microdroplets of various size. The authors showed that the extract could undergo several mitotic oscillations, and described the influence of the size of the compartments on the period [et al. investigated the influence of surface-to-volume ratio of microcompartments on the efficiency of lysate-based TXTL, suggesting a deleterious interaction between membrane lipids and translation machinery [The reduced size of microdroplets is particularly interesting to study physical effects such as confinement. Guan e period . Sakamotachinery .et al. showed that noise in transcription reaction in systems displaying reduced diffusion resulted in gene expression bursts, which are likely to occur in cells but are not observable in bulk PURE-based TXTL reactions [Macromolecular crowding effects, which emerge in highly concentrated media such as the cytosol, can be artificially induced in TXTL mixtures using crowding additives inside microdroplets. Molecular crowding has been shown to influence the spatial segregation of biomolecules and kinetics of cell-free TXTL reactions ,48. In aeactions .et al. first implemented lysate-based TXTL reaction in a liposome permeated by haemolysin pores, which enabled the exchange of nutrients and by-products with a feeding solution, resulting in long-term gene expression [et al. tested various transcriptional cascades inside liposomes [et al. implemented a lysate-based TXTL liposome sensitive to osmotic changes via expression of the MscL calcium channel [et al. to serve as containers for the production and intratumoural delivery of a toxin protein [et al. recently described the first liposomes capable of isothermally replicating DNA using self-encoded proteins in PURE system, constituting a major step towards the construction of a true synthetic cell capable of full replication [Given that droplets are intrinsically closed systems, they cannot be utilised for long-term protein expression, nor as analogues of semi-permeable lipid bilayers for mimicking the cell membrane. Noireaux pression . Later, iposomes \u2022\u2022 , which induced the production of an enzyme disrupting an ongoing communication between an emitter bacterium (P. aeruginosa) and a receiver bacterium (engineered E. coli), a phenomenon also known as quorum quenching.Schwarz-Schilling bacteria . Each po species (Figure Cell-free TXTL reactions are becoming an essential tool to prototype novel genetic circuits and can facilitate their integration into living hosts. In combination with microfluidic technologies and the development of novel semipermeable microcapsules, TXTL reactions constitute a unique platform for the study of gene expression and coupling to other cellular processes in a biochemically relevant microenvironment.It has been established that size and confinement have a considerable impact on TXTL reactions ,46. For Current efforts are exploring possibilities to establish communication between artificial and natural systems, working towards therapeutic applications wherein information exchange between living cells are mediated or altered. We anticipate that micro-compartmentalised TXTL\u2014by accelerating the characterisation of novel genetic circuits and orthogonal molecular communication channels\u2014will have a compelling contribution to the field of synthetic biology.Nothing declared.\u2022 of special interest\u2022\u2022 of outstanding interestPapers of particular interest, published within the period of review, have been highlighted as:"} +{"text": "Infantile amnesia, a failure in early childhood to retain episodic memory for long periods, is a paradoxical phenomenon widely accepted as an everyday life fact, but its causes are extensively ignored. Recently, Josselyn and Frankland proposedAfter Freud and Brill mentioneRecently, Akers et al. probed tThe study of McGreevy et al. shows thLow levels of postnatal neurogenesis can also occur; maternal stress (Belnoue et al., per se. Infantile amnesia possibly allows the retention of only more critical memories, filtering those with a robust emotional component (Richardson et al., Even though higher rates of memory retention in young ages appear to be an adaptive advantage, hippocampal neurogenesis and infantile amnesia are widespread among mammals and are probably adaptive Growing evidence suggests that hippocampal neurogenesis is critical for spatiotemporal contextualization and emotional responses associated with memory processing even in humans (See references in Kempermann et al., AM-C proposed the idea. AM-C, GL-O, and PD wrote the paper. GL-O prepared the figure.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Pterosaurs are an extinct group of Mesozoic flying reptiles, whose fossil record extends from approximately 210 to 66 million years ago. They were integral components of continental and marginal marine ecosystems, yet their diets remain poorly constrained. Numerous dietary hypotheses have been proposed for different pterosaur groups, including insectivory, piscivory, carnivory, durophagy, herbivory/frugivory, filter\u2010feeding and generalism. These hypotheses, and subsequent interpretations of pterosaur diet, are supported by qualitative and/or quantitative evidence. Pterosaur dietary interpretations are scattered throughout the literature with little attention paid to the supporting evidence. Reaching a robustly supported consensus on pterosaur diets is important for understanding their dietary evolution, and their roles in Mesozoic ecosystems. A comprehensive examination of the pterosaur literature identified 314 dietary interpretations (dietary statement plus supporting evidence) from 126 published studies. Multiple alternative diets have been hypothesised for most principal taxonomic pterosaur groups. Some groups exhibit a high degree of consensus, supported by multiple lines of evidence, while others exhibit less consensus. Qualitative evidence supports 87.3% of dietary interpretations, with comparative anatomy most common . More speciose groups of pterosaur tend to have a greater range of hypothesised diets. Consideration of dietary interpretations within alternative phylogenetic contexts reveals high levels of consensus between equivalent monofenestratan groups, and lower levels of consensus between equivalent non\u2010monofenestratan groups. Evaluating the possible non\u2010biological controls on apparent patterns of dietary diversity reveals that numbers of dietary interpretations through time exhibit no correlation with patterns of publication (number of peer\u2010reviewed publications through time). 73.8% of dietary interpretations were published in the 21st century. Overall, consensus interpretations of pterosaur diets are better accounted for by non\u2010biological signals, such as the impact of the respective quality of the fossil record of different pterosaur groups on research levels. That many interpretations are based on qualitative, often untestable lines of evidence adds significant noise to the data. More experiment\u2010led pterosaur dietary research, with greater consideration of pterosaurs as organisms with independent evolutionary histories, will lead to more robust conclusions drawn from repeatable results. This will allow greater understanding of pterosaur dietary diversity, disparity and evolution and facilitate reconstructions of Mesozoic ecosystems. I.et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., Pterosaurs are an extinct clade of flying Mesozoic reptiles, with a 150\u2010million\u2010year fossil record from the Late Triassic to the latest Cretaceous in their gut and throat (content fossils) are interpreted as direct evidence of diet and oxygen (18O/16O) ratios from bone apatite and tooth enamel can reveal whether animals inhabited terrestrial or marine environments, allowing some limited dietary inferences to be drawn Preondactylus buffarinii and Quetzalcoatlus northropi and all its descendants of Scotland has been considered as a basal pterosauromorph et al. i) insectivory: insects and unarmoured terrestrial invertebrates; (ii) piscivory: fish and other nektonic organisms such as cephalopods carnivory: terrestrial vertebrates (predation and/or scavenging); (iv) durophagy: consumption of organisms with hard shells or armour herbivory/frugivory: fruits and plant matter; (vi) filter\u2010feeding on planktonic crustacean, mollusc and/or fish larvae; (vii) generalists: where authors explicitly mention this dietary category.We used seven principal dietary categories: (i) content fossils, including coprolites; (ii) spatiotemporal associations with taxa and/or depositional environments; (iii) ichnofossils; (iv) comparative anatomy; (v) functional morphology; (vi) stable isotope analysis.We assign the evidence used to support dietary interpretations to six categories: ((3)(a)The primary data set for this study is a compilation of interpretations, each comprising a statement regarding the diet of a taxon that either is one of the recognised principal taxonomic groups, or falls within one of those groups. Each interpretation was unambiguously assigned to one dietary category and to one evidence category. Dietary statements without supporting evidence were excluded.Dietary interpretations were compiled from the literature. Publications citing previous interpretations but lacking novel data or reasoning were excluded. One hundred and twenty six publications contained at least one novel dietary interpretation. When a publication identified the same dietary and evidence categories for multiple taxa within the same principal group, this was treated as a single novel interpretation. When a publication provided a single dietary statement for a single group but supported it with more than one evidential category, this was tabulated under each evidence category.Differences in the taxonomic content of principal groups between the phylogenies led to differences in the numbers of identified interpretations. For example, a publication assessing diet for two species from the same principal group in one phylogeny was treated as one interpretation. If these two species then fell within different groups in a different phylogeny, this interpretation was counted twice, once for each group. As a result, 314 interpretations were identified for the Unwin phylogeny, 311 for the Kellner phylogeny and 301 for the Andres phylogeny.Details of interpretations for each phylogeny are included as online Supporting Information (see online Appendix S1).(b)et al. A data set of all pterosaur\u2010focused publications was compiled to estimate research effort through time, from 1784 to February 2017. The sum of all publications (1828) is likely a slight underestimate.(4)et al., P < 0.05), and therefore a Spearman's rank correlation was used when comparing dietary groups and species counts.Numbers of interpretations for each dietary and evidence category were summed for each principal group. Total percentages of each evidence category were calculated. To investigate possible biological and non\u2010biological drivers of the diversity of dietary interpretations, numbers of species from each principal group were compared to numbers of assigned dietary categories. More speciose groups are hypothesised to have greater dietary diversity because they exhibit more morphological variation late 18th and 19th centuries (N = 3 from two publications); (ii) 1900\u20131969 (N = 17 from eight publications); (iii) 1970\u20131999 (N = 61 from 22 publications); (iv) 2000\u2013present (N = 233 from 94 publications).To assess changes in dietary consensus through time, dietary interpretations for each principal group were assigned to one of four different stages of pterosaur research history Witton, : (i) latIII.(1)Total numbers of dietary interpretations for each principal group Fig. show lar(2)There is a large disparity in the numbers of interpretations supported by qualitative and quantitative approaches, with the former supporting 87.3% of all interpretations Fig. . Compara(3)N = 16, P = 0.031) .There is a moderate positive correlation between pterosaur species per principal group and dietary categories for respective principal groups when Ornithocheiridae is excluded as an outlier The phylogenetic distributions of dietary interpretations for particular groups are broadly consistent among the three phylogenies . However, \u2018basal\u2010most pterosaurs\u2019 and Rhamphorhynchidae in the Andres phylogeny contain substantially more interpretations than their equivalents in the other phylogenies.(5)P = 0.302) Fig. A. The nu02) Fig. A. There 02) Fig. A. Publis02) Fig. A. This d02) Fig. A. The nu02) Fig. B. The ea(6)During the 19th century, 0.97% of dietary interpretations were proposed, 5.5% were proposed between 1900 and 1969, 19.7% between 1970 and 1999, and 73.8% since 2000 et al. c. 215\u2013190 Ma) of the UK and Italy digital reconstructions from \u2018mathematical slices\u2019 of specimen illustrations do not support this hypothesis et al., Anurognathids are known from the Middle\u2013Upper Jurassic 161\u2013145 Ma) of Germany, Central Asia and China ichnofossils Fig. . AnurognBennett, , 2007. SBennett, , 2007. ABennett, . High stBatrachognathus volans and Jeholopterus ningchengensis are known from lacustrine deposits and exhibit slightly recurved teeth c. 215\u2013176 Ma) of Central Europe and Greenland (a)et al., Rhamphorhynchines are from the Middle\u2013Upper Jurassic of Europe and China , and a rhamphotheca at the anterior end of the jaw, possibly for skim\u2010feeding on fish et al., Scaphognathines are from the Middle\u2013Upper Jurassic of Europe, Asia and Cuba and albatrosses (Diomedeidae) et al., et al., et al., et al., Basal monofenestratans are from the Middle\u2013Upper Jurassic 165\u2013151 Ma) of China et al., Istiodactylids are from the Lower Cretaceous 130\u2013112 Ma) of the UK and China . The proportional lengths of these bones are reportedly similar to modern chameleons (Chameleonidae), and Liaoxipterus was thus reasoned to have caught insects with the aid of a projectile tongue et al., Ornithocheirids are from Lower\u2013Upper Cretaceous 140\u201395 Ma) deposits on all continents except Antarctica (a)et al., Pteranodontids are from the Lower\u2013Upper Cretaceous 100\u201380 Ma) of North America and Europe possess similarly narrow jaws et al., Nyctosaurus gracilis exhibiting an \u2018antler\u2010like\u2019 head crest of Mid\u2010West USA, Mexico and Brazil (a)et al., Pterodactylus species exhibit straight, thin jaws with around 50\u201370 small, conical teeth of Germany et al., Ctenochasmatids are from the Lower\u2013Upper Cretaceous 152\u2013100 Ma) of Argentina, Central and Western Europe, Morocco, and East Asia Fig. . FunctioPiscivory interpretations are based on comparative anatomy Fig. . Sharp, Pterodaustro exhibit short, rounded teeth in their upper jaws, perhaps for crushing hard\u2010shelled crustaceans Lonchodectids are poorly known pterosaurs known from the Lower\u2013Upper Cretaceous 140\u201390 Ma) of China, the UK and Brazil (a)et al., Germanodactylus for example, exhibits edentulous jaw tips and low\u2010crowned teeth, supposedly for selecting and crushing bivalve and crustacean shells of Central and Western Europe, USA and Tanzania et al., et al., et al., et al., Dsungaripterids are from the Lower Cretaceous 145\u2013100 Ma) of China, Mongolia and South America et al., et al., Tapejarids are from the Lower\u2013Upper Cretaceous 130\u201393 Ma) of Brazil, Spain and China et al., Chaoyangopterids are from the Lower Cretaceous 130\u2013112 Ma) of China and Brazil, and had 1\u20134 m wingspans and \u2018scissor\u2010like\u2019 edentulous jaws et al., et al., Thalassodromids are from the Lower Cretaceous of Brazil 125\u2013100 Ma) et al., et al., Azhdarchids are from the Upper Cretaceous (99\u201366 Ma) of North America, North Africa, Eastern Europe and Asia Overall, there is limited consensus on diets for most pterosaur groups. Most dietary interpretations are supported by qualitative evidence, most commonly from comparative anatomy. There is strong consensus on diets for some pterosaur groups, such as insectivory in Anurognathidae and piscivory in Ornithocheiridae and Pteranodontidae, supported by one or several evidential categories. For other groups there is little consensus: in both basal ctenochasmatoids and Azhdarchidae, for example, six distinct diets have been suggested. In general, pterosaur principal groups with more species exhibit a higher diversity of dietary hypotheses. Choice of phylogeny has some impact on consensus, with the largest differences observed between phylogenies for non\u2010monofenestratan groups. Numbers of dietary interpretations exhibit small increases for much of pterosaur research history, with most interpretations proposed in the 21st century. Changes in the number of dietary interpretations through time do not correlate with numbers of pterosaur publications.et al., That the majority of dietary interpretations are qualitative is unsurprising because such interpretations can be readily proposed without rigorous experiments or analyses. Comparative anatomy uses extrapolations from observational, and occasionally experimental, studies of extant organisms Aerts, . Associaet al., i) they often require specialist technology and/or equipment; (ii) they require thorough understanding of experimental design and hypothesis testing; (iii) they can require destructive sampling of specimens et al., The range of methods employed in analysis of pterosaurs has proven highly successful in generating interpretations of diet. What is less clear, however, is how well they have performed in differentiating between alternative interpretations, and this partly explains why, for most pterosaur groups, multiple interpretations have been proposed. Greater use of analytical and experiment\u2010led methodologies (Veldmeijer (a)et al., Comparative anatomy is fundamental for analysis of homology and character evolution but has limitations as a tool for analysis of function and diet. Its application in this context is based on the assumption that convergence of morphological structures between extant and extinct taxa allows us to infer similar functional roles for structures in extinct taxa, including foraging and feeding ecology et al., Despite the straightforwardness of using associations, deriving dietary interpretations from simple presence and/or absence of contemporaneous fossils amounts to little more than speculation. Likewise, solely examining depositional environments does not account for organism dispersal abilities and taphonomic biases such as carcass transportation (Veldmeijer (c)et al., in vivo data) crania, for example, found that models which accounted for the heterogeneous nature of material properties in bone across the cranium exhibited fewest discrepancies with experimental outputs , aerial and aquatic\u2010based (diving and wading) behaviours can provide new constraints, or corroborate existing constraints, on possible foraging strategies and food\u2010acquisition behaviours. Locomotory models, however, are heavily influenced by mass estimations Witton, . Greater(d)et al., post mortem artefacts, with items introduced via water flows et al., via alignment of photographs taken from numerous angles 15N and/or \u03b4 13C. In the context of extant animals, isotopic approaches to dietary reconstruction are not without methodological limitations . Analysis of carbon isotopes from tooth enamel is possible, but this is primarily used to infer the relative proportions of C3 and C4 plants consumed et al., et al., Only one study has examined dental wear patterns on pterosaur teeth and used this to infer aspects of diet and the material properties of food \u0150si, . Dietaryet al., et al., et al., et al., et al., A more robust approach involves quantitative analysis of the sub\u2010micron scale 3D surface textures of teeth, known as dental microwear texture analysis A range of diets have been proposed for pterosaurs including insectivory, piscivory, carnivory, durophagy, filter\u2010feeding and generalism.(2) Most pterosaur dietary interpretations are supported by qualitative evidence including comparative anatomy, associations, content fossils and ichnofossils with a minority supported by quantitative evidence from functional morphology and isotope analyses.(3) Some pterosaur principal groups exhibit high levels of consensus regarding diet, supported by several evidential categories; others exhibit lower levels of consensus, with different interpretations inferred from conflicting evidence of the same categorical type, and typically poorly constrained analogy drawn from comparative anatomy.(4) More speciose pterosaur groups exhibit higher diversity of hypothesised diets. Whilst this may reflect biological signals such as ecological radiations or niche partitioning, non\u2010biological causes, such as historical biases in the data, quality of the fossil record and research intensity are more likely. These biases mean it is currently difficult to reliably test the hypothesis that the apparent patterns of pterosaur dietary diversity and disparity are biologically controlled.(5) Examining patterns of dietary diversity using different phylogenies reveals higher consensus among monofenestratan groups and lower consensus in non\u2010monofenestratan groups. Better resolution on the membership of pterosaur groups would assist in uncovering true diets for respective groups.(6) Numbers of pterosaur publications per year and dietary interpretations do not correlate through time. The majority of interpretations were proposed in the 21st century. The almost exponential rise in the number of publications containing dietary interpretations since the 1980s coincides with discoveries of new Lagerst\u00e4tten and other exceptionally preserved sites, as well as applications of new techniques to pterosaurs.(7) Many dietary interpretations are based on simple extrapolations and comparisons with modern biology with little scope for testing. Qualitative methods can serve as starting points for generating hypotheses, but quantitative tests provide more robust analyses and insights into dietary diversity, evolution and the ecological roles of pterosaurs. Improvements to current methods and application of novel methods to pterosaurs will provide better constraints on diets in pterosaurs with low levels of consensus, and better tests of dietary hypotheses in pterosaurs with high levels of consensus. This will allow reliable investigations into possible biological signals behind pterosaur dietary diversity and disparity, which will allow greater understanding of pterosaur dietary evolution and facilitate reconstructions of Mesozoic ecosystems.Appendix S1. Breakdowns of all pterosaur dietary interpretations, detailing dietary and evidential category assortments, for each of the Unwin, Kellner and Andres phylogenies. The Kellner and Andres pterosaur phylogenies and dietary interpretations for each pterosaur principal group are also included. Further information on phylogenies and the full list of 180 pterosaur species assorted into principal groups from the Unwin phylogeny is also provided.Click here for additional data file."} +{"text": "Dear Editor,A receptor. Therefore, endothelin-1 leads to a narrowing of the liver sinusoids and an increase of portal pressure . TGR5 ial., 2019). MoreovThe prevalence of liver diseases currently increases (Jansen et al., 2017; EkhlasiThe author declares no conflict of interest."} +{"text": "A and GABAB receptors . In immature networks and during numerous pathological conditions the strength of GABAergic synaptic inhibition is much less pronounced. In these neurons the activation of GABAAR produces paradoxical depolarizing action that favors neuronal network excitation. The depolarizing action of GABAAR is a consequence of deregulated chloride ion homeostasis. In addition to depolarizing action of GABAAR, the GABABR mediated inhibition is also less efficient. One of the key molecules regulating the GABAergic synaptic transmission is the brain derived neurotrophic factor (BDNF). BDNF and its precursor proBDNF, can be released in an activity-dependent manner. Mature BDNF operates via its cognate receptors tropomyosin related kinase B (TrkB) whereas proBDNF binds the p75 neurotrophin receptor (p75NTR). In this review article, we discuss recent finding illuminating how mBDNF-TrkB and proBDNF-p75NTR signaling pathways regulate GABA related neurotransmission under physiological conditions and during epilepsy.In the mature healthy mammalian neuronal networks, \u03b3-aminobutyric acid (GABA) mediates synaptic inhibition by acting on GABA A) receptors (GABAARs) causes membrane depolarization and Ca2+ influx in immature neurons , respectively is ~78\u201382 mV, close to the resting membrane potential hyperpolarization or depolarization. The activation of GABAARs during neuronal depolarization induced by the excitatory synapses allows massive Cl\u2212 entry that provides strong hyperpolarizing force and effectively compensates or diminishes the strength of the excitatory signal. The increased [Cl\u2212]i is rapidly extruded by electroneutral neuron-specific potassium-chloride cotransporter KCC2 complex which leads to a clathrin-mediated internalization and PKC pathways , a downstream target of PI-3 kinase signaling cascade . The surface expression of GABA transporter-1 (GAT-1), the major GABA transporter expressed by both neurons and astrocytes , attributed to the activity of KCC2 and not observed in BDNF KO mice (Fiorentino et al., BRs induce a calcium-dependent release of BDNF via the PLC-PKC signaling cascade and L-type voltage-gated calcium channels (Fiorentino et al., BRs also increased the phosphorylation levels of the \u03b1-CamKII, which play a critical role in BDNF release (Fischer et al., B-R-mediated release of BDNF. Interestingly, the regulated secretion of BDNF following GABAB receptor activation increases the number of GABAA \u00df2/3 subunits receptors at the postsynaptic membrane (Kuczewski et al., BRs activation and the subsequent BDNF secretion in developing hippocampal neurons contribute to the functional maturation of GABAergic synaptic transmission.Similarly to BDNF, a crucial factor regulating the development of inhibitory transmission is GABA itself (Ben-Ari et al., a et al. demonstr\u2212 homeostasis are considered to play a crucial role in epileptogenesis. Initial studies regarding the contribution of BDNF to epilepsy led to conflicting conclusions, with intrahippocampal BDNF perfusion or intraventricular injection of the BDNF scavenger TrkB-IgG, both being protective in a model of dorsal hippocampal kindling (Reibel et al., NTR are markedly increased after Pilocarpine-induced seizures. The elevated amounts of proBDNF following SE are associated with reduced proBDNF cleavage machinery that results from acute decreases in tPA/plasminogen proteolytic cascade and increases in API-1, an inhibitor of proBDNF cleavage (Reibel et al., NTR response following SE selectively downregulates KCC2, which in turn promotes a chloride homeostasis dysregulation leading to an excitatory action of GABAA receptors and facilitate epileptiform discharges (Kourdougli et al., NTR during the earliest phase of epileptogenesis restores KCC2 levels and reduces seizures frequency (Kourdougli et al., NTR play a critical role in the mechanisms of epileptogenesis (see Figure A receptor endocytosis and degradation (Riffault et al., ARs \u03b11 subunit gene through the activation of JAK-STAT pathway following SE (Lund et al., NTR signaling during the first postnatal weeks causing an impaired or delayed functional maturation of GABAergic inhibition. Alternatively, an excess of BDNF production and secretion associated with reductions in proBDNF cleavage in epileptic tissues (Ernfors et al., Epilepsy is a brain disorder characterized by the appearance of spontaneous recurrent seizures due to network hyperexcitability (Fischer et al., Unveiling the mode of action of BDNF in the development and functioning of the GABAergic network is a promising quest for developing new cures of a number of neurological diseases. BDNF influences the development and functioning of the GABAergic network which in turn controls BDNF levels. As a result of this interaction, impairment of one of the two systems will most disturb the other, and since each of them is fundamental to normal CNS functioning, this will potentially lead to a host of neurological conditions. As of today, there is hope that investigation of the molecular pathways mediating the trophic action of BDNF may provide new insights into the normal development of the GABAergic network, providing new therapeutic strategies to improve the symptoms in a broad spectrum of GABA-related pathologies.The review was conceptualized, written and edited by each of the authors. CP was the supervisor.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "The cerebellum plays a critical role in coordinating and learning complex movements. Although its importance has been well recognized, the mechanisms of learning remain hotly debated. According to the classical cerebellar learning theory, depression of parallel fiber synapses instructed by error signals from climbing fibers, drives cerebellar learning. The uniqueness of long-term depression (LTD) in cerebellar learning has been challenged by evidence showing multi-site synaptic plasticity. In Purkinje cells, long-term potentiation (LTP) of parallel fiber synapses is now well established and it can be achieved with or without climbing fiber signals, making the role of climbing fiber input more puzzling. The central question is how individual Purkinje cells extract global errors based on climbing fiber input. Previous data seemed to demonstrate that climbing fibers are inefficient instructors, because they were thought to carry \u201cbinary\u201d error signals to individual Purkinje cells, which significantly constrains the efficiency of cerebellar learning in several regards. In recent years, new evidence has challenged the traditional view of \u201cbinary\u201d climbing fiber responses, suggesting that climbing fibers can provide graded information to efficiently instruct individual Purkinje cells to learn. Here we review recent experimental and theoretical progress regarding modulated climbing fiber responses in Purkinje cells. Analog error signals are generated by the interaction of varying climbing fibers inputs with simultaneous other synaptic input and with firing states of targeted Purkinje cells. Accordingly, the calcium signals which trigger synaptic plasticity can be graded in both amplitude and spatial range to affect the learning rate and even learning direction. We briefly discuss how these new findings complement the learning theory and help to further our understanding of how the cerebellum works. It is widely recognized that the cerebellum is critical in coordinating muscles and learning novel movements with highly accurate and temporal precision. Even for a simple finger-to-nose task, to make different segments of hand and arm interact smoothly, humans need the cerebellum to precisely modulate the sequence and duration of elementary movements. The cerebellum has a relatively simple anatomy and the anatomic connections involved in its associated functions are also well known. In this context, the cerebellum becomes an ideal structure to explore learning rules and it also opens a window for us to begin to comprehend how the brain works.via excitatory synaptic connections. Small granule cells are electrically compact and they constitute the majority of neurons in the brain. Their axons project up into the molecular layer of the cerebellar cortex, bifurcate and form excitatory synapses onto Purkinje cell dendrites. As the sole output of the cerebellar cortex, each Purkinje cell is contacted by ~150,000 parallel fiber (bifurcations of granule cell axons) synapses. Meanwhile, parallel fibers also activate stellate cells and basket cells, which form inhibitory synapses with Purkinje cells, establishing a stereotypical feed-forward-inhibition circuit. Stellate cells tend to locate in the outer part of the molecular layer and mainly target Purkinje cell distal dendrites. In contrast, basket cells locate in the inner part of the molecular layer and mainly target Purkinje cell somas and axon initial segments (AIS).For decades, it has been of great interest to decipher cerebellar learning algorithms , adds to the confusion. In a recent experimental data-based theoretical study by Zang et al. (+ channels, and dendritic spike patterns.On one hand, a growing body of experimental evidence demonstrates the variability of climbing fiber responses, but on the other hand, it fails to provide a systematic explanation of somatic and dendritic spike initiation and variation and leads to many conflicting observations that stymie delineation of the functional role of climbing fibers in cerebellar learning. Furthermore, limited information extracted from noisy g et al. , the biog et al. . The som2+ influx can be graded by climbing fiber firing patterns, coincident background synapses, and voltage states in an analog manner. The conflicting spatial range of Ca2+ influx observed in different experiments can be accounted for by different voltage states (Miyakawa et al., 2+ channels, but also voltage-dependent K+ channels. Climbing fibers directly excite and depolarize proximal smooth dendrites (Palay and Chan-Palay, 2+ channels. When dendritic membrane potential is hyperpolarized, the large availability of K+ currents outweighs P-type Ca2+ current to \u201cbrake\u201d the initiation of dendritic spikes in distal parts. With depolarization, K+ currents inactivate and axial currents can trigger dendritic spikes in the whole dendrite. Theoretically, voltage-dependent climbing fiber responses also endow single Purkinje cells with the ability to overcome the credit assignment problem. Individual Purkinje cells can extract specific instruction information according to their firing states (equivalent to voltage states in vivo; Jelitai et al., In agreement with Ohtsuki et al. and Otsu2+ influx is larger than spontaneous Ca2+ spikes to signal the occurrence of an unexpected sensory event. Furthermore, Ca2+-based instruction signals in Purkinje cell dendrites contain analog information that encodes the strength of instructive stimuli trial-by-trial (Najafi et al., 2+ influx in Purkinje cells. Extracellular microelectrodes are usually used to extract simple spikes and complex spikes in vivo. Nonetheless, complex spike patterns are extremely variable in spikelet numbers and durations (Warnaar et al., in vivo noise. Thus, it is easier to sort complex spikes from Purkinje cells with low firing rates compared to Purkinje cells with high firing rates, and this causes unavoidable bias in statistical analyses. Sometimes, it is even impossible to separate a complex spike with its subsequent simple spike, when the complex spike lacks a significant pause. This situation mainly occurs when there is only one dendritic spikelet that fails to hyperpolarize dendrites (see Figure 6A in Davie et al., 2+ influx (Zang et al., Although Purkinje cells and climbing fibers are capable of encoding analog signals, does this really contribute to cerebellar learning? As reported by Najafi et al. , sensoryNoticeably, theoretical studies have also started to test the role of graded climbing fiber responses in cerebellar learning. Using a cerebellar network model with necessary climbing fiber-evoked burst-pause information in Purkinje cells (different with parallel fiber-evoked burst-pause by Steuber et al., 2+ induction threshold, usually with a climbing fiber as the polarity switch (2+ influx relative to the threshold, regardless of concurrent climbing fiber signals (Parallel fiber synapses exhibit bidirectional long-term plasticity (Coesmans et al., y switch . Howevery switch , and LTDy switch . Mathy ey switch requiredy switch . Pooling signals . In the signals . Nonethe signals . The aut signals demonstr signals and ther signals . In a mo signals demonstr2+ influx and consequently shifts plasticity from LTD to LTP.Although LTD is demonstrably critical in cerebellar learning, at least in some behavioral contexts, there is increasing awareness that it is not the sole mechanism. Accumulating evidence demonstrates that LTP of parallel fiber synapses also functions in procedural learning (Schonewille et al., 2+ influx is constrained to proximal dendrites in Purkinje cells showing decreased firing rates, as observed in vitro (Zagha et al., 2+ influx is still global in Purkinje cell dendrites, is it within the range of triggering LTP rather than LTD? In other words, does the directional change of Purkinje cell simple spike firing rates determine the polarity of parallel fiber synaptic strength changes, with LTD in \u201cbursting\u201d Purkinje cells, but LTP in \u201cpause\u201d Purkinje cells?In cerebellum-related behaviors such as saccadic eye movement (Herzfeld et al., 2+ threshold-dependent plasticity rule (Coesmans et al., A recent study proposed synaptic plasticity rules that strikingly contradict the current consensus of plasticity induction to solve the credit assignment problem (Bouvier et al., 2+ influx determines the computational unit of climbing fiber responses and constrains the learning capacity of individual Purkinje cells. Although Ca2+ influx due to strong parallel fiber-triggered dendritic spikes is constrained to branchlets (Hartell, 2+ signals was first shown to increase with distance from the soma in anesthetized rats (Kitamura and Hausser, 2+ channels with distance from the soma. In the physiologically detailed Purkinje cell model with homogeneous distributed Ca2+ channels, dendritic Ca2+ influx increases significantly with distance from the soma and shows significant variations in different branchlets at similar distances (Zang et al., in vivo, including firing state-related availability of K+ channels, concurrent synaptic input and compartment-specific dendritic excitability plasticity (Kitamura and Hausser, 2+ influx implies that both LTP and LTD can occur coincidently at different dendritic branchlets of a Purkinje cell. This spatio-temporal variability can be fine-tuned to modulate the distribution of branchlet-specific synaptic changes, rather than a homogeneous change. Accordingly, the learning capacity of single Purkinje cells can be significantly increased, which may be necessary for complex and arbitrary movement learning (Bouvier et al., Conventionally, dendritic trees are thought to just receive synaptic inputs from presynaptic cells and to convey them to the soma. In recent years, more and more experimental data have uncovered local computation in neuronal dendrites (Branco and H\u00e4usser, Hartell, , informaIn contrast to the idea of climbing fibers as teachers in the Marr-Albus-Ito theory, an alternative view of their role is to provide a \u201cmotor clock\u201d function in the initiation and timing of movements (Welsh et al., via complex spikes (Tang et al., Is there any connection between the \u201cmotor clock\u201d signal and instruction signal? The finding by Mathy et al. of climbDespite the growing awareness of multisite learning (Gao et al., YZ wrote the initial draft and revised it. ED edited the draft and revised it.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Al3+ has lethal effects on many aspects of the rhizobia/legume symbiosis, which include a decrease in root elongation and root hair formation, lowered soil rhizobial population, and suppression of nitrogen metabolism involving nitrate reduction, nitrite reduction, nitrogenase activity and the functioning of uptake of hydrogenases (Hup), ultimately impairing the N2 fixation process. At the molecular level, Al is known to suppress the expression of nodulation genes in symbiotic rhizobia, as well as the induction of genes for the formation of hexokinase, phosphodiesterase, phosphooxidase and acid/alkaline phosphatase. Al toxicity can also induce the accumulation of reactive oxygen species and callose, in addition to lipoperoxidation in the legume root elongation zone. Al tolerance in plants can be achieved through over-expression of citrate synthase gene in roots and/or the synthesis and release of organic acids that reverse Al-induced changes in proteins, as well as metabolic regulation by plant-secreted microRNAs. In contrast, Al tolerance in symbiotic rhizobia is attained via the production of exopolysaccharides, the synthesis of siderophores that reduce Al uptake, induction of efflux pumps resistant to heavy metals and the expression of metal-inducible (dmeRF) gene clusters in symbiotic Rhizobiaceae. In soils, Al toxicity is usually ameliorated through liming, organic matter supply and use of Al-tolerant species. Our current understanding of crop productivity in high Al soils suggests that a much greater future accumulation of Al is likely to occur in agricultural soils globally if crop irrigation is increased under a changing climate.Recent findings on the effect of aluminium (Al) on the functioning of legumes and their associated microsymbionts are reviewed here. Al represents 7% of solid matter in the Earth\u2019s crust and is an important abiotic factor that alters microbial and plant functioning at very early stages. The trivalent Al (Al Legumes, therefore, play a major role in reducing poverty, improving human health and nutrition and enhancing ecosystem functioning. With more than 78.3\u00a0million\u00a0ha of land planted to legumes, these species provide over 35% of the world\u2019s protein intake by legumes is therefore a major source of N for agriculture stress 2+, Al(OH)2+, Al(OH)3 and Al(OH)4\u2212 2+ and Al(OH)2+ species are formed as the soil pH increases 4\u2212] dominates under alkaline conditions and phosphoenol pyruvate carboxylase (PEPC), which catalyse the formation of carboxylic acids and Cd (0.3\u20130.5\u00a0mM) gene clusters have been discovered in Rhizobium leguminosarum bv. viciae and other members of the Rhizobiaceae that are expressed in response to heavy metal concentrations . We postulate that organic acids (OAs) secreted by roots and cluster roots chelate with active Al to form inactive complexes in the rhizosphere. We also suggest that these OAs inside roots and cluster roots form complexes with incoming active Al ions to form inactive Al-OA complexes that are stored in non-toxic forms in roots and cluster roots. This model could explain why the Al concentrations in below-ground organs such as roots and cluster roots are many folds greater than Al levels in above-ground shoots. In our view, this constitutes the mechanism by which A. linearis can thrive in Al-rich, highly acidic soils in the Cape Fynbos of South Africa. Taken together, these biochemical subtleties in Al tolerance support A. linearis as a natural system for studying metal tolerance in nodulated perennial legumes the selection and use of legume/rhizobia symbioses resistant to metals, (ii) the use of mixed inoculants containing metal-resistant rhizobia and plant growth-promoting rhizobacteria and (iii) plant inoculation with a mixture of rhizobia and mycorrhizae (Pajuelo et al. Furthermore, the ability of legumes such as 2 fixation of A. linearis (Muofhe and Dakora Interestingly, while there is evidence of acid-tolerant genes in symbiotic rhizobia (Dilworth et al. 2+ to overcome inhibition of root elongation by Al. Thus, even though tap roots might extend into acidic soil zones, development of lateral roots for nutrient and water capture could still be limited (Ferrufino et al. Al phytotoxicity can be amended through liming with calcium carbonate, addition of organic matter and/or by use of Al-tolerant species (Mokolobate and Haynes + and Al+3 activities (Sanzonowicz et al. +3 activity at root cell plasma membrane, thus preventing the disruption of cell expansion and cell division commonly induced by Al toxicity (Kochian Liming has also been found to increase Ca availability to rhizobia and the symbiosis (Hungria and Vargas Kochian . Similar Kochian . Applied Kochian .Organic matter can also be used to overcome Al toxicity in plants and microbes Foy . During 2 fixation and decreased crop yield. Therefore, selecting legume/rhizobia symbioses that are tolerant of Al toxicity is the easiest way to increase crop yields in Al-rich acidic soils. A better understanding of legume exudation in response to Al toxicity and the mechanisms underlying rhizobial tolerance of Al stress is crucial for increasing yield of grain and pasture legumes. Furthermore, understanding gene expression in the presence of added Al may be a strategy for identifying rhizobial genes and legume traits that permit high N2 fixation in the presence of Al stress.Taken together, Al stress is a major abiotic factor affecting plant growth and productivity. With 40% of the world\u2019s arable land consisting of acid soils and Al toxicity being associated with low pH, global legume production is likely to be hugely constrained. This is because Al toxicity in soils can inhibit root elongation, lateral root development, root hair growth, rhizobial infection of the roots, Nod factor production and nodule development, resulting in low N"} +{"text": "ARs) are the major mediators of synaptic inhibition in the brain. Aberrant GABAAR activity or regulation is observed in various neurodevelopmental disorders, neurodegenerative diseases and mental illnesses, including epilepsy, Alzheimer\u2019s and schizophrenia. Benzodiazepines, anesthetics and other pharmaceutics targeting these receptors find broad clinical use, but their inherent lack of receptor subtype specificity causes unavoidable side effects, raising a need for new or adjuvant medications. In this review article, we introduce a new strategy to modulate GABAeric signaling: targeting the intracellular protein interactors of GABAARs. Of special interest are scaffolding, anchoring and supporting proteins that display high GABAAR subtype specificity. Recent efforts to target gephyrin, the major intracellular integrator of GABAergic signaling, confirm that GABAAR-associated proteins can be successfully targeted through diverse molecules, including recombinant proteins, intrabodies, peptide-based probes and small molecules. Small-molecule artemisinins and peptides derived from endogenous interactors, that specifically target the universal receptor binding site of gephyrin, acutely affect synaptic GABAAR numbers and clustering, modifying neuronal transmission. Interference with GABAAR trafficking provides another way to modulate inhibitory signaling. Peptides blocking the binding site of GABAAR to AP2 increase the surface concentration of GABAAR clusters and enhance GABAergic signaling. Engineering of gephyrin binding peptides delivered superior means to interrogate neuronal structure and function. Fluorescent peptides, designed from gephyrin binders, enable live neuronal staining and visualization of gephyrin in the post synaptic sites with submicron resolution. We anticipate that in the future, novel fluorescent probes, with improved size and binding efficiency, may find wide application in super resolution microscopy studies, enlightening the nanoscale architecture of the inhibitory synapse. Broader studies on GABAAR accessory proteins and the identification of the exact molecular binding interfaces and affinities will advance the development of novel GABAAR modulators and following in vivo studies will reveal their clinical potential as adjuvant or stand-alone drugs.\u03b3-aminobutyric acid type A receptors (GABA ARs) are the principal mediators of phasic and tonic inhibition in the human brain, being a vital part of the molecular machinery that creates cognition, behavior, and consciousness domain carrying proteins. These showed that through modulation of receptor-scaffolding protein interactions a variety of responses could be achieved, ranging from disruption of glutamate signaling to neuroprotective effects in ischemic brain damage is a guanine nucleotide exchange factor for Cdc42, a gephyrin binding partner (Kins et al., AR subtypes and GABAAR scaffolds. Neuroligin dysfunction has been implicated in autism (Pettem et al., AR, remain uncharacterized. These molecular insights could greatly contribute to our understanding of the development of the inhibitory synapse, as well as the underlying molecular causes of developmental diseases. Neuroligin family members exert distinct roles in the formation and stabilization of inhibitory and excitatory synapses and display distinct cellular and subcellular distributions. Accordingly, molecules that interfere with their isoform-specific interactions could act as highly cell-type selective modulators of neurotransmission.Proteomic studies (Kang et al., ARs. The loss of gephyrin clusters following the loss of the GABAAR \u03b32 subunit (Essrich et al., AR clusters upon gephyrin deficiency (Kneussel et al., AR subunits could be confirmed (Tretter et al., in-silico studies (Pennacchietti et al., AR complexes and demonstrated that at least the GABAAR \u03b11\u20133 and GlyR \u03b2 subunits bind to an overlapping site within gephyrin in a mutually exclusive fashion (Maric et al., ARs and GlyRs (Specht et al., Gephyrin is a prime candidate for the role of master regulator of neuronal function at inhibitory sites (Tyagarajan and Fritschy, AR diffusion and contributing to input-specific adaptations at postsynaptic sites (Chen et al., AR synapse dynamics remain to be explored in a comprehensive approach that includes an extensive alternative splicing and complex post-translational modification patterns of this region. Identification of the targeted binding pockets and insights into the binding affinities of the modified and unmodified peptide regions within the central region of gephyrin could shed light on the enigmatic molecular mechanisms of gephyrin multimerization, degradation and the tuning of its ligand binding affinities. Additionally, gephyrin isoforms are tissue-specific (Paarmann et al., Gephyrin itself is dynamically regulated, affecting GABAvia gephyrin was achieved more than a decade ago using intrabodies (Zacchi et al., ARs. One such example is artemisinins [AR signaling. Kasaragod et al. (ARs clustering, making artemisinins the first small molecule lead compounds for a new class of inhibitory neurotransmission modulators. Strikingly, the druggable artemisinin-binding pocket overlaps with the universal receptor binding region of gephyrin, which is critical for the interaction with GABAA and glycine receptors (Kasaragod et al., A and glycine receptors that bind to gephyrin. Biomimetic optimization of the \u201chotspots\u201d amino acid sequence, enhanced the affinity of the resulting peptide ligands 46,000-fold compared to the corresponding native peptides (Maric et al., in vitro applications of these new super binder peptide reduced GABAAR \u03b12 conductivity and clustering, providing evidence that GABAAR-associated proteins can be successfully targeted with modified peptides to modulate fast synaptic inhibition (Maric et al., Gephyrin\u2019s crucial role in glycinergic and GABAergic transmission made it a major pharmacological target. The modulation of synaptic responses d et al. identifiAR trafficking is pivotal for the plasticity (Luscher et al., AR cycling is involved in various neurological disorders (Smith and Kittler, AR sites, that are involved in the trafficking of the receptors, has been identified to control receptor numbers and their concentration at synaptic sites (Comenencia-Ortiz et al., AR trafficking, endocytosis, degradation or recycling, is a promising pharmacological strategy. The proposed direct protein interactors are numerous, among them are muskelin (Heisler et al., GABAAR interaction rapidly modulates synaptic GABAAR numbers, inhibitory synaptic strength, neuronal excitability, and notably, affects animal behavior (Kittler et al., AR subunits (AR derived peptides, that effectively compete with AP2 binding, were successfully used to block the receptor internalization in hippocampal neurons, increasing surface concentration of GABAAR clusters by 50% (Smith et al., AR interactions with its intracellular trafficking partners is an alternative way to influence GABAergic signaling.The AP2-GABAsubunits . Short GAR clustering, like artemisinins or \u201csuper binding peptides,\u201d could reduce the GABAAR synaptic concentration and function. On the other hand, peptides hampering receptor interaction with AP2 trafficking protein increased the synaptic receptor levels. In theory, these approaches could be applied together to achieve bi-directional modulation of inhibitory neurotransmission, promoting a shift in the dynamic equilibrium from phasic to tonic neuronal response.Ongoing research uncovered original, seemingly contrasting, strategies of GABAergic signaling modulation. On the one hand, ligands disrupting gephyrin-GABAAR associated scaffold or trafficking proteins could be applied wherever abnormal GABAergic activity or regulation is involved in pathogenesis. In status epilepticus patients develop a time-dependent pharmacoresistance to GABAergic agents, probably, due to GABAAR internalization (Naylor et al., ARs (Nicholson et al., ARs and both could potentially be alleviated by targeting GABAAR-associated proteins. Stabilization of the gephyrin-receptor scaffolds at inhibitory postsynapses with molecules that mimic the stabilizing action of CB (Saiepour et al., AR loss and preserve inhibitory neurotransmission, alternatively, applying AP2 inhibitors could reduce GABAAR internalization and reverse the loss of postsynaptic GABAARs. Those examples illustrate the potential of GABAergic modulators as adjuvants ameliorating the effect of existing potent drugs, whereas in epilepsy or other diseases involving deregulation of inhibitory neurotransmission they could be applied as stand-alone therapeutics.Those new strategies of GABAergic neurotransmission modulation possess an untapped clinical potential. Agents targeting GABAAR intracellular interactors, accelerated by in-silico predictions and high throughput approaches, will lead to the discovery of novel GABAergic modulators. Affinity, selectivity, bioavailability and immunogenicity of these compounds would have to be optimized for clinical applications, where peptide-based ligands could be further evolved by the introduction of unnatural amino acids, cyclization and other chemical modifications.We expect that the study of GABAMicroscopy is an additional intriguing application of these molecules. The enhanced affinity and specificity of the engineered peptide-based compounds allowed to pioneer their use as fluorescent probes [AR associated proteins could prove to be a versatile pharmacological strategy with clinical potential. Further, we suggested that when combined with state-of-the-art super-resolution microscopy methods, the peptide-based fluorescent probes may resolve the nanoscale architecture of synapses in unprecedented detail. We anticipate that the discovery of additional GABAAR interactors could open the way for the development of new imaging tools and alternative pharmacological approaches.Here, we discussed how the targeting of GABAVK and HM wrote the manuscript and prepared the figures.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "In a recent study, Tuuli Karhu and colleagues from Helsinki University compared a set of cell lines for their susceptibility towards eight GATA4 targeting compounds . GATA4 al., 2018; Kikuchial., 2018; Pikkaraal., 2018). The goal., 2018). Of couIn recent years, the development of stem cell based test systems has been a major focus of research (Leist et al., 2017; Godoy ein vivo relevance still remains a major challenge.This approach has been used for developmental neurotoxicity (Waldmann et al., 2014; Meganat"} +{"text": "Following systematic scrutiny of the evidence in support of the hypothesis that the cardioprotective mechanism of action of dexrazoxane is mediated by a \u2018depletion\u2019 or \u2018downregulation\u2019 of Top2\u03b2 protein levels in heart tissue, the author concludes that this hypothesis is untenable. In seeking to understand how dexrazoxane protects the heart, the outcomes of a customised association rule learning algorithm incorporating the use of antecedent surrogate variables reveal a previously unknown relationship between dexrazoxane and poly(ADP-ribose) (PAR) polymer. The author shows how this previously unknown relationship explains both acute and long-term cardioprotection in patients receiving anthracyclines. In addition, as a direct inhibitor of PAR dexrazoxane has access to the epigenome and this offers a new insight into protection by dexrazoxane against doxorubicin-induced late-onset damage . Notably, through this review article, the author illustrates the practical application of probing natural language text using an association rule learning algorithm for the discovery of new and interesting associations that, otherwise, would remain lost. Historically, the use of CEME enabled the first report of the capacity of a small molecule to catalyse the hybrid self-assembly of a nucleic acid biopolymer via canonical and non-canonical, non-covalent interactions analogous to Watson Crick and Hoogsteen base pairing, respectively. Dexrazoxane (ICRF-187) belongs et al throughout the 1970s and 1980s .\u22123 = \u03bcM) with concentrations in ex vivo tissue mass (\u03bcmol kg\u22121), then using the literature values of IC50 as a measure of toxicity, it can be concluded that for prolonged periods the cardiac concentrations of doxorubicin are sufficient to negatively impact upon cardiomyocyte viability. The limited animal data suggest that cardiotoxic levels of doxorubicin remain significantly above the median IC50 value of 1.67 \u03bcM (see above) for at least 24 hours and decline below this limiting concentration between 24 and 48 hours. In both animals and patients, there is convincing evidence for long-term (days/weeks) retention of doxorubicin within cardiac tissue. Accordingly, given the long-term accumulation of doxorubicin within the heart, if the short pre-administration single infusion of dexrazoxane exerts a cardioprotective effect by depleting Top2\u03b2 protein, then by necessity suppression of the level of Top2\u03b2 protein within the heart must be profound and long lasting. As the author argues, this seems a remote prospect given that by comparison with the slow elimination of doxorubicin, dexrazoxane has a remarkably short half-life. The logical corollary of this substantial difference in pharmacokinetics is twofold. First, dexrazoxane must exert long-lasting suppression that results in maintenance of Top2\u03b2 protein below threshold. Second, in the absence of long-lasting suppression, recovery of suppressed Top2\u03b2 protein (transcription) must be slow.Accepting the proportionality error in comparing concentrations in an aqueous buffer (t1/2 of 9.3 hours), and to the final hydrolysis product ADR-925 (t1/2 of 23 hours) . Despite the plethora of drugs that degrade Top2\u03b2 protein, dexrazoxane remains the only product that is licenced for the prevention of cardiotoxicity associated with the use of anthracyclines; et al ] , and as et al triggered the formation of a supramolecular self-assembly consisting of non-covalent interactions between polyacrylate-anchored melamine units and monomeric cyanuric acid.Cyanuric acid catalyses the formation of supramolecular self-assemblies of a naturally occurring adenine-containing nucleotide, polyadenine (poly(A)), in both its DNA and RNA forms; structurally, poly(A) is closely related to PAR. Cyanuric acid-catalysed self-assemblies of poly(A) can be modelled using either canonical Watson\u2013Crick base pairing or a combination of both canonical Watson\u2013Crick and non-canonical Hoogsteen base pairing. Spectral studies suggest that the preferred assembly of poly(A) strands with cyanuric acid monomers is a coiling triplex formation consisting of both canonical and non-canonical base pairing.Cyanuric acid-catalysed self-assembly using a synthetic adenine-containing peptide nucleic acid (PNA) in place of poly(A) has also been demonstrated.Intuitively, it is to be expected that low-energy conformations will exist when there is a maximum separation between the piperazine structures. Conformational analysis undertaken by the author (for details of methods see the appended Supplement) confirm that lower energy conformations are consistent with the maximum spacing between piperazine structures with each adopting a half-chair conformation at methylene carbon atoms 3,5 of each ring structure resulting in an elevated tertiary amine nitrogen atom. As a half-chair conformation, the remainder of the piperazine structure is planar with the keto group at the carbon atom in position 2 of each ring structure in alignment with the tautomeric nitrogen of the secondary amine group of the adenine base of each ADP-ribose unit of PAR , a pilot study was undertaken by the author to investigate the interaction between dexrazoxane and PAR. Using isolated rat liver mitochondria, we demonstrated for the first time that dexrazoxane inhibits PAR-induced release of AIF. Although the details of this methodology are not presented here, the western blots from this study, using anti-AIF antibody (Abcam ab32516) are reproduced in In collaboration with Ritchie et al . Notably, echocardiographic data suggest that dexrazoxane provides long-term cardioprotection, implying that prevention of cardiomyocyte damage during therapy can reduce the incidence of delayed doxorubicin-associated cardiomyopathy in long-term survivors without compromising the chances of oncological cure [The initial application in 2014 of CEME to dexrazoxane revealed an unequivocal signal defining a previously-unknown relationship between dexrazoxane and PAR polymer. Subsequently, the unique property of dexrazoxane to sequester PAR by base pairing was demonstrated using cal cure , 118.What does the proposed association between dexrazoxane and PAR mean to Oncologists?In addressing this important question some historical perspective must be introduced. Dexrazoxane reduces the incidence of anthracycline-induced heart failure by 80% and it is the only drug approved for its prevention . DespiteWhile beyond the scope of this review, the clinical consensus is that dexrazoxane does not compromise anti-tumour therapy. Indeed, in May 2017, the EMA Committee for Medicinal Products for Human Use approved the expansion of the Cardioxane Marketing Authorisation enabling increased use of dexrazoxane within the paediatric population for whom dexrazoxane had been previously contraindicated (dexrazoxane is marketed by Clinigen UK as a branded generic). A review of the evidence that historically led to a reassessment of the European Label for dexrazoxane found that dexrazoxane is not associated with an increased risk of second primary malignancies and that dexrazoxane therapy does not impair anthracycline\u2019s anti-tumour efficacy .Thus, if the above changes and revisions lead to an increase in the use of dexrazoxane throughout different patient populations, then the alert clinician should be responsive to novel/atypical/unexpected clinical benefits and outcomes if dexrazoxane is acting in some part by sequestering PAR by base-pairing. et al propose that impaired cardiac function in doxorubicin-treated childhood cancer survivors is partly mediated by disruption of mitochondrial energy production and that this damage is abrogated by dexrazoxane [Indeed, scrutiny of existing clinical outcomes supports an interaction between dexrazoxane and PAR. For example, Lipshultzrazoxane . In a loAt a median follow-up of 7.8 years after treatment, doxorubicin-treated survivors without dexrazoxane had increased PBMC mtDNA copies per cell and concomitant use of dexrazoxane was associated with lower mtDNA copies per cell. The investigators add that mtDNA copies per cell in those who received dexrazoxane were within the normal ranges observed in their previous studies. OXPHOS activity was not different between groups. The investigators propose that in patients treated with doxorubicin alone, impaired mitochondria may undergo clonal expansion of mtDNA that functionally compensates for mutations or deletions that results in normal OXYPHOS and ATP production within the myocardium. They add that their findings support the evidence that dexrazoxane adjuvant therapy in paediatric high-risk ALL patients offers systemic mitochondrial protection and suggest a possible role of dexrazoxane prior to anthracycline therapy as a general protectant of mitochondrial function in other healthy tissues that include the ovaries . et al [Previously, Lebrecht et al had prop et al .Notwithstanding alternative explanations, can the above effects upon mtDNA be reconciled with an interaction between dexrazoxane and PAR, and in the absence of dexrazoxane can PAR explain the delayed onset of anthracycline-associated cardiomyopathy? et al [ et al speculate that by contrast with the well-established positive regulatory role of PARP1 in maintaining nuclear DNA integrity, the addition of PAR groups on mtDNA repair enzymes may uniquely reduce their affinity to bind with DNA and thus may attenuate the rate of mtDNA repair.Using wild-type and PARP1-depleted A549 cells, Szczesny et al investig et al [ et al [Accepting the new hypothesis that dexrazoxane sequesters PAR by base pairing, then arguably this sequestration state is comparable/equivalent to the PARP1-depleted cells used by Szczesny et al and that et al . For exa et al . Accordi [ et al may be a [ et al .A further example serves to illustrate how the existing data can be re-evaluated in the light of an interaction between dexrazoxane and PAR. While the acute cardiotoxic effects of doxorubicin are consistent with a doxorubicin-induced DNA damage response resulting in both PARP1-dependent apoptotic and necrotic cell death , 126, deTwo isoforms of MHC are expressed in the mammalian heart, \u03b1-MHC and \u03b2-MHC. The \u03b1-MHC isoform has a higher ATPase activity than \u03b2-MHC, and the contractile velocity of the heart is correlated with the relative amount of each isoform; hearts expressing greater amounts of \u03b1-MHC having a more rapid contractile velocity \u2013138.The \u03b1- and \u03b2-MHC ratio correlates directly with the overall cardiac performance in both animals and in patients with cardiomyopathy and heart failure \u2013145. PatDe Beer\u2019s Group in the Department of Medical Physiology at Utrecht University observed that long-term administration of doxorubicin resulted in an impairment of the actin-myosin interaction in the hearts of male Wistar rats . Notably et al investigated whether the cardio-protective properties of dexrazoxane involve the prevention of the deleterious effects of doxorubicin upon the actin\u2013myosin contractile machinery [Following their earlier observations of the effects of chronic treatment with doxorubicin upon the contractile function of the heart, de Beerachinery . They ob et al do not support a role for free radicals in mediating the deleterious effects of doxorubicin upon the actin\u2013mysoin contractile machinery [Interestingly, there are no reports within the literature of an association between Top2\u03b2 and MHC isoforms, and additional studies by de Beerachinery . Howeverachinery . MoreoveAre doxorubicin-induced alterations at the epigenome as manifest by a shift in the ratio between MHC isoforms and the effects of dexrazoxane in abrogating this shift, consistent with an interaction between dexrazoxane and PAR? et al in the Division of Cardiovascular Medicine at Stanford University School of Medicine show that Brahma-related gene 1 (Brg1), a chromatin-remodelling ATPase protein subunit of the BRG-1-Associated Factor (BAF complex), interacts with two other classes of chromatin-modifying enzymes, histone deacetylase (HDAC) and PARP, to regulate gene expression during cardiac growth, differentiation and hypertrophy [ et al show that in hypertrophic hearts PARP1 is bound to the proximal promoters of \u03b1-MHC and \u03b2-MHC, and that inhibition of PARP1 activity by the phenanthridinone-based PARP inhibitor PJ34 reduced both Brg1-mediated \u03b1-MHC repression and \u03b2-MHC activation in reporter assays, indicating that Brg1 and PARP1 cooperate to regulate MHC expression [Hangertrophy . In adulertrophy \u2013155. In pression .Mahesh Gupta\u2019s Group in the Department of Cardiothoracic Surgery at The University of Chicago demonstrate that nuclear extracts treated with anti-ADP-ribose antibodies reveal increased poly-ADP-ribosylation (PARylation) of nuclear proteins in failing hearts by comparison with controls, thus confirming the presence of PAR polymer [\u2212/\u2212 mice was also evident from the analysis of hypertrophic marker genes. In PARP+/+ aortic artery-banded mice, the levels of \u03b2-MHC mRNA were highly elevated, whereas \u03b1-MHC levels were repressed, as expected. By contrast, in PARP\u2212/\u2212 aortic artery-banded mice, no repression of \u03b1-MHC levels was observed, and the levels of \u03b2-MHC were increased to a much lesser extent.Notably, polymer . From thIn their concluding remarks, they propose that PARP inhibitors may be useful agents for managing cardiomyopathies and heart failure.Taken together, these and other data are provocative and indicate that in some part, the cardiotoxic effects of doxorubicin and the cardioprotective effects of dexrazoxane, converge upon the epigenome. By comparison with acute damage, delayed-onset toxicity is consistent with a phenotypic switch that results from doxorubicin alteration(s) at the epigenome. Sequestration of PAR through both canonical Watson\u2013Crick base pairing, and non-canonical Hoogsteen base-pairing provides a mechanism for the cardioprotective effects of dexrazoxane against the toxic effects of doxorubicin in the acute term, and abrogation of delayed-onset cardiomyopathy. et al have previously suggested a possible role of dexrazoxane prior to anthracycline therapy as a general protectant of mitochondrial function in healthy tissues in addition to the heart [ et al [very important neuroprotective properties\u2019.Given the focal role of PAR in an overwhelming number of diseases, it is indeed thought provoking that dexrazoxane offers, for the first time, a unique opportunity to investigate the impact of sequestration of PAR, by comparison with inhibition of PARP, within a remarkably broad context. By way of example, and whilst beyond the scope of this review, the interaction between dexrazoxane and PAR offers the prospect that dexrazoxane can function as a cytoprotectant within other physiological systems (Lipshultzhe heart ). Neurophe heart . In that [ et al concludepreviously unknown relationships\u2019 within the published literature for a nominated drug within an elected therapeutic category. By comparison with the kind of data stored in structured databases, natural language text within the published literature is unstructured, amorphous and difficult to mine using the traditional algorithms. In practise, the greatest obstacle is missing data within a dataset that is small (thousands of data pieces) by comparison with the much larger size (millions of data pieces) of dataset sets mined using methods traditionally applied in commercial operations such as mining high street supermarket transactions. CEME overcomes the problem of missing data by adapting the use of a statistical method known as \u2018antecedent surrogate variables\u2019. Specifically, in exploring the mechanism of action of dexrazoxane as a cardioprotectant, the task was to characterise as exhaustively as possible cell signalling within all known molecular phenotypes (as determined by extracellular first messengers) of anthracycline-exposed cardiomyocytes. In practise, this is an extremely demanding operation that requires the manual input for all known molecular phenotypes of all known second messengers, enzymes, receptors, transcription factors and so on. To reduce the possibility of a type I error , in addition to dexrazoxane, the CEME process should be repeated for all known cardioprotectants of anthracycline-exposed cardiomyoctes; the list includes beta blockers, angiotensin inhibitors, angiotensin-converting enzyme inhibitors and statins. That is, previously unknown relationships between each cardioprotectant and cell signalling within the anthracycline-compromised cardiomyocyte should be explored. For practical reasons, this was not undertaken.The CEME algorithm is a well-validated methodology that has been successfully applied by McCormack Pharma to many drugs throughout more than three decades \u2013134, 187In silico modelling studies demonstrate that dexrazoxane catalyses the formation of a hybrid self-assembled supramolecular structure between adjacent strands of PAR. These assemblies depict an antiparallel orientation of canonical Watson\u2013Crick base pairing of dexrazoxane with adenine bases. In addition, these in silico studies show that dexrazoxane can also base pair with PAR strands by non-canonical Hoogsteen base pairing. Importantly, these supramolecular structures employing either canonical or non-canonical base pairing and combinations of the two, accord with the theoretical expectation of low-energy thermodynamically stable entities. Moreover, that such supramolecular assemblies are possible is strongly suggested by the work of other groups using molecules that are closely analogous to both dexrazoxane and PAR [in vivo conditions is lacking. Initially, aqueous stability could be explored by utilising in vitro methods such as thermal denaturation using circular dichroism and ultraviolet\u2013visible spectroscopy [ and PAR , 104\u2013107troscopy . These cin vitro study show that consistent with expectation, dexrazoxane prevents PAR-induced AIF release from isolated mitochondria. However, dose-ranging studies are needed, and equilibrium times require investigation. The utilisation of exogenous PAR as used by the author is not a paradigm of a deep compartment for the accumulation of dexrazoxane and consequently does not adequately represent the in vivo situation. Preferably, further studies should incorporate the use of whole cells, such as isolated neonatal rat cardiomyocytes together with the use of ionising radiation to induce DNA damage with subsequent elaboration of endogenous PAR.The results of a preliminary The conformational relationship of the non-covalent interaction between dexrazoxane and PAR was explored using a modified version of Allinger\u2019s Molecular Mechanics MM2 force field (for review see ). Accord"} +{"text": "With the exponential growth of the Internet of Things (IoT) and cyber-physical systems (CPS), a wide range of IoT applications have been developed and deployed in recent years. To match the heterogeneous application requirements in IoT and CPS systems, many resource-constrained IoT devices are deployed, in which privacy and security have emerged as difficult challenges because the devices have not been designed to have effective security features. With the exponential growth of the Internet of Things (IoT) and cyber-physical systems (CPS), a wide range of IoT applications have been developed and deployed in recent years. To match the heterogeneous application requirements in IoT and CPS systems, many resource-constrained IoT devices are deployed, in which privacy and security have emerged as difficult challenges because the devices have not been designed to have effective security features.Despite many security solutions being developed for the Internet, there are major concerns regarding the resource-constrained environment in IoT, including data encryption, privacy preservation, vulnerabilities, threats, attacks, and controls, among others. To address these privacy and security challenges, appropriate technologies have to be developed for resource-constrained environments in IoT.The basic objective of this Special Issue is to report the most recent research efforts dedicated to strengthening the security and privacy solutions for resource-constrained devices in IoT and CPS systems. Specifically, each manuscript was carefully reviewed by at least two independent experts. All submissions were evaluated for their rigor and quality and their relevance to the topics proposed in this Special Issue. After a rigorous review process, 16 high-quality papers were accepted and included in this Special Issue. We will now briefly introduce the accepted papers.In the paper entitled \u201cA Lightweight Cipher Based on SALSA20 for Resource-Constrained IoT Devices\u201d , Lara etIn the paper entitled \u201cAn Incentive Mechanism in Mobile Crowdsourcing Based on Multi-Attribute Reverse Auctions\u201d , Hu et aBouaynaya et al. investigated formal methods to quantify and evaluate the risks associated with information systems in the paper \u201cExploring Risks Transferred from Cloud-Based Information Systems: A Quantitative and Longitudinal Model\u201d . FurtherThe paper entitled \u201cEfficient Privacy-Preserving Access Control Scheme in Electronic Health Records System\u201d focused on the constrained device access control in e-health records (HER) systems by reducing the computational resources . The proSun et al focused on a compressed-sensing-based fault-tolerant data aggregation in the paper \u201cCS-FCDA: A Compressed Sensing-Based on Fault-Tolerant Data Aggregation in Sensor Networks\u201d . The effIn the paper entitled \u201cBeeKeeper 2.0: Confidential Blockchain-Enabled IoT System with Fully Homomorphic Computation\u201d , Zhou etRahman et al. focused on the joint relay selection and power allocation for cooperative cognitive radio networks in the paper entitled \u201cJoint Relay Selection and Power Allocation through a Genetic Algorithm for Secure Cooperative Cognitive Radio Networks\u201d , which iAlromih et al. investigated a randomized watermarking technique for resource-constrained devices in the paper \u201cA Randomized Watermarking Technique for Detecting Malicious Data Injection Attacks in Heterogeneous Wireless Sensor Networks for Internet of Things Applications\u201d . The expFang, Yang, and Wu went through secure cost-aware data communication in IoT in the paper entitled \u201cSecurity Cost Aware Data Communication in Low-Power IoT Sensors with Energy Harvesting\u201d . In thisLuo et al. focused on the low-cost security and data integrity scheme in an air quality monitoring system in the paper entitled \u201cOn the Security and Data Integrity of Low-Cost Sensor Networks for Air Quality Monitoring\u201d .Alabdulkarim et al. developed a privacy-preserving single decision tree algorithm for resource-constrained IoT devices in the paper entitled \u201cPPSDT: A Novel Privacy-Preserving Single Decision Tree Algorithm for Clinical Decision-Support Systems Using IoT Devices\u201d . This woLara-Nion et al. investigated efficient scalar multiplication for resource-constrained devices in the paper \u201cEnergy/Area-Efficient Scalar Multiplication with Binary Edwards Curves for the IoT\u201d . In thisIn the paper entitled \u201cFPGA Modeling and Optimization of a SIMON Lightweight Block Cipher\u201d , Abed etQin et al. proposed a lightweight anomaly detection system for IoT in the paper titled \u201cIMLADS: Intelligent Maintenance and Lightweight Anomaly Detection System for Internet of Things\u201d . This woAl-Otaibi et al. aimed at developing a privacy-preserving vehicular rogue node detection scheme for light devices in IoT and fog computing in the paper entitled \u201cPrivacy-Preserving Vehicular Rogue Node Detection Scheme for Fog Computing\u201d . The simShifa et al. proposed a lightweight cipher for H.264 video in IoT in the paper entitled \u201cLightweight Cipher for H.264 Videos in the Internet of Multimedia Things with Encryption Space Ratio Diagnostics\u201d . This woIn this Special Issue, a wide range of topics are reported that cover ongoing research interests regarding security and privacy solutions for resource-constrained devices in IoT and CPS systems. The team hopes that this Special Issue will make contributions to encouraging IoT security and privacy issues.In conclusion, we sincerely thank all researchers for their sharing of their research works to this special issue and the reviewers for volunteering their time and expertise to carefully reviewing and commenting on all submissions. We would like to thank the sensors EIC and the admin team for their continuous support and guidance."} +{"text": "Current developments and challenges for serial sample delivery at synchrotrons and X-ray free-electron lasers are reviewed, including the new megahertz repetition-rate machines, with an emphasis on liquid injection and high-viscosity extrusion. The high peak brilliance and femtosecond pulse duration of X-ray free-electron lasers (XFELs) provide new scientific opportunities for experiments in physics, chemistry and biology. In structural biology, one of the major applications is serial femtosecond crystallography. The intense XFEL pulse results in the destruction of any exposed microcrystal, making serial data collection mandatory. This requires a high-throughput serial approach to sample delivery. To this end, a number of such sample-delivery techniques have been developed, some of which have been ported to synchrotron sources, where they allow convenient low-dose data collection at room temperature. Here, the current sample-delivery techniques used at XFEL and synchrotron sources are reviewed, with an emphasis on liquid injection and high-viscosity extrusion, including their application for time-resolved experiments. The challenges associated with sample delivery at megahertz repetition-rate XFELs are also outlined. This type of mixing, combined with SFX data collection from a GDVN-generated liquid jet, has been used to capture structural intermediates of a riboswitch binding its ligand \u223c10\u2005s after reaction initiation , the mixing process must take place close to the injector tip such that the travel time of the sample from the point of reaction initiation to the X-ray interaction region is short. Moreover, the effect of a non-uniform velocity profile on sample transport must be minimized downstream of the reaction initiation. Interestingly, an investigation of microcrystal flow through capillaries indicated the presence of a depletion zone containing no crystals near the capillary wall, which introduces an intrinsic cutoff of low-speed tails and alleviates flow smearing . High pressures are generally required for this, often in excess of those that can be supplied by an HPLC-based liquid-delivery system. A piston-driven sample reservoir must then be installed between the HPLC and the nozzle, with the piston serving both as a pressure amplifier and as the means of separating the sample from the hydraulic fluid . The high viscosity of an HVE sample precludes GDVN gas focusing, meaning that the extruded free stream is roughly equal in diameter to the inner diameter of the sample capillary, which is typically 50\u2013100\u2005\u00b5m. Sample flow rates can be as low as tens of nanolitres per minute; thus, sample efficiency is much higher and sub-milligram sample quantities can be sufficient exposure time. Thus, the diffraction patterns are not \u2018smeared out\u2019 by crystal movement, as shown recently at the SLS or shorter lipids such as MAG 7.9 , (i) the very same segment must be in the X-ray interaction region simultaneously with the X-ray pulse and not yet have travelled beyond or not yet have reached it, and moreover (ii) it must have passed beyond the interaction region before the next probe pulse. For ultrafast time delays \u0394t, the jet is essentially still during \u0394t and only (ii) needs to be considered. For longer time delays both points (i) and (ii) matter. Point (ii) sets a minimal jet-speed limit and depends on the optical laser diameter and position. Point (i) sets the maximal and potentially another minimal jet speed and/or the distance between reaction initiation and X-ray interaction. For successful TR measurements, the jet speed must therefore be known and, together with other experimental parameters, adjusted where possible. Generally, the jet speed limits TR measurements in the free jet to a maximal time delay of at most a few microseconds for GDVNs, whereas a few seconds may be achieved with HVE.Jet speed is also important for optically triggered TR experiments as it can determine the accessible time delays and microscopic jet size, which means that tracking a feature over time requires fast time resolution both to prevent blurring and to capture the feature at two or more successive positions. Pulsed (laser) illumination of sufficiently short duration (a few nanoseconds or less) can be used to deliver sharp images of liquid jets and the displacement of a feature can be tracked without difficulties using a camera with a reasonable frame rate and magnification whether they can be used at room or cryogenic temperature.Fixed-target techniques are another means of serially delivering fresh sample for each X-ray exposure. Here, the crystals are immobilized on a substrate, which is then scanned through the X-ray beam. An inherent advantage of this approach is that in principle the geometry and crystal distribution can be arranged such that every crystal on the substrate is probed. Each step in a raster scan must clearly move beyond the area affected by previous probe pulses, which is particularly important at an XFEL. Numerous solid-support approaches have been developed over the years and, depending on the design, they mainly vary in (i) the X-ray background, which can be caused by the substrate itself and by excess mother liquor, (ii) the extent to which they support high-throughput data collection in terms of maximal data-collection rate and high hit rate, (iii) whether only specific crystal sizes or shapes can be accommodated, (iv) crystal handling, et al., 2014et al., 2016et al., 2019Goniometer-based approaches using standard loops and micromeshes or specialized high-density sample-mounting grids have successfully been used at XFELs with larger microcrystals (>20\u2005\u00b5m) at cryogenic temperature etched into silicon and crystals mixed with Paratone N \u2018painted\u2019 onto the remaining 50\u2005nm layer of silicon nitride as a support . These windows can be endowed with modified surface properties to promote random crystal orientations within a silicon mesh and thereby provide an efficient sampling of reciprocal space (Zarrine-Afsar et al., 2015et al., 2016et al., 2017et al., 2015et al., 2015et al., 2015et al., 2015et al., 2016et al., 2015et al., 2016et al., 2017et al., 2015et al., 2016et al., 2017et al., 2015et al., 2017et al., 2016et al., 2017et al., 2017et al., 2018et al., 2018To reduce background scattering even further, silicon chips can incorporate microscopic \u2018wells\u2019 (Mueller 4.Despite the increasing variety of sample-delivery techniques, there is no universal technique of choice for serial crystallo\u00adgraphic data collection at XFEL or synchrotron sources. Instead, the suitability of a specific technique strongly depends on the investigated system and the experimental aim and conditions. We expect serial sample delivery to remain a rapidly developing field as the number of next-generation synchrotrons and XFELs is increasing, as will the user community."} +{"text": "The structures provide insight into the regulation and specificity of mirolysin, and hint at how it might be inhibited for therapeutic effect.Guevara, Rodriguez-Banqueri Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia. These pathogens release \u2018virulence factors\u2019, molecules that aid their colonization and survival in the subgingival niche and their evasion of the host immune response. Synergistic interactions of the pathogenic microbes with normal oral microbiota lead to a sustained host inflammatory response, degrading periodo\u00adntal tissues, eroding bone, and ultimately leading to tooth loss. Severe periodo\u00adntitis affects around 10% of the human population, and health implications are not limited to oral health; the chronic inflammatory conditions precipitated by the disease can extend to systemic manifestations, contributing to risk of cardiovascular and respiratory diseases, diabetes, rheumatoid arthritis, and cancer (Hajishengallis, 2015et al., 2017Periodo\u00adntitis, commonly known as gum disease, is a chronic inflammatory disease characterized by dysregulation of the oral microbiota in the subgingival space below the gum line (Hajishengallis, 2015T. forsythia. Mirolysin cleaves multiple components of the complement pathway to protect the pathogen against complement-mediated killing, and degrades the antimicrobial peptide LL-37, a human host defense peptide secreted by epithelial and immune cells at sites of infection (Jusko et al., 2015et al., 2017T. forsythia survival and immune evasion, mirolysin offers a potential therapeutic target, yet until now the structural basis for its latency, activation and substrate specificity have not been defined.Proteases are amongst the most abundant virulence factors produced by periodo\u00adntal pathogens. These enzymes degrade host proteins to feed bacterial growth, and also serve as key mediators of immune-subversive mechanisms, for example by degrading or inactivating endogenous antimicrobial peptides and components of the host complement system (Hajishengallis, 2015IUCrJ, Guevara, Rodriguez-Banqueri et al. report high-resolution crystal structures of promirolysin, defining the mechanism of latency of the precursor protein (Guevara et al., 2020et al., 2018et al., 1999et al., 2018In this issue of 2 between these structural elements, greater than the average buried surface area for protein\u2013protein complexes, which is remarkable given the small size of the 34-residue pro-segment. The pro-segment and catalytic domain interact through a balance of favorable hydro\u00adphobic interactions complemented by an array of hydrogen bonds and salt bridges, the same forces that shape affinity and selectivity of intermolecular protein\u2013protein interactions (Xu et al., 1997in trans, and serving as a template for engineering protein-based mirolysin inhibitors of therapeutic utility. Similar approaches have shown promise for engineering therapeutic inhibitors of ADAM family metallopeptidases, based upon their much larger pro-domains (Moss et al., 2007et al., 2016While this basic cysteine switch mechanism of latency is well precedented, the promirolysin structure reveals a surprisingly extensive interface between the pro-segment and the catalytic domain. The zymogen structure reveals a buried surface area of 2302\u2005\u00c5et al. further report a high-resolution structure of active mirolysin bound to a peptide product, providing insight into determinants of substrate specificity of the protease (Guevara et al., 2020et al., 2017et al., 2019Guevara, Rodriguez-Banqueri"} +{"text": "Accumulation of dysfunctional and damaged cellular proteins and organelles occurs during aging, resulting in a disruption of cellular homeostasis and progressive degeneration and increases the risk of cell death. Moderating the accrual of these defunct components is likely a key in the promotion of longevity. While exercise is known to promote healthy aging and mitigate age\u2010related pathologies, the molecular underpinnings of this phenomenon remain largely unclear. However, recent evidences suggest that exercise modulates the proteome. Similarly, caloric restriction (CR), a known promoter of lifespan, is understood to augment intracellular protein quality. Autophagy is an evolutionary conserved recycling pathway responsible for the degradation, then turnover of cellular proteins and organelles. This housekeeping system has been reliably linked to the aging process. Moreover, autophagic activity declines during aging. The target of rapamycin complex 1 (TORC1), a central kinase involved in protein translation, is a negative regulator of autophagy, and inhibition of TORC1 enhances lifespan. Inhibition of TORC1 may reduce the production of cellular proteins which may otherwise contribute to the deleterious accumulation observed in aging. TORC1 may also exert its effects in an autophagy\u2010dependent manner. Exercise and CR result in a concomitant downregulation of TORC1 activity and upregulation of autophagy in a number of tissues. Moreover, exercise\u2010induced TORC1 and autophagy signaling share common pathways with that of CR. Therefore, the longevity effects of exercise and CR may stem from the maintenance of the proteome by balancing the synthesis and recycling of intracellular proteins and thus may represent practical means to promote longevity. Accrual and aggregation of these defunct components result in disruption of cellular homeostasis, progressive degeneration, and increases the risk of cell death is a central regulatory kinase that regulates cellular growth and protein synthesis. This complex is stimulated by nutrient availability , mechanical stress, and growth factors and is inhibited by nutrient deprivation, energetic stress, and the macrocyclic polyketide rapamycin has been shown to be a reliable method of lifespan extension and/or moderator of age\u2010related disease through modulation of autophagic activity in numerous model species ranging from yeast to humans is one of two functionally and compositionally distinct multi\u2010protein TOR complexes; the second being TOR complex 2 (TORC2). Both complexes are highly conserved in all known eukaryotic cells (Honjoh, Yamamoto, Uno, & Nishida, This evidence suggests that normal levels of autophagy may be sufficient to maintain cytosolic proteostasis if the rate of protein and organelle synthesis is reduced; however, it may also be possible that the reduced levels of autophagy observed in older cells may not be linked to aging, but simply offer an indirect reflection of excess TORC1 activity Pani, . At pres4S.\u00a0cerevisiae and C.\u00a0elegans to rodents and primates, including humans (Figure Lifespan extension via autophagy has been closely linked to CR (Bergamini, Cavallini, Donati, & Gori, s Figure (Colman s Figure , and reps Figure . Calorics Figure .Even modest implementations of CR, such as intermittent fasting protocols, can promote health (Brandhorst et al., +\u2010dependent protein deacetylase also sensitive to energetic challenges, has been implicated in mediating the aging process (Salminen & Kaarniranta, Indeed, it has been observed that long\u2010term CR is a strong physiological promoter of autophagy, resulting in an upregulation of a number of autophagy\u2010related modulators and transcripts (Mercken et al., ++ is released from the lysosome into the cytosol, activating calcineurin which dephosphorylates TFEB and promotes its translocation to the nucleus where it initiates the transcription of a number of Atgs and proteins (Palmieri et al., Additionally, TFEB, a transcription factor involved in coordinating the expression of lysosomal and autophagic genes, has been shown to be activated during energy deprivation (Medina et al., Increasingly, autophagic activity has been shown to act as a key mediator of the observed impact of CR on lifespan (Bergamini et al., Some of the first data of long\u2010term CR on autophagic function in humans were collected from 15 lean and weight\u2010stable members of the Calorie Restriction Society who had practiced 30% CR for an average of 9.6\u00a0years. Upregulation of a number of autophagy modulators and gene and protein expression was noted including AMPK and SIRT family transcripts, ULK1, ATG101, APG12, GAPRAP/GATE\u20106, beclin\u20101, autophagin\u20101, and LC3 gene expression, as well as protein expression of FOXOs, PGC1\u03b1, beclin\u20101, and LC3 compared to age\u2010matched controls practicing a typical Western diet (Mercken et al., It is also interesting to note that suppression of the TORC1 pathway has been shown to potentiate longevity beyond the maximum extension achieved with CR alone (Bjedov et al., 5As discussed, energetic stress is a potent stimulator of autophagy; accordingly, exercise has been shown to augment acute autophagic activity in skeletal muscle (Jamart, Benoit, et al., While some data do exist relating to other forms of autophagy (Li et al., +, and ROS levels also are strong instigators of autophagic activity (Vainshtein & Hood, During exercise, autophagy mediates the clearance of proteins and organelles damaged by heat, pH changes, or mechanical stress which likely acts to prevent accumulation of these cytosolic components and maintain myocyte function (Schwalm et al., +, respectively. AMPK and SIRT1 both act to upregulate expression of Atgs by activating FOXO1 and FOXO3, increasing PGC1\u2010\u03b1 activity, and inhibiting mTORC1 (Vainshtein & Hood, In part, exercise acts to initiate autophagy in skeletal muscle through the same pathways as CR; namely, AMPK and SIRT1 are sensitive to alterations in AMP and NAD++\u2010dependent calcineurin where it then activates the CLEAR gene network and the transcription of Atgs and proteins.mTORC1 has also been implicated in regulating autophagy activity through mediating TFEB localization which may be subsequently modulated by exercise and nutrient deprivation (Medina et al., In addition to serving as a means to meet the energetic demands of exercise, autophagy is understood to facilitate exercise in numerous ways in skeletal muscle (Dokladny et al., While exercise\u2010induced skeletal muscle autophagy is presently the most studied, there are data showing enhanced autophagic activity in other tissues, thus demonstrating acute exercise is capable of instigating a global autophagic response Figure He, Bas. In theiThese noted systemic autophagic effects suggest exercise could possess a role in modulating some of the age\u2010related pathologies that autophagy has been reported to be implicated in, which include type 2 diabetes (Gonzalez et al., 6Emerging evidence suggests that the autophagic response to exercise may occur in a biphasic manner in that acute cellular perturbations induce a precipitous increase in autophagic flux occurring acutely following insult and is mediated by posttranslational protein modification (Vainshtein & Hood, 2max has been shown to stimulate autophagic activity in skeletal muscle (Jamart, Benoit, et al., 2max (Moller et al., 2peak (Schwalm et al., Aerobic exercise for 60\u00a0min or greater at 55%\u201370% VO2max did not alter autophagic activity in healthy adults (Masschelein et al., 2max induced autophagy (Moller et al., 2max did not produce a response (Tachtsis et al., Conversely, 20\u00a0min of cycling at ~50% VOThese findings help highlight the importance of exercise duration and intensity in stimulating autophagic induction and point to a threshold for activation, likely involving AMPK\u2010mediated determination of energy insufficiency. Importantly, the extreme elevations in autophagic activity observed with ultra\u2010endurance performance are likely indicative of excessive muscle damage and energetic protein catabolism, thus offering intriguing implications regarding the J\u2010shaped relationship observed between mortality and exercise participation (Arem et al., 7Currently, the long\u2010term effects of exercise on autophagic activity are ill\u2010characterized; however, they appear mediated by activation of a transcriptional program (Vainshtein & Hood, Skeletal muscle autophagy has been studied following regular exercise. In mice, 3\u00a0months of endurance exercise has been reported to produce no changes in resting levels of LC3\u2010II/LC3\u2010I ratio within skeletal muscle (Grumati et al., It is also interesting to note that autophagic activity appears to be necessary for the normal adaptations of skeletal muscle (He, Bassik, et al., Given aging is an organismal phenomenon, it is pertinent to establish the global effects of long\u2010term exercise on autophagy and determine its role beyond exercised skeletal muscle. While evidences exist demonstrating acute exercise is capable of upregulating autophagic activity and/or Atg expression in a number of tissues apart from skeletal muscle including heart (He, Bassik, et al., Ghareghani et al. reported8Investigation into the mechanisms underpinning lifespan and longevity shows that the appropriate maintenance of the proteome and organelle population is key in the augmentation of lifespan and/or mitigation of many pathologies associated with the aging process (Balch, Morimoto, Dillin, & Kelly, None declared."} +{"text": "Neurobiological models of stress and stress-related mental illness, including post-traumatic stress disorder, converge on the amygdala and the prefrontal cortex (PFC). While a surge of research has reported altered structural and functional connectivity between amygdala and the medial PFC following severe stress, few have addressed the underlying neurochemistry.in vivo MR-spectroscopy (1H-MRS) measurements of glutamate in 26 survivors from the 2011 Norwegian terror attack and 34 control subjects.We combined resting-state functional magnetic resonance imaging measures of amygdala connectivity with et al., 2017) and together suggest long-term impact of a traumatic experience on glutamatergic pathways. Importantly, local glutamatergic metabolite levels predicted the individual amygdala\u2013aMCC and amygdala\u2013vmPFC functional connectivity, and also mediated the observed group difference in amygdala\u2013aMCC connectivity.Traumatized youths showed altered amygdala\u2013anterior midcingulate cortex (aMCC) and amygdala\u2013ventromedial prefrontal cortex (vmPFC) connectivity. Moreover, the trauma survivors exhibited reduced levels of glutamate in the vmPFC which fits with the previous findings of reduced levels of Glx (glutamate + glutamine) in the aMCC (Ousdal Our findings suggest that traumatic stress may influence amygdala\u2013prefrontal neuronal connectivity through an effect on prefrontal glutamate and its compounds. Understanding the neurochemical underpinning of altered amygdala connectivity after trauma may ultimately lead to the discovery of new pharmacological agents which can prevent or treat stress-related mental illness. Twenty-six survivors from the terror attack at Ut\u00f8ya and 34 healthy control subjects between 16 and 25 years were included in the study after giving written informed consent. All data were collected between 21 and 33 months after the terror attack. This study is part of a larger project assessing the effects of traumatic stress during late adolescence on cognition, behaviour and biological measures.et al., et al., The trauma survivors were recruited through written invitation sent out from the Resource Centre for Violence, Traumatic Stress and Suicide Prevention, Western Norway (64% response rate). The control sample was an age-, sex- and education-matched group, who were not involved in the trauma, and were not otherwise related to any of the survivors. In order to obtain information concerning participants\u2019 mental health, the Mini International Neuropsychiatric Interview . All volumes were realigned to the first volume . We used the software default measures, which included (1) >1.5% variance in global signal from scan to scan and (2) >0.5\u00a0mm/TR frame-wise displacement. Volumes that exceeded one of these cut-offs were replaced via interpolation. Subjects with more than 20% bad volumes in total were excluded . After artefact correction, all images were spatially normalized to a standard EPI template based on the Montreal Neurological Institute (MNI) reference brain . This method calculated the temporal correlation between brain activation from our seed region and all other brain areas using a General Linear Model (GLM) approach. Following the CompCor strategy as implemented in CONN, nuisance covariates including CSF, white-matter signals and the individual realignment parameters were modelled and regressed out from the analysis. In addition, the data were band-pass filtered (0.008\u20130.09\u00a0Hz).The goal of our analysis was to assess amygdala\u2013prefrontal resting-state connectivity due to the importance of these neurocircuits in stress-related psychiatric disorders -corrected significance of p\u00a0<\u00a00.05 at the cluster level. In addition, as we had a priori hypotheses regarding amygdala\u2013dACC/aMCC and amygdala\u2013vmPFC connectivity, small volume correction (svc) based on anatomically defined aMCC and vmPFC ROIs and peak-level FWE-corrected p values were used. The anatomically defined ROIs were created using the SPM Wake Forest University (WFU) Pickatlas toolbox .We tested for statistical significance using an initial voxel-wise threshold of et al., a priori for measurements of glutamate levels and related compounds. We obtained 1H-spectra from the vmPFC and the aMCC using a single voxel point resolved spectroscopy (PRESS) sequence. Glx data from the aMCC and the vmPFC voxels have been published previously were used from the LCModel output. There was an association between Glu/Cr and white as well as grey matter in the vmPFC MRS voxel (see online Supplementary Methods), thus we controlled for voxel white and grey matter in all analyses of the vmPFC Glu/Cr data. We did not obtain aMCC 1H-MRS spectra from three of the controls. Furthermore, vmPFC 1H-spectra data from one trauma survivor and two controls had to be excluded due to poor data quality. Thus, the final sample size for the aMCC 1H-MRS data included 24 controls and 23 trauma survivors, and for the vmPFC 1H-MRS data, 25 controls and 22 trauma survivors.The amygdala is closely connected to the vmPFC and the dACC/aMCC , amygdala\u2013dorsolateral PFC and amygdala\u2013cerebellum connectivity. The location of the aMCC cluster accords with a recent meta-analysis which showed that hyperactivity in the aMCC is among the most consistent finding in traumatized subjects which develop PTSD and amygdala connectivity while controlling for comorbid PTSD and panic disorder. The group differences were robust to adjustments for comorbidity (see online Supplementary Table S2). Except for the group difference in amygdala\u2013dorsolateral PFC connectivity, we also replicated the findings in un-scrubbed data (see online Supplementary Table S3). Thus, the main findings were not dependent on any adjustments made by the ArtRepair software. Finally, a comparison of BA31 functional connectivity between the groups revealed no whole-brain or small-volume significant clusters.Amygdala functional connectivity maps across groups are presented in online Supplementary Fig. S1 with corresponding statistics in online Supplementary Table S1. We observed whole-brain significant group differences in amygdala\u2013aMCC connectivity \u00a0=\u00a04.54, p\u00a0=\u00a00.04, partial \u03b72\u00a0=\u00a00.10, t45\u00a0=\u00a00.38, p\u00a0=\u00a00.71) in the aMCC. Mean Glu/Cr values for the vmPFC and the aMCC voxels are presented in online Supplementary Table S4.post-hoc analyses, we explored whether the glutamatergic compounds which displayed significant group differences also predicted inter-individual differences in amygdala functional connectivity. In the first analysis, we regressed individual vmPFC Glu scores onto the individual functional connectivity maps while controlling for vmPFC white and grey matter. The analysis revealed a negative association between vmPFC Glu and amygdala\u2013vmPFC functional connectivity , and should therefore be interpreted with caution. Based on a previous finding of reduced levels of aMCC Glx in the trauma survivors after controlling for the effect of group.In two separate r\u00a0=\u00a0\u22120.51, p\u00a0=\u00a00.02), supporting that the reduction of vmPFC Glu developed over time. Previous analyses revealed no association between aMCC Glx and time elapsed since the traumatic event . A second regression showed that the group was associated with aMCC Glx . aMCC Glx was also associated with amygdala\u2013aMCC connectivity . Importantly, adding the aMCC Glx levels as a second predictor of the amygdala\u2013aMCC connectivity removed the effect of the group , and the indirect effect of aMCC Glx on amygdala\u2013aMCC connectivity was significant and the amygdala in the aftermath of significant stress exposure (Gee et al., et al., et al., et al., et al., et al., et al., et al., In addition to reduced negative connectivity between vmPFC and the amygdala, we found significantly reduced positive connectivity between the amygdala and aMCC in the trauma survivors. The result is in line with studies in subclinical and clinical PTSD groups also finding compromised resting-state amygdala\u2013dACC/aMCC connectivity (Thomason et al., et al., et al., et al., et al., et al., et al., et al., 1H-MRS, which restricts measurements to \u2018bulk\u2019 levels of metabolites, and the technical challenges especially related to acquisitions in the most ventral parts of the PFC (de Matos et al., et al., et al., Mounting evidence from animal studies suggests that acute and chronic stress affect Glu neurotransmission in the PFC (Popoli et al., et al., The directionality of the associations is worth reiterating. We have previously reported that traumatic stress was associated with a reduction of aMCC Glx (Ousdal et al., et al., et al., per se. As such, future studies should investigate potential associations between vmPFC Glu and amygdala connectivity in both PTSD patients and trauma-exposed control groups, to understand the association between stress exposure, stress-related psychopathology and amygdala\u2013vmPFC glutamatergic connections. Finally, the association between amygdala functional connectivity and prefrontal glutamatergic metabolites were based on correlations, which do not imply causation. Thus, the present results should be interpreted with caution, bearing in mind the study design and analytic approaches.We acknowledge a potential limitation of the present study rests in the relative small group sizes and the heterogeneity related to the trauma group. This is always likely to be a problem in these types of studies given the variability of response to stressors. Furthermore, we acknowledge that the use of an amygdala ROI and not an independent component analysis when processing the rsfMRI data may be less sensitive to physiological noise (Van Dijk In conclusion, we found that traumatic stress influences functional connectivity between amygdala and medial prefrontal cortical regions, which are regions implicated in emotion generation and regulation. More specifically, trauma-exposed individuals had less positive amygdala\u2013aMCC connectivity and less negative amygdala\u2013vmPFC connectivity compared with a matched group without any trauma exposure. Overall, the results support a model in which traumatic stress is associated with reduced regulation of amygdala responses, both directly and indirectly. The amygdala\u2013aMCC connectivity pattern was mediated by Glx, suggesting that the compromised connectivity in trauma survivors may be secondary to trauma-induced changes in prefrontal glutamatergic pathways. Identifying the neurochemical underpinning of the observed connectivity changes in trauma-exposed individuals may ultimately contribute to new pharmacological treatments of stress-related mental illness."} +{"text": "Capripoxvirus-induced diseases of cattle and sheep, respectively. Despite the extensive vaccination program adopted by Egyptian veterinary authorities, LSD and sheep pox are still prevalent and spread throughout the whole country. The current study was designed for molecular characterization and phylogenetic analysis of LSD virus (LSDV) and Sheep pox virus (SPPV) recovered from field cases in Egypt along with vaccinal strains to assess their genetic relatedness.Lumpy skin disease (LSD) and sheep pox are economically important Skin biopsies were collected from naturally infected cases of LSD in Ismailia (n=3 farms) and Beni-Suef (n=2 farms) Governorates and sheep pox in Beni-Suef (n=1 flock). Virus isolation was carried out on primary ovine fetal kidney and heart cell cultures. DNA was extracted from infected materials as well as LSDV Neethling vaccine strain and Romanian SPPV vaccine strain. Polymerase chain reaction was performed using oligonucleotide primers targeting the entire open reading frame of G protein-coupled receptors (GPCR) gene and gene sequences were analyzed.Virus isolation on primary ovine fetal kidney and heart cell culture revealed a cytopathic effect at the third passage characterized by rounding of infected cells and margination of nuclear chromatin. Comparative sequence analysis of GPCR gene revealed that Egyptian LSDV isolated from Ismailia and Beni-Suef shared 99:100% nucleotide and amino acid (AA) identities with each other. In comparison to the vaccinal strains, Egyptian LSDV isolates shared 98:99 nucleotide and AA identities with LSDV Neethling vaccine strain and 93:94% with SPPV Romanian vaccine strain. No differences at the nucleotide or AAs were observed between the SPPV vaccine and virulent strains (100% identity). Phylogenetic analyses revealed that LSDV Neethling vaccine strain is more related to field Egyptian LSDV and clustered within the LSDV group while Romanian SPPV vaccine strain clustered in a separate clade with SPPV field isolates.Comparative sequencing and phylogenetic analyses of the GPCR gene reveal a minimal genetic variation between LSDV field isolates from different locations and a close relationship between virulent field strains and homologous vaccines. Capripoxvirus (CaPV)-induced diseases of cattle and sheep, respectively. LSD virus (LSDV) and Sheep pox virus (SPPV) are categorized within the genus CaPV in the family Poxviridae [Lumpy skin disease (LSD) and sheep pox are economically important xviridae .LSD is an acute to subacute disease of cattle characterized by fever, rapid eruption of numerous circumscribed skin nodules, and generalized lymphadenitis -5. The cSheep pox is a highly contagious disease of small ruminants characteThere is no doubt that vaccination is the most effective way to control CaPV diseases . Due to In Egypt, the Kenyan SGP O-240 vaccine was used during LSD incursion in 2005-2006 . At presIn the current study, G protein-coupled receptors (GPCR) gene was used for molecular characterization and phylogenetic analysis of LSDV and SPPV circulating in the field along with vaccinal strains to assess their genetic relatedness.All animal handling procedures, as well as samples collection and disposal, were approved by the animal care and use committee of the Faculty of Veterinary Medicine, University of Sadat City, Egypt, according to the guidelines and recommendations of the European Communities Council Directive 1986 (86/609/EEC).From June 2015 to September 2016, LSD and sheep pox were suspected among dairy cattle herds located in private farms belonging to Ismailia (n=3) and Beni-Suef (n=2) Governorates and one sheep flock in Beni-Suef Governorate, respectively. All farms were clinically examined and a total of five nodular skin lesions were collected from diseased cattle with skin eruption all over the body while skin lesions were collected and pooled from infected sheep pox flock showing generalized pock lesions. The collected samples were kept at \u221220\u00b0C for virus isolation and polymerase chain reaction (PCR).4 TCID50 of freeze-dried, live, attenuated virus (SIS Neethling-type)from the Republic of South Africa and Romanian SPPV vaccine strain provided from the Pox Department, Veterinary Serum and Vaccine Research Institute, Abbassia, Cairo, Egypt, was used in the molecular study (103 TCID50/ml).South African Neethling vaccine strain each 1 mL (1 dose) of the vaccine contains 10et al. [Skin biopsies were used for the isolation of LSDV and SPPV according to Tuppurainen et al. . BrieflyDNA was extracted from skin lesions, infected cell culture, LSDV Neethling vaccine strain, and Romanian SPPV strain using a DNA/RNA Extraction Kit according to the manufacturer\u2019s instructions.et al. [The primers were developed to amplify the entire GPCR gene at position 6961-8119 according to Le Goff et al. . The priet al. [http://Zwww.ncbi.nlm.nih.gov/blast) was initially implemented to establish sequence identity to GenBank accessions. Phylogenetic tree and sequence alignments were generated using MEGAVersion X software. The tree was generated by the neighbor-joining method based on 1000 bootstrapped data sets [PCR amplicons were purified using QIAquick PCR Purification Kit and dispatched to Macrogen\u2122, Seoul, Korea, for DNA sequencing using two additional primers(5\u00b4-GATGAGTATTGATAGATACCTAGCTGTAGTT-3\u00b4and 5\u00b4-TGAGACAATCCAAACCACCAT-3\u00b4) according to Le Goff et al. . BLAST aata sets .Fever, skin lesions scattered in different parts of the body , and enlVirus isolation revealed CPE at the third passage on primary ovine fetal kidney and heart cells, characterized by retraction of the cell membrane from surrounding cells, rounding of cells, and margination of the nuclear chromatin .Using primer sequences targeting the entire GPCR gene, a fragment of 1158 bp has been amplified from all DNA extracts . SequencRegarding Romanian SPPV vaccine strain, it was found that AA residues situated in position 10-16 (SATMYNS) in LSDV field isolates are missed in SPPV vaccine; moreover, there are 27 variances in AA motifs between LSDV field isolates and SPPV vaccines were observed along GPCR gene and 2. OTo represent the evolutionary relationships among field and vaccinal strains of LSDV and SPPV sequenced in this study and available CaPVs in the database, a GPCR-based phylogenetic tree was generated using the neighbor-joining method on nucleic acid sequences. The tree showed three tight genetic clusters . LSDV faCapripoxviruses field and vaccinal strains based on sequence analysis of GPCR gene.LSD and sheep pox diseases are now considered as endemic diseases in Egypt. Despite the extensive vaccination program adopted by Egyptian veterinary services, LSD and sheep pox are still prevalent and spread throughout the whole country, thereby the present study provides a molecular characterization of LSDV and SPPV recovered from field cases in Egypt and a comparison of A total of five LSDV and one SPPV were isolated from naturally infected animals with typical clinical features of LSD and sheep pox, respectively, after being confirmed by PCR.The complete open reading frames of the GPCR gene of isolated viruses were sequenced along with vaccine strains. Comparative sequence analysis revealed that all of the five LSDV isolates are closely related to each other with a nucleotide and AA identity ranged from 99% to 100% in between confirming the circulation of the same virus strain.Egyptian LSDV field isolates were found more related to LSDV Neethling vaccine where it differs only in four AA substitutions and an AA deletion at positions 30-33.et al. [et al. [The comparative sequence analysis revealed that the 5-end of GPCR gene of SPPV vaccine was characterized by deletion of 21 nucleotides (7-aa) when it compared with LSDV field isolates. This sheep pox gap was recorded in all isolates and vaccine strains located in the database and was considered as a specific signature for SPPV as reported previously by Le Goff et al. . Many va [et al. and El-T [et al. . Intereset al. [et al. [et al. [Phylogenetically, CaPV was delineated into three clades LSDV, GTPV, and SPPV as previously reported by Rouby et al. , Rouby [et al. , Rouby aet al. , and Maf [et al. . LSDV fa [et al. . The oth [et al. and rece [et al. .Comparative sequencing and phylogenetic analyses of GPCR gene revealed a minimal genetic variation between different LSDV isolates from different locations and a close relationship between LSDV Neethling vaccine strain and Egyptian field LSDV isolates. GPCR gene possesses specific signatures for LSDV and SPPV at both nucleotide and AA sequences level and cluster them separately according to their host origin.ME designed the study. SRR and AB performed PCR, sequence analysis, and wrote the initial draft of the manuscript. MW and ME revised the manuscript. All authors read and approved the final manuscript."} +{"text": "The aim of this review was to summarize key developments in classification and diagnosis of the idiopathic inflammatory myopathies (IIMs).The recently published European League Against Rheumatism/American College of Rheumatology (EULAR/ACR) classification criteria for the IIMs provide a comprehensive, accurate and data-driven approach to identification of IIM cases appropriate for inclusion in research studies. Further, recent studies have advanced understanding of clinical manifestations of the IIMs and delineated the role of imaging, particularly magnetic resonance.The recent publication of the EULAR/ACR classification criteria will potentially greatly improve IIM research through more accurate case identification and standardization across studies.Future inclusion of newly recognized clinical associations with the MSAs may further improve the criteria's accuracy and utility. Clear and comprehensive understanding of associations between clinical manifestations, prognosis and multisystem involvement can aid diagnostic assessment; recent advances include delineation of such associations and expansion of the role of imaging. The idiopathic inflammatory myopathies (IIMs) are a group of autoimmune diseases characterised by chronic muscle inflammation (myositis), internal organ inflammation and significant morbidity and mortality . The widThis article aims to summarize recently published research pertinent to advances in IIM classification and diagnosis. A Medline search for research articles published between January 2017 and May 2018 was carried out using the MeSH term \u2018myositis\u2019. Articles primarily focussing on myositis-specific autoantibodies were excluded, as they will be reviewed in detail in a separate article.\u00a0The IIMs have traditionally been classified and diagnosed according to the criteria by Bohan and Peter Table since pu\u25aa\u25aa]. Initial methodology identified 93 candidate variables for inclusion in the classification criteria. Variable domains included pattern of weakness, dermatological manifestations, disease course, systemic manifestations, response to treatment, pattern of muscle biopsy abnormalities, presence of MSAs, electromyogram (EMG) and MRI features.These drawbacks have limited accurate identification of well defined populations suitable for IIM research studies. Therefore, in 2004, the International Myositis Classification Criteria Project (IMCCP) was established with the aim of developing new IIM classification criteria. The IMCCP working committee was formed of experts from adult and paediatric rheumatology, neurology, dermatology, epidemiology and biostatistics. In 2017, the newly developed European League Against Rheumatism/American College of Rheumatology (EULAR/ACR) classification criteria were published [4Using data from 976 IIM cases and 624 comparators, the combination of candidate variables that could most accurately distinguish between IIM and non-IIM participants was identified. The variables included in the final classification criteria are displayed in Table The likely IIM subtype of each case can also be ascertained, according to the published classification tree Fig. . Subtypeet al. [et al. [www.imm.ki.se/biostatistics/calculators/iim.The EULAR/ACR criteria demonstrated high sensitivity 93%) and specificity 88%) when muscle biopsy data were included. Sensitivity and specificity also remained high when muscle biopsy data were not included: 87 and 82%, respectively. The accuracy of the newly created classification criteria was compared against previously developed criteria, including those by Bohan and Peter [% and speet al. , Targoff [et al. , Dalakas8% when mThe criteria have a number of major strengths. Their development methodology included a large IIM population with non-IIM comparators and considered inclusion of a wide variety of candidate variables. The ease of use and provision of a website-based calculator are also major strengths. Drawbacks include the fact that a majority of the development population were white, thus potentially limiting the criteria's validity in Asian and African populations. Further, MRI and electromyography data were only available in 38 and 29% of cases, thus potentially excluding these variables from the criteria only due to missing data. A limitation of subtype identification is the inability to separately identify IMNM and ASS cases. The inclusion of only one myositis-specific autoantibody (anti-Jo-1) in the classification criteria is a major limitation, as recent studies have illustrated the important and distinctive clinical and subtype associations . TherefoIn summary, the newly developed EULAR/ACR classification criteria for the IIMs provide an accurate method through which clearly defined study populations can be formed, thus potentially improving validity of IIM research.The newly developed classification criteria provide robust methods for identifying IIM cases for research purposes; however, their use is not designed nor recommended for use in clinical practice.Accurate diagnosis of an IIM is key to appropriate treatment instigation, prognostication and prevention of complications. However, diagnosis and subtype identification in clinical settings can be challenging, in part due to potential multisystem involvement and wide variations between subtype manifestations. Currently, no clear diagnostic criteria for the IIMs exist. However, findings from clinically focused research studies can aid a clinician's diagnostic accuracy, identification of factors associated with prognosis and guide investigation of multisystem involvement.et al.[N\u200a=\u200a2145). Interestingly, seven of the nine cases also fulfilled criteria for ASS and six cases were positive for anti-Jo-1 antibodies. This study highlights the importance of foot examination in IIM cases when trying to identify cutaneous manifestations. Mamyrova et al.[et al.[Findings from epidemiological and observational studies can inform clinical practice and guide the diagnostic process. A number of observational studies have recently been published and these will be summarized, with a particular focus upon potential clinical applications. Cox et al. recentlyva et al.\u25aa identifA recent study compared the utility of muscle testing via hand-held dynamometry against manual muscle testing in myositis cases . They reet al.[et al.[\u221703, thus indicating a genetic interaction with smoking and IIM development. These studies therefore highlight the importance of ascertaining a patient's smoking history due to its potential diagnostic utility and estimation of ILD risk.Interstitial lung disease (ILD) is an important extramuscular manifestation to consider during the diagnostic process and subsequent assessments. A number of studies have recently investigated risk factors for ILD. A recent study by Schiffenbauer et al. has highl.[et al. in 2012,et al.[et al.[Investigation for predictors of poor survival in 497 IIM-associated ILD cases was carried out by Sato et al.. They idl.[et al. reportedThe role of imaging in IIM diagnosis has expanded in recent years and has a number of capabilities. Imaging techniques, such as MRI, can help identify the presence of myositis, delineate its extent and assess treatment response, through serial scans and also help focus appropriate areas for muscle biopsy, limiting the likelihood of a false negative sample.et al.[et al.[et al.[Studies by both Yao et al.\u25aa and Andl.[et al. have recl.[et al.\u25aa also coet al.[et al.[Pinal-Fernandez et al. reportedl.[et al. in 12 biet al.[et al.[Focused MRI scanning can identify focal areas on myositis; however, whole-body MRI offers the ability to completely delineate all muscle groups affected and potentially identify secondary organ involvement and the presence of associated malignancy. Whole-body MRI, as opposed to focused imaging, was advocated by Elessawy et al.\u25aa as the l.[et al. also repet al.[Other imaging modalities also offer utility in IIM diagnosis. Burlina et al.\u25aa recentlet al.[Computed tomography (CT) scanning offers detailed assessment of the presence of ILD. A recent study by Ungprasert et al. investiget al.[et al.[The ability to identify cardiac involvement (an uncommon but important extramuscular manifestation) and distinguish from other heart disease has undergone advances recently. The utility of MRI in identifying cardiac involvement has been confirmed in recent years by a small number of studies ,29. Receet al. have recl.[et al. aimed toet al.[Huber et al.\u25aa confirmAccurate case identification is key to IIM research and the recent publication of the EULAR/ACR classification criteria will potentially greatly improve IIM research through accurate case identification and standardization across studies. Clear diagnosis of the IIMs is important to ensure appropriate diagnosis and treatment instigation. Recent advances in knowledge of clinical features will aid the clinician in prognostication, treatment stratification and investigation for multisystem involvement.None.This work was supported by Medical Research Council (MRC) UK grant MR/N003322/1. The views expressed in this publication are those of the author(s) and not necessarily those of the NHS, the National Institute for Health Research or the Department of Health.There are no conflicts of interest.\u25aa of special interest\u25aa\u25aa of outstanding interestPapers of particular interest, published within the annual period of review, have been highlighted as:"} +{"text": "To conduct advanced psychometric analysis of Primary Care Assessment Tool (PCAT) in Tibet and identify avenues for metric performance improvement.Measuring progress toward high-performing primary health care can contribute to the achievement of sustainable development goals. The adult version of PCAT is an instrument for measuring patient experience, with key elements of primary care. It has been extensively used and validated internationally. However, only little information is available regarding its psychometric properties obtained based on advanced analysis.We used data collected from 1386 primary care users in two prefectures in Tibet. First, iterative confirmatory factor analysis examined the fit of the primary care construct in the original tool. Then item response theory analysis evaluated how well the questions and individual response options perform at different levels of patient experience. Finally, multiple logistic regression modeling examined the predicative validity of primary care domains against patient satisfaction.A best final structure for the PCAT-Tibetan includes 7 domains and 27 items. Confirmatory factor analysis suggests good fit for a unidimensional model for items within each domain but doesn\u2019t support a unidimensional model for the entire instrument with all domains. Non-parametric and parametric item response theory analysis models show that for most items, the favorable response option (4 = definitely) is overwhelmingly endorsed, the discriminability parameter is over 1, and the difficulty parameters are all negative, suggesting that the items are most sensitive and specific for patients with poor primary care experience. Ongoing care is the strongest predictor of patient satisfaction. These findings suggest the need for some principles in adapting the tool to different health system contexts, more items measuring excellent primary care experience, and update of the four-point response options. Values were also imputed for the \u2018not sure/don\u2019t remember\u2019 response option as an alternative to attributing the pre-set value of 2.5. In general, estimates produced with listwise deletion are less efficient than other methods of handling missing data. Therefore, we reported only results using database with ML imputation imputation method was used to replace the missing values; match age, sex, education, self-rated health status; 295 respondents were excluded because of missing values for the matching variables, leaving 1091 respondents in subsequent analyses. Those excluded were more likely to be female and less educated. To examine the robustness of our conclusion, we excluded all respondents who had at least one missing value on any item (listwise deletion) and repeated all data analyses by domain was conducted to flag items with low correlations (Pearson <0.20) or low factor loading (<0.30) for potential deletion. We repeated the exploratory factor analysis after deleting each item with low factor loading until all retained items had a factor loading of at least 0.30. The results of exploratory factor analysis guided subsequent confirmatory factor analysis.Then confirmatory factor analysis using structural equation modeling was used to test the goodness of fit of the items to the theoretical domains of the original PCAT. Subsequently, confirmatory factor models were adjusted iteratively based on fit and judgment until the goodness-of-fit statistics were optimized. We also assessed the entire instrument including all domains in our data analysis. First, we included all domains in confirmatory factor analysis. Then we only included the four core domains in confirmatory factor analysis. The following goodness-of-fit statistics were used: normed fit index (NFI) \u22650.9 indicating good fit, comparative fit index (CFI) \u22650.9 indicating good fit, standardized root mean square residual (SRMR) \u22640.05 indicating acceptable fit.For each domain confirmed as being unidimensional, we examined the distribution of response options of individual items within each domain based on domain performance using nonparametric item response theory analysis. However, we cannot get the exact information of each item performance. To be more precise, two parameter estimates (discriminability and difficulty) were subsequently generated using Samejima\u2019s grade reWe estimated the correlations between domains to examine how domains were related and whether they demonstrated distinctiveness.Finally, we used logistic regression modeling to examine how primary care domains were associated with patients\u2019 satisfaction with service attitude (one item) and perceived technical quality (one item) of their primary care provider. The five-point Likert response scale for the two items was dichotomized to indicate satisfaction (very satisfied or satisfied) versus dissatisfaction. All domains were put in the same model, and age, sex, education, and self-rated health status were included in the model as covariates. Education level was categorized into three groups: illiterate, primary school, junior high school and above. Self-rated health status was measured by a visual analogue scale with end points of 0 and 100, where 0 corresponds to \u2018the worst health status\u2019, and 100 corresponds to \u2018the best health\u2019.Descriptive, correlation, and exploratory factor analysis were conducted with SPSS 22.0. ML imputation and confirmatory factor analysis were conducted with LISREL9.1. Nonparametric and parametric item response theory analyses was conducted with SPSS 22.0 and MULTILOG 7.03.Table S1.For first contact access, two of the six items were deleted because of the unacceptable goodness-of-fit statistics (FCA5 and FCA6); and two items fit better in first contact utilization (FCA1 and FCA4), leaving only two items in first contact access and four in first contact utilization. In the eight-item ongoing care, two items were deleted because of unacceptable goodness-of-fit statistics (OC1 and OC7); and a further item (OC8) was removed to improve the goodness-of-fit statistics, leaving a five-item ongoing care scale. The eight-item comprehensiveness subscale showed unacceptable goodness-of-fit statistics on a single factor ; and after removing four of the poorest fitting items, a final four-item comprehensiveness subscale demonstrated good fit . There is no change in items in other domains. The detailed iterative process to finalize the original factor structure was reported in Supplemental Table Table However, either the model including all domains or the model including only the four core domains shows unacceptable goodness-of-fit statistics on a single factor, which suggest that the domains included in original PCAT may not measure a common single construct in Tibet context, and it is not appropriate to report a total score.\u03b1 is over 0.70, indicating good internal consistency of items for all domains except first contact access (0.66) and ongoing care (0.66).The mean of each domain score is lower than the median and negatively skewed, indicating most of patients reporting favorable response answers. The first contact utilization score is the highest (3.66\u00b10.48), while the first contact access score is the lowest (2.94\u00b10.95). Cronbach Non-parametric item response theory graphs were modeled on each unidimensional domain to provide further insight into item performance and reliability. In most items, the option characteristic curve for the response option \u20182 = Probably not\u2019 is overshadowed by other options and ongoing care (0.60). First contact utilization is also highly correlated with family centeredness (0.54) and ongoing care (0.55). First contact access, comprehensiveness, and community orientation have lower correlation with other domains.The Pearson correlations between the domains indicate the distinctiveness of each domain. Correlation coefficients between domains range from 0.23 to 0.61 and are lower than Cronbach Finally, Table This advanced psychometric analysis of the PCAT-Tibetan versions provides further insight into some of the problematic psychometric properties found in the initial validation analysis, and it suggests avenues to improve the metric performance of the tool. Despite the metric problems, the PCAT-Tibetan domains of first-contact, ongoing care, and coordination with specialists are associated with an increased likelihood of patient satisfaction. This illustrates the potential of the PCAT and underlines the importance of improving the tool to address some of the metric problems. Some of the problems, such as the skewness of item response distribution found in many items, are shared with the original PCAT and other versions; and our results suggest some solutions that could improve performance. Others may be specific to the PCAT-Tibetan version \u2013 such as the non-optimal resolution of items relating to First Contact constructs \u2013 suggest the need for some principles in adapting the tool to different health system contexts.et al., et al., et al., et al., et al., et al., et al., et al., The widespread use of the PCAT to evaluate primary care in many countries provides an opportunity to compare primary care across different contexts and to support a worldwide movement to improve primary care. Our results show not only the association of the PCAT-Tibetan with patient satisfaction but also other analysis that demonstrated the capacity to distinguish between health-care organizations in Tibet and other regions in China (Wang et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., The negative skewness of item response distribution in many items was noted in the original validation of the long PCAT version (Shi et al., et al., et al., et al., et al., et al., et al., Another metric issue results from offering the \u2018not sure/don\u2019t remember\u2019 option. The high rate of endorsing this response option is common in many language versions, especially in Asian countries including Korean, Japanese and Chinese. (Lee et al., et al., et al., et al., et al., \u03b1 was only 0.38 (Mei et al., et al., et al., et al., et al., et al., et al., Collaborative international work could also address principals for measuring and/or comparing domains that affected health system specificities, for instance, in the access domain. First contact in the PCAT-Tibetan fails to meet optimal psychometric standards of construct validity and internal consistency. Similar problems were also found in other PCAT validation studies in China (Yang et al., et al., et al., In contrast, the domain of ongoing care most strongly predicts patient satisfaction with primary care provider in Tibet. This is consistent with a previous systematic review showing that the most important determinants of satisfaction are the interpersonal relationships and their related aspects of care (Crow This study contributes to the growing international work supporting the relevance and need for valid and reliable measures of the patient experience of health care. Several lessons from PCAT-Tibetan validation study may be shared with our colleagues in low- and middle-income countries. First, the items or content of instruments from developed countries may not be appropriate in other countries. Some specific features of local primary care system may not be reflected in the translated instruments. For example, no items in PCAT could reflect the feature of geographical accessibility, which is a major aspect in Tibet. Some items in comprehensiveness domain are not appropriate in Tibet. Therefore, instead of adapting existed instrument directly, qualitative research is needed to understand the local population preference of primary care first. Second, more items to measure excellent primary care experience should be developed. Most existing items have adequate capacity to discriminate between poor and average performance on different primary care domains. However, items that discriminate clearly between average and good primary care are needed. Further research is required to explore the characteristics of some exemplars with good primary care experience and what the good primary care is from their narratives. Third, the choice of response categories should be careful. The current four-point response scale and its wording in PCAT may not be appropriate in some countries. Several factors could be considered when exploring the appropriate response categories, such as literacy level, response tendency, and judgment making of local population. This could be done through qualitative research.Finally, a summary score of overall primary care experience including all domains, which is often the most used metric when assessing a health system, is not supported by our analysis of PCAT-Tibetan version. However, this psychometrically validated 27-item Tibetan version of PCAT will be useful in monitoring and evaluating the performance of primary care system in Tibet in specific areas, especially in accessibility, continuity, and coordination, which are the priorities of current health reform efforts in Tibet. The health service research is underdeveloped in Tibet, and there is no instrument measuring patient experience that could be used when this study was conducted. We hope this study could bring more researchers\u2019 attention into primary care performance evaluation in Tibet. We also recognize that different policy interventions to achieve primary care functions are inter-related, but each policy has its own priorities. For example, the family doctor contract service model is being developed and expanded now to improve performance in accessibility and continuity; and the transformation of tiered health service delivery system aims to promote collaboration between different health-care providers and to improve coordination. Under this context, PCAT-Tibetan version is a potential useful instrument to evaluate the effectiveness of these policy interventions."} +{"text": "Hyaluronan and proteoglycan link protein 2 (Hapln2) is important for the binding of chondroitin sulfate proteoglycans to hyaluronan. Hapln2 deficiency leads to the abnormal expression of extracellular matrix (ECM) proteins and dysfunctional neuronal conductivity, demonstrating the vital role of Hapln2 in these processes. Studies have revealed that Hapln2 promotes the aggregation of \u03b1-synuclein, thereby contributing to neurodegeneration in Parkinson\u2019s disease (PD), and it was recently suggested to be in intracellular neurofibrillary tangles (NFTs). Additionally, the expression levels of Hapln2 showed lower in the anterior temporal lobes of individuals with schizophrenia than those of healthy subjects. Together, these studies implicate the involvement of Hapln2 in the pathological processes of neurological diseases. A better understanding of the function of Hapln2 in the central nervous system (CNS) will provide new insights into the molecular mechanisms of these diseases and help to establish promising therapeutic strategies. Herein, we review the recent progress in defining the role of Hapln2 in brain physiology and pathology. Parkinson\u2019s disease (PD), Alzheimer\u2019s disease (AD) and schizophrenia are neurological diseases characterized by the dysfunction of certain types of neurons , is vital for neuronal conductivity and the formation of the extracellular matrix (ECM), and besides its potential role in the UPP, it has been identified as a contributor to the pathological processes in several neurological disorders. For example, Martins-de-Souza et al. showed tHAPLN2 revealed seven exons encoding a polypeptide with an estimated molecular mass of 38 kDa (Hirakawa et al., The structure and roles of Hapln2 in the CNS have been studied since around the turn of this century, when an analysis of the gene now known as The hyaluronan and proteoglycan link family of proteins consists of four members: Hapln1, Hapln2, Hapln3, and Hapln4 (Spicer et al., HAPLN2 mRNA is expressed at high levels in the human hippocampus, medulla oblongata, putamen, SN, thalamus, and spinal cord but at lower levels in the cerebellum, cerebral cortex, frontal lobe, and nucleus accumbens. By in situ hybridization, we detected high expression levels of Hapln2 mRNA in the SN, olfactory bulb, red nucleus, cerebellum, brain stem, and hippocampus but relatively weak signals in the cerebral cortex of adult rat brain (Wang et al., Northern blot analyses by Hirakawa et al. showed tLecticans such as brevican and aggrecan mainly bind to the hyaluronans, which are the major components of the ECM in the brain (Yamaguchi, + channels antibody (Oohashi et al., + channels and the contactin-associated protein (Rasband et al., Northern blot analyses have shown that Hapln2 expression in the mouse brain begins at 20 days after birth and increases with age (Hirakawa et al., + channel clustering coincides with myelination (Bekku et al., + channels. Of note, the clustering of Na+ and K+ channels is critical for saltatory conduction (Oohashi et al., + and K+ channels, flash visual evoked potentials had a longer latency and smaller amplitude in recordings from Hapln2 knockout mice compared with those from wild-type mice (Bekku et al., + channels (Susuki et al., + channel, nodes formation and AP propagation. However, more studies should be performed to clarify the details about the relationship between Hapln2 and the paranodal barriers or the CS in these processes during the development.As NaAs mentioned above, Hapln2 deficiency decreased the expression of ECM-associated proteins (Bekku et al., PD is characterized by the formation of Lewy bodies and selective loss of dopamine neurons in the SN (Liu et al., via proteasome inhibition resulted in the degeneration of dopaminergic neurons in vitro and in vivo (Kikuchi et al., Flow cytometry and immunostaining analyses showed that the overexpression of Hapln2 increased the death of MES23.5 and primary neuronal cells (Wang et al., Immunostaining analysis of MES23.5 cells overexpressing Hapln2 also showed an accumulation of \u03b1-synuclein, aggregates of which were found to colocalize with Hapln2 (Wang et al., A variety of environmental and genetic factors have also been implicated in the pathology of AD (Chin-Chan et al., via disruptions of the UPP. Postmortem tissue samples from the hippocampus, superior and middle temporal gyri, parahippocampal gyri, and the inferior parietal lobes of AD patients exhibit signs of reduced proteasome activity compared with those from control subjects (Keller et al., Notably, both AD and PD are age-associated neurodegenerative diseases with characteristic protein aggregates (Bridi and Hirth, in situ hybridization (Wang et al., Although high levels of Hapln2 in the hippocampus were measured by via the degradation of particular substrate proteins (Tsukamoto and Yokosawa, Although the pathophysiology of schizophrenia has not been fully defined, genetic factors, epigenetic elements, and abnormal neurotransmission in the hippocampus are important contributors (Kim et al., Moreover, it has been reported that ECM plays important roles in regulating neurogenesis, axonal outgrowth, synaptogenesis and cell migration (Bandtlow and Zimmermann, As a brain specific-hyaluronan and proteoglycan link protein, Hapln2 plays vital physiological roles in the formation and maintenance of the ECM scaffold. Studies in knockout mice have also revealed that Hapln2 influences neuronal conductivity. Several studies have revealed the involvement of Hapln2 in the pathogenesis of neurological diseases including PD, AD and schizophrenia, which provided new insights into the underlying molecular mechanisms of brain disorders .Current research suggested that Hapln2 was involved in the formation of \u03b1-synuclein aggregates in PD pathology and study in AD suggested that Hapln2 might be the component of NFTs aggregates (Minjarez et al., QW, CW, BJ, and CY wrote the manuscript. JC and JZ edited the manuscript. All the authors approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "The mental and physical health of individuals with a psychotic illness are typically poor. Access to psychosocial interventions is important but currently limited. Telephone-delivered interventions may assist. In the current systematic review, we aim to summarise and critically analyse evidence for telephone-delivered psychosocial interventions targeting key health priorities in adults with a psychotic disorder, including (i) relapse, (ii) adherence to psychiatric medication and/or (iii) modifiable cardiovascular disease risk behaviours.Ten peer-reviewed and four grey literature databases were searched for English-language studies examining psychosocial telephone-delivered interventions targeting relapse, medication adherence and/or health behaviours in adults with a psychotic disorder. Study heterogeneity precluded meta-analyses.Twenty trials [13 randomised controlled trials (RCTs)] were included, involving 2473 participants . Five of eight RCTs targeting relapse prevention and one of three targeting medication adherence reported at least 50% of outcomes in favour of the telephone-delivered intervention. The two health-behaviour RCTs found comparable levels of improvement across treatment conditions.Although most interventions combined telephone and face-to-face delivery, there was evidence to support the benefit of entirely telephone-delivered interventions. Telephone interventions represent a potentially feasible and effective option for improving key health priorities among people with psychotic disorders. Further methodologically rigorous evaluations are warranted. Life expectancy is 12\u201319 years shorter than that of the general population Laursen, , with CVet al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., Importantly, increasing evidence supports the role of psychological interventions for improving symptoms . The focus of this review will be on person-delivered interventions using the spoken word and one or more psychological strategies and the protocol has been published were assessed against the 11 item Physiotherapy Evidence Database (PEDro) scale was assessed using the Collaboration's Risk of Bias tool, as described in the A study was considered to have a positive outcome if more than 50% of the reported outcome measures (primary and secondary) demonstrated a between-group difference in favour of the telephone group at the treatment end. Positive maintenance outcome(s) were identified when this effect was evident at short and/or medium and/or long-term follow-up .Comparability of study design and outcome measures across studies was assessed by a consultant statistician to determine the possibility of conducting meta-analyses on RCTs to examine effects on relapse, medication adherence and smoking and other health behaviours and CVD risk. A narrative synthesis of the findings was conducted, structured around intervention type, outcome, population and methodological quality. As Clinical Guidelines recommend an improved focus on personally meaningful recovery relative to traditional clinical outcomes (e.g. symptoms and relapse) in mental health care, to help inform clinical practice, the assessment, reporting and/ or change in these additional outcomes is also central to the structure of the review.Across all studies, the total number of participants was 2473, with 867 in relapse prevention, 1273 in medication adherence and 333 in smoking and/or other health risk behaviour studies (see online Supplementary Table S1). The average age was 40.7 years . Overall, the percentage of males across the studies was 50.1%. However, there was a higher percentage of males in studies of schizophrenia samples (64.5%) compared with studies of bipolar (37.7%) and mixed samples (44.2%). No study used a first episode sample.et al. in favour of TAU has been argued to be a sufficient alternative (Hansson"} +{"text": "In the present contribution, we introduce the eLignin database, use its dataset to map the reported ecological and biochemical diversity of the lignin microbial niches, and discuss the findings.Lignin is a heterogeneous aromatic biopolymer and a major constituent of lignocellulosic biomass, such as wood and agricultural residues. Despite the high amount of aromatic carbon present, the severe recalcitrance of the lignin macromolecule makes it difficult to convert into value-added products. In nature, lignin and lignin-derived aromatic compounds are catabolized by a consortia of microbes specialized at breaking down the natural lignin and its constituents. In an attempt to bridge the gap between the fundamental knowledge on microbial lignin catabolism, and the recently emerging field of applied biotechnology for lignin biovalorization, we have developed the eLignin Microbial Database ( The eLignin database was launched online in March 2017 and aims to bring together the bibliome of this field in one self-contained searchable platform, and thus fill a gap presently not covered by other online biological databases, as well as to demonstrate the high diversity of this microbial niche , as these play an important role in microbial degradation of native lignin and can be applied for enzymatic depolymerzation of technical lignins . Due to the nature of the data collection for eLignin (scientific publications), there will be some overlap with other biological databases such as MetaCyc , manually curated and supplemented with links to relevant entries in other well-established biological and chemical databases and two fungal phyla (Ascomycota and Basidiomycota) (see Tables Proteobacteria (114 species/strains), Basidiomycota (58 species/strains),\u00a0Actinobacteria (31 species/strains), Ascomycota (27 species/strains), and Firmicutes (22 species/strains) (Tables Euryarchaeota) is also beginning to emerge lignin signified by its brown color (hence the name of this group of wood degraders); they are primarily found in softwood ecosystems currently listed in eLignin are distributed between ta Table . The cliWhen it comes to lignin-degrading activity, fungi tend to be more studied than bacteria because of their higher prevalence of lignolytic secretomes , with Proteobacteria dominating the list with its 114 entries , followed by \u03b2-Proteobacteria (27 species/strains), \u03b1-Proteobacteria (18 species/strains), and \u03b4-Proteobacteria (3 species/strains), again highlighting that certain types of microbes are greatly enriched in the eLignin bibliome. It can also be noted that many of the organisms in this particular niche have undergone one or several taxonomical reclassifications since they were first isolated and described , with the remainder being Gram-positive (46 species/strains) and unknown/Gram-indeterminate (4 species/strains). This may have implication on studies focusing on, e.g., transport of compounds over membranes (discussed in a separate section below), or when expanding a species\u2019 substrate range by metabolic engineering. In the latter case, the difference in total GC content in the genome that is in general seen between Gram-positives and Gram-negatives (Muto and Osawa Although fungi are known as the main degraders of the lignin macropolymer (as described in the previous subsection), there are a substantial number of studies that describe delignifying bacteria. Tian et al. reviewed the topic and performed phylogeny on 57 lignin-degrading and 463 laccase-encoding prokaryotes that led them to propose that screening for laccases genes may be a good way to detect new lignin-degrading species extension of the lignin microbial niche.Five archaeal isolates\u2014classified in niche subgroup 2 via acetyl-CoA to evaluate the physiology of the microbial niche monoaromatic compounds tend to be more commonly studied across all phyla. Note that there are no Acidobacteria or Spirocheates in the eLignin bibliome that have been reported to degrade natural/technical lignins and di-/oligoaromatics.Similar to how fundamental and applied studies on lignin focus on a few model organisms, many studies use a few common model aromatic model compounds that represent different funneling pathways , coniferyl alcohol , and p-coumaryl branch (no methoxy groups) they catabolize: the sinapyl branch (two methoxy groups), coniferyl branch (one methoxy group), and the see Fig. . Within see Fig. . Funnelisee Fig. , is anotIn a lot of bibliome studies, the substrate specificity of a species is presented without going into the intracellular conversion mechanisms nor reporting evidence of a specific funneling pathway. Therefore, in order to be able to use the eLignin dataset to look at pathway diversity, we developed a prediction algorithm to infer funneling branches from reported substrates from the literature. This is possible since many of the funneling branches are linear, e.g., ferulic acid is degraded via vanillin, and any species that have been reported to grow on these compounds and their intermediates can then be theoretically inferred to have the coniferyl branch Fig. . Cinnamip-coumaric (H) branches seem by far to be the most abundant in niche 2. This might be correlated to the number of methoxy groups -derived monomers. Spruce lignosulfonate has for instance been reported to yield vanillin, guaiacol, acetovanillone, and vanillic acid (P\u00e9rez and Tuck Amycolatopsis sp. ATCC 39116 (Pometto III et al. Comamonas sp. B-9 (Chen et al. Rhodotorula rubra IFO 889 (Huang et al. Sphingobium sp. SYK-6 (Katayama et al. Acetobacterium woodii NZva16 (Bache and Pfennig Rhizobium sp. YS-1r (Jackson et al. Oceanimonas doudoroffii JCM21046T (Numata and Morisaki Exophiala jeanselmei CBS 658.76 (Middelhoven Many lignin valorization studies apply chemical depolymerization since microbial enzymatic breakdown of lignin is a very slow process taking many weeks (Fackler et al. Although the chemical structure of many aromatic compounds allow them to passively diffuse through the lipid bilayers of biological membranes (Engelke et al. Acinetobacter baylyi ADP1 (Collier et al. Bradyrhizobium japonicum USDA110 (Michalska et al. Klebsiella pneumoniae M5a1 (Xu et al. Pseudomonas putida KT2440 (Nishikawa et al. Rhodopseudomonas palustris CGA009 (Giuliani et al. Sinorhizobium meliloti 1024 (Michalska et al. Sphingobium sp. SYK-6 (Mori et al. Corynebacterium glutamicum ATCC 13032 (Chaudhry et al. Lactobacillus plantarum WCFS1 (Rever\u00f3n et al. Rhodococcus jostii RHA1 (Otani et al. Pseudomonas isolate that excreted vanillyl alcohol during growth on vanillin as a tolerance mechanism to handle excess vanillin that was not catabolized to vanillic acid fast enough, but the mechanism by which vanillyl alcohol was transported out of the cell has not been elucidated yet (Ravi et al. Current knowledge on bacterial aromatic transporters mostly focuses on Gram-negative bacteria which have a cell envelope with two lipid bilayers separated by a periplasmic space: the outer and the inner membrane Nikaido . Some GrThe interest for lignin as an underexploited carbon source has markedly increased during the last two decades, as evidenced by the exponential increase in published papers on lignin valorization (Abej\u00f3n et al. The microbiological aspects of lignin and aromatics degradation have a long history with a vast bibliome, and the need for resources such as the eLignin database will continue to grow as the field expands. In the future, we expect to further implement in eLignin a number of discussed features including improved prediction algorithms, lignolytic communities, and substrates that cannot be converted by a given organism. Economically feasible lignin valorization will require advanced metabolic engineering and thorough knowledge on microbial physiology. In that context, the objective of eLignin is not only to generate new overviews of the field but also to fuel new research ideas and engineering strategies and thus become an operational tool for studies on the microbiological aspect of lignin degradation, catabolism, and valorization."} +{"text": "Synapse loss has detrimental effects on cellular communication, leading to network disruptions within the central nervous system (CNS) such as in Alzheimer\u2019s disease (AD). AD is characterized by a progressive decline of memory function, cognition, neuronal and synapse loss. The two main neuropathological hallmarks are amyloid-\u03b2 (A\u03b2) plaques and neurofibrillary tangles. In the brain of AD patients and in mouse models of AD several morphological and functional changes, such as microgliosis and astrogliosis around A\u03b2 plaques, as well as dendritic and synaptic alterations, are associated with these lesions. In this review article, we will summarize the current literature on synapse loss in mouse models of AD and discuss current and prospective treatments for AD. Synapse loss has harmful effects on cellular communication, leading to network disruption in the central nervous system (CNS). The communication of billions of neurons within the mammalian brain generates and controls memory, thoughts and emotions. In a neuronal network with different cells, the transfer of information is coordinated at specialized compartments such as the synapse. Synapses are contact points between two neurons, where they communicate by passing ions or neurotransmitter across the synaptic cleft. Synapses can have excitatory or inhibitory effects on the target cells, depending on the released signals. The formed synapses are not rigid but rather dynamic and can either strengthen, shrink or even get lost. Considering the critical role of synapses under physiological conditions, it is not surprising that a severe loss of synaptic integrity can cause substantial disorders such as neurodegenerative diseases which currently affects 46 million people worldwide Prince, . Over a Besides aging, new genetic risk factors for AD were reported recently in GWAS, such as ApoJ/Clusterin, PICALM, complement receptor 1 (CR1), TREM2 and sialic-binding immunoglobulin (Ig)-like lectin CD33 (Kuchibhotla et al., Astrocytes represent the most abundant cell type in the brain. They are involved in synapse formation and elimination, synaptic plasticity and activity. Due to their essential role in brain function it is likely that astrocyte dysfunction results in progression of neurodegenerative diseases. Similar to microglia, reactive astrocytes surround senile A\u03b2 plaques in the brain of AD patients and in mouse models of AD. They become reactive as indicated by their hypertrophic processes and increased expression of GFAP (Wisniewski and Wegiel, Further investigations of neuron-glia signaling pathways and their disruption in neurodegenerative diseases are necessary for the development of new successful therapies that are promising due to the early involvement of glia in the disease process.Although our knowledge regarding the mechanism underlying AD pathogenesis has improved over the last decades, there is still no cure available. Moreover, open questions concerning memory and synapse loss, as well as gliosis and related neuronal damage, still remain (De Strooper and Karran, In vivo imaging of 3xTg-AD mice revealed spine loss on dystrophic dendrites positive for hyperphosphorylated tau in areas without plaques (Bittner et al., Most current therapeutic approaches focused on the reduction of A\u03b2 levels and A\u03b2 plaque load by inhibiting or modifying the generation of A\u03b2. Other attempts tried to target the tau protein instead (Roberson et al., In vivo 2-photon imaging allows to explore structural plasticity of synapses in living mice, even for long-time periods (Grutzendler et al., in vivo 2-photon imaging and followed dendritic spines and axonal boutons over the course of several weeks in APPS1 mice. Pre- and postsynaptic structures showed an enhanced instability in the vicinity of A\u03b2 plaques (Grutzendler and Gan, Prime targets for AD therapies are \u03b2- and \u03b3-secretase inhibitors. Numerous inhibitors currently undergo clinical trials (May et al., in vivo imaging experiments (Filser et al., Unfortunately, the inhibition of BACE1 is known for its mechanism-based side-effects. Conditional deletion of BACE1 in 5xFAD mice resulted in reduced A\u03b2 plaque load and improved synaptic function, determined by LTP and contextual fear conditioning experiments (Hu et al., The oligomeric form of A\u03b2 is often considered as the toxic form. Immunotherapy against A\u03b2 oligomers had little effect on synapse loss in the vicinity of A\u03b2 plaques but abolished synapse loss further away from plaques (Dorostkar et al., To date, it remains an open question whether such A\u03b2 lowering strategies will be successful. Therefore, alternative treatment options should be considered. Mice exposed to an environmental enrichment developed enhanced numbers of new dendritic spines, excitatory synapses and dendritic branches on pyramidal neurons (Mora et al., Besides the physical degeneration of synapses in AD and other neurodegenerative diseases, it is unclear which role glial cells play during the process of synapse loss. Further research will hopefully provide more insights into the role of glial cells and their contribution to synapse loss, in particular at earlier pre-depositing stages when synapses are already vulnerable. Future preclinical treatment approaches should combine pharmacological, non-pharmacological and behavioral studies.SZ-W and MM-L contributed equally to this work, wrote the manuscript, read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Ocular surface disease is an umbrella term that includes a variety of complex pathologies such as Stevens\u2013Johnson syndrome, mucous membrane pemphigoid, limbal stem cell insufficiency, dry eye disease, and ocular graft-versus-host disease. Regardless of the underlying disease process, ocular surface failure may result in inflammatory and infectious complications and potentially devastating visual loss. Early diagnosis and appropriate treatment of ocular surface disease require a high level of expertise as these conditions can be extremely challenging even for experienced clinicians. Although key issues in the management of patients with complex ocular surface disease still remain controversial, recent advances in diagnostics and therapeutics have enhanced our armamentarium and allow for improved clinical outcomes.in vivo confocal microscopy, the morphology of two types of vortex keratopathy: amiodarone-induced keratopathy and the Fabry disease-associated keratopathy. Finally, Moschos et al. explore the psychological aspects and the incidence of depression in patients with symptomatic keratoconus.In our special issue on ocular surface disease, Wr\u00f3bel-Dudzi\u0144ska et al. report on the clinical efficacy of platelet-rich plasma in the management of neurotrophic corneal ulcer, showing promising clinical results. Hazarbassanov et al. assess the effect of osmoprotection in the management of dry eye disease after refractive surgery in a randomized controlled double-blind clinical trial, while Moussa et al. investigate the effect of different prostaglandin analogues on the ocular surface of patients with primary open-angle glaucoma. Krysik et al. report on indications, outcomes, and complications of penetrating keratoplasty for ocular surface disease-related pathologies in a tertiary referral center, whereas Sun et al. analyze the therapeutic effects of corneal debridement combined with intrastromal voriconazole in a patient series with recalcitrant fungal keratitis. Ikegawa et al. investigate, with the aid of Zisis GatzioufasSamer HamadaSotiria PaliouraThe authors declare that they have no conflicts of interest."} +{"text": "Innate immunity against pathogen infection by membrane-localized receptors is evolutionarily conserved among eukaryotes and receptor-like proteins (RLPs) ; and (2) VIGS in N. benthamiana avoids gene function redundancy and allows for simultaneous silencing of multiple homologous genes (Wang et al., The VIGS-based approach for identification of PRRs has advantages over methods that rely on map-based cloning and Arabidopsis T-DNA insertion lines (Zipfel et al., N. benthamiana recognizes XEG1, a widely distributed MAMP in microbial taxa (N. benthamiana can identify XEG1 family proteins secreted by various microbes. Therefore, RXEG1 could potentially be used to protect a broad range of plants, especially other solanaceous plant species such tomatoes, whereby high disease resistance might be achieved through genetic engineering or by spraying.In addition to identifying and characterizing PRRs, elucidating the mechanisms by which PRRs perceive microbial attack will significantly advance our understanding of plant innate immunity. The comprehensive and intensive work of Wang et al. revealedial taxa . When miial taxa . Wang etial taxa demonstrWang et al. identifiCurrently, the recognition of certain MAMPs remains restricted to solanaceous plants (Wang et al., WW drafted the manuscript. SW and WW revised the manuscript. SW draw The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Geriatrics infusion and transformation of community-based settings to support \u201caging in place\u201d is complex. It requires a customized approach that engages multiple stakeholders who are invested in systems redesign and process change. Rowan University School of Osteopathic Medicine\u2019s NJ Geriatrics Workforce Enhancement Program, in partnership with an affordable housing facility operated by Fair Share Housing/Northgate II, and Rutgers University School of Nursing (RSoN), implemented a Resident Health Risk Assessment (RHRA) tool as part of an interprofessional community-based training experience for health professions students. The goal was to identify health risks that impact a resident\u2019s ability to age in place and implement a person-centered intervention plan. Multi-level stakeholder engagement and ongoing rapid cycle quality improvement catalyzed changes in structure and process for all partners. Results included refinement of an interprofessional clinical rotation, introduction of competency attainment into the orientation process, and resource reallocation to support data collection."} +{"text": "Fungi are eukaryotic heterotrophs present in virtually every environment, with potentially more than 5 million individual species . DespiteThe constant exposure of humans to both commensal and environmental fungi requires a competent immune system for tolerance, protection, and/or clearance, while limiting collateral damage caused by excessive or detrimental inflammation. Innate responses to fungal pathogens are initiated by fungal component recognition via an array of pathogen-associated molecular pattern recognition receptors (PRRs) \u201311. RecoIncreased understanding of these host-fungal interactions and the mechanisms that favor protective immunity over immune pathology could provide targets for novel therapeutic approaches that complement the limited repertoire of existing antifungal drugs. The aim of this research topic is to explore the host and fungal pathways that program innate and adaptive immunity and the immune cells, molecules, and regulatory pathways that comprise protective or detrimental responses to fungal exposure or infection. Researchers from 15 countries in North and South America, Europe, Asia, and Australia contributed 36 review and original research articles that cover a wide range of fungal pathogens, disease models, and effector and regulatory cell and molecular pathways of host immune responses to fungal exposure and infection. Here, we present an outline of the findings, perspectives, and reviews contained in this research topic.Tang et al.. Although the important role of the \u03b2-1,3-glucan receptor dectin-1 in antifungal immunity is well appreciated, the \u03b1-mannan-binding dectin-2 and dectin-3 also influence responses to fungal exposure and infection and their roles are less clear. In support of these studies, Preite et al. show that dectin-2 and dectin-3 mediate antifungal activity and cytokine secretion of human plasmacytoid dendritic cells (pDCs) in response to the endemic dimorphic fungal pathogen Paracoccidioides brasiliensis via a pathway mediated by the CLR-associated signaling molecule Syk. In addition, de Castro et al. show that mouse bone marrow-derived macrophages (BMMs) and DCs (BMDCs) produce IL-1\u03b2 and IL-18 in response to Fonsecaea pedrosoi, the main causative agent of chromoblastomycosis, in a dectin\u22121, \u22122, and \u22123-dependent manner. A role for the NOD-Like Receptor P3 (NLRP3), an intracellular protein complex that controls activation of inflammatory caspases and cytokine production, is shown by Feriotti et al. as important for the development of protective immunity to pulmonary infection with P. brasiliensis. \u03b2-glucan stimulation of NLRP3 inflammasome-mediated IL-1\u03b2 secretion in B cells reported by Ali et al. shows innate antifungal cytokine production in an adaptive lymphocytes that were also capable of producing IgM in an NLRP3-dependent manner.The major class of pattern recognition receptors, the C-type lectin receptors (CLRs), including molecules critical for the initiation of inflammation and the development of adaptive immunity to fungi, are reviewed by Aspergillus fumigatus . Circulating monocytes are subsequently recruited to sites of infection in response to inflammatory signals produced by tissue-resident macrophages. T\u00f3th et al. report that the exposure of hyphae of the dematiaceous mold Curvularia lunata to human THP-1 monocytes resulted in increased inflammatory IL-8 and regulatory/anti-inflammatory IL-10, suggesting a possible mechanism for the ability of this species to cause chronic infections in immune competent individuals. Landgraf et al. report that dihydrolipoyl dehydrogenase secreted by P. brasiliensis may act as an exoantigen that enhances macrophage phagocytosis. Mukaremera et al. report that hypha of the yeast Candida albicans induced low levels of cytokine secretion from human monocytes, with the highest levels from yeast and intermediate levels from pseudohypha, and cell wall mannan depletion partially reversed these responses. Using fluorescence yeast cell wall-labeling to measure yeast division within macrophages, Dagher et al. show that activation of the tyrosine kinase Syk is critical for macrophage control of phagocytosed C. glabrata. Together, these studies provide new insights into the roles for monocytes and macrophages in induction and regulation of antifungal inflammation and fungal clearance.PRR-expressing tissue-resident macrophages are part of the first line of defense against fungal infections cells in innate antifungal immunity is reviewed by Schmidt et al.. These works further illustrate diverse mechanisms and roles for innate myeloid and lymphoid cells in antifungal immunity.Inflammatory cytokine production by macrophages leads to localized inflammation with production of chemokines that attract additional innate immune cells. Maldonado and Fitzgerald-Bocarsly. Xu et al. investigated the role of macrophage receptor with collagenous structure (MARCO) on pDC recruitment and induction of antifungal adaptive immunity to C. neoformans infection, and report that expression of MARCO promoted recruitment of lymph node DCs and skewed local and systemic T helper cell responses away from protective Th1 responses and toward non-protective Th2 responses. Wong et al. demonstrate a role for leucine-rich repeat kinase 2 (LRRK2) in translocation of NFAT to the nucleus in DCs in the early stages of the non-canonical autophagic response to A. fumigatus conidia, thus connecting LRRK2 with NFAT-mediated IL-2 expression in early antifungal immunity. Hellmann et al. compared immune responses to A. fumigatus in human and mouse DCs, macrophages, and neutrophils, and observed that human DCs exhibited more significant increases in maturation markers and phagocytic ability than murine DCs, while murine neutrophils and macrophages displayed more reactive oxygen species production after A. fumigatus exposure. Collectively, these works provide support for additional mediators of DC function that shape antifungal adaptive immunity, and suggest distinct roles for these cells in human fungal disease and experimental animal models.Dendritic cells (DCs) are multifaceted professional antigen presenting cells that integrate antigen uptake and local inflammatory signals in order to effectively prime adaptive antifungal immune responses upon migration to site-draining lymph nodes . The rolTrist\u00e3o et al. report that these cytokines, along with the IL-17 receptor A, were necessary for protective lung granuloma formation in mice infected with P. brasiliensis. IL-1\u03b1/\u03b2 also influence adaptive immune responses to fungi, such as the dimorphic Cryptococcus spp., as Shourian et al. observed that IL-1 receptor type 1-deficient mice had increased cryptococcal burden, eosinophilia, M2 macrophage polarization, and recruitment of CD4+IL-13+ T cells, with a concomitant reduction in pro-inflammatory Th1 and Th17 cytokines. However, excessive inflammation and Th1 activation in C. neoformans infection is also detrimental, and was reported by Fa et al. to be mediated by the cell death regulatory genes FADD (Fas-associated death domain) and RIP3K (receptor interacting protein kinase 3). The results of these studies support roles for Th1 and Th17 responses in antifungal immunity, as well as a requirement for regulation of cell death to limit excessive inflammation.After DCs migrate to draining lymph nodes, they present fungal antigen to na\u00efve T cells, inducing antifungal adaptive immunity. This process is influenced by cytokines in the microenvironment. Formation of the antifungal Th17 subset of T helper cells is promoted by the inflammatory cytokines IL-6 and IL-23, and A. fumigatus; findings that shed light on this host-pathogen dynamic are reviewed by Choera et al.. De Ara\u00fajo et al. report that DCs from an infected P. brasiliensis-susceptible mouse strain exhibit IDO activity and promote inflammation, while DCs from resistant mice phosphorylate IDO and promote a tolerogenic phenotype. The same group further compared P. brasiliensis infection in IDO-deficient and wild-type mice, and observed increased mortality, fungal burden, histopathology, and Th17 cell recruitment and activation in IDO-deficient mice with concomitant decreases in Th1 and Treg cells de Ara\u00fajo et al.. In addition to the enzymatic activity and signaling by IDO, antifungal immune responses are regulated at the post-transcriptional level by microRNAs (miRNAs); these molecules and their associated pathways are reviewed by Croston et al.. Zimmerman et al. report that patients with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) exhibit decreased levels of the signaling molecule STAT1, in contrast with patients with STAT1 gain-of-function mutations, despite similar disease phenotypes. These works highlight findings of enzymatic, post-translational, and signal transduction in the regulation of antifungal immunity and fungal disease susceptibility.Cell and molecular regulation of immunity occurs at multiple levels from induction to resolution of immune responses. An enzyme that limits available tryptophan and thus dampens immune cell proliferation and effector function, indoleamine 2,3-dioxygenase (IDO), is produced by mammalian hosts and the fungal pathogen Kernien et al. review host immune recognition of fungal biofilms and biofilm properties that inhibit host recognition and clearance. Zhang et al. explore how immune interactions with respiratory and gastrointestinal fungal microbiota contribute to chronic airway inflammatory disease. Sparber and LeibundGut-Landmann more specifically discuss host immune responses to skin-colonizing Malassezia species, yeasts that are involved in a variety of skin disorders. Finally, Casadevall et al. review the evidence that C. neoformans mediates host damage at the molecular, cellular, tissue, and organism levels, in some instances with formation of large fungal masses in brain tissue. These reviews highlight emerging areas of fungal immunology that consider fungal interactions with the host immune system beyond the level of cell and molecular recognition.Although spore/hyphal recognition by innate cells drives the development of immunity to fungal pathogens, host-fungal pathogen interactions are also considered at the macroscopic level of microbial communities and host tissues. Elsegeiny et al. review studies of immune pathology in cryptococcal infection and discuss the need for immune targeting therapies that suppress immune-mediated damage without further compromising host protection. Van Dyke et al. demonstrate that combining non-lethal C. neoformans challenge with local IFN-\u03b3 production elicits an immune response that protects mice from subsequent lethal infection. Tso et al. summarize efforts and obstacles in the development of anti-Candida vaccines, and discuss the potential use of trained immunity in the development of strategies against opportunistic fungal infections. Finally, Kumaresan et al. review the development of CD8+ T cell therapy for the control of invasive fungal infections, with a focus on the use of chimeric antigen receptor (CAR) T cells that target \u03b2-glucan. These reviews underscore the importance of current and future efforts to enhance immune protection while limiting immune pathology in patients that may not respond to traditional antifungal therapies.Despite significant advances in our understanding of host immunity to fungal exposure and infection, treatment of fungal diseases has not progressed beyond the use of a limited repertoire of antifungal drugs that are rendered increasingly ineffective by emerging fungal resistance. Since appropriate antifungal immunity is critical for protection from fungal disease, complementary therapies that target immune pathways represent areas of considerable interest for fungal immunologists. Collectively, the studies described in original research and review articles in this topic provide optimism for the future of antifungal immune therapy. Recent advances by fungal immunologists have greatly increased our understanding of the basic mechanisms of innate immune recognition, inflammation, adaptive immunity, and regulation of antifungal immune responses at molecular, cell, tissue, and organismal levels. We hope these articles will stimulate further research with the ultimate goal of improving outcomes for patients with fungal diseases.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "People with diabetes mellitus (DM) have a higher-than-average risk of having a heart attack or stroke , 2. In fDe Rosa et al., from Magna Graecia University, elegantly illustrated fundamental genetic and epigenetic mechanisms linking cardiovascular disease and DM; similarly, Pordzik et al. identified the functional role of specific platelet-related microRNAs in the pathophysiology of cardiovascular events in high-risk populations, including diabetic patients.Soares Felicio et al. demonstrated an association between reduced levels of Vitamin D and the presence and severity of diabetic kidney disease in type 1 DM (T1DM); the molecular mechanisms underlying diabetic nephropathy have been also explored by Zou et al. in streptozotocin-induced DM. Arcangeli et al. found a significant association between the number of circulating endothelial progenitor cells (cEPCs) and the age and duration of the disease in T1DM patients: indeed, young T1DM patients have significantly higher levels of cEPCs compared to adult T1DM patients; of note, such difference is also maintained when the disease lasts for more than 10 years. The Authors propose that maintaining a high number of cEPCs, possibly through an efficient glycemic control, would contribute to contain the cardiovascular burden in T1DM. Notably, in vitro experiments performed by Lin et al. at New York University have shown how to ameliorate purification and maturation of human induced pluripotent stem cell (iPSC)-derived cardiomyocytes through means of culture in glucose-depleted medium supplemented with fatty acids (oleic acid and linoleic acid) and 3,3\u2032,5-triiodo-L-thyronine (T3).Infante et al. revealed a greater severity of coronary artery disease in type 2 diabetes (T2DM) patients compared to non-diabetic individuals; equally important, van Bussel et al. from Maastricht University Medical Center, highlighted the actual advantages of multiparametric neuroimaging in the clinical evaluation of cognitive decline in T2DM.Applying comprehensive analyses based on imaging and molecular biology, Orosz et al. in subjects with impaired glucose tolerance, a prediabetic condition, have shown that prediabetes is associated with repolarization instability, indicated by elevated values of beat-to-beat short-term QT interval variability, thereby suggesting that an impaired autonomic control precedes the actual onset of diabetes. Last but not least, Altara et al. validated the key importance of targeting microvascular disease, common in both diabetes and obesity, in order to treat heart failure with preserved ejection fraction (HFpEF). Microvascular disease is a growing public health problem, accounting for approximately half of hospital admissions of individuals with heart failure (The studies performed by failure , 11, 12.In summary, the present Research Topic indicates that the exceptional advances achieved in the last decade in understanding the molecular alterations involved in the pathophysiology of both DM and cardiovascular disease are opening new therapeutic opportunities for the treatment of these disorders and, potentially, their future application to the clinical scenario might result to further enhancements in patient care. Furthermore, the exciting findings discussed herein might foster community awareness of these important diseases and stimulate further research in the field.The author confirms being the sole contributor of this work and has approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Numerous studies have shown that infiltration of lymphocytes into tumor tissue is associated with better prognosis (Bindea et al., 2013; SchmidtThis result is of relevance, because it shows that immune modifiers have different roles in NSCLC of smokers and non-smokers. It will be important to consider this difference in therapy studies targeting the PD-1/PD-L1 axis. Predicting prognosis and response to therapy remains a challenging task (Hellwig et al., 2016; Lohr etThe author declares no conflict of interest."} +{"text": "Cervico-vaginal mucus (CVM), the product of epithelial cells lining the uterus, cervix and vagina, is secreted to facilitate uterine lubrication and microbial clearance. Predominantly composed of water and mucins, CVM also contains high levels of immuno-active proteins such as immunoglobulin A (IgA), lactoferrin and lysozyme which protect against infection by blocking adhesion and mediating microbial killing. The repertoire of cytokines, chemokines and antimicrobial peptides is predominantly generated by the secretions of endometrial epithelial cells into the uterine lumen and concentrated in the CVM. The quantity and relative proportions of these inflammatory biomarkers are affected by diverse factors including the estrus cycle and health status of the animal and therefore potentially provide important diagnostic and prognostic indicators. We propose that measuring molecular signatures in bovine CVM could be a useful approach to identifying and monitoring genital tract pathologies in beef and dairy cows. Cervico-vaginal mucus (CVM) represents a mixture of vaginal, cervical and uterine mucus and is composed of 92\u201395% of water, ions and 5\u20138% solid matter multiplication in the uterus are secreted directly into the endometrial lumen while TNF-\u03b1, IL-1 and IL-8 are concentrated in uterine mucus after infiltrating through uterine wall (Carneiro et al. IL8 in the endometrium 2\u00a0weeks after calving, and intrauterine infusion of exogenous IL-8 reproduces the disease (Chapwanya et al. IL-1 is the key mediator of uterine inflammation that is secreted as a result of tissue damage associated with parturition (Adnane et al. E. coli infection (Chapwanya et al. E. coli have high systemic levels of AGP at 21 and 28 DPP (Williams et al. Trichomonas foetus or endometritis (Adnane et al. APPs are highly responsive proteins, secreted by the liver in response to injury or infection in an effort to restore homeostasis. Extra-hepatic sources of APPs have also been identified, including in endometrial cells (Chapwanya et al. Members of the S100 family chelate calcium and therefore regulate many cellular processes including microbial viability (Corbin et al. Here we reviewed the measurement of inflammatory biomarkers in CVM early postpartum as an alternative method for the prognosis of genital tract pathology in cows and other species. CVM molecular signatures are specific to uterine health status may also reflect local microbiome diversity. They provide a convenient, cost effective and welfare-friendly method for timely detection of uterine inflammation in order to reduce the impact of endometritis on dairy cow production. CVM therefore is a potentially valuable resource to investigate the diversity of bacteria in cows with uterine disease as many of these bacteria are trapped by the mucus structure."} +{"text": "DiMarco et al., Chem. Sci., 2018, DOI: 10.1039/c7sc03839a.Correction for \u2018Dye-sensitized electron transfer from TiO The authors regret that The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "Microglia are the resident immune cells of the central nervous system and have many functions including neuroinflammation, axonal guidance and neurotrophic support. We aimed to provide a quantitative review of g). The difference in 18-kDa translocator protein (TSPO) binding between patients with schizophrenia and healthy controls, as quantified by either binding potential (BP) or volume of distribution (VT), was used as the main outcome. Sub-analysis and sensitivity analysis were carried out to investigate the effects of genotype, ligand and illness stage.Demographic, clinical and imaging measures were extracted from each study and meta-analysis was conducted using a random-effects model but no significant differences when VT was used .In total, 12 studies comprising 190 patients with schizophrenia and 200 healthy controls met inclusion criteria. There was a significant elevation in tracer binding in schizophrenia patients relative to controls when BP was used as an outcome measure, (Hedge's In conclusion, there is evidence for moderate elevations in TSPO tracer binding in grey matter relative to other brain tissue in schizophrenia when using BP as an outcome measure, but no difference when VT is the outcome measure. We discuss the relevance of these findings as well as the methodological issues that may underlie the contrasting difference between these outcomes. However, findings have been inconsistent and so far they have only been partially reviewed quantitatively . In order to extract data from studies where data were available only in a plot format, we have used the plot digitiser software (http://plotdigitizer.sourceforge.net/).The main outcome measure was the difference in the TSPO imaging index between patients with schizophrenia-spectrum disorders and healthy controls. For all studies, we extracted the following variables: authors, year of publication, subject characteristics for the patient and healthy control group , imaging characteristics and modelling method. The estimation of pooled standard deviation was performed using the statstodo software . The DVR method used in this study is not equivalent to the measures used in the other studies. Also Ottoy et al. . Therefore, the main analysis of VT values included six studies using the 2TCM model.The main outcome measure was the effect size determined using the TSPO tracer measure and quantified by either BPd Hickie , and alt express both mixed affinity for TSPO (MAB), while those without the polymorphism (HH) have high-affinity binding (HAB) for TSPO . Leave-one-out sensitivity analyses were conducted to investigate the potential effect of an individual study on the outcome measure. A p value <0.05 (two-tailed) was taken as a significance level. The statistical analysis of the extracted data was conducted using the R statistical programming language version 3.2.2 with the \u2018metafor\u2019 package.Publication bias was assessed using visual inspection of funnel plots as well as regression test. Where potential publication bias was suspected, trim and fill analysis was conducted to correct for putatively missing studies. Heterogeneity was estimated using the The PubMed, EMBASE and PsycINFO databases were searched without language restrictions. The electronic search using EMBASE and PsycINFO were carried out together using Ovid. The following keywords were used: \u2018(Positron Emission Tomography OR PET OR Single photon emission tomography OR SPET OR Single Photon Emission Computed Tomography OR SPECT) AND (schizophrenia OR schizophreniform OR psychosis) AND \u2019. Review papers were also screened to search for additional studies.The inclusion criteria were: original studies in (1) patients with a diagnosis of schizophrenia or related psychotic diagnoses , (2) reporting PET measures using a TSPO-specific ligand and (3) reporting data for the whole grey matter or grey matter regions. Studies that did not have a control group were excluded. Where there was sample overlap between studies, we included the largest one and excluded the other to avoid double counting.VT). As these give different information, we conducted separate meta-analyses of these outcomes. The studies that used BP as an outcome measure have used either values obtained using microparameters from Simplified Reference Tissue Model or volume of distribution 0.02\u20130.6] . Visual inspection of the funnel plot suggested asymmetry . The regression test was significant . The trim and fill analysis showed three putatively missing studies on the left side. The results were not significant after correcting for these studies . The results were significant in two out of six in the leave-one-out analysis, with effect sizes varying from 0.27 to 0.47. Five out of the six studies used the [11C]-PK11195 ligand, with the sub-analysis of these studies revealing a significant elevation of BP in patients with schizophrenia with an effect size of 0.35 .Six studies reported outcome measures as BP. Our results showed that BP was significantly elevated in patients with schizophrenia when compared with healthy controls with an effect size of 0.31 [Hedge's .02\u20130.6] . The I2 VT. VT in patients with schizophrenia when compared with healthy controls . The I2 test revealed moderate\u2013high heterogeneity . Visual inspection of funnel plot suggested asymmetry . However, the regression test was not significant . Trim and fill analysis showed two missing studies in the right side. The effect sizes varied from \u22120.08 to \u22120.37 in the leave-one-out analysis. We extracted data for high- (HABs) and mixed-affinity binders (MABs) separately from these studies to conduct a sub-analysis stratified by genotype. Bloomfield et al. reported only one patient who was a MAB, precluding accurate estimation of the effect size. Thus, this study was not included in the sub-analysis of MAB subjects . However, there was a significant difference in the MAB .Six studies reported the outcome measure as g\u00a0=\u00a00.31; p\u00a0=\u00a00.03), but there is no significant difference when the tracer volume of distribution (VT) is used as the outcome measure . In the following section, we consider methodological factors and the implications of our findings.Our main findings are that TSPO PET tracer binding is significantly elevated in patients with schizophrenia relative to controls when BP is used as an outcome measure, with a small-to-moderate effect size is negative, the results using this tracer are not an outlier, suggesting findings may not entirely be accounted for by tracer differences. It has been suggested that TSPO expression may change during the course of the disorder, which could account for the differences in findings between studies measures the tracer binding in the tissue of interest relative to another brain region selected to have negligible specific binding, to give specific binding in the region of interest measures the total amount of tracer in the brain region relative to that in the blood that are known to be elevated in schizophrenia (Telford et al., et al., et al., VT, and potentially mask an elevation in the patients\u2019 brain as compared with controls. There is inconsistency in findings in schizophrenia, with one study showing elevation in the plasma concentration levels of [11C]-PBR28 in patients with schizophrenia relative to controls (Bloomfield et al., 18F]-FEPPA and [11C]-PBR28 show no differences in plasma concentration levels (Hafizi et al., et al., et al., et al., et al., VT values in the disorder, but it should be recognized that there is no direct evidence that plasma binding alters VT values in humans (Cumming et al., VT changes are not explained by an effect of protein binding (Collste et al., VT in schizophrenia. Nevertheless, while this is a potential concern for studies using VT as the outcome measure, blood binding should not affect the studies that use a ratio approach, as these methods report tracer uptake relative to another brain region rather than blood. Furthermore, TSPO is expressed on the endothelial cells of brain blood vessels as well as on the outer layer of mitochondria in microglia (Rizzo et al., et al., The non-displaceable BP (BPet al., et al., et al., et al., et al., Taken together, our meta-analytic findings suggest an elevation in TSPO tracer binding in total grey matter relative to other brain tissue, but not relative to blood, with the caveat that the relative increase is largely based on studies using PK11195, which has a lower specific signal. Thus, this could reflect an increase in TSPO in grey matter or a reduction in TSPO in the reference region, or altered non-specific binding in the brain (Cumming VT as the outcome measure, showed a reduction in VT in patients relative to healthy controls (Plaven-Sigray et al., VT in our meta-analysis, our meta-analysis included one more second-generation study showing no differences in VT between schizophrenia patients and healthy controls (Ottoy et al., in utero and early development may predispose to schizophrenia (Meyer, VT approach (Bloomfield et al., VT as the outcome measure, using the tracer [18F]-FEPPA and [11C]-PK11195, showed no differences in TSPO binding between healthy controls and in individuals at ultra high risk of psychosis for psychosis (Hafizi et al., et al., et al., et al. (et al. (et al. (Our different findings depending on the outcome measure used point towards the existence of potential methodological problems in TSPO imaging, raising questions over the interpretation of the elevation in grey matter TSPO binding relative to a reference region in patients with schizophrenia when compared with healthy controls. A recent individual participant data meta-analysis of second-generation radioligand studies, with a Meyer, . However, et al. and van (et al. suggest (et al. b using tVT is the outcome measure used. These results suggest that potential methodological differences between TSPO studies need to be accounted for and addressed in future studies and keep open the discussion over the existence of an increase in microglia activity in patients with schizophrenia-spectrum disorders.In conclusion, there is evidence for a moderate effect size elevation in TSPO tracer binding in grey matter in schizophrenia-spectrum disorders when using BP as an outcome measure, but no changes when"} +{"text": "Purified proteins offer a homogeneous population of biological nanoparticles, equipped in many cases with specific binding sites enabling the directed self-assembly of envisaged one-, two- or three-dimensional arrays. These arrays may serve as nanoscale biotemplates for the preparation of novel functional composite materials, which exhibit potential applications, especially in the fields of nanoelectronics and optical devices. This review provides an overview of the field of protein-mediated biotemplating, focussing on achievements made throughout the past decade. It is comprised of seven sections designed according to the size and configuration of the protein-made biotemplate. Each section describes the design and size of the biotemplate, the resulting hybrid structures, the fabrication methodology, the analytical tools employed for the structural analysis of the hybrids obtained, and, finally, their claimed/intended applications and a feasibility demonstration (whenever available). In conclusion, a short assessment of the overall status of the achievements already made vs. the future challenges of this field is provided. Purified proteins offer a homogeneous population of biological nanoparticles, equipped in many cases with specific binding sites enabling directed self-assembly into one-, two- or three-dimensional arrays. These arrays may serve as biotemplates for the preparation of novel functional composite materials on the nanoscale, using either specific or site-directed chemical binding, with the potential for expansion by genetic or chemical manipulations.The potential inherent in protein-mediated biotemplating has attracted much attention throughout the past two decades . SeveraThis review provides an overview of the field of protein-mediated nanoscale biotemplating, focused on the achievements made throughout the past decade. The review is organized into sections according to the size and configuration of the protein-made biotemplate ranging from single protein soluble molecules and supramolecular protein \u201ccages\u201d to two-dimensional (2D) protein arrays, protein fibers and tubes, viral envelopes, three-dimensional (3D) protein crystals, and fragments of microbial cells.Each section describes the structure and size of the biotemplates employed, the fabrication methodology, the analytical tools used for its structural analysis, its claimed/intended applications and a feasibility demonstration (whenever available).Dagan-Moscovich et al. initiateThe construction of a conducting metallic silver coating on the surface of the oxidoreductase enzyme glucose oxidase (GOx), a 5 \u00d7 8 nm \u2018peanut-like\u2019 dimer, enabled the nanowiring of its active site to platinum electrodes, resulting in the oxygen-independent direct determination of glucose by electrochemical biosensors , as scheThe biotemplating approach for electroless metal deposition directed to the surface of single soluble protein molecules was readily expanded from silver deposition to the deposition of other metals . GlucoseBy changing the biologically active protein core from GOx to the binding protein avidin, which is capable of specifically binding the vitamin biotin, additional applications of metallic silver-coated soluble protein molecules became available: imaging by HRTEM without staining. Mor et al. demonstrThe range of applications of silver\u2013enzyme hybrids was expanded further to antimicrobial applications aiming at the prevention of microbial biofilm formation, by affecting the on-site enzymatically attenuated release of silver ions, generating in situ antimicrobial activity. \u2018Zone of inhibition\u2019 antimicrobial studies demonstrated the feasibility of using these hybrids as wide-spectrum antimicrobial agents, especially as potential antifungal agents against skin and hair fungal contaminations .Veelders and Essen demonstr2S nanoparticles (NPs) ~20 nm in diameter, enabled by RNase A capping, as hollow or full spheres with a claimed potential application as a drug delivery vehicle. The structural characterization of the QDs and Ag2S NPs thus obtained was carried out by high-resolution electron microscopy and spectral analysis.Kong et al. describeThree recent reports have described biomineralization processes directed by protein molecules and designed for biomedical applications, including (i) the formation of albumin-mediated platinum nanocrystals for tomography imaging ; (ii) siFerritin\u2014a natural, spherical, protein-made nanoscale \u201ccage\u201d\u2014serves as an iron storage protein in bacteria, plants, animals and humans. In eukaryotic cells, ferritin is comprised of 24 subunits forming a hollow spherical shell with an inner diameter of 8 nm ,18). TheThe currently available biochemical data on the molecular mechanisms involved in these processes has inspired an exploration of the potential biomedical applications of a series of ferritin and mutated ferritin as protein cages for the preparation of hybrids by biotemplating. The templating of metals, alloys and metal ion salts, including iron, silver, gold, palladium, cadmium, cobalt, platinum and copper, were described . It Listeria innocua [The demonstrated feasibility of using the cavity, 7 nm in diameter, of natural ferritin for biotemplating has triggered efforts to develop analogous, smaller protein cages made of other proteins, resulting in CdS and CdSe NPs biotemplated inside the 4.5 nm diameter cavity of a cage-shaped protein derived from innocua ,24, as wThe use of the cage-forming protein clathrin as a biotemplate for the synthesis of metallic NPs was also described, enabling the preparation of either homogeneous or bimetallic silver\u2013gold or silver\u2013copper NPs, albeit in sizes larger than those available by ferritin-mediated biotemplating (characterized by HRTEM) .2 molecules by using a small de novo designed protein, self-assembled as a \u201cnanoreactor\u201d for biomineralization. Behrens et al. [Voet et al. demonstrs et al. describeZhou et al. describeThe pioneering work of Slytr et al. on the iThe elucidation of the mechanism underlying the biotemplating of FePt NPs from the gas phase into a 2D array on the surface of a bacterial S-layer was presented by Queitsch et al. and veriValero et al. demonstrTwo-dimensional protein arrays other than bacterial S-layers were also used as biotemplates: Shindel and coworkers ,38,39 ex2 nanotubes enabled directed gold deposition, resulting in a hybrid exhibiting enhanced electron transfer between heme proteins and electrodes [Bovine serum albumin adsorbed as a 2D monolayer on the surface of TiOectrodes .Magnetospirillum magneticum for biotemplating of magnetic nanoparticle arrays; characterized by TEM, SEM and AFM, with potential applications in the manufacture of electric circuits.Galloway et al. and BirdThe data available in the literature on self-assembly of proteins into fibers and tubes has paved the way for the exploitation of the inherent potential of using a series of readily available self-assembled protein fibers as biotemplates.Colby et al. describeMalisauskas et al. describeMethanocaldococcus jannaschii as a biotemplate decorated with silver, gold, palladium or platinum NPs, by electroless deposition using NaBH4 as a reducer of pre-adsorbed metal ions. The coated protein filaments thus obtained were characterized by TEM, spectral and electrical analysis, with the intended application of metal nanowires of defined length.Slocik et al. describeIonov et al. describep-nitrophenol to p-aminophenol by NaBH4. The gold nanoparticle-decorated lysozyme fibrils obtained were subjected to structural analysis by TEM and X-ray diffraction (XRD) analysis, and the feasibility of their use as a catalyst was demonstrated by kinetic spectrophotometric analysis.Juarez et al. describeLeroux et al. demonstr3O4\u2013protein hybrids thus obtained were characterized by HRTEM, XRD and Fourier transform infrared spectroscopy (FTIR) analysis, and the feasibility of their magnetization demonstrated, leading towards potential applications as MRI contrast agents.The preparation of magnetic nanowires was also described by Juarez et al. . The coaGeobacillus stearothermophilus into tubular structures serving as biotemplates for the chemical deposition of platinum. The resulting hybrid was characterized by HRTEM, SEM and fluorescence analysis, in preparation for an intended application as nanowires.Korkmaz et al. describe2 and TiO2 nanowires were grown on the biotemplate by monomer polymerization. The structures obtained were characterized by TEM and field-emission scanning electron microscopy (FESEM), and the feasibility of their use for biosensors based on the enzymatic activity of acetylcholine esterase was demonstrated and characterized.Padalkar et al. describep-nitrophenol by NaBH4 to 4-aminophenol was described by Batzli and Love [Insulin fibers were coated with palladium by electroless deposition. The nanostructure and electrical properties of the palladium hybrids thus obtained were characterized by AFM, TEM, X-ray photoelectron spectroscopy (XPS) and scanning capacitance microscopy (SCM) analysis . The useand Love ; see alsand Love .Methanocaldococcus jannaschii, and its use as the major component of a biotemplate has been constructed in combination with other protein-made connecting units .The highly-ordered 3D protein envelopes of viruses, especially of plant viruses and bacteriophages, offer a \u2018natural\u2019 biotemplating tool, which is available in several geometrical shapes and sizes , used as a biotemplate for electroless deposition mediated by reduction with NaBH4 of cobalt and platinum ions within the channel. Nanowires ranging from 50 to 100 nm were identified by HRTEM analysis, as well as by EDXS and magnetometry measurements.Tsukamoto et al. describe2 nanocrystals biotemplated onto the surface of M13 bacteriophages, significantly improving parameters such as operational and storage stability.Neltner et al. describeLim et al. describeThe production of 3 nm aligned magnetic NPs within the central channel of mutated tobamovirus was described by Kobayashi et al. . Energy-Nuraje et al. demonstrThe controlled preparation of a 3D super lattice of metallic nanocrystals was demonstrated by Alloyeau et al. , combiniZhou et al. describeFlexible electroactive nanomaterials were prepared by Vera-Robles et al. by biote2+ cations [Piezoelectric nanowires were biotemplated on a genetically engineered M13 bacteriophage coating protein surface, displaying triglutamate residues for the periodical positioning of Pb cations . A latti cations and tuna cations .As the structural complexity degree of biotemplated arrays grows, it should be mentioned that new characterization tools are also gradually becoming available for the biotemplating arena, as demonstrated by the recent report by Carreno-Fuentes et al. on the cProtein crystals, routinely prepared for the elucidation of protein 3D structures by X-ray crystallography, present a highly ordered 3D array of protein molecules. Along with the formation of this array, a complementary 3D array of nanosize voids is also formed; with patterns, geometry and dimensions depending on the protein \u2018building block\u2019 and its mode of \u2018packaging\u2019 within the crystal .The composites obtained by \u2018filling\u2019 these voids of protein crystals as biotemplates have potential in providing nanostructured, 3D, highly accurate arrays of hybrid materials for applications in fields such as electronic devices and speThe exploitation of the potential inherent to 3D protein crystal biotemplating calls for the availability of complex infrastructures, including the means, know-how, and the control of protein crystallization and protein crystal stabilization to prevent crystal dissolution; as well as structural analysis by XRD and related advanced spectrophotometric methods in order to monitor the preservation of the parent 3D crystalline structure throughout the templating procedure, and the prevention of distortion resulting from the impact on the template array by the templated material.The feasibility of using such an infrastructure for the monitoring of the stability of a crosslinked lysozyme crystal throughout biotemplating of a crosslinked hydrogel inside its internal voids array was successfully demonstrated in our laboratory . FollowiStabilization of protein crystals by chemical crosslinking is a pre-requisite to controlled 3D biotemplating, as the stability of the crystalline protein template should be preserved throughout the process. This stabilization is mostly affected by glutaraldehyde crosslinking . In vieThe elucidation of the glutaraldehyde crosslinking mechanism and an understanding of its progress throughout the protein crystal structure paved the way for the potential inherent to site-preferred partial crosslinking, enabling the isolation of the homogeneous population of protein products \u201cliberated\u201d from partially crosslinked crystals by re-dissolution, as demonstrated in our laboratory .A large spectrum of 3D void arrays, exhibiting different shapes and sizes, is available for exploitation from the Protein Data Bank (PDB). Their use is, however, often limited by the commercial availability of an envisaged protein or tedious crystallization procedures and their structural analysis. An alternative route for fishing data out from the PDB might be the use of a \u201cparent\u201d protein building block for the preparation of a series of protein crystals, each exhibiting a different 3D void array. This approach may be materialized by affecting different crystallization conditions: (i) modification of salt and alcohol concentrations, (ii) \u201cdirecting\u201d the crosslinking of binding proteins by bi-ligand as demonstrated by crosslinking of concanavalin A, yielding \u2018diamond-like\u2019 crystals by crosslinking with monosaccharide bi-ligands ; or (iiiAcetivibrio cellulolyticus) as illustrated in To illustrate the feasibility of the latter approach, Wine and coworkers ,83 used An alternative route to affect parent protein\u2013protein molecular interactions affecting crystal \u201cpacking\u201d was demonstrated by Cohen-Hadar et al. , using tTakeda et al. suggeste4, indicating the superiority of smaller crystals for this application.He and colleagues ,87 demonThe feasibility of using bacterial flagella, either extracted from bacterial cells or re-assembled from purified flagellin, was demonstrated by several groups.Salmonella typhimurium . Oxide formation on the surface of a series of biological templates, including flagella mechanically detached from S. typhimurium was also demonstrated by this laboratory [Mao and colleagues demonstrboratory , using cS. typhimurium flagella reconstructed from isolated and purified flagellin. The hybrid obtained was characterized by HRTEM, X-ray and Raman spectroscopy, followed by electrochemical I-V measurements, indicating that electrical conductivity of the hybrid prepared from reconstructed flagella was superior to the conductivity exhibited by directly extracted flagella.Gopinathan et al. demonstrProtein-mediated biotemplating has attracted much attention throughout the past decade, culminating in data proving the feasibility of a wide spectrum of biotemplating systems demonstrated in one, two and three dimensions. All these data are supported by advanced, state-of the-art characterization technologies.The preliminary feasibility demonstrations of several applications made available by protein-mediated biotemplating was also demonstrated with an emphasis on nanoelectronics and biosensing. It appears, however, that most protein-mediated biotemplating systems described so far did not culminate in feasibility demonstrations for their potential applications. Therefore, it appears that protein-mediated biotemplating-related research and development (R+D) efforts should address the challenges of the identification and demonstration of new applications, and focus on further developing the already described initiations.Future advancement of such a technology should also aim to solve important practical aspects as bringing results from bench to practice, including the provision of the means of contact or connection of the hybrids thus obtained to working/detecting systems, as well as data on storage and operational stability. The process of coping with future R+D challenges may benefit from further exploitation of the potential inherent to the incorporation of additional protein engineering and chemical modification-based methods for the manipulation and performance improvement of protein templates."} +{"text": "The hypothalamic non-apeptide oxytocin (OT) has several physiological functions, ranging from lactation to social attachment neurons, showed that OT induces membrane hyperpolarization and consequently inhibits the nociceptive transmission by Ca2+-dependent mechanisms neurons and ex vivo patch-clamp as well as behavioral analyses, suggest that OT inhibits the nociceptive input either directly , for which OT has a physiologically relevant affinity, cannot be ruled out abolished the OT-induced Ca2+ influx. These results were confirmed by replicating both the nociceptive DRG neurons isolated from wild-type animals and the modest OT-induced analgesia observed in TRPV1 knock-out mice. Further electrophysiological, planar lipid bilayer, and in silico experiments showed that OT acts as a partial agonist of TRPV1 channels and induces a strong desensitization of these channels, under nociceptive stimulation. This pioneering study strongly suggests that at peripheral level, OT-induced analgesia passes at least in part, through a direct TRPV1 interaction.Nersesyan et al. was first to show that aversive behaviors induced by an intracutaneous (i.c.) injection of capsaicin into an animal hindpaw, were reduced in animals concomitantly treated with i.c. OT. One can therefore propose that the OT effect is a result of OTR activation at the peripheral nerve endings mediated by GABA. These data suggest that OT recruits GABAergic interneurons, which in turn, activate GABAB receptors and consequently inhibit the capsaicin-sensitive neurons. Interestingly, TRPV1 and GABAB receptors were found to be coexpressed in primary nociceptive neurons. Additionally, the TRPV1 spinal cord expression was enhanced in neuropathic rats, an up-expression corrected by intrathecal OT treatment. Accordingly, the OT-induced analgesia was partially reduced in TRPV1 knockout mice. Like the proposal of Nersesyan et al., this study points out that OT-induced analgesia may rely on both GABA release and modulation of TRPV1 activity.To further explore if OT-induced analgesia in neuropathic pain may occur via TRPV1, Sun et al. first confirmed that intrathecal OT diminishes the aversive behavior in neuropathic animals (Miranda-C\u00e1rdenas et al., B receptors, which in turn modulate the TRPV1 function during neuropathic pain. This finding is in line with the observation that GABAB receptors revert the TRPV1 sensitization by noncanonical signaling under pathological pain (Hanack et al., In the past decades, OT has slowly emerged as an important mediator of endogenous analgesia (Poisbeau et al., An intriguing point in Sun et al. study is how exactly, at the spinal level, OT engages the intracellular machinery to modulate the TRPV1 expression. At least two mechanisms can be proposed. The first might be a direct action of OT on TRPV1, as suggested by Nersesyan et al. The second might be that the OT-induced activation of OTR, leads to an intracellular cascade capable of modulating the expression or epigenetic modifications of TRPV1, as suggested by Sun et al. It remains unclear however, how exactly OT modulates the TRPV1 functions and whether it remains true at physiological concentrations. Indeed, while Nersesyan et al. clearly show a direct interaction between OT and TRPV1, neither them nor Sun et al. were able to reveal a strong, behaviorally relevant, OT-mediated TRPV1 modulation. Since OTR was recently involved in functional heterodimers, such as vasopressin or dopamine receptors (Terrillon et al., Finally, although the sources of OT and its spinal effects are neuronal and seem to play a role in endogenous analgesia (Eliava et al., In conclusion, a collection of evidence, briefly explained here, suggests that a triad between OT, GABA, and TRPV1 does exist and sheds a light on new mechanisms involved in OT-induced analgesia, which ultimately is much more complex than initially expected.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Rhodovulum sulfidophilum, a marine purple non-sulfur bacterium. Several types of artificial RNAs have been successfully produced in R. sulfidophilum. Purple photosynthetic bacteria produce hydrogen via nitrogenase, and genetic engineering strategies have been investigated to enhance the hydrogen production. This mini review describes the microbial production of these high-value compounds using purple photosynthetic bacteria as the host microorganism.Photosynthetic microorganisms can serve as the ideal hosts for the sustainable production of high-value compounds. Purple photosynthetic bacteria are typical anoxygenic photosynthetic microorganisms and are expected to be one of the suitable microorganisms for industrial production. Purple photosynthetic bacteria are reported to produce polyhydroxyalkanoate (PHA), extracellular nucleic acids and hydrogen gas. We characterized PHA production as a model compound in purple photosynthetic bacteria, especially focused on marine strains. PHA is a family of biopolyesters synthesized by a variety of microorganisms as carbon and energy storage materials. PHA have recently attracted attention as an alternative to conventional petroleum-based plastics. Production of extracellular nucleic acids have been studied in Cyanobacteria, algae and plants have two photosystems (photosystem I and II), extract electrons from water, and evolve oxygen as a byproduct , extracellular nucleic acids and hydrogen gas as shown in Cupriavidus necator, a hydrogen-oxidizing bacterium, is the most studied bacterium for PHA production and produced about 90% of dry cell weight (wt%) PHA through three enzyme reactions . KetothiA. vinosum, a purple sulfur bacterium, has Class III type PhaC and extensively studied its biological activity was produced by a cell free protein expression system and characterized its activity -3-hydroxybutyryl-CoA (3HB-CoA) and did not saturate, suggesting that the PhaCRs was not saturated due to low affinity for the substrate. Generally, PhaC is thought to exist as monomeric and dimeric forms in equilibrium and dimerization of PhaC induced by substrate binding facilitate the PHA polymerization affect the cell growth and PHA production in R. sulfidophilum among three types of wavelengths we studied. We found that marine purple non-sulfur bacteria strains hardly accumulated PHA (<5 wt%) under aerobic conditions in the presence of malate and pyruvate. Interestingly, the addition of acetate induced high PHA production (33 wt%) under aerobic conditions. The expression of isocitrate dehydrogenase in the tricarboxylic acid (TCA) cycle decreased under aerobic conditions in the presence of malate and pyruvate and upregulated by the addition of acetate. Considering these results, we proposed that low PHA production under aerobic conditions is caused by low activity of the TCA cycle and its activity was enhanced by the addition of acetate. We found that the expression of PdhR, which is a transcriptional repressor of the pyruvate dehydrogenase complex, was upregulated upon the addition of acetate. The changes in the metabolic state might be induced by the addition of acetate under aerobic conditions and PdhR is involved in this regulation.PHA production was examined in marine purple non-sulfur bacteria under various growth light and oxygen conditions [P(3HB-co-3HV)], has a lower melting temperature and higher biodegradability compared to P(3HB) depending on the polymer composition and 3-hydroxyvalerate (3HV). PHA composition affects the mechanical and thermal properties of PHA. Poly(3-hydroxybutyrate) [P(3HB)], homopolymer of 3HB, is a highly crystalline and brittle material. Melting temperature of P(3HB) is around 180\u00b0C Rehm, . The copE. coli that do not have PHA degradation pathway have been found in natural conditions such as freshwater, seawater, and soil and it is reported that some bacteria produced nucleic acids extracellularly are considered to be involved in the production of extracellular nucleic acids in in vitro transcription . Purple non-sulfur bacteria is known to produce hydrogen via nitrogenase (Franchi et al., A variety of kinds of large-scale photobioreactor has been investigated for industrial photohydrogen production in purple non-sulfur bacteria. Reactor design, culture light condition and nutrient sources have been investigated and are reviewed elsewhere (Eroglu and Melis, 2, N2 and sunlight energy can be used as a culture medium, biological sources and energies. Genetic tools such as synthetic promoter and transformation method have not been fully established yet in marine purple photosynthetic bacteria, even though we recently developed a transformation method using chemical competent cells of marine purple photosynthetic bacteria (Higuchi-Takeuchi et al., Marine purple photosynthetic bacteria are environmentally friendly microorganisms and can produce high-valuable compounds such as PHA, extracellular nucleic acids, and hydrogen gas. Previously, we demonstrated that marine purple photosynthetic bacteria were able to produce PHA even in seawater (Higuchi-Takeuchi et al., MH-T and KN conceptualized and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Electronic health tools are becoming increasingly popular for helping patients\u2019 self-manage chronic conditions. Little research, however, has examined the effect of patients using eHealth tools to self-report their medication management and use. Similarly, there is little evidence showing how eHealth tools might prompt patients and health care providers to make appropriate changes to medication use.The objective of this systematic review was to determine the impact of patients\u2019 use of eHealth tools on self-reporting adverse effects and symptoms that promote changes to medication use. Related secondary outcomes were also evaluated.MEDLINE, EMBASE, and CINAHL were searched from January 1, 2000, to April 25, 2018. Reference lists of relevant systematic reviews and included articles from the literature search were also screened to identify relevant studies. Title, abstract, and full-text review as well as data extraction and risk of bias assessment were performed independently by 2 reviewers. Due to high heterogeneity, results were not meta-analyzed and instead presented as a narrative synthesis.A total of 14 studies, including 13 randomized controlled trials (RCTs) and 1 open-label intervention, were included, from which 11 unique eHealth tools were identified. In addition, 14 RCTs found statistically significant increases in positive medication changes as a result of using eHealth tools, as did the single open-label study. Moreover, 8 RCTs found improvement in patient symptoms following eHealth tool use, especially in adolescent asthma patients. Furthermore, 3 RCTs showed that eHealth tools might improve patient self-efficacy and self-management of chronic disease. Little or no evidence was found to support the effectiveness of eHealth tools at improving medication recommendations and reconciliation by clinicians, medication-use behavior, health service utilization, adverse effects, quality of life, or patient satisfaction. eHealth tools with multifaceted functionalities and those allowing direct patient-provider communication may be more effective at improving patient self-management and self-efficacy.Evidence suggests that the use of eHealth tools may improve patient symptoms and lead to medication changes. Patients generally found eHealth tools useful in improving communication with health care providers. Moreover, health-related outcomes among frequent eHealth tool users improved in comparison with individuals who did not use eHealth tools frequently. Implementation issues such as poor patient engagement and poor clinician workflow integration were identified. More high-quality research is needed to explore how eHealth tools can be used to effectively manage use of medications to improve medication management and patient outcomes. Use of the internet has increased considerably since the early 1990s. The World Bank reports that almost 44% of people across the globe used the internet in 2015, compared with 0.25% in 1993 , while aWell-functioning eHealth tools can help patients better understand their health and may The ability of patients to use eHealth tools to better manage medication by reporting feedback on symptoms and use of medications directly to health care providers has not been comprehensively explored in the literature. Similarly, there is little evidence showing how eHealth tools might provide prompts to patients and health care providers to make appropriate changes to medication use based on this feedback. A synthesis of this literature will provide greater understanding of what eHealth tool design features may be helpful in patient reporting of medication-related experiences and outcomes.The objective of this systematic review was to determine the impact of patients\u2019 use of eHealth tools on self-reporting adverse effects and symptoms that promote changes to medication use. The PICO model was used to focus the objective of the review, as seen in This systematic review was performed following steps outlined by Cochrane\u2019s Effective Practice and Organization of Care group and reported based on the Preferred Reporting Items for Systematic Reviews and Meta-Analyses statement . A totalFor the purposes of this review, an eHealth tool was considered to be any internet-based intervention, including mobile health apps, used by patients for clinical purposes that focused on improving patient health and clinical outcomes. The term PHR refers to an eHealth tool wherein a patient has access to and can enter or edit their own health data. The population investigated was community-dwelling individuals of any age in an outpatient setting.For a study to be included, the eHealth tool must have allowed patients (or caregivers) to enter information directly ; included self-reporting functionalities focusing on medication monitoring, contain a medication monitoring or use component, or specifically incorporating the option for the patient or caregiver to enter symptoms including adverse effects; and needed to focus specifically on medication use, clinical outcomes, or symptom reporting following use of the eHealth tool. Any eHealth tools involving changes in medication reconciliation and recommendations made to changes in drug therapy were also included.Exclusion criteria were conference abstracts; qualitative studies; articles without a comparator group; articles that did not report on at least one medication-related outcome; articles where self-management strategies focused on lifestyle modification, behavioral interventions, or nondrug interventions; articles focused solely on the validation of an eHealth tool; articles focused on methodological or technical aspects of eHealth interventions; articles containing nonempirical information; articles that synthesized information about multiple eHealth tools in an article ; and eHealth tools used by regulatory agencies to report adverse drug events (ADEs).All potentially relevant articles were uploaded into DistillerSR software, which was used throughout the selection process. Potentially relevant articles underwent title, abstract, and full-text review. Articles that met inclusion criteria proceeded to data abstraction and risk of bias assessment. Articles not meeting inclusion criteria were excluded at both levels. Title and abstract review were performed independently by 2 reviewers from a pool of 5 reviewers. Of these, 1 reviewer went through the reference lists of all the articles included in this study. Another reviewer went through reference lists of relevant systematic reviews identified during the literature search. Potentially relevant articles were identified. These articles went through abstract review by 2 reviewers. Studies found not to fit inclusion criteria after abstract review were excluded. Full-text review was performed independently by 5 reviewers. The kappa scores were calculated to determine agreement among reviewers who conducted review of titles and abstracts. All kappa scores calculated were greater than .93. Conflicts were resolved by consensus.Data extraction and risk of bias assessment were performed for each study independently by 2 reviewers. Data extracted included study design and setting, participant demographics, number of participants in each group, intervention components, comparator group components, eHealth tool functionality measured, and results and significance levels for each outcome measure. Conflicts in data extraction were resolved by consensus. Risk of bias assessment used questions recommended by the Agency for Healthcare Research and Quality\u2019s 2014 publication Methods Guide for Effectiveness and Comparative Effectiveness Reviews and was The primary and secondary outcomes are listed in Subgroup analyses were performed to investigate differences in treatment effect present because of (1) age of participants, (2) patients with specific conditions targeted by intervention, and (3) different features and functionalities of the included eHealth tools.A total of 3515 articles were generated from database and reference searching, resulting in 2489 potential articles that were screened based on their titles and abstracts, after duplicates were removed. Furthermore, 75 full-text articles were assessed for eligibility, of which 14 were included in this systematic review -31 and 1Of the 13 RCTs included in this review, 2 studies were cluster RCTs ,26. The A total of 4 RCTs focused on pediatric and adolescent asthma patients ,23-25; 1double-dummy style intervention, where both groups used Web-based PHRs to record information that differed only in content. Cho et al [In 3 studies, use of an eHealth tool in the intervention group was compared with usual care plus links to relevant websites ,25,28. Sho et al comparedho et al utilizedho et al used a sho et al developeho et al used a mho et al . Carlsenho et al used an From the 14 included studies, 11 unique eHealth tools were described. The 2 RCTs by Joseph et al ,25 utiliAll 11 eHealth tools from all 14 studies included a component where patients could self-report medication management information or changes, including symptoms, health data, adverse effects, or ADEs. A total of 12 studies included Web-based patient questionnaires or surveys ,21,23-32The results of each study by outcome can be seen in A total of 6 RCTs -22,24,27P=.035). A nonsignificant trend approaching significance was seen for decreased number of over-the-counter medications used in the eHealth tool group (P=.05) [P<.001). They also found that a significantly higher proportion of patients in the intervention group had medications initiated or dosages changed for hypertension and hyperlipidemia .Chrischilles et al found a (P=.05) . Grant e (P=.05) found a P=.01). Simon et al [21=10.5, P=.001). Carlsen et al [P<.001) [Joseph et al found thon et al found then et al showed ten et al showed t[P<.001) .P=.36). Fiks et al [In contrast, Cho et al found noks et al provided1c) [A total of 9 RCTs ,27,30,311c) , and 2 fP=.001) and that the active group had less frequent flare-ups . Gustafson et al [P=.01).Fiks et al reportedon et al found anP=.009), symptom days , days of restricted activity , and school days missed . In another study, Joseph et al [P=.01). Following subgroup analysis, it was found that teenagers with moderate to severe asthma had fewer symptom days , total school days missed , school days missed because of asthma , and days of restricted activity .Joseph et al found thph et al reportedP=.04). Ahmed et al [1c ; however, they did find a statistically significant decrease in waist circumference between intervention and control . Cho et al [1c in the active group at 30 months. Grant et al [1c and for percentage of patients at target HbA1c levels. Similarly, Carlsen et al [P=.18).Simon et al reporteded et al reporteded et al found noho et al did findnt et al found noen et al found noA total of 5 RCTs measured this outcome ,23,26,30P=.01). They also found a positive significant effect of intervention on ACQ score when mediated by information competence . Schnipper et al [P<.001). Ahmed et al [Gustafson et al found ther et al found thed et al found thed et al found thed et al found noA total of 3 RCTs measured this outcome, all using measures of medication adherence as a surrogate for medication use behavior ,23,24. NP=.01). In addition, Schnipper et al [P=.04). The number of medication discrepancies per patient with the potential for harm approached significance as a result of using eHealth tools (P=.05) [A total of 3 RCTs reported on this outcome ,20,26. OCho et al acknowleP value: .02 to <.001) relative to usual care. Descriptive evidence from Fiks et al [A total of 1 open-label intervention and 5 RCks et al showed 1ks et al providedks et al or ADEs ks et al ,28 foundP=.01). The remaining 5 studies [A total of 6 RCTs reported health service utilization outcomes ,27,28,30 studies ,27,28,30A total of 4 studies measured this outcome ,30-32, a21=8.38, P=.004). Fiks et al [A total of 2 RCTs ,27 and 1ks et al providedA total of 4 RCTs investigated the use of eHealth tools in children and teens with asthma ,23-25. TSubgroup analysis also found that multifaceted interventions combining use of eHealth tools with clinician support or case management and eHealth tools utilizing direct patient-provider communication might be more effective at improving some aspects of patient self-management and self-efficacy ,26,30,31Many studies reported barriers to the implementation of eHealth tools. The most common barrier was lack of participant engagement, resulting in low eHealth tool utilization rates. This was reported by 9 of the 14 studies -27,31,32Evidence from 4 RCTs and 1 open-label intervention ,24,27,32Evidence was found that eHealth tools improved the outcome of patient self-management and self-efficacy. Subgroup analysis found that eHealth tools that allow patients and clinicians to communicate directly, and multifaceted interventions combining eHealth tools with clinician support and case management might lead to greater increases in patient self-management and self-efficacy. It is notable that more significant improvements were found for more objective outcome measures, such as number of medication changes and clinical signs, and less were found for more subjective outcome measures such as self-management and self-efficacy. It may also be that sample sizes were too small to detect differences, particularly if this was not the primary objective for these studies. It is likely that the eHealth tools under investigation either did not provide effective content or functionalities to help participants improve self-management and medication management in participants or the tools used to measure these outcomes were not able to detect any differences between groups. Another possibility is the lack of patient understanding of chronic disease and poor perception of health goals.It was thought that eHealth tools that focus on improvement of patient self-efficacy and self-management might lead to improved medication-use behavior, which in turn may lead to changes in medication use, identification of real or potential ADEs, improvement in signs and symptoms, and overall improvement in HRQoL. However, there is not enough evidence to draw conclusions as to the effectiveness of eHealth tools for identification of adverse effects, improving medication-use behavior, increasing recommendations to medication therapies and improving medication reconciliation, improving health service utilization, and improving overall health status and patient satisfaction. Only a small number of included studies investigated these outcomes; it is likely that with such a small overall sample size, it was not possible to find differences between groups.As with most systematic reviews on the subject of eHealth tools ,33,54-57A 2012 systematic review by Ammenwerth et al found that use of patient portals linked to a PHR led to significant increases in medication adjustments in diabetic patients . Other rOverall, this review supports findings by Ammenwerth et al that intTo our knowledge, this is the first systematic review of eHealth interventions focusing on patient self-reporting of symptoms and adverse effects. The review\u2019s search strategy was augmented by reference searching. This review was limited to studies that included medication-related outcomes. The majority of studies included in this review were RCTs, most of which were of moderate quality.eHealth and believe future research should focus on establishing better consensus for this term. There was considerable variety among the interventions in the studies, some of which included features such as direct health care provider follow-up, thus making it more complicated to determine which outcomes could be specifically attributable to using an eHealth tool. As this review also examined different populations of varying sample sizes and medical conditions for eHealth tools, it may be difficult to detect differences and generalize findings and conclusions. We did not include qualitative studies in our review because the goal of our study was to better understand the effectiveness and impact of changes to medication regimens based on quantifiable differences in using eHealth tools for self-reporting adverse effects and symptoms that promote changes to medication use versus a comparator. We value the insight of qualitative studies that have been investigated elsewhere [This review also has a number of weaknesses. It was limited to studies published in English, which may have excluded relevant articles. No searching of grey literature was performed, as we focused on empirical work published in academic journals, and so it is possible that some early non\u2013peer-reviewed reports may have been missed. We acknowledge the lack of definitional clarity surrounding the term lsewhere ,60. AddiWhere possible, health care providers should encourage patient use of eHealth tools for symptom and adverse effect self-reporting. eHealth tools may be especially useful for reducing symptoms in certain populations, for example, children and teenagers with asthma. eHealth tools might also encourage patients to improve self-management behaviors and participate in shared decision making with clinicians. Having information from the EMR entered directly into the eHealth tool may reduce the burden on the patient to routinely update their clinical information (something that only highly motivated patients are likely to do regularly) . CliniciThere is a paucity of primary research articles investigating eHealth tools and their impact on medication use. Studies are generally small and of moderate quality. Large-scale RCTs focusing on the use of eHealth tools for medication and symptom management should be undertaken to establish more high-quality evidence. This is especially important given how ubiquitous the use of medication is. Furthermore, the effects of patient self-management and self-efficacy on medication use and symptom experience are not well studied; more research in this area could help drive creation of medication-focused eHealth tools. Low patient engagement and eHealth tool utilization were commonly noted implementation barriers; it could be that patients were not engaged in eHealth tool use enough for them to feel an impact on their satisfaction with health care or overall quality of life. Descriptive evidence shows low proportions of patients felt that eHealth tools improved their care or communication with providers, indicating that development of eHealth tools should focus on functionalities and outcomes that are important to the patient. This may be achieved by utilizing research on patient motivation and behavior change to increase patient engagement ,24,25.The results of this review show initial and promising findings that specialized eHealth tools can be used for reporting and monitoring of symptoms and medication-related adverse effects and some evidence that use of eHealth tools have the potential to identify instances where changes in medication use may be appropriate. A modest amount of mixed evidence was found, demonstrating that eHealth tools can improve patient self-management and self-efficacy. Very little or no evidence was found to demonstrate that use of eHealth tools could increase numbers of medication recommendations or improve medication-taking behavior, health services utilization, identification of adverse effects, overall health status, and patient satisfaction. eHealth tools may be more effective at promoting medication changes and improving patient self-management and self-efficacy if they provide mechanisms for direct patient-provider communication and may be more effective in certain populations such as children and teenagers with asthma."} +{"text": "Recently Christian Hudert and colleagues from the Charit\u00e9 in Berlin published a study about genetic determinants in pediatric non-alcoholic liver disease . Non-alDue to the current increase in the incidence of liver diseases a better understanding of their pathophysiology is of major importance (Jansen et al., 2017; Vartak"} +{"text": "Sci., 2019, DOI: ; 10.1039/c8sc05721d.Correction for \u2018Facile N-functionalization and strong magnetic communication in a diuranium( The authors regret that The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "Alzheimer's disease (AD), the most common type of dementia among the elderly, is caused by progressive neural death that results in impaired memory, thinking skills and, eventually, the ability to carry out simple tasks. Unfortunately, approximately 5% of people over the age of 65 suffer from AD, and the prevalence of AD increases with aging (Udeochu et al., Investigators have proposed that the onset and progression of AD is contributed by pathogenic microbes although definitive evidence has not been presented (Sjogren et al., Based on unbiased approaches and large-scale data sets from several brain banks and cohort studies, this is the first study to provide strong evidence supporting the controversial hypothesis that viruses play an essential role in regulatory genetic networks that are believed to lead to AD. Identifying links to viruses may help scientists interested in developing potential new treatment strategies.Several important questions are raised from this phenomenal study. First, does herpes virus cause the onset or progression of AD? Eimer and colleagues recently reported that A\u03b2 traps herpes viruses in insoluble amyloid, and active herpes infections in brain accelerate amyloid deposition, indicating that herpes infection may promote AD pathology directly via amyloid-mediated pathological pathways (Eimer et al., Second, whether herpesviruses regulate, or are regulated by AD-associated genes? Will anti-herpes drugs be effective against early onset of AD? This study established a strong connection between multiple viruses, especially HHV-6A, and AD risk genes, including PSEN1, BACE1, and APBB2 which are implicated in regulation of A\u03b2 production. Besides, several recent studies show that A\u03b2 is an antimicrobial protein of the body's innate immune system, capable of providing immediate, effective protection from infection with pathogens like herpes viruses in both cultured human brain cells and animal models of AD (Kumar et al., Third, is miR-155 a regulator of anti- or pro-viral activity in early AD? Could miR-155 gain-of-function help with A\u03b2 clearance and neuroprotection in AD? MiR-155 has been reported as an important regulator of T cell and microglia in response to neurodegeneration (Song and Lee, Lastly, there remain some open issues that future studies will need to address about the Readhead et al. findings. For example, HHV-6A/B and HHV-7 are considered lymphotropic rather than neurotrophic (Berneman et al., X-WS drafted an initial version of this commentary. C-ML and Z-QT revised and finalized the text. All authors approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} diff --git a/PMC_clustering_418.jsonl b/PMC_clustering_418.jsonl new file mode 100644 index 0000000000000000000000000000000000000000..493a303570d52d29abb5047ba9e0cac52c3b778b --- /dev/null +++ b/PMC_clustering_418.jsonl @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:8d727d70dad56fb7418918105490e289dd48e558da9cb401d265f1d06d38a62a +size 55396937 diff --git a/PMC_clustering_419.jsonl b/PMC_clustering_419.jsonl new file mode 100644 index 0000000000000000000000000000000000000000..621823cf81f2b2602965e8f6d81b6535061c0260 --- /dev/null +++ b/PMC_clustering_419.jsonl @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:81249551df756ec97e87c3f6154397c359dce19db72b6338abf27b9543d81cb3 +size 26377334 diff --git a/PMC_clustering_420.jsonl b/PMC_clustering_420.jsonl new file mode 100644 index 0000000000000000000000000000000000000000..a2d3ad4aec7ff91ac574de7c6c911758c105bd50 --- /dev/null +++ b/PMC_clustering_420.jsonl @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:6e213adb0379dd77bc9cd3b1ee3414f5a49821308d4330cea623d860595700f1 +size 90895727 diff --git a/PMC_clustering_421.jsonl b/PMC_clustering_421.jsonl new file mode 100644 index 0000000000000000000000000000000000000000..60377522b5d8750b2d9b60e9f91ea6fe0d36e1f1 --- /dev/null +++ b/PMC_clustering_421.jsonl @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:0c50d1cbd77c6860cc4a35f05c5e7084e617d14aeea712b9a929ab68930076ed +size 15583004 diff --git a/PMC_clustering_422.jsonl b/PMC_clustering_422.jsonl new file mode 100644 index 0000000000000000000000000000000000000000..82e2199a29cac8876baacfd2927815192982645a --- /dev/null +++ b/PMC_clustering_422.jsonl @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:0feb0068b772590525dc87e9cfb50df9b13d42e1606a4ed96adf4e0de12cddde +size 52503191 diff --git a/PMC_clustering_423.jsonl b/PMC_clustering_423.jsonl new file mode 100644 index 0000000000000000000000000000000000000000..cf5cbb45d7db8b65bd950c6afa1fdd5268b2aa08 --- /dev/null +++ b/PMC_clustering_423.jsonl @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:34a4c19d4d2c581ca4b886c34a047753f50da580347316667672bc7c02836e9f +size 44665666 diff --git a/PMC_clustering_424.jsonl b/PMC_clustering_424.jsonl new file mode 100644 index 0000000000000000000000000000000000000000..f61c15947b11a7ba568fc849a5e6791cd613157c --- /dev/null +++ b/PMC_clustering_424.jsonl @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:d07d163e5c85210cb817d9a3a9d7bef43fa780a03f3fe2d836875e7f12b24135 +size 88900503 diff --git a/PMC_clustering_425.jsonl b/PMC_clustering_425.jsonl new file mode 100644 index 0000000000000000000000000000000000000000..5aefbc052af685b27078779a2e8958e20a7fc6e5 --- /dev/null +++ b/PMC_clustering_425.jsonl @@ -0,0 +1,604 @@ +{"text": "This article was republished on August 13, 2020, to correct errors in the figures that were introduced during the typesetting process. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on July 10, 2020, to correct an error in the placement of the tables and figures: they were erroneously placed ahead of its first in-text citation during the typesetting process. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "There is an error in the Funding statement. The correct numbers for National Institutes of Health are R01AI112619, R01AI133976, R01AI100989, R21AI125907, and T32AI007509.The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated."} +{"text": "An incomplete, earlier version of this article was published in error. The Conclusion section was erroneously omitted at the end of the article. The publisher apologizes for the error. This article was republished on March 8, 2021, to correct for this error. Please download the article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on May 22, 2020, to correct errors in the byline that were introduced during the typesetting process. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "An incorrect version of Figs 1 and 5 was published in error. The publisher apologizes for this error. This article was republished on Jun 26, 2020, to correct for this error. Please download this article again to view the correct version.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "February 5, 2021, to correct the erroneous inclusion of figure text in the Discussion section. This error was introduced during the typesetting process. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.This article was republished on S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on November 5, 2020, to correct errors throughout the article that were introduced during the typesetting process. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on August 12, 2020 to replace a corrupted supporting information file. No changes were made to the rest of the article. The publisher apologizes for the error. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "An incomplete, earlier version of this article was published in error. The publisher apologizes for the error. This article was republished on February 19, 2020 to correct for this error. Please download the article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on January 14, 2021, to replace an incorrect Fig 4 image included in the article PDF file. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "The affiliation for the 1st author is incorrect. Bing Min Tsai is affiliated with both #1 and #5. Affiliation #5 is: Institute of Emergency and Critical Care Medicine, National Yang Ming University, Taipei City, Taiwan."} +{"text": "This article was republished on February 26, 2020, to correct errors introduced during the typesetting process in the captions for Figs 1 and 6 as well as in the Results, Discussion, and Materials and Methods sections. The publisher apologizes for these errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "The correct affiliation is not indicated. Tie-jian Feng is not affiliated with #1 but with: Shenzhen Center for Disease Control and Prevention, Shenzhen, China.The affiliation for the 12th author is incorrect. Min Zhang is not affiliated with #1 but with #2: Shenzhen Nanshan Maternity & Child Healthcare Hospital, Shenzhen, China.The affiliation for the 13"} +{"text": "This article was republished on July 16, 2020, to correct errors throughout the article that were introduced during the typesetting process. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on April 2, 2020, to correct errors in Fig 1 that were introduced during the typesetting process. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on January 30, 2020, to remove an erroneous sentence from the Introduction and to correct the Academic Editor\u2019s affiliation. The publisher apologizes for these errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on December 11, 2020, to correct errors in the Qualitative interviews subsection of the Results section that were introduced during the typesetting process. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "Scott, was originally published published Online First without Open Access. After publication online Allergan Sales, LLC requested that the article be Open Choice to make the article an open access publication. Post-publication open access was funded by Allergan Sales, LLC. The article is forthwith distributed under the terms of the Creative Commons Attribution 4.0 International License (The original article has been corrected."} +{"text": "This article was republished on July 20, 2020, to correct Fig 2. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on August 28, 2020, to correct an error in the greater-than or equal to, and less than or equal to symbols. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "An incomplete, earlier version of this article was published in error. The publisher apologizes for the error. This article was republished on January 24, 2020, to correct for this error. Please download the article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on November 19, 2020, to correct errors in the tables that were introduced during the typesetting process. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on May 8, 2020, to correct an error in the heading levels in the Findings subsection of the Results. Please download the article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "The following information is missing from the Funding statement: This study was supported by NIH grant P50 AI150481.The full funding statement should read:https://www.nih.gov/) grants R21 AI150384, R56 AI076121, and P50 AI150481 to CA. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.This study was funded by National Institutes of Health ("} +{"text": "This article was republished on August 3, 2020 to correct an error in the title. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "PLOS ONE\u2019s Editorial Board confirmed that the updated figure supports the results as reported in the original article.In ftshi4 mutant , and the details of those antibodies were described in a previous study [Except for the image data provided here in support of The authors apologize for the error in the published article.As of the date of this notice, the corresponding author (Chunyi Zhang) is affiliated with the Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing 100081, P. R. China.S1 File(TIF)Click here for additional data file.S2 File(TIF)Click here for additional data file.S3 File(TIF)Click here for additional data file.S4 File(TIF)Click here for additional data file.S5 File(TIF)Click here for additional data file.S6 File(TIF)Click here for additional data file."} +{"text": "Cancer Medicine, published online on 5 October 2016 on Wiley Online Library (wileyonlinelibrary.com) and in Volume 5, pp. 3023\u20133030, has been retracted by agreement between the Authors, the journal Editor\u2010in\u2010Chief and John Wiley & Sons Ltd. The retraction has been agreed due to major overlap with a previously published article.The above article from"} +{"text": "Scientific Reports 10.1038/srep24811, published online 04 May 2016Correction to: The Supplementary Information file is missing from this Article and can be found below.Supplementary Information"} +{"text": "This article was republished on February 12, 2020, to correct errors in the Introduction and author byline. The publisher apologizes for these errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on April 17, 2020, to correct an error in the Data Availability statement. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on January 24, 2020, to correct errors that were introduced during the typesetting process. The publisher apologizes for this error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on March 12, 2020, to correct formatting errors introduced during the typesetting process. The publisher apologizes for these errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on November 16, 2020, to correct an error in the title that was introduced during the typesetting process. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.This article was republished on September 24S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on August 14, 2020, to correct an error in the title. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on February 26, 2020, to correct errors in the order of the figures and in Table 7. The publisher apologizes for this error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on February 19, 2020, to correct errors in Fig 1. The publisher apologizes for this error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on August 21, 2023, to correct capitalization in the title. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on July 20, 2023, to replace the incorrect editor (\u201cR&R PLOS\u201d) with the correct editor (Collins Otieno Asweto). The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on August 30, 2023, to correct missing content from Table 2. The publisher apologizes for the error.In addition, the republished version of the article reflects the addition of an author, who was inadvertently excluded from the original publication. Corrections were also made to the author contributions.Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on May 10, 2023, to include an author who was inadvertently omitted from the byline in the final stages before publication. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.[S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on December 7, 2022 to replace the incorrect editor (\u201cPLOS Staff Editor\u201d) with the correct editor (Julia Robinson). The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on August 30, 2023, to correct the copyright statement to reflect the WHO-affiliated copyright. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "The affiliations for the second and eleventh authors are incorrect. Sameh Awwad is not affiliated with #2 and Mohammed AlMotairi not affiliated with #8. Both authors are affiliated with #1: Pharmaceutical Service Department, Main Hospital, King Fahad Medical City, Riyadh, Saudi Arabia."} +{"text": "This article was republished on March 10, 2023, to address figure errors. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on August 8, 2023, to correct an author\u2019s name. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on November 22, 2023, to correct the error in the affiliation that was introduced during the typesetting process. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.ReferenceS1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on April 17, 2023, to add Bryan Lanning as the third author. The Funding Statement, About the Authors, and citation details were updated accordingly. Please download this article again to view the correct version.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on August 30, 2023, to correct the affiliation of the editor, which incorrectly was typeset as the authors\u2019. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on December 19, 2022 to replace the incorrect editor (\u201cPLOS Manuscript Reassignment\u201d) with the correct editor (Julia Robinson). The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on December 19, 2022 to replace the incorrect editor (\u201cPLOS Editor Invitation\u201d) with the correct editor (Collins Otieno Asweto). The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "EquatioA correction has been made to Section 2. Methods, Section 2.4. Optical Parameter Model, Equation (1). The correct equation is:The authors state that the scientific conclusions are unaffected. This correction was approved by the Academic Editor. The original publication has also been updated."} +{"text": "This article was republished on December 19, 2022 to replace the incorrect editor (\u201cPLOS Manuscript Reassignment\u201d) with the correct editor (Julia Robinson). The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on December 7, 2023, to correct errors in the figures that were introduced during the typesetting process. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on July 31, 2023, to correct for errors in the Data Availability statement. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on October 16, 2023, to correct errors in formatting that were introduced during the typesetting process. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on October 24, 2023, to correct the error in the title that was introduced during the typesetting process. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "The About the Authors section and citation details were updated accordingly. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.This article was republished on September 12RD and JPA stand by the original author list.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "Journal of Public Health, Volume 44, Issue Supplement_1, November 2022, Pages i3\u2013i7, https://doi.org/10.1093/pubmed/fdac110This is a correction to: Public health professionals delivering better health for all\u201450 years of the Faculty of Public Health, In the originally published version of this manuscript the author Farhang Tahzib was inadvertently omitted from the author byline. The publisher apologizes to the author for this omission.This error has been corrected online."} +{"text": "This article was republished on September 15, 2023, to correct an error in the fourth sentence of the Correction notice. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "The fifth author\u2019s name was spelled incorrectly. This article was republished on June 01, 2023 to correct for this error. Please download this article again to view the correct version.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on December 7, 2022 to replace the incorrect editor (\u201cPLOS Manuscript Reassignment\u201d) with the correct editor (Prabhdeep Kaur). The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on September 12, 2023 to add Yushuf Sharker as the eighth author. The author contributions and Acknowledgments in the original publication did not correctly indicate Yushuf Sharker\u2019s contributions. The About the Authors section was updated accordingly. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on May 17, 2022, to correct the XML version of the article which displayed an incorrect notation beside two authors names in the byline. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "After publication of this article , concernIn In Regarding Regarding The corresponding author additionally stated that the AKT panel in In addition, the primary data underlying results in this article were not included with the published article, although the Data Availability Statement for this article declared that all relevant data are within the paper.With this Correction, the authors provide corrected panels for Figs The authors apologize for the errors in the published article.S1 File(XLSX)Click here for additional data file.S2 File(PPTX)Click here for additional data file.S3 File(XLSX)Click here for additional data file.S4 File(TIF)Click here for additional data file."} +{"text": "After this article was publThe authors apologize for the error(s) in the published article.erin.lloyd@uwa.edu.au.During follow-up, the authors informed the journal that corresponding author GJP is deceased. At the time of publication of this notice, the corresponding author for this article is updatS1 File(JPG)Click here for additional data file.S2 File(JPG)Click here for additional data file."} +{"text": "This article was republished on April 24, 2023, to correct an author\u2019s name which was misspelled. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on July 31, 2023, to correct for errors in the Data Availability statement. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on December 7, 2022 to replace the incorrect editor (\u201cPLOS Manuscript Reassignment\u201d) with the correct editor (Julia Robinson). The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on July 12, 2023, to correct errors in the title. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on December 19, 2022 to replace the incorrect editor (\u201cPLOS Manuscript Reassignment\u201d) with the correct editor (Stephen J. Kerr). The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "Notice of RepublicationThere were numerous errors throughout the article introduced during production, regarding the linguistic symbols. The article was republished on March 17, 2014. Please download the article again to view the correct version. The publisher apologizes for these errors.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on March 21, 2014, to replace an incorrectly published version. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on February 28, 2014, to replace an incorrectly published version. The new version includes the figures in the correct order, addressing all issues noted in the previous Formal Correction. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected articleClick here for additional data file.File S2Republished, corrected articleClick here for additional data file."} +{"text": "The affiliation for the tenth author was incorrect. Yong Zhang is not affiliated with #2 but with #1 The Shenzhen Proteome Engineering Laboratory, BGI Shenzhen, Shenzhen, P. R. China. The PDF version of the article is correct."} +{"text": "This article was republished on March 12, 2014, due to the omission of Equation 1. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "Notice of RepublicationThis article was republished on March 7th, 2014, due to the figures missing in the PDF version of the previously published article. Please download the PDF again to view the correct figures. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article(PDF)Click here for additional data file.File S2Republished, corrected article(PDF)Click here for additional data file."} +{"text": "Notice of RepublicationThis article was republished on March 17, 2014, to replace an incorrectly published version. In the original article, the signs did not appear correctly throughout the text. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished corrected article.(PDF)Click here for additional data file."} +{"text": "The affiliation for the last author was incorrect. Flaminio Squazzoni is affiliated with: Department of Economics and Management, University of Brescia, Brescia, Italy.In Table 7 and Table 8, the Greek letters indicating the parameters are missing.Please see the corrected Table 7 here: Please see the corrected Table 8 here: Figure 2 has been reformatted for better readability. Please see Figure 2 here:"} +{"text": "Institutional affiliation 1 for authors Ximing Chen and Jianhua Xiao is incorrect. The correct affiliation is: 1. Medical College of University of South China\uff0c Hengyang, China. In addition, the Corresponding Author for the article was incorrectly listed as Ximing Chen. The Corresponding Author is Jianhua Xiao, who can be reached at JianhuaXia2010@163.com"} +{"text": "This article was republished on February 10, 2014, to replace an incorrectly published version. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article(PDF)Click here for additional data file.File S2Republished corrected article(PDF)Click here for additional data file."} +{"text": "This article was republished on March 13 2014, to include the supplementary information that were corrupt in the original version. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on March 24, 2014 to correct an error in the formatting of Figure 4. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.Click here for additional data file.File S2Republished corrected article.Click here for additional data file."} +{"text": "The email address of the Corresponding Author is incorrect. The correct email address is: alexandergamisch@gmx.at.In addition, there was an error in Figure 2B. A corrected version of Figure 2B can be viewed here:"} +{"text": "This article was republished on February 21, 2014 to replace an incorrectly published version. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished corrected article.(PDF)Click here for additional data file."} +{"text": "Pavkov. The changes were made to our website on February 5, 2013, and appear online at"} +{"text": "This article was republished on March 17, 2014, because of errors that prevented Figure 2 from opening properly. The publisher apologizes for these errors. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.Click here for additional data file.File S2Republished corrected article.Click here for additional data file."} +{"text": "This article was republished on March 19, 2014, to replace an incorrectly published version. The publisher apologizes for these errors.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on February 28, 2014, to replace an incorrectly published version. The new version includes all figures in the correct order and addresses the figure issues noted in the previous Formal Correction. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "Notice of RepublicationThis article was republished on February 7, 2014, to update the figure legends for Figures 2-4, which were incorrectly labeled in the original version of the article. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.Click here for additional data file.File S2Republished corrected article.Click here for additional data file."} +{"text": "Supporting Table S1, Bacterial Strains and Plasmids, does not appear. Please view Table S1 here: Click here for additional data file.The legend for Figure 9 is: Bacterial strains and plasmids used in this work."} +{"text": "This article was republished on March 14, 2014, to replace Tables 1 and 2, which were originally published incorrectly. The publisher apologizes for these errors. Please download this article again to view the correct version."} +{"text": "Notice of RepublicationThis paper was republished on February 5, 2014, due to italicization being lost during the production process. The publisher apologizes for the mistake. Please download the paper again to view the corrected version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article(PDF)Click here for additional data file.File S2Republished, corrected article(PDF)Click here for additional data file."} +{"text": "This article was republished on March 27, 2014, to correct spacing errors that were introduced during the production process. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.Click here for additional data file.File S2Republished, corrected article.Click here for additional data file."} +{"text": "Some information is missing from the Funding statement. The following should be appended to the end of the first sentence:\"and EUCLIDS (Grant number: FP7 GA no. 279185). Work in SML\u2019s laboratory is funded by the Oxford Martin School.\"Also, Supplementary Tables S3, S4 and S5 are formatted incorrectly, with a page missing for each. The correct files can be found below. Please view Table S3 here:Click here for additional data file.Please view Table S4 here:Click here for additional data file.Please view Table S5 here:Click here for additional data file."} +{"text": "This article was republished on March 27, 2014, to correct the figure order. Please download the article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on March 20, 2014, to correct the ordering of figures. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on January 27, 2014, due to missing symbols in the text. Please download the article again to view the correct version. The originally published, uncorrected article is provided here as File S1 for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file."} +{"text": "Notice of RepublicationThis article was republished on March 11, 2014, due to incorrect figure order. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on March 18, 2014, to replace an incorrectly published version. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished corrected article.(PDF)Click here for additional data file."} +{"text": "Notice of RepublicationThis article was republished on March 7, 2014 to correct for formatting issues in Table 1. The publisher apologizes for these errors. Please download this article again for the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2 Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on March 20, 2014, to replace Figures 2-6 and Table 1, which were originally published incorrectly. This republication also resolves the initial correction published on November 8, 2013. The publisher apologizes for these errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished corrected article.(PDF)Click here for additional data file."} +{"text": "Notice of RepublicationThis article was republished April 3, 2014, to correct numerous spacing errors throughout the text that were introduced during the typesetting process. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference. The publisher apologizes for this error.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on March 17, 2014, to replace an incorrectly published version. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on July 28th, 2014, to replace an erroneously published version. The publisher apologizes for this error. Please download this article again to view the corrected article. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article(PDF)Click here for additional data file.File S2Republished, corrected article(PDF)Click here for additional data file."} +{"text": "This article was republished on April 10, 2015, to correct Figs. 5, which were distorted during the production process. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on July 7, 2014, to correct an error in the byline; the publisher apologizes for the error. In addition, Figures 1-6 were resized. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on May 19, 2014, to correct errors in equations that were introduced during the typesetting process. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference. The publisher apologizes for these errors.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on August 20, 2015, to correct a paragraph of the Results section, which was mistakenly incorporated into the legend of Table 1. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on April 6, 2015, due to the text of the original submission being published, rather than the text of the revised manuscript. The incorrect submission file was uploaded during the production process. Please download this article again to view the correct, revised version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 Original Article(PDF)Click here for additional data file.S2 Revised Article(PDF)Click here for additional data file."} +{"text": "This article was republished on September 24, 2014, due to Table S1 being erroneously removed. The publisher apologizes for this error. Please view this article again for the Supporting Information file. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article(PDF)Click here for additional data file.File S2Republished, corrected article(PDF)Click here for additional data file."} +{"text": "This article was republished on July 22, 2015, to correct an error in the title introduced during the typesetting process. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on June 22, 2015, to correct Figs 1 and 2, which were darker than intended. The publisher apologizes for the error. Please download this article again to view the corrected version."} +{"text": "This article was republished on July 2, 2014, to correct a typographical error in the title that was introduced during the publication process. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article(PDF)Click here for additional data file.File S2Republished, corrected article(PDF)Click here for additional data file."} +{"text": "This article was republished on February 11, 2016, to correct figure readability errors in the PDF that were introduced during the typesetting process. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on June 27, 2014, to correct an error in the citation. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on May 16, 2014, to include missing formulas. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on September 29, 2015 to correct errors in the order of figures that were introduced during the production process. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on July 30, 2014, to correct the placement of the figures which were mistakenly published as Supporting Information. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on April 13, 2016, to correct errors in the figures in the PDF that were introduced during the typesetting process. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.This article was republished on September 12File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on May 5, 2015, to correct errors throughout the paper that were introduced during the typesetting process. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference. This republication has addressed the issues in the posted correction.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on May 29, 2014, due to figure errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on May 5, 2014, to correct the images and legends of Figures 5 and 6, and hyperlinked text in the Materials and Methods. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on June 18, 2014, due to the figures missing in the PDF version of the previously published article. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on May 23, 2014, to replace an incorrectly published version in which there were symbol errors throughout the PDF and XML of the article. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on April 3, 2014, to include an image which was missing from Figure 1. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on May 26, 2015, to include details of the editor who handled the submission, which had been omitted due to an error in production. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on November 21st, 2014, due to multiple figures being posted incorrectly. The publisher apologizes for this error. Please download this article again to view the corrected version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article(PDF)Click here for additional data file.File S2Republished, corrected article(PDF)Click here for additional data file."} +{"text": "This article was republished on September 15, 2015, to replace the name of the 21st author, Hagos Godefay, which was missing from the byline. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on October 14, 2014, to correct errors that were introduced during the typesetting process. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on April 21, 2014, to correct errors in number symbols that were introduced during the typesetting process. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on August 12, 2015, to correct typographical errors throughout the text, in the Abstract, and in Table 2 that were introduced during the typesetting process. The errors consisted of un-subscripted characters and omitted Greek symbols. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on June 16, 2014, due to an error in the title. The publisher apologizes for this error. The citation has been updated to match the corrected title. Please download this article again to view the corrected title and citation. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article(PDF)Click here for additional data file.File S2Republished, corrected article(PDF)Click here for additional data file."} +{"text": "This article was republished on October 14, 2015, to correct the order of Figs 8\u201313. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on April 22, 2014, to correct errors in Figures 3, 4, and 5 that were introduced during the production process. Please download this article again to view the correct figures.PLOS style does not allow for the code in the PDF version of this article to be displayed as the authors intended. Please see the author-provided PDF attached to this notice to view the correctly formatted code.The publisher apologizes for the figure errors and the code formatting in the PLOS PDF.The originally published, uncorrected article and the republished article with corrected figures are also provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished article with corrected figures.(PDF)Click here for additional data file.File S3Author-provided PDF.(PDF)Click here for additional data file."} +{"text": "This article was republished on May 26, 2015, to include details of the editor who handled the submission, which had been omitted due to an error in production. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on January 5, 2015, to correct errors in the text of the article and figures. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference. The publisher apologizes for the errors that were introduced during the typesetting process.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on July 3rd, 2014, due to Figure 1 and Figure 2 being ordered incorrectly. Please download the PDF again to view the corrected article. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on April 11, 2014, to correct formatting errors in genus and species names that were introduced during the typesetting process. These names should have been italicized throughout the article. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on September 12, 2014, to correct typesetting errors with the figures. The publisher apologizes for these errors. Please download this article again to view the corrected figures. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected articlePDFClick here for additional data file.File S2Republished, corrected articlePDFClick here for additional data file."} +{"text": "This article was republished on June 26, 2015, to correct Fig 4, which was darker than intended. The publisher apologizes for the error. In addition, the family names \"Scolopacidae\" and \"Charadriidae\" have been un-italicized in Table 1, and tracked changes have been removed from S1 File. Please download this article again to view the corrected version."} +{"text": "This article was republished on July 9, 2015, to replace text and equations in the Methods section that were lost during the production process. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on June 27, 2014, to correct an error in the byline. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on August 7, 2014, to replace incorrectly changed character in the byline, citation, and references. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on January 2, 2015, to include the Supporting Information files, which were mistakenly left out of the original publication. Please download this article again to view the correct files. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on December 30, 2014, to replace Figure 1, which was incorrect. The publisher apologizes for the error."} +{"text": "This article was republished on July 1st, 2014, due to formatting errors in the Materials and Methods section of the text. The publisher apologizes for this error. Please download the PDF again to view the corrected article. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on July 25th, 2014, due to incorrect information being published in the Funding statement. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on May 26, 2015, to include details of the editor who handled the submission, which had been omitted due to an error in production. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on May 26, 2015, to replace an incorrect Fig 1. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on March 11, 2015, to include a missing paragraph in the Results section that was removed in the typesetting process. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on January 30, 2015, to correct errors in Table 3. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on April 30, 2014, to include figures in the main article that were previously published as Supporting Information. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on April 10, 2015, to correct figure quality errors that were introduced during the typesetting process. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on November 3, 2014, to correct errors in equations and in the text of the paper that were introduced during the typesetting process. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on July 2nd, 2014, due to an error in the title. The publisher apologizes for this error. Please download this article again to view the corrected title and citation. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on April 6, 2015, to correct two errors with the Fig 5 legend. The errors were introduced during the typesetting process, and the publisher apologizes for the errors. Please download the article again and view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on April 14, 2014 to replace an incorrectly published version where Equations 10 and 11 were typeset incorrectly. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on May 28, 2014, to correct errors in several author names and affiliations. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on June 25, 2014, to correct errors in the figures that were introduced during the production process. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on February 2, 2016, to correct errors in the figure order and to correct author affiliation errors that were introduced during the production process. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on April 8, 2015, to correct figures that were distorted during the production process. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on August 7, 2014, to replace incorrectly changed characters in the byline. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on June 3rd, 2014, due to the figures being ordered incorrectly. The publisher apologizes for this error. Please download the PDF again to view the corrected article. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on August 6, 2014, to replace chi and phi characters that were changed in the paper. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on September 1, 2014, to correct errors in the references and in the legend for Figure 10 that were introduced during the typesetting process. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.Files S1Originally published, uncorrected article.(PDF)Click here for additional data file.Files S2Republished corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on April 8, 2015, to correct figures that were distorted during the production process. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on June 16, 2016, to correct errors in the figure legends that were introduced during the typesetting process. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on January 26, 2016, to correct errors in the figure order that were introduced while preparing the paper for production. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on December 3, 2014, to correct errors in Figures 3, 4, and 5 that were introduced during the typesetting process. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on January 7, 2016, to correct spacing errors in the text that were introduced during the pre-production process. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on June 23, 2015, to correct errors in Tables 1 and 2 that were introduced during the typesetting process. The publisher apologizes for the errors. Please download this article again to view the correct version. The corrected article is provided here for reference.S1 File(PDF)Click here for additional data file."} +{"text": "This article was republished on June 3, 2015, to correct an error in the title. The correct title is: The Effect on Mortality of Fluconazole or Echinocandins Treatment in Candidemia in Internal Medicine Wards. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on May 9th, 2014, due to Figure 1, Figure 2, and Figure 3 being ordered incorrectly. Please download the PDF again to view the corrected article. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on August 5, 2014 to correct the figure sizing. The publisher apologizes for this error. Please download this article again to view the corrected figures. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on September 15, 2014, to correct typesetting errors involving missing minus symbols throughout the text and tables. The publisher apologizes for these errors. Please download this article again to view the article. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected articlePDFClick here for additional data file.File S2Republished, corrected articlePDFClick here for additional data file."} +{"text": "This article was republished on July 24, 2014, to correct Figure S1, which would not open. The publisher apologizes for the error. Please view this article again to download the file."} +{"text": "This article was republished on July 10, 2014, due to Figure 2 being erroneously removed from the PDF. The publisher apologizes for this error.File S1Originally published, uncorrected article.(pdf)Click here for additional data file.File S2Republished, corrected article.(pdf)Click here for additional data file."} +{"text": "This article was republished on October 10, 2014, to correct errors in the formatting of the equations and paragraphs. These errors were introduced during the typesetting process. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on September 30, 2014, to correct errors in the authorship list and citation that were introduced during the typesetting process. The second author\u2019s name was spelled incorrectly. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on February 1, 2016, to correct errors in figure captions that were introduced during the typesetting process. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "Haemophilus influenza\u201d. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.This article was republished on May 13, 2016, to correct the title which was incorrectly published as \u201cTransformed Recombinant Enrichment Profiling Rapidly Identifies HMW1 as an Intracellular Invasion Locus in S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on June 5, 2014, to correct the images of Figures 6 and 7, which were switched during the production process. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.This article was republished on the 14File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on June 9, 2015, to replace an incorrectly published version of the manuscript. In addition to textual changes, the correct version includes author Shing-Hong Liu. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on May 22, 2015, to correct Figures 5 and 6, which were both resized during the typesetting process. The publisher apologizes for the errors. Please download this article again to view the correct version."} +{"text": "This article was republished on June 5, 2014, to replace an incorrectly published version. During the typesetting process, an equation in the Results section was removed from the XML and PDF of the article. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on Jul 31, 2014, to replace figures that were previously unreadable. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished corrected article.(PDF)Click here for additional data file."} +{"text": "Please download the PDF again to view the corrected article. The originally published, uncorrected article and the republished, corrected article are provided here for reference.This article was republished on September 25File S1Originally published, uncorrected article(PDF)Click here for additional data file.File S2Republished, corrected article(PDF)Click here for additional data file."} +{"text": "This article was republished on April 27, 2015, to correct figures that were distorted during the production process. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "Macaca radiata\" was changed to \"Macaca radiate.\u201d The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.This article was republished on April 8, 2015, to correct an error in the title and abstract that was introduced during the production process: \"S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on August 8, 2016, to correct errors in the title and Table 2. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on June 29, 2015, to correct an error in the title that was introduced during the typesetting process. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on March 20, 2015, to correct an error introduced during the typesetting process. The legend for Figure 4 was misplaced within the Results section of the paper. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on October 28, 2014, due to multiple instances of \"Gnptab-/-\" being published as \"Gnptg-/- \". The publisher apologizes for this error. Please download the article again to view the corrected version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article(PDF)Click here for additional data file.File S2Republished, corrected article(PDF)Click here for additional data file."} +{"text": "This article was republished on April 10, 2015, to correct Figs. 2 and 3, which were distorted during the production process. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on October 9, 2015, to correct errors in equations and formatting that were introduced during the typesetting process. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on April 18 2014, to correct the spelling error in the title. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on June 24, 2015, to remove the captions for S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on May 15, 2015, to correct the legend for Table 1, which was mistakenly incorporated into the body text during the production process. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "The affiliation for the first author is incorrect. Fl\u00e1vio Ricardo Guilherme is not affiliated with #1 but with #3 Department of Physical Education, State University of Maringa, Parana, Brazil."} +{"text": "This article was republished on July 25, 2014, due to an error in the title. The publisher apologizes for this error. Please download this article again to view the corrected title and citation. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article(PDF)Click here for additional data file.File S2Republished, corrected article(PDF)Click here for additional data file."} +{"text": "This article was republished on June 10, 2016, to correct a textual error in the body of the correction article. Alain Vaguet was spelled incorrectly in the correction text, appearing as: Alain Vague. The correction article meant to specify only the order of authors. The publisher apologizes for misspelling the author name. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on May 14, 2014, due to multiple instances of \"\u00b1\" being published as \"+.\" The publisher apologizes for this error. Additionally, some symbols were missing from Figure 3. Please download the PDF again to view the corrected article and Figure 3. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on April 2nd, 2015, to correct errors in the footer year, the citation year and copyright year that were introduced during the typesetting process. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on May 18, 2016, to fix an issue with broken supporting information hyperlinks that was introduced during the typesetting process. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on August 1, 2016, to correct errors that were introduced during the typesetting process: in several places throughout the article the \u03b1 was omitted from \u03b1-MSH. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on April 25, 2014, to correct errors in the title that were introduced during the typesetting process. The correct title of the article is \u201cCirculating Ang-2 mRNA Expression Levels: Looking Ahead to a New Prognostic Factor for NSCLC.\u201d Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on August 7, 2014, to replace incorrectly changed characters in the byline, citation, copyright statement, and Acknowledgments. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on April 10, 2015, to correct figures that were distorted during the production process. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on May 21, 2014, to correct a typesetting error in the author byline and citation. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on November 24, 2014, to correct multiple errors in the order of the figures. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on August 14, 2015, to correct errors in the order of the figures. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.This article was republished on April 10File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on November 20, 2014 to correct typographical errors in Table 1, which were introduced during the typesetting process. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.PDFClick here for additional data file.File S2Republished, corrected article.PDFClick here for additional data file."} +{"text": "The affiliation for the second author is incorrect. Md Asri Ngadi is not affiliated with #2 but with #1 Department of Computer Science, Universiti Teknologi Malaysia, 81310 Johor Bahru, Malaysia."} +{"text": "This correction was republished on August 31, 2015, to replace an incorrectly uploaded file and include the correct supplementary file. The publisher apologizes for the error. Please download this correction again to view the correct version. The originally published correction and the republished correction are provided here for reference.S1 PDF(PDF)Click here for additional data file.S2 PDF(PDF)Click here for additional data file."} +{"text": "This article was republished on September 30, 2014, to correct a typesetting error involving a missing equation in the Statistical Analysis subsection of the Materials and Methods section. The publisher apologizes for this error. Please download this article again to view the corrected version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article(PDF)Click here for additional data file.File S2Republished, corrected article(PDF)Click here for additional data file."} +{"text": "Please download this article again to view the corrected version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.This article was republished on May 30File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on July 15, 2015, due to errors in Figs. 1\u20134 and 8\u201310. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on September 23, 2014, to correct the publication year in the footer of the PDF. The publisher apologizes for these errors. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article(PDF)Click here for additional data file.File S2Republished, corrected article(PDF)Click here for additional data file."} +{"text": "This article was republished on May 20th, 2014, due to Figure 16, Figure 17, and Figure 18 being ordered incorrectly. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on November 7, 2014, to correct errors in Figure 3 and the Author Contributions that were introduced during the preparation of the manuscript. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on August 11, 2014, to replace incorrectly changed characters in the byline and citation. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on December 15, 2014 to correct an error with Figure 6. The prior version contained a copy of Figure 8 in place of Figure 6. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on May 16, 2014 to include figures that were removed during the typesetting process. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on April 10, 2015 to correct an error in the sizing of Figure 1 that was introduced during the production process. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on May 5, 2015, to correct Figs 1\u20133, which were missing several panels. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on April 25, 2016 to correct errors in the figures that were introduced during the typesetting process. Supplemental Figures 1\u20135 were published in place of Figures 1\u20135. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on August 11, 2014, to replace incorrectly changed characters in the byline and Competing Interests statement. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on August 27th, 2014, to correct an error where Figure 1 was posted as a duplicate of Figure 2. Please download the PDF again to view the corrected article. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article(PDF)Click here for additional data file.File S2Republished, corrected article(PDF)Click here for additional data file."} +{"text": "This article was republished on April 6, 2016, as all instances of the character \u03bc were erroneously dropped during the typesetting of the article for publication. The publisher apologizes for the errors.Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on May 20, 2014, due to an error in the title. The citation has been updated to match the corrected title. Please download this article again to view the corrected title and citation. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on April 8, 2015, to correct figures that were distorted during the production process. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on August 26, 2015, to correct the correction published on July 31, 2015. An incorrect version of Fig 3 was inadvertently used in the original correction. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected correction and the republished, corrected correction are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on June 17, 2014, to correct an error in the title. The publisher apologizes for the error. In addition, a typographical error was corrected in the Abstract. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on July 10, 2014, to correct an error in the citation. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on April 10, 2015, to correct Figure 5, which was distorted during the production process. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on January 1, 2015, to correct errors in equations that were introduced during the typesetting process. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on May 20th, 2014, due to Figure 4, Figure 5, and Figure 6 being ordered incorrectly. Please download the PDF again to view the corrected article. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on April 21, 2014 to replace an incorrectly published version where an equation in the Materials and Methods section was omitted in the PDF and XML during the typesetting process. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on June 3rd, 2014, due to the figures being ordered incorrectly in the previously published article. Additionally, the name of the fourth author now reads Christopher J. B. Nicol. Please download the PDF again to view the corrected article. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on September 12, 2014, to correct the figure sizing. The publisher apologizes for this error. Please download this article again to view the corrected figures. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article(PDF)Click here for additional data file.File S2Republished, corrected article(PDF)Click here for additional data file."} +{"text": "This article was republished on June 3, 2014, to correct errors in the title that were introduced during the typesetting process. The publisher apologizes for these errors. The republication encompasses the errors addressed in the correction notice, published on May 2, 2013.Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on September 12th, 2014, due to Figure 3 and Figure 4 being published as duplicates of Figure S3 and Figure S4. Please download the PDF again to view the corrected article. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article(PDF)Click here for additional data file.File S2Republished, corrected article(PDF)Click here for additional data file."} +{"text": "This article was republished on March 16, 2016, to remove instances of the word \u2018black\u2019 that appeared throughout the text as a result of a LaTeX file conversion error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on March 9, 2015, to correct errors in equations that were introduced during the typesetting process. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on April 11, 2014, to correct an error that marked coauthor Tim D. Smith as deceased. The publisher apologizes for this error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on June 19, 2014, due to multiple typesetting errors. The publisher apologizes for these errors. Please download the PDF again to view the corrected article. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article(PDF)Click here for additional data file.File S2Republished, corrected article(PDF)Click here for additional data file."} +{"text": "This article was republished on July 29, 2014, to correct errors in Tables 1 and 2 and Equation 2. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on July 7, 2014, to correct several errors that were introduced during the typesetting process. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF).Click here for additional data file.File S2Republished corrected article.(PDF).Click here for additional data file."} +{"text": "This article was republished on June 23, 2015, to replace an incorrectly published version. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on May 26, 2015, to include details of the editor who handled the submission, which had been omitted due to an error in production. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on April 15, 2015, to correct errors in tables and the body of the article that were introduced during the typesetting process. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on June 24, 2015, to replace an incorrectly published version. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on May 13, 2016, to correct errors that were introduced during preparation of the manuscript for publication: Figs 5\u20137 were swapped and S1 Fig was incorrect. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on June 9, 2014, to correct the ordering of the images of Figures 1-3. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on May 27, 2015 to correct words that were inadvertently merged during the typesetting process. The publisher apologizes for the errors. Also, grammatical errors, hyperlinks and the author contributions were corrected. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on April 10, 2014, to correct formatting errors that were introduced during the typesetting process. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on April 10, 2015 to correct an error in the first author\u2019s name that was introduced during the typesetting process. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on April 25th, 2014, due to figures and tables being incorrectly left out of the article or duplicated in the Supporting Information. The publisher apologizes for this error. Please download the PDF again to view the corrected article. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished corrected article.(PDF)Click here for additional data file."} +{"text": "Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.This article was republished on July 30File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on June 23, 2016, to correct errors introduced while preparing the manuscript for publication. IFN-\u0251 appeared incorrectly throughout the article. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on June 1, 2016, to correct an error in the title. The word \u201cMulberry\u201d was misspelled. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on April 23, 2015, to correct errors in figures that were introduced during the typesetting process. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "The affiliation for the sixth author is incorrect. Dr. France Kittel is not affiliated with #2 but with #3 School of Public Health, Universit\u00e9 libre de Bruxelles, Brussels, Belgium."} +{"text": "This article was republished on July 2, 2015, to replace supporting information file S1 Software, which would not open. In addition, the republication addresses the errors in the author names noted in the correction published on June 22, 2015. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on August 7, 2014, to replace incorrectly changed characters in the byline, citation, and references. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on August 5, 2014 to correct the figure sizing. The publisher apologizes for this error. Please download this article again to view the corrected figures. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on June 2, 2014, to correct a typographical error in the title. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on February 3, 2015, to replace the missing Equation 5 in the online version and to correct errors in the legend of S1 Table in both the online and PDF versions. The publisher apologizes for the errors. The S1 Table legend has now been moved to within the file itself. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on April 3, 2014, to correct affiliations for all authors and correct legends for Figures 4B and 4C. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1 Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on May 30, 2014, to replace an incorrectly published version where Table 4 was missing in the XML and PDF of the article. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on May 27, 2014, to include Checklist S1, the PRISMA checklist, which was mistakenly omitted during the production process. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on May 14, 2015, to correct an error in the title: \u201cBovis Bacillus\u201d was incorrectly displayed as one word. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on May 26, 2015, in order to correct headings for Table 1 which were incorrect. The headings for Table 1 are now correct in the republished manuscript. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on June 24, 2014, to replace missing Greek characters throughout the text. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on May 1, 2015, to correct Fig 1, which was distorted during the production process. The publisher apologizes for the error. Please download this article again to view the correct version."} +{"text": "This correction was republished on January 19, 2016, to correct errors in the author byline. The publisher apologizes for the errors. Please download this correction again to view the correct version. The originally published, uncorrected correction and the republished, corrected correction are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on April 28, 2014, to correct errors in the order of the figures. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on June 25, 2014, to remove copyrighted or confidential material. Please view this article again to see the corrected version.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on November 6, 2014, to include the correct versions of Figures 5 and 6. Tables 1 and 2 in the original article were also changed into Figures 4 and 7, respectively, in the republished article. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on June 18, 2014, to correct Figure 1, which was missing panels B-D. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on July 29, 2015, to correct errors in equations that were introduced during the typesetting process. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference. The publisher apologizes for the error.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on April 8, 2015, to correct errors in the PDF layout that affected the scientific understanding of the article. The publisher apologizes for these errors that were introduced during the typesetting process. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on May 26, 2015, to include details of the editor who handled the submission, which had been omitted due to an error in production. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on May 9th, 2014, due to several Supporting Information files being erroneously removed. The publisher apologizes for this error. Please view this article again for all Supporting Information files. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "The publisher apologizes for the error. Please view the originally published, uncorrected article and the republished, corrected article here.This article was republished on September 21, 2015, to correct an error in the title that was introduced during production of the article: S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on April 21, 2014 to correct errors in the title. The correct title is \u201cThe Effect of Paternal Age on Offspring Intelligence and Personality when Controlling for Parental Trait Levels.\u201d Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on October 28, 2014, due to an error in the title. The publisher apologizes for this error. The citation has been updated to match the corrected title. Please download this article again to view the corrected title and citation. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on June 19, 2014, to correct errors in symbols that were introduced during the typesetting process. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on June 18, 2014, to replace Figure 3, which was an accidental duplication of Figure 2. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on March 15, 2016, to correct errors in the article layout that were introduced during the typesetting process. Specifically, the abstract should have only contained a single paragraph, however the first two paragraphs of the main article text were also included in this. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on December 15, 2014, to fix incorrect characters in the General Methods section and missing italicization throughout the article. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on August 7, 2014, to replace incorrectly changed characters in the byline and citation. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on May 26, 2015, to include details of the editor who handled the submission, which had been omitted due to an error in production. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on February 9, 2015, to remove a paragraph in the Results section, which was an erroneous duplication of the Figure 6 legend. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on June 08, 2015, to correct errors in the funding information and the abstract. This republication addresses the issues noted in the correction published on April 14, 2015. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on July 11th, 2014, due to Figure 4 and Figure 5 being ordered incorrectly. Please download the PDF again to view the corrected article. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(pdf)Click here for additional data file.File S2Republished, corrected article.(pdf)Click here for additional data file."} +{"text": "The affiliation for the tenth author is incorrect. Achille Patrizio Caputi is not affiliated with #2 but with #3 Clinical and Experimental Medicine Department, University of Messina, Messina, Italy."} +{"text": "This article was republished on August 11, 2014, to replace incorrectly changed characters in the byline, citation, and references. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on April 15, 2015 to correct an error where the Table 1 legend was included in the main text of the article. The publisher apologizes for these errors, which were introduced during the typesetting process. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on April 6, 2015, to correct the captions for Tables 1 and 2, which were mistakenly incorporated into the body text during the production process. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on June 17, 2014, due to an error in the title. The publisher apologizes for this error. Please download this article again to view the corrected title and citation. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article(PDF)Click here for additional data file.File S2Republished, corrected article(PDF)Click here for additional data file."} +{"text": "This article was republished on May 4, 2015, due to figures 8, 9, and 10 being ordered incorrectly. The publisher apologizes for this error. Please download the PDF again to view the corrected article. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on August 7, 2014, to replace incorrectly changed characters in the byline and citation. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on September 8, 2015, to correct the order of Figs. 3\u20135. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on June 29, 2016, to correct the order of images for Figs 2\u20139, which were published out of order. The author apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on April 25, 2016, to correct an error in Figure 5 which was introduced during the typesetting process. The publisher apologises for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on June 20, 2014, due to spaces in the text being lost during the typesetting process. The publisher apologizes for these errors. Please download the PDF again to view the corrected article. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article(PDF)Click here for additional data file.File S2Republished, corrected article(PDF)Click here for additional data file."} +{"text": "This article was republished on March 2, 2015 to correct an error in Figure 4. The bottom panel was previously omitted. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on April 18, 2016, to correct text errors in the Methods, Results and Discussion sections that were introduced when an incorrect version was provided during the production process. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on June 6, 2014, to correct missing text in the article. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on June 20, 2014, due to a typesetting error. The publisher apologizes for this error. Please download the PDF again to view the corrected article. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article(PDF)Click here for additional data file.File S2Republished, corrected article(PDF)Click here for additional data file."} +{"text": "This article was republished on November 23, 2015, to correct the Supporting Information files, which were labeled incorrectly. In addition, Fig 4 was replaced with a corrected version. The publisher apologizes for the errors. Please download this article and its Supporting Information files again to view the correct versions.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on July 1st, 2014, due to panel B of Figure 3 being erroneously removed during the production process. The publisher apologizes for this error. Please download the PDF again to view the corrected article and the complete Figure 3. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article(PDF)Click here for additional data file.File S2Republished, corrected article(PDF)Click here for additional data file."} +{"text": "This article was republished on May 26, 2015, to include details of the editor who handled the submission, which had been omitted due to an error in production. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on June 12, 2015, to include details of the editor who handled the submission, which had been omitted due to an error in production, and to correct a misspelling of the fifth author\u2019s name. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on May 26, 2015, to include details of the editor who handled the submission, which had been omitted due to an error in production. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on February 12, 2015, to replace Figure 1, which was an erroneous duplication of Figure 2. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on August 7, 2014, to replace incorrectly changed characters in the byline. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on March 14, 2016, to correct the captions of Figs 3\u20137 and Figs 9\u201310 and figure citations in the Discussion section. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on April 8, 2015, to correct figures that were distorted during the production process. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on July 1st, 2014, due to the legends of Figure 6, Figure 7, and Figure 8 being ordered incorrectly. Please download the PDF again to view the corrected article. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on May 23, 2014, to correct Figure 1, which was an accidental duplication of Figure S1. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on June 27, 2014, due to the exclusion of Figure 1. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on July 28, 2014, due to correct the figure sizing. The publisher apologizes for this error. Please download this article again to view the corrected figures. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on July 3rd, 2014, due to Figure 1 and Figure 2 being ordered incorrectly. Please download the PDF again to view the corrected article. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on July 24, 2015, to correct errors in to correct errors in Figs 2 and 4\u20138 that were introduced during the production process. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on April 23, 2015, to correct figures that were distorted during the production process. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on December 12, 2014 to correct errors in the figure captions that were introduced during the typesetting process. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on May 21st, 2014, due to the figures being published as Supporting Information. The publisher apologizes for this error. Please download the PDF again to view the figures in the corrected article. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article(PDF)Click here for additional data file.File S2Republished, corrected article(PDF)Click here for additional data file."} +{"text": "This article was republished on August 13, 2014, to correct errors in the References section that were introduced in the typesetting process. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on March 30, 2015, to correct the sizing and placement of the figures; none of the article content was changed. The publisher apologizes for the original layout errors. Please download this article again to view the corrected version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on May 26, 2015, to include details of the editor who handled the submission, which had been omitted due to an error in production. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on August 8, 2014, to correct errors in the author byline, citation, and references that were introduced during the production process. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on August 7, 2014, to replace incorrectly changed characters in the byline, citation, and references. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on October 14, 2015, to correct formatting errors in the figures and tables. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on June 15, 2015, to correct an error in the title. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on April 27, 2015, to insert information omitted by the publisher, specifically an additional author, Figure S7, and some text in the Results section. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on December 22, 2014 to correct errors in the body of the paper and in Tables 2 and 3 that were introduced during the typesetting process. The publisher apologizes for these errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "Please download this article again to view the corrected version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.This article was republished on July 25, 2014, to correct the spelling of the 4File S1Originally published, uncorrected article(PDF)Click here for additional data file.File S2Republished, corrected article(PDF)Click here for additional data file."} +{"text": "This article was republished on January 8, 2016, to correct errors in the values throughout the article as well as well as an error in the affiliations. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on May 14, 2015, to correct Fig 1, which was distorted during the production process. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on June 1, 2016, to correct errors in the in-text references throughout the article. The in-text references did not accurately correspond to the references 14\u201378 in the Reference list. The authors apologize for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on October 19, 2015, to correct errors in equations that were introduced during the typesetting process. Specifically, the exponent in the fourth equation in the Material and Methods should be -8.821, not 8.821, and this has been corrected. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on February 25, 2015, to correct an error in the byline of the online version and the affiliations in the online and PDF versions. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "An affiliation is missing for the fourth author. Lisa Nupen is affiliated with #1 and with #3 National Zoological Gardens of South Africa, Pretoria, Gauteng, South Africa."} +{"text": "This article was republished on June 27, 2015, to correct an error in the title. The title erroneously said \"The Return Trip Is Felt Longer\" instead of \"The Return Trip Is Felt Shorter.\" Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on June 30, 2014, due to some characters in the Abstract and byline being erroneously changed during the typesetting process. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on April 24, 2014, to correct errors in in-text references and the missing Figure 4 in the PDF version of the article. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished corrected article.(PDF)Click here for additional data file."} +{"text": "Cervus elaphus\u201d in the title, abstract, and citation. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.This article was republished on October 21, 2015, to correct the spelling of \u201cS1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on June 3, 2014, to replace Files S1-S18, which had file types unsupported by the PLOS website. The publisher apologizes for the issue. Please view this article again to download the files in ZIP format. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on December 15, 2014, to correct an error in the title. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on August 7, 2014, to replace incorrectly changed characters in the byline and citation. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on April 11, 2015, to correct errors to figure files that were introduced during the typesetting process. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on June 12, 2015 to correct the omission of Corresponding Author Yu Wang and his affiliation from the Author byline. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on July 9, 2015, to replace an incorrectly published version. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on April 21, 2014, to correct the figure order. Please download the article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on September 30, 2014, to correct an error in the third author\u2019s affiliation and in the Author Contributions section that were introduced during the production process. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on July 29, 2014, to correct the PDF, which used an incorrect version of the manuscript. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on April 10, 2015 to fix errors with the reference links within the article that were introduced at copyedit. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on August 1, 2014, due to the errors in Figure 5 and Table 2. The publisher apologizes for these errors. Please download the PDF again to view the corrected article. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article(PDF)Click here for additional data file.File S2Republished, corrected article(PDF)Click here for additional data file."} +{"text": "This article was republished on July 23, 2015, to correct errors in Fig 5\u201310 that were introduced during the production process. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on April 27, 2015, to correct errors in figures that were introduced during the typesetting process. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.Please note that the republication addresses the issues in the correction posted on April 8, 2015.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on September 1, 2015, to correct errors that occurred during the final formatting of the manuscript. The authors apologize for these formatting errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on May 26, 2015, to include details of the editor who handled the submission, which had been omitted due to an error in production. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on June 10, 2016, to correct errors in the references that were introduced during the typesetting process. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on April 17, 2014, to correct the figure order. Please download the article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on August 11, 2014, to replace an incorrectly changed character in the byline. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on October 16, 2014, to correct the order and numbering of Figures 5-7, as well as one in-text figure citation. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on May 26, 2015, to include details of the editor who handled the submission, which had been omitted due to an error in production. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on May 6, 2014, to correct the order of Figures 1-3. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on August 5, 2014, to replace the file for Table S3. Please view this article again to download the correct file."} +{"text": "This article was republished on November 17, 2015, to replace incorrectly published Supporting Information files. The originally published, uncorrected Supporting Information files and the republished, corrected Supporting Information files are provided here for reference.S1 Zip(ZIP)Click here for additional data file.S2 Zip(ZIP)Click here for additional data file."} +{"text": "This article was republished on August 19, 2014, to correct the figure sizing. The publisher apologizes for this error. Please download this article again to view the corrected figures. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article(PDF)Click here for additional data file.File S2Republished, corrected article(PDF)Click here for additional data file."} +{"text": "This article was republished on April 6, 2015, to correct errors that were introduced during the typesetting process. Fig 4 was incorrectly cropped, and we have also corrected some typographical errors in the text. The publisher apologizes for these errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "Table 1 has been corrected for improved readability. The publisher apologizes for the error. Please see the complete, corrected Table 1 here."} +{"text": "This article was republished on April 21, 2015, to correct figures that were distorted during the production process. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "The fifth author\u2019s name is spelled incorrectly. The correct name is: Philip Jonsson.The affiliation for the fifth author is incorrect. Philip Jonsson is not affiliated with #1 but with #5: Department of Radiation Oncology, Center for Molecular Oncology, Memorial Sloan Kettering Cancer Center, NY, 10022, USA"} +{"text": "This article was republished on August 5, 2014 to correct the figure sizing. The publisher apologizes for this error. Please download this article again to view the corrected figures. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on September 17, 2015, to correct formatting errors to Tables 1\u20133 and symbol errors in figure legends. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on May 26, 2015, to include details of the editor who handled the submission, which had been omitted due to an error in production. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on August 25, 2014, to correct Figure 4, which was erroneously resized during the typesetting process. The publisher apologizes for the errors. Please view this article again to download the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on October 19, 2015, to correct editor comments that were erroneously included in the main text. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.In addition, the authors wish to make a correction: The original article duplicated Fig 2 under the caption for S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "The first affiliation for the fifth author is incorrect. Willibald Zeck is not affiliated with #2, but with #1: UNICEF Philippines, Makati City, Philippines."} +{"text": "Figures S1, S2 and S3 are omitted from the Appendix S1 file in the Supporting Information. They can be viewed below.Figure S1Basic structure of HIV simulation model.See text for details.(TIF)Click here for additional data file.Figure S2Typical patient histories with and without pipeline drugs. See text for details.(TIF)Click here for additional data file.Figure S3The Quantile-Quantile plot for pipeline arrival process. Quantile-Quantile plots are used to compare a dataset to a theoretical distribution. It provides an assessment of graphical goodness of fit. If the points lie on the line, the probability distribution is acceptable.(TIF)Click here for additional data file."} +{"text": "This article was republished on September 24, 2014 to correct errors in the Competing Interests Statement. Please download this article again to view the correct version. The corrected article is provided here for reference.File S1Republished corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on June 19, 2014, due to the figures being ordered incorrectly. Please download the PDF again to view the corrected article. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article(PDF)Click here for additional data file.File S2Republished, corrected article(PDF)Click here for additional data file."} +{"text": "This article was republished on October 17, 2014 to include the supporting information files, which were omitted in the original version. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on November 19th, 2014, due to Figure 3 being erroneously published as a duplicate of Figure 7. The publisher apologizes for this error. Please download the PDF again to view the corrected article. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article(PDF)Click here for additional data file.File S2Republished, corrected article(PDF)Click here for additional data file."} +{"text": "This article was republished on April 8, 2015, to correct figures that were distorted during the production process. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on August 7, 2015, to replace Fig 2, which was incorrect. The publisher apologizes for the error. Please download this article again to view the correct version."} +{"text": "This article was republished on March 12, 2015, to correct a spelling error in the title that was introduced during the production process. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on October 21, 2015, to replace the original Figs 2\u20135 with Tables 2\u20135 in order to correct formatting errors, and to remove the tracked changes from S1 and S4 Tables. The article was republished again on November 9, 2015, to correct formatting errors in Table 2; the publisher apologizes for this error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on July 25th, 2014, to include missing panels from several figures. The publisher apologizes for this error. Please download this article again to view the corrected version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article(PDF)Click here for additional data file.File S2Republished, corrected article(PDF)Click here for additional data file."} +{"text": "This article was republished on July 9, 2014, due to an error in the title. The publisher apologizes for this error. Please download this article again to view the corrected title and citation. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(pdf)Click here for additional data file.File S2Republished, corrected article.(pdf)Click here for additional data file."} +{"text": "This article was republished on August 11, 2014, to replace incorrectly changed characters in the byline, citation, and copyright statement. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on May 23, 2014, due to the exclusion of Figure 6 from the PDF. The publisher apologizes for the error. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1. Originally published, uncorrected article.(PDF)Click here for additional data file.File S2. Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on May 13, 2015, because radical signs used to differentiate native and radical forms of species were omitted throughout the paper. These errors were introduced during the typesetting process. The publisher apologizes for these errors. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on June 18, 2014, due to Figure 6 and Figure 7 being ordered incorrectly. Please download the PDF again to view the corrected article. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article(PDF)Click here for additional data file.File S2Republished, corrected article(PDF)Click here for additional data file."} +{"text": "This article was republished on July 21, 2015 to correct errors in the figure order that were introduced during the typesetting process. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on May 26, 2015, to include details of the editor who handled the submission, which had been omitted due to an error in production. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "Tursiops truncatus\" was incorrectly changed to \"Tursiops truncates.\" The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.This article was republished on April 27, 2015, to correct the distorted Fig 1 and the misspelled species name in the Abstract: \"S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on May 30, 2014, to correct the order of Figures 1-3. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.This article was republished on June 9S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on January 25, 2016, to correct capitalization errors in notations throughout the paper that were introduced during the typesetting process. In addition, a sentence was corrected in the Discussion section, and Table 6 was replaced with S1 Table. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on August 7, 2015, to replace Fig 2, which was incorrect. The publisher apologizes for the error. Please download this article again to view the correct version."} +{"text": "PLOS ONE Staff, and to note the publisher as responsible for the error. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.This article was republished on July 27, 2015, to change the byline of the correction, which should have been The S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on April 2, 2014, to correct Roman letters that were substituted for Greek letters in the Introduction, Results, Discussion, Materials and Methods, and Figure 2 Legend. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on May 22, 2014, to correct errors in figure legends that occurred during the typesetting process. The publisher apologizes for these errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on August 5, 2016, to correct errors in Table 3 that occurred during composition of the article for publication. The colors displayed in the \u201cStimulus\u201d column of Table 3 were incorrect. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "The correct name is: Roland Spie\u00df. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.This article was republished on August 7File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on April 14th, 2014, due to the figures being ordered incorrectly in the previously published article. Please download the PDF again to view the corrected article. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished corrected article.(PDF)Click here for additional data file."} +{"text": "There is an error in affiliation 1 for author Yuhua Ji. Affiliation 1 should be: Key Laboratory of Neuroregeneration, Nantong University, Nantong, China. The publisher apologizes for the error."} +{"text": "This article was republished on July 2, 2015 to correct errors in figures that were introduced during the typesetting process as well as errors in the Author Contributions. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on April 30, 2014, correct the figure order. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on June 5, 2014, to remove copyrighted information. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished corrected article.(PDF)Click here for additional data file."} +{"text": "This article was republished on August 20, 2015, because the legend for Table 2 was erroneously typeset as part of the body text of the article. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on June 29, 2016, to correct errors in Table 1 that were introduced during the typesetting process. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on June 11, 2015, to correct the Results section, in which degrees of freedom were mistakenly hyperlinked to references. The publisher apologizes for the error. S1 and S2 Tables were also corrected to remove tracked changes. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on June 3, 2014, to correct Figure 1, which was missing panels d-k. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished, corrected article.(PDF)Click here for additional data file."} +{"text": "After this article was published, similarities were noted between this article and submIn response to queries about these concerns, the first author provided the underlying data in The authors commented on aspects of how data were collected and analyzed for this study, but overall their responses did not fully resolve the concerns.PLOS ONE Editors issue this Expression of Concern.In light of these issues, the S1 File(XLSX)Click here for additional data file.S2 File(RM5)Click here for additional data file.S3 File(RAR)Click here for additional data file."} +{"text": "This article was republished on January 9, 2023 to correct for errors in the Data Availability statement. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on December 29, 2022, to correct for errors in the Data Availability statement. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on January 12, 2023 to correct for errors in the Data Availability statement. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on December 29, 2022, to correct for errors in the Data Availability statement. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on April 1, 2022, to update the author byline and list all members of the group consortium. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "Correction: BMC Palliat Care 21, 200 (2022)https://doi.org/10.1186/s12904-022-01090-4Following the publication of the original article , the autThe correct referencing for Table 2 is as follows:The correct referencing for Table 3 is as follows:The correct referencing for Table 4 is as follows:The correct referencing for Table 5 is as follows:The correct referencing for Table 6 is as follows:The correct referencing for Table 7 is as follows:The original article has been updated to correct this."} +{"text": "PLOS ONE website on May 11, 2022. The Retraction [At the time of retraction, the article remained"} +{"text": "This article was republished on January 17, 2023, to correct for errors in the Data Availability statement. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This error does not have consequences for the validity of the model, nor the statistical analysis in the original submission. Please download the article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.This article was republished on May 9S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on December 22, 2022, to correct for errors in the Data Availability statement. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on December 27, 2022, to correct for errors in the Data Availability statement. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "In an articleIn Figure 5, Figure 5F have been replaced, the correct figure is presented below.The authors apologize for the error."} +{"text": "This article was republished on December 29, 2022, to correct for errors in the Data Availability statement. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on December 22, 2022, to correct for errors in the Data Availability statement. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on April 17, 2021, to remove identifying information that was incorrectly included in the \"Author Response\" attachment in the \"Peer Review tab\" on their published article. The publisher apologizes for the errors. An incorrect version of Fig 2 was also published in error; the authors have provided a corrected version in the republished article. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on April 22, 2021, to correct inaccurate formatting in the term \u201crest:activity\u201d found in the title and throughout the article. The publisher apologizes for the errors introduced during the typesetting process. Please download this article again to view the correct version.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on November 2, 2021, to correct errors in the affiliations that were introduced during the typesetting process. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "The affiliation for the tenth author is incorrect. Mushref B. Assas is not affiliated with #3 but just with #5: Faculty of Applied Medical Sciences, King AbdulAziz University, Jeddah, Saudi Arabia."} +{"text": "There is an error in affiliation 2 for author Fauzia Mohsin. The publisher apologizes for the error.The correct affiliation 2 is: Department of Biology, Government College for Women Chungi No. 14, Multan, Pakistan."} +{"text": "This article was republished on February 25, 2022, to correct the order in which Dr. Kraus\u2019 affiliations are listed. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "An incorrect version of the Abstract and Introduction was published in error. This article was republished on April 16, 2021, to correct for this error. The publisher apologizes for the error. Please download the article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on April 22, 2021, to correct an error in the Participants subsection of the Methods introduced in the typesetting process. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on April 8, 2021, to correct errors in the Data Availability Statement that were introduced during the typesetting process. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "Scientific Reports 10.1038/srep16917, published online 23 November 2015Correction to: The Supplementary Information file published with this Article is incomplete, where the supplementary figures and tables are omitted.The correct Supplementary Information file is now linked to this correction notice.Supplementary Information."} +{"text": "Supplementary Figure S1, Supplementary Figure S2, and Supplementary Figure S3, respectively. The correct images for Due to a production error, there was a mistake in The publisher apologizes for this mistake. The original version of this article has been updated."} +{"text": "This article was republished on April 6, 2021, to correct the second author\u2019s affiliations and contributions, which were erroneously left blank in the originally published version of the article. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on March 10, 2022, because an incorrect version of the manuscript was mistakenly used in the original publication. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on February 10, 2022, to correct errors throughout the article that were introduced during the revision process. The authors apologize for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on November 1, 2021 to correct an error in the title. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on March 26, 2021 to correct an error in the Author Contributions. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on November 24, 2021, to correct errors introduced during typesetting. The conclusion section was erroneously removed. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "An incorrect file was mistakenly included in File S1. This article was republished on February 2, 2022, to correct for this error. Please download this article again to view the correct version."} +{"text": "This article was republished on December 10, 2020, to remove an erroneous \u2019Paperpile\u2019 link that appeared in the Introduction paragraph of the online and PDF versions of the article. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "Scientific Reports 10.1038/srep40384, published online 11 January 2017Correction to: This Article contains an error in Figure 4D, where the image for the blank condition was inadvertently duplicated for the NC panel. The correct Figure"} +{"text": "This article was republished on July 21, 2021 to correct errors in the article text that were introduced during the typesetting process. The publisher apologizes for the error. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on July 7, 2021, to correct errors in equations that were introduced during the typesetting process. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on April 8, 2021, to correct errors in the Data Availability Statement that were introduced during the typesetting process. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "The source code link provided in Methods section is wrong in 3 occurrences. The correct link to the full source code is Methods, Statistical Analysis, First Paragraph:A correction has been made to https://doi.org/10.26208/pykx-8s25.Full source codes used for the statistical analyses are publicly available with this publication at Methods, Scenario Analysis, Last Paragraph:A correction has been made to https://doi.org/10.26208/pykx-8s25.Full source codes used for the statistical analyses are publicly available with this publication at Methods, Data Extraction, Last Paragraph:A correction has been made to https://doi.org/10.26208/pykx-8s25.A complete list of all included and excluded studies is publicly available at The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated."} +{"text": "This article was republished on April 5, 2021, to correct errors in the author byline that were introduced during the typesetting process. The publisher apologizes for the errors. The second and fourth authors\u2019 names were spelled incorrectly. The correct names are: Md. Ashraful Alam and Md. Ahshanul Haque. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on October 8, 2020, to correct errors in equations and headings in the article PDF that were introduced during the typesetting process. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on December 7, 2021, to correct errors in the figure placements. Multiple figures were switched, causing the figures to appear in the incorrect order. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on April 12, 2021, to correct errors in Table 1 that were introduced during the typesetting process. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on April 5, 2021 to correct errors in the heading levels in the Performance structure of esports\u2013theory, Results, and Discussion sections that were introduced during the typesetting process. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.1S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "PLoS Biology, volume 3, issue 3: DOI: 10.1371/journal.pbio.0030068In In the caption for Figure 5A, the formula for the lognormal fit was given as follows:.The second squaring exponent should have been inside the parentheses, so it reads as follows:."} +{"text": "This article was republished on January 3, 2017, to correct an error in the author order that was introduced during the typesetting process. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on June 19, 2017, to correct an error that was introduced in the title during the typesetting process. The publisher apologizes for the error.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "The name of the third author was incorrectly left off the authorship list. The byline should appear as above.Volume 8, no. 6, 2017,"} +{"text": "This article was republished on April 23, 2018, as the original Supporting Information file S1 Text was published in error. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "The affiliation for the fourth author is incorrect. Bernt Lindtj\u00f8rn is not affiliated with #1 but with #2 University of Bergen, Faculty of Medicine, Center for International Health, Bergen, Norway."} +{"text": "This article was republished on September 6, 2016, to correct an error in the article title that was introduced during the typesetting process. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "The affiliation for the second author is incorrect. Antonio Mart\u00ednez-Sabater is not affiliated with #2 but with #1 Nursing Department, University of Valencia, Valencia, Spain."} +{"text": "This article was republished on March 15, 2017, to correct errors in figures that were introduced during the typesetting process. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on September 19, 2016, to correct an error in the figures provided in the correction article. Figure 1 in the correction article was incorrect. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "There is an error in affiliation 1 for all authors. Affiliation 1 should be:Precision Agriculture Research Chair, King Saud University, Riyadh, Saudi Arabia.The publisher apologizes for the error."} +{"text": "This article was republished on May 29, 2018 to correct a technical error present in the original article acceptance date. The publisher apologizes for the error. Please download this article again to view the corrected version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on February 3, 2017, to correct errors in figure dimensions for Figs 2, 3, 7 and 9 that were introduced during the typesetting process. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on January 25, 2018, to correct formatting errors that were introduced during the typesetting process. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on October 13, 2016 to correct errors that occurred during the typesetting process. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This correction was republished on January 25, 2017, to correct errors that were introduced during the typesetting process. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on March 29, 2018, to correct errors in the statistics as well as the editor and guest editor lists. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected editor lists and the republished, corrected editor lists are provided here for reference.S1 Editor List(PDF)Click here for additional data file.S1 Guest Editor List(PDF)Click here for additional data file.S2 Editor List(PDF)Click here for additional data file.S2 Guest Editor List(PDF)Click here for additional data file."} +{"text": "This article was republished on October 21, 2016 to correct errors in figures that were introduced during the typesetting process. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on December 7, 2016, to correct errors in the order of the figures. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on March 6, 2017, to correct an error in the author order that was introduced during the typesetting process. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "The image for Fig 4 is a duplicate of the image for Fig 6. Please see the correct The image for Fig 5 is a duplicate of the image for Fig 8. Please see the correct Brasiliensis should be lowercase. Please see the corrected caption here.In the figure title of Fig 6, the word The image for the S1 Fig is incorrect. Please see the correct image for the S1 Fig10.1371/journal.pone.0168658.s001.Photography by J\u00falia Morais Fernandes. doi:(JPG)Click here for additional data file."} +{"text": "This article was republished on July 3, 2017, to correct errors in the formatting that were introduced during the typesetting process. The publisher apologizes for the errors.http://datadryad.org/resource/doi:10.5061/dryad.64r0j).The Data Availability statement for this paper is incorrect. The correct statement is: New sequences generated for this study are deposited in GenBank under accession numbers KY809160\u2013KY809180 for the fungal cultivar and KY828479\u2013KY828592 for the ants. Nexus and tree files can be found in Dryad Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "Specifically, the last paragraph in box one was not separated from the preceding paragraph, and the subheading \u2018Regression model\u2019 was formatted incorrectly. The publisher apologizes for the errors. The originally published, uncorrected article and the republished, corrected article are provided here for reference.This article was republished on 23S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on April 5, 2018, to correct an error in Fig 11. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on October 12, 2016, to correct errors that were introduced during the typesetting process. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on November 22, 2016, to correct errors in Fig 1 that were introduced during the technical check process. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This Correction was republished on October 14, 2016, to correct errors in the citation that had been incorrectly excluded from the Correction. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on August 25, 2015, to correct an error in the display of the sixth author's name. Author Domenico Del Turco was erroneously noted as Turco DD, rather than Del Turco, D. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article is provided here for reference.S1 File(PDF)Click here for additional data file."} +{"text": "This article was republished on March 31, 2017, to correct an erroneous image within the correction of the original article. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference. The publisher apologizes for the error.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on November 18, 2016, to correct an error in the title that was introduced during the typesetting process. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on September 6, 2016, to correct an error in the article title that was introduced during the typesetting process. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on January 25, 2018, to correct an error in denoting the corresponding author that was introduced during the typesetting process. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on March 21, 2018, to include the Abstract that was omitted during the typesetting process. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on September 6, 2016, to correct errors in formatting that were introduced during the typesetting process, as well as correct an error in Table 1. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on January 8, 2016, to correct an error in the legend of Fig 1 that was introduced during the typesetting process. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on April 11, 2017, to replace Fig 2. An incorrect figure was typeset as Fig 2 during the production process. Please download this article again to view the correct version, with the correct Fig 2 included. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on March 15, 2017, to correct errors that were introduced during the typesetting process. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on November 20, 2017 to correct errors in the author list that were introduced during the production process. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article, and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on November 3, 2016, to correct an error in the article title that was introduced during the typesetting process. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on April 3, 2017, to correct an error in the author order that was introduced during the typesetting process. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on March 27, 2014, due to the figure and tables being published as Supporting Information. Please download the PDF again to view the corrected article. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on October 20, 2016, to correct an error in the title that was introduced during the typesetting process. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This correction was republished on March 9, 2017, to correct an error in Fig 5 within the correction article. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on February 14, 2017, to correct an error in the author list. The publisher apologizes for the error. Please download the article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on October 11, 2017, to correct errors in the article title that were introduced during the typesetting process. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article, and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on September 8, 2016, to correct errors in the article title that were introduced during the typesetting process. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on August 30, 2016 to correct errors that were introduced during the typesetting process. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on September 8, 2016, to correct an error in the article title that was introduced during the typesetting process. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on November 28, 2016, to correct an error in Figure 9 that was introduced during the typesetting process. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on February 10, 2017, to correct figure errors that were introduced during the composition process. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on September 8, 2016, to correct an error in the article title that was introduced during the typesetting process. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "The affiliation for the second author is incorrect. Tiziana Leone is not affiliated with #1 but with #2 Department of International Development, LSE, London, United Kingdom. The publisher apologizes for this error."} +{"text": "This article was republished on November 20, 2017 to correct errors in figures that were introduced during the typesetting process. The publisher apologizes for the errors. Please download this article again to view the correct figures. The originally published, uncorrected article and the republished article with corrected figures are also provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on December 17, 2015, to correct errors in Fig 2, 3, and 4 that were introduced during the typesetting process. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on January 10, 2018, to correct errors in the references. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on January 5, 2018, to correct errors with the supplementary data files during the typesetting process. The publisher apologizes for the errors. Please download this article again to view the correct version.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on October 3, 2016, to correct for errors in the corrected Funding section. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on November 4, 2016, to correct errors in the order of the figures. The authors apologize for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on October 26, 2017, to correct errors in Figs 3, 4, 5, 8, and 9 that were introduced during the typesetting process. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "The affiliation for the sixth author is incorrect. Frans C. H. Bakers is not affiliated with #3 but with #4 Department of Radiology, Maastricht University Medical Centre+, Maastricht, The Netherlands."} +{"text": "This article was republished on November 4, 2016, to correct errors in the figure order. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on December 15, 2016, to correct an error in the author order that was introduced during the typesetting process. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "There is an error in affiliation 2 for author Tzvi Dwolatzky. Affiliation 2 should be: The Rambam Health Care Campus and Technion, Haifa, Israel."} +{"text": "This article was republished on November 18, 2016 to correct errors in the author byline. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on April 27, 2017 to correct an error in the title introduced during the typesetting process. The publisher apologizes for this error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "PLOS ONE team would like to express our sincere gratitude to all of you who lent us your valuable time and expertise as an academic editor, as a guest editor, or as a peer reviewer in 2016. We are fully dependent on your voluntary efforts to publish PLOS ONE for the larger academic community, and we could not carry forward our mission without you. In 2016, PLOS ONE depended upon over 5,000 academic and guest editors to manage the nearly 50,000 articles submitted, which were assessed by nearly 70,000 reviewers. Together, your efforts enabled the publication of more than 22,000 rigorously reviewed Open Access papers.PLOS and the The names of our editors that handled submitted manuscripts in 2016 appear in the Supporting Information as S1 Editor List(PDF)Click here for additional data file.S1 Reviewer List(PDF)Click here for additional data file.S2 Reviewer List(PDF)Click here for additional data file.S3 Reviewer List(PDF)Click here for additional data file.S4 Reviewer List(PDF)Click here for additional data file.S5 Reviewer List(PDF)Click here for additional data file."} +{"text": "This article was republished on October 3, 2016, to correct an error in the title that was introduced during the typesetting process. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on April 14, 2017 to correct some textual errors that were introduced during the typesetting and proof processes. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on January 17, 2020, to correct errors in the author list. The publisher apologizes for this error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on August 31, 2018 to correct for an error involving the Fig 1 caption that was introduced during the typesetting process. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on December 21, 2018 to correct an error in the author byline introduced during the typesetting process. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on May 7, 2019 to correct for errors to the References and reference citations introduced during the typesetting process. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on February 25, 2019 to correct for errors introduced during the typesetting process. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on May 3, 2019, to correct errors in the figures and supporting figures. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on September 12, 2017, to correct an error in the author byline and article citation. The publisher apologizes for the error. Please download this article again to view the correct version.S1 File(PDF)Click here for additional data file."} +{"text": "This article was republished on June 26, 2018, to correct an error in the ORCID iD for the corresponding author. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on September 18, 2015, to correct errors that were introduced during the typesetting process. The publisher apologizes for the errors. Please download this article again to view the correct version.S1 File(PDF)Click here for additional data file."} +{"text": "An incomplete, earlier version of this article was published in error. The publisher apologizes for the error. This article was republished on May 21, 2019 to correct for this error. Please download the article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on January 16, 2018, to correct information provided in the Data Availability statement. Please download this article again to view the correct version.S1 File(PDF)Click here for additional data file."} +{"text": "The following information is missing from the Funding statement: MEXT-Supported Program for the Strategic Research Foundation at Private Universities S1511018 to Dr. Satoshi Okumura. Mitsui Life Social Welfare Foundation to Satoshi Okumura. An Academic Contribution from Pfizer Japan to Satoshi Okumura. The publisher apologizes for the error.The Supporting Information figure legends for S1-S7 Fig are omitted from the manuscript. The authors have provided the S1-S7 Fig legends as an additional Supporting Information file. It can be viewed below.S1 Supporting information figure legends(PDF)Click here for additional data file."} +{"text": "This article was republished on October 24, 2018 to include Supporting Information files S1 Table and S2 Table, which were omitted from the originally published article. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "Incorrect versions of Figs 1, 6, and 7 were published in error. The publisher apologizes for the errors. This article was republished on August 15, 2019 to correct for these errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on November 24, 2015, to correct errors in the Supporting Information. The publisher apologizes for the errors. Please download this article again to view the correct version.S1 File(PDF)Click here for additional data file."} +{"text": "Supplementary Tables S5-S8 were mislabelled due to errors introduced during the typesetting process. The publisher apologizes for the errors. This article was republished on July 5, 2019 to correct for these errors. Please download this article again to view the correct version."} +{"text": "This article was republished on December 17, 2018 to correct for Unicode character errors introduced during the typesetting process. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on June 21, 2019, to correct errors in the display of the Greek symbol \u03b2 throughout the article. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "Many of the equations had an erroneous symbol at the end (#). These have been removed. The publisher aplogises for the errors. Please download this article again to view the correct version. The orignally published, uncorrected article and the republished corrected article are provided here for reference.This article was republished on the 19S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on March 27, 2019 to correct for significant typographical errors introduced during the typesetting process. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on October 30, 2019, to replace a corrupted version of S1 Fig. The publisher apologizes for this error. Please download this article again to view the correct version."} +{"text": "The Data Availability statement for this paper is incorrect. The correct statement is: The data underlying this study have been uploaded to GenBank and are available using the following accession numbers: MG808039.1, MF592987.1, and MG808038.1."} +{"text": "An incomplete, earlier version of this article was published in error. The publisher apologizes for the error. This article was republished on November 19, 2018 to correct for this error. Please download the article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on February 21, 2019, to correct for errors in the Data Availability statement introduced during the production process. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on November 8, 2019 to correct errors in the Author Contributions, typographical errors in the Introduction and Methods sections, and errors affecting Figs 1, 5, 6, and 7 that were introduced during the typesetting process. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on November 21, 2018, to correct an error introduced during the typesetting process in which a portion of the Results section was included in the Table 1 caption. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "An incomplete, earlier version of this article was published in error. The publisher apologizes for the error. This article was republished on October 1, 2019 to correct for this error. Please download the article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.This article was republished on January 18, 2019 to correct errors with the presentation of the S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "An incomplete, earlier version of this manuscript was published in error. The publisher apologizes for the error. This article was republished on August 22, 2018 to correct for this error. Please download the article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on July 30, 2018, to correct for errors in the Data Availability statement introduced during the production process. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "Fig 1 was published in error. This article was republished on 21 November 2018, to correct Fig 1. The publisher apologizes for the error. The correct figure is available in the Supporting Information of this article.S1 File(TIF)Click here for additional data file."} +{"text": "This article was republished on June 24, 2019 to correct for errors in the title introduced during the typesetting process. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "The penultimate sentence of the second paragraph of the Introduction is incorrect. The correct sentence is: For each competition, the horses are separated by horse group , sex and age , and evaluated by three judges.S3 VideoReprinted under a CC BY license, with permission from H\u00e9ctor Barriga Torres, original copyright [2018].(MP4)Click here for additional data file."} +{"text": "This article was republished on February 15, 2019 to correct for errors in Fig 3 introduced during the typesetting process. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on October 26, 2018 to correct for an error in the title that was introduced during the typesetting process. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on December 17, 2018 to correct an error in the title introduced during the typesetting process. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on July 2, 2019 to correct for missing units of measurement that were incorrectly removed during the typesetting process. The publisher apologizes for these errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on January 9, 2020, to correct errors in the author byline and citation affecting all author names. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "An incomplete, earlier version of this manuscript was published in error. The publisher apologizes for the error. This article was republished on October 16, 2018 to correct for this error. Please download the article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on October 16, 2018 to correct for formatting errors in the Materials and methods, Results, and Discussion sections introduced during the typesetting process. The publisher apologizes for these errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on October 18, 2017, to correct errors that were introduced during the typesetting process. The publisher apologizes for the errors. Please download this article again to view the correct version.S1 File(PDF)Click here for additional data file."} +{"text": "This article was republished on May 3, 2019, to correct errors in the article text. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on August 14, 2018, to correct for errors in the Data Availability statement introduced during the production process. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on August 11, 2016, to correct an error with Fig 4 that was introduced during the typesetting process. The publisher apologizes for the errors. Please download this article again to view the correct version.S1 File(PDF)Click here for additional data file."} +{"text": "This article was republished on March 26, 2019 to correct an error in the ORCID iD of the corresponding author introduced during the typesetting process. The publisher apologizes for the errors. Please download this article again to view the correct version.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on September 7, 2018, to correct the article type to \"Methods and Resources\". The publisher apologizes for this error. The scientific content of the article has not changed.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on January 17, 2018, to correct errors that were introduced during the typesetting process. The publisher apologizes for the errors. Please download this article again to view the correct version.S1 File(PDF)Click here for additional data file."} +{"text": "This article was republished on November 2, 2018, to replace the supporting information file S3 Video, which had previously been unavailable for download. The caption remains the same. The publisher apologizes for this error."} +{"text": "This article was republished on October 5, 2018 to correct for an error to the author byline introduced during the typesetting process. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "An incorrect version of https://osf.io/dey8g/.In addition, the following information is missing from the Data Availability statement: All recordings, transcripts, computed features, and computed scores are also publicly available from: The publisher apologizes for these errors.S1 Fig(PDF)Click here for additional data file."} +{"text": "This article was republished on April 23, 2015, to correct errors in the equations that were introduced during the typesetting process. The publisher apologizes for the errors. Please download this article again to view the correct version.S1 File(PDF)Click here for additional data file."} +{"text": "The original Supporting Information file was published in error. The file was removed in the HTML and PDF versions of this article on July 6, 2018. The article was republished again on July 13, 2018 to update the Data Availability statement for accuracy. Please download this article again to view the correct version. The republished article from July 6, 2018 and the current, republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "The affiliation for the fourth author is incorrect. Claudia Niccolai is not affiliated with #1 but with #2: IRCCS Fondazione Don Carlo Gnocchi, Florence, Italy."} +{"text": "An incomplete, earlier version of this article was published in error. The Materials and methods incorrectly included URLs that did not relate to the article content. The publisher apologizes for this error. This article was republished on October 16, 2019 to correct for this error. Please download the article again to view the correct version.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "The affiliation for the 15th author is incorrect. Min Zhang is not affiliated with #2 but with #1: Shenzhen Nanshan Center for Chronic Disease Control, Shenzhen, China."} +{"text": "Incorrect versions of Figs 1\u20133 were published in error. The publisher apologizes for the errors. This article was republished on February 6, 2019, to correct for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on April 9, 2019 to correct for significant typographical errors introduced during the typesetting process. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "Incorrect versions of Supporting Information files S1 Fig, S4 Table, and S6 Table were published in error. This article was republished on July 06, 2018 to correct for these errors. Please download this article again to view the correct version."} +{"text": "This article was republished on February 21, 2019, to correct for an error in the Data Availability statement introduced during the production process. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "There is an error in affiliation 2 for author Giorgio Arcara. The correct affiliation 2 is: IRCCS San Camillo Hospital, Venice, Italy. The publisher apologizes for the error."} +{"text": "This article was republished on December 14, 2018, to correct errors in the Supporting Files that were introduced during the typesetting process. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "Masao Nagasaki is not affiliated with #2 but with #3 Tohoku Medical Megabank Organization, Tohoku University, Sendai, Japan.The affiliation for the 16"} +{"text": "This article was republished on July 26, 2016, to correct errors in Tables 1 and 2 that were introduced during the typesetting process. The publisher apologizes for the errors. Please download this article again to view the correct version.S1 File(PDF)Click here for additional data file."} +{"text": "This article was republished on December 5, 2019 to correct errors in the Funding statement and to add an ORCID iD for the last author. The publisher apologizes for these errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "This article was republished on December 18, 2019, to correct errors in the title that were introduced during the typesetting process. The publisher apologizes for this error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "The publisher apologizes for this error.This article was republished on 30Please download this article again to view the correct version. The originally published, uncorrected article is provided here for reference.S1 File(PDF)Click here for additional data file."} +{"text": "This article was republished on February 06, 2019 to correct for errors in the Data Availability statement introduced during the typesetting process. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "An incorrect version of Fig 1 was published in error. The publisher apologizes for this error. This article was republished on March 5, 2019 to correct for this error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} diff --git a/PMC_clustering_426.jsonl b/PMC_clustering_426.jsonl new file mode 100644 index 0000000000000000000000000000000000000000..97bc488368feba5ee6965bfa0d8283fa8eb20805 --- /dev/null +++ b/PMC_clustering_426.jsonl @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:21d85647cfc6f931483be7c6b3c6b748a1d9224fa140dc13fc17ff4e0aab2530 +size 93108355 diff --git a/PMC_clustering_427.jsonl b/PMC_clustering_427.jsonl new file mode 100644 index 0000000000000000000000000000000000000000..589e5049858c8b381908b3d9ca1992823fc8271c --- /dev/null +++ b/PMC_clustering_427.jsonl @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:9b446346911858fc8fb1fd0f3698c2dece42359d5a3e309034dfcad73854567e +size 31583422 diff --git a/PMC_clustering_428.jsonl b/PMC_clustering_428.jsonl new file mode 100644 index 0000000000000000000000000000000000000000..57dba6fcae2a55aeb2ea23ea3fade931687dd9f3 --- /dev/null +++ b/PMC_clustering_428.jsonl @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:d5ad9322d36c8911004efee9ac1591d38fd774d56e595498a14a70a1d2a2e638 +size 115085909 diff --git a/PMC_clustering_429.jsonl b/PMC_clustering_429.jsonl new file mode 100644 index 0000000000000000000000000000000000000000..9804e0892a36472c744796fc372c15ca51f7c877 --- /dev/null +++ b/PMC_clustering_429.jsonl @@ -0,0 +1,811 @@ +{"text": "Nature Communications 10.1038/s41467-020-14489-5, published online 7 February 2020.Correction to: The original version of this Article omitted the following from the Acknowledgements: \u201cFWF QuantERA via QTFLAG I03769\u201d, which incorrectly read as \u201cQuantERA via QTFLAG\u201d in the original version. This has now been corrected in both the PDF and HTML versions of the Article."} +{"text": "Nature Methods 10.1038/s41592-019-0658-6, published online 28 November 2019.Correction to: This paper was originally published under standard Springer Nature copyright (\u00a9 The Author(s), under exclusive licence to Springer Nature America, Inc.). As of the date of this correction, the paper is available online as an open-access paper under a Creative Commons Attribution 4.0 license."} +{"text": "Nature Communications 10.1038/s41467-017-01460-0, published online 23 November 2017.Correction to: The original version of this article omitted the following from the Acknowledgements: \u2018This project has received funding from the European Union\u2019s Horizon 2020 research and innovation programme under the Marie Sk\u0142odowska-Curie grant agreement No 641458\u2019. This has now been corrected in both the PDF and HTML versions of the article."} +{"text": "Scientific Reports 10.1038/s41598-019-56734-y, published online 27 December 2019Correction to: In the Supplementary Information file originally published with this Article \u201cSupplementary Figure 6\u201d was omitted. This error has been corrected in the Supplementary Information file that now accompanies the Article."} +{"text": "Correction to: Obesity Surgery (2020) 30:3354\u20133362.10.1007/s11695-020-04539-8This article was originally published electronically on the publisher\u2019s internet portal on May 5, 2020 without open access. With the author(s)\u2019 decision to opt for Open Choice the copyright of the article changed on August 6, 2020 to \u00a9 The Author(s) 2020 and the article is forthwith distributed under a Creative Commons Attribution."} +{"text": "Scientific Reportshttps://doi.org/10.1038/s41598-020-70875-5, published online 20 August 2020.Correction to: This Article contains an error in Figure\u00a07 where the representative images for MS group are duplicates of the representative AFR images and MSEE DAPI does not match the Merge panel. The correct Figure\u00a07 appears below as Figure"} +{"text": "Nature Communications 10.1038/s41467-017-01339-0, published online 30 October 2017.Correction to: The original version of this Article contained two errors in Fig.\u00a02h, in which the photoluminescence spectra curve of the \u201cPVK:OXD-7\u201d was plotted incorrectly and the figure legends were incorrectly given. The correct version is:which replaces the previous incorrect version:This has now been corrected in both the PDF and HTML versions of the Article."} +{"text": "Correction to: Molecular Psychiatry10.1038/s41380-019-0439-8 published online 05 June 2019This article was originally published under standard licence, but has now been made available under a [CC BY 4.0] license. The PDF and HTML versions of the paper have been modified accordingly."} +{"text": "Correction to: BMC Health Serv Res 20, 1040 (2020)https://doi.org/10.1186/s12913-020-05906-yFollowing publication of the original article , the EdiThe article title currently reads:BMC health services research title: the 2020 blast in the port of Beirut: can the Lebanese health system \u201cbuild back better\u201d?The article title should\u00a0read:The 2020 blast in the Port of Beirut: can the Lebanese health system \u201cbuild back better\u201d?The original article has been corrected."} +{"text": "Correction to: Journal of Human Hypertension10.1038/s41371-019-0196-9This Article was originally published under Nature Research\u2019s License to Publish, but has now been made available under a [CC BY 4.0] license. The PDF and HTML versions of the Article have been modified accordingly."} +{"text": "Correction to:Leukemia32; 1920\u20131931 (2018);10.1038/s41375-018-0122-0;published online: 30 March 2018Following the publication of this article, the authors noted that the following should be included in the Acknowledgements section: \u201cMA is the recipient of a START scholarship (0785) from FNP\u201d. The authors wish to apologise for any inconvenience caused."} +{"text": "Translational PsychiatryCorrection to: 10.1038/s41398-020-01093-w published online 24 November 2020p\u2009<\u20090.01)\u201d. The authors apologize for the mistake. The original article was revised.The original version of this article unfortunately contained an error in the abstract. The correct sentence should be \u201cWe showed that T-PRS was associated with widespread reductions in neural response to neutral faces in women and increases in neural response to emotional faces and shapes in men (multivariate"} +{"text": "Scientific Reports 10.1038/s41598-018-25688-y, published online 09 May 2018Correction to: The Acknowledgements section in this Article is incomplete.\u201cThis work was performed within the framework of the project No. UMO-2015/17/N/NZ4/02738. This work was supported by equipment bought for the purposes of the Project: \u201cSilesian BIO-FARMA Center for Biotechnology, Bioengineering, and Bioinformatics\u201d co-financed by European Regional Development Fund within the framework of Innovative Economy Operational Programme 2007\u20132013.\u201dshould read:\u201cThis work was performed within the framework of the project No. UMO-2015/17/N/NZ4/02738 financed from the funds of National Science Center. This work was supported by equipment bought for the purposes of the Project: \u201cSilesian BIO-FARMA Center for Biotechnology, Bioengineering, and Bioinformatics\u201d co-financed by European Regional Development Fund within the framework of Innovative Economy Operational Programme 2007\u20132013.\u201d"} +{"text": "British Journal of Cancer 10.1038/s41416-019-0565-8, published online 4 September 2019Correction to: This Article was originally published under Nature Research\u2019s License to Publish, but has now been made available under a CC BY 4.0 license.This has now been corrected in both the PDF and HTML versions of the Article."} +{"text": "The third author\u2019s name should have read S. V. Subramanian. This article has been corrected.1In the Original Investigation titled \u201cAssessment of Knowledge of HIV/AIDS and Association With Socioeconomic Disparities Among Young Women in Low- and Middle-Income Countries, 2003 to 2018,\u201d"} +{"text": "Scientific Reports\u2009https://doi.org/10.1038/s41598-018-24553-2, published online 18 April 2018Correction to: The Supplementary Information file that accompanies this Article contains technical errors, where mathematical symbols and equations are not displayed correctly.The correct Supplementary Information file is linked to this correction notice.Supplementary information."} +{"text": "Scientific Reports 10.1038/srep46250, published online 07 April 2017Correction to: This Article contains an error in Figure 1B where the incorrect image was shown for PEAL-LA/VEGFab NPs. The correct Figure"} +{"text": "Correction to: BMC Infect Dis (2020) 20:786https://doi.org/10.1186/s12879-020-05507-4Following publication of the original article , the autThe sentence currently reads:\u201c The in-hospital mortality rate was significantly lower in the IVIg group compared to the control group . \u201c.The sentence should read:\u201c The in-hospital mortality rate was significantly lower in the IVIg group compared to the control group . \u201c.Furthermore, the correct Table The original article has been"} +{"text": "Correction to: Gene Therapy;10.1038/s41434-018-0043-6; published online 26 Sept 2018This Article was originally published under Nature Research\u2019s License to Publish, but has now been made available under a CC BY 4.0 license. The PDF and HTML versions of the Article have been modified accordingly."} +{"text": "Scientific Reports 10.1038/s41598-019-51037-8, published online 08 October 2019Correction to: This Article contains a typographical error in the Acknowledgements section.\u201cS.B. has received founding from the European Research Council (ERC) under the European Union\u2019s Horizon 2020 research and innovation program (grant agreement No 670787).\u201dshould read:\u201cS.B. has received founding from the European Research Council (ERC) under the European Union\u2019s Horizon 2020 research and innovation program (grant agreement No 724690).\u201d"} +{"text": "Scientific Reports 10.1038/s41598-020-73329-0, published online 01 October 2020Correction to: This Article contains a typographical error in the Code availability section.https://public.cranfield.ac.uk/e102081/DeepMP/.\u201d\u201cThe code used for the simulations is freely available under the GNU General Public License v3 and can be downloaded from the Cranfield repository should read:http://public.cranfield.ac.uk/e102081/DeepMP/.\u201d\u201cThe code used for the simulations is freely available under the GNU General Public License v3 and can be downloaded from the Cranfield repository"} +{"text": "Nanomaterials [The authors wish to add the following information to the acknowledgements section of their paper published in aterials . This research was funded by national funds by Funda\u00e7\u00e3o para a Ci\u00eancia e a Tecnologia (FCT), Foundation for Science and Technology (I.P.) through the Associated Laboratory (IN) and by European funds through Fundo Europeu de Desenvolvimento Regional (FEDER) , by the Operational Programme for Competitiveness and Internationalisation (COMPETE2020), under project PTDC/CTM-NAN/3146/2014 (POCI-01-0145-FEDER-016623)."} +{"text": "Scientific Reportshttps://doi.org/10.1038/s41598-020-67709-9, published online 02 July 2020Correction to: In this Article, the weblink for readers to access the raw metabolomics data is incorrect.As a result, within the Results section under the subheading \u2018Metabolome\u2019,https://dev.metabolomicsworkbench.org:22222/data/DRCCMetadata.php?Mode=Project&ProjectID=PR000932&access=LduV2970.\u201d\u201cAll of the raw metabolome data are available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench: current project ID is PR000932, and DOI number is 10.21228/M8710D. The weblink for the data is should read:\u201cAll of the raw metabolome data are available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench: current project ID is PR000932. The data can be accessed directly via it's Project DOI: (10.21228/M9710D). This work is supported by NIH grant U2C-DK119886.\u201d.Also, within the Data Availability section:https://dev.metabolomicsworkbench.org:22222/data/DRCCMetadata.php?Mode=Project&ProjectID=PR000932&access=LduV2970.\u201d\u201cAll of the raw metabolome data are available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench: current project ID is PR000932, and DOI number is 10.21228/M8710D. The weblink for the data is should read:\u201cAll of the raw metabolome data are available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench: current project ID is PR000932. The data can be accessed directly via it's Project DOI: (10.21228/M9710D). This work is supported by NIH grant U2C-DK119886.\u201d."} +{"text": "Scientific Reports 10.1038/s41598-019-47899-7, published online 09 August 2019Correction to: The original version of this Article contained extensive errors in the Reference list. Reference 46 was incorrectly listed as Reference 92, and References 47-92 were incorrectly listed as References 46-91 respectively.Additionally, there was a typographical error in the Methods.47\u201349 were compared with the detected species from metabarcoding data.\u201d\u201cRecords from rapid assessment surveys previously conducted for non-native invertebrates at the sample sitesshould read:48\u201350 were compared with the detected species from metabarcoding data.\u201d\u201cRecords from rapid assessment surveys previously conducted for non-native invertebrates at the sample sitesThese errors have now been corrected in the PDF and HTML versions of the Article."} +{"text": "Scientific Reports 10.1038/s41598-020-65452-9, published online 29 June 2020Correction to: In the original version of this Article, the email address for the corresponding author Laurent Pr\u00e9t\u00f4t was listed as: pretot@bc.eduCorrespondence and requests for materials should instead be addressed to: laurent.pretot@gmail.comThis error has now been corrected in the PDF and HTML versions of this Article."} +{"text": "Correction to:Journal of Human Hypertension10.1038/s41371-019-0261-4published online 2 October 2019This article was originally published under Nature Research\u2019s License to Publish, but has now been made available under a [CC BY 4.0] license. The PDF and HTML versions of the article have been modified accordingly."} +{"text": "Cell Death & DiseaseCorrection to: 10.1038/s41419-020-02779-1 published online 27 July 2020The original version of this Article omitted the statement \u201cThese authors contributed equally: Jianjun Qi and Ningning Zhou\u201d. This has now been corrected in both the PDF and HTML versions of the Article."} +{"text": "Correction to: Oncogene10.1038/s41388-019-1023-zThis article was originally published under Springer Nature\u2019s License to Publish, but has now been made available under a CC BY 4.0 license. The PDF and HTML versions of the paper have been modified accordingly."} +{"text": "Scientific Reports 10.1038/s41598-020-61254-1, published online 9 March 2020Correction to: This Article contains errors in Table 1, where the data for the rows 'Paratyphi C' and 'Poano' have been omitted. The correct Table The Supplementary file that accompanies this Article also contains an error in which Supplementary Table S1 is missing. The correct Supplementary Table S1 appears below as Table"} +{"text": "Correction to: Scientific Reportshttps://doi.org/10.1038/s41598-020-68258-x, Published online 09 July 2020This Articlecontains errors in Supplementary Figure S5, where an incorrect scale is given for the x axis in the inset graphs in panels a) and b). The correct Supplementary Figure S5 appears below as Figure"} +{"text": "Cell Death & DifferentiationCorrection to: 10.1038/s41418-020-0584-2 published online 08 July 2020This Article was originally published under Nature Research\u2019s License to Publish, but has now been made available under a [CC BY 4.0] license. The PDF and HTML versions of the Article have been modified accordingly."} +{"text": "Nature Communications 10.1038/s41467-018-04736-1, published online 11 June 2018.Correction to: The original PDF version of this Article contained errors in Eqs.\u00a0The correct forms of Eqs.\u00a0The pdf has been updated to include these corrections. The original HTML version was correct and has not been changed."} +{"text": "Correction to: British Journal of Cancer 10.1038/s41416-019-0457-y, published online 24 April 2019This Article was originally published under Nature Research\u2019s License to Publish, but has now been made available under a CC BY 4.0 license. The PDF and HTML versions of the Article have been modified accordingly."} +{"text": "British Journal of Cancer (2020); 10.1038/s41416-020-0773-2, published online 5 March 2020Correction to: Since the publication of this paper the authors noticed an error in the name of Maria Letizia Taddei, where Professor Taddei\u2019s surname was incorrectly listed as Professor Letizia Taddei. This has been corrected above."} +{"text": "Correction to: Scand J Trauma Resusc Emerg Med https://doi.org/10.1186/s13049-020-00773-2In Fig.\u00a0\u03ba).In the \u2018Study selection\u2019 section, the symbol for the Kappa Coefficient shows up blank instead of (Following the publication of the original article , the aut"} +{"text": "Scientific Reports 10.1038/s41598-019-52727-z, published online 08 November 2019Correction to: The Acknowledgements section in this Article is incomplete.\u201cWe thank Emily Khuc and Selene M. Clay for collecting patients samples for the original genome-wide DNA methylation assay.\u201dshould read:https://www.rpbusa.org/rpb/), an RPB Unrestricted Grant , the National Institutes of Health , and That Man May See (http://thatmanmaysee.org/).\u201d\u201cWe thank Emily Khuc and Selene M. Clay for collecting patients samples for the original genome-wide DNA methylation assay. This study was supported by funds from a Research to Prevent Blindness (RPB) Physician-Scientist Award (to MFC,"} +{"text": "OncogenesisCorrection to: 10.1038/s41389-020-00301-y published online 9 January 2021The original version of this article unfortunately contained a mistake. The following correction has therefore been made in the original: Affiliations 1 and 3 were interchanged and affiliation 2 was incomplete. The original article has been corrected."} +{"text": "Scientific Reportshttps://doi.org/10.1038/s41598-020-66272-7; published online, 10 June 2020.Correction to: The Data Availability section of this Article was incomplete.\u201cThe data used in this study were obtained under license and so cannot be publicly available\u201d.now reads:\u201cThe data used in this study were obtained under license and so cannot be publicly available. Data are however available from the authors upon reasonable request, and with authorization of Nellore breeding programs.\u201d"} +{"text": "Nature Biotechnology 10.1038/s41587-020-0446-y, published online 2 March 2020.Correction to: This paper was originally published under standard Springer Nature copyright (\u00a9 The Author(s), under exclusive licence to Springer Nature America, Inc.). It is now available as an open-access paper under a Creative Commons Attribution 4.0 International license. The error has been corrected in the HTML and PDF versions of the article."} +{"text": "Funding statement appears below.In the original article, we neglected to include the grant number \u201cUID/BIA/04004/2020\u201d for the Portuguese Foundation for Science and Technology (FCT). The corrected This research was supported by POPH/FSE funds by the Portuguese Foundation for Science and Technology (FCT) with a doctoral grant to MC (SFRH/BD/89910/2012), a starting grant and exploratory project to SC (IF/01267/2013) and the project UID/BIA/04004/2020, by Xunta de Galicia with the grant to MS (ED431B 2018/36), and by Project RENATURE financed by the \u201cPrograma Operacional Regional do Centro 2014-2020 (Centro2020) \u2013 CENTRO-01-0145-FEDER-000007\u201d.The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated."} +{"text": "Correction to:Neth Heart J 202010.1007/s12471-020-01531-wThe original version of this article unfortunately contained a\u00a0mistake. The title was incomplete; the corrected title is given above. The original article has been corrected."} +{"text": "Scientific Reportshttps://doi.org/10.1038/s41598-020-73483-5, published online 09 October 2020Correction to: This Article contains a typographical error in the Results section. The subheading \u2018Agonist and inverse aFree energy landscapes suggestgonist establish differential interactions with the receptor\u2019should read:\u2018Agonist and inverse agonist establish differential interactions with the receptor\u2019"} +{"text": "Correction to: Leukemia10.1038/s41375-019-0662-yThis article was originally published under NPG\u2019s License to publish, but has now been made available under a CC BY 4.0 license. The PDF and HTML versions of the paper have been modified accordingly."} +{"text": "Scientific Reports 10.1038/s41598-018-29718-7, published online on 27 July 2018Correction to: The Acknowledgements section in this Article is incomplete.https://www.scar.org/science/pais/).\u201d\u201cThis work is supported by the PNRA national Italian project PNRA16_00016, \u201cWest Antarctic Ice Sheet History from Slope Processes \u2013 Eastern Ross Sea (WHISPERS)\u201d and by the MAE bilateral Italy-US project US16GR04, \u201cGlobal Sea Level Rise & Antarctic Ice Sheet Stability predictions: guessing future by learning from past (GSLAISS)\u201d. We acknowledge the CMCC Foundation for providing computing resources. This research is a contribution to the SCAR PAIS program (should read:https://sdls.ogs.trieste.it) for providing open access to international marine seismic data. This research is a contribution to the SCAR PAIS program (https://www.scar.org/science/pais/).\u201d\u201cThis work is supported by OGS and CINECA under HPC-TRES Program Award Number 2018-01, by the PNRA national Italian projects PNRA16_00016 WHISPERS and PNRA16_00293 GLEVORS, and by the MAE bilateral Italy-US Project US16GR04, \u201cGlobal Sea Level Rise & Antarctic Ice Sheet Stability predictions: guessing future by learning from past (GSLAISS)\u201d. We acknowledge the CMCC Foundation for providing computing resources. We acknowledge the Antarctic Seismic Data Library ("} +{"text": "Scientific Reports 10.1038/s41598-019-44440-8, published online 27 May 2019Correction to: This Article contains an error. A Data Availability section was originally not included \u2013 it should appear as below:Data AvailabilityRaw data generated for this study is in GenBank under accession number LR596613.1 and Short Read Archive with accession numbers ERX3426322 and ERX3426323."} +{"text": "Scientifc Reports 10.1038/s41598-020-69434-9, published online 28 July 2020Correction to: The original version of this Article contained an error.The heading for Figure\u00a05 was incorrect. The text,\u201cLevel2 GO terms of Melopsittacus_undulates\u201d.now reads:\u201cLevel2 GO terms of Sclerotinia sclrotiorum\u201d.This has now been corrected in the PDF and HTML versions of the Article."} +{"text": "Nature Communications 10.1038/s41467-019-11559-1, published online 20 August 2019.Correction to: Supplementary Data 2.The original version of the Supplementary Data associated with this article duplicated Supplementary Data 1 for both Supplementary Data 1 and Supplementary Data\u00a02. The correct file can be found here: This has now been corrected in the HTML version."} +{"text": "Correction to: BMC Public Health (2020) 20:327https://doi.org/10.1186/s12889-020-8391-8It was highlighted that the original article containeIncorrect sentence.The literature review conducted in Phase 1 found eight different theoretical models for work ability and two Correct sentence.In the literature review conducted in Phase 1 we found several different theoretical models for work ability and two models for functioning. In a recent Finnish review , eight m"} +{"text": "Scientific Reports 10.1038/s41598-019-40632-4, published online 14 March 2019Correction to: The link to the original data is incomplete. The following Data Availability text should be included:\u201cThe mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD020493.\u201d"} +{"text": "Salvage treatment for refractory or relapsed acute myeloid leukemia: a 10-year single-center experienceIn the article Figure 1 for:Replace"} +{"text": "Nature Communications 10.1038/s41467-020-16723-6; published online 9 June 2020.Correction to: The original version of this Article contained an error in the title, which was previously incorrectly given as \u2018Fossilized solidifications fronts in the Bushveld Complex argues for liquid-dominated magmatic systems\u2019. The correct version states \u2018Fossilized solidification fronts in the Bushveld Complex argue for liquid-dominated magmatic systems\u2019.This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Nature Methods 10.1038/s41592-020-0796-x, published online 6 April 2020.Correction to: This paper was originally published under standard Springer Nature copyright (\u00a9 The Author(s), under exclusive licence to Springer Nature America, Inc.). It is now available as an open-access paper under a Creative Commons Attribution 4.0 International license. The error has been corrected in the print, PDF and HTML versions of the article."} +{"text": "Gastroenterology Research and Practice has retracted the article titled \u201cInfluence of DPYD Genetic Polymorphisms on 5-Fluorouracil Toxicities in Patients with Colorectal Cancer: A Meta-Analysis\u201d . This arTwo similar meta-analyses were not discussed , 4. The"} +{"text": "Scientific Reports 10.1038/s41598-020-66611-8, published online 12 June 2020Correction to: The Acknowledgements section in this Article was omitted. The Acknowledgements section should read:\u201cThe work has been funded by the Operational Programme Human Capital of the Ministry of European Funds through the Financial Agreement 51675/09.07.2019, SMIS code 125125.\u201d"} +{"text": "Correction to:Journal of Perinatology10.1038/s41372-019-0358-1published online 02 April 2019This article was originally published under a standard License to Publish, but has now been made available under a CC BY license. The PDF and HTML versions of the paper have been modified accordingly."} +{"text": "Correction to: International Journal of Impotence Research;10.1038/s41443-018-0075-x;published online 13 September 2018This Article was originally published under Nature Research\u2019s License to Publish, but has now been made available under a CC BY 4.0 license. The PDF and HTML versions of the Article have been modified accordingly."} +{"text": "Scientific Reports 10.1038/s41598-019-46411-5, published online 09 July 2019Correction to: The Supplementary Information file that accompanies this Article contains an error in Supplementary Table S2, where the reference numbers shown in the table are incorrect. The correct Table S2 appears below as Table"} +{"text": "In: Beach Elizabeth Francis, Cowan Robert, O\u2019Brien Ian. Regulations to reduce risk of hearing damage in concert venues. Bull World Health Organ. 2020 May 1;98(5):367\u2013369 On page 368 and 369, the citation date for all references should read as follows: [cited 2020 Jan 18]."} +{"text": "Scientific Reports 10.1038/s41598-020-59019-x, published online 11 February 2020Correction to: The Acknowledgements section in this Article is incomplete.\u201cThis work was supported by NSF grants BCS-1461088 and BCS-1921678. E.S. is a funded by the Human Frontier Science Program and the Zuckerman STEM Leadership Program.\u201dshould read:\u201cThis work was supported by NSF grants BCS-1461088 and BCS-1921678 and by the Mind Science Foundation. E.S. is funded by the Human Frontier Science Program and the Zuckerman STEM Leadership Program.\u201d"} +{"text": "Correction to: Journal of Human Hypertension10.1038/s41371-019-0203-1This article was originally published under Nature Research\u2019s License to Publish, but has now been made available under a [CC BY 4.0] license. The PDF and HTML versions of the article have been modified accordingly."} +{"text": "Scientific Reportshttps://doi.org/10.1038/s41598-020-69258-7, published online 23 July 2020Correction to: This Article contains an error in Figure 1b and c, where the bars for minimum and maximum standing carbon stock and sequestration rate have been flipped vertically. The correct data are reported in Table 1 and the corrected Figure appears below as Figure"} +{"text": "Scientific Data 10.1038/s41597-020-0376-z, published online 11 February 2020Correction to: Following publication it was found that co-author Aleksandr Ananin\u2019s second affiliation was missing. The additional affiliation has now been added to both the HTML and PDF versions of the Data Descriptor."} +{"text": "Nature Methods 10.1038/s41592-019-0612-7, published online 21 October 2019.Correction to: This paper was originally published under standard Springer Nature copyright (\u00a9 The Author(s), under exclusive licence to Springer Nature America, Inc.). As of the date of this correction, the paper is available online as an open-access paper under a Creative Commons Attribution 4.0 license."} +{"text": "Correction to: Oncogene 3810.1038/s41388-019-1003-3This article was originally published under Springer Nature\u2019s License to Publish, but has now been made available under a CC BY 4.0 license. The PDF and HTML versions of the paper have been modified accordingly."} +{"text": "Correction to:Cancer Gene Therapy (2018);10.1038/s41417-018-0039-9;published on 13 August 2018.This article was originally published under Nature Research\u2019s License to Publish, but has now been made available under a [CC BY 4.0] license. The PDF and HTML versions of the article have been modified accordingly."} +{"text": "Publisher correction to: Chinese Neurosurgical Journal (2019) 5https://doi.org/10.1186/s41016-019-0163-xChinese Neurosurgical Journal. This article was published in volume 5 with a duplicate citation number.An error occurred during the publication of one article in In this correction article the old and new citation metadata are published in Table\u00a0The original article has been updated. The publisher apologizes for the inconvenience caused to our authors and readers."} +{"text": "Scientific Reports 10.1038/s41598-019-46022-0, published online 03 July 2019Correction to: This Article contains typographical errors in the Acknowledgements section.\u201cThis research is supported by the Singapore Ministry of Health\u2019s National Medical Research Council under its NMRC/OFYIRG/NOV006/2016.\u201dshould read:\u201cThis research is supported by the Singapore Ministry of Health\u2019s National Medical Research Council under its NMRC/OFYIRG/0034/2017.\u201d"} +{"text": "Scientific Reportshttps://doi.org/10.1038/s41598-020-66698-z, published online 18 June 2020Correction to: The Acknowledgements section in this Article was omitted. The Acknowledgements section should read:\u201cThis project was supported by European Research Council Consolidator awarded to JS (WANDERINGMINDS \u2013 646927).\u201d"} +{"text": "Scientific Reports 10.1038/s41598-020-64544-w, published online 15 May 2020Correction to: The Supplementary information5 originally published with this Article contained redundant data. This redundant data has been removed from the Supplementary Information5 that now accompanies the Article."} +{"text": "Journal of Translational Medicine entitled \u201cEmerging trends and research foci in gastrointestinal microbiome\u201d [Huang et al. recently published a paper in robiome\u201d . Huang eThere is no \u201cWeb of Science Core Collection (WoSCC) of Thomson Reuters\u201d but Web of Science Core Collection of Clarivate Analytics (formerly known as the Thomson Reuters and the Institute for Scientific Information). The data were collected on June 23 in 2018. A bias might be obtained as some publications in 2018 that have not yet been updated in the Web of Science Core Collection. Using the same method as mentioned in the original paper with searching keywords \u201cgastrointestinal microbiomes\u201d in Topic [Web of Science Core Collection includes.1. Science Citation Index Expanded (SCI-EXPANDED).2. Social Sciences Citation Index (SSCI).3. Arts & Humanities Citation Index (A&HCI).4. Conference Proceedings Citation Index\u2014Science (CPCI-S).5. Conference Proceedings Citation Index\u2014Social Science & Humanities (CPCI-SSH).6. Book Citation Index\u2014Science (BKCI-S).7. Book Citation Index\u2014Social Sciences & Humanities (BKCI-SSH).8. Emerging Sources Citation Index (ESCI).Web of Science Core Collection: Citation Indexes includes.1. Current Chemical Reactions (CCR-EXPANDED).2. Index Chemicus (IC).Web of Science Core Collection: Chemical Indexes.https://liu.brooklyn.libguides.com/az.php?a=e) [The subject of gastroenterology and hepatology can only be found in SCI-EXPANDED. In addition, since there are many levels of databases as mentioned above, the authors should choose the appropriate databases for their research . For insphp?a=e) . SSCI, Aphp?a=e) .Journal of Translational Medicine, this may result in misleading the journal readers. The authors could have provided a greater accuracy and information about their data if they understood Web of Science Core Collection beforehand. In addition, the fact that only 339 documents were published in 21\u00a0years (1998\u20132018) is not legitimate for a statistics point of view.From my view, Huang et al. used inappropriate searching keywords and method to publish bibliometric paper in"} +{"text": "Scientific Reports 10.1038/s41598-019-40421-z, published online 07 March 2019Correction to: In the Supplementary Information file originally published with this Article, Supplementary Figures and Supplementary Tables (Tables S1\u20134) were omitted. These errors have been corrected in the Supplementary Information that now accompanies the Article."} +{"text": "Correction to: Laboratory Investigation10.1038/s41374-020-0406-7This article was originally published under Nature Research\u2019s License to Publish, but has now been made available under a [CC BY 4.0] license. The PDF and HTML versions of the article have been modified accordingly."} +{"text": "European Journal of Clinical NutritionCorrection to: 10.1038/s41430-020-0580-0This article was originally published under Nature Research\u2019s License to Publish, but has now been made available under a CC BY 4.0 license. The PDF and HTML versions of the article have been modified accordingly."} +{"text": "Nature Communications 10.1038/s41467-020-19015-1, published online 6 November 2020.Correction to: The original version of this Article contained an error in the Acknowledgements:\u2018This work was supported by the European Union\u2019s Horizon 2020 research and innovation programme under grant agreement 668858\u2019.The number 668858 should have read 826121.This has now been corrected in both the PDF and HTML versions of the Article."} +{"text": "Nature Structural & Molecular Biology 10.1038/s41594-020-0466-9, published online 3 August 2020.Correction to: This paper was originally published under standard Springer Nature copyright (\u00a9 The Author(s), under exclusive licence to Springer Nature America, Inc.). It is now available as an open-access paper under a Creative Commons Attribution 4.0 International license, \u00a9 The Author(s). The error has been corrected in the print, HTML and PDF versions of the article."} +{"text": "Nature Communications 10.1038/s41467-020-19852-0, published online 30 November 2020.Correction to: The original HTML version of this Article was updated shortly after publication because the previous Peer Review file was mistakenly labelled as the Source Data file, and the Source Data file was mistakenly labelled as Peer Review file."} +{"text": "Scientific Reports 10.1038/s41598-018-31577-1, published online 03 September 2018Correction to: The article contains errors in Table\u00a0In Table\u00a0In Table\u00a0TI: Time interval between correct clicks (TI for the first correct click is computed from the replication period inset time) CC: Number of correct clicks RP: Replication period IC: number of incorrect clicks PP: Number of pattern points DC: number of clicks on the distracting point\u201d\u201cshould read:TI: Time interval between correct clicks (TI for the first correct click is computed from the replication period onset time) CC: Number of correct clicks RP: Replication period IC: number of incorrect clicks PP: Number of pattern points DC: number of clicks on the distracting point RTRP: Remaining Time of the RP\u201d\u201cThe correct Table\u00a0Finally, in the Reference list, the title is missing from Reference 20. The correct Reference 20 appears below as Reference"} +{"text": "Scientific Data 10.1038/s41597-020-0429-3, published online 11 March 2020Correction to: Following publication of this Data Descriptor it was found that co-author Gilberto Saccorotti\u2019s surname was spelled incorrectly. This has now been corrected in both the HTML and PDF versions."} +{"text": "Nature Communications 10.1038/s41467-020-14309-w, published online 24 January 2020.Correction to: The original version of this Article contained an error in the Methods section, when reporting ethical approval related to the generation of hESCs, which read: \u2018hESC (obtained from WiCell) were generated by the originating institute with informed consent and ethical approval from the Robert-Koch Institut, Berlin (Az.3.04.02/0101) and NIH (NIHhESC-10-0043). Studies with hESC (WA01/H1) were approved by the BC Children\u2019s and Women\u2019s Hospital Human Research Ethics Board \u2019. However, the cells were purchased from WiCell (Wisconsin) and the Robert-Koch Institute and NIH were not involved in the study. The sentence has now been corrected to state: \u2018hESC were purchased from WiCell (Wisconsin) . Studies with hESC (WA01/H1) were approved by the BC Children\u2019s and Women\u2019s Hospital Human Research Ethics Board \u2019. This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Nature Communications 10.1038/s41467-020-15778-9, published online 24 April 2020.Correction to: The original version of the Supplementary Information associated with this Article contained an error in the Supplementary Data 2 file, which arose during manuscript preparation. The values in column D were sorted in descending order, but columns A\u2013C were not re-ordered accordingly. The HTML has been updated to include the correct Supplementary Data 2 file."} +{"text": "The article titled \u201cThe Role of Antioxidants in Skin Cancer Prevention and Treatment\u201d was founSignificant text overlap was also identified with the following sources: Santosh An article posted in Life Extension Magazine, January 2003 (https://www.lifeextension.com/magazine/2003/1/cover_skinaging/Page-01). . An artic McArdle The authors apologize for this overlap."} +{"text": "Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR\u2019 by Corman et al. published on 23 January 2020, the correct affiliation of Marco Kaiser was added and the remaining affiliations were renumbered on 29 July 2020.In the article \u2018"} +{"text": "Endocrine Reviews. 2020;41(4):594\u2013609; doi: 10.1210/endrev/bnaa016), the following error occurred in the published paper: \u201cIn In the above-named article by Severinsen MCK and Pedersen BK (In the Graphical Abstract, the upward-pointing arrow next to the word \u201cappetite\u201d has been changed to a downward-pointing arrow online.The legend to The legend to 10.1210/endrev/bnaa016Doi:"} +{"text": "Scientific Reports 10.1038/s41598-019-39337-5, published online 06 March 2019Correction to: The Supplementary Information file originally published with this Article was incorrectly published as a \u2018.tex\u2019 file. The correct Supplementary Information file now accompanies the Article."} +{"text": "Nature Communications 10.1038/s41467-019-09965-6, published online 13 May 2019Correction to: The original version of the Source Data file associated with this Article contained errors in the data underlying Figure 1i and Figure 5a. The HTML has been updated to include a corrected version of Source Data file."} +{"text": "Nature Communications10.1038/s41467-020-16816-2, published online 19 June 2020.Correction to: The original version of this article omitted the following from the Acknowledgements:\u2026 R01GM125629\u2026This has now been corrected in both the PDF and HTML versions of the article."} +{"text": "Genetics in Medicine 10.1038/s41436-018-0268-1; published online 12 September 2018Correction to: This article was originally published under Nature Research\u2019s License to Publish, but has now been made available under a CC BY 4.0 license. The PDF and HTML versions of the Article have been modified accordingly."} +{"text": "Scientific Reports 10.1038/s41598-019-56044-3, published online 23 December 2019Correction to: A supplementary file containing Figure S1 was inadvertently omitted from the original version of this Article. This error has now been corrected in the HTML and PDF versions of the Article."} +{"text": "Scientific Reports 10.1038/s41598-020-74055-3, published online 08 October 2020Correction to: The Acknowledgements section in this Article is incomplete.https://www.editage.co.kr) for English language editing.\u201d\u201cWe would like to thank Editage (should read:https://www.editage.co.kr) for English language editing.\u201d\u201cThis research was supported by the Basic Science Research Program through the NRF, funded by the Ministry of Science, ICT & Future Planning (NRF-2019R1C1C1002830); a grant from the National R&D Program for Cancer Control, Ministry of Health & Welfare, Republic of Korea (1520120). We would like to thank Editage ("} +{"text": "Scientific Reportshttps://doi.org/10.1038/s41598-020-69796-0, published online 04 August 2020Correction to: This Article contains an error in the legend of Figure\u00a01, where57, edited with ArcMap 10.458).\u201d\u201cLocation of the Ammer River catchment with research area marked and the channelized river-section indicated by levees along the research section on Ammer River .\u201cLocation of the Ammer River catchment with research area marked and the channelized river-section indicated by levees along the research section on Ammer River (Sources:"} +{"text": "Correction to: BMC Pulm Med 17, 68 (2017)https://doi.org/10.1186/s12890-017-0413-7Following publication of the original article , the autThe error is that the plot of panel \u2018C\u2019 has been duplicated as the plot of panel \u2018A\u2019.Please see the corrected figure in this correction article.The authors apologize for any inconvenience caused."} +{"text": "Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR\u2019 by Corman et al. published on 23 January 2020, a conflict of interest statement was added for authors Olfert Landt and Marco Kaiser on 29 July 2020.In the article \u2018"} +{"text": "Scientific Reports 10.1038/s41598-019-56007-8, published online 27 December 2019Correction to: The link to the original data is incomplete. The following Data Availability text should be included:\u201cThe grizzly proteomics data is available at the ProteomeXchange Consortium (PXD016974), and the grizzly RNA-seq data is available at Gene Expression Omnibus (GSE63864).\u201d"} +{"text": "Correction to:Strahlenther Onkol 201810.1007/s00066-018-01423-4The article was initially published with incorrect copyright information or changed later to incorrect copyright information.Upon publication of this Correction, the copyright of the article changed to \u201cThe Author(s)\u201d.The original article has been corrected."} +{"text": "Scientific Reportshttps://doi.org/10.1038/s41598-020-64984-4, published online 26 May 2020Correction to: The original HTML version of this Article contained atypographical error in Equation\u00a01.now reads:This error has now been corrected in the HTML version; the PDF version was correct from the time of publication."} +{"text": "Scientific Reports 10.1038/s41598-020-62556-0, published online 27 March 2020Correction to: The Acknowledgements section in this Article was omitted. The Acknowledgements section should read:\u201cThe authors would like to acknowledge the Clinical Team at the Outpatient Addiction Services, Cambridge Health Alliance for dedicated clinical support provided on this study and Delany Berry for her assistance with editing and formatting this manuscript.\u201d"} +{"text": "The EudraCT trial registration number given as 2015-001463-39 should have been 2015-004347-39. This article has been corrected.1In the Original Investigation \u201cComparison of Lemborexant With Placebo and Zolpidem Tartrate Extended Release for the Treatment of Older Adults With Insomnia Disorder: A Phase 3 Randomized Clinical Trial,\u201d"} +{"text": "Genetics in Medicine ; 10.1038/s41436-018-0088-3; published online 22 July 2018Correction to: This Article was originally published under Nature Research\u2019s License to Publish, but has now been made available under a [CC BY 4.0] license. The PDF and HTML versions of the Article have been modified accordingly."} +{"text": "Scientific Reportshttps://doi.org/10.1038/s41598-020-63099-0, published online 23 April 2020Correction to: The Supplementary Information file that accompanies this Article contains a typographical error in the second line of Equation\u00a05 whereshould read:The correct version of Equation\u00a05 is below:This correction does not affect the conclusions of the Article."} +{"text": "Correction to: Inj Epidemiol (2020) 7:9https://doi.org/10.1186/s40621-020-0235-6The equations were not adequately presented since they all Greek symbols disappeared in the PDF version of the article.The correct presentation of the equations is given below.Following publication of the original article The grey horizontal lines were missing in Fig.\u00a0The original article (Chien et al."} +{"text": "Correction to: BMC Musculoskelet Disord 21, 358 (2020)https://doi.org/10.1186/s12891-020-03320-3i)n\u2009=\u2009363) and have corrected the numbers (n\u2009=\u2009337). Patients from the cohort Nilsson-Helander et al. [The authors noticed that the reported number of patients that were treated surgically were too many New England Journal of Medicine [With updated guidelines on how to perform non-inferiority studies having been published in the Medicine , since tFollowing publication of this article , the aut"} +{"text": "Scientific Reports 10.1038/s41598-020-67216-x, published online 02 July 2020Correction to: In the Supplementary Information file originally published with this Article, Supplementary Figures 3 and 4 were omitted. These errors have been corrected in the Supplementary Information that now accompanies the Article."} +{"text": "Scientific Reports 10.1038/s41598-023-41878-9, published online 05 September 2023Correction to: The Data availability section in the original version of this Article was incorrect.\u201cThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.\u201dnow reads:https://databases.lovd.nl/shared/variants/0000932922\u201d\u201cWe deposited the variant into the LOVD locus specific database, which was already revised and accepted by the curator. The reference number: The original Article has been corrected."} +{"text": "Nature Communications 10.1038/s41467-023-37211-7, published online 27 March 2023Correction to: In the Acknowledgements section of this article, the grant number relating to US National Cancer Institute\u2019s Clinical Proteomic Tumor Analysis Consortium given for Li Ding was incorrectly given as U24CA210976 and should have been U24CA210972. The original article has been corrected."} +{"text": "British Journal of Cancer 10.1038/s41416-023-02174-5, published online 10 February 2023Correction to: In this article the data availability statement was incorrectly given. \u201cEGAS0000100682\u201d should have been \u201cEGAS00001006821\u201d.The correct statement as follows.The datasets generated during and/or analysed during the current study are deposited at the European Genome-phenome Archive (EGA) under the accession numbers EGAS00001006821 and EGAD00001009790.The original article has been corrected."} +{"text": "Sci.: Atmos., 2023, 3, 399\u2013407, https://doi.org/10.1039/D2EA00133K.Correction for \u2018Ring-opening yields and auto-oxidation rates of the resulting peroxy radicals from OH-oxidation of \u03b1-pinene and \u03b2-pinene\u2019 by Ben H. Lee The authors regret that a grant was missing in the acknowledgements section of the published article. The corrected acknowledgements section is shown below:Acknowledgementsx generation, and Henrik G. Kj\u00e6rgaard (University of Copenhagen) and Kristian H. M\u00f8ller (University of Copenhagen) for their suggestions on theoretical calculations.This work was funded by National Science Foundation Environmental Chemical Sciences (grant no. CHE-1807204 and CHE-2003359) and the European Research Council under the European Union's Horizon 2020 research and innovation programme under Grant No. 101002728. The authors acknowledge Ezra Wood (Drexel University) for his instructions on HOThe Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "Correction to: European Journal of Pediatrics\u00a0https://doi.org/10.1007/s00431-023-04990-6The authors under the \"Keep Me Safe\" study group in the original published version of the above article should have been presented in the authorship section.The original article has been corrected."} +{"text": "Correction to: Journal of Computer-Aided Molecular Design10.1007/s10822-023-00513-5In the original article, the Data availability statement was missing and should have read as below.\u2018Data availabilityFiles with structures and data produced with the different calculations methods can be found in following the link:https://github.com/ankolocouris/dlpno_extrapolated_dt. And there is also available the sdf file with all tested organic molecules structure outputs.\u2019The original article has been corrected."} +{"text": "Scientific Reportshttps://doi.org/10.1038/s41598-023-30176-z, published online 20 February 2023Correction to: The original version of the Article contained errors. The Data Availability section was incomplete,\u201cAll data generated or analyzed during this study are included in this published article and its supplementary information files.\u201dnow reads:https://github.com/nctu-dcs-lab/predatory_journals_detection.\u201d\u201cAll data generated or analyzed during this study are included in this published article and its supplementary information files. The underlying source code is available at Additionally, in the original version of the Supplementary Information, the legends for the Supplementary Tables were missing.The Article and accompanying Supplementary Information file have been corrected."} +{"text": "Correction to: BMC Genomics24, 83 (2023).10.1186/s12864-022-09096-1Following the publication of the original article , the autThe incorrect author\u2019s name is: Hadi Alipoor.The correct author\u2019s name is: Hadi Alipour.The author group has been updated above and the original article has been"} +{"text": "Correction: Journal of Eating Disorders (2022) 10:97\u00a010.1186/s40337-022-00621-xThe original article has incoFundingThis work was supported by the National Institute on Aging (NIA) [K76AG060003-A1]. The article has received sponsorship from Takeda Pharmaceutical Australia to cover the Article Processing Charge.The original article has been"} +{"text": "Scientific Reportshttps://doi.org/10.1038/s41598-023-38922-z, published online 21 July 2023Correction to: The original version of this Article contained an error in Figure\u00a015, where the values for vertical and horizontal speed in the cruise phase and the vertical speed in the hover phase for landing were incorrect.The original Figure\u00a0The original Article has been corrected."} +{"text": "Correction to: Urolithiasis (2023) 51:80 10.1007/s00240-023-01453-3The original version of this article unfortunately contained a mistake. The copyright holder for this article was incorrectly given as\u00a9 This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may applybut should have been\u00a9 The Author(s) 2023The original article has been corrected."} +{"text": "Oncogenesis 10.1038/s41389-020-00274-y published online 10 October 2020Correction to: Following the publication of this article an error was noted in figure assembly for Figure 2E, where an image from the control group had been mistakenly included to represent EV treatment.The correct figure showing EV treatment is presented below.The original article has been corrected."} +{"text": "Nature Structural & Molecular Biology 10.1038/s41594-022-00893-6. Published online 23 December 2022.Correction to: This paper was originally published under a standard Springer Nature license (\u00a9 The Author(s), under exclusive licence to Springer Nature America, Inc.). It is now available as an Open Access paper under a Creative Commons Attribution 4.0 International license, \u00a9 The Author(s). The error has been corrected in the HTML and PDF versions of the article."} +{"text": "Scientific Reportshttps://doi.org/10.1038/s41598-023-40277-4, published online 25 August 2023Correction to: The original version of this Article contained errors in Table 2, where the data in the columns \u2018Purified antibody\u2019 and \u2018Affinity-purified antibody\u2019 was interchanged for the Antigen \u2018RBD\u2019. The correct and incorrect data appears below.Incorrect:Correct:The original Article has been corrected."} +{"text": "Correction to: Journal of Comparative Physiology B (2020) 190:521\u2013534 10.1007/s00360-020-01296-zThe Fig.\u00a03 of the original publication is wrong and replaced by a new version.The Correct figure is given below:The original article has been corrected."} +{"text": "Nature Neuroscience 10.1038/s41593-021-00947-w. Published online 8 November 2021.Correction to: This paper was originally published under a standard Springer Nature license (\u00a9 The Author(s), under exclusive licence to Springer Nature America, Inc.). It is now available as an open-access paper under a Creative Commons Attribution 4.0 International license, \u00a9 The Author(s). The error has been corrected in the print, HTML and PDF versions of the article."} +{"text": "Scientific Reports 10.1038/s41598-023-33825-5, published online 01 May 2023Correction to: The Supplementary Information file published with this Article contained a text error in the Figure S1 legend and a stray object in Figure S4. That Supplementary Information file is provided below.The errors have now been corrected in the Supplementary Information file that accompanies the original Article.Supplementary Information."} +{"text": "Scientific Reports, 10.1038/s41598-023-37913-4, published online 1 July 2023Correction to: The Funding section in the original version of this Article was incomplete. It now reads:\u201cOpen Access funding enabled and organized by Projekt DEAL. This publication was funded by the German Research Foundation (DFG).\u201dThe original Article has been corrected."} +{"text": "Nature Communications 10.1038/s41467-023-38742-9, published online 12 June 2023Correction to: The original version of this Article contained an error in Fig. 3, in which figure panels (i)\u2013(n) were replicas of panels (c)\u2013(h). Panels (i)\u2013(n) have now been replaced by the correct figure panels. This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Correction to: Journal of Thrombosis and Thrombolysis (2022) https://doi.org/10.1007/s11239-022-02714-5The name of Aishah Z Aishah (2) has been duplicated for both name and surname; the correct author name is \u201cAishah Z Mughal\u201dIn the original version of the article:These have been corrected with this erratum."} +{"text": "This article has been corrected.1In the Original Investigation titled \u201cEffect of Internet-Based vs Face-to-Face Cognitive Behavioral Therapy for Adults With Obsessive-Compulsive Disorder: A Randomized Clinical Trial,\u201d"} +{"text": "Scientific Reports 10.1038/s41598-023-36816-8, published online 28 June 2023Correction to: The Funding section in the original version of this Article was omitted. The Funding section now reads:www.savemedcoasts2.eu) funded by the European Commission through the DG-ECHO.\u201d\u201cThis research was funded by Pianeta Dinamico Project, directed by INGV under the umbrella of the Italian Ministry of Research and SAVEMEDCOASTS2 Project (number 874398 The original Article has been corrected."} +{"text": "Scientific Reports 10.1038/s41598-022-21551-3, published online 08 November 2022Correction to: The Acknowledgements section in the original version of this Article was incomplete.\u201cThis work was supported by the Zahedan University of Medical Sciences, Zahedan, Iran [Grant No. 10588].\u201dnow reads:\u201cThe authors would like to thank Zahedan University of Medical Sciences for funding this research. .\u201dThe original Article has been corrected."} +{"text": "Correction: Child and Adolescent Psychiatry and Mental Health (2023) 17:23https://doi.org/10.1186/s13034-023-00568-0Following publication of the original article, the author noticed the induced errors occurred in the published version. The typesetter has inadvertently removed the captions of Figure 2 and Table 2 during correction stage. Figure\u00a0The original article has been corrected."} +{"text": "Scientific Reports 10.1038/s41598-023-44250-z, published online 13 October 2023Correction to: The original version of this Article contained errors. In Figures 2 and 3 the confidence intervals were inadvertently omitted during typesetting from all scatterplots.The original Figures\u00a0The original Article has been corrected."} +{"text": "Nature Biomedical Engineering 10.1038/s41551-023-01061-x, published online 6 July 2023.Correction to: This article was originally published under a standard Springer Nature license (\u00a9 The Author(s), under exclusive license to Springer Nature Limited 2023). It is now available as an open-access paper under a Creative Commons Attribution 4.0 International license, \u00a9 The Author(s). The error has been corrected in the HTML and PDF versions of the article."} +{"text": "Scientific Reports 10.1038/s41598-023-41940-6, published online 13 September 2023Correction to: In the original version of this Article, the Data availability section was incorrect,\u201cOn acceptance, the data used in this study would be archived in Dryad repository and the data DOI included in the article.\u201dnow reads:https://doi.org/10.5061/dryad.hhmgqnknq.\u201d\u201cData supporting the results are archived in the following Dryad data repository: DOI: The original Article has been corrected."} +{"text": "DOI: 10.20945/2359-3997000000258Arch Endocrinol Metab. 2020;64(4):462-78Where you read (TITLE):Reference values of 25-hydroxyvitamin D revisited: a position statement from the Brazilian Society of Endocrinology and Metabolism (SBEM) and the Brazilian Society of Clinical Pathology/Laboratory Medicine (SBPC)Should read:SBPC/ML )Reference values of 25-hydroxyvitamin D revisited: a position statement from the Brazilian Society of Endocrinology and Metabolism (SBEM) and the Brazilian Society of Clinical Pathology/Laboratory Medicine ( Where you read (TEXT):[anti-25(OH)D or DBP antibodies] .These assays include a first step in which 25(OH)D is dissociated from its carrier proteins. In a second step, the 25(OH)D in the sample competes with an analogue for the same sites of the ligand's assay Should read:[anti-25(OH)D antibodies and DBP] .These assays include a first step in which 25(OH)D is dissociated from its carrier proteins. In a second step, the 25(OH)D in the sample competes with an analogue for the same sites of the ligand's assay Where you read:Should read:Where you read:Correspondence to:Carolina MoreiraRua Le\u00e3o J\u00fanior, 28580060-000 \u2013 Curitiba, PR Brasilcarolina.aguiar.moreira@gmail.comShould read:Carolina MoreiraAv Agostinho Le\u00e3o J\u00fanior, 28580060-000 \u2013 Curitiba, PR Brasilcarolina.aguiar.moreira@gmail.com"} +{"text": "Cancer Cell International (2020) 20:28710.1186/s12935-020-01387-5The Editors-in-Chief have retracted this article. Concerns were raised regarding Fig.\u00a02d. This figure appears to overlap with Fig.\u00a04f in an article by different authors that was simultaneously under consideration with another journal . The EdiThe authors have not responded to correspondence regarding this retraction."} +{"text": "Scientific Reports 10.1038/s41598-023-38310-7, published online 12 July 2023Correction to: The original version of this Article contained an error in the name of author Jesse D. Roberts Jr., where the name suffix \u2018Jr.\u2019 was inadvertently omitted.The original Article has been corrected."} +{"text": "Correction to: Cell and Tissue Research (2022) 390:113\u2013129 https://doi.org/10.1007/s00441-022-03658-1The authors regret that Fig.\u00a02 showed in the article was the wrong one.The original article has been corrected."} +{"text": "Scientific Reports 10.1038/s41598-023-36093-5, published online 08 June 2023Correction to: The original version of this Article contained an error. The way the individual values were represented in Figure\u00a06 did not represent the individual results correctly. The original Figure\u00a0The original Article has been corrected."} +{"text": "Scientific Reports 10.1038/s41598-023-28187-x, published online 16 January 2023Correction to: The original version of this Article contained an error, where the incorrect file was provided as the Supplementary Information 1 file.The Supplementary Information 1 file that accompanies the original Article has now been corrected."} +{"text": "Nature Communications 10.1038/s41467-023-38746-5, published online 25 May 2023Correction to: The Supplementary Information associated with this Article contained text highlights corresponding to tracked changes that were made during revision. The HTML has been updated to include the correct version of the\u00a0Updated Supplementary InformationIncorrect Supplementary Information"} +{"text": "Correction to: Infectious Agents and Cancer (2023) 18:1810.1186/s13027-023-00495-xFollowing publication of the original article , the autThe statement in the \u2018Data availability\u2019 section originally read: All data are available in the manuscript.https://zenodo.org/record/7741988#.ZBNQm3bMK3A.The statement should read: All data are available in the manuscript and at link The original article has been"} +{"text": "Correction to: Current Oncology Reports10.1007/s11912-023-01434-0The original version of this article contained a mistake in the authors\u2019 names. Many of the authors\u2019 names were reversed .The authors\u2019 names shown above are correct, and the original article has been corrected."} +{"text": "Retraction Note: BMC Public Health 9, 241 (2009)10.1186/1471-2458-9-241The Editor has retracted this article due to the author\u2019s inability to provide documentation of approval from an ethics committee.Ramesh Adhikari does not agree with this retraction. Jyotsna Tamang has not responded to correspondence from the Editor about this retraction."} +{"text": "This is a peer-review report submitted for the paper \"Risk Factors of SARS-CoV-2 Infection: Global Epidemiological Study\".Thank you for your submission . I don\u2019t"} +{"text": "Nature Communications 10.1038/s41467-023-38866-y, published online 08 June 2023Correction to: In this article the grant number R21AG080435 relating to the National Institute on Aging of the National Institutes of Health for Fr\u00e9d\u00e9ric A. Meunier was omitted. The original article has been corrected."} +{"text": "Nature Communications 10.1038/s41467-023-38294-y, published online 05 May 2023Correction to: The Source Data file and the Reporting Summary were missing from this article and have now been uploaded. The original article has been corrected.Further information on research design is available in the\u00a0Reporting Summary"} +{"text": "Nature Communications 10.1038/s41467-023-42080-1, published online 16 October 2023Correction to: An incorrect version of the Source Data file was originally published with this article; it has now been replaced with the correct one.The original article has been corrected.Updated Source Data"} +{"text": "British Journal of Cancer 10.1038/s41416-023-02178-1, published online 01 February 2023Correction to: The original version of this article unfortunately contained errors in figures. After publication, the authors found that the downloaded version of PDF has several distorted figures. This is due to an error in typesetting. The list of distorted figures is:Figure 1d-e: The heatmaps are distorted and lack some annotation panels (top of heatmaps)Figure 2b: The heatmap lacks some annotation panels Figure 4b: The heatmap lacks some annotation panels Figure 7b: The heatmap lacks some annotation panels The correct figures can be found above. The original article has been corrected."} +{"text": "Correction to: Stress Biol 1, 21 (2021)https://doi.org/10.1007/s44154-021-00023-0The name of the 2nd author should be \u2018Suomeng Dong\u2019 and this has been reflected in this Correction;The reference \u2018Hout B et al., 2014\u2019 and its corresponding citation should be removed.Following publication of this article (Wang & Dong, The original article has been updated."} +{"text": "BMC Public Health (2023) 23:232.10.1186/s12889-023-15127-7.Due to an error in the publication process the values in Tables\u00a01 and 3 are not properly aligned. The original article has been updated to correct this."} +{"text": "This article has been corrected to include the following funding disclosure which was accidentally omitted by the submitting author at the time of submission:\"Funding required for this study was made available by Banaras Hindu University under a seed grant for new faculties under IoE to Dr. Chetan Sahni. The letter number is \"R/Dev/D/IoE/Equipment/Seed Grant-II/2O22-23/48676\", dated 07.06.2022.\"The above statement is now visible in the 'Payment/services info' section of the\u00a0Ethics Statement and Conflict of Interest Disclosures. The submitting author deeply regrets this omission."} +{"text": "Correction to: BMC Zoology (2021) 6:810.1186/s40850-021-00073-xFollowing publication of the original article , the linWe have therefore added the link and changed the section \u2018Nomenclature acts\u2019 from:\u201cThe electronic edition of this article conforms to the requirements of the amended International Code of Zoological Nomenclature, and hence the new name contained herein is available under that Code from the electronic edition of this article.\u201dto:http://zoobank.org/), the online registration system for the ICZN and can be accessed under http://zoobank.org/urn:lsid:zoobank.org:pub:BE76B7A1-8FF7-4903-A1F7-9D52D03EAD83.\u201d\u201cThe electronic version of this article is considered a published work according to the International Commission on Zoological Nomenclature (ICZN). Therefore, the new name contained in the electronic version is officially published under the ICZN code (see articles 8.5\u20138.6) alone. The work and its content have also been registered in ZooBank (The original article has been"} +{"text": "Nature Communications 10.1038/s41467-023-36376-5, published online 28 February 2023Correction to: The original version of this Article contained an error in Equation (2) for fluidic pumping, and incorrectly read: The original version of the\u00a0Supplementary information"} +{"text": "Nature Communications 10.1038/s41467-023-41235-4, published online 14 September 2023Correction to: The original version of the Peer Review File associated with this Article contained 13 pages. The Peer Review File was updated shortly after publication to remove page 13 that had been erroneously added to the original file.Updated Peer Review file"} +{"text": "Correction to: The Cerebellumhttps://doi.org/10.1007/s12311-021-01360-6The original version of this article unfortunately contained some errors.Some editorial comments were inserted into figure legend 3 by mistake and the intended corrections in legend subpanels were not made.The original article has been corrected."} +{"text": "Correction:J Exp Clin Cancer Res38, 39 (2019)https://doi.org/10.1186/s13046-019-1052-zFollowing publication of the original article , an overThe images of Oct4 in TS and TS\u2009+\u2009DMSO groups were replaced and the correct Fig. The correction does not affect the overall results or conclusion of the article."} +{"text": "Correction to: Archives of Orthopaedic and Trauma Surgery 10.1007/s00402-022-04396-3The original version of this article unfortunately contained a mistake. All authors\u2019 first name and last name have been swapped.Viktoria HerterichLuzie HofmannWolfgang B\u00f6ckerHans PolzerSebastian Felix BaumbachHere is the correct first name and last name:The original article has been corrected."} +{"text": "Correction to: Pediatric Radiology (2023) 53:1659\u20131668https://doi.org/10.1007/s00247-023-05638-1The original article contains an error in the supplementary materials. The file originally titled \u201cSupplementary file1\u201d was an incorrect file \u2013 this has been removed. The order of the supplementary material has been corrected, and two additional previously missing supplemental files.The original article has been corrected."} +{"text": "Correction: Clinical and Experimental Medicine 10.1007/s10238-023-01006-3The original version of this article unfortunately contained a mistake. Author\u2019s family name Ianiro was incorrectly written as Laniro.The correct name should beGianluca Ianiro"} +{"text": "Correction: BMC Pediatr 23, 279 (2023)10.1186/s12887-023-04101-2Following publication of the original article , the autCorrect Tables\u00a02 and 3 captions:Table\u00a02: The effect of non-verbal music on the level of anxiety of hospitalized children.Table\u00a03: The effect of non-verbal music on vital signs of hospitalized children.Correct reference citations:A direct relationship between anxiety levels and vital signs such as systolic blood pressure, heart rate, and respiratory rate has been reported in various studies .The original article has been corrected."} +{"text": "Correction: Infection (2022) 50:1565\u20131572 10.1007/s15010-022-01938-0The original version of this article unfortunately contained a mistake. In the author list, the first and last names were incorrectly structured. The corrected author list is given above.Further in Fig.\u00a01 of this article there was an error: 7 (not 1) (87.5%) were HDV-RNA negative; the figure (Fig.\u00a0The original article has been corrected."} +{"text": "Cell Death and Disease 10.1038/s41419-022-04905-7, published online 09 May 2022Correction to: In this article the author\u2019s given and family name of Scaffa Cono have been interchanged. It should be read: Cono Scaffa.The original article has been corrected."} +{"text": "Scientific Reports 10.1038/s41598-023-40903-1, published online 21 August 2023Correction to: The Acknowledgements section in the original version of this Article was omitted. The Acknowledgements section now reads:\u201cThe researchers would like to acknowledge the Deanship of Scientific Research, Taif University for funding this work.\u201dThe original Article has been corrected."} +{"text": "Correction to: Virchows Archivhttps://doi.org/10.1007/s00428-023-03633-3The article title of the published version of the above article was incomplete. The correct title is shown as follows:\u201cThe many faces of nodal and splenic marginal zone lymphomas. A report of the 2022 EA4HP/SH lymphoma workshop\u201dThe original article has been corrected."} +{"text": "Nature Communications 10.1038/s41467-023-38887-7, published online 09 June 2023Correction to: This Article omits a declaration from the Competing Interests statement, which should have included the following: \u2018A patent \u201cInhibitors of ACK1/TNK2 Tyrosine Kinase\u201d covers (R)-9b compound. NPM and KM are named as inventors. These patents have been licensed by TechnoGenesys Inc. KM and NPM are co-founders of TechnoGenesys Inc., own stocks, and serve as consultants for TechnoGenesys Inc. The other authors declare no potential conflicts of interest.\u2019These errors have now been corrected in either the PDF or HTML versions of the article."} +{"text": "Scientific Reports 10.1038/s41598-021-90650-4, published online 07 June 2021Correction to: The original version of this Article contained an error in the Methods section, where the equation to convert from Watts to photons was incorrect.Under the sub-heading \u2018Quantifying photoreceptor isomerization rates for each light level\u2019The original Article has been corrected."} +{"text": "Correction to: Administration and Policy in Mental Health and Mental Health Services Research (2023) 50:520\u201353310.1007/s10488-023-01255-0The original version of the article unfortunately contained a mistake in Table\u00a03, which was introduced during formatting. The correct version of Table"} +{"text": "Scientific Reportshttps://doi.org/10.1038/s41598-023-34932-z, published online 24 May 2023Correction to: The original version of this Article contained an error in the Data availability section.\u201cAll the datasets used and/or analysed during the current study available from the corresponding author on reasonable request.\u201dnow reads:\u201cAll the datasets used and/or analysed during the current study available from the corresponding author on reasonable request.\u201dThe original Article has been corrected."} +{"text": "Scientific Reportshttps://doi.org/10.1038/s41598-023-30572-5, published online 07 March 2023Correction to: The original version of this Article contained an error in the Methods, under the subheading \u2018Governing equations\u2019, where the notation of Equation\u00a06 was incorrect.The original Article has been corrected."} +{"text": "Nature Communications 10.1038/s41467-023-41431-2, published online 19 September 2023Correction to: The original version of the\u00a0https://www.sasbdb.org/data/SASDQQ8/]\u2019. This has been corrected in both the PDF and HTML versions of the Article.The original version of this Article contained an error in the Data Availability statement, which incorrectly omitted the following: \u2018The SAXS data has been submitted to SASBDB with the deposition ID of SASDQQ8 [Updated Supplementary Information"} +{"text": "Cabeza et al., Chem. Sci., 2023, https://doi.org/10.1039/D3SC02709K.Correction for \u2018Fast and scalable solvent-free access to Lappert's heavier tetrylenes E{N(SiMe The originally published \u03b7solv = 1* , was omitted.In In The updated The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "Scientific Reports 10.1038/s41598-023-33641-x, published online 10 May 2023Correction to: The original version of this Article contained an error in the name of author Waleska Teixeira Caiaffa which was incorrectly given as Waleska Teixeira.The original Article has been corrected."} +{"text": "Nature Communications 10.1038/s41467-022-33798-5, published online 13 October 2022Correction to: The original version of this Article contained an error in the Acknowledgements, which incorrectly read \u2018B.L. and M.J.O. acknowledge support from the Oregon Department of Transportation from SPR807 and SPR 808 and National Science Foundation Grant ECI1929304.\u2019 The correct version states \u2018CMMI-2050047\u2019 in place of \u2018ECI1929304\u2019. This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Scientific Reports 10.1038/s41598-023-36332-9, published online 6 June 2023Correction to: The original version of this Article contained an error in Figure\u00a04 where the legend of the plot lines was incorrect in panel (b).The original Figure\u00a0The original Article has been corrected."} +{"text": "Correction to: Neurological Sciences (2023)10.1007/s10072-023-06598-yThe above article was published with error in author names. The correct capturing of the author name \u201cAna Lu\u00edsa de Almeida Marcelino\u201d has been updated.The Original article has been corrected."} +{"text": "Correction to: European Journal of Pediatrics https://doi.org/10.1007/s00431-023-04886-5DDC gene variant for patient 8 was incorrect in Fig.\u00a0In the original published version of the above article, the The original article has been corrected."} +{"text": "Correction to: European Journal of Ageing (2023) 20:1https://doi.org/10.1007/s10433-023-00749-yFollowing publication of the original article [1], the authors would like to change the corresponding author from Mia Niemi to Marjaana Sepp\u00e4nen.The original article has been corrected."} +{"text": "Retraction Note : Cellular & Molecular Biology Letters (2019) 24:13 10.1186/s11658-019-0137-1The Editor in Chief has retracted this article because of concerns about the data presented. Specifically, the flow cytometry plots presented in Fig.\u00a01E show a significant similarity to the plots presented in a previously published paper with no common authors . The Edi"} +{"text": "Cell Death and Disease 10.1038/s41419-022-05475-4 published online 16 December 2022Correction to: The original version of this article contained an error in an author name. The correct form should be Xin M. Liang not Xinmin Liang. The original article has been corrected."} +{"text": "Scientific Reports 10.1038/s41598-023-29034-9, published online 01 February 2023Correction to: The Supplementary Information file published with this Article contained an error. The Supplementary Information file did not include information specifying what was the patient treatment allocation: the alternate day group or the daily dose group. The original Supplementary Information file is provided below.This error has now been corrected in the Supplementary Information file that accompanies the original Article.Supplementary Information."} +{"text": "Scientific Reports 10.1038/s41598-023-45118-y, published online 26 October 2023Correction to: The original version of this Article contained errors in Figure 2 where the gray data curves were incorrectly captured in panels (a) and (b).The original Figure\u00a0The original Article has been corrected."} +{"text": "Scientific Reportshttps://doi.org/10.1038/s41598-023-27835-6, published online 14 February 2023Correction to: The original version of this Article contained an error in Figure 1 where the light-brown triangle indicating Gruppo Foca Monaca (2020) and the yellow square indicating positive samples were omitted in the legend. The original Figure\u00a0The original Article has been corrected."} +{"text": "Correction:J Exp Clin Cancer Res39, 63 (2020)10.1186/s13046-020-01567-1This study was supported by the National Natural Science Foundation of China (81873186).\u2019 to \u2018None.\u2019Following publication of the original article , the autThe correction does not affect the overall result or conclusion of the article. The original article has been corrected."} +{"text": "Cell Death and Disease 10.1038/s41419-023-06114-2, published online 04 September 2023Correction to: Due to a technical problem the uncorrected proof has been published online. The online PDF version of this article has been updated now."} +{"text": "Scientific Reportshttps://doi.org/10.1038/s41598-023-35282-6, published online 23 May 2023Correction to: The Acknowledgements section in the original Article was incomplete.https://www.g-red.eu/ in the framework of the \u201ceXperimental jOint inveRsioN project\u201d. This work was partly funded by CNES in the framework of the project \u201cExploitation de la mission Swarm\u201d. We thank the WDMAM working group for making the map available at www.wdmam.org.\u201d\u201cThis work was partially funded by Geomatics Research & Development srl now reads:https://www.g-red.eu/ in the framework of the ESA funded \u201ceXperimental jOint inveRsioN project\u201d. This work was partly funded by CNES in the framework of the project \u201cExploitation de la mission Swarm\u201d. We thank the WDMAM working group for making the map available at www.wdmam.org.This work was partially funded by Geomatics Research & Development srl The original Article has been updated."} +{"text": "Extensive acute cutaneous graft versus host disease: A rare case report of survival by Prajwal Pudasaini et al., https://doi.org/10.1002/ccr3.7545The cover image is based on the Case Report"} +{"text": "Cell Discovery (2023) 9:84Correction to: 10.1038/s41421-023-00567-7 published online 08 August 2023After the publication of this article, it was noticed that the ethical document was published as a Supplementary file by mistake. The ethical document has been removed now. We apologize for this error."} +{"text": "The correct Funding statement is: This research received financial support from Climate and Land Use Alliance (CLUA) (Grant Number G-2007-56990) and Brazilian National Cancer Institute Jos\u00e9 Alencar Gomes da Silva (INCA). We thank the Public Labour Prosecution Office for paying the publication fees through public civil action No. 0100263\u201341.2021.5.01.0005."} +{"text": "Scientific Reportshttps://doi.org/10.1038/s41598-023-34383-6, published online 02 May 2023Correction to: The original version of this Article contained a spelling error in the name of the author As\u2019ad Alizadeh which was incorrectly given as Asad Alizadeh.The original Article has been corrected."} +{"text": "Scientific Data 10.1038/s41597-022-01840-2, published online 24 November 2022.Correction to: The Acknowledgements section was missing from this article and should have read \u2018This work is supported by Youth Innovation Promotion Association CAS.\u2019"} +{"text": "Scientific Reports 10.1038/s41598-023-36244-8, published online 09 June 2023Correction to: The original version of this Article contained an error in the spelling of the author Pasquale D\u2019Angelo which was incorrectly given as Passquale D\u2019Angelo.The original Article has been corrected."} +{"text": "Correction to: European Journal of Applied Physiology 10.1007/s00421-023-05186-4The original version of this article unfortunately contained a mistake. Both Figs.\u00a03 and 4 are the same.The Fig.\u00a0The original article has been corrected."} +{"text": "Correction: Health Justice 11, 34 (2023)10.1186/s40352-023-00237-6This note is to inform readers that an incorrect earlier version of the article was erroneously published due to an error by the publisher. In the original article (Mutz & M\u00fcller, The html version of the figures in the original article (Mutz & M\u00fcller,"} +{"text": "Correction: Malaria Journal (2023) 22:48 https://doi.org/10.1186/s12936-023-04475-9An. stephensi larvae\u2019, they had referred to VectoBac WDG instead of FourStar\u00aeBriquets; in the Discussion and the Conclusion, they had referred to \u2018Bti VectoBac\u2019 where it should just say \u2018Bti\u2019. The authors thank you for reading and apologize for any inconvenience caused.Following publication of the original article , the aut"} +{"text": "Correction to: European Journal of Nuclear Medicine and Molecular Imaging10.1007/s00259-023-06273-6The authors regret that the \u201cMethods\u201d section of the abstract in the original article is incorrect. The correct \u201cMethods\u201d section appears below:Methods This study included a total of 3020 asymptomatic subjects who underwent whole-body [18F]FDG PET/MRI and chest HRCT examinations. All subjects received a 2\u20134-year follow-up for cancer development. Cancer detection rate, sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the [18F]FDG PET/MRI with or without chest HRCT were calculated and analyzed.The original article has been corrected.The original article can be found at 10.1007/s00259-023-06273-6."} +{"text": "Correction to: Applied Psychophysiology and Biofeedback10.1007/s10484-023-09588-0The original version of this article unfortunately contained a typo in co-author name.The second author\u2019s name should be spelled as Elena Franco instead it was published incorrectly as Eleno Franco.The original article has been corrected."} +{"text": "Correction to: European Journal of Orthopaedic Surgery & Traumatology 10.1007/s00590-022-03397-7The original version of this article unfortunately contained a mistake. One of the author\u2019s name and affiliation are incorrect. The correct author\u2019s name and affiliation should beChikumbutso C. MpangaQueen Elizabeth Central Hospital, Malawi."} +{"text": "Correction: Marine Life Science & Technology 10.1007/s42995-022-00160-zIn this article the graphics relating to Figs.\u00a02 and 3 captions had been interchanged; the figure(s) should have appeared as shown below.The original article has been corrected."} +{"text": "The correct corresponding author is Woo-Young Ahn. Woo-Young Ahn\u2019s email address is:"} +{"text": "Scientific Reportshttps://doi.org/10.1038/s41598-023-46790-w, Published online 13 November 2023Correction to: The original version of this Article contained an error in Table 2, where the legend of Figure 4 was incorrectly duplicated in the title of Table 2. The correct title of Table 2 is \u2018Chlorella cell counts\u2019.The original Article has been corrected."} +{"text": "Scientific Reports 10.1038/s41598-023-44822-z, published online 21 October 2023Correction to: The original version of this Article contained an error in the spelling of the author Jo\u00ebl Allanguillaume, which was incorrectly given as Jo\u00ebl Allanguillame.The original Article has been corrected."} +{"text": "Pediatric chance fracture with seatbelt syndrome: A case report by Hirohito Hirata et al., https://doi.org/10.1002/ccr3.7886The cover image is based on the Case Report"} +{"text": "Correction: J Nanobiotechnol (2021) 19:209 10.1186/s12951-021-00958-6Following publication of the original article , the autThis error does not affect the conclusions of this research. The authors apologize for not noticing this error before publication, and for any inconvenience caused.\u00a0The original article has been corrected."} +{"text": "Correction: Molecular Biology Reports (2022) 49:11037\u201311,04810.1007/s11033-022-07765-8.In this article, the statement in the Funding information section was incorrectly given as \u2018This study was supported by grants from the Zheji-ang Provincial Basic Technology Foundation of China (Grant No.GC20H160003)\u2019.The correct statement should read as \u2018This study was supported by grants from the Zhejiang Provincial Basic Technology Foundation of China (Grant No.LGC20H160003)\u2019. The Acknowledgements section is removed.The original article has been corrected."} +{"text": "Correction to:European Journal of Nutrition 10.1007/s00394-023-03141-9The original version of this article unfortunately contained a mistake. The x-axis visualization is somewhat blurred or diffused in Fig.\u00a01.The corrected Fig.\u00a0The original article has been corrected."} +{"text": "Scientific Reports 10.1038/s41598-022-12645-z, published online 24 May 2022Correction to: The original version of this Article contained an error in the spelling of the author Miroslaw Woloszyn which was incorrectly given as Miroslaw Wo\u0142oszyn.The original Article has been corrected."} +{"text": "Correction to:Neth Heart J 202310.1007/s12471-023-01795-yFigure\u00a02 was inadvertently missing from this article; the figure should have appeared as shown below. The caption to Fig.\u00a02 was inadvertently published below the infographic.The original article has been corrected."} +{"text": "Correction to: Journal of Cancer Educationhttps://doi.org/10.1007/s13187-023-02285-wThe original version of this article unfortunately contained a mistake.The author name Celia D\u00edez de los R\u00edos de la Serna is now corrected in the author group.The original article has been corrected."} +{"text": "Correction: European Journal of Medical Research (2023) 28:51210.1186/s40001-023-01476-xIn the original publication of the article, the \u201cEthics approval and consent to participate\u201d statement was incorrectly given as \u201cAll patients signed the informed consent. The present study obeyed Declaration of Helsinki. Study approval was obtained by the ethical committee of the authors\u2019 afliation (No. HPHP-2018-0095)\u201d. The corrected statement should read as \u201cAll patients signed the informed consent. The present study obeyed the Declaration of Helsinki. Study approval was obtained by the ethical committee of Hunan Provincial People\u2019s Hospital [2023]-59\u201d. The original article ["} +{"text": "Correction: European Journal of Medical Research (2023) 28:418 10.1186/s40001-023-01393-zIn the original version of this article, the given and family names of all the authors were swapped and published incorrectly as Ko\u0161ir Miha, Mo\u017eina Hugon and Podbregar Matej. The corrected author names should read as Miha Ko\u0161ir, Hugon Mo\u017eina and Matej Podbregar. The original article has been"} +{"text": "Scientific Reports 10.1038/s41598-021-85401-4, published online 16 March 2021Correction to: The Acknowledgements section in the original version of this Article was incomplete. It now reads:\u201cThis work was financed through grant FAF-C/2018/1190 of the Foundation against Cancer. Annick Van Greveling has been funded by a grant of Think-Pink. Liv Veldeman is recipient of post-doctoral clinical mandate of Foundation against Cancer. Prototype research is funded by StarTT 241 grant of the Industrial Research Fund, Ghent University). Dr Parkes was funded by a Marie Sklodowska-Curie Individual Fellowship \u201cMechanical ventilation for radiotherapy\u201d (894619) from the European Commission.\u201dThe original Article has been corrected."} +{"text": "Scientific Reports 10.1038/s41598-022-09675-y, published online 13 April 2022 Correction to: The Funding section in the original version of this Article was omitted. The Funding section now reads:\u201cAdrian Galdran is funded by the EU's Horizon 2020 R&I programme under the MSC grant agreement No 892297.\u201dThe original Article has been corrected."} +{"text": "Experimental & Molecular Medicine 10.1038/s12276-023-01078-x, published online 23 August 2023Correction to: After online publication of this article, the authors noticed an error in the \u201ccuproptosis\u201d section.The correct statement of this article should have read as below.\u201cThis was first described and the term was initially defined by Tsvetkov, P. et al. in 2019 .\u201dThe authors apologize for any inconvenience caused. The original article has been corrected."} +{"text": "Scientific Reports 10.1038/s41598-023-39894-w, published online 04 August 2023Correction to: The original version of this Article contained an error in Figure\u00a0The original Article has been corrected."} +{"text": "The correct author name is Tisha Mentnech.\u201cDetermining COVID-19's impact on an academic medical library's literature search service,\u201d 2022;110(3):316-22The PDF as published contained an incorrect author last name. The correct author name is David Petersen.\u201cIn Memoriam: Virginia M. Bowden,\u201d 2022;110(3):381-82The article was published with an incorrect HTML title and incorrect author affiliation information. The correct affiliation for Janna C. Lawrence is Director."} +{"text": "Correction: Andrews et al. BMC Med Inform Decis Mak (2021) 21:28210.1186/s12911-021-01640-5After the publication of the original article , the autFurthermore, the Conclusions section reads: \u201cRMT was used by more than three quarters of their patients\u201d. The statement should have read: \u201cRMT was used by patients of more than three quarters of respondents\u201d.The original article has been"} +{"text": "Correction to: Molecular Biology Reports 10.1007/s11033-023-08273-zIn the published article, the statement \u201cLili Zhang and Xiaojing Sun contributed equally to this work\u201d was missed.The original version of the article has been corrected."} +{"text": "Correction: BMC Ecology and Evolution (2022) 22:121 10.1186/s12862-022-02073-ySites and sampling.Following publication of the original article , the autThe sentence currently reads:Nine of these dates (Apr 16th\u2013May 6th) were subsequently analyzed for this part of the study based on visual confirmation of the period when eggs were present at regional spawning areas.The sentence should read:(Apr 18th\u2013May 7th) were subsequently analyzed for this part of the study based on visual confirmation of the period when eggs were present at regional spawning areas.Nine of these dates Moreover, the authors identified an error in Fig.\u00a02. The correct figure (Fig. The original article has been"} +{"text": "Acta Neuropathologica Communications (2023) 11:7910.1186/s40478-023-01569-yFollowing publication of the original article [1], the authors reported incorrect information in the \u2018Acknowledgements\u2019 section.The first part of the \u2018Acknowledgements\u2019 section originally read: Work was supported by Ministerio de Ciencia e Innovaci\u00f3n and FEDER funds: CP21/00116 and PI22/01171 to RG.The sentence should read: This study has been funded by Instituto de Salud Carlos III (ISCIII) through the project \u201cCP21/00116 and PI22/0117\u201d and co-funded by the European Union to RG.The original article has been"} +{"text": "BMC Psychology (2023) 11:160.10.1186/s40359-023-01202-6.Following publication of the original article, it was brought to the journal\u2019s attention that the following had been erroneously incorporated into the author list: \u2018Orcid Number of & Orcid Number\u2019. The author list has since been corrected. The publisher thanks you for reading and apologizes for any inconvenience caused by this formatting error."} +{"text": "Signal Transduction and Targeted Therapy 10.1038/s41392-023-01560-y, published online 9 August 2023Correction to: 1 the authors noticed one inadvertent mistake in Fig. 2b. The image of Sirus Red Dark in ApoE\u2212/\u2212 NPRC\u2212/\u2212 group was mistakenly inserted as the image of Sirus Red Bright in ApoE\u2212/\u2212 group during the final revision process. The correct figure was provided as follows.After online publication of the article,The original article has been corrected."} +{"text": "Nature 10.1038/s41586-022-05169-z Published online 5 October 2022Correction to: This paper was originally published under a standard Springer Nature license (\u00a9 The Author(s), under exclusive licence to Springer Nature Limited). It is now available as an Open Access paper under a Creative Commons Attribution 4.0 International license, \u00a9 The Author(s). The error has been corrected in the HTML and PDF versions of the article."} +{"text": "Communications Biology 10.1038/s42003-022-03730-0, published online 24 August 2022Correction to: In this article, the funding from \u2018Biotechnology and Biological Sciences Research Council\u2019 was omitted. The original article has been corrected."} +{"text": "Correction to: Virchows Archiv10.1007/s00428-023-03599-2\u201cCavity\u2011based lymphomas: challenges and novel concepts. A report of the 2022 EA4HP/SH lymphoma workshop\u201dThe article title of the original published version of the above article was incomplete. The correct title is shown as follows:The original article has been corrected."} +{"text": "Scientific Reports 10.1038/s41598-023-42660-7, published online 16 September 2023Correction to: The Acknowledgments section in the original version of this Article was omitted. The Acknowledgments section now reads:\u201cThis work was carried out as part of the ENIGMA project, a veterinary contingency project funded by the Danish Veterinary and Food Administration.\u201dThe original Article has been corrected."} +{"text": "Communications Biology 10.1038/s42003-022-04245-4, published online 24 November 2022.Correction to: In this article the hyperlink provided in the Code Availability statement in the sentence beginning \u2018The DrugnomeAI package\u2026\u2019 was incorrect. The original article has been corrected."} +{"text": "Scientific Reports 10.1038/s41598-022-07585-7, published online 08 March 2022Correction to: The Funding section in the original version of this Article was omitted. The Funding section now reads:\u201cF.R. acknowledges support from the European Union Horizon 2020 research and innovation programme (Grant Agreement No. 857470) and from the European Regional Development Fund via the Foundation for Polish Science International Research Agenda PLUS programme (Grant No. MAB PLUS/2018/8).\u201dThe original Article has been corrected."} +{"text": "Nature Communications 10.1038/s41467-023-38002-w, published online 25 April 2023Correction to: The original version of this Article contained an error in Figure 3f. The data for \u201cMECOM g-2\u201d was omitted. This has been corrected in the PDF and HTML versions of the Article."} +{"text": "Correction to: BMC Public Health23, 669 (2023)10.1186/s12889-023-15565-3The original publication of this article contained 2 errors:The affiliation \u201cShenzhen Center for Chronic Disease Control\u201d was not shown for Bingliang Lin.The acknowledgement section did not corresponding with the funding section.The original article has been updated."} +{"text": "Scientific Reports 10.1038/s41598-023-42106-0, published online 15 September 2023Correction to: The original version of this Article contained an error. In the original version of this article, the Acknowledgements section was missing. The Acknowledgements section now reads:\u201cThe researchers would like to acknowledge the Deanship of Scientific Research at Taif University for funding this work.\u201dThe original Article has been corrected."} +{"text": "Scientific Reportshttps://doi.org/10.1038/s41598-020-68275-w, published online 09 July 2020Correction to: The original version of this Article contained an error in Figure 3, panel (a), where the positioning of the atomic structure overlaid on the HAADF STEM image was incorrect.The original Figure\u00a0The original Article has been corrected."} +{"text": "Correction to: Clinical Oral Investigationshttps://doi.org/10.1007/s00784-022-04599-3In the list of authors, the surnames and first names are in the wrong sequence. The correct forms of author names are: Katri Croft, Sari Kervanto-Sepp\u00e4l\u00e4 and Eero Kerosuo.The original article has been corrected."} +{"text": "Nature Structural & Molecular Biology 10.1038/s41594-023-00997-7. Published online 18 May 2023.Correction to: In the version of this article initially published, Supplementary Table 1 was missing the \u201cHalf-life (h) DMSO\u201d column that now appears in the updated file available in the HTML version of the article."} +{"text": "Scientific Reportshttps://doi.org/10.1038/s41598-023-43399-x, published online 10 October 2023Correction to: The Funding section in the original version of this Article was omitted. The Funding section now reads:\u201cFujian Provincial Clinical Medical Research Center for First Aid and Rehabilitation in Orthopaedic Trauma (2020Y2014).\u201dThe original Article has been corrected."} +{"text": "Scientific Reports 10.1038/s41598-023-39829-5, published online 16 August 2023Correction to: The original version of this Article contained an error in the upper inset of Figure 4, where the atomic model was missing. The original Figure\u00a0The original Article has been corrected."} +{"text": "Nature Ecology & Evolution 10.1038/s41559-023-02115-8, published online 6 July 2023.Correction to: This paper was originally published under a standard Springer Nature license (\u00a9 The Author(s), under exclusive licence to Springer Nature Limited 2023). It is now available as an open-access paper under a Creative Commons Attribution 4.0 International license, \u00a9 The Author(s). The error has been corrected in the HTML and PDF versions of the article."} +{"text": "Nature Communications 10.1038/s41467-023-36834-0, published online 25 February 2023Correction to: The wrong Supplementary Movie 1 was originally published with this article; it has now been replaced with the correct. The original article has been corrected."} +{"text": "Correction: Yang et al. BMC Medical Informatics and Decision Making (2022) 22:23010.1186/s12911-022-01976-6After the publication of the original article , the autXn3 in the equation is incorrect as it should have been Xnn. Equation\u00a01 should have been written as follows:The original article has been"} +{"text": "Correction: Journal of Eating Disorders (2017) 5:50https://doi.org/10.1186/s40337-017-0180-0Following publication of the original article , the autThe incorrect author\u2019s name is Andrea Philipou. The correct author\u2019s name is Andrea Phillipou. The author group has been updated above and the original article has been"} +{"text": "Scientific Reports 10.1038/s41598-020-73267-x, published online 01 October 2020Correction to: The Acknowledgements section in the original version of this Article was incomplete.\u201cThe work was funded by the IVCC.\u201dnow reads:\u201cThe work was funded by the IVCC (supported by the Bill and Melinda Gates Foundation under Grant Number OPP1148615), and Medical Research Council (Grant Ref: MR/V001264/1).\u201dThe original Article has been corrected."} +{"text": "Correction: BMC Oral Health (2023) 23:576https://doi.org/10.1186/s12903-023-03275-6The figure legends for Figs. n\u2009=\u2009121), whereas the newer version is correct and corresponds with the content of the article (n\u2009=\u2009130).The \u2018number of cases\u2019 in Fig.\u00a0In the original publication of the article , the autThe corrected Figs. The original article has been corrected."} +{"text": "Correction to: Neuroinformatics10.1007/s12021-022-09613-3The original version of this article was revised to update the Figure 8 (panel B) image. The correct image should have a yellow shape as presented below.The original article has been corrected."} +{"text": "Helmet ventilation for pediatric patients during the COVID-19 pandemic: A narrative review By Mu S-C, Chien Y-H, Lai P-Z and Chao K-Y (2022) Front. Pediatr. 10:839476. doi: 10.3389/fped.2022.839476A Corrigendum on In the published article, there was a mistake in the \u201cExhaled Air Dispersion Distance\u201d section. This sentence previously stated: \u201cPlacement of nasogastric or orogastric tubes is a common measure for preventing aerophobia caused by NIV .\u201dThe corrected sentence appears below: \u201cPlacement of nasogastric or orogastric tubes is a common measure for preventing aerophagia caused by NIV .\u201dThe authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated."} +{"text": "Nature 10.1038/s41586-023-06442-5 Published online 23 August 2023Correction to: In the version of the article initially published, Gal Eyal\u2019s first affiliation\u2014The Mina & Everard Goodman Faculty of Life Sciences, Bar Ilan University, Ramat Gan, Israel\u2014was missing. This has now been added to the HTML and PDF versions of the article."} +{"text": "Correction to: Journal of Immigrant and Minority Health10.1007/s10903-023-01521-1The original version of this article unfortunately contained a typo in co-author name.The co-author\u2019s name should be spelled as Carolyn Beukeboom instead it was published incorrectly as Caroline Beukeboom.The original article has been corrected."} +{"text": "Correction to: Archives of Dermatological Research 10.1007/s00403-023-02586-6In this article the wrong figure appeared as Fig. 2; the Fig.\u00a0The original article has been corrected."} +{"text": "Correction: Nutrition & Metabolism (2023) 20:38https://doi.org/10.1186/s12986-023-00756-xbold\u00a0typeface.Following the publication of the original article , the autThe sentence currently reads:The total vegetarian score was calculated by summing all of the points from each respective section to generate a composite score ranging in values from 0 to 80.The sentence should read:14.The total vegetarian score was calculated by summing all of the points from each respective section to generate a composite score ranging in values from 0 to The incorrect Table The correct Table The original article has been"} +{"text": "Correction to: Khodayari et al. Respiratory Research (2022) Sep 6;23(1):23210.1186/s12931-022-02161-zAfter the publication of this original article [1], the authors identified an error in Fig.\u00a0The correct version of figure is given below."} +{"text": "Nature 10.1038/s41586-022-05675-0 Published 8 March 2023Correction to: This paper was originally published under a standard Springer Nature license (\u00a9 The Author(s), under exclusive licence to Springer Nature Limited). It is now available as an open-access paper under a Creative Commons Attribution 4.0 International license, \u00a9 The Author(s). The error has been corrected in the HTML and PDF versions of the article."} +{"text": "Incorrect FundingIn the published article, there was an error in the Funding statement. The Funding section misses the funding agency for AKP. The correct Funding statement appears below.FUNDINGAOP acknowledges funding from the Dutch Medical Research Council (ZonMw), grant no. 09120011910035. CVL and OR acknowledge funding from the Belgian National Fund for Scientific Research ; the French INSERM agency \u201cANRS/Maladies infectieuses \u00e9mergentes\u201d; the \u201cT\u00e9l\u00e9vie\u201d program of the F.R.S.-FNRS; ViiV Healthcare; the \u201cFondation Roi Baudouin\u201d; the Internationale Brachet Stiftung (IBS); the Walloon Region (\u201cFonds de Maturation\u201d); The \u201cAmis des Instituts Pasteur \u00e0 Bruxelles\u201d, asbl.; the University of Brussels (ULB - Action de Recherche Concert\u00e9e (ARC) grant); the NEAT (European AIDS Treatment Network) program; the University of Brussels (Action de Recherche Concert\u00e9e ULB grant); the Marie Sk\u0142odowska-Curie COFUND action; and the European Union\u2019s Horizon 2020 research and innovation program under grant agreement No 691119-EU4HIVCURE-H2020-MSCA-RISE-2015. CVL is \u201cDirectrice de Recherches\u201d of the F.R.S-FNRS. The laboratory of CVL is part of the ULB-Cancer Research Centre (U-CRC) . AKP acknowledges funding from the National Science Centre, Poland (Sonata Bis Grant UMO2018/30/E/NZ1/00874).The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated."} +{"text": "Correction: World Journal of Emergency Surgery (2023) 18:1https://doi.org/10.1186/s13017-022-00467-3Following publication of the original article [1], in PubMed the author name Daniele Bissacco under Team Dynamics Study Group has not been tagged and now it has been rectified.The original article has been corrected."} +{"text": "Correction to: BMC Surgery (2023) 17:110.1186/s12893-023-02201-5Following publication of the original article [1], the given and family names of Mari\u00e1n Kohut were incorrectly structured. The name was displayed correctly in all versions at the time of publication.The original article has been corrected."} +{"text": "Nature 10.1038/s41586-023-06352-6 Published online 30 August 2023Correction to: This paper was originally published under a standard Springer Nature license (\u00a9 The Author(s), under a exclusive licence to Springer Nature Limited). It is now available as an open-access paper under a Creative Commons Attribution 4.0 International license, \u00a9 The Author(s). The error has been corrected in the HTML and PDF versions of the article."} +{"text": "Correction to: Techniques in Coloproctology (2020) 24:965\u2013969 10.1007/s10151-020-02268-9In this article the wrong figure appeared as Fig.\u00a05; the figure should have appeared as shown below.The original article has been corrected."} +{"text": "Correction to: J Patient Rep Outcomes7, 33 (2023).10.1186/s41687-023-00576-wFollowing publication of the original article , the autThe incorrect author\u2019s name is: Joan Menauthoril.The correct author\u2019s name is: Joan Mendivil.The author group has been updated above and the original article has been"} +{"text": "Retraction Note: Orphanet Journal of Rare Diseases (2022) 17:6410.1186/s13023-022-02221-zThe Editor-in-Chief has retracted this article after the authors discovered 14 data calculation errors in Table\u00a01. The authors have been invited to submit a new manuscript for peer review. Kaiyue Hu disagrees with this retraction. None of the other authors has responded to correspondence from the Publisher about this retraction."} +{"text": "Communications Chemistry 10.1038/s42004-020-0259-4, published online 24 January 2020.Correction to: In this article the funding from \u2018the Natural Science Foundation of Guangdong Province for Distinguished Young Scholars (2019B151502051)\u2019 was omitted from acknowledgement. The original article has been corrected."} +{"text": "Scientific Reports 10.1038/s41598-023-30652-6, published online 02 March 2023Correction to\u00a0: The original version of the Article contained an error in the Author Information section, where the authors Laurent Freoa and Luis\u2011Miguel Chevin were incorrectly tagged as authors who jointly supervised the work.The original Article has been corrected."} +{"text": "Correction to: Acta Diabetologica (2023) 60:687\u2013695 10.1007/s00592-023-02046-7Author would like to update below correctionsThe second author\u2019s name is Liliana (name) Indelicato (surname) and not Indelicato Liliana as it appears at the moment.Affiliation number 3 is incorrectly updated for all authors instead of updating only for the author Maddalena Trombetta.The original article has been corrected."} +{"text": "Scientific Reportshttps://doi.org/10.1038/s41598-022-12376-1, published online 17 May 2022Correction to: The original version of this Article contained an error in the spelling of the author Dinesh Nath which was incorrectly given as Dinesh Nash.The original Article and accompanying Supplementary Information 1 file have been corrected."} +{"text": "Scientific Reports 10.1038/s41598-022-18554-5, published online 25 August 2022Correction to: The Funding section in the original version of this Article was omitted. The Funding section now reads:\u201cThe authors would like to thank the funding sources for this project, the UK Engineering and Physical Science Research Council and Ultraleap Ltd who part-funded L.C.\u2019s CASE studentship, and the EPSRC funder Grant Number\u2014EP/N509619/1.\u201dThe original Article has been corrected."} +{"text": "Correction: Parasites & Vectors (2023) 16:209 10.1186/s13071-023-05827-9Mustela lutreola\u2019 had been erroneously used instead of \u2018Mustela vison\u2019 in Table 1 and the graphical abstract of the article. The article has since been updated to correct this. The authors thank you for reading and apologize for any inconvenience caused.Following publication of the original article , the aut"} +{"text": "BMC Psychology (2023) 11:28110.1186/s40359-023-01336-7Following publication of the original article [1], the authors flagged the following error in the Results section of the Abstract: where it now says \u2018Metacognition, experiential avoidance, and the non-judging subscale of FFMQ-24 constituted\u2019, \u2018Mindfulness\u2019 had erroneously been written in place of \u2018Metacognition\u2019. The published article has since been corrected. The authors thank you for reading this erratum and apologize for any inconvenience caused."} +{"text": "The anion connects three AgI atoms through its O and two N-donor atoms. One neutral ligand functions in a monodentate mode; the other functions in a bridging mode, binding though its two N atoms. The coordination geometry of the two independent metal atoms is T-shaped; the manner of bridging gives rise to a layer motif parallel to (100). In the crystal, the nitrate ion is disordered over two positions in a 1:1 ratio, and is sandwiched between adjacent layers. O\u2014H\u22efO hydrogen bonding is present between nitrate ions and layers, and also between adjacent layers.The title coordination polymer, {[Ag DOI: Click here for additional data file.10.1107/S1600536812046090/xu5648Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "There was an error in the Funding statement. The correct statement is available below:The authors would like to thank the Liga Portuguesa Contra o Cancro-Centro Regional do Norte (Portuguese League Against Cancer) and Funda\u00e7\u00e3o para a Ci\u00eancia e Tecnologia (FCT), this project was partially sponsored by an unrestricted educational grant for basic research in molecular oncology from FCT (PTDC/SAU-FCF/71552/ 2006). This project was partially funded by FCT and by Operational Programme \"Factores de Competitividade\" (COMPETE) (PTDC/SAU-FC/71552/2006 and FCOMP-01-0124-FEDER-011113). ALT is a Doctoral degree grant holder from FCT (SFRH/BD/47381/2008). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."} +{"text": "The water O atom is invoved in three different hydrogen-bonding interactions, as donor to the acetate O atom and to the the ligand O atom and as acceptor to a ligand N atom.In the binuclear centrosymmetric title compound, [Cu DOI: 10.1107/S1600536812023070/gw2117Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "The Co2+ cations are connected by bridging formate anions into a three-dimensional coordination network in which the K+ cations are embedded. The asymmetric unit consits of one Co2+ cation located on a center of inversion, one K+ cation located on a twofold axis and two crystallographically independent formato anions, of which one is located on a twofold axis and the other occupies a general position.In the crystal structure of the title compound, poly[tri-\u03bc-formato-cobalt(II)potassium], [CoK(CHO DOI: 10.1107/S1600536811008737/kj2172Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In \"Migratory Passerine Birds as Reservoirs of Lyme Borreliosis in Europe,\" by P\u00e4r Comstedt et al., an error occurred in the second sentence of the first paragraph of Acknowledgments. The sentence should read \"This is report no. 214 from the Ottenby Bird Observatory.\"http://wwwnc.cdc.gov/eid/article/12/7/06-0127_article.htm.The corrected text appears in the online article at We regret any confusion these errors may have caused."} +{"text": "Three of the four O atoms of the chromate anion accept these bonds; the remaining Cr\u2014O bond length is notably shorter than the others. In the crystal, the anions and cations stack in layers lying parallel to (100): the hydrogen-bonding pattern leads to a three-dimensional network.In the title mol\u00adecular salt, (C DOI: 10.1107/S1600536814002463/hb7193Isup2.hklStructure factors: contains datablock(s) I. DOI: CCDC reference: crystallographic information; 3D view; checkCIF reportAdditional supporting information:"} +{"text": "The charge compensation is ensured by Li+ cations sharing a tetra\u00adhedral site with Co2+ ions . The anionic unit is formed by two octa\u00adhedra and three tetra\u00adhedra linked only by corners. The CoM1M2As2O19 units associate to an open three-dimensional framework containing tunnels propagating along the a-axis direction. One Na+ cation is located in the periphery of the tunnels while the other two are situated in the centres: all Na+ cations exhibit half-occupancy. The structure of the studied material is compared with those of various related minerals reported in the literature.The title compound, tetrasodium lithium cobalt aluminium hexa\u00ad(orthoarsenate), was synthesized by a solid state reaction route. In the crystal structure, Co DOI: 10.1107/S1600536813025233/vn2076Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "The PtII atom is in a distorted square-planar environment and is bound to the ligand via the P and amine N atoms in a cis fashion, with the chlorine atoms located at the two remaining sites.The title compound, [PtCl DOI: 10.1107/S1600536811040086/go2028Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "There is an error in the legend for Figure 7, \"Forest plot on recurrence.\" The correct Figure 7 legend is: 7a: forest plot on recurrence. Random-effects model. Figure 7b: forest plot on recurrence. Fixed-effect model."} +{"text": "The following information is missing from the Funding section: This study was partly funded by grants from the Lundbeck Foundation, DK nr R44-A4384 (Lars Hestbjerg Hansen), The Danish Council for Independent Research, Natural Sciences (S\u00f8ren S\u00f8rensen), and The Danish Council for Independent Research, Technology and Production, ref no: 09-090701 (Mette Burm\u00f8lle)."} +{"text": "There is an error in the left column of line 15 in Table 1. \"% HLA-DR+ lymphocytes\" is incorrect. The correct text for this column is \"% HLA-DR+ monocytes.\""} +{"text": "Both constituents are completed by crystallographic inversion symmetry. In the dimeric cation, the CoII atom is surrounded in a distorted octa\u00adhedral coordination by four N atoms from two chelating 2,2\u2032-bipyridine ligands and by two O atoms from the chelating oxalate anion. The Lindqvist-type anion exhibits the characteristic W\u2014O bond-length distribution, with the shortest bonds being the W\u2014Oterminal bonds and the longest being those to the central O atom.The asymmetric unit of the title compound, [Co DOI: 10.1107/S1600536810023007/wm2358Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "One SmIII ion is nine-coordinated by one N and five O atoms from the three ptc ligands and three aqua ligands in a distorted monocapped square antiprismatic geometry, and the other is eight-coordinated by one N and five O atoms from three ptc ligands and two aqua ligands in a 4,4\u2032-bicapped trigonal anti\u00adprismatic geometry. The ptc ligands brigde the SmIII ions into a three-dimensional polymeric framework. Extensive O\u2014H\u22efO hydrogen bonding is observed in the crystal structure.The asymmetric unit of the title compound, {[Sm DOI: 10.1107/S1600536812002462/cv5233Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "The complete cation is generated by crystallographic twofold symmetry, with the V atom lying on the rotation axis. The unusual ligand arose from nucleophilic attack on the coordinated nitrile by the thiol\u00adate precursor and reduction of nitrile to the imidate.In the title compound, [N(C DOI: 10.1107/S1600536810022014/hb5425Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The asymmetric unit consists of three crystallographically independent CoII cations, six thio\u00adcyanate anions and six coordinating bpe ligands in general positions. Additionally, three non-coordin\u00adating bpe ligands are present in the asymmetric unit with two of them located on a center of inversion. The CoII cations are connected by the bpe ligands into layers parallel to the bc plane. The crystal investigated was non-merohedrically twinned, with a fractional contribution of 0.261\u2005(2) for the minor domain.In the crystal structure of the title compound, [Co(NCS) DOI: 10.1107/S1600536812019009/bt5886Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "The cations are arranged in a head-to-tail fashion and form stacks along [100]. The central CuII atom of the anion is in a distorted tetra\u00adhedral environment.The asymmetric unit of the title compound, (C DOI: 10.1107/S1600536811012840/gk2356Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The number of the grant from Taipei Medical University (beginning \"TMU...\") is incorrect. The correct grant number is: TMU99-AE1-B11."} +{"text": "The crystal under investigation was twinned by pseudo-merohedry with a twofold rotation around the c axis as an additional twinning operation. The crystal structure is built of layers of distorted edge- and corner-sharing CeF8 square-anti\u00adprisms. The Cs+ cations are located between the layers and exhibit coordination numbers of nine. Upon compression, CsCeF5 undergoes an irreversible phase transition at about 1\u2005GPa.Single-crystals of CsCeF DOI: 10.1107/S1600536814003286/fj2660Isup2.hklStructure factors: contains datablock(s) I. DOI: CCDC reference: crystallographic information; 3D view; checkCIF reportAdditional supporting information:"} +{"text": "Two errors were introduced in the preparation of this article for publication.The title of Table 1 is missing. It should read: \"Table 1: NOHA-induced changes in mitochondrial proteins in MDA-MB-468 cells.\"Figure S1 was replaced by Figure 1. Please find a correct version of Figure S1 here: Click here for additional data file."} +{"text": "There was a typographical error in the second author's name. Fredrik Mattsson is correct. The correct citation is:Lagergren J, Mattsson F (2012) Diverging Trends in Recent Population-Based Survival Rates in Oesophageal and Gastric Cancer. PLoS ONE 7(7): e41352."} +{"text": "Two other coordination sites are occupied by the O atoms of a chelating carboxyl unit of another citrate; one of these atoms is additionally involved in bridging. The seventh coordination site is occupied by the O atom of the formally double-bond O atom of a neighboring citrate. The remaining two coordination sites are occupied by water mol\u00adecules. The citrate functions in a \u03bc3-bridging mode, connecting the metal atoms into a ribbon structure parallel to [010]. The structure is consolidated into a three-dimensional network by O\u2014H\u22efO hydrogen bonds.In the coordination polymer, {[Pr(C DOI: 10.1107/S1600536811023804/si2362Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "There is an error in the entry for Reference 6. The correct Reference 6 is:\u201cHarding EJ, Paul ES, Mendl M (2004) Animal behaviour - cognitive bias and affective state. Nature 427:312.\u201d"} +{"text": "In Table 1, data was omitted from the first section. Please see the corrected Table 1 here: The email address for Corresponding Author Mauricio Rodriguez-Lanetty is missing. His complete email address is: rodmauri@fiu.edu"} +{"text": "In our publication \"Effect of low tidal volume ventilation and inflammation in mice\" an errorThe pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2466/12/7/prepub"} +{"text": "This correction article relates to the response by Alan Barclay and Jennie Brand-Miller that was published as part of a research article authoredThe pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/13/1134/prepub"} +{"text": "The ZnII atoms are connected by the benzene-1,3-dicarboxyl\u00adate anions and the nitro\u00adgen ligand into layers parallel to the ac plane. The asymmetric unit consits of two crystallographically independent ZnII cations, two BDC anions and two water mol\u00adecules in general positions, as well as one-half of the BTB ligand that is completed by inversion symmetry.In the crystal structure of the title compound, [Zn DOI: 10.1107/S1600536810051433/nc2208Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "This compound is stable in the air for several hours, but rapidly decomposes at room temperature in solution. The cobalt(I) atom has s trigonal\u2013bipyramidal coordination enviroment in which the cyano group and one of the PMe3 groups are in the axial positions.The title compound, [Co(CN)(C DOI: 10.1107/S160053681101083X/om2401Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The cations and anions are linked in a complex three-dimensional framework by three types of strong hydrogen bonds , which form various ring and chain patterns of up to the ternary graph-set level.In the title compound, (C DOI: 10.1107/S1600536810045435/nk2068Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The HgII ions are four-coordinated in a distorted tetra\u00adhedral geometry, the coordination demand being satisfied either by four bridging iodide ligands or by three iodide ligands and a thio\u00adcarbonyl S atom. Due to the bridging nature of the dithione ligand, the coordination polymer has a two-dimensional structure, built up of undulated layers parallel to (001). There is an inversion center at the mid-point of the central C=C double bond.The title compound, poly[(\u03bc DOI: 10.1107/S1600536811006556/fi2103Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The asymmetric unit consists of half of the complex and one water mol\u00adecule (the full comlex being completed by application of inversion). In the crystal, the water mol\u00adecules participate in the formation of an intricate three-dimensional network of hydrogen bonds involving the coordinated water mol\u00adecule and the carboxyl\u00adate groups.In the title compound, [Co(C DOI: 10.1107/S1600536811053414/vm2142Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The alkali cations are occupationally disordered. The two K+ cations are situated in the inter\u00adlayer space, whereas the smaller Na+ cations are located in the cavities of the anionic framework. The K+ cations are surrounded by six and seven O atoms, the Na+ cations by seven and nine O atoms. The resulting coordination polyhedra of the two types of cations are highly distorted.The title compound, sodium potassium trialuminium tetra\u00adkis\u00ad(orthoarsenate), was prepared by solid-state reactions. The anionic framework consists of corrugated layers parallel to (010) and is made up of corner-sharing AlO DOI: 10.1107/S1600536813033850/wm2786Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supporting information:"} +{"text": "In the Funding section, the number of the grant from the European Community's Seventh Framework Programme was missing. The sentence should read \u201cThis was partly supported by the European Community's Seventh Framework Programme (FP7/2007\u20132013) ENGAGE (contract no. HEALTH-F4-2007-201413)\""} +{"text": "Additional author: During the editing process of the article \u201cInduction of labour with a Foley catheter or oral misoprostol at term: the PROBAAT-II study, a multicentre randomised controlled trial\u201c an authoThe pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2393/13/183/prepub"} +{"text": "The asymmetric unit contains a half-cation and a half-anion. The atoms of the cation occupy general positions about an inversion centre, which is located at the midpoint of the central C\u2014C bond. The Co atoms lie on a twofold rotation axis. The slightly distorted tetra\u00adhedral coordination environment around the metal atom consists of two Cl atoms and their symmetry-related pairs.The crystal structure of the title compound, (C DOI: 10.1107/S1600536811050744/hp2021Isup2.hkl Structure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The following information was missing from the Funding section: J. Duch and M. Sales-Pardo\u2019s work have been partially supported by the Spanish DGICYT under project FIS2010-18639."} +{"text": "After publication of this work , we noteThe correct data are shown in the following revised Table two Table\u00a0 here:The revised text in the results should read:Patients had a median of 1.0 comorbidity recorded at baseline admission, although the range was wide , with some evidence of an increase in comorbidity burden over time Table two (Table The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6963/13/179/prepub"} +{"text": "The anion lies on a crystallographic inversion center, which is located at the mid-point of the central C\u2014C bond. The K+ cation is coordinated by six O atoms, two from the chelating carboxyl\u00adate group of the anion and four from four neighboring and monodentately binding anions, giving rise to an irregular [KO6] coordination polyhedron. The coordination mode of the cation leads to the formation of K/O layers parallel to (100). These layers are linked by the nearly coplanar anions into a three-dimentional network.The title salt, [K DOI: 10.1107/S1600536812008513/wm2592Isup2.hklStructure factors: contains datablock(s) I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "The trimer unit has an Au\u22efAu distance of 3.1687\u2005(3)\u2005\u00c5. In both the monomeric and trimeric units, the AuI atoms are also bonded to two S atoms. Within the trimeric unit, the AuI atoms exist in differing environments; one Au atom has a T-shaped three-coordinate geometry while the other has a square-planar four-coordinate geometry. The AuI atom of the monomer adopts a linear two-coordinate geometry. The extended structure can be described as a three-dimensional coordination polymer consisting of chains of Ba atoms bridged by thio\u00adcyanate N atoms. These chains are cross-linked via the gold monomeric and trimeric units.The noteworthy structural feature of the title complex, poly[acetonitrile\u00adtetra-\u03bc DOI: 10.1107/S1600536810021276/bg2340Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Protocol for the isotoxic intensity modulated radiotherapy (IMRT) in stage III non-small cell lung cancer (NSCLC): a feasibility study. BMJ Open 2016;6:e010457. This paper was published under an incorrect license. The correct license is CC BY and the following statement should be applied:Haslett K, Franks K, Hanna GG,"} +{"text": "The first sentence of the second paragraph of the \u201cAssemblies\u201d subsection of the Methods should have cited reference 34 instead of 33. The correct sentence should read: The Illumina datasets were assembled using SOAPdenovo 1.05 [34] using following parameters: \u2018\u2018-K 23 -L 70 -M 3 -u -R -F\u2019\u2019.The fourth sentence of the first paragraph of the \u201cImportance of Quality Control for Illumina Data\u201d subsection of the Results should have cited reference 34 instead of 33. The correct sentence should read: However, the most commonly used assemblers for Illumina data (SOAPdenovo [34], Velvet [45], SSAKE [46] do not use quality values for assembly."} +{"text": "Drs. Yang, Zeng, and Zhang share first authorship. The article has since been corrected online.In the article \u201cSurgical Treatment and Clinical Outcome of Nonfunctional Pancreatic Neuroendocrine Tumors: A 14-Year Experience From One Single Center\u201d,"} +{"text": "Yeh, Chen, and Yaw-Bin Huang was originally presented incorrectly. The article has since been corrected online.In the article \u201cEpidemiology and Medication Utilization Pattern of Aortic Dissection in Taiwan: A Population-Based Study\u201d,"} +{"text": "In the Funding section, a grant number from the funder National Institute of Neurological Disorders and Stroke (NINDS) is missing. The additional grant number is: #1R56NS091616-01."} +{"text": "This corrigendum presents correct sentences of background and conclusions parts of abstract which contained two typographical errors.Background: Chronic hepatitis B is one of the world's major health concerns.Conclusions: There was no association between TGF-\u03b21 -509C/T and +915G/C polymorphisms with chronic hepatitis B and it seems that these changes do not play a significant role in increasing the risk of chronic infection in Iranian patients.Corresponding author:Dr. Seyed Reza Mohebbi"} +{"text": "Nature Communications7: Article number: 10959 10.1038/ncomms10959 (2016); Published: 03242016; Updated: 06272016In this Article, there is an error in the labelling of the western blot in Fig. 3f. The labels indicating the presence of pterosin B in each lane should read \u2018\u2212++ +'. The correct version of Fig. 3 appears below.Figure 3"} +{"text": "The Data Availability Statement for this paper is incorrect. The correct statement is: Data are available at MG-RAST (4676501.3\u20134676559.3), project 16202."} +{"text": "The publisher apologizes for the error.There is an error in the title of the correction published October 2, 2015. The correct title is: Correction: Glucose-Dependent Insulin Secretion in Pancreatic \u03b2-Cell Islets from Male Rats Requires Ca"} +{"text": "In the Funding section, the grant number from the funder Ministerio de Econom\u00eda y Competitividad is listed incorrectly. The correct grant number is: SAF/FEDER 2013-49076-P."} +{"text": "Ming-Chia Lin's affiliation was not reported in full. Dr. Lin is affiliated with the E-Da Hospital. The articles have since been corrected online.In the articles \u201cHigh Risk of Depressive Disorders in Patients With Gout: A Nationwide Population-Based Cohort Study\u201d,"} +{"text": "Nature Communications6: Article number: 885510.1038/ncomms9855 (2015); Published 12042015; Updated 02152016In Fig. 4k of this Article, the first row in the far-right column of the table contains a typographical error and should read \u2018VS3'. The correct version of Fig. 4 appears below.Figure 4"} +{"text": "Ming-Chia Lin's affiliation was not reported in full. Dr. Lin is affiliated with the E-Da Hospital. The articles have since been corrected online.In the articles \u201cAtrial Fibrillation is Associated With Morphine Treatment in Female Breast Cancer Patients: A Retrospective Population-Based Time-Dependent Cohort Study\u201d,"} +{"text": "Scientific Reports6: Article number: 24592;10.1038/srep24592 Published online: 04192016; Updated: 06022016The Acknowledgements section in this Article is incomplete.\u201cThe authors gratefully acknowledge Raffaele Colombelli for precious suggestions and insights on the application of coupled-mode models to strongly-coupled systems\u201d.should read:\u201cThe authors gratefully acknowledge Raffaele Colombelli for precious suggestions and insights on the application of coupled-mode models to strongly-coupled systems. This work was in part supported by the European Research Council through the Advanced Grant SOULMAN (ERC-FP7-321122)\u201d."} +{"text": "Sergentomyia huberti comb. nov.\u201dThe type-species is not designated in the published article. The type-species should appear in the Results as: \u201cType-species of this new subgenus:"} +{"text": "Tae-Hyun Kim's name was originally misprinted as Tae-Hyun Lee. The article has since been corrected online.In the article \u201cBeyond Volume: Hospital-Based Healthcare Technology for Better Outcomes in Cerebrovascular Surgical Patients Diagnosed With Ischemic Stroke: A Population-Based Nationwide Cohort Study From 2002 to 2013\u201d,"} +{"text": "The authors would like to add the following Acknowledgements section:\u201cAcknowledgementsThis work was funded by the National Science Centre in Poland grant UMO-2012/05/E/NZ6/03314.\u201d"} +{"text": "Hypercohones A-C (1\u20133), along with five other known hypercohones (4\u20138), were isolated from the aerial parts of Hypericum cohaerens. The structures of 1\u20133 were elucidated on the basis of comprehensive spectroscopic analysis. The inhibitory activities of these isolates against five human cancer cell lines in vitro were tested. Three new Supplementary material is available for this article at 10.1007/s13659-013-0032-9 and is accessible for authorized users. Supplementary material, approximately 1.91 MB."} +{"text": "The article was republished on April 14, 2015 to include a reference that was missing from the published article. The reference should be: [11] Thomson RC, Wang IJ, Johnson JR (2010) Genome-enabled development of DNA markers for ecology, evolution and conservation. Molecular Ecology 19: 2184\u20132195.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "Oskar\u201d should be \u201coskar.\u201d The correct title is: Translational Activation of oskar mRNA: Reevaluation of the Role and Importance of a 5' Regulatory Element. The correct citation is: Kanke M, Macdonald PM (2015) Translational Activation of oskar mRNA: Reevaluation of the Role and Importance of a 5' Regulatory Element. PLoS ONE 10(5): e0125849. doi:10.1371/journal.pone.0125849In the title of this article, \u201cThe publisher apologizes for this error."} +{"text": "Scientific Reports6: Article number: 1979910.1038/srep19799; published online: 02022016; updated: 03012016The Acknowledgements section in this Article is incomplete.\u201cWe thank Carmen Christoph for excellent technical assistance. This work was supported by the F\u00f6rderverein des Tumorzentrums Erlangen, funding grants from National Science Fund for Excellent Young Scholars China (No. 31422049) Hubei Science Fund for Excellent Scholars China (No. 2015CFA042) and Sino Kazakh inter governmental scientific and technological cooperation project to Z Cao.\u201dshould read:\u201cWe thank Carmen Christoph for excellent technical assistance. This work was supported by the F\u00f6rderverein des Tumorzentrums Erlangen, funding grants from National Science Fund for Excellent Young Scholars China (No. 31422049) Hubei Science Fund for Excellent Scholars China (No. 2015CFA042) and Sino Kazakh inter governmental scientific and technological cooperation project to Z. Cao. T. Xu performed the present work under supervision of Dr. Nicolai Savaskan in fulfillment of the requirements for obtaining the degree Dr. med. at the Friedrich-Alexander University of Erlangen-N\u00fcrnberg (FAU). We acknowledge gratefully support by the Friedrich-Alexander-University of Erlangen-N\u00fcrnberg (FAU) and the German Research Council (DFG) for patronizing the \u2018Open Access Publication\u2019 project.\u201d"} +{"text": "Nature Communications6: Article number: 879910.1038/ncomms9799 (2015); Published 11192015; Updated 12142015Due to errors in the production process data were missing in the original version of Fig. 4b. The correct version of this figure appears below.Figure 4"} +{"text": "Nature Communications6: Article number: 783010.1038/ncomms8830 (2016); Published 07222015; Updated 02152016The original version of this article was published with an incorrect label for Fig. 4c. The labels \u2018no tacrine' and \u20183\u2009mM tacrine' were associated with the incorrect fluorescence micrographs. The correct version of Fig. 4 appears below.Figure 4"} +{"text": "Scientific Reports6: Article number: 19845; 10.1038/srep19845 published online: 01222016; updated: 03042016.The original version of this Article contained an error in the spelling of the author Gorka Zamora-L\u00f3pez, which was incorrectly given as Zamora-L\u00f3pez Gorka. This has now been corrected in the PDF and HTML versions of the Article."} +{"text": "The funding statement for this article should read as follows:http://www.esf.org/) and the KU Leuven (www.kuleuven.be) for the IDO-BioCo3 project and KU Leuven Excellence Center project PF/2010/007. EDG acknowledges the KU Leuven for the Grant F+/11/033. MD acknowledges the Fonds de la Recherche Scientifique\u2014FNRS (F.R.S.\u2013FNRS\u2014www.fnrs.be) and the European Research Council (ERC\u2014erc.europa.eu) ERC Advanced Grant \u201cE-SWARM: Engineering Swarm Intelligence Systems\u201d (grant 246939). AET acknowledges the Scientific and Technological Research Council of Turkey (Tubitak\u2014www.tubitak.gov.tr/en) grant TUBITAK-2219. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.\u201d\u201cEF acknowledges the Fund for Scientific Research (FWO)\u2013Flanders (grant 12N7515N). EF, AET and TW acknowledge the European Science Foundation \"H2Swarm\" program ("} +{"text": "Medicine, Dr. Shih's name was originally spelled incorrectly. The article has since been corrected online.In the article \u201cAssociation of Brachial-Ankle Pulse Wave Velocity and Cardiomegaly With Aortic Arch Calcification in Patients on Hemodialysis\u201d,"} +{"text": "The following information is missing from the Funding section: This work is funded by National Funds through FCT\u2014Foundation for Science and Technology under the Strategic Project PEst-OE/AGR/UI0115/2014.The complete, correct funding statement should read: Carla Marisa R. Varanda is the recipient of a Post doctoral fellowship from Funda\u00e7\u00e3o para a Ci\u00eancia e a Tecnologia (FCT), SFRH/BPD/76194/2011. This work has been supported by FEDER and National funds, through the Programa Operacional Regional do Alentejo (InAlentejo) Operation ALENT-07-0262-FEDER-001871/Laborat\u00f3rio de Biotecnologia Aplicada e Tecnologias Agro-Ambientais. Additionally, this work is funded by National Funds through FCT\u2014Foundation for Science and Technology under the Strategic Project PEst-OE/AGR/UI0115/2014. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."} +{"text": "The ninth author\u2019s name is spelled incorrectly. The correct name is: Nobukata Kazawa.In Table 3 the description of the dose of pirfenidone is incorrect. The correct dose of pirfenidone is: 600 mg (n)/1200mg(n)/1800mg(n). Please see the corrected"} +{"text": "The following information is missing from the Funding section: CNPq grant (PDJ 150069/2014-6) to RNL."} +{"text": "Ming-Chia Lin's affiliation was not reported in full. Dr. Lin is affiliated with the E-Da Hospital. The articles have since been corrected online.In the articles \u201cDry Eye Syndrome Risks in Patients With Fibromyalgia: A National Retrospective Cohort Study\u201d,"} +{"text": "Scientific Reports5: Article number: 1615610.1038/srep16156; published online: 11042015; updated: 12092015In the HTML version of this Article, there is an error in Table. 1\u201cStructure-activity relationship of Btk inhibitors.\u201dshould read:\u201cMean Reaction Times (ms) and SE for cued and uncued trials as a function of response (yes/no) and congruency between first shape and target (congruent=same shape incongruent=different shape) in the three experiments.\u201d"} +{"text": "Balakrishnan et al. (2013) should have been cited in the first paragraph of \u201cMethods\u201d for HAP (household air pollutants). The correct sentence and reference are provided below.In \u201cAn Integrated Risk Function for Estimating the Global Burden of Disease Attributable to Ambient Fine Particulate Matter Exposure\u201d by Burnett et al. [Environ Health Perspect 122:397\u2013403 (2014);\u20022.5 exposure from HAP was estimated for study subjects using coal or biomass for cooking and for those study subjects using cleaner fuels to integrate this information into our IER risk model.The equivalent long-term PM2.5 from solid cookfuel use: results from measurements and modeling in India for estimation of the global burden of disease. Environ Health 12:77; doi:10.1186/1476-069X-12-77.Balakrishnan K, Ghosh S, Ganguli B, Sambandam S, Bruce NG, Barnes DF, et al. 2013. State and national household concentrations of PMThe authors regret the error."} +{"text": "This erratum corrects \u201cPredictors of malignancy in pancreatic head mass: za prospective study\u201d. The Pan African Medical Journal. 2011;9:30. The original version of this article containe"} +{"text": "Nature Medicine 10.1038/s41591-021-01544-x, published online 9 December 2021.Correction to: This paper was originally published under standard Springer Nature license (\u00a9 The Author(s), under exclusive licence to Springer Nature America, Inc.). It is now available as an Open Access paper under a Creative Commons Attribution 4.0 International license, \u00a9 The Author(s). The error has been corrected in the HTML and PDF versions of the article."} +{"text": "Communications Biology 10.1038/s42003-022-03193-3, published online 15 March 2022.Correction to: In this Q&A the affiliation details for Victor Garcia were incorrectly given as \u201cAssociate Professor at New York Medical College\u201d but should have been \u201cAssistant Professor at New York Medical College\u201d.Both errors have now been corrected in the HTML and PDF versions of the Q&A."} +{"text": "Scientific Reports 10.1038/s41598-022-11433-z, published online 17 May 2022Correction to: The original version of this Article contained an error in the Data Availability section where\u201cThe stimuli and processed data files will be available available at: figshare.\u201dnow reads:https://doi.org/10.6084/m9.figshare.19780264.\u201d\u201cThe stimuli and processed data files are available at The original Article has been corrected."} +{"text": "Correction to: Biological Trace Element Researchhttps://doi.org/10.1007/s13187-022-02196-2The original version of this article unfortunately contained a mistake. The name of \u201cJohnathan Soggee\u201d is now corrected in the author group.The original article has been corrected."} +{"text": "Nature Communications 10.1038/s41467-022-32076-8, published online 29 July 2022Correction to: The original version of this Article incorrectly cited reference 85 instead of reference 84 in the methods section \u2018An. stephensi immune-responsive gene expression\u2019. This has now been corrected in both the PDF and HTML versions of the Article."} +{"text": "Correction: Chiropractic & Manual Therapies (2022) 30:51https://doi.org/10.1186/s12998-022-00460-2Following the publication of the original article , two errCracking joints header:Under the 4\u00a0mm; a relatively large separation for an MCP joint!After this crack, with increasing load the line returns to a near horizontal gradient once again, until it reaches a maximum separation of approximately Fig.\u00a011:In the legend of Kawchuk et al. [33], was \u201cT1 static images of the left hand in the resting phase before cracking (left). The same hand following cracking with the addition of a post-cracking distraction force (right). Note the dark, intraarticular void (yellow arrow)\u201d.MR images before and after cavitation in an MCP joint. The original caption of this figure, reproduced from The original article has been"} +{"text": "Correction to: Nature Communications 10.1038/s41467-022-33470-y, published online 01 October 2022The original HTML version of this Article was updated shortly after publication because the previous HTML version linked to the LaTeX source file of the\u00a0Updated Supplementary Information"} +{"text": "Scientific Reports 10.1038/s41598-022-22687-y, published online 21 October 2022Correction to: The original version of this Article contained an error in the spelling of the author Mohammad Hossein Modarressi, which was incorrectly given as Mohammad\u2011Hossein Modarresi.The original Article has been corrected."} +{"text": "Correction: Int J Equity Health 21, 56 (2022)https://doi.org/10.1186/s12939-022-01661-0After publication of this article , the autIt was also reported that the copyright holder for this article was incorrectly given as 'The Author(s)', but should have been 'WHO'.The original article has been"} +{"text": "Correction to: Langenbeck's Archives of Surgeryhttps://doi.org/10.1007/s00423-022-02638-xThe original version of this article unfortunately contained a small mistake on page 11, the in-text citation [17] should be changed to [14].The corrected in-text citation is shown below:Heimann et al. indicated that number of bowel resections prior to hernia repair predicted recurrence of IH [14].The original article has been corrected."} +{"text": "Nature Microbiology 10.1038/s41564-021-01049-w, published online 31 January 2022.Correction to: This paper was originally published under a standard Springer Nature license (\u00a9 The Author(s), under exclusive licence to Springer Nature America, Inc.). It is now available as an Open Access paper under a Creative Commons Attribution 4.0 International license, \u00a9 The Author(s). The error has been corrected in the HTML and PDF versions of the article."} +{"text": "Scientific Reports 10.1038/s41598-022-24307-1, published online 17 November 2022Correction to: The original version of this Article contained errors in Figure\u00a04 where the upper panels did not match the legend by colour. In the right lower panel, the order of estimates was incorrectly switched. The original Figure\u00a0The original Article has been corrected."} +{"text": "Correction: BMC Med Educ 22, 726 (2022)https://doi.org/10.1186/s12909-022-03776-yFollowing publication of the original article , the autThe incorrect author name is: Miguel K\u00fcnder Kl\u00fcnderThe correct author name is: Miguel Kl\u00fcnder Kl\u00fcnderAlso, they informed us that first name and last name of author Eduardo Bracho Blanchet has been incorrectly tagged.The correct first name is: EduardoThe correct last name is: Bracho BlanchetThe author group has been updated above and the original article has been"} +{"text": "Nature Methods 10.1038/s41592-022-01423-4, published online 8 April 2022.Correction to: In the version of this article initially published, the Acknowledgements section for Ryan M. Layer did not include the funding information \u201cNIH/NCI grant no. UO1 CA231978.\u201d The grant has been included in the HTML and PDF versions of the article."} +{"text": "Scientific Reports 10.1038/s41598-021-03781-z, published online 20 December 2021Correction to: The Acknowledgements section in the original version of this Article was incomplete.\u201cThis study was funded by a psychological/neurological disease research grant (Grant number: 2-4) provided by the National Center of Neurology and Psychiatry, Japan. We are indebted to Mr. Ben Cooper for his medical writing services.\u201dnow reads:\u201cThis study was funded by a psychological/neurological disease research grant (Grant number: 2-4) provided by the National Center of Neurology and Psychiatry, Japan and supported by SHISEIKAI Scientific Award. We are indebted to Mr. Ben Cooper for his medical writing services.\u201dThe original Article has been corrected."} +{"text": "Correction: BMC Cardiovascular Disorders (2023) 23:33https://doi.org/10.1186/s12872-023-03042-zFollowing the publication of the original article , the autAlso, in this article the Ethics statement was incorrectly given as \u2018Ethical approval was obtained from the University and Hospital Human Research Ethics Committee .\u2019 but should have been \u2018Ethical approval was obtained from the Hospital Human Research Ethics Committee \u2019.The original article has been corrected."} +{"text": "Scientific Reportshttps://doi.org/10.1038/s41598-022-05331-7, published online 04 February 2022Correction to: The original version of this Article contained errors in Table 1, where the Metric \u2018Daily survey: available observations per subject\u2019 was incorrect for the Mean (column \u2018M\u2019) and the Median (column \u2018Med\u2019). The correct and incorrect values appear below.Incorrect:Correct:The original Article has been corrected."} +{"text": "In the published article, there was an error in the funding statement. The grant number for the Excellence Initiative of the German Federal and State Governments was listed as \u201cG:(DE-82) EXSSF-SFSynt001\u201d but should be \u201cG:(DE-82) EXS-SF-SFDdM013\u201d. The correct Funding statement appears below."} +{"text": "Nature Communications 10.1038/s41467-022-30098-w, published online 11 May 2022Correction to: \u201cThe original version of this Article contained an error in Fig. 5, in which the figure was inadvertently replaced with an earlier version containing incorrect AFG values. The correct version of Fig. 5 is:which replaces the previous incorrect version:This has been corrected in both the PDF and HTML versions of the Article.\u201d"} +{"text": "Scientific Reportshttps://doi.org/10.1038/s41598-023-28142-w, published online 17 January 2023Correction to: The original version of this Article contained an error in the Acknowledgements section.\u201cThis research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.\u201dnow reads:\u201cThis work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. 2022R1F1A1062794).\u201dThe original Article has been corrected."} +{"text": "Communications Biology 10.1038/s42003-022-03777-z, published online 17 August 2022.Correction to: In the Acknowledgements section, \u201cThis work was supported by the Translational Research Institute for Space Health award FIP0005 (to Dr. Goukassian)\u201d should have read \u201cThis work is supported by the Translational Research Institute for Space Health through NASA Cooperative Agreement NNX16AO69A \u2013 FIP0005\u201d.The original article has been corrected."} +{"text": "International Breastfeeding Journal (2022) 17:63 10.1186/s13006-022-00500-wFollowing publication of the original article [1], the authors flagged that \u2018Formerly\u2019 had been erroneously included in affiliation 3. The published article has since been corrected and the corrected affiliation may be seen in this erratum."} +{"text": "Scientific Reportshttps://doi.org/10.1038/s41598-022-15877-1, published online 11 July 2022Correction to: The original version of this Article contained an error in Figure 1, where the labels for \u2018Flag %\u2019 and \u2018No Flag %\u2019 were interchanged in the legend of the graphs. The original Figure\u00a0The original Article has been corrected."} +{"text": "Retraction Note: Stem Cell Research & Therapy (2021) 12:85https://doi.org/10.1186/s13287-021-02154-7The authors have retracted this article. After publication the authors noted that they used the incorrect \u03b2-actin protein expression data in Fig.\u00a01E. Yao Fu did not participate in the experiments presented and were included in the author list without their permission. Meng Zhou, Yao Fu and Zi Wang agree to this retraction. Mingjiao Chen and Jin Li do not agree to this retraction."} +{"text": "Scientific Reports 10.1038/s41598-022-25801-2, published on 9 December 2022Correction to:The original version of this Article omitted the Funding section. The Funding section now reads:\u201cWe thank VT\u2019s OASF for supporting us in publishing this article.\u201dThe original Article has been corrected."} +{"text": "Correction to Current Nutrition Reports 10.1007/s13668-023-00456-1The author \u201cLudovica Verde\u201c should be retained as secondly listed in the authorgroup instead of last based on the accepted manuscript.The original article has been corrected."} +{"text": "Nature Communications 10.1038/s41467-019-12536-4, published online 24 October 2019Correction to: The original version of this Article contained an error in Table\u00a0which replaces the previous incorrect version:This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Correction to: BMC Medical Genomics (2021) 13:19610.1186/s12920-021-01054-2Following publication of the original article [1], it was reported that this article should have been published in Volume 14, Supplement 3.The details of the supplement in which this article ought to have been published are given below:About this supplementhttps://bmcmedgenomics.biomedcentral.com/articles/supplements/volume-14-supplement-3This article has been published as part of BMC Medical Genomics Volume 14 Supplement 3 2021: Selected articles from the 19th Asia Pacifc Bioinformatics Conference (APBC 2021): medical genomics The full contents of the supplement are available at The publisher apologizes for any inconvenience caused."} +{"text": "Nature Communications 10.1038/s41467-022-29310-8, published online 23 March 2022Correction to: In this article the funding from \u2018European Union\u2019s Horizon 2020 research and innovation program under VEO grant agreement No. 874735\u2019 was omitted. The original article has been corrected."} +{"text": "Mucosal Immunology 10.1038/s41385-022-00557-0, published online 05 September 2022Correction to: The original version of this article unfortunately contained a mistake in the figure legends as the following statement was missing \u201cCreated with BioRender.com\u201d. The original article has been corrected."} +{"text": "Nature Communications 10.1038/s41467-022-30773-y, published online 27 July 2022Correction to: In this article the funding from \u2018FOREUM Foundation for Research in Rheumatology\u2019 was omitted. The original article has been corrected."} +{"text": "Discussion section at the sentence that reads \u201cThe balance between OPG and RANKL activity is thus considered a major factor controlling bone remodeling. Loss of mature B cells impairs OPG/RANKL ratio leading to decreased cortical bone in four-month old \u00b5MT-/- C57BL/6 mice (4)\u201d. This was incorrect and missing the correct reference and it should be changed to a new reference : 3839\u20133848. doi: 10.1182/blood-2006-07-037994. PMCID: PMC1874582 PMID: 17202317)]. The citation has now been inserted in the Discussion section, and the paragraph should read:In the published article error[s] and state that this does not change the scientific conclusions of the article in any way. The original article has been updated."} +{"text": "This article has been corrected.1In the Original Investigation titled \u201cEffect of Internet-Based vs Face-to-Face Cognitive Behavioral Therapy for Adults With Obsessive-Compulsive Disorder: A Randomized Clinical Trial,\u201d"} +{"text": "Dear Editor,we would like to share ideas on the publication \u201cPrevalence of COVID-19 Vaccine Side Effects among Early-Vaccinated Healthcare Workers in Eastern Ethiopia . Another"} +{"text": "Nature Communications 10.1038/s41467-022-31266-8, published online 25 June 2022Correction to: The original version of the Supplementary Information associated with this Article included an incorrect Description of Additional Supplementary Files, which was missing all the movie legends. The HTML has been updated to include a corrected version of the\u00a0Updated Supplementary Information"} +{"text": "BMC Psychology (2023) 11:2110.1186/s40359-023-01052-2Following publication of the original article [1], the authors flagged that a 'model consent form\u2019 completed in the Dutch language had been erroneously added to Figure 2. The figure has since been corrected to remove the form. The authors thank you for reading this correction and apologize for any inconvenience caused."} +{"text": "Legends for \u201cFigures 3\u20138\u201d as published. The correct Legends appear below.In the original article, there was a mistake in the (A) Comparisons of overall survival (OS) and disease-free survival (DFS) time among the BC subtypes by Kaplan\u2013Meier curves. The log-rank test p values are shown. Comparisons of the proportion of high-grade (G3) tumors, the proportion of late-stage (stage III\u2013IV) tumors (B), and proportions of HER2+, TNBC, HR + tumors (C) among the BC subtypes in METABRIC. The Fisher\u2019s exact test p values are shown.\u201d\u201cFIGURE 3 | Comparisons of clinical features among the BC subtypes. (A), SCNA scores (B), and global methylation levels (C) among the BC subtypes. The one-tailed Mann\u2013Whitney U test p values are shown in . (D) Prediction of the scores (high (>median) versus low ( PMID: 28538834.The authors would like to retract the article \u201cCharacteristics of liver fibrosis with different etiologies using a fully quantitative fibrosis assessment tool\u201d that was published in volume 50 no. 6 (2017) in the"} +{"text": "Nature Communications8:366 10.1038/s41467-017-00446-2; Article published online: 28 August 2017This Article contains two typesetting errors. In the penultimate sentence of the first paragraph of the Results section,\u00a0the equation should be \u2018"} +{"text": "Scientific Reports7: Article number: 4399210.1038/srep43992; published online: 03092017; updated: 04062017The original version of this Article contained an error in the spelling of the author Aleix Tarr\u00e9s-Sol\u00e9, which was incorrectly given as Aleix Tarr\u00e9s-Soler. This error has now been corrected in the PDF and HTML versions of the Article."} +{"text": "Scientific Reports 10.1038/s41598-017-13651-2, published online 16 October 2017Correction to: The Acknowledgements section in this Article was omitted. The Acknowledgements section should read:\u201cThis work was supported by the National Natural Science Foundation of China (No. 51479022).\u201d"} +{"text": "The TenoRes Study Group. Global epidemiology of drug resistance after failure of WHO recommended first-line regimens for adult HIV-1 infection: a multicentre retrospective cohort study. Lancet Infect Dis 16: 565\u2013752016; \u2014In this Article, the affiliation for Rami Kantor was incorrect. This correction has been made to the online version as of May 23, 2016."} +{"text": "Nature Communications8:1073 10.1038/s41467-017-01074-6; Article published online: 20 October 2017The financial support for this Article was not fully acknowledged. The Acknowledgements should have included the following:This study was in part supported by the Swiss National Foundation Grant No.: 31003A-156023 to Alessandro Sartori."} +{"text": "Scientific Reports 10.1038/s41598-017-14595-3, published online 27 October 2017Correction to: The Acknowledgements section in this Article is incomplete.\u201cThis work is supported by the Mayo Clinic Center for Individualized Medicine.\u201dshould read:http://cancergenome.nih.gov/\u201d\u201cThis work is supported by the Mayo Clinic Center for Individualized Medicine. The results published here are in whole or part based upon data generated by the TCGA Research Network:"} +{"text": "Scientific Reports 10.1038/s41598-017-18894-7, published online 24 January 2018Correction to: An additional Supplementary Information file containing programme codes was omitted from the original version of this Article. This has been corrected and the additional Supplementary Information accompanies this Article."} +{"text": "Scientific Reports 10.1038/s41598-017-09121-4, published online 17 August 2017Correction to: The Acknowledgements section in this Article is incomplete.\u201cThe author\u2019s laboratory is supported by DST grant of Govt. of India.\u201dshould read:\u201cAuthors would like to thank Prof. A. K. Gupta for his support and valuable inputs. The author\u2019s laboratory is supported by DST grant of Govt. of India.\u201d"} +{"text": "Geng's affiliation was originally given incorrectly. The article has since been corrected online.In the article \u201cOutcomes of off- and on-hours admission in ST-segment elevation myocardial infarction patients undergoing primary percutaneous coronary intervention: A retrospective observational cohort study\u201d,"} +{"text": "J. Epidemiol. (2016) 45 (4): 1006-1010; doi:This commentary was originally published with errors in an equation on p.1007 in section \u2018Total and joint effects\u2019.The equation initially was shown as When it should have been This has now been corrected. The publishers apologise for the error."} +{"text": "This is an Open Access article and should have contained the following copyright line: \u00a9 The Author(s) 2018. Published by Oxford University Press for the Infectious Diseases Society of America. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.\u201cSafety of single dose primaquine in G6PD-deficient and G6PD-normal males in Mali without malaria: an open- label, phase 1, dose-adjustment trial\u201d by Chen et al. [J Infect Dis 2018; doi:The authors regret the error."} +{"text": "The Funding statement is incorrect. The correct statement is: Natural Sciences and Engineering Research Council of Canada (NSERC), ANR-15-CE32-0005 \u00ab Convergenomix \u00bb. Part of the analyses were run on the PRABI cluster."} +{"text": "The incorrect file for S3 FigShown are the best-fitting line, Pearson\u2019s correlation coefficient and p-value of correlation tests. The mean correlation between two methods was 83% .(TIFF)Click here for additional data file."} +{"text": "Nature Communications8: Article number: 14358; DOI: 10.1038/ncomms14358 (2017); Published 02062017; Updated 11292017.This Article contains a typographical error in the sentence preceding Eq. 4. This sentence should read, \u2018Under the secular approximation and working in the limit There is a further error in Fig 4. The label on the upper state of the right-most transition in Fig. 4b should be \u201c"} +{"text": "Scientific Reports7:565; doi:10.1038/s41598-017-00628-4; Article published online 03 April 2017The original Supplementary Information file published with this Article was incorrect. This version included errors in the titles of references 8 and 9 and a number of typographical errors. The correct Supplementary Information file now accompanies the Article."} +{"text": "Correction to:Oncogene (2018) 37, 1062\u20131074; doi: 10.1038/onc.2017.368; published online 6 November 2017This article was originally published under NPG's License to Publish, but has now been made available under a CC BY license. The PDF and HTML versions of the paper have been modified accordingly."} +{"text": "The correct date was December 21, 2015. The article has since been corrected online.In the article \u201cPolysplenia Syndrome With Splenic and Skeletal Muscle Metastases From Thyroid Carcinoma Evaluated by FDG PET/CT: Case Report and Literature Review: A CARE-Compliant Article\u201d,"} +{"text": "There is grant information missing from the Funding Disclosure. The statement should read: This work was supported by the S\u00e3o Paulo Research Foundation (FAPESP). Grants were provided by the following Brazilian agencies: FAPESP , CAPES and CNPq (300553/2013-9)."} +{"text": "Nature Communications9:641; 10.1038/s41467-017-02715-6; Published online 13 February 2018Correction to: +\u2019 rather than an \u2018N\u2019. The correct version of Fig. 4c is:The original version of this Article contained an error in Fig. 4c, in which the right-most chemical structure included an \u2018Nwhich replaces the previous incorrect version:This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "The impact of demand management strategies on parents\u2019 decision-making for out-of-hours primary care: findings from a survey in The Netherlands. BMJ Open 2017;7:e014605. doi: 10.1136/bmjopen-2016-014605Giesen M, Keizer E, van de Pol JThe article has been corrected since it first published. \u2018CI\u2019 has been changed to \u201895% CI\u2019 several times throughout the paper and reference 24 has been added."} +{"text": "Erratum to:npj Genomic Medicine (2017); doi:10.1038/s41525-016-0002-3; Published 09 January 2017The following text was erroneously included in the final publication : \u201cSince the references were not in order it is has been rearranged it\u201d. This text has now been deleted in the HTML and PDF versions of this article."} +{"text": "Scientific Reports 10.1038/s41598-017-18685-0, published online 10 January 2018Correction to: This Article contains an error in the Acknowledgements section.\u201cThis work was supported by the National Science Foundation of China under grant no. 50875006. The authors thank the technical support from Professor Pingheng Tan.\u201dshould read:\u201cThis work was supported by the National Science Foundation of China under grant no. 51575013. The authors thank the technical support from Professor Pingheng Tan.\u201d"} +{"text": "Scientific Reports 10.1038/s41598-017-09497-3, published online 29 August 2017Correction to: The Acknowledgements section in this Article is incomplete.\u201cATJ was supported by the ESRC grant ES/K001477/1 (\u201cThe hearing body\u201d) and by a Spanish \u201cMinisterio de Econom\u00eda y Competitividad\u201d Ram\u00f3n y Cajal research contract (RYC-2014\u201315421). DB was supported by PSI2014-56301-R Ser Einstein: La Influencia de Internalizar un Cuerpo Virtual en la Inteligencia, Ministerio de Econom\u00eda, Industria y Competitividad of Spain. This work was also supported by the Virtual Embodiment and Robotic Re-Embodiment (VERE) Integrated Project funded under the European Seventh Framework Programme, Future and Emerging Technologies, Grant Agreement 257695. We thank Torsten Marquardt for his help with the experimental design, Professor Mark Huckvale for his help with the design of the the auditory stimuli and for provinding the real-time voice-transformation system and Andrea Yeung for her assistance with the vocal production analysis.\u201dshould read:\u201cATJ was supported by the ESRC grant ES/K001477/1 (\u201cThe hearing body\u201d) and by RYC-2014\u201315421 and PSI2016-79004-R , Ministerio de Econom\u00eda, Industria y Competitividad of Spain. DB was supported by PSI2014-56301-R Ser Einstein: La Influencia de Internalizar un Cuerpo Virtual en la Inteligencia, Ministerio de Econom\u00eda, Industria y Competitividad of Spain. This work was also supported by the Virtual Embodiment and Robotic Re-Embodiment (VERE) Integrated Project funded under the European Seventh Framework Programme, Future and Emerging Technologies, Grant Agreement 257695. We thank Torsten Marquardt for his help with the experimental design, Professor Mark Huckvale for his help with the design of the auditory stimuli and for providing the real-time voice-transformation system and Andrea Yeung for her assistance with the vocal production analysis.\u201d"} +{"text": "The authors of \u201cMaking Air Pollution Visible: A Tool for Promoting Environmental Health Literacy\u201d :e16) would like to make a correction to the text in Reference 26. It should read:26. An interactive map of traffic pollution for Boston Chinatown.\u00a0URL: http://chinatown.oicweave.com [accessed 2017-08-14] [WebCite Cache ID 6siTaGLNk]The corrected article will appear in the online version of the paper on the JMIR website on December 20, 2017, together with the publication of this correction notice. Because this was made after submission to PubMed Central, the corrected article also has been re-submitted to PubMed Central."} +{"text": "The basal positions are occupied by three donor atoms from the tridentate Schiff base ligand and by one N atom from a bis\u00admethane ligand. The apical position is occupied by the N atom of the other ligand of this type. There are only van der Waals contacts in the crystal structure.In the title compound, [Cu(C DOI: 10.1107/S1600536808037264/bq2096Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "We noticed a one-digit error on the tandem repeat number at the MIRU 20 after the publication of our article . This waWe do apologize to readers for any misunderstanding or inconvenience caused by this error.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/10/79/prepub"} +{"text": "The Eu atom is located on a threefold rotation axis and is nine-coordinated by three tridentate Schiff base ligands in a distorted tricapped trigonal-prismatic geometry. The O atom at the phenol hydr\u00adoxy group is partially deprotonated and the H atoms are modelled with one-third occupancy according to the space group R The title compound, [Eu(C DOI: 10.1107/S1600536809017206/bq2130Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the Funding section a digit was missing from one of the grant numbers. NIH grant U54-CA11296 should read U54-CA112967."} +{"text": "The imidazole ligand is disordered over two orientations of equal occupancy. Two of the perchlorate ion sites are located on a twofold rotation axis, and one of is disordered over two sites of equal occupancy. In the crystal structure there is a two-dimensional infinite network of hydrogen-bonded mol\u00adecules parallel to the ab plane.In the title complex, [Cu(C DOI: 10.1107/S1600536808017273/kj2084Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The triethano\u00adlamine ligands coordinate via each axial N atom and two of the three O atoms, while the third arm of the ligand has the hydroxyl group pointing away from the metal centre. The sulfate anions are hydrogen bonded to the coordinated hydroxyl groups and also to the free arm, forming a two-dimensional supra\u00admolecular hydrogen-bonded network expanding parallel to (010).The title compound, [Ni(C DOI: 10.1107/S1600536809023046/pk2167Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The three-dimensional network leads to two different tunnels occupied statistically by Na+ and Ca2+. One As and one Mg atom lie on twofold rotation axes; one Na and one Ca are disordered over two sites with occupancies of 0.7 and 0.3 and these sites lie on a twofold rotation axis and an inversion centre, respectively.The title compound, sodium calcium trimagnesium tris\u00ad(arsenate), an alluaudite-like arsenate, was prepared by solid-state reaction at high temperature. The structure is built up from edge-sharing MgO DOI: 10.1107/S1600536808014633/br2069Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Disorder is noted in the amine portion of the ligand and this was modelled over two sites, with the major component having a site-occupancy factor of 0.794\u2005(14).The Hg atom in the title complex, [HgCl DOI: 10.1107/S1600536808000858/tk2241Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The FeII atom is four-coordinated in a tetra\u00adhedral geometry by the O and N atoms of the two Schiff base ligands. The O atom of the furan substituent in the ligand unit is not involved in coordination to the Fe atom.The Fe atom of the title compound, [Fe(C DOI: 10.1107/S1600536808015559/sj2505Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The asymmetric unit consists of one iron cation located on a crystallographic center of inversion, as well as one iron cation, three thio\u00adcyanate anions and four pyrimidine ligands occupying general positions. The structure consists of square secondary building units (SBUs) with an Fe atom at each corner, which are \u03bc-N 1:N 3-bridged by the pyrimidine ligands. The SBUs are linked into infinite chains running in the c-axis direction via common opposite corners.In the crystal structure of the title compound, [Fe DOI: 10.1107/S1600536809005509/im2102Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The CuII atom lies on an inversion center N,O,N\u2032-chelated by two di-2-pyridylmethane\u00addiol ligands in a tetragonally distorted octa\u00adhedral geometry. The tartrate anion is also located on an inversion center and has disordered hydroxyl groups, each with an occupancy factor of 0.5. The hydroxyl groups of the complex cation are hydrogen bonded to the carboxyl\u00adate groups of the anion, thus connecting the two building units. The reaction of di-2-pyridyl ketone with copper dichloride dihydrate and tartaric acid in water afforded the title compound, [Cu(C DOI: 10.1107/S1600536808034983/hy2156Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The cations and ferrate anions are linked into a layered structure; the layers are connected through the uncoordinated water mol\u00adecules into a hydrogen-bonded three-dimensional supra\u00admolecular structure. One of the uncoordinated water molecules is disordered around an inversion centre and was refined with half-occupancy for each position.In the title salt, (C DOI: 10.1107/S1600536809010514/ng2562Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "In the crystal structure, the acidic H atom is located on the Schiff base N atom and hydrogen bonded to the phenolate O atom. The coordination environment of the EuIII ion is nine-coordinate by three chelating methoxy\u00adphenolate pairs of O atoms and three N-atom terminals of the thio\u00adcyanate ions. The compound is isostructural with the CeIII analogue [Liu et al. al. 2009. Acta Cr DOI: 10.1107/S1600536809049125/zl2242Isup2.hklStructure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:"} +{"text": "Its solid-state structure is defined by a three-dimensional framework of numerous electrostatic-supported N\u2014H\u22efCl hydrogen bonds between the ionic components of the compound. Within this framework, layered arrangements of the complex ions on one hand and of the protonated amines and chloride ions on the other hand, can be recognized. The octahedral hexa\u00adchloridorhodate(III) anion resides on a Single crystals of the new title compound , [H DOI: 10.1107/S1600536807067293/gk2126Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The two five-membered chelate rings in the dimeric mol\u00adecule both adopt twist conformations. Each VV atom is six-coordinated by one oxide group and by two N and one O atom of the tridentate Schiff base ligand, and is bridged by two additional oxide atoms. The metal centre has a distorted octa\u00adhedral coordination. The monoanionic ligands occupy one equatorial and two axial positions.In the crystal structure of the title compound, [V DOI: 10.1107/S1600536808014396/xu2414Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The asymmetric unit has four independent cadmium atoms, two of which lie on special positions of 2 site symmetry. The tartrate anions all lie on general positions. All hydroxyl groups are engaged in O\u2014H\u22efO hydrogen-bonds, one of which is also bifurcated. The non-coordinating water molecule is situated on a site with half-occupation.The title compound, {[Cd(C DOI: 10.1107/S1600536809016882/ci2796Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "We compared population-based incidence rates for classical Kaposi's sarcoma and multiple myeloma. Neither for men (Spearman's rank correlation coefficient (r) = 0.01, P = 0.97, 13 pairs) nor for women did the incidences of the two conditions correlate. This absence of correlation does not support the hypothesis that Kaposi's sarcoma and multiple myeloma share a common aetiology, such as HHV-8."} +{"text": "Am J Trop Med Hyg 82(3): 501\u2013504 by Taketa-Graham and others, one of the author's names is misspelled. The last author's name should be Bagher Forghani, not BagHer Forghani. The journal regrets this error.In Am J Trop Med Hyg 83(4): 834, the financial support section was incomplete. It should have read as follows: Financial support: This study was supported by the Young Researcher Program (ID 098-2007) and the contract of Instituto Colombiano para el Desarrollo de la Ciencia y la Tecnolog\u00eda.In"} +{"text": "The x-axis and y-axis labels were omitted from Figure 2. Please view the corrected figure at:"} +{"text": "There was an in error in Figure 3. Panels (S-U) were not included. The corrected figure is available here:"} +{"text": "The PbII atom is chelated by acetate and substituted quinolin-8-olate anions; the O atoms of the quinolin-8-olates also bridge to confer a five-coordinate status to each metal center. The geometry approximates a distorted \u03a8-fac octa\u00adhedron in which one of the sites is occupied by a stereochemically active lone pair.The mol\u00adecule of the title compound, [Pb DOI: 10.1107/S1600536809003559/tk2365Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Nature Communications7: Article number: 10144; DOI: 10.1038/ncomms10144 (2016); Published 01112016, Updated 07242018The original version of this Article omitted the following from the Acknowledgements: \u2018This work was also financed by an ERC starting grant \u2018SM-IMPORT\u2019 (No. 638536 to T.C.)\u2019. This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Correction to: BMC Infect Dishttps://doi.org/10.1186/s12879-019-4374-8After publication of the original article , we wereThe correct version can be found below:Furthermore, the words \u201cHiv specific antibody levels and\u201d should be deleted from the \u2018Case presentation\u2019 section in the Abstract.The original article has been corrected.The publisher apologies for the inconvenience."} +{"text": "Correction to: Heredity;10.1038/s41437-019-0187-1; published online 08 February 2019This article was originally published under standard License to Publish, but has now been made available under a CC BY 4.0 license. The PDF and HTML versions of the paper have been modified accordingly."} +{"text": "Nature Communications 10.1038/s41467-018-06812-y. published online 26 Oct 2018.Correction to: The original version of this Article contained an error in the Data Availability Statement. The accession code indicated \u201853V\u2019 and should have read \u20185X3V\u2019. This has been corrected in both PDF and HTML versions of the Article."} +{"text": "Correction to: Bioethical Inquiry (2018) 15:269\u201327810.1007/s11673-018-9845-xThere was a spelling error in the second author\u2019s last name in the original publication. The name is correct in this erratum."} +{"text": "Nature Communications 10.1038/s41467-018-06764-3; published online 09 October 2018Correction to: The original version of this Article contained an error in Fig.\u00a0which replaces the previous incorrect version shown below as Fig. This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Nature Communications 10.1038/s41467-019-11161-5, published online 30 July 2019.Correction to: y-axis labels incorrectly included \u2018Emissions reduction (million ton)\u2019 rather than the correct \u2018Total emissions (million ton)\u2019. This has been corrected in both the PDF and HTML versions of the Article.The original version of this Article contained an error in Figs.\u00a03 and 4, in which all"} +{"text": "Nature Communications; 10.1038/s41467-018-04372-9; published online 15 May 2018Correction to: The original version of this Article omitted the following from the Acknowledgements: Z. Leghtas\u02bc primary affiliation is Centre Automatique et Syst\u00e8mes, Mines ParisTech. This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Nature Communications 10.1038/s41467-019-09186-x, published online 13 March 2019Correction to: The original version of this Article contained an error in Acknowledgements, which incorrectly omitted the following: \u2018This work was also supported by NHLBI grant P01HL132825 to A.-L.B.\u2019 This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Scientific Reports 10.1038/s41598-018-26406-4, published online 23 May 2018Correction to: The Acknowledgements section in this Article was omitted. The Acknowledgements section should read:\u201cThis study was supported by Grants-in-Aid for Scientific Research 15H02652 and 15H04425 (K.T.) from the Japan Society for the Promotion of Science.\u201d"} +{"text": "Correction to: Spinal Cord Series and Cases 5:3010.1038/s41394-019-0173-0published online 15 March 2019This article was originally published under standard licence, but has now been made available under a [CC BY 4.0] license. The PDF and HTML versions of the paper have been modified accordingly."} +{"text": "Nature Communications ; 10.1038/s41467-018-05475-z; published online 6 August 2018Correction to: The original version of this Article omitted the following from the Acknowledgements:\u2018This work was support by EPSRC grant EP/K504336/1 and Leverhulme Trust grant RPG-2016-017.\u2019This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Scientific Reports 10.1038/s41598-018-19824-x, published online 23 January 2017Correction to: The original version of this Article contained an error in the title of the paper, where \u201cnanospheres enhances\u201d was incorrectly given as \u201cnanospheresenhances\u201d.In addition, this Article contained an error in the Acknowledgements section,\u201cand the Next-Generation BioGreen 21 Program (No.PJ000000), Rural Development Administration, Republic of Korea.\u201dnow reads:\u201cand the Next-Generation BioGreen 21 Program (No.PJ01122202), Rural Development Administration, Republic of Korea.\u201dThese errors have now been corrected in the PDF and HTML versions of the Article."} +{"text": "Correction to:Cell Death and Disease (2018) 9: 67910.1038/s41419-018-0714-7published online 07 June 2018Since publication of this paper, the authors have noticed that there were errors in Fig.\u00a0The authors would like to apologize for any inconvenience this may have caused."} +{"text": "The following information is missing from the Funding statement: Dr. Zsuzsanna Izsv\u00e1k was funded by European Research Council-2011-ADG-TRANSPOSOstress\u2013 294742. Additional information was added to the Funding statement in the Correction published in 2013 ["} +{"text": "Scientific Reports 10.1038/s41598-019-39611-6, published online 04 March 2019Correction to: In the Supplementary Material file originally published with this Article, text was omitted from the \u2018Text S2\u2019 section. This error has been corrected in the Supplementary Information that now accompanies the Article."} +{"text": "Correction to:Journal of Exposure Science & Environmental Epidemiology10.1038/s41370-018-0099-9published online: 8 January 2019This paper was originally published under a standard licence. This has now been amended to a CC BY licence in the PDF and HTML."} +{"text": "Journal of Perinatology; 10.1038/s41372-018-0112-0; published online 24 May 2018Correction to: This Article was originally published under Nature Research's License to Publish, but has nowbeen made available under a [CC BY 4.0] license. The PDF and HTML versions of the Articlehave been modified accordingly."} +{"text": "Correction to: Infect Agents Cancerhttps://doi.org/10.1186/s13027-019-0259-0In the original publication of this article several Table 1 Packages for womenTable 2 Packages for menFigure 1 Cancer Screening Algorithm for Cancer Prevention and Control Service at ALIADA"} +{"text": "There are errors in the Funding statement. The correct Funding statement is as follows: This study was funded in part by the Malaysian Ministry of Education via the Fundamental Research Grant Scheme (FRGS)-FRGS/2/2014/ICT07/MUSM/03/1. No additional external funding was received for this study."} +{"text": "Correction to:Cell Death and Disease;10.1038/s41419-018-1036-5;published online 9 October 2018The article published with errors in the authors\u2019 information. The correct\u00a0ordering and designations for corresponding /first authors are shown here."} +{"text": "Correction to: Virology Journal (2019) 16:41https://doi.org/10.1186/s12985-019-1148-2Virology Journal guidelines for the citation of unpublished data, all authors request to delete it from their article.In the original publication of the article , as the The largest number of farmed Atlantic salmon tested for PRV in BC-Canada found PRV in approximately 80% of 146 pooled samples from 539 Atlantic salmon tested in 2010 .\u201d\u201c"} +{"text": "Nature Communications 10.1038/s41467-018-07010-6, published online 01 November 2018.Correction to: The original version of this Article contained an error in the Data availability statement. The accession code for TSS-seq data was incorrectly indicated as \u2018GSE113677\u2019 but should have read \u2018GSE119304\u2019. This has been corrected in both PDF and HTML versions of the Article."} +{"text": "Correction to: Raihan et al. BMC Cancer (2019) 19:315.https://doi.org/10.1186/s12885-019-5516-5Conclusion section was incorrect. The sentence should read as follows:Following publication of the original article , it was \u201cHowever, further experimental studies need to be carry out in vitro and in vivo to decipher the molecular mechanism underlaying tumour cytolysis and cytokines released due to NDV infection\u201d.The original article has been"} +{"text": "G3: Genes|Genomes|Genetics 7: 3073-3082) entitled \u201cGenome-Wide Transcriptional Dynamics in the Companion Bacterial Symbionts of the Glassy-Winged Sharpshooter Reveal Differential Gene Expression in Bacteria Occupying Multiple Host Organs\u201d on page 3076 in the Data Availability Statement, there was an error in the GenBank Single Read Archive (SRA) database accession numbers. The text has now been corrected to cite SRR4241705-SRR4241717.In the article by G. M. Bennett and R. A. Chong ("} +{"text": "Scientific Reports 10.1038/s41598-018-21680-8, published online 20 February 2018Correction to: The Acknowledgements section in this Article was omitted. The Acknowledgements section should read:\u201cThis publication was funded by the German Research Foundation (DFG) and the University of Wuerzburg in the funding programme Open Access Publishing.\u201d"} +{"text": "Future Science OA 2[2], FSO124 [2016]; DOI 10.4155/fsoa-2016-0031), has had the title updated to:The following publication of the Company Profile by IA Blair, C Mesaros, P Lilley & M Nunez, (which appeared in a 2016 issue of Company profile: BluePen Biomarkers LLC \u2013 integrated biomarker solutionsFuture Science OA would like to sincerely apologize for any confusion or inconvenience this may have caused our readers.The editors of"} +{"text": "Clinical and Translational Gastroenterology;10.1038/s41424-018-0037-0; published online 06 July 2018Correction to: This Article was originally published under Nature Research\u2019s License, but has now been made available under a CC BY 4.0 license. The PDF and HTML versions of the article have been modified accordingly."} +{"text": "Nature Communications 10.1038/s41467-018-07106-z, published online 07 November 2018.Correction to: The original version of the Supplementary Information associated with this Article contained several errors. The molar ratio values on the y-axis scale of Supplementary Figure 6 (left panel) were incorrectly labelled to show the data as ranging between 17.2\u201368.5 rather than between 46.6\u2013185.8 as it should have been. The correct version of Supplementary Figure 6 (left panel) is:which replaces the previous incorrect version:\u22121) and right panels (\u03bcg.mg\u22121)). The HTML has been updated to include a corrected version of the Supplementary Information.Units were missing on the y-axes labels of Supplementary Figure 7 (middle (mg.mg"} +{"text": "This article has been corrected: Due to a software malfunction while cropping the western blot image into the PDF, the previous image for \u03b2-action (in IGF-1+Dex group) and for Akt T308 -P (in Dex group) in Figure 6A is incorrect. The corrected Figure 6A is shown below. The original image for western blot is also attached. The authors declare that these corrections do not change the results or conclusions of this paper.26966-26978. https://doi.org/10.18632/oncotarget.9034Original article: Oncotarget. 2016; 7:26966\u201326978."} +{"text": "Genetics in Medicine 2019; 10.1038/s41436-019-0649-0, published online 11 September 2019Correction to: This Article was originally published under Nature Research\u2019s License to Publish, but has now been made available under a [CC BY 4.0] license. The PDF and HTML versions of the Article have been modified accordingly."} +{"text": "Sci., 2016, DOI: ; 10.1039/c6sc00100a.Correction for \u2018Superior upconversion fluorescence dopants for highly efficient deep-blue electroluminescent devices\u2019 by Yi-Hsiang Chen Modified versions of The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "Scientific Reports 10.1038/s41598-019-52831-0, published online 07 November 2019Correction to: The Acknowledgements section in this Article was omitted. The Acknowledgements section should read:\u201cThis work was supported by the Sao Paulo Research Foundation \u201d."} +{"text": "The following information is missing from the Funding statement: Dr. Zsuzsanna Izsv\u00e1k was funded by European Research Council-2011-ADG-TRANSPOSOstress\u2013 294742. Additional information was added to the Funding statement in the Correction published in 2013 ["} +{"text": "In the Funding section, the first grant number from the funder Carlos III Institute of Health, Spain, and Comunidad Aut\u00f3noma de Madrid S2010/BMD-2378 is listed incorrectly. The correct grant number is: PI12/01328."} +{"text": "Nature Communications; 10.1038/s41467-018-03906-5, published online: 30 April 2018Correction to: The PDF version of this Article was updated shortly after publication following an error which resulted in the \u03a6 symbol being omitted from the left hand side of equation 8. The HTML version was correct from the time of publication."} +{"text": "Correction to: Genetics in Medicine; 10.1038/s41436-018-0132-3 published online 27 July 2018This Article was originally published under Nature Research\u2019s License to Publish, but has now been made available under a [CC BY 4.0] license. The PDF and HTML versions of the Article have been modified accordingly."} +{"text": "This corrigendum for \u201cOverexpression of a fusion defensin gene from radish and fenugreek improves resistance againstleaf spot diseases caused by Cercospora arachidicola and Phaeoisariopsis personata in peanut\u201d : 139-149; doi: 10.3906/biy-1412-46) is issued to replace a figure. This figure is of the amplification of theselectable marker used in the gene construct. While preparing Figure"} +{"text": "Scientific Reports 10.1038/s41598-019-46160-5, published online 04 July 2019Correction to: This Article contains errors in Figure 4D, where the incorrect image is shown for passage 60 of mTert/mTert embryonic stem cells. The correct Figure appears below as Figure\u00a0The conclusions of the Article are unaffected by these changes."} +{"text": "China. Hang-ying Qu's affiliation should appear as The Department of Oncological Surgery, Shaanxi University of Chinese Medicine.In the article, \u201cPrognostic role of pretreatment neutrophil to lymphocyte ratio in breast cancer patients: A meta-analysis\u201d"} +{"text": "Correction to: BMC Med Res Methodol (2019) 19:116https://doi.org/10.1186/s12874-019-0755-3There is a middle initial in John P. Greenwood.An institution\u2019s name in the Funding section should be changed from \u201cNIHR Bristol Biomedical Research Unit for Cardiovascular Disease\u201d to \u201cNIHR Bristol Biomedical Research Centre\u201d.In the original publication of this article , the autThe original article has been corrected.The authors apologize for the inconvenience caused to our readers."} +{"text": "The Surgery Journal(10.1055/s-0039-1700497). The correct figure has now been updated as given below.It has been brought to our attention that"} +{"text": "Scientific Reports 10.1038/s41598-018-25778-x, published online 10 May 2018Correction to: This Article contains an error in Figure 4 where the key is incorrect in panels (d), (e), and (f). The correct Figure 4 appears below as Figure"} +{"text": "Scientific Reports 10.1038/s41598-017-07522-z, published online 09 August 2017Correction to: The Acknowledgements section in this Article is incomplete.\u201cThis research was supported by an Independent Starting Grant from the European Research Council to A. Olsson and a post-doctoral grant from the Swedish Science foundations (2015-00312) to A. Golkar.\u201dshould read:\u201cThis research was supported by an Independent Starting Grant from the European Research Council, and the Knut and Alice Wallenberg Foundation (KAW 2014.0237) to A. Olsson and a post-doctoral grant from the Swedish Science foundations (2015-00312) to A. Golkar.\u201d"} +{"text": "Scientific Reports 10.1038/s41598-018-36710-8, published online 24 January 2019Correction to: The Acknowledgements section in this Article was omitted. The Acknowledgements section should read:\u201cThis project was supported by grants from the U.S. National Institutes of Health and from JSPS KAKENHI Grant Number 17K14937 and the Kato Memorial Bioscience Foundation support to HM.\u201d"} +{"text": "Correction to: Clin Epigeneticshttps://doi.org/10.1186/s13148-019-0609-1FADS2 associates with FADS2 genotype\u201d.Following publication of the original article , the autIt has been corrected in the original article as well.The publisher apologizes for any inconvenience caused by this error."} +{"text": "Correction to: Heart Fail Rev10.1007/s10741-019-09828-8The original version of this article unfortunately contained a mistake. Unfortunately one of the author\u2019s last name is misspelled: the correct name is Viviana Maestrini. The original article has been corrected."} +{"text": "Chem. Chem. Phys., 2018, DOI: 10.1039/c8cp05517c.Correction for \u2018Self-adaptive multiscaling algorithm for efficientsimulations of many-protein systems in crowded conditions\u2019 by Sergio A. HassanThe author would like to correct The Royal Society of Chemistry apologises for these errors and any consequentinconvenience to authors and readers."} +{"text": "Correction to: Molecular Psychiatry10.1038/s41380-019-0441-1;published online 14 June 2019In the original version of this article, panel labels in the legend of figure\u00a01 were incorrectly positioned alongside non-corresponding panel descriptions. The legend of figure\u00a01 has now been updated. This has been corrected in both the PDF and HTML versions of the article."} +{"text": "Correction to: Genome Biology (2019) 20:82https://doi.org/10.1186/s13059-019-1679-2di in the \u201cScans for local adaptation\u201d subsection in the Method section, was identified. The correct formula should beFollowing publication of the original article , a typogThe authors apologize for any confusion this error may have caused."} +{"text": "Nature Communications 10.1038/s41467-019-13234-x, published online 20 November 2019.Correction to: 56-69 incorrectly stated \u2018PDB ID: 6GIG \u2018. The correct version reads \u2018PDB ID: 6DIG\u2019.The original version of this Article contained an error in the Data Availability statement. The accession code for the X-ray structural data of DQ6-HCRTThis has now been corrected in both the PDF and HTML versions of the Article."} +{"text": "Scientific Reports 10.1038/s41598-018-28566-9, published online 16 July 2018Correction to: This Article contains typographical errors in the Acknowledgements section.\u201cThis study was supported by a research grant (No. K1220301) from Korea University College of Medicine .\u201dshould read:\u201cThis study was supported by a research grant (No. K1517861) from Korea University College of Medicine .\u201d"} +{"text": "Correction to: Genome Biolhttps://doi.org/10.1186/s13059-019-1806-0Following publication of the original article , the fol\u201cBenchmarking dimensionality reduction methods for scRNA-seq\u201d, the brackets should be removed from inside the square roots. The correct equation is shown below:1) In the method section titled \u201cBatch effect correction\u201d, the position of superscript in the subscript are incorrect. The correct equation is shown below.2) In the method section titled"} +{"text": "Correction to: Trials (2018) 19: 633https://doi.org/10.1186/s13063-018-2993-9and presence of FBSS or notAllocation will be stratified by centre and minimised on patient age (\u2265 65 or\u2009<\u200965\u2009years), gender, Following publication of the original article , we have"} +{"text": "Correction to:British Journal of Cancer (2018) 119:96\u2013104; 10.1038/s41416-018-0141-7; http://www.bjcancer.com; published online 19 June 2018This article was originally published under the standard License to Publish, but has now been made available under a CC BY 4.0 license. The PDF and HTML versions of the paper have been modified accordingly."} +{"text": "The Polish National Science Center grant number should be 2017/25/B/NZ6/01463. The article has been corrected online ("} +{"text": "Scientific Reports 10.1038/s41598-019-39709-x, published online 06 March 2019Correction to: The information in this Article is incomplete. The text for the Competing Interest statement,\u2018The authors declare no competing interests.\u2019should read:\u2018The authors declare no competing interests. Frontier Scientific Services intends to market this diet.\u2019The proportions of all ingredients in the NCRMO-1 diet formulation are provided in Table\u00a0Referenceet al. Diet improvement for western corn rootworm (Coleoptera: Chrysomelidae) larvae. PloS one12, e0187997 (2017).1 Huynh, M. P."} +{"text": "Scientific Reports 10.1038/s41598-017-00461-9, published online 28 March 2017Correction to: The Acknowledgments section of this Article is incomplete, where:\u201cJian Wang is deceased. This paper is dedicated to his memory\u201dshould read:\u201cThis research was supported by the Lilly research fund and the National Natural Science Foundation of China (Grant No. 81541009). Jian Wang is deceased. This paper is dedicated to his memory\u201d"} +{"text": "Correction to:Wien Klin Wochenschr 201810.1007/s00508-018-1340-1The original version of this article unfortunately contained a\u00a0mistake. The presentation of the sentence \u201climiting on-duty working hours to 58h per week.\u201d was incorrect. The correct limitation of the on-duty working hours is 48\u202fh per week. The original article has been corrected."} +{"text": "Heredity; 10.1038/s41437-018-0119-5; Published online: 6 July 2018Correction to: This article was originally published under standard licence, but has now been made available under a [CC BY 4.0] license. The PDF and HTML versions of the paper have been modified accordingly."} +{"text": "It was highlighted that the original article containeCorrect FundingThis work has received funding from the European Union\u2019s Horizon 2020 research and innovation programme under Grant Agreement No. 681002 (EU-ToxRisk) and from the LIFE Project LIFE15 ENV/ES/000416 (LIFE-COMBASE).Incorrect FundingThis study was funded by EU-ToxRisk (Project Reference: 681002) and LIFE-COMBASE (LIFE15 ENV/ES/000416)."} +{"text": "Nature Communications 10.1038/s41467-019-09468-4, published online 29 March 2019.Correction to: The original version of this Article contained errors in the References.The citation currently given as reference [13] was previously incorrectly given as reference [14], and vice versa.The citation currently given as reference [31] was previously incorrectly given as reference [37]. As a consequence of this, current references [32\u201337] were previously incorrectly numbered [31\u201336], respectively.This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Nature Communications 10.1038/s41467-018-03291-z; published online 02 March 2018Correction to: The originally published version of this article contained an error in the subheading \u2018Heme is required for CO-dependent channel activation\u2019, which was incorrectly given as \u2018Hame is required for CO-dependent channel activation\u2019. This has now been corrected in both the PDF and HTML versions of the Article."} +{"text": "Sci., 2015, 6, 1115\u20131119.Correction for \u2018Cobalt co-catalysis for cross-electrophile coupling: diarylmethanes from benzyl mesylates and aryl halides\u2019 by Laura K. G. Ackerman Figure 1 in our original article contained an error. The words oxidation and reduction were exchanged on entries 3 and 4. The corrected figure appears below.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "Scientific Reports 10.1038/s41598-018-29495-3, published online 24 July 2018Correction to: The Acknowledgements section in this Article is incomplete.\u201cWe thank the subjects for their committed participation in this study. The project was financed by a National Science Centre award based on decision number DEC-013/09/B/NZ9/02365.\u201dshould read:\u201cWe thank the subjects for their committed participation in this study. The project was financed by a National Science Centre award based on decision number DEC-2013/09/B/NZ9/02365.\u201d"} +{"text": "Collecting and exploiting data to understand a nation\u2019s sexual health needs: Implications for the British National Surveys of Sexual Attitudes and Lifestyles . Sexually Transmitted Infections 2019;95:159\u2013161.Mercer CH, Clifton S, Prior G, The license type of the paper has changed from CC BY-NC to CC BY."} +{"text": "Bench press sub-section of the Results section is incorrect. The value should instead be \u20187.7%\u2019.The original article contains"} +{"text": "Sci., 2018, DOI: ; 10.1039/c7sc04813k.Correction for \u2018Single quantum dot-based nanosensor for sensitive detection of 5-methylcytosine at both CpG and non-CpG sites\u2019 by Zi-yue Wang The authors regret that the incorrect The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "Nature Communications 10.1038/s41467-019-12657-w, published online 15 October 2019.Correction to: The original HTML and PDF of this Article contained errors in Figs.\u00a03 and 4. All panels for Figures\u00a03 and 4 were inadvertently inverted. This has been corrected in both the HTML and PDF versions of the Article."} +{"text": "BioMed Research International and Evidence-Based Complementary and Alternative Medicine, respectively.\u201cThe State of Exosomes Research: A Global Visualized Analysis\u201d and \u201cCur\u2217 AND publishing year\u2009=\u2009(from 1994 to 2017) AND Language\u2009=\u2009(only English) AND Document types\u2009=\u2009(REVIEW OR ARTICLE)\u201d [Wang et al. stated in Section 2.1 that \u201cPublication information from the Web of Science (SCI-Expanded)\u201d and in Section 2.2 that \u201cthe research terms\u201d were as follows: theme\u2009=\u2009exosomeKeyWords Plus were taken into consideration.Using the methods applied by Wang et al. , we founChen et al. stated in 2.1. Search Strategy that \u201cPapers published from 2000 to 2016 were tracked by the keywords \u201cTraditional Chinese Medicine\u201d, \u201cTraditional Chinese Medicine Formula\u201d, and \u201cChinese Herb Formula\u201d for inclusion in analysis and summarization, based on their presence in the Web of Science database [Web of Science from 2000 to 2016 were applied as mentioned in the original paper in the ohttps://clarivate.com/products/web-of-science/databases/) includesWeb of Science Core Collection\u2009 BIOSIS Citation Index\u2009 BIOSIS Previews\u2009 Biological Abstracts\u2009 Zoological Record\u2009 MEDLINE\u2009 CAB Abstracts, CABI Global Health\u2009 Inspec\u2009 FSTASpecialist Collection\u2009 Chinese Science Citation Database\u2009 Russian Science Citation Index\u2009 KCI Korean Journal Database\u2009 SciELO Citation IndexRegional Collection\u2009 Data Citation IndexData Collection\u2009 Derwent Innovations Index (DII)Patent CollectionWeb of Science databases (Science Citation Index Expanded (SCI-EXPANDED)Social Sciences Citation Index (SSCI)Arts and Humanities Citation Index (A&HCI)Conference Proceedings Citation Index-Science (CPCI-S)Conference Proceedings Citation Index-Social Science and Humanities (CPCI-SSH)Book Citation Index-Science (BKCI-S)Book Citation Index-Social Sciences and Humanities (BKCI-SSH)Emerging Sources Citation Index (ESCI)Web of Science Core Collection: Citation Indexes includesCurrent Chemical Reactions (CCR-EXPANDED)Index Chemicus (IC)Web of Science Core Collection: Chemical Indexeshttps://clarivate.com/products/web-of-science/databases/).Web of Science Core Collection includes multidisciplinary information from over 18,000 high-impact journals, over 180,000 conference proceedings, and over 80,000 books from around the world (http://liu.brooklyn.libguides.com/az.php?a=e). Web of Science Core Collection: Chemical Indexes as well as SSCI, A&HCI, ESCI, CPCI-S, CPCI-SSH, BKCI-S, and BKCI-SSH are inappropriate for \u201cCurrent Research Trends in Traditional Chinese Medicine Formula: A Bibliometric Review from 2000 to 2016\u201d [Authors should choose appropriate databases based off of their topic instead of choosing random databases for their bibliometric research. For instance, Emerging Sources Citation Index (ESCI) complements the highly selective indexes by providing earlier visibility for sources under evaluation as part of SCIE, SSCI, and A&HCI's rigorous journal selection process (KeyWords Plus, were irrelevant to \u201cexosomes\u201d in Wang et al.'s paper [The SCI-EXPANDED is designed mainly for researchers to find published literature studies, not for bibliometric studies \u20136. Thus,'s paper and \u201ctra's paper . Due to 's paper \u201310.In the case of Wang et al.'s paper , 1,371 pIn the case of Chen et al.'s paper , after oRadix scutellariae extract and Huang-Lian-Jie-Du-Tang to rats\u201d [Qingkailing injection by high-performance liquid chromatography with diode array detection\u201d [Furthermore, typical evidence was found in Table 7 . The topto rats\u201d ; and \u201cAntection\u201d .Environmental Science and Pollution Research [Renewable and Sustainable Energy Reviews [Journal of Soils and Sediments [Journal of Foot and Ankle Surgery [International Journal of Environmental Research and Public Health [Sustainability [Journal of Cleaner Production [Environmental Science and Pollution Research [Published articles by for example Li and Nan , Liu andResearch , Renewab Reviews , Journalediments , and Jou Surgery . Howeverc Health , 27, Susnability , 29, Jouoduction , 31, andResearch , 33. EdiBioMed Research International and Evidence-Based Complementary and Alternative Medicine. This may result in misleading readers of the journals. From my prospective, authors should have used an appropriate bibliometric method and filter for their studies, thereby providing greater accuracy results for their bibliometric studies.Wang et al. and Chen"} +{"text": "Nature Communications 10.1038/s41467-019-10619-w, published online 18 June 2019.Correction to: GSE130287\u2019. This has been corrected in both PDF and HTML versions of the Article.The original version of this Article contained an error in the Data availability statement. The accession code indicated \u2018GSM130287\u2019 and should have read \u2018"} +{"text": "In the original article \u201cWon Fen Wong\u201d was incorrectly assigned as the second corresponding author. The sole corresponding author is \u201cPei Pei Chong.\u201dIn addition, we neglected to include the funder \u201cTaylor's University, TRGS/ERFS/2/2018/SBS/025\u201d to Pei Pei Chong.Funding statement:A correction has therefore been made to the \u201cThis work was supported by the Taylor's Research Grant Scheme (TRGS), Taylor's University, Malaysia (Award No. TRGS/MFS/2/2016/SOM/013) and Taylor's University, (Award No. TRGS/ERFS/2/2018/SBS/025).\u201dThe authors apologize for these errors and state that they do not change the scientific conclusions of the article in any way. The original article has been updated."} +{"text": "Scientific Reports 10.1038/s41598-019-46380-9, published online 10 July 2019Correction to: This Article contains an error in the Data Availability section, where the link to the code repository is missing. As a result,\u201cThe datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.\u201dshould read:https://github.com/saibalmars/GraphRicciCurvature).\u201d\u201cThe datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. Code for graph Ricci curvature and Ricci flow computation is available on GitHub ("} +{"text": "Nature Communications 10.1038/s41467-018-06713-0; published online 12 Oct 2018Correction to: The original version of this Article contained an error in Fig.\u00a04. In panel i, the lower CYA and \u03b1-SMA images were inadvertently inverted. This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Scientific Reports 10.1038/s41598-018-29491-7, published online 30 July 2018Correction to: The Acknowledgements section in this Article is incomplete.\u201cPartially supported by Maryland Stem Cell Research Fund\u201d.should read:\u201cPartially supported by Maryland Stem Cell Research Fund. The work is also partially supported by Science & Technology Project No. B2017179 from Hubei Provincial Department of Education to H.Z.\u201d"} +{"text": "Scientific Reports 10.1038/s41598-019-46146-3, published online 12 July 2019Correction to: This Article contains an error in the legend of Figure 2 and Figure 3.The legend of Figure 2:\u201cPairwise difference plots for Alpha diversity metrics. Treatment group is represented on the x-axis and pairwise change between pre and post measurements are demonstrated on the y-axis. The asterisk denotes significance (p\u2009=\u20090.05). (A) Pairwise difference comparison in observed OTUs (Alpha diversity). (B) Pairwise difference comparison of community richness using Shannon indices (Beta diversity).\u201dshould read:Figure 2. Pairwise difference plots for Alpha diversity metrics. Treatment group is represented on the x-axis and pairwise change between pre and post measurements are demonstrated on the y-axis. The asterisk denotes significance (p\u2009=\u20090.05). (A) Pairwise difference comparison in observed OTUs between micronutrient and placebo group. (B) Observed OTUs for each time point of the micronutrient and placebo groups.\u201d\u201cAdditionally, the legend of Figure 3:\u201cPairwise difference plots for Alpha and Beta diversity metrics. Treatment group is represented on the x-axis and pairwise change between pre and post measurements are demonstrated on the y-axis. The asterisk denotes significance (adjp\u2009=\u20090.05). (A) Pairwise difference comparison in observed OTUs (Alpha diversity). (B) Pairwise difference comparison of community richness using Shannon indices (Beta diversity).\u201dshould read:Figure 3. Pairwise difference plots for Beta diversity metrics. Treatment group is represented on the x-axis and pairwise change between pre and post measurements are demonstrated on the y-axis. The asterisk denotes significance (adjp\u2009=\u20090.05). (A) Pairwise difference comparison in Shannon indices between micronutrient and placebo group. (B) Comparison of community richness using Shannon indices for each time point of the micronutrient and placebo groups.\u201d\u201c"} +{"text": "Publisher Correction to: J Clin Mov Disord (2019) 6https://doi.org/10.1186/s40734-019-0077-yAn error occurred during the publication of an article in Journal of Clinical Movement Disorders. This article was published in volume 6 with a duplicate citation number.In this correction article the old and new citation metadata are published in Table\u00a0The original article has been updated. The publisher apologizes for the inconvenience caused to our authors and readers."} +{"text": "Correction to: BMC Med Educhttp://www.biomedcentral.com/1472-6920/14/255Following publication of the original article , we\u2019ve bTable 1 to be obscure. The legend to be replaced by the following text:N\u00a0=\u2009170). This table been removed because the authors have not obtained permission to reproduce the Maslach Burnout Inventory Educator Survey (MBI-ES). The results presented in this table are available by contacting the authors.Summary of the 22-item factor analysis loadings for the MBI-ES in veterinary medical students ("} +{"text": "Am J Trop Med Hyg 99 (1): 171\u2013179 (https://doi.org/10.4269/ajtmh.17-0648) Saadiyah Bilal and Eric Nelson should have been denoted as co-first authors on the manuscript. In addition, the following line should have been included in the financial support section of the manuscript: \u201cSoftware development was supported by the National Institutes of Health [DP5OD019893] to EJN.\u201dIn"} +{"text": "Correction to:npj Aging and Mechanisms of Disease3, 17 (2017); 10.1038/s41514-017-0016-9; Published online 24 November 2017Methods Mol. Biol. 1077, 167\u2013177 (2013).\u201d The citation for reference 20 has been changed to \u201cCheng, Y. et al. SIRT1 activation by pterostilbene attenuates the skeletal muscle oxidative stress injury and mitochondrial dysfunction induced by ischemia reperfusion injury. Apoptosis21, 905\u2013916 (2016).\u201d The original version of the published article did not list a source for the placebo pills or the investigational product NRPT in the Intervention section of Methods. The last sentence of the Intervention section of Methods now lists the source for the placebo pills and the investigational product NRPT as follows: \u201cThe matched placebo pills and the investigational product (NRPT) were provided by Elysium Health .\u201d This has now been corrected in the PDF and HTML versions of the article.The original version of the published article contained an incorrect citation for reference 20 \u201cHubbard, B. P. & Sinclair, D. A. Measurement of sirtuin enzyme activity using a substrate-agnostic fluorometric nicotinamide assay."} +{"text": "Scientific Reports 10.1038/s41598-018-22834-4, published online 15 March 2018Correction to: The Acknowledgements section in this Article was omitted. The Acknowledgements section should read:\u201cThis project was funded in part under a grant with the Pennsylvania Department of Health (#SAP 4100070267). The Department specifically disclaims responsibility for any analyses, interpretations or conclusions.\u201d"} +{"text": "Funding statement. The correct Name for the \u201cChinese National Major Projects\u201d is the \u201cNational Program Project.\u201dThere is an error in the Funding statement. The correct numbers for the \u201cNational Program Project\u201d are \u201cGrants 2018ZX10731301-006-001 (HS/SPH), 2013ZX10003009-002 (HS/IPS).\u201d The correct number for the \u201cClinical Research Plan of SHDC\u201d is \u201c16CR1028B (WS/SPH),\u201d and the correct numbers for the National Institutes of Health R01 grants are \u201cOD015092/RR13601, HL64560, and HL129887 (ZC).\u201dThere are also errors in the grant numbers in Funding statement:A correction has therefore been made to the \u201cThis work was supported by the National Program Project Grants 2018ZX10731301-006-001 (HS/SPH), 2013ZX10003009-002 (HS/IPS), Clinical Research Plan of SHDC 16CR1028B (WS/SPH), and the National Institutes of Health R01 grants OD015092/RR13601, HL64560, and HL129887 (ZC).\u201dThe authors apologize for these errors and state that they do not change the scientific conclusions of the article in any way. The original article has been updated."} +{"text": "Correction to:British Journal of Cancer; 10.1038/s41416-019-0506-6; published online 28 June 2019.This article was originally published under a standard license to Publish, but has now been made available under a CC BY license. The PDF and HTML versions of the paper have been modified accordingly."} +{"text": "Oncogenesis; 10.1038/s41389-017-0008-4; published online 28 November 2017Correction to: This article was originally published under Nature Research's License to Publish, but now has been made available under a CC BY 4.0 license. The PDF and HTML versions of the Article have been modified accordingly."} +{"text": "Scientific Reports 10.1038/s41598-019-46168-x, published online 08 July 2019Correction to: This Article contains an error in Figure\u00a08d, where the incorrect brightfield image is shown for the DMSO control embryo at 0\u2009hours. The correct brightfield image appears below.The conclusions of the Article are unaffected by these changes."} +{"text": "The authors regret that some of the information in Table\u00a02 is incorrect. The correct text should read:CWK-F12 Recombinant fusion protein. Amino acids 256\u2013505 of human wt ERb Mouse Monoclonal C-terminus WB, IP, IHC.The authors would like to apologise for any inconvenience caused."} +{"text": "Virtual Reality-Based Exercise with Exergames as Medicine in Different Contexts: A Short Review2019, 15: 15-20Clinical Practice & Epidemiology in Mental Health, The corrections are provided and replaced online which is mentioned as under:Original:The name of coauthor was C\u00e9sar Augusto Ottero VaghettiCorrected:The name of coauthor has been revised as C\u00e9sar Augusto Otero Vaghetti"} +{"text": "Nature Communications; 10.1038/s41467-018-04914-1; published online 10 July 2018Correction to: The original version of this Article omitted the following from the Acknowledgements:\u2018J. Ma\u2019s primary affiliation is Shanghai Jiao Tong University.\u2019This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Cell Death and Disease (2018); 10.1038/s41419-018-0510-4; published online 03 May 2018.Correction to: Since the publication of this Article, the authors reported that duplication had mistakenly occurred in Fig.\u00a03a and d\u2014panels that display H&E.The corrected Article appears online together with this erratum."} +{"text": "Correction to: Horticulture Research (2019)6:6110.1038/s41438-019-0142-6 Published online 01 May 2019Since the publication of this article, the authors have noticed that the GeneIDs from new and original genome annotations don\u2019t match in Table S6, the correct Table S6 is given here.The authors would like to apologize for this error.Supplementary Information"} +{"text": "Scientific Reports 10.1038/s41598-019-48985-6, published online 29 August 2019Correction to: In the Supplementary Information file originally published with this Article, the dataset \u2018Supplementary Information 4\u2019 was omitted. This error has been corrected in the Supplementary Information file that now accompanies the Article."} +{"text": "Nature Communications 10.1038/s41467-018-06057-9; published online 07 September 2018Correction to: The original version of this article contained an error in Fig. 3. In panel c, the labels \u2018mdx\u2019 and \u2018mdx Ripk3-/-\u2018 were inadvertently inverted. This has now been corrected in the PDF and HTML versions of the article."} +{"text": "Correction to: British Journal of Cancer (2019) 120, 139\u2013140; 10.1038/s41416-018-0353-x; published online 11 December 2018.Since the publication of this paper, the author noticed that the funding information was not complete. The correct funding information is now shown in the Funding section.FundingFB is funded by the AIRC\u2014Fondazione AIRC per la Ricerca sul Cancro | AIRC."} +{"text": "Correction to: Cell Death & Disease10.1038/s41419-019-1888-3, published online 11 September 2019.Since online publication of this article, the authors noticed that the Supplementary Tables S1\u2013S8 were absent from the published version. The article has now been corrected to include these. The authors apologize for any inconvenience caused."} +{"text": "This article has been corrected: Due to errors in document preparation, The NOKs used in the original version of this manuscript were in fact N/Tert-1 ; the manuscript is now corrected accordingly. None of the results or the conclusions from the manuscript are in any way altered by this different genetic background; HPV16 reprograms N/Tert-1 cells similarly to HPV16 in head and neck cancer as determined by analysis of The Cancer Genome Atlas (TCGA). The HPV16 immortalized human tonsil keratinocytes remain a bridge between the results with HPV16 positive N/Tert-1 and TCGA data. The authors apologize for this error.https://doi.org/10.18632/oncotarget.18328Original article: Oncotarget. 2017; 8:81892\u201381909."} +{"text": "Correction to: BMC Pregnancy and Childbirth (2018) 18:405https://doi.org/10.1186/s12884-018-2039-zFollowing publication of the original article , the aut1. The correct name of the last author is Margaret Gyapong and not Margarete Gyapong.2. The word \u201cordered\u201d in the 3rd line under statistical methods should be removed.The original article has been corrected."} +{"text": "Correction to: International Journal of Obesity42, 1823\u20131833;10.1038/s41366-018-0230-y;published online 09 October 2018This paper was originally published under a standard licence. This has now been amended to a CC BY licence in the PDF and HTML."} +{"text": "Can India\u2019s primary care facilities deliver? A cross-sectional assessment of the Indian public health system\u2019s capacity for basic delivery and newborn services.\u00a0BMJ Open 2018;8:e020532. doi: 10.1136/bmjopen-2017-020532.Sharma\u00a0J, Leslie HH, Regan M, The previous version of this manuscript contains some error in affiliation section namely the affiliation for Dr. Devaki Nambiar\u2019s institute. It should read as:The George Institute for Global Healthinstead ofGeorge Institute for Health, New Delhi, India"} +{"text": "TH Open 2018; 02(04): e399-e406) it has come to the authors attention that the wrong corresponding author was listed. The amended corresponding author address is:In the Original Article by Choi et al. \u201cComparison of ACUITY, CRUSADE, and GRACE Risk Scales for Predicting Clinical Outcomes in Patients Treated with Dual-Antiplatelet Therapy\u201d (Address for correspondenceProf. Moo Hyun KimDepartment of CardiologyCollege of MedicineDong-A UniversityBusanRepublic of KoreaKimMH@dau.ac.kr)(e-mail:"} diff --git a/PMC_clustering_430.jsonl b/PMC_clustering_430.jsonl new file mode 100644 index 0000000000000000000000000000000000000000..8208cd8a9b2ac6f727ef938975eb1e002b6073c1 --- /dev/null +++ b/PMC_clustering_430.jsonl @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:f1303104858ab00f5533ce9ffcdae670a9b088d50d7fb13d72fac8c199e50d42 +size 79312154 diff --git a/PMC_clustering_431.jsonl b/PMC_clustering_431.jsonl new file mode 100644 index 0000000000000000000000000000000000000000..3fb5b1fabf085b63b451a07f6842d4b5d16dc064 --- /dev/null +++ b/PMC_clustering_431.jsonl @@ -0,0 +1,3 @@ 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sha256:965991b12cadcf15c2e619b896a8791297a79e47c2ce18edbe582f28e0ceab9f +size 56313058 diff --git a/PMC_clustering_442.jsonl b/PMC_clustering_442.jsonl new file mode 100644 index 0000000000000000000000000000000000000000..5731930b246ff7c1663d5fa0c113e4d4bbd45f06 --- /dev/null +++ b/PMC_clustering_442.jsonl @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:df679d2c4ed11514ced148780a826779e55ef7481020e74a21cebec88a6c7a71 +size 91864292 diff --git a/PMC_clustering_443.jsonl b/PMC_clustering_443.jsonl new file mode 100644 index 0000000000000000000000000000000000000000..7bfbf766df33f1ec79d485931928536766c7497d --- /dev/null +++ b/PMC_clustering_443.jsonl @@ -0,0 +1,154 @@ +{"text": "Protagonists in the standard SCR reaction have been caught in the act by modulation excitation IR & XAFS spectroscopy. x) by ammonia (NH3). In this work, Cu-SSZ-13 has been studied at 250 \u00b0C under high conversion using a modulation excitation approach and analysed with phase sensitive detection (PSD). While the complementary X-ray absorption near edge structure (XANES) spectroscopy measurements showed that the experiments were performed under cyclic Cu+/Cu2+ redox, Diffuse Reflectance Infrared Fourier Transform Spectroscopy (DRIFTS) experiments provide spectroscopic evidence for previously postulated intermediates Cu\u2013N scale\" fill=\"currentColor\" stroke=\"none\">O)\u2013NH2 and Cu\u2013NO3 in the NH3-SCR deNOx mechanism and for the role of [Cu2+(OH\u2013)]+. These results therefore help in building towards a more comprehensive understanding of the reaction mechanism which to date has only been postulated in silico.The small pore zeolite Cu-SSZ-13 is an efficient material for the standard selective catalytic reduction of nitrogen oxides (NO ZSM-5 and beta).3-SCR is complicated and as of yet, not fully rationalised in terms of reaction kinetics, temperature regimes, and perhaps more relevant here, reaction intermediates. A number of plausible mechanisms have been proposed on the basis of spectroscopic and theoretical data ,x species to be converted to N2. Operando spectroscopic studies have proven to be successful at providing insight into both active sites and intermediate species for NH3-SCR.operando conditions with many of the pertinent results outlined in recent reviews.in situ NH3-SCR studies.3, NH4+, NO3\u2013) which renders the detection of short-lived, transient, species difficult. Similarly, for X-ray absorption near edge structure (XANES) spectroscopy, which provides a method to determine the oxidation and coordination state of copper within the sample (e.g. Cu2+/Cu+), it can be challenging to deconvolute signals relevant to the active site from those that originate from other sites in the catalyst that may not be pertinent to the reaction; the large majority of spectroscopic studies are equilibrium-based experiments and many phenomena such as adsorption of reactants, desorption of products and the formation of spectator species occur within similar time frames.3-SCR catalysts, as demonstrated for V-based and Cu-SSZ-13 catalysts using DRIFTS/UV-Vis and XANES, respectively.Ammonia selective catalytic reduction (NHConcentration modulation excitation (ME) represents an approach to enable the detection of dynamic species directly involved in a reaction,3-SCR reaction scale\" fill=\"currentColor\" stroke=\"none\">O)\u2013NH2) and copper bidentate nitrate species (Cu\u2013NO3), while highlighting the regeneration and importance of [Cu2+(OH\u2013)]+ sites in the catalytic process. Ancillary ME XANES experiments confirmed that the measurements were conducted in the presence of the Cu2+/+ redox behaviour in the cycle.Importantly the concentration modulation excitation DRIFTS data augmented with the application of Density Functional Theory (DFT) allowed us to observe the formation of key intermediates including a copper nitrosamine and was connected to heated stainless-steel gas supply lines. The outlet of the cell was connected to a FTIR spectrometer (Bruker Alpha equipped with a 70 mm path length gas cell heated to 150 \u00b0C). The sieved sample was placed in the sample cup of the cell . Prior to the experiments, the sample was dried in situ in 10 vol% O2/N2 (100 ml min\u20131) at 400 \u00b0C for 2 h. The overall flow was kept at 100 ml min\u20131 (100\u2009000 h\u20131) and the temperature at 250 \u00b0C. All DRIFT spectra were obtained by accumulating 10 interferograms at 4 cm\u20131 resolution and 80 kHz scanner velocity (0.9 s per spectrum). During a concentration modulation excitation experiment, solenoid valves were used to automatically switch between gases and were operated by the OPUS software (Bruker). The following pulse sequences were used; 20 cycles of 120 s with 60 s of 500 ppm NO flow on followed by 60 s of NO flow off, into a constant stream of 500 ppm NH3, 10\u2009000 ppm O2 make up N2 . The sets of time-resolved DRIFTS spectra obtained from the modulation experiments at equilibrium conditions spectra were measured using a Bruker Vertex 70 spectrometer equipped with a Praying Mantis mirror unit (Harrick) and a liquid-Nnditions were timnditions . A concem/z 2 (H2), 18 (H2O), 28 , 30 (NO), 32 (O2), 44 (N2O/CO2), and 46 (NO2). Prior to the operando experiments, the calcined catalysts were subjected to high temperature activation in an O2 atmosphere. Samples were heated to 400 \u00b0C at 10 \u00b0C min\u20131 and held at this temperature until no more changes were observed in the XAS spectra, the sample was then allowed to attain the desired temperature. During the ME experiments the zeolite catalysts were exposed to a similar pulse sequence at 250 \u00b0C as outlined above for the DRIFTS experiments, although 10 cycles of 40 s were used in which there was a 20 s NO on pulse followed by 20 s NO off. XANES data processing was performed using IFEFFIT with the Horae package (Athena).Cu K-edge XAS studies were performed at the Swiss Light Source (SLS) at the Paul Scherrer Institute, Switzerland, on the SuperXAS beamline.k-point at the gamma point.\u20131. The converged bulk energies are within 10\u20134 eV. The vibration frequencies were calculated using finite differences to determine the second derivatives. A cube of 8 unit cells of the CHA structure was constructed, which has dimensions of 18.69 \u00c5 \u00d7 18.69 \u00c5 \u00d7 18.69 \u00c5 containing 94 Si atoms, 2 Al atoms and 192 O atoms. The Cu is placed in the 8 ring as a Cu2+ with the framework acting as counter charge due to the acid site hydrogen atoms being removed. In calculations on structures where the Cu2+ has been reduced to Cu+, an additional NH4+ cation has been included to maintain charge neutrality for the model.The VASP code using the Perdew\u2013Burke\u2013Ernzerhof (PBE) functional was employed for the Density Functional Theory simulations. The projector augmented wave (PAW) method with a plane-wave cut-off of 450 eV was used with single 3 coordinated to Cu2+ ions (see 2+(OH\u2013)]+ centres, showing the formation of adsorbed complexes on these particular sites. The region below 2000 cm\u20131 is, in contrast, dominated by alternating positive\u2013negative signals, attributable to the interaction of NH3 molecules, rendering the identification of changes around other features challenging.The 2400 spectra collected during the ME DRIFTS experiments (2+(OH\u2013)]+ species is completely reversed. For clarity, the time-resolved response of several of the relevant features and regions of interest in the ME data have been expanded in 3 measured online to the DRIFTS cell confirms that the ME experiment was performed under actual SCR conditions (The corresponding phase-resolved spectra (nditions .\u20131) that appear in the time-resolved data, such as NH4+ ions formed on the Br\u00f8nsted acid sites and NH3 adsorbed on Cu2+. In addition, bands at 1812 and 2158 cm\u20131 are also observed that were previously attributed to Cu+-NO and NO+ (i.e. formed by either NO2 disproportionation or NO oxidation on Cu2+ sites), respectively. It is important to note, however, that the most intense feature in the phase-resolved spectra is that at 3655 cm\u20131, characteristic of [Cu2+(OH\u2013)]+ sites, presenting a larger variation than those corresponding to either silanol groups and bridging hydroxyls. This observation can be attributed to a response of this species to the variation of gas composition, pointing to an active role of this species in the catalytic mechanism, as is clearly illustrated in 2+(OH\u2013)]+ species to be affected during the entirety of the NO pulse, which is clear evidence for its consumption and subsequent regeneration under SCR conditions.\u20131, which on first sight appears to be due to NH4+ species . Upon closer inspection, this band is clearly red-shifted from the centre of the band due to adsorbed ammonia species, observed in the time-resolved spectra, by approximately 20 cm\u20131. There have been previous studies into the nature and evolution of the broad feature associated with the NH4+ species, notably by Giordanino et al. and Lezcano-Gonzalez et al.4+ ions (i.e. NH4+\u00b7nNH3 associations) and the two bending vibrations of NH4+ ions. Desorption of solvating NH3 molecules with increasing temperatures was seen to lead to a gradual intensity decrease and shift to lower wavenumbers of the component at 1463 cm\u20131, and eventually to the appearance of a single broad band at 1430 cm\u20131 due to un-solvated NH4+ ions attached to the zeolite framework. Lezcano-Gonzalez et al. showed that at 250 \u00b0C, these NH4+ ions slowly react under a flow of NO and O2, concluding that these species are not an intricate part of the NH3-SCR mechanism and more likely remain as a \u2018reservoir\u2019 of NH3.\u20131 band, it seems unlikely that this is due to the consumption and regeneration of NH4+ ions. Nevertheless, to further probe the origin of the band at 1436 cm\u20131, a series of additional time-resolved pulse experiments were conducted.A second interesting feature observed in the phase-resolved data concerns a band centred at 1436 cm\u20131 region the consequent desorption of NH3 . While the features at 1460 and 1436 cm\u20131 share the same response when NO is omitted from the catalyst bed scale\" fill=\"currentColor\" stroke=\"none\">O)\u2013NH2), with an N PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O stretching (with some Cu\u2013N component) frequency determined to 1431.2 cm\u20131 scale\" fill=\"currentColor\" stroke=\"none\">O)\u2013NH2 species at 1258 cm\u20131. Upon (re)inspection of the PSD data there is a weak band in the spectra that evolves and is consumed at exactly the same time scale\" fill=\"currentColor\" stroke=\"none\">O)\u2013NH2 is a key intermediate postulated in the recent works of Janssens et al. and Paolucci et al.,3-SCR reaction mechanism.To understand the possible origin of the 1436 cm1.2 cm\u20131 . In additime see and phas3 and NO+ (2160 cm\u20131).2+(OH\u2013)]+ or any feature close to the signal observed at 1436 cm\u20131 in the experiments described above, thereby supporting the proposal that Cu\u2013N scale\" fill=\"currentColor\" stroke=\"none\">O)\u2013NH2 intermediate species is part of the SCR catalytic cycle. In addition, the results confirm that the various species observed in the NO pulse ME experiment are consumed and regenerated during the SCR reaction and that they are not spectator species or by-products caused by unselective NO oxidation.To validate further the findings of the ME DRIFTS experiment, additional modulation experiments were conducted in the absence of NHand NO+ 20 cm\u20131.16ca. 1606 cm\u20131. Although the exact origin of this band is unclear, its position falls in the range characteristic of nitrate/nitrite and Cu-amine species, also postulated as relevant intermediates in the SCR mechanism.17Another feature that shows a strong response to the ME concentration stimulation (NO) is a band centred at PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O stretching frequencies ca. 1595 cm\u20131 (\u00b130 cm\u20131). Of the three structures, the bidentate Cu\u2013NO3 structure A scale\" fill=\"currentColor\" stroke=\"none\">O stretching frequency closest to that observed in the phase-resolved IR spectrum. The observation of only one IR active stretch mode (E mode) also implies that the D3h symmetry of the NO3\u2013 molecule is preserved on coordination with the Cu ion; the absence of two bands with similar stretching frequencies (\u03bdas/\u03bds modes) suggests the absence of a nitrate species of monodentate geometry (C2v symmetry). We note, however, that there have been many studies performed on Cu-containing zeolites and that a number of possible nitrates/nitrites have been proposed, including some similar to the structures shown here , further suggesting that the accurate assignment of nitrates/nitrites is not trivial.x\u2019 only occurs after the system has moved away from SCR conditions (i.e. after NH3 flow is switched off).i.e. different temperatures and conversion regimes, but also of their fraction being too short lived for XAS to be detected compared to all other species and thus of the different sensitivity of the two spectroscopic techniques. This observation allows us to conclude that there is probably a difference between the nitrates that play a role in the catalytic cycle as seen here, compared with those that form in the absence of NH3 (i.e. those that form slowly during NO oxidation).Examination of possible stable species predicted using DFT suggests a few candidate mono, bidentate nitrate and nitrite species with typical NA see ESI possesse\u20131, which can be assigned to the stretching modes of both NH4+ ions and adsorbed NH3 on Cu2+, respectively, together with a broad feature at 2156 cm\u20131 due to NO+.\u20131 are also seen to be present, which have previously been assigned to hydrogen-bonded coordination spheres around NH4+ (2800 cm\u20131) and H3O+ ions (3025 cm\u20131).3 before the SCR reaction begins with the loss of at least one NH3 ligand around the Cu ions necessary for the SCR reaction to take place. The absence of the more commonly observed, yet weaker NH3/NH4+ bending modes in the phase-resolved data suggests that these species undergo only minor modulation during the SCR reaction, which is largely due to their playing an indirect role in the reaction; solvating NH3 and NH4+ species are proposed to provide a reservoir of ammonia for the reaction although as has been shown previously for this catalyst, NH4+ ions are not particularly reactive.17Additional weak bands are seen in the phase-resolved data of et al. ]+ and is followed by the formation of Cu\u2013N scale\" fill=\"currentColor\" stroke=\"none\">O)\u2013NH2.3 followed by NO+ (2160 cm\u20131) from the disproportionation of NO2, is observed at its maximum intensity with roughly the same phase angle as those associated with Cu\u2013NH3 stretch (3182 cm\u20131) which suggests a complex is formed containing both Cu\u2013NH3 and NO2 towards the end of the cycle. Indeed, if we normalise the phase angle to the start of the catalytic cycle (i.e. presence of [Cu2+(OH\u2013)]+ as shown in The intermediate species seen in this study have previously been proposed as protagonists in the reaction mechanism proposed by Janssens al. see .10 Howev2+, followed by an evolving feature with time attributed to a 1s\u20134p dipole transition at 8982 eV due Cu+.2+ and Cu+ species in the time-resolved data much more clearly. Significantly, the maximum amplitude of the Cu+ feature correlates exactly with the minimum amplitude for the Cu2+ feature. This experimental observation is a clear indication of the accompanying Cu2+/Cu+ redox process occurring during the SCR catalytic cycle in these experiments ]+ is consumed/reduced to form the transient Cu\u2013N scale\" fill=\"currentColor\" stroke=\"none\">O)\u2013NH2 species. We note that the NO stretch of this particular species is red-shifted by \u223c400 cm\u20131 with respect to where stretches are typically found for heteronuclear N PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019Ox species and which, we argue, is caused by electron density removal from the N PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O double bond brought about by the formation of an L-type ligand interaction between Cu and N(O) leading to a distribution of 7 electrons over 4 bonds. It is interesting to note that we do not see the initial Cu\u2013NH3 or Cu\u2013NO(OH) interactions suggesting that although these must take place they are simply too fast to be observed in this experiment. Indeed, it is well known that Cu+/2+\u2013NH3 and Cu+\u2013NO interactions occur readily, which for NH3 even leads to reaction inhibition at low temperatures.\u20131, which could be tentatively attributed to nitrate-type species, and subsequently the appearance of NO+ (which has been proposed to be formed through the disproportionation of NO2) suggests an important role in the catalytic cycle also for nitrates and nitrites, particularly for completing the Cu redox cycle as has been previously postulated for both NH3-SCR in Cu zeolites and observed in hydrogen-promoted hydrocarbon SCR on silver\u2013alumina catalysts.operando DRIFTS experiments as the spectra are dominated by species originating from NH3. Interestingly the identification of these intermediates indicates a pathway through which gas phase NO2 can be produced in the cycle to circumvent the need to co-feed it to effect fast SCR as is typical in Fe-based systems.2+/Cu+ redox cycle operating at the same frequency as the concentration pulse. It is interesting to note that previous experiments under steady-state, high-temperature (i.e. >250 \u00b0C) operational conditions contain no evidence for Cu reduction although it is to be expected that this must occur.et al., although we do note that such species appear in a number of proposed cycles to date.The concentration ME experiments have provided key mechanistic details from both DRIFTS and XANES experiments. The DRIFTS data suggest that the catalytic cycle follows a reaction pathway whereby [Cu3-SCR process on copper containing zeolites. The ME DRIFTS and XANES data reveal the start of the reaction to involve [Cu2+(OH\u2013)]+, highlighting two key intermediates, Cu\u2013N scale\" fill=\"currentColor\" stroke=\"none\">O)\u2013NH2 and Cu\u2013NO3, that would be very difficult to observe using standard operando DRIFTS measurements. Using the mechanism in 2), and with the latter species, show how the Cu ion reoxidation occurs. Furthermore, within this mechanism, these species also help to rationalise how NO2 is produced circumventing the necessity to utilise additional NO2 for effective NH3-SCR behaviour. These results, moreover, are in line with predictions from theory, which helps benchmark the ME approach for obtaining potential mechanistic insight importantly obtained under more relevant conditions.10Cu-SSZ-13 has been studied by concentration ME using both DRIFTS and XANES. The data obtained have provided mechanistic insight into the NHi.e. Cu2+(NH3)3(NO3)).x applications, perhaps using cationic components that could be more active or at least more environmentally benign or at risk than copper.Despite the new insight obtained, we observe that the ME technique is not able to allow us to rationalise fully all the steps in the catalytic mechanism at this temperature or to discriminate definitively between active and spectator species that may evolve at the same rate as the dose response. It may be that better resolution regarding these steps could be realised by performing experiments at lower conversions and/or at lower temperatures. We note, furthermore, that the temperature employed for this study is also unable to discriminate between the low and high temperature mechanism because of the Cu loading, but as it was performed at 250 \u00b0C, these results are likely to be more pertinent to a high temperature mechanism; indeed some of the intermediates observed here are different from those recently reported at much lower temperatures (There are no conflicts to declare.Supplementary informationClick here for additional data file."} +{"text": "A series of molybdenum pincer complexes has been shown for the first time to be active in the catalytic hydrogenation of amides. Mo-1a proved to be particularly well suited for the selective C\u2013N hydrogenolysis of N-methylated formanilides. Notably, high chemoselectivity was observed in the presence of certain reducible groups including even other amides. The general catalytic performance as well as selectivity issues could be rationalized taking an anionic Mo(0) as the active species. The interplay between the amide C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O reduction and the catalyst poisoning by primary amides accounts for the selective hydrogenation of N-methylated formanilides. The catalyst resting state was found to be a Mo\u2013alkoxo complex formed by reaction with the alcohol product. This species plays two opposed roles \u2013 it facilitates the protolytic cleavage of the C\u2013N bond but it encumbers the activation of hydrogen.A series of molybdenum pincer complexes has been shown for the first time to be active in the catalytic hydrogenation of amides. Among the tested catalysts, This is in sharp contrast with the results obtained with Fe pincer complexes, in which formanilide derivatives give the highest conversion.6a) and simple benzanilide (7a) were employed and results comparable to formanilide (4a) were obtained. Likewise, only low conversions and yields were obtained in the case of N-iPr- (4b) and N-allylformanilide (4c), respectively. Surprisingly, when N-allylformanilide was tested as substrate, the formation of N-allylaniline was only observed in traces. The main product was identified to be aniline, thus hinting at a deallylation pathway that additionally takes place to the envisaged hydrogenolysis. In contrast, N,N-diphenylformanilide (4d) was reduced smoothly and N,N-diphenylamine was isolated in excellent yield. Next, the hydrogenation of N-methylacetanilide (5) and the more activated 2,2,2-N-methyl-trifluoroacetanilide (6b), respectively, were attempted. In either case, only poor conversions were determined demonstrating the high preference of this complex for specific formanilides. This was further supported by the low reactivity of N-methylbenzanilide (7b) and some aliphatic formamides with extremely high preference. Notably, the reaction still proceeded with 80% conversion with respect to N-methylformanilide (1a). To further highlight the scope of our system, we designed model substrate 9 combining two amide functionalities in one structure. After 24 h reaction, the intended hydrogenolysis of the formamide moiety in 9 had occurred smoothly and the target molecule 10 was isolated in a very high yield (92%). Notably, no cleavage of the benzamide was observed.Based on these observations, we were curious to demonstrate selective formamide reduction in the presence of other amide moieties. In a proof of concept experiment, the hydrogenation of the benchmark amide in the presence of benzamide We believe these results could pave the way towards new and selective deprotection strategies in organic synthesis mediated by this base metal PNP pincer complex.Mo-1a and its performance with different amides, DFT calculations and supporting experiments were conducted. Mo-1a with NaBHEt3 resulted in rapid hydrogen evolution. The nature of the gas was determined in a scale up experiment (100 \u03bcmol of Mo-1a) using GC-analysis. This observation prompted us to assume that the obtained reaction product was likely to be a pincer amido species such as Mo-3, in which Mo(i) has been reduced to Mo(0). This conclusion was further supported by HR-ESI mass spectrometry of the corresponding reaction mixture. When the distinct reactivity of the catalyst towards formanilide was studied, we isolated Mo-4 in form of colorless needles from the reaction mixture center. This is also consistent with the EPR-silent nature of the product formed in the activation of Mo-1a by NaBHEt3.Notably, the crystal structure of Mo-4 featuresMo-4 suggests that the Mo(0)-complexes Mo-3 and Mo-5, shown in Scheme 5, are presumably the main catalytic intermediates. Similar species have been proposed for the isoelectronic Fe(ii)-complexes Fe-2, Fe-3 and the Mn(i)-complex Mn-2 scale\" fill=\"currentColor\" stroke=\"none\">O reduction, C\u2013N bond protonolysis of the formed hemiaminal, and aldehyde C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O reduction. These steps were computed for N-methylformanilide and the energy profiles for the preferred pathways are given in As represented in PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O hydrogenation by Mo-5 consists of the hydride transfer from Mo to the amide carbonyl group (Mo-ts-6-7), followed by proton transfer from the ligand nitrogen to the amide oxygen (Mo-ts-7-8). This pathway was computed for formanilide . Interestingly, these energies are lower than those reported by us for the analogous Fe catalyst with formanilide . An increase of less than 1 kcal mol\u20131 is observed by changing the substrate to N-methylacetanilide (Me\u2013NMeMo-ts-C).The mechanism for the C\u2013N bond cleavage from the formed hemiaminal was alsots-CH\u2013NH .15 With N-methylformanilide vs. 20% Conv. in N-methylacetanilide). In addition, the lower energy barriers obtained with Mo compared to Fe are inconsistent with the higher H2 pressure and time required to accomplish amide hydrogenation with Mo-1a compared to Fe-3.14The similar energy barriers obtained with these substrates did not account for the large differences in yield observed experimentally (99% Conv. in Mo-3 with methanol leading to the Mo-methoxy intermediate Mo-9a (Mo-ts-3-9a), has a low energy barrier and is highly exergonic . The formation of related M-methoxy species have been observed for similar Fe, Ru, Os and Mn PNP-pincer complexes.N-protonation of the hemiaminal intermediate (Mo-ts-11-9a). The highest energy of this process is 10.8 kcal mol\u20131, which corresponds to the zwitterion hemiaminal intermediate interacting with the methoxide\u2013Mo complex (Mo-11). This energy is lower than the energy barrier for the hydride transfer , indicating that the C\u2013N bond cleavage is not the rate limiting step once MeOH is formed than with MeOH increasing the global energy barrier for the hydride transfer from 10.6 to 26.8 kcal mol\u20131 with formanilide. This energy may increase to 31.4 kcal mol\u20131 by reaction with BEt3 (Mo-4). In contrast, with N-methylformanilide, the only penalty to pay is the addition of MeOH. Therefore, the energy barrier for the hydride transfer increases from 13.1 to 22.9 kcal mol\u20131, which is lower than the barrier for formanilide, consistent with the larger conversion obtained with N-methylformanilide. In the case of N-methylacetanilide, the addition of ethanol instead of methanol is expected. The higher stability of the ethoxide complex Mo-9b compared to Mo-9a by ca. 2 kcal mol\u20131 by acting as a proton-shuttle with a global energy barrier of 23.0 kcal mol\u20131. Similar mechanisms have been proposed with Ru\u2013N and Fe\u2013N complexes scale\" fill=\"currentColor\" stroke=\"none\">O reduction (blue cycle). This reaction yields amine and formaldehyde, which is reduced to alcohol by the catalyst Mo-5 in a subsequent reaction (in red). In the presence of alcohol, a Mo-alkoxo intermediate is formed, Mo-9a. This species, which becomes the catalyst resting state, is involved in the hemiaminal C\u2013N bond cleavage. Finally, the catalyst recovery takes place by the displacement of alcohol by H2. The nature of the catalyst resting state may change with secondary amides, which reacts with the catalyst forming an adduct that hampers the reaction.The results from the computational study can be summarized in the catalytic cycle represented in 3, NaHBEt3, and KHBEt3. Carrying out the benchmark reaction at 80 \u00b0C, 5 mol% of the alkali metal hydrides were added to activate Mo-1a. It could be shown, that for NaBHEt3 and KBHEt3 similar conversions of N-methylformanilide (1a) and yields of 2a were obtained. However, when LiBHEt3 was used, only 10% conversion of 1a and 9% yield of N-methylaniline 2a was obtained. These results were in agreement with the trends on the energy barriers obtained for the amide C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O reduction step, which are 22.9, 23.0 and 28.8 kcal mol\u20131 with Na+, K+ and Li+, respectively, taking Mo-9a as energy reference. The stronger electrostatic interaction of Li+ with the methoxide intermediate (LiMo-9a), accounts for the highest energy barrier predicted for this system active species, the role of the counter-cation in this reaction was explored computational and experimentally by using LiHBEtm see ESI.N-methylformanilide (1a) and 93% product yield were obtained. However, the addition of 200 mol% resulted in a sharp decrease in conversion and yield . Thus, it was concluded that ethanol has a detrimental effect on the performance of the catalytic system. Notably, these trends were reproduced with a microkinetic model based on the general mechanism represented in Next, the role of the alcohol was explored by adding different amounts of ethanol to the benchmark system. In the presence of 50 mol% of EtOH, 96% conversion of N-Alkylated and N-arylated formamides can be hydrogenated to the corresponding products in good to high yields. Applying complex Mo-1a high selectivity for the hydrogenation of formamides was observed in the presence of other reducible groups. These results pave the way for potential applications of this type of complexes in synthetic methodologies.Well-defined molybdenum\u2013PNP pincer complexes have been used for the first time in the hydrogenation of a range of amides to the corresponding alcohols and amines. Mo-5) reduces the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O group of the amide through low-energy barriers, compared to Fe-based systems. However, the alcohol product and secondary amides react with the catalyst forming stable adducts encumbering catalyst recovery and increasing the overall barrier for the reduction of the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O group. These results suggest that further catalyst design should focus on preventing the formation of these adducts, while keeping the high hydricity of the complex.The DFT study shows that the active Mo(0) species (All hydrogenation reactions were set up under Ar in a 300 mL autoclave (PARR Instrument Company). In order to avoid unspecific reductions, all catalytic experiments were carried out in 4 mL glass vials, which were set up in an alloy plate and placed inside the autoclave.Mo-1a . Toluene (2 mL) was added and the corresponding brown suspension was treated with NaBHEt3 . The reaction mixture was stirred for 10 minutes and the corresponding substrate was subsequently added. Afterwards, the vial was capped and transferred into an autoclave. Once sealed, the autoclave was purged three times with 10 bar of hydrogen, then pressurized to the desired hydrogen pressure (50 bar), and placed into an aluminum block that was preheated to the desired temperature (100 \u00b0C). After 24 h, the autoclave was cooled in an ice bath and the remaining gas was released carefully. The solution was subsequently diluted with ethyl acetate and filtered through a small pad of Celite (1 cm in a Pasteur pipette). The Celite was washed with methanol (2 mL) and the combined filtrates were subsequently evaporated to dryness. The remaining residue was purified by column chromatography . In the case of substrate 7, the purified product was dissolved in 5 mL of Et2O and subsequently treated with 1 mL of HCl (2 M in Et2O). The reddish precipitate was filtered off, washed three times with 5 mL of Et2O and finally dried in vacuo. For the characterization of the products of the catalysis, see ESI.In a glove box, a 4 mL glass vial containing a stirring bar was charged with complex + cation was evaluated in some of the intermediates, and the preferred position is represented in figures and schemes of the manuscript (see ESIMo-1a and Mo-4 were consistent with a doublet and singlet ground state, respectively (see ESIp = 50 atm and T = 373.15 K. The energy of the optimized geometries was refined by single point calculations with triple-z quality basis sets, including the LANL2TZhttps://iochem-bd.bsc.es/browse/handle/100/193698.DFT calculations were carried out with Gaussian 09\u2009t see ESI. The geoy see ESI. VibratiThere are no conflicts to declare.Supplementary informationClick here for additional data file."} +{"text": "Antibodies conjugated to a photoactive transition metal carbonyl complex afford antigen-directed delivery of cytotoxic carbon monoxide to ovarian cancer cells. in vitro. The results described here provide the first example of an \u201cimmunoCORM\u201d, a proof-of-the-concept antibody-drug conjugate that delivers a gaseous molecule as a warhead to ovarian cancer.Carbon monoxide (CO)-releasing antibody conjugates were synthesized utilizing a photoactivatable CO-releasing molecule (photoCORM) and mouse monoclonal antibodies linked by a biotin-streptavidin system. Different monoclonal antibodies raised against different surface-expressed antigens that are implicated in ovarian cancer afforded a family of antibody-photoCORM conjugates (Ab-photoCORMs). In an immunosorbent/cell viability assay, Ab-photoCORMs accumulated onto ovarian cancer cells expressing the target antigens, delivering cytotoxic doses of CO Carbon monoxide (CO), while long recognized as a toxic gas, is an endogenously produced gasotransmitter that regulates immune/inflammatory processes and vascular tone through reactions with heme-containing proteins.2The challenge of delivering efficacious concentrations of CO to a target tissue has been approached by our group and others by synthesizing CO-releasing molecules (CORMs) with properties necessary for a potential therapeutic, including water solubility,versus non-targeted tissues. With this in mind, we sought to conjugate a photoCORM to a monoclonal antibody with the goal of improving target specificity of CO-release. Antibody-drug conjugates (ADCs) are fast emerging as an effective strategy for anticancer therapies. In most cases small molecule drugs are combined with monoclonal antibodies to achieve high selectivity.i.e. the warhead) to monoclonal antibodies using a biotin-streptavidin linker is a novel, currently unexplored and potentially effective strategy that could be employed for the controlled delivery of CO to specific tissues.Although a number of CORMs and photoCORMs has been developed in recent years,Herein we report the successful conjugation of a biotinylated-photoCORM to streptavidin-conjugated mouse monoclonal immunoglobulin G (IgG) antibodies to isolate Ab-photoCORMs for the controlled delivery of CO to ovarian cancer cell cultures with high specificity. Utilizing different monoclonal antibodies, a family of Ab-photoCORMs was synthesized with the goal of localizing and delivering cytotoxic levels of CO to ovarian cancer cells expressing different tumor-specific surface antigens. To the best of our knowledge, this communication is the first report of an antibody-drug conjugate in which the drug is a gaseous molecule, namely CO.3(phen)(4-pyAl)](CF3SO3) as the photoactivatable CO donor. Biotinylation of this photoCORM (1) was achieved through reaction with biotin-hydrazide in trifluoroethanol at room temperature (Scheme S11 was confirmed by electrospray ionization Fourier Transform mass spectrometry (ESI FTMS); (M+) m/z = 666.13539 scale\" fill=\"currentColor\" stroke=\"none\">OC bands at 2039 and 1939 cm\u20131, characteristic of the manganese tricarbonyl moiety, and one \u03bd PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019OC band at 1685 cm\u20131 derived from the biotin unit exhibited a broad absorbance band in the visible region between 320 and 450 nm , broadband, visible light Scheme S1. The com) Fig. S1, and 1H ctrum ESI. The inft Fig. S2. Electrom Fig. S3. Exposura Fig. S4 arising S Fig. S4. Complext Fig. S5. Furthery Fig. S6.1 upon illumination with visible light, significantly reduced cell viability in two ovarian cancer cell lines OVCAR-5 and SKOV-3 (ED50 = 48 and 25 \u00b5M respectively) assayed 24 h post-treatment and stability of the streptavidin-biotin interaction.2) for cellular studies.A streptavidin-biotin strategy was used to link Complex 2 with excess Complex 1 afforded the antibody-photoCORM conjugate (Ab-photoCORM) through a streptavidin-biotin interaction (1 by size-exclusion chromatography (Reaction of Complex eraction ESI\u2020). . 2 with tography .1 (M+) incorporated into the Ab-photoCORM was observed in the full MS scan was also synthesized for application in cell viability experiments in order to account for any non-CO-mediated effects of the antigen-specific Ab-photoCORMs.A family of Ab-photoCORM conjugates was synthesized using th\u20131 of Ab-photoCORMs for 60 min in the dark and then washed 3 times with 1x PBS to remove any non-specific association. Next fresh media was added to the cells and they were exposed to low-power visible light for 30 min for CO photorelease. After an incubation period of 24 h, cell viability was assessed by cellular reduction of MTT. The viability study clearly demonstrated that treatment of OVCAR-5 and SKOV-3 cells with Ab-photoCORM conjugates recognizing epitopes expressed in those ovarian cancer cell lines delivered cytotoxic levels of CO and dramatically decreased cell viability conjugated to an antibody also requires that drug release is not dependent on linker cleavage or through complete degradation of the antibody within the tumor cell. The cleavable linkers impart small molecule drug ADCs with poor pharmacokinetics and circulation instability.Conventional ADCs require a number of specific properties in order to exhibit sufficient potency and stability. As one of the main mechanisms of drug resistance is ADC eflux,The authors declare no conflict of interest.Supplementary informationClick here for additional data file."} +{"text": "An Fe-based heterogeneous catalyst allows for the synthesis of cyclopropanes via a carbene transfer reaction, a transformation usually belonging to the homogeneous domain. in situ-generated iron/phenanthroline complexes in the presence of a carbonaceous material leads to specific supported nanosized iron particles, which are effective catalysts for carbene transfer reactions. Using olefins as substrates, cyclopropanes are obtained in high yields and moderate diastereoselectivities. The developed protocol is scalable and the activity of the recycled catalyst after deactivation can be effectively restored using an oxidative reactivation protocol under mild conditions.The first examples of heterogeneous Fe-catalysed cyclopropanation reactions are presented. Pyrolysis of Similarly, substrates with electron-donating groups (1f\u20131g) afforded the corresponding cyclopropanes in very high yields. Also halogen-containing substrates were well tolerated, maintaining intact the C\u2013X bond, which can allow further functionalization. To our surprise, pentafluoro styrene (1l) as an example of an electron-poor substrate afforded the corresponding product in a good yield. Notably, more challenging aliphatic substrates such as 1-octene (1t) and \u03b2-pinene (1v) gave the products in satisfactory yields. Using vinyl cyclohexene (1u) the cyclopropanation reaction occurred regioselectively on the terminal olefinic bond maintaining intact the internal one. The selectivity for the terminal double bond in 1u is explained by the lack of activity of the catalyst in the case of internal olefins .Under the optimized conditions, the scope of the diazo compound was also examined . Mono-suth run (D.1 in ca. 0.1%) loss of iron from the catalyst, so this cannot be the reason for deactivation.In order to demonstrate the practical utility of this protocol, the scalability and recyclability of the system were studied. The model reaction was successfully scaled-up to 15-fold without significant variations of yield and diasteroisomeric ratio compared to the small-scale run. Regarding the recyclability, a significant decrease of the activity was observed after the 42O2, the initial catalyst activity was restored (R.2 in e.g. fouling or poisoning).In order to make the whole process both efficient and effective, two routes of re-activation of the spent catalyst were explored. The material obtained after 5 runs (Fe/Phen@C-800_S) was thermally treated under an inert atmosphere R.1 in . Howeverd R.2 in ! The read R.2 in , cycle 7n+/H2O2 system (known as Fenton's reagent) is also effective in these processes thanks to the generated HO\u02d9 radicals.2O2 and related Fenton-like reactions are responsible for the removal of the oligomers/polymers from the spent catalyst.Note that most oxidative reactivation processes are based on the use of air at high temperatures, but such a treatment would result in the complete burning of the carbon support of our catalyst. Hydrogen peroxide has been reported as a milder alternative in a few cases.x centers, pyrrolic-N, and graphitic-N, respectively. PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C/C\u2013H (284.8 eV), C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N or C\u2013O (approximately at 285 eV), and C\u2013N or C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O (285\u2013291 eV) functionalities can be detected.3/2 region showed four pairs of peaks which can be attributed to various Fe states. Peaks at around 708 eV, 711 eV, and 714 eV in the region correspond to Fe(0), Fe(ii) and Fe(iii), respectively.x bonds which is in agreement with the previous results from N 1s spectra.43To further elucidate the reason for the declined activity, the fresh, spent and regenerated catalysts were characterized by X-ray photoelectron spectroscopy (XPS) and scanning transmission electron microscopy (STEM). XPS N 1s analysis showed common peaks at around 399 eV, 400 eV, and 401 eV for all the materials in the material. The spent catalyst (Fe/Phen@C-800_S) showed similar structures: still defined Fe-based NPs enveloped in carbon shells and dispersed iron clusters can be clearly observed (Finally, the interpretation of the O 1s region is not trivial because of the large amount of oxygen functionalities both derived from oxygen groups present in the carbonaceous matrixobserved . Finallyobserved .2O2 treatment, rather than by a structural change of the catalyst itself.The general morphology and distribution of Fe, N, O and C remained similar between the three different states of the catalyst as shown by the STEM data. Combined with the small changes in the electronic structure revealed by XPS, the data are consistent with the deactivation of the catalyst being due to fouling, which is removed by the HIn conclusion, here we report the first iron-based heterogeneous catalyst for cyclopropanation reactions. While in the past, this class of non-noble metal catalysts was generally employed for reduction or oxidation reactions, they are indeed effective catalysts also for carbene transfer reactions. The developed protocol allows obtaining several cyclopropanes from aromatic and aliphatic olefins and different diazo compounds in a practical and efficient manner. The heterogeneous nature of the catalyst and its robustness make it also an ideal candidate for flow chemistry applications. The deactivation of the catalyst has also been studied and an oxidative regeneration protocol was effectively developed, which may be of more general use even for other reactions.There are no conflicts to declare.Supplementary informationClick here for additional data file."} +{"text": "The Canadian Assessment of Physical Literacy (CAPL) is the first comprehensive protocol designed to assess a child\u2019s level of physical literacy. Current approaches to analyzing CAPL-2 raw data are tedious, inefficient, and/or can lead to computation errors. In this paper we introduce the capl R package (open source), designed to compute and visualize CAPL-2 scores and interpretations from raw data. The capl package takes advantage of the R environment to provide users with a fast, efficient, and reliable approach to analyzing their CAPL-2 raw data and a \u201cquiet\u201d user experience, whereby \u201cnoisy\u201d error messages are suppressed via validation. We begin by discussing several preparatory steps that are required prior to using the capl package. These steps include preparing, formatting, and importing CAPL-2 raw data. We then use demo data to show that computing the CAPL-2 scores and interpretations is as simple as executing one line of code. This one line of code uses the main function in the capl package (get_capl) to compute 40 variables within a matter of seconds. Next, we showcase the helper functions that are called within the main function to compute individual variables and scores for each test element within the four domains as well as an overall physical literacy score. Finally, we show how to visualize CAPL-2 results using the ggplot2 R package. Physical literacy is defined as \u201cthe motivation, confidence, physical competence, knowledge, and understanding to value and take responsibility for engagement in physical activities for life\u201d . It has The Canadian Assessment of Physical Literacy (CAPL) was the first comprehensive protocol designed to assess a broad spectrum of skills and abilities that contribute to and characterize the physical literacy level of a participating child . The CAPbmcpublichealth.biomedcentral.com/articles/supplements/volume-18-supplement-2). Data in each paper included approximately 10,000 children aged 8 to 12 years, recruited from several provinces across Canada. The CAPL-2 manual and materials have been translated in five languages and have been used internationally \"age\"\u00a0\u00a0\u00a0\u00a0\"gender\"\u00a0\u00a0\u00a0\u00a0\"pacer_lap_distance\"#> [4] \"pacer_laps\"\u00a0\u00a0\u00a0\u00a0\"plank_time\"\u00a0\u00a0\u00a0\u00a0\"camsa_skill_score1\"#> [7] \"camsa_time1\"\u00a0\u00a0\u00a0\u00a0\"camsa_skill_score2\"\u00a0\u00a0\u00a0\u00a0\"camsa_time2\"#> [10] \"steps1\"\u00a0\u00a0\u00a0\u00a0\"time_on1\"\u00a0\u00a0\u00a0\u00a0\"time_off1\"#> [13] \"non_wear_time1\"\u00a0\u00a0\u00a0\u00a0\"steps2\"\u00a0\u00a0\u00a0\u00a0\"time_on2\"#> [16] \"time_off2\"\u00a0\u00a0\u00a0\u00a0\"non_wear_time2\"\u00a0\u00a0\u00a0\u00a0\"steps3\"#> [19] \"time_on3\"\u00a0\u00a0\u00a0\u00a0\"time_off3\"\u00a0\u00a0\u00a0\u00a0\"non_wear_time3\"#> [22] \"steps4\"\u00a0\u00a0\u00a0\u00a0\"time_on4\"\u00a0\u00a0\u00a0\u00a0\"time_off4\"#> [25] \"non_wear_time4\"\u00a0\u00a0\u00a0\u00a0\"steps5\"\u00a0\u00a0\u00a0\u00a0\"time_on5\"#> [28] \"time_off5\"\u00a0\u00a0\u00a0\u00a0\"non_wear_time5\"\u00a0\u00a0\u00a0\u00a0\"steps6\"#> [31] \"time_on6\"\u00a0\u00a0\u00a0\u00a0\"time_off6\"\u00a0\u00a0\u00a0\u00a0\"non_wear_time6\"#> [34] \"steps7\"\u00a0\u00a0\u00a0\u00a0\"time_on7\"\u00a0\u00a0\u00a0\u00a0\"time_off7\"#> [37] \"non_wear_time7\"\u00a0\u00a0\u00a0\u00a0\"self_report_pa\"\u00a0\u00a0\u00a0\u00a0\"csappa1\"#> [40] \"csappa2\"\u00a0\u00a0\u00a0\u00a0\"csappa3\"\u00a0\u00a0\u00a0\u00a0\"csappa4\"#> [43] \"csappa5\"\u00a0\u00a0\u00a0\u00a0\"csappa6\"\u00a0\u00a0\u00a0\u00a0\"why_active1\"#> [46] \"why_active2\"\u00a0\u00a0\u00a0\u00a0\"why_active3\"\u00a0\u00a0\u00a0\u00a0\"feelings_about_pa1\"#> [49] \"feelings_about_pa2\"\u00a0\u00a0\u00a0\u00a0\"feelings_about_pa3\"\u00a0\u00a0\u00a0\u00a0\"pa_guideline\"#> [52] \"crf_means\"\u00a0\u00a0\u00a0\u00a0\"ms_means\"\u00a0\u00a0\u00a0\u00a0\"sports_skill\"#> [55] \"pa_is\"\u00a0\u00a0\u00a0\u00a0\"pa_is_also\"\u00a0\u00a0\u00a0\u00a0\"improve\"#> [58] \"increase\"\u00a0\u00a0\u00a0\u00a0\"when_cooling_down\"\u00a0\u00a0\u00a0\u00a0\"heart_rate\"capl package is also equipped with the get_capl_demo_data function. This function allows users to randomly generate demo raw data and takes one parameter, n (set to 500 by default). This parameter is used to specify how many rows of demo raw data to generate and must, therefore, be an integer and greater than zero. Users, for example, can randomly generate demo raw data for 10,000 participants by executing a single line of code:The get_capl_demo_data(n = 10000)capl_demo_data2 <- R str function can be called to verify how many rows of data were created.The base str(capl_demo_data2)#> \u2019data.frame\u2019:\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a010000 obs. of 60 variables:#> $ age:\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0int 10 10 9 12 9 12 10 11 9 11 \u2026#> $ gender:\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0chr \"f\" \"Boy\" \"g\" \"Female\" \u2026#> $ pacer_lap_distance:\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0num 20 20 15 20 15 20 15 20 15 20 \u2026#> $ pacer_laps:\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0int 75 93 102 131 96 151 129 150 127 10 \u2026#> $ plank_time:\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0int 132 125 120 38 37 173 164 137 267 38 \u2026#> $ camsa_skill_score1:\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0int 8 NA 7 5 6 6 10 4 10 7 \u2026#> $ camsa_time1:\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0int 19 16 34 NA 32 16 19 20 25 25 \u2026#> $ camsa_skill_score2:\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0int 7 10 9 6 10 8 6 6 7 11 \u2026#> $ camsa_time2:\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0int 25 32 35 23 35 14 29 27 18 24 \u2026#> $ steps1:\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0int 19261 22363 1181 5950 7020 21141 22435 18804 16575 \u2026### For complete output, refer to the capl vignetteexport_capl_data function allows them to export data objects to Excel.If users prefer to examine the CAPL-2 demo raw data in a workbook, the export_capl_dataget_capl) in the capl package to compute CAPL-2 scores and interpretations, they must ensure their variable names match the names of the 60 required variables. Users can rename their variables by calling the rename_variable function to be renamed, and the replace parameter must be a character vector representing the new names for the variables specified in the search parameter. Below we show how to rename variables using a fake dataset called raw_data.If users import their own raw data and plan to use the main function to help provide a \u201cquiet\u201d user experience:There are eight validation functions included in the \u25cb validate_age\u25cb validate_character\u25cb validate_domain_score\u25cb validate_gender\u25cb validate_integer\u25cb validate_number\u25cb validate_scale\u25cb validate_stepsUsers can learn more about these functions by accessing the documentation within the R environment.?validate_age?validate_character?validate_domain_score?validate_gender?validate_integer?validate_number?validate_scale?validate_stepsSections 2.7.2 and 2.7.3 illustrate examples of validation.age variable to execute a computation , the age variable is validated via the validate_age function.The CAPL-2 is currently validated with 8- to 12-year-old children. However, when a function requires the validate_age)validated_age <- Notice the NA values in the results.validated_age#> [1] NA 8 9 10 11 12 NA NA NA 12 8The first element is NA because the original value is 7 and the next five elements are identical to their original values because they are integers between 8 and 12. Recall that the CAPL-2 is validated with children aged 8 to 12 years, hence why the first value is NA . The next two elements because the original values (\"\" and NA) are obviously invalid. The last element is 8, but notice that the original value is a decimal. Because 8.5 is between 8 and 12, it is considered valid but the floor of the value is returned since CAPL-2 performs age-specific computations based on integer age.gender variable to execute a computation, the gender variable is validated via the validate_gender function.The CAPL-2 is currently validated for children who identify as boys or girls. When a function requires the validate_gender)validated_gender <- validated_gender#> [1] \"girl\" \"girl\" \"girl\" \"girl\" \"girl\" \"girl\" \"girl\" NA\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0NA\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"girl\"The validate_gender function behaves in a similar fashion for the male gender; it also accepts a number of case-insensitive options and returns a standardized \u201cboy\u201d value.Notice the results again. This function accepts a number of case-insensitive options for the female gender and returns a standardized \u201cgirl\u201d value. The two elements that are returned as NA have original values that are obviously invalid (\"\" and NA). validate_gender)validated_gender <- validated_gender#> [1] \"boy\" \"boy\" \"boy\" \"boy\" \"boy\" \"boy\" \"boy\" NA\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0NA\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"boy\"www.capl-eclp.ca/capl-manual):The CAPL-2 scoring system is outlined in capl package is the get_capl function. This function takes two parameters, raw_data and sort. It computes the CAPL-2 scores in capl package. The raw_data parameter must be structured as a data frame and contain the raw data. The sort parameter is set to \u201casis\u201d by default. This means the new computed variables will be added to the data frame as they are computed. If sort is set to \u201cabc\u201d, all variables will be sorted alphabetically whereas if sort is set to \u201czyx\u201d, all variables will be sorted in reverse alphabetical order. Once the raw data has been imported, computing the CAPL-2 scores and interpretations is as simple as executing one line of code:The main function in the get_caplcapl_results <- str function , the physical competence score is computed by summing the plank, PACER and CAMSA scores:As illustrated in pacer_laps_20m function is used to convert PACER (Progressive Aerobic Cardiovascular Endurance Run) 15-metre shuttle run laps to 20-metre shuttle run laps. If laps are already 20-metre laps, the data are returned as is unless outside the valid range (1-229). This variable is used to compute the PACER score.The get_pacer_20m_lapscapl_demo_data$pacer_laps_20m#> [1] 18 31 169 38 63 12 25 110 33 NA 127 62 39 19 NA 84 145 166#> [19] 108 125 98 147 85 49 4 118 144 85 122 85 197 5 184 19 63 112#> [37] 89 46 178 35 69 122 54 79 120 85 1 187 59 178 47 55 89 98#> [55] 79 119 11 70 89 88 68 82 116 38 152 195 4 69 100 99 NA 88#> [73] 57 43 98 125 127 5 16 173 20 33 89 99 39 35 43 100 177 15#> [91] 141 141 39 NA 8 41 43 2 101 NA 54 78 90 176 40 2 122 58#> [109] 98 5 51 112 101 122 12 177 38 92 31 53 102 200 138 166 62 31#> [127] 86 75 87 56 147 140 45 51 136 74 148 194 67 142 172 121 NA 78#> [145] 40 18 59 190 78 69 33 78 78 58 84 122 27 199 147 75 180 84#> [163] 36 129 14 133 70 65 8 43 110 64 69 120 125 28 142 115 108 14### For complete output, refer to the capl vignetteget_pacer_score function computes a PACER score that ranges from zero to 10 based on the number of PACER laps run at a 20-metre distance. This score is used to compute the physical competence domain score variable.The get_pacer_score(capl_demo_data$pacer_laps_20m)capl_demo_data$pacer_score <- capl_demo_data$pacer_score#> [1] 3 6 10 7 10 2 5 10 6 NA 10 10 7 3 NA 10 10 10 10 10 10 10 10 9 0#> [26] 10 10 10 10 10 10 1 10 3 10 10 10 9 10 7 10 10 10 10 10 10 0 10 10 10#> [51] 9 10 10 10 10 10 2 10 10 10 10 10 10 7 10 10 0 10 10 10 NA 10 10 8 10#> [76] 10 10 1 3 10 4 6 10 10 7 7 8 10 10 3 10 10 7 NA 1 8 8 0 10 NA#> [101] 10 10 10 10 8 0 10 10 10 1 10 10 10 10 2 10 7 10 6 10 10 10 10 10 10#> [126] 6 10 10 10 10 10 10 9 10 10 10 10 10 10 10 10 10 NA 10 8 3 10 10 10 10#> [151] 6 10 10 10 10 10 5 10 10 10 10 10 7 10 2 10 10 10 1 8 10 10 10 10 10#> [176] 5 10 10 10 2 10 10 8 10 10 10 10 10 10 10 10 10 5 4 8 10 10 3 10 10#> [201] 3 5 10 10 10 10 10 3 NA 10 10 6 7 10 10 NA 5 9 10 10 7 9 NA 10 9#> [226] 10 10 9 10 10 NA 6 10 7 10 5 10 10 10 10 10 10 NA 10 10 10 1 10 NA 10### For complete output, refer to the capl vignetteget_capl_interpretation function computes an age- and gender-specific CAPL-2 interpretation for a given CAPL-2 protocol or domain score.The get_capl_interpretationcapl_demo_data$pacer_interpretation#> [1] \"beginning\" \"beginning\" \"progressing\" \"beginning\" \"beginning\"#> [6] \"beginning\" \"beginning\" \"progressing\" \"beginning\" NA#> [11] NA \"beginning\" NA NA NA#> [16] \"progressing\" \"beginning\" \"progressing\" \"beginning\" \"progressing\"#> [21] \"beginning\" \"beginning\" \"progressing\" NA NA#> [26] NA \"beginning\" NA \"beginning\" \"progressing\"#> [31] \"progressing\" \"beginning\" \"beginning\" NA \"beginning\"#> [36] NA NA \"beginning\" \"progressing\" \"beginning\"#> [41] \"beginning\" \"progressing\" \"progressing\" \"beginning\" \"beginning\"#> [46] \"progressing\" NA \"progressing\" NA NA### For complete output, refer to the capl vignetteget_plank_score function computes a plank score that ranges from zero to 10 based on the duration of time (in seconds) for which a plank is held. This score is used to compute the physical competence domain score.The get_plank_score(capl_demo_data$plank_time)capl_demo_data$plank_score <- capl_demo_data$plank_score#> [1] 10 10 0 10 10 9 1 10 0 3 10 10 10 10 10 10 10 10 0 10 10 5 3 10 9#> [26] 10 10 0 10 5 0 0 5 10 5 10 4 7 10 3 6 10 0 10 8 6 10 10 0 10#> [51] 5 9 10 10 10 10 10 10 10 10 10 8 0 10 4 10 2 9 0 6 10 10 7 10 4#> [76] 10 10 10 6 0 10 10 10 10 9 10 10 10 10 5 0 10 10 10 1 8 10 10 8 2#> [101] 4 10 0 10 2 10 9 10 3 10 10 5 10 8 4 10 10 10 10 0 10 10 0 10 10#> [126] 10 10 10 10 10 5 10 3 10 0 10 7 0 10 7 0 10 10 10 6 10 10 10 0 1#> [151] 5 10 10 5 2 10 4 10 10 10 10 1 10 0 8 10 10 0 7 7 10 10 1 10 10#> [176] 10 10 10 10 10 1 0 9 10 10 10 10 10 10 10 6 3 3 10 10 1 10 10 10 8#> [201] 0 0 10 4 10 3 10 10 3 0 10 3 10 10 10 7 10 0 10 10 10 1 10 10 0#> [226] 10 10 10 2 4 10 0 4 0 10 10 0 6 10 10 10 0 10 10 8 10 10 10 0 10### For complete output, refer to the capl vignetteget_capl_interpretation function computes an age- and gender-specific CAPL-2 interpretation for a given CAPL-2 protocol or domain score.The get_capl_interpretationcapl_demo_data$plank_interpretation#> [1] \"excelling\" \"excelling\" \"beginning\" \"excelling\" \"excelling\"#> [6] \"excelling\" \"beginning\" \"excelling\" \"beginning\" \"progressing\"#> [11] NA \"excelling\" NA NA \"excelling\"#> [16] \"excelling\" \"excelling\" \"excelling\" \"beginning\" \"excelling\"#> [21] \"excelling\" \"achieving\" \"progressing\" NA NA#> [26] NA \"excelling\" NA \"excelling\" \"progressing\"#> [31] \"beginning\" \"beginning\" \"progressing\" NA \"progressing\"#> [36] NA NA \"achieving\" \"excelling\" \"progressing\"#> [41] \"progressing\" \"excelling\" \"beginning\" \"excelling\" \"achieving\"#> [46] \"progressing\" \"excelling\" \"excelling\" NA NA### For complete output, refer to the capl vignetteget_camsa_time_score function computes the CAMSA (Canadian Agility and Movement Skill Assessment) time score that ranges from one to 14 based on the time taken (in seconds) to complete a trial capl_demo_data$camsa_score#> [1] 5.357143 3.571429 9.285714 5.000000 6.785714 NA NA#> [8] 7.857143 4.285714 8.571429 5.357143 8.571429 8.571429 9.285714#> [15] 6.428571 9.285714 NA 6.428571 3.214286 3.571429 6.428571#> [22] 5.714286 8.928571 7.500000 NA 8.571429 NA 8.214286#> [29] 10.000000 2.857143 NA 5.714286 NA 8.571429 10.000000#> [36] 7.500000 7.857143 6.428571 7.142857 6.428571 5.714286 NA#> [43] 6.071429 5.714286 9.642857 2.142857 7.500000 8.214286 8.571429#> [50] 7.857143 7.500000 10.000000 7.142857 7.142857 NA 4.285714#> [57] 6.785714 6.428571 7.857143 6.428571 3.928571 4.285714 10.000000#> [64] 5.714286 NA 7.500000 6.071429 NA 7.500000 NA### For complete output, refer to the capl vignetteget_capl_interpretation function computes an age- and gender-specific CAPL-2 interpretation for a given CAPL-2 protocol or domain scoreThe get_capl_interpretationcapl_demo_data$camsa_interpretation#> [1] \"beginning\" \"beginning\" \"excelling\" \"beginning\" \"progressing\"#> [6] NA NA \"progressing\" \"beginning\" \"excelling\"#> [11] NA \"achieving\" NA NA \"progressing\"#> [16] \"excelling\" NA \"progressing\" \"beginning\" \"beginning\"#> [21] \"progressing\" \"beginning\" \"excelling\" NA NA#> [26] NA NA NA \"excelling\" \"beginning\"#> [31] NA \"beginning\" NA NA \"excelling\"#> [36] NA NA \"progressing\" \"progressing\" \"progressing\"#> [41] \"beginning\" NA \"progressing\" \"beginning\" \"excelling\"#> [46] \"beginning\" \"progressing\" \"achieving\" NA NA### For complete output, refer to the capl vignetteget_pc_score function computes a physical competence domain score that ranges from zero to 30 based on the PACER, plank and CAMSA scores. If one protocol score is missing or invalid, a weighted domain score is computed from the other two protocol scores. This score is used to compute the physical competence domain score.The get_pc_scorecapl_demo_data$pc_score#> [1] 19.5 24.0 15.0 22.0 30.0 16.5 9.0 30.0 9.0 NA 30.0 30.0 25.5 19.5 NA#> [16] 30.0 30.0 30.0 15.0 30.0 30.0 22.5 19.5 28.5 13.5 30.0 30.0 15.0 30.0 22.5#> [31] 15.0 1.5 22.5 19.5 25.0 30.0 21.0 24.0 30.0 15.0 24.0 30.0 15.0 30.0 27.0#> [46] 24.0 15.0 30.0 15.0 30.0 21.0 29.0 30.0 30.0 30.0 30.0 18.0 30.0 30.0 30.0#> [61] 30.0 27.0 20.0 25.5 21.0 30.0 3.0 28.5 15.0 24.0 NA 30.0 25.5 27.0 21.0#> [76] 30.0 30.0 16.5 13.5 15.0 21.0 24.0 30.0 25.0 24.0 25.5 27.0 30.0 30.0 12.0#> [91] 15.0 30.0 25.5 22.5 3.0 24.0 27.0 15.0 27.0 NA 21.0 30.0 15.0 30.0 15.0#> [106] 15.0 28.5 30.0 19.5 16.0 30.0 22.5 30.0 27.0 11.0 30.0 25.5 30.0 24.0 15.0#> [121] 30.0 30.0 15.0 30.0 30.0 24.0 30.0 30.0 30.0 30.0 22.5 30.0 18.0 30.0 15.0#> [136] 30.0 25.5 15.0 30.0 25.5 15.0 30.0 NA 30.0 21.0 19.5 30.0 30.0 15.0 16.5### For complete output, refer to the capl vignetteget_capl_interpretation function computes an age- and gender-specific CAPL-2 interpretation for a given CAPL-2 protocol or domain score.The get_capl_interpretationcapl_demo_data$pc_interpretation#> [1] \"achieving\" \"excelling\" \"progressing\" \"excelling\" \"excelling\"#> [6] \"progressing\" \"beginning\" \"excelling\" \"beginning\" NA#> [11] NA\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"excelling\" NA\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0NA\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0NA#> [16] \"excelling\" \"excelling\" \"excelling\" \"progressing\" \"excelling\"#> [21] \"excelling\" \"excelling\" \"achieving\" NA\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0NA#> [26] NA\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"excelling\" NA\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"excelling\" \"achieving\"#> [31] \"progressing\" \"beginning\" \"excelling\" NA\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"excelling\"#> [36] NA\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0NA\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 \"excelling\" \"excelling\" \"progressing\"#> [41] \"excelling\" \"excelling\" \"progressing\" \"excelling\" \"excelling\"#> [46] \"excelling\" \"progressing\" \"excelling\" NA\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0NA### For complete output, refer to the capl vignetteget_capl_domain_status function computes the status of a CAPL domain.The get_capl_domain_statuscapl_demo_data$pc_status#> [1] \"complete\"\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"complete\"\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"complete\"#> [4] \"complete\"\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"complete\"\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"missing protocol\"#> [7] \"missing protocol\"\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"complete\"\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"complete\"#> [10] \"incomplete\"\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"missing interpretation\"\u00a0\u00a0\u00a0\u00a0\"complete\"#> [13] \"missing interpretation\"\u00a0\u00a0\u00a0\u00a0\u00a0\"missing interpretation\"\u00a0\u00a0\u00a0\u00a0\u00a0\"incomplete\"#> [16] \"complete\"\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"missing protocol\"\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"complete\"#> [19] \"complete\"\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"complete\"\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"complete\"#> [22] \"complete\"\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"complete\"\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"missing interpretation\"#> [25] \"missing interpretation\"\u00a0\u00a0\u00a0\u00a0\"missing interpretation\"\u00a0\u00a0\u00a0\u00a0\u00a0\"missing protocol\"#> [28] \"missing interpretation\"\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"complete\"\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"complete\"### For complete output, refer to the capl vignettewww.capl-eclp.ca/capl-manual), the formula for computing the daily behaviour score is:As illustrated in get_step_average function computes the daily arithmetic mean of a week of steps measured by pedometry. This variable is used to compute the step score.The get_step_average(step_df <- \u00a0\u00a0\u00a0\u00a0capl_demo_data)get_step_average function returns a data frame with nine columns: steps1 , steps2 , steps3 , steps4 , steps5 , steps6 , steps7 , valid_days and step_average capl_demo_data$step_score <- capl_demo_data$step_score#> [1] 25 25 19 21 23 22 23 15 22 23 25 12 19 20 9 24 24 22 24 21 21 25 23 22 23#> [26] 25 21 23 22 22 23 23 18 22 20 25 25 25 25 23 19 25 23 25 22 16 13 24 21 12#> [51] 25 25 24 20 23 25 14 23 25 20 25 20 13 25 21 22 5 21 25 18 24 14 25 19 20#> [76] 22 20 8 25 21 25 25 19 21 14 25 25 25 16 25 21 20 21 25 25 25 25 23 25 25#> [101] 24 22 23 25 23 13 11 25 NA 23 25 10 25 25 19 13 24 21 16 18 25 25 24 20 19#> [126] 22 11 23 14 15 17 24 24 14 23 25 21 20 22 13 18 25 20 18 19 12 14 25 23 25#> [151] 22 17 22 18 22 23 25 23 14 19 18 25 25 17 25 16 25 22 19 22 22 6 22 16 25#> [176] 24 22 23 22 24 15 21 25 25 22 25 24 25 22 22 23 14 22 14 22 22 24 19 23 16#> [201] 14 17 4 24 22 25 23 20 25 19 24 22 25 24 15 25 22 21 25 22 16 23 16 25 25#> [226] 25 19 13 20 25 20 25 21 25 25 22 21 18 18 24 21 15 23 18 21 25 24 25 25 25### For complete output, refer to the capl vignetteget_self_report_pa function computes a score that ranges from zero to five based on the response to \u201cDuring the past week (7 days), on how many days were you physically active for a total of at least 60 minutes per day? ?\u201d in the CAPL-2 Questionnaire (www.capl-eclp.ca/wp-content/uploads/2018/02/CAPL-2-questionnaire.pdf). This score is used to compute the daily behaviour domain score.The get_self_report_pa_score(capl_demo_data$self_report_pa)capl_demo_data$self_report_pa_score <- capl_demo_data$self_report_pa_score#> [1] NA 1 1 3 2 4 NA 5 5 5 NA 3 1 4 5 5 0 5 5 5 5 5 5 NA 5#> [26] 3 4 5 4 1 2 0 5 5 5 5 4 3 4 2 4 5 5 5 2 5 5 NA 5 5#> [51] 1 5 3 1 0 5 3 2 5 1 2 0 5 NA 3 5 4 0 3 NA 5 4 1 NA 0#> [76] 5 0 2 0 NA 5 5 1 1 3 3 5 2 5 4 5 5 4 3 1 4 2 1 5 5#> [101] 5 2 1 4 3 2 5 5 3 2 2 5 5 5 2 NA 5 1 5 5 NA 5 5 1 0#> [126] 4 5 4 0 0 5 4 5 0 5 5 5 5 2 5 5 5 2 5 1 3 5 5 NA 5#> [151] 5 0 2 1 0 5 3 3 2 0 5 1 5 4 0 3 5 5 3 5 5 5 0 5 4#> [176] NA 5 5 4 5 3 4 3 0 2 1 5 3 0 5 1 5 5 5 5 0 2 3 0 1#> [201] 0 1 5 5 5 2 5 3 4 2 5 5 5 5 2 5 5 1 0 1 5 4 1 5 3#> [226] 4 3 5 0 5 2 5 0 NA 5 3 3 NA 2 0 4 5 5 5 5 4 0 3 3 2### For complete output, refer to the capl vignetteget_db_score function computes a daily behaviour domain score that ranges from 0 to 30 based on the step and self-reported physical activity scores. This score is used to compute the overall physical literacy score.The get_db_scorecapl_demo_data$db_score#> [1] NA 26 20 24 25 26 NA 20 27 28 NA 15 20 24 14 29 24 27 29 26 26 30 28 NA 28#> [26] 28 25 28 26 23 25 23 23 27 25 30 29 28 29 25 23 30 28 30 24 21 18 NA 26 17#> [51] 26 30 27 21 23 30 17 25 30 21 27 20 18 NA 24 27 9 21 28 NA 29 18 26 NA 20#> [76] 27 20 10 25 NA 30 30 20 22 17 28 30 27 21 29 26 25 25 28 26 29 27 24 30 30#> [101] 29 24 24 29 26 15 16 30 NA 25 27 15 30 30 21 NA 29 22 21 23 NA 30 29 21 19#> [126] 26 16 27 14 15 22 28 29 14 28 30 26 25 24 18 23 30 22 23 20 15 19 30 NA 30#> [151] 27 17 24 19 22 28 28 26 16 19 23 26 30 21 25 19 30 27 22 27 27 11 22 21 29#> [176] NA 27 28 26 29 18 25 28 25 24 26 29 28 22 27 24 19 27 19 27 22 26 22 23 17#> [201] 14 18 9 29 27 27 28 23 29 21 29 27 30 29 17 30 27 22 25 23 21 27 17 30 28#> [226] 29 22 18 20 30 22 30 21 NA 30 25 24 NA 20 24 25 20 28 23 26 29 24 28 28 27### For complete output, refer to the capl vignetteget_capl_interpretation function computes an age- and gender-specific CAPL-2 interpretation for a given CAPL-2 protocol or domain score.The get_capl_interpretationcapl_demo_data$db_interpretation#> [1] NA\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"achieving\" \"progressing\" \"achieving\" \"achieving\"#> [6] \"achieving\" NA\u00a0\u00a0\u00a0\u00a0\"progressing\" \"excelling\" \"excelling\"#> [11] NA\u00a0\u00a0\u00a0\u00a0\"progressing\" NA\u00a0\u00a0\u00a0\u00a0NA\u00a0\u00a0\u00a0\u00a0\"progressing\"#> [16] \"excelling\" \"achieving\" \"excelling\" \"excelling\" \"excelling\"#> [21] \"achieving\" \"excelling\" \"excelling\" NA\u00a0\u00a0\u00a0\u00a0NA#> [26] NA\u00a0\u00a0\u00a0\u00a0\"excelling\" NA\u00a0\u00a0\u00a0\u00a0\"excelling\" \"achieving\"#> [31] \"achieving\" \"achieving\" \"achieving\" NA\u00a0\u00a0\u00a0\u00a0\"excelling\"#> [36] NA\u00a0\u00a0\u00a0\u00a0NA\u00a0\u00a0\u00a0\u00a0\"excelling\" \"excelling\" \"achieving\"#> [41] \"achieving\" \"excelling\" \"excelling\" \"excelling\" \"achieving\"#> [46] \"progressing\" \"progressing\" NA\u00a0\u00a0\u00a0\u00a0NA\u00a0\u00a0\u00a0\u00a0NA### For complete output, refer to the capl vignetteget_capl_domain_status function computes the status of a CAPL domain.The get_capl_domain_statuscapl_demo_data$db_status#> [1] \"incomplete\"\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"complete\"\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"complete\"#> [4] \"complete\"\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"complete\"\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"complete\"#> [7] \"incomplete\"\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"complete\"\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"complete\"#> [10] \"complete\"\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"incomplete\"\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"complete\"#> [13] \"missing interpretation\" \"missing interpretation\" \"complete\"#> [16] \"complete\"\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"complete\"\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"complete\"#> [19] \"complete\"\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"complete\"\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"complete\"#> [22] \"complete\"\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"complete\"\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"incomplete\"#> [25] \"missing interpretation\" \"missing interpretation\" \"complete\"#> [28] \"missing interpretation\" \"complete\"\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"complete\"### For complete output, refer to the capl vignettewww.capl-eclp.ca/capl-manual), the formula for computing the motivation and confidence score is:As illustrated in get_predilection_score function computes a predilection score that ranges from 1.8 to 7.5 based on responses to three items from the Children\u2019s Self-Perception of Adequacy in and Predilection for Physical Activity as they appear in the CAPL-2 Questionnaire (www.capl-eclp.ca/wp-content/uploads/2018/02/CAPL-2-questionnaire.pdf). This score is used to compute the motivation and confidence domain score.The get_predilection_scorecapl_demo_data$predilection_score#> [1] 3.6 4.9 5.5 3.0 NA 3.0 6.2 4.9 4.8 4.9 3.0 3.0 5.6 4.9 5.5 5.5 4.9 4.3#> [19] 5.6 3.6 4.2 3.6 6.1 3.6 4.3 4.8 5.6 3.0 4.3 4.3 6.1 4.9 6.2 3.6 6.8 5.5#> [37] 5.5 6.2 3.0 2.4 4.3 3.6 5.5 6.1 NA 4.3 2.4 4.9 4.9 NA 5.5 4.3 4.2 4.2#> [55] 4.9 6.2 4.3 5.5 1.8 3.7 4.9 3.7 6.8 4.8 6.2 4.2 3.7 1.8 5.5 3.0 5.5 4.2#> [73] 4.8 5.5 4.9 3.6 4.9 3.7 3.6 3.6 4.9 6.2 4.2 3.7 3.7 3.6 3.0 2.4 4.2 4.2#> [91] 3.0 5.4 5.5 6.2 4.3 3.6 5.6 6.1 4.9 NA 4.2 5.5 6.8 4.8 4.9 4.9 4.9 2.4#> [109] 4.8 3.0 3.6 3.6 4.9 3.6 4.3 3.7 2.4 3.0 3.6 4.2 4.3 4.3 6.8 5.6 4.9 6.2#> [127] 3.0 3.0 5.5 3.6 4.2 3.7 6.1 3.7 3.6 6.2 6.1 4.8 3.6 4.9 1.8 5.6 3.0 4.9#> [145] 4.9 4.8 3.0 4.8 4.2 4.9 2.4 5.5 3.7 4.3 3.6 3.0 4.8 5.5 7.5 4.9 4.8 4.2#> [163] 3.0 2.4 5.5 2.4 5.5 6.1 3.6 5.5 4.3 4.2 4.3 NA 6.1 3.6 4.9 5.6 3.7 2.4### For complete output, refer to the capl vignetteget_adequacy_score function computes an adequacy score that ranges from 1.8 to 7.5 based on responses to three items from the Children\u2019s Self-Perception of Adequacy in and Predilection for Physical Activity as they appear in the CAPL-2 Questionnaire (www.capl-eclp.ca/wp-content/uploads/2018/02/CAPL-2-questionnaire.pdf). This score is used to compute the motivation and confidence domain score.The get_adequacy_scorecapl_demo_data$adequacy_score#> [1] 2.4 5.5 7.5 4.3 5.5 5.5 4.2 4.3 3.6 4.3 6.2 4.9 4.9 4.2 4.3 5.6 4.2 4.8#> [19] 4.8 6.2 4.3 4.8 4.9 3.6 6.8 4.3 6.2 6.2 4.3 7.5 6.1 5.6 4.9 6.2 6.2 2.4#> [37] 4.2 5.6 5.6 4.2 5.5 4.9 5.5 4.9 4.2 6.2 5.6 6.2 4.9 4.2 3.6 3.7 3.6 6.8#> [55] 1.8 3.6 5.6 4.9 3.6 3.0 3.6 4.9 3.7 4.9 3.6 3.7 4.2 5.6 6.1 4.9 3.6 3.0#> [73] 2.4 4.3 6.1 3.0 3.0 3.7 4.9 4.3 5.4 5.5 3.0 2.4 6.1 5.6 5.5 3.7 4.2 2.4#> [91] 5.6 4.9 3.0 5.6 4.9 3.6 4.9 6.8 4.9 2.4 5.6 4.8 4.2 3.0 4.9 3.0 3.0 3.7#> [109] 4.9 3.7 6.2 4.9 5.5 5.6 5.6 4.8 3.7 6.2 3.0 5.4 4.9 3.6 5.4 3.0 4.8 3.6#> [127] 4.8 4.2 4.2 2.4 4.9 4.3 6.2 2.4 4.9 4.9 4.9 4.2 4.9 5.5 4.9 5.5 7.5 5.6#> [145] 5.5 3.0 4.9 6.1 4.9 5.5 3.0 5.6 6.2 4.9 4.3 4.9 5.6 4.2 2.4 5.5 4.9 3.7#> [163] 4.3 4.3 4.9 4.3 6.1 4.8 4.9 5.5 6.2 6.1 3.6 2.4 3.6 4.9 6.2 4.3 4.3 4.2### For complete output, refer to the capl vignetteget_intrinsic_motivation_score function computes an intrinsic motivation score that ranges from 1.5 to 7.5 based on responses to three items from the Behavioral Regulation in Exercise Questionnaire (BREQ) as they appear in the CAPL-2 Questionnaire (www.capl-eclp.ca/wp-content/uploads/2018/02/CAPL-2-questionnaire.pdf). This score is used to compute the motivation and confidence domain score.The get_intrinsic_motivation_scorecapl_demo_data$intrinsic_motivation_score#> [1] 6.0 4.5 5.0 4.5 4.0 5.5 6.5 NA 5.5 NA 6.5 5.0 4.0 3.5 NA 6.0 4.5 4.5#> [19] 5.0 3.0 2.0 2.5 3.5 NA 5.0 4.0 4.5 7.0 3.5 4.0 NA 6.0 5.0 3.5 NA 4.5#> [37] 4.5 NA 5.0 5.0 4.0 3.5 6.5 4.0 NA 4.5 3.0 NA 5.0 5.0 4.0 6.5 NA 4.0#> [55] 5.0 3.5 6.5 2.5 6.5 5.0 6.0 5.5 4.5 3.5 3.0 2.5 4.5 4.5 4.5 4.5 4.5 3.0#> [73] 4.5 4.5 5.0 5.0 2.5 6.5 5.5 4.5 6.0 2.5 4.0 7.0 4.5 NA 3.5 NA 3.5 3.0#> [91] 4.0 4.0 5.0 5.0 2.5 NA 3.5 6.0 1.5 6.5 5.5 5.5 5.5 4.0 4.0 4.0 NA 3.0#> [109] NA 4.0 5.0 5.5 3.5 4.0 3.0 4.0 6.5 3.5 6.0 3.0 4.5 6.0 NA 3.0 3.5 NA#> [127] 5.0 3.0 NA 5.5 5.5 6.5 4.0 NA 5.0 5.0 5.0 NA 3.0 2.5 NA 4.0 6.0 5.0#> [145] 5.0 3.5 4.5 5.0 NA 4.5 2.5 4.0 4.0 5.5 3.0 7.0 5.0 5.0 5.0 NA 5.5 7.5#> [163] 4.0 2.5 3.5 5.5 NA 4.5 6.0 5.0 5.0 5.5 2.5 4.5 5.5 2.0 5.0 7.0 NA 6.0### For complete output, refer to the capl vignetteget_pa_competence_score function computes a physical activity competence score that ranges from 1.5 to 7.5 based on responses to three items from the Behavioural Regulation in Exercise Questionnaire as they appear in the CAPL-2 Questionnaire (www.capl-eclp.ca/wp-content/uploads/2018/02/CAPL-2-questionnaire.pdf). This score is used to compute the motivation and confidence domain score.The get_pa_competence_scorecapl_demo_data$pa_competence_score#> [1] 5.5 5.0 3.0 5.0 4.0 2.5 4.5 4.5 4.5 6.0 4.5 4.0 3.5 4.0 4.0 3.5 5.0 3.5#> [19] 5.0 6.0 5.5 4.0 NA 4.5 5.5 NA 5.0 4.0 5.0 5.0 4.0 NA 3.0 NA 4.5 3.0#> [37] 6.0 NA 1.5 3.5 4.5 6.5 2.5 5.5 3.5 6.0 4.0 6.5 5.5 5.0 6.0 NA 3.0 5.5#> [55] 5.0 5.0 5.5 NA 5.0 5.0 5.0 NA 4.5 5.0 4.0 7.0 3.5 5.5 4.5 4.5 NA 2.5#> [73] 4.0 3.5 4.5 5.0 4.0 4.0 3.5 3.5 2.5 5.0 5.0 3.0 5.0 3.0 5.0 NA 3.0 3.5#> [91] 3.5 5.0 5.0 NA NA 5.0 3.5 5.0 5.0 4.0 NA 3.5 5.0 4.5 5.5 7.5 4.0 5.0#> [109] NA 4.0 6.0 NA 5.5 6.0 5.5 3.0 4.0 3.5 4.0 6.0 3.5 4.0 NA 5.0 6.0 7.0#> [127] 4.0 4.5 4.5 2.5 NA 2.5 7.5 4.5 4.0 4.0 NA 3.0 4.0 5.5 NA 4.0 3.5 4.5#> [145] 3.5 5.0 3.0 NA 5.5 4.5 5.5 3.5 6.0 5.0 6.5 6.0 4.0 2.0 4.0 NA 2.5 4.5#> [163] 4.5 5.0 6.0 5.5 2.0 7.0 NA 3.5 3.5 NA 5.0 NA 5.5 6.0 5.5 5.0 4.0 4.0### For complete output, refer to the capl vignetteget_mc_score function computes a motivation and confidence domain score that ranges from zero to 30 based on the predilection, adequacy, intrinsic motivation and physical activity competence scores. If one of the scores is missing or invalid, a weighted domain score is computed from the other three scores. This score is used to compute the overall physical literacy score.The get_mc_scorecapl_demo_data$mc_score#> [1] 15.333333 20.533333 21.333333 16.400000 NA 14.666667 19.866667#> [8] 18.266667 17.200000 20.266667 18.266667 15.866667 18.666667 17.466667#> [15] 18.400000 19.466667 18.800000 16.800000 20.533333 21.066667 18.666667#> [22] 16.533333 NA 15.600000 22.133333 NA 22.400000 17.600000#> [29] 18.133333 22.400000 21.600000 NA 18.800000 NA 23.333333#> [36] 14.533333 20.933333 NA 13.466667 13.466667 19.066667 20.000000#> [43] 18.000000 22.000000 NA 22.000000 16.000000 23.466667 20.400000#> [50] NA 20.133333 NA 14.400000 22.000000 15.600000 19.733333#> [57] 20.533333 NA 13.866667 15.600000 18.000000 NA 20.000000#> [64] 19.600000 18.400000 19.866667 15.200000 17.200000 21.466667 16.533333### For complete output, refer to the capl vignetteget_capl_interpretation function computes an age- and gender-specific CAPL-2 interpretation for a given CAPL-2 protocol or domain score.The get_capl_interpretationcapl_demo_data$mc_interpretation#> [1] \"beginning\" \"progressing\" \"progressing\" \"progressing\" NA#> [6] \"beginning\" \"progressing\" \"progressing\" \"progressing\" \"progressing\"#> [11] NA\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"beginning\" NA\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0NA\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"progressing\"#> [16] \"progressing\" \"progressing\" \"progressing\" \"progressing\" \"progressing\"#> [21] \"progressing\" \"progressing\" NA\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0NA\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0NA#> [26] NA\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"progressing\" NA\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"progressing\" \"progressing\"#> [31] \"progressing\" NA\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"progressing\" NA\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"achieving\"#> [36] NA\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0NA\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0NA\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"beginning\" \"beginning\"#> [41] \"progressing\" \"progressing\" \"progressing\" \"progressing\" NA#> [46] \"progressing\" \"beginning\" \"achieving\" NA\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0NA### For complete output, refer to the capl vignetteget_capl_domain_status function computes the status of a CAPL domain.The get_capl_domain_statuscapl_demo_data$mc_status#> [1] \"complete\"\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"complete\"\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"complete\"#> [4] \"complete\"\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"missing protocol\"\u00a0\u00a0\u00a0\u00a0\u00a0 \"complete\"#> [7] \"complete\"\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"missing protocol\"\u00a0\u00a0\u00a0\u00a0\u00a0 \"complete\"#> [10] \"missing protocol\"\u00a0\u00a0\u00a0\u00a0\u00a0\"missing interpretation\" \"complete\"#> [13] \"missing interpretation\" \"missing interpretation\" \"missing protocol\"#> [16] \"complete\"\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"complete\"\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"complete\"#> [19] \"complete\"\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"complete\"\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"complete\"#> [22] \"complete\"\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"missing protocol\" \"missing interpretation\"#> [25] \"missing interpretation\" \"missing interpretation\" \"complete\"#> [28] \"missing interpretation\" \"complete\"\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"complete\"### For complete output, refer to the capl vignettewww.capl-eclp.ca/capl-manual), the formula for computing the knowledge and understanding score is:As illustrated in get_binary function computes a binary score for a response to a questionnaire item based on the value(s) set as answer(s) to the item.The get_binary_score)capl_demo_data$pa_guideline_score <- capl_demo_data$pa_guideline_score#> [1] 0 1 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0#> [26] 1 1 0 0 0 0 0 0 1 1 0 0 0 0 1 0 0 0 1 0 1 0 0 1 0#> [51] 0 0 0 0 0 0 0 0 1 1 0 0 0 0 0 0 0 1 0 0 0 0 1 0 1#> [76] 0 0 0 1 1 1 0 0 1 0 0 1 0 0 1 0 1 0 0 1 1 0 0 0 1#> [101] 0 0 1 0 1 0 1 0 0 0 0 0 0 0 0 1 0 0 0 1 1 1 1 0 1#> [126] 0 0 0 0 0 1 0 0 0 0 0 1 0 0 1 1 0 0 1 1 1 0 0 0 0#> [151] 0 0 0 0 0 0 1 1 0 0 0 1 0 1 0 0 0 0 0 1 0 1 0 0 0#> [176] 0 1 0 1 0 0 1 1 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 1#> [201] 0 1 0 0 0 0 1 1 0 0 0 0 0 0 1 0 0 0 1 0 0 0 0 0 0#> [226] 0 0 0 0 0 0 1 1 0 0 0 0 0 0 0 1 0 0 1 1 1 0 0 1 0### For complete output, refer to the capl vignetteget_binary function is also called to analyze responses for Q2, Q3, and Q4.The get_binary_score)capl_demo_data$crf_means_score <- capl_demo_data$crf_means_score#> [1] 0 0 0 1 1 0 1 0 0 0 1 1 0 0 0 1 0 0 0 0 0 1 0 0 0#> [26] 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0#> [51] 0 0 0 1 0 1 1 1 0 0 0 0 0 0 1 0 0 0 1 1 0 1 1 0 1#> [76] 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 1#> [101] 1 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 1 0 1 0 0 0 0 0 0#> [126] 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 1 1 0 1 1 0 0 0 0 1#> [151] 0 0 0 1 0 1 0 0 0 1 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0#> [176] 0 0 1 1 0 0 0 0 0 0 0 1 1 0 0 0 1 1 1 0 0 0 1 0 0#> [201] 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 1 0#> [226] 0 1 0 1 1 0 0 0 0 1 1 0 0 0 0 1 0 0 0 0 0 0 0 0 1### For complete output, refer to the capl vignetteget_binary_score)capl_demo_data$ms_means_score <- capl_demo_data$ms_means_score#> [1] 0 0 1 0 0 1 1 0 0 0 1 1 1 0 1 0 0 0 0 0 0 0 0 0 0#> [26] 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 1 0 0 0 0 0#> [51] 0 1 1 0 1 0 1 1 0 1 0 0 0 0 1 1 1 1 0 1 1 1 0 1 0#> [76] 0 0 0 0 0 0 0 1 0 0 0 0 1 0 0 1 0 1 0 1 0 0 0 0 0#> [101] 1 0 0 0 0 0 0 0 0 0 1 0 0 1 0 1 1 0 0 0 1 1 1 0 0#> [126] 0 0 1 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0#> [151] 0 0 0 1 0 1 0 0 1 1 0 0 1 0 0 1 1 0 0 0 0 0 0 1 1#> [176] 1 0 0 0 0 1 1 0 1 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0#> [201] 0 0 0 0 0 0 0 0 1 0 0 0 0 0 1 NA 0 0 0 0 0 1 0 1 0#> [226] 0 0 1 0 0 1 0 1 0 0 0 1 0 0 0 1 1 0 0 1 0 1 0 0 0### For complete output, refer to the the capl vignetteget_binary_score)capl_demo_data$sports_skill_score <- capl_demo_data$sports_skill_score#> [1] 0 1 1 0 0 0 0 0 1 0 1 0 0 0 1 0 0 1 1 0 0 0 0 0 0 0 0 1 1 0 0 1 0 0 1 0 0#> [38] 0 0 0 0 0 0 0 0 1 0 1 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 1 1 1 0 0 0 0 0#> [75] 1 0 0 1 0 1 0 0 0 1 0 0 1 1 0 0 0 0 1 1 0 0 0 1 0 1 0 0 1 1 0 0 1 0 0 1 0#> [112] 0 0 0 1 1 0 0 0 0 1 1 0 0 0 0 0 1 0 0 0 0 1 0 0 0 0 0 1 0 0 0 0 1 0 1 0 0#> [149] 0 0 0 0 0 0 0 0 0 1 0 0 0 0 1 0 0 0 1 0 0 0 1 0 0 0 0 1 0 0 0 0 0 0 0 1 0#> [186] 0 0 0 0 0 1 0 1 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 1 0 1 0 0 0 0 0#> [223] 0 1 0 0 0 0 0 1 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 1 0 0 0 0 0 0 0 1#> [260] 0 0 1 0 1 0 0 0 0 0 0 0 0 1 0 0 0 1 1 1 1 0 0 0 0 0 1 1 0 0 0 0 0 1 0 0 0#> [297] 0 0 0 0 0 0 0 0 0 0 1 0 0 0 1 0 1 0 0 0 0 0 0 0 1 0 0 1 0 0 0 0 0 1 0 0 1#> [334] 0 0 0 1 0 0 0 1 1 0 0 0 1 1 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0### For complete output, refer to the capl vignetteget_fill_in_the_blanks_score function computes a score that ranges from zero to five for responses to the fill in the blanks question. This score is used to compute the knowledge and understanding domain score.The get_fill_in_the_blanks_scorecapl_demo_data$fill_in_the_blanks_score#> [1] 1 5 2 5 5 3 3 3 3 4 5 4 3 4 4 3 2 2 3 4 3 3 4 1 5 4 6 4 4 4 4 4 1 4 4 2 4#> [38] 4 3 5 4 3 4 3 5 5 4 5 2 3 4 4 2 3 6 3 3 5 5 2 6 2 2 4 2 4 3 3 6 2 3 5 5 3#> [75] 3 2 5 2 3 3 5 4 4 4 5 5 2 3 4 3 4 5 3 1 5 5 5 4 4 4 5 3 4 2 0 3 3 4 4 3 3#> [112] 5 3 4 4 2 2 3 6 4 3 5 5 2 3 3 5 5 3 6 4 3 2 3 3 0 3 4 3 3 3 3 4 3 3 2 5 5#> [149] 4 5 4 3 3 5 0 3 3 4 4 2 2 3 4 1 3 3 2 3 6 3 4 4 4 6 2 4 2 3 2 5 4 3 2 5 5#> [186] 5 3 2 5 3 3 3 3 5 6 2 4 4 3 4 4 2 5 2 4 3 4 4 1 4 3 5 4 3 3 4 3 2 0 5 4 4#> [223] 4 5 3 6 2 5 4 2 3 3 2 4 3 4 3 2 5 3 3 5 3 3 4 5 0 3 4 4 4 4 1 2 4 4 2 3 1#> [260] 3 5 2 3 5 4 1 3 3 3 4 3 3 3 4 3 4 5 0 5 3 3 3 3 5 6 5 3 4 3 3 6 6 2 1 2 2#> [297] 3 5 4 4 2 1 1 1 5 2 2 5 4 3 5 4 6 3 5 2 5 3 2 5 4 5 4 4 1 3 4 6 3 3 3 5 5#> [334] 2 3 4 3 5 5 4 4 0 4 3 4 3 3 3 4 2 3 2 3 6 5 3 5 2 4 4 5 3 2 4 3 2 3 3 5 2### For complete output, refer to the capl vignetteget_ku_score function computes a knowledge and understanding domain score that ranges from zero to 10 based on the physical activity guideline (Q1), cardiorespiratory fitness means (Q2), muscular strength and endurance means (Q3), sports skill (Q4) and fill in the blanks (Q5) scores. If one of the scores is missing or invalid, a weighted domain score is computed from the other four scores. This score is used to compute the overall physical literacy score.The get_ku_scorecapl_demo_data$ku_score#> [1] 1.000000 7.000000 4.000000 6.000000 6.000000 4.000000 6.000000 3.000000#> [9] 4.000000 4.000000 8.000000 6.000000 4.000000 4.000000 6.000000 4.000000#> [17] 2.000000 3.000000 4.000000 4.000000 3.000000 4.000000 4.000000 1.000000#> [25] 5.000000 5.000000 7.000000 5.000000 5.000000 5.000000 4.000000 5.000000#> [33] 1.000000 5.000000 6.000000 2.000000 4.000000 4.000000 3.000000 6.000000#> [41] 4.000000 3.000000 5.000000 4.000000 6.000000 7.000000 4.000000 7.000000#> [49] 3.000000 3.000000 4.000000 5.000000 3.000000 4.000000 7.000000 4.000000#> [57] 6.000000 7.000000 6.000000 4.000000 6.000000 2.000000 2.000000 4.000000#> [65] 4.000000 5.000000 5.000000 6.000000 8.000000 4.000000 4.000000 7.000000#> [73] 7.000000 4.000000 6.000000 2.000000 5.000000 3.000000 4.000000 5.000000### For complete output, refer to the capl vignetteget_capl_interpretation function computes an age- and gender-specific CAPL-2 interpretation for a given CAPL-2 protocol or domain score.The get_capl_interpretationcapl_demo_data$ku_interpretation#> [1] \"beginning\" \"achieving\" \"beginning\" \"progressing\" \"progressing\"#> [6] \"beginning\" \"progressing\" \"beginning\" \"beginning\" \"beginning\"#> [11] NA\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"progressing\" NA\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0NA\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"progressing\"#> [16] \"beginning\" \"beginning\" \"beginning\" \"beginning\" \"beginning\"#> [21] \"beginning\" \"beginning\" \"beginning\" NA\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0NA#> [26] NA\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"progressing\" NA\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"beginning\" \"progressing\"#> [31] \"beginning\" \"beginning\" \"beginning\" NA\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"progressing\"#> [36] NA\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0NA\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"beginning\" \"beginning\" \"progressing\"#> [41] \"beginning\" \"beginning\" \"progressing\" \"beginning\" \"progressing\"#> [46] \"achieving\" \"beginning\" \"achieving\" NA\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0NA### For complete output, refer to the capl vignetteget_capl_domain_status function computes the status of a CAPL domain.The get_capl_domain_statuscapl_demo_data$ku_status#> [1] \"complete\"\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"complete\"\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"complete\"#> [4] \"complete\"\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"complete\"\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"complete\"#> [7] \"complete\"\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"complete\"\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"complete\"#> [10] \"complete\"\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"missing interpretation\" \"complete\"#> [13] \"missing interpretation\" \"missing interpretation\" \"complete\"#> [16] \"complete\"\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"complete\"\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"complete\"#> [19] \"complete\"\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"complete\"\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"complete\"#> [22] \"complete\"\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"complete\"\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"missing interpretation\"#> [25] \"missing interpretation\" \"missing interpretation\" \"complete\"#> [28] \"missing interpretation\" \"complete\"\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"complete\"### For complete output, refer to the capl vignetteget_capl_score function computes an overall physical literacy score that ranges from zero to 100 based on the physical competence, daily behaviour, motivation and confidence, and knowledge and understanding domain scores. If one of the scores is missing or invalid, a weighted score is computed from the other three scores.The get_capl_scorecapl_demo_data$capl_score#> [1] 54.28571 76.90000 60.00000 68.80000 79.00000 63.00000 52.00000 71.26667#> [9] 58.40000 74.66667 83.14286 67.90000 67.50000 65.10000 54.85714 83.60000#> [17] 74.60000 77.10000 68.40000 78.80000 75.00000 71.40000 70.83333 64.42857#> [25] 68.10000 80.46667 83.30000 68.20000 74.10000 71.30000 65.60000 51.50000#> [33] 65.60000 69.23333 79.33333 77.40000 74.20000 80.00000 77.10000 61.10000#> [41] 69.30000 81.50000 68.00000 84.50000 81.42857 73.00000 52.00000 86.38095#> [49] 64.30000 68.93333 70.10000 83.33333 74.40000 75.50000 79.86667 82.30000#> [57] 62.90000 79.20000 86.13333 71.70000 82.50000 67.80000 59.50000 68.14286#> [65] 65.80000 79.40000 32.90000 76.30000 71.60000 64.14286 73.04762 67.70000#> [73] 71.20000 69.71429 67.50000 75.60000 69.40000 47.40000 60.00000 51.28571### For complete output, refer to the capl vignetteget_capl_interpretation function computes an age- and gender-specific CAPL-2 interpretation for a given CAPL-2 protocol or domain score.The get_capl_interpretationcapl_demo_data$capl_interpretation#> [1] \"progressing\" \"excelling\" \"progressing\" \"achieving\" \"achieving\"#> [6] \"progressing\" \"beginning\" \"achieving\" \"progressing\" \"excelling\"#> [11] NA \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0progressing\" NA\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0NA\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"progressing\"#> [16] \"excelling\" \"achieving\" \"excelling\" \"progressing\" \"excelling\"#> [21] \"achieving\" \"achieving\" \"achieving\" NA\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0NA#> [26] NA\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"excelling\" NA\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"achieving\" \"achieving\"#> [31] \"progressing\" \"progressing\" \"progressing\" NA\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"excelling\"#> [36] NA\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0NA\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"excelling\" \"excelling\" \"progressing\"#> [41] \"achieving\" \"excelling\" \"achieving\" \"excelling\" \"excelling\"#> [46] \"achieving\" \"progressing\" \"excelling\" NA\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0NA### For complete output, refer to the capl vignetteget_capl_domain_status function computes the status of a CAPL domain.The get_capl_domain_statuscapl_demo_data$capl_status#> [1] \"missing protocol\"\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"complete\"\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"complete\"#> [4] \"complete\"\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"complete\"\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"complete\"#> [7] \"missing protocol\"\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"complete\"\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"complete\"#> [10] \"missing protocol\"\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"missing interpretation\" \"complete\"#> [13] \"missing interpretation\" \"missing interpretation\" \"missing protocol\"#> [16] \"complete\"\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"complete\"\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"complete\"#> [19] \"complete\"\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"complete\"\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"complete\"#> [22] \"complete\"\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"complete\"\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"missing interpretation\"#> [25] \"missing interpretation\" \"missing interpretation\" \"complete\"#> [28] \"missing interpretation\" \"complete\"\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\"complete\"### For complete output, refer to the capl vignettecapl package makes use of the famous ggplo2 R package to create custom functions that render beautiful plots for visualizing CAPL-2 results.The get_capl_bar_plot function. The mean score for each interpretative category appears above each bar \")colors argument \",c\u00a0\u00a0\u00a0\u00a0colors = )export_capl_data function allows them to export their data to Excel or SPSS.If users want to export their data, the export_capl_dataexport_capl_datacapl package developed for use in the R environment. The primary motivation for developing the capl package was to offer interested users \u2013 most likely researchers \u2013 a fast, efficient, and reliable approach to analyzing CAPL-2 raw data. We begin this paper by discussing several preparatory steps that are required prior to using the capl package. These steps include preparing, formatting, and importing CAPL-2 raw data. We then use demo data to show that computing the CAPL-2 scores and interpretations is as a simple as executing one line of code. This one line of code uses the main (wrapper) function in the capl package (get_capl) to compute 40 variables. Next, we introduce each helper function that is called within the main function to explain how to compute individual variables and scores for each test element within the four domains as well as how to calculate an overall physical literacy score. Finally, we show how to visualize CAPL-2 results using the ggplot2 R package.In this paper we introduce the capl package is that it is specifically built for CAPL-2 raw data, and therefore not fully accessible to users with earlier versions of the CAPL. In the future, we intend to make the capl package available to users across all versions of the CAPL. The future version of the capl package will also include more data visualization features. We also plan to release an R Shiny application that runs locally in a web browser, providing users with a web-based interface for the capl package .One limitation of the current capl package, users are no longer required to perform a large number of computations nor are they burdened with the monotonous task of entering data individually for each participant via the CAPL-2 website. Furthermore, we carefully crafted the package to create a \u201cquiet\u201d user experience, whereby \u201cnoisy\u201d error messages are suppressed via validation. Instead of throwing noisy errors that halt code execution, the capl package returns missing or invalid values as NAs. The release of the capl package will contribute to the growing and popular topic of physical literacy, and will not only support current users of CAPL-2 but may also attract new users to this area of research.With the development of the"} +{"text": "A covalent organic framework-based nanoplatform has been developed for cancer imaging, photodynamic therapy, and prognosis. In vitro and in vivo experiments revealed that the nanoplatform has a high specificity and inhibition effect toward cancer cells and solid tumors. Interestingly, prognostic evaluation was also realized with COF-survivin. This work will offer new insights into COF-based probes and inspire the development of more versatile tools for biomedical applications.Covalent organic frameworks (COFs) have emerged as a kind of promising material for analytical and biomedical purposes. However, simultaneous cancer diagnosis and therapy with COFs remain a challenge. We report here a COF-based theranostic nanoplatform by integrating a dye-labeled oligonucleotide onto porphyrin-based COF nanoparticles for highly efficient cancer diagnosis and therapy. The fluorescence of the dye was effectively quenched by the COF through fluorescence resonance energy transfer (FRET). In the presence of biomarker survivin mRNA, more stable duplexes were formed and separated from the COF NPs, enabling the recovery of the fluorescence signal and selective cancer imaging. Under NIR laser irradiation, COF NPs generated abundant reactive oxygen species (ROS) to induce cancer cell apoptosis owing to their crystalline reticular structure. Covalent organic frameworks (COFs), as a class of emerging crystalline porous polymeric materials, possess tremendous potential for application in a variety of fields owing to their facile design and well-defined structure.Photodynamic therapy (PDT), employing a specific laser to excite a photosensitizer (PS) to generate reactive oxygen species (ROS) and kill malignant cells, is highly spatiotemporally controlled and has been approved for clinical cancer treatment.Traditionally, cancer diagnosis and treatment are individual and separate processes.Herein, we developed a nanoscale COF-based theranostic nanoplatform. A nanoscale porphyrin-based COF was prepared and a TAMRA-labeled survivin antisense strand (TSAS) was integrated onto the COF NPs to give rise to the nanoplatform (termed COF-survivin). The crystalline reticular structure endowed the COF with better stability and higher ROS generation ability in aqueous solution than those of the porphyrin monomer, while the large planar structure composed of the strong \u03c0-electron system makes it easy to absorb DNA single strands and quench the fluorophore to form a stable nanoplatform. The TSAS could readily form a duplex structure with survivin mRNA , PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O stretching band at 1660 nm is decreased, while a new peak at 1601 nm appeared corresponding to the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N stretching band, demonstrating the successful condensation between amino and aldehyde groups. Moreover, multiple intense peaks were observed in the PXRD pattern, indicating the successful preparation of crystal COF structures , one of the most toxic ROS,COF NPs were prepared based on a solvothermal strategy.s Fig. S1. The TEMs Fig. S1 and SEM s Fig. S1 results s Fig. S1 of COF Ns Fig. S1 of COF N1.000000,.000000 s\u20131 of different groups was determined. Prominent \u0394\u03a8m loss was detected in the therapy group as shown in Subsequently, the in vivo experiments were carried out. We first investigated whether the nanoplatform could differentiate tumor tissue from normal tissue. The result of in vivo fluorescence imaging by integrating a TAMRA-labeled survivin antisense oligonucleotide onto a nanoscale porphyrin-based COF. The crystalline structure characteristics of the COF endowed the nanoplatform with excellent stability and ROS generation capability. In the presence of cancer biomarker survivin mRNA, the readily formed duplex detached from the COF, which could restore the fluorescence by FRET prohibition and realize selective cancer imaging. Under NIR laser irradiation, the COF could generate abundant All the animal experiments were conducted and agreed with the Principles of Laboratory Animal Care (People's Republic of China) and the Guidelines of the Animal Investigation Committee, Biology Institute of Shandong Academy of Science, China.The authors declare no competing financial interest.Supplementary informationClick here for additional data file."} +{"text": "Borata-alkenes can serve as anionic olefin equivalent ligands in transition metal chemistry. A chelate ligand of this type is described and used for metal coordination. 2P(CH2)2B(C6F5)2 frustrated Lewis pair in the \u03b1-CH[B] position gave the methylene-bridged phosphane/borata-alkene anion. It reacted with the [Rh(nbd)Cl] or [Rh(CO)2Cl] dimers to give the respective neutral chelate [Rh] complexes. The reaction of the \u2013 anion with [Ir(cod)Cl]2 proceeded similarly, only that the complex underwent a subsequent oxidative addition reaction at the mesityl substituent. Both the resulting Ir(iii)hydride complex 15 and the P/borata-alkene Rh system 12 were used as hydrogenation catalysts. The Rh(nbd) complex 12 served as a catalyst for arylacetylene polymerization.Borata-alkenes can serve as anionic olefin equivalent ligands in transition metal chemistry. A chelate ligand of this type is described and used for metal coordination. Deprotonation of the Mes PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019B bond lengths around 1.45 \u00c5 were found in these systems. In addition, a variety of related boryl-carbanion \u2194 borata-alkene systems were in situ generated and employed as reagents e.g. in borata-Wittig olefination chemistry.3Carbanions in the \u03b1-position to boryl groups show a conjugative interaction with the adjacent Lewis acid. Such systems can be described as borata-alkenes. Borata-alkenes derived from some alkyldiarylboranes had previously been prepared.6F5)2 group resulted in a markedly increased \u03b1-CH acidity in the respective boranes. A DFT study had revealed that e.g. H3C\u2013B(C6F5)2 showed a pKa-value comparable to that of cyclopentadiene.3C\u2013B(C6F5)2 borane must be considered >10 pKa values more C\u2013H acidic than the related H3C-BMes2 borane. Consequently, R\u2013H2C\u2013B(C6F5)2 systems were easily deprotonated to give the corresponding \u2013 borata-alkene systems. Several of such systems were isolated as their Li+ salts. Some were used in borata-Wittig olefination reactions.It was recently shown that the presence of the strongly electron-withdrawing \u2013BC6F2 group r3-borata-allyl metal complexes and related systems known.et al. had prepared the borata-alkene tantalocene complex 4 scale\" fill=\"currentColor\" stroke=\"none\">B(C6F5)2]\u2013 ligand with the neutral \u03b72-olefin analogues. The Piers group developed some follow-up chemistry of complex 4.et al. have just recently described a conceptually related borata-phenanthrene gold complex.Neutral bora-alkene compounds had previously been used as ligandsomplex 4 9 and emp PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CH2\u2013 terminus by a Mes2P\u2013CH2-substituent now gave an anionic system that served as a chelate ligand in Rh and Ir coordination chemistry.Formal substitution of a hydrogen atom of the borata-alkene 7.2P-vinyl (5) with Piers' borane [HB(C6F5)2] (6)15We started our phosphane/borata-alkene chelate ligand synthesis from the ethylene-bridged frustrated P/B Lewis pair (FLP) 7 at the \u03b1-position to the boron atom by treatment with LDA . Compound 7 is \u03b1-CH acidic, but it is also an active boron Lewis acid that is able to abstract hydride from amines in the \u03b1-position to nitrogen with iminium salt formation.7 generated the respective imine. This is found as a component in the product 8 that we isolated from the reaction mixture as a white solid in 67% yield 2 backbone. The boron atom shows a pseudotetrahedral coordination geometry (\u03a3B1CCC 333.2\u00b0); it has hydride attached. The lithium cation shows contacts to the [B]-H moiety, the phosphorus atom and one ortho-C6F5 fluorine atom. The Li cation has the newly formed imine moiety N-coordinated. In solution compound 8 shows a 11B NMR [B]-H doublet at \u03b4 \u201318.4 with a 1JBH \u223c70 Hz coupling constant and a 7Li NMR (C6D6) signal at \u03b4 1.4. The \u2013N PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CMe2 imine 13C NMR resonance occurs at \u03b4 173.5.We first attempted deprotonation of 7% yield . Compounanalysis . It show7 with the LiTMP reagent, a base that has no CH groups \u03b1 to nitrogen. The reaction was carried out with the in situ generated FLP 7. Treatment with LiTMP in pentane for 16 h at room temperature followed by workup gave the methylene-linked phosphane/borata-alkene product 9 \u00c5, which is much shorter than the adjacent boron-aryl bonds (B1\u2013C31: 1.607(3)\u00c5, B1\u2013C41: 1.602(3)\u00c5). The boron coordination geometry in compound 9 is trigonal-planar (\u03a3B1CCC 360.0\u00b0). The B1\u2013C2\u2013C1 angle amounts to 126.4(2)\u00b0. The lithium ion in 9 shows contacts to the borata-alkene unit as well as to the phosphorus atom and one ortho-C6F5 fluorine atom. The lithium atom Li+ also has the HTMP amine ligand bonded to it that had been formed in the deprotonation process (Compound process .8) compound 9 features a typical borata-alkene 11B NMR signal at \u03b4 18.6. The 31P NMR signal is at \u03b4 \u201320.6 and the \u2013CH2\u2013CH PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019 backbone shows 1H NMR resonances at \u03b4 4.21 ) and \u03b4 3.36 . The 19F NMR spectrum of compound 9 shows two sets of o,p,m-resonances of the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019B(C6F5)2 moiety .In solution 2 reagent.10 (isolated as a yellow solid in 52% yield). It was characterized by C,H-elemental analysis, by spectroscopy and by its reaction with dihydrogen (see below). Compound 10 is a typical intramolecular FLP, showing a P\u2013B interaction with one boron atom and having the other one free. However, the temperature dependent 19F NMR spectrum showed exchange between the pair of B(C6F5)2 groups at e.g. 299 K. Only at low temperature (e.g. 203 K) we observed a set of three broad 19F NMR resonances of a free trigonal planar B(C6F5)2 unit and a set of ten separate signals of the rotationally hindered P\u00b7\u00b7\u00b7B(C6F5)2 group. The 31P NMR (299 K) signal of compound 10 is at \u03b4 16.3 and the \u2013CH2\u2013CH PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019 backbone shows 1H NMR features at \u03b4 3.55 and \u03b4 4.30, respectively ).We briefly investigated the nucleophilic property of the borata-alkene unit in compound 10 reacted rapidly with dihydrogen under mild conditions to give the phosphonium/hydridoborate dihydrogen splitting product 11 (isolated as a solid in 71% yield). The X-ray crystal structure analysis (CCC 339.7\u00b0) and the newly formed hydride-bridged bis-borane moiety. In solution (CD2Cl2) the phosphonium [P]-H unit showed up at \u03b4 7.58 (1H NMR) and \u03b4 \u20133.7 , respectively. We recorded a broadened 11B NMR signal at \u03b4 \u201318.1 with a corresponding broad 1H NMR [B](\u03bc-H) feature at \u03b4 5.45. The 19F NMR spectrum of compound 11 shows two equal-intensity sets of o,p,m-C6F5 signals of the pair of B(C6F5)2 groups and we observed the 1H/13C NMR signals of the \u2013CH2\u2013CH backbone at \u03b4 2.84/27.8 (PCH2) and \u03b4 1.80/7.6(br)(BCH), respectively.Compound analysis showed t9 as a chelate ligand in Rh chemistry. For that purpose, we treated the (norbornadiene)RhCl dimer with the prefabricated borata-alkene reagent 9 for 18 h in toluene solution at room temperature. Workup then gave the respective neutral chelate phosphane/borata-alkene(norbornadiene)Rh complex 12 in >60% yield scale\" fill=\"currentColor\" stroke=\"none\">B system serves as a chelate ligand. It is unsymmetrically \u03b72-coordinated through both backbone atoms of the borata-alkene moiety and \u03baP-bonded to the attached phosphanyl group. As the P/C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019B ligand is mono-anionic, the resulting Rh complex is neutral. The C2\u2013B1 bond is only marginally elongated, it is still within the typical C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019B distance of borata-alkene examples12 the phosphane donor exhibits a stronger structural trans-effectWe used the methylene-bridged phosphane/borata-alkene anion of the lithium salt 0% yield . Suitabl0% yield . Compoun12 shows a 31P NMR signal (CD2Cl2) at \u03b4 \u201389.0 with a 1JRhP \u223c 120 Hz coupling constant. This changed only marginally when the spectrum of 12 was recorded in d8-THF solution. Compound 12 shows a 11B NMR signal at \u03b4 24.3, a value that is similar to that of the uncomplexed borata-alkene anion 9 (see above).19F NMR spectrum of 12 shows two sets of o,p,m-C6F5 signals for the pair of pentafluorophenyl substituents at boron. We observed the 1H NMR signals of the chelate ligand backbone at \u03b4 4.14/3.90 (PCH2) and \u03b4 3.68 scale\" fill=\"currentColor\" stroke=\"none\">CH\u2013), respectively ), and there are the 1H/13C NMR signals of the coordinated norbornadiene ligand at rhodium Rh dimer was carried out at r.t . Workup involving extraction with pentane and crystallization gave the neutral chelate [P/borata-alkene]Rh(CO)2 complex 13 as a yellow crystalline solid in 46% yield. The X-ray crystal structure analysis scale\" fill=\"currentColor\" stroke=\"none\">B unit . Compound 13 shows strong IR CO bands at \u03bd = 2069 and 1997 cm\u20131.2Cl2 solution it shows a 11B NMR signal at \u03b4 27.3, i.e. in the typical borata-alkene range. The 31P NMR resonance was located at \u03b4 \u2013105.0 with a 1JRhP = 88.5 Hz coupling constant. The borata-alkene unit in complex 13 shows 19F NMR signals of a pair of inequivalent C6F5 substituents at boron.The reaction of the P/borata-alkene lithium salt analysis showed a9 and the iridium(cyclooctadiene)chloride dimer was carried out similarly . It gave a slightly different outcome. We assume that initially a Ir(cod) complex 14 was generated, analogous to the formation of the Rh system 12. However, it was apparently not persistent under the prevailing reaction conditions but underwent intramolecular C\u2013H bond activationortho-methyl group of a mesityl substituent at phosphorus to give the oxidative addition product 15 .The reaction between the borata-alkene reagent oduct 15 . It was 15 revealed that the iridium atom has undergone oxidative addition at a mesityl group at phosphorus, with formation of a new benzylic \u2013CH2\u2013Ir\u2013H moiety the iridium complex 15 shows four olefinic 1H NMR signals of the coordinated cyclooctadiene ligand. It also features four arene CH 1H NMR signals of the mesitylene and the CH-activated Mes substituents at phosphorus. Complex 15 shows a broadened 11B NMR resonance at \u03b4 \u201317.5. The 31P NMR signal is observed at \u03b4 \u2013104.0. It shows coupling to the Ir\u2013H moiety (2JPH \u223c 70 Hz).\u03b4 \u201310.4 with ca. 70 Hz coupling to phosphorus complex 15 was actually formed by an oxidative addition reaction at a mesityl methyl group at the stage of the analogous intermediate 14. We carried out some preliminary investigation toward the use of the new chelate phosphane/borata-alkene complexes in catalysis. For this reason, we performed two sets of catalytic reactions using either of the complexes 12 and 15. We first turned to alkene and alkyne hydrogenation catalysis.15 to dihydrogen revealed the stoichiometric formation of cyclooctane, the reduction product of the cod ligand of the Ir complex 15. Consequently, we employed compound 15 as a catalyst in our hydrogenation experiments. The hydrogenation of styrene is a typical example. With both 1 or 0.5 mol% of 15 quantitative hydrogenation to ethylbenzene was achieved analogue of the Rh complex 12 (see above).Our study has shown that the methylene linked phosphane/borata-alkene anion of the salt achieved ; with 0.15 derived catalyst system with 1 mol% of vinylcyclohexane or cyclohexene, as well. The more sterically encumbered 1-methylcyclohexene substrate gave only a 39% conversion under these conditions and phenylacetylene eventually furnished a ca. 3\u2009:\u20091 mixture of styrene and ethylbenzene with a combined conversion of 77% after 16 h.Quantitative alkene hydrogenation was found at the 12 under our standard conditions . The bulkier 1-methylcyclohexene was not hydrogenated at the Rh catalyst system 12 under our typical conditions.Styrene was quantitatively hydrogenated to ethylbenzene with 0.5 mol% of the Rh catalyst nditions . With 0.12 or 15, but we presently cannot rule out an alternative \u201cFLP-like\u201d metal/borane dihydrogen splitting reaction.24So far we assume a conventional pathway of dihydrogen activation at the metal centre in the complexes 12.12 was carried out in the non-polar solvent benzene or in ethereal solution (diethylether or tetrahydrofuran). We carried out the phenylacetylene polymerization at room temperature for a duration between 30 min (in ether) or 2 h (in benzene). With decreasing catalyst amounts an almost quantitative amount of polyphenylacetylene was isolated from the reaction in benzene. Even with 0.025 mol% as well as 0.01 mol% of the catalyst poly(phenylacetylene) was isolated, albeit in lower yields . The obtained polymer was similar in appearance (yellow to orange solids) as the poly(phenylacetylene) obtained by Noyori et al. at the remotely related neutral [(Ph3P)n(nbd)Rh-CCPh] (n: 1 or 2) derived catalysts, so we assume it has a similar structure.p-fluorophenylacetylene and p-methoxyphenylacetylene at the catalyst system 12 (0.1 mol%) and isolated the respective polyacetylenes in close to quantitative yields sample was characterized by MALDI-TOF mass spectrometry, which showed the regular sequence of signals separated by the mass of the respective monomer unit of 132 (depicted in the ESIp-anisylacetylene) sample obtained in ether with the Rh complex 12 derived catalyst we found a molecular weight of Mn \u2265 100000. The respective poly(phenylacetylene) sample had an about twice as high Mn, and the poly(p-fluorophenylacetylene) had the highest measured Mn in the series of >400000 scale\" fill=\"currentColor\" stroke=\"none\">B(C6F5)2\u2013 ligand was generated by a typical organometallic reaction pathway within the coordination sphere of the metal . Since we had found about the vastly increased \u03b1-CH acidity of the B(C6F5)2 boranes2C,B-borata-alkene complexes has become evident: deprotonation2\u2013CH2\u2013B(C6F5)2 borane gave the borata-alkene in an independent initial step. Our syntheses of the methylene-bridged chelate phosphane/borata-alkene Rh and Ir complexes serve as examples of this development. The complexes are readily prepared, although the Ir system undergoes a subsequent rearrangement reaction. This new approach will probably allow for some variation on the ligand side, and it may open pathways to choosing variations on the metal side. The P/C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019B ligands in the here reported complexes do not interfere with catalytic features in our examples. To us this indicates that the readily available borata-alkenes might see useful applications as polar alkene ligand analogues in organometallic and coordination chemistry as well as in catalysis.Our study has shown that the seminal study published by Piers There are no conflicts to declare.Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "The first broad spectrum dendralene synthesis permits the widest structural and substituent variation and promotes applications in step economic synthesis. E)/(Z)-stereoselective syntheses of dendralenes are reported (twenty-eight examples). The approach involves twofold Pd(0)-catalyzed Negishi couplings of 1,1-dibromoalkenes with alkenylzinc reagents, and exploits both substrate- and catalyst-controlled aspects of chemo-, regio- and stereoselectivity in the two C(sp2)\u2013C(sp2) bond forming steps. The value of the new hydrocarbons in rapid structural complexity generation is demonstrated through their deployment in unprecedented diene- and triene-transmissive pericyclic reaction sequences.The first general synthetic approach to substituted [3]- and higher dendralenes is reported. Fifty-one mono- through to penta-substituted dendralenes carrying alkyl-, cycloalkyl-, alkenyl-, alkynyl-, aryl- and heteroaryl-substitutents are accessed, and the first ( We provide solutions that are of broad scope, allowing selective access to both E- and Z-diastereomers of an internally-substituted dendralene from the same 1,1-dibromoalkene precursor.Building upon the foundations of previous, narrow-scope cross-coupling methods for dendralene synthesis, PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond. Thus, 1,1-dibromoalkenes 1 undergo twofold Negishi C(sp2)\u2013C(sp2) coupling2, where the two newly introduced alkenyl substituents are the same. 1 and four different alkenyl nucleophiles, to demonstrate the broad scope of this lynchpinThe first group of [3]dendralenes reported here lack stereogenicity about the central C2a\u2013w with substitution at all possible sites on the dendralene framework. Substituents incorporated at the central methylene position of the [3]dendralene (substituents colored blue in 2(dppf)] was superior for twofold cross-coupling.The twofold Negishi cross-coupling protocol works well with vinylzinc bromide and its alkyl and aryl-substituted congeners, to access mono- to penta-substituted [3]dendralenes 1 with different alkenylzinc reagents. It is well established that aldehyde-derived 1,1-dibromoalkenes undergo chemo- and regio-selective single cross-coupling with an alkenyl nucleophile to replace the bromine trans- to the carbon-based substituent (1 \u2192 3).18To achieve the first diastereoselective synthesis of dendralenes, we envisioned two successive cross-couplings of a 1,1-dibromoalkene stituent , 1 \u2192 3.13)4] was the most consistent performer in the trans-selective mono-coupling of 1,1-dibromoalkenes (1 \u2192 3). In all but one case examined, a clear preference for the mono-coupled product was observed and, in every case, only the (1Z)-2-bromo-1,3-butadiene diastereomer was detected.e.g. stoichiometry of coupling partners, temperature) for optimal results. In our hands, as catalyst. We are delighted to report that the same ligand also brings about Pd(0)-catalyzed stereo-retentive cross-couplings of (1Z)-2-bromo-1,3-dienes with alkenyl nucleophiles to permit the first stereoselective dendralene synthesis (3 \u2192 4).Negishi reported that cross-couplings of the resulting -2-bromo-1,3-butadienes. Inconsistent with Negishi's findings, however, are couplings of alkyl-substituted systems , which generally give mixtures of E and Z-diastereomers in the second cross-coupling (see ESI2(dppf)] as pre-catalyst, which proceed with stereochemical inversion3 \u2192 4).Consistent with the results of the Negishi group with non-olefinic nucleophiles,t-Bu3P-catalyzed stereo-retentive cross-coupling (2(dppf)] pre-catalyst in which the order of addition of the two non-equivalent alkenyl-zinc bromides is reversed. As shown in Overall, the sequence involving Pd(0)/coupling has broacatalyst . NineteeZ4e- and Z4l- sequences to form octalins. The present work significantly extends the scope of the DTDA process since, as we demonstrate here for the first time in Z4a- and E4a- reacts with the dienophile N-methylmaleimide with complete selectivity for the 1,3-butadiene site that lacks the inside-1,3-butadiene R substituent.Z4a- gives semicyclic diene 6, which reacts on, in situ, with a second NMM molecule to furnish 1-methyl-\u03941(9)-octalin 7 as the major product. The semicyclic diene 8 derived from diastereomeric dendralene E4a- undergoes a second NMM cycloaddition under high pressure conditions to form 10-methyl-\u03941(9)-octalin 9, possessing an angular methyl substituent. Octalin and decalin ring systems are extremely common structural motifs in natural products and medicinal agents.22Thus, each diastereomeric [3]dendralene Z10a-, by simply deploying 2-zinc bromide as a coupling partner scale\" fill=\"currentColor\" stroke=\"none\">C unit of [4]dendralene Z10a- is essential for the first twofold, triene transmissive 6\u03c0\u20136\u03c0 electrocyclization sequence (Z10a- \u2192 11 \u2192 12). The execution of a pair of electrocyclizations in this interconnected manner is without precedent. Several variations upon this original theme can be envisaged, which has potential for development into a broad scope, new method for step economic polycycle synthesis.The freshly minted PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C and C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C through-conjugation, as evidenced by the preparation of 23 new compounds containing this feature. We venture that the findings described herein, when combined with the strategies recently introduced for the preparation of the unsubstituted higher [n]dendralenes (n = 5\u201312),In conclusion, the first broad-spectrum synthesis of substituted dendralenes has been demonstrated, and unprecedented domino sequences for polycycle construction proven. [3]Dendralenes bearing from one to five alkyl, cycloalkyl, alkenyl, alkynyl, aryl and heteroaryl substituents have been prepared. Substitution at every conceivable position on the [3]dendralene framework has been realized. The previously unsolved problem of diastereoselective synthesis of internally-substituted systems has been solved. The method has been shown to work also with 4]dendralenes. Importantly, the approach represents the first general synthesis of dendralenic structures with extended CdendralenThere are no conflicts to declare.Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "Tailoring the graphene-covered Fe with Cs modifies the surface electronic properties of the catalysts such that selective C\u2013O bond cleavage of phenol is achieved in liquid phase by inhibiting the facile tautomerization followed by ring saturation. i.e., selective removal of oxygen without aromatic ring saturation) under liquid-phase conditions is highly challenging. Here, we report an efficient approach to engineer earth-abundant Fe catalysts with a graphene overlayer and alkali metal , which produces arenes with 100% selectivity from liquid-phase hydrodeoxygenation (HDO) of phenolics with high durability. In particular, we report that a thin (a few layers) surface graphene overlayer can be engineered on metallic Fe particles (G@Fe) by a controlled surface reaction of a carbonaceous compound, which prevents the iron surface from oxidation by hydroxyls or water produced during HDO reaction. More importantly, further tailoring the surface electronic properties of G@Fe with the addition of cesium, creating a Cs-G@Fe composite catalyst in contrast to a deactivated Cs@Fe one, promotes the selective C\u2013O bond cleavage by inhibiting the tautomerization, a pathway that is very facile under liquid-phase conditions. The current study could open a general approach to rational design of highly efficient catalysts for HDO of phenolics.Development of inexpensive sulfur-free catalysts for selective hydrogenolysis of the C\u2013O bond in phenolics ( Since its discovery,5i.e., removing oxygen-containing functional groups from bio-oil. During the HDO of phenolics, selective removal of the oxygen without saturation of the aromatic ring not only minimizes the consumption of valuable hydrogen, but also produces a gasoline blending stock with high octane number, which is critical to the efficient upgrading of bio-oil.via tautomerization.17Hydrodeoxygenation (HDO) plays pivotal roles in biomass conversion, 2 (H2-TPSR), in situ X-ray photoelectron spectroscopy (XPS), and in situ attenuated total reflectance Fourier-transform infrared spectroscopy (ATR-FTIR) reveal that the thin graphene overlayer prevents the Fe surface from oxidation, making the graphene/Fe composite (G@Fe) a durable Fe-based catalyst resistant to oxidation in HDO of phenol. More importantly, upon modification of this graphene/Fe composite with an alkali metal , the graphene overlayers could also regulate the Fe\u2013alkali metal interactions. As such, rather than a poison of Fe surface on Cs@Fe, tailored surface electronic properties were observed on the Cs-G@Fe composite, leading to exclusive arene production in HDO of phenolics in liquid phase via inhibiting tautomerization and aromatic ring saturation of phenolics.Motivated by the protection of transition metals by graphene overlayerI2D/IG ratio and the width of 2D band2 bonding.2/N2. If the Fe surface is not fully covered by the graphene overlayers, oxidation of Fe by O2 would be inevitable. From 2-TPSR profiles of the Fe and G@Fe samples after oxygen exposure . For the Fe sample, formation of surface iron oxides after exposure to oxygen was confirmed by both Raman . In contrast, both Raman for the selective C\u2013O bond cleavage, as suggested by homogeneous catalysis for the selective hydrogenolysis of aromatic ethers,The G@Fe catalyst was synthesized with a chemical vapor deposition method reported elsewhere,n Fig. S4 and H2-Tn Fig. S4 and H2-TS42-TPSR charactein situ pretreatment with H2 at 300 \u00b0C (simulating the surface under HDO reaction). Over G@Fe, C 1s spectrum displays a peak at 284.8 eV dominate on these catalysts under the liquid-phase conditions. Herein, using phenol as a probe molecule, we evaluated the bare Fe (BET surface area: 9.2 m2 g\u20131), G@Fe (BET surface area: 4.5 m2 g\u20131) and Cs-G@Fe (BET surface area: 4.5 m2 g\u20131) in liquid-phase HDO. As shown in via a mediated Fe\u2013alkali metal interaction, the reason of which remains unclear and subjects to further studies. Regardless, we first applied a similar approach used in homogeneous catalysis to the heterogeneous analogues and achieved the selective C\u2013O bond cleavage of phenolics in HDO. It should be mentioned that the addition of alkali metal resulted in the decrease of apparent rate for benzene production as shown in st cycle, the catalyst remained relatively stable afterwards for 4 additional testing cycles. More importantly, the benzene selectivity remained at 100% in all the tested cycles. It should also be mentioned that, due to the magnetic nature of the catalyst, the catalysts can be readily separated from the liquid phase by applying a magnetic field as demonstrated in the cyclic stability tests of a reaction.Ea of the reactions on the G@Fe and Cs-G@Fe (Ea of HYD (89 kJ mol\u20131) is much lower than that of DDO (143 kJ mol\u20131). This is consistent with the higher bond dissociation energy of Caromatic\u2013Oi.e. Cs significantly increased Ea for ring saturation) and the apparent Ea of direct C\u2013O bond cleavage is 141 kJ mol\u20131 which is comparable with that of DDO on G@Fe (143 kJ mol\u20131). This implies that Cs may not be directly involved in C\u2013O bond cleavage, but instead, is primarily responsible for inhibition of the ring-saturation pathway.The binding energy of Fe is lowered by 0.6 eV after adding Cs, as shown in Cs-G@Fe . The rea Cs-G@Fe . Over G@Ea for DDO remains the same after Cs addition, it is hypothesized that the addition of Cs largely inhibits the tautomerization reaction pathway but has a minimal effect on the C\u2013O bond cleavage. To further verify this hypothesis, in situ ATR-FTIR was employed to investigate the phenol adsorption over G@Fe and Cs-G@Fe. As shown in \u20131 (stretching vibrations of the aromatic ring)\u20131 (stretching vibration of the C\u2013O bond),\u20131, which can be assigned to stretching vibration of C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O bond, PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond,2 , and depositing Cs may selectively block the one for tautomerization with the other one being exposed to phenol for C\u2013O cleavage. To test this hypothesis, we further compared the performances of other alkali metal doped catalysts with same molar loading is well correlated with the capability of electron donation of alkali metali.e. graphene-covered Fe) may play pivotal roles to inhibit the functionality for catalyzing tautomerization of phenol. While allowing the electron tunneling, graphene overlayers may mediate the interaction between Fe and alkali metal/substrate oxygen to prevent Fe from deactivation.Previous resultsC bond,2 of surfa Fig. S12. Given ti.e., phenol). To our best knowledge, this is the first sulfur-free inexpensive catalyst reported for exclusive hydrogenolysis of the C\u2013O bond in phenolics under liquid-phase conditions. Moreover, the catalyst also offers other beneficial properties, i.e. magnetic material for a facile separation from reaction slurry, making it a promising catalyst for liquid-phase reactions. This work could also lead to a general methodology for rational design of heterogeneous catalysts for selective HDO of oxygenates.Our results present an approach to protect Fe with graphene and further tune the G@Fe catalyst with alkali metal for selective hydrogenolysis of C\u2013O bond in HDO of phenol. Graphene overlayers on Fe protect Fe from oxidation while, at the same time, maintaining the nature of Fe as confirmed by its catalytic activity resembling that of bare Fe. More importantly, analogous with homogeneous catalysis, alkali metals such as Cs could be added to tailor the surface electronic properties of G@Fe, leading to the selective inhibition of tautomerization and thus exclusive C\u2013O bond cleavage in the heterogeneously catalyzed HDO of phenolics (There are no conflicts to declare.Supplementary informationClick here for additional data file."} +{"text": "Unprecedented photoluminescence, AIE and heat-induced emission characteristics of non-conjugated thermoresponsive PNVCL were unveiled for the very first time which eventually empowered PNVCL to act as an intracellular thermometer. N-vinylcaprolactam) (PNVCL) devoid of any classical fluorophore entity. PNVCL underwent a coil to globular conformational transition in an aqueous medium and appeared to be fluorescent above its lower critical solution temperature (LCST) near body temperature (38 \u00b0C). Eventually, this intriguing aspect enabled higher cellular uptake of PNVCL at the LCST boundary. By virtue of the AIE effect, the thermo-induced aggregation phenomenon has been ingeniously utilized to apply PNVCL as a novel fluorescent thermometer for intracellular temperature determination.Since temperature is one of the most significant physiological parameters that dictate the cellular status of living organisms, accurate intracellular temperature measurement is crucial and a valuable biomarker for the diagnosis and treatment of diseases. Herein, we introduce the foremost example of a non-conjugated polymer as a next generation fluorescent thermometer which is capable of addressing the key shortcomings including toxicity and thermal-induced fluorescence quenching associated with \u03c0\u2013\u03c0 conjugated system-based thermometers developed so far. We revealed, for the first time, the unique photophysical and aggregation-induced emission (AIE) characteristics of well-known thermoresponsive poly( Temperature is an important physiological parameter that governs a broad range of biological activities, especially all biological reactions within living cells.In this context, an astonishing evolution of numerous fluorescent thermometers has been witnessed over the past few years.11The discovery of the aggregation-induced emission (AIE) phenomenon by Tang's group20 PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C, C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O and C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N functionalities, have been reported to act as nonconventional luminophores upon aggregation or clustering.N-vinylcaprolactam) (PNVCL) is expected to exhibit interesting photophysical behavior. As anticipated, for the very first time, we have revealed the intrinsic photoluminescence characteristics of well-known thermo-responsive PNVCL and its AIE activity. The temperature-driven aggregation behavior and AIE characteristics have judiciously been exploited to use PNVCL as a potential fluorescent polymeric thermometer for intracellular temperature measurement. Overall, the present non-conjugated macromolecule has the ability to replace the current traditional-fluorophore functionalized fluorescent polymeric thermometer, because PNVCL has multiple fascinating features such as AIE, temperature-sensitive fluorescence enhancement, and excellent cytocompatibility, which are rare in a single polymeric system.Electron-rich heteroatoms, such as nitrogen, oxygen, phosphorous, sulfur, and/or unsaturated CN-vinylcaprolactam (1H NMR spectroscopy and size exclusion chromatography (SEC) were employed to characterize PNVCL. A typical 1H NMR spectrum shown in Fig. S1Mn,SEC) of 9500 g mol\u20131, with dispersity (\u00d0) = 1.95 , dioxane, methanol (MeOH), ethanol (EtOH), acetone, etc., except in hexane. Moreover, the polymer was soluble in aqueous media owing to the strong H-bonding between the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O groups of lactam and water molecules.Initially, PNVCL was synthesized in one step by conventional free radical polymerization (FRP) of commercially available rolactam . 1H NMR 5 Fig. S2. PNVCL w\u03bbex) of 339 nm in different solvents to understand their emission characteristics. As illustrated in Fig. S4,\u20131) exhibited stronger fluorescence in THF than in any other common organic/aqueous solvents. Thus, PNVCL possessed a higher quantum efficiency (\u03c6F at \u03bbex 350 nm) and average fluorescence lifetime ( 350 nm) and average fluorescence lifetime (\u3008\u03c4\u3009) in THF in THF than in water yielded a low photoluminescence (PL) signal with an almost flat line parallel to the abscissa and obviously no emission was observed upon irradiation to the good solvent (THF) containing the polymer, without increasing the polymer concentration Fig. S5. A dilutadiation . But wit mg mL\u20131 . A simil mg mL\u20131 , with a mg mL\u20131 . As the mg mL\u20131 . This contration . The higolutions .i.e. any sort of \u03c0-conjugated system, it produces bright blue emission in the concentrated or aggregated state. This fact can be attributed to the clustering-triggered emission (CTE) mechanism. PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C, C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O, C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N, etc. PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O groups with \u03c0-electrons/a lone pair of electrons and N atoms with a lone pair of electrons of one cyclic amide come in close proximity to another amide to form a cluster in the aggregated state. This clustering results in chromophores with effective through-space electronic communication giving rise to extended electron delocalization and simultaneous rigidified conformation. The rigidity of the molecular conformation restricts vibration and molecular rotation which efficiently suppresses the non-radiative relaxation.Although the present polymer lacks conventional chromophores, Notably, progressive enhanced absorption with red shifting of absorption maxima was observed as the polymer concentration increased , indicat\u03bbex values of 365 (UV), 460 (blue) and 530 nm (green) was consistent with the preceding observations , where there is more opportunity for the chromophores to approach closer and provide discernible emission. Surprisingly, direct observation of the PNVCL film under a fluorescence microscope showed tunable photoluminescence from blue and green to red upon altering the emission channels . Furtherinset of . Apart fvia UV-Vis spectroscopy.T) was observed until 35 \u00b0C; an abrupt decrease due to the LCST was observed at 37.5 \u00b0C scale\" fill=\"currentColor\" stroke=\"none\">O groups of lactam units and O\u2013H groups of water molecules at a higher temperature.Dh) of the polymer in response to heat were assessed by DLS measurements below and above the LCST. The Dh values in the temperature window 25\u201335 \u00b0C (below the LCST) were found to be 6\u20138 nm and increased up to 170\u20131000 nm above the LCST and normal lung epithelial cells (WI-38) by performing MTT assay in a dose-dependent manner Fig. S9. The celi.e. 38 \u00b0C . Cultured cells after treatment with PNVCL were incubated for 24 h at 25, 35, and 38 \u00b0C . The CLSe. 38 \u00b0C , when obe. 38 \u00b0C . Howevere. 38 \u00b0C .As shown in \u20131 for different incubation times followed by staining of nuclei using DAPI (blue emission). At 38 \u00b0C (above the LCST) and at a fixed polymer concentration (250 \u03bcg mL\u20131), with an increasing incubation time ranging from 4 to 24 h the average emission intensity obtained from MCF-7 cells observed under the green channel was found to increase , it exhibited significant emission upon increasing the concentration, and also in the solid and film state. This unprecedented light emission could be attributed to the AIE or CTE mechanism. Interestingly, the PNVCL film displayed distinct blue, green and red emission upon illumination with different excitation wavelengths, demonstrating the coexistence of multiple emissive species with different energy levels. Moreover, the PNVCL solution exhibited a bright blue emission upon heating above its LCST (37.5 \u00b0C), which was attributed to the heat-assisted aggregation-induced emission phenomenon. Capitalizing on the aforesaid aspects, a higher cellular internalization of PNVCL was attained at 38 \u00b0C (above the LCST) compared to 35 \u00b0C (below the LCST) with significant light-up fluorescence confirmed through CLSM images. We believe that the present findings disclosed the potentiality of the non-toxic biocompatible PNVCL to be a next generation fluorescent thermometer by detecting minor temperature changes, beneficial for the early detection of diseases and local hyperthermia treatment.40In summary, unique emission behaviour of non-conjugated PNVCL without traditional chromophores has been investigated. Although PNVCL is non-fluorescent in dilute solution (up to 0.1 mg mLThere are no conflicts to declare.Supplementary informationClick here for additional data file."} +{"text": "Catalytic bicyclization of 1,5-enynes anchored by \u03b1,\u03b2-conjugates with arylsulfonyl radicals was established using TBAI and Cu(OAc)2 as co-catalysts. in situ from sulfonyl hydrazides has been established using TBAI (20 mol%) and Cu(OAc)2 (5 mol%) as co-catalysts under convenient conditions. In addition, the use of benzoyl peroxide (BPO) as the oxidant and pivalic acid (PivOH) as an additive was proven to be necessary for this reaction. The reactions occurred through 5-exo-dig/6-endo-trig bicyclizations and homolytic aromatic substitution (HAS) cascade mechanisms to give benzo[b]fluorens regioselectively. A similar catalytic process was developed for the synthesis of \u03b3-ketosulfones. These reactions feature readily accessible starting materials and simple one-pot operation.A catalytic bicyclization reaction of 1,5-enynes anchored by \u03b1,\u03b2-conjugates with arylsulfonyl radicals generated Herein,s (HASs) .p-tolyl)prop-2-en-1-one 1a was selected as a benchmark substrate to investigate the additions by sulfonyl radicals. With 20 mol% tetrabutylammoniumiodide (TBAI) as the catalyst, the reaction of substrate 1a with tosylhydrazide 2a was performed in CH3CN in the presence of benzoperoxide (BPO) (4.0 equiv.) as an oxidant at 70 \u00b0C under air conditions, affording the expected benzo[b]fluorens 3a, albeit with a low yield of 18% (3CN showing the best performance (entries 2\u20134). Raising the reaction temperature to 100 \u00b0C slightly ameliorates the yield of 3a (entry 5). A subsequent investigation of other catalysts was conducted in CH3CN. As illustrated in entries 6\u20138, different types of catalysts including I2, KI, and CuI were employed in the model reaction, and it turned out that I2 and KI hardly facilitate the reaction (entries 6 and 7), while CuI as a catalyst only led to a poor yield of 16%. Next, we turned our attention to evaluating different additives (entries 9\u201311). We found that the addition of PivOH (1.0 equiv.) delivered 3a in a 35% yield (entry 11). Notably, the reaction of 1a and 2a in the presence of 2.0 equiv. of PivOH gave 3a in a 71% yield using a co-catalyst of TBAI (20 mol%) and Cu(OAc)2 (5 mol%) with complete consumption of the starting material 1a (entry 15). Without PivOH, the yield of the expected product 3a decreased remarkably (entry 17). Further screening of other oxidants, such as TBHP (64% yield), DTBP (very poor yield) and H2O2 (no product) for this transformation showed that BPO was the best choice phenyl)-1- at the para position of the aromatic ring (Ar1) directly bound to the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond gave the corresponding sulfonated products 3e and f in 55% and 68% yields, respectively. Alternatively, a naphthalen-1-yl substituent linked to the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond was also well-tolerated, affording the product 3g in a 60% chemical yield. Similarly, either electron-donating (methyl) or electron-withdrawing (bromo) groups (R1) at the para position of the phenyl ring tethered to the enone unit were well-suited for these radical 1,5-enyne-bicyclizations (3a\u20133k). 1,5-Enynes 1 carrying electron-neutral groups were also smoothly converted into the corresponding sulfonated benzo[b]fluorens 3l\u20133n in 39\u201367% yields. Notably, 2-naphthalenylethanone-derived 1,5-enynes furnished the unprecedented pentacyclic indenophenanthren-7-ol 3n in a 67% chemical yield though sulfonyl radicals triggered the 1,5-enyne-bicyclization. Unfortunately, a bulky ortho-Br substituent and benzylsulfonyl hydrazide did not work at all (3o and 3p). Besides the NMR and HR-MS spectroscopic analysis for benzo[b]fluorens 3, the X-ray diffraction for product 3f has been performed as shown in With the optimized reaction conditions in hand, we examined the substrate scope of the sulfonyl hydrazides nynes 1a . As antib]fluorens 3, we reasoned that in the absence of alkyne moieties, chalcones 4 would be able to accept sulfonyl radicals via typical 1,4-additions, which would expand their utility for the synthesis of \u03b3-ketosulfones. We thus explored this possibility through a one-pot reaction of (4-chlorophenyl)-3-phenylprop-2-en-1-one (4a) with 2a under the conditions described above. The expected \u03b3-ketosulfone 5a was obtained but with a lower yield (15%) initially. After careful optimizations were performed, we found that although Cu(OAc)2 and PivOH did promote this catalytic process, the use of co-oxidants of BPO (2.0 equiv.) and TBHP in 20 mol% of TBAI proved to be suitable for the current hydrosulfonylation, furnishing product 5a in a 77% yield. Subsequently, we further studied the reaction scope by reacting arylsulfonyl hydrazides 2 with various chalcones 4 under these conditions was proven not to be an adaptable substrate for this reaction, which may be ascribed to the relative instability of the sulfonyl radicals generated in situ from aliphatic sulfonyl hydrazides. Joining previously reported work,In view of our success with the synthesis of functional benzofluoren product 6 (eqn (3)). These control experiments suggest that BPO is essential for the catalytic cycles and the in situ generated sulfonyl radical triggers a 5-exo-dig/6-endo-trig bicyclization cascade.To understand the mechanism, several control experiments were conducted. The treatment of 1,5-enyne A from the sulfonyl hydrazide using the benzoyloxy radical generated in situ from the I\u2013 anion-assisted decomposition of BPO. The intermolecular addition of the resulting sulfonyl radical A and the 1,5-conjugated enyne 1 followed by a 5-exo-dig cyclization gives intermediate B, in which the homolysis of carbon\u2013copper(iii) affords vinyl radical C. Intermediate C is converted into aryl radical Dvia a 6-endo-trig cyclization. Intermediate D undergoes SET (single electron transfer) oxidation and subsequent deprotonation to provide intermediate F. The tautomerization of F leads to the formation of benzo[b]fluorens 3. Although the generation of sulfonyl radicals triggered by various oxidants has been achieved well,via sulfonyl radical initiated bifunctionalization of enynes is very rare in organic chemistry as mentioned earlier.On the basis of the above observations and those reported in literature,in situ generated sulfonyl radicals across the activated double bond is able to trigger a cascade 5-exo-dig/6-endo-trig bicyclization and HAS sequence, delivering tetracyclic sulfonylated benzo[b]fluorens in a successive C\u2013S and C\u2013C bond-forming process. Using chalcones as replacements for 1,5-conjugated enynes, this reaction enables the hydrosulfonylation of alkenes to form \u03b3-ketosulfones with good to excellent yields. These two methods allow easy access to important functional sulfones for potential applications in organic and medicinal chemistry.In summary, we have discovered new 1,5-enyne-bicyclization and hydrosulfonylation reactions of \u03b1,\u03b2-conjugates under convenient co-catalytic conditions. The addition of Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "Azulene is a convenient platform for accessing heterobimetallic complexes and self-assembled monolayers of a \u03c0-linker with asymmetric junctions. PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C) terminated conducting \u03c0-linkers are often employed in the ever-growing quest for organoelectronic materials. While such systems typically involve symmetric dimercapto or diisocyano anchoring of the organic bridge, this article introduces the chemistry of a linear azulenic \u03c0-linker equipped with one mercapto and one isocyano terminus. The 2-isocyano-6-mercaptoazulene platform was efficiently accessed from 2-amino-6-bromo-1,3-diethoxycarbonylazulene in four steps. The 2-N PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C end of this 2,6-azulenic motif was anchrored to the [Cr(CO)5] fragment prior to formation of its 6-SH terminus. Metalation of the 6-SH end of [(OC)5Cr] (7) with Ph3PAuCl, under basic conditions, afforded X-ray structurally characterized heterobimetallic Cr0/AuI ensemble [(OC)5CrAuPPh3] (8). Analysis of the 13C NMR chemical shifts for the [(NC)Cr(CO)5] core in a series of the related complexes [(OC)5Cr] scale\" fill=\"currentColor\" stroke=\"none\">C, Br, H, SH, SCH2CH2CO2CH2CH3, SAuPPh3) unveiled remarkably consistent inverse-linear correlations \u03b4(13COtrans) vs. \u03b4(13CN) and \u03b4(13COcis) vs. \u03b4(13CN) that appear to hold well beyond the above 2-isocyanoazulenic series to include complexes [(OC)5Cr(CNR)] containing strongly electron-withdrawing substituents R, such as CF3, CFClCF2Cl, C2F3, and C6F5. In addition to functioning as a sensitive 13C NMR handle, the essentially C4v-symmetric [(\u2013NC)Cr(CO)5] moiety proved to be an informative, remote, \u03bd PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CN/\u03bd PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019OC infrared reporter in probing chemisorption of 7 on the Au(111) surface.Mercapto (\u2013SH) and isocyano (\u2013N PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C) substituents are among particularly popular anchoring groups in coordination and surface chemistry as they are well-known to provide stable junctions at metal/organic interfaces. PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C functionalities in the same molecule are not presently known and constitute a formidable synthetic challenge. Indeed, a mercapto group is incompatible with reaction conditions commonly employed to form an isocyano substituent, PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C or the \u2013SH terminus of such a hypothetical linker prior to forming and tethering its other end. There is only one related example in the literature, albeit not involving a mercapto group per se but rather its disulfide surrogate.via the 4-isocyanophenylthiolate bridge, Kubiak and coworkers attached both \u2013N PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C ends of otherwise non-isolable 1,2-bis(4-isocyanophenyl)disulfide to trinuclear nickel clusters in the \u03bc3,\u03b71 fashion.3(\u03bc3-I)(\u03bc2-dppm)3 scale\" fill=\"currentColor\" stroke=\"none\">NC6H4S\u2013)}2]2+(I\u2013)2 (dppm = bis(diphenylphosphino)methane), underwent homolysis of its S\u2013S moiety upon exposure to a gold surface to give rectifying, presumably ionic, monolayer films.26Mercapto (\u2013SH) and isocyano scale\" fill=\"currentColor\" stroke=\"none\">N) junctions for accommodating the asymmetric anchoring on the premises that the \u2013SH and \u2013C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N termini would facilitate alignments of a linker's HOMO and LUMO , respectively, through Fermi level pinning.2Earlier this year, Ratner and van Dyck proposed a new paradigm for the design of efficient molecular rectifiers that involved two \u03c0-conjugated units asymmetrically anchored to metallic electrodes and separated by a decoupling bridge.and isocyano anchoring groups. The linker's core is comprised of the non-alternant aromatic framework of azulene, a substitution-free molecular diode which has, among other unusual physico-chemical characteristics, complementary orbital density distributions within its Frontier molecular orbitals . As illustrated in 1 and 2: 2-isocyano-6-mercapto-1,3-diethoxycarbonylazulene (3a) and 2-mercapto-6-isocyano-1,3-diethoxycarbonylazulene (3b). Among these two hybrids, 3a is particularly interesting because each substituent in its structure reinforces the molecular dipole of the azulenic framework. In fact, our Density Functional Theory (DFT) calculations suggest that the dipole moment of the \u201cparent\u201d azulene molecule should increase nearly 10-fold upon incorporation of all substituents to form 3a disulphide (vide supra),255 is thermally and air-stable for practical purposes and can be stored under ambient conditions for at least a few weeks without spectroscopically detectable deterioration. Compound 5 reacted with Cr(CO)5(THF) via its 2-NC end to form orange Cr0 adduct 6. No product featuring the thioether S \u2192 Cr(CO)5 interaction5] moiety of 6 tolerated the basic environment and subsequent acidification of the reaction mixture used to convert 6 into auburn organometallic thiol 7, which constitutes 3a with its 2-NC terminus anchored to the 16-e\u2013 [Cr(CO)5] fragment. Metalation of the 6-SH end of 7 with PPh3AuCl under basic conditions yielded orange-red crystals of heterobimetallic Cr0/AuI complex 8 after a simple workup.Our synthetic approach to constructing and metalating 8\u00b7\u00beCH2Cl2 features two very similar but crystallographically independent molecules of 8 in the asymmetric unit that are linked together via a weak Au\u00b7\u00b7\u00b7Au interaction8 undergoes donor\u2013acceptor face-centred stacking3 ligand giving the intercentroid distances8 may be viewed as a hybrid of our X-ray structurally characterized mononuclear Cr0 and AuI adducts of 1 and 2, respectively, depicted in 9 ,ca. 166.5\u00b0) in 8 is undoubtedly a consequence of the Au\u00b7\u00b7\u00b7Au bonding reinforced further by the \u201caromatic donor-acceptor interactions\u201d.8 compared to that in 10, which are ca. 107.8\u00b0 105.0\u00b0, respectively. Notably, the solid state structure of 10 exhibits neither aurophilic nor aromatic stacking interactions akin to those observed for 8.29The solid-state structure of ted in 9 and 10 (in 9 (10 ). While 5] core in 8 are quite similar to those observed for 9 scale\" fill=\"currentColor\" stroke=\"none\">N bond distances8 and 9 scale\" fill=\"currentColor\" stroke=\"none\">C groups at position 6 of the azulenic scaffold. However, this suggestion should be taken cum grano salis as such subtle variations in d(Cr\u2013CN) and d scale\" fill=\"currentColor\" stroke=\"none\">N) are statistically ambiguous, especially under the 3\u03c3 criterion. More drastic changes in the electronic nature of the isocyanide ligand's substituent do lead to significant alterations in the Cr\u2013CN and C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N bond lengths in (RNC)Cr(CO)5 as illustrated in tBu scale\" fill=\"currentColor\" stroke=\"none\">CF2.43The metric parameters for the octahedral core in 6, 7, 8, and 9, we noticed that they were predictably sensitive to the nature of the substituent at position 6 of the azulenic scaffold. To further validate this initial observation, we expanded the above family of four related complexes [(OC)5Cr] scale\" fill=\"currentColor\" stroke=\"none\">C) to include species with X = H (11) and Br (12). The top six rows in 13C NMR data pertaining to the [(NC)Cr(CO)5] moiety in this series of six 2-isocyanoazulenic adducts. All of these 13C NMR measurements were performed for samples dissolved in CDCl3.Whilst considering net electron-releasing ability of X decreases scale\" fill=\"currentColor\" stroke=\"none\">C), the \u03b4(13CN) value for the isocyano carbon resonance increases in the range spanning ca. 8 ppm, thereby signifying gradual drop in the \u03c3-donor/\u03c0-acceptor ratio of the 2-isocyano-6-X-azulene ligand. Concomitantly, both \u03b4(13COtrans) and \u03b4(13COcis) values decrease, albeit in tighter chemical shift ranges , indicating reduction in the electron richness of the Cr-centre. Even though the 13C chemical shifts of terminal CO and CNR ligands in low-valent complexes are influenced considerably by the paramagnetic shielding term, \u03c3para, which reflects the degree of \u03c0-backbonding,\u03b4(13CN) and \u0394\u03b4(13CO) as a combined \u03c3-donor/\u03c0-acceptor effect.From 13C NMR data in the top six rows of \u03b4(13COtrans) vs. \u03b4(13CN) and \u03b4(13COcis) vs. \u03b4(13CN), as illustrated in trans-CO ligand to a greater extent than the cis-CO's of the [(NC)Cr(CO)5] moiety. Would the trends depicted in 5 species containing strongly electron-withdrawing substituents R, for which 13C NMR data acquired in the same solvent (CDCl3) were available (bottom four rows in \u03b4(13COtrans) vs. \u03b4(13CN) and \u03b4(13COcis) vs. \u03b4(13CN) plots that, in addition to the 2-isocyanoazulenic complexes, include (OC)5Cr(CNR) with R = C6F5,2F3,2Cl,3 (\u03b4(13CN) and \u0394\u03b4(13CO) windows.Closer examination of the F3,2Cl,3 are show\u03b4(13CO)/\u03b4(13CN) NMR analysis serves as a convenient tool for quantifying even subtle electronic influence of a CNR ligand's substituent R. In this regard, it offers a simple alternative to the well-established method involving correlation the carbonyl 13C chemical shifts with the corresponding CO force constants (kCO) for complexes (RNC)Cr(CO)5.kCO due to mild electronic perturbations of the R group are often not clearly discernible.kCO's under the C4v symmetry for complexes (RNC)5Cr(CO)5 using the Cotton\u2013Kraihanzel (C\u2013K) approximation\u03bd PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019OC vibrational profile\u0393\u03bdCO = 2A1 + B1 + E, e.g., 5 species, the lower energy \u03bd PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019OC(A1) band is often obscured by the intense \u03bd PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019OC(E) band,\u03bd PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019OC(A1) value (vide infra).The above \u03b4(13CN) for the (RNC)Cr(CO)5 adducts in 13C NMR resonance for the terminal C-atom in the available uncoordinated 2-isocyanoazulenes moves upfield upon increasing electron-donating power of the substituent X at the azulenic 6-position scale\" fill=\"currentColor\" stroke=\"none\">C, Br, H, SCH2CH2CO2CH2CH3, respectively). Yet, the \u03bd PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CN stretching frequency for these free 2-isocyano-azulenes (2126 \u00b1 1 cm\u20131 in CH2Cl2) is insensitive to the nature of the group X. However, upon proceeding from 8 to to 12 to 9, the \u03bd PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CN band undergoes a small red shift scale\" fill=\"currentColor\" stroke=\"none\">CN bands at 2583 and 2140 cm\u20131, respectively, it features a typical pattern in the \u03bd PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019OC stretching region for a LM(CO)5 species.\u20131 corresponds to the \u03bd PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019OC mode A1(1) where all five CO ligands vibrate in-phase scale\" fill=\"currentColor\" stroke=\"none\">OC vibration of B1-symmetry, which is IR-forbidden under the strict C4v symmetry but gains slight intensity because of minor deviations of the structure from the idealized C4v geometry. The intense \u03bd PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019OC band at 1958 cm\u20131 chiefly represents the doubly degenerate vibration of E-symmetry. This \u03bd PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019OC(E) band obscures the remaining IR-active \u03bd PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019OC mode A1(2). Interestingly, perturbations of the local C4v symmetry in 7 through crystal packing interactions in the solid state are sufficient to split the E-mode into two separate \u03bd PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019OC peaks while unmasking the original A1(2) mode (phase cf.. The ver(2) mode .ca. 1 \u00d7 1 cm2 gold substrates to a 2 mM solution of 7 in CHCl3 without protection from air and ambient lighting reproducibly afforded self-assembled monolayer (SAM) films of 7 on the Au(111) surface. This chemisorption process is presumably accompanied by formation of the thiolate junction and the release of H2.7 on Au(111) is shown in \u03bd PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CN absorption at 2135 cm\u20131, it features two \u03bd PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019OC bands. The \u03bd PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019OC region in this RAIR spectrum, however, is quite different from that in \u03bd PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019OC band in the solution IR spectrum of 7, which is primarily attributed to the \u03bd PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019OC mode of E symmetry, practically vanishes upon the SAM formation, while simultaneously uncovering the hidden A1(2) band of much lower intensity. This observation implies approximately parallel orientation of the cis-CO ligands with respect to the gold surface. Indeed, surface IR selection rules7 is expected to be essentially linear, the appearance of the RAIR spectrum in i.e., straight C\u2013S\u2013Ausurface angle) of the molecules in the SAMs of 7.Exposing via S(3p)\u2013Au \u03c0-bonding.5] unit. The A1(1) and A1(2)\u03bd PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019OC bands at 2058 and 1995 cm\u20131 in the RAIR spectrum in \u03bd PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019OC peaks in the solution FTIR spectrum of 7 mode, to be attributed solely to differences in intermolecular interactions within the SAM vs. solution of 7. The larger change in energy of the \u03bd PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019OC A1(2) mode compared to that of the A1(1) mode upon chemisorption of 7 stems from the greater contribution of the trans-CO stretch to the former.62The \u201chollow-linear\u201d coordination of organic thiolates in their SAMs on Au(111), akin to that depicted in 7 provided consistent SAM thickness values that nicely corroborate the monolayer nature of these films and upright orientation of the molecules on the gold surface (7 and 9 on Au(111) differ only in the surface anchoring group (thiolate vs. isocyanide) and appear to exhibit essentially identical thicknesses.7 on Au(111) would be consistent with the \u201con-top-bent\u201d7 invoking a bent C\u2013S\u2013Ausurface geometry. The ellipsometric measurements on SAM films formed from our recently reportedThe tilt angle of the aromatic moiety in SAMs of benzenoid mercaptoarenes on Au(111) can be highly variable. surface . In term13C NMR signatures of the octahedral [(\u2013NC)Cr(CO)5] core in related complexes 6, 7, 8, 9, 11, and 12 provided a sensitive spectroscopic handle for tuning electron richness of the Cr0-centre through mediation by the 2,6-azulenic framework. Moreover, the remarkably consistent inverse-linear trends \u03b4(13COtrans)/\u03b4(13CN) and \u03b4(13COcis)/\u03b4(13CN) for a wide spectrum of complexes (RNC)Cr(CO)5 offer a simple and more accurate alternative to the \u03b4(13CO)/kCO strategy in quantifying electronic influence of the substituent R in isocyanide ligands. This 13C NMR approach utilizes feedback from the entire [(\u2013NC)Cr(CO)5] unit rather than focusing on the [Cr(CO)5] fragment in the \u03b4(13CO)/kCO method. In addition, the C4v-symmetric [(\u2013CN)Cr(CO)5] moiety served as a distinctly informative \u03bd PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CN/\u03bd PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019OC infrared reporter for probing self-assembly of the 6-mercaptoazulenic motif on the Au(111) surface. We hope that the chemistry of the 2-isocyano-6-mercaptoazulenic platform introduced herein will facilitate further development and experimental validation of the emerging concept of asymmetric anchoring relevant to the design of organic electronics materials. Efforts to access and isolate completely free 3a are currently in progress.The asymmetric nonbenzenoid aromatic framework of azulene proved to be a convenient platform for accessing the first \u03c0-linker terminated with both mercapto and isocyano junction moieties. Anchoring the 2-isocyano end of this linker was an important prerequisite to successfully installing its 6-mercapto terminus. The Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "Background: A recent bioinformatics technique involves changing\u00a0nucleotide sequences into 2D speckles. This\u00a0technique produces\u00a0speckles\u00a0called\u00a0GB-speckles (Gene Based speckles). All classical strategies of speckle-optics, namely speckle-interferometry, subtraction of speckle-images as well as speckle-correlometry\u00a0have been inferred for processing of GB-speckles. This indicates the considerable improvement in the present\u00a0tools of bioinformatics.\u00a0\u00a0Methods: Colour\u00a0s-LASCA\u00a0imaging of virtual laser GB-speckles, a new method of high discrimination and typing of pathogenic viruses, has been developed. This method has been adapted to the detecting of natural mutations in nucleotide sequences, related to the\u00a0spike\u00a0glycoprotein\u00a0(coding the gene \u00abS\u00bb) of SARS\u2013CoV-2 gene as the molecular target.\u00a0\u00a0\u00a0Results: The rate of the\u00a0colouring\u00a0images of virtual laser GB-speckles generated by\u00a0s-LASCA\u00a0can be described by the specific value of\u00a0R. If the nucleotide sequences compared utilizing this approach the relevant images are completely identical, then the three components of the resulting\u00a0colour\u00a0image will be identical, and therefore the value of\u00a0R\u00a0will be equal to zero. However, if there are at least minimal differences in the matched nucleotide sequences, then the\u00a0value\u00a0of\u00a0R\u00a0will\u00a0be\u00a0positive.\u00a0\u00a0\u00a0Conclusion: The high effectiveness of an application\u00a0of the\u00a0colour\u00a0images of GB-speckles that were generated by\u00a0s-LASCA- has been demonstrated for discrimination between different variants of the SARS\u2013CoV-2\u00a0spike\u00a0glycoprotein\u00a0gene. Recently, the possibility of transforming a nucleotide sequence into a pattern of 2D speckles had been demonstrated. This new type of speckle pattern has been called \u201cGB-speckles\u201d (gene-based speckles). Changes within in the structure of the GB-speckles can reflect even negligible changes in the nucleotide sequence, caused by inartificial mutations. This allows detection of single-nucleotide polymorphisms (SNP) using virtual GB-speckles with outstanding precision. In addition, it offers unlimited potential of improving the diagnosis\u2019 accuracy by increasing the Fourier transform area.As it is well known, if laser light diffracts on random objects, then laser speckles are formed. implementation of speckle-optics methods, like speckle-interferometry and subtraction of speckle-images as well as speckle-correlometry for processing of GB-speckles, provides considerable progress in the current bioinformatics toolbox. This can become crucial to significantly improve existing routine methods of laboratory diagnostics of infectious diseases. GB-speckles as a technique opens the door to the new horizons in digital biology.Essential advancement in the area of GB-speckles has been reported in previous years. According to previously published reports,omp1 genes for two different ofChlamydia spp., such asChlamydia trachomatis andChlamydia psittaci of at least six genovars have been composed. Probability density functions and correlation properties of spatial intensity fluctuations for the relevant GB-speckle patterns have been studied. As it has been shown in previous studies, the presence of inartificial mutations in analysed strains, including single SNP cases, can be easily defined using methods of speckle-optics. More recently, the encoding algorithm\u2019s optimization for nucleotide sequences ofC. trachomatis into two-dimensional GB-speckle pattern had been carried out; and speckle-interferometric technique may give rise the ultra-fast optical processors of DNA sequences. This is ensured by the development of the exclusive system of interferential fringes which are generated by the model interference pattern led by the existence of any type of mutations. Additionally, the method of virtual phase-shifting speckle-interferometry was reported to be efficacious to investigate of polymorphism of theC. trachomatis omp1 gene. This approach allowed the detection of theC. trachomatis omp1 gene with SNPs, including both a single SNP and a combination of several SNPs in the bacterial strains with genetic mutations had been developed.Recently, model GB-speckle patterns of nucleotide sequences of theEnterobacteriaceae. These proteins have been found on the surface of several bacterial agents causing different enteric infections, such as salmonellosis, shigelosis, yersiniosis, and escherichiosis. Further, the phase and the relevant two-dimensional distributions of the intensity of GB-speckles in various strains of viral pathogens, namely of lumpy skin disease virus of cattle, LSDV, and also for sheep-pox virus, SPPV have been obtained. Additionally, interference patterns for generated the specific superposition in the relevant fields of GB-speckle and the certain difference in their images have been successfully investigated to reveal a minimal discrimination between the initial viral nucleotide sequences.The format of GB-speckles had been successfully applied to transform the nucleotide sequences of the genes expressing the serine proteases, the well-known Omptin family proteins within the GB-speckles processing via ans-LASCA technique application. As it had been demonstrated, it is possible to extend affectability of the proposed approach comparing to current bioinformatics strategies usings-LASCA imaging in the GB-speckles\u2019 processing. It had been shown in Ref.s-LASCA imaging method are very effective to analyze nucleotide polymorphism in several genes ofC. trachomatis.A new bioinformatics approach has been proposed very recently:s-LASCA imaging of GB-speckles. Such a technique is an improved version of previously suggested \u201cgreyscale\u201ds-LASCA imaging that was recently developed especially for GB-speckles. Nucleotide sequences for some target genes SARS\u2013CoV-2 have been successfully processed using coloureds-LASCA-imaging. Natural mutations in the comparing genes have been reliably and accurately detected.This paper is devoted to development of advantageously new technique: the colouredSeven nucleotide sequences of spike glycoprotein of SARS-CoV-2, namely:the gene#1. hCoV-19/cat/USA/TX-TAMU-078/2020 (Accession ID: EPI ISL 699509),the gene#2. hCoV-19/cat/Russia/RII-LEN-22246S/2021 (Accession ID: EPI ISL 811147),the gene#3. hCoV-19/cat/Greece/2K/2020 (Accession ID: EPI ISL 717979),the gene#4. hCoV-19/Wuhan/WIV04/2019 (Accession ID: EPI ISL 402124),the gene#5. hCoV-19/England/QEUH-B11766/2020 (Accession ID: EPI ISL 642476),the gene#6. hCoV19/South Africa/KRISP-EC-K005299/2020 (Accession ID: EPI ISL 678597),the gene#7. hCoV-19/Russia/MOS-CRIE-13604226/2020 (Accession ID: EPI ISL 754198).GISAID database.have been compared on the base of analysis of GB-speckles. The official reference sequences were taken from thes-LASCA imaging techniqueAlgorithm for the total conversion of a nucleotide sequence to a colour GB speckle structure, processed byFirst, the sequence of the letters derived from the original one-dimensional nucleotide sequence was converted into the sequence of numbers in accordance with the following rule: thus, other rules could have been applied to the encoding, for instance:It is critical to emphasize that the specific relationship between the letters and numbers in this case is not critical as used earlier;Next, all possible triad combination are generated. As a result, a complete set of all triads is formed:The number of all possible combinations of four numbers combined in triads is 64. This algorithm was implemented in Matlab R2015a (RRID:SCR_001622); an open access alternative isJulia. The value of h is a positive integer, varying in the range from 1 to 64. In this case, each triad from the original nucleotide sequence is associated with only one h value. So, for example, the combination (1 1 1) conforms to the value h = 1, (1 1 2) corresponds to h = 2, (1 1 3) conforms to h = 3, (1 1 4) conforms to h = 4, (1 2 1) conforms to h = 5, (1 2 2) conforms to h = 6, and so on. Finally, the latest combination (4 4 4) conforms to the value h = 64. Finally, a square matrix Hn,m was formed by a one-dimensional array h. The physical significance of the shaped matrix Hn,m is that each of its elements represents the local height of some virtual rough surface corresponding to the local content of the analyzed genetic construction. The resulting virtual rough surfaces could be used to model original speckle structures corresponding to diverse particular nucleotide sequences.Then, a discrete magnitude, h, is allotted to each triad in accordance with the simple algorithm described previously.n,m. At each point of the virtual diffuser (in the beam scattering plane), some phase modulation Un,m = exp is introduced (j is an imaginary unit). The surface is illuminated at the normal incidence of the beam; the phase in the illuminating beam was a constant value.The two-dimensional speckle patterns that corresponded to each specific sequence was generated with the use the diffraction of a coherent beam with a square cross-section profile on a virtual scattering surface with a microrelief described by the matrix HXo andYo are the coordinates in the observation plane,z is the distance between the scattering plane and the observation plane,\u03bb is the wavelength. The illuminating radiation is completely monochromatic, thus,\u03bb = const. In this situation, the structure of speckles does not depend on the wavelength andz. Only the sizes of GB-speckles depend on these values, the average size of which is determined by the ratio:a is the size of the illuminated fragment of virtual surface. It is important to emphasize that the ratio\u03bb/a characterizes the diffraction angular divergence of a laser beam in the far field, and the product of this divergence angle by the light traveled distancez is equal to the lateral size of the beam. Thus, it can be seen that the diameter of the undisturbed laser beam is shown below.The original nucleotide sequence is as follows:ATGTTTGTTTTTCTTGTTTTATTGCCACTAGTCTCTAGTCAGTGTGTTAATCTTACAACCAGAACTCAATTACCCCCTGCATACACTAATTCTTTCACACGTGGTGTTTATTACCCTGACAAAGTTTTCAGATCCTCAGTTTTACATTCAACTCAGGACTTGTTCTTACCTTTCTTTTCCAATGTTACTTGGTTCCATGCTATACATGTCTCTGGGACCAATGGTACTAAGAGGTTTGATAACCCTGTCCTACCATTTAATGATGGTGTTTATTTTGCTTCCACTGAGAAGTCTAACATAATAAGAGGCTGGATTTTTGGTACTACTTTAGATTCGAAGACCCAGTCCCTACTTATTGTTAATAACGCTACTAATGTTGTTATTAAAGTCTGTGAATTTCAATTTTGTAATGATCCATTTTTGGGTGTTTATTACCACAAAAACAACAAAAGTTGGATGGAAAGTGAGTTCAGAGTTTATTCTAGTGCGAATAATTGCACTTTTGAATATGTCTCTCAGCCTTTTCTTATGGACCTTGAAGGAAAACAGGGTAATTTCAAAAATCTTAGGGAATTTGTGTTTAAGAATATTGATGGTTATTTTAAAATATATTCTAAGCACACGCCTATTAATTTAGTGCGTGATCTCCCTCAGGGTTTTTCGGCTTTAGAACCATTGGTAGATTTGCCAATAGGTATTAACATCACTAGGTTTCAAACTTTACTTGCTTTACATAGAAGTTATTTGACTCCTGGTGATTCTTCTTCAGGTTGGACAGCTGGTGCTGCAGCTTATTATGTGGGTTATCTTCAACCTAGGACTTTTCTATTAAAATATAATGAAAATGGAACCATTACAGATGCTGTAGACTGTGCACTTGACCCTCTCTCAGAAGCAAAGTGTACGTTGAAATCCTTCACTGTAGAAAAAGGAATCTATCAAACTTCTAACTTTAGAGTCCAACCAACAGAATCTATTGTTAGATTTCCTAATATTACAAACTTGTGCCCTTTTGGTGAAGTTTTTAACGCCACCAGATTTGCATCTGTTTATGCTTGGAACAGGAAGAGAATCAGCAACTGTGTTGCTGATTATTCTGTCCTATATAATTCCGCATCATTTTCCACTTTTAAGTGTTATGGAGTGTCTCCTACTAAATTAAATGATCTCTGCTTTACTAATGTCTATGCAGATTCATTTGTAATTAGAGGTGATGAAGTCAGACAAATCGCTCCAGGGCAAACTGGAAAGATTGCTGATTATAATTATAAATTACCAGATGATTTTACAGGCTGCGTTATAGCTTGGAATTCTAACAATCTTGATTCTAAGGTTGGTGGTAATTATAATTACCTGTATAGATTGTTTAGGAAGTCTAATCTCAAACCTTTTGAGAGAGATATTTCAACTGAAATCTATCAGGCCGGTAGCACACCTTGTAATGGTGTTGAAGGTTTTAATTGTTACTTTCCTTTACAATCATATGGTTTCCAACCCACTAATGGTGTTGGTTACCAACCATACAGAGTAGTAGTACTTTCTTTTGAACTTCTACATGCACCAGCAACTGTTTGTGGACCTAAAAAGTCTACTAATTTGGTTAAAAACAAATGTGTCAATTTCAACTTCAATGGTTTAACAGGCACAGGTGTTCTTACTGAGTCTAACAAAAAGTTTCTGCCTTTCCAACAATTTGGCAGAGACATTGCTGACACTACTGATGCTGTCCGTGATCCACAGACACTTGAGATTCTTGACATTACACCATGTTCTTTTGGTGGTGTCAGTGTTATAACACCAGGAACAAATACTTCTAACCAGGTTGCTGTTCTTTATCAGGGTGTTAACTGCACAGAAGTCCCTGTTGCTATTCATGCAGATCAACTTACTCCTACTTGGCGTGTTTATTCTACAGGTTCTAATGTTTTTCAAACACGTGCAGGCTGTTTAATAGGGGCTGAACATGTCAACAACTCATATGAGTGTGACATACCCATTGGTGCAGGTATATGCGCTAGTTATCAGACTCAGACTAATTCTCCTCGGCGGGCACGTAGTGTAGCTAGTCAATCCATCATTGCCTACACTATGTCACTTGGTGCAGAAAATTCAGTTGCTTACTCTAATAACTCTATTGCCATACCCACAAATTTTACTATTAGTGTTACCACAGAAATTCTACCAGTGTCTATGACCAAGACATCAGTAGATTGTACAATGTACATTTGTGGTGATTCAACTGAATGCAGCAATCTTTTGTTGCAATATGGCAGTTTTTGTACACAATTAAACCGTGCTTTAACTGGAATAGCTGTTGAACAAGACAAAAACACCCAAGAAGTTTTTGCACAAGTCAAACAAATTTACAAAACACCACCAATTAAAGATTTTGGTGGTTTTAATTTTTCACAAATATTACCAGATCCATCAAAACCAAGCAAGAGGTCATTTATTGAAGATCTACTTTTCAACAAAGTGACACTTGCAGATGCTGGCTTCATCAAACAATATGGTGATTGCCTTGGTGATATTGCTGCTAGAGACCTCATTTGTGCACAAAAGTTTAACGGCCTTACTGTTTTGCCACCTTTGCTCACAGATGAAATGATTGCTCAATACACTTCTGCACTGTTAGCGGGTACAATCACTTCTGGTTGGACCTTTGGTGCAGGTGCTGCATTACAAATACCATTTGCTATGCAAATGGCTTATAGGTTTAATGGTATTGGAGTTACACAGAATGTTCTCTATGAGAACCAAAAATTGATTGCCAACCAATTTAATAGTGCTATTGGCAAAATTCAAGACTCACTTTCTTCCACAGCAAGTGCACTTGGAAAACTTCAAGATGTGGTCAACCAAAATGCACAAGCTTTAAACACGCTTGTTAAACAACTTAGCTCCAATTTTGGTGCAATTTCAAGTGTTTTAAATGATATCCTTTCACGTCTTGACAAAGTTGAGGCTGAAGTGCAAATTGATAGGTTGATCACAGGCAGACTTCAAAGTTTGCAGACATATGTGACTCAACAATTAATTAGAGCTGCAGAAATCAGAGCTTCTGCTAATCTTGCTGCTACTAAAATGTCAGAGTGTGTACTTGGACAATCAAAAAGAGTTGATTTTTGTGGAAAGGGCTATCATCTTATGTCCTTCCCTCAGTCAGCACCTCATGGTGTAGTCTTCTTGCATGTGACTTATGTCCCTGCACAAGAAAAGAACTTCACAACTGCTCCTGCCATTTGTCATGATGGAAAAGCACACTTTCCTCGTGAAGGTGTCTTTGTTTCAAATGGCACACACTGGTTTGTAACACAAAGGAATTTTTATGAACCACAAATCATTACTACAGACAACACATTTGTGTCTGGTAACTGTGATGTTGTAATAGGAATTGTCAACAACACAGTTTATGATCCTTTGCAACCTGAATTAGACTCATTCAAGGAGGAGTTAGATAAATATTTTAAGAATCATACATCACCAGATGTTGATTTAGGTGACATCTCTGGCATTAATGCTTCAGTTGTAAACATTCAAAAAGAAATTGACCGCCTCAATGAGGTTGCCAAGAATTTAAATGAATCTCTCATCGATCTCCAAGAACTTGGAAAGTATGAGCAGTATATAAAATGGCCATGGTACATTTGGCTAGGTTTTATAGCTGGCTTGATTGCCATAGTAATGGTGACAATTATGCTTTGCTGTATGACCAGTTGCTGTAGTTGTCTCAAGGGCTGTTGTTCTTGTGGATCCTGCTGCAAATTTGATGAAGACGACTCTGAGCCAGTGCTCAAAGGAGTCAAATTACATTACACATAA (7)After converting a sequence of letters into a sequence of numbers in accordance with the algorithm described by rule (1) described previously, the nucleotide sequence takes the following form:143444344444244344441443221241342424134213434344114244121122131124211441222224321412124114424442121234334344414412224312111344442131422421344441214421124213312443442441224442444422114344124433442214324141214342424333122114334124113133444314112224342241221444114314334344414444324422124313113424112141141131332433144444334124124441314423113122213422241244144344114112324124114344344144111342434311444211444434114314221444443334344414412212111112112111134433143311134313442131344414424134323114114432124444311414342424213224444244143312244311331111213334114442111114244133311444343444113114144314334414444111141414424113212123224144114441343234314242224213334444423324441311221443341314443221141334144112142124133444211124441244324441214131134414443124224334314424424421334433121324334324321324414414343334414244211224133124444241441111414114311114331122144121314324341312434321244312224242421311321113434123443111422442124341311111331142414211124424112444131342211221121311424144344131444224114144121112443432224444334311344444112322122131444321424344414324433112133113131142132112434344324314414424342241414114422321421444422124444113434414331343424224124111441114314242432444124114342414321314421444341144131334314311342131211142324221333211124331113144324314414114414111441221314314444121332432344141324433114424112114244314424113344334334114414114412243414131443444133113424114242111224444313131314144421124311142414213322334132121224434114334344311334444114434412444224441211421414334442211222124114334344334412211221412131341341341244424444311244241214321221321124344434331224111113424124114443344111112111434342114442112442114334441121332121334344244124313424112111113444243224442211211444332131312144324312124124314324342234314221213121244313144244312144121221434424444334334342134344141121221331121114124424112213344324344244414213334344112432121311342224344324144214321314211244124224124433234344414424121334424114344444211121234321332434441141333324311214342112112421414313434312141222144334321334141432324134414213124213124114424224233233321234134341324134211422142144322412124143421244334321311114421344324412424114112424144322141222121114444124144134344122121311144241221343424143122113121421341314434121143412144434334314421124311432132114244443443211414332134444434121211441112234324441124331141324344311211312111112122211311344444321211342111211144412111121221221144111314444334334444114444421211141441221314221421111221132113133421444144311314241244442112111343121244321314324332442142111211414334314432244334314144324324131312242144434321211113444112332244124344443221224443242121314311143144324211412124424321243441323334121142124424334433122444334321334324321441211141221444324143211143324414133444114334144331344121213114344242414313112211111443144322112211444114134324144332111144211312421244424422121321134321244331111244211314343342112211114321211324441112123244344111211244132422114444334321144421134344441114314142244421234244312111344313324311343211144314133443142121332131244211134443213121414343124211211441144131324321311142131324424324114244324324124111143421313434341244331211421111131344314444434331113332414214244143422442224213421321224214334341342442443214343124414342224321211311113112442121124324224322144434214314331111321212444224234311334342444344421114332121212433444341121211133114444414311221211142144124121312112121444343424334112434314344341141331144342112112121344414314224443211224311441312421442113313313441314111414444113114214121421221314344314441334312142424332144114324421344341112144211111311144312232242114313344322113114441114311424242142314242211311244331113414313213414141111433221433412144433241334444141324332443144322141341143343121144143244432434143122134432434134434242113332434434424434331422432432111444314311312312424313221343242111331342111441214412121411 (8)As a result of diffraction of coherent beam on the phase screen ) with a square cross-section is formed GB-speckle-structure of two-dimensional intensity distribution, seeImportant to emphasize, that experimental studies were not carried out in this work, only computer modeling. The scheme for calculating GB-speckles during radiation diffraction on a virtual scattering surface is described in detail in the work.s-LASCA strategy has been connected for handling of GB-speckles. The strategy ofs-LASCA is based on the examination of an individual realization of static speckles., In this case, the whole realization of the speckle field is divided into square zones; typically, each counting 5\u00d75 or 7\u00d77 pixels.For each zone, the contrast of GB-speckles was calculated using the simplest formula:I was the varying intensity of GB-speckles, changing from point to point;I\u03c3 was the standard deviation of the intensity of fluctuations. After the contrastC is calculated in each point,LASCA image is developed. Here, the size of subarea for the local contrast calculating was 2\u00d72 pixels. As it has been demonstrated this size of subarea is close to optimal.whereTo generate three two-dimensional implementations of GB speckles built for different genetic sequences, it is necessary to construct a colour image, where each colour component has its own GB speckle structure. When all three speckle structures were totally indistinguishable, the colour images look grey-scale. If the colour components differ from each other, then, as a result, colouring will appear in the image.InInIt is quite obvious that in the case under consideration, there is a pronounced colouring over the whole image for the field of GB-speckle.Thus, the obtained colour image for the intensity and phase of GB speckles is a reliable diagnostic sign of the presence of polymorphism.s-LASCA image is obtained for each of the three components of the matched genetic sequence, the final colour image can be constructed. An example of such an image is shown inOnce anIt is obvious that the image shown inwherei is the pixel number,M andN are the number of rows and columns of the analyzed image,N \u00d7M is the total number of pixels in the image.is the average intensity value in each pixel,s-LASCA imaging of GB-speckles are completely identical, then the three components of the resulting colour image will be identical, and therefore the value ofR will be equal to zero. However, if there are at least minimal differences in the compared nucleotide sequences, then the value ofR will take a positive value. Thus, the value ofR calculated for theObviously, if the nucleotide sequences compared usingR calculated forInR is that this parameter characterizes the degree of coloring of the picture (GB-speckle- pattern). The bioinformatic (molecular biology) value ofR is that it takes positive values, even in the case of the appearance of a one SNP in the analyzed nucleotide sequences. Thus, the minimum natural mutations of the virus can be determined using the parameterR.The physical meaning of the introduced parameterR equals to 0.596 for this case.Finally, three SARS\u2013CoV-2 genes are reflected inR calculated forR at least in two times higher for GB speckles, processed bys-LASCA imaging technique.It is important to note that the value ofR is positive for all images inR is an important diagnostic feature when detecting the presence of SNPs in SARS\u2013CoV-2 genes. This is the main result of this paper.Evidently,s-LASCA \u2018imaging technique\u2019 generating original GB-speckles. It is established that even one SNP can be reliably detected. It has been demonstrated that suggested technique is very effective tool for discrimination between different variants of the SARS\u2013CoV-2 spike glycoprotein gene.A fundamentally new bioinformatics technique for reliable detection of single SNPs is proposed. The new method is based on the applying of theGISAID Gene: hCoV-19/cat/USA/TX-TAMU-078/2020. Accession number EPI ISL 699509;GISAID Gene: hCoV-19/cat/Russia/RII-LEN-22246S/2021. Accession number EPI ISL 811147;GISAID Gene: hCoV-19/cat/Greece/2K/2020. Accession number EPI ISL 717979;GISAID Gene: hCoV-19/Wuhan/WIV04/2019. Accession number EPI ISL 402124;GISAID Gene: hCoV-19/England/QEUH-B11766/2020. Accession number EPI ISL 642476;GISAID Gene: hCoV19/South Africa/KRISP-EC-K005299/2020. Accession number EPI ISL 678597;GISAID Gene: hCoV-19/Russia/MOS-CRIE-13604226/2020. Accession number EPI ISL 754198.GISAID public database.Sequences are available after registration at the I am very grateful for the chance to take part in the review of the article for your respected journal. The current manuscript is devoted to demonstrating the application of the s-LASKA method for discrimination of different variants of the SARS\u2013CoV-2\u00a0spike\u00a0gene. Fortunately, the authors consider this approach as very attractive and available for precise diagnostics of the infection with the use of a new generation of medical devices. In fact, this can be recognized as the new direction for bioscience.via ans-LASCA technique\u2026\u2019 \u2013 via should not be italic;p. 3: \u2018A new bioinformatics approach has been proposed very recently: 14 GB-speckles processingp. 7-8: the legends for Figures 1-2 contain no indication for which genes the GB-speckles were generated, as well as it is pointed in the legends for Figures 3a, 3b, and 3c. It is critical for the extended auditorium of readers to clearly understand what is demonstrated in Figures 1-2. These points need a correction. I have no additional proposals for edits. The revised version of the article can be recommended for indexing with no further reviewing. In my point of view, this is very interesting and important research that contains several new findings. The article is clearly written, conclusions correspond to data obtained, and statistical treatment is appropriate. However, the paper needs minor revision before it can be indexed. The following are my concerns about the article:Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:laser physics, biomedical scienceI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Dear Prof\u00a0Dmitry A. Zimnyakov, we highly appreciate your review for our manuscript. All your recommendations were accepted. All necessary corrections have been done in the text of the paper. Thank you very much once more. The authors. The paper can de accepted for indexing in the present new form.Is the work clearly and accurately presented and does it cite the current literature?PartlyIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?PartlyAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Singular and Correlation OpticsWe confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. The article is devoted to an extremely relevant and very\u00a0 interesting topic \u2013 an application of GB- speckles, in particular, processed by the s-LASCA imaging method, in relation to the discrimination of SARS\u2013CoV-2 strains. GB-speckle is a new word in the field of bioinformatics. In the future, these optical virtual speckle structures can be effectively used as an alternative to classical bioinformatics methods. The usage of GB-speckles can be considered as a replacement for traditional computer methods of sequencing of any nucleotide sequences. As it follows from the analysis of the literature, the authors of this article have successfully used these methods, based on the processing of virtual speckles, to the analysis of natural mutations in the comparing genes.s-LASCA\u00a0\u2018imaging technique\u2019 generating original GB-speckles. The article demonstrates that colored s-LASCA-imaging can be successfully used for the diagnosis and differentiation of various strains of SARS-CoV-2. Also, this article presents a fundamentally new method, based on the using of colored GB-speckles, processed by s-LASCA-imaging technique. The new method is based on the applying of the\u00a0The authors need to check carefully the reference(s) and link(s) for the bioinformatic tools used.After the providing of clear the answer to this question, the reviewed article can be accepted for indexing without any additional changes and without the additional cycles of reviewing. The utilization of GB-speckles in diagnostics is an absolutely new direction, which has been developed and is being successfully promoted by the authors of this article. I have no questions about the application of speckle interferometry methods in molecular biology, reflected in this work. I only have some minor criticisms, regarding bioinformatics:Is the work clearly and accurately presented and does it cite the current literature?PartlyIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:NAI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. The authors are extremely grateful to the respected reviewer Alexey Bashkatov for a detailed analysis of our article. In accordance with the recommendations of the reviewer, all references and links are checked, thank you. Any optical speckle is the result of the interference of waves scattered by inhomogeneities, and to obtain a speckle pattern, the wavelength is decisive. Thus, the question arises about the concrete wavelengths used in the study and their relationships with pseudo inhomogeneities. It would be interesting to know about the coherence of the sources used the modelling.\u00a0Please present an experiment setup, and the results of experimental modelling.It would be interesting to conduct a complete analysis of the entered parameter R, to represent its physical, biochemical meaning. The virus is constantly mutating, how is it possible to estimate the degree of virus mutation using this parameter. The paper can be indexed after reworking.The paper is devoted to the investigations of virtual Gene Based speckles for the determination the difference between SARS-Cov-2 spike glycoprotein genes. As it was noted by the authors the given method can use the classical approaches of speckle-optics. But there are some questions:\u00a0Is the work clearly and accurately presented and does it cite the current literature?PartlyIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?PartlyAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Singular and Correlation OpticsI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Any optical speckle is the result of the interference of waves scattered by inhomogeneities, and to obtain a speckle pattern, the wavelength is decisive. Thus, the question arises about the concrete wavelengths used in the study and their relationships with pseudo inhomogeneities. It would be interesting to know about the coherence of the sources used the modelling.\u00a0Response: This is really reasonable and very important question.\u00a0Fragment below is added to the new version of the text: It is assumed that speckles are formed in the far diffraction zone and described in the Fraunhofer approximation. In this case, the expression for the amplitude of the scattered field is the Fourier transform of the field in the diffraction plane, evaluated at frequencies spaces\u00a0Reply to the comments of reviewer Prof. Oleg\u00a0Angelsky: Fx=Xo/( z* \u03bb),\u00a0\u00a0\u00a0Fy=Yo/( z* \u03bb),\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0(*)\u00a0 where Xo and Yo are the coordinates in the observation plane, z is the distance between the scattering plane and the observation plane,\u00a0\u03bb\u00a0is the wavelength. The illuminating radiation is completely monochromatic, thus,\u00a0\u03bb\u00a0=const.\u00a0McGraw Hill Companies, New York\u00a0(1988).\u00a0 Reference to Goodman, J. W. Introduction to fourier optics.\u00a0 In this situation, the structure of speckles does not depend on the wavelength and z. Only the sizes of GB-speckles depend on these values, the average size of which is determined by the ratio:\u00a0 d~3*z* \u03bb /a\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0(**)\u00a0 where a is the size of the illuminated fragment of virtual surface. It is important to emphasize that the ratio\u00a0\u03bb/a characterizes the diffraction angular divergence of a laser beam in the far field, and the product of this divergence angle by the light traveled distance z is equal to the lateral size of the beam.\u00a0 New reference: M. Francon. La granularite laser (speckle) et ses applications en optique. Masson, Paris, New York, Barcelone, Milan 1978.\u00a0 Thus, it can be seen that the diameter of the undisturbed laser beam ) and the average speckle size are approximately equal to each other in any observation plane.\u00a0 In other words, when the parameters z and\u00a0\u03bb\u00a0change, a proportional change in the size of all speckles occurs synchronously. At the same time, the structure of speckle-patterns in all observation planes are completely similar, only their scale changes from plane to plane, but not the shape of the speckles or their location in the speckle pattern.\u00a0Please present an experiment setup, and the results of experimental modelling.\u00a0Response:\u00a0Experimental studies were not carried out in this work, only computer modeling. The scheme for calculating GB speckles during radiation diffraction on a virtual scattering surface is described in detail in the work:\u00a0 Ulianova OV, et al.: Speckle-interferometry and speckle-correlometry of GB-speckles. Frontiers in Bioscience-Landmark 2019; 24, 700-711.\u00a0This fact is mentioned in the new version of this article.\u00a0It would be interesting to conduct a complete analysis of the entered parameter R, to represent its physical, biochemical meaning. The virus is constantly mutating, how is it possible to estimate the degree of virus mutation using this parameter.\u00a0\u00a0Response: The physical meaning of the introduced parameter R is that this parameter characterizes the degree of coloring of the picture (GB-speckle- pattern). The bioinformatic (molecular biology) value of R is that it takes positive values, even in the case of the appearance of a one SNP in the analyzed nucleotide sequences. Thus, the minimum natural mutations of the virus can be determined using the parameter R.\u00a0This circumstance is also noted in the Conclusions of new version of this article.\u00a0 \u200b\u200b\u200b\u200b\u200bThe authors are extremely grateful to the respected reviewer, whose critical comments have significantly improved the quality of this article."} +{"text": "A carbon catalyst prepared by air oxidation of woody biomass hydrolyses woody biomass, and the reaction residue is transformed back to the catalyst by the same air oxidation method. Eucalyptus) provides a carbonaceous material bearing an aromatic skeleton with carboxylic groups (2.1 mmol g\u20131) and aliphatic moieties. This catalyst hydrolyses woody biomass to sugars in high yields within 1 h in trace HCl aq. Furthermore, after the reaction, the solid residue composed of the catalyst and insoluble ingredients of woody biomass is easily transformed back to fresh catalyst by the same air oxidation method. This is a self-contained system using woody biomass as both the catalyst source and substrate for realising facile catalyst preparation and recycling.Biomass is the sole carbon-based renewable resource for sustaining the chemical and fuel demands of our future. Lignocellulose, the primary constituent of terrestrial plants, is the most abundant non-food biomass, and its utilisation is a grand challenge in biorefineries. Here we report the first reusable and cost-effective heterogeneous catalyst for the depolymerisation of lignocellulose. Air oxidation of woody biomass ( Production of biofuels and bio-chemicals from lignocellulose, the most abundant non-food biomass, is a grand challenge in biorefineries.The use of heterogeneous catalysts is desired for the efficient depolymerisation of lignocellulose as they are non-corrosive and can be separated from product solution.Regardless of the preferable characteristics of heterogeneous catalysts, the contamination of the catalyst with solid lignin after the reaction prevents their application in the depolymerisation of real lignocellulose. Removal of lignin from solid catalysts is often challenging, essentially rendering the catalyst useless after the first reaction. Hence, the lignin fraction must be removed by pretreatment such as the kraft process before applying lignocellulose to the hydrolysis reaction.via oxidation. Thus, we can expect that the carbon material prepared by air oxidation hydrolyses lignocellulose. In this way, the catalyst is readily prepared, and more importantly the used catalyst and residual lignin can be together transformed into fresh catalyst by the same air oxidation method.Our idea for resolving the issues of conventional heterogeneous catalysts is to produce a weakly acidic carbon catalyst through simple air oxidation of lignocellulose and lignin residue. Organic materials thermally decompose to form carbonaceous material at an elevated temperatureEucalyptus by air oxidation, which is necessary only once. The second part (Part 2) is a cyclic process consisting of milling pretreatment, hydrolysis of Eucalyptus to glucose and xylose in trace HCl, and transformation of the solid residue to fresh catalyst by the same air oxidation. We used Eucalyptus as both a catalyst source and biomass substrate to make this a self-contained system. Eucalyptus is a fast-growing and inexpensive plant [<0.1 pounds (GBP) kg\u20131] that has been cultured as a major feedstock for pulping.Eucalyptus to produce a catalyst. Eucalyptus powder was first washed with boiling water and dried , xylan hemicellulose (10 wt%) and lignin (32 wt%). Ash was present in a small amount (0.12 wt%) and mainly composed of Ca salts was used to clarify the structural change of Eucalyptus by air oxidation gave major peaks at 110\u201350 ppm, mainly ascribed to cellulose and hemicellulose.Caromatic\u2013O at 160\u2013140 ppm, Caromatic\u2013C and Caromatic\u2013H at 140\u2013110 ppm).CO2R (170 ppm), Caromatic\u2013O (150 ppm) and sp3 carbons (<100 ppm).E-Carbon in a transmission mode scale\" fill=\"currentColor\" stroke=\"none\">O) (1770\u20131720 cm\u20131), \u03bd scale\" fill=\"currentColor\" stroke=\"none\">C, aromatic) (1610 cm\u20131), \u03b4(C\u2013H) (1470\u20131370 cm\u20131) and a mixture of various vibrations such as \u03bd(C\u2013O) (1350\u20131000 cm\u20131),2R at 288.6 eV (13 \u00b1 1%), C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O at 287.2 eV (3 \u00b1 2%), C\u2013O at 286.2 eV (20 \u00b1 5%), and C\u2013C and C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C at 284.6 eV (65 \u00b1 5%) . It was confirmed that the aromatics and acidic sites were not only derived from lignin but also from cellulose fractions; air oxidation of cellulose gave a similar carbon material 1470\u201310 cm\u20131 an) Fig. S3.38 The ce Fig. S5. This reEucalyptus under N2 at 573 K as a control. This material had significantly weaker aromatic peaks in the NMR gives aromatic precursors at 473\u2013573 K for the manufacture of carbon fibres.2-treated Eucalyptus, only weak C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O peaks were observed in the IR spectrum ] were milled together, named mix-milling,E-Carbon (50 mg) and Eucalyptus (324 mg) was subjected to a hydrolysis reaction in a 120 ppm HCl aqueous solution (pH 2.5) at 488 K .The mix-milled solid containing at 488 K . This recalyptus , entry 4Eucalyptus, we assume that E-Carbon and the mild acidic solvent (pH 2.5) synergistically accelerate the formation of monomeric sugars. It has been reported that trace HCl hydrolyses cellulose to produce soluble oligosaccharides, which enables the subsequent hydrolysis of oligosaccharides by solid acid catalysts.Eucalyptus, reactions in the absence of HCl or carbon catalyst provide unpractical yields of monomeric sugars (entries 1\u20133). HCl can be neutralised after the reaction with very low economic impact, as the acid concentration is less than 1/50 of conventional mineral acid processes.In the hydrolysis of entries \u20133. HCl cE-Carbon. Since a reaction in aq. HCl without E-Carbon afforded glucose in 32% yield and xylose in 26% yield (entry 3), E-Carbon increases the yield of glucose by 46% and yield of xylose by 68% (subtraction of yields in entry 3 from those in entry 4). The increase corresponds to a turnover number of carboxylic acid of 5.6. The result indicates that E-Carbon acts as a catalyst for the hydrolysis of cellulose and hemicellulose in Eucalyptus. We also found that the air-oxidised carbon prepared from cellulose worked in this reaction similarly to E-Carbon as shown in entry 7 . This shows that a cellulose-derived part also constitutes the active catalytic domain in E-Carbon. Contrastingly, the Eucalyptus-based catalyst prepared by N2 treatment was inactive . Therefore, it is concluded that the air oxidation of woody biomass provides active catalysts for the hydrolysis of lignocellulose.Controlled experiments were performed to reveal the important parameters influencing the catalytic activity of E-Carbon can be converted to a fresh catalyst again by air oxidation as shown in Eucalyptus (mainly lignin) and 0.51 g of E-Carbon, obtained in a large-scale experiment] to a black powder of 1.14 g. Accordingly, the catalyst weight increased from 0.51 to 1.14 g after one cycle in this system. The surplus residue can be used as fuel to power the process, since the solid is derived only from woody biomass and air. The 13C CP/MAS NMR spectrum of the regenerated catalyst contained a strong aromatic carbon peak with small fractions of \u2013CO2R, Caromatic\u2013O and aliphatic groups for the preparation of catalyst, which is in sharp contrast to conventional catalytic processes that require removal of the contaminant.The solid residue recovered after the reaction with 9% yield , entry 5 Table S4. It is tE-Carbon with reported catalysts, the active site is slightly similar to those of enzymes (cellulase).E-Carbon can work under harsher conditions, which enables the rapid hydrolysis of lignocellulose in trace HCl aq. at high temperature. Moreover, our catalyst is reusable and the price (ca. 0.1 GBP kg\u20131) is two-orders lower than that of cellulase (6.5\u201326 GBP kg\u20131).Comparing ce ca. 0. GBP kg\u20131Eucalyptus, produces a carbon-based catalyst overcoming the limitations of conventional catalysts used for the hydrolysis of woody biomass. The catalyst quickly converts lignin-containing Eucalyptus to glucose and xylose in high yields. Lignin remains as a solid together with the catalyst after the reaction; however, this solid mixture is a source for fresh catalyst and fuel. Therefore, the E-Carbon system drastically reduces the preparation and post-treatment costs of the catalyst. In general, the deactivation or spoiling of catalyst by contaminant is often a major issue in catalytic reactions. Hence, our idea that converts contaminant to a catalyst can be a useful strategy for improving the efficiency of catalytic processes.The air oxidation of biomass feedstock, Eucalyptus powder was washed with boiling water prior to use for all purposes in this study. 4.00 g of dried Eucalyptus powder was spread with a thickness of 3 mm on a Pyrex dish (\u00f8130) to uniformly prepare the catalyst and avoid hot spots. The sample was calcined under air at atmospheric pressure in an electric furnace with the following program: 298 to 573 K by 5 K min\u20131 and 573 K for 1 h. In the case of reaction residue, the residue of 1.63 g was calcined under the same conditions. The temperature inside the sample was monitored using a thermocouple (\u00f80.5) equipped with a quartz tube (ca. \u00f81).Eucalyptus (5.0 g) and catalyst (0.77 g) were milled together in an Al2O3 pot (250 mL) with Al2O3 balls using a Fritsch P-6 planetary ball mill. Milling conditions were 500 rpm for 2 h with a 10 min interval after every 10 min of milling.Eucalyptus was performed in a hastelloy C-22 high-pressure reactor equipped with an agitator operating at 600 rpm and a thermocouple. Mix-milled sample (374 mg) and 40 mL of 120 ppm HCl aq. were added into the reactor. The temperature of the reaction mixture was elevated from 298 K to 488 K in ca. 17 min and then quickly lowered to 298 K. Soluble products were analysed by high-performance liquid chromatography (Shodex SUGAR SH1011 and Phenomenex Rezex RPM-Monosaccharide Pb++ columns with refractive index detectors).The hydrolysis of"} +{"text": "The correct list of authors\u2019 names is provided and replaced online which is mentioned as under:Consuelo Sanavia1#*Marco Tatullo2#Jessica Bassignani1Silvia Cotellessa1Giulia Fantozzi1Giovanna Acito1Alessia Iommiello1Lorella Chiavistelli1Silvia Sabatini1Gianna Maria Nardi13The published list of authors was: Consuelo Sanavia1,\u00a0#,\u00a0*,\u00a0Marco Tatullo2,\u00a0#,\u00a0Jessica Bassignani1,\u00a0Silvia Cotellessa1,\u00a0Giulia Fantozzi1,\u00a0Giovanna Acito1,\u00a0Alessia Iommiello1\u00a0Lorella Chiavistelli1\u00a0Alessia Iommiello1\u00a0Silvia Sabatini1\u00a0Gianna Maria Nardi1\u00a03"} +{"text": "Targeted C(sp3)\u2013H activation or nucleophilic substitution reactions have been achieved through the interaction of a diborane dianion with haloalkanes. 1H2 and its B PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019B-bonded formal deprotonation product Li2[1] can activate the particularly inert C(sp3)\u2013H bonds of added H3CLi and H3CCl, respectively. The first case involves the attack of [H3C]\u2013 on a Lewis-acidic boron center, whereas the second case follows a polarity-inverted pathway with nucleophilic attack of the B PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019B double bond on H3CCl. Mechanistic details were elucidated by means of deuterium-labeled reagents, a radical clock, \u03b1,\u03c9-dihaloalkane substrates, the experimental identification of key intermediates, and quantum-chemical calculations. It turned out that both systems, H3CLi/1H2 and H3CCl/Li2[1], ultimately funnel into the same reaction pathway, which likely proceeds past a borylene-type intermediate and requires the cooperative interaction of both boron atoms.Organoboranes are among the most versatile and widely used reagents in synthetic chemistry. A significant further expansion of their application spectrum would be achievable if boron-containing reactive intermediates capable of inserting into C\u2013H bonds or performing nucleophilic substitution reactions were readily available. However, current progress in the field is still hampered by a lack of universal design concepts and mechanistic understanding. Herein we report that the doubly arylene-bridged diborane(6) As prominent examples, 9,10-dihydro-9,10-diboraanthracenes (DBAs) catalyze inverse electron-demand Diels\u2013Alder reactions of 1,2-diazines1H2/Li[1H]/Li2[1] together with a dianionic species ([1]2\u2013). As a decisive difference, however, the boron atoms in [DBA]2\u2013 are linked by two o-phenylene rings, whereas in [1]2\u2013 they are directly connected by a double bond. Both systems thus possess different frontier orbitals and should exhibit different reactivities.With the triad ]/Li2[1] , we rece1H]\u2013 and [1]2\u2013 are accessible in good yields via alkali-metal reduction of 1H2.1]2\u2013 back to [1H]\u2013 and finally 1H2.1H2 to afford [1H]\u2013 is also quantitative, provided that the sterically demanding bases (Me3Si)2NLi and (Me3Si)3CLi are used. In case of the smaller nBuLi, the deprotonation reaction (20%) is accompanied by the formation of an anionic diborylmethane featuring a boron-bridging hydrogen atom .1H2 activate C(sp3)\u2013H bonds of added alkyllithium reagents RCH2Li? (ii) Will [1]2\u2013 show nucleophilic behavior also toward electrophiles other than the proton ?The anions /Li2[1] as a perfect platform for further studies into boron-promoted C\u2013H-activation processes and boron-centered nucleophiles. Herein we present evidence that the reactions of 1H2 with RCH2Li indeed proceed through C(sp3)\u2013H-cleavage steps and that the boron-bridging H atoms in the diborylmethane products stem from the organolithium reagents and are not remains of 1H2 (cf.3H7). We also show that the B PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019B double bond of the dianion [1]2\u2013 behaves as a closed-shell nucleophile toward organohalides and that specifically H3CCl/Li2[1] and H3CLi/1H2 funnel into the same reaction channel. When H3CCl is replaced by an excess of H3C\u2013I, C\u2013H-activation is completely suppressed by a second nucleophilic substitution reaction to afford 2 equiv. of 9-methyl-9-borafluorene (3C). Employing \u03b1,\u03c9-dihaloalkanes X(CH2)nX and Li2[1], we gained further insight into the competition between the nucleophilic substitution and C\u2013H-activation scenarios as well as the cooperativity of the two adjacent boron centers .Before the background provided by the literature and our own previous results, we regarded the triad fluorene ; R = H3C1H2 with nBuLi furnish not only the deprotonation product Li[1H], but also the diborylmethane-hydride adduct shown in 3H7)?We started our study by addressing the question: why and how does the reaction of 3CLi in place of nBuLi maintains the same general reactivity . The 1H NMR spectroscopic monitoring of the reaction in a sealed NMR tube showed no free H2 (\u03b4 4.55 ppm),1H2 and H3CLi contain a sum of five BHB/H3CLi protons, of which only three remain in the product Li[2].First, we confirmed that a simplified system using Hactivity . From eq3CLi/1H2 or H3CLi/1D2 combinations furnished isotopically pure Li[2-d3] or Li[2], respectively (\u03b4(1H) 0.49 ppm, d), but also the boron-bridging hydrogen atom (\u03b4(1H) 1.94 ppm, br) in Li[2] originate from the organolithium reagent. None of the two BHB atoms of 1H2 is still present in the product Li[2-d3] (see the ESI1H] nor Li[2] (or their partly deuterated counterparts) and are consequently accountable for the missing 50% product yield (see below).Deuterium-labeling experiments with Dectively . Thus, n1H2 with H3CLi will be described the fate of the boron-bonded hydrogen atoms of 1H2, and (iii) the combined yield of only 50% for Li[1H] and Li[2]: similar to the case (Me3Si)3CLi/1H2, the reaction H3CLi/1H2 starts with the deprotonation of 1H2 to afford Li[1H]. The byproduct CH4 was detected by 1H and 13C{1H} NMR spectroscopy; when D3CLi was employed as the Br\u00f8nsted base, we instead observed the formation of D3CH 3CLi/1H2, the reaction involving H3CLi does not necessarily stop at the stage of Li[1H], because the small [H3C]\u2013 ion also has the potential to act as a Lewis base. Nucleophilic attack of H3CLi on a boron atom of Li[1H] establishes a B\u2013CH3 bond and shifts the boron-bridging hydrogen atom to a terminal position. The structural motif of the resulting intermediate [3]2\u2013 has precedence in the crystallographically characterized dianion [8]2\u2013,2[3] rearranges to Li2[4] through a 1,2-phenyl shift, accompanied by a 1,2-hydride shift. Again, a comparable hydrogen-containing species Li2[9] exists (2[9] can isomerize to Li2[FluB(H)\u2013(H)BFlu] (BFlu = 9-borafluorenyl),2[3] to Li2[4]. The latter reaction continues with an LiH-elimination step to generate Li[5], which possesses a three-coordinate boron atom with a vacant pz orbital and therefore easily undergoes a 1,2-phenyl shift to produce Li[6]. The anion [6]\u2013 can be viewed as the [H3C]\u2013 adduct of a diborane(4) containing two 9-borafluorene units that are linked by a B\u2013B single bond. Only the sp3-hybridized boron atom has acquired an electron octet, however, also the B(sp2) center might gain some electron density from an agostic interaction with the methyl group and thereby reduce its strong Lewis acidity.2]. It is well known that B(sp2)\u2013B(sp3) diboranes readily undergo B\u2013B-bond heterolysis and thereby act as mild sources of nucleophilic boron.2]\u2013 and [6]\u2013 are isoelectronic with protonated cyclopropane [C3H7]+. This cation has been thoroughly investigated by experimental6]\u2013 rearranging to [2]\u2013. At this stage, the dynamic behavior comes to an end, because, contrary to the case of [C3H7]+, the three corners of [2]\u2013 are not equivalent and the BHB bridge should be thermodynamically favored over alternative BHC bridges.Contrary to the case 4][2]; tBu substituents. The computed parent systems will be denoted with a superscript \u2018c\u2019 . The 1,2-phenyl shift in [5c]\u2013 proceeds viaTS1 with an activation barrier of \u0394G\u2021 = 9.9 kcal mol\u20131 and is endoergic by \u0394GR = 5.9 kcal mol\u20131. The resulting open-chain rearrangement product [6c-open]\u2013 features a large B\u2013B\u2013CH3 bond angle of 121\u00b0 and the vacant pz orbital of the B(sp2) atom is oriented almost orthogonal to the B\u2013CH3-bond vector, which precludes an agostic interaction in this isomer. To establish the B\u2013H\u2013C bridge proposed above, the tricoordinate borafluorene fragment must be rotated by approximately 70\u00b0 and the B\u2013B\u2013CH3 bond angle contracted \u2013 ultimately to a value of 68\u00b0 in the local-minimum structure [6c]\u2013. The conversion of [6c-open]\u2013 to the cyclic isomer [6c]\u2013viaTS2 is associated with a moderate energy penalty of \u0394GR = 4.6 kcal mol\u20131. The actual C\u2013H-activation process involves the transition state TS3 in which the B\u2013B bond and one C\u2013H bond are concertedly cleaved and a new B\u2013C bond is formed .In addition to the qualitative comparison with the all-carbon model system [Culations . Apart f2c-open]\u2013 is thermodynamically favored by \u201314.1 kcal mol\u20131 and \u20133.6 kcal mol\u20131 compared to [6c]\u2013 and [5c]\u2013, respectively. A further stabilization is achievable through rotation about a B\u2013C bond and placement of the hydrogen atom into a boron-bridging position to obtain the final product [2c]\u2013 . In summary, the reaction cascade from [5c]\u2013 to [2c]\u2013 possesses an overall activation barrier of \u0394G\u2021 = 14.9 kcal mol\u20131, which is easily surmountable at room temperature. An appreciable thermodynamic driving force is provided by the exergonicity of the [2c]\u2013 formation .The primary, open-chain activation product [1H] as the first intermediate along the pathway from 1H2 to Li[2], we treated an isolated sample of Li[1H] with 1 equiv. of H3CLi in THF. Even though the reaction started as expected, it stopped at the stage of Li2[4] (which enabled us to record a 1H NMR spectrum of this compound). The elimination of LiH from Li2[4] is thus not a spontaneous process, but apparently requires a hydride-trapping reagent. Compound 1H2 constitutes an ideal candidate for this purpose and, indeed, after the addition of 1 equiv. of 1H2, Li2[4] quantitatively vanished and Li[2] formed instead. Moreover, we found two sets of proton resonances that are assignable to two isomeric hydride-trapping products of 1H2 in 5] should be more facile on a B(sp2)\u2013B(sp3) rather than a B(sp3)\u2013B(sp3) scaffold .2] possesses an average C2v symmetry in solution, whereas a pending C3H7 substituent reduces the symmetry to Cs. Consequently, the 1H NMR spectrum of Li[2] contains only one set of signals for all four tBu-C6H3 rings. The corresponding spectrum of its Cs-symmetric congener features two sets of resonances,2] and thus likely assignable to those halves of the 9-borafluorene subunits, which point into the same direction as the proton residing on the methylene bridge. A similar interpretation is valid for the 13C{1H} NMR spectrum of Li[2]. Single crystals of [Li(thf)4][2] suitable for X-ray analysis were grown from THF-hexane .angement . For com10] readily isomerizes to the secondary hydride-trapping product Li[7], which we have isolated and characterized by NMR spectroscopy as well as X-ray crystallography. The anion of [Li(thf)3(Et2O)][7] consists of one 9-borafluorenyl and one BH2 fragment that are linked by a \u03bc-H atom and a 2,2\u2032-biphenylylene bridge .At room temperature, Li\u2013 and the known anion [9]2\u2013 are essentially superimposable, apart from the fact that the latter features a covalent B\u2013B bond (1.810(5) \u00c5) instead of the \u03bc-H atom , whereas the third set consists of very broad signals, each of them integrating 2H (thf)][Li(thf)2][11]; 11]2\u2013.Turning our attention from the products of the reaction CH2[11] are reasonably close to those of Li2[4] (cf. the ESI1H NMR spectra). Remarkably, Li2[11] is also accessible via a different approach, starting from the doubly boron-doped dibenzochrysene Li2[1] and tBuCCH, the conjugate weak acid of [tBuCC]\u2013 scale\" fill=\"currentColor\" stroke=\"none\">B double-bonded species. As mentioned above, the intermediate Li[6] of the reaction H3CLi/1H2 can be regarded as the [H3C]\u2013 adduct of a diborane(4). Conceptually, it should be possible to arrive at the same molecule by formally transferring two electrons from the carbon nucleophile to the redox-active organoborane and thus starting from methylium-ion sources and the anion [1]2\u2013 (39The facile protonationon [1]2\u2013 .392[1] is stirred at room temperature under a blanket of H3CCl gas (1 atm), a quantitative conversion to Li[2] occurs scale\" fill=\"currentColor\" stroke=\"none\">B fragment of Li2[1] likely acts as a nucleophile toward H3CCl to form [12]\u2013, which carries a boron-bonded methyl substituent and contains a central B\u2013B single bond. The B(sp2)\u2013B(sp3) species Li[12] then undergoes a 1,2-phenyl shift to afford Li[5] and thereby funnels into the reaction cascade outlined above for the formation of Li[2] from H3CLi/1H2 , the outcome is a mixture of Li[2], 9-methyl-9-borafluorene (13), and residual Li2[1] and (H3C)BFlu .When Hl Li2[1] . After iBFlu 13; . [BFlu]\u20133C\u2013I/Li2[1], the methyl group initially gets attached to only one of the symmetry-related boron centers, but the other is equally important for the subsequent C\u2013H-activation and nucleophilic substitution steps. The degree of B\u2013B cooperativity in Li2[1] as well as the insertion vs. nucleophilic behavior of [BFlu]\u2013 thus deserve a detailed assessment. To this end, we conducted a systematic study using 1\u2009:\u20091 mixtures of Li2[1] and \u03b1,\u03c9-dihaloalkanes X(CH2)nX with chain lengths in the range of n = 2\u20136 and leaving groups of different qualities . In these experiments, smaller alkylidene linkers are supposed to mimic higher local concentrations of the electrophile. As summarized in n = 2 and 3, cf.14C2 and 14C3; 1,3-dichloropropane leads to a complex mixture of products). Clean C\u2013H-activation reactions occur with the long-chain substrates (n = 5 and 6) to afford the haloalkyl species Li/Li and Li/Li. The medium-chain substrates (n = 4) mark the switching point between both scenarios: with the worse chloride leaving group, C\u2013H-activation is preferred over the twofold substitution. The reverse is true in the case of the better bromide leaving group. The solid-state structures of 14C2\u00b7thf, 14C3 2] 4], and [Li(thf)4] are supported by X-ray diffraction studies, however, due to disordered haloalkyl chains, tBu groups, and THF molecules, the quality of these three structures prevents their inclusion into this publication.41In case of the system Hhf, 14C3 , 14C4, a15C5,Cl] were chaThe observed chain-length dependence of the product distribution suggests that the carbene-type insertion and the second nucleophilic substitution both follow an intramolecular pathway involving two cooperating boron atoms.2X center and the BCH2 group are similarly close to the B\u2013B bond, the nucleophilic process occurs at a higher rate than the carbene-type C\u2013H-activation. As the alkylidene spacer grows, the second electrophilic functionality moves further apart whereas the reactive \u03b1-CH2 unit stays in place such that the C\u2013H-activation becomes more and more relevant until it finally takes over.If the remaining CH2[1] and, e.g., H3C\u2013I can convincingly be rationalized by assuming a nucleophilic pathway, the possible operation of a radical mechanism remains to be ruled out. We first note in this context that 1,2-dihaloethane in the presence of Li2[1] did not undergo reductive dehalogenation with ethene formation. Yamashita, Nozaki et al. have treated their boryllithium compound with methyl trifluoromethanesulfonate (H3COTf)2[1] (3COTf showed the same reactivity as described above for H3C\u2013I (cf. Li[2] and 13); BnBr (as well as BnCl) gave the C\u2013H-activation product Li[16] rather than any haloboranes, as confirmed by NMR spectroscopy and X-ray crystallography on [Li(thf)4][16].Although the reaction between LiOTf)2[1] , middle:2[1] to 1 equiv. of (bromomethyl)cyclopropane, a well-established radical clock . The absence of the ring-opened olefin derivative Li[18] in the reaction mixture strongly supports the proposal of a closed-shell scenario in contrast to an open-shell process.As the ultimate test, we added Lial clock , bottom. PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019B double bonds, but open new access routes to ditopic boranes of high Lewis acidity. Molecules containing two or more potentially cooperating boron sites are of great current interest, inter alia, as organocatalysts14nC already constitute free Lewis acids, but do not contain functional groups amenable to further derivatization.The results collected thus far are not only fundamentally interesting with respect to the reactivities of electron-rich B15n,XC]. Here, the terminal halogen atoms provide ample opportunities, e.g., for grafting the organoboron units onto polymers, dendrimers, or surfaces, but the Lewis acids need to be activated through LiH elimination prior to use.The opposite is true for the salts Li to its conjugate acid 14C1 to the tricoordinate spectral region (14C1: \u03b4 45 ppm).While the bulky hydride scavenger \u2013H activation and nucleophilic substitution reactions have been performed on the same redox-active diborane platform. We propose that the doubly 2,2\u2032-biphenylylene-bridged diborane(6) 1H2 reacts with H3CLi to furnish the rearranged B(sp2)\u2013B(sp3) intermediate Li[FluB\u2013BFlu(CH3)] . Li[6] also forms via an umpolung approach starting from H3CX and the B PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019B bonded, nucleophilic Li2[1], a compound which can be regarded as the product of a double deprotonation of 1H2 . Li[6] readily undergoes B\u2013B-bond heterolysis to formally give the [BFlu]\u2013 anion and (H3C)BFlu (13). The final product distribution depends on the relative amount of H3CX and the leaving-group qualities of X, because [BFlu]\u2013 can either insert into a C(sp3)\u2013H bond of 13 or replace the halogen atom of a second equivalent of H3CX. The product of the carbene-type C\u2013H insertion is Li[FluB(\u03bc-CH2)(\u03bc-H)BFlu] (Li[2]) while the nucleophilic substitution on C\u2013X generates 2 equiv. of 13. Further insight into the competition between the two scenarios was gained with the help of \u03b1,\u03c9-dihaloalkanes X(CH2)nX . In the resulting intermediates Li[FluB\u2013BFlu((CH2)nX)], both possible follow-up reactions should be intramolecular processes. A longer alkylidene chain corresponds to a lower local concentration of the electrophile, while the BCH2 groups are always similarly close to the reactive B\u2013B bond. Consequently, short chains result in double substitution products FluB(CH2)nBFlu and long chains in C\u2013H-activation products Li[FluB(\u03bc-C(H)(CH2)n\u20131X)(\u03bc-H)BFlu]. In the case of the intermediate chain length n = 4, a mixture of both compounds is obtained: the worse leaving group X = Cl leads to a higher proportion of the C\u2013H-activated species, the better leaving group X = Br furnishes more FluB(CH2)4BFlu. We finally note that the B\u2013B-bond heterolysis of Li[6] with concomitant transfer of a reactive [BFlu]\u2013 moiety is reminiscent of the reactivity patterns of the widely used alkoxy-diborane(4) adducts [pinB\u2013Bpin(OR)]\u2013.\u2013 appears to be considerably more reactive than in situ-generated [Bpin]\u2013, because C\u2013H-insertion reactions of the latter are so far unknown.In summary, C(spThere are no conflicts to declare.Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "The novel rectification\u2013insertion mechanism for the polymerization of polar norbornenes: making alternating copolymers from a single monomer. endo position. We have examined the polymerization mechanism of NBEs bearing one or two CO2Me groups either in exo or endo position catalyzed by the so-called naked allyl Pd+ SbF6\u2013 catalyst (1). Although endo dimethyl ester of 5-norbornene-2,3-dicarboxylic acid (NBE(CO2Me)2) is polymerized by 1, two endo units are never inserted consecutively along the polymer chain. Indeed, 1 is a tandem catalyst which not only catalyzes the insertion of the monomer but also the isomerization of endo and exo isomers. Thus, the polymerization of endo monomers proceeds via a novel mechanism, coined rectification\u2013insertion mechanism, whereby half of the endo monomers are rectified into exo ones prior insertion, leading to the formation of an alternating endo\u2013exo copolymer using an endo only feedstock. With this mechanism, the lack of reactivity of endo norbornenes is bypassed, and the polymerization of predominantly endo polar NBEs bearing a variety of functionalities such as esters, imides, acids, aldehydes, alcohols, anhydrides, or alkyl bromides proceeds with catalyst loadings as low as 0.002 mol%.The catalytic 1,2-insertion polymerization of polar norbornenes (NBEs) leads to the formation of functional rigid macromolecules with exceptional thermal, optical and mechanical properties. However, this remarkable reaction is plagued by the low reactivity of the polar monomers, and most notably of those bearing a functional group in Furthermore, Lewis acids such as alkyl aluminums, methylalumoxane or fluorinated boranes which are cocatalysts of early transition metal catalysts for NBE polymerization usually react with polar monomers.et al. demonstrated that [Pd(CH3CN)4][BF4]2 promoted the living polymerization of a series of esters of bicyclo[2.2.1]hept-5-ene-2-exo-methanol.3-allyl)palladium compounds with BF4\u2013 or SbF6\u2013 counter ions were able to catalyze the polymerization of bicyclo[2.2.1]hept-5-ene-2-carboxylic acid methyl ester (NBECO2Me).2H) was also reported. In 1995, Novak et al. reported that neutral Pd(ii) complexes bearing an hexafluoroacetyl acetonate and a \u03c3\u2013\u03c0 bicyclic alkyl ligands catalyze the living polymerization of NBE and a substituted oxanorbornene.2Me but a cationic analog was found to be active for the polymerization of NBECO2Me and the copolymerization of NBECO2H with NBE in a non-living manner, with either BF4\u2013,6\u2013 (i.e. diluted) with NBE or with ethyleneendo isomer is practically non-reactive and it deactivates the catalyst. The lack of reactivity of the endo substituted polar NBEsvia the exo face,endo face of the monomer, as shown by Sen et al. through the X-ray structure of an homologous Pt complex.et al. has ruled out the formation of chelate for naked cationic Pd complexes stabilized by the tBu3P phosphine and found that endo isomers are as reactive as exo ones.Functional macromolecules are essential components for the formation of complex nanostructures with defined shape and functionality with applications in optical and electronic materials, catalysis, recognition/separation technologies and drug delivery. BF4\u2013,6\u2013 or MAO30endo isomer is due to a series of factors . Furthermore, the catalyst promotes the rectification of the monomer . Numerous functional PNBEs 4\u2013, SbF6\u2013) have been investigated in the past for the polymerization of NBE, and we have selected SbF6\u2013 as it is more bulky than BF4\u2013 and PF6\u2013, and therefore putatively less coordinating, but it does not require handling pyrophoric MAO and B(C6F5)3.Our initial work on the polymerization of polar norbornenes was inspired by the discovery that cationic \u2018naked\u2019 Pd and Ni catalysts1, [PdS2]+SbF6\u2013 (S = solvent).2Me) (73% endo) occurs in low to moderate yield (0\u201356%) at high catalyst loadings (\u22650.2 mol%) in the majority of solvents becomes feasible even with a catalyst loading as low as 0.003 mol% which contrasts with past results reported in literature whereby catalyst loadings are typically comprised between 0.5 and 1%. In this study, all mechanistic studies have been performed in nitromethane as it keeps the polymer in solution without coordinating too strongly catalyst 1.When the polymerization is performed neat , the yield is limited to 65% . This limitation is purely physical in nature, and corresponds to a vitrification phenomenon: a mixture of 65% of polymer and 35% of monomer is so viscous that the monomer cannot diffuse to reach the active site. Thus, the yield of the neat polymerization modestly decreases when the catalyst loading is lowered, so that the homopolymerization of NBE(COcis-NBE(CO2Me)2 (abbreviated NBE(CO2Me)2) and trans-NBE(CO2Me)2 (one endo and one exo CO2Me group) are illustrated in endo content, the kinetics of trans isomer being comprised between the one of 35% endo and of 75% endo, and therefore being comparable to 50% endo. When the endo content in the monomer is greater than 25%, the polymerization is zero order in monomer, as shown by a linear evolution of the conversion vs. time, a behavior which is also observed for the polymerization of other monomers with 1 2]/[1] = 10 2 were inserted in 1, leading to the formation of catalysts 2, 3 and 4 respectively and by X-ray crystallography 2 is rapid, i.e. it is quantitative in less than a minute at room temperature 2 occurs in a cis fashion on the exo face, as shown in X-ray structures. The 3J coupling value between protons H2 and H3 is comprised between 6 and 8 Hz which is characteristic of a cis coupling are consistent with an exo placement of the Pd atom, ruling out the presence of a directing effect. Addition of NBE(CO2Me)2 (trans) to 1 results in the formation of two products (4X and 4N) in 50\u2009:\u200950 mol% ratio, as shown by 1H NMR product, the Pd atom is on the same side of the bridge as the exo (resp endo) CO2Me. The fact that 4X and 4N are in equal proportion is another indication that the endo ester does not act as a directing group for catalyst addition. In solution, the ester groups of the inserted NBE(CO2Me)2 are not coordinated, as shown by 13C NMR chemical shifts comprised between 173.2 and 175.5 ppm for C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O of complexes 2, 3, 4N and 4X, which correspond to the usual chemical shifts of CO2Me esters. For the sake of comparison, in a norbornane ring bearing two CO2Me groups in exo (resp trans), the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O resonates at 173.1 ppm (2Me either on these naked Pd complexes (see below) or on cationic Pd diimine complexes are found at lower field (177\u2013195 ppm).52In order to clarify the mechanism of polymerization, ectively , which wlography . The rea Fig. S11. In the 73.1 ppm . Structure 2s is a tetrameric macrocycle whereas 3s and 4Ns are polymeric (2O2-H and 3-THF) are obtained. The tetrameric solid 2s is soluble in non-coordinating solvents such as dichloromethane, chlorobenzene and tetrachloroethane however, polymeric 3s and 4Ns are insoluble in such solvents. Therefore, when the polymerization is performed in a non-coordinating solvent, the insertion of endo and the trans monomer leads to the formation of an insoluble active species (3s and 4s). Thus, the lack of reactivity of endo isomers in chlorinated solvents is in part explainable by the loss of Pd-containing species by precipitation. Interestingly, characteristic bond lengths and bond angles of complexes 2s, 3s, 4Ns, 2O2-H and 3-THF 2 at room temperature result in the immediate formation of 5 and 6 . In these complexes, endo-NBE(CO2Me)2 is chelated via its endo face to the Pd complex, as deduced by an unexpected downfield resonance for the HC13 PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C14H moiety . The 13C carbonyl resonances are also shifted downfield (177.5 and 177.1 respectively for 5 and 6vs. 172.7 ppm for the uncoordinated monomer). This chelate is labile and rapidly exchanging within NMR timescale. For example, from 7.05 ppm in 5, the H13/14 resonance is displaced to 6.83 ppm for a 1\u2009:\u20091 mixture of 5 and endo-NBE(CO2Me)2, and to 6.48 ppm for a 1\u2009:\u200910 mixture of 5 and endo-NBE(CO2Me)2. Such exchange phenomenon is not observed when an excess of exo monomer is added to 5 or 6 as shown by separate resonances for the chelated endo monomer and free exo-NBE(CO2Me)2 2, polymerization is quantitative within 9 hours, as shown by the decrease of the olefin resonances in 1H NMR at 6.35 ppm. To our surprise, although no endo monomer was added to this reaction, the presence of the chelated complex 5 is also clearly detected in the reaction mixture, as shown by the presence of (1) a new downfield olefin resonance (6.8\u20137.0 ppm) characteristic of the endo chelated double bond, (2) a OCH3 resonance at 3.81 ppm which is neither observed in 2 (3.74 ppm) nor in the exo growing polymer chain (3.74 ppm) and (3) a characteristic H17 bridge proton for the chelated endo monomer at 1.71 ppm which could account for a maximum of 20% of the chelated Pd complexes . However, as much as 70% of the Pd atoms are found to be chelated. Therefore, the endo isomer must be generated during the polymerization reaction. Lewis acids are known to catalyze direct and retro Diels\u2013Alder reactions,exo monomer in endo monomer. Although is it well known that the exo monomer (thermodynamic product) is more stable than the endo monomer (kinetic product), DFT calculations indicate that the gain of stability is only 2.7 kJ mol\u20131. Therefore, the equilibrium constant between both isomers is 3, and the exo\u2013endo monomer distribution at equilibrium is 75\u2009:\u200925. Thus, even when starting from 100% exo monomer, endo isomer is generated during the polymerization. It will be seen below that the reverse situation is also true, that is to say that when starting from pure endo monomer, exo monomer is generated during the reaction.When Fig. S26. The exoexo monomer is polymerized by 2, the Lewis-acid catalyzed formation of the endo isomer leads to the generation of chelated 5 which is significantly less active for polymerization. Thus, only a fraction of the catalyst is 'naked' and active, resulting in the formation of polymers with molecular weights which are higher than expected (2. The initiation of the polymerization of exo-NBE(CO2Me)2 by 2 (rate constant ki) is slow relative to subsequent insertions (kp) as the Pd\u2013C2\u2013C3\u2013C10\u2013C11\u2013C12 6-member chelate must be broken during the first insertion. When polymerizing endo-NBE(CO2Me)2, the propagation rate (kp) is greatly decreased, thus the discrepancy between kp and ki is less noticeable. As the result, experimental and theoretical molecular weights are in good agreement for endo monomers (in situ formation of chelated 5 during the polymerization of exo-NBE(CO2Me)2 explains the deviation from linearity observed in the polymerization kinetics at high conversion 2 or mixtures of endo and exo NBE(CO2Me)2, as the catalyst is then entirely chelated from the onset of the polymerization.When the exo-NBE(CO2Me)2. However, it is desirable to polymerize monomers directly obtained by Diels\u2013Alder reaction, that is to say rich in endo isomer in order to avoid a painstaking separation step between endo and exo isomers. In this case, due to the presence of excess endo isomer, the catalyst is entirely chelated. Therefore, we will now concentrate on the reactivity of catalysts 5 and 6. Using 1H NMR, one can assess the regiochemistry of the last inserted monomer unit. Indeed, the methine proton in \u03b1 of Pd 2 2 (experiment B), insertion of a first exo monomer occurs within minutes , whereas the addition of two consecutive endo units is very slow (experiment A). Using the usual denomination for copolymerization rate constants, kendo,exo \u226b kendo,endo. The value of kendo,exo is too high to be measured precisely via1H NMR, but a lower limit for kendo,exo could be determined on the account that the insertion of one exo monomer by 6 proceeds in less than 10 minutes 2 in endo-NBE(CO2Me)2 is clearly observed in experiment B , but soon after, 1.6 equivalents of endo monomer are present , or that the insertion of endo monomer is immediately followed by the insertion of an exo monomer, due to the high value of kendo,exo. To lift this ambiguity, we have examined the reaction of 5 with 10 equivalents of endo-NBE(CO2Me)2 2 is inserted, as shown by the apparition over a few hours of a characteristic doublet at 4.3 ppm corresponding to H2 in an endo unit. Thus, the endo monomer is reactive and kexo,endo is non null. The low stability of catalyst 5 in solution is another indication that the endo isomer can be inserted after an exo unit. Indeed, when a solution of 5 in CD3NO2 ([5] = 0.032 mol L\u20131) is left for 7 hours, 30% of the chelated endo monomer is inserted 2, Fig. S33kexo,endo, obtained from the slope of the kinetic profiles, are respectively 3.6 \u00d7 10\u20132, 4.1 \u00d7 10\u20132 and 4.9 \u00d7 10\u20132 h\u20131, which yield an average value of kexo,endo of 4.2 \u00d7 10\u20132 h\u20131 , solutions of 6 are stable indefinitely 2 into exo-NBE(CO2Me)2 before polymerization can proceed. The value of kexo,exo was measured in two separate kinetic experiments , the insertion of two exo monomers consecutively is highly unlikely due to the low concentration of exo monomer in solution. Thus, the polymerization of the endo monomer leads to the formation of an alternating endo\u2013exo copolymer. The catalyst has rectified 50% of the less reactive endo monomers into more reactive exo monomers. We have coined such mechanism rectification\u2013insertion.Contrasting with the lack of stability of Fig. S31, indicaty ratios , it is cxo chain . When an1H NMR or 13C NMR has been hampered by the broadness of the peaks caused by the rigidity of polynorbornenes in solution 2, the 13C spectrum is constituted of very broad resonances, with a single peak observed at 173.1 ppm for C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O scale\" fill=\"currentColor\" stroke=\"none\">O and two resonances for exo C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O scale\" fill=\"currentColor\" stroke=\"none\">O are upfield relative to exo ones in 2, 3, 5 and 6). The presence of two exo C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O peaks in same proportion could arise from two different tacticities arising from the placement of consecutive NBEs.Microstructural analysis of these polymers, either by Fig. S39 could coendo isomer must be interconverted into exo. It is also well established that Lewis acids (such as 1) catalyze the Diels\u2013Alder and retro Diels\u2013Alder reaction.1 . Interestingly, freshly cracked cyclopentadiene can also be polymerized with 1, leading to a polymer which is not entirely similar to polydicyclopentadiene by 1H NMR . Second, kendo,endo must be significantly lower than the other propagation rate constants. Preliminary theoretical calculations indicate that kendo,endo is very low because of the large steric hindrance between the endo substituents of the penultimate inserted unit and the active site when two endo monomers are inserted in a row. Several elements point toward the fact the rectification\u2013insertion mechanism is not only prevailing with NBE(CO2Me)2 but also with other functionalized norbornenes. Induction periods are observed for the polymerization of predominantly endo monomers 2, NBE(imide), and NBE(CHO)). As a proof of the unique versatility of this reaction, the polymerization of aldehyde containing monomer NBE(CHO) was found to proceed in high yield which contrasts with radical, cationic and anionic polymerizations which are usually not efficient to prepare linear polymers containing pendant aldehyde groups.2H)2.Thanks to this mechanism, catalyst monomers , 2 and 4exo content (PNBE(imide) 5% endo, PCA, PNBE(CO2H)2) lead to polymers with a molecular weight higher than expected. As shown above, this behavior is a consequence of the rectification\u2013insertion mechanism: even when starting from pure exo monomer, endo chelated species can be formed, and only the remaining (unchelated) fraction is rapidly polymerizing. When the monomer contains high amount of endo isomer, experimental molecular weights are commensurate with theoretical values, which is again indicative of a high degree of control for the polymerization. All these polymers exhibit Tg which are higher than 300 \u00b0C with virtually identical yields to those obtained under inert atmosphere. For example, the polymerization of NBE(CO2H) occurs in 40% and 42% yield when performed respectively under inert atmosphere and in air.When the polymerization is performed in the absence of solvent, very low amounts of catalyst can be used (as low as 0.002 mol%), which is indicative of the exceptional robustness of the active species. The polymerization is then controlled by the drastic increase of viscosity associated with the formation of high Tg polymers, a physical limitation which could for example be mitigated by the use of heterophase processes. The polymers have in general low polydispersity indices (1.1 \u2264 PDI \u2264 1.6), indicating some degree of livingness for this type of polymerization (a feature which will be further explored in a subsequent report). Monomers with high Fig. S48, which iendo-rich monomers directly obtained via Diels\u2013Alder reaction with no need for cumbersome and time-consuming separation of both isomers. Furthermore, catalyst loadings as low as 0.002 mol% can be used, and both the monomer preparation and the polymerization can be performed in the absence of any solvent. Thus, the preparation of these rigid macromolecules is an archetypal example of green chemical process. This study has also aimed at clarifying the mechanism of polymerization of substituted NBEs. The endo isomers deactivate the catalyst because the endo active species are less soluble than the exo ones and the endo monomer forms a chelate with the naked catalyst. However, these limitations can be counteracted by the judicious choice of polymerization conditions, and, most importantly, by the action of the rectification\u2013insertion mechanism. Thus, the naked Pd complex has a tandem role of polymerization catalyst and of endo/exo isomerization catalyst. As two endo units cannot be inserted consecutively, it is possible to prepare alternating endo\u2013exo copolymers when starting from an endo monomer only. We envision that this novel mechanism could be easily exploited further, for example by adding a separate Lewis acid which could catalyze the retro/direct Diels\u2013Alder reaction, which should putatively lead to a rate acceleration and the disappearance of the induction period. Furthermore, due to high degree of control of these polymerization, we believe that this mechanism open the way to the formation of a wealth of hierarchical nanostructures generated upon self-assembly of rigid functional amphiphilic block PNBEs.The novel rectification\u2013insertion polymerization mechanism is a powerful mechanism for the preparation of rigid macromolecules obtained from polar NBEs, yielding functional polymers bearing highly valuable functional groups such as aldehydes, anhydrides, alcohols, alkyl halogens and carboxylic acids. Thus, this method offers the same level of versatility and practicality as highly popular chain-growth polymerizations such as ROMP or radical polymerizations. The reaction readily proceeds with Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "Half-sandwich RhIII anticancer complexes with activated Cp* rings not only undergo sequential CH3 H\u2013D exchange, but also react with biological dienes, generating RhI Diels\u2013Alder adducts in aqueous media at ambient temperature. x C\u2013H protons in certain organometallic RhIII half-sandwich anticancer complexes [(\u03b75-Cpx)RhCl]+, where Cpx = Cp*, phenyl or biphenyl-Me4Cp, and N,N\u2032 = bipyridine, dimethylbipyridine, or phenanthroline, can undergo rapid sequential deuteration of all 15 Cp* methyl protons in aqueous media at ambient temperature. DFT calculations suggest a mechanism involving abstraction of a Cp* proton by the Rh\u2013hydroxido complex, followed by sequential H/D exchange, with the Cp* rings behaving like dynamic molecular \u2018twisters\u2019. The calculations reveal the crucial role of p\u03c0 orbitals of N,N\u2032-chelated ligands in stabilizing deprotonated Cpx ligands, and also the accessibility of RhI\u2013fulvene intermediates. They also provide insight into why biologically-inactive complexes such as [(Cp*)RhIII(en)Cl]+ and [(Cp*)IrIII(bpy)Cl]+ do not have activated Cp* rings. The thiol tripeptide glutathione and the activated dienophile N-methylmaleimide, (NMM) did not undergo addition reactions with the proposed RhI\u2013fulvene, although they were able to control the extent of Cp* deuteration. We readily trapped and characterized RhI\u2013fulvene intermediates by Diels\u2013Alder [4+2] cyclo-addition reactions with the natural biological dienes isoprene and conjugated -linoleic acid in aqueous media, including cell culture medium, the first report of a Diels\u2013Alder reaction of a metal-bound fulvene in aqueous solution. These findings will introduce new concepts into the design of organometallic Cp* anticancer complexes with novel mechanisms of action.The Cp These were sensitive to traces of water.et al. have shown that deuteration of the Cp* in [(Cp*)RhD2(BPin)] (BPin = bis(pinacolato)diboron) occurs readily in MeOD-d4 and synthesized the deuterated compound [(D15Cp*)RhD2(BPin)], under reflux in D2O/OD\u2013 for 3 days.et al. showed that deuteration of [(Cp*)RhCl(PTA)2]+ is assisted by the basic centers in the PTA ligand,I\u2013fulvene species, but no experimental evidence for the fulvene complex was obtained.In an early example, Kang and Maitlis deuterated the methyl groups in the dinuclear Rhet al.et al.I catalyst [(Cp*)Rh(bpy)] can lead to formation of H2 in dry acetonitrile. In the latter case, [(Cp*H)Rh(bpy)Cl], generated by reduction of the RhIII complex [(Cp*)Rh(bpy)Cl]+ followed by acidification in ether, was isolated, and shown to reduce NAD+ to NADH. The Cp* methyl protons underwent exchange with deuterium. Peng et al. have recently characterized the RhI phenanthroline analogue containing protonated Cp*, [(Cp*H)Rh(phen)Cl].et al.4-pentamethylcyclopentadiene\u2013RhI intermediates under protic conditions.Quintana 1H NMR peaks of some [(Cp*)RhClCl]+ complexes which we had synthesized as potential anticancer agents and transfer hydrogenation catalysts, rapidly lost intensity, suggesting activation towards H/D exchange under mild conditions. We have investigated the conditions under which this occurs, including the influence of solvent, aquation, and pH, and monitored the reactions by both 1H NMR spectroscopy and high resolution FT ICR MS spectrometry. We have also investigated the influence of substituents on the Cp* ligand (Cp* versus CpxPh and CpxPhPh), influence of the N,N\u2032-chelating ligand versus aromatic diamines bipyridyl (bpy), 4,4\u2032-dimethyl-2,2\u2032-bpyridine (mbpy), and phenanthroline (phen)), as well as the metal ion (RhIIIversus IrIII). The X-ray crystal structures of 8 new complexes are reported.Our own investigations began with observations that in aqueous solutions at ambient temperature, methyl x C\u2013H activation reactions. Importantly the DFT calculations led to the prediction of accessible RhI\u2013fulvene states and so we investigated the trapping of such intermediates by addition reactions with thiols (glutathione), dienes and dienophiles, l-Glu-l-Cys-Gly) is present in cells at millimolar concentrations, and might be expected to undergo addition reactions with double bonds such as those in a fulvene, as might NMM which contains an activated double bond. Fulvene is known to undergo Diels\u2013Alder reactions with both dienes and dienophiles.Z,11E)-linoleic acid -Linoleic acid is a common dietary conjugated fatty acid, of particular interest for its potential role in the prevention and treatment of a variety of diseases,We have used density functional theory (DFT) calculations to elucidate the mechanism of these Cpeic acid . IsoprenI\u2013fulvene trapping reactions under mild conditions of biological relevance is likely to open up new avenues of investigation into reactions of this class of half-sandwich organometallic complexes and influence their design as biologically-active agents. Our discovery of Diels\u2013Alder reactions of a metal-bound fulvene under mild conditions in aqueous solutions, appears to be unprecedented.The surprising success of these RhIII complexes 1 and 3\u201312 , and a monodentate chloride ligand were synthesized by the reaction of the appropriate chloride-bridged dimer and chelating ligand, except complex 12 which was prepared by substitution of chloride in complex 1 by pyridine. They were fully characterized by 1H NMR spectroscopy, ESI-mass spectrometry, and elemental analysis Rh(phen)Cl]SO3CF3,1, 3 and 4 are similar to those reported for [(Cp*)Rh(bpy)Cl]SO3CF3.12, the Rh\u2013N(pyridine) bond (2.127(4) \u00c5) is longer than other two Rh\u2013N (bipyridine) bonds (ca. 2.105 \u00c5).Organo\u2013Rhand 3\u201312 and IrII1 and 3\u201311 in 20% methanol-d4/80% D2O was confirmed by comparing 1H NMR spectra before and after removal of the chloride ligand by reaction with AgNO3. Complexes 1, 3\u201311 show fast hydrolysis at 310 K, reaching equilibrium in <10 min. Complete conversion of chloride complex 11 to the aqua adduct was observed, whereas 1, and 3\u201310 reached equilibrium with 30\u201360% formation of the aqua species , but resonances corresponding to the methyl protons in the Cp*, CpxPh and CpxPhPh ligands were not observed as the expected sharp singlets, but as broad signals, difficult to distinguish from the baseline Rh(bpy)(py)](PF6)2 (12). The X-ray crystal structure . Quantitative full deuteration was achieved at ambient temperature by the time the first 1H NMR spectrum was recorded (ca. 12 min). In contrast, when the sample of 1 was made acidic with DNO3, no deuteration was observed after 12 h. These results are compatible with Rh-OD acting as a base for proton abstraction and subsequent deuteration of the Cp*.Since deprotonation of Rh\u2013OHd Fig. S8. Complex15\u03b75-Cp*)Rh(bpy)Cl]PF6 (1D15) and used 2H NMR to show that, as expected, back D/H exchange was slow (ca. 50%) after 3 days in 60% MeOH/40% H2O involving the Cp* protonation.4Cp PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CH2]2\u2013 which could then rapidly gain a deuteron from solvent to generate a \u2013CH2D substituent. Rapid methyl and ring rotation can lead to the sequential deuteration of all 15 methyl protons. We modeled the hydroxido adducts using the CAM-B3LYP functional with a CEP-31G basis set. In the cases of both RhIII and IrIII bpyridine complexes [M(Cp*\u2013)(bpy)OH]+, the optimized structures show a relatively close M\u2013O(H)\u00b7\u00b7\u00b7HCH2 contact between the bound hydroxide and a Cp* ring methyl (11), the hydroxide is oriented so as to optimize H-bonding with an en NH2 proton, Rh\u2013O(H)\u00b7\u00b7\u00b7HNH(en) 2.053 \u00c5, These were performed to gain insight into the mechanism of deuteration and the difference in behavior between the bpy and phen complexes, and inactive Rhg methyl , being sg methyl . In cont4Cp PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CH2]2\u2013 and H2O ligands, \u20131 higher in energy than the original R\u2013OH/Cp*-state scale\" fill=\"currentColor\" stroke=\"none\">CH2]2\u2013/H2O-coordinated chelates lay 28 kJ mol\u20131 for Rh and 62 kJ mol\u20131 for Ir above Cp*/M\u2013OH. In the aqua adduct of the en Rh-complex, the H2O ligand is effectively outside the first coordination sphere , which together with rapid methyl rotation, would allow sequential deprotonation and deuteration of each of the ring methyl protons.Next we determined the energies of the structures after transfer of a proton from a Cp* methyl group to coordinated hydroxide, giving coordinated dianionic [Me Table S5. The com3 character of the CH2 carbon (H2O) scale\" fill=\"currentColor\" stroke=\"none\">CH2)]+. Thus, deprotonation of Cp* might introduce new pathways of activity including addition reactions of a bound fulvene, and redox reactions of rhodium.Remarkably, the comparison of the geometries of (a) free deprotonated Cp* ligand (optimized with CAM-B3LYP/CEP-31G), (b) deprotonated Cp* in the optimized structures of the complex, and (c) that of the neutral fulvene Cp* derivative (optimized with CAM-B3LYP/CEP-31G) suggest that the latter two are close in energy to each other, while the former reveals an spN,N\u2032-chelated ligand plays an important role in stabilizing the aqua/dianionic 2\u2013 intermediate in which H/D exchange can readily occur. Within the series of chelates of varying \u03c0-character of the N,N\u2032 ligands, the stability of the intermediates increases with increasing \u03c0-acceptor character of the ligand. When we increased the \u03c0-acceptor strength by adding strongly electron-withdrawing groups as in 1,2-diimino-4,5-dicyanobenzene, the energy difference decreased to only 13 kJ mol\u20131 above the OH adduct (Table S5).\u20131 higher in energy than that for the corresponding Rh complexes (Table S5I/IrI fulvene complex) is related to delocalization of the negative charge onto the N,N\u2032-chelated ligand via the d-orbitals of the metal. This is facilitated by the \u03c0-acceptor character of the N,N\u2032 ligand and is less effective for Ir than for Rh. Alternatively, the enhanced \u03c0-acceptor character of the N,N\u2032-chelate stabilizes the lower RhI oxidation state.Our calculations suggest that the \u03c0-acceptor strength of the Table S5. Thus, Dl-Glu-l-Cys-Gly) is prevalent in cells at millimolar concentrations, reactions of GSH were studied with 1 under conditions in which activation of Cp* ring methyls was expected. A solution of 1 and excess GSH at pH 7 gave rise only to MS peaks assignable to [(\u03b75-Cp*)Rh(bpy)(SG)]. Interestingly, the presence of GSH restricted, but did not prevent, ring methyl deuteration [(Cp*)Rh(bpy)(OH)]+/GSH, (c) [Rh scale\" fill=\"currentColor\" stroke=\"none\">CH2)(bpy)(OH2)]+/GSH, and (d) [Rh scale\" fill=\"currentColor\" stroke=\"none\">CH2)(bpy)]+/GSH/H2O, revealed [Rh(bpy)Cp*(SG)] as the most stable complex .Since ene\u2013thiol reactions are well known, and glutathione Rh(bpy)Cl]PF6, since with 2 mol equiv. of N-methylmaleimide present in 60% MeOD-d4/40% D2O, complete deuteration was not observed even after 7 days, rather MS peaks for all D1 to D15 sequential steps scale\" fill=\"currentColor\" stroke=\"none\">O\u00b7\u00b7\u00b7H\u2013OH bond of 1.68 \u00c5 rather than attacking the fulvene double bonds -linoleic acid, were used in attempts to trap the possible fulvene intermediate as [4+2] cyclo-addition Diels\u2013Alder adducts. These are both biologically-important, natural dienes. Isoprene is the most abundant hydrocarbon in human breath, with an estimated human production of 17 mg per day.Z,11E)-Linoleic acid is a common dietary conjugated fatty acid of much interest because of its potential health effects.[4+2] Diels\u2013Alder reactions with the exocyclic double bond of fulvene as dienophile have been reported in the literature.1 in 60% MeOD-d4/40% D2O at 310 K and isoprene was studied, since isoprene was expected to offer minimal steric hindrance for the reaction with the proposed exocyclicfulvene C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CH2. The ESI-MS spectra clearly indicated formation of a [4+2] cyclo-addition adduct, with the peaks at m/z = 497.1225 assignable to the RhI complex (13+H)+ in a RhI\u2013(Cp*H) complex.I system compared to RhIII, making a square-planar RhI (4d8) adduct accessible + . HR-MS d Fig. S17.m/z of 461.16 assignable to [13-Cl]+, as was also observed by ESI-MS.Adduct formation was also evident from LC-MS data Fig. S18, showing1H NMR studies. Time-dependent 600 MHz 1H NMR spectra of the reaction mixture of complex 1 and isoprene (10 mol equiv.) in 60% MeOD-d4/40% D2O showed a decrease in the intensity of the isoprene peaks with time, Fig. S19,13). The new peaks due to formation of the [4+2] cyclo-addition adduct are assigned in 1H COSY and NOESY data in RPMI-1640 cell culture medium supplemented with 10% fetal calf serum, a typical medium used for culture of cancer cells Rh(bpy)OH]+. The formation of the Diels\u2013Alder adduct from the fulvene species [ scale\" fill=\"currentColor\" stroke=\"none\">CH2)RhI(bpy)OH2]+ is energetically very favourable (106 kJ mol\u20131), while the corresponding end product [(adduct)RhI(bpy)]+ is presumably driven by entropic effects, with a calculated energy difference between it and the [(adduct)RhIII(bpy)](OH2) of only 3 kJ mol\u20131. The change of the oxidation state is suggested by the calculated Rh\u2013N bond lengths of 2.141 and 2.192 \u00c5 for the species with coordinated water, and 2.106 and 2.102 \u00c5 for the final product.The reaction of [(Cp*)Rh(bpy)Cl]ulations . The res1 with the conjugated fatty acid, -linoleic acid, under similar experimental conditions in water, and in RPMI-1640 cell culture medium supplemented with 10% of fetal calf serum, were studied. The HRMS and MS-MS data again clearly indicated the formation of the fulvene-diene cyclo-addition adduct scale\" fill=\"currentColor\" stroke=\"none\">CH2, or with the dienophile N-methylmaleimide, although they can control the extent of ring deuteration -linoleic acid formed readily in aqueous solution. They might easily form in cells, and influence the redox state of cells and biological activity of this class of organo\u2013rhodium anticancer complexes. The Diels\u2013Alder adducts formed readily in biological media, e.g. cell culture medium (Fig. S22 and S23x rings (Table S3Adducts with isoprene, an abundant diene in human breath, and with the common dietary diene conjugated (9 Table S3. The posThere are no conflicts to declare.Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "Using the \u201ccrystal sponge\u201d approach, weak organic electron donor molecules were impregnated and evenly distributed in a crystal of a metal\u2013organic framework (MOF), with the self-assembly of the donor\u2013acceptor pairs with electron acceptor ligands. The nanopores of the MOF confined them and induced a charge transfer phenomenon, which would not occur between donor and acceptor molecules in a bulk scale. N,N,N\u2032,N\u2032-tetramethyl-1,3-propanediamine (TMPDA), was realized in a structurally flexible metal\u2013organic framework, {Mn76(DMA)6}\u221e , with electron-accepting anthraquinone groups, generating two MOF guest charge transfer complexes: {Mn76(DMA)6(TTF)5} and {Mn76(DMA)4(H2O)2(TMPDA)7}. Using a mild impregnation procedure, single crystals of the target complexes were obtained via a crystal-to-crystal conversion, and the crystals were suitable for structural analysis. Single crystal X-ray analysis demonstrated the different arrangements of these intercalated donor molecules: some donor molecules interacted with the anthraquinone groups and formed infinite D\u2013A\u2013A\u2013D stacks, some appeared beside the anthraquinone groups but only formed donor\u2013acceptor pairs, and the remainder of the molecules simply filled the space. The charge transfer between the guests and the framework was spectroscopically confirmed, and the radical densities on the organic species were estimated using magnetic susceptibility measurements. Compared with a solid-state mixture of anthraquinone and donor molecules, the evenly distributed donor molecules in the micropores of the MOF resulted in a \u201csolid solution\u201d state and significantly promoted the degree of charge transfer between donors and acceptors. Such an encapsulation process may be adopted as a new strategy for post-modification of the electronic and magnetic properties of MOFs, as well as for generating new semiconducting charge-transfer complexes.A spontaneous entrapment of electron-donating small guest molecules, including tetrathiafulvalene (TTF) and A few elegant examples have been reported on this topic: Allendorf et al. reported the synthesis of TCNQ@HKUST-1 2}), and the conductivity of HKUST-1 was improved by six orders of magnitude with the infiltration of TCNQ.et al. illustrated VOC (volatile organic compound) detection with thin-film devices made of two-dimensional conductive MOFs.et al. demonstrated the iodine oxidation of TTF -based MOFs, and found that the oxidation modulated the spin-crossover property of the MOF.Metal\u2013Organic Frameworks (MOFs) are a series of polymeric coordination compounds with intrinsic porosity and crystallinity.To understand how guest molecules have tuned the electron transfer ability of MOFs, an important lesson should be learned from the studies of charge transfer molecular conductors such as TTF\u2013TCNQet al. have provided a straightforward yet universal strategy for this goal\u2014namely, the \u201ccrystal sponge\u201d strategy.via this mild guest inhalation process. Such a gentle self-assembly process may not only lead to the impregnation of structurally resolvable guest molecules in a confined space, but may also provide an environment for maximizing the interaction between donor\u2013acceptor pairs and optimizing their packing motifs 6(DMA)6} (Mn-MOF), that was previously reported by our group,76(DMA)6(TTF)5} and {Mn76(DMA)4(H2O)2(TMPDA)7} . They were composed of organic donor guests and an Mn-MOF with electron-accepting anthraquinone (AQ) groups. The crystal structures were successfully acquired through a spontaneous crystal-to-crystal transformation using the \u201ccrystal sponge\u201d method, and the charge transfer between the MOF scaffold and organic guest donor molecules was evidenced by a series of physical property measurements.Fujita g motifs . Herein,The preparation of a Mn-MOF was first described by our group.1 and 2. Single crystal XRD data collection was first carried out on these crystals using a Rigaku RA-micro7 Mercury CCD diffractometer equipped with a Mo K\u03b1 light source (\u03bb = 0.71073 \u00c5) at \u2013150 \u00b0C. Two hemispheres were scanned for each crystal. The frames were integrated with Rigaku Crystal Clear 2.0, and the solving and refinement of structures were completed with Rigaku Crystal Structure 2.0. Based on the crystallographic results from single crystal XRD, we concluded that the data quality of 2 was not adequate for scientific publication. To overcome this problem, synchrotron radiation diffraction was also performed on a crystal of 2 at the Aichi Synchrotron Radiation Center, with beamline BL2S1 (\u03bb = 0.75000 \u00c5). The instrument conditions of the beamline were formerly reported,2. The powder X-ray diffraction patterns were collected on a Rigaku MultiFlex X-ray diffractometer, and the crystals of the as-prepared Mn-MOF guest complexes were roughly ground by sandwiching them between two glass slides to create a crystalline powder. A simulated powder diffraction pattern was generated from the crystal structure using Diamond 3.0 crystallographic imaging software for comparison.X-ray diffraction analysis (XRD) was performed on the single crystals of 4 powder with a weight ratio of 1\u2009:\u200910. The spectrum was taken within a wavelength range of 200\u20131400 nm. To verify the effect of micropores on the charge transfer degree between the donor molecules and functional groups on the MOF, we also performed a reaction between the donor molecules and anthraquinone-containing ligands by dissolving them in hot DMF with a 1\u2009:\u20091 molar ratio. The DMF solution was kept at 120 \u00b0C for 1 hour and the solid-state products were collected by evacuating all the DMF solvent. These products were also examined by solid-state UV-vis diffuse reflectance spectroscopy under the same conditions.A series of physical properties were characterized to investigate the existence of charge transfer phenomena between the host scaffold and the encapsulated guest molecules. The as-prepared Mn-MOF, organic donor molecules, and Mn-MOF guest crystals were analysed by solid-state UV-vis-NIR diffuse reflectance spectroscopy using a JASCO V-570 UV-Vis-NIR spectrophotometer. Due to the limited amount of Mn-MOF guest crystals, the diffuse reflectance measurements were performed by mixing the crystals with BaSO\u03c7\u2013T plot data sets were fitted using the Curie\u2013Weiss law, and the Curie constants were used to calculate the magnetic contribution of the organic radicals. To confirm the presence of organic radicals and exclude the contribution of paramagnetic impurities, temperature variable electron paramagnetic resonance (EPR) measurements were also performed on Mn-MOF and Mn-MOF guest crystals. A JEOL JES-FA200 continuous wave X-band EPR instrument was used to complete this task.The charge transfer degree between the donor molecules and anthraquinone groups was evaluated by temperature variable magnetization measurements, which were performed with a superconducting quantum interference device (SQUID) Quantum Design MPMS system. DC measurement was performed with a magnetic field of 1000 Oe, and the temperature range was 2\u2013300 K. The 2Cl2 solution of these donors and evaporating the solvent. This procedure yielded MOF guest complexes. Fortunately, high quality single crystals could be obtained for both charge transfer complexes, which makes it easy to inspect the structural features of the donor\u2013acceptor pairs in the Mn-MOF guest crystals. After the insertion of the donor molecules, the skeleton of Mn-MOF was not significantly perturbed; however, astonishingly complicated arrangements of organic donor molecules were observed in the structures of both 1 and 2 scale\" fill=\"currentColor\" stroke=\"none\">C double bond are still coplanar, while the five-membered rings are bent along the dithiole line with a torsion angle of \u223c30\u00b0. Such distortion should be a result of weak attractive intermolecular S\u00b7\u00b7\u00b7S interactions, as the S\u00b7\u00b7\u00b7S distance between the adjacent TTF molecules is 3.916 \u00c5. PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C double bonds of TTF molecules were also examined, since these bond lengths are benchmarks for predicting the oxidation states of TTF species. Interestingly, abnormalities were observed in various types of TTF molecule in the structure. Because they are adjacent to an AQ group, the Type I and Type III TTF molecules were expected to possess a positive charge, and the central C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond was expected to be elongated by the single bond component from the resonance structure compared to that in the neutral, space-filling Type II TTF molecules. In reality, the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond lengths of Type I and Type III TTF were 1.334 \u00c5 and 1.335 \u00c5, respectively, and Type II TTF had a central C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond length of 1.383 \u00c5. As a reference, this bond length was 1.337 \u00c5 in neutral TTF and 1.369 \u00c5 in TTF\u2013TCNQ (\u03b4TTF = +0.59).1, it is inappropriate to simply predict the charge transfer degree with bond lengths, and the bond lengths of impregnated TTF are determined by not only the oxidation states, but also the steric effects arising from the limited space in the nanopores in the MOF structure.As shown in 2 .1 and 2 showed a self-arranged placement of donor molecules in these MOF\u2013guest complexes. With the \u201ccrystal sponge\u201d approach, donor molecules will automatically find acceptors, forming columns that are potentially capable of electron transfer.A few of the aforementioned structural features were also observed in compound 2 . For inse Fig. S4. This fi1 and 2 have provided quite a few hints of charge transfer taking place between AQ groups and donor molecules, evidence from physical measurements is still essential to prove the existence of organic radical species. We therefore conducted solid-state diffusive reflectance UV-vis spectroscopy measurements on the original MOF, donor molecules and MOF\u2013guest complexes. By overlaying the absorbance spectrum of the complexes with those of the donor molecules and Mn-MOF, broad new bands were observed for both 1 and 2 at 740 nm and 790 nm, respectively cations. This small Curie constant could be simply explained by the antiferromagnetic (AF) exchange coupling between the Mn(ii) cations within one secondary building unit (SBU). To simplify the analysis, we decided to use the giant spin approximationS = 13/2, g = 2.04 and zJ = \u20130.177 cm\u20131 0.15+ Mn-MOF0.15\u2013 and (TMPDA7)7+ Mn-MOF7\u2013. Although this estimation ignored the potential AF interactions between AQ\u02d9\u2013 and the donor\u02d9+, as well as the AF interactions between metal clusters and organic radicals, it was still qualitatively shown that the impregnated TTF molecules were almost neutral, and the main character of the TMPDA-AQ charge transfer pairs was ionic.Although the crystal structures of ectively . These br Fig. S9. For the1 and 2 were fitted to be \u201315.0 K, \u201315.5 K and \u201321.4 K, respectively. For Mn-MOF and 1, it was suggested that there were antiferromagnetic interactions between the SBUs. Since the skeleton of the framework in all three compounds is nearly identical, a slightly increased critical temperature of 2 might be a sign of the additional antiferromagnetic interactions between the SBUs and organic radicals. We confirmed this assumption with temperature variable EPR measurements , the line width was notably broadened, suggesting that an increased population of excited states was generated by the interaction between the SBUs. A similar feature was also noticed for 1, since the TTF species were almost neutral and contributed no perturbation to the spin states of the Mn7 cluster. In contrast, although the g value was not changed, the line width of 2 was slightly narrower at high temperature, and only slightly broadened when the temperature was lower than the Tc and organic radicals (S = 1/2). Hence, the number of microstates generated by the interactions was dramatically reduced and the 1st oxidation potential of TTF and TMPDA (E1/2 = 0.45 V and 0.37 V vs. EAg/AgCl), the potential differences between the donor and acceptors were larger than 1.0 V in both cases. According to empirical rules, the combination of these donor molecules and AQDC molecules will not lead to a spontaneous formation of charge transfer salts but rather neutral complexes.The cyclic voltammetry experiments of the AQDC ligand, TTF and TMPDA Fig. S2 uneartheBy post-synthetically inserting donor molecules into a flexible MOF with electron-accepting groups, we successfully achieved two MOF guest charge transfer complexes, and the structures of these complexes could be fully resolved using the \u201ccrystal sponge\u201d approach. The nanopores or nanochannels in this MOF played a critical role in this process. The confinement of functional species in a limited space facilitated the guest-to-host electron transfer and yielded ionic charge transfer complexes, even when the redox potential gap between the donor and acceptor species was vast. Meanwhile, an even distribution of the donor molecules in the MOF structure maximized the contact between the donor and acceptor species, which also boosted the degree of charge transfer. The even distribution and confinement could be synergistically achieved in one MOF material, and this synergic effect can be summarized as \u201cMOF-induced charge transfer\u201d (see computational discussion in Section S11, ESIThere are no conflicts to declare.Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "Intramolecular N-to-S or N-to-O acyl shifts in peptides are of fundamental and practical importance, as they constitute the first step in protein splicing and can be used for the synthesis of thioester-modified peptides required for native chemical ligation. anti to the carbonyl oxygen, as in a cis amide. Despite the importance of such reactions, an understanding of this geometric restriction remains obscure. Here we argue that the empirical requirement for positioning the nucleophile is a stereoelectronic effect arising from the ease of approach of the nucleophile to a carbonyl group, not ground-state destabilization. DFT calculations on model amides support our explanation and indicate a significant decrease in both the transition-state energy and the activation energy for a cis amide. However, the approach of the nucleophile must be anti not only to the carbonyl oxygen but also to the nitrogen. The direction of approach is expressed by a new, modified B\u00fcrgi\u2013Dunitz angle. Our data shed light on the mechanisms of acyl shifts in peptides, and they explain why a cis peptide might be required for protein splicing. The further implications for acyl shits in homoserine and homocysteine peptides and for aldol condensations are also considered.Intramolecular N-to-S or N-to-O acyl shifts in peptides are of fundamental and practical importance, as they constitute the first step in protein splicing and can be used for the synthesis of thioester-modified peptides required for native chemical ligation. It has been stated that the nucleophile must be positioned N-terminal residue),8Protein splicing is the posttranslational excision of an internal polypeptide sequence, the intein, followed by ligation of the C-terminal and N-terminal segments, thereby generating the spliced extein.anti to the carbonyl oxygen for the N-to-S or N-to-O acyl shift to take place.cis (E) amide, even though the product ester is the same from either . PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O. The difference between cis and trans must be sought elsewhere.These acyl shifts are classified as allowed 5-trans amide reacts more slowly because its geometry restricts the nucleophilic O or S from approaching the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O from the preferred direction. To test this proposal, we have calculated structures and energies for the intramolecular reactions of cis and trans acetamides 1 with O, S, and Se anions , via transition states 2, leading to tetrahedral intermediates 3 /6-311++G calculations were performed with Gaussian 09 software Revision C.01.26\u03c6BD, PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O directions scale\" fill=\"currentColor\" stroke=\"none\">O direction and the projection of the C\u2013Nu direction onto the plane containing the C and the two attached groups , which we designate as \u03c6\u2032BD.However, these angles are not appropriate for specifying the preferred direction of approach to an amide. Whereas the nucleophile approaches \u03c6\u2032BD was calculated as follows: first Xnormal, the normal to the OCN plane containing C, O, and N, was calculated as (XO \u2013 XC) \u00d7 (XN \u2013 XC), the cross product between the C\u2013O and C\u2013N vectors. Next PNu, the projection of XNu onto the NCO plane, was calculated as XNu + Xnormal/Xnormal \u00d7 Xnormal. Finally, cos(180\u00b0 \u2013 \u03c6\u2032BD) was evaluated as the normalized dot product (XNu \u2013 XC)(PNu \u2013 XC)/|XNu \u2013 XC||PNu \u2013 XC|.The modified B\u00fcrgi\u2013Dunitz angle cis and trans stereoisomers of extended-chain amide 1, tetrahedral intermediate 3, and transition state 2 connecting them, for X = O, S, and Se, along with the activation energies Ea = E(2) \u2013 E(1). For X = S and Se either HF or HCl, respectively, was coordinated to the carbonyl oxygen in order to converge addition to the amide, which otherwise is thermodynamically unfavorable because RS\u2013 and RSe\u2013 are such stable anions and because C\u2013S and C\u2013Se bonds are weak. Besides, the coordinated acid can mimic the \u201coxyanion hole\u201d, which stabilizes the tetrahedral intermediate in some enzyme-catalyzed reactions.trans amides are a reasonable 2 kcal mol\u20131 more stable than the cis, and the open-chain amides 1 are calculated to be more stable than the high-energy tetrahedral intermediates 3. Moreover, nearly the same results for X = O are obtained with the M06-2X method, which accounts for dispersion,Because the coordination to HF or to HCl, the use of anionic nucleophiles, and the use of PCM are all devices to facilitate the calculations, the absolute energies in cis transition state for both X = O (without HF) and X = S (with HF), by \u223c2.5 kcal mol\u20131. The differences in activation energies are slightly larger, 4\u20135 kcal mol\u20131. These differences thus reproduce the faster cyclization seen for cis amides. However, the faster cyclization is not merely because of the destabilization of a cis amide, but because of the lower energy of the cis transition state, consistent with the Curtin\u2013Hammett Principle. The case of X = Se, omitted from 3. The lengthening is greater for 2trans, especially for X = O or N-methylacetamide HCl. The smaller angles in the cyclic transition states and especially in the trans transition states represent a greater displacement of the nucleophile from the \u03c0* MO of the amide group is due to the greater proportion of a stereoisomer that cyclizes more rapidly, the reason for the faster cyclization is the ease of nucleophilic approach. Moreover, the approach is not simply anti to the carbonyl oxygen, but anti to both O and N.The data in trans amide is a consequence of a greater restriction on the ability of the nucleophile to reach the carbonyl carbon, as manifested by \u03c6\u2032BD < optimum. This is a constraint of the five-membered ring being formed. It is an unusual example of angle strain that differs between cis and trans, even though they are both 5-exo-trig.Approach control has previously been recognized as arising from steric repulsions in the transition state, as in hydride reduction of cyclohexanones.This difference in angle strain is recognizable even with a simple molecular-model kit. Therefore we expect that the order of relative energies in trans is substantially higher than that for the cis. This is simply because of the ground-state steric destabilization of the cis, as originally proposed to explain its greater reactivity. However, the data also show that the transition-state energies for cis and trans differ by less than 1 kcal mol\u20131, much less than the 4\u20135 kcal mol\u20131 for N-to-O and N-to-S shifts. Those shifts of a trans amide are retarded by the inability of the nucleophile to reach the carbonyl carbon. However, because the C\u2013Se bond is longer, the nucleophilic selenium has less difficulty in reaching the carbonyl carbon. However, according to the values in \u03c6\u2032BD is again significantly smaller in the trans transition state, just as for X = O or S, so that this parameter does not reflect the slight difference in transition-state energies. This may be a consequence of C\u2013Se\u2013C angles in both transition states that are constrained near 80\u00b0, thereby distorting the five-membered ring.The N-to-Se acyl shift in a selenocysteine residue provides an instructive contrast. According to the calculated energies in trans mono(selenylethyl) peptide might suffice, although it would be retarded by the lack of the ground-state steric destabilization of the cis isomer.As a corollary, there may be no strong constraint on the approach of the selenium to the carbonyl carbon. Although a bis(selenylethyl) peptide readily undergoes a N-to-Se acyl shift,4 (X = O) or 4 HF (X = S) are nearly equal for cis and trans amides \u03c6FL is calculated to be 55\u00b0 for cis and 50\u00b0 for trans, not far from the 52\u00b0 for OH\u2013 addition to N-methylacetamide. With a six-membered ring there is little restriction on the ability of the nucleophile to reach the carbonyl carbon. Indeed, the N-to-S acyl shift in a homocysteine residue is facile, without any necessity for N-alkylation or population of the cis amide.36In further contrast, according to the data in 7 and its derivatives to the corresponding 8 are known,9 to the corresponding 10. This could be due simply to a lack of demand for 10, but it can also be explained by the difficulty for the enolate carbon of 9 to reach the carbonyl carbon, whereas 7 can twist to allow its enolate carbon to achieve the preferred approach to the carbonyl, as described by the B\u00fcrgi\u2013Dunitz and Flippin\u2013Lodge angles.These results also have implications for aldol condensation . Althougcis acylcysteine or acylserine toward N-to-S or N-to-O acyl shift. The reactivity difference between cis and trans can be attributed to the ease of approach to the carbonyl carbon by the nucleophile, not to ground-state destabilization. This represents an extension of Baldwin's rules to two distinguishable cases of 5-exo-trig ring closures. Moreover, there is no large reactivity difference in an N-to-Se acyl shift or in 6-exo-trig ring closures. We thus have provided a better understanding of the geometric constraints required for the N-to-S or N-to-O acyl shift in cysteine and serine peptides. We expect that this information will enable better design of peptides, especially ones containing homocysteine, that generate thioesters useful for chemical ligation techniques.DFT calculations can reproduce the greater reactivity of a There are no conflicts of interest to declare.Supplementary informationClick here for additional data file."} +{"text": "Sham electroacupuncture (EA) control is commonly used to evaluate the specific effects of EA in randomized-controlled trials (RCTs). However, establishing an inert and concealable sham EA control remains methodologically challenging. Here, we aimed to systematically investigate the sham EA methods. Eight electronic databases were searched from their inception to April 2015. Ten out of the 17 sham EA methods were identified from 94 RCTs involving 6134 participants according to three aspects: needle location, depth of needle insertion and electrical stimulation. The top three most frequently used types were sham EA type A, type L and type O ordinally. Only 24 out of the 94 trials reported credibility tests in six types of sham EA methods and the results were mainly as follows: sham EA type A (10/24), type B (5/24) and type Q (5/24). Compared with sham EA controls, EA therapy in 56.2% trials reported the specific effects, of which the highest positive rate was observed in type N (3/4), type F (5/7), type D (4/6) and type M (2/3). In conclusion, several sham EA types were identified as a promising candidate for further application in RCTs. Nonetheless, more evidence for inert and concealable sham EA control methods is needed. A randomized controlled trial (RCT) has been the cornerstone of medical clinical research since the first RCT paper entitled \u201cStreptomycin treatment of pulmonary tuberculosis: a Medical Research Council investigation\u201d was published in 1948RCTs for acupuncture appeared in 1970s89Electroacupuncture (EA) is an extension technique based on traditional acupuncture combined with modern electrotherapy1113Eight electronic databases, including Cochrane Controlled Trials Register, PubMed, EMBASE, AMED, China National Knowledge Infrastructure (CNKI), VIP Journals Database, Wanfang database, and Chinese Biomedical Database (CBM) were searched from their inceptions to April 2015. The search terms were confined to \u201cElectroacupuncture\u201d AND \u201csham acupuncture OR placebo acupuncture\u201d AND \u201crandomized controlled trial (RCT)\u201d. All searches were limited to studies on human.RCTs concerning the effects of EA on any kind of diseases with at least one control group receiving sham EA were included, regardless of publication status and languages. Quasi-RCTs and non-RCTs were excluded.The studies were eligible if EA therapy alone or adjunct therapy were given in treatment group and secondly, if sham EA or any type of faked manipulation mimicking real EA in aspects of acupoint, penetration and electro-stimulation were given in control group. There were no restrictions on needle parameters or intensity, frequency and mode of stimulation. Studies that compared EA with transcutaneous nerve electrical stimulation (TNES), another acupuncture plus sham EA or placebo medications were excluded. If three or more groups were designed in one study, only real EA versus sham EA groups were included.Two authors reviewed the titles and abstracts of the potential references independently. All the potentially relevant studies were marked and their full articles were retrieved. Further examinations were carried out to make a final selection decision. The same two authors performed the data extraction independently for the predefined items: author, year, country, EA indications, sample size, the characteristics of interventions, outcome measures, results and dropouts. The disagreements were resolved through consulting a third part (GQZ).Two authors (ZXC and YL) performed the methodological quality assessment of each included trial independently based on the Cochrane Collaboration\u2019s tool for assessing risk of biaset al.The sham EA methods used in each control group were examined and the details were extracted according to three respects: needle location, depth of needle insertion and electrical stimulation. Partially based on the previous sham acupuncture type I~V classification published by Dincer Considering wildly varying outcome measures across different disease conditions, treatment efficacy was evaluated for each study according to the modified method based on a previous publicationThe credibility test was formally performed in validation studies to assess the blinding effect of sham acupuncture based on credibility questionnaire and statistical analysis1516A total of 679 potentially relevant articles were identified. By reviewing titles and abstracts, 374 papers were excluded for at least one of following reasons: (1) not clinical trials; (2) case report, comment, review, letter, news or editorial; (3) not in contrast with sham EA; (4) lack of EA intervention. After examining the full content of the remaining 305 articles, we removed 211 records, of which 127 articles were due to lack of sham EA controls, including electro-acupuncture (14 studies), manual acupuncture (13 studies), TNES (7 studies), other treatment 69 studies) or no treatment 24 studies); 49 articles removed for lack of real EA groups, with their target intervention designed as manual acupuncture (10 studies), TNES 38 studies) or periosteal stimulation therapy (PST) (1 study); 10 articles were not RCTs; 13 articles were double publications; 12 articles were cross-over design. Ultimately, 94 studies181920212223 studies;25262728293031323334353637 studies 394041424344454647484950515253545556575859606162636465666768 studies 707172737475767778798081828384858687888990919293949596979899100101102103104105106107108109110The 94 included articles were published from 1992 to 2015. Among them, 5 studies29549095222425273031384547525356575867687172737477788183849293971041051061101819202123262832333435363739404142434446484950515559606162636465666970757679808285868788899194969899100101102103108181921242627313336374042454751525660666772768284889092969710311046507374868994252829346210523535965683249546198996495223938356379834377deqi\u201d sensation was required in 65 real EA groups18192021222324252629303133343536394041434447485051565758596061626364666769717577787980818283858687909193989910010110210410510610961782021235260636719202122262728313233343537394042434446475051586061626465667071777879838589921001041052429545659808286879095989910210710833373839464750515272737782848688899495969710010110227467276777989909727467276778997EA treatment alone was adopted in 55 trials1920212223242526282930313435363738394243535558606166686971727577787980838485868788899091959899100101103104107108110244754384361639899254666899010510610826495476959697Ten different types of sham EA methods used in the trials were identified as follows: (1) sham EA type A were used in twenty-six control groups1819202122313337406066677374768194969710210310410610810923245965848828444749517529436264723861717898992627394346485053546177798589989942686383903032354145565758708086879125343638525563100107For the needle location, 48 sham EA groups252627303234353638394142434546485052535455565758616368697077798083858687899091929598991001011073861717898992627283839424344464748495051535461686971757778798285899899293032354143455657586263647072808386879091929321232531365559636566243719204060671822333876737494961021041051061071081091108423242627383943464850535459616365717778798384858889909598991011819202122252829303132333435363738404142434445474951525556575860626364666768697072737475768081828687919293949697100102103104106107108109110The number of items complied with the criteria varied from 3/8 to 7/8 with the average of 5.2. All 94 studies declared randomization and 63 studies reported the details. Among them, 49 studies18192223242527293536373842434445474951555960616263646566677071728188909192939697989910210610710810911032404150525769737485838718192023252930353637434445555758606163646566676971727679818486889091939899105106107109110222325343645525759606365728188971021031051061071082530456065697980919293979899103105106181920212224252627282930313233343536373839404143444647495051525354555657585960616263646566676870717273747576777879818283848586878889909192949596971001011021031041051081091102342454869809398991063639414244596364757980869193981819202122232425262728293031323334353637383940414243444546474849505152535455566061626364656667687072737475767980838485868788899192939495969799101103104105109575859697881829098102106107108Only 24 out of the 94 studies reported the credibility of blinding in participants by conducting the creditability test in six types of sham EA methods. Twenty-three studies222325343645525759606365728188971021031051061071082260668197102103106108235965882534365245All 94 studies involving 105 comparisons of real and sham EA groups provided the information for between-groups analyses. Among them, 59 real EA groups18192122242830333435373840414243444849515354575960616263646569707172737577787983848687889091929495979910010450Compared with sham EA controls, EA therapy in about 56.2% (59/105 comparisons) of comparisons reported the specific effect. Correspondingly, the real EA was superior to sham EA for type N , type F , type D and type M . The lowest percentage of positive efficacy result was 44.4% (8/18 comparisons) in sham EA type L. The positive rate of efficacy for the three most often used sham EA methods were 50% (13/26 comparisons) for sham EA type A, 44.4% (8/18 comparisons) for sham EA type L and 64.3% (9/14 comparisons) for sham EA type O.The type of sham EA methods varied across different EA indications. The sham EA type A was most commonly used in RCTs for pain, anesthesia and osteoarthritis. The sham EA type D and sham EA type Q were applied mainly in stroke studies. The sham EA type B was commonly applied to RCTs on depression. The sham EA type L and sham EA type O were commonly performed in trials on obesity. The sham EA type F and sham EA type L were commonly used in studies on primary dysmenorrhea.To our knowledge, this is the first systematic analysis to address sham EA methods in RCTs. The numbers of publications and sham EA methods have been increasing every decade. We summarized seventeen kinds of sham EA methods according to three aspects as needle location, depth of needle insertion and electrical stimulation, whereas only ten types of sham EA methods were identified from 94 included RCTs involving 6134 participants. The three predominant types of sham EA methods used were sham EA type A, type L and type O ordinally. Only 24 out of 94 trials reported credibility test with the results of 23 success and 1 failure using six types of sham EA methods mainly as follows: sham EA type A (10/24 with 1 failure), type B (5/24) and type Q (5/24). The remaining 3 sham EA methods were only tested in 4 trials. About 56.2% of comparisons provided the evidence of specific effect of EA therapy, and the four types of Sham EA controls with highest positive rate of efficacy result were type N , type F , type D and type M ordinally. However, all types of Sham EA controls were used in a small number of trials. Thus, the evidence was insufficient to recommend any type of sham EA control despite of the high positive rate. The sham EA control was frequently used in RCTs for pain, anesthesia, stroke, depression, obesity and primary dysmenorrheal/menstrual pain, suggesting that these diseases are particularly worthy of further EA RCTs.et al.et al.The ideal design of sham acupuncture method remains methodologically challenging2627The main purpose of RCTs on EA is to evaluate its specific effect. An optimal sham acupuncture technique must be biologically inactive and psychologically credible181921202123526063671822et al.The top three types of sham EA methods used were sham EA type A, type L and type O. The most frequently applied sham EA method was type A, accounting for a popular belief in its inertness based on its absence of key EA components as needle stimulation and electrical stimulation as well as its indistinguishable manipulation on same therapeutic acupoints. In the present study, the validation of credible participant blinding of this sham EA type was reported by most credibility tests. The debate emerged over the past decades over the inertness of non-penetrating procedure since the slight acupressure effects and physiological activity might be evoked by the tactile stimulation from blunt needle tips even without skin penetration112Sham EA type Q is deemed to be the most inert type of sham EA control because it avoids all therapeutic components, which also probably makes this sham method perceptually and operationally distinguishable from real EA intervention and to some extent results in problematic credibility of blinding in participant. In the present study, the credibility of participant blinding of this sham EA type was endorsed by five studies with credibility test25343652et al.et al.et al.et al.et al.et al.The second commonly used sham EA method was type L. It was found that the differential effects of real EA and sham EA, which were attributed to point location, was not consistent across studies and conditions within this sham EA type, suggesting that EA on non-acupoints might be efficacious as EA on therapeutic acupoints. Furthermore, the improvements from baseline were also observed by Sahin et al.et al.et al.The third commonly used sham EA was type O. During the procedure, the shallow insertion was applied to simulate deep skin penetration and to ensure the blinding of participant. In the present study, the validation of participant blinding of this sham EA type was endorsed by two studies4532465657et al.123124In the present study, six types of sham EA method were reported as successful in blinding. However, further investigations are needed for confirmation, since half of the tested types of sham EA controls were reported in a small number of trials. It should be noted that studies included did not provide sufficient evidence of blinding in acupuncturist. Vase RCTs are generally recognized as the gold standard for the efficacy of clinical interventions by excluding the non-specific effect via a placebo controlIn the present study, 43/105 comparisons reported that EA has no specific effects compared with sham EA controls202324252627293132363943444546475255565863666768768081828589939698101102103105106107109127There are several weaknesses in the present study. Firstly, the search and screen procedure were limited to randomized, parallel-controlled trials published in English. Thus, those trials with cross-over design or published in other than English language were omitted. Secondly, with the aim of evaluating the sham method in RCTs on EA, a generous criterion was established to select eligible studies. Therefore, it was not easy to examine the specific effect of EA by data synthesis from different outcomes and indications because of the heterogeneity of trials. Finally, the reported credibility test addressed blinding effects in participant rather than in both participant and acupuncturist. The credibility tests were not reported in all studies and the number of studies using sham EA types was small, and therefore the conclusion should be interpreted with cautions.Ten types of sham EA methods were identified based on our scheme classification. Generally, sham EA type A, type L and type O were frequently used. Yet, further clinical trials are recommended to maintain standard methodology of concealable and inert placebo EA techniques. Only 24 out of 94 trials were reported as positive credibility test in six types of sham EA methods, where sham EA type A, type B and type Q were highly practiced. It is worthy to study further about the importance of concealable sham EA types. EA therapy in approximately, 56.2% of comparisons provided the specific effects. The four types of sham EA represented the highest positive rate of efficacy results. However, progressive evidences on specific effects are mandatory. The sham EA control was observed frequently in pain, anesthesia, stroke, depression, obesity and primary dysmenorrhea RCTs. Also, broader studies in these predominant diseases are advised.How to cite this article: Chen, Z. et al. Sham Electroacupuncture Methods in Randomized Controlled Trials. Sci. Rep.7, 40837; doi: 10.1038/srep40837 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations."} +{"text": "FeIV=O and/or FeV=O intermediates are suggested to be involved in water oxidation with [NH4]2[CeIV(NO3)6], NaIO4, or Oxone catalyzed by [FeIII(L1)Cl2]+ (1) on the basis of spectroscopic measurements and DFT calculations. III(L1)Cl2]+ pyridinophane) complex is an active catalyst for the oxidation of water to oxygen using [NH4]2[CeIV(NO3)6] (CAN), NaIO4, or Oxone as the oxidant. The mechanism of 1-catalysed water oxidation was examined by spectroscopic methods and by 18O-labelling experiments, revealing that FeIV PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O and/or FeV PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O species are likely to be involved in the reaction. The redox behaviour of 1 and these high-valent Fe PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O species of L1 has been examined by both cyclic voltammetry and density functional theory (DFT) calculations. In aqueous solutions, the cyclic voltammograms of 1 at different pH show a pH-dependent reversible couple (E1/2 = +0.46 V vs. SCE at pH 1) and an irreversible anodic wave (Epa = +1.18 V vs. SCE at pH 1) assigned to the FeIII/FeII couple and the FeIII to FeIV oxidation, respectively. DFT calculations showed that the E value of the half reaction involving [FeV(L1)(O)(OH)]2+/[FeIV(L1)(O)(OH2)]2+ is +1.42 V vs. SCE at pH 1. Using CAN as the oxidant at pH 1, the formation of an FeIV PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O reaction intermediate was suggested by ESI-MS and UV-vis absorption spectroscopic measurements, and the rate of oxygen evolution was linearly dependent on the concentrations of both 1 and CAN. Using NaIO4 or Oxone as the oxidant at pH 1, the rate of oxygen evolution was linearly dependent on the concentration of 1, and a reactive FeV PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O species with formula [FeV(L1)(O)2]+ generated by oxidation with NaIO4 or Oxone was suggested by ESI-MS measurements. DFT calculations revealed that [FeV(L1)(O)2]+ is capable of oxidizing water to oxygen with a reaction barrier of 15.7 kcal mol\u20131.The macrocyclic [Fe E = +1.23 V vs. NHE at pH 0) involving the simultaneous removal of four electrons and four protons from two water molecules to give one oxygen molecule.Water oxidation is an energetically uphill reaction (ii) and organometallic iridium(iii) complexes, and also complexes of 1st-row transition metals such as manganese, iron and cobalt bearing chelating N and/or O ligands, have been reported for water oxidation.3In the literature, many examples of molecular catalysts, particularly polypyridyl ruthenium(4]2[CeIV(NO3)6]) or NaIO4,4,Because of the high earth abundance of iron and the recent impressive advances made in the isolation and characterization of non-heme iron\u2013oxo complexes, notably by Nam, Que, and co-workers,5 ligands,Me2Pytacn, tpa, and bqen (cis labile sites (the abovementioned 14-TMC ligand forms trans complexes such as trans-[Fe(14-TMC)(OTf)2] which was found to be unreactive for catalytic water oxidation with CAN or NaIO4 (II(mcp)(OTf)2] + NaIO4\u2019 system reported by Costas, Lloret-Fillol, and co-workers formed oxygen with a turnover number (TON) of up to >1000 at pH 2.iv) scale\" fill=\"currentColor\" stroke=\"none\">O) intermediates supported by Me2PytacnIV(bqen)(O)(OH2)]2+ may be involved in O\u2013O bond formation,II\u2013Me2Pytacn system favoured FeIV PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O reactive intermediates. PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019FeIV\u2013O\u2013CeIV species in the water oxidation with CAN catalysed by [FeII(mcp)(OTf)2].v) scale\" fill=\"currentColor\" stroke=\"none\">O) species are proposed to be the active intermediates directly responsible for O\u2013O bond formation in water oxidation reactions,III\u2013TAML,II\u2013Me2Pytacn,II\u2013mepV PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O species, which were generated by oxidation with peracids, H2O2 or tBuOOH in organic solvents, have been reported in the literature.V(TAML)(O)]\u2013,V(Me2Pytacn)(O)(OH)]2+,V(L)(O)(S)]3+ .V(TAML)(O)]\u2013 (H2O)]\u2013 with \u2018[RuII(bipy)3]2+ + Na2S2O8\u2019 in 50% MeCN\u2013borate buffer mixture, was reported to be an active intermediate in the [FeIII(TAML)(H2O)]\u2013-catalysed photochemical water oxidation.V PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O intermediates with neutral chelating N ligands and bqen , which aor NaIO4 b). Notab(O)]\u2013 I, generateands II, remain eN,N\u2032-Dimethyl-2,11-diazapyridinophane Cl2]+ (1).III(L1)Cl2][FeCl4] (1\u00b7FeCl4) is an efficient catalyst for the cis-dihydroxylation of alkenes using Oxone as the oxidant, and the generation of an [FeV(L1)(O)2]+ intermediate in the reaction was inferred from high resolution ESI-MS analysis and DFT calculations.1 toward water oxidation. It is noted that ligand L1 has also been employed for developing the oxidation chemistry of other transition metal complexes, including [OsIII(L1)(OH)(OH2)]2+, which can catalyse the cis-dihydroxylation of alkenes by H2O2via a reactive [OsV(L1)(O)(OH)]2+ intermediate,II(L1)(Me)2], which reacts with oxygen to generate [PdIII(L1)(Me)2(OO\u02d9)] followed by protonation to give [PdIV(L1)(Me)2(OOH)]+.2O)2]2+ and electrochemical oxidation of water catalysed by [Mn(L1\u2032)(H2O)2]2+ (L1\u2032 = the N-tBu counterpart of L1) have been reported as well.17hane L1, ,15a a neIII(L1)Cl2]+ (1) for water oxidation with Oxone, as well as CAN and NaIO4, under mild conditions, together with mechanistic studies by means of high-resolution ESI-MS, UV-vis absorption spectroscopy, 18O-labelling experiments, kinetic studies, cyclic voltammetry, EPR analysis, and DFT calculations. During the course of this study, Sun and co-workers communicated their findings on water oxidation with CAN catalysed by [FeII(L1)(MeCN)2]2+.V PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O species responsible for O\u2013O bond formation in water oxidation catalysed by Fe\u2013L1 systems.In the present work, we report the use of [FeCl4] (1\u00b7FeCl4) exhibited the best performance, affording oxygen with TONs of up to 41 and 32 in 0.1 M HNO3 and in pure water, respectively, after 30 minutes with CAN (840 equiv.) as oxidant .At the outset, a series of iron complexes bearing N1-catalysed water oxidation was investigated by using a mixture of H216O and H218O as the medium and by analysing the gaseous product(s) using GC-MS. With CAN as oxidant, the gaseous products obtained in the first 5 minutes were a mixture of 16O2/16O18O/18O2 with a ratio of 30.0\u2009:\u200949.6\u2009:\u200920.4, which is close to the theoretical value of 26.5\u2009:\u200950.0\u2009:\u200923.5 \u2009:\u2009(7.6\u201339)\u2009:\u2009(0.51\u201312) reported for water oxidation with Oxone catalysed by a MnIII(O)2MnIV complex.19The origin of the oxygen gas produced from the reaction mixture of 0\u2009:\u200923.5 b expecte1-catalysed water oxidation with CAN in 0.1 M HNO3 using different concentrations of CAN (12.5\u2013125 mM) and 1 (6.25\u201325 \u03bcM) were examined; the plots of oxygen evolution (measured by GC) over time are depicted in Fig. S1 and S2 in the ESI.1 was observed, as depicted in The time courses of 1-catalysed water oxidation with NaIO4, the time course (0\u201360 min) plots of oxygen evolution at different concentrations of NaIO4 (12.5\u2013125 mM) and 1 (6.25\u201325 \u03bcM) in 0.1 M HNO3 are shown in Fig. S3 and S4,1 but not on the concentration of NaIO4 in 0.1 M HNO3 catalysed by n Fig. S8, which i2]+ and Oxone (8 equiv.) in MeCN\u2013H2O (5\u2009:\u20091 v/v) by high-resolution ESI-MS revealed a cluster peak at m/z 356.0981, attributed to [FeV(L1)(O)2]+.1 with CAN and NaIO4 in aqueous solution, we performed ESI-MS measurements on the corresponding reaction mixtures.In our previous work, analysis of a reaction mixture of + based on the isotopic distribution, collision-induced dissociation, and 18O-labelling for 30 seconds; the spectrum showed three new cluster peaks at m/z 357.0992, 402.0854 and 448.0815 (O)(OH)]+, [FeIV(L1)(O)(NO3)]+, and [FeIII(L1)(NO3)2]+, respectively. The ESI-MS spectrum for the reaction in 0.1 M HNO3 was conducted in H218O (instead of H216O); the corresponding isotopic patterns and collision-induced dissociation spectrum obtained in the 18O-labelling study are shown in Fig. S17\u2013S19.Before examination of the reaction intermediates in Fig. S13B, assignaFig. S13C resemble1 and NaIO4 (800 equiv.) in 0.1 M HNO3 during the first 30 seconds of the reaction revealed new species at m/z 356.0944 (V(L1)(O)2]+ and [FeIII(L1)(OO\u02d9)(OH)]+. Collision-induced dissociation spectra of the two species are depicted in Fig. S21 and S22.1 with NaIO4 (800 equiv.) was conducted in H2O under similar conditions, a new peak at m/z 356.0937 assignable to [FeV(L1)(O)2]+ was formed within 10 seconds with 5 equiv. of CAN in 0.1 M HNO3 at room temperature was monitored by UV-vis absorption spectroscopy. This reaction immediately (within 10 seconds) generated a new species with \u03bbmax 830 nm and a shoulder near 540 nm with 5 equiv. of NaIO4 or Oxone in 0.1 M HNO3, UV-vis measurements revealed different phenomena from that observed for the \u20181 + CAN\u2019 system. Upon treatment of 1 with NaIO4, a new species with \u03bbmax 830 nm and a shoulder near 540 nm was gradually formed over 10 minutes in 0.1 M HNO3 at room temperature followed by immediate cooling to 6 K is depicted in gavg = 5.54 and the other with g \u2248 2.EPR spectroscopy was employed to examine the reaction mixture of 4\u2013 anion in 1\u00b7FeCl4, we used the ClO4\u2013 salt of 1 and examined its electrochemical properties in 0.1 M HNO3 at pH 1 and in buffered solutions at various pH by means of cyclic voltammetry and rotating disk voltammetry using glassy carbon as the working electrode. The cyclic voltammograms of 1\u00b7ClO4 at pH 1\u20136 are depicted in E1/2 +0.46 V (pH 1) to +0.12 V (pH 6) vs. SCE, a small irreversible oxidation wave II at Epa +1.18 V (pH 1) to +0.86 V (pH 6) vs. SCE, and the onset of a catalytic oxidation wave at +1.4 V (pH 1) to ca. +1.1 V (pH 6) vs. SCE. Both E1/2 of the reversible couple I and Epa of the irreversible wave II shift cathodically by \u223c67 mV per pH unit upon increasing the pH of the redox process corresponding to the reversible couple I increased linearly with the square root of the rotation rate (\u03c91/2). A plot of iL against \u03c91/2 (Levich plot) shows a straight line with R2 = 0.999 in different oxidation states, together with that of the FeIII/FeII couple of 1, were estimated using DFT calculations. To avoid direct calculation of the proton free energy and systematic errors, we computed the redox potentials through isodesmic reactions using the electrochemical proton-coupled electron transfer (PECT) reactions of the cis-(dioxo)ruthenium(vi) complex [RuVI(L2)(O)2]2+ V/FeIVvs. RuV/RuIV, FeIV/FeIIIvs. RuIV/RuIII, and FeIII/FeIIvs. RuIII/RuII) are comparable and can be cancelled out in the calculations of \u0394\u0394G1, \u0394\u0394G2, and \u0394\u0394G3 depicted in reactions (1a)\u2013(3a) and (1b)\u2013(3b).The electrochemical potentials of iron\u2013oxo complexes [Fe(L1)(O)(X)]V/RuIV, RuIV/RuIII and RuIII/RuII couples,V/FeIV, FeIV/FeIII and FeIII/FeII couples can be calculated as:E(FeV/FeIV) = E(RuV/RuIV) + \u0394\u0394G1E(FeIV/FeIII) = E(RuIV/RuIII) + \u0394\u0394G2E(FeIII/FeII) = E(RuIII/RuII) + \u0394\u0394G3where E(RuV/RuIV) = +0.72 V, E(RuIV/RuIII) = +0.63 V, and E(RuIII/RuII) = +0.26 V at pH 1 according to the experimental findings.20From the DFT computed \u0394\u0394G1, \u0394\u0394G2 and \u0394\u0394G3 values, and the experimental redox potentials of the RuIII/FeII and FeIV/FeIII redox potentials at different pH by using three commonly used density functionals (DFs): BPW91 (pure-GGA), B3LYP (hybrid-GGA), and M06L (meta-GGA). A correlation between the experimental redox potentials of FeIII/FeII and FeIV/FeIII and those calculated using these DFs is depicted in note of caution: calculated redox potentials are thermodynamic values). It is evident that BPW91 and B3LYP led to marked-to-severe (up to ca. +0.57 V) overestimation of the E1/2 of FeIII/FeII, although B3LYP showed good performance in the prediction of the electrochemical potential of FeIV/FeIII. Only M06L gave good estimations of the electrochemical potentials of both FeIII/FeII and FeIV/FeIII at different pH with a linear fit (E(M06L) = E(expt) + 0.06, R = 0.998, SD = 0.03) between the calculated redox potentials and the experimental data. Hence, M06L was chosen for the subsequent DFT calculations in this work. In the literature, the M06L functional has been reported to show good performance in modelling water oxidation by rutheniumWe calculated the FeKa values, as an example, the pKa of [FeV(L1)(O)(OH)]2+ was calculated based on the following pair of isodesmic reactions (reactions (4a) and (4b)).Ka([FeV(L1)(O)(OH)]2+) = pKa([RuV(L2)(O)(OH)]2+) \u2013 \u0394\u0394G4/2.303RTwhere pKa([RuV(L2)(O)(OH)]2+) = 1.8 according to the experimental study.20For the calculation of the pV/FeIV, FeIV/FeIII and FeIII/FeII couples, including those of [Fe(L1)(O)(X)]n+ , along with the calculated pKa values, are depicted in Ka of the [FeV(L1)(O)(OH)]2+/[FeV(L1)(O)2]+ equilibrium is 3.0; (2) the calculated E1/2 of [FeV(L1)(O)(OH)]2+/[FeIV(L1)(O)(OH2)]2+ (+1.42 V vs. SCE at pH 1) is comparable to the E1/2 of CeIV/CeIII (+1.38 V vs. SCE) and close to the calculated E1/2 values of [FeV(L)(O)(OH)]2+/[FeIV(L)(O)(OH2)]2+ with L = Me2Pytacn or mep ; (3) the calculated E1/2 of [FeIV(L1)(O)(OH2)]2+/[FeIII(L1)(OH)(OH2)]2+ (+1.25 V vs. SCE at pH 1) matches the experimental Epa value of FeIV/FeIII ; (4) the calculated E1/2 of the FeIII/FeII couple is +0.49 V at pH 1, which matches the experimental E1/2 value for FeIII/FeII well; (5) the calculated E1/2 of the FeV/FeIII couple (E1/2 = +1.34 V vs. SCE at pH 1) coincides with the catalytic oxidation wave at +1.4 V observed in the cyclic voltammogram of 1 at pH 1. It is noted that there is just 170 mV difference in the calculated E1/2 values of [FeV(L1)(O)(OH)]2+/[FeIV(L1)(O)(OH2)]2+ and [FeIV(L1)(O)(OH2)]2+/[FeIII(L1)(OH)(OH2)]2+ at pH 1.The calculated redox potentials for various FeV(L1)(O)2]+ is a quartet state with three unpaired electrons; the lowest-lying excited state is a sextet state which is 2.8 kcal mol\u20131 higher in energy than the ground state. A quartet ground state for an FeV PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O species has previously been predicted by DFT calculations for [FeV(Me2Pytacn)(O)(OH)]2+. PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O distance in [FeV(L1)(O)2]+ in the quartet state (4FeV) is 1.614 \u00c5, which is shorter than that in the sextet state due to the antibonding character of the d1 and d2 orbitals scale\" fill=\"currentColor\" stroke=\"none\">O distances were reported for [FeV(L1)(O)2]+ (1.613\u20131.638 \u00c5), computed at the B3LYP/6-31(G) (lanl2dz) level in our previous work, and for [FeV(Me2Pytacn)(O)(OH)]2+ (1.63 \u00c5)V(TAML)(O)]\u2013 (1.60 \u00c5).V(L1)(O)2]+ calculated in this work is shown in V(L1)(O)2]+ (3 configuration in accordance with the assignment of the Fe(v) oxidation state.The ground state of + , right rIV(L1)(O)(OH)]+ is a quintet state with four unpaired electrons, and the triplet state is 16.0 kcal mol\u20131 higher in energy. The computed Fe PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O distance of [FeIV(L1)(O)(OH)]+ (5FeIV) in the ground state is 1.632 \u00c5, very close to the experimental Fe PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O distance in [FeIV(N4Py)(O)]2+ (1.639(5) \u00c5).5IV(L1)(O)(OH)]+. Fe has the largest spin density of 3.1; the oxo and hydroxyl groups have spin densities of 0.59 and 0.18, respectively. The MO diagram of [FeIV(L1)(O)(OH)]+ oxidation state.The ground state of [FeO)(OH)]+ , right rS = 3/2, 4FeV) and sextet states of [FeV(L1)(O)2]+ were considered in the mechanism studies. The doublet state (S = 1/2) of [FeV(L1)(O)2]+ was found to be the most energetically unfavourable and was not considered in this work. 4RC1/6RC1, formed between 4FeV/6FeV and the substrate (water molecule), which proceeds to the transition state 4TS1/6TS1 in which an O\u00b7\u00b7\u00b7O bond and an O\u00b7\u00b7\u00b7H bond are formed. The ground state is 4RC1, which is 2.8 kcal mol\u20131 lower in energy than 6RC1. The free energy barrier (\u0394G\u2021) for the quartet state is 15.7 kcal mol\u20131, lower than that for the sextet state . Both activation barriers are comparable to those for the similar oxidation of water by [FeV(TAML)(O)]\u2013 V(Me2Pytacn)(O)(OH)]2+ .6INT1 being 6.4 kcal mol\u20131 more stable than the quartet state 4INT1. The location of the minimum energy crossing point (MECP) between the sextet and quartet states was found using the code developed by Harvey and co-workers.\u20131 higher than that of 4INT1, suggesting that spin state reversion can easily occur, leading to the more stable complex 6INT1.Both the quartet (6INT1 complex can subsequently be oxidized by [FeV(L1)(O)2]+ (4FeV) to give the superoxo complex [FeIII(L1)(OO\u02d9)(OH)]+ (7INT2/5INT2). Meanwhile, 4FeV is reduced to [FeIV(L1)(O)(OH)]+ (5FeIV). The subsequent reaction steps include exchange of an oxygen molecule by a water molecule and the release of 3O2/1O2 (reaction (5)). The search for the transition state has not been successful. A barrier of 8.8 kcal mol\u20131 for the release of O2 from [TAML\u2013FeIV\u2013OO\u02d9]\u2013 was reported by Crammer and co-workers.2, complex [FeII(L1)(OH2)(OH)]+ (5FeII) with a quintet state ground state is formed; this complex can further comproportionate with [FeIV(L1)(O)(OH)]+ (5FeIV) to regenerate two [FeIII(L1)(OH)2]+ (6FeIII) molecules.The cis non-planar coordination geometry. The macrocyclic N4 ligand L1 scale\" fill=\"currentColor\" stroke=\"none\">O species of L1, including [FeV(L1)(O)2]+, in the cis-dihydroxylation of alkenes with Oxone catalysed by [FeIII(L1)Cl2]+ (1),V PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O species were proposed to be the reactive intermediates in iron-catalysed water oxidation reactions reported in the literature.The main objective of this work is to gain insight into the mechanism of water oxidation by iron complexes of tetradentate macrocyclic ligands with a 4 or Oxone was used as oxidant.During optimization of the reaction conditions, it was noted that although the turnover number of oxygen increased with oxidant concentration, the oxidant efficiency (defined in note c of 2] + CAN\u2019 system (which can oxidize water to O2 in non-buffered aqueous solution but not in 0.1 M HNO3 solution),1 + CAN\u2019 system produced oxygen with comparable TONs in water and in 0.1 M HNO3 (OTf)2]II(L1)(MeCN)2]2+ following the order Oxone > CAN > NaIO4 obtained in the 18O-labelling studies, the oxygen evolved in the \u20181 + Oxone\u2019 system originated not only from Oxone but also from water (OTf)eCN)2]2+ g used asII(mcp)(OTf)2] for water oxidation, as reported by Costas, Lloret-Fillol and co-workers,II(mcp)Cl2]III(L1)Cl2]+ (1) for water oxidation. Under our reaction conditions with catalyst/oxidant = 1\u2009:\u2009840, the amounts of oxygen formed in [FeII(mcp)Cl2]-catalysed water oxidation in 0.1 M HNO3 with CAN and NaIO4 as oxidants were approximately 1.4 times (TON = 56) and 2.9 times (TON = 35) the amounts formed in the 1-catalysed reactions, respectively. Changing the catalyst/oxidant ratio from 1\u2009:\u2009840 to 1\u2009:\u200910\u2009000 led to an increase in the TON for oxygen formation from 41 to 93 for \u20181 + CAN\u2019, from 56 to 177 for \u2018[FeII(mcp)Cl2] + CAN\u2019, from 12 to 44 for \u20181 + NaIO4\u2019, and from 35 to 114 for \u2018[FeII(mcp)Cl2] + NaIO4\u2019. These results showed that [FeII(mcp)Cl2] is more active than 1 in catalysing water oxidation.Considering the excellent catalytic ability of 2+/[FeII(L1)(OH2)2]2+ redox couple and the assignment of wave II to the oxidation of [FeIII(L1)(OH)(OH2)]2+ to [FeIV(L1)(O)(OH2)]2+. Despite the low scan rate of 5 mV s\u20131 for the rotating disk voltammetry, the magnitude of the current recorded for the [FeIV(L1)(O)(OH2)]2+/[FeIII(L1)(OH)(OH2)]2+ oxidation wave is not the same as that recorded for the [FeIII(L1)(OH)(OH2)]2+/[FeII(L1)(OH2)2]2+ redox process. As the oxidation of FeIII\u2013OH to an FeIV PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O species involves the deprotonation of an Fe\u2013OH unit, the electrochemical oxidation would be kinetically slow and highly sensitive to the electrode surface. Indeed, the electrochemical oxidation of Ru\u2013OH2/Ru\u2013OH to a Ru PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O species is well documented to be significantly affected by the nature/surface of the working electrode.IV/FeIII) and couple I (FeIII/FeII) is about 700 mV. For a ruthenium complex supported by the mcp ligand,IV(mcp)(O)(OH2)]2+/[RuIII(mcp)(OH)(OH2)]2+ and [RuIII(mcp)(OH)(OH2)]2+/[RuII(mcp)(OH2)2]2+ couples is 550 mV, which is 150 mV smaller than that between the iron analogues and can be attributed to the stronger \u03c0-bond formed in the RuIV PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O moiety. There is a previous report on the observation of a pH-dependent reversible couple assigned as a proton-coupled electron transfer FeIV/FeIII couple for [FeIV(N4Py)(O)]2+ -bis(2-pyridyl)methylamine) in buffered aqueous solution (pH 1.5\u20134) with an E1/2 value of +0.41 V vs. SCE at pH 4 .III/FeII couple was not reported for [FeIV(N4py)(O)]2+. The E1/2 value of +0.41 V for the [FeIV(N4py)(O)]2+/[FeIII(N4py)(OH)]2+ couple in aqueous solution at pH 4 is unexpectedly low. Should this be the case, the corresponding FeIII/FeII couple would have to occur at a potential less than 0.0 V vs. SCE based on the DFT calculations in this work. Our DFT calculations using the M06L functional gave redox potentials of +1.21 V vs. SCE for the [FeIV(N4py)(O)]2+/[FeIII(N4py)(OH)]2+ couple and +0.3 V vs. SCE for the [FeIII(N4py)(OH)]2+/[FeII(N4py)(OH2)]2+ couple at pH 1. The observation of a catalytic wave for 1 (IV/FeIII) at, for example, Epa +1.18 V vs. SCE (pH 1), which is comparable to the DFT-calculated potential of +1.25 V vs. SCE (pH 1) for the [FeIV(L1)(O)(OH2)]2+/[FeIII(L1)(OH)(OH2)]2+ couple, corroborates the finding that 1 can catalyse water oxidation through iron\u2013oxo species at oxidation states beyond FeIV.The cyclic voltammetric and rotating disk voltammetric measurements of ve for 1 and S27\u20201 in water (OH)2]+, suggests rapid exchange of the Cl\u2013 ligands of 1 with solvent. The new signals, generated upon addition of CAN, at m/z 357.0992, 402.0854, and 448.0815 can be attributed to [FeIV(L1)(O)(OH)]+ (O)(NO3)]+ (NO3)2]+ (18O)(18OH)]+ (18O)(N16O3)]+ (18O)(16OH)]+ and [FeIV(L1)(16O)(18OH)]+ were not detected in the experiment, indicating that the oxygen atoms of the hydroxide and oxo ligands in [FeIV(L1)(O)(OH)]+ come from water and not from CAN.ESI-MS analysis of a solution of 1 with CAN scale\" fill=\"currentColor\" stroke=\"none\">O species of L1, with reference to the characteristic bands of FeIV PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O species (ranging from 700\u2013850 nm) reported in the literature.IV(L1)(O)(OH)]+ and [FeIV(L1)(O)(NO3)]+ by ESI-MS. We further examined the intensity of the cluster peak at m/z 357.0992, attributed to [FeIV(L1)(O)(OH)]+, at different reaction times. The ion count of this signal decreased with reaction time, as depicted in Fig. S32,\u03bbmax 830 nm assigned to the FeIV PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O species in the UV-vis spectra shown in IV PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O species of chelating N-donor ligands have been reported to be reasonably stable, with half-lives generally ranging from 2 to 60 hours.IV(mcp)(O)(H2O)]2+ species (generated in situ from [FeII(mcp)(OTf)2] and CAN in aqueous solution), which could persist under catalytic conditions with a half-life of 2.4 hours,IV PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O species of L1 decayed with a half-life of 107 seconds under these conditions scale\" fill=\"currentColor\" stroke=\"none\">O species upon oxidation of 1 with CAN in aqueous solution, as supported by ESI-MS and UV-vis analysis, together with the absence of an induction period for oxygen evolution, an FeIV PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O species is suggested to be involved in water oxidation by the \u20181 + CAN\u2019 system. Kinetic studies revealed a linear dependence of the initial rate of oxygen evolution on the concentrations of both CAN and 1 (IV(L1)(O)(OH)]+, and one equivalent of CAN. One of the possibilities is the oxidation of [FeIV(L1)(O)(OH)]+ by CAN to give FeV PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O species, such as [FeV(L1)(O)(OH)]2+ or [FeV(L1)(O)2]+, and another possibility is the reaction of [FeIV(L1)(O)(OH)]+ with CAN to form an O PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019FeIV\u2013O\u2013CeIV species similar to that recently reported for the \u2018[FeII(mcp)(OTf)2] + CAN\u2019 system,V PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O and O PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019FeIV\u2013O\u2013CeIV species have not been clearly detected in ESI-MS analysis of the reaction mixture of 1 with CAN in H2O.Given the instant generation of an FeAN and 1 , which s1 + NaIO4\u2019 system, the new signals at m/z 356.0944 (major) and 373.0940 (minor), revealed by ESI-MS analysis of a mixture of 1 and NaIO4 in 0.1 M HNO3 (V(L1)(O)2]+ and [FeIII(L1)(OO\u02d9)(OH)]+ , respectively. The assignment of [FeV(L1)(O)2]+ is supported by 18O-labelling studies, revealing a shift of the signal at m/z 356.0944 to m/z 360.1042 attributable to [FeV(L1)(18O)2]+ upon changing the reaction medium to H218O (18O)2]+ could come from H218O directly and/or from 18O-incorporated IO4\u2013. Changing the reaction medium from 0.1 M HNO3 to pure water reduced the intensity of the signal assigned to [FeV(L1)(O)2]+; this observation is in line with the smaller TON of oxygen produced in pure water than in 0.1 M HNO3 (O)2]+ in this work and on [FeV(Me2Pytacn)(O)(OH)]2+ in the literatureV PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O species supported by these neutral chelating N ligands all adopt a quartet ground state (S = 3/2), different from the doublet ground state (S = 1/2) of [FeV(TAML)(O)]\u2013 complexes, which bear tetraanionic tetraamide ligands and have been detected by EPR spectroscopy.V(TAML)(O)]\u2013 using the M06L functional, which revealed a doublet ground state for this species, consistent with previous DFT calculations reported in the literature.V PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O species with S = 3/2 ground state has been characterized by EPR spectroscopy. The X-band EPR spectrum of the reaction mixture of 1 with NaIO4 in 0.1 M HNO3 species whereas the latter probably arose from decomposed NaIO4, as a similar S = 1/2 signal was observed in the X-band EPR spectrum of a solution of NaIO4 in 0.1 M HNO3 recorded at 7 K (O)2]+ species by EPR since the signal of this S = 3/2 FeV PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O species, which is likely to have a low concentration, could easily be masked by the S = 5/2 signal.The DFT calculations on [Fe1 M HNO3 is domin Fig. S33. EPR sigV(L1)(O)2]+ from the oxidation of 1 with NaIO4 resembles the generation of [FeV(L1)(O)2]+ from Oxone;4 and Oxone are typically two-electron oxidants and can directly oxidize Fe(iii) to Fe(v). UV-vis spectroscopy revealed that the reaction of 1 with NaIO4 or Oxone is different from the reaction of 1 with CAN, as exemplified by the immediate formation of a new band at \u03bbmax 830 nm attributable to an FeIV PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O species in the \u20181 + CAN\u2019 system scale\" fill=\"currentColor\" stroke=\"none\">O species.Generation of + and [FeV(L1)(O)2]+ based on ESI-MS analysis; their protonated forms [FeIV(L1)(O)(OH2)]2+ and [FeV(L1)(O)(OH)]2+ could be involved as well (O)2]+, [FeV(L1)(O)(OH)]2+ also adopts a quartet ground state, with the doublet state being \u223c15 kcal mol\u20131 higher in energy, according to DFT calculations. The possible involvement of the FeV PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O reactive species in the reaction is supported by the reasonably low reaction barrier for the oxidation of water by [FeV(L1)(O)2]+ of 15.7 kcal mol\u20131 obtained by the DFT calculations (V(L1)(O)(OH)]2+, the reaction barrier for O\u2013O bond formation with water was calculated to be 21.0 kcal mol\u20131 (O)2]+. The FeV PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O species is suggested to be attacked by water molecules, assisted by hydrogen-bond interactions, leading to the formation of an O\u2013O bond, analogous to the O\u2013O bond formation in water oxidation by MnV PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O,IV PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019FeIV\u2013O\u2013CeIV speciesIV(L1)(O)(OH2)]2+/[FeIII(L1)(OH)(OH2)]2+ and +1.42 V for [FeV(L1)(O)(OH)]2+/[FeIV(L1)(O)(OH2)]2+ at pH 1. The resulting iron(iii)\u2013peroxo intermediate in 4, or Oxone) to give [FeIII(L1)(OO\u02d9)(OH)]+ (OO\u02d9)(OH)]+/[FeIII(L1)(OOH)(OH)]+: +0.64 V vs. SCE), the presence of which is supported by ESI-MS analysis (OO\u02d9)(OH)]+ with water; the resulting [FeII(L1)(OH)(OH2)]+ is oxidized by the sacrificial oxidant to regenerate the catalyst [FeIII(L1)(OH)2]+ (OH)2]+/[FeII(L1)(OH)(OH2)]+: +0.26 V vs. SCE).On the basis of the above findings, a mechanism involving Feulations . For + and/or iron(v)\u2013oxo species such as [FeV(L1)(O)2]+, or their protonated forms [FeIV(L1)(O)(OH2)]2+ and/or [FeV(L1)(O)(OH)]2+, in the 1-catalysed water oxidation reactions.A mononuclear iron(Supplementary informationClick here for additional data file."} +{"text": "The ambient temperature hydrometallations of a variety of unactivated alkene and alkyne substrates using two-coordinate hydrido-tetrylenes, :E(H)(L\u2020) , are reported. \u2020) (SiPri3); Ar\u2020 = C6H2{C(H)Ph2}2Pri-2,6,4), with a variety of unactivated cyclic and acyclic alkenes, and one internal alkyne, lead to the rapid and regiospecific hydrometallation of the unsaturated substrate at ambient temperature. The products of the reactions, [L\u2020E(C2H4R)] , [L\u2020E{CH(CH2)3(CH2)n}] and , include the first structurally characterised examples of two-coordinate amido/alkyl germylenes and stannylenes. The cycloalkene hydrometallation reactions are cleanly reversible under ambient conditions, a process which computational and experimental van't Hoff analyses suggest proceeds via \u03b2-hydride elimination from the metal coordinated cycloalkyl ligand. Similarly, the reactions of :Ge(H)(L\u2020) with 1,5-cyclooctadiene and 2-methyl-2-butene, both likely proceed via \u03b2-hydride elimination processes, leading to the clean isomerisation of the alkene involved, and its subsequent hydrogermylation, to give [L\u2020Ge(2-cyclooctenyl)] and [L\u2020Ge{C2H4C(H)Me2}], respectively. Reactions of [L\u2020GeEt] and [L\u2020Ge(C5H9)] with the protic reagents, HCl, NH3 and EtOH, lead to oxidative addition to the germanium(ii) centre, and formation of the stable chiral germanium(iv) complexes, [L\u2020Ge(C5H9)(H)Cl] and [L\u2020Ge(Et)(H)R] (R = NH2 or OEt). In contrast, related reactions between [L\u2020SnEt] and ButOH or TEMPOH proceed via ethane elimination, affording the tin(ii) products, [L\u2020SnR] (R = OBut or OTEMP). In addition, the oxidation of [L\u2020Ge(C6H11)] and [L\u2020Sn(C2H4But)] with O2 yields the oxo-bridged metal(iv) dimers, [{L\u2020(C6H11)Ge(\u03bc-O)}2] and [{L\u2020(ButC2H4)Sn(\u03bc-O)}2], respectively.Reactions of the solution stable, two-coordinate hydrido-tetrylenes, :E(H)(L While much less studied than boranes, a variety of electron deficient, polar hydride complexes of aluminium, the heavier group 13 metals,The 1,2-addition of element-hydrogen bonds across the carbon\u2013carbon unsaturations of alkenes and alkynes is of immense importance to organic synthesis. In this respect, and since Brown's seminal work on the hydroboration of alkenes in the 1950's,iv) hydrides do not possess any vacant valence orbitals, it is not surprising that they are poorly effective for the hydroelementation of alkenes and alkynes, at least in their own right. However, reactions of this type (particularly hydrosilylations) are of considerable synthetic importance, and can proceed, for example, in the presence of transition metal catalysts or radical initiators; and/or when subjected to UV irradiation or elevated temperatures.Considering that neutral group 14 element(ii) hydride complexes, a small number of which hydride, I, has been shown to hydrosilylate cyclopentene and a series of terminal olefins at elevated temperatures (70\u2013120 \u00b0C) and in the presence of large excesses of the alkene substrate.anti-Markovnikov product predominates. In one case, i.e. the reaction with trimethylsilylethylene, the reaction proceeds via an isolated [2 + 1] cycloadduct, viz. the silirane , which exists in equilibrium with I and free H2C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C(H)(SiMe3) at ambient temperature. With respect to hydrogermylation and hydrostannylation reactions, the three-coordinate species, II and III, have been shown to cleanly hydrometallate activated (ester substituted) terminal and internal alkynes at ambient temperature.ii) hydride complexes, IV and V, react with tert-butylethylene at ambient temperature over 48 hours to give the alkyl/aryl substituted ditetrelenes [{Ar\u2032E(CH2CH2But)}2] 2-2,6; E = Ge or Sn). Contrastingly, after 48 hours, the reaction of IV with excess cyclopentene at ambient temperature yielded only a mono-hydrogermylation product, viz. the hydrido-digermene, (Cp = cyclopentyl).IV to the two-coordinate hydrido-germylene, Ge(H)Ar\u2032, in solution is minimal.It would be a significant advantage if the addition of group 14 element-hydrogen bonds to unsaturated hydrocarbons could be effected in the absence of catalysts or initiators, and in a facile manner under ambient conditions. The first hints that this might be possible came with the kinetic stabilisation of group 14 elementGe PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019Ge(H)L\u2020] 1 (L\u2020 = \u2013N(Ar\u2020) (SiPri3); Ar\u2020 = C6H2{C(H)Ph2}2Pri-2,6,4),e.g. [L\u2020Sn(\u03bc-H)2SnL\u2020] 2.\u2020) (E = Ge 3 or Sn 4), in hydrocarbon solutions. Subsequently, two-coordinate hydrido-germylenes, bearing even bulkier amide ligands, e.g. [:Ge(H)(LOBut)] (LOBut = \u2013N(Ar\u2020){Si(OBut)3}) were isolated in the solid state.3 and 4 possess empty p-orbitals at their metal centres (cf. boranes) it was proposed that they would act as effective reagents for the hydrometallation of unsaturated substrates. This was shown to be the case for aldehydes and ketones, and, indeed, 3 and 4 were also shown to be highly efficient catalysts for the hydroboration of the same substrates.Recently, we have utilised extremely bulky amide ligands, developed in our group,(i)3 (as an equilibrium mixture with 1) with 1\u20131.5 equivalents of a range of unactivated terminal or cyclic alkenes, or 1 atm. of ethylene, led to almost instantaneous changes in the colour of the reaction solutions from orange to yellow at ambient temperature. This indicated that the hydrometallation reactions were complete in well under 1 minute. 1H NMR spectroscopic analyses of the reaction mixtures after ca. 10 min confirmed that the hydrometallation reactions were essentially quantitative after that time, affording the amido/alkyl germylenes, 5\u201310 (ii) hydride 4 (as an equilibrium mixture with 2) were carried out at low temperature (\u201380 \u00b0C) due to the mild thermal instability of 4 at room temperature . It is also of note that the hydrometallations of all of the terminal alkenes regiospecifically yielded the anti-Markovnikov product, as is typically the case with alkene hydroborations.The facility of these uncatalysed olefin hydrogermylation and hydrostannylation reactions is unprecedented, and is likely a result of them preferentially involving the monomeric hydridoterylenes, 5\u201313 are thermally stable in the solid state. The solution state NMR spectroscopic data for the compounds are fully consistent with their proposed monomeric structures, and do not warrant further comment here. X-ray crystallographic studies were used to confirm the monomeric nature of all compounds, which represent the first structurally characterised examples of two-coordinate amido/alkyl germylenes and stannylenes. Illustrative examples of the molecular structures of the compounds are depicted in 8, 10, 11 and 13), while selected geometrical parameters are collected in 7 and 12 confirmed the molecular connectivity of the compounds, they are not of a quality suitable for publication, and their geometrical parameters will not be discussed here.5\u201310 . The clC (3.87 \u00c5), which 3 and 4 as reagents for the hydrometallation of alkenes, it seemed likely that they would also be very reactive toward unactivated alkynes. To assess this, and to investigate if the metal hydrides could doubly hydrometallate alkynes, 1-phenyl-1-propyne was treated with two equivalents of either 3 or 4. Analysis of the reaction mixtures indicated that only one hydrometallation event occurred in both cases, and within several minutes at ambient temperature. These reactions afforded compounds 14 and 15 respectively, in close to quantitative NMR spectroscopic yields, and moderate isolated yields. The reactions proceeded with complete regiospecificity, giving the cis-isomer with the L\u2020E fragment bonded to the phenyl substituted alkenenic carbon which reveal both to be monomeric in the solid state with the alkeneic phenyl and methyl substituents cis- to one another. Their C(45)\u2013C(46) distances reflect localised double bonds, while the geometries about the metal centres are similar to those in 5\u201313. That is, the CNSi and NEC fragments are close to co-planar with one another, which allows for the possibility of N \u2192 E \u03c0-bonding in the compounds. In contrast, their C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C units are close to orthogonal to the NEC fragments, which discounts the possibility of any \u03c0-delocalisation over those fragments.Both (ii)9 and 10, it was noticed that 1H NMR spectra (C6D6) of pure samples of the compounds reproducibly exhibited signals due to the presence of small amounts of the germanium hydride equilibrium mixture, 1 and 3, and the free cycloalkene. In addition, recrystallisation of the compounds, and 8, repeatedly led to co-crystallisation with small amounts of 1. Moreover, C6D6 solutions of the tin cyclopentyl compound, 13, decomposed over several days at ambient temperature , yet were stable for extended periods, even at 80 \u00b0C, in the presence of excess cyclopentene. All of these observations point to the hydrometallation products from the reactions of 3 and 4 with cycloalkenes being in equilibria with significant amounts of those reactants at room temperature (see 3 and 9). The net decomposition of 13 can be explained by the mild instability of the tin hydride 4 at ambient temperature, which upon decomposition, inextricably leads to loss of 13 from the equilibrium mixture in that case.During characterisation of the cycloalkene hydrogermylation products 9 was explored by a VT 1H NMR spectroscopic study of a sample of the compound which was prepared by reaction of CyMgBr (Cy = cyclohexyl) with L\u2020GeCl. This ensured the absence of any 1 in the purified sample of 9 used for the experiment. Despite this, dissolution of the compound in C6D6 again revealed the presence of a small amount (ca. 5% as determined by 1H NMR spectroscopy) of the 1/3 equilibrium mixture, and cyclohexene, as determined by a 1H NMR spectrum acquired at 20 \u00b0C. Heating the solution to 60 \u00b0C led to an increase in the quantities of these starting materials, which decreased when the solution was again cooled back to 20 \u00b0C. A van't Hoff analysis of this reversible process over the temperature range 304\u2013314 K (see H\u00b0 = \u2013172 kJ mol\u20131) with a relatively large entropic factor (\u0394S\u00b0 = 395 J mol\u20131). Accordingly, the Gibb's free energy for the exergonic hydrogermylation reaction at 298 K is fairly small (\u0394G = \u201354 kJ mol\u20131), and therefore the weakly endergonic reverse reaction might be expected to be become more pronounced at elevated temperatures.The reversibility of the reaction that gave 14 K see revealed8\u201310 and 13. Indeed, inspection of the crystal structures of the compounds revealed, in each case, that the distance between the metal centre and the closest cycloalkyl \u03b2-hydrogen atom (range: 2.62\u20132.99 \u00c5) is significantly less than the sum of the van der Waal's radii for E and H scale\" fill=\"currentColor\" stroke=\"none\">CBut, by a three-coordinate germanium(ii) hydride, [(MesNacnac)GeH] (MesNacnac = [(MesNCMe)2CH]\u2013, Mes = mesityl), which reversibly affords the phosphaalkenyl complex, .MesNacnac)GeC(But) PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019PH].It is possible that the observed reverse reactions are due to \u03b2-hydride elimination processes, which are enabled by the coordinatively unsaturated nature of the two-coordinate metal centres in n 3.28 \u00c5 ). As far8\u201310, DFT calculations were carried out at several levels of theory on the hydrogermylation reaction that gave 9 (see ESIG = \u201342.3 kJ mol\u20131 at M06-2X+D3/def2-TZVPP//TPSS+D3/def2-TZVPP) that is small, and not dissimilar to that found from the experimental van't Hoff analysis of the reaction. Importantly, the reverse reaction was, indeed, found to proceed via a \u03b2-hydride elimination process, involving a transition state with a four-membered GeC2H ring (see \u2021G = 76.6 kJ mol\u20131), is fully consistent with the experimentally observed equilibrium for the reaction that gave 9.To explore the possibility of facile \u03b2-hydrogen elimination processes being the origin of the reversibility of the reactions that gave ring see and ESI\u2020via \u03b2-hydrogen elimination processes, comes from the reactions of 3 with 1,5-cyclooctadiene and 2-methyl-2-butene scale\" fill=\"currentColor\" stroke=\"none\">CR2 (R = Me or Ph). These observations presumably result from the considerable steric bulk of the monomeric hydridogermylene, 3.Further evidence that the reversibility of the cyclic alkene hydrogermylation reactions proceed 2-butene . In both16 proceeds via the expected hydrogermylation product, 18, as an intermediate. This then undergoes a \u03b2-hydrogen elimination to give 3 and 1,4-cyclooctadiene . Hydrogermylation of this by 3, and another \u03b2-hydrogen elimination event, yields 3 and 1,3-cyclooctadiene , the latter of which is then hydrogermylated to give the observed product, 16. Similarly, hydrogermylation of 2-methyl-2-butene affords the initially expected product, 19, which \u03b2-hydride eliminates to give 3 and 3-methyl-1-butene. Hydrogermylation of this olefin then leads to the observed product, 17. The facility of these reactions highlights the potential that 3, and related reagents, have for the selective stoichiometric isomerisation of alkenes. While such isomerisations are common for transition metal systems,It is possible that the formation of 16 and 17 were crystallographically characterised, and their molecular structures are depicted in 5\u201310, reported here. In the case of 16, the presence and location of the residual double bond of its cyclooctenyl moiety is confirmed by the shortness of the C(51)\u2013C(52) linkage (1.372(6) \u00c5).Both (iii)2 and hydridic reagents 3SiH and DIBAL) to 5\u201313 was explored, but in no case was a reaction observed under ambient conditions. Attention then turned to the reaction of stoichiometric amounts of protic reagents with 5, 8 and 11. In the case of the germylenes, the oxidative addition of HCl, NH3 or EtOH to the Ge centres of 5 or 8 occurred, to give a few crystals of 20 , and good isolated yields of 21 and 22, respectively (21 and 22 are resistant to reductive elimination (RE) of ethane or cyclopentane, even when heated to 50 \u00b0C for one hour. It is noteworthy that the reactions that gave 20\u201322 are comparable to related oxidative additions of HF,3Preliminary further reactivity studies were carried out on examples of the amido/alkyl germylene and stannylene complexes prepared here, with a view to utilising these compounds in catalytic synthetic protocols. Initially, the oxidative addition (OA) of Hectively . Solutio11 with protic reagents. These were not clean, and typically generated product mixtures that contained significant amounts of the secondary amine, L\u2020H. The two exceptions here were the reactions with stoichiometric amounts of the bulkier reagents TEMPOH and ButOH. These afforded moderate to good isolated yields of the piperidinyl N-oxide product, 23, and the known tin tert-butoxide, 24,1H NMR spectroscopy, the generation of significant amounts of ethane was observed. It cannot be sure if these reactions proceed via initial oxidative additions of the O\u2013H bond of the reagents to the SnII center of 11, prior to reductive elimination of ethane, but given the formation of the stable germanium(iv) ethoxide, 22, this is certainly a possibility (cf. related \u201cOA/RE\u201d reactions of H2 and NH3 with Ar\u20322Sn: (Different outcomes resulted from the reactions of the stannylene Ar\u20322Sn: ).2Ge PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O),2Sn PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O), toluene solutions of the germylene, 9, and stannylene, 12, were reacted with excess O2. Instead of yielding monomeric products, the dimeric oxo-bridged species, 25 and 26, were obtained in moderate isolated yields 2}2Sn(OTEMP)2],25 and 26 was confirmed by X-ray crystallographic studies (see 26), which also showed their oxide ligands to essentially symmetrically bridge two distorted tetrahedral metal centres.No spectroscopic data could be obtained for the HCl oxidative addition product, via \u03b2-hydride elimination from the cycloalkyl ligand, regenerating the cycloalkene and hydrido-tetrylene starting materials. Further evidence for this proposal comes from the reactions of a hydrido-germylene with 1,5-cyclooctadiene and 2-methyl-2-butene, both of which seemingly proceed via intermediate \u03b2-hydride elimination processes, and the clean isomerisation of the alkene involved, prior to its ultimate hydrogermylation. In addition, the element-hydrogen bonds of several protic compounds have been shown to oxidatively add to the germanium(ii) centre of two of the amido/alkyl germylenes prepared in this study, while similar reactions with an equivalent stannylene proceed via alkane elimination, and generation of tin(ii) products. Oxidations of two amido/alkyl tetrylenes with O2 have been shown to give four-coordinate, oxo-bridged metal(iv) dimers. We continue to explore the stabilisation and synthetic utility of low oxidation state group 14 element compounds.In summary, reactions of solution stable two-coordinate hydrido-tetrylenes with a variety of unactivated cyclic and acyclic alkenes, and one internal alkyne, lead to the unprecedentedly rapid and regioselective hydrometallation of the unsaturated substrate at ambient temperature. The products of the alkene hydrometallations represent the first structurally characterised examples of two-coordinate amido/alkyl germylenes and stannylenes. In the cases of the cycloalkene hydrometallations, the reactions were shown to be cleanly reversible under ambient conditions. The results of computational and experimental van't Hoff analyses of one such reaction, strongly suggest that its reversal proceeds Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "PIP\u2013TPE\u2019s fluorescence turns on blue due to the large viscosity of lysosomes which restricts intramolecular motions but it red-shifts in the bulk. Lysosomes are involved in a multitude of cellular processes and their dysfunction is associated with various diseases. They are the most acidic organelles with the highest viscosity (47\u2013190 cP at 25 \u00b0C) in the cell. Because of their acidity, pH dependent non-AIE active fluorescent lysosomal probes have been developed that rely on protonation inhibited photoinduced electron transfer (PET). In this work, an acidic pH independent lysosome targetable piperazine\u2013TPE (PIP\u2013TPE) AIEgen has been designed with unique photophysical properties making it a suitable probe for quantifying viscosity. In a non-aggregated state PIP\u2013TPE shows deep-blue emission as opposed to its yellowish-green emission in the bulk. It possesses high specificity for lysosomes with negligible cytotoxicity and good tracing ability due to its better photostability compared to LysoTracker Red. In contrast to most known lysosome probes that rely solely on PET, restriction of intramolecular motion (RIM) due to the larger viscosity inside the lysosomes is the mechanism responsible for PIP\u2013TPE\u2019s fluorescence. PIP\u2013TPE\u2019s high selectivity is attributed to its unique molecular design that features piperazine fragments providing a perfect balance between lipophilicity and polarity. Thus, the working concentration of LysoTracker Red is usually low. Furthermore, it is not photostable due to its BODIPY-based structure, which makes it unsuitable for long-term lysosome trace tracing. Moreover, the LysoTracker probes also have small Stokes shifts (less than 20 nm) which is highly disadvantageous for bioimaging applications. On the contrary, the development of AIEgens (aggregation-induced emission luminogens) allows a higher working concentration to be used with good photostability. PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C twisting in MeOH with different fractions of glycerol increase as the content of glycerol increases Fig. S4, its PL = 509 nm . Similars Fig. S5. Thus, t\u03bbem = 420 nm) is not sensitive to pHs below 7 (\u03bbem = 509 nm) and turns on when its fluorescence quantum yield is above 1.8% in the aqueous buffer solution of pH 7.13, and the quantum yield maximum value reaches 12.7% with a large extent of increased fluorescence intensity in the basic aqueous solution of pH 13.03 in vitro. Since protonated PIP\u2013TPE molecules would naturally display a lower propensity to form large aggregates in acidic media (vide supra), we assume that in lysosomes they will primarily exist in the form of individual protonated molecules and/or nano-scale aggregates with the upper size limit determined by the size of the lysosomes (100 nm to 1.2 \u03bcm).e.g., outside lysosomes vide supra). However, PIP\u2013TPE\u2019s fluorescence does turn on in lysosomes due to its large viscosity.e.g. PET).It is difficult to precisely replicate intralysosomal conditions -2,5-diphenyltetrazolium bromide) assay. The result Fig. S9 shows thThe commercial lysosome probe LysoTracker Red cannot be used to trace the migration of lysosomes due to its serious photobleaching after a second time of excitation.In an attempt to gain more insight and explore possible fluorescence mechanisms operative in PIP\u2013TPE in lysosomes and in the bulk, DFT/TD-DFT calculations were performed using the B3LYP functional47Ka values of the structurally very similar dimethylpiperazine (pKa = 8.3) and diethylamine (pKa = 6.6) in water reported elsewhere,+ would be the actual predominant photoactive species that is observed in lysosomes. We thus decided to use it in our computational studies, and will henceforth refer to it in short as PIP\u2013TPEH+(soln). On the other hand, for the calculations of PIP\u2013TPE in the bulk, we took crystal clusters to consist exclusively of neutral PIP\u2013TPE molecules, and assumed a neutral PIP\u2013TPE molecule imbedded in such a cluster to be the photoactive species. We will thus use PIP\u2013TPE(bulk) to refer to the QM PIP\u2013TPE molecule in such a cluster in all further discussions of our computational results.Given the lysosomes\u2019 acidic milieu, in lysosomes PIP\u2013TPE will exist for the most part in its protonated form. Based on the p+(soln) in the ground state and then geometry optimization of PIP\u2013TPEH+(soln) in the first excited state, and studied any major changes in certain relevant metric parameters of these molecules yields a structure that displays no pronounced changes in the metrics in comparison to the original PIP\u2013TPE (see \u03c4 (C9\u2013C10\u2013N1\u2013C19)). However, expectedly marked changes in bond lengths, angles and dihedral angles of the TPE-fragment and protonated piperazine moiety in PIP\u2013TPEH+(soln) upon photoexcitation are predicted (\u03c4 (C2\u2013C3\u2013C6\u2013C7) from 23.44\u00b0 in the ground state to 82.41\u00b0 in the first excited state is anticipated, indicating that in the excited state the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond of the TPE-fragment is significantly more twisted. Furthermore, in the excited state this C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C double bond is also significantly elongated and loses its \u201cdouble bond\u201d character upon excitation. Based on these predictions, one might speculate that this twisting around the C3 PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C6 double bond in the excited state of PIP\u2013TPEH+(soln) as well as the rotation of the phenyl groups and piperazine moieties are all possible relaxation channels that are responsible for the radiationless decay in non-viscous media but get blocked in the more viscous lysosomal environment causing the fluorescence to turn on (i.e. to be visible to the naked eye).49The optimized ground state geometry of PIP\u2013TPE obtained at the DFT level of theory reproduces its single crystal X-ray structure well. Ground state geometry optimization of PIP\u2013TPEH\u2013TPE see except fredicted . Distincredicted as well redicted . Also, a1) of PIP\u2013TPEH+(soln) show that it is dominated by a single excitation from the HOMO to the LUMO in this molecule. Both the HOMO and the LUMO in PIP\u2013TPEH+(soln) are located mainly on the carbon atoms of the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C double bond contributing the most with an additional less substantial contribution from the four phenyl rings of the TPE-moiety (see +(soln) are an integral part of the HOMO but not the LUMO. We further note that the same holds true for the HOMO and LUMO distributions of PIP\u2013TPE and PIP\u2013TPE(bulk) is caused by a twisted intramolecular charge transfer (TICT) process.(bulk), an assumption that is substantiated by the results of the calculations showing that the optimized ground state (S0) and first excited state (S1) geometries of PIP\u2013TPE(bulk) both have a dihedral angle \u03c4 (C2\u2013C3\u2013C6\u2013C7) similar to the one calculated for PIP\u2013TPE (0) and first excited state (S1) geometries of PIP\u2013TPE(bulk) reveals only negligible differences in the metrics of these structures observed for PIP\u2013TPEH+(soln) in polar milieu can be attributed to the higher twisting degree of the molecule in the excited state that would automatically result in less efficient conjugation, while the red-shifted yellowish-green emission of PIP\u2013TPE(bulk) could be attributed to the higher degree of conjugation preserved in the excited state of the molecule upon excitation.(bulk) result in a \u03bbPL,max = 471 nm that is in good agreement with experiment and appears to confirm the proposed model.The results of the TD-DFT calculations for the first excited state , Dulbecco\u2019s modified Eagle\u2019s medium (DMEM), phosphate buffered saline (PBS), fetal bovine serum (FBS), penicillin and streptomycin, LysoTracker\u00ae Red DND-99, and -2,5-diphenyltetrazolium bromide) were purchased from Invitrogen. Buffer solutions (pH = 1\u201313) were purchased from Fisher Scientific. Chloroquine was purchased from Bide Pharmatech Ltd. Milli-Q water was supplied by Milli-Q Plus System .1H and 13C NMR spectra were measured on a Bruker ARX 400 NMR spectrometer using CDCl3 as the solvent and tetramethylsilane (TMS: \u03b4 = 0 ppm) as an internal standard. High-resolution mass spectra (HRMS) were recorded on a Finnigan MAT TSQ 7000 Mass Spectrometer System operating in MALDI-TOF mode. Suitable single crystals of PIP\u2013TPE were selected under oil under ambient conditions. Single crystal X-ray diffraction intensity data were collected in a stream of cold nitrogen at 100 K on a SuperNova, Dual, Mo at zero, Atlas diffractometer. Using Olex2,The et al.tert-butylphosphonium tetrafluoroborate (P(tBu)3HBF4), 3.600 g (11.00 mmol) cesium carbonate (Cs2CO3), and 20 mL dry and degassed toluene were added and stirred for 1 h at r.t. under nitrogen. Then 0.097 g (0.43 mmol) Pd(OAc)2 was added. The reaction mixture was stirred for another 0.5 h under N2. Upon the formation of an orange suspension, 1.000 g (2.04 mmol) 1,1\u2032-bis(4-bromo)-benzene and 0.5 mL (4.51 mmol) of 1-methylpiperazine were added, and a yellowish green suspension formed immediately. The reaction mixture was allowed to reflux under N2 for 2 days. After the reaction was cooled down to r.t., the toluene was removed; 50 mL EtOH and 10 mL acetone were added to dissolve the crude product. The suspension was filtered, concentrated under reduced pressure and purified via silica gel flash chromatography (chloroform/MeOH = 40\u2009:\u20091). 0.162 g of yellow powder of PIP\u2013TPE was obtained. Yield: 15%. Pale yellow block crystals were grown from acetone/hexane via solvent diffusion. 1H NMR , \u03b4H : 7.12\u20136.99 , 6.94\u20136.87 , 6.67\u20136.59 , 3.25\u20133.04 , 2.59\u20132.42 , 2.32 . 13C NMR , \u03b4C : 149.27, 144.72, 140.51, 138.28, 135.08, 132.40, 131.46, 127.63, 125.83, 114.58, 55.15, 48.60, 46.22, 11.20. MALDI-TOF-MS: m/z calcd for [C36H40N4]: 528.33; found 528.3274.The starting material 1,1\u2032-bis(4-bromo)-benzene was synthesized according to the procedure published by Li 1H NMR , \u03b4H : 12.99 , 7.15\u20137.05 , 7.04\u20136.96 , 6.96\u20136.82 , 6.81\u20136.53 , 3.91\u20133.28 , 3.26\u20132.97 , 2.86 . MALDI-TOF-MS: m/z calcd for [C36H40N4H+]: 529.33; found 529.3319.In a 4 mL screw top vial, PIP\u2013TPE was dissolved in dichloromethane (2 mL) to form a yellow solution. Hydrochloric acid was added and a white suspension formed immediately. After stirring for a few seconds, the suspension dissolved and the color of the solution changed to pale yellow. The vial was capped and the reaction mixture was stirred overnight. The next day, the layer of dichloromethane solution was pipetted out and the remaining aqueous layer was extracted with dichloromethane (10 times with 2 mL). Then, all dichloromethane layers were combined and after rotoevaporation, a pale yellow powder of the protonated PIP\u2013TPE was obtained (10.0 mg). Yield: 89%. ca. 1 mL of each sample. The viscosity was measured as a function of shear rate in the range from 100.0 to 0.01 s\u20131. In The viscosities of the aqueous buffer\u2013glycerol mixtures were measured using a TA ARES-G2 rotational rheometer at a temperature of 25 \u00b1 0.5 \u00b0C. Each measurement used \u20131 streptomycin, in a humidity incubator with 5% CO2 at 37 \u00b0C. Before experiment, the cells were pre-cultured until confluence was reached.HeLa cells were cultured in MEM and DMEM, respectively. All the cells were cultured in media supplemented with 10% heat-inactivated FBS, 100 units per mL penicillin and 100 \u03bcg mLAll the cells were grown overnight on a 35 mm Petri dish with a cover slip or a plasma-treated 25 mm round cover slip mounted to the bottom of a 35 mm Petri dish with an observation window. The live cells were incubated with 1 \u03bcM PIP\u2013TPE (2 \u03bcL of a 1 mM stock solution of PIP\u2013TPE in DMSO was diluted to 2 mL culture medium) for 15 min, and then were imaged under fluorescence microscopy. For co-staining experiments, the cells were co-stained with PIP\u2013TPE (1 \u03bcM) and a commercial biomarker (LysoTracker Red DND-99) for 15 min. The cells were then imaged using a laser-scanning confocal microscope (LSM7 DUO) using a 405 nm and 561 nm laser as the excitation light. The spectral collection region was 425\u2013545 nm and 575\u2013625 nm, respectively.\u20131 in phosphate buffer solution) was added into each well. After incubation for 4 h at 37 \u00b0C, the remaining medium in each well was totally removed and then replaced by 50 \u03bcM DMSO. With mixing for 15 min, the absorbance of each well at 595 nm was recorded by the plate reader (Thermo Scientific\u2122 Varioskan\u2122 LUX multimode microplate reader). Each experiment was performed at least 6 times as parallel tests.The HeLa cells were seeded in 96-well plates at a density of 5000 cells per well. After overnight culture, the medium in each well was replaced by fresh medium containing different concentrations (0.1/0.2/0.5/1/2/5 \u03bcM) of PIP\u2013TPE. After 24 h of treatment, 10 \u03bcL MTT solution scale\" fill=\"currentColor\" stroke=\"none\">C twisting of its protonated species in the excited state upon excitation. Based on the aforementioned facts, this molecular design can be used as a guide to develop new promising lysosomal pH stable fluorescent AIE probes, which would allow monitoring of some important fundamental microenvironmental parameters with no or little influence from pH.In conclusion, we have developed an acidic pH independent lysosome targetable AIEgen, PIP\u2013TPE, which displays fluorescence emission of different colors depending on its aggregation state: deep-blue for non-aggregates and yellowish-green for the bulk solid. Experiments suggest that protonation of PIP\u2013TPE has no significant influence on its photophysical properties but does affect its scale of aggregation. PIP\u2013TPE has proved to have high specificity to lysosomes with a good signal-to-noise ratio and negligible cytotoxicity. PIP\u2013TPE\u2019s good selectivity towards lysosomes can be attributed to the piperazine functional groups and higher viscosity of the intralysosomal milieu and renders it a good lysosomal tracing agent with better photostability than the commercial alternatives such as LysoTracker Red. PIP\u2013TPE\u2019s turn-on fluorescence in lysosomes can be attributed to the viscosity restricting intramolecular motion (RIM). PIP\u2013TPE\u2019s blue emission in lysosomes can be attributed to the high degree of CThere are no conflicts to declare.Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "C\u2013H activation of methane followed by dehydrocoupling at room temperature led ultimately to the formation of the olefin H2C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CHtBu via the addition of redox-active ligands (L) such as thioxanthone or 2,2\u2032-bipyridine (bipy) to (PNP)Ti PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CHtBu(CH3) (1). 2C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CHtBu via the addition of redox-active ligands (L) such as thioxanthone or 2,2\u2032-bipyridine (bipy) to (PNP)Ti PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CHtBu(CH3) (1). Using both of these exogenous ligand systems, we could trap the titanium fragment via an insertion reaction with these two substrates to afford species of the type (PNP)Ti(L)(LH). A combination of computational and isotopic labeling studies reveals that the L ligand promotes the C\u2013C bond forming step by migration of the methyl moiety in 1 to the \u03b1-alkylidene carbon by producing a Ti(iii) species (PNP)Ti{CH(CH3)tBu}(L). In the case of L = thioxanthone, \u03b2-hydrogen abstraction gives an olefin, whereas with 2,2\u2032-bipyridine \u03b2-hydride elimination and migratory insertion lead to (PNP)Ti(L)(LH). These redox-active ligands play two important roles: (i) they accept an electron from the Ti-alkylidene fragment to allow the methyl to approach the alkylidene and (ii) they serve as traps of a hydrogen atom resulting from olefin elimination. These systems represent the first homogeneous models that can activate methane and selectively dehydrocouple it with a carbene to produce an olefin at room temperature.C\u2013H activation of methane followed by dehydrocoupling at room temperature led ultimately to the formation of the olefin H PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bonds at the expense of breaking the stronger C\u2013H bonds of methane or another alkane. Surface supported organometallic reagents and heterogeneous catalysts with a sulfur-based hydrogen acceptor were reported previously, but they require high temperatures with concurrent release of toxic H2S.2Sc(CH3)3)(\u03b73-CH2CHCMe2).2Sc(CH3) at room temperature.4 + H2C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CHCH3 \u2192 H3CCH(CH3)2. More recently, Legzdins and co-workers found that addition of CO under high pressure to Cp*W(NO)(CH3)(\u03b73-CH2CHCMe2), a species derived from methane activation, could result in insertion into the methyl ligand ultimately leading to extrusion of a mixture of \u03b2,\u03b3-unsaturated ketones.9The non-oxidative conversion of methane to an olefin is fundamentally an endergonic process, and hence only a handful examples exist, where this remarkable reaction was seen. PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CHtBu(CH2tBu) can eliminate H3CtBu to form transient (PNP)Ti PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CtBu (A), which then activates CH4via 1,2-CH bond addition to form the neopentylidene\u2013methyl (PNP)Ti PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CHtBu(CH3) (1) .1 can also extrude CH4 to reform A, this process was found to be much slower with an associated \u0394G\u2021 of 28.1 kcal mol\u20131 .1 tautomerizes to the methylidene (PNP)Ti PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CH2(CH2tBu) (B), but does so also very slowly with a \u0394G\u2021 > 28.1 kcal mol\u20131 .4 can take place competitively. More evidence for B being the more reactive tautomeric form, was derived from an independent synthesis involving \u201cCH2\u201d group transfer from a phosphorus ylide to the titanium olefin complexes of the type (PNP)Ti(CH2tBu)(\u03b72-olefin).2\u201d, B is not observed, but instead tautomerizes quickly to 1 corroborating our claim that B lies 7.8 kcal mol\u20131 higher in energy to 1 .We previously reported how complex (PNP)TiCH3) (1) .10,11 ThB since it contains the dehydrocoupled form of methane and may allow for accessing olefins via reductive coupling.iv) center without promoting \u03b1-hydrogen abstraction to form A by losing CH4. Unfortunately, complexes 1 and intermediates A and B pose some challenges since they contain reactive and nucleophilic alkylidyne and alkylidene moieties that may engage in Wittig-like chemistry.1, albeit slowly to carry out the expected Wittig-like chemistry and produce tBuHC PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C13H8O as shown in PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O(CH3)] could not be detected and instead, a mixture of metal-based products was observed by 31P NMR spectroscopy. PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O(CH3) was not unexpected since the close analogue, (PNP)Ti PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O(CH2tBu), is known to decompose rather quickly in solution. PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CC12H8S) is used instead, we observed some Wittig-like reactivity along with an additional olefin, which was identified to be H2C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CHtBu on the basis of 1H NMR spectroscopy and GC-MS scale\" fill=\"currentColor\" stroke=\"none\">CHtBu was unambiguously confirmed when compared to an independently prepared sample. Since the reaction mixture contained some unreacted 1, performing the same transformation using a 2 equiv. of thioxanthone produced higher yields of tBuHC PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C13H8S and H2C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CHtBu in approximately 1\u2009:\u20099 based on the 1H NMR spectrum. Under these conditions, we were also able to isolate the titanium complex (PNP)Ti scale\" fill=\"currentColor\" stroke=\"none\">CC12H8S)(OCHC12H8S) (2) in 52% yield and carbon Ti\u2013OCH at 83.8 (13C NMR) ppm. The most notable features in the solid-state structure of 2 are the presence of a \u03b72- bound thioxanthone in addition to a coordinated thioxanthoxide resulting from a hydrogen adding to the ketone carbon of the formal thioxanthone scale\" fill=\"currentColor\" stroke=\"none\">CHtBu indicated that both methyl and neopentylidene ligands have undergone dehydrocoupling in 1. To circumvent the Wittig-like reaction observed between 1 and thioxanthone, we resorted to a ligand that lacked the ketone unit but which could be resistant to alkyl and alkylidene units. Treatment of 1 with two equiv. of 2,2\u2032-bipyridine (bipy) in benzene over 24 hours resulted in the formation of H2C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CHtBu along with the titanium complex (PNP)Ti(bipy)(bipyH) (3), that was isolated in 33% yield , which we propose contributes to the violet color.3 having a Ti(iii) radical antiferromagnetically coupled to a bipy\u02d9\u2013.Complex xanthone , left. T3% yield . Akin toly bipyH , right. 2 and 3, as well as propose the most likely pathway to C\u2013C bond formation, we conducted isotopic labelling studies using the 13C and 2H isotopomers (PNP)Ti PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CHtBu(13CH3) (1-13C) and (PNP)Ti PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CHtBu(CD3) (1-D3), respectively, with 2 equiv. of bipy and thioxanthone.1-13C irrefutably revealed the formation of H213C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CHtBu and 3 scale\" fill=\"currentColor\" stroke=\"none\">CHtBu (3-D1) based on a combination of 1H, 13C, and 2H NMR spectra.2C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CHtBu and that methyl migration to the neopentylidene most likely occurs by Path 1, as opposed to the less plausible Path 2 scenario involving tautomerization to B followed by neopentyl migration ; 1H NMR spectrum when 1-13C is used in the presence of bipy and thioxanthone (b); 1H NMR spectrum when 1-D3 is used in the presence of bipy and thioxanthone (c); and 2H NMR spectrum when 1-D3 is used in the presence of bipy and thioxanthone (d).To establish the origin of the inserted hydrogen in both Bu and 3 whereas >CHtBu and to afford the intermediates B1 and T1 at 18.7 and 5.3 kcal mol\u20131, respectively. These six-coordinate complexes containing a methylidene-alkyl or a methyl-alkylidene moieties may then undergo the reductive C\u2013C coupling reaction traversing the triplet transition states 3B1-TS and 3T1-TS associated with reaction barriers of 30.8 and 14.1 kcal mol\u20131, respectively. These calculations suggest that the reductive C\u2013C coupling is most easily initiated from complex 1, rather than its tautomer B consistent with the isotopic labeling studies (vide supra).To better understand the mechanism, quantum chemical calculations based on density functional theory (DFT) were carried out. The free energy profiles for the two possible pathways of reductive migration of the methyl moiety are illustrated in A at room temperature was investigated previously1. As shown in T1, which undergoes irreversible reductive methyl migration to form 3T2. Interestingly, we found that the reduction of the metal is accompanied by a singlet to triplet spin-crossover, as the newly formed Ti(ii)-d2 center adopts a high-spin triplet configuration. In good agreement with experimental results, the barrier of this key step associated with 3T1-TS is only 14.1 kcal mol\u20131, suggesting that the methyl migration will be much faster than \u03b1-hydrogen abstraction to produce CH4 and A, which typically requires much higher activation energies in excess of \u223c30 kcal mol\u20131. Such a low barrier for a C\u2013C forming reaction is rareThe activation of methane by T1, the T-ligand is weakly bound with a Ti\u2013O distance of 2.61 \u00c5 and is arranged in trans disposition to the alkylidene fragment. As the reductive C\u2013C coupling traverses through the transition state 3T1-TS, significant structural and electronic rearrangements take place. First and foremost, the T-ligand binds much more tightly displaying a Ti\u2013O distance of 1.90 \u00c5, whereas the double-bond between Ti and the alkylidene-carbon is notably lengthened from 1.85 to 1.99 \u00c5. The T-ligand exerts a strong trans-effect by removing electron-density from the Ti-alkylidene bond into a stronger Ti\u2013O \u03c3-bond and allowing for an easier activation of the Ti PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond.3 bond is formally cleaved to transiently give a methyl-anion, which may attack the Ti-alkylidene double-bond. As shown in 1T1-TS. The two main orbital interactions are drawn as MO-A, the in-phase combination of the methyl lone-pair orbital with the \u03c0-orbital of the Ti-alkylidene moiety, and as MO-A*, the corresponding antibonding combination. Both orbitals are of course occupied, since these are interactions of two filled fragment orbitals. We were unable to locate this putative transition state, because the \u03c0*-orbital of the thioxanthone ligand T\u2013\u03c0* is the lowest unoccupied molecular orbital (LUMO) of the T1 complex and will also be low enough in energy in the putative 1T1-TS which will be lower than MO-A*. Consequently, calculations naturally converge to a state where an intramolecular electron-transfer from the highly unfavorable MO-A* orbital to the low-lying T\u2013\u03c0* orbital will afford a much more favorable electronic structure. As the T\u2013\u03c0* orbital places the electron far away from the metal center, the metal-containing frontier orbitals will move to lower energies, giving the final orbital energy ordering found in 3T1-TS. What was labeled as MO-A is found in 3T1-TS as HOMO\u20133 at \u20136.62 eV and the corresponding antibonding combination, conceptually labeled MO-A*, is found as SOMO1 at \u20134.29 eV (The electronic distortion accompanying the spin-crossover is illustrated in \u20134.29 eV . Interesiii)-complex formally. In order to test the mechanistic role of the thioxanthone ligand identified in our calculations, we carried out a series of experiments. First, phosphine (PMe3) and pyridine ligands were used as exogenous ligands with the expectation that they will be unable to function in a similar manner as an electron acceptor if the computational results are correct. And, indeed, when complex 1 is treated with either ligand only gave activation of solvent is observed via \u03b1-hydrogen abstraction to form CH4 and alkylidyne A.\u20131 and 35.0 kcal mol\u20131, respectively, emphasizing the magnitude of the impact that the electronic reorganization has on the transition state energy. Details are given in the ESI.This conceptual MO-analysis is helpful, because it provides a compelling and simple explanation for why the computed barrier for the reductive C\u2013C coupling is so low. The thioxanthone ligand acts as a temporary electron storage unit where one of the electrons can be placed during the C\u2013C coupling reaction. Once the insertion completes and the C\u2013C bond is formed, the SOMO1 becomes a classical Ti-centered frontier orbital to afford the one-electron reduced Ti(L)M scale\" fill=\"currentColor\" stroke=\"none\">CH2)(CH3)]+ can engage in methyl migration to form a C\u2013C bond and proposed that a subsequent \u03b2-hydride elimination may generate H2C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CH2 and [M\u2013H].3T2 and found the transition state 3T2-TS to have a barrier of 29.3 kcal mol\u20131. In search of an alternative, lower energy transition state, we considered the singlet spin state analogue T3, which was found 1.2 kcal mol\u20131 lower in energy. And, indeed, we were able to locate the transition state T3-TS that gives a barrier of 25.0 kcal mol\u20131 for the dehydrogenation of the methyl group on the singlet potential energy surface. Interestingly, this transition state does not lead to the anticipated \u03b2-hydride elimination to give a metal-hydride. Instead, the hydride is transferred concertedly to the carbonyl-carbon of the thioxanthone functionality, as illustrated in T4 is formed. Our calculations suggest that this step is most difficult energetically with a barrier of 25 kcal mol\u20131, which is in good agreement with the experimental observation that this reaction does occur at room temperature. The final steps of the reaction involve product release and addition of another equivalent of the thioxanthone substrate, where either the singlet or triplet spin configurations are adopted to lower the total energy.As illustrated in d that , which is expected to quickly decompose in solution. Mechanistically, the Wittig reaction will likely invoke a [2 + 2] cycloaddition between the alkylidene and the ketone, which may subsequently undergo bond metathesis, as illustrated in \u20131, as the transition state 3T1-TS is lower in energy than W2-TS. Curiously, the calculated energy ordering of the two analogous transition states are reversed when X is used and the Wittig-like reaction pathway is predicted to be favored by \u223c2 kcal mol\u20131, as shown in As mentioned above, the Ti-alkylidene intermediate T1 \u2192 3T1-TS and X1 \u2192 3X1-TS. With T, this transformation is 6.9 kcal mol\u20131 uphill, whereas 9.4 kcal mol\u20131 is found for X. The presence of the sulfur atom in T makes the C\u2013C coupling transition state 2.5 kcal mol\u20131 lower electronically when compared to that of xanthone. This energy difference can be broken down into chemically meaningful components, as shown in T1 and X1, we calculated the \u201csnap dissociation energy\u201d where the (thio)xanthone and the titanaalkylidene fragments are dissociated without each of the fragments being allowed to change their structure, which were 18.3 and 19.7 kcal mol\u20131, respectively. Next, we evaluated the energy required to change the geometry of the Ti-alkylidene fragment to that found in the transition state and they were 46.5 and 46.6 kcal mol\u20131, respectively, marked as [Ti] \u2192 [Ti]* in \u20131, respectively. Lastly, the structural distortion of the T or X ligands was found to by uphill by 8.9 and 8.0 kcal mol\u20131, respectively. Taken together, these energy components add to afford 177.2 and 176.5 kcal mol\u20131, respectively, and represent the energy that must be invested to take the intermediates T1 and X1 to the C\u2013C coupling transition states. It is interesting that all the slight differences cancel and the energetic costs are essentially identical with the numerical difference being only 0.6 kcal mol\u20131.In order to better understand the origin of the computed energy differences, we first examined the different components of the solution phase free energies and found that the entropy and solvation energy components do not give any meaningful difference. Next, we analyzed the electronic energy differences by deconstructing the total energies into chemically meaningful energy components, as illustrated in \u20131 for thioxanthone and xanthone, respectively. The interaction energy between the two molecular fragments are computed to be \u2013148.1 and \u2013147.4 kcal mol\u20131, respectively. Obviously, the most important difference in energy stems from the reduction of the T and X substrates, which act as redox-active ligands and temporarily accommodate one electron, as explained above. In ii)-center becomes formally a Ti(iii)-center with one electron moved to the ligand-based SOMO. Comparing T to X, it is easy to understand the energetic difference discussed above. The contribution of the 3px orbital of sulfur is 6.0%, whereas 2px of oxygen contributes only 3.1%. This result of course reflects on the fact that sulfur is much more polarizable than oxygen and, thus, better in accommodating the excess charge than oxygen would. And whereas this difference is small, it is responsible for lowering the transition state by \u223c2.6 kcal mol\u20131 when T is employed compared to when X is used. This energy difference is enough to invert the energetic ordering of the transition states of the olefination and Wittig-like reaction, as illustrated in As represented in \u2013, but participates actively in the C\u2013C coupling by acting as a reservoir of the excess electron density at the transition state. The structurally related X substrate is incapable of promoting the C\u2013C coupling, because the lack of the sulfur heteroatom reduces its ability to accommodate the excess electron thus resulting in a higher transition state of \u223c2.6 kcal mol\u20131. The carbonyl functionality present in the substrate gives rise to a competing reaction channel, where a [2 + 2] cycloaddition may afford a Wittig-like product. Our calculations show that the barriers of this reaction are very similar to those of the C\u2013C coupling reactions, especially in the case of T. Such an additional redox stabilization of the T ligand can push the reaction to be more selective for C\u2013C coupling, whereas in X only the [2 + 2] cycloaddition product is observed.In conclusion, our computational studies highlight how spin-crossover at the metal site and the redox-activity of the substrate work in concert to couple a methyl group to an metalla-alkylidene with a very low barrier. This process leads ultimately to the olefination of methane, and requires the removal of two electrons and two protons overall. We found that T is capable of acting not only as the sacrificial oxidant that becomes reduced during the process to form TH1 to form Y1, where one phosphine arm of PNP ligand must dissociate to accommodate the chelating nature of the bipy ligand. In the C\u2013C coupling transition state 3Y2-TS the bipy functionality acts as an electron-reservoir and becomes formally reduced, confirming our expectation that bipy is a competent redox-active ligand. Unlike what was observed previously, the C\u2013H activation is accomplished via a classical \u03b2-hydride elimination mechanism when the transition state 3Y2-TS is traversed with a calculated barrier of 26.8 kcal mol\u20131, which produces the olefin product H2C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CHtBu. This barrier is slightly higher than the \u223c25 kcal mol\u20131 observed for T and is in good agreement with the observation that with bipy, the reaction is slower. Once 3Y3 is formed, it may bind another equivalent of bipy to afford 3Y4. Finally, the hydride migrates to the newly added bipy functionality traversing the transition state 3Y4-TS, and a spin-crossover to the singlet surface affords the diamagnetic final product complex Y6. Thus, the bipy substrate behaves as expected based on the general mechanistic understanding and is capable of promoting the methane olefination reaction, albeit with a slightly higher barrier and with minimally different mechanistic features that were easy to be anticipated.Given the detailed insights about the mechanism summarized above for T and X, bipy substrate offers several interesting features that will enhance our understanding of the mechanism. First, the lack of the carbonyl functionality prevents Wittig-like chemistry. Second, the bipy ligand is widely known to be redox active and we anticipate the that it too may act as an electron reservoir just like thioxanthone and, third, we expect that the lack of flexibility in structure and binding geometry will limit the possible spin-crossover scenarios. Thus, we examined the mechanism of methane olefination using bipy in detail and the computed reaction energy profile is shown in 4 can be activated by (PNP)Ti PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CHtBu(CH2tBu) via transient titanium alkylidyne intermediate at room temperature to generate (PNP)Ti PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CHtBu(CH3) and how methyl migration can be promoted with exogenous redox-active ligands to ultimately yield the dehydrocoupled product H2C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CHtBu. In addition, whereas thioxanthone resulted in the formation of H2C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CHtBu via reductive C\u2013C coupling, the xanthone ligand only produced the olefin from a Wittig-like reaction. By combining computational and experimental methods of mechanistic inquiry, we revealed a complete pathway that unifies experimental observations and computational results. We found that the critical role of thioxanthone and bipyridine is to become redox active during the course of the reaction and accommodate an electron to enable reductive methyl migration to form a C\u2013C bond and ultimately H2C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CHtBu. The methane olefination is therefore facilitated by the redox-active ligand which acts as an electron reservoir to avoid a filled\u2013filled interaction between the two Lewis basic fragments in the putative singlet transition state. As the C\u2013C coupling takes place, one electron from the Ti-alkylidene \u03c0-orbital is removed and placed in the \u03c0*-orbital of the redox-active ligand. Experimentally observed chemoselectivity between thioxanthone and xanthone was also scrutinized and explained using fragment analysis. The resulting C\u2013C coupled product then forms the olefin H2C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CHtBu by hydride elimination. In the case of thioxanthone, \u03b2-hydrogen abstraction promotes olefin formation whereas for bipy, the more classical \u03b2-hydride elimination ensues. Several spin-crossover events are proposed along the reaction trajectory that helps to lower the energies of intermediates and transition states. Our strategy therefore provides a mild route to make C\u2013C bonds with methane using an electropositive base metal that generally do not engage in two-electron redox processes.We have shown how CHThere are no conflicts to declare.Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "Theory meets experiment for the simplest model of alcohol\u2013alkene hydrogen bonding and both support a close to harmonic description. \u20131), the small change in diagonal anharmonicity (\u20133 cm\u20131) and the overtone intensity attenuation (2 \u00d7 102-fold) together with theoretical predictions for the preferred structural arrangement and the zero-point-corrected dissociation energy (8 kJ mol\u20131) may thus be regarded as definitive reference values for related systems and for more approximate computational methods. In particular, MP2 calculations are shown to fail for this kind of weak intermolecular interaction.An FTIR spectroscopic study of the elusive hydrogen-bonded methanol\u2013ethene complex, the most elementary example for weak intermolecular alcohol hydrogen bonding to a \u03c0 cloud, is presented. By isolating the complex in a supersonic jet, the rigorous comparability to high-level quantum chemical calculations is ensured. In stark contrast to classical hydrogen bonds, experimental overtone analysis reveals the harmonic oscillator approximation for the OH red shift to be accurate. Harmonic calculations up to explicitly correlated local coupled-cluster level are thus found to agree very well with experiment. The experimental OH values for the red shift (45 cm This holds not only for strong hydrogen bonds to heteroatoms, but also for weak OH\u00b7\u00b7\u00b7\u03c0 interactions which have been associated with olfactory processes.via overtone bands\u20131 red shift in the prototypical methanol dimer into its harmonic and anharmonic contributions, and high-level quantum chemical calculations have shown that many popular theoretical methods are inadequate for a quantitative description of the harmonic component.Quantum chemical calculations are customarily used to suggest structural motifs, dissociation energies and assignments to observed spectral features. Direct comparison between theory and experiment is typically hampered by the fact that anharmonic vibrational treatments are challenging except for small systems and rather simple methods. In addition, an experimental determination of anharmonicity PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond is perpendicular to the mirror plane of the methanol molecule through which a stream of helium is directed, and by admixture of ethene in helium stored in a gas cylinder at 50 bar.The jet-FTIR experiments were carried out using the \u201cfilet\u201d jet, which has been described in detail elsewhere.2.2Quantum chemical calculations were carried out using the MOLPRO 2012.1 and GAUSGAUSSIAN09 was used for canonical MP2 and B2PLYP-D3BJ calculations cc-pVnZ,nZ\u201d. For the explicitly correlated calculations, the VDZ-F12 basis set was used.nZ/JKFITnZ/MP2FIT38Most In the current study, we refer to the LCCSD(T*)-F12a(int)/VDZ-F12 method as our benchmark level of theory, as it has been found to be essentially converged to the basis set limit in the methanol dimer33.1PS = 0.75 bar. Lower stagnation pressures down to 0.40 bar were also used to decrease the amount of larger aggregates. We identify the mixed ME dimer band at 3641 cm\u20131 .20One further analysis involves the observed intensities of the fundamental and overtone bands, with the fund\u2009:\u2009ot ratio predicted to increase with stronger hydrogen bonds . While this is qualitatively expected for a weak OH\u00b7\u00b7\u00b7\u03c0 bond, it represents an interesting case where the observable red shift can be explained to a good approximation by harmonic effects alone, given that diagonal and off-diagonal anharmonic contributions are small and mutually canceling. Together with the sensitive ethene torsion preference, the ME dimer thus provides a nice accuracy test for quantum chemical methods without the need to evaluate anharmonic effects.One important contribution to the overall experimental OH red shift in the methanol homodimer is the anharmonic cross-term that couples the stretching motion and the hindered rotation (libration) in the dimer. Since the latter motion tends to weaken the hydrogen bond, xOH,i of the donor OH stretching vibrations in MM and ME are given in Perturbational anharmonic treatments are available in the GAUSSIAN program package\u03bd\u0303OH = 3564 cm\u20131. Closer inspection reveals that this is in part due to the difficult ethene torsion which is predicted at a harmonic wavenumber of \u03c9tors \u2248 1 cm\u20131. While the corresponding stretching-ethene torsion coupling term xOH,E-tors amounts to about 0.5 and \u20130.03 cm\u20131 in the robust B2PLYP-D3BJ/VTZ and MP2/VTZ calculations, respectively, it is \u201398 cm\u20131 at this faulty level of theory. We attribute this to a BSSE effect caused by diffuse functions on the hydrogen atoms. When neglecting this error, the overall MP2/aVTZ anharmonic correction is about \u2013177 cm\u20131, in agreement with the robust calculations ,Q in the methanol monomer and the two dimers. We fit a modified Morse potential of the formC and b1 through b5 left free in the fit. We refrain from denoting the prefactor as a dissociation energy, since the resulting potential is not strictly dissociative anymore. Solutions to the vibrational Schr\u00f6dinger equation were found by numerical variational calculations with a basis set of Gaussian functions distributed along the coordinate Q, using the reduced masses from the respective normal modes. The results are displayed in \u20132 cm\u20131 with respect to changes in number, spacing and width of the basis functions. Harmonic wavenumbers at the equilibrium position provide a consistency check with the normal-mode calculations, showing deviations up to 3 cm\u20131. We attribute these to fitting errors and assume the same variations for the calculated energy levels. Among our three test cases, the most interesting system is the methanol monomer, since the torsional perturbations of the OH oscillator \u2013 which cannot be captured with a 1-D model \u2013 are smallest there. The experimental wavenumbers are reproduced well by the benchmark method; conversely, the MM wavenumbers are underestimated due to the lack of this specific coupling. Still, the results are compatible with the blue-shifting xOH,lib \u2248 60 cm\u20131 coupling suggested by the VPT2 calculations. Overall, the diagonal anharmonicities of the OH stretching oscillator from variational and VPT2 calculations show a satisfying agreement with the experiment across our methods even when the corresponding harmonic results are unreliable.Estimating anharmonicity constants with our explicitly/locally correlated benchmark method is difficult due to the lack of a comparable implementation in the MOLPRO program package. We thus calculated potential energy curves along the (donor) O\u2013H stretching normal modes De = 11.4 kJ mol\u20131. This corresponds to a variation of only 0.5 kJ mol\u20131 when compared to the double-zeta result. It shows the good convergence of the value relative to the basis set. Given that these are all coupled cluster values, the error bar for De should be around 1 kJ mol\u20131. This is a rather conservative estimate. Adding the harmonic zero-point energy corrections, we obtain a value of Dh0 = 8.2 kJ mol\u20131. In order to obtain a more reliable estimate of the spectroscopic dissociation energy, accurate anharmonic calculations for the zero-point energy would be required. However, these are extremely challenging given the large amplitude motions present in the system.As previously noted, the computed dissociation energies for the methanol\u2013ethene system can be found in 4\u03bd\u0303OH = 45 cm\u20131 from the monomer reference is reduced by about 60% from that of the homodimer. The weakness of this prototypical OH\u00b7\u00b7\u00b7\u03c0 contact is further attested by the minute change in diagonal anharmonicity of \u0394xOH,OH \u2248 \u20133 cm\u20131 and moderate 170(70)-fold intensity attenuation of the overtone with respect to the fundamental.We have recorded FTIR spectra of methanol\u2009:\u2009ethene mixtures in supersonic expansions, assigning the fundamental and overtone transitions of the mixed dimer. The observed OH stretching red shift \u2013\u0394\u03c9OH = 45 cm\u20131 which coincides with the experimental anharmonic value. Assuming the chosen method to be robust, the observed wavenumber shift is thus mostly a harmonic effect, indicating that diagonal and off-diagonal anharmonic corrections closely cancel each other. As in the methanol homodimer,\u20131, providing another measure for the weakly perturbing character of the intermolecular interaction. Likewise, the harmonic zero-point dissociation energy at our best level of theory is Dh0 = 8.2 kJ mol\u20131, 55% less than in the methanol dimer (Dh0 = 18.3 kJ mol\u20131).\u20131 for the spectroscopic dissociation energy of ME appears justified. Microwave verification of the subtle structural preference of the methanol\u2013ethene complex for a perpendicular arrangement of the C\u2013O and C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C axes would be welcome.High-level quantum chemical calculations with local and explicit electron correlation treatment predict a harmonic red shift of \u2013\u0394We reiterate our previous findings that the MP2 method is inadequate for harmonic wavenumber predictions in alcoholic hydrogen bonds, significantly overestimating the red shift in canonical and local correlation treatments. However, SCS-LMP2 fares well in this regard both for the weak OH\u00b7\u00b7\u00b7\u03c0 methanol\u2013ethene and stronger OH\u00b7\u00b7\u00b7O methanol\u2013methanol contacts, at the well-known45"} +{"text": "A Ru/La0.5Ce0.5O1.75 catalyst pre-reduced at an unusually high temperature (650 \u00b0C) catalyses ammonia synthesis at a high rate under mild conditions. 0.5Ce0.5O1.75 pre-reduced at an unusually high temperature (650 \u00b0C) catalysed ammonia synthesis at extremely high rates under mild conditions; specifically, at a reaction temperature of 350 \u00b0C, the rates were 13.4, 31.3, and 44.4 mmol g\u20131 h\u20131 at 0.1, 1.0, and 3.0 MPa, respectively. Kinetic analysis revealed that this catalyst is free of hydrogen poisoning under the conditions tested. Electron energy loss spectroscopy combined with O2 absorption capacity measurements revealed that the reduced catalyst consisted of fine Ru particles (mean diameter < 2.0 nm) that were partially covered with partially reduced La0.5Ce0.5O1.75 and were dispersed on a thermostable support. Furthermore, Fourier transform infrared spectra measured after N2 addition to the catalyst revealed that N2 adsorption on Ru atoms that interacted directly with the reduced La0.5Ce0.5O1.75 weakened the N PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N bond and thus promoted its cleavage, which is the rate-determining step for ammonia synthesis. Our results indicate that high-temperature pre-reduction of this catalyst, which consists of Ru supported on a thermostable composite oxide with a cubic fluorite structure and containing reducible cerium, resulted in the formation of many sites that were highly active for N2 reduction by hydrogen.Ammonia is an important feedstock for producing fertiliser and is also a potential energy carrier. However, the process currently used for ammonia synthesis, the Haber\u2013Bosch process, consumes a huge amount of energy; therefore the development of new catalysts for synthesising ammonia at a high rate under mild conditions (low temperature and low pressure) is necessary. Here, we show that Ru/La Approximately 60% of the energy consumed by the process is recovered and stored as enthalpy in the ammonia molecule; but the remaining energy is lost, mostly during hydrogen production from natural gas, ammonia synthesis, and gas separation. The development of methods for reduction of the energy used by this process has been the goal of a considerable amount of research. PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N bond of N2 (945 kJ mol\u20131).2, which weakens the N PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N bond and promotes its cleavage.2O.+, Ru, and MgO possesses high ammonia-synthesis activity2+, Ru, and activated carbon has been used in industrial-scale commercial processes.24Al28O64]4+(e\u2013)4 (Ru/C12A7:e\u2013) and Ru/Ca(NH2)2, also show high ammonia-synthesis activity.2)2 is higher than the activities of any previously reported Ru catalysts, as well as the activities of 3d transition metal\u2013LiH composites, which are a new class of non-Ru ammonia-synthesis catalysts.Ammonia has been synthesised under ambient conditions with organometallic catalysts, but strong reducing agents and proton sources are generally needed, and the ammonia production rate is too low for practical applications.et al. found that rare earth oxides, such as CeO2 and La2O3, are effective supports for Ru catalysts.2O3 exhibits high ammonia-synthesis activity.et al. reported that the rate of ammonia synthesis over Ru/CeO2 is high when the catalyst has been pre-reduced at 500 \u00b0C.4+ is reduced to Ce3+, and thus an electron is transferred to Ru and then to adsorbed N2 molecules. However, the ammonia synthesis rate is slower over a catalyst that has been pre-reduced at a temperature higher than 500 \u00b0C, owing to structural changes associated with sintering of the support. To increase the specific surface area of the catalysts, as well as the reducibility of the Ce4+, various investigators have used composite-oxide supports, such as CeO2\u2013La2O3,2,2,2\u2013ZrO2,2O3\u2013CeO2,et al., the pre-reduction temperature for these catalysts is kept below 500 \u00b0C to minimize aggregation of the Ru particles.26In the 1990s, Aika 0.5Ce0.5O1.75, a catalyst consisting of Ru supported on a La0.5Ce0.5O1.75 solid solution, which is a composite oxide of CeO2 and La2O3. After pre-reduction at the unusually high temperature of 650 \u00b0C, the catalyst exhibited high ammonia-synthesis activity at reaction temperatures from 300 to 400 \u00b0C; the activity was the highest among oxide supported Ru catalysts and comparable to that of the most active Ru catalysts reported to date. The thermostable oxide support, which had an average composition of La0.5Ce0.5O1.64 after pre-reduction at 650 \u00b0C, consisted of fine Ru particles strongly anchored to the reduced support and had numerous active Ru sites. The dependence of the catalyst structure and state on the reduction temperature was elucidated by means of various characterisation techniques, including energy electron loss spectroscopy (EELS) and scanning transmission electron microscopy (STEM). This catalyst has the advantages of being easy to prepare and stable in the atmosphere, which makes it easy to load into a reactor.Herein, we report the ammonia-synthesis activity of Ru/La0.5Ce0.5O1.75 was measured at 1.0 MPa after pre-reduction of the catalyst at 450, 500, 650, or 800 \u00b0C. Under the reaction conditions, the equilibrium ammonia-synthesis rate and the ammonia yield at 400 \u00b0C are 127 mmol g\u20131 h\u20131 and 7.91%, respectively. At all reaction temperatures, the ammonia-synthesis rate was markedly higher over the catalyst pre-reduced at 650 \u00b0C than over the catalysts pre-reduced at 450 \u00b0C reached 31.3 mmol g\u20131 h\u20131 and was much higher than the rates over the other tested catalysts, such as Ru/CeO2_650red (17.2 mmol g\u20131 h\u20131) and Ru/La2O3_500red (10.8 mmol g\u20131 h\u20131), whose supports each contain one of the rare earth elements in La0.5Ce0.5O1.75, and Ru/Pr2O3_500red (15.7 mmol g\u20131 h\u20131),0.5Ce0.5O1.75_650red was approximately 7.6 times that over Cs+/Ru/MgO_500red (4.1 mmol g\u20131 h\u20131), a well-known catalyst that is often used as a benchmark and that is more active than Ba2+/Ru/activated carbon0.5Ce0.5O1.75_650red was comparable to that over 10 wt% Ru/Ca(NH2)2 .24We also compared the ammonia-synthesis rates over various other supported 5 wt% Ru catalysts at 350 \u00b0C and 1.0 MPa . Each of0.5Ce0.5O1.75_650red and Cs+/Ru/MgO_500red with the use of the rates at 300, 325, 350, and 375 \u00b0C (Ea) calculated for Ru/La0.5Ce0.5O1.75_650red (64 kJ mol\u20131) was much lower than that for Cs+/Ru/MgO_500red (100 kJ mol\u20131), and was comparable to that reported for 10 wt% Ru/Ca(NH2)2 (59 kJ mol\u20131).0.5Ce0.5O1.75_650red was responsible for the high ammonia-synthesis rate.We prepared Arrhenius plots for ammonia-synthesis reactions catalysed by Ru/Lad 375 \u00b0C . To avoi+/Ru/MgO_500red.2 molecules (a phenomenon referred to as hydrogen poisoning), which is a typical drawback of conventional Ru catalysts.0.5Ce0.5O1.75_650red was 13.4 mmol g\u20131 h\u20131, which is the highest value reported for Ru catalysts to date; and the rate increased to 31.3 and 44.4 mmol g\u20131 h\u20131 when the pressure was increased to 1.0 and 3.0 MPa, respectively. Hence, we assumed that hydrogen poisoning did not occur over Ru/La0.5Ce0.5O1.75_650red at the tested temperature. To confirm this assumption, we performed kinetic analysis at 350 \u00b0C and 0.1 MPa. For that purpose, reaction orders for N2, H2, and NH3 were determined with the assumption of the rate expression (1) scale\" fill=\"currentColor\" stroke=\"none\">N bond cleavage, which is the rate-determining step for ammonia synthesis, is relatively promoted over Ru/La0.5Ce0.5O1.75_650red. Moreover, stability of Ru/La0.5Ce0.5O1.75_650red at 350 \u00b0C under 3.0 MPa was examined. When an inline gas purifier was installed for cleaning the H2/N2 mixture , and the elemental distributions and valence states of the Ce ions were evaluated by means of STEM spectrum imaging of simultaneous energy dispersive X-ray (EDX) mapping and EELS. Because the elemental states and the structure of the catalyst might be changed by exposure to air, we carried out these analyses in the absence of air using a special holder with a gas cell to transfer the sample from an inert gas environment to the inside of the TEM column. Comparison of the high-angle annular dark-field (HAADF) STEM images and 901.8 eV and at 885.6 and 903.5 eV, respectively.4+ predominated in the centre region (green square) of the thick catalyst particle, whereas Ce3+ predominated at the edge (blue square) of the thick catalyst particle, and the proportion of Ce3+ was highest at the centre (red square) of the thin catalyst particle. EELS maps of Ce in the thick and thin particles clearly showed the same tendency; that is, Ce3+ was enriched near the surface of the catalyst particles pattern of Ru/La2O3, many peaks assignable to LaOOH and La(OH)3 were observed, in addition to small peaks assigned to La2O3 molar ratios for fresh Ru/LayCey1\u2013Oy2\u20130.5 (0 \u2264 y \u2264 0.5) was linear after pre-reduction at 500 \u00b0C were finer than those that formed on La2O3 (mean diameter = 7.8 nm) and on CeO2 (mean diameter = 2.4 nm) see . In addi5_500red was 3.5 Table S2. These r0.5Ce0.5O1.75 and decreased the specific surface area of the catalyst from 42 to 21 m2 g\u20131. On the other hand, the H/Ru ratio decreased gradually as the pre-reduction temperature was increased from 500 to 800 \u00b0C. Note that when the reduction temperature was increased from 500 to 650 \u00b0C, the H/Ru ratio decreased from 0.46 to 0.35, but the mean diameter of the Ru particles remained unchanged. These results indicate that the surface Ru atoms were partially covered with partially reduced support material, at least after reduction at 650 \u00b0C, owing to the SMSI phenomenon, which is consistent with the EDX and EEL spectra , and to the formation of oxygen vacancies. Specifically, the lattice parameter of the cubic fluorite structure of La0.5Ce0.5O1.75, as measured by in situ XRD analysis, increased from 0.5577 nm at room temperature to 0.5596 and 0.5603 nm after treatment with H2 at 500 and 650 \u00b0C, respectively and in the mean diameter of the Ru particles (to 2.7 nm) . PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N bond cleavage, which is the rate-determining step for ammonia synthesis over Ru/La0.5Ce0.5O1.75, we determined the state of the adsorbed N2 molecules by means of Fourier transform infrared (IR) spectroscopy. The IR spectra measured after addition of 14N2 or 15N2 to Ru/La0.5Ce0.5O1.75_500red and Ru/La0.5Ce0.5O1.75_650red at room temperature are shown in \u20131 and a broader peak at around 1700\u20131900 cm\u20131. Note that the wavenumber of the broader peak decreased from 1883 to 1844 cm\u20131 when the pre-reduction temperature was increased from 500 to 650 \u00b0C. In the spectra measured after 15N2 adsorption, the two peaks were observed at lower wavenumbers (2091 and 1819 cm\u20131) relative to those for the 14N2 spectra, and the wavenumbers were in good agreement with those estimated by consideration of the isotope effect:\u20131 \u00d7 (14/15)1/2 = 2091 cm\u20131 and 1883 cm\u20131 \u00d7 (14/15)1/2 = 1819 cm\u20131. Similar peak shifts ascribable to the isotope effect were observed in the spectrum after adsorption of 15N2 on Ru/La0.5Ce0.5O1.75_650red. Therefore, all of the peaks were assignable to the stretching vibration mode of N2 adsorbed in an end-on orientation on the Ru particles. The peak at 2164 cm\u20131, the location of which was independent of reduction temperature, was assigned to N2 adsorbed on Ru atoms that interacted only weakly with the reduced support scale\" fill=\"currentColor\" stroke=\"none\">N bond of N2 was weakened by the contribution of SMSI even after reduction at 500 \u00b0C, and when the reduction temperature was increased to 650 \u00b0C, the contribution of SMSI was greater. That is, the partially reduced support, which is enriched in electrons owing to the reduction of Ce4+ to Ce3+ and to the formation of oxygen vacancies, partially covered the Ru particles. As a result, electron transfer from the reduced support to the Ru metal was greatly enhanced, and the electrons were transferred to the antibonding \u03c0-orbitals of N2; thus, the N PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N bonds of N2 adsorbed on Ru atoms that interacted directly with the reduced support were further weakened. The ratio of the peak area of the higher-wavenumber peak to that of the lower-wavenumber peak decreased when the pre-reduction temperature was increased from 500 to 650 \u00b0C, which is consistent with an increase in the contribution of the SMSI.Our results indicate that the N\u20131) were abundant (H/Ru = 0.35) after pre-reduction at 650 \u00b0C explains the high ammonia-synthesis rate (31.3 mmol g\u20131 h\u20131) over Ru/La0.5Ce0.5O1.75_650red. In contrast, after pre-reduction at 800 \u00b0C, the Ru sites were very active (TOF = 0.108 s\u20131), but the number of active Ru sites was small (H/Ru = 0.11); thus the ammonia-synthesis rate over Ru/La0.5Ce0.5O1.75_800red (20.6 mmol g\u20131 h\u20131) was lower than that over Ru/La0.5Ce0.5O1.75_650red. Note that the specific surface area of Ru/CeO2_650red was only 20 m2 g\u20131, the mean diameter of the Ru particles was 3.1 nm, and H/Ru was 0.17, which indicates that sintering of the Ru particles and La0.5Ce0.5O1.75 was retarded in the case of Ru/La0.5Ce0.5O1.75_650red, and thus the H/Ru ratio for this catalyst remained high.These results demonstrate that pre-reduction at high temperature induced SMSI and enhanced the turnover frequency (TOF) but decreased the number of Ru active sites because the Ru particles became partially covered by partially reduced support. The fact that active Ru sites (TOF = 0.051 s2O and CO2) on the surface of the fresh catalyst are removed. However, pre-reduction at an excessively high temperature results in sintering, which decreases the number of active sites. Here, we found that 400\u2013450 \u00b0C was usually sufficient to reduce Ru3+. However, pre-reduction of Ru/La0.5Ce0.5O1.75 at the unusually high temperature of 650 \u00b0C produced a catalyst that showed a high ammonia-synthesis rate under mild reaction conditions . This catalyst consisted of fine Ru particles anchored on a heat-tolerant complex-oxidic support. During pre-reduction, the particle size of the Ru particles remained unchanged, but the particles became partially covered with partially reduced La0.5Ce0.5O1.75. A strong interaction between the Ru active sites and the reduced support accelerated the rate-determining step of ammonia synthesis, that is, N PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N bond cleavage. We suggest that this simple strategy for the design of Ru catalysts\u2014that is, using a thermostable composite oxide containing a redox-active rare earth element in a cubic fluorite structure as a support, and pre-reducing the supported catalyst at high temperature\u2014will lead to the development of a more energy efficient ammonia-synthesis process, thus reducing global energy consumption and facilitating the eventual use of ammonia as an energy carrier.Pre-reduction of conventional supported-metal catalysts is crucial for their activation, because active metal sites are formed on the surface by reduction of metal oxides, and because adsorbates (such as HThere are no conflicts to declare.Supplementary informationClick here for additional data file."} +{"text": "A chiral Lewis acid-promoted cyclopropanation using a phenyliodonium ylide as the carbene precursor was developed. An EPR spectroscopy study supported a stepwise biradical mechanism. A chiral Lewis acid-promoted enantioselective cyclopropanation using phenyliodonium ylide as the carbene precursor was developed. A variety of spirocyclopropane-oxindoles with contiguous tertiary and all carbon quaternary centers were obtained in excellent outcomes . EPR spectroscopy study supported a stepwise biradical mechanism. PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C double bond provides efficient access to optically active cyclopropane derivatives that are essential motifs in natural productsi)-, Rh(ii)-, Ru(ii)-, and Co(ii)-complexes with diazo compounds, permits such cylcopropanations in high yields and selectivities.2{(S)-nttl}4]i)-bis(oxazoline) complexes.via enantiocontrol of an olefin substrate in the carbene transfer step, but the difficulty of gaining high stereoselectivity and yield lies in the background reaction and the readiness of carbene dimer formation.The catalytic asymmetric cyclopropanation of a Cvia the carbene transfer could also give access to these targets.N,N\u2032-dioxide complexesN,N\u2032-dioxide could bind 3-alkenyl-oxindoles into a perfect chiral environment, benefiting the cyclopropanation of a free carbene generated from spontaneous decomposition of phenyliodonium ylide malonate 2 complex catalyzed asymmetric cyclopropanation of 3-alkenyl-oxindoles with phenyliodonium ylide. Excellent diastereo and enantioselectivity were achieved for a variety of substituted spirocyclopropane-oxindoles under mild reaction conditions. Free carbene species formation was confirmed from EPR and HRMS analysis of the reaction system.Spirocyclopropane-oxindoles are versatile building blocks for the synthesis of natural products and pharmaceuticals.malonate . Herein,E)-N-Boc-3-alkenyl-oxindole 1a as the model substrate with phenyliodonium ylide malonate 2 in CH2Cl2 at 25 \u00b0C. The known enantioselective activation of the substrate 1a by metal complexes of chiral N,N\u2032-dioxide 2L-PiPr inspired us to examine it as the supporting ligand.2 obtained a trace amount of the desired spirocyclopropane-oxindole 3a (2 and Zn(OTf)2 enabled access to the product 3a in moderate yields and enantioselectivities yet high diastereoselectivities . The results were appealingly consistent with our Lewis acid-activation pathway. The following survey of the ligands coordinated with Ni(OTf)2 showed that other N,N\u2032-dioxides, including L-PiPh, 2L-PrPr and 2L-RaPr, were less competent than 2L-PiPr in terms of the reactivities and enantioselectivities (entry 4 vs. entries 5\u20137). The reaction was further optimized through systematic study of several parameters. An improved enantioselectivity and moderate yield was observed in toluene (entry 8). An encouraging 85% yield with 98% ee of the product was given in Et2O (entries 9 and 10). It was suspected that Et2O may effectively prevent dimerization of the phenyliodonium ylide, but the poor solubility of the catalyst in Et2O led to the low performance of this catalytic system. Considering this question, a mixed solvent system was tested to improve the situation. A mixed solvent of CH2Cl2/Et2O (v/v = 1\u2009:\u20094) was selected, and the asymmetric cyclopropanation gave a substantial improvement in catalytic yield with excellent diastereo and enantioselectivity .Our investigation commenced with the cyclopropanation of (E)-3-ester-substituted methyleneindolinone derivatives was next surveyed under the optimized reaction conditions . The electron-donating substituents at the C5-position of the oxindole ring were well-tolerated, giving slightly higher enantioselectivities than the electron-withdrawing ones . A 6-bromo-substituent resulted in a good yield . 3-Acyl substituted methyleneindolinone derivatives produced the corresponding spirocyclopropane products 3m\u2013p in excellent yields and enantioselectivities at lower reaction temperature. The alkyl-substituted alkenes, such as propyl, cyclohexyl and cyano, could also undergo this reaction smoothly, affording the desired adducts in moderate yields and good enantioselectivities . However, the reaction between (E)-1-Boc-3-tert-butylideneindolinone and phenyliodonium ylide 2 remained challenging due to the steric hindrance (4a\u2032\u2032).The generality of the catalytic cyclopropanation with a range of . It is noteworthy that no diastereomers were detected in most cases (>19\u2009:\u20091 d.r.), except for benzo[d]dioxole substituted 4o and 2-naphthyl substituted 4p. The sense of diastereoselectivity in the latter two cases was appreciably decreased, and a trace amount of the diastereomer was confirmed by 1H NMR spectroscopy (19\u2009:\u20091 d.r.), whereas the enantioselectivity was unaffected. As a representative substituted, 3-benzylidene-indolinone underwent efficient cyclopropanation, giving 4q in excellent yields. The absolute configuration of the product 3k and 4b was determined to be by X-ray analysis.4r\u20134s). Benzofuran-2(3H)-one enabled access to the desired product with an excellent yield; however, the outcome of enantiocontrol was disappointing (4t). Compared with the N-Boc oxindoles, the loss of the necessary bidentate manner of two carbonyl groups might have led to poor chiral induction.However, the deprotection of the Boc-group of the 3-aryl-substituted methyleneindolinone derivatives occurred, which prevented the cyclopropanation process. Changing the ratio of the substrates stigated . The rea1a with phenyliodonium ylide malonate 2 was carried out on a gram scale. The desired product 3a was generated in 99% yield, >19\u2009:\u20091 d.r. and 99% ee through a Lewis acid catalyzed nucleophilic ring-opening reaction using aniline as the nucleophile -N-Boc-3-alkenyl-oxindole 1a and phenyliodonium ylide malonate 2 proceeded smoothly in the absence of the catalyst, giving the desired product in 40% yield; neither the N,N\u2032-dioxide nor Ni(OTf)2 could substantially enhance the reaction. The outcomes of the reaction were unaffected when carried out in the dark. Clearly, the catalytic system of N,N\u2032-dioxide\u2013Ni(OTf)2 is a ligand-accelerated process in view of the excellent yields previously discussed. The Ni(ii)-complex of 2L-PiPr has been confirmed by X-ray analysis in our early study.1a or phenyl substituted 3-alkenyl-oxindole 1b\u2032 to the metal cation of the chiral catalyst was detected from ESI-MS spectra. Peaks at m/z 1200.4629 and 1176.4937 were assigned to [Ni2+ + 2L-PiPr + 1a + TfO\u2013]+ and [Ni2+ + 2L-PiPr + 1b\u2032 + TfO\u2013]+, respectively 2 showed no signals, indicating that there is no unpaired electron on the nickel(ii) center due to the strong coordination of the supporting ligands. Interestingly, the EPR spectrum of the mixture of oxindole 1a and phenyliodonium ylide 2 with or without the catalyst exhibits a similar rhombic band and is centered around g = 2.003 2 initially. The cyclopropanation was slower than intersystem crossing to the more stable triplet carbene 3:C(CO2Me)2, which exhibits two unpaired electrons. In this circumstance, the cyclopropanation occurs through a stepwise mechanism involving an analogous biradical intermediate. The unresolved hyperfine structure implied the interaction of 3:C(CO2Me)2 with the substrate.To determine the carbene intermediate, the reaction system was further characterized by EPR spectroscopy. The EPR X-band spectrum of = 2.003 . The int = 2.003 vs.1b. Tvia a free carbene intermediate center in a bidentate manner with two carbonyl groups. The facial-control of the carbene addition was directed by the blocking of the amide unit underneath the ligand. Initially, the decomposition of the phenyliodonium ylide generated a more stable triplet carbene. It would prefer electronic addition to the outer C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond because of the low steric hindrance and the stability of the triplet biradical intermediate. Due to the steric hindrance of the substituents on the biradical intermediates, the C\u2013C bond rotation is slower than spin flip of the intermediate. Therefore, high diastereo and enantioselectivity of the products were given.Therefore, in view of the aforementioned consequences as well as the structures of the catalystrmediate . SubstraN,N\u2032-dioxide/Ni(OTf)2 complex exhibited excellent performance in the reaction of 3-alkenyl-oxindoles with phenyliodonium ylide malonate under mild reaction conditions. The desired spirocyclopropane-oxindoles with contiguous tertiary and all carbon quaternary centers were attained in high yields and stereoselectivities . At the same time, when the catalytic system was applied to other non-oxindolic olefins, coumarins were also able to provide the bridge ring derivatives with good yields and enantioselectivities. A stepwise biradical process is suggested based on EPR spectroscopy. Further application of iodonium ylides and chiral N,N\u2032-dioxide\u2013metal complexes in asymmetric transformations are underway.In conclusion, we have developed a new asymmetric catalytic strategy for cyclopropanation of olefins. The chiral Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "A complex with single, double and triple bonds between nitrogen and the same metal center has been synthesized, [NCr(NPh)(NPri2)2]\u2013. The complex shows differential activity, with some electrophiles attacking the imido and others the nitrido. t)(CHBut)(CH2But)(dmpe), has been prepared. The new complex is the nitrido, imido, amido anion [NCr(NPh)(NPri2)2]\u2013, which was structurally characterized with the [K(crypt-2.2.2)]+ counterion. The \u201cCr\u2013N 1-2-3\u201d complex was prepared from NCr(NHPh)(NPri2)2, which exists as this nitrido\u2013amido tautomer, rather than the bis(imido) Cr(NH)(NPh)(NPri2)2. By selection of electrophile, the nitrido\u2013imido salt K[NCr(NPh)(NPri2)2] can undergo reaction at either the imido or the nitrido to form unusual examples of nitrido or bis(imido) complexes.A nitrogen-based analogue of the Schrock and Clark \u201cyl-ene-yne\u201d complex, W(CBu The structure is shown schematically in 3Nitrogen-based complexes containing a variety of multiple bond types in the same complex are less explored. For example, while complexes with multiple imido groups are numerous, there are few examples of terminal nitrido\u2013imido complexes in the Cambridge Structural Database. One example of such a complex is the fascinating manganese-based nitrido complex [Li(OEti2)2] ] (2) in 76% yield as a dark red complex (1 with KH and 1 equiv. of (2.2.2)-cryptand in THF sequesters the potassium cation and liberates the anion for structural study. The amber product, [K(crypt-2.2.2)][NCr(NPh)(NPri2)2] (CrN123), was structurally characterized, and the anion is shown in Treatment of complex . The comCrN123, the average Cr\u2013NPri2 distance increased significantly over the value in 1 to 1.871(3) \u00c5, consistent with the stronger donor ability of imide relative to phenylamide. The Cr\u2013N(nitrido) distance, however, is the same as 1 within error. The N\u2013Cr\u2013N angles in CrN123 are all close to the tetrahedral angle and range from 106.0\u00b0 to 110.8\u00b0.In the solid-state structure of CrN123 exhibits fast diisopropylamide rotation consistent with imide being an extremely good donor as would be expected. Using 14N NMR spectroscopy one can easily distinguish the three different nitrogens in the complex along with the nitrogen in the cryptand at 560 ppm.In solution, cryptand at \u223c40 pt)(CHBut)(CH2But)(dmpe) and the anion of CrN123. The calculated W\u2013C bond orders for the neopentylidyne, neopentylidene and neopentyl groups were 2.65, 1.68 and 0.67, respectively. In the CrN123 anion, the Cr\u2013N bond orders to the nitrido, imido and amido were calculated as 2.84, 1.73, and 0.78, respectively, and are remarkably similar to the W\u2013C bond orders in the Schrock complex (CHBut)(CH2But)(dmpe) and the anion of CrN123 were examined by local Natural Resonance Theory (NRT) using NBO6, which are also shown parenthetically in trans-influencing alkylidene ligand having a lower order bond.In addition, Schrock's W equilibrium above. The preferred site of attack is the imide Ph](NPri2)2.The reactions of potassium salt he imide when mete.g., enolate attack by simple electrophiles generally occurs at oxygen.2 gave back 1 in 80% yield 2 (3).\u00b6The product from acyl chloride reaction was the same spectroscopically, but the product was not as clean as with acetic anhydride. That the electrophilic attack occurs at the imide rather than the nitride was definitively assigned by 14N NMR; 3 shows a distinct nitride resonance at 1011 ppm along with amidinate and diisopropylamide resonances at 402 and 203 ppm COMe bond of 1.971(2) \u00c5 relative to the diisopropylamide bonds of 1.823(2) \u00c5 due to the strongly electron-withdrawing group on the former. That the acyl group strongly interacts with the amido nitrogen can be seen in the short N\u2013C(acyl) bond distance of 1.348(3) \u00c5. There seems to be a weak Cr\u2013O(acyl) interaction as well with a distance of 2.753 \u00c5 pseudo-trans to the nitride with an N(nitrido)\u2013Cr\u2013O angle of 152\u00b0.Acyl-containing 3 gave a value of 15.09 kcal mol\u20131 for the N(Ph)C(O)Me amidinate fragment. This LDP value, for reference, is similar to chloride , but there may be steric influences in the LDP value resulting from the bidentate nature of the ligand, which would raise the LDP over its electronic value.An LDP measurement on a crude solution 2 with pivaloyl chloride leads to reaction at the nitrido nitrogen and formation of the green bis(imide) Cr[NC(O)But](NPh)(NPri2)2 (4), as shown in 14N NMR spectrum is definitive for this structure, with two imido resonances at 522 and 493 ppm Me and ClC(O)Bu4 is a rare, structurally characterized example of a transition metal imido complex bearing a carboxyl group on the nitrogen, and the first structurally characterized example of such a complex with chromium PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019NC(O)But unit is slightly but significantly longer than in the Cr PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019NPh moiety at 1.688(3) and 1.646(3) \u00c5, respectively. The average Cr\u2013NPri2 distance in 4 is similar to 1 at 1.838(3) \u00c5. The N\u2013Cr\u2013N angles are fairly close to tetrahedral and range from 111.6(1)\u00b0 to 106.3(1)\u00b0.Complex CrN123. As is often the case, exploration of the syntheses required for the production of the complex led to unusual intermediates as well, and, once formed, the nitrido\u2013imido\u2013amido complex exhibits interesting reactivity commensurate with its unusual structure.Here, we have described the first example of a complex with nitrogen triple, double and single bonds to the same metal center, Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "Quinoidal-vs.-aromatic synergy: Cyclopentadithiophene-vinylene oligomers with different sizes, in different oxidation states and as pro-aromatic quinoidals are studied considering balanced contributions of aromatic and quinoidal forms. H-cyclopentadithiophene vinylene repeating unit has been prepared and characterized by X-ray, electrochemical, spectroscopic and density functional theory methods. The oligomers in their neutral, oxidized and reduced forms have been investigated. The neutral compounds show a longer mean conjugation length than oligothiophenes and oligothiophene-vinylenes and display very rich redox chemistry with the stabilization of polycationic states of which the radical cations and dications are strong NIR absorbers, the latter displaying singlet diradicaloid character. An interesting complementarity between the sequence of aromatic-quinoidal structural segments in the radical cations and dications has been described and interpreted. Two derivatives with the 4,4 dihexyl-4H-cyclopentadithiophene vinylene unit, disubstituted either with electron donor, bis(triaryl amino) groups, or acceptors bis(dicyano-methylene) caps enforcing a quinoidal structure in the dithiophene-vinylene bridge, have been also synthesized and characterized. The radical cation of the triarylamine compound and the radical anion of the tetracyano compound similarly display hole and electron charge localization, or confinement, in the nitrogen and dicyano surrounding parts, or class II mixed valence systems, while their dication and dianion species, conversely, are open-shell diradical and closed-shell , respectively. The preparation of these new \u03c0-conjugated oligomers gives way to the realization of compounds with new electronic properties and unique structures potentially exploitable in organic electronics.A new series of \u03c0-conjugated oligomers based on the 4,4 dihexyl-4 Therefore, it is important to investigate the optoelectronic and structural properties of new oligomers as a function of oligomer length, ,CPDT unit alternating with double bonds to create a new oligomeric series from a dimer to a hexamer except for compound 5. In this case the synthesis was performed in 72% yield by using n-BuLi and DMF . This longest wavelength absorption band is due to a one-electron excitation from the HOMO to the LUMO (\u03bbmax for 6CPDTV.the LUMO . Whereas2CPDTV has been carried out by measuring the fluorescence spectra in solution at room temperature and at liquid nitrogen temperature (0 \u2192 S1 excitation (S1 \u2192 S0 emission). The small Stokes shift is an indication of the small conformational reorganization upon excitation, as it is expected for a molecule with only two degrees of dihedral rotation. At 77 K, the vibronic structure is better resolved and the Stokes shift has almost vanished due to minimization of motion around the vinylene bridge. In this context, the X-ray structure of 2CPDTV-Br has been resolved and is shown in 2\u2013Csp2 bond lengths (1.328(5)\u20131.455(5) \u00c5) consistent with a high degree of \u03c0-conjugation. In addition, nearly orthogonal orientations of the hexyl side chains effectively prevent \u03c0-stacking in the solid-state.An emission study of perature . The emi\u20131 and another one around 1400 cm\u20131. The 1600 cm\u20131 band is associated with the stretching vibration of the central C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C, or \u03bd scale\" fill=\"currentColor\" stroke=\"none\">C), which displays a progressive frequency downshift of 12 cm\u20131 from 2CPDTV \u2192 6CPDTV (8 cm\u20131 on 2CPDTV \u2192 3CPDTV) due to a progressive enlargement of the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C vinylene bonds upon increasing the chain length. This is a clear fingerprint of the increasing of \u03c0-electron conjugation. The most intense Raman band of the spectra appears around 1400 cm\u20131 and correspond to a \u03bd scale\" fill=\"currentColor\" stroke=\"none\">C) of the thiophene rings of the CPDT unit. This band also experiences a frequency downshift with increasing oligomer size and this trend is consistent with other \u03c0-conjugated systems fibers on mica. This self-assembly is driven by \u03c0\u2013\u03c0 intermolecular interactions and by the interplay with the HOPG. In this case the nearly co-planar structure of the \u03c0-conjugated core offers a large available \u03c0-surface which promotes the formation of these long fibers.For most of the nCPDTVs . The \u03bbmax of these absorptions are represented in n where both bands fit linearly, indicating that their spectral properties are the result of the effective extension of the \u03c0-electron structure in the radical cation state along the increasing molecular platforms (not the whole structure) when enlarging the molecular size.The oxidation processes have been analyzed by nCPDTV\u02d9+ are shown in \u03bd scale\" fill=\"currentColor\" stroke=\"none\">C) band, appearing by nearly 30 cm\u20131 lower than in the neutral case. This is a clear indication that the first one-electron oxidation greatly affects the central vinylene, due to the partial C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C \u2192 C\u2013C transformation, or weakening, of the double bond. Changes in the frequencies of the thiophene bands are much smaller than those in the vinylene stretching band. Nonetheless, by progressing to the radical cations of the longer oligomers, the frequency of the vinylene Raman band upshifts from 1564 to 1568 cm\u20131 on +3CPDTV\u02d9 \u2192 +4CPDTV\u02d9 due to the further dissemination of the polaronic structural defect in a larger \u03c0-conjugated system. This effect reaches a plateau on +5CPDTV\u02d9/+6CPDTV\u02d9 for which the deformation is almost constant.The Raman spectra of the \u03bbmaxversus 1/n scale\" fill=\"currentColor\" stroke=\"none\">C\u2013C \u2192 C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C\u2013C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C evolution.Additional one-electron oxidation of the radical cation produces the progressive disappearance of the pair of radical cation bands and the continuous rise of a strong absorption at wavelengths in between the two of the radical cations. The wavelength position of the rsus 1/n also fol2+6CPDTV where, as well as the strongest band at 2121 nm, another medium intensity band at 1178 nm is detected. This double-band pattern in the dications of oligothiophenes has been associated with the presence of polaron-pair defects, where the dication, instead of being embedded in one single structural bipolaron deformation, prefers to generate two radical cation structural defects by putting away the two charges.2+6CPDTV, thanks to its size and structure, is a candidate to stabilize the dicationic structure in a polaron-pair format rather than in a bipolaron, which is preferentially the case of the smaller oligomers, such as 2+3CPDTV.In addition, a new band appears at higher energies together with the strongest one just described. This is clearly seen in the case of nCPDTV2+ series (n = 4\u20136) are displayed in \u03bd scale\" fill=\"currentColor\" stroke=\"none\">C) band appears at 1568 cm\u20131 in 2+4CPDTV and displays the same frequency than the radical cation (+4CPDTV\u02d9 at 1568 cm\u20131) suggesting that the perturbation of the central vinylene is similar in the dication than in the cation. This is in consonance with the repulsion effect between the charges, which pushes these towards the terminal rings. In the two longer dications, 2+5CPDTV and 2+6CPDTV, the vinylene \u03bd scale\" fill=\"currentColor\" stroke=\"none\">C) bands are at almost the same values than in 2+4CPDTV (1567 cm\u20131 in both cases). The invariance of these Raman frequencies, among dications and also from the radical cations to dications of the longer oligomers, indicates that in the monovalent species, the structural alteration affects the central part of the molecule while in the divalent species these structural modifications do not happen in the center but are placed at the terminal rings leaving the middle site with a partial affectation . This spectroscopic-structural interpretation is in agreement with the formation of a polaron-pair structure in longer dications. In short, since the polaron-pair is composed of two separated radical cations, it is expected that these show similarities with the radical cations per se.11The Raman spectra of the EOC, are represented in 2+2CPDTV and 2+3CPDTV, \u0394EOC = 0 kcal mol\u20131, indicating that the stable form is the closed-shell configuration, or bipolaron structure. From 2+4CPDTV, \u0394EOC starts to be negative suggesting the preference for the open-shell form or polaron-pair \u2013 a preference that is more accentuated in the longer compounds.A polaron-pair charge defect can be described as an open-shell diradical dication and can be compared, by quantum chemistry, to the bipolaron charge defect, which corresponds only to one structural deformation and therefore it is a closed-shell system. Thus, we have calculated at the DFT/(U)B3LYP/6-31G** level the energy difference between the closed- and open-shell structures, both as singlet states, as a function of the oligomer size for the dications and these differences, \u0394EOC, the triplet state is progressively getting closer to the singlet ground electronic state due to the transformation of this into an open-shell diradical. The nature and consequences on the closed-shell to open-shell transition from the data provided by quantum chemical calculations in correlation with the experimental spectroscopic data in the case of 2+6CPDTV is now discussed in more detail.For these dications, we have also calculated the difference between the singlet ground electronic state and the first energy lying triplet. Following the tendency of \u0394+6CPDTV\u02d9 and of 2+6CPDTV from the B3LYP/6-31G** optimized structures) are represented which respectively depict a full quinoidal structure in the middle thiophenes for the radical cation and a pseudo-aromatic form for these also in the middle for the dication. In the dication, a full quinoidal structure can be formulated for the thiophene ring (closed-shell structure). However, this is unstable at the UB3LYP/6-31G** level and evolves by the rupture of one bond of this quinoidal structure giving way to the diradical species stabilized by the aromaticity recovery in the central thiophenes .6CPDTV (summarized in In DA(2CPDTV), disubstituted with triphenylamine groups at the terminal positions of the cyclopentadithiophenes to study the role of the nCPDTV bridge upon charge transfer from the donor amine groups. Compound 9 was prepared in 75% yield by the Horner\u2013Wadsworth\u2013Emmons reaction between 7 and 8.9 and 10 afforded DA(2CPDTV) in 66% yield (see Section 2 of ESI file for further detailsDA(2CPDTV) yields the radical cation, +DA(2CPDTV)\u02d9, which is characterized by the appearance of two very intense bands at the expense of the bands of the neutral at 514 nm (+2CPDTV\u02d9 and +3CPDTV\u02d9 and very close to those of +4CPDTV\u02d9 indicating the role of the triphenylamines in extending \u03c0-conjugation.One-electron oxidation of t 514 nm . These t+DA(2CPDTV)\u02d9 at 1879 nm emerging from a HOMO \u2192 SOMO excitation where the HOMO is mainly placed at the terminal phenyl amino rings, whereas the SOMO shifts the electron density mostly on the center of the dithiophene-vinylene core. The large red-shift on +2CPDTV\u02d9 \u2192 +DA(2CPDTV)\u02d9 is caused by the charge resonance of the positive charge of the radical cation between the two nitrogens of the triphenyl amines through the 2CPDTV bridge during the photoinduced absorption. However, in +2CPDTV\u02d9 the charge and its corresponding excitations are placed in the center of the molecule. This band is the so-called intramolecular charge transfer band or ICT band. From its spectrum, one can infer about the type of mixed valence system involved, either class II or class III. For class III, the band might show a vibronic component and a cut-off on the low-energy side, while class II systems exhibit Gaussian-shape bands without fine structure.+DA(2CPDTV)\u02d9 could be described as a class II mixed valence compound where the positive charge in the ground electronic state is mainly residing in one nitrogen partially stabilized towards the bridge.The lowest-energy lying band at 1848 nm is predicted by TDDFT/(U)B3LP/6-31G* in +DA(2CPDTV)\u02d9 yields the dication species, 2+DA(2CPDTV), whose electronic absorption spectrum in 2+6CPDTV, due to an open-shell polaron-pair dication. In this case, the great resonance stabilization of one charge in each nitrogen atom, together with the aromatization of the central dithiophenes, might justify the formation of this diradical species in this rather small compoundPost-oxidation of nCPDTV, Q(2CPDTV), together with its synthesis was accomplished using the well-established procedure: (i) Pd-catalyzed Takahashi couplingQ(2CPDTV) in good yield.Oligomers of this type are of interest as tetracyanoquinodimethanes oligothiophenes are being extensively exploited in the field of organic conjugated molecules.Q(2CPDTV) in the neutral state; the absorption spectra of the two relevant reduced states by spectroelectrochemistry are shown in Q(2CPDTV), in contrast with 2CPDTV, shows a two-electron reduction at \u20130.12 V and two one-electron oxidations displaced at higher anodic potential in comparison to the aromatic analogues, at 1.10 and 1.38 V. Neutral Q(2CPDTV) shows one very strong absorption band at 744 nm with a vibronic satellite at 679 nm (nCPDTV which exhibit the same pattern of bands (for instance in 2+3CPDTVhas a strong peak at 1157 nm and a satellite at 1012 nm) although in the case of the charged species the absorptions are displaced at longer wavelengths indicative of the smaller HOMO\u2013LUMO gaps. This is in accordance with a well defined closed-shell thienoquinoidal structure for Q(2CPDTV) whereas the dications display a transition between closed to open-shell structure such as discussed before.t 679 nm as a function of the temperature in solution are shown in Q(2CPDTV).nCPDTV and has been also described in other quinoidal oligothiophenes resulting from J-aggregates.The absorption spectra of Q(2CPDTV) have been carried out at reducing potentials. One-electron reduction gives rise to the progressive disappearance of the neutral band and the emergence of two new weak features at 1001 and 1889 nm (vibronic shoulder at 1585 nm). TDDFT/(U)B3LP/6-31G* of \u2013Q(2CPDTV)\u02d9 . Furthermore, this band is predicted to have a very low oscillator strength which is consistent with its weak absorbance in the experiment. The lowest-energy absorption band of \u2013Q(2CPDTV)\u02d9 resembles very much the ICT band of +DA(2CPDTV)\u02d9 either in their wavelength positions (1848 and 1889 nm respectively) or in their shapes (both are Gaussian type bands), indicating that the radical anion is also a class II mixed valence compound in which the additional negative charge resides in the dicyano-methylene partially invading the bridge (see +DA(2CPDTV)\u02d9, the HOMO \u2192 SOMO excitation represents an electron density displacement from the external sites to the internal core, producing a large variation of the dipolar momentum (and of the transition momentum). In \u2013Q(2CPDTV)\u02d9, the same excitation invokes a reorganization of the electron density in the whole molecule with a much smaller impact in the variation of the dipolar momentum of the transition (see \u2013Q(2CPDTV)\u02d9 gives rise to the dianion species which is characterized by a band at 586 nm and is typical of those of the aromatic nCPDTV revealing the full aromatization of the bithiophene moieties upon double charging \u02d9\u2013 allows tidge see . Both bacharging .Q(2CPDTV) in \u03bd scale\" fill=\"currentColor\" stroke=\"none\">C) band at 1543 cm\u20131, largely downshifted relative to the values in nCPDTV at 1590 cm\u20131, as a result of the weakening of the vinylene bond upon tetracyano substitution scale\" fill=\"currentColor\" stroke=\"none\">C) frequency at 1602 cm\u20131 in neutral 2CPDTV and at 1543 cm\u20131 in Q(2CPDTV) might represent the limits of a thienoquinoidization scale of the CPDTV \u03c0-conjugated structure, the former being the frequency of the full aromatic structure scale\" fill=\"currentColor\" stroke=\"none\">C) bands at 1564 cm\u20131 for +3CPDTV\u02d9 yields a quinoidization extension of 64% in line with the aromatic \u2192 quinoidal transitional character of the radical cations. For the dications, the quinoidal percentage is almost identical although the structure is not the same since the structural alteration is distributed in a polaron-pair form. \u03bd scale\" fill=\"currentColor\" stroke=\"none\">C) frequency values positions regarding the values in neutral Q(CPDTV2) and 2CPDTV. These observations show how the radical cations and dications both occupy a middle region between the full aromatic and full quinoidal points revealing the transitional character of their structures. Nevertheless, the neutral oligomers logically reside in a part of the quinoidal scale, closer to the aromatic fingerprint.The Raman spectrum of Fig. S69. TherefoH-cyclopentadithiophene vinylene repeating units has been prepared and characterized by spectroscopic and modelling methods. The neutral compounds display sizeable \u03c0-conjugation with increasing molecular size and a longer mean conjugation length than their oligothiophene and oligothiophene vinylenes homologues. Consequently the new compounds show rich redox chemistry with the stabilization of polycationic states of which the radical cations and dications are strong NIR absorbers \u2013 the latter displaying singlet diradicaloid states due to the gaining or recovery of aromaticity in the thiophene rings from the unstable quinoidal bipolaronic structures. Radical cations are described as having a quinoidal structure placed in the molecular center and flanked by two aromatic structures in the terminal parts. Conversely, the dications, as a result of their diradical character, display the reversed trend with aromatic segments in the molecular center surrounded by two quinoidal external moieties.A new series of \u03c0-conjugated oligomers based on the 4,4\u2032 dihexyl-4i.e., hole) or negative transport, two more derivatives were prepared. The CPDTV cores were substituted, either with electron donor triphenyl amino groups or with bis(dicyano-methylene) caps, enforcing a quinoidal structure in the dithiophene-vinylene bridge. The positive charge of the radical cation of the triarylamine compound is localized in the amine groups, and behave as class II mixed valence systems. The radical anion of the tetracyano compound, meanwhile has the same confinement effect of the charge in the environment of the electron accepting groups. The dication and dianion of these two compounds are open-shell biradical or polaron pair and closed-shell or bipolaronic structures respectively.To better investigate the potential charge transport properties as channels for either positive (The rich redox chemistry using these bridging groups, together with the adequate substitution of these cores, produce \u03c0-conjugated compounds with a variety of structures and properties. They can be exploited to function as materials for organic electronics but these materials are also of importance to gain insights into the intricate electronic structure of highly \u03c0-electron conjugated molecules.There are no conflicts to declare.Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "Aerobic, site-selective C(sp3)\u2013H oxygenation using a novel N-oxyl radical directing activator is described. 3)\u2013H functionalization. The use of a radical N-oxyl directing activator promoted the aerobic oxygenation of benzylic, propargylic, tertiary, and unactivated acyclic methylene C(sp3)\u2013H bonds in aliphatic alcohols with \u03b3- (or \u03b4-) selectivity under mild conditions (room temperature to 50 \u00b0C). The reaction was unaffected by the presence of various oxidation-sensitive functional groups, which proved to be problematic in previously reported studies on the oxidation of C(sp3)\u2013H bonds. Structural modifications on the directing activator altered the regioselectivity, and thus provided an ultra-remote aerobic C(sp3)\u2013H oxygenation. The observed reactivity and regioselectivity could be rationalized in terms of the intramolecular conformational accessibility of the N-oxyl radical and the electronic characteristics of C(sp3)\u2013H bonds.Chemically reactive directing groups (directing activators) represent a promising strategy for mild and regioselective C(sp Especially for the streamlined synthesis of complex drug lead molecules, containing a multitude of Csp\u2013H bonds,3)\u2013H bonds located remotely from the hydroxy group,2) as the oxidant.3)\u2013H bonds;3)\u2013H bonds at a specific position, while simultaneously overriding the innately higher reactivity of the C\u2013H bonds at \u03b1-position with respect to the oxygen atom;2 in initiation of the oxygenation reaction.We were thus interested in a site-selective oxygenation of alcohols that is able to target C(sp3)\u2013H bonds \u2013H bonds .11N-hydroxyphthalimide (NHPI) chemistry \u2013H bonds homolytically to produce a carbon radical. By covalently attaching the DA to the hydroxy group of substrate alcohols, this C(sp3)\u2013H activation step should become an intramolecular process. Thus, it should be possible to selectively cleave specific C(sp3)\u2013H bonds that can engage in suitable spatial contact with the in situ-generated N-oxyl radical of the DA.2 to produce the corresponding oxygenated alcohols or ketones.14We devised a novel DA inspired by Ishii's hemistry ,12 whichN-hydroxyisoindolinone structure. As the reactivity of N-oxyl radicals towards C\u2013H cleavage follows their electron-deficiency,1 = CF3) was effective. Then we screened suitable metal catalysts that could promote N-oxyl radical formation from the N-hydroxy group as the solvent (2 (entry 6) and Mn(OAc)3\u00b72H2O (entry 8) were effective. Although the \u03b1-C(sp3)\u2013H bond adjacent to the ether oxygen atom is the innately more reactive site,3) atom that was predominantly oxygenated. Metal salts bearing counterions other than acetate showed very low reactivity.2 and Mn(OAc)3\u00b72H2O, and the desired C\u2013H oxygenation product 2a was obtained in 67% NMR yield (entry 10). The catalyst loading could be reduced to 5 mol% of each metal without loss of efficiency (entry 11). A DA with a simple alkyl substitution (R1 = Et), instead of a CF3 group, did not show satisfactory performance (entry 12). Introduction of another CF3 moiety on the aromatic ring of the DA did not improve the result (entry 13). Other types of DA modification resulted in production of complex reaction mixtures.16Based on this reaction design, we began our investigation of aerobic C\u2013H oxygenation by modifying the DA structure and performing a screening of oxygenation conditions using DA-bound alcohol substrates . In orderoup see using 1-2O entry were effN-hydroxy-3-trifluoromethylisoindolinone moiety and reaction conditions using Co(OAc)2 (5 mol%), Mn(OAc)3\u00b72H2O (5 mol%), and O2 (1 atm) in TFE (0.1 M) at 40 \u00b0C represent optimal conditions (condition A). The use of a fluoroalcohol solvent was crucial for high reactivity, as fluoroalcohols are able to stabilize radicals, dissolve molecular oxygen, and are resistant to oxidation.17On the basis of this study, we established that a DA containing an 3)\u2013H bonds, however, resulted in the formation of complex product mixtures. For example, exposing 1i to condition A afforded 2i and C\u2013C bond-cleaved products , Me2S (1.2 equiv.), and O2 (1 atm) in TFE (0.1 M) at 40 \u00b0C (condition B) as the best set of conditions for the oxygenation of tertiary C(sp3)\u2013H bonds.Applying the cobalt-catalyzed conditions to the oxygenation of tertiary C(spproducts .18 As th3)\u2013H bonds of aliphatic alcohols (1a\u20131g) regioselectively into the corresponding C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O bonds (2a\u20132g). Using condition B, the oxygenation of tertiary C(sp3)\u2013H bonds proceeded generally in higher yield (2h\u20132r). The condition B was applicable to a gram-scale reaction of 1h without significant loss of efficiency.3)\u2013H bonds, which proceeded rapidly, even at lower temperature (2s\u20132ae).20Using these conditions allowed the oxygenation of a broad variety of DA-bound alcohols as shown in N-oxyl radical moiety in DA. For example, the very challenging substrate 1d possesses a flexible alkyl chain, but was converted into a 1.2\u2009:\u20091 mixture of \u03b3-oxo (2d) and \u03b4-oxo (2d\u2032) products. Conversely, the corresponding \u03b1-, \u03b2-, and \u03b5-oxo products were not detected or detected only in trace amounts. Oxygenation of substrates 1e\u20131g, containing an ester or a phthalimide moiety, occurred exclusively at the \u03b3-position.2ivs.2j; 1j was unreactive in 24 h at 40 \u00b0C). Benzylic C\u2013H oxygenations also showed a similar reactivity tendency.R)-1k, 1l, 1z, 1aa, and 1ac afforded products that were selectively oxygenated at the \u03b3-position. For DA-bound (+)-menthol, i.e. a diastereoisomer mixture of (R)-1k and (S)-1k, containing three tertiary C(sp3)\u2013H bonds, only one specific C\u2013H bond of (R)-1k was converted into a C\u2013OH bond. The corresponding product (R)-2k exhibited a partial isopulegol hydrate structure and was obtained within 2 h in almost quantitative yield. However, diastereomer (S)-1k was completely unreactive. The contrasting reactivity between these two diastereomers is probably due to the accessibility of the C\u2013H bond to the intramolecular N-oxyl radical moiety, as suggested by the X-ray structure of O-(4-nitrobenzyl)-(R)-1k and molecular modeling.N-oxyl radical: in case of 1o, oxygenation was selective towards a \u03b3-tertiary C\u2013H bond rather than towards a propargyl C\u2013H bond, whereas oxygenation of 1ac was selective towards a propargyl C\u2013H bond rather than towards a tertiary C\u2013H bond.Our approach, based on using a radical DA and molecular oxygen, thus provided access to previously unattained C\u2013H oxidation protocols. Especially the following three points should be worth noting: firstly, the C\u2013H oxygenation proceeded only at specific and predictable positions depending on the accessibility of the 2u, 2ad and 2ae), haloarenes (2v and 2x), a silyl arene (2y), aryl and alkyl boronates (2q and 2w), a terminal hydroxy group (2m), an acetal (2n), ethers , a C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C double bond (2p), and a C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C triple bond (2o and 2aa\u20132ac).Secondly, various oxidation-sensitive functional groups were tolerated, due to the mild reaction conditions employed that avoid the use of reactive oxidants. Thus, C\u2013H oxygenation could be conducted in the presence of electron-rich (hetero)aromatic rings (R)-1k differed from that in the previously reported Fe- or Ru-catalyzed C(sp3)\u2013H hydroxylation of O-acylmenthol.1m, 1n, 1o, and 1p, even though those compounds contain more reactive \u03b1-hydroxy (1m), acetal methylene (1n), propargylic (1o), and allylic (1p) C\u2013H bonds.Thirdly, the DA approach was able to override the innate reactivity difference between C\u2013H bonds. The observed regioselectivity in the reaction of (via the linker structure. As shown in 3), thus demonstrating complementary regioselectivity to the oxygenation of 1z (with use of molecular oxygen has not yet been reported in the literature. This result thus provides an opportunity for a controlled switching of the oxygenation site by designing a suitable linker between the target C(sp3)\u2013H bond and the reactive N-oxyl radical moiety.20The regioselectivity can be changed on of 1z . The DA-3)\u2013H oxygenation, we conducted several control experiments -3) promoted by an equimolar amount of DA-bound methanol (1af) or acetophenone (1ag) produced a significantly different profile compared to oxygenation, shown in O-Me-1a in the presence of 1af resulted in complete recovery of unchanged O-Me-1a after 20 h at 80 \u00b0C -3 with 1ag required a raised temperature (50 \u00b0C), and the position selectivity was moderate species, which are generated through the reaction between Co(ii) and O2.N-oxyl radical 5 abstracts a hydrogen atom of a C\u2013H bond at a proximate position, generating carbon radical species 6. Trapping 6 with molecular oxygen, the resulting alkyl peroxy radical 7 is quenched by a hydroperoxy radical generated in the initiation step to produce alkyl peroxide 8, or through intramolecular hydrogen abstraction from DA, generating 9. In the case of tertiary C\u2013H oxygenation, undesired decomposition pathway from 8 \u2013H oxygenation of aliphatic alcohols, using an N-oxyl radical group as a directing activator. Benzylic, propargylic, tertiary, and even the very challenging acyclic methylene C(sp3)\u2013H bonds were thus converted to C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O or C\u2013OH bonds under mild conditions (room temperature to 50 \u00b0C), while high functional group tolerance was maintained. Molecular oxygen was used as the stoichiometric oxidant, and the reactions proceeded regioselectively at the \u03b3 (and \u03b4) position(s), whereas the \u03b1, \u03b2, and other positions beyond the \u03b4 position remained intact. This regioselectivity can be explained in terms of the intramolecular accessibility of the reactive N-oxyl radical site, despite the low regioselectivity between \u03b3 and \u03b4 positions in electronically non-biased substrates is a current limitation that must be solved in future works. Preliminary structural tuning of the DA led to an alteration of the regioselectivity, providing a selective ultra-remote aerobic C\u2013H oxygenation. Although laborious synthesis of DA-bound substrates has remained problematic at this stage, devising catalytic applications of DAs will overcome this limitation. Efforts in such a direction are currently ongoing in our laboratory.In conclusion, we have developed a method for the regioselective C(spSupplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "Simple acid\u2013base properties explain the differences in amide and imide dimerisation, and represent an alternative to the secondary interactions hypothesis. PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019OS) and H-bonded scale\" fill=\"currentColor\" stroke=\"none\">OHB) carbonyl groups in imides. Notwithstanding the considerable body of experimental and theoretical evidence supporting the JSIH, there are some computational studies which suggest that there might be other relevant intermolecular interactions than those considered in this model. We conjectured that the spectator carbonyl moieties could disrupt the resonance-assisted hydrogen bonds in imide dimers, but our results showed that this was not the case. Intrigued by this phenomenon, we studied the self-association of a set of amides and imides via1H-NMR, 1H-DOSY experiments, DFT calculations, QTAIM topological analyses of the electron density and IQA partitions of the electronic energy. These analyses revealed that there are indeed repulsions of the type OS\u00b7\u00b7\u00b7OHB in accordance with the JSIH but our data also indicate that the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019OS group has an overall attraction with the interacting molecule. Instead, we found correlations between self-association strength and simple Br\u00f8nsted\u2013Lowry acid/base properties, namely, N\u2013H acidities and C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O basicities. The results in CDCl3 and CCl4 indicate that imides dimerise less strongly than structurally related amides because of the lower basicity of their carbonyl fragments, a frequently overlooked aspect in the study of H-bonding. Overall, the model proposed herein could provide important insights in diverse areas of supramolecular chemistry such as the study of multiple hydrogen-bonded adducts which involve amide or imide functional groups.Amides dimerise more strongly than imides despite their lower acidity. Such an unexpected result has been rationalised in terms of the Jorgensen Secondary Interactions Hypothesis (JSIH) that involves the spectator (C Hydrogen bonds (HBs) in amides and imides are ubiquitous directional forces in nature. HBs in these functional groups are responsible for the secondary interactions, along with other interactions of the tertiary and quaternary structures of proteins.The formation of amide and imide dimers is frequently studied by NMR or IR spectroscopy to determine self-association constants in solution.(i)Kdimer of amides as compared to imides in spite of their lower acidic character.HB\u00b7\u00b7\u00b7H contacts as shown in HB\u00b7\u00b7\u00b7OHB) and hydrogens (H\u00b7\u00b7\u00b7H) involved in HBs between the monomers (black double arrows in the above-mentioned figure). Nevertheless, the imide dimers present two additional repulsive interactions OHB\u00b7\u00b7\u00b7OS (green arrows), wherein OS denotes the oxygen atom in a spectator carbonyl group. The repulsions OHB\u00b7\u00b7\u00b7OS are also consistent with the notion of two nearly parallel repulsive C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O electric dipoles in the imide dimers (purple arrows). There is a considerable body of theoretical and experimental work which is concordant with the JSIH. For example, crystallographic data of some imides such as maleimide, trans-3,4-diphenylsuccinimide, and 1-methyl-hydantoin show that the separation between OS and OHB is very long (>4 \u00c5)The Jorgensen Secondary Interactions Hypothesis (JSIH),1.000000,.000000 s(ii)As schematised by the black arrows in (iii)An analysis of the inductive and resonance effects in these compounds suggests not only a higher acidity of the protons in imides but also a smaller basicity of their oxygens, because of the distribution of the negative charge of the zwitterionic structure across two carbonyl groups as illustrated in 1H-NMR titrations, 1H-DOSY experiments, electronic structure calculations as well as quantum chemical topology tools, namely, the Quantum Theory of Atoms in Molecules (QTAIM)HB and OS, but these interactions are more than compensated by other intermolecular attractions. Moreover, we found that criterion (iii) is the most suitable to explain the experimental tendencies of the self-association of imides and amides through an interplay of the respective basicity and acidity of the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O and N\u2013H groups. In other words, a weak HB acceptor carbonyl (as it is the case in many of the examined imides) can significantly weaken the investigated self-association processes despite a strong acidity of the imidic hydrogen and vice versa. Our results not only explain the studied phenomenon but also provide a model which could be exploited in diverse areas of supramolecular chemistry, such as the study of multiple hydrogen-bonding complexes which entail the amide and imide functional groups.Given this background, we performed a combined experimental and theoretical study to elucidate the factors governing the self-association of amides and imides. We analysed hypotheses (i)\u2013(iii) through A1), an archetype for the study of the self-association of the functional groups concerned in this investigation,d, acetonitrile-d3 and DMSO-d6, and also considered the reported value in CCl4 in order to select the most suitable solvent for this research. Another feature of 2-pyrrolidone which makes it particularly suitable for this purpose is the fact that it is liquid at room temperature and thereby its self-association is easier to study in deuterated solvents which cover a broad range of polarity. As expected, Kdimer diminishes with the polarity of the medium of the studied amide and imide dimers as reported in rcalc). As expected, structurally similar compounds have close values for rH and rcalc, e.g., the radii of amides A1, A2 and imide I4 are alike because all of them are unsubstituted five-membered rings. A4, A5 and I1 are likewise six-membered heterocycles with closely related structures as reflected in their values of rcalc and rH. Nevertheless, the cyclic chains in the uracil derivatives I5 and I8 and the benzo-derivatives A6, A7 and I3 introduce discrepancies between rH and rcalc. This condition occurs because the spherical particle model employed in the estimation of rH does not represent the oblate spheroid character of these dimers. Despite these differences, the experimental rH values are always smaller than the computed rcalc data . These results indicate that the investigated amides and imides do not form trimers or larger clusters to an appreciable extent.Next, we employed e.g., A5vs.I1, A1vs.I2, A6vs.I3 and A4vs.I6\u2013I13. These results point out that an increase in acidity does not necessarily lead to a stronger self-association. For example, the decreasing orders of acidity of the structurally related imides (I7\u2013I11) are I7 \u2248 I10 > I11 > I8 > I9, while those of dimerisation are backwards. We found, however, also exceptions to this behaviour, for instance A5vs.A4 and I5vs.I8 in which acidity and self-association increase in the same direction. Based on the above experimental results, now we proceed to examine the three considered hypotheses concerning the comparison of the self-association between amides and imides.Once we chose a suitable solvent and verified that the dimeric species are indeed predominantly formed in solution, we consider now the compounds shown in (i)A5 and the imide I1 dimers are only HBs. QTAIM shows no repulsion of either kind OHB\u00b7\u00b7\u00b7OHB, H\u00b7\u00b7\u00b7H or OHB\u00b7\u00b7\u00b7OS. In particular, we did not detect either attractive or repulsive interactions involving the spectator carbonyl moieties in the analysed imide dimers (Table S1 in the ESII1 molecule displayed in green) with the atoms of another I1 monomer (shown in orange). We note that the interaction between OHB and OS is indeed strongly repulsive . After considering all the atoms of the neighbouring molecule, the atom OS has an overall repulsion with the interacting monomer . The nearly parallel C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019OHB and C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019OS dipoles also present a slight repulsive interaction between them .We test here the hypothesis that repulsions involving the spectator carbonyl groups are responsible for the smaller degree of dimerisation of imides as compared with that of amides. For this purpose, we used electron density topology analyses in accordance with the QTAIM theory and the IQA approach. The results shown in \u20131 + 10.7 kcal mol\u20131 = \u20131.8 kcal mol\u20131). We computed the same value for the other spectator carbonyl group. This attraction between the spectator carbonyl moiety and the whole interacting molecule evidences the numerous relevant intermolecular atomic pairwise interactions in the system.The same analysis can also be carried out for every atom in one monomer which included up to terms which depend on R\u20136 (e.g. dipole\u2013hexadecapole and quadrupole\u2013octupole interactions), R being the distance between two multipoles. One of the main conclusions of this study concerns the difficulty to explain the relative stability of H-bonded clusters by only considering a subset of atomic pairs located in the boundaries between the interacting monomers as opposed to taking into account the whole set of intermolecular pairs of atoms in the system.The importance of the intermolecular interactions not considered by the JSIH in hydrogen-bonded dimers had already been pointed out by Popelier and Joubert.(ii) PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O bonds as schematised in VXC| of these bonds. The results shown in the bottom of VXC| values and the \u0394DIs for the C\u2013N and C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019Os bonds are barely affected. Moreover, in most cases the \u0394DI of the spectator C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O and the absolute value of VXC increase slightly because of the formation of the RAHB in imide dimers as opposed to the flux of electrons suggested at the top of QTAIM analyses allowed us to consider the potential disruption of the resonance-assisted hydrogen bonds in imide dimers by virtue of their spectator carbonyl groups top of . The eve PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019Os interactions. These data point out that the disruption of the RAHB in imides is not the factor which explains the larger self-association of amides with respect to imides, a condition consistent with the observation that electron delocalisation is not the most stabilizing effect in resonance-assisted hydrogen bonds.44Additionally, the changes in the DIs and distances of the bonds involved in this RAHB are substantially larger than those of the neighbouring C\u2013N and C(iii)E(A), and C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O protonation, E(B), as shown in E(A)| while a high value of |E(B)| indicates a strong basicity of the oxygen in the carbonyl group. The deprotonation energies for a wide variety of species which include imides, amides, (thio)ureas,Ka values, ditto for |E(B)| and pKBH+, which indicates the pKa of the conjugate acid of the species under consideration | and |E(B)| for compounds A1\u2013A7 and I1\u2013I13 | and |E(B)| whose distribution of points adjusts to a first-degree polynomial model. The species A2 was not contemplated in the correlation because it is a cyclic carbamate that can form bifurcated hydrogen bonds with CHCl3 as indicated by DFT geometry optimisations and schematised in Fig. S31 in the ESI.A2 in comparison with the rest of the studied compounds. Indeed, the exclusion of A2 improved the value of r2 in E(B)| and |E(A)| (CE(B)|| and CE(A)||) are positive and negative respectively. These conditions support the model that self-association increases with the acidity of the N\u2013H moiety and the basicity of the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O group. Besides, |CE(B)||| > |CE(A)|||, and hence the dimerisation processes of the examined compounds are more sensitive to changes in the basicity of the carbonyl group than to modifications of the acidity of the amidic or imidic hydrogen. In general, this model points out that a high acidity or basicity by itself does not ensure a large association constant since there must be a balance between these properties to observe a substantial value of Kdimer. In other words, very poor acceptor or donor features of a system can substantially hinder its self-association process as it is the case for imides and basic amides A3 and A5 respectively. This analysis indicates that the low basicity of the carbonyl groups in imides allows us to explain why these compounds dimerise less strongly than amides notwithstanding their larger acidic character. The model in 3. For example, the lowest value of the dimerisation constant corresponds to maleimide I4, a compound with high acidity but the one with the smallest basicity among the analysed systems. On the other hand, A4 undergoes the strongest self-association among the molecules of interest. It has the highest basicity and an acidic character similar to the examined imides. These results point out that the occurrence of an aromatic sextet scale\" fill=\"currentColor\" stroke=\"none\">O fragments in the self-association of amides and imides is also observed for the uracil analogues I6\u2013I13. These imides are more basic than other compounds with the same functional group and hence dimerise more strongly. Furthermore, the consideration of the basicity of the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O groups is also useful to rationalise which fragments are involved in the dimerisation. For example, it is well known that uracil derivatives form hydrogen bonds with the oxygen at C-4 rather than the one at C-2.via a C-5, C-6 double bond. When this is no longer the case, e.g.I5 and I11, the most basic carbonyl is the one at C-2 as illustrated in the middle and bottom of I5 scale\" fill=\"currentColor\" stroke=\"none\">O basicities. In addition, there is a good correlation between the experimental values of ln\u2009Kdimer with those predicted in the model of Following the same arguments concerning the relative importance of basicity and acidity of the examined compounds, we can explain why among the studied five-membered heterocycles, amide 4 as well.3 ln\u2009Kdimer and (ii) the basicity of the carbonyl group together with the acidity of the N\u2013H moiety as shown in CE(B)|| > 0, CE(A)|| < 0 and |CE(B)||| > |CE(A)|||. The last-mentioned condition indicates once again that the dimerisation constant of amides and imides is more sensitive to the basicity of the carbonyl group than to the acidity of the N\u2013H moiety, hence the larger association of amides in comparison to imides.We considered amide and imide dimerisation constants reported in CCls well.3 Fig. 8)4 as wellKheter indicates that hetero-association between A5 and I1 is stronger than in each homodimer as illustrated in 1H-NMR spectroscopy results. The change in the N\u2013H chemical shift due to complexation, \u0394\u03b4N\u2013H, is reported to increase with the strength of the association.A5\u2013I1 adduct involved in the heterodimer is smaller than the corresponding value for the HB in the imide homodimer. Fig. S15A1 and I2 heterodimers. The measurement of Kheter was performed with a constant total amount of amide A1 while imide I2 was added to the system. The value of \u03b4N\u2013H diminished through the titration, a condition indicative of a weakening of the HB entailing the amidic hydrogen. In the opposite experiment the value of \u03b4N\u2013H for the imidic proton increased more than in the corresponding homodimers, evidencing the strengthening of the hydrogen bond of the heterodimer which involves this proton. The last-mentioned effect is also observed in the A5\u2013I1 molecular cluster.The examination of heterodimers gives further insights about the dimerisation of amides and imides. The determination of HB-1 in is largeA5\u2013I1 and A1\u2013I2 heterodimers, the strongest HB-1 is formed between the most acidic proton (the one in the imide) and the most basic carbonyl (found within the amide). On the other hand, the weakest HB takes place in the interaction of the less acidic proton, i.e. N\u2013H of amides A5 and the less basic carbonyl (the one in the imide). The strengths of the HBs in the amide and imide homodimers are between these two extremes. These results along with those of DFT geometry optimisations of the A5\u2013I1 heterodimer show the following trend of descending HB strength:Concerning the A5 homodimer > HB in I1 homodimer > HB-2HB-1 > HB in A5 as opposed to HB-2 which does not present this effect. Despite the aforementioned agreement of the JSIH with structural data, this hypothesis is not completely consistent with the H-bond strength and distance patterns found in the heterodimers. In contrast, the consideration of the acidity and the basicity of the N\u2013H and C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O fragments explains the observed association constants, hydrogen bond distances and chemical shift patterns in these systems. The QTAIM and IQA analysis of the intermolecular interactions within the heterodimers A5\u2013I1 scale\" fill=\"currentColor\" stroke=\"none\">O in the imide presents a strong repulsion with the oxygen of the neighbouring carbonyl involved in the HB and with the rest of the molecules of the amide. These repulsions are even stronger than in the case of the I1 homodimer. Still, the hydrogen bond closest to the OS atom in the A5\u2013I1 heterodimer is the strongest of the system. The relative strength of these HBs is revealed by their lengths as previously discussed and other indices used in the study of hydrogen bonds which include (i) Espinosa's empirical formula PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O group has an overall attractive interaction with the neighbouring imide or amide in a similar fashion to the corresponding homodimers. These IQA homo- and heterodimer results point out that the OHB\u00b7\u00b7\u00b7OS repulsions or those between the corresponding dipole carbonyls are not the decisive factor for the energetics of the amide and imide dimerisation.This order is similar to that found by Jorgensenrs A5\u2013I1 and A1\u2013IKdimer for these systems. The interplay of acidity and basicity allows us to explain other baffling results e.g. the fact that 2-ethyl-2-methylsuccinimide self-associates much more strongly (by a factor of 15) than tetrafluorosuccinimide.Kdimer. Likewise and according to the JSIH, the fluorinated compound should dimerise to a larger extent because the charges in the carbonyl oxygens are less negative than those in the alkylated succinimide. The alternative explanation that we offer is that the fluorine atoms reduce substantially the basicity of the carbonyls, thereby impairing the self-association of this compound.Our investigation provides an alternative model to the JSIH to rationalise experimental observations concerning the homo- and heterodimerisation of amides and imides, for example, the hydrogen bond distance pattern in amide/imide heterodimers and the relative magnitude of PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O moiety than it is to the acidity of the N\u2013H group. Hence, the modification of the basicity of this carbonyl represents a good opportunity in the modulation of the strength of the non-covalent interactions established by these groups. For example, the change of a carbonyl in a crucial amide for a more basic imino fragment in vancomycin enhances the association properties with the cell wall of bacteria immune to this antibiotic.Additionally, our results can be useful in understanding why amides are much more used in many technologies such as crystal engineering, development of materials and pharmaceuticals than imides.Finally, the results of our investigation suggest a different approach for the analysis of the stability of multiple hydrogen-bonded systems such as uracil\u2013diamino purine, cytosine\u2013guanine and ADA\u2013DAD systems.General experimental and computational details are given in the ESI. PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O group, the acidity of the N\u2013H moiety and Kdimer resulted in a first-degree model suggesting that the proton acceptor capacity of the carbonyl group is more important than the acidity of the amidic or imidic hydrogens for the self-association of the investigated compounds. This model also indicates that there must be a balance between the respective acidity and basicity of the N\u2013H and C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O fragments to observe a substantial self-association of these systems. Particularly, amides exhibit larger self-association constants because of their higher basicities in comparison with imides. Similar conclusions were drawn from systems investigated in CCl4. Our results also explain the hydrogen bond distance patterns found in amide and imide homo- and heterodimers. The acidity/basicity balance in the dimerisation of amides and imides is an alternative approach to the JSIH in order to explain this phenomenon. Overall, we expect that the application of the insights presented herein will prove valuable to understand and modulate hydrogen bonds between the investigated functional groups which are present in a wide variety of supramolecular systems throughout biology and chemistry.We investigated the reasons underlying the stronger self-association of amides as compared to imides. Our results indicate that the spectator carbonyl presents indeed repulsions with the oxygens involved in the HB but these repulsive interactions are not the key factor in the energetics of the H-bonded systems examined herein. This statement is based on the relevance of the pairwise interactions which are not accounted for by the JSIH and the experimental and theoretical examination of amide\u2013imide homo- and heterodimers. Our results also reveal that the spectator carbonyl groups in imides do not interfere with the resonance-assisted hydrogen bonds in the dimers of these species. Then, we consider the Br\u00f8nsted\u2013Lowry acid/base properties of the HB donors and acceptors involved in the interaction within these molecular aggregates. The examination of the basicity of the CThere are no conflicts to declare.Supplementary informationClick here for additional data file."} +{"text": "Diastereoselective aerobic dehydrogenative cyclization of hydrazones is described via a copper-catalyzed sp3 C\u2013H functionalization process. 3\u2013H bond of amines. Among this reaction category, copper-catalyzed selective C\u2013C bond formation under atmospheric O2 is of considerable research interest and significant progress has been achieved in recent years. In comparison, development of the intramolecular version of this transformation is still in its infancy. Furthermore, diastereoselective cyclization with this transformation has not been achieved. Here, we describe the highly diastereoselective intramolecular dehydrogenative cyclization of N,N-disubstituted hydrazones by a copper-catalyzed sp3 C\u2013H bond functionalization process. The reaction protocol utilizes O2 as the oxidant and shows great functional group compatibility. Computational studies suggest that a 5-center/6-electron disrotatory cyclization mechanism is probably involved in the process for controlling the diastereoselectivity. This work represents the first example of a copper-catalyzed, direct intramolecular diastereoselective coupling reaction via an iminium ion intermediate. Additionally, it provides an environmentally friendly and atom efficient approach to access substituted pyrazolines, an important structural unit in many biologically active compounds.Transition metal-catalyzed cross dehydrogenative coupling is an important tool for functionalization of the \u03b1 Csp Representative examples, which have expanded product diversity, include oxidative dimerization of phenols, naphthols and electron-deficient arenes, cross coupling of terminal alkynes with electron-deficient arenes, and intramolecular dehydrogenative cyclization of anilides.via an sp3 C\u2013H bond functionalization process has also been established on tertiary amines by Miura, Li, and others.via oxidation of the corresponding amine. This intermediate then serves as an active electrophile for the subsequent nucleophilic addition reaction.N,N-dimethylaniline derivatives have been reported as suitable substrates. Furthermore, this transformation is limited to the intermolecular version, in part due to the non-selective oxidation of the amine over the internal nucleophile.8The oxidative dimerization of terminal alkynes, the Glaser reaction reported in 1869, is the first example of copper-catalyzed aerobic dehydrogenative coupling.We envisaged that the use of an enamine-type motif as an internal nucleophile could potentially overcome this drawback and extend the reaction scope to intramolecular cyclizations.123 C\u2013H functionalization strategy was thoroughly examined and further explored to expand its scope of synthetic utility. To provide important mechanistic insights into various factors that control the product distribution, density functional theory (DFT) calculations were further conducted to rationalize the diastereoselectivity observed. The combined work of chemical synthesis, NMR, and computation revealed how the interplay of molecular configurations, steric effects, and the symmetry requirement of electronic structure reactivity, can be utilized to precisely control the diastereoselectivity in the products, thereby providing a valuable guidance to rational designs of the related synthetic routes.Here we report detailed studies in which the copper-catalyzed sp1a) under atmospheric O2 2 and stoichiometric KI, KOAc, and DBU in NMP with O2 as the external oxidant (entry 5). Interestingly, this reaction showed high diastereoselectivity, with the anti isomer as the major product. Further optimization showed that this reaction was improved by using NMP and tAmOH as co-solvent (entry 8). It was then noticed that the reaction yield was significantly increased by extending the reaction time to 12 h from 4 h (entry 9). In addition, the replacement of DBU with DBN significantly increased the reaction rate, and the desired product was obtained in 86% yield within 4 h (entry 10). Moreover, the cyclization could also be effectively catalyzed by other CuI or CuII sources under the above modified conditions (entries 13\u201317).Our study commenced with copper-catalyzed dehydrogenative cyclization of 1,1-dibenzyl-2-(1-phenylpropylidene)hydrazine (heric O2 . After a2a\u2013h). Interestingly, the reaction showed a substrate-dependent diastereoselectivity pattern, and the anti-isomers of 2a and 2b were obtained as the major products while the syn-isomers of 2c\u2013g were the major forms. It is noteworthy that the current methods for the synthesis of syn-4,5-pyrazolines rely primarily on the [3 + 2]-cyclization of a diazo compound and an olefin, which often suffers from poor regioselectivity.N-benzyl group could also be replaced with another alkyl group (2i\u2013n), in which the aromatic hydrazones favored the anti-isomers, and the aliphatic substrates favored the syn-isomers. Additionally, an N-phenyl substituted hydrazone yielded exclusively the syn-isomer 2o, providing an important and tunable strategy to selectively access either anti or syn pyrazolines. Moreover, 1,2,3,4-tetrahydroquinoline is also an effective substrate (2p).With the optimized conditions in hand, the scope of hydrazone substrates was studied . As expeN-cyclohexylidene-3,4-dihydroisoquinolin-2(1H)-amine (via an oxidative sp3 C\u2013H activation process: mercuric oxide-mediated dimerization and Rh(iii)-catalyzed cycloaddition.In our previous study of dehydrogenative cyclization/aromatization of 1-benzyl-1-isopropyl-2-(1-phenylethylidene)hydrazine, it has been demonstrated that both electron-donating and electron-withdrawing groups on the phenyl rings were compatible.H)-amine . For eacsyn diastereoisomers as the major products (5b\u2013o). Generally, electron-withdrawing group-substituted substrates provided better yields compared with electron-donating substituents in the same position. Considering that an iminium ion intermediate is formed prior to cyclization, these results are not surprising since an electron-withdrawing group increases the electrophilicity of the iminium ion and thus facilitates the cyclization. Additionally, an electron-withdrawing group may retard the further oxidation of pyrazolines and thus minimize formation of the by-product pyrazoles. Furthermore, a steric effect was observed for this reaction: C8-substituted hydrazones gave lower yields of desired products compared with the C5-, C6-, or C7-substituted substrates. Finally, halogens are also well tolerated, which provides the opportunity for further product manipulation.As shown in 1 of 5a and H12a, H12b were observed. Additionally, there was no observable NOE between H1 and H13, or H14 and H12a or H12b. These results indicate that H13 and H14 have the syn relationship.NOESY analysis was carried out to confirm the relative configuration of the product . NOEs be5q and 5r). The 5-membered ring substrate also provided a good yield of product 5p under the modified reaction conditions. Furthermore, \u03b1,\u03b2-unsaturated hydrazones are also compatible (5u and 5v), which allows for further transformation of the initial products. As expected, good to high yields of products were obtained on substrates with substituted cyclohexylidenehydrazine moieties . In addition, good to excellent diastereoselectivity was observed with \u03b1-substituted cyclohexylidenehydrazine, favoring the anti-products (5w\u2013y). In comparison, \u03b3-substituents on the cyclohexylidenehydrazine moiety did not significantly affect the relative diastereoselectivity (5z and 5aa). Additionally, linear hydrazones are also compatible with the oxidative conditions, favoring the formation of the syn-isomers. However, in this case, the ratio of diastereoselectivity was dramatically decreased. Considering that there are two possible conformers present in the iminium ion intermediate oxidized either from 5z or 5aa, the above result is not surprising.Next, an imine substrate scope study was carried out . The rea1 generates the radical cation Avia an SET process. The radical cation A is then converted into the iminium ion intermediates B1 and B2via either an oxidation/deprotonation or a homolytic cleavage process. Tautomerization of the imine moiety on B1 and B2 to enamine-type diastereoisomers C1, C2, C3 and C4, followed by subsequent nucleophilic addition provides the intermediate D1 and D2, which then give pyrazoline 2 upon deprotonation. The rationale of high syn over anti diastereoselectivity is also proposed based on the previous reports by Hoffmann and List.C will behave as an sp2 hybridized atom. Therefore, the lone-pair of electrons on the nitrogen will participate in the \u03c0-conjugation together with the four \u03c0 electrons in the double bonds, resulting in a 5-center/6-electron system, similar to the situation found in a pentadienyl anion. For such homoconjugated systems, the HOMO is actually a \u201csymmetric\u201d nonbonding orbital, which is 1,5-bonding with the preferred U planar configuration. For thermally induced electrocyclic ring closures, the symmetric HOMO requires a disrotatory mechanism, which results in the corresponding product 2. It should be mentioned that \u03b1-alkyl Cu species could potentially be formed from deprotonation of the intermediates B1/B2 under basic conditions. Intramolecular nucleophilic addition of these Cu species could also provide the desired products.On the basis of the above results and our previous report,2a, 2f, 2p, 5a, and 5ab).Toward molecular-level understanding of diastereoselective 5-center/6-electron cyclization mechanism displayed in these copper-based C\u2013H functionalization reactions, we performed density functional theory (DFT) investigations on five representative systems related to diastereoselective cyclization in TS) from C \u2192 D, which seems to be the most relevant step in determining the diastereoselectivity of the products, were especially emphasized, and the transition states from B \u2192 C are outside the scope of this work and thus were excluded. The influence of the solvent environment on the reaction was evaluated by using the polarizable continuum models (PCM) methodN-methylpyrrolidone (\u03b5 = 32.2) and 2-methyl-2-butanol (\u03b5 = 5.78) are not available in Gaussian 09, we chose ethanol as the solvent (\u03b5 = 24.55). Frequency analysis was carried out to verify the optimized geometry as minima or transition state, and to obtain free energy of each species. The intrinsic reaction coordinate (IRC)All the calculations were carried out for the five systems by using Gaussian 09 program suites.1), the iminium ion (B), the enamine-type intermediate (C) and the charged pyrazoline product (D). For 2f, 2p, 5a, and 5ab, C1/C2 represents the enamine-type intermediates with trans/cis conformation for the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C double bond; D1/D2 represent the syn/anti charged pyrazoline intermediates. For 2a, B1/B2 is the iminium ion with trans/cis conformation for the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N+ double bond; D1/D2 are the anti/syn charged pyrazoline intermediates. Given the conformational combinations of the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N+ and C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C double bonds, four enamine-type intermediates are considered for this system, which are referred to as C1 , C2 , C3 and C4 , respectively. The transition state of a disrotatory/conrotatory reaction is specified by \u201ca\u201d/\u201cb\u201d, for example, TS2a/TS2b is the transition state of the disrotatory/conrotatory reaction of C2. The proposed mechanism, optimized geometries, some free energy profiles, and original data are shown in ESI.In all systems, the symbols used to represent the related species are: hydrazine scale\" fill=\"currentColor\" stroke=\"none\">N+ bond is \u20130.769e in C1, \u20130.609e in TS1a, and \u20130.466e in TS1b, respectively. The charge of the terminal carbon atom in the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond is 0.916e in C1, 0.679e in TS1a, and 0.729e in TS1b, respectively. As shown in Fig. S1,D scale\" fill=\"currentColor\" stroke=\"none\">N+\u2013N\u2013C) and D scale\" fill=\"currentColor\" stroke=\"none\">C) of the pyrazoline ring in TS1a are 22.1\u00b0 and \u201319.6\u00b0, respectively. The C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N+ and C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bonds form a pseudo-plane with the dihedral angle D scale\" fill=\"currentColor\" stroke=\"none\">N+\u00b7\u00b7\u00b7C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C) to be 3.1\u00b0, and the neutral nitrogen atom is a little out of that plane. For TS1b, the values of D scale\" fill=\"currentColor\" stroke=\"none\">N+\u2013N\u2013C), D scale\" fill=\"currentColor\" stroke=\"none\">C) and D scale\" fill=\"currentColor\" stroke=\"none\">N+\u00b7\u00b7\u00b7C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C) are 35.0\u00b0, 7.5\u00b0, and 40.2\u00b0, respectively, implying that the pyrazoline ring in TS1b is twisted and it looks like a helical folding conformation. Clearly, compared with the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N+\u2013N\u2013C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C in TS1b, the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N+\u2013N\u2013C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C in TS1a is closer to a coplanar conformation. The distance R scale\" fill=\"currentColor\" stroke=\"none\">N+\u00b7\u00b7\u00b7C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C) between the two terminal carbon atoms of the pyrazoline ring is 2.25 \u00c5 in TS1a and 2.15 \u00c5 in TS1b. These show that the C \u2192 D reaction is a 5-center/6-electron cyclization constituted by C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N+\u2013NH\u2013C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C, although this system does not completely satisfy the H\u00fcckel rule .Based on the complexity of these systems, the computational results of C is less stable than its tautomer B by 10.9 kcal mol\u20131. The reaction is endothermic by 1.9 kcal mol\u20131 for C \u2192 D1 process, and exothermic by 4.3 kcal mol\u20131 for C \u2192 D2 process. Even though the conformation of the cyclohexane is chair in D1 and twist boat in D2, D2 (anti) shows more stability than D1 (syn). The reason may be that the two bulky substituents are on the same side of the pyrazoline ring in D1, while the two substituents are on the different side of the pyrazoline ring in D2, causing the steric repulsion being stronger in D1 than in D2. However, the active free energy generating D1 is 1.6 kcal mol\u20131 lower than that generating D2, indicating that C prefers to undergo disrotatory process (TS1a) to form D1, rather than conrotatory process (TS1b) to afford D2. This may be because the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N+\u2013N\u2013C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C (5-center/6-electron) in TS1a is more coplanar and more satisfies the H\u00fcckel rule compared with that in TS1b. These are in good agreement with the diastereoselectivity observed in the copper-based C\u2013H functionalization reactions (syn-D1 is major product).In the free energy profiles , C is leTS1a and TS1b were compared. As shown in PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N+ bond overlaps with the under lobe of the orbital of the terminal C atom in the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond, and C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N+\u2013NH constitutes a delocalized system in TS1a. In TS1b, the under lobe of the orbital of the C atom in the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N+ bond overlaps with the upper lobe of the orbital of the terminal C atom in the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond. However, the delocalized orbitals of the N\u2013NH bond do not overlap with the C atom in the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N+ bond, generating an orbital nodal surface between the C atom and the N atoms. These demonstrate that TS1b is less stable and more difficult to cross than TS1a, supporting that the intermediate C tends to undergo a disrotatory process to form D1, rather than a conrotatory process to afford D2.To better understand this point, HOMOs of C \u2192 D1/D2 processes are exothermic and the active free energy barriers are easier to overcome, shown in parentheses in The presence of a solvent can obviously improve the reaction process scale\" fill=\"currentColor\" stroke=\"none\">N+ bond is \u20130.464e in C1, \u20130.321e in C2, \u20130.313e in C3, \u20130.579e in C4, \u20131.407e in TS1a, \u20130.388e in TS1b, \u20130.983e in TS2a and \u20130.656e in TS2b, respectively. The charge of the terminal carbon atom in the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond is separately 0.139, 0.337, 0.641, \u20130.077, 0.131, 0.297, 0.042 and \u20130.161e for the corresponding species. Seen from Fig. S2,D scale\" fill=\"currentColor\" stroke=\"none\">N+\u2013N\u2013C) and D scale\" fill=\"currentColor\" stroke=\"none\">C) of the pyrazoline ring in TS1a are 30.0\u00b0 and \u201333.8\u00b0, respectively. The C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N+ and C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bonds form a pseudo-plane with the dihedral angle D scale\" fill=\"currentColor\" stroke=\"none\">N+\u00b7\u00b7\u00b7C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C) of \u20132.4\u00b0, and the neutral nitrogen atom is a little out of that plane. The corresponding values are separately \u201344.1\u00b0, 4.3\u00b0 and \u201337.7\u00b0 in TS1b, also suggesting that the pyrazoline ring in TS1b is a pseudo-plane. However, this pyrazoline ring in TS1b is slightly apart from the coplanar conformation compared with that in TS1a. In TS2a, the values of D scale\" fill=\"currentColor\" stroke=\"none\">N+\u2013N\u2013C), D scale\" fill=\"currentColor\" stroke=\"none\">C) and D scale\" fill=\"currentColor\" stroke=\"none\">N+\u00b7\u00b7\u00b7C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C) are separately 37.6\u00b0, 23.3\u00b0 and 56.5\u00b0, implying that the pyrazoline ring is twisted and it looks like a helical folding conformation. The corresponding values are separately \u201329.2, \u201318.9\u00b0 and \u201343.7\u00b0 in TS2b, showing that the pyrazoline ring in TS2b is twisted with the opposite direction, but the twist with the opposite direction would increase the steric hindrance between the methyl group and the benzyl group. The distance R scale\" fill=\"currentColor\" stroke=\"none\">N+\u00b7\u00b7\u00b7C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C) between the two terminal carbon atoms of the pyrazoline ring is 2.36 \u00c5 in TS1a, 2.32 \u00c5 in TS1b, 2.34 \u00c5 in TS2a, and 2.34 \u00c5 in TS2b. These also demonstrate that the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N+\u2013NH\u2013C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C constitutes a 5-center/6-electron system, but not a typical H\u00fcckel system (not completely coplanar).The charge of the carbon atom in the CC1 and C2 are more stable than C3 and C4, supporting that C1 and C2 are major intermediate compared with C3 and C4. The anti product D1 is more stable than the syn product D2 in the gas phase, which is also attributed to the steric repulsion between the two bulky substituents on the same side of the pyrazoline ring in D2 compared with D1. TS1a is \u20133.3 kcal mol\u20131 relative to TS1b, and TS2a is \u20133.1 kcal mol\u20131 relative to TS2b, showing that C tends to undergo a disrotatory (TS1a/TS2a) process to give the product, rather than a conrotatory process (TS1b/TS2b) to afford the product. The reason may be that the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N+\u2013N\u2013C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C in TS1a is closer to the H\u00fcckel system compared with that in TS1b (5-center/6-electron and coplanar conformation), and the steric hindrance between the methyl group and the benzyl group in TS2a is smaller than that in TS2b. Compared with C3 and C4, C1 and C2 overcome less of a free energy barrier, determining the diastereoselectivity of this reaction. Similar to 5a, the presence of a solvent can obviously improve the reaction process (the C \u2192 D1/D2 processes are exothermic and the active energy barriers become easier to overcome), but the overall diastereoselective trend is the same as that captured from gas-phase calculations. The main reason for such a huge improvement of the reaction is also the solvation of ions.As shown in TS1a, TS1b, TS2a and TS2b were compared. Seen from PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N+ bond overlaps with the under lobe of the orbital of the terminal C atom in the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond, and C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N+\u2013NH\u2013C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C and phenyl group constitute a delocalized system in TS1a. In TS1b the under lobe of orbital of C atom in C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N+ bond overlaps with the under lobe of the orbital of the terminal C atom in the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond, but the delocalized system only consists of C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N+\u2013NH and phenyl groups. The delocalized system in TS1b is smaller than TS1a, suggesting that TS1b is less stable and more difficult to overcome than TS1a. Compared with the orbital in TS2a, the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N+\u2013NH\u2013C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C in TS2b does not constitute a complete delocalized orbital, and two orbital nodal surfaces are found between the C and N+\u2013NH, and N+\u2013NH and C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bonds, also suggesting that TS2b is less stable and more difficult to cross than TS2a. Therefore, C1 (C2) goes through a disrotatory process to form D1 (D2), instead of a conrotatory process to afford D2 (D1).To better understand this point, HOMOs of 2f, two enamine-type iminium ion intermediates are formed in the cyclization process, resulting in two different diastereoisomers. Based on the calculation (see ESIE)-isomer is more stable, resulting in syn-2f as the major product via a 5-center/6-electron disrotatory cyclization process. In the case of 2p, the intermediate with the methyl and phenyl groups on the opposite side is the major isomer, and thus the anti-isomer becomes the major product. For 5ab, the intermediate with two alkyl groups on the same side becomes the major isomers, and thus the syn-5ab is the major product. Compared with 5ab, the diastereoselectivity in 5ac is lower because the ratio of the major isomer is decreased due to the stronger steric effect by the isopropyl group. The combined results from experimental and computational studies suggest that the diastereoselectivity of products is possibly determined by the relative stability of the enamine-type iminium ion intermediates via a 5-center/6-electron disrotatory cyclization process.Furthermore, computational studies of three more representative substrates were also carried out to account for the diastereoselective outcome . For 2f,n see ESI, the (E)N,N-disubstituted hydrazones with good functional group tolerance was developed using a copper-catalyzed double sp3 C\u2013H bond functionalization process. The observed diastereoselectivity was rationalized by a 5-center/6-electron disrotatory cyclization mechanism, which was supported by computational studies. As the first example of the copper-catalyzed aerobic diastereoselective intramolecular cyclization of amines via an iminium ion intermediate, this transformation provides a straightforward, environmentally friendly and atom efficient access to pyrazolines with up to five fused-ring systems. The enantioselective version of this transformation is currently under study in our laboratory.In summary, a highly diastereoselective aerobic intramolecular dehydrogenative cyclization of CCR2CC chemokine receptor 2CCL2CC chemokine ligand 2CCR5CC chemokine receptor 5TLCThin layer chromatographySupplementary informationClick here for additional data file."} +{"text": "Assembly of bimetallic complexes on boron clusters by coordination of coinage metalcations to Ru\u2013B single bonds. i)cations insert into B\u2013Ru bonds of the (BB)\u2013carborynecomplex of ruthenium with the formation of four-membered B\u2013M\u2013Ru\u2013Bmetalacycles. Results of theoretical calculations suggest that bonding within thesemetalacycles can be best described as unusual three-center-two-electron B\u2013M\u00b7\u00b7\u00b7Ruinteractions that are isolobal to B\u2013H\u00b7\u00b7\u00b7Ru borane coordination for M = Cu and Ag, orthe pairs of two-center-two electron B\u2013Au and Au\u2013Ru interactions for M = Au.These transformations comprise the first synthetic route to exohedral coinage metal borylcomplexes of icosahedral closo-{C2B10} clusters,which feature short Cu\u2013B (2.029(2) \u00c5) and Ag\u2013B (2.182(3) \u00c5) bondsand the shortest Au\u2013B bond (2.027(2) \u00c5) reported to date. The reportedheterometallic complexes contain Cu(i) and Au(i) centers inuncharacteristic square-planar coordination environments. These findings pave the way torational construction of a broader class of multimetallic architectures featuringM\u2013B bonds.In this work, we introduce a novel approach for the selective assembly of heterometalliccomplexes by unprecedented coordination of coinage metal cations to strained singleruthenium\u2013boron bonds on a surface of icosahedral boron clusters. M( Synthesis and reactivity of bimetallic complexes containing late transition metals andcoinage metals have attracted considerable attention due to the discovered cooperativereactivity in cross-coupling reactions, transmetallation processes, and the relevance toheterogeneous catalysis.20closo-C2B10H12.Transition metal boryl complexes have been extensively studied because of both fundamentaland applied reactivity potential. Success in metal-catalyzed borylation of a variety oforganic substrates led to the enormous growth in studies of structure, bonding, andreactivity of complexes, containing metal\u2013boron bonds. PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1100000000000000000000000000000000 1111110000000000000000000000000000 0011111111000000000000000000000000 0000001111111100000000000000000000 0000000000111111110000000000000000 0000000000000011111111000000000000 0000000000000000001111111100000000 0000000000000000000000111111110000 0000000000000000000000000011111111 0000000000000000000000000000001111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000001111 0000000000000000000000000011111111 0000000000000000000000111111110000 0000000000000000001111111100000000 0000000000000011111111000000000000 0000000000111111110000000000000000 0000001111111100000000000000000000 0011111111000000000000000000000000 1111110000000000000000000000000000 1100000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019Ru metalacycle can be described as a metalacycloboropropane with two highlystrained 2c-2e B\u2013Ru \u03c3 bonds. The significant distortion of the exohedral bonds ofthe carborane cage resulted in enhanced reactivity associated with these bent B\u2013Rubonds, which themselves served as nucleophilic reaction centers with organic substrates,exhibiting metal\u2013ligand cooperative activity. We conjectured that the bonding pair ofthe distorted electron-rich B\u2013Ru bond in the BB-carboryne metallacycle could beaccessible for interaction with inorganic electrophiles such as coinage metal cations.The exohedral metal center on a carborane cage can be simultaneously connected to twovertices giving rise to carborynes, which are inorganic analogs of benzynes.i),Ag(i), and Au(i) to a single Ru\u2013B metal\u2013boryl bond and theformation of the heterobimetallic complexes featuring unique B\u2013M\u2013Ru\u2013Bbridging interactions 2-m-2,6-dehydrocarborane)3, and Au(SMe2)Cl in1\u2009:\u20091 ratio led to the selective formation and isolation of complexes2-Cu, 2-Ag, and 2-Au, respectively. The31P and 11B NMR spectra of crude reaction mixtures reflected cleantransformation of 1 to new products, which possessed lower symmetry of aboron cage according to 11B NMR spectra, consistent with interaction of coinagemetal cations with one of the B\u2013Ru bonds of the parent carboryne 1. The11B and 11B{1H} spectra of products contained pairs ofsignals corresponding to two different metalated boron atoms of the icosahedral cage,indicating the persistence of such interactions in solution. The complexes2-Cu, 2-Ag, and 2-Au were isolated in70\u201380% yields and were found to be moderately stable in air in the solid state inthe absence of light. The single crystal X-ray diffraction study revealed the molecularstructures of 2-Cu, 2-Ag, and 2-Au (Reaction of the (POBBOP)Ru(CO)and 2-Au . In all 2-Cu crystallized as a chloride-bridged dimer from anacetonitrile/hexanes mixture \u00c5, which is only slightly longer than 2c-2e Cu\u2013B bonds in therecently reported Cu(i) boryl complexes (1.980(2)\u20132.002(3) \u00c5).2-Cu is 2.630(1) \u00c5, which is longer thanCu\u2013Ru bonds in bimetallic complexes (2.439(1)\u20132.552(7) \u00c5)The complex mixture . The copi). The distortion from the planarityfor the Cu(Cl)2(B)(Ru) unit is remarkably small with the\u03c44 value of 0.17 . To the best of our knowledge, this is one of the lowest values ofthe \u03c44 parameter for Cu(i) complexes reported todate.Two bridging chloride ligands (Cu\u2013Cl distances are 2.321(1) \u00c5 and 2.329(1)\u00c5) complete the coordination sphere of the copper center to four-coordinate planargeometry, which is uncharacteristic for Cu cation inserted into one of the B\u2013Ru bonds of thecarboryne complex. The Au1\u2013B2 bond length is 2.027(2) \u00c5, which is the shortestAu\u2013B distance reported to date, as it is slightly shorter than 2c-2egold\u2013boron bonds in the previously disclosed gold boryl complexes(2.069(3)\u20132.144(4) \u00c5).2-Cu, the relativelylong Ru1\u2013B2 distance of 2.723(3) \u00c5 implies no significant bonding while theRu1\u2013B1 bond length (2.133(2) \u00c5) is within the typical range for rutheniumboryls. to 2-Cu . The Au2(B)(Ru) moiety in 2-Au exhibits four-coordinate planarconfiguration that is unusual for Au(i)\u03c44 = 0.11) as indicated by nearly linearRu1\u2013Au1\u2013Cl1A and B2\u2013Au1\u2013Cl1 angles (167.5(1)\u00b0 and177.5(1)\u00b0, respectively).Similarly to the copper coordination environment in 2-Ag, crystallized from anacetonitrile/hexanes mixture as a monomeric acetonitrile adduct (i) cation inserted into one of the B\u2013Rubonds of the carboryne complex. The Ag1\u2013B1 bond length is 2.182(3) \u00c5, whichis, to the best of our knowledge, the third example of the silver\u2013boryl bond andthis distance is comparable to those in two previously reported silver boryl complexes(2.118(2) \u00c5 and 2.122(4) \u00c5).61The light-sensitive silver insertion product, e adduct . The Ag(+ acrossthe ruthenium\u2013silicon double bond is 2.681(1) \u00c5.2-Cu.The Ru1\u2013Ag1 distance is 2.750(1) \u00c5, which is longer than the previouslyreported distances in unsupported bimetallic complexes (2.608(1)\u20132.709(1)\u00c5)1 to 2-M complexes. The values of \u03bd(CO) =2010 and 1958 cm\u20131 (\u03bd(CO)average = 1984cm\u20131) for the BB-carboryne complex 1 can be compared tothe corresponding parameters in coinage metal insertion products (see ESI\u03bd(CO)average values for these complexes are 1995cm\u20131 for 2-Cu, 2002 cm\u20131 for2-Au, and 2017 cm\u20131 for 2-Ag. Thus, in allthree cases, the \u03bd(CO)average increased reflectingcoordination of Lewis acidic cations to one of Ru\u2013B bonds in 1.Trends in values of stretching frequencies of carbonyl ligands of the ruthenium centercan provide an insight into changes of its electronic structure upon conversion of11B NMR spectra of 1 and 2-M inC6D6 showed changes of chemical shifts of metalated boron atomsupon coordination of coinage metals. The starting complex 1 exhibited asignal for the (BB) PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1100000000000000000000000000000000 1111110000000000000000000000000000 0011111111000000000000000000000000 0000001111111100000000000000000000 0000000000111111110000000000000000 0000000000000011111111000000000000 0000000000000000001111111100000000 0000000000000000000000111111110000 0000000000000000000000000011111111 0000000000000000000000000000001111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000001111 0000000000000000000000000011111111 0000000000000000000000111111110000 0000000000000000001111111100000000 0000000000000011111111000000000000 0000000000111111110000000000000000 0000001111111100000000000000000000 0011111111000000000000000000000000 1111110000000000000000000000000000 1100000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019Ru metallacycle at \u20132.8 ppm while the corresponding signals for theboron atom of the B\u2013Ru bond are at \u20131.5 ppm for 2-Cu, +2.1 ppmfor 2-Au, and \u20132.7 ppm for 2-Ag. The signals of boronatoms of B\u2013M bonds are at \u201310.3 ppm for 2-Cu, \u20139.7 ppm for2-Au, and \u201311.9 for 2-Ag. The 31P spectra of2-M complexes are similar with a signal in the 204\u2013205 ppm range .The comparison of ii) center making it heptacoordinate. Inthe structure C, coinage metal forms the 2c-2e electron bond to one of the boron atom ofthe cage, while the structure D features coordination of the B\u2013M bond to theruthenium center. The structure E is derived from the structure C by addition of thedative M\u2013Ru interaction.Several resonance structures can be considered to describe bonding inB\u2013Ru\u2013M\u2013B metallacycles in these complexes . The strThe presence of unique B\u2013M\u2013Ru\u2013B metallacycles in the reported newcomplexes prompted us to examine the bonding situation in more detail using the analysisof the electron density in the framework of the quantum theory of atoms-in-molecules(QTAIM)2-Cu, 0.74 for 2-Ag, and 0.75 for2-Au. These values are also close to DI values corresponding to 2c-2emetalboron bonds in the starting carboryne complex 1 (DI = 0.69).Topological analysis revealed the presence of the Ru\u2013B1 bond path in all complexes.The bonding between ruthenium and the boron atom B1 exhibits concentration of the electrondensity (blue contours) that is bent outward the Ru\u2013B1 bond path, indicating thestrain of this metal\u2013boron bond. The Ru\u2013B1 bonding in all three structures canbe considered a 2c-2e bond with delocalization indices of 0.73 for 2-Cu and2-Ag as demonstrated by DI values of 0.31 (2-Cu) and 0.33(2-Ag). The ruthenium\u2013coinage metal interactions in these complexesdo not have bond paths for 2-Cu and 2-Ag. The DI values of theseinteractions are 0.42 for 2-Cu, 0.40 for 2-Ag. Such an extensive\u201csharing\u201d of electrons between Ru, B2, and Cu or Ag is indicative of3-center-2-electron bonding . Note that the depletion ofthe electron density on B2 also results in the weakening of the B1\u2013B2 bonding.The Ru\u2013Au\u2013B2 bonding situation is noticeably different. First, the QTAIManalysis revealed the presence of the Ru\u2013Au bond path with a DI value of 0.58. TheDI value of Au\u2013B2 bond is 0.81, which is also higher than that for Cu\u2013B2 andAg\u2013B2 bonds. At the same time, the Ru\u2013B2 delocalization index in\u03b7a; the maximum ELF values for each basin) and basinpopulations for selected valence basins ofinterest. For 2-Cu and 2-Ag, the ELF analysis revealed thepresence of one disynaptic valence basin V (which is similar to that in thestarting complex 1) and one trisynaptic V or V basin,respectively. Attractors of trisynaptic V and V basins are located closeto the centers of Ru\u2013M\u2013B2 triangles (but somewhat displaced closer to B2).Populations of these basins are 2.6 e in 2-Cu and 2.0e in 2-Ag.Electron localization function (ELF) is visualized by 3D representations , and its2-Au reveals a substantial change in the bondingsituation. The trisynaptic basin observed in 2-Cu and 2-Ag issplit into two independent disynaptic basins V and V. The population value\u03a9 for the V interaction is 1.9 e, which is similar to that ofthe V basin and corresponds to the localized 2c-2e bonding. The V basin hasthe smaller \u03a9 value of 0.7 e indicating a weaker 2-center bondinginteraction.The ELF analysis of 2-Cu complex. The transition ofthe trisynaptic basin in 2-Cu and 2-Ag complexes indicative of2c-3e bonding to two disynaptic basins in 2-Au as found in the ELF analysisagrees with the results of the QTAIM analysis discussed above. As there is no directRu\u2013B2 bonding, the value of the delocalization index of Ru\u00b7\u00b7\u00b7B2 interaction can beused as an indicator of the 3-center boding. In 2-Cu and 2-Agthis DI value is rather large, 0.31 and 0.33, respectively, whereas in 2-Auit is reduced to 0.10. In parallel, DI/DI values are increased from0.40\u20130.42/0.66\u20130.63 in 2-Cu and 2-Ag to 0.58/0.81 in2-Au. Thus, both QTAIM and ELF analyses show a transition from the lesslocalized three-center B\u2013M\u00b7\u00b7\u00b7Ru bonding in 2-Cu and 2-Agto the more localized distinct two-center Ru\u2013Au and Au\u2013B2 interactions in2-Au.Notably, the sum of the V and V basin populations is close to theV basin population in the analogous 2-Cu and 2-Ag complexes is the 3c-2e B\u2013M\u00b7\u00b7\u00b7Ru interactiondescription that is often used for bridging B\u2013H\u00b7\u00b7\u00b7M interactions in boranes and boronclusters while bonding in 2-Au is closer to the localized 2c-2e Au\u2013Bbond. These findings are consistent with the Au\u2013B bond length in 2-Aubeing the shortest reported to date. The bonding of Cu and Ag to boron atoms in2-Cu and 2-Ag is also unusually strong with B\u2013M distancescomparable to the recently reported isolated 2c-2e bonds of coinage metals with nucleophilicboryls. Importantly, the formation of these complexes represents a unique synthetic strategyfor generation of the first examples of the exohedral coinage metal\u2013boryl bonds in{C2B10} carborane clusters as the direct activation of theirB\u2013H bonds by Group 11 metals remains unknown.The results reported herein uncover unusual reactivity of the ruthenium BB-carboryne withinorganic electrophiles and introduce a new approach for the rational construction ofmultimetallic complexes supported on the surface of polyhedral boron cages.+, Ag+, and Au+ into strained Ru\u2013Bsingle bonds in 1 is unprecedented. Notably, coordination of coinage metalcations to alkylidenes, silylenes, and borylenes/boryls has been reported.+ to a platinum\u2013aryl bond has beendisclosed.1 has not been reported. It also remains to be seen if similartransformations can be observed in the case of other metal boryls or in the case of otherstrong inorganic Lewis acids thus providing an alternative synthetic access to novelmultimetallic architectures featuring M\u2013B bonds.Insertion of CuThere are no conflicts to declare.Supplementary informationClick here for additional data file.Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "Synthesis and transformations of AgNO3 complexes of trans-cycloheptene (TCH) and trans-1-sila-4-cycloheptene (Si-TCH) derivatives are described. 3 complexes of trans-cycloheptene (TCH) and trans-1-sila-4-cycloheptene (Si-TCH) derivatives is described. A low temperature flow photoreactor was designed to enable the synthesis of carbocyclic TCH derivatives due to their thermal sensitivity in the absence of metal coordination. Unlike the free carbocycles, TCH\u00b7AgNO3 complexes can be handled at rt and stored for weeks in the freezer (\u201318 \u00b0C). Si-TCH\u00b7AgNO3 complexes are especially robust, and are bench stable for days at rt, and for months in the freezer. X-ray crystallography was used to characterize a Si-TCH\u00b7AgNO3 complex for the first time. With decomplexation of AgNO3in situ, metal-free TCO and Si-TCH derivatives can engage in a range of cycloaddition reactions as well as dihydroxylation reactions. Computation was used to predict that Si-TCH would engage in bioorthogonal reactions that are more rapid than the most reactive trans-cyclooctenes. Metal-free Si-TCH derivatives were shown to display good stability in solution, and to engage in the fastest bioorthogonal reaction reported to date (k2 1.14 \u00d7 107 M\u20131 s\u20131 in 9\u2009:\u20091 H2O\u2009:\u2009MeOH). Utility in bioorthogonal protein labeling in live cells is described, including labeling of GFP with an unnatural tetrazine-containing amino acid. The reactivity and specificity of the Si-TCH reagents with tetrazines in live mammalian cells was also evaluated using the HaloTag platform. The cell labeling experiments show that Si-TCH derivatives are best suited as probe molecules in the cellular environment.A photochemical synthesis of AgNO The unique reactivity of trans-cycloalkenes has produced an impressive collection of applications in synthesis, including reactions with dienes,trans-cycloalkenes can serve as excellent ligands for transition metals. In the field of bioorthogonal chemistry,trans-cycloalkenes hold special significance due to their particularly fast kinetics in cycloaddition reactions.trans-cyclooctene, the chemistry of the homolog trans-cycloheptene is less explored. trans-Cycloheptene was first trapped with diphenylisobenzofuran by Corey and Winter from trans-1,2-cycloheptenethionocarbonate through treatment with P(OMe)3.cis/trans equilibria could be driven by selective addition reactions of trans-cycloalkenes.For nearly seventy years,trans-Cycloheptene was first spectroscopically characterized by Inoue via singlet sensitized photoisomerization of cis-cycloheptene at \u201335 \u00b0C.trans-cyclooctene, which is stable at room temperature, trans-cycloheptene undergoes rapid isomerization under ambient conditions via a proposed \u2018interrupted dimerization\u2019 mechanism.30trans-Cycloheptene has also been prepared via ligand exchange from a trans-cycloheptene\u00b7CuOTf complex.trans-cycloheptene and trans-cycloheptenone derivatives are also known to be thermally unstable at ambient temperature, but can be trapped in situ.trans-cycloheptene is thermally labile, it has been demonstrated in several studies that metal complexes can be isolated. CuOTf has been proposed to catalyze photodimerization reactions of cyclic olefins via photoinduced cis\u2013trans isomerization,trans-cycloheptene\u00b7CuOTf complex has been prepared through irradiation of cis-cycloheptene\u00b7CuOTf, but a yield for the process was not reported.2 complex of trans-cycloheptene has been prepared by irradiation of the corresponding ethylene complex in the presence of cis-cycloheptene under singlet sensitized conditions.4 complexes of 3-methoxy-trans-cycloheptene and 6-methoxy-(Z),4(E)-cycloheptadiene.4\u00b73-methoxy-trans-cycloheptene complex was combined with a number of dienes to give the products of metal decomplexation and [4 + 2] cycloaddition.While the parent trans-cycloalkenes.E)-1,1,3,3,6,6-hexamethyl-1-sila-4-cycloheptene was synthesized, resolved, and characterized crystallographically.trans-alkene. Recent studies by Woerpeltrans-cycloalkene derivatives in synthesis. Woerpel has elegantly developed a general method for the preparation of trans-oxasilacycloheptenes\u2014seven membered rings that contain trans-alkenes and siloxy bonds in the backbone.Because C\u2013Si bonds are long, the inclusion of silicon into the cyclic backbone can alleviate olefinic strain and impart stability to trans-cycloheptene derivatives. In 1980, Jendralla showed that silver complexes of 3-methoxy-trans-cycloheptene are isolable, and can reversibly dissociate and in unspecified yields undergo cycloaddition reactions.trans-oxasilacycloheptenes, and recently has reported the Diels\u2013Alder reactions of trans-oxasilacycloheptenes with furan and tetrazine derivatives.trans-cyclooctene (\u2018s-TCO\u2019) derivative.s-tetrazine in benzene at rt in less than 10 min and in 90% NMR yield.48There has been little investigation into the reaction chemistry of trans-cyclooctene derivatives, whereby selective complexation with AgNO3 is used to drive the formation of trans-isomer from cis-cyclooctene.trans-cycloheptene (TCH) and trans-1-sila-4-cycloheptene (Si-TCH) derivatives via flow photochemical synthesis. The derivatives described are especially stable as their AgNO3 metal complexes, which can be stored neat or in solution for long periods. With decomplexation of AgNO3in situ, metal-free TCH and Si-TCH derivatives can engage in a range of cycloaddition reactions as well as dihydroxylation reactions. Unlike the carbocycles, Si-TCH derivatives display good stability in solution and are shown to engage in the fastest bioorthogonal reaction reported to date. Decomplexation of AgNO3 can be carried out in situ directly in cell media for bioorthogonal protein labeling in live cells.Our group has described a closed-loop flow reactor for the synthesis of trans-cyclooctene derivatives, the trans-cycloalkene is first scavenged on AgNO3/SiO2 and then liberated through treatment with aqueous or methanolic ammonia.cis-isomers.trans-cyclooctenes could be enhanced by storing the cycloalkenes as their AgNO3 complexes, and that the free alkenes could be liberated in situ through treatment with NaCl in aqueous solution or cell media.For the photochemical synthesis of 3 derivatives were carried out at rt using the previously described flow-photoisomerization apparatus,3 complexes were directly isolated from SiO2 without Ag-decomplexation. The metal complexes that were obtained were stable in neat form for >1 month in the freezer. However, it was necessary to alter our reactor design for the synthesis of carbocyclic trans-cycloheptenes due to their thermal lability was positioned before the photowell, and the photoisomerization was conducted in a coil of optically transparent FEP tubing.3/SiO2. In our standard setup, an inline thermometer was included to measure the temperature for the flowing mixture either before or just after the UV lamp. The temperature was measured as 0 \u00b0C before entering the Rayonet photoreactor, and as 20 \u00b0C after exiting the photoreactor. With this apparatus, TCH\u00b7AgNO3 complexes were eluted from the column, and isolated as semisolids that are moderately stable at rt but stable for weeks in the freezer.Photoisomerizations to form Si-TCH\u00b7AgNOlability . As withtrans-cycloheptene (1a) and trans-5-hydroxymethylcycloheptene (1b) were prepared in 53% and 64% yields, respectively. These TCH\u00b7AgNO3 complexes are stable enough to handle on the bench for modest periods (hours), and to longer-term storage in the freezer (\u201318 \u00b0C). NMR monitoring showed 90% fidelity for a CD3OD solution of 1a after 10 days storage in freezer (\u201318 \u00b0C), and 92% fidelity for a CD3OD solution of 1a after 10 hours at rt on the bench.The scope of TCH and Si-TCH synthesis is shown in 3OD solution of 2a after 8 days at rt, and 96% fidelity for 2a after storage for 1 month in the freezer . The photoisomerization method could be used to produce diphenyl (2a) or dialkyl (2b\u2013g) substituted silacycles. Cyano (2b) and hydroxyl (2c\u2013e) groups were tolerated, as were NHS ester (2f) and chloroalkane (2g) groups that could be used to enable conjugation to fluorophores and HaloTag3 and Si-TCH\u00b7AgNO3 complexes were isolated as semisolids that contained 20\u201330% free AgNO3. The isolated yields were corrected by measuring the 1H NMR against an internal standard.The silver complexes of Si-TCH are much more stable. NMR monitoring showed 93% fidelity for a CDtrans-cyclooctene coordinates with AgNO3 as a 1\u2009:\u20091 complex.2a were grown from ethyl acetate/methanol. Selected bond lengths and angles are displayed in i) nitrate complex of the equatorial diastereomer of 5-hydroxy-trans-cyclooctene 3 scale\" fill=\"currentColor\" stroke=\"none\">C\u2013C dihedral angle for 3 (136.7\u00b0) is smaller than metal-free TCO 4 (139.1\u00b0). PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C\u2013C dihedral angle for 2a (126.3\u00b0) was smaller than that of metal free Si-trans-cycloheptene 5 (130.9\u00b0),6 (126.1 \u00b0C)i) alkene complexes, the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond length for the Ag(i) complexes was very similar to that of the metal-free complexes 4\u20136 (1.331\u20131.335 \u00c5).Previously, a crystal structure showed that octene 3 . Here, t4OH or aq. NaCl, followed by extraction with organic solvent. For example, silver complex 2a upon treatment with aq. NH4OH was extracted with C6D6 to give a solution of Si-TCH 7a (98% trans isomer). Consistent with previous reports on a biomolecular mechanism for TCH isomerization,cis-isomer of 7a was observed when 7a was concentrated to dryness on the rotovap. However, 7a displayed high stability when maintained in solution, with only 8% isomerization observed for a 100 mM solution of 7a that was stored for 24 hours at room temperature, and <5% isomerization for a similar solution that was stored for 24 hours in a freezer (\u201320 \u00b0C).Silver-free Si-TCH compounds could be prepared by treating their corresponding silver nitrate complexes with an excess of aq. NHi)-complex 2d could also be freed of metal to give alkene 7d as a mixture of diastereomers scale\" fill=\"currentColor\" stroke=\"none\">C double bond stretch of 7d at 1624 cm\u20131 is shifted to 1559 cm\u20131 for the Ag(i) complex 2d. This 65 cm\u20131 shift is consistent both in magnitude and direction for a Ag(i) alkene complex.i)-complexation leads to signature shifts of alkene resonances in both the 1H and 13C NMR spectra with dichloroketene, benzylazide or diazomethane only returned unreacted starting material. An X-ray structure was obtained for the dichloroketene adduct 13 . As discussed below, this fluorophore conjugate finds utility for bioorthogonal labeling in live cells. We also demonstrated that the equatorial allylic alcohol 17(eq), derived from Ag-complex 2e, could be elaborated to the carbamate 18 through treatment with benzylisocyanate level, and compared to previous calculations on trans-cyclooctene and s-TCO 20. PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C\u2013C dihedral angle for 19 (128.8\u00b0) is smaller than that for trans-cyclooctene (137.7\u00b0) or 20 (131.9\u00b0). M06L/6-311+G calculations were also carried out to compare the Diels\u2013Alder reactivity of Si-TCH 19 to trans-cyclooctene and s-TCO 20 9.91 kcal mol\u20131 and \u0394H\u2021 8.87 kcal mol\u20131. The barrier is significantly lower than that of trans-cyclooctene with 3,6-diphenyl-s-tetrazine \u20133.99 kcal mol\u20131, \u0394\u0394H\u2021 4.06 kcal mol\u20131). The barrier is also lower than that calculated for s-TCO 20 \u20130.59 kcal mol\u20131, \u0394\u0394H\u2021 \u20130.72 kcal mol\u20131). The computations prompted us to conduct experimental investigations into the utilization of Si-TCH complexes for applications in bioorthogonal chemistry.Anticipating that Si-TCH compounds may be useful in bioorthogonal chemistry, computation was used to study the structure and Diels\u2013Alder reactivity of Si-TCH 19 . We comps-TCO 20 . These cs-tetrazine in MeOH at 25 \u00b0C with s-TCO,trans-cyclooctene,\u20131 s\u20131, respectively. Si-TCH 7c was liberated from AgNO3, and in agreement with computational prediction, was found to react with diphenyl-s-tetrazine with a rate constant of 4360 (+/\u2013430) M\u20131 s\u20131\u20141.4 times faster than sTCO, 8.4 times faster than dTCO, and 228 times faster than trans-cyclooctene itself M\u20131 s\u20131. This is the fastest rate constant reported to date for a bioorthogonal reaction.Under aqueous conditions, tetrazine ligations are accelerated due to the hydrophobic effect. We previously had studied the reactions of s-TCO, d-TCO and in vitro and in vivo cycloaddition of SiTCH and a green fluorescent protein with an unnatural tetrazine-containing amino acid , encoded via the procedure of Mehl and coworkers.s-tetrazin-3-yl)phenylalanine was site-specifically introduced into a C-terminally hexahistidine-tagged GFP (sfGFP-150TAG-His6) via orthogonal translation using the evolved aminoacyl-tRNA synthetase MjRS/tRNACUA pair. Co-expression of these components in E. coli resulted in the amino acid-dependent synthesis of full-length recombinant GFP-Tet of E. coli overexpressing GFP-Tet by measuring the increase in whole-cell fluorescence upon addition of 7c. At room temperature, a second-order rate constant of 155\u2009000 \u00b1 20\u2009000 M\u20131 s\u20131 was measured for the in vivo reaction, which is 62% as rapid as the in vitro ligation. The modest reduction in rate is in line with previous observations with TCO-based dienophiles.7c is capable of crossing the bacterial cell membrane and engaging in rapid, high yielding conjugation inside a living cell.Although BS at rt . The reaBS at rt , and is trans-cycloalkene\u2013AgNO3-complexes liberate AgNO3 immediately in cell media due to the high NaCl content, and perform identically to their metal-free analogs in cell labeling experiments.AgSiTCH-Halo) and methyl-tetrazine and used these clickable HaloTag ligands to covalently label HaloTag protein expressed in HEK293T cells with the clickable tag. In a competitive pulse-chase experiment, it was shown that 10 \u03bcM of these HaloTag ligands completely blocked incorporation of BODIPY-Halo substrate for different times (2\u201390 min) . The rea\u201390 min) , and in-SiTCH-Halo with MeTz-BODIPY was complete within 15 minutes completely isomerizes in 2 h at 22 \u00b0C in CD3OD with mercaptoethanol (30 mM), whereas as control sample without thiol was >98% stable under these conditions. At \u201317 \u00b0C, 7b isomerizes more slowly in the presence of 30 mM mercaptoethanol, with 47% isomerization after 24 h. By comparison, the trans-cyclooctenes d-TCO, s-TCO, and oxo-TCO are much more stable toward thiol promoted isomerization. In CD3OD with mercaptoethanol (30 mM) at room temperature, s-TCO (30 mM) isomerized only after an 8 hour induction period, with complete conversion to the cis-isomer after 4 additional hours.3OD over a 22 hour period at room temperature.72Compared to the periods . By contprotocol .56 SpeciTogether, the labeling experiments in bacteria and HEK293T cells show that SiTCH derivatives can serve as useful probe molecules in the cellular environment, as the unprecedented speed of the bioorthogonal reactions of SiTCH are much more rapid than competing deactivation pathways. However, the utility of SiTCH derivatives as protein tagging molecules, where extended incubation in the cellular environment takes place prior to bioorthogonal reactivity, appears much more limited in utility plausibly due to alkene isomerization in the cellular environment.3 complexes of trans-cycloheptene and trans-1-sila-4-cycloheptene derivatives have been prepared via a flow photochemical synthesis, using a new low temperature flow photoreactor to enable the synthesis of carbocyclic TCH derivatives. TCH\u00b7AgNO3 complexes can be handled for brief periods at rt and stored for weeks in the freezer (\u201318 \u00b0C). Si-TCH\u00b7AgNO3 complexes are especially stable, and can be stored on the bench stable for >1 week at rt, and for months in the freezer. X-ray crystallography was used to characterize a Si-TCH\u00b7AgNO3 complex for the first time. With decomplexation of AgNO3in situ, metal-free TCO and Si-TCH derivatives can engage in a range of cycloaddition reactions as well as dihydroxylation reactions. Computation predicted that Si-TCH would display faster bioorthogonal reactions toward tetrazines than even the most reactive trans-cyclooctenes. Metal-free Si-TCH derivatives were shown to display good stability in solution, and to engage in the fastest bioorthogonal reaction reported to date (k2 1.14 \u00d7 107 M\u20131 s\u20131 in 9\u2009:\u20091 H2O\u2009:\u2009MeOH). Utility in bioorthogonal protein labeling in live cells is described, including labeling of GFP with an unnatrual tetrazine-containing amino acid. The reactivity and specificity of the Si-TCH reagents with tetrazines in live mammalian cells was also evaluated using the HaloTag platform. The cell labeling experiments show that Si-TCH derivatives are suitable as highly reactive probe molecules in the cellular environment.In conclusion, AgNOThere are no conflicts to declare.Supplementary informationClick here for additional data file.Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "Formal addition of diazomethane's terminal nitrogen atom to the 9,10-positions of anthracene yields H2CN2A . 2CN2A . The synthesis of this hydrazone is reported from Carpino's hydrazine H2N2A through treatment with paraformaldehyde. Compound 1 has been found to be an easy-to-handle solid that does not exhibit dangerous heat or shock sensitivity. Effective umpolung of the diazomethane unit imbues 1 with electrophilicity at the methylene carbon center. Its reactivity with nucleophiles such as H2CPPh3 and N-heterocyclic carbenes is exploited for C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond formation with elimination of dinitrogen and anthracene. Similarly, 1 is demonstrated to deliver methylene to a nucleophilic singlet d2 transition metal center, W(ODipp)4 (2), to generate the robust methylidene complex . This behavior is contrasted with that of the Wittig reagent H2CPPh3, a more traditional and Br\u00f8nsted basic methylene source that upon exposure to 2 contrastingly forms the methylidyne salt [MePPh3].Formal addition of diazomethane's terminal nitrogen atom to the 9,10-positions of anthracene yields H An initial survey of the reactivity patterns of 1 has revealed it not to be a simple substitute for diazomethane, instead characterizing it as a unique electrophilic methylene source. Its electrophilicity differentiates 1 from common metal-free methylene transfer reagents such as diazomethane and methylene triphenylphosphorane.Diazomethane is infamous for the dangers associated with its use.1 proceeded from Carpino's hydrazine H2N2A upon paraformaldehyde treatment in a biphasic diethyl ether\u2013water mixture,11Synthesis of hydrazone 1 was found to be an air-stable and crystalline solid, easily manipulable and displaying no propensity for detonation upon heating or shock. The solid was found to be volatile by thermogravimetric analysis (TGA), which showed gradual sample evaporation up to 120 \u00b0C without any discrete mass-loss events that would be expected from its fragmentation into diazomethane and anthracene. Within a sealed capillary, 1 melted without explosion (116\u2013119 \u00b0C). After heating the melt to 140 \u00b0C, NMR spectroscopic analysis of the resolidified solid showed 74% recovery of 1 with 26% anthracene production. Its behavior in solution was similar, evincing only slow fragmentation into anthracene at temperatures greater than 120 \u00b0C. The volatility of this compound foiled attempts at analysis of its thermal behavior by molecular beam mass spectrometry (MBMS), limiting our ability to comment on the fragments directly produced by its thermal fragmentation.Hydrazone 1 to pose a low explosion risk, we were encouraged to proceed to test its reactivity as a methylene synthon. Our initial investigations rapidly uncovered contrasting reactivity patterns vis-\u00e0-vis those characteristic of diazomethane. For example, methylation of carboxylic acids, a hallmark of diazomethane reactivity,1.Having established 1 did not demonstrate nucleophilicity. Such behavior is not unexpected, as the \u03c0CN is known to be polarized away from the carbon center, although less so than an imine \u03c0CN or a ketone \u03c0CO bond.1 should be expected to exhibit moderate electrophilicity at its methylene carbon. This would effectively induce umpolung of the diazomethane unit as diazomethane generally reacts as a carbon nucleophile.15Hydrazones are known to be carbon ambiphiles;1 and H2CPPh3. Combination of these two reagents in benzene-d6 yielded ethylene in 21% yield over 12 h in concert with anthracene, triphenylphosphine, and, presumably, dinitrogen. The reaction was found to produce several unidentified byproducts by NMR spectroscopy, explaining the low yield of ethylene; however, isotopic labelling of the ylide led to H2C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-201913CH2 from 1 and H213CPPh3, and H2C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CD2 from 1 and D2CPPh3, confirming ethylene formation through the unification of the electro- and nucleophilic methylene units. Although the yield was low, this mode of reactivity was instructive for our further studies.The predicted reversal of philicity was initially confirmed by successful methylene transfer in the reaction between 1 lent itself well to the synthesis of N-heterocyclic olefins from N-heterocyclic carbenes (NHCs).6 solution, 1 reacted with nucleophilic IPr imidazol-2-ylidene) to yield the corresponding olefin in 70% yield after 13 h at 80 \u00b0C.N,N\u2032-diamidocarbene (\u201cDAC\u201d) was found to react in essentially quantitative yield to form a new C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond over 24 h at 22 \u00b0C. PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N\u2013N PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C moiety.1 with triphenylphosphine or tricyclohexylphosphine has not yielded the analogous yieldes, suggesting a modest Lewis acidity at the carbon center of 1.The electrophilicity of 1 and diazomethane thus encouraged us to attempt the use of 1 in methylidene complex synthesis to see if engagement of the terminal nitrogen in bonding to anthracene subdues deleterious alternate reaction pathways.It is rare for diazomethane to be used in transition metal chemistry for the synthesis of a stable methylidene complex.4] 2 transition metal complex well poised to behave as a methylene acceptor.2 is synthetically easy to access, and its square-planar geometry features a nucleophilic lone pair of electrons housed in a metal-centered dz2-like orbital, analogously to related tantalum and molybdenum singlet d2 species.2 with excess 1 gave facile formation of the anticipated methylidene complex after mild heating in benzene to 55 \u00b0C for 35 h for 35 h . Charact2 PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CH2] (\u03c4 = 0.48), PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C double bondvi) methylidenes.2 PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CH2] was not found to react productively with ethylene or 1-hexene upon heating to 70 \u00b0C in benzene-d6 for 18 h, confirmed by a lack of isotopic migration from to the olefins.2 PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CH2] also did not react with mesitaldehyde or 4,4\u2032-dimethylbenzophenone to form and the corresponding olefins. Despite this, is notable as an example of a methylidene complex with aryloxides as the exclusive supporting ligands. As such, it is an interesting structural model for methylidene complexes supported by silica or alumina surfaces implicated in alkane or olefin metathesis.Crystallization from pentane at \u201335 \u00b0C overnight enabled an X-ray diffraction study of [g>CH2] , left th1 was particularly satisfying after discovery of the contrasting behavior of H2CPPh3, a known reagent for CH2 delivery to transition metal centers.2 with H2CPPh3 (1 equiv.) in THF at 25 \u00b0C rapidly consumed 50% of 2 and formed the methylidyne salt [MePPh3]. Doubling the amount of H2CPPh3 gave total consumption of 2 and provided [MePPh3] in 49% isolated yield scale\" fill=\"currentColor\" stroke=\"none\">CH2] formation, indicating competitive deprotonation of intermediate by Br\u00f8nsted basic H2CPPh3. Such acid-base chemistry is postulated to play a critical role in the formation of surface-bound alkylidenes and alkylidynes for alkane and olefin metathesis,2 PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CH2] serves also as an interesting reactivity model for alkylidyne synthesis mediated through proton transfer. This was corroborated by independent deprotonation of with H2CPPh3, and highlights the utility of 1 as a weakly Br\u00f8nsted basic source of methylene. Protonation of [MePPh3] using lutidinium triflate presents a complementary route to .The reactivity of ed yield . Variati3] revealed a W\u00b7\u00b7\u00b7C interatomic distance of 1.749(1) \u00c5 and a square pyramidal (\u03c4 = 0.21) coordination geometry about tungsten. A search of the CSD revealed this to be the first catalogued example of a structurally characterized metal methylidyne in an all-oxygen ligand environment, and the first catalogued example of a tungsten(vi) methylidyne complex.An X-ray crystallographic study of [MePPh1 can be further exploited in their syntheses. Compound 1 has also shown promise in formation of new C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bonds with H2CPPh3 and NHCs, and may find use in construction of terminal olefins.As interest in metal methylidene species is rapidly growing both in homogeneous and heterogeneous catalysis,There are no conflicts to declare.Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "We herein present a simple methodology to systematically expand the scope of maleimide-based dyes and also provide an insight into the relationship between substitution pattern and optical properties. A series of maleimide derivatives were systematically designed and synthesized with tunable fluorescent properties. The facile modifications herein provide a simple methodology to expand the scope of maleimide-based dyes and also provide insight into the relationship between substitution pattern and optical properties. The development of novel organic fluorophores is of great interest in the areas of biological labels and probes,DTM)ABM),2 or N2.33Unsubstituted maleimides have been extensively reported as effective fluorescence quenchers through direct conjugation to fluorophores.Despite the recent advances in this field, there has been little work on varying the maleimide structures and studying the subsequent impact on their fluorescence. PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O groups and the group on the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C moiety act as the electron acceptor and donor respectively. Our initial strategy began with reacting three different pro-fluorescent precursors: dibromo (DBM), dichloro (DCM) and diiodo (DIM) maleimide to aminobromomaleimide (ABM), through single substitution, with an amine, has been previously reported by our group.21The design concept relies on the typical electron donor and acceptor mechanism in maleimide-based fluorophores where the Cde 1a\u2013c, . The conDCMs and DIMs). The reaction resulted in the formation of a singly substituted product for all precursors. The optical properties of these three different amino substituted halogeno maleimides (2a\u2013c) were investigated. In diethyl ether, the three compounds (2a\u2013c) exhibited two similar absorption peaks around 238 nm and 370 nm assigned to an n\u2013\u03c0* and a \u03c0\u2013\u03c0* transition, respectively and aminobromomaleimide showed similar green emission ca. 475 nm while the aminoiodomaleimide produced a slightly red shifted emission (ca. 487 nm). The fluorescence quantum yields (\u03a6f) of these products were measured using quinine sulfate as a reference, to establish any effect of the halogen.\u03a6f decreased from Cl through Br to I in diethyl ether and compared this to the analagous chloro derivative (2d).\u03a6f of 2d was 42% in diethyl ether and 65% in cyclohexane, which is the highest \u03a6f of all reported amino halogeno maleimides.Based on this method, we expanded the scope by reacting the amine with other halogeno maleimides exhibited a similar change in fluorescent emission across the solvent series and the emission wavelengths were shifted to higher wavelengths. This is consistent with previous work which shows that the hydrogen bonding between protic solvents and the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O group in maleimides causes quenching effects through electron driven proton transfer from the solvent to the fluorophore.\u03a6f was observed, likely as a consequence of the suppression of twisted intramolecular changer transfer (TICT) in the maleimide rings.9The solvatofluorochromic properties were further investigated in solvents across a wide polarity range. All compounds . Following the route of Booker-Milburn et al., sodium alkoxide was used to catalyze a substitution reaction with DCM and DBM and a series of alcohols.in situ formation of the products, 3a\u2013f, proceeded successfully for both aryl and alkyl alcohols at room temperature.Looking at these results it was clear that the optical properties are closely related to the electronegativity of the halogen substituents, with increased electronegativity correlating with increased 3a\u2013d, the typical \u03c0\u2013\u03c0* transition peak shifted from ca. 360 nm to ca. 335 nm, while also reducing in intensity (2b) to an ethoxy (3b) group also lead to a dramatic decrease in \u03a6f from 30% to 10% (in diethyl ether). This indicates that a strong electron donating group on the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond is critical in obtaining a high fluorescence intensity. Looking at the series in more detail, both the OCM and OBM substituted ethoxy (3a\u2013b) and benzyloxy (3c\u2013d) maleimides showed similar emission wavelengths, whereas no emission was observed for the phenoxy (3e\u2013f) derivatives . As the optical properties are closely related to modifications made to this series of singly substituted maleimides, it was hypothesized that more remarkable changes may be realized by exploring disubstituted maleimides. Previously, it has been demonstrated that DBMs were capable of undergoing an efficient addition-elimination reaction with thiols resulting in a disubstituted product using thiol groups.ABM scaffold using microwave irradiation at 50 \u00b0C.ABMs altered the electronic nature of the neighbouring site, such that a second amine addition does not occur at room temperature. Conversely, we were inspired by the fast and efficient double addition elimination reaction with thiols and hence attempted to introduce a second thiol group on an ABM. A similar compound has been reported to date, by Naka et al., where thiophenol was able to undergo reaction with an aniline substituted maleimide. However, due to the aromatic nature of the aniline, almost no fluorescence was observed in solution.ATM) were synthesized and their optical properties explored. ATMs were generally synthesized from ABMs through the addition of 1 equivalent of thiol and sodium hydroxide at 80 \u00b0C in DMF. Following the successful synthesis of a series of ten ATMs (4a\u2013j), the optical properties were characterized. The excitation wavelength of the ATM compounds remained in the 360\u2013380 nm range (in diethyl ether) similar to ABMs, while the emission shifted to higher wavelengths compared with previous substituted maleimides, which could make them useful in F\u00f6rster Resonance Energy Transfer (FRET) based applications. The solvafluorochromic properties of ATM (4h) was compared with ABM (2e) in eight common solvents , the emissions of ATM (4h) were higher in wavelength (green to yellow range). Similar with ABMs, fluorescence quenching of ATM was observed in protic solvents which is attributed to hydrogen bonding between the protic solvent and the carbonyl.In the absorbance spectra of ntensity . MeanwhiATMs with varying thiol substituents (4a\u2013d) were compared. The emission of two alkyl thiol substituted compounds (4a and 4b) showed a relatively high emission wavelength (ca. 560 nm) which approached the red region of the spectrum , the emission maxima blue-shifted with an unexpected increase in quantum yield. Furthermore, strong emission was also observed in the solid state of thiophenol functionalized ATM (4h) compared to their analogous ABM (2e). Comparing the absolute \u03a6f in solid states, 4h processes a higher \u03a6f (4h 8% versus2e 1%) and longer fluorescence lifetime and dichloromethane (good solvent). The fluorescence of 4h gradually increased with the addition of non-solvent while aminomaleimide 2e did not present the same trend showed an increased \u03a6f compared with compounds with methyl at R1 (4a\u2013g). As expected, the direct conjugation of an aniline group at the R2 position (4g) also caused dramatic quenching of the fluorescence (\u03a6f < 0.1%), consistent with previous reported aminobromomaleimides.ATMs originates from the aminomaleimide moiety analogous to ABMs, in which the amino group on C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C double bond acts as the donor moiety in a donor\u2013acceptor skeleton.Following the study on the effect of thiol groups in the R3 position, substituents on the R1 and R2 positions were also found to affect the optical properties significantly /6-311G level using time-dependent density functional theory scale\" fill=\"currentColor\" stroke=\"none\">C bond positively affect fluorescence. Building upon this work and further exploring the relationship between the structure of maleimides and their optical properties is essential for full understanding of the fluorescence mechanism. This, in turn, will aid the design of future maleimide fluorophores with small size, multi-functionalization and high fluorescence efficiency for biological and chemical sensing applications.In conclusion, we successfully synthesized a library of maleimide fluorophores with remarkable fluorescent properties such as tunable emissions among the visible colour range (blue to yellow), high fluorescence quantum yields (up to 64%) and solvafluorochromism. Significantly, we investigated the effect on optical properties when altering the halogen group and the donor groups . The fluorescence results provide evidence of the previously hypothesized push\u2013pull model and computational results further confirmed that smaller charge differences on the CThe authors thank the University of Warwick, the ERC (grant no. 615142), Ministerio de Econom\u00eda y Competitividad (MINECO) of Spain (project CTQ2016-80375-P) and EPSRC for financial support. The authors acknowledge the computational resources and technical and human support provided by DIPC.There are no conflicts to declare.Supplementary informationClick here for additional data file."} +{"text": "Molecular cage-bridged gold nanoclusters with well-defined hotspots were demonstrated as novel plasmon-assisted nanoreactors. Creating well-defined plasmonic hotspots with enormous field enhancements as well as the capability of selectively trapping targeted molecules into hotspots is of critical importance and a prerequisite for numerous plasmon-assisted applications, but it represents a great challenge. In this work, a robust molecular cage decorated with thioether moieties at the periphery was designed and synthesized. By using the synthesized cage as a linker, a series of molecular cage-bridged plasmonic structures with well-defined nanogaps (hotspots) were fabricated in an efficient and controllable fashion. It was found both experimentally and theoretically that the nanogaps of about 1.2 nm created by the molecular cage in the resultant plasmonic structures led to a strong plasmon coupling, thus inducing great field enhancement inside the nanogaps. More importantly, the embedded molecular cages endowed the formed hotspots with the capability of selectively trapping targeted molecules, offering huge opportunities for many emergent applications. As a demonstration, the hotspots constructed were used as a unique nanoreactor, and under mild conditions two types of plasmon-driven chemical transformation were successfully performed. All the results clearly indicate that the integration of the host\u2013guest chemistry of the molecular cage with the plasmon-coupling effect of metal particles afforded a new class of plasmonic structures, showing great potential for facilitating a broad variety of plasmon-based applications. In recent years, it was elucidated that the enormous field enhancement could also lead to the efficient generation of hot charge carriers,Noble metal nanoparticles (NPs) support unique localized surface plasmon resonances (LSPRs) through coherent oscillations of free electrons in metallic nanostructures under light illumination, and a strong electromagnetic field is induced in the close vicinity of the metal surface.et al., organic thiol-linkers were used to direct the organization of NPs.et al. have prepared well-organized NP ensembles with complex and hierarchical nanostructures via DNA.5Generally, top-down and bottom-up strategies are employed for creating or engineering hotspot nanostructures.2L4 metal\u2013organic molecular cage decorated with thioether moieties at the periphery under mild conditions as well as the cycloaddition from 4-ethynylphenol (4-EP) and 4-azido-phenol (4-AP) without catalysts were successfully realized. All the results indicate that the molecular cage-bridged plasmonic clusters described are new plasmonic nanostructures with advanced functionalities.In this work, we describe a new Meriphery . We founL was synthesized from 2,6-dibromoisonicotinic acid (Scheme S1L (2 equiv.) and [Pd(CH3CN)4](X)2 (1 equiv.) at room temperature (X= BF4\u2013 or OTf\u2013), the colorless cage single crystal was achieved after three days with good yields of 79\u201393% than the free ligand L (D = 1.9 \u00d7 10\u20139 m2 s\u20131), which is indicative of the formation of larger chemical species n)(4\u2013]n+ (n = 2\u20134) platinum(ii) (DCTP) and 1,4-phenyldiamine hydrochloride (PDA) using 1H NMR, UV-Vis and ESI-MS of the suspension of CP in the CD3CN solution of the cage. As shown in Fig. S3,\u03b4 = 0.06 ppm) and a broadening of the internal proton Ha of the cage were detected in the NMR spectrum of the mixed solution, indicating that the guest CP was trapped in the molecular cage. Indeed, the ESI-MS result 2]4+ complex with an intense peak at m/z = 544.28, confirming that two cisplatin molecules were trapped in the cage. In fact, the same encapsulation behavior of the CP was also observed in a similar molecular cage only without the thioether moieties at the periphery.8The host\u2013guest behavior of the synthesized molecular cage was studied using five guest molecules with different size, shape and charge, including CP, 4-NP, HQ, t Fig. S4 shows th2L4 cage was examined using 1H NMR, ESI-MS and UV-Vis methods. Due to the formation of hydrogen bonding between the guest and the pyridine moieties of the cage, the protons (Ha and Hb) of the cage were most shifted upon HQ and 4-NP encapsulation @(4-NP)2]3+ with the peak at m/z = 648.8 in the ESI-MS spectrum was also detected for the case of 4-NP n@guest]n)+(4\u2013 (n = 1\u20133) peaks along with the peaks due to fragmentation of the cage under a certain spray voltage. In fact, a similar phenomenon was also observed in the literature.For the case of guest HQ and 4-NP, the trapping ability of the PdQ Fig. S8. Similar Fig. S14. In our 2L4(BF4)n) absorption at 525 nm. As shown in The assembly of Au NPs with the aid of the Pdca. 1.2 nm scale\" fill=\"currentColor\" stroke=\"none\">C, C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C and C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N bands or NaOH solution (pH = 10), the fabricated substrate structure gradually diminished, which was indicative of the instability of the embedded cages in acidic and alkaline solutions for a given time (2 h), and after being thoroughly washed with DMSO and ethanol the substrates were checked using the SERS technique. \u20131 (asymmetric and symmetric stretching doublet vibrations \u03bdPt-NH3) appeared in the SERS spectrum . 4-NP was firstly chosen as a substrate to be converted into the product dihydroxylazobenzene (DHAB), as shown in \u20131 (\u2013NO2 \u03b3 stretching) was detected. After the irradiation of plasmonic clusters under laser irradiation (1 mW) at 633 nm for a given time, three bands associated with DHAB at \u03bd1 = 1481 cm\u20131, \u03bd2 = 1509 cm\u20131 scale\" fill=\"currentColor\" stroke=\"none\">N\u2013stretching) and \u03bd3 = 1652 cm\u20131 (\u03bdCC stretching) appeared,\u03bd1 = 1400 cm\u20131 and \u03bd2 = 1453 cm\u20131 were detected,2 stretching remain unchanged and no bands associated with \u2013N PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N stretching appeared over time, indicating that no reaction occurred in the molecular cages in the absence of gold nanoparticles under identical conditions. Similar results were also observed for the case of the cycloaddition reaction separation, but can also simultaneously impart the capability of trapping low affinity guests in the created hotspots, which could greatly expand the applications of plasmon-assisted chemistry. Ideally, the blank experiment of reactions will need to be performed with gold nanoparticles and cages that are not linked. In our system, however, due to the presence of thioether moieties at the periphery of the used cage, it is impossible or difficult to create an ideal system consisting of gold nanoparticles and cages that are not linked to perform the desired control experiments. Thus, in the future, we will perform a systematic study to address this issue through the synthesis of the same molecular cage without thioether or thiol groups.The unique capacity of plasmonic nanostructures to trap molecules that have a low affinity for metal surfaces and to selectively place the target molecules into hotspots could offer tremendous opportunities for numerous plasmon-assisted applications. Fig. S35. These rCreating well-defined plasmonic hotspots with enormous field enhancement as well as the capability of selectively trapping targeted molecules into hotspots is critical for numerous plasmon-assisted applications, but represents a great challenge. In this work, based on the assembly of Au NPs using a metal\u2013organic molecular cage as a linker, a series of cage-bridged plasmonic nanostructures were developed. It was found that the integration of host\u2013guest chemistry of the molecular cage with the plasmon coupling of Au NPs enables the formed plasmonic structures to address the mentioned challenges in terms of the formation of well-defined hotspots, the capture of molecules with lower affinities to the metal surface, the favourable molecule-accessibility, and the selective placement of target molecules in hotspots. The chosen applications above clearly highlight the significant value and tremendous potential of the cage-bridged plasmonic structures. Although one molecular cage was demonstrated in our case, the strategy described is generally applicable to other molecular cages for creating desired plasmonic nanostructures. As the described molecular cage is constructed through the coordination of organic linkers and metal ions, a careful selection of ligands and metal ions permits control of the geometric and host\u2013guest characteristics of the formed cages in a broader range. Such attractive attributes imply that the precise design and tuning of hotspots in plasmonic structures and their molecule-trapping properties are possible. Thus, our work opens a new avenue for engineering plasmonic architectures and affords a new class of plasmonic nanostructure, which could offer huge opportunities for numerous plasmon-based applications.C. W. planned and performed the experiments, analysed the data and wrote the paper. L. T., N. G. and K. Z. assisted in the experimental work. S. W. assisted in the theoretical simulation. W. Z., X. Y., and W. Z. discussed the results. G. L. guided the project and wrote the paper.There are no conflicts to declare.Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "A \u03b2-diketiminato n-butylmagnesium complex is presented as a selective precatalyst for the reductive hydroboration of organic nitriles with pinacolborane (HBpin). n-butylmagnesium complex is presented as a selective precatalyst for the reductive hydroboration of organic nitriles with pinacolborane (HBpin). Stoichiometric reactivity studies indicate that catalytic turnover ensues through the generation of magnesium aldimido, aldimidoborate and borylamido intermediates, which are formed in a sequence of intramolecular nitrile insertion and inter- and intramolecular B\u2013H metathesis events. Kinetic studies highlight variations in mechanism for the catalytic dihydroboration of alkyl nitriles, aryl nitriles bearing electron withdrawing (Ar(EWG)CN) and aryl nitriles bearing electron donating (Ar(EDG)CN) substitution patterns. Kinetic isotope effects (KIEs) for catalysis performed with DBpin indicate that B\u2013H bond breaking and C\u2013H bond forming reactions are involved in the rate determining processes during the dihydroboration of alkyl nitriles and Ar(EDG)CN substrates, which display divergent first and second order rate dependences on [HBpin] respectively. In contrast, the hydroboration of Ar(EWG)CN substrates provides no KIE and HBpin is not implicated in the rate determining process during catalysis. Irrespective of these differences, a common mechanism is proposed in which the rate determining steps are deduced to vary through the establishment of several pre-equilibria, the relative positions of which are determined by the respective stabilities of the dimeric and monomeric magnesium aldimide and magnesium aldimidoborate intermediates as a result of adjustments to the basicity of the nitrile substrate. More generally, these observations indicate that homogeneous processes performed under heavier alkaline earth catalysis are likely to demonstrate previously unappreciated mechanistic diversity.A \u03b2-diketiminato Ca < Sr < Ba) whereas our own studies of the homoleptic bis(trimethylsilyl)amides [Ae{N(SiMe3)2}2]2 have evidenced more subtle kinetic dependences upon the identity and characteristics of the Ae2+ cation.Our own research has focussed on the development of a homogeneous catalytic chemistry for complexes, LAeX , derived from the heavier alkaline-earth elements.V) for the hydroboration of aldehydes and ketones,2 aminopyridines.F) requires the consumption of two molecules of HBpin and the intermediacy of an initial magnesium-bound imide (A), a borylated imine (B) as the product of initial B\u2013H/Mg\u2013N metathesis and an amide species (D) formed by formal Mg\u2013H insertion of the borylimine (B). The additional complexity introduced by the requirement for two-fold nitrogen functionalisation, thus, raises a number of issues regarding validity of such a stepwise process and the identity of other potential intermediates such as the borate species shown as C and E shown in vide infra).We have previously reported the use of the magnesium alkyl precatalyst , which is stable to both Schlenk-type redistribution equilibria and to chemical degradation under conditions of the catalysis, allows for an assessment of the effects of gradual substrate adjustment which suggests that every reaction must be treated on its merits.With these broader considerations in mind we now describe a magnesium-centred protocol for the hydroboration of organic nitriles with the unactivated borane reagent pinacolborane (HBpin). Our selection of a single \u03b2-diketiminato magnesium precatalyst amine product within 30 minutes. The clean formation of this new species was clearly apparent through the emergence of a (2H) triplet methylene resonance in the 1H NMR spectrum at \u03b4 3.42 ppm synchronous with a new singlet signal in the 11B NMR spectrum at \u03b4 29.5 ppm. In contrast an analogous reaction performed without the addition of V evidenced less than 5% consumption of the propionitrile substrate when heated at 60 \u00b0C for 16 hours. Encouraged by this result the conditions of the catalytic study were extended to the successful di-hydroboration of the range of alkyl and aryl nitriles summarised in An initial catalytic reaction using 10 mol% of PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C-bound carbon centre of aliphatic nitriles wherein a 10-fold increase in reaction time is observed across the transition from propionitrile (entry 1) to iso-propylnitrile (entry 2) to tert-butylnitrile (entry 3). Aryl nitriles (entries 5\u201315) also provided clean reactions with no side products, but with markedly longer reaction times. Notably varied reaction times were also required with changing aryl substituent patterns. Although in the case of o-tolunitrile (entry 6) the slower reaction may again be attributed to an increase in substituent steric hindrance proximal to the reaction centre, any minor variability across the range of meta-and para-substituted arylnitriles is more realistically attributed to electronic adjustments across the pi-conjugated substrate or intermediate structure (vide infra). The identities of the products from this catalysis were confirmed by 1H, 11B and 13C NMR spectroscopy and a single crystal X-ray diffraction analysis performed upon the product (compound 1) isolated from the catalytic dihydroboration of propionitrile. The outcome of this latter experiment is shown in 3 hybridisation at the C1 carbon atom.In common with our earlier reports of the magnesium-catalysed hydroboration of aldehydes, ketones, imines and isonitriles,A\u2013E in t-BuCN was particularly informative due to the ready discrimination of the diagnostic singlet tert-butyl resonances in the 1H NMR spectra. We have earlier reported that V and HBpin react to form a magnesium hydride/borohydride species along with BuBpin within minutes at room temperature.t-BuCN provided complete C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N insertion into the magnesium hydride bond, which was clearly apparent after heating for 12 hours at 60 \u00b0C through the formation of a single new compound designated as an aldimide derivative analogous to the species A depicted in \u03b4 7.84 ppm assigned as the aldimide methine signal and a set of resonances associated with a single new \u03b2-diketiminate ligand environment in the 1H NMR spectrum (see Scheme S1m- and p-MeOC6H4CN. While both of the resultant compounds (2 and 3) again displayed characteristic 1H aldimide methine singlet resonances in their respective 1H NMR spectra, in these latter cases crystallisation from benzene solutions also provided samples of both compounds suitable for single crystal X-ray diffraction analysis. The results of these analyses are presented in 2 and 3 are the first magnesium aldimide complexes to be characterised in the solid state, their structures are otherwise analogous to a previously described calcium benzaldimide derivative supported by the identical \u03b2-diketiminate ligand.2 and 3 are dimeric with bridging Mg\u2013N\u2013Mg interactions provided by the aldimide ligands. Whereas the asymmetric unit of compound 2 comprises half of a dimer which straddles a crystallographic inversion centre, each half of the dimeric unit of compound 3 is unique. The gross features of both structures are, however, very similar wherein the magnesium centres are bridged by unsymmetrical Mg\u2013N\u2013Mg interactions. The magnesium to aldimide nitrogen bond lengths in 2 and 3 are shorter than the magnesium to amide contacts observed within topologically related dimeric magnesium benzylamide and pyrollidide derivatives [2.1251(16) and 2.117(2) \u00c5 respectively], both of which contain four-coordinate magnesium centres supported by the identical \u03b2-diketiminate ligand.3 to sp2 hybridisation at the bridging nitrogen centres in comparison to these previously described compounds is also reflected by the more obtuse Mg\u2013N\u2013Mg bond angles within compounds 2 and 3. As previously highlighted in Harder and co-workers' discussion of their calcium benzaldimide species,2 and 3 are virtually co-planar with the planes formed by the magnesium and the aldimide nitrogen and sp2 carbon centres. A similar in-plane conformation is also a common feature of dimeric diorganoaluminum benzaldimide species, 2, PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CH2]2, to lie some 17 kcal mol\u20131 lower in energy than the alternative conformation in which the methylene unit is perpendicular to the Li2N2 plane.2 and 3 indicate that this stabilisation is further enhanced by delocalisation across the aryl substituents. Although such data should be treated with caution due to the possibility of solid-state crystal packing and dispersion effects, we ascribe the greater co-planarity of 2 in comparison to 3 to the mesomeric influence of the respectively stabilizing 3-methoxy- and destabilising 4-methoxyphenyl substituents. While only providing a minor solid-state effect, we suggest that modulation of aldimide resonance stabilisation is a significant factor during the magnesium-catalysed hydroboration of the range of aryl nitriles listed in vide infra).To provide further insight into the course of these successful catalytic reactions and the viability of intermediate species such as those shown as t-BuCN did not provide any initial evidence of B\u2013H/Mg\u2013N metathesis and production of a borylated imine derivative and more typical of four-coordinate boron.vide infra) allowed the isolation of the aldimidoborohydride derivative, compound 4, which was fully characterised by multinuclear NMR spectroscopy and a further single crystal X-ray experiment. The results of this latter analysis are presented in 4 provides the first crystallographic evidence for the intermediacy of borohydride intermediates during any magnesium-mediated hydroboration catalysis. The C(3)\u2013N(3) distance (1.226(2) \u00c5) of the formal C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N double bond, although shorter than the aldimide C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N distances, comprising 2-coordinate nitrogen, within compounds 2 and 3 (2: 1.263(2); 3: 1.262(2), 1.271(2) \u00c5), is closely comparable to previously reported, albeit exclusively transition metal-coordinated, imine-derived borate anions.25Counter to the expectation illustrated in t 1JBH = 5 Hz and 4 in C6D6 were observed to undergo continued reaction on standing at room temperature over a period of 12 hours; clearly observed by monitoring of the time-resolved 1H NMR spectra through the disappearance of the downfield 1H aldimido proton signal and the simultaneous appearance of a new singlet resonance at \u03b4 3.35 ppm with an ultimate 2H intensity by integration. The corresponding 11B NMR spectra over this period evidenced a small downfield shift to \u03b4 6.3 ppm with loss of the doublet signal multiplicity. We suggest that these observations are a consequence of an intramolecular hydride shift resulting in reduction of the aldimide residue and production of a magnesium borylamide derivative directly analogous to species D shown in F in E in Samples of compound 2 and 3 provided no evidence for similar borate formation and provided resonances consistent with the persistence of the unreacted starting compounds. DOSY NMR analysis of these reactions also evidenced no change in the solution diffusion coefficient attributed to the dimeric species even with increasing temperature and the addition of further equivalents of HBpin. We attribute this observation to the additional conjugative stability provided to the dimeric unit by the co-planarity of the 3- and 4-methoxyphenyl substituents with the C\u2013N\u2013Mg linkage.Notably, analogous addition of one equivalent of HBpin to solutions of compounds 2\u20134 could themselves be utilised as precatalysts for the hydroboration of m- and p-MeOC6H4CN and t-BuCN respectively. These reactions provided conversions and reaction times that were broadly commensurate with the reactions initiated by V were isolated from the unstirred reaction mixture. Although this new species was insufficiently soluble to allow characterisation by solution-state NMR spectroscopy, a single crystal X-ray diffraction analysis revealed compound 5 as the unusual dinuclear magnesium complex shown in 5 are connected through bridging interactions provided by both oxygen atoms of a [H2Bpin]\u2013 anion, in a manner reminiscent of that observed in both a previously reported trimeric calcium species, [LCa(H2Bpin)]3 (where L is as defined in R1 = 10.31%) X-ray diffraction analysis.pseudo-tetrahedral N3O-coordination sphere, with ligation provided by the bridging borate and a single \u03b2-diketiminate anion, the nature of the fourth nitrogen-centred ligand differs across the two Mg centres of the molecule. Whereas Mg(1) is coordinated by a molecule of diphenylacetonitrile, the coordination sphere of Mg(2) is completed by a diphenylketeniminate anion, generated by deprotonation of the nitrile starting material. Magnesium and barium derivatives of the identical diphenylketeniminate anion are precedented by complexes in which the alkaline earth centres were further coordinated by sterically demanding bis(imino)acenaphthene (Dipp-BIAN) radical anions, in which case the magnesium complex was prepared by deprotonation of diphenylacetonitrile by [(Dipp-BIAN)MgMe].5 is formed in a similar manner, in a process which is detrimentally competitive with the desired hydroboration reactivity under catalytic conditions , using the standard catalytic reaction of 10 mol% of the precatalyst V in conjunction with a 1\u2009:\u20092.1 ratio of propionitrile (0.40 M) to HBpin (0.82 M). While the reactions displayed apparent pseudo-zero order kinetic behaviour signified a first order dependence on [HBpin] of 2.79. Although relatively small, this value would constitute a very large secondary effect and is significantly in excess of comparable results (kH/kD = 1.62) reported by Hartwig et al. during studies of catecholborane metathesis at ruthenium(ii) alkyl centres, in which B\u2013H bond breaking processes are integral to the progress of the reaction.4 do not persist under catalytic conditions but are consumed by intramolecular hydride transfer from HBpin to the metal-bound aldimide fragment.We interpret the suppression of reaction rate at high substrate concentrations on the ability of both reagents to coordinate to the metal centre, either as a neutral nitrile donor or through the likely formation of borohydride species. We suggest that this deduction is further supported by the first order dependence of the reaction rate on [HBpin] in the presence of excess EtCN wherein increasing concentration will favour the displacement of the neutral nitrile from the magnesium coordination sphere allowing the onward formation of intermediate aldimidoborohydride species akin to compound pseudo-zero order kinetics displayed during the hydroboration of propionitrile, experiments performed to interrogate the rates of reactions displayed by the array of aryl nitriles employed in the study = \u20131.46; \u03c1(EWG) = +0.68] are relatively small and must be interpreted with caution,In contrast to the he study evidencehe study , the obsp-MeOC6H4CN) and electron withdrawing (m-MeOC6H4CN) aryl substitution. For the hydroboration of p-MeOC6H4CN a first order dependence on the concentration of [catalyst] .This apparent change in mechanism was interrogated through kinetic studies of the hydroboration of nitrile substrates bearing representative electron donating into a hydride intermediate (G) through metathesis of the magnesium\u2013butyl bond with HBpin (designated as the circled reaction 1 in H and I) exemplified by the isolation of compounds 2 and 3 appears facile, irrespective of nitrile substrate identity (reaction 2). Whilst the activation of organonitriles toward different nucleophiles \u2013(c) may be discriminated.H (i.e. k\u20133 \u226b k3). The assembly of aldimidohydridoborate anions (J) such as that confirmed by the crystallographic characterisation of compound 4 requires the displacement of pre-coordinated nitrile by the HBpin substrate amine product (M) via the assembly of a further borate intermediate (L) J K\u20131 mol\u20131) of the free energy of activation for the alkyl nitrile hydroboration catalysis.The more basic character of alkyl nitriles ensures that the monomer/dimer equilibrium depicted as reaction 3 in k5 > k\u20134 yieldingH to J and its subsequent consumption through B\u2013H transfer to the coordinated aldimide fragment. The observed rate of catalysis for alkyl nitrile hydroboration is, thus, dictated by not only the ability of HBpin to replace nitrile in the magnesium coordination sphere but also the consequent ease of intramolecular C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N hydride reduction.The catalytic hydroboration of alkyl nitriles is consequently determined by the pre-equilibration of 2 and 3, magnesium aldimide derivatives bearing N-aryl substitution will benefit from considerably enhanced conjugative stability in comparison to those bearing alkyl residues of comparable steric demands. We suggest, therefore, that the kinetic profile observed for the hydroboration of p-MeOC6H4CN reflects the resistance to intramolecular hydride transfer of aldimidoborate species analogous to compound 4 scale\" fill=\"currentColor\" stroke=\"none\">N reduction suggests that the sequence of reactions 5\u20137 in J with a further molecule of HBpin (shown as reaction 8 in S\u2260 = \u2013174.8 (\u00b118.2) J K\u20131 mol\u20131) is also congruent with pre-equilibration of Ar(EDG)CN and HBpin and the onward reaction with a second molecule of HBpin. Consideration of the entire process illustrated by As highlighted by the isolation of compounds I) with a solution structure similar to that depicted for 2 in H* (vide infra). Use of the steady state approximation allows the derivation of a rate law that is dependent on [I] and may be identified as the rate determining process in a hydroboration catalysis in which k3 \u226b k\u20133 \u226a k4.For 3-methoxybenzonitrile, Mg\u2013H insertion will provide a dimeric aldimide (H*). Under this regime the role of the nitrile substrate is encapsulated by the experimentally deduced second order dependence on initial precatalyst concentration, [V], and is simply reflective of the involvement of two magnesium centres in the formation of H*.We suggest that this process, therefore, occurs with only partial rupture of the dimeric unit as illustrated in V has been demonstrated as an active precatalyst for the HBpin-derived hydroboration of a range of alkyl and aryl nitriles to form bis(boryl)amines. Catalysis proceeds under mild conditions, with reasonable catalyst loadings and is proposed to occur through a sequence of magnesium-mediated B\u2013H insertion and metathesis steps that are crucially dependent on a variety of pre-equilibration steps that are dictated by minor variations in substrate basicity and the stability of mono- and dimeric intermediates. These observations indicate that, somewhat counter to historical prejudice, there is likely to be considerable variation across even superficially identical reactions when catalysed by alkaline earth reagents.In conclusion, the \u03b2-diketiminato magnesium species Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "The first reactivity of the hydroborylene ligand is described, giving access to a remarkable range of unprecedented boron-centered species. II complex [{cat(TMSL)Si}(Cl)Ni\u2190:BH(NHC)2] 1 (Dipp); Dipp = 2,6-Pri2C6H3; NHC = :C[(Pri)NC(Me)]2) to form the hydride-bridged hydroborylene-NiII complexes 2. The reaction of 1 with isoelectronic CO, however, is reversible and furnishes the related unprecedented hydride- and CO-bridged hydroborylene NiII complex 2-CO, which undergoes isomerisation through silyl/NHC exchange at ambient temperature to afford the corresponding hydro(silyl)boryl NiII complex 3. Markedly, 2 readily and quantitatively react with one further molar equiv. of isocyanide to give, under borylene liberation and H/Cl ligand exchange, boraketiminium species, which represent cationic BI complexes. These latter compounds are highly reactive in solution, and can undergo quantitative transformation into previously unknown cyanoborenium cations.For the first time, the reactivity of the metal- and N-heterocyclic carbene-supported monovalent hydroborylene is reported. Isocyanides react with the hydroborylene Ni C. Brown in the synthesis and reactivity of hydroboranes,I centre .16 Thus,1 contains a three-coordinate, 16-electron NiII centre,1 at \u201378 \u00b0C resulted in an immediate color change to deep orange. A single-crystal X-ray diffraction analysis of deep orange-yellow crystals grown from this reaction mixture revealed that 2-Cy (11B NMR spectrum of 2-Cy (\u03b4 = \u201343.0 ppm) clearly correlates to a signal in its 1H NMR spectrum as shown through 1H,11B HMQC NMR analysis = 2.230(2) \u00c5, MBOB\u2013Ni = 0.48; For 1: d(B1\u2013Ni1) = 2.015(2), MBOB\u2013Ni = 0.76). This results in a tetrahedral BI centre whose lone-pair of electrons is directed towards the \u03c0*-orbital of the CNCy ligand (d(B1\u2013C44) = 1.637(3) \u00c5, MBOB\u2013C = 0.75), resulting in considerable C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N bond weakening in this fragment. The calculated HOMO for 2-Cy comprises largely of this bonding interaction, as well as bonding contributions from Ni scale\" fill=\"currentColor\" stroke=\"none\">NC = 1626 cm\u20131) when compared with related reported species (viz. [CpMn(CO)2-\u03b72(CNR)-B(But)(NHC\u2032)], \u03bd PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019NC = 1856\u20131930 cm\u20131; R = Me, Cy; NHC\u2032 = C[(Me)NC(Me)]2).14 PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019NC = 1655 cm\u20131, in 2-Cy is in line with that observed experimentally.\u00a7The calculated value, \u03bd The low Mayer bond order (MBO) for the B\u2013C(NCy) bond suggests little degree of CyNC:\u2192B donation. Conversely, some back-bonding from the NiII centre to the CyNC ligand in 2-Cy is observed (d(Ni1\u2013C44) = 1.820(2) \u00c5, MBOC\u2013Ni = 1.12), further evidenced by the relatively linear Ni1\u2013C44\u2013N6 angle (\u2220NiCN = 153.0(1) \u00b0) when compared to the B1\u2013C44\u2013N1 angle of 126.3\u00b0. Thus, the bonding model outlined in 2-Cy.Compound hat 2-Cy , Fig. 2 i.e. CNR with R = 2,6-Xyl, Bz) to THF solutions of 1 resulted in the formation of essentially isostructural \u03bc-hydride, \u03bc-CNR hydroborylene NiII complexes, 2-Xyl and 2-Bz, respectively scale\" fill=\"currentColor\" stroke=\"none\">NC = 1634 cm\u20131; 2-Bz: \u03bd PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019NC = 1627 cm\u20131). Their 11B NMR spectra are also very similar to that for 2-Cy, each showing a single broad resonance. Complexes 2 are poorly soluble in common organic solvents, and decompose to a complex mixture over the course of one day, but are stable in the solid state for an indefinite period of time. Important geometrical and spectroscopic parameters are summarised in 1H,11B HMQC NMR analyses (see above).\u00b6B\u2013H stretching vibrational bands were not observed for these species, presumably as they were too weak. The presence of the B\u2013H fragments was confirmed with Addition of related ligands . The stretching vibrational frequency for the CO ligand in 2-CO indicates significant lowering of the CO bond order (\u03bdCO = 1694 cm\u20131) relative to free CO. Despite this, the reaction of 1 with CO is reversible, with storage of suspensions of 2-CO in THF yielding small amounts of crystalline 1 after one day. Observation of the frontier orbitals of 2-CO reveals that both the HOMO and HOMO-5 involve considerable donation to the \u03c0* orbital of the CO ligand , leading to the four-coordinate NiII complex IM1 . Subsequent coordination of the BI centre to the CO ligand leads to weakening of the B\u2013Ni bond and B\u2013C bond formation, generating 2-CO . The negligible thermodynamic barriers and minimal energy gain upon binding CO, then, explain the reversibility of this reaction. Whilst some degree of B\u2013Ni bonding is present in 2-CO, calculated bond orders for this interaction (Wiberg Bond Index (WBI)B\u2013Ni = 0.17; MBOB\u2013Ni = 0.30) are considerably lower than those for both the B\u2013C and Ni\u2013C bonds . Notably, these calculated MBO values for the Ni\u2013C and B\u2013C bonds in 2-CO are somewhat lower than those for the closely related bonding interactions in 2-Cy, again yielding an explanation for the reversibility of the reaction of 1 with CO.In contrast to reactions with isocyanides, uct 2-CO as an eqO ligand , in a si with CO indicate2-CO is stable in the solid state for weeks. However, upon warming THF solutions of in situ generated 2-CO to ambient temperature, a new compound is formed as a mixture with 1 after 2 h. Repeated re-crystallisations of this reaction mixture led to crystalline samples of the new product contaminated with small amounts of 1 , considerably more shielded than that for 2-CO most likely due to a greater charge density residing on the formally anionic boron centre in 3. The CO stretching vibrational band in the IR spectrum of 3 is higher relative to that in 2-CO indicative of reduced back-bonding from boron to CO in transitioning from a borylene to a boryl ligand. The greater stability of 3 over 2-CO may be owed to the NHC ligand now on NiII, whose Pri groups allow for an octahedral geometry at nickel through anagostic interactions with two flanking CH3 groups.19s formed , Fig. 4.1 leads to the formation of dark precipitates and silent 11B NMR spectra. Conversely, addition of two molar equiv. of CNR to 1 gives rise to complete borylene liberation as well as an unexpected H/Cl ligand exchange, giving facile and quantitative access to extraordinary three-coordinate boraketiminium complexes, [{(NHC)2BCNR}+Cl\u2013] , which can also be described as three-coordinate BI cations.SiLNiH] complex fragment = 1.467(4); t4-Bu: d(B1\u2013C23) = 1.433(2) \u00c5) and C\u2013N (4-Cy: d(C23\u2013N5) = 1.221(4); t4-Bu: d(C23\u2013N5) = 1.220(2) \u00c5) bonds are also in keeping with this, supported by a DFT analysis of the frontier orbitals in 4-Cy, which are indicative of \u03c0-bonding between these centres boraketiminium form is more prominent (45.3%), corroborating that 4-Cy indeed has a degree of BI character. The striking further reactivity of t4-Bu, which is reminiscent of low-valent boron and group 14 chemistry,1H NMR spectrum of CD2Cl2 solutions of t4-Bu indicates the loss of isobutene, and the clean formation of a single species containing a B\u2013H fragment , giving strong evidence for the formation of the cyanoborenium cation, [{(NHC)2B(H)(CN)}+Cl\u2013] 5-H. Remarkably, addition of two equiv. of CNBz to 1 directly leads to the benzyl derivative of 5-H, [{(NHC)2B(Bz)(CN)}+Cl\u2013] 5-Bz, via C\u2013N bond cleavage of the boraketiminium/borylene cation intermediate. The molecular structure of 5-Bz confirms the formation of a terminal cyanoborenium complex (d = 1.142(7) \u00c5) when compared with the terminal C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N bond in t4-Bu (d(C23\u2013N5) = 1.220(2) \u00c5). The formation of these complexes represents a new entry into NHC-cyanoborane chemistry, species which are typically extremely challenging to access.1.Braunschweig and co-workers have previously reported that addition of multiple equivalents of CNR or CO to borylene TM complexes can affect complete borylene-TM bond cleavage.fragment undergoe centres . Surprisnds.4-Cy suggests complex , with a Cy: dB1\u2013C = 1.467(II complex 1 with one molar equiv. of isocyanides or CO has given access to a new hydride-bridged isomeric form in hydroborylene transition-metal chemistry in complexes 2, as well as the hydride- and CO-bridged hydroboryl complex 3. In addition, the unprecedented boraketiminium and cyanoborenium salts 4 and 5, respectively, resulted from reaction of 1 with two molar equiv. of isocyanides in good yields. As such, this chemistry demonstrates the potential utility of the hydroborylene ligand in HB:\u2192TM complexes for the realisation of new functional groups in boron chemistry.In summary, the striking reactivity of the hydroborylene ligand in the HB:\u2192NiThere are no conflicts to declare.Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "Room-temperature metal-free hydrogenation catalysis. tBu)HB(C6F5)22 prompts intramolecular C\u2013H bond activation to give (CHN)2(tBu) (CMe2CH2)CB(C6F5)23, defining an upper limit of Lewis acidity for FLP hydrogenation catalysis. A series of seven N-heterocyclic carbene\u2013borane (NHC\u2013borane) adducts ((R\u2032CNR)2C)(HBC8H14) and ((HC)2(NMe)(NR)C)(HBC8H14) are prepared and converted to corresponding borenium salts. These species are evaluated as catalysts for metal-free imine hydrogenation at room temperature. Systematic tuning of the carbene donor for the hydrogenation of archetypal substrate N-benzylidene-tert-butylamine achieves the highest reported turn-over frequencies for FLP-catalyzed hydrogenation at amongst the lowest reported catalyst loadings. The most active NHC\u2013borenium catalyst of this series, derived from 10a, is readily isolable, crystallographically characterized and shown to be effective in the hydrogenation catalysis of functional group-containing imines and N-heterocycles.This manuscript probes the steric and electronic attributes that lead to \u201cfrustrated Lewis pair\u201d (FLP)-type catalysis of imine hydrogenation by borenium ions. Hydride abstraction from (I The hydrogenation of unsaturated bonds is one such chemical transformation that is employed on a terrific industrial scale.8H14] [B(C6F5)4] can be used as a catalyst for the metal-free hydrogenation of imines and enamines.2. This affords an NHC\u2013borane that delivers hydride to a transient iminium ion with HB(C6F5)2 led to the formation of NHC\u2013borane adduct (ItBu)HB(C6F5)22 (NHC\u2013B bond length of 1.645(4) \u00c5 and an average B\u2013C6F5C bond length of 1.639(4) \u00c5 22 , which w639(4) \u00c5 . The exp2via treatment with the hydride abstraction reagents [Ph3C][B(C6F5)4], Me3SiOTf or HOTf showed no reaction. This stands in contrast to the facile hydride donation typically demonstrated by NHC\u2013boranes.2 with HNTf2 in toluene to >100 \u00b0C for four days the clean conversion to a new product was evident from the appearance of the 11B resonance at \u201314.8 ppm. 1H NMR spectroscopy showed sharp singlet resonances at 0.86 ppm and 1.04 ppm and a broad singlet resonance at 1.80 ppm integrating in a 9\u2009:\u20096\u2009:\u20092 ratio. These combined NMR data suggest the new species (CHN)2(tBu) (CMe2CH2)CB(C6F5)23 is derived from C\u2013H activation of a tert-butyl substituent (3 confirmed its bicyclic nature (2(tBu)(CMe2CH2)CBBr2\u20092(tBu)(CMe2CH2) CB(tBu)Cl3 is thought to proceed via transient generation of a cation and subsequent C\u2013H activation imidazol-2-ylidene (Idipp) at 60 \u00b0C for one hour to afford Idipp\u2013borane adduct 4a in 79% yield 4] at 45 \u00b0C overnight results in the generation of Ph3CH and the quantitative conversion of the NHC\u2013borane to a new species as evidenced by 11B NMR signals at 82.6 ppm and \u201316.6 ppm. These are consistent with the formation the borenium\u2013borate salt [(Idipp)BC8H14][B(C6F5)4] 4b. Alternatively, treatment of 4a with tBuN PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CHPh and the addition of a stoichiometric equivalent of [tBu3PH]][B(C6F5)4] results in generation of 4b with concurrent reduction of the imine as evidenced by 1H NMR spectroscopy. This observation prompted efforts to employ 4b in an FLP hydrogenation of tBuN PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CHPh. However combination of excess tBuN PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CHPh and 4b under 102 atm H2(g) showed no evidence of reduction of the imine 2]. This latter one-pot approach is similar to that described by Brahmi et al. to prepare a series of NHC\u2013BH3 compounds.2C)HBC8H14 and ((HC)2(NMe)(NR)C)HBC8H14 were prepared 4] to give the corresponding borenium salts [((R\u2032CNR)2C)BC8H14] [B(C6F5)4] and [((HC)2(NMe)(NR)C)BC8H14] [B(C6F5)4] concomitant with the generation of a stoichiometric amount of Ph3CH (11B resonance in the range of 81\u201388 ppm attributable to a three-coordinate B center. The expected resonances for the [B(C6F5)4]\u2013 anion were seen at \u201316.7 ppm. The species 10b was isolated as colorless crystals in 72% yield via recrystallization from CH2Cl2/pentane at \u201335 \u00b0C. Crystallographic data (NHC bond length of 1.5768(3) \u00c5 similar to that observed for 1b (1.580(3) \u00c5).67Each of these adducts reacts with [Phof Ph3CH . The moshic data revealed3P)(HBC8H14) (11) was also synthesized and isolated as colorless crystals in 82% yield. The 11B NMR signal was observed at \u201314.9 ppm and exhibited both B\u2013H coupling of 88 Hz and B\u2013P coupling of 48 Hz. The 31P{1H} resonance for 11 is at \u201313.0 ppm and possesses similar B\u2013P coupling. Single crystal X-ray diffraction confirmed the formulation (see ESI11 with stoichiometric [Ph3C][B(C6F5)4] gave a complex mixture of products as evidenced by 31P{1H} and 11B NMR-spectroscopy.For comparative purposes the phosphine\u2013borane adduct scale\" fill=\"currentColor\" stroke=\"none\">CHPh as a comparative screen. A solution of each was generated in situ, added to the imine substrate and pressurized with 102 atm H2(g) for 30 minutes. After the reaction, the extent of conversion to amine was assessed by 1H NMR spectroscopy. These data reveal an inverse correlation between the steric demands of the NHC and the hydrogenation activity of the borenium catalyst of 940 h\u20131. A slight increase of catalyst loading to 0.15 mol% and an extension of the reaction time to 2 h at room temperature under 102 atm H2(g) led to complete conversion to tBuNHCH2Ph and the product could be isolated in 83% yield scale\" fill=\"currentColor\" stroke=\"none\">NtBu, is readily reduced (p-(MeO2C)C6H4CH PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019NtBu (6F5)3.6H5CH PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019NCHPh2 slow imine reduction scale\" fill=\"currentColor\" stroke=\"none\">NPh and p-EtOC6H4(Me)C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019NPh are readily hydrogenated to corresponding amines scale\" fill=\"currentColor\" stroke=\"none\">NCH2Ph was observed (1b,6710b smoothly catalyzes the hydrogenation of 8-methylquinoline to 1,2,3,4-tetrahydro-8-methylquinoline (Comparison of the isosteric catalysts d yields . In thes reduced , entry 1g>NtBu , entry 2eduction , entry 3observed , entry 6observed , entry 7observed , entry 8uinoline , entry 9via an FLP mechanism, perturbations that enhance the Lewis acidity at B or the steric demands of the NHC ligand can serve to deactivate the catalyst. At the same time, sterically unencumbered NHCs bearing electron withdrawing substituents enhance catalyst activity. Crudden and co-workers10b is an effective catalyst for imine and N-heterocycle reduction at low catalyst loadings and it affords the highest TOF yet reported for metal-free hydrogenation catalysis. Efforts are continuing to systematically develop borenium-based metal-free hydrogenation catalysts and to further broaden their applications.In this manuscript we have probed the electronic and steric parameters that impact on the ability of ligand stabilized borenium cations to act as metal-free hydrogenation catalysts. Although this catalysis proceeds Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "Photocyclized intermediate formation and quasi C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C twisting are the dominant processes behind the AIE. i.e. formation of intermediate or rotation around the elongated C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond, is responsible for the AIE effect, which is strongly structure-dependent but not related to structural rigidity.Aggregation-induced emission (AIE) is the long-sought solution to the problem of aggregation-caused quenching that has hampered efficient application of fluorescent organic materials. An important goal on the way to fully understand the working mechanism of the AIE process was, for more than a decade, and still remains obtaining more comprehensive insights into the correlation between the ultrafast excited-state dynamics in tetraphenylethylene (TPE)-based molecules and the AIE effect in them. Here we report a number of TPE-based derivatives with varying structural rigidities and AIE properties. Using a combination of ultrafast time-resolved spectroscopy and computational studies, we observe a direct correlation between the state-dependent coupling motions and inhibited fluorescence, and prove the existence of photocyclized intermediates in them. We demonstrate that the dominant non-radiative relaxation dynamics, E\u2013Z isomerizationIn less than two decades, the seminal discovery of aggregation-induced emission (AIE) PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C twisting35The excited-state dynamics in TPE and TPE derivatives, including the role of phenyl rotation,1\u20136 scale\" fill=\"currentColor\" stroke=\"none\">C twisting) in 1\u20136, with the formation of a photocyclized intermediate in all compounds on different timescales, and the dominant relaxation channels responsible for the AIE effect (or its absence) in every particular case being strongly structure-dependent. The implications of these observations for the working mechanism of AIE are fully explained.In our work to restrict certain relaxation channels in the excited state and investigate their role in the AIE effect, we synthesized a set of TPE-based derivatives, 1\u20136 , with va1\u20136 with increased structural rigidity and their corresponding photocyclized phenanthrene derivatives 1-PC\u20136-PC were synthesized according to the synthetic routes shown in 1H-NMR, 13C-NMR and high resolution mass spectrometry. The structures were determined by single-crystal X-ray diffraction between geminal phenyl groups display only one type of phenyl orientation , giving rise to what we will refer to from now on as a quasi C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond. In contrast, the bonds connecting the peripheral phenyl groups with the C1\u2013C2 bond shorten (e.g. d2\u2013C15C) to fall into the bond length range that is between that of a typical single C\u2013C bond and a normal C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C double bond. The twisting of the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond and torsion of the phenyl rings in 1\u20136 are analyzed metrically in terms of the dihedral angles \u03c421\u2013C1\u2013C2\u2013C15C and \u03c41\u2013C2\u2013C15\u2013C20C, respectively. For 1\u20136 in solution, each molecule has its unique set of restricted degrees of freedom with varying degrees of rigidity. The absolute change of the dihedral angle around the C1\u2013C2 bond (|\u0394\u03c421\u2013C1\u2013C2\u2013C15C|) in TPE derivatives in solution upon excitation decreases in the order 1\u20133 and 5\u20136 do effectively restrict the motion of the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond. We note that this trend persists for the changes of the dihedral angles of the phenyl torsion (|\u0394\u03c41\u2013C2\u2013C15\u2013C20C|) in 1\u20133 after photoexcitation. Two ethylene tethers in geminally locked structure 5 and vicinally locked structure 6 have similar restriction effects on the phenyl torsion with the smallest change of the dihedral angles (ca. 20\u00b0). Fig. S26. To expl and 5\u20136 , which i1\u20136, compounds 1 and 2 are archetypal TPE-based AIEgens showing intense turn-on fluorescence upon aggregation (see the fluorescence quantum yields (QY) in 3 shows unexpectedly highly fluorescence in solution, and becomes even more fluorescent upon aggregation making it AEE (aggregation-enhanced emission)-active. Contrary to our expectations, the significantly more rigid structures 4\u20136 have low fluorescence quantum yields in solutions, among which 5 is an AIEgen, while 4 and 6 are not AIE-active. These findings clearly demonstrate that structural rigidity is not directly correlated with the AIE properties and the RIM paradigm needs to be revisited.The RIM mechanism dictates a reciprocal correlation between the rigidification of the structure and possible AIE properties. For molecules 1\u20136 in dilute solutions and film are shown in 1\u20133 in solution display similar UV absorption peaks at around 310 nm, the UV absorption maxima of 4 and 5 are markedly blue shifted (<270 nm), and the absorption maximum of 6 is notably red shifted (368 nm). All compounds 1\u20136 in dilute solutions undergo photocyclization upon UV excitation derivatives.3 undergoes photocyclization, as evidenced by the emergence of similar new emission peaks at 361, 378 and 400 nm scale\" fill=\"currentColor\" stroke=\"none\">C bond twisting, phenyl torsion and photocyclization) in 1\u20136 upon excitation, and to determine their contribution to the photophysics of the AIE mechanism, probing the molecular dynamics in the excited state is necessary. Prior to this, developing a quantitative understanding of the coupling relationship between the relaxation channels directly related to the molecular motions is imperative. To that end, we have constructed the 3D potential energy surface (PES) of 1 in solution as a function of the aforementioned modes using DFT calculations ) has a molecular geometry corresponding to ca. 8\u00b0 twisting angle along the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond twisting mode, ca. 50\u00b0 torsion along the phenyl torsional coordinate, and potential energy whose value is set to 0 kcal mol\u20131. The coupling between the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond twisting and phenyl torsion along the minimum energy path (MEP) of 1 on the ground state PES scale\" fill=\"currentColor\" stroke=\"none\">C bond twisting (<9\u00b0) and potential energy , indicating that under standard conditions the torsion of the phenyl rings dominates the ground state dynamics in 1. This is further corroborated by the thorough analysis of the intrinsic reaction coordinate (IRC) calculations scale\" fill=\"currentColor\" stroke=\"none\">C bond twisting scale\" fill=\"currentColor\" stroke=\"none\">C bond twisting mode in 1 in the ground state from one minimum energy geometry through the highly twisted transition state to the other minimum energy geometry scale\" fill=\"currentColor\" stroke=\"none\">C bond twisting is associated with significant changes in the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond length (\u223c0.07 \u00c5), and a marked increase in potential energy . In the excited state, the elongation (\u223c0.12 \u00c5) and partial loss of the double bond character of the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond in 1 triggers the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond twisting as the dominant motion which is coupled with the phenyl torsion scale\" fill=\"currentColor\" stroke=\"none\">C bond twisting mode in 1 on the first excited state PES reveals that on the way from the Frank\u2013Condon (FC*) geometry to the minimum energy geometry , the quasi C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond twists ca. 50\u00b0, which is accompanied by the phenyl torsion with an amplitude of less than 25\u00b0. Although their coupling relationship in the excited state is well fitted by the quadratic function \u0394 twisting = 0.0671 (\u0394 torsion)2 \u2013 3.9048 \u0394 torsion \u2013 2.7952, the PES around the S1,min is rather shallow and simple harmonic oscillations PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond twisting may easily take place with the damping process, resulting in the relaxation to the S1 min spectrum corresponds to the statistically most probable geometry of the molecules on a specific timescale in the excited state. Thus, the photoinduced excited state structural dynamics associated with molecular motions is reflected in the evolution of TA spectra.1\u20136 flows mainly from the central C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond to the adjacent C\u2013C (Ph) bonds scale\" fill=\"currentColor\" stroke=\"none\">C bond and shortening of peripheral bonds, which is accompanied by C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond twisting coupled with phenyl torsion. This process in 1 is reflected in the excited state absorption spectra shown in 1 transitions from Sn to the emissive state S1 due to the weak stimulated emission as evidenced by the band at 500 nm with a negative amplitude at 1.3 ps. PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond elongation PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond twisting. On the picosecond timescale between 1.3 ps and 3.79 ps scale\" fill=\"currentColor\" stroke=\"none\">C bond twisting, PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond took place. After 3.79 ps scale\" fill=\"currentColor\" stroke=\"none\">C bond elongation (\u03c41), the sequential dominant motion of the quasi C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond twisting (\u03c42), and the last motion dominated by the phenyl torsion (\u03c43), respectively. It should be noted that both processes corresponding to \u03c41 and \u03c42 are coupled with phenyl torsion, as proved by the band at 430 nm and predicted by the calculation (1-IM (\u03c44) having a new bond connecting atoms C20 and C22 directly scale\" fill=\"currentColor\" stroke=\"none\">C bond elongation corresponding to the population of transients in the emissive state . In contrast to 1\u20132, the decay of the band at 610 nm with growing intensity of the band at 430 nm accompanied by a blue shift scale\" fill=\"currentColor\" stroke=\"none\">C bond slows down. This is probably due to the steric hindrance from the ortho-position substituents in 3. The depopulation of the band at 428 nm scale\" fill=\"currentColor\" stroke=\"none\">C elongation and quasi C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond twisting) of 4 upon excitation have similar dynamics and time constants to those of 1\u20132 scale\" fill=\"currentColor\" stroke=\"none\">C bond elongation in 5 upon excitation in solution is reflected by the population of the 628 nm band with its red shift before 1.03 ps scale\" fill=\"currentColor\" stroke=\"none\">C bond twisting and phenyl torsion take place at the same time. Concomitantly, the intensified red-shifted band at 330 nm indicates that a new species is formed, which is further proved by the emergence of a band at 438 nm scale\" fill=\"currentColor\" stroke=\"none\">C elongation in 6 upon excitation was not observed in the fs-TA spectra because of its negligible increase of only 0.03 \u00c5 (6 upon excitation has a short d20\u2013C22C distance (1.70 \u00c5). This means that the vicinally bi-locked structure of 6 restricts the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C elongation and the freedom of torsion for all phenyl rings with short distances, which facilitates the ultrafast formation of the photocyclized intermediate on the subpicosecond timescale, and thus 6 barely fluoresces in solution. After 4.68 ps, the photocyclized intermediate of 6 undergoes structural relaxation scale\" fill=\"currentColor\" stroke=\"none\">C bond elongation with a time constant of \u03c41 < 0.4 ps scale\" fill=\"currentColor\" stroke=\"none\">C bond twisting with a time constant of 1\u20132 ps , or (b) the ultrafast formation of the photocyclized intermediate (IM) on the sub-picosecond timescale , or they do fluoresce (c) due to the structural stability of the emissive state of the molecule on a relatively longer timescale (3). Thus, the dynamics of the quasi C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond twisting (\u03c42) with the possibility of intermediate formation in S* is the key factor that determines the fluorescence properties of TPE derivatives in solution. When the non-radiative relaxation pathways for TPE derivatives in solution are blocked in the solid state via two different routes: relaxation to the original ground state structure and formation of a singlet ground state IM with a long-lived lifetime (\u03c44) that either (1) reverts back to the original compound or (2) is oxidized to form the corresponding photocyclized product (PC) scale\" fill=\"currentColor\" stroke=\"none\">C bond twisting, the potential energy hypersurface, the Gibbs free energy, the geometry optimization of TPE derivatives in solution and in the solid state (both in the ground state (S0) and excited state (S1)), and the UV/vis and Raman spectra of photocyclized intermediates 1-IM\u20136-IM, were performed at the DFT level of theory using the M062X functionalDetailed information on the synthesis and characterization of 2) and time-resolved resonance Raman (ns-TR3) experiments were carried out using the same experimental setups and methods as described previously.3 measurements was 266 nm. Two probe wavelengths (355 nm and 309.1 nm) were used for the ns-TR3 experiment. The ns-TR2 data were obtained by using the difference between the Raman spectra obtained at different power values of the 266 nm pump laser. Compounds 1\u20136 in MeCN solution were studied in a flow-through 2 mm path-length cuvette with an absorbance of 0.5 at 267 nm throughout the data acquisition. More details are available in ESIFemtosecond transient absorption (fs-TA), nanosecond transient absorption (ns-TA), femtosecond time-resolved fluorescence (fs-TRF), nanosecond transient resonance Raman scale\" fill=\"currentColor\" stroke=\"none\">C bond elongation, quasi C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond twisting, phenyl torsion and photocyclization are the sequentially dominant processes in TPE derivatives upon photoexcitation in solution. Their state-dependent coupling motions derived from ultrafast time-resolved spectroscopy show that (a) the lifetime of the species with electronic configuration conducive to fluorescence is the determining factor for the observed fluorescence quantum yields, (b) in less rigid structures, the rotation of the elongated C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond is the dominant motion that eventually leads to the non-radiative decay (e.g. AIE-active TPE), and (c) in the more rigid structures, the torsion of phenyl rings will dominate the relaxation dynamics and, if possible, lead to the formation of singlet ground state photocyclized intermediates (e.g. AIE-active or AIE-inactive bi-locked TPE derivatives). Thus, while the dominant intramolecular motion that serves as a non-radiative relaxation channel being restricted upon aggregation in every particular case is strongly structure-dependent, counter-intuitively, the AIE effect (or absence thereof) is not directly related to rigidity as seemingly implied by the RIM paradigm.TPE-based derivatives There are no conflicts to declare.Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "Bisdititanium Ti22 trimerises carbon suboxide scale\" fill=\"currentColor\" stroke=\"none\">C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O) to form [{Ti22}{\u03bc-C9O6}], which contains a 4-pyrone core, via the monoadduct [Ti22 (\u03b72-C3O2)]. syn-bimetallic bisdititanium complex Ti22 2) 1 with carbon suboxide scale\" fill=\"currentColor\" stroke=\"none\">C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O, C3O2) results in trimerisation of the latter and formation of the structurally characterised complex [{Ti22}{\u03bc-C9O6}]. The trimeric bridging C9O6 unit in the latter contains a 4-pyrone core, a key feature of both the hexamer and octamer of carbon suboxide which are formed in the body from trace amounts of C3O2 and are, for example, potent inhibitors of Na+/K+-ATP-ase. The mechanism of this reaction has been studied in detail by DFT computational studies, which also suggest that the reaction proceeds via the initial formation of a mono-adduct of 1 with C3O2. Indeed, the carefully controlled reaction of 1 with C3O2 affords [Ti22 (\u03b72-C3O2)], as the first structurally authenticated complex of carbon suboxide.The reaction of the CO and CO2) promoted by well-defined molecular complexes,3O2 is the first in the series of the synthetically available carbon sub-oxides featuring an odd number of carbonse.g. ylides3O2 (hereafter referred to as carbon suboxide) is relatively unstable (it auto-polymerises above 0 \u00b0C but can be stored indefinitely below \u201335 \u00b0C), it is moderately straightforward to prepare via the dehydration of malonic estersUnlike the plethora of catalytic and stoichiometric transformations of carbon's most common oxides . It is then rapidly oligomerised into macrocyclic structures, predominantly cyclic hexamers and octamers .Carbon suboxide is also formed in small quantities octamers , which c3C3O2 to produce C3O2 involved a coordination complex of Ag,3O2 towards Pt(0), Pt(ii) and Rh(i) complexes by Pandolfo et al. proposed the formation of C3O2 complexes but lack of structural data plagued these early investigations.3O2 with organometallic fragments by isolating, for example, the products of its insertion into M\u2013H bonds. A main problem of these early studies was the propensity of C3O2 to act as a source of ketene scale\" fill=\"currentColor\" stroke=\"none\">C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O) and CO. Thus, in the presence of phosphorous containing ligands in the coordination sphere of the metal centre, this led to the formation of the corresponding phosphorous-ylides, as shown by Hillhouse et al. by the reaction of C3O2 with WCl2(PMePh2)4 furnishing WCl2(CO)(PMePh2)2{C,C\u2032:\u03b72-C(O)CPMePh2}.3O2 can displace COD in (PPh3)2Ni(COD) to yield (PPh3)2Ni{C,C\u2032:\u03b72-C3O2} where the C3O2 ligand coordinates via the central and one of the terminal carbons,3O2 to late transition metal centres have also been reported, but, and to the best of our knowledge, there have been no other reports investigating the interaction of C3O2 with organometallic or other coordination compounds. Undoubtedly one of the reasons is its capricious nature, which has favored in silico studies of its reactivity especially towards transition metals.3O2 is one of the least explored \u2018small molecules\u2019 from a synthetic chemist's point of view, a fact underlined by only two short reviews in the current literature.In terms of coordination chemistry, it was proposed that the thermal decomposition of Agsyn-bimetallic complex [Ti22] (Pn\u2020 = C8H4(SiiPr3)2) (1) towards CO, CO2, and heteroallenes and therefore envisioned that (1) might be a good candidate for the binding and activation of C3O2. Herein we present the unprecedented trimerization of C3O2 promoted by (1), We have previously reported on the synthesis,1) to C3O2 at \u201378 \u00b0C, instantly produced a homogeneous brown solution which, upon warming to \u201335 \u00b0C and then slowly to room temperature, deposited some C3O2 polymer, together with a brown supernatant. Filtration of the reaction mixture and work up of the filtrate afforded a brown-green solid, which was isolated in moderate to good yields , and proved to be a diamagnetic, spectroscopically pure new compound (2). The 1H-NMR spectrum of (2) consisted of 16 doublets in the aromatic region signifying the formation of a dimer exhibiting four inequivalent pentalene environments; this was further substantiated by the observation of eight peaks in the 29Si{1H}-NMR spectrum of (2).Exposure of a crimson-red toluene solution of (ctrum of (2) cons13C{1H}-NMR spectrum of (2) displayed 41 resonance in the region between 389\u201396 ppm, 32 of which were assigned to the four inequivalent pentalene environments and Th(iv) featuring dihaptoacyl ligands with substantial oxy-carbene character.2) showed a strong absorption at 2061 cm\u20131 characteristic of a C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O moiety along with bands at 1658, 1591 and 1532 cm\u20131 characteristic of carbonyl functionalities, but also in agreement with haptoacyl ligands with a strong oxo-carbene character.2) (3O2 (9O6] core between the two [Ti2Pn\u20202] moieties found in (2) (The 145 ppm ), with tacter.2) , which i.2) (3O2 . At thisd in (2) and 5, wd in (2) , all of 2) (2.4648(14) and 2.4834(16) \u00c5) are retained but have been slightly elongated in comparison to that in (1) (2.399(2) \u00c5).2 fashion. This bonding mode is best described as an haptoacyl with a considerable carbenoid contribution to the resonance structure. We base this on the observed metrics of the corresponding bond lengths and angles (Ti2\u2013C1: 2.17(3) \u00c5, Ti2\u2013O: 2.13(3) \u00c5, Ti3\u2013C7: 2.316(17) \u00c5 Ti3\u2013O: 2.133(19) \u00c5, C1-01/C7\u2013O5: 1.30(3)/1.28(3) \u00c5; C7\u2013Ti3\u2013O: 33.1(7)\u00b0, C1\u2013Ti2\u2013O: 35.3(8)\u00b0 C7\u2013O\u2013Ti3: 81.3(12)\u00b0 Ti2\u2013O\u2013C1: 74.1(16)\u00b0) which compare well with those crystallographically determined for [(\u03b75-C5H5)2Ti(\u03b72-COMe)Cl] (Ti\u2013C: 2.07(2) \u00c5, Ti\u2013O: 2.194(14) \u00c5, C\u2013O: 1.18(2) \u00c5; C\u2013Ti\u2013O: 32.0(4)\u00b0, Ti\u2013O\u2013C: 68.3(7)\u00b0, Ti\u2013C\u2013O: 79.7(6)\u00b0),13C{1H}-NMR spectroscopic data discussed above. Furthermore, the bonding of these haptoacyl moieties to the pyrone heterocycle of the [C9O6] core (i.e. C2\u2013C1 and C5\u2013C7. 1.38(3) \u00c5 and 1.46(3) \u00c5 respectively) are in good agreement with the CO\u2013CH3 groups found in [(\u03b75-C5H5)2Ti(\u03b72-COMe)Cl] (C\u2013C: 1.47(3) \u00c5).9O6] core in (2) is that the pyrone 6-membered ring is not planar scale\" fill=\"currentColor\" stroke=\"none\">O bond distances (i.e. C6\u2013O4: 1.207(18) \u00c5 vs. 1.253(12) \u00c5 in 4-pyrone) are similar within esd's. Unfortunately, due to the mixed occupancy of the CCO moiety and O6 over the two sides of the [C9O6] core in (2) and the resulting crystallographic restraints used to model this disorder, we cannot talk with certainty about the bond lengths and angles of these two ligating moieties to this 6-member ring. Nevertheless, upon inspection of the corresponding bond lengths of these two atoms to the Ti centres, we can deduce that the bonding situation is far from straightforward. For instance, the Ti1\u2013C\u2032 bond resembles the ones found in Ti\u2013NHC complexes, although closer to the high end of the spectrum (2.2\u20132.35 \u00c5),3O2\u20093O2) while the C\u2013O bond remains unchanged (1.442(13) \u00c5 in free C3O2). The same trend (i.e. C\u2013C elongation) applies when compared with the corresponding bond lengths found in ketene scale\" fill=\"currentColor\" stroke=\"none\">C: 1.314 \u00c5, C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O: 1.162 \u00c5). PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O\u2013Ti dative interaction, e.g. in [cis-Ti(OEt2)22].2) is diamagnetic and the elongation of the Ti\u2013Ti bonds might suggest an increase of the formal oxidation state of each Ti centre by one (i.e. Ti(ii) in (1) to Ti(iii) in (2)). However, the oxy-carbene character of the C7\u2013O5 and C1\u2013O1 units as well as the non-aromatic 6 membered heterocycle of the [C9O6] core suggest that complicated resonance structures are in play, and assignment of formal oxidation state and Ti\u2013Ti bond order in (2) is therefore problematic.The Ti\u2013Ti bonds in (2), the reaction was probed computationally. Density functional calculations using the ADF program suite (BP86/TZP) were carried out on model systems in which the SiiPr3 groups on the pentalene ligands were replaced by H atoms to increase computational efficiency. The computational analogues of experimental structures are denoted by italics; calculations on the analogue of the starting material 1, Ti2(C8H6)21, have been described previously.3O2 to 1, Ti2(C8H6)2(C3O2), led to two local minima, 3 and 3\u2032 . The alternative structure, 3\u2032, could possibly be formed as a kinetic product. The structure of 3 clearly suggests that formation of 2, with two C atoms and one O atom bound to the two Ti atoms, proceeds by the left hand side of the molecule depicted in Isomer 3O2 proposed here differs from some others calculated which indicate bonding primarily to the central carbon. In the cases of metal carbonyls2Pn2 has very high energy electrons and acts as a electron pair donor through its Ti\u2013Ti bond.3O2 has electron density on the central C can be found in that described for the adduct of (1) with CO2 [Ti22(\u03bc-CO2)] (6) which has been studied computationally due to the instability of (6) in solution .3O2 in (3) reveals a similar picture to the one found in (6). The Ti\u2013O distance in 3 (2.19 \u00c5) is shorter than that of 6 (2.27) indicating increased donation to O. This analogy between (3) and (6) is further reflected by the short Ti\u2013Ti bond distances that are characteristic of both these computational models. It should be noted that the HOMO-3 retains Ti\u2013Ti bonding character hence there is only a slight lengthening of Ti\u2013Ti distance from 1 to 3 (2.37 \u00c5 to 2.41 \u00c5). A CBC3 has an arrow going from the Ti PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019Ti double bond to C3O2 acting as a Z ligand, in the same way as CO2 behaves in [Ti22(\u03bc-CO2)].3O2 in this model are in good agreement with the ones determined crystallographically vide infra.The HOMO of orbital . The HOM orbital is respo2, and to investigate possible intermediates in the reaction, the geometries of Ti2(C8H6)2(C3O2)2, 4, Ti2(C8H6)2(C3O2)3, 5, and 2 were optimised (In order to examine the energetics for the formation of ptimised . Key bonptimised .3O2 and to 5 reacting with 1 appear to be purely entropic.The free energies for possible reaction pathways are shown in 2): (a) the formation of the intermediate [Ti22] (3) (i.e. the adduct between (1) and C3O2 that would arise from the addition of 1 eq. of the latter to the former) and (b) the termination of the consecutive two additions of C3O2 to (3) by the capping of (5) by (1) or alternatively (c) the reaction of (4) with (3). The barriers for the two pathways, (b) and (c), are too similar to distinguish between them energetically. In all possible pathways leading to (2), the formation of adduct (3) is the common denominator. The high activation energy calculated for the reaction of (3) with a further molecule of C3O2 to form (4) indicates that (3) should be isolable at low temperature. Indeed, repeating the reaction in the same manner as for the synthesis of (2), but removing volatiles at ca. 0 \u00b0C, resulted in the formation of no carbon sub-oxide polymer and 1H-NMR analysis showed the formation of an extra species along with (2), exhibiting two inequivalent pentalene ligand scaffolds (i.e. 8 doublets in the aromatic region). Encouraged by this observation, the reaction between (1) and C3O2 was repeated under higher dilution conditions to prevent the last step of the formation of (2) and the reaction mixture was kept below \u201310 \u00b0C throughout. Upon removing volatiles at low temperature (ca. \u201325 \u00b0C), and lyophilising the residue with benzene (below \u201310 \u00b0C), this new species was isolated in almost quantitative yields and with spectroscopic purity of >98%. More conclusive evidence that (3) is indeed that predicted by calculations was provided by 13C{1H}-NMR spectroscopy. The most salient features of this spectrum are three resonances located at 159.8, 260.4 and 7.03 ppm which all correspond to quaternary carbons and which we assign to coordinated C3O2 : 129.74 (OCCCO) and \u201314.62 (OCCCO)et al. and Pandolfo et al. using 13C-CP/MAS NMR spectroscopy, while the latter is significantly shifted downfield in comparison with these two literature examples -C3O2)(PPh3)2] with M = Ni, Pt respectively).3O2 in (3) is found at much lower field (7.03 ppm) compared with the ones assigned to the terminal carbons (see above) and follows the trend observed in previous studies -C3O2)(PPh3)2] with M = Ni, Pt respectively); it has to be noted though that is shifted downfield compared to these reported values. These discrepancies are expected as the documented examples concern electron rich monometallic metal fragments of d10 transition metals, unlike the present case where a syn-bimetallic Ti\u2013Ti core is involved. The coordination of C3O2 was further corroborated by IR spectroscopy (thin film) that showed characteristic bands for CCO (2060 cm\u20131) and CO functionalities (for free C3O2 2280 cm\u20131) which are in good agreement with values reported for the complexes [M-C3O2)(PPh3)2] .3) even in the solid state. Nevertheless, based on the spectroscopic data discussed above, (3) was assigned as the adduct of C3O2 with (1), i.e. the first intermediate towards the formation of (2). This was unequivocally established by a single crystal XRD study and \u03b71via that same carbon to the other one (Ti1). The latter also coordinates to the central carbon (C2) of the C3O2 ligand. The molecular structure of (3) represents the first example of a crystallographically authenticated example of C3O2 coordination and confirms the coordination modes of C3O2 predicted by Pandolfo and Hillhouse based on spectroscopic evidence.3)2C3O2 (2.4293(14) \u00c5) is similar to the one found in parent (1) (2.399(2) \u00c5) within esd's; a similar invariance in the Ti\u2013Ti bond length has been observed in the adducts of (1) with CO 2] dTi\u2013Ti = 2.4047(5) \u00c5; [Ti22(CO)2] dTi\u2013Ti = 2.4250(10) \u00c5).3O2 has a profound effect on its bond angles, with the most prominent changes being the significant deviation of the O1\u2013C1\u2013C2 and C1\u2013C2\u2013C3 angles from linearity (179.93(11)\u00b0 and 178.32(12)\u00b0 respectively in free C3O2\u2009vs. 179.57(12)\u00b0 in free C3O2\u20093O2 are similar within esd's to the ones found in free C3O2 (C1\u2013O1: 1.372(12)/1.1479(12) \u00c5; C1\u2013C2: 1.291(13)/1.2564(15) \u00c5; C2\u2013C3: 1.300(13)/1.2475(15) \u00c5; C3\u2013O2: 1.175(9)/1.1442(13) \u00c5) with the exception of the C1\u2013O1 bond distance which is elongated (1.372(12) \u00c5 in (3) vs. 1.1479(12) \u00c5 in free C3O2\u20092) (i.e. C2\u2013C3) is shorter (1.300(15) \u00c5 vs. 1.40(3) \u00c5 in (2)) while the C\u2013O bond lengths are the same within esd's. In the case of the corresponding angles, the CCO angle in both (2) and (3) are identical (172.4(16) \u00c5 and 172.0(10) \u00c5 respectively) (As can be seen from .3)2C3O2 is closey (179.93\u00b0 and 178y (179.93\u00b0 and 178y (179.93\u00b0 and 178 C3O2\u20092) , the corctively) .3O2 promoted by a well-defined molecular complex leading to the formation of (2). The core structure between the two [Ti2Pn\u20202] moieties is reminiscent of biologically relevant compounds responsible for the regulation of ion concentrations in cells. This transformation was studied computationally revealing that the first step is the formation of (3), which was confirmed experimentally by its isolation and structural characterization.In conclusion, we report the first example of the trimerisation of CThere are no conflicts of interest to declare.Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "We definitively show that the CO stretching response to metal coordination is driven exclusively by \u03c0 polarization, which quantitatively correlates with \u03c0 back-donation and changes in CO bond length and frequency. nM(CO)]m complexes (L is an auxiliary ligand) is investigated in relation to the \u03c3 donation and \u03c0 back-donation components of the M\u2013CO bond and to the electrostatic effect exerted by the ligand\u2013metal fragment. Our analysis encompasses over 30 carbonyls, in which the relative importance of donation, back-donation and electrostatics are varied either through the ligand in a series of [(L)Au(CO)]0/+ gold(i) complexes, or through the metal in a series of anionic, neutral and cationic homoleptic carbonyls. Charge-displacement analysis is used to obtain well-defined, consistent measures of \u03c3 donation and \u03c0 back-donation charges, as well as to quantify the \u03c3 and \u03c0 components of CO polarization. It is found that all complexes feature a comparable charge flow of \u03c3 symmetry (both in the M\u2013CO bonding region and in the CO fragment itself), which is therefore largely uncorrelated to CO response. By contrast, \u03c0 back-donation is exceptionally variable and is found to correlate tightly with the change in CO bond distance, with the shift in CO stretching frequency, and with the extent and direction (C \u2192 O or C \u2190 O) of the CO \u03c0 polarization. As a result, we conclusively show that \u03c0 back-donation can be an important bond component also in non-classical carbonyls and we provide the framework in which the spectroscopic data on coordinated CO can be used to extract quantitative information on the \u03c0 donor properties of metal\u2013ligand moieties.The CO stretching response upon coordination to a metal M to form [(L) In most metal\u2013carbonyl complexes the CO bond appears weakened, i.e., the stretching frequency decreases (\u0394\u03bdCO = \u03bdCO \u2013 \u03bdfree-CO < 0) and the bond distance increases (\u0394rCO = rCO \u2013 rfree-CO > 0), but in a minority of complexes, which are sometimes termed \u201cnon-classical\u201d,\u03bdCO > 0 and \u0394rCO < 0). These differences in the CO stretching response to the M\u2013CO bond formation in metal carbonyl complexes are commonly explained in terms of the relative importance of the DCD constituents of the M\u2013CO bond. In particular, M \u2192 CO \u03c0 back-donation is represented as exerting a bond-weakening effect on CO, while M \u2190 CO \u03c3 donation is thought to act in the opposite way.On the experimental side, discussions on the nature of the M\u2013CO bond are mostly based on the analysis of the variation in the CO stretching frequency nM(CO)]m complex, three VB structures differing for the extent of \u03c0 back-donation can be written:(a) \u2013M\u2013C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O+ \u2194 (b) M PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 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0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O \u2194 (c) +M PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C\u2013O\u2013One way to schematically depict M(CO) bonding resorts to a simple Valence Bond (VB) picture. Focusing on the M(CO) moiety of a generic [(L)nM]m fragment. At the same time, the electronic structure of CO is also affected by the electric field generated by this fragment, especially in those cases when m \u2260 0. For CO in the presence of an electric field generated, for instance, by a positively charged metal fragment (exemplified here with the symbol \u2295), three analogue VB structures can be written:(d) \u2295 \u2013C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O+ \u2194 (e) \u2295 C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O \u2194 (f) \u2295 +C\u2013O\u2013In going from structure (a) to structure (b) and (c), where one has zero, one and two \u03c0* orbitals of CO engaged in back-bonding, the CO bond multiplicity goes from three to two to one. The relative weight of each structure will of course depend on the \u03c0 donor properties of the specific + 2Pz)3]\u2013,3C6H2, SIdipp for 1,3-bisimidazolin-2-ylidene, Idipp for 1,3-bisimidazol-2-ylidene and [HB2Pz)3]\u2013 is a fluorinated tris(pyrazol)borate ligand. They all exhibit blue shift of the CO frequency and therefore are classified as non classical. This has been taken by some authors as proof that the gold fragment gives poor or no back-donation.i) complex showing \u0394\u03bdCO < 0 has been fully characterized,o-carborane diphosphine (DPCb) as an ancillary ligand. Such an \u201cexception\u201d, which is even more singular when considering that the formal positive charge should strengthen the CO bond, made the authors speak of \u201cenhanced \u03c0 back-donation\u201d from the [(DPCb)Au]+ fragment. For the reader's convenience, an overview of the experimentally characterized systems, with the reported \u0394\u03bdCO values and reference to the original papers, is displayed in i) system, [{MeB[3-(Mes)Pz]3}Au(CO)], has been preliminarily reported as red-shifted in Still, however, carbonyl complexes showing blue shifted (\u0394via charge-displacement (CD) analysis,The relationship of the DCD constituents of coordination bonds, determined unambiguously \u03bdCO and \u0394rCO and the charge displacements of \u03c3 and \u03c0 symmetry along the M\u2013C\u2013O axis in response to the M\u2013CO bond formation in metal carbonyl complexes. We carry out our analysis first on an exhaustive series of 23 gold(i) carbonyls of formula [(L)Au(CO)]0/+, where L is a varying auxiliary ligand (including none), which includes 8 of the experimentally characterized complexes and which is evenly partitioned between charged and neutral complexes, as well as between classical (CO bond elongated and frequency red-shifted) and non-classical (CO bond shortened and frequency blue-shifted). The choice of binary gold complexes seems to be particularly simple and useful, as it permits to isolate and study systematically the effect of the trans ligand across a wide variety of metal binding properties and electronic effects. We begin our analysis (Section 3.1) by studying in greater detail the two extreme cases of \u201cnaked\u201d Au+, [Au(CO)]+, which displays the experimentally largest blue-shift, and of [(DPCb)Au(CO)]+, which is the only known case of a positively charged but significantly red-shifted gold(i) complex. Having thus highlighted the main findings, we then thoroughly confirm them by extending the study to the whole series of complexes (Section 3.2). To complete the work we then also investigate the role of the metal itself in driving CO response to coordination, by studying a series of homoleptic [(CO)nM(CO)]m complexes, with M including Hg, Ir, Ni, Fe, Cr, Mo, Co, Ru (Section 3.3). Finally, an ad hoc study of CO in a uniform axial electric field (Section 3.4) concludes the work, in order to isolate the impact of CO polarization and of its \u03c3 and \u03c0 components on CO stretching response.We thus investigate the relation between \u03942\u03c1 between the electron density of the adduct AB and that of the two non-interacting fragments A and B frozen at their in-adduct geometries. A partial progressive integration of \u0394\u03c1 along a suitably chosen bond axis z yields the so called charge-displacement function (CDF)42In the charge-displacement (CD) analysis framework, a chemical bond A\u2013B is analyzed in terms of the difference \u0394z, the exact amount of electron charge displaced from right to left (the direction of decreasing z) upon bond formation through a plane perpendicular to the z axis through the point z . If both the adduct and its constituting fragments have proper symmetry, \u0394\u03c1 can be decomposed into additive components of \u03c3 and \u03c0 symmetry with respect to the bond axis z ]m. Since the M\u2013CO bond is under investigation, the appropriate fragments are the ligand\u2013metal moiety [(L)nM]m and carbon monoxide CO, and the z reference axis joins the M and C centres. For the purpose of separating the \u03c3 and \u03c0 components of \u0394\u03c1, we group the orbitals of adduct and fragments according to the irreducible representations of the complex (and fragments) symmetry groups, which in the present cases are either the C2v group or the C3v group . No CO orbital is of A2 symmetry, therefore this representation is not relevant for the DCD analysis of the M\u2013CO bond and is found to represent only a (minor) rearrangement internal to the ligand\u2013metal fragment. Among the gold(i) complexes considered, [(PF3)Au(CO)]+, [(PH3)Au(CO)]+, [(P(CH3)3)Au(CO)]+, [(CF3)Au(CO)] and [(CH3)Au(CO)] belong to the C3v point group. All others have C2v symmetry. The symmetry point groups of the homoleptic complexes considered in Section 3.3 are listed in C2v and C3v have been used to separate the \u03c3 and \u03c0 components of the electron density difference also for these complexes.All of the complexes studied in this work have general formula + (Section 3.1). We then extend the analysis to a whole series of 21 [(L)Au(CO)]0/+ complexes (Section 3.2) and, finally, to a series of nine homoleptic complexes of general formula [(CO)nM(CO)]m (Section 3.3). The full list of complexes considered is in As mentioned in the Introduction, we first describe here a detailed investigation of the M\u2013CO bond in [Au(CO)]3)2Pz)3]\u2013 and Mes3P do not satisfy the symmetry requirements discussed in Section 2. [(DPCb)Au(CO)]+, however, is only slightly asymmetric in its minimum configuration and has been here constrained to C2v symmetry . The other two have been excluded from our analysis because they are much more asymmetric and to constrain them to C3v symmetry would probably alter their properties significantly.Three of the experimentally characterized gold complexes, with ligands DPCb, [HBAu(CO)], [(Cl)Au(CO)] and [(Br)Au(CO)] (the first of which is measured in the solid state and the others in solution) is actually blue-shifted rather than red-shifted as the calculations consistently suggest for all the neutral systems (the computed vfree-CO is 2143 cm\u20131). Regarding this apparent inconsistency, Frenking et al. recently found that the experimental blue shift is actually due to the presence of intermolecular interactions and not to the properties of the single molecule.3)Au(CO)] and of [(Cl)Au(CO)] and finding that the frequency increases from smaller to larger values than that of free CO. Indeed, Au\u2013Au interactions have been experimentally observed for these two complexes in the solid state\u03bdCO and \u0394rCO (computed rfree-CO = 1.137 \u00c5). In fact, we shall most often refer to the latter parameter only, because the non-uniform influence of vibrational mode coupling, and the more complicated CO vibration modes in the homoleptic carbonyls, make \u0394\u03bdCO a less reliable parameter than \u0394rCO for a quantitative analysis of its relation with the M\u2013CO bond characteristics.As 3.1+ and [(DPCb)Au(CO)]+. As mentioned in the Introduction, among the experimentally characterized gold carbonyl complexes, these two systems display the most different spectroscopic properties. [Au(CO)]+ (observed in neon matrix\u03bdCO = 94 cm\u20131) while [(DPCb)Au(CO)]+ (\u03bdCO = \u201330 cm\u20131). The computed values ]Au(CO)]+ represen+, showing in z, a positive CDF value corresponds to a charge flow from right to left while a negative value corresponds to a charge flow in the opposite (Au+ \u2192 CO) direction. The total CDF is positive over both the Au\u2013C and C\u2013O bond regions and also at the oxygen far side of CO, indicating a continuous flow of electrons in the direction from CO towards gold. The negative values of the curve on the left side of Au+ indicate a rearrangement in the opposite direction, which was shown in 1 component which is large and positive in the Au\u2013carbon region (identifying \u03c3 donation) and a B1 + B2 component which is negative in the same zone (identifying \u03c0 back-donation) plus a negligible A2 component. These components are easily recognized in the isodensity plots of the respective density difference shown at the top of the figure.We focus first on [Au(CO)]net from CO to Au+ amounts to 0.16e resulting from a donation component CT\u03c3don of 0.34e and a back-donation component CT\u03c0back of 0.18e. The first important comment here is that, in a system like this showing a large blue-shift of the CO stretching frequency, back-donation is actually a significant component of the interaction, estimated to be more than half as large as the donation.The net charge transfer CTEorb was found to be surprisingly large . The authors were cautious, however, in attributing such contribution exclusively to \u03c0 back-donation, as \u0394Eorb not only accounts for genuine inter-fragment orbital interactions but also for the polarization of the orbitals within each fragment.An analogous significant contribution from the electron charge rearrangement of \u03c0 symmetry was also recently highlighted in rCO/2 = 0.16e, resulting from a \u03c3 contribution of 0.07e and a \u03c0 contribution of 0.09.This uncertainty may be dissolved here, because, as discussed in Section 2, the interfragment charge transfer and its components are automatically separated from the corresponding components of CO polarization in the CDF picture. Inspection of +, with its CDFs reported in 1 (dashed blue line) and B2 (dotted-dashed line) components are not identical and are shown separately in the plot. We notice an immediate striking contrast with the previous [Au(CO)]+ case, in that the back-donation components globally dominate over \u03c3 donation in the coordination bond region, so that the total CDF is negative everywhere, indicating a continuous, though modest, flow of electrons from [(DPCb)Au]+ to CO. This confirms the already cited findings of 2 component. The net charge transfer at the inter-fragment boundary is \u20130.06e, resulting from a \u03c3 donation component of 0.26e (A1) and a \u03c0 back-donation component of \u20130.32e (\u20130.07 due to the B1 component and \u20130.25 due to the B2 component).We now turn to [(DPCb)Au(CO)]+. In analogy with [Au(CO)]+, the \u03c3 CDF remains positive in the CO region and the B1 component turns positive at the C site, reflecting the polarization of the CO bonding orbitals due to the electrostatic effect of the metal fragment. However, by contrast, the B2 component maintains its negative sign also in the CO region, i.e. the back-donation it represents is so pronounced that it penetrates the CO region and extends even beyond the oxygen. As a consequence, the CO bond is on the whole slightly polarized in the C \u2190 O direction (CTrCO/2 = 0.03e), resulting from a \u03c3 polarization in the same direction and a \u03c0 polarization in the opposite C \u2192 O direction .The polarization of the electron cloud in the carbonyl region also differs remarkably from that in [Au(CO)]+ behaves non-classically (blue-shifted \u0394\u03bdCO), while [(DPCb)Au(CO)]+ behaves classically (red-shifted \u0394\u03bdCO). The CD analysis reveals that the \u03c3 donation component of the metal\u2013CO bond is roughly comparable in the two cases (CT\u03c3don 0.34 vs. 0.26e), while \u03c0 back-donation is almost twice as large in [(DPCb)Au(CO)]+ (CT\u03c0back 0.32 vs. 0.18e) and its extent substantially reduces the C \u2190 O polarization of the CO bond. The polarization of the CO \u03c3 bonding orbitals is comparable in the two complexes ( 0.07 vs. 0.05e), but that of the \u03c0 bonding orbitals is not ( 0.09 vs. \u20130.02e). These findings suggest that \u03c0 electron displacement upon coordination is the main factor driving CO bond response. In particular, if the presence of the metal fragment, especially if positively charged, is capable of polarizing the \u03c0 CO bonding orbitals, even in the presence of a significant back-donation, the CO bond is strengthened; if, on the other hand, \u03c0 back-donation is strong and extended enough to contrast CO polarization, even in the presence of an equally cationic metal fragment, the CO bond is weakened.It is worth deepening the comparison between the two complexes examined so far. In both, the metallic fragment bears a formal positive charge. However, [Au(CO)]3.20/+ complexes listed in \u03bdCO and \u0394rCO as well as the various computed CT figures. The complexes are listed in order of increasing \u0394rCO and the experimentally characterized compounds are those shown in boldface. As briefly discussed at the beginning of Section 3, it is seen that, according to our computed shifts, the neutral complexes plus [(DPCb)Au(CO)]+ behave classically, while the remaining cationic complexes behave non-classically. The \u03c3 donation and \u03c0 back-donation CDFs for these complexes are collected, respectively, in the top and bottom panel of \u03bdCO, blue lines are for those showing blue shift.We now need to verify if the above preliminary surmise stands the test of a wider series of carbonyl compounds. To this end, we have extended the analysis to all 23 [(L)Au(CO)]+, of the inert ligands Ne and Xe, and of the anomalous [(DPCb)Au(CO)]+, even the net ligand-to-metal \u03c3 donation, CT\u03c3don, varies by only 0.05e across the whole series of ligands. On the contrary, the \u03c0 CDF all invariably exhibit a flow of \u03c0 electrons in the C \u2190 O direction , due to the positively charged metallic fragment, while the complexes showing red-shifted \u03bdCO (red lines) exhibit a negative , i.e., charge flows in the opposite C \u2192 O direction .Two eye-catching features emerge upon inspection of i) carbonyls: (i) \u03c3 donation is much less tunable than \u03c0 back-donation, being very little dependent on the nature and the charge of the ligand; (ii) whereas the net CO bond polarization turns out to be invariably oriented in the C \u2190 O direction (CTrCO/2 > 0), the direction of its \u03c0 density component can vary and appears to be tightly connected with the direction of the CO stretching shift and bond-length change. These findings are given a definitive illustration in rCO with CTnet, CT\u03c3don, CT\u03c0back, CTrCO/2, and is reported. In both figures, black triangles are used for the overall CT, red squares for its \u03c3 component and blue circles for its \u03c0 component. Empty symbols are for the neutral species, filled ones are for the cationic species.It thus appears quite clearly that in the series of gold can be seen between \u0394rCO and CT\u03c0back, a trace of which remains in the plot of \u0394rCOvs. CTnet. The same bond weakening effect of \u03c0 back-donation is also evident in the plot of \u0394\u03bdCOvs. CT\u03c0back (see ESIR2 = 0.849). rCO with CTrCO/2 and its components and . Not surprisingly, as these quantities are all directly related to the charge rearrangement of the CO bond itself, correlations are here quantitatively better (R2 is 0.970 for that with ). Obviously, as \u0394rCO correlates well with both \u03c0 back-donation and CO \u03c0 electron polarization, the latter two quantities are also in mutual correlation.Focusing first on k see ESI, though 3.3i) complexes where the donor/acceptor properties of the M\u2013CO bond were varied through the ligand L. We now extend the analysis to a series of homoleptic carbonyls of formula [(CO)nM(CO)]m, where the relative extent of the DCD constituents of the M\u2013CO bond and CO polarization are varied essentially by changing the metal. The full list of the considered homoleptic complexes is in rCO. We omit for brevity a presentation of the complete CDFs. The computed structures for these systems are in agreement with experimental X-ray data where available.22+ and Ir(CO)63+, both cationic, behave non classically, with experimental blue-shifted \u03bdCO at 2279.5 cm\u20131 for the former and at 2254, 2276 and 2298 cm\u20131 for the latter.42\u2013 at 1730 cm\u20131 5 ,4 and Cr(CO)6. The complexes present therefore a wide range of \u03bdCO variation but \u0394\u03bdCO turns out not to be a good parameter for analyzing correlations with the CD data because normal-mode coupling varies significantly with the different structure of the complexes. We therefore base our discussion, as already done for the gold(i) complexes, on the computed \u0394rCO. This varies in a range of 0.087 \u00c5 over the series, from \u20130.018 to 0.069 \u00c5 (In the previous sections we considered a series of gold( 0.069 \u00c5 .e) is much larger than that of \u03c3 donation (0.15e). In particular, almost no back-donation is found for Hg(CO)22+ while CT\u03c0back for [Fe(CO)4]2\u2013 is as high as 0.71e. This picture is consistent with the simple VB view discussed in the Introduction, in that we go from a purely \u03c3 M\u2013CO bond (structure a) for Hg(CO)22+ to a situation in which all \u03c0* CO orbitals are engaged in back-bonding (structure c) for [Fe(CO)4]2\u2013. Also the charge rearrangement (polarization) in the carbonyl region is seen to follow a similar trend, with a much narrower range of values (between 0.02 and 0.10e) than that of (from 0.21 to \u20130.30e). As before, no clear correlation can be discerned between \u0394rCO and the \u03c3 CT data, while CT\u03c0back and values are seen to decrease almost monotonically as \u0394rCO increases.The table shows that also in this series of compounds the range of variation in \u03c0 back-donation series, appears in fact to suggest, because the range of variation is now significantly enlarged, that a quadratic fit, rather than a linear one, may better represent the actual correlation , i.e. the cationic Hg(CO)22+ and Ir(CO)63+, show a flow of \u03c0 electrons in the C \u2190 O direction. All other complexes, where the CO bond weakens (red-shifted \u0394\u03bdCO and positive \u0394rCO) show opposite-direction flows.Once again, in the homoleptic series, the carbonyl complexes featuring CO bond strengthening C \u2192 O (negative) polarization, C\u2013O bond length increases quadratically and \u03c0 polarization is seen to increase much more rapidly than \u03c3 polarization. Conversely, as the field increases on the right, inducing C \u2190 O polarization, the C\u2013O bond shortens (much less rapidly).Let us focus first on the stretching response to the electric field. When the field is absent, the system corresponds to free CO and \u0394rCO and the \u03c3 and \u03c0 components of CO polarization induced by metal coordination, rather than by an applied field (disconnected circles in the figure), we notice immediately that the \u03c0 circles follow quite closely the correlation between field-induced polarization and stretching, while, in striking contrast, the \u03c3 circles deviate from the field-induced line and, moreover, span a very narrow range of (positive) polarization, essentially without any correlation with the widely varying \u0394rCO. This is indeed a very strong confirmation that the CO stretching response to any solicitation causing electron charge rearrangement, be it the formation of a M\u2013CO coordination bond or the effect of an external electric field, is driven essentially exclusively by the charge rearrangement of \u03c0 symmetry: whether induced by an external electric field or by metal coordination, C \u2192 O (C \u2190 O) polarization of the \u03c0 bond orbitals invariably and tightly correlates with bond lengthening (shortening).When we now compare these curves with the relation observed between \u03944nM(CO)]m metal carbonyl complexes, with the aim of elucidating on quantitative grounds the \u03c3 donation and \u03c0 back-donation effects on the CO stretching response, in particular the change in bond length \u0394rCO, to the M\u2013CO bond formation. The analysis was carried out for a large variety of carbonyls, in which the relative extent of the DCD constituents were varied both through L in a series of [(L)Au(CO)]0/+ gold(i) carbonyl complexes and through M in a series of anionic, neutral and cationic [(CO)nM(CO)]m homoleptic carbonyls. Crucially, for the purpose of this investigation, reliable and consistent measures, not only of \u03c3 donation and \u03c0 back-donation charges but also of the \u03c3 and \u03c0 components of CO polarization were obtained by the well-established charge-displacement analysis of electron-density differences, as resulting from accurate DFT calculations. The nature of the M\u2013CO bond in the considered complexes was found to range smoothly between the two extreme cases of an almost purely \u03c3 bonded complex (Hg(CO)22+, CT\u03c0back = 0.02e) and of a strongly back-bonded complex ([Fe(CO)4]2\u2013, CT\u03c0back = 0.71e). Conversely, all complexes were found to feature a narrowly comparable \u03c3 donation component, with CT\u03c3don values ranging from 0.14 to 0.34e. The same picture holds accurately for the electron cloud rearrangement over the carbonyl region: all considered complexes feature a comparable \u03c3 polarization of CO and a much more variable \u03c0 polarization. Quite remarkably, no correlation is found between \u0394rCO and the \u03c3 displacements, while \u0394rCO, \u03c0 back-donation and CO \u03c0 polarization all correlate tightly with one another.In this work we have carried out an in-depth analysis of the M\u2013CO bond in [(L)+ complex, where \u03c0 back-donation is so strong as to invert the polarization of the \u03c0 CO bonding orbitals in the C \u2192 O direction despite the formal positive charge on the ligand\u2013metal fragment, making it the only example of a cationic gold(i) carbonyl complex with classical behavior (\u0394rCO > 0). An ad hoc study of CO in a uniform axial electric field demonstrates that it is indeed the polarization of the \u03c0 CO bonding orbitals, no matter how induced (whether by the coordination bond to M or by an electric field), that drives direction and magnitude of the CO stretching response to the M\u2013CO bond formation.These results show that the driving force of the CO stretching response to the M\u2013CO bond formation is provided exclusively by the changes taking place in the \u03c0 electron density. In the complexes studied, such \u03c0 charge rearrangement is found to result from the interplay between \u03c0 back-donation (structures a\u2013c of the Introduction) and the electrostatic effect (structures d\u2013f) exerted by the metal\u2013ligand fragment. In particular, cationic metal\u2013ligand fragments polarize the \u03c0 CO bonding orbitals in the C \u2190 O direction, thus shortening the bond and enhancing the covalency, as highlighted in rCO in metal carbonyl complexes, we conclude that the value of \u0394rCO quantifies to an excellent extent the \u03c0 back-donation component of the M\u2013CO bond, since such component directly correlates with the \u03c0 polarization. In particular, where changes its sign , CT\u03c0back is approximately as high as the average extent of \u03c3 donation among the complexes herein considered. This indicates that \u03c0 back-donation is an important component also in the class of non-classical complexes, as those of gold(i) considered in this work.Regarding the fundamental question of what can be inferred on the nature of the M\u2013CO bond from the analysis of \u0394Supplementary informationClick here for additional data file."} +{"text": "Water-enhanced oxidation of graphite via a modified Hummers method can produce graphene oxide with controlled species of oxygenated groups. Graphene oxide (GO) sheets with controlled species of oxygen-containing groups are important for fabricating graphene materials with desired structures and properties. Here we report a water-addition modified Hummers method to prepare GO sheets with tunable amounts of hydroxyl and epoxide groups without destroying their structural integrity. This method is simple, effective, and efficient. It can be applied to the mass-production of GO with controlled amounts and species of oxygenated groups, and improve the yields of synthesizing high-quality GO at low temperatures. However, the use of NaNO3 leads to the formation of NO2/N2O4 toxic gases, and introduces Na+ and NO3\u2013 ions to the waste water. Recently, Tour et al. improved the Hummers method by excluding NaNO3, increasing the amount of KMnO4, and performing the reaction in a 9\u2009:\u20091 H2SO4/H3PO4 mixture for a prolonged time.3 from the chemical recipe of the Hummers method did not affect the yield and oxidation degree of GO.GO can be prepared by oxidation and exfoliation of graphite. The Hummers method is the most widely employed technique for this purpose.On the other hand, GO with a high degree of oxidation usually has a high content of permanent defects.vii) compound (3 that is generated by the oxidation of water with Mn(vii) compound in the H2SO4 solution of KMnO4.In this paper, we report that heavily oxidized GO with good structural integrity can be produced in a high yield by adding a certain amount of water to the reaction system of our modified Hummers method.compound . This mevia a modified Hummers method4 (3.0 g) in 46 mL concentrated H2SO4 containing n mL water at 40 \u00b0C for 2 h, and the resulting GO is nominated as GO-n and the corresponding reduced GO is named rGO-n. A control GO sample, GO-0-95, was synthesized in the system without the initial addition of water. However, after the oxidation process, 100 mL water was slowly added into the reaction system, and kept at 95 \u00b0C for 15 min. The corresponding reduced GO is called rGO-0-95.GO samples were prepared All of these GO samples were carefully purified for characterization scale\" fill=\"currentColor\" stroke=\"none\">C (284.6 eV), C\u2013O (286.6 eV), C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O (287.8 eV), and O\u2013C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O (289.0 eV).IOC/ICC) of oxygenated carbon atoms scale\" fill=\"currentColor\" stroke=\"none\">O, and O\u2013C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O) and intact carbon scale\" fill=\"currentColor\" stroke=\"none\">C) reflects the oxidation degree of GO,IOC/ICC becomes less pronounced as the water volume > 10 mL. The \u2018water-enhanced oxidation\u2019 was also confirmed by the magic-angle spinning 13C solid-state nuclear magnetic resonance (ssNMR) spectra of the as-prepared GO samples. The 13C ssNMR spectrum of GO mainly has the following signals: epoxide , hydroxyl , graphitic sp2 carbon scale\" fill=\"currentColor\" stroke=\"none\">C, \u223c133 ppm), carboxylic acid carbonyl scale\" fill=\"currentColor\" stroke=\"none\">O, \u223c167 ppm), and ketone carbonyl scale\" fill=\"currentColor\" stroke=\"none\">O, \u223c191 ppm).2 carbon at 133 ppm scale\" fill=\"currentColor\" stroke=\"none\">O and C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O are mainly located at the edges of basal-plane vacancies or at the periphery of the GO sheets; PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O and C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O groups, reflecting a better structural integrity. According to PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O (\u223c169 ppm) in its ssNMR spectrum.The delocalized \u03c0-conjugated structure of a graphene sheet is gradually fragmented to smaller domains upon functionalization, weakening its absorption of visible light. Thus, the color difference between GO samples can be used to qualitatively compare their functionalization degree. 133 ppm . Among t PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O (1740\u20131720 cm\u20131)/H2O (\u223c1620 cm\u20131) peaks remains nearly unchanged in the spectra of GO-0 to GO-12, while this ratio for the spectrum of GO-0-95 is much higher, indicating the last sample has the highest content of carbonyl groups. However, the other oxygenated groups exhibited comparable intensities in all of the IR spectra.\u03b8 = 10.65\u00b0, and their d-spacings were calculated to be around 8.30 \u00c5. This value is much larger than that of natural graphite (3.35 \u00c5), indicating the successful functionalization of graphene by oxygenated groups.14The modulation of the oxidation degree and functional groups of GO by adding water has also been confirmed by ATR-FTIR spectral studies . The int\u20131) is associated with the defect-activated breathing modes of six-membered carbon rings, and the G-band (1580\u20131600 cm\u20131) is assigned to the E2g phonons at the Brillouin zone center.ID/IG, reflects the average distance between defects (LD) in graphene. For graphene and its derivatives, the value of ID/IG initially increases with increasing LD , followed by a decrease .LD derived from ID/IG. However, the relative amounts of these two types of defects in GO have to be evaluated by combining the ssNMR and XPS spectra of GO and the Raman spectrum of the corresponding rGO (indicative of the permanent defects).Raman spectroscopy is a powerful tool for studying the structures of CMGs. The typical Raman spectrum of CMG sheets consists of the D-, G-, and 2D-bands of carbon. The D-band , while its LD (1.26 nm) is the smallest among these GO samples. This result indicates that the \u2018defects\u2019 of GO-0-95 are mainly originated from permanent vacancies, implying that the reaction at 95 \u00b0C severely destroyed the graphitic domains of the GO sheets. This conclusion was also supported by its relatively higher content of carboxyl groups that usually locate at the permanent defects (vacancies and edges).In ondingly . Considen were measured to be 19.6, 18.0, 16.5, and 16.2 \u03bcm, respectively. The gradual decrease of size was caused by the unavoidable cutting of GO sheets upon water-enhanced oxidation.vii) species and/or decomposition at an elevated temperature of 95 \u00b0C.The severe structural damage of the GO-0-95 sheets was also indicated by the sheer decrease in their lateral dimensions. The sizes of the GO-0 to GO-12 sheets have a wide distribution from <5 up to over 50 \u03bcm, mainly (>90%) in the range 5\u201340 \u03bcm . The aveTd) of a GO sample increased with its oxidation degree. This trend agrees well with that of partially reduced GO samples.2, and H2O.Td of GO increase with its oxidation degree. The Td of GO-0-95 was measured to be 208.5 \u00b0C or GO-0-95, indicating that water in the reaction system significantly promoted the hydrolysis of organosulfate.Interestingly, thermogravimetric analysis TGA, demonstrC Fig. S5, between\u03b8 = 23.8\u201324.0\u00b0, d-spaces = 3.71\u20133.74 \u00c5). However, the XRD peak of rGO-0-95 is much broader (FWHM = 4.0\u00b0) than those of rGO-n (FWHM = 1.7\u20132.3\u00b0), also reflecting its higher content of residual oxygenated groups. The typical Raman spectra of all the rGO papers led to forming relatively more permanent defects to decrease the LD of rGO. The structural difference between the rGO papers is also reflected by their conductivities (\u20131) > rGO-4 (595 \u00b1 18 S cm\u20131) > rGO-8 (554 \u00b1 13 S cm\u20131) > rGO-12 (510 \u00b1 11 S cm\u20131). Accordingly, a small amount of water addition (\u22644 mL) during oxidation has negligible effect on the structural integrity of GO and the corresponding rGO, while a large amount of water (\u22656 mL) led to a slight decrease of their quality because of excess oxidation. Notably, rGO-0-95 exhibited a much lower conductivity (257 \u00b1 8 S cm\u20131), only about 40% of that of the rGO-0 papers. This is mainly due to the smaller sizes and much higher content of the permanent defects of rGO-0-95.rGO papers were prepared by reducing GO papers with HI dissolved in a water/ethanol mixed solvent (v/v = 1\u2009:\u20091). XPS C 1s analysis indicates that most of the oxygenated groups of GO have been removed upon reduction . The speO papers and S8\u2020 tivities and S9\u2020.2SO4 with water, lowering the intercalation efficiency of H2SO4/KMnO4 between the graphene layers of graphite.2SO4 has alternate graphene and H2SO4 layers; it is an intermediate of converting graphite to GO.2SO4 is usually formed by electrochemically or chemically increasing the electrochemical potential of graphite. The potential of stage I GIC increases, while the oxidizing potential of KMnO4 decreases upon reducing the concentration of H2SO4 (C2SO4H). Hence, stage I GIC could not be formed by oxidation with KMnO4 as C2SO4H < 14.4 M , because EGIC-I < E4KMnO.The yields of different GO samples have a large difference. The yield increased from 69 \u00b1 2% for GO-0 to 131 \u00b1 4% for GO-4, followed by a gradual decrease to 67 \u00b1 2% for GO-14 . This trOn the other hand, the relatively high yield of GO-0-95 is attributed to the in-plane vacancies and holes formed during the destructive oxidizing of GrO at 95 \u00b0C, facilitating the osmotic swelling and exfoliation of GrO to individual GO sheets.2-carbon content,The \u2018water-enhanced oxidation\u2019 of graphite to GO in our systems is an unusual phenomenon, because water is an ineffective oxidant for graphite. Moreover, it was reported that \u2018pristine GO\u2019 reacted with water to form conventional GO with an increase of spn and GrO-0-95) showed large variations (n (n = 4 to 14) were measured to be 50\u2013150 mL, much larger than those of GrO-0, GrO-2, and GrO-0-95 (42\u201344 mL). These volumes were also much larger than that of graphite powder , O3 was generated via oxidizing water by multi-nuclear Mn(vii) clusters as reported in literature.3 gas can also account for the larger volume expansions of GrO-n (n = 4\u201314) than those of the other GrO pastes.In fact, GO samples were formed by the exfoliation of their GrO precursors was also evidenced by monitoring the corresponding ultraviolet-visible (UV-Vis) spectra. The UV-Vis spectrum of a fresh concentrated H2SO4 solution of KMnO4 (3 g KMnO4 in 46 mL H2SO4) did not show a O3 band centered at 255 nm with peaks at 300 and 460 nmvii) by water, although the dilution of the solutions by adding water also had some contributions (only a maximum decrease of 16.3% by diluting with 12 mL water). The volume of the O3 gas formed in the solution also increased with its content of water, indicated by the numbers of gas bubbles generated in the solution filled in a capillary tube (inset of vii) (mainly from MnO3+). However, the solutions with water addition contain brown particles of MnO2 formed by reducing Mn(vii) with water.40The formation of O for 2 h , possiblinset of . The sol3 is a well-known strong oxidant with a standard oxidizing potential (2.08 V) higher than that of MnO4\u2013 (1.68 V).2 formed by reducing Mn(vii) with water is an effective catalyst for decomposing O3 to atomic oxygen.O3 can be enhanced by both water and newly-formed MnO2 .3 (vii) and H2SO4 .3 , atomic nd H2SO4 . The oxy2O2 (30%) was added to convert the remaining Mn species completely to soluble Mn(ii) ions in the system of preparing GO-0. However, in the system of preparing GO-0-95, only about 0.5 mL of H2O2 was consumed. Consequently, GrO-0-95 was further destructively oxidized by the residual Mn(vii) compound at an elevated temperature of 95 \u00b0C in a diluted H2SO4 solution (about 6.0 M). This destructive oxidation led to the formation of more permanent defects capped by carboxyl groups.On the other hand, the mechanism for the destructive oxidation process at 95 \u00b0C is elucidated as follows. Experimentally, 2.2\u20132.4 mL Hn. The electrical conductivity of rGO-4-0-48 h (894 \u00b1 26 S cm\u20131) is much higher than that of rGO-0 (605 \u00b1 20 S cm\u20131). Therefore, water addition is also an effective method to increase the oxidation degree of GO at low temperatures for producing high-quality GO in a much higher yield.The \u2018water-enhanced oxidation\u2019 was observed to be more pronounced by lowering the temperature of the oxidizing graphite ESI. For exavia a modified Hummers method can increase the oxidation degree of GO sheets. This approach can also modulate the content of hydroxyl and epoxide groups on GO sheets without sacrificing their structural integrity, and greatly increase the yield of high-quality GO prepared at a low temperature of 0 \u00b0C. The selective formation of carboxyl groups on GO sheets has been realized by the destructive oxidizing of GO at a high temperature of 95 \u00b0C. This work provided a simple and scalable technique for producing GO with controlled species of functional groups.The addition of a certain amount of water into the system of synthesizing GO Supplementary informationClick here for additional data file."} +{"text": "Accessing highly electron deficient partially alkylated tungsten hydrides on silica via controlled hydrogenolysis of surface organometallic complex scale\" fill=\"currentColor\" stroke=\"none\">Si\u2013O\u2013)W(Me)5. PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019Si\u2013O\u2013)W(Me)5], 1, is an excellent precatalyst for alkane metathesis. The unique structure of 1 allows the synthesis of unprecedented tungsten hydrido methyl surface complexes via a controlled hydrogenolysis. Specifically, in the presence of molecular hydrogen, 1 is quickly transformed at \u201378 \u00b0C into a partially alkylated tungsten hydride, 4, as characterized by 1H solid-state NMR and IR spectroscopies. Species 4, upon warming to 150 \u00b0C, displays the highest catalytic activity for propane metathesis yet reported. DFT calculations using model systems support the formation of [ scale\" fill=\"currentColor\" stroke=\"none\">Si\u2013O\u2013)WH3(Me)2], as the predominant species at \u201378 \u00b0C following several elementary steps of hydrogen addition (by \u03c3-bond metathesis or \u03b1-hydrogen transfer). Rearrangement of 4 occuring between \u201378 \u00b0C and room temperature leads to the formation of an unique methylidene tungsten hydride [ scale\" fill=\"currentColor\" stroke=\"none\">Si\u2013O\u2013)WH3 scale\" fill=\"currentColor\" stroke=\"none\">CH2)], as determined by solid-state 1H and 13C NMR spectroscopies and supported by DFT. Thus for the first time, a coordination sphere that incorporates both carbene and hydride functionalities has been observed.The well-defined single-site silica-supported tungsten complex [( In this regard, the immobilized tungsten hydrides serve as multifunctional precatalysts that engage in separate, successive elementary steps. These involve propagative species that are proposed to display both hydridic (for the C\u2013H bond activation and olefin hydrogenation) and carbene functionalities (for the olefin metathesis steps). PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019Si\u2013O\u2013)W(Me)5] (1) (Me = CH3) is a precatalyst for the metathesis of propane.1 should take place more readily than for the analogous Schrock species,2 surface.We recently demonstrated that the silica-supported tungsten pentamethyl .Because tungsten hydrides are expected to be highly fluxional on the NMR time scale, and are known to be found within a wide inset of reveals i.e., relativistic) effects, with variation in the other magnetic shielding mechanisms being on the order of a few tenths of one ppm. This aspect is discussed more fully in the ESI, Section 6.1H signals at 11.6 and 18.8 ppm are assigned to surface WHx, consistent with the results of the DFT calculations. This is also in agreement with the presence of multiple IR bands of 2 assigned for \u03bd(W\u2013H) in the range 1993\u20131905 cm\u20131 scale\" fill=\"currentColor\" stroke=\"none\">Si\u2013H and PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019Si\u2013CH3 moieties observed respectively by FT-IR and 1H solid-state NMR demonstrate that hydrogenolysis at 150 \u00b0C leads to the formation of a variety of species with little control on structure and identity of the ensuing surface complexes.Intriguingly, the variation in these chemical shifts appears to be nearly fully attributable to spin\u2013orbit (905 cm\u20131 . The pre13C cross-polarization (CP)/MAS NMR spectrum of 2 scale\" fill=\"currentColor\" stroke=\"none\">Si\u2013H moieties of 2. Furthermore, the solid-state 13C CP/MAS NMR spectrum of 3 shows signals at 42 and 47 ppm which are left as a consequence of the partial hydrogenolysis of 1.The rum of 2 , preparem Fig. S6 attribut4 was prepared at low temperature, we recorded the solid-state 1H NMR spectrum using a low-temperature probe, at 100 K. The spectrum displays multiple signals in the low-frequency range of 0\u20132 ppm, and at high frequency, weak signals, at 8.7 and 16.3 ppm. Assignments of these signals are presented after discussion of the various hydrogenolysis pathways starting with 1.Because species PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019Si\u2013O\u2013)W(Me)5], 1, takes place, we proceeded with a DFT approach using a cluster model of the silica support. To ensure that our relatively small cluster model could be used to simulate the reactions on the silica surface, we validated it against periodic DFT and using slab silica models scale\" fill=\"currentColor\" stroke=\"none\">Si\u2013O\u2013)W(Me)5] precursor 1 depicted as I in \u03b72\u2013H2) heptacoordinated tungsten species, with predicted W\u2013H distances of 1.84 \u00c5, and the H\u2013H bond elongated to 0.86 \u00c5 from 0.741 \u00c5 in gaseous H2.2 coordination is endergonic by 14.8 kcal mol\u20131. The hydrogenolysis of I occurs via transition state (TS) [I\u2013IV]\u2021 and requires an activation energy of 15.9 kcal mol\u20131, making this process possible at room temperature. This step corresponds to a \u03c3-bond metathesis event with the release of a CH4 molecule and formation of the tungsten monohydride [ scale\" fill=\"currentColor\" stroke=\"none\">Si\u2013O\u2013)WH(Me)4], IV, which is 18.2 kcal mol\u20131 lower in free energy than I.Starting from the silica-supported [ scale\" fill=\"currentColor\" stroke=\"none\">Si\u2013O\u2013)WH2(Me)3], VIII, is more stable than IV by 18.0 kcal mol\u20131. Similar to the previous findings for I, the formation of a methylidene species (VI) or an ethyl hydride (VII) requires too high of an activation energy and these processes can thus be excluded from further consideration. However, in contrast to I, IV can undergo reductive elimination to form a triplet W(iv) species [ scale\" fill=\"currentColor\" stroke=\"none\">Si\u2013O\u2013)W(Me)3], V, with the release of one methane molecule. This process is favoured thermodynamically, with a free energy change of \u201323.1 kcal mol\u20131. The associated energy barrier estimated at the minimum energy crossing point (MECP) along the CH3\u2013H(W) bond stretching mode turned out to be only 14.8 kcal mol\u20131. However, because direct hydrogen addition occurs via a free energy barrier lower by \u223c3.0 kcal mol\u20131, the conversion of IV to VIII is the most likely route for further hydrogenolysis. The addition of the third, fourth, and fifth hydrogen atoms occurs via direct successive hydrogen additions. All other competing reactions again were found to require higher activation energies than the hydrogenation steps. Even reductive elimination, with formation of a W(iv) complex , requires 33.3, 38.4, and 26.3 kcal mol\u20131 for complexes VIII, XII, and XVI, respectively. Eventually, the final step is the formation of a silica-supported tungsten pentahydride [ scale\" fill=\"currentColor\" stroke=\"none\">Si\u2013O\u2013)WH5], XVIII, which was found to be the most stable species of all those considered, with a Gibbs free energy of \u201377.9 kcal mol\u20131 relative to I.The subsequent transformation of I to XVIII suggests that hydrogenolysis of I is an exergonic process , leading to the formation of silica-supported tungsten pentahydride as the thermodynamic product. The proposed mechanism is a cascade or sequence of hydrogen addition reactions forming the series of supported metallo hydrides scale\" fill=\"currentColor\" stroke=\"none\">Si\u2013O\u2013)WHx(Me)y], x = 1\u20135, y = 4\u20130) along with the elimination of free methane molecules. The lowest barrier corresponds to the reaction with the second H2 molecule. The highest barrier was calculated for the addition of the fourth hydrogen molecule (during transformation of XII to XVI) and amounts to more than 20 kcal mol\u20131, suggesting that this step might be rate determining. On the basis of these calculations it is possible to suggest that the silica-supported [ scale\" fill=\"currentColor\" stroke=\"none\">Si\u2013O\u2013)WH3(Me)2] (XII) might represent the predominant surface species if 1 is hydrogenolysed to afford 4 at \u201378 \u00b0C. Other mechanistic pathways involving the reduction of W(vi) species have been found to be characterized by higher activation barriers.The above characterization of transformations from 2\u20134, i.e., 104) was obtained with 2 (prepared at 150 \u00b0C) (entry 1) compared with the tungsten hydrides prepared from a grafted neopentyl organometallic precursor.3 (prepared at 25 \u00b0C), a further increase in activity for propane metathesis was observed . This result could be attributed to a higher number of active tungsten hydride sites at this treatment temperature (25 \u00b0C), inasmuch as no apparent hydride transfer to the silica surface was observed by solid-state 1H NMR and IR spectroscopy , gave the best activity for this transformation . Significantly, the catalytic activity of species 4, when it was allowed to warm to room temperature scale\" fill=\"currentColor\" stroke=\"none\">Si\u2013CH3 scale\" fill=\"currentColor\" stroke=\"none\">Si\u2013CH3 or of a different kind of methyl group functionalities implies that the initial monopodal species 4 might have partially rearranged into a bipodal species under the experimental conditions by the transfer of a methyl group to the silica support. PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019Si\u2013O\u2013)W(Me)5] (vide infra). Allowing the rotor sample to warm to room temperature for 10 min (outside the NMR probe) and then reintroducing it into the cold NMR probe led to the complete disappearance of the 1H chemical shift at 0.8 ppm and to the increase of the signal at 0.4 ppm ).13Second, this process was monitored by solid-state at 100 K . In the evident . The 1H Si\u2013CH3 and to t 0.4 ppm . Additio 0.4 ppm . All theperature and withs Fig. S7 as we fi1H NMR spectrum in 1H\u201313C HETCOR moieties, and a distinct signal at 231 ppm. The 1H peak at 7.3 ppm correlates only with the carbon resonance at 231 ppm in the 2D 1H\u201313C HETCOR NMR spectrum scale\" fill=\"currentColor\" stroke=\"none\">CH2) moiety.13C solid-state NMR spectrum of species 3 generated directly at room temperature scale\" fill=\"currentColor\" stroke=\"none\">CH2 and W\u2013H signals, the formation of a methylidene hydride from partially alkylated W-hydrides is supported by DFT calculations (vide infra). The W-alkylidene species formed was found by 1H NMR spectroscopy to be stable at temperatures up to 150 \u00b0C, but it decomposed readily upon introduction of D2 gas at 80 \u00b0C. Moreover, at room temperature we observed a proton chemical shift at 0.0 ppm corresponding to PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019Si\u2013CH3 moieties. This result is supported by the appearance of a peak at \u201312 ppm scale\" fill=\"currentColor\" stroke=\"none\">Si\u2013CH3) in the 29Si CP/MAS NMR spectrum of 4 recorded at room temperature scale\" fill=\"currentColor\" stroke=\"none\">Si\u2013O\u2013)WH3(Me)2] is in agreement with the inferred structure as a main surface component. Considering the low hydrogenolysis temperature of \u201378 \u00b0C, the structural proposal is also in agreement with the DFT calculations that show a relatively high energy barrier of 20.3 kcal mol\u20131 for the reaction of [ scale\" fill=\"currentColor\" stroke=\"none\">Si\u2013O\u2013)WH3(Me)2] with the fourth molecule of hydrogen , are depicted with the selected characteristic NMR signals assigned 2] ; whereasH3(Me)2] and by sH3(Me)2] and 6a, assigned .4 when it is allowed to warm to room temperature. To provide further evidence for this chemical transformation, the evolution of 1 as a model compound was monitored by X-ray absorption spectroscopy (XAS) at room temperature with the sample in a stream of dry helium. As reported previously, microelemental analyses of 1 indicate that the tungsten pentamethyl moiety is anchored via only one isolated silanol group of the surface, maintaining its hexacoordination around the W center.The transformation of the well-defined monopodal species into a bipodal surface complex, accompanied by the transfer of methyl groups to the silicon atoms of the support, is invoked in the text as a major decomposition pathway for III-edge EXAFS scan of 1 in flowing helium strongly support the inference of two absorber\u2013backscatterer pairs, W\u2013C and W\u2013O, with internuclear distances consistent with typical sigma bonds. The W\u2013C and W\u2013O coordination numbers were found to be 4.9 and 1.0, respectively. Within the expected error (\u00b120%), these values are consistent with the initial supported species 1 being monopodal on the surface. Furthermore, there was no detectable W\u2013W contribution in the fit, indicating that, within error, the tungsten species remained mononuclear.The structural parameters determined by the best fit of the data of the first W L1 was present in the flow-through cell in the presence of helium (flow rate: 1.0 mL min\u20131), with continuous exposure to the X-ray beam, changes in the X-ray absorption near edge structure (XANES) region of the X-ray absorption spectrum show that the supported tungsten species was transformed.When The XANES region shows thE0 value determined in the best fit of the W\u2013C shell of species 1 after 45 min in contact with flowing helium , but the associated activation barrier for the direct conversion via transition state [XII\u2013XIV] is quite high to be accessible under the reaction conditions used. In contrast, the conversion of XVI to XIV is exergonic by only 2.3 kcal mol\u20131, and the associated free energy barrier for the direct conversion via transition state [XVI\u2013XIV] and the release of a H2 molecule amounts to just 22.3 kcal mol\u20131. This barrier can be overcome at room temperature, suggesting that the dominant route for the formation of XIV starts from XVI.Motivated by the structural rearrangements in the structure of XII is the predominant species at low temperatures according to our spectroscopic investigation and DFT calculations, species XVI can be formed by a less than stoichiometric conversion in the hydrogenolysis of XII to XVI, which we calculated scale\" fill=\"currentColor\" stroke=\"none\">Si\u2013O\u2013)W(Me)5] (1) has been investigated at various temperatures. The hydride products have been characterized by means of elemental microanalysis, IR and solid-state NMR spectroscopies revealing that hydrogenolysis is temperature dependent and leads to different structural features. The results demonstrate that the hydrogenolysis of [ scale\" fill=\"currentColor\" stroke=\"none\">Si\u2013O\u2013)W(Me)5] at \u201378 \u00b0C leads to an unprecedented silica-supported partially methylated tungsten hydride species. On the basis of DFT calculations, we propose that [ scale\" fill=\"currentColor\" stroke=\"none\">Si\u2013O\u2013)WH3(Me)2] is the predominant surface species at this temperature. The thus-synthesized tungsten hydride represents the precursor of the most active catalyst for propane metathesis yet reported. The thermal rearrangement of this partially methylated species, when allowed to warm to room temperature, has been observed to proceed via divergent pathways. This change involves methyl transfer to the silica surface, evidenced by the presence of PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019Si\u2013CH3 species indicated by solid-state NMR spectroscopy, transforming the initial monopodal surface complex 4 into a bipodal species. Such a change in podality was indicated by EXAFS spectra determined with 1 as a model substrate. Furthermore, the formation of a tungsten alkylidene (methylidene) hydride intermediate, which has always been considered in alkane metathesis but never been observed, has been proposed on the basis of solid-state NMR spectroscopy and DFT calculations.The controlled hydrogenolysis of silica-supported tungsten pentamethyl [(Supplementary informationClick here for additional data file."} +{"text": "A porous aromatic framework with mesopores was used as a platform for an immobilized Pd catalyst with superb catalytic activity and size selectivity for the Suzuki\u2013Miyaura coupling reaction. 2PAF70-NH) to immobilize a palladium (Pd)-based molecular catalyst has been developed. The resulting immobilized catalyst PAF70-Pd, in which the framework is entirely constructed by phenyl rings linked with stable carbon\u2013carbon bonds, has high structural rigidity and stability. Compared with the known porous organic material immobilized Pd-based catalysts, PAF70-Pd has the highest Pd content so far. Moreover, PAF70-Pd has extremely high catalytic activity with good size selectivity and very easy recyclability in catalyzing the Suzuki\u2013Miyaura coupling reaction. In the current system, the catalyst loading could be as low as 0.001 mol% and the TOF value could go up to 28\u2009800 h\u20131 which is far higher than those of the known porous organic material immobilized Pd-based catalysts. In order to elucidate the particularly high catalytic efficiency of PAF70-Pd, we prepared PAF1-Pd from 2PAF1-NH for comparison. PAF1-Pd has a higher Pd content than PAF70-Pd. However, due to the absence of large enough mesopores in 2PAF1-NH, PAF1-Pd has almost no catalytic activity under the same conditions, which definitely demonstrated that the intrinsic mesoporosity of 2PAF70-NH plays a crucial role in the superb catalytic efficiency of PAF70-Pd. This strategy to immobilize Pd-based molecular catalysts has very good expansibility to be applied in the immobilization of different organometallic catalysts into the pores of PAFs, which also has very high potential in the chemical and pharmaceutical industry.A strategy using a mesoporous amine-tagged porous aromatic framework ( The excellent catalytic activity of the organometallic catalysts has attracted intensive interest from chemists in diverse research fields. Thereinto, palladium (Pd)-based organometallic catalysts are versatile tools that can catalyze various organic reactions such as the Suzuki\u2013Miyaura coupling reaction and Heck reaction.etc. Owing to their robust structure together with high stability in most organic solvents, PAFs are extremely suitable platforms for the catalysis of organic reactions. It\u2019s worth noting that, due to the presence of the Pd center and organic ligand, Pd-based organometallic catalysts usually have relatively large sizes. Hence, immobilization of Pd-based molecular catalysts into the porous materials often needs a large enough pore size. Most of the reported porous organic material immobilized Pd-based catalysts always suffer from low Pd utilization efficiency which might be due to that the pore space after introduction of the Pd-based catalyst is too small to accommodate the catalytic reaction. Apparently, for application of PAFs as the platforms for Pd-based organometallic catalysts, PAFs with large enough mesopores are needed. However, the synthesis of narrowly distributed mesoporous PAFs is still a challenge because of the interpenetration while using large-size monomers. Thus using PAFs as the platforms for covalent anchoring of organometallic catalysts into the pores still remains rare up to now. In this paper, we will make an attempt in this area.Using porous materials such as metal\u2013organic frameworks (MOFs), covalent organic frameworks (COFs) or porous organic polymers (POPs) as supported materials began to appear in the last few decades, which is a good idea because of their porosity and high surface area.2PAF70-NH, an amine-tagged PAF with narrowly distributed mesopores which was recently reported by our group,2PAF70-NH was used for the synthesis of our desired material, and the catalytic performance of the desired material (PAF70-Pd) was systematically studied. In order to further demonstrate the importance of the mesopores in 2PAF70-NH, another amine-tagged PAF (2PAF1-NH) without mesopores was also used as a platform to immobilize the same Pd-based molecular catalyst, affording PAF1-Pd for comparison with PAF70-Pd.Considering the need for large enough pore space for accommodating Pd-based molecular catalysts and the subsequent catalysis, in this paper, 2.2.1via the pre-modification procedure used in our previous literature report,2PAF70-NH, which contains mesopores with 3.8 nm diameter and amine anchors in the pores. Then, via the amine anchors using a condensation reaction with picolinaldehyde, the chelating ligand unit for Pd was introduced into the material, yielding the PAF which was named PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CPyPAF70-N. After a second post-treatment of PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CPyPAF70-N with palladium acetate, the PAF material containing the Pd-based molecular catalyst was obtained, which was named PAF70-Pd. It\u2019s worth noting that the N,N-bidentate ligand is one of the most versatile coordination systems in organometallic catalysis, which can coordinate with various metal ions and has been widely used in homogeneous catalysis. In addition, the post-synthesis modification was very facile and efficient. The above features of our synthetic method could expand the application value of the PAF material. In addition, for the purpose of comparison, we prepared 2PAF1-NH according to the literature report,PAF1-Pd (the counterpart of PAF70-Pd) was prepared and the appearance of the characteristic peaks of the Schiff base (the new peaks at around 1600 cm\u20131) in the FT-IR spectrum of PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CPyPAF70-N indicated the formation of an imine bond and thus the successful construction of PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CPyPAF70-N. In comparison with PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CPyPAF70-N, in the FT-IR spectrum scale\" fill=\"currentColor\" stroke=\"none\">O stretching vibration and the new peaks at 1321 cm\u20131 could be attributed to the C\u2013O stretching vibration, which obviously indicated that PAF70-Pd was successfully obtained through our strategy. In addition, the successful synthesis of PAF1-Pd was also confirmed through a similar analysis of the FT-IR spectra scale\" fill=\"currentColor\" stroke=\"none\">CPyPAF70-N and PAF70-Pd all showed sharp uptakes, indicating the existence of micropores in the materials. It\u2019s worth noting that, in the desorption branch of 2PAF70-NH, a relatively sharp hysteresis demonstrated the presence of narrowly distributed mesopores. Compared with 2PAF70-NH, the corresponding hysteresis disappeared in the desorption branches of PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CPyPAF70-N and PAF70-Pd, which indicated the disappearance of the mesopores after post-modification of 2PAF70-NH. The apparent surface area calculated from the Brunauer\u2013Emmett\u2013Teller (BET) model was 599 m2 g\u20131 for 2PAF70-NH, 263 m2 g\u20131 for PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CPyPAF70-N, and 172 m2 g\u20131 for PAF70-Pd. Through the change of pore size distributions calculated by non-local density functional theory (NLDFT), it was clear that the mesopores with a pore width of 3.8 nm of 2PAF70-NH disappeared in PAF70-Pd and PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CPyPAF70-N (red curve) showed similar TGA curves. There is almost no weight loss before 300\u00b0C, which suggested the high thermal stability of 2PAF70-NH and PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CPyPAF70-N. At about 400\u00b0C, the framework decomposition started and when the temperature was above 500\u00b0C the decomposition became obvious. The 3.96 wt% residue for 2PAF70-NH and 2.15 wt% residue for PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CPyPAF70-N at 800\u00b0C could be ascribed to some palladium oxide residue which originated from the Pd catalysts in the preparation process of 2PAF70-NH. As shown in PAF70-Pd (blue curve) had a 56% weight loss at 277\u2013320\u00b0C. This weight loss could be attributed to the decomposition of both N,N-bidentate ligand and AcO\u2013 species which were directly connected to the Pd center. Compared with PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CPyPAF70-N, PAF70-Pd showed lower stability, which might be due to that the Pd species could catalyze the cleavage of carbon\u2013carbon bonds around the Pd centers in the PAF material.etc. The high thermal stability and chemical stability made PAF70-Pd fully satisfy the demands of catalysis. The TGA analysis of PAF1-Pd can be found in the ESI was performed to test the thermal stabilities of the above PAF materials. As shown in Fig. S17. The Pd PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CPyPAF70-N and PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CPyPAF1-N, X-ray photoelectron spectroscopy (XPS) was performed. As shown in 5/2 orbital, indicated that the Pd species in PAF70-Pd and PAF1-Pd are present in the +2 state. Compared with the BE of 338.55 eV for free Pd(OAc)2, the BE for Pd species in PAF70-Pd and PAF1-Pd negatively shifted by 0.75 eV. This negative shift indicated that Pd(OAc)2 has strong coordination with the N,N-bidentate ligand in PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CPyPAF70-N and PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CPyPAF1-N.In order to further investigate the incorporation of palladium within PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CPyPAF70-N, some evenly distributed black dots with a mean diameter of about 1 nm emerged in the TEM images of PAF70-Pd, indicating that the Pd species are uniformly dispersed in the frameworks of the PAF material, which was in accordance with the above analysis of the TGA curve of PAF70-Pd. Similarly, compared with PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CPyPAF1-N images obviously showed the successful introduction of Pd species into the PAF materials. As shown in vg>CPy , the TEM, EtOH gave the best results in terms of the reaction rate and yield of the current catalytic Suzuki\u2013Miyaura coupling reaction. Increasing the reaction temperature from 25\u00b0C to 80\u00b0C improved the reaction rate significantly scale\" fill=\"currentColor\" stroke=\"none\">CPyPAF70-N or the substituted aryl bromides with either an electron-withdrawing group or an electron-donating-group CHCH3, entries 6\u20138) afforded the cross-coupling products in excellent yields (up to >99%) with high turnover frequency (TOF) values , demonstrating the wide generality and functional tolerance of the current system.The catalytic performance of PAF70-Pd, some contrast tests were performed as shown in entries 9\u201311 of 9a, which could smoothly transform to 9b completely . These indicated that there is very low metal leaching during the reaction process. The results demonstrated that PAF70-Pd could undergo at least 3 cycles of the reaction without obvious loss of catalytic activity.For heterogeneous catalysts, recyclability is an important factor. Hence the recyclability of 2.4PAF70-Pd, PAF1-Pd and previously reported porous organic material immobilized Pd catalysts are given in PAF70-Pd and PAF1-Pd have the highest Pd contents, which might be due to the high effective surface areas of our materials. Moreover, PAF70-Pd showed far higher TOF values than other catalysts in PAF70-Pd gave a rare example of a catalytic system with size selectivity in this field , by a post-synthesis method, PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CPyPAF70-N with an N,N-bidentate ligand was successfully obtained. After a second post-treatment with palladium acetate, the PAF material containing the Pd-based molecular catalyst, named PAF70-Pd, was prepared. Because the narrowly distributed mesopores (3.8 nm) in 2PAF70-NH endow sufficient pore space for immobilizing the Pd-based molecular catalyst with a relatively large size, the resulting immobilized catalyst PAF70-Pd has evenly distributed Pd species and high Pd content (23.0 wt%). Furthermore, PAF70-Pd showed superb catalytic activity for catalyzing the Suzuki\u2013Miyaura coupling reaction with good size selectivity and very easy recyclability. Compared with the reported porous organic material immobilized Pd catalysts, PAF70-Pd has the highest Pd content and exhibits a far higher TOF value when catalyzing the same Suzuki\u2013Miyaura coupling reaction. By comparison with PAF1-Pd, it was clearly demonstrated that the mesopores in 2PAF70-NH are very important for the high activity of PAF70-Pd. After the introduction of Pd species with a relatively large size, PAF70-Pd still has large enough pore space to accommodate the catalyzed reaction. This could significantly enhance the utilization efficiency of the Pd catalyst in the material. Our strategy supplied a versatile method for the immobilization of different organometallic catalysts into the pores of PAFs, which will promote the development of PAF-based organometallic catalysts.Based on the mesoporous PAF (There are no conflicts to declare.Supplementary informationClick here for additional data file."} +{"text": "Strongly phosphorescent hetero-metallic [2]catenanes, including bimetallic scale\" fill=\"currentColor\" stroke=\"none\">C)12Au6M6 (M = Ag or Cu), scale\" fill=\"currentColor\" stroke=\"none\">C)12Au10Ag2 and trimetallic scale\" fill=\"currentColor\" stroke=\"none\">C)12Au6CunAgn6\u2013, were obtained. PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C\u2013 ligand; Dtbp = 3,5-di-tert-butylphenyl), Au\u2013Ag scale\" fill=\"currentColor\" stroke=\"none\">C\u2013 ligand), and Au\u2013Cu, Au\u2013Ag scale\" fill=\"currentColor\" stroke=\"none\">C\u2013 ligand; C6-Fluo = 9,9-dihexyl-9H-fluoren-2-yl) complexes as well as a trimetallic Au\u2013Ag\u2013Cu scale\" fill=\"currentColor\" stroke=\"none\">C\u2013 ligand) complex, which feature [2]catenane structures. The formation of the [2]catenane structure is significantly affected by the coinage metal ion(s) and change of the structure of the alkynyl ligand. These hetero-metallic [2]catenane structures are strongly luminescent with tunable emission \u03bbmax from 503 to 595 nm and \u03a6 values up to 0.83.Homo-metallic metal alkynyl complexes exhibit interesting catenane structures, but their hetero-metallic catenane counterparts are under-developed. In this work, we report rare examples of bimetallic Au\u2013Cu (DtbpC Various strategies, including \u03c0\u2013\u03c0 stacking, hydrogen bonding, metal templating, and hydrophobic interactions, have been developed to direct the assembly of interpenetrated structures.n n = 10,) which fI-alkynyl catenanes is the presence of two linear RC PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C\u2013Au\u2013C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CR units in the locking center. PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C\u2013M\u2013C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CR species commonly seen in the literature,I\u2013alkynyl system, PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C\u2013 , 9,9-dihexyl-9H-fluoren-2-yl (C6-Fluo), or tBu) to construct novel assemblies of hetero-metallic alkynyl complexes. Herein, we described the formation of five hetero-metallic alkynyl [2]catenanes molar ratio, and the trimetallic complexes 3 and 6 were prepared by mixing the homoleptic gold, silver and copper alkynyl complexes in a 2\u2009:\u20091\u2009:\u20091 molar ratio or by mixing the hetero-metallic Au\u2013Cu and Au\u2013Ag alkynyl complexes in a 1\u2009:\u20091 molar ratio. Complexes 1\u20133 were also accessible from the reactions of alkynes with Au(SMe2)Cl, AgOTf and/or [Cu(MeCN)4]PF6 (2\u2009:\u20091\u2009:\u20091 for 1 and 2 and 3\u2009:\u20091\u2009:\u20091\u2009:\u20091 for 3) in the presence of Et3N (yields: 27\u201378%). X-ray diffraction-quality crystals of 1\u20137 were obtained by the slow evaporation of the CH2Cl2/MeCN, chlorobenzene or toluene/MeCN solutions and their structures were determined by X-ray crystallography scale\" fill=\"currentColor\" stroke=\"none\">C)12Au6Cu6, has a crystallographic D2 symmetry and features two twisted scale\" fill=\"currentColor\" stroke=\"none\">C)6Au3Cu3 rings that are interlocked to form a [2]catenane structure scale\" fill=\"currentColor\" stroke=\"none\">C\u2013Au\u2013C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CDtbp units and three Cu ions with relatively weak Au\u2013Cu (2.9632(10) \u00c5) and Au\u2013Au (2.9625(3)\u20133.1497(8) \u00c5) interactions .1- and \u03b72-modes, respectively. The M\u2013C distances (Au\u2013C 1.979(7)\u20132.009(6) \u00c5 and Cu\u2013C 2.018(7)\u20132.285(14) \u00c5) are comparable to those in the homo-metallic [2]catenane [ scale\" fill=\"currentColor\" stroke=\"none\">CAu)6]2 (Au\u2013C 1.85(4)\u20132.26(3) \u00c5)1 as a hetero-metallic [2]catenane. In contrast, the PhC PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C\u2013 counterpart of 1, scale\" fill=\"currentColor\" stroke=\"none\">C)12Au6Cu6, adopts a non-catenane structure in which the six AuI ions are co-planar and the six CuI ions form a trigonal prism with the PhC PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C\u2013 ligands situated on the two opposite sides, rendering the Ph groups (for each side) rather close to each other1 in the assembly/stabilization of the [2]catenane structure of the Au6Cu6 alkynyl complex. It is probable that the non-catenane structure of scale\" fill=\"currentColor\" stroke=\"none\">C)12Au6Cu6, upon changing its alkynyl ligands to the bulkier and more basic DtbpC PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C\u2013 ligands, is destabilized owing to the increased steric hindrance [resulting from the bulkiness of the electron-donating tBu substituents and stronger metal-alkynyl binding (cf. Au\u2013C 2.004(6)\u20132.045(5) \u00c5 in scale\" fill=\"currentColor\" stroke=\"none\">C)12Au6Cu6 (vs. 1.979(7)\u20132.009(6) \u00c5 in 1)], and as such the steric hindrance is minimized in the [2]catenane structure of the Au\u2013Cu complex 1.The Au\u2013Cu complex tructure . Each riu 2.88 \u00c5 ), which u (2.9632 \u00c5) and A12Au6Cu6 i vs. 1.9 PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C\u2013 ligand unchanged, a non-catenane complex scale\" fill=\"currentColor\" stroke=\"none\">C)16Cu8Ag8 (2) was obtained. Complex 2 has a structure with an approximate S4 symmetry scale\" fill=\"currentColor\" stroke=\"none\">C)16Au8Ag8, also with a non-catenane structure.3, its structure scale\" fill=\"currentColor\" stroke=\"none\">C)16Au8Ag8 molecule PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C)16Au8Ag8, 3 features central Au\u2013Au distance (Au2\u2013Au5 2.9591(9)vs. Au3\u2013Au7 3.1825(4) \u00c5) and substantially larger C\u2013M\u2013C angles (139.4(5)\u2013142.6(4)\u00b0 11vs. 172.4(3)\u2013177.0(4)\u00b0).By changing Au\u2013Cu to Cu\u2013Ag, but with the same DtbpCtructure features PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C\u2013 from Dtbp to the bulkier C6-Fluo resulted in the formation of bimetallic scale\" fill=\"currentColor\" stroke=\"none\">C)12Au6Cu6 (4) and scale\" fill=\"currentColor\" stroke=\"none\">C)12Au6Ag6 (5) and trimetallic scale\" fill=\"currentColor\" stroke=\"none\">C)12Au6CunAgn6\u2013 (6), and all of the three complexes adopt a [2]catenane structure scale\" fill=\"currentColor\" stroke=\"none\">C\u2013Au\u2013C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CCR units in 4\u20136 is similar to that in 1; the connection of the C6-FluoC PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C\u2013Au\u2013C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CC6-Fluo units by \u03c0-C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C\u2013Cu/Ag coordination forms the [2]catenane structures. The Cu and Ag atoms in 6 are in substitutional disorder scale\" fill=\"currentColor\" stroke=\"none\">C\u2013M is partially occupied by Cu and Ag atoms, the occupancy of the Ag atom of the outlier positions 1 and 2 is slightly higher (4 and 5 used in the preparation of 6. The average Cu/Ag\u2013C(\u03b1) distance in 6 is 0.14 \u00c5 longer than the average Cu\u2013C(\u03b1) distance in 4 and 0.12 \u00c5 shorter than the average Ag\u2013C(\u03b1) distance in 5, while the difference of the average Au\u2013C(\u03b1) distances between 4\u20136 is <0.03 \u00c5.Changing the R group of RCtructure . The arrdisorder : each \u03c0-y higher , and the6 can be formed by mixing the [2]catenanes 4 and 5 in solution, we also mixed the previously reported [2]catenane [ scale\" fill=\"currentColor\" stroke=\"none\">CAu)6]2 scale\" fill=\"currentColor\" stroke=\"none\">CAg]n (molar ratio 5\u2009:\u20091), and obtained 7, adopting a [2]catenane structure similar to that of [ scale\" fill=\"currentColor\" stroke=\"none\">CAu)6]2 except for the replacement of one bis scale\" fill=\"currentColor\" stroke=\"none\">C) coordinated Au ion in each ring by one Ag ion (As [2]catenane CAu)6]2 with + scale\" fill=\"currentColor\" stroke=\"none\">C\u2013 signals were observed at room temperature , which were broadened into one set upon increasing the temperature to 353 K and were then recovered by cooling back to room temperature scale\" fill=\"currentColor\" stroke=\"none\">C\u2013 belong to a single complex (diffusion constant D = 8.32 \u00d7 10\u201310 m2 s\u20131), thus providing additional evidence for the purity of 1 in solution.We examined the solution behavior of the hetero-metallic [2]catenanes \u223c10\u20132 M, , three s1 as an example. The DFT-optimized geometry of 1 is comparable to that determined by X-ray crystal analysis. For example, the computed structure of 1 features average values of Au\u2013C 1.997 \u00c5, C\u2013Au\u2013C 176.2\u00b0, and Cu\u2013C 2.053 \u00c5; these values compare well with the corresponding ones in the crystal structure of 1 \u00c5, C\u2013Au\u2013C 176.9(8)\u00b0 and Cu\u2013C 2.097(7) \u00c5). To gain insight into why the [2]catenane of scale\" fill=\"currentColor\" stroke=\"none\">C)12Au6M6 was obtained for M = Cu (1) but not for M = Ag, we attempted to perform DFT optimization of scale\" fill=\"currentColor\" stroke=\"none\">C)12Au6Ag6 with a hypothetical similar [2]catenane structure, which did not converge. The highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) of 1 are depicted in z2 orbitals of the two Au atoms in the locking center of the [2]catenane structure, while the LUMO is distributed on the empty 6p orbitals of the same two Au atoms.DFT calculations were performed to examine the electronic details of the hetero-metallic [2]catenanes using 1\u20137 are emissive in the solid state (1 and 4\u20136 exhibit moderate yellow to strong orange emissions in the solid state (\u03a6 = 0.37\u20130.83). Changing the ligand from DtbpC PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C\u2013 to C6-FluoC PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C\u2013 resulted in a bathochromic shift in emission energy and a significant improvement in the quantum yield. Notably, the emission energy and efficiency show only minor variation with the metal compositions of 4\u20136 . The comparison of the emission spectra of scale\" fill=\"currentColor\" stroke=\"none\">C)12Au8Ag8 . The excitation of 7 in the solid state gave a strong green emission at \u03bbmax 503 nm with a tail up to 710 nm (\u03a6 = 0.82). The wide span in emission energy (\u03bbmax from 503 to 595 nm) and high solid state emission quantum yields highlight the prospect of hetero-metallic [2]catenanes based on a coinage metal alkynyl system as useful photo-functional molecular materials.Complexes id state . In view= 489 nm b) and 3 PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C\u2013 ligand to result in the assembly of hetero-bimetallic and hetero-trimetallic [2]catenanes scale\" fill=\"currentColor\" stroke=\"none\">C)12Au6M6 (M = Cu 4 and Ag 5) and scale\" fill=\"currentColor\" stroke=\"none\">C)12Au6CunAgn6\u2013 (6) is remarkable. As 4\u20136 are nearly isostructural, it appears that their C6-FluoC PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C\u2013 ligands dominate the intermolecular interactions, with the effect of the Ag and Cu ions being minor in these cases. As revealed by the crystal structures of 4 and 5, replacing the Cu ions with Ag ions slightly expands the metallacycle core owing to the longer Ag\u2013C than Cu\u2013C distances scale\" fill=\"currentColor\" stroke=\"none\">C\u2013 ligand with flexible C6-alkyl chains is likely to restrict such tendency. For the complexes of the DtbpC PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C\u2013 ligand, which is sterically less demanding and relatively rigid, the replacement of the Cu ions by Ag ions leads to a core enlargement from M12 (1) to M16 (3). On the other hand, an Ag ion, compared with a Cu ion, is a stronger Lewis acid and is inclined to form weak interactions with more alkynyl ligands scale\" fill=\"currentColor\" stroke=\"none\">C\u2013Ag interactions may distort the ring unit and then break the [2]catenane structure. Moreover, the preference of AuI for a linear two-coordinate configuration should also play an important role in the assembly of the hetero-metallic [2]catenanes in view of the core enlargement from M12 (1) to M16 (2), upon replacing the Au ions with Ag ions, and the presence of linear RC PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C\u2013AuI\u2013C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CR units in all of the [2]catenanes 1, 4\u20136, and 7.The use of the C6-FluoC PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C\u2013, C6-FluoC PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C\u2013, and tBuC PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C\u2013 ligands. The structure of the trimetallic [2]catenane 6 is analogous to its corresponding Au\u2013Ag bimetallic [2]catenanes with some of the Ag atoms replaced by Cu atoms; mixing the Au\u2013Cu and Au\u2013Ag complexes is a feasible and efficient method to prepare the trimetallic complex. The formation of [2]catenanes relies upon a delicate balance between various intermolecular interactions. The structural characterization of 1\u20137 provides useful insight into a better understanding of hetero-metallic coinage metal alkynyl complexes.We have prepared and structurally characterized five hetero-metallic [2]catenanes based on coinage metal alkynyl complexes, including bimetallic Au\u2013Cu and Au\u2013Ag complexes and a trimetallic Au\u2013Cu\u2013Ag complex, by employing bulky DtbpCThere are no conflicts to declare.Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "The metal centre remains a spectator through the peripheral mechanism investigated by a combined spectroscopic, crystallographic, and computational analysis. ii) catalyst through oxidative addition of a hydrosilane. Neither the hydrogen atom nor the silicon atom bound to the iron(ii) centre are subsequently transferred onto the carbonyl acceptor, instead remaining at the sterically inaccessible iron(ii) atom throughout the catalytic cycle. A series of labelling, crossover and competition experiments as well as the use of a silicon-stereogenic hydrosilane as a stereochemical probe suggest that the iron(ii) site is not directly involved in the hydrosilylation. Strikingly, it is the silyl ligand attached to the iron(ii) atom that acts as a Lewis acid for carbonyl activation in this catalysis. The whole catalytic process occurs on the periphery of the transition metal. Computation of the new peripheral as well as plausible alternative inner and outer sphere mechanisms support the experimental findings.Combined experimental and theoretical analysis of the carbonyl hydrosilylation catalysed by an iron(0) pincer complex reveals an unprecedented mechanism of action. The iron(0) complex is in fact a precatalyst that is converted into an iron( Iron-catalysed carbonyl hydrosilylation can be traced back to seminal reports by Brunnerinner sphereouter sphereinner sphere mechanisms with \u03c3-bond-metathesis-type Si\u2013H bond cleavage at an iron\u2013oxygen bondouter sphere mechanisms with iron acting as a Lewis acid,Mechanisms of transition-metal-catalysed hydrosilylations exhibit a wide variety of modes of activation.1 and 2 (ii) hydride in 1 was postulated to be relevant in the catalytic cycle.2 was, in turn, believed to be a precatalystinner nor outer sphere but on the periphery of the metal centre without its direct involvement.Driess and co-workers recently introduced silylenes as \u03c3-donor ligands in iron-based catalysis, and iron(0) complexes 1 and 2 were app2 (2.5 mol%) was found to catalyse the hydrosilylation of various acetophenones 3a\u20133i with silane 4a at elevated temperatures2N group in the para position (entry 2). The reaction was, however, sensitive toward steric hindrance. Substituents in the ortho position significantly lowered the yield (entries 7\u20139), and a 2,6-disubstituted substrate did not react . Benzophenone reacted readily while propiophenone and isobutyrophenone afforded 18 and 16% yield, respectively. Hydrosilylation of cyclopropyl substituted ketone proceeded efficientlyThe SiNSi pincer complex 2 and hydrosilanes 4a\u2013c suitable for X-ray diffraction analysis, and that confirmed the molecular structure deduced from the NMR analysis. The structure shows a distorted octahedral iron(ii) coordination environment. The hydride was located tilted toward one of the silylene donor arms deviated from the trans coordination to the Me3P ligand (P\u2013Fe\u2013H1 170.20(5)\u00b0), a situation similar to that of known iron(ii) hydride pincer complexes.t-Bu groups encage the iron hydride with a Si1\u2013Fe\u2013Si2 angle far from the linearity, 144.54(3)\u00b0.To gain insight into the mechanism, we investigated the reaction between iron(0) complex ii) complex 7 is the active catalyst, we measured the kinetic profiles for the hydrosilylation of 3a with hydrosilane 4a catalysed by 2 or 7a complex 7 as the active catalyst.To validate whether the thus formed iron( 2 or 7a . Conversii) hydrides 7 in hand, we had a closer look at the hydride transfer. Maintaining 7b and deuterium-labelled hydrosilane 4b-d1 in THF at 70 \u00b0C resulted in slow H/D exchange, visible both at the silicon and iron atoms (ii) hydride 7b, hydrosilane 4b-d1 (>95% deuteration grade), and ketone 3e was puzzling though yielded 8eb with little deuterium incorporation at 19% conversion (H/D = 90\u2009:\u200910). That ratio subsequently decreases to 78\u2009:\u200922 to reach equilibrium after 24 hours. These results reveal that even though the hydride at the silicon atom in 4 is exchanging with the iron-bound hydride in 7, hydride transfer to the carbonyl carbon atom of 3 most likely occurs from the hydrosilane 4 and not from complex 7.vide infra).With the iron hydride 7b indeed led to H/D scrambling. Conversely, no erosion of the enantiomeric purity was seen when subjecting enantiopure silyl ether (S)-8eb to the typical protocol -8eb suggests that the hydride transfer itself is irreversible, and a concerted mechanism involving frontside attack at the asymmetrically substituted carbon atom is needed to explain the hydrogen atom exchange between the catalyst and the product.The possible H/D exchange at the methine position of silyl ether g 8eb-d1 , top. Trvia a silylene-assisted concerted mechanism are energetically accessible under the reaction conditions.After the transition state, the silylene-bound hydrogen atom migrates back to the silicon and carbon atom, respectively. The activation barriers of the scrambling reactions 3P (6-d9) was observed did not lead to the formation of the silyl ether 8eb is significantly slower in the presence of ketone 3e. The formation 11 was also investigated computationally POCOP-pincer complex is thwarted by additional Me3P (6), indicating dissociation of one of the phosphine ligands as part of the catalytic cycle.3P to the reaction mixture, the reaction was unaffected catalyses the hydrosilylation of 3e with MePh2SiH (4c) with hardly any incorporation of the Me2PhSi moiety into the product; silyl ether 8ec rather than 8eb is formed almost exclusively (ii) complexes 7 are the actual catalysts, originating from oxidative addition of hydrosilanes 3 to the iron(0) complex 2; 2 is a precatalyst. During the crossover experiment no changes in the characteristic signals of complex 7b in the 1H and 31P NMR spectra were detected. However, when the assumed inability of 7b and 4c to exchange their silyl groups was examined with another control experiment were observed, indicating that the exchange (7b to 7c) is in fact a side product of the decomposition rather than simple scrambling of the silyl groups.As is to be expected from the above observations, the hydride complex lusively , right. 7b promoted the reaction between highly enantioenriched hydrosilane (SiS)-4d (e.r. > 95\u2009:\u20095) and ketone 3e but conversion was slow as expected from the data obtained with achiral triorganosilane 4b -8ed in 31% yield; diastereoselection was poor. The enantiomeric ratio of unreacted (SiS)-4d was found to be unaffected. Subsequent reductive cleavage of the Si\u2013O bond in (SiR)-8ed (known to proceed with stereoretention at silicon atomSiS)-4d with overall retention of the stereochemistry at the silicon atom (e.r. > 95\u2009:\u20095). Hence, the hydrosilylation step involves frontside attack at the silicon atom, and that makes a mechanism involving Lewis-acid activation of the hydrosilane unlikely.18With sufficient knowledge of the active catalyst, we decided to analyse the stereochemical course at the silicon atom of the reacting hydrosilane .18 Catalii) catalyst 7 is generated from the iron(0) precatalyst 2 by oxidative addition of hydrosilane 4 to the zero-valent iron atom (ii) centre in 7a (grey box), we did not locate any structure resulting from direct insertion of the ketone C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O group into the iron hydride 7a or the silylene ligands (not shown). Instead, we were able to find a minimum structure for the phosphine-dissociated complex cis-14a . The intermediate cis-14a readily coordinates THF forming the adduct 17a. This intermediate is however a resting state if not a \u201cmechanistic dead-end\u201d. Ketone coordination to the iron centre of cis-14a gives intermediate 19oa with activated carbonyl group. The catalytic cycle is closed by an outer sphere concerted hydrosilane addition \u202120oa to the ketone with an activation barrier of 33.7 kcal mol\u20131.Based on the combined findings, we propose that the active iron to afford the iron alkoxide 25oa. The silylated alcohol is released by an inner sphere silylation through \u202126oa with an energy barrier of 34.9 kcal mol\u20131. An alternative inner sphere mechanism could be a reductive elimination from the intermediate 25oa. However, the energy barrier for the transition state \u202127oa was found to be high, and the resulting iron(0) complex 28 is energetically disfavoured. Recoordination of phosphine 6 gives iron(0) complex 29 which oxidatively adds to a silane 4a to form 7a.Isomerisation of ficantly , right. outer nor inner sphere mechanisms give satisfactory fits to the experimental evidence. Hence, we looked for an adduct of 7a and 3o wherein the silyl group would act as a Lewis acid led to intermediate 31oa with a pentacoordinate silicon atom, and the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O double bond being significantly elongated compared to its equilibrium distance from 1.211 to 1.251 \u00c5, indicating activation.4a to the carbonyl group in 31oa, and the hydrosilylation event releases 8oa through transition state \u202132oa with retention at the silicon atom. In accordance with our labelling experiments (cf.\u20131). To further validate this, we conducted a competition experiment between electron-rich 3a and electron-deficient 3f is more positively polarised accelerating the hydride transfer, than that of donor-substituted 3a (X = OMe). The reactivity is also in agreement with the proposed KIE based on the control reactions with deuterated silane 4b-d1 (In addition to the high energy barriers, neither wis acid .8,18,20 ments cf., this iscient 3f . The parne 4b-d1 .i.e., on the periphery of the transition metal. Coincidentally, the mechanism becomes outer sphere at silicon.inner or outer sphere mechanisms do not apply to this unique catalyst.The value of the present study is that it demonstrates, to our knowledge for the first time, an unusual case where the transition metal of a catalyst complex is not directly involved in the catalytic process. Activation of both substrate and reagent as well as the bond-forming and -breaking events happen in the ligand sphere, Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "We have developed a novel AND logic based fluorescence probe for the simultaneous detection of ONOO\u2013 and GSH (GSH-PF3). \u2013 and GSH (GSH-PF3). The GSH-PF3 probe was synthesised over three steps starting from commercially available fluorescein. The probe was constructed by attaching the GSH reactive motif, 2,4-dinitrobenzenesulfonyl, to the previously reported boronate fluorescence based probe, PF3. GSH-PF3 produced only a small fluorescence response towards the addition of GSH or ONOO\u2013 separately. However, when the probe was exposed to both analytes, there was a significant (40-fold) fluorescence enhancement. GSH-PF3 demonstrated an excellent selectivity towards both GSH and ONOO\u2013. In cellular imaging experiments the probe was shown to be cell permeable with no \u2018turn-on\u2019 response observed for the addition of either GSH or ONOO\u2013 separately. However, in the presence of both analytes, a clear fluorescence response was observed in live cells. GSH-PF3 was further able to monitor the co-existence of metabolically produced ONOO\u2013 and GSH by exogenous stimulation.We have developed a novel AND logic based fluorescence probe for the simultaneous detection of ONOO GSH is the predominant form, existing at millimolar concentrations in most cells.\u2013 therefore acting as a cellular defence by serving as an ONOO\u2013 scavenger. Elevated levels of GSH are common in cells under oxidative stress and the susceptibility of a cell towards ONOO\u2013 largely depends on the concentration of intracellular GSH.Peroxynitrite and reduced by GSH (turn \u201coff\u201d). The fluorescence of the CyPSe probe is initially quenched by a photoinduced electron transfer (PET) process. The presence of ONOO\u2013 results in the oxidation of selenium to CyPSe PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O causing the fluorescence emission to be \u201cturned on\u201d. Then in the presence of biological thiols such as cysteine and GSH, the CyPSe PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O probe is reduced back to its non-fluorescent selenide form. A similar reversible NIR Tellurium-based fluorescence probe was later developed for monitoring the redox cycles between ONOO\u2013 and GSH. This probe was successfully applied for the visualisation of the redox cycles of ONOO\u2013 and GSH in live cells and animals.et al. are turn \u201con\u201d with ONOO\u2013 and \u201coff\u201d with GSH. As far as we are aware, there are currently no AND logic based fluorescence probes for GSH \u201cand\u201d ONOO\u2013.Currently, only a few reversible fluorescence based probes for the detection of ONOO\u2013 logic based fluorescence probe and H2O2.PF3 would produce a selective GSH-ONOO\u2013 AND logic based fluorescence probe, GSH-PF3 (GSH-PF3 was readily synthesised in three steps. Fluorescein was triflated using N-phenyl bis(trifluoromethanesulfonamide) to afford fluorescein mono-triflate in good yield. Suzuki\u2013Miyaura conditions were then carried out to provide fluorescein mono-boronate, PF3. The 2,4-dinitrobenzenesulfonyl unit was then attached to PF3 using 2,4-dinitrobenzenesulfonyl chloride, CH2Cl2 and NEt3 at 0 \u00b0C. Using these conditions GSH-PF3 was prepared in a reasonable yield of 52%.With this research, we aimed to develop a GSH \u201cand\u201d ONOOce probe . We iden GSH-PF3 . GSH-PF3GSH-PF3 in hand, fluorescence experiments for the detection of GSH and ONOO\u2013 were performed. As shown in GSH-PF3 was initially non-fluorescent and with the addition of ONOO\u2013 (10 \u03bcM), a small fluorescence increase was observed. However, incremental additions of GSH resulted in a much larger increase in fluorescence intensity was added to GSH-PF3, and the probe was incubated for 10 min. Remarkably, this only led to a small increase in fluorescence intensity and the subsequent additions of ONOO\u2013 resulted in a large fluorescence increase \u2013 see ESI Fig. S3.GSH-PF3 displayed an excellent selectivity against other amino acids including serine, methionine and lysine. The probe demonstrated excellent selectivity for ONOO\u2013 over many other ROS including H2O2 \u2013 or by addition of exogenous GSH. As shown below in \u2013 led to a small fluorescence response in cells. This observation is believed to be due to the presence of a low levels of endogenous biological thiols in the cells, resulting in the activation of the probe's fluorescence. As predicted, treatment of cells with both GSH and SIN-1 led to a clear fluorescence increase in RAW264.7 cells with GSH-PF3, in clear agreement with the analytical data obtained on the fluorimeter.The macrophage cell line \u2013 RAW264.7 \u2013 was used for cell imaging experiments. The cells were incubated with GSH-PF3 for monitoring the co-existence of metabolically produced ONOO\u2013 by lipopolysaccharide (LPS) simulation\u2013 in macrophage, which is a signature of inflammation, whereas caffeic acid (CA) is commonly used to treat inflammation through the augmentation of intracellular GSH. Consequently, treatment with LPS, followed by the addition of increasing CA (0\u2013100 \u03bcM) led to a gradual enhancement in the fluorescence intensity of GSH-PF3 led to the suppression of probe's fluorescence, which is believed to be due to the production of an excess of GSH, resulting in quenching of the ONOO\u2013 , since it is known that high levels of ONOOGSH-PF3 is an easy-to-prepare fluorescence based probe providing a platform for the development of other novel AND logic based fluorescence imaging probes for use in medical diagnostics. We are currently exploring the use of 6-amino/carboxyfluorescein, which we believe could provide the opportunity to attach additional fluorophores in order to develop ratiometric fluorescence sensorsIn summary, No conflicts of interest.Supplementary informationClick here for additional data file."} +{"text": "Thioamide substitution into macrocyclic peptides increases the conformational rigidity of the backbone resulting in enhanced biological activity and metabolic stability. PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019S group and the surrounding atoms are the major driving force in inducing the conformational restriction, resulting in well-defined structures of these cyclic peptides with static 3-D presentation of the pharmacophores. Utilizing this property of thioamides, we report the development of a superactive antagonist of pro-angiogenic \u03b1v\u03b23, \u03b1v\u03b25 and \u03b15\u03b21 integrins, which are responsible for cancer cell proliferation and survival. Using simple thio-scanning and spatial screening of a non-efficacious and conformationally flexible cyclic peptide, we could achieve a more than 105 fold enhancement in its efficacy in cellulo via a single O to S substitution. The developed peptide shows better efficacy in inhibiting the pro-angiogenic integrins than the drug candidate cilengitide, with a significantly enhanced serum half-life of 36 h compared to that of cilengitide (12 h). The long shelf-life, absence of non-specific toxicity and resistance to degradation of the thioamidated macrocyclic peptides in human serum suggest the promise of thioamides in markedly improving the affinity, efficacy and pharmacology of peptide macrocycles.We show that substituting a single atom, O to S (amide to thioamide), in a peptide bond results in global restriction of the conformational flexibility in peptide macrocycles with minimal perturbation of the parent conformation. The van der Waals interactions between the C The high-resolution structures of the thionated cyclic pentapeptides indicated the coexistence of \u03b2II\u2032-type and \u03b3-type turns. The \u03b3-type turn is in equilibrium between an open (indicated by the NOE between the Ala4HN\u2013Ala5HN protons) and a closed form with similar global conformations but different \u03c6 and \u03c8 values (ESId-Ala1\u2013Ala2 and the \u03b3-turn is centered about Ala4.3 resulted in a conformation with the \u03b2II\u2032-type turn centered about Ala2\u2013Ala3 and the \u03b3-turn centered about Ala5 (3).To initially determine the conformational impact of thioamide substitution in peptide macrocycles, we synthesized a regioisomeric library of monothionated analogs of the parent cyclic peptide cyclo(\u2013ype turn .14 The 1ation ESI. Utiliziation ESI. The int bonding ) as demo1.000000,.000000 salues ESI, where t1.000000,.000000 s PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O groups in the cyclic pentapeptide are at d-Ala1, Ala2 and Ala4, and upon thionation did not perturb the conformation of the parent peptide. However, thionation at the internally oriented C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O groups, in the case of Ala3 (3), completely changes the conformation of the peptide; this is also observed for Ala5 (5), albeit to a lesser extent. These results indicate that thioamide substitution at a site where the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O group does not engage in intramolecular hydrogen bonding can retain the conformation of the parent macrocyclic peptide. Thus, we note that thioamides can be valuable probes for identifying the hydrogen bond involved in stabilizing the conformation of a macrocycle.3 and wondered whether this was a result of the enhanced hydrogen bond (H-bond) donating properties of the thioamide, as noted by Kessler et al.1H chemical shifts. The upfield shift of the amide resonances in Ala3HN and Ala5HN indicate their involvement in intramolecular hydrogen bonding, as opposed to d-Ala1HN, Ala2HN, and Ala4HN in all the peptides except 3 Cept 3 ESI.2HN being involved in a \u03b3-turn with Ala5CO in 1,2 and 4 did not indicate a significant increase in the thioamide H-bond donation. This suggests that the increased thioamide H-bond donor propensity3 and 5, instead, the van der Waals interactions between S scale\" fill=\"currentColor\" stroke=\"none\">S bond) and the surrounding atoms are crucial in dictating the conformation of the peptide, as predicted from the computational studies of thioamides containing dipeptides by Lipton et al.3CO completely flips outward on thio substitution in 3, presumably due to the 1,2 and 1,3-diaxial strain between S and the flanking Ala3 and Ala4C\u03b2. However, owing to the flanking heterochiral residues, thio substitution of Ala5CO allows for a partial flip of the thioamide bond.It was interesting to note that despite the possibility of Ala13C spin-lattice relaxation time (T1) of the backbone C\u03b1 in these cyclic peptides, which provides qualitative information on the segmental motion of amino acids in peptides.i + 1 residue in the \u03b2-turn displayed the lowest T1 compared to the other residues in the same macrocycle indicating its segmental rigidity (T1 is not feasible due to differences in the concentration and viscosity of the samples).T1 of the i + 1 residue with the T1 of the alanine about which the flexible \u03b3-turn is centered structural rigidity in cyclic peptides.To understand whether the thioamide substitution results in reduced flexibility of the cyclic peptide backbone, we determined the rigidity (compari6),d-Phe\u2013Val and a \u03b3-turn about Gly as observed in 1. Interestingly, we noted that 6 displays two conformations in the ratio 1\u2009:\u20092 on the NMR time scale .t) (8) underwent spontaneous acid catalyzed degradation.t), c(KGDfVt) and c(NleGDfVt) (Nle: norleucine). It was surprising to note that all of these protected cyclic peptides underwent spontaneous acid catalyzed degradation as observed in 8 (ESI1\u20135) did not show any trace of thio to oxo conversion even after 24 months at 25 \u00b0C in DMSO solution, indicating the stability of the thiopeptides in an oxidizing environment.To validate our observation of enhanced structural rigidity in peptide macrocycles through thionation in aqueous solution , we resorted to a moderate affinity cyclic peptide integrin antagonist cyclo(RGDfV) scale\" fill=\"currentColor\" stroke=\"none\">S group and not the improved thioamide H-bonding ability.9, its temperature coefficient is \u20136.8 ppb K\u20131, indicating the absence of intramolecular hydrogen bonds, as opposed to d-PheHN (\u20132.4 ppb K\u20131) and ArgHN (\u20131.6 ppb K\u20131), which are involved in \u03b3-turns (11 (d-PheHN) is also solvent exposed (\u20135.0 ppb K\u20131) and was not involved in stabilizing the \u03b3-turn about Asp with GlyCO.To demonstrate the uniform behavior of the thioamides in dictating the conformation of the cyclic peptide, we analyzed the solution conformations of 7\u201311 . It was \u03b3-turns . LikewisN-methylation, which introduces severe conformational heterogeneity into macrocyclic peptides via cis\u2013trans isomerism ,cis\u2013trans rotational barrier of the thioamide bond.3 and 9), the structural rearrangement results in a parent-like conformation, but with an altered side chain identity. These two observations suggested that (i) thioamides are valuable tools to reduce the conformational entropy in peptide macrocycles and (ii) thioamide substitution is an ideal modification for spatial screeningWe were quite excited to observe that unlike N-methylated cyclic peptide, cilengitide (Cilen), unfortunately failed in phase III clinical trials against glioblastoma.24To validate our hypothesis and demonstrate the potential of thioamides in delivering selective and high-affinity ligands, we chose to target the cell-adhesion receptors, integrins.in vitro competitive solid phase binding assay.6, showed a moderate affinity towards the pro-angiogenic integrins due to the conformational flexibility, which does not allow the static presentation of the pharmacophores responsible for tight binding. On the contrary, the conformationally rigid thio analogs yielded very interesting results. 7, which can be directly compared with Cilen, where an N-methylated amide bond is substituted with a thioamide bond, displayed a higher IC50 than 6 against \u03b1v\u03b23. However, remarkably it showed a better IC50 than Cilen against \u03b1v\u03b25 and \u03b15\u03b21 in the in vitro assays with purified integrins.The inhibitory potency of the peptides to block the association between the extracellular matrix and the integrins overexpressed on cell-surfaces was determined using an in vitro data and determine the efficacy of the peptides against the pro-angiogenic integrins in a complex cellular milieu, we subjected the peptides to a competitive cell-adhesion assay . This suggests the high specificity and selectivity of the thioamidated analogs towards the pro-angiogenic integrins in the presence of complex cellular components, which can otherwise sequester the peptides, reducing their efficacy.To validate our on assay utilizin7 strongly competed with vitronectin (Vn) in inhibiting the adhesion of MDA-MB-231 cells, suggesting its strong affinity towards \u03b1v\u03b25. A relatively lower potency of 7 was observed when inhibiting U-87 MG cell-adhesion to Vn (Vn binds to \u03b1v\u03b23 and \u03b1v\u03b25),7 towards \u03b1v\u03b23. 11 was the other member of the thio-scan library that showed better affinity than 6 towards the pro-angiogenic integrins in vitro, which was also reflected by its efficacy in the cell-adhesion assay.We were quite excited to note that as detected in the solid phase assay, 9 with thionation at Arg, which led to a complete reshuffling of the residues about which both the turns in 9 were centered affinity and efficacy of 9b in comparison to 9, by simple alteration in the spatial positioning of the pharmacophores on the thioamide backbone. To emphasize the effect of thio substitution, we synthesized compound 12 on the NMR time scale, as observed in 6, whereas 9b showed a single conformation, further reinforcing the rigidifying role of thioamides in peptide macrocycles.The spatial screening of in 9a\u20139d , of whicg of 9b) , which s9 and the spatial screen analogs (9a\u20139c) were comparable, except for 9d, which did not undergo the conformational switch scale\" fill=\"currentColor\" stroke=\"none\">S group and the surrounding atoms are critical in dictating the conformation of thioamidated macrocycles.Interestingly, the backbone conformations of l switch . This isin vitro and in cellulo activity of the synthesized peptides between H\u03b5 of Phe and one of the carboxylate oxygens of (\u03b2)-Asp126 . . in vitr)-Asp126 . The C\u2013H11. However, the lower potency of 11 stems from the loss of some crucial interactions with the receptor mediated by the \u2013RGD\u2013 motif scale\" fill=\"currentColor\" stroke=\"none\">S group compared to the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O group.32The docked poses of all the other thioamidated peptides do not show such a C\u2013H\u00b7\u00b7\u00b7O interaction, except for motif ESI. Likewis9b and 7, which differ only in the chirality about the phenylalanine, show comparable affinity for \u03b1v\u03b25 and \u03b15\u03b21, but >103 fold difference in \u03b1v\u03b23 affinity. Docking of 7 revealed that this difference is due to the smaller distance between Arg and Asp C\u03b2 compared to Cilen and 9b, leading to the loss of end-on hydrogen bonding between Arg and (\u03b1)-Asp150 -Asp150 and the )-Asp150 . This re mode ESI. The comCilen was required to achieve the half maximal inhibitory concentration (IC50). This clearly suggested that incorporation of thioamides into macrocyclic peptides does not result in unspecific cytotoxicity even after 48 h, which is an important concern for the use of thioamides in lead design. We also noted that glioblastoma cells were more sensitive towards these antagonists than breast cancer cells, presumably due to the expression of all the pro-angiogenic integrins, despite the known resistance of U-87 MG cells against detachment-induced apoptosis.6 and 12 displayed minimal detachment-induced apoptosis in breast cancer cells compared to the potent thioanalogs.Since integrin antagonists are known to inhibit the proliferation of cancer cells,Cilen (N-methylated peptide) and the most potent thioanalogs in human serum ex vivo for 72 h (6 showed slightly better metabolic stability (t1/2: 9 h) than 12 (t1/2: 7 h) due to the presence of d-Phe. It is interesting to note the significant improvement in metabolic stability of all the thioamidated analogs throughout the course of the experiment as observed previously in linear thionated peptides.11 and 9b showed a significantly improved serum half-life (t1/2: 36 h each) compared to that of the clinical candidate Cilen (t1/2: 12 h). These data collectively suggest that thioamidation could have a stronger influence in improving the metabolic stability of macrocyclic peptides than d-amino acid incorporation.One major drawback to translating peptides as drugs is their inherent metabolic instability due to the action of endo- and exoproteases.for 72 h . As expe PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O to C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019S substitution in peptide macrocycles dramatically reduces the conformational flexibility while minimally perturbing the global conformation as observed in the model alanine, thio-scan and spatial screen integrin antagonists. Thus, thioamide incorporation leads to predictable changes in the conformation of the peptide backbone, unlike other amide bond modifications. Thioamide incorporation fine tunes the 3D-pharmacophore orientation, which qualifies thioamides as important bioisosteric mimics of peptide bonds.in vitro and in cellulo data against the pro-angiogenic integrins demonstrates the specificity of these thioamidated macrocyclic peptides against their endogenous target, which can be extended to other bioactive peptides as well. Thioamidated macrocyclic peptides are non-cytotoxic and, presumably due to their marked conformational rigidity, display enhanced stability against degradation by proteases,36In summary, we have demonstrated that macrocyclic peptides exhibit conformational flexibility in both apolar and polar environments (integrin antagonists), which tends to reduce their bioactivity.10, the conformational restriction imparted by the thioamidation of glycine is also predicted to stabilize the local conformation in the vicinity of the active site in methyl-coenzyme M reductase, leading to the increased half-life of the protein.i.e. conformational flexibility and metabolic instability, will accelerate the discovery of peptide-based drugs and antibacterials.in vivo and are currently being investigated.It is curious to note that few thioamide containing peptide natural products, such as thioviridamide, thioholgamides, and their related analogs,There are no conflicts of interest to declare.Supplementary informationClick here for additional data file."} +{"text": "Treatment of an anionic dimanganaborylene complex with cationic coinage metal complexes led to the coordination of the incoming metal and displacement of dimethylsulfide in the formation of hexametalladiborides. 2Mn}2B]\u2013) with coinage metal cations stabilized by a very weakly coordinating Lewis base (SMe2) led to the coordination of the incoming metal and subsequent displacement of dimethylsulfide in the formation of hexametalladiborides featuring planar four-membered M2B2 cores comparable to transition metal clusters constructed around four-membered rings composed solely of coinage metals. The analogies between compounds consisting of B2M2 units and M4 units speak to the often overlooked metalloid nature of boron. Treatment of one of these compounds (M = Cu) with a Lewis-basic metal fragment (Pt(PCy3)2) led to the formation of a tetrametallaboride featuring two manganese, one copper and one platinum atom, all bound to boron in a geometry not yet seen for this kind of compound. Computational examination suggests that this geometry is the result of d10\u2013d10 dispersion interactions between the copper and platinum fragments.Treatment of an anionic dimanganaborylene complex ([{Cp(CO) Dating from the early work of Lipscomb and fellow main group pioneers, the bonding arrangements behind the clustering of boron atoms into three dimensions has been methodically explored.The relationship between molecular clusters and solid state materials has drawn interest and engendered discussions.2Mn}2B]\u2013, 1). PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019B PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019Mn] unit of the borylene either in a position equidistant from the two Mn centers, in an arrangement held together by intermetallic bonds between the incoming metal and Mn, or spanning one of the B PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019Mn bonds, using the \u03c0-system as a side-on ligand with interactions roughly described by the Dewar\u2013Chatt\u2013Duncanson bonding model , 1,3-bis(4-tolyl)imidazol-2-ylidene (ITol), and 1--3,3,5,5-tetramethylpyrrolidin-2-ylidene (CAAC), had a direct influence on the bonding preferences of their accompanying coinage metals. Despite the observed differences, each of the three ligands explored in Which of these bonding geometries a complex took, was found to depend on the combination of the metal's identity and its stabilizing base. The magnitudes of both the \u03c3-basicity and \u03c0-acidity of the three ligands studied, tricyclohexylphosphine . As this chemical shift fell near the range established as normal for trimetallaborides (\u223c209\u2013216 ppm),To assess the effects of a highly \u03c3-acidic metal stabilized by a weak \u03c3-donor, we synthesized AuCl and CuCl stabilized by dimethyl sulfide (DMS) and reacted each with 1 . In both2 and 3 consist of planar M2B2 units with [Cp(CO)2Mn] fragments bridging each M\u2013B bond, alternating in positions above and below the M2B2 plane. This framework is reminiscent of a class of transition metal cluster complexes constructed around homometallic four-membered planar cores of naked coinage metal ions, with each M\u2013M bond similarly bridged by a transition metal fragment.162 and 3 can be directly compared to two such compounds, the Cu4[(CO)4Co]4 and Au4[Cp(CO)2Mo]4 clusters reported by Kl\u00fcfers and Braunstein, respectively and 2.8030(9) \u00c5. The Cu\u2013Cu bond length in 2 (2.4730(5) \u00c5) is substantially shorter than the edge Cu\u2013Cu bonds found in Kl\u00fcfer's complex (2.703(4)\u20132.731(4) \u00c5), and even shorter than bonds comprising various triangular arrangements of Cu atoms (\u223c2.58\u20132.67 \u00c5),Both ectively .16b,e Thi.e., open valence shells, Lewis acidity, lower electronegativity than hydrogen, etc.) have been noted, and comparisons have been drawn between the chemical behaviors of boron and metals, both in bulk materials and discrete molecules.3Fe3-core of a [FeBH2]3 compound reported by Walter ,The mimicry of the coinage metals by boron is interesting. Despite its position on the periodic table within the span occupied by elements commonly thought of as \u201cmetalloids\u201d, boron has most often been treated solely as a non-metal.2 and 3 are assumed to follow a pathway involving the initial formation of the triangular trimetallaboride complex (DMS1\u00b7M) followed by loss of DMS and dimerization . In contrast, the analogous release of an N-heterocyclic carbene ligand or simple phosphine from a the Au complex requires 33.2 kcal mol\u20131 (IMe) and 20.7 kcal mol\u20131 (PMe3), and 30.3 kcal mol\u20131 (IMe) and 18.5 kcal mol\u20131 (PMe3) from the Cu complex. As the \u0394G values for the dimerization of two liberated fragments in the formation of 2 and 3 are \u201323.9 and \u201327.8 kcal mol\u20131, respectively, the process of ligand-loss and subsequent dimerization is only downhill for the DMS-bound metals, explaining the fact that such dimerization has not been previously observed in reactions with either phosphine- or NHC-stabilized trimetallaborides. The treatment of 2 and 3 with PCy3 led to separation of each complex into two equivalents of PCy31\u00b7M, in line with the calculated thermochemistry. The computed energies of each compound are compiled in Table S1.The syntheses of rization . Dative 1 for Cu , leaving01\u00b7M complexes are themselves stronger ligands for open coordination sites on coinage metal cations than DMS, but weaker ligands than NHCs or phosphines. Such reactivity, of course, requires them to be amphoteric in a Lewis sense. In the case of Au, these fragments are in the proper orientation for direct dimerization via simple HOMO/LUMO interactions 2. The addition of two equivalents of Pt(PCy3)2 to a toluene solution of 2 led to the formation of a new product with an 11B NMR peak at 215 ppm. Crystallographic isolation indeed showed the splitting of 2, but instead of addition to the Lewis acidic Cu atom, Pt(PCy3)2 was found to have donated one phosphine to Cu and inserted along the adjacent Mn\u2013B bond, immediately next to Cu, yielding a tetrametallaboride , The easy separation of 4 along the plane formed by the B, Cu and Pt atoms (2. An abbreviated structure 2* (the ligand architecture has been removed from two of the four Mn atoms) is displayed with the line-of-sight along the Cu\u2013Cu\u2013B plane. The above- and below-plane positions of Mn1 and Mn2 in 4 mimic the positions of Mn1 and Mn2 in 2*, while Mn3 and Mn4 in 2* have been replaced by PCy3 ligands in 4. This observation lends support to the notion that B PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019Mn \u03c0-bonds, such as those in 1, may act as \u03c3-donating side-on ligands in their interactions with metals.A view of compound Pt atoms shows th1 coordinated to Pt(PCy3) and Au(ITol)+ has been reported, but in an altogether different geometry.5) was formed through the reaction of [{Pt(PCy3)}{Cp(CO)2Mn}2B]\u2013 (6)3\u2013CuCl and 6 again yielded 4 (eqn (3)). When the ligand on copper was changed for an NHC (ITol\u2013CuCl) the same reaction (eqn (4)) led to the formation of a new product with an 11B NMR peak at 226 ppm, close to the shift reported for 6 (224 ppm). Single crystal X-ray analysis confirmed the distorted planar geometry of the boride, with the platinum fragment situated symmetrically between the Mn atoms, and the Cu bridging one of the two B\u2013Mn bonds scale\" fill=\"currentColor\" stroke=\"none\">Mn \u03c0-bond and participates in back bonding with a \u03c0* orbital + fragment bridges the B\u2013Mn1 bond in an arrangement typical of Class A bonding, while on the other the Pt(PCy3) fragment adopts a Class B geometry, bending the [Mn\u2013B\u2013Mn] backbone (168.2(3)\u00b0) to maximize intermetallic bonding of both Mn1 and Mn2 with Pt. As was found typical of Class A bonding, the Cu-bridged Mn1\u2013B bond (1.970(5) \u00c5) was elongated with respect to the Mn2\u2013B bond (1.907(5) \u00c5). As typical for Class B bonding, the Mn1\u2013Pt (2.707(1) \u00c5) and Mn2\u2013Pt (2.731(1) \u00c5) bonds were similar, and both shorter than the sum of the covalent radii of Mn and Pt (2.75 \u00c5) as determined by Cordero.As mentioned, we recently described two different bonding motifs assumed by d orbital , Class An number , Class B3) fragment as Lewis acidic is at odds with previous descriptions of the behavior of this fragment in similar trimetallaborides. Calculations on a compound composed of a Pt(PCy3) fragment complexed to a dimetallaborylene backbone consisting of a boron atom between iron and platinum (3) as a Lewis base stabilizing an electrophilic boron.7 are both shorter than the sum of covalent radii, the Pt\u2013Pt distance in the complex with the [Fe\u2013B\u2013Pt] backbone is roughly 3.3 \u00c5, significantly longer than the 2.72 \u00c5 sum of covalent radii, indicating little metallophilic interaction between the incoming Pt(Cy3) fragment and the Pt in the borylene backbone. Subsequent reports from our group used this description of Pt(Cy3) as a base in conjunction with the [Mn\u2013B\u2013Mn] backbone;This description of the Pt, the cve is tabulated as 98. If instead the four lighter metals (Mn) use 9 AO and two heavier metals (Cu and Au) use 8 AO, the cve counts become 94. Cluster 4 is unique in that it possesses 60 cve; however, if the same approach is applied as for 2 and 3, it yields 66 cve, in the case where all metals use 9 AO, and 62 when Mn uses 9 and Pt/Cu use 8 AO. The extra electrons are perhaps more localized on the metal centers and thus are not involved in skeletal bonding. It is common for heavier transition metals, e.g. Pt and Au, to form complexes with either 16 or 14 valence electrons,2\u20134.The comparison made in 4 and 7 are perplexing. It is perhaps attractive to view the systems as examples of four-coordinate boron exiting in both distorted planar (7) and distorted tetrahedral (4) geometries; however, the distortions from ideal tetrahedral geometry in 4 are rather large, with angles as large as 148\u00b0 and as small as 70\u00b0. Another possible explanation of the observed differences stems from the possibility of metallophilic interactions between the Cu and Pt in 4, and a lack of these interactions in 7. Closed shell, d10\u2013d10 interactions involving these two nuclei are rare in comparison to examples involving gold;i) interaction has been suggested as possible by a combined HF-DFT-MP2 study of simple model compounds.4 (2.668(3) \u00c5) is slightly less than the sum of covalent radii of the two metals (2.68 \u00c5),7, featuring identical metals, seemingly contradicts this argument. Optimization of 4 using the OLYP functional failed to accurately reproduce the experimental Pt\u2013Cu bond length, instead giving a much longer distance (2.83 \u00c5). As it is well known that standard DFT methods do not adequately treat dispersion interactions,3)+ fragment in 4 was replaced by a Cu(IMe)+ fragment (IMe4)4 than a compound with an NHC-stabilized Cu atom.The differences between 2 dimers as compared to a range of other ligands, including phosphines.2 dimers, these Au\u00b7\u00b7\u00b7L interactions were found to be the strongest when L = NHC, but only in conformations where the planes of the NHCs were parallel to one another. In other conformations, the stabilizing interaction is far smaller, falling below the computed strength of the Au\u00b7\u00b7\u00b7PH3 interaction in the [PH3\u2013AuCl]2 dimer. Calculations at the MP2 level have indicated that in the case of a [PH3\u2013CuCl]2 dimer the Cu\u00b7\u00b7\u00b7PH3 contribution to dispersion is the dominant term.IMe4, which perhaps limits the strength of the dispersion forces. Both the [Cu(PCy3)]+ and [Cu(ITol)]+ fragments are bulky, and from a strictly steric standpoint would favor inhabiting opposite sides of the molecule from the likewise bulky [Pt(PCy3)] fragment (the distorted square planar arrangement); however, the significant dispersion forces calculated for 4 may play the deciding role in the compound's observed geometry.The influence of stabilizing ligands on the dispersion forces between closed-shell metal ions is a matter of open debate. In comparing model L\u2013AuCl systems, Pyykk and coworkers found NHCs to promote the strongest dispersion forces in [L\u2013AuCl]4 is the seeming lack of them in 2 and 3, especially when considering the rather short Cu\u00b7\u00b7\u00b7Cu and Au\u00b7\u00b7\u00b7Au distances. The optimized geometries for both of these compounds give M\u00b7\u00b7\u00b7M lengths of 2.48 and 2.82 \u00c5 for 2 and 3, respectively, which are slightly greater than, but still in the range of, their experimental values (2.4730(5) \u00c5, 2; 2.803(1) \u00c5, 3). The application of Grimme's dispersion correction changed the optimized bond distances only slightly . Though confirmation of this through ab initio methods is still needed, these findings suggest that the geometries of 2 and 3 are dictated by covalent bonding in the Mn4M2B2 core rather than by closed-shell dispersion interactions.Perhaps even more interesting than the presence of strong dispersion forces in 3)2, giving structures that seem to rely on dispersion-type d10\u2013d10 interactions for their shape.The use of coinage metal ions with easily-displaced ligands provided a route to expanded metallaboride compounds containing six metal atoms. Systems such as these may find use in the future as mimics for the surfaces of boron-containing bulk materials. In these compounds the metalloid nature of boron is on display. These hexametalladiborides were split into tetrametallaborides through treatment with Pt(PCyElucidation of the transition from boron-rich metallaboranes to metal-heavy transition metal borides may well lead to the discovery of bulk materials with many possible applications. Knowledge regarding the systematic syntheses of compounds within this continuum, such as the findings described here, are important tools in these efforts.Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "Utilization of tellurite anion as template, led to the formation of unprecedented oxothiometalate based building blocks and the spontaneous manifestation of chirality. 2S2O2(H2O)6]2+ and a tellurite anion led to the formation of three new clusters, 1\u20133, with unique structural features. The tellurite anion not only templated the formation of [(Mo2O2S2)4(TeO3)(OH)9]3\u20131 and [(Mo2O2S2)12(TeO3)4(TeO4)2 (OH)18]10\u20133, but also the in situ generation of two different types of dimeric {Te2O6} based moieties induced the spontaneous assembly of the chiral [(Mo2O2S2)10(TeO3)(Te2O6)2(OH)18]8\u2013 anionic cluster, 2.Utilization of [Mo Polyoxometalates (POMs) are metal oxide-based molecular clusters that have attracted a lot of attention in the last three decades due to their structural versatility42\u2013 or WO42\u2013),2O2S2]2+ which forms the basis of the work reported herein.The principal factor that distinguishes POTMs from conventional POMs is the unique set of building blocks from which these compounds are derived. Whereas conventional POMs are synthesised in most cases directly from mononuclear metal oxide anions 32\u2013) or tellurite (TeO32\u2013) anions30In the absence of any other structure directing agents, the structures formed by the condensation of the [Mo2O2S2]2+ cationic dimers and form intricate architectures, it has been the only inorganic species observed to template the formation of building blocks on their own. In an effort to expand and diversify the existing building block library, it was hypothesised that the larger atomic radius of the tellurite anion, TeO32\u2013, its tendency for aggregation and difference in redox properties32\u2013 anions could influence the assembly process and potentially trigger redox processes which could lead to the generation of new building block libraries and facilitate the formation of new species.While it has been demonstrated that the selenite anion can template constructively [Mo3[(Mo2O2S2)4(TeO3)(OH)9]\u00b720H2O (1), K8[(Mo2O2S2)10(TeO3) (Te2O6)2(OH)18]\u00b745H2O (2) and (C4H12N)K9[(Mo2O2S2)12(TeO3)4 (TeO4)2(OH)18]\u00b748H2O (3) . Interes2O2S2]2+ solution gave rise to a series of new building blocks \u20131.921(9) and 1.831(8)\u20131.865(7) \u00c5; Mo\u2013Mo bond lengths within the [Mo2O2S2]2+ unit are found to be 2.831(1)\u20132.866(1) and 2.807(1)\u20132.848(1) \u00c5; Mo\u2013S bond lengths vary from 2.231(3)\u20132.360(3) and 2.306(3)\u20132.344(3) \u00c5; and Mo PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O bond lengths vary from 1.664(8)\u20131.695(7) and 1.666(9)\u20131.693(8) \u00c5, respectively. Finally, the Mo\u2013O(Te) distances are found to be 2.142(7)\u20132.499(8) and 2.198(7)\u20132.373(7) \u00c5 while the distance between the Mo centres and the hydroxo bridges Mo\u2013O(H) spans the ranges 2.056(7)\u20132.171(7) and 2.100(8)\u20132.149(8) \u00c5.The presence of the tellurite anion in the [Mog blocks . The bui2O2S2]2+ dimer units gave rise to the formation of two fundamentally new building blocks, [(Mo2O2S2)3(Te2O6)(OH)8]4\u2013C and D. The discovery of these two new building blocks was possible due to the ability of the tellurite anion to aggregate into small clusters which can then template further the formation of [Mo2O2S2]2+ building blocks. This chemical behaviour has been observed before where small multi-nuclear tellurite fragments were trapped by vanadium-based polyoxometalate fragments as reported by Norquist et al., although organic species have been known to trap these types of moieties as well.C and D building blocks might look similar, there is a crucial difference of the connectivity between the tellurium atoms. In C, the tellurium atoms are linked through two bridging oxygen atoms with varying Te\u2013O bond lengths, 1.924(6)\u20132.270(7) \u00c5 while building block D exhibits only a single bridging oxygen atom between the two tellurium atoms with the relevant Te\u2013O bond lengths falling within the range of 1.872(6) and 2.220(6) \u00c5. The difference in connectivity which is reflected in the bond distances, and causes significant distortion in the \u201copen-ring\u201d structure and asymmetry in the available coordination sites is proven to be crucial during the assembly process, which will be discussed below.Interestingly, the interaction of tellurite anions with the 4\u2013 anion. It is important to note here that the observed change in coordination mode is not associated with a change in its oxidation state as shown by bond valence sum (BVS) calculations. The [TeIVO4]4\u2013 template adopts a \u201csee-saw\u201d configuration; the two \u201carm\u201d oxygen atoms display an angle of 163.2(3)\u00b0 while the \u201cpivot\u201d oxygen atoms display an angle of 98.6(4)\u00b0. The \u201carm\u201d oxygen atoms are coordinated to one Mo-centre each, where one of the \u201cpivot\u201d oxygen atoms is coordinated to two and the other one is free. In this case, the Te\u2013O bonds appeared to be elongated as expected; the Te\u2013O(arm) bond lengths fall in the range 2.034(9)\u20132.042(9) \u00c5, while the Te\u2013O(pivot) (coordinated) and Te\u2013O(pivot) (uncoordinated) bonds were found to be 1.864(9) and 1.940(10) \u00c5, respectively. Finally, the rest of the bond distances appeared to be in agreement with the previously discussed A\u2013D building blocks; the Mo\u2013Mo, Mo\u2013S and Mo PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O distances were found to be 2.819(1)\u20132.826(1), 2.315(3)\u20132.334(3) and 1.683(9)\u20131.701(9) \u00c5, respectively. Finally, the bonding distances between the oxygen of the template and the Mo centres range from 2.142(8)\u20132.155(9) \u00c5 [Te\u2013O(arm)] and 2.290(9)\u20132.297(9) \u00c5 [Te\u2013O(pivot)].Finally, building block A\u2013E building blocks in the reaction mixtures became evident also during the course of the electrospray ionization mass spectrometry (ESI-MS) studies in solution. It was possible to identify not only the intact 1\u20133 species but also their building blocks generated by partial fragmentation of the clusters into their most stable components and 60.45\u00b0 to the B 12(TeO3)4(TeO4)2(OH)18]10\u2013, is the largest member of this family of clusters and is constructed from four type A and two type C building blocks. These building blocks form a large ring and are arranged in an alternating fashion, with each one rotated 180\u00b0 relative to the two on either side of it. This gives rise to a structure reminiscent of the chair-conformer of cyclohexane. The central cavity of the molecule is 8.97 \u00c5 in diameter, with all lone pairs on the Te atoms pointing towards the centre of the cavity. Crystallographic studies revealed the presence of disordered electron density within the cavity attributable to the presence of potassium cations. The ring shaped topology in combination with the directionality of the tellurium based lone pair electrons makes a cluster framework that resembles an inorganic \u201ccrown ether\u201d topology with the ability to bind different cations in its central cavity.Compound 3 are considered relatively symmetric, the overall structure exhibits less symmetry elements than excepted due to the relevant orientation between the two C-type and four A-type building blocks. Thus, the point group of cluster 3 is lowered to CS, which exhibits only a reflection plane and involves the Te-atoms of the two C-type building blocks. Very subtle and localised changes in coordination geometry and orientation of the building blocks drastically reduced the symmetry of the entire molecule. It is worth noting at this point a few synthesis considerations which influence the formation of 1\u20133. In each case, the reaction takes place under the same conditions. However, there is a narrow range of pH values (6.8\u20137.8) which distinguishes the different assembly processes 3(Te2O6)(OH)8} C and {(Mo2O2S2)3(Te2O6)(OH)8} D. Interestingly, the ability of the tellurite anion to aggregate into small clusters, {Te2O6}, not only templated the formation but also induced asymmetry to the discovered building blocks and influenced further their connectivity and assembly. This resulted finally in the spontaneous assembly of the second example2, and dramatically lowered the overall symmetry to CS for 1 and 3. In future work, we will attempt to generate new templates which will give rise to new building blocks and will also extend our symmetry breaking approach to gain access to new POTM structural features, nuclearities and symmetries.In conclusion, we have discovered and characterised the first members of a new family of POTM clusters which are constructed using [MoThe authors declare no conflicts of interest.Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "The Cr-catalyzed [aziridine + CO2] coupling to form oxazolidinone was found to exhibit excellent selectivity for the 5-substituted oxazolidinone product in the absence of any cocatalyst. 2] coupling to form oxazolidinone was found to exhibit excellent selectivity for the 5-substituted oxazolidinone product in the absence of any cocatalyst. Quantum mechanical calculations suggest that the preferential opening of the substituted C\u2013N bond of the aziridine over the unsubstituted C\u2013N bond is a key factor for this selectivity, a result that is supported by experiment with several phenyl-substituted aziridines. In the presence of external nucleophile such as dimethyl aminopyridine (DMAP), the reaction changes pathway and the ring-opening process is regulated by the steric demand of the nucleophile.The Cr-catalyzed [aziridine + CO Traditional oxazolidinone syntheses often rely on the use of phosgene and reactive derivatives of carbonic acid, which is not atom-economical and can limit the scope of their utility.2 is an attractive alternative that can exploit the easy accessibility of a broad range of substituted aziridines and CO2.2.Oxazolidinones constitute an important class of organic molecules with significant biological relevance,2 into 4-substituted oxazolidinone or an unselective mixture of 5- and 4-substituted oxazolidinones. We also reported the use of [CrIIICl + DMAP] catalyst in the facile conversion of a range of aziridines into 5-substituted and 4-substituted oxazolidinones with selectivity up to 20\u2009:\u20091 favoring the 5-substituted isomer (eqn (1)).2, the observed selectivity was only moderately in favor of 4-substituted oxazolidinone, consistent with the opening of the aziridine ring at the less substituted position.IIICl + DMAP] catalyst system is quite unique and we proposed that this is a consequence of a Lewis-acid activation that favored ring-opening at the carbon stabilized by the aryl substituent.30During the past decade, a handful of catalysts\u2014including DMAP,IIICl was even more selective for reaction 1 in the absence of DMAP cocatalyst: the conversion of N-propyl-2-phenylaziridine to the corresponding 5-substituted oxazolidinone proceeds with a selectivity of 40\u2009:\u20091, albeit with a slightly slower rate than that for the DMAP-cocatalyzed reaction.N-propyl-2-(p-methoxyphenyl)aziridine (see below). These data are in stark contrast to the analogous [epoxide + CO2] coupling2 to the CrIII center CrIII(aziridiniumcarbamate) intermediate (3). The CO2-derived oxygen nucleophile of the carbamate moiety can then intramolecularly ring-open the tethered aziridine substrate. The uniqueness of this mechanism lies in the key presence of the CO2-coordinated intermediate 2 and the ability of the carbamate oxygen nucleophile to regulate the oxazolidinone selectivity by preferentially opening one of the two available C\u2013N bonds in an intramolecular fashion, depending on the ability of the substituents at the aziridine C2 to stabilize the developing cationic charges.Interestingly, CrI center . This ac2 can weakly bind to the highly Lewis-acidic CrIIICl center and cause a slight polarization of the electron density in the coordinated C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O bond. This results in an increase in the electrophilicity of the CO2 carbon and renders it susceptible to a nucleophilic attack by the phenyl aziridine substrate to form intermediate 3 CrIIICl center results in a polarization of the coordinated C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O bond, which slightly elongates (1.17 \u00c5) over the other C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O bond (1.16 \u00c5).2 carbon -fitted charge increases to 0.73 from 0.69), rendering it easier to undergo attack by the phenyl aziridine substrate. Our quantum mechanical calculations suggest that this event is favored enthalpically by 7.5 kcal mol\u20131, but is canceled out by the translational entropic penalty to afford a solvation-corrected Gibbs free energy of \u20130.1 kcal mol\u20131, measured from the initial lowest-energy reference state of the system .As mentioned above, the binding of CO2 by CrIIICl, as shown in 2 employed in our experiments, it can be inhibited by the direct binding of the Lewis-basic aziridine substrate to the Cr center. Such coordination can competitively retard the rate of the catalytic cycle, especially in the absence of a cocatalyst that can ring-open the coordinated substrate. Indeed, our calculations reveal that while the Lewis acid\u2013Lewis base interaction between CrIIICl and the aziridine substrate does significantly activate the aziridine ring CrIIICl-coordinated alkoxide intermediate 3 for this process can be located at an energy of 14.6 kcal mol\u20131. Consistent with such a nucleophilic attack, the linear CO2 becomes significantly bent to 157.8\u00b0, with noticeable elongations of both \u201cC PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O\u201d bonds (\u223c0.03 \u00c5). The formation of intermediate 3 is energetically uphill by 8.7 kcal mol\u20131 from the initial lowest-energy state. At this intermediate stage, the coordinated aziridine retains its three-membered ring structure despite significant elongations of both substituted and unsubstituted C\u2013N bonds compared to the unsubstituted N\u2013C3 bond (to 1.48 \u00c5 from 1.45 \u00c5). This differential elongation is larger than the bond lengths change when aziridine binds directly to the CrIII center CrIIICl center and the aziridine substrate, does not require the opening of a new coordination site from distorting the salen ligand, as proposed by Luinstra and coworkers for the coupling of CO2 and epoxide.The nucleophilic attack of the aziridine substrate on the activated COediate 3 , where C\u2013N bonds . Notably2 bond in the aziridine-attacked intermediate 3 should logically lead to a selective formation of the 5-oxazolidinone product. To verify whether the observed selectivity of reaction 1 has a thermodynamic component, we evaluated the difference in ground-state energies of nine pairs of 5- and 4-aryl-N-propyl oxazolidinones comprising a broad range of para (p)-substituted phenyl groups. The apparent insensitivity of this difference to electronic changes in the aryl substituent . Since this process is completely intramolecular, the carbon with higher partial positive character is more likely to undergo nucleophilic attack. Thus, the presence of electron-donating (or -withdrawing) p-substituents on the aziridine phenyl rings can be expected to greatly influence this process via stabilization (or destabilization) of the developing carbocationic charge at the C2 center. To verify this, we evaluated the differences in charges between C2 and C3 centers for five different analogs of 3 where the phenyl groups of the coordinated aziridine rings possess p-substituents ranging from electron-donating to -withdrawing , effectively stabilizing the developing positive charge of the \u201cincipient carbocation\u201d. Indeed, the corresponding molecular orbital picture of major3-TS for the C2\u2013phenyl bond, to 1.29 from 1.02, that is considerably higher than that of a single bond (\u223c1.00). Upon free-geometry optimization, the located major3-TS easily leads to intermediate 4, indicating a direct connection between it and the 5-substituted oxazolidinone product.Having verified that ring-opening is favored at the Cediate 3 , we locaaction 1 , the rin3 are equally efficient for the subsequent nucleophilic aziridine ring-opening. The alternative ring-opening transition state major3-TS\u2032, where O4 is the nucleophile, is electronically only 1.45 kcal mol\u20131 higher in energy than major3-TS and has essentially the same solvation-corrected free energy for this reaction, the amount of free chloride ion CrIIICl catalyst) that can promote the ring-opening of any activated aziridine through a \u201cbimolecular\u201d mechanism would be negligible. The low level of chloride can be attributed to a combination of low catalyst loading (\u22641 mol%) and the strong bond between the anionic chloride ligand and the cationic Cr(III) center: our calculations suggest that the dissociation of chloride ion from CrIIICl would cost a sizable energy penalty of 26.5 kcal mol\u20131. The catalyst, CrIIICl itself, being a very poor nucleophile, also cannot open the activated aziridine ring in a bimetallic reaction mode in this asynchronous TS are quite similar. In the gas phase, the electronic energy of minor3-TS was \u223c7.3 kcal mol\u20131 higher than that of major3-TS, consistent with the disfavored formation for the 4-substituted oxazolidinone that was experimentally observed.\u20131) is higher than our expectation, it reproduces well the trend in favor of the major product.Interestingly, transition-state searches for the path that leads to the 4-substituted oxazolidinone minor product revealed an minor3-TS starts with rot3, a rotamer of intermediate 3 where proper alignment of the respective interacting groups have been attained scale\" fill=\"currentColor\" stroke=\"none\">O bond is elongated considerably (from 1.21 to 1.24 \u00c5), a direct consequence of its rehybridization into a nucleophilic C\u2013O moiety. This change can be quantified by the change in the Mayer\u2013Mulliken BO for the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O group, which is reduced to 1.56 in minor3-TS from an initial value of 1.81 in rot3. Upon closer inspection, it becomes evident that the unstabilized incipient carbocation at C3 is so electron-deficient that the phenyl group on the adjacent C2 carbon is taking part in anchimeric assistance. As in the case of major3-TS, minor3-TS also adopts an envelope structure that is characteristic of five membered rings. Given the high energy of minor3-TS, we surmise that the small amount of 4-substituted oxazolidinone minor product observed under our experimental condition does not arise from the intramolecular opening of the aziridine ring by the carbonyl functionality of 3. Instead, an alternative pathway may be operative where a CrIII-bound aziridine is opened by another aziridine molecule, in a manner similar to DMAP in the mechanism shown in 2 and the minor product forms via ring-closing (see discussion below).It is noteworthy that the pathway leading to attained . The req3 that eventually leads to the enhanced formation of 5. Such differences in charge development should be apparent through product selectivity in a Hammett-type investigation. To this end, we examined the CrIIICl-catalyzed coupling of CO2 with several p-substituted N-propyl-2-arylaziridines in the competitive presence of the parent N-propyl-2-phenylaziridine and in the absence of DMAP (eqn (2)). Notably, the experimental selectivity for the 5-substituted isomer was found to vary over almost an order of magnitude, with as high as 80\u2009:\u20091 for N-propyl-2-(p-methoxyphenyl)aziridine and as low as 12\u2009:\u20091 for N-propyl-2-(p-bromophenyl)aziridine.Thus far, our theoretical analyses predict a differential activation of the two carbons on the activated aziridine ring in intermediate \u03c3p+ values, the ratio of oxazolidinone products (5-substituted/4-substituted) obtained from the corresponding aziridines in reaction 2 afford an excellent linear correlation (R2 = 0.98), signifying a clear influence of substrate electronic effects on product selectivity. In addition, the moderate magnitude of this negative \u03c1 (\u20131.28) is consistent with the presence of an incipient cationic character2 in the aziridine ring-opening step. This fits well with our computational results that the aziridine N\u2013C2 bond becomes polarized upon attacking the coordinated CO2 moiety on the coupling.Because reaction 1 passes through a concerted, synchronous transition state CrIII(aziridiumcarbamate) intermediate. Theoretical modeling and transition state search reveal that such a process is only possible through an initial key coordination of the CO2 molecules to the CrIII center, which activates the CO2 carbon for nucleophilic attack by the aziridine substrate. In the resulting CrIII(aziridiumcarbamate) intermediate, either of the two carbamate oxygen atoms can act as an intramolecular nucleophile to ring-open the aziridine moiety, allowing for an exquisite control of the regioselectivity. Together, these results also shed light on the erosion of selectivity for the 5-substituted isomer when the CrIIICl-catalyzed [aziridine + CO2] coupling is carried out in the presence of the DMAP cocatalyst.In summary, we presented the complete mechanism of the Cr2 allows for the substituents of the aziridine substrates to have a substantial influence on both their reactivities with CO2 and the selectivities for the final product. Indeed, the broad range of selectivity for 5- vs. 4-substituted oxazolidinone varies almost over an order of magnitude for different aziridines (80\u2009:\u20091 for N-propyl-2-(p-methoxyphenyl)aziridine to 12\u2009:\u20091 for N-propyl-2-(p-bromophenyl)aziridine).Notably, we showed through a detailed Hammett study that while there is not a significant formal charge development in the aforementioned ring-opening process, its intramolecular nature and the incipient cationic nature of the aziridine CSupplementary informationClick here for additional data file."} +{"text": "The trifunctional P/B/B frustrated Lewis pairs 11a\u2013c featuring bulky aryl groups at phosphorus were synthesized. Compounds 11a,b react with carbon monoxide and form the macrocyclic dimers 17a,b, while the carbonylation reaction of the Mes*P/B/B FLP 11c gives the macrocyclic trimer 18c. 11a\u2013c featuring bulky aryl groups at phosphorus react with H2 by heterolytic hydrogen splitting followed by cleavage of HB(C6F5)2 to give the zwitterionic six-membered heterocyclic PH phosphonium/borate products 14a\u2013c. Compounds 11a,b react with carbon monoxide by means of a Lewis acid induced CO insertion/rearrangement sequence that eventually results in the formation of the macrocyclic dimers 17a,b. The respective carbonylation reaction of the Mes*P/B/B FLP gives the macrocyclic trimer 18c. The new products were characterized spectroscopically and by X-ray diffraction and the reaction mechanism was analyzed by DFT calculations.The trifunctional P/B/B frustrated Lewis pairs Macrocyclic compounds have very interesting structural features.We have now found that Lewis pair formation might favor intermolecular cyclooligomerization in cases where the direct internal interaction of the Lewis acid and base functionalities is effectively precluded by specific geometric restrictions. We have found that this may selectively lead to cyclodimeric and even cyclotrimeric ring systems in a rather simple experimental procedure. First examples will be presented and discussed in this account.6F5)2] (2) occurs readily.1) hydroboration compound PhCH2CH2B(C6F5)2 (3), however, do not readily insert CO at ambient conditions 3. The difference is even more pronounced with vicinal P/B frustrated Lewis pairs (FLPs), such as compound 6, which readily reacts with CO, but does not form the CO insertion product into the [B]\u2013CH2\u2013 linkage but rather undergoes cooperative 1,1-P/B addition to the carbon atom of carbon monoxide to yield the CO-bridged product 7. A number of related P/B FLPs show a similar behavior.Alkyl boranes are important building blocks in organic synthesis. Many such systems are readily available by convenient hydroboration routes.6F5)2 Lewis acid unit making the alkyl migration step to carbon monoxide unfavorable. Introduction of a second B(C6F5)2 group into the system might potentially provide a way out of this behavior: specifically located it could function as an activator for the P/B bonded carbonyl unit and thus initiate the otherwise unfavorable CO insertion reaction in such systems. This turned out to be a successful concept and, in addition, it opened an easy pathway to several rather unusually structured macrocyclic ring systems. Three such examples with some of their remarkable characteristic features will be reported in this account.We reasoned that this behavior might originate from the very special properties of the strong B(C8a\u2013c by treatment of the respective ArPCl2 precursors8c with one molar equiv. of HB(C6F5)2 which had given the unique zwitterionic methylene phosphonium product 10cvia internal B(C6F5)2 addition to the adjacent vinyl phosphane and 2,4,6-triisopropylphenyl (Tipp) aryl groups at phosphorus, respectively. Compound 11a also features an equilibrating dynamic structure in solution analogous to the previously described behavior of 11c. We had shown that the P/B/B system 11c splits dihydrogen in the presence of the external base tBu3P to give 12c.49We have prepared the aryldivinylphosphanes hane see .48 Additcopy see .49f We h11a\u2013c to dihydrogen in the absence of the external base and found a markedly different behavior which indicated a surprising mode of participation of the extra \u2013B(C6F5)2 Lewis acid. Typically, the in situ generated system 11a was exposed to a H2 atmosphere (1.5 bar) in dichloromethane solution for 30 min at r.t. to give a mixture of the zwitterionic heterocyclic phosphonium/borate product 14a (Ar: Dmesp) and HB(C6F5)2. The latter was removed from the mixture by the hydroboration reaction with 1-pentene converting it to pentane soluble pentyl-B(C6F5)2. It was isolated and identified as its pyridine adduct 15py . The 13C NMR spectrum shows signals of the six-membered core unit at \u03b4 19.0 and \u03b4 20.8 , respectively 2 Lewis acid becomes actively involved and forms the six-membered P/B heterocycles 14 by a \u03c3-bond metathesis type reaction6F5)2. The formation of the products 14 and HB(C6F5)2 (2) gave us a strong indication of the active role of the additional-B(C6F5)2 Lewis acid in the compounds 11. This we used advantageously in the reaction of the P/B/B FLPs 11a\u2013c with carbon monoxide.The P/B/B compounds 11b (Ar: Tipp) in situ 2 functionalityvia16A might initiate the kinetically facile and thermodynamically feasible formation of the CO insertion product3216B. Isomerization forms the P/B Lewis pair. In 16C the direct internal interaction of the carbonyl oxygen with the pendent borane Lewis acid is geometrically precluded; the system may serve as an active C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O/B frustrated Lewis pair. This leads to dimerization giving the observed macrocyclic reaction product 17b.We assume a reaction pathway as it is depicted in PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O\u00b7\u00b7\u00b7borane interactions. The remaining boron atoms form Lewis pair interactions with their adjacent phosphane Lewis bases. In this situation two diastereoisomers are possible due to the phosphorus chirality;C2-symmetric rac-structure in the crystal = 1588 cm\u20131 [\u03bd\u0302(13CO) = 1547 cm\u20131] carbonyl stretching band.In solution, compound 11a reacts analogously with CO. We isolated the C2-symmetrical dimer 17a in 61% yield and characterized it by C,H-elemental analysis, by NMR and IR spectroscopy (\u03bd\u0302(CO) = 1579 cm\u20131) and by X-ray diffraction (for details see the ESI17b. Compound 17a lost CO upon heating to 50 \u00b0C (12 h) in dichloromethane solution to give the starting material 11a.The Dmesp substituted P/B/B system 11c took a slightly different course. Exposure of the in situ generated Mes*P/B/B system 11c to CO gave compound 18c (81% isolated) (see 11c in solution (CD2Cl2). Therefore, the solution NMR data were monitored using in situ generated samples at low temperature and a cis-, trans-, trans-isomer. The latter structural situation is found in the crystal of compound 18c. Each of the three symmetry inequivalent but chemically closely related subunits features a five-membered P/B containing heterocyclic carbonyl moiety. The C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O group is used for bridging to the pendent \u2013B(C6F5)2 Lewis acid of the next monomeric subunit (see The carbonylation reaction of the Mes*P/B/B system ted) see . The comunit see .18c). We note an almost co-linear arrangement for the pair of Tipp-P units in the dimer 17b. The respective P1\u2013C11\u2013C14 angles for the pair of crystallographically independent Tipp-P subunits were found at 174.4\u00b0 and 173.1\u00b0. We find a slightly bent structure for the more bulky Dmesp-P groups in compound 17a with P1\u2013C11\u2013C14 angles of 171.0\u00b0 and 166.2\u00b0, respectively, but we note a rather extreme bending of the Mes*-P moiety. In the Mes*-PCl2 reagent the P1\u2013C11\u2013C14 type angle amounts to 156\u00b0.18c we find this distortion of the (aryl)C\u2013P moieties being increased further by ca. 10\u00b0 to P1\u2013C11\u2013C14 values of 144.1\u00b0, 147.9\u00b0, and 146.1\u00b0, respectively. 18c, which visualizes this strong distortional effect. This might actually have created an overall conformational situation that may have contributed to determining the observed chemistry of this system to a considerable extent, especially the specific association behaviour of the monomeric subunits forming the observed cyclotrimer 18c.Arylphosphanes usually have the C(aryl)-P vector oriented in line with the aryl plane. Very bulky arylphosphanes may deviate from this behavior structure of the cyclotrimer 18c containing three different phosphorus atoms. Consequently, three 31P NMR signals at 12, 13 and 15 ppm were observed which are broadened and additionally split by the indirect 31P\u201311B spin\u2013spin coupling (1J(31P\u201311B) \u223c 80 Hz). As illustrated in 11B and 1H decoupling enhances the resolution of the 12 pairs of diastereotopic CH2 hydrogen atoms as well as 12 methylene 13C NMR signals of the core ring carbons. There are nine separate 1H NMR t-Bu singlets and the 19F NMR signals of 12 C6F5 groups at the boron atoms of compound 18c. The 13CO derived isotopologue showed three 13C NMR carbonyl signals 2 Lewis acid function influences the reaction of the internal ethylene-bridged P/B FLP functionality of the compounds 11 in two decisive ways: activation of the carbonyl group at the stage of the conventional cooperative P/B CO addition intermediate 16A2\u2013B(C6F5)2 group feasible. Our DFT analysis of the CO insertion step of the Mes*P/B/B system revealed an exergonic formation of the intermediate 16Bc = 0]. Once the carbonyl compound is formed by C\u2013C bond formation it is prone to rearrangement generating a monomeric intermediate 16C featuring both an organic carbonyl function and a remote free \u2013B(C6F5)2 Lewis acid, a combination which paves the way to formation of the unique macrocyclic oligomers 17 and 18 by Lewis adduct formation between these pairs of functional groups.It seems that the presence of the additional B in the Tipp substituted system vs. the cyclotrimer (18c) in the case of the Mes* containing system: in the Tipp containing system we find an energetic preference of the formation of the cyclodimer of ca. 5 kcal mol\u20131 over the trimer, whereas in the case of the more bulky Mes* system this becomes reversed and the cyclotrimer is favored by ca. 10 kcal mol\u20131 over the dimer (see the ESI18c might indeed point to a marked influence of specific conformational features introduced by the very bulky aryl Mes* substituent into this chemistry.Why are the macrocyclic dimers and even a cyclotrimer formed in our examples instead of the alternative linear oligomers? Actually, we do not know for sure, but we may speculate that this has to do with the special properties encountered in phosphane/borane frustrated Lewis pair chemistry. This chemistry is governed by van der Waals interactions between the bulky protagonists and it becomes increasingly apparent that conformational features strongly determine frustrated Lewis pair behavior.11 emphasizes the potential that frustrated Lewis pair chemistry has for discovering surprisingly facile pathways to unusual products formed under mild reaction conditions.The formation of the macrocyclic dimers and trimers from our carbonylated P/B/B FLP systems may place some frustrated Lewis pair reactions into the group of macrocyclic ring closure procedures that show a \u201cnatural\u201d tendency of favoring the internal bond formation in cases of a suitable general design.There are no conflicts to declare.Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "We report a monometallic dysprosium(iii) single molecule magnet with record energy barriers and unusual spin relaxation behaviour. iii) bis(methanediide) single molecule magnet (SMM) where stabilisation of the highly magnetic states and suppression of mixing of opposite magnetic projections is imposed by a linear arrangement of negatively-charged donor atoms supported by weak neutral donors. Treatment of [Ln(BIPMTMS)(BIPMTMSH)] with benzyl potassium/18-crown-6 ether (18C6) in THF afforded [Ln(BIPMTMS)2][K(18C6)(THF)2] . AC magnetic measurements of 2Dy in zero DC field show temperature- and frequency-dependent SMM behaviour. Orbach relaxation dominates at high temperature, but at lower temperatures a second-order Raman process dominates. Complex 2Dy exhibits two thermally activated energy barriers (Ueff) of 721 and 813 K, the largest Ueff values for any monometallic dysprosium(iii) complex. Dilution experiments confirm the molecular origin of this phenomenon. Complex 2Dy has rich magnetic dynamics; field-cooled (FC)/zero-field cooled (ZFC) susceptibility measurements show a clear divergence at 16 K, meaning the magnetic observables are out-of-equilibrium below this temperature, however the maximum in ZFC, which conventionally defines the blocking temperature, TB, is found at 10 K. Magnetic hysteresis is also observed in 10% 2Dy@2Y at these temperatures. Ab initio calculations suggest the lowest three Kramers doublets of the ground 6H15/2 multiplet of 2Dy are essentially pure, well-isolated |\u00b115/2, |\u00b113/2 and |\u00b111/2 states quantised along the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019Dy PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C axis. Thermal relaxation occurs via the 4th and 5th doublets, verified experimentally for the first time, and calculated Ueff values of 742 and 810 K compare very well to experimental magnetism and luminescence data. This work validates a design strategy towards realising high-temperature SMMs and produces unusual spin relaxation behaviour where the magnetic observables are out-of-equilibrium some 6 K above the formal blocking temperature.We report a dysprosium( Proposals have been made for devices employing the quantum effects of molecular magnets,iii).Ueff, for thermal relaxation processes. Furthermore, owing to the high symmetry of the LF potential there should be no mixing between components of opposite magnetic projection, therefore disallowing short-cuts through or under the barrier.iii), where equatorial LF potentials are required; this alternative approach has recently yielded an excellent result in the form of [Er(COT)2]\u2013.Ueff barriers.Many interesting SMMs have been reported based on a single lanthanide centre,Ueff.et al. reported the bis(methanediide) complex [Zr{C(PMe2NSiMe3)2}2], where the two ligands are orthogonal due to the \u2018locking\u2019 effect of the imino arms to avoid steric clashing.Simple electrostatic considerations imply that the strength of the axial LF depends on the charge on the donor atoms on the axis, and hence we proposed that the use of dianionic methanediides would optimise iii) which has a Ueff value of 813 K, the largest for any monometallic dysprosium(iii) complex. This complex also possess rich magnetisation dynamics where out-of-equilibrium magnetisation is observed below 16 K yet TB appears to be 10 K. Although the bis(methanediide) complex is not a two-coordinate linear system, it is clear that there is significant charge accumulation along the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019Dy PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C axis which effectively mimics the linear arrangement we ultimately seek. Thus, this work experimentally validates our proposition that a linear arrangement of negative charges in a dysprosium(iii) complex should lead to very large energy barriers to magnetic relaxation, and provides a promising direction to making high-temperature SMMs a reality.Here we report the synthesis, structure, theoretical characterisation and magnetic studies of a bis(methanediide) complex of dysprosium(iii) complex is shown in 2Ph)3(THF)3]TMSH2 [BIPMTMSH2 = H2C(PPh2NSiMe3)2] in toluene produces an orange solution, which when briefly heated fades to yellow. Work-up and recrystallisation from toluene affords colourless crystals of the mixed methanediide\u2013methanide complex [Dy(BIPMTMS)(BIPMTMSH)] (1Dy) in 63% isolated yield. Alternatively, treatment of [Dy(BIPMTMS)(CH2Ph)(THF)] with one equivalent of BIPMTMSH2 also affords 1Dy in 63% yield. Complex [Dy(BIPMTMS)(CH2Ph)(THF)] was reported previously by us,TMS)(CH2Ph)(THF)] that effects metallation of free BIPMTMSH2 when heated. We previously showed that early, large lanthanides (La\u2013Gd) spontaneously form the mixed methanediide\u2013methanide combination irrespective of reactant ratios, presumably via highly reactive [Ln(BIPMTMS)(CH2Ph)(THF)] complexes due to the large metal size, whereas the later, smaller lanthanides like Dy and Er form isolable methanediide\u2013benzyl combinations. The formulation of 1Dy is supported by IR, CHN, and Evans method magnetic moment (\u03bceff = 11 \u03bcB), but the 1H NMR spectrum is broad and uninformative due to the paramagnetic DyIII ion.The route to a bis(methanediide) dysprosium derivative. Treatment of 1Dy with benzyl potassium in THF gave an orange suspension, which yielded a yellow solution after stirring. Following addition of 18-crown-6 ether (18C6) and concentration, yellow crystals of the bis(methanediide) complex [Dy(BIPMTMS)2][K(18C6)(THF)2]\u00b72THF (2Dy) were obtained in 43% isolated yield. The identity of 2Dy is supported by IR, CHN, and Evans method magnetic moment data (\u03bceff = 11 \u03bcB); however, as for 1Dy the 1H NMR spectrum of 2Dy is uninformative. For the purposes of doping 2Dy into a diamagnetic host we prepared the yttrium bis(methanediide) analogue [Y(BIPMTMS)2][K(18C6)(THF)2]\u00b72THF (2Y) in 60% yield from [Y(BIPMTMS)(BIPMTMSH)] (1Y). The interaction of the methanediide centres in 2Y with yttrium can be seen in the 13C NMR spectrum, which exhibits a single triplet of doublets at 53.70 ppm showing that the methanediides are magnetically equivalent on the NMR timescale; this can be compared to the 13C NMR spectrum of 1Y which exhibits a triplet at 19.87 ppm (JPC = 114.25 Hz) and a triplet of doublets at 66.50 ppm for the methanide and methanediide centres respectively. This suggests a significant interaction between the yttrium and methanediide centres in 2Y, and by inference a similar situation for the dysprosium and methanediide centres in 2Dy, which is important for generating a largely axial LF at dysprosium.With 1Dy and 2Dy were determined by X-ray crystallography and are illustrated in 1Dy crystallises as discrete monomers where the dysprosium centre is six-coordinate. The C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019Dy\u2013C angle is 158.25(6)\u00b0, and the N\u2013Dy\u2013N angles are 129.51(5) and 110.51(5)\u00b0 for the methanediide and methanide ligands, respectively. The Dy PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C and Dy\u2013C bond lengths are 2.3640(17) and 2.9001(18) \u00c5, respectively, which reflects the formal double and single bond character of these linkages. The Dy\u2013N distances are longer in the methanediide [range: 2.4587(15)\u20132.4786(15) \u00c5] than the methanide [range: 2.3903(15)\u20132.4092(15) \u00c5]. The methanediide centre adopts an essentially T-shaped planar geometry [\u03a3\u2220 = 355.06(16)\u00b0] whereas the methanide is puckered to accommodate the hydrogen atom. Complex 1Dy does not approach the linear arrangement of highly charged donor atoms that we seek.The solid state structures of 2Dy crystallises as a solvent separated ion pair and there are no significant contacts between the [Dy(BIPMTMS)2]\u2013 anion and the [K(18C6)(THF)2]+ cation components. Complex 2Dy has the on-axis C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019Dy PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C arrangement of highly charged donor atoms required to test our proposal for high-temperature SMMs. The dysprosium ion is six-coordinate and the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019Dy PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C angle is almost linear at 176.6(2)\u00b0. The methanediide centres adopt planar T-shaped geometries [av. \u03a3\u2220 = 357.1(6)\u00b0] and, importantly, the two C(PN)2Dy planes are disposed essentially orthogonal to each other [89.47(12)\u00b0]. The Dy PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond distances of 2.434(6) and 2.433(6) \u00c5 are statistically identical, and longer than the Dy PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C distance of 2.364(2) \u00c5 in four coordinate [Dy(BIPMTMS)(CH2Ph) (THF)] and in six-coordinate 1Dy, reflecting the trans-disposition of the two methanediide centres and the electron-rich, anionic formulation of the dysprosium fragment in 2Dy. The Dy\u2013N bond lengths in 2Dy average 2.461(9) \u00c5, which is consistent with the analogous methanediide-derived Dy\u2013N bond lengths in 1Dy. All other bond distances and angles in the (BIPMTMS)2\u2013 are unexceptional for this ligand in its dianionic state.Complex 1Dy and 2Dy were measured as neat polycrystalline powders dispersed in eicosane and flame sealed in a quartz NMR tube. The room temperature value of \u03c7mT of 2Dy is 13.5 cm3 mol\u20131 K, around 10% lower than expected for a free DyIII ion . Magnetisation (M) versus field (H) curves at 1.8 K saturate at a value of \u223c5.1 \u03bcB mol\u20131, confirming a well isolated |\u00b115/2 ground state.The magnetic properties of 2Dy in zero DC field show temperature and frequency dependent behaviour, characteristic of slow magnetic relaxation over two thermal barriers, \u03c4) at high temperatures is indicative of a dominant Orbach relaxation mechanism, whilst at lower temperatures its curvature suggests competing relaxation processes are active. As a temperature independent regime is not reached, this cannot be attributed to QTM and we therefore interpret this as a second order Raman process.iii) Kramers ion in the absence of a static magnetic field. A second-order Raman process is invoked because measurements were performed in zero DC field.\u00a7The first-order process should not be active for a Dy(AC magnetic measurements performed on Ueff(1) = 721(1) K (501 cm\u20131), \u03c40(1) = 1.11(3) \u00d7 10\u201312 s, C(1) = 3.01(7) \u00d7 10\u201311 s\u20131 K\u20138, n(1) = 8, \u03b1(1) = 0.01\u20130.03, Ueff(2) = 813(1) K (565 cm\u20131), \u03c40(2) = 5.65(20) \u00d7 10\u201313 s, C(2) = 3.55(10) \u00d7 10\u20139 s\u20131 K\u20136, n(2) = 6 and \u03b1(2) = 0.11\u20130.21. The values of \u03c40 are of the correct order of magnitude expected for an Orbach relaxation process over a large barrier (\u03c40 \u223c (10\u20135 to 10\u20133)/Ueff3)C and n are as expected for the second-order Raman process for Kramers ions.27iii) complex where the phonon spectrum is likely to be more complex. The fitted data were measured over a temperature range of 22\u201341 K where the approximations inherent to the phonon spectrum treatment mean that a simple T9 law cannot be expected to hold for the two-phonon process of a Kramers ion. For further details see pg 564\u2013565 of n are: non-Kramers doublet, n = 7; Kramers doublet, n = 9; multiplet with small splitting, n = 5. These exponents are based on a number of approximations that are parameterised overall by two numbers and thus represent a guide and not absolutes. For further details see pg 65\u201366 of \u00b6The Raman pre-factor ranges are wide but are close to predictions. The theoretical framework is based on simple crystal lattices with Debye-like phonon spectra; here we have an isolated 6-coordinate Dy, Fig. S3,Fitting the two data sets with eqn (1) yields TB) is conventionally defined as the maximum in the ZFC susceptibility;et al. have pointed out that for SMMs a second temperature, TIRREV, is also important which is the point where the FC and ZFC susceptibilities diverge, as this is the temperature below which the magnetic observables are out-of-equilibrium and show history dependent behaviour.TB and TIRREV are very similar, and observed differences have been assigned to a distribution of relaxation times.TB is the temperature where the relaxation time is 100 s.The blocking temperature (TB and TIRREV for 2Dy. Magnetic hysteresis is observed in M(H) loops for 10% 2Dy@2Y to at least 10 K for a sweep rate of 3.5 mT s\u20131 (2Dy gives a relaxation time of 100 s at 12 K. FC(c)/zero-FC (ZFC) measurements for 2Dy diverge at temperatures up to 16 K ; these data also diverge from FC(c) at temperatures up to 16 K depending on rates. Unusually, for any rate that we measured, the FC(w) data go above the FC(c) before reaching equilibrium. Such behaviour would normally be associated with a metastable state arising from the phenomenon of magnetostriction,We have used DC and AC magnetic measurements to establish both 5 mT s\u20131 and S4\u2020; to 16 K , with thTB and TIRREV, usually explained as owing to a range of relaxation times, is very large and does not appear to have precedent in SMMs. Accounting for these two observations, we can only suggest that multiple relaxation processes are competitive at low temperatures, including Raman and QTM mechanisms, which gives rise to this strange behaviour.Furthermore, the discrepancy of up to 6 K between M(H) even at the lowest temperature measured (1.8 K), Fig. S4 and S5.M = 0.15 \u03bcB after 10 hours. This is only 3% of the saturation magnetisation, but clearly some component of the system has a very long lifetime.There remains a significant, sweep-rate dependent, loss of magnetisation at zero-field in \u03c7mT of 1Dy is 13.7 cm3 mol\u20131 K at room temperature and is weakly temperature dependent until below 70 K where it starts to gradually fall, followed by a larger drop at very low temperatures at 1.8 K saturates at a value of \u223c5.2 \u03bcB mol\u20131 vs. 1/T curve at high temperatures, whilst at lower temperatures we observe a transition to a temperature independent regime vs. 1/T plot still curves at lower temperatures K (177 cm\u20131), \u03c40 = 3.55(9) \u00d7 10\u201312 s, C = 1.46(3) \u00d7 10\u20135 s\u20131 K\u20137, n = 7, \u03c4QTM = 9.26(10) \u00d7 10\u20133 s and \u03b1 = 0.06\u20130.22. The values of \u03c40 are of the correct order of magnitude expected for an Orbach relaxation process over a large barrier and the values of C and n are as expected for the second order Raman process for Kramers ions.27The best-fit parameters are 1Dy and 2Dy, we performed ab initio calculations of the CASSCF/RASSI/SINGLE_ANISO variety with MOLCAS 7.8.\u03c7mT vs. T plots for both compounds are almost identical to that obtained experimentally, save for the sub-16 K data for 2Dy scale\" fill=\"currentColor\" stroke=\"none\">Dy PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C axis. The result of this is that thermal relaxation via the second and third states is quenched. The fourth and fifth doublets are strongly mixed, and have main magnetic axes perpendicular to that of the ground state, allowing efficient relaxation (Ueff(1) = 721 K and Ueff(2) = 813 K.Based on the X-ray crystal structures of for 2Dy and S7\u2020,lets see and S1\u2020.\u2013\u2013P+(R)2\u2013C2\u2013\u2013P+(R)2\u2013N\u2013\u2013R resonance form of the (BIPMTMS)2\u2013 dianion is known to be the most appropriate way to formulate the formal charge distribution of this ligand,v) centres withdraw electron charge from the nitrogen centres rendering them relatively soft donors more in keeping with the imino character that is drawn in Lewis-style depictions. Although the phosphorus(v) centres do polarise some of the methanediide charge, it is evident from extensive studies of early metal BIPMTMS chemistry that the majority of the dianion charge remains at carbon available for donation to a coordinated metal.13C NMR chemical shift of the methanediide centres in 2Y is consistent with charge accumulation at these carbon centres and by inference this should be the case for 2Dy also. Thus, and in accord with experimental observations, the symmetrical disposition of the four nitrogen donors, which reside away from the formal equatorial plane due to the bite angle of the BIPMTMS ligand, imposes an axially symmetric equatorial potential (approximate S4 symmetry) which reduces the strength of, but does not destroy, the axial potential of the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019Dy PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C motif in 2Dy. It is germane to note that although the dysprosium centre in 2Dy is of pseudo-octahedral geometry, an effectively linear charge build-up is obviously felt by the dysprosium centre, resulting in strong axial anisotropy, as evidenced by the magnetic behaviour of this system. If the pincer nitrogen donors could be replaced by more weakly coordinating groups, the Ueff value(s) would be even higher,Whilst the RN1Dy the ground doublet is largely |\u00b115/2 with a small admixture of |\u00b111/2, while the second doublet is more significantly mixed but still shows a dominant 81% |\u00b113/2 contribution scale\" fill=\"currentColor\" stroke=\"none\">Dy\u2013C(H) angle = 158.3\u00b0] and has a weaker axial potential due to the mono- and di-anionic ligands vs. the bis(di-anionic) set of 2Dy. The result is that the third doublet has a main magnetic axis perpendicular to the ground state and shows significant transverse g-factors, thus providing an efficient thermal relaxation pathway, Fig. S13 and Tables S5 and S6.Ueff value of 245 K agrees very well with the experimentally determined value of Ueff = 255 K.For 2Dy; we report results recorded at 13 K. After excitation with UV irradiation, sensitised complexes of DyIII are known to exhibit luminescence in the optical region owing to radiative decay from the 4F9/2 multiplet to the 6HJ multiplets, with photon energies (wavelengths) of approximately 21\u2009100 cm\u20131 (475 nm), 17\u2009500 cm\u20131 (570 nm) and 15\u2009200 cm\u20131 (660 nm), for the J = 15/2, 13/2 and 11/2 multiplets, respectively.2Dy exhibits strong emission at 20\u2009000\u201321\u2009000 cm\u20131 (J = 15/2) and 16\u2009700\u201317\u2009700 cm\u20131 (J = 13/2) but only weak signals are observed around 15\u2009000 cm\u20131 (J = 11/2), To further test and corroborate the validity of our model, variable temperature optical emission spectroscopy has been performed for 4F9/2 term, the only reliable transition is that of lowest energy into each multiplet, corresponding to a transition from the lowest lying 4F9/2 state into the highest energy states of the 6HJ multiplets. Therefore, using the low energy edge of the emission band to fix the location of the highest energy Kramers doublet in both the 6H15/2 and 6H13/2 multiplets, we compare the observed transitions to the calculated energy levels (6H15/2 multiplet (\u223c20\u2009490 cm\u20131) corresponds well to the calculated position of the third excited state, which suggests that the strong mixing of this state results in an enhanced transition probability. Conversely, transitions into the first excited and ground states of the 6H15/2 multiplet are expected to be much weaker as these are virtually pure |\u00b113/2 and |\u00b115/2 states. These conclusions are supported by the ab initio calculated transition probabilities between the lowest lying 4F9/2 state and the eight Kramers doublets of the 6H15/2 multiplet complex to date, where a Ueff of 481 K was previously the highest found in a Dy-salen-type Schiff base complex,4K2O(OtBu)12],2] derivative.3)2)3]Ueff = 122 K seems much more compatible with relaxation via the second state at a spectroscopically-determined 158 K.50The AC data are unequivocal that relaxation between 20 and 40 K occurs by Orbach and Raman mechanisms alone; the Orbach process going 2Dy. Firstly, there is a large step at zero-field in M(H) plots, which is conventionally explained as the hallmark of QTM. Our calculations predict a virtually pure |\u00b115/2 ground state and for such a state QTM should have a vanishingly small probability, therefore more work is required to investigate the relaxation mechanisms which cause this phenomenon. This step remains in the 3% diluted sample, and therefore it is possible that this is not dilute enough to completely remove transverse dipolar fields from nearby paramagnetic complexes. An alternative explanation is that nuclear hyperfine interactions may be responsible for this fast relaxation \u2013 a mechanism not accounted for in our purely electronic calculations. Such arguments have been made in Ho-SMMs,et al., who show that isotopic enrichment with nuclear-spin-free 164Dy provides a significant opening of the hysteresis loop compared to the I = 5/2 161Dy isotope.Below 16 K there are features we do not presently understand for 2Dy which contains two methanediide centres that are disposed trans to one another. Although the pincer arms coordinate in off-axial positions, and in principle may carry some charge, it is clear that overall the dysprosium(iii) bis(methanediide) complex 2Dy possess a strong axial LF due to significant negative charge accumulation along the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019Dy PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C axis. We find that the weak equatorial donors do not destroy this strong axial LF; this is in contrast to the recent report of Zhang et al. who show that erbium(iii) complexes are strongly affected by weak axial donors, lowering the Ueff = 122 K of [Er{N(SiMe3)2}3] to Ueff = 34 K for [Er{N(H)Dipp}3(THF)2].48In the continued absence of true two-coordinate bis lanthanide complexes, we have prepared complex 2Dy in zero DC field show temperature- and frequency-dependent SMM behaviour. Orbach relaxation dominates at high temperature, but a second-order Raman process becomes important as the temperature is lowered. We find thermal energy barriers (Ueff) of 721 and 813 K for two distinct processes, the largest Ueff values reported for any monometallic dysprosium(iii) complex.45AC magnetic measurements of Ab initio calculations, which independently model the magnetic data remarkably well and are in good agreement with experimental optical spectra, suggest that the bottom three Kramers doublets of the ground 6H15/2 multiplet of 2Dy are essentially pure, well-isolated |\u00b115/2, |\u00b113/2 and |\u00b111/2 states quantised along the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019Dy PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C axis. Thermal relaxation via the second and third states is quenched, and relaxation occurs via the fourth and fifth states because they are strongly mixed, with calculated Ueff values of 742 and 810 K that compare very well to experimental values.2Dy suggest that TB = 10\u201312 K, yet the FC/ZFC data show a clear divergence at TIRREV = 16 K. Compound 2Dy is therefore a peculiar molecule where the magnetism is history dependent at a temperature significantly above the conventional \u201cblocking\u201d temperature. Previous in-depth studies to assess the competing relaxation mechanisms2Dy move us into a new area where chemical control of molecular geometry generates new and intriguing electronic structures. Despite a mature understanding of the microscopic origins of magnetic relaxation in complexes of the 3d metals,58Magnetic measurements of PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019Dy\u2013F\u2013Dy PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C unit, which should provide a platform to examine in great detail the exchange interactions between pure mJ states. PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019Dy PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C units could be coupled through a radical ligand bridge which also represents a promising direction for Ln SMMs.62Given the properties of the molecules presented herein, realised by following a simple design strategy, we anticipate that such motifs could be employed with other contemporary Ln chemistry, using the idea of \u2018building-block engineering\u2019.TMSH2, [K(CH2Ph)], [Ln(CH2Ph)3(THF)3] and [Ln(BIPMTMS)(CH2Ph)(THF)] were prepared as described previously.261H, 13C, 29Si, and 31P NMR spectra were recorded on a Bruker 400 spectrometer operating at 400.2, 100.6, 79.5, and 162.0 MHz respectively; chemical shifts are quoted in ppm and are relative to TMS and 85% H3PO4 (31P). FTIR spectra were recorded on a Bruker Tensor 27 spectrometer. Variable-temperature magnetic moment data for 2Dy, 10% 2Dy@2Y and 3% 2Dy@2Y were recorded in an applied dc field of 0.1 T on a Quantum Design MPMS XL5 superconducting quantum interference device (SQUID) magnetometer using doubly recrystallised powdered samples. Samples were carefully checked for purity and data reproducibility between several independently prepared batches for each compound examined. Care was taken to ensure complete thermalisation of the sample before each data point was measured and samples were immobilised in an eicosane matrix to prevent sample reorientation during measurements. Diamagnetic corrections were applied using tabulated Pascal constants and measurements were corrected for the effect of the blank sample holders and eicosane matrix. Solution magnetic moments were recorded at room temperature using the Evans method. CHN microanalyses were carried out by Tong Liu at the University of Nottingham. The compounds described herein can be classed as moderately air-/moisture-sensitive, but with adequate precautions they can be handled under a dry nitrogen atmosphere for extended periods with no sign of decomposition.All manipulations were carried out using Schlenk techniques, or an MBraun UniLab glovebox, under an atmosphere of dry nitrogen. Solvents were dried by passage through activated alumina towers and degassed before use or were distilled from calcium hydride. All solvents were stored over potassium mirrors except for THF which was stored over activated 4 \u00c5 sieves. Deuterated solvent was distilled from potassium, degassed by three freeze\u2013pump\u2013thaw cycles and stored under nitrogen. BIPMTMSH2 in toluene (10 ml) was added dropwise to a precooled (\u201378 \u00b0C) suspension of [Dy(CH2Ph)3(THF)3] in toluene (15 ml). The resulting orange suspension was warmed to room temperature with stirring over 16 h then refluxed for 10 minutes to afford a yellow solution. Volatiles were removed in vacuo and the resulting yellow residue recrystallised from hot toluene (4 ml) to afford colourless crystals of 1Dy on cooling to room temperature. Yield: 2.02 g, 63%. Anal. calcd for C62H77DyN4P4Si4: C, 58.33; H, 6.08; N, 4.39%. Found: C, 58.38; H, 6.05; N, 4.36%. FTIR \u03bd/cm\u20131 (Nujol): 1306 (w), 1106 (m), 1047 (w), 841 , 697 (w), 610 (w), 553 (w), 522 (w). Magnetic moment : \u03bceff = 10.61 \u03bcB.BIPMTMSH2 in toluene (10 ml) was added dropwise to a precooled (\u201378 \u00b0C) suspension of [Y(CH2Ph)3(THF)3] in toluene (15 ml). The resulting orange suspension was warmed to room temperature with stirring over 16 h then refluxed for 10 minutes to afford a yellow solution. Volatiles were removed in vacuo and the resulting yellow residue recrystallised from hot toluene (4 ml) to afford colourless crystals of 1Y on cooling to room temperature. Yield: 2.91 g, 48%. Anal. calcd for C62H77N4P4Si4Y: C, 61.90; H, 6.45; N, 4.66%. Found: C, 61.74; H, 6.45; N, 4.57%. 1H NMR : \u03b4 0.31 3), 0.39 3), 2.40 P2), 7.06 , 7.25 , 7.79 , 7.94 ppm. 13C{1H} NMR : \u03b4 5.11 (NSi(CH3)3), 6.16 (NSi(CH3)3), 19.87 P2), 66.50 , 126.88 (Ar-C), 127.68 (Ar-C), 128.50 (Ar-C), 130.57 (Ar-C), 132.32 (Ar-C), 134.10 (Ar-C), 135.08 (Ar-C), 141.81 ppm. 31P{1H} NMR : \u03b4 7.50 , 21.74 P2) ppm. 29Si{1H} NMR : \u03b4 \u20138.34 (NSi(CH3)3), \u20132.59 (NSi(CH3)3) ppm. FTIR \u03bd/cm\u20131 (Nujol): 1305 (w), 1242 (w), 1105 (m), 1045 (m), 841 , 696 (s), 597 (w), 522 (m).BIPMTMS)(BIPMTMSH)] and [K(CH2Ph)] . The resulting orange suspension was allowed to slowly warm to room temperature with stirring over 16 h to afford a yellow solution. 18C6 in THF was then added and the resulting yellow solution stirred for 2 h. The solution was then reduced in volume to ca. 2 ml, which afforded yellow crystals of 2Dy on standing at room temperature. Yield: 0.30 g, 43%. Anal. calcd for C82H116DyKN4O8P4Si4: C, 57.16; H, 6.78; N, 3.25%. Found: C, 56.67; H, 6.67; N, 3.39%. FTIR \u03bd/cm\u20131 (Nujol): 1350 (w), 1303 (w), 1070 (s), 959 (m), 848 (m), 760 (m), 744 (s), 699 (m), 634 (m), 523 (s). Magnetic moment : \u03bceff = 11.16 \u03bcB.THF (15 ml) was added to a precooled (\u201378 \u00b0C) mixture of [Dy(BIPMTMS)(BIPMTMSH)] and [K(CH2Ph)] . The resulting orange suspension was allowed to slowly warm to room temperature with stirring over 16 h to afford a yellow solution. 18C6 in THF was then added and the resulting yellow solution stirred for 2 h. The solution was then reduced in volume to ca. 2 ml, which afforded yellow crystals of 2Y on standing at room temperature. Yield: 1.26 g, 60%. Anal. calcd for C82H116KN4O8P4Si4Y: C, 59.71; H, 7.09; N, 3.40%. Found: C, 59.01; H, 6.92; N, 3.56. 1H NMR : \u03b4 1.84 3), 4.93 2O)6) 7.30 , 8.05 . 13C{1H} NMR : \u03b4 6.11 (NSi(CH3)3), 53.70 P2), 70.13 (((CH2)2O)6), 127.58 (Ar-C), 128.76 (Ar-C), 134.00 (Ar-C), 145.19 ppm. 31P{1H} NMR : \u03b4 1.55 ppm. 29Si{1H} NMR : \u03b4 \u201311.34 (NSi(CH3)3) ppm. FTIR \u03bd/cm\u20131 (Nujol): 1351 (w), 1303 (w), 1067 (s), 960 (m), 848 (m), 760 (m), 743 (s), 700 (m), 635 (m), 523 (s).THF (15 ml) was added to a precooled (\u201378 \u00b0C) mixture of [Y(BIPM\u20136 mbar or better was maintained. The sample temperature was monitored via silicon PIN diode and controlled using an Oxford Instruments ITC 503 Intelligent Temperature Controller. Photo-excitation was provided, off-axis by a 10 mW 375 nm coherent cube laser diode with an unfocussed spot size of \u223c4 mm. The photoluminescence was collected using a collimating lens and focussed onto the slit of a 1 m, single 1200 lines mm\u20131 grating spectrometer. The signal was detected using a Hamamatsu photon counting head and Stanford Research Systems SR400 gated photon counter from where it was read-in and displayed on a PC using a custom built LabView program.The sample was installed in a custom bored-out copper support and sealed with a sapphire window in a glove box environment before being transferred to the cold finger of a recycling He cryostat in which a vacuum of 109 configuration of DyIII was modelled with a complete active space of 9 electrons in 7 orbitals, where 21 sextets, 224 quartets and 158 doublets were included in the orbital optimisation and 21 sextets, 128 quartets and 130 doublets were mixed by spin-orbit coupling. The ab initio results were then parameterised by a set of crystal field parameters,12For all calculations the Dy atoms were treated with the ANO-RCC-VTZP basis, the N and C donors and the P atoms with the ANO-RCC-VDZP basis, while all other atoms were treated with the ANO-RCC-VDZ basis.Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "We compiled a list of 11\u2009016 unique structures of small-molecule ligands bound to proteins representing 750\u2009873 protein\u2013ligand atomic interactions, and analyzed the frequency, geometry and the impact of each interaction type. The most frequent ligand\u2013protein atom pairs can be clustered into seven interaction types. As the protein databank (PDB) recently passed the cap of 123\u2009456 structures, it stands more than ever as an important resource not only to analyze structural features of specific biological systems, but also to study the prevalence of structural patterns observed in a large body of unrelated structures, that may reflect rules governing protein folding or molecular recognition. Here, we compiled a list of 11\u2009016 unique structures of small-molecule ligands bound to proteins \u2013 6444 of which have experimental binding affinity \u2013 representing 750\u2009873 protein\u2013ligand atomic interactions, and analyzed the frequency, geometry and impact of each interaction type. We find that hydrophobic interactions are generally enriched in high-efficiency ligands, but polar interactions are over-represented in fragment inhibitors. While most observations extracted from the PDB will be familiar to seasoned medicinal chemists, less expected findings, such as the high number of C\u2013H\u00b7\u00b7\u00b7O hydrogen bonds or the relatively frequent amide\u2013\u03c0 stacking between the backbone amide of proteins and aromatic rings of ligands, uncover underused ligand design strategies. Significant progress in high-throughput X-ray crystallography9Several approaches have been developed for large-scale analysis of protein\u2013small molecule interactions, such as SuperStar, or the method implemented to build the Relibase database.A statistical analysis of the nature, geometry and frequency of atomic interactions between small molecule ligands and their receptors in the PDB could inform the rational optimization of chemical series, help in the interpretation of difficult SAR, aid the development of protein\u2013ligand interaction fingerprints, and serve as a knowledge-base for the improvement of scoring functions used in virtual screening. To the best of our knowledge, such public resource is currently missing.Here, we analyze the frequency of common atomic interactions between protein and small molecules observed in the PDB. We find that some interactions occur more frequently in fragments than drug-like compounds, or in high-efficiency ligands than low-efficiency ligands. We next review in detail each of the most frequent interactions and use matched molecular pairs to illustrate the impact of these atomic interactions on binding affinity.We extracted from the PDB all X-ray structures of small-molecules in complex with proteins, with a resolution \u22642.5 \u00c5, resulting in a collection of 11\u2009016 complexes. To be considered as a ligand, the compound had to meet several criteria such as being a small molecule and be of interest for medicinal chemistry applications for compounds with high FQ are 27 and 1.7, respectively, and 21 and 0.2, respectively, for compounds with low FQ. Both groups showed similar profiles for other properties like polar surface area (median PSA: 95.3 vs. 89.8 \u00c52), hydrogen bond acceptors (median HBA: 5 for both), and hydrogen bond donors (median HBD: 2 for both). Taken together these results show that small-molecule ligands that bind their target with high efficiency are more hydrophobic, and that hydrophobic interactions are a driving factor for the increased ligand efficiency.We find that hydrophobic interactions are more frequent in high-efficiency ligands. In particular, the frequency of hydrogen bonds is reduced from 59% to 34% of that of hydrophobic contacts in efficient binders, and the frequency of salt bridges is more than halved, from 13% to 7% . This obSince fragments are typically binding their targets with higher ligand efficiency than larger ligands, we asked whether hydrophobic interactions were also more frequent in protein-fragment complexes. The frequency of each interaction type was calculated for two random groups of 1500 protein\u2013ligand complexes, one with fragment molecules (HA \u2264 20), the other with drug-like molecules (30 \u2264 HA \u2264 50) . Unlike 23The higher prevalence of polar interactions in fragments compared to drug-like compounds could be seen as a requirement for high solubility, as fragments are tested at high concentrations. It also reflects the fact that fragments are freer than larger compounds to adopt binding poses that will optimally satisfy the geometric constraints of high-efficiency interactions, such as electrostatic or hydrogen-bonds.24Together, these results show that fragments are using polar interactions to gain maximum binding efficiency from a limited number of interactions, but as small-molecule ligands are optimized, geometric constraints associated with polar bonds are more challenging to satisfy, and the contribution of hydrophobic interactions increases.To gain further insight, we next analyzed in detail the composition, geometry, frequency, protein side-chain preference, and impact towards binding affinity of each protein\u2013ligand interaction type in the PDB.From our analysis, hydrophobic contacts are by far the most common interactions in protein\u2013ligand complexes, totalizing 66\u2009772 contacts between a carbon and a carbon, halogen or sulfur atom . Hydroph\u20131 compared with a purely hydrophobic interaction.27Contacts involving an aromatic or aliphatic carbon in the receptor and an aliphatic carbon in the ligand were observed in 8899 and 8974 instances, respectively Table S2. We obse\u20131 or a 3.2-fold increase in binding constant per methyl group.29Hydrophobic interactions are the main driving force in drug\u2013receptor interactions. The benefit of burying a solvent-exposed methyl group on a ligand into a hydrophobic pocket of a protein is about 0.7 kcal molvs. 7551, respectively). Proteins were more often hydrogen-bond donors than acceptors . Surprisingly, glycine was the most frequent hydrogen-bond acceptor, and the second most frequent donor, probably due to the absence of side-chain to mask backbone atoms, and increased backbone flexibility to better satisfy the spatial constraints of hydrogen-bonds .We find that hydrogen bonds were the second most frequent type of interactions observed in our collection of protein\u2013ligand complexes, with a total of 28\u2009577 . N\u2013H\u00b7\u00b7\u00b7Os Fig. S6. Arginins Fig. S6. Among Os Fig. S6. The moss Fig. S6. Serine s Fig. S6. Finally PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O and OH/NH.We found that heavy atoms in N\u2013H\u00b7\u00b7\u00b7O, N\u2013H\u00b7\u00b7\u00b7N, and O\u2013H\u00b7\u00b7\u00b7O hydrogen bonds were all separated by similar median distances of approximately 3.0 \u00c5 . This va\u20131 to \u20134.7 kcal mol\u20131.39Hydrogen bonds are the prevailing directional intermolecular interactions in biological complexes,Among numerous examples, a series of potent thrombin inhibitors shows a remarkable increase in binding affinity (>500-fold) through simple addition of hydrogen-donating ammonium group .40 In th41The third most frequent protein\u2013ligand contacts in the PDB were aromatic interactions . Interac6 (IC50 = 7 nM), the phenyl ring is positioned to allow \u03c0-stacking interaction with H524 without the phenyl ring is l00-fold less potent. While \u03c0-stacking interaction can increase the binding affinity of the inhibitor for its target, it has been pointed out that reducing the number of aromatic rings of a molecule might improve its physicochemical properties, such as solubility.Interactions involving aromatic rings are major contributors to protein\u2013ligand recognition and concomitantly to drug design.ith H524 , while avs. 708 interactions, Table S2The fourth most frequent interactions (13\u2009600 contacts) were C\u2013H\u00b7\u00b7\u00b7O hydrogen bonds, the existence of which is well documented .48,49 WhThe median distance of the C\u2013H\u00b7\u00b7\u00b7O hydrogen bonding was 3.4 \u00c5, which is 0.4 \u00c5 longer than traditional hydrogen bonds , and distances separating the two heavy atoms were rarely lower than 3.2 \u00c5 . The ang50\u03b1\u2013H\u00b7\u00b7\u00b7O PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C interactions are about one-half the strength of an NH\u00b7\u00b7\u00b7O PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C hydrogen bond.\u03b1\u2013H\u00b7\u00b7\u00b7O hydrogen should be better interpreted as secondary interactions, as they are frequently accompanied by bifurcated N\u2013H\u00b7\u00b7\u00b7O hydrogen bonds.2 by a methyl group on the thiazole ring of compound 8 was the fifth most frequent interaction type in our analysis (7276 interactions) .The contact between a positively charged nitrogen and a negatively charged oxygen (actions) . The numN,N-dimethylamino tail of 10 forms a salt bridge with D831 in the kinase domain of epidermal growth factor receptor (EGFR) potency was reduced by more than 800-fold.71Salt bridges contribute little to protein stability as the favorable binding energy obtained from forming a salt bridge is not sufficient to offset the energetic penalty of desolvating charged groups.r (EGFR) . When thInteractions between an amide group and an aromatic ring were the sixth most frequently observed . In thesKi between a matched pair of oxazole-containing factor Xa inhibitors was attributed to the influence of the dipole of the oxazole ring on the amide\u00b7\u00b7\u00b7\u03c0 interaction with the trimethylated ammonium group (compound 17), indicated that the cation\u2013\u03c0 interaction contributed to a 60 fold increase in potency (Many drug\u2013receptor interactions involve cation\u2013\u03c0 interactions. One of the earliest examples is the recognition of acetylcholine (ACh) by the nicotinic acetylcholine receptor (nAChR). Similarly, GABA,3 receptor potency .Although specific interactions involving halogen atoms were much less frequent than the other interactions discussed above we included them in our analysis as the impact of these interactions is regularly debated among medicinal chemists.We found 351 interactions of the type C\u2013X\u00b7\u00b7\u00b7Y where Y was either from protein side chain or backbone. These halogen bonding (XB) interactions96From the 351 interactions, those involving a chlorine atom were the most frequent (222 interactions), followed by bromine (91 interactions) and iodine (38 interactions) Table S2. This isHalogen bonds are well-characterized intermolecular interactions in small molecules, and have many applications in fields as diverse as crystal engineering and supramolecular chemistry.102a]pyrimidine PDE2a inhibitors.meta position of inhibitor 19 scale\" fill=\"currentColor\" stroke=\"none\">O (or N\u2013C) and X\u00b7\u00b7\u00b7C\u20131) associated with fluorine multipolar interaction.108Compared with other interactions, little attention has been given to the role of multipolar interactions in molecular recognition events of chemical and biological systems.24 (IC50 = 106 nM) by fluorine in 25 (IC50 = 14 nM) improved the potency by approximately 8-fold (para-position of the ring in the crystal structure of 24 confirms a short distance from the peptide carbonyl carbon and amide nitrogen of L104 and V105, respectively, indicative of a multipolar interaction (A series of p38\u03b1 inhibitors recently illustrated the potential impact of a fluorine multipolar interaction.y 8-fold . Introdueraction .We presented here a statistical analysis of the nature, geometry and frequency of atomic interactions between small molecule ligands and their receptors available in the PDB. The enrichment of polar interactions in bound fragments, but hydrophobic contacts in optimized compounds reflects the challenge of overcoming desolvation penalty during lead optimization. This unbiased census recapitulates well-known rules driving ligand design, but also uncovers some interaction types that are often overlooked in medicinal chemistry. This analysis will help in the interpretation of difficult SAR, and may serve as a knowledgebase for the improvement of scoring functions used in virtual screening.The author declare no competing interests.Supplementary informationClick here for additional data file.Supplementary informationClick here for additional data file."} +{"text": "Binding of C2H2 in MFM-300(VIII) showing interactions to O\u2013H, carboxylate O-centres and intermolecular packing. III) {[VIII2(OH)2(L)], LH4 = biphenyl-3,3\u2032,5,5\u2032-tetracarboxylic acid} is accompanied by deprotonation of the bridging hydroxyl groups to afford isostructural MFM-300(VIV), [VIV2O2(L)]. The precise role of the hydroxyl groups, O-carboxylate centres and \u03c0\u2013\u03c0 interactions in the supramolecular binding of C2 hydrocarbons in these materials has been determined using neutron diffraction and inelastic neutron scattering, coupled with DFT modelling. The hydroxyl protons are observed to bind to adsorbed unsaturated hydrocarbons preferentially in MFM-300(VIII), particularly to C2H2, which is in a sharp contrast to MFM-300(VIV) where interactions with O-carboxylate centres and \u03c0\u2013\u03c0 interactions predominate. This variation in structure and redox leads to notably higher separation selectivity for C2H2/CH4 and C2H4/CH4 in MFM-300(VIII) than in MFM-300(VIV). Significantly, owing to the specific host\u2013guest interactions, MFM-300(VIII) shows a record packing density for adsorbed C2H2 at 303 K and 1 bar, demonstrating its potential for use in portable acetylene stores.Fine tuning of host\u2013guest supramolecular interactions in porous systems enables direct control over the properties of functional materials. We report here a modification of hydrogen bonding and its effect on guest binding in a pair of redox-active metal\u2013organic frameworks (MOFs). Oxidation of MFM-300(V Porous materials have shown great promise for substrate binding, storage, separation, and delivery due to their unique host\u2013guest interactions.via binding of the unsaturated species to metal centres.2 hydrocarbons, in particular for distinguishing between alkenes and alkynes.Metal\u2013organic frameworks (MOFs) with extraordinary tuneability of the pore geometry and surface functionality can facilitate control over adsorptive separation of small molecule hydrocarbons, and their high surface areas generally give rise to a large working capacity for dynamic separation.III), {[VIII2(OH)2(L)] LH4 = biphenyl-3,3\u2032,5,5\u2032-tetracarboxylic acid}, and its oxidised iso-structural counterpart MFM-300(VIV), [VIV2O2(L)], provide a unique platform to study the precise role of the \u2013OH group in the supramolecular binding of hydrocarbon molecules.via neutron powder diffraction (NPD) and inelastic neutron scattering (INS), coupled with DFT modelling. These complimentary studies find that the hydroxyl protons play an important role in binding of C2H2/C2H4 molecules in the pores in contrast to the case of MFM-300(VIV) where adsorbed C2H2/C2H4 molecules are found to be remote from the bridging oxy groups. The pore environment of MFM-300(VIII) is found to be optimal for binding of C2H2 and leads to an exceptional packing density of up to 0.38 g cm\u20133 of adsorbed C2H2 at 303 K and 1 bar.We were interested in assessing the role of hydroxyl groups within porous MOFs for the selective binding of light hydrocarbons for potential adsorptive separation. We sought to modify the hydroxyl groups within the pore while still retaining the overall structure and porosity of resultant MOF. MFM-300(VIII) was synthesised following our previously reported methodI4122. Adjacent pair of VIII centres are bridged by two carboxylates and a \u03bc2-hydroxyl group forming an extended chain of [V2(OH)2O4]\u221e along the c axis. The carboxylate ligands further bridge the [V2(OH)2O4]\u221e chains to give a three dimensional network with square-shaped channels decorated with hydroxyl groups pointing into the pores (IV) was obtained by heating the as-synthesised MFM-300(VIII) sample at 150 \u00b0C under a flow of O2 for 16 h. MFM-300(VIV) retains the space group I4122 and the overall framework structure. The V\u2013O bond length involving the bridging oxygen reduces upon oxidation, and loss of the hydroxyl hydrogen atom in MFM-300(VIV) is confirmed. Analysis of the high pressure CO2 adsorption isotherm at 273 K by DFT/Monte-Carlo methods gives surface areas of 1892 and 1565 m2 g\u20131, pore size distributions (PSD) centred at 5.2 and 5.4 \u00c5, and cumulative pore volumes of 0.490 and 0.481 cm3 g\u20131 for MFM-300(VIII) and MFM-300(VIV), respectively. Another pair of MOFs, MIL-47(VIII) and MIL-47(VIV), has structural similarity to MFM-300(V). However, MIL-47(VIII) and MIL-47(VIV) show significantly different porosity owing to framework flexibility as confirmed by CO2 and H2O adsorption,cis-\u03bc2-OH groups thus yielding highly robust isostructural frameworks for both VIII and VIV materials.MFM-300 is 8.1 and 6.9 mmol g\u20131 at 273 and 303 K, respectively, and those for MFM-300(VIV) are 7.8 and 6.1 mmol g\u20131 under the same conditions (2H2 in MFM-300(VIII) is calculated to be 0.38 g cm\u20133 at 303 K, which is 184 times higher than the safe compression limit for C2H2 storage at 2.0 bar. This value is comparable to the highest value of 0.38 g cm\u20133 observed in [Zn2(adc)2(dabco)] octane] but at a lower temperature of 296 K,\u20133 at 296 K)\u20133 at 296 K).2H2 uptake of MFM-300(VIII) is 0.3 mmol g\u20131 higher than that of MFM-300(VIV) at 1.0 bar, while the difference is significantly greater (\u0394 = 1.7 mmol g\u20131) in favour of MFM-300(VIII) at 0.1 bar than in MFM-300(VIV) at low surface coverage; with increasing gas loading, less surface sites are available and guest\u2013guest interaction start to be significant in driving the gas adsorption, leading to a reduced difference in gas uptake.Gravimetric adsorption isotherms for CH73\u2013303 K . At 1.0 nditions . The denr Fig. S1. Given t2 hydrocarbons of both MFM-300(VIII) and MFM-300(VIV) track the order of C2H2 > C2H4 > C2H6 at 1.0 bar and 273\u2013303 K (III) follows the order of C2H2 > C2H6 > C2H4, while that in MFM-300(VIV) follows C2H6 > C2H2 > C2H4. At low surface coverage, the effect of MOF\u2013gas interaction is more pronounced in driving physical adsorption processes. The inversion of uptakes of C2H6 and C2H2 in MFM-300(VIII) and MFM-300(VIV) indicates that the host\u2013guest binding mechanism is very sensitive to the presence of hydroxyl groups in the pore and to the small difference in chemical structure of these hydrocarbon molecules.The uptake capacities of C73\u2013303 K . This is2Hn/CH4 for MFM-300(VIII) and MFM-300(VIV) were calculated at 303 K and 0\u20131 bar (III) outperforms MFM-300(VIV) for the selectivities of C2H2/CH4 and C2H4/CH4 by ca. 40%. This is likely due to the presence of stronger binding sites to unsaturated hydrocarbons in MFM-300(VIII). The C2H6/CH4 selectivity of MFM-300(VIII) is between 22\u201314, and is nearly identical to that of MFM-300(VIV) under same conditions. The C2H6/CH4 selectivity of MFM-300(V) is comparable with the benchmark MOFs, [Fe2(dobdc)] (20 at 313 K and 1.0 bar)2H6 in MFM-300(VIII) is as high as 5.0 mmol g\u20131 at 303 K and 1.0 bar, which is comparable to that of MOF-74(Fe) (5.0 mmol g\u20131 at 318 K and 1.0 bar) and much higher than that of UTSA-35a (2.4 mmol g\u20131 at 296 K and 1.0 bar), suggesting that MFM-300(VIII) is a promising material for separation of C2H6 from CH4.The IAST selectivities of equimolar mixture of C 0\u20131 bar . MFM-300Qst, for C2H2 in MFM-300(VIII) is centered at 32.0 \u00b1 0.1 kJ mol\u20131 without notable variation as a function of surface coverage. In contrast, the Qst of C2H2 in MFM-300(VIV) is estimated as 26.0 \u00b1 0.4 kJ mol\u20131 at low pressure and increases gradually with increasing gas loading to 30.1 \u00b1 0.1 kJ mol\u20131 at 4.0 mmol g\u20131. Interestingly, the Qst value for C2H2 in MFM-300(VIII) is significantly higher than that of MIL-53(Al) (19.2 kJ mol\u20131), which adopts similar structural feature but with trans-orientated hydroxyl groups at the metal centre.\u20131) incorporating open CuII center.Qst values of C2H6 for both compounds are similar and lie within the range of 28\u201332 kJ mol\u20131. The Qst value of C2H4 in MFM-300(VIII) (28\u201331 \u00b1 0.1 kJ mol\u20131) is also higher than that of MFM-300(VIV) (24\u201326 \u00b1 0.1 kJ mol\u20131). Finally, the Qst values of CH4 are similar for both compounds and within the range of 17\u201319 kJ mol\u20131.The isosteric heat of adsorption, i.e., C2H2 and C2H4), the uptake capacity and Qst are much higher in MFM-300(VIII) than in MFM-300(VIV) at low pressure, indicating the presence of possible \u2013OH\u00b7\u00b7\u00b7C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C/C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C interactions. In comparison, for saturated hydrocarbons , the Qst in both compounds are similar, suggesting the presence of similar host\u2013guest binding mechanisms for saturated hydrocarbons in MFM-300(VIII) and MFM-300(VIV). The observed undulations in the Qst plots may suggest a packing effect reflecting optimum packing of the substrate within the pore at specific loadings.For unsaturated hydrocarbons , NPD experiments were carried out for gas-loaded samples of MFM-300(VIII) and MFM-300(VIV) at 7 K. Comparison of the NPD patterns for bare and gas-loaded MOFs indicated that no major structural phase change took place on gas-loading, and NPD data enabled full structural analysis via Rietveld refinement to yield positions, orientations and occupancies of adsorbed gas molecules within the framework hosts.In order to determine the preferred binding sites for C2D2/V, analysis of the NPD data revealed two crystallographically independent sites for C2D2 (I and II) scale\" fill=\"currentColor\" stroke=\"none\">CC distance of 3.016(1) \u00c5 (c.g. = centre of gravity), indicating a moderate hydrogen bond between the \u03c0-electrons of C2D2 and the HO\u2013V moiety. C2D2II interacts with the carboxylate O atoms with a D\u00b7\u00b7\u00b7O distance of 2.608(1) and 2.871(1) \u00c5. The HOH\u00b7\u00b7\u00b7c.g. PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CC distance is shorter than that found in the C2D2-loaded MFM-300(Al) [3.26(1) \u00c5] studied by NPD at 10 K.III) is likely attributed to the difference in the acidity between the Al\u2013OH and V\u2013OH bridges, and is consistent with the observation in the corresponding CO2-loaded MFM-300 systems where adsorbed CO2 binds to V\u2013OH groups more strongly than to the Al\u2013OH group.2D2I and the aromatic benzene rings in the carboxylate ligand, with a c.g. PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CC\u00b7\u00b7\u00b7c.g.phenyl distance of 3.591(2) \u00c5. Further dipole interactions are observed between adsorbed C2D2 molecules on sites I and II in a T-shape arrangement with a D\u00b7\u00b7\u00b7c.g. PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CC distance of 3.190(1) \u00c5 scale\" fill=\"currentColor\" stroke=\"none\">CC = 3.178(1) \u00c5].2D2 molecules was obtained by free refinement against the NPD data. The occupancies of C2D2I and C2D2II are found to be 0.55 and 0.45, respectively, at a loading of 1.0 C2D2/V, indicating that site I which contacts to the hydroxyl group directly is more favourable at low pressure. As the loading increases to 1.76 C2D2/V, the occupancies of C2D2I and C2D2II are found to be 0.82 and 0.94, respectively, indicating the presence of intermolecular cooperative binding between adsorbed gas molecules.At a loading of 1.0 C and II) . C2D2I i2D2-loaded MFM-300(VIV) also shows two independent binding sites for adsorbed C2D2 molecules with retention of the framework structure. C2D2I\u2032 interacts with the carboxylate group with D\u00b7\u00b7\u00b7OCOO distances of 2.34(1) and 2.43(1) \u00c5, which are slightly longer than the H2H2C\u00b7\u00b7\u00b7OCOO hydrogen bond distance (2.2 \u00c5) observed in [Cu2(pzdc)2(pyz)] at 170 K ,COO separation of 3.12(1) \u00c5 found in C2D2-loaded MFM-300(VIII). This indicates a shift in the nature of the primary binding site on going from C2D2-loaded MFM-300(VIII) to C2D2-loaded MFM-300(VIV) scale\" fill=\"currentColor\" stroke=\"none\">CC\u00b7\u00b7\u00b7c.g.phenyl distance of 3.78(1) \u00c5. The shortest separation between the adsorbed C2D2 molecules and the bridging oxy group is 4.83(1) \u00c5.Analysis of the NPD data of C300(VIV) . \u03a0\u00b7\u00b7\u00b7\u03c0 ie.g., MgO) by forming hydrogen bonds between the acidic C\u2013H groups and the basic sites on the material surface. In this study, the absence of any notable binding interactions between adsorbed C2D2 molecules and the surface oxy groups is a direct result of the weak basicity of the oxy bridges in MFM-300(VIV), which, in turn, reflects the relative acidity of the hydroxyl bridges in MFM-300(VIII), consistent with the binding observed in C2D2-loaded MFM-300(VIII). In contrast to C2D2-loaded MFM-300(VIII), \u03c0\u00b7\u00b7\u00b7\u03c0 interactions are observed between the adsorbed C2D2I\u2032 and C2D2II\u2032 molecules in MFM-300(VIV) with a c.g.I\u2032\u00b7\u00b7\u00b7c.g.II\u2032 distance of 3.45(2) \u00c5 .Acetylene or propyne are frequently used as probes to determine the basicity of oxide materials (2D2 molecules within the two frameworks is compared in III) while the guest\u2013guest interaction is stronger in MFM-300(VIV). This result suggests that the proton on the bridging oxy group can not only affect the host\u2013guest binding through fine-turning of the pore surface chemistry, but also alter the subsequent guest\u2013guest interaction, thus controlling the gas adsorption and substrate packing within the pores.The packing of adsorbed C2D2-loaded MFM-300(VIII) and MFM-300(VIV) have also been optimised by DFT calculations at a loading of 2.0 C2H2/V (III) confirms that the adsorbed C2H2 molecules (i) interact with the hydroxyl groups in a side-on model scale\" fill=\"currentColor\" stroke=\"none\">CC = 2.56 \u00c5); (ii) form \u03c0\u00b7\u00b7\u00b7\u03c0 bonds with the phenyl rings of ligands scale\" fill=\"currentColor\" stroke=\"none\">CC\u00b7\u00b7\u00b7c.g.phenyl = 3.86 \u00c5); (iii) bind with the carboxylate oxygen atom via dipole interactions (H2H2C\u00b7\u00b7\u00b7Ocarboxylate = 2.88 \u00c5). In contrast, the DFT calculation for MFM-300(VIV) confirms (i) an absence of binding interaction between the adsorbed C2H2 molecules and the bridging oxy group; (ii) presence of \u03c0\u00b7\u00b7\u00b7\u03c0 interaction between C2H2 molecules and the phenyl rings of ligands scale\" fill=\"currentColor\" stroke=\"none\">CC\u00b7\u00b7\u00b7c.g.phenyl = 3.89 \u00c5); (iii) the adsorbed C2H2 molecules interact strongly with the carboxylate oxygen atom (H2H2C\u00b7\u00b7\u00b7Ocarboxylate = 2.77 \u00c5). The shift of the binding strength for host\u2013guest and guest\u2013guest interaction on going from MFM-300(VIII)\u00b74C2H2 to MFM-300(VIV)\u00b74C2H2 was clearly observed in the DFT calculation \u00b72.12C2H4 shows two binding domains for C2D4 (2D4I (occupancy = 0.56) is located close to the hydroxyl group with a c.g. PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CC\u00b7\u00b7\u00b7HOH distance of 3.737(1) \u00c5, which is much longer than the corresponding value of 3.016(1) \u00c5 observed in MFM-300(VIII)\u00b72.0C2D2. This result suggests the presence of a weaker hydrogen bond due to the reduced \u03c0-electron density in C2D4 compared to C2D2. C2D4II molecules (occupancy = 0.50) contacts with the carboxylate O atoms with D\u00b7\u00b7\u00b7Ocarboxylate distances in the range of 2.64(1)\u20133.03(2) \u00c5. The NPD analysis of C2D6-loaded MFM-300(VIII) suggests two sites for adsorbed C2D6 molecules, C2D6I molecule is aligned at a long distance to the \u2013OH group [distance c.g.C\u2013C\u00b7\u00b7\u00b7HOH = 3.87(1) \u00c5] as a result of no \u03c0-electron density and notable repulsion between the host and guest hydrogen atoms confirms no specific host\u2013guest interactions but van der Waals interactions between adsorbed CD4 molecules and the MOF interior [distance c.g.C\u2013C\u00b7\u00b7\u00b7HOH = 3.59(2) \u00c5] The Qst values for C2H2 and C2H4 are higher in MFM-300(VIII) than those in MFM-300(VIV) at low surface coverage, which is due to the stronger binding interaction between adsorbed C2H2 and C2H4 molecules with the framework host of MFM-300(VIII), particularly with the free acidic hydroxyl groups via hydrogen bonds. (ii) The Qst for C2H2 increases with increasing gas loading in MFM-300(VIV), while that for MFM-300(VIII) remains near-constant over the entire loading, which is due to the stronger intermolecular interaction between adsorbed C2H2 molecules within the pore of MFM-300(VIV) as a result of lack of specific surface binding sites. (iii) The Qst values of C2H6 and CH4 are similar for both MFM-300(VIII) and MFM-300(VIV) because of the absence of host\u2013guest hydrogen bonds via the hydroxyl groups and the van der Waals interaction drives the adsorption of C2H6 and CH4 in both materials.Combining the results of NPD experiments and DFT calculations, the differences in measured 2H2, C2H4, C2H6 and CH4 molecules in MFM-300(VIII) and C2H2 in MFM-300(VIV) shows a series of peaks in the range of 40\u201360 MeV attributed to the deformational modes of the hydroxyl group, and the disappearance of these peaks in the INS spectrum of MFM-300(VIV) unambiguously confirms the deprotonation of the bridging hydroxyl group upon oxidation.In addition to static crystallographic studies, we have combined INS and DFT to directly visualise the binding dynamics of adsorbed C300(VIV) . INS is III) to MFM-300(VIII)\u00b73C2H2 . On convergence, excellent agreement between the calculated and experimental structural models were obtained K to minimize the thermal motion of the adsorbed hydrocarbons and the host, reveals five major changes in peak intensity on going from bare MFM-300(VI)\u00b73C2H2 . Peaks Iobtained . Peaks Ine rings . The sig2H2 in MFM-300(VIV) is accompanied by the appearance of four new peaks in the INS spectrum . No major changes in the intensity at 40\u201370 MeV, which is in a sharp contrast to that in the INS study of MFM-300(VIII) .2H4-loaded MFM-300(VIII), small changes in intensity of peaks at 47 and 115 MeV were observed upon addition of C2H4, which are attributed to the interaction of adsorbed C2H4 with the hydroxyl and C\u2013H groups from phenyl rings, respectively (2H6-loaded MFM-300(VIII), it is clear that all peaks are ascribed to the adsorbed C2H6 molecules, and no specific host\u2013guest interaction was seen (4 in MFM-300(VIII) does not induce an obvious change in the INS spectra either , in excellent agreement with the NPD study and the Qst analysis.In the case of Cectively and S17\u2020was seen and S18\u2020III), both \u2013OH and \u2013CH/phenyl ring are active binding sites for adsorbed C2H2 molecules; (ii) in MFM-300(VIV), \u2013CH/phenyl ring is the sole binding sites for adsorbed C2H2 molecules via formation of supramolecular contacts. These observations are entirely consistent with the NPD studies and confirm the significant effects of bridging hydroxyl groups on the guest binding in the host on a molecular level.Thus, the INS and DFT results confirm that (i) in MFM-300(VIII/IV), incorporating redox-active vanadium centers. The oxidation of V centers from III to IV induces deprotonation of the bridging hydroxyl group, achieving fine-tuning of the pore environment. It has been confirmed in this study that these protons play a key role in adsorption of unsaturated hydrocarbons (both uptakes and isosteric heats of adsorption). A comprehensive study combining NPD, INS and DFT modelling has unambiguously determined the preferred binding sites and structural dynamics for these host\u2013guest systems. The differences in C2Hn/CH4 selectivities between MFM-300(VIII) and MFM-300(VIV) are fully rationalised. The acidic bridging hydroxyl groups in the pore provides specific binding activity to unsaturated hydrocarbons via formation of hydrogen bonds, leading to both high selectivity and packing efficiency of adsorbed gas molecules.We report the adsorption of light hydrocarbons in a pair of iso-structural MOFs, MFM-300(VThe authors declare no competing financial interests.Crystal structure dataClick here for additional data file.Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file.Crystal structure dataClick here for additional data file.Crystal structure dataClick here for additional data file.Crystal structure dataClick here for additional data file.Crystal structure dataClick here for additional data file.Crystal structure dataClick here for additional data file.Crystal structure dataClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "Structural, spectroscopic and magnetic methods have been used to characterize the tris(carbene)borate compound PhB(MesIm)3Mn PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N as a four-coordinate manganese(iv) complex with a low spin (S = 1/2) configuration. 3Mn PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N as a four-coordinate manganese(iv) complex with a low spin (S = 1/2) configuration. The slow relaxation of the magnetization in this complex, i.e. its single-molecule magnet (SMM) properties, is revealed under an applied dc field. Multireference quantum mechanical calculations indicate that this SMM behavior originates from an anisotropic ground doublet stabilized by spin\u2013orbit coupling. Consistent theoretical and experiment data show that the resulting magnetization dynamics in this system is dominated by ground state quantum tunneling, while its temperature dependence is influenced by Raman relaxation.Structural, spectroscopic and magnetic methods have been used to characterize the tris(carbene)borate compound PhB(MesIm) Strong spin\u2013orbit coupling can lead to SMM properties in complexes having f1 electron configurations. For example, the SMM behaviour of the 5f1 U(v) complex, (trenTIPS)U(O) (trenTIPS = {N(CH2CH2NSiiPr3)3}3\u2013) has been attributed to an energy gap between the MJ = \u00b13/2 ground Kramers doublet and the lowest-lying excited Kramers doublet (either MJ = \u00b11/2 or MJ = \u00b15/2).1 Ce(iii) complexes have been similarly rationalized.9 SMM, [Ni(6-Mes)2]+ -3,4,5,6-tetrahydropyrimidin-2-ylidene),S = 1/2 Ni(i) complexes attributed the observed SMM properties to direct and Raman processes.S = 1/2 SMM systems is often difficult to establish as it can be induced by different mechanisms ,Since the discovery of a four-coordinate iron tris(carbene)borate complexes.ST = 1/2) Fe(v) complex, + borate ligands.vg>N]+ .16 Detaiiv) nitride, PhB(MesIm)3Mn PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N (PhB(MesIm)3\u2013 = phenyltris(3-mesitylimidazol-2-ylidene)borato) which shows similar structural and spectroscopic properties to the Fe(v) complex. Magnetic measurements reveal that this new manganese complex shows slow relaxation of its magnetization, which is unexpected for a low spin (ST = 1/2) d3 configuration. A combined approach using a detailed experimental study of the relaxation time (in temperature and dc field) and electronic structure theory has been used to delineate the origin of the observed magnetization dynamics in this new SMM.Building from this work, we report in this contribution the synthesis, characterization, spectroscopic and magnetic properties of the isoelectronic Mnborate ligand precursor, PhB(MesImH)3OTf2, was prepared according to a literature procedure.131H NMR data were recorded on a Varian Inova 400 MHz spectrometer at 20 \u00b0C. Resonances in the 1H NMR spectra are referenced to residual C6D5H at \u03b4 = 7.16 ppm. IR spectra were recorded on a Perkin Elmer Spectrum Two spectrometer in THF solution. Cyclic voltammograms were measured using a CH Instruments Model 600B Series Electrochemical Analyzer/workstation in a glovebox with a glassy carbon working electrode. Elemental analysis data were collected by Midwest Microlab, LLC .All manipulations were performed under a nitrogen atmosphere by standard Schlenk techniques or in an MBraun Labmaster glovebox. Glassware was dried at 150 \u00b0C overnight. Diethyl ether, 3(OTf)2 in Et2O (50 mL) at \u201378 \u00b0C. The resulting mixture was stirred at \u201378 \u00b0C for 15 min and then slowly warmed to room temperature. After stirring until the reaction mixture became golden yellow, the solvent was removed in vacuo. Tetrahydrofuran (15 mL) was added to the resulting yellow solid, followed by MnCl2 . The reaction was stirred at room temperature overnight and then dried under vacuum. After washing with Et2O and drying under vacuum, the product was obtained 3(OTf)2) as white solid. Colorless crystals were obtained by diffusion of pentane into a THF solution of the product at \u201335 \u00b0C. \u03bceff = 6.1(3) \u03bcB [\u03c7T = 4.6(1) cm3 K mol\u20131]. Elemental analysis calcd for C42H44BMnCl: (%) C 68.79, H 6.04, N 11.45 found (%) C 68.70, H 6.04, N 11.39.Lithium diisopropylamide was added to a precooled slurry of PhB(MesImH)1 , NaN3 and THF (100 mL). The mixture was stirred overnight under UV irradiation to yield a yellow solution. The solvent was removed in vacuo. Minor impurities were removed by washing with Et2O. The remaining solid was extracted into THF and filtered through Celite to yield a yellow solution. The solvent was removed in vacuo to afford a yellow solid 3MnCl). X-Ray quality crystals were obtained by the slow diffusion of n-pentane into a THF solution of the product at \u201335 \u00b0C. 1H NMR : \u03b4 12.8 ; 10.8 ; 9.3 ; 8.9 ; 7.0 ; 2.7 ; \u20133.2 ; \u201311.9 . Elemental analysis calcd for C42H44BMnN7\u00b70.5C4H8O (%) C 71.35, H 6.53, N 13.24; found (%) C 70.56, H 6.51, N 13.32.A 250 mL quartz round-bottom-flask was charged with 1 was measured using a Bruker APEX II Kappa Duo diffractometer equipped with an APEX II detector at 150(2) K. Complex 2 was investigated with synchrotron radiation at 100(2) K at the ChemMatCARS 15IDB beamline at the Advanced Photon Source at Argonne National Lab, Chicago. Additional details of the data collection and refinement are included in the ESI.Complex 1 were collected on a modified Bruker ESP-300 spectrometer with 100 kHz field modulation (4 G modulation amplitude) at 20 K through the utilization of an Oxford Instruments liquid helium flow cryostat. Simulations of EPR spectra were performed using the MATLAB EasySpin (v4.5) toolbox .17Continuous-wave (CW) X-band (9.32 GHz) EPR spectra of The magnetic measurements were carried out with the use of Quantum Design MPMS-XL SQUID magnetometer and PPMS-9 susceptometer. These instruments work between 1.8 and 400 K with applied dc fields ranging from \u20137 to 7 T (MPMS).2 sealed in a polyethylene bag and covered with mineral oil or directly in their frozen THF mother liquor within a sealed straw to prevent desolvation of the solid. Only experiments done with 2 maintained in frozen mother liquor and prepared under nitrogen atmosphere led to reproducible dc and ac magnetic data. No evaporation of the mother liquor was observed during these measurements. The mass of the sample was determined after the measurements and subsequent mother liquor evaporation. Prior to the experiments, the field-dependent magnetization was measured at 100 K in order to confirm the absence of any bulk ferromagnetic impurities. Ac susceptibility measurements were made with an oscillating field of 1 to 6 Oe with a frequency from 10 to 10\u2009000 Hz (PPMS). The magnetic data were corrected for the sample holder, mineral oil, mother liquor and the intrinsic diamagnetic contributions.Measurements were performed on a polycrystalline samples of Versa Probe II instrument equipped with monochromatic Al K source. The X-ray power of 50 W at 15 kV was used for 200 micron beam size. The instrument work function was calibrated to give a binding energy (BE) of 84.0 eV for Au 4f7/2 line for metallic gold and the spectrometer dispersion was adjusted to give BEs of 284.8, 932.7 and 368.3 eV for the C 1s line of adventitious carbon presented on the non-sputtered samples, Cu 2p3/2 and Ag 3d5/2 photoemission lines, respectively. The PHI dual charge compensation system was used on all samples. XPS spectra with the energy step of 0.1 eV were recorded using software SmartSoft-XPS v2.0 and processed using PHI MultiPack v9.0 at the pass energies of 46.95, 23.5, 11.75 eV for Mn 2p and Mn 3s, for N 1s, and for C 1s regions, respectively. Peaks were fitted using GL line shapes, i.e., a combination of Gaussians and Lorentzians with 0\u201350% Lorentzian content. Shirley background was used for curve-fitting.XPS experiments were performed using PHI ab initio ligand field theory (AILFT) approach.21Electronic structure calculations were performed using the ORCA 3.0.3 software package and MOLCAS 8.0.3Mn PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N (2) is accessible by the same synthetic pathway used to prepare the related Fe(iv) nitrides (ii) complex PhB(MesIm)3MnCl (1) reveal 2 to be a four-coordinate Mn(iv) nitride complex with a low spin (ST = 1/2) d3 electron configuration that is subject to a Jahn\u2013Teller distortion. The molecular structure of 2 has been determined by single crystal X-ray diffraction (Table S1iv) nitrides,iv) centre. The manganese ion lies ca. 0.1 \u00c5 out of the plane defined by the carbon atoms of the tris(carbene)borate ligand, which is similar to the equivalent distance observed in the iron analogues. Similarly to the isoelectronic + complex,2 and the related Mn(iv) nitride + (TIMENxyl = tris[2-(3-xylylimidazol-2-ylidene)ethyl]-amine)xyl)Mn PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N]+, which has a relatively flexible tris(carbene)amine ligand, significant elongation of one Mn\u2013C bond (by 0.15 \u00c5) occurs to lower the local symmetry at the Mn site. The greater rigidity of the tris(carbene)borate ligand in 2 evidently hinders such a distortion, and all Mn\u2013C distances are similar in length. Instead, the B\u2013Mn\u2013N angle in 1 is bent away from 180\u00b0 (B\u2013Mn\u2013N = 174.7\u00b0), whereas the equivalent angle in + is almost linear (179.4\u00b0).The manganese nitride complex, PhB(MesIm)nitrides .22 SpeciMnCl (1) in the p Table S1, reveali Table S1, that cr Table S1. The Mn\u20132 has also been spectroscopically characterized. The solution 1H NMR spectrum reveals eight paramagnetically-shifted resonances with relative integration appropriate for a three-fold symmetric complex. The solution magnetic moment, as determined by the Evans' method (\u03bceff = 2.2(3) \u03bcB; \u03c7T = 0.6(1) cm3 K mol\u20131), is consistent with a single unpaired electron and unquenched spin\u2013orbit coupling seen in the solid state (see below).Complex 2 have been investigated by cyclic voltammetry. As with the structural data, interesting differences with + are observed (2 and + can be reversibly reduced on the CV timescale, only the latter can be oxidized to Mn(v).v) state for the TIMENxyl ligand is in part due to the ability of apical bridgehead nitrogen atom of this ligand to bind to Mn in this higher oxidation state, forming a five-coordinate complex. Such additional stabilization is not possible with the tris(carbene)borate ligand.The redox characteristics of observed , likely 2 have been obtained from EPR spectroscopy. The frozen solution EPR spectrum (iv), I = 5/2, centre. The |MI\u3009 = |\u22125/2\u3009 and | = |\u20135/2\u3009 = |\u22125/2\u3009 and | and |MI = |\u20133/2 = |\u22123/2\u3009 manifolds at the low magnetic field edge of manifolds at the low magnetic field edge of g\u2225 are well resolved, and simulated with an A1(55Mn) = 300 MHz coupling. The g\u22a5 values are slightly split, with anisotropic 55Mn hyperfine couplings, as determined by simulation of the EPR spectrum, yielding g values and 55Mn couplings . The average g value, gav = [(g1 + g2 + g3)/3] = 2.096, is in agreement with the g factor, 2.1(1), determined from the magnetic susceptibility measurements detailed below. The electronic structure of 2 and EPR parameters remarkably resemble those of other low-spin trigonal d3 centres (Mn(iv) and Fe(v)) with tris(carbene) ligands.2 was also prepared for EPR characterization by suspending the solid in pentane to form a slurry. The X-band EPR spectrum of this slurry feature is too broad and not observed, however, the A355Mn hyperfine splitting of 204 MHz is distinctly observed in the EPR spectrum of the slurry parameters of the slurry sample match those observed for the solution. EPR spectra of this slurry collected at various temperatures (3.6 to 20 K) exhibit only the S = \u00bd Mn(iv) complex identifiable by the 55Mn hyperfine structure (see ESI3 Mn(iv) nitride are the same in both solution and the solid state.More detailed insights into the electronic structure of spectrum , top incs slurry , middle s slurry , top. The see ESI. In shor2. This oxidation state assignment has been confirmed using X-ray Photoelectron Spectroscopy (XPS). The standard position of the 2p3/2 peak for the Mn(iv) state is accepted to be in the range from 641.1 to 642.5 eV with the spin\u2013orbit splitting of 11.7 eV between Mn 2p3/2 and Mn 2p1/2 levels. The measured binding energies of Mn 2p3/2 for 2 are well within this range (2+ ions) is observed for MnPO4, but not for Mn2O3, despite the Mn(iii) state of both compounds.iv) complex, which clearly are more resolved for the Mn(ii) complex 1 ions in a SnO2 matrix.2 in comparison to that of 1 d3 electron configuration.In summary, the combined characterization data reveal that 2 have been studied by dc and ac techniques. Perfectly reproducible data were obtained when the compound was maintained below 200 K during the measurements and in its THF mother liquor, which prevents loss of solvent from the polycrystalline sample. At 200 K, the \u03c7T product has a value of 0.47 cm3 K mol\u20131 in good agreement with a magnetically isolated low-spin (ST = 1/2) Mn(iv) centre . At lower temperatures and as already detected by EPR (vide supra), the marked decrease of the \u03c7T product reveals the presence of antiferromagnetic interactions between Mn(iv) complexes. These intermolecular interactions were evaluated at \u20130.6(1) K (zJ/kB) by simulating the experimental data in the frame of the mean-field approximation\u03c7T vs. T values (red line in S = 1/2 species (M = 1.05 \u03bcB at 7 T & 1.85 K). The fit of the experimental data with an S = 1/2 Brillouin function confirms an average g factor around 2.10(2), which is in perfect agreement with that deduced from EPR .The magnetic properties of ) centre . When lo\u03c7\u2032) susceptibility consistent with the dc susceptibility (\u03c7\u2032\u2032). However, application of a dc field leads to the detection of a relaxation process in both components of the ac signal indicating a weak distribution of the relaxation time (\u03c4) and thus a relaxation mode that is dominated by a single relaxation process. The characteristic frequency of this relaxation mode continuously decreases when applying higher fields (to about 1000 Hz at 1 T) while the amplitude of the mode (\u03c70 \u2013 \u03c7\u221e) exhibits a maximum around 0.45 T suggests the presence of a thermally activated (Orbach) process of relaxation with a pre-exponential factor, \u03c40, of 5(1) \u00d7 10\u20136 s and an energy gap of only 5.1(5) K (3.5 cm\u20131). While the origin of the relaxation process will be discussed in more detail below, it is important at this stage to note the unusually small energy barrier and the large value of \u03c40 .The magnetization dynamics of this manganese nitride complex were probed by ac susceptibility measurements. In the absence of a dc field, the ac data, above 1.8 K and for frequencies up to 10 kHz, display a frequency independent in-phase K .5 cm\u20131. 2 were further analysed by means of an ab initio multireference methodology. A symmetrized model complex was first studied to obtain a qualitative description of the ground state nature of 2 and then these conclusions were corroborated by calculations of the full complex.The magnetic properties of the low-lying states of C3v group. Initial CASSCF calculations for the model system using the ORCA code indicate the following orbital sequence : dxy and dx2\u2013y2 at a reference energy (i.e. 0.0 cm\u20131), dz2 at 31\u2009000 cm\u20131 and at 32\u2009500 cm\u20131 leads to the correct spin state ordering and a NEVPT2 correction that preserves the ground state for the model structure. The lower energy orbitals in the CASSCF calculations are still , with a doubly degenerate ground state that corresponds predominantly (81% weight in both wavefunctions) to the and configurations. The next excited state is 7300 cm\u20131 higher in energy (10\u2009100 cm\u20131 in NEVPT2) and is not relevant for discussing the SMM properties of 2. Thus, magnetic anisotropy in this system emerges from the quantum mixing of the degenerate ground state by the spin\u2013orbit coupling (SOC), given that the dx2\u2013y2 and dxy orbitals are connected by the z component of the angular momentum operator.S = 1/2 states, separated by 470 cm\u20131 (676 K). The ground doublet of the model system presents a markedly uniaxial g tensor with gz = 5.15, gx = gy = 0.15. This strong anisotropy is significantly reduced in the full system due to the deviations from trigonal symmetry that breaks the degeneracy between dx2\u2013y2 and dxy orbitals, partially quenching the SOC mixing. In the full complex, the calculated ground state is split to an energy difference of 2103 cm\u20131 calculation including only doublets). This splitting leads to a marked decrease of the g tensor anisotropy of the ground doublet to values of gx = 1.940, gy = 1.942 and gz = 2.674, yielding a gav of 2.185, in satisfactory agreement with the values obtained for magnetization and EPR measurements. Equivalent CASSCF + RASSI calculations performed with MOLCAS code provide similar values with a first excited Kramers doublet at 1932 cm\u20131 (2800 K) and gx = 1.927, gy = 1.933 and gz = 2.790 values , direct and Raman mechanisms.The model complex was constructed from its original geometry, where the aryl groups were replaced with methyl substituents, symmetrizing the structure to the 2, which (i) should be dominated by QTM, direct and/or Raman mechanisms and (ii) cannot involve Orbach processes. With these elements in mind, the experimental relaxation time has been further analysed starting from its field dependence at 1.8 K (\u03bcBH \u226a kBT), most of the processes inducing a magnetization relaxation are weakly field dependent and thus they have been included as constant, k(T), in eqn (1).B1 = 24\u2009800(50) s\u20131, B2 = 15.6(5) T\u20132 and k(T) = 5427 s\u20131) confirming the key role of the quantum tunnelling of the ground doublet in the relaxation mechanism, in agreement with the theoretical predictions (TH4 term in eqn (1). The fit of the experimental data ) and including thermally active processes, which are either thermally activated (Orbach) or following a power law of the temperature for Raman mechanisms (with exponents ranging from 1 to more than 9).The temperature dependence of the relaxation at 0.45 T was analysed analogously, considering n) of 2.93(5) (with b = 1105 s\u20131 K\u20132.93 and \u03c4QTM fixed at 1.67 \u00d7 10\u20134 s). As discussed recently by Sessoli etal. for an ST = \u00bd VIV complex,ca. 2 \u00d7 10\u20134 s. Nevertheless, this relaxation mechanism is clearly assisted by Raman processes that rationalize the thermal dependence of the relaxation time.Remarkably, eqn (2) is able to reproduce the experimental data with a single power law and an exponent (iv) complex PhB(MesIm)3Mn PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N (2) is a rare example of a low spin (S = 1/2) d3 complex. Its degenerate electron configuration is subject to a Jahn\u2013Teller distortion, which is manifested in 2 by bending of the B\u2013Mn\u2013N vector, similarly to the isoelectronic Fe(v) complex, +.\u20131 above the ground state, SMM properties observed by ac susceptibility measurements cannot rely on an Orbach mechanism and even if the traditional semi-logarithm \u03c4 vs. T\u20131 presentation of the experimental data could suggest the contrary. A detailed analysis of the field and temperature dependence of the relaxation time supports the theoretical CASSCF + RASSI calculations, and highlights the key role of the quantum tunnelling mechanism in the slow dynamics of the magnetization in this S = \u00bd species. Additionally, the signature of Raman processes could be detected in the thermal variation of the relaxation time. Since theoretically the Jahn\u2013Teller distortion significantly activates the quantum tunnelling mechanism, we anticipate that complexes where the structural distortion is smaller than in 2 will have much larger relaxation times. Investigations aimed at testing this hypothesis are currently in progress.Structural and spectroscopic methods reveal that the Mn(Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "EPR spectroscopy and DFT calculations show that the site of reduction of porphyrinato gold(iii) complexes depends on the counterions X, the meso substituents R and the solvent. Meso tetraarylporphyrinato gold(iii) cations bearing different substituents at the aryl substituents were prepared and characterised. Their reversible one-electron reductions were studied by (spectro)electrochemical means as well as by selective chemical one-electron reduction using cobaltocene. The preferred location of the spin density, namely gold centred or porphyrin centred, was probed by electron paramagnetic resonance spectroscopy as well as by density functional theory calculations (spin densities). In all cases studied experimentally and theoretically, the gold(ii) valence isomer (5d9 electron configuration) is preferred over the porphyrin \u03c0 radical anion. In the hexafluorophosphate salt of the nitro derivative a further nitro \u03c0 radical anion valence isomeric species is significantly populated. In the presence of chloride ions this nitro \u03c0 radical anion/AuII valence isomeric equilibrium evolves towards the porphyrin \u03c0 radical anion. The electronic structures of the nitro \u03c0 radical and the AuII \u03c3 radical valence isomers could be calculated by DFT methods. The electron transfer pathway between the nitro \u03c0 radical anion and the AuII valence isomer is well described by the location of the hexfluorophosphate counterion, the Au\u2013N distances , the symmetric stretching mode of the NO2 substituent and a meso-nitrophenyl rotation. The specific geometric and electronic properties of the favoured gold(ii) \u03c3 radical valence isomer, namely counterion dislocation and \u03c3 symmetry of the redox orbital, might stabilise charge-shifted states [(gold(ii) porphyrin)-donor\u02d9+] by retarding the back electron transfer to give the ground state (gold(iii) porphyrin)-donor. This will guide the design of (photo-induced) electron transfer pathways with tetraarylporphyrinato gold(iii) complexes as electron acceptors. Based on early UV/Vis spectroscopic and theoretical studies the products of the reduction of gold(iii) porphyrins had been described as porphyrin-centred \u03c0 radical anions.H]+[A to HA is metal centred giving gold(ii) porphyrins . Disproportionation and dimerisation of [AuII(en)2]2+2+D has been suppressed by encapsulation in the pores of a zeolite (en = ethylenediamine).15The site of gold(rphyrins .9 Only aii) porphyrin HA (iii) porphyrin cation +[AH] with the strongly reducing naphthalene radical anion yielded a broad EPR resonance centred at gav = 2.06.197Au has been reported for the central g line .ii) complex B of hematoporphyrin IX with g\u22a5 = 2.035, g\u2225 = 1.970 and A\u22a5(197Au) = A\u2225(197Au) = 15 G at 130 K suggesting a less pronounced metal character (+]*[BF4] followed by disproportionation to AuIII and Au0 was unsuccessful as well (tht = tetrahydrothiophene).+[2a] was prepared via metallation and ion exchange of nitroporphyrin ester Ia to give 6][1a] with SnCl2/HCl to give the aurated amino-substituted porphyrin [2a]Cl (iii) ion was neither reduced nor removed. Hence, the AuIII porphyrins are stable under protic conditions. Counterion exchange of [Au(porph)][AuCl4] or [2a]Cl with KPF6 yielded the corresponding soluble hexafluorophosphate salts which are conveniently purified by column chromatography.Auration of the amino-substituted porphyrin n [2a]Cl . During [1a][PF6]\u2013[3a][PF6] are sufficiently soluble in dichloromethane. However, THF is required for acids 6][4b][PF and 6][4c][PF and even methanol is necessary for 6][4a]\u2013 counterions show the characteristic septet at \u03b4 = \u2013144 ppm in the 31P NMR spectra. Upon auration the characteristic high-field pyrrol NH resonances of the free-base porphyrin disappear. Furthermore, auration of the free-base porphyrins consistently shifts the pyrrole CH proton resonances to lower field by 0.5 ppm, in accordance with the positive charge of the metal centre. In the IR spectra, characteristic absorptions for group vibrations are found for the ester, amine, amide, nitro, trifluoromethyl and acid substituents at around 1719, 1618, 1690, 1520/1346, 1324 and 1716 cm\u20131, respectively. The [PF6]\u2013 counterions display absorptions for the PF stretching and deformation modes at 835\u2013843 and 556\u2013558 cm\u20131, respectively. ESI mass spectra fully confirm the integrity and stability of the complex cations displaying peaks at m/z values corresponding to the intact complex cation (see Exp. section).Ester-substituted complexes 3 = OnBu-substituted complex +[4b] all gold(iii) porphyrinato complexes +[1a], +[2a], +[3a], +[4a] and +[4c] show hypsochromically shifted Soret bands as compared to their corresponding free-base porphyrins Ia, IIa, IIIa, IVa and IVc porphyrinato complexes are non-emissive at room temperature in fluid solution as exemplarily checked for 6][1a][3a][PF and 6][4c][PF.With the exception of the electron-rich R+[1a], +[2a], +[3a], +[4b] and +[4c] 10\u20133 M in 0.1 M [nBu4N][PF6]/THF solution than +[Au(TPP)] to Au(TPP) (\u20130.97 V), while +[2a] (\u20130.99 V) is more difficult to reduce. Similarly, the potentials shift to more negative values in the series +[4c] (\u20131.00 V), +[4a] (\u20131.02 V) and +[4b] (\u20131.08 V), which is again explicable by the increasing electron donating nature of the substituents . Similar to the corresponding free-base porphyrins the shifts are only small.2 substituent . The nitro derivative +[1a] shows even further reversible reductions. Hence, one of the +[1a] reductions might be associated to the nitro substituent itself (vide infra).Several reversible reductions are observed for cations [1a]+, a+, [3a]+solution . For solobserved , Table 1KC > 1010 for the neutral complexes.The differences between the first and second reduction potentials amount to 0.60\u20130.68 V which corresponds to very high comproportionation constants of iii) complexes were reduced electrochemically to the neutral species in an optically transparent thin layer electrochemical (OTTLE) cell using THF as solvent (MeOH for +[4a]). In all cases, isosbestic points were observed corroborating the reversible nature of the first reduction process (+[Au(TPP)]/Au(TPP) process in THF porphyrin complexes were dissolved in CH2Cl2 (6]\u2013[1c][PF6][1a][4b][PF/6][4c][PF) or MeOH (6][4a] by CoCp2 in CH2Cl2 shows a well-resolved EPR pattern which could be reasonably simulated by a rhombic g tensor with hyperfine interaction to a single 197Au nucleus scale\" fill=\"currentColor\" stroke=\"none\">; natural abundance 100%) and superhyperfine coupling to four 14N nuclei . The high resolution allows a very good estimation of the high-field parameters while the low-field parameters are less well-resolved (Cu(TPP) complex scale\" fill=\"currentColor\" stroke=\"none\">; combined natural abundance 100%; g1 = 2.197, g2 = g3 = 2.054)A1 *[C with strongly electron donating meso substituents at the gold porphyrin a much larger hfc to 197Au has been reported [A1(197Au) = 180 G].2+D with the pure \u03c3 donor ligand ethylenediamine features a significantly larger hyperfine coupling to 197Au than Au(TPP) as well.Indeed, resolved , Fig. 4. = 197 G d] is sig1.000000,.000000 s1a\u20133a the broad EPR resonance corresponding to the AuII valence isomer is less well resolved due to the lower symmetry and hence different superhyperfine interactions . These data fit to gold(iii) porphyrin radical anions 2a\u2032 and 3a\u2032. For 4a\u20134c prepared in THF or MeOH, the corresponding gold(iii) porphyrin radical anions 4a\u2032, 4b\u2032 and 4c\u2032 are only present in negligible amounts (ii) valence isomers 2a\u20134c and their corresponding porphyrin radical anions 2a\u2032\u20134c\u2032 is in favour of the gold(ii) isomers. The very strong preference of 4a\u20134c over 4a\u2032\u20134c\u2032 independent of the meso substituents might be due to a solvent effect overwhelming the substituent effects. Indeed, in THF or in MeOH solvent-separated ion-pairs +//[PF6]\u2013[4a\u20134c] should be present while in CH2Cl2 solution contact ion pairs of 6][2a][PF or 6][3a][1a][PF in CH2Cl2 . This is in good accordance with a nitroarene radical anion.1a\u2032\u2032. The radical distribution 1a\u2009:\u20091a\u2032\u2009:\u20091a\u2032\u2032 is estimated as 78\u2009:\u20093\u2009:\u200919. The decomposition into the component spectra is displayed in the ESI.nBu4N]Cl to the solution prior to reduction of the gold(iii) porphyrin with CoCp2. No significant changes are observed for Au(TPP), 3a (CH2Cl2) or 4c (THF) in the presence of chloride. However, the presence of chloride transforms the 1a\u2009:\u20091a\u2032\u2009:\u20091a\u2032\u2032 radical mixture almost completely into a 1a\u2009:\u20091a\u2032 mixture (65\u2009:\u200935) as only the gold(ii) resonance and the porphyrin radical anion resonance are observed under these conditions radical (1a), the porphyrin based \u03c0 radical (1a\u2032) and a further nitro group based \u03c0 radical (1a\u2032\u2032). Assuming, that rapid freezing does not strongly affect the equilibria of valence isomers, we can conclude that the environment, namely anions and the solvent, appears to influence these valence isomeric equilibria significantly. The substituents influence the equilibria as well, especially, when a strongly electron accepting nitro group is present. A conceivable intervalence transition between 1a and 1a\u2032/1a\u2032\u2032 is not detected in the UV/Vis spectrum by comparison with the spectra of 2a and 3a radicals 1a\u20134c, the valence isomeric equilibrium 1a/1a\u2032\u2032 and the effect of counterions will be addressed by theoretical methods in the next section.The electronic structure of the gold(iii) porphyrins +[Au(TPP)], +\u2013[4c]+[1a] and the structures of all corresponding neutral species Au(TPP), 1a\u20133a and 4a\u20134c were optimised by DFT methods complexes and their neutral congeners are found in the Au\u2013N distances which increase by ca. 4% from 2.051 to 2.124 \u00c5 in all cases (B2u) distortion is noted in the neutral complexes bond distances which is not observed. The calculated Mulliken spin densities are in full accordance with these structural parameters. In all neutral complexes the majority of the spin density is located at the metal centre (Mulliken spin density at Au: 0.44), especially in the 5dx2\u2013y2 orbital . Compared with the isoelectronic Cu(TPP) the spin densities are more delocalised onto the nitrogen atoms which is in agreement with the EPR results as well optimised structure of 1a\u2032\u2032 as shown in 1a\u2032\u2032 are fully consistent with a gold(iii) oxidation state (+[1a] and 1a the C6H4NO2 torsion angle with respect to the porphyrin plane C5\u2013C12\u2013C38\u2013C43 is significantly reduced from 66.1\u00b0 and 62.4\u00b0 to 50.8\u00b0 suggesting a conjugative electron withdrawing effect of the gold(iii) porphyrin as expected for a \u03c0-centred radical. The spin density is mainly located at the NO2 substituent and partially delocalised over the \u03c0-system of the porphyrin. The Mulliken spin density at the gold atom in 1a\u2032\u2032 is essentially zero but only hydrogen-bonded to two CH groups of the aryl substituents fully agree with a gold(iii) porphyrin but not with a gold(ii) porphyrin . The N\u2013O distances have increased from 1.284 \u00c5 in 6][1a\u00b7\u00b7\u00b7PF\u2013 to 1.315 \u00c5 in 6]\u2013[1a\u2032\u2032\u00b7\u00b7\u00b7PF as expected for population of N\u2013O antibonding orbitals. The spin density is largely confined to the NO2 substituent and partially delocalized to the \u03c0-system of the porphyrin. The C5\u2013C12\u2013C38\u2013C43 torsion angle of the nitrophenyl substituent decreases from 61.4\u00b0 (6]\u2013[1a\u00b7\u00b7\u00b7PF) to 50.2\u00b0 (6]\u2013\u2013[1a\u00b7\u00b7\u00b7PF and 6]\u2013[PF6] as the supporting electrolyte in THF (MeOH). Potentials are given relative to the ferrocene/ferrocenium couple. Spectroelectrochemical experiments were performed using a thin layer quartz glass (path length 1 mm) cell kit equipped with a Pt gauze working electrode, a Pt counter electrode and a Ag/AgNO3 reference electrode . X-band CW EPR spectra were measured on a Miniscope MS 300 . g-Values are referenced to external Mn2+ in ZnS . Simulations were performed with the program package EasySpin.Porphyrins H2Cl2) was employed. No (symmetry) constraints were imposed on the molecules, except for the NO distance constraint for 1a\u2032. The presence of energy minima of the ground states was checked by analytical frequency calculations.Density functional calculations were carried out with the Gaussian09/DFT seriesiii) porphyrin complex (c = 5 \u00d7 10\u20133 M) in CH2Cl2 (6][Au(TPP)][1a][2a][3a][4a][PF) or THF was treated with 0.95 equivalents of cobaltocene CoCp2. The X-band EPR spectrum of the sample was measured immediately after freezing the solution to 77 K. The effect of chloride was measured by addition of 2.0 equivalents of [nBu4N]Cl prior to the reduction.Under an inert atmosphere a solution of the respective gold(iii) and sodium acetate were dissolved in glacial acetic acid (20 mL). The reaction mixture was heated to reflux for 20 h, allowed to cool to room temperature, and diluted with dichloromethane (100 mL). The mixture was washed with water (2 \u00d7 50 mL), saturated sodium carbonate solution (2 \u00d7 50 mL) and water (1 \u00d7 50 mL), dried over anhydrous magnesium sulfate and filtered. The filtrate was evaporated to dryness and the residue dissolved in dichloromethane (50 mL). The organic phase was stirred with a saturated aqueous solution of potassium hexafluorophosphate (10 mL) for 72 h. The mixture was diluted with dichloromethane (100 mL) and washed with water (2 \u00d7 50 mL), dried over anhydrous magnesium sulfate, and filtered. The filtrate was removed under reduced pressure and the residue purified by chromatography over silica to yield 6][Au(TPP)]+. HR-MS (ESI): m/z 809.1993 . CV : E\u00bd/V \u20132.350, \u20131.650, \u20130.975.5,10,15,20-Tetraphenylporphyrin , potassium tetrachloridoaurate, potassium tetrachloridoaurate(iii) and sodium acetate were dissolved in glacial acetic acid (40 mL). The reaction mixture was heated to reflux for 22 h, allowed to cool to room temperature, and diluted with dichloromethane (200 mL). The mixture was washed with water (2 \u00d7 100 mL), saturated aqueous sodium carbonate solution (2 \u00d7 100 mL) and water (1 \u00d7 100 mL), dried over anhydrous magnesium sulfate and filtered. The filtrate was evaporated to dryness and the residue dissolved in dichloromethane (100 mL). The organic phase was stirred with a saturated aqueous solution of potassium hexafluorophosphate (20 mL) for 72 h. The mixture was diluted with dichloromethane (100 mL) and washed with water (2 \u00d7 50 mL), dried over anhydrous magnesium sulfate, and filtered. The filtrate was removed under reduced pressure and the residue purified by chromatography over silica to yield 6][1a]+. HR-MS (ESI): m/z 912.1905 . CV : E\u00bd/V \u20132.300, \u20131.795, \u20131.560, \u20130.920.10,20-Di(phenyl)-15-(4-(methoxycarbonylphenyl))-5-(4-nitrophenyl)porphyrin iii) hexafluorophosphate 6][1a] to yield 6][2a]+. HR-MS (ESI): m/z 882.2163 . CV : E\u00bd/V \u20132.500 (irrev.), \u20132.280, \u20131.645, \u20130.990.gold(N-Acetylaminophenyl))-10,20-di(phenyl)-15-(4-(methoxycarbonylphenyl))porphyrin IIIa , potassium tetrachloridoaurate(iii) and sodium acetate were dissolved in glacial acetic acid (20 mL). The reaction mixture was heated to reflux for 24 h, allowed to cool to room temperature, and diluted with dichloromethane (100 mL). The mixture was washed with water (2 \u00d7 50 mL), saturated aqueous sodium carbonate solution (2 \u00d7 50 mL) and water (1 \u00d7 50 mL), dried over anhydrous magnesium sulfate and filtered. The filtrate was evaporated to dryness and the residue dissolved in dichloromethane (50 mL). The organic phase was stirred with a saturated aqueous solution of potassium hexafluorophosphate (10 mL) for 72 h. The mixture was diluted with dichloromethane (50 mL) and washed with water (2 \u00d7 50 mL), dried over anhydrous magnesium sulfate, and filtered. The filtrate was removed under reduced pressure and the residue purified by chromatography over silica to yield 6][3a]+. HR-MS (ESI): m/z 924.2229 . CV : E\u00bd/V \u20132.490 (irrev.), \u20132.300, \u20131.630, \u20130.990.5-(4-(N-Acetylaminophenyl))-10,20-di(phenyl)-15-(4-(carboxyphenyl))porphyrin IVa , potassium tetrachloridoaurate(iii) and sodium acetate were dissolved in glacial acetic acid (20 mL). The reaction mixture was heated to reflux for 24 h, allowed to cool to room temperature and diluted with dichloromethane (100 mL). The mixture was washed with water (2 \u00d7 50 mL), saturated aqueous sodium carbonate solution (2 \u00d7 50 mL) and water (1 \u00d7 50 mL), dried over anhydrous magnesium sulfate and filtered. The filtrate was evaporated to dryness and the residue dissolved in dichloromethane (50 mL). The organic phase was stirred with a saturated aqueous solution of potassium hexafluorophosphate (10 mL) for 72 h. The mixture was diluted with dichloromethane (50 mL) and washed with water (2 \u00d7 50 mL), dried over anhydrous magnesium sulfate, and filtered. The filtrate was removed under reduced pressure and the residue purified by chromatography over silica to yield 6][4a]+. HR-MS (ESI): m/z 910.2115 . CV : E\u00bd/V \u20131.030.5-(4-(N-Acetylaminophenyl))-10,20-di((4-butoxy)phenyl)-15-(4-(carboxyphenyl))porphyrin IVb , potassium tetrachloridoaurate(iii) and sodium acetate were dissolved in glacial acetic acid (40 mL). The reaction mixture was heated to reflux for 24 h, allowed to cool to room temperature, and diluted with dichloromethane (200 mL). The mixture was washed with water (2 \u00d7 100 mL), saturated aqueous sodium carbonate solution (2 \u00d7 100 mL) and water (1 \u00d7 100 mL), dried over anhydrous magnesium sulfate and filtered. The filtrate was evaporated to dryness and the residue dissolved in dichloromethane (100 mL). The organic phase was stirred with a saturated aqueous solution of potassium hexafluorophosphate (20 mL) for 72 h. The mixture was diluted with dichloromethane (100 mL) and washed with water (2 \u00d7 50 mL), dried over anhydrous magnesium sulfate and filtered. The filtrate was removed under reduced pressure and the residue purified by chromatography over silica to yield 6][4b]+. HR-MS (ESI): m/z 1054.3218 . CV : E\u00bd/V \u20132.450, \u20131.745, \u20131.070.5-(4-(N-Acetylaminophenyl))-10,20-bis(4-(trifluoromethylphenyl))-15-(4-(carboxyphenyl))porphyrin IVc , potassium tetrachlorido aurate(iii) , and sodium acetate were dissolved in glacial acetic acid (20 mL). The reaction mixture was heated to reflux for 24 h, allowed to cool to room temperature and diluted with dichloromethane (100 mL). The mixture was washed with water (2 \u00d7 50 mL), saturated aqueous sodium carbonate solution (2 \u00d7 50 mL) and water (1 \u00d7 50 mL), dried over anhydrous magnesium sulfate and filtered. The filtrate was evaporated to dryness and the residue dissolved in dichloromethane (50 mL). The organic phase was stirred with a saturated aqueous solution of potassium hexafluorophosphate (10 mL) for 72 h. The mixture was diluted with dichloromethane (50 mL) and washed with water (2 \u00d7 50 mL), dried over anhydrous magnesium sulfate and filtered. The filtrate was removed under reduced pressure and the residue purified by chromatography over silica to yield 6][4c]+. HR-MS (ESI): m/z 1046.1863 . CV : E\u00bd/V \u20132.300, \u20131.590, \u20130.990.5-(4-(meso-tetraaryl substituted AB2C porphyrins with KAuCl4 in the presence of HOAc/NaOAc cleanly gives the corresponding gold(iii) porphyrinato complex cations. Amino-substituted porphyrins are N-acetylated under these conditions and have to be prepared from the corresponding nitro-substituted gold(iii) porphyrins by reduction with SnCl2/HCl. The gold(iii) complexes can be reduced at least three times. The potentials slightly depend on the electron withdrawing and donating nature of the substituents. The first reduction is addressed by UV/Vis spectroelectrochemistry and by EPR spectroscopy. Upon one-electron reduction, the Soret band experiences a small bathochromic shift. The intensity of the Soret band of the electron rich complexes +[2a] (R2 = NH2) and +[4b] (R3 = OnBu) slightly increases upon reduction while all other neutral complexes feature less intense Soret bands as compared to their parent AuIII complexes. These spectral data clearly suggest the presence of an unreduced porphyrinato ligand in all cases under these conditions. Chemical one-electron reduction of the porphyrinato gold(iii) hexafluorophosphate salts by cobaltocene yields the corresponding AuII porphyrin complexes with a characteristic EPR pattern displaying hyperfine coupling to 197Au and 14N. The degree of 197Au hfc and g anisotropy places the gold contribution to the spin density in (tetraphenylporphyrinato)gold(ii) complexes in between that of [Au(en)2]2+ porphyrins bearing carboxylic acid, amine and amide substituents, as introduced in this report, with light-harvesting porphyrins and electron donating porphyrins via amide connectivityAuration of (en)2]2+ and the Supplementary informationClick here for additional data file."} +{"text": "Synthesis of strained five-membered cyclic sulfamides has been achieved for the first time by intramolecular 1,5-C(sp3)\u2013H amination via Co(ii)-based metalloradical catalysis. ii)-based metalloradical catalysis (MRC) proves effective for intramolecular 1,5-C\u2013H amination of sulfamoyl azides under neutral and nonoxidative conditions, providing a straightforward approach to access strained 5-membered cyclic sulfamides with nitrogen gas as the only byproduct. The metalloradical amination system is applicable to different types of C(sp3)\u2013H bonds and has a high degree of functional group tolerance. Additional features of the Co(ii)-catalyzed 1,5-C\u2013H amination include excellent chemoselectivity toward allylic and propargylic C\u2013H bonds. The unique reactivity and selectivity profile of the Co(ii)-catalyzed 1,5-C\u2013H amination is attributed to the underlying radical mechanism of MRC.Co( If successful, the catalytic C\u2013H amination process \u2013H amination to prepare N-heterocycles of variable ring size and functionality.via metal-catalyzed intramolecular C\u2013H amination.3)\u2013H amination for the formation of 5-membered cyclic sulfamides is a challenging process, which is presumably attributable to the potentially strained [3.1.0]-bicyclic transition state associated with the asynchronous concerted mechanism that is shared by most catalytic C\u2013H amination systems via metallonitrene intermediates.A) followed by low-barrier radical substitution,ii)-based metalloradical system exhibits excellent chemoselectivity and high functional group tolerance.A number of different metal-based catalytic systems have been developed for regioselective intramolecular C\u2013H amination via MRC \u2013H bond, affording the strained 5-membered cyclic sulfamide 2a in 51% yield. Further optimization experiments indicated that [Co(P1)], which is supported by the D2h-symmetric amidoporphyrin 3,5-DitBu-IbuPhyrin (P1), was a superior metalloradical catalyst for the radical C\u2013H amination reaction, leading to the formation of the desired 2a in 90% yield (P1)] over [Co(TPP)] is ascribed to the stabilization of the key \u03b1-Co(iii)-aminyl radical intermediate by the amide functionalities through hydrogen-bonding interaction (A).At the outset of this project, the sulfamoyl azide via MRC . Initial0% yield . The enheraction , A.5a,b,P1)]-based metalloradical system was shown to be effective for intramolecular 1,5-C(sp3)\u2013H radical amination of a wide range of sulfamoyl azide substrates (ii)-based catalytic system could efficiently aminate \u03b1-C(sp3)\u2013H bonds of heteroaromatic rings such as furan (entry 5) and thiophene (entry 6), without complication from potential reactions with the heteroatoms. As exemplified by the high-yielding formation of trans-cyclic sulfamide 2c, excellent diastereoselectivity could be achieved (entry 3). The metalloradical amination by [Co(P1)] could also be applied for non-benzylic C\u2013H substrates, as demonstrated with the successful formation of the cyclic sulfamide 2g and bicyclic sulfamide 2h in respectable yields (entries 7 and 8), along with the corresponding 6-membered structure product formation.3)\u2013H substrates, such as \u03b1-C\u2013H bonds of esters and amides, could be aminated smoothly, producing \u03b1,\u03b2-diamino acid derivatives (entries 9 and 10).3)\u2013H bonds as well (entries 7 and 11). It is notable that the \u03b1,\u03b2-diamino acid derivative 2k bearing a quaternary \u03b1-carbon center could be synthesized in near quantitative yield (entry 11). Different N-substituents in the azide substrates were effectively tolerated in the C\u2013H amination process. For example, sulfamoyl azides containing both electron-donating and electron-withdrawing N-substituents, such as N-benzyl (entry 1), N-methyl (entry 2), N-Boc groups (entry 11), and N-4-methoxybenzyl (entry 15), groups proved to be suitable substrates.Under the optimized conditions, the [Co]-catalyzed 1,5-C\u2013H radical amination system exhibited excellent chemoselectivity towards allylic C\u2013H bonds without affecting the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C \u03c0 bonds.2l and 2m without observation of the corresponding aziridination products (entries 12 and 13). Furthermore, this amination process was shown to be stereospecific regarding the stereochemistry of the alkene units as exemplified by the catalytic reactions of both trans- and cis-alkene-derived sulfamoyl azide substrates (entries 14\u201316). Under the standard conditions, the expected allylic C\u2013H amination products 2n, 2o and 2p were formed in high yields with excellent stereospecificity as well as chemoselectivity. The fact that no olefin isomerization was observed during these catalytic amination reactions suggests the 5-exo-tet radical cyclization of the corresponding \u03b5-Co(iii)-allylic radical (B to 2) proceeds with a low barrier and even faster than the facile trans- and cis-C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C \u03c0 bond isomerization.ii)-based MRC is among the few catalytic systems that are effective for amination of propargylic C\u2013H bonds without affecting the electron-rich C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C \u03c0 bonds.2t in 89% yield.The [Co]-catalyzed 1,5-C\u2013H amination made it possible for late-stage functionalization of complex molecules in a predicable fashion. For example, when stigmasterol-based azide 1u, which was directly prepared from the corresponding amine by a one-step procedure scale\" fill=\"currentColor\" stroke=\"none\">C bonds was chemoselectively achieved, providing the fused multicyclic sulfamide 2u in 70% yield ], the propargylic C\u2013H bond was selectively aminated to afford the deoxyuridine-derived 5-membered cyclic sulfamide 2v in 95% yield ).The demonstrated chemoselectivity and functional group tolerance of is an effective catalyst with the capability of activating a broad scope of sulfamoyl azides for intramolecular 1,5-amination of different types of C(sp3)\u2013H bonds with high stereospecificity, providing straightforward access to the potentially bioactive 5-membered cyclic sulfamide compounds in high yields. The Co(ii)-based catalytic system can be simply operated under neutral and non-oxidative conditions without the need for any additives, generating nitrogen gas as the only byproduct. Furthermore, this 1,5-C(sp3)\u2013H amination process features excellent chemoselectivity and functional group tolerance, allowing for late-stage functionalization of complex molecules. The success in addressing this challenging amination process by Co(ii)-MRC is believed to be directly related to the underlying radical mechanism involving the key \u03b1-Co(iii)-aminyl radical intermediate.In summary, by applying the concept of metalloradical catalysis (MRC), a new approach has been successfully demonstrated for addressing the challenges of intramolecular 1,5-C(spSupplementary informationClick here for additional data file.Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "We report an unusual reaction design in which a chiral bis-cyclometalated rhodium(iii) complex enables the stereocontrolled chemistry of photo-generated carbon-centered radicals and at the same time catalyzes an enantioselective sulfonyl radical addition to an alkene. iii) complex enables the stereocontrolled chemistry of photo-generated carbon-centered radicals and at the same time catalyzes an enantioselective sulfonyl radical addition to an alkene. Specifically, employing inexpensive and readily available Hantzsch esters as the photoredox mediator, Rh-coordinated prochiral radicals generated by a selective photoinduced single electron reduction are trapped by allyl sulfones in a highly stereocontrolled fashion, providing radical allylation products with up to 97% ee. The hereby formed fragmented sulfonyl radicals are utilized via an enantioselective radical addition to form chiral sulfones, which minimizes waste generation.We report an unusual reaction design in which a chiral bis-cyclometalated rhodium( In this respect, some elegant protocols have been disclosed in enantioselective transformations of photo-generated allylic carbon radicals. In 2013, MacMillan and co-workers introduced a radical\u2013radical recombination process by the combination of a chiral amine and photoredox catalyst in which only one example with moderate enantioselectivity was reported showing the challenge of stereocontrol over such radical intermediates 15 as a photoredox mediator for the generation of radical species under mild conditions generates the key Rh-coordinated radical intermediate B, which is trapped by an electron-deficient allyl sulfone 2 delivering the secondary radical intermediate C. The subsequent fragmentation11 of C provides the sulfonyl radical E and enolate intermediate D, the latter of which yields the C\u2013C bond formation product 3 upon protonation. Meanwhile, the sulfonyl radical E undergoes a stereocontrolled radical addition16 to A in a reversible fashion11 and a subsequent HAT followed by ligand exchange provides the C\u2013S bond formation product 4.Our design is based on our recently introduced bis-cyclometalated chiral rhodium-based Lewis acids (LAs)nditions . Initial1 to ensure a highly chemoselective reduction. Secondly, the radical trapping and subsequent fragmentation process should be fast enough to compete with the protonation of intermediate B which would generate undesirable free \u03b2-carbonyl carbon radicals. The reaction of such free radicals with 2 would compromise the enantioselectivity of product 3. Therefore, this design with the utilization of the leaving sulfonyl radicals, which otherwise would lead to by-products, is very attractive not only from the perspective of green and sustainable chemistry17 but also for suppressing side reactions of the sulfonyl radical and shifting the equilibrium of a potentially reversible radical fragmentation.11Two key challenges are needed to be solved to achieve a high asymmetric induction. Firstly, a robust and effective chiral Lewis acid catalyst is required to control the stereochemistry of two mechanistically distinct processes as well as reduce the reduction potential of substrate N-acylpyrazole 1a with allyl sulfone 2a under visible light irradiation employing a stoichiometric amount of the Hantzsch ester HE-1 as the photoredox mediator and reductant.15 Although our well-established iridium catalyst \u0394-IrS18 could not give any detectable product . Interestingly, the related \u0394-RhO provided 3a in much higher ee indicating that the mechanism differs from our previous reports about Giese-type radical reactions in which RhS, which features a higher steric congestion, works better than RhO. Notably, our recently developed chiral rhodium complexes show a unique reactivity for this reaction. Other Lewis acids such as Sc(OTf)3 gave very low efficiency while LiBF4 could not even catalyze the process (entries 4 and 5) and no conversion was observed without catalyst (entry 6). Other substituted HE species also worked very well (entries 7 and 8), whereas DIPEA, which is widely used as a sacrificial reductant in photoredox catalysis, could not accomplish the transformation (entry 9). These results highlight the multiple functions of the HE in our system acting as a photoredox mediator as well as an electron donor and proton source. Furthermore, on illumination with blue LEDs, which do not emit any UV light along with the recycled C\u2013S formation products 4a\u2013h in good yields and ee (up to 89% ee) (entries 1\u20138). Intriguingly, a lower yield and slightly lower ee were observed for the radical functionalization product 3b when allyl sulfone bearing a less electron deficient ester group was employed (compare entries 9 with 1). It is noteworthy that functional groups including a C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C triple bond, a C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C double bond and an imide are well tolerated under these mild conditions (entries 11\u201313). As a limitation, substrates with a long chain at the \u03b2-position (1b\u2013d) produced the radical allylation products 3f\u2013h with decreased ee (eqn (1)) and \u03b2-aryl \u03b1,\u03b2-unsaturated N-acylpyrazole could not afford any expected product. Furthermore, the alkenyl sulfone 5 was proven to be competent, providing the radical alkenylation product 6 in 54% yield with 93% ee (eqn (2)).With the optimized conditions at hand, we next investigated the substrate scope with respect to radical acceptors . A wide 1a + 2a \u2192 3a + 4a in the presence of a series of common chemical functionalities as additives containing azido, cyano, and carbonyl groups that are vulnerable to reductive conditions can be recovered in high yields under the standard conditions (entries 1\u20134). Importantly, several heterocycles which might competitively coordinate to the catalyst did not erode the enantiomeric excess of products (entries 3\u20139). Several natural products, including coumarin, caffeine, and (\u2013)-citronellol, were found to have little influence on the reaction outcomes (entries 7\u201310). Furthermore, N-acylpyrazoles known as a useful and reactive synthetic building block can be easily converted into other compounds, such as alcohols or amides (eqn (3) and (4)). Overall, these results highlight the potential of this protocol for further applications in the synthesis of complex molecules.To further evaluate the functional group tolerance and robustness of this catalytic system, we conducted the reaction nalities and ESI\u2020HE as a visible light harvesting antenna being consistent with recent reports and RhO-1a (\u20131.62 V vs. Fc/Fc+), thus making highly selective SET between RhO-1a and the excited state of HE-1 (E+/HE*)(HE\u02d9 = \u20132.23 V vs. Fc/Fc+) feasible or 2,2,6,6-tetramethyl-piperidinooxy (TEMPO) as radical scavengers. When the reaction was monitored by electron paramagnetic resonance (EPR) using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as a free radical spin-trapping agent, mixed signals containing two radical species were observed, one of which was identified as a phenyl sulfonyl radical (Fig. S8 in ESI22 Finally, a quantum yield of 0.09 was determined for the reaction 1a + 2a \u2192 3a + 4a, which is consistent with the proposed mechanism being devoid of any chain process (see ESI for detailsA number of experiments support the proposed mechanism . Firstly23In conclusion, we here have introduced an unusual reaction scheme in which a chiral rhodium complex enables the catalytic enantioselective functionalization of a photo-generated carbon radical employing cheap and readily available Hantzsch esters as a photoredox mediator and reductant. Intriguingly, in this radical allylation reaction using allyl sulfones as reagents, the generated sulfonyl radical by-product can be trapped by electron deficient alkenes and transformed into valuable enantioenriched S-containing building blocks thereby minimizing waste generation. The simple reaction setup and the mild reaction conditions as well as the demonstrated compatibility with a wide range of functionalities render this robust catalytic system an appealing process. Further investigations on the stereocontrolled chemistry of prochiral radicals are ongoing in our laboratory.There are no conflicts to declare."} +{"text": "Moderate variations in the fuel structure cause large changes in the rate of the back and forth motions experienced by a chemically fuelled catenane-based switch. para-substituents in the order Cl > H > CH3 > OCH3 (\u03c1 = +5.2). Thus, the time required to complete a full cycle was almost two days for the OCH3 derivative and dropped to a few minutes for the Cl derivative. These results show for the first time that the rate of operation of a molecular switch can be regulated by variations in the fuel structure.This work deals with the use of 2-cyano-2-arylpropanoic acids as chemical fuels for an acid\u2013base operated molecular switch that consists of a Sauvage-type catenand composed of two identical macrocycles incorporating a phenanthroline unit. When used as a base promoter of the decarboxylation of propanoic acid derivatives, the switch undergoes large amplitude motion from the neutral catenand to a protonated catenate and back again to the neutral state. The rate of back proton transfer, which determines the rate of the overall process, was markedly affected by While B\u2032 and B\u2032\u2032 are well-defined states with the two phenanthroline subunits tightly held together by interactions with the shared proton, state A is not co-conformationally well-defined. The fuel, 2-cyano-2-phenylpropanoic acid undergoes base-promoted quantitative decarboxylation via a carbanion intermediate, which is rapidly formed (step B\u2032 \u2192 B\u2032\u2032) and slowly transformed (step B\u2032\u2032 \u2192 A) to the \u201cwaste\u201d product 2-phenylpropanonitrile by back proton transfer.It is only lately that molecular machines operated by the irreversible reaction of a single chemical fuel have been reported by uspara positions of 2-cyano-2-(p-chlorophenyl)propanoic acid , 2-cyano-2-(p-methylphenyl)propanoic acid and 2-cyano-2-(p-methoxyphenyl)propanoic acid on the rate of the fuelled switching of catenand 1.Since the stability and reactivity of carbanions are strongly structure dependent,2 were prepared according to the same procedure adopted for the preparation of the parent acid 2 (X = H) (6Compounds (X = H) .62\u2013 bond, the Et3N promoted decarboxylation of the acid derivatives was monitored by 1H NMR spectroscopy in CD2Cl2 at 25 \u00b0C for comparison with the corresponding reaction of the parent acid 2 (X = H). For the latter it has been established2 (X = H) to Et3N generates a hydrogen bonded ion pairinter alia, with the finding that the time required to complete the decarboxylation of 2 (X = H) dropped from about 3 h to 10 min when Et3N was replaced by the proton sponge 1,8-bis(dimethylamino)naphthalene the concentration of free ions is most likely negligibly small.\u00b6Because of the low polarity of dichloromethane becomes the slow step in the decarboxylation of the parent acid 2 (X = H).61H NMR spectra of a 1\u2009:\u20091 mixture of 1 and 2 (X = H) . The exceptional slowness of the proton removal from the tetrahedral cavity defined by the four N atoms1H+ is such a weakly interacting anion as CF3CO2\u2013, the equivalence of the two phenanthroline units in the 1H NMR spectrum in dichloromethane at 25 \u00b0C were monitored by 1H NMR spectroscopy , the spectra showed that in all cases the sole reaction product was the corresponding decarboxylated compound 3, besides the catenane in its original neutral state. Furthermore, the 1H NMR spectra recorded in the course of the reactions confirmed the presence of transient species, characterized by patterns of signals very similar to each other, as well as to those displayed by the reaction of the parent acid (B\u2032\u2032 in 2 (X = H) led us t Quite remarkably, the time required to achieve quantitative transformation to 3 amounts to almost two days for the OCH3 derivative and decreases in the order OCH3 > CH3 > H > Cl, becoming as short as a few minutes for the Cl derivative.The reactions of ent acid . Clearlyd B\u2032\u2032 in are invo2 (X = OCH3) . The trace at t = 2.5 min shows that 1 is no longer in its original, neutral state, but the signals of the ion pair B\u2032\u2032 are still barely perceptible. This means that the major component of the reaction mixture is the salt denoted by B\u2032 in 1H+ and its carboxylate counterion. The concentration of this transient species is still significant at t = 14.5 min and becomes negligibly small in the next spectrum at 34.5 min.The spectra related to the early stages of the sluggish reaction of CH3) see , particu 5.5 ppm , gave in\u03bbmax = 375 nm) belonging to transient species. The latter were identified with intermediates B\u2032\u2032 or, more precisely, with their carbanion components, since the absorbance of 0.30 mM 1H+ is 0.30 at 350 nm, drops to 0.05 at 375 nm and becomes negligibly small at 380 nm and beyond. Indeed, the spectra related to the reaction of 2 (X = OCH3) (B\u2032\u2032 as monitored by 1H NMR spectroscopy (2 (X = CH3 and H) the maximum is reached in about 3 and 1.5 min, respectively, which means that the build-up of B\u2032\u2032 was complete, or very nearly so, when the first 1H NMR spectrum in Fig. S7 and S6,Corroborating evidence came from investigation of the time-dependent UV-vis spectra of the reaction mixtures, which was prompted by the observation that the solutions became yellow in the course of the reactions, but were colourless at the end. The four sets of time-dependent UV-vis spectra are char = OCH3) show thatroscopy . In the 2 (X = OCH3 and CH3), the absorbance growth and subsequent decay were treated as two separate first-order reactions with rate constants k\u2032 and k\u2032\u2032, respectively. k\u2032 and k\u2032\u2032.For the reactions of 1 and 2 (X = OCH3) was varied over the range 0.30\u20131.50 mM , A set of four kinetic runs in which the initial concentration of 0 mM ESI, page S184NBr on the rate of reaction of 0.30 mM 2 (X = H) with equimolar 1 was investigated. UV-vis monitoring of the reactions showed in all cases a rapid growth followed by a slower decay of the absorption band centered at 375 nm, the shape and maximum intensity of which were hardly affected by the added salt, even at the highest salt concentration of 5.0 mM increased with increasing salt concentration and showed a marked tendency to saturate. B\u2032\u2032 (Kass = 1600 M\u20131), which is significantly more reactive than uncomplexed B\u2032\u2032 .1H+Br\u2013 and Bu4N+R\u2013. It should be stressed that this mechanism, which is almost kinetically indistinguishable from that in \u2021An alternative mechanism involves fast and reversible double exchange between ion pair partners, followed by a rate limiting second-order reaction between .In a last set of rate measurements the effect of added Bu Fig. S10. Again, B\u2032\u2032, namely 1H+ and the carbanion derived from 2 (X = H), are brought into an orientation more adapted to proton transfer when B\u2032\u2032 becomes a component of an ion quartet as a result of association with Bu4NBr. However, due to the lack of structural information on the ion quartet, a detailed interpretation of the rate enhancing effect exerted by the added salt does not appear to be accessible.It seems likely that the components of the ion pair 1H NMR and UV-vis spectroscopy strongly supports the operation of the reaction mechanism outlined in B\u2032 and B\u2032\u2032 as reaction intermediates.To sum up, the combination of data from 1H NMR spectroscopy. Firstly, the transient absorption band at 375 nm provided direct evidence of the existence and kinetic behavior of the carbanion intermediates, whose signals in the 1H NMR spectra were hardly visible. Secondly, the kinetics based on UV-vis spectroscopy gave qualitative information about the substituent effects on the rate of step B\u2032 \u2192 B\u2032\u2032. The reactivity order Cl > H > CH3 > OCH3 is the same as that observed in the reactions promoted by Et3N, but the rupture of the R\u2013CO2\u2013 bond is much faster in the reactions promoted by 1 because any stabilization of R\u2013CO2\u2013 by hydrogen bonding is hardly conceivable when 1H+ is the countercation.Data from UV-vis spectroscopy gave pieces of information which could not be obtained from B\u2032\u2032 \u2192 A), whereas only a qualitative reactivity order could be obtained from the 1H NMR spectra. The Hammett plot of log (k\u2032\u2032x/k\u2032\u2032H) vs. \u03c3p reported in \u03c1 = 5.2 (r = 0.98). Such a large \u03c1 value is indicative of a much larger fraction of negative charge on the benzyl carbon atom in the transition state, which is equivalent to saying that the kinetic basicity of the anionic component in B\u2032\u2032 is enhanced by EWGs and depressed by EDGs. Unusual as it might seem, such a behavior is not unprecedented.et al.2 (12 substituents). They found that the rates of protonation of ArCMe PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019NO2\u2013 were enhanced by EWGs and retarded by EDGs (\u03c1 > 0) and concluded that there is a very little delocalization of the fractional negative charge to the oxygen atoms in the transition state, whereas the negative charge is mostly localized on the oxygen atoms of the nitronate anions.Finally, kinetic treatment of the UV-vis data provided a quantitative estimate of the effect of the substituents on the rate of the back proton transfer with Bu4NOH.2 benzylic carbon atom, as well as significant delocalization to the nitrogen atom of the cyano group. In our case, a high negative charge density on the nitrogen atom of the CN group would guarantee a strong electrostatic stabilization of B\u2032\u2032, whose shape is supposed to be one in which the group PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N\u2013 points to the proton embedded in the two-phenanthroline core.Although a cyano group is less effective than a nitro group in stabilizing a negative charge on an adjacent carbon atom, we suggest that a similar interpretation applies to the protonation of our cyano-stabilized carbanions. Accordingly, it seems likely that the canonical structure I , unlike 1H NMR spectra were recorded on a 300 MHz spectrometer. The spectra were internally referenced to the residual proton signal of the solvent at 5.30 ppm. Spectrophotometric measurements were carried out on a diode array spectrophotometer equipped with a thermostated cell compartment. Mass spectrometric measurements were carried out using an ESI-TOF spectrometer.2Cl2 and CD2Cl2 were flushed through basic alumina immediately prior to use. The same batch of solvent was used in each of the sets of kinetic measurements. Et3N was distilled from metallic sodium before use. Catenane 1 was available from a previous investigation.2 (X = H) was available from a previous investigation2 were prepared from the corresponding ethyl esters14All reagents and solvents were purchased at the highest commercial quality and were used without further purification unless otherwise stated. CH1H NMR \u03b4: 7.55\u20137.52 , 7.46\u20137.43 , 3.52 , 1.99 .This compound had a m.p. of 81\u201382 \u00b0C, lit. 81\u201382 \u00b0C.151H NMR \u03b4: 7.46\u20137.43 , 7.24\u20137.22 , 4.05 , 2.36 , 1.97 . 13C NMR \u03b4: 171.2, 139.1, 131.8, 129.8, 125.7, 119.0, 47.7, 24.2, 20.9. UV-vis (CH2Cl2): \u03bbmax (\u03b5) = 230 nm (3200 cm\u20131 M\u20131), 262 nm (400 cm\u20131 M\u20131), 285 nm (200 cm\u20131 M\u20131). ESI-MS (negative-ion-mode): 144 (M \u2013 H+ \u2013 CO2).This new compound had a m.p. of 86\u201388 \u00b0C (dec.). 1H NMR \u03b4: 7.51\u20137.46 , 6.99\u20136.93 , 4.95 , 3.82 , 1.97 . 13C NMR \u03b4: 171.3, 160.0, 127.1, 126.7, 119.2, 114.3, 55.3, 47.1, 23.8. UV-vis (MeOH): \u03bbmax (\u03b5) = 230 nm (13\u2009900 cm\u20131 M\u20131), 274 nm (1600 cm\u20131 M\u20131), 281 nm (1300 cm\u20131 M\u20131). ESI-MS (negative-ion-mode): 160 (M \u2013 H+ \u2013 CO2).This new compound had a m.p. of 87\u201389 \u00b0C (dec.). para-substituent of the 2-cyano-2-arylpropanoic acid used as a fuel. The time taken to complete a full cycle decreased in the order OCH3 > CH3 > H > Cl. It was as long as two days for the OCH3 derivative, and dropped to a few minutes for the Cl derivative. Fine tuning of the motion rate could be further achieved by the addition of Bu4NBr. A detailed kinetic investigation has shown the intermediacy of ion pairs B\u2032 and B\u2032\u2032, which differ in the nature of the anionic component. The negative charge of the anion in B\u2032\u2032 was suggested to be mostly localized on the nitrogen atom of the CN group, and strongly shifted to the benzyl carbon atom in the transition state of the rate-determining proton transfer step.In this work we have described the first example of a chemically operated molecular switch in which the rate of the back and forth motion could be regulated within wide limits by variations in the fuel structure, namely by variations in the nature of the There are no conflicts to declare.Supplementary informationClick here for additional data file."} +{"text": "To date, enantiomerically enriched molecules containing gem-diaryl containing tertiary or quaternary stereogenic centers have been readily accessed by transition metal-catalyzed enantioselective or stereoconvergent aryl transfer reactions. gem-diaryl containing tertiary or quaternary stereogenic centers are present in many natural products and important pharmacophores. While numerous catalytic asymmetric methods enable access to 1,1-diaryl motifs, transition metal-catalyzed asymmetric arylations (TMCAAr) are one of the most powerful methods to prepare enantiopure gem-diarylalkane compounds. The main methodology includes enantioselective 1,2- or 1,4-additions across C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O, C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N and C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bonds by arylmetallic reagents; aryl cross-couplings of olefins, benzylic (pseudo)halides and aziridines; asymmetric aryl substitution reactions of allylic substrates; and isotopic benzylic C\u2013H arylation.Chiral Thus, the development of effective methods to access enantiomerically enriched diaryl structural motifs will play a significant role in both academic and industrial settings. Enantiomerically pure drugs or their precursors are usually produced by the chiral kinetic resolution technique. However, access to 1,1-diarylalkanes with a high level of optical purity using this technique is challenging because little differentiates the two aryl groups installed on the stereogenic center electronically and sterically. This issue can be solved by asymmetric synthetic methods through either stereospecific or enantioselective transformations. In the last few decades, an array of catalytic enantioselective approaches towards the construction of nonracemic gem-diaryl compounds have been developed, including asymmetric Friedel\u2013Crafts reactions, asymmetric aryl transfer reactions (arylations), asymmetric hydrogenation of 1,1-diarylalkenes, asymmetric C\u2013H functionalization of enantiotropic diarylalkanes and so on. Among them, transition metal-catalyzed asymmetric arylations (TMCAArs), which install an aryl group onto the benzylic position of substrates in an enantioselective or stereoconvergent manner, represent the most powerful method. In this field, development of new reactions, chiral ligand families and metal complexes has enabled the precise construction of various chiral diaryl motifs, including dibenzyl alkanes and alkenes, 1,1-diarylmethanols, 1,1-diarylmethylamines and so on. To the best of our knowledge, TMCAAr for the synthesis of gem-diaryl compounds includes nucleophilic 1,2- or 1,4-additions of arylmetallic reagents across C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O, C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N and C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bonds; aryl cross-couplings to olefins, benzylic (pseudo)halides and aziridines; asymmetric aryl substitution reactions of allylic substrates; isotopic benzylic C\u2013H arylation and so on scale\" fill=\"currentColor\" stroke=\"none\">C, C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O and C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N bonds represent a highly efficient method to construct tertiary or quaternary stereogenic centers, concomitant with the formation of Csp3\u2013Csp2 bonds. These transformations are frequently used to prepare important chiral gem-diaryl containing compounds from activated styrene and aryl-substituted carbonyl substrates. Gem-diaryl stereogenic centers are generated in the key step of aryl migratory insertion across the unsaturated C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C(O or N) bonds, followed by the hydrolysis or \u03b2-H elimination of the metal-binding intermediate -catalyzed highly enantioselective 1,4-addition of arylboronic acids to \u03b2-aryl substituted unsaturated carbonyl derivatives using a carvone-derived chiral diene ligand (L1)tert-butyl 3,3-diarylpropanoates were afforded with 89\u201393% ee. Miyaura found that both Rh(i)ii)S,S)-chiraphos ligand are competent catalysts for TMCAAr of \u03b2-aryl-\u03b1,\u03b2-unsaturated ketones and esters (R)-Segphos catalyst to provide enantiomerically pure 4-arylchroman-2-ones. The product, (R)-6-methyl-4-phenylchroman-2-one, was readily converted in two steps into (R)-tolterodine, an important urological drugi) complex of sulfoxide\u2013phosphine was an appropriate catalyst to afford chiral 1,3,3-triarylpropan-1-ones with up to 98% ee.7In 2005, Carreira successfully realized Rh . NonraceN,N-dimethylsulfamoyl imino esters with a new bicyclic bridgehead phosphoramidite ligand (L3). Chiral (Z)-\u03b3,\u03b3-diaryl-\u03b1,\u03b2-dehydroamino esters were afforded with excellent yields and enantioselectivities (75\u201396% ee) .N-heterocyclic carbene ligand (L4) (i) mediated the catalytic cycle that consists of transmetalation/insertion/ligand exchange. Zhou and coworkersL5) efficiently promoted the enantioselective 1,4-addition of chalcones with arylboroxines and a direct 1,4-insertion mechanism was proposed and supported by DFT calculations and natural-abundance 13C KIE experiments , top. Theriments , bottom.2.2L6 could give 69% conversion but a low ee value (\u20137%), while the more sterically hindered L7 gave 28% ee but a low conversion (4%) (L6 and L7 in a 1\u2009:\u20091 ratio could improve the conversion (92%) as well as the enantioselectivity (31%). In 2013, IulianoL8) significantly improved the enantioselectivities (94\u201399%) as well as the yields (82\u201398%) of the desired products , top. Inproducts , bottom.L9). In 2013, Wu and coworkersL10) in the arylation of nitroalkenes with high enantioselectivities (89\u201397%) , right. L11L12 ligand L11L12 , middle.L14) as a chiral ligand, enantioenriched 2,2-diaryl nitroalkanes can be produced in high yields and good enantioselectivities in air induces the protonation of the alkylrhodium intermediate faster than the \u03b2-H elimination process, thus selectively forming the addition product instead of the substitution product. Later on, Xu employed the chiral phosphine\u2013olefin ligand (L16) in the same asymmetric reaction to achieve generally high yields and ee valuesIn contrast to the extensive studies on conjugate arylations of nitroalkene substrates, successful conjugate additions of sulfonyl olefins have rarely been reported.2.3syn-diastereoselective arylfluorination of chromenes with arylboronic acids and selectfluor.S)-4-tert-butyl-2-(2-pyridyl)oxazoline (L17) as the chiral ligand, a wide spectrum of enantioenriched 2-fluoro-4-phenylchromanes were produced with up to 96% ee, albeit in moderate yields -catalyzed electrophilic arylation of allylic amidesIn contrast to nucleophilic arylation, Gaunt recently reported a novel copper/bisoxazoline isoborneol (L19)iPr)4 also promoted the enantioselective addition of Ph3Al, ArTi(OiPr)3 and ArMgBr to ketones, producing chiral diaryl alkyl carbinols.31For ketone arylations, Fu reported the first enantioselective 1,2-addition of PhL20) as the chiral ligand, albeit with only 39% ee (L21) was demonstrated to promote the addition of arylborons to cyclic or acyclic arylketones with up to 68% eeL22 complex, which produced a range of chiral diaryl alkyl carbinols with excellent ee (95\u201399%) , right. i)-spirophosphite (L23) catalystN-(sulfinyl)cinnamylamine ligand (L24) in the arylation of \u03b1-ketoesters and \u03b1-diketones.2(p-cymene)]2 and -Me-BIPAM (L25) could also promote the asymmetric addition of arylboronic acids to \u03b1-ketoesters with high enantioselectivities.33For the 1,2-arylation of activated ketones, Xie and Zhou developed the first highly enantioselective addition of arylboronic acids to \u03b1-ketoesters using a chiral Rh/phosphoramidite (L26) catalysed 1,2-addition of arylboronic acids to 2,2,2-trifluoroacetophenones was highly effective in the Rh-catalyzed arylation of trifluoroacetophenones ,ii),i)ii)R)-MeO-mop as a chiral ligand. A variety of optically active 3-hydroxy-3-aryl-2-oxindoles were afforded in good to excellent yields (49\u201398%) with high enantioselectivities (72\u201391%) (i)-catalyzed arylation of NH isatin but obtained a poor enantioselectivity (55%). Liao and co-workersL28 is also compatible with the NH isatin arylation process and gives an improved efficiency , left. Mficiency , right.N-tosyl ketimines with sodium tetraarylborates by employing a chiral diene ligand (L29) . The met3 group are attractive to organic and medicinal chemists. In 2013, Xu and coworkers developed a rhodium-catalyzed asymmetric addition of arylboronic acids to CF3- or alkoxycarbonyl-substituted cyclic ketimines.L30) which they developed themselves to provide such molecules in high yields with excellent enantioselectivities and phosphine-oxazoline (L31) ligands, respectively (ii)/L33 complex and enables the synthesis of enantioenriched 3-amino-3-aryl-2-oxindoles with high ee. Zhang also demonstrated the first Ni(ii)-catalyzed asymmetric addition of arylboronic acids to cyclic imines using a tropos phosphine-oxazoline biphenyl ligand.41Pd-catalyzed enantioselective additions of arylboronic acids to cyclic ectively . Analogo3i)/L34 complexes, which afforded the \u03b3 product with a high enantioselectivity but moderate \u03b3-regioselectivity as the promoter. The formal SN2-substituted products, gem-diarylpropenes, were obtained with excellent ee reactions of cinnamyl electrophiles are one of the most important strategies to access chiral 1,1-diarylpropene molecules. Although the transfer of aryl groups to \u03b3-aryl substituted substrates resulted mainly in the achiral \u03b1 product with palladium catalysis, the \u03b3-regioselectivity is facile for iridium and copper catalysis. In 2007, Alexakisectivity , top. Rellent ee , bottom.i)-catalyzed AAAr,N-heterocyclic carbenes displayed remarkably high \u03b3-regioselectivity as well as excellent enantioselectivity reagents can couple with cinnamyl bromides or carbonates to construct tertiary and quaternary gem-diarylmethine stereogenic centres.In the field of Cu/L39 catalyst. The method was applied to a gram-scale synthesis of (S)-sertraline tetralone from the available racemic 4-hydroxy-4-phenylbutanoate. Other efforts to attempt the enantioconvergent arylation of racemic benzylic chloride or trifluoroborate, also by Ni(ii)/bis(oxazoline) catalysis, revealed the moderate stereoselectivity. In 2017, ReismanL40) as the chiral ligand as the reductant was the best ligand for the asymmetric transformation.The catalytic asymmetric \u03b1-arylation of styrenyl aziridines is one of the most important methods to access nonracemic 2,2-diarylethylamine derivatives. However, successful cross-coupling reactions rely on the stereospecific transformation of enantiomerically enriched aziridines. Recently, Sigman and Doyle developed an elegant Ni-catalyzed stereoconvergent reductive cross-coupling of racemic eductant , bottom.4.2gem-diarylalkanes, wherein the precoordination of the metal catalyst with prochiral substrates in a bidentate or monodentate manner is usually demanded. In 2015, DuanL41) into the Pd(ii)-catalyzed direct \u03b2-arylation of aminoquinoline derived aliphatic amides with aryl iodides. An array of \u03b2,\u03b2-diaryl carboxylic acid derivatives were produced in moderate to good enantiomeric ratios catalysts. In the end, both high yields and enantioselectivities were obtained using a substoichiometric amount of chiral phosphoric acid (L42) under solvent-free conditions \u2013H arylation of benzaldehydes via the precoordination of Pd(ii) with the in situ generated imine intermediatel-tert-leucine, 10 mol% Pd(OAc)2 and 3 equiv. H2O, o-alkyl benzaldehydes reacted with a wide range of aryl iodides to produce 1,1-diaryl alkanes in moderate yields with high enantiomeric ratios.In 2016, Yu employed chiral \u03b1-amino acids as transient directing groups in the enantioselective benzylic C(spL43) ligands in the Pd(ii)-catalyzed monodentate auxiliary directed C(sp3)\u2013H arylation of aliphatic amides.Soon afterwards, the same group employed chiral acetyl-protected aminoethyl quinoline -segphos as a chiral ligand. Enantioenriched 3-aryl-3-fluorooxindoles including a chiral quaternary center were obtained in high yields with excellent enantioselectivities trifluoroacetate and a chiral spiro phosphoric acid (SPA) (2(TFA)4 catalyst is responsible for the generation of the zwitterion (I). The 1,2-proton shift occurs via a proton shuttle model, which is mediated and stereochemically controlled by the chiral SPA (L44).In 2015, Zhu and Zhouid (SPA) . Chiral 5gem-diaryl moieties. The conceptual strategy of this method involves the enantioselective formation from the styrene and stereospecific coupling of metal bound benzyl intermediates. These species are either nucleophilic or electrophilic depending on the nature of the initiator (M1-R\u2032) .Inspired by the efficiency of direct aryl-benzyl coupling, transition metal-catalyzed three-component cross-coupling reactions of olefins have been developed as an important and complementary method in the construction of (M1-R\u2032) . In this5.12 atmosphereL45) could give the best enantioselective induction (up to 64%).In 2010, the Sigman group initially studied the palladium-catalyzed asymmetric hydroarylation of styrenes with arylboron esters in the presence of an i-PrOH solvent and in an Ovia double aryl cross-coupling to acrylates.L46 and Pd2(dba)3, optically active 3,3-diaryl esters with a high enantioselectivity were produced (ii)\u2013H complex intermediate.In 2016, Sigman and Toste developed an elegant enantioselective 1,1-diarylation method produced . The pro2SiH proceeded smoothly to produce enantioenriched 1,1-diarylethanes in good yields with good to excellent enantioselectivities.Recently the Buchwald group developed an alternative strategy to realize highly enantioselective hydroarylation of styrenes through CuH/Pd(0) cooperative catalysis5.2L47) was compatible with a variety of 1,2-bisubstituted alkyenylarene substrates with excellent diastereo- and enantioselectivities. The syn/trans selectivity of the arylboration addition of 1,2-dihydronaphthalene was facilely switched by changing the achiral ligands on the Pd(ii)-complex to promote the Cu/Pd-catalyzed enantioselective arylboration of terminal vinylarenes with aryl iodides under mild conditions /L49 catalyst, the enantioselective trifluoromethyl and aminoarylation of styrenes proceeded smoothly and afforded gem-diarylethane derivatives in moderate to high yields and with good ees to provide nonracemic gem-diarylalkane compounds. Due to distinguishing features including the wide range of substrate scope, good functional group tolerance and the use of easily accessible substrates, the related methodologies have received increasing interest from synthetic and pharmaceutical chemists, aiding the latter in synthesising medicinal molecules in a highly efficient manner.In this review, a large number of TMCAAr reactions, which target the construction of chiral gem-diaryl molecules has recently been highlighted and will be the focus of continuous research. The present methods, including hydro- or borylarylation, direct benzyl C\u2013H bond arylation and so on, need improvement of the efficiency and broadening of the substrate scope, and their use in the construction of quaternary carbon stereogenic centres remains challenging.Predictably, the development of strategies that transform commercially available feedstocks to highly valuable There are no conflicts to declare."} +{"text": "The general capability of graphene quantum dots to serve as capping ligands exchanging native organic stabilizers for various types of semiconductor nanoparticles affords the opportunity to engineer functional nanocomposites with remarkable thermoelectric properties. Graphene quantum dots (GQDs) are shown to serve as phase transfer agents to transfer various types of nanoparticles (NPs) from non-polar to polar solvents. Thorough characterization of the NPs proves complete native ligand exchange. Pellets of this GQD\u2013NP composite show that the GQDs limit the crystal size during spark plasma sintering, yielding enhanced thermoelectric performance compared with NPs exchanged with inorganic ions. A photoluminescence study of the GQD\u2013NP composite also suggests energy transfer from GQDs to NPs. Herein, we present the use of graphene quantum dots (GQDs) as ligands to stabilize nanoparticles.Recent advances in nanocomposite materials allow for a scalable methodology to generate multifunctional materials with properties stemming from both their individual components and, more interestingly, their synergistic interactions.GQDs can be viewed as a derivative of the extensively studied two-dimensional material graphene.Recent incorporation of GQDs into nanocomposite materials also offers the opportunity to take advantage of their unique charge carrier extraction capability for better solar cell efficiency.Inorganic NPs were made following well-developed wet-chemistry methods.vs. Cd) with GQDs. The TEM images before and after phase transfer indicate that the NPs\u2019 shape and size are preserved scale\" fill=\"currentColor\" stroke=\"none\">O and C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C, respectively, indicating the presence of GQDs after ligand exchange. Furthermore, 1H nuclear magnetic resonance (NMR) spectroscopy clearly proves the exclusive removal of alkyl H atoms in the range of 0.8\u20132.5 ppm and alkene H atoms at 5.3 ppm (The Fourier transform infrared spectroscopy (FTIR) spectrum of the NP dispersion after phase transfer shows a greatly reduced peak for the C\u2013H stretching mode at \u223c2900 cm\u20131 and S3\u2020, 5.3 ppm and S4\u2020; 5.3 ppm in whichX-ray diffraction (XRD) patterns for PbTe NPs before and after GQD ligand exchange are shown in Fig. S5a.in situ. \u03b6-potential in the insets of via increasing their energy of solvation. The proposed scenario is presented in in situ details of their status in the GQD\u2013NP complexes, which still remains a major challenge for almost all types of ligands on NP surfaces.It would be interesting to learn how the GQDs assemble near the surface of the NPs to form a stable dispersion in a polar solvent. We therefore performed a dynamic light scattering (DLS) study to reveal this behaviour \u03c41 = 1.34 ns and \u03c42 = 6.77 ns to \u03c41 = 0.98 ns and \u03c42 = 6.56 ns, respectively (To further prove the binding of GQDs to NPs, we studied the PL of GQD-capped CdSe NPs dispersed in solution .43,44 Cdectively , which sectively for CdSeComposites made from NPs are promising materials for thermoelectric applications, because of their inherent low thermal conductivity and enhanced Seebeck coefficients that result from quantum confinement and energy filtering effects.The thermoelectric measurements highlight the advantages of using GQDs over SCNs as capping agents for both electrical conductivity and Seebeck coefficient . The fin2 NPsThe prepared GQD\u2013NP composite may also be applied when the collective properties of different components are desired. We have demonstrated the effect of GQDs in lessening the sintering of PbTe NPs for enhanced thermoelectric performance. Another application is likely to be photovoltaic materials. There are reports of mixing GQDs with TiOIn conclusion, for the first time, we reported the general capability of GQDs to serve as capping ligands exchanging native organic stabilizers for various types of semiconductor NPs. The FTIR, NMR, TEM and XRD characterization results proved that the ligand exchange is complete and that the integrity of the NPs is preserved. Thermoelectric measurement of GQD\u2013PbTe composites revealed that the GQDs play a crucial role in limiting crystal size leading to an enhanced Seebeck coefficient, and thus a considerable ZT value of 0.46, without tuning the composition or doping level of the NPs. The PL lifetime of the GQD\u2013NPs indicated efficient energy transfer between the GQD ligands and the NP cores. Given the many and yet tunable properties of GQDs, we anticipate that versatile properties could be engineered from this novel type of GQD\u2013NP composite and to benefit various applications, including photovoltaic and thermoelectric devices, and catalysis.Supplementary informationClick here for additional data file."} +{"text": "Enantioselective 3-exo iodo-cycloetherification of allyl alcohols was realized by employing a novel ion-pair organocatalyst. exo iodo-cycloetherification of allyl alcohols was achieved using NIS as a halogen source. Based on this reaction, one-pot asymmetric 3-exo iodo-cycloetherification/Wagner\u2013Meerwein rearrangement of allyl alcohols en route to enantioenriched 2-iodomethyl-2-aryl cycloalkanones was subsequently developed. Due to the participation of adjacent iodine, the Wagner\u2013Meerwein rearrangement of 2-iodomethyl-2-aryl epoxide proceeds with unusual retention of stereoconfiguration.By designing a novel chiral ion-pair organocatalyst composed of chiral phosphate and DABCO-derived quaternary ammonium, highly enantioselective 3- In this regard, although 3-exo halo-cycloetherification of allyl alcohols has long been known,exo halocyclization, which impedes the development of an asymmetric version of this reaction.Halogenative functionalization of olefins is one of the most important transformations in organic synthesis, as it not only provides a versatile handle for further derivatization, but also delivers highly diastereoselective ring closure when the nucleophile and alkene are tethered together.via combinational approaches, which greatly accelerates the catalyst screening process. Inspired by Toste's recent workWith the advent and booming of organocatalysis,exo iodo-cycloetherification of allyl alcohols using commercially available NIS as a halogen source. Additionally, this protocol provides direct access to enantiopure 2-iodomethyl epoxides,15Herein, we would like to report the success of implementation of the ion-paring strategy, leading to the discovery of a novel ion-pair organocatalyst. This unprecedented organocatalyst enables the first enantioselective 3-exo-iodocyclization of allyl alcohol 1a was explored using an ion-pair organocatalyst generated in situ by combining silver phosphate with DABCO-derived quaternary ammonium salt for convenience of catalyst screening . Further structural modification of ammonium salt A3 revealed that A8 was the optimal cation fragment for the ion-pair organocatalyst, furnishing epoxide 2a with 92% ee (entries 3\u20139). As for the anion fragment, 8H-R-TRIP-OAg provided a better result than any other chiral silver phosphate evaluated were also surveyed under identical reaction conditions but gave no desired cyclization product, with the starting material being fully recovered . As a slight excess of A8 was used in the in situ procedure, we reasoned that A8 might be an effective promoter for this reaction. Indeed, comparable enantioselectivity was obtained by adding a catalytic amount of A8 to the reaction. It is postulated that A8 might act as a Lewis base to stabilize the iodonium intermediate PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019PPh3 and electron-donating groups (2ag-2ah and 2ca\u20132ch) on the phenyl moiety were tolerated, affording the corresponding epoxides with good to excellent enantioselectivities (87% to 99% ee). Gem-substituents were crucial for the reaction, as 2f lacking gem-substituents was obtained in only 41% yield and 63% ee. Epoxides with cyclic gem-substituents were obtained with higher enantioselectivities (2c-2ch and ESI2a and 2b). A 2-alkyl substituted allyl alcohol was also smoothly converted to epoxide 2g, albeit with low enantioselectivity (37% ee). Furthermore, gram syntheses of epoxides 2a and 2c\u20132e were also smoothly realized by using 5 mol% C1 without affecting enantioselectivities, and the catalyst loading could even be reduced to 1 mol% affording comparable results . Surprisingly, the absolute configuration of 3c was established to be S by X-ray crystallographic analysis of hydrazone 4 derived from 3c,TS2, which then rearranged to ketone 3c with double inversion of configuration. Furthermore, derivatizations of 3c were also performed to demonstrate its synthetic utility. Substitution of the iodide with NaN3 provided azide ketone 5 smoothly, and the iodide could also be converted to an alcohol via formyloxylation/hydrolysis6 in satisfactory yield. It is noteworthy that no erosion of enantiopurity was detected in all these reactions.Next, Wagner\u2013Meerwein rearrangementn center . BF3\u00b7Et2r see ESI, deliverexo iodo-cycloetherification/Wagner\u2013Meerwein rearrangement was also developed . Different substituents on the phenyl group were found to be compatible with the one-pot process, affording the corresponding cyclohexanones 3c\u20133f in satisfactory enantiopurities. Furthermore, seven-membered cycloketone 3g could also be obtained via this one-pot cascade reaction with 91% ee (comparable with that of the corresponding epoxide 2d), providing a complementary route to previous protocols involving enantioselective halonium-induced semi-Pinacol rearrangement for the enantioselective construction of halogenated cycloheptanones.To simplify the operation, one-pot asymmetric 3-eveloped . Fortunaexo iodo-cycloetherification of allyl alcohols using NIS as a halogenating reagent. By employing this novel catalyst, a variety of enantiopure 2-iodomethyl-2-aryl epoxides were successively prepared with good to excellent enantioselectivities, even on a gram scale. Subsequently, one-pot asymmetric 3-exo iodo-cycloetherification/Wagner\u2013Meerwein rearrangement of 2-aryl-2-propen-3-ol was explored, which provided direct access to chiral 2-iodomethyl-2-aryl cycloalkanones with good enantioselectivities. Unusual retention of configuration owing to the assistance of the adjacent iodide was also observed in the Wagner\u2013Meerwein rearrangement.In conclusion, a novel ion-pair organocatalyst comprised of chiral phosphate and DABCO-derived quaternary ammonium was designed, which enabled the first asymmetric 3-Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "Covalent organic frameworks with high porosity and crystallinity have been synthesized, through macrocycle-to-framework strategy, using shape-persistent arylene-ethynylene macrocycles as the key components to control the topology and modulate the porosity. Macrocycle-to-framework strategy was explored to prepare covalent organic frameworks (COFs) using shape-persistent macrocycles as multitopic building blocks. We demonstrate well-ordered mesoporous 2D COFs (AEM\u2013COF-1 and AEM\u2013COF-2) can be constructed from tritopic arylene-ethynylene macrocycles, which determine the topology and modulate the porosity of the materials. According to PXRD analysis and computer modelling study, these COFs adopt the fully eclipsed AA stacking mode with large accessible pore sizes of 34 or 39 \u00c5, which are in good agreement with the values calculated by NLDFT modelling of gas adsorption isotherms. The pore size of COFs can be effectively expanded by using larger size of the macrocycles. Provided a plethora of polygonal shape-persistent macrocycles with various size, shape and internal cavity, macrocycle-to-framework strategy opens up a promising approach to expand the structural diversity of COFs and build hierarchical pore structures within the framework. Mesoporous 2D COFs with high surface area, large pore volume, good thermal stability and high crystallinity were successfully prepared from arylene-ethynylene macrocycles. By varying the size of macrocycles, the pore size of the COFs can be systematically tuned.Covalent organic frameworks (COFs) represent a novel class of porous crystalline polymers, in which the building blocks are assembled into two- or three-dimensional architectures through covalent bonds. COFs possess rigid structures, high thermal stabilities, and low densities. Since the pioneering work of Yaghi and co-workers,vC),1 and AEM-2, which closely resemble the commonly used trigonal connector, 2,3,6,7,10,11-hexahydroxytriphenylene (HHTP), were prepared (1 and AEM-2 have similar triangle shape as HHTP (7 \u00c5) but with increased sizes, having the extended lateral lengths of approximately 9 \u00c5 and 13 \u00c5 respectively. Gram-scale AEM-1 and AEM-2 were obtained from simple dipropynyl monomer 1, or 2 through acyclic diyne metathesis macrocyclization (ADIMAC), followed by deprotection of TBS groups in high yields. Highly active multidentate triphenolsilane-based Mo(vi) carbyne complexAmong various SPMs, arylene-ethynylene macrocycles (AEM) are of our particular interest, since they are perfectly planar and rigid and their size and geometry can be easily tailored.prepared . AEM-1 a1 and AEM-2 as a novel type of multitopic building units, which can modulate pore size/distribution of COFs. We fixed the length of the linker using the same simple 1,4-benzenediboronic acid (BDBA), and varied the size of multitopic connectors: HHTP (7.1 \u00c5), AEM-1 (9.3 \u00c5), and AEM-2 (13.2 \u00c5). For the comparison purpose, COF-5, which was previously reported by Yaghi,1 was obtained in mesitylene/dioxane by heating the reaction mixture at 100 \u00b0C for 7 days without stirring. Although AEM\u2013COF-2 shares a similar structure motif with AEM\u2013COF-1, it requires a different solvent combination. A low surface area material was obtained when AEM-2 and BDBA were heated (120 \u00b0C) in mesitylene/dioxane for 7 days. Among various solvent systems we tested , the combination of DMAc/DCB provided crystalline AEM\u2013COF-2 with the highest surface area under conventional heating or microwave heating . AEM\u2013COF-1 and AEM\u2013COF-2 were isolated as yellow microcrystalline powders through centrifugation followed by successive washing with anhydrous acetone. Both COFs are insoluble in common organic solvents such as alkanes, arenes, acetone, ethers, and N,N-dimethylformamide.Most COFs are generally constructed from two types of building blocks: symmetric multitopic connectors and ditopic spacers. The multitopic connectors not only determine the topologies of the COFs, but also work in tandem with the spacers to determine the pore sizes, pore volumes, surface areas and functions of the COFs. Since there are a rich diversity of ditopic spacers readily available, a common strategy to enlarge pore apertures of COFs with a given topology has been to increase the length of the rigid ditopic linkers.1 and AEM\u2013COF-2 were characterized by FT-IR, 13C-MAS NMR, elemental analysis, TGA, SEM and PXRD analysis. The FT-IR spectra of AEM\u2013COF-1 and AEM\u2013COF-2 show stretching bands of B\u2013O at 1335 cm\u20131 and 1323 cm\u20131, respectively. We also observed broad absorption band around 3430 cm\u20131, which likely corresponds to the residual hydroxyl groups of the macrocycles and boronic acids. In the magic angle spinning (MAS) solid-state 13C NMR spectrum of AEM\u2013COF-1, we observed a single peak at 92.6 ppm which can be assigned to the carbons of triple bonds, indicating the uniformity of the chemical environment around C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bonds. The 13C NMR spectrum of AEM\u2013COF-2 shows C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond carbon peak at 90.7 ppm. Thermogravimetric analysis (TGA) of AEM\u2013COF-1 and AEM\u2013COF-2 shows <10% weight loss at 400 \u00b0C and <30% at 800 \u00b0C under a nitrogen atmosphere characterization measurement. The PXRD patterns of the COFs exhibit intense peak at 2\u03b8 = 2.9\u00b0 and 2.2\u00b0, for AEM\u2013COF-1 and AEM\u2013COF-2, respectively, along with some other peaks with lower diffraction intensities, indicating long-range molecular ordering in both COFs. We did not observe diffraction peaks that are characteristic for the starting materials a fully eclipsed model with an AA stacking (space group P6/mmm), and (ii) a staggered model with an AB stacking (space group P63/mmc). Each layer was translated from the next one by one-half of the a and b lattice parameters. A geometrical energy minimization was performed using the universal force-field implemented in the forcite module to optimize the geometry of the building molecules, as well as the unit cell parameters. The powder diffraction patterns for the models were then calculated and compared with the experimental ones. We found the simulated PXRD patterns of the fully eclipsed models of AEM\u2013COF-1 and AEM\u2013COF-2 are in excellent agreement with experimental results, indicating the eclipsed stacking mode of the layers ; and AEM\u2013COF-2: a = b = 40.935 \u00c5, c = 3.257 \u00c5 , both of which agree well with the observed reflections. Therefore, similar to COF-5, AEM\u2013COF-1 and AEM\u2013COF-2 adopt eclipsed stacking of the layers, which lead to 1D mesopores with theoretical diameters of 34 \u00c5 and 39 \u00c5 respectively.The crystallinity of AEM\u2013COF-e layers . A full 1 and AEM\u2013COF-2 were then investigated by N2 adsorption isotherms at 77 K and the results are summarized in P/P0 = 10\u20135 to 10\u20132) followed by a second stage pore filling starting around P/P0 = 0.05, which levels off at a relative pressure of P/P0 = 0.18, 0.25 and 0.35 for COP-5, AEM\u2013COF-1 and AEM\u2013COF-2, respectively. The gradual shift of the step positions suggests the increasing sizes of the pores in these three COFs. Calculations based on the non-local density functional theory (NLDFT) also reveal the trend of increasing pore sizes in the series, showing a narrow pore-size distribution (PSD) centered around 2.6 nm for COF-5, 3.2 nm for AEM\u2013COF-1, and 3.8 nm for AEM\u2013COF-2 predicted from the modelling based on XRD crystal packing. Correspondingly, we observed increasing pore volumes (Vp), which were calculated to be 0.828 cm3 g\u20131 (COF-5), 1.15 cm3 g\u20131 (AEM\u2013COF-1), and 1.38 cm3 g\u20131 (AEM\u2013COF-2) at P/P0 = 0.90. No or little hysteresis loops were observed in the whole range of adsorption\u2013desorption isotherms in all three frameworks. The absence of hysteresis loop has been observed for similar mesoporous MCM-41 with tubular hexagonal pores of sizes <40 \u00c5 at temperatures above 77.4 K.2 g\u20131 (correlation coefficient = 0.998), which is in good agreement with the reported literature value (1590 m2 g\u20131).1 and AEM\u2013COF-2 . As shown in macrocycle-to-framework strategy to construct ordered crystalline COFs with tunable pore diameters and volumes by varying the dimensions of tritopic macrocyclic building units.The porosities of the frameworks AEM\u2013COF-EM\u2013COF-2 . These v2, which contains AEM-2 with interior void of 5.8 \u00c5. However, we did not observe micropores below 1 nm range. Although the X-ray diffraction data is in excellent agreement with the perfectly eclipsed model of the AEM\u2013COF-2 layered structure, there might be slight offset between the adjacent interlayers, leading to the restricted accessibility of such micropores. In order to obtain COFs with multiple-type pore structures, the use of macrocycles with large intrinsic pores are desired.Initially, we expected hierarchical pore structures in the case of AEM\u2013COF-e.g. arylene-vinylene macrocycles (AVM),macrocycle-to-framework strategy opens up new avenues in the synthesis of COFs with intriguing architectures, properties and applications.We have demonstrated that arylene-ethynylene macrocycles (AEMs) can be utilized as well-defined building blocks for construction of COFs with high thermal stability, permanent porosity, and high crystallinity, either under conventional solvothermal conditions or microwave heating. The \u03c0\u2013\u03c0 interactions between rigid arylene-ethynylene backbones likely contribute considerably to the eclipsed packing of the layers as well as the formation of ordered crystalline materials. Our study shows that the customizable SPMs can be effectively utilized as a new type of multitopic connectors to control the topologies of the COFs and tune the surface area, pore size, and pore volume of the COFs. Given the vast availability of SPMs with different backbones and properties, Supplementary informationClick here for additional data file."} +{"text": "A non-oxido V(v) complex with glutaroimide-dioxime (H3L), a ligand for recovering uranium from seawater, was synthesized from aqueous solution as Na[V(L)2]\u00b72H2O, and the structure determined by X-ray diffraction. v) complex with glutaroimide-dioxime (H3L), a ligand for recovering uranium from seawater, was synthesized from aqueous solution as Na[V(L)2]\u00b72H2O, and the structure determined by X-ray diffraction. It is the first non-oxido V(v) complex that has been directly synthesized in and crystallized from aqueous solution. The distorted octahedral structure contains two fully deprotonated ligands (L3\u2013) coordinating to V5+, each in a tridentate mode via the imide N (RV\u2013N = 1.96 \u00c5) and oxime O atoms (RV\u2013O = 1.87\u20131.90 \u00c5). Using 17O-labelled vanadate as the starting material, concurrent 17O/51V/1H/13C NMR, in conjunction with ESI-MS, unprecedentedly demonstrated the stepwise displacement of the oxido V PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O bonds by glutaroimide-dioxime and verified the existence of the \u201cbare\u201d V5+/glutaroimide-dioxime complex, [V(L)2]\u2013, in aqueous solution. In addition, the crystal structure of an intermediate 1\u2009:\u20091 V(v)/glutaroimide-dioxime complex, [VO2(HL)]\u2013, in which the oxido bonds of vanadate are only partially displaced, corroborates the observations by NMR and ESI-MS. Results from this work provide important insights into the strong sorption of vanadium on poly(amidoxime) sorbents in the recovery of uranium from seawater. Also, because vanadium plays important roles in biological systems, the syntheses of the oxido and non-oxido V5+ complexes and the unprecedented demonstration of the displacement of the oxido V PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O bonds help with the on-going efforts to develop new vanadium compounds that could be of importance in biological applications.A non-oxido V( Sorption of these cations on poly(amidoxime) sorbents follows the order: vanadium(v) \u226b iron(iii) > uranium(vi).\u20131, 37 nM)v) from the sorbent for reuse are much harsher than those used to elute uranium and other cations and ultimately destroy the sorbent.Although these results are promising, studies also reported significant co-sorption of iron(vi) and Fe(iii) complexes with glutaroimide-dioxime (iii) much more strongly than U(vi) as manifested by the shorter Fe\u2013O and Fe\u2013N bond lengths relative to the corresponding U\u2013O and U\u2013N bond lengths (even after taking into consideration the difference in ionic radii between Fe3+ and UO22+). The shorter bond lengths in the Fe(iii) complex were attributed to the higher charge density of Fe(iii) as well as its larger orbital participation in bonding relative to uranium. The higher thermodynamic stability and shorter bond lengths of the Fe3+/glutaroimide-dioxime complexes were postulated to be responsible for the higher sorption of Fe3+ compared to UO22+ in marine tests.Structural studies can be used to provide valuable insights into the coordination behavior of vanadium and other metal cations with amidoxime ligands and can also help explain their subsequent sorption behavior with poly(amidoxime) sorbents. For example, the crystal structures and thermodynamic stability constants have been reported for U with glutaroimide-dioxime has not been reported, reasonable speculations about its structure can be made using information obtained from the known V(v) crystal structures. Based on the reported structures of V(v) complexes with organic ligands prepared from aqueous solutions (or ionic liquid equilibrated with water), it is known that the VO2+ moiety with two short oxido V PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O bonds scale\" fill=\"currentColor\" stroke=\"none\">OV = 1.60\u20131.63 \u00c5) usually remains intact.22+ cation which possesses a linear trans dioxido configuration that allows two tridentate ligands to bind in the equatorial plane to form a strong 1\u2009:\u20092 U(vi)/L complex,2+ cation with its bent cis dioxido configuration cannot accommodate two such ligands due to steric hindrance and insufficient coordination sites.Though the crystal structure of V is sorbed much more strongly than U(vi) by the amidoxime sorbents. One hypothesis that could explain the much stronger complexation of V(v) is that V(v) exists in the glutaroimide-dioxime complex as a non-oxido, \u201cbare\u201d V5+ ion coordinated with the ligand(s). A non-oxido V5+ cation could have a very high affinity for O and N donor ligands due to its high charge density and could easily accommodate two tridentate ligands in a mode similar to that in the Fe3+/glutaroimide-dioxime complex.4+ complexes with ligands such as 1,3,5-triamino-1,3,5-trideoxy-cis-inositol (taci)N-hydroxy-iminodiacetate5+ complexes from aqueous solutions are extremely rare. One non-oxido V5+ complex, [PPh4][\u0394-V-HIDPA)2]\u00b7H2O dipropionic acid, H3HIDPA), was crystallized as the oxidized analogue of the naturally-existing Amavadiniv) complex by Ce(iv).5+ complexes directly synthesized from oxido V(v) species scale\" fill=\"currentColor\" stroke=\"none\">V PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O]+ or vanadates) and crystallized from aqueous solution. In addition, the formation of non-oxido V5+ complexes in aqueous solutions via the displacement of the oxido V PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O bonds by chelating ligands complex with 4-hydroxy-dipicolinic acid , a ligand that is structurally similar to glutaroimide-dioxime, was shown to exhibit insulin mimetic behavior in vivo.v) complexes could prove useful for the design of improved insulin mimetic compounds.Although complexation of vanadium with Schiff bases such as glutaroimide-dioxime is problematic for the extraction of uranium from seawater, such complexes are currently of great interest for a variety of biological applications. For example, the V(v)/glutaroimide-dioxime complexes and characterize their crystal- and solution structures by single-crystal X-ray diffraction (XRD), multinuclear nuclear magnetic resonance (NMR), electrospray ionization mass spectrometry (ESI-MS), and electron paramagnetic resonance (EPR). This work represents the synthesis and identification of the first non-oxido V(v) complex that was directly synthesized from an oxido V(v) species and crystallized from aqueous solution. The displacement of oxido V PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O bonds by chelating ligands that leads to the formation of a non-oxido V(v) complex in aqueous solution has been unprecedentedly demonstrated by concurrent 51V/17O NMR experiments. Results from this work provide important insights into the strong sorption of vanadium on poly(amidoxime) sorbents in the recovery of uranium from seawater.In an effort to provide structural insights into vanadium complexation with amidoxime ligands, the present work has been conducted to synthesize crystals of V(2]\u00b72H2O(cr) consists of a \u201cbare\u201d V5+ center bound to two fully deprotonated glutaroimide-dioxime ligands (L3\u2013), through one nitrogen and two oxygen atoms of each ligand, along with a sodium ion and two water molecules (P1[combining macron] (a = 7.9375(3) \u00c5, b = 8.7365(4) \u00c5, c = 12.1972(5) \u00c5, \u03b1 = 102.684(2)\u00b0, \u03b2 = 107.187(2)\u00b0, \u03b3 = 103.796(2)\u00b0. The bond lengths for the V\u2013N bonds are 1.9557(8) and 1.9551(8) \u00c5 while those for the V\u2013O bonds are 1.8667(8), 1.8741(7), 1.9039(6), and 1.9024(8) \u00c5. The extended crystal structure can be considered as successive [V(L)2]\u2013 complexes bridged by sodium atoms via N(2) and N(5) to form a one dimensional chain. The chains are then linked via bridging water molecules (O(1W)) between the sodium atoms to form a ribbon \u2013N(3)*, O(2W)\u2013O(2)*, and O(2W)\u2013N(6)*, where the superscript * denotes symmetry related positions. Tables S1 and S2 in the ESI2]\u00b72H2O.The asymmetric unit of Na[V(L)olecules . The bin macron] with unia ribbon . The rib2]\u00b72H2O(cr) are within the range of V\u2013O bond distances reported for other non-oxido V5+ compounds obtained from non-aqueous solutions (1.8\u20132.0 \u00c5), PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O double bonds (\u223c1.6 \u00c5).The V\u2013O bond distances in Na(cr).The 1\u2009:\u20091 V complex with glutaroimide-dioxime can be synthesized and crystallized from aqueous solution. In other words, the glutaroimide-dioxime ligand can displace the oxido V PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O bonds in vanadate and form a \u201cbare\u201d V5+ complex. In addition, the crystallization of Na[VO2(HL)] suggests that an intermediate 1\u2009:\u20091 complex, in which the oxido V PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O bonds in vanadate are only partially displaced by glutaroimide-dioxime, may also exist in aqueous solution. To verify the structure of the unusual non-oxido V5+ complex and demonstrate the stepwise displacement of the oxido V PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O bonds in aqueous solutions, we hypothesized a reaction scheme ]\u2013 in the crystal structure (OH)L]\u2013 or [VO2(HL)]\u2013 does not alter the validity of the discussions below.The successful synthesis of Na\u2013 complex is the only vanadium species present. At this point, all of the V = 17O bonds of the starting vanadate would be displaced by the donor atoms of glutaroimide-dioxime and there would be no 17O atoms in the [V(L)2]\u2013 complex.17O NMR experiments have shown no oxygen exchange between the 17O-enriched water and the glutaroimide-dioxime ligand under the experimental conditions within 12 days.\u2021Prior Concurrently, the 51V NMR signal for the vanadate (with V\u2013O coupling) should disappear and a new 51V NMR signal for the [V(L)2]\u2013 complex with no V\u2013O coupling would appear.As shown in 51V/17O NMR spectra of a series of solutions with [L]/[V] ratios ranging from 0 to 3 are shown in 51V NMR spectrum of a D2O solution of Na[V(L)2]\u00b72H2O(cr) was collected to help confirm the assignment of the vanadium signal and is also shown in 51V NMR spectrum of the initial solution (a) in the absence of glutaroimide-dioxime shows the peaks for the vanadates (VO43\u2013 and HVO42\u2013) at \u03b4 = \u2013537, \u2013561 ppm. The vanadate peak has broad shoulders indicating the spin\u2013spin coupling with 17O shows a broad peak at \u223c560 ppm for the vanadate species , with an apparent linewidth of 5250 Hz due to coupling with the spin-7/2 51V nucleus. These 17O/51V spin\u2013spin coupling features agree with those reported for 17O-labelled NaVO3 in the literature.29The 51V and 17O signals for vanadates disappeared. In addition, a new 51V signal in the 51V spectra began to appear at \u03b4 = \u2013410 ppm (\u25bf) and achieved maximum intensity at [L]/[V] = 1 (51V spectrum b), diminished as [L]/[V] was increased to 2 (51V spectrum c), and nearly disappeared as [L]/[V] was further increased to 3 (51V spectrum d). Concurrently, a new peak appeared in the 17O spectra around \u03b4 = 905 ppm (\u25bf) and achieved maximum intensity at [L]/[V] = 1 (17O spectrum b), diminished at [L]/[V] = 2 (17O spectrum c), and completely disappeared at [L]/[V] = 3 (17O spectrum d).As different equivalents of glutaroimide-dioxime were added to the vanadate solution, both the \u2013, that is hypothesized in 17O signal for the intermediate 1\u2009:\u20091 V/L complex (\u25bf) suggests that, in this complex, the glutaroimide-dioxime ligand only partially displaces the oxido V PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O bond(s) from the initial 17O-labelled vanadate, which is consistent with 2(HL)] and 2 (17O spectrum c) were noted to be slightly different. The difference probably results from different degrees of protonation in the [V(O)(OH)L]\u2013 species due to slight differences in pH between the two solutions .Based on the changes in the peak intensities with the increase of [L]/[V] and the occurrence of the maximum intensity at [L]/[V] = 1, it is reasonable to assign these peaks (\u25bf) to a 1\u2009:\u20091 intermediate complex, such as [V(O)(OH)L]h 2(HL)] . The 17O51V peak at \u03b4 = 740 ppm (\u25a1) appears at [L]/[V] = 1 (51V spectrum b), intensifies at [L]/[V] = 2 (51V spectrum c), and achieves maximum intensity at [L]/[V] > 2 (51V spectrum d). The chemical shift is identical to that of the 51V peak in spectrum e for the solution of Na[V(L)2]\u00b72H2O, implying that this peak (\u25a1) can be assigned to the 1\u2009:\u20092 V/L complex, [V(L)2]\u2013, hypothesized in 51V peak for the 1\u2009:\u20092 complex should not show 17O/51V spin\u2013spin coupling features because the ligands in the 1\u2009:\u20092 complex completely displace the oxido V PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019*O bonds of the initial 17O-labelled vanadate. However, the large linewidth of the 51V signal resulting from the low symmetry of the complex precludes the verification of the absence or presence of the coupling features for the 51V NMR signal of the 1\u2009:\u20092 (\u03b4 = 740 ppm) or 1\u2009:\u20091 complex (\u03b4 = \u2013410 ppm). Nevertheless, the absence of NMR signals on the 17O spectrum d clearly indicates that the 1\u2009:\u20092 complex does not contain oxido V PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019*O bonds and is a \u201cbare\u201d V5+ complex.Accompanying the appearance and disappearance of the peaks (\u25bf) for the 1\u2009:\u20091 V/L complex, a new and extremely shifted 51V NMR signal for the final complex at [L]/[V] > 2 remained unchanged beyond 12 days, which suggests that vanadium remained in the V(v) oxidation state in the solution at neutral to slightly alkaline pH. If reduction of V(v) to the paramagnetic V(iv) species were to occur, it would diminish and eventually \u201cwash-out\u201d the 51V NMR signal. Further reduction to V(iii) is very unlikely: V(iii) is generally much less stable in aqueous solutions, and no signals were observed in the lower 51V chemical shift range of below \u03b4 = \u20131000 ppm.28The intensity of the 51V/17O NMR experiments in acidic solutions were not performed in this study because (1) [V(L)2]\u2013 may not be the dominant and most stable complex in acidic regions and (2) preliminary experiments suggested that redox reactions could occur between V(v) and glutaroimide-dioxime in more acidic solutions. The redox reactions between V(v) and the ligand are the subject of a future study.51V/17O NMR experiments have unprecedentedly demonstrated that the displacement of oxido V PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O bonds in vanadates by glutaroimide-dioxime leads to the formation of a non-oxido V5+ complex in aqueous solution. The 51V chemical shift of the complex is identical to that of the solution of Na[V(L)2]\u00b72H2O(cr), suggesting that the complex in solution is probably [V(L)2]\u2013. Further verification of the stoichiometry by 1H NMR and ESI-MS is described below.To summarize, concurrent 1H and 13C NMR spectra of the V(v)/glutaroimide-dioxime solutions used in the 17O/51V experiments , as well as a solution of only glutaroimide-dioxime (a\u2032), were acquired. A 1H COSY spectrum of solution c was also acquired to confirm the peak assignments. The 1H NMR and COSY spectra are shown in 13C NMR spectra are provided in ESI /glutaroimide-dioxime solutions show two sets of signals at \u03b4 = 2.5\u20132.8 ppm and \u03b4 = 1.8\u20132.1 ppm, respectively. In each set, there are three signals that were straightforward to assign to the free glutaroimide-dioxime (\u25cb), the 1\u2009:\u20091 V/L complex (\u25bf), and the 1\u2009:\u20092 V/L complex (\u25a1), respectively, based on the NMR spectrum of the pure ligand, the COSY spectrum, the spin\u2013spin coupling patterns, and the intensity changes as a function of the [L]/[V] ratio. The signals for the 1\u2009:\u20091 complex (\u25bf) achieve maximum intensity at [L]/[V] = 1 (spectrum b) and diminish as [L]/[V] is increased to 2 and higher (spectra c and d), while the signals for the 1\u2009:\u20092 complex (\u25a1) are weak at [L]/[V] = 1 (spectrum b), intensify as [L]/[V] is increased to 2 (spectrum c), and achieve a maximum at [L]/[V] > 2 (spectrum d). These observations support the proposed structures of the 1\u2009:\u20091 and 1\u2009:\u20092 V(v)/glutaroimide-dioxime complexes, corroborate the 17O/51V NMR data, and validate the hypothesized stepwise displacement of the oxido V PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O bonds leading to the formation of the non-oxido [VL2]\u2013 complex in aqueous solution.The 1H spectra of the complexes showed that the equivalencies of the H atoms in the free ligand remain unchanged in the 1\u2009:\u20091 and 1\u2009:\u20092 complexes (1H resonances (two) with the same spin\u2013spin coupling fine structures is observed for the complex and the free ligand, which agrees with the coordination modes of the ligand in the complexes hypothesized in 5+/glutaroimide-dioxime complex. The same analysis can be made with the 13C NMR spectra with [L]/[V] = 1 and 2 are shown in m/z = 223.8 and 251.8, respectively. The peak at 223.8 corresponds to the intermediate 1\u2009:\u20091 [V(O)(OH)L]\u2013 complex hypothesized in m/z = 251.8 corresponds to a 1\u2009:\u20091 [V(O)(OCH2CH3)L]\u2013 complex . Evidently, ethoxide (OCH2CH3\u2013) from the electrospray solvent substituted the hydroxide (OH\u2013) of the [V(O)(OH)L]\u2013 complex during the dilution and/or electrospray process. The solution with [L]/[V] = 2 (lower spectrum) shows a single peak with m/z = 330.8 corresponding to [V(L)2]\u2013 , confirming the formation of the 1\u2009:\u20092 V/L complex. Simulations of the collected spectra for the regions containing the peaks at m/z = 223.8, 251.8, and 331.0 are provided in the ESI section, Fig. S2.The negative mode ESI-MS spectra for two aqueous solutions . Consequently, the initial vanadate peak corresponding to an isotopologue containing one 18O, should be observed if the vanadium complex still contains an oxido V PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019*O bond from the vanadate and, more importantly, the (m +2) peak should be absent if all oxido V PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019*O bonds of the vanadate are displaced by the glutaroimide-dioxime ligand.According to the manufacturer's specifications, the 10% vanadate was actum/z = 330.8 does not show the unnatural (m + 2) isotopic pattern that could indicate the presence of one 18O atom (or two 17O atoms with a much lower probability) in the 1\u2009:\u20092 complex scale\" fill=\"currentColor\" stroke=\"none\">*O bonds of the initial 17,18O-labelled vanadate are displaced by the ligands to form the non-oxido 1\u2009:\u20092 V(v)/glutaroimide-dioxime complex in solution. The presence of a small (m +1) peak at m/z = 331.8 is in accord with the natural 13C/15N abundances.Notably, the base peak at \u2013 and [V(O)(OCH2CH3)L]\u2013) show unnatural (m + 2) peaks at 225.8 and 253.8, respectively, corresponding to the presence of one 18O atom (or two 17O atoms with a much lower probability) in the complex. The presence of the (m + 2) peak indicates incomplete displacement of the oxido V PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019*O bonds of the initial 17,18O-labelled vanadate in the intermediate 1\u2009:\u20091 complex, in agreement with m + 1) peaks include the contributions from the natural 13C/15N abundances, and the additional contribution from the isotopologue containing one 17O atom.In contrast, the two base peaks for the 1\u2009:\u20091 complexes ([V(O)(OH)L]m + 2) feature that corresponds to the non-oxido complex, [V(L)2]\u2013, and a peak at 238.0 with a prominent (m + 2) feature that corresponds to a 1\u2009:\u20091 complex, [V(O)(OCH3)L]\u2013, containing one 18O. In the 1\u2009:\u20091 complex, it is the methoxide that substitutes the hydroxide of [V(O)(OH)L]\u2013 during the dilution and/or spray process.Two ESI-MS spectra obtained by using a different diluent (methanol) on a different spectrometer (Finnigan LTQ FT mass spectrometer) are shown in ESI, Fig. S3.2CH3)L]\u2013 in 3)L]\u2013 in Fig. S2,\u2013 by ethoxide and methoxide, respectively. This is consistent with the existence of the 1\u2009:\u20091 V(v)/glutaroimide-dioxime complex as [V(O)(OH)L]\u2013 in aqueous solution as hypothesized in 2(HL)]\u2013 observed in solid. The exact mechanism of substitution is unclear, but it is reasonable to assume that, energetically and kinetically, substitution of a V PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O bond in [VO2(HL)]\u2013 is less favorable than that of a V\u2013OH bond in [V(O)(OH)L]\u2013.Interestingly, the ethoxide and methoxide adducts, [V(O)2]\u2013, in aqueous solution via the displacement of the oxido V PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019*O bonds. The presence of an intermediate 1\u2009:\u20091 complex that still contains oxido V PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O bonds, [V(O)(OH)L]\u2013, in solution has also been confirmed.To summarize, all of the ESI-MS data have validated the hypothesized reaction scheme and confg = 2.00 and no hyperfine coupling was observed, which is due to the presence of organic radicals. This signal is frequently observed due to the high sensitivity of EPR spectroscopy. The lack of hyperfine coupling and the fact that the g value is quite different from that typical for V(iv), 1.95, strongly suggest that only V(v) is present at low temperature.iv) is only a minor component at this temperature. Overall, the EPR spectra are consistent with a V(v) ground state and indicate the potential presence of a low lying charge transfer state that could be populated at high temperatures.EPR spectra of powdered crystals were recorded at 300 K and 4 K to poly(amidoxime) sorbents in marine tests was reportedly much higher than that of Fe(iii) and U(vi), following the order: V(v) \u226b Fe(iii) > U(vi). Useful structural insights into the higher sorption of V(v) can be gained by comparing the structural parameters and coordination modes of the glutaroimide-dioxime complexes with V(v), Fe(iii), and U(vi), as shown in 2]\u00b72H2O(cr) and Fe(H2L)(HL)\u00b78H2O(cr) are non-oxido metal (V5+ or Fe3+) complexes in distorted octahedral environments with similar O\u2013V\u2013N and O\u2013Fe\u2013N bond angles of approximately 73\u201375\u00b0. The average bond distances of V\u2013O and V\u2013N in Na[V(L)2]\u00b72H2O(cr) are 1.8868 \u00c5, and 1.9554 \u00c5, respectively, and are shorter than those of Fe\u2013O and Fe\u2013N in Fe(H2L)(HL)\u00b78H2O(cr) by 0.16 \u00c5 and 0.06 \u00c5, respectively. Taking into consideration the fact that the ionic radii for V(v) (0.54 \u00c5) and low spin Fe(iii) (0.55 \u00c5) are nearly identical,5+ forms a stronger complex with glutaroimide-dioxime than Fe3+ assuming a predominantly ionic bonding model. The formation of stronger V5+ complexes is most probably responsible for the higher sorption of V(v) than Fe(iii) by poly(amidoxime) sorbents.As previously mentioned, the sorption of V(2(H2L)(H2L)\u00b7H2O(cr) complex is very different from those of Na[V(L)2]\u00b72H2O(cr) and Fe(H2L)(HL)\u00b78H2O(cr). In the U(vi) complex, the UO22+ moiety maintains its linear di-oxido configuration and the two ligands coordinate to U via its equatorial plane. Evidently, glutaroimide-dioxime is not sufficiently strong to displace the oxido U PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O bonds to form a \u201cbare\u201d U6+ complex in aqueous solutions. However, it is interesting to note that the existence of a non-oxido U5+/U4+ couple was reported in the aqueous solutions of redox systems containing the unsaturated polyoxometalate anions \u03b1-[P2W18O62]6\u2013, [P2W17O61]10\u2013, and [SiW11O39]8\u2013. PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O bonds (from U5+ to UO2+) that results in the existence of a non-oxido U5+ complex in aqueous solutions containing the U5+/U4+ couple.The structure of the UO3L) in the three complexes decreases in the order: V(v) > Fe(iii) > U(vi). In Na[V(L)2]\u00b72H2O(cr), both ligands are triply deprotonated whereas in Fe(H2L)(HL)\u00b78H2O(cr), one ligand is doubly deprotonated and the other is singly deprotonated. Lastly, in UO2(H2L)(H2L)\u00b7H2O(cr), both ligands are singly deprotonated. Since each batch of crystals was obtained from solutions prepared at or near neutral pH where the ligand had the same protonation state (H3L), it is evident that V(v) competes the most effectively with protons for the ligand under these conditions. In conjunction with the parallel trend in bond lengths discussed above, this observation corroborates the suggestion that vanadium(v), in the form of the \u201cbare\u201d V5+ ion, forms the strongest complex with glutaroimide-dioxime by complete deprotonation of the ligand.The degree of deprotonation of glutaroimide-dioxime (as Hv) by the poly(amidoxime) sorbents is probably due to the formation of the very stable non-oxido V5+ complex with glutaroimide-dioxime. To improve the selectivity of the sorbent for U(vi) over V(v), an ideal ligand would be the one(s) with a binding ability that is sufficiently high for U(vi) but not high enough to displace the oxido V PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O bond(s) in the V(v) species. Starting with the cyclic glutaroimide-dioxime platform, adding electron-withdrawing groups to the platform could reduce the basicity of the imide and oxime groups and \u201cfine-tune\u201d the binding ability of the ligand(s).In summary, the extremely strong sorption of V(v) complex from aqueous solution is a very rare occurrence. To the best of the authors' knowledge, only one other non-oxido V(v) complex has been reported. The non-oxido V(v) complex, [PPh4][\u0394-V(HIDPA)2]\u00b7H2O(cr), can be synthesized by oxidizing Amavadin, which is itself a non-oxido V(iv) complex of -2,2\u2032-(hydroxyimino)dipropionic acid (H3HIDPA), and subsequently precipitating it from an aqueous solution containing a tetraphenylphosphonium (PPh4+) salt.2\u2013 complex contains two tetradentate ligands that coordinate via a central nitrogen and three oxygen donors unlike [V(L)2]\u2013, in which tridentate bonding is observed. As 2]\u00b72H2O(cr) are significantly shorter than the analogous bonds for the V(HIDPA)2\u2013 complex by 0.075 \u00c5 and 0.055 \u00c5, respectively. The shorter bond lengths coupled with the fact that the oxime moieties of glutaro-imidedioxime are more basic (pKa \u2248 11\u201312) than the carboxylate moieties (pKa \u2248 4\u20135) in HIDPA implies that [V(L)2]\u2013 is likely a stronger complex.The isolation of a \u201cbare\u201d non-oxido V(v)- and 1\u2009:\u20092 non-oxidovanadium(v)\u2013glutaroimide-dioxime complexes could help to understand and develop vanadium(v) compounds that mimic the effects of insulin in the treatment of diabetes. It is known that vanadium plays very important roles in biological systemsv) organic complexes, such as the aforementioned K[VO2(Dpa-OH)] complex, exhibit insulin mimetic behavior.2(Dpa-OH)]\u00b7H2O(cr) and the two V(v)\u2013glutaroimide-dioxime complexes.In addition to helping improve the extraction of uranium from seawater, the structural information for both the 1\u2009:\u20091 oxidovanadium(2(HL)](cr) are shorter than the analogous bond distances in K[VO2(Dpa-OH)]\u00b7H2O(cr) by 0.10 \u00c5 and 0.06 \u00c5, respectively, implying stronger bonding in the glutaroimide-dioxime complex. Interestingly, the oxido V\u2013O bonds in VO2(HL)\u2013 are slightly longer than the oxido bonds in the Dpa-OH complex, which implies weaker V PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O bonds and may explain the ability of the second glutaroimide-dioxime ligand to subsequently displace the two oxido oxygens. In fact, the non-oxido [V(L)2]\u2013 complex formed upon addition of a second ligand to VO2(HL)\u2013 leads to an even more significant reduction of bond lengths in the Na[V(L)2]\u00b72H2O crystal. The V\u2013N and average V\u2013O bonds in [V(L)2]\u2013 are the shortest of all three complexes by 0.13 \u00c5 and 0.12 \u00c5, respectively, compared to VO2(Dpa-OH)\u2013. In this case, the higher charge density of V5+ (compared to the VO2+ moiety) coupled with the very short bond lengths indicate that [V(L)2]\u2013 is a much stronger complex than VO2(Dpa-OH)\u2013.As shown in the table, the V\u2013N bond and the average V\u2013O bond distances in Na exhibits insulin mimetic behavior in vivo, the mechanism of action could be different from that of the dissociated VO2(Dpa-OH)\u2013 complex, making Na[VO2(HL)] a worthy candidate for further investigation.Concurrent solution showed tiv) complexes, very compelling evidence was provided suggesting that only those complexes that transformed to their vanadyl (oxido) form at physiological pH exhibited insulin mimetic behavior.v) complexes partly because very few non-oxido V(v) complexes have been identified. The non-oxido and oxido V(v) complexes with glutaroimide-dioxime could provide a unique opportunity to investigate the in vivo behavior of intact oxido- and non-oxido V(v) complexes containing the same ligand, binding motif, and overall charge at physiological pH. The results of these studies could help corroborate the hypothesis of Yoshikawa et al. regarding the requirement for an oxido (or dioxido) vanadium moiety to observe insulin mimetic behavior.Lastly, in a detailed study carried out by Yoshikawa and co-workers of six different crystalline non-oxido V(v) complex with glutaroimide-dioxime (H3L), Na[V(L)2]\u00b72H2O(cr), was crystallized from aqueous solution and characterized via X-ray diffraction. The complex was found to contain two fully deprotonated L3\u2013 ligands bound to the bare V5+ cation via two oxime oxygens and the imide nitrogen. An intermediate complex, Na[VO2(HL)](cr), was also isolated and found to contain the typical VO2+ moiety present in many V(v) complexes.A rare, non-oxido V\u2013glutaroimide-dioxime complex. ESI-MS studies of V(v)\u2013glutaroimide-dioxime solutions allowed the identification the intermediate 1\u2009:\u20091 M\u2009:\u2009L complex as well as the bare [V(L)2]\u2013 complex at m/z = 330.8.Further characterizations using v) to amidoxime-based sorbents relative to U(vi) and Fe(iii) were gained by comparing the structural parameters of the V(v)\u2013glutaroimide-dioxime complex with the analogous U(vi)\u2013 and Fe(iii)\u2013glutaroimide-dioxime complexes. For these complexes, the degree of protonation of the ligand was found to decrease from U(vi) to V(v). In conjunction with the substantially shorter bond lengths observed for the V(v) complex relative to the other complexes, this implies stronger bonding in the V(v) complex and higher thermodynamic stability. In fact, the trend in binding strengths parallels the observed trend in sorption of these cations to poly(amidoxime) sorbents in marine tests.Structural insights into the much higher sorption of V(v) compounds suitable for the treatment of diabetes, the structural studies with glutaroimide-dioxime are useful for aiding the development of new, highly stable organic V(v) compounds. In fact, the high solubility of Na[V(L)2]\u00b72H2O in aqueous and ethanol solutions coupled with its stability at physiological pH could make it a potential candidate for use in diabetic treatment studies.Lastly, as there are ongoing studies to synthesize vanadium(2]\u00b72H2O(cr) were prepared at Lawrence Berkeley National Laboratory (LBNL). The glutaroimide-dioxime ligand was synthesized, and its purity was verified as described previously.3 (0.2 mmol), NaCl (12 mmol), and 0.5 mmol glutaroimide-dioxime was slowly evaporated over the course of a week to generate shiny, dark brown/black acicular crystals. The crystals are very soluble in water, fairly soluble in ethanol, and less soluble in acetonitrile and methanol. Interestingly, it was observed that prolonged heating of aqueous Na[V(L2)] solutions at \u223c50\u201360 \u00b0C resulted in the apparent decomposition of the complex as evidenced by the fading color of the solution from dark brown to a yellow-orange color. However, no further efforts were made to ascertain whether the apparent decomposition was due to either partial oxidation of glutaroimide-dioxime by V(v) or to other mechanisms.Single crystals of Na[V(L)\u03bb = 0.7749 \u00c5 were calculated using the Brennan method in XDISP2]\u00b72H2O(cr) are provided in ESI, Table S1.A single crystal was selected, removed from Paratone oil with a MiTiGen microloop, and mounted on to a Bruker goniometer equipped with a PHOTON100 CMOS detector and Oxford Systems Cryostream 800 series on beamline 11.3.1 of the Advanced Light Source at LBNL. The data were collected at 100K using the Bruker APEX2 software2(HL)] were prepared at Pacific Northwest National Laboratory (PNNL). Glutaroimide-dioxime3 was added, resulting in a dark brown solution immediately. After stirring for 5 h, the solution was filtered to remove any undissolved solids prior to removing the solvent. The residue was then re-dissolved in ethanol and filtered as before. Orange crystals were obtained from vapor diffusion of hexane into the ethanol solution. Note that the undissolved solids remaining after either filtration were not characterized.Single crystals of Na[VO2(HL)](cr) are provided in ESI, Table S3.A Bruker-AXS Kappa Apex II CCD diffractometer with 0.71073 \u00c5 Mo K\u03b1 radiation was used for data collection. Crystals were mounted on a MiTeGen MicroMounts pin using Paratone-N oil. Data were collected at 100 K. The software used for data analysis includes Br\u00fcker APEX II17O-labelled solutions for NMR experiments was performed at LBNL. NMR data were collected at University of California, Berkeley (UCB) and LBNL.Preparation of the 17O-enriched water was purchased from Cambridge Isotope Laboratories, Inc. (Lot # I1-3969). 3.67 mg (0.296 mmol) NaVO3 was dissolved in 2.0 mL 17O-enriched H2O, followed by adding 50.7 mg 40% NaOD (in D2O) solution. The colorless solution was agitated and set aside for 2\u20133 days at room temperature to allow 16O/17O exchange. The solution was checked by 17O NMR after 2 and 3 days to confirm the oxido ligand exchange.217O solutions precluded accurate pH measurements, the pH of the solutions were determined to be 7.5 (b), 8.5 (c) and 8.7 (d) by using H2O solutions of a larger volume (4.0 mL) containing the same concentrations of vanadate and glutaroimide-dioxime as the H217O solutions. These solutions were allowed to equilibrate for one day after acid additions before acquisition of NMR spectra. The final colors of solutions b, c, and d were amber, brown, and dark brown, respectively.The above-described vanadate solution was equally divided into four solutions for multinuclear NMR experiments. Different quantities of glutaroimide-dioxime were added into solutions b, c, and d to obtain an [L]/[V] ratio of 1, 2 and 3 for solutions b, c, and d, respectively. At this time, solution a (with vanadate only) remained colorless, but solutions b, c, and d (with vanadate and glutaroimide-dioxime) became pale yellow. A total of 0.12 mL 0.980 M HCl was added in two portions into each of solutions b, c, and d to adjust the pH of the solutions to around 8. Because the small volume (0.5 mL) of the H217O solutions of V(v)/glutaroimide-dioxime described above , one D2O solution of pure glutaroimide-dioxime (a\u2032) and one D2O solution of the Na[V(L)2]\u00b72H2O crystal (e) were also prepared for 1H/13C and 51V NMR experiments. Detailed information on the conditions of solutions a, b, c, d, e, and a\u2019 is provided in ESI, Table S3.In addition to the four H51V spectrum of the D2O solution of Na[V(L)2]\u00b72H2O(cr) was acquired at LBNL on a Bruker AV-300 spectrometer referenced to an external standard of VOCl3 in C6D6. All other 17O, 51V, and 13C NMR spectra were acquired at UCB on a Bruker DRX-500 spectrometer equipped with a Z-gradient broadband probe. The 1H spectra were acquired at UCB on a Bruker AV-500 spectrometer equipped with a Z-gradient triple broadband inverse detection probe using WATERGATE solvent suppression. The 1H, 13C, and 51V spectra were referenced to an external standard of VOCl3 in C6D6 and the 17O spectra were referenced to the H217O water resonance.All NMR spectra were acquired at 20\u201322 \u00b0C. The \u20131. The ESI-MS experiments with methanol spray were conducted on a Finnigan LTQ FT mass spectrometer (Thermo) at the QB3/Chemistry Mass Spectrometry Facility (UCB). Aliquots of the 1\u2009:\u20091 and 2\u2009:\u20091 [L]/[V] samples were taken and diluted in methanol. The samples were injected directly via a syringe at a flow rate of 5 \u03bcL min\u20131 with a spray voltage of 3.5 kV.Two sets of ESI-MS experiments were performed using different spray solutions on two different instruments. The ESI-MS experiments with the ethanol/water mixture were performed using an Agilent 6340 quadrupole ion trap mass spectrometer with a micro-ESI source at LBNL. Aliquots of the solutions with [L]/[V] at 1\u2009:\u20091 and 2\u2009:\u20091 were diluted in (90/10) ethanol/water and injected into the instrument and sprayed in the negative ion mode at 1 \u03bcL ming = 2.0036).EPR spectra were obtained at LBNL at room temperature and at 4 K with a Varian E-12 spectrometer equipped with liquid helium cryostat, an EIP-547 microwave frequency counter, and a Varian E-500 gaussmeter, which was calibrated using 2,2-diphenyl-1-picrylhydrazyl and participated in unlabeled 51V/1H/13C NMR and EPR experiments. B. F. Parker conducted the unlabeled and 17O-labeled 51V/17O/1H/13C NMR and ESI-MS experiments (methanol spray). S. J. Teat collected and analyzed the structure data for Na[V(L)2]\u00b72H2O(cr). Z. Zhang participated in 17O-labeled 51V/17O/1H/13C NMR and ESI-MS experiments. W. W. Lukens collected and analyzed the EPR data. P. D. Dau conducted the ESI-MS experiments with ethanol spray. J. Arnold and J. K. Gibson supervised the research of B. F. Parker and P. D. Dau, respectively. L. Rao, B. F. Parker, and Z. Zhang designed the concurrent 17O/51V/1H/13C NMR experiments. L. Rao supervised the research of C. J. Leggett and Z. Zhang, and organized the preparation of the manuscript, to which all authors contributed. S. M. Peterson and M. G. Warner designed the experiments for synthesizing Na[VO2(HL)](cr) and S. M. Peterson conducted the synthesis. A. J. P. Cardenas collected and analyzed the crystal structure data for Na[VO2(HL)](cr).C. J. Leggett synthesized the glutaroimide-dioxime ligand and Na[V(L)Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "The formal cyclopentane-1,3-diyl derivatives [E1(\u03bc-NTer)2({E2C} = NDmp)] were prepared by 1,1-insertion of CNDmp into the N\u2013E2 bond of [E1(\u03bc-NTer)2E2] . 1(\u03bc-NTer)2({E2C} = NDmp)] were prepared by 1,1-insertion of CNDmp into the N\u2013E2 bond of [E1(\u03bc-NTer)2E2] . The insertion does not occur for E1 = E2 = As or E2 = Sb. Dependent on the choice of formal radical centres E, either a biradicaloid or a zwitterion was obtained. The biradicaloid features a P and an As radical center and its biradical character was established by computations as well as characteristic reactivity with respect to the formation of a housane derivative and the activation of molecules bearing multiple bonds, which was demonstrated using the example of PCtBu. In contrast, the formally N,As- and N,P-centered biradicaloids are better regarded as zwitterionic species in accord with computations and diminished reactivity, as neither housane formation nor activation of multiple bonds could be observed.The formal cyclopentane-1,3-diyl derivatives [E To date, several heteroatom-substituted derivatives of cyclopentanediyl bearing different substituents are known (selected examples: B\u2013D).Biradicals and biradicaloids are highly reactive species that can occur in the processes of bond formation and bond breaking. They were discussed as intermediates even in Diels\u2013Alder reactions by M. Dewar 2P] and [E1(\u03bc-NTer)2E2] ]2,2 ,2 phenyl),2Ge2 phenyl).2(\u03bc-NTer)2N], only diminished reactivity was observed, hence these are better regarded as zwitterionic compounds than as biradicaloids in agreement with computational studies. In the case of [Sb(\u03bc-NTer)2P], the biradicaloid was found to be a transient intermediate, whose existence could be proven by trapping experiments.34Until recently, all known cyclobutane-1,3-diyl derivatives incorporated equivalent radical centres, even though several examples investigated by the groups of Power and Yoshifuji are known featuring differing bridging moieties. PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O, into cyclodiphospha diazanediyl, [P(\u03bc-NTer)]2 (1PP), which afforded species E scale\" fill=\"currentColor\" stroke=\"none\">N\u2013R 2, Ter; Ter = 2,6-dimesityl-phenyl, Dmp = 2,6-dimethylphenyl), with diphosphadiazanediyl 1PP. By variation of the organic substituent, steric and electronic properties of the isonitrile could be varied. These could be adjusted to cleanly afford the cyclopentane-1,3-diyl derivative, when 2,6-dimethylphenyl-isonitrile was utilized by reaction of the available group 15 cyclobutanediyl derivatives (1E21E) with a selected isonitrile, C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N\u2013Dmp .2N3H and the product 2NP could not be isolated. Upon insertion of the isonitrile, the colour of the solution changed from yellow (1NP) to red .tBu did not alter any of these characteristics, indicating that no reaction with 2NP occurred in accord with a rather small biradical character (see below).On the contrary, the ring expansion reaction of the triazenide-derived cyclobutanediyl derivatives smoothly when a s1NAs with CNDmp similarly resulted in a change of colour from initially yellow (\u03bbmax = 379 nm) to red, indicating the presence of 2NAs . Similar to 2NP, 2NAs features a \u03bd scale\" fill=\"currentColor\" stroke=\"none\">N) vibration at 1612 in the Raman and at 1610 cm\u20131 in the IR spectrum which is significantly different from the \u03bd scale\" fill=\"currentColor\" stroke=\"none\">N) vibration of pure CNDmp exhibiting a CN triple bond (2123 cm\u20131). Crystals suitable for single X-ray studies were obtained after concentration at 4 \u00b0C in good yields (83%). Red needle-shaped crystals of 2NAs decompose above 141 \u00b0C and are moisture and air sensitive. Like 2NP, 2NAs does not react with PCtBu also displaying diminished biradical character. The molecular structure of 2NAs (3CAs heterocycle. The As\u2013N bond of 1.875(3) \u00c5 is considerably longer than in the known tetrazarsole galliumtrichloride adduct Mes*N4As\u00b7GaCl3 (1.784(2), 1.805(2) \u00c5; cf. \u03a3rcov(As\u2013N) = 1.91 \u00c5) indicating single bond character.rcov(As\u2013C) = 1.97 \u00c5). The N\u2013N distances in 2NAs of 1.316(4) and 1.349(4) \u00c5 are between the sum of covalent radii for a double and a single bond , indicating delocalized double bond character, while the C49\u2013N3 bond length (1.428(5) \u00c5) corresponds to a single bond (\u03a3rcov(C\u2013N) = 1.46 \u00c5) contrary to the exocyclic C49\u2013N4 bond (1.293(5) \u00c5) which is in the typical range of a C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N double bond.54The reaction of of 2NAs features1PAs in benzene with CNDmp. Within 10 minutes the insertion of CNDmp into the dark purple 1PAs (\u03bbmax = 550 nm)2PAs, which is of green colour vibration is dramatically shifted from 2123 to 1633 cm\u20131, as expected for the transition from a C\u2013N triple to double bond. Crystals of 2PAs decompose above 122 \u00b0C.In a next series of experiments we treated a solution of 13C NMR data, in which the former isonitrile carbon atom gives rise to a resonance at 184.98 ppm, which appears as doublet with a small JCP = 9.9 Hz, indicating a 2J coupling. X-ray diffraction experiments on single crystals of 2PAs revealed a secondary irradiation-induced isomerization to the housane isomer 2\u2032PAs scale\" fill=\"currentColor\" stroke=\"none\">C triple bond. The initially green solution of 2PAs in benzene quickly turned yellow upon addition of the phosphaalkyne and formation of 3PAs was observed in good yields , indicating that exclusively one isomer was formed. The strong JPP coupling of 260 Hz is characteristic for a 1JPP coupling constant. Single crystal X-ray structure elucidation unequivocally revealed the exclusive formation of one isomer . It is interesting to note that for the cyclobutanediyl derivative [As(\u03bc-NTer)2P], the regioselectivity was opposite and only the C\u2013P and P\u2013As bonded isomer was observed due to thermodynamic preference of a C\u2013P over C\u2013As bond.32The biradical character of lds 78%, , Fig. 3.2PAs/2\u2032PAs and 3PAs suitable for structure analysis were obtained from benzene solutions. The most prominent structural feature of 2PAs represents the almost planar five-membered heterocycle with rather short P\u2013N bond lengths (P1B\u2013N1 1.636(2), P1B\u2013N2 1.691(2) \u00c5) in the range of PN double bonds scale\" fill=\"currentColor\" stroke=\"none\">N) = 1.62 \u00c5), while the As\u2013N (As1B\u2013N1 1.874(2) \u00c5) and As\u2013C bonds (As1B\u2013C49 1.937(2) \u00c5, 2NAs, Single crystals of 2\u2032PAs. The transannular P\u2013As distance is shortened from 3.049(2) to 2.2920(7) \u00c5 clearly indicating the presence of a transannular P\u2013As single bond (\u03a3rcov(P\u2013As) = 2.32 \u00c5). Additionally, the P\u2013N\u2013As angle strongly decreases from 120.5(1) to 77.10(6)\u00b0. The three-membered As\u2013N\u2013P ring is almost perpendicular condensed to the four-membered As\u2013P\u2013N\u2013C ring. These experimental structural parameters are in good agreement with those of DFT computations \u00c5. The P\u2013C bridging bond length amounts to 1.673(2) \u00c5 in accord with a P PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C double bond.The phosphaalkyne addition product 1E22E five-membered heterocycles was computed for 1E22E and CASSCF computations were carried out (CASSCF = complete active space self-consistent field). Experimentally, biradicaloids 1E22E show no EPR signal and 1H, 13C, and 31P NMR signals. All 1E22E compounds have a singlet ground state in accord with rather large \u0394ES\u2013T values computations confirmed the biradicaloid nature of 1E22E. The dominant contributions to the CI wave function arise from the HOMO/LUMO exchange. The biradicaloid character can be estimated by using the formula: \u03b2 = 2c22/(c12 + c22).1 the biradical character is preserved compared to the starting material 1E21E. Therefore, as illustrated in 1 = As < P < N and E2 = Sb < As < P. For example, a biradical character \u03b2 of only 13% was computed for 2NP and 11% for 2NAs, respectively , coefficients of main contributions 0.946, \u20130.250 for 1NP and 0.956, \u20130.229 for 1NAs).2PAs features substantial biradical character of \u03b2 = 24% , c1 = 0.916 and c2 = \u20130.339), in agreement with the experimental fact that this species is capable of activating molecules containing triple bonds (vide supra) contrary to 2NP or 2NAs. Moreover, the larger zwitterionic character of 2NAs compared to 2PAs is also manifested by the HOMO of 2NAs featuring very large coefficients at As and very small ones at N, while for 2PAs the contributions are distributed almost evenly.To investigate the biradicaloid character of T values signific\u03b2 and Wiberg bond index (WBI) between the two radical centers (1\u2013E2) of all considered species 1E22E ranges from 0.262 to 0.434, which originates from partial occupation of the transannularly bonding LUMO. This reflects strong antiferromagnetic coupling between the radical centers E1 and E2. NICS values decrease when heavier elements are incorporated in the five-membered ring, indicating less stabilization by electron delocalization within the heterocycle, which is due to diminished orbital overlap between E and the adjacent N or C atoms.The computational data show a correlation between biradical character centers . The WBI1PP and the reaction of 1PP with different isonitriles, we suggest a mechanism involving the formation of a [1.1.1]bicyclic intermediate, which subsequently rearranges to give the cyclopentanediyl derivative < P (82.0) < Sb (126.8 kJ mol\u20131) for E. For the heavier homologues with E1 being P, it is exothermic and the reaction energy increases in the same order: E2 = As (\u201368.8) < P (\u201349.9) < Sb (\u201328.9 kJ mol\u20131). The second reaction step, the rearrangement from the [1.1.1]bicycle to the planar five-membered ring, is exothermic in every case. In this case, there is a tendency of the reaction becoming less exothermic as the pnictogen E2 becomes heavier .2PAs as well as 2\u2032PAs. However, upon UV irradiation, decomposition occurred, preventing the isolation of the housane species 2\u2032PAs.Finally, we want to address the issue of housane formation. Computational studies indicate, that 2\u2032PAs, which also features a localized double bond aside the P\u2013As axis of ble bond .e.g. within the homologous series 2NP, 2NAs, 2NSb), the distance between the radical centres is large and hence the orbital overlap is small, thereby reducing the stability of the heavier cyclopentane-1,3-diyl species. This is reflected in the decreasing relative stability of the singlet ground state compared to the lowest lying triplet state.In summary, the ring expansion reaction of cyclobutanediyls with isonitriles enabled the synthesis of three new group 15 derivatives of cyclopentane-1,3-diyl featuring N/P, N/As, or P/As atoms as radical centres within the five-membered heterocycle. However, there are limitations to this insertion reaction, and the preparation of cyclopentane-1,3-diyls bearing N/Sb, As/As, or P/Sb radical centres remains a challenge. Two reasons can be accountable for this: (i) in the zwitterionic border case, due to strong polarization the valence electron density distributed far from equally between the formal radical centres, so the biradical reactivity is diminished, and (ii) by incorporating heavier elements in the heterocycle (3 moiety (E1 = N) have strongly polarized N\u2013E2 bonds, a rather small biradical character and therefore are better referred to as zwitterions, which is also manifested by their inability to activate molecules bearing multiple bonds. In contrast, the P/As centered biradicaloid 2PAs exhibits a considerable biradical character, higher reactivity and can be isomerized to the short-bond species 2\u2032PAs or be utilized in small molecule activation.The new cyclopentane-1,3-diyl derivatives containing an NSupplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "The syntheses and 1,2-addition reactivities of nontrigonal phosphazenes supported by trianionic tricoordinating chelates of the type L3P PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019Ndipp (3: L3 = N[CHC(tBu)O]23\u2013; 4: L3 = N(o-NMeC6H4)23\u2013; dipp = 2,6-diisopropylphenyl) are reported. 3P PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019Ndipp (3: L3 = N[CHC(tBu)O]23\u2013; 4: L3 = N(o-NMeC6H4)23\u2013; dipp = 2,6-diisopropylphenyl) are reported. These compounds are characterized by multinuclear NMR and single-crystal X-ray diffraction experiments. Distorted phosphazenes 3 and 4 are shown to add B\u2013H, B\u2013O, and Si\u2013H bonds across the formal P PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N double bond, and their reactivities are contrasted with acyclic analogues. Derivatives of phosphazene 3 bearing sterically unencumbered N-substitutents readily dimerize to form the corresponding cyclodiphosphazanes; compounds with sterically demanding N-substituents are interconvertible between their monomeric and dimeric forms. The enhanced electrophilicity of the phosphorus center in nontrigonal phosphazenes 3 and 4 is rationalized by DFT calculations. Gas phase fluoride ion affinities are computed to be markedly higher for distorted phosphazenes, while proton affinities are largely unaffected by geometric distortion. These results are interpreted to suggest that distortion from pseudotetrahedral geometry results in stabilization of the P-based LUMO, while HOMO energies are essentially unchanged.The syntheses and 1,2-addition reactivities of nontrigonal phosphazenes supported by trianionic tricoordinating chelates of the type L PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N unit proves quite inert; indeed, the robustness of the P PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N moiety forms the basis for the many remarkable applications of polyphosphazene inorganic/organic hybrid materials.2Phosphazenes, \u03c3 PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N double bond scale\" fill=\"currentColor\" stroke=\"none\">NR\u2032 \u2194 R3P+\u2013N\u2013R\u2032) also gives rise to numerous applications for phosphazenes as nitrogen-based electron pair donors. For instance, phosphazenes are known to be strong donor ligands for transition metals.t-Bu-P4 displays exceptionally high Br\u00f8nsted\u2013Lowry basicity and has been investigated for a variety of base-mediated transformations.4As might be expected on the basis of the differing electronegativities of phosphorus and nitrogen, however, the polarization of the formal P PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N unit. Chief among this class of reactions are metathetical transformations stemming from formal (2 + 2) addition/elimination of unsaturated organic compounds at the phosphazene P PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N moiety, of which the aza-Wittig7Apart from this nitrogen-based reactivity, phosphazenes also have been employed in a number of transformations that leverage the vicinal ambiphilic character of the P1 at phosphorus through a ligand-cooperative mechanism.In addition, we reported the synthesis of mpound 2 , top and1 and 2 might range beyond the tricoordinate state to support interesting chemical properties of their distorted \u03c34,\u03bb5-phosphazene derivatives. In this study, we report a combined theoretical and experimental treatment of phosphazenes based on supporting structures 1 and 2 which validate the hypothesis that imposition of a geometric constraint at positions ancillary to the P PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N unit enhances vicinal ditopic ambiphilicity of these phosphazenes. We also show that 1,2-reactivity of the P PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N unit leads to facile addition of \u03c3-bonded B\u2013H, B\u2013O, and Si\u2013H reagents across the P PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N moiety. In total, the results establish a rational framework for the design of bespoke phosphazenes with novel properties and reactivities that expand the functional role of this important class of main group compounds.Based on this precedent, we wished to ascertain the extent to which the distinctive reactivity traits of nontrigonal phosphorus compounds 2.2.1.3P PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019NH (1\u2013P\u2013H3 (\u03b1) in the range 90\u00b0 < \u03b1 < 180\u00b0 at the M06-2X/def2-TZVP level of theory2\u2013P\u2013N\u2013H4 was relaxed to minimize energy associated with rotation about the P PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N formal double bond; bond lengths were held constant at values obtained from the equilibrium geometry of H3P PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019NH ; these parameters are in good agreement with those found in previous computational studies of H3P PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019NH.17A computational appraisal of the consequence of molecular distortion on phosphazene frontier electronic structure illustrates the theoretical framework underlying our experimental study. With the parent phosphazene Hsvg>NH , inset a1.000000,.000000 s\u03b1 = 97.9\u00b0) predictably lead to increases in overall energy (\u03b5HOMO) remains more or less constant across the scanned coordinate. Visual inspection of the relevant orbitals provides for a qualitative interpretation of this observation; highest occupied molecular orbitals are found to be mostly nitrogen-based, corresponding largely to the N lone pair (nN) with minor contributions from the ancillary phosphorus substituents.Deviations from the equilibrium geometry (\u03b5LUMO) decreases as the bond angle \u03b1 increases, ultimately resulting in stabilization of more than 3 eV as LUMO takes on increasing s-orbital character in the distortion to seesaw geometry (\u03b1 = 180\u00b0). This electronic picture suggests that the nontrigonal distortion of phosphazenes should retain the Lewis basicity of the N position but dramatically increase the Lewis acidity of the P position. We posit that the juxtaposition of donor and acceptor character at adjacent atoms should lead to an increase in 1,2-ambiphilic reactivity of the P PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N unit, in analogy to well-established chemistry of early transition metal imido scale\" fill=\"currentColor\" stroke=\"none\">NR) complexes.By contrast, the energy of the lowest unoccupied orbital , followed by removal of volatiles and trituration of the residue in pentane, produced a white solid whose 31P{1H} NMR spectrum displayed a resonance at \u03b4 7.3 ppm; the observed chemical shift is consistent with compositionally similar O,N,O-substituted phosphazenes previously reported in the literature.1H NMR spectrum, both vinylic protons (\u03b4 5.49 ppm) and tBu groups (\u03b4 0.91 ppm) on the O,N,O-support scaffold of 3 give rise to a single resonance, respectively, suggesting a time-averaged molecular geometry of Cs molecular symmetry or higher. In contrast to the planar starting material 1, the 3JP\u2013H scalar coupling constant between phosphorus and the vinylic hydrogen nuclei for 3 (3JP\u2013H = 29 Hz) is discernible and modest in magnitude. Arduengo previously noted3JP\u2013H value and the extent of molecular folding of the O,N,O-supporting ligand in related \u03c34-P compounds. On this basis, we infer that the bicyclic O,N,O ligand backbone is distorted from planarity by folding along the P\u2013N axis. We therefore assigned the structure of 3 as in The requisite phosphazenes are easily prepared by Staudinger imination4 was prepared by the reaction of 2 with 1 equiv. of 2,6-diisopropylphenyl azide and subsequent recrystallization from a solution of pentane and dichloromethane. The 31P{1H} NMR spectrum of this compound showed a resonance at \u03b4 14.5 ppm, consistent with typical values for tetraazaphosphazenes. The spectroscopic equivalence of both ligand N-methyl substituents in the 1H NMR spectrum suggest time-averaged Cs or higher symmetry, as with compound 3.Similarly, compound 7 and 8 were likewise synthesized from the corresponding phosphorus compounds 5 and 6 and N,N,N- (8)Crystalline solids of termined , Table 13 displays substantial opening of the O1\u2013P1\u2013O2 bond angle relative to the acyclic phosphazene 7; the bond angle in 3 (112.62(6)\u00b0) is 16\u00b0 wider than that of 7 (96.70(8)\u00b0). The increase in O1\u2013P1\u2013O2 bond angle in 3 is complemented by a moderate (ca. 5\u201310\u00b0) decrease in internal bond angles O1\u2013P1\u2013O2. Additionally, the N1\u2013P1\u2013N2 angle likewise increases in 3 (124.96(7)\u00b0) relative to 7 (116.46(11)\u00b0) in order to restrict unfavorable steric interactions between the N-dipp substituent and the ligand backbone. Thus, the solid state structure of phosphazene 3 corresponds to a deviation from idealized pseudo-tetrahedral geometry along the distortion coordinate illustrated in The solid state structure of N,N,N-phosphabicyclic phosphazene 4 has an expanded N3\u2013P1\u2013N4 bond angle of 4 (119.73(6)\u00b0), 14\u00b0 greater than the average angle in 8 (105.15(15)\u00b0). Furthermore, the N2\u2013P1\u2013N3 and N3\u2013P1\u2013N4 endocyclic angles were generally smaller relative to 8. As with constrained phosphazene 3, there was also an expansion of the N1\u2013P1\u2013N2 bond angle in 4 (129.22(6)\u00b0) versus8 (109.99(15)\u00b0, average of 3 angles), constituting a difference of 19\u00b0; this difference is greater than that of the 3/7 pair and is likely a result of increased steric congestion imposed by the N-methylanilides of 4.Similarly, via a bicyclic ligand framework results in a nontrigonal geometry along the distortion coordinate toward see-saw molecular structures.These solid state structures confirm our hypothesis that imposing geometric constraints 2.2.33, 4, 7, and 8 at the M06-2X/def2-TZVP level of theory as a way of quantifying their respective Lewis acidities and basicities and proton affinity (PA) calculations on 3 and 4 are significantly greater than their unconstrained analogues 7 and 8. This result conforms with qualitative predictions from the model system H3P PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019NH ; upon distortion from pseudo-tetrahedral geometry, the phosphorus-based LUMOs decrease in energy, allowing for stronger interactions of exogenous anions like F\u2013 with nontrigonal 3 and 4 than with trigonal compounds 7 and 8.Fluoride ion affinities for constrained phosphazenes 3 and 7 are 238 kcal mol\u20131 and 239 kcal mol\u20131, respectively. For compounds 4 and 8, the PAs are 246 kcal mol\u20131 and 258 kcal mol\u20131, respectively. Given the magnitude of the calculated proton affinities, the difference in calculated PAs for 3/7 and 4/8 does not exceed 6% overall. This outcome is in accord with the interpretation from calculations in Section 2.1. in which molecular distortion would not be expected to affect significantly the N-based HOMOs responsible for Lewis basicity.Differences in proton affinities, on the other hand, are much smaller in magnitude. For instance, the computed PAs for compounds 2.3.2.3.13 could be prepared and characterized as indicated above, prolonged standing of solutions (ca. 3 d) at 60 \u00b0C led to the emergence of a new species with a low-field 31P NMR resonance (\u03b4 = \u201326.9 ppm). The formation and intensity of this new signal followed a concentration dependence in [3], suggesting a possible bimolecular origin. In accord with this observation, the identity of the new species was ultimately confirmed by single crystal X-ray diffraction analysis to be that of a head-to-tail homodimeric 1,3-diaza-2,4-diphosphetidine (cyclodiphosphazane) 3\u2032, which could be selectively crystallized from benzene solutions of 3/3\u2032 \u00c5) than the equatorial P\u2013N bonds (dP\u2013N = 1.665(3) \u00c5). Both bond distances are elongated as compared to monomeric 3 (dP\u2013N = 1.5069(14) \u00c5). In short, the structural metrics are consistent with a superposition of the Lewis structures for 3\u2032, displayed in Cyclophosphazane cal site . The mos3\u2032 in C6D6 at ambient temperature resulted in repopulation of the mixture containing both dimer 3\u2032 and monomer 3 as judged by 1H and 31P NMR spectroscopy. Consequently, we conclude that the formal 2 + 2 dimerization of 3 to 3\u2032 is reversible, as illustrated in 3 and 3\u2032 in the 31P NMR spectra is most consistent with a slow interconversion relative to the NMR timescale.Dissolution of a single-crystalline sample of O,N,O-phosphazene 3, dimerization of the constrained N,N,N-phosphazene 4 was not observed under any conditions.N-substituent prohibits close approach of a second phosphazene as would be necessary for dimer formation. Additionally, the phosphorus center of 4 is less Lewis acidic than that of 3 , so it is possible that decreased electrophilicity at P precludes sufficient driving force for phosphazene dimerization.In contrast to the deformed 2.3.2N-substituent has a controlling effect on the rate and position of monomer\u2013dimer equilibrium for phosphazenes based on O,N,O-platform 1. Whereas the N-dipp substituted phosphazene could be isolated in either monomeric (3) or dimeric (3\u2032) forms, O,N,O-phosphazenes bearing less sterically encumbered N-substituents could be isolated only in their dimeric cyclodiphosphazane forms moieties are detected as transient intermediates in the overall process, converting ultimately to the dimeric cyclodiphosphazane . By contrast, phosphazenes with electron-withdrawing phenyl, 10\u2032, \u03b4 \u201344.0 ppm) and alkyl N-substituents were observed only in their dimeric cyclodiphosphazane forms.The identity of the imino ne forms . In a tyosphorus . In situ31P NMR resonances of cyclodiphosphazanes 9\u2032\u201312\u2032 appear at noticeably lower field compared to 3\u2032, suggesting a geometric distinction between the very bulky 2,6-diisopropyl-substituted cyclodiphosphazane and less sterically demanding congeners. Single crystals of dimeric N-p-tolyl derivative 9\u2032 were obtained by slow evaporation of a benzene solution, and the structure was interrogated by X-ray diffraction \u00c5) and equatorial P\u2013N (dP\u2013N = 1.635(3) \u00c5) bond lengths for N-p-tolyl cyclodiphosphazane 9\u2032 are approximately 0.04 \u00c5 shorter than for N-dipp cyclodiphosphazane 3\u2032. We infer that the reduced steric congestion about the P2N2 core for 9\u2032 permits tighter association of the monomer subunits in a head-to-tail fashion that is manifest in the 31P isotropic chemical shielding differences. Congruent with this assessment, attempts to access monomeric phosphazenes 9\u201312 by heating of benzene solutions of 9\u2032\u201312\u2032 were unsuccessful; formal (2 + 2) dimerization appears to be prohibitively downhill in enthalpy and, therefore, irreversible for these less sterically congested cyclodiphosphazanes.The fraction . Globall PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N units is well-precedented in the literature, specifically for phosphazenes bearing strongly electron-withdrawing P-substituents or where relief of ring strain provides a driving force.O,N,O-phosphazenes (9\u2032\u201312\u2032) readily dimerize is thus a qualitative indication of the marked Lewis acidity of these phosphorus centers as compared to their acyclic congeners. Furthermore, the sum of the observations concerning the monomer\u2013dimer speciation of distorted phosphazenes 3/3\u2019 evidences a propensity for 1,2-ambiphilic reactivity of the P PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N unit, suggesting that intermolecular 1,2-additions of exogenous reagents might be feasible.Formal [2 + 2]-cyclodimerization of phosphazene P2.4.2.4.1 PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N unit in deformed phosphazenes 3 and 4, we elected to attempt the 1,2-addition of \u03c3-bonded E\u2013H small molecules. To this end, treatment of O,N,O-phosphazene 3 with 1 equiv. of pinacolborane (HBpin) in C6D6 at room temperature resulted in rapid consumption of the starting materials and formation of a single new compound with a 31P NMR resonance at \u03b4 \u201343.7 ppm presenting as a doublet of triplets . The magnitude of the larger coupling constant is indicative of a direct P\u2013H linkage; existence of a P\u2013H moiety was confirmed in 1H NMR spectra by the appearance of a doublet centered at \u03b4 9.37 ppm with complementary coupling (1JP\u2013H = 837 Hz). Although attempts to obtain a solid state structure of 13 from X-ray diffraction analysis were unsuccessful, these spectroscopic signatures are consistent with formation of hydrido amido phosphorane 13 in which the O,N,O-chelate spans two apical and one equatorial position about a phosphorus-centered trigonal bipyramid, and the hydride and borylamide substituents reside in the equatorial plane. Further support for this assignment comes by way of analogy to previous results from our group. We reported previously that hydrido amido phosphoranes related to 13 are accessible via intermolecular N\u2013H oxidative addition to 3.3 gave a crystallographically characterized oxidative addition product (i.e. the des-boryl congener of 13) that exhibits spectroscopic features in close agreement with those obtained for 13. In short, the combined data lend strong evidence to the structural assignment of the B\u2013H addition product 13.To investigate reactivity of the PN,N,N-phosphazene 4 with 1 equiv. of pinacolborane in C6D6 at ambient temperature quickly consumed starting materials to yield a species 14 with a doublet 31P NMR signal at \u03b4 \u201337.6 ppm . Complementary coupling was observed in the 1H NMR spectrum with a doublet resonance at \u03b4 6.36 ppm. As with the reaction of 3 and HBpin, these spectral data are characteristic of pentacoordinated hydrido amido phosphorane featuring a direct P\u2013H bond. By analogy to previous results on the intermolecular N\u2013H oxidative addition of 2,6-diisopropylaniline to 4,14 as in N,N,N-ligand, equatorial N-dipp substituent, and an axial hydride.Likewise, treating 3 and 4, acyclic O,N,O- and N,N,N-phosphazenes 7 and 8 were unreactive with respect to pinacolborane under identical reaction conditions. Additionally, none of the dimeric cyclodiphosphazanes 9\u2032\u201312\u2032 were found to undergo reaction with HBpin. We conclude, therefore, that the ability of the phosphazenes 3 and 4 to undergo 1,2-addition of H\u2013Bpin is dependent on the distorted structure enforced by the trianionic heteroatom supporting structures, and that access to the monomeric form of the phosphazene is critical for intermolecular addition.By contrast to 2.4.2O,N,O-phosphazene 3 was also able to add B\u2013O \u03c3 bonds across the P PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N unit. Addition of 1 equiv. of n-butoxy catecholborane to a C6D6 solution of 3 at room temperature , which can be attributed to coupling between the phosphorus center and the vinylic protons of the O,N,O-ligand. The magnitude of this coupling is smaller than would be expected for \u03c35-phosphorus compounds with a planar, meridional O,N,O-chelate. This result suggests that antiperiplanarity between P and H has been lost; that is, the dihedral angle between the phosphorus and vinylic hydrogen atoms has decreased from 180\u00b0 in accordance with the Karplus equation for 3J scalar coupling,O,N,O-framework adopts a folded geometry.The deformed perature resultedO,N,O-folding (15 is the unexpectedly short distance between the ligand amido N atom and the N-dipp-bound boron (dN2\u2013B1 = 1.637(3) \u00c5), consistent with the presence of a dative interaction N2 \u2192 B1 in 15; pyramidalization of the boron atom evident in the solid state structure further evidences this conclusion. By consequence of this interaction, compound 15 may be viewed as a trigonal bipyramidal phosphorus compound supported by a tetracoordinating boroazaphosphatrane ligand, where the fifth apical binding site of the trigonal bipyramid is occupied by the n-butoxy substituent. Indeed, the apical bond distance dP1\u2013N2 is quite long (1.9392(19) \u00c5), conforming to precedent from cationic phosphatranescf. dP\u2013N = 1.986(5) \u00c5 for [HP(OCH2CH2)3N][BF4];29dP\u2013N = 1.967(8) \u00c5 for [HP(MeNCH2CH2)3N][BF4]15 is substantially longer than the equatorial P1\u2013N1 bond (1.6195(18) \u00c5), comprising a difference of more than 0.3 \u00c5. As a consequence, the trans apical P1\u2013O3 bond is found to be quite short (1.5906(16) \u00c5); in fact, the apical P\u2013O bond is shorter than the equatorial P\u2013O bonds (1.6242(16) \u00c5 and 1.6252(16) \u00c5). This observation runs counter to typical trigonal bipyramidal geometries, where the 3-center, 4-electron apical bonds are usually longer than the 2-center, 2-electron equatorial bonds. These unusual structural features of the B\u2013O adduct of phosphazene 3 likely arise from molecular constraint imposed by the O,N,O-ligand and by the Lewis acidic boron atom.A single crystal suitable for X-ray diffraction was grown from benzene solution, and the solid state structure corroborates the above conclusion regarding -folding . A disti3, N,N,N-ligated phosphazene 4 did not react with n-butoxy catecholborane, perhaps again due to increased steric congestion about the P PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N unit. Likewise, acyclic analogues 7 and 8 were unreactive to B\u2013O bonds.Unlike phosphazene 2.4.33 was reactive toward the Si\u2013H bond of phenylsilane. Treating phosphazene 3 with phenylsilane in C6D6 at 50 \u00b0C for 16 h . Likewise, a doublet resonance with a large coupling constant centered at \u03b4 9.44 ppm (1JP\u2013H = 802 Hz) was observed in the 1H NMR spectrum. Another complementary doublet signal, corresponding to ligand vinylic protons, appeared at \u03b4 5.34 ppm (3JP\u2013H = 31 Hz). Analogous to other \u03c35,\u03bb5-phosphorus compounds synthesized here (vide supra), the coupling constant of 31 Hz is indicative of a planar O,N,O-chelate, as in PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N bond of 3 to give a hydrido phosphorane with a N-silyl substituent.In addition to activating B\u2013H and B\u2013O bonds, phosphazene for 16 h producedN,N,N-Phosphazene 4 also reacted with phenylsilane, but at a slower rate than 3. The conversion of 4 and phenylsilane to hydrido phosphorane 17 was completed after 48 h of heating at 80 \u00b0C in C6D6. The resulting 31P NMR spectrum showed one signal centered at \u03b4 \u201329.1 ppm ; the magnitude of the scalar coupling constant is characteristic of a P\u2013H bond, consistent with the structure of the \u03c35-phosphorus Si\u2013H addition product 17. By analogy to other N,N,N-ligated compounds synthesized earlier, we expect the trianionic chelate to adopt a folded structure, as in 7 and 8 were found to be unreactive to Si\u2013H addition. The fact that compounds of this type are robust to silane addition has been exploited by Fontaine, who has shown that phosphazenes similar to 8 can be used as Lewis base-catalysts for catalysed hydrosilylation of CO2.30Acyclic phosphazenes PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N unit of a phosphazene has often been invoked, albeit usually in tandem with subsequent elimination from P(v) and without direct observation of the elementary step. Kawashima has studied the intramolecular addition of a Si\u2013H bond to a functionalized phosphazene.in situ reduction of a phosphazene, presumably initiated by 1,2-addition of hydrosilane.via 1,2-addition via a trigonal bipyramidal hydrido amido phosphorane intermediate, but despite the prevalence of the 1,2-addition proposal, there do not exist discrete, well-characterized analogues of this key step. Our current results represent a rare well-defined addition reaction giving rise to stable pentacoordinate adducts that substantiate the notion of 1,2-addition of hydrosilanes to phosphazenes.The 1,2-addition of hydrosilane across the P3.O,N,O- and N,N,N-ligands exhibit increased cyclodimerization and 1,2-addition reactions of B\u2013H, B\u2013O, and Si\u2013H \u03c3 bonds as compared to acyclic congeners. The results of the combined experimental and theoretical studies above support the conclusion that nontrigonal distortion of phosphazenes leads to an enhancement of the 1,2-ambiphilic reactivity of the P PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N unit. In view of the multivarious roles of phosphazenes, the ability to master electronic structure and reactivity as a function of a modifiable parameter contributes to the discovery of novel applications. In conjunction with established approaches to synthetic tuning through substituent effects, the current results establish a rational geometry-based framework for modulating the reactivity of this important class of main group compounds, which may be leveraged in the design of functionally novel entities.Constrained phosphabicyclic phosphazenes supported by trianionic scaffolding 4.Full experimental details are available in the online ESI.4.1.6D6 (1 mL) was stirred at 40 \u00b0C for 16 h. All volatiles were removed in vacuo, and the resulting residue was triturated with pentane. The crude product was obtained after filtration, and pure 3 was isolated as a white solid by recrystallization from a 10\u2009:\u20091 dichloromethane/pentane solution . 1H NMR : \u03b4 7.23\u20137.21 , 7.12\u20137.07 , 7.07 , 5.49 , 3.83 , 1.40 , 0.91 ppm. 13C NMR : \u03b4 154.19, 141.29 , 123.03, 121.98, 112.43 , 32.64 , 29.35, 26.89, 23.75 ppm. 31P NMR : \u03b4 7.21 ppm. MS (ESI) calc'd for C24H37N2O2P (M+) 416.2593, found 416.2596.A solution of 1 with 2,6-diisopropylphenyl azide in C4.2.6D6 (1 mL) was stirred under ambient temperature for 12 h. The solvent was removed in vacuo, and the resulting solid product was recrystallized from a 10\u2009:\u20091 dichloromethane/pentane solution . 1H NMR : \u03b4 7.26 , 7.16 , 7.09\u20137.02 , 6.98\u20136.85 , 6.77 , 6.23 , 3.65 , 2.73 , 1.22 ppm. 13C NMR : \u03b4 141.38, 141.00 , 138.09 , 134.58 , 124.56, 123.26, 121.34, 120.14, 116.34 , 108.66 , 29.29 , 23.75 ppm. 31P NMR : \u03b4 14.54 ppm. MS (ESI) calc'd for C26H31N4P (M+) 430.2286, found 430.2290.A solution of 2 with 2,6-diisopropylphenyl azide in C4.3.6D6 (0.3 mL) was stirred at ambient temperature for 15 min. The solvent was removed in vacuo to afford the crude product . 1H NMR : \u03b4 9.38 , 7.27\u20137.25 , 7.08\u20137.04 , 5.49 , 3.62\u20133.56 , 1.53 , 1.19 , 1.15 ppm. 13C NMR : \u03b4 150.73 , 147.14, 126.64, 124.52 , 124.02, 123.42, 100.51 , 82.52, 31.69, 28.25, 27.51, 24.66, 23.62, 23.26 ppm. 31P NMR : \u03b4 \u201343.02 ppm. MS (ESI) calc'd for C30H49BN2O4P (M\u2013H+) 543.3518, found 543.3523.A solution of 3 and HBpin in C4.4.6D6 (0.3 mL) was stirred at 80 \u00b0C for 48 h. All volatiles were removed in vacuo to afford 20 as a yellow oil . 1H NMR : \u03b4 7.32 , 7.14\u20136.99 , 6.95 , 6.87 , 6.73 , 6.37 , 5.75 , 5.05 , 3.45\u20133.33 , 2.49 , 1.15 , 1.06 ppm. 13C NMR : \u03b4 148.30 , 137.93, 135.77 , 134.65 , 134.25, 133.46, 129.01, 127.16 , 123.90 , 121.42, 119.38, 113.89 , 108.72 , 29.69 , 27.61, 26.24, 23.84 ppm. 31P NMR : \u03b4 \u201328.54 ppm. MS (ESI) calc'd for C32H40N4PSi (M + H+) 539.2760, found 539.2763.A solution of 4 and phenylsilane in CThere are no conflicts to declare.Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "The neutral molecule NPrO and its anion NPrO\u2013 are characterized to be linear pentavalent praseodymium nitride-oxides that possess Pr PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N triple bonds and Pr PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O double bonds. \u2013 are produced via co-condensation of laser-ablated praseodymium atoms with nitric oxide in a solid neon matrix. Combined infrared spectroscopy and state-of-the-art quantum chemical calculations confirm that both species are pentavalent praseodymium nitride-oxides with linear structures that contain Pr PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N triple bonds and Pr PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O double bonds. Electronic structure studies show that the neutral NPrO molecule features a 4f0 electron configuration and a Pr(v) oxidation state similar to that of the isoelectronic PrO2+ ion, while its NPrO\u2013 anion possesses a 4f1 electron configuration and a Pr(iv) oxidation state. The neutral NPrO molecule is thus a rare lanthanide nitride-oxide species with a Pr(v) oxidation state, which follows the recent identification of the first Pr(v) oxidation state in the PrO2+ and PrO4 complexes . This finding indicates that lanthanide compounds with oxidation states of higher than +IV are richer in chemistry than previously recognized.The neutral molecule NPrO and its anion NPrO Combined matrix-isolation infrared absorption spectroscopy and sophisticated quantum chemistry studies reveal that both species are linear pentavalent compounds with Pr PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N triple bonds and Pr PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O double bonds, and that the neutral NPrO molecule, which is isoelectronic to the PrO2+ cation, is also a pentavalent praseodymium species with a Pr center in the highest +V oxidation state. The NPrO species thus provides additional evidence that lanthanides can form complexes with oxidation states higher than IV.Herein, we report a combined experimental and theoretical study on the neutral NPrO molecule and its anion NPrO15NO were used without further purification. The as-deposited samples were subjected to annealing and photolysis experiments to initiate diffuse and photo-induced reactions. The infrared absorption spectra of the products were recorded in the mid-infrared region (4000\u2013450 cm\u20131) using a Bruker Vertex 80V spectrometer at a resolution of 0.5 cm\u20131 using a liquid nitrogen cooled HgCdTe (MCT) detector.Praseodymium nitride-oxide species were prepared by reactions of praseodymium atoms and nitric oxide in solid neon, and were studied by infrared absorption spectroscopy as described in detail previously.ab initio single-reference WFT method of coupled-clusters with singles, doubles and perturbative triples (CCSD(T))16Quantum chemical calculations were performed at the density functional theory (DFT) and wavefunction theory (WFT) levels to gain insight into the geometries, electronic structures, bonding and oxidation states of the observed species. The hybrid B3LYP functionalIn addition, multi-configurational complete active space self-consistent-field theory and complete active space with second-order perturbation theory (CASSCF/CASPT2) methods2\u20134d10] for Pr, and [1s2] for O and N. Kohn\u2013Sham molecular orbital (MO) analysis and calculations of Mayer bond order indexes25Electronic structure and bonding analyses were performed at the B3LYP level using the ADF 2013 program.\u2013 species in a solid argon matrix but did not address their oxidation states.2, (NO)2+ and (NO)2\u2013 absorptions,\u20131. This band can be attributed to diatomic PrO absorption, which is observed at 817.0 cm\u20131 in solid argon and at 826 cm\u20131 in the gas phase.\u20131 were also observed upon sample deposition, sharpened and decreased upon sample annealing at 10 and 12 K, and diminished under \u03bb > 800 nm light irradiation. Three additional groups of absorptions were produced when the sample was annealed at high temperatures (10 and 12 K) at the expense of NO absorption. The first group involves two doublet absorptions at 926.2/918.5 and 762.2/755.9 cm\u20131, which slightly decreased under \u03bb > 800 nm irradiation. The second and third groups each involve three absorptions at 1862.6, 886.6 and 751.6 cm\u20131 and 1825.2, 1720.6 and 826.3 cm\u20131, respectively. The latter two groups of absorptions were destroyed upon \u03bb > 800 nm light irradiation, during which absorption at 747.3 cm\u20131 was produced, which can be assigned to the antisymmetric OPrO stretching vibration of the weakly perturbed PrO2 complex, as this absorption shows no shift with 15NO with a band position very close to that of PrO2.15NO and 14NO + 15NO samples. Selected regions of the isotopic spectra are shown in 26The reaction products from the co-deposition of laser-ablated praseodymium atoms with nitric oxide in excess argon were previously studied using Fourier transform infrared absorption spectroscopy, which indicated the formation of NPrO and NPrO\u20131 absorptions are assigned to the NPrO molecule trapped in solid neon in two trapping sites. The upper doublet shifted to 903.9/896.6 cm\u20131 with 15NO. The observed quite large 15N-isotopic shift indicates that this mode is mainly a Pr\u2013N stretching vibration. The low doublet shifted to 757.0/750.7 cm\u20131 with 15NO, and the quite small 15N-isotopic shift implies that this mode is largely due to a Pr\u2013O stretching vibration. Analysis of the spectrum obtained from the experiment using the mixed 14NO + 15NO sample confirms the involvement of only one N atom and one O atom in this molecule. The two stretching vibrational modes of NPrO were observed at 900.8 and 742.0 cm\u20131 in solid argon,\u20131, respectively, thus indicating significant argon matrix effects that are non-negligible. The Pr\u2013N stretching frequency of NPrO is higher than that of diatomic PrN, which was reported at 857.9 cm\u20131 in solid argon.\u20131 in Ne and 817.0 cm\u20131 in Ar). The NPrO absorptions increased markedly upon annealing in both neon and argon matrices, indicating that the ground state praseodymium atoms can insert into the N\u2013O bond of nitric oxide spontaneously with negligible activation energy required.The 926.2/918.5 and 762.2/755.9 cm\u20131 absorptions observed right after sample deposition correspond to the absorptions observed at 718.2 and 612.3 cm\u20131 in solid argon, which were attributed to the NPrO\u2013 anion species.\u20131 absorption shows almost no shift with 15NO, thus indicating that this band is a pure Pr\u2013O stretching mode. In contrast, the 730.9 cm\u20131 band shifted to 709.2 cm\u20131 with 15NO, and the isotopic 14N/15N frequency ratio of 1.0306 implies that it is a Pr\u2013N stretching mode. The argon-to-neon shifts of 12.7 and 11.6 cm\u20131 are only about half of those of the neutral NPrO, thus suggesting that the NPrO\u2013 anion is less affected by the noble gas atoms. The NPrO\u2013 anion is presumably formed via electron capture of the neutral NPrO molecule during the co-condensation process. It is well-known that laser ablation of a metal target can produce not only neutral metal atoms but also metal cations and electrons.\u20131 at the CCSD(T) level of theory , which is consistent with the experimental observation that the NPrO\u2013 anion is photobleached upon \u03bb > 800 nm light irradiation.The rather weak 730.9 and 623.9 cm\u20131 absorptions are assigned to an NPrO(NO) complex. The upper mode shifted to 1829.8 cm\u20131 with 15NO, with the band position and isotopic shift indicating a terminally bound nitrosyl stretching vibration. The 886.6 and 751.6 cm\u20131 bands are assigned to the Pr\u2013N and Pr\u2013O stretching modes, respectively, which are slightly red-shifted from those of the NPrO molecule. The 1825.2, 1720.6 and 826.3 cm\u20131 absorptions are assigned to an NPrO(NO)2 complex, with the two upper absorptions corresponding to N\u2013O stretching vibrations. The spectrum of the mixed 14NO + 15NO sample indicates that two equivalent NO subunits are involved in these two modes. The 826.3 cm\u20131 absorption exhibits less 15N-isotopic shift (9.1 cm\u20131) than the Pr\u2013N stretching mode of NPrO (22.3 cm\u20131), thus implying that the 826.3 cm\u20131 absorption can instead be assigned as an antisymmetric NPrO stretching mode. Calculations also suggested that the symmetric stretching is too weak to be observed.The 1862.6, 886.6 and 751.6 cm\u20131 lower in energy than the singlet state at the B3LYP level, but is 10.0 kcal mol\u20131 higher than the latter at the more accurate CCSD(T) level. Both the quintet and triplet states can thus be ruled out as they are clearly excited states. The calculated Pr\u2013N and Pr\u2013O stretching vibrational frequencies are compared to the experimental values in Theoretical calculations were performed to elucidate the structure, oxidation states and bonding of the observed species. Geometry optimizations were performed at both the B3LYP and CCSD(T) levels of theory on the neutral NPrO molecule with spin multiplicity of 1, 3 and 5, and the results are listed in 22+) and the PrO2+ ion, the neutral NPrO molecule in the singlet ground electronic state also prefers a linear structure in order to optimize the overlap of the Pr 5d and 4f orbitals with those of the N and O atoms. The Pr\u2013N and Pr\u2013O bond lengths are predicted to be 1.677 and 1.765 \u00c5, respectively, at the B3LYP level of theory, and CCSD(T) calculations give slightly higher values (1.697 and 1.775 \u00c5). The Pr\u2013N bond length is about 0.14 \u00c5 (B3LYP) or 0.12 \u00c5 (CCSD(T)) smaller than the sum of the triple-bond covalent radii of Pr and N proposed by Pyykk\u00f6 et al.2+ calculated at the same level of theory.Similar to the isovalent uranyl ion (UO1\u03a3 singlet ground state of NPrO is isoelectronic to PrO2+ and has a ground electronic configuration of [core](1\u03c0)4(1\u03c3)2(2\u03c0)4(2\u03c3)2(4f5d)0. As shown in 0d0) configuration and that Pr\u2013N is triple bonded and Pr\u2013O is double bonded, which is consistent with the Lewis electron-pair model. Accordingly, the linear singlet NPrO neutral molecule can be classified as a pentavalent praseodymium species with an oxidation state of +V as the PrO2+ cation. Natural bond orbital (NBO) analyses scale\" fill=\"currentColor\" stroke=\"none\">N and Pr PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O multiple bonding in NPrO with two sets of localized \u03c32\u03c04 bonds. The Pr\u2013N \u03c3 bond is composed of 60.9% Pr and 36.8% N character, with a Pr 5d\u2009:\u20094f contribution of 21\u2009:\u200974 and an N 2s\u2009:\u20092p contribution of 13\u2009:\u200987. The two degenerate \u03c0 bonds are each composed of 32.5% Pr and 67.5% N character, with a Pr 5d\u2009:\u20094f contribution of 41\u2009:\u200958. The Pr\u2013O \u03c3 bond is composed of 30.9% Pr and 67.3% O character, with a Pr 5d\u2009:\u20094f contribution of 26\u2009:\u200971 and an O 2s\u2009:\u20092p contribution of 13\u2009:\u200987. Each of the two degenerate Pr\u2013O \u03c0 bonds that represent one covalent and one dative bond are composed of 17.1% Pr and 82.8% O character, with a Pr 5d\u2009:\u20094f contribution of 44\u2009:\u200955. These bonding analyses clearly show that the Pr\u2013O bonding interaction is much more strongly polarized than the Pr\u2013N bonding interaction, which is consistent with the electronegativity difference and the lower Pr PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O bond order. The greater covalency of the Pr\u2013N interaction results in a shorter Pr\u2013N bond (1.677 \u00c5 at B3LYP and 1.697 \u00c5 at CCSD(T)) than the Pr\u2013O bond (1.765 \u00c5 at B3LYP and 1.775 \u00c5 at CCSD(T)), albeit the atomic radius of oxygen is slightly smaller than that of nitrogen. As has been discussed before,2+ and NPrO species. In contrast, our preliminary calculations show that the analogous protactinium nitride-oxide NPaO species is a Pa(v) complex with a slightly bent structure because of the significant participation of Pa 5f orbitals in addition to the 6d/7s ones in the chemical bonding.The 2+ and NPrO, in terms of O2\u2013 and N3\u2013, with PrO3+. In this diagram, only the 2p atomic orbitals of the N and O atoms are shown, with the 2s ones omitted for clarity. The bonding interactions in these two isoelectronic species are in general quite similar. The interactions of the three 2p atomic orbitals of O2\u2013 or N3\u2013 with the highest fully occupied 1\u03c0 and 1\u03c3 MOs of PrO3+ lead to the six highest fully occupied MOs of PrO2+ and NPrO level. The calculated red-shifts from the corresponding modes of neutral NPrO are 192.3 and 174.8 cm\u20131 at the CCSD(T) level and 207.2 and 185.2 cm\u20131 at the B3LYP level, which are in reasonable agreement with the experimentally observed shifts of 195.3 and 138.3 cm\u20131 (for the major site absorptions of NPrO in solid neon) when considering that no matrix effects are accounted for. The NPrO\u2013 anion in the 2\u03a6 ground electronic state has an electronic configuration of (core) (1\u03c0)4(1\u03c3)2(2\u03c0)4(2\u03c3)2(1\u03c6)1. The unpaired electron is located on the 1\u03c6 MO that is a non-bonding Pr-based 4f atomic orbital, which is consistent with Mulliken population analysis which shows that the spin density is located on the Pr center. Therefore, the anion can be regarded as being formed by adding an electron to the 1\u03c6 LUMO of the neutral NPrO molecule. The extra electron reduces the natural charge of the Pr center from +1.49 in NPrO to +1.14 in the NPrO\u2013 anion, which significantly increases the ionic radii and reduces the electrostatic interaction in the anion. The Pr center in the 2\u03a6 state of NPrO\u2013 has an (f\u03c61d0) electronic configuration, and thus corresponds to a Pr(iv) oxidation state. Natural bond orbital analyses indicate that despite the Pr(iv) oxidation state in NPrO\u2013, the 2\u03a6 ground state NPrO\u2013 anion is also a pentavalent species with Pr PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N and Pr PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O multiple bonds scale\" fill=\"currentColor\" stroke=\"none\">N and Pr PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O bonds, respectively or 7.0 kcal mol\u20131 (CCSD(T)) higher in energy than the 2\u03a6 ground state. This 2\u0394 state NPrO\u2013 anion has a Pr(f\u03b41d0) configuration and an oxidation state of +IV as well. The most stable quartet state with a linear structure, an (f\u03b41f\u03c61\u03c31) electronic configuration and a Pr(iii) oxidation state is 15.4 (B3LYP) or 43.3 kcal mol\u20131 (CCSD(T)) higher in energy than the ground state.The NPrOtructure . The Pr\u2013 Table S3, which aectively . Another\u2013 species, additional single-point multi-configurational SCF calculations using CASSCF were performed on the optimized structures of the linear singlet neutral NPrO molecule and the doublet NPrO\u2013 anion at the B3LYP level of theory. The CASSCF calculations, with 12 electrons in 12 orbitals including six bonding orbitals and six antibonding orbitals (f0d0) configuration from the CASSCF calculations has a dominated weight of 86.7%, the sum of the natural orbital occupation numbers (NOONs) of the six low-lying orbitals amounts only to 0.29 e\u2013 in total. Moreover, the T1-diagnostic value of 0.056 obtained from the single-determinant CCSD(T) calculation also implies that the wavefunction does not exhibit particularly large multi-reference features (f1d0) configuration has a dominated weight of 88.8% and that the sum of the NOONs of the five low-lying orbitals amounts only to 0.27 e\u2013. These results indicate that the single-reference DFT and CCSD(T) calculations are reasonably reliable, and the assignments of the oxidation states of Pr(v) in neutral NPrO and Pr(iv) in the NPrO\u2013 anion are appropriate due to the single reference features of their wavefunctions and the calculated state energies.To assess the potential single-reference nature of the electronic structures of the NPrO and NPrOs Fig. S4 on the sfeatures . Similars Fig. S5 found th\u2013 molecules, our experiments show that the NPrO molecule further reacts with nitric oxide to form NPrO(NO) and NPrO(NO)2 complexes spontaneously upon annealing. The NPrO(NO) complex is predicted to have a doublet ground state with a Cs symmetry 2 complex is predicted to have a singlet ground state is reduced to Pr(iv) and that the complexes are converted to PrO2 and N2 under near infrared light irradiation.In addition to the NPrO and NPrOy Fig. S6, which ie Fig. S6, which c2 complexes indicates that NPrO is a Lewis acid, which points to the possibility of forming complexes with noble gas atoms. DFT/B3LYP calculations with or without dispersion correction (D3) were thus performed on the complexes formed between NPrO and noble-gas atoms for comparison. The total Ng binding energies are calculated as the energy change for the process: (NPrO)Ngn \u2192 NPrO + nNg. The calculated binding energies are listed in Table S26 complex, which is predicted to have a C6v structure with all the argon atoms equatorially coordinated to the metal center. The total binding energy of the six argon atoms is 12.7 kcal mol\u20131 with dispersion correction. Upon argon atom coordination, the Pr\u2013N and Pr\u2013O stretching modes red-shifted by 11.9 and 7.9 cm\u20131, respectively. This result implies that the NPrO molecule trapped in a solid argon matrix may be regarded as a matrix-isolated NPrO(Ar)6 complex, as in the case of the argon-coordinated CUO molecule.5 complex has a slightly smaller binding energy , which might be partially responsible for the different matrix sites of NPrO observed. However, consistent with the much lower coordination ability or Lewis basicity of neon, the binding between Ne atoms and NPrO is rather weak, which leads to a total binding energy of only 7.2 kcal mol\u20131 for the NPrO(Ne)6 complex, thus suggesting that the coordination effect of neon atoms compared to argon atoms is somewhat more insignificant.Observation of the NPrO(NO) and NPrO(NO) PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N and Pr PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O multiple bonds. Thus, the neutral NPrO molecule is another pentavalent species with a Pr(v) oxidation state, which follows the recently reported PrO2+ complexes with a Pr(v) center.\u2013 anion is also formed via electron capture of the neutral molecule during the co-condensation process. Although the anion exhibits quite large red-shifted Pr\u2013N and Pr\u2013O stretching frequencies relative to those of the neutral molecule, the anion is characterized to be another pentavalent praseodymium species with Pr PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N and Pr PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O multiple bonds. Especially noteworthy is the Pr(iv) oxidation state of this pentavalent NPrO\u2013 complex. Evidence is also presented for the formation of the NPrO(NO) and NPrO(NO)2 complexes, which convert to PrO2 complexes with a Pr(iv) oxidation state under \u03bb > 800 nm light irradiation. Theoretical calculations suggest that NPrO is weakly coordinated by noble gas atoms in solid noble gas matrices. The present study together with our previous work on PrO2+ and PrO4 complexesv) oxidation state are plausible in both oxides and nitrides. Further investigations into lanthanide oxofluorides, nitride-oxides, halides and carbides would be interesting to explore the undeveloped pentavalent lanthanide chemistry.The reactions of praseodymium atoms with nitric oxide are studied using matrix-isolation infrared absorption spectroscopy. The ground state Pr atoms react with NO to spontaneously form an inserted NPrO molecule upon annealing in solid neon, with the molecule characterized to have a linear structure with both PrSupplementary informationClick here for additional data file."} +{"text": "A systematic, efficient route to the first heavier tetrylidyne complexes of niobium starting from the carbonyl niobate (NMe4)[Nb(CO)4(\u03ba2-tmps)] is presented. 4)[Nb(CO)4(\u03ba2-tmps)] (1) (tmps = MeSi(CH2PMe2)3) with a suitable organotetrel(ii)halide. Compound 1 was obtained from (NMe4)[Nb(CO)6] and the triphosphane tmps by photodecarbonylation. Reaction of 1 with the disilene E-Tbb(Br)Si PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019Si(Br)Tbb in the presence of 4-dimethylaminopyridine afforded selectively the red-brown silylidyne complex methyl)phenyl). Similarly, treatment of 1 with E(ArMes)Cl afforded after elimination of (NMe4)Cl and two CO ligands the deep magenta colored germylidyne complex (3-Ge), and the deep violet, light-sensitive stannylidyne complex (3-Sn), respectively. Formation of 3-Sn proceeds via the niobiastannylene [(\u03ba3-tmps)(CO)3Nb\u2013SnArMes] (4-Sn), which was detected by IR and NMR spectroscopy. The niobium tetrylidyne complexes 2-Si, 3-Ge and 3-Sn were fully characterized and their solid-state structures determined by single-crystal X-ray diffraction studies. All complexes feature an almost linear tetrel coordination and the shortest Nb\u2013E bond lengths (d(Nb\u2013Si) = 232.7(2) pm; d(Nb\u2013Ge) = 235.79(4) pm; d(Nb\u2013Sn) = 253.3(1) pm) reported to date. Reaction of 3-Ge with a large excess of H2O afforded upon cleavage of the Nb\u2013Ge triple bond the hydridogermanediol Ge(ArMes)H(OH)2. Photodecarbonylation of [CpNb(CO)4] (Cp = \u03b75-C5H5) in the presence of Ge(ArMes)Cl afforded the red-orange chlorogermylidene complex (5-Ge). The molecular structure of 5-Ge features an upright conformation of the germylidene ligand, a trigonal\u2013planar coordinated Ge atom, and a Nb\u2013Ge double bond length of 251.78(6) pm, which lies in-between the Nb\u2013Ge triple bond length of 3-Ge (235.79(4) pm) and a Nb\u2013Ge single bond length (267.3 pm). Cyclic voltammetric studies of 2-Si, 3-Ge, and 3-Sn reveal several electron-transfer steps. One-electron oxidation and reduction of the germylidyne complex of 3-Ge in THF are electrochemically reversible suggesting that both the radical cation and radical anion of 3-Ge are accessible species in solution.A systematic, efficient approach to first complexes containing a triple bond between niobium and the elements silicon, germanium or tin is reported. The approach involves a metathetical exchange of the niobium-centered nucleophile (NMe Whereas earlier work concentrated exclusively on group 6 metals, recent studies have shown that also group 7, PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019E functionality. Group 5 metal complexes featuring a triple bond to the heavier tetrels (E = Si\u2013Pb) are presently not known, and even compounds with a M PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019E double bond are very scarce and poorly characterized illustrating the challenge to make such compounds. PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019E (E = Si\u2013Sn) triple bonds.Complexes of the general formula were prepared, following the method developed by J. E. Ellis et al.m-terphenyltetrel(ii)halides E(ArMes)Cl = 2,4,6-trimethylphenyl).4)[Nb(CO)6] with Ge(ArMes)Cl in refluxing toluene did not provide any evidence for a conversion of the niobate even after prolonged heating, probably due to the poor nucleophilicity of [Nb(CO)6]\u2013. Therefore, as next we turned our attention to niobates containing ligands with a higher \u03c3-donor/\u03c0-acceptor ratio than CO, such as trialkyl- or triarylphosphanes. Various carbonyl(phosphane) niobates of the general formula [Nb(CO)4L2]\u2013 (L2 = bidentate di- or oligo-arylphosphane ligand) have been accessed from [Nb(CO)6]\u2013 upon photolytic CO substitution.2PMe2)3 (tmps).12We decided to apply the first method, given the availability of anionic niobium carbonyl complexes.4)[Nb(CO)6] was carried out in the presence of one equivalent of tmps in THF at room temperature. A high-power blue light LED (\u03bb = 465 nm) was used instead of a high-pressure mercury UV-lamp. The use of a nearly monochromatic source with an exciting wavelength close to the longest-wavelength absorption maximum of [Nb(CO)6]\u2013 (\u03bbmax = 440 nm in CH2Cl2)11Photolysis of (NMe4(\u03ba2-tmps)]\u2013 proceeding via the pentacarbonyl intermediate [Nb(CO)5(\u03ba1-tmps)]\u2013 (\u03bd(CO) in THF: 1966 (m), 1821 (vs) cm\u20131). After work-up the salt (NMe4)[Nb(CO)4(\u03ba2-tmps)] (1) was isolated in nearly quantitative yield (97%) as an orange, analytically pure, very air-sensitive powder, which decolorizes immediately upon exposure to air. The salt decomposes upon heating at 142 \u00b0C to a dark brown mass, and is well soluble in acetonitrile and tetrahydrofurane (THF), but only moderately soluble in benzene, toluene, and diethyl ether. Attempts to grow suitable single crystals of 1 for an X-ray diffraction study failed, however unambiguous proof for the composition and structure of 1 was provided by elemental analysis, IR spectroscopy and 1H, 13C{1H}, 31P{1H} and 29Si{1H} NMR spectroscopy. The IR spectrum of 1 in THF displays four \u03bd(CO) absorption bands at 1900, 1787, 1764 and 1732 cm\u20131 4 fragment.\u03bd(CO) bands of 1 are shifted to lower frequencies than those of [Nb(CO)4(Ph2PCH2CH2PPh2)]\u2013 (\u03bd(CO) in THF = 1908, 1806, 1782 and 1746 cm\u20131) or related disubstituted arylphosphane-carbonyl niobates.1, which enhances the electron density at the metal center and leads to a stronger Nb(d\u03c0) \u2192 CO(\u03c0*) backbonding and softening of the CO bonds in 1. The NMR spectra of 1 corroborate the presence of an overall Cs symmetric complex, in which one of the arms of the tripodal ligand tmps is pendant and the other two arms are bonded to the niobium center. For example, the 31P{1H} NMR spectrum of 1 displays a sharp singlet for the 31P nucleus of the pendant CH2PMe2 arm, which appears at almost the same position (\u03b4(PA) = \u201355.8 ppm in benzene-d6) as that of the non-coordinated (\u201cfree\u201d) tmps (\u03b4(P) = \u201355.1 ppm in benzene-d6), and a very broad signal for the two symmetry-equivalent Nb-bonded 31P nuclei at considerably lower field (\u03b4(PB) = \u201311.6 ppm in benzene-d6) = 696 Hz) is caused by the quadrupole moment of the 93Nb nucleus and its effect on the relaxation time.29Si{1H} NMR spectrum of 1, which shows a sharp signal for the bridgehead Si atom, that is split to a doublet of triplets = 14.7 Hz, 2J = 8.2 Hz). A positional exchange of the pendant and the Nb-bonded arms of the tmps ligand in 1 was not observed in solution at 298 K.In fact, IR-monitoring of the reaction revealed a slow, but very selective conversion into the tetracarbonyl niobate chloride Ge(ArMes)Cl in toluene at \u201340 \u00b0C followed by warming to room temperature afforded rapidly and selectively the germylidyne complex (3-Ge) , that is moderately soluble in benzene and toluene, and well soluble in THF. No evidence for the formation of the putative metallogermylene intermediate [(\u03ba3-tmps)(CO)3Nb\u2013GeArMes] could be obtained during IR monitoring of the reaction of 1 with Ge(ArMes)Cl in toluene, the reaction starting at \u201335 \u00b0C and proceeding rapidly with CO evolution below 0 \u00b0C.Similarly, treatment of complex ] (3-Ge) . Compounm-terphenyltin(ii)chloride Sn(ArMes)Cl with 1 in toluene afforded after stirring at ambient temperature the brick-red metallostannylene [(\u03ba3-tmps)(CO)3Nb\u2013SnArMes] (4-Sn) with a small amount of the stannylidyne complex (3-Sn) . Prolong4-Sn to afford 3-Sn is a remarkable, new type of reaction in the chemistry of metallostannylenes. In fact previous attempts to transform the metallostannylenes [Cp(CO)3M\u2013SnR] ,2Fe\u2013SnR] 3W\u2013Sn(IDipp)]+ (Idipp = C[N(Dipp)CH]2, Dipp = C6H3\u20132,6-iPr2)4-Sn and decreases thereby the barrier for a CO dissociation. In addition, formation of a strong Nb PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019Sn triple bond resulting from the higher energy and larger radial extension of the d orbitals, which are engaged in the Nb(d\u03c0) \u2192 SnR(\u03c0*) back bonding, may be also a driving force for the reaction.Decarbonylation of 2-Si, 3-Ge and 3-Sn were characterized by elemental analyses, IR spectroscopy and 1H, 13C{1H}, 31P{1H}, 29Si{1H} and 119Sn{1H} NMR spectroscopy. In addition their molecular structures were determined by single-crystal X-ray crystallography (3-bonded) tmps ligand, which spans three facial coordination sites with the P\u2013Nb\u2013P bite angles varying in a small range (85.3\u201387.9\u00b0). A view along the Si\u00b7\u00b7\u00b7Nb vector reveals that the CH2 groups connecting the bridgehead Si atom with the P donors are twisted out creating a local C3 symmetric, right or left-handed conformation, which reduces the bite of the chelating triphosphane ligand and optimizes the bonding with the niobium center pm) is ca. 28 pm shorter than the Nb\u2013Si single bonds of silyl complexes (d(Nb\u2013Si)mean of 28 structurally characterized complexes = 261.3 pm),3-Ge (235.79(4) pm) ca. 31 pm shorter than a Nb\u2013Ge single bond (d(Nb\u2013Ge)mean = 267.3 pm).3-Sn (253.3(1) pm) is ca. 30 pm shorter than a Nb\u2013Sn single bond (d(Nb\u2013Sn)mean 282.9 pm).2-Si, 3-Ge and 3-Sn with those of related molybdenum tetrylidyne complexes scale\" fill=\"currentColor\" stroke=\"none\">Si) in = 222.41(7) pm;1d scale\" fill=\"currentColor\" stroke=\"none\">Ge) in (R = C(SiMe3)3, ArMes, ArTrip) = 227\u2013228 pm;d scale\" fill=\"currentColor\" stroke=\"none\">Sn) in trans- = 248\u2013249 pm) PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019E triple bond lengths compare reasonably well with the difference (7 pm) of the metallic radii of the two elements .et al.2-Si, 3-Ge and 3-Sn, are however, longer than the sum of the theoretically predicted triple bond radii scale\" fill=\"currentColor\" stroke=\"none\">E)calc = Si: 218 pm, Ge: 230 pm, Sn: 248 pm).The tetrylidyne complexes 2-Si: 159.2(2)\u00b0, 3-Ge: 164.0(1)\u00b0, 3-Sn: 160.9(3)\u00b0). Bending occurs in all cases towards the CO ligands. It is presently unclear, whether this phenomenon, which is also observed in a series of group 6 metal dicarbonyl ylidyne complexes, is of steric or electronic origin or both. No clear evidence for steric congestion is at least provided by the molecular structures of 2-Si, 3-Ge and 3-Sn. For example, the closest van der Waals contacts were found in 2-Si between the methyl groups of the tmps ligand and the SiMe3 methyl groups of the Tbb substituent (d(H\u00b7\u00b7\u00b7H) = 244 pm). These contacts are longer than twice the van der Waals radius of hydrogen (rvdW(H) = 110 pm). PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019E\u2013R atom sequence from linearity does not require a lot of energy, indicating that subtle electronic effects may cause such a bending.26In all complexes the tetrylidyne ligand is slightly bent at the tetrel center as evidenced by the bonding angle Nb\u2013E\u2013C1 bands of almost equal intensity, which are typical for cis-dicarbonyl complexes and can be assigned to the in-phase (A\u2032 symmetric) and out-of-phase (A\u2032\u2032 symmetric) CO stretching modes assuming local Cs symmetry of the M(CO)2 fragment (\u03bd(CO) bands of 3-Sn appear at lower frequencies (1851 and 1791 cm\u20131 in toluene) than those of 3-Ge (1868 and 1805 cm\u20131 in toluene), which suggests that the stannylidyne ligand SnArMes has a higher \u03c3-donor/\u03c0-acceptor ratio than the germylidyne ligand GeArMes. Notably, the \u03bd(CO) bands of 2-Si appear also at lower wavenumbers (1855 and 1790 cm\u20131 in toluene) than those of 3-Ge. This shift can be rationalized with the stronger +I effect of the Tbb substituent, leading to a higher \u03c3-donor/\u03c0-acceptor ratio of the silylidyne ligand SiTbb than that of the germylidyne ligand GeArMes. The low-frequency position of the \u03bd(CO) bands of 2-Si, 3-Ge and 3-Sn suggests the presence of an electron-rich Nb center that is engaged in strong Nb(d\u03c0) \u2192 CO(\u03c0*) backbonding. Additional evidence for a strong Nb(d\u03c0) \u2192 CO(\u03c0*) backbonding is provided by the 13C{1H} NMR spectra, which all display a broad CO signal at even lower field (\u03b4CO = 238.7 ppm (2-Si), 239.2 ppm (3-Ge), 238.9 ppm (3-Sn)) than that of 1 (\u03b4CO = 226.5 ppm).Cs symmetry of the tetrylidyne complexes in solution and a rapid rotation of the tetrel-bonded aryl group about the E\u2013Caryl bond. The signals of all nuclei directly attached to the quadrupolar 93Nb nucleus are significantly broadened due to fast relaxation (vide supra). For example, the 29Si{1H} NMR spectrum of 2-Si displays at 298 K a very broad signal (\u0394\u03bd1/2 = 130 Hz) for the Nb PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019Si nucleus at \u03b4 = 267.8 ppm, for which the 2J coupling could not be resolved. In comparison, the remote positioned bridgehead Si atom of the tmps ligand and the SiMe3 groups of the Tbb substituent give rise to sharp signals at \u03b4 = \u20130.7 ppm and +1.5 ppm, respectively, with the first of these signals being split into a quartet due to coupling to the three 31P nuclei = 9.7 Hz) and \u201310.7 ppm (\u0394\u03bd1/2 \u2248 187 Hz at 283 K), respectively, instead of two 31P NMR signals expected for an AX2 spin system coupling of 20.9 Hz as illustrated by the 31P NMR spectrum of 3-Ge at 193 K at \u03b4 = 829.7 ppm . Similan system . Broadneat 193 K . Taking . S36 ESI).3-Ge and the related molybdenum germylidyne complexes (R = C(SiMe3)3, ArMes, ArTrip). Thus treatment of with H2O or MeOH (one equiv.) in diethyl ether at 0 \u00b0C followed by warming to ambient temperature afforded within one hour selectively the brown hydroxy/methoxygermylidene complexes , which were fully characterized.3-Ge with H2O (one equiv.) was observed in THF even at 60 \u00b0C. The inertness of 3-Ge can be rationalized with the stronger metal-germylidyne Nb(d\u03c0) \u2192 GeR(\u03c0*) back bonding, which reduces the electrophilicity of the Ge center in 3-Ge, and increases in combination with the steric protection of the metal center by the tridentate tmps ligand the activation barrier for the H2O addition at the Nb PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019Ge bond. In fact, a large excess of water (925 equiv.) and prolonged heating (3 h) was necessary to effectuate a full conversion of 3-Ge accompanied by a color change of the reaction solution from magenta to orange. IR monitoring of the reaction did not provide any evidence for the formation of the anticipated H2O addition products. Instead, a continuous decrease in intensity of the two \u03bd(CO) bands of 3-Ge was observed suggesting the formation of mainly CO-free products. Benzene extraction of the orange-brown solid obtained after solvent evaporation afforded a benzene soluble, pale-orange part containing mainly the germanediol Ge(ArMes)H(OH)2, as well as a benzene-insoluble brownish part. The unprecedented hydridogermanediol1H NMR spectroscopy. Its IR spectrum displays two \u03bd(OH) bands at 3600 and 3398 cm\u20131 and a characteristic \u03bd(Ge\u2013H) band at 2104 cm\u20131, the latter one appearing at a close position to that of GeBr2HMes (\u03bd(Ge\u2013H) = 2105 cm\u20131).1H NMR spectrum a distinctive doublet signal is observed for the Ge(OH)2 protons at \u03b4 = 0.91 ppm and a triplet signal for the Ge\u2013H functionality at \u03b4 = 5.61 ppm = 3.5 Hz) in the integral ratio of 2\u2009:\u20091. Notably, the Ge\u2013OH protons of the germanetriol Ge(ArTrip)(OH)3 have a similar chemical shift (\u03b4 = 0.77 ppm in CDCl3).29First studies reveal a marked difference in the reactivity of the niobium germylidyne complex 4]\u03bb = 465 nm) in the presence of one equivalent of Ge(ArMes)Cl. IR monitoring of the reaction revealed a quite selective decarbonylation leading to the chlorogermylidene complex 5-Ge, which after work-up was isolated as red-orange, air-sensitive crystals in 25% yield (5-Ge and to form the putative germylidyne complex cation + were not successful so far. For example, no reaction of 5-Ge with Na[B(ArF)4] 2) was observed in C6H5F at room temperature.Attempts were also undertaken to access cationic tetrylidyne complexes. For this purpose, (d(Ge\u2013Cl) = 232.2(1) pm),3)3Ni PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019Ge(ArMes)Cl] (d(Ge\u2013Cl) = 230.03(8) pm)3)3Pd PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019Ge(ArMes)Cl] (d(Ge\u2013Cl) = 227.3(1) pm),5-Ge provides a rationale for its inertness towards mild chloride abstraction reagents.The germylidene ligand adopts an upright conformation, with the Are (251.78 pm) of 5e (251.78 pm) of 55-Ge are fully consistent with its solid-state molecular structure. Thus, the IR spectrum of 5-Ge in THF displays three intense \u03bd(CO) absorption bands at 1980, 1910 and 1899 cm\u20131, as expected for a Nb(CO)3 fragment with local Cs symmetry, which are assigned to the A\u2032 , A\u2032 and A\u2032\u2032 symmetric (two COlat modes out-of-phase) CO stretching modes, respectively. The \u03bd(CO) absorption bands of 5-Ge are high-frequency shifted compared to those of [CpNb(CO)3THF] (\u03bd(CO) in THF = 1961, 1840 cm\u20131)3PEt3] (\u03bd(CO) in THF = 1953, 1850 cm\u20131),3N2] (\u03bd(CO) in n-heptane = 1991, 1905 cm\u20131)MesCl and the N2 ligand. The 1H and 13C{1H} NMR spectra also confirm the Cs symmetry of 5-Ge in solution. Rotation of the m-terphenyl substituent about the Ge\u2013Caryl bond occurs fast on the NMR time-scale at ambient temperature leading to an exchange of the two diastereotopic ortho and meta positions of the enantiotopic mesityl substituents. Therefore, only one singlet signal is observed in the 1H NMR spectrum of 5-Ge for the C2,6-bonded methyl groups and C3,5-bonded protons of the mesityl substituents, respectively.The solution IR and NMR spectra of 2-Si, 3-Ge and 3-Sn were carried out using cyclic voltammetry to elucidate the redox properties of these compounds. All complexes display a rich electrochemistry involving several electron-transfer steps of \u20132.612 mV and \u2013405 mV vs. the dmfc1+/0 redox couple (dmfc = decamethylferrocene), respectively ). Evidence that the redox process at E1/2 = \u2013405 mV involves a one electron oxidation of 3-Ge was provided by chemical means. Thus, no reaction of 3-Ge with the one-electron reducing agent cobaltocene (E1/2 of CoCp2 in DME = \u2013740 mV) was observed in fluorobenzene even at 70 \u00b0C, whereas an instantaneous oxidation of 3-Ge occurred upon treatment with one equivalent of [Fe(\u03b75-C5Me5)2][B(ArF)4] in fluorobenzene solution at \u201330 \u00b0C. Unfortunately, attempts to isolate the putative germylidyne complex radical cation [(\u03ba3-tmps)(CO)2Nb(GeArMes)]+ failed so far.36 Notably, the redox potential for the one-electron oxidation of 3-Ge is slightly lower than that of the molybdenum tetrylidyne complexes trans- verifying the presence of an electron-rich Nb center in 3-Ge.In comparison, the corresponding redox steps of 4)[Nb(CO)4(\u03ba2-tmps)] allowed to explore its reactivity towards a series of organotetrel(ii) halides, which lead to the isolation of the first niobium complexes featuring triple bonds with the elements Si, Ge and Sn. Photochemical CO substitution in [CpNb(CO)4] (Cp = \u03b75-C5H5) by Ge(ArMes)Cl afforded also the novel chlorogermylidene complex . The structural, spectroscopic and electrochemical data of the tetrylidyne complexes (2-Si), (3-Ge) and (3-Sn) suggest the presence of an electron-rich metal center that is engaged into strong metal (d\u03c0) \u2192 ER(\u03c0*) and metal (d\u03c0) \u2192 CO(\u03c0*) back bonding. Remarkably, one-electron oxidation and reduction of the germylidyne complex 3-Ge are electrochemically reversible.The synthesis of the tailor-made carbonyl-niobate (NMeSupplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "Establishing the structure\u2013property relationship for multi-stimuli responsive mechanochromic materials based on charge transfer luminogens. i.e., cross packing), and subsequently, does not give rise to mechanochromism. The Hirshfeld surface analysis from a single crystal infers that non-covalent interactions are extremely important to yield mechanochromism under external force. Correlating all solid-state behavior with the molecular structure, we conclude that the synergistic effect between the twisting and conformational flexibility of donor moieties along with numerous non-covalent interactions gives rise to multi-stimuli responsive behaviors. Finally, the newly designed molecules are found to be highly emissive in solution and potentially applicable in fluorescence thermometer construction, lighting up cells, acid\u2013base sensors and rewritable devices.Prediction of multi-stimuli responsive behavior in newly developed luminogens is an appealing yet challenging puzzle, since no concrete design strategy has been developed so far. In this article, we demonstrate a potent strategy to gain a deep understanding of the structure\u2013property relationship to design multi-stimuli responsive mechanochromic materials. To achieve our goal, a variety of new isoindolinone core based charge transfer luminogens exhibiting aggregation-induced emission (AIE) have been prepared through C\u2013H bond activation using a cost-effective ruthenium (Ru) metal catalyzed one-pot synthetic strategy. We have shown that slight tuning of the donor moiety is found to be highly effective in controlling molecular packing and metastable energy states in solid states, and thus, optical properties and multi-stimuli responsive behaviors. The flexibility and twisting of donor moieties afford a loosely bound \u2018herringbone\u2019 packing, enabling reversible transformation under multiple mechanical stimuli. The cyclized derivative of the donor exhibits a completely different packing mode ( However, the PVS attached isoindolinone framework , which exhibits flexibility, and twisting nature (CNC angle 123.9\u00b0).i.e., carbazole is a well-known moiety having interesting material properties, such as charge transport, luminescence and thermal stability.According to the focus of this work, we have incorporated three common frameworks in each molecule: (1) D\u2013A framework, (2) twisted conformation of the donor moiety and (3) multiple non-covalent interaction sites. In addition, attention has been paid to the tuning of the donor moiety to control the flexibility and molecular packing. After a careful search, we have selected isoindolinone as the core moiety for this work. Isoindolinone, a typical planar, electron deficient molecule, consists of a fused phenyl and pyrrolidinone ring, and hence, it may be well suited for the \u03c0\u00b7\u00b7\u00b7\u03c0 stacking. A slight modification of the five-membered heterocyclic pyrrolidinone ring of isoindolinone has been performed by introducing a phenyl vinyl sulfone (PVS) group }2 (5.0 mol%), silver salt (AgSbF6 (20 mol%)), Cu(OAc)2, and H2O (2.0 equiv.) in acetic acid (AcOH) solution at 120 \u00b0C for 36 h (for details see ESIin situ synthetic route results in the cyclization product (Z)-3-((phenylsulfonyl)methylene)isoindolin-1-one (PMI) (3a) selectively as a Z-stereoisomer with high product yield (72%). In the same manner, substituted benzonitrile (2b\u2013d) efficiently undergoes an in situ cyclization reaction with PVS (4) resulting in (Z)-5-(diphenylamino)-3-((phenylsulfonyl)methylene)isoindolin-1-one (DPAPMI), (Z)-5-(9H-carbazol-9yl)-3-((phenylsulfonyl)methylene)isoindolin-1-one (CPMI), and (Z)-5-(dimethylamino)-3-((phenylsulfonyl)methylene)isoindolin-1-one (DMAPMI) (3b\u2013d) with 63%, 66% and 53% product yield, respectively catalyzed, step economical one-pot synthetic procedure using C\u2013H bond activation as a key step. To the best of our knowledge, this is the first ever report on synthesizing mechanoactive molecules based on Ru metal catalyzed C\u2013H bond activation. A brief representation of the synthetic scheme and mechanism is outlined in Scheme S2. In brieScheme S2. The funE) of donor substituted molecules narrows down compared to the parent PMI molecule. The calculated HOMO energies of DPAPMI, CPMI and DMAPMI are \u20135.73, \u20135.49 and \u20135.72 eV, respectively.To assess the viability of the molecular design and the electronic effect of donor substituents on the parent PMI molecule, we have conducted density functional theory (DFT) calculations at the B3LYP/6-31G level (see ESIf = 0.208) to assign the LE and CT emission peaks and at 505 nm (CT state), respectively in bulk THF. Upon gradual addition of water (poor solvent) into the THF, the emission becomes gradually weaker for both PMI and DMAPMI with a concomitant redshift of \u223c45 nm only for DMAPMI . The formation of nano-aggregates is confirmed by DLS, FE-SEM and AFM studies at higher water content like normal rigid fluorophores. Surprisingly, despite the presence of DMA substitution, DMAPMI shows ACQ nature in the aggregated state. This observation suggests that the smaller size of the DMA group is not sufficient enough to disturb the \u03c0\u00b7\u00b7\u00b7\u03c0 stacking interactions between the PMI moieties in the aggregated state. For DPAPMI and CPMI, probably the twisting conformations of the molecules do not allow them to be involved in effective stacking interactions in the aggregates. This is also evident from the crystal-induced enhanced (CIE) emission observed in the condensed state for both the compounds. Generally, the intramolecular rotations decrease the emission efficiency from the CT state in bulk solution medium, whereas in the CT process in the aggregate, the intramolecular rotation is restricted, thereby, causing an increase in the efficiency of CT emission. As a result, the emission from the CT state gets a boost by the aggregation induced emission (AIE) process for both DPAPMI and CPMI molecules.Z-conformation Z-conformer creates a one-dimensional planar sheet aided by multiple hydrogen bond scale\" fill=\"currentColor\" stroke=\"none\">O\u00b7\u00b7\u00b7H\u2013C) interactions in the symmetric repetitive fashion at 20 \u00b0C for all luminogens , the acceptor PMI part sits just above the DPA donor unit of the lower DPAPMI molecule \u03b82), while the other aryl group highly twists to \u223c77.78\u00b0 (\u03b83) with respect to the PMI core to fit into the crystalline lattice PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O\u00b7\u00b7\u00b7H\u2013C (2.56 \u00c5 and 2.45 \u00c5), C\u2013H\u00b7\u00b7\u00b7\u03c0 (2.89 \u00c5), C\u2013H\u00b7\u00b7\u00b7O PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019S , C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O\u00b7\u00b7\u00b7\u03c0 (3.15 \u00c5), etc. , etc. . Moreove\u00c5), etc. taking a\u00c5), etc. . Emissio\u00c5), etc. , with tri.e., DMAPMI also displays a \u2018herringbone\u2019 packing like DPAPMI (\u03bc) value of \u223c9.41 D , and the partially double bond character of the C\u2013N bond (dC\u2013N = 1.366 \u00c5) between the donor (DMA) and the acceptor (PMI) moiety column in a symmetrical fashion scale\" fill=\"currentColor\" stroke=\"none\">S (2.631 \u00c5), C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O\u00b7\u00b7\u00b7H\u2013C (2.464 \u00c5), S PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O\u00b7\u00b7\u00b7\u03c0 (3.211 \u00c5 and 3.072 \u00c5), C\u2013H\u00b7\u00b7\u00b7H\u2013C (2.266 \u00c5), C\u2013H\u00b7\u00b7\u00b7\u03c0 (2.602 \u00c5), etc. , etc. and S4\u2020.\u20131) of stabilization. Interestingly, the C\u2013H\u00b7\u00b7\u00b7\u03c0 (% C\u00b7\u00b7\u00b7H) interaction contributed substantially in DPAPMI and DMAPMI luminogens, while the \u03c0\u00b7\u00b7\u00b7\u03c0 (% C\u00b7\u00b7\u00b7C) interaction has a minor contribution for all donor substituted luminogens to mechanochromism (for details see Note S2 in the ESIminogens and 7. Cgens (\u20131 ) and its\u03c1 values obtained for DPAPMI and DMAPMI are 7.2 and 5.86 respectively, inferring the herringbone packing for both these molecules. However, CPMI exhibits the lowest \u03c1 value of \u223c1.5 owing to its highest % of \u03c0\u00b7\u00b7\u00b7\u03c0 (% C\u00b7\u00b7\u00b7C in 3) despite the presence of multiple non-covalent interactions and a twisted carbazole ring. This is probably because CPMI contains four molecules per unit cell than CPMI but nearly the same void space as DPAPMI, as it contains flexible acyclic donor (DMA) substitution, like the DPAPMI molecule.The Fig. S13, while aSince some of our designed molecules have flexibility and void space, their solid state emission properties should depend on the alteration of molecular arrangements in response to external mechanical treatment (mechanochromism), temperature (thermochromism) and exposure to solvent (vapochromism). The pristine powder of the parent PMI molecule exhibits non-emissive behavior Fig. S14. Even af\u03c6pristine = 65 \u00b1 10%) having an intense peak at \u223c545 nm (CT) and a peeping peak at \u223c425 nm (LE) , although the peeping peak (LE state) remains nearly unaltered and \u223c77.78\u00b0 (\u03b83)) compared to the PMI core 3). Hence, grinding results in the release of twisting stress and rupturing of non-covalent interactions, which probably leads to more planarized individual DPAPMI molecules. As a result, the CT state is getting more stabilized due to the increased orbital overlap between the donor and the acceptor; hence, a redshift is observed under high grinding conditions compared to the pristine powder. Taking together the molecular conformation and emission color change upon stepwise grinding of DPAPMI, it is clearly understandable that two metastable states are generated due to the presence of two flexible aryl rings with different twisting angles in DPAPMI, the good solvent (DCM) molecule can access inside that accessible void space resulting in the rearrangement of the crystalline state of the luminogen molecules. The PXRD measurements reveal the transformation from the metastable semi-crystalline state to the crystalline state upon DCM treatment Fig. S21. However Fig. S21. Moreove Fig. S21. The sha Fig. S21. Most in Fig. S21. Once itIn modern technological applications, mechanochromic luminogens are mostly used as thin films, where they often stay as thin layers or in segregated states.3), which rarely allows the molecule to take different metastable energy states under mechanical stress and external stimuli. Thus, we conclude that it is not possible to disturb the architecture of highly stable \u2018cross mode\u2019 molecular arrangements of CPMI with the aid of any external stimulus and stress, and hence, CPMI is considered as a mechano-inactive molecule. It is also clear from PXRD data that the crystalline feature of CPMI before and after the grinding almost remains intact plays a significant role in designing a mechanochromic material, since the twisting angle of the donor is the only structural difference between them 3) owing to its cyclized donor unit and at the same time, it is also a mechano-inactive molecule. Thus, the mechano-inactivity of CPMI compared to its acyclic analogue DPAPMI suggests that a flexible donor unit is desirable to construct a mechanochromic material. Thirdly, Hirshfeld surface analysis of mechanochromic DPAPMI suggests that both C\u2013H\u00b7\u00b7\u00b7\u03c0 and \u03c0\u00b7\u00b7\u00b7\u03c0 interactions are important, however, it seems that the C\u2013H\u00b7\u00b7\u00b7\u03c0 interaction is dominant over the \u03c0\u00b7\u00b7\u00b7\u03c0 interaction to yield mechanochromism. To provide further insight, we have extensively mapped the Hirshfeld surface over the shape index and curvedness to get specifically the \u03c0\u00b7\u00b7\u00b7\u03c0 interaction region. Interestingly we have noticed that the mechanoactive DPAPMI molecule contains the \u03c0\u00b7\u00b7\u00b7\u03c0 interaction region in a small part residing at the central aryl ring with a minimum percentage of 4.1%, while other luminogens exhibit the \u03c0\u00b7\u00b7\u00b7\u03c0 interaction in a much wider region with higher percentage play major roles in mechanochromism. The importance of the twisting of the donor moiety in mechanochromism can be understood by looking at the molecular structure, packing style and Hirshfeld surface analysis of DPAPMI and DMAPMI. Hirshfeld surface analysis reveals nearly the same % C\u2013H\u00b7\u00b7\u00b7\u03c0 and % \u03c0\u00b7\u00b7\u00b7\u03c0 interactions for DPAPMI and DMAPMI . Moreovef = 0.003) upon excitation at 350 nm to the PMI core (A). Here, It is pertinent to mention that unlike other conventional ICT/TICT molecules, where the CT state is generated from the LE state, here the CT state can be generated by direct excitation at \u2265405 nm , the molecule probably reaches a high energy state, in which the diphenyl rings are not properly oriented to have an efficient charge transfer process. Now, the excited molecule can come back to the ground state by a radiative transition at 400\u2013430 nm from that higher energy state. Alternatively, it can also reach the other stabilized state having a CT character. When the molecule emits from this stabilized CT state, it shows a red-shifted emission (CT) compared to the LE state. The formation of the CT character has also been justified by a redshift and dramatic decrease in the emission intensity of the CT peak in highly polar solvents. When the solvent polarity is further increased (\u0394f > 0.22), the emission profile of DPAPMI consists of only a redshifted LE peak , the lower energy peak shows a gradual red shift with increment of solvent polarity (\u0394f > 0.014). In highly polar solvents (\u0394f > 0.31), the lower energy peak exhibits a usual redshift and higher energy emission appears as a shoulder. The high energy peak in n-heptane may be attributed to the pyramidal conformation of the \u2013N(CH3)2 group, which consists of a less charge character, as the \u2013N(CH3)2 group is out of resonance with the PMI moiety. This claim is further supported by the absorption spectrum, where the absorption for the CT peak (>400 nm) is very much less. The lower energy peak is attributed to the CT emission and this CT nature is further verified by the emission profile collected upon selective excitation of the charge transfer band, i.e., at 405 nm 2 and PMI stay in a perpendicular geometry, resulting in maximum charge transfer between donor and acceptor moieties.When two phenyl moieties are replaced by methyl groups, then the solvatochromic behavior dramatically changes. The methyl substituted derivative DMAPMI exhibits a low intensity peak at \u223c430 nm and 470 nm in non-polar solvents, like Fig. S25. Althoug Fig. S28. The exii.e., CPMI also exhibits unique solvatochromic behavior. CPMI shows an emission maximum at \u223c430 nm upon photo-excitation at 350 nm in non-polar solvent n-heptane , dual emission peaks appear in the emission profile, where the CT peak dominates the LE peak suggesting that LE and CT states are proximately closer in energy in this polarity region sensors and security ink. Moreover, highly emissive CPMI may be useful as a possible candidate for organic lasing materials and OLED fabrication. Here, we have demonstrated the applications of these newly developed luminogens in fluorescence thermometers, lighting up cells, rewritable media and acid\u2013base induced fluorescence switching media.Charge transfer (CT) luminogens having fluorescence switching ability between LE and ICT states by varying temperature are ideal for the construction of fluorescence thermometers, which are often used in industrial, atmospheric and deep-sea research, where conventional thermometers can't be used.Luminogens with aggregation-induced emission (AIE) have been recognized as potential candidates in recent years to light up a targeted part of the cell.To demonstrate rewritable media application, we have chosen DPAPMI, as this molecule only shows mechanoactivity. For this purpose, \u2018IISERP\u2019 has been written with a metal spatula on a glass substrate and the grinding induced emission color was monitored under UV irradiation . The wri3 vapor .Finally, dynamic fluorescence on\u2013off switching of DPAPMI and CPMI has been monitored under TFA and NH Fig. S30. Owing ti.e. CPMI) leads to a completely different packing mode (cross mode), which subsequently gives rise to the mechano-inactive properties of the luminogen. Surprisingly, although DMAPMI (having a dimethylamine group as a donor) exhibits a comparable packing style and non-covalent interactions to DPAPMI, it does not show mechanoactivity like its close analogue. Besides, Hirshfeld surface analysis specifically infers that non-covalent (C\u2013H\u00b7\u00b7\u00b7\u03c0 and \u03c0\u00b7\u00b7\u00b7\u03c0) interactions are also responsible for the observed mechanochromic properties. Considering all these aspects, we conclude that the loose molecular packing, conformational twisting and flexibility of the donor along with numerous non-covalent interactions are crucial for designing efficient CT mechanochromic luminogens. Moreover, these results establish the unique role of the flexible propeller-shaped donor unit in the self-reversible mechanochromism of CT luminogens. Although, we have chosen CT luminogens to establish the structure\u2013property relationship for designing multi-stimuli responsive materials, we believe that this strategy may also be beneficial to use as a general strategy to obtain mechanochromism. Finally, the designed molecules are found to be potentially applicable for fluorescence thermometer construction, lighting up cells (HEK 293), rewritable devices, acid\u2013base induced fluorescence switching, etc.This article provides a new avenue regarding the structure\u2013property relationship in order to design mechanochromic materials based on CT luminogens. To achieve our goal, we have developed a series of new isoindolinone based D\u2013A dyes using a one pot synthetic strategy through a Ru metal catalyzed C\u2013H bond activation approach. Slight tuning of the donor moiety is found to be very effective in controlling the molecular packing, and thus, the solid-state optical properties and mechanochromism as well. The crystal-induced emission enhancement (CIEE) effect is believed to be responsible for the observed high quantum yields of two of our synthesized luminogens, namely, DPAPMI and CPMI in the solid state. In DPAPMI (consisting of a diphenylamine group as a donor), the herringbone packing along with multiple non-covalent interactions and the flexible donor unit affords a loose herringbone molecular packing, enabling it to undergo reversible transformation under multiple stimuli. Cyclization of the donor unit (There are no conflicts to declare.Supplementary informationClick here for additional data file.Supplementary movieClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "Rare and amphoteric intermediates, in situ generation: controlled reactivity in diverse cascade reactions, over 100 examples. C-substituted isocyanates, nitrogen-substituted isocyanates (N-isocyanates) are rare. Their high reactivity and amphoteric/ambident nature has prevented the scientific community from exploiting their synthetic potential. Recently, we have developed an in situ formation approach using a reversible equilibrium, which allows controlled generation and reactivity of N-isocyanates and prevents the dimerization that is typically observed with these intermediates. This blocked (masked) N-isocyanate approach enables the use of various N-isocyanate precursors to assemble heterocycles possessing the N\u2013N\u2013C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O motif, which is often found in agrochemicals and pharmaceuticals. Cascade reactions for the rapid assembly of several valuable 5- and 6-membered heterocycles are reported, including amino-hydantoins, acyl-pyrazoles, acyl-phthalazinones and azauracils. Over 100 different compounds were synthesized using amino-, imino- and amido-substituted N-isocyanates, demonstrating their potential as powerful intermediates in heterocyclic synthesis. Their reactivity also enables access to unprecedented bicyclic derivatives and to substitution patterns of azauracils that are difficult to access using known methods, illustrating that controlled reactivity of N-isocyanates provides new disconnections, and a new tool to assemble complex N\u2013N\u2013C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O containing motifs.In contrast to normal Classical rearrangement reactions such as the Curtius, Schmidt, Lossen and Hoffman rearrangements are commonly used to form isocyanates in situ. In contrast, blocked (masked) isocyanates are simple precursors releasing isocyanates through a chemical equilibrium. Such blocked isocyanates\u2014typically generated from an isocyanate and a blocking group e.g. thermolysis, base, acid, and metal catalyzed formation).2The high reactivity of isocyanates is essential to many industrial processes but their promiscuous nature can be problematic. Therefore, for a variety of applications the use of C-substituted isocyanates.N-isocyanates) are a class of heterocumulene possessing comparable synthetic potential to C-substituted isocyanates. However, despite the early discovery of N-isocyanates,N-isocyanates results in difficult syntheses and a propensity for these intermediates to homodimerize or oligomerize, even at temperatures as low as \u201340 \u00b0C.4Isocyanates attached to heteroatoms are also known, but are less developed and used than normal N-isocyanates, and only explored their reactivity.N-isocyanates under UV photolysis conditions. Despite this pioneering work, the reaction conditions required to form these amphoteric intermediates, and their propensity to dimerize, severely limited their synthetic applications. To date, only a few reactions are reported using N-isocyanates or their blocked derivatives. After an extensive literature search, we only found 57 publications either forming, studying, using or suggesting the formation of N-isocyanates and N-isothiocyanates.N-isocyanate intermediates. In contrast, there are over 100\u2009000 publications and patents on C-isocyanates, and over 7000 on blocked C-isocyanates. The difference between the synthetic uses of C-isocyanates vs. N-isocyanates is inherently proportional, and highlights the need for a convenient procedure to form N-isocyanates and control their reactivity. Recently we have developed several blocked N-isocyanate precursors as part of our efforts on intra- and intermolecular alkene aminocarbonylation reactions (eqn (1)). These reactions involve N-isocyanates as key intermediates in a reaction sequence allowing the transformation of alkenes into valuable \u03b2-aminocarbonyl motifs. This work required the development of practical reagents: the use of hydrazide and hydrazone derivatives as N-isocyanate precursors emerged as a practical and general approach for these reactions. Most importantly, it allowed the desired concerted [3 + 2] alkene cycloaddition to occur in high yield, with little N-isocyanate dimerization or decomposition .C-isocyanate literature: (1) they appear to follow similar deblocking temperature trends; (2) base catalysis is also possible; (3) observations support a reversible equilibrium favoring the hydrazide and hydrazone starting materials.Much of the early work studied the formation of N-isocyanates, for example with alcohols, amines, and thiols as nucleophiles. This encouraged us to investigate the generation and reactivity of N-isocyanate precursors with simple nucleophiles. Gratifyingly, the substitution reactions proceeded efficiently under stoichiometric conditions using both hydrazonesN-isocyanates scale\" fill=\"currentColor\" stroke=\"none\">O motif in complex bioactive molecules, including several marketed agrochemicals and pharmaceuticals scale\" fill=\"currentColor\" stroke=\"none\">O containing substructures: fully or partially incorporated within a heterocycle, or in acyclic molecules.In parallel to this aminocarbonylation work, we noticed the paucity of simpler reactions of cyanates . A compaeuticals . There a PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O motif in complex molecular scaffolds is highly desirable. However, it is a considerable challenge due to the diversity of motifs present and due to intrinsic chemoselectivity issues associated with hydrazine derivatives: the presence of two nitrogen atoms that can react and lead to different products.N-isocyanate chemistry and the diversity of N\u2013N\u2013C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O motifs present, it was hypothesized that N-isocyanates could provide the missing link for a unified approach to N\u2013N\u2013C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O incorporation in heterocyclic chemistry; provided that their reactivity could be controlled enough to allow for new cascade reactions. This article constitutes a detailed account of our work toward this goal.Therefore, the development of strategies for the incorporation of the N\u2013N\u2013CN-isocyanates for the synthesis of heterocyclic molecules. In addition to previously communicated work toward saturated 5- and 6-membered azacycles and nitrogen-substituted hydantoins, PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O motif in several different orientations. N-Isocyanates were used to assemble 5- and 6-membered aromatic heterocycles including acyl-pyrazoles, acyl-phthalazinones and azauracils. This novel synthetic approach gives rise to substitution patterns that have otherwise been difficult or impossible to access, and allows the formation of new bicyclic heterocycles. With over 100 new compounds spanning 6 heterocyclic classes assembled using cascade reactions of amphoteric N-isocyanate intermediates, this article highlights that highly controlled reactivity is possible through the use of blocked (masked) N-isocyanate precursors.Herein, we discuss the first cascade reactions developed using amino-, imino- and amido-substituted N-isocyanate precursors could lead to efficient cascade reactions, we first targeted a reaction sequence in which generation of the N-isocyanate and reaction at its electrophilic carbon would occur first, followed by cyclization. It was expected that the substitution of nitrogen nucleophiles (e.g. amines) on N-isocyanates would be essentially irreversible. Building on our expertise in metal-free hydroamination reactions of hydrazine derivatives,N-isocyanate addition/Cope-type hydroamination cascade for the formation of saturated nitrogen heterocycles (eqn (2)), illustrated below using blocked N-isocyanate 1a.7To acquire proof of concept results to validate that the controlled reactivity of N-isocyanate precursors allowed the formation of several 5- and 6-membered nitrogen heterocycles incorporating one nitrogen atom (\u03b2N) of the amino-isocyanate in the desired heterocycle (in situ generated N-isocyanate to form the corresponding semi-carbazide (A), which then undergoes a Cope-type hydroamination to form the nitrogen heterocycle. Since isocyanate generation/addition occurred rapidly (ca. <10 minutes at 80 \u00b0C), the hydroamination reaction was rate limiting and the build-up of the unsaturated semi-carbazide A was observed when monitoring these reactions. However, upon heating at temperatures allowing hydroamination to occur, this cascade allowed the synthesis of semi-carbazide-based pyrrolidines , piperidines and piperazine (2c) using pyrrolidine as the nucleophilic amine. As expected, substitution was well tolerated on the alkenyl chain, and incorporation of a Thorpe\u2013Ingold bias was beneficial to achieve cyclization at a lower temperature (2d) or to reduce the time required for reaction completion (2e). Unfortunately, the incorporation of a small chiral centre on the alkenyl chain didn't result in any diastereoselecitivty . The cascade reaction also allowed cyclization via the more challenging hydroamination of an internal alkene (2h). A protected alcohol on the alkene chain was also tolerated (2g) and could allow further functionalization of the desired product. In addition to providing a cascade for the rapid assembly of molecular complexity, this data showed that semi-carbazide formation is essentially irreversible at temperatures up to 175 \u00b0C, a useful finding for the development of other cascade reactions.Gratifyingly, erocycle . This reN-isocyanate precursors in cascade reactions allows the formation of multiple hydroamination substrates from a common precursor, followed by intramolecular hydroamination events. This approach led to a diversity-oriented synthesis of pyrrolidines shown in Before developing other cascade reactions, we decided to address an important limitation of intramolecular hydroamination reactions.N-isocyanate addition/Cope-type hydroamination cascade ten different semi-carbazide-based pyrrolidines were synthesized from the same carbazate precursor , and piperidine derivatives (3c\u2013g) yielded the desired products in good to excellent yields. (S)-Prolinol was also a competent nucleophile but only showed modest diastereoselectivity. Product 3g bearing a bromine atom was also formed to highlight the potential of this metal-free method. The medicinally relevant 2-oxopiperazine demonstrated chemoselectivity for the most nucleophilic nitrogen, providing the desired heterocycle 3h in 85% yield. While both acyclic and cyclic secondary amines proved competent reaction partners, the result with primary amine 3j led to a modest, but preparatively useful yield. We attribute this difference of reactivity to a more challenging hydroamination step, due to the relative population of the E- and Z-conformers of the semi-carbazide.Using this rsor 1a, . Cyclic E hydrazide conformer is thermodynamically favoured.i.e. destabilizing interaction between R2 and \u03b2N in the E-conformer). Thus, the Z-conformer of these intermediates is thermodynamically favoured. Previous DFT studies suggest that the Z-conformer is the reactive conformer in Cope-type hydrohydrazidations , with an alkene present on the N-isocyanate substrate. To further develop cascade reactions of N-isocyanates, we were drawn to different cascade reactions in which cyclization would occur on a functional group (FG) present on the incoming nucleophile. Recently, we reported such a cascade reaction using \u03b1-amino esters to rapidly assemble N-substituted hydantoins would yield the 5-membered amino-hydantoin, while cyclization using the distal nitrogen (\u03b2N) would yield the 6-membered aza-diketopiperazine.o-esters .3N-substituted glycine esters . Alanine (5d), leucine (5e) and proline (5f) esters also cyclized in moderate to good yields. However, racemization occurred under these reaction conditions. In addition, we were pleased to observe the formation of a dihydrouracil ring (5g) in moderate yield using a N-benzyl \u03b2-aminoester as the reaction partner. Since the reaction was completely selective for the cyclization on the proximal nitrogen (\u03b1N), we decided to expand this study to include other N-isocyanate precursors. As indicated in the introduction, differences are expected and observed for the formation and reactivity of diverse N-isocyanates. In the context of this cascade reaction we wondered if blocked precursors of other amino-isocyanates , imino-isocyanates , and amido-isocyanates would be suitable reaction partners. The results using various N-isocyanate precursors are presented in N-alkyl glycine esters . We were also pleased that electron-rich and electron-poor aromatic carbazones both led to efficient product formation (7c\u2013e). We then surveyed the reactivity of bulky keto-carbazones: acetophenone, fluorenone and diisopropyl ketone-derived reagents afforded the cyclized products in good yields (7f\u2013h). A heteroaromatic carbazone also produced the desired heterocycle in good yield (7i). We also investigated the use of several N-glycine esters using the aldcarbazone derived from 4-methoxybenzaldehyde as a test substrate. The somewhat hindered N-isopropyl glycine ester afforded the desired hydantoin 7j in moderate yield. Functional groups such as nitriles (7k) and esters (7l) were tolerated on the nitrogen substituent. Even N-aryl glycine esters provided the N-aryl substituted amino-hydantoins. This indicates that electron-rich (7n), electron-neutral (7m) and even electron-poor (7o) anilines are competent nucleophiles under the reaction conditions. We then used this late-stage functionalization strategy to synthesize 5 azumolene analogues (7s\u2013w), without the use of chromatography (i.e. purified by filtration). Finally, we performed exploratory attempts toward three related cascades. These proved rewarding as we showed that: (1) imidazolidinone (7p) formation was possible if ring closure was achieved via 1,4-addition , using an \u03b1,\u03b2-unsaturated amino-ester as reagent; (2) an N-isothiocyanate precursor also engaged in a related cascade7q); (3) amide-substituted hydantoin (7r) could be synthesized using an amido-isocyanate precursor. Collectively, this data suggested that a variety of N-isocyanate precursors could engage in cascade reactions and display similar reactivity.Aliphatic carbazones afforded the desired hydantoins in excellent yields ).Considering the encouraging results obtained with the two reaction sequences presented above, we felt confident that we could expand this chemistry to different synthetic targets. The diversity of NCO (eqn ).4aq4i.e. NH2vs. NHR, previously) would result in a greater propensity to dimerize. We thus became interested in achieving even milder reactivity through the use of base catalysis. Previous studies conducted in the context of our alkene aminocarbonylation work showed that bases (e.g. Et3N) led to imino-isocyanate formation under milder conditions.C-isocyanatesN-isocyanate precursors, which could prove an asset for the development of other cascade reactions. Gratifyingly using 20 mol% of DBU with O-phenyl carbazate proved to be a convenient way of generating NH2\u2013NCO at room temperature. This was performed in the presence of a nucleophilic amine (1.1 equiv.) and afforded the desired semicarbazide products . Both primary and secondary benzylic amines proved competent reactants yielding the desired semi-carbazide in good to excellent yield. Propargylamine underwent the substitution reaction providing the propargylic semi-carbazide in modest yield . In general, both acyclic and cyclic secondary amines were tolerated . An ester functionality was also tolerated to yield the substituted piperidine based semi-carbazide . Finally, we were pleased to observe that double substitution could also be achieved using piperazine . Overall, the data shown in N-isocyanates could also benefit from base catalysis, with no detectable dimerization or oligomerization occurring under the reaction conditions. This new reactivity also provided a new route to semi-carbazides that could serve as building blocks for more complex derivatives.16Using this base catalysis procedure, we studied the reaction of different amines with O-phenyl carbazate could also engage in established cascade reactions. The advantage of this strategy is the ability to provide a free NH2 group for further derivatization reactions. We were quite pleased to see that the reaction with N-methyl glycine ethyl ester provided the NH2-substituted hydantoin in 91% yield on gram scale (eqn (5)). We then used the NH2 group to form pyrrole-substituted hydantoin 10b in 90% yield (eqn (6)). We were also able to synthesize an imidazolidinone derivative through a N-isocyanate cascade exploiting addition/cyclization by 1,4-addition (eqn (7)).A natural extension was to explore if N-isocyanates to form saturated heterocycles, amino-hydantoins and imidazolidinones, we sought to develop reaction cascades forming aromatic heterocyclic compounds possessing the N\u2013N\u2013C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O motif. One could expect that the aromaticity of the product should prove advantageous by either facilitating the cyclization event or simply by forming stable products that do not interfere with the cascade reaction. However, this strategy also inherently implied the use of precursors at a higher oxidation state, with the unsaturations required for aromatization being present in their structure.Having established the potential of N-isocyanate precursor , morpholine and an ether-containing proline derivative yielding the desired heterocycle efficiently after heating for 18 h. Cascade reactions of secondary acyclic amines required prolonged heating (48 h) but yielded the corresponding phthalazinones in almost quantitative yields . Symmetrical and unsymmetrical amines afforded the desired products, but mixtures of semi-carbazide rotamers were observed by 1H NMR for the adducts of unsymmetrical amines. Unfortunately, all attempts to use primary amine nucleophiles resulted in the free N\u2013H phthalazinone. This observation strongly suggested that the desired product was formed, but then acted as a blocked C-isocyanate precursor by forming the isocyanate upon thermal extrusion of N\u2013H phthalazinone. Nevertheless, despite being limited to secondary amines this cascade provided us with the first cascade reaction forming a heteroaromatic core using N-isocyanate intermediates. The hydantoin and phthalazinone work showcased the cyclization potential of carbazone-derived N-isocyanates on esters.Pleasingly, optimization provided the desired cascade reaction, and carbamoyl-substituted phthalazinones were formed with several secondary amines at 100 \u00b0C. The reaction was tolerant of secondary cyclic amines such as pyrrolidine , morpholine (14b) and piperazine derivatives (14c and d) all cyclized in high yield. A halogen substituted aromatic group was tolerated in the reaction (14c), which highlights the possibility of further functionalization. Acyclic amines were also good reaction partners for the synthesis of acyl pyrazoles (14e\u2013l). Secondary amines yield the desired heteroaromatic core in high yield at room temperature. In contrast, primary amines required gentle heating at 50 \u00b0C but also provided the desired pyrazoles in good yield. Interestingly, benzylamine (14g) and furfurylamine (14j) did not require higher temperature to form the corresponding pyrazoles. Anilines could also be used as nucleophiles (14k). Given the low nucleophilicity of anilines, their use in this cascade reaction at 50 \u00b0C again supports the formation of a reactive N-isocyanate intermediate as the participating electrophile. The reaction could also be highly chemoselective for the most nucleophilic amine when using diamines, as demonstrated by the selective formation of adduct 14l. In parallel to efforts using different amines the cascade reaction was also performed with several carbazones, using pyrrolidine as a representative nucleophile (1) substituent on the outcome of the cascade reaction proved minimal. Products containing both small and large substituents were formed in high yield. The result obtained with the methyl-substituted carbazone was especially noteworthy. Indeed, both E and Z isomers of the carbazone starting material were present, favoring (ca. 9\u2009:\u20091 by 1H NMR) the E isomer which was not the appropriate configuration to cyclize. Thus the high yield supports that carbazone or imino-isocyanate isomerization occurred under the reaction conditions to form the Z-isomer required for cyclization on the alkyne. Alkyne substitution (R2) was also well tolerated and allows the formation of 1,3,5-trisubsituted pyrazoles under similar conditions (14p\u2013r). Finally, it should be noted that product formation for these 1,3,5-trisubtituted entries was observed at room temperature but that yields were typically higher at 50 \u00b0C, and that this cascade reaction is also scalable (eqn (8)).Gratifyingly, the y amines .22 A widy amines , top. Cyleophile , bottom.14 (R4 = H) are known to be blocked (masked) C-isocyanate precursors.3N-treated silica gel was also needed, suggesting that mildly acidic conditions could promote isocyanate formation. Overall, this data illustrated the usefulness of milder conditions for the development of new reaction cascades.It was imperative to use basic conditions for the formation of these acyl pyrazoles due to the labile nature of products formed with primary amines. Indeed, attempts to form acyl pyrazoles upon heating in the absence of base led to formation of free N\u2013H pyrazoles, since the acyl-pyrazoles products N-isocyanates could engage in cascade reactions forming 5-membered and 6-membered aromatic heterocycles, we sought to develop a cascade in which the amine nucleophile would be incorporated within the aromatic heterocycle formed. Strategically, this represented the most difficult cascade reaction targeted with N-isocyanates. After surveying potential scaffolds that could be obtained using this approach, the 6-azauracil ring system stood out as an excellent synthetic target due to reported biological activities7 receptor antagonists,1A receptor agonists.N-isocyanates to readily form semi-carbazones, we envisioned the use of carbazones derived from \u03b1-keto-esters.After showing that As illustrated in eqn (9), using this approach would provide the ability to incorporate the primary amine reagent at the 3 position of the azauracil compounds, upon cyclization of the incoming-nitrogen atom on the ester group of the parent carbazone.A under mild conditions. We believed that both E and Z isomers of A would be in equilibrium thus allowing for complete conversion to the stable aromatic product. However, we expected a strong conformational preference for this intermediate that would make the cyclization step difficult, noting that related cyclizations (R2 = H) typically only proceed at high temperatures.N-isocyanate addition products (semi-carbazone A) were observed at temperatures below 150 \u00b0C. However cyclization was typically observed around 150 \u00b0C, and further optimization showed that the desired azauracils formed in good yields upon heating at 175 \u00b0C. With these conditions in hand, we explored the scope of this cascade reaction: the results are displayed in Initially, we were confident about the ability to access semi-carbazone 15a (R1 = Me). We were pleased that a variety of primary amines, a hydrazine and a hydroxylamine could form azauracil products in moderate to high yield (35\u201385%). The reaction tolerated the use of hindered amines such as cyclohexylamine , of less nucleophilic amines such as anilines (16f), and of primary amines with a proximal electron-withdrawing group . While conducting the reaction with 4-bromoaniline, a crystalline product (16p) was obtained in 77% yield, and X-ray analysis secured the structural assignment , an ester (16v) and a tetrahydrofuryl group at the R1 position. These substitution patterns have medicinal relevance, for example product 16u is a C-linked nucleoside analogue. Finally, this cascade tolerated a diverse set of functional groups, including a free hydroxyl (16h), allyl (16o) and propargyl (16b) groups, a nitrile (16i), ethers (16t and 16u), an ester (16v), heteroaromatic rings such as thiophene (16x) and N\u2013H indole (16j), a free amide (16k), and aromatic bromides (16p) and fluorides (16e). Collectively, these results highlight that this cascade reaction has broad applicability to rapidly assemble 6-azauracil compounds.Fortunately, the cascade reaction forms a variety of substituted 6-azauracils effectively . First, via an intramolecular condensation, rather than form a bis-azauracil through cyclization of each nitrogen atom precipitated out of the reaction upon heating in acetonitrile, and cooling at the end of the reaction. In addition to encouraging results to form tricyclic systems using 1,2-aminoaniline (entries 1 and 2), we were pleased that 1,3-diaminopropanes yielded the corresponding 6,6-bicyclic compounds in good yields (entries 3\u20135). Surprisingly, this ring system had not been described in the literature, despite decades of work on the synthesis of purine analogues,26c further highlighting that cascade reactions of N-isocyanates can provide access to new heterocycles through simple reaction sequences.Encouragingly, several diamines engaged in a cascade reaction forming bicyclic or tricyclic systems. Relatively high yields (55\u201376%) were obtained considering that product formation involves N-isocyanates are powerful intermediates in heterocyclic chemistry. Our data shows that the use of N-isocyanates provides a versatile strategy to assemble heterocyclic compounds possessing N\u2013N\u2013C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O motifs, which are common in agrochemicals and pharmaceuticals. Various heterocycles could be assembled by taking advantage of the controlled reactivity provided by the use of blocked (masked) precursors that reversibly form the desired N-isocyanates upon heating or in the presence of catalytic bases such as DBU. This reactivity also demonstrated the ability of different N-isocyanates\u2014amino-, imino-, and amido-isocyanates\u2014to engage in cascade reactions and allowed a comparison of their reactivity. We also demonstrated the use of O-phenyl carbazate as a precursor for the simplest N-isocyanate, NH2\u2013NCO. Over 100 new heterocyclic products were formed using new reaction cascades, including new heterocycles and heterocyclic products with substitution patterns that are either difficult to prepare or that have not been reported in the literature. Beyond providing a new tool in heterocyclic chemistry, this work addresses an important void in the isocyanate literature: the lack of reactions exploiting the reactivity of N-isocyanates. This scarcity is surprising considering that the applications of C-substituted isocyanates are extremely well developed. We hope that this first thorough study on the synthetic uses of N-isocyanates will encourage others to develop reactions of N-isocyanates, also taking advantage of the blocked N-isocyanate approach to overcome dimerization. Efforts along these lines are ongoing in our laboratories and will be reported in due course.In summary, we have demonstrated that despite their amphoteric nature and reported propensity to dimerize, Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "For the first time, \u03b1-formyldiazoacetates (FDA), have been successfully applied for asymmetric olefin cyclopropanation via Co(ii)-based metalloradical catalysis. via Co(ii)-based metalloradical catalysis. The cobalt(ii) complex of the D2-symmetric chiral amidoporphyrin [Co] is an effective metalloradical catalyst that can activate \u03b1-formyldiazoacetates to cyclopropanate both aromatic and aliphatic olefins with varied electronic properties, affording the synthetically useful 1,1-cyclopropaneformylesters in high yields with both high diastereo- and enantioselectivity.For the first time, \u03b1-formyldiazoacetates have been successfully applied for the asymmetric cyclopropanation of alkenes As stable metalloradicals, cobalt(ii) complexes of the D2-symmetric chiral amidoporphyrins [Co(D2-Por*)] have emerged as a class of effective catalysts for asymmetric olefin cyclopropanation through a distinct radical process involving the catalytic generation of \u03b1-metalloalkyl radicals as the key intermediates (A).5 It has been suggested that the unusual capability of [Co(D2-Por*)] in activating acceptor/acceptor-substituted diazo reagents as well as regulating the reactivity and selectivity of the radical processes is further enhanced by the postulated double hydrogen-bonding interactions between the amide N\u2013H donors on the amidoporphyrin ligand and the two acceptors on the C-centered radical moiety -based metalloradical catalysis (Co(ii)-MRC), we were attracted to the possibility of accessing a new type of \u03b1-metalloalkyl radical bearing both \u03b1-formyl and \u03b1-alkoxycarbonyl functionalities from the metalloradical activation of \u03b1-formyldiazoacetates (FDA).6 Despite the fact that free \u03b1-formylalkyl radicals are scarce and prone to H-atom abstraction because of the weak aldehydic C\u2013H bonds,7 we reasoned that this type of \u03b1-metalloalkyl radical might be accessible on the basis of the combined effects of metal stabilization, double H-bonding interaction, and protection by the well-defined cavity of the ligand system (A). Assuming that the \u03b1-formyl-\u03b1-alkoxycarbonyl-\u03b1-Co(iii)-alkyl radicals (A) are capable of undergoing stereoselective radical addition with olefins, followed by the effective 3-exo-tet radical cyclization8 of the corresponding \u03b3-Co(iii)-alkyl radicals (B), we anticipated the potential development of a new catalytic process for the asymmetric synthesis of optically active cyclopropanes bearing both aldehyde and ester functionalities, which would be valuable for stereoselective organic synthesis . The development of this catalytic process apparently confronts the formidable challenges associated with the inherent low reactivity of the acceptor/acceptor-substituted diazo reagents and the incompatibility of the aldehyde functionality with existing catalytic systems.11 Recently, Fokin and coworkers developed a Rh2-catalyzed system for a highly asymmetric cyclopropanation with N-sulfonyl-1,2,3-triazoles for the production of cyclopropyl imines, which could be subsequently transformed into the corresponding formyl cyclopropane derivatives.12 While this offers a valuable alternative for the preparation of optically active formyl cyclopropanes, the direct synthesis via asymmetric cyclopropanation with \u03b1-formyl diazo reagents is an appealing process that remains to be developed. As a new application of Co(ii)-MRC, we herein wish to report the first catalytic system based on [Co(D2-Por*)] that is highly effective in activating FDA for asymmetric cyclopropanation. This asymmetric radical process is generally applicable for a broad scope of alkenes, offering a direct method for the high-yielding synthesis of 1,1-cyclopropaneformylesters with excellent control of the diastereo- and enantioselectivity. The products can be readily transformed into other chiral 1,1-bifunctionalized cyclopropanes and chiral dihydrofurans.The catalytic asymmetric cyclopropanation of alkenes with diazo reagents represents the most general approach for the stereoselective synthesis of optically active cyclopropanes.ii)-MRC (2(OAc)4] was indeed incompatible (entry 1), [Co(TPP)] only produced trace amounts of the corresponding cyclopropane from ethyl \u03b1-formyldiazoacetate (EFDA) (entry 2). Remarkably, when the Co(ii) complex of the D2h-symmetric achiral amidoporphyrin [Co(P1)]13 was used as the catalyst, the reaction proceeded successfully to form the desired (E)-1,1-cyclopropaneformylester in a 46% yield (entry 3). The dramatic difference in the catalytic activity between [Co(TPP)] and [Co(P1)] is in alignment with the hypothesized role of the double H-bonding interaction in activating EFDA and stabilizing the resulting intermediate A (P2)],14 the reactivity was further enhanced with the observation of a significant level of enantioselectivity (entry 4). Of the solvents examined, toluene was proven to be the medium of choice (entries 4\u20138). Lowering the reaction temperature further increased the enantioselectivity, but decreased the yield (entries 8\u201310). The diastereoselectivity was greatly improved when the bulkier tert-butyl \u03b1-formyldiazoacetate (t-BFDA) was used, affording cyclopropane 1a in a 78% yield with 95\u2009:\u20095 dr and 96% ee (entry 11). The product yield could be further improved to 84% by increasing the catalyst loading to 5 mol% while maintaining the high level of diastereo- and enantioselectivity (entry 12).Initial experiments were carried out with styrene as the model substrate to examine the suitability of FDA for the catalytic radical cyclopropanation by Co(ii)-MRC . While [ediate A . By switii)-based asymmetric radical cyclopropanation was investigated (P2)] with t-BFDA. For example, p- and m-alkyl styrenes were cyclopropanated to formylcyclopropanes 1b\u20131d in high yields with excellent diastereo- and enantioselectivity (entries 1\u20133). Halogenated (entries 4\u20136) and electron-deficient (entries 7 and 8) styrene derivatives could also undergo high-yielding cyclopropanation, producing 1e\u20131i with high stereoselectivities. The configurations of the two contiguous chiral centers in 1h were established as by X-ray crystal structural analysis (see ESI1j (entry 9). In addition, 1,1-disubstituted olefins such as \u03b1-methylstyrene could also be effectively employed, affording (E)-formylcyclopropane 1k in a 93% yield with remarkable control of both the diastereo- and enantioselectivity of the two newly-generated contiguous all-carbon quaternary stereogenic centers (entry 10). To demonstrate the functional group tolerance of the Co(ii)-based radical cyclopropanation, m-formylstyrene could be effectively cyclopropanated to cyclopropane 1l in a high yield with high diastereo- and enantioselectivity (entry 11). Notably, the two unprotected formyl groups were well tolerated by the metalloradical system. It is also worth mentioning that the Co(ii)-catalyzed cyclopropanation process could be scaled up ten-fold as demonstrated with the high-yielding synthesis of the cyclopropane 1d on a 1.0 mmol scale without affecting the excellent stereoselectivity (entry 3).Under the optimized conditions, the scope of this Co(stigated . Like sts see ESI. The cycii)-based radical cyclopropanation was further highlighted for its exceptional reactivity toward electron-deficient olefins, which are typically problematic substrates for catalytic systems involving electrophilic metallocarbene intermediates. For example, [Co(P2)] could catalyze the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C cyclopropanation of acrylonitrile with t-BFDA to form 1,1,2-cyclopropaneformylesternitrile 1m in a high yield with good enantioselectivity (entry 12), leaving the cyano group untouched. In marked contrast, when treated with Rh2-based catalyst, acrylonitrile was previously shown to react with the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N bond of FDA to form oxazoles.15 Other electron-deficient olefins such as ethyl and methyl acrylates could also be cyclopropanated to form the 1,1,2-cyclopropaneformyldiesters 1n and 1o in good yields with 96% ee and 97% ee, respectively, although with diminished control of diastereoselectivity (entries 13 and 14). The presence of three electron-withdrawing groups in the cyclopropanes 1m\u20131o renders them highly electrophilic, making them valuable intermediates for synthetic applications.16 Furthermore, aliphatic olefins, another class of challenging substrate for asymmetric cyclopropanation, could also be cyclopropanated by [Co(P2)] as exemplified by the high-yielding reaction of 1-octene under neat condition, forming 1p with high stereoselectivity (entry 15).The Co(E)-1a could be transformed into a trans-vinyl unit via the Horner\u2013Wadsworth\u2013Emmons reaction, affording (E)-1,1-cyclopropanevinylester 2 in a 78% yield with full retention of both the relative and absolute configurations (eqn (1)). When treated with Bestmann\u2013Ohira reagent, the formyl group in (E)-1a could be smoothly converted to a terminal alkyne functionality, resulting in chiral (E)-1,1-cyclopropaneethynylester 3 in a 70% yield without any diminishment of the original stereochemistry (eqn (2)). This transformation provides an alternative way to direct asymmetric cyclopropanation with \u03b1-ethynyldiazoacetates for chiral 1,1-cyclopropaneethynylesters.17 It is noted that \u03b1-ethynyldiazoacetates containing terminal alkyne units seem synthetically inaccessible. While the [Co(P2)]-catalyzed cyclopropanation with FDA generally forms (E)-cyclopropanes, the (Z)-diastereoisomers could be conveniently accessed through the stereospecific epimerization previously reported.5 As demonstrated with (E)-1a, treatment with 5 equivalents of NaI at room temperature resulted in the formation of (Z)-1a as the major diastereomer with only partial loss of the original optical purity (eqn (3)). Interestingly, when (E)-1g was treated with 10 equivalents of NaI at an elevated temperature, a ring-expansion involving the formyl group occurred instead, affording 2,3-dihydrofuran 4 in a 74% yield (eqn (4)). In the absence of any external chiral induction, the enantiopurity appeared to be largely retained during the rearrangement.As an initial exploration of applications, the formyl unit of the resulting chiral 1,1-cyclopropaneformylesters could be readily converted into other functional groups, forming various cyclopropane derivatives while retaining high enantiopurity. For example, the formyl group in (P2)] can effectively activate \u03b1-formyldiazoacetates (FDAs) for a highly asymmetric olefin cyclopropanation, without affecting the otherwise reactive aldehyde functionality. This represents the first application of \u03b1-formyldiazo reagents for metal-catalyzed asymmetric cyclopropanation. The Co(ii)-based radical cyclopropanation with FDA can be successfully applied to a broad scope of olefin substrates, permitting the direct synthesis of chiral 1,1-cyclopropaneformylesters in high yields with high diastereo- and enantioselectivity. Given that the resulting enantioenriched cyclopropanes contain two contiguous chiral centers in the ring structure, including one all-carbon quaternary stereogenic center bearing both aldehyde and ester functionalities, this new Co(ii)-based asymmetric radical cyclopropanation process should find wide applications in stereoselective organic synthesis.In summary, we have demonstrated that the metalloradical catalyst [Co("} +{"text": "A multitechnique investigation of an evaporable vanadyl spin system with long-lived quantum coherence that self-assembles on gold. i.e. the logic units of quantum computers. These have to retain memory of their quantum state for a sufficiently long time allowing quantum operations to be performed. For molecular based spin qubits, strategies to increase phase coherence by removing nuclear spins are rather well developed, but it is now crucial to address the problem of the rapid increase of the spin\u2013lattice relaxation rate, T1\u20131, with increasing temperature that hampers their use at room-temperature. Herein, thanks to the combination of pulsed EPR spectroscopy and AC susceptometry we evidence that an evaporable vanadyl complex of formula VO(dpm)2, where dpm\u2013 is the anion of dipivaloylmethane, presents a combination of very promising features for potential application as molecular spin-qubit. The spin\u2013lattice relaxation time, T1, studied in detail through AC susceptometry, decreases slowly with increasing temperature and, more surprisingly, it is not accelerated by the application of an external field up to several Teslas. State-of-the art phase memory times for molecular spin systems in protiated environment are detected by pulsed EPR also in moderate dilution, with values of 2.7 \u03bcs at 5 K and 2.1 \u03bcs at 80 K. Low temperature scanning tunnel microscopy and X-ray photoelectron spectroscopy in situ investigations reveal that intact molecules sublimated in ultra-high vacuum spontaneously form an ordered monolayer on Au(111), opening the perspective of electric access to the quantum memory of ensembles of spin qubits that can be scaled down to the single molecule.Electronic spins in different environments are currently investigated as potential qubits, T1, which corresponds to the lifetime of a classical bit that can assume either the |0> or the |1> value; ii) the characteristic time in which the spin loses the memory of the phase of the superposition state in which it has been prepared. A lower estimation of this decoherence time, T2, can be extracted by the memory time, Tm, which is commonly measured with pulsed EPR or NMR: the ratio of Tm over the time necessary for an individual quantum operation has to be larger than 104 to allow for fault tolerant quantum computing.The realization of a quantum computer is expected to trigger a second revolution in information and communication technology,Tm, the research in this field has recently focused back on the simplest spin S = 1/2 systems constituted either by organic radicalsTm, in particular at high temperature, because there are no excited spin levels that can foster the magnetic relaxation when thermally populated. In these systems the interaction of the electronic spin with the nuclear spins is the most relevant source of decoherence. Outstanding results have very recently been obtained with vanadium(iv) ions assembled with nuclear spin-free ligands.2, Tm approaches one millisecond at low temperature,T2.T1, thus resulting in enhanced coherence time at high temperatureT1, are still poorly investigated.In the field of electron spin-based qubits nitrogen vacancies in diamondiv) by the combination of AC magnetic susceptometry to study spin\u2013lattice relaxation with pulsed EPR spectroscopy to characterize the spin coherence. The two techniques can in fact shed light on different contributions to the relaxation but their association is unprecedented in the search for potential spin-based qubits.In this study we have investigated the magnetic relaxation of a simple mononuclear complex of vanadium scale\" fill=\"currentColor\" stroke=\"none\">O bond is expected to increase the rigidity of the coordination sphere with a reduction of spin\u2013lattice relaxation efficiency. The presence of \u03b2-diketonate ligands in a neutral complex imparts a high volatility that can be exploited to deposit the molecule on surfaces. An unexpected long T1 over wide field and temperature ranges has been found to accompany Tm values that are among the longest ones observed in molecular species surrounded by spin active nuclei. The in situ morphological and spectroscopic characterization of a monolayer deposit of VO2(dpm)2 on Au(111) suggest that the molecules are intact on the surface, making these simple units potential candidates as molecular qubit individually addressable by scanning probe techniques.The vanadyl complex VO(dpm)2 was achieved according to an earlier reported procedure,2, prepared in the crystalline form and characterized by X-ray diffractometry scale\" fill=\"currentColor\" stroke=\"none\">O double bond (1.59 \u00c5 vs. an average of 1.964 \u00c5 for the V\u2013O single bonds). Deviations from tetragonal symmetry are already visible in the first coordination sphere in both bond lengths and bond angles. Given that the system crystallizes in the monoclinic P21 space group, two sets of molecules with the V PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O directions forming an angle of 64.1\u00b0 are present in the crystal lattice. The strongly axial ligand field produced by the short V PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O bond removes orbital degeneracy with the dxy orbital being the lowest in energy and the only one to be half occupied. Vanadyl systems are therefore well described by a spin S = 1/2 with slightly anisotropic g tensor close to the free electron value. The most abundant isotope of vanadium, 51V (99.75%), is characterized by I = 7/2 thus the S = 1/2 doublet is further split in 16 states by hyperfine interaction as schematized in The synthesis of crystalline VO(dpm)y see ESI, presenty see ESI with a s2, hereafter 1bulk, revealed no imaginary component of the susceptibility in zero static field down to the lowest investigated temperature (1.9 K). The application of a weak field induced however slow relaxation of the magnetization with the concomitant decrease of the real component \u03c7\u2032 and the appearance of a peak in \u03c7\u2032\u2032 component , evidencing also a gradual increase in the width of the distribution on lowering the temperature corresponds to the direct mechanism, dominating at low temperature, and the second one (b = 0.052 s\u20131 Kn\u2013) to a Raman-like, i.e. a multiphonon process involving virtual excited states.n = 3.22 \u00b1 0.02 is much smaller than the value of 9 or higher expected for the Raman process,34Maxima in i.e. up to 8.8 T, for three different temperatures, 5, 10, and 15 K. Notice that in this temperature range the direct process dominates as indicated by the almost linear dependence of \u03c4\u20131 on T. The corresponding relaxation times are reported in \u03c4 for weak applied field is followed by an almost flat region that extends up to ca. 4 T, followed by a rapid decrease at higher fields. Data of S = 1/2 systems. According to the seminal work done by de Vroomen et al. on the Cu2+ Tutton salt,To shed light on the mechanisms of magnetic relaxation the AC susceptibility was investigated in a wide field range, S = 1/2 should not be able to relax in zero field. The second term takes into account a sort of internal field whose origin can be either intramolecular (i.e. hyperfine interactions) or intermolecular (i.e. due to dipolar or exchange interactions). The latter is responsible for the efficient relaxation in zero field and presents, for the direct mechanism,The first term represents the direct mechanism between the two states split by the Zeeman energy, which is expected to vanish in zero field as a result of the Kramers theorem.S = 1/2 spin and reflects the fact that the larger is the Zeeman splitting between the states the higher is the density of phonons matching it. In the second one the d term represent the zero field relaxation rate, similar to the tunnelling rate in SMMs,f parameter takes into account the ability of the external field to suppress these mechanisms, while the e parameter, strongly dependent on the concentration of the spin centres, takes into account the field effects on the relaxation of interacting spins.c parameter for T = 5 K should be considered with caution because only a small fraction of the susceptibility is detected at such high fields. The field range where the relaxation remains slow is really remarkable, suggesting that the direct mechanism of relaxation is not very efficient.The first term is the typical field dependence of the direct process for a \u03b1 of the extended Debye formula 2 in favour of the dimeric one [TiO(dpm)2]2,1 in polystyrene with mass ratio 1\u2009:\u20095, 1PS1\u2009:\u20095, and 1\u2009:\u200910, 1PS1\u2009:\u200910, as well as a frozen 200 mM solution of 1 in a 2\u2009:\u20093 CH2Cl2\u2009:\u2009toluene mixture (1sol200 mM), were prepared and investigated by AC susceptometry vs. log(T) plot of \u03c4\u20131 \u221d Tn, with n = 1.49 \u00b1 0.04 for 1PS1\u2009:\u20095 and n = 1.86 \u00b1 0.04 for 1sol200 mM. Exponents larger than one for the direct mechanism are generally attributed to spin\u2013phonon bottleneck effects,\u03c4 of 1sol200 mM was also investigated at T = 5 K (see The long spin\u2013lattice relaxation time of VO(dpm)y Fig. S5. This watization : polymer 5 K see and reve1 (1sol1 mM) is shown in 1bulk, 1PS1\u2009:\u20095, 1PS1\u2009:\u200910, and 1sol200 mM spectra available in ESI Fig. S6The low temperature CW-EPR X-band spectrum of a frozen 1 mM solution of I = 7/2 nuclear spin of 51V: at the high and low field extreme region, peaks due to the parallel components of the hyperfine structure are observed, whereas in the centre the closely spaced perpendicular ones are evident, as schematized by the resonant fields in The spectra clearly show the features due to the anisotropic hyperfine coupling of the electron spin to the gx = 1.9880(2); gy = 1.9815(3); gz = 1.9490(2) and Ax = 0.0056(1) cm\u20131 (167.9 MHz); Ay = 0.0063(3) cm\u20131 (190.4 MHz); Az = 0.0170(2) cm\u20131 (509.6 MHz). These parameters are in the range previously reported for VO2+ \u03b2-diketonate-type derivativesSpectral simulations were performed1sol200 mM, 1sol1 mM, and 1PS1\u2009:\u200910 diluted samples (2 sample is experiencing the detected coherence.An echo-detected field-swept EPR spectrum (EDFS) was recorded using the standard Hahn sequence see ESI for 1sol samples . The obs1 as molecular qubit was performed by measuring the coherence time, Tm, as a function of temperature and field position for 1sol1 mM to reduce spin\u2013spin interactions. To maximize the observed echo the temperature dependence of Tm has been investigated on the so-called powder like line evidenced by an arrow in Determination of the potential applicability of diluted samples of The echo decay traces were theTm values ,T1 between 5 K and 110 K in 1sol1 mM, for which the AC susceptibility technique does not have the necessary sensitivity.Since earlier studies revealed strong correlation between the spin\u2013lattice relaxation time \u03b21 being in the range 0.6\u20130.9 in the investigated temperature range 2 in H2O:glycerol solution,T1 are observed at low temperature (50 ms at 4 K) and on heating T1 tends toward Tm (6 \u03bcs at 110 K). Interestingly, a Tn\u2013 dependence with n = 3.2 \u00b1 0.2 is observed above 40 K, with a gradual decrease of n at lower temperatures, in agreement with AC susceptibility results.Given the large range of relaxation times two different experimental procedures were applied: at low temperature (5\u201360 K) the echo saturation by fast repetition, suitable for long relaxation times1sol200 mM by pulsed EPR: the obtained results are consistent with those obtained by AC susceptibility deposit assembled on the Au(111) surface. A complete in situ X-ray photoelectron spectroscopy (XPS) and low temperature scanning tunnelling microscopy (STM) characterization was carried out, while the stability of the sample toward oxidation was investigated by exposing a thick film to air. T = 30 K for sub-ML coverage. As observed for other complexes with dpm\u2013 ligands,The high volatility of VO(dpm)2. By increasing the deposition time full coverage was achieved: regularly packed molecules still revealing the herringbone structure underneath were found scale\" fill=\"currentColor\" stroke=\"none\">OV), in good agreement with what observed for vanadyl phthalocyanine, VOPc, on Ag(111),\u2013 ligands scale\" fill=\"currentColor\" stroke=\"none\">OC). The ratio of the area of the two peaks is close to 1\u2009:\u20094, as expected for the stoichiometry of the molecule, thus confirming the integrity of the complex on surface. An analogous analysis allows to distinguish three components contributing to the C 1s region: the carbonylic carbon (287.2 eV), methyl carbon (285.4 eV) and the third one regrouping the remaining carbons .The observed broad O 1s peak around 532 eV was reproduced by considering two components. The smaller one at 531.3 eV was attributed to the oxygen of the vanadyl group scale\" fill=\"currentColor\" stroke=\"none\">OV 1s \u2013 V 2p3/2) of 14.9 eV which well compares with that observed in VOPc monolayer and multilayers scale\" fill=\"currentColor\" stroke=\"none\">OV 1s \u2013 V 2p3/2) = 14.6 eV).Even more interestingly the vanadium photoelectron peaks allowed to provide specific hints on the oxidation state of this element and thus on possible interaction with the metal surface. The V 2p2 molecules can be deposited intact on the surface and features a weak interaction with the gold substrate as the only occupied d orbital, dxy, is expected to lie flat with limited overlap with the substrate orbitals. A similar scenario was observed for copper(ii)phthalocyanine molecules that are known to retain their unpaired electron in the dx2\u2013y2 orbital.S = 1/2 of VIV compared to thicker films. It is therefore reasonable to envisage that VO(dpm)2 molecules retain their paramagnetic nature when in contact with the gold substrate. This system represents therefore an appealing alternative to the use of N-donors phthalocyanine- and porphyrin-based systems for deposition on surfaces, though VO(dpm)2 films resulted somehow instable in air: ex situ prepared thick films of about 150 nm showed a partial surface oxidation as suggested by the decrease in the scale\" fill=\"currentColor\" stroke=\"none\">OV 1s \u2013 V 2p3/2)) value accompanied by a progressive shift of the V 2p peak et al.S = 3/2 of CoII in Co(acac)2(H2O)2.S = 1/2 due to time reversal symmetry. VO(dpm)2 corresponds exactly to the hyperfine-split S = 1/2 model recently developedI = 7/2 gives origin to two sets of states characterized by F = |S \u00b1 I| with multiplicity 9 and 7, respectively, as can be observed in F = 4 and F = 3 states, which are however further split by the anisotropic components of the hyperfine tensor (see eqn (4)). The application of a weak static field has a different effect when applied along the molecular z direction, the one of largest hyperfine interaction corresponding to the V PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O bond direction, or perpendicular to it, as also indicated in the eigenvectors composition 2pseudo-spin S = 1/2 system, some striking quantitative differences are evident. The first one is that the relaxation time remains long over a wide field range, ca. 30 times larger in VO(dpm)2 compared to the CoII derivative. At 5 K, where the relaxation is still governed by the direct mechanism, the relaxation rate starts to grow above 3 T, to be compared to the drastic 0.1 T upturn observed for CoII at 1.8 K.II spins2 is unprecedented. This is extremely appealing for technological applications as it allows to exploit higher frequencies to coherently manipulate the spins, e.g. at W-band, corresponding to 95 GHz, with significant improvement of sensitivity. Moreover, working at W-band was also shown to increase Tm in samples of Yb3+ diluted in CaWO4, though at the same time the larger field was found to reduce T1. This serious drawback of the use of high frequencies is not expected for VO(dpm)2.55If similar features were already observed in the Co(acac)T1 of VO(dpm)2 remains long over a wide temperature range. For instance, a relaxation time of 2 ms is observed at 6 K for diluted Co(acac)2(H2O)2 but at temperatures as high as 40 K in concentrated VO(dpm)2. The anomaly arises from the small exponent of the Tn dependence of the Raman-like mechanism of relaxation. Such low exponents are relatively common for S = 1/2 states with small orbital contributions comprising light elements and have been associated to the soft character of the molecular lattices.The origin of the striking difference between the two compounds can be associated to the reduced efficiency of the direct mechanism of relaxation, which relies on the spin\u2013phonon coupling. The latter is mediated by the spin\u2013orbit coupling, which is significantly lower for such a light transition metal as vanadium. As a result, Tm values observed for a frozen solution of VO(dpm)2 are slightly longer \u2013 in the whole investigated temperature range \u2013 than those reported for a dispersion in protiated solvents at the same concentration of a vanadium complex with nuclear-spin free ligands:2 diluted in deuterated solvents. A substantially unchanged Tm was detected . We must stress that the processability and the surface stability of this \u03b2-diketonate complex are comparable to those of metal porphyrins, without the drawback of introducing 14N magnetic nuclei. This is not only relevant for reducing the efficiency of decoherence; as suggested by Freedman et al.,g is practically zero and therefore are weakly affected by changes in the local field. Enhanced coherence time for these clock transitions have been recently observed in Bi doped silicon enriched in 29Si nuclei.2 for which clock transitions are expected at fields where the magnetization dynamics is already rather slow.The observed decoherence times for VO(dpm)Tm of VO(dpm)2 is compared with relaxation times for a copper dithiolate complex with deuterated PPh4+ cation reported by van Slageren et al.et al., is expected to increase Tm of VO(dpm)2 as well as its T1.On the other hand, when T1, in terms of both temperature and field dependence, is mandatory for the realization of molecular spin qubits that can be operated at room temperature. AC susceptibility gives easily access to the field dependence of T1, in contrast to EPR, which relies on the resonance condition. Though T1 extracted with the two techniques are exactly the same only in the case of a S = 1/2 with no hyperfine splitting, a close relation exists also for systems with more than two levels. The simple molecule we have picked up with this approach, though not yet optimized for coherent manipulation of the spin state, presents state-of-the-art phase memory times combined with additional interesting features. The spin\u2013lattice relaxation remains slow even in strong fields, allowing the use of higher frequencies for coherent spin manipulation without losses in performances.We have shown here that a more rational search for potential qubits can significantly benefit from the combination of AC susceptometry with pulsed EPR techniques. This multitechnique approach is of particular relevance to define synthetic strategies because the optimization of PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O bond needs to be further investigated by extending the approach developed here to other and more promising systems.Ab initio modellization of the spin relaxation could also help to identify which structural features can favour long T1, and consequently long Tm, at high temperature.A particularly low efficient spin\u2013phonon coupling appears to be at the basis of this behaviour and the potentially positive role played by the strong Vg-factors.61Even if the crucial aspect of qubits entanglement has not been addressed in this work it can be easily achieved through connection of \u03b2-diketonate pockets in more complex architectures.2 molecules, retaining their paramagnetic nature thanks to the reduced interaction of the orbital carrying the unpaired electron with the substrate. Metallic nanostructures can be easily decorated with a monolayer of ordered VO(dpm)2 molecules, allowing to investigate the response of an ensemble of identical molecular qubits, whose size can be easily controlled by lithographic exposure of the metallic substrate. Thin films of VO(dpm)2 could be evaporated directly on a \u03bc-SQUID to detect by AC susceptometry the effects of surface confinement on the dynamics of the magnetization, as already done on SMMs.2 could be also a good candidate to investigate quantum coherence at the single molecule level thanks to the recently developed approach based on spin-polarized scanning tunnelling microscopy, employed at very low temperature on single Fe atoms deposited on a MgO surface.63Of great relevance is the possibility to obtain monolayers of ordered arrays of intact VO(dpm)T2 in nuclear spin free environments with the possibility to control the spin\u2013lattice relaxation through a rational synthetic design is foreseen to boost the interest for molecular spin systems as potential qubits.Combining the optimization of Supplementary informationClick here for additional data file."} +{"text": "The M-(\u03b72-BMn) complex [(\u03b75-C5H5)(OC)2Mn{\u03bc-B(Cl)(tBu)Au(PPh3)}] (2) can be functionalized via halide substitution reactions to afford isostructural complexes [(\u03b75-C5H5)(OC)2Mn{\u03bc-B(R)(tBu)Au(PPh3)}] . 2-BMn) complex [(\u03b75-C5H5)(OC)2Mn{\u03bc-B(Cl)(tBu)Au(PPh3)}] (2) can be functionalized via halide substitution reactions to afford isostructural complexes [(\u03b75-C5H5)(OC)2Mn{\u03bc-B(R)(tBu)Au(PPh3)}] . It also reacts with coinage metal complexes [MCl(PPh3)] in the presence of halide abstraction reagents to afford borylene-bridged heteromultinuclear complexes [{(\u03b75-C5H5)(OC)2Mn}2{\u03bc2-B(tBu)}2M][BArx4] 2). Experimental characterization as well as computational studies revealed that these complexes are best viewed as transition metal borylene complexes side-on coordinated to monovalent coinage metal cations, thus representing the first boron analogs of Stone's alkylidyne-bridged multinuclear complexes.The M-(\u03b7 PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N(SiMe3)2.These complexes possess a distinctly different structural architecture to the relatively small library of multi- and di-nuclear borylene complexes, all of which have been described in terms of [BR] bridging. PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019B(R)] as a ligand, which reveals that it serves as a side-on \u03c0-ligand that resembles classical metal olefin or alkyne \u03c0-interactions and thus also their isolobal alkylidyne complexes (OC)2Mn PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019BtBu] (1) reacted with gold(i) chloride complexes to form heterodinuclear complexes [(\u03b75-C5H5)(OC)2Mn{\u03bc-B(Cl)(tBu)}Au(L)] 2}), which are best viewed as \u03c3-coordinated transition metal haloboryl complexes.3) affords the best yield and therefore complex 2 has been used for subsequent studies , the reaction mixture afforded orange crystals 5a and 5b in moderate yields after cooling at \u201330 \u00b0C overnight. The 11B NMR spectrum of 5a showed a broad singlet at 121 ppm, slightly downfield-shifted from that observed for 2 (108 ppm). The chemical shift of the 11B NMR signal of 5b is not significantly different from that of its precursor, lying at 107 ppm. Both signals of 5a and 5b fall within the range expected for transition metal boryl complexes,Intuitively, the reactive boron\u2013halogen bond in haloboryl complexes should be relatively straightforward to functionalize, yet in reality, examples of such a reaction remain rare. Most of these transformations introduce \u03c0-donating \u2013OR and \u2013NReactions . Upon ad5a and 5b to be [(\u03b75-C5H5)(OC)2Mn{\u03bc-B(R)(tBu)}Au(PPh3)] , which are isostructural to 2, apart from the second anionic substituent at boron.5a and 5b are 2.156(3) and 2.157(9) \u00c5, respectively, very similar to each other and both slightly longer than that observed in 2 (2.126(9) \u00c5). Concurrently, the Au\u2013B bond also elongates slightly upon halide replacement, exhibiting Au\u2013B distances of 2.356(2) and 2.326(8) \u00c5 for 5a and 5b, respectively, compared to 2.303(9) \u00c5 of 2. The Mn\u2013Au distances (5a: 2.548(1) \u00c5; 5b: 2.547(2) \u00c5), on the other hand, are barely altered by halide substitution (cf. 2.553(1) \u00c5). As observed in 2, the boron centers of 5a and 5b adopt an almost planar geometry when the gold atom is disregarded .Single-crystal X-ray diffraction studies have confirmed the structures of 2 with [NBu4][NCS] led to a less selective reaction, from which a small amount of pale orange crystals (5c) were isolated from a complex mixture. Single-crystal X-ray diffraction studies confirmed the structure of this compound to be [(\u03b75-C5H5)(OC)2Mn{\u03bc-B(NCS)(tBu)}Au(PPh3)] (5c), isostructural to 5a and 5b with an unusual boron-bound isothiocyanate substituent (5c are 2.113(5) \u00c5 and 2.320(5) \u00c5, noticeably shorter than those observed in 5a and 5b. The B\u2013N distance of 1.532(7) \u00c5 falls between those established for aminoboranes R2B PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019NR2 and amine-borane adducts R3B\u2013NR3, indicating significant \u03c0-donation from the nitrogen to boron. This is also reflected by the linear geometry (B\u2013N\u2013C 177.0(5)\u00b0 and N\u2013C\u2013S 178.3(5)\u00b0) of the BNCS moiety. To the best of our knowledge, complex 5c represents the first structurally characterized example of a transition metal complex bearing an isothiocyanate\u2013substituted boryl ligand. The 11B NMR spectrum of 5c showed a more upfield-shifted singlet at 95 ppm, which also falls within the range expected for transition metal boryl complexes.Similar reactions of stituent .29 The M3 shows similar reactivity towards nucleophiles. However, the expected products [(\u03b75-C5H5)(OC)2Mn{\u03bc-B(R)(tBu)}Au(PCy3)] of the analogous reactions were in all cases found to be in equilibrium with the parent borylene complex 1 and [AuR(PCy3)] in the reaction mixtures, and thus could not be isolated. This liability of the [AuR(PCy3)] moiety in the [(\u03b75-C5H5)(OC)2Mn{\u03bc-B(Cl)(tBu)}\u2013Au(PCy3)] system was confirmed from the reaction of 3 with the platinum (0) complex [Pt(PCy3)2] and the isocyanide ligand CNMes* (OC)2Mn{\u03bc-CO}{\u03bc-B(tBu)}Pt(PCy3)] (6)5-C5H5)(Mes*NC)(OC)Mn{(CO)B(tBu)(CNMes*)}] (7)3)] (1 and the gold acetylide complex [Au(CCPh)(PPh3)], which afforded the acetylide\u2013borylene coupling product 5b in 61% yield (34Complex ] (7)3)] . These o1% yield . Gold ac2 was also probed with sodium tetraarylborates for halide abstraction reactions. Upon addition of Na[BArCl4] to a toluene solution of 2, with shaking, a cloudy orange solution was formed. After filtration and cooling at \u201330 \u00b0C over a week, the reaction mixture afforded orange crystals, which are insoluble in hexane, benzene or toluene, and moderately soluble in dichloromethane. The 11B NMR spectrum obtained from a CD2Cl2 solution showed a broad signal at 144 ppm and a sharp singlet at \u20137.04 ppm. The former is identical to that of the free borylene complex 1 (144 ppm), and the latter is attributed to the boron nuclei of the tetraarylborate counterion.Complex 5-C5H5)(OC)2Mn}{\u03bc-B(tBu)}Au(PPh3)][BArCl4] (ArCl = C6H3Cl2), single-crystal X-ray diffraction studies revealed the structure of these orange crystals to be [{(\u03b75-C5H5)(OC)2Mn}2{\u03bc-B(tBu)}2Au][BArCl4] ([8][BArCl4]), comprising two borylene moieties coordinated to a single gold(i) cation (2\u20135 (2.110\u20132.157 \u00c5), and only marginally longer than the parent borylene complex 1 (1.810(9) \u00c5). This suggests that the Mn\u2013B interaction of [8]+ retains most of its multiple bond character while coordinated to the gold(i) cation, as also suggested by its 11B NMR signal. The Au\u2013Mn distance of 2.619(1) \u00c5, is noticeably longer than those observed for 5a\u2013c (2.548\u20132.555 \u00c5), however, shorter than that observed in the neutral trimetallic gold boride complex [{(\u03b75-C5H5)(OC)2Mn}2{\u03bc-B(Au(PPh3))}] (2.651(4) \u00c5), in which the Au(PPh3) moiety migrates between the two Mn\u2013B parts rapidly even at \u201390 \u00b0C.5a\u2013c (2.320\u20132.356 \u00c5). The angle between the two Mn\u2013B\u2013Au planes is 72.8\u00b0.Instead of the expected product [{(\u03b7) cation . The Mn\u20138][BArCl4] was somewhat puzzling. Therefore, the reaction was repeated with an alternative halide abstracting agent Na[BArF4] (ArF = C6H3(CF3)2). From this reaction, besides the analogous product [{(\u03b75-C5H5)(OC)2Mn}2{\u03bc-B(tBu)}2Au][BArF4] 2), crystals of another product were also obtained, which was found to be [Au(PPh3)2][BArF4] (12) by X-ray crystallography. From this we proposed that the formation of [8][BArx4] (Arx = ArCl and ArF) proceeds via the expected cationic halide abstraction intermediate, followed by a ligand exchange, a route that has been reported for the analogous alkylidyne complexes ] in toluene led to formation of an inseparable mixture. However, in the presence of a stoichiometric amount of the halide abstracting agent Na[BArCl4], a mixture of 2 equiv. of 1 with [AgCl(PPh3)] led to formation of a mixture of colorless and orange crystals after being stored at \u201330 \u00b0C for 3\u20134 days. These crystals are extremely light-sensitive and thermally-unstable. Even in the strict argon atmosphere of a glovebox, black colloidal silver was observed inside and on the surface of crystals after being stored at room temperature. X-ray crystallographic studies revealed that this crystal mixture contained both the intended silver(i) product [{(\u03b75-C5H5)(OC)2Mn}2{\u03bc-B(tBu)}2Ag][BArCl4] ([9][BArCl4]) and the by-product [Ag(PPh3)3][BArCl4] was found to be possible in the presence of a non-coordinating counterion. The reaction of 1 and Ag[BArCl4] led to clean formation of [9][BArCl4] in 90% yield of [9]+ were found in both coplanar and staggered geometries from different crystal samples. This suggests that the energy difference between these two geometries is insignificant, which has been confirmed by computations (vide infra). In the staggered form, the angle between the two Ag\u2013B\u2013Mn planes is 67.8(8)\u00b0.The solid-state structures of [1 with [CuCl(PPh3)] and sodium tetraarylborate Na[BArCl4] in toluene or fluorobenzene were less selective compared to those involving silver(i) complexes. In both solvents, small yields of crystals were isolated. X-ray crystallography revealed the solid state structure of these crystals to be [11][BArCl4], where [11]+ consists of two metal\u2013borylene-coordinated copper moieties connected by a [MnH(\u03b75-C5H5)(CO)2] fragment, which makes it a diamagnetic compound (CO)3] was added to the reaction of 1 with [CuCl(PPh3)] and Na[BArCl4] in toluene. This reaction afforded low yields of orange crystals, which was confirmed to be the originally-intended product [{(\u03b75-C5H5)(OC)2Mn}2{\u03bc-B(tBu)}2Cu][BArCl4] ([10][BArCl4]) by X-ray crystallography. Crystals of 10 and 11 are sparingly soluble in benzene and toluene. Although moderately soluble in dichloromethane, the solutions decompose within a minute at room temperature. Due to this extreme instability and low yields of the products, no useful NMR data could be obtained. Subsequently both [10][BArCl4] and [11][BArCl4] could only be characterized by X-ray crystallography \u00c5), [10]+ (1.850(6) \u00c5 and 1.857(5) \u00c5) and [11]+ (1.865(4) \u00c5) are only slightly longer than that of the terminal borylene complex 1 (1.810(9) \u00c5) and much shorter than a Mn\u2013B single bond.8]+\u2013[11]+ range from 156.4\u00b0 to 162.7\u00b0 (cf. in 1: 174.3\u00b0). These structural features are in contrast to all other homo- and hetero-dinuclear bridging borylenes containing the same [MnBR] fragment, in which the Mn\u2013B distances are considerably longer (1.91\u20132.02 \u00c5) and the Mn\u2013B\u2013C angles are significantly more acute (135.2\u2013145.8\u00b0).5-C5H5)(OC)2Mn(\u03bc-BtBu)] moiety, (i.e. the borylene moiety is more semi-bridging), which is consistent with the 11B NMR data. Furthermore, The Mn\u2013M distances of [9]+ (M = Ag) and [10]+ (M = Cu) are 2.683(1) \u00c5 and 2.447(1) \u00c5 respectively (cf. Mn\u2013Au 2.619(1) \u00c5 in [8]+), consistent with the trend of the atomic radii of Au, Ag and Cu.8]+, [9]+, and [10]+ are listed in 2Au(\u03bc-CC6H4Me-4)2(CO)4(\u03b7-C5H5)2]+] (62\u00b0).41The important bond distances angles of the cations +\u2013[10]+ corresponded to the observed non-planar geometry of the M(MnB)2 cores. The optimization and subsequent harmonic frequency calculations of the coplanar Ci structure observed for [9]+ showed it to be a transition state on the energy hypersurface. The energy difference between the co-planar and staggered form is very small . This is presumably due to the closed-shell d10 centre of Ag+, the geometry of which is not dictated by crystal field effects.To gain more insight into the bonding of complexes ns orbital of coinage metal, which leads to its smaller overlap with the \u03c0-type orbital of the borylene unit. For the backbonding, the coinage metal delivers electrons from its (n \u2013 1)d orbital to the empty \u03c0* orbital of borylene. In the case of copper the 3s,p and 3d orbitals have similar size, thus the 3d \u2192 \u03c0* interaction is weakened by repulsion with the 3s,p orbitals, which have symmetries incompatible with this interaction. When changing to Ag, the orthogonality of the 4d and 3d orbitals causes an increase of the radii of the 4d orbitals. The spatial separation of the 4s,p and the 4d orbitals is larger and thus the repulsion is smaller, while the orbital overlap is better. In the case of Au, the relativistic contraction additionally amplifies this effect and therefore results in the strongest backdonation.43No localized two-center, two-electron bonds between the central metal and the borylene ligands have been found using the Natural Bond Orbital (NBO) method. However, strong donor\u2013acceptor interactions have been found using the second-order perturbation analysis. The dominating interactions are the donations from the Mn\u2013B bonds to a low-populated coinage-metal-centred orbital. The backdonation to the borylene from the coinage metals is an order of magnitude weaker. Computations carried out employing gross populations of fragment orbitals have shown that charge donation from the borylene ligands to the coinage metals decreases from Cu to Au , whereas the backdonation grows from Cu to Au . The first of these effects can be ascribed to the increasing size of the PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019B bond and large Mn\u2013B\u2013R angle). In turn, the semibridging geometry and the slight preference of the staggered geometry may play a role in maximising backdonation from the coinage metal cations to the borylene in a similar way to the bisphosphine gold(i) system [(DPCb)AuCO]+ (DPCb = o-carborane diphosphines) reported by Amgoune, Bourissou and co-workers. It has been observed that ligands in Au(i) complexes that deviate from linear geometry cause the energies of the 5d orbitals to rise, which strengthens the backdonation from the Au(i) center to the ligand (CO).+, alkylidynes also exhibit larger M\u2013\u03bcC\u2013R angles and shorter M\u2013\u03bcC bonds than bridging alkylidynes with most other metals, where the \u03bcCR vector is closer to perpendicular to the M\u2013Au bondThe weak backdonation from the coinage metal cations to borylene may be responsible for its semibridging geometry and preserved strong borylene character scale\" fill=\"currentColor\" stroke=\"none\">B \u2192 \u03c3-Cu+) and the minor backdonation from the copper HOMO into the LUMO of the borylene moieties scale\" fill=\"currentColor\" stroke=\"none\">B). This is in contrast to the semi-bridging aminoborylene complexes [LnM{\u03bc-BN(SiMe3)2}M\u2032(CO)5] (LnM = (\u03b75-C5Me5)(OC)Ir, M\u2032 = Cr, W) and +\u2013[10]+. Despite the bonding interactions between the Mn and M evident in the structural data, no bond critical points (BCP) between the manganese and the coinage metal cations, nor any ring critical points, could be found for the three-membered M{MnB} rings, similar to previously reported multi- and dinuclear borylene-bridged complexes.2,8]+\u2013[10]+ are strongly bent towards the manganese atom, suggesting that the interactions between the boron and coinage-metal atoms are not covalent but rather \u03c3-donation from \u03c0-orbitals of the manganese borylene unit. The valence shell charge concentration (VSCC) close to the boron atom is extended over both metal\u2013boron BCPs coordinated coinage metal complexes have been presented and their bonding has been thoroughly examined using DFT calculations, in which the terminal metal\u2013borylene moieties are treated as a ligand as a whole for the first time.Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "An unexpected N-heteroacene with a slipped two-dimensional ladder-like packing feature shows a hole mobility up to 0.3 cm2 V\u20131 s\u20131, while theoretical calculations suggest that this compound possesses potential well-balanced ambipolar charge-transport characteristics. b]phenazine) (2BPP) consists of two identical backbones piperazin-3-onephenazine), which are fused together through a C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C double bond and two intramolecular H-bonds. A study on the charge carrier transport indicates that a 2BPP single crystal has a hole mobility up to 0.3 cm2 V\u20131 s\u20131, while theoretical calculations suggest that this compound might possess potential well-balanced ambipolar charge-transport characteristics.An unexpected \u201ckinked\u201d N-heteroacene with a slipped two-dimensional ladder-like packing feature is produced from the conventional condensation reaction. The as-obtained compound bipiperazin-3-one[2,3- To date, most of the explored N-heteroacenes are linearly-fused systems, which can be prepared through the condensation reaction between ortho-diamine based acenes and ortho-diketone, ortho-dihydroxy, ortho-dicyano, or ortho-dihalogen substituted acenes and N-heteroacene derivatives have attracted a lot of attention, being widely investigated for applications in organic field-effect transistors (OFETs), organic resistance memories (ORMs), organic light-emitting diodes (OLEDs) and organic photovoltaics (OPVs).d acenes .6 HoweveTHNQ (shown in BTDP) and hexaketocyclohexane (HKCH), in which we believe that the steric effect of the TIPS group plays a crucial role.HKCH.BTDP and HKCH. In this situation, we might miss some important unknown N-heteroacene products. Thus, we reinvestigated this type of reaction and discovered a meaningfully \u201ckinked\u201d compound bipiperazin-3-onephenazine) (2BPP). To the best of our knowledge, this is the first report of the generation of a meaningfully \u201ckinked\u201d N-heteroacene through the conventional substitution reaction.In our previous study, we have already reported that linearly-fused N-heteroacene shown in can be p2BPP might undergo an unstable intermediate (UI) step, in which the diketone could be eliminated instead of participating in the further condensation reaction with the amine groups. The as-formed intermediate could be converted into the final product 2BPP through a rotation and radical pathway (initiated by light). The possible mechanism has been provided in the ESI (Scheme S12BPP was obtained in a low yield of 2.3% and was fully characterized by high-resolution mass (HR-MS) spectrometry, 1H NMR and 13C NMR spectroscopy, and single-crystal analysis. It is noteworthy that two 5,12-bis(TIPS)piperazin-3-onephenazine moieties are linked together by one double bond, which makes 2BPP fully \u03c0-conjugated along the backbone. In addition, the expected H-bonds along both sides were assumed to stabilize the large heteroacene system.As shown in Scheme S1. Note th14206812BPP suitable for single-crystal X-ray diffraction analysis were obtained by diffusing the poor solvent acetonitrile into a toluene solution. 2BPP crystallizes in a triclinic unit cell, space group P1(2). The molecular structure of 2BPP is shown in PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond (1.35 \u00c5), suggesting that the two piperazin-3-onephenazine, BPP) moieties are connected by a C2 PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C2\u2032 bond. The distance between H1 and O1 or H1\u2032 and O1\u2032 is 1.95 \u00c5, indicating the existence of an intramolecular H-bond. Clearly, the formation of the intramolecular H-bonds with a six-member ring configuration is helpful for stabilizing the planar molecular shape and supports the electron delocalization. In fact, the as-prepared molecule possesses a good planarity from the side view scale\" fill=\"currentColor\" stroke=\"none\">C2\u2032 bond is \u223c2.42\u00b0. As shown in 2BPP exhibits a slipped 2-D \u03c0-stacking motif, similar to that observed for some soluble TIPS-pentacene derivatives. The interplanar distance in 2BPP is \u223c3.27 \u00c5 along the b axis, less than that of the typical distance for van der Waals interactions, while the centre-to-centre distance between two adjacent molecules is \u223c14.65 \u00c5. The slipping angle of two adjacent \u03c0-conjugated BPPs is \u223c45\u00b0, contributing to a significant molecular overlap and ensuring the strong \u03c0\u2013\u03c0 interaction. While viewing along the a axis, the distance between two adjacent molecules is \u223c3.33 \u00c5 and in this stacking mode, the characterized centre-to-centre distance is about 17.61 \u00c5 with a much smaller slipping angle (\u223c30\u00b0) between two interactional individual BPPs leading to poorer electronic coupling. 2BPP molecules interact with each other to form two-layer BPP units. The face-to-face stacking mode results in overlapping between the second layer units and the first layer BPPs can be determined to be 1.71 eV. The strong absorption from 650 nm to 725 nm is similar to that of other hydro-azaacenes, and probably comes from the intramolecular charge transfer. The electrochemical properties of 2BPP were investigated by cyclic voltammetry of \u20131.29 V, which determines the LUMO level of 2BPP to be \u20133.51 eV. The HOMO level was calculated to be \u20135.22 eV from the LUMO level and Eg. The geometry structure of 2BPP was optimized by DFT calculations (B3LYP/6-31G*),2BPP. In both the experimental and theoretical calculated results, 2BPP has relatively high HOMO levels and a moderate band gap, which indicates 2BPP could be used as a promising suitable semiconducting material.etry CV, . Unlike 2BPP were grown on an octadecyltrichlorosilane (OTS)-treated SiO2/Si substrate by a drop-casting method. OTS was used to form a siloxane self-assembled monolayer (SAM) on the SiO2 layer, which could promote and facilitate molecular self-organization during the growth of ribbons. The optical and AFM images in 2BPP ribbons display a rhombic shape with unambiguous boundaries, indicating the high quality of the ribbons. The as-obtained micro/nanoribbons are several to tens of micrometers in width, and several tens to hundreds of micrometers in length. Three sharp and strong diffraction peaks at 2\u03b8 = 5.19, 10.32, and 15.45 degrees are observed in the out of plane X-ray diffraction patterns (XRD) (c-axis (17.27 \u00c5), meaning that the molecules stand up along the c-axis with an angle of 73\u00b0 on the substrate. The major driving force for the self-assembly of the \u03c0-conjugated material is the \u03c0\u2013\u03c0 interaction from the adjacent molecules, which causes the superior growth direction along either the a-axis or b-axis. The measured angle of the lamellar crystal is 100.4\u00b0, which is consistent with the 100.2\u00b0 dihedral angle between the (100) and (010) planes in the crystal structure. From the crystal morphology, it can be clearly seen that the primary growth direction is along the \u03c0-stacking direction (b-axis). In this direction, the slipping angle between the adjacent overlapped 2BPP units and the close contact between the adjacent \u03c0-scaffolds contributes to the significant molecular overlap and ensures the effective charge transport in the \u03c0\u2013\u03c0 stacking , which cstacking . In addi\u03bc) was extracted from the saturation regime, and the mobility was calculated by the linear fitting of (IDS)1/2vs. VG curves. Nearly 50 transistors have been measured and all the devices exhibited good gate modulation. According to the transfer characteristics, the mobility was probed in the range of 0.008\u20130.3 cm2 V\u20131 s\u20131 along the b-axis, the best hole mobility could reach 0.3 cm2 V\u20131 s\u20131 with a threshold voltage (VT) of \u201310 to \u201325 V and on-to-off current ratios (Ion/Ioff) > 105.The crystalline ribbons grown on the substrates were fabricated as top-contact, bottom-gate configuration transistors. The gold source and drain electrodes were deposited with copper masks covering the selected ribbons. With this simple method, 50 nm of Au was deposited on the covered substrate; and then, the masks were removed from the ribbon surface and the ribbon devices with a length of about 20 \u03bcm were manufactured. The electrical characteristics of the devices were measured at ambient conditions. The transfer and output curves are displayed in 2BPP, Marcus electron transfer theory and an incoherent Brownian motion model have been employed to calculate the hole and electron mobilities which has a transfer integral of ca. 60 meV. Although the material has been predicted to possess a well-balanced ambipolar property, electron transport was not apparently observed and the measured mobility was relatively low. However, we believe that with proper modification of this specific heterocyclic molecule or tuning of the device fabrication conditions, ambipolarity and good transport character could be realized.To understand the structure\u2013property relationship of s see ESI based on2BPP), in which two BPP units are fused together through a C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C double bond and two H-bonds. Single crystal X-ray studies have demonstrated that 2BPP is a near coplanar molecule with close intermolecular interactions. For the double-layer structure, the face-to-face stacking mode results in an overlap between the second layer BPP unit and the first layer unit of the second BPP molecule, forming a ladder-like corrugate. The electronic structure calculations suggest the unique large heterocyclic molecule could exhibit a good intrinsic ambipolar charge transport property. Experimentally, single-crystal FETs with charge carrier mobilities of 0.3 cm2 V\u20131 s\u20131 and current on/off ratios of 105 have been realized. Further studies on the mechanism of this unusual compound as well as the hydrogen bonding supramolecular synthons would provide more insights to design and prepare novel large conjugated heteroacenes with unique properties.In conclusion, we have presented the synthesis and full characterization of an unexpected \u201ckinked\u201d N-heteroacene (Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "The facile non-radiative decay for gold(iii) complexes is due to the thermally accessible 3LLCT, but not the usually assumed 3dd excited state. iii) arylacetylide complexes, , with different extents of \u03c0-conjugation at the doubly C-deprotonated [C^N^C] ligand via replacement of one of the phenyl moieties in the non-conjugated CH^N^C ligand (1) by a naphthalenyl (2) or a fluorenyl moiety . Conforming to the conventional wisdom that extended \u03c0-conjugation imposes rigidity on the structure of the 3IL(\u03c0\u03c0*(C^N^C)) excited state , the calculated Huang\u2013Rhys factors for the 3IL \u2192 S0 transition follow the order: 1 > 2 > 3-exo \u223c 3-endo, which corroborates qualitatively the experimental non-radiative decay rate constants, knr: 1 \u226b 2 > 3-exo, but not 3-endo. Density Functional Theory (DFT) calculations revealed that there is an additional triplet excited state minimum of 3LLCT character scale\" fill=\"currentColor\" stroke=\"none\">CPh-4-OMe) \u2192 \u03c0*(C^N^C)]) for complexes 1 and 3-endo. This 3LLCT excited state, possessing a large out-of-plane torsional motion between the planes of the C^N^C and arylacetylide ligands, has a double minimum anharmonic potential energy surface along this torsional coordinate which leads to enhanced Franck\u2013Condon overlap between the 3LLCT excited state and the ground state. Together with the larger spin\u2013orbit coupling (SOC) and solvent reorganization energy for the 3LLCT \u2192 S0 transition compared with those for the 3IL \u2192 S0 transition, the calculated knr values for the 3LLCT \u2192 S0 transition are more than 690- and 1500-fold greater than the corresponding 3IL \u2192 S0 transition for complexes 1 and 3-endo respectively. Importantly, when this 3LLCT \u2192 S0 decay channel is taken into consideration, the non-radiative decay rate constant knr could be reproduced quantitatively and in the order of: 1 \u226b 3-endo, 2 > 3-exo. This challenges the common view that the facile non-radiative decay rate of transition metal complexes is due to the presence of a low-lying metal-centred 3dd or 3LMCT excited state . By analysis of the relative order of MOs of the chromophoric [C^N^C] cyclometalated and arylacetylide ligands, one may discern why complexes 1 and 3-endo have a low-lying 3LLCT excited state while 3-exo does not.We have performed theoretical analyses of the photophysical properties of a series of cyclometalated gold( One of the impediments to the progress of photoluminescence of gold(iii) complexes is the high electrophilicity of the gold(iii) ion and the presence of a low-lying Au(5d\u03c3*) orbital. In effect, the deactivating ligand-to-metal charge transfer (LMCT) and/or dd ligand-field excited states become close in energy to the emitting excited state, leading to efficient luminescence quenching in gold(iii) complexes.N-heterocyclic carbenes (NHC), to the gold(iii) cyclometalated complexes; these complexes were reported to be weakly emissive in solution (\u03c6 < 0.01) at room temperature.Gold(-lying Aud\u03c3* orbitiii) cyclometalated complexes with different extents of \u03c0-conjugation at the [C^N^C] ligand).iii) complexes with a fluorenyl moiety incorporated into the doubly deprotonated [C^N^C] ligand.iii) cyclometalated complexes in solution reach 0.58, and the corresponding non-radiative decay rate constant (knr) falls to 1.74 \u00d7 103 s\u20131 is replaced by a fluorenyl moiety.ii) complexes when compared with the non-conjugated CH^N^C analogue;To enhance the emission quantum yield, the structural distortion between the emitting excited state and the ground state must be minimized, thereby decreasing the non-radiative decay rate.endo\u201d in the gold(iii) pincer complex and a nearly 40-fold increase in the non-radiative decay rate constant (knr \u223c 6.76 \u00d7 104 s\u20131) when compared with its \u201cexo\u201d analogue (endo\u201d complex). This means that, even with a seemingly suitable cyclometalated ligand , the phosphorescence efficiency of gold(iii) complexes is not necessarily high. Thus, for effective design of functional luminescent molecules, it is important to understand the effect of \u03c0-conjugation in the C-deprotonated cyclometalated [C^N^C] ligand on the excited state properties of these luminescent gold(iii) complexes.Interestingly, when the fluorenyl moiety is disposed in such a fashion that the long alkyl chains are \u201ciii) complexes with different [C^N^C] cyclometalated ligand scaffolds (H^N^C (1) and the \u03c0-conjugated Cnp^N^C (2) and Cfl^N^C (3-exo and 3-endo); complexes 2 and 3-exo (and 3-endo) have one of the phenyl moieties of 1 replaced by a naphthalenyl (np) or a fluorenyl (fl) moiety respectively. The ancillary ligand, p-methoxyphenyl acetylide scale\" fill=\"currentColor\" stroke=\"none\">CPh-4-OMe]\u2013) is kept the same for all four complexes. A detailed list of definitions and abbreviations is provided in the appendix.In this work, we have performed a detailed theoretical analysis of four gold is in equilibrium with the ground state electron density of the solute, while the solvent nuclear polarization (the \u201cslow\u201d component) remains equilibrated with the excited state electron density of the solute. For this reason, we have employed the state-specific (SS) approach to account for the dynamical solvent effect. Within the SS scheme, rather than using the ground state electronic density as in LR-TDDFT and \u0394SCF, the electronic density of the emitting excited state is used to compute the ground state energy.ESSem) is given by:EESEQ(QES0) is the energy of the excited state (ES) with equilibrium solvation at the optimized excited state geometry (QES0), and EGSNEQ(QES0) is the energy of the ground state (GS) with non-equilibrium solvation at (QES0) and time-dependent DFT (TDDFT) are the commonly used tools to study the ground state and excited state properties of medium- to large-sized molecules. In the literature, computation of emission energies in solutions is performed using either linear response TDDFT (LR-TDDFT) or the \u0394SCF method. For both types of calculations, both the excited state of interest and the ground state are calculated with equilibrium (EQ) solvation. However, in an emission process, the ground state should be treated with solvent polarization in the non-equilibrium (NEQ) regimet (QES0) .11\u03bbs), which is the ground state energy difference calculated with non-equilibrium solvation (EGSNEQ(QES0)) and with equilibrium solvation (EESEQ(QES0)) at the optimized excited state geometry (QES0) :112\u03bbs \u03bbSSV) is given by:EGSEQ(QGS0) is the energy of the ground state computed with equilibrium solvation at the optimized ground state geometry QGS0 to the S0 state vibrational manifolds is given by the sum of individual radiative decay rate constants (denoted kr\u03b1(\u03bd\u0303)), each corresponding to a single vibronic transition, T1\u03b1(\u03c5\u2032 = 0) \u2192 S0(\u03c5\u2032\u2032), with photon energy, \u03bd\u0303, and vibrational quantum number for the T1 and S0 states, \u03c5\u2032 and \u03c5\u2032\u2032, respectively:The total radiative decay rate constant from the vibrational ground state of the emitting T\u03b7 is the solvent refractive index, \u03bd\u0303 is the triplet emission energy (in cm\u20131), and MT\u03b1(Q) is the transition dipole moment of the T1\u03b1 \u2192 S0 transition (in ea0), and the prefactor 8\u03c02/3\u03b50\u0127 = 2.0261 \u00d7 10\u20136.The radiative decay rate constant for the single vibronic transition can be calculated from the Einstein coefficient of spontaneous emission:12i.e., MT\u03b1(Q) \u2248 MT\u03b1(QT10) with QT10 being the optimized T1 excited state geometry) and combining eqn (4) and (5), the total radiative decay rate constant, kr\u03b1, is given by:13\u03c7\u03c5\u2032\u2032 and \u03c7\u03c5\u2032 are the vibrational wavefunctions of the S0 and the T1 states respectively.By invoking the Condon approximation can be accounted for by the Franck\u2013Condon factors ((|\u222b\u03c7*\u03c5\u2032\u2032\u03c7\u03c5\u2032dQ|2). In general, one may approximate the last term in the summation as:13I(\u03bd\u0303) being the emission intensity at \u03bd\u0303 (corrected to the number of photons emitted per unit wavenumber). The emission intensity can be obtained either from experiment or by computational simulation. The total radiative decay rate constant for the T1\u03b1 \u2192 S0 transition may then be written as:Unless the emission spectrum is sharply peaked, as in an atomic emission spectrum, one should not take the integral in eqn (6) as unity and replace the summation in eqn (6) by the emission peak maximum, MT\u03b1(QT10) could be obtained by first-order perturbation interactions between the T1\u03b1-spin sub-state and the singlet excited state via spin\u2013orbit coupling (SOC):12Mm,jS is the j-axis projection of the Sm \u2192 S0 transition dipole moment, E(T1) and E(Sm) are the energies of the T1 and the mth singlet (Sm) excited states, respectively, and T1\u03b1|HSOC|Sm are the SOC matrix elements between the T1\u03b1-spin sub-state and the Sm excited state.The transition dipole moment 1\u03b1-spin sub-states is less than 5 cm\u20131, all sub-states should be equally populated at room temperature. Therefore, the average radiative decay rate constant kr is given by:As the energy splitting between the three Tknr) of the T1 \u2192 S0 transition can be estimated by application of the Fermi's Golden Rule expression, assuming that both electronic states are harmonic:14In the limit of the Franck\u2013Condon approximation in the non-adiabatic regime, the non-radiative decay rate constant intraligand vibrational modes (\u0127\u03c9M > 1000 cm\u20131), typically corresponding to the aromatic CC/CN stretching modes (\u0127\u03c9M \u223c 1200\u20131500 cm\u20131) and C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C stretching modes scale\" fill=\"currentColor\" stroke=\"none\">CC \u223c 2200\u20132300 cm\u20131) if the acetylide ligand is involved in the complex; \u03bbS is the solvent reorganization energy and may be obtained from eqn (2); \u0394E is given byE00 being the zero-point energy difference between the T1 and S0 states and \u03bblf being the reorganization energy contributed by the low-frequency (lf) modes of the complex . Assuming that all the normal modes are harmonic oscillators,Sj, mj, and \u0394Qj are the Huang\u2013Rhys factor, the reduced mass, and the equilibrium displacement of the jth normal mode \u03c9j, respectively; SM and nM are the Huang\u2013Rhys factor and the number of quanta of the effective high frequency mode \u0127\u03c9M (corrected to the nearest integer), respectively:This expression can be applied when \u03bbFCv, could be estimated as:\u03c9j.Under the harmonic oscillator approximation, the intramolecular reorganization energy, 2Cl2; \u03b7 = 1.424).0) and the lowest triplet excited state (T1) were respectively carried out using restricted and unrestricted density functional theory formalisms without symmetry constraints. Frequency calculations were performed on the optimized structures to ensure that they were minimum energy structures by the absence of imaginary frequency . Stability calculations were also performed for all the optimized structures to ensure that all the wavefunctions obtained were stable.In this work, the hybrid density functional, PBE0,E(Sm), the associated transition dipole moment of the Sm \u2192 S0 transition Mm,jS , and the coefficients necessary to compute the SOC matrix elements of Au in the MO relevant to the coupling excited states and the corresponding CI coefficients), were all obtained from a state-specific approach using \u201cExternalIteration\u201d implemented in G09.Vertical transition energies were computed using the linear response approximation for absorption, but the state specific approach for emission.Sj (using eqn (12c)) for the normal mode j\u03c9 may be obtained by performing a Franck\u2013Condon calculation implemented in G09 via \u201cfreq = fc\u201d and \u201cprtmat = 2\u201d. The simulated emission spectrum allows one to calculate the Franck\u2013Condon factor-weighted emission energy \u03bd\u0303fcf (using eqn (7)). The high-frequency normal modes (1000 < \u0127\u03c9m \u2264 1800 cm\u20131) can be characterized by a mean frequency \u03c9M and an effective electron-phonon coupling strength (or Huang-Rhys factor) SM:21The Huang\u2013Rhys factor Further computational details can be found in the ESI.1, 3-exo, and 3-endo are in good agreement with the X-ray crystallography data (<0.05 \u00c5 and 8.5\u00b0) except for the dihedral angle between the planes of the [C^N^C] ligand and the phenyl ring of the acetylide ligand (\u03b4); calculations revealed a nearly coplanar geometry (\u03b4 \u223c 5.7\u00b0 and \u20130.27\u00b0 for 1 and 3-exo respectively) whereas experimentally determined \u03b4 values are 66.1\u00b0 and 54\u00b0 respectively.3-endo (\u03b4 \u223c 130\u00b0), the corresponding X-ray data is only \u223c59\u00b0 (see ESI3-endo). In addition, the Au\u2013C(acetylide) distance for 1 was calculated to be 1.950 \u00c5 while the corresponding distance from the crystallography data is 2.009 \u00c5.H^N^C)AuIIIC PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CPh-4-Y] (Y is a substituent) complexes are in the range of 1.945\u20131.980 \u00c5;In general, the optimized ground state structures of 0 geometries of the four complexes studied herein. A full list of the TDDFT results can be found in the ESI.1 is 1LLCT in nature (LLCT = ligand-to-ligand charge transfer), with a calculated vertical excitation energy at \u03bb = 408 nm (f = 0.23).53-exo, the first singlet excited state (S1) is a 1LLCT excited state, derived mainly from the HOMO \u2192 LUMO transition, 1[\u03c0 scale\" fill=\"currentColor\" stroke=\"none\">CPh-4-OMe) \u2192 \u03c0*(C^N^C)] in character (>80%); this 1IL excited state is derived from the H \u2013 1 \u2192 LUMO transition and is a 1\u03c0\u03c0*(C^N^C) excited state. The difference in the nature of the S1 excited state among the four complexes can be rationalized as follows: upon increasing the \u03c0-conjugation along the series 1, 2, 3-endo, and 3-exo, H \u2013 1 is destabilized and the MO splitting (\u0394\u03b5) between HOMO and H \u2013 1 decreases from 0.62 eV (1) to 0.26 eV (3-endo) and 0.20 eV (3-exo), , . This de Table S9. As a reiii) complexes are listed in The experimental photophysical data regarding the emissions of the four gold.As depicted in 2 and 3-exo, only one triplet excited state, 3\u03c0\u03c0*(C^N^C) IL excited state, was found. On the other hand, two triplet excited state minima, one 3IL in character and the other 3LLCT scale\" fill=\"currentColor\" stroke=\"none\">CPh-4-OMe) \u2192 \u03c0*(C^N^C)]), were found for both 1 and 3-endo. The electron difference density maps (eddms) for the calculated triplet excited states, together with the relative energy splitting between the 3IL and 3LLCT excited states for complexes 1 and 3-endo, are presented in To understand the emission properties of the four complexes depicted in E00), vertical emission energies , and radiative decay rate constants of the optimized T1 excited states of the four gold(iii) complexes studied herein. \u0394ESSem, , Franck\u2013(i)2, there is generally a close correlation between the experimental solution emission maxima (\u03bbmax) at room temperature and the calculated \u0394E00 of the 3IL excited states of the gold(iii) complexes in 1, 3-exo, and 3-endo, the emission maximum may correspond to the 0\u20130 transition of 3IL \u2192 S0. The experimental emission maximum of 2 is at a lower energy than that of 3-exo (ii) [C^N^C] cyclometalated complexes, the one with a naphthalene moiety at the [C^N^C] ligand displays a higher energy emission peak than the one with a fluorene unit .ii) [C^N^C] cyclometalated complexes: \u0394E00 of the gold(iii) complexes is in the order 1 > 2 > 3-endo \u223c 3-exo. This trend is a manifestation of the increase in \u03c0-conjugation at the [C^N^C] cyclometalated ligand when one goes from 1 to 2 to 3-endo and 3-exo. Increasing \u03c0-conjugation destabilizes the \u03c0(C^N^C) orbital, .With the exception of of 3-exo . For relsee also , thereby(ii)1 excited states of the four complexes. Although the kr values of the 3IL excited states are slightly underestimated by a factor of \u223c2.7\u20133.1, they are consistent with the experimental kr values except in the case of 2 complexes .2 at 298 K and 77 K originated from different excited states. However, no other triplet excited state minimum was found for complex 2 using the present DFT/TDDFT method. compare with 4. perature . However(iii)3IL excited states of the four gold(iii) complexes. As depicted in SM) are in the order: 1 > 2 > 3-exo \u223c 3-endo. This trend is in line with the S0 to T1 structural distortion of the following organic molecules in the order: benzene > naphthalene > carbazole (carbazole is isoelectronic to fluorene).3IL excited states of these four gold(iii) complexes are mainly localized on the phenyl, naphthalenyl, and fluorenyl moieties, respectively to the H \u2013 1 (HOMO for 3-exo and 3-endo), at their optimized T1 geometries, are 4.18 (1), 0.36 (2), 1.94 (3-exo), and 1.07 (3-endo), respectively. As SOC is mainly brought about by the gold(iii) ion, the larger the coefficient of Au(d) in the H \u2013 1/HOMO, the larger should be the SOC matrix element, 3IL|HSOC|S02. The Au(d) character in the H \u2013 1/HOMO of the gold(iii) complexes studied herein is related to the nature of the HOMO of the C-deprotonated moiety in the [C^N^C] ligand. For complex 2, the H \u2013 1 is mainly localized on the long molecular axis of the naphthalene fragment (np^N^C] ligand to have little interaction with the gold(iii) ion and therefore, the smallest cd in the H \u2013 1 orbital of 2. On the other hand, the corresponding orbital of complex 3-exo is along the short molecular axis of the fluorene fragment, thus the [Cfl^N^C] ligand could have a stronger interaction with the gold(iii) ion, and hence, a larger cd in the HOMOs of complexes 3-exo and 3-endo.Besides, the magnitude of the SOC matrix element between the fragment , thus reSM and the SOC between the T1 and S0 states are largest for 1, the calculated non-radiative decay rate constant knr for the 3IL \u2192 S0 transition is smaller than that of 3-exo, a result contrary to the order of experimental knr values; knr: 2 > 3-exo > 1; knr(expt): 1 \u226b 2 > 3-exo. This is because 1 has a much larger energy gap between the 3IL and S0 states than the other three gold(iii) complexes (knr (3IL \u2192 S0) of 1. Similarly, the calculated non-radiative decay rate constant for 3-endo is \u223c1.25 \u00d7 103 s\u20131, which is also smaller than that of 3-exo, and is inconsistent with the experimental data scale\" fill=\"currentColor\" stroke=\"none\">CPh-4-OMe) \u2192 \u03c0*(C^N^C)], excited state. This 3LLCT excited state displays a large amplitude motion along the dihedral angle between the [C^N^C] plane and the arylacetylide plane (\u03b4): from \u223c\u20134.132\u00b0 (S0) to \u201388.739\u00b0 (3LLCT) for 1 and from 130.381\u00b0 (S0) to 92.352\u00b0 (3LLCT) for 3-endo (see 0 and 3LLCT excited states for complexes 1 and 3-endo). Because of this large amplitude motion, we refrained from performing a Franck\u2013Condon calculation on the 3LLCT \u2192 S0 transition, as we have performed for that of the 3IL \u2192 S0. This is because, for the Franck\u2013Condon calculation implemented in G09, the normal modes are represented in Cartesian coordinates. Cartesian coordinates are inadequate to describe large amplitude motions, such as torsions, as this could lead to artificial bond breaking and bond forming at its extreme.3LLCT to the S0 state. Moreover, such fictitious bond breaking and bond forming will lead to a diffuse Duschinsky matrix, which could lead to an incorrect interpretation of the fast non-radiative decay rate constant due to a large Duschinsky effect.Although both the effective Huang\u2013Rhys factor omplexes , making compare and 5. Fas found . This tr PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C stretching normal mode is decoupled from the other normal modes, as reflected by the Duschinsky matrix elements of the 3LLCT \u2192 S0 transition; \u03c9 PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CC is the only normal mode that has the diagonal matrix element equal to 1. Therefore, we estimated the non-radiative decay rate constants of the 3LLCT excited state by replacing all the Huang\u2013Rhys factors (Sj) of the 3LLCT \u2192 S0 transition with those of the 3IL \u2192 S0 transition, but keeping the Huang\u2013Rhys factor of the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C stretching normal mode from a Franck\u2013Condon calculation of the 3LLCT \u2192 S0 transition. Such an assumption is based on the fact that both the 3LLCT and 3IL excited states of the gold(iii) complexes involve changes in electron density at the [C^N^C] ligand.Nevertheless, the C3LLCT excited states of 1 and 3-endo are noted: (a) The solvent reorganization energy (\u03bbS) of 3LLCT is much larger than that of 3IL. This is attributed to the dipole moment of the 3LLCT being much larger than that of 3IL and the ground states (see \u03bcT1) and \u03bcGS)). In the framework of the SS approach, solvent reorganization energy is proportional to the square of the difference in dipole moments between the T1 excited state and the S0 ground state, i.e., \u03bbs \u221d (\u03bcT1 \u2013 \u03bcGS)2.3LLCT and the S0 potential energy surfaces (PESs) at the equilibrium geometry of the 3LLCT excited state. Thus, fewer quanta of the high-frequency vibrational mode (nM) are needed (see eqn (12e)) and the activation energy ) is smaller as this energy term is inversely proportional to the solvent reorganization energy; (b) the square of the HSOC matrix element between the 3LLCT excited state and the S0 ground state is larger than that between the 3IL excited state and the ground state calculation of the 3LLCT \u2192 S0 transition. (We have used the Huang\u2013Rhys factor of the 3IL \u2192 S0 transition where there is no such large amplitude torsion.) We have undertaken a rigid scan along the torsional coordinate (\u03b4) for 1. \u03b4 for the ground state, 3IL excited state, and 3LLCT excited state of complex 1. The potential energy minimum is roughly harmonic for both the ground state and the 3IL excited state but anharmonic for 3LLCT excited state. As the 3LLCT excited state has a double minimum potential while the ground state is approximately harmonic, the Franck\u2013Condon factor (FCF) between 3LLCT and S0 is expected to be larger than that between the 3IL and S0 states, where both PESs are harmonic along the torsion coordinate \u03b4. This may be rationalized as illustrated in 3LLCT excited state) than that with a harmonic PES \u223c 1.6 \u00d7 107 s\u20131 and knr(expt) \u223c 5.9 \u00d7 107 s\u20131). In other words, the major deactivating channel for the emissive excited state of 1 is not 3dd or 3LMCT, as is usually ascribed to efficient non-radiative decay for luminescent transition metal complexes, but 3LLCT due to a large SOC, a large solvent reorganization energy, and the non-planar torsional motion between the [C^N^C] and arylacetylide ligands. For 3-endo, the 3LLCT excited state is calculated to be \u223c1400 cm\u20131above that of the 3IL state. Therefore, the re-estimated knr becomes \u223c1.5 \u00d7 104 s\u20131, which is in good agreement with the values derived from the experimental measurements in solutions at 298 K (knr(expt) \u223c6.8 \u00d7 104 s\u20131).If one supposes that the torsional motion increases the FCF of the 3LLCT excited state that contributes to the very fast non-radiative decay rate. The relative order of the 3LLCT and 3IL excited states would thus be important in determining the phosphorescence efficiency. In the present series of gold(iii) complexes, this relative order can be understood from the relative energies of the \u03c0(C^N^C) and \u03c0 scale\" fill=\"currentColor\" stroke=\"none\">CPh-4-OMe) MOs. As the LLCT excited state is a charge transfer excited state, while the IL excited state is localized, the singlet\u2013triplet splitting of LLCT excited states (E(1LLCT)\u2013E(3LLCT)) would be smaller than that of IL excited states (E(1IL)\u2013E(3IL)). In the case of 1, due to the large orbital energy difference (\u0394\u03b5) between the \u03c0(CH^N^C) and \u03c0 scale\" fill=\"currentColor\" stroke=\"none\">CPh-4-OMe) MOs MOs , the 1ILed state .3IL\u20133LLCT gap of 2 should fall between that of 1 and 3-endo, as deduced from the relative order of the \u03c0(Cnp^N^C) and \u03c0 scale\" fill=\"currentColor\" stroke=\"none\">CPh-4-OMe) MOs depicted in 3LLCT excited state was located in the course of LR-TDDFT optimization; subsequent SS-TDDFT calculation at this geometry showed that this 3LLCT excited state is lower-lying than the 3IL one. However, global hybrid density functionals, generally underestimate the energy of charge transfer excited states within the TDDFT framework. Thus, we performed UDFT optimization starting from these TDDFT-optimized structures (which have a stable wavefunction) to see if there is a 3LLCT excited state minimum. Unfortunately, UDFT optimization starting from the TDDFT-optimized 3LLCT excited state went back to the 3IL excited state. It is likely that this 3LLCT excited state is metastable and exhibits vibronic coupling with other close-lying excited states.Based on the above rationale, it is speculated that the iii) [C^N^C] cyclometalated complexes with different extents of \u03c0-conjugation. It is commonly prescribed that a rigid ligand in a transition metal complex can minimize structural distortion between the emitting triplet excited state and the ground state, thereby decreasing the non-radiative decay rate. Franck\u2013Condon analyses on the 3\u03c0\u03c0*(C^N^C) IL \u2192 S0 transitions of the four gold(iii) complexes confirmed that an increase in \u03c0-conjugation at the [C^N^C] cyclometalated ligand results in a more rigid transition metal complex, as reflected by the effective Huang\u2013Rhys factor, SM: 1 > 2 > 3-exo and 3-endo. Although this trend correlates with the experimentally determined non-radiative rate constants, 1 \u226b 2 > 3-exo, the calculated knr of the 3IL \u2192 S0 transition is inconsistent with the experimental data if one also takes into consideration the 3IL\u2013S0 energy gap. DFT/TDDFT calculations reveal that there is an additional triplet excited state minimum, 3[\u03c0 scale\" fill=\"currentColor\" stroke=\"none\">CPh-4-OMe) \u2192 \u03c0*(C^N^C)] LLCT, for complexes 1 and 3-endo, but not for 3-exo. It was found that the non-radiative decay rate constant for this 3LLCT \u2192 S0 transition exceeds 107 s\u20131, which is more than three orders of magnitude faster than the knr for the 3IL \u2192 S0 transition. More importantly, if the relative splitting between the 3LLCT and 3IL excited states was included in estimating the knr of complexes 1 and 3-endo, the calculated and experimental knr are in quantitative agreement. Based on the analysis of the relative order of \u03c0(C^N^C) and \u03c0 scale\" fill=\"currentColor\" stroke=\"none\">CPh-4-OMe) MOs, one could rationalize why complexes 1 and 3-endo, but not 3-exo, have low-lying 3LLCT excited states. Our present analysis highlights the importance of the relative order of the frontier MOs of the coordinating ligands in multi-chromophoric transition metal complexes in designing strongly luminescent transition metal complexes. It also challenges the presumption that the low phosphorescence efficiency of transition metal complexes is due to the close proximity of the dd ligand-field state to the emitting triplet excited state.We have carried out a detailed theoretical study on four gold(Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "Aziridine aldehydes participate in a multicomponent reaction with \u03b1-amino amides and isocyanides to generate reactive imidoanhydride intermediates. Aziridine aldehyde dimers, peptides, and isocyanides participate in a multicomponent reaction to yield peptide macrocycles. We have investigated the selectivity and kinetics of this process and performed a detailed analysis of its chemoselectivity. While the reactants encompass all of the elements of the traditional Ugi four-component condensation, there is a significant deviation from the previously proposed mechanism. Our results provide evidence for an imidoanhydride pathway in peptide macrocyclization and lend justification for the diastereoselectivity and high effective molarity observed in the reaction. The vast majority of chemical processes rely on polar reactions between nucleophilic (Nu) and electrophilic (E) functional groups.91 . This pathway differs substantially from conventional Ugi MCR reactivity. We present evidence that implicates imidoanhydrides in aziridine aldehyde-driven macrocyclization of peptides. Our study underscores the notion that kinetically amphoteric molecules provide fertile grounds towards the deployment of underexplored intermediates and their application in the synthesis of valuable products.In this paper, we evaluate multicomponent reactivity between isocyanides, aziridine aldehydes, and linear peptides towards peptide macrocycles, an increasingly important class of molecules.Since 2010, we have been investigating the utility of aziridine aldehydes in the synthesis of chiral molecules.429, suggested that the rate-determining step was the formation of open dimer 4 (6 (the iminium ion produced from compounds 4 and 5), is a concerted asynchronous process with carboxylate group engagement that takes place at the same time as the isocyanide addition, directing the face of isocyanide attack to form mixed anhydride 8 LY(tBu)F in a TFE/DCM (1\u2009:\u20091) mixture.13C-labelled PS(tBu)LY(tBu)F precursor 10 reacted with excellent selectivity for macrocycle 12 as the parent sequence, which contained all hydrocarbon side chains. During the course of these studies, two products were identified in addition to the aziridine amide-bearing macrocycle (B). These were assigned to a bridged-amidine (C) and an amidine (D). Bridged-amidine products (C) failed to react with several different nucleophiles, confirming that no aziridine ring was present in the structure, while amidine products (D) were isolated after telescopic ring-opening with thioacids and desulfurization.I* has also been isolated in select instances and PH\u03b1 resonances were missing. We identified three new signals were found around 45 and 20 ppm for the methine and methylene positions, respectively. In contrast, the 13C and 1H\u201313C HSQC NMR spectra of 18C were devoid of these resonances, ruling out the possibility that 18C still contained an aziridine unit.The structure of the bridged-amidine products was assigned by a combination of 1D and 2D NMR. In the signals instead R see ESI, the conne group . The 13CC\u03b1 was designated as a quaternary center in 18C as no corresponding PH\u03b1 resonance could be observed in either the 1H or 1H\u201313C HSQC NMR. Another diagnostic difference in the 1H\u201313C HMBC NMR was the fact that the F PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019OC and P PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019OC chemical shifts were observed at 173 ppm and 169 ppm, respectively. The F PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019OC chemical shift was notably more upfield than a typical carbonyl of an aziridine amide (>180 ppm),18C.P15N, 1H\u201315N HSQC, and 1H\u201315N HMBC NMR . An 15N-labelled bridged-amidine peptide, 25C, was isolated and studied by 15N NMR. Gratifyingly, a 15N signal at 103 ppm was observed for the labelled compound 25C in DMSO-d6 with 0.1% formic acid , and too far upfield to be consistent with an amide (110\u2013130 ppm), but was in good agreement with reported chemical shifts for protonated amidines. PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019NC was also in line with the literature reported chemical shift of amidine carbons.49The identity of the nitrogen atoms was confirmed by R see ESI. In all mic acid . By 1H\u201311H\u201313C HMBC NMR . Using a similar set of NMR methods as those used for the structure elucidation of 18C and 25C scale\" fill=\"currentColor\" stroke=\"none\">O2C.To isolate the non-bridged amidine products, we employed the telescopic methodC see ESI, we dedu amidine . The 1H\u201322D and other amidine derivatives, we noticed that two peaks corresponding to the amidine product could be resolved, yet after purification they had a tendency to coalesce into one product by NMR. The PH\u03b1 atom is allylic with respect to the amidine moiety and thus potentially labile and prone to epimerization to a more thermodynamically stable epimer. Using 2D ROESY NMR, the major isomer of 22D was assigned as epi-22D and amidine (D) products involved the proline amide, we sought to perturb the system by varying either the N-terminal proline residue or the amino acid adjacent to proline and studying the effects on the cyclization outcome. First, we employed N-methylated amino acids in an attempt to affect the nucleophilicity of the proline amide. We observed no productive reaction from sarcosine- or N-methyl-leucine-containing peptides P-Sar-LGF and P-MeLeu-LGF and could not isolate neither the peptide macrocycles nor the amidine and bridged-amidine products (see ESIAs the formation of both the bridged-amidine (s see ESI.18). (l-\u03b2-homoPro)-GLGf failed to produce any of the expected products (see ESIl-\u03b1-MePro)-GLGf, formation of the amidine product was observed by NMR, but no bridged-amidine product was observed or isolated LY(tBu)F linear peptide 10 to obtain finer detail on the features that govern productive macrocyclization. (l-\u03b2-HomoPro)-S(tBu)LY(tBu)F failed to react productively to make the macrocycle or either of the amidine products , only trace aziridine amide peaks were detected by 13C NMR LY(tBu)F F 10, , where tBu)F 10, . The ratpecies 4 . While i32 and aziridine aldehyde dimer 3 would form the nitrilium intermediate, 33 or in a concerted addition, similar to the piperazinone chemistry .The imidoanhydride intermediate C and D, . If we cidine 39 . Aziridi39 and acidity of the PH\u03b1 would enable direct deprotonation of PH\u03b1 to make enamine 43 or imidate-containing (40) intermediates , the reaction also failed, presumably due to the comparably disfavoured seven-membered ring formation requirement.Further proof for the involvement of nditions . When pr47 and 48, we conclude that the proline amide is vital to the cyclization and these results imply that imidoanhydride 37 would be the first expected intermediate and the driving force for the diastereoselectivity (37 at either of the two positions, one of which would generate the macrocycle precursor 34 or the 14-membered ring 45. Compound 45 is yet another mixed anhydride which can undergo intramolecular transacylation with the aziridine to generate the macrocycle. The aziridine can also attack the other position of mixed anhydride 45 to generate 46, the aziridine amidine that is the precursor to the amidine products. Intermediate 46 can also be formed directly from 37 as presented in the amidine pathways (Based on the unfavourable cyclizations of ectivity . From thpathways .37, N-terminus and match of stereochemistry between the N-terminal amino acid and aziridine aldehyde dimer.37 is formed at the N-terminus, the linear peptide tail has to fold in for attack by the C-terminal carboxylate. While the experimental evidence obtained in the course of our work is consistent with the intermediacy of 37, parallel macrocyclization pathways cannot be discounted (In order to rationalize favourable conditions and a peptide sequence that will result in a macrocycle, it is important to note the duality of bond-forming events in the proposed mechanism . The firscounted .The abundance of intramolecular pathways leading to amidine and bridged-amidine products and 10 eN-terminal amide acting as a kinetic trap to avoid intermolecular reactivity with the nitrilium ion, the occurrence of multiple intramolecular pathways from the imidoanhydride intermediate avoids the oligomerization issues seen with conventional Ugi reactions to make macrocycles. This serves to increase the effective molarity of the reaction. The amide participation suggests a way for selecting a cyclization-friendly sequence. These considerations will help improve the cyclization process and enable pre-selection of cyclization-friendly linear peptide sequences. In broader terms, the interception of nitrilium ions by nucleophilic amides to yield imidoanhydrides may play a role in other MCRs,We have probed the mechanism of the aziridine aldehyde-promoted macrocyclization reaction. This investigation unveiled the features governing both diastereo- and chemoselectivity of the process. Through kinetic analysis and product characterization, we have found evidence for an imidoanhydride-driven pathway during macrocyclization with aziridine aldehydes. With the Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "This side-by-side reactivity study of a borylene and a diborene with the same empirical formula demonstrates their non-interconvertibility. 4 -3,3,5,5-tetramethylpyrrolidin-2-ylidene) and its diborene relative, (II), both react with disulfides and diselenides to yield the corresponding cAAC-supported cyanoboron bis. Furthermore, reactions of I or II with elemental sulfur and selenium in various stoichiometries provided access to a variety of cAAC-stabilized cyanoboron\u2013chalcogen heterocycles, including a unique dithiaborirane, a diboraselenirane, 1,3-dichalcogena-2,4-diboretanes, 1,3,4-trichalcogena-2,5-diborolanes and a rare six-membered 1,2,4,5-tetrathia-3,6-diborinane. Stepwise addition reactions and solution stability studies provided insights into the mechanism of these reactions and the subtle differences in reactivity observed between I and II.The self-stabilizing, tetrameric cyanoborylene [(cAAC)B(CN)] Having failed thus far to realise the interconversion of I and II through photolysis and/or heating, we were eager to test their respective reactivity with suitable reagents in order to define any differences or similarities, and hopefully gain definitive proof of their ability (or inability) to interconvert. Herein we present the reactivity of I and II with elemental chalcogens and dichalcogenides, based on our recently-reported reactions of these chalcogen reagents with B\u2013B multiply-bonded species.I and its formal dimer II, and they may therefore be viewed as close relatives, subtle differences in reactivity confirm that no Wanzlick-type equilibrium exists between the two.Our recent synthesis of the aforementioned tetraborylene macrocycle [(cAAC)B(CN)]4 I; , left9 aAC)] II; , right11I with four equivalents of diorganyldichalcogenides, E2R2 in benzene proceeded selectively to the corresponding cAAC-supported cyanoboron dichalcogenides, [(cAAC)B(CN)(ER)2] , which were isolated in moderate to good yields 2 (\u201318.4 ppm) and 3 (\u201314.4 and \u201315.8 ppm) exhibiting shifts significantly downfield from the bis(sulfide) 1 (\u20139.6 ppm). Furthermore, at room temperature the bis(phenylselenide) 3 displayed highly broadened 1H NMR ligand resonances as well as two distinct, very broad 11B NMR resonances (\u201314.4 and \u201315.8 ppm), which coalesced upon heating to 70 \u00b0C, indicating hindered rotation, presumably owing to steric interactions between the bulky cAAC substituents and the large phenylselenide ligands.The reaction of tetrameric borylene d yields . WhereasII and Ph2S2 or Ph2Se2 showed the monoboron bis 1 and 3 to be sole products of these reactions, independent of the stoichiometry used scale\" fill=\"currentColor\" stroke=\"none\">B double bond and, subsequently, across the remaining B\u2013B single bond. Although both compounds I and II reacted with Ph2Te2 at high temperatures, these reactions were rather unselective and did not yield any tractable products.NMR spectroscopic data of reaction mixtures of diborene try used . These r1\u20133 readily crystallized from THF at room temperature, 1 as a colorless crystalline solid, and 2 and 3 as yellow crystals.2 repeatedly crystallized as extremely thin, overlapping yellow plates, which could not be separated. X-ray crystallographic analysis of these provided proof of connectivity but data were of insufficient quality for further structural discussions \u00c5) is slightly elongated compared to that in diselenide 3 (1.597(9) \u00c5). The B\u2013CCN bond lengths (1: 1.584(2); 3: 1.580(9)) are similar to those found in other cAAC-supported cyanoboranes (1.574(5)\u20131.589(3) \u00c5).d]dithiaboroles (B\u2013S: 1.899(5)\u20131.930(2) \u00c5)ortho-carboranes (B\u2013Se: 2.031(6)\u20132.065(5) \u00c5).1\u20133 represent the first examples of boron chalcogenides synthesized by the atom efficient insertion of a borylene into the E\u2013E bond of a diorganodichalcogenide.Compounds I with elemental sulfur in a 1\u2009:\u20091 boron-to-sulfur ratio in benzene for 5 d at room temperature resulted in a yellow suspension, which upon filtration and slow evaporation yielded compound 4 as a yellow crystalline solid . The analogous reaction with elemental selenium at 60 \u00b0C for 3 d similarly provided compound 5 as an orange crystalline solid in 70% yield. Compound 5 was isolated as a single species with an 11B NMR singlet at \u201333.5 ppm, shifted ca. 25 ppm upfield from that of 4, and a highly shielded, broad 77Se{1H} NMR resonance at \u2013143.1 ppm. In solution at room temperature 5 partially isomerized to a second species presenting an 11B NMR shift at \u201331.8 ppm and slightly shifted 1H and 13C NMR resonances. High resolution mass spectrometry experiments performed on 4 and 5 provided molecular masses consistent with dimeric compounds of the formula [(cAAC)B(CN)E]2 .Stirring of a dark red suspension of tetrameric borylene ne solid . Compoun4 and 5 crystallized in near-identical triclinic unit cells as centrosymmetric species presenting planar 1,3-dithia- and 1,3-diselena-2,4-diboretane cores, respectively. The B2S2 ring in 4 is an approximate square with four almost identical B\u2013S bonds (1.939(3) and 1.940(3) \u00c5) and near-perpendicular B\u2013S\u2013B and S\u2013B\u2013S angles (85.21(12) and 94.80(12)\u00b0, respectively), whereas the planar B2Se2 ring in 5 displays two slightly different B\u2013Se bond lengths (2.069(4) and 2.100(4) \u00c5) as well as near-perpendicular B\u2013Se\u2013B and Se\u2013B\u2013Se angles (85.13(17) and 94.87(17)\u00b0, respectively). There are only a couple of structurally characterized 1,3,2,4-dichalcogenadiboretanes in the literature, all displaying sp2 hybridized boron centers stabilized by \u03c0-donating amino substituents.2S2 and B2Se2 heterocycles are ca. 0.08\u20130.10 \u00c5 shorter than in 4 and 5, while the B\u2013E\u2013B and E\u2013B\u2013E angles are ca. 3\u00b0 narrower and wider, respectively.18This was confirmed by X-ray crystallographic analyses, the results of which are displayed in es , 5: 1.606(5) \u00c5) suggest that the cAAC ligand functions as a pure \u03c3-donor ligand to the sp3 borane, unlike in borylene I, where a significant contribution of \u03c0-backbonding from the electron-rich B(i) center shortens the bond (ca. 1.47 \u00c5). It is noteworthy that both the B\u2013CcAAC and B\u2013CCN bond lengths are significantly longer in 4 than in 5 (B\u2013CCN: 4 1.597(4), 5 1.577(6) \u00c5).For both compounds the plane consisting of the cyanoboron moiety and the \u03c0-framework of the cAAC ligand forms a 4 proved indefinitely stable in solution at room temperature, the 11B NMR spectrum of an analytically pure sample of 5 in CD2Cl2 left at room temperature for 3 d showed the partial disappearance (<10%) of 5 concomitant with the appearance of a new boron-containing species at \u201322.8 ppm (2B2(CN)2Se] (6). X-ray crystallographic analysis of this compound showed that 6 is indeed a bis(cAAC)-stabilized 2,3-dicyano-2,3,1-diboraselenirane . The disordered 2,3-dicyano-2,3-diboraselenirane cores of both parts were freely refined. Fig. S45\u00a7The solid-state structure of molecule The structure of 6 is similar to that of the bis(NHC)-stabilized 2,3-dithienyl-2,3,1-diboraselenirane obtained by our group upon reaction of one equivalent of selenium with the corresponding diborene precursor,trans-arrangement of the cyano and cAAC ligands with respect to the B2Se core. Attempts to generate 6 from I using a 2\u2009:\u20091 boron-to-selenium ratio failed, resulting instead in 50% conversion to the diselenadiboretane 5, with the remaining 50% of I left unreacted at room temperature in C6H6, as shown by the appearance of the 11B NMR shift around \u201322 ppm at room temperature in C6H6 yielded the corresponding 2,3-dicyano-1,2,3-thiadiborirane 7 H2B\u2013B(CO)(cAAC)], which we reported recently.19The reaction of 33.5 ppm . Similarretane 4 . But whe7 showed a structure very similar to that of its selenium analogue 6, displaying the same trans-arrangement of the cyano and cAAC ligands with respect to the B2S core (7 1.777(6); 6 1.757(6) \u00c5) enforced by the significantly shorter B\u2013E bonds and the slightly wider B\u2013E\u2013B angle (6: 56.9(2); 7: 51.31(18)\u00b0). Unlike the IMe-supported dithienyldiborene2Mn PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019BtBu(IMe)] (Cp = cyclopentadienyl),I or II showed any reactivity toward elemental tellurium in toluene, even after prolonged heating in benzene at 80 \u00b0C.Single crystals of B2S core . The onl4 the refinement of the data generated some residual electron density around the B2S2 ring that hinted at its overlap with a five-membered 1,2,4-dithia-3,5-diborolane ring representing less than 5% of the structure. This was consistent with the observation that recrystallized samples of 4 always showed some contamination with another species showing an 11B NMR resonance at \u20139 ppm, and that closer inspection of the mass spectrum of 4 revealed traces of a compound with the formula [(cAAC)2B2(CN)2S3]. Based upon this observation, attempts were made to obtain this compound by addition of sulfur to 4. However, even in the presence of excess sulfur with prolonged heating at 60 \u00b0C, only starting material was recovered of the reaction of borylene I with selenium in a 2\u2009:\u20093 boron-to-selenium ratio in benzene at 60 \u00b0C diastereomer, the observation of two non-exchanging isomers of 8 in solution suggests that the meso diastereomer of 8 may also be formed. The formation of a single diastereomer of 9 may be attributable to the higher reaction temperature favoring the thermodynamic diastereomer. The structure of 8 is reminiscent of that of the bis(NHC)-stabilized 3,5-dithienyl-1,2,4-trithia-3,5-diborolane obtained by the reductive insertion of three sulfur atoms into the B PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019B double bond of a diborene precursor, with very similar B\u2013S bond lengths and B\u2013S\u2013B and S\u2013B\u2013S angles.9 is reminiscent of that obtained by Tokitoh and co-workers upon irradiation of a boron bis(methylselenide) bound to a very bulky aryl ligand 2H)3C6H2).3 hybridization of the cAAC-supported boron atoms in 9, however, the B\u2013Se bonds (2.068(4), 2.086(4) \u00c5) are slightly elongated and the Se\u2013B\u2013Se angle (109.4(2)\u00b0) is significantly more acute than in [(Tbt)2B2Se3] (B\u2013Se: 1.942(7), 1.926(8) \u00c5; Se\u2013B\u2013Se: 118.8(4)\u00b0).21Like their Bructures show a f8 revealed the presence of another boron-containing species presenting an 11B NMR singlet at \u201311.2 ppm and a single symmetrical cAAC ligand environment in its 1H NMR spectrum. Surmising that this may be a tetrathiadiborinane resulting from a 1\u2009:\u20094 boron-to-sulfur reaction, a scaled-up reaction with this stoichiometry was carried out -stabilized 3,6-dicyano-1,2,4,5-tetrasulfa-3,6-diborinane displaying a cis-arrangement of the cyano and cAAC ligands with respect to the central B2S4 ring, which displays a boat conformation . Attempts to insert a fourth sulfur atom into the isolated 1,2,4-trithia-3,5-diborolane 8 failed to yield 9, suggesting that, like 8, compound 9 forms directly from I, presumably by dimerization of a monomeric [(cAAC)B(CN)S2] intermediate. While 1,2,4,5-tetrasulfa-3,6-diborinanes have been postulated as minor reaction products by both Lappert and later N\u00f6th, based solely on elemental analysis, mass spectrometry and IR spectroscopy data,10 constitutes the first structurally and NMR-spectroscopically characterized B2S4 heterocycle.Multiple recrystallizations of 2B2Se3] from [(Tbt)B(SeMe)2].2]. This monomeric borylene may then dimerize to an electron-deficient diborene (A), which then undergoes the reductive insertion of three atoms of selenium atoms with full cleavage of the B PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019B bond. For path B, the authors proposed a monomeric intermediate resulting from the reaction of \u201c(Tbt)B:\u201d with Se, which dimerizes to a 1,3-diselena-2,4-diboretane and finally inserts the third selenium atom. Regarding path A, numerous attempts on our part have failed to convert borylene I into its diborene relative, compound II, under thermal and/or photolytic conditions. The fact that the three-membered B2E heterocycles 6 and 7 can only be accessed directly from diborene II, but not from borylene I, is further evidence that, despite extensive overlap of reactivity outcomes, I and II do not interconvert.With all these data in hand, it was now possible to reassess the viability of the mechanism proposed by Tokitoh and co-workers for the formation of [(Tbt)B(Tbt)] , path A,I by Lewis bases showed that only a relatively small and strong Lewis base \u2013 the NHC 1,3,4,5-tetramethylimiazol-2-ylidene \u2013 is able to break up the tetramer and generate the mixed base-stabilised [(cAAC)(NHC)B(CN)] borylene.I instead occurs by association of the reactant with the tetramer itself, ultimately causing it to deaggregate into mononuclear intermediates and products. For reactions involving I, the generation of intermediate [[B] PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019E] monomers following path B therefore seems the most likely. While the dimerization of via bridging chalcogen atoms provides compounds 4 and 5, the latter do not insert another chalcogen atom to form 8 and 9, respectively, under the reaction conditions employed herein scale\" fill=\"currentColor\" stroke=\"none\">E] yields the B2E3 heterocycles 8 and 9. Similarly, the B2S4 heterocycle 10 would result from the dimerization of [[B]S2].Our previous report on the deaggregation of tetramer d herein . This me11B NMR spectroscopic analysis of the final reaction mixture, the reaction of I with sulfur in a 2\u2009:\u20093 boron-to-sulfur ratio yielded 8 in 70\u201385% selectivity at most, alongside the smaller and larger heterocycles 4 and 10, whereas the analogous reaction with selenium provided 9 in near-quantitative yield. The increased selectivity in the formation of 9 may be ascribed to the possibility of selenium de-insertion, which allows any B2Se2 heterocycle (5) formed in the course of the reaction to lose a Se atom, forming the diboraselenirane 6, which can in turn be converted to 9 (2S2 (4) and B2S4 (10) heterocycles formed in the course of the reaction are inert towards sulfur de-insertion and will therefore remain as by-products. Finally, the fact that the four- and five-membered B2E2 and B2E3 heterocycles are inert towards chalcogen insertion, whereas the three-membered B2E heterocycles may be converted to both the larger B2E2 and B2E3 heterocycles, suggests that ring-expansion only proceeds by insertion of a single chalcogen atom or an E2 unit into any remaining B\u2013B bonds.It is noteworthy that, while the selenium-based reactions were highly selective, those based on sulfur always yielded a mixture of products. For example, based on ted to 9 . In conti) compounds of the same empirical formula, the tetrameric, self-stabilizing cyanoborylene I and its dicyanodiborene relative II, has demonstrated that, while both compounds provide access to the same products in many cases, this occurs via different pathways as shown in This first comparative study on the reactivity of two boron as sole products scale\" fill=\"currentColor\" stroke=\"none\">B double bond, resulting in full B\u2013B bond cleavage is most likely at work.In reactions with dichalcogenides, both products . In the I or II employing a 1\u2009:\u20091 or 2\u2009:\u20093 boron-to-chalcogen ratio yielded the corresponding 1,3-dichalcogena-2,4-diboretanes or 1,2,4-trichalcogena-3,5-diborolanes, respectively. A unique 1,2,3-thiadiborirane could only be accessed from the reaction of diborene precursor II with sulfur in a 2\u2009:\u20091 boron-to-sulfur ratio, whereas the corresponding diboraselenirane was accessible both directly from the analogous reaction with selenium, and indirectly by de-insertion of one selenium atom from the four-membered 1,3-diselena-2,4-diboretane Borylene-based reactions do not proceed (ii) Ring-expansion reactions can only proceed by insertion of chalcogens into existing B\u2013B bonds;(iii) Ring-contraction is possible in the case of boron\u2013selenium heterocycles only by de-insertion of Se atoms.I and II provide confirmation that no Wanzlick-type equilibrium exists between a putative monomeric form of I and its B\u2013B bonded dimer, diborene II, the question remains as to whether this is a result of the extremely stable, tetrameric constitution of borylene I. To date, I and II represent the only existing borylene/diborene pair with the same empirical formula, however, recent advances in the synthesis of borylenes will hopefully enable a more definitive answer to the question of a possible interconversion between [LRB:] borylenes and diborenes. Beyond the interest of such an interconversion from a fundamental point of view, its undeniable potential for providing a new, more reliable route towards hitherto inaccessible diborenes should continue to stimulate research into this area.Although the subtle divergences in reactivity between The authors declare no conflicts of interest.Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "Three endogenous biothiols in single cells were simultaneously quantified by plasmonic Raman probes and quantitative principal component analysis (qPCA). i.e. cysteine (Cys), homo-cysteine (Hcy) and glutathione (GSH), and quantify their concentrations within single living cells, using one platform of Raman probe. By monitoring the reaction kinetics of biothiols with Raman probes and discriminating their products with a quantitative principal component analysis (qPCA) method, these three biothiols could be simultaneously quantified in both cell lysis and single living cells. The concentrations of Cys, Hcy and GSH in single Hela cells were 158 \u00b1 19 \u03bcM, 546 \u00b1 67 \u03bcM and 5.07 \u00b1 0.62 mM, respectively, which gives the precise concentrations of these three biothiols at a single cell level for the first time. This method provides a general strategy for discriminating each component from a mixed system and has potential for quantifying any biomolecules within an in vitro or in vivo biological environment.Intracellular biothiols mediate many important physiological and pathological processes. Due to their low content and competing thiol-reactivity, it is still an unmet challenge to quantify them within a complicated intracellular environment. Herein, we demonstrated a strategy to discriminate three biothiols, Comparatively speaking, Raman spectroscopy with a very narrow bandwidth (i.e. 1\u20132 nm) and a wealth of fingerprint information can provide much more molecular structural information.via the one probe platform.Over the past few decades, a number of techniques have been exploited to detect biothiols, including high performance liquid chromatography (HPLC),N-(2-cyanobenzo[d]thiazol-6-yl)-5-pentanamide, TA-CBT) obtained by 2-cyano-6-aminobenzothiazole (CBT-NH2) and thioctic acid (TA) via a one-pot procedure, and the chemical structure was fully characterized by 1H/13C NMR (nuclear magnetic resonance) spectroscopy and liquid chromatography-mass spectrometry (LC-MS) analysis with high reactivity, but it reacts with free thiol groups (e.g. GSH) with much lower reactivity. PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N) can react with the sulfydryl groups (S\u2013H) of the biothiols to form products with alternative structures scale\" fill=\"currentColor\" stroke=\"none\">N at 2235 cm\u20131, PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N at 2235 cm\u20131 and S\u2013H at \u223c2543 cm\u20131,The Raman probe was composed of polyethyleneglycol (PEG)-modified 60 nm gold nanostars (AuNSs) and Raman reporter molecules, denoted as plasmonic Raman probes (PRPs) . Scheme k\u2032mix = [kCys + kHcy(y/x) + kGSH(z/x)][Cys]0, where k\u2032mix is the pseudo-first-order reaction rate constant of a mixture with unknown [Cys]0, [Hcy]0 and [GSH]0, kCys, kHys and kGSH are the second-order rate constants for Cys, Hcy and GSH, respectively, and [Cys]0\u2009:\u2009[Hcy]0\u2009:\u2009[GSH]0 = x\u2009:\u2009y\u2009:\u2009z. From the above equation, [Cys]0, [Hcy]0 and [GSH]0 could be calculated if k\u2032mix and x\u2009:\u2009y\u2009:\u2009z were given, since kCys, kHys and kGSH do not depend on the concentration of the reactants. In this work, k\u2032mix could be experimentally determined by monitoring the reaction kinetics of PRPs with a mixture of Cys, Hcy and GSH, and x\u2009:\u2009y\u2009:\u2009z was obtained by running a qPCA analysis on the Raman spectra of their products scale\" fill=\"currentColor\" stroke=\"none\">N in the PRPs disappeared gradually with the extension of the reaction time, so we could monitor the reaction kinetics by following the Raman band intensity of C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N. Firstly, we examined the kinetic changes of the reaction with a relatively large excess of Cys, Hcy or GSH over PRPs in pH 7.4 PBS at 37 \u00b0C. With varying concentrations of Cys, Hcy or GSH, we collected the real-time Raman spectra of PRPs at different time intervals (k\u2032) was calculated on the basis of the exponential fitting of C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N over time (k), and then we could calculate the second-order reaction rate constants for the reaction of PRPs with Cys, Hcy and GSH: (2.22 \u00b1 0.02) \u00d7 10\u20134 min\u20131 \u03bcM\u20131, (1.74 \u00b1 0.14) \u00d7 10\u20134 min\u20131 \u03bcM\u20131, and (3.53 \u00b1 0.49) \u00d7 10\u20136 min\u20131 \u03bcM\u20131, respectively (x\u2009:\u2009y\u2009:\u2009z) in a mixed system , Hcy (0.1\u20131 mM) and GSH (1\u201310 mM) reported in previous literature, PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N over time was calculated by fitting the changes of C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N over time scale\" fill=\"currentColor\" stroke=\"none\">N changes over time in the real-time Raman spectra of PRPs reacted with biothiols from single living cells (k\u2032cell) was calculated are expected to be precisely revealed in the near future.We demonstrated a strategy to discriminate and quantify three endogenous biothiols within single cells by one platform of Raman probe. For the first time, the precise concentrations of Cys, Hcy and GSH were simultaneously obtained at the single cell level. This strategy combining reaction kinetics with the unique qPCA method provides potential for extracting and measuring biomolecules in complex biological environments, 4\u00b73H2O), cysteine (Cys), homocysteine (Hcy) and glutathione (GSH) were purchased from Sigma-Aldrich . Silver nitrate (AgNO3), hydrochloric acid (HCl), ascorbic acid (AA), and trisodium citrate were purchased from Aladdin Chemistry Co. Ltd . Polyethyleneglycol was obtained from Jenkem Technology Co. Ltd . All chemicals were used without further purification, unless otherwise stated.Isobutyl chloroformate, thioctic acid, 4-methylmorpholine (MMP), tetrahydrofuran (THF), 2-cyano-6-aminobenzothiazole, tetrachloroauric acid trihydrate and 50 \u03bcL of 1 M HCl were added to 50 mL of 0.25 mM HAuCl4 solution at room temperature under moderate stirring. Then, 1 mL of 0.5 mM AgNO3 and 0.25 mL of 0.1 M AA were added simultaneously and then stirred for 30 s.AuNSs were synthesized by a seed-mediated growth method.1H NMR \u03b4 10.40 , 8.76 , 8.18 , 7.72 , 3.64 , 3.16 , 2.42 , 1.88 , 1.65 , 1.44 ; 13C NMR \u03b4 171.75, 147.39, 139.76, 136.71, 134.70, 124.71, 120.58, 113.58, 110.92, 56.05, 38.08, 36.28, 34.13, 28.28, 24.73. LC-MS: m/z calcd for C16H18N3OS3+ [(M + H)+]: 364.06; found 364.00. The details are shown in Scheme S1 and Fig. S2\u2013S4.Isobutyl chloroformate was added to a mixture of thioctic acid and MMP in THF at 0 \u00b0C. The mixture was stirred for 30 min, to which 2-cyano-6-aminobenzothiazole was then added, and then stirred at room temperature overnight. After the reaction, the solvent was removed under vacuum, and the residue was purified by flash chromatography on silica gel to yield the desired product Raman reporter as a white yellow solid . The AuNSs (50 mL of 0.1 nM) were first incubated with 100 \u03bcL of 0.05 mM PEG in a shaker for 12 h and then purified by centrifugation at 8000 rpm for 10 min. Subsequently, these PEGylated AuNSs (4 mL of 0.5 nM) were treated with 200 \u03bcL of 0.05 mM reporter and purified using centrifugation at 8000 rpm for 10 min to yield plasmonic Raman probes (PRPs). These PRPs were re-dispersed in phosphate-buffered saline for subsequent use and kept at 4 \u00b0C for long-term storage.via the reaction with different concentrations of Cys, Hcy and/or GSH in pH 7.4 PBS at 37 \u00b0C. All of the Raman spectra were obtained using a Renishaw inVia-Reflex Raman spectrometer equipped with a 785 nm excitation laser.The three products were obtained by reaction with a large excess of Cys, Hcy or GSH over PRPs in pH 7.4 PBS at 37 \u00b0C. By varying the concentrations of Cys, Hcy or GSH reacted with PRPs, a series of samples were prepared in pH 7.4 PBS at 37 \u00b0C. Multiple mixed products were gained 2 and 95% air.The HeLa cells were cultured in Dulbecco\u2019s Modified Eagles\u2019 Medium , supplemented with 10% fetal bovine serum , and 1% antimycotic solution (KeyGEN BioTECH) at 37 \u00b0C in a humidified incubator containing 5% COCell lysis was obtained by cells treated with NP-40 lysis , and then reacted with PRPs. The pH of cell lysis was adjusted by 0.1 M NaOH to 7.4. For single living cells, the cells were pre-cultured in 12-well cell culture plates for 24 h and to these 0.5 nM PRPs were added. After incubation for 0.5 h at 4 \u00b0C, the cells were incubated at 37 \u00b0C for another 2 h to allow the internalization of PRPs, and then washed with pH 7.4 PBS three times. Then the coverslips were placed in a homemade live cell chamber with pH 7.4 PBS maintained stable humidity and 37 \u00b0C temperature.via a commercial software package . The dielectric constant of gold was from Johnson and Christy.x-polarized and y-polarized incident plane wave propagating along the z-axis.The electromagnetic field distribution of the AuNS was simulated by FDTD vs. PC2 scores was utilized to classify the four products.We developed a quantitative principle component analysis (qPCA) method to quantify each of the components in a mixed system. The full proof and details about the mathematical model and calculation strategy are provided in the ESI.There are no conflicts to declare.Supplementary informationClick here for additional data file."} +{"text": "We present herein a nickel-catalyzed dicarbofunctionalization of alkenes using readily available organoboronic acids and organic halides in a three-component fashion. N-allylpyrimidin-2-amine is achieved when aryl and methyl iodides are utilized. In contrast, the use of alkyl bromides with \u03b2-hydrogens results in 1,3-hydroarylation or oxidative 1,3-diarylation. Preliminary mechanistic studies suggest an isomerization involving nickel hydride in the 1,3-difunctionalization reactions. On the other hand, the use of alkenyl or alkynyl halides promotes alternative regioselectivities to deliver 1,2-alkenylcarbonation or intriguing 2,1-alkynylcarbonation products. Such 2,1-alkynylarylation is also applicable to N-allylbenzamide as a different class of substrates. Overall, this nickel-catalyzed process proves to be powerful in delivering versatile difunctionalized compounds using readily available reagents/catalysts and a simple procedure.Efficient difunctionalization of alkenes allows the rapid construction of molecular complexity from simple building blocks in organic synthesis. We present herein a nickel-catalyzed dicarbofunctionalization of alkenes using readily available organoboronic acids and organic halides in a three-component fashion. In particular, an unprecedented regioselectivity of the 1,3-dicarbofunctionalization of To this end, the transition-metal-catalyzed dicarbofunctionalization of alkenesvia metal catalyzed C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond isomerization , has also attracted much attention.In a related area of research, remote alkene functionalization, which selectively installs a functionality distal to the initial alkene moiety Our group has been previously interested in the carbofunctionalization of alkenes under base metal catalysis.2.2.11a) and N-allyl aniline (1b) using iodobenzene and phenylboronic acid. Under various nickel-catalyzed conditions, unfortunately, no diarylation product was detected at all or an ester (1e) as the directing group proved futile (entries 3\u20135), we were excited to observe the formation of a desired diarylation product in 23% isolated yield from the use of 1f (entry 6), together with 42% yield of a Suzuki coupling side product. A screening of common phosphine ligands was carried out, from which dppm provided the optimal 47% isolated yield (entry 9 vs. 7 and 8). Here again, Suzuki cross-coupling consumed the reagents to a large extent (40% yield of biphenyl). Intriguingly, the product was determined to be a mixture of 2f and an unexpected 1,3-difunctionalization product 2f\u2032, which represents an unprecedented regioselectivity in alkene difunctionalization. Gratifyingly, when 1g bearing 2-aminopyrimidine was examined next, the 1,3-diarylation product 2g\u2032 was isolated exclusively with an improved yield of 68% (entry 10). Moreover, the undesired Heck and Suzuki products were minimized to 5% and 15% respectively. Other substrates, bearing related directing groups (1h\u2013j), were also examined and suffered from either low conversion or deallylation of the substrate (entries 11\u201313). Different metal precursors were also examined under the optimized conditions. Ni(ii) species in the presence of a reductant proved to be much less effective (entries 14 and 15). Importantly, the use of a palladium catalyst resulted in the deallylation of the substrate and undesired Suzuki coupling without any difunctionalization (entry 16).We initiated our investigation with the dicarbofunctionalization of simple alkenes such as allylbenzene , alkene (3g), ester (3f) and ketone (3h) groups. A higher temperature was required for the ortho-substituted aryl iodides probably due to steric hindrance (3i and 3j). In the case of 4-iodo-benzonitrile, a lower temperature of 85 \u00b0C proved to be important to produce 3d in a good 65% yield, while higher temperature led to much decomposition. Unfortunately, heteroaryl iodides like 2-iodo thiophene, pyridine, and pyrimidine gave only trace amounts of the desired product. A wide range of aryl boronic acids bearing bromo, vinyl, fluoro and trifluoromethyl substituents could also be used to deliver a range of diarylation products 3l\u2013u with good efficiency. Notably, alkenyl boronic acids could also be employed under the same conditions to afford the arylalkenylation products 4a\u2013e in good to high yields, which dramatically expanded the scope of this 1,3-dicarbofunctionalization of alkenes. However, heteroaryl boronic acids led to only trace amounts of the products owing to the decomposition of these boronic acids under the diarylation conditions.With the optimal conditions in hand, we moved on to examining the substrate scope of this 1,3-difunctionalization reaction. Initial efforts were focused on the variation in the substitution pattern of the alkene substrate. Unfortunately, various internal alkenes including l and 1m all fail2.3tert-butyl-p-cresol) or \u03b1-cyclopropylstyrene 8 on the difunctionalization reaction catalyst.Based on these data, we proposed a domino Heck\u2013isomerization\u2013Suzuki reaction pathway for the 1,3-diarylation as shown in 2.45a\u2013f). Besides, dialkenylation could also be realized to deliver 5g.When alkenyl bromides were examined under the same conditions, the related alkenylarylation proceeded smoothly, but with an interesting switch to the 1,2-regioselectivity . Under tvs. 1,3-difunctionalization (r.r.) for most 5 products was >10/1 (as determined by GC-MS), the electronic property of the styryl halides had a significant influence on the regioselectivity. As shown by the comparison of 5c and 5d, the change of the electron-withdrawing CF3 to the electron-donating OMe increased the ratio from 7.7/1 to >100/1. We assume that this 1,2-regioselectivity for alkenylarylation could be attributed to the coordinating effect of the alkenyl unit on nickel scale\" fill=\"currentColor\" stroke=\"none\">C bond from the electrophile would coordinate to nickel and suppress the isomerization. When an electron-deficient alkenyl halide is used, it presumably does not coordinate to nickel so effectively, leading to a higher ratio of isomerization and in turn 1,3-difunctionalization.10It is worth noting that while the ratio of 1,2 n nickel . Once in2.51g with various aryl and alkenyl halides, a three-component reaction between 1g, (bromoethynyl)benzene and PhB(OH)2 was tested under the previously optimized conditions (6a using Ni(COD)2/dppm. The use of alkynyl chloride and iodide also yielded 6a, albeit in lower yields. To our great surprise, both NMR and X-ray structure analysis confirmed an unexpected reverse regioselectivity. In contrast to the 1,2-alkenylarylation in PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond while the phenyl groups were added on to the terminals. To further optimize this intriguing transformation, various bisphosphine, monophosphine, phosphite and bipyridine ligands were screened (6a could be improved to 85% by using PCy3 as the ligand and toluene as the solvent. It is noteworthy that a good yield of 77% could be obtained for 6a even in the absence of a ligand in dioxane (<2% in toluene). The coordinative solvent presumably played an important role in stabilizing the nickel species in this case. On the other hand, the combination of Ni(ii) precursors with PCy3 or the use of a Pd complex were much less effective for this 2,1-alkynylarylation (<11% for 6a).Alkynyl halides are versatile and powerful reagents for the introduction of alkynyl groups into organic molecules.nditions . A moderscreened . The yie6b), trifluoromethyl (6c), acetyl (6d) and chloro (6e) substituents on the phenylboronic acids could be well-tolerated. Alkenylboronic acids also worked well to yield 6f and 6g in good yield.The scope of this 2,1-alkynylation was extended to various alkynyl bromides and boronic acids under optimized conditions . Bromo (6h), the perturbation of the electronic property of aryl-alkynyl bromides and the use of alkyl-substituted alkynyl bromide all resulted in no conversion to the desired product. While the previous 1,3- and 1,2-dicarbofunctionalizations necessitate the use of pyrimidine directing groups, we were curious whether the 2,1-alkynylarylation could overcome this limitation. To our excitement, simple N-allylbenzamide 1c was identified to be a suitable substrate for this reaction. As shown in 7 was obtained in a good yield of 56% at a slightly higher temperature. Further exploration of the scope of this transformation is ongoing.This catalytic system, however, showed a narrow scope for the alkynyl reagents. Other than electron-neutral aryl-substituted alkynyl bromides 2 to form B, which featured two distinct carbanion ligands on nickel. Selective insertion of 1g into the aryl/alkenyl\u2013nickel bond led to the formation of intermediate C, which delivered the 2,1-alkynylarylation products through reductive elimination. The key chemo-selectivity in the carbonickelation step was likely due to the fact that nickel\u2013alkynyl bonds are much stronger than the Ni\u2013aryl and Ni\u2013alkenyl counterparts.12It is noteworthy that in the previous 1,3-diarylation and 1,2-alkenylcarbonation reactions, a small amount of Heck product was isolated as the side product. In contrast, no Heck product was observed in the 2,1-alkynylcarbonation. Instead, a small amount of Suzuki product was detected. This observation was further confirmed in the attempted two-component couplings under standard conditions ; the alk2.61g, the 1,3-methylarylation (8a) or 1,3-methyl alkylation (8b) products could be obtained in 41% and 39% yield by using phenyl or styryl boronic acid, respectively. In addition, an unexpected oxidative diarylation product (2g\u2032) was also obtained.Alkyl halides are usually poor electrophiles in transition-metal catalyzed cross-coupling reactions due to their slow oxidative addition and facile \u03b2-hydride elimination.8c) was detected using GC-MS, while 2g\u2032 was obtained as the major product in 35% yield dibromide (B) by the release of ethylene gas.B with organoboronic acid then generated the key organo-nickel intermediate C. The next steps will then follow the pathways for either 1,3-diarylation was attempted under similar conditions. To our excitement, the desired product 8a was successfully obtained, albeit in a moderate yield . On the other hand, the removal of the pyrimidine moiety was also attempted under various conditions, which only led to the cleavage of the aminopyrimidine completely to deliver hydrocarbon products scale\" fill=\"currentColor\" stroke=\"none\">C bond coordination helped to anchor the Ni(ii) intermediate, resulting in less of a tendency towards isomerization and the formation of 1,2-difunctionalization instead. The reaction with alkynyl halides produced a reversed 2,1-regioselectivity, presumably resulting from an alternative sequence of transmetalation followed by selective C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C insertion into the weaker Ni(ii)-carbon bond. Alkyl halides with \u03b2-hydrogen furnished the 1,3- or 1,4-hydrofunctionalization product. In addition, dihaloethane worked as an oxidant under the same conditions to form the oxidative dicarbofunctionalization products. These methodologies can be used for the synthesis of 2-aminopyrimidine motifs as attractive pharmacophores. Current efforts in our laboratory are directed towards the dicarbofunctionalization of more diverse alkenes and enantioselective variants of these transformations.In summary, we have realized the first nickel-catalyzed dicarbofunctionalization of There are no conflicts to declare.Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "A highly oriented 2D nanosheet metal\u2013organic framework membrane is fabricated by a direct growth strategy. 2(bIm)4 (bIm = benzimidazole) ZIF nanosheet tubular membrane, based on graphene oxide (GO) guided self-conversion of ZnO nanoparticles (NPs). Through our approach, a thin layer of ZnO NPs confined between a substrate and a GO ultrathin layer self-converts into a highly oriented Zn2(bIm)4 nanosheet membrane. The resulting membrane with a thickness of around 200 nm demonstrates excellent H2/CO2 gas separation performance with a H2 performance of 1.4 \u00d7 10\u20137 mol m\u20132 s\u20131 Pa\u20131 and an ideal separation selectivity of about 106. The method can be easily scaled up and extended to the synthesis of other types of Zn-based MOF nanosheet membranes. Importantly, our strategy is particularly suitable for the large-scale fabrication of tubular MOF membranes that has not been possible through other methods.Highly oriented, ultrathin metal\u2013organic framework (MOF) membranes are attractive for practical separation applications, but the scalable preparation of such membranes especially on standard tubular supports remains a huge challenge. Here we report a novel bottom-up strategy for directly growing a highly oriented Zn On the other hand, Gascon et al.2/CH4 separation performance.In recent years, as a new member of the 2D family, 2D MOF nanosheets are receiving increasing attention because of their large internal surface areas, uniform channels, (sub)nanometer-sized cavities, thermal stability, and chemical tailorability.In fact, the top-down preparation method inevitably involves the exfoliation step which usually causes some fragmentation and morphological damage of the detached sheets.tubular substrate for H2/CO2 separation by the GO-guided self-conversion of ZnO NPs. As shown in 2 domains, and thus it can act as structural nodes and participate in bonding interactions with MOFs.In our previous study, a 2D ZIF nanosheet membrane was achieved by a self-conversion growth of ZnO nanoparticles (NPs).2(bIm)4 (Zn2(benzimidazole)4) as an example to demonstrate our GO-guided metal oxide self-conversion strategy, as the Zn2(bIm)4 units are considered as subunits of the 2D structure, in which the apices are occupied by Zn atoms and the sides are formed by benzimidazole ligands. Its aperture size, a four-membered ring, is no more than 0.21 nm. Thus, the 2D Zn2(bIm)4 membrane can demonstrate excellent performance in separating H2/CO2 mixtures and have potential applications in some small-molecule gas separations.19We chose Zn2.2.1The commercial porous \u03b1-alumina tubes with 4 mm OD (outer diameter), 3 mm ID (inner diameter) and an average pore size of 100 nm as substrates were purchased from Hyflux Ltd. Co. The tubes were cut into 60 mm lengths and washed sequentially with distilled water and ethanol several times. The tubes were then dried at 100 \u00b0C for 5 h before calcination at 550 \u00b0C for 6 h.3H8O2, EGME, 99.0%), toluene (\u226599 wt%), ammonium hydroxide , N,N-dimethylformamide , monoethanolamine and anhydrous methanol (\u226599.5%) purchased from Sinopharm Chemical Reagent Co., Ltd. Benzimidazole , phthalic acid and 1,3,5-benzenetricarboxylic acid were supplied by Sigma-Aldrich Chemical. Co. Ltd. Zinc oxide nanoparticles were purchased from Beijing Nachen S&T Ltd.The chemicals used in the membrane preparation include zinc nitrate hexahydrate (\u226599.0%), zinc acetate (98.0%), ethylene glycol monomethyl ether with a molar composition of 1\u2009:\u20091\u2009:\u200945\u2009:\u200945 was first prepared for the formation of ZIF nanosheets. Then, the tube with both ZnO NP and GO layers was placed in the synthesis solution at 100 \u00b0C for the desired reaction time. After the synthesis, the membrane was taken out, immersed in methanol for 2 days and then thoroughly rinsed with methanol. Finally, the membrane was dried at room temperature overnight and then stored in a desiccator for later use.According to our previous studies,2.5\u20131) were recorded on a Bruker Equinox 55 spectrometer in KBr plates. Thermal gravimetric analysis (TGA) in a DTU-2 was done in flowing air from 25 \u00b0C to 700 \u00b0C at 10 \u00b0C min\u20131. AFM micrographs were recorded with a Veeco Multimode Nanoscope 3A microscope operating in tapping mode. The MOF samples were applied to a previously annealed mica wafer substrate. Before microscopy inspection, a couple of drops from a suspension of nanosheets in methanol were applied and allowed to dry over the mica substrate. The surface elemental composition was obtained by X-ray photoelectron spectroscopy using a monochromatic Al X-ray source (1486.6 eV).X-ray diffraction (XRD) measurements were performed on a D/max-2400 X-ray diffractometer using Cu K\u03b1 radiation in the range of 3\u201360\u00b0 operating at 20 kV/100 mA. Scanning electron microscopy (SEM) images were obtained with a NOVA NANOSEM 450 SEM (FEI Company) and a Tecnai F30 transmission electron microscope (TEM). The settings of the SEM were as follows: high voltage (HV) 5\u201315 kV, working distance (WD) 5\u201310 mm and spot size 3.0. Fourier transform infrared (FTIR) spectra (4000\u2013400 cm2 (0.29 nm), CO2 (0.33 nm), N2 (0.36 nm) and CH4 (0.38 nm), while the separation of binary gas mixtures was investigated for H2/CO2, H2/N2 and H2/CH4. The experimental setup is shown in Fig. S1 of the ESI.Pi, is defined by following eqn (1): where Ni is the permeation rate of component i (mol s\u20131), \u0394Pi is the trans-membrane pressure difference of i component (Pa), and A is the membrane area (m2). Fi is the flux of component i (mol m\u20132 s\u20131). The ideal separation factor is calculated as the ratio of permeance Pi and Pj in eqn (2).Gas permeances of the highly oriented nanosheet membranes achieved were measured for single gases including H2/CO2, H2/N2 and H2/CH4). The feed gas was constant at a 1\u2009:\u20091 volume ratio. The total pressure on each side of the membrane was atmospheric. Nitrogen was used as the sweep gas on the permeate side for H2/CO2 and H2/CH4 mixtures, while methane was used for the H2/N2 mixture. The gas composition was analyzed using an online gas chromatograph (GC7890T). The separation selectivity \u03b1i/j for the binary gas mixtures was defined as the following eqn (3): where Xi and Xj are the molar fractions of components i and j in the retentate stream which is similar to the feed component, while, Yi and Yj are the molar ratios of components i and j on the permeate side.The separation properties were investigated for equimolar binary gas mixtures . As shown in 2(bIm)4 structure, indicating that this membrane is not oriented. This mainly results from the reason that the nucleation and self-conversion growth of ZnO NPs is able to take place in any direction on the substrate surface in the absence of orientational control, thus resulting in the formation of a layer of randomly oriented multiple nanosheets. Therefore, to date, few continuous 2D nanosheet MOF membranes have been successfully prepared using the direct growth method.For comparison, a thin layer of ZnO NPs coated on a porous substrate was directly converted to grow a ZIF nanosheet membrane in the ligand synthesis solution for 5 h without using any GO. As shown in via some reactions in the alkaline synthesis solution.2(bIm)4 of a highly oriented nanosheet membrane formed by self-conversion of ZnO NPs in a confined space of GO.Here, to obtain an oriented ZIF membrane, a thin layer of GO acting as orientational control was employed to coat the top of the ZnO NP layer supported on the porous substrate to form a sandwich-like structure in which the ZnO NP layer is sandwiched between the GO and surface of the substrate, realizing an oriented growth of a nanosheet membrane. As shown in via coating to form a ZnO NRs@GO layer supported on the substrate for growing a nanosheet membrane under the same synthesis conditions. As shown in To further verify the guiding function of the GO layer on the top of the ZnO NP layer, a layer of ZnO nanorods (NRs), instead of ZnO NPs, was grown on the porous substrate, followed by a thin layer of GO According to the crystallographic data calculated from XRD patterns, the 3D crystalline structure of the ZIF nanosheet was achieved on the surface modified with ZnO NPs .2(bIm)4 consisting of 2D layers is in agreement with that of the substrate. The aperture size of the ZIF is estimated to be 0.21 nm as shown in Fig. S8.The pore direction of the Zn3.22(bIm)4 nanosheet membrane using this bottom-up method. To investigate the role of the GO layer during the preparation of the membrane and further clarify the formation mechanism of the highly oriented membrane, a series of characterizations of the samples were carried out by using FTIR and XPS techniques. The FTIR spectra of Zn2(bIm)4 nanosheets, GO, and Zn2(bIm)4 grown for 9 h (M-9) achieved after the reaction for 9 h are shown in \u20131, representing C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O in carbonyl, and C\u2013O and C\u2013O\u2013C in the epoxy group, respectively, are obviously present.2(bIm)4 nanosheets and Zn2(bIm)4 M-9 have similar FTIR spectra. However, in the spectra of the Zn2(bIm)4 M-9, the intensities of the peaks at 1930 cm\u20131, 1900 cm\u20131 and 1780 cm\u20131 decreased and that of the peak at 470 cm\u20131 increased, showing the formation of a chemical covalent bond of Zn\u2013O between GO and Zn2(bIm)4 because the carboxyl groups and epoxy groups of GO can interact with Zn2+ to form Zn\u2013O bonds.\u20131) related to the coplanar vibration of sp2 bonded carbon atoms and the D band (1340 cm\u20131). The D (associated with the order/disorder of the system) and G (an indicator of the stacking structure) bands of the Raman spectra are the dominant vibrational modes observed in graphitic structures.ID/IG) is often used as a means of determining the number of layers and their overall stacking behavior in a graphene sample. A high ID/IG ratio indicates a high degree of exfoliation/disorder. It is found that the ID/IG value of the Zn2(bIm)4 membrane-9 increased from 0.96 to 1.28 compared with GO. This should be ascribed to the presence of abundant covalent bonds of Zn\u2013O formed by the interaction of C\u2013O and C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O of GO with ZnO NPs.The above discussion indicates that the GO layer plays a key role in guiding the oriented growth of the ZnFrom the above analysis of FTIR and Raman spectra, it is obvious that the GO on the top of the ZnO NP layer can interact with ZnO to produce Zn\u2013O covalent bonds in the ligand solution, which favors the nucleation and oriented growth of the ZIF membrane.2(bIm)4/GO composite. The C 1s peaks of GO consist of three components arising from C\u2013C/C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C, C\u2013O and C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O. PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C peaks of the Zn2(bIm)4/GO composite become predominant, while the peaks of carbon atoms bonded to oxygen (C\u2013O) are weakened remarkably, implying the conversion of the C\u2013O group into the Zn\u2013O bond by interaction with ZnO in the solution. From 2(bIm)4/GO, the peak in the O 1s spectrum displays a skewing to low binding energy, yet the binding energies of Zn-based bonds of the Zn2(bIm)4/GO composite are larger than those of Zn2(bIm)4. This should be ascribed to the fact that the electron cloud of bonds is shifted from Zn to O atoms, thus confirming that a Zn\u2013O coordination bond between the O of the GO and Zn in Zn2(bIm)4 is formed.2(bIm)4 nanosheets and the formation of the membrane because GO with abundant oxygen groups can play a seed role. Based on the above characterizations and analyses, a possible formation mechanism for the highly oriented Zn-based MOF nanosheet membrane is proposed as indicated in 2(bIm)4 nanosheets should start from the interface between GO and the top of the ZnO NP layer because the C\u2013O/C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O groups of the GO surface easily react with Zn2+ from ZnO NPs in the ligand solution. The ZnO NPs coming into contact with GO will preferentially react with the ligands to generate Zn2(bIm)4 nanosheets. Then, the formed nanosheets act as a seed-like template to induce the conversion of the bottom ZnO NPs into nanosheets downward under the GO restriction. With the reaction time, the nanosheets are gradually produced and then extended in a horizontally oriented direction along the surface of the GO sheet layer to form a continuous nanosheet membrane. Moreover, under the restricted state of the GO layer, the nanosheets in a parallel direction can only grow down toward the ZnO NP layer and stack into a multilayered nanosheet Zn2(bIm)4/GO membrane. Simultaneously the ZnO NPs are also consumed gradually and are finally used up with the increase of the nanosheet membrane thickness. Thus, in this method, the thickness of the ZnO NP layer determines that of the corresponding nanosheet membrane, while the GO layer on the top of the ZnO NP layer plays a key role in restricting and guiding the growth direction of the nanosheet membrane. Both these aspects lead to a highly oriented nanosheet MOF membrane. During the assembly of the nanosheet membrane, the part of the GO layer acting as nanosheets is possibly interweaved with ZIF nanosheets to form a ZIF nanosheet membrane interlaced with GO. Therefore, GO may remedy some defects possibly generated from the nanosheet membrane, thus leading to a high-performance membrane compared with the membrane obtained without any GO layer.To further confirm the reaction between the functional groups of GO and ZnO NPs, XPS spectra for the samples were recorded as shown in 3.32, N2, CO2 and CH4) through the membrane decreased rapidly with increasing the kinetic diameter of gas molecules. The smallest H2 gas showed the highest permeance among these gases, while the permeances of larger gases were far lower than that of H2. This indicated that the as-prepared membrane exhibited excellent molecular sieve performance for H2 over other gases. The ideal selectivities were as high as 106, 126 and 256 for H2/CO2, H2/N2 and H2/CH4, respectively, which are far beyond their corresponding Knudsen selectivities . Moreover, the molecular sieve performance of the M-9 membrane was confirmed by the separation of equimolar mixtures of H2 and CO2, N2 and CH4, and the separation selectivities for H2/CO2, H2/N2 and H2/CH4 at 30 \u00b0C reached about 89, 103 and 221, respectively. This showed that the nanosheet membrane gave excellent molecular sieve performance for H2 over other gases, especially for H2 over CO2, demonstrating the ultrahigh separation function of the nanosheet membrane for small-molecule mixtures. In addition, to further assess the mechanical stability of the obtained membrane, the permeation performances of gas molecules were measured at different trans-membrane pressure drops as shown in Fig. S9.2/CO2 separation selectivity through the nanosheet membrane could almost remain unchanged when the H2 partial pressure increased from 0.5 to 1.5 bar, thus demonstrating excellent mechanical stability. The permeances of all the gases through the membrane are independent of the trans-membrane pressure drop. This implies that the prepared nanosheet membrane is compact and contains no pinholes or defects.To evaluate the entire quality of the oriented nanosheet tubular membrane achieved after the solvothermal growth of 9 h (denoted as M-9), gas permeation performances with single and binary mixtures were measured, respectively. 2/CO2 and the long-term permeation performances of gas molecules for the achieved nanosheet tubular membrane were also investigated to evaluate its thermal and operating stability as shown in 2 and CO2 hardly changed within the temperature range from 30 to 150 \u00b0C. However, both the permeance of H2 and separation selectivity of H2/CO2 slightly increased at elevated temperature as shown in 2 is larger than the aperture size of the nanosheets, this tiny change of pore size can cause a slight increase in the permeance of H2 and hardly has an influence on the permeance of CO2, thus resulting in a slight increase in the separation selectivity of H2/CO2 at elevated temperature.19The influence of temperature on the gas permeance and selectivity of H2/CO2 binary mixture through the membrane. Then, the membrane was heated to 100 \u00b0C and exposed to equimolar H2/CO2 binary gas mixtures for 100 h. After that, the membrane was heated up to 150 \u00b0C and tested for 100 h before cooling down to room temperature. It is seen that there are barely changes for the permeances of both H2 and CO2 during the operating period of 400 h within the temperature range from 30 to 150 \u00b0C. No degradation in membrane performance was observed within the entire test except that there was a slight increase in the gas selectivity of H2/CO2 with elevated temperature because of a tiny increase of H2 permeance. Hence, this long-term permeation result showed that the nanosheet tubular membrane has excellent thermal and operating stability.2(bIm)4 nanosheet tubular membrane with some membranes reported in previous references for H2/CO2 separation was also made. As shown in Table S12/CO2, and the black dashed line represents the 2010 upper bound of microporous inorganic membranes for the separation of H2/CO2 mixtures, our prepared nanosheet membrane far surpassed the trade-off line and had much better separation performance for H2/CO2 than most of the other reported microporous molecular sieve membranes including zeolites, MOFs and polymers, demonstrating that our highly oriented nanosheet membrane is of high quality and has excellent gas separation performance for H2/CO2 mixtures. This suggests that highly oriented nanosheets play an important role in transporting gases through the molecular sieve membranes. Compared to the Zn2(bIm)4 nanosheet membrane with around 5 nm thickness supported on a porous disc by the exfoliation\u2013deposition method reported by Yang's group,2/CO2. The differences may mainly result from the substrate difference for membrane preparation. It is well recognized that the substrate quality seriously influences the membrane quality and its separation performance.2/CO2 gas separation performance.tubular substrate than a planar one. Unfortunately, by this strategy, we have not achieved a much thinner nanosheet membrane yet by now. Nevertheless, the separation performance of our nanosheet membrane is still much higher than those of other molecular sieve membranes reported to date and ZnBTC , were chosen to prepare their corresponding nanosheet membranes as shown in Fig. S11.4.i.e. bottom-up method) to prepare a highly oriented ZIF nanosheet tubular membrane. Excellent gas separation performance with a H2 permeance of 1.5 \u00d7 10\u20137 mol m\u20132 s\u20131 Pa\u20131 and a H2/CO2 ideal separation selectivity of 106 was achieved through our membrane. The method is based on self-conversion of a thin layer of ZnO NPs confined between the surface of a tubular substrate and an ultrathin layer of GO into a nanosheet membrane. The ZnO NP layer acts as seeds and anchoring sites for the membrane growth, while the GO layer on the top of the ZnO NP layer can confine and guide the oriented growth of nanosheets due to the interaction of the abundant carboxylate groups at the surfaces and edges of the GO layer with the Zn2(bIm)4 nanosheets generated at the initial stage. The highly oriented nanosheet membrane achieved is like a window-frame structure exhibiting excellent operating stability. Moreover, this method can also be used for the synthesis of other Zn-based MOF nanosheet membranes such as ZnBDC and ZnBTC. It is believed that this work opens up a simple and scalable direct growth route for achieving a highly oriented nanosheet MOF membrane, especially a tubular membrane for potential industrial applications.In conclusion, we for the first time developed a direct growth strategy (There are no conflicts to declare.Supplementary informationClick here for additional data file."} +{"text": "Luminescent platinum(ii) complexes show anti-cancer and pH-dependent self-assembly and sustained-release properties under physiological conditions. ii) complexes [Pt(C^N^Npyr) scale\" fill=\"currentColor\" stroke=\"none\">NR)]+ [HC^N^Npyr = 2-phenyl-6-(1H-pyrazol-3-yl)-pyridine)] containing pincer type ligands having pyrazole moieties. These Pt(ii) complexes exert potent cytotoxicity to a panel of cancer cell lines including primary bladder cancer cells and display strong phosphorescence that is highly sensitive to the local environment. The self-assembly of these complexes is significantly affected by pH of the solution medium. Based on TEM, SEM, ESI-MS, absorption and emission spectroscopy, and fluorescence microscopy together with cell based assays, [Pt(C^N^Npyr) scale\" fill=\"currentColor\" stroke=\"none\">NR)]+ complexes were observed to self-assemble into orange phosphorescent polymeric aggregates driven by intermolecular Pt(ii)\u2013Pt(ii) and ligand\u2013ligand interactions in a low-pH physiological medium. Importantly, the intracellular assembly and dis-assembly of [Pt(C^N^Npyr) scale\" fill=\"currentColor\" stroke=\"none\">NR)]+ are accompanied by change of emission color from orange to green. These [Pt(C^N^Npyr) scale\" fill=\"currentColor\" stroke=\"none\">NR)]+ complexes accumulated in the lysosomes of cancer cells, increased the lysosomal membrane permeability and induced cell death. One of these platinum(ii) complexes formed hydrogels which displayed pH-responsive and sustained release properties, leading to low-pH-stimulated and time-dependent cytotoxicity towards cancer cells. These hydrogels can function as vehicles to deliver anti-cancer agent cargo, such as the bioactive natural products studied in this work.Supramolecular interactions are of paramount importance in biology and chemistry, and can be used to develop new vehicles for drug delivery. Recently, there is a surge of interest on self-assembled functional supramolecular structures driven by intermolecular metal\u2013metal interactions in cellular conditions. Herein we report a series of luminescent Pt( PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019NR1)]Cl complex undergoes aggregation in cancer cells, showing persistent 3MMLCT emission in the cytoplasm.6Self-assembly of \u03c0-conjugated organometallic complexes driven by non-covalent interactions, such as metal\u2013ligand coordination, metal\u2013metal and ligand\u2013ligand interactions, has spurred extensive studies by virtue of the intriguing optical, electronic and chemical properties resulting from these interactions.III(C^N^C)(4-dpt)]OTf , which displayed anti-proliferative and sustained release properties in biological systems.There has been a growing interest in developing metal complexes which target DNA as well as proteins of relevance to cancers.ii) complexes are well documented to have rich luminescent properties, and more recently, to show anti-cancer properties. Indeed, examples of pincer type platinum complexes which display promising in vivo anti-tumor effect in animal model studies have recently been disclosed.ii) complexes (ii) complexes which form poly-electrolytes having hydrogel properties have been reported, an example of which is the dinuclear complex having [Pt(C^N^N) scale\" fill=\"currentColor\" stroke=\"none\">NR1)]+ motifs covalently connected to oligo(oxyethylene) chains.ii) isocyanide complexes containing the C-deprotonated C^N^Npyr ligand (HC^N^Npyr = 2-phenyl-6-(1H-pyrazol-3-yl)-pyridine) scale\" fill=\"currentColor\" stroke=\"none\">NR1)]Cl, [Pt(C^N^Npyr) scale\" fill=\"currentColor\" stroke=\"none\">NR1)]Cl, 1a (1a-hydrogel); (2) its self-aggregation and emission properties in aqueous solutions are pH-dependent; (3) it selectively forms aggregates in low-pH lysosomes in cancer cells; the accumulation of Pt(ii) complexes in lysosomes is suggested to contribute to its cytotoxicity towards cancer cells; (4) 1a-hydrogel exhibits sustained-release cytotoxicity towards cancer cells. Notably, the release activity of 1a-hydrogel can be stimulated by acidic environment; (5) 1a-hydrogel can encapsulate and deliver bioactive natural products, an example of which is the anti-metastatic, berberine.Pincer type platinum(omplexes . Platinuyridine) . Compare)]Cl, 1a , displayii) complexes containing various isocyanide ligands (pyr)Cl] with excess isocyanide ligand at room temperature for 12 h, and were characterized by 1H NMR spectroscopy, ESI-MS and elemental analyses \u2192 \u03c0*(L)) transitions of the C^N^Npyr ligand while the lower energy absorption beyond 375 nm can be assigned to singlet d\u03c0(Pt) \u2192 \u03c0*(L) metal-to-ligand charge transfer (1MLCT) transition.\u03bbmax at 503\u2013507 nm and lifetimes in the range of 7.4\u201315.6 \u03bcs, which are attributed to triplet excited states with mixed 3IL and 3MLCT character.The cyclometalated platinum were examined (1a displays green emission (\u03bbmax = 503 nm) in MeOH. Upon increasing the H2O content, the emission color gradually changes to yellow-orange \u2192 \u03c0*(C^N^N)] excited state. Parallel study using UV/Vis absorption spectroscopy also revealed the appearance of a new absorption band at \u223c375 nm, concomitant with the decrease of absorbance at \u223c350 nm were measured C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019NR1]+ cation at m/z 546.1 together with a singly charged cation at m/z 1091.2 attributed to the dimeric species (2[M]+ \u2013 H). All these findings are congruent with the formation of Pt(ii) aggregates in water, reminiscent of the previously reported self-aggregation behavior of [Pt(C^N^N) scale\" fill=\"currentColor\" stroke=\"none\">NR1)]Cl.The spectroscopic properties of examined . Complexw-orange . In 80\u20139 99% H2O ; this lom Fig. S4. The cryas anion displaysmeasured . At 298 1a could form hydrogels since the pyrazole moiety supports an intermolecular H-bonding interaction. Upon heating a suspension of 1a in water to 80 \u00b0C, the complex completely dissolved to give a clear solution complexes having different isocyanide ligands, 1b and 2\u20135, could form different nano-aggregates, including nanorods (1b), nanocubes (2), nanospheres (3), and nanofibers by evaporation of their ethanol solutions . Lam, Wong and coworkers reported that low pH (favoring the R2NH form) facilitates intramolecular Pt\u2013Pt interactions in dinuclear [Pt2(C^N^Npyr)2(\u03bc-dppm)]2+ while high pH (favoring R2N\u2013) does not.pyr)isocyanide]+ complexes in phosphate buffered saline solutions having different pHs . Taking 2 as an example, at pH 8, the complex in a mixture of buffer/acetonitrile showed a vibronic structured emission (\u03bbmax = 504 nm) which is typical of the emission of mononuclear [Pt(C^N^Npyr)isocyanide]+ complexes complexes were observed to form aggregates displaying red-shifted emissions at low pH containing single stranded DNA . The anionic phosphate backbone in ssDNA is known to facilitate assembly of cationic Pt(ii) complexes.1a shows a broad low-energy emission peaked at \u223c640 nm at pH 4 in the presence of ssDNA; however, there is no significant low-energy red emission in the absence of ssDNA or in the presence of peptide nucleic acid (PNA) which does not contain the anionic backbone. The broad prominent peak at \u223c640 nm observed at low pH is indicative of the self-assembly of the positively charged 1a ion on the nucleic acid backbone. Upon an increase in pH, the intensity of the long wavelength emission band decreases, which can be accounted for by the disassembling of surface bound Pt(ii) aggregates. It is conceivable that at high pH, deprotonation of pyrazole-H weakens the ionic binding interactions between the surface bound Pt(ii) complex and anionic ssDNA backbone, thus disrupting the intermolecular Pt\u2013Pt interactions along the surface of the DNA backbone. In contrast, the cationic form of the Pt complex in low pH undergoes self-assembly, which is synergistically enhanced by the ionic interactions between the cationic Pt complex and anionic DNA complexes accumulate in acidic lysosomes by fluorescence microscopy. After treatment of HeLa cells with 1a or 2 (10 \u03bcM) for 20 min, green emission could be detected in the cytoplasm or nuclei (Hoechst 33342) (R < 0.15). The existence of both green and orange emission in low-pH lysosomes is consistent with the aforementioned emission measurements in solutions of different pH.Lysosomes are acidic cellular compartments (pH = \u223c5) harboring acid hydrolases for the degradation of obsolete biomolecules and unwanted materials. We tested whether the Pt complexes in lysosomes, HeLa cells were incubated with nigericin, a H+/K+ ionophore that equalizes the pH across the lysosomes and cytosol to pH 8, in phosphate buffered saline (pH = 8), for 10 min after treatment of the cells with 2 for 1 h. As depicted in 2 for 1 h, the emission color in cells with nigericin treatment turned green. The disappearance of the orange-yellow emission after equalizing the cellular pH to 8 further suggests that formation of the intracellular aggregates was induced in acidic compartments such as lysosomes in the cancer cells. Complexes 1a, 1b, 3, 4, and 5 all demonstrated similar intracellular localization complexes in lysosomes was associated with an increase in lysosomal membrane permeability (LMP). HeLa cells were first stained with acridine orange, a cell-permeable dye which gives red fluorescence in acidic lysosomes but exhibits green emission in the cytoplasm and nucleus. The cells were then treated with 1a or 2 at different concentrations.1a or 2 at 5 or 10 \u03bcM led to dose-dependent reductions of red fluorescence, indicative of increased LMP.ii) complexes towards different cancer cells by means of MTT -2,5-diphenyltetrazolium bromide) under similar conditions. Complex 1a (2 \u03bcM) also inhibited anchorage-independent colony formation in HeLa cells under the same conditions.We then tested whether accumulation of platinum(bromide) and naphbromide) assays. Fig. S14. More im1a-hydrogel possesses sustained release properties in buffered solutions of varying pH by UV/Vis absorption spectroscopy. As shown in Fig. S16,1a-hydrogel showed time-dependent release activity at all pHs. It is noteworthy that 1a-hydrogel showed much faster release activity at pH 5 and 3 compared to pH 7.4 and 9. The sustained cytotoxicity of 1a-hydrogel towards HeLa cells was tested in double-chambered Transwell\u00ae 24-well plates (1a-hydrogel (100 \u03bcM) at different time intervals, the upper chambers were removed and the HeLa cells were further incubated in the absence of the hydrogel. After a total of 72 h, the cell survival percentage was quantified by MTT assay. As shown in 1a solution in the upper chamber caused the cell survival rate to rapidly drop to \u223c15% after incubation for 1 h in the lower chambers, and then putting the 1a-hydrogel to carry other therapeutic drugs such as berberine was examined. Berberine is a natural product with extensive pharmacological applications; its potential anti-cancer properties, especially its anti-metastatic property is well documented.1a-hydrogel) could also be formed after cooling a heated mixture of 1a and berberine in water to room temperature in the upper chamber for 4 h and 8 h. The wound of HeLa cells without treatment showed 93% recovery of the scratched area complexes containing C-deprotonated pincer ligands with pyrazole groups, which display self-assembly and anti-cancer properties. These Pt(ii) complexes form aggregates in low-pH buffer solutions, accumulate in acidic lysosomes, increase lysosomal membrane permeability and exert cytotoxicity towards different immortalized cancer cells and a primary cancer cell. One of these complexes, 1a, forms hydrogels in water, which displays sustained and pH-responsive release properties. The anti-cancer active 1a-hydrogel could also be used to deliver berberine, and, in principle, other therapeutic agents to achieve dual therapeutic effects.In summary, we have developed a class of platinum(Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "To further our understanding of the role of solution chemistry in directing nucleation processes new experimental and computational data are presented on the solution and crystallisation chemistry of tolfenamic acid (TA), a benchmark polymorphic compound. via dimers and clusters and raise experimental questions about how best to undertake relevant crystallisation studies.To further our understanding of the role of solution chemistry in directing nucleation processes new experimental and computational data are presented on the solution and crystallisation chemistry of tolfenamic acid (TA), a benchmark polymorphic compound. With these, and previously published data, we were able to establish that TA is rapidly fluctuating between conformers in solution with either solvated monomers or dimers present depending on the solvent. Hence, despite the fact that conformational polymorphs can be obtained from crystallisations in ethanol, we found no links between solution chemistry and crystallisation outcomes. We discuss the implications of these conclusions for the nature of the nucleation pathway R,S mandelic and benzoic acids crystallised from alcohols) yield harvested crystals which contain centres of symmetry despite the fact that strong solvation prevents the formation of dimers in the solution-phase.e.g. ethenzamide)In our quest to understand the molecular pathways involved in the nucleation of organic crystals from solutions, significant progress has been made over the last decade.In this contribution we explore further this central question of how much can really be learnt about nucleation from solution-phase experiments and associated computational studies. In particular we consider the essential levels of theory needed in the computational elements of such work; we question how best to characterise the solution species present; we also reflect on the question of how to measure the crystallisation outcomes of experiments so as to shed light on nucleation.et al.twisted or T hereafter) and a second more planar-like . These molecular conformations differ, as seen in \u03c4) which is \u00b174.95\u00b0 in Form I and \u00b1142.63\u00b0 in Form II. Although TA does not contain a chiral centre, its conformers are chiral. Because of the presence of an inversion centre in both polymorphs each crystal structure comprises both enantiomers (hence the \u00b1 sign for \u03c4). The enantiomers can in principle interconvert by rotation about \u03c4, but it is unlikely that this occurs spontaneously due to the high energy barrier. Further to this, it is also apparent that the transfer of the acid proton between oxygen atoms would offer the possibility of the existence of two tautomeric forms. Whilst both tautomeric forms have been studied in this work, the data for the metastable tautomer B is given in the ESIA, found in the solid state, is always the most stable in crystal, liquid and gas phases.As a vehicle for this journey we utilise tolfenamic acid (TA), 2-[(3-chloro-2-methylphenyl) amino] benzoic acid . This isFrom earlier studies,ca. 140 M\u20131 and 31 M\u20131 at 25 \u00b0C by the two techniques. These were interpreted in terms of the existence of carboxylic acid hydrogen-bonded dimers (HBD) in the ethanolic solutions. The reported relationship between supersaturation and the relative appearance of the two forms was then rationalised through the idea that increasing solute concentration creates increasing numbers of dimers and that, based on computational results, the most stable dimer is that in which the molecules adopt the Form I conformation (twisted). Thus the rationale was that at low supersaturations the solutions are rich in monomers displaying the conformer of Form II (planar) whilst at high supersaturations the solutions are rich in dimers displaying the conformer of Form I (twisted). Nucleation was then assumed to dominate the crystallisation outcome and the observations related directly to the solution chemistry.Mattei and Li have made an intensive study of the solution chemistry and crystallisation behaviour of TA Forms I and II.In our exploration of the solution chemistry \u2013 nucleation relations in this system we were first interested in re-examining the conformational energy relationships at varying levels of theory for monomer and dimers in both vacuum and solvent environments. Secondly, we wanted to review and extend the available spectroscopy in order to be clear about the nature of the solution species. Finally we wanted to extend the crystallisation experiments of Mattei and Liet al.Conformer energies were computed in the gas-phase and with various implicit solvation models using GAUSSIAN09.\u03c4 was generated by optimising molecular geometries with \u03c4 constrained every 10\u00b0 between \u2013220\u00b0 to 220\u00b0. All calculations were performed in an SMD solvation model for ethanol at the B97D/6-31+G level of theory.The potential energy surface (PES) of TA about G) is the sum of the electronic energy plus the thermal free energy (G(T) = Ee + Gcorr(T) where Gcorr(T) is calculated from the frequency analysis). Ee was re-computed via a single point energy calculation of the optimised geometries with the same functional but a larger basis set (B97D/def2QZVPP). The use of the large basis sets for this calculation ensures minimisation of the basis set superposition error. To minimise computational costs, the Gcorr term was computed with the smaller basis set B97D/6-31+G model only. The free energies of the dimers were then calculated at different temperatures as the difference between the free energy of the dimer (with either planar or twisted conformation) minus the free energy of two monomers with the planar conformation (since this is the most stable conformer according to our models).Gd(T) = Gdimer-planar/twisted(T) \u2013 Gmono-planar(T) \u2013 Gmono-planar(T).\u00a7\u0394 Similar models have recently been used for the computational study of self-association of various carboxylic acids in solution.20Geometry optimisations and frequency calculations of several dimer and monomer models were computed, free of constraints, at the B97D/6-31+G level of theory in the gas-phase and in the six different SMD solvent models. The Gibbs free energy was purchased from Sigma Aldrich and used without further purification. TA Form II was crystallised by crash cooling an ethyl acetate solution (3.45 g TA Form I and 50.00 g of ethyl acetate) to 10 \u00b0C. Both forms were isolated as pure phases as judged by their powder X-ray diffraction (PXRD) patterns. Ethyl acetate (EtOAc), ethanol and 2-propanol were purchased from VWR International Ltd. (UK), toluene from Fischer Chemicals and deuterated ethanol (EtOD) from Sigma Aldrich (>99.5%D). All solvents were of analytical reagent grade and the molar purities were >99.5%.Powder X-ray diffraction (PXRD) was performed using a Rigaku miniflex X-ray powder diffractometer at a wavelength of 1.5406 \u00c5 controlled by DIFFRACPLUS software from 4\u00b0 to 40\u00b0 with a step size of 0.03\u00b0.\u20131 resolution. The spectra were corrected for the solvent contribution by recording solvent spectra in the same liquid cell and subtracting these from the solution spectra.The FTIR spectra of solutions of TA in EtOD and deuterated toluene were recorded in 0.50 or 1.00 mm thick liquid-sample cells, using a Perkin Spectrum Two spectrometer with 2 cm\u20131 was used.Differential Scanning Calorimetry (DSC) experiments were performed using either a Mettler Toledo DSC 30 instrument controlled by Mettler TC15 complete with a liquid nitrogen cooling system with data analyzed by STARe software v.610 or a TA DSC Q100 with software universal analysis 2000 v. 4.5A. A heating rate of 10 K minThe crystallisation of TA was investigated in crash cooling experiments in toluene, ethylacetate, 2-propanol and ethanol. These experiments were carried out using a 50 mL jacketed vessel with an overhead 2-blade impeller stirring at 200 rpm. Solutions at different concentrations were prepared by dissolving the corresponding amount of TA Form I in 40 g solvent. The solutions were kept at 60 \u00b0C for 1 h to ensure that all the crystals were dissolved completely. 10 mL aliquots of the solutions were then withdrawn and filtered through a pre-heated 0.2 \u03bcm syringe filter, transferred to the jacketed vessel pre-set to the desired crystallisation temperature . The crystals were filtered immediately after nucleation and dried at room temperature for 0.5 h. Each experiment was repeated 5 times and both PXRD and visual observation (colour) were used to identify the polymorphic forms of the product crystals.\u20131 at 10 \u00b0C.Lattice energy calculations, thermal analysis, slurry and solubility measurements were used to determine the thermodynamic relationship between Forms I and II TA see ESI. Forms Iet al.22\u20131 .The relative stability of the TA conformers and the energy barriers for conformational change are key for the understanding of the role played by conformational flexibility during crystallisation. We have computed the stability of the various possible monomeric species of TA using several computational methods and compared the results with previous literature reports in the ESI.\u03c4 in ethanol also for tautomer A and for both enantiomers of TA about the rotatable bond ntiomers . The enevia stacking as presented in Considering the crystal structures of Forms I and II TA as guides, two types of self-assembly modes may be envisaged for TA : (1) thrIt is clear that solvatioIn comparing the relative free energies of HBDs to SDs at room temperature, it appears that HBDs are always preferred in non-polar media while both types of dimer are similarly stable in alcohols and DMSO. Only in water do SDs become more stable than HBDs. With respect to the molecular conformation, dimers with the planar conformer usually appear to be more stable than those with the twisted one. The energy differences, however, are small so dimerization is unlikely to be a cause of conformational restriction.et al.et al.1H-NMR spectra of TA in acetone were recorded at various temperatures. They concluded that the spectra did not show significant variations in the range 210\u2013290 K, indicating that TA does not exist in any favoured conformation under those conditions. Mattei et al.1H-NMR chemical shifts in ethanol with temperature and concentration and observed only very small variations in chemical shifts. This was particularly true as a function of concentration where the observed dependencies were interpreted in terms of molecular dimerisation in solution at increasing concentrations and decreasing temperatures. It was explicitly assumed that this dimer was identical to the H-bonded dimer found in the crystal structures and, following their computational results, that such a solution dimer should bear the twisted conformation. We have reexamined the available experimental data15Proton NMR experiments of TA solutions have been performed by Andersen \u03c4 in 10\u00b0 increments. It is clear from \u03c4 values between 110\u00b0 and 135\u00b0.To understand how the chemical shifts of the different protons change with conformation, we computed NMR chemical shifts for all conformations studied in the PES of TA at values of \u03b4exp = a\u03b4pred + b. This procedure was done twice, first with the monomeric species and second with the dimer species .In order to compare how well the chemical shift predictions for the two conformers of TA reproduce the experimental values, we carried out linear regression on the predicted against the experimental chemical shifts . NMR chea and the good of fitness R2 are presented in \u03c4 and that there is no preferred conformation.The resulting values for With respect to aggregation, the experimental H-NMR chemical shifts of low and high concentrated solutions hardly change see . The cal PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O group (\u223c1700 cm\u20131) of TA in ethanol as a function solution concentration. The C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O-stretch mode is a very sensitive probe of carboxylic-acid dimerization. This is partly because of hydrogen-bond induced weakening of the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O bond, but mainly because transition-dipole coupling between the two C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O bonds in a dimer results in a Raman-active symmetric and an IR-active antisymmetric C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O-stretch mode which both have frequencies very different from that of the monomer. PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O-stretch of a dimer has a frequency 40\u201350 cm\u20131 lower than the monomeric C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O-stretch . PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O-stretch and the dimer antisymmetric C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O-stretch), and their relative intensities depend strongly on concentration: at low concentration the monomer peak dominates, at high concentration the dimer peak. In the IR spectra of TA in ethanol scale\" fill=\"currentColor\" stroke=\"none\">O-stretch peaks, but the frequency splitting is only 20 cm\u20131, much less than would be expected for a monomer-dimer difference. More importantly, the intensity ratio of the two peaks is completely independent of concentration, which we varied over more than an order of magnitude scale\" fill=\"currentColor\" stroke=\"none\">O-stretch peaks cannot be due to TA dimerization. We ascribe this difference to a TA\u2009:\u2009ethanol hydrogen-bonding equilibrium scale\" fill=\"currentColor\" stroke=\"none\">O-stretch frequencies corresponding to hydrogen-bonded and non-hydrogen-bonded CO groups, respectively). In fact, a solution of N-methylacetamide in methanol has a similar, concentration-independent two-peak C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O-stretch spectrum due to the formation of C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O\u00b7\u00b7\u00b7H\u2013O hydrogen bonds by part of the molecules,\u20131, exactly the same value as observed here.In order to shed further light on this issue we have used FTIR to monitor the band associated with stretching of the C ethanol we obseragnitude . Hence, Our overall conclusion from both the existing NMR data and new FTIR results is that ethanolic solutions of TA are unlikely to contain hydrogen-bonded or stacked dimers, rather the solutions are populated with solvated monomeric species in which the chlorine-containing ring oscillates between the twisted and the planar conformers. The calculations carried out in the previous section also suggest that monomers are preferred over dimers in ethanol solutions.We have proven in the previous sections that TA exists as a monomer in ethanol solutions. But, what species should be favoured for TA in toluene? Intuitively, toluene is a non-polar aromatic solvent so we might expect it to interact more strongly with the aromatic side of the TA molecule but not with the carboxylic acid group. In fact, the calculations in PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O band for TA solutions of various concentrations in toluene scale\" fill=\"currentColor\" stroke=\"none\">O-stretching peaks at \u223c1700 cm\u20131 and \u223c1660 cm\u20131 respectively. The intensity of the dimer band at \u223c1660 cm\u20131 increases relative to the intensity of the monomer band at \u223c1700 cm\u20131 in going from a 2 mM to a 4 mM solution of TA in deuterated toluene scale\" fill=\"currentColor\" stroke=\"none\">O-stretch spectrum are due to water-vapour absorption and supersaturations (S) were studied. Detailed results are given in the ESIvia transformation.The polymorphic outcomes of crash cooling crystallisation in toluene, ethyl acetate, 2-propanol and ethanol at different temperatures to the most polar solvent (ethanol) solutions will go from being dimer to monomer rich. Using this insight, together with the data in Firstly it seems clear from both the computations and our comments on the available NMR data that for TA, conformation change is facile so that a crystal nucleating or growing from a monomer rich solution will be insensitive to the existence of the two conformers. Hence each molecule is a potential growth unit and we would expect no conformationally driven outcomes when crystallisation occurs from monomeric solutions. If crystallisation takes place from a dimer-rich solution then the situation is unchanged since the computations suggest that H-bonded dimers (like monomers) show no significant conformational preference. Our analysis shows no experimental evidence from NMR to support the existence of a preferred conformation in ethanol solutions.Considering dimerization, on the one hand ethanol solutions appear to favour the solvation of the acid group and are hence, monomer rich. Toluene solutions, on the other hand, seem to favour the formation of HB dimers. Crystallisation results, however, show that both monomer and HB dimer containing solutions follow Ostwald's Rule of Stages.In considering the creation of crystal nuclei it is worth noting two points. Firstly there is nothing about the solution chemistry that would select a particular conformer for incorporation into a critical cluster. Secondly, while the existence of solution phase hydrogen-bonded dimers between enantiomer pairs could offer a pathway for the creation of the required crystallographic inversion centre, their absence means that both the conformation and the centre of symmetry must be determined at some other stage during nucleation. How this occurs remains an open question. For example, the expulsion of solvent from a disordered aggregate might enable the formation of dimers: with HB dimers a molecule has only to find its mirror image to begin the process of crystallisation. However, if we make an analogy with micellisation, a clustering phenomenon of amphiphiles in polar solvents, then we might imagine that a critical assembly of TA molecules has an aromatic core with acid groups at its surface, making use of the solvating power of the solvent. In this situation the drive towards crystalline order may be initiated by the attainment of stacking interactions. However since stacked dimers as found in Forms I & II comprise only one enantiomer, the centre of symmetry must be created through a subsequent self-assembly process between stacks and this might be seen as less likely. The development of H-bonded dimers and consequent centres of symmetry Finally, we note that other type of aromatic interactions between two TA molecules can be generated by inversion and are observed in Forms III and IV. However, these are less energetically favoured than the SDs considered here as found in Forms I & II. Interestingly, Forms III & IV can only be nucleated on surfaces of non-polar aromatic polymers.12In attempting to understand the relationship between crystal nucleation, solution chemistry and molecular conformation, it may be considered that TA is a suitable benchmark system. Prior to our current work, existing publications suggested that all that should be done on this system had been done \u2013 the crystal structures of two conformationally distinct forms had been reported, phase behavior and solubilities had been measured, crystallisation outcomes were recorded under well-defined conditions and significant investigations of solution chemistry made. All this had been combined with computational chemistry to develop a self-consistent interpretation relating crystallisation to dimerisation and molecular conformation.no energetic preference for the solid state conformers and in which there are no dimers to offer a pathway for symmetry and structural control during the self-assembly process. Importantly, it is worth considering how to proceed both experimentally and computationally when exploring such problems and what data we lack in trying to resolve the molecular issues surrounding the nucleation transition state.Having repeated and extended both experimental and computational aspects of this system, it now appears that there is no link between its solution chemistry and conformational polymorphism. Both additional experiments and higher-level computations have shed new light on the energetics of TA conformers and on the species existing in ethanolic solutions. It now appears that a comprehensive nucleation model for this system must be able to explain how the conformational polymorphs arise from a solution in which there is The initial impetus for study of this system came from the apparently anomalous outcome of the original crystallisation experiments which we were unable to repeat. Crystallisation is a response to an intimate combination of phase equilibria and kinetics and so we are not surprised that different workers, in different labs, using different equipment, chemicals of different purity and ethanol of different water contents should obtain different results. This possibility is well known to those active in the field and reflected in so-called \u2018disappearing polymorphs\u2019.28J and the growth rate constants, k3, of the two polymorphs that determines the experimental outcome. Subsequent workers, however, have continued to use the final, macroscopic outcome of crystallisation experiments to infer structural information about nucleation, ignoring the contribution made by growth.35Perhaps a more important consideration is the question of exactly which crystallisation experiments should be pursued to define the nucleation characteristics of a given system. Workers in the field have become used to the idea that structural characterisation of the final macroscopic crystals in any given experiment provides a link to nucleation. In their 1983 paper concerning the crystallisation of stearic acid polymorphs from cyclohexanone, Sato and BoistelleThe computational results of different workers should not be subject to the same variation in outcomes as the experiments. Our work confirms this but additionally highlights the need to perform calculations at suitable levels of theory in order to achieve the most reliable results, at least for this molecule in which intra- and intermolecular interactions need to be accurately modeled.a priori that a motif present in a crystal structure is also present in solution is a very dangerous assumption to make.Finally we note the difficulty in characterizing uniquely the state of molecular assembly in solutions. Solutions and their dynamic nature are of course fully characterised only in terms of the radial distribution functions describing the environment of the different atoms on the solute/solvent pair. Techniques such as NMR will only be useful in cases where dimers or conformers are particularly stable and abundant. FTIR offers a different view on the state of interactions of specific functionalities with their environment but as yet from the position and intensity of a particular absorbance it is hard to make a unique assignment concerning the intermolecular interactions involved. Certainly, to assume Supplementary informationClick here for additional data file."} +{"text": "New cyclooctenes have been synthesized under continuous-flow conditions and applied in ring opening metathesis polymerization, providing highly functionalized materials. Functionalized cyclooctenes (FCOEs) are important monomers in ring-opening metathesis polymerization (ROMP). Herein, a new library of disubstituted FCOEs bearing adjacent heteroatoms were synthesized and applied in ROMP. To address the issues associated with the handling of the reactive thienyl chloride intermediate, a two-step continuous flow method has been developed to prepare 5-thio-6-chlorocyclooctene compounds from abundant cyclooctadiene starting materials. These newly synthesized FCOE monomers were subsequently polymerized through ROMP, giving rise to a range of functionalized polymers with high molecular weights. Furthermore, we demonstrated that the thermal properties of these polymers could be fine-tuned by changing the functional groups in the FCOE monomers. We expect that this functionalization-polymerization strategy will enable the preparation of a range of polymeric materials with complex structures. The development of synthetic methods to access functionalized polymers is of considerable interest due to the interesting physical and chemical properties associated with these materials. As a result, extensive efforts have been made to accomplish this task by designing well-tailored monomers for different synthetic methods, such as controlled radical polymerizationvia C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond addition of cyclooctadienes (CODs),9FCOE derivatives are a class of the most widely used monomers for ROMP.In contrast to monosubstituted FCOEs, polysubstituted FCOEs are much less investigated for ROMP reactions. PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond addition to prepare FCOEs from cis,cis-1,5-COD . Although the thienyl chloride (RSCl) species has been known for over half a century, the explosive natureIn this regard, we have designed a two-step sequence of thienyl chloride formation/C1.000000,.000000 sp-toluenethiol 1a as the model substrate. In the flow setup, a solution of 1a in anhydrous dichloromethane (DCM) was mixed with SO2Cl2 in anhydrous DCM and introduced into a tubing reactor (R1) immersed in a cooling bath. After the arylthiol was completely converted, as monitored by thin layer chromatography (TLC) analysis, R1 was assembled with the following setup of step II via a T-mixer, allowing the solution from R1 to combine with the COD (3) solution in-line. The resultant mixture was further delivered into the second tubing reactor (R2), which was submerged in another cooling bath, to perform the direct difunctionalization of the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C double bond. After the reaction, the mixture was collected and directly analyzed without the isolation of 4a. Upon investigating a variety of reaction parameters, we determined that the synthesis of 4a proceeded in good yield with a 1/1.05/4 ratio of 1a/2/3, and two reactors cooled at 0 \u00b0C and \u201320 \u00b0C respectively (tR) of less than 4 min.4a investigated in 5b\u20135g in about 4 h of reaction time (5b\u20135g in satisfactory yields (55\u201370%). Notably, since aryl halides (e.g. Cl and Br) are versatile functional groups in metal-catalysed cross-coupling reactions, the incorporation of such groups (5c and 5d) would bring in reactivity orthogonal to the substituent on the COE backbone.1 and OMe into the COEs.With the method established for the preparation of 5, a three-step continuous-flow setup has been developed in DCM at room temperature (step IV).5a), an electron-withdrawing group , or a phenyl group , affording a variety of functionalized polymers in high yields (6a\u20136f: 90\u201396% yields) following isolation via a three-time precipitation from methanol. Similar to the Ru-promoted ROMP of alkylthio mono-substituted COEs reported by Noels and coworkers,nC12H25S,6-MeO-COE was used a decreased polymerizing reactivity was observed, providing 6g with 36% monomer conversion in 48 h of reaction time. This is probably due to the increased coordinating effect of an alkylthio group to the metal center compared to that of an arylthiol group. For all examples (5a\u20135g) investigated in Mn,GPC = 106\u2013311 kg mol\u20131, \u0110 = 1.49\u20131.73) were obtained, further confirming the reliability of the ROMP of these new FCOEs EtOAc in petroleum ether as an eluent. During the column chromatography process, 4a underwent a full hydrolysis within 30 min, resulting in the cyclic olefin 7a which has a hydroxy handle. Upon reaction with different electrophiles (step IV), the hydroxy handle was readily connected to a t-butyldimethylsilane , a benzyl , or an acetyl group. Additionally, the chloro group was also converted to an N-heteroatom containing substituent by simply reacting with a nucleophile . Although 3\u20134 steps were employed, compounds 7a to 7f were isolated in good overall yields, and these compounds were characterized by NMR, IR and HRMS analysis scale\" fill=\"currentColor\" stroke=\"none\">C double bond keeps a cis configuration, the SAr group and the morpholine group are trans to each other. This is consistent with the vicinal SR group assisted substitution process, which could proceed through a thiiranium ion intermediate.A solution containing COE 7a\u20137h) were next polymerized in the presence of G2 at room temperature . Although decreasing the monomer/G2 ratio to 20/1 led to complete monomer conversion within 24 h, 8a\u2032 with a much lower Mn,GPC of 6.8 kDa was provided (entry 2), with a \u0110 value similar to 8a . We hypothesized that the improved monomer conversion was due to less of the transition-metal being poisoned by increasing the G2/monomer ratio. When the reaction temperature was increased from room temperature to 45 \u00b0C, poly(FCOE)s were generated with a similar Mn and slightly improved control over the molecular weight distributions . When the third-generation of Grubbs carbene complex (G3) was used to initiate the ROMP of 7b ([7b]/[G3] = 200/1) at room temperature, the corresponding polymer was produced with \u0110 = 1.65 and Mn = 94 kDa at >99% conversion.The newly synthesized FCOE monomers . When the SR1 group was adjacent to a morpholine group instead, polymer 8f was isolated in 82% yield . Both NMR and IR analyses clearly demonstrate that both types of functional group have been successfully incorporated in polymers 8a\u20138f s with high molecular weights, a monomer/G2 ratio of 500/1 was used during the ROMP reaction of the other FOCEs. When 5a and 7f were employed. As shown in Mn,GPC values, while the \u0110 values stayed at a similar level s can be generated at the desired Mn by choosing a proper [M]/[G2] ratio within the investigated range.22To investigate the ROMP of difunctionalized FCOEs at different monomer/G2 ratios, 6a\u20136g and 8a\u20138f were analyzed by differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA). The summarized results of their glass-transition temperature (Tg) and decomposition temperature (Td) are shown in 6a to 6g, while keeping the MeO group constant, changing the alkylthio side chains to arylthio chains resulted in polymers possessing increased Tg values (6g: \u201356 \u00b0C vs.6a\u20136f: \u201312 \u00b0C to 35 \u00b0C). Among 6b\u20136g, an increased functional group size on the aryl ring or an increased degree of conjugation led to increased Tg values. These results are in agreement with the sidechain influence on the glass transition temperature, as observed by others.8b\u20138e, when the hydroxy side groups were protected with groups larger than methyl, the resultant Tg values were higher than 6b (8b\u20138e: 0\u201318 \u00b0C vs.6b: \u201312 \u00b0C). Replacement of the MeO group with a morpholine group also led to an increased glass-transition temperature (8f: 45 \u00b0C vs.6a: 4 \u00b0C). The thermogravimetric analysis in 6a\u20136g and 8a\u20138f possess Td values ranging from 225 \u00b0C to 350 \u00b0C at 5% weight loss.The thermal properties for the polymers 6e was conducted to demonstrate the preparation of linear polyolefins possessing two different side chains on every seventh and eighth backbone carbon, from the corresponding poly(FCOE)s. The hydrogenation reaction was performed using p-toluenesulfonylhydrazide as the reductant and tri-n-hexylamine as the base with a catalytic amount of 2,6-di-t-butyl-4-methylphenol (BHT) in o-xylene solvent.9 was obtained in 88% isolated yield via precipitation from methanol. As shown in the 1H NMR spectra no new shoulder peaks in the GPC traces, suggesting that the polymer backbone remains intact during the reduction process. Moreover, the hydrogenated polymer 9 has a lower Tg value than 6e s.Finally, the hydrogenation of polymer spectra , during 6e and 9 show: in anhydrous DCM, and fitted to the syringe pump. Another syringe was loaded with a solution of 2 in anhydrous DCM, and fitted to a same syringe pump. The third syringe was loaded with a solution of COD in anhydrous DCM, and fitted to the second syringe pump. Following the setup as shown in 1a and 2 were mixed and reacted in the tubing reactor R1 submerged in a cooling bath. When the reaction was complete, the resultant solution was mixed with the solution of COD and reacted in the tubing reactor R2 submerged in another cooling bath. After the reaction, the resultant mixture was passed through a back-pressure regulator before collection. After reaching steady state (waiting for 12 min), 1.0 mmol samples (10 mL reaction solution) were collected into an oven-dried vial equipped with a stir bar.The experimental procedure for the preparation of via syringe at room temperature. When the reaction was completed, as monitored by TLC analysis, the mixture was treated with DCM (150 mL) and NaHCO3 saturated aqueous solution (20 mL). The separated organic layer was washed with brine two times (2 \u00d7 10 mL), dried over Na2SO4 and then concentrated under vacuum. The residue was purified by column chromatography (eluting with 0\u20132% EtOAc in petroleum ether) to afford 5a in 64% isolated yield.Anhydrous MeOH (10 mmol) was added into the vial 5a (0.5 M) in anhydrous DCM under N2. The G2 compound solution was added via micro syringe into the vial at room temperature. After stirring for 24 h, the mixture was concentrated and then added dropwise into MeOH with vigorous stirring. The solid compound was collected and re-dissolved in a minimal amount of DCM. The precipitation procedure was repeated three times in total to afford the target product. The produced polymer was characterized by 1H NMR, 13C NMR, FT-IR, GPC, DSC and TGA analysis.An oven-dried vial equipped with a stir bar was charged with a 1.0 mL solution of There are no conflicts to declare.Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "Quantitative dihydroboration of cyclic dienes leading to new, tunable boron-containing hydrocarbon polymeric materials. 11B NMR to identify the actual boron species formed, as opposed to the most common analysis of the resultant oxidation products. Quantitative dihydroboration was achieved for the full range of cyclic dienes investigated including dienes, which were previously reported to be resistant to dihydroboration, leading to the formation of new boron-containing polymeric materials. The conditions favoring dihydroboration are reported as well as full characterisation of the materials. Furthermore, a hydroboration cascade mechanism is proposed for the formation of such boron-containing polymers, supported by both experimental and theoretical data.The hydroboration 1,3- and 1,4-cyclic dienes has been systematically investigated. The behavior of such dienes towards mono and dihydroboration was monitored directly by We found the exceptional resistance of smaller rings towards dihydroboration, even in the cases where excess hydroborating agent was used, rather puzzling. Although a diene is certainly a more electron rich system when compared to an alkene, the non-conjugated nature of the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond present in the formed monohydroboration products could potentially compel them to be more reactive than the starting diene, on account of the non-conjugated double bond compared to the initial system. Additionally, both the cyclic diene and the borane reagents (both the starting borane and the formed products) are capable of undergoing further hydroboration reactions. Thus, one might expect the formation of polymeric and/or network materials, as was previously reported in the case of straight-chain dienes31It is possible that dihydroboration of cyclic dienes to generate diols has not been thoroughly explored as a result of the strong and contradicting conclusions drawn in a series of published studies by H. C. Brown i.e. by identifying the boron species present.1 was used.26As a result of our interest in preparing substituted diboranes, we aimed to systematically investigate the hydroboration of cyclic dienes in order to identify the suitable conditions for dihydroboration of smaller rings. While the results and conclusions by Brown 1 utilized by H. C. Brown et al.3\u00b7SMe2vs. B2H6 prepared from NaBH4 and BF3\u00b7OEt2).We first focused our attention on the number of equivalents of diene present in the reaction, as the large excess of PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bonds were only observed in entry 1, while the borane starting reagent was fully consumed. Analysis of the filtrates obtained for entries 2\u20134 , revealed large amounts of unreacted starting borane, seen as a quartet at \u201320 ppm in the 11B NMR. Furthermore, the absence of any C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bonds in the 1H NMR for these entries suggested that full dihydroboration has taken place utilising all C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bonds.In order to fully characterise the reaction, the precipitates were collected by filtration (under nitrogen) and the filtrates were analysed by NMR. All B\u2013H bonds appeared to undergo hydroboration, as unsaturated Cin situ, the same trend was observed gas production. This was achieved by either adding BF3\u00b7OEt2 to a solution containing both NaBH4 and 1, resulting in a slow release of the desired gas (as the reaction proceeds via the formation of the NaBH4BH3 complex first), or by addition of NaBH4 to a solution containing both BF3\u00b7OEt2 and diene 1, which resulted in the instantaneous release of diborane(6). As in the case of BH3\u00b7SMe2, all reactions yielded a white precipitate, with only NaBF4 being observed in the filtrate, appearing at approximately \u20130.4 ppm in the 11B NMR, indicating reaction completion.When the source of borane was changed to diborane(6), produced rved see . As dibo3\u00b7Lewis base adducts,4 serving as indirect evidence that boron hydrides are present in these materials indicating the presence of trapped solvents, which was confirmed by solid state NMR. Moreover, this strongly suggested the formation of a polymeric network. Addition of Lewis bases such as PPh2.1.1\u20131), terminal B\u2013Ht (2600\u20132500 cm\u20131), bridged B\u2013Hb (1600\u20131500 cm\u20131) and B\u2013C (1200\u2013800 cm\u20131) bonds and the absence of any C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bonds (1800\u20131700 cm\u20131). All solids analysed resulted in similar spectra despite originating from a number of different reaction conditions. The FT-IR spectra of four selected materials are shown in vide infra). The B\u2013Ht bond stretches could indicate either presence of a R2BHBH3 species (see 2BH)2 species and unreacted borane or diborane(6) starting material, due to R2BHBH2R or RBH2BH2R, or unbridged R2BH species or even due to BH3 trapped inside the material network. The latter option, however, could be dismissed based on solid state NMR data scale\" fill=\"currentColor\" stroke=\"none\">C double bonds in both the filtrate and the solid confirmed that full dihydroboration had taken place.Analysis of the materials by FT-IR under anaerobic conditions indicated the presence of C\u2013H .8 remained soluble in ether and could be isolated upon concentration followed by recrystallisation (+81 ppm in solution 11B NMR). The dominant formation of dicyclohexylborane 9 (R2BH)2, especially at the earlier stages of hydroboration, suggested that it was the kinetic product, which could be converted to the thermodynamic tricyclohexylborane 8 over time as opposed to the instantly pyrophoric and highly unstable tricyclohexylborane 8. The direct dependence on the rate of reagent addition towards the formation of kinetic product 9vs. thermodynamic product 8 led to materials with different physical properties and reactivities. For example, materials which are mainly consisted of the kinetic products bearing the boron hydride bridges are highly moisture sensitive but not pyrophoric. When these materials where exposed to moisture they transformed from solid to liquid clearly breaking the B\u2013H\u2013B bridges which hold the insoluble polymeric network together. We are currently investigating the applications of these materials especially as instant moisture scavengers.Three equivalents of cyclohexene 2.1.311B NMR analysis of model compounds 8 and 9 indeed allowed for deconvolution and assignment of the NMR spectra obtained from the white insoluble materials prepared by hydroboration of cyclohexadiene 1. As seen in 11B resonance from tricyclohexylborane 8 has a relatively broad line shape due to large anisotropic interactions , which are not efficiently reduced by magic angle spinning at 10 kHz. The isotropic chemical shift of +82 ppm fits well with the value measured in solution 11B NMR scale\" fill=\"currentColor\" stroke=\"none\">C bonds critically points to quantitative dihydroboration of 1 using a variety of borane sources and reaction conditions.The remaining signals appearing at approximately +50 and +18 ppm most likely correspond to other boron hydride species such as RR2BHBH2R . This is2.1.412 was observed in the absence of any unsaturated C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bonds. The formation of this alcohol could be attributed to a diborane elimination side reaction similar to what is shown in 10, is highly unlikely in the absence of a driving force, such as a Lewis base. Perhaps, SMe2, THF (if present), diglyme or even a species formed during the reaction could mediate such an elimination. Protonolysis of the \u03b1-boron atom, in the 1,2-disubstituted diborane case, could also possibly lead to the formation of an oxidised alkylborane which resembles a carboxylic acid ([(RO)2B(OR)OH]\u2013) thus eliminating one of the boron atoms and replacing it with a hydride. However, we believe that this is unlikely due to the large amounts of cyclohexanol 12 observed in some cases and the highly uncontrolled nature of this hydroboration system leading to a statistical mixture of 1,2-, 1,3- and 1,4-substitution patterns. Furthermore, during our studies we observed that \u03b2-hydride elimination of diboranes is possible, however, such an elimination followed by oxidation would not result in the formation of cyclohexanol but rather the unsaturated compound 2-cyclohexene-1-ol 2. Finally, elimination reactions caused by oxidation would also produce alcohols with C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C double bonds rather than cyclohexanol and therefore must also be discounted. We are currently further investigating the mechanism of this elimination.Basic oxidation of these insoluble materials with hydrogen peroxide, adapted from literature procedures1 combined with the trifunctional nature of BH3 and the energetic stabilisation gained from the formation of B\u2013H\u2013B bridges is akin to an A2 + B3 step-growth polymerisation leading to insoluble, crosslinked networks. Moreover, the solids readily transform into liquids upon exposure to air, further suggesting that oxidation leads to facile disruption of the borane-hydrocarbon polymeric network. Finally, the dependence of the materials' physical appearance on the reaction solvent (glassy in THF and a white powder in diglyme) together with the high solvent content present in these materials are also indicative of the formation of a polymer network.Several attempts were made to avoid the formation of the insoluble precipitates including lowering the reaction temperature (\u201378 \u00b0C), decreasing the concentration, or carrying out the reaction in non-ethereal solvents, known to considerably slow down the rate of hydroboration, such as DCM.2.2via a cascade of hydroboration reactions. Detailed inspection of the boron species formed during hydroboration by NMR gave rise to the following proposed mechanism shown in 1 undergoes monohydroboration to form either an allyl RBH2 species 13a or homoallyl RBH2 species 13b. The ratio of 13a to 13b is likely to be highly dependent on the particular diene structure and is beyond the scope of this study. However, it was expected that the allyl RBH2 species 13a is slightly more stable compared to the homoallyl 13b on account of conjugation with the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond. Indeed, during our calculations we found that the BH2 group is stabilised in an axial position, when SMe2 is absent, by interactions with the adjacent double bond that partly delocalises to the boron 2p orbital , the equatorial conformation is preferred. It is worth noting, that the formed RBH2 species most likely exist as dimers, which contain B\u2013H\u2013B bridges providing substantial stabilisation,We believe that these materials are polymeric species formed 1 and borane. This was especially evident on account of the unreacted borane observed in solution after the consumption of all C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bonds, entries 2\u20134 in 1 leads to the formation of an isolated C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond, which was observed to be highly reactive (at least under our conditions) compared to the conjugated starting material 1, leading to a cascade of hydroboration reactions and concomitant formation of a cross-linked polymeric network as the monohydroboration species continue to react preferentially.Once hydroboration occurs, both the monohydroboration and dihydroboration species are much more reactive than the starting materials, conjugate diene 3 (or substituted RBH2 species) by breaking the solvent\u2013borane adduct or the borane dimer. The second step is the addition of the BH3 to the double bond and usually has a smaller barrier.3 intermediate is stabilized by short-lived intermediate complexes formed with the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond containing reaction partner. We found that the barrier of the first step is reduced from +26.1 kcal mol\u20131 to +14.6 kcal mol\u20131 using BH2-cyclohexene, to +15.0 kcal mol\u20131 using 1,3-cyclohexadiene 1, and to +13.8 kcal mol\u20131 using cyclohexene are used with electron-donating R groups, even when the free RBH2 is not further stabilised by adduct formation with a solvent molecule or a C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond. With R = cyclohexyl (or cyclohexenyl), the barrier to generate a free BRH2 is +13.3 kcal mol\u20131 , which is +12.7 kcal mol\u20131 smaller than for the dissociation of the BH3\u00b7SMe2 complex can further react rapidly and hydroborate another cyclohexadiene molecule . It is worth noting that the second hydroboration reaction can produce eight distinct diborane cyclohexane isomers in general if the chair-boat conformational change is not hindered, and many more in the conformationally restrained polymers. The formation of each isomer has different potential reaction mechanisms and corresponding rates. Interestingly, the intermolecular hydroboration, with the bridged cis-1,2-cyclohexane scale\" fill=\"currentColor\" stroke=\"none\">C bond, in relation to the sterically demanding group, was crucial in the formation of these materials, which were not limited by ring size.Intrigued by the results of our study on 1,3-cyclohexadiene, we expanded the substrate scope to include a range of different cyclic dienes including: \u03b1-terpinene, 1,3-cycloheptadiene, 1,3-cyclooctadiene, 1,3,5,5-tetramethyl-1,3-cyclohexadiene, \u03b3-terpinene, 1,4-cyclohexadiene, 1,2,4,5-tetramethyl-1,4-cyclohexadiene, 1,5-cyclooctadiene and the acyclic 2,3-dimethyl-1,3-butadiene as a control and direct comparison to the reported polymer structures (see ESI S9, Fig. S493et al.To conclude, boron containing polymers form when cyclic 1,3- or 1,4-dienes, free of sterically demanding groups are fully hydroborated, irrespective of the cyclic diene size, borane equivalents and mode of addition. When steric hindrance is present or cyclic borane species are favourably formed, as for example when straight chain of 1,5-dienes are utilised, clear solutions are observed. These materials were characterised by solid state FT-IR and NMR and were found to consist of boron hydride bridged species due to stabilisation reasons and the synthetic time scale utilised. Further supported by oxidation analysis, it was clear that hydroboration proceeds further than the monosubstituted borane adducts to form higher substituted, more stable, bridged borane species. Therefore, our results are in disagreement with the published conclusions on the study of dihydroboration of cyclic dienes by H. C. Brown Supplementary informationClick here for additional data file.Supplementary informationClick here for additional data file.Supplementary informationClick here for additional data file.Supplementary informationClick here for additional data file.Supplementary informationClick here for additional data file.Supplementary informationClick here for additional data file.Supplementary informationClick here for additional data file."} +{"text": "Amination of a silylated ester generates an intermediate urea that transfers an aryl ring to the aminated centre and cyclises to a hydantoin. PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N bond of an azocarboxamide, generating a N-amino-N\u2032-aryl urea derivative of a substituted aminoester. Treatment with a base forms an ester enolate which undergoes arylation by intramolecular migration of an aryl ring to the \u03b1-position of the ester. The product undergoes ring closure to a hydantoin, which may itself be deprotected and functionalised. Aryl migration is successful with rings of various electronic character and with esters bearing functionalised and unfunctionalised chains, and the products have features in common with several bioactive compounds.5,5-Disubstituted hydantoins, formally the cyclisation products of quaternary amino acids, were formed connectively from simple ester-derived starting materials by a one-pot tandem method. Amination of the silyl ketene acetal derivative of a methyl ester takes place by silver-catalysed addition to the N Hydantoin rings, formally the cyclocarbonylation products of amino acids, are found in a number of medicinally significant molecules .1,2 For 6Methods for the synthesis of substituted hydantoinsWe have shown that intramolecular migration of an aryl ring to the \u03b1-position of an amino acid-derived urea can provide a general method for making substituted hydantoins20N\u2032-aryl migration to the \u03b1-position of the resulting ester followed by ring closure would give a hydantoin.Our initial plan for a direct route to structurally diverse 5,5-disubstituted hydantoins is illustrated in Three major challenges need to be overcome. Regioselective addition of the silyl ketene acetal to the azocarboxamide must lead to a 2-ureido ester to allow the subsequent aryl migration step. Secondly, the enolate of the product must undergo rearrangement rather than any other alternative reaction (such as substitution or elimination), and finally the product must cyclise to a hydantoin. All these steps ideally should occur in a single, tandem process.1 were made by acylation of hydrazine with N-methyl-N-arylcarbamoyl chloride followed by oxidation with NBS.1a and the silyl ketene acetal 2a with AgOTf (20 mol%) in dichloromethane gave the addition product 3 in 68% yield . Increasing the amount of KHMDS to 3.0 equiv. gave clean product 4a in 75% yield (entry 4).We started by exploring the amination step with a symmetrical azodicarboxamide to allow us to study the viability of the rearrangement while avoiding issues of regioselectivity. Silver-catalysed aminations of silyl ketene acetals were known using azodicarboxylates,8% yield , entry 11b\u2013g were made, and likewise treated with silyl ketene acetals 2a and 2b .The products S see ESI, we wereioisomer , entry 1N-Boc-protected aminohydantoin 7a (entries 3\u20135) took place, in parallel with the results seen using the symmetrical aminating agent 1a. With 2.0 equiv. of KHMDS, warming the reaction to \u201340 \u00b0C for 2 h, hydantoin product 7a was formed in 20% yield (entry 3), increasing to 72% yield on warming to \u201320 \u00b0C (entry 5). Other unsymmetrical aminating agents were also explored, including N-benzoyl, N-tert-butyl-carboxamido and N-methyl-N-tert-butyl carboxamido substituted azo compounds. Although intermediate aminated products corresponding to 6 were obtained, treatment with the base led only to decomposition.When KHMDS was added directly to the crude reaction mixture containing the amination product, arylation and cyclisation to the 5 and silyl ketene acetals 2 and the antibacterial drug nitrofurantoin (9b). Alternatively, the N\u2013N bond of the product could be cleaved to reveal the parent hydantoins 10. Several methods were screened for this transformation, and we found that treatment of a selection of products 7 with sodium nitrite in 3\u2009:\u20091 acetic acid/1 M HCl at 110 \u00b0C scale\" fill=\"currentColor\" stroke=\"none\">O stretching absorptions at 1745 cm\u20131 (ester), 1720 cm\u20131 (carbamate) and 1678 cm\u20131 (urea). A transforms initially into an intermediate which we assign as the enolate B, consistent with the disappearance of the ester C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O stretch at 1745 cm\u20131, and the appearance of peaks we assign to the enolate function at 1640\u20131660 cm\u20131 plus a peak at 1604 cm\u20131 corresponding to the anionic carbamate. The enolate evolves to a species that has C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O stretching absorptions at 1764 cm\u20131 and 1710 cm\u20131, typical of a hydantoin,\u20131. We assign these peaks to species D, the conjugate base of the ultimate product 7i. The rearrangement of B to D presumably passes through an undetectable transient intermediate C that cyclises rapidly to D. Evidence from related reactions suggests that the formation of the new C\u2013C bond and breakage of the old C\u2013N bond during the formation of the proposed intermediate C are to some extent concerted.The course of the reaction between model substrates PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N bond of a new class of unsymmetrical azocarboxamides, and the arylation takes place by base-promoted intramolecular N to C migration within the N\u2032-aryl urea linkage that results from the amination step. The hydantoin then forms directly from the product of enolate arylation. In situ infra-red spectroscopy reveals four successive species on the reaction pathway from the amination step to the hydantoin ring closure. The one-pot protocol allowed the connective synthesis of a range of 5,5-disubstituted hydantoins bearing electronically diverse aryl substituents, compounds which have potential applications in the construction of biologically active molecules.In conclusion, 5,5-disubstituted hydantoins may be formed by a tandem amination-intramolecular arylation sequence of silyl ketene acetals. The amination entails silver-catalysed regioselective addition to the NThere are no conflicts of interest to declare.Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "An efficient aerobic unactivated C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond cleavage process was achieved, in which the succinimide or glutarimide derivatives could be prepared directly from alkenyl amides. PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond double cleavage are always attractive but very challenging. We report herein a chemoselective approach to valuable cyclic imides by a novel Cu-catalyzed geminal amino-oxygenation of unactivated C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bonds. O2 was successfully employed as the oxidant as well as the O-source and was incorporated into alkenyl amides via C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond cleavage for the efficient preparation of succinimide or glutarimide derivatives. Moreover, the present strategy under simple conditions can be used in the late-stage modification of biologically active compounds and the synthesis of pharmaceuticals, which demonstrated the potential application.The transformations of unactivated alkenes through C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C double bond cleavage. PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond cleavage,via chemoselective C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C double bond cleavage would be highly promising to produce succinimides in the presence of oxygen (ii)-catalyzed intramolecular aza-Wacker-type cyclization. PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond cleavage was successfully achieved but in two steps. DABCO was required as a base with the formation of \u03b3-lactam products. To the best of our knowledge, the chemoselective cleavage of C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C double bonds in alkene-tethered amides for cyclic imide synthesis has not been accomplished yet.Recently, Of oxygen . To dateN-methoxy alkenyl amide 1a. After a lot of experiments, we were surprised to find that the unactivated C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C double bond could be cleaved with the incorporation of one oxygen atom using O2. Encouraged by the copper catalyzed olefin amino-oxygenation which delivered 2a in 47% yield (Ligand II) as the ligand the unactivated C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C double bond geminal amino-oxygenation reaction in toluene proceeded well and produced the desired succinimide product 2a with excellent efficiency while substrates bearing bulky groups showed poor conversion (2d and 2e). When a hydrogen atom (2f) or benzyl group (2g) was attached to the amide nitrogen, the reaction did not work. The reason is that the alkyl-metal intermediate formation might be favored with the assistance of alkoxy protecting groups.2h\u20132o), producing polysubstituted and spiro-succinimides in moderate to good yields. It is noteworthy that the reaction could contain one of the identical allyl groups specifically to give the allylic imide in 44% yield (2o). The mono-methyl or benzyl substituted enamides were also tolerated, and the desired products could be obtained in fair yields (2p and 2q). To our delight, the vinylcyclohexane derived enamide underwent the process smoothly to afford the corresponding imide 2s. 2-Vinylbenzamide was also compatible to give the synthetically important phthalimide 2t albeit the efficiency is a little bit low because the conjugated alkenes would undergo unwanted oxidation. Unfortunately, the alkene-tethered amide without alkylation of the backbone did not work. Notably, the glutarimide derivatives 4a\u20134d were also obtained in moderate yields with hex-5-enamides as an anti-inflammatory, antipyretic and analgesic agent, could deliver succinimide nditions . The stri and methsuximide ii , which possesses antiepileptic effects. This method avoids the use of highly toxic hydrocyanic acid in industrial production. Similarly, the methsuximide 17 could also be obtained from succinimide 15 in good overall yield.Furthermore, our strategy can be applied to the synthesis of two pharmaceutical compounds ethosuximide imide ii . As show18 could be obtained in 87% yield, with the formation of 2a in only 10% yield scale\" fill=\"currentColor\" stroke=\"none\">C bond cleavage, 2-pyrrolidinone 19 was employed under standard conditions. The formation of 2r with some unconsumed raw materials compared with the results in 18O2 delivered the labeled succinimide [18O]-2a in 80% yield (67% 18O) due to the exchange with H2O is oxidized to copper(ii) by O2 in the initial step. Then, copper(ii)-catalyzed alkene cis-amidocupration affords an unstable organocopper(ii) intermediate B. Primary radical C, which could be trapped by TEMPO enolate G which undergoes formal [2 + 2] cycloaddition with another molecule of oxygen to give the 1,2-dioxetane Jvia radical species H and the cyclic peroxo intermediate I.2a and release carbon monoxide.Based on previously reportedby TEMPO , is subs TEMPO B. could novia a chemoselective cyclization/C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond cleavage processes that revealed an efficient approach to polysubstituted succinimides and glutarimides. Our reaction exhibited good functional group tolerance under simple conditions. The success of this protocol in the late-stage modification of biologically active compounds and the synthesis of pharmaceuticals would motivate further exploration of the transformations of unactivated alkenes.In summary, we developed a novel molecular oxygen mediated geminal amino-oxygenation of unactivated olefins in alkene-tethered amides The authors declare no competing financial interest.Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "Crystalline and porous covalent organic frameworks (COFs) with donor-acceptor moieties in their backbone are utilized as initiators for visible light induced radical polymerization. The COFs are efficient photoinitiators, maintaining their structural integrity for several cycles. b]thiophene-2,5-dicarbaldehyde or -5,5\u2032-dicarbaldehyde linkers are presented. The resulting crystalline and porous COFs have been applied as photoinitiator for visible light induced free radical polymerization of methyl methacrylate (MMA) to poly-methyl methacrylate (PMMA). These results pave the way to the development of robust and heterogeneous systems for photochemistry that offers the transfer of radicals induced by visible light.Covalent organic frameworks (COFs) are promising materials for applications in photocatalysis, due to their conjugated, porous and chemically stable architectures. Alternating electron donor\u2013acceptor-type structures are known to enhance charge carrier transport mobility and stability in polymers and are therefore also interesting building units for COFs used as photocatalysts but also as photoinitiator. In this work, two donor\u2013acceptor COFs using electron deficient 4,4\u2032,4\u2032\u2032-trianiline and electron rich thiophene-based thieno[3,2- Covalent organic frameworks (COFs) have recently received increasing interest due to their intriguing properties such as low density, high porosity, good crystallinity and the possibility to introduce a range of organic functional moieties into chemically stable frameworks.To further improve the catalytic activities, strategies such as introduction of heteroatoms (N and S),b]thiophene-2,5-dicarbaldehyde or -5,5\u2032-dicarbaldehyde , respectively, in presence of 6 M acetic acid (0.5 mL) and mesitylene/dioxane (3 mL) as the solvents [Section S2, ESI13C solid-state cross-polarization magic angle spinning nuclear magnetic resonance (CP/MAS-NMR) spectroscopy, N2 sorption analyses, scanning electron microscopy (SEM), in situ as well as ex situ electron paramagnetic resonance (EPR) spectroscopy, and ultraviolet-visible spectroscopy (UV-vis) analyses.Considering the beneficial properties of donor\u2013acceptor based materials for charge generation, mobility and stability, we have attempted the synthesis of donor\u2013acceptor COFs as photoinitiators for visible light induced free radical polymerization .35,36 TT\u03bb = 1.5418 \u00c5) was performed to assess the crystallinity of the as-synthesized COFs. The PXRD patterns for TTT\u2013DTDA and TTT\u2013BTDA are dominated by an intense reflection in the low-angle region, at 2.68 and 2.52 2\u03b8 degrees for TTT\u2013DTDA and TTT\u2013BTDA, respectively, which can be assigned to the (100) facet of a primitive hexagonal lattice (\u03b8 degrees for TTT\u2013DTDA and TTT\u2013BTDA COFs that can be assigned to the (001) facet, confirms the formation of two-dimensional (2D) COFs in a crystalline and \u03c0\u2013\u03c0 stacked form. The structural models for TTT\u2013DTDA and TTT\u2013BTDA were constructed by generating the probable 2D hexagonal layers with hcb topology with either eclipsed (AA) or staggered (AB) stacking arrangement analyses with Cu K\u03b1 radiation scale\" fill=\"currentColor\" stroke=\"none\">N bonds, confirming the condensation reaction of aldehyde (TTT) and primary amines (DTDA or BTDA). The signals at \u223c110, 125, 130 and 138 ppm can be ascribed to the carbon atoms of the phenyl groups, while the sharp peak at \u223c165 ppm is attributed to the carbon atoms of core triazine ring from TTT linkers in the COFs, validating the presence of intact triazine moieties in the backbones. The formation of frameworks in TTT\u2013DTDA and TTT\u2013BTDA was further corroborated by Fourier transform infrared (FT-IR) spectroscopy. In the FT-IR spectra the presence of a strong vibration at 1582 cm\u20131 can be assigned to the stretching mode of the \u2013C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N bonds and amino (\u223c3450 cm\u20131) stretching bands of respective starting materials completely disappeared after COF formation and Langmuir (440 m2 g\u20131) surface areas. In case of TTT\u2013BTDA, most probably, due to the partial offset stacking of adjacent layers seen in the less defined PXRD pattern in comparison to TTT\u2013DTDA, just moderate surface area values were observed \u2013COOCH3. The signal recorded after 160 min reaction time (g = 2.0048 and superhyperfine structure (shfs) constants for the coupling of the electron at C\u03b1 with the three CH3 protons and the two protons at C\u03b2, A3CH = 22.2 G, A\u03b21H = 14.7 G, A\u03b22H = 7.9 suggesting a light-driven process as well as involvement of a radical polymerization mechanism to poly-methyl methacrylate (PMMA), where COFs play a key role in the generation of holes that induce hydrogen abstraction from the amine co-initiator. The facile separation of these COF-photoinitiators from the MMA/PMMA mixture and their recyclability for multiple radical polymerization cycles together with their capability to initiate radical reactions under visible-light, make these materials very promising photoinitiators for a range of photochemical reactions.There are no conflicts to declare.Supplementary informationClick here for additional data file."} +{"text": "Just boil it in cumene! A general metal-free oxidation method is described. 2 and initiate radical chain reactions in catalyst-free conditions. In the absence of additional substrates, these processes can lead to acetophenones. In the presence of substrates, the cumene oxidation process can be intercepted in various chain reactions, affording very simple protocols for functional group oxidation.Due to the relative stability of the cumyl radical, cumenes and \u03b1-methyl-styrenes are ideally structured to directly harvest the oxidizing reactivity of O PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond cleaving oxidation of styrenes with O3),2, such as in the Wacker process, PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond cleaving oxidation of styrenes towards the corresponding phenones while using an iron(iii)-triflate catalyst bearing a tridentate amine ligand, with O2 as the terminal oxidant .4 = Ac, 2j), make the cumenes unreactive, likely by preventing the first oxidation step: the cumyl H-atom abstraction. For halides, methoxy, and phenyl functional groups, the tolerance is generally acceptable. A mechanism is proposed in 2 in a Hayashi-like mechanism.tert-butylphenol added to the reactor as a radical inhibitor suppresses acetophenone formation oxidizing strategy .2 system would also be competent for simple functional group oxidation. We therefore considered the oxidation of a series of organic substrates under those conditions. For example, benzhydrol 9 yields benzophenone . Diphenylmethane 10 also yields benzophenone , while xanthene (11) and fluorene (12) produce xanthone 16 and fluorenone 17 with 93% and 74% yield respectively. Finally 1,2-diphenylhydrazine (13) yields azobenzene 18 (93%). All were efficiently transformed while simply being heated up in cumene under 1 atm of O2 at 150 \u00b0C, without any further additive. These trivial oxidation processes are among the most studied reactions in the literature.The following reaction conditions were eventually selected: cumene, 14 mmol, is placed in an 85 mL glass reactor, which is then flushed with Oormation . This restrategy . One metydrazine yields a2 towards the corresponding phenones can proceed efficiently without a catalyst. These typically require significantly higher temperatures however than under catalytic conditions.2 mediated oxidation of cumene can be intercepted in the frame of cross dehydrogenative couplings, or the simple oxidation of diverse functional groups. Clearly, cumene and its congeners are well suited for the organic activation of O2 towards oxidative applications.In conclusion, the oxidative C\u2013C bond cleaving oxidation of cumenes or \u03b1-methyl-styrenes with OSupplementary informationClick here for additional data file."} +{"text": "The synthesis and isolation of two-electron reduced naphthalenediimides is reported. A doubly zwitterionic structure is observed for the first time in a naphthalene moiety, which aids in its stabilization. \u2013) reduced, highly electron-rich naphthalenediimides (NDIs). A doubly zwitterionic structure is observed for the first time in a naphthalene moiety and validated by single crystal X-ray crystallography and spectroscopic methods. The synthesis avoids hazardous reducing agents and offers an easy, high-yielding route to bench-stable di-reduced NDIs. Notably, we realized high negative first oxidation potentials of up to \u20130.730 V vs. Fc/Fc+ in these systems, which establish these systems to be one of the strongest ambient stable electron donors. The study also provides the first insights into the NMR spectra of the di-reduced systems revealing a large decrease in diatropicity of the naphthalene ring compared to its 2e\u2013 oxidized form. The NICS, NICS-XY global ring current, gauge-including magnetically induced current (GIMIC) and AICD ring current density calculations revealed switching of the antiaromatic and aromatic states at the naphthalene and the imide rings, respectively, in the di-reduced system compared to the 2e\u2013 oxidized form. Notably, the substituents at the phosphonium groups significantly tune the antiaromatic\u2013aromatic states and donor ability, and bestow an array of colors to the di-reduced systems by virtue of intramolecular through-space communication with the NDI scaffold. Computational studies showed intramolecular noncovalent interactions to provide additional stability to these unprecedented doubly zwitterionic systems.The di-reduced state of the naphthalene moiety and its congeners have long captivated chemists as it is elusive to stabilize these intrinsically reactive electron-rich \u03c0-systems and for their emergent multifaceted properties. Herein we report the synthesis and isolation of two-electron (2e Although through-space charge delocalization has been elegantly formulated in NDI cyclophanes, ambient stabilization of 2e\u2013 reduced individual NDIs has not been possible.\u2013 uptake and release as well as the tunability of the aromatic naphthalene \u03c0-backbone and the nonaromatic imide rings. Thus, redox-assisted inter-conversion between the aromatic and nonaromatic states can be envisioned.Isolation of a two-electron reduced naphthalene dianion and its \u03c0-extended systems is a challenge being pursued over last several decades. In 1965, a naphthalene dianion was realized in the solution state1\u20135) with the first oxidation potential ranging from \u20130.724 to \u20130.263 V vs. Fc/Fc+ . We observed that second reduction potential of the dicationic NDI (2e\u2013 oxidized form) plays a key role during the synthesis of di-reduced compounds. EWGs at the axial- and the core-positions of the NDI led to a significant contrast between the first and second reduction potentials, which significantly favoured the one-step synthesis of 3\u20135. The reduction potentials and the corresponding LUMO levels of the parent dicationic compounds are listed in Herein we report the first synthetic protocol towards the isolation of ambient stable NDI-based di-reduced compounds at the axial positions , which is shifted upfield by 1.61 ppm compared to the same protons of 2+1 that appear at 8.94 ppm (J = 11 Hz). This signifies a significant contribution of the paratropic ring current in the naphthalene ring and decrease in diatropicity. This upfield 1H NMR chemical shift is observable even for the protons not directly attached to NDI scaffold. For example, the protons Hf and Hg of the ethyl groups linked to the phosphonium groups in 1 appear at 2.34 and 1.13 ppm, respectively, while in 2+1 they appear at 2.75 and 1.30 ppm, respectively. The axial group protons, He (5.30 ppm) and Hb\u2013d (7.28\u20137.15 ppm), of 1 also show measurable upfield chemical shifts compared to the same protons in 2+1 appearing at 5.41 and 7.48\u20137.27 ppm, respectively.In the presence of an external magnetic field, aromatic systems sustain a diatropic ring current while antiaromatic systems sustain a paratropic ring current.e of 12+ . In the 1H NMR spectra was also observed for 2\u20135. The phenylene/naphthalene protons in 2 solution.To have the first insight into the structural characteristics of the di-reduced compounds, we sought to crystallize 2+1 and 2+2 were grown from DCM: toluene (2\u2009:\u20091) solution at room temperature and the structural parameters compared with 1 and 2.23Further, single crystals of 1 crystallizes in the monoclinic space group \u201cC2\u201d with one molecule per asymmetric unit scale\" fill=\"currentColor\" stroke=\"none\">O bond length of 1 compared to 2+1 and the longest C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O bond was found to be 1.252 \u00c5, suggesting its partial double bond character. In addition, bond lengthening in the annular C5\u2013C5\u2032 and the transverse bonds of the naphthalene moiety along with shortening in the P1\u2013C2, C2\u2013C3, C1\u2013C6 and C4\u2013C7 bonds was observed indicating delocalization of the two additional electrons over the NDI scaffold (ESI Table S12+1 (vide infra). PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O groups and shortening of the P\u00b7\u00b7\u00b7O distance suggest significant polarization of the positive and negative charges over the P atom of the phosphonium group and the O atom of the imide groups, respectively. A \u03c0-conjugated betaine-type di-zwitterionic structure is therefore pertinent from the crystallographic insight.ric unit . We obse2+1 crystallizes in the triclinic space group \u201cP1[combining macron]\u201d with a half molecule per asymmetric unit scale\" fill=\"currentColor\" stroke=\"none\">O adjacent to the phosphonium group shows a bond distance of 1.212 \u00c5, while the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O bond situated remote to the phosphonium group had a distance of 1.219 \u00c5. The intramolecular P\u00b7\u00b7\u00b7O non-bonded distance was found to be 2.811 \u00c5. The BF4\u2013 counter anions show strong anion\u2013\u03c0 interaction with a small shift towards the imide region of the NDI scaffold. The distance between the F atom of anion and the C atoms of the naphthalene moiety is in the range of 3.113\u20133.451 \u00c5 and the distance from the centroid of naphthalene (Ct) to the B atom of the anion was found to be 3.524 \u00c5.ric unit . The imi2 crystallizes in the triclinic space group \u201cP1[combining macron]\u201d with a half molecule per asymmetric unit scale\" fill=\"currentColor\" stroke=\"none\">O bond length of 1.224 \u00c5 in comparison to 2+2 (1.213 \u00c5) corroborates partial double bond character. The C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O group remotely placed with respect to the phosphonium group also showed bond elongation (1.245 \u00c5) in comparison to 2+2 (1.208 \u00c5) scale\" fill=\"currentColor\" stroke=\"none\">O adjacent to the phosphonium group shows a bond length of 1.213 \u00c5, slightly longer than the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O bond (1.208 \u00c5) situated remote to the phosphonium group. The distance between P\u00b7\u00b7\u00b7O was found to be 2.738 \u00c5, which is 0.073 \u00c5 shorter than that in 2+1, corroborating a stronger P\u00b7\u00b7\u00b7O donor\u2013acceptor type interaction when the phosphonium groups are substituted with phenyl rings. Moreover, both the BF4\u2013 anions form anion\u2013\u03c0 interactions and are situated exactly on the top and bottom of the naphthalene ring with a distance of 2.968\u20134.010 \u00c5 between the naphthalene carbons and the F atom of BF4\u2013 anions and differential pulse voltammetry (DPV) experiments were performed for the di-reduced and dicationic forms in DCM reaching 3.3 \u00d7 104 L mol\u20131 cm\u20131 . In toluene\u2009:\u2009DCM (97\u2009:\u20093 v/v), the t1/2 was found to be 6.1 months, which is the highest stability amongst any reported di-reduced NDI molecules calculations. The negative NICS values indicate a diatropic ring current, while positive NICS values indicate a paratropic ring current.ectively . NICS values for the same was found to be 0.8 and 1.6 ppm respectively, which indicates the nonaromatic nature of the naphthalene moiety.However, the positive NICS (0) values of 4.0 ppm and 4.9 ppm for the naphthalene ring in the case of the di-reduced systems 2+1 and 2+2 comprising of the six-membered rings shows positive NICS (0) values of 4.7 and 5.2 ppm, respectively, indicating antiaromaticity. The NICS (1) values for the same were found to be 0.3 and 0.7 ppm, respectively, indicating nonaromatic nature. However in 1 and 2, the NICS (0) turns negative with values of \u20134.3 and \u20133.5 ppm, indicating its weakly aromatic nature, while NICS (1) turns negative with with values of \u20135.9 and \u20135.4, suggesting its aromatic nature. These findings indicate that in the di-reduced compounds, the naphthalene moiety nurtures antiaromatic/nonaromatic states and the imide rings provide the aromatic/nonaromatic state. Thus, a 2e\u2013 redox reaction can switch the antiaromatic\u2013aromatic sites in these electron-rich and electron-deficient systems.In contrast, the imide regions of XY scans for the di-reduced systems and compared with the corresponding dications.XY scan for 1 and 2+1 in different directions keeping NICS probes (dummy atoms) at 1.7 \u00c5 above the molecular plane under examination at 0.1 \u00c5 intervals and scanned along the X-axis value of the carbonyl group remote to the phosphonium group, respectively. This clearly suggests the bond lengthening of carbonyl groups adjacent to the phosphonium group due to the strong P\u00b7\u00b7\u00b7O non-bonding interactions as validated from crystallographic studies scale\" fill=\"currentColor\" stroke=\"none\">O bonds of the dication also shows antiaromatic nature basis set using the IEFPCM model in DCM. The axial groups in all the cases were replaced by the methyl group to save computational cost. The calculated HOMO and LUMO energies of 1 were found to be \u20134.145 and \u20131.436 eV, respectively, while for 2 they were \u20134.225 and \u20131.628 eV, respectively. For 2+1, the HOMO and LUMO levels were \u20138.372 and \u20134.903 eV, respectively, and for 2+2 they were \u20138.063 and \u20134.894 eV, respectively. The calculated HOMO energy levels of di-reduced and LUMO energy levels of dications are in excellent correlation with electrochemically obtained HOMO\u2013LUMO values distribution of m groups .Natural population analysis (NPA) = 2.24 kcal mol\u20131 for 2+2 and E(2) = 4.44 kcal mol\u20131 for 2 per P\u00b7\u00b7\u00b7O interaction was estimated using second-order perturbation theory. This stronger P\u00b7\u00b7\u00b7O interaction in 2 further supports greater distribution of the negative charge through the imide carbonyl groups due to the additional two electrons in the di-reduced compounds.Natural bond orbital (NBO) analysis1, 2 and its dicationic precursors.Atoms in molecule (AIM) analysis2\u03c1(rb) P\u00b7\u00b7\u00b7O, E(rb)P\u00b7\u00b7\u00b7O, G(rb)P\u00b7\u00b7\u00b7O, and V(rb)P\u00b7\u00b7\u00b7O for the P\u00b7\u00b7\u00b7O interactions are summarized in \u03c1(rb), values at the BCP of the P\u00b7\u00b7\u00b7O are in the range of H-bonding interactions (\u03c1H-bond \u2248 0.002\u20130.04 a.u.).\u03c1(rb) for 1 (0.021 a.u.) and for 2 (0.025 a.u.) are higher than that for 2+1 (0.018 a.u.) and 2+2 (0.018 a.u.) validating a stronger P\u00b7\u00b7\u00b7O interaction in the di-reduced compounds. The Laplacian density, \u22072\u03c1(rb), is also in the range of strong H-bond interactions.The 2D critical bond paths are shown in E(rb) for molecule 2+2 indicates its dominant electrostatic nature and the negative E(rb) values for 2 indicate its partial covalent nature [E(rb) value at a specific BCP indicates whether the interaction is electrostatic dominant E(rb) > 0 or covalent dominant E(rb) < 0]. The positive value of G(rb) and negative value of V(rb) support its stable, bound stationary states reduced, highly electron-rich, bench-stable NDIs. A doubly zwitterionic structure is observed in a naphthalene moiety and validated by single crystal X-ray crystallography. This new genre of compounds was achieved in high-yields via a solvent-free synthetic protocol. The di-reduced systems endow exceptional inherent stability with a half-life time of more than six months in toluene under ambient conditions. Notably, high negative first oxidation potentials up to \u20130.730 V vs. Fc/Fc+ and HOMO levels extending to \u20134.360 eV were realized.We demonstrated isolation of two-electron (2e\u2013 oxidized form, suggesting a large decrease in the diatropicity of the naphthalene ring. The NICS, NICS-XY, GIMIC and AICD calculations revealed redox switching of the antiaromatic and aromatic states at the naphthalene and the imide rings, respectively, in the di-reduced system compared to the corresponding rings of the 2e\u2013 oxidized form. The substituents at the axial- and core-positions tune the antiaromatic\u2013aromatic states and the electron donor ability. Further an array of diverse colors was achieved for the di-reduced systems. The through-space non-covalent interactions are key elements assisting this tunability. Isolation of this new class of ambient stable systems should have fascinating implications in controlling electron transfer reactions and development of new switchable materials.The study offers the first insights into the NMR spectra of the di-reduced systems revealing large upfield chemical shifts for the NDI-core atoms and the adjoining groups compared to their 2eThere are no conflicts to declare.Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "Dual modulated luminescence of carbogenic nanodots derived from zeolites has been acquired by controlling the concentration and pH value of CND aqueous dispersions. N-methylpiperazine-templated zeolite precursor by calcination and NaOH treatment. The isolated CNDs exhibit tunable photoluminescence according to the concentration and pH value of aqueous dispersions of the CNDs. Fine-tuning of the fluorescence emission wavelength across the entire visible spectrum can be easily achieved by varying the concentration of the CND dispersions. Meanwhile, both the emission wavelength and intensity of the photoluminescence can be tuned by controlling the pH value of the CND dispersion. The pyrolysis of organic templates confined in nanoporous zeolites represents a new approach to controlling the optical properties of CNDs, which may open more opportunities in applications such as multimodal sensing and full-color displays.Hydrophilic N-doped carbogenic nanodots (denoted as CNDs) have been prepared from a Recently, the pyrolysis of organic templates confined within zeolites has been found to be an efficient approach for preparing well dispersed CNDs with uniform size, benefiting from the well-confined pore space of zeolites.Carbogenic nanodots (denoted as CNDs) are defined as small graphene-based oxygenous carbon nanoparticles with a size of less than 10 nm. As a new member of the nanocarbon family, luminescent CNDs have attracted increasing attention due to their intriguing advantages when compared with conventional semiconductor quantum dots, such as their low toxicity, biocompatibility, low cost and chemical inertness.etc., may affect the PL properties of CNDs, some alternative methods have been used to adjust the PL of CNDs. For example, Li et al. have reported the size-dependent photoluminescence of CNDs by controlling the current intensity of an electrochemical oxidation method.et al. have reported the PL modulation of graphene quantum dots through a surface chemistry route.et al. have observed that the fluorescence intensity of CNDs decreased when the pH value of the solution was shifted from 7.0 - regardless of whether it was increased or decreased.et al. observed that the emission intensities of CNDs were similar within the pH range of 4\u201312, but decreased abruptly at lower pH values (<4).et al. and Hu et al. have presented CNDs with full-color emissions by varying the reagents and reaction conditions.Tunable photoluminescence (PL) is one of the attractive properties of CNDs for their practical application, in particular, CNDs with full-color emissions are highly desirable.N-methylpiperazine (NMP) confined in zeolite MgAPO-44 with CHA zeotype topology. The isolated CNDs exhibit excellent aqueous dispersibility and stability. Interestingly, the PL emission of aqueous dispersions of the CNDs can be simply modulated by controlling the concentration of CNDs, which display full-color emissions. In addition, not only the intensity of the photoluminescence, but also the emission wavelength can be tuned by varying the pH value of the CND dispersions. This ability to control the optical properties of CNDs may offer more opportunities in applications such as multimodal sensing and full-color displays.In this work, we present the preparation of uniform N-doped CNDs by the pyrolysis of 2O3\u2013P2O5\u2013NMP\u2013H2O. Typically, pseudoboehmite and magnesium acetate tetrahydrate (Mg(CH3COO)2\u00b74H2O, 99.0%) were dispersed in a solution of orthophosphoric acid in water under vigorous stirring at room temperature. Then, NMP was added to this mixture. After stirring for one hour, a homogeneous gel with an overall molar composition of 1.0 Al2O3\u2009:\u20091.0 MgO\u2009:\u20092.2 P2O5\u2009:\u20095.0 NMP\u2009:\u2009350 H2O was formed, which was heated under autogenous pressure at 180 \u00b0C in a 15 mL Teflon-lined stainless steel autoclave for 3 days. The crystals were washed in distilled water and dried at 60 \u00b0C overnight.The reagents and solvents employed in the synthesis were commercially available and used as received without further purification. The magnesium aluminophosphate zeolite MgAPO-44 was prepared under hydrothermal conditions in a reaction system of MgO\u2013Al\u20131 in air, followed by a 4 h isothermal hold at this temperature. Carbonization of NMP was occurring during this time, resulting in a CNDs@MgAPO-44 composite material.The as-synthesized zeolite product was placed into a normal furnace and heated from room temperature to 400 \u00b0C at a temperature ramp rate of 2 \u00b0C min\u20131. The R-CNDs sample was diluted to 0.92, 0.68, 0.22 and 0.04 g L\u20131 samples . To study the influence of the pH value on the PL properties of the CNDs, NaOH and HCl aqueous solutions were used to adjust the pH value of the G-CND solution to 8\u201314 and 1\u20136, respectively.400 mg of CNDs@MgAPO-44 sample was dissolved in 2.5 mL of 2.5 M NaOH aqueous solution at room temperature, and ultrasonic treatment was used to accelerate the dissolution. Then 5 mL of deionized water was added and insoluble residues were centrifugally separated from the solution. The dark colored supernatant was collected and neutralised with HCl solution to a pH value of about 7, it was then further centrifugally separated and purified by dialyzing with a cellulose ester membrane bag (molecular-weight cutoff = 1000). The initial purified CNDs were denoted as R-CNDs with a concentration of around 1.36 g L13C solid-state MAS NMR were performed on an Infinity Plus400 spectrometer operating at B0 = 9.7 T. 27Al solid-state MAS NMR were recorded on a Bruker AVANCE III 400 WB spectrometer operating at B0 = 9.4 T. Total Organic Carbon (TOC) analysis was performed on an Elementar Vario TOC cube. The absolute fluorescence quantum yields were measured on an Edinburgh FLS920 fluorimeter. Photoluminescence spectra were recorded on a Fluoromax-4 spectrofluorometer (Horiba Jobin-Yvon). Nano-second fluorescence lifetime measurements were performed using a time-correlated single-photon counting (TCSPC) system under right-angle sample geometry. A NanoLED-340 was used to excite the samples. The white light used in this work was obtained by irradiation with a High Power Xenon Lightsource (HPX-2000).Powder X-ray diffraction (PXRD) data were collected on a Rigaku Ultima IV diffractometer. The CND ethanol solution was spotted onto a salt plate and the spectrum was measured on a Bruker FTIR IFS-66V/S. A baseline correction was applied after measurement. UV-vis adsorption spectra were obtained on a Shimadzu UV-2550 spectrophotometer. The TEM and HRTEM images were taken on a FEI Tecnai G2 S-Twin F20 transmission electron microscope. The X-ray photoelectron spectroscopy (XPS) measurements were performed using a Thermo Escalab 250 spectrometer with monochromatized Al K\u03b1 excitation. CHA zeotype structure.13C MAS NMR pattern of MgAPO-44 images reveal their lattice spacings to be 0.21 nm, which is consistent with the lattice spacing of the (100) plane of graphene.TEM images of the isolated CNDs are shown in PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bonds (284.7 eV), C\u2013N/C\u2013O bonds (286.2 eV) and C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O bonds (287.9 eV). The N 1s spectra can be deconvoluted into two peaks of pyridinic type (399.0 eV) and pyrrolic type (400.1 eV) N atoms. The O 1s spectra are assigned to O PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bonds (531.0 eV), O\u2013C bonds (532.8 eV) and adsorbed water (535.6 eV). PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O bonds spectra of the B-CNDs and R-CNDs are shown in C bonds 5.0 eV, O\u2013In previous reports, near zero or only a few percent (about 2%) N content has been found in CNDs obtained by the pyrolysis of templates confined in zeolites.ca. 18.7% (B-CNDs) without any further functionalization, which is higher than most of the bare and raw CNDs, which have typical values below 10%.Strikingly, the isolated CNDs not only exhibit excitation-dependent luminescence, but also display concentration-modulated luminescence. The as-made CND dispersions are stable for over six months, and no obvious PL changes are observed (Fig. S7, ESINotably, such concentration-modulated luminescence of CNDs across the entire visible spectrum is distinguished from previous reports, which tune emissions by controlling the reaction conditions or subsequent oxidation/reduction operations.The PL decay curves of the obtained CNDs were measured, and the decay fitting results are listed in Table S1.i.e., blue, green and green-yellow, can be observed at pH values of 2, 6 and 10 respectively, upon irradiation with white light (The influence of the pH value on the PL properties of the CNDs has also been investigated. As shown in te light . PreviouFTIR and UV-vis spectra of the CND dispersions at pH values of 2, 6, and 10 have been obtained (Fig. S11 and S12, ESIN-methylpiperazine occluded in the MgAPO-44 zeolite. Abundant N/O-containing hydrophilic groups are in situ generated on the surface of the as-made CNDs, endowing these CNDs with excellent aqueous dispersibility and stability, as well as a high PL quantum yield of up to 18.7% without any further functionalization. These CNDs exhibit interesting concentration-dependent PL properties, including full-color emissions when excited by white light. Such tunable PL of CNDs might mainly result from the energy transfer between multiple emitters in the nanoclusters that form in the highly concentrated CND dispersions. Meanwhile, pH-sensitive color and PL intensity modulation of the CND dispersions have also been observed. The detailed mechanism needs to be further investigated. This work provides a new approach for facilely controlling the optical properties of CNDs through the pyrolysis of organo-templated zeolites, which may open more opportunities in the application of CNDs in multimodal sensing and full-color displays.In summary, we have successfully developed fluorescent CNDs responsive to the concentration and pH value of their CND dispersions. The CNDs are prepared by the pyrolysis of Supplementary informationClick here for additional data file."} +{"text": "Through mixed metal cooperativity, alkali metal magnesiates efficiently catalyse the cyclisation of alkynols. PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond. In order to optimise the reaction conditions and to garner vital catalytic system requirements, a series of alkali metal magnesiates were enlisted for the catalytic intramolecular hydroalkoxylation of 4-pentynol. In a prelude to the main investigation, the homometallic magnesium dialkyl reagent MgR2 (where R = CH2SiMe3) was utilised. This reagent was unsuccessful in cyclising the alcohol into 2-methylenetetrahydrofuran 2a or 5-methyl-2,3-dihydrofuran 2b, even in the presence of multidentate Lewis donor molecules such as N,N,N\u2032,N\u2032\u2032,N\u2032\u2032-pentamethyldiethylenetriamine (PMDETA). Alkali metal magnesiates MIMgR3 performed the cyclisation unsatisfactorily both in the absence/presence of N,N,N\u2032,N\u2032-tetramethylethylenediamine (TMEDA) or PMDETA. When higher-order magnesiates were employed, in general a marked increase in yield was observed for MI = Na or K; however, the reactions were still sluggish with long reaction times (22\u201336 h). A major improvement in the catalytic activity of the magnesiates was observed when the crown ether molecule 15-crown-5 was combined with sodium magnesiate Na2MgR4(TMEDA)2 furnishing yields of 87% with 2a\u2009:\u20092b ratios of 95\u2009:\u20095 after 5 h. Similar high yields of 88% with 2a\u2009:\u20092b ratios of 90\u2009:\u200910 after 3 h were obtained combining 18-crown-6 with potassium magnesiate K2MgR4(PMDETA)2. Having optimised these systems, substrate scope was examined to probe the range and robustness of 18-crown-6/K2MgR4(PMDETA)2 as a catalyst. A wide series of alkynols, including terminal and internal alkynes which contain a variety of potentially reactive functional groups, were cyclised. In comparison to previously reported monometallic systems, bimetallic 18-crown-6/K2MgR4(PMDETA)2 displays enhanced reactivity towards internal alkynol-cyclisation. Kinetic studies revealed an inhibition effect of substrate on the catalysts via adduct formation and requiring dissociation prior to the rate limiting cyclisation step.Mixed s-block metal organometallic reagents have been successfully utilised in the catalytic intramolecular hydroalkoxylation of alkynols. This success has been attributed to the unique manner in which these reagents can overcome the challenges of the reaction: namely OH activation and coordination to and then addition across a C Being 100% atom efficient,2-vinylidene metal complex intermediate (I), which is then susceptible to nucleophilic addition to generate a Fischer oxacarbene (II) that ultimately yields the endocyclised product (III).A catalogue of catalysts has been developed for the cyclisation of alkynols, including various transition metal complexes as well as alkaline earth and f-block compounds. Transition metals previously used to promote this transformation are mostly precious metalsrmediate , I, whicacarbene , II that product , III.53\u2013Besides the numerous examples of transition metal catalysts, the use of f- and s-block catalysts for this reaction has been investigated. In terms of f-block chemistry, Marks has utilised lanthanide amide catalysts,via exocyclisation, although with alkaline earth metal amides two different product isomers were observed, an internal and external alkene. Since Hill's initial study, several papers focusing on alkali metal and alkaline earth catalysts have been published. Liu has shown that potassium tert-butoxide is an effective and selective catalyst for cyclisation of aromatic alkynylamines and alkynols,cis-6-hydroxy- and cis-6-acyloxyhex-2-en-4-ynals to 2-acylfurans and 2-furans is achievable using calcium catalysis.These particular studies highlight that transformations proceed selectively PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bonds. Combining kinetic experiments with reactivity studies, here we provide informative mechanistic insights on how cooperative effects can be maximised as well as on the key role that each metal plays in these novel magnesiate-catalysed transformations.Expanding the scope of s-block cooperative catalysis, here we report the first catalytic applications of alkali metal magnesiates to promote cyclisation reactions, focusing on intramolecular hydroalkoxylation of alkynols. Benefitting from the enhanced metallating and nucleophilic abilities of these synergistic systems, we envisaged that they could play a dual role in overcoming the main challenges encountered in these transformations, facilitating not only OH activation, but also the required addition across C2SiMe3)2, containing thermally stable monosilyl groups, as a potential pre-catalyst. Using 4-pentynol (1) (as previously employed by Marks and Hill) for benchmarking with a catalytic quantity of the magnesium reagent, no reaction was observed even when the mixture was heated to 75 \u00b0C for 36 h magnesium reagent Mg2 could be due to the magnesium centre not being large enough to simultaneously bind to the oxide anion and be in close proximity to the \u03c0-density of the carbon\u2013carbon triple bond.This raises the question, \u201cwhy is magnesium unable to mediate this cyclisation whereas calcium can?\u201d Major contributory factors are likely due to the significant difference in ionic radii between the two metal cations and the low \u03c0-philicity of magnesium.2SiMe3) and Na(CH2SiMe3) were employed in the same reaction; however, the conversions were poor (<5%) (see ESI2SiMe3) (2SiMe3), Na(CH2SiMe3) and Mg(CH2SiMe3)2 can perhaps be attributed to the larger ionic radius of potassium versus the other metals allowing it to interact with the carbon\u2013carbon triple bond, activating it for cyclisation.We next focused on testing homometallic alkali metal organometallics, which, being significantly more polar than their Mg counterparts, have recently found applications in hydrophosphination catalysis.) see ESI. Using tI2SiMe3) , entry 3i.e., promote a dual-activation where a magnesium alkoxide forms, thus preparing one end of the substrate for cyclisation while the alkali metal could then interact with the \u03c0-system of the carbon\u2013carbon triple bond. As alluded to previously, this latter feature is essential as the magnesium alone cannot provide this interaction. LiMg(CH2SiMe3)3 . Moving to sodium (2SiMe3)]. When the potassium magnesiate KMg(CH2SiMe3)3 (2SiMe3) (11%). So, in summary, lower order magnesiate reagents are generally poor at catalysing the hydroalkoxylation/cyclisation reaction.Next, we investigated the catalytic ability of a lower-order magnesiate. It was envisaged that a bimetallic magnesiate could function cooperatively, 2SiMe3)3 , entry 5o sodium , entry 62SiMe3)3 , entry 8N,N,N\u2032,N\u2032-tetramethylethylenediamine) and PMDETA , were employed to completely fill the coordination sphere of the alkali metals. Again, starting with the lithium derivative, the higher-order magnesiate (2MgR4(TMEDA)2 was employed (2MgR4(PMDETA)2 is used . In addition, they continue to have a high isomer selectivity with up to 90% of the external alkene isomer being produced. The remarkable alkali metal effect observed between Li2MgR4(TMEDA) and K2MgR4(PMDETA)2 is even more striking when considering that both mixed-metal species are isostructural.3(TMEDA) and KMgR3(PMDETA) (2MgR4(PMDETA)2 is unaffected when the bidentate Lewis base TMEDA is used in place of its tridentate analogue PMDETA (2MgR4(TMEDA)2 displayed the same reaction time and selectivity as its PMDETA variant, demonstrating the reaction rate to be unaffected by the choice of Lewis base.In the hope of improving this situation, higher-order magnesiate reagents were considered. The Lewis basic proligands, TMEDA (3\u2013 or Mg(OR)42\u2013 fragment, of the now solvent-separated complex, was not able to perform the catalysis on its own. Surprisingly, in contrast, the results obtained in these reactions showed a dramatic improvement in reactivity!To establish whether or not both metals need to be present for the magnesiate to function, a catalytic amount of crown ether was added as a co-catalyst. As a crown ether of appropriate size is often able to sequester an alkali metal, the intention here was to ascertain, if the alkali metal was captured (non-cooperative), that the remaining anionic Mg(OR)Adding catalytic quantities of 15-crown-5 or 18-crown-6 to the sodium and potassium systems respectively dramatically increased the activity of the magnesiate catalysts, with the sodium higher-order magnesiate now nearing completion in 5 h . Therefore, these results suggest that the alkali metal is intimately involved and plays a key role in the cyclisation reaction. To ascertain whether the 18-crown-6 is catalytically active, when it is combined with only Mg(CH2SiMe3)2 the reaction fails (entry 2).Due to the significant difference in completion rates of these two reactions (5 h 2a is produced by employing La[N(SiMe3)2]3.3)2}2 where Ae = Ca, Sr, Ba], where reaction times ranged from 2.5\u20136 h, exhibiting a 2a isomer selectivity of 90\u201397% , where the carbon\u2013carbon triple bond has moved from a terminal to an internal location (Z-enynols (7 and 9). In 7) employing the optimised conditions we see the production of an aromatic furan product (8). This product differs from the product previously reported using heavier alkaline earthHaving established the optimal conditions for cyclisation of 4-pentynol, the scope of the reaction was evaluated. Terminal and internal alkynes were tested to assess the range and robustness of the catalytic system and 3. Ilocation , entry 1location , entry 23 or CN functional groups were incorporated into the substrate. Looking at 5-phenylpent-4-yn-1-ol (11) and an internal alkene (12c) (note these three products are still exocyclic). In this work, it was also noted that the product ratio appeared to have a degree of temperature dependence and required significantly harsher reaction conditions .In order to further probe the functional group tolerance of the catalytic magnesiate system, a range of internal alkynes were synthesised with different pendant functional groups, including halogens, and electron-donating and electron-withdrawing groups. It transpired that no side reactions took place when MeO, F, Cl, CF-ol (11) , entry 1t-butyl group , the same isomer ratios were obtained for all the reactions which tended towards completion show nicely the benefit of using a bimetallic system for these cyclisation reactions. With the heavier alkaline earth metal amides and lanthanide amides, substrate 11 is more challenging to cyclise, taking 16 h at 90 \u00b0C with Ca(HMDS)2 and about 15 h at 120 \u00b0C with La(HMDS)2. Marks3 is sterically driven by the phenyl substituent that prevents interaction between the metal and the internal alkyne. Using a bimetallic system, it appears that this steric clash is somewhat circumvented giving rise to a faster reaction time than the other two systems mentioned.This set of substrates based on 5-phenylpent-4-yn-1-ol (1), as this was used for the initial parameterisation studies. A plot of concentration of 1 against time displays a linear fit until half conversion when a rate acceleration is observed ), while at high concentrations of 1 (k\u20131[1] \u226b k2) eqn (3) rules this process. Alternatively, a pre-equilibrium approximation in which dissociation of 1 from magnesium does not influence the reaction rate leads exclusively to eqn (3), and hence to a good inverse first order relationship in 1 at any concentration of this ligand. The plot of the observed initial rates vs. 1/[1] during the course of the reaction only occurs upon its decoordination.Overall the deduced mechanism starts with formation of the active catalyst from the magnesiate pre-catalyst. This involves the deprotonation of four alcohol substrate molecules to form a magnesiate alkoxide, liberating tetramethylsilane. This \u2018active catalyst\u2019 is then involved in a coordination/decoordination process with an additional substrate molecule as suggested by the kinetic studies. This additional molecule of In cycle A the carbon\u2013carbon triple bond of the alkyne is directly inserted into the metal\u2013oxygen bond, leading to the formation of only one product upon protonolysis. Cycle B on the other hand involves an equilibrium isomerisation from alkyne to allene which upon insertion into the metal\u2013oxygen bond, and subsequent protonolysis, can yield two product isomers. Protonolysis releases the cyclised products and reforms the active catalyst completing the cycle. PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bonds of the substrate. Thus, coordination of the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond to the larger more \u03c0-philic potassium centre enables further activation of this unsaturated group and brings it into close proximity to the di-anionic (magnesiate) {Mg(OR)4}2\u2013 component, which is significantly more nucleophilic than a charge-neutral magnesium compound, facilitating intermolecular ring-closure to furnish the relevant oxygen-heterocycle.Disclosing a unique cooperative behaviour under catalytic conditions, each of the metals plays a key role in this process, by simultaneously activating the O\u2013H and C2SiMe3)2, K(CH2SiMe3), LiMg(CH2SiMe3)3, NaMg(CH2SiMe3)3, KMg(CH2SiMe3)3, Li2Mg(CH2SiMe3)4(TMEDA)2, Na2Mg(CH2SiMe3)4(TMEDA)2, K2Mg(CH2SiMe3)4(TMEDA)2 and K2Mg(CH2SiMe3)4(PMDETA)2] were prepared following literature procedures2SiMe3), 1 and 5 were obtained from Sigma-Aldrich; 1 and 3 from Alfa-Aesar; and 7 from Fluorochem. Substrates 11, 13, 15, 17, 19 and 21 were synthesised based on literature procedures13\u201322 can be found in the ESI1H, 100.6 MHz for 13C or 128.4 MHz for 19F.All reactions were carried out under a protective atmosphere of argon using standard Schlenk techniques. Non-deuterated solvents were dried by heating to reflux over sodium ketyl radicals under nitrogen and deuterated solvents were degassed and stored over molecular sieves. Pre-catalysts and with the oil bath temperature being changed where necessary based on observed reaction times. 0.5 mmol alkynol substrate was used with the monometallic species and lower-order magnesiates, and 0.6 mmol with higher-order magnesiate pre-catalysts. 0.025 mmol pre-catalyst was employed in all cases, quantities shown in 2Mg(OAlk)4(18-c-6)2 resting-state\u2019.In the catalytic cyclisation of 4-pentynol (1) with K2Mg(CH2SiMe3)4(PMDETA)2 + 18-c-6, 4-pentynol was placed in a Young's tap NMR tube with C6D6 (0.6 mL), 1,2,3,4-tetraphenylnaphthalene and 18-crown-6 . To this K2Mg(CH2SiMe3)4(PMDETA)2 was added. The reaction was maintained at 70 \u00b0C in the NMR spectrometer and was monitored by 1H NMR spectroscopy. Yields were calculated using NMR spectroscopic integrals characteristic of 2a relative to the internal standard, 1,2,3,4-tetraphenylnaphthalene. This procedure was repeated with 0.90, and 1.20 mmol alkynol (1) to provide the data presented in Fig. S1In the kinetic studies to determine the rate dependence of alkynol in the cyclisation of 4-pentynol 4(PMDETA)2 were employed, necessitating 0.05, 0.06 and 0.07 mmol 18-crown-6 co-catalyst. The data from these experiments with varying catalyst concentration are presented in Fig. S2The same procedure was applied to the investigation of the dependence of the initial rates of the reaction on the catalyst. The same concentration of 2K2Mg(CH2SiMe3)4 paired with 18-crown-6 has been shown to function well with both terminal and internal alkyne substrates having a range of functional groups. Kinetic studies have revealed an inhibition effect of substrate on the catalyst under standard conditions by the formation of a coordination adduct which requires dissociation prior to the cyclisation step. Initial reactivity studies suggest that coordination of the 18-crown-6 to K finely tunes the reactivity of the bimetallic system, probably minimising the coordination of additional substrate molecules to the catalyst.In summary, alkali metal magnesiates have been shown to successfully promote the catalytic intramolecular hydroalkoxylation of alkynols through cooperative bimetallic catalysis. The roles of both magnesium and potassium components are crucial for the success of the process, affording a unique type of substrate activation that is not possible in conventional single-metal systems. Through cooperativity, the utilisation of an alkali metal magnesiate has overcome the inherent problems associated with homometallic magnesium systems. The optimised catalyst system, (PMDETA)There are no conflicts to declare.Supplementary informationClick here for additional data file."} +{"text": "The important role of hydrogen bonding in the interactions of cationic-amphiphilic polymers with bacterial membranes has been reported. N-alkyl maleimide based amphiphilic polymers. Our studies reveal that amide polymer (AC3P) is a potent antibacterial agent with high membrane-disrupting properties compared to its ester counterpart (EC3P). To understand these differences we performed bio-physical experiments and molecular dynamics (MD) simulations which showed strong interactions of AC3P including hydrogen bonding with lipid head groups of bacterial model lipid bilayers, that are absent in EC3P, make them selective for bacterial membranes. Mechanistic investigations of these polymers in bacteria revealed specific membrane disruptive activity leading to the delocalization of cell division related proteins. This unprecedented and unique concept provides an understanding of bacterial membrane interactions highlighting the role of hydrogen bonding. Thus, these findings will have significant implications in efficient design of potent membrane-active agents.Biomimetic antibacterial polymers, the functional mimics of antimicrobial peptides (AMPs), targeting the bacterial cell membrane have been developed to combat the problem of antibiotic resistance. Amphiphilicity, a balance of cationic charge and hydrophobicity, in these polymers has been shown to be pivotal for their selective interactions with anionic lipid membranes of bacteria instead of zwitterionic mammalian (human erythrocyte) membranes. However, it is unclear if and to what extent hydrogen bonding in amphiphilic antibacterial polymers contributes to this membrane binding specificity. To address this, we employ isosteric substitution of ester with amide moieties that differ in their potency for hydrogen bonding in the side chains of This amphiphilicity which is a fine balance of hydrophobicity and cationic charge has been shown to play a key role in the selective interaction of these polymers with the bacterial membranes instead of mammalian based amphiphilic polymers prepared from their maleic anhydride precursor polymer. Substitution of an ester with an amide is isosteric and also varies in the potency of hydrogen bonding. We found that the amide polymers (AC3P) displayed high antibacterial activity compared to their ester (EC3P) counterparts. To probe these differences, we performed biophysical experiments and molecular dynamic (MD) simulations on bacterial model lipid bilayers. Collectively, our studies provided the evidence that AC3P, but not EC3P, form strong interactions including hydrogen bonding with the phosphate head groups of bacterial model lipid bilayers based amphiphilic polymers. An amide bond is isosteric to an ester because the \u2013O\u2013 in the ester is of near-equal size as the \u2013NH\u2013 in the amide moiety.1H NMR analysis as described previouslyalt-N--maleimide) (PIBMI) based quaternized amide (A) and ester (E) appended alkyl side chains respectively. A control polymer, HexP was weakly antibacterial with MIC = 125\u2013250 \u03bcg mL\u20131 against both E. coli and S. aureus has potent antibacterial efficacy with MIC of 31 \u03bcg mL. aureus .\u20131 and 3 \u03bcg mL\u20131 for AC3P whereas EC3P showed 16 \u03bcg mL\u20131 and >200 \u03bcg mL\u20131 against S. aureus and E. coli, respectively (Table S150 > 1000 \u03bcg mL\u20131 (50 = 30 \u03bcg mL\u20131) leading to low selectivity (HC50/MIC) against E. coli and S. aureus at 25 \u03bcg mL\u20131. AC3P has higher depolarization than the corresponding ester, EC3P (E. coli and S. aureus. Kinetics of membrane permeabilization was studied by measuring the uptake of the fluorescent probe propidium iodide (PI) against E. coli and S. aureus at 25 \u03bcg mL\u20131. Amide containing polymer has higher ability to permeabilize the bacterial cell membrane than ester polymer showed higher release of ATP levels compared to its ester counterpart, EC3P (50 \u03bcg mL\u20131) in both E. coli and S. aureus , the dissipation of membrane potential resulted in the complete delocalization of MinD, MreB and FtsZ. CCCP had been used as a positive control to see the effect of dissipating the membrane potential on delocalization of MinD /(I440 + I490)).To emphasize the fact that the interaction of the polymers is indeed with the cell membrane, we used model lipid bilayers. Liposomes were made using DPPG\u2009:\u2009DPPE (88\u2009:\u200912) and DPPC mimicking the bacterial and human erythrocyte membranes respectively with Laurdan , a hydrophobic dye encapsulated in them .34 Due ti.e. by gradually reducing the concentration of the free polymer in the solution (lipid\u2009:\u2009polymer = 30\u2009:\u20091). The interaction of the polymers with a DPPG\u2009:\u2009DPPE (88\u2009:\u200912) model lipid bilayer was found to be an entropy-driven endothermic process. AC3P had a complete and spontaneous entropy-driven interaction with the bacterial model lipid bilayer (within 20 injections) having \u0394G = \u201311.03 kcal mol\u20131 but with a lower positive \u0394S = 43.9 cal mol\u20131 K\u20131 suggest the \u201chydrophobic effect\u201d explained by the loss of water as the amphipathic molecule enters the lipid bilayer.To delineate the molecular understanding of interactions between the model lipid bilayers and polymers, we used isothermal titration calorimetry (ITC) for obtaining the thermodynamics of interactions. DPPG\u2009:\u2009DPPE (88\u2009:\u200912) or DPPC model lipid bilayers were injected into the polymer solution, l mol\u20131) . The conn Fig. S3. On the n Fig. S3. These rn Fig. S3. PositivN-alkylmaleimide and isobutylene moieties) simulations. The interactions were modelled using a mixture of (POPE\u2009:\u2009POPG = 7\u2009:\u20093).oieties) which weoieties) . Conformoieties) . Even Heoieties) . These rSuch differences in conformations indicate that these isosteric amide and ester polymers differ in the favourability of their interactions with bilayer lipids. To investigate the same, their interaction energies with the lipid bilayers were computed. The interaction energies have been computed in per sequestered side chain basis to keep them in same footing, since various polymers have different number of sequestered side arms after 150 ns of simulations. It can be seen from g(r)). Radial distribution functions have been computed between the amide and ester groups' hydrogen bond formers and the same from lipid head group atoms and are interpret in the following as their relative affinities in forming hydrogen bonds. The amide group scale\" fill=\"currentColor\" stroke=\"none\">O moieties) of the amide polymers interacted through strong hydrogen bonds with the oxygen atoms of the phosphate head groups of the POPG lipid molecules as seen in The differences in the ability of isosteric polymer species to form hydrogen bonds with the lipid head group atoms were probed through direct computations of number of hydrogen bonds. The positional order in the spatial distribution of hydrogen bond forming lipid head group atoms in the neighbourhood of amide and ester moieties has also been calculated. Conventionally, in atomistic MD simulations, hydrogen bonds are calculated using geometric criteria and the same was used in the present cases (donor\u2013acceptor distance \u2264 4.0 angstrom (\u00c5) and donor\u2013H\u2013acceptor angle \u2264 60\u00b0). The amide polymers have displayed a greater propensity to form hydrogen bonds compared to their ester counterparts, both in the overall number of hydrogen bonds formed and the number of sequestered side arms observed to be involved in the formation of said bonds Table S2. The proThe discernible differences in lipid\u2013amide polymer and lipid\u2013ester polymer interactions described above are further reflected in the relative ability of isosteric polymers in inducing structural re-organization of the lipid bilayer. The number density distribution of polymers and POPG molecules in the upper leaflet is shown in 20\u20131) and the amide (\u2013CONH\u2013) and the ester (\u2013COO\u2013) regions in the polymers (1600\u20131800 cm\u20131).To gain further insight into the hydrogen bonding interactions of polymers with lipid molecules, Raman spectroscopic studies were performed. The organic solution of the lipid + polymer was drop-casted onto a substrate, dried and studied using a Raman spectrophotometer. The full Raman spectra are provided in ESI Fig. S9. Since t\u20131 vibrational mode is due to \u2013P\u2013O\u2013 (DPPG head group), 1100 cm\u20131 is due to PO2\u2013 and 1129 cm\u20131 is due to \u2013P\u2013O\u2013 (DPPG tail group) (blue curve) for DPPG.\u20131 vibrational mode splits into doublet upon interacting with AC3P and EC3P scale\" fill=\"currentColor\" stroke=\"none\">P\u2013O\u2013 has the ability to resonate, hence the Fermi resonance (expected in AB2) doublet does not exist in PO2\u2013 of DPPG and the higher frequency mode is due to the P PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O group (1100 cm\u20131). Interestingly, the relative intensities of the two peaks in doublet is higher in AC3P + DPPG than in EC3P + DPPG suggesting the strong interactions of amide polymer with the lipid compared to the ester polymer of the spectra as shown in \u20131) and the side chain ester C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O vibrational mode (1740 cm\u20131). PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O of DPPG scale\" fill=\"currentColor\" stroke=\"none\">O (imide) at around 1770 cm\u20131. It is interesting to see that amide I mode (1680 cm\u20131) disappears in DPPG + AC3P (green curve \u20131) peak has shifted showing some weak interaction between DPPG and HexP as observed earlier.The polymer region (1600\u20131800 cmTm) of DPPG is 41 \u00b0C) (\u20131) band above 35 \u00b0C suggested the breaking of hydrogen bond. The strong 1740 cm\u20131 also shows the emergence of long chain ester C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O vibration of the lipid. Similar experiments in the DPPG + EC3P spectrum bring the emergence of a strong 1740 cm\u20131 similar to DPPG + AC3P indicated the breaking of ester polymer C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O hydrogen bond with DPPG . The eme Fig. S10. Heating Fig. S10.The above results clearly suggests that the difference between the interaction of AC3P and EC3P with DPPG is that the amide provides strong hydrogen bonding interactions with DPPG compared to EC3P probably through the two hydroxyl groups of the DPPG head group. These results also correlated with MD simulations which showed the hydrogen bonding interactions of amide polymers with the hydroxyl and phosphate oxygen atoms of POPG in bacterial model lipid bilayers. Taken together, these data suggested that despite similar electrostatic interactions, the lack of strong hydrogen bonding interactions can render weaker binding of ester polymers with lipid bilayers compared to amide polymers.In conclusion, isosteric substitution of ester with amide moiety in cationic-amphiphilic polymers influences the bacterial membrane interactions. With the unique concept of amide and ester moiety bearing polymers, for the first time we provided evidence for the role of hydrogen bonding in bacterial membrane interactions. However, the differences in the binding affinity of the amide and ester polymers with the bacterial membranes might not be solely due to the hydrogen bonding. We believe that this understanding will aid in bacterial membrane specific design of membrane-active molecules in future.alt-maleic anhydride) , 3-aminopropyldimethylamine and 1-propyl amine were purchased from Sigma-Aldrich and used as received. 1-Propanol was obtained from Spectrochem (India) and used as received. Culture media and the antibiotics were from HIMEDIA (India) and Sigma-Aldrich respectively. NMR spectra were recorded using Bruker AMX-400 (400 MHz for 1H) spectrometer. The chemical shifts (\u03b4) are reported in parts per million downfield from the peak for the internal standard TMS for 1H NMR. Infrared (IR) spectra of the solid compounds were recorded on Bruker IFS66 V/s spectrometer using KBr pellets. IR spectra of the compounds soluble in low-boiling solvents were recorded with the same instrument using NaCl crystal. Mass spectra were recorded on a Micromass Q-ToF micromass spectrometer. Optical density and absorbance were measured by Tecan InfinitePro series M200 Microplate Reader.All the solvents were of reagent grade and dried prior to use wherever required. Bromoacetyl-bromide, poly(isobutylene-S. aureus (MTCC 737) and E. coli (MTCC 443 or ATCC 25922) were purchased from MTCC . The antibacterial activity of the polymers was done against both Gram-negative (E. coli) and Gram-positive (S. aureus) bacteria. E. coli was cultured in Luria Bertani broth . For solid media, 5% agar was used along with above mentioned composition. The bacterial samples were freeze dried and stored at \u201380 \u00b0C. 5 \u03bcL of these stocks were added to 3 mL of the nutrient broth and the culture was grown for 6 h at 37 \u00b0C prior to the experiments.Bacterial strains Synthesis and characterization are provided in ESI.Antibacterial activity, hemolytic activity, cytoplasmic membrane depolarization and permeabilization were performed as described earlier.8\u20139 CFU mL\u20131) were harvested and washed twice with 10 mM TRIS buffer (pH = 7.5) and were resuspended in the same buffer. Then, 150 \u03bcL of bacterial suspension and 50 \u03bcL of test drugs were added to the micro centrifuge tube and incubated at 37 \u00b0C for 15 min. After 15 min, the bacterial suspension was centrifuged and 50 \u03bcL of the supernatant was transferred into a Corning 96 well black plate with clear bottom to find out the released ATP levels using ATP Bioluminescence Assay Kit (Sigma Aldrich) as per the manufacturer's instructions. A standard curve for ATP levels was generated using the ATP standards provided in the kit in the range of 1 \u00d7 10\u20136 to 1 \u00d7 10\u201311 moles of ATP levels. Relative ATP levels both in the standard curve and the test sample measurement were measured by subtracting the background ATP levels from the test sample ATP levels as per the manufacturer's instructions. All measurements were performed in duplicates using Tecan InfinitePro series M200 Microplate Reader.Mid-log phase bacteria (\u223c10B. subtilis strains were cultured in LB medium. Overnight cultures from a single colony were diluted and grown at 30 \u00b0C. Xylose (0.1\u20130.5%) was added to induce the GFP fusion proteins. At OD600 \u223c 0.2, 200 \u03bcL of cells were incubated with 25 \u03bcg mL\u20131 of polymers or positive control for 10 minutes. The last 3 minutes, 1 \u03bcg mL\u20131 Nile red (Sigma) was added for membrane staining. Microscope slides were covered with 1% agarose in deionized water. 0.4 \u03bcL of cells were spotted onto the agarose layer and excess liquid was allowed to evaporate before a 0.31 mm cover slip (VWR) was placed on top. Cells were observed with a Nikon Eclipse Ti inverted microscope with Nikon plan apo 100\u00d7 1.45 oil objective, and Hamamatsu ORCA Flash UBB 3.0 camera. Images were processed using Nikon and ImageJ software.Lipids (0.5 mM of DPPC or 0.5 mM DPPG\u2009:\u2009DPPE (88\u2009:\u200912)) and Laurdan dye (5 \u03bcM) were taken in round-bottom glass vials in chloroform. Thin films were made under dry argon gas, and films were dried under vacuum for complete drying. Lipid films were hydrated with 1\u00d7 PBS (pH = 7.4) for overnight. Hydrated films were then processed for 10 freeze thaw cycles from 70 to 4 \u00b0C with intermittent vortexing. Multilamellar vesicles were then sonicated at 70 \u00b0C for 15 min to get unilamellar vesicles.I440 \u2013 I490)/(I440 + I490)wherein I440 and I490 represent the fluorescent emission intensity at 440 nm and 490 nm respectively.2 mL of Laurdan embedded liposome dispersion (lipid\u2009:\u2009dye = 100\u2009:\u20091) containing the polymer solution (lipid\u2009:\u2009polymer = 7.4\u2009:\u20091) in PBS (pH = 7.4) was taken in a fluorescence cuvette. The fluorescence emission intensity was measured at 440 nm and 490 nm by using the excitation wavelength at 350 nm. The measurements were performed at 37 \u00b0C using Water Peltier system attached PerkinElmer LS-55 Luminescence Spectrometer. Laurdan, a hydrophobic dye detects changes in the membrane-phase properties through its sensitivity to the polarity of environment in the lipid bilayer. Polarity changes are shown by shifts in the Laurdan emission spectrum, which are quantified by calculating the generalized polarization (GP). Then, membrane hydration was determined using perturbations in Laurdan dye fluorescence due to the result of hydration of the lipid bilayer and quantified by calculating general polarization (GP). GP was calculated by using the following equation.GP = with 10 min intervals to ensure that the titration peak returned to the baseline before the next injection was done. Each injection lasted for 10 s and the stirring speed was kept at 307 rpm for homogenous mixing. A background titration was performed under same conditions with the buffer placed in the calorimetric cell instead of the polymer solution. This result was subtracted from each polymeric sample titration to account for the heat of dilution. The change in enthalpy (\u0394H) and the association constant (Ka) were obtained by fitting the data to one set of sites model using Origin 7 software from Micro Cal Inc. The Gibbs free energy change (\u0394G) and change in entropy (\u0394S) were calculated using the following equation\u0394G = \u2013RT\u2009ln(55.5Ka) = \u0394H \u2013 T\u0394Swherein, R is the universal gas constant , factor 55.5 is to account for the molar concentration of water and T is the absolute temperature in K.All experiments were performed at 37 \u00b0C using isothermal titration calorimeter . Reference cell was filled with double distilled water. All the experiments were performed in 10 mM HEPES and 0.14 M NaCl (pH = 7.4) buffer. The liposomal suspensions (DPPC or DPPG\u2009:\u2009DPPE (88\u2009:\u200912)) were made in the above buffer at 1 mM and polymers were also dissolved in the same buffer and used at a concentration of 50 \u03bcg mL+, Cl\u2013). Equilibrium structures of the 12-monomer length polymers in aqueous environment were obtained by simulating single polymers in 150 mM NaCl solutions for 50 ns. Initial system sizes for such simulations were \u223c68 \u00d7 68 \u00d7 68 \u00c53 containing \u223c9500 water molecules. Polymer properties in aqueous environment were computed over the last 30 ns (20\u201350 ns) of simulation data. The polymer configurations at the end of 50 ns were extracted, replicated and used to construct the polymer-bilayer systems. To construct each of the polymer-bilayer systems, four polymers were placed dispersed in the bilayer plane \u223c(12\u201318) \u00c5 away along the bilayer normal from one of the bilayer leaflets (referred to as \u201cupper leaflet\u201d). Additional water molecules were added so as to prevent the polymers from interacting strongly with the other bilayer leaflet (referred to as \u201clower leaflet\u201d), at least at the initial stages of simulations. Further, Na+ and Cl\u2013 ions were added to neutralize the excess charges in the systems and set salt concentration at 150 mM. The number of atoms for the polymer-bilayer systems simulated was \u223c86\u2009000. These systems were each simulated for 150 ns and stationary properties were computed over the last 20 ns (130\u2013150 ns) of simulation, unless stated otherwise. All simulations were performed under isothermal\u2013isobaric (NPT) conditions at a temperature of 310.15 K. Pressure was maintained at 1 atm using Langevin Piston.Classical atomistic molecular dynamics (MD) simulations were performed using simulation package NAMD (versions 2.8 and 2.9)\u20131) of sample was drop coated onto Sapphire substrate, dried under vacuum and studied using a Raman spectrophotometer.Measurements were performed using WiTec Raman spectrometer (UHTS600 SMFC) equipped with CCD (CCD-17531). The excitation source was 532 nm with a power of \u223c20 mW at the sample. A 60\u00d7 objective (Nikon make) with a numerical aperture (NA) 0.8 was used for normal Raman and 50\u00d7 ultra long working distance objective with 0.45 NA was used for temperature dependent studies for focusing the laser and collecting scattered light in 180\u00b0 back scattering geometry. The typical accumulation times were 600 seconds. Temperature dependent measurements were performed using Linkam cryostage (Linkam Scientific Instrument). 20 \u03bcL of methanolic solution (400 \u03bcg mLSupplementary informationClick here for additional data file."} +{"text": "The structures of metabolites produced in microgram quantities by enzymatic reductions with baker's yeast were analyzed using the crystalline sponge method. The crystalline sponge method coupled with HPLC purification would be a useful method for metabolic analysis and drug discovery. The structures of metabolites produced in microgram quantities by enzymatic reductions with baker's yeast were analyzed using the crystalline sponge method. The X-ray data provided reliable structures for trace metabolites including their relative and absolute stereochemistries that are not fully addressed by conventional NMR and LC-MS analyses. Technically, combining two or more chromatographic purification techniques is essential because, unlike abundant synthetic compounds, extracted metabolites contain many low level UV-silent impurities. The crystalline sponge method coupled with HPLC purification (LC-SCD) would thus be a useful method for metabolic analysis and drug discovery. NMR and MS) and separation techniques (e.g. GC and LC) have been frequently used and established to elucidate the structures of scarce metabolites from a mixture. However, full characterization of their structures, including their absolute stereochemistry, is still a laborious task because of limited sample supply.The structural information of metabolites is key to understanding enigmatic cellular processes and for drug design.The crystalline sponge method2)3(tpt)2\u00b7(c-C6H12)x]n -1,3,5-triazine),2) by reductases found in baker's yeast. When compound 2 (100 mg) was treated with fermenting baker's yeast (dry yeast (11.2 g) and sucrose (23.5 g) in 100 ml of water) at 30 \u00b0C for 1 week, a small amount of a metabolic product (\u223c1.3% yield based on UV spectroscopy) was detected in the ether extract obtained from the mixture. LDI-TOF MS spectrometry indicated the loss of one chlorine atom from 2, suggesting the formation of 1,1-bis(4-chlorophenyl)-2,2-dichloroethane . The formation of 3 in a low yield is consistent with the previous report.4, cannot be excluded as the product based solely on MS information.As a model case of metabolic analysis using a crystalline sponge [(ZnItpt)2\u00b7c-CH12x]n using PTLC, the analytical sample was further purified using HPLC with a narrow collection time window \u00b7(C7H4Cl2)\u00b72.5(C3H6), M = 2230.02, colorless block, 0.23 \u00d7 0.11 \u00d7 0.09 mm3, monoclinic, space group C2/c, a = 35.7365(13) \u00c5, b = 15.0610(5) \u00c5, c = 30.9345(11) \u00c5, \u03b2 = 104.141(7)\u00b0, V = 16\u2009145.2(11) \u00c53, Z = 8, Dc = 1.835 g cm\u20133, T = 93(2) K, 2.989\u00b0 < \u03b8 < 27.468\u00b0, 17\u2009652 unique reflections out of 88\u2009524 with I > 2\u03c3(I), GoF = 1.063, final R factors R1 = 0.0780, and wR2 = 0.2736 for all data, CCDC deposit number 1451761.\u2021Crystallographic data for One is located near a tpt ligand where guest 3 was observed with 100% occupancy . A better resolution was obtained for A, and the electron density map F0 clearly shows the structure of 3 were obtained presumably due to very minor disorder in 3, which cannot be modeled yet contributes to the residual electron density significantly because of the heavy atom (Cl).The crystallographic analysis confirmed the structure of the metabolite to be 3 . The cryure of 3 . Slightl13C or 2D NMR) become difficult even for determination of the relative stereochemistry. The absolute stereochemistry can hardly be addressed by any spectroscopic methods unless empirical rules or data for authentic compounds are available. Thus, we applied the LC-SCD analysis for the full structural characterization (including absolute stereochemistry) of a metabolite from tetralone 5. This compound exists as a racemate due to rapid enolization at C1 and can give four possible stereoisomers (including enantiomers) upon reduction of the carbonyl group at C2.When a prochiral functional group is enzymatically reduced to a chiral one, the stereochemical issues are of major concern for the structural characterization. Moreover, with the product(s) isolated only in microgram quantities, NMR analyses with baker's yeast (11.2 g) for 18 h gave analytically pure chiral alcohol 6 in 10 \u03bcg quantity after isolation using HPLC. The relative stereochemistry of 6 was confirmed to be cis by comparison with both an authentic cis\u2013trans mixture obtained by the NaBH4 reduction of 5 and literature reported spectroscopic data.cis isomer 6. An inclusion crystal 1\u00b76 was prepared by soaking a crystal of 1 in a cyclohexane/1,2-dichloroethane (v/v = 9\u2009:\u20091) solution of 6. The crystal structure was solved with a non-centrosymmetric space group C2.1\u00b76: C72H48I12N24Zn6\u00b73.79(C13H16O4)\u00b71.93(C6H12), M = 4220.24, colorless rod, 0.18 \u00d7 0.11 \u00d7 0.06 mm3, monoclinic, space group C2, a = 36.3783(8) \u00c5, b = 14.6755(2) \u00c5, c = 31.2278(6) \u00c5, \u03b2 = 103.184(2)\u00b0, V = 16\u2009232.2(5) \u00c53, Z = 4, Dc = 1.727 g cm\u20133, T = 100(2) K, 4.1720\u00b0 < \u03b8 < 74.0140\u00b0, 31\u2009961 unique reflections out of 69\u2009980 with I > 2\u03c3(I), GoF = 1.031, final R factors R1 = 0.0511, and wR2 = 0.1396 for all data, Flack parameter (Parsons) = 0.010(4), CCDC deposit number 1451762.\u00a7Crystallographic data for A reasonable Parsons' Flack parameter value of 0.010(4) was obtained and the final R1 and wR2 values were 0.0511 and 0.1396, respectively. In an asymmetric unit, three independent guest molecules 6 (G1\u2013G3 in 6 (G1\u2013G3) show a 1R,2S absolute configuration, consistent with a previous report,6. From this, a plausible reaction mechanism to give 6 is suggested to involve reduction from the Re face of a 1R isomer of 5 that is kinetically resolved within the enzyme pocket.Treating tetralone G1\u2013G3 in were cleG1\u2013G3 is worthy of additional discussion. The methoxycarbonyl groups in G1 and G3 are located at the pseudo-axial position, while that in G2 is at the pseudo-equatorial position. For G2, intramolecular hydrogen bonding between the OH and COOMe groups was indicated by the short OO\u2013H\u2013O PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019OC distance (\u223c2.8 \u00c5) that may stabilize this conformation. Presumably, this conformer exists in solution and the crystalline sponge traps both conformers at different binding sites, enabling the concomitant observation of both conformers.Conformational analysis of the observed guests 7) provided a more challenging task because three prochiral carbonyl carbons at C3, C11, and C17 can in principle generate 26 different compounds upon reduction of the carbonyl group(s). When steroid 7 was metabolized by baker's yeast on a 200 \u03bcg scale, HPLC analysis revealed the exclusive formation of a single metabolic product (hereafter denoted as 8).The structural and stereochemical analysis of a metabolite from adrenosterone . The increase in mass unit by 2.0 Da from 7 suggests a mono-hydrogenation of 7 at either a carbonyl or C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C group. The 1H NMR spectrum of 8 was, unfortunately, not conclusively able to determine its structure and stereochemistry because of overlapping signals. The unequivocal structure determination of 8 can be achieved, given that 8 is obtained only in microgram quantity, most reliably by the LC-SCD analysis.The parent ion peak of metabolite ca. 20 \u03bcg of a crude mixture obtained from the ether extract was purified using HPLC and subjected to guest soaking with a crystal of 1. After guest soaking at 50 \u00b0C for 2 d, the crystallographic analysis revealed three independent molecules of 8 (H1\u2013H3 in 1\u00b78: C72H48I12N24Zn6\u00b72.8(C19H26O3)\u00b71.5(C6H12), M = 4147.41, colorless plate, 0.28 \u00d7 0.08 \u00d7 0.07 mm3, monoclinic, space group C2, a = 34.7707(6) \u00c5, b = 14.8406(3) \u00c5, c = 31.2152(5) \u00c5, \u03b2 = 102.601(2)\u00b0, V = 15\u2009719.6(5) \u00c53, Z = 4, Dc = 1.749 g cm\u20133, T = 93(2) K, 2.9030\u00b0 < \u03b8 < 73.3970\u00b0, 29\u2009574 unique reflections out of 106\u2009306 with I > 2\u03c3(I), GoF = 1.005, final R factors R1 = 0.0788, and wR2 = 0.2259 for all data, Flack parameter (Parsons) = \u20130.008(8), CCDC deposit number 1451763.\u00b6Crystallographic data for All the structures revealed that the carbonyl group at C17 in 7 is stereoselectively reduced to a hydroxy group with an S configuration. The two carbonyl groups at C3 and C11 remain intact: typical C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O distances (1.19(3) and 1.20(3)) were observed at C3 and C11, respectively, and these carbon atoms still adopt a trigonal planar geometry. In contrast, elongation of the C\u2013O bond length of 1.41(3) \u00c5 and a tetrahedral geometry at C17 were observed, indicating that the carbonyl reduction took place only at C17. Notably, favorable host\u2013guest interactions were observed. For example, guest H1 is trapped by the host framework of 1 with C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O\u00b7\u00b7\u00b7H\u2013C or C\u2013H\u00b7\u00b7\u00b7I hydrogen bond interactions.Thus, H1\u2013H3 in in an as1 often preferentially absorbs minor components, even low-level impurities may disturb efficient guest soaking. Given that trace metabolites separated using HPLC are normally contaminated with many impurities, great care should be taken in the purification steps. Therefore, pre-purification with PTLC or LC\u2013LC is highly recommended. Second, pre-analysis of the abundant parent compounds would be beneficial because soaking conditions optimized for the parent compounds can usually be applied to the analysis of their metabolites. Third, having successful results in this study, we are more convinced that the crystalline sponge method, coupled with HPLC separation, will innovate the structural analysis of scarce amounts of microbial products in natural product chemistry as well as in drug discovery.In conclusion, LC-SCD analysis has been demonstrated as a useful tool for structural analysis of trace microbial metabolites. Some important lessons are learned in this study working with trace microbial metabolites, which we have not noticed in the previous analysis of abundant synthetic compounds. First, a key to the success of the analysis is to obtain high purity samples of the microbial metabolites by HPLC separation. As the crystalline sponge Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "Nitric oxide reacts with a NiSOD model complex to yield a thiolate-ligated/N-nitrosated {NiNO}10 species with unusually labile Ni\u2013NO bonds. + and NO with the model complex, Et4N[Ni(nmp)(SPh-o-NH2-p-CF3)] picolinamide; \u2013SPh-o-NH2-p-CF3 = 2-amino-4-(trifluoromethyl)benzenethiolate) and its oxidized analogue ox1, respectively. The ultimate products of these reactions are the disulfide of \u2013SPh-o-NH2-p-CF3 and the S,S-bridged tetrameric complex [Ni4(nmp)4], a result of S-based redox activity. However, introduction of NO to 1 affords the green dimeric {NiNO}10 complex (Et4N)2[{Ni(\u03ba2-SPh-o-NNO-p-CF3)(NO)}2] (2) via NO-induced loss of nmp2\u2013 as the disulfide and N-nitrosation of the aromatic thiolate. Complex 2 was characterized by X-ray crystallography and several spectroscopies. These measurements are in-line with other tetrahedral complexes in the {NiNO}10 classification. In contrast to the established stability of this metal-nitrosyl class, the Ni\u2013NO bond of 2 is labile and release of NO from this unit was quantified by trapping the NO with a CoII\u2013porphyrin (70\u201380% yield). In the process, the Ni ends up coordinated by two o-nitrosaminobenzenethiolato ligands to result in the structurally characterized trans-(Et4N)2[Ni(SPh-o-NNO-p-CF3)2] (3), likely by a disproportionation mechanism. The isolation and characterization of 2 and 3 suggest that: (i) the strongly donating thiolates dominate the electronic structure of Ni-nitrosyls that result in less covalent Ni\u2013NO bonds, and (ii) superoxide undergoes disproportionation via an outer-sphere mechanism in NiSOD as complexes in the {NiNO}9/8 state have yet to be isolated.Nitric oxide (NO) is used as a substrate analogue/spectroscopic probe of metal sites that bind and activate oxygen and its derivatives. To assess the interaction of superoxide with the Ni center in Ni-containing superoxide dismutase (NiSOD), we studied the reaction of NO Additionally, NO, while not isoelectronic with O2\u02d9\u2013, has the same ground state electronic structure with a singly occupied \u03c0* MO. Thus, NO interactions with the active sites of O2-activating/ROS-breakdown enzymes report coordination (inner-sphere substrate binding) and the extent of substrate bond activation from vibrational spectroscopic measurements of the N\u2013O and M\u2013NO stretching frequencies.Nitric oxide (NO) and its derivatives (termed reactive nitrogen species or RNS) play a vital role in a variety of mammalian physiological and pathological processes.III/II-coordination to cysteinato-S (CysS) and peptido-N donors (2\u02d9\u2013) and products (O2 and H2O2) of the SOD catalyzed reaction.3S2 donor set found in the active site.III oxidation state due to redox associated with the coordinated thiolates. One model from our lab, namely Et4N[Ni(nmp)(SPh-o-NH2-p-CF3)] picolinamide; see vs. Fc/Fc+ in DMF) that, based on EPR, UV-vis, MCD, and DFT computations, represents the electrochemical conversion from NiII in 1 to a NiII-thiyl \u2194 NiIII-thiolate resonance species termed ox1.2\u02d9\u2013 probe to define potential intermediates that may be traversed in the NiSOD mechanism. We report here, for the first time, the reactions and product characterization of NO (and NO+) with 1 and the well-defined analogue of NiSODox (ox1). NO/NO+ oxidize the aromatic thiolate ligand in ox1 and 1, respectively. However, introduction of NO to 1 affords the green dimeric {NiNO}10 complex (Et4N)2[{Ni(\u03ba2-SPh-o-NNO-p-CF3)(NO)}2] (2) via NO-induced loss of nmp2\u2013 as the disulfide and N-nitrosation of the aromatic thiolate .N donors , the forthiolate . While 21 with NOBF4 and in situ prepared ox1 with NO then reacts with NO to form the Ni-nitrosyl, formally a {NiNO}8 complex, assuming binding of NO and no other coordination sphere changes, using the notation defined by Enemark and Feltham.ox1 to generate the same species. Mixing a DMF solution of 1 with NOBF4 (1\u2009:\u20091) resulted in instantaneous bleaching of the solution, consistent with oxidation of the RS\u2013 ligand to disulfide (RSSR), and the appearance of a dark-red precipitate that was spectroscopically identified to be the neutral S,S-bridged tetramer [Ni4(nmp)4] classification,9/8 species is not entirely unrealistic in light of the strong donors present in 1 and in NiSOD, i.e., peptido-N and alkyl-thiolato-S.In the anticipation of isolating a Ni-nitrosyl as an analogue of a potential Ni-superoxo/peroxo catalytic intermediate of NiSOD, we examined the reaction of with NO . In theo4(nmp)4] .16 This \u2013NO bond .27 OveraII complex 1 with NO. In general, NO does not react with square-planar [Ni(nmp)(SR)]\u2013 complexes due to their diamagnetic nature. However, when R contains a potentially bidentate chelate, as in 1, a different course takes place. For instance, exposing a DMF solution of 1 with NO(g) for 30 s resulted in a gradual change of the solution from dark-red to green over several minutes. Workup of this reaction indicated a Ni-nitrosyl based on the strong double-humped peak in the N\u2013O stretching (\u03bdNO) region of the IR spectrum (vide infra). Subsequent crystallization of the bulk material from MeCN/Et2O at \u201320 \u00b0C resulted in green crystals of a dinuclear thiolate-bridged {NiNO}10 complex (Et4N)2[{Ni(\u03ba2-SPh-o-NNO-p-CF3)(NO)}2] (2) as depicted in 2 are distorted tetrahedral ,2 is analogous to the limited number of four-coordinate/S-bound Ni-nitrosyls,thiolate bonds3Ni\u2013NO/L2XNi\u2013NO complexes exhibit similar metric parameters. The coordinated nitrosamine is bent (N\u2013N\u2013O: 115.4\u00b0), i.e., sp2-hybridized nitroso-N, with N\u2013N and N\u2013O distances of 1.299 and 1.269 \u00c5, respectively. These values suggest a small degree of delocalization in the R\u2013N\u2013N\u2013O unit. However, the structure is more biased towards the nitrosamino R\u2013N\u2013\u2013N PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O versus diazoate R\u2013N PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N\u2013O\u2013 resonance form. To compare, the structure of syn-methanediazoate reflects the true double bond character in an authentic R\u2013N PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N\u2013O unit.3)(ON2Ph-p-NO2)] (I)vs. amine-N as in 2) also afford similar structural parameters in the RNNO.III(P)(ONNR2)2]+ (P = porphyrin) complexes are nearly identical and result in a single 15N-sensitive peak in the IR due to overlapping \u03bdNN/\u03bdNO modes.As a control, we also explored the reaction of Ni4 = 0.73 ) resulti2 was characterized by a variety of spectroscopic methods. The solid-state IR spectrum (KBr matrix) of 2 exhibits two closely spaced, but well-resolved, \u03bdNO at 1759 and 1743 cm\u20131 , two IR-active N\u2013O vibrational modes are expected. The other feasible isomer of 2 would be of Ci symmetry and would display one IR-active N\u2013O stretch. Indeed, the IR spectrum of 2 in DMSO exhibits one \u03bdNO at 1784 cm\u20131 suggesting possible cis/trans-NO conversion in solution or thiolate-bridge splitting to yield a four-coordinate mononuclear {NiNO}10 with DMSO as the fourth ligand, i.e., [Ni(\u03ba2-SPh-o-NNO-p-CF3)(DMSO)(NO)]\u2013. The 1H NMR spectrum of 2 in CD3CN are similar and thus do not distinguish any of the proposed structures. Comparable IR spectral changes in the opposite direction are observed for the one other thiolate-supported anionic dinuclear {NiNO}10, (Et4N)2[Ni2(NO)2(\u03bc-SPh)2(SPh)2] (II), with trans NO ligands .10 complex.15N-sensitive peaks in the IR of 2 at 1342 and 1258 cm\u20131 are assigned as \u03bdNO and \u03bdNN, respectively. In comparison, a series of secondary nitrosamines display \u03bdNO: 1428\u20131463 cm\u20131 and \u03bdNN: 1035\u20131154 cm\u20131 in CCl4.2 to cause the corresponding downshift in \u03bdNO/upshift in \u03bdNN. While no paramagnetically shifted resonances are observed in the 1H NMR (CD3CN) of 2, several species are indicated in freshly prepared solutions . The 15N NMR spectrum of 15NO2- confirms multiple solution speciation with four major peaks in the range for nitrosamines and linearly coordinated NO 2[Ni(NO)(SPh)3] (III) and an uncharacterized [Ni(NO)(SPh)] species.2 is centered at m/z: 248.960 ]2\u2013 species through loss of the Ni-coordinated NO and one Ni (vide infra).Complex N Fig. S6 or DMSO-s Fig. S6 that are2, especially in donor solvents such as MeCN or DMF, gradually lose their green color to give red-brown solutions more reminiscent of square-planar NiII\u2013N2S2 complexes.3CN solutions of 2 exhibit multiple peaks in the 1H/15N NMR, and ESI-MS shows a new species with a Ni isotope pattern at m/z \u223c 249 (vide supra). This change is enhanced when vacuum is applied and FTIR spectra of these reaction mixtures lack any \u03bdNO suggesting the loss of coordinated NO from 2 to generate a new Ni species. Slow diffusion of Et2O into MeCN solutions of 2 that have been left standing for several weeks result in crystals of a square-planar (\u03c44 = 0.12) NiII compound where two N,S-chelating o-nitrosaminobenzenethiolato ligands bind to Ni in a trans configuration, viz. trans-(Et4N)2[Ni(SPh-o-NNO-p-CF3)2] (3) .i.e., short C\u2013S, C\u2013N distances of the coordinated o-aminobenzenethiolate3 is unremarkable from 2. 1H and 15N NMR of crystals of 3 are consistent with the X-ray structure and analogous to other nitrosamines confirm this formulation with peaks corresponding to [M\u20132Et4N]2\u2013 as the prominent peak 2] (3) . The bon3)2] (3) are simi2 (forming 3 among other products), solutions of 2 were mixed with the NO(g) trap [Co(T(-OMe)PP)] (T(-OMe)PP = 5,10,15,20-tetrakis(4-methoxyphenyl)-21H,23H-porphine).2 and the CoII\u2013P (1\u2009:\u20092) in CH2Cl2 at RT for 24 h resulted in the {CoNO}8 complex [Co(T(-OMe)PP)(NO)] in \u223c70% avg. yield as quantified by 1H NMR (CD2Cl2) and further verified by IR spectroscopy using 15NO2- via1H NMR to confirm the fate of the {NiNO}10 complex 2. To eliminate bimolecular NO-transfer via a putative Co\u00b7\u00b7\u00b7NO\u00b7\u00b7\u00b7Ni intermediate, NO(g) release was further verified by vial-to-vial trapping reactions wherein a CH2Cl2 solution of the CoII\u2013P was separated from an MeCN solution of 2 to generate the {CoNO}8 porphyrin complex (80% avg. yield) as shown by 1H NMR and IR measurements Cl] , a common HNO (or NO\u2013) trap.10 has not been characterized as a particularly labile EF notation, we note that the majority of these complexes are cationic or neutral without coordinated thiolate ligands.10 complex III photochemically releases NO to [Co(TPP)] in MeCN suggesting some lability in the Ni\u2013NO bond. Furthermore, the RN\u2013NO bond is quite stable (as noted by formation of 3) and the energetically stabilized MOs that contribute to the electronic structure of 2 and 3 where HOMO\u20133 represents a bonding MO with primary contributions from \u03c3-NR and \u03c3-NO orbitals is released from Fig. S24. In cont Fig. S25.2 and 3 at the OLYP/def2-TZVPP level of theory. Pure functionals such as BP86 and OLYP were used for geometry optimization and single point energy calculations, respectively, as these functionals have been established to deliver better matches with experimental geometries in MNO systems.2 was performed with coordinates from the crystal structure to yield DFT-optimized complex 2* are slightly beyond the allowable tolerances for satisfactory DFT performance in small molecules .2. The computations also reasonably match the two closely spaced N\u2013O stretching frequencies for the symmetric and asymmetric \u03bdNO in the IR at 1730 and 1708 cm\u20131, respectively. The \u223c30 cm\u20131 downshift from 2 is likely due to a slight overestimation of Ni\u2013NO bond covalency arising from Ni-d\u03c0 backbonding. Previous calculations on three-10 complexes support a NiII\u20133NO\u2013 oxidation state assignment. This is comparable to high-spin nonheme {FeNO}7 systems that are classified as FeIII\u20133NO\u2013 (Stot = 3/2).3NO\u2013 serves as a strong \u03c0-donor to afford a highly covalent Fe\u2013NO bond.3NO\u2013 to result in diminished M\u2013NO bond covalency. This property has been established in the {FeNO}7 case, but not yet for {NiNO}10. Indeed, examination of the frontier MOs of 2* show that, much like other {NiNO}10 systems with Tp ligands2Ni core. The HOMO is antibonding in nature and suggests a thermally unstable structure. As expected from analogous {FeNO}7 systems, based on the increased donor strength of the anionic nitrosamine-N/thiolate-S supporting ligands in 2*, the covalency in the Ni\u2013N\u2013O unit is less than in TpNi\u2013NO complexes and rationalizes the observed lability of the Ni\u2013NO bond and the Ni(\u03bc-SR)2Ni core in 2.Density functional theory (DFT) computations have provided a deeper understanding of the electronic structure of a variety of metal nitrosyls,mplex 2* . Structu3* were performed in the same fashion as for 2*. Geometry optimized 3* is square-planar (\u03c44 = 0.09) with metric parameters on-par with the X-ray structure of 3 and within the error of the DFT method /S(p\u03c0) contributions (SR)]\u2013 complexes at least in the solid-state. Introduction of NO(g) can then result in either: (i) reduction of NiII to NiI and formation of NO+ that nitrosates the coordinated amine, or (ii) nitrosylation of Ni to yield {NiNO}10 with the electron originating from the coordinated thiolate of nmp2\u2013 to result in the disulfide. Our results do not differentiate either of these transformations, but the disulfide of nmp2\u2013 . In ligand rearrangement, the products would be a NiI\u2013N2S2 precursor to 3 (3-PC), an L\u2013NiI\u2013NO species (L = solvent), and free NO. Ultimately this NiI intermediate oxidizes 3-PC to generate NiII complex 3 and an L\u2013Ni0\u2013NO complex that would presumably release NO(g) as evidenced by the NO(g) trap experiments (vide supra). While the reaction mechanism for the conversion of 2-to-3 is likely more complex, similar chemistry has been proposed for N-heterocyclic carbene (NHC) Ni-nitrosyls.The formation of shown in to yield1 reacts with NO(g) in the NiII state to form the metastable {NiNO}10 dimeric complex 2via loss of the nmp2\u2013 ligand as the disulfide and N-nitrosation of the o-aminobenzenethiolate ligand. Reaction of NO with ox1, or NO+ with 1, only yields the S,S-bridged tetrameric compound [Ni4(nmp)4] through oxidation of the aromatic thiolate ligand. While any reaction with NO (S = 1/2) is generally unexpected for square-planar (S = 0) NiII complexes, this Ni-nitrosyl likely forms due to an equilibrium mixture of 1 and a tetrahedral (S = 1) or five-coordinate derivative (9 (reaction of 1 with NO) or {NiNO}8 (reaction of ox1 with NO) oxidation levels have yet to be defined and support an outer-sphere superoxide interaction in NiSOD. Although these EF notations have yet to be accessed, one would propose that NiSOD mimetics, especially with strong-field carboxamido-N and alkyl-thiolato-S donors, would surely stabilize such an electron poor species. Furthermore, the properties of complexes such as 2 extend to biology, where analogous S-bridged mononitrosyl species, i.e., Fe\u2013S clusters and tetrahedral (RS)3Fe\u2013NO complexes are proposed as intermediates in the repair of NO-damaged clusters.2 is stable in the solid-state but breaks down slowly in solution causing rupture of the Ni(\u03bc-SR)2Ni core and release of NO that was trapped in near quantitative yield with a CoII\u2013porphyrin receptor. The resulting NiII\u2013N2S2 complex 3 (coordination of two o-aminobenzenethiolate in trans configuration) was isolated and structurally/spectroscopically characterized as the ultimate Ni breakdown product with the nitrosamine unit still intact. This release may take place through a disproportionation mechanism (or through ligand rearrangement), as has been proposed in other Ni-nitrosyls, to a yet ill-defined Ni0 complex is high or generate other reactive intermediates of environmental significance such as hyponitrite (N2O22\u2013) in five-coordinate {NiNO}10 speciesIn conclusion, NiSOD model complex rivative . Even ifplex see .28,78 Heal mol\u20131 ) compareThere are no conflicts to declare.Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "Post-polymerization ladderization is explored as a promising technique to boost the photo-catalytic activity of conjugated polymers. via post-polymerization annulation and oxidation techniques to generate rigidified, planarized materials bearing dibenzothiophene (cLaP1) and dibenzothiophene sulfone subunits (cLaP2). The high photocatalytic activity of cLaP1 (1307 \u03bcmol h\u20131 g\u20131) in comparison to that of cLaP2 (18 \u03bcmol h\u20131 g\u20131) under broadband illumination (\u03bb > 295 nm) in the presence of a hole-scavenger is attributed to a higher yield of long-lived charges , as evidenced by transient absorption spectroscopy. Additionally, cLaP1 has a larger overpotential for proton reduction and thus an increased driving force for the evolution of hydrogen under sacrificial conditions.Conjugated ladder polymers (cLaPs) are introduced as organic semiconductors for photocatalytic hydrogen evolution from water under sacrificial conditions. Starting from a linear conjugated polymer (cLiP1), two ladder polymers are synthesized Building block 1 was synthesized from 1,4-dibromobenzene -catalyzed Suzuki\u2013Miyaura cross-coupling reaction gave the parent polymer cLiP1.+-MecLaP) was dealkylated using NEt4Br to give the ladderized polymer cLaP1. Further oxidation of cLaP1 with H2O2 in glacial acetic acid gives cLaP2, which is a ladder-type analogue of the linear dibenzothiophene sulfone polymer (P10).301H{13C} NMR and IR spectra for previously reported compounds scale\" fill=\"currentColor\" stroke=\"none\">OS) and, in the fingerprint region, sharp peaks corresponding to wagging C\u2013H and C\u2013C vibrations for 1,4- and 1,2,4,5-substituted benzene subunits are observed and 1156 cm\u20131 scale\" fill=\"currentColor\" stroke=\"none\">S PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019OO), see also Fig. S11\u20131 consistent with a polymer containing 1,2,4,5-substituted benzene subunits. The signals of the triflate anion scale\" fill=\"currentColor\" stroke=\"none\">OS)) after the ladderization could not be detected due to the overlapping signals of the triflate anion. Thus, no further conclusions on the success rate of the annulation or the presence of defects (non-annulated aryl sulfoxide groups) could be made at this point. Upon oxidation to cLaP2, the spectrum shows two strong stretching vibrations scale\" fill=\"currentColor\" stroke=\"none\">S PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019OO) indicating the oxidation of the dibenzothiophene moiety to dibenzothiophene dioxide was used to analyze the insoluble polymers: Fig. S10. Upon la Fig. S12. AdditiocLiP1 has limited long-range order, while cLaP1 and cLaP2 are amorphous can be found in the ESI.\u00a7UV-vis and fluorescence spectra for and this clearly shows that the system has a higher degree of conjugation as the annulation of neighbouring conjugated units reduces their respective torsion angles close to zero degrees as the system is rigidified show that polymer Fig. S22. UV-vis id-state : polymercLaP2 and cLaP1 compared to cLiP1 , as observed previously.31The fluorescence emission spectra in the solid state show the same bathochromic shifts as the UV-vis absorption spectra. Moreover, a smaller Stokes\u2019 shift and resolved vibronic coupling in the excitation spectrum of \u03bb > 295 nm and \u03bb > 420 nm; cLiP1 showed very limited activity (15 \u03bcmol h\u20131 g\u20131), even compared to poly(p-phenylene) (232 \u03bcmol h\u20131 g\u20131).cLaP1, the photocatalytic activity increased dramatically to 1307 \u03bcmol h\u20131 g\u20131. When the material was recovered and used again as a photocatalyst, an increase in the hydrogen evolution rate to over 2000 \u03bcmol h\u20131 g\u20131 was observed .The photocatalytic activity of these materials for hydrogen evolution from water in the presence of triethylamine (TEA) as a sacrificial electron donor was studied under broad-spectrum and visible light irradiation . Similarly, cLaP2 showed a 10-fold increase in activity (from 18 to 272 \u03bcmol h\u20131 g\u20131) under broadband illumination with platinum as co-catalyst .All of the materials were tested as synthesized without the addition of any additional metal co-catalyst. However, it has been shown that residual palladium originating from the Suzuki\u2013Miyaura coupling reaction can act as a co-catalyst.cLaP1 in H2O/TEA/MeOH mixtures using monochromatic light and these show that the hydrogen production is indeed photocatalytic . This is higher than previously reported for P1 (EQE420 nm = 0.4%)P12 (EQE420 nm = 1.4%)B-BT-1,4 (EQE420 nm = 4.0%)b,d]thiophene sulfone P7 (EQE420 nm = 7.2%)2O/TEA/MeOH mixture.External quantum efficiencies (EQEs) were estimated for Fig. S31. At 420 cLiP1, cLaP1 and cLaP2 in the presence of a H2O/TEA/MeOH mixture under a nitrogen atmosphere (cLaP1 was far greater (\u00d75\u201310) than those of cLiP1 and cLaP2, indicating an increase in long-lived photogenerated species. The TA spectrum of cLaP1 contains two distinct absorptions centred at ca. 500 and 630 nm that decay at different rates , 25 \u03bcs (630 nm) under N2).\u00b6We note that the lifetimes of all of the bands are dependent upon the history of the sample and tend to decrease after prolonged experiments. However, the signal at 500 nm is consistently shorter-lived compared to the signal at 625 nm. In the absence of methanol and TEA the long-lived TA bands at 500 and 630 nm are no longer observed spectroscopy. TA has been shown to be an effective tool for studying the formation and lifetime of electron\u2013polaron states in polymer photocatalysts for hydrogen evolution.mosphere . All throbserved . The rol Fig. S37. The obscLaP2 are far smaller than those of cLaP1 on the \u03bcs timescale, the kinetic traces recorded at 500 nm for cLaP2 do indicate an extremely long-lived (t50% = 0.3 s) photogenerated species. The presence of extremely long-lived charges, potentially with a low thermodynamic driving force, may also be a factor behind the low HER observed for cLaP2. To explore this observation further, Pt was added as a co-catalyst in the hope that it may be able to either intercept photoelectrons and prevent trapping or offer suitable catalytic sites to facilitate charge transfer into solution. cLaP2@Pt does show an improvement in HER, increasing from 18 \u03bcmol h\u20131 g\u20131 to 272 \u03bcmol h\u20131 g\u20131 + H+ and II: TEAR + h+ + H2O \u2192 diethylamine + acetaldehyde + H+), then the fact that cLaP1 is predicted to have only negligible driving force for the first step, in contrast to cLaP2, might suggest that differences in catalytic activity between cLaP1 and cLaP2 stems from differences in the driving force for proton reduction. In line with experimental UV-vis absorption spectra, ladder polymers cLaP1 and cLaP2 are predicted to have optical gaps that are \u223c1.0 and \u223c0.8 eV lower for cLaP1 and cLaP2, respectively, than for cLiP1 is also in line with experimental spectra.To gain insights into changes in the thermodynamic driving force for proton reduction and TEA oxidation within the investigated series of polymers, the IP and EA levels as well as optical gaps for varying oligomer lengths were estimated using a family of recently developed semi-empirical density functional tight-binding methods.cLaP1 outperforms its non-annulated parent polymer, cLiP1, significantly, and the optical properties of the material after annulation show a red-shift in the absorption onset. The ability of cLaP1 to absorb more photons while maintaining a hydrogen reduction driving force at least partially explains its higher photocatalytic activity, especially under filtered visible light. No significant changes in the optical properties were observed upon oxidation of cLaP1 to cLaP2, but the resulting dibenzothiophene sulfone material is almost inactive. This low activity of cLaP2 is surprising since TCSPC shows that cLaP2 has the longest weighted average lifetime of the exited state; significantly longer than those of cLiP1 and cLaP1. Also, the introduction of dibenzothiophene sulfone motifs into other polymer photocatalysts has been reported to give materials with high photocatalytic activity.cLaP2 has a reduced overpotential for proton reduction relative to cLaP1, while cLaP1 and cLaP2 both have a reasonable driving force for TEA oxidation. From a thermodynamic perspective, cLaP1 thus appears to be the best material in terms of combining thermodynamic driving force with light absorption. This is supported by TA measurements, which show the highest yield of long-lived charges in the case of cLaP1. From the TA data, it is clear that there is a direct correlation between the yield of long-lived charges present and the measured hydrogen evolution rate, suggesting that electron polaron states with lifetimes on the \u03bcs to ms timescale are required in order for hydrogen evolution to occur. This might rationalize the higher hydrogen evolution yields for cLaP1. The greater yield of long-lived photoelectrons may be related to more efficient hole scavenging at early timescales. The catalytic activity of the materials does not correlate with the residual palladium content, but it is unclear whether the threshold for an effect of the residual palladium on the photocatalytic performance has been reached.cLaP2 shows a persistent long-lived feature, which could be attributed to a deep-trapped charge . In both cases, we observed an increase in photocatalytic performance, but there was no evidence for higher charge carrier yields in the TA spectrum of the platinized cLaP2.The ladder polymer d charge , right, cLiP1, cLaP1 and cLaP2 can be rationalized by comparison of charge carrier lifetimes, light absorption, and thermodynamic driving forces. Compared with related linear polymers, such as P6/P60 and P7/P10 (cLaP1 (1307 \u03bcmol h\u20131 g\u20131) were found to be similar to those of its linear, non-ladderized analogues P6 (1660 \u03bcmol h\u20131 g\u20131) and P60 (1295 \u03bcmol h\u20131 g\u20131). However, when Pt was used as a co-catalyst, cLaP1@Pt outperformed P60@Pt under both broadband (2297 vs. 1703 \u03bcmol h\u20131 g\u20131) and visible light irradiation (1489 vs. 213 \u03bcmol h\u20131 g\u20131).In summary, the differences in photocatalytic activity in the series of d P7/P10 , the ideb,d]thiophene and dibenzothiophene sulfone polymers, we set out to synthesize a new class of ladderized conjugated polymer photocatalysts for photocatalytic evolution of hydrogen from water. Through post-polymerization ladderization, a planarization of the polymer chain and expansion of the \u03c0-system could be achieved, as evidenced by the bathochromic shift of the absorption edge. A significant increase in photocatalytic activity was measured for one ladder polymer (cLaP1) while the other (cLaP2) remained almost inactive. The difference in photocatalytic activity could be rationalized by analysis of the charge carrier lifetimes via TA spectroscopy and comparison of the driving forces derived from calculations. These results suggest that post-polymerization ladderization could be a valuable technique in the preparation of efficient photocatalysts and that ladder polymers containing other photocatalytically active subunits might be considered for future studies.Inspired by photocatalytically active dibenzo[There are no conflicts to declare.Supplementary informationClick here for additional data file."} +{"text": "A modular dynamic covalent approach towards rigid aryleneethynylene covalent organic polyhedrons (COPs) and the mechanistic features were explored. D2h symmetric), dimer, or interlocked complex can be formed from monomers with the same face-to-edge angle but in different sizes. As alkyne metathesis is a self-exchange reaction and non-directional, the cyclooligomerization of multi-alkyne monomers involves both intramolecular cyclization and intermolecular metathesis reaction, resulting in complicated thermodynamic process disturbed by kinetic competition. Although a tetrahedron-shaped tetramer (Td symmetric) has comparable thermodynamic stability to a D2h symmetric tetramer, its formation is kinetically disfavored and was not observed experimentally. Aryleneethynylene COPs consist of purely unsaturated carbon backbones and exhibit large internal cavities, which would have interesting applications in host\u2013guest chemistry and development of porous materials.A dynamic covalent approach towards rigid aryleneethynylene covalent organic polyhedrons (COPs) was explored. Our study on the relationship of the COP structures and the geometry of their building blocks reveals that the topology of aryleneethynylene COPs strongly depends on the size of the building blocks. A tetramer ( In addition, ethynylene linked COPs exhibit much higher stability, compared to those linked by C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N bonds or B\u2013O bonds, which are usually labile and prone to hydrolysis. Ethynylene-linked COPs have generally been prepared through kinetically-controlled Sonogashira or Glaser-type coupling.Molecular cages have shown great potential in molecular recognition, chemical sensing, catalysis, and gas adsorption/separation.COP-I and interlocked dimer complex (COP-II) through one-step alkyne metathesis cyclooligomerization of simple building blocks (1 provides high yielding of COP-I (72%) without any noticeable amount of interlocked species, monomer 2 predominantly forms interlocked complex COP-II (59%) along with a small amount of single dimer (6%). Intrigued by these results, we replaced the central panel of the monomers with a benzene ring, the smallest possible planar moiety available, wondering what would be the assembly product, interlocked complex or a dimer cage COP-III. The synthesis of tricarbazolyl-substituted benzene monomer 4 is straightforward scale\" fill=\"currentColor\" stroke=\"none\">\u2014PPT) and drive the metathesis equilibrium to completion. The alkyne metathesis of the monomer 4 was completed within 2 h under the catalysis of molybdenum(vi) carbyne complex prepared from molybdenum(vi) trisamide precursor EtC PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019Mo[N(t-Bu)Ar]3 and triphenolamine ligand (LN).Previously, we reported covalent assemblies of dimeric , and 2) .50,51 Botforward . . These 1H NMR patterns are highly similar to the D2h symmetric tetrameric cage (COP-V), whose structure has been unambiguously determined by single crystal X-ray analysis .6 produces symmetrical dimer COP-VII instead of a D2h symmetric tetramer in excellent isolated yield (84%), whereas 7 produces unknown precipitates with a trace amount of a dimer species, which was only observed in the MALDI-MS spectrum of the crude product mixture (16H33) are attached to the monomer in order to prevent the premature precipitation of the oligomeric/polymeric products from the reaction medium. Many attempts under various conditions failed to provide a soluble discrete species from monomer 7.It is unexpected to obtain mixture . In bothin situ by mixing Mo(vi) carbyne precursor Et PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019Mo[NAr(tBu)]3 with multidentate triphenolsilane (LSi) or triphenolamine (LN) ligands. Both catalytic systems are highly active and have long lifetime, thus suitable for the cage synthesis.We used molybdenum carbyne catalytic system containing multidentate ligand in the alkyne metathesis cyclooligomerization approach. The catalyst solution was prepared Alkyl substituents are attached to prevent precipitation of the reaction intermediates from the solution. In order to reach the equilibrium with the predominant formation of the desired cage products, precipitation (kinetic traps) should be avoided, which cannot further participate in the dynamic equilibrium process. We tested various alkyl chains, linear or branched, long or short. We found alkyl substituents have little effect on the control of cage topologies, that being said, their main contribution is to maintain good solubility of reaction intermediates.Alkyne metathesis is an exchange reaction involving redistribution of alkylidyne units between two alkynes through the proposed formation of metallacyclobutadienes followed by cycloreversion . Since aCOP-I), an interlocked complex consisting of two dimer cages (COP-II), or a tetrameric cage with D2h symmetry (COP-IV) were obtained as the assembly products, respectively (e.g.COP-III) similar to COP-I from all the monomers with face-to-edge angle of 90\u00b0. At first glance, such dimers would be entropically- and enthalphically-favored, since they consist of the fewest possible monomer units connected with minimum angle strain. Therefore, we were surprised to observe the predominant formation of tetrameric COP-IV with D2h symmetry from monomer 4. However, when we further scrutinized the reaction pathway, we found the possible steric reasons responsible for the disfavored formation of a dimer. As shown in PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C, there are two possible metathesis outcomes: nonproductive and productive pathways. In order to form a dimeric cage, in the final cyclization step, the two arms have to approach each other in head-to-head conformation to undergo a productive metathesis COP-V and our original target, tetrahedron-shaped COP-VI.The next puzzling observation that attracted our attention was the formation of COP-V and COP-VI. It should be noted that the pendant arms (carbazole or benzene) attached to the central panels (porphyrin or benzene) can freely rotate in all the monomers tested and are not preorganized to form a particular product. Since monomers are multitopic and self-reacting, the initial formation of the dimeric intermediate I[1+1] likely triggers intramolecular cyclization to form macrocyclic intermediates II[1+1] and further to form cages , interlocked dimer complexes (COP-II) or a tetramer COP-IV or COP-V are kinetically preferred. Unless there is a strong thermodynamic driving force towards COP-VI, its formation could thus be limited. When a benzene ring is used as the planar central piece in a monomer, the pendant third arms on the macrocyclic intermediate II[1+1] prefer intermolecular cyclization to form a tetramer COP-IV or COP-V rather than forming a dimer cage, presumably due to the significant angle strain built up in the latter case as discussed previously. The assemblies of building blocks vary significantly with the choice of connecting chemical bonds that display different properties, such as kinetics, directional/non-directional, bond angles, and torsional/rotational freedoms. The dynamic assembly of COPs through alkyne metathesis, which is self-reacting, represents a complex system in which both thermodynamic and kinetic factors have to be considered.Intrigued by these observations, we next investigated the possible kinetic competition between the formation of rm cages . We indeI[1+1] dimer, followed by a series of subsequent intramolecular cyclizations rather than intermolecular reactions, would be preferred. Therefore, the formation of tetrahedron-shaped COPs (e.g.COP-VI), which involves more intermolecular metathesis steps, is kinetically disfavored. However, formation of such a cage structure can be predominant when there is a strong thermodynamic preference. The dynamic assembly through non-directional alkyne metathesis is complicated, in which both thermodynamic stability and kinetic factors could play critical roles in determining the final product. Although a general predictable relationship between building blocks and the assembled structures can be deduced by exploiting analogy between supramolecular and covalent assembly, accurate assessment of such a relationship still remains challenging considering the diversity of dynamic reactions and the complexity of dynamic assembly process. We carefully design building blocks based on literature precedents and experience, however, all too often our planning instead brings something surprising, yet logical. Our study will shed some light on this intriguing dynamic covalent assembly, which has shown tremendous potential in chemistry, material science and biology.The design and syntheses of aryleneethynylene COPs through dynamic alkyne metathesis cyclooligomeriztion approach and the effect of the size and geometry of the building units on the final outcome of assembly process were systematically investigated. Alkyne metathesis is a non-directional exchange reaction, which requires regioselective arrangement of ethynylene groups in order to proceed the productive pathway. As a result, the dynamic assembly through alkyne metathesis is significantly influenced by the geometrical properties of building blocks. Our study indicates the size of building blocks plays a significant role in determining the topology of the assembly structure. Depending on the size of top and bottom panels, dimeric cage, tetrameric cage, or interlocked dimeric cage were obtained from the monomers with the same face-to-edge angle. The starting materials for the cage products are designed to be multitopic and self-reacting, which inevitably raise the kinetic competition between intramolecular and intermolecular cyclization. The initial bimolecular reaction to form Supplementary informationClick here for additional data file."} +{"text": "We report a 2D manganese benzoquinoid network that undergoes simultaneous redox switching of magnetic order and electrical conductivity. 4N)2[MnII2(L2\u2013)3] , as synthesized via post-synthetic counterion exchange. This material is paramagnetic above 1.8 K and exhibits an ambient-temperature electrical conductivity of \u03c3295 K = 1.14(3) \u00d7 10\u201313 S cm\u20131 (Ea = 0.74(3) eV). Upon soaking in a solution of sodium naphthalenide and 1,2-dihydroacenaphthylene, this compound undergoes a single-crystal-to-single-crystal (SC\u2013SC) reduction to give Na3(Me4N)2[Mn2L3]. Structural and spectroscopic analyses confirm this reduction to be ligand-based, and as such the anionic framework is formulated as [MnII2(L3\u2013\u02d9)3]5\u2013. Magnetic measurements confirm that this reduced material is a permanent magnet below Tc = 41 K and exhibits a conductivity value of \u03c3295 K = 2.27(1) \u00d7 10\u20138 S cm\u20131 (Ea = 0.489(8) eV), representing a remarkable 200\u2009000-fold increase over the parent material. Finally, soaking the reduced compound in a solution of [Cp2Fe]+ affords Na(Me4N)[MnII2(L2\u2013)3] via a SC\u2013SC process, with magnetic and electrical properties similar to those observed for the original oxidized material. Taken together, these results highlight the ability of metal benzoquinoid frameworks to undergo reversible, simultaneous redox switching of magnetic order and electrical conductivity.Materials with switchable magnetic and electrical properties may enable future spintronic technologies, and thus hold the potential to revolutionize how information is processed and stored. While reversible switching of magnetic order or electrical conductivity has been independently realized in materials, the ability to simultaneously switch both properties in a single material presents a formidable challenge. Here, we report the 2D manganese benzoquinoid framework (Me Materials with switchable magnetic order or values of electrical conductivity underpin the realization of modern electronic and spintronic technologies.4via pre-synthetic design or post-synthetic modification.2Despite this potential, the realization of inorganic materials that possess switchable magnetic order associated with large changes in electrical conductivity faces several key challenges.As an alternative to inorganic compounds, metal\u2013organic frameworks (MOFs) represent a powerful platform to design materials with tailored conductive and magnetic properties, owing to their extraordinary chemical versatility and tunability.nBu4N)[CrIIIMnIIL3] 2 paddlewheel-TCNQ derivative.Tc = 10 to 33 K, whereas the latter undergoes switching between a paramagnet and a permanent magnet with Tc = 88 K. In addition, MOFs with high electrical conductivities have only very recently been realized.Although early examples of MOFs were nearly exclusively electrically insulating due to their composition of diamagnetic, redox-inert carboxylate linkers and metal nodes,0.9Co0.1O2 can be controlled through electrolyte gating, where a maximum of 6-fold change in conductivity is observed.3GeTe2, where a similar up to 6-fold change in conductivity was observed in a 70-layer device.3 in several studies,4N)2[Mn2L3], wherein reversible, post-synthetic chemical reduction transforms a paramagnet into a permanent magnet with Tc = 41 K, with a concomitant 200\u2009000-fold enhancement of electrical conductivity. To our knowledge, this material is unique in its ability to simultaneously undergo redox switching of permanent magnetism and variation in electrical conductivity of several orders of magnitude.The coexistence of switchable magnetic order and electrical conductivity is exceedingly rare in any class of materials. For example, the magnetic ordering temperature of a dilute magnetic oxide Ti4N)2[Mn2L3]\u00b7xDMF (1) as a platform to target switching of magnetism and conductivity, as it features exclusively S = 0 L2\u2013 linkers between S = PBM data was replaced with SVG by xgml2pxml: 0001111111111110000000000000000001 0011000000000000000000000000000011 0011000000000000000000000000000110 0011000000000000000000000000001100 0011000000000000000000000000011000 0011000000000000000000000000110000 0011000000000000000000000001100000 0011111111100000000000000011000000 0000000000111100000000000110000000 0000000000000110000000001100000000 0000000000000011000000011000000000 0011000000000011000000110000000000 0011000000001100000001100000000000 0000110000001100000011000000000000 0000011111111000000110000000000000 0000000000000000001100000000000000 0000000000000000011000000000000000 0000000000000000110000000000000000 0000000000000001100000000011110000 0000000000000011000000001100001100 0000000000000110000000110000000011 0000000000001100000000110000000011 0000000000011000000000000000000011 0000000000110000000000000000000011 0000000001100000000000000000000011 0000000011000000000000000000001100 0000000110000000000000000000110000 0000001100000000000000000011000000 0000011000000000000000001100000000 0000110000000000000000011000000000 0001100000000000000000110000000000 0011000000000000000000110000000000 0110000000000000000000110000000000 1100000000000000000000111111111111\tCreated by potrace 1.16, written by Peter Selinger 2001-2019MnII centres and should not exhibit magnetic order at finite temperatures owing to weak metal\u2013metal interactions solution of (Me4N)BF4 at 75 \u00b0C for 40 h, followed by subsequent rinses with DMF and diethyl ether (Et2O), produced (Me4N)2[Mn2L3]\u00b73.2Et2O (2) suitable for single-crystal X-ray diffraction analysis 2[Mn2L3]\u00b73.9THF (3). Taken together, these data establish the composition of the 2D framework in 3 to be [Mn2L3]5\u2013. Considering the Mn\u2013O distance in 3 (see below) and the rare presence of MnI in molecular compoundsII remains unchanged upon reduction. Instead, each L2\u2013 undergoes a one-electron reduction to give the semiquinoid radical L3\u2013\u02d9, as is supported by the Raman spectroscopy and magnetometry described in detail below.To determine the chemical formula and to tentatively assign the oxidation states of Mn and organic linker in the reduced compound, we employed inductively coupled plasma optical emission spectroscopy (ICP-OES), NMR spectroscopy, and combustion elemental analysis. First, ICP-OES analysis gave a Na\u2009:\u2009Mn ratio of 2.99\u2009:\u20092, consistent with a three-electron reduction per Mn3 in a solution of 3.3 equivalents of [Cp2Fe](BF4) in THF/CH3CN at ambient temperature for 6 days afforded brown hexagonal plate-shaped crystals of the re-oxidized material Na(Me4N)[Mn2L3]]\u22195.5THF\u22190.8CH5.5THF]\u22195.5THF\u22190.8CH0.8CH3CN (4). The Na\u2009:\u2009Mn ratio in 4 was determined to be 1.03\u2009:\u20092 by ICP-OES, indicating removal of only 2/3 of the original Na+ counterions. From a charge-balance standpoint, a Me4N+ counterion is expected to remain in the compound, and its presence in 4 was confirmed by NMR spectroscopy transformation mechanism. Second, according to optical microscopy images, shown as insets of Fig. S11\u2013S13,2\u20134 across the ab planes are approximately 100 \u03bcm, and this unchanged crystal size and morphology further supports a SC\u2013SC mechanism. Third, the reduction from 2 to 3 was performed in THF. Again, a dissolution\u2013recrystallization mechanism is unlikely due to the poor solubility of the Mn2+, Me4N+, and L2\u2013. Finally, according to the 13C NMR spectrum of the reaction supernatant from 3 to 4 \u00c5 relative to that of 2.158(3) \u00c5 for 1 indicates the retention of the high-spin MnII upon counterion exchange \u00c5 that is consistent with PXRD analysis (see below). The Me4N+ counterions occupy the same positions as in 2. The Na+ ions could not be found in the difference electron density map, suggesting they are not ordered on any lattice positions. Bond length analyses further support the reduction of the ligand. As shown in 2\u2013 to L3\u2013\u02d9.24A single crystal of the reduced framework compound 3 to 4, the diffraction quality of 4 dramatically improved . The Me4N+ ion is situated at the same position as in 2 and 3, but with 50% crystallographic occupancy. Similar to 3, the remaining Na+ was not located in the electron density map, suggesting it is disordered within the pore space. While there is no contraction of the Mn\u2013O bond, the C\u2013O and C\u2013C bonds are shortened and lengthened, respectively peak shifts slightly toward higher angle, from 8.510\u00b0 to 8.675\u00b0. This shift indicates a minor shortening of the interlayer distance by 0.20(3) \u00c5, likely due to the smaller size of the Me4N+ in 2 compared to Et4N+ in 1. The reduction from 2 to 3 results in an even more pronounced shift of the (001) peak, corresponding to a shortening from 10.18(2) to 9.35(1) \u00c5. This interlayer contraction likely stems from increased electrostatic interactions between [Mn2L3]5\u2013 layers and five interlayer cations, compared to two cations with [Mn2L3]2\u2013 in 2. As expected, the oxidation from 3 to 4 alters the charge of the framework from [Mn2L3]5\u2013 to [Mn2L3]2\u2013, and therefore causes an increase of the interlayer distance from 9.35(1) to 10.20(2) \u00c5, identical to that of 2.In order to probe the phase purity of crystalline ysis see . The pea3, which is most prominent in the range 2\u03b8 = 19\u201323\u00b0. Furthermore, in stark contrast to 2, in which the high-angle diffraction peaks are well preserved upon counterion exchange, the high-angle peaks in 3 are completely extinguished upon reduction. For 3, the increase in peak full width at half the maximum intensity (FWHM) at higher angle appears to be more pronounced than what is described by the Scherrer equation for crystallite-size related broadening, where FWHM is expected to be inversely proportional to cos\u2009\u03b8. Instead, such a significant increase in FWHM seems proportional to tan\u2009\u03b8 2[ZnII2(L2\u2013)3],2\u2013 in 1, 2, and 4 are unambiguously confirmed. Upon reduction, the C\u2013C bonds and the C\u2013O bond are contracted and elongated, respectively, in 3, as determined above from X-ray diffraction. Consequently, the corresponding vibrations should shift toward higher and lower energy, respectively. Only one prominent band is present in the spectrum of 3, centred at 1426 cm\u20131. This band most likely corresponds to overlapping peaks for both C\u2013C and C\u2013O stretches of L3\u2013\u02d9. Although these vibrations in 3 are slightly shifted relative to those of \u03bdC\u2013C = 1390 and \u03bdC\u2013O = 1488 cm\u20131 observed for (Cp2CoIII)1.43(Me2NH2)1.57[FeIII2(L3\u2013\u02d9)3],\u20131 for the dinuclear complex [CrIII2(tren)2(L3\u2013\u02d9)]3+ (tren = tris(2-aminoethyl)amine).2\u2013 in 1, 2, and 4, and as L3\u2013\u02d9 in 3.To confirm the ligand oxidation state assignments for nd 4 see . Based o2\u20134 at 295 K \u00d7 10\u201313 and 1.45(2) \u00d7 10\u201313 S cm\u20131, respectively. The nearly identical conductivity values for 2 and 4 support the conclusion that the redox switchability of conductivity is close to completely reversible. These low conductivity values fall in the typical range observed for MnII-based MOFs with diamagnetic linkers.26To probe the impact of the ligand oxidation state on the electronic communication and transport in these frameworks, two-contact-probe conductivity measurements were carried out for pressed pellets of 95 K see , upper. 3 exhibited a conductivity of \u03c3295 K = 2.27(1) \u00d7 10\u20138 S cm\u20131, representing a 200\u2009000-fold increase relative to the parent compound 2. To our knowledge, this is the largest change in electrical conductivity stemming from ligand-based redox chemistry in a MOF. This large increase upon chemical reduction indicates that reduction effectively injects electrons into the framework and thereby dramatically improves its charge carrier density. Notably, the ambient temperature conductivity value of 3 is among the highest of structurally-characterized Mn-based MOFs, and is only eclipsed by the value of \u03c3 = 8(1) \u00d7 10\u20135 S cm\u20131 reported for a single crystal of Mn2(TTFTB) . Note that the observed pressed-pellet conductivity values for low-dimensional materials, such as 3, are typically several orders of magnitude lower than single-crystal measurements, due to the anisotropic conducting pathway and grain boundaries.3 is likely much higher than the value for the pellet.The reduced compound 3 is comparable to the 1D radical-bridged chain compound (Cp2Co)Mn , which exhibits an ambient-temperature conductivity of \u03c3 = 8.6 \u00d7 10\u20138 S cm\u20131.3 is 2.2 \u00d7 104 times lower than that of the related compound (Cp2Co)1.43(Me2NH2)1.57[FeIII2(L3\u2013\u02d9)3], which exhibits a pressed-pellet conductivity of \u03c3300 K = 5.1(3) \u00d7 10\u20134 S cm\u20131.3+/Fe2+ and L2\u2013/L3\u2013\u02d9 pairs relative to Mn3+/Mn2+ and L2\u2013/L3\u2013\u02d9 pairs. In particular, the better energetic match of redox potential in the former pair might allow ligand radical electrons to transport through the Fe3+ centres via a transient valence tautomerism mechanism,The conductivity value of 2, and 275\u2013375 K for 3, respectively. As depicted in the lower panel of 2 and 3, suggesting the presence of thermally-activated charge transport in both materials. Fitting the data using the Arrhenius equation (see ESIEa = 0.74(3) eV for 2 and 0.489(8) eV for 3, respectively. The lowering of the activation energy upon reduction is likely due to the Fermi level rising closer to the conduction band as a consequence of doping electrons into the material.30To further probe the origin of the electrical conductivity, variable-temperature two-contact-probe pressed-pellet conductivities were measured in the temperature range of 310\u2013385 K for 4N)2[Mn2L3] is remarkable among magnets. As noted above, the ability to modulate electrical conductivity values over several orders of magnitude in permanent magnets constitutes a significant synthetic challenge, as low values of on/off ratio typically observed for inorganic compounds have hampered their practical use in field-effect transistors. For example, a six-fold change in conductivity was observed at 300 K for the dilute magnetic oxide Ti0.9Co0.1O2 upon applying a gate voltage of 3.8 V through an ionic liquid,3GeTe2 upon applying a 5 V bias through an ionic gate.8The 200\u2009000-fold redox switching of electrical conductivity in scale\" fill=\"currentColor\" stroke=\"none\">MnII ions per formula unit with g = 2. Upon lowering the temperature, \u03c7MT for 2 and 4 remains nearly constant down to ca. 150 K. Below 150 K, \u03c7MT for 2 undergoes a monotonic decrease to reach a minimum value of \u03c7MT = 0.74 cm3 mol\u20131 K at 2 K, indicating relatively weak antiferromagnetic superexchange coupling between S = PBM data was replaced with SVG by xgml2pxml: 0001111111111110000000000000000001 0011000000000000000000000000000011 0011000000000000000000000000000110 0011000000000000000000000000001100 0011000000000000000000000000011000 0011000000000000000000000000110000 0011000000000000000000000001100000 0011111111100000000000000011000000 0000000000111100000000000110000000 0000000000000110000000001100000000 0000000000000011000000011000000000 0011000000000011000000110000000000 0011000000001100000001100000000000 0000110000001100000011000000000000 0000011111111000000110000000000000 0000000000000000001100000000000000 0000000000000000011000000000000000 0000000000000000110000000000000000 0000000000000001100000000011110000 0000000000000011000000001100001100 0000000000000110000000110000000011 0000000000001100000000110000000011 0000000000011000000000000000000011 0000000000110000000000000000000011 0000000001100000000000000000000011 0000000011000000000000000000001100 0000000110000000000000000000110000 0000001100000000000000000011000000 0000011000000000000000001100000000 0000110000000000000000011000000000 0001100000000000000000110000000000 0011000000000000000000110000000000 0110000000000000000000110000000000 1100000000000000000000111111111111\tCreated by potrace 1.16, written by Peter Selinger 2001-2019 MnII ions. As such, 2 is a paramagnet at temperatures down to at least 2 K. Conversely, in the case of 4, \u03c7MT undergoes a slight increase below 90 K to a maximum value of \u03c7MT = 9.59 cm3 mol\u20131 K at 40 K, followed by a monotonic decrease to reach a minimum value of \u03c7MT = 1.09 cm3 mol\u20131 K at 2 K. This slight upturn in 4 upon lowering the temperature is most likely due to the contribution from a minuscule amount of reduced species remaining in the sample (see below).To probe the magnetic behaviour of the manganese-quinoid compounds, variable-temperature dc magnetic susceptibility data were collected for 3, the value of \u03c7MT = 9.60 cm3 mol\u20131 K at 300 K is relatively close to that of 9.875 cm3 mol\u20131 K expected for magnetically isolated two S = PBM data was replaced with SVG by xgml2pxml: 0001111111111110000000000000000001 0011000000000000000000000000000011 0011000000000000000000000000000110 0011000000000000000000000000001100 0011000000000000000000000000011000 0011000000000000000000000000110000 0011000000000000000000000001100000 0011111111100000000000000011000000 0000000000111100000000000110000000 0000000000000110000000001100000000 0000000000000011000000011000000000 0011000000000011000000110000000000 0011000000001100000001100000000000 0000110000001100000011000000000000 0000011111111000000110000000000000 0000000000000000001100000000000000 0000000000000000011000000000000000 0000000000000000110000000000000000 0000000000000001100000000011110000 0000000000000011000000001100001100 0000000000000110000000110000000011 0000000000001100000000110000000011 0000000000011000000000000000000011 0000000000110000000000000000000011 0000000001100000000000000000000011 0000000011000000000000000000001100 0000000110000000000000000000110000 0000001100000000000000000011000000 0000011000000000000000001100000000 0000110000000000000000011000000000 0001100000000000000000110000000000 0011000000000000000000110000000000 0110000000000000000000110000000000 1100000000000000000000111111111111\tCreated by potrace 1.16, written by Peter Selinger 2001-2019 MnII ions and three S = \u00bd L3\u2013\u02d9 radicals per formula unit scale\" fill=\"currentColor\" stroke=\"none\">MnII centres that are antiferromagnetically coupled to three S = \u00bd L3\u2013\u02d9 radical ligands, giving a net repeating spin of and g = 2.00.To confirm the quantitative ligand-based reduction and to further investigate the nature of magnetic coupling in II and L3\u2013\u02d9 in 3 are clearly illustrated in the variable-temperature magnetization plot, obtained from data collected under an applied dc field of H = 10 Oe. As shown in 3 gradually increases, following a Curie\u2013Weiss relationship to ca. 50 K and out-of-phase (\u03c7\u2032M) components of the ac susceptibility are observed below 42 K, establishing an ordering temperature of Tc = 41 K.\u03c6 = 0.0085, indicating the possible presence of some glassy behaviour or other complicated magnetization dynamics.31To precisely determine the ordering temperature of 3 was probed by collecting variable-field magnetization data at selected temperatures. As shown in 3 at 1.8, 10, 20 and 25 K were determined to be HCi = 300, 100, 40, and 12 Oe, respectively, with field-sweep rates of 0.6, 0.6, 0.2, and 0.1 Oe s\u20131. These HCi values of 3 are significantly lower than the iron analogue (Cp2CoIII)1.43(Me2NH2)1.57[FeIII2(L3\u2013\u02d9)3], which features HCi = 4520 and 2270 Oe at 1.8 and 10 K, respectively. This difference likely stems from the negligible magnetic anisotropy associated with high-spin MnII. Indeed, the behaviour of 3 as a soft magnet may render the compound useful in spintronics applications, where the direction of its spin polarization can be easily manipulated with a weak external magnetic field.1Finally, the presence of magnetic hysteresis of 3 and Fe3GeTe2, capacitive post-synthetic doping of charge carriers can tune ordering temperature and coercivity.Tc. Similarly, post-synthetic modulation of Tc has been demonstrated in the metal\u2013organic permanent magnets (nBu4N)[CrIIIMnIIL3],2NH2)2[Fe2L3],nBu4N)2[Fe2(dhbq)3].2 is a paramagnet above 1.8 K, whereas the reduced compound 3 is a permanent magnet below Tc = 41 K, and this redox process is chemically reversible. To our knowledge, the only other MOF that can undergo reversible switching between paramagnetism and magnetic order is a Ru2 paddlewheel-TCNQ derivative.The ability to switch between a paramagnet down to 1.8 K and a permanent magnet is nearly unprecedented. In oxide-based dilute magnetic semiconductors or other 2D solid-state inorganic materials such as CrIgnets nBuN[CrIIIMn4N)2[MnII2(L2\u2013)3], accessed through SC\u2013SC cation exchange, behaved as a paramagnet above 1.8 K with an ambient-temperature electrical conductivity of \u03c3295 K = 1.14(3) \u00d7 10\u201313 S cm\u20131 (Ea = 0.74(3) eV). Subsequent post-synthetic chemical reduction afforded the reduced semiquinoid compound Na3(Me4N)2[MnII2(L3\u2013\u02d9)3] via a SC\u2013SC process. This latter species was found to be a permanent magnet with a characteristic temperature of Tc = 41 K, and its ambient-temperature conductivity of \u03c3295 K = 2.27(1) \u00d710\u20138 S cm\u20131 (Ea = 0.489(8) eV) represents a 200\u2009000-fold increase relative to the oxidized form. Finally, the redox process was shown to be chemically reversible, with oxidation of the semiquinoid compound affording Na(Me4N)[MnII2(L2\u2013)3]. Future efforts will target related compounds that undergo simultaneous switching of magnetism and conductivity, but with higher magnetic ordering temperatures and higher values of electrical conductivity.The foregoing results demonstrate the ability of metal\u2013organic frameworks to undergo reversible redox-switching of magnetic order and electrical conductivity. The 2D manganese-benzoquinoid framework compound (MeThere are no conflicts to declare.Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "Organic isocyanates are readily converted to methyl amine products through their hydroboration with HBpin in the presence of a \u03b2-diketiminato magnesium catalyst. N-,O-bis(boryl)hemiaminal species have been identified as intermediates during the reductive catalysis, the overall reduction and C\u2013O activation is metal-mediated and proposed to occur through the further intermediacy of well-defined magnesium formamidato, formamidatoborate and magnesium boryloxide derivatives. Examples of all these species have been identified and fully characterised through stoichiometric reactivity studies and the stability of the borate species leads us to suggest that, under catalytic conditions, the onward progress of the deoxygenation reaction is crucially dependent on the further activation provided by the Lewis acidic HBpin substrate. These deductions have been explored and ratified through a DFT study.Organic isocyanates are readily converted to methyl amine products through their hydroboration with HBpin in the presence of a \u03b2-diketiminato magnesium catalyst. Although borylated amide and We have shown that use of compound 1 enables the heterofunctionalisation and hydroboration of an assortment of multiply bonded and heteroatomic C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019E scale\" fill=\"currentColor\" stroke=\"none\">E has attracted increasing attention given its potential applications in biofuels and fine chemical syntheses.1 was able to effect ester cleavage during the hydroboration of 3-methylnicotinate with pinacolborane (HBpin). PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O bond and the formation of the methanol-equivalent H3COBpin through the use of a modified catalyst system derived from 1 and a B(C6F5)3 co-catalyst. PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O activation. Although a Schwartz reagent-mediated reduction of isocyanates has been described as a straightforward and direct route to otherwise inaccessible formamides and the in situ hydroboration of t-BuN PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O with HBpin catalysed by [Mg(THF)6][HBPh3] has been very recently reported to provide the bis-borylated hemiaminal, t-BuN(Bpin)CH2OBpin,Hydrodeoxygenation of carbonyl-containing compounds oxide, O(Bpin)2, through comparison with literature data PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O bond within the substrate with consequent formation of the N-borylated N-methyl isopropylamine 2 by-product in the 11B NMR spectra. Contrary to expectation, increased RNCO substituent steric demands provided generally faster reaction times; R = i-Pr (entry 1) and t-Bu (entry 5) evidenced complete consumption of the isocyanate in less than 4 hours compared to Et (entry 3) and n-Pr (entry 4) both of which required 21 hours. The increased reactivity associated with enhanced alkyl substituent steric demands also coincided with the onset of secondary side reactions. Although attempts to identify the products of these side reactions were unsuccessful it is likely that oligomerisation of the isocyanates is competitive with the reductive C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O cleavage.N-phenyl and N-mesityl (Mes) isocyanate substitution provided ca. 90% conversion in 24 hours (entries 7 and 8), while an increase of the N-aryl substituent's steric demands to 2,6-di-isopropylphenyl (Dipp) resulted in a significantly reduced conversion during the same time period (entry 9).Extension of this reactivity to other commercially available alkyl isocyanates provided similar reactivity to i-PrNCO. Entries 1 to 6 in 1 at room temperature leads to stoichiometric formation of n-BuBpin and a may act as a source of a heteroleptic magnesium hydride species.2, the formation of which was clearly apparent from the appearance of a new downfield (1H by relative integration) singlet resonance at \u03b4 7.92 ppm in the resultant 1H NMR spectrum. Crystallisation of compound 2 direct from the reaction solution provided crystals suitable for a single crystal X-ray diffraction analysis. The results of this analysis was readily located and freely refined, clearly demonstrating that there was no significant Mg\u2013H interaction in the solid state. The structure of compound 3 is reminiscent of a previously described borate species resulting from addition of HBpin to a magnesium formamidinate supported by the same \u03b2-diketiminate ligand.3Addition of one equivalent of HBpin to a solution of compound N-borylated formamidine, DippN(Bpin)HC(O) , by prolonged (>24 hours) heating of samples of compound 3 in d8-toluene either at 60 \u00b0C or 80 \u00b0C failed to provide any evidence of onward reactivity. Similar treatment of 3 at the higher temperature of 100 \u00b0C for 16 hours, however, resulted in the production of a single new \u03b2-diketiminato species (4) along with the methylene imine, DippN PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CH2, which was tentatively identified through the appearance of an AB spin system in the 1H NMR spectrum with signals at \u03b4 6.90 and 7.25 ppm. The identity of compound 4 was confirmed through the isolation of single crystals suitable for an X-ray diffraction experiment. The results of this analysis revealed that 4 was a dimeric magnesium boryloxide species scale\" fill=\"currentColor\" stroke=\"none\">CH2 as the mode of C\u2013O activation, the elevated temperature required for this reaction militates against its operation during the catalytic reactions summarised in 3 and a single equivalent of HBpin was, thus, undertaken. Although no consumption of 3 took place at room temperature, heating of this d8-toluene solution at 60 \u00b0C for 8 hours resulted in the predominant formation of DippN(Me)Bpin along with a variety of \u03b2-diketiminato magnesium species which could not be identified with any meaningful level of confidence. In an attempt to shed further light on this reactivity, compound 1 was reacted with 3 molar equivalents of HBpin and 2 equivalents of DippNCO. This reaction at room temperature surprisingly resulted in the generation of the amidate derivative (2) along with the concomitant production of a single new compound (5). This latter species was identified as the bis-borylated hemiaminal, Dipp(pinB)NCH2OBpin, through the appearance of a distinctive methylene singlet resonance at \u03b4 5.35 ppm in the 1H NMR spectrum and two broad signals of equal intensity at \u03b4 28.3 and 24.8 ppm in the corresponding 11B NMR experiment. Furthermore, continued treatment of this reaction mixture with additional equivalents of HBpin and DippNCO in a 2\u2009:\u20091 ratio provided for the catalytic production of compound 5.While the formation of compound 1 to provide compound 5 and the analogous N-mesityl species (6) within 10 minutes at room temperature oxide, O(Bpin)2, by-product.Attempted extension of this bis-borylation protocol to PhNCO and the range of 6 and Jones' \u03b2-diketiminato magnesium hydride complex, [CH{C(Me)NDipp}2MgH]2.1H NMR spectroscopy demonstrated that heating to 60 \u00b0C resulted in the generation of the N-borylated methyl amine, pinBN(Me)Mes, along with the magnesium boryloxide (4) (eqn (2)). A further reaction performed by addition of compound 6 to a d8-toluene solution of the pre-catalyst (1) and HBpin provided a similar result. Subsequent addition of HBpin to either of these reaction mixtures or to a solution of an isolated sample of compound 4 in d8-THF resulted in the consumption of the magnesium boryloxide with concomitant production of (pinB)2O and the regeneration of [CH{C(Me)NDipp}2MgH]2 (eqn (3)). Although these routes to compound 4 and its reactivity with HBpin suggest that the C\u2013O cleavage reaction is primarily magnesium mediated, it is notable that heating of a solution of compound 6 at 60 \u00b0C for 12 hours in the absence of any other reagents also resulted in the consumption of ca. 50% of the hemiaminal and production of (pinB)2O and the methylene imine, MesN PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CH2.To provide further insight into the role of such hemiaminal species as potential intermediates during the course of the C\u2013O activation catalysis, a reaction was carried out between compound 5. Although a 1H NMR spectrum recorded five minutes after the initiation of the catalytic reduction of DippNCO with two equivalents of HBpin catalysed by 1 demonstrated the facile production of compound 5, the formation of this species was accompanied by a new compound (7) characterised by the appearance of a high frequency singlet resonance at \u03b4 9.04 ppm in the 1H NMR spectrum. Compound 7 was assigned as the N-borylated formamidine, DippN(Bpin)HC(O), which was completely consumed upon completion of the reaction. Several attempts to generate compound 7 by the reaction of a single equivalent of HBpin and DippNCO in the presence of 10 mol% 1 resulted in the consumption of only 50% of the isocyanate starting material and the production of compound 5. A similar reaction performed at lower catalyst loading (0.1 mol%) and in the minimum amount of toluene solvent, however, resulted in the formation of a significant quantity of crystalline material. Mechanical separation of single crystals formed within this material enabled a series of single crystal X-ray diffraction analyses which showed it to be primarily a mixture of compound 5 and compound 7. The results of the analysis of compound 7 are shown in An additional intermediate was identified during our investigation of the catalytic production of compound in situ monitoring (1H NMR spectroscopy) of a reaction of HBpin and i-PrNCO catalysed by 10 mol% 1 at 60 \u00b0C. en route to the ultimate methyl amine product HC(O), analogous to compound 7, was observed to accumulate during the initial 15 minutes of the reaction prior to any observable methyl amine formation . After this time, however, it was consumed simultaneously with the appearance of significant quantities of a bis-borylated hemiaminal derivative, i-Pr(pinB)NCH2OBpin, analogous to compounds 5 and 6, which was apparent as a persistent singlet resonance at \u03b4 5.08 ppm . A further low field singlet was observed at \u03b4 7.80 ppm . Once evolved, this latter resonance persisted at an effectively constant intensity throughout the course of the catalysis. This chemical shift is reminiscent of the signal associated with the amidato methine proton observed for compound 3 (\u03b4 7.69 ppm) and leads us to postulate that a borate species with a comparable constitution is also formed in a steady state concentration as a key intermediate during the reduction of i-PrNCO. The initial appearance of this species was accompanied by the emergence of a clear AB spin system , which may be cautiously assigned to the methylene imine, i-PrN PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CH2. This small molecule is evidently consumed, however, at more mature phases of the catalysis.The significance of these observations with respect to the catalytic reactivity was assessed by ), i-Pr(pinB)NCH2OBpin and i-PrN(Bpin)Me during the reduction of i-PrNCO. N-borylated amidine production in our recent report of carbodiimide hydroboration catalysed by 1.3 in the absence of further HBpin.Evidence for a sequential mechanism was provided by examination of the temporal evolution of the signals assigned to i-PrN(Bpin)HC(O) to provide the transient magnesium hydride (or borohydride) necessary to generate formamidate and borate intermediates analogous to compounds 2 and 3. The apparent stability of this latter compound mitigated against its direct thermal conversion to boron-containing small molecules at temperatures relevant to the catalysis. We, thus, suggest that the most kinetically accessible cycle necessitates breakdown of a similar borate species to produce RN(Bpin)HC(O).Although these observations attest to a complex mechanistic landscape, we postulate that the gross features of this reactivity may be rationalised by the generic mechanism for the reduction of RNCO illustrated in N-borylated formamidine were observed to accumulate during the early stages of the catalytic reduction of i-PrNCO, this species is apparently consumed through its onward reaction with magnesium hydride. We have not identified any resultant magnesium hemiaminalate species but suggest that compounds of this type will be rapidly consumed through further B\u2013H/Mg\u2013O metathesis to yield R(pinB)NCH2OBpin. The ultimate production of RN(Bpin)Me and closure of the catalytic cycle are then predicated upon a sequence of C\u2013O/Mg\u2013H and Mg\u2013O/B\u2013H metathesis steps reminiscent of the stoichiometric reactivity summarised for the isolable species 6 and 4 in eqn (2) and (3).We postulate that this process occurs by the necessary activation of the borate intermediate through its interaction with a further equivalent of Lewis acidic HBpin prior to intramolecular boron to magnesium hydride transfer. This process is, thus, reminiscent of our recent reports on related magnesium borate species identified during the hydroboration of organic carbodiimides and nitriles. PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CH2. It is notable that appreciable quantities of i-PrN PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CH2 were only observed after the generation of significant amounts of i-Pr(pinB)NCH2OBpin during the reduction of i-PrNCO. We infer, therefore, that this small molecule is generated by the direct decomposition of i-Pr(pinB)NCH2OBpin rather than any higher energy magnesium-centered process and implicates a second plausible pathway for the production of i-PrN(Bpin)CH3.Although our observations suggest that this reaction sequence predominates under the mild conditions applied during catalysis, the mechanism must also reconcile the production and consumption of RN2 and 3 were found to be formed by an overall exothermic sequence of isocyanate Mg\u2013H insertion and subsequent reaction of the as-formed magnesium amidate with HBpin. As deduced from the stability of compound 3, the production of a borylated formamide analogous to compound 7 entails the interaction of the magnesium amidatoborate with a second equivalent of HBpin to enable the necessary boron to magnesium hydride transfer. While the production of compounds such as 7 requires the traversal of a several higher energy transition states and intermediates, mainly associated with the HBpin insertion into the Mg\u2013O bond of 3 at a cost of 5.0 kcal mol\u20131 and the need for B\u2013O bond disruption , these processes occur prior to a kinetically facile and highly exothermic re-insertion of the carbonyl function into the magnesium hydride bond. The subsequent generation of bis(boryl)hemiaminals such as 5 and 6 occurs via B\u2013H/Mg\u2013O sigma bond metathesis, which incurs an enthalpic penalty of some 10.2 kcal mol\u20131 associated to the formation of a monomeric Mg\u2013H complex rather than its very stable dimeric form. However, while this is lower than the entrance channel , 5 can only be considered as an intermediate in the course of the formation of methyl amine product in line with the proposed catalytic cycle. The ultimate cleavage of the C\u2013O bond and production of the methyl amine product requires traversal of an energy barrier of some 40.0 kcal mol\u20131 involving the further interaction of these reduced intermediates with a magnesium hydride, whereupon the formation of a boryloxide derivative similar to compound 4 is significantly exothermic . Dimerisation and a further Mg\u2013O/H\u2013B metathesis with a further molecule of HBpin yield (pinB)2O with the regeneration of the magnesium hydride scale\" fill=\"currentColor\" stroke=\"none\">O bond. The sequential nature of this reactivity indicates that it may be further generalised to the targeted cleavage of the C\u2013O bonds in alternative molecular and, potentially, even macromolecular species. We are continuing to study these possibilities.In conclusion, we describe a mild protocol for the catalytic transformation of the isocyanate function to a methyl amine. The activation of the heterocumulene occurs through a magnesium-centered hydroboration and ultimately the complete cleavage of the CSupplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "The unique abilities of homo-oligo-adamantyl peptides to adopt \u03b1- and \u03b3-turn conformations are demonstrated by X-ray diffraction, and NMR and FT-IR absorption spectroscopies. N\u03b1-formyl-adamantyl tripeptide iso-propyl and tert-butyl amides are respectively found to adopt an isolated \u03b1-turn and an incipient \u03b3-helix conformation by X-ray diffraction crystallography. The shortest example of a single \u03b1-turn with ideal geometry is observed in the crystalline state. In solution both peptides predominantly assume \u03b3-helical structures.The unique abilities of homo-oligo-adamantyl peptides to adopt \u03b1- and \u03b3-turn conformations are demonstrated by X-ray diffraction, and NMR and FT-IR absorption spectroscopies. Assembled by an Ugi multiple component reaction strategy, PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O\u00b7\u00b7\u00b7H\u2013N hydrogen bonds and minimization of side-chain steric interactions. PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O and NH groups, respectively, of residues i and i + 4 (\u03b1-turn13). On the other hand, a single \u03b1-turn in short linear peptides (\u22645 amino acid residues), unambiguously authenticated by X-ray diffraction analysis, is rare,i + 2 \u2192 i or C7) \u03b3-turn structures5Helices constitute the most abundant peptide and protein secondary structures in nature and play vital roles in protein\u2013protein recognition.\u03b1,\u03b1-Disubstituted glycines are known to be able to constrain peptides to adopt distinct secondary structures.\u03c6 and \u03c8 torsion angles adopted by these residues are typically contingent on the substituent size and nature. Specifically, C\u03b1,\u03b1-dimethylglycine and C\u03b1-methylated analogs of proteinogenic amino acids favor folded backbone conformations in the 310-/\u03b1-helical region ,10-helices,16 homo-oligomer.n-propyl, phenyl or benzyl substituents adopt predominantly fully-extended (C5) conformations .Ci.e., N\u03b1-unprotected N-carboxyanhydride, N\u03b1-acetyl/trifluoroacetyl 5(4H)oxazolones and EDC (N-ethyl-N\u2032-3-(dimethylaminopropyl)carbodiimide)/HOAt ] to provide simple dipeptides with N-terminal Adm residues.3) precursor of the \u03b1-amino group,12Although homo-oligomers of Aib have been commonly prepared and studied,\u03b1,\u03b1-disubstituted glycines with very bulky side chains, the Ugi reactions of adamantan-2-one has previously delivered -Adm-Gly- dipeptides, albeit in modest yields.3-NHR tripeptides 1 and 2 . A concomitant combination of X-ray diffraction crystallography and spectroscopic methods has demonstrated that the Adm residue can favor the elusive single 13-membered intramolecularly hydrogen-bonded \u03b1-turn, as well as the almost completely developed \u03b3-helix conformations.Among the potentially useful, alternative methods for synthesizing CN\u03b1-formyl-Adm homo-tripeptides 1 and 2 began respectively by reacting adamantan-2-one with iso-propyl and tert-butyl isocyanides 3 in methanol (MeOH) with ammonium formate dissolved in the minimum amount of water between \u20135 to \u201310 \u00b0C for 1\u20132 hours. After aqueous workup and column chromatography, isocyanides 5 were isolated as solids in \u226590% yields. Exposure of isocyanides 5 to similar Ugi conditions as those described above gave homo-dipeptides 6 as solids in \u226585% yields. Subsequently, formamide to isocyanide conversion went smoothly using the POCl3 conditions to afford isocyanides 7. Finally, the desired homo-tripeptides 1 and 2 were synthesized using the Ugi approach. However, the reaction was relatively sluggish, and required heating for 2 days in the presence of excess ammonium formate to provide solid products after chromatography in about 60% yields.The synthesis of 1 and 2 were determined by single crystal X-ray diffraction (1 (R = iPr) and ESI\u20201 was also crystallized from solvents of varying polarity within the limits imposed by solubility. Crystals suitable for X-ray diffraction analysis were obtained from three different systems; however, all gave the identical \u03b1-turn structure, within experimental error \u00c5 from the nitrogen atom of Adm(3), suitable for forming a 10-membered intramolecular hydrogen bond (\u03b2-turn),al error .1, the average \u03c6 and \u03c8 torsion angles over the three Adm residues differ only by 6\u00b0 and 10\u00b0, respectively, from the canonical values based on statistical analysis of \u03b1-helices in crystalline peptides.1 represents the shortest peptide sequence giving rise to an isolated \u03b1-turn of regular geometry.As stated above, crystallography of single \u03b1-turns is very unusual. \u201357\u00b0, \u20135\u00b0 differ 2, the backbone torsion angles .\u03c61 value, compared to that of \u03c62, in contrast to the less than 2\u00b0 difference in the values of \u03c81 and \u03c82. The distortion of \u03c61 weakens the N2\u2013H\u00b7\u00b7\u00b7O0 intramolecular hydrogen bond, but enables the N1\u2013H group to participate in an intermolecular hydrogen bond with the O2 atom of a symmetry related molecule (ESI\u03c8 torsion angle of Adm(3) is forced to adopt a value (\u2013113.2\u00b0) which prevents the C-terminal amide NH group from approaching the carbonyl oxygen of Adm(2) at a suitable hydrogen-bond distance.In the X-ray diffraction structure of tripeptide n angles and distn angles and ESI\u2020 \u03c6, \u03c8 = 7\u00b0,\u201375\u00b0.5 1 (1.254 g cm\u20133) was less than that of 2 (1.302 g cm\u20133). The packing mode of tripeptide 1 features one N\u2013H\u00b7\u00b7\u00b7O PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C hydrogen bond, three C\u2013H\u00b7\u00b7\u00b7O contacts within the distance of 2.66 \u00c5,2, intermolecular interactions include one N\u2013H\u00b7\u00b7\u00b7O PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C hydrogen bond, as well as two C\u2013H\u00b7\u00b7\u00b7O contacts and nine aliphatic H\u00b7\u00b7\u00b7H contacts in the range 2.10\u20132.39 \u00c5. Within the sum of the van der Waals radii, the latter may stabilize the packing and account for the higher density of crystals of 2 through London dispersion.21The calculated density for the crystals of 1 and 2 in solution, NMR and FT-IR absorption spectroscopic methods were employed.To study the conformation of peptides 1 and 2 in CDCl3 were achieved by a combination of COSY and HMBC experiments, the latter optimized to identify long-range, through-bond J couplings caused significant downfield shifts for only the formamide NH proton signal, suggesting that this group is exposed to solvent in contrast to the other NH proton signals which exhibited limited variations in chemical shifts (0.02 to 0.22 ppm). This different behavior of the formamide NH proton is already evident at the very beginning (2% DMSO in CDCl3) of the solvent titration curves the formamide NH signal relative to those of the other amide protons. The NH chemical shifts of the latter protons varied less with temperature in the solvent of lower polarity CDCl3 (\u0394\u03b4/\u0394K = \u20130.3 to \u20131.5 ppb) than in DMSO (\u0394\u03b4/\u0394K = \u20131.0 to \u20133.3 ppb K\u20131).Solvent shielded and exposed NH protons were examined by studying influences of changes in environment on the chemical shifts of their signals . Amide pn curves . Similar2 grown from DMSO (bis-DMSO solvate) highlighted the presence of only one \u03b3-turn in each of the two independent but similar peptide conformers A and B in the crystal matrix (\u03c6 and \u03c8 torsion angles of Adm(1) in the two peptide conformers were \u201375.4\u00b0, 74.8\u00b0, and 76.6\u00b0, \u201369.9\u00b0, respectively. Three intermolecular hydrogen bonds were observed between each peptide and two co-crystallized DMSO solvent molecules: one between the Adm(1) NH group and the oxygen atom of one molecule of DMSO, and the other two between the Adm(3) and tert-butyl amide NH groups together with the oxygen atom of a second DMSO molecule. In each peptide conformer, Adm(2) and Adm(3) were helical but of opposite screw sense: \u03c62, \u03c82, \u03c63, \u03c83 = 59.8\u00b0. 64.1\u00b0, \u201359.0\u00b0, \u201355.5\u00b0 (in A); \u201360.0\u00b0, \u201366.9\u00b0, 61.1\u00b0, 58.5\u00b0 (in B).Notably, an X-ray diffraction analysis of crystals of peptide l matrix and ESI\u2020\u20131), the FT-IR absorption spectra of peptides 1 and 2 in CDCl3 solution accompanied by a much broader band with maximum near 3300 cm\u20131 (hydrogen-bonded NH groups).versus free N\u2013H stretching bands were found over the concentration range 10.0 mM\u20130.1 mM is likely responsible for the outcome in the crystal states (\u03b1-turn or \u03b3-turn). An example has already been reported of two homo-peptides differing only in their C-terminal group (ethyl ester vs. tert-butyl ester) giving rise to two different conformations in the crystal state, but adopting apparently the same conformation in solution.Considering the marginal role of the crystallization solvent properties to significantly bias the conformations of peptides \u03b1,\u03b1-disubstituted glycines bearing bulky side chains using the Ugi multiple component reaction has been achieved and applied in the synthesis of N\u03b1-formyl adamantyl tripeptide amides 1 and 2. Conformational analysis has revealed the potential for the Adm residue to favor the unprecedented single \u03b1-turn with ideal \u03c6 and \u03c8 torsion angles and an incipient \u03b3-helical structure. Notably, the overwhelming majority of C\u03b1-tetrasubstituted \u03b1-amino acids prefer 310-helices, which are generated by consecutive type-III (III') \u03b2-turns, and subsequently \u03b1-helix conformers.10-helix appears to be unavailable to the Adm residue. In the \u03c6 and \u03c8 space, the most significant difference between the two conformations is the value of the \u03c8 torsion angle. For the Adm residue, typical \u03c8 values for right- and left-handed 310-helices have never been observed, likely due to a destabilizing aliphatic C\u2013H\u00b7\u00b7\u00b7O interaction between the pro-R or pro-S \u03b3-CH2 groups with the carbonyl oxygen atom, which would require an energetically disfavored H\u00b7\u00b7\u00b7O separation of the order of 2.10 \u00c5.\u03c8 values to those observed in the \u03b1-helical turn of peptide 1 (in the range \u201346.5\u00b0 to \u201359.6\u00b0) which resulted in H\u00b7\u00b7\u00b7O separations between 2.23 and 2.33 \u00c5, and to a larger extent in a \u03b3-turn conformation (peptide 2), in which the observed H\u00b7\u00b7\u00b7O separations are >2.40 \u00c5.To summarize, a novel method for assembling sterically congested peptides from C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O)\u2013 derivatives] have already found important applications in medicinal chemistry as drugs to treat parkinsonism and related syndromes, and as antiviral agents to combat type-A influenza virus in humans.26Considering the power of the Ugi multiple component method reported in this work for synthesizing sterically hindered peptides, potential now exists to harness bulky residues for various studies in peptide science. The unique \u03b3-helical folding pattern which was observed both in solution and in the crystal state, as well as the fascinating diamondoid hydrocarbon side chains may similarly lend themselves for novel applications. In this connection, adamantane-functionalized compounds [particularly its \u2013NH- and \u2013C(There are no conflicts to declare.Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "New tris-amidinate actinide complexes containing a rare O-bound terminal phosphaethynolate (OCP\u2013) ligand were synthesized and fully characterized. tris-amidinate actinide complexes containing a rare O-bound terminal phosphaethynolate (OCP\u2013) ligand were synthesized and fully characterized. The cyanate (OCN\u2013) and thiocyanate (SCN\u2013) analogs were prepared for comparison and feature a preferential N-coordination to the actinide metals. The Th(amid)3(OCP) complex reacts with Ni(COD)2 to yield the heterobimetallic adduct (amid)3Th(\u03bc-\u03b71(O):\u03b72-OCP)Ni(COD) featuring an unprecedented reduced (OCP\u2013) bent fragment bridging the two metals.New Origtris-amidinate chloride precursors MCl(amid)3 benzamidinate) and Na(OCP)(dioxane)2.9 affords the desired phosphaethynolate complexes M(OCP)(amid)3 as block-shaped crystals in 76% and 63% isolated yields, respectively is significantly shifted downfield (\u03b4 = \u2013334 ppm) compared to that reported for Re scale\" fill=\"currentColor\" stroke=\"none\">C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O)(CO)2(triphos)\u03b4 = \u2013398 ppm) and alkali and alkaline earth phosphaethynolate salts (\u03b4(31P) range: \u2013362 to \u2013397 ppm).iv) center, the 31P NMR signal for 3 is observed at even higher frequency (\u03b4 = \u2013285 ppm). Compounds 3 and 4 feature strong IR absorption bands at almost identical wavenumbers corresponding to the C\u2013O stretching vibrational mode of the OCP\u2013 ligand. This feature appears at lower energy than that found in Re scale\" fill=\"currentColor\" stroke=\"none\">C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O)(CO)2(triphos) (1860 cm\u20131) and alkali OCP\u2013 salts (1730 to 1780 cm\u20131), which indicates weakening of the C\u2013O bond order. As evidenced both by the low wavenumber IR absorption of \u03bdC\u2013O and the downfield 31P NMR chemical shift, the OCP\u2013 moiety in 3 and 4 is best described as a phosphaalkyne-type limiting resonance structure scale\" fill=\"currentColor\" stroke=\"none\">C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O)(CO)2(triphos) PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019P bond upon coordination to the oxophilic actinide centers.Salt-metathesis reactions between the ectively . Both coture see in contr\u2013 fragment is confirmed by X-ray crystallography scale\" fill=\"currentColor\" stroke=\"none\">P motif pointing to two different directions in a 45\u2009:\u200955 ratio. The discussion of metrical parameters for 4 is, therefore, performed on the non-disordered molecule only. The thorium and uranium analogs adopt C3-symmetry, with the three bidentate amidinate ligands wrapping around the actinide in a propeller-like geometry, and the OCP\u2013 ligand pointing in the axial position. The O\u2013C\u2013P (179.1(4)\u00b0 in 3; 179.7(4)\u00b0 in 4) and An\u2013O\u2013C (170.9(3)\u00b0 in 3; 176.4(3)\u00b0 in 4) angles are close to linearity. The C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019P (1.576(5) \u00c5 in 3; 1.561(4) \u00c5 in 4) and C\u2013O (1.219(6) \u00c5 in 3; 1.246(4) \u00c5 in 4) bond lengths are in the expected range scale\" fill=\"currentColor\" stroke=\"none\">P = 1.579(3) \u00c5; C\u2013O 1.212(4) \u00c5 in [K([18]crown-6)][PCO]), PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019P triple bond and a weakening of the C\u2013O bond when compared with the metrical parameters computed for the OCP\u2013 anion scale\" fill=\"currentColor\" stroke=\"none\">P = 1.625 \u00c5; C\u2013O 1.203 \u00c5).iv).amid and Th\u2013Namid bond distances average respectively 2.44(3) \u00c5 and 2.49(3) \u00c5 and compare well with related compounds.The O-coordination of the OCPlography -top. Com5\u20138 symmetric species in solution. The IR C\u2013N stretches for compounds 5\u20138 are of high intensities and fall in the range of previously reported N-bound cyanate5\u20138, (uranium derivatives shown in OCN (2.340(3) \u00c5 in 5, 2.410(2) \u00c5 in 6) and An\u2013NSCN (2.385(4) \u00c5 in 7, 2.428 (4) \u00c5 in 8) bond distances compare well with those reported for structurally characterized An(iv) cyanate and thiocyanate complexes.iv) and Th(iv). The most striking difference is the preferred O-coordination for OCP\u2013vs. N-coordination in the case of OCN\u2013 and SCN\u2013.The cyanate and thiocyanate counterparts 5\u20138 were pre\u2013 binds actinides through the N-terminus, at first glance, one could have expected OCP\u2013 to behave similarly and bind through the pnictide donor, as is the case in Re(XCO)(CO)2(triphos) .\u2013 anion (q(O) = \u20130.65; q(P) = \u20130.44) and in the OCN\u2013 anion (q(O) = \u20130.75; q(N) = \u20130.81);Two major limiting resonance structures have to 4 is 7.7 kcal mol\u20131 lower in energy than in the hypothetical P-bound analogue (\u20131 lower for the O-bound complex 4 (1666 cm\u20131) than for the P-bound (1901 cm\u20131). In the case of the NCO ligand, the computed CO stretching frequency of Th\u2013NCO (2230 cm\u20131) is 15 cm\u20131 lower than in Th\u2013OCN (2245 cm\u20131). In the same way, for SCN, the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N stretch is computed to be lower by 132 cm\u20131 for Th\u2013NCS (2016 cm\u20131) over Th\u2013SCN (2148 cm\u20131). These two sets of calculations fit with the experiment.DFT calculations are in line with the experimental observations and show that the N-bound mode is preferred with cyanate and thiocyanate anions, while the O-bound one is favored for the phosphaethynolate anion. This observation contrasts with the few previous studies which reported that the P-bound products are thermodynamically preferred;analogue . IR calc4 indicates that the O-bound complex is preferred over the P-bound analog because of the donation from the lone pairs of the oxygen atom to the empty hybrid d/f orbital of the metal (Wiberg index of 0.42). It is worthy to note that there is no interaction between the C\u2013P and the C\u2013O bonds; the molecular orbitals within the OCP\u2013 unit are localized onto either one or another. Contrarily, when OCP\u2013 is coordinated through the lone pair of the phosphorus, a more covalent interaction is observed (Wiberg index of 0.90). Moreover, there is also a strong interaction between the lone pairs of the oxygen and the Th\u2013P bond, giving rise to the formation of an allylic-type interaction between the three centers . As thorium prefers to be rather ionic, the O-bound configuration is the most energetically prominent isomer.NBO analysis of \u2013 anions , the N-bound derivatives are respectively 13.4 kcal mol\u20131 and 17.5 kcal mol\u20131 lower in energy than the O- and S-bound ones for Th . The computed \u0394E are surprisingly greater for OCN\u2013 than for OCP\u2013 given that the terminus charge density difference is more pronounced in OCP\u2013 compared to OCN\u2013 and the hard/soft mismatch is stronger for An\u2013P bonding. This suggests that the P- vs. O-coordination selectivity is subtle in phosphaethynolate metal derivatives, with OCP\u2013 behaving as an ambident Lewis base depending on the nature of the metal.For the XCN4. Formation of SCP\u2013 has been observed upon reaction of alkali salts of the OCP\u2013 anion with CS2; compound 4, however, is stable with respect to [2 + 2] cycloaddition with CS2. Since the P-atom in 4 is protruding above the TMS groups, we reasoned that it could be accessible and act as a soft-donor to bind late transition metals. Accordingly, treatment of 4 with one equivalent of Ni(COD)2 results in a strong darkening of the solution and leads to the heterobimetallic adduct (amid)3Th(\u03bc-\u03b71(O):\u03b72-OCP)Ni(COD) 9 (9 is the sole product resulting from the reaction of 4 with Ni(COD)2, NMR studies performed in benzene solution show that these species are in equilibrium. Unfortunately, these compounds exhibit similar solubility in common solvents, therefore preventing the quantitative isolation of 9 in pure form. The 31P NMR spectrum of 9 shows a drastic downfield shift of the signal (\u03b4(31P) = \u20137.7 ppm) compared to 4 which is indicative of strong rearrangement of the phosphaethynolate moiety. Strong deshielding of the phosphorus atom is typically observed in related \u03b72-phosphaalkene derivatives.9 is confirmed by 1H NMR which features two vinylic resonances at \u03b4 = 6.1 and 5.5 ppm.We have carried out preliminary reactivity investigations involving i(COD) 9 . While 99 established unequivocally the formation of a three-membered nickel phosphametallacycle (C1\u2013P1\u2013Ni1 = 57.4(2)\u00b0) resulting from the addition of the Ni(0) center across the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019P bond. The most striking feature of this structure (depicted in \u2013 moiety (P\u2013C\u2013O angle = 148.1(3)\u00b0; Th\u2013O\u2013C angle = 157.5(3)\u00b0) bridging the two metals in an unprecedented \u03bc-\u03b71(O):\u03b72 fashion. This is indicative of strong backbonding from the square-planar nickel center into the \u03c0* orbital of the ligated C\u2013P unit. While these structural features are reminiscent of Ni(0) activation of phosphaalkynes\u2013 moiety in 9 is unique. Electron-donation to the antibonding \u03c0* orbital results in significant elongation of the coordinated C\u2013P bond length (1.660(4) \u00c5) from the corresponding value of 1.561(4) \u00c5 found in 4 and falls in the range (1.630 to 1.694 \u00c5) of side-on coordinated phosphaalkynes to nickel2NMe)(\u03b72-(Ph3C)C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019P)]4, while the Th\u2013O distance (2.279(3) \u00c5) is shortened which further indicates higher electron density on the OCP\u2013 moiety.Single-crystal X-ray diffraction analysis of 9 by DFT scale\" fill=\"currentColor\" stroke=\"none\">P orbital overlaps with a d-orbital of the nickel to give the HOMO seen in Examination of the structure of n as a salt-metathesis reagent for accessing phosphaethynolate actinide complexes. Unlike the P-bound product favored with rhenium, O-bonding is preferred with actinides while cyanate and thiocyanate anions adopt N-bonding. Actinide coordination polarizes the OCP\u2013 moiety and enhances its phosphaalkyne character. Addition of Ni(0) across the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019P bond of the Th-bound phosphaethynolate results in the formation of an unprecedented reduced OCP\u2013 moiety of bent-geometry bridging the two metals. These preliminary results pave the way towards the development of metal phosphaethynolate complexes both for reactivity purposes and to generate original heteropolymetallic architectures. Studies aiming at expanding actinide phosphaethynolate chemistry and uncovering the full range of reactivity of the metal-bound OCP\u2013 moiety are ongoing in our group.In summary, this study has proven the utility of Na(OCP)(dioxane)Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "Sodium phosphaethynolate reacts with [MCl(PDI)] to give metallaphosphaketenes, which in the case of iridium rearranges into a dimetalladiphosphene. via CO migration from phosphorus to the metal. Two different bonding modes of the PCO anion to CAAC-coinage metal complexes [CAAC: cyclic (amino)(carbene)] are reported, one featuring a strong Au\u2013P bond and the other an \u03b72 coordination to copper. The gold complex appears to be mostly unreactive whereas the copper complex readily reacts with various organic substrates. A completely free PCO anion was structurally characterized as the [Cu(La)2]+ (OCP)\u2013 salt. It results from the simple displacement of the PCO unit of the cationic (CAAC)Cu(PCO) complex by a second equivalent of CAAC.Sodium phosphaethynolate reacts with [MCl(PDI)] to give metallaphosphaketenes, which in the case of iridium rearranges into a dimetalladiphosphene, NoteworD to E, and the displacement of the PCO unit of the copper complex by a CAAC, which leads to a PCO anion with no coordinating solvents or binding agents.Herein we describe salt metathesis reactions leading to both unstable and stable terminal PCO transition metal complexes, featuring different coordination modes, and reactivity. We also report the experimental demonstration of the predicted conversion of Because of the strongly reducing character of Na(OCP),14iPr)] complex 1 was reacted with Na(OCP) in THF at \u201330 \u00b0C the color changed from pink to deep purple, and a single broad resonance in the 31P NMR spectrum at \u03b4 = \u2013226 ppm [vs. \u03b4 = \u2013392 ppm for Na(OCP)] indicated quantitative conversion. The metallaphosphaketene 3 was isolated in 61% yield and fully characterized. The IR spectrum showed the asymmetric stretching frequency of the phosphaketene unit at \u03bdasym = 1851 cm\u20131, intermediate between Na(OCP) (\u03bdasym = 1755 cm\u20131) and Ph3Sn\u2013P PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O (\u03bdasym = 1946 cm\u20131) PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O. This is confirmed by a single crystal X-ray diffraction analysis ] 2 with the less sterically encumbered PDIMe ligand reacts with Na(OCP) at low temperatures to cleanly give product 4. A 31P NMR resonance at \u03b4 = \u2013316.7 ppm indicates a metallaphosphaketene scale\" fill=\"currentColor\" stroke=\"none\">C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O) featuring a highly covalent metal phosphorus bond. Complex 4 could not be isolated. Keeping a THF solution at 20 \u00b0C for about 6 h leads cleanly to complex 5 , which was isolated as red crystals. A single crystal X-ray structure analysis shows compound 5 to be a dimetalladiphosphene scale\" fill=\"currentColor\" stroke=\"none\">P\u2013R. The iridium centers are bound to a redox-inactive PDI ligand with short C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N bonds and a carbonyl ligand. The rearrangement of 4 into 5 is the experimental confirmation of the computationally predicted transformation of D to E.3, to a transient terminal nitrido complex, Ir PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N, which could be spectroscopically characterized but also dimerizes to an Ir\u2013N PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N\u2013Ir complex.16The corresponding iridium complex and C\u2013O bond lengths are similar, and the M\u2013P\u2013C angle is slightly more acute for the copper complex 9 .Monitoring by studies . Only ve8a and 9. The NBO charges of Au and PCO in 8a are +0.39 and \u20130.56 a.u., respectively, whereas those of Cu and PCO in 9 are +0.58 and \u20130.68 a.u., suggesting that the PCO anion in 9 is more ionic and \u201cfree\u201d than that in 8a. This is in agreement with the different 31P NMR chemical shifts of 8a (\u03b4 = \u2013360 ppm) and 9 (\u03b4 = \u2013387 ppm). The NBOs corresponding to the M(PCO) (M = Au or Cu) fragments are quite different as shown in 8a forms three bonds analysis at the M06/6-311++G+SDD//M06/6-31+G(d)+LANL2DZ(+f) level of theory shows significant differences between the electronic structures of d P\u2013C \u03c0) . In cont \u03c0 bonds . Moreoveonds 3P], as determined by an X-ray diffraction study, is reminiscent of the rearrangement product of Ph3Sn(PCO), namely (Ph3Sn)3P.31P NMR spectrum displays a signal at \u03b4 = \u2013200 ppm, which is considerably high-field shifted compared to alkyl and aryl phosphines. The electron rich nature as well as the steric crowding around the P center could make 10 an interesting redox active ligand for transition metals.18The different bonding modes in perature . The tri8a,b are rather inert and do not react with heavier group 14 element halides to give R3E-PCO derivatives. Equally, no reaction with N,N\u2032-dicyclohexylcarbodiimide or with carbene La are observed. On the contrary, the copper salt 9 does react with these reagents similarly to Na(OCP) (see the ESI9 reacts with carbene La to afford the cationic bis(CAAC)Cu complex 11, in which the PCO fragment is the anionic counterpart. The 31P NMR signal appears at \u03b4 = \u2013400 ppm, which is more downfield shifted than Na(OCP) (\u03b4 = \u2013392 ppm), implying that PCO\u2013 is less coordinated. The IR spectrum showed the asymmetric stretching frequency of the PCO unit at \u03bdasym = 1791 cm\u20131, suggesting a more cumulenic nature than in the two crystalline forms of Na(OCP) (\u03bdasym = 1780 or 1755 cm\u20131).The gold complexes 8a and 9 are mainly localized on the PCO fragments (see ESI8a and \u20130.59 a.u. in 9). Thus, we were curious to see if the terminal oxygen atom could react with a Lewis acidic borane. Indeed, adding one equivalent of B(C6F5)3 to either 8b or 9 led to the same type of heterocycle 12 and 13, respectively CoCl 1,MePDI)IrCl 2,a,bAuCl 6a,baCuOtBu 71H, 13C, 11B, 19F, and 31P NMR spectra were recorded on a Varian VX 500, Bruker 300, Bruker 500 and Jeol 500 spectrometer at 25 \u00b0C. All 1H and 13C NMR chemical shifts are reported relative to SiMe4 using the 1H and 13C chemical shifts of the solvent as a secondary standard. NMR multiplicities are abbreviated as follows: s = singlet, d = doublet, t = triplet, sept = septet, m = multiplet, br = broad signal. Chemical shifts are given in ppm and coupling constants J are given in Hz. Peak widths at half heights (in Hz) are given for broad signals. Infrared spectra were collected on a Perkin-Elmer-Spectrum 2000 FT-IR-Raman and Bruker ALPHA FT-IR spectrometer. Elemental analyses were performed at the Mikrolabor of ETH Z\u00fcrich. Single crystals suitable for X-ray diffraction were coated with polyisobutylene oil in a dry-box, transferred to a nylon loop and then transferred to the goniometer of a Bruker X8 APEX2 diffractometer equipped with a molybdenum X-ray tube (\u03bb = 0.71073 \u00c5) or on a Bruker Apex II-CCD detector using Mo-K\u03b1 radiation (\u03bb = 0.71073 \u00c5) or Cu-K\u03b1 radiation (\u03bb = 1.54178 \u00c5). The data were processed using the Bruker SAINT+ program and corrected for absorption using SADABS. The structures were solved using direct methods (SHELXS) completed by Fourier synthesis and refined by full-matrix least-squares procedures. Mass spectra were performed at the UC San Diego Mass Spectrometry Laboratory. Melting points were measured with an electrothermal MEL-TEMP apparatus.All air- and moisture-sensitive manipulations were carried out using standard vacuum line Schlenk techniques or an MBraun dry-box under argon. THF was distilled over sodium benzophenone-ketyl before use. THF-iPrPDI)CoCl 1 and 10 mL of THF. The solution was cooled to \u201335 \u00b0C and Na(OCP) was added portion-wise over the course of 5 minutes, eliciting a color change from pink to dark purple. The reaction was placed in the freezer at \u201335 \u00b0C for one hour then filtered through Celite. The solution was concentrated, layered with hexane and placed at \u201335 \u00b0C. This gave 0.152 g (48%) of a purple crystalline solid identified as [(iPrPDI)Co(PCO)] 3. The mother liquor was placed back in the freezer to obtain another 42 mg (13%) of product. X-Ray quality crystals were grown from the second fraction. Analysis for C34H43CoN3OP, 599.64 g mol\u20131, calc.: C, 68.10; H, 7.23; N, 7.01 found: C, 66.05; H, 7.35; N, 6.85. IR (powder): \u03bd PCO = 1851 cm\u20131. 1H NMR : \u03b4 = 9.66 , 7.47 , 7.35 , 7.05 , 3.32 ), 1.13 ), \u20130.19 ; 13C NMR : \u03b4 = 181.2 , 168.1, 153.4, 150.5, 140.0, 125.1, 124.0, 116.4, 28.6 (Ar-CH3), 24.0 (Ar-CH3), 23.3, 21.8; 31P NMR : \u03b4 = \u2013225.8 ppm (lb = 634 Hz).In the glove box, a 20 mL scintillation vial was charged with 0.200 g (0.347 mmol) of (2 and 5 mL of THF and cooled in a dry-ice/acetone bath. A solution of Na(OCP) in THF (3 mL) was syringed into the stirring iridium solution, immediately causing a color change to dark purple. The reaction was warmed to room temperature, whereupon the color changed to deep pink, and stirred for an additional hour. The reaction was then filtered through Celite and then concentrated to roughly 3 mL. Storing at \u201335 \u00b0C overnight produced a solid that was collected on a glass frit and dried under reduced pressure, yielding 0.076 g (74% yield) of red crystalline solid 5. X-Ray quality crystals were grown from the slow evaporation of the mother liquor at room temperature overnight. NMR analysis was performed in CD2Cl2 due to the poor solubility of 5 in ethereal or aromatic solvents, but the compound slowly decomposed (if left overnight) in methylene chloride. Analysis for C52H55Ir2N6O2P2, calcd: C 50.27, H 4.46, N 6.76; found: C 49.96, H 4.63, N 6.31. IR (powder): \u03bd CO = 1967 cm\u20131. 1H NMR : \u03b4 = 8.12 , 7.26 , 7.07\u20136.93 , 2.3 , 1.63 , 1.43 ; 13C NMR : \u03b4 = 187.50 (CO), 152.66, 149.21, 143.12, 131.89, 130.36, 128.28 (Ar-CH), 127.96 (Ar-CH), 126.02, (Ar-CH), 123.97 (m-Py-CH), 116.16 (m-Py-CH), 20.66 (Ar-CH3), 18.31 (Ar-CH3), 15.51 (CN\u2013CH3); 31P NMR : \u03b4 = 683.2.A 20 mL Schlenk flask was charged with 0.100 g (0.167 mmol) of (MePDI)IrCl 6a and [Na(PCO)(dioxane)2.5] was cooled to \u201378 \u00b0C before THF (10 mL) was added. The mixture was stirred for 15 minutes and then warmed to room temperature. After 30 min, the solvent was removed under vacuum and the resulting brown solid was extracted with 15 mL of benzene. After removing the solvent, 8a was obtained as a light yellow solid . Colorless single crystals of 8a were obtained by vapor diffusion of pentane into a saturated benzene solution of 8a in the dark. IR (C6H6): \u03bd PCO = 1887 cm\u20131. M.P. = 193 \u00b0C (dec.). 1H NMR : \u03b4 = 7.13 , 7.00 , 2.72 , 1.63 , 1.54 , 1.42 , 1.08 , 0.85 ; 13C {1H} NMR : \u03b4 = 253.5 , 183.0 scale\" fill=\"currentColor\" stroke=\"none\">O d, JPC = 100.4 Hz), 146.1, 135.3, 130.9, 126.0, 81.0, 63.0, 43.0, 32.4, 20.1, 29.5, 27.7, 23.7, 10.3; 31P {1H} NMR \u03b4 = \u2013359.5. HRMS was attempted but a peak corresponding to M+ could not be located, probably due to the weak P metal bond.A mixture of (CAAC)AuCl 6b and [Na(PCO) (dioxane)2.5] was cooled to \u201378 \u00b0C before THF (10 mL) was added. The mixture was stirred for 15 minutes and then warmed to room temperature. After 30 min, the solvent was removed under vacuum and the resulting brown solid was extracted with 15 mL of benzene. After removing the solvent, 8b was obtained as a light yellow solid . Colorless single crystals of 8b were obtained by vapor diffusion of (TMS)2O into a saturated benzene solution of 8b in the dark. IR (C6H6): \u03bd PCO = 1889 cm\u20131. M.P. = 221 \u00b0C (dec.). 1H NMR : \u03b4 = 7.14 , 7.00 , 3.17 , 2.79 , 2.05 , 1.95 , 1.88 , 1.82 , 1.69 , 1.58 , 1.52 , 1.26 , 1.09 , 0.93 , 0.87 ; 13C {1H} NMR : \u03b4 = 253.5 , 182.5 scale\" fill=\"currentColor\" stroke=\"none\">O d, JPC = 101.1 Hz), 146.2, 145.8, 136.1, 130.6, 129.2, 125.8, 77.6, 65.4, 53.4, 52.0, 50.0, 36.4, 31.3, 30.1, 29.8, 28.8, 28.0, 27.3, 25.6, 23.8, 23.7, 23.6, 20.7; 31P {1H} NMR \u03b4 = \u2013364.2. HRMS was attempted but a peak corresponding to M+ could not be located, probably due to the weak P metal bond.A mixture of (CAAC)AuCl 7 and [Na(OCP) (dioxane)2.5] was stirred for 10 minutes in 3 mL of benzene at room temperature. The solvent was removed under vacuum and the resulting brown solid was washed with 10 mL of pentane. After drying under vacuum, 9 was obtained as a light yellow solid . Colorless single crystals of 9 were obtained were obtained by vapor diffusion of (TMS)2O into a saturated toluene solution of 9 at \u201340 \u00b0C. IR (C6H6): \u03bd PCO = 1849 cm\u20131. M.P. = 173 \u00b0C (dec.). 1H NMR : \u03b4 = 7.12 , 7.00 , 2.75 , 1.68 , 1.43 , 1.38 , 1.08 , 0.93 , 0.85 ; 13C {1H} NMR : \u03b4 = 251.8 (Ccarbene), 175.2 scale\" fill=\"currentColor\" stroke=\"none\">O d, JPC = 97.8 Hz), 145.9, 135.5, 130.7, 125.6, 81.2, 63.1, 43.1, 31.9, 29.9, 29.4, 27.9, 23.1, 10.4; 31P {1H} NMR \u03b4 = \u2013387.4. HRMS was attempted but a peak corresponding to M+ could not be located, probably due to the weak P metal bond.A mixture of (CAAC)CuOtBu 8b was left standing in 2 mL of THF for 1 week under visible light. White crystals of 10 were generated and washed with 5 mL of pentane . M. P. = 293 \u00b0C (dec.). 1H NMR : \u03b4 = 7.39 , 7.18 , 2.72 , 2.28 , 1.88 , 1.78 , 1.67 , 1.34 , 1.24 , 1.13 , 0.98 , 0.90 , 0.82 ; 13C {1H} NMR : \u03b4 = 260.3 , 146.3, 145.7, 135.3130.2, 125.5, 125.2, 79.0, 78.9, 66.5, 66.4, 52.9, 50.5, 36.5, 30.5, 30.4, 30.0, 29.9, 29.5, 29.0, 28.9, 28.5, 25.2, 25.1, 24.5, 23.3, 23.2, 21.3; 31P {1H} NMR \u03b4 = \u2013200.2. HRMS: m/z calculated for [C81H130N3Au3P]+ (M + H)+ 1766.8999; found 1766.8980.Complex 9 and carbene La was stirred for 2 min in benzene (0.5 mL). The suspension was filtered and the colorless powder was washed with benzene (1 mL), yielding 11 . Single crystals were obtained by slow evaporation of a saturated benzene solution of 11. IR : PCO \u03bd = 1791 cm\u20131. M.P. = 158 \u00b0C (dec.). 1H NMR : \u03b4 = 7.08 , 6.97 , 2.70 , 1.54 , 1.42 , 1.32 , 1.05 , 0.84 , 0.81 ; 13C {1H} NMR : \u03b4 = 253.3 (Ccarbene), 145.8, 135.3, 130.7, 125.5, 80.8, 63.0, 43.6, 31.6, 30.0, 29.4, 28.0, 23.1, 10.2; 31P {1H} NMR \u03b4 = \u2013399.5. HRMS: m/z calculated for [C44H70CuN2]+ 689.4835; found 689.4847.A mixture of 8b and B(C6F5)3 was stirred in 2 mL of benzene for 5 minutes. The resulting yellow suspension was filtered and the yellow residue was washed with benzene (0.5 mL), then dried under vacuum, yielding 61 mg (64%) of a bright-yellow powder. Yellow single crystals of 12 were obtained in the filtrate in less than 1 min. M.P. = 230 \u00b0C (dec.).A mixture of 9 and B(C6F5)3 was stirred in 1 mL of toluene for 5 minutes. The solvent was removed under reduced pressure and the residue was washed with pentane (2 mL), yielding 68 mg (62%) of a light-yellow powder. Colorless single crystals were obtained by vapor diffusion of (TMS)2O into a saturated toluene solution of 13. M.P. = 160 \u00b0C (dec.). 1H NMR : \u03b4 = 7.08 , 6.93 , 2.59 , 1.40 , 1.31 , 1.12 , 1.08 , 1.01 , 0.85 ; 13C {1H} NMR : \u03b4 = 248.8 (Ccarbene br), 149.5 , 149.0 , 145.5, 138.1 , 135.1, 131.2, 125.8, 82.5, 62.8, 43.0, 31.5, 29.7, 29.2, 27.7, 22.7, 9.9; 31P {1H} NMR \u03b4 = 260.6 (br), 136.2 (br). HRMS was attempted but a peak corresponding to M+ could not be located, probably due to the weak P metal bond.A mixture of Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "Evidence for a transient, highly reactive ThNTh nitride is presented, in contrast to uranium analogues that are stable and isolable. Surprisingly, computational studies reveal a \u03c3 > \u03c0 energy ordering for all these bridging nitride bonds, a phenomenon for actinides only observed before in terminal uranium nitrides and uranyl. DMBS)(I)] 3}3\u2013) with NaN3 or KN3, respectively, affords very rare examples of actinide molecular square and triangle complexes [{M(TrenDMBS)(\u03bc-N3)}n] . Chemical reduction of 3 produces [{U(TrenDMBS)}2(\u03bc-N)][K(THF)6] (5) and [{U(TrenDMBS)}2(\u03bc-N)] (6), whereas photolysis produces exclusively 6. Complexes 5 and 6 can be reversibly inter-converted by oxidation and reduction, respectively, showing that these UNU cores are robust with no evidence for any C\u2013H bond activations being observed. In contrast, reductions of 4 in arene or ethereal solvents gives [{Th(TrenDMBS)}2(\u03bc-NH)] (7) or [{Th(TrenDMBS)}{Th(N[CH2CH2NSiMe2But]2CH2CH2NSi[\u03bc-CH2]MeBut)}(\u03bc-NH)][K(DME)4] (8), respectively, providing evidence unprecedented outside of matrix isolation for a transient dithorium-nitride. This suggests that thorium-nitrides are intrinsically much more reactive than uranium-nitrides, since they consistently activate C\u2013H bonds to form rare examples of Th\u2013N(H)\u2013Th linkages with alkyl by-products. The conversion here of a bridging thorium(iv)-nitride to imido-alkyl combination by 1,2-addition parallels the reactivity of transient terminal uranium(iv)-nitrides, but contrasts to terminal uranium(vi)-nitrides that produce alkyl-amides by 1,1-insertion, suggesting a systematic general pattern of C\u2013H activation chemistry for metal(iv)- vs. metal(vi)-nitrides. Surprisingly, computational studies reveal a \u03c3 > \u03c0 energy ordering for all these bridging nitride bonds, a phenomenon for actinides only observed before in terminal uranium nitrides and uranyl with very short U\u2013N or U\u2013O distances.Molecular uranium-nitrides are now well known, but there are no isolable molecular thorium-nitrides outside of cryogenic matrix isolation experiments. We report that treatment of [M(Tren PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019PH, and bridging ThP(H)Th, ThAs(H)Th, ThAs(H)K, ThPTh and ThAsTh units.2 linkages are highly unstable and readily decompose. In contrast, most bridging UNU units seem to be relatively stable,Building on our prior work on terminal uranium-nitrides,Here, we report the synthesis and characterisation of two triamidoamine uranium- and thorium-azides. Despite marginal differences in the covalent radii of these metals, the uranium complex is a rare example of an actinide molecular square whereas the thorium analogue is a molecular triangle. Reduction of the uranium-azide complex generates diuranium-nitrides, with two charge states of a UNU core being accessible, and interchangeable, with no evidence of C\u2013H activation chemistry even under photolytic conditions. However, a ThNTh complex could not be isolated. Instead, the isolation of two ThN(H)Th complexes, which is an unprecedented linkage in thorium chemistry and rare in f-block chemistry generally,3 reduction approach using the TrenDMBS {N(CH2CH2NSiMe2But)3}3\u2013 ligand as this was anticipated to be sterically open enough to allow any nitrides to bridge, whereas the bulkier TrenTIPS {N(CH2CH2NSiPri3)3}3\u2013 variant stabilises ThPTh and ThAsTh linkages but terminal UN for uranium. Accordingly, treatment of [M(TrenDMBS)(I)] 3 or KN3 affords [{M(TrenDMBS)(\u03bc-N3)}n] as green-yellow and colourless crystalline solids after work-up in isolated yields of 35 and 86%, respectively, 3 and 4, in particular the ATR-IR spectra of 3 and 4 both exhibit strong absorptions at 2131 cm\u20131, which is characteristic of actinide-bound bridging-azide ligands.3, \u03bcB per molecule decreasing smoothly to 0.78 \u03bcB at 2 K and tending to zero as expected for a tetrametallic UIV complex, since UIV usually has a magnetic singlet ground state at low temperature.In order to prepare MNM linkages we pursued a M\u2013N3 and 4 we determined their solid-state structures, 1 and 2, and chloride analogues, are monomers, 3 and 4 are tetrameric and trimeric in the solid-state. Such molecular squares and trianglesC3v symmetry of TrenDMBS is lowered to Cs the cleft that opens up allows two azides to enter the coordination sphere of uranium in 3 at an approximate right angle (\u223c85\u00b0) whereas for the larger thorium in 4 the azides approach at a slightly more acute N\u2013Th\u2013N angle (\u223c79\u00b0), which seems to be enough to switch from tetramer to trimer. It would seem that the N\u2013Th\u2013N angle can close at the larger metal without as much inter-azide clashing due to longer Th\u2013N bonds placing the azides further apart from one another, which accounts for the aggregation states of 3 and 4. The U\u2013 and Th\u2013Nazide distances in 3 and 4 are longer than in terminal azide complexes,trans to a TrenDMBS amide centre ; 4, 2.609(8) \u00c5 av.) than amine centre ; 4, 2.478(7) \u00c5 av.), possibly implying a trans-influence. All other bond lengths are within normal ranges and do not suggest any strong activation of the azides.In order to confirm the formulations of 3 and 4 in-hand we investigated their reduction chemistries. The reaction of 3 with KC8 always produces the diuranium(iv/iv)-nitride [{U(TrenDMBS)}2(\u03bc-N)][K(THF)6] (5) and the mixed-valence diuranium(iv/v)-nitride [{U(TrenDMBS)}2(\u03bc-N)] (6) in \u223c42% overall yield , with concomitant elimination of N2 and KN3.5\u2009:\u20096 varies from 77\u2009:\u200923 to 50\u2009:\u200950 as the KC8 ratio is varied from 5 to 3 equivalents but is independent of the solvent used . Other KC8 ratios gave intractable product mixtures. Complexes 5 and 6 can be separated by fractional crystallisation, however we find that 6 can be cleanly prepared in 45% isolated crystalline yield by photolysis of 3 with a 125 W Hg-lamp for 7 hours. Gratifyingly, 6, or a known mixture of 5 and 6, can be reduced with KC8 to give solely 5, and 5 or a known mixture of 5 and 6 can be oxidised with AgBPh4 to give exclusively 6, 5 and 6 are always formed in the respective reduction and oxidation reactions, irrespective of the amounts of KC8 (2\u20138 equiv.) and AgBPh4 (2\u20133 equiv.) used, and we found no evidence for further reductions or oxidations, respectively.With 5 and 6 are distinct, reflecting their UIV/UIV and UIV/UV formulations, respectively. The 1H NMR spectra of 5 and 6 span the ranges +96 to \u201334 and +25 to \u201313 ppm, respectively, reflecting their 5f2/2vs. 5f2/1 natures. The solution Evans method (298 K) gives magnetic moments of 4.0 and 3.5 \u03bcB per molecule of 5 and 6, whereas the solid-state magnetic moments, \u03bcB, respectively. These values decrease to 1.00 and 1.07 \u03bcB at 2 K, respectively. For 5 the respective values per uranium ion are 3.39 (298 k) and 0.74 \u03bcB (2 K), which per ion are slightly higher than the corresponding values for 3 reflecting their nitride and azide formulations.6 are consistent with its UIV/UV combination,V ion has a magnetic doublet ground state at all temperatures, and anti-ferromagnetic U\u2013U coupling is suggested by a maximum at \u223c60 K in the \u03c7 vs. T plot of 5.V in 6 is unequivocally confirmed by EPR spectroscopy (S- and X-bands) at 5 K, g-values with geff = 3.13, 0.95, 0.50, and 2.70, 0.74, 0.43; these data reflect the presence of two conformational isomers in the solid-state structure of 6 due to positional disorder of three of the six SiMe2But groups, and we note that the effective g-values of spin\u2013orbit doubles are extremely sensitive to small changes in structure.E1/2 \u20131.4 V (vs. [Fc(Cp)2]0/+1) for the [UIV/UV]/[UIV/UIV]\u2013 redox couple is found, 3 and 4 which do not exhibit any electrochemical events in the solvent-accessible window of 2.5 to \u20133.0 V. The chemical inter-conversion of 5 and 6 suggests the presence of robust UNU cores, as was found for [{U3}2(\u03bc-N)]n which can exist in three charge states, PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019U angles of 5 compared to 6 (see below).As expected, the characterisation data for 5 and 6 were determined, 5 resides on a crystallographic 3-fold rotation axis and therefore the U\u2013N\u2013U and Nnitride\u2013U\u2013Namine angles are rigorously 180\u00b0, however in 6 the U\u2013N\u2013U angle is bent at 161.2(2)\u00b0. In 5 the U1/2\u2013N nitride, amide, and amine distances are 2.0648(2), 2.343(3), and 2.733(5) \u00c5, respectively; these distances reflect the bridging nature of the nitride, that is consistent with other UNU distances,IV complexes, and possibly a strong trans-influence from the nitride since the amine distances are quite long like in related ThPTh and ThAsTh complexes,vi)-nitride.6 the U\u2013Nnitride distances are now inequivalent at 2.081(5) and 2.136(5) \u00c5, and the U\u2013Namide and U\u2013Namine distances (av. 2.287(5) and 2.649(5) \u00c5) are now shorter than in 5, presumably reflecting the neutral formulation of 6 and a reduced nitride trans-influence since the Nnitride\u2013U\u2013Namine angles are now \u223c159\u00b0.The solid-state structures of 4, in contrast to 3, gives two distinct products, in addition to N2 and KN3, that are exclusive to the solvent media, DMBS)}2(\u03bc-N)][K] the parent imido [{Th(TrenDMBS)}2(\u03bc-NH)] (7) is isolated in 52% crystalline yield. When ethereal solvents are used the cyclometallated tuck-in-tuck-over,DMBS)}{Th(N[CH2CH2NSiMe2But]2CH2CH2NSi[\u03bc-CH2]MeBut)}(\u03bc-NH)][K(DME)4] (8) is isolated in 46% crystalline yield. The 1H NMR spectrum of 7 reveals a resonance at 5.55 ppm that corresponds to one N\u2013H proton; this resonance disappears when the reaction is conducted in D8-toluene, suggesting the source of H is aromatic solvent with K\u2013C6H5/\u2013CH2Ph as by-products. In-line with this, 7 does not react with benzyl potassium. The presence of the N\u2013H group is confirmed by a broad absorption at 3390 cm\u20131 in the ATR-IR spectrum of 7. The 1H NMR spectrum of 8 is now more complicated due to the desymmetrisation of one of the TrenDMBS ligands, but the N\u2013H proton resonance can be observed at 5.39 ppm.The reduction of 7 and 8 were determined, 7 the Th\u2013Nimido\u2013Th angle is 145.96(19)\u00b0 and the imido adopts a trigonal planar geometry in contrast to ThP(H)Th and ThAs(H)Th linkages2-NH dianion but p-orbital-dominated bonding of PH and AsH dianions. The Th\u2013Nimido distances of 2.331(4) and 2.312(4) \u00c5 are similar to the Th\u2013Namide distances (\u223c2.330 \u00c5) and \u223c0.3 \u00c5 longer than Th PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019NR terminal imido bonds.8 the Th\u2013Nimido\u2013Th angle is 120.9(7)\u00b0, reflecting the presence of the tuck-in-tuck-over cyclometallate enforcing a constrained C\u2013Th\u2013N\u2013Th four-membered ring. Despite this, the Th\u2013Nimido distances of 2.309(15) and 2.264(15) \u00c5 are essentially the same as those in 7. The Th\u2013C distances of 2.88(2) and 2.78(2) \u00c5 are long, as observed in other Th\u2013TrenDMBS cyclometallates.28The molecular structures of 5 and 6, especially the latter under photolytic conditions, is significant because photolysis of terminal uranium(vi)-nitridesiv)-nitride generated transiently by reduction5 and 6 contain quite robust, redox inter-convertible UNU cores, and when N2 is eliminated from 3 the nitride secures stabilisation by two uranium Lewis acid centres rather than instigating C\u2013H activation reactions.The formation and isolation of 4 does not lead to the isolation of dithorium-nitrides, the isolation of 7 and 8 is instructive. Like 5 and 6, reduction of an azide precursor, 4, results in the formation of a ThNTh unit, except in both cases this is protonated. For 7, the potassium from reduction has been exchanged for a proton suggesting that a transient nitride [{Th(TrenDMBS)}2(\u03bc-N)]\u2013 (\u20139) has C\u2013H activated arene solvents, and we note the yield of 7 does not vary when changing the solvent from benzene to toluene. Likewise, in the formation of 8 a \u03bc-NH unit forms again, but this time in the absence of deprotonatable arene solvent, and with the potassium cation sequestered by ethereal solvent, the transient nitride has C\u2013H activated the TrenDMBS to form a tuck-in-tuck-over cyclometallate3 species that also cyclometallate Tren-ligands due to proximity and entropy effects,5 and 6. Indeed, DFT calculationsDMBS)}2(\u03bc-N)]\u2013 (\u20135) and \u20139 suggest that the latter contains more polar and ionic metal-nitride linkages, but importantly the frontier molecular orbitals that principally comprise the ThNTh bonding interactions in \u20139 are destabilised by \u223c1.1 (\u03c3) and \u223c0.4 (\u03c0) eV compared to the corresponding UNU orbitals of \u20135. This results in a more basic, effectively superbasic, nucleophilic nitride in \u20139, as experimentally inferred by the isolation of 5versus7 and 8, and shown computationally where the anion of 8 is found to be 19.8 kcal mol\u20131 more stable than \u20139 and formed via a transition state with an experimentally accessible barrier of 16.4 kcal mol\u20131.18Although reduction of 8 parallels the reactivity of a transiently formed uranium(iv)-nitride that undergoes ligand C\u2013H activation to give a cyclometallated ligand and a uranium(iv) parent imido functionality by 1,2-addition,vi)-nitridesVIvs. MIV ions in this context generally, The formation of IVNUIV and ThIVNThIV -nitrides. Attempts to prepare a dithorium-nitride complex resulted in the isolation of two parent imido complexes, in-line with the paucity of isolable molecular thorium-nitrides to date. However, the two dithorium-imido products suggest for the first time that reduction of thorium-azides can generate nitrides, and provides evidence that a transient and highly reactive dithorium-nitride is formed, but that this linkage is highly basic and nucleophilic so is capable of activation C\u2013H bonds of arene solvent or the supporting TrenDMBS ligand. The contrasting stabilities of UNU and putative ThNTh units reported here may be related to the general tendency of uranium to engage in more covalent bonding than thorium, on a like-for-like basis. The results here suggest a general pattern of actinide-nitride reactivity where metal(iv)-nitrides, bridging or terminal, activate C\u2013H bonds to produce imido-alkyl combinations, whereas metal(vi)-nitrides produce alkyl-amide linkages, which can be related to the range of accessible oxidation states of these ions during reactions. Lastly, computational studies surprisingly reveal a \u03c3 > \u03c0 energy ordering for the bridging nitride linkages in this study, a phenomenon so far only found in terminal uranium-nitrides and uranyl complexes with very short U\u2013N/\u2013O distances.In conclusion, the synthesis of two uranium- and thorium-azide complexes has provided rare examples of actinide molecular square and triangle complexes. We have prepared two diuranium-nitride complexes in different charge states; these UNU complexes are quite robust, and do not engage in C\u2013H activation chemistry, even under photolytic conditions, unlike terminal uranium(There are no conflicts to declare.Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "Stepwise reaction of an indyl-anion with organic azides initially forms the indium imide, which undergoes (2 + 3)-cycloaddition to generate the indium tetrazenide. Ar)] (NONAr = [O(SiMe2NAr)2]2\u2013, Ar = 2,6-iPr2C6H3) and its reactivity with organic azides RN3 is reported. When R = 2,6-bis(diphenylmethyl)-4-tBu-phenyl, a dianionic alkyl-amide ligand is formed via C\u2013H activation across a transient In\u2013Nimide bond. Reducing the size of the R-group to 2,4,6-trimethylphenyl enables oxidation of the indium and elimination of dinitrogen to afford the imide species, K[In(NONAr)(NMes)]. The anion contains a short In\u2013Nimide bond, shown computationally to contain appreciable multiple bond character. Reaction of isolated imides with an additional equivalent of azide generates tetrazenido-indium compounds K, shown by X-ray crystallography to contain planar InN4 heterocycles in the anion.The synthesis of a new potassium\u2013indyl complex, K[In(NON These ligands are typically bulky, a requirement to limit (or prevent) aggregation and protect the metal from unwanted redox chemistry. This concept is best illustrated in the context of this work by the series M(BDIAr) ]\u2013, Ar = 2,6-iPr2C6H3), for which mono-metallic Al,The most common members of this class of compound are represented by the general formula M(X), where the charge on the metal is balanced by a mono-anionic ligand, [X]2]2\u2013 to support M(I) metal centres, generating an overall negative charge on the metal-containing species, [M(X2)]\u2013. Whilst this class of compound has been well studied for gallium,2)]\u2013 and [In(X2)]\u2013 systems indicate significant lone-pair character at the metal , with preliminary reactivity consistent with an Al(i) or In(i) nucleophile. We report in this contribution an investigation of the reducing potential of a new potassium indyl compound towards organic azides. This class of substrate was selected to target synthetically challenging indium imide species.A recent development in the chemistry of mono-valent aluminium and indium is to employ a dianionic ligand III\u2013VI was achieved through kinetic stabilization of the M\u2013Nimide bonds using sterically demanding ligands that prevent formation of ring- and cage-structures containing \u03bc2- and \u03bc3-NR ligands.imide functional group is highly reactive.15The isolation of 2 and oxidation of the metal M(III), which occurs with a concurrent increase in the coordination number of the metal. It is of note that, if insufficient steric protection is provided during synthesis, in situ addition of unreacted azide in the reaction mixture to the transient \u2018M PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019NR\u2019 bonds can occur ,A general synthetic methodology to group 13 imides is the reduction of organic azides by a monovalent M(I) metal complexes .13b\u2013d,16an occur .17 For Aiii) imide, shown crystallographically and computationally to contain In\u2013Nimide multiple bonds. Reaction of isolated examples with additional azide proceeds via a (2 + 3)-cycloaddition pathway to generate tetrazenido-indium salts, containing the first structurally characterized examples of the InN4-heterocycle.In this contribution we report a new potassium indyl salt and its controlled (stepwise) reactivity with organic azides. The initial products are characterised as a new class of anionic indium(Ar-ligand (NONAr = [O(SiMe2NAr)2]2\u2013; Ar = 2,6-iPr2C6H3) stabilizes anionic indyl species as the indyllithium complex In(NONAr)(Li{THF}3), or in the ion-separated salt [K(crypt-222)][In(NONAr)] (crypt-222 = [2.2.2]-cryptand).The NON2[(NONAr)(THF)n] (1), is readily obtained from the reaction of KH with the ligand pre-cursor (NONAr)H2.n = 1), and the reagent can be used without further purification. However, crystallization from THF affords the Tris\u2013THF adduct (n = 3) which forms a dimer [1_{THF}3]2 in the solid-state (Et2O)2] which forms a polymer in the solid-state adduct that crystallizes as the dimer [K2{(NONAr)(18-c-6)}]2 Cl\u2019, elemental analysis was inconsistent with this formula and the compounds was therefore analysed by single-crystal X-ray diffraction (The reaction of fraction .2 contains the four-coordinate indium anion [In(NONAr)Cl2]\u2013 Cl2]\u2013 . The chaolecules .2 with two equivalents of potassium yields the new indyl compound K[In(NONAr)] (3) as a hexane soluble, yellow solid. The 1H NMR spectrum of 3 displays a single resonance for the SiMe2 substituents, indicative of a symmetrical environment for the NONAr-ligand. There are no resonances attributable to In\u2013H hydride ligands ]\u2013 anions linked by potassium cations that are involved in \u03c0\u2013aryl interactions to flanking Ar groups (C\u00b7\u00b7\u00b7K distances 3.109(4)\u20133.346(3) \u00c5). The In\u00b7\u00b7\u00b7In separation is 4.710(3) \u00c5, with In\u2013N bond lengths (2.182(3)\u20132.240(3) \u00c5) consistent with anionic In(i) metal centres.The molecular structure of 3 is remin3 was made using an equimolar amount of the sterically demanding 2,6-bis(diphenylmethyl)-4-tBu-phenyl azide and a reduction in the intensity of the CHPh2 resonance . A new peak at 3.24 ppm {C6H2(CPh2)(CHPh2)-tBu-2,6,4} ligand chelating to a four-coordinate, anionic indium(iii) centre scale\" fill=\"currentColor\" stroke=\"none\">NAr\u2021\u2019 bond of a transient indium imide.The structure of ) centre . The pot1.000000,.000000 s3) under the conditions described above (5). The 1H NMR spectrum is consistent with a symmetrical NON-backbone and a freely rotating Mes group .To mitigate complications from ligand activation, the reaction was repeated with the sterically less intrusive 2,4,6-trimethylphenyl azide \u20133.296(3) \u00c5). The indium centres are distorted trigonal planar (\u03a3angles 358.7\u00b0), with In\u2013Nimide bond lengths of 1.986(2) and 1.999(2) \u00c5 to N3 and N6, respectively. These represent an average shortening of 3.6% compared with the In\u2013N bonds in the three coordinate amide In(NHMes*)3 ,VIb (1.928(3) \u00c5)The solid-state structure of complex . The comimide bonds in 5 may be influenced by interactions with the potassium cations (N3\u2013K1 2.661(3) \u00c5, N6\u2013K2 2.646(3) \u00c5), which are located closer to the nitrogen atom than in other potassiated imides (range 2.732(3)\u20133.069(11) \u00c5).5 was crystallized in the presence of [2.2.2]-cryptand. The crystal structure of the product confirmed formation of the separated ion-pair [K(crypt-222)][In(NONAr)(NMes)] \u00c5) remains unchanged (within 3\u03c3) and the In\u2013N\u2013C angle is still bent (In\u2013N3\u2013C29 127.43(14)\u00b0), suggesting that the N\u00b7\u00b7\u00b7K interactions in 5 have little effect on the structural component of the indium\u2013nitrogen bond.The length of the In\u2013NMes)] 6, . Interesimide unit in 5 and the isolated anion [In(NONAr)(NMes)]\u2013 from compound 6 ([6]\u2013) that are substantially higher than the In\u2013N bonds to the NONAr-ligand .Optimisation and subsequent analysis using density functional theory confirmed the multiple-bond character of the In\u2013N) see ESI. This isVIb,A\u2013D, C did not yield a viable solution by this method. However, structures A (triple bond), B (double bond) and D (single bond) all have a low n-L contribution to their overall NBO solution (Table S2A and B (n-L = 1.965% and 1.990% respectively), while D was only slightly less well localised (n-L = 2.094%). Although it is difficult to extract a precise numerical value for the multiplicity of the In\u2013Nimide bond from these computational data, the results confirm a strong multiple-bond component in accordance with crystallographic results, and observed reactivity (vide infra).To explore the nature of this bond in more detail, plausible resonance structures analogous to those examined for Table S2. The bes5 and [6]\u2013 has been performed. The bond critical point between the In and Nimide bonds have a low ellipiticity (\u03b5) of 0.079 and 0.072 for 5 and [6]\u2013, respectively, inconsistent with a conventional In PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N double where a larger value (>0.25) is predicted. These data suggest a non-elliptical cross-section of electron density in the In\u2013N bond vector. This is consistent with a model proposed by Power and co-workers in related gallium imides related to VIa,i) species interacts with a singlet nitrene, with incomplete donation of electron pairs (represented by dashed lines in Quantum Theory of Atoms In Molecules (QTAIM) analysis of IV\u2013VI, we wished to determine whether the In\u2013Nimide bond in 5 was available for controlled reactivity studies. Inspired by the proposed formation of metallotetrazenes from group 13 metal imides .As the imido-mesityl substituents are considerably less bulky than the terphenyl groups in l imides , we inve3 to an orange solution of 5 at room temperature resulted in decolorization over an approximate 5 minute period (7) or SiMe3 (8) groups, consistent with their incorporation in the product. In agreement with these data, the composition of the products as the first examples of indium tetrazenido compounds was confirmed by X-ray crystallography scale\" fill=\"currentColor\" stroke=\"none\">NMes\u2019, and that the formation of the unsymmetrical tetrazene 8 strongly endorses the previously assumed (2 + 3)-cycloaddition pathway of transient imides.Addition of a solution of RNe period . NMR spelography , Table 47\u00b7(toluene)]2 lies on a two-fold rotation axis that forms a dimeric unit, with K\u00b7\u00b7\u00b7\u03c0\u2013aryl interactions between Mes-groups and an incorporated toluene molecule that encapsulates the cation (C\u00b7\u00b7\u00b7K distances 3.006(5)\u20133.993(5) \u00c5). Crystallization of [7\u00b7(toluene)]2 in the presence of 18-crown-6 (18-c-6) disrupts dimer formation to afford [K(18-c-6)][In(NONAr)] (7 (18-c-6)). This salt forms a contact ion-pair linked by K\u00b7\u00b7\u00b7N interactions to the two central nitrogen atoms of the InN4-tetrazene ring (2.820(2) \u00c5 and 2.939(2) \u00c5, Fig. S27Ar)] also crystallizes as the dimer [8]2, with the potassium cations linking non-symmetry related units through a combination of K\u00b7\u00b7\u00b7N (2.641(6)\u20132.913(6) \u00c5) and K\u00b7\u00b7\u00b7\u03c0\u2013aryl (3.024(9)\u20133.264(8) \u00c5) interactions.The dimesityl derivative [VII, 7 and 8 represent the first structurally characterized indium compounds containing the tetrazenide ligand, and are unique examples where the MN4-heterocycle is a component of an anionic species.In all cases the anion comprises two approximately orthogonal rings fused at a four-coordinate indium centre. The metallotetrazene rings are essentially planar, with nitrogen\u2013nitrogen bond lengths indicating double-bond character between atoms in the 3- and 4-positions of the heterocycle see VII, . These pi) to In(iii). The isolated compounds have been structurally verified as a new class of anionic indium imide, shown computationally to contain In\u2013Nimide multiple bonds. Furthermore, we demonstrate that the reduced size of the imide-substituent in this work compared with previous examples allows access to the In\u2013Nimide bond, demonstrated by the reaction with additional equivalents of azide. The products from this (2 + 3)-cycloaddition are the first time that this reaction has been extended to indium, and crystallographic analysis confirms a planar InN4-heterocycle as a component of the anion.This work describes the first detailed reactivity study of an indyl-anion. We confirm that the negative charge associated with the indium centre does not adversely affect their ability to act as a reducing agent towards organic azides. The reactions proceed cleanly with elimination of dinitrogen and oxidation of the indium(There are no conflicts to declare.Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "A combination of MS and computation on \u03bc-nitrido bridged diiron complexes reveals H2O2 binding to the complex and generates an oxidant capable of oxidizing methane. iv)\u2013oxo species have been identified as the active intermediates in key enzymatic processes, and their catalytic properties are strongly affected by the equatorial and axial ligands bound to the metal, but details of these effects are still unresolved. In our aim to create better and more efficient oxidants of H-atom abstraction reactions, we have investigated a unique heteroleptic diiron phthalocyanine complex. We propose a novel intramolecular approach to determine the structural features that govern the catalytic activity of iron(iv)\u2013oxo sites. Heteroleptic \u03bc-nitrido diiron phthalocyanine complexes having an unsubstituted phthalocyanine (Pc1) and a phthalocyanine ligand substituted with electron-withdrawing alkylsulfonyl groups (Pc2RSO) were prepared and characterized. A reaction with terminal oxidants gives two isomeric iron(iv)\u2013oxo and iron(iii)\u2013hydroperoxo species with abundances dependent on the equatorial ligand. Cryospray ionization mass spectrometry (CSI-MS) characterized both hydroperoxo and diiron oxo species in the presence of H2O2. When m-CPBA was used as the oxidant, the formation of diiron oxo species (Pc2RSO)FeNFe(Pc1) PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O was also evidenced. Sufficient amounts of these transient species were trapped in the quadrupole region of the mass-spectrometer and underwent a CID-MS/MS fragmentation. Analyses of fragmentation patterns indicated a preferential formation of hydroperoxo and oxo moieties at more electron-rich iron sites of both heteroleptic \u03bc-nitrido complexes. DFT calculations show that both isomers are close in energy. However, the analysis of the iron(iii)\u2013hydroperoxo bond strength reveals major differences for the (Pc1)FeN(Pc2RSO)FeIIIOOH system as compared to (Pc2RSO)FeN(Pc1)FeIIIOOH system, and, hence binding of a terminal oxidant will be preferentially on more electron-rich sides. Subsequent kinetics studies showed that these oxidants are able to even oxidize methane to formic acid efficiently.Iron( For instance, iron(iv)\u2013oxo intermediates contribute as the active oxidant in heme monoxygenases, like the cytochromes P450, whereas in the structurally analogous peroxidases the detoxification of hydrogen peroxide to water is catalyzed.iv)\u2013oxo active species in their catalytic cycle to transfer an oxygen atom to substrates with functions ranging from DNA base repair and the biosynthesis of antibiotics.iv)\u2013oxo species have been identified as originating from the metal-substrate orbital-interactions and the electron-transfer pathways during the chemical reaction.iv)\u2013oxo intermediates in chemistry and biology, many studies have been devoted to the preparation and spectroscopic characterization of these short-lived species using enzymesiv)\u2013oxo species on the porphyrin,10High-valent ironFeNFe(Pc)], IIIFeIV and FeIVFeIV oxidation states.2O2 and m-chloroperbenzoic acid (m-CPBA), to form a high-valent diiron\u2013oxo species, i.e. . The addition of H2O2 to \u03bc-nitrido diiron tetra-t-butylphthalocyanine 8, [(PctBu)FeNFe(PctBu)] (tBu)FeIVNFeIII(PctBu)\u2013OOH], followed by the generation of the diiron\u2013oxo species, , as identified by electrospray ionization-mass spectrometry (ESI-MS) using labelled H218O2.IVNFeIV(TPP+\u02d9) PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O] with m-CPBA oxidant at \u201390 \u00b0C, and characterize it by ESI-MS, low-temperature UV-vis, electron paramagnetic resonance (EPR) and M\u00f6ssbauer spectroscopic techniques.\u03bc-Nitrido diiron phthalocyanines belong to the class of stable binuclear single atom bridged complexes that are known to bind metal ions in high oxidation states,(PctBu)] , resulteiv)\u2013nitrido group affect the catalytic functions of the iron(iv)\u2013oxo moiety and how can these be modified? In order to answer these questions, we decided to synthesize localized structures with non-equivalent iron sites, where each of the iron atoms is placed into macrocyclic ligands with very different electronic properties. Our approach opens interesting possibilities but also leads to deeper insight into the formation of the active species responsible for the catalysis. In particular, since such complexes with localized structure have two possible sites where an iron(iv)\u2013oxo species can be formed. Therefore, we designed unique chemical complexes that are based on the same phthalocyanine ligand structure, but exhibiting different electronic properties due to different substitution patterns. In particular, we created structures with one macrocyclic ligand with an electron-poor phthalocyanine group with four or eight alkylsulfonyl substituents, whereas the other macrocyclic ligand contained a more electron-rich unsubstituted phthalocyanine macrocycle. We report here the first synthesis and characterization of this series of unique heteroleptic structures scale\" fill=\"currentColor\" stroke=\"none\">O9) or hydroperoxo (9\u2013OOH) bound. The studies show that the electron-withdrawing character of the equatorial ligand, i.e. the phthalocyanine moiety, affects the strength of the iron(iii)\u2013hydroperoxo bond, and, thereby determines the site where the oxo group will bind. The work reveals the subtleties of ligand binding on the chemical and ultimately catalytic properties of metal\u2013oxo complexes. The ESI-MS and DFT results suggest the higher catalytic activity for more electron-rich iron sites. This prediction was confirmed in the catalytic heterogeneous oxidation of methane with H2O2 using three \u03bc-nitrido diiron phthalocyanine complexes with different substitution patterns.The macrocyclic \u03bc-nitrido diiron phthalocyanine provides a unique possibility to probe the differences in chemical properties of the two iron centers within the same molecule. The question, therefore, is: How do the functional properties of the ironFe(iv) unit are not necessarily inequivalent in terms of the oxidation state,i.e. with different electronic properties,iii) complexes, which are then mixed in a solution with sodium azide and \u03b1-chloronaphthalene to form the \u03bc-nitrido diiron complexes.Tetrapyrrolic ligands, such as porphyrin and phthalocyanine, can be used to form dimeric complexes.5 is prepared by a modified procedure4 with H2O2 in acetic acid at 79% yield. Iron(ii) octahexylsulfonyl phthalocyanine 6 is obtained by heating 5 in an o-dichlorobenzene\u2009:\u2009dimethylformamide (3\u2009:\u20091) mixture commonly used for alkylsulfonyl phthalonitriles2 (ii) phthalocyanine in \u03b1-chloronaphthalene in the presence of NaN3.3 and 7 were prepared by using a 10-fold excess of unsubstituted iron phthalocyanine 1 relative to the monomeric alkylsulfonyl phthalocyaninatoiron(ii) 2 or 6, respectively. These conditions allow the formation of the desired heteroleptic complexes . The lower yield of 7 with respect to 3 is attributed to a steric hindrance of the octasubstituted ligand 6.The synthesis of alkylsulfonyl substituted phthalonitriles is achieitriles2 . The preomplexes together3 and 7 was confirmed by FT-IR, UV-vis and EPR spectroscopy and ESI-MS studies. The ESI-MS spectra of these mixed ligand complexes exhibit prominent molecular peaks corresponding to the expected values of the molecular ion: m/z = 1943.4 [M]+ for 3 and m/z = 2335.6 [M]+ for 7 ] at 930 cm\u20131 and for homoleptic complex [(Pc)FeNFe(Pc)] at 915 cm\u20131.The successful preparation of 3 and homoleptic complex 8 as reference. Interestingly, the XES spectra of 3 and 8 are virtually identical in the solid phase module peaks of 3 for the first coordination shell. This is most likely due to differences in their Debye\u2013Waller factors, as a result of more position isomers for 8 as compared to 3. The effective disorder is therefore higher in 8, and manifests itself in the EXAFS spectra with increased Debye\u2013Waller parameters. Note that asymmetrization of the Fe\u2013N\u2013Fe fragment would lead to the decrease of Fe\u2013N\u2013Fe related features, as a result of weakening of the multiple scattering effects,3 and 8 reveals a symmetric Fe\u2013N\u2013Fe fragment in both complexes despite differences in the ligand properties in 3.No significant differences in the interatomic distances of the heteroleptic complex spectra in eithe3 and 8, we performed a series of XANES experiments. In contrast to the XAS, XES and EXAFS studies reported above, the XANES studies reveal a difference between the two structures \u2013oxo species readily in a reaction with active oxygen atom donors. The formation of the iron(iv)\u2013oxo species, therefore, comprises of two fundamental steps: (i) coordination of the oxygen donor to the iron and (ii) heterolytic cleavage of the O\u2013O bond of the peroxide unit. In the homoleptic complex both iron sites are identical and, as such, only one isomeric iron(iv)\u2013oxo species can be formed. In the heteroleptic complex, by contrast, there are, in principle, two isomeric iron(iv)\u2013oxo products possible (iv)\u2013oxo complex? In order to find out whether a site-specific iron(iv)\u2013oxo complex will be formed and what the properties of the ligands are that determine these issues, we performed a detailed combined ESI-MS and DFT study.Despite the initial high oxidation state of iron, the \u03bc-nitrido diiron phthalocyanine complexes form the high-valent iron(possible . The que2) was determined to be \u223c60 eV for the fragmentation of the diiron species into monomeric units without consecutive dissociation patterns. Fragmentation patterns of the heteroleptic compounds (iv)\u2013oxo species, whether it is electron-rich or electron-deficient. Indeed, the analysis of the initial fragmentation pattern provides a composition of two different phthalocyanine species with or without oxo and/or nitrogen ligands, which enables us to conclude whether the oxo species is attached to the electron-deficient Fe(Pc2RSO) ligand or to the more electron-rich Fe(Pc) fragment and the results are schematically depicted in To a \u223c103 + HOOH]+ core at m/z 582\u2013585 and 603 containing N, OH and H2O2 ligands are much larger than those with Fe(Pc2) fragments that are found in the m/z range of 1376\u20131379 and 1397. The intensity of naked [Fe(Pc1)]+ ion at m/z 568 obtained after loss of oxygen ligands is also much higher than that of protonated [Fe(Pc2)+H]+ ion at m/z 1362.The principal ions formed upon fragmentation of [+ HOOH]+ are ions3 + O]+ are identical to those theoretically predicted for the diiron\u2013oxo complex scale\" fill=\"currentColor\" stroke=\"none\">O3), which confirms its formation. Upon CID MS/MS conditions, the PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O3 ion behaves very similarly to [3 + HOOH]+ FeNFe(Pc2)]+ (3) at m/z 1943.46. Importantly, the fragmentation of [3 + O]+ produces almost exclusively [Fe(Pc1)]+ ions at m/z 568, 585, 600, whereas only minor amounts of [Fe(Pc2)]+ ions are detected at m/z 1362.The exact molecular mass and isotopic distribution pattern of + and + ions on the basis of exact molecular mass and isotopic distribution of the cluster. A large signal at m/z 585 is attributed to either + or [(Pc1)(N)Fe\u2013OH]+ ion formed by hydrogen atom abstraction from organic solvent by the oxo species with strong oxidizing properties. As expected, all these Fe(Pc1) oxygen containing species lose oxygen and nitrogen ligands to form naked [Fe(Pc1)]+ ions under CID-MS/MS conditions. The analysis of the fragmentation of [3 + H2O2]+ and [3 + O]+ ions suggests that high valent diiron species containing oxo ligands preferentially bind at more electron-rich iron sites such as Fe(Pc1).It should be noted here that fragments containing oxo ligands were only detected at the unsubstituted, PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O3 was also obtained using m-chloroperbenzoic acid as the oxygen donor. Importantly, the CID-MS/MS spectrum of PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O3m-CPBA shows only fragments derived from the Fe(Pc1) core (1) fragments bearing an oxo-ligand at m/z 585 and 600 are much stronger scale\" fill=\"currentColor\" stroke=\"none\">O32O2H species fragments with oxo-ligands are observed core . The sigstronger compared species . This su9, which is an analogue of 3 whereby the adamantylsulfonyl groups are replaced by methylsulfonyl: [(Pc1)FeNFe(Pc2MeSO)]. In addition, we calculated the iron(iv)\u2013oxo form of 9, i.e. , and finally the iron(iii)\u2013hydroperoxo complex [9\u2013OOH]\u2013. Although we calculated all structures in the lowest lying doublet, quartet and sextet spin states, actually in all cases the doublet spin state is the ground state and well separated from the other spin states. We, therefore, will focus in the main text on the doublet spin state only; all other results can be found in the ESITo support the experimental results and gain insight into the relative stability of \u03bc-nitrido diiron phthalocyanines with different substitution patterns of the distal ligand, we performed a detailed density functional theory (DFT) study on structure 9 with UB3LYP and UBP86 methods and the structures (1) group has a spin of 0.32, whereas the other iron atom has a spin of only 0.29. This small change in radical character between the two iron atoms, due to the electron-withdrawing/donating groups attached to the phthalocyanine ligands may be responsible for the preferred site of hydrogen peroxide binding. In particular, the larger radical character of the Fe(Pc1) group will favour the binding of an active oxidant over the Fe(Pc2MeSO) group.We initially optimized the geometry of doublet spin ructures show onl9\u2013OOH]\u2013 and reoptimized all geometries in the gas phase, see 1)FeIIINFeIV(Pc2MeSO)]\u2013, designated [9\u2013OOH]A\u2013, and [(Pc1)FeIVNFeIII(Pc2MeSO)OOH]\u2013 or [9\u2013OOH]B\u2013. Similarly to previous studies on iron(iii)\u2013hydroperoxo porphyrin complexes of mononuclear iron systems, the doublet spin state is the ground state in all cases.iv)\u2013nitrido group with an unpaired electron on the iron(iii)\u2013hydroperoxo group. Although the energy gap between the two structures is small, the most stable isomer is structure [9\u2013OOH]A\u2013 by 1.8 kcal mol\u20131 over [9\u2013OOH]B\u2013. Therefore, the iron(iii)\u2013hydroperoxo is preferentially bound to the site with electron-donating substituents attached to the phthalocyanine manifold. This is a surprising result as previous computational and experimental studies found very little effect, for instance, of meso-substitution of porphyrin rings on the electron affinity of the iron(iv)\u2013oxo group.9\u2013OOH]A\u2013versus [9\u2013OOH]B\u2013 confirms the experimental MS results described above that more fragmentation is found from the [9\u2013OOH]A\u2013 isomer.Subsequently, we added a hydroperoxo group to form [9\u2013OOH]A\u2013 and [9\u2013OOH]B\u2013 show some critical differences between the two structures. Thus, the iron(iii)\u2013hydroperoxo group has larger radical character on the metal when it has a ligand with electron-donating substituents (Pc1) than one with electron-withdrawing substituents (Pc2MeSO): \u03c1Fe1 = \u20130.80 for [9\u2013OOH]A\u2013 and \u03c1Fe1 = \u20130.75 for [9\u2013OOH]B\u2013. The same is true for the iron(iv)\u2013nitrido group that sees the spin density polarize more towards the iron with the Pc1 ligand rather than the Pc2MeSO ligand. Therefore, electron-withdrawing substituents on the phthalocyanine scaffold create metal centers with lesser radical character and as such should be less reactive.The group spin densities reported in \u20131, however, it is not ruled out that a mixture of isomers will be formed in the process dependent on temperature, pressure, solvent environment etc. Indeed, the mass spectra show the presence of both type A and type B complexes as major and minor species, respectively.In summary, the computational results implicate preferential binding on the iron center with electron-donating substituents by a few kcal moli.e. FeIIIFeIV.18 On the other hand, studies on related \u03bc-oxo/\u03bc-hydroxo diiron porphyrin complexes show that they often have non-equivalent iron sites, whereby their oxidation states are very sensitive to structural and environmental perturbations.3 and reference homoleptic 8 complexes are practically the same, which indicates little or no effect of desymmetrization of the diiron unit. However, the CID-MS/MS data on clearly indicate the preferential formation of an iron(iv)\u2013oxo species at the more electron-rich, i.e. unsubstituted, phthalocyanine unit (Pc1). Noteworthy, the same trend was observed with either heteroleptic dimer 3 or 7 and too was found to be independent on the nature of the oxygen donor, i.e. H2O2 or m-CPBA.High-valent iron complexes bearing iron\u2013oxygen PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O9] isomers, namely or A and or B. The structures of A and B were calculated with B3LYP and BP86. The B3LYP and BP86 optimized structures show dramatic differences in chemical structure but follow the same trends upon adding substituents to one of the phthalocyanine scaffolds. Firstly, the iron\u2013oxo bond is short with B3LYP scale\" fill=\"currentColor\" stroke=\"none\">O9]A and 1.652 \u00c5 for structure B), which implicates an iron(iv)\u2013oxo double bond. These values match computational and experimental measurements of mononuclear iron(iv)\u2013oxo complexes of porphyrin systems excellently.iv)\u2013oxo bond length drops by 0.005/0.004 \u00c5 at B3LYP/BP86 level of theory when electron-withdrawing substituents are added at a distance of more than 8 \u00c5 from the iron center in the equatorial plane. Consequently, a significant difference in iron\u2013oxo bond length is obtained between structures A and B, which leads to stability differences and may be the primary reason for the difference in stability between doublet A and B structures. A change of this magnitude was not observed previously in meso-substituted iron porphyrins.iv)\u2013oxo species.To find out, whether there are stability differences for \u03bc-nitrido diiron\u2013oxo complexes with heteroleptic ligand systems as well, we decided to do an additional DFT study into the two [1)FeNFe(Pc2MeSO)], we decided to calculate the bond dissociation energy (BDE) of the hydroperoxo unit in [9\u2013OOH]A\u2013 and [9\u2013OOH]B\u2013 based on eqn (1). The calculated values for both complexes are given in FeOOH into different contributions as explained below. Of course, the BDEFeOOH difference between [9\u2013OOH]A\u2013 and [9\u2013OOH]B\u2013 is the same as the stability difference reported in To establish the origin of the hydroperoxo binding to A\u2013 and [9\u2013OOH]B\u2013 gives the effect of the second Pc unit on the BDE values for eqn (1), EPc, namely through \u03c0\u2013\u03c0 stacking interactions. As can be seen, the size and shape of the second Pc group has a major impact on the BDEFeOOH value, even though this bond is at a distance of well over 4 \u00c5. Thus, the BDEFeOOH is strongly increased for the (Pc2MeSO)FeOOH system as compared to the (PcH)FeOOH system by more than 20 kcal mol\u20131.Subsequently, we removed the phthalocyanine group of the N PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019FeIV(Pc) unit altogether and replaced it by a point charge. Again a single point calculation was performed to determine the relative BDEFeOOH values using eqn (1). These values establish the quantum mechanical effect (EQM) of the axial ligand upon the Fe\u2013OOH binding energy. Thus, the axial N PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019FeIV(Pc2MeSO) group gives a quantum mechanical effect that is dominated by the presence of the Pc2MeSO interaction with the rest of the system and has little contribution from the FeIV PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N moiety. By contrast, the removal of the N PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019FeIV(PcH) group has a major effect on the BDEFeOOH value and the system is considerably stabilized. Clearly, the equatorial ligands have a major contribution towards the binding of hydroperoxo groups to an iron scaffold and strengthen its bonding. This further shows that electron-withdrawing and electron-donating substituents can be used to manipulate the binding site of (hydro)peroxo moieties, and affect their stability and ultimately their catalytic properties.In a final set of calculations, we removed the N2tBu)4]2N, (FePc)2N and [FePc(tBu)4]2N (8) in the oxidation of methane. The complexes were supported onto silica and the heterogeneous oxidation of methane was performed in water containing 75 mM H2SO4 at 60 \u00b0C 4]2N < (FePc)2N < [FePc(tBu)4]2N the values of total turnover numbers strongly increase: 32.1 \u2192 102.9 \u2192 223.4. These turnover numbers reflect the difference in O\u2013H bond strength of iron(iii)\u2013hydroxo complexes with different phthalocyanine groups. Thus, previously we showed that the rate constant of hydrogen atom abstraction reactions correlates linearly with the O\u2013H bond dissociation energy of the corresponding iron(iii)\u2013hydroxo complex formed.tBu)4]2N complex bearing donating alkyl substituents showed an excellent performance in the mild methane oxidation providing a very high turnover number of 223.With all the catalysts, methane was efficiently oxidized to formic acid with 88\u201397% selectivity. Methanol was detected by GC-MS in trace amounts and formaldehyde is readily oxidized under the reaction conditions. Interestingly, with growth of the electron-donating properties of phthalocyanine ligand along the series 2N,Solvents were dried and distilled according to published procedures.4) was stirred in acetic acid (50 mL) at 90 \u00b0C, and subsequently a 33% solution of hydrogen peroxide (23 mL) was added in 1 mL portions over 4 h. The resulting mixture was allowed to cool to room temperature and stirred overnight. The resulting white precipitate was collected by filtration, washed with water and crystallized from ethanol (30 mL) to give 5 as a white powder. Yield 79% (1.4 g). Mp 113\u2013114 \u00b0C. Anal. calcd for C20H28N2O4S2: C, 66.62; H, 7.83; N, 7.77; S, 17.79. Found: C, 67.20; H, 7.70; N, 7.93; S, 17.57. 1H NMR (500 MHz CDCl3): \u03b4 = 0.8 , 1.3\u20131.7 , 3.7 , 8.6 ppm. ESI-MS: m/z: 447.23 [M + Na]+; IR (KBr) \u03bd (cm\u20131): 3097, 2959, 2928, 2861, 2244, 1545, 1464, 1336, 1314, 1149, 1125, 919, 795, 728, 648, 570, 525, 512, 474.1,2-Dicyano-4,5-bis(hexylthio)benzene (n-hexylsulfonyl)benzene (5) was heated at 130 \u00b0C in a mixture of o-dichlorobenzene\u2013DMF (3\u2009:\u20091) under argon for 8 h in the presence of iron(ii) chloride (0.15 mmol). The solvent was then concentrated under reduced pressure and the resulting green solid was recovered with dichloromethane and washed with water. Phthalocyanine 6 was purified by chromatography on silica gel using a mixture of di-chloromethane\u2009:\u2009ethanol (100\u2009:\u20091) as the eluent. Yield: 23% (61 mg). ESI-MS: m/z calc. for C80H112FeN8O16S8, 1172.5; found 1753.3 [M + H]+. UV-vis (CHCl3): \u03bbmax (log\u2009\u03b5) = 670 (5.2), 354 (5.0) nm; IR (KBr) \u03bd (cm\u20131): 1310, 1100, 1070, 808, 740.A mixture of 1,2-dicyano-4,5-bis(1 (1 mmol), sodium azide (1.5 g) and alkylsulfonyl phthalocy-aninato-iron(ii) (0.1 mmol) were suspended in \u03b1-chloronaphthalene (20 mL) under argon. The mixture was heated for 24 h at 190 \u00b0C under intensive stirring. The solvent was removed under reduced pressure. The remaining blue solid was diluted in CH2Cl2 (100 mL), filtrated and concentrated to be loaded on a silica gel column first eluted with CH2Cl2 to remove impurities. Then, the desired heteroleptic dimer was collected using CH2Cl2\u2009:\u2009EtOH (10\u2009:\u20091) as the eluent. Evaporation of solvent afforded the pure heteroleptic dimer as a dark blue powder.FePcm/z calc. for C104H88Fe2N17O16S8, 1943.8; found, [M]+, dicharged species calc. for 971.2287; found 971.2332. UV-vis (CHCl3): \u03bbmax (log\u2009\u03b5) = 635 (4.5), 338 (4.3) nm. IR (KBr) \u03bd (cm\u20131): 929 scale\" fill=\"currentColor\" stroke=\"none\">N\u2013Fe).Yield: 64% (124 mg). ESI-HRMS: m/z calc. for C112H128Fe2N17O16S8, 2334.6186; found, 2334.6091 [M]+. UV-vis (CHCl3): \u03bbmax (log\u2009\u03b5) = 645 (4.3), 338 (4.1) nm. IR (KBr) \u03bd (cm\u20131): 935 scale\" fill=\"currentColor\" stroke=\"none\">N\u2013Fe).Yield: 52% (120 mg). ESI-HRMS: 2tBu)4]2N, (FePc)2N and [FePc(tBu)4]2N complexes were supported onto silica with loading of 20 \u03bcmol g\u20131 according to a published procedure.The [FePcFeIIINFeIII(Pc2MeSO)], whereby the adamantyl-sulfonyl groups were replaced by SO2CH3 as shown in iv)\u2013oxo group, namely scale\" fill=\"currentColor\" stroke=\"none\">O9A) and scale\" fill=\"currentColor\" stroke=\"none\">O9B). Furthermore, structures were generates with an iron(iii)\u2013hydroperoxo group, i.e. [(PcH)FeIIINFeIII(Pc2MeSO)OOH] (9\u2013OOHA) and [HOO(PcH)FeIIINFeIII(Pc2MeSO)] (9\u2013OOHB). All individual structures were calculated in the lowest energy doublet, quartet, and sextet spin states, but the doublet spin state is the ground state in all cases.Density functional theory (DFT) procedures were employed using previously calibrated and benchmarked methods\u03b6 type basis set on iron (LACV3P+) and 6-311+G* on the rest of the atoms: basis set BS2. Finally, solvent corrections were obtained from single point calculations in Jaguar using the polarized continuum model with a dielectric constant mimicking dichlorobenzene. However, none of these calculations changed the spin state ordering or site preference, see ESITo test the effect of the basis set on the spin state ordering and relative energies, we calculated single points with a triple-via a harmonic frequency calculation, which gave real vibrational frequencies only.These DFT methods have been extensively used in calculations of iron\u2013porphyrin complexes and were shown to reproduce experimental rate constants and spin state orderings in good agreement with experiment.Supplementary informationClick here for additional data file."} +{"text": "PBM data was replaced with SVG by xgml2pxml:000000000000000000000000000000000000111111111111000000000000111111111111000000000000000000000000000000000000Created by potrace 1.16, written by Peter Selinger 2001-2019C), imine (CN), methylenephosphine (CP), iminophosphine (NP), diazene (NN), diphosphene (PP) and cyclopropane (\u0394). The data were obtained from ab initio geometric optimization and frequency calculations at HF, B3LYP, MP2 and CCSD levels of theory on 6\u2013311++G basis set. Input structures were generated by shell scripts and run by Q-Chem quantum chemical package. The output files were processed to extract geometric and energetic information by Wolfram Mathematica.This article presents theoretical data on geometric and energetic features of halogenated compounds of ethene (C The data in this paper were generated and optimized in vacuum by C), imine (CN), methylenephosphine (CP), iminophosphine (NP), diazene (NN), diphosphene (PP) and cyclopropane (\u0394) where substitutions are via halogenation with all degrees of substitution from mono- to tetra-substitution. The total numbers of all possible compounds are as follows: 175 for CC were generated partly by using a Unix shell script previously described elsewhere . The ab package to optim package to extra"} +{"text": "Chiral C2- and C1-symmetric BINOL-derived bis(phosphoric acid) catalysts facilitated the enantioselective aza-Friedel\u2013Crafts reaction of 2-methoxyfuran with \u03b1-ketimino esters. C2- and C1-symmetric BINOL-derived bis(phosphoric acid) catalysts, which have OP scale\" fill=\"currentColor\" stroke=\"none\">O)(OH)2/OP scale\" fill=\"currentColor\" stroke=\"none\">O)(OH)(OR) moieties at the 2,2\u2032-positions, were developed and used for the enantioselective aza-Friedel\u2013Crafts reaction of 2-methoxyfuran with \u03b1-ketimino esters for the first time. The intramolecular conjugated double hydrogen bond network is a key to increasing the Br\u00f8nsted acidity and preventing deactivation of the catalysts. Highly functionalized \u03b1-amino acid derivatives with a chiral quaternary carbon center could be transformed into versatile optically active N- and O-heterocycles and an \u03b1-aryl-substituted serine.Chiral In such a BBA system, one hydrogen atom of an XH group might participate in an intramolecular hydrogen bond with the other XH group, which might be activated and thus used for activation of the substrate. Since 2003, when Rawal reported the first example of an intramolecular single hydrogen bonding network in chiral TADDOLs 2. In sharp contrast, we envisioned that an intramolecular double hydrogen bond network may represent a new strategy for the design of chiral Br\u00f8nsted acid catalysts. However, a simple and closed double hydrogen bond network, as seen in a dimeric structure of two molecules of carboxylic acids, would lose both the Br\u00f8nsted acid- and base-functions upon neutralization. Therefore, here we developed chiral C2-symmetric BINOL-derived bis(phosphoric acid) catalysts (R)-5 as bis(diprotic acid)s R*(XH2)2, which have two OP scale\" fill=\"currentColor\" stroke=\"none\">O)(OH)2 moieties at the 2,2\u2032-positions of the chiral binaphthyl backbone (R)-5, chiral C1-symmetric catalysts (R)-10, which have OP scale\" fill=\"currentColor\" stroke=\"none\">O)(OH)2/OP scale\" fill=\"currentColor\" stroke=\"none\">O)(OH)(Oi-Pr) moieties, were also developed. Remarkably, outside of the conjugated intramolecular double hydrogen bond network, the Br\u00f8nsted acid moiety would still exist and work as an active center by the BBA methodology.The hydrogen bond network of chiral multiprotic acid catalysts plays an important role in activating Br\u00f8nsted acidity, controlling conformational flexibility, and producing high enantioselectivity.backbone . Based o2 (1a1a and particularly 2 under acidic conditions (entries 4\u20136). Fortunately, phosphoric acids could be used without the serious generation of byproducts due to their suitable Br\u00f8nsted acidity for this reaction, although the yields of product 3a were moderate (entries 7 and 8). Next, we examined conventional chiral phosphoric acid (R)-4a (entry 9), which would be less aggregatable than the less bulky achiral phosphoric acids in entries 7 and 8. As a result, although the pKa of (R)-4a would be similar to those of the achiral phosphoric acids in entries 7 and 8, the catalytic activity was greatly improved (see the ESIR)-4b with electron-withdrawing CF3 groups in its 3,3\u2032-diaryl moieties was used, the reaction was accelerated, although the enantioselectivity was still low (40% ee) (entry 10). Moreover, well-acknowledged bulky (R)-4c was much less active than (R)-4a and (R)-4b (entry 11). In contrast, chiral C2-symmetric bis(phosphoric acid) (R)-5aR)-4a\u2013c, and 3a was obtained in 82% yield with 70% ee within 5 h (entry 12).R)-5b derived from (R)-3,3\u2032-2C6H3)2-BINOL improved the enantioselectivity (76% ee) of 3a (entry 13). (R)-5c with a 5,5\u2032,6,6\u2032,7,7\u2032,8,8\u2032-H8-binaphthyl backbone showed lower catalytic activity than (R)-5a and (R)-5b, and a prolonged reaction time (24 h) was needed (entry 14). Moreover, we optimized \u03b2,\u03b3-alkynyl-\u03b1-imino esters 1 with the use of (R)-5b -3b was obtained in 83% yield with 91% ee (eqn (1)). Remarkably, we could perform a 1.89 g-scale synthesis of (S)-3b (77% yield with 91% ee), and 99% of (R)-5b could be recovered.We initially examined the aza-Friedel\u2013Crafts (FC) reaction of 2-methoxyfuran 2 with \u03b2,\u03b32 1a. As a reR)-5c\u00b7(pyridine)2 was crystallized, and the results of an X-ray analysis are shown in PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O)(OH)2 moieties at the 2,2\u2032-positions of the H8-binaphthyl backbone coordinate with each other, and the conjugated double hydrogen bond network is unambiguously formed at the center of the monomeric molecules. Pyridines are coordinated BBA-activated protons, which would be considered to activate the substrate in a similar way. In this regard, the role of the two outside protons was next examined with the use of monomethyl-protected (R)-6a and dimethyl-protected (R)-6b (R)-6a showed almost the same catalytic activity (81% yield and 91% ee of 3b) as (R)-5b, whereas (R)-6b gave a poor result (22% yield and 34% ee of 3b) in the reaction of 2 with 1b. This result strongly suggests that two activated protons in (R)-5b should be independent of each other, although the absence of both protons results in a loss of catalytic activity. In contrast, the protons in the tight structure of a double hydrogen bond network might not be directly involved in promoting the reaction as possible Br\u00f8nsted acids.We now turn our attention to mechanistic aspects. It is important to identify the intramolecular double hydrogen bond network in the catalysts. Fortunately, (d (R)-6b . As a re2 with 1a with the use of (R)-5b or (R)-4a (R)-5c\u00b7(pyridine)2, a linear relationship was observed for (R)-5b, and the yields were almost constant (71\u201375%) (R)-4a (R)-4a unlike (R)-5b. Indeed, although both (R)-5b and (R)-4a have almost the same sterically hindered 3,3\u2032-diaryl moieties, the strongly Br\u00f8nsted basic P PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O moiety is still free in (R)-4a. It is quite unlike the situation in (R)-5b, which has a core hydrogen bond network through the \u201cintramolecular dimerization\u201d of two P scale\" fill=\"currentColor\" stroke=\"none\">O)(OH)2 moieties. Thus, the much better catalytic activity of (R)-5b compared to (R)-4a might be attributed to not only the stronger acidity of (R)-5b due to a BBA system but also the monomeric active species of (R)-5b due to the closed P PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O moieties.20Moreover, a non-linear effect was examined in the reaction of r (R)-4a . As expe(71\u201375%) . In cont) (R)-4a .18 This 1a and 1b. Due to their synthetic importance, we chose aryl \u03b1-ketimino esters, such as 7a (R)-5b was not effective for the reaction of less reactive 7a, unlike more reactive 1a and 1b, and (R)-8a was barely obtained with 15% ee at slightly higher temperature (\u201360 \u00b0C) (entry 3). To improve the enantioselectivity, stereocontrol by more bulky substituents at the 3,3\u2032-positions of the C2-symmetric catalysts (R)-5 should be needed. However, we could not introduce phosphoric acid moieties at the 2,2\u2032-positions of the bulky 3,3\u2032-Ar2-BINOL-skeleton . Instead, an extremely bulky C1-symmetric catalyst (R)-9c with 2,4,6-Cy3C6H2 at the 3-position could be readily prepared as well as (R)-9a and (R)-9b . However, since two different active Br\u00f8nsted acid centers would compete, the enantioselectivity was low (11\u201318% ee), as expected (entries 4\u20136). Based on the above consideration of C2-symmetric catalysts (R)-5, we designed the site-selectively mono-i-Pr-capped catalysts (R)-10.R)-10c with extremely bulky 2,4,6-Cy3C6H2 at the 3-position, the reaction proceeded smoothly, and (R)-8a was obtained in 93% yield with 95% ee (entry 9). The bulkiness of the aryl moiety at the 3-position is important, since slightly less hindered (R)-10b with 2,4,6-i-Pr3C6H2 was less effective (entry 8), and much less hindered (R)-10a with 3,5-Ph2C6H3 was entirely ineffective (entry 7). Conventional chiral phosphoric acids, such as (R)-4b and (R)-4c, were not effective . When we investigated simple o-, m-, and p-tolyl-substituted substrates (7i\u2013k), p-tolyl 8i and m-tolyl 8j were obtained in high yields with high enantioselectivities , whereas o-tolyl 8k could not be obtained probably due to steric hindrance. Indeed, less sterically hindered o-F-C6H48c and 1-naphthyl 8m were obtained with high enantioselectivities . 2-Naphthyl 8l and heteroaryl 8n were also obtained successfully . Unfortunately, however, a low-reactive aliphatic substrate 7o could not be used, and no reaction proceeded. Moreover, instead of 2-methoxyfuran 2, 2-ethoxyfuran8p was obtained in 94% yield with 94% ee.With the optimized catalyst in hand, we next examined the scope of aryl \u03b1-ketimino esters 7 . As a reC2-symmetric (R)-5b and C1-symmetric (R)-10c. As seen above, (R)-5b-catalysis of 1b and 2 provided (S)-3b, and (R)-10c-catalysis of 7a and 2 provided (R)-8a. Therefore, the observed absolute stereochemistries in 5b and 8a were opposite each other. This changeover might be caused by the geometry of \u03b1-ketimino esters (E)-1bZ)-7aS)-3b was obtained in 14% yield with 6% ee by using (R)-10c (eqn (2)), whereas (R)-8a was obtained in 66% yield with 21% ee by using (R)-5b (eqn (3)). Overall, chiral C2- and C1-symmetric bis(phosphoric acid) catalysts were complementary, and either catalyst that was suitable for one reaction would not be suitable for the other reaction from the viewpoint of yield and enantioselectivity. Although preliminary possible transition states are considered as a working model to give 11 quantitatively. Next, 11 was reduced under typical reaction conditions. Wilkinson's catalyst completely reduced the acetylene moiety of 11, and 12 was obtained quantitatively. Lindlar's catalyst facilitated the selective hydrogenation of acetylene at 0 \u00b0C to give vinyl compound 13 in 94% yield, whereas both acetylene and the Cbz moieties of 11 were reduced at room temperature and 14 was obtained quantitatively. Furthermore, the NH2 moiety of 14 was protected by di-tert-butyl dicarbonate (Boc2O) in 83% yield, and the corresponding product was consequently treated with N-bromosuccinimide (NBS) to give 1,4-dicarbonyl compound 15 in 68% yield via furan cleavage. Finally, chemoselective reduction of the keto moiety of 15 with the use of CeCl3/NaBH4 gave \u03b3-butenolide 16 in 84% yield with a diastereomeric ratio of 80\u2009:\u200920.12Since optically active l groups . First, 11 to some optically active N- and O-heterocycles. Sonogashira coupling of 11 with 2-iodophenol proceeded in the presence of Pd(PPh3)4/CuI catalysts, and novel 2-substituted benzofuran 17, which has a chiral quaternary carbon center with substitutions of furan, ester, and carbonate moieties, was obtained in 36% yield . Moreover, a Diels\u2013Alder reaction of the furan moiety of 18a with benzyne gave the corresponding adduct, which, without purification, was treated with HCl/acetic acid to give 19a in 98% yield with a catalyst loading as low as 0.2 mol%. The obtained 8e was then reduced by NaBH4, and compound 20 was obtained in 84% yield. Compound 20 was highly crystalline, and a single recrystallization increased the enantiopurity up to >99% ee. Finally, the 2-methoxyfuran moiety of 20 was oxidized by RuCl3-catalyzed NaIO4-oxidation,21 (0.89 g) was obtained in 89% yield.Since optically active \u03b1-aryl-substituted serines are synthetically useful,8e . Before C2- and C1-symmetric bis(phosphoric acid) catalysts. The conjugated double hydrogen bond network was key to increasing the Br\u00f8nsted acidity and preventing dimerization/deactivation of the catalysts. In particular, we developed a highly enantioselective aza-Friedel\u2013Crafts reaction of 2-methoxyfuran with \u03b1-ketimino esters for the first time. By taking advantage of the highly functionalized products, some transformations to versatile N- and O-heterocycles and \u03b1-aryl-substituted serine with a chiral quaternary carbon center could be achieved. The further application of these catalysts in other asymmetric catalyses is underway.In summary, we have developed chiral BINOL-derived There are no conflicts to declare.Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "Allene reacts with the strongly electrophilic halogenoboranes XB(C6F5)2 (X: Cl or Br) by forming a mixture of 1,3,5-trimethylenecyclohexane and the stoichiometric halogeno-borated tetramerization products. 6F5)2 react with allene to give a mixture of the cyclotrimer 1,3,5-trimethylenecyclohexane (1) and the related halogenoborylated cyclotetramerization products 3a and 3b. Alkyl-substituted allenes were catalytically cyclotrimerized metal-free by XB(C6F5)2 to give cis,trans-2,4,6-trialkyl-1,3,5-trimethylenecyclohexanes under mild conditions.The halogenoboranes XB(C The resulting products contained a halide substituent at one end of the oligoacetylene chain and the B(C6F5)2 functional group at the other.2a and b. We have performed these reactions and here report about their surprising outcome.Allenes serve as important organic building blocks.6F5)2 (2a) in d8-toluene in a Young NMR tube at room temperature. After 7 h reaction time we monitored the NMR features of a mixture that contained the products 1,3,5-trimethylenecyclohexane (1) and the chloroborylated allene tetramer 3a in a ca. 1\u2009:\u20091 ratio (both present in ca. 8 mol% in the mixture) plus unreacted 2a (ca. 10 mol%) and allene (ca. 75 mol%). After 24 h the amount of the pair of allene cyclooligomerization products had almost doubled and there remained only ca. 3 mol% of the chloroborane 2a. That was almost completely consumed after 48 h reaction time at room temperature. The products 1 and 3a were identified spectroscopically from the mixture . Compound 3a shows the typical olefinic exo-methylene pairs of 1H NMR resonances at \u03b4 5.08, 4.84 scale\" fill=\"currentColor\" stroke=\"none\">) and \u03b4 4.63, 4.48 scale\" fill=\"currentColor\" stroke=\"none\">) as well as the AX-spin system of the diastereotopic 3,7-CH2 pairs and the AB pattern of the 5-CH2 at \u03b4 2.58, 2.54. The 1-CH2 group at boron and the 8-CH2 unit give rise to 1H NMR signals at \u03b4 2.18 and \u03b4 2.26, respectively 2 (2b) was carried out analogously. The reaction was directly followed by NMR spectroscopy. It resulted in the formation of the products 1 and 3b in a ca. 1\u2009:\u20092 ratio . Compound 3b was characterized from the mixture by NMR spectroscopy. It shows similar spectra as its chloro-substituted analogue 3a .We prepared compound 3% yield . It was in situ prepared 3b with a slight excess of pyridine gave the adduct 4b, which we isolated in 72% yield as a white solid on a 100 mg scale. The X-ray crystal structure analysis scale\" fill=\"currentColor\" stroke=\"none\">C double bonds annulated at carbon atoms C4 and C6 and it has the CH2\u2013CBr PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CH2 unit, derived from the fourth connected allene unit, attached at the ring carbon atom C2. This carbon atom also bears the CH2\u2013B(C6F5)2 substituent, which has the pyridine donor added to its boron atom. The central six-membered carbocycle of compound 4b features a distorted chair-like ring conformation.Treatment of analysis showed t2Cl2) compound 4b shows the 1H NMR PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CH2 signals of the symmetry equivalent pair of exo-methylene groups at the ring carbons C4 and C6 and the pair of signals of the C9 PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CH2 moiety. The C3/C7 CH2 hydrogen atoms are pairwise diastereotopic and the C8 and C1 CH2 groups both show 1H NMR singlets. Compound 4b shows a 11B NMR resonance in the typical tetra-coordinated borane range at \u03b4 \u20131.4 .In solution 2 elimination provides an attractive pathway to the observed product 1,3,5-trimethylenecyclohexane (1). This would in principle constitute a cyclotrimerization of allene catalysed by the XB(C6F5)2 reagents. However, the intramolecular allylboration of 8, giving the other experimentally observed products 3, represents a competing stoichiometric reaction branch that eventually removes the XB(C6F5)2 reagent from the system they react by ring closure12 . Workup with pentane and dichloromethane in this case gave a ca. 79\u2009:\u200921 mixture of compound 12 (characterization see below) and the [tBu3PH+]Cl\u2013 phosphonium salt 11a .Compounds wis base they realosure12 . As a ty3b and tBu3P was carried out similarly . Workup in this case gave the pure compound 12 as a white solid, which we isolated in 65% yield. It was characterized by C, H, N elemental analysis, by spectroscopy and by X-ray diffraction . The X-ray crystal structure analysis shows the newly formed borataspiroundecene framework (tBu3P-substituent attached at the C(9) PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C(10) carbon\u2013carbon double bond. The adjacent six-membered ring shows carbon atoms C4 and C6 which serve both as the ring sp2-carbons of the pair of exo-methylene C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CH2 groups.The reaction between the more reactive borane ramework which wa2Cl2) compound 12 shows the typical 1H NMR doublet of the tBu3P-substituent. It features the PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CH signals of the newly formed internal olefinic moiety at \u03b4 8.21 . The BCH2 group shows up as a 1H NMR signal at \u03b4 1.20 and the C(8)H2 methylene group as a doublet at \u03b4 2.06 (3JPH = 4.5 Hz). The pair of olefinic exomethylene groups of the adjacent carbocyclic six-membered ring shows typical 1H NMR signals at \u03b4 4.61/4.41 and the resonances of the pairwise diastereotopic methylene hydrogen atoms at the C3/C7 pair and at C5 , which we prepared according to a procedure reported by Ma et al.6F5)2 (2a) in d8-toluene solution at 60 \u00b0C. Workup after 48 h reaction time involving purification by chromatography gave the cyclotrimer 14c as the major product scale\" fill=\"currentColor\" stroke=\"none\"> unit shows one 1H NMR resonance, whereas the 3,5-H2C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019 moieties showed two due to their stereochemically unsymmetrical environment (see the ESI6F5)2 borane is an equally efficient metal-free cyclotrimerization catalyst for the n-octylallene 13c. The catalytic reaction was carried out under analogous conditions and gave the product 14c in 46% yield after workup (some minor byproduct was isolated (ca. 20%) but not positively identified as yet). We performed the XB(C6F5)2 catalysed alkylallene cyclotrimerization reaction for a second example: the reaction of n-dodecylallene (13d) with either of the Cl/BrB(C6F5)2 borane gave the tri-substituted cyclotrimer 14d with cis,trans-attachment of the long chained alkyl groups as the major product. We isolated it as a colorless oil in 40% yield from the ClB(C6F5)2 catalysed reaction [54% with BrB(C6F5)2] (see the ESI14d).We treated product . Compoun6F5)2 serves as an efficient metal-free catalyst for the cyclotrimerization of allene to 1,3,5-trimethylenecyclohexane and of cyclohexylallene to cis,trans-2,4,6-tricyclohexyl-1,3,5-trimethylenecyclohexane.6F5)2 in principle can induce the same catalytic reaction. Both these strongly electrophilic halogeno-boranes serve as catalysts for the cyclotrimerization of long-chain n-alkylallenes 13c,d to give the respective cyclotrimers 14c,d as the major products. Ring-closure and elimination is sufficiently effective to close the catalytic cycle with liberation of the respective XB(C6F5)2 catalyst 2 catalyzed allene cyclotrimerization opens attractive pathways to the synthesis of interesting highly substituted arene products.We have started to use the allene cyclotrimerization products sitylene . We alsoThere are no conflicts to declare.Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "Blue-yellow fluorescence\u2013phosphorescence dual emission from single-component white emissive W-CNQDs with a high PLQE of 25% is reported for the first time. Developing efficient single-component white light-emitting diodes (WLEDs) is extremely challenging due to the issue of Kasha's rule. Here we report the first demonstration of blue-yellow fluorescence\u2013phosphorescence dual emission from our newly minted single-component white emissive carbon nitride quantum dots (W-CNQDs). The W-CNQDs deliver an overall photoluminescence quantum efficiency of 25%, which is the highest value among white-emitting materials reported to date, based on utilizing both singlet and triplet states. Experimental and theoretical investigations reveal that the carbonyl groups at the rim of the W-CNQDs play a key role in promoting intersystem crossing and inducing intermolecular electronic coupling, affording intensive yellow phosphorescence. Efficient white emission is achieved with a phosphorescence quantum efficiency of 6% under ambient conditions. A WLED is fabricated by integrating W-CNQD phosphors into a UV-LED chip, which shows favorable white light characteristics with CIE coordinates and a CRI of and 85, respectively, demonstrating good color chromatic stability. This work opens up new opportunities for exploring dual emission mechanisms and designs to facilitate the development of efficient single-component WLEDs. Urea, which is widely used for preparing traditional g-C3N4, was selected as the precursor for synthesizing W-CNQDs.9The emerging carbon quantum dots (CQDs) have recently been demonstrated to be a superior fluorescence\u2013phosphorescence dual emitter for realizing efficient single-component WLEDs.3N4 are comparatively illustrated in 2) at the rim of the W-CNQDs under these mild reaction conditions, which plays a key role in promoting intersystem crossing (ISC) and inducing intermolecular electronic coupling, affording intense yellow phosphorescence.3N4 can be found in ESI .3N4 with its negligible oxygen content scale\" fill=\"currentColor\" stroke=\"none\">N (285.2 eV) and C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O (288 eV) bonds in the W-CNQDs scale\" fill=\"currentColor\" stroke=\"none\">C (399.5 eV) scale\" fill=\"currentColor\" stroke=\"none\">O scale\" fill=\"currentColor\" stroke=\"none\">C, and C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O, respectively. Furthermore, solid-state 13C-NMR spectra of the W-CNQDs further confirm that the carbonyl groups from the precursor were successfully produced at the rim of the W-CNQD networks. The clearly observable peak located at 210 ppm is indicative of the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O bond, which fundamentally differs from the negligible resonance signals in the range of 160\u2013220 ppm of the previously reported g-C3N4 scale\" fill=\"currentColor\" stroke=\"none\">C bonds, while no signals from saturated sp3 carbon atoms were observed pattern, demonstrating the typical feature of CNQDs with two peaks resulting from the graphite structure and tri-t Fig. S4, as prevt Fig. S4. The XPSt Fig. S4 reveal t W-CNQDs .12 The N99.5 eV) . The O 1/svg>O .12 The p/svg>O .12 The W4 Fig. S5. And theobserved .13 The a2 \u03c0-conjugation domains and C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O/C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N bonds contained in the W-CNQDs, respectively of W-CNQDs are comparable to their corresponding radiative decay rates (kF = 6.6 \u00d7 107), indicating an efficient ISC process to produce sufficient triplet excitons responsible for the efficient phosphorescence with a \u03a6P of 6% under ambient conditions, which is indeed the highest reported so far, thus enabling efficient white emission. In addition, typically small singlet\u2013triplet energy gaps (\u0394EST < 0.4 eV) are required to facilitate the ISC process in phosphorescence emitters;EST as 0.30 eV, which is far less than that of traditional g-C3N4 (\u0394EST = 0.46 eV) lamp irradiation and yellectively . Fig. 3dectively . The delectively , which r Table S2. The ISC1.000000,.000000 s3N4 for comparison were performed to confirm the mechanism above , which is smaller than those of traditional g-C3N4 , which potentially enables effective ISC from S1 to T1 states of the W-CNQDs based on theoretical calculation is close to the experimental value (\u0394EST = 0.30 eV). Furthermore, for the W-CNQDs, the numbers of energy transition channels (five channels) increased in comparison to traditional g-C3N4 (three channels) , also resulting in enhanced ISC (E1S = 3.4271 eV and E1T = 3.1071 eV) compared with those of traditional g-C3N4 , also enhance the yellow phosphorescence according to the energy gap law. In addition, we analyzed the characters of the excited states using the natural transition orbitals (NTOs)3N4 and increased numbers of energy transition channels (seven channels) in contrast to the isolated W-CNQDs above (five channels), resulting in enhanced ISC 3N4 . It is w T4, T5) , suggest Fig. S11. Indeed,The highly efficient blue-yellow fluorescence\u2013phosphorescence dual emission of W-CNQDs, coupled with their low cost and environmental-friendliness are compelling for their application in high performance single-component WLEDs. Due to the poor solubility of W-CNQDs, a UV-pumped WLED was fabricated for the first time by directly coating W-CNQD phosphor with cyanoacrylate (Super Glue) onto the surface of a commercial 380 nm UV-LED . Efficievia dual emission strategies. We anticipate that further improving the \u03a6P value of yellow phosphorescence emissive CNQDs and red-shifting their emission will lead to greatly improved performance for carbon-based phosphor single-component WLEDs; this research is underway in our laboratory and will be reported in due course.In summary, we report the first successful demonstration of blue-yellow fluorescence\u2013phosphorescence dual emission for single component white light emission with an overall PLQE as high as 25% and a relatively high yellow phosphorescence quantum efficiency of 6% under ambient conditions based on W-CNQDs. The key role of the carbonyl groups at the rim of the large \u03c0-conjugated structure in W-CNQDs has been uncovered, assisting the ISC arising from carbonyl (n\u03c0*) mediated intermolecular (\u03c0\u03c0*) electronic coupling. A WLED was fabricated by integrating W-CNQD phosphors in a UV-LED chip, which showed favorable white light characteristics with CIE coordinates and a CRI of and 85, respectively. This work opens up new opportunities for designing single-component WLEDs There are no conflicts to declare.Supplementary informationClick here for additional data file."} +{"text": "The twisting of an amide bond provides a new driving force for living ring-opening metathesis polymerization through resonance destabilization. \u20131 of the overall 12.0 kcal mol\u20131 ring strain. The twisted amide polymerization is capable of preparing high molecular weight polymers rapidly at room temperature, and post-polymerization modification combined with 2D NMR spectroscopy confirms a regioirregular polymer microstructure.The living ring-opening metathesis polymerization (ROMP) of an unsaturated twisted amide using the third-generation Grubbs initiator is described. Unlike prior examples of ROMP monomers that rely on angular or steric strain for propagation, this system is driven by resonance destabilization of the amide that arises from geometric constraints of the bicyclic framework. Upon ring-opening, the amide can rotate and rehybridize to give a stabilized and planar conjugated system that promotes living propagation. The absence of other strain elements in the twisted amide is supported by the inability of a carbon analogue of the monomer to polymerize and computational studies that find resonance destabilization accounts for 11.3 kcal mol Ring-strain is a common driving force for chemical reactions in both small molecule and macromolecular synthesis. This is the origin of remarkable carbon\u2013carbon bond cleavage mechanisms of cyclopropane rings,N-to-\u03c0* PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019OC orbital overlap, twisting hinders this interaction and results in nominal amides with unusual reactivity profiles. Hydrolytic stability and nitrogen basicity of these amides has been directly correlated to the degree of distortion, defined by the twist angle (\u03c4) and nitrogen pyramidalization (\u03c7N) parameters introduced by Winkler and Dunitz,12Twisted amides constitute an unusual class of molecules that have distorted, nonplanar amide N\u2013C(O) bonds as a result of geometric, steric, or electronic effects.1 was explored in HaRP and only resulted in oligomers with broad molecular weight distributions .1 to polymerize under ROMP conditions, the Winkler\u2013Dunitz parameters significantly deviate from planarity.N-to-\u03c0* PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019OC overlap causes the twisted amide to be higher in energy than a planar amide, and it was proposed that 1 would be competent in ROMP through the gain of resonance energy after ring-opening and planarization of the nitrogen atom to an sp2 hybrid (1 using the 3rd generation Grubbs initiator (G3) .1 was prepared following the two-step protocol described by Grigg starting from 2-iodobenzoic acid and tetrahydropyridine.1 was performed with G3 in DCM at room temperature targeting a degree of polymerization (DP) of 100 (ROMPP1 with a number-average molecular weight (Mn) of 15.2 kg mol\u20131 and dispersity (\u00d0) of 1.20. By varying the monomer-initiator ratio, different molecular weights (DP = 10 to 500) were accurately targeted with high monomer conversions and unimodal elution peaks by size-exclusion chromatography (SEC) analysis (\u00d0 = 1.26 and 1.46) implying some secondary metathesis of the backbone olefins occurred, which was supported by experiments at extended reaction times after full conversion was reached is several orders of magnitude lower than the norbornene derivatives commonly used in ROMP (e.g. 63.2 M\u20131 s\u20131 at 25 \u00b0C for exo-butyl norbornene imide).H\u2021 = 84.0 kJ mol\u20131, and entropy of activation \u0394S\u2021 = \u201330.8 J mol\u20131 K\u20131.in situ NMR studies were unable to conclusively demonstrate the coordination state of the chain-end of 100 , entry 4analysis . Higher analysis . A singlehaviour and a liehaviour . To bett2 in which the bridgehead nitrogen of twisted amide 1 is replaced with a methine carbon , no reaction was observed after three hours at room temperature that is consistent with inability of 2 to polymerize. To separate the contribution of resonance destabilization from the overall ring strain in 1, the carbonyl substitution nitrogen atom replacement (COSNAR) method developed by Greenberg was applied.1 and ring-opened amide 3 were calculated to be 8.7 kcal mol\u20131 and 20.0 kcal mol\u20131, respectively . This indicates that resonance destabilization is responsible for over 90% of the ring strain in 1 , further supporting gain of resonance energy as the primary driving force for polymerization.Computational studies were performed at the B3LYP-D3MBJ/6-311++G level of theory to gain more insight into the differences in ring strain between monomers ethylene .21 Analoectively . The 11.1 through HaRP and ROMP should give the same polymer structure. While only oligomers formed under HaRP conditions, slight differences were noted in the 1H NMR spectra of HaRPP1 and ROMPP1 , head-to-head (HH) or tail-to-tail (TT) connectivity , unique signals at 7.0 ppm and 2.7 ppm for 2-P1ROMPH (red highlight), as well as clear differences in the 3.1\u20133.9 ppm and 7.3\u20137.5 ppm regions (ROMPP3 (blue & red) correlated to head-to-tail and tail-to-tail connectivity at the benzylic position, implying indiscriminate ring-opening of the monomer from either side of the olefin showed slightly lower thermal stability of Fig. S19, indicat1 has been described that lacks the angular and steric strain elements traditionally found in ROMP monomers. In contrast, this system leverages resonance destabilization of an amide bond to promote living polymerization to high molecular weights. This is supported by the inability of the bicyclic ketone analogue 2 to polymerize under standard ROMP conditions and computational experiments that highlight the central role of resonance destabilization in the overall monomer ring strain. The microstructure of the resultant polymer ROMPP1 was determined to be regioirregular through hydrogenation experiments coupled to 2D NMR analysis. The monomer orientation had a minimal effect on the glass transition temperature of the polymer but was found to lower overall thermal stability. Future work is underway to design new unsaturated twisted amide structures for ROMP that are promoted by resonance destabilization, as well as exploring applications of this new materials class.The ring-opening metathesis polymerization of the unsaturated twisted amide monomer There are no conflicts to declare.Supplementary informationClick here for additional data file."} +{"text": "Acyl(chloro)phosphines RC(O)P(Cl)(t-Bu) have been prepared by formal insertion of tert-butyl phosphinidene (t-Bu\u2013P) from t-BuPA (A = C14H10 or anthracene) into the C\u2013Cl bond of acyl chlorides. t-Bu) have been prepared by formal insertion of tert-butyl phosphinidene (t-Bu\u2013P) from t-BuPA (A = C14H10 or anthracene) into the C\u2013Cl bond of acyl chlorides. We show that the under-explored acyl(chloro)phosphine functional group provides an efficient method to prepare bis(acyl)phosphines, which are important precursors to compounds used industrially as radical polymerization initiators. Experimental and computational investigations into the mechanism of formation of acyl(chloro)phosphines by our synthetic method reveal a pathway in which chloride attacks a phosphonium intermediate and leads to the reductive loss of anthracene from the phosphorus center in a P(v) to P(iii) process. The synthetic applicability of the acyl(chloro)phosphine functional group has been demonstrated by reduction to an acylphosphide anion, which can in turn be treated with an acyl chloride to furnish dissymmetric bis(acyl)phosphines.Acyl(chloro)phosphines RC(O)P(Cl)( Collectively, these data support the formulation of 1 as (tert-butylchlorophosphanyl)(phenyl)methanone, PCl(t-Bu)(C(O)Ph).Treatment of at 23 \u00b0C led to a1 could not be isolated as single crystals suitable for structural analysis, a transition metal complex of this phosphine was prepared. Treatment of 1 with half an equivalent of [Ru(p-cymene)Cl2]2 in tetrahydrofuran led to the formation of coordination complex 2. Layering the reaction mixture with pentane at 23 \u00b0C gave 2 as a red-orange crystalline material in 77% yield. Multinuclear NMR spectroscopy studies, in addition to a single crystal X-ray diffraction study confirmed the formulation of 2 as Ru(p-cymene)Cl2(PCl(t-Bu)(C(O)Ph)) , 1.875(4) and 2.3178(8) \u00c5 respectively, which are in line with the expected values.iii) ligands.As C(O)Ph)) . RacemicA compound is not \u03c0-donating.via nucleophilic attack by phosphorus at the electrophilic carbonyl group of benzoyl chloride. We envisioned that replacement of the chloride for a less nucleophilic anion may lead to the isolation of the putative cationic acylphosphonium intermediate. To this end, the reaction of equimolar solutions of t-BuPA and benzoyl triflate (PhC(O)OTf, OTf = CF3SO3\u2013)3[OTf] scale\" fill=\"currentColor\" stroke=\"none\">O absorption peak at 1636 cm\u20131. These data are consistent with the formulation of 3[OTf] as [AP(t-Bu)(C(O)Ph)][OTf].Previous studies on thermal phosphinidene transfer have established that thermal loss of a phosphinidene does not occur when the R-group of an RP\u2013)3[OTf] as a whi3[OTf] decomposes slowly in solution at ambient temperature, yielding an anthracenyl(acyl)hydridophosphonium triflate salt as the major product in chloroform at 23 \u00b0C quantitatively generated 1, [TBA][OTf] and anthracene product has previously been described in the literature.I2, see ESI3 and is the formal reverse of the McCormack reaction.I2 to be 65.0 kcal mol\u20131 which is significantly higher than either TS1 of TS2, ostensibly ruling it out as an intermediate in the reaction mechanism as determined by DFT calculations.The higher Gibbs free energy of the first transition state compared to the second is consistent with experiment, where a phosphonium intermediate is not observed when benzoyl chloride is used as the acylating reagent. Thus it is indicated that, when switching from chloride to triflate, there is a change in the rate determining step from 1 were undertaken by reduction of the P\u2013Cl bond. Treatment of compound 1 with 2 equivalents of freshly-prepared sodium naphthalenide4 as a crystalline orange powder in 49% yield. The connectivity of anion 4 as [OC(Ph)Pt-Bu]\u2013 was confirmed by a single crystal X-ray diffraction experiment scale\" fill=\"currentColor\" stroke=\"none\">O) orbital.Efforts to further derivatise periment . In the 4a, 4b, To quantify the degree of electron delocalization over the acylphosphide functional group, the electronic structure was evaluated using natural bond orbital (NBO) methods.4 and AdC(O)Cl (Ad = 1-adamantyl) in tetrahydrofuran generated 5, which was isolated as a crystalline yellow solid in 55% yield and 214 ppm (JPC = 46 Hz), which are assigned to the two acyl carbon atoms. The IR spectrum of 5 shows two C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O absorption peaks at 1665 and 1629 cm\u20131. These spectroscopic data inform our formulation of 5 as OC(Ph)P(C(O)Ad)t-Bu. Single crystals of 5 were grown from a concentrated pentane solution and analyzed by an X-ray diffraction study, which featured 5 as a single (R)-enantiomer (see ESIP21 (The combination of [Na(15-crown-5)]5% yield . In the r see ESI in the ce ESIP21 .5 from [Na(15-crown-5)]4 provides an approach for preparing dissymmetric bis(acyl)phosphines.5) P-protected acylphosphide anion describes C\u2013O instead of C\u2013P bond formation upon treatment with an acyl chloride, to give an ester-containing phosphaalkene.57The formation of t-BuPA with acyl chlorides to give acyl(chloro)phosphine-containing products, which were not accessible previously.In summary, we have described a novel reaction of There are no conflicts to declare.Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "Reaction of [3-N2-o-C2B10H11][BF4] with various kinds of nucleophiles gives a very broad spectrum of cage B(3)-substituted o-carborane derivatives, 3-X-o-C2B10H11 2, etc.), and serves as a simple and efficient method for multiple functionalization of o-carborane. o-carborane is described. Reaction of [3-N2-o-C2B10H11][BF4] with various kinds of nucleophiles gave a very broad spectrum of cage B(3)-substituted o-carborane derivatives, 3-X-o-C2B10H11 2, etc). This reaction may serve as another efficient [18F]-radiolabeling method of carborane clusters for positron emission tomography applications.A simple and efficient method for selective cage B(3) multiple functionalization of Carboranes, 3-dimensional relatives of benzenes, are a class of boron hydride clusters in which one or more BH vertices are replaced by CH units.2+X\u2013) constitute an important group of intermediates that have found wide applications in organic synthesis.o-carboranyl diazonium salts are non-isolable, can only be prepared in situ and undergo substitution reactions with the reaction solvent, usually inorganic acids, in the presence of copper salts.o-carboranyl diazonium salt, [3-N2-o-C2B10H11][BF4].o-carborane12Diazonium compounds -arylation of o-carborane can be achieved via the aromatic ene reaction of 1,3-dehydro-o-carborane or a visible-light mediated B\u2013C(sp2) coupling of a carboranyl boron-centered radical. However, the substrate scope is only limited to arenes.On the other hand, it has been reported that B(9)-carboranyl iodonium salt can react with nucleophiles.o-carboranes was prepared in 77% isolated yield, by treatment of 3-amino-o-carborane with 1.5 equivalents of nitrosonium tetrafluoroborate.1 is dependent upon the counterions used and BF4\u2013 offers the highest thermal stability of the salt among the anions examined, such as PF6\u2013 and Cl\u2013. A 1.0 g batch of carboranyl diazonium salt 1 stored at \u20135 \u00b0C showed no signs of decomposition over four months.3-Diazonium-1 reacted rapidly with various nucleophiles (2) in acetonitrile, providing the corresponding B(3)-substituted o-carboranes in good to excellent yields nucleophiles, such as halide ions, gave the corresponding halogenated carboranes in excellent yields in <5 min -functionalized o-carboranes 3\u201314 -substituted o-carboranes in moderate to good yields bonds. More importantly, various functional groups that were previously unable to be introduced into the carborane unit can now be installed in a very simple and efficient manner. For instance, common functional groups can be easily installed on the 17 were produced in 81\u201398% yield scale\" fill=\"currentColor\" stroke=\"none\">O and S PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O double bonds was also examined. For example, the reaction of dimethyl sulfoxide furnished compound 20 after hydrolysis -oxygenated carboranes 1H, 13C, and 11B NMR spectroscopy as well as HRMS spectrometry.All new compounds were fully characterized by 4 and 6 were further confirmed by single-crystal X-ray analyses.17The molecular structures of compounds 1 did not react with anhydrous ether position, was formed quantitatively (vide infra).Interestingly, precursor N1 type of mechanism carboranes are promising radiotracers in Positron Emission Tomography (PET). Previously, 18[F]-fluorination of o-carborane was achieved by nucleophilic substitution of a B(9)-carboranyl iodonium bromide.1 as the starting material. Under similar reaction conditions to those reported in the literature,3a was formed quantitatively within 1 min and it can be easily purified (eqn (5)).The present strategy provides a straightforward and practical access to cage boron functionalized o-carborane has been developed. By utilizing B-carboranyl diazonium salt as a synthon, a large class of o-carborane derivatives bearing previously inaccessible functional groups can now be efficiently prepared, which may find applications in materials sciences.A practical method for selective cage boron functionalization of o-carborane,o-carboranes.This work demonstrates that B-carboranyl diazonium salt can serve not only as a source of boron-centered radicals18F-labelled o-carborane derivatives for medical applications.21Compared to aryl diazonium salts, the exceptionally high reactivity of B-carboranyl diazonium salt may be due to the lack of conjugation between the carborane cage and the diazonium group. Such a method may find useful applications in the efficient and fast synthesis of Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "Head-to-tail peptide macrocyclisations are significantly improved, as measured by isolated yields, reaction rates and product distribution, by substitution of one of the backbone amide C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O bonds with an oxetane ring. PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O bonds with an oxetane ring. The cyclisation precursors are easily made by standard solution- or solid-phase peptide synthesis techniques. Macrocyclisations across a range of challenging ring sizes are enabled by incorporation of this turn-inducing element. Oxetane incorporation is shown to be superior to other established amino acid modifications such as N-methylation. The positional dependence of the modification on cyclisation efficiency is mapped using a cyclic peptide of sequence LAGAY. We provide the first direct experimental evidence that oxetane modification induces a turn in linear peptide backbones, through the observation of dNN and d\u03b1N NOEs, which offers an explanation for these improvements. For cyclic peptide, cLAGAY, a combination of NMR derived distance restraints and molecular dynamics simulations are used to show that this modification alters the backbone conformation in proximity to the oxetane, with the flexibility of the ring reduced and a new intramolecular H-bond established. Finally, we incorporated an oxetane into a cyclic pentapeptide inhibitor of Aminopeptidase N, a transmembrane metalloprotease overexpressed on the surface of cancer cells. The inhibitor, cCNGRC, displayed similar IC50 values in the presence or absence of an oxetane at the glycine residue, indicating that bioactivity is fully retained upon amide C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O bond replacement.Cyclic peptides are an important source of new drugs but are challenging to produce synthetically. We show that head-to-tail peptide macrocyclisations are greatly improved, as measured by isolated yields, reaction rates and product distribution, by substitution of one of the backbone amide C Many of the substrates were made in solution using Bn ester/Z-protection of the C- and N-termini to allow the synthesis of salt-free precursors by use of a final hydrogenolysis step. This enabled accurate comparisons in product yields to be made across different substrates without having to correct for the impact of salts on the cyclisations. The synthesis of oxetane modified peptides was also achieved using the more practical approach of solid-phase peptide synthesis (SPPS). In most cases, control peptides without the oxetane modification were made for comparison purposes see ESI.in situ coupling of the resulting amine to the next protected amino acid, preactivated as its succinyl ester.1 conjugate addition of the N-terminus of the growing peptide to 3-(nitromethylene)oxetane; (ii) nitro group reduction and ester.1 . Alternater.1 and corresponding oxetane modified WLGOxG (7) (where GOx = oxetane modified glycine) were undertaken. For this investigation, substrates containing a tryptophan residue were used to allow quantitative monitoring by UV spectroscopy. Both substrates were subjected to the DEPBT method and conversions monitored over 74 h. From these data, it is clear that the initial rate of formation of oxetane containing cyclic peptide 9 is considerably faster than 8, even though both linear precursors 6 and 7 are consumed at similar rates . In a striking illustration of the power of this new method, cyclisation to tetrapeptide 10 was achieved in 54% yield in the presence of the oxetane modification, however cyclodimerisation to octapeptide was the predominant reaction pathway (20%) for the unmodified system. The successful formation of 12 containing an alanine modified residue confirms that this methodology is not simply limited to glycine substitution. Generally, DEPBT was favoured as the activating agent as it gave efficient cyclisations and minimised formation of difficult to separate diastereomers arising from epimerisation at the C-terminus prior to cyclisation. In some instances, impurities derived from the DEPBT reagent proved hard to remove, in which case PyBOP was used as an alternative.To test the generality of these initial findings, the head-to-tail cyclisation of a variety of oxetane modified substrates was studied and the isolated yields compared with those for the unmodified systems . Oxetane18 in an impressive 39% yield from commercial H-Trp(Boc)-2-ClTrt (15) . First, approach and ESI\u20202, whose synthesis was described in 22 after reverse-phase HPLC purification. Importantly, the four-membered oxetane ring survives the strongly acid conditions required to globally deprotect the side-chains including the arginine Pbf group. Smaller 11- and 14-membered macrocycles 23 and 24 were also conveniently produced in good yields using this methodology.Macrocyclisations not involving head-to-tail ring closure have also been examined. Specifically, the formation of disulphide containing macrocycles through oxidative cyclisation of cysteine side chains has been explored . TreatmeN-Methyl glycine, 2-methylalanine (Aib), ethylenediamine, dimethylethylenediamine and \u03b2-alanine were all introduced in place of the glycine introducing a potentially beneficial Thorpe\u2013Ingold effect. In fact, only the oxetane modification led to marked improvement in isolated yield of the derived cyclic peptides, suggesting that oxetane introduction is particularly beneficial.To understand how significant these improvements are in the broader context of peptide macrocyclisation, a series of alternative modifications were made to the central G residue of LAGAY and the efficiencies of the ring closures compared. glycine . These m13 by formation of all four possible amide bonds was examined to see whether the location of the oxetane relative to the amide bond being formed is important scale\" fill=\"currentColor\" stroke=\"none\">O system leading to higher product yields. However, larger improvements are seen when the modification is more centrally located along the precursor backbone.Macrocyclisation to pentapeptide mportant . The yie1H\u20131H TOCSY and NOESY spectra were acquired as detailed in the ESI1H signals in both peptides and probe changes in conformation, especially near the site of modification. As shown in i.e. residue i to residue i + 1) and medium-range NOEs that are commonly observed in \u03b1-helices, \u03b2-sheets, and turns.d\u03b1N, dNN, d\u03b2N), medium-range NOEs are only observed in peptide LAGOxAY-OMe containing the modification. Specifically, dNN and d\u03b1N NOEs are clearly observed in the oxetane modified peptide between residues Gly3 and Tyr5 indicating their close proximity, data consistent with the formation of a turn. These NMR studies provide the first direct experimental evidence that the oxetane modification is indeed turn-inducing, and brings the peptide termini closer in space to facilitate efficient cyclisation and cLAGOxAY (13) in DMSO. 2D 1H\u20131H NOESY spectra were used to compile a set of nuclear Overhauser enhancement (NOE) \u2013 derived experimental distance restraints that were incorporated into MD simulations using the CHARMM forcefieldTo gain insights into how oxetane modification impacts the structure of the derived cyclic peptides, molecular dynamics (MD) computer simulations with distance restraints derived from NMR experiments were carried out on cLAGAY , are presented in 13 explores less of the \u03a6/\u03c6 space than 25, which supports our observation above that 13 is more rigid. The Ramachandran plots also highlight the effect of the oxetane introduction on the structure of the residues in proximity to the modification, with Gly3, Ala4 and Tyr5 shifting to different regions of \u03a6/\u03c6 space following the introduction of the oxetane, which is in agreement with the visual representation of the backbone in Cluster analysis was performed on 12\u2009500 structures extracted from the trajectories at 40 ps intervals. Five and four distinct structures were found for peptides y see ESI. Ten repAla4 see . The bac26) is known to target Aminopeptidase N (APN), a transmembrane zinc-dependent metalloprotease involved in a variety of processes, including blood pressure regulation, cell migration, viral uptake, cell survival, and angiogenesis.22 and cCNGRC (26) were examined for their inhibitory activity towards porcine APN using a spectrophotometric assay oxetane, or alternatively through coupling preformed Fmoc-protected dipeptide building blocks containing this modification. The first is convenient for the insertion of glycine modifications, whereas the second is more general, being suitable for introduction of oxetane modified derivatives of other l-amino acids was demonstrated. It can also be applied to side-chain to side-chain macrocyclisations, specifically oxidative ring closure of cysteine residues by disulphide bond formation. As a tool to improve head-to-tail macrocyclisations, our evidence suggests that oxetane incorporation is superior to a number of other common backbone modifications. Importantly, OMCPs are compatible with the harsh acidic conditions needed to deprotect amino acid side chains.Oxetane incorporation led to improvements in all the head-to-tail cyclisations studied as assessed by isolated yields, reaction rates and analysis of product distributions. The method works across a range of ring sizes and enabled the synthesis of a range of cyclic tetra-, penta-, hexa- and heptapeptides. Major improvements in yields are seen using glycine modified residues, with levels of epimerisation and dimerisation significantly reduced. The extension of this method to other modified dNN and d\u03b1N NOEs. The formation of a turn presumably helps bring the termini closer together enabling the macrocyclisation.A key question we sought to address in this study was why does oxetane introduction improve macrocyclisations? For maximum benefit, we have shown that the oxetane is best located centrally along the peptide backbone, an observation consistent with the hypothesis that this modification is turn-inducing.13. The full retention of bioactivity of 22 relative to cCNGRC suggests that the replacement of a C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O amide bond by an oxetane ring has little impact on binding, making it a viable bioisosteric replacement. Next steps will involve a fuller assessment of the properties of OMCPs and their application in drug discovery programmes.To be useful in drug discovery programmes as well as being easy to make, OMCPs must also have useful properties. Intuitively, one might expect oxetane introduction to only alter the backbone configuration of the cyclic peptide close to the site of modification. Indeed, this was borne out in detailed NOE-restrained MD simulations conducted on There are no conflicts to declare.Supplementary informationClick here for additional data file."} +{"text": "We introduce a modular synthetic procedure to produce a new class of synthetic oligomers called peptidines composed of repeating di-substituted glycine-derived amidines. N-alkyl glycine oligomers is replaced with a functionalized N-atom. Compared to peptoids or peptides, the presence of this amidine N-substituent in peptidines effectively doubles the number of diversification sites per monomeric unit, and can decrease their overall conformational flexibility. We have developed iterative solution- and solid-phase protocols for the straightforward assembly of peptidines containing diverse backbone and amidine substituents, derived from readily available primary and secondary amines. We have also performed crystallographic and computational studies, which demonstrate a strong preference for the trans (E) amidine geometry. Given their straightforward synthetic preparation and high functional group density, peptidines have the potential to serve as useful tools for library generation, peptide mimicry, and the identification of biologically active small molecules.Efforts to emulate biological oligomers have given rise to a host of useful technologies, ranging from solid-phase peptide and nucleic acid synthesis to various peptidomimetic platforms. Herein we introduce a novel class of peptide-like oligomers called \u201cpeptidines\u201d wherein each carbonyl O-atom within poly- Oligomer-based synthesis is central to all known life processes. In particular, the structural and functional variety found in proteins is derived from the assembly of only 20 amino acid building blocks. Efforts to emulate this diversity have led to a range of oligomer-based peptidomimetic strategies, including oligopeptides, peptoids, peptidosulfonamides, sulfonylpeptides, polypyrroles and others. PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O, with C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019NR thus doubling the accessible diversity for a given oligomer length. By varying the size and electronics of amidine N1 (backbone) and N2 (amidino) substituents, one can also modulate N-lone pair basicity, and backbone geometry.Herein we introduce a new class of oligomeric scaffolds that we term \u201cpeptidines\u201d. Peptidines are oligomers composed of repeating di-substituted glycine-derived amidines . AlthougN1 and N2 substituents to derive modularly from the large pool of commercially available primary amines. Using this route, we have been able to produce peptidines ranging in size from 2- to 4-mers, appended with sterically and electronically diverse substituents at both N1 and N2 positions. These syntheses proceed in short order, and with excellent yields in both solution and solid phases. Crystallographic and computational studies have demonstrated that amidines present within the peptidine scaffold prefer the trans-(E) geometry of the N1 substituent with respect to the N2 nitrogen. Peptidines therefore adopt discrete conformations as a function of both H-bonding effects and non-bonding interactions. In light of their facile preparation and high potential for chemical conformational diversification, we envision that peptidines will provide a useful scaffold for the preparation of novel and diverse structures with a range of chemical and biological applications.Thus, we have developed a concise peptidine synthesis protocol that allows both 1) with an imidoyl chloride (2) to produce an \u03b1-chloro amidine (3); second is an amination step (reaction 2), wherein the chloride atom in 3 is displaced by a primary amine (4) to form an \u03b1-amino amidine (5). Iteration of reactions 1 and 2 affords peptidine oligomers (6). In turn, the \u03b1-chloro imidoyl chlorides (2) used in reaction 1 are generated from commercially available primary amines (7), which are acylated to produce chloroamides (8), and then chlorinated to form imidoyl chlorides (2) (Our strategy for preparing peptidines is outlined in ides (2) .8a\u2013i) to the corresponding imidoyl chlorides successfully afforded sulfonyl- (3a\u2013d), aryl (3e\u2013f), carbamoyl-, and urea-derived (3g\u2013h) amidines. These yields were universally high (>70%) throughout a range of N2-substituents and as expected, reactions with sulfonamides proceeded faster (14 h versus 2 h), and in higher yields, than aryl and acyl derivatives. Furthermore, despite the possibility for reaction at the alkyl chloride position, we observed complete chemoselectivity for displacement at the acylimino carbon for all substrates examined, with no evidence of double addition. Similar chemoselectivities have been observed previously in condensation reactions between bis-electrophiles such as \u03b1-chloro-acid chlorides and secondary amines.2i did appear to provide the corresponding \u03b1-chloro-amidine (3i) following diisopropylamine treatment, attempts to purify this compound were hampered by the presence of inseparable amounts of diisopropylammonium chloride and diisopropylamine. However, by carrying out the chlorination reaction at room temperature to avoid decomposition (likely via polymerization), followed by trapping with diethylamine, we were able to access alkyl amidine 37 , we next focused our efforts on displacing the pendant chloride with amine nucleophiles (5a\u2013h) in excellent yields.p-nitrophenyl (entry 5) derivatives to give \u03b1-amino amidines (5a\u2013e) in near quantitative yields. Interestingly, the phenyl derivative 5f was found to decompose rapidly as the free base, however addition of hydrochloric acid in ether to this compound immediately after silica gel purification facilitated its isolation as the stable HCl salt. Furthermore, the reaction of 3h with benzylamine produced cyclic amidine 5h . Taken together, these results confirm that the peptidine core can be constructed through a three-step procedure involving: (1) amide chlorination, (2) amidination of a secondary amine, and (3) \u03b1-halide displacement with a primary or secondary amine.We next analyzed how varying the structure of the amine nucleophile would affect the chloride displacement process using substrate 5b, and using imidoyl chloride 2b and benzylamine as nucleophile, we were able to access 2-mers 10 and 11, and 3-mer 12 in only three steps and 80% overall yield by simply repeating chloride displacement and amidination steps. Notably, none of these transformations proceeded in lower than 90% yield. Furthermore, using this protocol, we have been able to prepare 12 in quantities greater than 1 g demonstrating the scalability of this procedure.Our next goal was to iterate the above three-step sequence to access longer oligomeric peptidines. We therefore chose to target a simple 3-mer composed of identical repeating monomeric units . Startin12 into longer oligomers gave rise to several notable findings. For example, treatment of 12 with benzylamine led exclusively to cyclic product 13 -methylbenzylamine afforded no cyclic product upon reaction with 12, but instead exclusively provided the expected linear product 14 (S)-methylbenzylamine provides sufficient steric encumbrance to prevent internal cyclization. Switching the nucleophile to Bn2NH provided linear 3-mer 15. We also succeeded in producing a linear 4-mer peptidine (16) via our two-step elongation protocol . These results demonstrate that by regulating the steric environment around backbone N1 substituents, we can access both linear and cyclic peptidines.Efforts to advance 3-mer oduct 13 , entry 1oduct 14 , entry 2protocol , entry 438) to the corresponding cyclohexylamine derivative to the synthesis of peptidines containing between one and four monomeric units .Given the suitability of oligomer-based synthetic strategies for the preparation of one-bead-one-compound libraries,ic units . After oe.g.12 \u2192 13, 24\u201326. Upon resin cleavage, however, linear intermediates underwent rapid cyclization. Compound 27 is an example of such a situation, and was initially observed as a mixture with its uncyclized counterpart 43 , and provided 228 mg of pure product, demonstrating the scalability of our solid-phase platform. N2-carbamoyl and carbonyl substituents could also be incorporated into our solid-phase platform ; however, as observed in analogous solution phase experiments, N2-carbamate derivative 34 readily underwent intramolecular cyclization to give an imidazolone substructure, similar to compound 5h. Amines containing acid-labile side-chain protecting groups, similar to those employed in Fmoc SPPS , were readily incorporated into peptidines, and unmasked after resin cleavage to give carboxylate and amine functional groups (as demonstrated by the synthesis of 35). Employing homo-\u03b2-alanine as the C-terminal group allowed us to access 2-mer 36 in good yield and purity tended to exhibit broadened peaks in 1H and 13C NMR spectra during routine characterization. This phenomenon was not surprising, as other oligomeric scaffolds, such as peptoids, can be very flexible and often exist in multiple different conformations at room temperature.12 demonstrated peak sharpening with increasing temperatures interacts with atoms at both termini of the molecule. Additionally, a hydrogen bond between the terminal ammonium N\u2013H group and the adjacent sulfonamide oxygen atom , psi (\u03c8), and omega (\u03c9) (\u03c4), referring to the position of the N2-substituent relative to the N1-nitrogen (\u03c4 angles exist in a trans conformation (\u03c4 = 180\u00b0 \u00b1 15\u00b0), despite each being in a different steric environment. This observation is consistent with previous literature reports,\u03c9 angles in 28, on the other hand, proved slightly more variable, demonstrating a preference for either cis or trans conformations , reminiscent of disubstituted amides found in peptides and peptoids.\u03c9 \u2248 180\u00b0 or 0\u00b0; \u03c4 \u2248 180\u00b0), there is a high degree of co-planarity surrounding each amidine in 28 . Additionitrogen . Perhapsne in 28 . The mosN2-substituent to prefer the trans geometry (\u03c4 = 180\u00b0 \u00b1 15\u00b0), we performed quantum chemical energy calculations and amidine (N2) nitrogen atoms. Also, we have obtained a crystal structure of 3-mer 28, which demonstrates all amidine motifs to adopt a trans geometry about the \u03c4 angle. This geometry conforms to structural constraints predicted by computational studies, and suggests that peptidines have the potential to project functionality in an ordered array. Furthermore, because peptidines possess two sites of diversity per monomeric unit (as opposed to only one present in peptides and peptoids), they may prove useful for library generation as greater diversity could be generated with shorter length oligomers. Peptidines therefore have significant potential to serve as useful tools for small molecule synthesis, peptidomimicry, and library generation.Herein we have introduced a novel class of glycine-amidine-based oligomers, which we term \u201cpeptidines\u201d readily afforded through modular synthetic approaches using both solid and solution phase chemistry. These synthetic protocols are high yielding, readily scalable, and enable straightforward installation of a variety of substituents onto backbone (Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "Palladium-catalyzed direct intermolecular coupling of o-carboranes with alkynyl bromides or terminal alkynes has been achieved, for the first time, with the help of a traceless directing group \u2013COOH, leading to the synthesis of a series of new cage B(4)-alkynylated-o-carboranes in high yields with excellent regioselectivity. o-carboranes has been achieved for the first time using two different catalytic systems. In the presence of 5 mol% Pd(OAc)2 and 3 equiv. of AgOAc, the reaction of 1-COOH-2-R1-C2B10H10 with R3SiC PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CBr in ClCH2CH2Cl gives 4- scale\" fill=\"currentColor\" stroke=\"none\">C)-2-R1-o-C2B10H10 in moderate to high yields. This reaction is compatible with alkynes possessing sterically bulky silyl groups such as iPr3Si or tBuMe2Si. Meanwhile, another catalytic system of Pd(OAc)2/AgOAc/K2HPO4 can catalyze the direct B(4)-alkynylation of 1-COOH-2-R1-C2B10H10 with terminal alkynes R2C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CH in moderate to high yields. The latter has a broader substrate scope from bulky silyl to aromatic to carboranyl substituents. Desilylation of the resultant products affords carboranyl acetylene 4- scale\" fill=\"currentColor\" stroke=\"none\">C)-2-R1-o-C2B10H10 which can undergo further transformations such as Sonogashira coupling, dimerization and click reactions. It is suggested that the above two catalytic systems may proceed via Pd(ii)\u2013Pd(iv)\u2013Pd(ii) and Pd(ii)\u2013Pd(0)\u2013Pd(ii) catalytic cycles, respectively. In addition, the silver salt is found to promote the decarboxylation reaction and thereby controls the mono-selectivity.Pd-catalyzed carboxylic acid guided regioselective alkynylation of cage B(4)\u2013H bonds in The development of efficient synthetic methodologies to incorporate alkyne motifs has received broad interest, as they are not only important building blocks in natural products, pharmaceuticals and materialso-carborane, followed by Pd(0)-catalyzed cross-coupling with alkynyl Grignard reagents,via cage B\u2013H activation.Though cage boron alkynylated carboranes can be prepared by two-step reactions, such as the selective iodination of an o-carboranes is very rare.23Directing groups are essential in transition metal catalyzed C\u2013H activation due to their ability to chelate the metal catalyst, position it for selective C\u2013H cleavage, and reduce activation energy by stabilizing the metallacycle intermediates.o-carboranes, in which the carboxyl group is removed in a one-pot fashion. Inspired by these results and other cage B\u2013H activation reactions,ii)\u2013Pd(iv)\u2013Pd(ii) catalytic cycle and by terminal alkynes via a Pd(ii)\u2013Pd(0)\u2013Pd(ii) catalytic cycle. These new findings are reported in this article with iPr3SiC PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CBr in the presence of 10 mol% Pd(OAc)2 and 1 equiv. of AgOAc in toluene at 90 \u00b0C for 6 h did not give any of the desired product scale\" fill=\"currentColor\" stroke=\"none\">C)-2-CH3-o-C2B10H10 in 40% GC yield were examined under the chosen optimal reaction conditions, and the results are compiled in 3 in high isolated yields afforded the product 3l in 54% yield gave 3k in only 40% isolated yield , the scope of R2 is highly limited in such a coupling reaction. tBuMe2SiC PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CBr worked well to give 3p in 70% isolated yield scale\" fill=\"currentColor\" stroke=\"none\">CBr was not reactive, probably due to its propensity to coordinate with a Pd center via the \u03c0 bond scale\" fill=\"currentColor\" stroke=\"none\">CBr as reagents. PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CBr and tBuC PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CBr were not compatible with this reaction.In contrast to R3-o-C2B10H10 (1a) with iPr3SiC PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CH under the aforementioned optimal reaction conditions. No reaction was observed in the absence of a base scale\" fill=\"currentColor\" stroke=\"none\">C\u2013C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CiPr3Si as the side product scale\" fill=\"currentColor\" stroke=\"none\">CH was added slowly via a syringe pump, leading to a significantly increased yield of 3a to 56% GC yield . The results are compiled in 1 = alkyl groups, the isolated yields of 3 are comparable to those observed in 1 = aryl unit such as 1g, the isolated yield of 3g is 30% and 1o (R1 = Me3Si) give 3n in 35% and 74% yields, respectively 2C6H3, 2-iPrC6H4 to 1-nC6H13-o-C2B10H10, affording the corresponding products, 3s, 3t, 3u and 3v, in 65%, 73%, 80% and 82% isolated yields, respectively to afford quantitatively the terminal alkyne 4a in 84% isolated yield. A click reaction of 4a with phenyl azide afforded carborane-functionalized 1,2,3-triazole (7a) in 95% isolated yield.To demonstrate the applications of the resultant compounds lkyne 4a . Like ot3 and 4a\u20137a were fully characterized by 1H, 13C, and 11B NMR spectroscopy as well as high-resolution mass spectrometry (HRMS).4a and 6a were further confirmed by single-crystal X-ray analyses and are shown in All new compounds 1a was treated with 1 equiv. of iPr3SiC PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CBr in the presence of 20 mol% Pd(dba)2 (dba = dibenzylideneacetone) in DCE at 90 \u00b0C for 6 h in the absence of AgOAc. On the other hand, under the same reaction conditions, replacement of Pd(dba)2 with Pd(OAc)2 gave the alkynylation product 3a in 30% GC yield scale\" fill=\"currentColor\" stroke=\"none\">CH afforded 3a in 16% GC yield without AgOAc as the oxidant. While, no 3a was observed when 20 mol% Pd(dba)2 was used instead of Pd(OAc)2 (ii) not Pd(0).To gain some insight into the reaction mechanism, the following control experiments were carried out. No reaction was observed if GC yield . SimilarPd(OAc)2 . These r1b and 3b\u2013COOH) was also examined site can induce the decarboxylation, and the addition of a silver salt can accelerate such decarboxylation, which is crucial for controlling the mono-selectivity.Decarboxylation of carboranyl carboxylic acids (examined . Compounii)\u2013Pd(iv)\u2013Pd(ii) catalytic cycle: an exchange reaction of 1 with Pd(OAc)2, followed by regioselective electrophilic attack at the more electron-rich cage B(4) site yields the intermediate A as the charge distribution on the cage follows the trend B > B > B > B.2C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CBr affords a Pd(iv) intermediate B.C, which undergoes a salt metathesis reaction, protonation and decarboxylation to give the final product 3 and regenerates the catalyst Pd(OAc)2. Meanwhile, another catalytic system involves a Pd(ii)\u2013Pd(0)\u2013Pd(ii) cycle. An acid\u2013base reaction between K2HPO4 and carboranyl carboxylic acid 1 gives the potassium salt 1\u2032.1\u2032 to the Pd(ii) center, followed by subsequent regioselective electrophilic attack at the more electron-rich cage B(4) site generates the intermediate D. Ligand exchange by acetylide gives a carboranyl-palladium acetylide intermediate E.F and Pd(0). Decarboxylation of F results in the formation of the final product 3, meanwhile Pd(0) is oxidized by AgOAc to regenerate Pd(OAc)2. It is noted that AgOAc acts as a bromide captor in the Pd(ii)\u2013Pd(iv)\u2013Pd(ii) catalytic cycle, but as an oxidant to regenerate Pd(ii) from Pd(0) in the Pd(ii)\u2013Pd(0)\u2013Pd(ii) catalytic cycle. However, in both cross-coupling reactions, AgOAc plays a crucial role in promoting decarboxylation and thereby controlling the mono-selectivity.On the basis of the aforementioned experimental data, two plausible reaction mechanisms are proposed in o-carboranes using alkynyl bromides or terminal alkynes as alkynylating agents, where \u2013COOH acts as a traceless directing group. A series of new cage B(4)-alkynylated o-carborane derivatives has been prepared for the first time, which could find many applications in the synthesis of carborane-based materials.We have developed two catalytic systems for regioselective and efficient alkynylation of cage B(4)\u2013H bonds in ii)\u2013Pd(iv)\u2013Pd(ii) cycle for using alkynyl bromides as coupling agents and a Pd(ii)\u2013Pd(0)\u2013Pd(ii) cycle for employing terminal alkynes as coupling partners. The latter has a broader substrate scope than the former. This work also gives some hints for the development of new catalytic systems for the functionalization of carboranes.On the basis of control experiments and literature work, two catalytic cycles are proposed for the above two reactions: a Pd(Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "We have developed a simple and versatile strategy for in situ growth of MnO2 on the surfaces of oleic acid-capped upconversion nanoparticles by optimizing the component concentrations in the Lemieux\u2013von Rudloff reagent. in situ growth of MnO2 on the surfaces of oleic acid-capped hydrophobic upconversion nanoparticles (UCNPs) by optimizing the component concentrations in the Lemieux\u2013von Rudloff reagent. The oxidation time was shortened by a factor of two compared to that of the reported method. This oxidation process has no obvious adverse effects on the phases of UCNPs. STEM, X-ray photoelectron spectroscopy (XPS), Fourier transform infrared (FTIR) and energy-dispersive X-ray analysis (EDX) characterization demonstrated the successful growth of MnO2 on the surfaces of UCNPs. Furthermore, when the weight ratio of MnO2/UCNPs reached (147.61 \u00b1 17.63) \u03bcg mg\u20131, 50% of the initial upconversion luminescence of UCNPs was quenched, as revealed by fluorescence and inductively coupled plasma optical emission spectrometry (ICP-OES) results. The presence of the surface MnO2 precipitate not only confers high dispersity of UCNPs in water, but also allows further activatable magnetic resonance imaging (MRI) and fluorescence multimodal imaging after reduction to Mn2+ by intracellular glutathione (GSH). A novel targeted drug carrier nanosystem was prepared to protect MnO2 from early decomposition in blood circulation by coating with mesoporous silica and capping with a gelatin nanolayer. Aptamer sgc8 was then attached to the surface of the gelatin nanolayer by covalent crosslinking to achieve targeted drug delivery. The results suggest that this nanosystem shows promise for further applications in cancer cell imaging and therapy.We have developed a simple and versatile strategy for To our knowledge, there have been no reports of MnO2 growth on the surfaces of UCNPs using the Lemieux\u2013von Rudloff reagent to achieve oxidation and MnO2 formation of UCNPs in one pot.Because OA-capped monodisperse UCNPs are synthesized in organic media, their transfer to the aqueous phase and their functionalization are challenging. To solve these problems, many researchers have focused on transferring hydrophobic nanoparticles to the aqueous phase by ligand exchange, silanization, or hydrophobic\u2013hydrophobic interactions.4 and NaIO4 in a mildly alkaline solution (pH 7.7) to accelerate the oxidation of OA, and this oxidation was accompanied by the growth of MnO2 on the surfaces of UCNPs molecules participate in many physiological processes not only in the cells, but also in blood with a concentration range from 0.8 to 15 mM.2 nanosheets can be converted to Mn2+via GSH reduction, but it is impossible for MnO2 to differentiate between the intracellular GSH and extracellular GSH in vivo. Hypothetically, MnO2 nanosheets were intravenously injected in in vivo experiments, MnO2 nanosheets may be reduced partly by GSH in blood, which would lead to false signal imaging or early cargo release before arriving at the tumor, decreased therapeutic efficiency, and side effects. So it is considerably necessary to protect MnO2 in the blood circulation, and to promise that MnO2 decomposition only happens in target cells or tissues. In the present work, a novel targeted drug carrier nanosystem was successfully fabricated to protect MnO2 from early decomposition in blood circulation by coating with mesoporous silica and capping with a gelatin nanolayer. More detailed information about this nanosystem is in the application part.Moreover, our group has reported the studies on using simple MnO4:Yb/Gd/Er UCNPs were synthesized according to a reported procedure.4 changed . Pictures of the reaction mixture at different times are shown in Fig. S3.NaYF Fig. S2a after 60 Fig. S2a. The flu2 nanosheets were prepared as a positive control according to a reported method,3/2 at 642.2 eV and 2p1/2 at 653.8 eV further indicate the presence of MnO2.2. XRD patterns of both the as-prepared and oxidized UCNPs were studied to investigate the phase effect of the oxidation process. From the XRD result shown in The identity of the precipitate formed on the surfaces of UCNPs was determined by energy-dispersive X-ray spectroscopy (EDX), scanning transmission electron microscopy (STEM), and X-ray photoelectron spectroscopy (XPS). Compositional analysis by EDX indicated the presence of a new element (Mn) not observed in the EDX data for UCNPs Fig. S4. The exi2 nanosheets, the as-prepared UCNPs and oxidized UCNPs at different reaction times all exhibit a broad band at around 3430 cm\u20131, corresponding to the O\u2013H stretching vibration. A peak at 3020 cm\u20131 attributed to the PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C\u2013H stretching vibration can be observed in the spectrum of UCNPs and UCNPs@MnO2 (12 h),2 (24 h), suggesting that all of the OA ligands on the surfaces of UCNPs were oxidized to azelaic acid ligands after 24 h. In addition, bands at 1557 and 1468 cm\u20131 observed in the spectrum of the UCNPs are attributed to the asymmetric and symmetric stretching vibrations of the carboxylate group of the OA ligand. However, in the two cases of the oxidized samples, bands corresponding to the carboxylate group are found at 1637 and 1563 cm\u20131, and 1632 and 1563 cm\u20131, respectively. The obvious changes of bands at around 810 cm\u20131 and 727 cm\u20131 observed in the spectrum of the as-prepared UCNPs and the oxidized UCNPs samples are associated with the external deformation vibration of PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C\u2013H, which decreases in intensity with oxidation time. These results suggested the cleavage of the \u2013HC PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CH\u2013 group of the bound OA. The FT-IR spectrum also showed the characteristic absorbance of the Mn\u2013O stretching vibration at around 470 cm\u20131 in the spectrum of MnO2 and both of the oxidized samples,2 on the surfaces of the oxidized UCNPs. On the basis of the above FTIR results, it can be deduced that the OA ligands on the surfaces of UCNPs were oxidized to azelaic acids, and the oxidation process was accompanied by the successful growth of MnO2 on the surfaces of UCNPs.The capping ligands on the surfaces of UCNPs were identified by FTIR spectra in 2 samples, thermogravimetric analysis (TGA) was performed decreased to 7.2%, which was consistent with the calculated result (see the ESI2 (12 h) decreased to 8.7%, which was between 7.2% and 10.5%, indicating that 56.2% of OA ligands were oxidized to azelaic acid, which was consistent with the FTIR result.To evaluate the ligand content in the as-prepared UCNPs and oxidized UCNPs@MnOd Fig. S7. The oxi% Fig. S7, so we c2 growth on the surfaces of UCNPs, control group 3 , then the rapid IO4\u2013 cleavage of the hydroxyketone products (step 2), and finally MnO4\u2013 oxidation of the second stage products to carboxyl groups (step 3). In control group 1 \u03bcg mg\u20131, 50% of the initial upconversion luminescence of UCNPs was quenched. The luminescence of UCNPs was recovered in the presence of GSH characterized by TEM, EDX and FT-IR following the reported method,Kd = 0.8 \u00b1 0.09 nM),2@mSiO2@gel-sgc8) through the bifunctional cross-linker sulfosuccinimidyl 4-[N-maleimidomethyl] cyclohexane-1-carboxylate (sulfo-SMCC)A novel targeted drug carrier nanosystem Scheme S1 was succ Fig. S15.38 Then 2 from early decomposition, we tested the longitudinal relaxation rate (1/T1) of UCNPs@MnO2 and the gelatin-protected nanosystem when exposed to normal human whole blood. The results and MRI e (1/T1) of the a2 nanolayer is reduced to Mn2+ ions by intracellular GSH, which, in turn, facilitates the fluorescence recovery of UCNPs. Meanwhile, Mn2+ ions can act as the contrast agent for MRI, giving an activatable upconverted fluorescence signal and magnetic resonance signal.This protecting ability can be reversed by the acid-responsive properties of gelatin. After crosslinking, at neutral pH, the charge of the cross-linked gelatin was positive (4.7 mV) Fig. S18, and theT1-weighted MRI images compared to CEM cells alone and Ramos cells treated with the sgc8-nanosystem. The amounts of Mn2+ in each CEM cell and Ramos cell treated with 50 \u03bcg mL\u20131 sgc8-nanosystem were 0.073 and 0.009 pg, respectively, tested by ICP-OES. These results demonstrated that our designed nanosystem can be used as a luminescent probe and as an MRI contrast agent for live cell imaging. As a proof-of-delivery concept, Dox was loaded into the nanosystem (sgc8-nanosystem-Dox). To investigate the acid-induced and controlled-release properties, the sgc8-nanosystem-Dox was exposed to buffers with different pH values at 4.0, 5.0, 6.0 and 7.4, as shown in Fig. S21.CCRF-CEM cells with high membrane PTK7 expression were chosen as the target cancer cell line and Ramos cells without membrane PTK7 expression were used as a negative control cell line.2 nanolayer on the surfaces of the oxidized UCNPs. The oxidation time was shortened to half the time of the reported method. Detailed investigations confirmed the existence of the MnO2 precipitate and provided information about the degree of oxidation at different oxidation times. It should be noted that our finding on the color change when using the Lemieux\u2013von Rudloff reagent can be used as an indicator to track the oxidation process. Moreover, we believe that this MnO2 growth method is not limited to hydrophobic UCNPs. It can be a common strategy applied to other hydrophobic nanoparticles synthesized by high-temperature thermolysis, such as semiconductor and metal nanoparticles, where only the coordinating ligands will be oxidized by the Lemieux\u2013von Rudloff reagent. Coincidentally, the surface MnO2 precipitate can act as a common quencher of the fluorescence of the host nanoparticles. The fluorescence can be totally recovered by reducing agents such as GSH and DTT, indicating potential for activatable fluorescence imaging application.In conclusion, by optimizing the component concentrations in the Lemieux\u2013von Rudloff reagent, we have broadened the application of this reagent in transferring OA-capped UCNPs into an aqueous phase, to include the direct formation of a MnO2 from early reduction by GSH in blood by coating with an acid-responsive gelatin nanolayer for more accurate imaging signals. The successful growth of MnO2 on the surfaces of UCNPs and the fabrication of the targeted delivery and imaging system suggest that it is possible to expand the application of the Lemieux\u2013von Rudloff reagent and these UCNPs@MnO2 carriers as activatable luminescent labels and contrast agents to other biological fields, such as bioimaging and cancer cell therapy.Last, but also important, we provided a method to protect MnOThere are no conflicts to declare.Supplementary informationClick here for additional data file."} +{"text": "The phosphaethynolate anion (OCP)\u2013 has three different functions in the reaction with 2,4,6-trimethylbenzoyl chloride (MesCOCl): it acts as a nucleophile, as an en-component in [2 + 2] cycloadditions and as a formal P\u2013 transfer reagent. \u2013 anion under the loss of carbon monoxide to yield a five-membered ring anion. Subsequently, the nucleophilic attack of the formed heterocyclic anion on a second acyl chloride molecule results in the 1,2,4-oxadiphosphole. The transient acyl phosphaketene is conserved during the reaction in the form of four-membered ring adducts, which act as a reservoir. Consequently, the phosphaethynolate anion has three different functions in these reactions: it acts as a nucleophile, as an en-component in [2 + 2] cycloadditions and as a formal P\u2013 transfer reagent.The reaction of Na(OCP) with mesitoyl chloride delivers an ester functionalized 1,2,4-oxadiphosphole in a clean and P-atom economic way. The reaction mechanism has been elucidated by means of detailed NMR-spectroscopic, kinetic and computational studies. The initially formed acyl phosphaketene undergoes a pseudo-coarctate cyclization with an (OCP) PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O have been described.via the cleavage of the PC double bond \u2013 transient phosphinidenes or phosphinidene complexes, respectively. Recently, we reported the synthesis of several hetero-phosphaketenes employing the phosphaethynolate (OCP)\u2013 anion as a phosphorous nucleophile. Furthermore, the ambident character of this anion has been demonstrated.1b is slightly larger than that of 1a (1c contributes to the electronic ground state. It describes the (OCP)\u2013 anion as a donor\u2013acceptor complex of a P\u2013 ion and carbon monoxide. Similar to the description of a transition metal carbonyl complex, the CO unit acts as a \u03c3-donor and a \u03c0-acceptor.Decarbonylation reactions, such as the transformation of aldehydesat of 1a .7b Besid\u2013 ion, however, the contribution of the mesomeric structure 1c suggests that this anion may act as a P\u2013 transfer reagent. Indeed, we could demonstrate that the reaction of an imidazolium salt with Na(OCP) forms the adduct of the parent phosphinidene (P\u2013H) with the corresponding N-heterocyclic carbene. PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O) is formed as an intermediate, which delivers the PH fragment in a concerted reaction step under the extrusion of CO.\u2013 anion was found to be a useful synthon to obtain heterocycles and cages, PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C\u2013CO2Et),4(COOEt)4]\u2013 was also obtained, accompanied by the loss of carbon monoxide.\u2013 and two equivalents of isocyanate formed five-membered azadiphospholides, which were found to be active catalysts for isocyanate trimerization via spiro phosphoranides.The delocalization of the negative charge hampers the spontaneous decarbonylation of the (OCP)1.000000,.000000 s\u2013 anion as both P nucleophile and P\u2013 transfer reagent.Here, we report the results of our investigations on the reactivity of Na(OCP) PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O), which are the heavier analogues of acyl isocyanates. Knowing that sterically demanding groups are needed to stabilize a phosphaketene in a monomeric form,2.5 was reacted with the rather bulky 2,4,6-trimethylbenzoyl chloride, MesCOCl, in a 1\u2009:\u20091 ratio is \u2013110 ppm at the B3LYP/aug-cc-pVDZ level of theory). Instead, two doublet resonances at \u03b4 = 112 and 253 ppm with a coupling constant of JPP = 46 Hz were observed. The 13C-NMR spectrum and in addition X-ray diffraction analysis revealed the formation of the acyloxy substituted 1,2,4-oxadiphosphole 3 shows a bond critical point between P1 and O3 and double bonds (C1\u2013P1 and C2\u2013P2), some extent of bond length equalization can be observed, which indicates a moderate aromatic delocalization. This is in agreement with previous theoretical studies, which stated that the aromaticity of the parent 1,2,4-oxadiphosphole is just slightly smaller than that of furan and other phosphasubstituted furans.d O3 see . The corfour equivalents of reactants are involved [2 MesCOCl + 2 Na(OCP)], a rather complex reaction mechanism with a number of steps must be involved in the formation of 3. To explore the reaction sequence, low temperature NMR measurements were performed. At room temperature the reaction is complete within mixing time of the reagents. A THF solution containing stoichiometric amounts of the starting materials was gradually warmed from \u201350 \u00b0C to room temperature and the progress of the reaction monitored using 31P-NMR spectroscopy. At \u201335 \u00b0C the parallel formation of two intermediates A and B was observed. NMR investigations at different temperatures indicated that B is in equilibrium with A and an (OCP)\u2013 anion. Intermediates A and B, the final product 3 and Na(OCP) are the only species which can be detected using 31P-NMR spectroscopy. NMR experiments using different stoichiometric ratios of the starting materials did give the same results.Since A and dianion B were identified using GIAO chemical shift calculations, which are shown in A and B are adducts of a mesitoyl phosphaketene molecule with one and two (OCP)\u2013 ions, respectively. This indirectly indicates the initial formation of mesitoyl phosphaketene, MesCO-P PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O. The common structural motif of A and B is the four-membered P(CO)2P ring, which was first described for [O2C\u2013P(CO)2P]2\u2013, the adduct of a CO2 molecule and two (OCP)\u2013 ions.31P-NMR chemical shifts of this adduct (\u03b4 = 279 and 102 ppm) are indeed very similar to those of the rings in A and B. The 31P-NMR chemical shift of the P5 (80 ppm) atom in B matches well with that of bismesitoyl phosphide [P(COMes)2]\u2013, (\u03b4 = 86 ppm).26Anion A and B are involved in the formation of 3. We therefore reacted 2,4,6-trimethylbenzothioyl chloride (4) with Na(OCP) in the hope of identifying another possible intermediate. When Na(OCP) and 4 were reacted at \u201378 \u00b0C in a ratio of 2\u2009:\u20091, again gas evolution indicated the formation of carbon monoxide. Using NMR spectroscopy we could observe the formation of the anion 5, which was isolated in low yield \u2013 ion, leading to IM2. From intermediate IM2, which was not observed experimentally, the cyclic anions A, B and IM3 can be formed. A [2 + 2] ring closure of IM2 leads to its isomer A, while a formal cycloaddition of IM2 with another equivalent of (OCP)\u2013 anion delivers intermediate B. Presumably, the reactions leading from IM1 to IM2 as well as from IM2 to the intermediates A and B are reversible. This is supported by the experimentally observed equilibrium between A, an (OCP)\u2013 anion and B (vide supra).IM2 can undergo a cyclization under the loss of CO to yield IM3. In the final step, the nucleophilic substitution on an acyl chloride with IM3 gives the final product 3.In the first step, the acyl phosphaketene forms as intermediate 31P-NMR spectroscopy and the concentrations of Na(OCP), A, B and the final product 3 were determined from the relative integrals of the corresponding peak areas (see Under the assumption that the reaction mechanism shown in reas see . Applyinreas see . As cert\u2013 in mesitoyl chloride for (OCP)\u2013 is slower than the subsequent reaction of the activated acyl phosphaketene MesCO\u2013P PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O IM1 with another equivalent of (OCP)\u2013. Indeed, this reaction between IM1 and (OCP)\u2013 is the fastest of all and explains why IM1 is not observed. Intermediate IM2 \u2013 likewise not observed \u2013 is consumed by four competing reactions: the back reaction to IM1 and (OCP)\u2013, the reversible formation of A, the reversible formation of B, which is faster than that of A (i.e. k4 > k3), and the irreversible formation of IM3 which is faster than that of A (i.e. k5 > k3) but smaller than that of B (i.e. k5 < k4). That A and B are transient but observable species is due to the slow back reaction to IM2, especially for B \u2192 IM2 which has the smallest rate constant, k4r. Consequently, the concentration of B increases steeply at the beginning of the reaction (in the first ca. 15 hours) and serves as a reservoir for intermediate IM2. Similarly, A is a conserved form of IM2, however in this case the accumulation effect is much less pronounced (k3 is similar to k3r) and the concentration of A is low and remains approximately constant throughout the reaction. IM2 is consumed irreversibly under loss of CO to give IM3, which rapidly and irreversibly reacts with acyl chloride to give the final product 3.In this model, the nucleophilic substitution of ClIM2 to A and IM2 to B were not investigated computationally, since similar transformations were subjects of a previous study.IM3 from IM2, however, deserves a closer look. High-level calculations revealed that this reaction follows a concerted mechanism and the activation barrier was found to be low /aug-cc-pVDZ//B3LYP/6-31+G* level of theory). The structure of the corresponding transition state is shown in PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C double bond of the phosphaketene moiety occurs simultaneously with the formation of the P\u2013O bond. A continuous change in the atom distances was observed along the reaction coordinate. In this process, the starting material, which consists of a phosphaketene unit and a separate delocalized OCPCO moiety, transforms to a moderately aromatic system. The increase in aromaticity is reflected by the NICS(0) values depicted as a function of the reaction coordinate, which become continuously more negative.In the present work, the reactions leading from IM3 is stabilized by a M\u00f6bius type eight-electron interaction and the coarctation of the orbital loop is at the P atom (see (IM2\u2192IM3) is almost planar and the barrier is very small, we propose that this reaction has pseudo-coarctate character. This is further supported by the ACID (anisotropy of induced current density) plot32Since there is an exocyclic part involved in this bond transformation process, we propose that this reaction is of coarctate type. Besides classical linear and pericyclic reactions, a third group of reactions was classified by Herges as coarctate.atom see . Coarcta\u2013 anion was found to react as a nucleophile and P\u2013 transfer reagent. The stepwise reaction mechanism was deciphered using low temperature 31P-NMR spectroscopy, kinetic measurements and theoretical calculations. Several [2 + 2] cycloaddition products were observed as intermediates using 31P NMR spectroscopy and identified with the help of chemical shift calculations, which demonstrate the performance of these theoretical methods. The formation of another, not yet observed, intermediate was made plausible through the reaction of Na(OCP) with a thioacyl chloride which gave a stable sodium 1,2,4-thiadiphosphol-3-olate. As observed previously in reactions with isocyanates\u2013 salts and heterocumulenes is the equilibrium, X PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019Y PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019Z + (OCP)\u2013 \u21c6 [XY(PCO)Z]\u2013, which indicates the stability of the (OCP)\u2013 anion with respect to its addition products. The final formation of the oxadiphosphole ring proceeds in a concerted, pseudo-coarctate reaction step. This transformation is reminiscent of reactions seen with \u03b1,\u03b2-unsaturated organic azides and indicates a possible resemblance between the isovalence electronic (OCP)\u2013 and (N3)\u2013 anions.In conclusion, we have presented a one-pot synthesis for substituted 1,2,4-oxadiphospholes, utilizing Na(OCP) as the phosphorous source. The reaction is remarkably selective and atom economic with respect to phosphorous and excellent yields can be obtained. The (OCP)Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "Formation and mode-specific autodetachment from a dipole-bound state in a radical anion dimer is observed in the frequency and time-domains. 0 dimer radical anion and its exited state dynamics. In the ground electronic state, the excess electron is localised on one monomer with a planar para-quinone ring, which is solvated by the second monomer in which carbonyl groups are bent out of the para-quinone ring plane. Through the \u03c0-stacking interaction, the dimer anion exhibits a number of charge-transfer (intermolecular) valence-localised resonances situated in the detachment continuum that undergo efficient internal conversion to a cluster dipole-bound state (DBS) on a \u223c60 fs timescale. In turn, the DBS undergoes vibration-mediated autodetachment on a 2.0 \u00b1 0.2 ps timescale. Experimental vibrational structure and supporting calculations assign the intermolecular dynamics to be facilitated by vibrational wagging modes of the carbonyl groups on the non-planar monomer. At photon energies \u223c0.6\u20131.0 eV above the detachment threshold, a competition between photoexcitation of an intermolecular resonance leading to the DBS, and photoexcitation of an intramolecular resonance leading to monomer-like dynamics further illustrates the \u03c0-stacking specific dynamics. Overall, this study provides the first direct observation of both internal conversion of resonances into a DBS, and characterisation of a vibration-mediated autodetachment in real-time.Isolated \u03c0-stacked dimer radical anions present the simplest model of an excess electron in a \u03c0-stacked environment. Here, frequency-, angle-, and time-resolved photoelectron imaging together with electronic structure calculations have been used to characterise the \u03c0-stacked coenzyme Q These interactions govern, for example, anion-recognition processes in supramolecular assemblies,0 dimer radical anion, (CQ0)2\u2013, the calculated minimum energy structure of which is shown in 0)2\u2013 is a useful prototype system because of the strong gas phase stability of one conformer, the well-understood dynamics of the isolated monomer radical anion, CQ0\u2013,para-quinones are often considered as prototypical electron acceptors due to their ubiquity in biological and technological electron transfer systems.0 is the smallest member in the coenzyme-Q series, where the subscript in CQ0 refers to the length of an isoprenyl tail attached to the para-quinone ring that serves to enhance lipid miscibility. It is thought that the synergetic co-operation of hydrogen bonding and \u03c0-stacking of the para-quinone ring in biological systems play important roles in facilitating electron transfer.para-quinones have been proposed as possible bypasses of the inverted Marcus region,Here, we consider the coenzyme Qpara-quinone radical anions have shown that photoexcitation cross-sections to quasi-bound \u03c0*-resonances can be larger than direct photodetachment.\u201314 s lifetimes); and Feshbach resonances, in which the corresponding neutral core is predominately in an electronically excited state . We have recently shown that anion photoelectron (PE) imaging is ideally suited to probe the dynamics of anion resonances,Recent gas phase studies on a series of monomer 0)2, with an electric dipole moment, |\u03bc| > 2.5 D, can support a dipole-bound state (DBS),\u03bc. Due to the diffuse nature of a DBS, the direct photoexcitation cross-section from a valence-bound state is usually very small. In contrast, photodetachment cross-sections of a DBS can be very large and increase with decreasing photon energy.Neutral molecules or clusters, such as (CQ0)2\u2013 is presented. The frequency- and angle-resolved dimensions involve recording single-photon PE images (spectra) at many different photon energies (h\u03bd) to identify trends and fingerprints of resonances and their associated dynamics. A selected resonance can then be photoexcited and the resulting dynamics monitored in real-time using time-resolved PE imaging. Supporting ab initio calculations using multi-state XMCQDPT2 theory with a large CASSCF reference space allow clear assignment of the experimental dynamics.h\u03bd = 3.10 eV with high intermolecular charge-transfer character leads to the formation of a DBS on a \u223c60 fs timescale, which then undergoes vibration-mediated autodetachment on a 2.0 \u00b1 0.2 ps timescale. This determination represents the first direct observation of the conversion of above-threshold valence-localised population to a DBS, and the first real-time characterisation of vibration-mediated autodetachment of a DBS. At slightly higher h\u03bd, a competition between non-adiabatic dynamics leading to the DBS and autodetachment dynamics has been observed. The competition is assigned to the interplay between dimer and monomer-like dynamics, and further highlights the role of \u03c0-stacking in influencing the excited state dynamics.Here, a combined frequency-, angle-, and time-resolved photoelectron imaging (FAT-PI)0)2\u2013 are shown in h\u03bd: (a) 4.66 eV (266 nm); (b) 4.13 eV (300 nm); and (c) the average of 2.53 eV (490 nm) to 3.02 eV (405 nm). The 4.66 eV and 4.13 eV spectra are also compared with selected PE spectra of CQ0\u2013 that extend to the same maximum in electron kinetic energy (eKE).0\u2013 and (CQ0)2\u2013 of \u223c1.2 eV. The spectra for (CQ0)2\u2013 and CQ0\u2013 broadly exhibit similar spectral features, except the dimer generally shows an increased yield of low-eKE electrons. The PE spectrum in h\u03bd, all of which are identical within noise. These spectra show three (perhaps four) reproducible partially resolved features that have the appearance of vibrational structure. It is remarkable that vibrational-like structure is discernible for such a large molecular system at 300 K, and implies a mode-specific detachment process.Three example PE spectra of , all PE spectra are essentially identical in h\u03bd \u2265 4.5 eV the PE spectra resemble that of the isolated monomer (0)2\u2013 has an increased yield of PE signal in the eKE \u2264 0.2 eV range.ical see . Between monomer ,28 althoh\u03bd, while the centre of the PD feature increases linearly with h\u03bd. The relative contributions of the three channels are shown in h\u03bd = 4.66 eV PE spectrum in h\u03bd < 3.0 eV, while the PD channel becomes dominant for h\u03bd > 3.0 eV. The modulation in region (ii) is reproduced between the PD and DBD channels. For h\u03bd > 3.5 eV, the DA channel becomes available and the contribution of the DBD channel is minimal (\u223c5% at h\u03bd = 4.66 eV).To analyse the detachment channel contributions in The adiabatic detachment energy (ADE) was determined in the global fit by extrapolating the rising edge of the PD feature for all frequency resolved PE spectra. Similarly, the vertical detachment energy (VDE) was determined from the maximum of the PD feature in the global fit. These data are tabulated in \u03b22 parameter (\u20131 \u2264 \u03b22 \u2264 2),\u03b22 values of \u20131 and +2 correspond to electron ejection perpendicular and parallel to the laser polarisation, \u03b5, respectively. In contrast to CQ0\u2013,PE angular distributions associated with h\u03bd < 4.0 is given in h\u03bd \u223c 3.75 eV, with its red edge overlapping with the modulations. While the total PE yield shows some reproducible closely-spaced oscillations, their extent does not account for the observed channel modulation. Instead, the modulations appear to result from a competition between processes yielding the two detachment channels rather than any sharp changes in the total photodetachment cross-section. Hence, the presentation of normalised PE spectra in To further investigate the DBD and PD channel modulation in region (ii) , the tot0)2\u2013 is shown in para-quinone ring is 3.9 \u00c5, which is the same as the neutral \u03c0-stacked benzene dimer.0)2\u2013 equilibrium geometry scale\" fill=\"currentColor\" stroke=\"none\">O) bond lengths of \u223c1.24 \u00c5. The right monomer has a non-planar geometry, in which the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O groups are bent out of the ring plane by \u223c12\u00b0, and the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O bond lengths are \u223c1.20 \u00c5. In contrast, both monomers are planar with all C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O bond lengths of \u223c1.20 \u00c5 in the optimised (CQ0)2 geometry.The calculated minimum energy geometry of support the structure shown in 490)2\u2013] \u2248 BDE[(CQ0)2\u2013] + ADE[CQ0\u2013], where ADE is the adiabatic detachment energy and ADE[CQ0\u2013] = 1.60 \u00b1 0.06 eV.28Calculated photodetachment energetics (including zero-point energy) are summarised in 0)2\u2013 are summarised in 0\u2013.h\u03bd range. The 42[F] and 82[S] resonances predominantly involve intramolecular excitation processes on the planar monomer, while the 52[F] and 72[S] have a significant intermolecular or charge-transfer character. The 12A excited state is a bound charge-resonance state, corresponding to the radical anion localised almost exclusively on the non-planar monomer.Valence-localised vertical excited states of have been excluded because they are of \u03c0* \u2190 O(p) character, are optically inactive, and played no clear role in the dynamics of CQ0\u2013.The first excited neutral state is vertically situated at 2[S] (oscillator strength \u223c 0.05) and intramolecular 82[S] (oscillator strength \u223c 0.22) resonances. The spectrum has therefore been modelled using two Gaussians, plus an underlying baseline for prompt detachment and 92[F] contributions at high h\u03bd.The resonance energetics and oscillator strengths allow the broad feature in the photodetachment yield spectrum to be as2[F] or 72[S] resonances involves high excitation of carbonyl wagging, ring puckering, C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O and C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C stretching modes, which are those required to achieve approximate 52[F]/32[F] and 72[S]/52[F] conical intersection geometries scale\" fill=\"currentColor\" stroke=\"none\">C and C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O stretching modes localised on the parent planar monomer.From the CASSCF wavefunction characters see ESI, Franck\u2013s see ESI. In cont\u03bc| in the ground electronic state of (CQ0)2 are \u223c6.9 D and \u223c5.5 D at the optimised (CQ0)2\u2013 and (CQ0)2 geometries, respectively, supporting DBSs with binding energy of \u223c150 meV and \u223c50 meV. However, the oscillator strength for direct photoexcitation of the DBS is \u223c10\u20134 to 10\u20135, which is small compared with those for valence-localised resonances. \u03bc at the (CQ0)2\u2013 and (CQ0)2 geometries. At the (CQ0)2\u2013 geometry, \u03bc is oriented between the \u03c0-stacked monomers and close to parallel with the planar monomer ring and the chord joining the two carbonyl groups on the non-planar monomer. In contrast, at the (CQ0)2 geometry the orientation of \u03bc is almost orthogonal to the monomer ring planes. Calculations connecting the ground electronic state (CQ0)2\u2013 to neutral geometries reveal that changes of the carbonyl tilt angle, \u03b8, associated with wagging modes of the non-planar monomer scale\" fill=\"currentColor\" stroke=\"none\">O bonds on the planar monomer), are the principal geometrical changes responsible for the large change in orientation of \u03bc. These C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O wagging modes are also FC active following intermolecular photoexcitation.Calculated values of |0)2 state, 11A at h\u03bd \u223c 4.5 eV from each of \u0394t \u2265 0 spectra. t, which reveals two timescales. The total PE signal was fitted with two functions: a Gaussian cross-correlation convoluted with an exponential decay for the fast component; and a cross-correlation function convoluted with an exponential rise and decay for the slow component. The fast component lifetime, t1 \u223c 60 fs, is limited by the experimental cross-correlation. The slow component reaches a maximum contribution after the fast component has decayed, and subsequently decays with a lifetime of t2 = 2.0 \u00b1 0.2 ps. From t1 is associated with the development and evolution of a broad PE feature into two narrow features, peaking at low-eKE and eKE = 1.6 eV. The high eKE feature is very close to the 1.55 eV probe energy and has a highly anisotropic PE angular distribution with \u03b22 \u223c +2 (see inset). The anisotropy suggests that the outgoing photoelectron has p-wave character, and therefore that the original orbital from which the electron is detached has s-character.t2 is associated with the concerted decay of both pump-probe features.Results of the 3.10 + 1.55 eV (pump + probe) time-resolved measurements are summarised in t1) to a narrow distribution. This PE feature is again situated at the probe photon energy with \u03b22 \u223c +2 angular character, and decays on a t2 \u223c 2.0 ps timescale. These observations are in agreement with the 1.55 eV probe experiments. However, concerted with the changes at high-eKE, there is now a bleach of the eKE < 0.25 eV signal, which recovers on the same t2 \u223c 2.0 ps timescale. t.Similar time-resolved measurements were performed with a 1.05 eV probe, which are summarised in 0\u2013)CQ0, composed of a localised planar monomer anion solvated by the second non-planar monomer. The frequency-resolved PE spectra broadly support this conclusion based on the similarity with CQ0\u2013 spectra when h\u03bd is red-shifted by \u223c1.2 eV 2 optimised geometry. Thus, ideal conditions for internal conversion to a DBS are achieved: the original FC photoexcitation modes of 52[F] modulates the 32[F] state over the DBS, which facilitates a curve crossing. The subsequent t2 = 2.0 \u00b1 0.2 ps lifetime of the DBS can be assigned to its autodetachment lifetime and leads to the vibrational structure observed in the DBD channel as seen in Before the DBS is formed, the time-resolved measurements showed a transient broad PE feature that sharpens to the DBS feature on a t1 timescale corresponds to only a few vibrational periods, implying that extensive intramolecular vibrational relaxation away from FC modes is unlikely.The overall time-resolved dynamics are summarised schematically in \u03bc 2\u2013.The vibrational structure associated with the DBD channel in the frequency-resolved spectra indicate\u03bc see , which i\u03bc see have bee3CN, CH3NO2, or nucleobases.3CN (|\u03bc| \u223c 3.9 D), which does not support a valence-bound anion, exhibits DBS autodetachment on a 4\u2013900 ps timescale.3NO2 (|\u03bc| \u223c 3.5 D) converts on a \u223c400 fs timescale to a valence-bound anion situated \u223c100 meV lower in energy, which is facilitated by a similar vibrational wagging and modulation of \u03bc to that for (CQ0)2\u2013.2[F] or 52[F] resonance) can also evolve into a DBS. However, in contrast to the Neumark studies, (CQ0)2\u2013 does not undergo internal conversion from the DBS to the lower-lying 12A and X2A states, probably because any coupling would require very large geometrical distortions (that may not support a DBS), and will be unlikely on the \u223c2 ps DBS autodetachment lifetime. It can therefore be concluded that internal conversion between a valance-localised state and a DBS, in either direction, requires near degeneracy. These trends provide further confirmation that the 32[F] resonance is likely involved in formation of the DBS rather than direct internal conversion from the photoexcited 52[F] resonance.Time-resolved dynamics involving DBSs have been implicated by the Neumark group in their femtosecond PE spectroscopy experiments following photoexcitation of iodine anions coordinated to CH0)2\u2013 is evidence of the extent to which the non-adiabatic dynamics are altered by \u03c0-stacking. Specifically, although the electronic ground state of the dimer represents a localised monomer that is merely solvated, a range of new non-adiabatic dynamics in the continuum are accessed due to the availability of charge-transfer excitations and a cluster DBS. This situation is likely to be common to other cluster anions with similar chromophore/electrophore groups, and means that the general extrapolation of monomer to cluster dynamics is not trivial. Nevertheless, as will be described next, monomer-localised dynamics can be observed in some circumstances.One of the key outcomes from , there should be photoexcitation features in 2[S] and 82[S] resonances. This conclusion is supported by three additional observations. First, h\u03bd > 3.8 eV the DBD channel yield diminishes, even though 82[S] is still strongly photoexcited. Second, from 2[S] photoexcitation profile. Third, for h\u03bd > 4.0 eV, the PE spectra of (CQ0)2\u2013 have a similar appearance to those for CQ0\u2013,The modulation between the DBD and PD channels in region (ii) of 2[S] resonance at h\u03bd \u223c 3.6 eV involves similar intermolecular FC modes as the 52[F] resonance. In contrast, photoexcitation of the 82[S] resonance almost exclusively involves intramolecular population of \u03c0*CC orbitals localised on the planar monomer. A conical intersection, detailed in the ESI,2[S] and 52[F] resonances are non-adiabatically connected along the FC modes in a similar way to the 52[F] and 32[F] resonances. Thus, a fast internal conversion of 72[S] population to 52[F] could be expected. In turn, the 52[F] resonance can form the DBS as above. Interestingly, the observed modulations in \u20131.Our calculations suggest that photoexcitation of the 7h\u03bd < 3.8 eV, the 82[S] resonance is predominantly photoexcited since it has a much higher oscillator strength. As this resonance is predominantly localised on the planar monomer, photoexcitation will involve intramolecular FC modes. From 2[S] photoexcitation does not result in efficient internal conversion to the DBS, rather the PD channel dominates. For h\u03bd > 3.75 eV, in which 82[S] is exclusively photoexcited, the competition between DBD and PD channels is no longer observed. Instead, the DA feature centred at eKE = 0.22 \u00b1 0.04 eV becomes available. The DA feature energetically correlates with a delayed autodetachment from the 42[S] resonance, which is also predominantly localised on the planar monomer. The assignment of DA to the 42[S] resonance suggests an internal conversion route from 82[S] photoexcitation. Because extensive nuclear motion (or energy redistribution) cannot occur on the ultrafast lifetimes of resonances, the internal conversion will be facilitated through intramolecular FC modes. In accord, the non-planar monomer effectively acts as a spectator so the detachment dynamics are monomer-like in character.h\u03bd > 3.75 eV PE spectra, which have a similar appearance to those of CQ0\u2013,For 3.55 < h\u03bd \u2265 3.8 eV range likely results from a mode-specific competition between non-adiabatic decay pathways of the 72[S] and 82[S] resonances. Such dynamics can only be uncovered by frequency-resolved PE spectroscopy combined with relative cross-section measurements. A detailed theoretical account of such dynamics will likely be very challenging, particularly due to the participation of the continuum. However, despite the interplay and competition of multiple channels, it is remarkable and encouraging that the technique of frequency-, angle-, time-resolved imaging can provide such rich insight. Overall, to the best of our knowledge, this study presents the first characterisation of a non-adiabatic dynamics competition between resonances with varying inter- and intramolecular character.In summary, the modulation between the DBD and PD channels in the 3.4 \u2265 We have demonstrated that intermolecular or charge-transfer photoexcitation can play a significant role in the excited state non-adiabatic dynamics of a \u03c0-stacked dimer radical anion. We have provided the first direct and real-time evidence of internal conversion of above-threshold resonances into a cluster-supported DBS, and its subsequent vibration-mediated autodetachment. Formation of the DBS is facilitated through charge-transfer photoexcitation by virtue of \u03c0-stacking. However, despite the additional complexity introduced by dimerization, monomer-like dynamics can also be observed following photoexcitation of resonances primarily localised on the monomer that supports the excess electron in the dimer ground electronic state. When both inter- and intra-molecular resonance photoexcitation profiles overlap, a remarkable competition between \u2018dimer\u2019- and \u2018monomer\u2019-like non-adiabatic dynamics has been observed. Such interplays between inter- and intramolecular non-adiabatic dynamics are likely to be common in other similar \u03c0-stacked cluster anions, and further illustrate the rich dynamics that can occur in the detachment continuum.Supplementary informationClick here for additional data file."} +{"text": "We report the first examples of 7-membered diazaphosphepines using phosphorus\u2013amine (P\u2013N) chemistry. d]-C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C double bond position of heteropine core allows us to effectively control the chemical and electronic structures in both the ground and excited states of these diazaphosphepines. This functionalization has led to a diverse set of crystal structures, which has in turn provided access to rich photophysical and redox properties. Of particular interest is the evidence for planar \u03c0-conjugated backbone in our non-aromatic heteropine and twisted intramolecular charge transfer, which have never been reported for heteropines. The introduction of electron-accepting substituents at [d]-position of diazaphosphepines results in heteropines that are more electron deficient than any heteropine reported to-date. As proof of concept, we have fabricated organic solar cells with heteropines as non-fullerene acceptors.We have designed and synthesized the first examples of 7-membered diazaphosphepines using phosphorus\u2013amine (P\u2013N) chemistry. Different from previous functional protocols of heteropines, the installation of \u03c0-conjugated substituents having diverse chemistries at the [ PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C double bond in the classical six-membered benzenoid building block with isoelectronic B\u2013N fragments has generated azaborine and borazine; materials comprising these substitutions display absorption and emission at energies lower than their pure carbon counterparts and, as such, have been incorporated in optoelectronic devices.\u03c0-Conjugated cyclic building blocks that contain heteroatoms have attracted a lot of attention due to their diverse chemical and electronic structures, and tunable electronic properties.Heteroatom-containing seven-membered cycloheptatrienes, namely heteropines, have not been studied extensively. Previous studies have focused on their fundamental characteristics, including their conformational change, chemical reactivity and electronic structure.b,d,f]-C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C double bonds is required to stabilize the seven-membered ring scale\" fill=\"currentColor\" stroke=\"none\">C double bond position of the heteropine core scale\" fill=\"currentColor\" stroke=\"none\">C bond accordingly. We thus expect BZ, FBZ, and BTD substituents to weaken electron delocalization in BZ-P, FBZ-P, and BTD-P, while CP, MI, and AN substituents to enhance electron delocalization in CP-P, MI-P, and AN-P. Electronic structure aside, we expect sterics to play an important role as well. Compared to five-membered ring substituents, like CP and MI, six-membered ring substituents, like BZ, FBZ, and BTD, as well as naphthalene-functionalized AN should exhibit stronger steric hindrance that will induce more twist when installed at the [d]-position of the diazaphosphepine core. This steric effect can also influence electron delocalization. The combination of both offers a powerful handle to fine-tune the intramolecular electronic communication of diazaphosphepines.We have chosen to introduce six \u03c0-conjugated substituents , includiBZ-, FBZ-, BTD-, CP-, MI- and -InAN are described in ESI.BZ-, FBZ-, BTD-, CP-, MI- and -InAN with PhPCl2 in the presence of NEt3 in anhydrous acetonitrile solution, resulting in BZ-, FBZ-, BTD-, CP-, MI- and \u2013PN in 55\u201380% yields. While the compounds were all obtained at reflux conditions, the ring-closing reaction to yield BTD-P and MI-P proceeds readily even at 0 \u00b0C, an indication of the ease with which these compounds can be made. Unique to this synthetic scheme are the mild reaction conditions and the absence of reactive intermediates, thereby enabling the installation of strong electron-withdrawing substituents, such as FBZ, BTD, and MI, which were inaccessible through previous synthetic protocols of heteropines and P-containing cyclic materials.The synthesis of diazaphosphepines is summarized in iii) center.2O2 (30% in H2O), BZ-, FBZ-, BTD-, CP-, MI- and -PAN are converted to their oxide derivatives in yields of 80\u201390%. The oxidation of these diazaphosphepines requires four days for complete conversion, which suggests their resistance against oxidation under ambient conditions. That our compounds are stable against moisture is perhaps not surprising given previous studies that have shown aminophosphines having pyrrole moieties to also be stable in water and alcohols. This phenomenon was attributed to the participation of N lone-pair electrons in the ring resonance.31P NMR spectra of the diazaphosphepines confirm the presence of two distinct classes of \u03c0-substituents in these species stemming from the different electronic character of the substituents; the 31P NMR spectra of BZ-, FBZ-, -PBTDs exhibit chemical shifts in the range of 38.5\u201338.7 ppm, which are up-field shifted compared to those of CP-, MI- and -PANs (40.3\u201344.4 ppm). All diazaphosphepines have been fully characterized, the results of which are summarized in ESI.Unlike carbon-based phosphepines, these diazaphosphepines are highly air-stable because the electronegative N atoms draw electron densities away from the P scale\" fill=\"currentColor\" stroke=\"none\">C double bond of BZ and BTD that now bridges the two ortho-indole moieties. This [d]-C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C double bond is, for example, 1.418(2) \u00c5 in BZ-P compared to 1.392 \u00c5 in free-standing benzene,BTD-P compared to 1.42 \u00c5 in free-standing benzothiadiazole.c,e]-C\u2013C single bonds that connect BT and BZ to the indole moieties in BZ-P (1.473(2) and 1.477(2) \u00c5) and BTD-P (1.468(1) and 1.471(1) \u00c5) are shorter than typical C\u2013C single bonds (1.533(3) \u00c5).BZ-P and BTD-P despite the lack of coplanarity.We were able to obtain high quality single crystals of of BZ-P and BTD-P scale\" fill=\"currentColor\" stroke=\"none\">C double bond position to be a promising route for modifying the chemical and electronic structures of heteropines.The crystal structures we have examined thus far are aligned with our expectation based on the electronics of the substituents. We are thus surprised that the crystal structure of MI-PO is substr Fig. S3, which aAN-P also reveals a twisted backbone scale\" fill=\"currentColor\" stroke=\"none\">C double bond and a shorter -C\u2013C single bond, suggesting stronger electronic communication in AN-P. These observations also imply the electronic structure of the [d]-substituents to dictate \u03c0-electron delocalization in diazaphosphepines, with sterics playing a secondary role. The crystal structure of AN-PO scale\" fill=\"currentColor\" stroke=\"none\">C double bond length and -C\u2013C single bond length with that of AN-P, suggesting oxidation of the P-center does not affect the electronic communication between AN and the indole moieties in AN-PO.The crystal structure of backbone , presumaO Fig. S2 shows coBZ-P and CP-P; these compounds comprise the simplest of the aromatic and non-aromatic substitutions, respectively. CP-P are red-shifted compared to those of BZ-P, despite the fact that BZ has the larger conjugated \u03c0-system of the two. Both the absorption and emission spectra of CP-P show strong vibronic structures; these vibronic structures are not observed in the absorption and emission spectra of BZ-P. Additionally, the spectra of CP-P (\u0394\u03bbmax = 974 cm\u20131) show a smaller Stokes shift compared to those of BZ-P (\u0394\u03bbmax = 5641 cm\u20131). The fluorescence quantum yield of CP-P (14.5%) is about three times higher than that of BZ-P (5.4%). Collectively, the photophysics data suggest CP-P to exhibit a more conjugated and rigid structure compared to BZ-P. This assertion is further consistent with our theoretical calculations that reveal CP-P to possess a smaller HOMO\u2013LUMO gap (3.52 eV) compared to BZ-P .We carried out photophysical studies to probe the electronic structures of these diazaphosphepines. We start by comparing d]-C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C double bond experiences strong electronic confinement. This electronic confinement prevents efficient intramolecular electronic communication between the two indole moieties. In contrast, the \u03c0-electrons of the same C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C double bond in CP are less electronically confined and should instead allow more efficient electronic communication between its two indole moieties. This hypothesis is further supported by the comparison between their precursors BZ-In and CP-In that is detailed in Fig. S7.BZ-In is blue-shifted compared to that of CP-In. In addition to electronic effect of the [d]-C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C double bond, the twisted structure observed in crystal of BZ-P could also limit efficient electron delocalization.Given the local aromaticity of BZ, the \u03c0-electrons of the -C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond, it thus exhibits the weakest C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C double bond character, as evidenced by its degenerate HOMO\u20131 calculations to verify the aromatic character of the central seven-membered P-ring in our diazaphosphepines at the GIAO/B3LYP/6-31+g(d) level of theory. The NICS(1) values are all near-0, implying the non-aromatic character of these P-containing seven-membered rings Table S2. This reBTD-P shows a stronger solvatochromic effect than MI-P seems counter-intuitive since MI is a stronger electron-accepting substituent than BTD.BTD-P and MI-P, we carried out theoretical calculations on their excited states. The excited-state structures were optimized by TD-DFT. Their HOMOs and LUMOs were calculated at the B3LYP/6-31+g(d) level. In MI-P shows enhanced planarization in the S\u20321 state along with an elongation of the [d]-C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C double bond, a shortening of the -C\u2013C single bonds and a decrease in the torsion angle compared to its S0 state. This comparison suggests enhanced \u03c0-conjugation in the excited state. The S\u20321 structure of BTD-P, on the other hand, is more twisted, with a shortening of its [d]-C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C double bond and an increase in its torsion angle compared to its S0 state. This opposite trend suggests instead decreased \u03c0-conjugation in the excited state of BTD-P. Looking more closely at BTD-P, photoexcitation leads to a conformational change from its symmetric structure in its S0 state to an asymmetric structure in its S\u20321 state. In its S\u20321 state, the HOMO of BTD-P is mainly localized on one indole moiety, while the LUMO of BTD-P is mainly localized on the BTD substituent. Accordingly, the excited-state molecular dipole moment (2.49 D) is larger than that of its S0 state (1.63 D). Both observations point to enhanced intramolecular charge separation . This difference is consistent with the stronger solvatochromic effect observed in the emission spectra of BTD-P compared to that of MI-P. Further, the excited- and ground-state oscillator strengths of BTD-P are smaller compared to those of MI-P, which is also in line with the lower fluorescence quantum yield observed in BTD-P compared to MI-P. Finally, the calculated Stokes shift of BTD-P (0.78 eV) is larger than that of MI-P (0.28 eV), which is also consistent with experimental data.That paration comparedand LUMO . These oBTD-P) and non-aromatic (MI-P) substituents, the conformation of BTD-P appears unique, even amongst the diazaphosphepines having aromatic substitutions. Whereas the conformations of the excited states of BZ-P and FBZ-P are more planar compared to their ground states scale\" fill=\"currentColor\" stroke=\"none\">C double bond character. In addition to modulating the ground-state electronic structure of diazaphosphepines, our calculations thus show that [d]-C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C engineering tunes the excited state character of these compounds.While the above comparison highlights the ground- and excited-state differences between diazaphosphepines having aromatic , CP-P shows a smaller oxidation potential at 0.65 V that is consistent with its higher theoretical HOMO energy level and stronger \u03c0-conjugation observed in our photophysical studies. Enhancing the electron-accepting characteristics by replacing BZ with FBZ and BTD reduces FBZ-P\u2019s and BTD-P\u2019s susceptibility to oxidation, with BTD-P's oxidation potential outside the scan window. Compared to BZ-P, FBZ-P, and BTD-P, the oxidation potentials of CP-P, MI-P, and AN-P show a weaker dependence on the electronic character of the substituents. In fact, the oxidation potential of MI-P is much lower than those of FBZ-P and BTD-P, despite electron-accepting character of MI is stronger than both of FBZ and BTD. Such easy oxidation of CP, MI, and AN derivatives implies that strong electron delocalization along the backbone of CP-P, MI-P, and AN-P dominates their redox character. This observation is consistent with the trend of theoretical HOMO energy levels determined for these compounds.The introduction of substituents at the -C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C double bond, providing access to electronic properties previously inaccessible in both the ground- and excited-states of heteropines. Particularly, our strategy allows us to access heteropines having much broader absorption and emission compared to the previous protocols. The MI-substituted diazaphosphepine exhibits planar \u03c0-conjugated structure that has not been accessed in non-aromatic heteropines. Having the weakest [d]-C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C double bond character of the current series, the BTD-substituted diazaphosphepine exhibits twisted ICT in its excited state. This excited-state conformation contrasts the planar conformations of previous heteropines in their excited states. With the same chemistry, we have introduced electron-accepting substituents at the [d]-C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C double bond, which has resulted in compounds having more electron deficient character than any previously reported heteropines. Despite their low photocurrents, the operation of devices comprising these materials demonstrates\u2014for the first time\u2014the viability of heteropines as electron acceptors for organic photovoltaics.By leveraging the versatility of P\u2013N chemistry, we demonstrated that we can engineer the [Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "This work reports new (4 + 2)-annulations of \u03b1-alkyl vinylgold carbenes with benzisoxazoles to afford 3,4-dihydroquinoline derivatives with high anti-stereoselectivity. anti-stereoselectivity. The annulations are operable with carbenes in both acyclic and cyclic forms. This reaction sequence involves an initial formation of imines from \u03b1-alkylgold carbenes and benzisoxazoles, followed by a novel carbonyl-enamine reaction to yield 3,4-dihydroquinoline derivatives. This system presents the first alkyl C\u2013H reactivity of \u03b1-alkyl gold carbenes with an external substrate.This work reports new (4 + 2)-annulations of \u03b1-alkyl vinylgold carbenes with benzisoxazoles to afford 3,4-dihydroquinoline derivatives with high D/A) types I whereas highly desirable \u03b1-alkyl metal carbenes II are less efficient because of a competitive 1,2-hydrogen shift to form olefins (eqn (2)).+ carbons are highly cationic.ii) catalysts focused on \u03b1-alkyl metal carbenes of D/A types.D/A carbenes II, aiming at two objectives: (i) suppression of a 1,2-H shift and (ii) an alkyl C\u2013H reaction with an external substrate.Metal carbenes are versatile intermediates to implement a vast number of useful reactions including cyclopropanation, X\u2013H insertion , skeletal rearrangement and annulation reactions eqn 1)).).1 Despi).).1 DesD/A-type benzyl \u03b1-oxogold carbene II\u2032 (R = Ph and EWG = CO2Et), yielding an olefin product III\u2032 efficiently (eqn (3)). We envisage that D/D type carbenes such as \u03b1-alkyl alkenylgold carbenes IV might be viable species to achieve new annulations with benzisoxazoles because their gold-stabilized allyl cation character IV is unfavorable for a 1,2-H shift. According to this hypothesis, this work reports novel intermolecular (4 + 2)-annulations between \u03b1-alkyl vinylgold carbenes and benzisoxazoles, thus manifesting an unprecedented C\u2013H reactivity of \u03b1-alkyl metal carbenes.Interest in the reactions of benzisoxazoles is rapidly growing in gold catalysis because of their various annulation modes with gold \u03c0-alkynes.A that were generated in situ from cyclopropene derivatives 1a\u20131b and gold catalysts.6, quinoline derivatives 3a and 3b were isolated in satisfactory yields (72\u201375%), together with enones 1a-O and 1b-O in minor proportions (17\u201319%). A 1,2-hydrogen shift was effectively suppressed with vinylcarbenes A, supporting our hypothetic role of gold-stabilized allyl cations A.As shown in eqn (5), we further tested the reaction of acyclic alkylgold carbenes via cascade reactions. VI-1)\u2013(VI-6) bearing a common tricyclic framework VI, which can be easily constructed from cyclopentenylgold carbene A\u2032 and benzisoxazole. Indenoquinoline (VI-1) showed antiproliferative activities against breast (MCF-7) and lung epithelial (A-549) cells.VI-2 and VI-3 served as 5HT2c agonists and CRTH2 receptor modulators, respectively.VI-4 and VI-5 were N-containing steroids found in higher plants.VI-6 is a key intermediate for the total synthesis of naturally occurring (\u2013)-isoschizogaline9Our primary interest is to construct complicated frameworks lial A-54 cells.9a4a and benzisoxazole 2a in dichloromethane (DCM) using various gold catalysts; species 4a serves as a precursor for cyclopentenylgold carbene A\u2032 ; the latter was derived from a 1,2-H shift of gold carbenes A\u2032 that was generated from cyclizations of gold-stabilized pentadienyl cation A-I. Notably, an increased gold loading (10 mol%) enhanced the yield of desired 5a up to 85%. Other gold catalysts LAuCl/AgSbF6 (L = P(OPh)3, PPh3 and P(t-Bu)2(o-biphenyl)) gave 5a in 40\u201382% yields with L = P(OPh)3 being the most effective (entries 3\u20135). For various silver salts as in IPrAuCl/AgX (X = OTf and NTf2), resulting 5a was obtained in 65% and 71% yields, respectively (entries 6\u20137). AgNTf2 was entirely inactive (entry 8). IPrAuCl/AgSbF6 in various solvents gave 5a in the following yields: DCE 70%, MeCN 20% and 1,4-dioxane 0 (entries 9\u201311). The molecular structure of compound 5a was characterized with X-ray diffraction,anti-configuration between the alcohol and phenyl groups.An initial test of IPrAuCl/AgSbF4b\u20134t catalyzed with IPrAuCl/AgSbF6 (10 mol%) in DCM. All resulting products 5b\u20135t assumed anti-configurations with the alcohol and R1 groups being mutually trans. We tested the reaction of trisubstituted vinylallenes 4b\u20134f bearing R1 = 4-MePh, 4-OMePh, 4-ClPh, 4-CF3Ph and n-Bu, yielding desired 5b\u20135f in 78\u201388% yields (entries 1\u20135). For species 4g and 4h bearing 3-phenyl substituents (X = OMe and Cl), their corresponding products 5g and 5h were obtained in 84% and 87% yields, respectively (entries 6 and 7). The reactions were extensible to other vinylallenes 4i\u20134k bearing 2-naphthyl, 2-furan and 2-thiophene, further delivering desired products 5i\u20135k in 82\u201384% yields (entries 8\u201310). We tested the reaction on vinylallene 4l bearing distinct R1 = Me and R2 = Ph, which yielded compound 5l with an anti-configuration in which the hydroxy and methyl groups are mutually trans (entry 11); this configuration was established by the 1H NOE effect. Additional alkyl-substituted vinylallenes 4m\u20134p yielded desired 5m\u20135p in satisfactory yields . Variations of the R2 group with an n-butyl group as in species 4q gave expected product 5q in 86% yield (entry 16). We prepared species 4r bearing varied R2 = Ph and R3 = n-butyl, producing compound 5r in 80% yield (entry 17). For 1,3-disubstituted vinylallenes 4s and 4t (R3 = H), their resulting compounds 5s and 5t were obtained in 82\u201383% yields (entries 18 and 19).6a\u20136g, bearing varied phenyl , 2-thienyl and isopropyl substituents; these enyne acetates can be catalyzed with the same gold catalyst to yield distinct \u03b1-alkylgold carbenes A\u2032 -annulation products 7a\u20137g in satisfactory yields (61\u201374%), further manifesting the reaction generality (entries 1\u20137). For unsubstituted propargyl acetate 6h (R = H), its reaction led to a 68% recovery of initial 6h (entry 8). Even if the reaction is successful, a dehydration of compound 7h would occur to give quinoline products. The molecular structure of compound 7a (R = Ph) was confirmed with X-ray diffraction analysis that revealed an anti-configuration (11We tested these new annulations on distinct substrates such as enynyl acetates s A\u2032 see .12 To ouguration .112b\u20132j substituted with the C(3), C(5) and C(6) carbons. Other C(5)-substituted benzisoxazoles 2b\u20132f maintained high efficiencies to deliver anti-configured products 8b\u20138f in 80\u201390% yields (entries 1\u20135). High reaction efficiencies were maintained also for C(6)-substituted benzisoxazoles 2g\u20132i that furnished products 8g\u20138i in 86\u201392% yields (entries 6\u20138). A final applicable reaction with a C(3)-substituted benzisoxazole 2j enabled the production of a tertiary alcohol 8j, reflecting the reaction feasibility (entry 9). 1H NOE spectra were recorded to verify the stereochemistry of compound 8j ).9b was characterized with X-ray diffraction.9a and 9b with a gold catalyst were unsuccessful because of the two different forms of the enol imines (eqn (6)). To rationalize the origin of the stereoselectivity, compound 5a was treated with Zn(OTf)2 (20 mol%) in refluxing DCE to examine the hydroxyl epimerization that turned out to be slow. An equilibrium, anti/syn = 4\u2009:\u20091, was attained for species 5a after reflux in DCE for 48 h (eqn (7)).Gold-catalyzed reactions of 3,5-dimethylisoxazole anti-5avia NaBH4 reductions and m-CPBA oxidations, respectively yielding compounds 5a-H and 5a-O as single diastereomeric products. The stereochemistries of compounds 5a-H and 5a-O were established with 1H NOE spectra. Likewise, the acetate species 7a was readily removed under basic conditions, yielding the enol form 7a\u2032 as shown by its NMR in CD3COCD3 and CDCl3. We also studied an O3-induced oxidative cleavage of the acetate derivative 5a-OAc to cleave the olefin group, yielding the peroxide 35a-O in 85% yield. The molecular structure of species 35a-O has been characterized by X-ray diffraction.11A and benzisoxazole, yielding 2-iminoyl benzaldehyde C. This hypothesis is supported by our observation of 3,5-dimethylisoxazole, depicted in eqn (6). A tautomerization of imine species C is expected to form enamines D bearing an NH\u00b7\u00b7\u00b7O PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C hydrogen bond. We believe that this enamine form, unlike other enamine-carbonyl couplings, PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond to enable a concerted process, analogous to the well-known carbonyl\u2013ene reactions. A boat-like conformation D is favorable to yield anti-5 stereoselectively.As depicted in 1.000000,.000000 s2 to form 3,4-dihydroquinoline derivatives. Gold carbenes in cyclic and acyclic forms are both applicable. In this reaction sequence, the gold complex catalyzes an initial formation of imines between alkylgold carbenesvia a hydrogen bond to induce a carbonyl-enamine reaction. Control experiments with 3,5-dimethylisoxazoles supported this postulated mechanism. This new synthetic design involving \u03b1-alkyl metal carbenes of D/D types will attract growing interest because of its distinct utility.This work reports novel gold-catalyzed (4 + 2)-annulations between alkylgold carbenes and benzisoxazoles There are no conflicts to declare.Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "DFT study to elucidate the mechanism of Rh-catalyzed C\u2013H aminations with N-mesyloxycarbamates and the pathway by which by-products formed. N-Mesyloxycarbamates are practical nitrene precursors that undergo C\u2013H amination reactions in the presence of rhodium dimer catalysts. Under these conditions, both oxazolidinones and chiral amines have been prepared in a highly efficient manner. Given the elevated reactivity of the intermediates involved in the catalytic cycle, mechanistic details have remained hypothetical, relying on indirect experiments. Herein a density functional theory (DFT) study is presented to validate the catalytic cycle of the rhodium-catalyzed C\u2013H amination with N-mesyloxycarbamates. A concerted pathway involving Rh\u2013nitrene species that undergoes C\u2013H insertion is found to be favored over a stepwise C\u2013N bond formation manifold. Density functional calculations and kinetic studies suggest that the rate-limiting step is the C\u2013H insertion process rather than the formation of Rh\u2013nitrene species. In addition, these studies provide mechanistic details about competitive by-product formation, resulting from an intermolecular reaction between the Rh\u2013nitrene species and the N-mesyloxycarbamate anion. Due to their high reactivity, only a limited number of discrete metal\u2013nitrene intermediates have been isolated.ii) dimer complexes, such as Rh2(OAc)4, using carbamate- and sulfamate-derived iminoiodinane reagents, a concerted asynchronous insertion of a singlet Rh(ii)\u2013nitrene has been found in previous computational studies,2(esp)2, an alternative one-electron mechanism involving Rh(ii)\u2013Rh(iii) intermediates has been proposed with a concerted C\u2013H insertion step.The synthesis of sulfonyl-protected amines has also been achieved ii)\u2013porphyrinvia a concerted or a stepwise mechanism. In the case of first row metal complexes reacting with azide reagents, experimental and computational studies imply an open-shell electronic state of the metal nitrene , the resting state of the catalyst, the rate-determining step, the mechanism (stepwise or concerted) and the pathway responsible for the formation of the by-products will be addressed in this computational study.Most computational studies had focused on the structure and reactivity of the metal nitrene species. As a result, little attention has been devoted to the formation of the metal nitrene species, and the type of nitrene precursors applied in C\u2013H amination reactions. Namely, the rate-determining step is unknown for pre-oxidized metal nitrene precursors, such as 2(OAc)4 crystal structure,vide infra) (A number of approximations were tested to determine their accuracy in reproducing the Rhe infra) . The purEst) of the dirhodium\u2013nitrene species ((HO(O)C)4Rh2 PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019NH) was calculated to be 1.4 kcal mol\u20131 with the Coupled-cluster (with) Single (and) Double (and Perturbative) Triple (excitations) (CCSD(T)) method,\u20131, the closest among all other computational methods studied. Conversely, hybrid functional methods afforded singlet\u2013triplet energy difference values superior to 10 kcal mol\u20131.\u20131 with PBE-DKH/BS3).\u03b5 = 5.98) on the gas-phase optimized geometries.The singlet\u2013triplet energy difference (\u03942(OAc)4 was chosen as the model for dirhodiumtetracarboxylate complexes. Although experimentally it was not the most active catalyst for the C\u2013H amination reaction with N-mesyloxycarbamates due to poor solubility, the catalyst provides the desired product with acceptable yields.N-mesyloxycarbamate (A) and n-hexyl N-mesyloxycarbamate (B) as the respective substrates (eqn (3) and (4)). The formation of the primary carbamate and the corresponding carbonyl compound was also studied, as these are known by-products identified namely in reactions with secondary N-mesyloxycarbamates (vide infra). (R)-Phenyl 2,2,2-trichloroethyl N-mesyloxycarbamate (C) is known to undergo C\u2013H amination with 2-phenylethane and was selected to model the intermolecular pathway (eqn (5)).In the interest of computational tractability, RhN-mesyloxycarbamate A and potassium acetate or sodium 2-ethylhexanoate (known to be soluble in organic solvents) produced the potassium or sodium N-mesyloxycarbamate salt as a white precipitate with a small energy barrier of 0.5 to 4.0 kcal mol\u20131 , leading to the most favoured TS and stable Salt-K. Similar results were obtained with sodium acetate and A with slightly higher values for the deprotonation TS , affording the sodium salt . Of note, it is known that lithium bases afford only trace quantities of the desired amination product, likely due to the poor solubility of these reagents. In addition, DFT calculations showed that the energy barrier for the deprotonation TS with lithium acetate and A was substantially higher than that with sodium acetate , confirming the experimental observations, a 7-membered ring coordinated lithium salt was observed 4, the potassium N-mesyloxycarbamate salt first forms a coordination complex (CO-K), in which the carbonyl of the carbamate is coordinated to the apical position of the rhodium complex . The CO-K complex affords the stable nitrenoid species (NRO-K) via the transition state TSCO-K, where both carbamate carbonyl and nitrogen are coordinated to rhodium. The calculated activation barrier is lower than the one for the deprotonation transition state. NRO-K is the most stable metal intermediate, displaying a strong interaction between the nitrogen atom and the rhodium center, with the leaving group (MsOK) still bound to nitrogen. The Rh\u2013nitrogen bond length of NRO-K is calculated to be 2.17\u20132.18 \u00c5 is due to the cleavage of the N\u2013O(Ms) bond to release the mesylate salt. Consequently, the formation of NR from NRO is entropy driven and the positive \u0394G value is a consequence of an increase of the enthalpy. Experimentally, we observed the precipitation of KOMs, which prevented the reversibility of the conversion of NRO-K to NR. The rhodium nitrene derived from N-mesyloxycarbamate C is the most stable, possibly due to a positive electronic effect of the trichloromethyl group.In the presence of Rh complex .83 The s7\u20132.18 \u00c5 . Conforme NRO-KA . Such ancies NR, . The depcies NR, . All thr1NR) and triplet (3NR) rhodium nitrenes for all three models has been calculated has two singly occupied MOs and the two unpaired electrons are mainly localized on the Rh\u2013Rh\u2013N moiety and 0.778 (C), and the configuration of the carbon atom changed from a pyramidal to a planar structure to produce INT. The result of the spin-density analysis shows that INT is a diradical (INT proceeds to the recombination transition state (TSr), the spin density of C decreases to 0.671 (A) and 0.766 (C). The configuration of the C atom returns to pyramidal with a shortening of the C\u2013N interaction to produce TSr. The relative reaction rates for the singlet pathways over the triplet may be estimated according to the transition-state theory (eqn (6)).The reaction barrier for rophilic . The LUMs of 1TS . There id manner . TripletN moiety . The foriradical . As INT ksinglet/ktriplet for the amidation of benzylic intramolecular and intermolecular C\u2013H bonds are 33.7 and 8.4 \u00d7 1010, respectively. Consequently, it appears that the singlet-concerted pathway is predominant and responsible for the formation of oxazolidinone and carbamate products.The relative reaction rates A, B & C) (A & B) are more facile than the intermolecular C\u2013H bond insertion (C) by 7.6 and 4.4 kcal mol\u20131, respectively, as a result of a favourable entropy. CTS is however an early, thus accessible transition state (TSn for the benzylic C\u2013H bond insertion (A) was determined to be 3.2 kcal mol\u20131 lower than the TSn for the singlet aliphatic intramolecular C\u2013H insertion (B), in agreement with experimental results reported. The structural analysis of the corresponding transition states shows that the Rh\u2013N(R) and C\u2013H bonds are 0.04 \u00c5 and 0.23 \u00c5, respectively, longer in BTS than ATS, whereas (H)C\u2013N and N\u2013H(C) bonds are 0.03 \u00c5 and 0.18 \u00c5 shorter, respectively is the resting state of the catalyst, i.e. the turnover frequency (TOF)-determining intermediate (TDI). The energetic span model was used to assess the kinetics of the catalytic cycle.NRO-K as the TDI, the transition state of the C\u2013H insertion (TSn) was established as the TOF-determining transition state (TDTS) or the transition state involved in the rate-limiting step. An energy range of 15.8 and 18.2 kcal mol\u20131 (\u0394\u0394G = \u0394GTSn \u2013 \u0394GNRO), corresponding to the apparent activation energies of the full cycle, was determined for, respectively, the intramolecular amination of benzylic C\u2013H bonds (A) and the intermolecular amination of C\u2013H benzylic bonds (C). The experimental and calculated kinetic isotopic effect also confirms TSn as the rate limiting step . The inton state . Among tectively . TSB is ing step . The proN-sulfonyloxycarbamates derived from secondary alcohols, the corresponding primary carbamate and carbonyl derivatives became the major products. For example, the reaction with trans-4-phenylcyclohexyl-N-tosyloxycarbamate D afforded the ketone and the corresponding primary carbamate in respectively 58% and 15% yields (eqn (7)). The formation of ketones has also been observed in rhodium-catalyzed C\u2013H amination reactions using iminoiodinanes as metal nitrene precursors.in silico data are available to support this hypothesis. With orthohalogenated N-mesyloxycarbamates such as substrate E, only the corresponding primary carbamate was isolated (eqn (8)). Primary carbamates have been observed in other catalyzed C\u2013H amination reactions. Initially, it was postulated that they arose from the homolytic cleavage of the [M]\u2013N bond, as they appeared only when the metal\u2013nitrene species are not rapidly intercepted by the substrate.via hydrogen atom abstraction processes from the substrate.72With a few substrates, namely A, but also from substrates D&E.1TSD is 4.1 kcal mol\u20131 more favourable than the \u03b1-C\u2013H amination 1TSD, leading to the formation of the corresponding ketone scale\" fill=\"currentColor\" stroke=\"none\">C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019NOMs to regenerate DSalt-K.D, this pathway displays an overall free activation barrier shorter than the Rh\u2013N bond (2.09 \u00c5) than the one calculated for A was calculated for a hydride transfer from ESalt-K, forming EINT\u2032 (The hydride transfer hypothesis between (2.09 \u00c5) . Donatioed for A . It was ng INT\u2032E .1NRE potential energy surface revealed an energy barrier of only +4.9 kcal mol\u20131 leading to a less stable imido intermediate EINT\u2032\u2032 with a new C\u2013I (C\u00b7\u00b7\u00b7I = 2.22 \u00c5) bond formed (via the oxygen of the carbamate moiety simultaneously with the C\u2013I cleavage (C\u00b7\u00b7\u00b7I = 2.67 \u00c5) and the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C formation of the alkene.However, if such a process is operative, the aldehyde would have been isolated experimentally. Ethyl acetate (solvent) was then suspected as the hydride source. The transfer to the d formed . The proGsol for the formation of the primary carbamate from 1NRE and EtOAc is \u201323.3 kcal mol\u20131. When compared to the hydride transfer pathway with ESalt-K, the kinetic process is 3.8\u00d7 faster with EtOAc. Given that EtOAc is also the solvent of the reaction, the pathway shown in 4 experimentally.Proton tautomerization affords the primary carbamate. The overall \u0394N-mesyloxycarbamates was presented, where we consider the concerted C\u2013H amination from a closed shell singlet Rh\u2013nitrene species and a stepwise radical mechanism starting from an open-shell triplet Rh\u2013nitrene species. The overall reaction is favoured by \u2248\u201350.0 kcal mol\u20131. The coordination of the anion of the N-mesyloxycarbamate with the catalyst takes place in an associative way, chelating the carbonyl function of the substrate to the catalyst. Upon coordination, the O\u2013Rh bond is substituted by the N\u2013Rh bond through a low activation barrier. With the inner nitrenoid complex identified as the TOF-determining intermediate, potassium plays a relevant role as the coordinative cation holding together the leaving group and the nitrene moiety giving rise to a stable nitrenoid complex. The potassium mesylate leaves the imido-complex through a late transition state where the barrier is reachable at room temperature. After the loss of the salt, the two spin states of nitrene species (singlet and triplet) are both capable of performing the C\u2013H insertion through two competitive paths. A concerted insertion through a singlet rhodium\u2013nitrene species is computed to have a smaller activation barrier, rather than a H-abstraction from the triplet nitrene species, affording the amine product and releasing the catalyst. Along with the kinetic isotopic effect, this computational study has clearly identified the C\u2013H insertion transition state as the one involved in the rate-limiting step. The rhodium catalyst is qualified as a more efficient catalyst for the C\u2013H intramolecular amination reaction under these conditions with a calculated TOF value of 2.3 \u00d7 102 h\u20131 compared to a calculated TOF value of 21 h\u20131 for the analogous intermolecular C\u2013H amination.A computational study of the rhodium-catalyzed C\u2013H amination with N-sulfonyloxycarbamate anion or ethyl acetate) affording the primary carbamate with or without the corresponding carbonyl by-product. Now that the pathway leading to their formation has been established, one can envision that the control of the reaction conditions may in some cases minimize the formation of these undesired products. Given that ketones had also been identified as by-products in other rhodium-catalyzed nitrene C\u2013H insertions,Furthermore, the DFT study has ruled out homolytic cleavage of the [M]\u2013N bond and \u03b1-intramolecular C\u2013H amination as pathways responsible for the formation of ketones and primary carbamates as by-products. There is instead a competitive intermolecular reaction of the singlet rhodium nitrene species with a hydride source (either the There are no conflicts to declare.Supplementary informationClick here for additional data file."} +{"text": "The first isolable germanium chalcogenide complexes 2\u20135 representing heavier congeners of CO and CO2 were synthesised from the germylone adduct 1. 2, their heavier mono- and dichalcogenide homologues, EX and EX2 , are important support materials and/or semiconductors and exist typically as insoluble crystalline or amorphous polymers under normal conditions. Herein, we report the first successful synthesis and characterisation of an extraordinary series of isolable monomeric GeX and GeX2 complexes , representing novel classes of compounds and heavier congeners of CO and CO2. This could be achieved by solvent-dependent oxidation reactions of the new zero-valent germanium (\u2018germylone\u2019)\u2013GaCl3 precursor adduct (bis-NHC)Ge0\u2192GaCl31 (bis-NHC = H2C2, Dipp = 2,6-iPr2C6H3) with elemental chalcogens, affording the donor\u2013acceptor stabilised monomeric germanium(iv) dichalcogenide (bis-NHC)GeIV scale\" fill=\"currentColor\" stroke=\"none\">X) PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019X\u2192GaCl3 and germanium(ii) monochalcogenide complexes (bis-NHC)GeII PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019X\u2192GaCl3 , respectively. Moreover, the reactivity of 4 and 5 towards elemental sulphur, selenium, and tellurium has been investigated. In THF, the germanium(ii) monoselenide complex 4 reacts with activated elemental selenium to afford the desired germanium(iv) diselenide complex 3. Unexpectedly, both reactions of 4 and 5 with elemental sulphur, however, lead to the formation of germanium(iv) disulfide complex 2 under liberation of elemental Se and Te as a result of further oxidation of the germanium centre and replacement of the Se and Te atoms by sulphur atoms. All novel compounds 1\u20135 have been fully characterised, including single-crystal X-ray diffraction analyses, and studied by DFT calculations.In contrast to molecular CO and CO Generally, they adopt polymeric structures owing to the relatively weak p\u03c0\u2013p\u03c0 bond between germanium and the respective chalcogen atom and the high polarity of the E\u2013X bond. It has been shown that the molecular variants of EX and EX2 can exist in condensed cryogenic matrices at very low temperature or diluted in the gas phase at high temperature. Thus they have solely been detected spectroscopically4The binary group 14\u201316 compounds, EX and EX PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019X 2 are still very rare. The first examples include the Sn PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O (I) and Pb PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O (II) units stabilised by a benzannulated bis-stannylene reported by Hahn and co-workers scale\" fill=\"currentColor\" stroke=\"none\">Se complex in {[K(18-crown-6)]-[K(en)2]K[Rh3(CN)2(PPh3)4(\u03bc3-Se)2(\u03bc-PbSe)]}2\u00b73en (III) as another type of EX coordinating to Rh sites complexes as precursors. By employing an N-heterocyclic carbene (NHC) (NHC = C:, Dipp = 2,6-iPr2C6H3), the disilicon(0) complex (NHC)Si PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019Si(NHC) was synthesised by Robinson and co-workers,2O4IV stabilised disilicon(0) complex (CAAC)Si PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019Si(CAAC),2 (V) and SiSe2 (VI) complexes could be obtained (2C2) we succeeded in the synthesis of a cyclic zero-valent monosilicon complex (\u2018silylone\u2019)3, namely (bis-NHC)SiS2 and (bis-NHC)Si scale\" fill=\"currentColor\" stroke=\"none\">S)SGaCl3 scale\" fill=\"currentColor\" stroke=\"none\">X and X PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019Ge PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019X complexes. Herein, we wish to present a series of unprecedented germanium analogues of both CO and CO2 utilizing the donor\u2013acceptor stabilisation strategy.During the last decades, the concept of kinetic and/or thermodynamic stabilisation has enabled great achievements in synthesising isolable low-coordinate group 14 element species as ligands in complexes. Several unusual compounds featuring elusive terminal E-workers .5i Very Rh sites .6g In thrs,2O4IV , a complobtained .8b,c In Cl3 VII; .11 In faB, B is sensitive not only towards air and moisture, but also to visible light. For its reactivity investigation, we introduced the Lewis acid GaCl3 to prepare the more stable germylone\u2013GaCl3 adduct 1 through a one-pot reaction, starting from the bis-NHC supported chlorogermyliumylidene chloride (bis-NHC)GeCl2A species (germylone C)GeCl2A .10 Accor work-up .1 reflects the coordination of the germanium centre via one of its lone pair of electrons to the GaCl3 moiety, thus lowering the symmetry of the molecule relative to that of its precursor germylone B. Therefore the spectrum of 1 exhibits four doublets for the methyl protons and two septets for the methine protons in the isopropyl groups. Moreover, the two geminal protons in the CH2 moiety on the backbone in 1 give two doublets with 2JHH = 13.3 Hz \u00c5 in 1 is longer than that in germylone B (Ge\u2013C 1.965(2) \u00c5). PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CH\u2013N(Dipp)]Ga\u2013Ge[N(Dipp)]2CN(iPr)2.12The proton NMR spectrum of ric unit . A singl1 is much more stable than its precursor B because the zero valent germanium center is coordinated to the Lewis-acid GeCl3 by donating one of the two lone pairs of electrons to the electron-deficient gallium atom. It is noteworthy that germylone B also reacts readily with other Lewis acids such as AlBr3, BCl3, but the desired products could not be isolated in pure form as yet. However, B reacts smoothly with GaCl3 to afford 1 as an isolable product. Compound 1 is soluble in THF and acetonitrile. Treatment of THF solutions of 1 with 1/4 molar equivalents of X8 leads to quantitative formation of 2 and 3, which could be isolated in excellent yields as colourless and pale yellow solids, respectively in THF or acetonitrile solutions of the respective bis(NHC) ligand in the presence of GaCl3 failed. Likewise, other alternative approaches to synthesize 2 or 3 from the respective bis(NHC)GeCl4 precursor and in situ prepared M2X salts in the presence of GaCl3 were also unsuccessful which highlights the benefit of the reported synthetic method to form isolable monomeric GeX2 complexes. Compounds 2 and 3 are insoluble in hydrocarbons and only scarcely soluble in polar organic solvents such as THF and CH3CN as shown by a series of ESI-MS experiments. Thus only their 1H NMR spectra could be recorded in solutions but their low solubility prevents VT-NMR spectroscopy at low temperature.Complex ectively . We note2 and 3 suitable for X-ray diffraction analyses were obtained in dilute THF solutions. Unexpectedly, compounds 2 and 3 exhibit higher symmetry than 1, that is, only two doublets for the methyl protons in the CH(CH3)2 groups, instead of four doublets as in 1, could be detected in their 1H NMR spectra, suggesting the statistically equal position of the GaCl3 moiety between two sulphur or selenium atoms in solution, respectively GeS2 + H]+) and at m/z = 703.08749 GeSe2 + H]+), respectively.Fortunately, single-crystals of y see ESI. The ESI2 and 3 crystallise isotypic in the monoclinic space group Cm with two lattice THF molecules in the respective asymmetric unit and the single-crystal X-ray diffraction analyses revealed them to be isostructural, with each of the germanium centres bound to two chalcogen atoms (2 and Se1\u2013Ge1\u2013Se2 angle of 115.2(1)\u00b0 in 3, respectively, are reminiscent of the corresponding S\u2013Si\u2013S value observed in (bis-NHC)Si scale\" fill=\"currentColor\" stroke=\"none\">S) PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019S\u2192GaCl3 (115.0(1)\u00b0).2 and 91.8(3)\u00b0 in 3 are larger than that in 1 (85.7(1)\u00b0): accordingly, the Ge\u2013C bond distances (1.998(3) \u00c5 in 2 and 1.987(5) \u00c5 in 3) are slightly shorter than that in 1 (2.038 \u00c5).Compounds en atoms . Thus th2 (2.087(1) vs. 2.198(1) \u00c5) and the two Ge\u2013Se bonds in 3 (2.214(1) vs. 2.326(1) \u00c5) show a significant difference. The Ge1\u2013S1 bond length of 2.087(1) \u00c5 in 2 is slightly longer than that in Tbt(Tip)Ge PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019S (2.049 \u00c5), PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019S double bonds in LGe scale\" fill=\"currentColor\" stroke=\"none\">S)SH (2.064(1) \u00c5) PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019S)\u2013SPh 2.071(1) \u00c5 ,3 is slightly longer than that in Tbt(Tip)Ge PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019Se methyl]phenyl, Tip = 2,4,6-triisopropylphenyl), PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019Se group in LGe scale\" fill=\"currentColor\" stroke=\"none\">Se)\u2013SePh (2.205(1) \u00c5, L = aminotroponiminato ligand),3 is only slightly shorter than the Ge\u2013Se single bond in the latter complex (2.367(1) \u00c5).2 and 3 can be understood in light of the calculations presented below.The two Ge\u2013S distances in 2 and 3 is still unknown, it is reasonable to propose that the dichalcogenide formation occurs stepwise via the respective monochalcogenides as shown in 1 by one chalcogen atom to yield the divalent GeX complex C. The subsequent GaCl3 migration from the Ge(ii) centre to the more Lewis-basic chalcogenide site affords D bearing a three-coordinate Ge(ii) centre. Subsequent oxidation of the latter from Ge(ii) to Ge(iv) by an additional chalcogen atom results in the final product 2 or 3, respectively. These suggestions are supported by results of DFT calculations.1 \u2192 2 (and 3) are shown in Fig. S7 in the ESI.3 fragment, C \u2192 D is 29.9 and 29.5 kcal mol\u20131 for X = S and X = Se, respectively, in the gas phase (see details in the ESID (X = S) to 2 is by 5.0 kcal mol\u20131 more exothermic than for D (X = Se) to 3 (gas phase). The overall reaction from 1 to 2 (X = S) is more exothermic than from 1 to 3 (X = Se) by 10.3 (gas phase) 11.3 (CH3CN), and 10.9 (THF) kcal mol\u20131, in line with the experimental observation of a faster reaction for X = S than for X = Se.14Although the mechanism of formation of t, C \u2192 D , is exotC and/or D, 1/8 equivalent of S8 and Se8 were employed for the reaction with 1 at \u201330 \u00b0C in THF solutions. However, even at low temperature, regardless of the ratio of the two reactants, the dichalcogenides 2 and 3 are the exclusive products. In contrast, by employing acetonitrile as solvent the reaction of 1 with 1/8 equivalents of S8 at room temperature became much slower than in THF, and the reaction afforded a mixture containing 2 and, presumably, C and/or D (X = S). Furthermore, the reaction of 1 with 1/8 molar equivalents of activated selenium Se8 at room temperature became so slow that the formation of the germanium(ii) monoselenide complex 4, the selenium version of D (4 could be isolated from the resulting solution as a colourless solid in moderate yield (64%). For the reaction of 1 with elemental tellurium, the reaction is even slower than that with selenium and furnishes in THF at room temperature only the germanium(ii) monotelluride complex 5 in the 1H NMR spectrum of 4 (AB-spin system). The ESI-MS spectrum of 4 (positive mode) shows an ion peak of [M \u2013 SeGaCl3 + Cl]+ at m/z = 577.21454 corresponding to the [(bis-NHC)GeCl]+ (+A) cation. Indeed, the facile formation of similar species with a [LECl]+ cation naphthalene, E = Si, Ge) have been described in our previous reports.15Compound 4, the 1H NMR spectrum of 5 exhibits four doublets for the methyl protons and two septets for the methine protons in the CHMe2 groups. Similarly, the bridging N\u2013CH2\u2013N protons are coupled with each other with 2JHH = 13.1 Hz (AB-spin system). Similar to that of 4, in the ESI-MS spectrum of 5 a signal at m/z = 577.21399 centre is stabilised by the chelating bis-NHC ligand and the Ge PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019Se moiety is supported by GaCl3 coordination, leading to a Ge\u2013Se\u2013Ga angle of 110.7(1)\u00b0. The three-coordinate Ge(ii) centre features a pyramidal coordination geometry with a sum of angle of 289.5\u00b0. The average Ge\u2013C distance of 2.050(3) \u00c5 in 4 is very close to that observed in 1 (2.038(3) \u00c5). The Se1\u2013Ga1 length of 2.337(1) \u00c5 is comparable to that in 3 (2.374(1) \u00c5) with a similar coordination environment. The Ge1\u2013Se1 distance (2.438(1) \u00c5) is significantly longer than the two Ge\u2013Se bonds in the four-coordinate germanium diselenide complex 3 (2.214(1) and 2.326(1) \u00c5). It is also longer than the Se\u2013Ge length of 2.346 \u00c5 in the (CO)5W PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019Ge(SeAr)2 germanium(ii) species with a three-coordinate planar Ge centre. PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019Se double bond character can not be concluded from the Ge\u2013Se distance. This is also manifested in the resonance structures 4\u2032\u2032 calculated by NRT which are discussed below.Compound ric unit , left. T5 suitable for X-ray diffraction analysis were obtained from THF solutions. 5 crystallises in the triclinic space group P1[combining macron] with three lattice THF molecules in the asymmetric unit. The latter analysis revealed a similar structure to that of 4 \u00b0. The germanium centre also adopts a trigonal\u2013pyramidal coordination geometry with a sum of bond angle of 285.8\u00b0 around the germanium atom. The average Ge\u2013C bond length of 2.037(4) \u00c5 in 5 is close to those in its precursor 1 (2.038(3) \u00c5) and in 4 (2.050(2) \u00c5). The Ge1\u2013Te1 distance of 2.654(1) \u00c5 is significantly longer than that in a four-coordinate germanium(iv) species LGe(R) PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019Te\u2013GeCl2 (2.461(7) \u00c5) .4, the Ge\u2013Te double bond character in 5 is rather low. It is noteworthy that the 125Te NMR resonance of 5, similar to the 77Se NMR resonances of 3 and 4, could not be observed, presumably owing to the zwitterionic nature of 3 and 4 to sulphur, spontaneously yielding F. Subsequent displacement reaction of the latter should afford 2 as the final product. The latter scenario is supported by DFT calculations. For instance, the calculated reaction free energy (at B3LYP-D3(BJ)/def2-SV using the PCM model for the solvents' effect) of 4 + 1/4 S8 \u2192 2 + 1/8 Se8 is \u201321.6 (gas phase), \u201325.2 (acetonitrile) and \u201322.6 (THF) kcal mol\u20131. The migration from E to F is nearly thermo-neutral (\u0394G = \u20130.74 (gas phase), \u20131.60 (acetonitrile), and \u20130.64 (THF) kcal mol\u20131).14Since the germanium centres in ectively . Althoug4 with activated selenium can afford compound 3 in CH3CN. However, after 24 h only 10% of 4 reacted. In contrast, in THF the reaction is complete after 2 h. This may explain why 4 could be isolated in CH3CN, whereas only 3 was isolable in THF. On the other hand, no reaction of 5 with elemental selenium in both THF and CH3CN could be detected after 24 h, confirming that 5 is less reactive than 4. Furthermore, the reactivity of 4 towards tellurium in THF at room temperature was probed, however, after three days, no reaction occurred.As expected, the reaction of 1\u20135 and for the respective model compounds 1\u2032\u20135\u2032 where the bulky Dipp groups are replaced by Ph groups.1\u20135 at B3LYP-D3(BJ)/def2-TZVPP and those of 1\u2032\u20135\u2032 optimized at B3LYP-D3(BJ)/def2-SV are in good agreement with the corresponding X-ray structures.To obtain better understanding of the electronic properties and reactions of the novel compounds described in this article we performed DFT calculations for the synthesised compounds 2\u2032, X = S, and 3\u2032, X = Se) of 2.091 \u00c5 and 2.231 \u00c5 are longer than those of Me2Ge PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019X , respectively. This trend is in line with the Ge\u2013X Wiberg Bond Indices (WBI)2Ge PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019X are larger than those of 2\u2032 (1.39) and 3\u2032 (1.36). The calculated Ge PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019X bond length and WBIs in linear X PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019Ge PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019X are 2.016 \u00c5 and 1.79, respectively, for X = S, and 2.145 \u00c5 and 1.82, respectively, for X = Se. The significantly smaller WBIs in 2\u2032 and 3\u2032 and the longer bond distances . The NCN bonds are 3-centre 4-electron bonds. This is manifested in the resonance structures which involve these electrons, i.e., N1 PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C:N2 \u2194 N1:C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N2, and which are reflected in the C\u2013N WBI of ca. 1.25, suggesting a partial double bond character. The Ge\u2013C bond length is 1.998 \u00c5 and 1.987 \u00c5 in 2 and 3, respectively. The small WBI may reflect the contribution of resonance structures in which the Ge is bound to only one carbene unit (see below). The Ge\u2013C bonds are longer than that in the germylone precursor B (average r(Ge\u2013C) = 1.963 \u00c5, exp.et al., of 1.940 \u00c5 and 1.954 \u00c5.20The calculated Ge1\u2013X1 distances , feature two resonance structure types which are responsible for ca. 75% of the total contributions scale\" fill=\"currentColor\" stroke=\"none\">Ge\u2013X\u2013GaCl3 subunit with a X1 PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019Ge double bond and where the Ge is bound to only one of the carbenes, accounting for 23%, for both X = S and X = Se, of the total. (b) Structures containing a X\u2013Ge\u2013X\u2013GaCl3 subunit with a bond between the Ge and each of the NHC units. These structures account for 53% (X = S) and 55% (X = Se) of the total. Many electron permutations in the NHC rings are possible for both resonance structure types and the values in The dichalcogenides ibutions : (a) str4 and 5 scale\" fill=\"currentColor\" stroke=\"none\">X double bond in 4 and 5. This is supported by the NRT calculations for 4\u2032\u2032 and 5\u2032\u2032 R = Me which show that the major contribution stems from the resonance structures shown in PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019X double bond.The relatively long Ge\u2013Se and Ge\u2013Te distances in the monochalcogenides 1\u2032\u20135\u2032 is shown in Fig. S6 in the ESI.IVX2GaCl3 and GeIIXGaCl3 fragments. The positive charge on the Ge atom decreases along the series X = S > Se > Te in 2\u2032, 3\u2032, 4\u2032 and 5\u2032, while the charge on the GaCl3 fragment increases along this series. Our results indicate that the chalcogen\u2013GaCl3 interaction is strong. In fact, attempts to remove the coordinated GaCl3 from 2\u20135 by using strong external Lewis bases such as \u2018free\u2019 NHC with methyl ligands at nitrogen or less substituted bis-NHCs or chelating bis-thiols had been performed, but no reaction could be observed. At elevated temperature decomposition occurred, leading to hitherto unidentified mixtures.The calculated Natural Population Analysis (NPA) charge distribution in compounds 1, the Ge(0) atom can be readily oxidised with elemental chalcogens to form, in solvent-dependent reactions, the first donor\u2013acceptor stabilised isolable monomeric germanium disulfide 2, diselenide 3, monoselenide 4, and monotelluride complex 5, respectively. They represent novel classes of heavier congeners of CO and CO2 complexes. Apparently, the presence of the GaCl3 Lewis acid is essential for the stabilisation of all monomeric species bearing highly polar Ge PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019X bonds. In THF, the germanium(ii) monoselenide complex 4 can further be oxidised with activated selenium to yield the corresponding germanium diselenide complex 3. Unexpectedly, both selenide and telluride compounds 4 and 5 react with elemental sulphur to produce 2 with liberation of elemental selenium and tellurium, respectively. The unusual structural, spectroscopic, and electronic properties of these novel species could be determined and analysed by combined experimental and computational investigations. One of the important lessons from the calculations is that the bonding framework in all these compounds is complex and cannot be described properly by a single valance bond structure. Currently, we continue to explore the synthesis of Lewis acid-free germanium and silicon chalcogenide complexes and to exploit their reactivity in the context of small molecule activation and ligand ability in metal coordination chemistry. The strategy of donor\u2013acceptor stabilisation is also expected to pave the way to isolable monomeric SiO and SiO2 complexes. Respective studies are currently in progress.Due to the substantial Lewis acid stabilisation of the Ge(0) atom in Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "Controlled oxidation of the terminal C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C triple bond using O2 (1 atm) as an oxidant and reagent. PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C alkynes to \u03b1-keto esters and quinoxalines via formation of phenylglyoxals as stable intermediates, under mild conditions by using molecular O2 as a sustainable oxidant. The current copper catalysed photoredox method is simple, highly functional group compatible with a broad range of electron rich and electron poor aromatic alkynes as well as aliphatic alcohols , providing an efficient route for the preparation of \u03b1-keto esters (43 examples), quinoxaline and naphthoquinone with higher yields than those in the literature reported thermal processes. Furthermore, the synthetic utility of the products has been demonstrated in the synthesis of two biologically active molecules, an E. coli DHPS inhibitor and CFTR activator, using the current photoredox process. In addition, we applied this methodology to the one-pot synthesis of a heterocyclic compound by trapping the intermediate phenylglyoxal with O-phenylenediamine. The intermediate phenylglyoxal can also be isolated and further reacted with an internal alkyne to form naphthoquinone. This process can be readily scaled up to the gram scale.Herein, we report a facile visible light induced copper catalyzed controlled oxidation of terminal C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C triple bonds by transition metal complexes in the presence of molecular O2 plays an important role in the chemical industry. PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C triple bonds to generate over-oxidized products.i)-catalysed cross-coupling and C\u2013H annulation reactions.i) phenylacetylide is the key photocatalyst involved in these visible light induced coupling reactions.i) phenylacetylide in the presence of molecular oxygen can generate Cu(ii)-phenylacetylide and a superoxide radical anion via the single electron transfer (SET) process. PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C triple bond of a terminal alkyne.via controlled oxidation. \u03b1-Keto ester analogues are considered to be valuable precursors and intermediates for various pharmaceuticals and bioactive molecules.et al. reported the photoredox catalyzed synthesis of \u03b1-keto esters via oxidation of \u03b1-aryl halo derivatives using an expensive ruthenium catalyst under sunlight irradiation controlled oxidation of the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C triple bond to phenylglyoxals, and thus no formation of ester or homo-coupling byproducts, and (d) a broad substrate scope and compatibility with a wide range of aromatic alkynes and 1\u00b0, 2\u00b0, or 3\u00b0 alcohols. To the best of our knowledge, the use of terminal alkynes as a key starting material for the synthesis of \u03b1-keto esters under visible light is yet to be reported.The significance of the present work includes the following: (a) this is the first example of oxidation of terminal alkynes to \u03b1-keto esters under visible light at room temperature under mild conditions; (b) low toxic, inexpensive CuI was used as a catalyst and abundant O1a) (0.5 mmol), MeOH (2a) (2 mL), copper iodide , and 2-picolinic acid (1.0 equiv.) as a ligand in CH3CN (4 mL) in the presence of molecular O2 was irradiated under blue LEDs at room temperature for 12 h, it furnished the desired \u03b1-keto ester (3a) with a yield of 86% was low and either the reaction failed or gave trace amounts of the desired product , n-propanol (2c), n-butanol (2d) and 2-methylpropan-1-ol (2e), and the desired product (3b\u2013e) was obtained in good yields at room temperature (2f) and benzyl alcohol (2g) providing \u03b1-keto esters (3f and 3g) in good to excellent yields under similar reaction conditions and tetrahydrofurfuryl alcohol (2i) reacted well with 1a to produce 3h and 3i in 84% and 68% yields, respectively, without cyclic ring opening. Next, 1a reacts with 2\u00b0 alcohols (2j\u20132l) smoothly to afford the corresponding \u03b1-keto esters (3j\u20133l) in good yields. Slightly strained or labile alcohols worked well in this protocol, without producing any cleavage products, which is not possible using the earlier thermal processes. Besides, 1a reacts with alicyclic 2\u00b0 alcohols (2m\u20132o) to afford the desired products (3m\u20133o) in good yields (p-benzoquinones in the presence of copper and O2.1a with tertiary butanol (2p) provided \u03b1-keto ester 3p in 70% yield with equal moles of 1\u00b0, 2\u00b0 and 3\u00b0 alcohols, such as MeOH (2a), isopropanol (2j) and tertiary butanol (2p), under standard conditions was surveyed, which afforded \u03b1-keto ester 3a as a major product in 73% yield derived from the 1\u00b0 alcohol, i.e., MeOH. Product 3j derived from the 2\u00b0 alcohol was formed in trace quantities without any \u03b1-keto ester 3p resulting from tertiary butanol. For nucleophilic attack on the glyoxal aldehyde, the 3\u00b0 alcohol is expected to be better than the 2\u00b0 alcohol and 1\u00b0 alcohol. This observed result clearly indicates that steric hindrance plays a more important role than the electronic factor, which leads to a predominance of the primary alcohol in the coupling reaction.Interestingly, cyclopropanemethanol (d yields . Unfortu0% yield . It is w4b\u20134j) with good to excellent yields as depicted in 3, CN, nitro, acetyl, ester, sulfone, and methoxy) groups showed excellent tolerance in the current photoredox protocol to give the corresponding \u03b1-keto esters (4k\u20134t) in good yields in moderate to good yields. However, heterocyclic alkynes ethynyl indole and ethynyl pyrimidine failed to give the desired \u03b1-keto esters under the current protocol. This protocol was successful in producing \u03b1-keto ester 4wa in 83% yield when heteroaryl alkyne 5-ethynyl-1,3-benzodioxole was used under similar conditions. Unfortunately, aliphatic terminal alkynes did not work for this protocol and failed to produce the corresponding \u03b1-keto esters as products. The reason for the failure of the aliphatic alkynes is most probably due to the lower acidity of aliphatic terminal alkynes as compared to aromatic ones, thus making the step of formation of copper phenylacetylide from aliphatic alkynes slower than that from aromatic alkynes.Note that when tertiary butanol was coupled with 1,3-dialkyne, only mono \u03b1-keto ester i)-catalyzed strategy was demonstrated for the expedient synthesis of compounds with biological activity, such as 1-benzyl-3-(3-nitrophenyl)quinoxalin-2(1H)-one 6n (a CFTR activator)5t (an E. coli DHPS inhibitor).6n could be carried out in 3 steps in 44% overall yield after irradiation with blue LEDs for 12 h at room temperature (ESI) and we have further evaluated and compared the green chemistry metrics o-phenylenediamines.2 as an oxidant. So, under the same reaction conditions, we added 1.0 equiv. of 4,5-dimethylbenzene-1,2-diamine (7) to the reaction solution and irradiated it with visible light for 12 h at room temperature (8), which is a biologically active FLT3 inhibitor,Synthesis of quinoxaline perature . Not sur-diamine to the ri)-phenylacetylide 1a\u2032 was reacted with MeOH, in the absence of CuI under similar reaction conditions, which produced the desired \u03b1-keto ester (3a) with 40% yield after 12 h of irradiation (eqn (1), To provide detailed insights regarding the reaction mechanism, we carried out several control experiments, as shown in i)-phenylacetylide powder exists in highly aggregated forms.in situ-generated Cu(i)-phenylacetylide is most probably the key light-absorbing photocatalyst involved in this oxidative coupling reaction. Next, we performed a short-time reaction of 3 h, under the optimal conditions, and we were delighted to isolate phenylglyoxal 13 as a stable intermediate in 62% yield (eqn (2), 2 mediated oxidation of substituted methyl ketones under harsh reaction conditions.13 for better understanding of the reaction mechanism. First, the reaction of phenylglyoxal with MeOH was carried out in the presence of light and O2, but in the absence of the CuI catalyst, which led to no formation of \u03b1-keto ester 3a (eqn (3), 2, but in the absence of the 2-picolinic acid ligand, only a trace amount of 3a was formed (eqn (4), 2, and blue light irradiation, 3a was produced in 90% yield (eqn (5), 2 atmosphere, no formation of 3a was observed (eqn (6), 2, and blue light irradiation all are very crucial factors for the formation of the \u03b1-keto ester product 3a.The reduced yield can be attributed to the fact that the isolated Cu scale\" fill=\"currentColor\" stroke=\"none\">C bonds via controlled oxidation to phenylglyoxal. It is documented in the literature that nitrogen containing ligands can reduce the formation of polymeric byproducts and Glaser alkyne\u2013alkyne homocoupling products.ii) bis-picolinate ligand, instead of a catalytic amount, is required to achieve the optimal product yield. Next, phenylglyoxal 13, isolated from the current photoredox process, could readily react with an internal alkyne for the synthesis of 1,2-naphthoquinone 16, via oxidative annulation reaction13 with MeOH was carried out under O2 in the absence of light, i.e., under dark conditions, which leads to no formation of \u03b1-keto ester 3a (eqn (9) and (10), i)-phenylacetylide, and is responsible for controlled aerobic oxidation of phenyl glyoxal to \u03b1-ketoesters.Selective oxidation of terminal alkynes to glyoxal, free from the subsequent over-oxidation to glyoxalic acid, is a very challenging reaction in synthetic chemistry.18O2 (98%), instead of 16O2 air, under the standard conditions -phenylacetylide (1a\u2032) triplet excited state Cu(i)-phenylacetylide (9)via ligand to metal charge transfer (LMCT).i)-phenylacetylide then donates an electron to molecular O2 to generate a superoxide radical anion (O2\u02d9\u2013) and an electron deficient Cu(ii)-phenylacetylide (10),ii)-phenylacetylide and subsequent reaction to molecular O2 results in the formation of copper(iii)-superoxo complex 11.iii)-peroxo complex (11)occurs with concurrent formation of a C\u2013O bond to form the intermediate (12).12) produces 2-oxo-2-phenylacetaldehyde (13) and CuII(pic)2 was eliminated as a blue ppt . When 4,5-dimethylbenzene-1,2-diamine (7) was present in the reaction mixture, it trapped the in situ-generated phenylglyoxal 13via intermolecular double condensation reaction to produce 6,7-dimethyl-2-phenylquinoxaline (8) in a one-pot manner, as shown in Based on the above control experiments and our previous studies,o-phenylene diamine acts as a better nucleophile (N is less electronegative than O) to phenylglyoxal than alcohol (2), thus favouring the formation of 2-phenyl quinoxaline (8), instead of the formation of hemiacetal (14).In the presence of 4,5-dimethylbenzene-1,2-diamine, the formation of \u03b1-keto esters was suppressed, due to the fact that PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C triple bond to phenylglyoxal at room temperature, followed by esterification, for the synthesis of \u03b1-keto esters that evades the need for a base, an expensive catalyst, strong oxidants, elevated temperatures and other harsh reaction conditions. The reaction proceeds easily with excellent functional group tolerance towards the electron donating and withdrawing terminal alkynes. Moreover, it is compatible with 1\u00b0, 2\u00b0, and 3\u00b0 alcohols and slightly strained or labile alcohols, which is not possible or difficult in thermal processes. The utility of this protocol has also been successfully applied for the synthesis of two biologically active molecules, i.e., 1-benzyl-3-(3-nitrophenyl) quinoxalin-2(1H)-one (a CFTR activator) and bis oxime ester (an E. coli DHPS inhibitor) on a gram scale with fewer steps and higher total yields than those in the literature reported processes. We have also demonstrated the one-pot synthesis of a pharmacologically active heterocyclic compound, i.e., 2-phenyl quinoxaline (an FLT3 inhibitor) via an unprecedented photoredox copper catalyzed process, as well as the synthesis of naphthoquinone using phenylglyoxal isolated from the current photoredox process.In summary, we have developed an unprecedented visible light induced copper catalyzed process for the controlled aerobic oxidation of the terminal CThere are no conflicts to declare.Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "A supramolecular-gel-based twenty-two-member sensor array has been created by introducing well-designed multi-competitive binding interactions into a supramolecular gel. \u2013, Cl\u2013, I\u2013, CN\u2013, HSO4\u2013, SCN\u2013, S2\u2013, OH\u2013, Al3+, Fe3+, Zn2+, Hg2+, Pb2+ and H+) in water. It's important to note that this sensor array needs only one synthesized receptor. Moreover, using this method, we also obtained a series of ion response fluorescent supramolecular materials, which could act as security display materials. Therefore, it's a novel and facile way for the design of a simple sensor array as well as ion response fluorescent supramolecular materials.Sensor arrays are a powerful tool for multianalyte sensing and the development of an efficient sensor array has become one of the most intriguing problems. However, sensor arrays often employ lots of receptors which need large amounts of work to synthesise. This study describes an efficient method for the fabrication of a simple sensor array based on the competitive binding in supramolecular gels. By rationally introducing various well-designed competitive binding interactions into the supramolecular gel, which is self-assembled from a naphthylhydrazone-based organogelator, a supramolecular gel-based twenty-two-member sensor array has been created. Interestingly, the sensor array has been shown to accurately identify fourteen kinds of important ions (F It is worth mentioning that the sensor array was constructed using only one receptor. Simply stated, in the twenty-two-member sensor array, the organogelator not only acts as a gelator, but also as a receptor. The selective sensing properties of the sensor array are accurately controlled by the various competitive binding interactions which we rationally induced in the gel.Fortunately, the rapid development of stimuli-responsive supramolecular gelsG by introducing a coordination site, multi-self-assembly driving forces, and fluorescent signal groups into the gelator molecule. For example, we introduced an acylhydrazone group and a hydroxy group as the coordination, hydrogen-bonding and recognition sites, a naphthyl group as the signal group and \u03c0\u2013\u03c0 stacking site and long alkyl chains as the strong van der Waals forces group. The gelation abilities of G were examined in various solvents by means of the \u201cstable to inversion of a test tube\u201d method (Table S1G showed excellent gelation abilities in various solvents such as dimethyl formamide (DMF), dimethyl sulfoxide (DMSO), acetone, ethanol, n-propyl alcohol, isopropanol, n-butyl alcohol, isoamyl alcohol, n-hexanol, cyclohexanol, ethyl acetate, CCl4, petroleum ether and so on. Among these solvents, the gelator G showed the lowest critical gelation concentration (CGC) and the highest gel\u2013sol transition temperature (Tgel) in n-butyl alcohol formed in n-butyl alcohol.Firstly, as show in Table S1. As we e Table S1. TherefoOG was investigated using 1H NMR, IR, X-ray and SEM. In the concentration dependent 1H NMR , \u2013NH (Hb) and \u2013N PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CH (Hc) resonance signals showed obvious downfield shifts as the concentration of G rose. These results revealed that in the gelation process, these groups formed hydrogen bonds with the \u2013C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O and \u2013N PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C groups on the adjacent gelators. Moreover, the IR studies also confirmed this result, as shown in Fig. S4a,G, the stretching vibration of \u2013OH and \u2013NH appeared as a broad peak at 3202 cm\u20131, while, owing to the formation of hydrogen bonds, in the organogel OG, these absorptions were shifted to 3380 and 3244 cm\u20131, respectively. On the other hand, with the gradual increase in concentration, the 1H NMR signal of the naphthyl protons showed an obvious upfield shift, indicating that the \u03c0\u2013\u03c0 stacking interactions between the naphthyl groups were involved in the gelation process.\u03b8 24.5\u00b0 (d = 3.96) also confirms the \u03c0\u2013\u03c0 stacking interactions. Thus, the gelator G self-assembled to organogel OG through the hydrogen bonds and \u03c0\u2013\u03c0 stacking as well as the vdW existing in the long alkyl chains. The morphological features of the organogel OG were studied using SEM G has no fluorescence in hot n-butyl alcohol solution (T > Tgel). However, with the temperature of hot n-butyl alcohol solution dropping below the Tgel of OG, the emission intensity at 475 nm showed a sudden increase and reached a steady state, which indicated that the turquoise fluorescence of OG was AIE. Meanwhile, this emission shows a large Stokes shift ca. 175 nm to OG generated the corresponding metallogels respectively in y Fig. S8. InteresOG-based metallogels. As shown in \u2013, Cl\u2013, Br\u2013, I\u2013, AcO\u2013, H2PO4\u2013, HSO4\u2013, N3\u2013, SCN\u2013, S2\u2013, ClO4\u2013, CN\u2013, and OH\u2013) into the metallogel FeG, only HSO4\u2013 could induce the metallogel FeG emitting a turquoise fluorescence at 470 nm immediately. While other anions such as F\u2013, Cl\u2013, Br\u2013, I\u2013, AcO\u2013, H2PO4\u2013, N3\u2013, CN\u2013, SCN\u2013, ClO4\u2013, S2\u2013 and OH\u2013 could not induce any significant emission changes. Therefore, FeG showed selective fluorescence \u201cturn-on\u201d sensing of HSO4\u2013 in water. The detection limits of FeG for HSO4\u2013 are 1.0 \u00d7 10\u20136 M in CuG, CrG, BaG, EuG, TbG and the fluorescence metallogels such as AlG, LaG and so on. From the results, CuG, CrG, BaG, EuG, and TbG could selectively fluorescence \u201cturn-on\u201d sense SCN\u2013, S2\u2013, F\u2013 and OH\u2013 into the metallogels. As a result, LaG and YG could selectively sense Hg2+ and Pb2+ in and Pb2+ while Ruand Zn2+ respecti2+, Ba2+, Fe3+, Cr3+ or Ru3+ could quench the AIE of OG while the addition of Ca2+, Al3+, La3+, and Y3+ could induce the AIE of OG taking place with obvious shifts or enhancement, we rationally induced two different kinds of metal ions into OG to control the AIE of the gel. Due to the different coordination capabilities of the bimetal ions, there is competitive coordination existing in the gelator and the two kinds of metal ions (competitive binding interaction (D) in 3+ and Fe3+ into OG, we could obtain the corresponding bimetallogel AlFeG. Using a similar method, we could obtain a series of bimetallogels such as AlHgG, AlCuG, AlCrG, BaMgG, BaCaG and so on. Interestingly, as we expected, the fluorescence properties of the bimetallogel depends on the metal ion which has the stronger coordination capability than the other metal ion. For example, because the coordination capability of Fe3+ is stronger than Al3+, the fluorescence properties of AlFeG are similar to those of FeG. Therefore, AlFeG is a no fluorescence metallogel. Similarly, AlHgG, AlCuG and AlCrG are no fluorescence metallogels while BaMgG and BaCaG are fluorescence metallogels.In light of the above results that the addition of metal ions such as CuAlFeG, AlHgG, AlCuG and AlCrG could selectively fluorescence \u201cturn-on\u201d sense CN\u2013 (by AlFeG and AlCuG), Cl\u2013 (by AlHgG) and S2\u2013 (by AlCrG) respectively; while, BaMgG and BaCaG could selectively fluorescence \u201cturn-off\u201d sense I\u2013 and HSO4\u2013 respectively.Then, we introduced competitive binding interactions among the bimetal ions, anions, and gelators (competitive binding interaction (E) in OG. As show in \u2013, Cl\u2013, Br\u2013, I\u2013, AcO\u2013, H2PO4\u2013, N3\u2013, SCN\u2013, S2\u2013, ClO4\u2013, CN\u2013, and OH\u2013) into OG respectively, only CN\u2013 could induce the AIE emission of OG to take place a significant red shift from 470 nm to 520 nm immediately. Meanwhile, turquoise fluorescence of OG changed to yellow. While other anions such as F\u2013, Cl\u2013, Br\u2013, I\u2013, AcO\u2013, H2PO4\u2013, N3\u2013, SCN\u2013, ClO4\u2013, S2\u2013 and OH\u2013 could not induce any significant emission or color changes. Therefore, OG could selectively fluorescence and colorimetric sense CN\u2013 in water.Moreover, we also introduced competitive binding interactions between the anions and gelators (competitive binding interaction (F) in OG + CN, as a result, as shown in OG + CN can selectively sense Fe3+.Finally, we introduced competitive binding interactions among the anions, metal ions and gelators (competitive binding interaction (G) in via fluorescence titrations , which is lower than the WHO guideline of 1.9 \u00d7 10\u20136 M.The detection limits of this supramolecular gel-based sensor array for the corresponding ions were investigated Fig. S11. As a revia IR and SEM. For instance, in the FT-IR of OG scale\" fill=\"currentColor\" stroke=\"none\">O and \u2013N PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CH\u2013 vibration absorption peaks appeared at 1641 and 1594 cm\u20131 respectively. While, with the addition of Fe3+ into OG and the formation of FeG, these two peaks shifted to 1647 and 1588 cm\u20131 respectively, which was attributed to the coordination of Fe3+ with the gelator via the acylhydrazone group of the gelator. As we expected, after the addition of HSO4\u2013 into metallogel FeG, the \u2013C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O and \u2013N PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CH\u2013 vibration absorption peaks returned to 1641 and 1594 cm\u20131 respectively, which indicated that the HSO4\u2013 competitively binds with Fe3+ and releases the acylhydrazone group of the gelator. This result indicated that the ion sensing mechanism of the above mentioned metallogels and bimetallogels is based on the competitive coordination which took place among the gelators, metal ions or anions in the supramolecular gel system. In the corresponding SEM scale\" fill=\"currentColor\" stroke=\"none\">CH protons on G appeared at \u03b4 9.51 ppm. After adding CN\u2013, this signal faded away, while, two new signals appeared at \u03b4 3.10 and 5.64 ppm, which were attributed to the formation of the NC\u2013CH\u2013 and \u2013NH groups, respectively. Meanwhile, in the FT-IR of OG scale\" fill=\"currentColor\" stroke=\"none\">CH vibration absorption at 1594 nm\u20131 disappeared and a new \u2013C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N vibration absorption appeared at 2176 cm\u20131. These results indicated that the CN\u2013 was added to the imines group on OGvia a nucleophilic addition reaction and formed a new organogelator OG + CN. During this process, the emission band of OG underwent a red shift, which is attributed to the ICT process.The further response mechanism of Fig. S12, the \u2013N Fig. S4c, the \u2013NFeG could selectively fluorescence \u201cturn-on\u201d sense HSO4\u2013, while, upon the addition of Fe3+ into the HSO4\u2013-containing FeG, the fluorescence of FeG was quenched, which was attributed to the Fe3+ coordination with the gelator again. These properties make FeG act as a HSO4\u2013 and Fe3+ controlled \u201cOFF\u2013ON\u2013OFF\u201d fluorescent switch. By alternating the addition of HSO4\u2013 and Fe3+, the switch could be reversibly performed for at least three cycles with little fluorescence efficiency loss (EuG and TbG could selectively fluorescence \u201cturn-on\u201d sense OH\u2013, while, upon the addition of H+ into the OH\u2013-containing EuG or TbG, the fluorescence of EuG or TbG was quenched (+ competitively binding with OH\u2013 and the Eu3+ or Tb3+ coordination with the gelator again. Meanwhile, EuG and TbG could act as H+ sensors.The competitive binding mechanism was also supported by the reversibility of the sensing process. For example, the metallogel ncy loss . MoreoveFeG film has no fluorescence emission, when writing on the film with a writing brush dipped in HSO4\u2013 water solution, a brilliant blue fluorescent writing appeared. This fluorescent image could be erased by brushing Fe3+ on the film again. Other supramolecular gels (Moreover, these supramolecular gels could act as ion response fluorescent materials. For example, by pouring a heated solution of these gels onto a clean glass surface and drying in the air, we could obtain the corresponding ion response supramolecular films. As shown in lar gels show sim\u2013, F\u2013, SCN\u2013, Hg2+, Pb2+etc. with high selectivity and sensitivity in a water solution. This sensor array needed only one synthesized receptor. Moreover, using this method, we also obtained a series of ion response fluorescent supramolecular materials, which could act as erasable security display materials. Therefore, this is an efficient and simple way to develop a sensor array as well as stimuli\u2013responsive supramolecular materials.In summary, by rationally introducing competitive binding interactions into a well designed supramolecular gel, a twenty-two member sensor array has been successfully developed. This sensor array could sense fourteen kinds of important ions such as CNSupplementary informationClick here for additional data file."} +{"text": "Hydrogen bonding-enabled highly regio- and stereo-selective hydrocarboxylation of alkynes has been successfully developed to afford 3-hydroxy-2(E)-alkenoates with up to 97% yield. syn-hydrocarboxylation of readily available 2-alkynylic alcohols with CO in the presence of alcohols with an unprecedented regioselectivity affording 3-hydroxy-2(E)-alkenoates. The role of the hydroxy group has been carefully studied. The synthetic potential of the products has also been demonstrated.Here we present an example of utilizing hydroxy groups for regioselectivity control in the addition reaction of alkynes\u2014a highly efficient Pd-catalyzed Surprisingly, the expected syn-hydrocarboxylation product (E)-2a\u2032E)-2a was exclusively formed unexpectedly together with 25% recovery of 1a (E)-2a -2a (E)-2a with 57\u201382% yields (E)-2a was improved to 90% but-3-yn-2-ol -2a . Varioussyn-hydrocarboxylation reaction delivered 3-hydroxy-2(E)-alkenoates as the sole product for various 2-alkynylic alcohols. Substitution of the pyridine ring at different positions made no difference (E)-2d in 71% yield -2h and (E)-2i in good yields -2j in 58% yield -2k in only 35% yield (1L), more rigid or more flexible backbone structures both made the reaction slower (1k (E)-2k could be obtained with the highest yield -2q was further confirmed by its single crystal X-ray diffraction analysis (E)-2s in 87% yield (E)-2t in 86% yield and 9% recovery of 1t (E)-2l in 89% yield -1\u2009E)-alkenoates with excellent ee values and high yields.Furthermore, as shown in E)-2l, Suzuki coupling reactions could easily afford (E)-7 in 80% yield.E)-8 in 80% yield,E)-9 in 80% yield.E)-10 in 94% yieldAs we know that 2-alkenoates are important intermediates in organic synthesis, their synthetic potential has been further demonstrated for the synthesis of different stereo-defined functionalized olefins. Owing to the presence of the C\u2013Br bond in -2k])} vs. reaction time , even with 10-fold excess of MeOH to ensure pseudo zero order in MeOH, indicating first-order dependence of the reaction rate with respect to propargylic alcohol -2k])} vs. reaction time, and show significantly different reaction rates, that is, the more electron-rich the substituent is, the faster the reaction rate is -2k and regenerate the Pd0 species to finish the catalytic cycle. Of course, further studies are needed to fully verify this mechanism.Based on these studies, a plausible mechanism is proposed . HydrogeE)-alkenoates and the observed regioselectivity may arise from hydrogen bonding, which needs further investigation. Due to the versatility of the functionality in the products, the importance of the stereo-selective construction of C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bonds, and the nature of regio-selectivity control, this method will be of high interest to organic and medicinal chemists. Further studies in this area are currently ongoing in our laboratory.In summary, we have developed hydroxy group-enabled highly regio- and stereo-selective hydrocarboxylation of 2-alkynylic alcohols, exploiting a previously unrecognized regioselectivity control strategy. The remarkable substrate scope, atom economy, and good to excellent yields make this reaction a facile synthetic method to produce highly functionalized 3-hydroxy-2(There are no conflicts to declare.Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "Computational pipeline for the accelerated discovery of organic materials with high refractive index via high-throughput screening and machine learning. The process of developing new compounds and materials is increasingly driven by computational modeling and simulation, which allow us to characterize candidates before pursuing them in the laboratory. One of the non-trivial properties of interest for organic materials is their packing in the bulk, which is highly dependent on their molecular structure. By controlling the latter, we can realize materials with a desired density (as well as other target properties). Molecular dynamics simulations are a popular and reasonably accurate way to compute the bulk density of molecules, however, since these calculations are computationally intensive, they are not a practically viable option for high-throughput screening studies that assess material candidates on a massive scale. In this work, we employ machine learning to develop a data-derived prediction model that is an alternative to physics-based simulations, and we utilize it for the hyperscreening of 1.5 million small organic molecules as well as to gain insights into the relationship between structural makeup and packing density. We also use this study to analyze the learning curve of the employed neural network approach and gain empirical data on the dependence of model performance and training data size, which will inform future investigations. I.The packing of atoms, molecules, and polymers in a given volume \u2013 either in crystalline or amorphous form \u2013 is a fundamental and long-standing issue that has been considered by various disciplines for over a century.e.g., Traditional, experimentally-driven trial-and-error searches for new compounds with desired sets of properties have proved to be time consuming and resource intensive. The advent of powerful modeling and simulation techniques as well as readily available time on high-performance computing systems have brought computational and computationally-guided studies to the forefront of the chemical and materials domain. These studies allow us to make increasingly accurate predictions for compounds of interest and uncover promising leads for experimentalist partners to follow up on via the Slonimskii method and the packing coefficient from a support vector regression machine learning model.i.e., different density regimes). We also evaluate the learning curve for the density prediction to assess the dependence of training set size and model accuracy.In this work, we develop a DNN prediction model for the packing density of small organic molecules in an amorphous bulk phase and conduct a hyperscreening of 1.5 million candidate compounds. Our interest in this target property originates from our ongoing In Sec. II, we detail the methods employed in our work. We describe the MD modeling protocol we use to compute the density values (Sec. IIA), discuss the molecular design space we consider and the application of our virtual high-throughput screening tools on the resulting compound library (Sec. IIB), introduce our DNN prediction model (Sec. IIC), and establish our pattern analysis approaches to mine the obtained results (Sec. IID). Sec. III presents and discusses the outcomes of our study, in particular the density predictions from MD and DNN (Sec. IIIA), the efficiency of the DNN approach (Sec. IIIB), and the emerging structure\u2013property relationships (Sec. IIIC). Our findings are summarized in Sec. IV.II.A.via steepest descent. We then compute the packing density with the general Amber force field (GAFF).3 simulation box and fill it with the pre-optimized target molecules. The number of molecules in the simulation box depends on the given molecule size, but a typical system contains around 1000 molecules . The system is first subjected to a minimization of the internal energy, which is associated with the relaxation of bonds, bond angles, and dihedral bond angles. This is followed by NVT and NPT equilibration steps for 100 and 240 ps, respectively. Both NVT and NPT ensembles use a Nos\u00e9\u2013Hoover thermostat at 298.15 K for temperature control. The NPT ensemble uses the Parinello\u2013Rahman barostat for pressure control. We conclude the MD protocol with a final 40 ps NPT production run. We use an MD timestep of 0.2 fs. We obtain the density by averaging the density values of the system at intervals of 0.2 ps during this final run. We note that this protocol is expected to yield kinetically stable amorphous phases rather than thermodynamically stable crystal structures or meta-stable polymorphs. GAFF is known to underestimate the density values compared to those from experiment, especially for high-density compounds.We employ the following MD modeling protocol to generate the data for the training and testing of the DNN density prediction model at the center of this work. Starting from the simplified molecular-input line-entry system (SMILES)40B.ChemLGi.e., a molecular weight within the range of 150 to 400 Dalton and limiting the number of ring-moieties to four. The hydrogen atoms in each building block are used as linker handles. Our proof-of-principle library is designed to feature different connections between simple moieties, most of which are commonly used in organic materials . We use the eToxPred software to compute the synthetic accessibility score (SAscore) of all 15 building blocksWe create a virtual library of 1.5 million small organic molecules using our library generator code via the MD modeling protocol introduced in Sec. IIA, we employ our automated virtual high-throughput screening framework ChemHTPS.ChemHTPS creates inputs for the MD simulations, executes the modeling protocol, monitors the calculations, parses and assesses the results, and extracts and processes the information of interest. Of the 1.5 million compounds in our screening library, we randomly select a subset of 100\u2009000 for study at the MD level.To facilitate the density evaluation for a large number of compounds C.ChemML,ChemML employs the scikit-learn 0.18.2 library for the multi-layer perceptron regressor 1.17.1 . We apply the bootstrapping method, i.e., for each training set size, we obtain the training set by randomly sampling the entire data set. The remaining data points serve as test set. For every training set size, we repeat the process (with replacement) 50 times, i.e., all 50 repetitions are independent of each other. We subsequently calculate statistics over the results of the 50 models that are based on these training sets for each training set size.We use the MD results for these 100,000 molecules as the ground truth for our data-derived density prediction model. For this, we pursue a DNN approach within a feature space of molecular descriptors. We build the DNN model using r 1.17.1 and 197 D.Z-scores (Zi) of each building block i used in the creation of the molecular library aswithwhere M is the total number of molecules in the entire library, m is the subset of molecules under consideration , Ki is the number of occurrences of building block i in M molecules and ki its occurrences in the subset of m molecules. A large Z-score indicates that a building block appears more frequently in that subset compared to the rest of the library (or a random sample). By applying the hypergeometric distribution analysis, we can thus identify the building blocks with the largest impact on the target property and the degree to which they correlate with desired density values. Furthermore, we identify the building blocks that are prominent in particular density regimes and assess Z-score trends in density-ordered candidate subsets across the entire density range. In addition, we compute the average density values of the candidates derived from each building block, and analyze this data for trends. We employ the ChemML package for all data mining and pattern recognition tasks.In addition to identifying candidates with particular density values from our MD screening and DNN hyperscreening studies, we mine the compiled results to better understand the correlation between molecular structure and packing density. One pattern recognition approach we pursue is the hypergeometric distribution analysis, in which we determine the R2, slopes, and offsets of linear regressions.The following metrics are used in the error analyses of our modeling approaches: mean absolute error (MAE), mean absolute percentage error (MAPE), root mean squared error (RMSE), root mean squared percentage error (RMSPE), mean error (ME), mean percentage error (MPE), maximum absolute error (MaxAE), and maximum absolute percentage error (MaxAPE). Aside from providing these direct measures, we also quantify the extent of correlations and systematic biases between results of different methods by listing the correlation coefficients III.A.\u20133. As shown in the first column of e.g., processing and ambient conditions) that are not accounted for in the simulations. We also note that the experimental data set is structurally more diverse than the screening library, i.e., it includes non-aromatic and aromatic moieties, halogens, and different functional groups such as OH, C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C, C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C, C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O, N PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O, etc. Despite this diversity, the comparison shows an R2 of 0.95, which underscores the utility of the employed MD approach. To test the accuracy of the MD density modeling protocol introduced in Sec. IIA, we compare its results against the experimentally known density values of 175 small organic molecules.R2 = 0.98, both MAPE and RMSPE are around 1%, and MaxAPE is just 5.5%. Most importantly, we find that the DNN prediction errors are significantly smaller than the intrinsic MD errors (by a factor of 4 to 6), which means that the DNN and MD results are statistically indistinguishable. The prediction quality for the MD training data set is essentially identical to the test set shown in R2 = 0.89 shows that the DNN model still captures the structure\u2013property relationships to a certain degree, and given appropriate training data, DNN should deliver predictive models for those compound pools as well.The trained DNN model mimics \u2013 by design \u2013 the MD simulations it is derived from. The second column of \u20133 with an average of 1384 kg m\u20133. The results show a t- or Gaussian-like distribution and most of the compounds in the library have density values between 1200 to 1600 kg m\u20133, with only very few examples at the extreme high and low density regime. It is worth noting that these extreme packing density values may be desirable for certain material applications . The sparsity of instances for extreme density values emphasizes the valuable role that high-throughput screening studies via physics-based modeling and/or data-derived prediction models can play in the discovery of suitable materials candidates.With the accuracy of the trained DNN model established, we apply it to the remaining 1.4 million compounds of the screening library introduced in Sec. IIB with the expectation of obtaining similar results as MD would yield. The DNN density predictions are summarized in B.ChemHTPS without manual intervention.) The demand on disk space is another issue, and we estimate a need for 120 terabytes for the entire library (15 terabytes without trajectories). The DNN prediction model produces essentially the same results in less than 10 core hours of compute time (without performance optimization), with all but 10 minutes of the time required to generate the feature matrix of the compound library. Disk use is marginal. This corresponds to a speed-up of about seven orders of magnitude, with negligible loss in accuracy. A speed-up of that magnitude allows a corresponding increase in the scale and scope that is affordable for screening studies.After confirming that the DNN prediction model can accurately reproduce MD-level results , we now investigate its efficiency, in particular relative to MD. Our MD calculations for the subset of 100\u2009000 molecules took a total of 5 million core hours of compute time on a modern high-performance computing cluster. For the entire screening library, this extrapolates to approximately 75 million core hours perform poorly. Models based on 2000 to 4000 data points offer acceptable accuracy. Those based on 4000 to 6000 data points offer very good accuracy, and at 10\u2009000 data points, the training is essentially saturated and the learning curve plateaus off. Additional training data does not lead to an improvement of the DNN model and is essentially wasted. Thus, we do not require a large data set of 100\u2009000 molecules to learn the packing density of organic molecules. We can develop an accurate model using MD data of just 5000 molecules (or more conservatively 10\u2009000). This reduces our demand of computing time from 5 million to less than 0.25 (or 0.5) million core hours , which has significant implications for the cost-benefit analysis and viability of this approach.The bottleneck of our DNN prediction model is the generation of the training data needed for its creation. It is worth noting, though, that this is a fixed cost rather than an effort that scales with the number of compounds studied. The size of the employed training set (100\u2009000 compounds corresponding to 5 million core hours) was originally chosen postmortem analysis of the learning curve as provided here, we will use an on-the-fly assessment of the learning curve combined with a just-in-time termination of the training data generation to minimize our data footprint in future studies.We stress that the data demand is highly dependent on the nature of the data and the employed machine learning approach (including the feature representation), and there are distinct limits to generalizing our findings. Instead of a C.cf.B7 and B12 have the highest average densities, while those that incorporate B1 (CH2-linker) and B9 (cyclopentane) exhibit the lowest values. Aside from the linker groups, there is a clear correlation between density value and the heteroatom type and fraction in a corresponding moiety.When considering the screening results, we are not only in a position to assess a large number of compounds, but we can also learn patterns from the data set in its entirety. Our analysis in Based on the construction of our library, more than 80% of the candidate compounds contain sulfur and more than 90% contain nitrogen. cf.Z-score analysis introduced in Sec. IID. i.e., the top 10% subset) with clear and distinct trends. Consistent with our previous analysis, we observe very large Z-score values for and thus a strong overexpression of B7 and B12. B13 (thiazole) also shows a large Z-score, and so do to a lesser extent B2 (S-linker) and B3 (O-linker) as well. These moieties are clearly favorable if high-density compounds are desired. In addition to assessing the high-density regime, we employ the hypergeometric distribution analysis to identify the prevalence of building blocks in the complete spectrum of density values. For this, we sort our virtual library by increasing density values, divide it into ten equal segments, and perform our analysis within each of these subsets as shown in Z-score of building blocks B2, B7, B12, and B13 increases with increasing density values, indicating a direct correlation, whereas it decreases for B1, B4, B9, B10, and B15, indicating an inverse correlation. The former are thus suitable to design organic molecules with higher density, and the latter could be used to achieve compounds with lower density. These findings are consistent with our prior analysis.While the average density values indicate the cumulative impact of a particular building block, we find relatively large standard deviations cf.. For a mIV.ChemLG molecular library generator code, the ChemHTPS automated high-throughput in silico screening program, and the ChemML machine learning package) in facilitating and supporting research efforts of this nature.The ability to predict the properties of novel compounds prior to synthesis, and to understand how these properties depend on their structure, is of considerable importance in materials discovery and design. In this paper, we showed that MD simulations can accurately predict experimental packing density values of small organic molecules and we provided corresponding benchmark results to quantify this finding. We conducted a high-throughput MD screening of 100\u2009000 compounds, which allowed us to train a DNN density prediction model. This DNN model accurately reproduces the MD data within the margins of MD's intrinsic error, while being nearly seven orders of magnitude faster than MD. This exceedingly efficient approach allowed us to rapidly obtain the density values of a 1.5 million compound screening library, which would have been prohibitively time consuming and well out of reach for MD. By analysing the large data set resulting from this study, we could elucidate structure\u2013property relationships that determine the density values. We identified prevalent moieties in the high and low density regime and could quantify the impact of heteroatoms (sulfur and nitrogen). Further, we evaluated the DNN learning curve for the density prediction with respect to the available training data and found a considerably lower data demand than we had anticipated. Following this lesson, we will in future studies employ an on-the-fly assessment of the learning curve and terminate the training data generation once we observe satisfactory saturation. This will allow us to alleviate the data generation bottleneck and make machine learning models an even more viable and attractive proposition. Overall, our study underscores the value of combining powerful machine learning approaches with traditional computational modeling for the generation of the necessary data. It also demonstrates the utility of our software ecosystem (including the The authors declare to have no competing financial interests.Supplementary informationClick here for additional data file.Supplementary informationClick here for additional data file.Supplementary informationClick here for additional data file."} +{"text": "Efficient removal of ammonia from air is demonstrated in a series of Br\u00f8nsted acidic porous polymers under dry and humid conditions. The impact of acidic group strength and their spatial distribution on the ammonia uptake is investigated systematically. 3) is also highly toxic and presents a substantial health and environmental hazard. The development of new materials for the effective capture and removal of ammonia is thus of significant interest. The capture of ammonia at ppm-level concentrations relies on strong interactions between the adsorbent and the gas, as demonstrated in a number of zeolites and metal\u2013organic frameworks with Lewis acidic open metal sites. However, these adsorbents typically exhibit diminished capacity for ammonia in the presence of moisture due to competitive adsorption of water and/or reduced structural stability. In an effort to overcome these challenges, we are investigating the performance of porous polymers functionalized with Br\u00f8nsted acidic groups, which should possess inherent structural stability and enhanced reactivity towards ammonia in the presence of moisture. Herein, we report the syntheses of six different Br\u00f8nsted acidic porous polymers exhibiting \u2013NH3Cl, \u2013CO2H, \u2013SO3H, and \u2013PO3H2 groups and featuring two different network structures with respect to interpenetration. We further report the low- and high-pressure NH3 uptake in these materials, as determined under dry and humid conditions using gas adsorption and breakthrough measurements. Under dry conditions, it is possible to achieve NH3 capacities as high as 2 mmol g\u20131 at 0.05 mbar (50 ppm) equilibrium pressure, while breakthrough saturation capacities of greater than 7 mmol g\u20131 are attainable under humid conditions. Chemical and structural variations deduced from these measurements also revealed an important interplay between acidic group spatial arrangement and NH3 uptake, in particular that interpenetration can promote strong adsorption even for weaker Br\u00f8nsted acidic functionalities. In situ infrared spectroscopy provided further insights into the mechanism of NH3 adsorption, revealing a proton transfer between ammonia and acidic sites as well as strong hydrogen bonding interactions in the case of the weaker carboxylic acid-functionalized polymer. These findings highlight that an increase of acidity or porosity does not necessarily correspond directly to increased NH3 capacity and advocate for the development of more fine-tuned design principles for efficient NH3 capture under a range of concentrations and conditions.Although a widely used and important industrial gas, ammonia (NH Solid-state adsorbents such as metal\u2013organic frameworks53) from air.3-based fuel cells after the gas decomposes to hydrogen.One example where this is of particular importance is in the removal of ammonia was recently investigated for NH3 capture.3 capacity was achieved using these materials, there was also a significant reduction in porosity upon functionalization with bulkier groups \u2013CO2H and \u2013SO3H, which hindered the accessibility and complete utilization of the acid sites. More recently, incorporation into a polymer membrane has been demonstrated to impart enhanced stability to the framework HKUST-1 under humid conditions, without diminishing NH3 capacity.14Ammonia can behave as both a Lewis base and a Br\u00f8nsted base, and therefore porous materials decorated with Lewis or Br\u00f8nsted acidic sites are promising targets for capture at particularly low concentrations.3 capture.\u20131 at an equilibrium concentration of \u223c500 ppm.\u20131 at \u223c900 ppm, in spite of its stronger Br\u00f8nsted acidity. This behavior was attributed to the presence of isolated and non-interacting acidic groups due to the non-interpenetrated structure of the material. Such a striking difference between these two polymers prompted us to further investigate the interplay between Br\u00f8nsted acidity and polymer structure in a systematic manner.Porous polymers have been investigated to a lesser degree in the context of low-pressure NH3Cl, \u2013CO2H, \u2013SO3H, and \u2013PO3H2 groups and P2-CO2H were recently reported,3H was achieved following a literature procedure.20The Br\u00f8nsted acidic porous polymers reported in this work were synthesized following either a postsynthetic functionalization strategy starting from PAF-1 or a 3Cl and P2-SO3H were synthesized protectiontert-butyl dicarbonate was followed by Miyaura borylation to afford 1a. The sulfonation of 1,4-dibromobenzene using chlorosulfonic acid (ClSO3H) provided 2,5-dibromobenzenesulfonyl chloride.1b. Suzuki polymerization of 1a and 1b with the tetrahedral precursor tetrakis(4-bromophenyl)methane using Buchwald's precatalyst (SPhos Pd G2) yielded polymers P2-NHBoc and P2-SO3Neo, respectively. The utilization of a 4 M HCl solution in 1,4-dioxane enabled the removal of the Boc group and protonation of the amino group simultaneously to yield the final polymer, P2-NH3Cl. In the case of P2-SO3Neo, hydrolysis of the neopentyl group was carried out using NaN3 in DMSO and subsequent acidification with 6 M HCl resulted in the polymer P2-SO3H.Porous polymers P2-NHthesized through y see ESI. The ter3Cl and P1-SO3H, the polymer P1-PO3H2 was prepared through postsynthetic modification 3, at elevated temperatures. The mild hydrolysis of the phosphonate ester groups into phosphonic acid moieties was readily accomplished using Me3SiBr, affording the polymer P1-PO3H2 in a quantitative manner.Similar to previously reported syntheses of P1-NHfication . The par1H and 13C NMR spectroscopy scale\" fill=\"currentColor\" stroke=\"none\">O and P\u2013O\u2013C stretching bands at 1240 and 960\u20131050 cm\u20131, respectively. The efficiency of this conversion was also reflected in the elemental analysis data scale\" fill=\"currentColor\" stroke=\"none\">O stretching band to 1140 cm\u20131 and the appearance of P\u2013O\u2013H bands in the range of 930\u20131000 cm\u20131. Complete hydrolysis of ester groups was also evidenced by the absence of P\u2013O\u2013C stretching bands. Similarly, complete removal of the Boc groups in P2-NH3Cl was unambiguously demonstrated by the disappearance of a C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O stretching band at 1713 cm\u20131 as well as the emergence of a broad N\u2013H band centered around 3345 cm\u20131. In the case of P2-SO3H, absorption bands at 960 cm\u20131 (S\u2013O\u2013C stretching) and at 1178 and 1350 cm\u20131 scale\" fill=\"currentColor\" stroke=\"none\">O stretching) were replaced by bands at 996 and 1020 cm\u20131 (S\u2013OH) and 1150 cm\u20131 scale\" fill=\"currentColor\" stroke=\"none\">O), respectively. Finally, the disappearance of C\u2013H stretching bands in the 2900\u20133000 cm\u20131 region also indicated the removal of neopentyl groups.The formation of porous polymers incorporating protected Br\u00f8nsted acidic groups and their subsequent hydrolysis was confirmed by Fourier transform infrared (FTIR) spectroscopy 1H NMR spectroscopy and cross-polarization magic angle spinning (CP/MAS) 13C NMR spectroscopy. Acidic protons appear as sharp, narrow peaks, especially in the cases of P1-PO3H2, P2-SO3H, and P2-CO2H in the solid-state MAS 1H NMR spectra, reflecting the high mobility of acidic protons in these materials was found to be 1.4 in P1-NH3Cl, while the number of \u2013SO3H and \u2013PO3H2 groups was lower in P1-SO3H (0.8) and P1-PO3H2 (0.7), respectively.Elemental analyses verified polymer conversion efficiency, with good agreement between experimental and calculated compositions in this series might be attributed to the presence of the sterically encumbering Boc groups used in the synthesis of the P2-NHBoc precursor, which could reduce the degree of interpenetration.25Surface area and porosity analyses were carried out using nitrogen gas adsorption isotherms collected at 77 K. Pore size distributions were calculated from the adsorption branch of isotherms employing a quenched solid-state DFT (QSDFT) model, which takes surface heterogeneity into account and assumes the presence of a mixture of slit, cylindrical, and spherical pores. All polymers display type I reversible isotherms, as typically observed for microporous materials are consistently higher than those for P2 polymers (<0.5 cm3 g\u20131), reflecting the influence of interpenetration in the latter frameworks. The microporosity of each sample was also further confirmed by t-plot curves , P1-NH3Cl displays an excess uptake of 0.37 mmol g\u20131 compared to 1.35 and 3.3 mmol g\u20131, for P1-SO3H and P1-PO3H2, respectively enables greater NH3 uptake at low pressures in P2-CO2H\u2014a comparison that also suggests the formation of strong binding sites with multiple weak acidic groups. At 1 bar, P2-NH3Cl and P2-CO2H display similar NH3 uptakes of 16.3 and 16.1 mmol g\u20131, respectively, whereas P2-SO3H has a lower uptake of 13.1 mmol g\u20131.Among the P1 polymers, P1-SOectively . Notably3 uptake . The NH33 concentration of 50 ppm a higher density of acidic sites, (ii) the bulkiness and flexibility of the \u2013CH2PO3H2 groups compared to \u2013SO3H groups, and/or (iii) a smaller surface area and pore volume. All of these factors render the acidic sites more proximal in P1-PO3H2 than in P1-SO3H.Perhaps more interestingly and relevant to permissible exposure limits, we now compare the uptake properties of these materials at a significantly lower NH P2-SO3H , it is c3 uptake in the cases of P1-PO3H2, P2-SO3H, and P2-CO2H can be drawn from some recent computational work.3H, it is possible that a low local polarity contributes to decreased ammonia affinity at these sites for concentrations as low as 50 ppm. Network interpenetration in the case of P2-SO3H and P2-CO2H and the reduced pore volume in P1-PO3H2 could, however, create a local dielectric polarization around each acidic site in the pores and therefore lead to stronger interactions with ammonia and enhanced capacities. Most notably, the performance of these latter materials at 50 ppm of dry ammonia is comparable to that of 5A zeolite (1.86 mmol g\u20131 at 58 ppm) and 13X zeolite (1.74 mmol g\u20131 at 41 ppm) and is significantly higher than sulfonated polymeric resin Amberlyst 15 (0.38 mmol g\u20131 at 71 ppm).26Another plausible explanation for the increased NHe.g., P1-SO3H and P2-SO3H), was compared after plotting gravimetric NH3 isotherms with respect to the number of acidic functionalities (mmol3NH mmolacid\u20131) determined by elemental analysis. The absolute pressure corresponding to the capture of one equivalent of ammonia per acid site was found to correlate well with the acid strength of functional groups within P1 and P2 was approximately 2.8 mbar.In order to gain further understanding of the NH3Cl and P2-NH3Cl, with the weakest Br\u00f8nsted acidic functionality, occurs much earlier than for the other polymers carrying sulfonic, phosphonic, or carboxylic acid groups within the same series. Also in agreement with the NH3 adsorption isotherms, P1-PO3H2 and P2-CO2H display highest uptake capacities of 5.2 and 6.7 mmol g\u20131, respectively, under dry breakthrough conditions.Under dry conditions, the trends in breakthrough saturation capacity and uptake are in excellent agreement with those obtained from gas adsorption measurements , the NH Fig. S41. Provide3H2 displays a broad desorption curve under humid conditions, the curve for P1-SO3H is rather steep ,3 uptake for these different acidic moieties. Spectral changes were also monitored upon desorption to assess the reversibility of ammonia binding.We sought to further probe the interaction of ammonia with Br\u00f8nsted acidic groups utilizing 3 adsorption the spectral changes all corresponded to those associated with the roto-vibrational profile of gaseous ammonia.We note that the parent material, PAF-1, does not exhibit any substantial spectral changes upon exposure to ammonia Fig. S37. The ini3H exhibits a series of very intense and sharp absorption bands in the 800\u20131800 cm\u20131 spectral range, which are ascribed to the vibrational modes of the aromatic structure and sulfonic acid group (\u20131 are generated by out-of-phase (\u03bdas) and in-phase (\u03bds) stretching vibrations of the SO2 group, and the OH bending mode of the S\u2013OH moiety, respectively.\u20131 region as a consequence of the formation of ammonium ions and overlaps with one of the peaks resulting from aromatic ring vibrations at 1465 cm\u20131. The absorption bands assigned to SO2 and S\u2013OH moieties in the activated material also disappear and two strong signals appear at 1035 and 1225 cm\u20131 due to \u03bds and \u03bdas modes of the newly formed sulfonate (\u2013SO3\u2013) group.3 adsorption, the sample was evacuated for 2 h at beam temperature to evaluate the reversibility of the proton transfer and \u03bds(SO2) modes of the sulfonic acid moieties could not be restored, even after prolonged outgassing.The spectrum of activated P1-SOin situ ammonia-dosed on P1-PO3H2 suggested a similar proton transfer between ammonia and phosphonic acid groups within the polymer. The activated spectrum of P1-PO3H2 exhibits typical features, in particular, strong and broad signals located at around 1000 and 1200 cm\u20131 and corresponding to P\u2013O(H) and P PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O stretching vibrations, respectively (\u03b4(NH4+) mode of the ammonium ion clearly appears as a broad component at \u223c1450 cm\u20131, while the P\u2013O and P PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O stretching modes are deeply perturbed, indicating deprotonation of one or both acidic protons scale\" fill=\"currentColor\" stroke=\"none\">O) stretching and \u03b4(C\u2013OH) bending vibrations of the carboxylic acid group, respectively (\u03b4(NH4+) mode at \u223c1458 cm\u20131 scale\" fill=\"currentColor\" stroke=\"none\">O) and \u03b4(C\u2013OH) band intensities decrease, and this change is accompanied by the appearance of two new bands at 1525 and 1378 cm\u20131 that can be ascribed to \u03bdas(OCO) and \u03bds(OCO) modes of the carboxylate (\u2013CO2\u2013) group, respectively.3H and P1-PO3H2, for P2-CO2H, the proton transfer to ammonia seems to be quite reversible (\u03b4(NH4+) and \u03bd(OCO) bands decrease dramatically and the characteristic signals of the carboxylic acid groups are partially restored.The spectrum of activated P2-CO2H upon ammonia exposure is the noticeable change in the scattering profile of the whole spectrum and one with (P2) framework interpenetration, wherein the strength of the acidic functional groups was systematically varied. Adsorption isotherms revealed that NHSupplementary informationClick here for additional data file."} +{"text": "An efficient method for the synthesis of the NHC-stabilised Si(i) halides Si2X2(Idipp)2 was developed, which involves the oxidation of Si2(Idipp)2 (1) with 1,2-dihaloethanes. Iodide abstraction from 2-I afforded the unprecedented silicon(i) salt [Si2(I)(Idipp)2][B(C6F5)4] (3). i) halides Si2X2(Idipp)2 CH]2) was developed, which involves the oxidation of Si2(Idipp)2 (1) with 1,2-dihaloethanes. Halogenation of 1 is a diastereoselective reaction leading exclusively to a racemic mixture of the RR and SS enantiomers of 2-X. Compounds 2-Br and 2-I were characterised by single-crystal X-ray crystallography and multinuclear NMR spectroscopy, and their electronic structures were analysed by quantum chemical methods. Dynamic NMR spectroscopy unraveled a fluxional process of 2-Br and 2-I in solution, which involved a hindered rotation of the NHC groups about the Si\u2013CNHC bonds. Iodide abstraction from 2-I by [Li(Et2O)2.5][B(C6F5)4] selectively afforded the disilicon(i) salt [Si2(I)(Idipp)2][B(C6F5)4] (3). X-ray crystallography and variable-temperature NMR spectroscopy of 3 in combination with quantum chemical calculations shed light on the ground-state geometric and electronic structure of the [Si2(I)(Idipp)2]+ ion, which features a Si PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019Si bond between a trigonal planar coordinated SiII atom with a Si\u2013I bond and a two-coordinate Si0 center carrying a lone pair of electrons. The dynamics of the [Si2(I)(Idipp)2]+ ion were studied in solution by variable-temperature NMR spectroscopy and they involve a topomerisation, which proceeds according to quantum theory via a disilaiodonium intermediate (\u201c\u03c0-bonded\u201d isomer) and exchanges the two heterotopic Si sites.An efficient method for the synthesis of the NHC-stabilised Si( PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019PR PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019Si(Br)R 2}2-4-tBu) were also isolated, fortifying the binding capacity of NHCs.II halides SiX2(NHC) and SiCl(R)(NHC) CMe]2) were shown to be valuable starting materials, which paved the way to new classes of unsaturated silicon compounds, including silylidyne complexes, zwitterionic silylidene complexes, metallosilylenes and metallosilanones.11The molecular chemistry of silicon has witnessed remarkable progress in recent years following the discovery that N-heterocyclic carbenes (NHCs) are particularly suitable Lewis-bases for the thermodynamic and kinetic stabilisation of highly reactive, low-valent silicon species.i) halides (B) has not been explored so far. This can probably be attributed to the severely limited access to this very reactive class of compounds, as revealed by the very low yield synthesis (6.1%) of the only presently known example Si2Cl2(Idipp)2 (2-Cl) upon the reduction of SiCl4(Idipp) with C8K.B can be viewed as bis(NHC) adducts of the disilynes Si2R2 2 is reported, facilitating the exploration of their reactivity. Moreover, iodide abstraction from 2-I is demonstrated to provide access to an unprecedented Si(i) salt containing the NHC-trapped [Si2I]+ cation.In comparison, the chemistry of NHC-stabilised silicon scale\" fill=\"currentColor\" stroke=\"none\">Si(Idipp) (1)i) halides, analogous to the well-known olefin halogenation in carbon chemistry.2H4X2 proved to be particularly suitable reagents to accomplish this transformation. In fact, the addition of a stock solution of 1,2-C2H4X2 to a dark red solution of 1 in THF or toluene at low temperature was accompanied by a rapid color change to red-orange and gas evolution (ethene), and selectively afforded the SiI halides 2-X in moderate to very good yields compound (Idipp)Si-I: 61%) .\u2020 Precis, Br, I) , which a1 by 1,2-C2H4X2 is a highly diastereoselective cis-addition leading exclusively to a racemic mixture of the RR and SS stereoisomers of 2-X (meso diastereoisomer (trans-addition product) was found, which, according to quantum chemical calculations at the B97-D3/I level of theory,RR/SS stereoisomers by 57 kJ mol\u20131 scale\" fill=\"currentColor\" stroke=\"none\">PR (R = C(SiMe3)3) with Cl2 was reported to give exclusively the meso diastereomer.Halogenation of s of 2-X . No evidI halides 2-X was also investigated, which involved comproportionation of the Si0 compound 1 with SiX2(Idipp). Whereas no reaction between 1 and SiBr2(Idipp) was observed at room temperature, heating a 1\u2009:\u20092 mixture of 1 and SiBr2(Idipp) in toluene at 85 \u00b0C afforded the SiI bromide 2-Br, as confirmed by NMR spectroscopy. However, conversion to the comproportionation product competed with the slow decomposition of 2-Br occurring under the same conditions (vide infra), leading finally to a mixture of 2-Br, SiBr2(Idipp) and Idipp. Whereas Idipp could easily be removed, separation of 2-Br from SiBr2(Idipp) proved to be difficult due to their similar solubility preventing the isolation of 2-Br in high-yield and pure form.An alternative approach to the Sii) halides 2-X were isolated as vermillion, extremely air-sensitive solids, which immediately decolourised when in contact with air, but can be stored indefinitely at room temperature under an atmosphere of argon. Compounds 2-Br and 2-I are thermally quite robust in the solid-state and decompose upon heating at a temperature (190 \u00b0C) similar to that previously reported for 2-Cl (184 \u00b0C).2-Br starts to decompose at a much lower temperature , and the decomposition leads to Idipp, SiBr2(Idipp) and an unknown toluene-insoluble solid bromide and iodide to be reported and were comprehensively characterised by single-crystal X-ray crystallography, NMR spectroscopy and quantum chemical calculations.Compounds n-hexane semisolvates 2-Br\u00b70.5(n-C6H14) and 2-I\u00b70.5(n-C6H14) were determined by single-crystal X-ray diffraction (2-X (X = Cl\u2013I) feature two stereogenic trigonal pyramidal silicon centers of the same configuration and display similar bonding parameters \u00b0,22-Br: \u201346.81(4)\u00b0, 2-I: 50.46(3)\u00b0) and CNHC\u2013Si\u2013Si\u2013CNHC torsion angles (2-Cl: \u2013162.9(3)\u00b0,22-Br: 161.5(1)\u00b0, 2-I: \u2013160.31(9)\u00b0), respectively (DP = 70\u201378%) (2-X compared to those in SiX2(Idipp), which was confirmed by comparative NBO analyses (2(Idipp) by the more electropositive substituent SiX(Idipp).2-X (2-Cl: 2.393(3) \u00c5,22-Br: 2.385(1) \u00c5, 2-I: 2.3909(9) \u00c5) are slightly longer than that in \u03b1-Si (2.352 \u00c5)I compounds C (2.413(2) \u00c5 and 2.489(2) \u00c5)D (2.331(1) \u00c5) at the silicon atoms .20 This 2(Idipp) . These t31(1) \u00c5) .14 Remar29Si{1H} NMR spectra in C6D6, the SiI halides display a characteristic singlet signal , which appears at a lower field than that of the corresponding Si(ii) halides SiX2(Idipp) .versus the N-heterocyclic rings and 13C (75.47 MHz) NMR signals of 2-Br were sharp at 298 K, several signals of 2-I were broadened under the same conditions, suggesting a dynamic behavior = 228 K; 2-I: \u0394G\u2260 = 51 kJ mol\u20131, Tc = 248 K).In the HC atoms . The 1H ic rings , which, 2-X contain many reactive sites for further functionalisation with the most appealing ones being the displacable halide and Idipp groups, which are not available in the silicon(i) congeners C and D (2O)2.5][B(C6F5)4] to a solution of 2-I in fluorobenzene at ambient temperature was accompanied by a colour change from bright to dark red and precipitation of LiI. Iodide abstraction from 2-I selectively afforded the disilicon(i) salt [Si2(I)(Idipp)2][B(C6F5)4] (3), as evidenced by NMR spectroscopy of the crude reaction mixture ) in 62% yield, and was comprehensively characterised.Compounds C and D . First r mixture . The sal3\u00b7(C6H5F) is an extremely air-sensitive solid, which is instantly degraded by air to a colourless powder. It is stable in THF-d8 solution for several days under strict exclusion of air, and decomposes upon heating in a sealed glass capillary tube under vacuum at 208 \u00b0C.Compound 3\u00b7(C6H5F) was determined by single-crystal X-ray diffraction and it is composed of well separated [Si2(I)(Idipp)2]+ and [B(C6F5)4]\u2013 ions.2(I)(Idipp)2]+ feature a trigonal planar coordinated Si1 atom (sum of the bond angles at Si1 = 359.7(1)\u00b0) and a two-coordinate Si2 atom with V-shaped geometry (Idipp atoms (C1 and C28). The Si\u2013Si bond of 3\u00b7(C6H5F) is considerably shorter (2.1739(9) \u00c5) than the Si\u2013Si single bond of 2-I (2.3909(9) \u00c5), and also shorter than the Si\u2013Si double bond of 1 (2.229(1) \u00c5), PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019Si bond lengths.2(I)(Idipp)2]+ (vide infra). The bulky NHC groups are trans-arranged at the Si\u2013Si double bond (torsion angles: C1\u2013Si\u2013Si2\u2013C28 = \u2013178.5(1)\u00b0 and I\u2013Si1\u2013Si2\u2013C28 = \u20136.71(9)\u00b0) and orthogonally oriented with respect to the planar core of the cation.1 (Si\u2013Si\u2013CNHC = 93.37(5)\u00b0), PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019PR \u00b0) PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019Si(Br)R 2}2-4-tBu; Si\u2013Si\u2013CNHC = 97.6(1)\u00b0).2(I)(Idipp)2]+, which indicates the presence of a stereochemically active lone-pair in an orbital of high s-character (77%) and Si2 hybrid orbitals of high p-character employed for the \u03c3-bonding to the Si1 atom and the NHC group scale\" fill=\"currentColor\" stroke=\"none\">SiTip2 (2.4520(7) \u00c5, Tip = C6H2-2,4,6-iPr3),2-I (Si1\u2013I1: 2.6036(6) \u00c5; Si2\u2013I2 2.5919(6) \u00c5) compared to that in 2-I (4%), and this is also reflected in the Si\u2013I Wiberg bond indexes (3: WBI (Si\u2013I) = 0.89; 2-I: WBI (Si\u2013I) = 0.78) (see NHC bond lengths of 3\u00b7(C6H5F) (1.901(2) and 1.931(2) \u00c5) have similar values to those of 2-I (1.943(2) \u00c5 and 1.939(2) \u00c5) and 1 (1.927(2) \u00c5) (2The solid-state structure of geometry . The two19(6) \u00c5) . This tr.78) see . The Si\u201327(2) \u00c5) .2a2(I)(Idipp)2]+ with the related cations [Si2(H)(Idipp)2]+ and [Si2(Me)(Idipp)2]+, the NHC-stabilised disilavinylidenes, the NHC-stabilised disilynes and the disilenide anions scale\" fill=\"currentColor\" stroke=\"none\">Si bond lengths and similar bond angles at the two-coordinate Si atom in THF-d8 revealed an interesting dynamic process leading to an exchange of the heterotopic Si sites. The degenerate isomerisation (topomerisation)via a NHC-stabilised disilaiodonium ion at 203 K (1H NMR spectrum of 3\u00b7(C6H5F) at 203 K displays a double set of resonance signals for the chemically different Idipp groups at 203 K shows a double set of signals for the inequivalent Idipp groups , which merge into one set of signals at 298 K are compatible with the results of the single-crystal X-ray diffraction and show an averaged Cs-symmetric structure of the cation [Si2(I)(Idipp)2]+ with fast rotating NHC substituents about the respective Si\u2013CNHC bonds.33Thus, two well separated at 203 K , right, regime) , right. 98 K see . Likewis4,5-H ring protons in the temperature range of 203\u2013263 K (k/T) against 1/T afforded a linear relationship (see Section 3 in the ESIR2 = 0.9966) and were found to be \u0394H\u2260 = 47.3 (\u00b10.7) kJ mol\u20131, \u0394S\u2260 = 1.39 (\u00b13.0) J K\u20131 mol\u20131 and \u0394G\u2260 (Tc = 235 K) = 47.0 (\u00b11.4) kJ mol\u20131.The rate constants of the dynamic process were determined by full line-shape analyses of the signals of the N-heterocyclic C03\u2013263 K . An Eyri2(I)(Idipp)2]+ was studied by quantum chemical calculations at the B97-D3/I level of theory3\u00b7(C6H5F) observed in solution. Geometry optimization of [Si2(I)(Idipp)2]+ afforded a \u201c\u03c3-bonded\u201d minimum structure with an excellent agreement between the calculated and the experimental bond lengths obtained for 3\u00b7(C6H5F) by single-crystal X-ray crystallography of the cation + , which lies at an energy 37.6 kJ mol\u20131 higher than the overall minimum structure calc3 . The most striking bonding parameters of calc3\u2032 are the elongated Si\u2013Si single bond (2.463 \u00c5), which is considerably longer than the Si PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019Si bond of calc3 (2.171 \u00c5), as well as the Si\u2013I bonds (2.696 \u00c5), which are longer than that of calc3 (Si\u2013I: 2.502 \u00c5). These bonding parameters suggest that calc3\u2032 can be better described as a NHC-stabilised disilaiodonium ion2(Idipp)2 (1) \u03c0-complex of I+. Notably, the structure of calc3\u2032 is reminiscent of those of the symmetrical 1,2-bridged halonium ions, which have been extensively studied in organic chemistry.35Furthermore, a \u201c\u03c0-bonded\u201d . The twore 3calc . The tra2(I)(Idipp)2]+ with those of the NHC-stabilised disilavinylidene (SIdipp)Si PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019Si(Br)R 2}2-4-tBu)4)RSi PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019SiR (IMe4 = C[N(Me)CMe]2, R = SiMe3)\u2013 (R = singly bonded substituent). In all cases, the HOMO is the Si PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019Si \u03c0-bonding orbital, which is followed by the lone-pair orbital at the two-coordinate Si atom (HOMO\u20131) (2(I)(Idipp)2]+ was analysed by the natural bond orbital (NBO) method and natural resonance theory (NRT) (see 2(I)(Idipp)2]+ scale\" fill=\"currentColor\" stroke=\"none\">Si, Si\u2013CNHC, and Si\u2013I bonds scale\" fill=\"currentColor\" stroke=\"none\">Si bond of calc3 and the high occupancies of its NBO lead to a high Wiberg bond index (WBI) of 1.81, which is twice as large as the WBI of the Si\u2013Si single bond of calc3\u2032 (0.89) and 2-I (0.96). These findings verify the presence of a Si PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019Si bond in the \u201c\u03c3-bonded\u201d isomer and a Si\u2013Si single bond in the \u201c\u03c0-bonded\u201d isomer of [Si2(I)(Idipp)2]+ or in 2-I, and are further confirmed by the NRT Si\u2013Si bond orders, which, in the case of calc3, is twice as large or 2-I (0.93) carries a lone pair of electrons in an NHO with high s-character (Idipp)2]+.A comparison of the frontier Kohn\u2013Sham orbitals of the ion +, as evidenced by the overall NPA charges of the NHCs (Si1-bonded: q(\u2211(NHC)) = 0.41; Si2-bonded: q(\u2211(NHC)) = 0.28) .i) halides Si2X2(Idipp)2 was developed, which involved a diastereoselective halogenation of Si2(Idipp)2 (1) with 1,2-dihaloethanes. This allowed the isolation of the first silicon(i) bromide (2-Br) and silicon(i) iodide (2-I) in high yield, enabling first reactivity studies of 2-I. The geometric and electronic structures of 2-Br and 2-I were comprehensively studied by experimental and theoretical methods. Iodide abstraction from 2-I selectively afforded the unprecedented disilicon(i) iodido salt [Si2(I)(Idipp)2][B(C6F5)4] (3), the geometric and electronic structure of which is isolobal to that of a NHC-stabilised disilavinylidene recently reported by our group. The topomerisation of the cation [Si2(I)(Idipp)2]+, leading to an exchange of the two heterotopic Si sites, was studied by variable-temperature NMR spectroscopy and the underlying dynamic process was analysed by quantum chemical calculations. The calculations suggest the intermediate formation of a C2-symmetric \u03c0-bonded isomer with homotopic Si sites reminiscent of the symmetrical 1,2-bridged halonium ions in organic chemistry. The present results corroborate the ability of N-heterocyclic carbenes to stabilise low-valent main-group element centers with unusual bonding features. Further studies addressing the reactivity of the NHC-stabilised Si(i) halides 2-X and the SiI salt 3 are currently underway.An efficient method for the synthesis of silicon(Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "The monomeric molecular aluminium(i) complex 1 [{(ArNCMe)2CH}Al] reacts with a series of terminal and strained alkenes including ethylene, propylene, allylbenzene and norbornene to form alkene bound products. i) complex 1 [{(ArNCMe)2CH}Al] reacts with a series of terminal and strained alkenes including ethylene, propylene, allylbenzene and norbornene to form alkene bound products. Remarkably all these reactions are reversible under mild conditions (298\u2013353 K) with alkene binding being disfavoured at higher temperatures due to the positive reaction entropy. Van't Hoff analyses have allowed quantification of the binding events with . Calculations and single crystal X-ray diffraction studies are consistent with the alkene bound species being metallocyclopropane complexes. Alkene binding involves a reversible redox process with changes from the +1 to +3 aluminium oxidation state. Under more forcing conditions the metallocyclopropane complexes undergo non-reversible allylic C\u2013H bond activation to generate aluminium(iii) allyl hydride complexes. This represents a rare example of redox-based main group reactivity in which reversible substrate binding is followed by a further productive bond breaking event. Analysis of the mechanism reveals a reaction network in which alkene dissociation and reformation of 1 is required for allylic C\u2013H activation, a realisation that has important implications for the long-term goal of developing redox-based catalytic cycles with main group compounds.The monomeric molecular aluminium( In 2009, following an unusual account of stannylene binding to a strained alkyne,i) reagent 1, first prepared by Roesky and co-workers,i) complex is necessary to liberate the reactive site and Frontier molecular orbitals involved in an intermolecular C\u2013H activation step. Our findings not only represent an important advance in transition metal mimetic behaviour of main group complexes, they also demonstrate the complementary mechanistic aspects of these two research fields.In this paper, we show that the aluminium(i) complex 1, known to activate a series of small molecules,2a complex and alkene binding as a redox process involving reversible oxidative addition and reductive elimination steps. In an effort to expand the scope of alkene binding the reaction of 1 with a series of simple and industrially relevant alkenes was investigated , propylene (1 bar), hex-1-ene, 3,3-dimethyl-1-butene (10 equiv.), allylbenzene (10 equiv.) and 4-allylanisole (10 equiv.) in C6D6 led to the formation of compounds 2b, 2c, 2d, 2e, 2f and 2g respectively. For example, 2b formed within 15 minutes at 298 K and the characteristic deep orange colour of 1 in benzene solution was seen to intensify upon alkene binding. In the 1H NMR spectrum, a new singlet peak is observed at \u03b4 = 0.67 ppm corresponding to the four protons of the newly formed and highly symmetric metallocyclopropane moiety of 2b. In all other cases, the 1H NMR data reflect the asymmetric nature of the metallocycle derived from substituted terminal alkenes. In 2f, magnetically and chemically inequivalent protons of the metallocyclopropane resonate at \u03b4 = 0.31 , 0.93 ppm (m) and 1.03 (m) ppm.The reaction of 1H NMR spectroscopy data on isolated samples of 2a\u2013f, remarkably in all cases alkene binding was found to be reversible. Although the metallocyclopropane is the dominant species in hydrocarbon solutions , the position of the equilibrium was found to be dependent on the nature of the alkene. At 298 K, in toluene-d8, 2a equilibrated to <1% of 1 and the non-coordinated alkene, whereas significantly more of 1 was observed to form from 2d center of 2a\u2013g compared to the Al(i) center of 1.Compounds lengths are all 1 there is an 18\u201321% increase in the C\u2013C bond length compared to the parent alkenes.3(\u03b72-CH2CH2)][K] the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond of bound ethylene elongates by 3% compared to that in free ethylene.2(\u03b72-CH2CH2)].1 and related main group systems,e.g. F, Cl, CF3, CN) to late transition metals. In these instances, back-donation from metal d-orbitals to the \u03c0*-orbital of the alkene is the dominant factor in bonding and alkene coordination can be conceptualised in terms of an oxidative addition to the transition metal.Upon ethylene or norbornene binding to 2b dimerises to form 3, a product which incorporates a bimetallocyclohexane ring confirm that 3 is thermodynamically favorable relative to 2 equiv. of 2b . The formation of 3 is non-reversible. While a series of related compounds were previously reported from the reaction of terminal alkenes with a bimetallic gallium complex, in this instance a metallocyclopropane intermediate could not be observed.2b to form 3 remains unclear and although it is tempting to suggest that this involves a simple bimolecular reaction of 2 equiv. of 2b, at this point the reversible formation of 1 as a reaction intermediate cannot be excluded (vide infra).In the presence of an excess of ethylene, ane ring . This difraction and DFT 2c, in the presence of excess alkene, at 353 K in C6D6 led to the loss of the dark orange colour and formation of the allylic C\u2013H activation product 4c and single crystal X-ray structure of 4f, which clearly showed the geometry of the double bond in the chain failed to provide any evidence for the reformation of 1.Further experiments showed that the formation of these C\u2013H activation products is non-reversible and that accessible allylic sp3 C\u2013H bonds of propene have a bond dissociation energy of 88.8 \u00b1 0.4 kcal mol\u20131 and while more reactive than those in propane, they are still challenging bonds to break with metal complexes.4 and 1 respectively has been reported and in the former case shown to be a reversible redox process.1 reacts with the C\u2013H bonds of benzene but only in the presence of a palladium catalyst.47The allylic sp1 in benzene-d6 was reacted with a 5 equiv. of allylbenzene, with full formation of 2f observed after 1 hour. The solution was heated to 343 K and monitored by 1H NMR spectroscopy over the course of 4 hours. Immediate formation of 4f was observed, along with small amounts of unreacted 1. Complex 1 remained present in low, but steady, concentration (<5%) throughout the reaction, suggesting that it may be a potential intermediate in C\u2013H activation.Kinetic data was obtained and modelled with Copasi software. These experiments were undertaken as a means to gain insight in to the reaction mechanism and establish the activation parameters for the C\u2013H activation step. A 0.018 M solution of 2f to 4f did not lead to reasonable activation parameters. The system was considered as an equilibrium between 1 and 2f, with non-reversible conversion of 1 to 4f (k\u20131/k1 = 0.056) and the irreversible C\u2013H activation (k2 = 3.0 \u00d7 10\u20133 s\u20131) at 343 K. An alternate kinetic model exists. The reaction network could also be considered as an equilibrium between 1 and 2f with non-reversible conversion of 2f to 4f. While the two kinetic scenarios involving a pre-equilibrium step cannot be differentiated from one another experimentally, calculations provide unambiguous support for the involvement of 1 as an intermediate and show that the direct conversion of 2f to 4f is in fact unfavourable (vide infra).Attempts to fit the data using pseudo-first order kinetics as a conversion from 1 to 4f . Copasi 1 to 4f, k2) yielded the activation parameters and , with a Gibbs activation energy of \u0394G\u2021298 K = 20.5 kcal mol\u20131 .Eyring analysis of the C\u2013H activation reaction over the temperature range 343\u2013363 K calculations were conducted using the hybrid basis set 6-31G**/SDD. A series of functionals were investigated and the M06L functional was found to best model the structural and thermodynamic parameters determined from experiment . At the same time the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C Wiberg bond indices decrease , while those of the Al\u2013C bonds increase . Endo-TS-1 is asymmetric being characterised by not only the displacement of the Al atom out of the plane of the \u03b2-diketiminate ligand but also two distinct Al\u00b7\u00b7\u00b7C distances which differ by \u223c0.3 \u00c5 scale\" fill=\"currentColor\" stroke=\"none\">C \u03c0-bond to the vacant p-orbital on Al with concomitant back-donation from the Al sp2 lone pair to the \u03c0* orbital of the alkene (vide infra).As the simplest substrate to undergo both binding and C\u2013H activation, propylene was made the focus of these studies. Alkene binding was determined to take place through an asynchronous concerted pathway involving two closely related transition states, ex Int-1 . NBO anay \u223c0.3 \u00c5 . Similar1. This pathway proceeds by TS-2 with an activation barrier of \u0394G\u2021 = 24.3 kcal mol\u20131. The energies of TS-1 and TS-2 are consistent with the metallocyclopropane 2c being formed as the kinetic product which ultimately converts to the thermodynamic product 4cvia reformation of 1. Formation of 4c is non-reversible and proceeds in an exergonic step, . In TS-2, propylene develops the character of an allylic ligand as it undergoes C\u2013H activation. The Al\u00b7\u00b7\u00b7H bond length (1.93 \u00c5) and C\u00b7\u00b7\u00b7H bond length (1.39 \u00c5) are consistent with a late transition state. The allylic character is evidenced by a the relatively short interaction between Al and the terminal alkene carbon of the propylene moiety (2.19 \u00c5). The NPA charge of Al increases as the C\u2013H bond breaks, consistent with an oxidative addition . IRC calculations confirm that TS-2 does not evolve from 2c, but instead is the result of C\u2013H activation directly from 1 and free propylene.A low energy activation pathway was identified involving intermolecular oxidative addition of the C\u2013H bond of propylene to iii) complex 2c could not be identified at this level of theory. This latter reaction pathway involves a \u03b2-hydride elimination step known to operate for simpler three coordinate aluminium(iii) alkyls at high temperatures,2 \u2192 4 could be identified and compared with that of 1 \u2192 4. With the \u03c9B97x functional, a hybrid basis-set adapted for solvent and dispersion (\u03c9B97xD) by single point corrections, the transition state for \u03b2-hydride elimination of 2f was located and found to be obstructively high in energy >40 kcal mol\u20131. For comparison at the same level of theory TS-2 is \u0394G\u2021 = 27.1 kcal mol\u20131. Related \u03b2-fluoride elimination pathways have been modelled in these systems and are lower in energy likely due to a large ionic component to Al\u2013F bonding and the fluorophilicity of the Al3+ ion.37A pathway for the direct intramolecular C\u2013H activation of the bound propylene of the aluminium(trans-4f was observed as the sole reaction product over a 343\u2013363 K temperature range. Comparison of the C\u2013H activation transition states explains the regioselectivity. The Gibbs free energy (at 298 K) for the transition state for the formation of trans-4f was \u223c3 kcal mol\u20131 lower in energy than the TS that leads to cis-4f, likely due to 1,3-allylic strain (A-strain) induced as the hydrogen atom is transferred to aluminium and the hydrocarbon ligand starts to adopt alkene character of 1 consist of a vacant 3p orbital (LUMO) and an orthogonal sp2-hybridised lone-pair (HOMO). Alkene binding proceeds via an asymmetric transition state that involves overlap of the fMOs of 1 with those of the unsaturated C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond reversible alkene binding to form a metallocyclopropane and (ii) direct intermolecular C\u2013H activation, C bond . The resH \u03c3-bond .i) centre, along with an extremely rare case of a reaction sequence involving reversible substrate binding and C\u2013H activation at a main group fragment. The simple realisation that the fMOs of aluminium(i) required for alkene binding are also those required for C\u2013H activation has broad implications for the development of catalytic cycles.In summary, we report the first examples of reversible alkene binding to an aluminium only allow for binding or activation of a single substrate at a time. In contrast, for transition metal catalysts often several d-orbitals of suitable energy and symmetry are unoccupied. Most transition metal based redox catalytic cycles involve the coordination or activation of two substrates , bringing them into close proximity and facilitating bond formation.Low-valent main group species with small HOMO\u2013LUMO gaps are often targeted as a first step to develop redox catalysis, but the reality is that the most common designs hemi-labile ligands to open up coordination sites on the main group fragment, or (ii) integration of redox steps with more common pathways of main group compounds such as hydroelementation, \u03c3-bond metathesis and nucleophilic addition.The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.There are no conflicts to declare.Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "The design of novel heterogeneous catalysts with multiple adjacent functionalities is of high interest to heterogeneous catalysis. N-phenylsilanamine\u2013silanol pairs. In contrast with ammonia treated surfaces, the material is stable under air/moisture. Advanced solid state MAS NMR experiments and dynamic nuclear polarization enhanced 29Si and 15N spectra demonstrate both the close proximity between the two moieties and the formation of a covalent Si\u2013N surface bond and confirm the design of vicinal acid\u2013base pairs. This approach was successfully applied to the design of a series of aniline derivatives of bifunctional SBA15. A correlation between the substituent effects on the aromatic ring (Hammett parameters) with the kinetics of a model Knoevenagel reaction is observed.The design of novel heterogeneous catalysts with multiple adjacent functionalities is of high interest to heterogeneous catalysis. Herein, we report a method to obtain a majority of bifunctional acid\u2013base pairs on SBA15. Aniline reacts with SBA15 by opening siloxane bridges leading to One of the major current challenges in heterogeneous catalysis is the ability to develop multifunctional catalyst systems where each active site plays a distinct role in the overall catalytic process (cascade approach). To date, two main approaches to introduce functionalities into mesoporous materials exist: the soft templating strategy for the synthesis of organic\u2013inorganic hybrid materialsvia an alkyl spacer, can be randomly distributed or organized.In the soft templating method, the inorganic materials provide the surface area and the porosity. The organic active site, linked to the surface via a multistep mechanism. However, the surface\u2013complex bond is usually a \u03c3-bonded oxygen ligand, e.g., siloxy [ scale\" fill=\"currentColor\" stroke=\"none\">Si\u2013O\u2013)MLn], with M = metal and L = ligands, in the primary coordination sphere and the requirement for oxygen limits the development of SOMC methods. It would then be highly desirable to tune the coordination sphere of the metal center by designing surface ligands in close proximity to the surface to preserve the rigidity of the ensemble \u201csurface ligand/complex\u201d. By tuning the electronic and/or steric properties of the surface ligand, new catalysts and new reactions will be discovered.In the SOMC methodology,via treatment with ammonia, by analogy with the ring opening of epoxides by ammonia. PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019Si\u2013NH2] surface groups is associated with experimental, economical and safety disadvantages, such as the need for a high flow rate (200 mL min\u20131) of pure, expensive and corrosive ammonia, and the resulting materials being moisture sensitive. Aside from that, further chemical modifications of SBA15 to offer opportunities to provide tunable steric and electronic properties were impossible without affecting the structural parameters of the materials. To face these issues, we investigate an alternative approach based on the chemisorption of dry aniline onto highly dehydroxylated SBA15 (1100 \u00b0C), which has never been reported and its large and uniform pore diameter (6 nm) and relatively thick walls (3 to 6 nm).\u20135 mbar) yields the condensation of adjacent silanols and generates a support that contains a surface of mainly strained siloxanes along with a small amount of isolated silanols (<0.4 OH per nm2).1100 with dry aniline was performed in a solution in toluene at 80 \u00b0C for 20 h. The resulting material 1 was evacuated at room temperature overnight under high vacuum (10\u20135 mbar) and characterized by FT-IR spectroscopy. Comparison of the FT-IR spectra of SBA151100 and 1 (\u03bds(OH)] band with a red shift from 3748 to 3745 cm\u20131. Additionally, the typical single sharp infrared bands characteristic of secondary amines,N-phenylsilylamine appears at 3435 and 1500 cm\u20131. They correspond to the [\u03bd(NH)] and [\u03b4(NH)], respectively. Vibrational bands of the aromatic group are clearly visible at 3089\u20133023 cm\u20131 [\u03bd(CH)], at 1606 and 1500 cm\u20131 [\u03b4 scale\" fill=\"currentColor\" stroke=\"none\">C)] (overlap a NH band). Finally, the shoulder in the range of 3690\u20133585 cm\u20131 is assigned to electronic interactions of the \u03c0 system of the aromatic group with the newly formed adjacent silanol (\u03c0\u2013OH interactions).\u20131 assigned to the N\u2013H stretching vibration of physisorbed aniline.Well-ordered hexagonal mesoporous silica, SBA15, was chosen as a support because of its high thermal stability (up to 1200 \u00b0C), its high surface area spectrum scale\" fill=\"currentColor\" stroke=\"none\">SiOH proton. Its value appears slightly downfield compared to the chemical shift of PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019SiOH (1.7 ppm) generated by treatment with ammonia. PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019Si\u2013NH\u2013Ph.43Solid state NMR was used to characterize the pairwise nature of the atomic-level organization of the supported organic functionalities, spectrum shows foortho (Ho) and para (Hp) positions. The proton in the meta position (Hm) appears at 7 ppm NMR spectrum scale\" fill=\"currentColor\" stroke=\"none\">SiOH at 1.9 ppm and the proton resonance of PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019SiNHPh at 3.4 ppm [5.3 ppm in F1: \u03b4H(OH) + \u03b4H(NH) = 1.9 + 3.4] scale\" fill=\"currentColor\" stroke=\"none\">SiOH and the aromatic proton resonances of the neighboring PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019SiNHPh at 6.6 ppm [8.5 ppm in F1: \u03b4H(OH) + \u03b4H(Ho) = 1.9 + 6.6] scale\" fill=\"currentColor\" stroke=\"none\">SiNHPh at 3.4 ppm and the proton of the aromatic ring in ortho position Ho at 6.6 ppm [10 ppm in F1: \u03b4H(OH) + \u03b4H(Ho) = 3.4 + 6.6] scale\" fill=\"currentColor\" stroke=\"none\">SiOH] (1.9 \u00d7 2 = 3.8 ppm in F1) as well as between two (2 \u00d7 3.4 = 6.8 ppm in F1) are observed clearly demonstrates that the majority of sites are isolated \u201cacid\u2013base\u201d pairs : .Interestingly, the fact that no correlations between two silanols , with E = Si or H (commonly dubbed Q4 and Q3) PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019SiNHPh].52Dynamic nuclear polarization surface enhanced NMR (DNP SENS)rum of 1 displays15N DNP SENS spectrum shows a single peak at 66 ppm in the 2\u03b8 range of 0.7\u20134\u00b0. They confirm the presence of a well-ordered hexagonal mesophase with a d100 spacing of 86.28 \u00c5 , and Transmission Electronic Microscopy (TEM). The small angle X-ray diffraction patterns of 1 of approximately 512 m2 g\u20131 (versus 679 m2 g\u20131 for SBA1100) and pore volumes of 0.65 cm3 g\u20131 (versus 0.9 cm3 g\u20131 for SBA1100). Also, 1 showed type IV isotherms and the dissociative chemisorption of aniline , the mesoporous structure is still regular over the whole particle of 1.Further evidence for a well-ordered hexagonal mesostructure is provided by the TEM images , which aKa = 13) scale\" fill=\"currentColor\" stroke=\"none\">C bond forming reactions. It produces several important key intermediates such as \u03b1, \u03b2 unsaturated products widely used for the synthesis of therapeutic drugs, functional polymers and fine chemicals.A materials with atomic organization of acid\u2013base pairs should exhibit cooperative catalytic behavior for the Knoevenagel condensation of benzaldehyde with diethyl malonate (pKa = 13) .55\u201357 Thet al. PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019Si\u2013OH are capped with trimethylsilyl group scale\" fill=\"currentColor\" stroke=\"none\">Si\u2013OSiMe3). So, weakly acidic silanols play a vital role in the cooperative catalytic cycle as well as the spatial organisation of the acid\u2013base functionalities.In the literature, several studies have revealed an efficient catalysis by cooperative acid\u2013base pairs well organized on mesoporous silica.1H-MAS solid state NMR spectroscopy (3745 cm\u20131), \u03bd(NH) and \u03b4(NH), 3435 and 1500 cm\u20131 respectively. Vibrational bands of the aromatic group are still present at 3089\u20133023 cm\u20131 [\u03bd(CH)], at 1606 and 1500 cm\u20131 [\u03b4 scale\" fill=\"currentColor\" stroke=\"none\">C)] (overlapping a NH band). The shoulder characteristic of the electronic interactions of the \u03c0 system of the aromatic group with the newly formed adjacent silanol (\u03c0\u2013OH interactions) in the range of 3690\u20133585 cm\u20131 is again observed for all catalysts .All the FT-IR spectra of catalysts 1H NMR spectra feature the characteristic signal of PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019SiOH and PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019SiNH at around 2 ppm and 3.5\u20133.9 ppm, respectively. As expected, the protons in ortho and meta position to electron donating (OMe) and electron withdrawing substituents show distinct upfield and downfield shifts is a weaker base than catalyst (1). A chloro group in the para position is a slightly EWG, so catalyst (3) exhibits better activity than (2) and is slightly less active than (1). Introducing an electron-donating group (EDG) as a p-methoxy group in the catalyst (4) enhances the catalytic performance in the Knoevenagel reaction. Among all these catalysts, (4) exhibits the best performance whereas (5) exhibits the lowest due to the base weakening effect.Their catalytic performance were tested (6), SBA15 where primary amine and silanol groups are proximal (7).6) shows no activity as no basic sites are present. (7) contains primary amines which are supposed to be the strongest base; yet it gives only 18% conversion after 24 h. (1) and (4) yield better conversion although their basicity is lower than that of (7). These results are explained by the higher stability of these catalysts under the experimental conditions (ethanol is the solvent and water is produced during the Knoevenagel reaction). Indeed, the PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019SiNH2 group is well-known to be easily hydrolyzed.Besides this, the catalytic results of this series of acid\u2013base paired catalysts (7) and (1) towards ethanol was monitored by FT-IR spectroscopy. After 5 min in contact with ethanol, the FT-IR spectrum shows complete disappearance of the characteristic bands of the PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019SiNH2 group . However the FT-IR spectrum of (1) shows the characteristic bands of PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019Si\u2013NHPh, [\u03bd(NH) = 3435 cm\u20131] even after 1 h in contact with dry ethanol. In addition, during the catalytic test with catalyst 1, no leaching of aniline was detected by both GC-FID and GC-MD scale\" fill=\"currentColor\" stroke=\"none\">Si\u2013O\u2013Si PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019) of mesoporous SBA151100. The generation of well-defined adjacent N-phenylsilanamine\u2013silanol pairs was unambiguously determined through FTIR, 2D solid state NMR, XRD, nitrogen sorption and TEM. This way to design bi-functionalized mesoporous surface offers new opportunities to modify the electronic and steric properties of mesoporous silica useful for heterogeneous catalysis.The opening siloxane bridges approach was successfully established to create an atomic organization of well-defined bi-functional acid\u2013base pairs on mesoporous SBA15. This approach is based on an analogy between organic epoxides and strained siloxanes (Supplementary informationClick here for additional data file."} +{"text": "The \u03b2-diketiminato magnesium hydride, [(BDI)MgH]]2, reacts with alkenes and catalyses their hydrosilylation with PhSiH3. 2, reacts at 80 \u00b0C with the terminal alkenes, 1-hexene, 1-octene, 3-phenyl-1-propene and 3,3-dimethyl-butene to provide the respective n-hexyl, n-octyl, 3-phenylpropyl and 3,3-dimethyl-butyl magnesium organometallics. The facility for and the regiodiscrimination of these reactions are profoundly affected by the steric demands of the alkene reagent. Reactions with the phenyl-substituted alkenes, styrene and 1,1-diphenylethene, require a more elevated temperature of 100 \u00b0C with styrene providing a mixture of the 2-phenylethyl and 1-phenylethyl products over 7 days. Although the reaction with 1,1-diphenylethene yields the magnesium 1,1-diphenylethyl derivative as the sole reaction product, only 64% conversion was achieved over a 21 day timeframe. Reactions with the \u03b1,\u03c9-dienes, 1,5-hexadiene and 1,7-octadiene, provided divergent results. The initial 5-alkenyl magnesium reaction product of the shorter chain diene undergoes 5-exo-trig cyclisation via intramolecular carbomagnesiation to provide a cyclopentylmethyl derivative, which was shown by X-ray diffraction analysis to exist as a three-coordinate monomer. In contrast, 1,7-octadiene provided a mixture of two compounds, a magnesium oct-7-en-1-yl derivative and a dimagnesium-octane-1,4-diide, as a result of single or two-fold activation of the terminal C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C double bonds. The magnesium hydride was unreactive towards internal alkenes apart from the strained bicycle, norbornene, allowing the characterisation of the resultant three-coordinate magnesium norbornyl derivative by X-ray diffraction analysis. Computational analysis of the reaction between [(BDI)MgH]2 and 1-hexene using density functional theory (DFT) indicated that the initial Mg\u2013H/C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C insertion process is rate determining and takes place at the intact magnesium hydride dimer. This exothermic reaction traverses a barrier of 18.9 kcal mol\u20131 and results in the rupture of the dinuclear structure into magnesium alkyl and hydride species. Although the latter three-coordinate hydride derivative may be prone to redimerisation, it can also provide a further pathway to magnesium alkyl species through its direct reaction with a further equivalent of 1-hexene, which occurs via a lower barrier of 15.1 kcal mol\u20131. This Mg\u2013H/C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C insertion reactivity provides the basis for the catalytic hydrosilylation of terminal alkenes with PhSiH3, which proceeds with a preference for the formation of the anti-Markovnikov organosilane product. Further DFT calculations reveal that the catalytic reaction is predicated on a sequence of Mg\u2013H/C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C insertion and classical Si\u2013H/Mg\u2013C \u03c3-bond metathesis reactions, the latter of which, with a barrier height of 24.9 kcal mol\u20131, is found to be rate determining.The dimeric \u03b2-diketiminato magnesium hydride, [(BDI)MgH] Compoun2 , reacts as a dimer via highly polarised pathways, even with unactivated terminal alkenes, to provide exceptionally potent calcium alkyl nucleophiles, [(BDI)CaR]2. PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019E scale\" fill=\"currentColor\" stroke=\"none\">E , PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bonded small molecule appears to have been described.28It has previously been shown that the \u03b2-diketiminato calcium hydride, [(BDI)CaH]E E = O, NR19\u201322 E E = O, NR26 bonNotwithstanding impressive recent advances in first row transition metal chemistry,3,6F5)3,3Si(C6H6)][B(C6F5)3],2][B(C6F5)4]\u2013 via silane hydride abstraction by the potent Lewis acid centre and sequential delivery of the silylium cation and hydride to the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C double bond.+[B(C6F5)4]\u2013 (5) as a catalyst for the hydrosilylation of alkenes and alkynes.5 is strictly isoelectronic to a monomeric unit of 4 and, although the exact details could not be elucidated, the authors suggested that the most likely mechanism involves alkene attack on an incipient silylium ion generated through hydride abstraction by the highly Lewis acidic aluminium cation scale\" fill=\"currentColor\" stroke=\"none\">C insertion into the M\u2013Si bond of a metal silanide, which is apparently formed by M\u2013C/silane metathesis in competition with the expected hydride intermediates. This supposition was vindicated by Okuda's subsequent development of several group 1 and calcium silanide and hydridosilicate species, which also yield the anti-Markovnikov products for the hydrosilylation of 1,1-diphenylethylene and similarly activated alkenes.e.g.3) may be converted to n-alkyl species by their reaction with unactivated alkenes.4 and that subsequent silane metathesis of the resultant organomagnesium species provides a basis for the hydrosilylation of C\u2013C unsaturated substrates.Hydrosilylation mediated by s-block centres was pioneered by Harder and co-workers who employed highly polar potassium, calcium and strontium benzyl species as pre-catalysts for the silane reduction of conjugated 1,1-diphenylethylene, styrene and diene substrates.1.000000,.000000 s4) and two molar equivalents of 1-hexene at 80 \u00b0C. Monitoring by 1H NMR spectroscopy over a period of 4 hours evidenced complete consumption of the starting materials and the production of a single new base-free \u03b2-diketiminato n-hexyl magnesium derivative , similar thermal treatment of both 3-phenyl-1-propene and 3,3-dimethyl-1-butene indicated that the facility of these C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C insertion reactions is significantly perturbed by the relative steric demands of the alkene substrate. In these latter cases, reactions at 80 \u00b0C between compound 4 and the unsaturated organic reagents required 2 days and 21 days to achieve complete conversion to the respective magnesium 3-phenyl-propyl (8) and 3,3-dimethyl-1-butyl (9) complexes, which were nevertheless isolated in analytically pure form.The successful synthesis of compound alkenes . Althoug4 with both styrene and 1,1-diphenylethene. Whereas the terminal hydride derivative 1 was reported to react readily with styrene within 2 hours at room temperature to yield compound 2 and 1-phenylethyl (10B) complexes, resulting from either 1,2- or 2,1-C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C insertion, respectively. This noteworthy kinetic effect was exacerbated by the introduction of additional phenyl substitution. Despite benefitting from the benzhydrylic substitution pattern that has previously been observed to greatly facilitate analogous reactions with molecular calcium hydrides,4, requiring 6 weeks at 100 \u00b0C to achieve 64% conversion to the 1,1-diphenylethyl complex (11). Although pure bulk samples of compounds 10A/B and 11 could not be isolated, the structures of these organomagnesium derivatives were assigned with a high degree of certainty by in situ studies of the two reactions by 1H and 13C{1H} NMR spectroscopy.These observations were underscored by the reactions of mpound 2 ,11 a sim3) with both 1,5-hexadiene and 1,7-octadiene proceed via initial formation of the respective open chain 5-alkenyl and 7-en-1-yl derivatives. The shorter chain species is rapidly consumed, however, through an intramolecular carbocalciation reaction to provide an isolable calcium cyclopentylmethyl derivative.4 at 80 \u00b0C similarly resulted in complete and selective conversion to a single new compound, which was identified as the magnesium cyclopentylmethyl derivative (12) resulting from facile 5-exo-trig cyclization and the dimagnesio-octane-1,4-diide (14) were formed in effectively equimolar quantities through complete consumption of the hydride reagent at 80 \u00b0C over the course of 5 hours. Although compounds 13 and 14 proved to be inseparable, irrespective of variations in the reaction stoichiometry, the compounds could be readily discriminated by both 1H and 13C NMR spectroscopy.A similar reaction performed between compound closure . Rather,3),4 was found to be completely unreactive towards the internal alkenes, 2,3-dimethyl-2-butene, cyclopentene and cyclohexene but to react smoothly, albeit slowly, with both the strained bicyclic alkene, norbornene (3 days at 80 \u00b0C) and the internal alkyne, diphenylacetylene (6 days at 80 \u00b0C), to provide the magnesium norbornyl (15) and (E)- (16) derivatives. Although monitoring of the reaction with norbornene revealed tentative evidence that the production of 15 occurs via the formation of a dinuclear hydridonorbornyl-dimagnesium intermediate and norbornene,15) was found to be thermally stable allowing its isolation in high (>90%) yield. The formation of compound 15 was signified in its 1H NMR spectrum by a new BDI methine singlet at \u03b4 4.93 ppm and a characteristic upfield doublet of doublets of doublets signal at \u03b4 \u20131.31 ppm, which emerged in a 1\u2009:\u20091 ratio by integration. The constitution of compound 16 was readily established by 1H NMR spectroscopy through the appearance of new BDI methine and vinylic C\u2013H singlet resonances, each of which developed simultaneously and with identical 1H integrals at \u03b4 4.93 and 5.85 ppm. Compound 15 was also characterised by single crystal X-ray diffraction analysis scale\" fill=\"currentColor\" stroke=\"none\">C insertion mechanism was provided by density functional theory (DFT) calculations carried out with the same computational approach scale\" fill=\"currentColor\" stroke=\"none\">C insertion profile shown in H = \u201314.1 kcal mol\u20131) 1-hexene insertion takes place into the dimeric magnesium hydride, 4(A), via an accessible barrier of 18.9 kcal mol\u20131. For comparison, we also computed an initial insertion step on the mononuclear magnesium compound (C) to form a proximal but non-bonding pair of dissimilar three-coordinate magnesium alkyl and hydrido complexes (D) is significantly exothermic . Although the hydridic component may redimerise to 4(A), the low coordinate magnesium hydride also holds the potential to undergo a second 1-hexene insertion via an accessible barrier of 15.1 kcal mol\u20131 scale\" fill=\"currentColor\" stroke=\"none\">C insertion at 4(A) and the three-coordinate hydride generated by its monomerisation, respectively. Although this latter observation appears somewhat surprising, it is possibly ascribed to a stabilising interaction between the hexyl and hydrido complexes at the transition state (TS-EF). A transition state corresponding to the second 1-hexene insertion into the dinuclear magnesium hydride complex C could be also located led us to discard this possible pathway. The dimerisation of complex F, yielding complex F1, was computed to be endothermic by 17.0 kcal mol\u20131 allowing us to also discount this possibility. Consistent with the experimental results, therefore, the overall result of Mg\u2013H/C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C insertion may be assessed to be a facile process affording three-coordinated, mononuclear alkyl species (F).Further insight into the Mg\u2013H/Ccompound , that isal mol\u20131 . This va Fig. S36, althoug4 with C\u2013C multiple bonds, we next turned our attention to its potential to mediate catalytic hydrosilylation of alkenyl substrates with phenylsilane. An initial reaction was performed in C6D6 between 1-hexene and PhSiH3 in the presence of 5 mol% 4. Monitoring of the reaction performed at 60 \u00b0C by 1H NMR spectroscopy indicated that, although somewhat sluggish, the reaction proceeded with complete conversion to the product of anti-Markovnikov silane addition, n-hexyl(phenyl)silane, over the course of one week. An otherwise identical reaction performed at 80 \u00b0C provided similar observations but in the shorter timeframe of 4 days. We suggest that the observation of the anti-Markovnikov product is consistent with both the high regioselectivity of the stoichiometric insertion reaction to provide the terminal n-hexylmagnesium product (6) and the operation of a mechanism dependent upon a sequence of Mg\u2013H/C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C insertion and Mg\u2013C/Si\u2013H metathesis events, which is broadly analogous to that envisioned for organolanthanide-based hydrosilylation catalysis (vide infra).Given the unexpectedly broad reactivity of compound 3 and a range of alkene substrates (3 and vinylsilanes (entries 4 and 5) were less successful, providing, at best, only stoichiometric (based on Mg) conversion to the unsymmetrical \u03b1,\u03c9-disilane product for the Ph3Si-substituted substrate (entry 4) and no evidence of any reaction for Me3Si scale\" fill=\"currentColor\" stroke=\"none\">CH2) (entry 5). In line with the expectation provided by the synthesis of compound 8, allylbenzene yielded, primarily, the anti-Markovnikov product (entry 6), while only the Markovnikov product was observed for the catalysis performed with 1,1-diphenylethene (entry 7). The observation of this latter product indicates that the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C insertion reaction is likely to be dictated by similar stereoelectronic considerations to those operant during Parkin and co-workers synthesis of compound 2 bias toward the production of the Markovnikov product, phenyl(1-phenylethyl)silane (entry 8).These deductions were borne out by a subsequent assessment of a series of catalytic hydrosilylation reactions performed with PhSiHbstrates . Consistmpound 2 . In the 3 provided an approximate 1\u2009:\u20093\u2009:\u20096 distribution of the open chain alkenylsilane, the symmetrical \u03b1,\u03c9-disilane and cyclised cyclopentylmethylsilane products (entry 9). Underlining the ease of formation of compound 12, the preponderance of this latter compound suggests that intramolecular carbomagnesiation is competitive with Mg\u2013C/Si\u2013H metathesis under the conditions of the catalysis. Similarly, although dimagnesiation of 1,7-octadiene to form compound 14 was competitive with its monomagnesiation under stoichiometric conditions, the product of its monohydrosilylation was found to predominate under the conditions of the catalysis (entry 10).The reaction of equimolar quantities of 1,5-hexadiene and PhSiH15, norbornene was the only internal alkene (entries 11\u201313) to undergo catalytic hydrosilylation in the presence of compound 4, providing the racemic exo-2-(silyl)norbornane product. The likely operation of an insertion-metathesis mechanism similar to that depicted in E)-(phenyl)silane as the sole reaction product.Consistent with the successful synthesis of compound 3 was studied by density functional theory calculations. i.e. from complex F. The associated transition state displays a classical Mg\u2013C/Si\u2013H metathesis arrangement, via a barrier of 24.9 kcal mol\u20131 (TS-GH). In accordance with the experimental observations, the hydrosilylation step is the rate determining process affording, in the case of 1-hexene, the experimentally observed anti-Markovnikov n-hexyl(phenyl)silane product exclusively. For completeness, we also computed the hydrosilylation reaction subsequent to the first 1-hexene insertion from the n-hexyl hydrido complex D and 35.4 kcal mol\u20131 (TS-F2F3), the magnitude of (TS-F2F3) allows us to discount the latter pathway. While (TS-D1D2) is competitive with that computed for (TS-GH), the barrier found for the subsequent necessary 1-hexene insertion is considerably lower than that associated with the Si\u2013H/Mg\u2013C metathesis reaction , suggesting that the latter step is rate determining and that the hydrosilylation process is, in any case, more likely to occur from F after complete alkylation of complex 4.The hydrosilylation of 1-hexene in the presence of PhSiH Fig. S37 and from Fig. S38. Althoug4) derivative reacts directly with terminal alkenes, the strained internal alkene, norbornene, and diphenylacetylene to provide the corresponding organomagnesium derivatives. Although the dinuclear structure of the magnesium hydride is retained during its initial reaction with 1-hexene, which occurs via a kinetic barrier comparable to that deduced for the analogous reaction of the hydridocalcium compound (3),via rate determining Si\u2013H/Mg\u2013C metathesis of the three-coordinate organomagnesium derivative.A dimeric \u03b2-diketiminato magnesium hydride (1) and compound 4 with styrene, however, hints that even more expansive substrate scope and catalytic activity may be achievable through the adoption of more sophisticated ligand design. Although not a central focus of our own research, we hope that our observations will prompt others toward a more wide-ranging and sustainable future.These observations reveal that hydridomagnesium compounds may display a much broader reactivity with alkenyl substrates than previously appreciated. The comparable reactivity of Parkin's terminal magnesium hydride (There are no conflicts of interest to declare.Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "Owing to the increased proton affinity that results from additional negative charges, multiply-charged anions are shown as a route to preparing powerful \u2018superbases\u2019. ortho-diethynylbenzene dianion (ortho-DEB2\u2013) and present observations of this novel species undergoing gas-phase proton-abstraction reactions. Using a theoretical model based on Marcus\u2013Hush theory, we attribute the stability of ortho-DEB2\u2013 to the presence of a barrier that prevents spontaneous electron detachment. The proton affinity of 1843 kJ mol\u20131 calculated for this dianion superbase using high-level quantum chemistry calculations significantly exceeds that of the lithium monoxide anion, the most basic system previously prepared. The ortho-diethynylbenzene dianion is therefore the strongest base that has been experimentally observed to date.Owing to the increased proton affinity that results from additional negative charges, multiply-charged anions have been proposed as one route to prepare and access a range of new and powerful \u201csuperbases\u201d. Paradoxically, while the additional electrons in polyanions increase basicity they serve to diminish the electron binding energy and thus, it had been thought, hinder experimental synthesis. We report the synthesis and isolation of the The strongest base prepared to date is the lithium monoxide anion (LiO\u2013).\u20131, LiO\u2013 supplanted the methide anion (CH3\u2013) at the top of the basicity scale in 2008, exceeding the proton affinity of the carbanion by approximately 40 kJ mol\u20131.3O\u2013.\u2013 and CH3\u2013 necessarily satisfy two essential requirements: they are the conjugate bases of very weak gas-phase acids and their neutral radicals have low electron affinities (EAs). Multiply-charged anions can also fulfil these thermochemical requirements, as the gas-phase acidity of an anion is inherently low while the electron affinity of an anion can be low or even negative. Despite their potential instability, such dianion systems have been observed because of a repulsive Coulomb barrier (RCB) that arises from the interaction between the local bound-potential of the functional group carrying the charge and the repulsive Coulomb potential between like charges.meta-DEB2\u2013) was postulated to be a gas-phase superbase with a calculated PA of 1796.6 kJ mol\u20131, approximately 15 kJ mol\u20131 greater than that of the lithium monoxide anion.In the gas phase, the proton affinity of an anion is equivalent to the enthalpy of deprotonation \u0394acidH29 of the cmeta-DEB2\u2013 in the gas phase along with the isomeric 1,2- and 1,4-diethynylbenzene dianions . Observation of proton-transfer reactions between these dianions and a number of weak acids demonstrates their behaviour as gas-phase bases. The calculated proton affinity of each of the DEB2\u2013 isomers exceeds that of the lithium monoxide anion, with ortho-DEB2\u2013 representing the strongest gas-phase base synthesised to date.In this article, we outline the synthesis of ortho-DEB2\u2013 dianion at a mass-to-charge ratio (m/z) of 62 was performed using tandem mass spectrometry in a linear quadrupole and followed the process outlined in m/z 106), which was mass-selected and subjected to successive collisional activation steps to remove the carboxylate groups while retaining both charges. Such decarboxylation processes that are accompanied by retention of charge have previously been noted for several organolithium compounds.meta- and para-DEB2\u2013, using the appropriate isomeric diacid precursor. In addition to the DEB2\u2013 dianion (m/z 62) and its associated proton-transfer product (m/z 125) observed in 2 and loss of an electron from m/z 84 (m/z 124), accompanied by a small amount of C2 loss from this ion (m/z 100).Synthesis of the m/z 62 dianions is retained from their precursor diacids . This is strongly supported by the differences in product ions and product ion abundances observed in the mass spectra at each step of the gas-phase preparation is diagnostic of deuteron abstraction from the heavy water. Because the performance of the ion-trap mass spectrometer is diminished at very low m/z (i.e. m/z < 20), the intensity of the m/z 18 peak appears artificially reduced compared with the intensity of m/z 126 and consequently a robust comparison of the product ion abundances is not possible. The presence of the m/z 125 ion in all spectra is evidence of proton abstraction from unlabelled water and other background gases present in the instrument.Mass selection allowed isolation of the m/z 125 . This prortho- and meta-DEB2\u2013 and adiabatic electron affinities of the corresponding singly-charged radical anions . Using a relation derived from Marcus\u2013Hush theory, the RCB height can be estimated from the calculated AEA and VDE values according to eqn (1).18For ta-DEB2\u2013 , proton ra-DEB2\u2013 , a produG* as the energy barrier for electron detachment, the reorganisation energy \u03bb as the energy difference between the VDE and AEA, and \u0394G\u00b0 as the AEA, an estimate of the RCB height can be ascertained from the computed minimum energy structures of diethynylbenzene as both singly- and doubly-charged anions. The results, compiled in ortho- and para-DEB\u02d9\u2013, suggesting that only the meta-DEB2\u2013 dianion is thermodynamically stable with respect to electron detachment. For the three dianions, however, electron ejection is inhibited by RCBs of 11.1 (ortho), 52.7 (meta) and 1.9 (para) kJ mol\u20131. These barrier heights, calculated from eqn (1), are consistent with the analytical determination of barrier heights obtained using a rectilinear projection of the singly- and doubly-charged anion geometries . These results provide experimental evidence that the DEB2\u2013 isomers are capable of deprotonating benzene and thus have proton affinities in excess of PA[C6H5\u2013] = 1678.7 \u00b1 2.1 kJ mol\u20131.2O and C6H6, the reactivity observed for ortho-DEB2\u2013 far exceeds that of the other two isomers, reinforcing the heightened basicity of this species compared with meta- and para-DEB2\u2013, which are essentially the same at this level of ion signal. No proton-transfer reactions were observed between DEB2\u2013 isomers and either dihydrogen or methane despite favourable thermodynamics for these processes, which may be attributed to the presence of a substantial barrier for proton abstraction from these acids and benchmark calculations show good agreement in the case of strong gas-phase bases for which experimental PA data exist calculated at the same level of theory. Most significantly, ortho-DEB2\u2013 has a computed proton affinity of 1843.3 kJ mol\u20131, making it the strongest base synthesised to date by some 65 kJ mol\u20131.The G4(MP2)-6X method has been shown to have good performance for computing PAs shows that the proton affinity of acetylide anions can be related to the homolytic bond dissociation enthalpy (BDE) of the C\u2013H bond and the AEA of the corresponding radical by the ionisation energy of a hydrogen atom (IE[H]).21 PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C\u2013(CH2)n\u2013C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C]2\u2013 (n = 1\u20134) scale\" fill=\"currentColor\" stroke=\"none\">C\u2013(CH2)n\u2013C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C]\u02d9\u2013 radical anions decrease from +86.1 kJ mol\u20131 (n = 4) to +1.4 kJ mol\u20131 (n = 2) before returning large negative values when only a single methylene separates the two acetylide groups: \u201377.1 kJ mol\u20131 (n = 1). The decrease in AEA is accompanied by a concomitant increase in the proton affinity for the 2\u2013 dianion from 1775.4 (n = 4) through to 1888.1 kJ mol\u20131 (n = 1). While the proton affinity can be enhanced by bringing the like charges together, synthesis and isolation of a dianion superbase requires an RCB sufficient to prevent electron detachment. The simple formalism of eqn (1) predicts that when n = 1 the RCB is very low (4.2 kJ mol\u20131), suggesting that, like para-DEB2\u2013 to yield the doubly-decarboxylated dianion at m/z 62 along with the singly-charged CO2 loss ion m/z 124 and a proton-transfer product (m/z 125). This protocol was used for all three isomers. A minor product at m/z 100, most likely C2 loss from m/z 124, was observed for ortho-DEB2\u2013 only.The dianions were synthesised by electrospray ionisation of a methanolic solution of 3,3\u2032-(phenylene)dipropiolic acid, basified with aqueous ammonia to aid deprotonation. Mass spectra were acquired using a dual linear quadrupole ion-trap mass spectrometer . Precursor dianions at 2O and C6H6). The vapour pressure of the neutral reagent at room temperature was sufficient to seed the helium buffer gas and was delivered to the high-pressure cell of the dual ion trap through the unmodified buffer gas inlet and split flow in the mass spectrometer. Reactions with gaseous reagents (D2 and CD4) were performed using a pre-made mixture of the deuterated reagents in UHP helium and delivered into the ion trap through the ion trap buffer gas He inlet. The proportions of each gas mixture were 1.6% by volume in helium for D2 and 0.14% by volume in helium for CD4, yielding estimated number densities of 1.42 \u00d7 1012 molecules per cm3 and 1.24 \u00d7 1011 molecules per cm3, respectively, at the \u223c2.5 mTorr pressure within the ion trap.2O, D2 and CD4 was necessitated by the presence of adventitious protonation reagents (such as H2O and CH3OH) in the vacuum system.Ion\u2013molecule experiments were conducted by passing the ion-trap He buffer gas over a small amount of the neutral reagent for the fully-optimised structures, we used BMK/6-31+G harmonic vibrational frequencies and appropriate literature scale factors .ortho-, meta- and para-diethynylbenzene, we have examined the potential energy surfaces for the mono- and di-anionic species along the path that connects the geometries of the two ions. This is accomplished using structures obtained through a linear combination of the optimised structures for the mono- and di-anions (see \u20131. Cartesian coordinates for all structures calculated are provided in the ESI as Table S4.Standard ions see . ImproveSupplementary informationClick here for additional data file."} +{"text": "A comprehensive experimental and quantum chemical study of the open-shell mixed valent disilicon hydride Si2(H)(Idipp)2 CH]2) is reported. II PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019Si0(Idipp)][B(ArF)4] (1H[B(ArF)4], Idipp = C[NCH]2, ArF = C6H3-3,5-(CF3)2) reveal a reversible one-electron reduction at a low redox potential (E1/2 = \u20132.15 V vs. Fc+/Fc). Chemical reduction of 1H[B(ArF)4] with KC8 affords selectively the green, room-temperature stable mixed-valent disilicon hydride Si2(H)(Idipp)2 (1H), in which the highly reactive Si2H molecule is trapped between two N-heterocyclic carbenes (NHCs). The molecular and electronic structure of 1H was investigated by a combination of experimental and theoretical methods and reveals the presence of a \u03c0-type radical featuring a terminal bonded H atom at a flattened trigonal pyramidal coordinated Si center, that is connected via a Si\u2013Si bond to a bent two-coordinated Si center carrying a lone pair of electrons. The unpaired electron occupies the Si PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019Si \u03c0* orbital leading to a formal Si\u2013Si bond order of 1.5. Extensive delocalization of the spin density occurs via conjugation with the coplanar arranged NHC rings with the higher spin density lying on the site of the two-coordinated silicon atom.Cyclic voltammetric studies of the hydridodisilicon borate [(Idipp)(H)Si Quantum chemical studies revealed the same sequence of frontier orbitals in +1H and its isolobal phosphorus counterpart +, according to which the HOMO\u20131 corresponds to the lone-pair orbital at the two-coordinated E atom , the HOMO is the E PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019E \u03c0-bonding orbital and the LUMO is the E PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019E \u03c0* orbital.+1H might be also reversibly reducible as the phosphanylphosphenium cation + .1H[B(ArF)4] in fluorobenzene at room temperature revealed a reversible one-electron reduction at a rather low half-wave potential (E1/2) of \u20131.63 V as well as an irreversible oxidation at +0.67 V versus the [Fe(\u03b75-C5Me5)2]+1/0 reference electrode scale\" fill=\"currentColor\" stroke=\"none\">Si0(Idipp)][B(ArF)4] (1Me[B(ArF)4])E1/2 = \u20131.85 V) than 1H[B(ArF)4]. Notably, reduction of +1H and +1Me occurs at much lower potentials than that of the cation + (E1/2 = \u20130.48 V).9The hydridodisilicon salt [(Idipp)(H)Silectrode and ESI\u20201H[B(ArF)4]. Indeed, vacuum transfer of THF to a 1\u2009:\u20091 stoichiometric mixture of 1H[B(ArF)4] and KC8 at \u2013196 \u00b0C followed by warming to \u201340 \u00b0C resulted in a distinct color change of the dark red solution of 1H[B(ArF)4] to give an intensely dark green solution, which after work-up and crystallization from n-hexane at \u201360 \u00b0C afforded Si2(H)(Idipp)2 (1H) as a dark green, almost black crystalline solid in 55% yield and 1 (5%).The CV results prompted us to attempt a chemical one-electron reduction of 5% yield (see ESI5% yield . Compoun1H [E1/2 in C6H5F = \u20132.15 V vs. [Fe(\u03b75-C5H5)2]+1/0 (Fc+/Fc)]E1/2 in THF = \u20132.30 V vs. Fc+/Fc)5-C5Me5)2] (E1/2 in MeCN = \u20131.91 V vs. Fc+/Fc),1H is a very strong one-electron reducing agent. Consequently, the radical 1H is selectively oxidized back to 1H[B(ArF)4] upon treatment with one equivalent of [Fe(\u03b75-C5Me5)2][B(ArF)4] in THF-d8 . The UV-Vis-NIR spectrum was also analyzed by time-dependent density functional theory (TdDFT) calculations against the absolute temperature (T) showed a linear correlation from which the effective magnetic moment \u03bceff was calculated after linear regression and found to be 1.68 \u03bcB (\u03bceff = 1.73 \u03bcB).Magnetic susceptibility measurements of solid 1.68 \u03bcB . This va1H was determined by single crystal X-ray crystallography. The radical features a crystallographically imposed inversion symmetry (space group: P21/c) in marked contrast to the C1-symmetric structure of +1H in 1H[B(ArF)4].R2 values. 1H features as 1H[B(ArF)4] and 1 a trans-bent planar CNHC\u2013Si\u2013Si\u2013CNHC core (1H (2.281(3) \u00c5) is considerably longer than that in 1H[B(ArF)4] (2.1873(8) \u00c5)1 (2.229(1) \u00c5) PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019Si double bond (2.20 \u00c5)e.g. 2.352 \u00c5 in \u03b1-Si).NHC bonds in 1H (1.873(4) \u00c5) are shorter than the Si\u2013CNHC bonds of the dicoordinated Si atoms in 1H[B(ArF)4] (1.940(2) \u00c5)1 (1.927(1) \u00c5)1H[B(ArF)4] (1.882(2) \u00c5).+1H results also in a distinct change of the conformation of the NHC substituents. Thus, both N-heterocyclic rings in 1H are arranged coplanar with the trans-bent CNHC\u2013Si\u2013Si\u2013CNHC core as evidenced by the dihedral angle \u03c6NHC of 3.3(2)\u00b0 (+1H one of the two N-heterocyclic rings (bonded to the two-coordinated Si atom) adopts an almost orthogonal orientation (vide infra). Thus, reduction of +1H leads to a population of the Si PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019Si \u03c0* orbital with one electron, reducing thereby the formal Si\u2013Si bond order from 2 in +1H to 1.5 in 1H as nicely reflected in the computed Si\u2013Si Wiberg bond indexes of +1H = 1.70; WBI(Si\u2013Si) of 1H = 1.17) scale\" fill=\"currentColor\" stroke=\"none\">Si \u03c0* orbital with \u03c0*(CN2) orbitals of the NHC substituents in the SOMO of 1H (NHC bonds and the concomitant elongation of the CNHC\u2013NNHC bonds of 1Hversus+1H (The molecular structure of NHC core . However 3.3(2)\u00b0 , whereasentation . All theMO of 1H , providiersus1H+ .1H displayed a \u03bd(Si\u2013H) absorption band at 2089 cm\u20131, which is characteristic for stretching vibrations of terminal Si\u2013H bonds band of Si2H is predicted at significantly lower wavenumbers (1592 cm\u20131 (2A1 state); 1491 cm\u20131 (2B1 state)),\u03bd(Si\u2013H\u2013Si) absorption bands of H-bridged silylium ions are shifted to much lower wavenumbers compared with the \u03bd(Si\u2013H) bands of the corresponding silanes (ca. 2150 cm\u20131).\u03bd(Si\u2013H) absorption band of 1H appears in-between that of 1H[B(ArF)4] containing a trigonal planar coordinated Si atom (\u03bd(Si\u2013H) = 2142 cm\u20131),ii)-hydride (IMe4)SiH(SitBu3) containing a strongly pyramidal bonded Si atom (IMe4 = C[N(Me)CMe]2: \u03bd(Si\u2013H) in KBr = 1984 cm\u20131).\u03bd(Si\u2013H) frequency decreases with increasing pyramidalization of the Si atom, which according to the quantum chemical calculations can be traced back to the decreasing s-character of the Si hybrid orbital in the Si\u2013H bond com1H was provided by continuous wave (cw) EPR spectroscopy at X-band frequencies. Spectra with a nicely resolved hyperfine coupling pattern could be obtained from samples of 1H in n-hexane solution at 336 K nucleus, two different 29Si (I = 1/2) and two pairs of two magnetically equivalent 14N (I = 1) nuclei, respectively hyperfine coupling constants (1.725 and 0.431 mT) were found, indicating an asymmetric spin density distribution over the Si atoms. Both values are smaller than those of other Si-based \u03c0 type radicals, such as the disilene radical cation [Si2(SitBu2Me)4]+ (2.30 mT)2R4]\u2013 1[B(ArF)4] (5.99 mT),vide infra). The two a(14N) hfcc's (0.246 and 0.100 mT) suggest a fast rotation of the magnetically different NHC substituents about the Si\u2013CNHC bonds on the EPR timescale occurring even at low temperature , whereas two almost degenerate minimum structures were obtained at the B97-D3/II level of theory contain a trigonal-pyramidal coordinated Si1 atom with a sum of angles of 335.51\u00b0 (B3LYP/I) and 342.58\u00b0 (B97-D3/II), respectively. Remarkably, the calculated structure of the diphosphanyl radical P2(Me)Mes*2, which is isolobal to 1H, displays a trigonal pyramidal geometry at the three-coordinated P atom (sum of angles: 337.5\u00b0),calc1H. In comparison, the second minimum structure obtained at the B97-D3/II level of theory is only 5.5 kJ mol\u20131 higher in energy than calc1H and contains the Si1 atom in a trigonal planar environment (sum of angles: 359.61\u00b0). A comparison of the structural parameters of calc1H and calc1H\u2032 with those obtained by single crystal X-ray diffraction reveals a good agreement of the calculated Si\u2013Si, Si\u2013CNHC and CNHC\u2013NNHC bond lengths of both minimum structures . All caldentical . These sructures . While tcalc1H at the B3LYP/I level of theory and of calc1H and calc1H\u2032 at the B97-D3/II level of theory are almost identical scale\" fill=\"currentColor\" stroke=\"none\">Si \u03c0* orbital, confirming that reduction of +1H leads to a population of the empty Si PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019Si \u03c0* orbital of +1H with one electron scale\" fill=\"currentColor\" stroke=\"none\">Si \u03c0 and the n(Si) lone pair orbital, respectively.The calculated quasi-restricted orbitals (QROs) of dentical . The SOMcalc1H revealed that the overall wave function is described by a major ground state configuration of [2-1-0] of the DOMO, SOMO and LUMO with 96% contribution, suggesting that static correlation can be neglected in the electronic description of 1H /def2-TZVP calculationsH see ESI.calc1H and calc1H\u2032 at the B97-D3/II level of theory are depicted in calc1H, 29% in calc1H\u2032), whereas the spin density at the Si1 atom is quite small , which is in full agreement with the observation of one large and one small a(29Si) hfcc in the experimental EPR spectrum of 1H (vide supra) and Si2-bonded NHC substituents, which explains the EPR-spectroscopic detection of two a(14N) hfcc's. The calculated giso values of calc1H (2.00483) and calc1H\u2032 (2.00454) agree well with the experimentally obtained giso value (2.00562).The calculated spin densities of 1H was provided by a natural bond orbital (NBO) analysis at the B3LYP/I level of theory scale\" fill=\"currentColor\" stroke=\"none\">Si \u03c0 bond with an occupancy of 1.95 and 0.82 electrons, respectively, which indicates indirectly a population of the Si PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019Si \u03c0* orbital with one electron leading thereby to a decrease of the formal Si\u2013Si bond order from 2 in +1H to 1.5 in 1H (vide supra). The Si2 atom in calc1H bears a lone pair of high s-character (72%) as similarly found for +calc1H (75%). Remarkably, both Si\u2013CNHC bonds in calc1H are composed of one doubly occupied Si\u2013CNHC \u03c3 NBO and one singly occupied Si PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CNHC \u03c0 NBO, of which the latter is absent in +calc1H. These additional Si\u2013CNHC \u03c0 contributions rationalize the shortening and strengthening of the Si\u2013CNHC bonds in 1H, which is also reflected in the higher Si\u2013CNHC WBI indexes (1H: WBI(Si\u2013CNHC) = 1.01 and 0.95; +1H: WBI(Si\u2013CNHC) = 0.86 and 0.74).Further insight into the electronic structure of calc1H and +calc1H at the B3LYP/I level of theory reveal that the positive partial charges at the Si atoms of +calc1H (q(Si1) = 0.27e, q(Si2) = 0.21e) are decreased by the reduction (1H: q(Si1) = 0.14e, q(Si2) = 0.03e) = 0.36e, q(NHC2) = 0.30e; 1H: q(NHC1) = 0.05e, q(NHC2) = \u20130.04e), whereas the hydridic character of the Si1-bonded H atom is retained = \u20130.14e; 1H: q(H) = \u20130.18e).Comparative analyses of the charge by natural population analyses (NPA) of 3.2H (1H) can be considered as a major advance in low-valent silicon hydride chemistry, given the intermediacy of Si2H in the chemical vapor deposition of amorphous hydrogenated silicon that is widely used in solar cell and thin film transistors technology. Whereas Si2H features a C2v-symmetric H-bridged ground state structure and is a \u03c3-type radical with a symmetric distribution of the spin density over the two silicon atoms, its NHC-trapped counterpart Si2(H)(Idipp)2 (1H) features a terminal Si\u2013H bond and is a \u03c0-type radical, in which the unpaired electron occupies the Si PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019Si \u03c0* orbital (SOMO), leading to a formal Si\u2013Si bond order of 1.5. Significant delocalization of the spin density into the NHC substituents occurs via \u03c0-conjugation of the Si PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019Si \u03c0* orbital with the \u03c0* orbitals of the coplanar arranged N-heterocyclic rings leading to a stabilization of the radical, in which the spin density is higher at the two-coordinated Si site. The mixed valent disilicon hydride 1H can be alternatively regarded as a H atom trapped in the closed shell compound Si2(Idipp)2. Implications of this view in hydrogen atom transfer chemistryThe isolation and full characterization of NHC-trapped SiSupplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "A reverse polarity photocatalysed Povarov reaction of imines and electron deficient alkenes is described. via a putative nucleophilic \u03b1-amino radical, generated by a proton coupled electron transfer process, addition to a range of conjugated electron deficient alkene substrates affords substituted tetrahydroquinoline products in high yields and with typically good to excellent diastereoselectivity in favor of the trans diastereoisomer. Sub-stoichiometric quantities of Hantzsch ester were found to be key to initiate the overall redox-neutral, free radical cyclization cascade. This new reaction complements existing two electron Lewis acid mediated variants and expands the capabilities of imine umpolung chemistry to synthetically relevant cyclisation methodology.A visible light mediated iridium photocatalysed reverse polarity Povarov reaction of aryl imines and electron deficient alkenes is described. Operating Photoredox catalysis, through its ability to generate reactive radical intermediates under mild yet highly tunable reaction conditions and experimental set-ups,7via the SET reduction of imine derivatives.15Photocatalytic approaches to the synthesis and functionalisation of amines and their derivatives \u2013 with direct impact on medicinal chemistry programmesin situ generated \u03b1-amino radicals from imines. Such a reversal of the natural imine polarity via the PCET manifold establishes a new umpolung approach for the synthesis of \u03b1-functionalised amines for the generation of \u03b1-heteroatom radicals,d amines , and creN,N-dimethylaniline and maleimide derivatives provided some precedent for the cyclisation pathway , phenyl vinyl sulfone as the Michael acceptor, (Ir[dF(CF3)ppy]2(dtbbpy))PF6 as photocatalyst and the commercial Hantzsch ester as a stoichiometric reductant, in DMSO, under blue LED light irradiation. Pleasingly, good reactivity was identified early and importantly cyclised product 3a \u2013 a reverse polarity Povarov productHE1. This was largely due to suppression of over-reduction and aza-pinacol side products (entry 2) and the use of substituted Hantzsch esters further suppressed their formation.Preliminary studies were carried out using fluorine tagged aldimine (3b\u2013d). Good reaction efficiency and excellent diastereoselectivity towards the trans diastereoisomeric product was noted in all cases.cis-configured stereoisomeric products often predominate.3e\u2013g, 3j\u2013n) whereas electron poor aromatics led to longer reaction times (3h) and even complete nullification of reactivity (3i). 3-Fluoro and 2-fluoro-substituted aldimines were tolerated in this chemistry however longer reaction times were required and yields diminished with proximity to the imine C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N bond (3p\u2013r). A substituted pyridyl substrate was also shown to be effective in the reaction mixture leading to an excellent yield of product (3s).With optimal conditions established, we looked to probe the scope of the photocatalytic reverse polarity Povarov reaction . Initial3t) even after prolonged reaction times. 4-Chlorophenyl vinyl sulfone was found to be an excellent electrophile (3ab). Similarly, maleimide and N-phenylmaleimide electrophiles were shown to be excellent coupling partners in the new cyclisation methodology, however diastereoselectivity was respectively reduced or absent in the reaction products (3ac\u2013ae). The chemistry was extendable to a three component one-pot process was resubmitted to the reaction conditions. Pleasingly an increase to 18\u2009:\u20091 was observed and mass balance was maintained ,4a, via initial reduction of the imine. This reinforces the proposal of PCET construction of the key \u03b1-amino radical.Recent investigations have shown that imines was replaced with 3a ppy]2(dtbbpy)PF6 (E01/2 = \u20131.37 V vs. SCE in CH3CN)A. As this methodology only requires sub-stoichiometric quantities of the substituted Hantzsch ester (HE4), we suggest this Povarov radical intermediate A can lose a proton to form radical anion intermediate B, in a base assisted homolytic aromatic substitution-type mechanism.1a and 2a, to be 18.3a) and regenerating the key \u03b1-amino radical.From these insights, we propose that PCET would enable Ir[dF(CFvia the PCET of imine derivatives. 10 mol% of a Hantzsch ester was found to be optimal as a reductive initiator, a feature which has not previously been disclosed in photoredox catalysis. Further investigations are ongoing to establish further coupling partners and scaffolds for this reverse polarity platform for the synthesis of \u03b1-functionalised amines.In conclusion we have developed a new photocatalytic reverse polarity Povarov reaction to construct decorated tetrahydroquinolines in high yield and diastereoselectivity. This polarity reversal was postulated to stem from an \u03b1-amino radical formed There are no conflicts to declare.Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "The deselenization of selenocysteine selectively removes the selenol group to give alanine under anaerobic conditions or serine under aerobic conditions (oxygen saturation). The development of native chemical ligation coupled with desulfurization has allowed ligation at several new ligation junctions. However, desulfurization also converts all cysteine residues in the protein sequence into alanine. Deselenization of selenocysteine, in contrast, selectively removes the selenol group to give alanine in the presence of unprotected cysteines. In this study we shed more light onto the deselenization mechanism of selenocysteine to alanine and provide optimized conditions for the reaction. The deselenization can be accomplished in one minute under anaerobic conditions to give alanine. Under aerobic conditions (oxygen saturation), selenocysteine is converted into serine. First, TCEP reduces selenocystine into selenocysteine, followed by deselenization with \u223c90% conversion after 24 hours. The slow rate of the reaction can be attributed to the acidic conditions (pH\u223c1). The observed 1H-NMR shift confirms both the conversion of selenocysteine to mono-deuterated Ala scale\" fill=\"currentColor\" stroke=\"none\">Se and TCEP PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O scale\" fill=\"currentColor\" stroke=\"none\">Se, and TCEP PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O , featuring a single Sec residue, was chosen as our control. Our starting conditions were phosphate buffer at room temperature, as previously reported.Following our initial investigation, we synthesized a series of peptides 1\u20138, and test1 led to a faster reaction where with 200 equiv. TCEP we observed complete deselenization within 1 min over peptide in 1 min and S13\u2020tBuSH) but was not completely eliminated. We therefore omitted any thiol additives from the following experiments.Under these ambient aerobic conditions, the Ser product was observed in significant amounts (20%) .33,51 Ine.g. temperature) play a more significant role in enhancing this reaction.Irradiation could in principle enhance the deselenization reaction by enhancing selenenyl radical formation, as suggested earlier for desulfurization reactions.1 increased significantly in the presence of the radical initiator VA-044, even when using a lower concentration of TCEP, and was completed within 1 min was almost completely halted even after 12 h scale\" fill=\"currentColor\" stroke=\"none\">Se (a product of the deselenization reaction) completely inhibits the deselenization reaction. PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019Se the reaction proceeded smoothly, and the deselenized Ala product formed within 30 min was slowed due to the low redox potential of the diselenide bond in the UXXU motif.7, the Cys analog of 3, required 200 equiv. TCEP and 10 equiv. VA-044 at 37 \u00b0C to give the doubly desulfurized product after 8 h in which Cys5 is substituted with Sec (BPTI(1\u201358)(C5U), peptide 1 was treated with TCEP at 0 \u00b0C in oxygen-saturated buffer, the Ser product was observed as the major product within 5 min was prepared together with the two possible products containing l-Ser 8a and d-Ser 8b leads to the formation of an alkyl radical on the \u03b2-carbon III, which abstracts a hydrogen to form the Ala product.The proposed radical deselenization mechanism is shown in II) becomes more favored. On the other hand, decreasing the temperature could slow the C\u2013Se bond cleavage and allow other reactions, such as the attack of the diradical oxygen molecule (if present), to take precedence. The inability of peptide 5 to form the Ser product, even under oxygen-saturated conditions, is consistent with our proposed mechanism, in which molecular oxygen is converted to a radical species via direct contact with the seleno-phoshoranyl radical II. This radical intermediate gives the peroxy-radical IV, which is reduced by TCEP to give Ser. In contrast, the alkyl radical on the \u03b2-carbon III (a common species in desulfurization and deselenization reactions) has little or no effect on molecular oxygen, thus no Cys to Ser conversion is observed with peptide 5. The driving force of the deselenization reactions is the formation of a strong P PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019Se bond in TCEP PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019Se.The formation of the Ser product is interesting, as this product can be completely eliminated under anaerobic conditions or selectively formed under oxygen-saturated conditions. As we noted initially, the deselenization rate of Sec to Ala increases with increasing the temperature of the reaction, suggesting that the homolytic C\u2013Se bond cleavage in the seleno-phosphoranyl radical (In this work, we have optimized both the selective deselenization reaction and provided considerable experimental evidence to support the previously suggested radical mechanism.We envision that selenocysteine modification reactions such as the deselenization reactions presented in this study will find future utility in chemical protein synthesis. This will enable the use of native cysteines for some ligation sites and selenocysteines at sites in which a non-chalcogen containing amino acid is desired.Supplementary informationClick here for additional data file."} +{"text": "We report the straight forward synthesis of a series of arene-borazine hybrids (BN-PAHs) called borazatruxenes; the DFT, solid state and solution characterisation are reported along with the separation and chiroptical studies of four optical isomers. We report the synthesis and characterization of a series of arene-borazine hybrids called borazatruxenes. These molecules are BN-isosteres of truxene whereby the central benzene core has been replaced by a borazine ring. The straightforward three step synthesis, stability and their chiroptical and electronic properties recommend them as new scaffolds for BN\u2013carbon hybrid materials. Computational studies at DFT level, closely matching the experimental data, provided insights in the electronic structure of these molecules. The interest in polycyclic aromatic hydrocarbons (PAHs) has grown over the past decade due to their stability, tuneable properties and versatility.3-symmetric PAH that has been intensively studied for the past 15\u201320 years.Truxene is a C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond, PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C with a B\u2013N in a PAH will lead to quasi-identical geometries but drastically different electronic structures.The B\u2013N bond is quasi-isosteric and isoelectronic with the C2 in close capped vials; they are stable in solution for at least two weeks with minimal decomposition.\u00a7The borazatruxenes are stable in solid state for at least one year if kept under N and easy to synthesise. Borazatruxenes are truxene analogues in which the central benzene core has been replaced by borazine. The B atom is directly linked to the phenylene rings which are connected via a methylene bridge to the N atom of the borazine core. This particular arrangement is responsible for the borazatruxenes' hydrolytic stability. Borazatruxenes 1\u20133 were synthesised according to the synthetic pathway illustrated in Herein we introduce a new class of borazine-PAHs that are moisture stablemeta- and para- substituted 2-formylbenzeneboronic acids were reacted with methoxyamine hydrochloride under refluxing conditions for 15 minutes in water (pH 7) to produce compounds 8\u201310 in high yields (70\u201390%). LiAlH4 in THF was added dropwise to a solution of phenylboronic acid derivatives 8\u201310 in THF at \u201378 \u00b0C, the mixture was allowed to warm to room temperature followed by heating to reflux for 3 hours. In all cases, the amine-borane11\u201313, a BN analogue of indane, was isolated in excellent yields (85\u201390%). Trimerisation11\u201313 under microwave-assisted conditions afforded the desired borazatruxenes 1\u20133 in 56\u201365% yields as white solids after a simple filtration/washing protocol.Commercially available 14\u201317 were synthesised from meta- and para- substituted benzonitriles. The benzonitriles were reacted with lithium tetramethylpiperidine (LTMP) and B(OiPr)3 at \u201378 \u00b0C, followed by slow warming to room temperature (20 \u00b0C) overnight to afford the respective ortho- substituted 2-cyanophenylboronic acid. The reaction was quenched using AcOH (2.2 equiv.) followed by an in situ protection of the boronic acid with 1,3-propanediol. The use of a protecting group is key for isolation of 14\u201317, and the choice of 1,3-propanediol allows the reduction to the corresponding amine-borane products 18\u201321 in very good yields (65\u201395%), without requiring a further deprotection step , respectively.Due to the limited number of commercially available 2-formylphenylboronic acids and their relatively lengthy syntheses, a second pathway was devised in order to obtain functionalised borazatruxenes . 2-Cyanoion step and ESI\u2020 8 and 9 , have be1 is soluble in organic solvents of medium polarity. This is in stark contrast to the parent truxene, which has very poor solubility in most common solvents.1, which is likely due to their increased molecular weight. The 1H-NMR of 1 indicates that the aromatic protons closest to the borazine rings are most deshielded (8.03\u20138.05 ppm) while the external ones experience a lower influence of the BN anisotropy with chemical shifts in the range of 7.42\u20137.59 ppm. The 11B NMR spectrum displays a peak resonating at 37.2 ppm which is in good agreement with a typical 11B chemical shift of a substituted borazine. The UV-vis spectrum of 1 shows a very intense and broad absorbance centered at 250 nm followed by three distinct peaks of lower intensity at 279.5, 272.0 and 265.0 nm. These peaks are blue shifted compared to corresponding truxene ones, thus highlighting that introducing BN bonds into the all-carbon system increases the HOMO\u2013LUMO gap isomer identical with the and isomers. Therefore, the only possible isomers are the enantiomeric pairs syn: and with all three methyl groups located on the same side of the borazatruxenes plane, and anti: and , where one methyl is on the opposite side of the plane with respect to the other two while the anti enantiomers are separated with a selectivity factor of 1.17 . The Circular Dichroism (CD) spectra of the two enantiomeric pairs syn and anti of chiral derivative 9 display strong Cotton effects on both the arene and borazine absorbance. The identification of all four isomers was possible by comparing the experimental (vide infra) CD spectra.There are four possible optical isomers of borazatruxene ther two . The synrimental with the11 (P2n1/ with four molecule units per unit cell packed in staggered array motif.The molecular structure of BN-indane 11 was obtaThe distance between boron and nitrogen atom is 1.637 \u00c5 consistent with an N \u2192 B Lewis-type interaction. There are two weak NH\u00b7\u00b7\u00b7\u03c0 intermolecular interactions between the amine group with the aromatic rings of two neighbouring molecules: N\u00b7\u00b7\u00b7Ph(C2\u2013C7) distance of 3.239 \u00c5 and N\u00b7\u00b7\u00b7Ph distance of 3.339 \u00c5.1 came from an X-ray diffraction analysis of single crystals grown by slow evaporation from a CH2Cl2 solution. 1, which crystallises in a P21/c space group. This compound forms a staggered L-shaped configuration in the unit cell -9 and -9 co-crystallised in the R3[combining macron] space group. In this structure the two enantiomers adopt a 60\u00b0 rotated face-on stacked arrangement with an intermolecular B\u2013N distance of 3.749 \u00c5 and the Me groups pointing outwards overlay of the experimentally determined (X-ray diffraction) co-ordinates and the computed ones. Geometry optimisations were performed using M06-2X,Gaussian 16 software. Also, TD-DFTversus the agreement to experimental X-ray data (Table S10, Fig. S20The X-ray determined structure of In summary, we have developed the synthesis of a new class of BN-PAHs that incorporate a borazine unit in place of the central benzene core of a truxene. These derivatives have higher solubilities than the parent all-carbon derivatives and can be synthesised in three steps from commercially available starting materials. These derivatives are air and moisture stable which is in contrast to the majority of non-sterically hindered borazines. We have synthesised and separated the first chiral derivatives of these molecules which also represents a premiere in the larger truxene family. The borazatruxenes molecules are likely to become important materials in molecular electronic devices due to their unique structure which combines areas of electron-conductance with electron-insulating domains. Further investigations into the synthesis of new borazatruxenes analogues are currently in progress in our group.There are no conflicts to declare.Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "Against expectations the covalency in a cerium(iv)\u2013carbon multiple bond interaction is essentially as covalent as the uranium(iv) analogue. TMS)(ODipp)2] whereas for M = Th the M PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond interaction is much more ionic. On the basis of single crystal X-ray diffraction, NMR, IR, EPR, and XANES spectroscopies, and SQUID magnetometry complexes 1\u20133 are confirmed formally as bona fide metal(iv) complexes. In order to avoid the deficiencies of orbital-based theoretical analysis approaches we probed the bonding of 1\u20133via analysis of RASSCF- and CASSCF-derived densities that explicitly treats the orbital energy near-degeneracy and overlap contributions to covalency. For these complexes similar levels of covalency are found for cerium(iv) and uranium(iv), whereas thorium(iv) is found to be more ionic, and this trend is independently found in all computational methods employed. The computationally determined trends in covalency of these systems of Ce \u223c U > Th are also reproduced in experimental exchange reactions of 1\u20133 with MCl4 salts where 1 and 2 do not exchange with ThCl4, but 3 does exchange with MCl4 and 1 and 2 react with UCl4 and CeCl4, respectively, to establish equilibria. This study therefore provides complementary theoretical and experimental evidence that contrasts to the accepted description that generally lanthanide\u2013ligand bonding in non-zero oxidation state complexes is overwhelmingly ionic but that of uranium is more covalent.We report comparable levels of covalency in cerium\u2013 and uranium\u2013carbon multiple bonds in the iso-structural carbene complexes [M(BIPM Nature of the Chemical Bond over 75 years ago, chemists have vigorously debated the nature of chemical bonding.4Ever since the publication of vs. 0.89 \u00c5 and 1.01 vs. 1.03 \u00c5 for the +IV and +III oxidation states, respectively);iii) complexes is generally described as being independent of the ligand environment and \u2018free-ion-like\u2019.iii) the [Xe]4f1 \u2192 [Xe]4f05d1 transition is found to depend strongly on the ligand field, varying from 49\u2009737 cm\u20131 for gaseous Ce3+, to 22\u2009000 cm\u20131 for Ce3+ doped into Y3Al5O12, to 17\u2009650 cm\u20131 for [Ce{\u03b75-C5H3(SiMe3)2}3].vs. 5f elements. Overall, for an isostructural pair of tetravalent uranium and cerium complexes, the order of covalency involving those metal centres would normally be expected to be uranium significantly greater than cerium. This is important to understand, from a fundamental perspective, but there are also practical implications; these three elements can be found in the presence of one another in spent nuclear fuel and future strategies to separate them might depend on exploiting differences in their covalent chemical bonding.A comparison of chemical bonding that is often made is between 4f cerium and 5f uranium, since according to Shannon their ionic radii are very similar carbene diaryloxide complex [Ce(BIPMTMS)(ODipp)2] .1 is notable for being a cerium(iv) organometallic and containing a Ce PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C multiple bonding interaction. Whilst dominated by electrostatics, this bond exhibits covalency according to NBO analysis of DFT-derived densities. There is reason to have confidence in such analysis as SAOP/ZORA/TZP TD-DFT calculations at the same level of theory reproduce very well the experimentally observed UV/Vis/NIR spectrum. NBO analysis identifies \u223c13% cerium character in each of two Ce\u2013C bonding interactions (\u03c3 + \u03c0). Non-aqueous cerium(iv)th ionisation energy of cerium is greater than the sum of the first three;1 in-hand, we surmised that as uranium(iv) and thorium(iv) are robust oxidation states, the synthesis of 1 presents an opportunity to directly compare the nature of the chemical bonding of cerium, uranium, and thorium. Here, we report the synthesis and characterisation of [M(BIPMTMS)(ODipp)2] ; the synthesis of 2 and 3 are straightforward, but importantly permit a comparison of the bonding of three isostructural complexes. Surprisingly, both DFT and CASSCF/RASSCF methods suggest that the covalency and f-orbital interactions for the cerium and uranium complexes are essentially the same, in contrast to the thorium complex that is essentially ionic. The emergence of these results is in contrast to almost all other examples of comparative studies of 4f and 5f covalency,iv) and uranium(iv) hexachloride dianion salts, where on the basis of orbital energy near-degeneracy similar levels of covalency between cerium(iv) and uranium(iv) have been proposed.1\u20133 are also consistent with experimental exchange reactions with metal tetrahalide salts of cerium, uranium, and thorium, further supporting our findings.Recently, as part of a wider effort to prepare lanthanide\u2013carbon multiple bonds,1, which required a multi-step preparation,2 and 3 was straightforwardly accomplished by installation of the BIPMTMS carbene then the two aryloxides onto uranium or thorium by sequential salt elimination reactions, 2 and 3 were isolated as brown and colourless crystals in 56 and 61% yield, respectively. The 1H NMR spectrum of 2 spans the range \u201319 to +17 ppm and the 31P NMR spectrum exhibits a broad resonance at \u2013293 ppm, consistent with the uranium(iv) formulation that is supported by a solution magnetic moment of 2.75 \u03bcB at 298 K. In contrast, the 1H NMR spectrum of 3 spans the range 0 to +8 ppm and the 31P NMR resonance appears at +4.7 ppm. The electronic absorption spectrum of 2 (see ESI\u03b5 < 80 M\u20131 cm\u20131) absorptions over the range 500\u20131900 nm that are characteristic of Laporte forbidden f\u2013f transitions for the 3H4 electronic manifold of the 5f2 uranium ion3 the spectrum is featureless over 400\u20132000 nm as expected for its colourless 6d05f0 nature. As reported previously, the electronic absorption spectrum of 1 exhibits two broad absorptions in the visible region , the broadness and resulting purple colour of which is a defining feature of many cerium(iv) complexes.23In contrast to 2 see ESI is chara2 and 3 were determined by single crystal X-ray crystallography, 1\u20133 is a monomeric formulation with terminal M PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bonds. The remaining coordination sphere of each metal is completed by two BIPMTMS imino chelate arms and the two aryloxide oxygen centres which enforce a pseudo square-based pyramidal geometry. We found a Ce PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C distance of 2.441(5) \u00c5 in 1; PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bonds reported in the theoretical models of CeCH2+ and Cp2CeCH2 complexes,2+ and Cp2CeCH2 are experimentally unknown, sterically unimpeded and, in the case of the former, benefit from the reduced electronic repulsion associated with a net positive charge. For experimentally realised compounds, the Ce PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C distance of 1 is amongst the shortest ever reported, except for the special case of fullerene encapsulated Ce2. PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C and Th PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C distances in 2 and 3 were determined to be 2.414(3) and 2.508(5) \u00c5, respectively; on the basis of Shannon's ionic radii PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C and Th PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bonds in BIPMTMS complexes.26The molecular structures of 1\u20133 are also consistent with solid state magnetic measurements, 2 has a room temperature \u03c7T of 0.93 cm3 K mol\u20131 and decreases rapidly on cooling tending to zero, which is typical for 5f2 uranium(iv) that is a magnetic singlet at low temperature.3 is diamagnetic, consistent with closed-shell thorium(iv). Studies of 1 give a very small magnetic moment (0.02\u20130.1 cm3 K mol\u20131 depending on temperature and diamagnetic corrections) that varies from batch to batch nor the difference between the 4f (Ce) and 5f (Th) ion diamagnetism. Because measured samples of 1 have a very weak paramagnetism, the precise nature of the \u03c7T(T) plot or other diamagnetic configurations (admixtures of cerium(iv) with singlet cerium(iii) + radical ligand configurations have been proposed for cerocene).ca. 5\u201315% of values expected for f1 cerium(iii) .1 decomposes to give cerium(iii) \u2013 we note the related complex [Ce(BIPMTMS)(ODipp)(THF)]geff,\u2225 = 3.7 and geff,\u22a5 = 0.85 , and therefore that comparisons to 2 and 3 are valid, we recorded the XANES spectrum of 1.The weak paramagnetic response from samples of 5 see ESI. That thIII-edge spectrum of 0.01 M Ce(NO3)3 in water, the cerium(iii) precursor to 1 [Ce(BIPMTMS)(ODipp)2K(THF)], CeO2, and 1 are illustrated in iii) complexes, the LIII-edge spectra of aqueous Ce(NO3)3 and [Ce(BIPMTMS)(ODipp)2K(THF)] both consist of a single peak, just above the absorption threshold, at \u223c5725.7 eV that is characteristic of cerium(iii).III-edge spectra of CeO2 and 1 both exhibit the characteristic double absorption features of cerium(iv) at \u223c5727.2 and \u223c5736.7 eV, that are similar to those of CeF4, Ce(SO4)2\u00b74H2O, and CeCl62\u2013.III-edge E1 absorption for 1 is \u223c1.5 eV higher in energy than the corresponding [Ce(BIPMTMS)(ODipp)2K(THF)] LIII-edge absorption, as found generally for cerium(iv) complexes.2 and 1 are both essentially 1\u2009:\u20091, as has been found for CeCl62\u2013 and CeF4,1 L\u20131 contribution to a multiconfigurational ground state,i.e. a multiconfigurational excited state.iv) complexes with oxide and halide ligands is due to final state effects,1 is certainly much more like that of CeO2, CeF4, Ce(SO4)2\u00b74H2O, and CeCl62\u2013 than cerocene; this observation is consistent with the premise that an open-shell singlet or triplet formulation of 1 should be regarded as less likely than a closed-shell singlet and so we conclude that the presence of cerium(iii) character in 1 can be excluded.The cerium L1 possesses formal cerium(iv) character, it can legitimately be compared to 2 and 3. We previously reported Natural Bond Orbital (NBO) data for 1 which returned \u03c3- and \u03c0-bonds composed of \u223c13% Ce and \u223c87% C character.2 by NBO, a similar breakdown is returned. Specifically, the \u03c3-bond is composed of 16% U and 84% C and the \u03c0-bond is made up of 14% U and 86% C character. The U contributions to the \u03c3- and \u03c0-bonds are 5f (87%) and 6d (12%), and 5f (77%) and 6d (22%), respectively. In contrast, the NBO data for 3 return ionic interactions with localised carbene lone pairs with no Th character as the contribution of the latter falls below the default cut-off of 5% in the NBO code. These calculations suggest that, whilst the bonding between cerium\u2013, uranium\u2013, and thorium\u2013carbon centres in 1\u20133 are dominated by ionic interactions, a modest and surprisingly comparable covalent contribution to the bonding is evident in 1 and 2 despite the commonly held view that lanthanide\u2013ligand chemical bonding is purely ionic.Since the XANES data suggest that PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C units in 1\u20133. These calculations employed the restricted-active-space self-consistent field (RASSCF) theory,via a configuration interaction approach. Whilst RASSCF is a powerful technique for elucidating the nature of metal\u2013ligand interactions in complexes such as those considered here, it is limited in the size of systems to which it can be applied. For this reason, complexes 1\u20133 were truncated in order to render RASSCF calculations computationally tractable by replacing P-phenyls with H, silyl-methyls with H, and the bulky Dipp groups by Me. This truncation retains the coordination environment of all atoms directly bonded to the metal: where hydrogen termination was employed, only the positions of the terminating hydrogens were optimised.Although the NBO calculations are internally consistent and well suited to describing covalency in molecules,iv) complexes have shown density-based analysis methods provide unambiguous electronic structure interpretations.\u03b4), a quantitative measure of the degree of electron sharing between two atomic centres, PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond critical point (\u03c1), an accepted measure of covalency. These two measures, while complementary, are not equivalent: \u03c1 provides a quantitative measure of charge accumulation in the bonding region, which is related to spatial overlap, whereas the delocalisation index, \u03b4, between two bonded atoms is maximised when electrons are shared equally which, in a monodeterminantal framework, is a manifestation of orbital degeneracy. This analysis therefore allows us to determine the variation in both of these phenomena when the metal centre is varied and has previously been reported in several studies of cerium and uranium complexes.\u03c1 and <3% in \u03b4 are observed when comparing full and truncated complexes, demonstrating that the quantitative bonding characteristics of the full complexes 1\u20133 is retained.In order to assess any truncation effects on the electronic structures, ground state electron densities were calculated at the PBE/TZVP level of theory. These densities were probed with the quantum theory of atoms in molecules (QTAIM) approachTMS favour f- over d-orbital participation in the bonding of this ligand to f-elements generally, but further studies will be required to confirm this. This definition of the active subspaces resulted in RASSCF calculations. The number of explicitly correlated electrons, n, was 22 for complexes 1 and 3 and 24 for complex 2. In all cases, calculations were performed in Cs symmetry.The electronic structures of the truncated complexes were then evaluated using the RASSCF methodology. These calculations employed three active spaces: RAS1, containing only occupied orbitals from the monodeterminantal reference wavefunction, RAS2, containing both occupied and virtual orbitals, and RAS3, containing only virtual orbitals. Full configuration interaction (CI) was performed in RAS2, while truncated CI, considering only singly and doubly excited configurations, was performed between the RAS1, RAS2 and RAS3 subspaces. All active space orbitals were optimised. Due to the large computational costs of such calculations, the RAS1, RAS2 and RAS3 subspaces were restricted to 11, 7 and 11 orbitals, respectively: the 7 RAS2 orbitals incorporated the 4f/5f manifold, whereas the RAS1 and RAS3 subspaces included all orbitals with significant carbon and nitrogen 2s and 2p character. The oxygen 2s and 2p orbitals could not be included in the active subspaces, since attempts resulted in the intrusion of phosphorus-based orbitals. It was observed, however, that oxygen 2s and 2p orbitals that were successfully stabilised in the active subspaces exhibited occupation numbers extremely close to integer values. Similarly, occupation numbers of formally unoccupied d-orbitals was effectively 0, indicating that the inclusion of these orbitals in the active subspaces is not required. It may be that the geometric constraints of BIPMiv) centres, in agreement with our experimental measurements, and these configurations contribute 89.0, 89.5 and 89.3% to the ground state RASSCF wavefunctions of 1\u20133 , respectively. Maximum deviations from integer values in natural orbital occupations were 0.032, 0.033, and 0.025, respectively, indicating rather weak multi-configurational character.2 configuration of the uranium compound) should be sufficient to accurately describe the M PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bonding interaction. Subsequent analysis of CASSCF-derived densities revealed them to be extremely similar to their RASSCF counterparts N-heterocyclic carbene complex [Ce(L){N(SiMe3)2}2F] [L = OCMe2CH2(CNCH2CH2N-Dipp)] a \u03c1 of 0.045 a.u. is found,\u03c1 was found to be 84.9% of the \u03c1 value. Similarly, calculations on cerocene\u03c1 to be 0.0395, which is 83.0% of the analogous value calculated for uranocene.\u03c1 for 1 to be 91.3% of the corresponding U value in 2. Comparison of \u03b4 can also be made with that in cerocene, where it was found to be 83.2% of the \u03b4 value in uranocene; here, we calculate \u03b4 for 1 to be 99.1% of the corresponding \u03b4 value in 2.The RASSCF-calculated wavefunctions were used to obtain explicitly correlated electron densities for subsequent QTAIM analysis. Metal charges are all significantly higher than those found using DFT, increasing by 0.68, 0.59 and 0.49 to give absolute values of +2.84, +2.89 and +3.02 a.u. for 1\u20133 and calibrate the above calculations, we investigated the exchange reaction chemistry of 1\u20133 since it is well known that covalency can drive exchange reactions. For example, rare earth tris-cyclopentadienyl complexes readily react with iron halides to afford ferrocene and rare earth halides and this reactivity, whilst undoubtedly reflecting the favourable formation of lanthanide\u2013halide bonds, is driven by the formation of highly covalent iron\u2013cyclopentadienyl bonding.43In order to experimentally probe the relative levels of covalency in 1, which is known to be unstable in solution, no reaction between 1 and [ThCl4(THF)3.5] is observed in benzene after a 24 hour stir (eqn (1)). After a 5 day stir 1 is completely decomposed to yield a species that exhibits a resonance at \u201334 ppm in the 31P NMR spectrum. Although we have not been able to isolate and identify this species its 31P NMR chemical shift is in the region where related cerium(iii) BIPMTMS complexes exhibit 31P NMR resonances.1 does not react with thorium tetrachloride and instead decomposes before any reactivity can occur. In the reverse scenario, eqn (2), treatment of 3 with [CeCl4(HMPA)] [HMPA = OP(NMe2)3] results in the loss of 1H NMR resonances attributable to 3 and evolution of the characteristic purple colour of 1 in the first 15 minutes. After 15 minutes the purple colour fades and an intractable mixture of products is formed. Given that the preparation of 1 is not straightforward it is not surprising that if formed under these less than optimal conditions it would decompose given its instability in solution, but the purple colour is certainly consistent with the exchange of BIPMTMS from thorium(iv) to cerium(iv) and in-line with the proposed differences in covalency.Apart from the onset of decomposition of 1 and [ThCl4(THF)3.5], eqn (1), we find that there is also no reaction of 2 with [ThCl4(THF)3.5], eqn (3). In the reverse situation, eqn (4), 3 does react with [UCl4(THF)3]. Unfortunately, an intractable product mixture is obtained, likely due to ligand scrambling under conditions that are by definition less controlled than the usual route to prepare 2. However, it is clear that 1H NMR resonances attributable to 3 are lost so the implication is that the BIPMTMS ligand is transferred to uranium. Irrespective of the precise outcomes, these reactions are consistent with uranium being more covalent than thorium.As with the absence of reaction between 1 is treated with [UCl4(THF)3], eqn (5), the intense purple colour of 1 fades within 30 minutes and is replaced by a green colour which is then replaced by a brown colour consistent with the formation of 2. In the reverse situation, eqn (6), 1H NMR resonances attributable to 2 are lost; no purple colour was observed, but it is clear that ligand exchange has occurred, and we note that after a 5 day stir the mixture exhibits a 31P NMR resonance at \u201334 ppm, which is indicative of a cerium(iii) BIPMTMS derivative.2 with neat HMPA in a control experiment and found that no reaction occurs. The uranium\u2013cerium exchange reactions are not clean, but it is evident that ligand exchange occurs to some extent. Although equilibria are to some extent established, 1 is not stable in solution for extended periods and the evidence suggests that eventually the cerium decomposes to the trivalent state, which then degrades the equilibria.When Hrxn) for the full, balanced versions of eqn (1)\u2013(6), eqn (7)\u2013(12), by calculating the gas phase geometry optimised structures of all the constituent components. A solvent continuum was not applied since the solvent for eqn (1)\u2013(6) was benzene, which could reasonably be expected to have systematically minimal interactions with the electropositive species in solution. Experimentally, [ThCl4(THF)3.5] is most likely a separated ion pair formula like related lanthanide triiodides,4(THF)3]. The calculations most likely carry absolute errors of 5\u201310 kcal mol\u20131, but, assuming that this is to some extent systematic, the relative errors will reduce to \u223c2\u20135 kcal mol\u20131. The calculations are thus clear-cut as they independently and correctly reproduce the experimental outcome in every case.To further support the above findings, we determined the theoretical bond enthalpy changes (\u0394iv) does not displace BIPMTMS from cerium(iv) or uranium(iv) whereas the latter pair do displace BIPMTMS from the former. When cerium(iv) or uranium(iv) derivatives are mixed it is evident that equilibria are established, but the reactions are not clean and the equilibria are disrupted due to the instability of 1. Although some of the products of these reactions are not known, the key point is whether a reaction occurs at all or not. The fact that distinct colour changes are observed, or not, suggests that the carbenes are, or not, transferred since it is the M PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bonds that contribute to absorptions in the visible part of the optical spectra for 1 and 2 and the M\u2013ODipp linkages absorb well into the UV-region. Therefore, the conclusion is that thorium(iv) is the most ionic in this context, whereas cerium(iv) and uranium(iv) do exhibit comparable covalency and these observations experimentally support the same theoretical proposition.Overall, these exchange reactions demonstrate that thorium scale\" fill=\"currentColor\" stroke=\"none\">C units in these complexes is predominantly ionic, we note a significant covalent contribution to these linkages for cerium and uranium. Significantly, the levels of covalency and f-orbital participation in the M PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bonds are remarkably similar for cerium and uranium, but different from thorium which is ionic. Importantly, the similar levels of covalency in the cerium(iv)\u2013 and uranium(iv)\u2013carbon multiple bonds in 1 and 2 manifests in more than one type of theoretical treatment , and most compellingly is supported by experimental exchange reactions that proceed as predicted from the above covalency arguments. It may be that the similar levels of covalency of cerium(iv) and uranium(iv) is a more general effect than currently recognised, but one that is relatively small and so has eluded detection in systems that exhibit minimal covalency. Since the synthesis of cerium(iv) complexes that go beyond simple salts is still in its infancy, and is experimentally challenging, it may be that more examples of cerium(iv) and uranium(iv) complexes containing similar levels of covalency await discovery. At the very least the results presented here provide a basis to question the established exclusive ionic bonding textbook description of the lanthanides in non-zero oxidation states, especially with reference to certain 5f metals.In summary, we have reported the synthesis of TMS)(ODipp)2] (1),3(BIPMTMS)Li(THF)2], and [Th(Cl)2(BIPMTMS)] were prepared by published methods.1H, 13C, 29Si, and 31P NMR spectra were recorded on a Bruker 400 spectrometer operating at 400.2, 100.6, 79.5, and 162.0 MHz respectively; chemical shifts are quoted in ppm and are relative to TMS and 85% H3PO4 (31P). FTIR spectra were recorded on a Bruker Tensor 27 spectrometer. UV/Vis/NIR spectra were recorded on a Perkin Elmer Lambda 750 spectrometer. Data were collected in 1 mm path length cuvettes loaded in an MBraun UniLab glovebox and were run versus the appropriate reference solvent. Solution magnetic moments were recorded at room temperature using the Evans method. Static variable-temperature magnetic moment data were recorded in an applied dc field of 0.1 T on a Quantum Design MPMS XL7 superconducting quantum interference device (SQUID) magnetometer using doubly recrystallised powdered samples. Care was taken to ensure complete thermalisation of the sample before each data point was measured and samples were immobilised in an eicosane matrix to prevent sample reorientation during measurements. Diamagnetic corrections were applied for using tabulated Pascal constants and measurements were corrected for the effect of the blank sample holders and eicosane matrix. Variable temperature (300\u20135 K) EPR spectra were measured at X-band on a Bruker Elexsys E580 spectrometer. Polycrystalline samples were sealed under vacuum in 1 mm i.d. silica tubing, and double-contained for EPR by insertion into an X-band silica tube or PTFE sleeve. CHN microanalyses were carried out by Tong Liu at the University of Nottingham. Cerium LIII-edge XANES measurements were performed using a Si(111) double-crystal monochromator on the Rossendorf Beamline at the European Synchrotron Radiation Facility . Higher harmonics were rejected by two Si coated mirrors. The spectra were collected using ionisation chambers filled with nitrogen and a 13-element Ge fluorescence detector. The samples were measured at 15 K in a closed-cycle He cryostat. The reference samples spectra of 0.01 M Ce(iii) nitrate in H2O and solid CeO2 were measured at room temperature in transmission mode.All manipulations were carried out using Schlenk techniques, or an MBraun UniLab glovebox, under an atmosphere of dry nitrogen. Solvents were dried by passage through activated alumina towers and degassed before use or were distilled from calcium hydride. All solvents were stored over potassium mirrors, except for ethers that were stored over activated 4 \u00c5 sieves. Deuterated solvent was distilled from potassium, degassed by three freeze\u2013pump\u2013thaw cycles and stored under nitrogen. [Ce(BIPMTMS)(Cl)3(Li)(THF)2] and [K(ODipp)] . The resulting brown suspension was allowed to warm to room temperature with stirring over 16 h to afford a brown solution. Volatiles were removed in vacuo and the resulting solid was extracted into toluene. Volatiles were removed in vacuo to afford a brown solid which upon recrystallisation from Et2O (2 ml) at \u201330 \u00b0C afforded 2\u00b7Et2O as brown crystals. Yield: 0.69 g, 56%. Anal. calcd for C59H82N2O3P2Si2U: C, 57.91; H, 6.76; N, 2.29%. Found: C, 57.76; H, 6.66; N, 2.33%. 1H NMR (C6D6): \u03b4 \u201318.94 3), \u20133.43 2), \u20133.25 2), 1.23 , 3.34 , 6.70 , 8.17 , 13.40 , 16.07 , 16.48 (Ar-H). 31P{1H} NMR (C6D6): \u03b4 \u2013293.42 (UCP2). FTIR \u03bd/cm\u20131 (Nujol): 1590 (w), 1539 (w), 1403 (w), 1330 (w), 1200 (s), 918 (w), 887 (w), 857 (s), 838 (s), 748 (w), 694 (w), 661 (w). Magnetic moment : \u03bceff = 2.75 \u03bcB.THF (15 ml) was added to a precooled (\u201378 \u00b0C) mixture of [U(BIPM2(BIPMTMS)] in THF (5 ml) was added to a solution of [ThCl4(THF)3.5] in THF (5 ml) at \u201378 \u00b0C. The pale yellow mixture was stirred at \u201378 \u00b0C for 30 minutes, then was allowed to warm to room temperature with stirring for 2 h. Volatiles were removed in vacuo and DippOK was added. Toluene (10 ml) was added slowly to the cold (\u201330 \u00b0C) stirring mixture, the resultant mixture was allowed to warm to room temperature with stirring for 1 h. After this time the mixture was filtered, and all volatiles were removed in vacuo. The product was recrystallised from a toluene/hexane mixture to yield 3\u00b70.5(toluene) as colourless crystals. Yield: 0.69 g, 61%. Anal. calcd for C62H80N2O2P2Si2Th: C, 60.27; H, 6.53; N, 2.27%. Found: C, 59.96; H, 6.64; N, 2.45%. 1H NMR (C6D6): \u03b4 0.14 3), 1.35 2), 3.74 2), 6.97\u20137.04 , 7.21 , 7.55\u20137.61 . 13C{1H} NMR (C6D6): \u03b4 3.18 3), 24.45 2), 28.14 , 67.04 scale\" fill=\"currentColor\" stroke=\"none\">CP2), 120.15, 123.26, 125.66, 128.53, 129.29, 130.11 (ArC), 131.36 , 131.43 , 136.94 , 139.01 , 139.49 , 161.26 . 31P{1H} NMR (C6D6) \u03b4 4.65 (s). 29Si{1H} NMR (C6D6) \u03b4 \u20137.20 , \u20137.24 . FTIR \u03bd/cm\u20131 (Nujol): 1589 (w), 1325 (w), 1260 (s), 1197 (m), 1100 , 1095 , 1042 (m), 1023 (m), 887 (w), 856 (m), 800 (m), 726 (m), 609 (m).A solution of [Li1, 2, and 3 and the components of the exchange reactions using coordinates derived from their X-ray crystal structures. No constraints were imposed on the structures during the geometry optimisations. The calculations were performed using the Amsterdam Density Functional (ADF) suite version 2010.01.et al.via single point energy (SPE) calculations, replacing the basis sets of the metal ions with the segmented all-electron relativistically contracted (SARC) basis sets,via the 2nd order Douglas\u2013Kroll\u2013Hess Hamiltonian.via the 2nd order Douglas\u2013Kroll\u2013Hess Hamiltonian. Topological and integrated atomic properties, obtained using the quantum theory of atoms in molecules (QTAIM), were performed using version 13.11.04 of the AIMAll software package.65Unrestricted and restricted geometry optimisations were performed as appropriate for full models of Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "Computations on the heavier group 14 dimetallenes [E{CH(SiMe3)2}2]2 and [E{N(SiMe3)2}2]2 and their respective monomers indicated that empirically observed dimerization is principally driven by attractive dispersion forces. 3)2}2]2 and [E{N(SiMe3)2}2]2 and their dissociation into :E{CH(SiMe3)2}2 and :E{N(SiMe3)2}2 monomers were studied computationally using hybrid density functional theory (DFT) at the B3PW91 with basis set superposition error and zero point energy corrections. The structures were reoptimized with the dispersion-corrected B3PW91-D3 method to yield dispersion force effects. The calculations generally reproduced the experimental structural data for the tetraalkyls with a few angular exceptions. For the alkyls, without the dispersion corrections, dissociation energies of \u20132.3 (Ge), +2.1 (Sn), and \u20130.6 (Pb) kcal mol\u20131 were calculated, indicating that the dimeric E\u2013E bonded structure is favored only for tin. However, when dispersion force effects are included, much higher dissociation energies of 28.7 (Ge), 26.3 (Sn), and 15.2 (Pb) kcal mol\u20131 were calculated, indicating that all three E\u2013E bonded dimers are favored. Calculated thermodynamic data at 25 \u00b0C and 1 atm for the dissociation of the alkyls yield \u0394G values of 9.4 (Ge), 7.1 (Sn), and \u20131.7 (Pb) kcal mol\u20131, indicating that the dimers of Ge and Sn, but not Pb, are favored. These results are in harmony with experimental data. The dissociation energies for the putative isoelectronic tetraamido-substituted dimers [E{N(SiMe3)2}2]2 without dispersion correction are \u20137.0 (Ge), \u20137.4 (Sn), and \u20134.8 (Pb) kcal mol\u20131, showing that the monomers are favored in all cases. Inclusion of the dispersion correction yields the values 3.6 (Ge), 11.7 (Sn), and 11.8 (Pb) kcal mol\u20131, showing that dimerization is favored but less strongly so than in the alkyls. The calculated thermodynamic data for the amido germanium, tin, and lead dissociation yield \u0394G values of \u201312.2, \u20133.7, and \u20133.6 kcal mol\u20131 at 25 \u00b0C and 1 atm, consistent with the observation of monomeric structures. Overall, these data indicate that, in these sterically-encumbered molecules, dispersion force attraction between the ligands is of greater importance than group 14 element\u2013element bonding, and is mainly responsible for the dimerization of the metallanediyls species to give the dimetallenes. In addition, calculations on the non-dissociating distannene [Sn{SiMetBu2}2]2 show that the attractive dispersion forces are key to its stability.The structures and bonding in the heavier group 14 element olefin analogues [E{CH(SiMe The mixing of a bonding and antibonding orbital generates a molecular orbital with lone-pair character, and hence a pyramidalized coordination for E. This interaction takes place more readily in the heavier main group element derivatives because the bonds become increasingly long (weaker) as a result of larger Pauli repulsions between core electrons as the group is descended. Consequently, the energy difference between the \u03c3* and \u03c0 orbitals also decreases, and hence the extent of their interaction increases. The trans-folding, which weakens the E\u2013E bond, is often sufficient to cause dissociation of the double bond of the heaviest tin and lead species to two monomeric metallanediyls. However, dissociation is less common in their germanium analogues and for the iconic disilenes, first reported in 1981,19The synthesis of the lower valent group 14 element dialkyls :E{CHSiMe2}2 3}2, which has a long central single C\u2013C bond of 1.67 \u00c5 and is stabilized via dispersion force interactions between the But groups, whereas the corresponding C\u2013C bonded, less hindered, unsubstituted species (CPh3)2 is unknown.26The investigation and rationalization of bonding in these and related multiple-bonded compounds has been a topic of broad interest3)2}2 and E{N(SiMe3)2}2 as well as the corresponding E\u2013E bonded dimers. The calculations without the inclusion of dispersion force effects revealed that the E\u2013E bonding energies are low for the tetraalkyls, and insufficient to stabilize their dimeric structures under ambient conditions. In contrast, the inclusion of dispersion force resulted in large increases in the binding energies. Application of the same protocols to the amido compounds also afforded lower binding energies that are insufficient to sustain the dimeric structures under ambient conditions.Despite these developments, the realization of importance of the dispersion interactions between the C\u2013H moieties of substituent ligands to the stability of inorganic and organometallic compounds is not widespread. However, several reports have shown that such forces are very important for the bonding and structure of a variety of species.H), entropy (S), and free energies (G) as well as zero point energies (ZPE) were estimated using calculated harmonic vibrational frequencies and BSSE corrected energies.All calculations were carried out using the Gaussian 09 program.3)2}2 (E = Ge or Sn) monomers have the syn, syn orientation of the \u2013CH(SiMe3)2 alkyl groups in the gas phase, as shown in 3)2}2 monomer. However, in 3)2}2, derived from a syn, syn Pb{CH(SiMe3)2}2 unit within the [Pb{CH(SiMe3)2}2]2 (3)2}2 (E = Ge or Sn)syn, anti conformation, as shown in 3)2}2 units within the lead \u2018dimer\u2019 have the aforementioned syn, syn configuration, as shown in i.e. the same configuration as those experimentally observed for the germanium and tin dialkyl monomers in the gas phase.9; Sn\u2013Sn = 2.768(1) \u00c510) are reproduced with good accuracy. Exceptions to this generalization involve the calculated torsion angles between the methane C\u2013H bonds and the central EC2 (E = Ge or Sn) plane in the germanium and tin monomers, and the C\u2013Ge\u2013C angle in the germanium monomer, which is somewhat high. For the torsion angle in the germanium and tin monomers, the discrepancy is almost 20\u00b0 in the germanium and 6\u20138\u00b0 in the tin monomer. Unfortunately, in the monomeric structures which are measured from GED (gas electron diffraction) data, no standard deviations were given. However, standard deviations of 2\u00b0 were listed for the EC2 angle, and it is reasonable to suppose the standard deviation for the torsion angle for the methane C\u2013H bonds would be larger than this value. We replicated the optimization with the HCECH torsion angles fixed at the experimental values of 2\u00b0 (E = Ge) and 15\u00b0 (E = Sn). As shown in Table S1,\u20131 for Ge, but only 2.5 kcal mol\u20131 for tin. Thus, the HCECH torsion angle has significantly larger effect in the germanium dimer, consistent with its more sterically-congested structure. It is worthwhile to recall that the calculations on the dimers are for the molecules in isolation (i.e. in the gas phase), and take no account of the effects of neighboring molecules (i.e. packing forces), which can also be expected to exert some effect on their structure.The calculated structural parameters at various levels of optimization for the alkyl-substituted germanium, tin, and lead monomers and dimers are presented in e3)2}2]2 \u2018dimer\u2019.3)2}2 dimer3)2}2 units in the crystal structure. Nevertheless, long interactions of this type between the heavier main group elements can be significant.3)2}2 units within the lead dimer are syn, syn monomers, that is to say the long Pb\u2013Pb interaction is ignored, the structural parameters given for Pb{CH(SiMe3)2}2 in 3)2}2 monomer (cf. C\u2013Sn\u2013C = 97(2)\u00b0 by GED). In effect, the data suggest that the Pb{CH(SiMe3)2}2 units within the lead dimer are behaving essentially as weakly interacting plumbylene monomers, rather than as a Pb\u2013Pb multiple bonded diplumbene.The calculated Pb\u2013Pb separation and C\u2013Pb\u2013C angle in the dimeric lead structure differ considerably from the experimentally measured values. The experimental Pb\u2013Pb distance of 4.129(1) \u00c5 in the crystal structure of the 2 \u2192 2P{CH(SiMe3)2}2 process upon relaxation of the configurations from syn, anti to syn, syn.3)2}2 unit steric crowding because of the larger sizes of germanium and tin in comparison to that of phosphorus.It was noted above that the experimentally-determined orientation of the \u2013CHSiMe2 groups 3)2 groups of the [Pb{CH(SiMe3)2}]2 dimer to the syn, anti orientation observed for the lighter congeners; the data in Table S4syn, syn orientation; the dispersion corrected distance of 2.956 \u00c5 (B3PW91-D3) approaches the sum of the covalent radii indicating a significant degree of Pb\u2013Pb bonding. Also noteworthy is the effect upon the C\u2013Pb\u2013C angle which is calculated to widen significantly (91.3 versus 106.6\u00b0 at the B3PW91-D3 level); likely a consequence of steric clash between the SiMe3 groups of the ligand. Such an angle, however, would also optimize Pb\u2013Pb bonding by increasing the p-character of the Pb lone pair. That the Pb{CH(SiMe3)2} dimer does not adopt such a conformation must suggest the nature of the Pb{CH(SiMe3)2}2 dimer is that of two weakly interacting plumbylene monomers wherein significant s-character of the lone pair enforced by the narrow C\u2013Pb\u2013C angles (93.4(2)\u00b0) lead to \u201cclosed-shell\u201d interactions between the Pb atoms.It is also informative to consider the effect of altering the orientation of the CH(SiMe3)2}2]2 to two E{CH(SiMe3)2}2 monomers are summarized in \u20131 for the germanium, tin, and lead dimers with BSSE and ZPE corrections. The low values result from steric repulsion between the \u2013CH(SiMe3)2 groups. However, inclusion of dispersion effects using the B3PW91-D3 approach dramatically increases the binding energies to 28.7, 26.3, and 15.2 kcal mol\u20131, respectively. With the B97-D3 method, the calculated energies are 33.9, 33.3, and 20.2 kcal mol\u20131. Single-point MP2-based calculations afford energies of 41.2, 41.5, and 10.3 kcal mol\u20131. Taken together, the calculated energies strongly suggest that the major portion of the binding energy for the germanium, tin, and lead dimetallenes are a result of attractive dispersion forces between the \u2013CH(SiMe3)2 ligands. Calculation of the free energy changes (\u0394G) at 25 \u00b0C and 1 atm for the dissociation of the dimers, without dispersion force correction, affords values of \u201317.8, \u201314.8, and \u20139.9 kcal mol\u20131 for the germanium, tin, and lead derivatives respectively. In other words, dissociation is favored in all cases. Application of the dispersion-corrected B3PW91-D3 method changes these \u0394G values to +9.4, +7.1, and \u20131.7 kcal mol\u20131. Thus, the dimeric structures become favored for the germanium and tin species, but remains slightly disfavored for lead. These findings are in accord with the relatively long element\u2013element distances experimentally observed for the germanium and tin dimers, and the very weak interaction in the case of the lead species. The \u0394G energies calculated for the dissociation of germanium and tin alkyls at the temperatures and pressures at which the GED data sets were collected, i.e. 428 K and 0.1 Torr for the germanium dialkyl and 393 K and 0.1 Torr for the tin dialkyl, were \u20137.2 and \u20136.1 kcal mol\u20131. These negative values are consistent with the monomeric structures observed in the vapor phase.The data for the dissociation of the dimeric [E{CH2) series.3)2}2]2 and the behavior of the 13C shifts for the methine carbons in the monomers and dimers. This allowed calculation of \u0394H = 12.8 kcal mol\u20131 and \u0394S = 33 cal K\u20131 mol\u20131 for the dissociation. These experimental values differ considerably from those calculated . At present, the reason for the discrepancy between the experimental and calculated values is unclear, and more data will be required to establish the expected values for similarly-substituted monomer\u2013dimer equilibria. For example, significantly higher \u0394S values of 75 and 66 cal mol\u20131 K\u20131 have been determined for the dissociation of the [M{N(SiMe3)2}2]2 (M = Fe or Co), which carry \u2013N(SiMe3)2 substituents that are isoelectronic to CH(SiMe3)2.6H2-2,4,6-Me3){C6H2-2,4,6-(CH(SiMe3)2)3}]2 was studied by electronic spectroscopy which revealed a \u0394H value of 14.7(2) kcal mol\u20131 and a \u0394S value of 42.4 cal mol\u20131 K\u20131.49There has been only one report of an experimental determination of energies associated with the monomer\u2013dimer equilibrium of the E3)2}2]2 dimers. The syn, anti conformation adopted by germanium and tin yields an increased interligand steric congestion,syn, syn orientation and consequent attenuation of metal\u2013metal bonding. This conclusion is supported by the fact that the seven-membered ring dialkyl lead(ii) species PBM data was replaced with SVG by xgml2pxml: 1111111111111111111111111111111111 1111111111111111111111111111111111 1100000000000000000000000000000000 1100000000000000000000000000000000 1100000000000000000000000000000000 1100000000000000000000000000000000 1100000000000000000000000000000000 1100000000000000000000000000000000 1100000000000000000000000000000000 1100000000000000000000000000000000 1100000000000000000000000000000000 1100000000000000000000000000000000 1100000000000000000000000000000000 1100000000000000000000000000000000 1100000000000000000000000000000000 1100000000000000000000000000000000 1100000000000000000000000000000000 1100000000000000000000000000000000 1100000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019PbC(SiMe3)2SiMe2CH2CH2SiMe2C PBM data was replaced with SVG by xgml2pxml: 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000011 0000000000000000000000000000000011 0000000000000000000000000000000011 0000000000000000000000000000000011 0000000000000000000000000000000011 0000000000000000000000000000000011 0000000000000000000000000000000011 0000000000000000000000000000000011 0000000000000000000000000000000011 0000000000000000000000000000000011 0000000000000000000000000000000011 0000000000000000000000000000000011 0000000000000000000000000000000011 0000000000000000000000000000000011 0000000000000000000000000000000011 0000000000000000000000000000000011 0000000000000000000000000000000011 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019(SiMe3)2,3)2}2]2 and a C\u2013Pb\u2013C angle of 117.1(2)\u00b0, has no Pb\u2013Pb contact shorter than 8.911 \u00c5. Furthermore, the Sn(ii) dialkyl PBM data was replaced with SVG by xgml2pxml: 1111111111111111111111111111111111 1111111111111111111111111111111111 1100000000000000000000000000000000 1100000000000000000000000000000000 1100000000000000000000000000000000 1100000000000000000000000000000000 1100000000000000000000000000000000 1100000000000000000000000000000000 1100000000000000000000000000000000 1100000000000000000000000000000000 1100000000000000000000000000000000 1100000000000000000000000000000000 1100000000000000000000000000000000 1100000000000000000000000000000000 1100000000000000000000000000000000 1100000000000000000000000000000000 1100000000000000000000000000000000 1100000000000000000000000000000000 1100000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019SnC(SiMe3)2(CH2)2C PBM data was replaced with SVG by xgml2pxml: 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000011 0000000000000000000000000000000011 0000000000000000000000000000000011 0000000000000000000000000000000011 0000000000000000000000000000000011 0000000000000000000000000000000011 0000000000000000000000000000000011 0000000000000000000000000000000011 0000000000000000000000000000000011 0000000000000000000000000000000011 0000000000000000000000000000000011 0000000000000000000000000000000011 0000000000000000000000000000000011 0000000000000000000000000000000011 0000000000000000000000000000000011 0000000000000000000000000000000011 0000000000000000000000000000000011 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019(SiMe3)2,3)2 moieties have a syn, syn-like conformation analogous to the vapor phase structure of Sn{CH(SiMe3)2}2 and the lead congener in all cases. These data suggest that a syn, syn orientation precludes strong M\u2013M contact and thus any dimerisation must emerge principally from dispersion interactions.A rationalization for the observed congeneric variation can thus be constructed wherein the interplay between the effects of dispersion interactions, steric congestion and metal\u2013metal bond strength define the observed structures of the 2 dimers at various levels of optimization are given in 3)2}2 units within the dimers are close to those calculated for the monomeric structures which match those experimentally determined by X-ray crystallography. The E\u2013E distances undergo large contractions when dispersion force corrections are included. However, the shortest distances calculated for the germanium and tin amido dimers, even with the inclusion of such forces at the B3PW91-D3 level, are significantly longer (by ca. 1.4 and 0.7 \u00c5) than the measured values and the values calculated at the same level for the alkyl dimers. The metal\u2013metal distance calculated for the amide-ligated lead dimer (3.714 \u00c5) by the B3PW91-D3 method is ca. 0.5 \u00c5 longer than the corresponding calculated distance (3.241 \u00c5) in the dialkyl lead dimer. These distances indicate considerably weaker interactions between the group 14 elements for the amido derivatives. Thermodynamic data 2 ligands. Further calculations on the thermodynamics of dissociation show that the \u0394G energies for the process are \u201316.4, \u201318.0, and \u201310.8 kcal mol\u20131 at 25 \u00b0C and 1 atm, so that dissociation and monomeric structures are favored under these conditions. These findings are, of course, consistent with the structural and physical data. The much weaker E\u2013E interactions in the amides in comparison with the alkyls is probably a result of electronic and steric factors. The more electronegative \u2013N(SiMe3)2 ligand causes a larger HOMO\u2013LUMO separation on the E atom, which would lower the extent of orbital interaction between the monomers. Also, the configurational differences between the \u2013CH(SiMe3)2 and \u2013N(SiMe3)2 may lead to greater steric hindrance, and hinder the association of the two monomeric fragments in the case of the amido ligand.The calculated structural parameters and dissociation energies of the putative [E{N4. While no full molecule calculations have been reported for [Sn{SiMetBu2}2]2 to investigate dispersion force contributions, a qualitative assessment can be made. One notable feature of dispersion force interactions is their dependence on length-scale and they are thought to be highly attenuated beyond the sum of the van der Waals radii of the interacting atoms.With these analyses in hand, it is informative to consider other stannylenes in the literature. Whilst a plethora of such species have been reported, the vast majority are monomeric in solution, and many with extremely sterically demanding ligands remain monomeric in the solid state.3)2}2]2 and [Sn{SiMetBu2}2]2 is informative 2}2 unit indicating that such a sterically unfavourable conformation is partly stabilised by dispersion interactions.As shown, analysis of the crystallographically defined structures of [Sn{CH2}2]2 species. Whilst the reporttBu3}2]2 provides an extensive electronic rationale for Sn\u2013Sn interactions being responsible for its observed dimeric structure and short Sn\u2013Sn distance, it is likely that dispersion forces are also of importance in stabilising the persistent dimeric nature of this compound.In the case of [Sn{SiMetBu2}2]2 confirm the presence of a shorter tin\u2013tin bond (2.647 \u00c5) than that in [Sn{CH(SiMe3)2}2]2. However, the calculations show that the binding energy increases from 25.8 to 46.8 kcal mol\u20131 with inclusion of dispersion effects, and that the \u0394G of dissociation at 25 \u00b0C and 1 atm is increased from 8.3 to a value of 26.5 kcal mol\u20131 upon inclusion of the dispersion correction, indicating that the dispersion force attraction is of key importance in maintaining the dimeric structure.To check this hypothesis, our initial full molecule calculations which are notable particularly for the \u2013CH(SiMe3)2 ligand. A similar analysis for the non-dissociating distannene [Sn{SiMetBu2}2]2 shows that in this molecule also, the dispersion forces are of key importance in its stabilization. These studies provide an initial framework for the analysis of metal\u2013metal bonding which takes into account these more subtle effects. Although much effort has been expended in development of bonding models for the multiple bonds between heavier main group elements, it seems both probable and ironic that the dispersion force attraction forces exceed those of the multiple bonds in many instances and that a variety of more subtle interactions including packing effects are of key importance for the understanding of their bonding.The calculations have shown that the interplay between dispersion force attraction, steric repulsion and element\u2013element bonding stabilize the dimeric structures. Although the E\u2013E distances indicate E\u2013E bonding is present in the germanium and tin dimers, and possibly the lead dimer, the bonding is weak, and represents a relatively small fraction of the binding energy. The results emphasize the importance of including attractive dispersion force interactions in consideration of multiple bonded heavier main group element species where sterically encumbering ligands are employed in their stabilization.Supplementary informationClick here for additional data file."} +{"text": "A new, highly active Ir catalyst for dehydrogenative borylation of terminal alkynes (DHBTA) has been identified. via the changes in the substitution at phosphorus led to the discovery of a catalyst whose activity, longevity, and scope far exceeded that of the original SiNN archetype. Several Ir complexes were prepared in a model PNP system and evaluated as potential intermediates in the catalytic cycle. Among them, the (PNP)Ir diboryl complex and the borylvinylidene complex were shown to be less competent in catalysis and thus likely not part of the catalytic cycle.Following the report on the successful use of SiNN pincer complexes of iridium as catalysts for dehydrogenative borylation of terminal alkynes (DHBTA) to alkynylboronates, this work examined a wide variety of related pincer ligands in the supporting role in DHBTA. The ligand selection included both new and previously reported ligands and was developed to explore systematic changes to the SiNN framework (the 8-(2-diisopropylsilylphenyl)aminoquinoline). Surprisingly, only the diarylamido/bis(phosphine) PNP system showed any DHBTA reactivity. The specific PNP ligand (bearing two diisopropylphosphino side donors) used in the screen showed DHBTA activity inferior to SiNN. However, taking advantage of the ligand optimization opportunities presented by the PNP system The products of dehydrogenative borylation of terminal alkynes (DHBTA), alkynylboronates, are versatile building blocks in synthetic chemistry. Their synthetic value derives not just from the direct use in C\u2013Cet al.: deprotonation of alkyne by n-BuLi, followed by reaction with a boric ester and quench with anhydrous acid.et al.et al. also recently demonstrated that certain borenium cations can react with terminal alkynes to give alkynylboronates.2 and sp3 C\u2013H bonds, catalysis of direct coupling of a C\u2013H bond with a B\u2013H bond (2)\u2013H and C(sp3)\u2013H bonds, for the relatively acidic C(sp)\u2013H bonds (pKa \u223c 25) of terminal alkynes, the C\u2013H activation itself is generally not a difficult task and C\u2013H bond selectivity would not typically be an issue. On the other hand, in contrast to the non-olefinic C(sp2)\u2013H and C(sp3)\u2013H substrates, a combination of a triple C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond, a B\u2013H bond and a metal catalyst is very likely to lead to hydroboration.2 (the by-product of DHBTA) may also be a concern.The classical synthesis of alkynylboronates was developed by Brown 1-Ir-COE and 21-Ir-Bpin, ca. 10 turnovers per min) with a variety of alkyl-, aryl- and silyl- terminal alkynes in high yield. However, the catalyst longevity was limited to ca. 100 turnovers. Very recently, Tsuchimoto et al. described DHBTA catalysis by Zn(OTf)2/pyridine using 1,8-nathpthalenediamidoborane.ca. 1 turnover per hour) even at 100 \u00b0C. Our group also reported that (POCOP)Pd complexes are modest DHBTA catalysts for some substrates.42In 2013, we reported the first example of catalytic DHBTA performed by Ir complexes of a SiNN pincerThe discovery of the prowess of the SiNN ligand in DHBTA was rather serendipitous, and we sought to explore the associated ligand space in a more systematic fashion. Here we report the exploration of a series of Ir complexes of related ligands as potential catalysts in DHBTA that has led to the discovery of a new highly active, much more long-lived catalyst with a broader scope, as well as to the insight into the role of possible intermediates in DHBTA.1-Ir-COE in DHBTA, we decided to examine a series of ligands that systematically explored variations of the SiNN ligand features or replaced it with hemilabile donors (3-H to 7-H). For 8-H and 9-H, the silane segment and the central amido donor were maintained while the quinoline moiety was eliminated (8-H) or substituted with a phosphine donor (9-H). We also included the PNP ligand (10-H) and the PCP/POCOP ligands (11-H to 14-H) because these are commonly used pincer ligands with a rich history of C\u2013H activation chemistry with Ir.In light of the success of features . From 2-i.e.2-H,473-H, 4-H, 5-H, 6-H,487-Hvia Buchwald\u2013Hartwig coupling of 8-bromoquinoline with various anilines or 8-aminoquinoline with various bromoarenes. 8-H was prepared via the intermediate S1. The synthesis of S1 relied on the same selective dilithiation of bis(2-bromo-4-methylphenyl)amine we previously used in the synthesis of S2,S1 was isolated in 86% yield by column chromatography. Treatment of S1 with n-BuLi, followed by addition of iPr2SiHCl and workup gave 8-H in 73% yield. The new SiNP ligand 9-H was prepared from S2 through a similar protocol.The syntheses of ligands used in the screening of DHBTA are shown in 1-Ir-COE precatalyst in situ from 1-Na and [(COE)2IrCl]2 . They were deprotonated with 1 equiv. of NaN(SiMe3)2in situ, allowed to react with 0.5 equiv. of [(COE)2IrCl]2 in C6D6 and the resultant solutions were tested for catalytic DHBTA activity. In the case of PCP/POCOP ligands 11\u201314, we isolated dihydride complexes (211-Ir-H212-Ir-H2H413-Ir-C and 14-Ir-COE) to be used in DHBTA testing.In our original DHBTA report,)2IrCl]2 produced2H413-Ir-C was obtained after modification of previously reported procedures for related compounds (13-H with [(COE)2IrCl]2 at 80 \u00b0C overnight in toluene resulted in a dark red solution that contained ca. 85% of the desired product (31P NMR evidence). Column chromatography allowed for the collection of 99% pure 13-Ir-HCl in 22% yield. This portion of 13-Ir-HCl was then treated with a slight excess of NaOtBu in toluene, degassed, and then stirred under an atmosphere of ethylene for 30 min. After filtration and removal of volatiles under vacuum, analytically pure 2H413-Ir-C was obtained as a dark brown solid in 66% isolated yield (based on 13-Ir-HCl). 14-Ir-COE was synthesized by reacting the previously reported 14-Ir-HCltBu and COE in C6D6 was selected as the alkyne for testing. We mimicked the conditions that were successful for the SiNN ligand 1, with 1 mol% Ir loading and 2 equiv. of HBpin used at ambient temperature in C6D6 solvent. The results are summarized in 10-H showed any DHBTA reactivity. In all other cases, no evidence for the DHBTA product A1-Bpin was visible by 1H NMR spectroscopy after 1 h. In general, sluggish and nonselective hydrogenation and hydroboration was observed for 2-H to 9-H, and for the iso-propyl PCP/POCOP iridium complexes (2H413-Ir-C and 14-Ir-COE). For tert-butyl PCP/POCOP iridium complexes (211-Ir-H and 212-Ir-H), a mixture of trans-alkenylboronate (A1-1) and cis-alkenylboronate (A1-2) were observed as major products. The use of 10-H resulted in 76% A1-Bpin after 10 min and 90% (NMR evidence) after 1 h, with about 3% of 4-ethyltoluene . We also tested one of the most active arene borylation catalyst systems ([(COD)Ir(OMe)]2 + 4,4\u2032-di-tert-butyl-bipyridine)4-Ethynyltoluene (210-Ir-HA1-H), trimethylsilylacetylene (A2-H), and 1-hexyne (A3-H) (210-Ir-H in the DHBTA of 4-ethynyltoluene (A1-H) was similar to the catalyst generated from 10-Hin situ. DHBTA of trimethylsilylacetylene (A2-H) was finished in 1 h and gave an excellent yield of A2-Bpin. The catalytic activity of 210-Ir-H towards A3-H was significantly lower than towards A1-H and A2-H and only 50% yield was achieved after 3 h. A small amount of the hydrogenation product A1-3 was observed in DHBTA of A1-H, but no hydrogenation products were detected in the reactions of A2-H and A3-H.With this lead in hand, we tested isolated e (A3-H) . The eff210-Ir-H fell somewhat short of the SiNN-based catalysis, where >95% yield of A1/2/3-Bpin was obtained in <10 min and without any hydrogenation side products. Nonetheless, we were encouraged by the results because the PNP framework offers facile opportunities for optimization of the ligand via substituent variation. We selected previously reported PNP ligands 15-H,5016-H,17-H15-H is an oil that is difficult to purify; however, the Li derivative (15-Li) could be isolated in in 56% yield as a pure solid. 15-Li was then reacted with 0.5 equiv. of [(COE)2IrCl]2 to yield 15-Ir-COE. 16-Ir-COE17-Ir-COE were synthesized via one-pot reactions by deprotonation of the neutral ligands in situ and treatment with [(COE)2IrCl]2.The effectiveness of 15-Ir-COE, 16-Ir-COE, 17-Ir-COE), 210-Ir-H, and the previously reported 1-Ir-COE were all tested in DHBTA by using A1-H as the substrate with 2 equiv. of HBpin at ambient temperature in C6D6 solvent. The results are summarized in 15-Ir-COE, 17-Ir-COE, 210-Ir-H, as well as 1-Ir-COE gave excellent yields of A1-Bpin at ambient temperature, whereas 16-Ir-COE did not (entry 4) and was eliminated from further consideration. At 0.25% Ir, 17-Ir-COE showed superior reactivity to 1-Ir-COE, 210-Ir-H, and 15-Ir-COE by producing 92% A1-Bpin in 10 min. 1-Ir-COE gave 43% yield after 1 h (entry 6) and the yield did not increase with longer reaction times, suggesting faster catalyst decomposition for the SiNN-based catalyst. 17-Ir-COE was able to effect 100% conversion of A-1H and 85% yield of A1-Bpin (NMR evidence) even at 0.025% loading in only hours at ambient temperature. The reaction rate was higher at 60 \u00b0C (entry 12) without loss in yield. The 84% yield of A1-Bpin at 0.025 mol% catalyst loading (entry 12) corresponds, impressively, to 3400 turnovers. Under incomplete conversion with 0.01 mol% loading (entry 13), a turnover number of 6500 was achieved after 2 h at 60 \u00b0C. In terms of chemoselectivity, 1-Ir-COE is superior in DHBTA of A1-H as it gave A1-Bpin as the product exclusively; 2\u201310% of hydrogenation product A1-3 was observed in all reactions catalyzed by the (PNP)Ir complexes Ir(COE) complexes , as well as 3-methyl-3-trimethylsiloxy-1-butyne (A5-H), trimethylsilyl propargyl ether (A6-H), 4-dimethylamino-phenylacetylene (A7-H), N-tosylated allyl propargyl amine (A8-H), and dimethyl 2-allyl-2-(prop-2-yn-1-yl)malonate (A9-H) were chosen as representative substrates for aromatic, silyl, aliphatic terminal alkynes, propargyl derivatives and 1,6-enynes, respectively and comparable yields/side product (A6-1 for A6-H) were obtained. No DHBTA products were observed for phenyl propargyl sulfide, 3-ethynylpyridine, 4-cyano-1-butyne, 3,3-diethoxy-1-propyne, and methyl propiolate with 0.1 mol% 17-Ir-COE. A1-Bpin, A5-Bpin and A8-Bpin could be easily purified by recrystallization and were isolated in good yields in preparative-scale reactions. In contrast, 1-Ir-COE requires 1% loading for high yields of A1-, A2-, A4-, and A5-Bpin, and is altogether ineffective for the synthesis of propargyl derivatives A6- and A8-Bpin . A mercury drop test17-Ir-COE loading and A1-H as substrate. No significant yield changes were observed for either A1-Bpin or the major side-product A1-3 which suggested that the catalysis is homogeneous.To further explore the catalytic reactivity of ectively . For A1-C2v-symmetric structure, and because a number of its iridium complexes are already known,10 for this study. We were particularly interested in determining the possible products arising from combining the 10-Ir fragment with HBpin, terminal alkynes, and alkynylboronates and their catalytic competence.In order to gain new insight into the reaction mechanism, we set out to examine conceivable intermediates in DHBTA. Because of its NMR-friendly 21-Ir-Bpin can be synthesized by reacting 1-Ir-COE with 5 equivalents of HBpin,21-Ir-Bpin exhibited the same catalytic activity as 1-Ir-COE. Treating 210-Ir-H with 5 equivalents of HBpin, however, led to a mixture of 3Bpin10-Ir-H and 10-Ir-HBpin in equilibrium with free H2 in its 1H NMR spectrum, and the peak sharpened upon 11B decoupling and \u201312.4 ppm in the 1H NMR spectrum at ambient temperature. The resonance at \u20135.3 ppm (\u03c91/2 = 35 Hz) sharpened upon 11B decoupling level of theory, see details in ESI10-Ir-p-tol; the vinylidene complex 10-Ir-v-tol; and the alkynyl boryl complex 10-Ir-ynlBpin-tol and 25.6 (11%) ppm respectively in the 31P{1H} NMR spectrum Ir precursor was reacted with A1-Bpin under milder conditions. We first attempted to mix 210-Ir-H with A1-Bpin with subsequent rapid removal of volatiles. The residue was redissolved in C6D6 and analyzed by 1H and 31P NMR spectroscopy. The major product was assigned as the alkynylboronate \u03c0-complex 10-Ir-p-tol of the 1H NMR resonances of the ortho-hydrogens of the p-tolyl group in the coordinated A1-Bpin. Such downfield chemical shift is characteristic of internal aromatic alkyne \u03c0-complexes.10-Ir-p-tol to 10-Ir-v-tol proceeded at an appreciable rate at ambient temperature (about 50% after 15 h) and precluded the isolation of pure 10-Ir-p-tol. We surmised that a more electron-poor alkyne should be thermodynamically less predisposed to form a vinylidene,A1-Bpin with A10-Bpin as the reactant than the analogous transformation of 10-Ir-p-tol. Similarly to 10-Ir-p-tol, the aromatic proton signals of the 2,6-positions on A10-Bpin in 3tol10-Ir-p-F were shifted downfield to 8.22 ppm in the 1H NMR spectrum. In the 13C NMR spectrum, the carbon signal of alkynyl\u2013C scale\" fill=\"currentColor\" stroke=\"none\">C\u2013B) in 3tol10-Ir-p-F (\u03b4 105.7) was slightly downfield of that in free A10-Bpin, as expectedEsteruelas and L\u00f3pezIr-p-tol , and itsreactant and mixe10-Ir-HBpin, 210-Ir-Bpin, 10-Ir-v-tol, and 3tol10-Ir-p-F in the solid state by X-ray diffractometry on corresponding single crystals. The structure of 10-Ir-HBpin analysis of 10-Ir-HBpin and 12-Ir-HBpin in the gas phase using the M06 functional was also performed. DFT calculations show 10-Ir-HBpin possesses shorter Ir\u2013B and Ir\u2013H bond distances and a B\u2013H bond distance 0.2 \u00c5 longer than 12-Ir-HBpin which was judged to be a \u03c3-borane complex, suggesting greater degree of B\u2013H bond activation in 10-Ir-HBpin. The structure of 210-Ir-Bpin ((amido) and the two boryls with an acute B\u2013Ir\u2013B angle (68.2\u00b0). The Y-shaped geometry is expected for a five-coordinate d6 complex(amido)) and two strong \u03c3-donors (two boryls). The two Ir-bound Bpin fragments display essentially the same metrics, and the associated Ir\u2013B distances are similar to the analogous Ir\u2013Bpin distances reported in the literature (2.02\u20132.07 \u00c5).2 plane are very close to the previously reported 21-Ir-Bpin (210-Ir-Bpin should be unambiguously viewed as an Ir(iii) diboryl complex.We were able to determine molecular structures of 10-Ir-v-tol and 3tol10-Ir-p-F 2N]Ir PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CH2 vinylidene complex reported by Fryzuk.3tol10-Ir-p-F, A10-Bpin is bound to iridium in an \u03b72 fashion (Ir\u2013C1: 2.165(11) \u00c5, Ir\u2013C2: 2.101(12) \u00c5) and the Ir\u2013C distances are within the range of other square planar Ir(i) alkyne complexes. PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond (1.304 \u00c5) and the bending of C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C\u2013Cipso (147.5(11)\u00b0) and C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C\u2013B (161.9(11)\u00b0) away from 180\u00b0 indicate back-donation from the iridium center to the \u03c0* orbitals of C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond.The coordination environment about Ir in the structures of -p-F3tol can be dMePNPiPr)Ir complexes played in DHBTA, these compounds were examined in reactions with the three components in DHBTA: a terminal alkyne (substrate), HBpin (substrate), and H2 (by-product). 10-Ir-HBpin was reacted with three different terminal alkynes to study the boryl transfer ability: A1-H, A2-H and A10-H and unidentified iridium compounds in 1 h at ambient temperature. After overnight, 210-Ir-H was the only observable species by 31P NMR spectroscopic analysis. Lack of observation of A1-4 in catalytic reaction mixtures suggested that 10-Ir-v-tol is not present in significant concentrations during catalysis. Treating 3tol10-Ir-p-F with 1 equivalent of HBpin at ambient temperature cleanly led to 83% 10-Ir-HBpin formation after 3 h was also observed.perature , top. Onfter 3 h , bottom;MePNPiPr)Ir compounds as pre-catalysts. The catalytic reactions were carried out in the fashion consistent with our other studies \u2013 the Ir compound was treated with 200 equiv. of HBpin, followed by 100 equiv. of the terminal alkyne . When 210-Ir-H was treated with excess HBpin, a yellow mixture of 10-Ir-HBpin and 3Bpin10-Ir-H immediately formed before the addition of alkyne. Not surprisingly, essentially identical yields of A1-Bpin and hydrogenation side-product A1-3 was observed when using 10-Ir-HBpin and 210-Ir-H as pre-catalysts. The use of 10-Ir-v-tol did lead to the formation of A1-Bpin, but in a significantly smaller yield than with 10-Ir-HBpin and 210-Ir-H. In contrast, 210-Ir-Bpin showed no DHBTA at all after the first 10 min and only gave 37% A1-Bpin after 3 h. The inertness of 210-Ir-Bpin in the DHBTA correlated with its lack of reactivity in the stoichiometric reactions described above. In the two reactions with A10-H, 210-Ir-H and 3tol10-Ir-p-F led to the same yield of A10-Bpin and the hydrogenation side-product A10-1 (4-CF3-C6H4-C2H5) at the 10 min and the 1 h mark.210-Ir-Bpin and the vinylidene complexes analogous to 10-Ir-v-tol can be firmly ruled out as intermediates in the DHBTA catalysis by (PNP)Ir complexes. Interestingly, this raises the question of whether the previously reported (SiNN)Ir catalysis actually requires 21-Ir-Bpin as an intermediate or if it is merely an off-cycle precursor that can access the catalytic cycle rapidly enough.On the basis of the stoichiometric and catalytic experiments the diboryl complexes analogous to 17-Ir-COE, useful yields were obtained with 0.025% loading of catalyst, corresponding to thousands of turnovers. Good to excellent yields were obtained under mild conditions for aryl-, silyl-, and alkyl-substituted terminal alkynes, even propargyl derivatives and 1,6-enynes. Unlike the strict chemoselectivity in the (SiNN)Ir system, <10% hydrogenation products were observed as the main side-products in all the (PNP)Ir systems with arylacetylene substrates. This has not precluded isolation of alkynylboronate products in 70\u201382% yields on preparative scale.Building on a recent report of successful dehydrogenative borylation of terminal alkynes (DHBTA) with a pincer iridium catalyst,10 were synthesized and examined as potential intermediates in the catalytic cycle via testing in stoichiometric and catalytic reactions. The vinylidene (10-Ir-v-tol) and diboryl (210-Ir-Bpin) complexes reacted too slowly with either terminal alkynes or HBpin under the conditions of catalysis, which ruled them out of the catalytic cycle of the 10-Ir system. The inactivity of 210-Ir-Bpin is in contrast to the analogous 21-Ir-Bpin10-Ir-HBpin) and the alkynylboronate \u03c0-complex (3tol10-Ir-p-F) showed nearly identical performance to 210-Ir-H indicating that they either are intermediates in the catalytic cycle or are connected to such via a low-barrier pathway. Although the full mechanistic picture remains uncertain, the presently reported results strongly suggest that a pincer ligand containing an amido donor is key to an active DHBTA catalyst with iridium. An intriguing possibility is that this is related to the facile migration of boryl from the metal to the amido nitrogen we recently discovered for 21-Ir-Bpin and 21-Rh-Bpin.80Several iridium complexes of the symmetric PNP ligand Supplementary informationClick here for additional data file.Supplementary informationClick here for additional data file.Supplementary movieClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "Controlling the steric environment in U2)(\u03b75-Cp*)] enables selective formation of either mononuclear U(v) or dinuclear U(iv) oxo and nitrido complexes. v) neutral terminal oxo complex and a U(v) sodium uranium nitride contact ion pair are described. The synthesis of the former is achieved by the use of tBuNCO as a mild oxygen transfer reagent, whilst that of the latter is via the reduction of NaN3. Both mono-uranium complexes are stabilised by the presence of bulky silyl substituents on the ligand framework that facilitate a 2e\u2013 oxidation of a single U(iii) centre. In contrast, when steric hindrance around the metal centre is reduced by the use of less bulky silyl groups, the products are di-uranium, U(iv) bridging oxo and (anionic) nitride complexes, resulting from 1e\u2013 oxidations of two U(iii) centres. SQUID magnetometry supports the formal oxidation states of the reported complexes. Electrochemical studies show that the U(v) terminal oxo complex can be reduced and the [U(iv)O]\u2013 anion was accessed via reduction with K/Hg, and structurally characterised. Both the nitride complexes display complex electrochemical behaviour but each exhibits a quasi-reversible oxidation at ca. \u20131.6 V vs. Fc+/0.The synthesis and molecular structures of a U( U(ii) PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O complexes requires the use of bulky supporting ligands.in situ generation and involvement in C\u2013H activation had been proposed and studied computationally.17The study of well-defined molecular complexes of uranium is a thriving field of research,1.000000,.000000 s2iii) mixed sandwich complexes of the general type [U{\u03b78-C8H6-2}(\u03b75-CpR\u2032\u2032)THF] (R = iPr (1), Me (2)). In particular, the reductive transformations of CO2 using the complexes [U{\u03b78-C8H6-2}(\u03b75-Cp4R\u2032Me)THF] (A) can be largely controlled by varying the size of R\u2032 .A that exhibit a clear trend between the effect of steric environment and the outcome of the possible reductive transformations, when the analogous complexes in which the SiMe3 group had been replaced by the bulkier SiiPr3 group were reacted with CO2, either intractable reaction mixtures were obtained or the reductive disproportionation of CO2 was promoted exclusively.e.g. RN PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 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0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O) as model substrates for CO2 might be informative.20We have previously demonstrated the significance of the steric environment around the uranium centre in controlling the reductive coupling of CO6D6 solution of [U{\u03b78-C8H6-2}(\u03b75-Cp*)THF] (1) with a slight excess (1.05\u20131.1 eq.) of tBuN PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O under an Ar atmosphere resulted in an immediate colour change to brown red. 1H-NMR spectroscopy showed complete consumption of (1) and the formation of a new uranium species and free tBuNC (further confirmed by GC-MS of the trapped volatiles of the reaction mixture). The 29Si{1H}-NMR spectrum of the product displayed a single resonance at \u201373 ppm, shifted downfield from \u2013129 ppm in (1) suggesting that a change in the formal oxidation state of the uranium centre of (III) to (V) had taken place, in accordance with the general trend observed by Evans et al.v) terminal oxo complex {U[\u03b78-C8H62](\u03b75-Cp*)O} (3), and was confirmed by X-ray crystallography (Reaction of a brown-olive green Clography .3) (1.826(3) \u00c5) is shorter than that found in the U(v) terminal oxo complex [Ph3PMe][U(O)(CH2SiMe2NR\u2032)(NR\u20322)2] (1.847(2) \u00c5),v) complexes [U(O)(NR\u20322)3] (1.817(1) \u00c5),TIPS(O)] (1.856(6) \u00c5, TRENTIPS = [N(CH2CH2NSiiPr3)3]3),RArO)tacn)U(O)] (1.848(8) \u00c5; R = tBu, Ad; tacn = triazacyclononane),2U(O)(ODipp) (1.859(6) \u00c5, Dipp = 2,6-iPr2-C6H3),tBu)3}4K] (1.825(2) \u00c5)4][trans-U(NR2\u2032)3(O)CN].iv) (137\u2013140\u00b0) and U(iii) (150\u2013155\u00b0) mixed sandwich complexes supported by these ligands;3) was further characterised by spectroscopic7D8) gave an effective magnetic moment (\u03bceff) of 2.49 \u03bcB, very close to the theoretical value of 2.54 \u03bcB for an f1 system (see below for further details and SQUID magnetometry).The U\u2013O bond length in (3) was repeated on a larger scale, a second species co-crystallised with (3), and fractional crystallisation produced a small crop of crystals suitable for single crystal X-ray diffraction. The latter revealed the product to be the tBuNC adduct of (3) [U(\u03b75-Cp*)O(\u03b71-CNtBu)] (4), and the molecular structure is shown in When the synthesis of (4) is the elongation of the U\u2013O bond by almost 0.1 \u00c5 as compared with that in (3), and also with the U(V) PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O bonds compared above, with the exception of that in [U(O)(NR\u20322)3] (R\u2032 = SiMe3).vide infra) revealed \u03bdNC at 2179 cm\u20131 for the isocyanide ligand in (4), a value very close to those observed in [UCp*2(NMe2)(tBuNC)2]BPh4 (3(CNC6H11)(NCMe)]+ cation;4) is also comparable (within esd's) to those in the latter complexes, while the small deviation of the C\u2013N\u2013C(tBu) from linearity presumably alleviates steric congestion around the metal centre. The Ct(COT)\u2013U1\u2013Ct(Cp*) angle is slightly more obtuse (ca. 2\u00b0) than that in (3), while the Ct(Cp*)\u2013U1 and Ct(COT)\u2013U1 distances are slightly elongated but within the range observed for previously reported complexes supported by these ligands.The most salient feature of (C)2]BPh4 and the 4) in better yields from the reaction of (1) with tBuNCO were unsuccessful, leading to mixtures of (3) and (4), and indeed the tBuNC ligand in (4) is very labile and any attempted isolation or manipulation of (4) via operations in vacuo invariably again led to mixtures of (3) and (4). In order to isolate (3) free from (4), the best route involved the reaction of (1) with tBuNCO followed by repeated dissolution in pentane and subsequent evaporation, a method used by Andersen and Evans et al. to obtain base-free Cp* lanthanide complexes,3) in 55% yield. Reaction of a C6D6 solution of the resultant microanalytically pure (3) with one equivalent of tBuNC resulted in small but discernible shifts of the resonances due to (3) in the 1H-NMR spectrum and which we ascribe to the formation of (4). Similarly, in situ IR spectroscopy showed that, upon reaction of (3) with 1 equivalent of tBuNC in methyl\u2013cyclohexane, two new peaks appeared, one at 2134 cm\u20131 (\u03bdNC in free tBuNC) and one at 2179 cm\u20131 assigned to \u03bdNC in (4).Attempts to isolate (v) terminal oxo complex (3) proceeds via the isocyanide adduct (4): the use of tBuNCO as an efficient oxygen transfer reagent1) and the formation of tBuNC and hence (4) (probably via a concerted reaction), and ultimately (3) after work-up (The above data suggest that the synthesis of the novel U( work-up .iv) complex {U[\u03b78-C8H62](\u03b75-Cp*)}2(\u03bc-O) (5) from the reaction of (1) with a mixture of NO/CO.5), the isolation of the mononuclear terminal oxo U(v) complex (3) would appear surprising. We therefore decided to investigate whether (3) could be prepared using alternative oxygen transfer reagents. Reaction of (1) with exactly 0.5 equivalents of N2O (administered accurately via a T\u00f6epler line) in C7D8 at \u201378 \u00b0C resulted in an immediate colour change to bright red, leading to the clean formation of (5) as evidenced by 1H and 29Si{1H}-NMR spectroscopy, and the \u03bc-oxo complex was isolated as the sole product in very good yields terminal oxo complex (3) and the U(iii) precursor (1) were mixed in C7D8, no reaction was observed at RT and conversion to the \u03bc-oxo complex (5) (ca. 25% spectroscopic yield relative to (1)) was observed only after heating at 45 \u00b0C over three days.4) indicate that these two reactions most likely proceed via different mechanisms. The case of N2O would be consistent with a concerted mechanism involving a dinuclear intermediate in which N2O bridges, and then eliminates N2 leading to a dinuclear \u03bc-oxo product. However for tBuNCO, the formation of mononuclear (4) after the oxo transfer step, stops any further reaction with (1) that could lead to (5), due to the steric congestion imposed by both the TIPS groups and the tBuNC ligand. To further test this hypothesis, the less sterically hindered homologue of (1), [U{\u03b78-C8H6-2}(\u03b75-Cp*)THF] (2) was reacted with tBuNCO. In this case the reaction furnished cleanly the dinuclear \u03bc-oxo U(iv) complex {U[\u03b78-C8H62](\u03b75-Cp*)}2(\u03bc-O) (6) as evidenced by its NMR spectroscopic data that were in excellent agreement with those previously reported.1) and (2) have very similar [U(III)] \u2194 [U(IV)] redox potentials highlights the importance of the steric hindrance imposed by the silyl substituents on the 8-membered ring in dictating the outcome of the reactions of (1) and (2) with tBuNCO. In the case of (1), reaction with tBuNCO results in a single 2e\u2013 oxidation of the metal centre leading to the U(v) complex (4), and hence (3), whereas in the case of (2) this reaction results in two 1e\u2013 oxidations leading to the dinuclear U(iv)\u2013U(iv) complex (6) .3) using other isocyanates or oxo transfer reagents were uniformly unsuccessful leading to intractable reaction mixtures. Interestingly when Me3SiNCO was reacted with (1), the U(iv) complex {U[\u03b78-C8H63)2](\u03b75-Cp*)(OSiMe3)} (7) was isolated as the sole product of the reaction.7) can reasonably be explained by the formation of a short-lived complex which, due to the oxophilicity of the SiMe3 group, undergoes a formal reduction to produce the observed U(iv) complex (7) and presumably cyanogen (CN)2 . Similar reactivity of U PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O bonds towards silicon electrophiles has been observed by Andersen et al.8Attempts to generate (3) suggested that the steric protection afforded by the U[\u03b78-C8H62](\u03b75-Cp*) mixed sandwich framework might be exploited to access the analogous uranium nitride. The highly reducing nature of (1) (UIII/UIV \u20132.13 V vs. Fc+/0), suggested reduction of N3\u2013 as a possible method for installing the nitride ligand.13Given the similarities between nitride and oxo ligands,1) with NaN3 were isolated in moderate yield (ca. 30%), together with other product(s) which could not be unambiguously characterised despite repeated attempts. X-ray diffraction studies showed (9) to be the nitride complex [U{\u03b78-C8H6-2}(\u03b75-Cp*)(\u03bc-N)(\u03bc-Na{OEt2}2)], best described as a sodium uranium nitride contact ion pair terminal nitride anion supported by the TRENTIPS ligand, as well as its U(vi) neutral analogue.9) is comparable to that in the U(v) nitride complex [U(TRENTIPS)N]\u2013[Na(12-c-4)2]+ (1.825(15) \u00c5) where the two ions are separated, but is shorter than the one found in [U(TRENTIPS)(\u03bc-N)(\u03bc-Na)]2 (1.883(4) \u00c5) where a N\u2013Na interaction is also present.6F5)3BNU(V)(NMestBu)3][NnBu] (1.916(4) \u00c5) and [(C6F5)3BNU(VI)(NMestBu)3] (1.880(4) \u00c5)vi) complex [U(TRENTIPS)N] the U\u2013N bond in (9) is similar within esd's.9) is shorter than the ones found in [U(TRENTIPS)(\u03bc-N)(\u03bc-Na)]2 (2.308(5) \u00c5)TIPS)(\u03bc-N)(\u03bc-Na{15-c-5})] (2.291(5) \u00c5),9) is elongated compared to (3) and (4) while the Ct(COT)\u2013U1\u2013N1 and Ct(Cp*)\u2013U1\u2013N1 angles are significantly more acute than the ones found for the corresponding angles Ct\u2013U1\u2013O1 angles in (3). The reason for these differences is unclear.Liddle 9) was further characterised by spectroscopic\u03bceff (Evans method) was determined to be 2.21 \u03bcB , which is in reasonable agreement with the value of 1.99 \u03bcB for [U(TRENTIPS)N]\u2013,v) complexes.36Complex (23Na NMR spectrum of (9) in THF revealed a single, very broad (\u0394\u03bd1/2 = 8300 Hz) resonance centred at ca. \u03b4 200 ppm suggesting that the interaction of the sodium cation with the paramagnetic uranium centre is maintained in solution (cf. (10), vide infra).The iii) complex [U{\u03b78-C8H6-2}(\u03b75-Cp*)THF] (2) affords the bridging \u03bc-oxo complex (6), the reaction of (2) with a slight excess of NaN3 (1.5 mol eq.) in a C7H8/THF solvent mixture (ca. 2\u2009:\u20091) was explored. Indeed, after work-up and re-crystallisation from THF/Et2O, brown-red crystals of the bridging nitride complex [{U[\u03b78-C8H62](\u03b75-Cp*)}2(\u03bc-N)]\u2013[Na(THF)6]+ (10) suitable for X-ray diffraction studies were isolated in 81% yield (\u03bc-N3)UCp*2]4,2(\u03bc-I)2}3(\u03bc3-N)] (2.152(3)\u20132.138(3) \u00c5). PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019U bonding interactions (1.95\u20132.12 \u00c5).9), that in (10) is significantly elongated as expected. The U\u2013N\u2013U bond in (10) has significantly deviated from linearity, which is a common structural motif for many bridging U nitride complexes(IV)\u2013N\u2013U(IV)]\u2013, [U(IV)\u2013N\u2013U(V)] and [U(IV)\u2013N\u2013U(VI)(O)]\u2013 complexes supported by bulky silyl amide ligands,tBu)3)]2 (106.1(2)\u00b0),10) is identical to the U\u2013O\u2013U bond angle found in the \u03bc-oxo complex {U[\u03b78-C8H62](\u03b75-Cp*)}2(\u03bc-O) (6)6) and that shortening might account for the slightly more acute Ct(COT)\u2013U\u2013Ct(Cp*) angles in (10) compared to the ones found in (6) (139.7(16)\u00b0 and 140.0(16)\u00b0).18The two U\u2013N bond lengths (N1\u2013U1 2.063(5) \u00c5, U2\u2013N1 2.066(5) \u00c5) in the anionic dimer are essentially the same, suggesting a delocalised [U \u2243 N \u2243 U] bonding interaction as in [Cp*10) readily loses its crystallinity due to loss of coordinated THF to yield [{U[\u03b78-C8H62](\u03b75-Cp*)}2(\u03bc-N)]\u2013[Na(THF)2]+ (10\u2032) as a well-defined product, as evidenced by microanalysis. As in the case of its \u03bc-oxo analogue [{U[\u03b78-C8H62](\u03b75-Cp*)}2(\u03bc-O)] (6), the 1H-NMR (C4D8O2) spectrum of (10\u2032) is consistent with a C2-symmetric structure that is retained in solution. In marked contrast to (9), the 23Na NMR spectrum of (10) in THF exhibited a sharp resonance (\u0394\u03bd1/2 = 78 Hz) at \u03b4 \u20137.94 ppm, parameters suggesting no interaction of the [Na(THF)6]+ counterion with the paramagnetic uranium anion.38Complex (2) with tBuNCO that yields the \u03bc-oxo complex (6), the bridging nitride complex (10) can be seen as the product of two 1e oxidations of the U(iii) precursor (vs. the one 2e oxidation that produces (9) in the case of the bulkier COT substituents), since the formal oxidation state of the uranium centres in (10) is +4. The \u03bceff for 10\u2032 was determined to be 3.64 \u03bcB for the dimer or 2.57 \u03bcB per uranium centre, a value consistent with a U(iv) ion (further details including SQUID magnetometry below).Similarly to the reaction of (\u03bceff for complexes (3), (9) and (10\u2032) at 300 K as determined in solution (Evans method), and in the solid state (SQUID under an applied field of 0.1 tesla); the values determined by these two methods are in fair agreement.3) exhibits a steady decline from the value of 2.16 \u03bcB at 300 K to 1.54 \u03bcB at 5 K terminal oxo complexes .The effective magnetic moment of (B at 5 K . This be9), its effective magnetic moment was found to be 2.00 \u03bcB at 300 K and 1.35 \u03bcB at 5 K (TIPS)][Na(12-crown-4)2] ,v) complexes more generally\u03c7m/T, \u03c7mT/T and \u03c7m\u20131/T).In the case of the nitride complex (B at 5 K . These v10\u2032) measured for zero-field cooled and field cooled samples coincided exactly, indicating the absence of long\u2013range interactions between spins on the two U(iv) centres. At 300 K the effective magnetic moment per U is 2.53 \u03bcB, and decreases to 0.69 \u03bcB at 2 K dianion [{3tacn)U}2(\u03bc-O)2]2\u2013 by Meyer et al. showed a \u03bceff per U of 2.73 \u03bcB at 300 K.\u03c7m) that obeys the Curie\u2013Weiss law, \u03c7m = C/(T \u2013 \u0398), where C is the Curie constant and \u0398 is the Weiss constant. The plot of \u03c7m\u20131vs. T 2](\u03b75-Cp*)}2(\u03bc-N)]\u2013 anion behaves as two non-interacting U(IV) centres. Furthermore, there is no maximum observed in the \u03c7mvs. T plot ((IV) ion (3H4 ground term) typically has minimal covalency, hence the two metal centres in 10\u2032 do not participate in exchange coupling.Magnetic susceptibility data sets for , (9) and (10\u2032) are presented in Finally, magnetic data for all three compounds (vi) complexes, the redox properties of (3), (9) and (10) were studied by cyclic voltammetry (C.V.).In order to gauge the potential for accessing terminal oxo and nitrido uranium(tBuArO)tacn)U(O)] complex reported by Meyer et al. that features a reversible oxidation,3) revealed only a quasi-reversible reduction process at \u20131.77 V vs. Fc+/0 did not alter the shape of the observed wave and no other processes were found to occur over the solvent window. This process is assigned to the [U(V)] \u2194 [U(IV)] couple, and based on this voltammogram, the reduction of (3) should be a chemically accessible process. Indeed, (3) can be chemically reduced with a slight excess of K/Hg (0.5% w/w) in the presence of 18-crown-6 in n-pentane/Et2O. The almost instantaneous reaction produced a red-pink solid that, after work-up and re-crystallisation from toluene, gave dark red rods suitable for X-ray diffraction studies which showed the product to be the U(iv) complex [U{\u03b78-C8H6-2}(\u03b75-Cp*)(\u03bc-O)K(18-c-6)] (11) (Changing the scan rate (50\u2013300 mV s6)] (11) .11) is longer than that in the U(v) complexes (3) (1.826(3) \u00c5) and [U(NR2\u2032)3O]iv) complex (4) (1.916(8) \u00c5). The K1\u2013O1 bond length is as expected shorter than the O PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019U PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O\u00b7\u00b7\u00b7K bonds (2.60\u20132.9 \u00c5)The U\u2013O bond length (1.891(4) \u00c5) in (11) was fully characterised by spectroscopic and analytical methods ), a value that is even more upfield than that for the U(iii) complex (1) (\u2013129 ppm), probably due to the anionic nature of (11).Complex in the anodic direction over several cycles revealed the existence of several processes in the accessible solvent window ] \u2194 [U(V)] couple. As can be seen from ca. \u20131.8 V vs. Fc+/0) is also present, which features an asymmetric current response that leads us to conclude that this is probably related to a short lived electrochemically generated species. The shape of the wave at \u20131.63 V did not change by variation of the scan rate (50\u2013350 mV s\u20131).C.V. scans of the nitride complex (s. Fc+/0 which we9) exhibits also another two irreversible processes: one anodic at 0.7 V and a cathodic one at \u20132.8 V (both vs. Fc+/0). The nature of these two irreversible processes cannot be unambiguously assigned, but they could be due to ligand activation involving the nitride moiety. Attempts to chemically oxidise (9) by reaction with mild oxidants such as I2 and AgBPh4 have thus far resulted in intractable mixtures from which only ligand decomposition could be observed spectroscopically (1H-NMR).In addition to this process, the (full) voltammogram of (10) revealed a quasi-reversible process (peak separation 87 mV) centred at \u20131.46 V vs. Fc+/0 (9) as well as for the {[U(IV)] PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019[U(IV)]}\u2013 \u2194 {[U(V)] PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019[U(IV)]} couple ([U] = U(NMestBu)3) reported by Cummins et al.(IV)]\u2013N\u2013[U(V)]} \u2194 {[U(IV)]\u2013N\u2013[U(IV)]} redox pair.Similarly, anodic scans of the bridging nitride ]\u2013N\u2013[U(V)]}, {[U(VI)]\u2013N\u2013[U(V)]} etc.), although other reasons (e.g. ligand activation) cannot be excluded. As in the case of (9) an irreversible reduction is also observed at ca. \u20132.5 V vs. Fc+/0 that as above could correspond to a mixed valence species (i.e. {[U(III)]\u2013N\u2013[U(V)]}) or arise from a ligand activation process. Given that similar processes appear in the case of (9), we envisage that they are more likely due to the latter rather than the former.Apart from this process, the voltammogram also displayed additional irreversible processes centred at anodic voltages or dinuclear U(iv) nitrido/oxo complexes depending on the size of the silyl substituents on the supporting ligands. This has led to the preparation of an anionic uranium(v) nitride complex (9) featuring a U\u2013N triple bond, as well as a neutral U(v) terminal oxo complex (3). Magnetic studies corroborate the formal oxidation states of these complexes further confirming that the 2e\u2013 oxidation leads to products featuring either one U(v) or two U(iv) metal centres depending on steric hindrance at the uranium centre. Cyclic voltammetry studies of complex (3) show that it can be readily reduced to the \u2013 anion (11), which has also been achieved chemically. Unlike (3), cyclic voltammetry studies have shown that the nitride complex (9) might be amenable to oxidation to the U(vi) species although initial attempts to do so have been unsuccessful thus far.In summary we have described how the steric environment around the metal centre can manipulate redox events at a uranium centre. This has been demonstrated by the isolation of either mononuclear U(Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "Polar and apolar boron-based triple bonds promote the single and double C\u2013H activation of acetone following similar coordination-deprotonation mechanisms. PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N and B PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019B triple bonds induce C\u2013H activation of acetone to yield a (2-propenyloxy)aminoborane and an unsymmetrical 1-(2-propenyloxy)-2-hydrodiborene, respectively. DFT calculations showed that, despite their stark electronic differences, both the B PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N and B PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019B triple bonds activate acetone via a similar coordination-deprotonation mechanism. In contrast, the reaction of acetone with a cAAC-supported diboracumulene yielded a unique 1,2,3-oxadiborole, which according to DFT calculations also proceeds via an unsymmetrical diborene, followed by intramolecular hydride migration and a second C\u2013H activation of the enolate ligand.B PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019NR\u2032 , PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N bond enables their participation in a vast array of spontaneous [2 + 2] cycloaddition6Due to their intrinsic electron deficiency, linear compounds containing a multiply bonded, sp-hybridised boron atom are far more reactive and difficult to isolate than isolobal carbon-based compounds. Owing to their ease of derivatisation, monomeric iminoboranes of the form RB PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019NR\u2032 compounds have been studied for over 30 years, isolobal LB PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019BL compounds displaying two dicoordinate, zero-valent boron atoms long eluded isolation. Since our report of the first stable diboryne, (IDip)B PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019B(IDip) -imidazolidin-2-ylidene),I are inert towards H2, PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019B(SIDep) -4,5-(dihydro)imidazolidin-2-ylidene), which is supported by saturated NHCs of intermediate \u03c0 acidity,2 at 80 \u00b0C to yield a 1,2-dihydrodiborene.MecAAC)\u00b7\u00b7\u00b7B PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019B\u00b7\u00b7\u00b7(MecAAC) -3,3,5,5-tetramethyl-pyrrolidin-2-ylidene),I and II, activates H2 at room temperature14While linear RB PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019NR\u2032 and LB PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019BL species B PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019NAr* 2-4-tBuC6H2); TMP = 2,6-tetramethylpiperidyl), towards acetone and show that, despite their marked electronic differences, compounds II and IV activate acetone following a similar mechanism, whereas cumulene II promotes an unprecedented spontaneous double activation of acetone.Intrigued by the seeming lack of reactivity overlap between isolobal linear RB species , we wereIV in hexanes with excess acetone overnight at 70 \u00b0C resulted in clean formation of the (2-propenyloxy)aminoborane 1 = 359.9(18)\u00b0) and N1 (B1\u2013N1\u2013C4 126.92(16)\u00b0) as well as the elongation of B1\u2013N1 to a single bond (1.424(3) \u00c5). The 2-propenyloxide ligand coordinated to B1 displays a C1\u2013O1 single bond (1.343(2) \u00c5) and a terminal C1 PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C2 double bond (1.320(3) \u00c5). Formally, compound 1 results from the addition of 2-propenol, the enol form of acetone, across the polar B PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019N triple bond of iminoborane IV. While there is, to our knowledge, no literature precedent for the reactivity of monomeric iminoboranes with enolisable ketones, the dimeric iminoborane 2 has been shown to undergo 1,4-enol addition to the ring-opened iminoborane dimer with acetone, acetophenone and 3,3-dimethylbutan-2-one.2,X-Ray crystallographic analysis of 1 confirms1.000000,.000000 sI proved unreactive towards acetone even under forcing conditions, diboryne II reacted rapidly with excess acetone in benzene at room temperature to yield the green-coloured 1,2-enol addition product 2 \u00c5 similar to that of its dihydrodiborene relative, (SIDep)HB PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019BH(SIDep) (1.589(4) \u00c5).3H5 moiety is twisted ca. 35.5\u00b0 out of the diborene plane and displays a pure \u03c3-donor interaction (B1\u2013C4 1.574(4) \u00c5). The planar 2-propenyloxide ligand lies at a ca. 58\u00b0 angle with respect to the diborene plane, and its bond lengths (O1\u2013C1 1.352(3), C1 PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C2 1.316(4) \u00c5) are similar to those of 1. With Kinjo and co-workers recently reporting the first diborene with two different donor ligands2 is only the second unsymmetrical diborene with respect to the anionic substituents.Whereas diboryne roduct 2 . Compoun2 at the (smd: n-pentane)lc-\u03c9PBE/6-311+g(d) level of theory provided a maximum UV-vis absorbance at 592 nm scale\" fill=\"currentColor\" stroke=\"none\">B double bond into the empty pz orbital of the carbene carbon of the SIDep ligand supporting the BOC3H5 moiety, and is responsible for the blue-green color of the compound.TDDFT calculations performed upon the optimised geometry of III with acetone did not yield the expected cAAC analogue of 2. Instead, 11B NMR data revealed a 92\u2009:\u20098 mixture of two sp2\u2013sp3 diborane products, the major one (3a) showing two broad singlets at 42.8 and \u20131.9 ppm (fwhm \u2248 130 Hz), and the minor (3b) presenting a very broad resonance at 63.0 ppm (fwhm \u2248 630 Hz) and a broad BH doublet at \u201315.0 ppm (1JB\u2013H = 50.8 Hz), suggesting a non-bridging hydride. The 1H NMR spectrum of the mixture showed very similar sets of resonances for 3a and 3b, which strongly suggests an isomeric relationship. Both compounds display one neutral cAAC ligand and one C1-protonated cAAC ligand as well as a single 1H alkene resonance . PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C2 double bond (1.3301(18) \u00c5, 2-bridged by a hydride (B1\u2013B2 B1\u2013H1 1.213(16), B2\u2013H1 1.485(17) \u00c5) positioned orthogonally to the B2C2O heterocycle 105.7(9)\u00b0) and shows a bond length of 1.721(2) \u00c5 typical of a diborane (5). The alkenylborane moiety around B1 is supported by a neutral cAAC ligand with a relatively short B1\u2013C4 bond (1.5295(19) \u00c5) and forms an angle of only ca. 19\u00b0 with the plane of the B2C2O heterocycle 14.4(2)\u00b0), which is indicative of \u03c0 conjugation. The enoxyborane moiety around B2 bears a protonated cAAC ligand displaying clear sp3-hybridisation at C24 (B2\u2013C2 4 1.6045(18), C24\u2013N2 1.4878(16) \u00c5). The structure of 3a is reminiscent of the products obtained from the reduction of (SIMes)BBr2BAr2 diborane (5) precursors . These display a central, \u03bc2-hydride-bridged, planar B2C5 heterocycle, resulting from the C\u2013H activation of one aryl substituent by an intermediate boraborylene, and coordinated on one side by a neutral SIMes ligand and on the other by the second aryl substituent.Single-crystal X-ray crystallography revealed a unique planar 2,3-dihydro-5-methyl-1,2,3-oxadiborole heterocycle displaying an endocyclic C1d do not fit the experimental data . Since 3a presents two stereocentres, one at B2, which is locked by the B2C2O ring and the asymmetrically bridging hydride, and one at the protonated cAAC carbon atom, the other possibility is that 3a and 3b could be diastereomers. This would also fit the observation that they do not exchange in solution even at high temperatures. To test this, the geometries and 11B NMR chemical shifts of the possible diastereomeric pairs derived from 3a were computed /-3a pair adequately match the experimentally-observed shifts \u2248 \u00b12 ppm). Calculations on the diastereomeric pair showed that a form with a non-bridging hydride is the most likely. This also correlates well with the observation that, unlike 3a, which shows two very broad 11B NMR resonances typical for a \u03bc2-hydride-bridged diborane, 3b shows a doublet at \u201315.0 ppm (1J11B\u20131H = 50.8 Hz), indicating a terminal hydride rather than a bridging one. The predicted 11B NMR chemical shifts for the /-3b pair are comparable to the experimental ones \u2248 \u00b13 ppm). The relative energy of /-3b, at 3.1 kcal mol\u20131 above /-3a, is consistent with the experimentally observed ratio of 92\u2009:\u20098.The predicted 3a/b is particularly remarkable in view of the fact that there is seemingly no literature precedent for a one-step, uncatalysed, 100% atom-efficient double C\u2013H activation of acetone or other enolisable ketones. We were therefore keen to investigate the mechanism of the formation of 3a/b and compare it to that of the boron enolates 1 and 2. While the reaction of dimeric iminoboranes with enolisable ketones always yielded the 1,4-enol addition products, Paetzold and co-workers showed that with acetophenone, which is less prone to enolisation, a [2 + 4] cycloaddition product can also be isolated.IV and diboryne II with acetone showed no evidence of [2 + 2] cycloaddition products or intermediates.The spontaneous formation of IV and at the ONIOM(M06-2X/6-311+G(d):PM6) level for II and III showed that acetone activation does not proceed via 1,2-enol addition, as the enol form of acetone lies 15.3 kcal mol\u20131 higher than the reactants, well above the activation energy for direct acetone addition level for IV two plausible mechanisms were investigated, the first via a 4,4-dimethyl-1,3,2-oxaboretidine [2 + 2] cycloaddition product (A), the second via concerted acetone coordination-deprotonation , 20.6 (II), 10.1 (III) kcal mol\u20131), respectively (IV) or the electron-rich, second boron centre (for II and III), to yield the cis-aminoborane 1I2, and the SIDep- and cAAC-supported cis-diborenes 2I2 and 3I2, respectively (\u0394G\u20212 = 4.0 (IV), 14.1 (II), 14.9 (III) kcal mol\u20131). Finally, the trans-aminoborane 1 and the trans-diborenes 2 and 3I4 are obtained by rotation around the B\u2013N and B\u2013B bond, respectively. Overall, the formation of 2 presents the highest energy barrier and is also the most exergonic , followed by that of 1 and 3I4 .Instead, for compounds ectively and 5. Tcis-diborenes 2I2 and 3I2 to the trans-diborenes 2 and 3I4, respectively, was further investigated to determine the rotation barrier in each case. Interestingly, DFT calculations showed two distinct mechanisms at work for the SIDep-stabilised and the cAAC-stabilised diborene, respectively scale\" fill=\"currentColor\" stroke=\"none\">B double bond into the \u03c0 backbonding to the unsaturated carbene ligands. The resulting transition state 2TS3 now displays a B\u2013B single bond, which allows facile rotation. The isomerisation process from 2I2 to 2 occurs with a low barrier of 9.7 kcal mol\u20131. In contrast, the lowest energy pathway for the cAAC analogue 3I2 proceeds via a 1,2-hydride shift from boron to the adjacent cAAC carbene carbon to yield the intermediate diborene 3I3 , in which the boron bearing the now protonated cAAC ligand is sp-hybridised. Rotation about this B\u2013CcAACH single bond and a second 1,2-hydride shift back to the boron centre then yield the trans-diborene 3I4 with a low barrier of 9.3 kcal mol\u20131. This pathway is assisted on the one hand by the facile 1,2-hydride shuttling chemistry displayed by cAAC hydroboron compounds3I3.The exergonic isomerisation step leading from the ectively . For theIII, however, the reaction does not stop at trans-diborene 3I4 . Coordination of the pendant terminal alkene to the two-coordinate boron yields adduct 3I5, which is 6.6 kcal mol\u20131 more stable. Subsequent C\u2013H activation of the methylidene moiety yields the bis(cAAC)-stabilised 1,2,3-oxadiborole 3I6 . This is the highest energy barrier in the entire reaction mechanism. 3I6 then tautomerises to compound 3a by concomitant migration of the hydride on B1 to the adjacent cAAC carbene centre and bridging of the hydride on B2 . Overall the formation of 3a from III and acetone is exergonic by 61.7 kcal mol\u20131, which explains why the intermediate diborene cannot be isolated.For cumulene rene I43 . The latIV, diboryne II and cumulene III, all activate acetone via a similar acetone coordination-deprotonation mechanism, regardless of their polar or nonpolar nature. For the iminoborane-based reaction, an enol addition mechanism and a mechanism proceeding via a [2 + 2] cycloaddition intermediate, as would normally be expected for such a polar compound, were both ruled out. For diboron compounds II and III the addition of acetone first yields a cis-diborene intermediate which isomerises to the thermodynamic trans-diborene product through a low energy barrier. Calculations showed that this isomerisation process heavily relies on the \u03c0-accepting nature of the carbene ligands, coupled, in the case of the cAAC-supported diborene, with a hydride shuttling mechanism from boron to the carbene carbon and back. These cAAC-specific properties also enable an unprecedented second C\u2013H activation of the enolate ligand to yield a novel 1,2,3-oxadiborole heterocycle, demonstrating once again the unique reactivity of cAAC-supported low-valent boron compounds.To conclude, we have shown that three linear, isolobal, multiply bonded boron compounds, iminoborane Overall this study should act as a reminder that the parallels all too eagerly drawn between organic compounds and their isoelectronic/isolobal inorganic p-block counterparts only rarely translate into actual organomimetic behaviour when it comes to reactivity or reaction mechanisms. Furthermore, this first example of reactivity overlap between polar and nonpolar boron-based triple bonds opens up new avenues for attempting reactions that may have been previously disregarded, such as the addition of nonpolar small molecules to iminoboranes or, alternatively, of polar molecules to diborynes.There are no conflicts to declare.Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "We report the synthesis and characterization of terminal uranium(iv) hydrosulfido and sulfido complexes, supported by the hexadentate, tacn-based ligand 3tacn3\u2013. iv) hydrosulfido and sulfido complexes, supported by the hexadentate, tacn-based ligand framework 3tacn3\u2013 -5-methyl-2-hydroxybenzyl)-1,4,7-triazacyclononane). The hydrosulfido complex [3tacn)U\u2013SH] (2) is obtained from the reaction of H2S with the uranium(iii) starting material [3tacn)U] (1) in THF. Subsequent deprotonation with potassium bis(trimethylsilyl)amide yields the mononuclear uranium(iv) sulfido species in good yields. With the aid of dibenzo-18-crown-6 and 2.2.2-cryptand, it was possible to isolate a terminal sulfido species, capped by the potassium counter ion, and a \u201cfree\u201d terminal sulfido species with a well separated cation/anion pair. Spectroscopic and computational analyses provided insights into the nature of the uranium\u2013sulfur bond in these complexes.Herein, we report the synthesis and characterization of a series of terminal uranium( PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019E compounds , enabling a more detailed insight into the electronic structure and degree of covalency in this structural motif.The anhydrous coordination chemistry of the light actinides has become an active field of research since the discovery of suitable starting materials.iii) to undergo one electron oxidation resulting in dinuclear, sulfido-bridged diuranium(iv/iv) complexes rather than stabilizing the terminal sulfido ligand, S2\u2013.iv) hydrochalcogenido complexes employing H2E as the chalcogenido ligand source.Ad,MeArO)3N)U\u2013SH(DME)] 3N3\u2013 = trianion of tris(2-hydroxy-3-(1-adamantyl)-5-methylbenzyl)amine). Due to their potential application as catalysts, transition metal hydrochalcogenido complexes have received considerable interest in recent years.iv) hydroxo complex, namely [3mes)-U\u2013OH], was found to be the key intermediate in the electrocatalytic production of dihydrogen from water.9In contrast to the rapidly increasing number of reported terminal uranium oxo complexes,iii) complexes [3tacn)U] 3tacn3\u2013 = trianion of 1,4,7-tris--1,4,7-triazacyclononane) and [3N)U(DME)] 3N3\u2013 = trianion of tris(2-hydroxy-3-(1-adamantyl)-5-methylbenzyl)amine) efficiently activate the elemental chalcogens 2E.via (poly-)chalcogenido as well as bis-hydrochalcogenido bridges was observed.Ad,RArO)3tacn)3\u2013 .iii) precursor [3tacn)U] (1) 3tacn3\u2013 = trianion of 1,4,7-tris(3-(1-adamantyl)-5-methyl-2-hydroxybenzyl)-1,4,7-triazacyclononane) allowed for synthesis of the here reported monomeric uranium (hydro-) chalcogenido complexes. More importantly, the bulky adamantyl groups effectively prevent dimerization upon deprotonation of the SH\u2013 ligand; thus, yielding the targeted uranium terminal sulfido complex for direct comparison to the bonding situation in U complexes with \u03b71-SH and \u03b71-S ligands. The presence of crown ethers or cryptands in the deprotonation step not only increases the solubility of the formed metal salts, but additionally allows for the quantitative evaluation of the bonding situation in a U\u2013S\u2013H versus a U\u2013S\u00b7\u00b7\u00b7K complex.We previously demonstrated that the uranium(iii) complex [3tacn)U] (1) with various S atom transfer reagents, such as Ph3P PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019S or elemental sulfur, does not yield terminal U PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019S complexes. Either a reaction was not observed at all or an intractable mixture of compounds without any isolable product was received. Finally, the synthesis of terminal uranium(iv) hydrosulfido and sulfido complexes was successfully achieved by treatment of complex 1 with one equivalent of H2S. The dropwise addition of 0.8 M H2S in THF to a red-brown solution of 1 in THF reproducibly affords the uranium(iv) hydrosulfido complex [3tacn)U\u2013SH] (2) in excellent yields with concomitant evolution of H2 gas . The mononuclear complex [3tacn)U\u2013SH] exhibits a seven-coordinate uranium ion in a face-capped octahedral coordination environment (2 were determined to be 2.844(4) and 2.775(2) \u00c5, respectively. This is in good agreement with other reported uranium\u2013sulfur single bonds (2.588(1)\u20132.907(3) \u00c5)\u2013 ligand is situated on the C3 axis of the molecule in the axial position, trans to the tacn anchor. Since the chalcogen-bound H atom could be located in the difference Fourier map, the U\u2013S\u2013H angle was determined to be 152\u00b0 and 156\u00b0, respectively. The U\u2013Oaryloxide distances are 2.152(4) \u00c5 and 2.188(3) \u00c5, respectively, and the U\u2013Ntacn bond lengths are 2.680(5) \u00c5 and 2.650(4) \u00c5. The uranium out-of-plane shift (Uoop), defined by the displacement of the uranium ion below the plane of the three aryloxide oxygen atoms, was measured to be \u20130.282 and \u20130.268 \u00c5, respectively. All these parameters are in good agreement with other uranium(iv) complexes supported by the 3tacn3\u2013 ligand system .Complex ironment .47 The Uiv) sulfido species, complex 2 was treated with potassium bis(trimethylsilyl)amide in THF to deprotonate the \u2013SH moiety. In order to encapsulate the potassium counterion, the reaction was performed in the presence of either dibenzo-18-crown-6 or 2.2.2-cryptand 3tacn)U PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019S\u00b7\u00b7\u00b7K(db-18-c-6)] (3) and [K(2.2.2-crypt)] (4) were carried out. The uranium(iv) sulfido complex 3\u00b70.62 benzene\u00b70.38 Et2O crystallizes in the monoclinic space group P21/c with one molecule per asymmetric unit, whereas 4 crystallizes in the chiral, hexagonal space group P63 with a third of one independent molecules per asymmetric unit. Both the uranium complex and the [K(2.2.2-crypt)] moiety were found on a crystallographic threefold axes. As anticipated, the sulfido ligand of the uranium(iv) complex (3) is capped by the [K(db-18-c-6)]+ cation, whereas complex [K(2.2.2-crypt)] (4) features a genuine terminal sulfido ligand with the [K(2.2.2-crypt)]+ cation in the outer coordination sphere of the complex anion . Single-ex anion .2, UIV complex 3 features a seven-coordinate uranium ion with the sulfido ligand occupying the axial position. The S\u2013K distance is 3.136(1) \u00c5, demonstrating a bonding interaction between the S2\u2013 ligand and the K+ counter ion 3tacn)U\u2013SH] av = 2.810(4) \u00c5), but slightly longer than those of other reported uranium(iv) sulfido complexes (2.442(2)\u20132.4805(5) \u00c5).1H NMR spectrum of 3 reveals a C3-symmetrical molecule in solution (vide infra), coordination of the [K(db-18-c-6)]+ crown ether leads to a loss of C3 symmetry in the crystal structure. The average U\u2013Oaryloxide distance of 2.219 \u00c5 and the mean U\u2013Ntacn bond length of 2.819 \u00c5 are slightly longer compared to UIV hydrosulfido complex 2. Interestingly, the U out-of-plane shift (Uoop) significantly decreases from \u20130.275 in 2 to \u20130.055 \u00c5 in 3; hence, the uranium center is positioned almost perfectly in the plane of the three oxygen donors. This observation is quite unusual for uranium(iv) ions in the tacn-based ligand system, and is typically only seen for high-valent UV and UVI complexes with strong \u03c0-donor ligands, such as the oxo and isoelectronic imido functionality.3 (vide infra).Like complex nter ion , left. T3O3S ligand donor set in the anionic complex \u2013 (4)\u2013 is analogous to that found for complex 3. In the case of 4, however, the potassium cation is encapsulated by the sterically encumbered 2.2.2-cryptand and located in the outer coordination sphere of the anionic UIV complex, leading to a discrete ion pair with isolated [K(2.2.2-crypt)]+ cations and \u2013 anions (4 exhibits a slightly longer uranium\u2013sulfido distance of 2.536(2) \u00c5 and\u2014along with the longer U\u2013S distance\u2014a slightly but noticeably larger Uoop of \u20130.086 \u00c5 compared to 3 (d(U\u2013S)av = 2.507(1) and Uoop = \u20130.055 \u00c5). It is suggested that the diphenyl-18-crown-6 moiety exerts a considerable steric strain that might push the sulfur atom slightly deeper into the cavity of the [((AdArO)3tacn)U] moiety, while at the same time, the uranium reduces its negative out-of-plane shift and moves closer to the sulfur atom in order to accommodate the sterically demanding potassium diphenyl-18-crown-6 moiety in the complex periphery. In addition, the seven-coordinate uranium center is chiral with an idealized C3 symmetry, affording a racemate of complex 4. After crystallization, a conglomerate of enantiomerically pure crystals was found for 4 with an A-configuration of the uranium center in the analyzed crystal.2\u20134 are stable in the solid form or in THF solution for at least 3 weeks without any notable decomposition.The connectivity of the N\u2013 anions , right. 1H NMR spectroscopy shows that compounds 2\u20134 possess C3 symmetry in solution, induced by coordination of the tacn ligand to the metal center with the hydrosulfido/sulfido ligand situated in the axial position on the C3 axis in order to investigate the complexes' symmetry at low temperatures. Upon cooling the 1H-NMR signals broaden and shift, but coalescence is not observed. The VT-NMR experiments thus suggest that, in solution, the crown ether complexated potassium ion remains in the vicinity of the paramagnetic uranium complex anion. It is further suggested that the potassium crown ether moiety fluctuates around the anion's threefold axis faster than the NMR timescale; even at very low temperatures. As a consequence, the U\u2013S bond distance of 3 relaxes in solution, leading to a weaker uranium\u2013sulfur bonding interaction .s see ESI. Hence, 2\u20134 was studied by UV/vis near-infrared spectroscopy. In the high-energy region (\u03bb < 600 nm) broad and rather intense ligand-based absorption bands as well as charge-transfer transitions are observed. In the UV region of the spectra, all three complexes exhibit an absorption band at 298 nm with an extremely high extinction coefficient ; 300 M\u20131 cm\u20131 (4)), absent in 2. This absorption band is most likely due to a metal to ligand charge-transfer transition (MLCT) of a metal-centered 5f electron into a sulfido-based orbital.3 and 4 compared to the pale blue-green color of 2. The visible near-IR electronic absorption spectra of complexes 2\u20134 in pyridine (5 mM) are shown in iv) complexes possess a 5f2 electron configuration, and therefore display rather complicated electronic absorption spectra with multiple low-intensity absorption bands and fine structure in the vis/NIR region.\u03b5 = 6\u2013116 M\u20131 cm\u20131) give rise to signature absorption bands characteristic of tetravalent uranium complexes. This is particularly true for a series of complexes, in which the symmetry around the metal center remains constant and the core structure is dominated by the ((ArO)3tacn)3\u2013 chelate, and thus, quite similar.3 and 4 exhibit 10 absorption bands with nearly identical absorption patterns in the vis/NIR region between 540 and 2100 nm, with three relatively strong absorption peaks at around 990, 1111, and 1990 nm. Noticeably, the molar extinction coefficients observed in the spectra of the separate ion pair 4 are consistently larger than those of complex 3, with the capped terminal sulfido ligand. The NIR spectra have been reproduced multiple times and the differences in extinction coefficient are significantly larger than the experimental error. Further inspection of the NIR spectra reveals approximately equal line width for the absorption bands in complexes 3 and 4; thus, excluding an intensity stealing mechanism.3: FWHM994 nm = 26 nm, FWHM1111 nm = 40 nm, FWHM1998 nm = 106 nm; complex 4: FWHM991 nm = 24 nm, FWHM1111 nm = 38 nm, FWHM1990 nm = 76 nm.\u2021Complex A reduced symmetry also cannot account for different extinction coefficients in 3 and 4, since both possess C3 symmetry in solution (as established by (VT) 1H NMR spectroscopy, vide supra). However, it is worth noting that the timescale of electronic absorption spectroscopy is significantly shorter compared to proton NMR spectroscopy, therefore complex 3 could lose its C3 symmetry. Regardless, in the latter case, absorption bands of complex 3 should be more intense than those of complex 4. Since the intensity of an electronic absorption band in the NIR region is considered indicative of the degree of covalency of the uranium ligand multiple bond in a conserved ligand field,iv) sulfido complex, 3. This is in contrast to the shorter U PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019S bond observed in the solid-state structure of 3 implying a stronger, more covalent bond compared to 4. Therefore, one can only conclude\u2014and reiterate\u2014that the mere bond distance is not a valid measure of covalency.The electronic structure of complexes Fig. S14. This spgion \u03bb < 0 nm broa2 shows about the same number of absorption bands as 3 and 4, but the f\u2013f transitions occur at slightly different energies and a charge-transfer transition is not observed. Additionally, the intensities of the bands are significantly lower (\u03b5 = 6\u201348 M\u20131 cm\u20131) and, in accordance with the lack of charge-transfer transitions, indicate the presence of a ligand with predominantly \u03c3-donor character.Hydrosulfido complex 1 and tetravalent 2\u20134 from 2 to 300 K (\u03bcS) and total angular momentum approximations (\u03bcJ), respectively, there is currently no approximation to predict the magnetic moment for actinide coordination complexes, since ligand-field effects cannot be ignored and spin\u2013orbit coupling is large.2 valence electron configuration, which results in a non-magnetic ground state at very low temperatures; and consequently, strongly temperature-dependent magnetic moments, \u03bceff, with values typically ranging from 0.3 \u03bcB at 2 K to 2.8 \u03bcB at room temperature.SQUID magnetization measurements were carried out to study the temperature behavior of trivalent to 300 K bottom. III ions (f3) possess a half integer spin with a doublet, EPR-active ground state , but the low temperature effective magnetic moment together with an EPR signal confirms a trivalent uranium ion in complex 1 andomplex 1 top.2\u20134 possess nearly the same magnetic moment with 2.85 \u03bcB, 2.90 \u03bcB, and 2.87 \u03bcB, respectively, but show significantly different temperature-dependent behavior. These results support the notion that the room temperature magnetic moments cannot be used to determine the oxidation state of the uranium ion, since trivalent 1 at room temperature shows nearly the same (or even slightly lower) magnetic moment as tetravalent 2\u20134. At 2 K, however, uranium(iv) complexes with the f2 ion typically show distinctively lower magnetic moments, which are due to the ions' non-magnetic singlet ground state.2 exhibits temperature-dependency overall typical for a uranium(iv) compound. The low magnetic moment, \u03bceff, of 1.03 \u03bcB at 2 K continually increases with increasing temperature. On the contrary, sulfido complexes 3 and 4 reveal an unusually strong temperature-dependency in the range of 2 to 50 K, with a subsequent moderate increase from 50 to 300 K. Notably, complex 3 shows a typically low magnetic moment of 0.84 \u03bcB at 2 K, whereas complex 4 possesses an unusually high \u03bceff value of 1.84 \u03bcB. Despite this high magnetic moment, complex 4 is EPR silent . Similar high magnetic moments have been observed for UIV complexes with separate ion pairs like [Cp*2Co]-[U(O)(N(SiMe3)2)3],5H10)5],4]-[U(CH2tBu)5], and [Li(DME)3]-[U(CH2SiMe3)5].67At room temperature, complexes 2 possesses a more isolated magnetic ground state, where the higher magnetic states slowly become thermally accessible with increasing temperature. Hence, the low-lying magnetic states of complexes 3 and 4 appear to be closer in energy, and are already thermally accessible at temperatures below 50 K. Consequently, the magnetic moment increases rapidly from 2 to 50 K, and merely increases with increasing temperatures above 50 K. The intriguing difference in the temperature dependency of the magnetic moments of complexes 2\u20134 is due to the different crystal-field-splitting caused by the purely \u03c3-type SH\u2013versus the \u03c3- and \u03c0-type S2\u2013 ligands.67On the other hand, complex 3 in THF in the presence of \u223c0.1 M [N(n-Bu)4][BPh4] electrolyte and the ferrocenium/ferrocene redox couple (Fc+/Fc) acting as internal standard. The cyclic voltammogram of 3 reveals a quasi-reversible redox process at a half-step potential, E1/2, of \u20131.494 V (iv/v) redox couple, with the half-step potential in the range of other published UIV/V couples (\u20131.81 to 0.12 V vs. Fc+/Fc).nP,MeArO)3tacn)U}2(\u03bc-O)2] (nP = neopentyl) shows a comparable UIV/V couple at a half-step potential of \u20131.55 V (vs. Fc+/Fc).50Cyclic and linear sweep voltammetry were performed on \u20131.494 V . A posit\u20131.494 V . Accordi2 and 4 in polar solvents, such as THF, cyclic voltammetry experiments could not be performed with these complexes. Given the lack of characterized terminal uranium(v) sulfido complexes in the literature, and the expectation that the covalency of the uranium\u2013chalcogenide bond increases with increasing valence,3 is desirable. Unfortunately, all attempts to chemically oxidize 3 and 4 have not yet been met with success and resulted in decomposition of the compounds.Due to the poor solubility of 2\u20134. Geometry optimizations were conducted on [3tacn)U\u2013SH] (2), (3), and [K(2.2.2-crypt)] (4) at the DFT level without any symmetry constraints. Subsequently, molecular orbital (MO) and natural bond orbital (NBO) analyses were performed.In order to gain further insight into the nature of the U\u2013S bond, theoretical investigations were carried out on complexes 2. The NBO analysis of 2 clearly indicates a single bond between U and S and a single bond between S and H (Wiberg bond indices (WBI) of 0.77 and 0.92, respectively). Accordingly, the molecular orbitals are consistent with a single U\u2013S bond (iv) hydrochalcogenido complex, namely [((tBuO)3SiO)4U(SH)]\u2013 obtained by Andrez et al., exhibits significant double bond character of the uranium sulfur interaction (determined by MO and WBI).2 and [((tBuO)3SiO)4U(SH)]\u2013, these two complexes were analyzed in more detail.Initially, bond analysis was carried out on the hydrosulfido species U\u2013S bond , reveali2 is strongly polarized with 10% uranium and 90% sulfur orbital character. The metal orbital is a hybrid sdf orbital with 12% 7s, 38% 6d, and 50% 5f contribution. This is comparable to the hybrid orbital composition of the hydrosulfido complex [((tBuO)3SiO)4U(SH)]\u2013 exhibiting a \u03c3 (and \u03c0) orbital with 14% uranium character (12% for the \u03c0) and a strongly hybridized orbital . As evidenced by the X-ray structure, the geometry of 2 differs significantly from the trigonal bipyramidal complex [((tBuO)3SiO)4U(SH)]\u2013. The computational analysis suggests that the pyramidalized uranium ion of 2 has an efficient overlap with the N donor atoms of the tacn ring. This, in turn, results in a trans-effect reducing the U\u2013SH bond strength, which is rather unusual for uranium complexes. In order to emphasize the importance of the trans-influence of the tacn ligand, a hypothetical tris(aryloxide) complex, 2* (without the triazacyclononane ligand) was also computed. Interestingly, this model complex adopts a tetrahedral geometry at the uranium center, and a U\u2013S double bond character is found (see ESIThe U\u2013S \u03c3-bond of complex iv) complex 3 clearly reveals a formal U PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019S triple bond with one \u03c3 and two \u03c0-type interactions (3 (and 4) illustrates that the uranium ion is situated almost perfectly in the trigonal plane of the three aryloxides with a weaker U\u2013Ntacn interaction and more efficient uranium\u2013sulfur orbital interaction resulting in the observed U\u2013S multiple bond. NBO analysis shows that the U\u2013S bond is strongly polarized with more than 75% charge on the sulfur. A \u03c3-bond is formed by an sp orbital of sulfur (77%) and a dz2/fz3 hybrid orbital of uranium (23%), and two \u03c0 orbitals are formed by the interaction of a p lone pair of sulfur and a hybrid d\u03c0/f\u03c0 orbital of uranium (23%). This formal uranium sulfur triple bond is virtually unaffected by the minor interaction of the sulfido ligand with the potassium counterion (WBI of 0.1). To further substantiate the effect of the weakly associated K+ ion in 3, the bonding analysis of 4 with an encrypted and well-isolated potassium ion was carried out. As expected, a triple bond between uranium and the sulfido ligand was found with the orbitals closely resembling those of 3 . The experimentally determined U\u2013S bond length of 4 (without the S\u00b7\u00b7\u00b7K interaction) is elongated compared to 3. However, this result is not reproduced by the calculations that show the bond in 3 to be slightly longer (0.02 \u00c5) than in 4 in 3 leads to a stronger negative charge on the sulfido ligand (\u20130.1 unit difference), which is formally interacting with two positively charged ions. Since the charge at the uranium ion is the same for 3 and 4, the coordination of the potassium ion leads to a higher negative charge on the sulfido ligand in 3, counterbalancing the charge. Consequently, a higher charge on the sulfido ligand in complex 3 leads to a smaller orbital overlap, and therefore less covalent interaction. In order to determine the nature of this discrepancy between experiment and theory, calculations were carried out on the putative anionic complex [3tacn)U(S)]\u2013 (4\u2013). The bonding analysis confirmed the negligible influence of the K+ ion on the electronic structure of the U PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019S bond of 3 and 4, but not on the U\u2013S bond length . Complex 4\u2013 possesses the shortest U\u2013S distance, in line with the influence of the K+ bonding, but contrary to the bond lengths observed in the solid state . Perrin et al. reported a similar effect for the distorted geometry of an amido lanthanide complex.71In the calculation, a weak S\u00b7\u00b7\u00b7K interaction scale\" fill=\"currentColor\" stroke=\"none\">S bond is obtained, which is strongly polarized towards S (between 70 and 75%) with hybrid s/d orbital involvement of the metal (roughly 80% 6d). Interestingly, the nature of the U PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019S bond of complexes 3 and 4 appears to be quite similar to other computed uranium(iv) chalcogenido complexes with different supporting ligand systems. PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019S entity, regardless of whether aryl-oxide, siloxide, or supporting amide ligands are applied. In all reported complexes, the geometries at the uranium center are either distorted tetrahedral or trigonal pyramidal. Quite surprisingly, the atomic 5f and 6d orbitals experience a very similar ligand field effect in all complexes.Based on all of these results, we assign a significant degree of covalency to the U\u2013S bond of complexes 3 and 4\u2013 were carried out. Based on the report by Andersen on a Cp*2UO compound, a more ionic bonding description can be expected for the oxo complex.\u20131, in line with a strong agostic interaction, see Table S4 ESIIn order to investigate the possible influence of the chalcogenido ligand, the bonding analyses of the oxo-homologs of 2\u20134, supported by the 3tacn3\u2013 ligand system. Proton NMR spectroscopy reveals C3 symmetry of the complexes in solution, and the vis/NIR electronic absorption spectroscopy, together with the SQUID magnetization measurements, allow for the unambiguous assignment of the uranium ion to the +IV oxidation state. The differences in temperature-dependency of complexes 3 and 4 at low temperatures (T < 50 K) also suggest a significant influence of the potassium counter ion on the crystal field splitting of the terminal sulfido complexes as well as the nature of the U\u2013S bond. DFT computational analyses further provided detailed insight into the bonding properties of complexes 2\u20134, and reveal a non-negligible degree of covalency in the uranium\u2013sulfur bond of 3 and 4. This is supported by the complexes\u2019 structural parameters, vis/NIR electronic absorption spectroscopy, and SQUID magnetometry. The electrochemical studies show that complex 3 can be electrochemically oxidized, most likely to a UV PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019S species, which is expected to exhibit an even greater degree of covalency of the uranium sulfur bond.v) sulfido complex led to decomposition products.In summary, we here present a new and high-yield synthetic protocol and the characterization of the terminal uranium hydrosulfido and sulfido complexes iv) complexes with terminal hydrochalcogenido and chalcogenido ligands is part of our on-going studies.The synthesis of a complete series of uranium(Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "The electronic effects induced by the synergy of stable C12A7:e\u2013 electride and bimetallic Ru\u2013Fe nanoparticles efficiently control the chemoselective reduction reaction. 24Al28O64]4+\u00b7(e\u2013)4, with extremely low work function, promotes the superior activity and selectivity of a Ru\u2013Fe nano-alloy for the conversion of \u03b1,\u03b2-unsaturated aldehydes to unsaturated alcohols in a solvent-free system. The catalyst is easily separable from the product solution and reusable without notable deactivation. Mechanistic studies demonstrate that electron injection from the electride to the Ru\u2013Fe bimetallic nanoparticles promotes H2 dissociation on the highly charged active metal and preferential adsorption of C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O bonds over C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019Cs bond of the unsaturated aldehydes, to obtain the thermodynamically unfavorable but industrially important product.Controlling the electronic structure of heterogeneous metal catalysts is considered an efficient method to optimize catalytic activity. Here, we introduce a new electronic effect induced by the synergy of a stable electride and bimetallic nanoparticles for a chemoselective reduction reaction. The electride [Ca Numerous efforts have been devoted to achieving a high yield of required chemicals without any byproduct using a heterogeneous catalyst.24Al28O64]4+\u00b7(e\u2013)4 (C12A7:e\u2013) is an inorganic electride with anionic electrons in the positively charged framework ([Ca24Al28O64]4+) that was created in 2003.\u2013 electride is its low work function (2.4 eV), comparable to that of alkali metals, but with higher chemical inertness, which makes this material a promising electron donor in chemical reactions.\u2013 alone, or in combination with Ru, functions as an effective catalyst in chemical reactions, such as ammonia synthesis and decomposition, and CO2 splitting.\u2013 as a catalyst for liquid phase reactions because they are chemically unstable in aqueous media and tend to release electrons into the solvent or moist environment. The C12A7:e\u2013 electride has recently been used as an electron generator in aqueous solution to facilitate the pinacol coupling reaction of aldehydes, and the chemoselective reduction and oxidation of ketones.Electrides are ionic crystals with cavity-trapped electrons that act as anions. Ca24Al28O44+\u00b7(e\u2013)4e\u2013)4 C12A:e\u2013 is an PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond over the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O bond is both thermodynamically and kinetically favored.e.g., the adsorption of a polar reactant is enhanced by a non-polar solvent, and vice versa.16Chemoselective hydrogenation of \u03b1,\u03b2-unsaturated aldehydes to the corresponding unsaturated alcohols is difficult but fundamentally important in both chemical research and industry.1.000000,.000000 s\u2013 electride-supported Ru\u2013Fe alloy nanoparticles (Ru\u2013Fe/C12A7:e\u2013) can act as a highly efficient and selective heterogeneous catalyst for the liquid phase chemoselective hydrogenation of \u03b1,\u03b2-unsaturated aldehydes under solvent-free conditions. After reaction completion, the solid Ru\u2013Fe/C12A7:e\u2013 can be easily separated from the reaction mixture and reused without decrease in catalytic efficiency. Detailed characterization indicates that the electronic effect induced by the electron donation of the C12A7:e\u2013 electride support and the charge transfer between nano-alloy particles is responsible for the superior catalytic performance for chemoselective hydrogenation. To the best of our knowledge, this is the first report on an electride-based heterogeneous catalyst in a liquid catalytic reaction with a solvent-free system.In this work we demonstrate that C12A7:e\u2013 catalysts were fabricated initially by conventional solid-phase reaction and subsequent chemical vapor deposition (CVD) of metal carbonyl complexes in a vacuum patterns .\u2013. X-ray photoelectron spectroscopy and Ru/C12A7:e\u2013 exhibited poor activity and selectivity for cinnamyl alcohol of C12A7:e\u2013 as a support . As expected, all substrates were quantitatively reduced to their corresponding alcohols under optimized catalytic conditions. The reaction was sensitive to steric hindrance of the substituents on the alpha sites, and as a result trans-2-methyl-2-pentenal and trans-2-methyl-2-butenal exhibited relatively low conversion under relatively rigorous conditions , but still with high selectivity. In contrast, the activity of the \u03b1,\u03b2-unsaturated ketone hydrogenation reaction was quite poor due to steric hindrance from the methyl group in the carbonyl component high-angle annular dark field scanning transmission electron microscopy (HAADF-STEM). \u2013 electride, which is consistent with scanning electron microscopy (SEM) analysis , which is consistent with the results of XRD measurement to understand the interaction between Ru and Fe in the alloy. As shown in ca. 140 \u00b0C, which is assigned to the reduction of RuO2.x species.2 into C12A7:e\u2013 cages as H\u2013 ions during the high temperature stage.8The reducibility of the catalysts was investigated at various metal weight ratios using hydrogen temperature-programmed reduction (H\u2013 electride was examined by diffuse reflectance infrared Fourier transform (DRIFT) spectroscopy using CO as a probe molecule. As shown in 2\u2013 exhibits main peaks at around 2100\u20132000 cm\u20131, which can be assigned to the C\u2013O stretching vibration of linearly adsorbed CO on Ru0 sites, indicating that there is no electron transfer from C12A7:O2\u2013 to the Ru metal nanoparticles. This is due to the fact that C12A7:O2\u2013 is a typical insulator material, in which O2\u2013 ions are incorporated as counter anions to the positively charged [Ca24Al28O64]4+ lattice framework composed of subnanometer-sized cages.\u20131 corresponds to tricarbonyl species on partially oxidized Ru sites.2\u2013 has no adsorption peaks, which indicates that the interaction between CO molecules and Fe is quite weak after Fe addition. The red-shift of the CO signal is clear evidence of the electronic interaction between Ru and Fe species. Electropositive Fe metal generally acts as an electron-donating ligand that increases the electron density of Ru, thereby favoring the back-donation of electrons to the 2\u03c0* antibonding orbitals of CO, which accounts for the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O bond weakening (red-shift of the CO stretching band). In contrast, the electron density of Fe atoms decreases due to the electron transfer from Fe to Ru. A similar electron transfer effect is demonstrated on a model surface of Pt80Fe20(111) by H\u00fcckel calculations. PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O bond weakening on both Ru/C12A7:e\u2013 and Ru\u2013Fe/C12A7:e\u2013. Compared to Ru/C12A7:O2\u2013 and Ru\u2013Fe/C12A7:O2\u2013, a clear red-shift is observed for the linear Ru0\u2013CO peak (2030 cm\u20131) of Ru/C12A7:e\u2013 and for that (2020 cm\u20131) of Ru\u2013Fe/C12A7:e\u2013, respectively scale\" fill=\"currentColor\" stroke=\"none\">O bond of a CO molecule adsorbed on Ru/C12A7:e\u2013 and Ru\u2013Fe/C12A7:e\u2013 is weakened by the electrons encaged in C12A7:e\u2013, which has an extremely low work function and metallic conductivity.\u2013 electride.The electron donation capabilities of Ru\u2013Fe nanoparticles on the C12A7:eharged CaAl28O644+ Fig. S10. Notablyectively , which i\u2013 is shown in PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C and C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O bonds can interact with the metal surface and the adsorption mode is strongly dependent on the surface of the metal catalyst. In our case, the electronic nature of the metal nanoparticles can be modified by the electron donation from C12A7:e\u2013, leading to the marked increase in the hydrogenation selectivity. The electron enrichment of the metal surface decreases the binding energy of the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond via increased repulsive interaction between metal d-orbitals and the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond, favoring a vertical adsorption configuration via the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O bond scale\" fill=\"currentColor\" stroke=\"none\">O bond. Additionally, electropositive Fe sites are formed in the Ru\u2013Fe bimetallic system as demonstrated in FT-IR analysis scale\" fill=\"currentColor\" stroke=\"none\">O bond via the lone electron pair of the oxygen atom, which leads to a weakening of the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O bond. PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O bonds is thus enhanced compared with the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bonds. There are two possible routes to cleave H2. H2 dissociation occurs preferentially on the Ru surface due to the low dissociation barrier of H2 on Ru, which results in the formation of nonpolar hydrogen species via homolytic cleavage of H2 scale\" fill=\"currentColor\" stroke=\"none\">C and C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O bonds, with the hydrogenation of C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond being thermodynamically favorable. However, the hydrogenation of C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O bond selectively occurs over Ru\u2013Fe/C12A7:e\u2013 because the unsaturated aldehyde molecules are adsorbed on the catalyst surface via the vertical configuration.The proposed reaction mechanism for the chemoselective hydrogenation of \u03b1,\u03b2-unsaturated aldehydes over Ru\u2013Fe/C12A7:eO bond .28 This O bond . Zero oranalysis . The Fe ge of H2 , Route 12 dissociation (H2 \u2192 H\u2013 + H+) at the interface between a metal and basic support, which would subsequently enhance selective hydrogenation reactions.4 and LiAlH4via nucleophilic attack by H\u2013 species to the positively charged carbon of the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O bond.\u2013, it can be expected that H\u2013 species are formed on electron-rich metal sites and H+ is simultaneously formed on a framework oxygen at the surface of C12A7:e\u2013. The resulting H\u2013 and H+ species react with the carbon and oxygen sites of C\u03b4+ PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O\u03b4\u2013 bonds, respectively scale\" fill=\"currentColor\" stroke=\"none\">C bonds. Although the detailed mechanism remains to be clarified at this stage, it is important to emphasize that electron transfer between electride and metal nanoparticles could be the key factor in the chemoselective reduction of unsaturated aldehydes.Another possibility is heterolytic Hectively , Route 2\u2013 electride were constructed to achieve highly efficient solvent-free hydrogenation of \u03b1,\u03b2-unsaturated aldehydes. The intrinsically low work function C12A7:e\u2013 injects electrons into the active Ru\u2013Fe nanoparticles, which leads to the formation of the unsaturated alcohol with outstanding catalytic activity and high selectivity, without the need for basic additives in the reaction solution. The synergistic effect of the alloy metal Ru and Fe offers atom-scale electron transfer to activate Ru and induces electrophilic activation towards C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O, both favoring chemoselective hydrogenation. The electride-based catalyst also exhibited excellent sustainability and superior chemoselectivity of over 95% during long-term cycling. These results could serve as inspiration for the further exploitation of electride-based metal or alloy catalyst interactions in the design and synthesis of novel heterogeneous catalysts. We consider that a family of electrides with high electron donation ability could find wide application in different fields of catalysis.In summary, Ru\u2013Fe alloy nanoparticles deposited on C12A7:eSupplementary informationClick here for additional data file."} +{"text": "The synthesis and catalytic reactivity of a class of water-tolerant cationic phosphorus-based Lewis acids is reported. v) cations of the type [ArP(cor)][B(C6F5)4] 2C6H3; cor = 5,10,15-(C6H5)3corrolato3\u2013, 5,10,15-(C6F5)3corrolato3\u2013) were synthesized and characterized by NMR and X-ray diffraction. The visible electronic absorption spectra of these cationic phosphacorroles depend strongly on the coordination environment at phosphorus, and their Lewis acidities are quantified by spectrophotometric titrations. DFT analyses establish that the character of the P-acceptor orbital comprises P\u2013N antibonding interactions in the basal plane of the phosphacorrole. Consequently, the cationic phosphacorroles display unprecedented stability to water and alcohols while remaining highly active and robust Lewis acid catalysts for carbonyl hydrosilylation, C3sp\u2013H bond functionalization, and carbohydrate deoxygenation reactions.The synthesis and catalytic reactivity of a class of water-tolerant cationic phosphorus-based Lewis acids is reported. Corrole-based phosphorus( A nontrigonal substitution at phosphorus. Specifically, we envisioned that a tetragonal substituent field would be less conducive to formation of a formal P PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O multiple bond, diminishing the propensity for phosphine oxide formation and thereby preserving the Lewis acidity at the cationic phosphorus center (v) corroles readily undergo apical halide/alkoxide exchange11We considered that an alternate approach to robust phosphorus-based Lewis acids might be accessible by deliberate alteration of the molecular geometry. Having previously demonstrated that molecular deformation of neutral phosphorus compounds allows for novel bond activation reactions and catalytic transformations,s center , bottom.v) cations are robust, tunable, and catalytically-active Lewis acids. We find that the rigid tetragonal geometry imparts stability to water and alcohols while maintaining Lewis acidity, enabling these compounds to effect transformations which were previously inaccessible to phosphonium catalysts. This desirable superposition of properties is rationalized within an electronic structure argument that advances our ongoing program to establish new reactivity for p-block elements by imposition of underexplored molecular geometries.We demonstrate here that square pyramidal corrole-based phosphorus(4) in the presence of triethylamine furnished an unstable intermediate, which upon the addition of [Bu4N][BH(OAc)3] yielded hexacoordinate 1\u00b7H as a chromatographically stable green solid (31P{1H} NMR spectrum of 1\u00b7H showed a resonance at high field (\u03b4 \u2013 231.3 ppm), consistent with compositionally similar hexacoordinate phosphorus compounds reported previously.31P NMR resonance evolves into a doublet of triplets, with coupling constants evidencing a direct P\u2013H bond (1JP\u2013H = 928.0 Hz) as well as longer range coupling to the ortho protons of the apical P-aryl moiety (3JP\u2013H = 25.1 Hz).1H NMR channel, the P\u2013H unit was observed with complementary coupling ; the rather high-field chemical shift of this 1H nucleus is attributed to shielding from the diamagnetic ring current of the corrole system,1\u00b7H as in Synthesis of the target cations was achieved in two steps from the freebase corrole. First, treatment of 5,10,15-triphenylcorrole with 1 equiv. of phenyl tetrachlorophosphorane 4] immediately produced a red solution, from which a new phosphorus-containing product was obtained by precipitation via slow addition of pentane. A 31P NMR spectrum of the resulting maroon solid displayed a single new triplet resonance downfield of the starting compound . This chemical shift is indicative of a pentacoordinate phosphorus center shielded by diamagnetic ring current;1JP\u2013H coupling and concomitant formation of triphenylmethane further evidence the formation of the hydride abstraction product +1. Related phosphacorroles +\u20134+2 were synthesized analogously from the corresponding triarylcorrole and aryl phosphorane as depicted in The apical hydride of hexacoordinate compound +1 was revealed by X-ray diffraction experiments (\u03c4 = 0.09),+4 (\u03c4 = 0.05)P center protrudes less from the binding pocket relative to +1 (\u0394d = 0.023 \u00c5). In both instances, the overall geometry imposed by the corrole ligand may be viewed as a monovacant octahedron about phosphorus. In conjunction with structural data for known hexacoordinate phosphorus corrole compounds ,4+ similarl+\u20134+1 for Lewis bases was initially assayed by recording 31P NMR chemical shift differences (\u0394\u03b4) for a phosphine oxide ((n-octyl)3P PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O) probe upon binding according to a modification of the Gutmann\u2013Beckett method.\u03b4 values (+1 < +2 < +3 < +4) as a function of increasing modular fluorination. A direct comparison of the Lewis acidity of +\u20134+1 to other Lewis acids on the basis of these \u0394\u03b4 values is tempting, but we caution against such a potentially specious interpretation in the present circumstance. In view of the diamagnetic ring current of the corrole moiety,\u03b4 values. Other NMR-based methods for the determination of Lewis acidity . The marked difference in color between cationic five-coordinate phosphacorroles (red) and neutral six-coordinate congeners (green) provided a convenient colorimetric method for measuring equilibrium binding in +\u20134+1. The sensitivity of the color dependence to the concentration of an exogenous Lewis base was demonstrated by titrating cationic phosphacorrole +4 with varying amounts of (n-octyl)3P PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O (e.g. \u03bb = 566 nm) confirms adduct formation free of decomposition or other deleterious reactivity.A unbiased quantification of Lewis acidity for /svg>O , top. Thn-octyl)3P PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O were fitted to the Hill equationKd //B3LYP/def2-TZVP leveliii) corroles,+\u20134+1 correspond to the corrole \u03c0 manifold, and are apparently not responsible for the experimentally observed Lewis acidity of cationic phosphacorroles.To further understand the varying Lewis acidities of +4, e.g. \u20132.31 eV for +4, see +\u20134+1 correlate with the experimental dissociation constants Kd for phosphine oxide binding, implying that Lewis acid/base interactions are hosted by this orbital cleanly gives P-hydroxide 3\u00b7OH (\u03b4 \u2013 200.2 ppm) in quantitative fashion as in +3 but instead gives an adduct that we formulate as +\u00b7OH23 via 1,5-hydride shift from a N,N-dialkylaniline donor to a malonate alkylidene acceptor followed by intramolecular cyclization to give 6 in good isolated yield (13C6-d-glucose (7) is exhaustively deoxygenated by a catalytic amount of +4 (5 mol%) in the presence of an excess of H2SiEt2 at room temperature to give a mixture of hexanes and hexenes (67% total yield, +4 is not degraded; instead, an approximately equimolar amount of 4\u00b7H and +4 were observed spectroscopically as the only 31P NMR resonances. The lack of reaction between +4 and H2SiEt2 in a control experiment further indicated that +4 is a true catalyst for this reaction. The persistence of +4 with respect to a substrate that under these conditions presents a 100\u2009:\u20091 ratio of free hydroxyl moieties to catalyst confirms the noteworthy chemical robustness inherent to tetragonal cationic phosphorus-based Lewis acids.The phosphacorrole cations ed yield .31 Furth+\u20134+1 as enforced by the tetraazamacrocycle, which produces an acceptor orbital that is distinct in character from prior trigonal electrophilic phosphonium cations and prohibits irreversible decomposition to phosphine oxides. The modularity of the corrole supporting framework allows the Lewis acidities of these electrophilic phosphorus species to be readily tuned. Together, these results establish within the family of designer main group Lewis acids a new structural type that extends the range of potential applications for this valuable class of compounds.In summary, we have shown that cationic phosphacorroles are potent Lewis acids that exhibit marked tolerance toward hydroxylic functionality including water. We propose that this useful property arises from the tetragonal geometry of There are no conflicts to declare.Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "Post synthetic linker exchange can be combined with Cu-catalyzed alkyne/azide click chemistry to enable orthogonal modification of known metal organic frameworks. via post synthetic linker exchange. Sequential and orthogonal click reactions could be performed on these modified MOFs by incubating the crystals with small molecule substrates bearing azide or acetylene groups in the presence of a copper catalyst. 1H NMR of digested MOF samples showed that up to 50% of the incorporated linkers could be converted to their \u201cclicked\u201d triazole products. Powder X-ray diffraction confirmed that the UiO-67 structure was maintained throughout all transformations. The click reaction efficiency is discussed in context of MOF crystallite size and pore size. As the incorporation of clicked linkers could be controlled by post synthetic exchange, this work introduces a powerful method of quickly introducing orthogonal modifications into known MOF architectures.Biphenyl-4,4\u2032-dicarboxylic acid derivatives containing either azide or acetylene functional groups were inserted into UiO-67 metal organic frameworks (MOFs) Metal organic frameworks (MOFs) have moved beyond \u2018simple\u2019 homogeneous materials into complex multiple-domain structures.orthogonal MOF modification. Orthogonal linker modification via a selective and stepwise tuning of specific subsets of linkers within a MOF would rapidly permit the construction of multi-functional MOFs. The vast majority of post synthetic MOF modifications methods do not discriminate within the target. For example, post synthetic metal ion exchangeOne desirable route for rapidly adding functionality is by coupling known MOF syntheses with unrelated yet well-established synthetic paradigms. We envisioned that post synthetic exchangeOrthogonal click chemistry is generally not possible through routine solution chemistry. If a mixture of substrates containing either acetylene or azide groups is exposed to catalytic conditions and a click reaction partner, the expected result is a statistical mixture of product and substrate cross-coupling. We expect that if two different click substrates are immobilized within the same MOF framework this unproductive cross-reaction can be avoided and so allow selective stepwise click reactions on the same material.i)-catalyzed azide\u2013alkyne cycloaddition (CuAAC) click chemistry within a MOF has been shown using a MOF built entirely of linkers bearing azide and acetylene functional groups. After synthesis, this UiO-68 (UiO = University of Oslo) type MOF could undergo sequential click reactions within the solid crystals in quantitative yield.Very recently orthogonal copper control over the final fraction of modified \u2013 and so clicked \u2013 linkers, (2) permitting the use of known MOFs, and (3) providing a way to introduce reactive sites that would not survive the initial MOF solvothermal synthesis. The last point is especially important, as it has been shown that the azide-functionalized linker we used here does not survive typical UiO-67 solvothermal conditions.26UiO-67 was chosen for this study due to its good bulk structural stability, relatively large pore size, and proven ability to engage in post synthetic linker exchange reactions.2O were prepared. To these were added -4,4\u2032-dicarboxylic acid functionalized with either an azide or acetylene group scale\" fill=\"currentColor\" stroke=\"none\">C respectively, as shown in 6-DMSO with aqueous HF for 1H NMR quantification scale\" fill=\"currentColor\" stroke=\"none\">C. Increasing the temperature to 40 \u00b0C did not appreciably change the incorporation yield for the azide linker. No thermocyclization of the azide linker, as seen for solvothermal synthesis of UiO-67 containing L-N3,3 and L-C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C were 33% and 26%, respectively. These exchange yields track with the lower incorporation of L-C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C observed in the single substitution experiment. 1H NMR of UiO-67 digested after simultaneous post synthetic exchange with both L-N3 and L-C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C.Crucially, both linkers could be introduced into the same UiO-67 material through this post synthetic exchange strategy . With 13ca. 10% modified linkers with either L-N3 or L-C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C. UiO-67 containing ca. 10% of modified linkers were used in subsequent click reaction experiments.As a MOF containing large numbers of modified linkers may result in excessive steric crowding during subsequent click reactions, we sought to generate UiO-67 with fewer substituted linkers. Indeed, a straightforward decrease in the net equivalents of both linkers present during exchange yielded UiO-67 with 3CN)4]PF6 and the fluorine-labeled substrates 1-ethynyl-4-(trifluoromethyl)benzene and 4-azido-1,1,1-trifluorobutane. 19F NMR was used to confirm that the MOF material after the click reactions contained the corresponding triazole products 4]PF6 in 1.5 ml THF and degassed for 5 minutes prior to the addition of the fluorinated click partners .Click reactions were performed under inert atmosphere at 50 \u00b0C in freshly distilled THF for 24 hours. In general, 20 mg of functionalized UiO-67 was mixed with 0.5 molar equivalent of PF6 catalyst, and 2 equivalents of click partners; in Zhang's work the reaction was carried out at 60 \u00b0C in DMF with CuI catalyst for 24 hours and unknown click partner stoichiometry.The difference in click conversion yields between this work and that of Zhang d4-1,4-dicarboxylic acid (H2BDC-d4) into ca. 100 \u03bcm crystals of UiO-66 was examined and a pronounced core\u2013shell structure was found, with exchange only occurring in the outer layers.33Individual crystallite size also matters: smaller crystals should facilitate access to interior click reaction sites. To our knowledge, there is no rigorous study available comparing post synthetic exchange into different sized crystals. However, one comparison can be made between two papers that both used UiO-66. In one study, the incorporation of benzene-2,3,5,6-ca. 1\u20133 \u03bcm across (via SEM, see Fig. S13\u2013S20The difference in crystallite size for this study and Zhang's UiO-68 work is not so pronounced. Here, crystals were Given these differences, a quantitative comparison is not straightforward; however, we suspect that the larger pore sizes of UiO-68 were the primary factor contributing to the reported quantitative click yields. Future studies comparing identical click reaction conditions using UiO-67 and UiO-68 crystals of similar size could address this question.known MOF materials are of special interest.MOFs have the potential to revolutionize industrial material science; as such, interest in new methods of functionalizing MOFs remains high. As there are thousands of MOF architectures to choose from, methods to easily modify Via the combination of these two methods, this work demonstrates a new approach to orthogonally modify MOFs. First, post synthetic exchange reactions showed that the native linkers in UiO-67 could be replaced with linkers bearing azide or acetylene groups. Stepwise incubation with a copper catalyst and small molecule click partners allowed the formation of two different triazole click products in a selective and orthogonal manner.Post synthetic exchange is firmly established as a reliable method of tuning known MOFs,via1H NMR revealed that up to 50% of the modified linkers could be converted to their clicked partners. PXRD analysis confirmed that the parent UiO-67 structure was maintained throughout all transformations.Control reactions established unambiguously that no click reactions occur if either the click partner or Cu catalyst is omitted. Quantification of click reaction yields Given the versatility of post synthetic exchange and power of click chemistry, we expect that this method of modifying MOFs in a stepwise and orthogonal fashion will be broadly useful. The ability to selectively change two different subsets of linkers within a MOF opens new avenues for rapid construction of complex functionality. From a fundamental perspective, the ability to selectively perform click chemistry on only one substrate while both azide and acetylene substrates are present is not normally possible in solution chemistry \u2013 it is through immobilization within a MOF framework that selective and non-statistical products can be obtained. Interesting questions remain regarding optimization of the click reaction efficiency, especially for MOFs with small pore diameters.UF performed research; UF and SO designed research and analysed data; BDM, UF, and SO wrote the paper.There are no conflicts to declare.Supplementary informationClick here for additional data file."} +{"text": "This paper presents a comparison of a graph-based genetic algorithm (GB-GA) and machine learning (ML) results for the optimization of log\u2009P values with a constraint for synthetic accessibility and shows that the GA is as good as or better than the ML approaches for this particular property. P values with a constraint for synthetic accessibility and shows that the GA is as good as or better than the ML approaches for this particular property. The molecules found by the GB-GA bear little resemblance to the molecules used to construct the initial mating pool, indicating that the GB-GA approach can traverse a relatively large distance in chemical space using relatively few (50) generations. The paper also introduces a new non-ML graph-based generative model (GB-GM) that can be parameterized using very small data sets and combined with a Monte Carlo tree search (MCTS) algorithm. The results are comparable to previously published results using a recurrent neural network (RNN) generative model, and the GB-GM-based method is several orders of magnitude faster. The MCTS results seem more dependent on the composition of the training set than the GA approach for this particular property. Our results suggest that the performance of new ML-based generative models should be compared to that of more traditional, and often simpler, approaches such a GA.This paper presents a comparison of a graph-based genetic algorithm (GB-GA) and machine learning (ML) results for the optimization of log\u2009 Within the past few years several papers have been published on using machine learning (ML) to generate molecules with the aim of optimizing their properties.P values with a constraint for synthetic accessibility and show that the GA is as good as or better than the ML approaches for this particular property. I also introduce a new non-ML graph-based generative model that can be parameterized using very small data sets and combined with a Monte Carlo tree search algorithm. The implementation of both methods relies on the open source RDKit cheminformatics package and the codes are made freely available.In this paper I present a comparison of graph-based GA and ML results for the optimization of log\u2009et al.et al.et al.et al.P scores.The graph-based genetic algorithm (GB-GA) combines the ideas from the algorithm developed by Brown et al.et al.X \u223c Y \u2192 X1 \u223c Z \u223c Y1 where \u201c1\u201d indicates that X and Y are bonded and \u201c\u223c\u201d indicates an arbitrary bond order.Tsuda and coworkers PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C\u2013C, which accounts for 45% of all 3-atom combinations in rings scale\" fill=\"currentColor\" stroke=\"none\">C\u2013C mutation is chosen 45% of the time, and similarly for the 34 other X \u223c Y \u223c Z combinations found in the data set. The site of insertion/creation is chosen randomly and excludes aromatic and six-membered rings. Similarly, for addition the most common bond involving at least one non-ring atom is C\u2013C, so a C \u2192 C\u2013C mutation is chosen more often scale\" fill=\"currentColor\" stroke=\"none\">C vs. C\u2013C, was considered but then the most probable bonding patterns are often not found in the early stages of molecule growth and the growth process effectively stops.In order to create realistic looking molecules, such as those in the ZINC data set, the mutations and choice of element are chosen with probabilities obtained by an analysis of a subset of the molecules in the ZINC dataset. An analysis of first 1000 molecules in the ZINC dataset shows that 63% of the atoms are ring atoms, so the ring-creation or ring-insertion mutation is chosen 63% of the time. The most common 3-atom combination in rings is Cin rings , so a C and rollout is terminated if the molecule exceeds the target size as described for the GB-GA. Any three- or four-membered alkene rings are subsequently expanded to five-membered rings by inserting C atoms. The reward function is 1 if the predicted J(m) value (see below) is larger than the largest value found so far and 0 otherwise. This reward function was found to work slightly better than the one used by Yang et al.2The GB-GM-MCTS code is a modified version of the mcts.py codeet al.et al.J(m):P(m) is the octanol\u2013water partition coefficient for a molecule (m) as predicted by RDKit, SA(m) is a synthetic accessibility score,Following G\u00f3mez-Bombarelli J(m) depends both on the number and types of atoms and can be made arbitrarily large by increasing the molecular size. Therefore it is important to limit the molecular size in order to make a fair comparison to previous studies. Yang et al.et al.J(m) scores for each simulation are averaged. The population size is 20 and 50 generations are used (i.e. 1000 J(m) evaluations per run). The initial mating pool is 20 random molecules sampled from the first 1000 molecules in the ZINC data set. The mean J(m)-score for this set is 0.2 and the maximum value is 3.6.Ten GA simulations are performed and the maximum J(m)-score for the GA is 6.8 \u00b1 0.7 and 7.4 \u00b1 0.9 using a 50% and 1% mutation rate, respectively -score evaluations. The latter took 8 hours each, while the GB-GA calculations took 30 seconds each on a laptop. These J(m)-scores are also significantly larger than those of the other ML-based methods tried by Yang et al.The average maximum ectively . For comJ(m)-scores found by the GB-GA. These scores, 8.8 and 8.5, are slightly larger than the three largest values (7.8\u20138.0) obtained by You et al.J(m) values of 0.9 and \u20132.4 value evolves with each generation for 10 different GB-GA runs. While most of the runs have mostly peaked after about 20 generations the three best performing runs show significant improvements between 30 and 40 generations, so running fewer than 50 generations cannot be recommended for J(m) maximisation. None of the runs increased J(m) significantly after periods of 20 generations with no or little change in J(m) (with the possible exception of run 7). So a good strategy may be to terminate the GB-GA run if the J(m) value has not changed for more than 20 generations (at least for J(m) maximisation). PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C\u2013C, which is reasonably close to the 45% in the reference set scale\" fill=\"currentColor\" stroke=\"none\">C\u2013\u201d atom will be added. It is thus a little bit more likely that a \u201c\u2013C\u2013\u201d atom will be \u201caccepted\u201d, compared to a \u201c PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C\u2013\u201d atom.As described in the Computational methodology section, the GB-GM method grows a molecule one atom at a time where the choice of bond order and atom type is chosen probabilistically based on a bonding analysis of the first 1000 molecules in the ZINC dataset (referred to hereafter as the reference set). The GB-GM model is tested by generating 1000 molecules using ethane as the \u201cseed\u201d molecule (which takes about 30 seconds on a laptop) and repeating the statistical bonding analysis. The average molecular size in the new data set is 23.2 \u00b1 4.1 atoms, which is nearly identical to that of the training set: 23.2 \u00b1 4.4 atoms. There are 2498 rings compared to 2768 in the reference set and 59% of the atoms are in rings, which also is close to the 63% in the reference set. 41% of the 3-atom combinations in rings is Cence set . This di PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019[*]\u2013[*] PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019[*]\u2013[*] PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019[*]1) as well as cyclopentane ([*]1\u2013[*]\u2013[*]\u2013[*]\u2013[*]1) and cyclohexane ([*]1\u2013[*]\u2013[*]\u2013[*]\u2013[*]\u2013[*]1) type rings, while there are more of most of the other types compared to the reference set. The reason is that the molecules in the ZINC data set are not made by randomly adding atoms, but by assembling larger functional groups such as aliphatic and aromatic rings. As a result the average ring composition does not reflect the most likely ring compositions. It is possible to scale the probabilities to skew the results towards one or the other ring-type. For example in the last column the probabilities are scaled such that the probability of X = Z\u2013Y is 80% rather than the 62% in the reference set, which increases the number of benzene-like rings from 479 to 850 at the expense of the aliphatic rings.Not surprisingly, there are bigger differences for the larger scale features not specifically encoded in the rules such as the type of ring . For exaJ(m)-scores for each simulation are averaged. The tree is traversed 1000 times, i.e. there are 1000 J(m) evaluations per run. For GB-GM-MCTS the average maximum J(m)-score value is 2.6 \u00b1 0.6, which is significantly lower than the lowest value in the study of Yang et al.et al. PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C\u2013C containing rings is increased from 62% to 80% then the average maximum J(m)-score increases to 3.4 \u00b1 0.6. Increasing the number of tree traversals to 5000 increases the value to 4.3 \u00b1 0.6, which is similar to the 4.9 \u00b1 0.4 obtained by Yang et al.et al.Ten GB-GM-MCTS simulations are performed using ethane as a seed molecule and the maximum et al.J(m)-scores can be obtained using long aliphatic chains, but this structural motif is relatively rare in the ZINC data set and therefore rarely suggested by the generative models.et al.,P values with a constraint for synthetic accessibility (J(m)) and shows that the GA is as good as or better than the ML-based approaches for this particular property. The GB-GA predicts maximum J(m)-values that, on average, are 1.3\u20131.8 units higher than the best ML-based results reported by Yang et al.,J(m)-scores that, depending on the method, often are several units larger than those found with ML-based approaches. These molecules bear little resemblance to the molecules used to construct the initial mating pool, indicating that the GB-GA approach can traverse a relatively large distance in chemical space using relatively few (50) generations.This paper presents a comparison of a graph-based genetic algorithm (GB-GA) and machine learning (ML) results, compiled by Yang et al.et al.J(m)-values of 4.3 \u00b1 0.6 compared to 4.9 \u00b1 0.6 found using 5000 property evaluations. While the results are slightly worse than the RNN results, the GB-GM-based method is several orders of magnitude faster. In both cases the MCTS approach essentially extracts the main hydrophobic structural motif (a benzene ring) found in the training set. The MCTS results thus seem more dependent on the composition of the training set than the GA approach for this particular property.The paper also introduces a new non-ML graph-based generative model (GB-GM) that can be parameterized using very small data sets and combined with a Monte Carlo tree search (MCTS) algorithm such as the one used by Yang et al.While the results are quite encouraging, it is important to perform similar comparisons for other properties before drawing general conclusions. In a very recent study Brown There are no conflicts to declare.Supplementary informationClick here for additional data file."} +{"text": "The synthesis and characterisation of a 1-titanacyclobuta-2,3-diene complex, an organometallic analog of elusive 1,2-cyclobutadiene, is presented. rac-(ebthi)Ti(Me3SiC3SiMe3)] ), a formal metallacyclic analogue of a non-existent four-membered 1,2-cyclobutadiene, is described. By variation of the cyclopentadienyl ligand of the titanocene precursor it was possible to stabilise this highly exotic compound which selectively reacts with ketones and aldehydes to yield enynes by oxygen transfer to titanium. Analysis of the bonding and electronic structure of the metallacycle shows that the complex is best described as an unusual antiferromagnetically coupled biradicaloid system, possessing a formal Ti(iii) centre coordinated with a monoanionic radical ligand.The synthesis of an unusual 1-metalla-2,3-cyclobutadiene complex [ Later, 2SiMe3 and \u03b1-CH2N(SiMe3)2 substituted titanocene alkyne complexes was found to be unsuccessful.3 framework prior to coordination to the metal. We have thus revisited and optimised the synthesis of a previously reported 1,3-dilithioallene precursor [Li2(Me3SiC3SiMe3)]2ZrCl2]. To our surprise, we obtained two unusual allenediide bridged dizirconocene complexes [Cp2Zr(RC3R)2ZrCp2] and [Cp2Zr(Cl)RC3R(Cl)ZrCp2] (R = SiMe3),2Zr(RC3R)2ZrCp2] degrades under mass spectrometry conditions into the desired mononuclear 1-zirconacyclobuta-2,3-diene compound. In this contribution, we report on the synthesis and characterisation of the first 1-metalla-2,3-cyclobutadiene complex of a group 4 metal as well as the reactivity of this unusual complex.To further explore the frontiers of the chemistry of group 4 metallacycles Jemmis, Schulz and Rosenthal have computationally investigated the possibilities to stabilise planar 1-metalla-2,3-cyclobutadienes and found that the incorporation of electron-donating substituents at \u03b1-carbon atoms could be beneficial.2TiCl2] with [Li2(Me3SiC3SiMe3)]. 1H NMR analysis of an aliquot taken after a reaction time of 16 hours at room temperature shows the formation of several Cp containing products along with a large number of resonances in the SiMe3 region of the spectrum, out of which we identified the coupling product 1 and observed a slow colour change from red to brown. NMR analysis of the reaction mixture shows the formation of unidentified titanocene species along with the coupling product 1 as the main species, which can be isolated after column chromatographic workup in 90% yield allenes supports this proposal.21To evaluate the influence of the metal centre we first adapted our previously reported procedure0% yield . The for and the allene precursor Me(Me3Si)C3(SiMe3)Me to form the desired four-membered metallacycle and ethane. This reaction is slightly exergonic and thus in principle feasible ] at low temperature we proposed formation of a 1-titana-2,3-cyclobutadiene (vide supra), which is in accordance with the computed exergonic reaction of Cp2TiMe2 and Me(Me3Si)C3(SiMe3)Me . The fact that a coupling reaction leading to 1 was observed in reactions with Cp and Cp* complexes indicates that at some stage of the reaction the interaction of an intermediately formed C3 framework with the Ti centre occurred. ansa-Cp complexes in many cases show reactivities that are comparable to Cp analogues and furthermore exhibit additional stabilisation of the metallocene through the bridging unit. Subtle differences in reactivity (e.g. formation of different products or faster conversion) are often referred to as the ansa effect.rac-(ebthi)TiMe2] and Me(Me3Si)C3(SiMe3)Me is even more thermodynamically favourable .In a previous study we had computed the isodesmic exchange reaction of rac-(ebthi)TiCl2] and [Li2(Me3SiC3SiMe3)] at room temperature in pentane can be explained by the formation of 1 as a byproduct which was identified by NMR spectroscopy and this nicely demonstrates the subtle differences in reactivity along the series Cp\u2013rac-(ebthi)\u2013Cp*. Single crystals suitable for X-ray analysis were obtained from a saturated pentane solution at room temperature.We have thus next reacted complex we have performed stability tests. Therefore, we exposed solutions of 2 to air and moisture and found that, in both cases, slow formation of the propyne Me3SiC2CH2SiMe3 takes place (see ESIiii) species takes place.To gain first insights into the reactivity of the isolable and unusual biradical coupling with formal insertion of the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O bond into the M\u2013C bond being the first step of the reaction. Further studies to understand the structural principles as well as further investigations regarding the reactivity of 2 and related complexes will be done in our lab in the future.For the first time, we have shown that through combination of suitable metal centre, cyclopentadienyl ligand and substituents at the CThere are no conflicts to declare.Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file."} +{"text": "The spectroscopic data base for cis-formic acid is considerably extended to make it fit for experimental benchmarking of vibrational calculation tools. cis\u2013trans isomerism of formic acid. It quintuples the previously available gas phase vibrational data base on this excited form of a strongly anharmonic planar molecule despite its limited spectral resolution. The newly determined cis-formic acid fundamentals allow for a balanced vibrational benchmark on both rotamers of formic acid. Assuming the adequacy of vibrational perturbation theory, it reveals weaknesses of standard methods for these systems like B3LYP-D3(BJ)/aVQZ VPT2 or PBE0-D3(BJ)/aVQZ VPT2. The functionals \u03c9B97-XD and M06-2X additionally suffer from severe integration grid size and symmetry dependencies. The vibrational benchmark suggests B2PLYP-D3(BJ)/aVQZ VPT2 and MP2/aVQZ VPT2 as partially competitive and in any case efficient alternatives to computationally demanding coupled cluster vibrational configuration interaction calculations. Whether this is due to fortuitous compensation between electronic structure and vibrational perturbation error remains to be explored.A new technique to rotationally simplify and Raman-probe conformationally and vibrationally excited small molecules is applied to the These have been surprisingly scarce until very recently, with a single exception.5Vibrational spectra of small molecules effectively probe the quality of potential energy hypersurface (PES) predictions, when combined with accurate anharmonic calculations.trans-form to the higher-energy cis-form has been of particular interest,cis-formic acid, matrix isolation has been the method of choice thus far, because the possibility of long irradiation times allows for a significant formation via laser excitation of the trans-form.cis-isomer thus suffer from a lack of gas phase experimental reference data. Two recent studies where this applies are by Tew and MizukamiAs the smallest carboxylic acid, the formic acid monomer has been addressed by a plethora of theoretical\u20131 between both rotamers of formic acid,cis-form are rare. The first gas phase band position of cis-formic acid has been published in 2006 by Baskakov and co-workers, who studied the out-of-plane bending vibration with high resolution FTIR spectroscopy. PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O stretch coupling in jet-cooled carboxylic acid dimers.cis-formic acid. It is based on a powerful new Raman scattering approach of thermally populated and rapidly re-cooled molecules. Instead of conserving the conformational excitation by cryogenic matrix trapping,trans-form is also not an issue on the time scale of the jet expansion, making it an \u201ceasy\u201d experiment.cis-formic acid vibrational frequencies after a decade of stagnation, we provide the first systematic access to the performance of quantum chemical methods towards this model system. This decreases the likelihood of accidental error compensation between electronic structure, vibrational treatment, and matrix shifts for cis-formic acid by orders of magnitude.Due to the fairly large energy difference of 1365 \u00b1 30 cmcis-formic acid were recorded, followed by a detailed analysis of the spectra and a first benchmark of vibrational perturbation theory and literature variational data against the experimental data. It is hoped that this progress will trigger further growth in the experimental data base and its use in benchmarking the global PES of formic acid and pentatomic vibrational treatments.The structure of this paper is as follows: we briefly illustrate the general approach of how the spectra of 22.1cis-Formic acid was formed in small quantities from the trans-rotamer by heating the nozzle and its feed line to temperatures between 100\u2013190 \u00b0C.A detailed description of the experimental set-up can be found in previous publications.2.2The quantum chemical calculations shown in this work were performed with the Gaussian 09 program package (revision E.01)cis-formic acid fundamentals has been supported by scaled, harmonic frequency calculations at the B3LYP-D3(BJ)/aVTZ level, which have proven to yield sufficient agreement in a previous study.The assignment of Cs symmetry as well as a finer integration grid (pruned super fine integration grid (150\u2009974)Additionally, exploratory VPT2 calculations utilising the 33.1cis-rotamer detection, the band positions and Raman scattering cross-sections have been predicted using B3LYP-D3(BJ)/aVTZ alongside those of the trans-form. The results are displayed in cis-formic acid vibrations with the largest scattering cross-sections are \u03bd1, \u03bd2, \u03bd3, and \u03bd6, namely the O\u2013H, the C\u2013H, the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O, and the C\u2013O stretching vibration. In fact, \u03bd6 is the only stretching vibration with a distinctly larger scattering cross-section compared to trans-formic acid.To choose suitable spectral regions for trans-monomer band of lowest intensity amongst the four. Consequently, any hot band, i.e., cis-formic acid or a non-isomeric hot band originating from thermally populated low-lying energy levels of trans-formic acid, should increase in intensity with nozzle temperature, whereas any formic acid cluster band will decrease due to thermal dissociation.The experimental spectra of these four vibrational modes of both rotamers can be found in \u20131. The band position is in good agreement with the harmonically calculated, \u03bd1(F)-scaled band position of cF with a deviation of only 5 cm\u20131. Either the anharmonicity of F and cF is similar or there is error compensation with the density functional used. Another way of validating this assignment is to compare the intensity ratio of the cF (3637 cm\u20131) and F (3570 cm\u20131) bands with the energy difference of both forms. The harmonically calculated energy difference of 15.9 kJ mol\u20131 (B3LYP-D3(BJ)/aVTZ with zero point energy correction) is just below the error bounds of the only experimental value of 1365 \u00b1 30 cm\u20131 by W. Hocking.cis-formic acid at 190 \u00b0C. After correction by the theoretical cross-section ratio, the ratio of the experimental band integrals provides a cis-abundance of 2%, thus reaffirming the cF assignment. The additional bands downshifted compared to the O\u2013H stretching vibration of trans-formic acid at 3560 cm\u20131 and 3566 cm\u20131 are most likely trans-formic acid combination bands of \u03bd2 with the lowest frequency vibrations \u03bd7 (3560 cm\u20131) and \u03bd9 (3566 cm\u20131), which benefit from the large Raman scattering cross-section of the C\u2013H stretching vibration. The former is in good agreement with the predicted values of Tew and Mizukami (3566 cm\u20131)\u20131)\u03bd2 + \u03bd9) in Fermi resonance with (\u03bd3 + 3\u03bd9) at 3571 cm\u20131 and 3579 cm\u20131.The spectra in the O\u2013H stretching region show one band that increases in intensity with temperature at 3637 cm\u03bd2 region is spectrally more congested due to its low sensitivity to hydrogen bonding. In the spectral windows 2970\u20132945 cm\u20131 and 2935\u20132925 cm\u20131, there are several bands that decrease in intensity with temperature, i.e., cluster bands. The broad underlying signal is due to rovibrational O and S branches of \u03bd2. As opposed to the O\u2013H stretching region, there are two distinct bands increasing in intensity with temperature at 2925 cm\u20131 and 2873 cm\u20131. The latter deviates from the predicted band position of cis-formic acid by 14 cm\u20131. The amount of cis-formic acid at 190 \u00b0C deduced from the integrated intensities of the bands amounts to 1%, which fits the energy difference, as detailed above. Therefore, the band at 2873 cm\u20131 can be assigned to cF. The second hot band at 2925 cm\u20131 is shifted by \u201317 cm\u20131 from the fundamental of F (2942 cm\u20131). For an assignment to F, two things need to be considered: firstly, the shift directly yields the off-diagonal anharmonicity constant xi2 between \u03bd2 and a low-lying energy level vi that is thermally populated. Secondly, the intensity ratio is dependent on the Boltzmann population of that level and yields the excitation energy of the latter. Hence, the assignment can be checked by comparing the experimentally determined anharmonicity constant and intensity with the calculated values for the lowest-lying energy levels of trans-formic acid. From the anharmonicity matrix elements in \u03bd7 will most likely overlap with the fundamental, whereas the hot band originating from \u03bd9 (and \u03bd6) could overlap with a cluster band at 2938 cm\u20131 causing the highest nozzle temperature spectrum (red) and the lowest nozzle temperature spectrum (black) to have similar intensities. However, due to the spectral congestion in this area, reliable assignments are not feasible. Additional depolarisation measurements to subtract the O and S branches from the sharp Q peak are currently ongoing and will be addressed in detail in a subsequent publication. Here we focus on the straightforward assignment of the 2925 cm\u20131 band. Its observed shift of \u201317 cm\u20131 perfectly matches the calculated anharmonicity constant x28. The expected intensity ratio at 190 \u00b0C of around 4% approaches the observed ratio of 3%, so that it can be assigned to \u03bd2 + \u03bd8 \u2013 \u03bd8.The \u03bd3 spectral region at nozzle temperatures of 23 \u00b0C, 110 \u00b0C, 140 \u00b0C, and 170 \u00b0C can be found in a previous publication.\u03bd3 region. Briefly, the cis-formic acid band can be seen at 1818 cm\u20131, which deviates from the calculated, \u03bd3(F)-scaled band position by 3 cm\u20131. The hot band downshifted by 7 cm\u20131 from F can most likely be attributed to \u03bd3 + \u03bd7 \u2013 \u03bd7, with a negligible (1 cm\u20131) deviation of the calculated anharmonicity constant x37 compared to the experimentally observed value and a reasonable Boltzmann population match (14% from the level energy and 10% from the Raman spectrum). The hot band intensity qualitatively rules out major contributions from higher energy levels such as \u03bd8. There are two weaker potential hot bands shifted from F by +6 cm\u20131 and \u201313 cm\u20131 with intensities of around 1\u20132% compared to F. An assignment is not possible since the shifts do not match the predicted anharmonicity constants scale\" fill=\"currentColor\" stroke=\"none\">O stretching vibration.A first analysis of the \u03bd6 of trans-formic acid at 1105 cm\u20131. The shifts amount to \u20134 cm\u20131, \u20137 cm\u20131, and \u201311 cm\u20131 with intensities of around 7%, 3%, and 7% of \u03bd6 at 190 \u00b0C. To assign \u03bd6 of cis-formic acid, the shifts are compared to the calculated anharmonicity constants xi6 in x67 and x68 agree (\u20133.7 cm\u20131). In addition, x69 and x66 are very similar (\u20135.6 cm\u20131 and \u20136.2 cm\u20131). Therefore, it seems likely that the bands at 1101 cm\u20131 and 1097 cm\u20131 are a result of overlapping hot bands. The slightly higher intensity of the former is a result of the greater overlap with the fundamental and the lower energy of \u03bd7 and \u03bd8 compared to \u03bd9 and \u03bd6. The next higher energy level is \u03bd5 with a predicted band position of 1219 cm\u20131. A hot band originating from \u03bd5 is expected to be shifted by \u201314.3 cm\u20131 from \u03bd6 of trans-formic acid -scaled harmonic B3LYP-D3(BJ)/aVTZ calculations. Additionally, the observed intensity matches the calculated energy difference between both rotamers, considering the four times larger predicted scattering cross-section of \u03bd6 of cis-formic acid compared to the trans-form determined from high resolution FTIR measurements\u20131 or +23 to \u201321 cm\u20131, dependent on the matrix site. This scatter is of a similar order of magnitude as the cis\u2013trans spectral differences themselves, which are also listed in 15The band positions of all stretching vibrations of 3.2cis-formic acid have been compared to vi(F)-scaled harmonic band positions calculated at the B3LYP-D3(BJ)/aVTZ level, which has shown to be quite valuable in supporting the assignment. The small size of the formic acid monomer and its structural rigidity enable anharmonic vibrational perturbation theory calculations (VPT2),trans-formic acid monomer at various levels of theory in a study of the trans-formic and -acetic acid monomers and their nitrogen clusters.trans-acetic acid monomer, where the presence of the floppy methyl groups resulted in instabilities such as a wavenumber increase of the lowest frequency vibration compared to the harmonic case. The newly determined band positions of cis-formic acid thus enable a significantly extended VPT2 benchmark involving both rotamers, which should not suffer from such methyl torsion instabilities.So far, the band positions of cis-formic acid as well as the band position difference between the cis- and trans-fundamentals. The methods tested are the same as in \u20131 . All other methods fail to predict the cis-formic acid band positions correctly despite generous experimental error bars for the stretching vibrations. An accurate prediction of \u03bd9 (and the respective shift) is evidently unrealistic due to the high accuracy of the high resolution measurements. The lower resolution Raman spectra are seen to be fully adequate to challenge theory on an absolute wavenumber scale. The vibrations where the shift is predicted within the experimental error for most methods are the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O and C\u2013O stretching vibrations, whereas the largest divergence is observed for the C\u2013H stretching vibration. This is not surprising as the C\u2013H stretching vibration is prone to stretch-bend Fermi resonance, although the VPT2 code employedcis-formic acid, which is amplified by Fermi resonance. If the band labels are switched, the agreement increases significantly \u2013 the severe underestimation of the band position of \u2013142 cm\u20131 and \u0394\u03bd\u0303i(cF\u2013F) clearly illustrates that there are few reliable methods. In case of PM3, this is not surprising. It is the only method that fails to predict the energetic order of the vibrations correctly with \u03bd4 and \u03bd6 switched. Other methods with particularly severe deviations from experiment are \u03c9B97-XD (cf. \u03bd1 and \u03bd9) and M06-2X . The large underestimation of \u03bd\u03032(cF) and \u0394\u03bd\u03032(cF\u2013F) of M06-2X is enhanced by a level switch between resonance partners, as discussed above. All other methods predict the correct sequence of fundamental and overtone. Another numerical or fundamental deficiency of M06-2X/aVQZ VPT2 is the incorrect sign of the total anharmonicity of \u03bd9 of cis-formic acid, which gives rise to a large overestimation of the anharmonic band position (+163 cm\u20131). In combination with an overestimation of the negative anharmonicity of \u03bd9 of trans-formic acid, this results in a severe overestimation of the shift (+308 cm\u20131) between both rotamers. As such, this data point has been omitted from \u03bd3 and \u03bd6), whereas B3LYP-D3(BJ) predicts the shifts correctly in three of the five cases . Both exhibit similar deviations with respect to the band positions. Since the rotational constant prediction of B3LYP-D3(BJ) is also more accurate, it is the overall better choice. MP2 is particularly good for the description of the lower frequency modes \u03bd6 and \u03bd9 and overshoots for \u03bd1 and \u03bd2. For \u03bd3, an agreement with the shift is reached with the largest basis set aVQZ. It is generally rewarding that basis set sensitive methods tend to move towards the experimental region with increasing basis set size and shows only small deviations for the other two. The band positions are slightly, but consistently underestimated, apart from \u03bd2, where a small overestimation occurs for the larger basis sets, and \u03bd9, which is slightly overestimated for all basis set sizes.A comparison of the individual performance of the methods for the determination of size cf.. Another\u03bd9 differs solely by \u20131 cm\u20131. With regard to the shifts, only one is predicted within the experimental uncertainty (\u03bd3), but the shift of \u03bd9 differs solely by about 1 cm\u20131. Deviations are generally small and on the same order as for B2PLYP-D3(BJ)/aVQZ VPT2 or MP2/aVQZ VPT2. The agreement of the MCTDH calculations of Richter and Carbonni\u00e8re\u03bd3, where the value is with 36 cm\u20131 just outside the experimental confidence interval (41 \u00b1 4 cm\u20131). The band position of the C\u2013H stretching vibration is predicted within the experimental accuracy and the \u03bd9 prediction deviates only by 2 cm\u20131. However, the latter gas phase value was the only band position of formic acid known in the gas phase before cis-values were true predictions for the isolated molecule.The band positions and shifts obtained from the VCI calculations of Tew and Mizukamicis-formic acid band position (ci) and the band position shift between cis and trans (\u0394i). The size of the deviation from experiment is encoded in the octagon size. A point on a node with the smallest octagon translates into theoretical agreement within the experimental error bars (\u00b12 cm\u20131 for the band position and \u00b14 cm\u20131 for the shifts). Correspondingly, a point on a node with the nth octagon implies a deviation of that value by up to n confidence intervals from experiment. The predicted band position for the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O stretching vibration of cis-formic acid by Tew and Mizukami (1824 cm\u20131)\u20131 from the Raman jet value of 1818 cm\u20131. Considering the experimental confidence interval of \u00b12 cm\u20131, the prediction for c3 lies on the third octagon, or in other words, three nodes away from the origin on the c3 axis. Note that the origin in these diagrams cannot be met due to the experimental uncertainty. The sign of the deviation is illustrated by the colour shade of the symbol, whereby a dark colour shows over- and a light colour underestimation. The intermediate shade represents an indeterminate sign of the deviation, which can be seen for the shift of the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O stretching vibration \u03943 of Tew and Mizukami. The aforementioned consistent overestimation of the VCI calculations of Tew and Mizukami (T & M) (apart from \u03943) can thus be directly seen by the otherwise dark-coloured symbols. The MCTDH method of Richter and Carbonni\u00e8re falls closer to the origin and varies more in sign. Therefore, it shows superior agreement with experiment compared to the results of Tew and Mizukami. The tendency of the B2PLYP-D3(BJ)/aVQZ VPT2 calculations to underestimate the band position ci as well as its ability to predict most shifts within the experimental accuracy is illustrated. Altogether, et al.trans-formic acid only improves slightly when moving from a matrix to the gas phase. PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O) stretching vibrations in an Ar matrix, which mimic the underestimation of these vibrations by the B2PLYP functional in the gas phase. Such good agreements for the wrong reason must be avoided in proper benchmarking. Only the gas phase comparison can provide a realistic picture of the electronic structure performance.Another way of visualising the agreement of the theoretical predictions of Tew and Mizukami, Richter and Carbonni\u00e8re, and results obtained with vibrational perturbation theory (B2PLYP-D3(BJ)/aVQZ VPT2) with experiment is shown in trans-formic acid, the coupling constants to levels with significant thermal population at 190 \u00b0C predicted with B3LYP-D3(BJ)/aVTZ (see \u20131 (x36), which is below the spectral resolution of the Raman experiment.With regard to the previous assignment of hot bands of aVTZ see are in g3.3Cs symmetry and a finer integration grid ).i.e., 10 values.As previously mentioned in Section 2.2, all production calculations have been carried out without the use of symmetry using the pruned ultra fine integration grid of Gaussian 09.\u20131) dependence on symmetry. The size of the deviation depends largely on the density functional theory method used as well as on the vibration. The most sensitive vibrations of the fundamentals discussed in this work are the O\u2013H stretching (\u03bd1) and out-of-plane bending vibration (\u03bd9), while the smallest deviations are observed for the C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O (\u03bd3) and C\u2013O stretching vibrations (\u03bd6). For B3LYP-D3(BJ), B2PLYP-D3(BJ), and PBE0-D3(BJ), these deviations are below \u00b110 cm\u20131, with mean absolute deviations of 2.5 cm\u20131, 1.7 cm\u20131, and 2.1 cm\u20131 for the ultra fine integration grid, respectively. Particularly severe divergence is observed for \u03c9B97-XD and M06-2X with discrepancies of up to \u201396 cm\u20131 and 133 cm\u20131, respectively. The mean absolute deviations for these methods are as large as 30.2 cm\u20131 (\u03c9B97-XD) and 59.5 cm\u20131 (M06-2X). These can be reduced by using the finer integration grid (super fine integration grid). This is illustrated in C1 and Cs symmetry is plotted for both grid sizes (black and blue squares). This decrease in divergence, however, occurs at the expense of distinctly higher computational costs. In case of \u03c9B97-XD and M06-2X, this leads to an mean absolute deviation of 2.8 cm\u20131 and 48.0 cm\u20131. The large value for M06-2X is caused by outliers where the deviation between calculations with Cs and C1 symmetry is enhanced by using the finer grid and \u03bd6(cF)).All density functional theory methods show deviations for anharmonic frequency (VPT2) calculations with and without the use of symmetry when the integration grid size is kept constant, whereas the results obtained with PM3 and MP2 have a negligible (\u22640.2 cmC1 or Cs), the band positions vary on average between 1\u20132 cm\u20131 for B3LYP-D3(BJ), B2PLYP-D3(BJ), and PBE0-D3(BJ). This is on the same order of magnitude as the symmetry effects discussed above. Again, a huge impact of the integration grid size is seen for \u03c9B97-XD and M06-2X, where mean absolute deviations of 28.6 cm\u20131 and up to 63.5 cm\u20131 are observed , B2PLYP-D3(BJ), and PBE0-D3(BJ). Nonetheless, one should keep in mind that individual outliers are slightly larger. Anharmonic frequency calculations with \u03c9B97-XD and M06-2X on the other hand, show substantial differences with regard to the symmetry and integration grid chosen, so that these results must be viewed with caution, as has been discussed before.Cs symmetry and the finer integration grid. Since the improvement of the accuracy is below the experimental confidence interval for the more reliable DFT methods, if present at all, Altogether, these symmetry and integration grid size dependent variations in anharmonic band positions of the fundamentals of 4cis-formic acid in a perturbation-free environment. These reference data points are essential for the validation and comparison of modern quantum chemical methods towards a more global description of this model system. Recent examples are VCI calculations of Tew and Mizukamicis- and trans-vibrations. A benchmark examining various levels of theory revealed the failure of methods like M06-2X/aVQZ VPT2 or \u03c9B97-XD/aVQZ VPT2 to give consistent results, partly due to numerical grid size and symmetry sensitivity. With the single gas phase value from 2006 compare223 cm\u20131 ), makingThere are no conflicts to declare."} +{"text": "The first catalytic \u03b1-alkynylation of cyclic amines with the help of the N-propargylic group with an exclusive high E-stereoselectivity has been realized. N-propargylic group to afford 2- N-allylic cyclic amines with an exclusive E-stereoselectivity for the in situ formed C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond has been realized. Based on mechanistic studies, it is proven that the reaction proceeds through metal-mediated anti-1,5-hydride transfer forming an iminonium intermediate, which accepts the addition of the in situ generated 1-alkynyl metal species. The synthetic application has also been demonstrated.The first catalytic \u03b1-alkynylation of cyclic amines with the help of the N-protected cyclic amines with terminal alkynes or 1-alkynyl trifluoroborate in the presence of a stoichiometric amount of an oxidant -allylic 2-alkynyl cyclic amines by using CdBr2 (or ZnI2) as the catalyst scale\" fill=\"currentColor\" stroke=\"none\">C bond , 2a (2 equiv.), and CdBr2 (10 mol%) in tBuOMe at 120 \u00b0C were defined as the optimized reaction conditions for further study of this reaction.Then solvents were screened: when 1a. Terminal aryl acetylenes bearing electron-donating p-Me and p-MeO, and electron-withdrawing and synthetically attractive p-F, p-Cl, m-Cl, p-NO2, p-EtOOC, p-CN and p-Ac groups on the aryl ring could all afford the corresponding product 3 in moderate yields (2k) and cyclohexylacetylene (2l), were found to be sluggish affording the corresponding products in 31% and 40% yields, respectively and 1-hexyne (2n) are also compatible of 2 may coordinate better with the C\u2013C triple bond to trigger the 1,5-H transfer reaction.Based on the above deuterium labeling experiments and the products in the amine 3 . In addi3ee and 3am 8 and 10 equiv. of DMSO afforded the Pauson\u2013Khand reaction product 8 in 45% yield.19Furthermore, diversified synthetic utilities of these two products were demonstrated. Suzuki coupling between 1% yield . Deprote diyne 7 . SequentN-propargylic cyclic amines, providing 1-(2(E)-alkenyl) 2- cyclic amines highly stereoselectively. Further studies on identifying the chiral catalyst, the scope of nucleophiles, and their applications to natural products are being actively pursued in the laboratory.In conclusion, we have succeeded in developing a catalytic \u03b1-alkynylation of There are no conflicts to declare.Supplementary informationClick here for additional data file.Supplementary informationClick here for additional data file."} +{"text": "Aza-analogues of carbocations inhibit \u03b4-cadinene synthase: 1,6-cyclisation. E,E)-FDP. Previous work with this enzyme using substrate analogues revealed the ability of DCS to catalyse both 1,10- and 1,6-cyclisations of substrate analogues. To test whether this apparent promiscuity was an artefact of alternate substrate use or an inherent property of the enzyme, aza analogues of the proposed \u03b1-bisabolyl cation intermediate were prepared since this cation would be formed after an initial 1,6-cyclisation of FDP. In the presence of 250 \u03bcM inorganic disphosphate both (R)- and (S)-aza-bisaboyl cations were potent competitive inhibitors of DCS . These compounds were also shown to be potent inhibitors of the 1,6-cyclase amorpha-4,11-diene synthase but not of the 1,10-cyclase aristolochene synthase from Penicillium roquefortii, demonstrating that the 1,6-cyclase activity of DCS is most likely an inherent property of the enzyme even when the natural substrate is used and not an artefact of the use of substrate analogues.\u03b4-Cadinene synthase (DCS) is a high-fidelity sesquiterpene synthase that generates \u03b4-cadinene as the sole detectable organic product from its natural substrate ( Terpene synthases catalyse some of the most complex reactions in the natural world. From a small pool of isoprenyl diphosphates they generate a myriad of hydrocarbons and alcohols that are often processed into thousands of terpenoids with diverse biological activities with many potential applications for instance as agrochemicals or therapeutic agents.1Aspergillus terreus in both closed and open conformations along with complexes containing the complete substrate , diphosphate anion and/or Mg2+ co-factors2+-ion is followed by coordination of the prenyl diphosphate substrate and a second Mg2+ ion; coordination of a third Mg2+ ion triggers active site closure to form the Michaelis complex.Abies grandis generate 34 and 52 products from farnesyl diphosphate (1), respectively.Gossypium arboreum is a high-fidelity sesquiterpene synthase that catalyses the formation of the bicyclic hydrocarbon (+)-\u03b4-cadinene (7),307DTYD311 on helix D, but instead of the usual characteristic NSE/DTE Mg2+ binding motif, DCS has a second aspartate rich motif D451DVAE455 on helix H.vide infra).R)-nerolidyl diphosphate ((3R)-NDP, (2)) as an enzyme-bound intermediate. In pathway (a), a 1,10-macrocyclisation occurs to generate cis-germacradienyl cation (4). A subsequent -hydride shift is followed by a 1,6-electrophilic ring closure to cadinenyl cation (6), from which \u03b4-cadinene (7) is formed after proton loss from C6. In pathway (b), a 1,6 ring-closure of 2 is followed by a -hydride shift from C1 to C7; subsequently a second ring closure and a -hydride shift lead to cadinenyl cation, an intermediate common to both pathways. In previous work, using substrate analogues we were unable to definitively rule out pathway (b) and indeed when 6-fluorofarnesyl diphosphate (6F-FDP) was used as a substrate analogue it proved to be a potent inhibitor (Ki = 2.4 \u03bcM), giving no detectable pentane-extractable products when incubated with DCS. This result is consistent with an initial 1,6-cyclisation pathway since it would be expected to undergo 1,10-ring closure and give an abortive product rather than inhibit the enzyme in the latter scenario. On the other hand, 2-fluorofarnesyl diphosphates (2F-FDP) and 10-fluorofarnesyl diphosphate (10F-FDP) gave products arising from 1,10- and 1,11 ring-closures, respectively, consistent with an initial 1,10-ring closure mechanism.12The details of terpene synthase chemistry2 hybridised carbocationic carbon of a given intermediate with an sp3 hybridised nitrogen in an amine analogue or with a sp2 hybridised nitrogen in an iminium ion. Although the tetrahedral tertiary ammonium ions inherently are imperfect geometric analogues of the planar carbocations, these aza-terpenoids are thought to mimic the topological and electrostatic properties of carbocations generated by these enzymes.Hence examination of the catalytic mechanism of DCS using FDP analogues has led to inconclusive, yet intriguing results, showing that this enzyme has the potential to use alternative reaction pathways. Yet the question arises, is this simply an artefact of the substrate used or is this an inherent property of the enzyme? The work described here provides alternative mechanistic data for the DCS-catalysed transformation of FDP to \u03b4-cadinene using aza-analogues of putative carbocation intermediates. Although the highly unstable carbocationic intermediates formed during terpene synthase catalysis, cannot be isolated, it is possible to replace the spS. Comparison of their effect upon catalysis by AS and amporpha-4,11-diene synthase (ADS), enzymes that follow 1,10- and 1,6-ring-closure mechanisms, validate the result that DCS has inherent 1,6- as well as 1,10 ring closure activity.Hence, the use of strategically designed aza-analogues may enable the disentanglement of the possible reaction mechanisms catalysed by DCS. Here we report the stereoselective synthesis of the two enantiomers of aza-bisabolyl cation and their kinetic evaluation as inhibitors of DC8 is a reaction intermediate on the pathway to \u03b4-cadinene (7), one or both of enantiomeric aza-analogues of 11 as a chiral auxiliary.3 as the base in CH2Cl2, diester 16 was isolated in 60% yield. Again, an asymmetric Diels\u2013Alder reaction with 2-methylbutadiene was carried out, this time at \u201310 \u00b0C in CH2Cl2 using TiCl4 as a Lewis acid catalyst yielding the R ester in 84% yield (ee = 92% and de = 97%).l-pantolactone not used for bulk preparation for the R-enantiomer of 18. Optical purity of all subsequent compounds was checked using chiral GC, HPLC and/or polarimetry and in all cases no loss of optical purity was detected in later synthetic steps.Both enantiomers of pared.18 . This waolactone as a chiS-enantiomer of 11. Hydrolysis of 14 using LiOH in an equivolume mixture of THF, water and methanol for 1 h at 50 \u00b0C gave carboxylic acid 18 in near quantitative yield. 18 was converted to p-methoxybenzyl urethane derivative 19 by treatment with diphenylphosphorylazide (DPPA) followed by a Curtius rearrangement in the presence of p-methoxybenzyl alcohol, which proceeded with strict retention of stereochemistry.19 was 60% over the two steps. Final conversion to (S)-11 was achieved first through reduction with LiAlH4 in anhydrous Et2O (50%) then HBTU mediated coupling to 4-methypent-3-enoic acid (70%) followed by a second reduction with LiAlH4 in Et2O. To prevent air oxidation upon storage the product was converted to its hydrochloride salt with HCl in ether, yielding (S)-11\u00b7HCl in 55% yield over the final two steps. The optical purity of (S)-11 was estimated to be \u226598% by comparison with previously reported data.R)-11.Both syntheses now proceeded in identical manner and Penicillium roqueforti (AS) and amorpha-4,11-diene synthase (ADS). These two enzymes are known to proceed via 1,10- and 1,6-cyclisations of the initial carbocation during their catalytic cycle -11 and (S)-11 were tested as inhibitors using a standard radiolabelled assay involving conversion of tritium labelled FDP by each enzyme and scintillation counting of the pentane extractable products.i pairs and inhibition was assessed both in the presence and absence of 250 \u03bcM diphosphate (11 with diphosphate has been observed previously for a variety of other terpene synthases.Recombinant AS and ADS were prepared and purified according to previously published procedureshosphate . Synergiv0 = kcat[E][S]/(KM + [S]). The mode of inhibition was determined by inspection of double reciprocal plots and observed to be competitive in all cases where inhibition was significant at low concentrations of 11. KI was determined from a plot of inhibitor concentration versus K\u2032M/(kcat[E]) where K\u2032M = KM(1 + [I]/KI).Kinetic data were fitted by non-linear regression to the Michaelis\u2013Menten equation (i had little effect on the ability to inhibit AS (KI > 200 \u03bcM in both the presence and absence of PPi for AS). Both enantiomers of 11 acted as competitive inhibitor of ADS, showing that they are able to compete effectively with the natural substrate FDP at the active site. As these aza-compounds cannot be turned over by ADS, these result support the intermediacy of an \u03b1-bisabolyl cation in the biosynthesis of amorpha-4,11-diene, in agreement with the findings of Picaud et al.R-enantiomer of the \u03b1-bisabolyl cation is the sole intermediate formed in the biosynthesis of amorpha-4,11-diene. Therefore, only the R enantiomer of 11 would be expected inhibit ADS; however, if the S-enantiomer was a slightly more potent inhibitor (Ki = 50 \u03bcM for (R)-11versus 25 \u03bcM for (S)-11) et al. showed that both enantiomers of the aza-analogue 11 were equally effective inhibitors of trichodiene synthase.i enhanced inhibition of ADS by both enantiomers, improving the Ki approximately 20-fold (Ki = 1.5 and 3.7 \u03bcM for the S and R enantiomers respectively) demonstrating that the active site of ADS prefers a cation\u2013anion pair in its active site.The inhibition data for AS and ADS validate both of these compounds as valuable mechanistic probes for the present investigation since they are poor inhibitors of AS and potent inhibitors of ADS. PP6) as previously described.6 in the presence of PPi but only poor inhibitors in its absence -\u03b4-cadinene (7), which would only be expected if DCS had a 1,6-cyclase activity. The use of a variety of substrate analogues possessing different stereochemistry and heteroatoms did not lead to clear results regarding whether DCS follow a 1,6 or 1,10 pathway.2) was suppressed using a fluorine atom at C2 then a 1,10 cyclisation was observed (25) was the DCS catalysed product from the transoid -2-fluorofarnesyl diphosphate (24) while the cisoid substrate analogue 26 gave the cisoid product 2F-helminthogermacrene A (27).7 along two distinct reaction paths.11 acts as a competitive inhibitor of the DCS catalysed conversion of FDP provides strong evidence that DCS can efficiently use a 1,6-cyclisation pathway.The aza-bisabolyl cations observed . 2-FluorR)-11 acts as a weak inhibitor in the absence of PPi is more difficult to explain. It was previously suggested that, in the DCS active site pocket, the alkenyl chain of (3R)-nerolidyl diphosphate (2) is ideally positioned to ensure the formation of the \u03b1-bisabolyl cation with an R configuration at C6 -9 formation is expected (R)-11 should mimic better the \u03b1-bisabolyl cation generated by this enzyme, and therefore act as competitive inhibitor with higher binding affinity when compared with the S-enantiomer. The evidence that both enantiomers of 11 are equally as effective in the presence of PPi is consistent with a permissive model of the active site structure, according to which an active site should accommodate a variety of rearranged intermediates of different shape and charge distribution without being rigidly complementary to a single intermediate. On the other hand, their lack of inhibitory effects on the 1,10-cyclase PR-AS shows that a major difference in the connectivity of the aza-analogue compared to the carbocation intermediate (i.e. bisabolyl cation rather than the 10-membered ring containing germacrenyl cation) renders them ineffective as inhibitors; hence the 1,6-cyclase activity of DCS postulated previouslyThe fact that inorganic diphosphate led to a more tightly bound active site carbocation/diphosphate ion pair is consistent with previous work where often the active site recognises a cation-PPi pair more effectively than the cation alone.on at C6 .12 The Cexpected . Hence tTerpene synthases can generate great structural and stereochemical complexity in one synthetic step and have therefore potential as powerful synthetic biocatalysts for the generation of many bioactive compounds.General experimental procedures, enzyme preparation and purification are described in ESIR)-pantolactone and Et3N in anhydrous CH2Cl2 (10 mL) at \u201324 \u00b0C. The resulting mixture was stirred for 5 h at \u201324 \u00b0C, and subsequently washed with aqueous 1 M HCl (10 mL). The aqueous phase was then extracted with CH2Cl2 (3 \u00d7 20 mL). The combined organic phases were washed with saturated NaHCO3 solution (3 \u00d7 20 mL), water (3 \u00d7 20 mL) and brine (3 \u00d7 20 mL). The organic phase was dried over MgSO4, concentrated under reduced pressure and the residue was purified by flash chromatography on silica (EtOAc\u2009:\u2009hexane 4\u2009:\u20096) to yield the pure compound as a yellow oil . \u03b4H 6.48 scale\" fill=\"currentColor\" stroke=\"none\">C), 6.18 scale\" fill=\"currentColor\" stroke=\"none\">CH2), 5.93 scale\" fill=\"currentColor\" stroke=\"none\">C), 5.40 scale\" fill=\"currentColor\" stroke=\"none\">OCH\u2013O), 4.03 scale\" fill=\"currentColor\" stroke=\"none\">O), 1.17 , 1.08 ; \u03b4C 174.7 scale\" fill=\"currentColor\" stroke=\"none\">OCHO), 172.5 scale\" fill=\"currentColor\" stroke=\"none\">OC PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019H2), 134.0 scale\" fill=\"currentColor\" stroke=\"none\">CHC PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O), 118.7 scale\" fill=\"currentColor\" stroke=\"none\">CHC PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O), 76.1 scale\" fill=\"currentColor\" stroke=\"none\">OCHO), 74.6 (OCH2CH), 40.2 (C\u2013(CH3)2), 23.4 (CH3), 23.0 (CH3). \u03b1D +10\u00b0 . Data are in agreement with previous work.Freshly distilled propenoyl chloride was added over 1 h to a stirred solution of in anhydrous CH2Cl2 (5 mL) at \u201310 \u00b0C, TiCl4 was added, and the resulting solution was stirred under argon at \u201310 \u00b0C for 1 h. 2-Methylbutadiene was then added over 5 min and the mixture was left stirring for 3 h at \u201310 \u00b0C. The reaction was quenched by addition of 10% Na2CO3 in water (5 mL). The aqueous phase was then extracted with CH2Cl2 (3 \u00d7 20 mL). The organic layers were combined, washed with H2O (3 \u00d7 10 mL), brine (3 \u00d7 10 mL), dried over anhydrous MgSO4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography on silica (EtOAc\u2009:\u2009hexane 2\u2009:\u20098) to yield pure 17 as a colourless oil . \u03b4H 5.32 scale\" fill=\"currentColor\" stroke=\"none\">O,) and scale\" fill=\"currentColor\" stroke=\"none\">), 3.98 , 2.69\u20132.51 scale\" fill=\"currentColor\" stroke=\"none\">O), 2.19 scale\" fill=\"currentColor\" stroke=\"none\">), 1.98 , 1.73 , 1.58 scale\" fill=\"currentColor\" stroke=\"none\">), 1.13 , 1.04 . \u03b4C 174.7 scale\" fill=\"currentColor\" stroke=\"none\">OCHO), 172.5 scale\" fill=\"currentColor\" stroke=\"none\">OC PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019H2), 134.0 scale\" fill=\"currentColor\" stroke=\"none\">CCH3), 118.7 scale\" fill=\"currentColor\" stroke=\"none\">CCH3), 76.1 scale\" fill=\"currentColor\" stroke=\"none\">OCHO), 74.6 (OCH2C), 40.2 (C\u2013(CH3)2), 38.9 scale\" fill=\"currentColor\" stroke=\"none\">O), 28.9 scale\" fill=\"currentColor\" stroke=\"none\">CCH2), 27.7 scale\" fill=\"currentColor\" stroke=\"none\">CHCH2), 25.2 scale\" fill=\"currentColor\" stroke=\"none\">CCH2CH2), 23.4 (CCH3), 23.0 (CCH3), 19.8 scale\" fill=\"currentColor\" stroke=\"none\">CH). LRMS (EI+) m/z: 252.13 (50%), 122.07 (20), 94.08 (100), 79.05 (30), 67.05 (10). \u03b1D \u201351.3 . Data are in agreement with previous work.16To a solution of S)-4-benzyloxazolidin-2-one in anhydrous THF (12 mL) at \u201378 \u00b0C, n-BuLi was added dropwise over 30 minutes and the mixture stirred for a further 3 h at \u201378 \u00b0C. Freshly distilled acryloyl chloride was added dropwise over 20 minutes and the reaction stirred for 2 h at \u201378 \u00b0C. The reaction was then allowed to warm to room temperature overnight. The reaction was quenched with sat. NH4Cl (20 mL) and extracted with diethyl ether (3 \u00d7 30 mL). The organic layer was washed with water (3 \u00d7 40 mL), saturated aqueous NaHCO3 (3 \u00d7 40 mL), dried (MgSO4), filtered, and concentrated under reduced pressure. Flash chromatography on silica gel (hexane\u2009:\u2009ethyl acetate 6\u2009:\u20094) afforded 13 as a colorless solid . \u03b4H 7.45 scale\" fill=\"currentColor\" stroke=\"none\">CH2), 7.23 , 6.54 scale\" fill=\"currentColor\" stroke=\"none\">CH2), 5.87 scale\" fill=\"currentColor\" stroke=\"none\">C), 4.68 , 4.14 , 3.29 scale\" fill=\"currentColor\" stroke=\"none\">CHHPh), 2.74 \u03b1D \u201386\u00b0 . Data are in agreement with previous work.14To a solution of at \u2013100 \u00b0C, were added 2-methylbutadiene in anhydrous CH2Cl2 (5.0 mL) and Et2AlCl . The reaction was stirred at \u2013100 \u00b0C for 30 min then the mixture was poured into ice cold aqueous hydrochloric acid . The mixture was extracted with CH2Cl2 (2 \u00d7 10 mL). The combined organic layers were dried over anhydrous MgSO4, filtered, and concentrated under reduced pressure. The product was purified by flash chromatography on silica (EtOAc\u2009:\u2009hexane\u2009:\u2009Et3N 92\u2009:\u20097\u2009:\u20091) to yield the Diels Alder adduct (14) as a white crystalline solid . \u03b4H 7.34\u20137.10 , 5.36 scale\" fill=\"currentColor\" stroke=\"none\">C), 4.63 , 4.23\u20133.95 , 3.60 scale\" fill=\"currentColor\" stroke=\"none\">O), 3.20 , 2.70 , 2.20\u20131.6 scale\" fill=\"currentColor\" stroke=\"none\">C), 1.64 \u03b1D +79 . LR-MS (EI+) m/z: 299.15 (100% M+), 300.16 (15), 269.06 (18), 267.07 (50), 232.10 (20), 178.08 (100), 146.07 (30), 140.03 (55), 122.07 (20), 91.00 (65), 63.00 (30). Data are in agreement with previous work.To a stirred solution of r adduct as a whi14 or 17 (0.32 mmol) in THF\u2009:\u2009MeOH\u2009:\u2009H2O , LiOH was added, and the resulting mixture was vigorously stirred for 1 h at 50 \u00b0C. The reaction was then cooled to room temperature and concentrated under reduced pressure. The resulting slurry was dissolved in H2O (10 mL) and extracted with CH2Cl2 (3 \u00d7 5 mL). The resulting aqueous phase was acidified to pH = 2 at 0 \u00b0C with 15% HCl, extracted with a mixture of n-pentane\u2009:\u2009CH2Cl2 (98\u2009:\u20092 3 \u00d7 10 mL), dried over anhydrous Na2SO4, and concentrated under reduced pressure to give 18 as a white powder . \u03b4H \u03b4 5.32 scale\" fill=\"currentColor\" stroke=\"none\">), 2.54\u20132.39 , 2.17 scale\" fill=\"currentColor\" stroke=\"none\">C), 1.93 , 1.69 , 1.59 . \u03b4C \u03b4 182.3 scale\" fill=\"currentColor\" stroke=\"none\">O), 133.8 scale\" fill=\"currentColor\" stroke=\"none\">CCH3), 119.0 scale\" fill=\"currentColor\" stroke=\"none\">CCH3), 39.0 (HC\u2013COOH), 29.13 scale\" fill=\"currentColor\" stroke=\"none\">CH3), 27.3 scale\" fill=\"currentColor\" stroke=\"none\">CCH3), 25.5 scale\" fill=\"currentColor\" stroke=\"none\">CH), 23.5 (CH3). (S)-18: \u03b1D \u201380.6 ; \u2013106.4 . (R)-18: \u03b1D +93 ; +105.5 . M.p. 82\u201392 \u00b0C. LRMS (EI+) m/z 140.06 , 136.06 (15), 125.05 (40), 122.06 (100), 95.87 (100), 94.06 (100), 93.07 (80), 79.04 (100), 77.03 (100), 68.06 (90), 67.04 (100) Data are in agreement with previous work.To a solution of 18 in anhydrous toluene (20 mL) at 0 \u00b0C, diphenylphosphoryl azide and Et3N were added. The resulting mixture was left stirring for 3 h at 100 \u00b0C before 4-methoxybenzyl alcohol was added, and the reaction was left to stir for 16 h at 100 \u00b0C. The reaction was then allowed to cool to room temperature and the solution was concentrated under reduced pressure. The residue was the purified by flash chromatography on silica gel (EtOAc\u2009:\u2009n-hexane 1\u2009:\u20099) to yield 19 as a yellow crystalline solid . \u03b4H \u03b4 7.23 , 6.81 , 5.21 scale\" fill=\"currentColor\" stroke=\"none\">C), 4.95 , 4.67\u2013460 , 3.74 , 2.29\u20132.20 , 1.93 , 2.01\u20131.86 , 1.55 scale\" fill=\"currentColor\" stroke=\"none\">CH). \u03b4C \u03b4 159.5 scale\" fill=\"currentColor\" stroke=\"none\">CO\u2013CH3), 155.8 scale\" fill=\"currentColor\" stroke=\"none\">O), 134.1 scale\" fill=\"currentColor\" stroke=\"none\">CCH3), 130.0 scale\" fill=\"currentColor\" stroke=\"none\">C aromatic), 128.7 (CCH2O), 118.3 scale\" fill=\"currentColor\" stroke=\"none\">CCH3), 113.9 scale\" fill=\"currentColor\" stroke=\"none\">C aromatic), 66.3 (CCH2O), 62.8 (CHN), 55.3 (OCH3), 31.9 (CH2CHN), 28.4 scale\" fill=\"currentColor\" stroke=\"none\">C), 28.0 scale\" fill=\"currentColor\" stroke=\"none\">C), 23.4 scale\" fill=\"currentColor\" stroke=\"none\">CCH3). \u03bdmax 3300 (N\u2013H stretch), 2900\u20132700 (C\u2013H stretch), 1650 scale\" fill=\"currentColor\" stroke=\"none\">O ester stretch), 1250 (C\u2013N stretch), 830 (aromatic CH bending); (S)-19: \u03b1D \u20139.3, (R)-19: \u03b1D +12 m.p. 69\u201371 \u00b0C LRMS (EI+) m/z: 275.15 (100% M+), 276.15 (20), 259.12 (18), 258.12 (60), 231.12 (25), 228.1128(12), 214.16 (100). HRMS (EI+) 275.1522; C16H21NO3 requires 275.1521.To a solution of 19 in anhydrous diethyl ether (7 mL) at 0 \u00b0C, was added LiAlH4 . The mixture was then heated to reflux for 5 h. The reaction was cooled to 0 \u00b0C before it was quenched by the addition of water (6 mL) and an excess of 15% NaOH solution (6 mL). The resulting mixture was left to stir at 0 \u00b0C for 1 h and the precipitate was removed by filtration through a Celite pad. The organic phase was extracted with water (2 \u00d7 10 mL) and the pooled organic layers were then washed with 10% HCl (2 \u00d7 10 mL) and the organic fraction was discarded. The combined aqueous layers were adjusted to pH 12 by dropwise addition of 10% NaOH (15 mL). The product was extracted with diethyl ether (4 \u00d7 15 mL), then dried over anhydrous MgSO4, and filtered. The product was then concentrated carefully under reduced pressure to give 20 as a volatile colorless oil . \u03b4H 5.24 scale\" fill=\"currentColor\" stroke=\"none\">C), 2.55 , 2.37 , 2.25\u20132.10 , 1.99\u20131.87 scale\" fill=\"currentColor\" stroke=\"none\">C), 1.87\u20131.66 scale\" fill=\"currentColor\" stroke=\"none\">CH), 1.61 scale\" fill=\"currentColor\" stroke=\"none\">CH), 1.60 scale\" fill=\"currentColor\" stroke=\"none\">CCH3), 1.45\u20131.26 scale\" fill=\"currentColor\" stroke=\"none\">CH). \u03b4C 134.0 scale\" fill=\"currentColor\" stroke=\"none\">CCH3), 119.1 scale\" fill=\"currentColor\" stroke=\"none\">CCH3), 54.8 (CHNH), 32.7 (CH2CHNH), 32.1 scale\" fill=\"currentColor\" stroke=\"none\">CHCH2CH), 29.7 (CH3N), 29.0 scale\" fill=\"currentColor\" stroke=\"none\">), 23.4 scale\" fill=\"currentColor\" stroke=\"none\">) (S)-20: \u03b1D \u201379 (R)-20: \u03b1D +84 Data in agreement with previous work.14To a stirred solution of carbamate 20 and DIPEA in anhydrous DMF (6 mL), HBTU was added and the resulting mixture was stirred at room temperature for 20 min before 20 was added. The reaction was then stirred for 24 h at room temperature. The mixture was concentrated under reduced pressure and the residue was dissolved in diethyl ether (20 mL). The solution was washed with water (2 \u00d7 25 mL), 10% NaHCO3 (2 \u00d7 25 mL), 10% HCl (2 \u00d7 10 mL), and brine (25 mL) before it was dried over anhydrous MgSO4 and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (EtOAc\u2009:\u2009hexane 4\u2009:\u20096) yielding 21 as a colorless oil . \u03b4H 5.24 scale\" fill=\"currentColor\" stroke=\"none\"> and PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CHCH2C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O), 4.70\u20134.54 , 3.75 , 3.01 scale\" fill=\"currentColor\" stroke=\"none\">O), 2.75 , 2.72 , 2.20\u20131.86 , 1.71\u20131.88 , 1.75 scale\" fill=\"currentColor\" stroke=\"none\">CHCH3), 1.68 scale\" fill=\"currentColor\" stroke=\"none\">CCH3), 1.66 scale\" fill=\"currentColor\" stroke=\"none\">CCH3). (S)-21: \u03b1D \u20139.5, (R)-21: \u03b1D +10, HRMS (EI+) 221.1779; C14H23NO requires 221.1780. Data are in agreement with previous work.14To a stirred solution of 21 in anhydrous diethyl ether at 0 \u00b0C, was added LiAlH4 . The mixture was heated to reflux for 6 h then allowed to cool to room temperature and stirred for a further 12 h. The reaction was quenched by the addition of water (6 mL) and 15% NaOH (6 mL) at 0 \u00b0C and stirred for 1 h at 0 \u00b0C. The white precipitate was removed by filtration on Celite and the filtrate was extracted with diethyl ether (2 \u00d7 25 mL). The combined organic layers were dried over anhydrous Na2SO4, concentrated under reduced pressure, and the residue was purified by flash chromatography on silica (Et2O\u2009:\u2009MeOH 1\u2009:\u20099) to yield the amine as a yellow oil . \u03b4H 5.27 scale\" fill=\"currentColor\" stroke=\"none\">CH), 5.03 2C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CH), 2.64\u20132.44 , 2.38 , 2.23 , 2.08 , 2.04\u20131.85 , 1.85\u20131.67 scale\" fill=\"currentColor\" stroke=\"none\">CHCH2CHN), 1.62 scale\" fill=\"currentColor\" stroke=\"none\">CH), 1.55 and 1.57 2C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CH). \u03b4C \u03b4 133.9 scale\" fill=\"currentColor\" stroke=\"none\">CCH3), 132.6 scale\" fill=\"currentColor\" stroke=\"none\">CCH3), 122.1 scale\" fill=\"currentColor\" stroke=\"none\">CCH3), 120.0 scale\" fill=\"currentColor\" stroke=\"none\">CCH3), 58.9 (CHN), 53.5 (CH2N), 37.9 (CH2CH2CHN), 30.8 scale\" fill=\"currentColor\" stroke=\"none\">CHCH2N), 27.2 (CH2CCH3), 26.5 (CH3N), 25.7(CH2CH2N), 25.6 (CH3CCH3), 23.2 (CH3CCH3), 17.8 scale\" fill=\"currentColor\" stroke=\"none\">). HRMS (EI+) 207.1990; C14H25N requires 207.1987. (S)-11: \u03b1D \u201361 (R)-11\u03b1D +63 . Data are in agreement with previous work.2O (1 mL) and HCl (1 M in anhydrous Et2O) was added slowly. A light yellow precipitate was formed. The ether was concentrated under reduced pressure and the salt stored in 1.2 mL of deionised water. \u03b4H 5.38 scale\" fill=\"currentColor\" stroke=\"none\">CH), 5.14 2C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CH), 3.58\u20133.42 , 3.32 , 3.25\u20132.97 , 2.85 , 2.59\u20132.39 , 2.39\u20132.26 , 2.26\u20132.05 scale\" fill=\"currentColor\" stroke=\"none\">CHCH2CH2N), 1.84 scale\" fill=\"currentColor\" stroke=\"none\">CHCH2), 1.76 scale\" fill=\"currentColor\" stroke=\"none\">CH), 1.72 2C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019CH). \u03b4C \u03b4 136.1 scale\" fill=\"currentColor\" stroke=\"none\">CCH3), 134.3 scale\" fill=\"currentColor\" stroke=\"none\">C(CH3)2) 117.6 scale\" fill=\"currentColor\" stroke=\"none\">CCH3) 116.5 scale\" fill=\"currentColor\" stroke=\"none\">CCH3), 61.8, 61.5 (CHN), 53.0, 52.5 (CH2N), 35.9, 35.2 (CH3N) 29.1, 29.0 (CH2CH2CHN), 25.7 scale\" fill=\"currentColor\" stroke=\"none\">CHCH2N), 24.5 (CH2CCH3), 24.2, 24.0 (NCH2CH2), 23.25, 22.64 scale\" fill=\"currentColor\" stroke=\"none\">), 21.64 (CH3CCH3), 16.65 (CH3CCH3). \u03bdmax 2972 , 1379 (C\u2013N stretch), 1161, 1051 and 1022 (C\u2013N stretch), 950, 879, 815; HRMS (APCI+) 208.2057, C14H26N requires 208.2065; m.p. 131\u2013133 \u00b0C, (S)-11\u03b1D \u201362.2 , (R)-11\u03b1D +57.1 . Data are in agreement with previous work.14To a stirred solution of There are on conflicts of interest to declare.Supplementary informationClick here for additional data file."} +{"text": "Ultra-permeable and robust membranes are prepared by creating a gradient nanoporous structure in low-cost phenolics, enabled by the spontaneous assembly of gradually enlarged phenolic nanoparticles. 2-accelerated thermopolymerization of resol in the progressive downward gelating polymer. Subsequent removal of the gelated polymer and ZnCl2 exposes the gradient nanopores. The gradient nanopores endow the phenolic structures with unprecedented permselectivity when used in membrane separation, totally rejecting fine particulates down to 5 nm dispersed in water or aggressive solvents while allowing water to permeate up to two orders of magnitude faster than other membranes with similar rejections. Our work opens up an avenue for the rational design and affordable synthesis of ultrafast membranes.Membrane technology is playing a pivotal role in providing potable water to our thirsty planet. However, the strong demand for highly permeable and durable membranes with affordable costs remains. Such membranes are synthesized herein by designing gradient nanopores in low-cost phenolics. The gradient nanopores are achieved by spontaneous assembly of phenolic nanoparticles with gradually enlarged sizes. These particles nucleate and grow as a result of ZnCl However, the selective layer also needs to be mechanically robust to withstand the pressure that drives the separation across the membrane. Therefore, asymmetric structures with a thin selective layer on top of a mechanically strong substrate are predominantly applied for membrane separation.From the Hagen\u2013Poiseuille equation which correlates the structural parameters of porous membranes with permeability (in situ performed on the surface of the polysulfone ultrafiltration (UF) membrane, producing a tight polyamide layer with a thickness of \u223c100\u2013400 nm on the UF substrate.3N4,Thin-film composite reverse osmosis (RO) membranes are best known for their asymmetric structures.via conventional methods. Alternatively, in precipitation polymerization, phenolic particles are precipitated from solution and form aggregates with certain porosity depending on the polymerization conditions. We expect that if the precipitation of phenolic particles occurs in a suitably controlled way, there might be a chance to have a well-organized assembly of phenolics with tunable porosity favoring fast mass transfer. Moreover, composite structures with a thin layer on top of a macroporous substrate have been widely used to improve the membrane permeance. And these composite structures frequently suffer from complicated preparation processes and poor interfacial adhesion between the two layers.To meet this demand, attention should also be paid to low-cost and easily available materials. Phenolic resins have long been used in many different fields because of their low cost and superior chemical, thermal, and mechanical robustness.2 and is coupled with the gelation of Pluronic polymer (PEO-block-PPO-block-PEO block copolymer) with the fast evaporation of the solvent. Under these conditions, phenolic nanoparticles nucleate and grow with gradually increased sizes from the top to the bottom and spontaneously assemble to form membranes with a gradient porous structure. Remarkably, the thus-produced phenolic membranes outperform not only commercial membranes (by a factor of 20\u201380 in terms of permeance) but also newly developed advanced membranes based on costly 2D materials. Also importantly, the phenolic membranes are selective and robust enough to completely reject fine particulates with a size down to a few nanometers dispersed either in water or in aggressive solvents. The aim of this work is to report an ultrafast membrane for water purification and also to demonstrate the unlimited potentials of low-cost materials in developing ultrafast membranes.Based on these considerations, we demonstrate herein the design and synthesis of phenolic-based advanced membranes by establishing a highly gradient nanoporous structure in phenolics. The precipitation polymerization of phenolics is accelerated by ZnCl2 was cast on the surface of a flat substrate and evaporated at 100 \u00b0C to produce a thin film supported on the substrate , Pluronic polymer (P123) and ZnCle Fig. S1. During Fig. S2a. Interes Fig. S2a. We thena. 67 nm . The pora. 67 nm . Spherica. 67 nm , formingd Fig. S3. This meWe tried to directly characterize the pore sizes. However, in the gradient structures, the pores are defined as gaps formed by stacking phenolic particles. These pores are interconnected with each other and do not have a measurable profile. Therefore, the sizes of such highly irregular pores cannot be estimated. Instead, the sizes of spherical phenolic particles from the top to the bottom of the membranes can be relatively easily measured, and the gaps between them are highly related to the particle sizes.2SO4 or water. As shown in \u20131 originate from hydroxyl groups,2.\u20131 in the spectrum of the as-synthesized phenolic film, which can be attributed to the interaction between ZnCl2 and phenolic group. With the introduction of ZnCl2, the aromatic C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019C bond in the phenolic film at 1605 cm\u20131 is enhanced due to the facilitated aromatization of the phenolic film by ZnCl2 while the deoxygenation effect of ZnCl2 weakens the characteristic \u2013C PBM data was replaced with SVG by xgml2pxml: 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000\tCreated by potrace 1.16, written by Peter Selinger 2001-2019O bond of the phenolic film at 1648 cm\u20131.\u20131. After the removal of ZnCl2 by water or H2SO4, the merged peak is decomposed to the initial two isolated peaks at 1605 cm\u20131 and 1648 cm\u20131. The peak of C\u2013O stretching of P123 was reported to be located at 1100 cm\u20131.\u20131 as a result of the complexation of Zn2+ to P123.2 by water, the peak recovers from 1075 cm\u20131 to 1100 cm\u20131. Moreover, a peak at 1100 cm\u20131 shows up with a weaker intensity for the water-soaked sample, indicating that P123 was only partially removed by water. In contrast, after soaking in H2SO4, the peak disappears, indicating the complete removal of P123.Infrared (IR) spectroscopy was applied to reveal the change in the chemical composition of the phenolic film before and after soaking in H2SO4 are able to completely remove ZnCl2 from the phenolic films. The peak around 533 eV originates from C\u2013OH of phenolic and ether groups of P1232, releasing some coordinated \u2013OH groups. The strong peak of this group is also achieved for the H2SO4-soaked film scale\" fill=\"currentColor\" stroke=\"none\">O) of the phenolic film2SO4 is capable of removing the oligomeric phenolic group and further facilitating the crosslinking of phenolics at 100 \u00b0C, the H2SO4-soaked film exhibits the strongest peak around 284.8 eV among all the samples. Meanwhile, the degradation of ester bonds takes place in H2SO4 at high temperature, resulting in the lowest peak intensity of around 286.5 eV for the H2SO4-soaked film.X-ray photoelectron spectroscopy (XPS) was further used to analyze the phenolic films before and after soaking. Zinc and chlorine elements are distributed on the surface of the as-synthesized phenolic film but absent in the water- and acid-treated films , confirmked film . However2 was not included in the simulation systems, and this simplification does not influence the reliability of the simulated results because ZnCl2, as a small molecule, would not evidently change the relative diffusivity and collision of solute molecules.We carried out molecular dynamics (MD) simulations to investigate the diffusion and reactions of resol molecules under fast evaporation conditions. We changed the concentrations of solutes (P123 and resol with a fixed molar ratio) to describe the progressive evaporation of ethanol. The concentration of solutes was progressively decreased from the top surface to the bottom with the top surface having a 100% concentration. 2SO4, both P123 and ZnCl2 are removed, leaving behind the network composed of interconnected phenolic particles as a nanoporous membrane with gaps among the particles as the pores tapered from the bottom side to the top side and cytochrome C were measured to be 96% and 95%, respectively .2, and MXene, our phenolic membranes with gradient porosities also show \u223c1.5\u20136 times higher permeabilities and similar rejections . The gol2SO4 soaking was a homogenous film composed of small phenolic particles without a noticeable gradient porous structure . For instance, the thickness is increased to 4.7 and 12.0 \u03bcm at a molar ratio of 70 and 280, respectively. Considering that the increase in thickness is caused by the formation of pores in the phenolic structure with the introduction of ZnCl2, we can easily estimate the porosity of the phenolic membranes by comparing their thicknesses with that of the phenolic structure produced in the absence of ZnCl2.2 dosages lead to larger porosities. Cross-sectional SEM examinations confirm that the phenolic membrane prepared at the molar ratio of 70 exhibits a relatively dense morphology while the membrane prepared at the molar ratio of 280 is highly porous .2. With the fast evaporation of the solvent, the ZnCl2-accelerated thermopolymerization of resol induces the nucleation of phenolics as nanoparticles. They continue to grow until the gelating P123 terminates the supply of resol, thus forming phenolic nanoparticles with increasing sizes from the top to the bottom. These phenolic particles further assemble to form a network with the stabilization effect of P123 and ZnCl2. After removal of P123 and ZnCl2, phenolics with a gradient nanoporous structure are formed. The thus-produced phenolic structures exhibit unprecedented separation performances when used as membranes. They show \u223c20\u201380 times higher water permeance than commercial membranes with similar rejections. Impressively, they also greatly outperform membranes based on expensive 2D materials by a factor of \u223c1.5\u20136 in terms of water permeance while their selectivities being comparable. Furthermore, the phenolic membranes are robust enough to operate in aggressive organic solvents. Such a gradient nanoporous structure combined with excellent chemical and thermal stability makes the thus-produced phenolic superstructures an exciting candidate for applications in other areas including batteries, supercapacitors, catalysis, etc.In summary, gradient nanopores are produced by controlling the thermopolymerization of resol in the mixture of Pluronic polymer (P123) and ZnClThere are no conflicts to declare.Supplementary informationClick here for additional data file."} diff --git a/PMC_clustering_444.jsonl b/PMC_clustering_444.jsonl new file mode 100644 index 0000000000000000000000000000000000000000..6e4c6c096e1cd32c401d1fff550e1b7a10828031 --- /dev/null +++ b/PMC_clustering_444.jsonl @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:a712199e1ce2937d404e4de8dc52bf21a858358676f991e8d841964ce31051f0 +size 28416986 diff --git a/PMC_clustering_445.jsonl b/PMC_clustering_445.jsonl new file mode 100644 index 0000000000000000000000000000000000000000..a1e9f05bb540f5b35d8174ed0bea5de2cae4987e --- /dev/null +++ b/PMC_clustering_445.jsonl @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:f622ef598638df2b73b79c9fe5cc27c6d2c89409a98e123162709b783f815cdf +size 83787217 diff --git a/PMC_clustering_446.jsonl b/PMC_clustering_446.jsonl new file mode 100644 index 0000000000000000000000000000000000000000..57028dd74c8fbd668c8f624ec07c58771727f68c --- /dev/null +++ b/PMC_clustering_446.jsonl @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:ccbe5aa2b570934ef030afd6bb907d1cf017722a15d8116a9a030352bbce57cf +size 24261972 diff --git a/PMC_clustering_447.jsonl b/PMC_clustering_447.jsonl new file mode 100644 index 0000000000000000000000000000000000000000..5c7820805e3bebb1eae604672710972e2e4a58cf --- /dev/null +++ b/PMC_clustering_447.jsonl @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:86ba6dab964d5b8a7cdb9e55e0daa88c3c749b8e544e420d444372b889c001b0 +size 113913134 diff --git a/PMC_clustering_448.jsonl b/PMC_clustering_448.jsonl new file mode 100644 index 0000000000000000000000000000000000000000..6e055cd4e5d0be81a7bd3adbdd1bcf9877f1328e --- /dev/null +++ b/PMC_clustering_448.jsonl @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:eeb84d73fdc3f506d0cb57f9224a35ce4c49a0952dca22d0a71e7da567c37b66 +size 17643464 diff --git a/PMC_clustering_449.jsonl b/PMC_clustering_449.jsonl new file mode 100644 index 0000000000000000000000000000000000000000..966154bfceda30b7e45fe368d5b53dda2c225569 --- /dev/null +++ b/PMC_clustering_449.jsonl @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:8080e25400d7c1f0f59721ff12621f6b8d660ae0d555f1b27b707cebf45cf930 +size 13894664 diff --git a/PMC_clustering_450.jsonl b/PMC_clustering_450.jsonl new file mode 100644 index 0000000000000000000000000000000000000000..6a9fcba2e42130f6b0520c7a1845fc61aa90a101 --- /dev/null +++ b/PMC_clustering_450.jsonl @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:0c9fd6190c3190fad085b968a0d8045186cce4f269f497ba407006dfa6b571c6 +size 60693141 diff --git a/PMC_clustering_451.jsonl b/PMC_clustering_451.jsonl new file mode 100644 index 0000000000000000000000000000000000000000..c044feec776824a8d8e161f737fff24ffc99fceb --- /dev/null +++ b/PMC_clustering_451.jsonl @@ -0,0 +1,1747 @@ +{"text": "The invasion of freshwater ecosystems by non\u2010native species can constitute a significant threat to native species and ecosystem health. Non\u2010native trouts have long been stocked in areas where native trouts occur and have negatively impacted native trouts through predation, competition, and hybridization. This study encompassed two seasons of sampling efforts across two ecoregions of the western United States: The Great Basin in summer 2016 and the Yellowstone River Basin in summer 2017. We found significant dietary overlaps among native and non\u2010native trouts within the Great Basin and Yellowstone River Basin ecoregions. Three orders of invertebrates composed the majority of stomach contents and were responsible for driving the observed patterns. Great Basin trout had higher body conditions (k), and non\u2010native Great Basin trout had higher gut fullness values than Yellowstone River Basin trout, indicating a possible limitation of food in the Yellowstone River Basin. Native fishes were the least abundant and had the lowest body condition in each ecoregion. These findings may indicate a negative impact on native trouts by non\u2010native trouts. We recommend additional monitoring of native and non\u2010native trout diets, regular invertebrate surveys to identify the availability of diet items, and reconsidering stocking efforts that can result in overlap of non\u2010native fishes with native cutthroat trout. Non\u2010native trout in the western United States pose a significant threat to native trout species. We found significant dietary overlaps among native and non\u2010native trouts within the Great Basin and Yellowstone River Basin ecoregions. Selectivity analysis also suggested that native trout were more selective and may be adapting to non\u2010native trout pressure by selecting different diet items. Our aim was to quantify dietary overlap and dietary variability among native and non\u2010native trout species occurring in both ecoregions. We hypothesized that (a) within ecoregions, native cutthroat trout would have specialist diets with high levels of dietary selectivity and lower levels of dietary overlap with non\u2010native trouts, (b) non\u2010native trout would have more generalist diets with lower levels of selectivity and higher levels of dietary overlap, and (c) cutthroat trout would maintain low variability in dietary selectivity between ecoregions, while non\u2010native trout species would have higher variability in dietary selectivity between ecoregions.22.1We sampled 23 sites in three rivers of the Great Basin: the Bear, Carson, and Humboldt Rivers, and 20 sites in three rivers of the Yellowstone River Basin: the Bighorn, Powder, and Tongue Rivers Figure\u00a0. Sites w2.22) of benthic invertebrates that was conducted at all study sites at the time of fish collection was used to determine the proportional environmental abundance of each diet item for selectivity analyses.Fish collections were performed during July and August 2016 in the Great Basin and July 2017 in the Yellowstone River Basin. At each site, fishes were collected from reaches measuring 20 times the average wetted width of the stream and mean prey abundance (Ni) were used to quantify diets of individual fishes. FO was calculated as follows:FO is the occurrence of a prey item Fi divided by the number of nonempty guts (P). The metric FO describes the percentage of individuals that have consumed a specific food item. While this metric does not provide details on amounts of items consumed, it is robust to limitations of other diet analysis challenges such as differences in prey condition and presence of unidentifiable tissues. was used to compare feeding behavior and diet composition among fishes were found in both ecoregions but excluded for low presence (3/23 sites (n\u00a0=\u00a022) in the Great Basin and 1/20 sites (n\u00a0=\u00a02) in the Yellowstone River Basin) and Paiute sculpin (Cottus beldingii) (n\u00a0=\u00a060) were captured in the Great Basin, but were excluded from these analyses due to a complete absence in the Yellowstone River Basin. We analyzed two native subspecies of cutthroat trout: Bonneville cutthroat trout (Oncohynchus clarkii utah) and Yellowstone cutthroat trout (Oncorhynchus clarkii bouvieri) and three non\u2010native species: brook trout , brown trout , and rainbow trout (Oncorhynchus mykiss). Of the 464 fishes analyzed, 15 individuals (~3%) had empty stomachs with all but one collected from the Great Basin. Despite the increased occurrence of empty guts, Great Basin trout had significantly higher body condition factors than trout in the Yellowstone River Basin .We captured and measured 1,102 fishes and processed 464 guts from four species of trout in the U.S. Great Basin and Yellowstone River Basin than in the Yellowstone River Basin . Great Basin brook trout and brown trout had significantly higher gut fullness than the same species in the Yellowstone River Basin, while rainbow trout and both subspecies of cutthroat trout did not differ among ecoregions. In the Great Basin, non\u2010native brook trout and brown trout had higher gut fullness values than native cutthroat but in the Yellowstone River Basin this relationship was reversed.Gut fullness was significantly higher in Great Basin fishes (ANOVA 3.2The frequency of occurrence (FO) of diet items provided the simplest quantification of diets. When we categorized diet contents to family level, we detected a wide range of relationships among fish species . In the Great Basin, Bonneville cutthroat trout diets significantly differed from brook trout diets . Within the Yellowstone River Basin, the greatest dissimilarity in diets was between Yellowstone cutthroat trout and rainbow trout . We did not find any significant differences when we compared Yellowstone cutthroat trout that co\u2010occurred with non\u2010native species to Yellowstone cutthroat trout that were collected at sites that did not have non\u2010native trout present .ANOSIM results suggested similar diet overlap trends as the NMDS plots. In the Great Basin and the Yellowstone River Basin ecoregions, brook trout, brown trout, and rainbow trout diets did not differ significantly . Brook trout and rainbow trout diets differed significantly, but these differences were minor and likely ecologically insignificant for invertebrate order abundances in fish guts with NMDS axis scores displayed a number of dietary trends Figure\u00a0. Cutthro4We found significant dietary overlaps among native and non\u2010native trouts within the Great Basin and Yellowstone River Basin ecoregions. Three orders of invertebrates composed the majority of stomach contents and were responsible for driving the observed patterns. Great Basin trout had higher body conditions, and non\u2010native Great Basin trout had higher gut fullness values than Yellowstone River Basin trout overall. Low body condition and gut fullness may indicate food limitation in the Yellowstone River Basin, and when considered in combination with dietary overlap, can suggest evidence for interspecific competition and the lowest gut fullness of all trout. Great Basin brown trout were less abundant but had excellent body condition (k\u00a0\u2265\u00a01.6) and the second highest overall gut fullness values of all sampled fishes. These physical factors suggest that Yellowstone River Basin brown trout may be experiencing intraspecific competition that affects growth ; Data curation (lead); Formal analysis (lead); Investigation ; Writing\u2010original draft (lead); Writing\u2010review & editing . Emily R. Arsenault: Conceptualization ; Investigation ; Writing\u2010original draft ; Writing\u2010review & editing . Bolortsetesg Erdenee: Investigation ; Methodology (supporting); Writing\u2010review & editing . Alain Maasri: Funding acquisition (lead); Investigation ; Writing\u2010review & editing . Mark Pyron: Conceptualization ; Funding acquisition (lead); Investigation ; Methodology ; Project administration ; Writing\u2010original draft ; Writing\u2010review & editing ."} +{"text": "Climate change is expected to alter the distributions of species around the world, but estimates of species\u2019 outcomes vary widely among competing climate scenarios. Where should conservation resources be directed to maximize expected conservation benefits given future climate uncertainty? Here, we explore this question by quantifying variation in fish species\u2019 distributions across future climate scenarios in the Red River basin south\u2010central United States. We modeled historical and future stream fish distributions using a suite of environmental covariates derived from high\u2010resolution hydrologic and climatic modeling of the basin. We quantified variation in outcomes for individual species across climate scenarios and across space, and identified hotspots of species loss by summing changes in probability of occurrence across species. Under all climate scenarios, we find that the distribution of most fish species in the Red River Basin will contract by 2050. However, the variability across climate scenarios was more than 10 times higher for some species than for others. Despite this uncertainty in outcomes for individual species, hotspots of species loss tended to occur in the same portions of the basin across all climate scenarios. We also find that the most common species are projected to experience the greatest range contractions, underscoring the need for directing conservation resources toward both common and rare species. Our results suggest that while it may be difficult to predict which species will be most impacted by climate change, it may nevertheless be possible to identify spatial priorities for climate mitigation actions that are robust to future climate uncertainty. These findings are likely to be generalizable to other ecosystems around the world where future climate conditions follow prevailing historical patterns of key environmental covariates. Expected change in the number of species within the Red River basin by the year 2050 across nine future climate scenarios, as projected by Maxent. The value for each raster 1/8\u00b0 cell represents the difference in species\u2019 probability of occurrence between the historical period and the year 2050, summed across all species. GBIF serves as one of the most extensive biogeographical resources in the world for each species. These fitted SDMs describe the historical probability of occurrence of each species across the Red River basin. Second, we projected the future distribution of each species under a range of potential climate scenarios by coupling our fitted species distribution models with projected values of climatic and hydrologic variables under future climate scenarios. Third, we summarized inter\u2010 and intraspecies variability in future stream fish distributions across these climate scenarios.We selected covariates for our SDM analysis by choosing environmental factors within the Red River Basin which are known to drive the distribution of stream fish species and are commonly used for modeling stream fish distributions . These nine scenarios were selected based on air temperature and precipitation biases over the south\u2010central United States to serve as a proxy for conductivity, which is known to be an important driver of fish assemblages in portions of the Red River basin , generalized additive models (GAMs), boosted regression trees (BRTs), and the MaxEnt maximum entropy model in the Red River Basin found that optimizing the regularization multiplier between 1.5\u00d7 and 2.0\u00d7 was necessary to prevent over\u2010prediction while staying under the target training omission rate of 30% . For other species, like Gambusia affinis and Lepomis cyanellus, changes to their projected distributional extent vary widely across the nine climate scenarios. For G.\u00a0affinis, its distribution is projected to increase to encompass an additional 7.6% of the basin under the most optimistic climate scenario (under GCM MIROC5 and RCP 4.5) in 2050 under MaxEnt. However, the most pessimistic climate scenario is dramatically different and suggests that its future distribution will contract and fail to include 34% of the basin where it historically occurred (under GCM MPI_ESM_LR and RCP 4.5).Species differed markedly in projected changes to their distributional range under future climate scenarios, and also in the variability of these projected outcomes across climate scenarios Figure\u00a0. For exaG.\u00a0affinis, L.\u00a0cyanellus, and Cyprinella lutrensis are all historically widely distributed across the basin, but are projected to occupy a much narrower portion of the basin in all future climate scenarios. Conversely, absolute changes to the distributional range of several species that were historically narrowly distributed were small because those species were rare to begin with. However, we did not find a significant correlation between historical distributional extent and proportional change in distributional extent in 2050 than for species that were not SGCN listed . We also found that the median outcome across climate scenarios was significantly better for pelagic\u2010broadcast spawners than for all other species . However, we did not find significant differences in outcomes among species when comparing all spawning modes ; here, we compared pelagic\u2010broadcast spawners , riverine spawners , egg burriers or attachers , and nesting species .We observed significant differences in distributional changes among groups of species. The median outcome across climate scenarios . In other cases, species considered as vulnerable by NatureServe , experienced dramatic range contraction across all climate scenarios. When considering conservation actions for imperiled species, conservation practitioners should recognize that future climate projections and SDM outputs entail multiple sources of error, and for some species model fit (as measured by AUC) was relatively lower. Thus, the precautionary principle species are projected to face the largest absolute changes in distributional extent Figure\u00a0 also undIn most cases, the relative importance of environmental covariates in our models Table\u00a0 mirrors Our analysis of projected fish species distributions under future climate scenarios highlights opportunities for conservation practitioners and decision makers to make pro\u2010active investments in fish conservation to mitigate potential climate impacts. Given the importance of stream temperature and flow, conservation practitioners might use our SDM projections to identify specific locations where species\u2019 distributions are likely to contract due to unfavorable stream temperature and flow. In these locations, practitioners may consider strategies to mitigate climate impacts by ensuring adequate instream flows ; Data curation (lead); Formal analysis (lead); Methodology ; Validation ; Visualization ; Writing\u2010original draft (lead); Writing\u2010review & editing . Rachel E Fovargue: Conceptualization ; Formal analysis ; Methodology ; Visualization ; Writing\u2010review & editing . Thomas Neeson: Conceptualization ; Formal analysis ; Funding acquisition (lead); Methodology ; Supervision (lead); Writing\u2010review & editing ."} +{"text": "A Caucasian male with known severe aortic stenosis was referred to our Ophthalmology Department after undergoing cardiac surgery using extracorporeal circulation. Signs of retinal ischaemia were found during fundus examination and neuroimaging showed posterior cerebral artery occlusion. Extracorporeal circulation involves a variety of changes in the organism\u2019s hemodynamics involving vasodilation and vasoconstriction. Retinal tissue is an easy target for complications to occur as its circulation is an end-arterial system.A 53-year-old Caucasian male was referred to our department after undergoing cardiac surgery as he reported that he was not able to see his left visual field. He suffered from severe aortic stenosis associated with mitral insufficiency and dilation of the ascending aorta. Cardiac surgery was performed using extracorporeal circulation and lasted for over 450 minutes. It involved a Bentall procedure and mechanical mitral valve implantation.Fig. 1), not present in preoperative ophthalmological examination. Visual acuity was 1 in both eyes using Snellen\u2019s visual acuity chart. Anterior segment slit lamp examination showed no pathological signs and intraocular pressure was not altered. Fundus examination revealed bilateral cotton-wool exudates and a white-centred retinal haemorrhage compatible with a Roth\u2019s spot in his right eye, suggesting retinal ischaemia . Neuroimaging was then requested and a right posterior cerebral artery occlusion was reported. However, these lesions did not explain the patient\u2019s clinical symptoms so a visual field campimetry and microperimetric examination using Macular Integrity Assessment were carried out. Left homonymous hemianopia was confirmed and microperimetric examination revealed altered values in the right eye\u2019s temporal area and in the left eye\u2019s nasal area, which were consistent with the visual field campimetry findings , although homonymous hemianopia persisted.Fundus lesions started to spontaneously remit, especially in his left eye, after the second week of follow-up (1] and cases of ischemic optic neuropathy have been reported as a result of hypoperfusion [2]. Retinal artery occlusion risk has also been proven to be higher in open-heart surgeries compared to those non open-heart surgeries [3].Cardiac surgery, especially when using extracorporeal circulation, has been linked to a variety of ocular post-operative manifestations. Optic nerve head blood flow has been proven to significantly decrease during these surgeries [4], simultaneous bilateral retinal detachment after coronary artery bypass graft [5], retinal microembolisms demonstrated by fluorescein angiography [6], flame-shaped haemorrhages and soft exudates [7].Other ophthalmological post-operative findings include diffuse chorioretinopathy [8].Impaired cerebral blood flow during cardiac surgery has also been described [A combination of vascular complications linked to cardiac surgery were presented in this case report, both of which affected the visual system but originated in different tissues: retinal ischaemia and posterior cerebral artery occlusion.Correlation between optic tract disease, visual field alterations and microperimetry changes were shown, highlighting the importance of microperimetry in order to determine the extent of macular involvement not clearly detailed in the visual field examination. This could have important prognostic implications as central values were altered in Macular Integrity Assessment tests, visual therapy techniques could be recommended in order to develop extrafoveal fixation and improve quality of life in these patients.Carrying out a systematic ophthalmological examination or a vision capacity questionnaire in patients undergoing these surgeries is of great importance as waiting for the patient to complain about visual symptoms may sometimes be too late to act. Complications described highlighted the necessity of a higher control of hemodynamic parameters during cardiac surgery, especially when using extracorporeal circulation, in order to minimize peripheral tissues from suffering.Conflict of interestAuthors declare no conflict of interest.FundingThere are no funders to report for this submission.Informed consentConsent was gathered from the patient in order to obtain and publish these images."} +{"text": "With five years gone since the adoption of the UN Agenda for Sustainable Development, the global community is moving far too slowly toward achieving its 2030 objectives for older persons. Achieving proper rights and wellbeing for older persons requires strategic and concerted action. The WHO Decade of Healthy Ageing initiative is one useful step toward defining the diverse physical, psychological, social and healthcare needs of older persons. But government leadership remains weak, and the nonprofit sector plays the key role spearheading advocacy efforts toward the SDG motto of \u201cleave no one behind.\u201d The urgent needs of older persons \u2013 whether fighting ageism, upholding the rights of people living with dementia, or promoting lifelong learning and contributions to society - can best be met by societies that \u201creframe aging\u201d and craft creative and innovative solutions. This presentation showcases NGOs that are leading the way in cultivating such transformative solutions."} +{"text": "Human programmed death\u20101 immune checkpoint inhibitor therapy is being rapidly introduced in metastatic renal cell carcinoma (mRCC) clinical practice in Japan, dramatically changing the therapeutic strategy for mRCC. The combination of radiation and immune checkpoint inhibitor therapies has gained particular interest because many reports have indicated that, in addition to radiation\u2010targeted lesions, non\u2010targeted distant lesions could also be shrunk by simultaneous or sequential combinations.IJU Case Reports, Nakajima et al. reported a possible abscopal effect of radiation therapy after discontinuation of nivolumab in mRCC.In this issue of in vitro experiments, which demonstrated that the RCC cell line was amongst the most radioresistant.RCC has historically been considered intrinsically radioresistant, and the radioresistance has been verified through The author declares no conflict of interest."} +{"text": "Knowledge graphs can support many biomedical applications. These graphs represent biomedical concepts and relationships in the form of nodes and edges. In this review, we discuss how these graphs are constructed and applied with a particular focus on how machine learning approaches are changing these processes. Biomedical knowledge graphs have often been constructed by integrating databases that were populated by experts via manual curation, but we are now seeing a more robust use of automated systems. A number of techniques are used to represent knowledge graphs, but often machine learning methods are used to construct a low-dimensional representation that can support many different applications. This representation is designed to preserve a knowledge graph\u2019s local and/or global structure. Additional machine learning methods can be applied to this representation to make predictions within genomic, pharmaceutical, and clinical domains. We frame our discussion first around knowledge graph construction and then around unifying representational learning techniques and unifying applications. Advances in machine learning for biomedicine are creating new opportunities across many domains, and we note potential avenues for future work with knowledge graphs that appear particularly promising. They have been used in social network mining to classify nodes Within a biomedical setting, some graphs can be considered knowledge graphs; although, precisely defining a knowledge graph is difficult because there are multiple conflicting definitions Biomedical knowledge graphs are often constructed from manually curated databases In this review we describe various approaches for constructing and applying knowledge graphs in a biomedical setting. We discuss the pros and cons of constructing a knowledge graph via manually curated databases and via text mining systems. We also compare assorted approaches for applying knowledge graphs to solve biomedical problems. Lastly, we conclude on the practicality of knowledge graphs and point out future applications that have yet to be explored.2Knowledge graphs can be constructed in many ways using resources such as pre-existing databases or text. Usually, knowledge graphs are constructed using pre-existing databases. These databases are constructed by domain experts using approaches ranging from manual curation to automated techniques, such as text mining. Manual curation is a time-consuming process that requires domain experts to read papers and annotate sentences that assert a relationship. Automated approaches rely on machine learning or natural language processing techniques to rapidly detect sentences of interest. We categorize these automated approaches into the following groups: rule-based extraction, unsupervised machine learning, and supervised machine learning and discuss examples of each type of approach while synthesizing their strengths and weaknesses.2.1Database construction dates back all the way to 1956 when the first database contained a protein sequence of the insulin molecule Semi-automatic methods are a way to accelerate the curation process Despite the negatives of manual curation, it is still an essential process for extracting relationships from text. This process can be used to generate gold standard datasets that automated systems use for validation 2.22.2.1Rule-based extraction consists of identifying essential keywords and grammatical patterns to detect relationships of interest. Keywords are established via expert knowledge or through the use of pre-existing ontologies, while grammatical patterns are constructed via experts curating parse trees. Parse trees are tree data structures that depict a sentence\u2019s grammatical structure and come in two forms: a constituency parse tree and a deGrammatical patterns can simplify sentences for easy extraction Pattern matching is a fundamental approach used to detect relationship asserting sentences. These patterns can consist of phrases from constituency trees, a set of keywords or some combination of both Rule-based methods provide a basis for many relationship extraction systems. Approaches in this category range from simplifying sentences for easy extraction to identifying sentences based on matched key phrases or grammatical patterns. Both require a significant amount of manual effort and expert knowledge to perform well. A future direction is to develop ways to automate the construction of these hand-crafted patterns, which would accelerate the process of creating these rule-based systems.2.2.2Unsupervised extractors draw inferences from textual data without the use of annotated labels. These methods involve some form of clustering or statistical calculations. In this section we focus on methods that use unsupervised learning to extract relationships from text.An unsupervised extractor can exploit the fact that two entities may appear together in text. This event is referred to as co-occurrence and studies that use this phenomenon can be found in Full text articles are able to dramatically enhance relationship detection Unsupervised extractors often treat different biomedical relationships as multiple isolated problems. An alternative to this perspective is to capture all different types at once. Clustering is an approach that performs simultaneous extraction. Percha et al.\u00a0used a biclustering algorithm on generated dependency parse trees to group sentences within PubMed abstracts Overall unsupervised methods provide a means to rapidly extract relationship asserting sentences without the need of annotated text. Approaches in this category range from calculating co-occurrence scores to clustering sentences and provide a generalizable framework that can be used on large repositories of text. Full text has already been shown to meaningfully improve the performance of methods that aim to infer relationships using cooccurrences 2.2.3Supervised extractors use labeled sentences to construct generalized patterns that bisect positive examples from negative ones . Most of these approaches have flourished due to pre-labelled publicly available datasets . These dSome supervised extractors involve the mapping of textual input into a high dimensional space. SVMs are a type of classifier that can accomplish this task with a mapping function called a kernel Deep learning is an increasingly popular class of techniques that can construct their own features within a high dimensional space Recurrent neural networks (RNN) are designed for sequential analysis and use a repeatedly updating hidden state to make predictions. An example of a recurrent neural network is a long short-term memory (LSTM) network Convolutional neural networks (CNNs), which are widely applied for image analysis, use multiple kernel filters to capture small subsets of an overall image Semi-supervised learning Weak or distant supervision takes a different approach by using noisy or even erroneous labels to train classifiers 3Knowledge graphs can help researchers tackle many biomedical problems such as finding new treatments for existing drugs 3.1Mapping high dimensional data into a low dimensional space greatly improves modeling performance in fields such as natural language processing 3.1.1Matrix factorization is a class of techniques that use linear algebra to map high dimensional data into a low dimensional space. This projection is accomplished by decomposing a matrix into a set of small rectangular matrices : nodes represent patients, drugs and diseases while edges represent relationships such as a patient being prescribed a treatment or a patient being diagnosed with a disease Early work in this field applied translational models and the National Institutes of Health (T32 HG000046)David Nicholson Funded by The Gordon and Betty Moore Foundation (GBMF4552) and the National Institutes of Health (R01 HG010067).Casey S. Greene Funded by DavidN.Nicholson: Conceptualization, Funding acquisition, Investigation, Writing - original draft, Visualization. Casey S. Greene: Conceptualization, Funding acquisition, Supervision, Writing - review & editing.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper."} +{"text": "Gaps exist in training medical residents to assess social determinants of health (SDOH) related to chronic conditions. To address the need for better screening, we partnered with two Internal Medicine (IM) residency programs based in Lansing and Flint (Michigan) to pilot the Caring for Patients with Chronic Conditions (CPCC) project. IM residencies train internists with expertise in diagnosis, treating chronic conditions, promoting health through wellness education, and preventing and managing diseases. CPCC incorporated information during didactic sessions that residents could apply during their clinical activities that can influence their current and future clinical practice patterns. Presentations and panels from local community organizations on specific topics were incorporated into the curriculum that address needs of patients age 50 and older. To build on this education, the residents adapted the Office- Guidelines Applied in Practice (Office-GAP) checklist to identify SDOH affecting a patient\u2019s ability to managed chronic conditions. Using this tool: 1) involves resident training; 2) provides a decision support checklist; 3) influences patient activation; and 4) increases provider and patient communication through shared decision making. The checklist includes questions for patient response pertaining to SDOH that prevents them from managing their chronic conditions in addition to the level of action the patient is willing to do. Areas identified are discussed between patient and resident increasing patient activation. Referrals to community-based resources to identified SDOH needs are guided by the clinic\u2019s care manager. The Office-GAP tool is administered during three subsequent visits to ensure that patients actually accessed the community resources."} +{"text": "Conservation of traditional poultry breeds is closely linked to product valorization considering the renewed interest in local poultry breeds production and consumers\u2019 orientation towards food items considered healthier and safer. The aim of the present research is to investigate the differences in egg physical parameters and fatty acid profile of three traditional chicken breeds and two commercial hybrids using marked-procured eggs. We evaluated the effects of the breeds and of the genetic origin on egg physical and chemical parameters, furthermore we analyzed the most influencing parameters and their effects on egg groups differentiation via Principal Component Analysis (PCA). Eggs produced by traditional breeds differentiate from eggs produced by commercial hybrids in physical and fatty acids parameters. The nutritional value of eggs obtained from traditional breeds has been demonstrated to be higher considering the yolk content, the Polyunsatured Fatty Acids - PUFA fraction, the n6/n3 ratio and the atherogenic and thrombogenic indexes. Commercial layers\u2019 eggs revealed their higher commercial value based on weight, albumen content and percentage of edible content.The aim of the present study is to investigate the physical parameters and fatty acid composition and related nutritional parameters of market-procured table eggs from Milanino, Mericanel della Brianza and Valdarnese Bianca hens compared to two commercial hybrid strains\u2019 eggs to determine characterizing quality traits for traditional breeds conservation and valorization through high quality niche products. Fifty-four market eggs by three traditional breeds and two commercial hybrid strains have been analyzed\u2014physical parameters, fatty acids profile and atherogenic and thrombogenic indexes were investigated. A General Linear Model\u2014GLM was applied to data analysis with breed and genetic origin as sources of variation. Two Principal Component Analyses (PCA) were carried out with physical parameters and fatty acid parameters as variables. Eggs produced by traditional breeds MRC MLN and VLD differentiate from eggs produced by commercial hybrids CHB and CHW in physical and chemical parameters (fatty acids parameters). The nutritional value of the traditional eggs has been demonstrated to be higher considering the yolk content, the PUFA fraction, the more favorable n6/n3 ratio and the atherogenic and thrombogenic indexes. Commercial layers\u2019 eggs revealed their higher commercial value based on weight, albumen content and percentage of edible content. Animal production has been characterized, in the past century, by a strong intensification of production systems and a dramatic reduction of traditional breeds. Traditional breeds well adapted after centuries of natural and artificial selection in specific geographic and climatic areas have been replaced with highly productive synthetic strains or commercial hybrids ,2.Two main sectors can be identified in the global poultry production\u2014the large scale production led by multinational corporations based on highly productive hybrid strains and the small scale production characterized by a variety of traditional breeds and populations (predominant in developing countries) . In deveGallus gallus domesticus egg physical parameters and fatty acid profile have been widely demonstrated to be discriminating genetic-based factors in breed differentiation [Traditional breeds and local populations are characterized by specific genetic backgrounds enabling them to adapt to specific conditions and to produce valuable products . Productntiation ,13,14,15ntiation .Mericanel della Brianza, Milanino and Valdarnese Bianca breeds are autochthonous Italian breeds from the Lombardy region and from the Tuscany region . Milanino and Valdarnese Bianca are mainly considered meat type traditional breeds, Mericanel della Brianza is mainly known for its brooding ability. No data are available about the characteristics of the table eggs from these breeds.The aim of the present study is to investigate the physical parameters and fatty acid composition and related nutritional parameters of table eggs from Mericanel della Brianza, Milanino and Valdarnese Bianca hens compared with two commercial hybrid strains\u2019 eggs. These parameters are used to characterize quality traits for traditional breeds conservation and valorization through high quality niche products. All eggs were purchased from commercial sources.Fifty-four market eggs from three traditional breeds and two commercial hybrid strains were analyzed. Birds\u2019 breeds, samples distribution, genetic origin, egg shell color, hen weight range and official recognition by the official poultry club recognized by Agriculture Ministry FIAV are listed in Physical parameters were recorded by individually weighing the whole egg (WEG), the yolk , the albumen (ALB) and the shell . Edible part , albumen, yolk, shell percentage and edible fraction percentage were calculated on the whole egg weight.The total lipids were extracted from singular yolk samples in a suitable excess of chloroform/methanol ,17. FattStatistical analysis was performed by the analysis of variance (ANOVA) using General Linear Model procedure of SPSS to assess the effect of the breed and of the genetic origin on egg physical parameters and fatty acids profile, the post hoc Bonferroni test was used to investigate the significant differences at breed and genetic origin levels . In ordeAll physical parameter variables were significantly influenced by the breed of the hen and the genetic origin. Physical parameters are presented in Egg weights differed among the breeds, Mericanel della Brianza (MRC), the only bantam breed, had significantly lighter eggs than those of all other breeds. Valdarnese Bianca (VLD) eggs were significantly lighter then Commercial Hybrid Brown (CHB) and Milanino (MLN) eggs. The genetic origin significantly influenced egg\u2019s weight, eggs produced by commercial hybrids HYB were the heaviest.Albumen weight was higher in the two hybrids strains than in the three traditional breeds. The highest albumen weight was recorded in CHB birds, the lowest in MRC.Yolk weight differentiated the breeds and their genetic origin. The highest yolk weight was recorded in Milanino (MLN) eggs. Traditional breeds (TRD) birds produced eggs with a heavier yolk weight.Eggshell weight was different in the five considered groups\u2014MLN birds produced the heaviest shell. The HYB eggs had heavier shells when compared with all breeds of TRD genetic origin.The edible part was heavier in the heaviest eggs\u2014CHB eggs, edible fraction weight was higher in HYB eggs than TRD eggs.Physical parameters percent proportions gave us a clearer definition of the occurring differences within the five groups\u2019 eggs considering the ratios between the different sections of the egg.Albumen percentage was higher in HYB eggs than in TRD eggs (62.60% vs. 48.38%); CHB eggs had the highest albumen content\u201463.77%, the lowest percentage was recorded in MRC eggs 42.14%.Yolk percentage is inversely proportional to albumen percentage\u2014TRD eggs were higher than HYB eggs (36.52% vs. 24.17%). MRC was the top breed for yolk percentage\u201441.15% almost doubling CHB (22.96%).Shell percentage is higher in TRD eggs than in HYB eggs (15.09% vs. 13.22%). MRC the highest shell proportion (16.70%) which highly differentiate it from CHB and CHW shell proportion .The edible part percentage is higher in HYB eggs and in CHW in particular.PCA scatter plot for physical parameters is presented in A significant effect of the breed on all the detected fatty acids composition in egg yolk was recorded . PalmitiPCA scatter plot for fatty acids parameters is presented in Egg physical parameters, fatty acids profile and nutritional values differentiate eggs from traditional breeds and commercial strains. The genetic origin influences egg quality parameters at physical and nutritional levels. Focusing on physical parameters whole egg weight is a basic quality characteristic which regulates egg commercial value according to European Union (EU) legislation about egg grading procedures (2295/2006/CE). CHB and CHW hens laid the heaviest eggs, within the traditional breeds only MLN showed similar weight values. MRC is a bantam breed and so egg weight is clearly lighter. The genetic origin of egg weight is well known together with hen age and with oviposition curve point of lay . Our resAlbumen and albumen content were significantly different in the studied groups\u2014HYB eggs were characterized by an higher proportion of albumen (62.60 vs. 48.38%). Our results are in accordance with those reported by Rizzi and Chiericato (2005) and Hocking et al. (2003), who described the same trend ,14. AlbuEggshell and eggshell percentage were significantly higher in TRD breeds; MRC showed the highest eggshell relative weight . No significant differences were recorded between CHB CHW and MLN and VDN eggs. Hocking et al. (2003) reported no difference in the relative weight of eggshell from local and commercial breeds . The samConsidering the PCA and the scatterplot in Egg lipids have high nutritional and biological values considering their function as major energy source and as provider of different essential components in embryo development and functionality . Egg yolThe PCA for fatty acid parameters revealed the 83% of the variance being related to PUFA and n6/n3 proportion. Traditional breeds\u2019 eggs differentiate from HYB eggs on the first component (55.07% of the variance). MRC and MLN eggs are totally overlapping while VLD eggs are close to CHW eggs, similar to the physical parameters\u2019 analysis. Except of MRC and MLN which cluster together VLN, CHW and CHB showed high clustering ability. Fatty acids profile is able, when considering PUFA and n6/n3 ratio in particular to differentiate eggs groups from different breeds and genetic origin.per se value of the traditional breeds is mainly based on adaptation ability and product quality. Objective and effective product characterization procedures could supply helpful tools in genetic reservoir valorization and in breeders\u2019 motivation to produce valuable niche products [In conclusion, market procured eggs produced by Mericanel della Brianza, Milanino and Valdarnese Bianca traditional breeds differentiate from eggs produced by brown and white commercial hybrids under physical and chemical parameters (fatty acids parameters). The nutritional value of the traditional eggs has been demonstrated to be higher considering the yolk content, the PUFA fraction and n6/n3 ratio and the atherogenic and thrombogenic indices. Commercial layers\u2019 eggs revealed their highest commercial value based on weight, albumen content and percentage of edible content. The products ."} +{"text": "Research in the nursing home industry shows more robust knowledge management activities are associated with the adoption of patient-centered, culture change initiatives among high Medicaid nursing homes. These findings are notable because they highlight the important role that knowledge management activities may play for improving quality of care in under-resourced nursing homes. They also raise important questions about the conditions that may support or hinder the use of these activities. Using survey responses from 393 nursing home administrators, we empirically examined whether two components of human resources \u2013 staffing levels and HRM practices \u2013 are associated with the level of knowledge management activities in high Medicaid census nursing homes. More robust HRM practices were associated with greater levels of knowledge management activities, as well as three separate domains of knowledge management . Staffing levels, in contrast, were not significantly associated with the level of knowledge management activities."} +{"text": "The described model combines sophisticated hepatic surgery with new insights regarding the physiology of livers perfused ex situ, and authors foresee its use in advanced cancer treatment including by partial liver autotransplantation.We read the publication by Mueller et al with great interest.Our team has used normothermic machine perfusion strategies as a platform for mechanistic research. In our setting, although we were unable to modify the liver resection technique to live donor-like dissection, our development objective was to establish models that are more applicable to wider research community. We present our experience focused on approaches to restore vascular inflow to partial human hemi livers, with vascular reconstruction for right and left hepatectomy specimens, as well as full-size diseased livers obtained from transplant recipient hepatectomies. Key perfusion characteristics hopefully act as a reference point to guide others in development of such research perfusion protocols.For the partial liver perfusion model, the important planning steps were the assessment of preoperative CT scans and discussion with the surgeon regarding the minor alterations of the hepatectomy procedure with respect to hepatic pedicle transection. Following failures to reconstruct hepatic arteries divided within parenchyma using stapler devices, we targeted operations where we envisaged: The right or left hepatic artery could be safely separated and divided outside the parenchyma; parenchyma transection likely to be performed without a Pringle\u2019s maneuver; and favorable tumor location to obtain representative tissue samples before commencing normothermic perfusion. n recipient hepatectomies, the explanted specimen was more standardized, and we targeted procedures without history of transarterial chemoembolization, with patent portal vein, and transplantations without long warm ischemia caused by routine creation of temporary portocaval shunt. For both types, the artery stump was flushed with heparinized saline before being closed with a bulldog clamp. If feasible, the liver was flushed with cold preservation solution immediately after being handed over by the operating team.2 The hepatic artery wall dissection was critical challenge to overcome and represented the principal cause for technical failure. Direct cannulation of the artery yielded poor flows during normothermic perfusion that were overcome by inclusion of arterial conduit. Satisfactory results were achieved with a donor artery or synthetic (PTFE or Dacron) vascular graft. Achieving a suitable diameter match was difficult for both these options with donor artery being preferred for partial livers inserted into the bile duct either on the back-table, if the bile duct was clearly identified, or after commencing the perfusion if the duct orifice could be identified by bile production.3 Similarly, it also allows for any bleeding from the resection plane to be recirculated. The technical success of our normothermic perfusion strategy was estimated based on the hepatic artery flows and liver ability to clearance perfusate lactate.4Experiments used the liver assist device (detail) that incorporates semiopen circuit. Importantly, this device did not require hepatic venous reconstruction as the perfusate is collected and recirculated via the device reservoir.The provided technical information is complimentary to Dr Mueller and colleagues\u2019 report and may allow wider adoption of long-term machine perfusion models as a flexible platform with extensive research applications including pharmacology, small molecules cellular therapy, and cancer studies."} +{"text": "Recovery narratives are an effective healing art medium that support individual development by virtue of the shared human experience transmitted through each story. Older adult recovery narratives are unique as they share their experiences of aging with a mental health conditions to support others with similar difficulties. Older adult recovery narratives offer encouragement of self-determination in late life, share lived experience of aging with a mental health condition, and promote age-related self-management skills development. The RecoverYdia smartphone app provides an online community for older adults with a lived experience of a mental health condition. RecoverYdia subscribers can search through hundreds of relevant videos and find the storyteller who tells the viewer\u2019s story, prompts them to reach out for help, and eventually inspires them to help others. This presentation will discuss the state of evidence regarding the evidence for recovery narratives across the globe and offer a RecoverYdia technology demonstration."} +{"text": "Research on the impact of age views on cognition has seen a strong momentum in recent years, fitting the stereotype embodiment theory prediction that the stereotypes taken in from a culture can impact older persons\u2018 cognition. These studies utilize experimental, longitudinal, and ecological momentary assessments (EMA), as well as a wide reach of cognitive outcomes. This symposium starts with two experimental studies. One demonstrates that negative age stereotypes reduce cognitive processing in older consumers . A second study strives to better understand the pathway by which age stereotypes influence cognitive outcomes by focusing on dysregulation of reward-seeking behaviors and the downregulation of the dopaminergic system . We next explore two longitudinal studies that reveal differential relations among views of aging and various cognitive indicators. The first study found that older persons with more positive age beliefs are less likely to develop dementia even in a high-risk gene subpopulation of older adults . The second study examined the association between awareness of age-related changes and cognitive scores Finally, L\u00fccke et al. examine in their EMA study with 6 measurement occasions per day across 7 days that such a fine-tuned seems not to clearly support a linkage among subjective age and working memory for which beginning but not consistent evidence has been reported previously. Brad Meisner will discuss contributions in the light of meta-analytic finding revealing that older persons\u2018 negative age stereotypes can impair whereas their positive age stereotypes can improve cognitive performance."} +{"text": "Although individualization of ovarian stimulation aims at maximal efficacy and safety in assisted reproductive treatments, in its current form it is far from ideal in achieving the desired success in women with a low prognosis. This could be due a failure to identify such women who are likely to have a low prognosis with currently used prognostic characteristics. Introduction of the patient-oriented strategies encompassing individualized oocyte number (POSEIDON) concept reinforces recognizing such low prognosis groups and stratifying in accordance with important prognostic factors. The POSEIDON concept provides a practical approach to the management of these women and is a useful tool for both counseling and clinical management. In this commentary, we focus on likely management strategies for POSEIDON group 2 criteria. The notable contribution to this success is the introduction of ovarian stimulation into ART in the early 1980s (Success following assisted reproductive treatments (ART) has improved significantly since the early years of ly 1980s . Soon afly 1980s . Since tA review in 2000 enlisted around 28 criteria used for the definition of POR and a laHowever, there has been skepticism whether the ESHRE consensus, the Bologna criteria for defining POR is fit for purpose and whether the new consensus definition mitigated clinical heterogeneity. The ESHRE consensus definition of POR considers proven poor responders based on previous cycle performances and predicted poor responders as one category, does not consider suboptimal response, does not factor in female age and the oocyte competence in terms of embryos aneuploidy rate . AdditioThe main objective of individualization of ovarian stimulation (OS) is to offer women the best treatment tailored to her own unique characteristics, thus maximizing the chances of pregnancy and eliminating the iatrogenic and avoidable risks resulting from ovarian stimulation . It currA systematic review and meta-analysis on predictive factors in IVF evaluated nine common predictors and found the following factors of female age, duration of infertility, basal follicle stimulating hormone (FSH) levels, the number of retrieved oocytes, and embryo quality to be associated with the chances of pregnancy . Older fYounger women have a favorable prognosis in achieving a live birth compared to older women. An important reason for this is the increase in aneuploid embryos and consequent decrease in euploid embryos with increasing female age. Whereas, female age influences the embryo euploidy rate, euploidy rate remains stable in relation to the embryo cohort sizes, thereby resulting in more euploid embryos with higher number of embryos . It therThe POSEIDON stratification is aimed at clinical management by considering the most important prognostic factors and stratifying women accordingly. Group 1: women aged <35 years with adequate ovarian reserve with an unexpected poor response (<4 oocytes) or a suboptimal response (4\u20139 oocytes); Group 2: women aged \u2265 35 years with adequate ovarian reserve with an unexpected poor response (<4 oocytes) or a suboptimal response (4\u20139 oocytes); Group 3: women aged <35 years with poor ovarian reserve ; Group 4: women aged \u2265 35 years with poor ovarian reserve .The aim of defining the POSEIDON groups is to individualize therapeutic approaches by fine tuning OS in terms of the right pituitary suppression regimen, the ideal gonadotrophin selection, along with dosage and optimize ovarian response and number of oocytes to obtain a euploid embryo with the highest implantation potential for transfer. POSEIDON classification reinforces the avoidance of iatrogenic suboptimal response underpinning likely genetic variants such as FSH receptor polymorphism , variantAs the aim is to improve egg numbers, these women may benefit from a higher gonadotrophin dose over the standard 150 IU \u2013 225 IU daily for OS agonist regimen is associated with a significantly higher oocyte yield over the short GnRH agonist regimen, and as such should be the preferred downregulation regimen with the use of GnRH agonists . Given ty for OS . There inant FSH dependinectively . WhetherP < 0.01) and euploid blastocysts after FPS compared to LPS. A systematic review which included eight studies and 338 women, reported no compromised in quality or quantity of oocytes retrieved following LPS compared to FPS is attempted in the same menstrual cycle . Earlierd to FPS . A \u201cfreed to FPS . CurrentOverall, such group of women in POSEIDON group 2 might benefit recognition and whether they could benefit from novel strategies or inteventions such as adjuvant androgen therapies, addition of growth hormone, preimplantation genetic testing for aneuploidy (PGT-A) warrants further research. Management options for POSEIDON group 2 women is summarized in Double stimulation protocol needs validation in POSEIDON group 2 population along with cost-effectiveness and safety data. SS drafted the manuscript with contribution from GR and MK. All authors approved final version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Lung transplant recipients are at particular high risk for postoperative respiratory failure as a result of poorly controlled pain, inadequate graft expansion, decreased cough, and reliance on systemic opioid therapy. Thoracic epidural and paravertebral blocks have been employed with the goal of improving postoperative pain control, improving pulmonary mechanics, and limiting the need for narcotic administration. These approaches require a needle position in proximity to the neuraxis and may cause significant hypotension that is poorly tolerated in transplant patients. Additionally, the use of anticoagulation or underlying clotting disorder limits the use of these regional blocks because of the concern of hematoma and subsequent neurologic injury. Ultrasound-guided continuous erector spinae plane (ESP) block has been shown to be efficacious for pain control following thoracotomy but has had minimal investigations following lung transplantation. In this study, we describe the effective use of a continuous erector spinae plane block to provide analgesia in a postoperative lung transplant recipient receiving systemic anticoagulation. The use of an ESP block with a more superficial needle tract that is further removed from the neuraxis allowed for a greater safety profile while providing efficacious pain control, decreased reliance on systemic narcotics, and improved oxygen saturation. The ESP block was effective in this case and thus may be a valuable alternative following lung transplantation for patients who are not candidates for thoracic epidural or paravertebral approaches. Thoracotomy for lung transplantation is associated with poorly controlled postoperative pain for a variety of reasons including preoperative deconditioning, elevated anxiety levels, established tolerance to analgesic medications, and dosing limitations of opioids as to avoid hemodynamic compromise . While t2) of 65% at 50 liters (L) to maintain her oxygen saturation >88% on continuous pulse oximetry. For the first 24 hours postextubation, the patient maintained adequate oxygen saturation on HFNC with a range of 40\u201370\u2009L and FiO2 of 45\u201365% without attempting to ambulate. Shortly following extubation, the patient reported uncontrolled pain, despite repeated administration of intravenous opioids (pain scores 5/10 at rest and 10/10 with exertion), with resultant difficulty participating in adequate pulmonary hygiene at fraction of inspired oxygen and continued to have respiratory compromise despite HFNC of 60\u201370% at high flows. During a 130-foot walk, the patient was unable to maintain oxygenation saturation levels above 87% despite continued use of HFNC with FiOThoracic epidural analgesia and thoracic paravertebral block are mainstays for management of postthoracotomy pain, but the ESP block can serve as an effective alternative \u201311. A grAn ESP block requires a relatively superficial needle path targeting a myofascial plane deep to the erector spinae muscle allowing for easier sonographic identification of the pertinent anatomy. Performing an ESP block may present a greater safety profile by virtue of being further removed from the pleura and spinal cord with less-associated hemodynamic disturbance and potentially less risk of bleeding leading to neurologic compromise. While there continues to be a lack of convincing data regarding the risk of hematoma formation for deeper blocks with a wide range of anticoagulation medications, the consequences of hematoma leading to cord compression are less significant as one travels further from the neuraxis. Whether to consider the ESP as a superficial or deep block in regards to risk of hematoma formation is unknown secondary to a lack or randomized controlled trials. Of note, one small case series reported the use of ESP in 5 patients with an international normalized ratio >1.5 without any recognizable bleeding or neurologic complications .While the available literature for the use ESP for patients undergoing thoracic surgical procedures is still limited, a growing body of evidence suggests that ESP is a viable alternative for thoracic surgical procedures. Further studies comparing ESP catheters to thoracic epidural and paravertebral approaches are needed to assess the optimal analgesic technique for patients undergoing lung transplantation.In this case, the use of a continuous erector spinae block improved pain control following single lung transplantation as evidenced by decreased pain scores and opioid administration while concomitantly improving oxygenation. Immediate recovery following lung transplantation is facilitated by adequate pain control thus allowing for improved respiratory mechanics. Considering that thoracic epidural and paravertebral blocks are frequently not a viable option in this patient population, we propose that an ESP block can serve as an effective regional technique that minimizes systemic opioids while improving deep breathing and graft expansion."} +{"text": "Housing is the main spatial context for aging, important for well-being, a sense of identity and independence in daily life. Yet, as people grow older housing needs change and knowledge about how people reason about their future home when they enter retirement age is lacking. This qualitative study presents findings that explored meaning of home and health dynamics in the present and in a projected future among community-living people aged 67 \u2013 70 years. Findings suggest that the home becomes progressively important after retirement. Not only the immediate home environment but also local neighborhoods influence perceptions about home. Home brings emotional and social benefits but also worries about how to cope with complex home ambivalence when reflecting upon future housing arrangements. The findings highlight the importance of considering perceived aspects of home and could be used to raise awareness among policymakers, housing authorities and professionals involved in housing-related counselling."} +{"text": "Within older adult marriages, spouses often help regulate one another\u2019s emotions, especially in times of stress . Although research shows that better emotion regulation skills are associated with better social skills , less is known about whether better emotion regulation skills in one spouse are associated with greater perceived relationship quality from their partner\u2019s point of view. Further, very little is known about these dynamics in the context of early stage dementia in which both spouses may be feeling more stress over time and need support from one another. In the present study, we hypothesized that (a) one spouse\u2019s greater emotion regulation skills would be associated with their own greater perceived emotional support from their partner and (b) their partner would also report greater perceived emotional support. We made the same hypothesis for both the person with dementia and their spouse without dementia. We used self-report baseline data from an intervention study of 45 older adult married couples (N=90) where one spouse has dementia. Spouses completed questionnaires that measured their emotional regulation habits and perceived emotional support from their partner . Results from the actor partner interdependence model showed that when both partners had high emotional regulation skills there were the highest levels of perceived partner support for each dyad member =-2.37, p=.023). Findings suggest couple-focused interventions to enhance emotion regulation skills are important for maintaining relationship quality in the early stages of dementia."} +{"text": "Neurological complications after cardiac catheterization are rare. We report an unusual case of isolated third cranial nerve palsy in a 72-year-old male patient whose past medical history was significant for diabetes mellitus and coronary artery disease (CAD). He presented for elective cardiac catheterization for stable angina, which revealed multivessel CAD and no intervention was done. Two hours after the procedure, the patient suddenly started complaining of new-onset double vision in his left eye. Ophthalmologic exam revealed ptosis of the left eye lid, sluggish pupillary reflex and impaired adduction of the left eye along with exotropia of the left eye on primary gaze, all findings consistent with the left third nerve palsy. Rest of the neurological exam and neuroimaging (CT angiogram of head and MRI brain) were normal. Embolic phenomenon has been described as a possible mechanism in such patients leading to small vessel ischemic disease and cerebral microinfarction. Neuro-ophthalmologic complications after cardiac catheterization are rare but devastating for the patients. These should be recognized promptly, and patients should undergo neuroimaging to evaluate for any identifiable causes. These patients should be treated with aspirin and statin therapy and evaluated by ophthalmology for correction with prism lenses if symptoms persist. Every year there are more than 1,000,000 cardiac catheterizations performed in the US . AlthougWe present a case of a 72-year-old male patient who presented for an elective cardiac catheterization for further evaluation of stable angina. His medical history was significant for diabetes mellitus and coronary artery disease (CAD) with a drug-eluting stent (DES) placed 16 years ago. He underwent cardiac catheterization through right radial artery approach which revealed multivessel CAD, no intervention was done and he was referred to cardiothoracic surgery team for evaluation of coronary artery bypass graft (CABG).Two hours after the procedure, the patient suddenly started complaining of persistent visual disturbance and described it as seeing everything double; he had never experienced similar complaints in the past. He denied any headache, dizziness, speech difficulty or motor or sensory weakness in his extremities. The patient was hemodynamically stable and found to be in sinus rhythm. Ophthalmologic exam revealed mild ptosis of the left eyelid, sluggish pupillary reflex in the left eye and exotropia of the left eye on primary gaze along with impaired adduction of the left eye Figures , 2.There were no abnormal findings in the right eye. The patient also complained of diplopia with downward and outward gaze, and these symptoms resolved transiently by covering his left eye. Rest of the exam for other cranial nerves was normal, and there were no focal motor or sensory deficits appreciated. Computed tomography angiography (CTA) of head and neck did not show any evidence of intracranial hemorrhage or territorial infarct, and revealed a small stable arteriovenous malformation (AVM) in right pons. MRI\u00a0of the brain did not show any abnormal enhancement and third cranial nerve appeared normal bilaterally, and again demonstrated small cavernous malformation in right pons Figure similar The patient continued to have persistent diplopia during his hospital stay, underwent CABG and was discharged 10 days later in stable condition. The patient followed up in ophthalmology clinic in four weeks; at this time, he complained of same visual complaints and there was no change in exam findings compared to his discharge from the hospital described above. The patient again followed up in clinic eight weeks later and his symptom of diplopia had now completely resolved. Ophthalmologic exam revealed his left eye ptosis had resolved, left pupil was round and reactive to light, and adduction of the left eye was normal.Our patient\u2019s ophthalmologic exam findings were consistent with third cranial nerve palsy on initial presentation. Neuro-ophthalmological syndromes after cardiac catheterization are rare and to our knowledge, only one case of isolated third cranial nerve palsy has previously been reported in literature which resolved spontaneously within a few days.The exact pathophysiology for neurological complications after cardiac catheterization remains unclear but embolism has been proposed as the primary mechanism, mainly cerebral embolism originating from large atherosclerotic vessels .\u00a0This hyNeuro-ophthalmologic syndromes after cardiac catheterization are extremely rare but can be devastating for the patients. Given preponderance for involvement of posterior cerebral circulation and risk for CVA, any neurological complaints including visual disturbance after cardiac catheterization should be promptly evaluated with appropriate imaging and followed up routinely for resolution of symptoms."} +{"text": "We have developed computational models of human aging that are based on complex networks of interactions between health attributes of individuals. Our \u201cgeneric network model\u201d (GNM) captures the population level exponential increase of mortality with age in Gompertz\u2019s law together with the exponential decrease of health as measured by the frailty index (FI). Our GNM includes only random accumulation of damage, with no programmed aging. Our GNM allows large populations of model individuals to be quickly generated with detailed individual health trajectories. This allows us to explore individual damage propagation in detail. To facilitate comparison with observational data, we have also developed and tested new approaches to binarizing continuous-valued health data. To extract the most information out of available cross-sectional or longitudinal data, we have also reconstructed interactions from generalized network models that can predict individual health trajectories and mortality."} +{"text": "Medicare home health providers are required to offer family caregiver training; however, there is little information regarding the impact of family caregiver training on patient outcomes in home health or other care delivery settings. A better understanding of this relationship is necessary to guide development of caregiver training interventions and inform policy discussions surrounding family caregiver training access. This research assesses whether and how unmet need for family caregiver training is associated with acute care utilization during Medicare home health. We examine 1,217 fee-for-service Medicare beneficiaries who participated in the National Health and Aging Trends Study (NHATS) and received Medicare-funded home health care between 2011-2016. We link NHATS data with home health patient assessments and Medicare claims, drawing measures of family caregivers\u2019 need for training from home health clinician reports and determining provision of training from Medicare claims. Using weighted, multivariable logistic regressions, we model the marginal change in probability of acute care utilization during home health as a function of family caregivers\u2019 unmet need for training. We found that older adults whose family caregivers had an unmet need for training had a probability of acute care utilization during home health that was 18 percentage points (p=0.001) greater than those whose family caregivers both needed and received training, holding all covariates at their means. Findings support the importance of connecting family caregivers to training resources and suggest one avenue by which investing in caregiver training may be cost-effective for integrated payers and providers."} +{"text": "Relationship quality is an important factor affecting care partners\u2019 health and wellbeing. Supportive marital relationships are associated with better physical and subjective health, whereas strain is associated with poorer health. Recent studies now indicate a dyadic effect of relationship quality on health outcomes, such that an individual\u2019s perceptions of their relationship also affects their partner\u2019s outcomes. Few studies have examined the dyadic effects of relationship quality on mental health among older cognitively intact caregiving couples. To address the lack of dyadic research about how perceived support from one\u2019s spouse related to experiences of depression for individuals and their care partners, we apply cross-sectional actor partner interdependence models (APIMs) to data from the Health and Retirement Study (HRS) (N=490 dyads). APIM regression models controlled for participant demographic characteristics, relationship length, and care recipient functional ability. Findings showed that positive perceived support from a spouse had a stronger negative association with one\u2019s own depression for care recipients than for caregivers. Similarly, greater negative perceived support from a partner was associated with higher levels of depression; whether the partner was the caregiver or care recipient did not make a difference in this model. Although there are hundreds of caregiver interventions to address caregivers\u2019 mental health, few have demonstrated improvement in care recipient outcomes. Observation of both actor and partner effects in this study suggests there may be opportunities to improve care recipient and caregiver mental health by targeting interventions to promote high quality relationships with caregivers or both members of the care dyad."} +{"text": "This study explores community leaders\u2019 understanding and attitudes toward depression among older Korean Americans. The community leaders who provide services for older adults play a significant role because these leaders typically engage with the target population by providing services. A qualitative method was applied for this study. A total of 12 community leaders were interviewed for 60 minutes using the semi-structured interview guide. The constant comparative approach was employed to analyze data using NVivo software. The findings indicated that the community leaders were aware of the severity of the issues, but lacked knowledge regarding depression and had stereotypes against depression. Those leaders identified negative personality issues of people with depression and desired to avoid discussing depression with older adults. The results suggest that interventions are essential to support community leaders and empower them to take actions to provide services to promote understanding of depression in older adults."} +{"text": "Students studying for health care professions have limited opportunities to learn about medication use and aging in an interprofessional experience. Health Care students who interact in a simulation of age-related sensory changes can identify adaptations for safe medication use and counseling necessary to promote healthy aging. This research assessed the impact of a simulated team experience on pharmacy and physician assistant students\u2019 confidence in understanding age related changes and in learning adaptations to promote safe medication use for older adults who may experience those changes. 63 pharmacy and 113 Physician Assistant students participated in 2-hour Interprofessional Education (IPE) sessions. The teams of pharmacy/physician assistant students utilized glasses to simulate changes in vision and gloves to simulate conditions of arthritis and neuropathy which increase in prevalence with age. Teams practiced skills of medication counseling and empathy towards their peers experiencing the simulations and learned medication administration adaptations for aging well. Pre survey results show a deficit of Pharmacist-Physician Assistant IPE with less than 20% of students reporting a strong understanding of the other profession\u2019s role in developing an older adult\u2019s care plan. Post survey results demonstrate an increase in students\u2019 confidence in both understanding how sensory impairments may affect a patient\u2019s ability to properly administer medication and confidence in counseling older adults on safe medication use. Descriptive data on learning in Interprofessional teams, Pre/Post comparison data and application to students studying other majors will be presented."} +{"text": "Expectations about aging are connected with both positive traits and negative traits . Adams-Price et al. (2016) created the Personal Longevity Scale (PLS), which measures positive expectations (Hope) and negative expectations (Dread). The purpose of this study was to examine how psychological benefits gained from participating in creative hobbies may positively impact middle-aged and older women\u2019s attitudes about aging. In addition, we examined if participant age moderated this relationship. A total of 198 women, aged 50 to 82 years old, completed the Personal Longevity Scale and the Creative Benefits Scale . The CBS includes four subscales: gaining a sense of identity (Identity), getting a sense of relaxation , feeling closer to God or nature , and receiving recognition from others for one\u2019s hobby (Recognition). Single moderation models suggest that higher Identity scores were linked to more positive attitudes about aging but did not significantly predict negative attitudes. Age provided a marginally significant moderation effect to this relationship such that older women who received more of a sense of identity from their hobby reported the most positive attitudes about aging. Spirituality was linked to more positive attitudes about aging but did not significantly predict negative attitudes. Neither Calming nor Recognition were predictive of either positive or negative attitudes about aging. Implications for women\u2019s development will be discussed from an Eriksonian perspective."} +{"text": "Two years of monthly observations of water stable isotopes in a temperate lowland catchment with extensive agrarian and forested landscapes in Latvia, River Salaca catchment. Observations include most significant water types within catchment: precipitation, raised bog, intermittent, groundwater and surface water at 15 observation points. The monthly data is supplemented by two intervals of one-month long sampling every second day for a subset of observation points. Water table, temperature and electrical conductivity co-observed as key variables helping to understand the isotope data. The data can be useful for in depth investigation of isotope hydrology and as background information for ecohydrological and other studies. The data is associated with the original research article \u201cAn insight into water stable isotope signatures in temperate catchment\u201d Specifications Table\u2022Presented 25-month time series provide unique insight into dynamics and seasonality of stable isotope ratios in different water types within temperate lowland catchment.\u2022Researchers working on hydrology related problems can used the data set as background information in planning their investigation or as input for global simulations of water using cycle using natural isotope tracer methods\u2022The data set can be used as input for compiling high resolution or temporal isoscapes of groundwater, wetlands, surface water and biota and to examine the secular variations of stable isotope ratios as water transitions between compartment of terrestrial hydrological cycle118O and \u03b42H at selected locations (see"} +{"text": "Haemoptysis is an uncommon presenting symptom in children and is usually caused by acute lower respiratory tract infection or foreign body aspiration. We report a rare case of right unilateral pulmonary vein atresia (PVA) as the underlying aetiology of recurrent haemoptysis in a child.A 4\u00a0years old girl presented with history of recurrent haemoptysis. Bronchoscopic evaluation excluded a foreign body aspiration but revealed right bronchial mucosal hyperaemia and varices. Diagnosis of right unilateral PVA was suspected on transthoracic echocardiography which demonstrated hypoplastic right pulmonary artery and non-visualization of right pulmonary veins. Final diagnosis was confirmed on cardiac CT angiography. A conservative treatment approach was opted with consideration for pneumonectomy in future when she is older.Rarer causes should be considered when investigating for recurrent haemoptysis in children. Bronchoscopy and cardiac imaging are useful tools to establish the diagnosis of unilateral PVA in our case. Haemoptysis is an uncommon presenting symptom in children. The most common aetiologies are acute lower respiratory tract infection and foreign body aspiration , 2. DiagWe report a rare case of right unilateral pulmonary vein atresia as the underlying aetiology of recurrent haemoptysis in a child.A 4\u00a0years old girl was referred to our hospital for suspected foreign body aspiration. She presented with 3\u00a0days history of cough and haemoptysis. The expectorant consisted of fresh bright blood mixed with small clots and mucus Fig.\u00a0a. There On physical examination, she was pink and not tachypnoeic. Her oxygen saturation was 98% under the room air. A grade 2 systolic murmur was detected over the right upper sternal border. There was reduced breath sound over the right lung field. Other organ system examination was unremarkable.Chest radiograph Fig. b showed The initial chest CT performed at referring hospital revealed an intratracheal linear structure suspicious of foreign body.An urgent flexible bronchoscopic evaluation was performed under general anaesthesia. No foreign body was detected but a prominent vertical mucosal fold was seen above the carina which explained the initial CT finding. The right main bronchus was significantly smaller in calibre compared to the left with mucosal hyperaemia, oedema and variceal engorgement of submucosal blood vessels Fig. c.Transthoracic echocardiogram was performed as part of the workout for heart murmur. It revealed left atrial isomerism and dextroposition of the heart. The right pulmonary artery was hypoplastic and the right pulmonary veins could not be traced. Otherwise, the rest of the intracardiac anatomy was normal and there was no evidence of pulmonary hypertension.Cardiac CT angiography confirmed the final diagnosis of right unilateral pulmonary vein atresia. The posterior left atrial wall at the expected site of right pulmonary veins entrance were completely smooth Fig.\u00a0. The rigAt 6 months after discharge, the patient remained symptom free.Haemoptysis is an uncommon but alarming presenting symptom in paediatric practice. The aetiologies differ significantly from that in adults. Haemoptysis in adults are commonly caused by tuberculosis, bronchogenic carcinoma and bronchiectasis but acute lower respiratory tract infection and foreign body aspiration constitute the two commonest causes of haemoptysis among children , 2. DiagIn our case, echocardiogram provided important clues to the diagnosis by inability to detect presence of right pulmonary veins draining into the left atrium with corresponding ipsilateral pulmonary artery hypoplasia. Bronchial varices detected on bronchoscopy is a hallmark clinical feature of this disease and finaUnilateral pulmonary vein atresia is an extremely rare congenital malformation due to failure of incorporation of the common pulmonary vein into the left atrium . BilaterA possible differential diagnosis to be considered in our case is Scimitar syndrome which is also associated with right lung hypoplasia, dextroposition of the heart and systemic collateral arterial supply to part of the right lung. However, Scimitar vein, the anomalous pulmonary vein which commonly drains into the inferior vena cava in Scimitar syndrome could not be demonstrated on the CT in our case.Without treatment, long term prognosis is usually guarded due to risk of massive pulmonary haemorrhage and development of pulmonary hypertension. Successful surgical repair has been reported in cases with well-developed pulmonary venous confluence to reconnect to the left atrium using sutureless pericardial marsupialization technique . PneumonIn conclusion, rarer aetiologies should be considered when investigating children presenting with recurrent haemoptysis. Presence of bronchial varices on bronchoscopy and failure to demonstrate the right pulmonary venous drainage with corresponding ipsilateral pulmonary artery hypoplasia on transthoracic echocardiogram have provided important clues leading to final diagnosis of unilateral pulmonary vein atresia by cardiac CT angiogram in our case."} +{"text": "This case reports on secondary extramedullary multiple myeloma within both breasts in the absence of axillary nodal involvement and discusses the difficulty in interpretation with clinical recommendations and learning outcomes.Differentiating plasmacytic lesions in the breast is often difficult as clinical and radiological appearances are known to mimic benignity and high-grade primary breast cancer.Extramedullary presentation can determine progression of the disease and can necessitate cross-sectional imaging. Therefore definitive diagnosis is essential as the clinical management of the patient may be altered. A 73-year-old female with a history of multiple myeloma was referred for breast assessment by her haematologist for a new superficial lump with associated bruising in the upper outer quadrant of the right breast (RUOQ). Additional information detailed a history of prior pulmonary embolisms and was therefore on long-term warfarin.Clinical breast examination revealed a suspicious lump in the RUOQ at the area of concern with overlying cutaneous bruising with two further similar masses palpated in the left outer breast (P4). The patient pathway and management is outlined in 1 this was graded indeterminate M3.In comparison with previous mammograms from 2014, multiple new bilateral predominantly well-defined masses with some subtle margin ill definition were identified showing indeterminate non-specific features requirinUltrasound (US) of the right breast demonstrated the clinically presenting index lesion to be a superficial well-defined, \u201cpseudocystic\u201d lesion with low level internal echoes, BI-RADS U3 . SonograExamination of the left breast identified two lesions, one a 7.5\u2009mm deeper seated non-specific hypoechoic mass with a surrounding hyperechoic penumbra . Subsequent core biopsy demonstrated heavy infiltration by neoplastic plasmacytoid cells forming sheets of moderately pleomorphic cells with enlarged rounded eccentrically placed nuclei highly suspicious of myeloma had a primary breast plasmacytoma, the other 45 cases (85%) had an extramedullary presentation of more widespread multiple myeloma. Most of the cases had a solitary presentation (66%) rather than multiple (34%), however the size of lesion ranged from 8 to 90\u2009mm.There is little peer-reviewed literature discussing breast plasmacytoma due to the rare presentation. Surov et al (2010) reviewed five cases within their institution between 1997 and 2008 with an additional 48 patients found in the literature between 1988 and 2010.14As with all focal breast lesions triple assessment is best practice. Cytology with FNA has been shown to demonstrate plasma cells at different stages of maturation; therefore core biopsy is not always necessary however adequate sampling is required to differentiate from primary carcinoma.15Lymphadenopathy associated with breast plasmacytoma is rare; however a case of a 78-yearold male has been reported with lymph node involvement and extracapsular extension of the tumour.16 This correlated with the literature suggesting that MRI is unnecessary providing mammography and ultrasound is combined with histopathology.The value of MRI of the breast for plasmacytoma is subjective as imaging features are often inconclusive. A case reported by Kocaoglu et al in 2003 concluded the benign features associated with plasmacytoma may be misleading despite MR contrast enhancement.17 MRI/CT may identify osteolytic lesions which even when unifocal should be regarded as meeting the CRAB criteria, regardless of whether they are seen on plain-film skeletal radiographs.17 Thus confirming a diagnosis of multiple myeloma and altering patient management.If extramedullary involvement is suspected recommendations from the International Myeloma Working group suggest MRI or CT combined with FDG-PET is indicated for the assessment of disease extent and monitoring of treatments.18 Destruction of the cortex can lead to local invasion of the soft tissues however metastatic soft tissue deposits are often an advanced presentation of myeloma and define extramedullary disease which correlates with the findings in our case.Medullary myeloma usually is identified radiologically by lytic lesions due to the dominance of osteoclast activity with suppression of osteoblast formation.Imaging appearances are varied ranging from superficial cystic type lesions to solid nodules with benign or ill-defined margins. Lymph node involvement is rare but can be present and MRI for breast assessment can be misleading. Presentation within the breast is unusual but can appear bilaterally and combined with benign features should not be falsely reassuring. Breast imagers need to be aware of this when imaging patients with a clinical history of myeloma.Demonstrating extramedullary soft tissue involvement can indicate disease progression and necessitate further cross-sectional imaging, potentially changing clinical chemotherapeutic management.Haematology clinicians need to be aware of the presentation which may manifest in the breast and include routine breast examination at follow-up clinical appointments.Triple assessment approach is advocated even when imaging appearances look benign. Core biopsy is preferred although adequate fine needle aspiration can be sufficient."} +{"text": "Research examining the relationship between subjective cognitive complaints and objective cognitive performance has been mixed. Despite the lack of clear evidence demonstrating an association, subjective cognitive complaints are used as a criterion for the diagnosis of mild cognitive impairment and is considered a risk factor for Alzheimer\u2019s disease. Cross-lagged panel analyses were used in the current study to examine the longitudinal relationships between subjective cognitive complaints (using the Memory Functioning Questionnaire) and objective cognition in healthy adults over 60 from the Virginia Cognitive Aging Project (N=441). Results indicated that objective and subjective cognition were only weakly related but that objective cognition is a stronger predictor of subjective cognitive complaints then vice versa. Although subjective cognitive complaints may be an early indicator of pathological aging, results indicate that subjective cognitive complaints may not be a valid predictor of objective cognitive decline."} +{"text": "Dear Editor,et al reported that temperature above 47oC for one minute is the critical threshold for bone necrosis1. Modification of drill bits designs , drilling techniques and use of coolant has been recommended to lower this risk2. Although many orthopaedic and trauma surgeons are faithfully irrigating the drill bit / drill sleeve with saline during bone drilling for this purpose, we do not even know whether the coolant ever reach the bone / drill bit interface, especially during minimally invasive percutaneous procedures. Benington et al and Sener et al showed that external saline irrigation was effective to prevent bone necrosis for dental procedures, but there was no similar study on clinical orthopaedic models4.We would like to share an unexpected finding related to a common orthopaedic procedure. Erickson We decided to investigate whether saline sprayed over the incision wounds or outer end of drill sleeves wound reach the interface between bone and drill bit during bone drilling . We chosWe noted that there was no trace of blue dye on the bone surface for the whole femur and proximal end of the tibia . Blue dy"} +{"text": "Balance and gait are modifiable targets for falls prevention and may play an important role in preventing falls in older visually impaired individuals. Balance and gait were objectively evaluated in the 239 Falls in Glaucoma Study participants damage). Greater sway, more time in double support and greater swing time variability were associated with higher fall rates, while higher gait velocity and faster cadence were associated with lower rates . Neither gait nor balance mediated the relationship between VF damage and fall rates, with VF damage remaining an independent predictor of fall in models including gait and/or balance features . While balance and gait measures are associated with fall rates, they do not explain why persons with greater VF damage fall more frequently, suggesting the importance of other factors such as hazard perception."} +{"text": "Anaemia is a pathology of too few red blood cells or too little haemoglobin, the oxygen-carrying molecule in blood, to deliver adequate oxygen for physiological needs . Low oxyEPAS1) encoding proteins regulating those pathways. Hypoxia inducible factors regulate the formation of red blood cells and haemoglobin [Ancient \u223c550 my) genetic and molecular oxygen homeostatic pathways regulate haemoglobin concentration in birds, fish, reptiles and mammals. Ethnic Tibetan highlanders have uniquely high frequencies of variant alleles at two loci (EGLN1 and moglobin that offmoglobin . Consequ50 my genAn evolutionary perspective explains that Andean and Tibetan highland populations represent different outcomes of the natural experiment of migration and adaptation to high altitude. Diagnostic criteria derived from one population may not apply to another. In the case of Tibetans such understanding may prevent unnecessary interventions and use of resources. Evidence that Tibetan men and women with elevated haemoglobin concentrations have poorer work capacity and preg"} +{"text": "From 1976 Australia has experienced seven highly pathogenic avian influenza (HPAI) outbreaks in poultry farms and there have been a total of 16 confirmed low pathogenic avian influenza (LPAI) cases in poultry in Australia at the time of writing. This paper describes all past LPAI and HPAI detections in Australian poultry and reviews avian influenza risk in the Australian commercial chicken industry. The factors that influence this risk are also discussed; notably the nomadic nature of Australian waterfowl, the increasing demand of free range poultry egg and meat production in Australia, and biosecurity practices implemented across farms including farm separations. \u2022Australia has experienced seven highly pathogenic avian influenza (HPAI) outbreaks in poultry farms\u2022There have been 16 confirmed low pathogenic avian influenza (LPAI) cases in poultry in Australia at the time of writing\u2022Australian waterfowl are nomadic in nature\u2022There is increasing demand of free range poultry production in Australia\u2022Mathematical models for avian influenza risk in Australia have been reviewed It is aThere are several factors that influence AIV outbreak risk in Australia. These include the presence of unique Australian viral lineages of LPAI virus in reservoir waterfowl populations . Austral2Dendrocygna arcuata) extend their distances to the Australo-Papuan region where travel from northern Australia to Asia occurs. Such movements are usually confined southeast of the Wallace line; a natural faunal boundary delineating Asian and Australasian flora and fauna that runs through Indonesia between Borneo and Sulawesi and through the Lombok Strait between Bali and Lombok [Australian waterfowl movements are nomadic; they are in response to rainfall and the presence of waterbodies and other resources . Such mod Lombok ,53,54. Id Lombok .Chicken is now the most consumed meat in Australia, surpassing beef, lamb and pork at 48.6\u00a0kg/person/year in 2019 . Egg coThe increase in free range production raises concerns due to the increased potential contact between wild birds and domestic poultry and the subsequent introduction of pathogens such as AIV ,51,52. V3Sterna vulgaris) trapped in a HPAI infected poultry shed in 1985 as these subtypes can cause moderate national socio-economic consequences and have the ability to mutate to HPAI virus. As HPAI has the potential to cause very severe production losses and significant impacts on the national economy, it is classed as a category 2 EAD in Australia [Reports of confirmed Australian LPAI cases in poultry are available from 1976. These confirmed cases are the result of passive surveillance (diagnostic submissions), active surveillance (primarily area surveillance during HPAI outbreaks) or incidental findings not associated with a disease or surveillance. At the time of writing, there have been a total of 16 confirmed LPAI cases in poultry in Australia with the latest case occurring in 2018 at the time of writing. Each case represents one farm where there have been positive LPAI virus PCR, virus isolation, or serological evidence of LPAI in poultry on that farm. Clinical signs in poultry in these LPAI events were largely mild, where some cases had no clinical signs apparent. Concurrent bacterial pathogens were associated in all LPAI events with clinically affected ducks. LPAI has never been detected on a single species commercial egg layer enterprise or on poultry farms in South Australia or Northern Territory . In 20104A quantitative exposure assessment estimated that the probability of a first LPAI virus exposure to an Australian commercial chicken farm from a single wild bird present on the farm at any point in time was extremely low. Free range layer farms were the most likely farm type to experience an LPAI virus introduction . HoweverThere was limited spread to other farms in all AIV outbreak detections in Australian poultry, attributable to the rapid stamping out response Table 1Table 1. The influence on farm type on AI outbreak risk in the Australian commercial chicken industry was assessed through branching process models. It was found that a 25% change in the proportion of farms in the Australian commercial chicken industry to free range farming would increase the probability of a HPAI outbreak by 6\u20137%, rising to 12\u201314% with a 50% change to free range farming . In addi5All past HPAI outbreaks in poultry in Australia were found to align with stochastic mathematical methods of frequent LPAI virus introductions with low probability of mutation. However, there are significant influences on LPAI virus introduction and HPAI outbreak occurrence risk. The review of risk assessments which used stochastic mathematical models have highlighted the importance of continued AIV wild bird surveillance, and to advocate good biosecurity practices including waterfowl deterrence on farms due to these factors' strong influence on LPAI virus introduction probability. The latter is particularly significant in compensating for the increase in HPAI outbreak risk that will occur from the increased proportion of free range commercial chicken farms in Australia. Further explorations of AIV infection dynamics in the Australian context can be conducted through validation of the models, such as through structured population-based surveillance of commercial meat or layer chickens at slaughter. It is important that ongoing research of AIV in the Australian context is performed to prepare for changes in AIV risk.Not applicable.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper."} +{"text": "The diverse needs of persons living with dementia in nursing home settings presents challenges for Certified Nursing Assistants (CNAs) to provide quality care. There is a lack of educational preparedness among nursing home CNAs regarding dementia knowledge and skills required to care for a person living with dementia. As direct caregivers for persons living with dementia, CNAs play an important role in long-term care. This pilot study evaluated the dementia knowledge and caregiving skills of newly trained CNA students. The students were trained by an instructor certified using Teepa Snow\u2019s Positive Approach to Care (PAC) curriculum. Conducted in a rural southwestern Indiana community, this study evaluated CNA students\u2019 knowledge and perception of dementia, as well as their skill performing the Positive Physical Approach\u2122 (PPA\u2122) technique to approach and connect. A 38-item knowledge and perception survey and a 12-step observed skills assessment using a standardized patient encounter were administered to CNA students. Data were analyzed using descriptive statistics and bivariate analysis. Preliminary results indicate that 100% of students correctly answered the survey item regarding non-confrontational body language, while 29% of students correctly performed the corresponding PPA skill. There is a statistically significant association between the knowledge that people find pressure in their palm comforting and the ability to perform the corresponding Hand-under-Hand\u00ae and PPA techniques. Incorporation of PAC into current CNA curriculum may equip CNAs with the knowledge and skills required to provide better care, with the potential to improve the overall quality of life for persons living with dementia."} +{"text": "But, molting is not the only energetically expensive task that adult female Weddell seals undergo within the short 7-week window of the Antarctic summer; they must also reproduce and rebuild their blubber stores for winter. If molting requires so much energy, why do Weddell seals do it Fur is an adaptation that insulates seals by trapping a warm layer of air close to skin. Molting replaces old, worn fur with a darker, higher quality fur that maintains efficient insulation. Between losing old fur and growing new fur, seals are temporarily left with little insulation, potentially leading to greater body heat loss to the environment. Weddell seals may then be forced to use more energy to maintain their high body temperature (~37\u00b0C), which presents a problem given the competing needs for energy during summer. Do the energetic requirements of molting then make Weddell seal populations more sensitive to changes in food availability or climate?Skyla Walcott and her colleagues designed a study to understand how environmental factors as well as physiology (e.g. molt stage) affect body heat loss and energetic costs. The researchers used infrared cameras and thermocouples to measure surface/core body temperature and heat loss in 93 adult female Weddell seals before and during molting. They used statistical models to determine which factors were most important in affecting body temperature and heat loss.Walcott\u2019s team found that seals maintained high skin temperatures regardless of molt stage, but seals in early molting lost 25% more heat compared to during the months before molting began. Not surprisingly, fur helped reduce body heat loss by 33%. Heat loss during molting led to a doubling of the energetic costs of thermoregulation (i.e. maintaining their core body temperature). The researchers also observed that lower air temperatures and higher wind speeds increased heat loss and further lowered skin temperatures. These findings suggest that natural selection drove molting to occur during the warm summer months. Higher summer temperatures help minimize heat loss during molt. Summer is also when food is most abundant and shallow in Antarctic waters, making for greater foraging success. These factors allow Weddell seals to recover critical blubber stores depleted by reproduction and supply energy for molting.Conditions in the Southern Ocean are changing rapidly. While climate change and the resulting warmer temperatures could reduce thermoregulatory costs for Weddell seals, climate change also decreases sea ice habitat and prey availability. With less sea ice, seals may spend more time in the water hunting and have fewer options for hauling out on land during reproduction and molting. If foraging takes longer due to reduced prey availability, it may delay molting into suboptimal cooler fall temperatures. And, we already know that late-molting females show lower success in reproduction, which further emphasizes that the timing of molting is crucial. This information regarding the timing and energy required of molting will help conservationists understand how the changing Antarctic ecosystem affects vulnerable Weddell seal populations.Illustration by Erin Walsh; Email: ewalsh.sci@gmail.com"} +{"text": "In the preoperative evaluation of the patient, the article incorrectly reported that the patient had severe heart failure with an ejection fraction of 33%. The patient did not have severe heart failure. The management method in perioperative time was incorrectly reported as of dexmedetomidine infusion. The patient was managed by propofol and remifentanil infusion.The article, \u201cSupra-inguinal fascia iliaca block under ultrasound guidance for perioperative analgesia during bipolar hip arthroplasty in a patient with severe cardiovascular compromise: A case report\u201d,"} +{"text": "Research conducted by the NYC Elder Abuse Center (NYCEAC) at Weill Cornell Medicine and colleagues found that concerned persons experience significant distress knowing about elder abuse and trying to assist victims. Data will be presented from a nationally representative survey which included items on concerned persons in elder abuse. Thirty-one percent of all respondents reported that they had a relative or friend who experienced elder abuse; of these, 61% had attempted to help the victim and over 80% reported the experience is very or extremely stressful (2017). By both knowing about and becoming involved in elder abuse situations, concerned persons experience significant emotional and practical problems and often need professional help. NYCEAC\u2019s Elder Abuse Helpline for Concerned Persons is the first of its kind in the country. The Helpline\u2019s services and structure will be explained, and possibilities for replication in other locations will be explored."} +{"text": "We report a patient with the triad of diabetic ketoacidosis, hypertriglyceridemia, and acute pancreatitis associated with computed tomography hypoperfusion complex and adrenal hyperdensity on abdominal imaging \u2013 an association not previously reported in diabetic ketoacidosis.Presence of computed tomography hypoperfusion complex with hyperdense \u2018Tubelight adrenals\u2019 in a patient with diabetic ketoacidosis is associated with poor prognosis and thus serves to guide clinicians towards early and aggressive management. A 27-year-old male with type 1 diabetes who was poorly compliant with insulin therapy presented to our emergency department (ED) with severe abdominal pain. His records revealed repeated ED visits for abdominal pain over the prior month. Based on laboratory findings the patient was diagnosed with diabetic ketoacidosis (DKA) . FurtherComputed tomography (CT) with intravenous contrast showed findings consistent with acute pancreatitis as well as enhancing bilateral adrenal glands with mucosal hyperenhancement of bowel loops and narrow caliber of abdominal aorta with imperceptible inferior vena cava, suggesting hypoperfusion complex . DespiteThe triad of diabetic ketoacidosis, hyperlipidaemia, and acute pancreatitis is important as it leads to profound hypovolemia comparable to post-traumatic shock, which leads to characteristic hypoperfusion complex on CT.3What do we already know about this clinical entity?Diabetic ketoacidosis when associated with acute pancreatitis and hypertriglyceridemia results in profound hypovolemic shock. Computed tomography (CT) finding in such patients corresponds to post-traumatic shock known as \u2018CT hypoperfusion complex.\u2019What is the major impact of the image(s)?Profound hypovolemia may result in CT hypoperfusion complex and hyperdense adrenal, or \u201ctubelight\u201d (fluorescent tube-shaped) adrenals.\u201d This CT finding indicates poor prognosis.How might this improve emergency medicine practice?Presence of this finding may guide physicians toward early and aggressive fluid management in these patients.The finding of adrenal hyperdensity, which we describe as \u201ctubelight adrenal sign\u201d [fluorescent-tube shaped] in our patient as a part of CT hypoperfusion complex is unique as it has not been reported in the setting of DKA and is associated with increased mortality. Early imaging for diagnosis of pancreatitis and associated CT hypoperfusion complex with hyperdense tubelight adrenals can aid in guiding treatment and prognosis in these patients. Presence of tubelight adrenal sign on CT must alert the clinicians to possible adverse outcome and these patients should be initiated with early and aggressive fluid therapy."} +{"text": "Nostalgia is a self-conscious, bittersweet but predominantly positive and fundamentally social emotion. The regulatory model of nostalgia suggests that experiencing nostalgia can buffer against social threat by providing individuals with sense of social connectedness . In the current research, we propose that this salutary effect of nostalgia may be stronger among older adults compared to younger adults because older adults value social meaningfulness to a greater extent. Fifty-nine younger adults and 56 older adults completed daily questionnaires three times a day for ten consecutive days, and reported their emotional experience and social activities. Results showed that perceiving social threat was positively correlated with nostalgia experience reported at the subsequent time point, and this association was stronger among older adults. In addition, nostalgia positively correlated with subsequent social activities among the older participants but not among the younger participants. These findings highlight that nostalgia brings beneficial psychological and behavioral outcomes to older adults."} +{"text": "Existing literatures yield established evidence about the heightened stress brought by multiple roles and potential role overload across work-family context, but little is known about the BMI levels of the \u201csandwich\u201d caregivers within families and the associated gender inequalities. Indeed, the Chinese pivotal generations are exposed to unshared stress and higher health risks considering that intergenerational support still predominates the caregiving patterns for the oldest old and dependent children under current socioeconomic backgrounds. Using 2011 and 2013 waves of China Health and Retirement Longitudinal Study , we examine associations of BMI and intergenerational caregiving patterns among the \u201csandwich generation\u201d aged 45 to 69. We find that the sandwich generation with at least one parent alive and one grandchild under 16 have higher BMI than their counterparts . A higher proportion of females act as caregivers and especially high-intensity caregivers than males, and they also score one-unit higher in BMI than males (23.4). Fixed effect regression results indicate that simultaneous caregiving to both parents and young grandchildren significantly advances individuals\u2019 BMI levels, while no evidence shows similar negative effect of providing care to one generation. Moreover, high-intensity caregiving (1000 hours and above during the past year) is associated with elevated BMI for females but not for males. The gendered caregiving patterns and health implications inform the physical and psychological vulnerability of the pivotal generation and the necessities of gender-specific intervention in middle and later life."} +{"text": "Older adults are assumed to be more averse to uncertainty than younger adults as economics and psychology studies based on prospect theory and related risk aversion theories show. Indeed, compared to younger adults, older adults engage in a smaller share of entrepreneurship. Yet adults 50 and over make up a steadily growing group of entrepreneurs in the US, and the 2008 financial crisis has not slowed them down. Sociological perspectives on older adults\u2019 entrepreneurship consider disruption in the structural conditions of the labor market as a result of the economic crisis and a culture of active aging to argue that older adults\u2019 entrepreneurial behaviors stem from structural and cultural roots. Analyses using 2003-2015 individual-level data from the Global Entrepreneurship Monitor show that an economic downturn did not thwart older adults\u2019 entrepreneurial activities any more than it affected younger adults\u2019 activities. Further, older adults\u2019 odds of planning to start a business and actually starting a business were higher after the crisis than before the crisis. A closer examination reveals that older adults with less wealth generally have higher odds of pursuing entrepreneurship than other older adults. Meanwhile, wealthier older adults have higher odds of pursuing opportunity-driven entrepreneurship. Evidence suggests that the saliency of labor market conditions or the culture of active aging depends on older adults\u2019 social location."} +{"text": "Background and Objectives: Even before the COVID-19 pandemic, older adults with cognitive impairment living alone were at high risk for negative health outcomes. There is an urgent need to learn how this population is managing during the pandemic. Research Design and Methods: This is a qualitative study of 24 adults aged 55 and over living alone with cognitive impairment from diverse racial/ethnic backgrounds. Participants\u2019 lived experiences during the pandemic were elicited via 59 ethnographic interviews conducted over the phone either in English, Spanish, or Cantonese. Using a qualitative content analysis approach, interview transcripts and fieldnotes were analyzed to identify codes and themes. Results: Qualitative analysis of transcripts revealed five themes: 1) fear generated by the pandemic; 2) distress stemming from feeling extremely isolated; 3) belief in misinformation, 4) strategies for coping during the pandemic; and 5) the importance of access to essential services. Discussion and Implications: This pandemic put a spotlight on the precarity and unmet needs of older adults living alone with cognitive impairment living. Findings underscore the need to expand access to home care aides and mental health services for this population."} +{"text": "This study explored the prevalence of positive attributes of aging among older adults in a nationally representative sample age 50-80 . Nearly 70% of older adults reported that people sought their guidance because of their wisdom and experience. Older adults reported that, as they have gotten older, they have become more comfortable with themselves (88%), have a strong sense of purpose (80%), feel more positively about aging (67%), and have found their life to be better than they had thought it would be (65%). Over half (52%) of those who said their lives were better than they thought reported very good or excellent physical health. Among those who disagreed, only one out of four (25%) reported very good/excellent physical health; similar results were found for mental health (48% vs. 22%). This session will describe positive attributes of aging and their association to physical and mental health."} +{"text": "Technology to support aging in place has been a topic of interest. Research indicates that older adults are increasingly using commercially available voice assistants in smart speakers. These devices enable non-visual interaction that does not require extensive expertise with traditional mobile or desktop computers, thus offering new possibilities of access to digital technology. We conducted two different studies with individuals aged 65 years old or above\u2014a three week smart speaker deployment study with individuals who did not use computing devices regularly and a workshop on customizing internet of things technology with tech savvy individuals. Our findings indicate specific ways that these voice technologies might support aging in place, including ease of use and due to their not being identified with aging-specific technologies. We observed that participants consistently used their voice agent for finding online information, particularly health-related, emphasizing the need to revisit concerns about credibility of information with this new interaction medium. And, although features to support memory were initially perceived as useful, the actual usage was unexpectedly low due to reliability concerns. Our work provides a basis to understand older adults\u2019 perceptions and usage of current voice technologies. We also identify opportunities for customizing voice technologies to better support aging in place."} +{"text": "The central and rapid accumulation of this metabolite represents a major factor involved in the process of fetal alcohol programming. According to recent investigations, it appears that ACD exerts early positive reinforcing consequences and antianxiety effects (negative reinforcement). Finally, this review also acknowledges human clinical and epidemiological studies indicating that moderate and binge-like drinking episodes during gestation result in neonatal recognition of EtOH\u2019s chemosensory properties coupled with a preference towards these cues. As a whole, the studies under discussion emphasize the notion that even subteratogenic EtOH exposure during fetal life seizes early functional sensory and learning capabilities that pathologically shape subsequent physiological and behavioral reactivity towards the drug.The anatomo-physiological disruptions inherent to different categories of the Fetal Alcohol Spectrum Disorder do not encompass all the negative consequences derived from intrauterine ethanol (EtOH) exposure. Preclinical, clinical and epidemiological studies show that prenatal EtOH exposure also results in early programming of alcohol affinity. This affinity has been addressed through the examination of how EtOH prenatally exposed organisms recognize and prefer the drug\u2019s chemosensory cues and their predisposition to exhibit heightened voluntary EtOH intake during infancy and adolescence. In altricial species these processes are determined by the interaction of at least three factors during stages equivalent to the 2nd and 3rd human gestational trimester: (i) fetal processing of the drug\u2019s olfactory and gustatory attributes present in the prenatal milieu; (ii) EtOH\u2019s recruitment of central reinforcing effects that also imply progressive sensitization to the drug\u2019s motivational properties; and (iii) an associative learning process involving the prior two factors. This Pavlovian learning phenomenon is dependent upon the recruitment of the opioid system and studies also indicate a significant role of EtOH\u2019s principal metabolite which is rapidly generated in the brain Two drug-related phenomena act as the foundation for the present review: (i) among other drugs of abuse, ethanol (EtOH) recruits non-associative and associative learning capabilities of the organism; and (ii) age of onset of EtOH-related experiences represents a critical factor determining later use and abuse of this psychotropic agent. Both issues are intrinsically related to the concept of early-life adaptations to environmental disturbances that increase susceptibility to diseases in later life , approximately 17,000 articles arise when linking the terms \u201cfetal\u201d and \u201calcohol.\u201d Within this considerable number of peer-reviewed studies, it is possible to detect an early publication that explicitly examined the consequences of fetal chronic exposure to EtOH upon subsequent intake patterns of the drug representing one of the major congenital causes of mental disabilities Finally, the last section of the review is devoted to highlighting human clinical and epidemiological studies endorsing the notion that prenatal EtOH experiences have a major impact on the development of subsequent EtOH affinity. As will be observed, this human section has been organized to depict the analogies and homologies existing between animal and human findings.The present review will be organized as follows: (i) a first section, primarily centered on preclinical studies, analyses different experimental strategies alluding to fetal capabilities of altricial mammals related to the detection and discrimination of EtOH\u2019s sensory attributes; (ii) a second item, also based in animal studies, incorporates the notion of EtOH\u2019s reinforcing effects that promote associative learning memories leading to heightened EtOH affinity; (iii) the third section emphasizes the need to consider the drug\u2019s principal metabolite (ACD) as a critical factor modulating EtOH\u2019s reinforcing effects; (iv) the role of the endogenous opiate system modulating prenatal EtOH reinforcement is additionally considered in the uterus including humans or a novel scent (lemon). Ten minutes later pups were delivered via cesarean surgical procedures via prenatal EtOH exposure and an intoxicated counterpart (demonstrator) which provides sensory information of the drug According to the information presented in the preceding item, fetuses detect EtOH\u2019s sensory attributes and memories arising from these early experiences allow latter discrimination of these cues and preference to such stimuli. Is sensory familiarization solely responsible for subsequent EtOH affinity? From a pharmacokinetic perspective, the studies previously reported based on maternal EtOH intoxication imply the juxtaposition of the drug in the amniotic fluid and its presence in fetal blood and brain. Hence, in terms of potential associative learning processes , the requirement of temporal contiguity between specific sensory cues and physiological effects of the drug is met.via direct EtOH contamination of the amniotic fluid while avoiding fetal intoxication with different biologically relevant events. Conditioned taste aversions have been reported in near term rat fetuses when pairing a tastant (apple juice) with the emetic effects of lithium chloride. These aversions are manifested in multiple ways: avoidance of maternal nipples scented with such tastant or spending less time over shavings that contain this gustatory cue that easily crosses the placenta and exerts no physiological consequences on the fetus. Cineol can be easily traced in terms of concentration and temporal duration in the amniotic fluid. The strategy was to explicitly associate or not the presence of this CS with the state of fetal EtOH intoxication. These experiences took place during gestational days of 17\u201320. Three weeks later pups originally exposed to the explicit association between cineol and EtOH intoxication exhibited conditioned orofacial responses when re-exposed to the odorant which were intraperitoneally injected. A positive antenatal history with the drug increased the magnitude of appetitive conditioned responses to the nipple mediated by EtOH intoxication and also increased the range of doses yielding a reinforcing effect served as a reinforcer. After operant training, a moderate EtOH dose (0.5 g/kg) was paired with the sweet taste. In pups prenatally exposed to EtOH, this association enhanced sucrose reinforcement and also increased resistance to extinction of the operant response deployed to stimulate the mammary gland and to facilitate nipple attachment increase as a function of intraoral delivery of maternal milk treated with a high EtOH dose (3.0 g/kg) later exhibit enhanced drug palatability and heightened predisposition to consume this psychotropic agent. At 10 days of age opposite outcomes (disgust reactions and intake decrements) are detected following toxic experiences generated by a similar dose that exerts positive reinforcing effects or an ACD dose (0.35 \u03bc mol) also known to exert psychomotor stimulating effects in adults. Both EtOH and ACD activate the mesolimbic dopamine system which is critical in mediating reinforcing effects of different drugs of abuse or 30% v/v EtOH solutions observed in cerebral hemispheres, striatum, cerebellum and brain stem were taken into account significantly decrease when delivering low to moderate EtOH doses exhibit poor performance in the San Diego Odor Identification Test. None of these subjects was diagnosed with FAS and interestingly, the evaluation did not include EtOH odor exert unconditioned motivational effects that are rapidly associated with the drug\u2019s sensory cues leading to long-lasting memories that impact upon later alcohol affinity even in terms of generating patterns of drug abuse. As discussed, the process of Fetal Alcohol Programming might occur independently from the teratological effects of the drug.Fetal Alcohol Programming should broaden our knowledge of potential alterations that are still not taken into account in the diagnostic criteria of FASD. Behavioral signs of Fetal Alcohol Programming have been already detected through relatively simple neonatal techniques developed for altricial mammals including humans (Abate et al., JM, RM-M, and PA conceptualized and designed the revision and drafted the sequential versions of the manuscript. GD\u2019A and FA participated in the writing process of the revision as well as in the appropriate selection of the studies under examination.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Family caregivers provide the lion\u2019s share of care that allows older adults with functional impairment to remain living at home. Yet as care needs grow, many older adults and their families turn to paid caregivers to provide additional support. While evidence suggests that paid and family caregivers work together to provide increasingly complex care at home, research that describes this important collaboration is limited. In this symposium, we present innovative and interdisciplinary research that highlights the overlap between family caregiving and long-term care workforce research. We begin by presenting two studies that focus on populations where paid caregivers may have outsized impact on family caregivers: Reckrey et al report that receipt of 20+ hours of paid caregiving per week was associated with less caregiver strain among family caregivers of those with advanced dementia and Falzarano et al report that home care hours mediated the association between caregiver stressors and negative effects of caregiving among long-distance family caregivers. Franzosa et al then describe home health aides\u2019 perceptions of relationship dynamics as aides and family members negotiate care tasks in the home. Finally, Gallopyn et al explore scenarios where paid and family caregiver roles blur . Taken together, these studies describe critical ways paid and family caregiver experiences are intertwined and highlight the importance of ongoing research about this collaboration."} +{"text": "Depression has been associated with greater risk of Alzheimer\u2019s disease (AD), and existing research has identified structural differences in brain regions in depressed subjects compared to healthy samples, but results have been heterogeneous. We sought to determine the effect of depression on regional brain volumes by cognitive and APOE e4 status. Secondary analysis of the National Alzheimer\u2019s Coordinating Center (NACC) Uniform Data Set was conducted using complete MRI data from 1,371 participants . Multiple linear regression was used to estimate the adjusted effect of depression (via the Neuropsychiatric Inventory Questionnaire) on regional brain volumes through measurement of 30 structural MRIs. Depression in the prior two years was associated with lower total brain, cerebrum,, and gray matter volumes and greater total brain white matter hyperintensities (p<.05). Greater volumes were also observed in all ventricular volume measures. Lower mean volumes were observed in six additional frontal lobe and parietal lobe cortical regions. Alternately, depression antecedent to the past 2 years correlated only with occipital lobe gray matter volumes . Our findings suggest that depression in the prior two years is associated with atrophy across multiple brain regions and related ventricular enlargement, even after controlling for intracranial volume and demographic covariates. The duration of depression influences results, however, as depression prior to 2 years before assessment was correlated with significantly fewer and different regional brain volume changes."} +{"text": "A knowledge of pulmonary vein anatomy variants allows an appropriate preoperative radiological assessment and safe surgical management of vascular anomalies in patients undergoing major lung resections. In our case, multiple pulmonary vein variations were identified pre\u2010 and intraoperatively in a patient undergoing thoracoscopic right lower lobectomy and included superior and common basal veins from the right lower lobe draining separately into the left atrium, middle lobe veins joining the superior segment right lower lobe vein and additional superior segment right lower lobe vein draining directly into the left atrium. The recognition of these anatomical abnormalities in pulmonary veins may help thoracic surgeons avoid surgical complications in patients undergoing anatomical lung resections. The great variability in pulmonary venous anatomy has been the subject of several radiologic studies as pulmonary veins play a critical role in the pathophysiology of atrial fibrillation and their anatomical variants are related to a higher arrhythmogenic potential. This important clinical issue in recent decades has also enabled anatomists, through dissection of human fixed cadaveric lungs, to identify variants in the number and course of pulmonary veins with particular care such as the medial and the lateral middle lobe pulmonary veins that drain, either as a common trunk or independently, directly into the left atrium or into the inferior pulmonary vein.A 79\u2010year\u2010old man was admitted to our operative unit for treatment of primary lung cancer located in the right lower lobe. He was classified as stage IIA, according to the eighth edition of the TNM staging system for lung cancer, and scheduled for video\u2010assisted thoracoscopic right lower lobectomy. Preoperative enhanced chest computed tomography (CT) scans showed a mass in the lower lobe of the right lung, measuring 48\u2009\u00d7\u200932\u2009mm and anatomical variations of pulmonary venous drainage including separate drainage of the superior segment right lower lobe vein (V6) from the basilar segments right lower lobe vein into the left atrium; middle lobe veins (V4 and V5) were found to empty into the V6 and accessory V6 passing behind the right lower lobe bronchus Fig and 2. DMost individuals have four pulmonary veins in total; two on the left and two on the right with two separate ostia.No authors do not report any conflict of interest."} +{"text": "The pathogenesis of intralobar pulmonary sequestration is not completely understood, either representing a congenital or acquired process. The presence of fungal organisms and aspiration has both clinical and etiologic significance. The pathogenesis of intralobar pulmonary sequestration is not completely understood, either representing a congenital or acquired process. The presence of fungal organisms and aspiration has both clinical and etiologic significance. We describe a case report of a young female presenting with intralobar sequestration associated with aspergillosis. There have been 38 prior cases, and none report additional findings of aspiration.Benign cystic congenital anomalies of the lung include congenital pulmonary airway malformation (CPAM) and bronchopulmonary sequestration (BPS). BPS is less common than CPAM and is divided into intralobar and extralobar types. By definition, BPS should have a systemic arterial supply and usually lacks connection with the bronchial airwaysBronchopulmonary sequestration is divided into intralobar and extralobar sequestration, whereas the former occurs in a normal lung lobe, while the latter contains a unique separate pleura.The purpose of this case report is to describe a rare association of aspergillosis and intralobar sequestration and the importance of pathologic examination to assess for aspiration.2An 18\u2010year\u2010old girl presented with recurrent lung infections for which she had been receiving antibiotics. The patient also had a history of asthma, and there was no reported history of aspiration. Computer tomography (CT) demonstrated findings suggestive of a sequestration with a direct arterial blood supply from the aorta and direct venous drainage from the azygos vein (Figure\u00a0The segment of lung parenchyma grossly demonstrated yellow\u2010tan areas and cystic change Figure\u00a0. Microsc3Although most intralobar sequestrations do not have a connection to the bronchopulmonary airways, it is reported that around 15% of cases may show a connection.The antecedent cases of aspergillosis associated with intralobar sequestration showed mostly noninvasive infection, while one case showed an acute presentation with fungal invasion.Grossly intralobar sequestration shows areas of cystic change and compact lung parenchyma.The pathogenesis of intrapulmonary sequestration is not fully agreed upon and, however, may occur early in lung development before separation occurs from the systemic blood supply. A study performed by Stocker and Malczak demonstrated systemic arteries arising from the thoracic aorta and traversing the pulmonary ligament in ten out of eleven random autopsy cases without congenital pulmonary or vascular disease.We have no financial disclosures or conflicts of interest and have not received any grant for this work.DJ.Z: contributed to write\u2010up of abstract, introduction, case presentation, discussion, and images. OE\u2010Z: reviewed and edited the manuscript."} +{"text": "Couples who are similar in their drinking behaviors often report better quality and longer marriages. These patterns of drinking may have negative implications for blood pressure, however, as individuals age. The present study examined the effects of alcohol use on blood pressure among couples and whether the associations varied by age. Participants included 2311 married/cohabiting couples followed from 2006 to 2014 in the Health and Retirement Study who reported their average drinks per week and had their blood pressure measured. Multilevel models revealed older men (aged 60+) who drank more had higher blood pressure irrespective of wives drinking whereas for younger men the link between their own drinking and increased blood pressure was greater when wives drank more. Although concordance may be beneficial for marriage, there may be a downside to drinking concordance for health especially among middle aged men. Part of a symposium sponsored by Dyadic Research on Health and Illness Across the Adult Lifespan Interest Group."} +{"text": "Million Hearts is a national initiative co-led by CDC and the Centers for Medicare &Medicaid Services that aims to prevent 1 million heart attacks, strokes, and otherrelated acute cardiovascular events by 2022 who had a rural-only service area and five health centers funded by the Health Resourcesand Services Administration. Among the 17 Champions, the median adult patient populationsize was 2,639 . The medThis national recognition program demonstrates that achieving high hypertension controlrates is possible across a range of health care settings and among patient populationsat high risk for uncontrolled hypertension ("} +{"text": "The established international program Opening Minds through Art (OMA) has been revised; this presentation reports evaluation of an early implementation with Gerontology university students and Alzheimer\u2019s nursing home patients. Ten patients were paired with student volunteers meeting once a week for eight weeks to co-create original artwork in structured one-hour sessions. Before and after art creation each volunteer recorded personal feelings and their partner\u2019s mood and satisfaction. Findings indicate the revised program is satisfying for patients and improves their mood. Families seeing the art expressed surprise and appreciation regarding patient creative capacity. Analysis of data indicates positive outcomes for student volunteers and Alzheimer\u2019s patients. Student volunteer reflections link program participation with expanded knowledge, insight, and especially empathy for Alzheimer\u2019s patients and their families. The current study contributes to robust support in the literature for efficacy of arts programming for student learning and the morale of Alzheimer\u2019s patients and their families."} +{"text": "Xenopus laevis using light microscopy of stained tissue sections and scanning electron microscopy (SEM) of vascular corrosion casts (VCCs). Results showed that bilaterally a vesical artery branched off the femoral artery. At the dorso\u2010lateral serosal surface of the body of the bladder each artery splitted within a short distance into up to five smaller arteries that supplied body and neck regions. Arteries gave off short and long terminal arterioles, which fed the mucosal capillary meshwork. Long terminal arterioles followed dimensional changes of the bladder, while short ones anchored the capillary network to the arterial system. Capillary mesh sizes and shapes varied according to the filling state of the urinary bladder. In the highly to moderately distended (filled) bladder, capillaries were rather straight or undulated only slightly, in the contracted (emptied) bladder they undulated strongly and lay side by side. Postcapillary venules formed by two equally sized capillaries or from capillaries, which serially drained into a small postcapillary venule. Vesical venules formed a large dorsal vesical and a varying number of smaller lateral and ventral vesical veins. The dorsal vesical vein drained either directly or via the posterior hemorrhoidal vein into the common pelvic vein. Lateral and ventral vesical veins also drained into the latter. The vascular patterns found were discussed in respect to the bladder spatial movements during distention (filling) and relaxation (emptying). Furthermore, it was hypothesized that an extensively filled bladder could compress the overlaying abdominal vein forcing part of the blood otherwise drained towards the liver to be detoured via the renal portal veins to the kidneys.We studied urinary bladders of adult male and female Xenopus laevis. SEM micrograph. Slightly undulating branches of vesical arteries indicate their capacity to straighten if distention (filling) of the bladder increases. Vesical veins (blue) are rather straight and adapt to a further increase in bladder volume by longitudinal stretching.Lateral view at a vascular cast of the urinary bladder of adult Her study was based on excellent dissections, but because of the limited depth of focus of the dissecting microscope she could not visualize the bladder microvascular anatomy and so studies on the microvascular anatomy of the anuran urinary bladder are still lacking. This strongly contrasts with the situation in mammals including humans, where we know the three\u2010dimensional microvascular anatomy of the healthy and diseased urinary bladder in detail . The high depth of focus and a sufficiently high resolution gained at an acceleration voltage of 10 kV in the conventional scanning electron microscope allowed (i) to identify cast capillaries as the blood vessels with the thinnest diameter, (ii) to differentiate arteries and veins by their characteristic endothelial cell nuclei imprint patterns displayed on the surface of vascular casts were studied. Adults were housed in aquaria (tap water depth: 15\u2009cm) equipped with aquarium filters and fed twice a week with either dried Gammarus pulex or ground beef heart.Ten adult individuals of both sexes of the African Clawed Toad, 2.2Two animals were euthanized by immersion into an 0.5% aqueous solution of MS 222 . After weighing, the heart was exposed, the sinus venosus opened and animals were perfused with amphibian Ringer's solution were studied. Euthanasia and rinsing perfusion were carried out in an identical manner to the histological study. When clear amphibian's Ringer's solution drained from the opened sinus venosus, 10 mL of Mercox CL\u20102B diluted with monomeric methylmethacrylate were injected with the infusor at a flow rate of 41\u2009mL/h. When the effluent resin became more viscous or the whole amount of resin had been perfused, the injection was stopped, and the animals were left for about 30\u2009minutes at room temperature to allow hardening of the injected resin. After tempering the injected resin by submerging the whole animal into a water\u2010bath , specimens were macerated in potassium hydroxide . Thereafter rinsed three times in distilled water, submerged in 2% hydrochloric acid, rinsed again three times in distilled water, and submerged in formic acid to remove any residual organic matter from the cast surfaces. Finally, specimens were rinsed for another three times in distilled water and frozen in fresh distilled water. Ice\u2010embedded casts were freeze\u2010dried in a Lyovac GT2 . Casts of the abdominal area with the urinary bladder in\u2010situ were mounted dorsal side down onto specimen stubs using the \u201cconductive bridge\u2010method\u201d of individual abdominal and urinary bladder vessels were exposed in some specimens by ripping\u2010off overlaying vessels with fine tipped insect pins under binocular control. Specimens then were sputter\u2010coated with gold and analyzed in the SEM.To allow less experienced readers to easily distinguish arteries from veins in SEM micrographs we color\u2010coded arteries (red) and veins (blue) by using Photoshop 7.0 .The study was approved by the Ethics Committee of the University of Salzburg, Austria and the Federal Government (BMBWK\u201066.012/0018\u2010BrGT/2006).3Xenopus laevis had an ovoid to spherical body specimen an almost perfect filling with few missing cast microvessels was gained. In all other specimens filling of vascular beds ranged from good to poor but allowed the recognition of individual differences between specimens in courses, calibers, branching and merging patterns of cast vesical arteries, veins and capillaries.3.2Xenopus laevis was by a single vesical artery from either side. This artery branched off the proximal portion of the femoral artery bladder\u2014for the aquatic frog Lithobates catesbeiana a mean internal bladder pressure of 0,16\u2009kPa was reported , smaller ones with strongly undulating vesical arteries and narrow capillary meshes were considered empty (contracted). Out of eight vascular castings three specimens presented distended bladders , one specimen possessed a moderately distended bladder , and four specimens had an empty (contracted) bladder . No obvious differences in the vascular anatomy of the bladders of female and male Xenopus laevis one vesical artery branched off the femoral artery bilaterally close to its origin from the common iliac artery. This contrasts with the situation in Rana esculenta to establish short arteriolar\u2010capillary transition times in all states of bladder filling and (ii) to anchor the mucosal capillary bed to the vesical arterial system.Of interest to mention are the few single, rather straight running capillary\u2010sized vessels found as the most abluminal located vessels Figure . They moThe formation of postcapillary venules varied greatly. Generally, capillaries formed small postcapillary venules, which gradually merged to form larger venules, small veins and vesical veins that emptied into the common pelvic veins. In some cases, however, capillaries emptied into veins having a much larger caliber, in others capillaries emptied bilaterally in series into a small postcapillary venule. The observation that postcapillary venules drained in an obtuse angle into a vein of much larger diameter is of interest. This mode of merging possibly builds up some backpressure, which most likely prolongs the arteriolar\u2010capillary\u2010venular transition time and so provides for a better blood supply to the mucosa.Xenopus laevis, ureters do not open into the urinary bladder but they open at the urogenital papilla or at the urinary papilla into the cloaca bladders to slightly undulating (in moderately filled) or even straight courses in distended (filled) bladders.In a Brown, . The moda Brown, . Accordia Brown, who studAlois Lametschwandtner: Conceptualization; investigation; methodology; visualization; writing\u2010original draft; writing\u2010review and editing. Bernd Minnich: Conceptualization; methodology; resources; visualization; writing\u2010original draft; writing\u2010review and editing.The authors have no conflict of interests to declare.https://publons.com/publon/10.1002/jmor.21310.The peer review history for this article is available at"} +{"text": "These results indicate that PDCs exhibit significantly less targetable genetic alterations in contrast to related TNBC and metaplastic breast carcinomas.BRCA1 gene indicates a potential benefit to platinum compounds and PARP inhibitors.All PDCs were confirmed to be of the breast origin and positive for epithelial markers . Estrogen receptor (ER), progesterone receptor (PR), and Her\u20102/neu protein were negative in all cases."} +{"text": "Chee et al enquiredA clinical study in patients with primary adrenal insufficiency aiming to compare the effects of continuous vs intermittent hydrocortisone delivery on hemodynamic parameters during elective surgery would be very challenging to execute and, in our opinion, only of theoretical benefit. There is no robust clinical evidence that short-term administration of stress dose hydrocortisone for prevention of adrenal crisis has significant adverse effects, while the potentially fatal consequences of glucocorticoid underreplacement in a stressed patient with adrenal insufficiency, in particular when paired with inflammation, are obvious. We think safety should prevail as first and foremost principle when looking after a patient with adrenal insufficiency who is exposed to major stress and we agree with Chee et al that ourChee et al. rightly enquired whether etomidate, an anesthetic agent that inhibits the crucial cortisol biosynthesis enzyme CYP11B1 , formed"} +{"text": "Previous research shows that adults with children experience poor sleep. We know that poor sleep is associated with experiencing more frequent and severe stressors the following day. This study examined whether the sleep\u2014stressor relationship is stronger for individuals with children than those without. Participants were 61 oncology nurses . Participants completed a background survey that assessed sociodemographic and work characteristics. Using 14 days of ecological momentary assessments, participants reported their sleep characteristics daily upon waking. Three times daily, they also reported whether they experienced any stressors and how severe those stressors were. Multilevel modeling was used to assess whether the sleep\u2014stressor relationship was stronger in nurses with children than those without. After controlling for sociodemographic covariates, poorer sleep quality was associated with more severe stressors. This daily association was moderated by the presence of children ; the association was apparent for individuals with children , but not for those without. The daily association for sleep quality and stressor frequency also differed by the presence of children , although the slope for individuals without children did not reach the statistical significance. These findings suggest that individuals with children are at risk for experiencing a stronger linkage between poorer sleep and greater stressor severity. Improving sleep health among adults with children is critical for stress management. Future studies should examine whether age of children or number of children further influences the sleep\u2014stressor relationship."} +{"text": "The current study examined whether partner substance use problems predict problem drinking and how gender and age moderate this relationship. Problem drinking refers to alcohol use resulting in alcohol dependence or health and social consequences. Participants were adults from Wave 2 of the Midlife Development in the United States (MIDUS) Study. Participants reported on both past 12-month problem drinking and partner substance use problems. Results indicated that 22.2% of the sample reported at least one problem drinking behavior in the past year. Multiple linear regression analysis revealed a significant interaction between gender and partner substance use problems such that for males having a partner with substance use problems was a risk factor for their own problem drinking. However, a three-way interaction with gender, age, and partner substance use problems indicated that partner substance use problems might have both gender and age-specific effects on problem drinking. Exploratory analyses of this interaction indicated that with age partner substance use problems might no longer promote risk for male problem drinking. Older adults are especially sensitive to the effects of alcohol for reasons such as lower tolerance, medication interaction, and health conditions. There is thus a need for identifying age-relevant factors associated with these drinking behaviors for intervention and prevention efforts."} +{"text": "Health literacy is becoming increasingly important in areas such as cancer care, where treatments are relatively difficult to navigate. This study aims to describe the how health literacy is associated with healthcare outcomes and health system usage among patients with lung cancer. Data include retrospective medical record data from 456 patients with lung cancer; half were age 70 and older. Patients were coded as having adequate or limited health literacy based on their response to their Single Item Literacy Screener (SILS). Data were collected from a 12 month period following diagnosis for each patient. One-third of patients had limited health literacy; this was significantly more common among adults age 70 and older. Patients with limited health literacy were more likely to have newly diagnosed lung cancers of stage 3B or higher compared to those with adequate health literacy. Patients with limited health literacy had higher median levels of depression based on the PHQ-9 questionnaire and a higher median number of emergency department visits and unplanned hospitalizations . Furthermore, patients with limited health literacy were more likely to have an emergency department visit or unplanned hospitalization sooner (p < 0.0001). These data illustrate a lower quality of life and a higher dependency on healthcare services for patients with limited health literacy. Assessment and interventions may be necessary to ensure access to quality healthcare."} +{"text": "Undergraduate nursing students are frequently exposed to older adults in the clinical setting, where they assess and manage their diseases and its consequences. But that is not enough! To support healthy aging, students need positive intergenerational learning experiences with older adults to discover the gifts of aging early in their curriculum. The goal of these experiences is to help students reflect on their thoughts about aging and reframe how they view older adults. During this presentation we will provide a tool kit based on our experience incorporating positive intergenerational learning early in our curriculum, offer practical guidelines and share constructive feedback."} +{"text": "A best evidence topic has been constructed using a described protocol. The three-part question addressed was: In patient undergoing open mesh repair of incisional hernia, is there any difference in the rate of seroma between Sublay and Onlay technique? The best evidence showed that Sublay repair has a lower seroma rate in comparison to onlay repair. \u2022Aim: Comparison of seroma rate among patients who underwent Onlay vs Sublay mesh repair of incisional hernia.\u2022Result: Six trials, including three randomized controlled.\u2022Conclusion: The best evidence showed that sublay mesh repair has lower rate of seroma compared to only repair. This fo2You are consenting a 45 year old female with recurrent incisional hernia for open mesh repair the pervious operation was complicated with seroma, the patient is wondering which technique provide a lower seroma rate onlay or sublay mesh repair ?3\u2022[In patient undergoing open mesh repair of incisional hernia]\u2022[is there any difference in the rate of seroma]\u2022[between Sublay and Onlay technique]?44.1[Incisional hernia] AND [mesh] AND [repair OR repairs] AND [onlay] AND [sublay] AND [seroama]B.Medline using the PubMed interface:[Incisional hernia] AND [mesh] AND [repair OR repairs] AND [onlay] AND [sublay] AND [ seroma]\u2022Inclusion criteria: all original articles in children or adults which compare the incidence of seroma among the Onlay and Sublay mesh repair of incisional hernia\u2022Exclusion criteria: case reports, letter to the editor, conference abstract, systematic review, articles not in English.5A total of 64 articles were identified after the removal of duplicates. Of these 40 were excluded because they are irrelevant on the basis of title and abstract. After full-text assessment of 24 articles another 16 articles were excluded because they met one of the exclusion criteria above. A total of 6 articles were identified to provide the best evidence to answer the question see .Fig. 1PR6Total of six articles were included in our review; three randomized control trials (RCTs), one prospective study and two retrospective studies. All the studies were conducted in a single centres except on was multicentre. Four out of six articles showed a statistically significant lower seroma rate among those patients who had Sublay mesh repair compared to Onlay technique.7Incisional hernia remains one of the most common postoperative complications following abdominal surgery, with incidences ranging from 11% to 20% . In addiIn this article, we have reviewed the best studies which compared the two most common modalities of open mesh repair of incisional hernia (Onlay and Sublay) in order to evaluate which techniques has the lower incidence of seroma formation.Two studies in our review showed no statistically significant difference in seroma rate between onlay and sublay mesh repair these studies were conducted by Kumar et al. and GleyIn contrast, the rest of the four studies showed statistically significant lower incidence of seroma among the sublay group of patients in comparison to the onlay group these include: three randomized control trials and one retrospective study .7.1According to the above articles, the best evidence showed a statistically significant low rate of seroma among sublay mesh repair of incisional hernia in comparison to Only repair.7.21.Small sample size in most articles: the largest sample size included was 125 patents which is relatively small number for a common problem such as incisional hernia2.All studies except were single centre studies: again this might be the reason behind the small sample size.3.In all articles there is nothing mentioned about the exact definition or classification of seroma and how it was diagnosed or treated.* in order to overcome this limitations; the authors do recommend a future large size multicentre well designed randomized controlled trail in order to draw a better conclusion about which techniques is associated with lower seroma rate.Not applicable.None.Rashid Ibrahim: conducted the literature search and wrote the paper. Sabry Abounozha: assisted in the literature search and Writing of paper. Adel Kheder: assisted in writing of paper. Talal Alshahri: assisted in the literature search, editing of writing.None."} +{"text": "The National Institute on Aging recognizes the importance of identifying promising non-pharmacological interventions (NPI) to promote health in individuals with Alzheimer\u2019s disease and related dementias. Several systematic reviews have been completed investigating exercise in this population resulting in mixed evidence regarding efficacy across functional domains. It is critical to investigate the methodological factors from the original interventions for a true understanding of these findings as to not outright dismiss exercise as beneficial. One example is Ohio\u2019s replication of Reducing Disability in Alzheimer\u2019s Disease (n=508), which resulted in no significant improvements in physical performance for individuals with dementia ((gait speed (p=.81), balance (p=.82), functional reach (p=.58)). In this investigation, along with many others, researchers were not guided by key principles of exercise science leading to critical intervention design and methodological flaws. Thus, exercise interventions for individuals with dementia need to include interpretations of non-findings and report key factors affecting the outcomes."} +{"text": "The plant hormone auxin acts as a signaling molecule to regulate a vast number of developmental responses throughout all stages of plant growth. Tight control and coordination of auxin signaling is required for the generation of specific auxin\u2010response outputs. The nuclear auxin signaling pathway controls auxin\u2010responsive gene transcription through the TRANSPORT INHIBITOR RESPONSE1/AUXIN SIGNALING F\u2010BOX pathway. Recent work has uncovered important details into how regulation of auxin signaling components can generate unique and specific responses to determine auxin outputs. In this review, we discuss what is known about the core auxin signaling components and explore mechanisms important for regulating auxin response specificity. A review of recent updates to our understanding of auxin signaling. Arabidopsis contains 22 full\u2010length The complexity of diverse auxin signaling responses can, at least in part, be regulated by unique characteristics imparted by three distinct protein domains within ARF family members\u2014the N\u2010terminal DNA\u2010binding domain (DBD), the variable middle region (MR) associated with activating or repressing activity, and the C\u2010terminal PB1 domain homologous to those found in Aux/IAA repressor proteins within auxin response genes.Sequence\u2010specific recognition and binding of the ARF transcription factors to auxin response genes is carried out by the DBD. Structural studies of the ARF1 and ARF5 DBD have revealed a composition of three distinct substructural components.AuxREAuxRE.AuxRE variants must still be validated, however, the presence of multiple target sites with varying ARF binding affinities may begin to help explain differences in ARF activity.Several ARF proteins were initially identified based on their ability to bind the canonical TGTCTC An additional layer of auxin\u2010responsive gene regulation by ARF transcription factors comes from the ability of these proteins to dimerize through the DD. The DBD of ARF1 and ARF5 form homodimers within the crystal structure with the B3 domains binding to an inverted repeat of the canonical TGTCTC AuxRE element.AuxRE site leads to reduced binding affinity, suggesting that dimerization of ARF proteins leads to cooperative binding at target sites.AuxREs with different spacing.Dimerization of ARF proteins allows for multi\u2010site recognition of auxin responsive motifs within target genes. Mutation of a single 4.2Whereas structural studies of the ARF DBD and PB1 domains have guided our understanding of the function of these domains, much less is known about the properties of the ARF middle region. This region has the most highly diverged amino acid composition and length amongst ARFs and thus it has been difficult to tease apart contributions of the MR in regulating ARF activity. The ARF MR alone is sufficient to confer transcriptional activator or transcriptional repressor activity.in planta.Although the precise mechanisms that regulate ARF activator and repressor activities are unclear, the MR of ARF proteins likely contain an intrinsically disordered region (IDR) that confers these activities that allow for auxin responsiveness.M. polymorpha to those of plant lineages with expanded auxin signaling protein families, such as P. patens and C. richardii, revealed that the number of ARF transcription factors scales with the number of auxin\u2010regulated genes.The presence of all necessary auxin signaling components can be found in the common ancestor of land plants, however, another important question is how this signaling pathway evolved in complexity to enable diverse auxin responses. Arabidopsis accessions have revealed interesting insights. These studies have uncovered evolved sequence variations that correspond to altered molecular phenotypes and underscore how small changes can have significant impacts on protein function and consequent auxin response.Not only have auxin signaling components been examined across evolutionary history,6Cis\u2010trans isomerization of prolines in Aux/IAA proteins is involved in auxin response, perhaps by regulating recognition by the SCF complex.Post\u2010translational modifications of each of the core auxin signaling components impart an additional level of regulation to auxin response outputs Table . For exaAlthough many of these modifications and their roles in regulating auxin outputs need to be confirmed in vivo, ARF7 and ARF19 are phosphorylated by the BRASSINOID\u2010INSENSITIVE 2 (BIN2) kinase to regulate lateral root organogenesis.BIN2\u2010mediated phosphorylation of ARF7 and ARF19 results in relief from Aux/IAA repression and enhances transcriptional activity by these ARF transcription factors.In addition, SUMOylation of ARF7 plays a role in hydropatterning of lateral roots.7Within the past several years, an increasing number of studies have begun to highlight the importance of biomolecular condensates as a means to regulate diverse biological functions within the cell . ARF3 does not contain a PB1 domain (reviewed in Refs. 9Taken at face value, the auxin signaling pathway seems a fairly straightforward mechanism for auxin perception and response; however, we are only beginning to understand the multiple layers of regulation necessary for generating distinct and dynamic auxin outputs. Diversity in signaling component family members allows for different combinations of protein interactions. Additionally, post\u2010translational modifications, establishment of transcriptional regulatory complexes, and interactions with components from other signaling pathways may play a role in auxin response specificity. Transcriptional regulation of auxin response components,The authors declare no potential conflict of interest."} +{"text": "Congenital intrahepatic portosystemic venous shunts are rare vascular malformations which are incidentally discovered on imaging or once hepatic encephalopathy becomes clinically apparent. Surgical ligation and endovascular embolization are potential treatments. Congenital intrahepatic portosystemic venous shunts are rare vascular malformations which are incidentally discovered on imaging or once hepatic encephalopathy becomes clinically apparent. Surgical ligation and endovascular embolization are potential treatments. They often go undiagnosed and are incidentally discovered on imaging or once hepatic encephalopathy becomes clinically apparent. We present two cases of incidental congenital portosystemic shunts in noncirrhotic patients who went on to develop refractory encephalopathy without having prior history of liver disease. Both patients were successfully treated with embolization. Familiarity with the pathogenesis and imaging features may enable prompt diagnosis and help guide appropriate patient endovascular or surgical management.Congenital intrahepatic portosystemic venous shunts (IPSVS) and extrahepatic portosystemic shunts make up the so called congenital portosystemic shunts (CPSS). Congenital portosystemic shunts are an important disorder in children and should be differentiated from metabolic deficiencies involving hyperammonemia or galactosemia.. These abnormalities include intrahepatic connections between branches of portal vein and hepatic veins.Congenital intrahepatic portosystemic venous shunts (IPSVS) have a prevalence of 1 in 30\u00a0000 births and are caused by abnormal involution of the fetal vasculature. In these patients, there is a persistent communication between vitelline veins of the omphalomesenteric system and the sinus venosus due to a focal absence of sinusoid formationsKanasawa et al proposed a classification based on the correlation of the severity of portal hypoplasia with portal venous pressure, histopathological findings, postoperative portal venous flow, and liver regeneration.Congenital IPSVS can present with various symptoms and ultimately can lead to long\u2010term complications by permitting hepatic bypass of mesenteric venous blood return.Over time, unprocessed portal metabolites result in hyperammonemia and subsequent hepatic encephalopathy.If the medical or operative treatment fails, liver transplantation is the only therapeutic option being cautious with major hemodynamic changes that can occur in the intraoperative period. Resection is the treatment of choice in patients when liver tumors are associated with extrahepatic portosystemic shunting.We describe two cases of congenital intrahepatic portosystemic shunts, discovered incidentally in noncirrhotic adult patients who develop refractory encephalopathy without having history of liver disease or prior surgical intervention. The two patients were successfully treated using transcatheter embolization with clinical improvement. Endovascular treatment offers a safe alternative of therapy after evaluation of anatomical and pathophysiological characteristics of the anomaly.1.1A 75\u2010year\u2010old woman with no significant history of liver disease or neurologic disorder was evaluated for altered mental status. Her initial ammonia level was 33\u00a0mmol/L , and liver function tests (LFTs) were within normal range. A CT of the abdomen was performed which demonstrated an intrahepatic shunt between the right posterior portal vein and the right hepatic vein was manipulated through the shunt into the main portal vein. Prior to embolization, a temporary balloon\u2010occlusion test of the shunt was performed using a 12\u2010mm Berenstein occlusion balloon catheter (Boston Scientific). The test did show a portal pressure of 9\u00a0mm\u00a0Hg before and 16\u00a0mm\u00a0Hg after balloon occlusion, and this lasts remaining unchanged after several minutes. The findings were interpreted as no significant hemodynamic changes or risk of portal hypertension that would preclude shunt occlusion. Therefore, the shunt was then successfully embolized with Penumbra POD coils (Penumbra). Due to the high flow nature of the shunt, it was necessary to use coils that were approximately 50% oversized for the targeted vessel diameter to prevent potential coil migration and nontarget embolization. Following POD embolization, an Amplatzer Vascular Plug II was deployed as a safety measure to further avoid nontarget coil migration into the right atrium and pulmonary vasculature.Following embolization, a contrast\u2010enhanced CT of the abdomen and pelvis was performed which demonstrated successful exclusion of the venous\u2010venous malformation with thrombosis of the posterior segment branch of the right portal vein Figure . Follow\u20101.2A 72\u2010year\u2010old man with no significant history of liver disease, neurologic disorder, or prior relevant surgical intervention presented with the acute onset of lower extremity weakness and confusion. A full neurological workup was performed which included a normal electro encephalogram (EEG) and an unremarkable MRI of the brain. An ammonia level of 145\u00a0mmol/L was measured which suggested hepatic encephalopathy as the cause of the patient's altered mental status. A contrast\u2010enhanced CT of the abdomen and pelvis was then performed which demonstrated an intrahepatic portosystemic shunt between the left portal vein and the left hepatic vein with multiple venous connections (Figure 2Intrahepatic portosystemic shunts are often incidentally detected on imaging or diagnosed after symptoms of hepatic encephalopathy develop. The incidence has been reported as 1/30\u00a0000. They have been associated with other types of anomalies such as cardiovascular, hepatobiliary, urogenital, and gastrointestinal among others. In addition, complications like portopulmonary hypertension, reported in 13%\u201066% in children,Multiple imaging modalities are useful for diagnosis of an intrahepatic portosystemic venous shunt including color Doppler ultrasonography, contrast\u2010enhanced CT, MRI or conventional catheter\u2010directed angiography. The imaging finding most consistent with diagnosis of a portosystemic venous shunt is visualization of a direct communication between portal and hepatic veins; however, this is not always demonstrated on imaging. Secondary findings on imaging suggestive of a shunt include abnormal blood pooling from a dilated portal branch with early visualization of the hepatic venous system.Multiple challenges can be present during the embolization including diminutive portal vein in large portosystemic shunts, difficulty to determine the anatomy especially in types II or IV if multiple venous communications can be present or large veins with potential risk of coils or plug migration. Optimal oversize of the plugs or coils is recommended to avoid any potential undesired migration to the systemic circulation. Patient follow\u2010up is mandatory to evaluate for the ammonia levels and to determine whether further embolization is required.3Intrahepatic portosystemic venous shunts are rare hepatic vascular malformations that often remain asymptomatic and are discovered incidentally on imaging. Alternatively, these patients may present with hyperammonemia and encephalopathy among other symptoms, and with proper endovascular management, this detrimental sequela is potentially reversible. Technical challenges during endovascular treatment could be overpassed with detailed evaluation of the anatomy as well as hemodynamic changes including identifying multiple potential venous communications, measuring portal pressure, and selection of the more appropriate embolization material.None declared.MPBM: served as the primary author; was responsible for accuracy and integrity of the case report; and involved in the conception of the article, performed the interventional radiology procedures of each case, collected the radiologic imaging studies, reviewed the manuscript drafts, and approved and modified all versions of the manuscript. AK: did the literature review and wrote the background of the manuscript. JCB: served as the coauthor; did the modifications and corrections suggested by the reviewers; complemented the background, results, and description of the cases; and additionally corrected and supplemented the references of the last manuscript. CL: served as the coauthor and helped to write the first draft of the manuscript. PC: served as the coauthor, and interpreted and read the diagnostic CT images. AS: is the corresponding author; involved in design and writing of drafts including all the manuscript sections; uploaded the images; collected the cases; and submitted the case report every time when needed. SB: served as the coauthor; reviewed the drafts; helped in the interpretation of interventional radiology images; and helped in maintaining the accuracy of all aspects of the manuscript."} +{"text": "Age stereotypes are complex and multifaceted: individuals can demonstrate and embody numerous and varied positive and negative stereotypes. Therefore, solutions to combat age stereotypes must also be complex and multifaceted. Additionally, both social and physical forms of age segregation are common in our society. This causes fewer and fewer opportunities for younger and older people to interact. Intergroup Contact Theory suggests age stereotypes can be reduced through increased intergenerational contact. One way to encourage contact between younger and older populations is through intergenerational programming. However, there is a lack of literature investigating the effects of intergenerational programs on perceptions of aging. The purpose of this paper was to critically review and explore literature on intergenerational programs to understand how they influence age stereotypes and ageist attitudes. The available literature suggests that intergenerational programs involving young children (ages 4-8), adolescents (ages 11-18), or emerging adults (ages 19-26) interacting with older adults (ages 65+) can significantly reduce age stereotypes towards older adults. Additionally, older adults (ages 65+) negative beliefs and attitudes towards younger people (ages 4-26) can also be deconstructed after participation in intergenerational programs. Intergenerational programs act to break down age barriers and promote connections and understandings between generations. These programs challenge the belief that older and younger people should live and participate in spaces that are separate from one another. Providing opportunities for younger and older people to participate in intergenerational programs is one way to promote respectful relationships and enhance the quality of life and health of all generations."} +{"text": "Mindfulness-based interventions (MBIs) in individual and group formats, have been shown to be effective for a variety of psychological disorders. Due to the promising evidence supporting the wide applicability of mindfulness skills, graduate student therapists were trained to deliver groups that attracted diverse individuals across the lifespan. In these groups, therapists noted how intergenerational dynamics facilitated group cohesion and allowed for increased normalization of common challenges related to practicing mindfulness skills. Therapists\u2019 prior training on cohort differences and treatment recommendations for older adults served as an important foundation to navigating these group interactions. Barriers to simultaneously collecting data and delivering intervention components were noted by the student therapists. Future research and therapist training gaps in knowledge related to effectively facilitating intergenerational groups were identified."} +{"text": "We enjoyed reading Povleson et al.\u2019s review entitled \u201cDiagnostic thoracic outlet syndrome: current approaches and future directions\u201d . The autIn the Povleson et al. review, brachial plexus ultrasound is identified as an emerging technology for neurogenic TOS. The authors highlight the relative low cost and availability as benefits of ultrasound over MRI (magnetic resonance imaging) for the investigation. To support this argument, the authors cite one study identifying an increased rate of neurogenic symptoms in patients with an atypical branching anatomy of the brachial plexus compared with those with a normal branching anatomy in a small feasibility study with a poor methodology ([4/8] 50% versus 5/35] 14%) [5 14%) [3To further explore the potential utility of ultrasound for neurogenic TOS, we attempted a comparative systematic review and meta-analysis assessing the diagnostic accuracy of both ultrasound and MRI evaluated against a surgical reference standard (PROSPERO Registration ID: 168479). All relevant articles up to 12 January 2020 in MEDLINE, EMBASE, Scopus, and the Cochrane Library were evaluated. A total of 178 potentially relevant articles were identified after duplicate removal. However, following the title and abstract review and subsequent full text review, no articles evaluating ultrasound for neurogenic TOS were available for inclusion. The results of this systematic review highlight the lack of available evidence to support the assertion that ultrasound could be used as a diagnostic test for neurogenic TOS. At present, the utility of ultrasound for this clinical scenario remains theoretical rather than emerging. Future studies evaluating the diagnostic accuracy of ultrasound for neurogenic TOS with a rigorous methodological standard including a reference standard that can minimize both selection bias and verification bias (such as electrodiagnostic studies and/or a combination of investigations) and reasonable sample size are needed."} +{"text": "Although the global community has discussed needs for establishing international standards of health care for immigrant older adults for decades, it is challenging for policy makers to consider international standards that could meet diverse needs for older adults from various migrant groups. The purpose of this symposium is to discuss challenges and possible strategies to develop global standards to protect immigrant older adults. There will be four presentations on the topic of various needs of older adults from different migrant groups. Noriko Toyokawa will present a study in diversity in parents\u2019 expectations on filial piety among immigrant older adults from different racial/ethnic groups in the Southern California. Weiyu Mao and her colleagues will present their study in the perceived neighborhood cohesion as a protective factor for older Chinese immigrants\u2019 oral health. Allen Glicksman and his colleagues will report the diversity across migrant groups and State Policies that create a challenge in using finding to establish global standards for best practices with older migrants based on a series studies on Mandarin speaking Chinese and Puerto Rican older immigrants. Finally, Mika Marumoto will suggest the \u2018reframing of aging initiative\u2019 as a possible means of leading the way of cultivating transformative solutions. Vivian Lou will comment on each presentation, discuss common themes among the presented studies, and address future research directions. With the audience, the presenters will discuss challenges in dealing with diversity issues and suggestions for a global initiative to protect human rights and health care accessibilities for immigrant older adults."} +{"text": "Populus angustifolia) with a double quantile regression approach. We show that intraspecific variation in plant size, growth, and leaf morphology corresponds with the species' total climate range and certain climatic limits related to temperature and moisture extremes. Moreover, we find evidence of genetic clines and phenotypic plasticity at environmental boundaries, which we use to create geographic predictions of trait variation and maximum values due to climatic constraints across the western US. Overall, our findings show the utility of double quantile regressions for connecting species distributions and climate gradients through trait\u2010based mechanisms. We highlight how new approaches like ours that incorporate genetic variation in functional traits and their response to climate gradients will lead to a better understanding of plant distributions as well as identifying populations anticipated to be maladapted to future environments.Global change is widely altering environmental conditions which makes accurately predicting species range limits across natural landscapes critical for conservation and management decisions. If climate pressures along elevation gradients influence the distribution of phenotypic and genetic variation of plant functional traits, then such trait variation may be informative of the selective mechanisms and adaptations that help define climatic niche limits. Using extensive field surveys along 16 elevation transects and a large common garden experiment, we tested whether functional trait variation could predict the climatic niche of a widespread tree species ( Accurately forecasting plant species distributions and range limits is a central challenge for understanding climate change effects on the landscape. Here, we extend a recently proposed quantile regression approach using within\u2010species functional trait variation across elevation gradients to predict a widespread tree species' climate niche. Collectively, these findings point towards the ecological and evolutionary drivers of climate range limits and demonstrate how this approach can improve current niche models by including intraspecific trait\u2010climate responses. Plant range limits reflect the biotic, energy, and resource constraints that determine where populations are no longer self\u2010sustaining . Specifically, we tested whether intraspecific trait variation could predict the species' climate range, if climate range limits impose constraints on the genetic variation of functional traits, and whether we can use this information to project how trait variation is constrained on the landscape. We surveyed functional trait variation (plant size/growth and leaf morphology) in the field across elevation gradients in distinct watersheds, treating each gradient as a subset of the overall species' phenotypic and climatic range. Then, using a common garden experiment with clonal replicates of P.\u00a0angustifolia cuttings to separate environmental and genetic effects on trait variation, we compared the response patterns in field traits (variation caused by environment and genetic effects) to common garden traits (variation caused by genetic effects). We hypothesized that:We extend the approach outlined by Stahl et al. that comHypothesis 1Plant size/growth and leaf morphology traits from field observations predict the species' climate range using double quantile regressionHypothesis 2Common garden trait\u2010climate relationships will show similar response patterns as field traits, indicating the presence of genetic clines that constrain trait variation at range limits.22.1Populus angustifolia James is a dominant riparian species with a wide geographic range throughout the Rocky Mountains were sampled and marked with GPS points (Oregon\u00ae 550t) (see Field sampling and common garden experiment below). Using the georeferenced sampling points, we gathered 15 climate variables at 30 arcsecond resolution from the Environmental Rasters for Ecological Modeling dataset . These variables encompass annual and monthly trends of temperature and moisture\u2010related factors are likely to be directly related to the ecophysiological processes that influence species geographic ranges, and in some cases outperform WorldClim variables in ENM , we sampled 5\u201310 putative genotypes of mature P.\u00a0angustifolia trees from three sites along an elevational transect in each watershed and collected cuttings from low\u2010hanging branches (2\u20132.5\u00a0m canopy height) for clonal propagation of putative genotypes in the common garden experiment (see below). Five fully flushed leaves were collected from multiple locations 360\u00b0 around the tree at the same canopy position (2\u20132.5\u00a0m canopy height). We calculated specific leaf area with scanned measurements of leaf area and leaf dry mass .We sampled natural occurring stands of a Figure . These ra Figure . Becausea Figure , we samd Figure . We measPopulus Genome Consortium and one from Tuskan et al., P.\u00a0angustifolia is known to hybridize with cooccurring Salicaceae species at lower elevations and form distinct hybrid zones to try and avoid collecting from the same genet, and subsequent microsatellite analysis was used to identity unique genotypes , potted in general potting mix , and randomized in the glasshouse. Cuttings were grown with no supplemental lighting, modest temperature control (within ~5\u00b0C compared to outdoor temperatures), and full watering thrice weekly, which allowed them to generally track the normal growing seasons with a dormancy period between November and March. In June of 2013, we measured the diameter of annual growth for plants in the common garden . In addition, five leaves were collected from multiple locations along the main stem of rooted cuttings for average leaf trait measurements . We calculated the broad\u2010sense heritability of these traits as the total amount of variance explained by plant genotype show a relationship between functional traits and the median climatic niche. More importantly, we interpret slopes that differ from zero at upper limits (90th and 95th) or lower limits (5th and 10th) as support for our first hypothesis by showing how functional trait variation predicts climate boundaries.To test the first hypothesis (that field trait variation will predict climate range limits), we explored relationships between DBH, leaf area, leaf mass, and SLA in the field and climate variables using double quantile regression. These are commonly measured functional traits that impact organismal performance and correspond to the morphological and physiological mechanisms that influence plant fitness and demography \u2014slopes from upper and lower trait\u2010climate relationships are significant in the same direction, creating a rhombus shape where a change in trait value does not correspond to a change in the climate range of the species.One\u2010sided (wedge)\u2014the slope of either the upper or lower quantile is significant, creating a wedge shape that indicates a constraint on trait values at one climate extreme.Reverse (acute triangle)\u2014both outer quantiles have significant slopes in opposing directions, creating an acute triangle shape that reflects a double\u2010sided constraint on traits from both ends of a climate gradient.The space between the outer quantiles reflects the climate niche that P.\u00a0angustifolia can change as trait values increase or decrease.One\u2010sided and reverse patterns (II and III) show how the possible climate range of Multivariate analysis of trait\u2010environment relationships; Figure To test the second hypothesis (that genetic variation from traits in the common garden experiment will predict climate range limits), we used the same double quantile approach described above using common garden traits . Similar response patterns between the trait\u2010environment relationships in the field and common garden would suggest that genetic clines influence functional trait variation at climate range limits and provide spatially explicit patterns of constraints on genetic variation in functional traits across climate gradients. All analyses were performed in R version 3.4.1 predict the species' climate range and show that genetic variation in functional traits is constrained at the species' climate limits. Annual growth diameter showed three obvious one\u2010sided response patterns that match the same patterns from DBH\u2010climate regressions . We find that tree diameter is predicted to be unconstrained at higher latitudes with areas in Montana and Wyoming having climates suitable for the widest range of P.\u00a0angustifolia DBH values (<20\u00a0cm to >60\u00a0cm). From here, \u201cno\u2010go\u201d areas for larger trees extend southward until ~20\u00a0cm is expected to be the maximum attainable DBH for trees in central Arizona along the Mogollon Rim . For instance, plants with thick leaves are often found in arid conditions to maintain photosynthesis while reducing transpirational water loss . Our results show that leaf mass variation . The number of inconsistent patterns between field and common garden trait\u2010climate relationships suggests that some level of phenotypic flexibility in plant growth and leaf morphology helps genotypes persist at contemporary climatic limits and annual growth diameter (common garden) to gridded climate data. When projected across the western US, we find that variation in growth decreases from north\u2010to\u2010south , and the maximum attainable DBH values decline from Montana to Arizona. In other words, the climate gradients covering these portions of the species range gradually impose \u201cno\u2010go areas\u201d for genotypes with growth traits above a certain DBH threshold, which decreases the potential range of trait values by limiting the maximum attainable DBH. This pattern suggests that southern populations may have insufficient genetic variation in growth traits for adaptive evolutionary responses to climate change that can greatly complement and improve upon existing ENM. We modeled how genetic clines in Populus tree species, but it should be noted that other studies exist with similar sampling designs for which this method could be applied with Clarkia , but that most other areas in the western US have at least one major climate pressure that constrains the lower limits of leaf mass variation. In summary, our approach shows the advantages in using double quantile regression for creating geographic predictions of genetically based functional trait variation. We demonstrate this for the first time using a single widespread 4.3Our study demonstrates how a widespread tree species' climate range is predicted by intraspecific trait variation. Because phenotypes are the level upon which plants physically respond and interact with their climatic , biotic are indicative of the physiological and evolutionary mechanisms that influence species range limits. Pairing this approach with ongoing advancements in ecological forecasting that includes an explicit consideration of genetic variation will significantly improve our ability to predict the adaptive capacity and local extirpation risk of marginal populations under future climate scenarios.None declared.MVN and JBV conceived the original ideas, analyzed the data, and wrote the first draft of the manuscript; IMW, LOM, SLJB, KKB, JAS, and JKB all contributed to the discussion of ideas and provided substantial edits to the manuscript.\u00a0Click here for additional data file."} +{"text": "Using the data of 2014 baseline survey of the China Longitudinal Aging Social Survey (CLASS), which provides a sample of older Chinese who had grandchild younger than 18 years old, this study examines the associations among grandchild care, social networks, and depressive symptoms among Chinese older adults. The older adults are divided into three groups basing on the frequency of their behaviors of taking care of grandchildren. The three groups are \u2018no care, providing care occasionally, providing care frequently\u2019. The mediating and moderating effects of social networks between grandchild care and depressive symptoms are tested. Results show that older adults who provide grandchild care report superior social networks and better mental health than those who don\u2019t provide grandchild (reference group). After controlling the related variables, the older adults who provide grandchild occasionally benefit more than those who take care of grandchild frequently. Grandchild care is related to larger social networks, and the social networks are fully mediating the association between grandchild care and depressive symptoms."} +{"text": "Genetic stock identification is a widely applied tool for the mixed\u2010stock management of salmonid species throughout the North Pacific Rim. The effectiveness of genetic stock identification is dependent on the level of differentiation among stocks which is often high due to the life history of these species that involves high homing fidelity to their natal streams.\u00a0However, the utility of this tool can be reduced when natural genetic structuring has been altered by hatchery translocation and/or supplementation. We examined the genetic population structure of ESA\u2010listed steelhead in the Snake River basin of the United States.\u00a0We analyzed 9,613 natural\u2010origin adult steelhead returning to Passive Integrated Transponder detection sites throughout the basin from 2010 through 2017. Individuals were genotyped at 180 single nucleotide polymorphic genetic markers and grouped into 20 populations based on their return location.\u00a0While we expected to observe a common pattern of hierarchical genetic structuring due to isolation by distance, we observed low genetic differentiation between populations in the upper Salmon River basin compared to geographically distant populations in the lower Snake River basin.\u00a0These results were consistent with lower genetic stock assignment probabilities observed for populations in this upper basin. We attribute these patterns of reduced genetic structure to the translocation of lower basin steelhead stocks and ongoing hatchery programs in the upper Salmon River basin. We discuss the implications of these findings on the utility of genetic stock identification in the basin and discuss opportunities for increasing assignment probabilities in the face of low genetic structure. The genetic population structure of ESA\u2010listed steelhead in the Snake River basin of the United States was examined using 9,613 natural\u2010origin adult steelhead returning to Passive Integrated Transponder detection sites from 2010 through 2017. We observed low genetic differentiation between populations in the upper Salmon River basin and geographically distant populations in the lower Snake River basin consistent with lower genetic stock assignment probabilities observed for populations in this upper basin. We attribute these patterns of reduced genetic structure to the translocation of lower basin steelhead stocks and ongoing hatchery programs in the upper Salmon River basin and discuss the implications of these findings on the implementation of genetic stock identification in the basin. Lower Granite Dam provides a sampling point for all steelhead migrating upstream in the Snake River, and PIT tag arrays allow assignment of spawning locations for presumed natural\u2010origin fish. Returning adult steelhead with both a genetic sample and PIT tag detection can be used to assess the genetic structure in the basin. To assess the impacts of translocations on genetic structure we quantified patterns of IBD at a basin\u2010wide scale including and excluding supplemented populations in the upper Salmon River watershed. Finally, we assessed the accuracy and precision of GSI assignments of samples from the upper Salmon River watershed relative to other populations throughout the Snake River basin.22.1O.\u00a0mykiss collected between 1999 and 2013 , and fish were genotyped at a panel of 180 single nucleotide polymorphisms (SNPs) used in the Columbia River steelhead GSI baseline 2010\u20132017. A small, nonlethal sample of fin tissue was collected from each fish for genotyping and subsequent GSI analyses. Wild adult steelhead that were not PIT tagged at time of capture in the adult fish facility had one inserted Ogden,\u00a0 prior toSamples were filtered to include only natural\u2010origin fish that successfully genotyped at \u226590% of the amplified loci. We identified fish as natural\u2010origin adult steelhead if they had no marks , no visible fin erosion . Within populations, only groups of genetically homogenous spawn year and PIT tag detection site combinations were used for analysis.Using PIT detections that reflected presumed natal origin, the corresponding genetic samples were grouped into 20 collections based on populations described in NMFS . We set For GSI assignments, we used the full Expectation\u2010Maximization algorithm maximum likelihood estimate option in the program gsi_sim .We calculated FST1\u2010FST as the response variable in a linear regression with stream distance between populations . We expected that successful steelhead translocations in the mid\u20101960s would reduce the overall genetic differentiation in the Snake River basin. This expected reduction in genetic differentiation is because the stream distance between most steelhead populations in the Snake River basin and Hells Canyon Dam is shorter than between these same populations and the upper Salmon River. We also expected that mimicking the historical association between genetic and geographic distance in the Snake River basin by analytically moving the upper Salmon River populations to Hells Canyon Dam would produce a similar pattern of IBD to the one observed when we removed the upper Salmon River populations from analysis if these translocations were successful. Therefore, our second test examined the difference between the patterns of IBD observed (a) after removing the populations from the upper Salmon River, and (b) after analytically moving these populations to Hells Canyon Dam.We used a statistical test for comparing the strength of IBD described in Powell that is We constructed a neighbor\u2010joining tree for the populations based on Cavalli\u2010Sforza Edwards chord distance . We also observed significant genetic differentiation across PIT tag detection sites within the lower Clearwater River population in 2013 and the Imnaha River population in 2011 . Individuals detected on arrays within the lower Clearwater River population in 2013 and the Imnaha River population in 2011 was removed from analysis. A total of 9,159 returning adult steelhead remained in the analysis following the removal of these individuals.Three loci were out of Hardy\u2013Weinberg equilibrium due to a deficit of heterozygotes in more than half of the populations analyzed in a given year and were removed from analysis. We observed significant genotypic differentiation across spawning run years and PIT tag detection sites within the lower Clearwater River (CRLMA\u2010s), Imnaha River (IRMAI\u2010s), and upper Grande Ronde River (GRUMA\u2010s) populations after excluding populations in the upper Salmon River from analysis clustered with the 2016 broodstocks from the Pahsimeroi, Sawtooth, and Oxbow fish hatcheries to compensate for the loss of anadromous steelhead populations in the middle and upper Snake River following the construction of Hells Canyon Dam, and (b) to provide hatchery steelhead to meet sport and tribal harvest mitigation goals in the upper Salmon River. To this end, Pahsimeroi Fish Hatchery was founded using steelhead trapped at Hells Canyon Dam indicate some limitations of differentiating stocks that have shared ancestries from translocation and supplementation efforts, there are opportunities to increase assignment accuracy by incorporating SNPs under selection ; formal analysis (lead); visualization (lead); writing \u2013 original draft (lead); writing \u2013 review & editing (supporting). Matthew R. Campbell: Conceptualization ; funding acquisition (lead); project administration (lead); resources (lead); supervision (lead); writing \u2013 original draft (supporting); writing \u2013 review & editing (lead)."} +{"text": "The Age-Friendly University (AFU) movement is specifically targeting one group of adult learners who are less represented within higher education -- individuals considered \u201colder adults,\u201d with five of the ten principles focused on promoting educational opportunities for older adult learners. However, there is less understanding within higher education for how to ensure inclusivity of this group. Importantly, some universities across the country have identified promising strategies for engaging older adult learners within higher education classrooms and supporting them beyond the classroom. As this intergenerational learning model continues to grow, there is much to learn from those who have begun efforts to appropriately utilize and engage older adult learners. This symposium will highlight examples from universities that have identified ways to create age-diverse programs within the university setting. The first paper will begin by discussing intergenerational learning opportunities for utilizing older adult learners in innovative ways to enhance university student experiences, and the second paper will specifically highlight successful activities used in a university class to engage older and younger adult learners. The third paper will examine ways in which a university and Osher Lifelong Learning Institute work together and promote research opportunities for both generations. The fourth paper will discuss research conducted to investigate how intergenerational classroom experiences are shaped by older adults. The fifth paper will describe the use of technology training workshops to promote service learning for university students and those in a retirement community. This would be a collaborative symposium between the AFU and ILRCE Interest Groups."} +{"text": "Research has shown that perceived discriminations impact physical and mental health in later life. Discrimination experiences could make older adults consider themselves as a social misfit and decrease their social interactions, which finally increases their loneliness. Religious behaviors has been reported as a key factor of a lower sense of isolation. Considering that religious behaviors provide opportunities to engage in more extensive social networks and have supportive social ties with community members, attending religious services might decrease the impact of older adults\u2019 perceived discrimination on loneliness. The current research aims to examine the moderating role of religious services attendance in the association between older adults\u2019 perceived discrimination and loneliness. We used data of 4,488 adults aged 50 to 80 from the Health and Retirement Study (HRS) collected in 2012 and 2014. Linear regression analysis was performed to investigate whether older adults\u2019 religious service attendance might decrease the impact of their perceived discriminations in daily life on the level of loneliness. The results indicated that more perceived discriminations older adults face on a daily basis were significantly associated with higher levels of loneliness. However, participants who frequently attended religious services showed a lower impact of perceived discriminations on their loneliness. These findings highlight the positive effects of engaging in religious activities on discriminated older adults\u2019 social well-being. These findings also emphasize the role of the religious community as a social resource for socially marginalized older adults."} +{"text": "To study use and completion of Life Sustaining Treatment (LST) templates across the Department of Veterans Affairs (VA) healthcare system, we designed a qualitative study to interview VA sites we identified who had high rates of LST template completion between July 1, 2018 and March 2019. We then conducted site visits with two VA sites and phone interviews with nine other VA site to better describe facilitators and barriers to implementation of this new practice and identified factors influencing high rates of LST documentation completion. Researchers who conducted the interviews analyzed interview data through applying the Consolidated Framework for Implementation Research. From these efforts, we identified best practices used, including the importance of a formally appointed implementation leader, gaining leadership support, and engaging staff through education. We will share these recommendations with VA sites across the healthcare system to improve LST documentation."} +{"text": "Mechanical prosthetic aortic and mitral valves preclude either a retrograde aortic or transseptal approach to the left ventricular (LV) endocardium. Several operators have reported on the application of nonconventional techniques for ventricular tachycardia (VT) ablation including transventricular septal puncture, epicardial approach, transmechanical valve approach, transcoronary venous approach, and transapical approach. Incorporating transventricular access to the LV under intracardiac echocardiography (ICE) guidance has been previously attempted in VT ablation procedures in patients with both aortic and mitral mechanical valves. However, while ICE is readily used in the United States, its use is less common in Europe, since the health insurance agencies largely do not cover the costs of ICE catheters. We therefore herein present a case of VT ablation in the LV using a transventricular approach in a patient who underwent mechanical double valve replacement performed under subcostal echocardiographic and fluoroscopic guidance. Several operators have reported previously on the use of nonconventional techniques during ventricular tachycardia (VT) ablation such as transventricular septal puncture, and 2, the interventricular septum was crossed with a Brockenbrough needle under uninterrupted warfarin and intravenous heparin therapy. It was difficult to dilate and advance the aforementioned Swartz\u2122 Braided SL1 sheath from the transventricular septum; however, the catheter manipulation was easy to perform after the crossing was complete. The three different VTs originating from the LV apical and posterobasal regions were induced and late potential substrate ablation during sinus rhythm and the areas of the middiastolic potentials during VT and 2C were ablated. In total, the procedure lasted about 5.3 hours without intraprocedural complications or residual ventricular septal defect . He recovered uneventfully and experienced one VT episode that responded to antitachycardia pacing therapy one day later. He was discharged five days after the procedure with heart failure therapy and mexiletine. At present, this patient is being followed up with in the outpatient clinic.A 69-year-old male patient with nonischemic dilated cardiomyopathy was referred for VT ablation due to electrical storm. He had a history of double valve replacement and biventricular pacemaker implantation. Since his documented VT morphologies were compatible with the LV posterobasal and apical regions, we chose to perform transventricular crossing, as it was thought that it could be potentially difficult to reach all parts of the LV apex using transapical access. The right internal jugular vein was accessed, and a Swartz\u2122 Braided SL1 Transseptal Guiding Introducer Sheath was advanced to the basal right ventricular septum. We preemptively prepared the Amplatzer\u2122 Muscular Ventricular Septal Defect Occluder device as a bailout plan for use if there was a catastrophic septal defect at the time of removal. After confirming there was a safe distance from the coronary septal perforator branches with both left and right coronary angiography and no entrapment of the tricuspid septal leaflet by subcostal echocardiography 8 Although epicardial VT ablation is a potentially useful method in patients with mechanical aortic and mitral valves,9 the coronary venous system approach6 or transventricular septal access2 have additionally been applied successfully in certain patient populations. Yamada et al.10 and Herweg et al.5 reported the successful ablation of LV VTs via a transseptal approach and the crossing of a mechanical mitral valve prosthesis.5 In the latter study, a recurrence of monomorphic VT at two months later required a second VT ablation procedure using the same transseptal\u2013transmitral approach.5 Transventricular septal access to the LV has been also reported in transcatheter aortic valve implantation procedures under intracardiac echocardiography (ICE) guidance.11 The use of ICE during transventricular septal puncture has been recommended from the viewpoint of safety; however, while it is generally used in the United States, it is not common in Europe, as health insurance agencies do not cover the costs of ICE catheters. Subcostal echocardiography was particularly useful for the confirmation of no entrapment of the tricuspid septal leaflet at the transventricular access point in the current case. Coronary angiography should be performed to assess the presence of large septal coronary artery perforators at the region of the midinterventricular septum, where safe access could be attempted.1 Transventricular access by subcostal echocardiographic guidance may be considered as an alternative route, particularly in critically ill patients when conventional percutaneous transaortic or transmitral valve access approaches are not possible.12In patients with mechanical double valve replacement and VT, catheter ablation may be prevented by limited access to the LV. However, direct access to the LV cavity by way of a percutaneous LV apical puncture through the intercostal space overlying the apex or through a left minithoracotomy or left lateral thoracotomy is a viable option for the mapping and ablation of LV VTs."} +{"text": "Most aging biomarkers such as DNA methylation and proteomic clocks have focused on measuring overall \u201cbiological age,\u201d a single number that predicts age-related morbidity and mortality better than absolute chronological age. While intuitive and interpretable, this single biological age number does not account for the possibility that different individuals may preferentially experience aging in different molecular and cellular pathways, and therefore does not suggest personalized aging interventions. We reasoned that a panel of biomarkers each capturing specific aging pathways, such as mitochondrial dysfunction or cellular senescence, may capture the heterogeneity of aging better than existing composite measures. To address this, we employed weighted gene co-expression network analysis to cluster tissue-specific transcriptomes and the serum proteome into specific modules with distinct biological functions and characterized how these modules change with age. We trained DNA methylation proxies of these functional modules that we then applied to independent validation data to identify associations with age-related morbidity and mortality. Clustering analysis using the DNA methylation biomarkers showed that different individuals show distinct patterns of aging. These pathway-specific biomarkers will elucidate how different aging mechanisms interact with each other to produce the larger phenomenon of aging, and for evaluating novel therapeutics targeting specific hallmarks of aging."} +{"text": "Enhanced NIR spectral dataset can be employed as a rapid, effective and non-destructive method to determine inner quality parameters of intact fruits.Presented manuscript aimed to describes enhanced near infrared spectral dataset used to improve prediction performances of near infrared models in determining quality parameters of intact mango fruits. The two mentioned quality parameters are total acidity (TA) and vitamin C which corresponds to main inner attributes of fruits. Near infrared (NIR) spectra data were acquired and recorded as absorbance spectral data in wavelength range from 1000 to 2500 nm. These data were then enhanced by means of several algorithms like multiplicative scatter correction (MSC), baseline linear correction (BLC) and combination of them (MSC+BLC). Prediction models, used to determine TA and vitamin C were established using most common approach: partial least square regression (PLS) based on raw and enhanced spectral data respectively. Prediction performances can be evaluated based on prediction accuracy and robustness, by looking statistical indicators presented as coefficient of determination (R Specifications Table\u2022Enhanced spectral dataset based on near infrared spectroscopy can be benefited for horticulture communities and industries especially for rapid quality inspection and evaluation.\u2022Datasets can be reused and remodelled to predict other quality parameters such as fibre and soluble solids content (SSC) of intact mango fruits by means of different regression approaches like support vector regression and artificial neural networks.\u2022Enhanced spectral data coupled with proper regression approach can be applied to predict inner quality parameters of intact fruits accurately.1Spectral data based on near infrared spectroscopy has become more attractive to be analysed and modelled due to its importance in many field of applications including agriculture. These data mainly can be used to determine inner quality parameters of agricultural products such as fruits, vegetables and their derivative products. In food and agriculture communities and practices, quality of their products is important issue. Consumers are gradually looking for agricultural products with good quality since it is related to health aspect of human body. They need to be ensured those agricultural products are in good condition physically and chemically. In order to determine fruits and other agricultural products quality, several methods like titrations and solvent extractions were widely employed. However, most of those methods are based on complicated and time consuming methods which also destructive in nature Near Infrared spectroscopy (NIRS) has been widely proposed and applied as an alternative method for determining inner quality parameters of foods and agricultural products, including fruits. Near infrared spectra data can be presented as reflectance, absorbance or transmittance spectral data As shown in To determine inner quality parameters of intact mango samples, spectra data either as raw spectra or enhanced spectra data must be modelled through a process called calibration using several regression approaches. The two most common methods in NIR calibration are principal component regression (PCR) and partial least square regression (PLS) On the other hand, the PLS is an approach with somehow close likely to PCR one. The main difference of the PLS method to PCR is that both spectra data of intact mango samples (X-variables) and their actual data of inner quality parameters like TA and vitamin C data (Y-variables) see , are proThe main advantage of PCR and PLS models compared to common multiple linear regression (MLR) is that the X-variables which corresponds to principal components or latent variables are uncorrelated, and the noise is filtered. Further, usually a small number of principal components and factors are preferable for the effectiveness of the prediction models The partial least square regression (PLS) attempted to find best correlation between near infrared spectra data and inner quality parameters of intact mango like total acidity and vitamin C. To date, the PLS method is still widely applied NIRS practice and application. The PLS approach seems to be fitted in many NIRS applications including in agriculture. In fact, near infrared spectra data of fruits and vegetables is essentially composed of a large set of overtones and combination bands. Spectra data are mainly influenced by wavelength dependant scattering effects, tissue heterogeneities of agricultural object, instrumental noise, ambient effects and other source of variability 22.1Datasets for near infrared spectra data and actual measured TA and vitamin C were obtained for a total of 58 mango fruit samples (cv. Kent) selected from two different origins: Brazil and Spain. All mango samples were obtained and purchased in local market in Goettingen, Gernany. Mango fruits were stored for 10 days at ambient temperature of 25\u00b0C and measured every two days in order to have samples with varied maturity, since mango is categorized as climacteric fruits..spa, and .csv extension file formats respectively. Moreover, standard laboratory method has been also prepared for actual total acidity (TA) and vitamin C measurements. Automatic titration equipment was used to determine actual TA, while standard titration method was applied to determine vitamin C of mango samples. On the other hand, the centrifuge was also used to obtain clarified sample juice and separate suspended solids of mango fruit samples.Near infrared spectroscopy instrument (Thermo Nicolet Antaris II) was used as main instrument for acquiring and recording spectra data. It was controlled using integrated software namely Thermo Integration\u00ae and Thermo Operation\u00ae. The workflow has been developed to run specified tasks for spectra data acquisition from which high resolution measurement with integrating sphere was chosen as a basic for obtaining spectra data. Sample labelling was also required every single measurement to distinct all 58 samples 2.2\u22121 and recorded as absorbance spectra data. Each sample was placed centrally upon fruit holder at the top of NIRS instrument. It placed exactly to the incoming light hole with 1 cm of diameter to ensure direct contact and minimize noises. Spectra data were acquired as a successive average of 64 scans per sample Near infrared spectra data for intact mango fruits were acquired in wavelength range from 1000 to 2500 nm or in wavenumber 4000 to 10 000 cm2.3Dichlorophenolindophenol as an indicator in titration method from which colour change from colourless to light red at the end of titration \u22121 fresh mass (FM). Moreover, to obtain total acidity (TA) data of mango fruit, another juices from 20 grams of fruit pulp sample and maximum 100 ml distilled water. In order to obtain clarified sample juice and separate suspended solids, the centrifuge was applied for about 10 minutes. A 10 mL filtered juice was taken and transferred into a beaker glass and titrated in automatic titrator equipped with 0.1 N NaOH to an end point of pH 8.1. The total acidity data can be read from the equipment display and it expressed as mg.100g-1 FM.Once after spectra data acquisitions were completed, actual total acidity and vitamin C of mango fruits were directly measured. The vitamin C content was performed firstly since this parameter is easily evaporated after pulp slicing. Five grams of fruit pulp sample was macerated, mixed and homogenized with a total of 20 ml meta-phosphoric acid into a beaker to prevent rapid oxidation. Distilled water was then added until 50 ml of volume was reached. The vitamin C content was quantified based on its reaction with the 2.4As mentioned previously, spectra data can be corrected and enhanced to improve prediction accuracy and robustness. Obtained spectra data sometimes contained noises and irrelevant background information due to internal and external factors such as light scattering, over-heated sensors and others. These noises can interfere quality properties information required to be revealed from spectra data. Therefore, it is recommended to correct and enhance spectra data prior to constructing prediction models. There are many spectra enhancement methods widely available with different algorithms such as derivation, normalization and transformation.ideal spectrum of the spectra data which is corresponds to the average spectrum. Moreover, baseline linear correction (BLC) is also commonly used for some users due to its ability to remove noises especially at the end of NIR wavelength at around 2300 \u2013 2300 nm. Spectra correction methods can also be combined among those methods to generate more accurate and robust prediction results as shown in Among those spectra enhancement methods, multiplicative scatter correction (MSC) is one of the most common and popular correction methods for NIRS users. The MSC is used to compensate for additive and multiplicative effects in the spectral data caused by physical effects. It also proven to be effective and robust spectra correction method among others. This method attempted to remove the effects of scattering by linearizing each spectrum to an 2 ellipse. Outliers are spectra data from fruit samples that considerably different from the other majority of remaining samples. They need to be treated carefully because they can influence whole prediction performance of NIRS prediction models. Dataset need to be improved by removing outliers and enhancing spectral data to achieve more accurate and robust prediction performances.In addition, spectra data can be observed firstly to detect some odd spectra data which are known as outliers. The most common method used to detect outlier data is a combination of principal component analysis (PCA) and Hotelling t2.5To obtain information about inner quality parameters of mango fruits which buried in the spectra data, calibration must be carried out to construct prediction models. These models were developed by means of specific multivariate analysis from which partial least squares regression (PLS) was applied. Another regression approach like principal component regression (PCR) or stepwise and backward linear regression can also be used in calibration to predict desired quality attributes of intact fruits 2) which essentially represents the proportion of explained variance of the response in the dataset, coefficient of correlation (r) between predicted and measured quality attributes (TA and vitamin C), prediction error which is defined as the root mean square error (RMSE), and the residual predictive deviation (RPD) defined as the ratio between standard deviation of the actual measured quality parameters and the root mean square error (RMSE), standard error of cross validation or prediction performance (RMSECV or RMSEP). The higher the value of RPD, the greater probability of the model to predict TA, vitamin C and other chemical constituent in samples set accurately.The performance of the prediction models were evaluated by using the following statistical indicators: the coefficient of determination and artificial neural networks (ANN) regression based"} +{"text": "Diagnosing the early onset of neuropathologies, such as mild cognitive impairment (MCI), requires repeated evaluation of cognitive skills several times per year -- a measurement design known as a \u201cburst design.\u201d Detecting the often subtle cognitive decline in the presence of retest effects requires careful statistical modeling. The double exponential model offers a modeling framework to account for retest gains across measurement bursts, as well as warm-up effects within a burst, while quantifying change across bursts in peak performance. This talk highlights how a Bayesian multilevel implementation of the double exponential model allows for flexible extensions of this framework in terms of accommodating different timescales (nesting) and person-level predictors and drawing intuitive inferences on cognitive change with Bayesian posterior probabilities. We will use reaction time data to show how individual differences in asymptotic performance and change can be related to predictors such as age and MCI status. Part of a symposium sponsored by the Measurement, Statistics, and Research Design Interest Group."} +{"text": "Thebeisan veins are microfistulous connections between a coronary arterial branch directly to a ventricular or atrial chamber. Extensive thebesian veins that empty into the left ventricle can cause typical chest pain symptoms, troponin elevation, and ischemic EKG changes from coronary steal leading to acute coronary syndrome in extreme cases. Literature review exposed a consistent pattern of EKG findings among patients with extensive thebesian veins involving all three major coronary arteries. We present a case study as an example of this rare anatomic finding of extensive thebesian veins draining into the left ventricle causing acute coronary syndrome in a symptomatic patient with elevated troponin and ischemic changes on EKG. This same EKG pattern that is present in our patient was discovered to be consistent among available case studies reviewed that had included an EKG tracing in their report. A newly proposed association between the ischemic changes on EKG due to extensive thebeisan veins and those of a severe proximal left anterior descending coronary artery stenotic lesion was discovered. The newly discovered consistency in the EKG pattern with acute coronary syndrome caused by extensive thebesian veins is the same pattern as that seen in Wellens Syndrome. A coronary artery fistula is an abnormal luminal connection between a coronary artery and either a vein, another artery, or even a ventricular chamber.When a significant number of microfistulae exist, left-to-left shunting occurs, meaning blood that should be continuing downstream in the arterial system has taken an alternative pathway creating a new circuit of blood flow that doubles back on itself. In this case, blood that should be conducted from the aorta to coronary arteries and coronary capillaries then to the coronary sinus and right atrium has found an abnormal alternative pathway.This alternative pathway involves blood flowing from the aorta to the coronary arteries back into the left ventricle (LV) and then the aorta again, creating a loop. Thus, blood circulation is diverted from a left sided structure (the coronary arteries), back into another left sided structure (the LV) and does not pass though the finer myocardial capillary network nor the pulmonary circulation.These extra pathways of blood flow can typically cause patients to experience anginal symptoms, dyspnea , exertional limitations, abnormal stress testing results, diastolic volume overload, and even myocardial ischemia with possible Acute Coronary Syndrome (ACS) .To have a single coronary artery in communication with a cardiac chamber is rare (0.08% - 0.3%),There are multiple ways for clinicians to discover a significant thebesian venous system, one of which is through coronary arteriography.Our patient was a black female in her early 60\u2019s who presented to the emergency room (ER) secondary to two days of progressive substernal chest pressure with associated mild shortness of breath. Her past medical history included Type 2 diabetes mellitus, hypertension, tobacco abuse, and stroke. This patient\u2019s physical exam was negative for any significant findings. Her electrocardiogram (EKG) was concerning for ACS. Troponin I level was elevated at 1.05 ng/ml . A prior myocardial perfusion test performed 12 months ago failed to identify any reversible or fixed cardiac defects.The patient was admitted with a diagnosis of ACS non-ST segment elevated myocardial infarction in which the EKG did not display ST segment elevations , but labs were consistent with myocardial injury (elevated troponin level). Her physicians were concerned that the culprit artery causing ischemic disease was the Left Anterior Descending (LAD) based on her EKG findings meeting criteria for Wellens Syndrome. She additionally reported currently undergoing an extremely stressful economic situation, which raised concern for possible Takotsubo Cardiomyopathy in which there is no coronary artery disease, but severe left ventricular myocardial dysfunction that is present with EKG changes mimicking ACS.She was taken for a diagnostic left heart catheterization which showed mild to moderate disease in multiple coronary arteries with severe stenosis of a small caliber mid right sided Posterior Descending Artery (rPDA) not amenable to percutaneous intervention. The cardiac catheterization also revealed an extensive network of microfistulae forming communications between all three major coronary arterial branches and the LV cavity . This patient\u2019s flow through her microfistulous communications was so brisk that a perfectly interpretable left ventriculogram was visualized with coronary angiographic imaging via a hand injection of contrast into the left coronary arterial system .It was not possible to quantitate this patient\u2019s amount of flow through her extensive thebesian venous network since it is generally extremely technically difficult to do so.Despite the known severe stenotic lesion of the rPDA, this patient\u2019s EKG did not correlate with the macrovascular disease found on angiography. The microfistulae were then suspected of being the cause of ACS due to coronary steal phenomenon.12 receptor inhibitor used to treat coronary artery disease by inhibiting normal platelet function), and low dose aspirin (81-100mg once per day). No intervention was performed on the stenotic coronary arterial segments since they were small caliber vessels not amenable to stenting. The patient\u2019s symptoms resolved prior to hospital discharge, and on the third day she was stable for discharge home. She was advised to follow up with Cardiology as an outpatient for continued care. This would entail optimizing her CAD medication regimen according to current guidelines, minimizing her CAD risk factors, and monitoring to ensuring she has no symptom recurrence.This patient was treated with metoprolol tartrate , isosorbide mononitrate , clopidogrel should be added and considered as a differential diagnosis when proximal LAD ischemia is suspected from a Wellens Syndrome EKG pattern. This type of ischemia to myocardial tissue can be due to the coronary steal phenomenon and mimic a severe proximal LAD stenotic lesion and a Wellens Syndrome EKG pattern.The most striking similarity between the found literature and the case presented here is the biphasic T wave seen in V3, as previously mentioned. All patients, except for one, also had some significant signs or symptoms of myocardial ischemia. This single case study that involved an asymptomatic patient with the EKG pattern described demonstrated a significant thebesian venous networked despite the absence of symptoms.This evidence should raise awareness for an extensive thebesian vein network being: 1) a possible mimicker of Wellens Syndrome, 2) capable of causing hemodynamically significant alterations in myocardial perfusion, 3) a cause of myocardial ischemia, and 4) the etiology of significant anginal signs and symptoms. Further research, including specific treatments for the clinical course of this condition, need to be considered to more fully evaluate optimal care approaches for these patients.The authors declare no conflict of interest."} +{"text": "GSA COVID-19 Task Force members collaborated with AGHE to develop multiple teaching briefs and biblio briefs on various topics to support faculty who had to adjust to online teaching quickly. Task Force members will review those documents, as well as the AGHE listening session to identify where educators and students need additional support."} +{"text": "Parental feeding practices significantly influence child eating behavior. The data for this article was from a cross-sectional case control larger study that aimed to record parental practices to manage feeding problems in children with typical development and children with gastrointestinal diseases. A set of 23 Likert-type questions was used to investigate parental practices. Demographic and anthropometric data were obtained via a structured set of questions. In total 765 parents of healthy children and 136 parents of children with gastrointestinal diseases aged one to seven years participated in the study. Healthy controls were recruited from kindergartens located in various geographical areas in Greece. Children with gastrointestinal diseases were recruited from a Pediatric Gastroenterology Outpatient Clinic. Descriptive measures alongside with statistical analysis measures are presented in this article. Chi-square tests and U-tests were performed for the purpose of the comparison between the two groups. Spearman's rho correlation coefficient was also calculated for inter-item correlations among the 23-items of the questionnaire. Specifications TableValue of the Data\u2022These data are useful because they describe parental practices when managing feeding problems and explore factors that may be associated with these practises.\u2022All researchers involved in child feeding can benefit from these data because they provide a thorough understanding of parental behavior during child feeding.\u2022Data can be used for further research on the management of feeding problems in children.\u2022Parental feeding practises data for this sample can be compared with that for other sample for further insight.\u2022Our data can be used to develop intervention programs aiming to address feeding problems in children.\u2022Further research could focus on the evaluation of the properties of this 23-item questionnaire (or a shorter version of it). The correlation matrix is provided for this purpose as an initial investigation step110.17632/c25bx2vpnw.12Parental feeding practices significantly influence child eating behavior The authors declare that they have no known competing financial interests or personal relationships which have, or could be perceived to have, influenced the work reported in this article."} +{"text": "The identification of vasa previa still remains difficult even for well\u2010trained obstetricians. Distinction of fetal from maternal veins is especially challenging because both have a slow blood velocity.Changes in venous flow with maternal respiration in vessels around the uterine cervix or lower uterine segment differentiate maternal from fetal vessel.Vasa previa (VP) is a serious obstetric complication that can result in fetal hemorrhage due to the laceration of unprotected fetal vessels, which run though the membranes near the internal cervical os.At the 29th gestational week (GW), a transvaginal ultrasound revealed venous flow in vessels near the internal cervical os which was difficult to identify whether they originated from the fetus or the mother. At color Doppler ultrasound, two types of venous flow were detected: one fluctuated simultaneously with maternal deep breathing Figure , and theIn order to identify VP in the presence of abnormal cord insertion , obstetricians should pay careful attention at adjusting machine settings , not only because the blood flows at a very low speed in fetal veins, but also because fetal vessels may run transversely near the internal cervical os as shown in our case 1, which might be to overlooked from the sagittal and parasagittal views commonly taken at transvaginal scans. Furthermore, retroplacental or periplacental maternal blood flow near the cervix should be distinguished from VP, since abnormal placentation is highly associated with VP.The authors declare no potential conflict of interest.No research data."} +{"text": "CMR adenosine first pass perfusion yields excellent results for the detection of significant coronary artery disease. As in patients after coronary artery bypass grafts (CABG) myocardial perfusion is more complex and additionally the kinetic of a first pass bolus may by altered due to different distances through the bypasses and/or native vessels, this patient group has been excluded from most published studies. Parameters like speed of contrast wash-in and peak enhancement are indirectly used for visual analysis and may be altered post CABG imitating perfusion defects without significant stenosis. In this case, adenosine perfusion would be an inadequate diagnostic test.Aim of the study was to evaluate semiquantitative perfusion parameters in patients after CABG in order to evaluate contrast kinetics in areas supplied by native coronaries and different bypass grafts.32 patients post CABG were included into the study consisting of adenosine first pass (0.05 mmol/kg Gd-DTPA) perfusion (3 short axis views/heart beat) and late Gadolinium enhancement before undergoing invasive coronary angiography. In invasive angiography, areas perfused by native coronaries and the different bypasses were identified. In these areas upslope, time to 50% peak enhancement, time to peak enhancement and relative peak enhancement were calculated using the ViewForum . Only segments without vessel stenosis and without LGE were used for final analysis.Results are displayed in Table Semiquantitaive parameters of first pass adenosine perfusion are similar in areas supplied by native vessels or by different bypass grafts. These parameters are indirectly used for visual analysis . Therefore the possible different contrast kinetic through grafts and native vessel does not seem to be a limiting factor for the accuracy of first pass adenosine perfusion in patients post CABG."} +{"text": "This presentation discusses amendments made to the Older Americans Act titles III, IV and V through the most recent reauthorization. Title III reauthorizes Title IV programs, Title IV reauthorizes title V programs and Title V reauthorizes title VI programs. The reauthorizations each include a seven percent increase in fiscal year 2020 and a six percent increase per year for the next four fiscal years. New in title III are an amendment that allows projects that address traumatic brain injury among older adults to be included in grant programs, an amendment that improves an existing transportation grant program and an amendment that improves an existing grant program for multigenerational collaboration. Additionally, existing falls prevention and chronic disease self-management programs are codified within title III. New in title IV is an amendment that allows eligible previously incarcerated individuals to be considered a prioritized population for the Senior Community Service Employment Program."} +{"text": "Beliefs about aging are grounded in social experience. This study considers the extent to which married older adults\u2019 shared beliefs about aging and markers of aging maintain a concurrent and enduring association with their partners\u2019 beliefs and markers of aging. Data from the 2010/2012 and 2014/2016 waves of the Health and Retirement Study provided measures of husbands\u2019 and wives\u2019 positive and negative beliefs about aging and internal (Cystatin C) and external (grip strength) markers of aging. Latent dyadic models parsed beliefs and markers into partners\u2019 individual and shared variance. Longitudinal analysis showed concurrent associations between shared beliefs and markers of aging to be stable over four years. Meanwhile, the enduring processes that connect beliefs and markers over time were best characterized as bidirectional. The study provides evidence that, within older couples, beliefs about aging are shaped in part through partners\u2019 co-experience of each other\u2019s biological aging."} +{"text": "The key imaging features of cerebral amyloid angiopathy (CAA) are lobar, cortical, or cortico-subcortical microbleeds, macrohaemorrhages and cortical superficial siderosis (cSS). In contrast, hypertensive angiopathy is characterized by (micro) haemorrhages in the basal ganglia, thalami, periventricular white matter or the brain stem. Another distinct form of haemorrhagic microangiopathy is mixed cerebral microbleeds (mixed CMB) with features of both CAA and hypertensive angiopathy. The distinction between the two entities (CAA and mixed CMB) is clinically relevant because the risk of haemorrhage and stroke should be well balanced if oral anticoagulation is indicated in CAA patients. We aimed to comprehensively compare these two entities.Patients with probable CAA according to the modified Boston criteria and mixed CMB without macrohaemorrhage were retrospectively identified from our database. Comprehensive comparison regarding clinical and radiological parameters was performed between the two cohorts.p\u2009=\u20090.036) and had a higher prevalence of cSS but a lower prevalence of lacunes and deep lacunes compared to patients with mixed CMB. Logistic regression revealed an association between the presence of deep lacunes and mixed CMB. The other collected parameters did not reveal a significant difference between the two groups.Patients with CAA were older contains supplementary material, which is available to authorized users. The key pathological feature of cerebral amyloid angiopathy (CAA) is amyloid deposition in small cerebral parenchymal and leptomeningeal vessels . In vivoWe hypothesized that differences between these two groups are already present in the absence of macrohaemorrhages. In this study, we aimed to determine the clinical and radiological differences between these two groups without macrohaemorrhages.The study was approved by the local ethics committee (Ethikkommission der Medizinischen Fakult\u00e4t der Christian-Albrechts-Universit\u00e4t zu Kiel). Patients consented to the retrospective use of acquired data at hospital admission. If consent for usage was not given, only anonymized data were used.We retrospectively included patients based on a search in our image database. Patients were included from 2014\u20132019 and there were no restrictions concerning the requesting department or clinic. Patients were included in the CAA group if they had an MRI at our institution including a susceptibility based sequence , for sequence parameters see Supplemental Table 1) and fulfilled at least the criteria for probable CAA (possible CAA excluded) according to the modified Boston criteria . The other collected parameters did not reveal a significant difference between the two groups. However, there was a trend (p\u2009<\u20090.10) toward high microbleed count (p\u2009=\u20090.065) and the presence of arterial hypertension in patients with mixed CMB.For the variables age, presence of lacunes, presence of deep lacunes and presence of cSS only the presence of deep lacunes was significantly associated with the diagnosis mixed CMB [The main finding from this study is that CAA and mixed CMB have a different clinical and radiological profile in the absence of macrohaemorrhages, although there is a large overlap between the two cohorts.This is the first comparison study between patients with CAA and mixed CMB without macrohaemorrhage. In a cohort of patients with sustained macrohaemorrhage, Pasi et al. could demonstrate differences mainly driven by vascular risk factors . They alPrevious studies demonstrated that lobar micro- and macrohaemorrhages might be caused by arterial hypertension which classically is thought to cause deep cerebral haemorrhages. Kim et al. demonstrated that amyloid burden and hypertensive small vessel disease have synergistic effects on the progression of lobar microbleeds . This miFurther, in our study the diagnostic value of convexity cortical superficial siderosis is reemphasized. Only supratentorial convexity cSS was present Fig.\u00a0 making iThe distinction between the two entities (CAA and mixed CMB) is clinically relevant because oral anticoagulation in patients with CAA should be carefully considered under certain circumstances and requires a differentiated look at the pattern and quantity of haemorrhage distribution . On the An alternative stroke prevention therapy in patients with atrial fibrillation and a high bleeding risk might be the use of a left atrial appendage occlusion . The treThe CSF profile might be used to identify CAA patients. Reduced A\u03b240 has been reported in patients with CAA when compared to healthy controls consistent with the pathological correlate of preferential amyloid A\u03b240 deposition in cerebral vessel , 31. HowRetinal abnormalities in the vasculature have been described in CAA patients . However11C-PiB (Pittsburgh Compound B) are not readily available.PET imaging with amyloid specific tracers provides insights into the presence and distribution of amyloid deposition , 33 but Notable strengths of our study are the strict application of the modified Boston Criteria for the definition of the CAA group and conversely for the mixed CMB group. We additionally excluded patients with previous surgery with heart\u2013lung-machine which can result in a radiological picture mimicking CAA or mixed CMB . FurtherA limitation of this study is its retrospective nature. Although we cannot exclude selection bias due to clinical presentation, symptom severity or delay from symptom onset to MRI, no such difference was seen in the group analysis. Compared to other prospectively maintained databases like the \u201cMassachusetts General Hospital Haemorrhagic Stroke Research Program\u201d our cohort is relatively small. However, it is unique when regarding the patients\u2019 disease progression with none of the patient having experienced symptomatic intracranial haemorrhage. Another drawback is the lack of pathological confirmation in our cohort. We are very well aware that other pathologies might explain the clinic-radiological picture in a part of the patients. However, we did not include the category \u201cPossible CAA\u201d according to the modified Boston Criteria and used additional exclusion criteria to keep diagnostic certainty as high as possible.CAA and mixed CMB show a different clinical and radiological profile in the absence of macrohaemorrhages. However, to differentiate these two entities more clinically available diagnostic parameters are necessary.Supplementary file1 (DOCX 12 kb)Below is the link to the electronic supplementary material."} +{"text": "The \"SORT maneuver\" was introduced in 2016 to ease nasogastric intubation for unconscious patients in operating theatre and critical care setting [From the practitioner\u2019s point of view, any procedure which is done in close contact with an infected patient\u2019s airway carries a higher risk of respiratory infection transmission which may not get totally eliminated after tracheal intubation , 3. WithOn the other hand, the management of the patients\u2019 coexisting problems such as cardiovascular disease and hemodynamic instability is a challenge during NGT intubation. Pharyngeal manipulation during NGT insertion is a potential treat for cardiac patients by increasing demand especially in hypertensive patients with uncontrolled blood pressure. These patients are the most vulnerable group to higher incidence of severe illness and worse outcome in COVID-19 as well , 11. SmoNGT tube insertion in intubated patient is a quite common procedure in operation theatre and intensive care settings. In patients with COVID-19 infection, SORT maneuver may protect both practitioners and patients from further avoidable hazards. I would encourage my colleagues to verify these proposals in their daily practice and by further investigations through clinical trials."} +{"text": "Aging parents\u2019 marital status shapes their ties to family members, but less is known about its link to their daily mood and interaction with grown children. This study examined married, widowed, or divorced/separated aging parents from the Family Exchanges Study, who completed a 7-day daily diary on their daily mood and interactions with the grown children . Findings from multilevel models indicated that widowed parents were more likely to report irritable interactions with their grown children than the married ones. Furthermore, married and widowed parents tended to report more negative mood, whereas separated parents tended to report less negative mood on days they had irritable interactions with grown children. This study highlights the centrality of aging parents\u2019 daily interaction with grown children and suggests that the dynamics of family composition warrant attention."} +{"text": "Cognitive impairment is a worldwide epidemic. Informed by NIA\u2019s Health Disparities Framework, this study investigated interpersonal, behavioral, and sociocultural risk and protective factors associated with cognitive health trajectories. Mixed models examined factors associated with cognitive health with data from the Health and Retirement Study among Whites, Blacks, and Hispanics . A majority of respondents who experienced everyday discrimination attributed it to ageism among this racially and ethnically diverse sample. Stratified mixed models of everyday discrimination by attribution (racism or ageism) revealed worse cognitive functioning. Major lifetime discrimination was not statistically associated with cognitive functioning. Economic factors and religious activity protected cognitive functioning and were particularly salient for Blacks and Hispanics. Strategies that bolster individual resilience as well as social policies that address discrimination and structural inequities will likely reduce health disparities and improve population health."} +{"text": "Ageism has been operationalized in a number of ways over the past half century, and several measures of ageism have been developed to reflect these conceptualizations. This presentation evaluates the extent to which existing ageism measures capture similar or different aspects of ageism. We examined the construct validity of several ageism measures in a sample of 473 undergraduate students (Mage = 19.5 years). Participants completed an online survey of ageism measures, as well as measures of convergent and discriminant validity . Ageism measures were generally positively associated with one another, though correlations were mostly weak to moderate in magnitude. There was also weak evidence of convergent and discriminant validity. These findings demonstrate conceptual problems with current ageism measures as they do not appear to reflect a common construct."} +{"text": "EClinicalMedicine underscores the importance of institutional action to overcome gender discrimination. But the progress resulting from gender equality initiatives is disappointing The recent issue on gender equality in Gender is a complex multidimensional phenomenon Inequality regimes are deeply ingrained in our universities and medical schools. In the absence of comprehensive institutional action, gender equality initiatives risk doing more harm than good. Research shows that those in power frequently compensate for the ostensibly \u201cunfair\u201d advantages women get from such initiatives, for instance by withholding resources and information"} +{"text": "This presentation will explore the development of AGHE\u2019s major contemporary contribution: AGHE\u2019s basic-competency guidelines and their role in program review and program enhancement. The presenter will describe in detail the steps in creating objective student learning outcomes and will explore how meeting these competencies improve instruction, self-reflection, program analysis and faculty discussion and ultimately gerontology programs. These guidelines and policies embody a living entity that is always evolving. Future iterations will be anticipated and discussed. Part of a symposium sponsored by the Geriatric Education Interest Group."} +{"text": "It is vital the workforce is prepared to meet the medical needs of our aging population. Asking older adults What Matters is an important aspect of excellence in clinical care. During a small group session in a two-year communication skills course, second year medical students (N=149) at Rush were taught how to ask What Matters as part of the 4Ms. Students then completed a video recorded Communication Skills Lab with a simulated older adult patient as they practiced how to discuss What Matters. Students then met with their instructors in individual feedback sessions to review the video and discuss strengths and areas for improvement in communicating with older adults. Students then completed a Clinical Skills Assessment for formal testing of their communication skills with older adults. Outcomes of the summative assessment will be presented and recommendations for integrating 4Ms into existing medical school and allied health curriculum will be discussed."} +{"text": "Fruit and vegetable consumption is associated with lower risk of chronic diseases and mortality. Despite the numerous health benefits, fruit and vegetable consumption of most older adults are below the daily recommendation. This paper aimed to investigate whether living alone and having children and friends nearby are associated with older adults\u2019 daily fruit and vegetable consumption using a nationally representative sample of older Americans. Daily fruit and vegetable consumption was measured using (1) daily serving and (2) daily recommendation . Poisson and logistic regression models were estimated using the HRS Health Care and Nutrition Study. The sample included 6,915 community-dwelling older adults. Older adults who were living alone had lower fruit and vegetable consumption and less likely to meet daily recommendation for vegetables, compared to those who were living with someone. Having friends nearby was positively associated with the outcomes, while having children nearby was associated with meeting daily recommendation for vegetables only among older adults living alone. Based on the findings, older adults who are living alone and do not have children and friends nearby may be at the risk of poor nutrition due to low levels of social support. Provision of help with grocery shopping and meal preparation as well as more social opportunities that can improve social support network and encourage healthy eating may increase daily fruit and vegetable consumption of older adults."} +{"text": "Aging is associated with a progressive decline in cellular health leading to dysfunction in organs with a high metabolic demand. A key feature of age associated cellular decline is impairment of mitochondrial quality control pathways such as mitophagy. Deficits in optimal functioning of these pathways results in a compromise in cellular bioenergetics that ultimately leads to mitochondrial dysfunction. Promising nutritional interventions have emerged that boost mitochondrial health such as nicotinamide riboside (vitamin B3 precursor) and Urolithin A (gut metabolite of compounds found in pomegranates), that act via different mechanisms of action to improve overall mitochondrial health. Recent literature on the evidence behind these interventions will be presented and discussed during this symposium. We will also share recent clinical evidence from double-blind placebo-controlled studies with Urolithin A. Our results suggest that nutritional interventions such as Urolithin A are promising approaches that can be employed to manage age associated cellular decline."} +{"text": "The nurse practitioner\u2019s (NP) clinical activities during the 12-month intervention period include 4 monthly in-home visits and 8 monthly telephone contacts. This presentation will detail the clinical assessments and activities conducted during the initial home visit, and how subsequent home visit activities and interventions are structured for older adults and their informal caregivers depending on whether older adults have dementia, depression, and/or recent delirium. Because the potential for medication-related problems is a critical concern for older adults with cognitive vulnerability, this presentation also will detail how the NP works with the 3D Team pharmacist to determine potential inappropriate medications through a review and reconciliation process, and how the NP and pharmacist summarize these results and correspond accordingly with the older adult\u2019s primary care physician. Finally, this presentation will explain how the NP manages communication among members of the 3D Team who provide interventions to the same older adult and caregiver."} +{"text": "The options around single use/multiple use, and the relative benefit of different types of catheter coatings are still debated. A recent, well done randomised trial in patients with spina bifida did not show a benefit in terms of UTI reduction over 8 weeks when intermittent catheters were reused compared to when catheters were only used once (Hydrophilic catheters have been introduced to the urological practice as an alternative to reduce the risk of recurrent UTI and urethral trauma . HoweverDespite the promising laboratory results published by Wilks et al, clinical studies are still needed to check whether diluted Milton strategy is effective and safe for patients. Bacterial biofilm still represents an important barrier for patients who reuse catheters . A gener"} +{"text": "Endobronchial\u2010invasive lung cancers are generally diagnosed at advanced stages and may require emergency treatment for airway obstruction. Stent implantation is a common intervention for such obstructed airways but certain subsets of patients cannot receive adequate treatment without respiratory support. Veno\u2010venous extracorporeal membrane oxygenation (ECMO) is a salvage therapy for respiratory failure but its usefulness in managing patients with advanced lung cancer remains unclear given the poor prognosis. In recent years, molecular targeted agents for patients with driver mutations offer rapid responses and may be administered even while under critical care. In this report, we describe the case of 39\u2010year\u2010old female who presented to our emergency department with severe respiratory distress. A computed tomography scan revealed a large mediastinal tumor invading the tracheal carina causing severe stenosis of the left main bronchus and right main pulmonary artery. ECMO support was required as the respiratory condition remained unstable despite high pressure ventilation. Under ECMO support, the patient underwent bronchial stent implantation and was successfully weaned off ECMO. The tumor was histologically diagnosed as pulmonary adenocarcinoma with anaplastic lymphoma kinase gene rearrangement. Treatment with a tyrosine kinase inhibitor, alectinib, induced a marked tumor reduction within a short period. The patient recovered well and is now in remission one\u2009year later. This case indicates that intensive respiratory support with ECMO may become a bridge through the critical period for selected patients with respiratory failure secondary to advanced lung cancer.ECMO was important to maintain oxygenation during airway intervention for acute respiratory failure due to critical lung adenocarcinoma with ALK gene rearrangement.With the development of targeted therapies and the improvement in therapeutic bronchoscopy, intensive respiratory support with ECMO may be helpful especially in selected lung cancer patients with oncogenic driver mutations. Respiratory support with extracorporeal membrane oxygenation (ECMO) in managing patients with advanced lung cancer remains unclear given the poor prognosis. Targeted agents for patients with driver mutations offer rapid responses and may be administered even while under critical care. In some patients with critical lung cancer, ECMO may become a bridge through the critical period with respect to effective anti\u2010cancer therapies. Due to possibly unfavorable prognoses, any indication of extracorporeal membrane oxygenation (ECMO) for patients with advanced lung cancer should be considered individually with respect to risks and benefits.ALK gene rearrangement was detected by fluorescence in situ hybridization analysis. The patient received a tyrosine kinase inhibitor (TKI), alectinib, via nasogastric tube from day 16, recovered well and was discharged from the ICU without any adverse events related to the targeted therapy. Six months later, a chest CT scan showed a marked reduction of the tumor revealed a large tumor primarily in the middle mediastinum invading the tracheal carina Fig . Furtherumor Fig . Since tAccording to previous studies, ICU and hospital mortality rates for patients with lung cancer admitted to emergency departments are estimated at 47% and 60%, respectively.This paradigm has gradually changed with both the development of targeted therapies for lung cancers with oncogenic mutations and the improvement of therapeutic bronchoscopy in the palliative setting of alleviating airway stenosis.In our case, there are three possible reasons for the successful treatment. First, the patient was a relatively young woman with good organ function and no comorbidities. Second, recovery from respiratory failure occurred as expected when the left main bronchial stenosis was relieved by endobronchial intervention. Third, the patient immediately underwent ALK inhibitors resulting in marked tumor regression within a short period. In contrast, there was also a major limitation regarding this clinical course as the histological diagnosis and mutation status were revealed only after the initiation of ECMO support. This delay in test results may confound treatment decisions as ECMO usage needs to be decided on an urgent basis. Accumulation of clinical experiences will thus be necessary to determine the appropriate indications for ECMO.In conclusion, here, we describe the successful ECMO support of a patient with malignant airway obstruction who achieved a rapid beneficial response to targeted therapy. Although ECMO support cannot be applied to the majority of lung cancer patients, this report can be a reference to consider the utility of ECMO for advanced malignancy.The authors have no conflicts of interest."} +{"text": "This international symposium brings together leading scholars in occupational therapy research whose shared aim is to support community mobility in older adulthood. in this session, five groups of researchers will share their collective and individual research outcomes supporting continued community mobility of older adults, especially when driving is no longer an option. The first presentation will be their collective international, cross sectional study of 247 older adults from seven countries. This study compared community destinations, methods of transportation and factors that influence community mobility. Each presentation that follows will highlight unique studies with the overarching goal of improving mobility options for older adults to \u201cage in place of choice.\u201d Our discussant will summarize the potential for expanding this research by engaging the audience through interactive questions, as to where local and global opportunities for innovations that support community mobility will be raised."} +{"text": "Breast cancer is the leading cause of cancer related death among women. Breast cancers are generally diagnosed and treated based on clinical and histopathological features, along with subtype classification determined by the Prosigna Breast Cancer Prognostic Gene Signature Assay . Currently the copy number alteration (CNA) landscape of the tumour is not considered. We set out to examine the role of genomic instability (GI) in breast cancer survival since CNAs reflect GI and correlate with survival in other cancers. We focused on the 70% of breast cancers classified as luminal and carried out a comprehensive survival and association analysis using Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) data to determine whether CNA Score Quartiles derived from absolute CNA counts are associated with survival. Analysis revealed that patients diagnosed with luminal A breast cancer have a CNA landscape associated with disease specific survival, suggesting that CNA Score can provide a statistically robust prognostic factor. Furthermore, stratification of patients into subtypes based on gene expression has shown that luminal A and B cases overlap, and it is in this region we largely observe luminal A cases with reduced survival outlook. Therefore, luminal A breast cancer patients with quantitatively elevated CNA counts may benefit from more aggressive therapy. This demonstrates how individual genomic landscapes can facilitate personalisation of therapeutic interventions to optimise survival outcomes. Breast cancer is one of the most common malignancies affecting women worldwide and is the leading cause of cancer related death among this group , 2. OverBreast cancer was previously treated as a single disease, but recent advances in areas such as next generation sequencing have led to it being regarded as a collection of highly heterogeneous diseases with distinct molecular and clinical phenotypes including disease progression rate, treatment response and survival \u20136. DespiHER2)-enriched and basal-like subtypes [The molecular classification of breast cancer currently makes use of PAM50 intrinsic subtyping determined by the Prosigna Breast Cancer Prognostic Gene Signature Assay based on gene expression profiling \u201310. Thissubtypes . The difsubtypes . Importasubtypes .Approximately 70% of breast cancers belong to the luminal subtypes lumA and lumB characterised by increased levels of estrogen receptor (ER) and progesterone receptor (PR) . LumA tuHER2 and tumour protein p53 (TP53) [Recent studies suggest that the relationship between lumA and lumB may be a continuum rather than a strict division of subtypes \u201313. It h3 (TP53) .At present, breast cancer diagnosis and treatment follows an integrative approach whereby both clinical and histopathological features such as age at diagnosis, tumour size, lymph node metastasis and histological grade are utilised alongside tissue derived biomarkers . HoweverSeveral studies have shown that the copy number landscape of a tumour can affect survival , 22, 23.These studies support the conjecture that the CNA landscape of a tumour is itself associated with both OS and DSS, and could provide a prognostic biomarker , 22, 23.We hypothesised that a more nuanced measure of CNA burden could provide additional prognostic information for the impact of GI on luminal breast cancer survival, and potentially be informative for the progression from lumA to lumB subtypes.A CNA Score metric was developed using the absolute CNA profiles of all luminal patients profiled within the METABRIC consortium. This was calculated by summing the absolute value of the scores for all genes. Cases were then assigned to ranked quartiles as a first-order means of segmentation for analysis .Survival analysis was carried out for these CNA Quartiles using associated clinical data to determine survival associated variables. Statistical association tests were then applied to validate that the association between a given CNA Quartile and its survival outcome was due to the CNA Quartile and not to a confounding variable. Finally, multivariable Cox proportional hazards (PH) models and the associated assumption tests were used to confirm that the survival outcomes are associated with GI in specific cohorts of luminal breast cancers and survival tree recursive partitioning was used to further explore survival interactions and cut-off points. In addition, quantile classification from the gene expression analysis of Tishchenko et al was utilMETABRIC provides a well-annotated dataset of over 2,000 breast cancer cases with long-term clinical follow-up data, transcriptomic and genomic data . Luminalsurvival, survminer and gglplot2 [maftools [partykit [Clinical data and CNA profiles were formatted and analysed using R (version 3.5.1) and RStudio (version 1.2.1335) with R packages gglplot2 \u201329. Addimaftools were alspartykit , 32 withA number of recent studies report that CNAs reflecting GI are associated with survival outcomes in several types of cancer , 22, 23.A strong association between breast cancer survival and clinical variables such as PAM50 subtype, clinical stage and age at diagnosis has been reported \u201335. A nu\u03c72 test was used to test the association between two categorical variables with sufficient cell sizes in the two-way table of categorical variables. Fisher\u2019s exact test was used in the case where any cell size was sufficiently small. The non-parametric Kruskal-Wallis test was used to determine if there were statistically significant differences between CNA Quartiles and continuous clinical variables associated with survival outcomes. These tests indicated that the CNA Quartiles are significantly associated with a number of clinical variables comprising approximately a quarter of METABRIC luminal patients each were defined by Tishchenko et al based onOur CNA Score is associated with the transcriptomic ranking of Tishchenko et al . Cross rWe analysed the effect of CNA burden on breast cancer survival by implementing a series of statistically robust tools for interrogation of the rich and well annotated METABRIC dataset. We assigned each patient to a group by quartile segmentation of the summed distribution profile of absolute CNA Score values, providing a first order measure of CNA burden. This metric is a more nuanced representation of GI compared to the binary segmentation used in previous studies . Our anaThe observed difference in survival outcomes could be the result of either a true association between survival and CNA Quartiles, or the result of confounding factors. A significant association was again observed between survival outcomes and CNA Quartiles for lumA patients utilising multivariable Cox PH models to address potential confounders. The association with DSS was linear in lumA patients where DSS outcomes decreased moving from CNA Q1 to Q4. Overall, CNA Quartiles were associated with breast cancer prognosis independent of other strong clinical predictors.Recent studies have proposed that lumA tumours may evolve into lumB tumours through the stochastic acquisitions of mutations in genes associated with worse prognosis , 16. TisThe lumA tumours identified in our analysis as associated with worse DSS outcomes largely correspond to Tishchenko q2, q3 and q4 of the Tishchenko et al study . This meLumA patients who belong to both higher CNA Quartiles and higher gene expression Tishchenko quantiles are at particular risk for long term survival outcome. This has potential clinical utility because these patients are potentially not well stratified by the PAM50 subtype but can be identified by the CNA Score. Therefore, patients classified as lumA that display gene expression levels more akin to lumB tumours and also have high CNA burden may benefit from the more aggressive treatment regime used for lumB patients in contrast to standard endocrine therapy for lumA patients .We defined the CNA Score metric as the sum of the absolute CNA values over all genes per patient. This definition enables unbiased analysis, maintains the easy interpretation of the data, and provides sufficient samples per CNA Quartile to implement meaningful statistical analyses. However, this definition is a simplification of the CNA landscape in tumour cells. Fine grained features including length of the CNA, whether it is an amplification or deletion, and the genomic location of the CNA are not considered. The analysis could potentially be made more sensitive with a richer metric, although the smaller sample groups available following such fine grained segmentation of the METABRIC luminal cohort would likely compromise rigorous statistical analysis. To avoid sample size limitations, expanded datasets that combine high quality genomic and transcriptomic profiling with long-term clinical follow-up are required to provide sufficient cases for independent discovery and validation.Overall, this work demonstrates a practical pathway towards personalised medicine using genomic characteristics. Such an individualised approach to classifying breast cancers could improve the success of treatment interventions by guiding tailored therapeutic strategies based on the genomic profile of an individual tumour . For exaIt is important to identify features of luminal breast cancer that have statistically robust prognostic value in order to identify patients with a greater risk of lethal disease because the number of women diagnosed is increasing and the majority of cases belong to luminal subtypes. We analysed freely available clinical and genomic patient data from the METABRIC dataset to study the impact of CNA burden on overall survival within the luminal subtypes. We observed that CNA Quartiles based on the sum of absolute CNA variations are a prognostic factor for breast cancer survival outcomes in a subset of patients with high GI suffering from lumA breast cancer. We further demonstrated that some of the lumA cases in our study lie in the ambiguous region between lumA and lumB subtype classifications identified in an earlier analysis of gene expression levels from the same METABRIC patient samples. Women diagnosed with lumA breast cancer who possess a CNA burden within our derived CNA Q3 or Q4 have reduced survival outcomes and may benefit from more aggressive therapy. This progresses efforts to incorporate individual genomic landscapes into more nuanced classifications of breast cancer cases, with the goal of personalising therapeutic interventions to optimise long term survival outcomes for patients.S1 File(PDF)Click here for additional data file."} +{"text": "Cerium oxide nanoparticles have been shown to sensitize cancer cells to radiation damage. Their unique redox properties confer excellent therapeutic potential by augmenting radiation dose with reactive oxygen species mediating bystander effects. Owing to its metallic properties, cerium oxide nanoparticles can be visualized inside cells using reflected light and optical sectioning. This can be advantageous in settings requiring none or minimal sample preparation and modification. We investigated the use of reflectance imaging for the detection of unmodified nanoceria in MDA MB231 breast cancer cells along with differential interference contrast imaging and fluorescent nuclear labeling. We also performed studies to evaluate the uptake capability, cellular toxicity and redox properties of nanocaria in these cells. Our results demonstrate that reflectance structured illumination imaging can effectively localize cerium oxide nanoparticles in breast cancer cells, and when combining with differential interference contrast and fluorescent cell label imaging, effective compartmental localization of the nanoparticles can be achieved. The total number of cells taking up the nanoparticles and the amount of nanoparticle uptake increased significantly in proportion to the dose, with no adverse effects on cell survival. Moreover, significant reduction in reactive oxygen species was also observed in proportion to increasing nanoceria concentrations attesting to its ability to modulate oxidative stress. In conclusion, this work serves as a pre-clinical scientific evaluation of the effective use of reflectance structured illumination imaging of cerium oxide nanoparticles in breast cancer cells and the safe use of these nanoparticles in MDA MB231\u00a0cells for further therapeutic applications. \u2022Internalized cerium oxide nanoparticles are imaged with reflected light in breast cancer cells for the first time.\u2022Cerium oxide nanoparticles demonstrated no toxicity in MDA MB231 breast cancer cells\u2022Cerium oxide nanoparticles modulated free radicals in MBA MB231 cells in a dose dependent manner The eff3+ and Ce4+ ions with the ability to have oxygen vacancies on their surface enabling modulation of free radicals according to the below redox chemistry - species in CeO2 nanoparticle-treated cells relative to untreated cells. The FL1 population was gated to include green fluorescent cells with a plot of the median fluorescent intensity (In order to examine the possible effects of intracellular cerium oxide nanoparticles on redox systems in MDA MB231\u00a0cells, we measured the levels of rhodamine 123 as an indicator of oxidation of dihydrorhodamine 123 by peroxynitrite anion [ONOO]ntensity A. The pentensity B. There in vivo for the treatment of breast cancer.In conclusion, we have shown for the first time that pure cerium oxide nanoparticles can be localized relatively easily in breast cancer\u00a0cells using reflectance confocal imaging. These cells can take up higher amounts of nanoceria without any adverse effects to cellular health by significantly downregulating reactive oxygen species levels, thus paving the way for its safe use as a potential radiosensitizer"} +{"text": "Keynote by Jenny Groh (Duke University) at the 20th European Conference on Eye Movement Research (ECEM) in Alicante, 19.8.2019Information about eye movements with respect to the head is required for reconciling visual and auditory space. This keynote presentation describes recent findings concerning how eye movements affect early auditory processing via motor processes in the ear . Computational efforts to understand how eye movements are factored in to auditory processing to produce a reference frame aligned with visual space uncovered a second critical issue: sound location is not mapped but is instead rate (meter) coded in the primate brain, unlike visual space. Meter coding would appear to limit the representation of multiple simultaneous sounds. The second part of this presentation concerns how such a meter code could use fluctuating activity patterns to circumvent this limitationVideo streamhttps://vimeo.com/356576513"} +{"text": "Transcatheter aortic valve implementation (TAVI) is currently the preferred treatment for patients with severe aortic valve stenosis and high surgical risk. Recent trials have found better clinical outcomes in patients with lower surgical risk scores compared to surgical aortic valve replacement. Consequently, TAVI indication is rapidly expanding to patients with intermediate and low surgical risk . PrevalePrevious studies have demonstrated that patients after TAVI have improved cognitive functioning. Khan et al. conducteThis overall improvement is interesting since TAVI can also result in cognitive decline due to cerebral (micro) infarction. Implementation of a new aortic valve releases debris and emboli from the native calcified valve. Up to 80% of TAVI patients show signs of these emboli as white matter hyperintensities on post-procedural brain imaging . Most ofSchoenenberger et al. found thAlterations in cardiac output have different effects on young and older brains. Aging comes with a larger cerebrovascular resistance because of arteriosclerosis and sustained cerebral vasoconstriction. As a result, cerebral perfusion becomes increasingly dependent on cardiac output in the elderly . TAVI enTAVI has the ability to restore cerebral autoregulation and improve cognition, particularly in those with impaired baseline cognition or insufficient cerebral perfusion. However, it is still unknown in which patients TAVR improves cognition and in which patients TAVI may even be cognitively harmful. Its expanding indication underlines the need to further investigate cognitive outcomes in younger and healthier patients with lower surgical risk, but also in older, frail patients. This will allow us to properly select patients that will benefit not only from better cardiac function but overall quality of life."} +{"text": "Variations in pulmonary venous anatomy should not be underestimated by thoracic surgeons prior to or during major lung resections in order to avoid serious surgical complications. Here, we report a case of middle lobe vein draining into a right inferior lobar vein formed by two anomalous trunks lying on the superior surface of the common basal bronchus: in such instance, to avoid compromising the middle lobe vein drainage during a thoracoscopic right lower lobectomy, the two main tributaries of the lower lobe vein were individually identified and dissected peripherally from the anterior aspect after division of the major fissure. A careful hilar dissection and a precise surgical strategy can help surgeons perform correct procedures in presence of pulmonary vascular anomalies. Here, we report a case of middle lobe vein draining into a right inferior lobar vein formed by two anomalous trunks lying on the superior surface of the common basal bronchus: in such instance, to avoid compromising the middle lobe vein drainage during a thoracoscopic right lower lobectomy, the two main tributaries of the lower lobe vein were individually identified and dissected peripherally from the anterior aspect after division of the major fissure. A careful hilar dissection and a precise surgical strategy can help surgeons perform correct procedures in presence of pulmonary vascular anomalies. The current scientific literature documents several imaging findings and systematic overview of pulmonary vein abnormalities provided by radiologists for a correct recognition of aberrant vessels and a more precise identification of pulmonary nodules or intersegmental lymph nodes near to vascular structures. In the last decade, also in other fields such as the thoracic surgery, many authors have reported a wide spectrum of venous pulmonary anomalies identified preoperatively or intraoperatively in order to make lung major resections safer during video\u2010assisted thoracoscopic surgery (VATS) where a short exposition of the pulmonary hilum can lead to mistakes due to incorrect identification of anatomical variations , 2, 3. TA 67\u2010year\u2010old man was admitted to our unit for treatment of non\u2010small cell lung cancer (NSCLC). Preoperative chest computed tomography (CT) scans showed a pseudo\u2010nodular thickening in the right lower lobe Fig. A and a vAlthough the middle lobe vein typically enters into the right superior pulmonary vein, some authors have described the veins from the medial and lateral segments of the middle lobe draining directly into the left atrium or into the right inferior lobar vein separately or as a single trunk . During Appropriate written informed consent was obtained for publication of this case report and accompanying images."} +{"text": "Cardiac gating leads to less artifacts and more accurate measurements of the thoracic aorta and root. Without gating the aortic root and ascending aorta are particularly prone to artifact. To date gating has lead to increased dose to patients who often have to undergo multiple studies.To investigate the effects of prospective cardiac gating and low kilovoltage parameters on image quality and radiation dose when acquiring CT angiography of the thoracic aorta (CTTA).Dual-source CTTA was performed on 60 consecutive patients. One group of thirty were examined with retrospective gating and standard parameters . The other thity were examined with prospective gating and low dosage parameters with the scanner in a Step and Shoot (SAS) mode that automatically recognizes and does not acquire data during ectopic heart beats. Qualitative analysis was performed by two blinded reviewers who assessed image quality and graded it on a three point scale. Sharpness of the thoracic aorta was also evaluated quantitatively at three levels by generating a line profile across the aortic vessel wall and calculating the point spread function. Both sides of the density profile were analyzed, averaged and then used to calculate sharpness (Hounsfield units/mm). Attenuation in the aorta and image noise was measured. Radiation dose was measured.Qualitative and quantitative analysis of sharpness of the thoracic aorta and image noise showed no significance difference (p > 0.05) between the two patient groups. Estimated effective radiation dose of the prospective low kilovoltage protocol (mean dose <3 mSv) was significantly lower (p < 0.01).Low dose imaging of the thoracic aorta with maintenance of image quality and sharpness is achievable using a prospective cardiac gated low kilovoltage technique."} +{"text": "The phyllosphere is an important microbial habitat, but our understanding of how plant hosts drive the composition of their associated leaf microbial communities and whether taxonomic associations between plants and phyllosphere microbes represent adaptive matching remains limited. In this study, we quantify bacterial functional diversity in the phyllosphere of 17 tree species in a diverse neotropical forest using metagenomic shotgun sequencing. We ask how hosts drive the functional composition of phyllosphere communities and their turnover across tree species, using host functional traits and phylogeny.Neotropical tree phyllosphere communities are dominated by functions related to the metabolism of carbohydrates, amino acids, and energy acquisition, along with environmental signalling pathways involved in membrane transport. While most functional variation was observed within communities, there is non-random assembly of microbial functions across host species possessing different leaf traits. Metabolic functions related to biosynthesis and degradation of secondary compounds, along with signal transduction and cell\u2013cell adhesion, were particularly important in driving the match between microbial functions and host traits. These microbial functions were also evolutionarily conserved across the host phylogeny.Functional profiling based on metagenomic shotgun sequencing offers evidence for the presence of a core functional microbiota across phyllosphere communities of neotropical trees. While functional turnover across phyllosphere communities is relatively small, the association between microbial functions and leaf trait gradients among host species supports a significant role for plant hosts as selective filters on phyllosphere community assembly. This interpretation is supported by the presence of phylogenetic signal for the microbial traits driving inter-community variation across the host phylogeny. Taken together, our results suggest that there is adaptive matching between phyllosphere microbes and their plant hosts.Video abstract. The phyllosphere\u2014the aerial surfaces of plants including leaves\u2014is a widespread microbial habitat that hosts a diversity of microorganisms that play key roles in plant ecology and evolution . PhyllosComparative studies of the taxonomic composition of phyllosphere microbial communities across plant hosts have demonstrated the importance of host identity as a key driver of variation in phyllosphere microbial taxonomic diversity. At fine taxonomic scales, the composition of these communities varies predictably across host plant species \u20139 and acDetermining whether plant\u2013microbe associations in the phyllosphere have an adaptive basis will require establishing how both plant and microbial functions are related across a range of host species. Plant functional traits\u2014measures of morphology and physiology that capture key axes of variation in plant life history and ecology \u2014have beeSeveral studies have reciprocally identified the broad-scale microbial functional categories and adaptations that epiphytic microbes possess for living on plants e.g. \u201319]. Fun. Fun19]]In this study, we quantified the functional repertoire of microbial communities on leaves of multiple tree species in a neotropical forest on Barro Colorado Island (Panama) using metagenomic shotgun sequencing. Sampling was performed in a 50-ha long-term plot of old-growth tropical forest within which ~300 tree species have been recorded, most of them evergreen . We askeOverall, we detected 4587 different functional genes across all samples based on the annotation of metagenomic shotgun sequencing of tropical tree phyllosphere communities. Functions related to metabolism were the most abundant overall in our dataset, making up 45% of all functionally annotated sequences Fig.\u2009. The priP\u2009<\u20090.05; Fig.\u2009The bacterial functions present on tree leaves were remarkably consistent among different samples. The vast majority of functional variation occurred within samples (>\u200997%), with a very small contribution of functional turnover among samples <\u20093%) to total functional diversity, regardless of the functional level under study. Most taxonomic diversity was also observed within samples, with a contribution of beta-diversity increasing from 1 to 4.4% of the total diversity with a refinement of the taxonomic scale utilized were most strongly associated with the first two axes of microbial functional variation. Leaf elemental concentrations of copper, aluminum, and manganese were also strongly correlated with these first dimensions. The plant trait gradients explained altogether ~17% of variation in functional composition among microbial communities. The vast majority of the microbial Tier 3 functions were more or less abundant than 95% of the values obtained from the null model keeping both the total abundance of a trait and the number of traits in a community constant Table\u2009. The filThe functional composition of tree phyllosphere microbial communities in a tropical forest in Panama is largely consistent with those reported in the literature, regardless of the type of plant studied, suggesting the presence of a core functional microbiota in phyllosphere microbial systems. Core functional microbiota in host-associated systems have also been reported for other hosts. Our study supports findings of an important role for the metabolism of carbohydrates and amino acids in bacterial survival in the phyllosphere , 28, 29 The low functional variability in microbiota observed among tree species represents a further line of evidence supporting the presence of a core phyllosphere functional microbiota. This low variability, observed even at fine functional levels, could be the consequence of essentially similar constraints imposed by the generally harsh leaf environment on its microbial communities, regardless of the specific physiological traits of the host plant species. This low functional turnover among communities was also associated with a low taxonomic turnover, contrasting with reports from phyllosphere-associated temperate systems where species identity was a strong driver of taxonomic composition of the microbial communities . These rDespite the high levels of convergence in microbial functions among the phyllospheres of different tree species, several lines of evidence support a role for plant species taxonomic and functional identity in driving microbial community assembly. Tree traits explained a notable portion of the functional turnover among microbial communities. Traits correlated with microbial functional turnover are mostly part of the leaf economics spectrum , a functOther lines of evidence support the idea that the plant host plays a selective role on microbial community assembly, such as the detection of bacterial traits that exhibit a strong phylogenetic signal with respect to the host plant phylogeny. While this pattern might arise from the filtering of microbes on phylogenetically structured selective plant traits or from co-evolution of the two partners, it is regardless indicative of an influence of the host on the functional makeup of bacterial phyllosphere communities. Interestingly, the set of pathways that are important in driving functional turnover among communities belong to the same functional categories as the ones that are phylogenetically structured among plant hosts, supporting the proposed match between these bacterial functions and their host\u2019s functional and taxonomic identity. The fact that the relative abundance of a large set of functions was different within communities than that expected by chance given their relative abundance across samples, also supports a role for individual tree species in structuring the functional composition of their phyllosphere bacterial communities. The higher filtering of most microbial taxa relative to microbial functions suggests a role for unmeasured trait variation in driving functional turnover among communities.The relatively small but significant contribution of functional turnover among microbial communities to the total functional diversity observed across samples suggests that the functions that are of importance in driving the distribution of bacteria across different host trees are actually relatively few compared to those enabling the bacteria to pass the overall \u201cphyllosphere filter\u201d that is needed to survive in the phyllosphere habitat. It remains unknown whether the majority of functional pathways that do not vary among trees are actually important for the ecology of the microbes, or if that trait variation is adaptively neutral within communities. It is also possible that some pathways important for microbial adaptations to leaf physiological gradients are not yet functionally described and are part of the large number of sequences that could not be functionally annotated. Ongoing efforts to better characterize gene functions will help improve the precision of ecological inferences in environmental metagenomes.In conclusion, we have identified a core functional microbiota in the phyllosphere of neotropical trees. While most functional variation was observed within individual microbial communities, we reveal a functional matching between the traits of microbes and the traits of plants across 17 tree species, emphasizing the role for energy metabolism, secondary metabolites, and antibiotic production as well as environmental sensing in mediating bacterial adaptation to leaf trait gradients in the canopy. Our identification of the adaptive drivers of phyllosphere microbial community composition in this neotropical ecosystem represents a good starting point for identifying the types of microbial traits that could be routinely studied by phyllosphere microbial ecologists to address global questions on the ecological and evolutionary dynamics of phyllosphere microbes. Empirical testing of the fitness consequences of variation in those traits will represent an important next step in understanding adaptive processes in the phyllosphere.g for 20\u2009min. DNA was extracted using MoBio PowerSoil DNA extraction kits and samples stored at \u2212\u200980\u2009\u00b0C for future analyses. We quantified DNA concentrations and sequenced both extraction negative controls and PCR negative controls for these samples as part of previously published analyses of bacterial 16S and fungal 28S amplicon sequencing of these samples [Microbial communities were collected from the leaves of 24 individual trees from 17 tree species (1\u20132 samples per species) in the tropical lowland rainforest of Barro Colorado Island, Panama, in December 2010. These samples were selected from a larger pool of samples , 26 for samples , 26; nonMetagenomic shotgun sequencing yielded 14,642,408 reads in total. We trimmed sequences to remove Illumina adapters and truncate end-bases with a quality score less than 20, and removed sequences shorter than 25\u2009bp, leaving 14,634,072 trimmed and quality-controlled reads. Taxonomic annotation of all sequences in each microbial community was performed to restrict functional analyses only to bacterial sequences. We annotated metagenomic reads using Kaiju, which annotates taxonomic identity of reads by comparing sequenced reads to the microbial subset of the NCBI BLAST non-redundant protein database [Functional annotation of microbial sequences was performed via protein homology searches using the KEGG annotation framework , 35 via We obtained measurements of plant functional traits for all plant species from a dataset collected previously on Barro Colorado Island . This tr\u03b3div\u2009=\u2009\u03b1div\u2009+\u2009\u03b2div [n\u2009=\u2009128) and rerunning the analyses. This subsampling did not affect our results . As such, we can assume that individuals from the same species are structuring their leaf microbial communities in a similar way and do not drive important functional differences among samples. All 24 samples were thus kept in this analysis.We performed a principal component analysis (PCA) of functional trait matrices and identified the functions contributing most to variation along the first axes of variation using R package FactoMineR . We fittmultiphylosignal from R package Picante [n\u2009=\u20099999 randomizations). We considered a microbial function to exhibit strong phylogenetic signal if it fell in the top 5% of the distribution of signal based on the randomization test (P\u2009<\u20090.05 according to randomization test). We selected a single random sample per host species for those host species with more than one sample prior to calculating phylogenetic signal. We repeated this for different random subsamples and it did not qualitatively change the results so we report phylogenetic signal for a representative random subsample.We quantified the phylogenetic signal in associations between microbial functions and host plant phylogeny using function Picante to calcuThe degree of host filtering on microbial communities was assessed by comparing the occurrence of traits in observed communities to those obtained from 9999 randomizations of community trait matrices. Host filtering was detected as an over- or under-representation of the given trait in individual communities. Randomizations were generated by permutations of the trait matrix preserving row and column totals. For each site and bacterial trait combination, we compare the observed frequency of the trait to the random values to assess whether it was lower or higher than expected by chance. To compare the strength of functional vs. taxonomic filtering, we applied the same procedure to the taxonomic datasets defined at each of six taxonomic levels, from the phylum to the species.Additional File 1. Supplementary Tables. This additional file contains 2 supplementary tables, referred to in the main text.Additional File 2. Supplementary Figures. This additional file contains 3 supplementary figures, referred to in the main text.Additional File 3. Host phylogeny. A phylogenetic hypothesis for host plant species obtained by grafting tree species from the study site onto a dated megatree of angiosperms (see Methods for details)."} +{"text": "Experiencing difficulties in performing basic activities of daily living poses significant challenges for older adults living with such limitations and also for their spouses. A growing body of evidence demonstrates cross-spousal linkages between activity limitations and depressive symptoms. However, under what conditions these linkages may be strengthened or weakened has received little attention in the literature. We addressed this gap by examining whether a) providing spousal caregiving and b) spousal pain moderated the link between spousal activity limitations and one\u2019s own depressive symptoms. We used seven waves of longitudinal household data from the Health and Retirement Study to estimate within-person associations between spousal activity limitations and depressive symptoms, focusing on the moderating roles of caregiving behavior and spousal pain. In particular, asymmetric fixed effects models were used to estimate the unique effects of transitioning into a spousal caregiver role in the context of spousal activity limitations. Results from multilevel models were gendered. For wives (but not for husbands), transitioning into a caregiver role to provide spousal care alleviated depressive symptoms associated with spousal activity limitations, whereas depressive symptoms were increased when husbands with activity limitations also reported frequent, moderate to severe pain. Our findings indicate that the link between spousal activity limitations and depressive symptoms is not uniform, and that the cross-spousal association may best be understood when relevant contextual factors are considered. The findings are also in line with recent studies showing that caregiving may also lead to enhanced well-being and reduced mortality risk under some circumstances."} +{"text": "Multi\u2010gene panel testing is replacing single\u2010gene testing for patients with suspected hereditary cancer syndromes. The detection of a hereditary cancer syndrome allows tested individuals to initiate enhanced primary and secondary prevention efforts\u2014where available\u2014with a view to reduce disease burden. Current policy prevents testing programmes from communicating genetic test results with potentially affected family members, yet it is well documented that tested individuals face multiple challenges in initiating such discussions with relatives.In response to this challenge, we sought patient recommendations about how to improve genetic risk communication to enhance interfamilial discussions about primary and secondary disease prevention.We conducted 25 semi\u2010structured interviews with individuals who received genetic testing through British Columbia\u2019s Hereditary Cancer Program between 2017 and 2018. Interviews were professionally transcribed and analysed using a constant comparative approach.Participants described difficulty engaging in conversations with relatives who were resistant to receiving genetic risk information, when communicating with younger relatives and where participants reported strained familial relationships. Participants recommended that testing facilities provide a summary of results and implications and that resources be made available to prepare patients for challenging discussions with family members.Our study demonstrates that individuals undergoing genetic testing for suspected hereditary cancer syndromes would benefit from additional supportive resources alongside genetic counselling. Providing this on\u2010going support will enhance the accurate and transparent communication of risk to facilitate the uptake of cascade testing and enhanced prevention strategies. The clinical application of multi\u2010gene panels is intended to guide decisions about enhanced primary and secondary prevention strategies as well as genetics\u2010informed treatment options. Due to the hereditary nature of certain cancer syndromes such as Lynch, Li\u2010Fraumeni and hereditary breast and ovarian cancer syndromes, test results carry implications for the first tested individual in the family\u2014probands\u2014as well as their genetic family members. Communicating accurate genetic risk information allows potentially affected family members to undergo testing and\u2014where appropriate\u2014benefit from enhanced prevention strategies.Current legislation within Canada and the United States does not require probands to discuss genetic test results with family members.In response to reported barriers, health\u2010care providers offer strategies such as allocating genetic counselling time to addressing family dynamics, as well as the provision of family letters and testing facility contact information to relatives.address barriers to effective communication. Enhancing communication between patents and relatives will increase the uptake of cascade testing. Determining what patients need to improve the communication process will help to meet this overarching objective will help to ensure equitable access to prevention strategies for high\u2010risk families.Tested individuals are the vehicle by which information about hereditary cancer susceptibility is relayed to family members. For this reason, there exists an unmet need to determine how to enhance the communication process, from the perspectives of those who have undergone testing. To date, there is a lack of patient\u2010directed guidance to mitigate challenges to communicating genetic risk to relatives. High\u2010quality patient decision support techniques present an opportunity to enhance shared decision making and supplement genetic counselling sessions, but do not yet exist for the purposes of enhancing interfamilial communication about hereditary cancers.1.1The current study was initiated in response to an unmet need to identify patient\u2010guided strategies to improve communication about hereditary cancer susceptibility. This work represents the preliminary phase of a larger investigation to develop a patient\u2010values informed decision support technique for hereditary cancer susceptibility genetic testing. The qualitative work was conducted specifically to inform the content of the decision support tool in the form of an e\u2010health application developed for feasible implementation. Here, we report on the aspect of patient interviews that pertain to communicating genetic testing information to family members.This study was conducted in collaboration with the British Columbia (BC) Cancer\u2019s Hereditary Cancer Program (HCP), the sole provider of publicly funded hereditary cancer genetic counselling and testing services across BC and the Yukon.22.1Our patient\u2010oriented approach involved on\u2010going consultation with a patient partner diagnosed with a hereditary cancer syndrome. Our patient partner was actively involved in protocol development, review of all study documentation, interview study design, interpretation of findings and review of research outputs. She attended all research team meetings either via teleconference or in\u2010person.2.2The study team developed an interview guide to explore patient experiences with the process of genetic testing and communicating test results with family members. The content of the interview guide was informed by previous qualitative studies examining patient opinions about genetic testing, the broad semi\u2010structured interview guide development literature, as well as consultation with our patient partner.2.3Patients eligible for this study were 19 years or older, received carrier or index genetic test results between 2017 and 2018, had previously consented to be contacted for future research studies and were able to complete an interview in English. Index testing occurs when the proband is tested in the absence of a previously identified hereditary cancer syndrome in his or her family. Carrier testing is undertaken when a familial cancer syndrome has already been identified in the family, and typically, an asymptomatic individual is tested for the presence of known syndrome\u2010specific pathogenic (disease causing) variants.To maximize participation, two recruitment approaches were taken. First, we identified a list of individuals having recently undergone genetic testing and had provided written consent to be contacted for future research. Potentially eligible participants were identified through the Hereditary Cancer Program database. Following the identification of eligible individuals, the study coordinator (SP) distributed a study invitation and consent letter via post. Non\u2010responders were contacted via telephone to assess interest after two weeks. Second, one investigator (SS) approached eligible patients in clinic to ascertain interest in the study. The study coordinator followed up with individuals who expressed interest in participating. Investigators applied a maximum variation sampling technique to ensure diversity in age, sex and personal experience with cancer. Participant recruitment continued until two reviewers (SP and SK) agreed that data saturation had been achieved.2.4A female PhD health researcher (SP) with qualitative research experience conducted all interviews. Participants were provided the option to be interviewed over the telephone or in\u2010person at the BC Cancer Research Centre in Vancouver, BC. The interviewer had no previous interaction with participants other than communicating the purpose of the research and obtaining consent in advance of the interviews. To the best of our knowledge, participants had no prior familiarity with the interviewer. No interviewer characteristics were provided beyond information about her participation in the research project. All interviews were audio\u2010recorded following written participant consent. The interviewer took minimal notes during the interview and documented field notes immediately following. For in\u2010person interviews, only the interviewer and participant were present in the room. A distress protocol was maintained during each interview.All interview transcripts were professionally transcribed, de\u2010identified and reviewed for accuracy prior to initiating the qualitative analysis. Transcripts and field notes were maintained on the research group\u2019s password\u2010protected secured drive with access limited to researchers listed on the ethics approval. Interview transcripts were not returned to participants.Two coders (SP and SK) applied a grounded theory approach to the qualitative analysis, using constant comparison for the development of the code book.3Between June and September 2018, 49 patients were invited to participate in the interview study. Of these, 26 consented to participate and 25 interviews were completed . A single interviewer (SP) conducted 21 telephone and 4 in\u2010person interviews. Interviews lasted approximately 45 minutes with a range of 30\u201070 minutes.The majority of participants were female (64%), married (72%), educated beyond high school equivalence (72%) and had a personal history of cancer (76%). Participants\u2019 age ranged from 32 to 78 years. Among patients with a self\u2010reported personal history of cancer, 70% (n=14) had experienced either breast or colon cancer, consistent with the distribution of patients who are referred to the HCP. Other diagnoses included lung cancer, malignant melanoma, testicular, kidney, bowel, uterine and endometrial cancer. Participants with a personal history of cancer reported between 1 and 3 primary cancer types.According to participant report, 21 underwent index testing and four participants were carrier tested. Just under half (43%) of participants reported having received a pathogenic variant. Three of four patients (75%) who underwent carrier testing reported that they received an uninformative result. A total of 10 participants (40%) reported the return of a variant of unknown significance or an inconclusive finding had chosen not to disclose the fact that they had undergone testing. One participant had made one attempt with the intention of broaching the topic again. Only one participant with a reported\u2010pathogenic variant explicitly stated that she had not attempted communicating her results with genetic family members. We also identified variation in terms of the timing of communication, often informed by participants\u2019 comfort level with raising the topic of genetic testing and disease status. Although some participants reported speaking with family members throughout the testing process and before the return of results, others waited until they had received their results to attempt discussions.Among patients who reported having initiated conversations with family either before or after testing, 7 referenced conversations with first\u2010degree relatives and an additional 4 participants specifically referenced conversations with second\u2010degree relatives . Eight participants specifically stated that at least one reason for receiving testing was to inform family members about their cancer risk. Other reasons for testing included a desire to inform treatment and prevention options, to assist in making family planning decisions, as well as general life planning.5Interview participants described a variety of experiences communicating genetic risk to family members. Communication challenges were highly contextualized given initial attempts at discussions about test results, relatives\u2019 perceptions about cancer risk, the presence of a family history of cancer, family culture, and interpersonal relationships with relatives. Here, we report barriers and facilitators identified through the qualitative synthesis, followed by a discussion of participant recommendations for facilitating constructive risk communication Table .5.1Interpersonal family relationships and culture played an important role in participant discussions about initiating conversations, and their perceptions about the success of attempts at communication. Participants described strained or distant relationships as well as family cultures wherein sensitive subjects were not discussed (quote 1\u20103). Although some participants expressed frustration about the lack of risk communication with relatives (quote 1), a minority appeared unbothered and unmotivated to overcome interpersonal barriers to communication (quote 2). In one case, family culture and perceptions about cancer were explicitly discussed as a primary barrier to initiating conversations about genetic test results.5.2Participants described relatives who would not engage in discussions about genetic risk as well as those who explicitly stated that they did not want to be informed about test results. Reasons for not wanting to receive risk information were multifold. For example, some characterized family members as fearful, overwhelmed or disinterested in health or genetic information (quotes 5 and 6). Others characterized family members as lacking interest in medical information (quote 9), as being distrustful of the health\u2010care system and health\u2010care providers (quote 11), or holding the preconceived opinion that cancer cannot be prevented (quote 13). Throughout these conversations, participants did not describe a lack of understanding of their own test results such that they were challenged by how to communicate risk and the value of cascade testing effectively. Rather, participants described feeling ill\u2010equipped to overcome relatives\u2019 unwillingness to engage in meaningful and constructive discussions.BRCA variant to her brother and children, each of whom had children of their own. This sentiment was shared by other participants when relaying information with younger family members (quote 5\u20107). In each of these discussions, participants expressed marked distress about the difficulty of ensuring that young family members would understand the implications of test results as well as the importance of taking preventative actions to reduce their own cancer risk.In a minority of instances, participants described specific challenges related to communicating test results to young family members. These conversations were met with the perception that young adult relatives considered themselves to be at an inherently low risk despite the presence of variant hereditary cancer syndrome in the family. One participant (quote 5) found it difficult to communicate information about her pathogenic Participants who experienced strained conversations with close family members struggled with the decision to inform second\u2010degree relatives. In two specific conversations (quotes 12 and 13), participants expressed a lack of clarity about the duty to inform family members. Although the duty to inform was discussed in a small subset of interviews, the topic was met with substantial distress and confusion regarding who should be informed, and how discussions with resistant family members should be broached. Participants whose attempts to relay genetic risk information were stonewalled by resistant family members expressed considerable frustration.5.3. Other times, the presence of a known family history of cancer helped to facilitate open and constructive discussions (quotes 14 and 15). In such instances, the presence of a strong family history of cancer was described as a common topic of conversation within families or as a feature that brought family members closer together (quotes 4).The presence of a known family history of cancer served to both facilitate and stifle constructive conversations. As shown in quotes 8, 9, 10 and 13, cancer was at times perceived as something fearful about which family members were unwilling to engage. One participant described her perception that that the detection of a hereditary cancer syndrome would be too much for her daughters to handle given her family\u2019s recent history of multiple cancer diagnoses (quote 10)6Following discussions about communicating genetic risk to family members, participants discussed ways in which an adjunct to genetic counselling could improve this process. Recommendations were not framed specifically in relation to the potential for an e\u2010health app. Rather, participants discussed recommendations in terms of broad strategies that would support the process of communication (quotes 18\u201023). Recommendations included a lay summary of test results and familial implications, alongside practical advice to facilitate constructive conversations. Participants recommended the potential for a lay summary of genetic test results and implications directed to family members. In some cases, participants reported that this could be used to supplement conversations with relatives (quotes 19 and 20). Participant perceived that documentation provided by testing programmes would facilitate and validate conversations and increase the likelihood that family members would initiate prevention strategies. Other participants recognized that a written report could be used in situations where family members reside in geographically disparate locations or within families where communication about sensitive subjects was particularly challenging (quote 19).The desire for practical advice about communication with family was articulated using multiple examples, such as the ability to connect with previously tested individuals to illustrate what successful conversations with family members might look like (quote 21). Participants spoke about the desire for examples or advice about how to broach the topic of genetic risk (quote 22 and 23). Among individuals who faced resistant family members, there was a lack of confidence about how to accurately relay the information in a manner that would be sensitive to the potential for worry, upset or guilt (quote 18). A key feature of participant recommendations was to better prepare probands for resistant family members and to provide them with the ability to manage negative reactions. Participants sought resources to prepare them for potentially difficult discussions, as well as on\u2010going support following unsuccessful attempts at communication Table .7Although previous investigations have elicited genetic counsellor practice patterns for enhancing family communication, to our knowledge, no patient\u2010reported recommendations to directly inform the development of a decision support tool exist.Throughout discussions, participants acknowledged the potential for worry and anxiety among family members. Participants discussed waiting to speak with family members until results had been returned in an effort to mitigate unnecessary concern. Participants also discussed the impact that worry and anxiety had on familial reactions to positive test results. Consistent with recent literature, these findings support the development of methods and resources to ensure that genetic risk information is relayed in a manner that will not overwhelm or overburden potentially affected relatives.individualized approach to encouraging discussions about genetic risk best aligns with patient preferences and experiences. Given the communication challenges faced by our participants, these findings further suggest that probands may be accepting of direct contact of potentially affected relatives by testing facilities, where conversations are challenging or infeasible.Our results further substantiate the individualized nature of successful communication. While the presence of a family history of cancer was at times a strong motivator and facilitator for effective communication, other participants experienced opposing reactions from relatives. This finding speaks to the need for an individualized approach to preparing patients for discussions, given their personal and familial experiences, perceptions and expectations. The implementation of an As evidenced here, genetic counselling for hereditary cancer testing does not always adequately prepare patients to relay their results to family members. In resource\u2010scarce health systems, genetic counselling sessions are typically limited in length and frequency, owing partially to a shortage of available counsellors. A substantial gap exists between the need for and availability of genetic counsellors to adequately guide patients through complex decision making.7.1The results of this work should be interpreted alongside limitations. Firstly, responses to the interview were self\u2010reported and therefore subject to reporting bias. To mitigate this potential bias, participants were informed that only de\u2010identified information would be reported and that all interview responses would remain confidential. Consent documentation provided assurance that responses would not impact clinical care.Further, we did not validate participant reported test results with individual patient test result reports. For this reason, we are unable to determine whether participants accurately understood their test results. While we report briefly on self\u2010reported results to frame discussions, the focus of this work centred on experiences communicating test results with relatives. Recognizing this limitation, in the second stage of this study\u2014following the development of the decision support tool\u2014we plan to validate self\u2010reported results with testing reports to determine the presence of recall bias or recall inaccuracies.Finally, our sample consisted of participants who had previously consented to be contacted for future research. The subset of individuals interested in research participation may differ systematically from the total population who underwent genetic testing. Despite the potential for selection bias, we sought to ensure a diversity of perspectives and experiences were captured, and recruitment continued until thematic saturation was reached. Qualitative research studies such as this do not seek sample representativeness and generalizability of findings. Given the diversity of communication experiences captured in this interview study, we are able to report on a substantial heterogeneity of experiences and challenges communicating genetic information to family.8Successful communication between probands and their family members is a highly individualized experience informed by multiple interpersonal factors. Our findings support the development of improved resources to assist patients through the entire trajectory of genetic testing. This on\u2010going support will ensure patients have the motivation, self\u2010efficacy, and informational resources for successful communication with their families. We further identify a need for practical guidance about how to broach conversations, and how to manage family dynamics when discussions are unsuccessful. While genetic counsellors have a responsibility to provide patients with adequate information that prepares them for constructive risk communication, they are facing time constraints in publicly funded health\u2010care systems. Additional supports that reduce genetic counsellor burden and enhance the familial communication process will better enable cascade testing programmes to benefit at\u2010risk families.The authors declare that they have no conflict of interest."} +{"text": "The national population of unhoused older adults is predicted to nearly triple by 2030. Unhoused older adults have a mortality rate four to nine times higher than housed populations, and face structural barriers during illness trajectory that likely influence both where care takes place and processes around attaining psychosocial later-life goals. This study aimed to (1) document patterns in the healthcare trajectories of unhoused older adults and (2) examine the role of care transitions in psychosocial goal attainment. Retrospective chart review was completed in partnership with a mobile homeless palliative care team; additional data was gathered from provider focus group. Through a content analysis of this data, it was discovered that older unhoused palliative care patients experienced more transitions, and that numerous care transitions were associated with disruptions to goals. The type of care transitions patients experienced also did not reflect their later-life goals. Patients\u2019 movements impacted the role of formal and informal care networks in care and highlighted the implications of place in common psychosocial goals, such as family reconciliation. Unconventional neighborhood supports were found to help facilitate treatment adherence. These findings offer translational opportunities for further research, including \u201crapid respite\u201d models and other innovations in mobile palliative care for unhoused and precariously-housed people with life-limiting illness."} +{"text": "To the editor,It is difficult to diagnose sepsis in critically ill patients admitted to the intensive care unit (ICU). A biomarker could help in sepsis identification and guide antibiotic use. A prognostic biomarker that identifies high-risk patients for the development of sepsis or nosocomial infections could help prevent those outcomes. However, current sepsis biomarkers often portray systemic inflammation and are unspecific to infection .The complement system is essential for defending against infections, yet it can also contribute to severe sepsis outcomes . A potenWe included 313 critically ill adult patients (defined as two or more systemic inflammatory response syndrome (SIRS) criteria upon admission) with an anticipated ICU stay of more than 24\u2009h and predictive role. Potentially, B2GPI can be helpful in diagnosing sepsis and stratifying ICU patients for infection risk."} +{"text": "The ubiquiotous nuclear protein HMGB1 is extracellularly released by dying cells or activated innate immunity cells to promote inflammation. Extracellular HMGB1 plays a prominent role in the pathogenesis of acute lung injury of infectious as well as sterile origin including hyperoxia. Excessive amounts of systemic HMGB1 and HMGB1-partner molecule complexes can be retained in the pulmonary circulation indicated by a substantial reduction of HMGB1 plasma levels in arterial versus venous blood. The cholinergic antiinflammatory mechanism ameliorates pulmonary inflammation by inhibiting HMGB1 release and HMGB1 receptor expression. This comprehension was recently reinforced by results reported in Molecular Medicine by Sitapara and coworkers demonstrating that administration of an \u03b17 nicotinic acetylcholine receptor agonist attenuated hyperoxia-induced acute inflammatory lung injury by alleviating the accumulation of HMGB1 in the airways and the circulation. Activating the cholinergic antiinflammatory path might be considered to alleviate severe COVID-19 with or without concurrent oxygen-induced lung injury. Excessive pulmonary inflammation causes acute lung injury (ALI) and acute respiratory distress syndrome (ARDS), two major problems with an unmet clinical need to control , and transported to the cellular endolysosomal system, which is disrupted by HMGB1 at high concentrations in the acidic intralysosomal environment . It is one of the most extensively studied DAMPs, present in high extracellular quantities and involved in the pathogenesis of many inflammatory diseases of infectious or sterile origin , hypoxia, hyperoxia, and barotrauma inflicted by mechanical ventilation (Kallet and Matthay What was then the rational for treating acute lung injury with an \u03b17nAChR agonist? The immune system is like all other organs controlled by the central nervous system. The vagus nerve-mediated inflammatory reflex with its efferent arm the cholinergic antiinflammatory pathway is so far the most extensively studied example of an immunoregulatory neural circuit (Chavan et al. Acetylcholine-mediated amelioration of inflammation may in addition be accomplished by electrical stimulation of the left vagus nerve (Chavan et al. The need for prolonged mechanical ventilation using high oxygen concentration is a major problem connected to the care for COVID-19 patients. The discovery by Sitapara and colleagues that hyperoxia-induced acute lung injury was ameliorated via \u03b17nAChR-agonist may offer important clinical progress if confirmed in future studies in patients. It should be noted that GTS-21 has been clinically tested in healthy adult volunteers and patients with schizophrenia demonstrating a favorable safety profile (Lewis et al."} +{"text": "In the article titled \u201cA Novel Method for Extraction of Galegine by Molecularly Imprinted Polymer (MIP) Technique Reinforced with Graphene Oxide and Its Evaluation Using Polarography\u201d , there wThe error was introduced during the production process of the article, and Hindawi apologises for causing this error in the article."} +{"text": "DNA damage occurs abundantly during normal cellular proliferation. This necessitates that cellular DNA damage response and checkpoint pathways monitor the cellular DNA damage load and that DNA damage signaling is quantitative. Yet, how DNA lesions are counted and converted into a quantitative response remains poorly understood. We have recently obtained insights into this question investigating DNA damage signaling elicited by single-stranded DNA (ssDNA). Intriguingly, our findings suggest that local and global DNA damage signaling react differentially to increasing amounts of DNA damage. In this mini-review, we will discuss these findings and put them into perspective of current knowledge on the DNA damage response. In order to ensure genome stability, cells need to detect DNA lesions such as DNA double-stranded breaks (DSBs) and signal their presence so that an appropriate cellular response is triggered marks sites of DNA damage and can be considered an upstream DNA damage signal that is recognized by proteins of the DNA damage checkpoint in order to transduce this upstream signal into a downstream checkpoint signal. While ssDNA is inherently generated during DNA replication and transcription, the presence of DNA lesions often triggers extensive ssDNA formation Zou . For exaWhat information does the amount of ssDNA in a cell hold? First, it gives information about the number of lesion sites, as many lesions will obviously expose more ssDNA than few. Second, it could potentially tell us about the persistence time of a given DNA lesion, at least in the case of DSBs (Pellicioli et al. How is the ssDNA signal read and translated into a downstream DNA damage checkpoint signal? Mechanistically, ssDNA in cells is bound by RPA Wold , which iThe concept of signaling thresholds separating different cellular responses can be observed already in the bacterial SOS response Michel . The SOSThe differentiation of signaling circuits brings up several interesting questions regarding the underlying molecular mechanisms. How is Mec1 kinase activity differentially channeled towards different Mec1 targets? How is the \u03b3H2A domain established efficiently with low levels of DNA damage-associated kinase? How are thresholds that are locally defined at every lesion site integrated over spatially distinct lesion sites?One fundamental difference between targets in the local and global circuits is their availability to phosphorylation by the Mec1 kinase. The global signaling cascade constituted by the 9-1-1 co-sensor, the Dpb11 and Rad9 scaffold proteins and the Rad53 effector kinase requires several steps of protein recruitment to the DNA lesion site, which itself is dependent on DNA end resection and Mec1 (Ira et al. Based on this model, the Mec1\u2013Ddc2 sensor module is critically involved in all ssDNA signaling circuits and likely involved in transducing the quantitative nature of the ssDNA signal. However, a second sensing mechanism might be required to confer the amount of ssDNA in the global signaling circuit. Currently, the best candidates for this second sensor are the 9-1-1 clamp and factors associated with it (Bantele et al. We therefore suggest that the cellular DNA damage response should not be viewed as a single pathway, but rather as separable signaling circuits. Different DNA damage scenarios trigger differential responses in these individual circuits. A quantitative understanding of the signal transduction in the individual circuits is therefore required to understand and manipulate cellular decision making upon DNA damage."} +{"text": "The cause-effect relationships between the various \u201challmarks of aging\u201d and chronic lung disease are not well understood. We have determined overlapping pathways involving deregulated nutrient sensing, mitochondrial dysfunction, and cellular senescence that may contribute to the evolution of chronic lung disease. In particular, I will discuss alterations in energy/metabolic sensing pathways and mitochondrial dysfunction as pathobiological mechanisms that may explain the age-related increased susceptibility to the development and progression of idiopathic pulmonary fibrosis (IPF), a disease of pulmonary aging. I will then broaden the discussion to include the potential role of these biologic alterations in other chronic lung disease which burden older adults."} +{"text": "Currently the term lamellar macular hole (LMH) alludes to a wide spectrum of macular conditions including distinct clinical entities with different pathomorphologies. Classifications into subtypes, tractional and degenerative or based on the associated preretinal tissue had been proposed. Recent insights suggest that only lesions with tissue loss should be considered \u2018true\u2019 LMH and not those morphological changes caused by tractional forces. Inclusion of lesions with foveoschisis with contractile epiretinal membrane (ERM) in earlier studies on LMHs has resulted in imprecise information about its clinical course. This review provides an overview of the evolving concepts of LMHs and analyses its natural history from study cases in previously published literature. There is currently no consensus about what constitutes \u2018lamellar macular hole\u2019 (LMH) and its definition. The term alludes to a wide spectrum of macular pathomorphologies. Gass in 1975 first used the term to describe complication of cystoid macular edema after cataract extraction proposedRecently Hubschman, Govetto and other members of an international panel of vitreoretinal experts proposed that only lesions with apparent tissue loss should be considered as LMHs and therefore presence of foveal cavity with undermined edges mandatory for its diagnosis along with irregular foveal contour and other signs of foveal tissue loss like thinning and pseudo-operculum . OptionaERM traction can cause foveoschisis and the appearance of an irregular foveal contour that can be confused with LMH . The patERM is seen more frequently than epiretinal proliferation and associated with a variety of clinical conditions. ERM appear as a whitish-gray translucent sheet on the retinal surface whereas epiretinal proliferation is typically not visible on ophthalmoscopy. On tomography ERM appear as highly reflective usually thin line whereas epiretinal proliferation is seen as usually thick homogenous material of medium reflectivity. On cursory examination the isoreflective epiretinal proliferation can be mistaken as part of the retina because of a thin hyperreflective line often covering it which is mistaken as ERM , 5, 10. Many studies have suggested that LMHs usually remain stable over time, very few evolving into FTMH , 6, 7, 9We present this case in which bilateral LMH with epiretinal proliferation progressed to FTMH in both eyes.A 72\u00a0year old male presented with increased blurring of vision in left eye since 1\u00a0week. He said he had blurred vision in both eyes since about a year. Best corrected visual acuity (BCVA) was 20/40 in right eye and 20/50 in left eye. Anterior examination showed pseudophakia both eyes, was otherwise unremarkable. Fundus examination showed FTMH in left eye, LMH in right eye with epimacular material in both eyes. OCT of left eye showed irregular margins of the macular hole with intraretinal edema. Epiretinal proliferation recognised as a homogeneous, isoreflective layer covered by a thin hyper-reflective line was seen at the edges of the hole contiguous with inner retina. Early ERM without tractional signs was also seen more centrifugally in their study group, there was successful macular hole closure after vitrectomy, ILM peel, intraocular tamponade and postoperative posturing. They observed that it is more difficult to start peeling LHEP than typical ERM describing it is more elastic and typically yellow . PeelingEntities referred to as \u2018tractional LMH\u2019 and \u2018LMH associated with tractional ERM\u2019 should be excluded from the definition of LMHs as they are clinically, morphologically and pathogenically distinct. The key feature of LMHs is foveal tissue loss evidenced on OCT as foveal cavity with undermined edges and central foveal thinning. They are usually associated with epiretinal proliferation. \u2018Mixed lesions\u2019 have both LMH and ERM. Analysing previous reports by excluding tractional entities from their study population show that true LMHs have a worse functional, morphological outcome and may have a propensity to progress to FTMH. Surgery may not be an appropriate therapeutic strategy. Prospective studies on the natural history of LMHs and evaluating the role of epiretinal proliferation in the progression to FTMH are warranted to improve management of these lesions."} +{"text": "Predicted climate change is widely cited to significantly reduce yields of the major cereal crop species in a period where demand is rapidly rising due to a growing global population. This requires exhaustive research to develop genetic resources in order to address the expected production deficiencies which will largely be driven by abiotic stress. Modification of multiple genes is an approach that can address the predicted challenges; however, it is time-consuming and costly to modify multiple genes simultaneously. Transcription factors represent a group of proteins regulating multiple genes simultaneously and are therefore promising targets to concurrently improve multiple traits concurrently, such as abiotic stress tolerance and grain size (a contributor to yield). Many studies have identified the complex role that transcription factors of multiple families have contributed toward abiotic stress tolerance or grain size, although research addressing both simultaneously is in its infancy despite its potential significance for cereal crop improvement. Here we discuss the potential role that transcription factors may contribute toward improving cereal crop productivity under adverse environmental conditions and offer research objectives that need to be addressed before the modification of transcription factors becomes routinely used to positively manipulate multiple target traits. In cereOsGLA1 confers a positive effect on grain length and weight driven by a single SNP influencing grain size have collectively been detected, yet only a small fraction of underlying candidate genes have been functionally annotated using advanced molecular approaches such as gene cloning with the majority of studies in rice . Broadlyngle SNP . Alterna pathway .Due to culinary preferences for different sized rice, the majority of grain size research has targeted this staple food crop, particularly the genetic engineering and functional analysis style research. cis-acting promoter elements of genes in complex regulatory networks treatment. Overexpression of TaNAC2-5A in Arabidopsis simultaneously improved drought, salinity, and freezing tolerance (TaNAC2-5A activity enhanced the expression of DREB2A and ABI5 transcription factors. It has been shown that stress-induced and constitutive overexpression of DREB2A in wheat and barley improved tolerance to drought and cold stress due to increased expression of late embryogenesis abundant (LEA) genes encoding dehydrins and cold-responsive proteins that contribute to membrane stability as well as other DREB family genes, further indicating the complexity of stress response and regulatory control for example, regulates cell elongation in the lemma/palea of rice by the heterodimerization of the two encoded proteins, regulating grain length (WIDE AND THICK GRAIN 1 (WTG1) in rice encodes an otubain-like protease involved in the ubiquitin\u2013proteasome pathway regulating grain size via cell expansion that is reportedly regulated by the transcription factors ABF1 and ABI5 (TaWTG1-7B) is highly correlated with the expression of ABF2 of the ABRE transcription factor family and D11 (positive regulator of grain length) in rice through the interaction of the mediator tail subunit of OsMAD15A with the two NAC transcription factors that promote the recruitment of additional transcriptional machinery to the promoters of OsNAC024/025 targets , project code UMU1903-003RSX.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "For older adults, a hospital stay can lead to loss of physical function and frailty. It is therefore important to investigate factors for recovery after hospitalization. Recent studies suggest negative self-perceptions of aging (SPA) as a potential risk factor in the context of serious health events. This ongoing longitudinal study investigates how negative SPA might contribute to worse physical recovery after hospital stay in a sample of 244 German adults aged 75 to 96. Preliminary mediation analysis based on available data of the first 50 participants indicate that negative SPA is related to increased fear of falling after 6 months, which predicts worse physical function one year after hospitalization . The results stress the importance of SPA for health recovery in old age and introduce fear of falling as a psychological pathway."} +{"text": "Fully adjustable articulators and pantographs record and reproduce individual mandibular movements. Although these instruments are accurate, they are operator-dependant and time-consuming. Pantographic recording is affected by inter and intra operator variability in the individuation of clinical reference points and afterwards in reading pantographic recording themselves. Finally only border movements can be reproduced.Bionic Jaw Motion system is based on two components: a jaw movement analyzer and a robotic device that accurately reproduces recorded movements. The jaw movement analyzer uses an optoelectronic motion system technology made of a high frequency filming camera that acquires 140frames per second and a custom designed software that recognizes and determines the relative distance at each point in time of markers with known geometries connected to each jaw. Circumferential modified retainers connect markers and do not cover any occlusal surfaces neither obstruct occlusion. The recording process takes 5 to 10\u2009s. Mandibular movement performance requires six degrees of freedom of movement, 3 rotations and 3 translations. Other robots are based on the so-called delta mechanics that use several parallel effectors to perform desired movements in order to decompose a complex trajectory into multiple more simple linear movements. However, each parallel effector introduces mechanical inter-component tolerances and mathematical transformations that are required to transform a recorded movement into the combination of movements to be performed by each effector. Bionic Jaw Motion Robot works differently, owing to three motors that perform translational movements and three other motors that perform rotations as a gyroscope. This configuration requires less mechanical components thus reducing mechanical tolerances and production costs. Both the jaw movement analyzer and the robot quantify the movement of the mandible as a rigid body with six degrees of freedom. This represents an additional advantage as no mathematical transformation is needed for the robot to reproduce recorded movements.Based on the described procedure, Bionic Jaw Motion provide accurate recording and reproduction of maxillomandibular relation in static and dynamic conditions.This robotic system represents an important advancement compared to available analogical and digital alternatives both in clinical and research contexts for cost reduction, precision and time saving opportunities. The first system that allowed to evaluate stone models statically at a given vertical dimension of occlusion (VDO) was described by Gariot in 1805 . Since tA substantial improvement regarding intra and inter operator agreement of recorded values was achieved with the introduction of the digital pantograph Denar Cadiax Compact (Teledyne Waterpik) and ArcuThe presented system is called Bionic Jaw Motion and it is composed of a Jaw movement analyzer and a robotic articulator. Since our study design is a report of a dental technique, no ethical approval was gained in accordance to EU regulations , 24. TheBJM differs from other digital robotic systems for acquisition and reproduction of mandibular movement because it is the first, at least to Authors\u2019 knowledge, that is capable to quickly record individual functional movement, analyze data, and reproduce it on a robot. Most published prototypes focus only or on movement recording or on robotic movement reproduction, usually using data arbitrarily inserted in a software and trying to reproduce them at best on a robot . The tecBJM quickly records and reproduces individual mandibular movements and overcomes many of the limitations of traditional pantograph-individual articulators systems. An intraoral reference system is adopted to avoid any possible mistake in clinical identification of extraoral landmarks whose univocal determination is nearly impossible. BJM also allows the recording of functional movement besides border movements.Additional file 1Additional file 2Additional file 3Additional file 4"} +{"text": "Middle East Respiratory Syndrome Corona Virus (MERSCoV) have been reported in Arabian peninsula and sporadic cases in Europe and Asia. This study was conducted to evaluate the genetic analysis of this virus in human and camel at the first time in Iraq. Two hundred samples were collected from camels and human who suffering from respiratory symptoms, these samples treated with RNA extraction kit then amplification the genetic material by PCR which give 5% positive results. The amplicon then sequenced, registration in gene bank of NCBI for getting accession numbers. The local strains give close relationship with neighbor countries as Saudi Arabia and Jordan strains when using MEGA analysis software."} +{"text": "Health benefits of marriage may stem in part from spouses discouraging unhealthy behavior and encouraging healthy practices. Although studies show spousal effects on health behaviors, few have assessed whether spousal effects vary by the quality of the marital relationship. Spouses in low-quality marriages may be less likely to engage in joint activities that promote health , make fewer attempts at monitoring their spouse\u2019s health behaviors, and be less successful in their attempts to intervene. Those in unhappy relationships may also use unhealthy behaviors as maladaptive coping strategies to deal with marital stress. We use dyadic data from couples over age 50 in the 2006 and 2008 waves of the Health and Retirement Study (HRS) to examine how both spouses\u2019 ratings of positive and negative dimensions of marital quality are associated with their own and their spouses\u2019 exercise and smoking . Using HLM software, we estimated actor-partner interdependence models (APIM). Results indicate that both own and husbands\u2019 ratings of positive marital quality are significantly associated with wives\u2019 odds of smoking. Own perceptions of negative marital quality and wives\u2019 perceptions of both positive and negative marital quality are associated with husbands\u2019 odds of smoking. For wives, neither own nor spousal marital quality is significantly related to exercise. For husbands, however, wives\u2019 higher positive marital quality and lower negative marital quality are associated with increased exercise. Strategies to improve marital quality may promote healthy behaviors among older adults, particularly for husbands."} +{"text": "These benefits are put into practice through an implementation of stability proofs for several examples in KeYmaera\u00a0X, a hybrid systems theorem prover based on dL.Stability is required for real world controlled systems as it ensures that those systems can tolerate small, real world perturbations around their desired operating states. This paper shows how stability for continuous systems modeled by ordinary differential equations (ODEs) can be formally verified in differential dynamic logic (dL). The key insight is to specify ODE stability by suitably nesting the dynamic modalities of dL with first-order logic quantifiers. Elucidating the logical structure of stability properties in this way has three key benefits:"} +{"text": "Many residents of Assisted Living (AL) confront serious illness and therefore might benefit from greater access to Palliative Care Services to improve quality of life. We surveyed resident records and AL nursing staff to identify patients in need of Palliative Care. Preliminary findings showed that nurses predicted 23% would not be alive and 49% would no longer live in AL. A majority of residents were over the age of 90, yet 30% did not have a reported code status. These findings suggest that a substantial portion of AL residents may have unmet needs with respect to palliative care. Future interventions are needed to support advance care planning conversations and make palliative care more accessible to this population."} +{"text": "Some studies have shown sensory impairment is associated with impaired cognitive test performance, however tests rely to varying degrees on hearing and vision. We hypothesized scores for cognitive tests whose administration depends on vision or hearing are biased among those with vision or hearing impairment, respectively, after controlling for underlying cognitive performance. We used item response theory methods to test for differential item functioning (DIF) by hearing and vision impairment in the Baltimore Longitudinal Study of Aging (BLSA) and the Atherosclerosis Risk in Communities study (ARIC). We identified DIF by hearing impairment for tests that do not rely on hearing and DIF by vision impairment for tests that do not rely on vision. We found no clinically meaningful differences between unadjusted and DIF-adjusted measures of cognition. Our finding that DIF by sensory domain was independent of administration modality suggests cognitive load may play a larger role than previously acknowledged."} +{"text": "Dear Editor,isolated arrhythmogenic left ventricular dysplasia, which, as they postulate, should be included separately in the differentiation between normal and abnormal imaging findings in athletes.We would like to thank Martin et alOur review was mainly focused on the summary and discussion of most common patterns of fibrosis observed in asymptomatic athletes described in the observational studies, including nonischemic insertion\u2010point fibrosis or small mid\u2010wall/subepicardial fibrosis and small ischemic\u2010type fibrosis (mostly in veteran athletes). Those patterns are usually found in athletes with otherwise normal hearts or presenting features of an athlete's heart defined as a physiological adaptation to physical exercise including balanced enlargement of heart chambers with or without mild left ventricular hypertrophy.isolated arrhythmogenic left ventricular dysplasia and for this reason, it is recently referred to as simply arrhythmogenic cardiomyopathy.In the last part of our review, we present how fibrosis detection and other cardiac magnetic resonance (CMR) markers can be used to distinguish between athlete's heart and pathology including arrhythmogenic right ventricular hypertrophy (ARVC). ARVC may include the cases of The authors declare no potential conflict of interest."} +{"text": "Personality can be an important resource as older couples cope with adverse life events. Analyzing 4,893 older couples from the Health and Retirement Study, this study examined how one\u2019s own and spouse\u2019s adverse life events occurring in the past two years are associated with changes in depressive symptoms. We further examined the moderating effects for this association of six dyadic personality profiles . We found significant actor and partner effects of health decline for increases in both spouses\u2019 depressive symptoms, and significant actor effects of a family death for husbands\u2019 increased depressive symptoms. For wives, having positive personality profiles buffered negative effects of one\u2019s own health decline and spouses\u2019 family death, whereas having negative profiles intensified negative effects of husbands\u2019 job exit and loss of wealth on the depressive symptoms for both spouses."} +{"text": "The growing international evidence on the impacts of social isolation and loneliness has profound implications for positive health status and wellbeing . This raises important questions about how we measure loneliness, particularly in extended family cultures where loneliness may be experienced differently from western more individualistic cultures. In this research, key questions around loneliness and social isolation were co-created with M\u0101ori Elders and responses were compared with a standard international loneliness scale to help identify universal aspects of loneliness and M\u0101ori specific aspects. The results demonstrated significant correlations between the co-created questions and the international scale. However, they also demonstrated substantial expressions of loneliness among older M\u0101ori that are not captured by standard scales showing the need for M\u0101ori specific scales which are more inclusive of their cultural norms and can provide more precise data for constructive policy making and service provision."} +{"text": "Voice First solutions offer the opportunity to promote social connectedness and independence. AARP Foundation has been successful in deploying Voice First devices in senior living and affordable housing communities and developing Voice First solutions through its Connect2Affect Connected Communities program. Connected Communities empowers older adults through voice first solutions to help them obtain the social support resources needed to stay socially connected, remain independent and age in place. AARP Foundation has developed three Voice First solutions 1) Community Hub, a voice application that encourages participation in group activities by voice-enabling a community\u2019s calendar of events and allowing older adults to discover, register and receive reminders for events, 2) A data collection voice application that administers the 10-item version of the Duke Social Support Index (DSSI) at regular intervals to assess an older adults\u2019 condition of isolation, 3) Social Check-in, a guided risk assessment for social isolation that helps older adult understand their individual risk factors."} +{"text": "Using smartphone sensing in real life, we examined conversational time travel , its functions and relation with positive affect . We used the Electronically Activated Recorder and collected a random sample of over 30,000 sound snippets (30 seconds long) from 61 young and 48 healthy older adults across four days. We transcribed and manually coded participants\u2019 speech. Multilevel models conducted in R showed that individuals tended to talk about their past with more social functions , whereas talked about their future for more directive purposes . Age group differences were minimal. We also found that individuals laughed two times more while talking about their past than their future. Results are discussed in relation to the functions of mental/conversational time travel in the context of healthy aging."} +{"text": "Family caregivers (FCs) of institutionalized noncommunicative older persons reported multiple unmet communication needs focusing on the need to receive reliable and regular updates on the patient\u2019s condition. We have developed a mobile app for improving communication between FCs and healthcare professionals (HPs), based on 152 interviews with FCs and 13 discussion groups with HPs from four Israeli geriatric facilities. Both parties participated in app planning, tailoring it to their needs and abilities. App use implementation encountered major obstacles including the bureaucratic process concerning signing contracts between the university and software development firms, which hindered the process for a full year; data security department required disproportionate security levels that interfered with user experience and delayed the development process; the study\u2019s definition varied across different ethics/Helsinki committees , which led to different demands, e.g., insurance for medical clinical trials although no drugs or medical device were involved; lack of cooperation by mid-level staff members despite the institutional adoption of the app project; low utilization by HPs resulted in FCs not receiving timely responses. Despite these and other obstacles, we tested app use for 15 months in one facility in a pre-post-design with intervention and control groups, and we have since begun testing it in another facility. FCs who had used the app had positive feedback and wished to continue using it. App use optimization requires implementation planning, assimilating changes in each facility\u2019s work procedures and HP\u2019s engagement and motivation and thus depends on institutional procedures and politics."} +{"text": "This report describes an isolated superior gluteal nerve injection injury following a corticosteroid injection for greater trochanteric pain syndrome. Ultrasound\u2010guided injections may be beneficial to target multiple pain\u2010producing regions of the hip while avoiding nerves and tendons. This report describes an isolated superior gluteal nerve injection injury following a corticosteroid injection for greater trochanteric pain syndrome. Ultrasound\u2010guided injections may be beneficial to target multiple pain\u2010producing regions of the hip while avoiding nerves and tendons. This report describes the clinical presentation of a 64\u2010year\u2010old female patient with an isolated right superior gluteal nerve injury following a landmark\u2010guided corticosteroid injection of the trochanteric bursae.Greater trochanteric pain syndrome (GTPS) presents as pain over the superior lateral region of the hip and tenderness to palpation over the greater trochanter. This term is considered preferable to greater trochanteric bursitis because there may not be signs of inflammation where the reported pain is located and pain may be related to numerus surrounding structures.Conservative treatments for GTPS include activity modification or rest, nonsteroidal anti\u2010inflammatory medications, physical therapy, and corticosteroid injections.Musculoskeletal injections are associated with potential complications. While complications are relatively rare, they may include allergic reactions, bleeding, infection, tendon rupture, or nerve injury.22 presented to clinic for evaluation of her right hip and leg after developing weakness following multiple greater trochanteric bursa injections by an orthopedic surgeon at an outside institution. She reported that right\u2010sided hip pain started 6\u00a0months prior after prolonged sitting on a two week bus tour. Pain was provoked during periods of standing. She worked as a floor nurse, where pain severely limited her ability to complete her tasks. She was evaluated by the surgeon and diagnosed with right hip greater trochanteric bursitis. The patient provided the history that the orthopedic surgeon injected corticosteroid into the trochanteric bursae using anatomic landmarks. She reported that her symptoms temporarily improved with the injection but reoccurred. She stated that she continued to receive landmark\u2010guided corticosteroid injections from this provider every 4\u20106\u00a0weeks. On the sixth landmark\u2010guided corticosteroid injection, she explained that the surgeon fanned out the injection from a single site in an effort to disperse the steroid over a larger area. The patient reported immediate pain and discomfort in the injection location. Within one week of the repeat injection, she experienced severe weakness of the right proximal leg. She was evaluated by a neurologist and had an electromyography (EMG) study performed. The EMG sampling included the right tibialis anterior but did not include the right gluteus medius; EMG results were unremarkable.A 64\u2010year\u2010old woman with no comorbidities and a body mass index of 24\u00a0kg/mUpon physical examination at our clinic, the patient had normal muscle bulk in the thighs and calves bilaterally. Muscle strength was normal, with the exception of 3/5 Medical Research Council (MRC) score for right hip abduction, compared to 5/5 on the left. Ankle dorsiflexion was 5/5 bilaterally. Gait evaluation was notable for a markedly positive right Trendelenburg sign. Magnetic resonance imaging (MRI) 6\u00a0months after the final injection showed diffuse fatty infiltration and gross muscle atrophy in the right gluteus medius and minimus compared to the contralateral side Figure\u00a0. We concFifteen years after the injury, she remains very active by working with a physical trainer multiple times per week. She has developed compensatory mechanisms to account for the atrophied muscles. She still struggles some with balance and isolated hip abduction is still weaker on the right compared to left side.3The report describes a patient that developed symptoms and clinical findings consistent with a superior gluteal nerve injection injury following an anatomic landmark\u2010guided injection for GTPS. Clinical correlations of the physical examination, MRI, and EMG are used to diagnose nerve injuries. The gluteus medius is not normally examined in a standard lower extremity EMG, which is why the nerve injury may have been initially missed. However, upon MRI examination, extensive fatty infiltration of the right gluteus medius and gluteus minimus was noted. These findings are consistent with chronic muscle denervation.Isolated superior gluteal nerve injuries have been rarely described,If there is clinical concern of multiple pain\u2010producing regions surrounding the hip, it may be beneficial to use ultrasound guidance during injections to avoid damaging anatomical structures and to confirm injection locations. Ultrasound\u2010guided injections can provide more precise injections into the greater trochanteric bursa as well as the subgluteus medius bursa.This report is limited by the fact that many of the initial diagnostic and treatment details are patient reported since the initial evaluation and injections were performed elsewhere and not available for our review. However, this report is applicable to clinical practice as some patients present to clinic without prior medical records and the evaluation and management is dependent on their reporting. Additionally, this information would not change the diagnosis of the superior gluteal nerve injection injury nor our conclusion that ultrasound\u2010guided injection may have prevented nerve injury by allowing the visualization of the needle during the injection administration.Isolated superior gluteal nerve injection injuries may occur with injections for greater trochanteric pain syndrome. Ultrasound\u2010guided injections may reduce superior gluteal nerve injection injuries by confirming injection locations and visualizing the needle throughout injection administration. However, when these injection injuries do occur, careful clinical correlation of physical, electrodiagnostic, and imaging findings should be used when evaluating patients that report lateral hip pain with abductor weakness following musculoskeletal injections.None declared.AT: conducted the literature search on the topic and drafted the initial version of the manuscript. EE: collected clinical information and provided critical revision of the manuscript for intellectual content."} +{"text": "An 81\u2010year\u2010old male with a history of systolic heart failure due to an underlying ischemic cardiomyopathy with a left ventricular ejection fraction of 13% and QRS duration of 130\u2009ms had undergone an uncomplicated cardiac resynchronization therapy defibrillator implantation , RA lead (Biotronik Safio S53) and RV shocklead (Biotronik Linox Smart S65 ProMRI) in 2015. During follow\u2010up, T\u2010wave oversensing (TWOS) was noticed. To avoid issues related to TWOS, the bradycardia sensitivity was adjusted successfully and TWOS was not seen afterwards. In 2020 the patient was evaluated on the implantable cardioverter defibrillator (ICD)\u2010outpatient clinic because of (near\u2010)syncope. Recent home monitoring reported non\u2010sustained oversensing (NSRVOS) in the VT2 zone (Near\u2010)Syncope caused by asystole/bradycardia due to inhibition of pacing.(2)Oversensing induced inappropriate tachy therapy or even shocks.The main concerns that remained were:The chosen solution was programming separate levels of sensitivity for the detection of bradycardia and tachycardia providing different responses to the same noise signal. The sensitivity threshold for bradycardia detection was elevated to a level above the amplitude of the recurrent noise signal . This significantly reduces the risk inappropriate inhibition of ventricular pacing resulting in asystole and danger of fainting. In addition, the sensitivity threshold for tachycardia detection was kept below the amplitude of the noise signal. By doing so the noise could still be monitored for change in amplitude or duration. And raising the sensitivity threshold for detecting tachycardia would also have introduced the danger of undersensing ventricular arrhythmias, especially ventricular fibrillation.In this particular case, the SecureSense algorithm was successful in distinguishing noise from true high rate ventricular episodes and thereby provided the ability to withhold tachycardia therapy in the presence of the recurrent interference signal if it were to last longer. The SecureSense algorithm is designed reduce inappropriate therapies by distinguishing noise from true VT/VF episodes and provides the ability to automatically withhold tachycardia therapy in the presence of lead noise.To further reduce the risk of inappropriate shocks the incount for ventricular tachycardia could be increased to 50 intervals. Although, increasing the incount in the VT2 zone does reduce the risk of inappropriate therapy, it could result in failure to treat life\u2010threatening arrhythmias due to undersensing during polymorphic VT/VF.3Recurrent noise with a certain stable signal amplitude can be dealt with by programming different levels of sensitivity for tachycardia sensing and bradycardia sensing. This could result in a remarkable device interrogation with both a ventricular sense and ventricular pace marked at the same moment. Furthermore, this separate interpretation by the ICD of the same signal can solve the interference by external noise."} +{"text": "Family caregivers of individuals living with Alzheimer\u2019s disease or related dementias (ADRDs) are exposed to unique stressors that put them at risk for depression and suicidal ideation. To date, little is known about contextual factors surrounding suicidal ideation among ADRD family caregivers. We investigated individual caregiver characteristics and daily environmental stressors associated with daily suicidal ideation using a micro-longitudinal design and ecological momentary assessment methods. Data were collected from a national sample of family caregivers (N=51) who completed daily diaries over 21 days (n=911). Suicidal ideation was endorsed on forty-seven days (5.16%) during the sampling period, with 11 participants (22%) endorsing suicidal ideation at least once. Suicidal ideation did not differ based on the caregiver\u2019s age and relationship to the care-recipient (spouse or child). Participants with a history of mild depression and anxiety endorsed more days with suicidal ideation. Finally, family caregivers were more likely to endorse suicidal ideation on a day when more than one type of BSD was reported and when BSDs were perceived as more bothersome than average . In this investigation, we identified descriptive and predictive factors that will inform the development of targeted interventions for ADRD caregivers at high risk of suicidal ideation."} +{"text": "Stress is an important correlate of cognitive aging that manifests in everyday life. Infrequent trait-based stress measures may not be as sensitive to mild cognitive impairment (MCI) as ecological momentary assessments (EMA). We compared EMA to global trait-based stress measures in discriminating MCI. A sample of 248 adults from the Einstein Aging Study were prompted to report whether a stressor occurred and to rate the severity up to four times daily for 14 days. Global perceived stress and neuroticism were assessed at baseline. Although MCI status was unrelated to stressor frequency (p>.05), individuals with MCI appraised their daily stressors as more severe than cognitively intact participants (p=.03). No MCI-related differences emerged on global stress or neuroticism assessments (ps>.05). Results suggest everyday stress markers may be more sensitive to differentiating MCI than global assessments and point toward their utility for early identification of pathological declines."} +{"text": "A pressure injury/ulcer (PrI) is a localized area of injured skin and tissue usually over a bony prominence and one of the highest priority problems identified in U.S. health care\u2019s federal quality initiatives; approximately 26.8 billion is spent for treatment each year. The problem is accentuated for nursing home residents who are often immobile/bedridden. Currently, resident repositioning/movement by nursing staff every 2-hours is the cornerstone of prevention care. Successful interventions must be nurse-led and designed to facilitate the prevention care of nursing staff on the front lines. My research focuses on integrating movement into everyday care for institutionalized older adults and is advancing the science of PrI prevention through testing of cueing interventions for nursing staff to improve the care delivery. My goals for innovating preventive care include enhancing our understanding of nursing subcultures\u2019 influence on care outcomes and leveraging emerging technology to enhance the care team\u2019s collaborative efforts."} +{"text": "To our knowledge, the exposed nerve roots in thoracic spine are usually sacrificed to facilitate osteotomy during posterior vertebral column resection (PVCR) for severe spinal deformity. Currently we report a case with severe spine deformity in which intraoperative neurological monitoring (IOM) loss after interrupting T8 nerve root finally led to spinal cord injury during PVCR surgery.The patient was a 14-year-old female with severe congenital kyphoscoliosis (CKS) without preoperative neurologic deficits. The IOM events (MEP loss and SSEP latency prolong) were showed when T8 nerve root at concave side was interrupted. And then we reduce the scope of osteotomy to control bleeding, raised blood pressure to increase blood supply for spinal cord, placed the bilateral rod to stabilized the spinal cord, used the methylprednisolone, explored the presence or absence of spinal cord compression, and prepared to change the surgical plan from PVCR to PSO. After that the IOM signals partial recovered from the lowest point. Postoperatively the patients showed transient motor function deficits of left lower limbs weak without somatosensory deficits, and come back to preoperative status 6\u2009months later.Interrupting the thoracic spine nerve root is danger to trigger the spinal cord injury during PVCR procedure of severe CKS. That probably because the increasing tension of contralateral anterior horn area of spinal cord via the nerve root pulling. Posterior vertebral column resection (PVCR) has become a useful technique for treatment of severe and rigid spinal deformity; meanwhile it is one of biggest spinal surgeries and at higher risk of excessive blood loss and neurological deficits \u20134. When The patient included in this study provided written informed consent in accordance with the principles of the Declaration of Helsinki and the study was approved by Ethics Committee. And all methods were carried out in accordance with relevant guidelines and regulations.A 14-year-old female with severe congenital kyphoscoliosis (CKS) without preoperative neurologic deficits Fig.\u00a0, and theAnd then we reduced the scope of osteotomy to control bleeding, raised blood pressure to increase blood supply for spinal cord, placed the bilateral rod to stabilized the spinal cord, used the methylprednisolone, explored the presence or absence of spinal cord compression, and prepared to change the surgical plan from PVCR to pedicle subtraction osteotomy (PSO). After those interventions the IOM signals partial recovered from the lowest point.Postoperatively the patients showed transient motor function deficit of left lower limb weak without somatosensory deficits, and then came back gradually to preoperative status 6\u2009months later Table\u00a0.Table 1In this case, the IOM events were happened 8\u2009min later after T8 nerve root interruption. So, the unresponsive IOM alerts during surgery were difficult to find the point to point reason, fortunately the surgeon soon recognized that the nerve root interruption during osteotomy will potentially increase the tension from the other side on the ventral horn of spinal cord. And then we rapidly reduced the scope of osteotomy to control bleeding, raised blood pressure MAP, 65\u201380) to increase blood supply for spinal cord 5\u201380 to i\u20139.According to many previous reports and our experience, among thoracic spine PVCR procedure interruption of several intercostal vessels and nerve roots was usually inevitable and low spinal cord injury risk \u201312. HoweThis case suggested that during patients with severe scoliosis originally the spinal cord tension at the apex was relatively high, and suddenly interrupting the thoracic spine nerve root on concave side was danger to trigger the stretching force increase on the other side of spinal cord by nerve root drag. So, we mightily agreed with the experience from Lenke et al. that temporary vascular clamps should be placed on the root sleeve for several minutes while paying closely attention to the IOM signal to make certain no changes in the potentials are seen before ligation .There are also some points should be noted to this case. On one hand, we did not record the preoperative IOM baseline especially SEP data. Although we performed clinical nervous system physical examination in detail, some hidden electrophysiological level abnormalities were still difficult to be found. So, the evaluation of preoperative spinal cord injury risk was not comprehensive enough. On the other hand, postoperative immediately imaging data especially MRI were absent for this case, which created that ischemic spinal cord injury could not be ruled out completely. According to our experience, the T4-T9 spinal cord segment was most likely to occurring cord ischemia on account of interrupting nerve root accompanying vessels. But the following 2 diagnostic evidence probably can help to prove our hypothesis.On one hand, we strictly followed up the patient\u2019s neurological function changes and found that was gradually recovery post operation. The patient could try to get out of bed and walk 1 month after surgery, and then the neurological function came back to preoperative status completely 6 months post operation. According to the neurological function\u2019s recovery the spinal cord injury was more like to mechanical injury of nerve pulling rather than ischemic injury. Because an ischemic insult can be more likely to lead to longer lasting or permanent injury than mechanical injury to the spinal cord \u201315.On the other hand, even more importantly, if the spinal cord injury is due to ischemic or other factors, both of the IOM data and postoperative neurological symptom should be happening in the ipsilateral rather than contralateral side. Nevertheless, according to the IOM data and postoperative neurological symptom, the side of spinal cord injury was on the convex side (left), which exactly adhere to our hypothesis that the neural jury is probably because increasing tension of contralateral nerve root.Interrupting the thoracic spine nerve root is danger to trigger the spinal cord injury during PVCR procedure of severe CKS. That probably because the increasing tension of contralateral anterior horn area of spinal cord via the nerve root pulling."} +{"text": "The refusal of Jehovah's Witnesses to use blood products can limit access to cardiac surgery, as patients may not be offered surgery for complex disease, especially revision surgery. We report a successful, complex adult congenital heart disease (ACHD) surgery with intraoperative and perioperative optimization. We have tried to highlight through this case that complex ACHD surgeries can be performed in\u00a0Jehovah's Witness patients with skilled perioperative and intraoperative management. The role of bovine hemoglobin in this population is being defined and was found helpful in this case. The refusal of Jehovah's Witnesses to use blood products can limit access to cardiac surgery. Usually, robust perioperative and intraoperative optimization is required in order to make the surgery successful . CardiacA 43-year-old adult Jehovah's Witness with tetralogy of Fallot and velocardiofacial syndrome had her first congenital heart surgery at the age of nine years, delayed due to blood product restriction. She presented with dyspnea and frequent palpitations. Electrocardiogram (EKG) was done, which revealed a wide QRS complex with right bundle branch block (RBBB) morphology Figure .Transthoracic echocardiogram was consistent with progressive right ventricular dilation dysfunction and normal left ventricular size and function. Magnetic resonance imaging (MRI) was performed for further elaboration of the right ventricle (RV) and the pulmonary arteries. It revealed a dilated and dysfunctional RV, pulmonic valve insufficiency with dilated main pulmonary artery,\u00a0focal stenosis of the left main pulmonary artery, and adhesions of heart to the chest wall Figures -3.Surgical options included hybrid pulmonary valve repair (PVR) with a pulmonary artery (PA) band and a catheter-based stent valve versus surgical replacement. Chest anatomy and adhesions elaborated by imaging eventually made us decide on surgical pulmonary valve replacement (PVR). Pre and postoperative erythropoietin were given. Congenital heart surgeons used cell saver technology, colloids, and bovine hemoglobin. The patient had a surgical PVR and bovine pericardial patch augmentation of the left pulmonary artery, which also secured the valve anteriorly. Time on cardiac bypass was minimized and heparin used intraoperatively was reversed using protamine.Patients who are Jehovah's Witnesses pose difficult ethical and moral dilemmas for surgeons because of their refusal to receive blood and blood products.\u00a0The reluctance to use whole blood and its main fractions has spurred the development of perioperative and intraoperative protocols to improve 'patients' outcomes particularly after complex surgeries including cardiothoracic surgeries ,4.\u00a0ProduPreoperative optimization of patients who are Jehovah's Witnesses may be achieved using intravenous iron infusions and erythropoietin-stimulating agents to augment erythropoiesis as was done in our patient\u00a0. ErythroA study comparing the outcomes of 31 Jehovah's Witness patients with a similar control group undergoing major cardiac surgeries showed similar results in terms of hospital stay and mortality in centers that practiced a rigorous blood product management protocol . A debriComplex ACHD surgery can be done in Jehovah's Witness patients with skilled perioperative and intraoperative management. The role of bovine hemoglobin in this population is being defined and was found helpful in this case. Here, we describe a successful pulmonary valve replacement (PVR) and left pulmonary artery (PA) augmentation in an adult Jehovah's Witness patient with prior tetralogy of Fallot repair and velocardiofacial syndrome. A multimodal strategy to optimize the intraoperative and perioperative blood loss usually makes complex surgeries, including complex cardiac surgeries in adult Jehovah's Witness patients, possible with favorable outcomes."} +{"text": "Neal Patel and colleagues from New York presented an analysis of SPARCS database for men who underwent inflatable penile prosthesis and/or artificial urinary sphincter insertion between 2000 and 2014.Compared with men who received a penile prosthesis alone those with a penile prosthesis and an artificial urinary sphincter (not necessarily done at the same surgery) had a higher likelihood of undergoing inflatable penile prosthesis reoperation at 1 year and 3 years .The authors concluded that combined inflatable penile prosthesis and artificial urinary sphincter insertion portends a higher likelihood of inflatable penile prosthesis reoperation at 1 and 3 years. However, artificial urinary sphincter outcomes remain comparable.These data are in opposition to 2013 publication on Journal of Urology by Dr. Segal and cols. ( One important limitation of all cited articles is the lacking on data concerning factors that could influence outcomes in prosthetic surgery such as surgeon volume, operative time, or antibiotic selection. And also the use of reoperation as a surrogate for device survival, since someone may elect not to undergo revision surgery despite mechanical failure.The decision to treat a man with DI or staged procedure is impacted by many factors including safety and durability, but patient preference and costs are essentials issues. A small retrospective study compare"} +{"text": "Diagnosis was made by nasoendoscopy and the infant underwent successful treatment by marsupialization via coblation technique. Images and videos were taken during the procedure both pre and post-operatively. This case highlights the need for an interdisciplinary evaluation of persistent neonatal stridor in newborn infants for early diagnosis and intervention to avoid critical airway obstruction and potentially fatal outcomes, DOI: Specifications table\u2022Congenital vallecular cyst is a rare cause of neonatal stridor, which could be fatal unless appropriate evaluation and intervention are done early.\u2022Increasing awareness of this rare congenital anomaly among the neonatal fraternity can facilitate early involvement of otolaryngologist, allowing quick assessment and required intervention.\u2022Pre- and post-intervention images and video of the vallecular cyst give a clear illustration of this congenital anomaly and the newer modality of surgical treatment for trainees in otolaryngology and pediatrics.1The data was collected from a term female newborn infant who presented with neonatal stridor. The infant presented with respiratory distress at 12 hours of life and required admission to neonatal intensive care unit for continuous positive airway pressure support. On day 2 of life, the respiratory distress persisted but the chest x-ray did not reveal any significant lung disease. Hence, an upper airway evaluation was undertaken by the otolaryngologists. Flexible nasoendoscopy showed a cystic mass at the vallecula abutting the epiglottis . An urge2A Parsons laryngoscope size 8 was used together with a Storz xenon light source for the laryngoscopy. A rigid 4mm telescope was mounted with Stryker endoscopic camera . Images and videos were captured with Stryker video tower. After confirmation of the diagnosis, a Lindholm laryngoscope was suspended and microscope was used during surgery. Raw images and videos were obtained."} +{"text": "We herein report a case of lung cancer with recurrent superior vena cava (SVC) syndrome, which was treated with additional stent placement. Our report suggests the possibility that additional SVC stent placement is an option for treatment of tumour ingrowth, even in patients with poor performance status. This is a case of lung cancer with recurrent superior vena cava (SVC) syndrome, which was treated with additional stent placement. A 78\u2010year\u2010old woman had previously received chemoradiotherapy for lung squamous cell carcinoma. She was hospitalized with dyspnoea and oedema of the upper body. Chest computed tomography (CT) Fig. revealedAppropriate written informed consent was obtained for publication of this case report and accompanying images."} +{"text": "Studies suggest spending more time interacting with and talking to others is associated with better well-being. Older adults with partners may spend more time with their romantic partners and rely on them for support, whereas older adults without partners may have a greater reliance on other family members and non-kin . Yet, we know little about how older adults\u2019 relationship status affects their time spending alone or with other social partners, and the frequency of conversation throughout the day. Adults aged 65+ (N = 313) completed an interview about their relationship status and social partners. They then reported social encounters in ecological momentary assessments every 3 hours for 5 to 6 days. Participants also wore Electronically Activated Recorders which captured snippets of their conversation throughout the day. Older adults with partners reported 85% of time was with their romantic partners. Multilevel models revealed that compared to older adults with partners, older adults without partners were more likely to spend time alone and have encounters with friends throughout the day. Older adults without partners also engaged in fewer conversations throughout the day. Further, older adults without partners talked significantly more when they encountered friends than did older adults with partners. Findings suggest that friends are important in older adults\u2019 social networks particularly for those who do not have romantic partners. Daily contact with social partners facilitates conversations and that could have implications for emotional or cognitive health."} +{"text": "We read with great interest the article by Jakeman et al, \u201cAddressing Latent Tuberculosis Infection Treatment Through a Collaborative Care Model With Community Pharmacies and a Health Department,\u201d which addressed latent tuberculosis infection (LTBI) treatment through a collaborative approach in New Mexico . The autThe study by Lal et al also found that having at least 4 visits with a health care provider increased the odds of completing treatment and that the time spent during the initial visit was an important factor in completing treatment . We suggWe congratulate the authors on their work in addressing LTBI treatment. Collaborative working relationships between community pharmacies and physicians have improved patient care in many communities . Moving"} +{"text": "Psychiatric disorders pose a unique risk for Alzheimer\u2019s disease (AD). Prior research indicates psychiatric disorders in MCI increase AD vulnerability. Less research has been done to understand how psychiatric disorders may affect the development of MCI. Understanding these potentially modifiable risk factors is important as they may represent a potential target of intervention for secondary prevention of AD. The present study examines the relationship between psychiatric disorders and amnestic MCI (aMCI) in a sample of Veterans with subjective memory complaints. The sample included 150 older adults with subjective memory complaints . aMCI diagnosis was based upon performance on the delayed recall trials of the Rey Auditory Verbal Learning Test and Logical Memory II of the Wechsler Memory Scale-4th edition. Psychiatric disorders were assessed using the Mini Neuropsychiatric Interview for DSM-IV. Logistic regression modeling demonstrated that diagnosis of anxiety disorders, but not mood or substance use disorders, was significantly associated with aMCI status. Specifically, older adults with an anxiety disorder were less likely to have aMCI than those older adults without an anxiety disorder. Additional analyses revealed that within those with aMCI (n=107), persons with a psychiatric disorder were significantly younger than those without a psychiatric disorder by an average of 6 years. These findings support prior research on the complex relationship of anxiety and cognitive impairment as well as suggest that those with psychiatric disorders may be at risk for developing aMCI at younger ages."} +{"text": "This dataset is intended to improve biomass and carbon estimates of forests in the northern ecotone region of Brazilian Amazonia, an area poorly known in terms of ecosystem dynamics.Wood density is expressed by the ratio between dry weight and fresh volume of a sample piece. The value of this measure is an important variable for assessing wood functional properties, successional stages and biomass/carbon stock estimates in different terrestrial ecosystems. Wood density data were collected for tree species from ecotone forests of the northern Brazilian Amazonia. We sampled 680 individuals with stem diameter \u226510\u202fcm. For each sampled individual measurements were taken for three stem variables: bark thickness (mm), bark density (g cm All samples to estimate the wood density of the different tree species occurring in the ecotone forests on eastern of Marac\u00e1 Island were obtained from a systematic sampling of 129 plots dispersed throughout the PPBio grid. These plots were intentionally established with small dimensions and with short between-plot distances to obtain high spatial resolution, and so better capture the microvariations in structural and species composition present across the island's altitudinal gradient; which defines the distinct hydro-edaphic conditions under which the different forest types of Marac\u00e1 Island occur. The minimum distance between the plots was 150\u202fm, based on the distance-markers located every 50\u202fm along the PPBio grid trails; all sampling plots are georeferenced in UTM and with topographically defined altitudes. All data and metadata related to trail topography is available on the official PPBio website Field collection and construction of the current Dataset were derived from an existing forest inventory"} +{"text": "This presentation features how 3D Team nurse practitioners (NP) use results of clinical assessments to determine whether older adults and caregivers enrolled in the study are referred to other Team members; these assessment results are called \u201cclinical triggers\u201d. Other team members who receive referrals based on NP-generated clinical triggers include: Licensed Clinical Social Workers, who deliver Problem Solving Therapy to older adults with significant depressive symptoms; Occupational Therapists, who deliver an evidence-based dementia care intervention; Physical Therapists, who deliver an adapted Otago exercise program; Registered Dietician, who provides nutrition and dietary instruction; and Community Health Educator, who provides community resource information to address social determinants of health. All clinical triggers will be detailed in this presentation, along with a description of each intervention delivered by other team members except the Community Health Educator. Case studies will be presented to illustrate how study participants receive multiple interventions from the 3D Team."} +{"text": "A presumed abnormal electrocardiogram (ECG) was obtained from an asymptomatic patient with a pacemaker. Systematic evaluation of the ECG revealed that the artifact was due to a physiological sensor in the pacemaker which was displayed when the enhanced pacemaker detection features on the ECG machine was activated. The article discusses the possible causes and an approach to similar artifacts. A presumed abnormal electrocardiogram (ECG) was obtained from an asymptomatic patient with a pacemaker. Systematic evaluation of the ECG revealed that the artifact was due to a physiological sensor in the pacemaker which was displayed when the enhanced pacemaker detection features on the ECG machine was activated. The article discusses the possible causes and an approach to similar artifacts. Tremor artifacts are continuous, low amplitude and rhythmic and thus not the cause of the observed artifact. While cardiac myoplasty stimulation may result in artifact associated with each QRS complex, one would not expect prolonged consistent high frequency signals with each QRS, nor would one expect a normal QRS morphology in this rare situation.Normal pacemaker function may produce ECG artifact related to physiologic sensors utilizing sub\u2010threshold pacing to measure intra\u2010cardiac and intra\u2010thoracic impedance. One such example is the closed loop stimulation sensor unique to Biotronik (Biotronik GmbH) pacemakers. This algorithm performs eight biphasic impedance measurements (obtained with 17 sub\u2010threshold paced beats) 50\u2010300\u00a0milliseconds after a ventricular event to determine intracavitary impedance to guide physiologic rate response.Further history confirmed the presence of a Biotronik pacemaker with activation of the closed looped stimulation algorithm. Additionally, interrogation of the ECG machine revealed utilization of the enhanced pacemaker detection feature during ECG acquisition. A repeat ECG after deactivation of this feature resulted in the absence of artifact.3This case highlights the value of a systematic approach to interpreting ECG artifacts and knowledge of the influence of enhanced pacemaker detection features on ECGs.Mr Jordan\u2010Watt is an employee of Biotronik, Canada. Drs. Kumar, Morgan and Singh have no relevant conflict of inflict to disclose."} +{"text": "In July 2020, the European Commission announced its \u20ac750 billion package to revive the postpandemic European economy, Next Generation EU. The programme comprises a number of loans and grants that will be funded by taking out European debt. Although the rules on liability sharing for Next Generation EU prevent a significant mutualisation of the debt, European leaders have taken the long-recognised significant first step towards European financial and political unification that stands in stark contrast to the misguided austerity programmes during the European sovereign debt crisis."} +{"text": "This case report demonstrates that atrioventricular and ventricular atrial conduction at rest may be unreliable in assessing the presence of reentrant atrioventricular nodal tachycardia. This case report demonstrates that atrioventricular and ventricular atrial conduction at rest may be unreliable in assessing the presence of reentrant atrioventricular nodal tachycardia. It was On the base of the tracing, a presumptive diagnosis of typical slow\u2010fast atrioventricular nodal tachycardia was done.Medical treatment consisted of sertraline and omeprazole.An electrophysiological study was performed. Para\u2010hissian ventricular pacing at baseline revealed absolutely no retrograde conduction Figure\u00a0. Atrial Atrial pacing induced a double ventricular response, one through the fast nodal anterograde pathway and one through the anterograde slow pathway followed by one retrograde fast pathway echo and a second ventricular echo with retrograde block in the fast pathway Figure\u00a0. A typicThe atrial activation during tachycardia was identical as the one during ventricular pacing when retrograde conduction was present, and it was not possible to advance the atrial electrogram when a ventricular pacing impulse was delivered during the refractory period of the His confirming the mechanism of the tachycardia. Five cryoablation lesions were delivered on the triangle of Koch on sites with an A/V ratio <1 and where rapid potentials suggesting the presence of the slow pathway were present. These lesions were delivered at the level of the upper part of the coronary sinus os and up to midway between the His recording and the coronary sinus os.This treatment abolished the conduction through the slow pathway with disappearance of the double response and also of the isolated nodal echoes induced during ventricular pacing on isoprenaline.After isoprenaline was stopped, all retrograde conduction disappeared again.There was no arrhythmia recurrence after a follow\u2010up of 2\u00a0years.2Atrioventricular nodal reentrant tachycardia is typically induced with anterograde block over the fast pathway and conduction over the slow pathway with subsequent retrograde conduction over the fast pathway.Complete absence of retrograde conduction at baseline is infrequent in the setting of reentrant atrioventricular nodal tachycardia. Complete absence of retrograde conduction at the baseline usually favors the diagnosis of atrial tachycardia.In previously described cases, the retrograde conduction can be absent during tachycardiaDual ventricular response during premature atrial stimulation inducing reentrant atrioventricular nodal tachycardia is another infrequent finding.It means that the fast anterograde pathway does not need to block anterogradely in order to trigger tachycardia. Very slow anterograde conduction through the slow nodal pathway finds the retrograde limb of the fast pathway excitable triggering slow\u2010fast reentry. During anterograde fast pathway conduction, a retrograde block is likely to be present on the slow pathway preventing retrograde concealed conduction at that level. The two observations, dual anterograde response and absence of retrograde conduction at baseline, are probably linked: bad retrograde conduction through the slow pathway is likely to be a prerequisite for dual response.Hence, complete absence of retrograde conduction and/or no anterograde jump at rest does not rule out the diagnosis of atrioventricular nodal reentrant tachycardia. In fact, atrioventricular and ventriculoatrial conduction at rest is unreliable in assessing the presence of reentrant atrioventricular nodal tachycardia.Isoprenaline infusion at increasing dosage should be used in these cases especially if tachycardia only occurs during exercise as was the case in this patient. Occurrence of the tachycardia during exercise also explains why the spontaneous tachycardia rate was also 42\u00a0bpm higher than the induced tachycardia.The dual response demonstrates that absence of anterograde fast pathway block during premature atrial stimulation does not rule out atrioventricular nodal tachycardia.Isoprenaline infusion allowed the tachycardia to be induced. Care should be taken when a high dosage is used because many side effects may occur.The situation described in this case report is different than the so\u2010called nonreentrant form of tachycardia due to dual ventricular response in sinus rhythm3This patient with typical atrioventricular slow\u2010fast reentrant nodal tachycardia showed a combination of electrophysiological features making it a very uncommon case: absence of baseline retrograde conduction and induction of tachycardia after a dual anterograde nodal response.This case report demonstrates that atrioventricular and ventricular atrial conduction at rest may be unreliable in assessing the presence of reentrant atrioventricular nodal tachycardia.None declared.TV: served as main electrophysiologist. JC: served as main electrophysiologist. TN, AA and MJ: served as attending electrophysiologists."} +{"text": "The 2019 novel coronavirus disease emerged in China in late 2019\u2013early 2020 and spread rapidly. China has been implementing emergency psychological crisis interventions to reduce the negative psychosocial impact on public mental health, but challenges exist. Public mental health interventions should be formally integrated into public health preparedness and emergency response plans. China was the first country affected by the pandemic of 2019 novel coronavirus disease (COVID-19), caused by severe acute respiratory syndrome coronavirus 2. Several unique characteristics of China\u2019s COVID-19 epidemic patterns and its management policy prompted a heightened public mental health crisis. First, many Chinese residents still remember the 2003 outbreak of severe acute respiratory syndrome (SARS) and its effect on China\u2019s social life and economy . The policy guidance also does not indicate operationalization of how various groups should be screened or assessed to determine the type and level of interventions to provide to each. This level of detail is needed because China lacks a well-established mental healthcare system and has no existing national-level emergency response system and designated workforce to provide the psychological crisis interventions during a national emergency or disaster might help, especially in low-resource areas (Given lessons learned from past outbreaks in China and other parts of the world, public mental health interventions should be formally integrated into public health preparedness and emergency response plans to effectively curb all outbreaks. The World Health Organization\u2019s strategic preparedness and response plan for COVID-19, however, has not yet specified any strategies to address mental health needs of any kind (Emergency psychological crisis interventions to address public mental health during the COVID-19 pandemic, China."} +{"text": "Sex and sexuality are important determinants of health and wellbeing across the life course. The desire and capacity for sexual intimacy and pleasure among older adults are neglected areas of research due to ageist assumptions that they no longer engage in sexual activity. These assumptions are most pronounced in HIV research, where we aggressively studied intimate details of sexual behaviors of people living with HIV until they became \u201cold.\u201d Interest in the sexual behaviors among older adults with HIV has waned in HIV prevention, suggesting an inherent ageism within the field. We will discuss emerging new HIV and STI risks for older adults, declining trends in gerosexuality funding, HIV media campaigns targeted for older adults, and new evidence that suggest that interventions that engage older adults with HIV in conversations about sexual health, menopause, and erectile dysfunction may be an effective strategy for promoting overall successful aging"} +{"text": "Idiopathic ventricular arrhythmia (IVA) is a term used to describe a spectrum of ventricular arrhythmia without structural heart disease (SHD). IVAs contain premature ventricular contractions (PVCs), nonsustained monomorphic ventricular tachycardia (VT), and sustained VT. Electrocardiography is a fundamental and important tool to diagnose and localize IVAs. More detailed, IVAs originating from different origins exhibit characterized ECGs due to their specific anatomic backgrounds. As catheter ablation becomes widely used to eliminate these arrhythmias, its high success rate is based on accurate localization of their origins. Therefore, these ECG characteristics show great importance for precise localization of their origins and subsequently successful ablation. This review aims to sum up ECG characteristics of IVAs based on anatomy and give brief introduction of mechanisms and treatment of IVAs. The LVOT can be further divided into supravalvular , infravalvular , and epicardial (the LV summit) portions.The mitral valve has two leaflets positioned anteriorly and posteriorly attaching to the corresponding papillary muscles mostly have an LBBB configuration. As the site of origin shifts more posteriorly from RCC, a transformation from LBBB to RBBB ensues accounts for about 8% of the IVAs and is further divided into the \u201cseptal\u201d and the \u201cfree\u2010wall\u201d portion for precise location has only been recognized recently as a site of origin of IVAs and a source of triggers of ventricular fibrillation(VF) is a distinct subgroup of right ventricular IVAs , followed by the LV summit(12%) and the LV ostium(about 5%\u201310%) in the left ventricle are connected respectively with the anterior and posterior leaflet of the mitral valve. IVAs originate predominantly from the posterior papillary muscle remains the presence of symptoms that are not improved by an explanation of their benign nature and reassurance from the physician. However, it has been validated that there exists a potential association between frequent PVCs burden and cardiomyopathy which is treatable by catheter ablation (Bogun et\u00a0al.,\u00a0Idiopathic VTs are basically monomorphic and hemodynamically stable Yamada,\u00a0. Most id6Surface ECG is a useful and convenient tool to identify the site of origin of IVAs, which usually exhibit characteristic ECGs based on their anatomical and mechanical background. Therefore, understanding special anatomical correlations is necessary to distinguish varied sites of origin. Special ECG characteristics of different origins have been summarized above but there remain some overlaps and difficulties for accurate identifications. More highly specific indications and molecular basis for IVAs are needed in the future research."} +{"text": "Ficedula hypoleuca). From numerous studies, we can conclude that the dark male color phenotype is adapted to a typical northern climate and functions as a dominance signal in male\u2013male competition over nesting sites, and that the browner phenotypes are favored by relaxed intraspecific competition with more dominant male collared flycatchers in areas where the two species co\u2010occur. However, the role of avoidance of hybridization in driving character displacement in plumage between these two species may not be as important as initially thought. The direction of female choice on male coloration in pied flycatchers is not simply as opposite in direction in sympatry and allopatry as traditionally expected, but varies also in relation to additional contexts such as climate variation. While some of the heterogeneity in the observed relationships between coloration and fitness probably indicate type 1 errors, we strongly argue that environmental heterogeneity and context\u2010dependent selection play important roles in explaining plumage color variation in this species, which probably also is the case in many other species studied in less detail.Understanding the origin and persistence of phenotypic variation within and among populations is a major goal in evolutionary biology. However, the eagerness to find unadulterated explanatory models in combination with difficulties in publishing replicated studies may lead to severe underestimations of the complexity of selection patterns acting in nature. One striking example is variation in plumage coloration in birds, where the default adaptive explanation often is that brightly colored individuals signal superior quality across environmental conditions and therefore always should be favored by directional mate choice. Here, we review studies on the proximate determination and adaptive function of coloration traits in male pied flycatchers ( We review numerous studies on both proximate and ultimate factors behind extensive plumage color variation in male pied flycatchers and particularly zoom in on the most unknown questions and suggest future research avenues. There is strong evidence that interspecific interactions with collared flycatchers are of crucial importance for explaining the origin and maintenance of plumage color variation in male pied flycatchers, but the role of avoidance of hybridization in driving character displacement in plumage between the two flycatcher species may not be as important as initially thought. Our review suggests that plumage traits should not be expected to signal high quality as such but rather different male competitive strategies, and a main take\u2010home message is that selection patterns acting on signaling traits are more diverse and fluctuating than generally expected. Hypotheses are tested and improved, thereby facilitating movement toward general principles see Box\u00a0. HoweverIn general, genetic variation in phenotypic traits that are linked to fitness is expected to be eroded by natural and sexual selection Fisher,\u00a0. Broad eIn birds, plumage coloration is often strikingly variable, but the processes promoting the maintenance of this variation are surprisingly poorly understood. Both the intensity of coloration and pattern formed by different colors can vary. Color itself is produced either by different pigments or by structure. Further, different types of coloration differ in many ways, such as the extent of genetically determined variance can reveal important information about the potential signaling functions of the trait .Hypothesis for adaptive functions of color variation below). Honest signaling requires that there is a cost associated with the signal , and older males have larger wing patches than young males and the trait has not been found to be affected by environmental factors during prebreeding molting on the wintering grounds were more likely to attack stuffed females than males during breeding or natural selection. 11 published studies have investigated potential relationships between melanin coloration and measures of breeding success Table\u00a0, age Al, male brHumidity and context\u2010dependent selection, above).Of the four studies that have investigated the relationship between breeding success and male forehead patch size, only one study found evidence for selection favoring larger patch size overlap in Central and Eastern Europe cope with variation in the biotic and abiotic environment, (2) communicate with conspecifics, and 3) communicate with heterospecifics ; funding acquisition (lead); methodology (lead); project administration (lead); visualization (lead); writing\u2014original draft (lead); writing\u2014review & editing . Anna Qvarnstr\u00f6m: Conceptualization (supporting); methodology (supporting); visualization (supporting); writing\u2014original draft (supporting); writing\u2014review and editing (supporting).Supplementary MaterialClick here for additional data file."} +{"text": "One modifiable risk factor of dementia is cognitive inactivity. Given cognitive ability is closely tied to continual performance of instrumental activities of daily living, cognitive training programs continue to be explored as a way to boost cognition and allow older adults to remain independent longer. While the efficacy of cognitive training is controversial, identifying activities older adults are willing to limit in exchange for cognitive training provides valuable information in relation to designing cognitive training programs that appeal to older adults. Using a qualitative approach, this study highlights activities older adults (ages 64+) noted as contributing to decreased gameplay of a cognitive training program on a tablet device. We found that respondents (61%) noted playing less as a result of entertainment activities , social activities (31%) and travel (27%). Findings have implications for device form factor in administering cognitive training and other programs."} +{"text": "Senior housing can be an ideal platform for high quality intergenerational programming. Often older adults who move to congregate housing settings experience feelings of loneliness and a loss of purpose. Creating long term partnerships with educational and youth-serving organizations can help senior housing providers expand residents\u2019 social networks and create meaningful civic engagement opportunities. A 3-year national initiative involving an environmental scan of intergenerational practice in senior housing communities, the development of a toolkit for senior housing providers, and the piloting of intergenerational partnerships and programs in six national housing communities was conducted by Generations United and Leading Age and supported by the Retirement Research Foundation. Promising practices, challenges, and lessons learned from this initiative will be shared and strategies for \u201cscaling\u201d this work will be discussed."} +{"text": "Varicella vaccine is a live attenuated varicella\u2010zoster virus.Varicella vaccine can enter latency and later reactivate as herpes zoster.Pseudorabies virus is another herpesvirus closely related to varicella\u2010zoster virus.The round trip model for pseudorabies virus explains pathogenesis of herpes zoster from vaccine virus. The neurovirulence of the live attenuated varicella vaccine virus is a subject of increasing interest.In the current report from the Journal of Medical Virology, the authors found one case of herpes zoster caused by varicella vaccine virus in a child immunized with varicella vaccine.The invasion of peripheral ganglia after PRV infection has been studied in the murine salivary gland model.The hypothesis of this commentary is that a PRV model for a severe dermatomal disease called mad itch explains severe dermatomal herpes zoster caused by the varicella vaccine virus. After injection of varicella vaccine, usually in the thigh, virus replicates locally and then infects sensory axons, before traveling in the retrograde direction to the lumbar DRG. (Note that 5 of the 6 cases of herpes zoster described in References Based on published reports about the severe\u2010combined immunodeficient (SCID) mouse model for varicella, we think it highly unlikely that subclinical spread of vaccine virus in skin immediately after immunization can account for subsequent severe dermatomal herpes zoster. Studies in the SCID model have established that vaccine virus does not spread widely after injection into human skin xenografts."} +{"text": "In the current study, we sought to examine how older patients incorporate the identity of a patient receiving autologous stem cell transplant (ASCT) for multiple myeloma (MM) into their daily lives. In this ethnographic study using interpretative phenomenological analysis, we observed pre-transplant education visits with 30 MM patients, followed by semi-structured interviews in their hospital rooms during transplant. The experience of receiving ASCT for MM required effort by patients to not only maintain their past identity but also establish a new patient identity. Reconciling these two identities required deliberate and emotionally draining effort from the patient. Results were organized into two overarching themes of social relationships and aesthetics with subthemes for each. Patients experienced challenges reconceptualizing their social support network to meet their changing needs; often with a spouse or child taking on a caregiving role. In regard to aesthetics, patients contended with the physical reminders of their new diseased identity, adopting various aesthetic strategies to either embrace or conceal bodily changes. Understanding methods MM patients who are receiving ASCT use to negotiate normalcy during treatment may be helpful for developing interventions for alleviating distress during this difficult time."} +{"text": "Social isolation has been linked to negative health outcomes, especially among older adults. Although ability to maintain social contact and existing ties to one\u2019s community is a primary benefit of receiving long-term supports and services in a community-based setting, few studies explored how geography might shape these residents\u2019 access to family members and friends. The current study explores this question in the context of adult foster homes (AFH), a type of family-style residential care licensed for five or fewer unrelated adults. Using cross-sectional data collected annually from 1,500 AFHs between 2015 and 2020, the study examines whether older adults residing in rural and urban AFHs in Oregon differ in terms of levels of distinct types of contact with their existing social networks. AFHs were designated as rural/urban at the zip code level using the definitions provided by the state coordinating organization for rural health. Results from negative binomial regression models show that rural residents were significantly less likely to receive help from their family members and friends in getting to medical appointments or outside activities or receive social visits or phone calls compared to their urban counterparts. Rural and urban residents had similar levels of help with personal care and taking medications. These results remained unchanged after accounting for a set of home and resident characteristics . These findings suggest important public health implications for improving rural residents\u2019 social connectedness and interventions aiming at improving social participation in long-term care residents."} +{"text": "The ocean plays critical roles in global carbon cycling and may provide a solution for climate change mitigation , including mangrove forests, salt marshes, seagrass meadows, and seaweed beds, constitute intense blue carbon sinks at the land\u2013ocean transition zones helps fulfill this function, transporting particulate organic carbon (POC) from the ocean surface to its interior and thus contributing to climate modulation on geological time scales that enhance marine aggregate formation and thus facilitate the BCP is sequestered for millennia , only a small fraction (<10%) is transported to the deep ocean 2 sinks) or heterotrophic is still being debated and MCP (~0.2 Gt C/year) may make similar contributions to long-term organic carbon sequestration (Guidi et al., The author confirms being the sole contributor of this work and has approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "This paper introduces the rapidly growing modern mixed methods research (MMR) and its application in a Chinese cancer screening program. While some previous researchers have incorporated quantitative and qualitative data in research, recent mixed methods developments have provided significant clarity that can guide those new to the MMR field. Understanding the context for using MMR and examining a complex mixed methods evaluation study in Taiwan can help illustrate opportunities for and application of mixed methods in Asians. The Taiwan Cervical Cancer Screening Education Program is used as an exemplar of a multi-phase complex mixed methods evaluation study showcasing various MMR designs. These include an exploratory sequential design to develop culturally sensitive study instrument, iterative concurrent and sequential mixed methods for intervention mapping, and an embedded mixed methods evaluation design to assess impact. Visual diagrams are introduced to facilitate communication of mixed methods design procedures and products in each phase."} +{"text": "The European SocieTy for Radiotherapy and Oncology Radiation Therapist Committee (ESTRO RTTC) published a guidance document and infographic providing recommendations to minimise risk of COVID-19 transmission in radiotherapy (RT) departments. The purpose of this study was to investigate the changes embedded in RT practice in the COVID-19 era and to recommend proactive measures to protect RT practice in future pandemics.The study was initiated by the ESTRO Radiation Oncology Safety and Quality Committee (ROSQC). A survey consisting of multiple choice, open ended and Likert scale questions was created to analyse the extent of changes embedded in RT practice in response to the COVID-19 pandemic under the four domains: patient care, RTT workflow, remote working and RT practice. This online survey was distributed globally in May 2020.229 respondents across 27 countries completed the survey. 60% of respondents reported continuing/commencing RT in COVID-19 patients. Routine testing of patients and RTTs was not common. Split teams' procedures, hot linacs and separate entrances were implemented by 50% of respondents. Remote working was implemented for RT team members where face to face patient contact was not essential. Lack of staff, connectivity issues and lack of confirmed positive cases in the department were the main reasons cited for not implementing recommended measures.It is suggested that RT departments have responded to the COVID-19 pandemic and implemented certain changes in RT practice. RT departments should act now to implement recommended proactive measures to protect patients and RTTs \u2013 frontline healthcare workers. COVID-19 is a highly infectious coronavirus first reported in December 2019 and characterised by the World Health Organisation (WHO) as a global pandemic in March 2020. By July 2020, over 14 million COVID-19 cases were confirmed worldwide with more than 600,000 deaths Radiotherapy (RT) plays a key role in cancer management, and is recommended as part of treatment for more than 50% of cancer patients Healthcare resources are limited, with PPE shortages widely reported due to ongoing or recurrent COVID-19 outbreaks The study was initiated by the ESTRO ROSQC in April 2020. During the development stage of the survey, the study set its sample population as RTT professionals. The survey was built (with full permission) on ongoing work carried out by the Canadian Association of Provincial Cancer Agencies. It consisted of both demographic questions and questions covering the 4 topics addressed in the ESTRO RTT COVID-19 guidance document and Infographic: patient care, RTT workflow, remote working and RT practice The questionnaire was circulated to RTTs identified from sources including the ESTRO RTT membership database and via a link to the survey posted on the American Society of Radiologic Technologists (ASRT) member forum and World Wide Radiation Therapists Facebook page. Participants were asked to complete the survey within three weeks with a reminder email sent 5\u202fdays prior to the closing date. After the closing date the raw data results of the survey were analysed by representatives from both ROSQC and RTTC.Between the 22nd May 2020 and 12th June 2020, 229 RTTs across 27 countries completed the survey . The hig82% of respondents stated that patients were checked for COVID-19 symptoms before entering the department, more than 60% of responses (140/229) suggested that patients were routinely asked to wear a mask in the department, less than half of the respondents (111/229) said that patients would be routinely tested for COVID-19 before commencing the radiotherapy planning process . 71 of tWhen the RTTs were asked if patients who tested positive for COVID-19 would be commencing/continuing their treatment, nearly 60% (133) of respondents answered \u201cYes\u201d 39 responses stated \u201cNot sure\u201d, 19 of them stated that they would not know the answer for this question because they had no positive COVID-19 cases at the time of survey. Reviewing the responses from LMICs, 92% of RTTs stated that COVID-19 positive patients would not commence or continue radiotherapy in their departments.On RT workflow procedures adopted for COVID-19 approximately half of the respondents stated their departments implemented approaches such as \u201csplit team\u201d (51%), \u201cstaggered appointment times\u201d (49%) and \u201chot linac\u201d (51%) to minimise the number of patients/staff in the department at the same time and avoid potential exposure and transmission across the entire RT team . ReviewiMasks and gloves were the most common types of PPE used by RTTs regardless of patient COVID-19 status. A greater variety and higher frequency of PPE use was reported by RTTs for COVID-19 positive/symptomatic patients. N95 masks were used more frequently for COVID-19 positive/symptomatic patients .Fig. 3A Nearly three quarters of the respondents were satisfied with the quality of the PPE used for both COVID-19 positive/symptomatic (73%) and COVID-19 negative/asymptomatic (76%) patients in their department.Over three quarters of responses reported opportunities for clinical staff to work remotely due to COVID-19 with remote working implemented for medical physicists (87%), treatment planning staff {RTTs and dosimetrists} (75%), and radiation oncologists (67%) . Over 80Overall respondents reported a low level of stress {average 3.1 (1\u201310)}, on not having physical access to staff members who were working remotely; and it was reported as straightforward to contact radiation oncologists (71%), medical physicists (84%) and treatment planning staff (80%) who were working remotely to query clinical issues.Routine screening of RTTs for COVID-19 was not common practice, with the majority (73%) of respondents stating \u2018No\u2019 . To miniCOVID-19 has had a severe effect globally on healthcare service delivery. Initial studies attribute a significant decline in the numbers of cancer patients being referred for treatment to; the reduction in cancer screening, patients not presenting to their primary care providers with suspicious symptoms Cancer patients are considered a high-risk group for contracting COVID-19 Measures recommended in the ESTRO guidance document such as symptom checks and mandatory mask wearing for patients Even though many patients attend as outpatients and are at risk of community transmission, symptom checks were carried out on their first visit to the RT department with no evidence of routine symptom checks throughout the treatment course.Recommendations:\u2022Mask wearing should be mandatory for all patients.\u2022Daily triage system should be in place for all patients entering the RT departments.\u2022Escalation procedures should be in place for patients that present with suspicious symptoms; testing procedures with swift turnaround and definitive guidelines for RTTs on which patient groups must commence/continue RT if tested positive.The ESTRO RTT guidance document recommended measures such as splitting teams and staggering patient appointment times to limit critical staff exposure and patient transmission Managers should be proactive and independently evaluate the potential effect of a further surge or future pandemic on their staff and how best to organise the workforce to ensure continuity of care. RTTs consistently voiced concerns on the anticipated increase in demand for RT services in the future and if their departments would cope. Delaying patients may not be an option if they have already experienced delays in the treatment process during the initial COVID-19 surge. Anderson et al gives detail of how they addressed workforce issues in his publication, and this provides an excellent guide to good practice in a pandemic setting WHO recommended PPE was routinely adopted into routine clinical RTT practice for COVID-19 positive/symptomatic patients [3]. HowRecommendations:\u2022Implementing team pods/split teams is recommended to avoid a situation where an entire RTT team would have to self-isolate resulting in cessation of all RTT services within that department.\u2022Adequate time for infection control measures between patients should be allowed when scheduling patient appointments. The use of catch-up slots in RT appointment scheduling could be considered.\u2022RTTs should be provided with adequate PPE for treating all patients regardless of COVID-19 status.Government directives recommend implementing a work from home policy wherever possible to minimise social interactions. RT is an essential service and as RTTs are frontline workers - remote working can only be considered at limited stages of the patient pathway such as treatment planning and review clinics Remote working in RT department future workforce planning, must be carefully considered. RTT practice is ever changing with an interdisciplinary approach essential to develop departmental protocols and processes. Research suggests that lack of face to face contact can reduce innovation and knowledge transfer in a workplace Recommendations:\u2022Impact of remote working on practice development in RT departments should be carefully considered and implemented where appropriate\u2022Decision making facilities of RTTs should be carefully considered if remote working remains standard practice in other RT groupsWithout significant vaccination coverage, preventive measures to limit disease transmission should be proactive rather than reactive to accommodate increasing patient numbers under the pandemic. Our data suggested that measures for reducing transmission (Perspex screens and separate entrances for COVID-19 positive patients) recommended in the ESTRO guidance document Some RT departments were operating a no waiting area policy with patients asked to wait in their cars until their appointment time and not have anyone accompany them into the RT department. This may isolate cancer patients and cause considerable psychological distress Less than 20% of the respondents reported seeing suspicious COVID-19 related changes on CBCT during RT treatment. This can be due to the lack of positive or symptomatic cases referred for RT. Respondents were not asked directly if these changes were detected on planning CT scans, but some responses indicated that RTTs had reported suspicious cases using planning CT images which were subsequently investigated. Some respondents did remark that CBCT image quality was too poor while some reported that it was not departmental practice for RTTs to interpret volumetric images in their department. Several publications discuss the increased use of hypofractionated radiotherapy during the COVID-19 pandemic Recommendations:\u2022Routine COVID-19 testing with swift turnarounds should be in place for RTTs.\u2022RT departments must review current RTT education/training and competency framework to ensure that staff are competent in IGRT execution and delivery.\u2022An escalation procedure should be in place for RTTs when suspicious COVID-19 related changes are detected on planning CT and CBCT scans.COVID-19 will remain a part of our lives until a significant percentage of the population are vaccinated. RT is an essential service and one that does not compete with healthcare resources for the pandemic. Our study demonstrates that RT departments have responded to the COVID-19 by implementing a certain level of changes in RT practice, and recommends a list of potential proactive measures under the four domains of patient care, RTT workflow, remote working and RT practice for future RTT practice.RTTs are frontline healthcare professionals, their relationship with patients is key to achieving optimum experience for patients. RT departments must consider the implications of potential further COVID-19 surges in service demands and implement proactive measures to protect RTTs and patients.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper."} +{"text": "Fisheries often combine high mortality with intensive size selectivity and can, thus, be expected to reduce body size and size variability in exploited populations. In many fish species, body size is a sexually selected trait and plays an important role in mate choice and mate competition. Large individuals are often preferred as mates due to the high fecundity and resources they can provide to developing offspring. Large fish are also successful in competition for mates. Fisheries\u2010induced reductions in size and size variability can potentially disrupt mating systems and lower average reproductive success by decreasing opportunities for sexual selection. By reducing population sizes, fisheries can also lead to an increased level of inbreeding. Some fish species avoid reproducing with kin, and a high level of relatedness in a population can further disrupt mating systems. Reduced body size and size variability can force fish to change their mate preferences or reduce their choosiness. If mate preference is genetically determined, the adaptive response to fisheries\u2010induced changes in size and size variability might not occur rapidly. However, much evidence exists for plastic adjustments of mate choice, suggesting that fish might respond flexibly to changes in their social environment. Here, I first discuss how reduced average body size and size variability in exploited populations might affect mate choice and mate competition. I then consider the effects of sex\u2010biased fisheries on mating systems. Finally, I contemplate the possible effects of inbreeding on mate choice and reproductive success and discuss how mate choice might evolve in exploited populations. Currently, little is known about the mating systems of nonmodel species and about the interplay between size\u2010selective fisheries and sexual selection. Future studies should focus on how reduced size and size variability and increased inbreeding affect fish mating systems, how persistent these effects are, and how this might in turn affect population demography. They showed that while the average juvenile body size did not change over time, the size variability decreased substantially were exposed to size\u2010selective harvesting or simply have higher fertility Table\u00a0. Our undIn zebrafish, females prefer large males for potentially two reasons: Large males are better able to acquire and defend territories than small males are females were paired with a small male, they delayed spawning allowing larger males the opportunity to displace the small courting male are known to attract females by producing a \u201cdrumming\u201d sound males typically form dominance hierarchies in which one or a few individuals control access to females have been shown to prefer large males early in the breeding season but reduce their choosiness as the breeding season progresses . These important asymmetries include, for example, fighting ability and body size. Body size is one of the most important factors in determining contest outcome in many fish species fisheries in Bristol Bay, Alaska, have disproportionally exploited more males than females landings on the east coast of the United States consist mostly of females fishery along the south Atlantic coast of the United States is heavily dependent on large females , winter flounder (Pseudopleuronectes americanus) in New York estuaries, and endangered dusky grouper (Epinephelus marginatus) in the central Mediterranean have been heavily exploited and consequently experienced moderate to severe inbreeding females can recognize their relatives using olfactory cues and adjust their behavior accordingly females have been shown to express postcopulatory inbreeding avoidance and reduce the amount of sibling sperm in their reproductive system compared to nonsibling males , inbreeding caused significant decreases in body length, weight, juvenile survival, and delayed spawning . However, it has been shown that mate choice is plastic and context dependent (Cotton, Small, & Pomiankowski,\u00a0Heterospecific factors can as well be important in shaping mate choice behavior. Sand goby females prefer large and brightly colored males (Forsgren,\u00a0When size\u2010selective fisheries reduce body size variability rapidly and unexpectedly, an individual's ability to discriminate among mates can be suddenly hampered (e.g., Francis & Barber,\u00a03Reduced size variability can be expected to disrupt mate choice and mate competition in systems where body size is a sexually selected trait. Given that size\u2010selective harvesting reduces size variability in exploited populations (Hutchings & Baum,\u00a0Small exploited populations may foster inbreeding via mating among relatives (Keller & Waller,\u00a0Fisheries present a relatively novel contemporary selection force that changes the environment rapidly by removing individuals that would otherwise likely be favored by natural and sexual selection. However, studies combining the often opposing effects of fisheries and sexual selection are rare (but see Hutchings & Rowe,"} +{"text": "Ultraviolet germicidal irradiation (UVGI) N95 filtering facepiece respirator (FFR) treatment is considered an effective decontamination approach to address the supply shortage of N95 FFRs during the ongoing Covid-19 pandemic. In this study, we investigated the nanomechanical and topographic properties of filtration fibers that have been exposed to different doses of UVC radiation. UVC exposure was shown to decrease both Young\u2019s modulus (E), hardness (H) and fiber width, as measured on individual polypropylene (PP) fibers. Our results also show that the PP microfiber layer loses its strength when N95 respirators are exposed to an accumulated UVC dose during the process of decontamination, and the PP fiber width also exhibits a logarithmic decrease during UVC exposure. The nanoscale measurement results on individual fibers suggest that maximum cycles of UVC disinfection treatment should be limited due to excessive accumulated dose, which has the potential to decrease the fiber breaking strength."} +{"text": "Older African Americans\u2019 (AA) participation in health-related research is severely limited; they are not involved in sufficient numbers and for sufficient duration to ensure the applicability of advancements in medical and behavioral health. This research participation gap exacerbates older AAs vulnerability to poor health outcomes and disparities. The Michigan Center for Urban African American Aging Research employs a progressive community-based participatory model that utilizes a structured community advisory board (CAB) of older AAs in metro Detroit to oversee the research recruitment and retention of fellow AA older adult research participants. CAB members are provided ongoing training on social and behavioral health research, supported in acting as a consultancy to outside researchers where they can be compensated for their expertise and engagement, and empowered as gatekeepers of a participant research registry of over 1000 AA older adults. This model has broad potential for advancing community engaged research with AA older adults."} +{"text": "Long-distance caregivers (LDCs) comprise a growing yet relatively understudied segment of the caregiving population. The current study (N=166) investigated the mediating role of home care hours on the association between primary caregiving stressors and LDCs\u2019 perceived strain (perceived interference of caregiving with other family responsibilities and work). Results from path analyses showed that home care hours fully mediated the negative effect of CRs\u2019 functional impairment on family interference and the negative effect of CRs\u2019 cognitive impairment on work interference. Further, the negative effect of CRs\u2019 cognitive impairment on family interference was partially mediated by home care hours. Thus, a mediating role of home care service hours in the associations between objective stressors and LDC strain was established for both family and work domains. Findings highlight the potential of home care services in alleviating strain in LDCs of community-dwelling CRs."} +{"text": "Immunotherapy targeting programmed death\u20101 or programmed death\u2010ligand 1 has become the standard of care for advanced non\u2010small cell lung cancer (NSCLC). Several recent clinical trials have investigated the efficacy of immune checkpoint inhibitors (ICIs) as neoadjuvant treatment for early NSCLC. However, the safety and feasibility of pulmonary resection after ICIs remain unclear. We herein report a patient in whom cohesion between the left main pulmonary artery and left upper bronchus was found during left upper lobectomy following neoadjuvant ICI combined with chemotherapy. After both central and peripheral sides of the left main pulmonary artery were clamped with the aim of controlling hemorrhage in case of vascular injury, the left main pulmonary artery and left upper bronchus were divided and individually cut with staplers. The thoracoscopic procedure was otherwise uneventful. The patient was discharged from our hospital with no postoperative complications. Thoracic surgeons should anticipate the possible need for management of cohesion between a pulmonary artery and bronchus in patients who have received immune checkpoint inhibitors preoperatively. We herein report a patient in whom cohesion between the left main pulmonary artery and left upper bronchus was found during left upper lobectomy following neoadjuvant immune checkpoint inhibitor combined with chemotherapy. The left main pulmonary artery and left upper bronchus were successfully divided and individually cut with staplers. Thoracic surgeons should anticipate the possible need for management of cohesion between a pulmonary artery and bronchus in patients who have received immune checkpoint inhibitors preoperatively. Immunotherapy targeting programmed death\u20101 or programmed death\u2010ligand 1 has become the standard of care for advanced non\u2010small cell lung cancer.A 74\u2010year\u2010old man with a 55\u2010pack\u2010year smoking history presented to our hospital. Computed tomography (CT) revealed a mass in the left upper lobe of the lung (maximum diameter: 3.2 cm) with left hilar lymph node swelling #10). He underwent a positron emission tomography (PET) scan and was clinically diagnosed as having left lower lobe lung cancer together with metastasis in a left hilar lymph node. Transbronchial lung biopsy resulted in a diagnosis of adenocarcinoma in the left upper lobe . He received four cycles of an ICI combined with cytotoxic chemotherapy as a participant in a clinical trial of neoadjuvant therapy examination had shown a single\u2010station bulky N1 lymph node metastasis (#10), we anticipated that it would be difficult to dissect the pulmonary artery from that lymph node. However, we did not expect any problem with dissection of the left main pulmonary artery because there were no adjacent lymph node metastases. In previous studies, simultaneous stapling of pulmonary vessel and bronchus have been performed because of previous surgery and/or silicotic lymphadenitis.According to previous reports on the safety of lung resection after administration of ICIs, cohesion between bronchus and pulmonary artery was not observed in patients who received ICIs that were not in a neoadjuvant setting.In conclusion, the possibility of cohesion between vessels and bronchi during thoracic surgery, as well as the attendant necessity for vascular management, should be considered in the planning of thoracic surgery in patients who have undergone immune checkpoint inhibitor and chemotherapy.None of the authors have conflicts of interest to declare in association with this study."} +{"text": "Pakistan and other developing countries need to address disparities in maternal health care and factors associated with it. This justifies tracking the progress on two important indicators \u2018spousal violence\u2019 and \u2018maternal health care utilization\u2019 to improve maternal health and achieve Sustainable Development Goals (SDGs) for these nations.The objective of this study is to compare the data from the latest two Demographic Health Surveys of Pakistan to identify trends in prevalence of various forms of spousal violence and maternal healthcare utilization and to determine the predictive role of spousal violence in poor maternal health.We conducted a retrospective analysis of nationally representative data from the 2012\u201313 and 2017\u201318 PDHS. The data used in this analysis is from the domestic violence module and core women\u2019s questionnaire. Spousal violence and sociodemographic background were predictor variables. Terminated pregnancy, number of pregnancy losses, number of antenatal visits for last birth and institutional delivery for last birth were taken as indicators of maternal health. Logistic regression analysis was conducted to test for association between maternal health indicators and various forms of spousal violence after controlling for sociodemographic variables.Almost one quarter of women experienced physical and emotional violence as revealed by both surveys. Binary analysis revealed that all forms of spousal violence significantly associate with maternal health variables in both surveys. The comparison of results on logistic regression analysis showed that odd ratios were relatively higher for 2012\u201313 as compared to 2017\u201318 PDHS. Logistic regression analysis from 2017\u201318 data showed that experience of less severe physical violence , severe physical violence , sexual violence , physical violence during pregnancy augment the risk of terminated pregnancy. Emotional violence decreases the likelihood for institutional delivery and above than four antenatal visits .Strategies to prevent spousal violence should be at the core of maternal health programs because health sector provides a platform to challenge social norms and promote attitudes that disapprove spousal violence which are essential for promoting gender equality, women empowerment (SDG 3) and improve maternal health (SDG 5). Maternal health is a well-recognized aspect of women\u2019s health. Women in developing countries more commonly suffer from pregnancy related complications, premature delivery of children and maternal death. This gap is as high as up to 33 percent between developed and developing countries . PakistaWomen in both developed and developing countries face various forms of violence among which spousal violence has emerged as a significant social nuisance and public health problem. World Health Organization (WHO) , definedIn this context, it is imperative to recognize that spousal violence is a major human rights issue but also a significant determinant of women\u2019s physical, reproductive, maternal and emotional health . The impWomen in developing countries frequently experience emotional violence, economic control as well as physical and sexual violence from their male partners. According to global and regional estimates on prevalence of Intimate Partner Violence (IPV), 30 percent of ever-partnered women at some point in their lives have experienced physical and/or sexual violence by an intimate partner and at least 10 percent of them faced IPV during their pregnancy . Women iStudies from other developing countries of South Asian region, such as in-depth analysis of data from 2005\u20132006 India\u2019s National Family Health Survey reported that almost 12% of women experienced severe physical violence and 14% of women had less severe injuries due to spousal violence . An analIn developing countries, spousal violence is rooted in a wider context of gender inequality. Gender-based violence and discrimination are major contributing factors of slow progress towards sustainable developmental goals as indicated by the latest rankings on Gender inequality index for Pakistan, which is 148 out of 149 countries . DespiteThis study aims to address this gap by doing analysis from the last two Pakistan Demographic Health Surveys conducted in four major provinces and capital city completed in years 2012\u201313 and 2017\u201318.This study has three objectives.To report the trends in prevalence of various forms of spousal violence and maternal health indicators.To estimate the association between experience of various forms of spousal violence and maternal health.To determine the predictive role of various forms of spousal violence with maternal health.https://dhsprogram.com. Demographic Health Surveys (DHS) are the biggest household surveys conducted four times in Pakistan and contains rich information on demography and maternal health variables for ever-married women aged (15\u201349 years). For the main survey, a nationally representative sample was obtained with a two-stage, stratified random sampling design. We selected these two surveys because spousal violence was investigated first time on a sub-sample in 2012\u201313 PDHS survey. In previous surveys of DHS in Pakistan, experience of spousal violence was not included. The PDHS 2012\u201313 obtained data about physical and emotional forms of spousal violence only. PDHS 2017\u201318 included data on experience of forms of spousal violence. The main survey comprises of larger sample size in PDHS 12\u201313 survey with a 95% confidence interval are given in Spousal violence also negatively influences antenatal care. The findings from PDHS 2012\u201313 shows that all forms of spousal violence significantly decrease the likelihood of more than four antenatal visits for the last birth. In PDHS 2017\u201318 dataset, \u2018less severe physical violence\u2019 and \u2018severe physical violence\u2019 significantly decrease the likelihood of more than four antenatal visits for the last birth with and respectively .Tables We examined the trends in prevalence of spousal violence and maternal health by using the data from the two most recent demographic health surveys 2012\u201313 (PDHS) and 2017\u201318 (PDHS). Besides, we conducted predictive analysis on these datasets to determine the impact of spousal violence on maternal health. Findings show that there is some decline (2% to 5%) in rates of spousal violence and slight improvement (5% to 7%) on maternal health indicators over the years between these two surveys. However, this is not a satisfactory progress towards achieving goals of sustainable development and a raise serious concern about disparities in women\u2019s access to maternal healthcare. 24% of women are still exposed to less severe physical violence; 51% of women had less than 4 ANC visits, 33% have not accessed institutional delivery and 32% had experienced terminated pregnancy. Current study findings consolidate the previous evidence that spousal violence significantly increases the risk of poor maternal health in women . FindingThis situation implies the need for more appropriate community health programs to outreach women and need to adopt more efficient ways to mobilize existing healthcare networks. The ground reality is that healthcare professionals are usually less sensitive and not trained to deal with spousal violence cases. Findings from a qualitative study showed that health care professionals are reluctant to inquire or screen women for spousal violence . They coAccording to WHO\u2019s Focused Antenatal Care (FANC), there should be minimum of four antenatal visits for a pregnant woman without any complications . Current study findings are alarming, as 24% of women reported no antenatal visits. Current findings also suggest that women who face violence made less number of antenatal visits which aligns with previous literature . Women wFindings of present analysis showed that women exposed to spousal violence are less likely to seek institutional services for the delivery of last childbirth. Previous literature though sThe important strength of this research is that we used data from demographic health survey, which included nationally representative sample from both urban and rural areas of Pakistan and findings are generalizable to other developing countries of this region with similar healthcare system.Among limitations, the survey did not have data on other indicators of women health such as maternal morbidity and mortality and not included in analysis. Secondly, keeping in view the social and cultural environment of Pakistani society, spousal violence is likely to be under-reported by participants in current survey. A large number of women participants were living in combined family systems and chances are high that they are reluctant to talk about experience of spousal violence.The impacts of spousal violence on maternal health are preventable through appropriate interventions, such as training health services staff in screening and early intervention in existing reproductive healthcare setting. The occurrence of spousal abuse is associated with number of social and cultural factors e.g. lack of economic independence, stigma and negative attitudes of people towards women who report abuse or seek help. There is need of increased political support and funding to implement variety of social, economic and health interventions. More outreach programs can ensure provision of antenatal care to pregnant women in the community. Psycho-behavioral interventions on family violence need to include educating families and husbands about repercussions of physical and emotional violence on mother health and child life prospects. Collective interventions at macro, meso and micro level in context of Pakistan and other developing countries are required to prevent spousal violence and improve indicators of maternal health.This study provides additional evidence about impacts of spousal violence on maternal health outcomes such as terminated pregnancy, deficient antenatal care and decreased likelihood of intuitional delivery. There is slight decrease in rates of spousal violence and slight improvement on maternal health indicators as indicated by the comparative analysis of 2012\u201313 and 2017\u201318 surveys. Nonetheless, this improvement is not substantial enough to achieve targets for sustainable development goals. These findings emphasize upon need for more integrative community interventions that should focus both on reducing spousal violence as well improving access to maternal health services in vulnerable populations.S1 Appendix(DOCX)Click here for additional data file."} +{"text": "Egg white proteins were digested following the INFOGEST gastrointestinal digestion protocol. Different time points of gastric and intestinal digestion were characterized regarding protein, peptide and amino acid content. Protein degradation was followed by SDS-PAGE where some electrophoretic bands were identified by MALDI-TOF/TOF after tryptic digestion. Moreover, the molecular weight distribution of egg white peptides found at different times of gastrointestinal digestion was performed using MALDI-TOF. Peptides identified from the most abundant egg white proteins by tandem mass spectrometry were represented using a peptide profile tool and raw data are given in table format. These results reveal the protein regions resistant to digestion and illustrate the free amino acid profile of egg white protein at the end of the digestion process. These data can be used for nutritional purposes and to identify allergen epitopes or bioactive sequences.These data are related to the research article entitled \u201cInduction of CCK and GLP-1 release in enteroendocrine cells by egg white peptides generated during gastrointestinal digestion\u201d. In this article, the peptide and free amino acid profile of egg white gastrointestinal Specifications Table\u2022The data provide the distribution of the nitrogen fraction into peptides and amino acids at the end of the gastrointestinal digestion of egg white. The profile of free amino acids that can be used for nutritional purposes is also given.\u2022in vivo data or with data obtained in dynamic systems.The here provided proteomic, peptidomic and amino acid profiles of egg white protein digests can be compared with \u2022Egg white protein domains resistant to gastrointestinal digestion are provided which could serve to detect allergen epitopes or peptides with biological activities.\u2022This peptidomic characterization was useful to identify peptides as inducers of incretin hormones, being relevant to control food intake and diabetes.Value of the data1in vitro digestion protocol [g over 20\u202fmin to separate soluble and insoluble fraction, followed by snap freezing in liquid nitrogen and freeze-dried. All digests were characterized regarding their protein, peptide and amino acid composition.Egg white protein was digested following a harmonized protocol ,2 where in vitro gastric and intestinal digestion was assessed by elemental analysis . Mass spectra were acquired in positive reflection mode and were collected from the sum of 1000 on average lasers shots. Monoisotopic peaks were generated by FlexAnalysis software. The monoisotopic peaks were organized and represented in a molecular weight distribution range. All other methods are described in The authors declare that they have no known competing financial interests or personal relation-ships that could have appeared to influence the work reported in this paper."} +{"text": "With great interest we read the article by Heinsar et al. discussing implications for extracorporeal membrane oxygenation (ECMO) during the coronavirus disease 2019 (COVID-19) pandemic when the number of patients reaches a level that demand may overwhelm available resources . Tho. Tho5]. Registries monitoring the availability of ICU beds and definition of regional clusters for patient allocation help to ease pressure from overburdened hospitals, too. This model could be established elsewhere.Triage committees or advanced prediction models may support decision makers in preparing for situations of scarcity; however, as soon as important resources are no longer available in sufficient quantities, structures and procedures must be in place that healthcare workers can access without any particular hurdles in order to refer or transfer patients to the places where the necessary resources are (still) available. In such situations triage committees or prediction models most likely cannot keep up with the pace and flexibility required."} +{"text": "Responding appropriately to exercise is essential to maintenance of skeletal muscle mass and function at all ages and particularly during aging. Here, a hypothesis is presented that a key component of the inability of skeletal muscle to respond effectively to exercise in aging is a denervation-induced failure of muscle redox signalling. This novel hypothesis proposes that an initial increase in oxidation in muscle mitochondria leads to a paradoxical increase in the reductive state of specific cysteines of signalling proteins in the muscle cytosol that suppresses their ability to respond to normal oxidising redox signals during exercise. The following are presented for consideration:Transient loss of integrity of peripheral motor neurons occurs repeatedly throughout life and is normally rapidly repaired by reinnervation, but this repair process becomes less efficient with aging. Each transient loss of neuromuscular integrity leads to a rapid, large increase in mitochondrial peroxide production in the denervated muscle fibers and in neighbouring muscle fibers. This peroxide may initially act to stimulate axonal sprouting and regeneration, but also stimulates retrograde mitonuclear communication to increase expression of a range of cytoprotective proteins in an attempt to protect the fiber and neighbouring tissues against oxidative damage. The increased peroxide within mitochondria does not lead to an increased cytosolic peroxide, but the increases in adaptive cytoprotective proteins include some located to the muscle cytosol which modify the local cytosol redox environment to induce a more reductive state in key cysteines of specific signalling proteins. Key adaptations of skeletal muscle to exercise involve transient peroxiredoxin oxidation as effectors of redox signalling in the cytosol. This requires sensitive oxidation of key cysteine residues. In aging, the chronic change to a more reductive cytosolic environment prevents the transient oxidation of peroxiredoxin 2 and hence prevents essential adaptations to exercise, thus contributing to loss of muscle mass and function.Experimental approaches suitable for testing the hypothesis are also outlined. \u2022It is hypothesised that denervation leads to failed redox signalling and attenuated muscle exercise responses in aging.\u2022Loss of peripheral motor neurons leads to increased mitochondrial peroxide in denervated and neighbouring muscle fibers.\u2022This peroxide stimulates increased expression of cytoprotective proteins to protect the fiber against oxidative damage.\u2022Increased mitochondrial peroxide does not increase cytosolic peroxide, but adaptations reduce the cytosol redox state.\u2022Key adaptations of skeletal muscle to exercise involve transient peroxiredoxin oxidation in the cytosol.\u2022Peroxiredoxin oxidation is suppressed during aging by a chronic change to a more reductive local cytosolic environment. The social and medical issues associated with increasing numbers of elderly members of populations are affecting most countries. The chronic conditions associated with musculoskeletal aging contribute to a heavy functional and economic burden for rapidly aging populations. Age is the greatest risk factor for the common musculoskeletal disorders, osteoarthritis, osteoporosis and sarcopenia and the number of older people with such disorders in countries such as the UK continues to increase dramatically . There r1In older people, declining muscle mass and function causes instability and increased risk of falls with a loss of independence . By age The loss of muscle that occurs with aging occurs in parallel with loss of motor units in both humans and rodents ,12. In y2Skeletal muscle adapts to different forms of exercise in many positive ways including an increase in aerobic capacity, increased muscle force generation, increased mass and decreased fatigability. These processes are essential for maintenance of muscle mass and function at all ages, but this becomes increasingly important with increasing age ,24. The 2O2), have been proposed as key factors that stimulate adaptive changes in contracting skeletal muscle . Th. Thex viUPRmt), mitochondrial metabolites and mitokines have also been proposed .HyPer 7 targeted to different cell compartments in K562\u00a0cells also found that increased mitochondrial H2O2 generation induced by addition of complex I inhibitors had no effect on the cytosolic H2O2 content , , 2O2 to Some studies have also recently indicated that Prxs can function as a signal peroxidase to activate specific pathways. Prx1 activates the transcription factor ASK1 and Prx2132O2. The effects were examined in an isolated FDB fiber model. Following commencement of contractile activity in fibers from adult mice, Prx1, 2 and 3 oxidised rapidly (shown by formation of dimers on non-reducing western blots) A and the14The effect of contractile activity on Prx oxidation was also examined in FDB fibers from old mice in comparison with the data obtained from FDB fibers of adult mice A. As preThis decrease in contraction-induced Prx2 oxidation in muscle fibers from old mice is consistent with the partial disruption of cell signalling and transcription factor activation and supp15\u2022Transient loss of integrity of peripheral motor neurons occurs repeatedly throughout life, but is normally rapidly repaired by reinnervation, including by collateral innervation from sprouting axons from neighbouring muscle fibers.\u2022Each transient loss of neuromuscular integrity leads to a large increase in mitochondrial peroxide production in the denervated fibers and in neighbouring fibers. This peroxide initially acts to stimulate axonal sprouting and regeneration to facilitate rapid reinnervation of the denervated fiber, but also leads to minor oxidative damage and increased expression of a range of cytoprotective proteins as a means of protecting the fiber and neighbouring tissues against severe oxidative damage A.Fig. 7A\u2022The increased peroxide within mitochondria does not lead to an increased cytosolic peroxide, but the increases in adaptive cytoprotective proteins include some located to the muscle cytosol which paradoxically modify the local cytosol redox environment to induce a more reductive state in key cysteines of specific signalling proteins.\u2022Key adaptations of skeletal muscle to exercise involve transient Prx oxidation as effectors of redox signalling in the cytosol. This requires sensitive oxidation of key cysteine residues. In aging, the chronic change to a more reductive cytosolic environment prevents the appropriate transient contraction-induced oxidation of key cysteines on Prx2 and hence prevents essential adaptations to exercise, thus contributing to loss of muscle mass and function muscle fibers and it could therefore be informative to study whether the hypothesis is sustained in slow twitch vs. fast twitch (type 2B) muscle fibers. The relative preservation of slow fibers in older individuals appears to be associated with less loss of the small motor neurons that innervate slow twitch muscle fibers in comparison with a greater loss of large fast \u03b1-motor neurons that innervate fast twitch muscle fibers . Type 1 h fibres . Thus, tIt is also implied from the hypothesis that interventions at multiple levels should preserve the essential adaptations to exercise that underpins improved maintenance of skeletal muscle mass and function during aging. Many previous studies have examined the possibility that loss of muscle mass and function in aging can be prevented by decreasing muscle oxidation through use of \u201cantioxidants\u201d, but our data indicate that key proteins in the cytosol of muscle are more reduced in old mice and this may attenuate redox-mediated responses to contractile activity. Thus, conventional non-specific antioxidants could not work in this situation.Some logical interventions in the pathway hypothesised (e.g. prevention of motor neuron loss) are generic and, if feasible, would most likely preserve muscle mass and function during aging whatever the downstream mechanisms. Direct testing of the hypothesis will therefore require more specific interventions:172O2 and other peroxides. One such approach is based the mito-targeted peptide SS-31 . This peptide rapidly and freely crosses the skeletal muscle plasma membrane and concentrates greater than 1000 fold in the mitochondria. It has been reported to improve muscle mitochondrial bioenergetics and exercise endurance capacity in old mice in addition to reducing muscle mitochondrial H2O2 generation and increasing the reduced glutathione status [MitoQ) act as mitochondria-selective antioxidants to scavenge ROS within the mitochondrial matrix and provide an alternative route to test the hypothesis [One such approach would be organelle-targeted reduction in mitochondrial peroxide production to prevent the induction of adaptations that lead to increased reduction of cysteines in the cytoplasm. There is also increasing interest in mitochondria-targeted therapeutics that target mitochondrial production of He status . In our e status . Other cpothesis .18Following this same approach, a potential mechanism can be identified from current data that may lead to the adaptive increased reduction of the cytosol and provides an intervention target to test the hypothesis. We previously reported an elevation in Trx1 (cytosolic) content in muscle from old in comparison with adult mice . Potenti"} +{"text": "Although the likelihood of developing a disability increases with age among all demographics, older adults of hispanic origin are more likely to experience vision and hearing impairment than both their white and black non-hispanic counterparts. Both hearing impairment and vision impairment are known risk factors for social isolation, yet little research has examined this association in Hispanic populations. Using data from 472 Hispanic and 5,186 White participants of the NHATS study, we examined 8-year trajectories of social isolation, along with how sensory impairment was associated with initial levels and change over time. Findings suggest that sensory impairments are linked with steeper increases over time among White participants. Among Hispanics vision and hearing impairments were linked with higher initial levels of social isolation, yet no associations were found across time. It may be that Hispanic older adults maintain social connections across time despite potentially isolating sensory impairments."} +{"text": "Hagimoto According to updated follow up data,We will have an increasing number of cases of mRCC with durable radiological response to systemic immunological treatment since we have currently more intensified treatment for mRCC including nivolumab plus ipilimumab, avelumab plus axitinib, and pembrolizumab axtinib. Ideally, metastasectomy with curable intent should be considered for those with originally oligometastatic (low metastatic burden) disease when the surgery is technically feasible and expectedly achieves radiologically disease\u2010free status.The author declares no conflict of interest."} +{"text": "Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of largely unknown pathophysiology, characterized by the progressive loss of motoneurons (MNs). We review data showing that in presymptomatic ALS mice, MNs display reduced intrinsic excitability and impaired level of excitatory inputs. The loss of repetitive firing specifically affects the large MNs innervating fast contracting muscle fibers, which are the most vulnerable MNs in ALS. Interventions that aimed at restoring either the intrinsic excitability or the synaptic excitation result in a decrease of disease markers in MNs and delayed neuromuscular junction denervation. We then focus on trans\u2010spinal direct current stimulation (tsDCS), a noninvasive tool, since it modulates the activity of spinal neurons and networks. Effects of tsDCS depend on the polarity of applied current. Recent work shows that anodal tsDCS induces long\u2010lasting enhancement of MN excitability and synaptic excitation of spinal MNs. Moreover, we show preliminary results indicating that anodal tsDCS enhances the excitatory synaptic inputs to MNs in ALS mice. In conclusion, we suggest that chronic application of anodal tsDCS might be useful as a complementary method in the management of ALS patients. Motoneuron activity is disrupted during Amyotrophic lateral sclerosis (ALS), depriving these cells of activity\u2010dependent neuroprotection. Anodal tsDCS can modify motoneuron excitability promising a new approach for ALS management. Most interestingly, interventions with pharmacological or chemogenetic tools that aim at correcting the firing of the most vulnerable MNs prove to have some beneficial impact on the disease. After reviewing these data, we focus on trans\u2010spinal direct current stimulation (tsDCS) as a potential alternative therapeutic method in ALS. Indeed, electrical polarization by direct current is well\u2010known to modify spinal networks. We review recent work suggesting that tsDCS could be used to compensate for the changes in intrinsic excitability of MNs, or synaptic excitation, and hopefully to deliver some neuroprotection in ALS.1.1In ALS, some motor pools are more vulnerable than others prolongs survival in patients with ALS, albeit modestly display a normal excitability. A similar conclusion was reached by Venugopal et al. . In these conditions, Delestr\u00e9e et al. \u2010derived MNs from human patients have consistently reported the same time\u2010dependent excitability changes as in ALS mice. After an initial hyperexcitability , or to glycine\u2010receptor transmembrane domain for neuronal hyperpolarization (to decrease the excitability) (either from Ia spindle afferents or from descending systems) are functionally depressed in spinal MN activated the cAMP/PKA signaling pathway eliciting membrane insertion of GluR4 subunits and restoration of excitatory synapses. This elicits an improvement of disease markers through the enhancement of neuronal firing massively and specifically target MN, (b) control the cellular and immune responses, and (c) avoid side effects such as neoplastic tumors. There is evidently a real interest to develop noninvasive methods that could restore intrinsic excitability or synaptic excitation of MNs. In this framework, we will present new data showing that tsDCS may actually induce long\u2010lasting restoration of MN excitability and synaptic inputs, with the hope that tsDCS has the potential to deliver some neuroprotection in ALS.1.2cranial direct current stimulation (tDCS) . Altogether these results indicate that in SOD1G93A mice tsDCS evokes polarity\u2010dependent MN plasticity. Although the functional and survival analysis of tsDCS effects on ALS mice are ongoing, these preliminary findings already provide a proof of concept for further tsDCS application in ALS management.The main advantage of DC polarization is that it is not invasive and can be easily used in humans. However preclinical animal studies have to be performed before starting clinical trials. Preliminary results indicate that anodal tsDCS can enhance the excitatory synaptic inputs to MNs in the SOD1S Figure . InteresS Figure . Moreove2G93A mice. Pharmacological and chemogenetics interventions that aimed at restoring either the intrinsic excitability or the synaptic strength were shown to ameliorate the disease phenotype. In parallel, other experiments showed that anodal tsDCS enhances the intrinsic excitability of spinal MNs and the effect outlasts the stimulation period. Moreover, preliminary results show that anodal tsDCS also elicits long\u2010lasting enhancement of EPSPs in SOD1G93A mice, compensating the EPSP impairment observed in these mice. We suggest that chronic anodal tsDCS may be useful in the management of ALS patients.Recent experiments suggest that vulnerable MNs become intrinsically hypoexcitable (as seen in a loss of their ability to discharge repetitively) and that their excitatory synapses are impaired in the SOD1"} +{"text": "Halotropism is a sodium specific tropic movement of roots in order to obtain the optimal salt concentration for proper growth and development. Numerous results suggest that halotropic events are under the control and regulation of complex plant hormone pathway. This minireview collects some recent evidences about sodium sensing during halotropism and the hormonal regulation of halotropic responses in glycophytes. The precise hormonal mechanisms by which halophytes plant roots perceive salt stress and translate this perception into adaptive, directional growth forward increased salt concentrations are not well understood. This minireview aims to gather recently deciphered information about halotropism focusing potential hormonal aspects both in glycophytes and halophytes. Advances in our understanding of halotropic responses in different plant species could help these plants to be used for sustainable agriculture and other future applications. Halotropism a relatively new discovered type of tropism in plants, allowing them to escape from high salt by bending. Plant roots have ability to move from high salinity to avoid growth retardation or cell death. However, recently new findings show that some halophyte plant species require to obtain optimal salt concentration for their optimal growth . ContinuBassia indica or Limonium bicolor species, the halophyte relatives of Arabidopsis revealed that the basal and salt stressed induced expression of SOS1 was higher compared to the glycophytes which could be involved in early salt stress responses of roots. Three candidate genes specific for halotropic movements were determined: CHX13, WRKY25 and DOB1. Arabidopsis thaliana WRKY25 is coding a salt-inducible transcription factor which can mediate oxidative stress tolerance and senescence in a redox-dependent manner and also required for halotropic events , a flavonoid hyper-accumulating turfgrass species showed halotropic movements exposed to NaCl concentration gradients , a gaseous molecule can be a good candidate for regulating multiple signal pathways during halotropism. It is accepted that NO has basic and essential role in root development and also under stress conditions . NO can t stress . Recentlotropism . ZwiewkaProsopis strombulifera is a sesquiterpene plant hormone involved in halotropism. It has many functions in plant development and abiotic stress tolerance as a general inhibitor of growth mechanisms, like primary root growth . ABA is pathways . GWAS stpathways . The halArabidopsis thaliana accessions studied by Ethylene play a central role in an orchestrated process cooperating with other hormones in case of primary root growth and development . GWAS ofArabidopsis thaliana was stated (Strigolactones (SLs) are new players in signaling pathways of plants . Their ps stated . Wang J.s stated Figure 2+ content in leaves without any toxicity in tomato is a plant hormone belonging to plant phenolic secondary metabolites could imn tomato . SA indut stress . New evi process . Also, S process , providiArabidopsis as essential polycations are regulators of a plethora of developmental and stress induced alterations . Emerginmulation Office by Hungarian Ministry under the grant number FK129061 and the University of Szeged Open Access Fund (4786). The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Gut microbiome though physically contained within the gut lumen is capable of exerting far reaching effects regulatiXiong et al. have shown that butyrate intervention preserved gut barrier integrity by maintaining tight junction protein expression and reduced pancreatic immune cell infiltration. Earlier research has also highlighted that butyrate mediated histone deacetylase inhibition maintains intestinal barrier by regulating the expression of cytokines and mucins could thus be a therapeutic strategy against diseases associated with dysbiosis. However, research is needed to investigate ways to modulate gut microbiota. In a similar quest in their original research article, Liang et al. explored gut microbiome restorative potential of polysaccharides extracted from a traditional herbal medicine Dendrobium candidum. Far reaching effects of the gut microbiome are re-affirmed in their model of atopic dermatitis where restoration of normal gut microbiome promoted not only intestinal homeostasis but also mitigated the symptoms of the disease. Biliary disease is another example of gut microbiome and remote organ crosstalk (Zhang et al. offer comparative evaluation of endoscopic retrograde cholangiopancreatography and laparoscopic transcystic common bile duct exploration approaches in the treatment of bile duct stones. Articles covered under this Research Topic indicate the role of the communication between endogenous gut microbial members and host tissues and suggest the potential of microbiota modulation strategies as therapeutic tools. We hope you share our excitement in reading these articles.Alterations in the functionality and composition of the gut microbiome are implicated in several disease pathologies including most debilitating intestinal diseases, inflammatory bowel disease and colorectal cancer . Role ofrosstalk . High in"} +{"text": "It explains cancer biology in a new way. EVs classified some significant subclasses such as macrovesicle (originated from plasma membrane), apoptotic bodies (originated from the plasma membrane and endoplasmic reticulum), and exosomes (originated from endosomes).The authors declare no conflict of interest.There is no funding for this study."} +{"text": "To the Editor: Vazquez et al. report a convincing case of relapsing fever caused by Borrelia lonestari bacteria ticks ticks and causes illness that differs epidemiologically from traditional TBRF is the name given to illness caused by several genospecies of relapsing fever Borrelia miyamotoi disease (BMD) is problematic because it is species specific and cannot accommodate the discovery of related pathogens transmitted by ixodid ticks, including potentially B. lonestari , for related agents transmitted by argasid ticks.In the absence of a formal nomenclature decision by the World Health Organization, the following terms are consistent with precedent, epidemiologically useful, linguistically sensible replacements for TBRF: hard tick relapsing fever (HTRF) for illness caused by relapsing fever\u2013clade"} +{"text": "The mechanisms leading to Oxytocin\u2019s differential effects among patients with borderline personality disorder have thus far been elusive.This study was aimed to explore the differential effect of OT administration among depressive patients with or without comorbid borderline personality disorder, and to explore the mediating role of attachment in these differential patterns.Patients treated with psychotherapy in an inpatient settings (N=58) were randomized and double-blindly allocated to receive oxytocin or placebo for a period of four weeks. The effect of OT on therapy process and outcome was examined among patients with (n=35) and without (n=23) borderline personality disorder. Moderated mediational models were estimated to explore whether attachment differentially affected the association between oxytocin and treatment outcomes.patients without BPD showed significantly larger improvements following OT administration as compared to placebo in OQ-45. On the other hand, patients with BPD showed no significant improvement following OT . The same pattern was observed in the HSCL, where patients without BPD demonstrated significantly larger improvements following OT administration as compared to placebo, while patients with BPD demonstrated no significant improvement . Moderated mediational models indicated no significant moderated indirect effect, however, a significant trend of indirect effect only in the BPD group was observed, whereby the no-BPD group showed a stronger direct effect , whereas the BPD group showed a stronger indirect effect .Patients with depression and comorbid BPD benefit less from OT administration as compared depressive patients without such comorbidity. It is possible that the involvement of the attachment system may be associated with the attenuation of OT\u2019s effect.None Declared"} +{"text": "Heat and moisture exchanger (HME) filters are commonly used as passive circuit humidifiers during mechanical ventilation, however, are only ~80% efficient. As a result, patients that undergo mechanical ventilation in critical care with HME filter circuits will be exposed to partial airway humidification. This is associated with detrimental effects including increased secretion load which has been shown to be an independent predictor of failed extubation. Nebulised normal saline is commonly utilised to supplement circuit humidification in ventilated patients with high secretion loads, although there are no randomised control trials evaluating its use. Novel vibrating mesh nebulisers generate a fine aerosol resulting in deeper lung penetration, potentially offering a more effective means of nebulisation in comparison to jet nebulisers. The primary aim of this study is to compare the viscosity of respiratory secretions after treatment with nebulised normal saline administered via vibrating mesh nebuliser or jet nebuliser.This randomised controlled trial is enrolling 60 mechanically ventilated adult critical care patients breathing on HME filter circuits with high secretion loads. Recruited patients will be randomised to receive nebulised saline via 3 modalities: 1) Continuous vibrating mesh nebuliser; 2) Intermittent vibrating mesh nebuliser or 3) Intermittent jet nebuliser. Over the 72-hr study period, the patients\u2019 sputum viscosity and physiological parameters will be recorded by an unblinded assessor. A median reduction in secretion viscosity of \u22650.5 on the qualitative sputum assessment score will be deemed as a clinically significant improvement between treatment groups at analysis.At the conclusion of this trial, we will provisionally determine if nebulised normal saline administered via vibrating mesh nebulisation is superior to traditional jet nebulisation in terms of reduced respiratory secretion viscosity in intubated patients. Results from this pilot study will provide information to power a definitive clinical study.NCT05635903).ClinicalTrails.Gov Registry ( Critically unwell mechanically ventilated patients have decreased ability to effectively clear their own secretions. This is due to multiple factors such as impaired cough, low conscious level and respiratory muscle weakness. Patients with intercurrent pulmonary infection and aspiration are also likely to have an increased overall secretion load, compounding the problem of poor clearance . In crit2O) Patient confidentiality will be maintained throughout the study as per the Data Protection Act (2018). Recruited patients will be assigned a unique study number. All data collection sheets, study reports and communications regarding the study will identify patients by study number and initials only.Any adverse event or adverse device event will be recorded on a case report form and reported to the patients\u2019 assigned consultant and the principal investigator (PI). All serious adverse events will be similarly recorded and immediately flagged to the patients\u2019 assigned consultant, the PI, the research and development office and the assigned ethics committee for further investigation. Adverse event analysis with take place as part of this study\u2019s secondary outcome analysis.A variation of the approved protocol will be considered a protocol deviation. Any protocol deviations will be recorded on case report forms and reported to the sponsor.This study was approved by the NHS Greater Glasgow & Clyde Research & Development Ethics Committee (reference 19/SS/0116) and will be carried out in accordance with the World Medical Associated Declaration of Helsinki (1964) and its revisions (Tokyo (1975), Venice (1983), Hong Kong (1989), South Africa (1996) and Edinburgh (2000)).Authorisation for all publications will be sought from the study PI and will be reviewed by the sponsor prior to publication. The results of completed analysis will be published in a peer-reviewed scientific journal and at national/international conferences.th November 2019) for the study Click here for additional data file.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "Clear cell renal cell carcinoma (ccRCC) is the most common form of kidney cancer, but a comprehensive description of its genomic landscape is lacking. We report the whole genome sequencing of 778 ccRCC patients enrolled in the 100,000 Genomes Project, providing the most detailed somatic mutational landscape to date. We identify new driver genes, which as well as emphasising the major role of epigenetic regulation in ccRCC highlight additional biological pathways extending opportunities for drug repurposing. Genomic characterisation identified patients with divergent clinical outcome; higher number of structural copy number alterations associated with poorer prognosis, whereas VHL mutations were independently associated with a better prognosis. The twin observations that higher T-cell infiltration is associated with better outcome and that genetically predicted immune evasion is not common supports the rationale for immunotherapy. These findings should inform personalised surveillance and treatment strategies for ccRCC patients."} +{"text": "To the Editor,The prognostic impact of implantable cardioverter defibrillator (ICD) therapy in patients with nonischemic dilated cardiomyopathy (DCM) has been of great interest. OmranThe clinical impact of ICD implantation as primary prevention in patients with nonischemic DCM is accumulating.Another concern is the protocol of their study. The baseline characteristics of the two groups were not statistically matched,Medical therapy is of great importance to improve clinical outcomes in patients with nonischemic DCM. However, up\u2010titration of beta\u2010blockers or any other heart rate\u2010lowering medications is sometimes difficult because of patients\u2019 sinus bradycardia or atrioventricular block. Cardiac implantable electronic devices, including ICDs and cardiac resynchronization therapy, can secure minimum heart rate and assist up\u2010titration of these medications, facilitating reverse remodeling and improving mortality and morbidity.The authors declare no conflicts of interest."} +{"text": "Pheidole displays a remarkable degree of morphological differentiation and task specialization among its workers. Notably, there is considerable variation in head shape within worker subcastes of Pheidole, which could affect the stress patterns generated by bite\u2010related muscle contraction. In this study, we use finite element analysis (FEA) to investigate the effect of the variation in head plane shape in stress patterns, while exploring the morphospace of Pheidole worker head shapes. We hypothesize that the plane head shapes of majors are optimized for dealing with stronger bites. Furthermore, we expect that plane head shapes at the edges of each morphospace would exhibit mechanical limitations that prevent further expansion of the occupied morphospace. We vectorized five head shapes for each Pheidole worker type located at the center and edges of the corresponding morphospaces. We conducted linear static FEA to analyze the stresses generated by mandibular closing muscle contraction. Our findings indicate that plane head shapes of majors exhibit signs of optimization to deal with stronger bites. Stresses are distinctly directed along the lateral margins of the head, following the direction of muscle contraction, whereas the stresses on the plane head shapes of minors tend to concentrate around the mandibular articulations. However, the comparatively higher stress levels observed on majors' plane head shapes suggest a demand for cuticular reinforcement, like increased cuticle thickness or sculpturing pattern. Our results align with the expectations regarding the main colony tasks performed by each worker subcaste, and we find evidence of biomechanical limitations on extreme plane head shapes for majors and minors.Food processing can exert significant evolutionary pressures on the morphological evolution of animal appendages. The ant genus Pheidole workers, we solve a set of finite element analyses while exploring variation in Pheidole worker head morphospace. Our results demonstrate that head shapes of majors show signs of optimization to deal with stronger bites given that stresses dissipate more evenly along the head than in minors. In addition, our results agree with the expectations regarding the main colony tasks performed by each worker subcaste, and we find some evidence of biomechanical limitations on extreme head shapes for majors and minors.To assess the role of head shape in stress patterns of Among bilateral animals, the mechanical processing of food usually happens with the aid of head structures are specialized in reproduction, and wingless individuals execute quotidian colony tasks opens the mandibles, whereas craniomandibulares internus (0md1) is responsible for closing them . We aimed to investigate how the mean plane head shapes of majors and minors\u2014which are good representatives of the most common head shapes observed among Pheidole workers\u2014differ in stress patterns and if plane head shapes located at the extremes of the occupied morphospaces show stress patterns that suggest some mechanical constraints, which potentially could explain the low frequency of such plane shapes in extant Pheidole lineages. We hypothesize that the mean plane head shape of Pheidole major and minor workers will display substantial differences in stress patterns, with majors showing patterns associated with the capacity to deal with stronger bite forces than minors. In addition, we expect that the plane shapes located on the morphospaces' extremes will show distinct stress patterns from the mean plane shapes, with signs of mechanical limitations that prevent its higher frequency on current Pheidole lineages. Given that the amount of head cuticle that shows any sculpturing pattern varies intra and interspecifically among Pheidole species , we also tested if such variation is associated with stress patterns generated by the 0md1 contraction, hypothesizing that majors have an increased area of the head dorsal wall covered with sculptures than minors.Our intention was to investigate the mechanical behavior of idealized plane head shapes while exploring the morphospace limits of 22.1Pheidole species with particularly extreme morphologies by exploring the morphospace inferred by Casadei\u2010Ferreira et al.\u00a0 axes and the mean shape for each subcaste , which are good representatives of the morphological variation of the plane head shapes here considered. We defined more simplistic loading and boundary conditions to perform the mesh convergence tests and analyzed the variation in Tresca equivalent nodal stress values with changes in mesh density. Once the error between the current and last mesh densities in nodal Tresca stress achieved <2% in three different nodes, we chose the coarser mesh of the converged pair to represent the final mesh density. Mesh convergence was achieved at the same density in all three plane head models . We define the mesh density after a mesh convergence procedure of three plane head shapes , considering the coefficient of determination (R2) to analyze the convergence of PC scores and P. epem121 (PC2min) showed a stress pattern similar to that of the mean shape, whereas Pheidole biconstricta (PC1min) and Pheidole pallidula (PC2max) exhibited slightly reduced stress levels along the head, especially on the lateral margins showed a considerable amount of stresses along the posterior margin of the head, and Pheidole casta showed proportionally higher stresses along the lateral margins of the head , which has a narrower head, an opposite pattern was observed, with a denser concentration of stress on the central head region, especially of compressive stresses along the x\u2010axis and P.\u2009kohli (PC1max) showed similar stress patterns, having in general higher stress levels than P.\u2009hercules and P.\u2009obtusospinosa, and proportionally higher levels of vertical compressive stresses along the lateral margins than P.\u2009grallatrix, although at substantially lower levels than what happens in majors from species with a larger area covered by the lowest stress interval . P. absurda showed the highest mean stress value among all shapes (0.78\u2009N/mm2), whereas P. kohli showed the lowest mean stress was only slightly lower than for P.\u2009obtusospinosa (0.69) Table\u00a0.3.4Pheidole workers differed in the amount of sculpture covering the dorsal head cuticle . Specifically, minors of Pheidole species tended to have sculpturing patterns of categories 0 and 1 , and majors patterns of category 3 , whereas there was no difference regarding category 2 . The head shape of P.\u2009flavens showed a propensity to dissipate stresses along a larger area of the head than the head shape of P.\u2009obtusospinosa, where stresses tended to concentrate more around the regions of mandibular articulation. Interestingly, such head shapes showed only a slight difference in the mean non\u2010normalized stress value, with P.\u2009obtusospinosa having a slightly higher value than P.\u2009flavens. Such differences involving the most common head shapes observed among current Pheidole lineages suggest that majors are more suited to dissipate stresses and avoid stress concentration, being able to withstand higher forces related to bite loading when no other morphological aspects are being taken into account .Our FEA simulations demonstrated that plane head shape variation affects stress patterns due to P.\u2009flavens showing a more homogeneous distribution of stress intervals than P.\u2009obtusospinosa, which showed a larger area of the head covered with intervals of low stress. On PC2, their separation is more evident, with P.\u2009flavens showing a larger area of the head covered with intervals of intermediate stress levels when compared to P.\u2009obtusospinosa, which showed a slightly higher amount of head area covered by the highest stress interval, which is located around the regions of mandibular articulation. Most major heads have a higher coverage of high\u2010stress intervals than minor worker heads, agreeing with the patterns observed on tensor plots and color maps, which suggest a more favorable stress dissipation in major worker plane head shapes. The plane head shapes of P.\u2009epem121 and P.\u2009absurda are particularly distinct because they depart even more from the remaining plane shapes regarding the percentage of area covered with intermediate toward high\u2010stress values. These shapes also showed higher mean values of Tresca equivalent stresses. P.\u2009absurda and P.\u2009epem121 represent plane head shapes located in more isolated regions of their morphospace ; formal analysis (lead); investigation ; methodology ; writing \u2013 original draft (lead); writing \u2013 review and editing (lead). Marco A. Argenta: Formal analysis ; methodology ; supervision ; writing \u2013 original draft ; writing \u2013 review and editing . Alexandre Casadei\u2010Ferreira: Conceptualization ; data curation ; writing \u2013 original draft ; writing \u2013 review and editing . Marcio R. Pie: Conceptualization ; supervision ; writing \u2013 original draft (lead); writing \u2013 review and editing (lead).We declare we have no competing interests.Figures S1\u2013S3Tables S1\u2013S4Click here for additional data file.Data S1Click here for additional data file.Data S2Click here for additional data file.Data S3Click here for additional data file.Data S4Click here for additional data file."} +{"text": "The COVID-19 pandemic prompted health care organizations to create or revisit crisis standards of care (CSC) guidelines. The goal of these plans is to preemptively determine the most effective way to allocate resources under circumstances in which not all patients can be treated in accordance with general standards of care. In 2020, the American Burn Association (ABA) released professional recommendations for the development of CSC guidelines. Specifically, the ABA provided triage tables that could be incorporated into organizational CSC guidelines with the intention of assisting providers by predicting mortality based on a patient\u2019s age and burn size. These tables are often misinterpreted by non-burn providers who tend to overestimate burn size and conclude that a patient has a higher predicted mortality than they actually do. Inaccurate predicted mortalities can result in inappropriate care plans and ultimately impact patient outcomes. We argue that the ABA predicted mortality tables are helpful when used by experienced burn providers, but that there are inherent ethical risks when they are utilized by providers who lack appropriate burn care experience.We conducted a literature review of CSC guidelines that included specific instructions for the triage of burn patients. We reviewed 15 publicly available CSC guidelines, 8 of which specifically mentioned burn patients, and examined each to see if/how they utilized ABA triage tables for predicting burn patient mortality.Of the 8 CSC plans that acknowledged burn patients, 5 utilized the mortality predictors outlined in the ABA triage tables to guide resource allocation. In the majority of plans, patients with a predicted mortality of >90% were not eligible to receive treatment and/or transfer. None of the plans specified the involvement of a specialized burn provider in assessing burns.Our findings indicate that the expert evaluation of burns and thus predicted mortality can be the difference between receiving and not receiving potentially life-saving interventions under CSC guidelines. If a non-burn provider overestimates a burn size, for example, a patient may be undertreated for potentially survivable injuries under some CSC guidelines. This may further perpetuate health disparities given vulnerable populations are at higher risk of suffering burn injuries and often face a higher risk of morbidity and mortality.This project demonstrates the need for expert input from specialized burn providers in assessing burns, especially in CSC scenarios, to ensure these assessments are reliable and accurate. Involving expert burn providers in this process will help ensure ethically appropriate clinical care decisions are being made which will improve patient outcomes and resource management in crises and decrease provider distress."} +{"text": "Cannabis policy liberalization has increased the availability of cannabis products for medical and recreational purposes, following a growing public demand. The large number of cannabinoid users reporting self-medication for several mental health-related problems and the limited medical indications for cannabis prescription have led to prescription dilemmas and confronted views between patients and clinicians. This discrepancy in perspectives grows together with a huge terminological confusion regarding medical cannabis, as some subjects use recreational products for medical purposes. In this session we will outline the current controversies of cannabis prescription in psychiatry and discuss how to deal with (il)legitimate patient needs and prescription barriers. Finally, we will discuss research approaches that could shed light on this controversial topic.None Declared"} +{"text": "Thoracotomy with posterolateral incision (PLI) is commonly used for surgical repair of patent ductus arteriosus (PDA) in extremely low birth weight (ELBW) infants. Some reports have described thoracotomy for PDA using an axillary skin crease incision (ASCI) in consideration of cosmetic problems such as surgical wounds and thoracic deformities, but the details remain unclear.In this study, we performed clipping ligation by thoracotomy with ASCI for ELBW infants with PDA from 2011 to 2015 for the purpose of improving cosmetic results, and retrospectively compared the results with those for conventional PLI cases performed from 2016 to 2020.ASCI was found to be associated with serious surgical complications and showed a significant difference in outcome parameters only for surgery time, suggesting a safety problem for ASCI. Considering these results, PLI allows clipping of the nearby PDA from the thoracotomy wound while looking straight ahead, whereas the PDA in ASCI is positioned deep and oblique to the thoracotomy wound, so the clipping angle is limited and accurate completion of the procedure is difficult.Regarding PDA repair in ELBW infants, ASCI shows a high risk of serious surgical complications. Conventional PLI remains preferable for safe and accurate results. Repair of patent ductus arteriosus (PDA) in neonates and infants is usually performed by clipping ligation under thoracotomy through posterolateral incision (PLI), bringing good results \u20133. Some This study was undertaken with the approval of the Ethics Committee for Clinical Research at Himeji Red Cross Hospital, Hyogo, Japan (registration no. 2022\u201331). Informed consent was obtained from the guardian of each patient prior to surgery.From 2011 to 2020, a total of 31 ELBW (birth weight\u2009<\u20091000\u00a0g) infants with PDA who became symptomatic after failure of indomethacin administration underwent clipping ligation by thoracotomy. Nineteen cases underwent clipping surgery by ASCI between 2011 and 2015 and the remaining 12 underwent PLI between 2016 and 2020 . Demographic factors, clinical findings and blood test data at surgery were compared between ASCI and PLI using the Mann\u2013Whitney test or chi-squared test using SPSS II Statistics software (SPSS Inc.). Significance was defined as a value of P\u2009=\u20090.037; Table Among the 31 cases, no complications were encountered in any of the 12 PLI cases (0%), whereas 6 of the 19 cases of ASCI (32%) developed serious surgical complications than in PLI than for PLI Table ,6.Table These findings clarified that the markedly higher incidence of surgical complications and long surgery time were attributable to the ASCI procedure itself.Surgical treatment for PDA in ELBW infants is performed when medical management cannot stabilize symptoms due to a hemodynamically severe defect . Video-aIt has been reported that ASCI has been applied to open surgeries for congenital esophageal atresia and congenital pulmonary airway malformation, and that excellent results have been obtained in terms of cosmetic appearance \u20136. AlthoIn considering potential causes, we found that the PDA with the ASCI approach was located deep and obliquely from the thoracotomy wound, severely limiting the angle of clipping, requiring more time for surgery and reducing the accuracy of maneuvers Fig.\u00a0a. On theBleeding is considered rare in both open and thoracoscopic repair of PDA , 12, butASCI is a useful procedure for improving the cosmetic appearance in thoracotomy, but in PDA clipping for ELBW infants this approach may develop serious surgical complications due to the limited clipping angle. We emphasize that in cases of open surgery, PDA repair should be performed safely and accurately using the PLI."} +{"text": "To the Editor: We read with great interest the recent article by Thomas et al. that catalyzed retinal oxidation in embryonic tissues (Evidence of genetic/environment factors in the etiology of clefts have been proposed, with ethanol acting as a competitive inhibitor of NAD tissues . This fu tissues . It has tissues . Tooth cBecause earlier evidence indicates that congenital dentofacial anomalies have been found in cases of maternal alcohol exposure, it is imperative to conduct rigorous future investigations to assess whether paternal alcohol exposure also results in a similar pattern."} +{"text": "To the Editor:Atherosclerosis is an immune disease that can lead to the formation of atherosclerotic plaques.The detailed methods are described in the Supporting Information. The single\u2010cell profiling of immune cells in coronary plaque obtained from GSE184073We achieved 426 differentially expressed immunologically relevant genes (DEIRGs) Figure\u00a0. We thenThen we reconstructed the differentiation trajectory. Through uniform manifold approximation and projection dimensionality reduction, we cannot obtain several T\u2010cell clusters due to limited cells Figure\u00a0. Then weLater, we used the bulk transcriptomic data to validate our results. A total of 1010 and 6373 dysregulated DEGs were retained in human peripheral blood mononuclear cells in GSE59867 and GSE62646, respectively Figure\u00a0. The fun+ immune cells from the coronary plaques from the patients with ACS and SAP. A total of five immune cell clusters were distinguished and monocytes and T cells were considered as the targets for immunotherapies. We also validated our results in the bulk transcriptomic data and preliminary explored the transcription factor\u2010related regulatory mechanisms. In conclusion, we used relatively standard analysis pipelines to uncover immune cells subpopulations and their transcriptomic signatures in patients with stable and unstable coronary plaques with some preliminary validation of selected clusters and mechanism exploration of transcriptional regulation. Cross\u2010talk between monocytes and T cells may enhance plaques vulnerability and CEBPB is involved in the transcriptional regulation of atherosclerosis. Our findings provide important information for understanding the cellular and molecular mechanisms of coronary plaques vulnerability and may contribute to the atherosclerotic therapy.Single\u2010cell RNA sequencing is an ideal method to map the cellular and molecular composition of atherosclerotic plaque and can help to find new precise immunotherapies.The authors declare no conflicts of interest.Supporting InformationClick here for additional data file.Supporting InformationClick here for additional data file."} +{"text": "It was adapted by the Aged Care Quality and Safety Commission from a successful initiative in England\u2019s care homes. We describe TDONTD adaptation and evaluation of TDONTD to support improvement in UTI management in Australian residential aged care facilities.Data were collected in 12 facilities, from November 2021 to July 2022. Baseline and 3 month interviews were conducted with nurse and pharmacist champions implementing TDONTD and antibiotic prescribing audits.Australia\u2019s most widely accepted national guideline for assessment of residents with suspected UTI discourages urine testing in asymptomatic persons, however, is not explicit on when to (or not) perform dipstick testing in residents who do not meet minimum criteria for UTI. We adapted key resources including case-based education, clinical pathway for assessing residents for suspected UTI and antibiotic audit tool to incorporate national guideline recommendations around dipstick testing. Surveys with nurse champions of urine dipstick practice showed improved practice in the facilities related to reduced testing for asymptomatic bacteriuria (ASB) and increased use of formal protocol to guide dipstick testing practice and UTI diagnosis. Qualitative interviews with champions identified: (i) increased knowledge around ASB, clinical signs and symptoms of suspected UTI; (ii) increased confidence in not relying on dipstick testing to diagnose suspected UTI; (iii) TDONTD challenged and changed nurse perceptions of how residents present with UTI; (iv) pressure from external healthcare professional providers and family to perform dipstick testing was common; and (v) TDONTD was a feasible intervention in Australian RACF setting. Oral antibiotic prescribing for all indications, including UTI, improved with the intervention as did prescribing appropriateness for UTI treatment but not prophylaxis .Table 1We found that TDONTD supported improved clinical management of residents with suspected UTI in an Australian aged care setting. Knowledge, skills and environmental context and resources contributed towards practice change. External healthcare professional providers and family were barriers to change suggesting that education around dipstick testing should be expanded to broader community and hospital healthcare settings in Australia."} +{"text": "Accelerated biological ageing might contribute to the higher prevalence of age-related diseases and excess mortality amongst individuals with mental disorders. Recent advances in machine learning and the collection of high-dimensional molecular \u201comics\u201d data allow for the quantification of biological age.The aim of this study was to use machine learning methods to predict biological age from nuclear magnetic resonance spectroscopy metabolomics data and to identify psychiatric traits associated with accelerated biological ageing.The UK Biobank is a multicentre community-based observational study that recruited >500,000 middle-aged and older adults. 168 metabolomic measures were quantified using the Nightingale Health platform. Phase 1 release of these data included a random subset of 118,462 UK Biobank participants. Metabolomic age delta (MetaboAge\u0394) was defined as the difference between predicted biological age and observed chronological age. We estimated group differences in MetaboAge\u0394 between individuals with and without mental disorders and examined whether polygenic scores for mental disorders predicted MetaboAge\u0394.Up to 110,780 participants with complete data on all metabolomic measures were included in the analysis. Individuals with a history of mental disorders had higher MetaboAge\u0394 values than people without a mental illness. For example, MetaboAge\u0394 suggested that the difference between predicted biological age and observed chronological age was about two-years greater amongst individuals with bipolar disorder than amongst people without mental illness. Polygenic scores for mental disorders were positively correlated with MetaboAge\u0394.These findings suggest that individuals with a history of mental disorders or with higher polygenic scores for mental disorders were biologically older than their chronological age.None Declared"} +{"text": "Patients living with HIV are a vulnerable population, with high burden of chronic diseases and challenging social determinants of health. Many receive care in Ryan White grant funded multidisciplinary clinics in the community providing an opportunity to reimagine care delivery using innovative practices like shared medical appointments (SMAs). We designed and implemented lifestyle medicine (LM) interventions using interprofessional teams including one of the first described SMA programs for patients with HIV. Our pilot describes an iterative patient-centered model transferable and scalable to other HIV clinics or FQHCs for delivering lifestyle medicine to patients with HIV and other chronic infections.Learning objectives of oral presentationLearning objectivesShared medical appointments recruitment flyerRecruitment flyerWe piloted LM interventions both in individual patient care and via SMAs using interprofessional teams . SMAs were 90 minutes long, designed to include 5-15 patients conducted monthly focusing on one pillar of LM at each event . A whole health questionnaire was completed at the initial session, and American College of Lifestyle Medicine (ACLM) self-assessment on various pillars of lifestyle completed each session. Participants created a goal at the end of each session, and shared progress and key takeaways on return. Session guides were created for each session.Lifestyle assessment page 1 of 2Lifestyle assessment page 2 of 2Serial assessments at SMAs demonstrated tangible and sustained changes in various pillars of LM. Patient feedback indicated high patient and provider satisfaction; patients found group visits \u201cgrounding\u201d in their lives. Disclosing HIV status to other patients was not considered a barrier by any attendees. Other key lessons learned include the benefit of social support for patients, ease of conducting repeated assessments of the six pillars of LM using ACLM form, benefit of creating session detailed guides for future iterations and scalability, and the importance of signing up an adequate number of patients for financial sustainability.Lifestyle medicine shared medical appointments outcomes graphicSMA OutcomesPre vs post program assessment data details for consenting patientsPre vs Post program assessment dataOur pilot provides an iterative patient-centered model of SMAs easily transferable and scalable to other practices for delivering lifestyle medicine care to impact chronic diseases affecting patients with HIV and other vulnerable populations.Rahul Anand, MD MBA MSCI, Paratek pharmaceuticals: Stocks/Bonds"} +{"text": "At a rural Appalachian health clinic in Kentucky, 20% of patients under 18 years were not up to date with the CDC-recommended immunization schedule. Reasons parents or caregivers chose to delay or refuse their child\u2019s immunizations were explored using the Caregiver Vaccination Attitude Scale. High levels of trust in the healthcare provider and self-reported vaccine knowledge highlight opportunities for rural healthcare providers to apply evidence-based communication strategies to address vaccine hesitancy and promote the safety and health of the entire community. Parental refusal or delay of childhood immunizations has raised significant concern on a national level over the past decade.2Parents\u2019 reported lack of trust in their healthcare provider influences their decision not to vaccinate.6A chart review of patients who visited a primary care clinic in rural Kentucky during 2020 determined that approximately 20% of their pediatric patients were not in compliance with the current laws concerning school attendance and immunization schedule and guidelines.10Seventy-five percent of the respondents reported delaying a vaccination, despite high levels of trust in their healthcare provider (70%), and knowledge about where (95%) and when (85%) to receive vaccines. A large number of those surveyed (80%) reportedly felt they should be able to selectively choose the vaccines they believe their child needs and that it is better to develop immunity by getting sick than through vaccination (45%).Seventy percent agreed that they trust the care and information offered by nurses and nurse practitioners (NPs), likely indicating this is not the most important factor influencing vaccine delay and refusal among this clinic population. Data highlighted that parents/caregivers did not agree with the prospect of multiple vaccines administered in a single setting and find the overall number of vaccines to exceed what they believe is necessary. Eighty percent of parents agreed that people in their rural Appalachian community have expressed concerns that a child might have a serious side effect from a vaccination.Understanding benefits and perceived barriers for patients in a rural Appalachian health clinic may enable providers to effectively impact vaccination by cueing action through personalized approaches to education and decision-making support. The effectiveness of any vaccine communication approach assumes the patient considers their healthcare provider a trusted source of information. The parents/caregivers at this clinic reported high levels of trust in their healthcare provider and self-reported vaccine knowledge, but the corresponding low uptake of vaccines may indicate the need for a more strategic response to delay and refusal based on information from this survey.The presumptive and participatory approaches are frequently used by clinicians in vaccine communication.12Nurses and NPs in rural healthcare settings can build on existing trust and attempt to increase compliance with vaccine recommendations in the long term by acknowledging the underlying power dynamics of the patient\u2013provider relationship, strengthening rapport through dialogue, and showing concern and empathy via answering questions."} +{"text": "Viral genomics and epidemiology have been increasingly important tools for analysing the spread of key pathogens affecting daily lives of individuals worldwide. With the rapidly expanding scale of pathogen genome sequencing efforts for epidemics and outbreaks efficient workflows in extracting genomic information are becoming increasingly important for answering key research questions.Here we present Genofunc, a toolkit offering a range of command line orientated functions for processing of raw virus genome sequences into aligned and annotated data ready for analysis. The tool contains functions such as genome annotation, feature extraction etc. for processing of large genomic datasets both manual or as part of pipeline such as Snakemake or Nextflow ready for down-stream phylogenetic analysis. Originally designed for a large-scale HIV sequencing project, Genofunc has been benchmarked against annotated sequence gene coordinates from the Los Alamos HIV database as validation with downstream phylogenetic analysis result comparable to past literature as case study.https://github.com/xiaoyu518/genofunc.Genofunc is implemented fully in Python and licensed under the MIT license. Source code and documentation is available at: The online version contains supplementary material available at 10.1186/s12859-023-05356-3. In the past decades, next generation sequencing and the evolution of computational biology have brought forth traction in the field of large scale genomics with public health implications , 2 alongHuman immunodeficiency virus (HIV) is a rapidly evolving RNA virus being the largest viral pandemic pre-Covid with more than 37 million infected individuals in 2020 based on UNAIDS . InitiatHere, we present Genofunc, a tool mainly for the annotating of raw sequences with protein coding features by aligning them in-frame based on reference sequence(s). In combination with several other command line functions such as feature extraction and filtering, key genomic features can be extracted for downstream phylogenetic analysis through tools such as Nextstrain augur or BEASTGenofunc is an easily utilizable single command line tool fully scripted using python3 and can be installed through Python Package Index (PyPI) through pip install Genofunc. We test the Genofunc pipeline using whole genome HIV sequences downloaded from the Los Alamos HIV database as well as benchmark the estimated annotations from Genofunc against the Los Alamos annotated information. We validate the pol dataset extracted and infer the HIV root date and location with estimated clock rate using BEAST and compare results to past literature , 16.To allow easy utility and installation of Genofunc, the toolkit can be installed through GitHub or pip. This toolkit is fully written in python3.9 with functions constructed in a sequential fashion for easy pipelining mechanics of manipulating large raw HIV whole genome sequence datasets. Genofunc includes single command line functions consisting of two main components, FASTA/metadata file manipulation and HIV sequence processing Fig.\u00a0. For theTo identify the versatility and accuracy of the annotation and extraction of gene features using Genofunc, we downloaded and tested on three viral datasets, the West Nile virus, the Zaire ebolavirus and the Monkeypox virus, from GenBank and one HIV-1 dataset from Los Alamos. All complete sequences with fully annotated gene features were used as the test dataset summing up to 1211, 512, 914 and 6030 sequences respectively. Accession ID HQ596519, KJ660348 and NC_003310 were used as reference sequences for West Nile virus, Zaire ebolavirus and Monkeypox virus according to past literature . SimilarNext, we test the efficiency of the Genofunc pipeline using 16,832 whole genome sequences from the Los Alamos HIV database . ThroughPhylogenetic analysis was done in another pipeline using augur startingAlthough Genofunc was written originally written for processing HIV genome sequences, the package can be easily utilised for other viral sequences with a given reference genome. This is shown through the benchmarking test of different viral datasets against another annotation tool Geneious and comparable to the annotation information on GenBank/Los Alamos. The results are robust not only for a diverse viral dataset such as HIV-1 using a short list of reference sequences but also proving true for viruses that are annotated using only a single reference for estimating gene feature coordinates with high accuracy over all genes analysed. Genofunc also remains robust for annotating larger DNA genomes but at a slightly lower accuracy compared to shorter RNA viruses for extracting specific gene features . Future development of Genofunc lies in improving algorithm for reducing runtime in analysing longer genomes, improving accuracy in annotating gene features and correcting artefactual frameshifts that may be due to errors in sequencing or consensus genome calling pipelines.https://github.com/xiaoyu518/genofunc.In summary, Genofunc is a single command line toolkit suitable for constructing an automated pipeline to process large raw virus sequence datasets efficiently and readily for large scaled phylogenetics. Genofunc is open source and available with documentation at Additional file 1. Supplementary Figures."} +{"text": "Central venous stenotic disease is reported in 7%\u201340% of patients needing a central venous catheter for dialysis and in 19%\u201341% of hemodialysis patients who have had a prior central venous catheter. Half of these patients will be asymptomatic. Venous Thoracic Outlet syndrome in hemodialysis (hdTOS) is part of this spectrum of disease. The extrinsic mechanical compression of the subclavian vein at the costoclavicular triangle between the clavicle and 1st rib results in an area of external compression with a predisposition to intrinsic mural disease in the vein. The enhanced flow induced by the presence of a distal arteriovenous access in all patients exacerbates the subclavian vein\u2019s response to ongoing extrinsic and intrinsic injury. Repeated endovascular interventions during the maintenance of vascular access accelerates chronic untreatable occlusion of the subclavian vein in the long term. Similar to patients with central venous stenosis, patients with hdTOS can present immediately after access formation with ipsilateral edema or longitudinally with episodes of access dysfunction. hdTOS can be treated in an escalating manner with arteriovenous access flow reduction to <1,500\u2005ml/min, endovascular management, surgical decompression by first rib resection in healthy patients and medial clavicle resection in less healthy patients followed by secondary venous interventions, or finally, a venous bypass. hdTOS represents a complex and evolving therapeutic conundrum for the dialysis community, and additional clinical investigations to establish robust algorithms are required. Central venous obstructive disease represents a continuing challenge to the maintenance of dialysis access in patients with end stage renal disease. Central venous stenotic disease is reported in 7%\u201340% of patients needing a central venous catheter for dialysis and in 19%\u201341% of hemodialysis patients who have had a prior central venous catheter predisposes the patient to intrinsic mural disease within the vein. Unlike classical venous TOS, the enhanced flow induced by the presence of a distal arteriovenous access in all patients and the presence of or a history of a central venous catheter in many patients exacerbates the subclavian vein\u2019s response to ongoing extrinsic and intrinsic injury . PatientAsymptomatic: On routine duplex imaging or venography CCJ compression is seen or is induced by TOS position of the arm. The patient does not have symptoms and dialysis access is unaffected.Arm Swelling: Patients may experience upper arm and forearm swelling due to venous hypertension as a result of the higher pressures and high flow induced by the AV access. which may be so significant as to effect arm function. Sudden onset of arm swelling may indicate subclavian vein deep venous thrombosis. Imaging will confirm CCJ compression or stenosis.Arm Pain: Patients may experience arm pain with or with arm swelling, which may be exacerbated while on hemodialysis. Imaging will confirm CCJ compression or stenosis.Arterio-venous Access Malfunction: The access may have increased pressures in the venous circuit or present with thrombosis leading to an inability to obtain effective dialysis. Imaging will confirm CCJ compression or stenosis.Patients with a patent access site and central venous stenosis can present in several ways and many of these symptoms can mimic patients with stenosis solely due to CCJ compression (hdTOS):Due to the fact that hdTOS can overlap central venous stenosis in presenting symptoms, an array of imaging modalities of the thoracic outlet in hemodialysis have been described to examine the subclavian vein and the anatomy of the thoracic outlet \u20137. It isThe indications for intervention are symptomatic arm swelling and arm pain or AV access malfunction. The treatment algorithm is shown in In good-quality vascular access circuits on the same side as the symptomatic HDTOS, the flow should be assessed and categorized as high flow (>1.5\u2005liters/min) or acceptable flow (<1.5\u2005liters/min). Access circuits with high flow (>1.5\u2005liters/min) should be considered for a flow reduction procedure to achieve a flow of less than 1.5\u2005L/min . These pThe commonest direct intervention for TOS compression and CCJ-induced stenosis is the use of various endovascular techniques to correct the stenosis encountered during an intervention for malfunction arteriovenous access/ occlusion or a subclavian DVT. Unfortunately, there is no specific data on treated CCJ-associated stenosis in the current clinical data sets because all central venous stenoses are combined in the reporting of the venous intervention. Outcomes for endovascular intervention show that primary angioplasty has an overall patency of 48% to 100%, while outcomes for conventional stenting range from 78% to 100%. Reintervention rates in these patients range from 2 to 2.7 per patient per year. Covered stent grafting has better outcomes . There iThe Hemodialysis Reliable Outflow (HeRO) device is a vascular access system consisting of a large bore central venous catheter that allows a bypass of central venous stenoses or occlusions and a connector that allows connection with arteriovenous autologous, allograft, or prosthetic access in the arm . This deJugular vein bypass: A alternative option to TOS decompression is to bypass the subclavian vein with a jugular vein turn down, a venous or prosthetic conduit bypass from the axillary vein to the jugular vein on the same side evaluating surgical thoracic outlet decompression for subclavian vein stenosis at the CCJ in an attempt to salvage a threatened hemodialysis access . There hThe current Kidney Disease Outcomes Quality Initiative (K-DOQI) guidelines from the National Kidney Foundation currently do not recommend intervention for physiological compression or mild subclavian vein stenosis . In patihdTOS represents a complex and evolving therapeutic conundrum for the dialysis community and additional clinical investigations to establish robust algorithms are required. Currently, hdTOS represents a critical issue for further investigation and decompression of TOS should only be performed in carefully selected patients where the risk benefit analysis is appropriate and should only be carried out in centers with substantial experience in advanced decompression of the thoracic outlet.Study design: MGD and JPH. Obtain data: MGD and JPH. Statistical analysis: MGD and JPH. Data interpretation: MGD and JPH. Manuscript draft: MGD and JPH. Critical revision: MGD and JPH. All authors contributed to the article and approved the submitted version,The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Dermochelys coriacea), loggerhead turtles (Caretta caretta), hawksbill turtles (Eretmochelys imbricata), olive ridley turtles (Lepidochelys olivacea) and green turtles (Chelonia mydas). Our approach combines freely available digital elevation models for continental and remote island beaches across different ocean basins with projections of field data and SLR. Our case study focuses on five of the seven living sea turtle species. Under moderate climate change scenarios, by 2050 it is predicted that at some sea turtle nesting habitats 100% will be flooded, and under an extreme scenario many sea turtle rookeries could vanish. Overall, nesting beaches with low slope and those species nesting at open beaches such as leatherback and loggerheads sea turtles might be the most vulnerable by future SLR scenarios.Sea level rise has accelerated during recent decades, exceeding rates recorded during the previous two millennia, and as a result many coastal habitats and species around the globe are being impacted. This situation is expected to worsen due to anthropogenically induced climate change. However, the magnitude and relevance of expected increase in sea level rise (SLR) is uncertain for marine and terrestrial species that are reliant on coastal habitat for foraging, resting or breeding. To address this, we showcase the use of a low-cost approach to assess the impacts of SLR on sea turtles under various Intergovernmental Panel on Climate Change\u00a0(IPCC) SLR scenarios on different sea turtle nesting rookeries worldwide. The study considers seven sea turtle rookeries with five nesting species, categorized from vulnerable to critically endangered including leatherback turtles ( By the end of this century it is projected that SLR will reach 82 cm2 and\u2014in extreme scenarios\u2014could exceed 2\u00a0m3 with the early onset of Antarctic ice sheet instability4. Regional variation in predictions in SLR show that tropical regions and small islands are among the most vulnerable5, threatening species that depend on these coastal habitats, such as sea turtles8. Sea turtle species exhibit natal philopatry, returning to the beach where they were born9 with exceptionally high precision for returns to island rookeries10. However, climate changes might be too rapid for sea turtles to respond through their ability to disperse or colonize new habitats11. These biological traits and their reliance on sandy beaches make them particularly vulnerable to changes in coastal areas, like those resulting from SLR.Climate change has accelerated sea level rise (SLR) since the 1970s and is now more rapid than the mean SLR rate recorded during the previous two millennia12. These previous studies have been mainly regionally-focused, including assessments from only one or two species16. This regional focus is likely a result from the challenges inherent in successfully assessing shoreline response to SLR18. Although most sea turtle assessments have been obtained from field survey methods, such studies of estimations of stream reach water surface slopes often have low accuracy19. Other approaches which couple LiDAR with biological data21 have higher accuracy, but are also more costly22. However, new methodologies, such as the use of open Digital Elevation Models (DEMs) might be a good proxy broadly applicable to assess SLR by satellite images23.As a result, concern exists on the potential impacts of SLR on sea turtles, however only a dozen studies to date have projected how SLR will impact them18 and the inability to couple projections with biological information such as sex ratios and reproductive success12. Here we present an assessment of the potential impact of SLR on sea turtle rookeries by applying a low-cost methodology to estimate the probability of flooding of nest locations under multiple IPCC SLR scenarios. This approach combines turtle nest locations, freely available DEMs and Climate Central maps under Coastal DEM predictions22. The study considers seven sea turtle rookeries with five nesting species, categorized from vulnerable to critically endangered24 including leatherback turtles (Dermochelys coriacea), loggerhead turtles (Caretta caretta), hawksbill turtles (Eretmochelys imbricata), olive ridley turtles (Lepidochelys olivacea) and green turtles (Chelonia mydas) and have different characteristics , which will allow us to estimate SLR effects on a wide range of nesting rookeries and highlights the broad applicability of our approach.Considering that most sea turtle rookeries across the globe are located in remote areas in low and middle-income countries, less costly approaches for field surveys are often preferred and can provide baseline data to identify areas at most risk. Indeed, the few studies assessing the impacts of SLR on sea turtles to date discuss the challenges inherent in successfully predicting shoreline response to SLR and storm activitiesFrom GPS locations of 2835 marine turtle nests belonging to five different species from seven study areas across the globe, we estimated the vulnerability of nests to flooding considering available projections of SLR caused by climate change from 2010 to 2100 Fig.\u00a0. In addiThen, we estimated the probability of flooding from GPS nest locations georeferenced on CoastalDEM maps may be at greater risk from SLR than other species.To determine differences between modelled nest flooding probabilities by species we assessed rookeries containing multiple species , nesting habitat preferences and suitable nesting areas33.In Ecuador, no differences in modelled nest flooding severity were found between olive ridley, greens and hawksbill species, potentially due to beaches at this location being steeper3 might impact the reproductive output of sea turtles at the rookeries included in our study. Recent predictions of accelerating global SLR due to rapid melting of ice in Greenland40 and the Antarctic41 in combination with ocean currents42 indicate that pessimistic scenarios could be more accurate than conservative scenarios43. Such scenarios support our projections by indicating that sea turtle nesting populations could be vulnerable to flooding under even moderate scenarios over the next decades.Our models of nest flooding validated by field data considered that even a moderate increase in greenhouse emissions (RCP 4.5)44, drones, photogrammetry and GPS have been adopted to assess impacts of SLR on sea turtle populations12. However, most highly accurate methodologies entail high costs limiting their use to more localised studies. Considering that most sea turtle nesting populations around the world are located in low and middle-income countries, local conservation projects cannot afford the costs of these intensive methodologies to assess the vulnerability of nesting beaches. We have demonstrated that a methodology based on low-cost technological models can be a useful tool for predicting possible future SLR scenarios in important sea turtle nesting areas. We highlighted the utility of global open DEM data with high accuracies for remote areas that could assist with estimation of the vulnerability of sea turtle nesting populations worldwide.Relatively recent methods of remote sensing and modeling including DEMs45. Such assessments will help identify conservation refugia and nesting beaches that have greater resilience to climate change36. Although sea turtles have been around for millions of years and would be present in several climate change events, we do not know how their populations might be affected by these projected rapid changes of high loss of nesting sites in the study areas by 2050. Thus, this demonstrates the urgency of developing a multi-species assessment at a global scale in order to develop conservation plans for the most vulnerable populations while there is still time. Conservation management strategies are already in place to enhance resilience to SLR at some nesting beaches, including sand refilling of nesting beaches46 such as in Raine Island, relocation of nests to safe places47 or the protection of hatcheries for rookeries with extreme erosion and flooding31. In addition, we highlighted the need for climate change adaptation measurements to be implemented in management plans considering estimated projections under moderate SLR scenarios.Scientific assessments are essential for prediction of the impacts of future climate scenarios and to assist stakeholders and managers in anticipating extreme scenarios of coastal erosion or flooding, and to predict areas at higher risk of flooding48 and existing management strategies may then be insufficient to protect the future of many sea turtle populations worldwide. In summary, our study predicts massive flooding at important rookeries in Australia, Dominican Republic, Costa Rica and the USA. These critical areas will face the effects of SLR in the next few decades, meaning that it is now urgent to reduce anthropogenic emissions to safeguard the future of sea turtle populations against climate change and associated sea level rise.However, if the world maintains current carbon dioxide emission rates, worst-case scenarios might be vastly underestimated by 3\u20134 timesSupplementary Information."} +{"text": "Assessing ileal conduit for double J stents removal after radical cystectomy is not always a straightforward task as navigation inside the ileal loop can be challenging to manage due to the difficulty to maintain a waterfilled environment and its long and tortuous aspect.We present a novel technique using a flexible ureteroscope that aims to ease this common demand with simple and readily available tools.This technique has been successfully utilized in 2 patients now. No complications were documentedWe propose a novel surgical technique to improve endoscopic navigation in incontinent ileal loop urinary diversion. \u2022Flexible ureteroscope may be easily used through a cut-end 3-way 24F Foley catheter to facilitate endoscopic navigation into an ileal conduit.\u2022This technique may specially benefit ureteral stents withdrawal.\u2022This surgical approach is easily reproducible and based upon ordinary endoscopic equipment.\u2022Navigation is easier than using a flexible nephroscope. Moreover, maneuvering the flexible scope into it requires patience and dexterity as the incontinent nature of the conduit doesn't allow saline distension of the loop making it even more time consuming.The present manuscript describes an original method to facilitate navigation and double J stent removal in a patient with ileal conduit following radical cystectomy that uses readily available urological devices.2A new endourological technique has been developed to expedite double J stent removal in ileal conduits.The technique was performed in an elderly 72 years-old male patient who underwent uneventful robotic radical cystectomy with total intracorporeal ileal conduit urinary diversion for treatment of muscle invasive bladder cancer. He had a 6Fr double J stent placed in each ureter before completion of ureteroileal anastomosis, and stent removal was carried out 8 weeks after initial surgical procedure.2.12) was then inserted through the main opening of the 24F Foley catheter while keeping the 3rd way closed after conduit filling. The ureteroscope could be smoothly inserted through the drainage channel of the Foley catheter with no resistance (Patient was positioned in dorsal decubitus under general anesthesia and proper surgical prep was done. A 24Fr 3-way Indwelling Foley catheter was carefully cut off at the very distal tip with care not to damage the balloon. The catheter was then introduced into the stoma until the balloon was completely inside the conduit, then it was inflated with up to 5 mL of distilled water to keep the catheter in place and to avoid liquid backflow around the catheter. The ileal conduit was then filled with saline through the irrigation channel of the catheter by using a IV tubing set or a 60mL syringe . Fillingsistance . It is hsistance . Endoscosistance . Stent esistance . PatientProcedure was concluded in less than 40 minutes with no complications.3In 1983, Jarowenko and Bennet described the use of a single J stent in order to avoid stricture formation6. It is well known the advantages of placing a ureteral stent through the ureteroenteric anastomosis to aid the healing process during periods of up to 8 weeks of time.The most common urinary diversion following radical cystectomy is the ileal conduit for its expeditious execution even if performed fully intracorporeally in robot assisted laparoscopic approach. When using single J catheter, the distal end of the 90 cm long stent comes out of the ileal loop allowing its removal by simple manual traction.,Removing the double J stents from an ileal conduit can be tricky and time-consuming as the intestinal loop is often tortuous and long, making it difficult to navigate with a flexible cystoscope. In an attempt of improving endoscopic maneuverability throughout the ileal conduit the use of a gastroscope or a duodenoscope has been reported.Our technique offers the advantage of using a common urological device, readily available for most urologists worldwide; therefore, there is no need to use instruments urologists are not familiar with and it does not add costs to the procedure. The idea of using a cut-end standard Foley catheter has been previously described for diagnostic purposes such as to delineate the ileal conduit anatomy, known as loopogram.4Double J stent removal from ileal conduits can be expedite with a new and practical endourological technique. Moreover, the present technique may facilitate navigation with flexible ureteroscope for diagnostic and therapeutic procedures in patients with ileal conduits.None."} +{"text": "Critical Care describing the correlation between circulating levels of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activity in critically ill adults and their risk of delirium or coma [We read with interest the article Hughes et al. recently published in or coma . The impDelirium is a state of acute brain failure seen commonly in critically ill individuals, variably associated with inattention, positive symptoms such as hallucinations or delusions, and altered level of consciousness, for which no treatment exists . AlthougCholinergic deficits are also thought to underlie cognitive symptoms in adults with several types of dementias, including Alzheimer\u2019s disease, dementia with Lewy bodies, and vascular dementia , and theSeveral randomized controlled trials have evaluated the ability of AChEi to prevent or treat delirium \u201313; howeIn support of the hypothesis that patients with preexisting cognitive impairment would benefit from AChEi during critical illness, we recently reported a retrospective analysis of the MIMIC database of nearly 60,000 ICU admissions at Beth Israel Deaconess Medical Center in Boston . When olWe propose a reexamination of the role for AChEi\u2019s in delirium prevention and treatment during critical illness Fig.\u00a0. Given tWe look forward to the next generation of studies that employ novel molecular diagnostics in high-risk populations to identify new treatments that can prevent or treat delirium during critical illness and improve outcomes among these vulnerable individuals."} +{"text": "Treatment adherence in patients living with Bipolar Disorders can influence prognosis and quality of life. It is associated with an increased morbidity and healthcare costs.The aim of our study was to evaluate treatment adherence in a sample of patients living with Bipolar disorders and to determine factors associated with poor adherence.We conducted a cross sectional study where we included bipolar patients being treated in psychiatry department A. We developed a survey containing sociodemographic and clinical features. We used the medical adherence rating scale to evaluate treatment adherence.Our sample consisted of 100 patients with a mean age of 47,5 years old. Sixty seven patients were being treated for bipolar disorder type 1. Medication adherence rate was 64%.Factors associated with poor medication adherence were being single, an early age of onset, comorbid substance abuse disorder, severe treatment side effects and poor insight.Poor medication adherence is a major issue for people living with Bipolar Disorders. Clinicians should pay more attention to sociodemographic and clinical factors to predict and enhance treatment adherence.None Declared"} +{"text": "Understanding the assembly of plant-pollinator communities has become critical to their conservation given the rise of species invasions, extirpations, and species\u2019 range shifts. Over the course of assembly, colonizer establishment produces core interaction patterns, called motifs, which shape the trajectory of assembling network structure. Dynamic assembly models can advance our understanding of this process by linking the transient dynamics of colonizer establishment to long-term network development. In this study, we investigate the role of intra-guild indirect interactions and adaptive foraging in shaping the structure of assembling plant-pollinator networks by developing: 1) an assembly model that includes population dynamics and adaptive foraging, and 2) a motif analysis tracking the intra-guild indirect interactions of colonizing species throughout their establishment. We find that while colonizers leverage indirect competition for shared mutualistic resources to establish, adaptive foraging maintains the persistence of inferior competitors. This produces core motifs in which specialist and generalist species coexist on shared mutualistic resources which leads to the emergence of nested networks. Further, the persistence of specialists develops richer and less connected networks which is consistent with empirical data. Our work contributes new understanding and methods to study the effects of species\u2019 intra-guild indirect interactions on community assembly. Colonizer establishment produces fundamental building blocks that shape the structure of assembling pollination networks. In this model, while colonizers leverage indirect competition to establish, adaptive foraging by pollinators maintains species coexistence which produces nested networks. Among those perturbed communities are plant-pollinator networks, which support terrestrial biodiversity and pollination services to crops5. Understanding and predicting the emergent structure and dynamics of novel plant-pollinator networks is critical to anticipating how global change will affect the ecosystem services these communities provide8. Both direct interactions between plant-pollinator pairs and indirect interactions within guilds contribute to colonizers\u2019 establishment11. Indirect interactions within a guild (pollinators or plants) occur when multiple species share the benefits of a mutualistic partner. By sharing mutualistic resources, species can have indirect competitive or facilitative effects on one another13. Over the course of assembly, colonizer establishment produces core interaction patterns, called motifs15 which function as the building blocks of ecological networks16.Global change is driving novel assemblages of ecological communities through species invasions, extinctions, and range shifts19, few studies have investigated the role of indirect interactions20. Indirect effects are difficult to detect empirically22 because they require increasing the ecological and temporal scales of study in order to consider more species and interactions24. Nevertheless, indirect interactions, that propagate through short or long paths, are critical to the complexity and biodiversity of mutualistic networks28. Previous studies have used mathematical methods to determine the strength of indirect effects by accounting for positive and negative feedbacks28. Network motifs function as a complimentary tool to those methods by establishing a connection between indirect effects and network structure14. The trade-off is that considering indirect effects through longer paths requires larger and more complex motifs which are more difficult to interpret. For simplicity, here we only consider indirect effects among species sharing a direct mutualistic partner (a path of length two). Particularly in communities where plant-pollinator mutualisms are obligate, species within a guild sharing mutualistic resources will boost or hinder each other\u2019s reproductive success33.While the assembly of mutualistic communities is often driven by direct interactions between well-connected species35 categorized as static or dynamic. Static assembly models encode simple rules for the attachment of colonizers to networks37. These static models have been successful at generating structures consistent with empirically observed food webs. Several static assembly models have also been developed for mutualistic networks. These models, based on either preferential attachment to abundant generalists38 or trait compatibility40, have reproduced nestedness. Nestedness is a feature of plant-pollinator networks in which a core of generalists interact with generalist and specialist species while specialists mostly interact with generalist species41. Both topics \u2014 the emergence and the dynamic stability of nestedness \u2014 have been widely debated43, and to date they have been studied separately.Numerous models have been developed for the study of food web assembly51. In these models, assembly is performed through a series of colonization or speciation events, while species turnover is governed by a separate population dynamics model. Dynamic assembly models developed for food webs found that complex community structure arises only when networks maintain variability in niche breadth rather than trending towards uniform generalism or specialism51. We are aware of only one dynamic assembly model developed for mutualisms52. Becker et al.\u2019s assembly model52 produced nested networks at intermediate stages of assembly but ultimately resulted in non-nested networks composed of only specialists in the absence of demographic noise. This is because specialist pollinators extracted resources most efficiently and excluded indirect generalist competitors within their guild. In the presence of demographic noise, Becker et al.\u2019s assembly model52 maintained variability in niche breadth but still resulted in non-nested networks. Here, we develop a dynamic assembly model from a consumer-resource model of plant-pollinator population dynamics that accounts for adaptive foraging by pollinators53. That is, the pollinators\u2019 capability to behaviorally increase their foraging effort on the plant species in their diet with the most floral rewards available. Adaptive foraging strongly influences plant-pollinator community dynamics by partitioning pollinators\u2019 niches and providing higher quality visits to specialist plants56.Dynamic assembly models allow network structure to emerge and evolve with population dynamics to highlight the trajectory rather than the endpoint of assemblyOur contribution investigates the role of intra-guild indirect interactions and adaptive foraging in shaping the structure of assembling plant-pollinator networks. We do so by evaluating how colonizers\u2019 intra-guild indirect interactions influence core motif development in dynamic assembly models with and without adaptive foraging. Specifically, we ask the following questions regarding colonizers that successfully establish in the network: 1) How do intra-guild indirect interactions affect colonizer establishment? (2) Do colonizers competitively exclude intra-guild indirect specialists? (3) Do colonizers facilitate the establishment of subsequent colonizing intra-guild indirect specialists? (4) What are the core motifs characterizing assembling networks? . A simulation begins with an empty network in which three plant and three pollinator species are introduced at low abundances (hereafter attempted colonizers) every 2000 timesteps for a total of 50 colonization events. Attempted colonizers are attached randomly to the network given their niche breadth type . Specialists are introduced with degree one and generalists are introduced with degree drawn randomly from a uniform distribution between two and the maximum number of species in the opposite guild. This split in degrees reflects qualitative differences between specialist and generalist species in the model, which can be generalized to specialist and generalist species in empirical systems: specialist plants offer the most exclusive floral rewards and specialist pollinators perform the highest quality of pollination services. However, specialists of both guilds are vulnerable to disturbance due to more inflexible niches. Each colonizer is also assigned values for each parameter in our model (see Methods) by sampling a uniform distribution of a given mean and variance Table\u00a0. Only a 5 timesteps that each of our simulations take corresponds to approximately\u00a0144 generations of pollinators (see Methods). Given that pollinators commonly reproduce annually, each simulation roughly spans 144 years and colonization events occur every 3 years. This timescale reasonably corresponds to novel species assemblages forming as a result of species invasions, extinctions, and range shifts \u2014 rather than considering the assembly of a community over the course of a season or over longer evolutionary timescales.Considering the doubling time of the population dynamic model given the parameter values used, the 10Binary interaction networks produced by the assembly model with adaptive foraging were significantly richer Fig.\u00a0, less coWe developed four motif groups see Fig.\u00a0 to trackAmong colonizing plant species in the assembly model with adaptive foraging, specialists who share their one pollinator species with only generalist plants (\u201cSpec-Gen\u201d) establish at the highest rate establish at the highest rate establish at the highest rate and because the networks assembled by our model with adaptive foraging were the most similar to empirical networks and bumblebee species (Bombus spp.) such as Bombus terrestris. These species are highly generalized in the communities they invade and suppress native pollinator activity due to exploitative competition65. In terms of colonizers\u2019 niche flexibility enabled by adaptive foraging, empirical literature also supports that pollinator species with high niche flexibility are highly successful colonizers66.We found that colonizers establish when they have a competitive advantage over species of the same guild that share their mutualistic resources. For pollinators, successful colonizers were generalists who are the most abundant and have the greatest niche flexibility. This finding is consistent with empirical literature on the two most well-known invasive pollinators in the United States: the European honeybee . However, future empirical studies could test the following key results: (1) colonizers contribute fundamentally competitive motifs to assembling plant-pollinator networks, and (2) pollinator niche flexibility allows those motifs to persist over time. The first result would require testing whether the colonizer increases (facilitative) or decreases (competitive) the population growth rate of resident species in their guild with whom they share mutualistic partners, or, as a proxy, the abundance of mutualistic resources/services available to those species. The latter combines the quantity of resources/services the colonizer consumes and the quantity they generate indirectly via increasing the population growth rate of shared mutualistic partners. To evaluate this empirically between colonizing and resident pollinator species, one could measure the quantity of nectar consumed and pollination services provided to shared plants. The effect of pollination services on plant demography would then need to be empirically measured to determine whether the colonizer contributed a net positive (facilitative) or negative (competitive) effect on the nectar available to resident pollinators. The second result would require empirically measuring whether resident pollinators redistribute their foraging effort among plant species in their niche in response to the colonizer\u2019s establishment. This could be done by evaluating whether resident pollinators have wider realized niche breadths than they did prior to colonization and/or whether the distribution of foraging efforts among species in their niche has shifted. Finding evidence for these two results would allow empiricists predict the trajectory of assembling network structure, dynamics, and stability.In conclusion, our work advances the field of community assembly by unveiling how the transient dynamics of colonizer establishment influence network structural development over long timescales. Specifically, we found that colonizers leverage competition with species in their guild for shared mutualistic resources to establish and that adaptive foraging maintains the persistence of inferior competitors. This produces motifs in which intra-guild indirect species possess variable niche breadths which are the building blocks for nested networks.53 to simulate the population dynamics of each plant . A specialist species has only one interaction upon introduction while the degree of a generalist species is drawn randomly from a uniform distribution ranging from two to the maximum number of partners in the network. Among the attempted colonizers, only a proportion successfully establish in the network and between each colonization event the model reaches a steady state. Following establishment, colonizers\u2019 degrees are unrestricted. As a result, many species gain interactions the longer they persist in the network, and generalist species added to larger networks are likely to have higher degrees.Therefore, the 10Four motif groups were developed to characterize the indirect interactions of colonizing species. They are distinguished by whether the colonizing species is generalist or specialist, and whether they interact indirectly with only generalists or at least one specialist; diagrams of each can be seen in Fig.\u00a0Motif development Fig.\u00a0 was eval5) we measured the network\u2019s binary interaction structure (121 networks per assembly model). This included plant degree distribution, pollinator degree distribution, richness, connectance, pollinator: plant ratio, and nestedness. Nestedness was calculated for each network with the NODFc metric to compare across networks of varying size and connectance55. We performed one-sided Welch t-tests to statistically evaluate the hypotheses that networks assembled from the model with adaptive foraging are richer, less connected, have a higher pollinator:plant ratio, and are more nested than networks assembled from the model without adaptive foraging. The Welch tests were paired to compare networks populated by specialist plants and specialist pollinators at the same probability.At the end of each simulation (timestep 10Further information on research design is available in the\u00a0Supplementary InformationPeer Review FileReporting Summary"} +{"text": "Myiopsitta monachus) calls recorded in Uruguay and the United States, respectively. We also evaluated whether the identity information emphasized in introduced range calls changed over time. To place our findings in an evolutionary context, we compared our results with another parrot species that exhibits well-established and distinctive regional vocal dialects that are consistent with signaling group identity. We found that both native and introduced range monk parakeet calls displayed the strongest convergence at the individual scale and minimal convergence within sites. We did not identify changes in the strength of acoustic convergence within sites over time in the introduced range calls. These results indicate that the individual identity information in learned vocalizations did not change over short evolutionary timescales in populations that experienced the social disruption of introduction. Our findings point to exciting new research directions about the robustness or responsiveness of communication systems over different evolutionary timescales.Animals can actively encode different types of identity information in learned communication signals, such as group membership or individual identity. The social environments in which animals interact may favor different types of information, but whether identity information conveyed in learned signals is robust or responsive to social disruption over short evolutionary timescales is not well understood. We inferred the type of identity information that was most salient in vocal signals by combining computational tools, including supervised machine learning, with a conceptual framework of \u201chierarchical mapping\u201d, or patterns of relative acoustic convergence across social scales. We used populations of a vocal learning species as a natural experiment to test whether the type of identity information emphasized in learned vocalizations changed in populations that experienced the social disruption of introduction into new parts of the world. We compared the social scales with the most salient identity information among native and introduced range monk parakeet ( In some avian and mammalian lineages, vocal communication partially depends on social learning. Learned vocalizations may carry information important to communicate to others, including individual identity or group membership. The information encoded in learned vocalizations may change under different social conditions, such as the number of individuals available for social interactions. We used populations of monk parakeets introduced to the United States of America as a natural experiment of social disruption. We tested the ideas that the type of identity information encoded in learned vocalizations could either remain the same or change in introduced populations compared to native range populations in Uruguay. Using computational approaches, we quantified patterns of acoustic variation linked to identity information in learned vocalizations of native and introduced range populations. We found that individual identity information was more pronounced than group membership in learned vocalizations in each of the native and introduced ranges. The type of identity information important for monk parakeets to communicate appears to have remained the same despite social disruption that occurred over the last 50 years. While socially learned traits are considered very flexible, our findings suggest that the type of identity information encoded in learned vocalizations can be robust to population disruption over cultural timescales. Animals can use communication signals to transmit social information, including group membership, individual identity, social status, sex, or other social characteristics ,2\u2060. The Tursiops truncatus) and green-rumped parrotlets (Forpus passerinus) can use vocal learning to produce distinctive individual signatures used for individual vocal recognition [Vocalizations are well-studied communication signals that can contain identity information. For example, voice cues arising from vocal tract filtering can provide receivers with information about individual identity \u20139. Howevognition \u201327.These findings from the same or closely related taxa suggest that changes in the social environment could influence the identity information that animals encode in learned vocalizations. For instance, living in large social groups or interacting repeatedly with different individuals may favor signaling individual identity information, due to either the pressure of providing sufficient information for receivers to discriminate among unique individuals , or the Amazona vittata) exhibit distinct vocal dialects that have arisen over only a few decades, and translocated individuals will switch between dialects to call in the dialect of the local population [Amazona auropalliata), a juvenile translocated between regional populations also switched to calling in the local vocal dialect [Mirounga angustirostris), increasing population size appears associated with a change in the type of identity information encoded in learned vocalizations over short evolutionary timescales. As recovering populations grew in size over 50 years, vocal dialects were replaced by more structurally complex calls that displayed greater individual distinctiveness, which may facilitate male signaling in more crowded social environments [Heterocephalus glaber), individuals learn colony-specific vocal dialects during development. However, the type of identity information emphasized in learned vocalizations appears sensitive to social stability conferred by the presence of a queen. In a colony that lost two queens within a year, individuals\u2019 chirps became less colony-specific and more individually distinctive during two periods of social instability [Short-term changes in the social environment can influence variation within or among types of identity information in learned vocalizations, which could reflect novel changes to the identity information used, or switching among historical forms of identity signaling. For instance, captive and wild Puerto Rican Amazon parrots a way to quantify the relative salience of different types of identity information in learned signals and 2) the potential to compare identity information across groups with different social characteristics.First, new tools are needed to better quantify the salient types of information in vocalizations. Computational approaches like machine learning can be applied within a conceptual framework that links patterns of vocal convergence to identity signaling. Individuals should use vocal learning to converge on vocalizations across different scales of social organization , and sucSecond, we can compare hierarchical mapping patterns among groups with variation in population stability to test whether identity information in learned vocalizations is robust or responsive to disruption of the social environment. We can leverage different types of natural experiments for this comparison, including the introduction of species to new parts of the world, which can cause founder effects that influence traits transmitted by genetic inheritance and by social learning in introduced populations ,41. IntrMyiopsitta monachus) to test how social disruption that occurred generations ago, over the course of the past 50 years, could cause changes in the type of identity information encoded in learned calls. Parrots are suitable for this research because they can use social learning to both acquire and modify \u201ccontact\u201d calls, which individuals are thought to use to maintain contact with their social companions while flying and foraging [In this study, we focused on native and introduced range populations of monk parakeets as independent replicates of populations established following social disruption. Recent work with monk parakeets supports the idea that the introduction process, including transport out of the native range and housing in long-term captivity, represents a form of extreme social disruption. Under naturalistic conditions, removing even a single individual from an established social group consistently disrupts monk parakeets\u2019 dominance ranks . In the We used contact call recordings to infer which type of identity information was most salient in learned monk parakeet vocal signals. We used this approach on both native and introduced range contact calls to test whether the type of identity information was the same or differed between the native and introduced ranges. Previous work demonstrated that the strongest acoustic convergence in monk parakeet contact calls occurs at the individual scale for native range populations in Uruguay . In addiThis research was conducted under an approved Institutional Animal Care and Use protocol and an animal care and use protocol approved by la Comisi\u00f3n de \u00c9tica en el Uso de Animales .We recorded contact calls from native range monk parakeets in 2017 at 37 sites across 7 departments in Uruguay in our previous work . Our intRecording sessions in 2004 used Marantz PMD670 or PMD690 recorders with Sennheiser ME67K6 shotgun microphones, and these recordings were digitized at 48000 Hz and 16 bit depth . In all We manually selected contact calls from our field recordings. For our introduced range recording sessions in later years, we selected contact calls using Raven version 1.4 , consistWe obtained contact calls at two different social scales for the purposes of this study: the individual scale, and a group scale that represented a higher level of social organization. To assess contact call convergence at the individual scale, we repeatedly sampled known individuals to obtain multiple exemplar contact calls produced by the same individual. This individual-level dataset included 229 total contact calls from 8 native range birds recorded at 3 different sites in 2017, and 9 introduced range birds recorded at 7 different sites in either 2004, 2011, or 2019 (see Table A5 in ). Each iTo address contact call convergence at a group scale, we recorded and compared contact calls across nesting sites. We used nesting sites as groups because parakeets likely interact often with other individuals at the same nesting site. Monk parakeet nesting sites include clusters of single or multi-chambered stick nests that are often built in close proximity , and parAfter pre-processing, our site scale dataset included 1353 total contact calls recorded at 63 sites (37 native range sites and 26 introduced range sites). Some introduced range sites were repeatedly sampled in different sampling years (see Tables A3 and A4 in ). This dTo compare hierarchical mapping patterns between the native and introduced ranges, we used 37 native range sites separated by 0.15\u2013513.59 km across 7 departments in Uruguay, and 18 introduced range sites across 5 U.S. states that were separated by 0.74\u20133502.98 km . In our We used contact call similarity measurements to quantify hierarchical mapping patterns across different social scales. For instance, if individuals were converging on shared contact calls within sites, then we expected that contact calls compared within the same site would exhibit high similarity measurements, and lower similarity measurements when compared to contact calls from different sites. We measured contact call similarity with spectrographic cross-correlation (SPCC) , which hWe also measured similarity among monk parakeet contact calls using a supervised machine learning approach that identifies biologically relevant patterns of variation in avian acoustic signals ,74,75. AWe built our first model with the full set of 1844 acoustic and image features. We built a second model by performing automated feature selection and using the most important features from that analysis to reduce the dimensionality of the SPCC and random forests similarity matrices, respectively, with the MASS R package over many permutations, in order to address whether vocalizations compared within categories are more similar than vocalizations among categories . We used the emdist R package to calcuWe compared the relative magnitudes of Earth Mover\u2019s Distance calculations over time in two U.S. cities to determine whether the strength of acoustic convergence at the site scale changed over time in the introduced range. For these analyses, we used introduced range populations that we had repeatedly recorded in Austin, Texas and New Orleans, Louisiana. We calculated Earth Mover\u2019s Distance with the emdist package with ourWe placed our results in a broader context by quantifying and directly comparing hierarchical mapping patterns of native and introduced range monk parakeets with the yellow-naped amazon, a species well-known for having regional group identity information in their contact calls. These amazon parrots imitate the contact calls of conspecifics and exhibit distinctive regional vocal dialects that are audibly perceptible to humans . Such voFor our comparative analyses, we quantified hierarchical mapping patterns over the individual and site social scales for native and introduced range monk parakeets (separately), and over the individual, site, and regional dialect social scales for yellow-naped amazons. For yellow-naped amazons, we used previously published contact calls recorded in Costa Rica in 1994 . We measWe also designed a customized bootstrapping approach to quantify the strength of acoustic convergence at each social scale for native range monk parakeets, invasive range monk parakeets, and yellow-naped amazons that complemented and validated our analyses with Earth Mover\u2019s Distance. In this analysis, we randomly selected 5 SPCC similarity values within the given categories and 5 SPCC similarity values among the given category in each bootstrapping iteration for known repeatedly sampled individuals indicated that parakeets in each of the native and introduced ranges consistently produced contact calls that were distinctive from those of other birds . This reOur supervised machine learning results also pointed to strong acoustic convergence at the individual scale. The final random forests model that we used for prediction displayed high classification accuracy during training. The model classified contact calls back to the individuals that we used for training with 97.44% accuracy (95% CI: 93.57\u201399.30%). The mean \u00b1 SE balanced accuracy of our model\u2019s classification performance per individual (representing the averaged sensitivity and specificity) was similarly high for the 4 native range (99.00% \u00b1 1%) and 4 introduced range training individuals (98.75% \u00b1 0.75%). Finally, our analyses of the strength of acoustic convergence at the individual scale with Earth Mover\u2019s Distance also supported strong individual signatures in native and introduced range contact calls. The Earth Mover\u2019s Distance values that we calculated at the individual scale in each of the native and introduced ranges were of similar magnitude ; Introduced range mean and 95% CI: 0.131 , Table B in We found that individuals at the same site did not produce similar contact calls . When weWe compared our Earth Mover\u2019s Distance results across the 3 site scale datasets to determine how keeping or filtering out contact calls of potentially repeatedly sampled individuals affected our results at this social scale. While the Earth Mover\u2019s Distance statistics for the 3 native range site scale datasets were consistently low, values for the introduced range varied more across the site scale datasets. The introduced range Earth Mover\u2019s Distance values for each site scale dataset were uniformly greater than those we obtained for the native range datasets using each similarity method occurred at the individual scale for native and introduced range monk parakeets . AltSignaling individual identity information in learned vocalizations could instead reflect a more fixed aspect of vocal communication systems, such as developmental constraints or genetic encoding of receivers\u2019 perceptual abilities. Future work could also address the stability of individual identity information encoding in learned contact calls across different social contexts, given that some vocal learning species exhibit rapid convergence or divergence that appears conditional on the social context \u201339, and We performed a comparative analysis with yellow-naped amazon contact calls to place our ecological comparison of native and introduced range monk parakeet contact calls in an evolutionary context. If introduced range monk parakeets switched to emphasizing group membership information in contact calls, then hierarchical mapping patterns in introduced range monk parakeet contact calls should have exhibited stronger convergence at a higher social scale. We used yellow-naped amazons as a baseline for comparison because this species exhibits strong acoustic convergence at a higher social scale with regional vocal dialects that are audibly and visibly distinctive to humans ,30,31,94Our comparative analysis also highlighted the importance of using quantitative tools to complement human perception of audible and visible variation in avian vocalizations. When relying on the human ear and eye, the variation among regional dialects in yellow-naped amazon contact calls is far more perceptible than individually distinctive monk parakeet contact calls. For example, the regional dialects that we recapitulated in the amazon contact calls are distinctive to the human ear , includiEupsittula canicularis), which display the greatest auditory sensitivity in a frequency band that overlaps with the greatest spectral energies in contact calls [Amazon vocal dialects may be more perceptible to humans than monk parakeet individual vocal signatures because of humans\u2019 limited abilities to perceive fine-scale temporal variation at higher frequencies ,96. Parrct calls . In addict calls ,98. OverWe combined computational tools with a conceptual framework of how hierarchical mapping patterns are connected to identity signaling in animal vocal signals. This combined approach allowed us to quantify hierarchical mapping patterns and then infer the most salient identity information encoded in vocal signals. Similar computational approaches could be applied to quantify hierarchical mapping patterns with existing datasets of animal signals to learn more about the social environments in which individuals communicate across a broader range of taxa, without depending on the time-intensive collection of social data from marked individuals. When communication signals are learned, hierarchical mapping patterns should capture overall patterns of acoustic variation that represent both active convergence or divergence within social groups, as well as the side-effects of learning from others in a given social group . Here, we used the social scale with the strongest acoustic convergence to infer which type of identity information animals are actively encoding in learned vocalizations (e.g. the type of identity information that is most important to communicate). In our conceptual framework, we considered stronger acoustic convergence as active convergence, and weaker patterns of acoustic convergence as stochastic outcomes associated with learning. For instance, monk parakeet contact calls recorded at the same site did display a degree of convergence (Table B in Acryllium vulturinum) and superb fairy-wrens , exhibit multilevel social structures in the wild, suggesting that hierarchical social structures may be more taxonomically widespread than traditionally thought [Whether and how animals perceive and use stronger or weaker patterns of acoustic convergence in learned vocalizations can be assessed experimentally using playbacks of contact call variants. Indeed, the hierarchical mapping patterns identified for a particular population or species can be used as an important foundation for designing biologically relevant playback experiments, which can be more time-consuming than recording communication signals, and are fundamental to understanding how receivers use the information that signalers communicate. Playback experiments are important because mismatches can occur between the social information encoded in signals and the information that receivers use for social recognition, especially when it is cognitively costly to track certain types of information ,99. Addr thought ,101.Eolophus roseicapillus) [While quantifying hierarchical mapping patterns can yield exciting insights into the identity information that may be important to communicate, researchers should be careful when using these patterns to inform new research directions about identity signaling and social systems. Recording unmarked individuals in natural populations provides only a snapshot of dynamic social interactions, as well as the social information conveyed in signals that is important in a given social environment. For instance, sampling a few vocalizations per individual over a short time frame makes it difficult to assess how identity information encoding may change during dynamic social interactions, such as the rapid vocal matching exhibited by wild orange-fronted conures and rose-breasted cockatoos (apillus) ,38,39. Iapillus) ,28,29,99We used native and introduced range monk parakeet contact calls to test whether the type of identity information encoded in learned vocalizations changed in populations that were established after social disruption that occurred over the last 50 years. We used computational tools, including supervised machine learning, to quantify and compare hierarchical mapping patterns in contact calls between the native and introduced ranges. We inferred that identity information encoding was robust to social disruption over short ecological timescales. By comparing hierarchical mapping patterns between monk parakeet and yellow-naped amazon contact calls, we found that identity information encoding in learned parrot vocalizations changed over longer evolutionary timescales. Our results suggest that signaling systems facilitated by socially learned vocalizations can be robust to changes in social conditions over short timescales, despite the flexibility generally attributed to socially learned behaviors. Taken together, our findings point to exciting new research directions on the flexibility or robustness of socially learned communication signals over short evolutionary timescales.S1 AppendixThis document provides more details about the datasets that we used as well as each of our customized analytical pipelines with monk parakeet and yellow-naped amazon contact calls. This appendix also contains Tables A through E.(PDF)Click here for additional data file.S1 FigAll 4 panels show SPCC acoustic space generated by multidimensional scaling (MDS) for contact calls of repeatedly sampled monk parakeets in each of the native and introduced ranges. Top left panel: 4 native range individuals that were used to train supervised random forests models. Bottom left panel: 4 introduced range individuals that we used to train supervised random forests models. Top right panel: 4 native range individuals were used to validate supervised random forests models. Bottom right panel: 5 introduced range individuals that were used to validate supervised random forests models. Blue palettes correspond to the native range and gold-brown palettes to the introduced range. In each panel, points represent different calls per repeatedly sampled individual. Individual identities are displayed through shapes and hues per range, and convex hull polygons demonstrate the area encompassed per individual in acoustic space. The acoustic space across all 4 panels can be interpreted on the same axes. Here, individuals were overdispersed in acoustic space, pointing to strong individual signatures in each range. These results were similar to our findings with random forests similarity .(TIFF)Click here for additional data file.S2 FigPlots of random forests acoustic space are shown by similarity method (columns), as well as the three datasets used to address repeated individual sampling in each of the native and introduced ranges (rows). Acoustic space for the clustering and visual classification datasets were generated by filtering multidimensional scaling (MDS) coordinates for the full dataset of calls. The 4 sites shown here and the aesthetics used per range are the same as in (TIFF)Click here for additional data file.S3 FigThese results were calculated using spectrographic cross-correlation similarity. The means and 95% confidence intervals (CIs) were obtained by summarizing across 100 resampling iterations for each of the 6 total bin numbers. The calculation used to report results in the main text (16 bins) is shown as a red \u201cX\u201d. The 95% CIs are small and are not visible around the mean.(TIFF)Click here for additional data file.S4 FigThese results were generated using spectrographic cross-correlation and random forests similarity, as well as the three site scale datasets used to address repeated sampling of unmarked individuals. The means and 95% confidence intervals (CIs) were obtained by summarizing across 100 resampling iterations for each bin number. The calculation used to report results in the main text (16 bins) is shown as a red \u201cX\u201d. The 95% CIs are small and are not visible around the mean.(TIFF)Click here for additional data file.S5 FigThese results were generated using spectrographic cross-correlation and random forests similarity, as well as the three site scale datasets used to address repeated sampling of unmarked individuals. The means and 95% confidence intervals (CIs) were obtained by summarizing across 100 resampling iterations for each bin number. The calculation used to report results in the main text (with 16 bins) is shown as a red \u201cX\u201d. These 95% CIs are also small and are not visible around the mean.(TIFF)Click here for additional data file.S6 FigThese results were generated using spectrographic cross-correlation and random forests similarity, as well as the three site scale datasets used to address repeated sampling of unmarked individuals. The means and 95% confidence intervals (CIs) were obtained by summarizing across 100 resampling iterations for each bin number. As above, the calculation used to report results in the main text (with 16 bins) is shown as a red \u201cX\u201d, and the 95% CIs are not visible around the mean.(TIFF)Click here for additional data file.S7 FigEach bar represents the mean percentage of monk parakeets observed relative to other species, averaged across weeks per year. The error bars denote the standard error. Gold rectangles highlight the sampling years in which monk parakeets were recorded in each city.(JPEG)Click here for additional data file.S8 FigPanels A, B, and C show density curves of SPCC values for native range monk parakeets, introduced range monk parakeets, and yellow-naped amazons, respectively. Each density curve was generated from the full symmetric matrix of similarity values for the given species and range . Panel D shows acoustic space for yellow-naped amazon contact calls, and points are colored by three regional dialects reported in Costa Rica by (Nor = N(JPEG)Click here for additional data file."} +{"text": "HIV testing is a critical strategy for the prevention of HIV/AIDS yet testing among sexually experienced adolescents remains low. This study examined the prevalence of HIV testing and its influential factors among high school students in the USA.We analyzed cross-sectional data from the 2019 National Youth Risk Behavior Survey. Analysis was limited to 3123 complete cases of sexually experienced high school students as measured by ever having sex. Chi-square and binary logistic regression analyses involved three models comprising predisposing, enabling and need factors were conducted to study lifetime HIV testing.The prevalence of lifetime HIV testing was 18.8% with females getting tested more than males. Female gender , being Black , and sexual debut below 13 years were positively associated with lifetime HIV testing. Further, we observed that having multiple sexual partners and mild physical activity were significantly associated with HIV testing. Lastly, we noted that Hispanics/Latinos and those using condoms were less likely to have reported ever having an HIV test.Our study revealed that HIV testing among sexually experienced high school students remains low and sexual risk behaviors that impede testing among students need to be addressed to effectively scale up testing for this vulnerable population. The implication of this study to the relevant stakeholders responsible for creating a healthier nation is to strengthen the current efforts targeting sexually experienced high school students to encourage HIV tests and linkage to care.All Authors: No reported disclosures"} +{"text": "Previous studies have documented geographic variation in preventable hospitalizations between rural and urban areas, but much less is known about preventable hospitalization patterns between heterogeneous rural areas. Unique challenges related to access of care and poverty may put the rural Appalachian Region at risk for higher rates of preventable hospitalizations.This study examines whether within-rural differences in Kentucky\u2019s preventable hospitalization rates exist and how these differences may be changing over time.Longitudinal and geographic trends in county-level preventable hospitalization rates were examined using Kentucky hospital discharge data from 2016 to 2019. Regression models were run to determine whether changes over time in preventable hospitalization rates led to an increasing or decreasing gap in outcomes between rural Appalachian counties and their urban and rural non- Appalachian counterparts.p < 0.01). A downward trend in overall preventable hospitalizations was observed for rural Appalachia over time, but trends were relatively stable for rural non-Appalachian and urban counties. Regression results indicate that there was no significant reduction in the \u201cAppalachian gap\u201d over time.Rural Appalachian counties consistently had significantly higher preventable hospitalizations rates compared to their rural non-Appalachian and urban counterparts ( The analyses confirm that rural areas within Kentucky experienced highly heterogeneous rates of preventable hospitalizations. Despite Medicaid expansion, there is little evidence of any narrowing of the \u201cAppalachian gap.\u201d Focus on improving access to care alone may be insufficient to improve outcomes. Alternative strategies that leverage population health approaches may improve capacity to address complex health and social needs in rural Appalachia. Preventable hospitalizations are costly to our healthcare system and negatively impact individual quality of life.4While previous studies have documented geographic variation in preventable hospitalizations between rural and urban areas, most research to-date has used a standard urban\u2013rural subgrouping to examine differences.5More research is needed to determine whether within-rural differences in preventable hospitalizations also exist for other conditions and populations and how these differences may be changing over time. To address this gap, this study examines geographic variation in four composite measures of preventable hospitalizations in Kentucky for the period 2016\u20132019. Kentucky contains diverse rural regions and ranks among the top five states with the highest rates of preventable hospitalizations.7additional files section for a list of conditions in each PQI). In alignment with AHRQ\u2019s methodology for examining preventable hospitalizations, PQIs were calculated at the county-level based on the patient\u2019s county of residence. The data include all preventable hospitalizations at inpatient facilities for individuals aged 18 or over in Kentucky.Public use hospital discharge data for the state of Kentucky from 2016 to 2019 were obtained directly from the Kentucky Cabinet for Health and Family Services. The Agency for Healthcare Research and Quality\u2019s (AHRQ) Quality Indicator (QI) software was used to calculate annual preventable hospitalization measures.Counties were classified as urban, rural Appalachian, or rural non-Appalachian to examine geographic variation in preventable hospitalization rates. Counties were categorized as rural based on their \u2013urban commuting area (RUCA) code. Although several rural classification systems exist, RUCA codes were selected to align with the Federal Office of Rural Health Policy\u2019s definition. Using RUCA codes also allows for a more granular approach to how rural and urban communities are identified, helping to eliminate some of the homogeneity in a strict urban v. rural classification. Appalachian status was identified using the Appalachian Regional Commission\u2019s listing of counties. Counties that were both urban and Appalachian were categorized as urban for the purpose of this analysis. The final Kentucky sample included four years of preventable hospitalization measures for 35 urban, 49 rural Appalachian, and 36 rural non- Appalachian counties.A longitudinal ecological study was conducted to examine trends in preventable hospitalization rates across the Kentucky regions. Trends in all four composite preventable hospitalization measures were examined by geographic region using mean values of each PQI measure in each of the three regions. Values were weighted based on the population size of the county. Models were run for each PQI composite to determine whether changes over time (year) led to an increasing or decreasing gap in outcomes between rural Appalachian counties and their urban and rural non-Appalachian counterparts.9Rural Appalachian counties consistently had significantly higher preventable hospitalizations rates compared to their rural non-Appalachian and urban counterparts . Results also confirmed that overall preventable hospitalizations and acute preventable hospitalizations were falling over time (p < 0.10), diabetes-related preventable hospitalizations were increasing over time (p < 0.01) and chronic preventable hospitalizations did not change significantly over time. Finally, results suggest that there was no significant reduction in the Appalachian gap over time.Results from the regression models confirmed trends observed in the longitudinal figures and provide insight on the statistical significance of changes over time. Specifically, results , p. 11 iAnalyses of Kentucky hospital discharge data for 2016\u20132019 confirm that rural areas within Kentucky experience highly heterogeneous rates of preventable hospitalizations. Specifically, there is significant evidence of an \u201cAppalachian gap\u201d between rural Appalachian and rural non-Appalachian counties. Rural non-Appalachian counties have outcomes far closer to urban Kentucky counties, apart from acute preventable hospitalizations. While the remoteness of Appalachian counties, along with their history of poverty, make this result somewhat intuitive, it is unclear how much attention additional inequities in the region have garnered among policymakers. In light of these findings, policies and programs that target rural areas may need additional tailoring to ameliorate the \u201cAppalachian gap.\u201d For example, efforts to enhance broadband connectivity in rural areas have experienced some success in improving telehealth access; yet they face significant logistical barriers in rural, mountainous areas. Similarly, transportation challenges in accessing health and social services may be particularly challenging because of the geographic landscape. Addressing the extreme poverty and associated unmet social needs in Kentucky\u2019s rural Appalachian counties may be essential to seeing any real progress in health outcomes. Additional research is needed to determine whether the identified in this study also exists in other states with heterogeneous rural areas.p < 0.10). Since Kentucky is a Medicaid expansion state, these results are consistent with previous work that found Medicaid expansion was associated with reductions in a variety of PQI measures across 36 states between 2009 and 2015.These analyses also highlight reductions in overall preventable hospitalizations and acute preventable hospitalizations over time p < 0.. Since KVery little evidence of any narrowing of the \u201cAppalachian Gap\u201d between 2016 and 2019 was found, despite the enhanced access to care associated with 2014 Medicaid expansion in Kentucky. While this is disappointing, other research on the impact of the Affordable Care Act (ACA) and Medicaid Expansion has made similar findings. That is, while ACA and Medicaid expansion have been associated with health improvements across population groups, the disparities in outcomes between resourced and underserved groups have not necessarily narrowed.As the results indicate, more tailored approaches could yield meaningful improvements in preventable hospitalizations in rural Appalachia. Emerging evidence suggests several possibilities, including investing in public health and social service systems and supporting multisector linkages to better address social determinants of health.What is already known about this topic?Preventable hospitalizations are costly, common and have been attributed to lack of healthcare access and inadequate disease management. Prior research has also documented higher preventable hospitalizations in low-income, rural U.S. counties.What is added by this report?This report explores whether there are significant differences in preventable hospitalizations between heterogenous rural areas. In Kentucky, rural Appalachian counties may be at particular risk for preventable hospitalizations because of unique problems with access to care and extreme poverty.What are the implications for future research?Findings show a significant \u201cAppalachian gap\u201d in preventable hospitalization rates and little evidence of any narrowing of this gap over time (2016\u20132019). More tailored approaches may be needed to close the gap in health outcomes between rural Appalachian and rural non-Appalachian areas."} +{"text": "Indwelling urinary catheters (IUC) are reinserted in patients who should initially be managed with intermittent straight catheterization for urinary retention. We hypothesize patients who have an IUC reinserted within 24 hours will be at higher risk of catheter-associated urinary tract infections (CAUTI) within 7 days compared to patients who were straight catheterized or did not have an IUC reinserted.A retrospective review of electronic health records (EHR) using Epic Systems was conducted for all inpatients at Stanford Hospital, Palo Alto, CA who had an IUC removed and a CAUTI defined by the National Healthcare Safety Network (NHSN) between January 1, 2020 to March 31, 2023. Patients with an IUC reinserted within 24 hours from initial removal of an IUC were compared to patients who had an IUC removed and did not have a reinsertion of an IUC within 24 hours. Relative risk of a CAUTI was the primary outcome metric.Between January 1, 2020 and March 31, 2023 there were 30,161 IUCs removed and 204 NHSN defined CAUTI identified within 7 days of removal that were included in the study. Patients who had an IUC reinserted within 24 hours had significant risk of having a CAUTI within 7 days post IUC removal compared to patients who did not have an IUC reinserted within 24 hours.Reinsertion of IUCs in patients with urinary retention following IUC removal should be managed with intermitted straight catheterization or more conversative methods if possible. Patients who are re-catheterized with an IUC within 24 hours of removal are at significantly higher risk of developing a CAUTI within 7 days compared to patients who are straight catheterized or do not have a Foley catheter reinserted. Healthcare providers should use alternative methods of bladder management to improve patient outcomes.All Authors: No reported disclosures"} +{"text": "An exam fee discount is available to Heart Rhythm Society members.Exam topics/questions strive to draw out evidence of the high level of competency expected of an arrhythmia specialist by drawing attention to areas of fundamentals of electrophysiology and electronics (devices) as well as the immensely multifaceted topic of rhythm recognition.\u2022Physics of electrophysiology\u2022Cardiac anatomy and physiology\u2022Pharmacology\u2022Fundamentals of electrophysiology\u2022Electrocardiogram and recognition of dysrhythmias\u2022Clinical assessment, laboratory considerations (CEPS)\u2022Invasive electrophysiology (CEPS)\u2022Implantable devices\u2022Applied science and technology (CCDS)\u2022Device and lead function/malfunction (CDRMS)\u2022Remote service management, device monitoring \u2022Real-time and diagnostic imaging\u2022Research methodology and interpretationThough not explicitly complete, the following list, as taken from the IBHRE handbook,Test candidates are encouraged to use exam prep resources provided on the IBHRE website, including suggested reading lists and links to websites providing further educational content. The importance of resourcing various textbooks, cardiac journals and articles, and individual clinical cardiac experience is emphasized. Consulting with a mentor from the IBHRE Ambassador program provides anecdotal advice derived from the personal experience of a highly knowledgeable and certified heart rhythm practitioner.The IBHRE has transitioned to a new approach of longitudinal competency validation through the C3 process: Continuous Competency, Continuous Learning, and Continuous Certification. The completely virtual process replaces the 10-year comprehensive exam requirement with a series of 12 brief online assessments to be completed every 2 years to maintain certification.Whether you pursue certification to gain a competitive career advantage, to elevate professional status, or purely to increase personal knowledge, the result of obtaining specialty certification for allied health professionals aligns with the mission of the IBHRE: to facilitate optimal outcomes for heart rhythm patients. If you are in the profession of caring for cardiac arrhythmia patients, certification is for you."} +{"text": "Specialist perinatal mental health services identify and treat women experiencing mental health conditions during pregnancy and up to one year post birth. There is limited knowledge about women\u2019s experiences of care from specialist services. Evaluation and optimisation of service delivery requires knowledge of women\u2019s care experiences. This review aimed to systematically identify, appraise, and synthesise qualitative evidence exploring women\u2019s experiences of specialist perinatal mental health services.A systematic literature search of five databases: Medline (OVID), EMBASE (Elsevier), PsycINFO (EBSCO), CINAHL (EBSCO) and Scopus (Elsevier), grey literature searching, and backward citation, identified a total of 1035 papers of which sixteen met inclusion criteria. Methodological quality of the included studies was assessed using the Critical Appraisal Skills Program (CASP) tool.Thematic synthesis identified three themes: connected relationships; new beginnings; and meaningful service delivery. Findings identified that relationships developed with clinicians were significant to women and their experience of care. Women valued continuity of care from dedicated non-judgemental clinicians. Peer support from other mothers was perceived as meaningful to women. Through service interventions women gained new insights into their infant\u2019s needs and grew in confidence as a mother.Women require provision of flexible and accessible specialist services with clinicians who are sensitive to their individual psychosocial needs and preferences. Examining discharge practices and continuing care needs is essential to ensure the best outcomes for women and their families. A QES is a systematic review of qualitative research evidence which enables the researchers to present a rich interpretation of the phenomenon and gain a greater appreciation of women\u2019s experiences, ideas, and priorities what factors influenced how women perceived their care and treatment? and (b) what research gaps, if any, warrant further investigation to inform evidence-based knowledge in specialist PMHS delivery.Inclusion of primary qualitative studies exploring women\u2019s experiences of care from a specialist PMHS during preconception, pregnancy or up to one year postpartum. Mixed methods studies presenting qualitative data were eligible for inclusion. Studies were excluded when (1) women received mental healthcare from a source other than a specialist PMHS (2) women were under 16 at the time of childbirth (3) papers were editorials, opinion papers, discussion papers, policy statements, or literature reviews and (4) published prior to January 2010.A preliminary literature search of the Cochrane library and PROSPERO was conducted to identify that no current listed reviews have, or were being, conducted on this research topic. To test for accuracy of the established search terms a search was conducted on Medline. An electronic database search of Medline (OVID), EMBASE (Elsevier), PsycINFO (EBSCO), CINAHL Complete (EBSCO) and Scopus (Elsevier) was conducted to identify studies which met eligibility criteria. Additionally, grey literature was searched using OpenGrey and Greylit. The search strategy included the use of Boolean operators \u2018AND\u2019 and \u2018OR\u2019, free text searching, synonyms, and MeSH index terms. The literature search timeline was from January 2010 to January 2022 as this has been a time of significant investment and development of specialist PMHS. No limitation was placed on studies location or language. Hand searching of included studies reference lists was also performed.Methodological quality of the included studies was assessed using The Critical Appraisal Skills Programme (CASP) qualitative checklist tool (Supplementary File 3). CASP items were scored independently by the first author [EM] in consultation with the review team. No papers were excluded based on the outcome of the assessment connected relationships, 2) new beginnings, 3) impactful service provision. Themes and subthemes are presented in a thematic map (Supplementary File 4). Direct participant quotes supporting the three themes is illustrated in Table This theme reports on various interpersonal relationships which were comprehensively described by women and significant to how care was experienced from specialist PMHS. Fourteen of the sixteen included studies contributed to this theme which is reported in three subthemes: relationships with clinicians; family involvement in care; and peer support.Relationships with PMHS clinicians was reflected in twelve papers and frequently outlined as being fundamental to women\u2019s experience of care. The relationship with clinicians facilitated personal change in the women\u2019s lives. Women valued being treated like ordinary mothers and felt clinicians had real insight and understanding of PMH conditions Clinical Practice Guideline COPE\u00a0 emphasisPeer relationships developed between mothers attending services were perceived as positive to women\u2019s service experience and offered them a valued sense of community and support. Peer support as an intervention strategy has the potential to address some of the key concerns associated with poor PMH such as isolation and poor social and emotional supports as a primary diagnosis among attending perinatal women (Kim et alRecently published guidelines by the World Health Organisation WHO, put forwDischarge from perinatal services was identified as challenging and anxiety provoking to women who felt they still required service supports (Myors et al. A rigorous and systematic approach was used in this QES to explore women\u2019s experiences of specialist PMHS. Five countries are represented in this review with the origin of studies reflecting where PMHS have been developed. As a result, the findings of this study may lack transferability to other contexts and regions. Consistent themes emerged from across the various specialist settings and interventions represented in this QES. Although the breath of services examined allowed for a wide range of women\u2019s experiences to be synthesised this may have limited the depth of understanding in specific perinatal contexts. Among the included studies which reported the socioeconomic status of women married and cohabiting women represented typically 80% of study participants therefore limiting representation of women who may have additional PMH risk factors and unique service experiences. This review recommends that a diverse sample of women be used in future exploration of PMHS experience.The evidence of this QES highlights various areas which require further research. Women\u2019s views, experiences, and outcomes of pre-conception specialist care were absent. Outside of one study exploring remote service delivery during COVID-19 restrictions and limited data on home visits, focused analysis on women\u2019s perceptions of remote PMHS delivery and telehealth are limited. Whilst this review found evidence of women\u2019s desire for increased family involvement in care various individual concerns were reported which warrant further qualitative exploration to determine how best to meet women\u2019s individual needs. Further qualitative evidence is also required to understand challenges women experience when being discharged from or transferred between perinatal services. With maternal mortality rates being a significant concern in the perinatal period, it is important to identify and plan for women\u2019s continuing care needs post discharge from specialist services, ensuring effective collaborative care planning practices are in place to enable women and their families to build a positive future. Future research is also required to capture women\u2019s experiences of newly established specialist PMHS e.g.: Ireland which will contribute to existing knowledge.In conclusion, women seek provision of continuity of care from supportive services which help them feel understood in a non-judgemental way. Greater provision of accessible and flexible services including access to local treatment intervention options would promote women\u2019s attendance at services and positively impact health outcomes. It is evident that there is a need to consider women\u2019s individual psychosocial circumstances, and preferences surrounding family inclusion, remote care, and discharge needs."} +{"text": "The conservative prosthodontic construction of an ocular prosthesis utilizing our novel threaded iris fabrication technique required high time and prosthodontic resource inputs and produced a lifelike aesthetic result.Patients with ocular defects frequently present with significant local anatomical deficiencies and complex histories and require extensive time and resource inputs to treat. This case report describes the conservative management of an ocular defect completed in a postgraduate prosthodontics clinical residency program utilizing a novel threaded iris fabrication technique. Initial presentation of the aquired eye defect with inflamed tissue and a contracted socket with superior sulcus deformity. The patThe treatment commenced with fitting an appropriately sized prefabricated impression tray selected from our laboratory's non\u2010proprietary impression tray range, marking the medial and lateral canthus and making an impression with light body polyvinyl siloxane impression material Figures\u00a0 and 3. TThe iris was fabricated using the novel threaded technique in preference to the painting technique. The correct iris size was firstly measured in reference to photographs, and a radiograph film trimmed to form the iris wheel. A central hole was created in the iris wheel (radiograph film) and a sewing needle was used to thread two layers of appropriate shade cotton around the iris wheel Figure\u00a0. The iriThe sclera was characterized guided by photographs and with the patient in attendance using monomer to thin the characterizing paint. A blue layer was firstly applied to add translucency with yellow, brown or red colors added as required. Small cut threads of red acrylic wool were added to simulate blood vessels and secured with unfilled resin Figure\u00a0. The proThe final prosthesis was delivered to the patient and instructions for placement, removal, cleaning and storage provided Figures\u00a0 and 9. T3The most common techniques used to construct an ocular prosthesis are empirical fitting of a stock eye, modifying a stock eye through making an impression of the ocular defect and the fully customized construction technique.The presented case was managed using traditional laboratory techniques some of which have similarities to denture fabrication procedures. The complexities in managing the case were highlighted by the focus on the laboratory stages of the treatment that required high time and resource inputs for a single prosthesis. The iris was constructed using our novel customized threaded iris technique which proposes more lifelike aesthetics due to the detailed construction inputs and serrated iris surface texture. The prefabricated painted ocular disc is the more traditional iris construction option with inferior optical properties and significantly shorter construction time. This case report represents the first known presentation of the threaded technique and certainly warrants further investigations.It has been proposed that digital pathways present improved economic and time efficiencies over traditional techniquesThe educational benefits and success of maxillo\u2010facial treatment completed in a postgraduate prosthodontics clinical residency program have been previously reported.James Dudley: Conceptualization; investigation; methodology; project administration; supervision; writing \u2013 original draft; writing \u2013 review and editing. Jane Pellew: Conceptualization; data curation; formal analysis; investigation; methodology; writing \u2013 original draft. Nafij Bin Jamayet: Conceptualization; data curation; methodology; project administration; supervision; writing \u2013 original draft.There are no funding sources for this research.The authors declare no conflict of interest regarding the publication of this paper.Informed consent was obtained from the patient.Written informed consent was obtained from the patient to publish this report in accordance with the journal's patient consent policy."} +{"text": "Prior findings relating secondhand tobacco smoke (SHS) exposure and internalizing problems, characterized by heightened anxiety and depression symptoms, have been equivocal; effects of SHS on neurodevelopment may depend on the presence of other neurotoxicants. Early life stress (ELS) is a known risk factor for internalizing symptoms and is also often concurrent with SHS exposure. To date the interactive effects of ELS and SHS on children\u2019s internalizing symptoms are unknown. We hypothesize that children with higher exposure to both prenatal SHS and ELS will have the most internalizing symptoms during the preschool period and the slowest reductions in symptoms over time.The present study leveraged a prospective, longitudinal birth cohort of 564 Black and Latinx mothers and their children, recruited between 1998 and 2006. Cotinine extracted from cord and maternal blood at birth served as a biomarker of prenatal SHS exposure. Parent-reported Child Behavior Checklist (CBCL) scores were examined at four timepoints between preschool and eleven years-old. ELS exposure was measured as a composite of six domains of maternal stress reported at child age five. Latent growth models examined associations between SHS, ELS, and their interaction term with trajectories of children\u2019s internalizing symptoms. In follow-up analyses, weighted quintile sum regression examined contributions of components of the ELS mixture to children\u2019s internalizing symptoms at each time point.p\u2009=\u20090.03). The interaction between ELS and SHS was also associated with a less negative rate of change in internalizing symptoms over time . Weighted quintile sum regression revealed significant contributions of maternal demoralization and other components of the stress mixture to children\u2019s internalizing problems at each age point .ELS interacted with SHS exposure such that higher levels of ELS and SHS exposure were associated with more internalizing symptoms during the preschool period (\u03b2\u2009=\u20090.14, Our results suggest that prior inconsistencies in studies of SHS on behavior may derive from unmeasured factors that also influence behavior and co-occur with exposure, specifically maternal stress during children\u2019s early life. Findings point to modifiable targets for personalized prevention.The online version contains supplementary material available at 10.1186/s12940-023-01012-8. Although the last 50 years has seen tremendous progress towards reducing smoking among American adults , 13% remprenatal SHS exposure.Equivocal evidence links prenatal SHS with internalizing symptoms. Some studies report strong associations between prenatal SHS and child internalizing problems, characterized by heightened anxiety and depression symptoms \u201312; howeGiven that maternal stress during children\u2019s early life is a known risk factor for internalizing symptoms , we propThe current study sought to address these gaps in knowledge by examining how combined exposure to prenatal SHS and maternal stress during children\u2019s early life increases internalizing symptoms and influences development in a prospective birth cohort of non-smoking Black and Latinx mothers and their children (N\u2009=\u2009482). Given prior findings from animal models and human epidemiologic studies, we hypothesized that prenatal exposure to SHS combined with maternal stress during children\u2019s early life would result in elevated internalizing symptoms in youth that would remain present over time.Columbia Center for Children\u2019s Environmental Health (CCCEH) Mothers and Newborns prospective birth cohort have been previously published [Detailed demographic and recruitment information regarding the ublished . To brieLongitudinal study visits began in the third trimester and occurred approximately every two years thereafter for each child in the cohort. Prenatal exposure to SHS was measured by cotinine in either maternal blood or cord blood samples taken at birth . The curChildren\u2019s behavior problems were measured via parent report on the Child Behavior Checklist . At child age 5, mothers completed a structured interview with a trained research assistant assessing demographics and six domains of early life stress exposure. These measures assess maternal experiences of stress during the child\u2019s early life; as such, we refer to them as early life stress (ELS) because they reflect the shared experience of stress between mother and child. Items were aggregated across domains of stress exposure to create a single composite score, as has been done in previous work . Items iCord blood samples were used whenever available N\u2009=\u2009500). If a cord blood sample was not available then SHS exposure was estimated using maternal blood samples (N\u2009=\u2009197). Reported maternal smoking during pregnancy was exclusionary for enrollment in the cohort. Children born with cotinine concentrations in their cord blood above 25 ng/ml (N\u2009=\u20096) were excluded from this analysis because they were likely exposed to active smoking during gestation [0. If a cWe estimated a latent growth model of children\u2019s internalizing symptoms assessed from preschool to age eleven years-old using the growth function of the lavaan package in R studio version 4.1.1 , 66. Of Prenatal cotinine, early life stress, and their interaction were included in latent growth models to examine our hypothesis that the SHS*ELS interaction would be associated with increases in children\u2019s internalizing symptoms and which would remain present over time. To control for potential confounding, we included as covariates the following variables which are known to be associated with SHS and ELS exposure: birthweight , child sTo explore effects of the individual components of the ELS composite while controlling for effects of SHS, weighted quantile sum (WQS) regression estimated associations between co-exposure to the six correlated measures of postnatal ELS and children\u2019s internalizing scores at each time point, controlling for sex, birth weight, maternal years of education, and cotinine. Separate WQS regressions were conducted for each age point and so leveraged all available data from participants with CBCL outcome data at each age point. The WQS index was constructed by summing the ranked concentrations (quintiles) of each individuals\u2019 exposures multiplied by the relative strength of each predictor variable\u2019s association with their internalizing scores. Importantly, WQS can determine the overall influence of the multiple early life stressors and identify the contribution of each of the individual stressors to the overall impact on internalizing symptoms , 79. A h2\u2009=\u20095.24, p\u2009=\u20090.02; Table 2\u2009=\u20095.97, p\u2009=\u20090.01; Table S2).Table\u00a0p\u2009=\u20090.03) and a slower decrease in internalizing problems over time when compared with children with lower SHS*ELS scores . Significant main effects show a positive association between ELS and internalizing problems during the preschool visit and a negative association between SHS and the slope of internalizing problems . No other significant findings were observed . Interaction effects were observed such that children with higher SHS*ELS had higher internalizing problems scores during the preschool visit increase in children\u2019s internalizing problems scores was detected increase in children\u2019s internalizing problems scores was detected increase in children\u2019s internalizing problems scores was detected which aCombined exposure to SHS and ELS was associated with a slower decrease in internalizing symptoms over time, consistent with our hypothesis that the interaction between SHS and ELS would disrupt normative patterns of reduced internalizing symptoms across childhood , 90. OurWe did not observe downward sloping trajectories of internalizing problems during pre-adolescence as has been observed in prior work \u201347. NotaExamining components of the ELS composite revealed that maternal demoralization and material hardship significantly contributed to internalizing problems at age eleven whereas the other four domains of stress did not. Maternal demoralization reflects mothers\u2019 subjective feelings of incompetence and associated feelings of psychological distress that havOur study is not without limitations. First, our cohort is not weighted for clinical outcomes, limiting our ability to examine trajectories of internalizing symptoms in both clinical and nonclinical populations. Future studies should examine these trajectories in a cohort weighted for clinical symptoms in order to assess these effects. Next, maternal demoralization is a proxy for maternal psychological distress, including maternal anxiety and depression; however, our study does not contain a clinical measure of maternal anxiety or depression symptoms. Future studies should include a measure of maternal psychopathology. Additionally, our stress composite score was measured during children\u2019s age 5 study visit; whereas, our first measure of children\u2019s internalizing symptoms occurred during their preschool (age 3\u20135) visit. Future studies should measure ELS prior to children\u2019s first visit to understand the temporally predictive effects of these associations. Finally, data missingness may limit generalizability. Primary reasons for data missingness include inability to participate in a particular study visit, participants\u2019 moving out of state, or loss of contact.Our study shows for the first time that combined exposure to prenatal SHS and maternal stress during children\u2019s early life increases children\u2019s internalizing symptoms in early childhood and that these effects persist across middle childhood. Clinically, our findings indicate that maternal demoralization and material hardship are important targets for personalized prevention and intervention to reduce the development of children\u2019s internalizing problems. The study has a number of strengths including the relatively large prospective longitudinal birth cohort design that includes individuals who have historically been excluded from developmental research, as well as using a biomarker of exposure. Importantly, our study helps to clarify previous equivocal findings linking internalizing symptoms and SHS exposure by examining the interactive effects of SHS and ELS. It is possible that these interactive effects offer one potential mechanism through which prenatal SHS exposure may prime vulnerability to ELS and increase risk for psychopathology. Further these findings point to a pathway through mental health inequities may arise in Black youth, whose mothers are differentially exposed to SHS. Better understanding of the underlying mechanism of action of these profiles is necessary and may help address the current public health crisis in adolescent mental health , 119.Below is the link to the electronic supplementary material.Supplementary Material 1"} +{"text": "Malawi continues to register HIV/AIDS mortality despite increased expansion of ART services and as well as advanced HIV screening as outlined in the 2020 -2025 Malawi National HIV Strategic Plan (NSP). This study aimed to explore factors influencing the implementation of the advanced HIV disease (AHD) screening package at Rumphi District Hospital, Malawi.We conducted a mixed method, convergent study at a secondary referral hospital with 8 659 clients on ART. Guided by a consolidated framework for implementation research (CFIR) we conducted semi-structured Interviews with healthcare professionals, purposively selected from various key departments that were actively involved in AHD screening. Transcripts were organized and coded using NVivo 12 software with thematically predefined CFIR constructs. Newly HIV-positive client records extracted from ART cards were analyzed using STATA 14 software.n\u2009=\u200961) of the newly diagnosed HIV clients had no documented results for CD4 Cell count. Barriers to AHD screening emerged from four major CFIR constructs: intervention complexity, communication, availability of resources and access to knowledge and information. The specific barriers included poor work coordination among implementers, limited resources to support the expansion of AHD screening, and knowledge gap among providers. External support from Ministry of Health implementing partners and the availability of committed focal leaders coordinating HIV programs emerged as major enablers of AHD screening package.One hundred one ART records met inclusion criteria for review and analysis of which 60% (The study has identified major contextual barriers to AHD screening including\u00a0knowledge gap, poor communication systems and inadequate supporting resources. Improving uptake of AHD screening services would therefore require overcoming the existing barriers by adopting a comprehensive approach in developing barrier-tailored strategies. Despite the significant progress in expanding access to antiretroviral therapy (ART) in Malawi, AIDS-related deaths plateaued over the recent years , 2. HIV The 2020/2021 Malawi Population based HIV Indicator Survey (MPHIA) findings reveal significant gains in the UNAIDS 95\u201395-95 targets in terms of ART treatment coverage 97.3%) and viral load suppression (96.9%) but lagging behind on the first target 88.3%) for HIV positive individuals who are aware of their status COVID -19 pandemic was evaluated as a strong barrier to AHD screening from the outer setting domain under critical incidents. Participants highlighted that the pandemic disrupted screening as less clients reported for HIV testing and less staff were available for duties due to imposed restrictions.\u201cWe have been given a CD4 register from the MoH, but it is missing these other tests . So if you are documenting these tests results, you will be using a separate improvised register which is cumbersome.\u201d [RDH/Lab tech 1].AHD screening process was described as complex as it involved various departments and multiple sequential tests which led to an increased patients waiting time. Additionally, results recording was reported to be a challenge, owing to a substandard integrated register for the three AHD rapid tests.\u201cThe government has delayed to provide the posters because we should have pasted some in the wards for our friends who have less knowledge to understand what\u2019s going on\u201d [RDH/Clinician 1].Respondents acknowledged the absence of supporting resources such as posters and testing algorithm which they said contributed to poor AHD screening. This was classified as a potential barrier to AHD screening. One respondent from ART clinic said:\u201cThe laboratory should continue providing the point of care tests (POCT ) up until the facility has enough resources to extend it to ART clinic\u201d [RDH/Nurse 2].Most respondents argued that it was difficult for other departments to start conducting the AHD diagnostic tests outside laboratory without the necessary laboratory equipment such as centrifuge and pipettes for sample processing as well as biosafety measures. Responding to the question as what could be the way forward, one of the nursing managers said:\u201cIn other sites they trained the HDAs (HIV Diagnostic Assistants) and ART Clerks so it becomes easy when all nurses and clinicians are not available\u201d [LH/Clinician 2]As a way of preparing the ART clinic for point of care testing (POCT) and to ensure that services are not interrupted due to staff engagements, a clinician working with lighthouse organization suggested the need to train lay cadres in conducting POCT at ART.\u201cIf the patient has to go to the lab to access testing services rather than within the ART clinic then it\u2019s no longer point of care.\u201d [LH Nurse 3].Physical barriers in the inner setting affected work coordination among teams involved in AHD screening. It was highlighted that the distance between ART clinic and the laboratory contributed to poor tracking of new ART clients undergoing AHD screening tests. One the providers argued that the current arrangement defeated the intended purpose of providing quick services to the clients which was against the policy that require testing services to be conducted at the ART clinic.\u201cSometimes there are stockouts of some reagents in the laboratory. It\u2019s very hard to know until you send a client and comes back without a test\u201d [RDH Nurse 1].Absence of such communication systems affected both formal and informal information sharing practices\u00a0which negatively impacted on work coordination among different working teams. Without ground telephone system in place, implementers used personal gadgets for communication.\u201cIn the male ward department, I think am the only one trained which means if am not available then we have problems [RHD/Clinician 4].Respondents reported knowledge gap as a barrier claiming that providers who never attended formal trainings lacked confidence to conduct AHD screening. One of the participants argued that trained AHD providers were not enough in most of the screening sites:\u201cThis kind of screening I think it was just delayed but it\u2019s very essential and it has helped us a lot in reversing some of the deaths which were imminent.\u201d [RDH clinician 3]Despite the complexity of the intervention, most participants perceived AHD screening package to have a relative advantage over the ordinary practice of care. They highlighted benefits such as improved diagnosis & proper management of opportunistic infections.This week a team from Department of HIV/AIDs has been going around in different health facilities carrying out mentorship programmes\u201d [RDH Clinician 3]\u201cThe facility was found to be well supported externally. Technical staff from Lighthouse organization facilitated integrated HIV programme review meetings at district level while MoH facilitated integrated HIV mentorships programmes and national bi-annual AHD review meetings.\u201cAs a trainer and Coordinator I make sure samples are collected adequately and run according to the specified guidelines. The ART coordinator makes sure that that new clients undergo CD4 test as the baseline test\u201d [RDH Coordinator 1].Respondents mentioned that the availability of two HIV programme coordinators for HIV Testing Services and ART, helped to spearhead the implementation of the AHD screening package.The Table rd Q, 38% [19/50]; 4th Q, 41% [21/51]}.Table Figure\u00a0This study sought to explore contextual factors that influence the implementation of AHD screening package at a secondary referral facility in Malawi using CFIR. 10 Key healthcare workers were interviewed whereas 101 ART data records were reviewed. Most of the barriers reported emerged from the inner setting and they related to complexity of the intervention due to multiple sequential steps involved, knowledge gap, poor communication systems and limited resources to support expansion of AHD screening among others.Findings of the review of 101 ART data records for PLWH suggest that the implementation barriers had a significant negative\u00a0effect on the delivery of AHD screening services provided at the laboratory. More than sixty percent of the eligible clients missed CD4 Cell count, a baseline test run before new clients are linked to treatment. This implies that most clients were not screened for TB and Cryptococcal meningitis using Urine LAM and serum CrAg respectively. Considering high number of cases of late HIV diagnosis presenting with AHD, newly HIV\u00a0diagnosed individuals may have\u00a0high risk of dying from life threatening conditions and possibly Immune reconstitution inflammatory syndrome (IRIS)\u00a0after being initiated on ART. We describe the barriers that led to screening under coverage as well as the facilitators and proposed strategies for scaling up the WHO recommended\u00a0AHD screening\u00a0package of care.Our findings suggest that lack of interpersonal-communication systems significantly contributed to poor work coordination among providers of AHD screening. All respondents from ART clinic highlighted similar communication challenges when dealing with laboratory staff. They further explained that laboratory staff were not communicating on time about stockouts and suspension of testing services, a practice that contributed to increased patient waiting time. It was established that that the facility had no any functional ground phone system nor WhatsApp group to facilitate information sharing and communication between the two crucial departments. Barriers of networks and communication within the implementing facilities often result in fragmented teams due to poor communication and frequent changes in personnel .Interpersonal communication is an important facilitator of effective delivery of HIV interventions and therefore measured must be put in place to sustain it. Tailored cost-effective measures such as regular inter-departmental meetings and WhatsApp group forums provides a simple, readily available means of facilitating consultations between healthcare providers in the management of patients where formal communication systems are inefficient , 20.Barriers due to incompatible work flow, congestion and as well as long distance between ART\u00a0and laboratory departments were also reported. These contributed to long AHD screening pathway that led to clients lost to follow-up and poor linkage to care. As a result, clinicians and nurses at ART department resorted to providing ARVs to their new clients before AHD screening, a practice that was not conforming to national standard guidelines. Corresponding findings are reported in a study in the Eastern Africa where overcrowding, long waiting times and lack of resources emerged as prominent barriers to implementation . To addrHowever, setting up a min-testing laboratory at the ART clinic like it is done in central hospital facilities, would require resources for biosafety and biosecurity infrastructure as well as devices for sample processing. In that regard, prior planning and budgeting should be considered. A recent study at a tertiary healthcare facility in Malawi estimated a capital investment cost of establishing and equipping an advanced HIV disease room for diagnostic tests to be U$10,708 . This isThis study also established that healthcare providers with less knowledge of the intervention lacked confidence and self-efficacy when conducting AHD screening. This led to low screening coverage. As a result, many eligible clients were missed out as evidenced by the findings of ART record review where 60% had no CD4 Cell count results. Moreover, all screening sites had no AHD posters for reference. Such findings are in agreement with other programme evaluation studies that highlight inadequately trained providers as a stumbling block to service delivery , 24. To Improving knowledge and information about an intervention through informal training is of great essence as well as it leads to improvement in provider\u2019s compliance to guidelines . As suchIn addition to formal and informal trainings, provision of posters displaying eligibility criteria as well as testing arigorithm may provide a reference for healthcare providers with less knowledge of the intervention. One advantage of using posters is that they are readily available, hence provides instant knowledge and awareness of the intervention to healthcare providers .It is important to highlight that provider\u2019s awareness of the interventional relative advantage emerged to be another facilitator of AHD screening. Respondents explained that trained healthcare providers perceived the AHD screening package of care to have more clinical benefits compared to the ordinary practice of care before the programme was rolled out in 2020. They further alluded that more deaths have been reversed with the coming of AHD diagnostic tests and treatment package. Currently, there is overwhelming evidence that AHD screening improves early diagnosis and management of opportunistic conditions, thereby contributing to general improvement of quality of life for PLWH who would otherwise succumb to death , 28\u201330. Regarding networks and partnerships, technical support from MoH and its implementing partners emerged to be strong enablers of AHD screening as they helped to fill in the gap of knowledge through capacity building. Respondents claimed that quarterly visits by MoH delegates conducting ART integrated supervisions improved overall facility performance in terms of screening and management of clients diagnosed with AHD and its associated life threatening conditions. HIV integrated review meetings as well as MoH led mentorship and supervision programmes are the drivers of implementation as they provide platforms for learning where implementers from different health facilities share successes and challenges faced in the implementation of AHD screening services.Internally, the presence of focal persons facilitate efficient delivery of health interventions. In this study, programmes coordinators for HIV Testing Services (HTS), and ART showcased extensive knowledge of the AHD management package of care with high level of commitment. Focal leads spearhead the implementation process thereby simplifying complex interventional packages which involve multiple steps and departments at a facility level . GloballIt is important to note that some of the implementation challenges faced can be partially addressed by integrating AHD screening services into the existing HIV testing services (HTS) and ART programmes which are sufficiently supported by externally and have already established structures for communication and expansion. Integration of related health programs reduces duplication of services, is cost-effective, and as well as efficient , 35.This study has potential limitations. Firstly, due to time and financial constraints, the study was not done on large scale involving multiple health facilities implementing the AHD screening. Different health facilities might present with different implementation challenges. Secondly, we did not include newly diagnosed-HIV positive clients for interviews to understand their experiences and challenges faced during AHD screening. Therefore, the findings of this study may not be generalizable considering the sample size. Nonetheless, the validity of the findings was partly achieved by the use of mixed methods design .In this study, poor coordination among providers, knowledge gap and lack of supporting resources were identified as major barriers to AHD screening, affecting the quality of care given to clients being linked to ART for the first time. For low income countries such as Malawi, improving access and coverage of AHD screening would therefore require overcoming the existing barriers in the facility set-up. One of the ways is to adopt cost effective communication approaches for improving work coordination and to maintain good institutional partnerships with MoH implementing partners to ensure continued technical support towards AHD management package of care. The study findings provide an insight of common contextual barriers and further suggest comprehensive approaches for HIV programme implementers to adopt and design barrier-tailored strategies for optimizing AHD screening and diagnostic services in healthcare facilities.To maximize screening coverage, MoH must provide all the necessary resources to ensure that departments such as ART Clinic have sufficient supporting materials to independently conduct all tests for AHD screening. Besides, the use of CFIR framework of analysis in evaluation of such complex HIV interventional programs to assess implementation progress highly recommended. Future studies should focus on understanding clients social-economic barriers and as well as testing acceptability and feasibility of tailored strategies highlighted in this study."} +{"text": "Health care in the United States is increasingly being delivered during ambulatory care visits.1 As new investment in health care shifts from the inpatient to outpatient setting, the role of ambulatory clinical pharmacists (CP) are expanding. While dispensing pharmacists provide clinical screening and patient education for potential drugrelated problems, CPs provide services that complement the care provided by dispensing pharmacists by directly advising and educating other health care providers and patients on appropriate pharmaceutical use and dosing."} +{"text": "Patients with symptomatic heart failure (HF) and left bundle branch block (LBBB) are currently treated with biventricular pacing (BiV) which has a Class IA recommendation. Given the possibility to re-establish the inter and intra-ventricular synchrony, BiV is commonly referred to as cardiac resynchronization therapy (CRT). This wording is widely utilized and over time the terms BiV and CRT have become interchangeable. Conduction system pacing (CSP) is emerging as a valid therapeutic opportunity to obtain CRT restoring the native conduction via the Purkinje network. Therefore the acronym CRT is no longer synonymous with BiV only but could also refer to CSP. A terminology update is needed to include the resource of CSP to ensure better communication among all the stakeholders involved in managing recipients of cardiac devices and should be a fundamental step in advancing the quality of patient care. Making use of the NBG code to describe the implantable cardiac device would ease such terminology update, since only the first three positions of the five letters NBG code are commonly utilized, while the last two are rarely used. Patients with symptomatic heart failure (HF) and left bundle branch block (LBBB) are currently treated with biventricular pacing (BiV) which has a Class IA recommendation given the morbidity and mortality benefit . As LBBB"} +{"text": "Obsessive compulsive symptoms may occur at any age. It was studied extensively in the youth contratry to the elderly that usually suffer for other health issues leading to misdiagnosis.We aim through this study to discover the particularities of OCD in elderly .Our literature review was based on the PubMed interface and adapted for 2 databases: Science Direct and Google Scholar using the following combination ( obsessive compulsive disorder [MeSH terms]) OR (OCD [MeSH terms]) AND (elderly [MeSH terms]) OR (dementia [MeSH terms]).Our review revealed 39 articles from which we selected 4 articles.We found that in aged adults over than 50 years experience mostly somatic symptoms, religiosity, and moral scrupulosity as obsessive thoughts.We also found that OCD can occur as a primary disorder in older women, whereas in men it either persists from, younger years or arises in the context of another psychiatric or medical disorderThe relationship between Obsessional illness, brain mechanisms and Cognitive disorders are not fully understood.Indeed, we noted a relative Impairment in executive function in older adults with OCD stressing the link with cognitive impairment. Moreover, Obessive compulsive symptoms may worsen cognitive functioning .Obsessive compulsive disorder presence in the elderly may be described as a primary health condition or be related to organic mental health issues in particular dementia. The physiopathology remains unclear therefore further studies are needed for better understanding and management.None Declared"} +{"text": "Staphylococcus aureus (MRSA) causes morbidity and mortality in premature hospitalized neonates. MRSA colonization is a risk factor for transmission and outbreaks within neonatal intensive care units. Decolonization aims to stop MRSA transmission and infection. National guidelines lack recommendations for which decolonization practices are the most effective in neonates. We hypothesize that both topical mupirocin and chlorhexidine (CHG) is more effective than mupirocin alone at preventing MRSA infection in neonates.Infection due to methicillin-resistant In this retrospective study, we compared the rates of MRSA infection among all colonized neonates admitted to a 42 bed NICU at Texas Children\u2019s Hospital from 2012 to 2020. Our surveillance and decolonization protocol was introduced in October 2016. The MRSA screening methodology initially consisted of culture, then was changed to PCR (reflexed to culture if MRSA was detected). We grouped colonized neonates as those who underwent no decolonization, partial decolonization (mupirocin only), and full decolonization (CHG+ mupirocin) and compared the rates of infection in each. Infection was defined by isolation of MRSA from any specimen in the context of illness. This study was approved through the Baylor College of Medicine Institutional Review Board.Figure 1MRSA surveillance and decolonization protocol7,980 neonates were admitted to the NICU from 2012-2020. Of those screened, 128 were MRSA-colonized. Eighteen were fully decolonized, 76 partially decolonized and 34 received neither (Table 1). Zero infants who underwent full decolonization had MRSA infection. Decolonized infants were less likely to develop infection compared to infants who did not undergo any decolonization. Neonates who received only mupirocin (due to age) were less likely to develop infection , however this reduction was not as impactful as with the combination of CHG and mupirocin .Table 1MRSA infection rates by category of decolonizationFigure 2Comparison of MRSA infection between decolonized vs not decolonized and mupirocin vs no mupirocin with odds ratio and relative riskNeonates who received CHG and mupirocin did not develop MRSA infection, and decolonization with mupirocin alone provides some but not as much protection from MRSA disease as does both mupirocin and CHG. Further studies should evaluate the safety of CHG wipes in neonates less than 36 weeks gestation/4 weeks chronologic age.All Authors: No reported disclosures"} +{"text": "Error in Figure/Table LegendIn the published article, there was an error in the legend for (This sentence previously stated: \u201c(F) mRNA levels of NUDT21 in 31 gastric cancer tissues from patents with metastasis and 39 gastric cancer tissues from patients without metastasis were analyzed by RT-qPCR\u201d.The corrected sentence appears below:(F) mRNA levels of NUDT21 in 31 gastric cancer tissues from patients with distant metastasis and 39 gastric cancer tissues from patients without distant metastasis were analyzed by RT-qPCR.The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.Text CorrectionIn the published article, there was an error.Abstract, paragraph one. This sentence previously stated:A correction has been made to \u201cThe expression levels of NUDT21 were also much higher in gastric cancer tissues from patients with tumor metastasis compared with those of patients without tumor metastasis.\u201dThe corrected sentence appears below:\u201cThe expression levels of NUDT21 were also much higher in gastric cancer tissues from patients with distant metastasis compared with those of patients without distant metastasis.\u201dThe authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.Text CorrectionIn the published article, there was an error.Introduction, paragraph three. This sentence previously stated:A correction has been made to \u201cThe expression levels of NUDT21 were also higher in gastric cancer tissues from patients with tumor metastasis compared with the tissues from patients without tumor metastasis.\u201dThe corrected sentence appears below:\u201cThe expression levels of NUDT21 were also higher in gastric cancer tissues from patients with distant metastasis compared with the tissues from patients without distant metastasis.\u201dThe authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.Text CorrectionIn the published article, there was an error.Results, Expression of NUDT21 in Human Gastric Cancer/Normal Gastric Tissue Samples and Cell Lines, paragraph one. This sentence previously stated:A correction has been made to \u201cMoreover, the mRNA levels of NUDT21 were dramatically higher in gastric cancer tissues from patients with tumor metastasis compared with gastric cancer tissues from patients without tumor metastasis (The corrected sentence appears below:\u201cMoreover, the mRNA levels of NUDT21 were dramatically higher in gastric cancer tissues from patients with distant metastasis compared with gastric cancer tissues from patients without distant metastasis (The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.Text CorrectionIn the published article, there was an error.Discussion, paragraph one. This sentence previously stated:A correction has been made to \u201cGastric cancer tissues from patients with tumor metastasis showed an elevated NUDT21 level compared with the tissues from patients without tumor metastasis.\u201dThe corrected sentence appears below:\u201cGastric cancer tissues from patients with distant metastasis showed an elevated NUDT21 level compared with the tissues from patients without distant metastasis.\u201dThe authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated."} +{"text": "Approximately 4% of patients with coronavirus disease 2019 (COVID-19) will require admission to an intensive care unit (ICU). Governments have cancelled elective procedures, ordered new ventilators and built new hospitals to meet this unprecedented challenge. However, intensive care ultimately relies on human resources. To enhance surge capacity, many junior doctors have been redeployed to ICU despite a relative lack of training and experience. The COVID-19 pandemic poses additional challenges to new ICU recruits, from the practicalities of using personal protective equipment to higher risks of burnout and moral injury. In this article, we describe lessons for junior doctors responsible for managing patients who are critically ill with COVID-19 based on our experiences at an urban teaching hospital. The COVID-19 pandemic has caused unprecedented disruption to health services worldwide. Approximately 4% of patients with confirmed COVID-19 become critically ill and require admission to the intensive care unit (ICU) for respiratory support.4Under normal circumstances, ICU medicine can be daunting for junior doctors. Notable challenges include managing multiple unwell patients simultaneously and different patterns of working alongside highly skilled nursing and ancillary staff. In the context of a pandemic, doctors working in critical care may experience additional strain as a result of staff absenteeism,Patients who are critically ill can deteriorate rapidly, and intensive care calls for detailed, thorough and repeated clinical assessments. A structured and systematic clinical assessment (such as FAST HUGS BID) ensures that pressing issues are identified and addressed promptly.8Critical care medicine requires close attention to detail and responsiveness to sudden changes in physiology and blood test results. Detailed ICU management of COVID-19 is described elsewhere.Cross-sectional imaging may be necessary to exclude differential diagnoses such as pulmonary embolism, but this is complicated by logistical challenges including transfers by anaesthetic staff and use of a dedicated machine. Junior staff can streamline imaging by liaising closely with radiologists, radiographers and anaesthetists. Finally, several studies have highlighted coagulopathy in severe COVID-19 and specialists have advocated aggressive anticoagulation strategies based on D-dimer results.The detail and complexity of critical care necessitates clear communication within the medical team and between doctors, nurses and allied health professionals. Visual aids including anonymised whiteboards displaying physiological parameters, test results and ceilings of care can maintain effective communication between shifts and within teams with high staff turnover.Infection prevention and control measures will lead to separation of team members in \u2018clean\u2019 and \u2018dirty\u2019 areas. Division of staff increases the risk of delays in the recognition, assessment and treatment of patients who may deteriorate rapidly. We have used remote communication aids including teleconferencing software and walkie-talkies in an attempt to bridge the gap between doctors providing direct care and their colleagues in \u2018clean\u2019 areas. This inevitably prolongs ward rounds and risks communication errors, which can be minimised by uninterrupted verbal handover and \u2018safety huddles\u2019.Two weeks into our pandemic response, we transitioned to a fully integrated electronic patient record. We had previously used a different electronic record for drug prescribing and medical notes, alongside bedside paper nursing charts. Initially, our new system caused some disruption to workflow, particularly for nursing staff responsible for inputting patient-level data. However, a fully integrated record has enabled medical staff and allied health professionals to rapidly review a patient\u2019s physiological parameters, drug chart and progress notes to make informed remote decisions about patient management. We have continued to use laminated bedside charts displaying daily medical management plans to reinforce key messages during this transition period.Friends and relatives can play an important role in the rehabilitation of patients who are critically ill by helping to prevent delirium21We keep track of daily discussions using a dedicated whiteboard and have used structured communication aids including an infographic developed by the palliative care team at an urban district general hospital23Healthcare workers are at increased risk of acquiring SARS-CoV-2 infectionIn the 2003 SARS outbreak, correct use of personal protective equipment (PPE) significantly reduced the risk of infection among healthcare workers.ICU is a surreal world in the COVID-19 pandemic, with redeployed doctors working for long periods on demanding rotas. Absenteeism due to ill health and childcare commitments can lead to unsustainably high workloads in ICU during pandemics.6In the UK, professional bodies have acknowledged that doctors must be prepared to work beyond their usual competencies in the pandemic response. Redeployed doctors may be concerned about negligence proceedings resulting from work in an unfamiliar setting.The Faculty of Intensive Care Medicine has created an online guide to support healthcare providers seeking to safely redeploy non-ICU-trained healthcare professionals to critical care.It is important to remember that your colleagues may have been redeployed to critical care from academic or community-based roles. They may take time to adjust to an intense new environment. We all have the same goal\u2014to achieve the best outcomes for our patients. Kindness and support can go a long way in helping your colleagues during a challenging time.The COVID-19 pandemic has significantly disrupted education and postgraduate training for junior doctors. Face-to-face teaching and postgraduate examinations39The pandemic has catalysed a rapid shift towards online medical education.Where possible, departmental teaching can and must shift to focus on areas relevant to COVID-19. Training in our Trust for redeployed doctors has included modified intubation protocols; basics of mechanical ventilation and ventilator troubleshooting; and use of inotropes and sedation. Early work suggests that in situ simulation for staff responsible for managing patients who are critically ill with COVID-19 can identify safety issues and optimise workflow.Junior doctors redeployed to ICU can also contribute to patient care through audit and quality improvement. In response to a clinical incident in which a patient\u2019s planned tracheostomy procedure was delayed by inappropriate anticoagulant administration, members of our team have developed a preoperative surgical tracheostomy checklist. This has been enthusiastically adopted by our specialist surgical teams and expanded to cover other ICU sites at two other hospitals within our multicentre National Health Service Trust. Quality improvement initiatives have also led to the development of similar checklists for intubation and proning. Frequent changes in clinical guidelines for COVID-19 management in critical care can make audit and quality improvement challenging. We recommend that trainees focus on core processes including completeness of daily medical assessments and communication with patient families.We have outlined six key lessons relevant to junior doctors redeployed to critical care during the COVID-19 pandemic. These are based on our collective experiences in an ICU at an urban teaching hospital within a multihospital Trust serving a diverse patient population.We recommend that senior clinicians and managers listen carefully to redeployed doctors\u2019 concerns and where possible arrange shadowing and simulation training prior to redeployment. Given the high mortality seen in patients with COVID-19 admitted to ICUs, and communication challenges posed by strict limitations on family visits, junior doctors should receive additional training and support in breaking bad news. Well-being is particularly important for redeployed doctors and simple measures such as introducing junior doctor forums can provide trainees with a space to reflect on stressful experiences with their peers. Future research should assess the impact of redeployment on postgraduate outcomes, burnout and morale among junior doctors.Despite considerable disruption to postgraduate training and education, redeployment to critical care offers unique opportunities for clinical and professional development. Senior support can help junior doctors acquire transferable skills that will enhance their performance in any field of medicine.In a pandemic setting, many junior doctors with little or no experience of critical care can expect to be redeployed to intensive care unit to enhance surge capacity.Although redeployment may disrupt training, teaching and postgraduate examinations, critical care is a rich learning environment for junior doctors.Senior colleagues should pay particular attention to junior doctor well-being, simulation training and interprofessional communication.Are there differences in mental health outcomes among junior doctors redeployed to critical care and their colleagues working in other specialties during the COVID-19 pandemic?Have junior doctor perceptions of critical care changed following redeployment?To what extent has redeployment to critical care during the COVID-19 pandemic disrupted career progression for junior doctors?"} +{"text": "In-vivo corneal confocal microscopy is a powerful imaging technique which provides clinicians and researcher with the capabilities to observe microstructures at the ocular surfaces in significant detail. In this Mini Review, the optics and image analysis methods with the use of corneal confocal microscopy are discussed. While novel insights of neuroanatomy and biology of the eyes, particularly the ocular surface, have been provided by corneal confocal microscopy, some debatable elements observed using this technique remain and these are explored in this Mini Review. Potential improvements in imaging methodology and instrumentation are also suggested. This Mini Review provides a focus on corneal confocal microscopy and insights into the neuroanatomy of the corneal surface using this technique and open debates are highlighted. Corneal confocal microscopy has provided an opportunity to image corneal microstructures non-invasively and monitor how these change overtime with exposure to various stimuli. This Mini Review summarises current iterations of corneal confocal microscopy instrumentation with particular focus on laser scanning confocal microscopy which is the most widely used form. In addition to the optics of confocal microscopy and image analysis, this Mini Review also explores the anatomical, neurobiological and immunological insights afforded by corneal confocal microscopy as well as the pervasive gaps in our knowledge. Limitations and future directions in the advancement of the corneal confocal microscopy technique will also be explored is defocused at the pinhole , the Confoscan 4 , and the Heidelberg Retinal Tomograph with Rostock Corneal Module . These microscopes differ from one another in terms of the intensity of the light source, magnification, image contrast, and image resolution. However, the HRT-RCM which uses a laser scanning confocal microscope provides higher resolution and magnification compared to earlier corneal confocal microscopy models6.While confocal microscopy is capable of imaging various regions of the ocular surface, it has been more widely used to characterise features of the cornea. Corneal epithelial cells, stromal keratocytes and endothelial cells can be imaged at high resolution which enable assessment of their integrity and shape. Cell densities, including resident immune cells, can also be monitored using corneal confocal microscopy7, and semi-automated tracing such as NeuronJ, a plugin in ImageJ . Automated procedures have also been proposed to substantially reduce the time needed to analyse corneal nerve parameters, although studies have shown that conventional automated procedures may underestimate these parameters compared to manual or semi-automated procedures9. The following section outlines further advancements in the analysis of corneal nerve parameters particularly with artificial intelligence (AI).Nerve fibres innervating the cornea form several plexi within anterior layers of the cornea, from the stroma to the epithelium. As the sub-basal nerve plexus is the densest and most homogenous of these nerve plexi, it has been widely studied particularly in ocular surface diseases. Currently, quantifying parameters such as corneal nerve fibre length or density are normally conducted using software which could include manual tracing or methods such as CCMetrics12. Automation provides clinicians with diagnostic leverage for inferring disease from measurable image structure using computational models developed in software. Two of the most popular geometric attributes that have been modelled using AI previously include nerve density or length and tortuosity of the sub-basal nerve plexus.Advances in AI have encouraged clinical neuroscience towards more objective systems for quantifying corneal nerve morphology from corneal confocal microscopy images2 or the \u2018sum of the nerve branches observed within a frame\u201913. Models that automatically estimate nerve density require images of corneal nerve fibres to be fully segmented. Any model fragmentation generated by false negatives would lead to underestimates of nerve density.Nerve density is commonly defined as the standardised distribution of nerve fibres over a finite area of image space in mm/mm12 used simple convolutional Gabor kernel filters in conjunction with image equalisation to enhance the nerve contours in corneal confocal microscopy images to improve nerve segmentation. They found the estimates of segmented nerve length from their model were correlated with the lengths of subjectively identified nerve contours, accounting for around 74% to 88% of subjective judgments of nerve length. Another recent approach used a multiscale dual-model approach that applied even-symmetric Gabor filters to the image in one step to then enhance contours and a background noise reduction operation in an alternate step14. Even symmetric Gabor filters model the behaviour of simple cells found in the primary visual cortex and thus model subjective gradings of corneal nerves. The success of this approach has been improved with the support of artificial neural networks15 and has been leveraged upon by end-user software packages like ACCMetrics16.Scarpa et al.17 devised a \u2018structure enhancement\u2019 model based on multiscale spatial Gabor filters and operations to eliminate vignetting of corneal confocal microscopy images. Although this approach is simple and principled in modelling human visual detection of image contours, it does not outperform ACCMetrics17. However, another recent proposal used U-Net convolutional neural networks to perform corneal nerve segmentation from corneal confocal microscopy images18. The sensitivity of their model performed at the level expected of human subjective graders, which has proved useful for segmenting corneal nerve contours in corneal confocal microscopy images to estimate nerve density. While deep learning algorithms continue to evolve and be developed to enhance nerve segmentation and evaluation, most are limited to research settings and require further validation before implementation in clinical practice20. A software using a customised deep learning-based approach known as deepNerve has recently been developed and used for animal and clinical human studies22, demonstrating the promising potential of artificial intelligence in this area.Kim and Markoulli13 proposed a grading system whereby a score of zero indicates nerve contours appear straight and a score of four indicates nerve contours frequently and largely change orientation across their length. A potential limitation of this definition is there is no clear guide on what an extreme example of tortuosity should look like and how intermediate levels perceptually scale. However, the advent of machine learning provides the ability to \u2018inversely\u2019 determine the image-based parameters that predict human observer gradings.In comparison to nerve density, nerve tortuosity is not as well defined. Oliveira\u2010Soto and Efron10 proposed a reliable automated model of tortuosity that was motivated by the need to account for the overall magnitude of changes in nerve orientation across the image and number of local \u2018twists\u2019\u2014denoted by the number of inflection points along a segmented nerve fibre\u2019s length. An alternative approach used Multinomial logistic ordinal regression to show that mean curvature at two different spatial scales was diagnostic of human subjective tortuosity gradings23. However, a more recent study by ref.\u200924 proposed that nerve tortuosity was best quantified directly using U-Net segmentation to assess adjacent angular detection of nerve contour orientations (USAAD). This model first obtains a local-scale index of tortuosity by determining the straight-line contours that connect every adjacent pair of nerve pixels segmented using the earlier-described U-Net segmentation algorithm. Reducing the number of key points allows the model to assess tortuosity over longer spatial ranges. This model was found to generate outstanding performance in accounting for subjective ratings. Evidence also suggested that subjective ratings of tortuosity is computed across multiple scales spanning the full range of nerve lengths available. Could this mean that tortuosity estimates are weighted towards longer nerves when they are visible? Consistent with this view, a simple model that weighted tortuosity estimates based on nerve fibre length was found to exhibit sound correspondence with human judgements of tortuosity and provided diagnostic leverage in the prediction of diabetes11.Using a generative model design, ref.\u2009In summary, there appears to be great benefit achieved thus far through image analysis techniques developed in previous literature. One potential limitation of these approaches is that ground truth is consistently referenced to subjective gradings made by human observers. However, this may not be a limitation as the end goal of AI is to optimally assist clinicians with their diagnostic and monitoring responsibilities, but not replace them.1. Both corneal confocal microscopy and immunohistochemical techniques have demonstrated distinct morphological patterns in this plexus, with some of the earlier montaged corneal confocal microscopy images showing nerve fibres converging towards a clockwise or anti-clockwise spiral25. This region has been termed the inferior whorl which is situated about 1\u20132\u2009mm inferonasal from the anatomical centre of the cornea. While various theories have been suggested for the spiral pattern of the sub-basal nerve plexus, recent studies have shown that interactions with corneal epithelial cells also migrating from limbal regions have substantial impact on dictating the course of nerve fibre migration27. Specifically, axon guidance ligands have been found on corneal epithelial cells including Semaphorins, Ephrins and Netrins28. Various studies have also indicated the potential for the inferior whorl to act as a landmark for monitoring neuronal changes particularly in peripheral neuropathic conditions30. However, there has been evidence of deviations from this spiral pattern associated with advancing age31 or neuronal damage32 which may confound identification of the inferior whorl region. While laser scanning confocal microscopy can provide substantial detail of corneal microstructures in high resolution and magnification, one of its limitations is the small field of view associated with each image frame which constitutes an area of 0.16\u2009mm2. In relation to quantitative sub-basal nerve plexus outcomes including corneal nerve parameters and dendritic cell density, various studies have shown that random sampling of a selected number of images from a total pool collected from areas of interest of the cornea may be sufficient in optimising reproducibility and accuracy of measures34. Widefield imaging capabilities could further facilitate analysis of nerve migration36 and monitor neuronal morphological changes in the sub-basal nerve plexus.Neural and immunological features are the two main components investigated using corneal confocal microscopy. As mentioned, the sub-basal nerve plexus is the one of the most studied part of the cornea given its uniform and dense nerve fibre distribution+ dendritic cells which are potent antigen presenting cells located mainly within the epithelial and anterior stromal layer of the cornea, as well as macrophages in the stroma37. Plasmacytoid dendritic cells, a subpopulation of bone marrow-derived dendritic cells, have also been shown to be present particularly in the peripheral regions with pivotal roles in inducing immune tolerance38 or wound healing39. While corneal confocal microscopy lacks the capability of providing functional characterisation of immune cells, researchers have used its high resolution and magnification to analyse morphological properties particularly with dendritic cells. Generally, dendritic cells with larger size and/or more dendrites are characterised as activated or mature41, indicating a more inflamed state in the cornea.Certain resident immune cells can also be imaged within the cornea through corneal confocal microscopy. This has been supported by earlier findings which identified the presence of bone marrow-derived CD11c44. Experimentally-induced corneal nerve cut or injury to one eye in animal studies have also affected corneal nerve function45 and increased CD11c+ or CD11b+ dendritic cells expressing co-stimulatory molecules in the unaffected eye46. Such interdependence between the two ocular surfaces may partly be due to a neurogenic inflammatory reflex mediated by activation of the transient receptor potential vanilloid 1 channel involved in nociceptive signalling and subsequent substance P release47. A corneal-trigeminal axis involving upregulation proinflammatory cytokines, substance P and infiltration of immune cells may also contribute to the propagation of inflammation from the affected corneal surface to bilateral trigeminal ganglia48.The biological interconnectedness between both eyes can also be demonstrated through the cornea. Recent studies using corneal confocal microscopy to investigate corneal infections including herpetic keratitis or cataract surgery in one eye have shown bilateral reduction in corneal nerves in both eyes50, but it is also thought to be able to detect signs of aberrant regeneration. Microneuromas are another neuronal feature that has been identified with corneal confocal microscopy and commonly described as terminal enlargements of a corneal nerve52. It is thought that these enlargements are associated with aberrant nerve regeneration and neuropathic pain following injury to the corneal nerve53. While severed nerves have historically demonstrated these abnormal neuronal growths54, recent studies have shown that some features previously identified as microneuromas with corneal confocal microscopy could potentially be corneal nerve stromal-epithelial nerve penetration sites55. The latter could be characterised as being diffuse hyperreflective sites as supported by immunohistochemical findings showing continuation between the sub-basal corneal nerve and underlying originating stromal nerve55. Emerging evidence from murine models demonstrated that the presence of these penetration sites may be elevated with metabolic stress or dysfunction56. However, standardisation in the identification of these neuronal features is still required.While corneal confocal microscopy has provided substantial information of anatomical structures and biological function in the cornea, certain gaps in knowledge remains pervasive. corneal confocal microscopy has been used to monitor corneal nerve recovery following therapeutic or surgical interventions in various ocular surface diseases57, dot-like features31 or round-shaped immune cells58. The evidence for the true nature and biological significance of these cells remain limited with little knowledge of immunohistochemical correlates. However, CD86+ round-shaped dendritic cells closely associated with sub-basal nerve fibre branching points have been observed by ref.\u200959. These cells penetrate the basement membrane into the stroma and may play a role in guiding nerve movement or trajectory59. Whether these cells represent those observed in corneal confocal microscopy remain unknown.Hyperreflective round cells have also been identified amongst the sub-basal nerve plexus particularly at the inferior whorl region. As their size and hyperreflectivity seem to reflect those of dendritic cells, they are considered to be a subtype of immune cells which lack dendrites. These cells have been given different labels including globular cells+ memory T-cells in constant motility following a resolution of local infection which also demonstrated dendritic morphology using immunostaining techniques60. Highly motile dendritiform cells were also observed with corneal confocal microscopy in humans by constructing single images of the same area imaged by corneal confocal microscopy in a time series60. These are reminiscent of \u2018immature\u2019 dendritic cells labelled in previous studies62, however whether corneal confocal microscopy can distinguish these immune cell types based on morphology alone requires further investigation. It is evident that more widefield in-vivo corneal imaging techniques which can also localise corneal nerve regions to be imaged could facilitate more thorough comparisons and precise monitoring of these corneal microfeatures.Cells with dendritiform morphology are often labelled as dendritic cells in studies using corneal confocal microscopy. A recent study identified the presence of resident CD866. Recent findings in corneal confocal microscopy have also been associated with systemic biological health such as higher corneal nerve parameters with higher serum levels of omega-3 polyunsaturated fatty acids particularly docosahexanoic acid67 and associations between reduced corneal nerve parameters in Alzheimer\u2019s disease or transgenic mice overexpressing human non-mutated tau68. Corneal confocal microscopy has mostly been studied in the context of diabetes, which is one of the most common causes of peripheral nerve injury in the distal extremities particularly the feet69. Its utility in diagnosing and predicting the development of diabetic peripheral neuropathy has been demonstrated in previous studies72 to facilitate follow-up assessments of the posterior pole, is yet to be devised for routine clinical use. More recently, OCT technology with real-time eye tracking has been adapted to provide non-contact visualisation of corneal nervesFurther information on research design is available in the\u00a0Reporting Summary"} +{"text": "The Australian healthcare system continues to work towards close the gap to improve and achieve equality in health and life expectancy for Aboriginal and Torres Strait Islander peoples. When culturally safe practice is forefront, it may be the driving force in improving Indigenous Australian healthcare outcomes. For students and practitioners to be equipped with the industry\u2010required cultural safety skills, we believe Indigenous Australian knowledge and perspectives must be effectively integrated into undergraduate education. A scoping review of the literature was conducted to identify the most effective teaching and learning methods and assessment tools to best integrate Indigenous knowledge and perspectives into undergraduate radiation therapy curriculum at Queensland University of Technology (QUT). Embase, CINAHL, Scopus and PubMed\u2010Medline were searched to access peer\u2010reviewed studies published between 2017 and 2022. A total of 591 articles were identified with 39 full\u2010text articles meeting the inclusion criteria. Methods of teaching and learning and independent assessment tools that promote cultural capability in undergraduate education were identified. Findings included intensive patient\u2010specific workshops, which were reported to provide students with an immersive learning experience, better preparing them for clinical placements and future practice. Additionally, other allied healthcare professions have utilised tools such as the Cultural Capability Management Tool (CCMT) and Trans\u2010Cultural Self\u2010Efficacy Tool (TSET) to quantitatively assess positive shifts and highlight developmental needs. These findings will inform current educational endeavours to promote cultural safety and the cultural capability continuum in the undergraduate radiation therapy curriculum. When culturally safe practice is forefront, it may be the driving force in improving Indigenous Australian healthcare outcomes. A scoping review was conducted to identify teaching and learning methods to best integrate Indigenous knowledge and perspectives into undergraduate radiation therapy curriculum. Our findings will inform current educational endeavors, and assist in promoting cultural safety and cultural capability in the undergraduate radiation therapy curriculum. To improve the quality of care for Aboriginal and Torres Strait Islander peoples and ultimately close the gap, it is imperative to recognise the unique considerations required for this patient demographic.Radiation therapists (RTs), alongside radiation oncologists, nurses and other allied health staff, work in a challenging environment to plan and deliver cancer treatments. For RTs to honour the responsibility of perpetuating culturally safe practices and promoting health\u2010seeking behaviours, foundational education should be well established during undergraduate education.Radiation therapy students graduate from university with the requisite knowledge and clinical skills for professional registration; however, the literature suggests a lack of Indigenous health foundational knowledge and subsequent practising confidence among health students and practitioners when required to provide care to Indigenous Australian peoples.Integrating Indigenous Australian knowledge and perspectives into the radiation therapy curriculum is a complex process. Health practitioners need to understand that Indigenous Australian people's health extends well beyond the physical well\u2010being of the patient. The construct of health is interconnected holistically, socially and spirituality to the whole Indigenous community.The aim of this scoping review was to identify the most effective teaching and learning methods to best integrate Indigenous knowledge and perspectives into tertiary curriculum.A scoping review of the literature was conducted which aimed to identify the most effective teaching and learning approaches and assessment tools for the integration of Indigenous health curriculum. Preferred Reporting Items for Systematic Reviews and Meta\u2010Analyses (PRISMA) guidelines for scoping reviews were abided by for this review.How can Indigenous knowledge and perspectives be best integrated into the undergraduate radiation therapy curriculum to promote better\u2010informed practice and cultural continuity?Embase, CINAHL, Scopus and PubMed\u2010MEDLINE were utilised from November 2021\u2013January 2022 to identify studies that reported teaching and learning strategies for Indigenous curriculum in educational and clinical settings. A search strategy was developed with key search terms , Trans\u2010Cultural Self Efficacy Tool (TSET) and a Cultural Competence Clinical Evaluation Tool (CCCET) were identified in the literature Table\u00a0. These wThe purpose of this scoping review was to identify methods of how to best integrate Indigenous Australian knowledge and perspectives into the undergraduate radiation\u2010therapy curriculum. The findings were largely collected from Australian university healthcare degrees and clinical settings due to the paucity of radiation therapy\u2010based studies.There was significant mention of low levels of Indigenous health knowledge among students and practitioners, along with feeling underconfident when required to provide care to Indigenous Australian peoples, these deficits require further investigation in a subsequent study. Immersive, transformative teaching and learning methods and assessment to specifically aid in increasing student knowledge of Indigenous Australian perspectives and to identify gaps within the undergraduate curriculum, to help fulfil professional registration requirements were identified.Facilitating cultural education is necessary to ensure culturally capable, responsive healthcare practice.Learning experiences with Indigenous Australian patient\u2010specific scenarios would provide students with a safe environment to identify the barriers and enablers to culturally safe care.Fleming and colleaguesAs awareness is the first step towards becoming culturally safe, we hope this is encouraged with opportunities for reflective practice and challenging discussions regarding power privilege, racism and ongoing disparities.This review identified tools designed for and implemented in podiatry and nursing undergraduates and health practitioners, used to consolidate theoretical knowledge and heighten self\u2010awareness skills with respect to Indigenous Australian knowledge, cultural safety and clinical practice. These tools are a means of promoting transformative learning aspects, by requiring users to self\u2010reflect to identify strengths and gaps in Indigenous Australian people's knowledge, therefore increasing self, and cultural awareness.The CCMT is a cultural capability tool developed based on undergraduate requirements of the National Framework for Aboriginal and Torres Strait Islander Health Curriculum in higher education and assesses the five stipulated domains in achieving cultural capability. These are Respect, Communication, Safety and Quality, Reflection and Advocacy.The Trans\u2010Cultural Self\u2010Efficacy Tool (TSET), which later prompted development of the Cultural Competence Clinical Evaluation Tool (CCCET) are established nursing tools designed and implemented to evaluate undergraduate students' confidence in transcultural nursing skills among diverse patient populations, well suited for the Australian population. The TSET utilises affective , cognitive and practical subsets to assess self\u2010efficacy strength and perception and had findings of high content validity and reliability.Findings suggest continuous formative evaluation is required of both students and the curriculum to guide future learning and course development and provide specific improvement of skills to build on the development of professional values as foundational practice to align with registration requirements.Education plays a pivotal role in providing foundational knowledge of Indigenous Australian history in efforts to consolidate culturally capable practice.We hope to implement intensive workshop sessions that include Indigenous Australian patient case studies, yarning circles and the use of the CCMT, TSET and CCCET for the duration of the course. Future studies may consider developing a radiation therapy\u2010specific cultural capability assessment tool through further research and adapting the tools as required.Closing the gap requires a culturally inclusive undergraduate health curriculum to promote self\u2010aware, culturally capable graduates and future practitioners.The authors declare no conflict of interest."} +{"text": "Current hepatitis C virus (HCV) testing guidance recommends a two-step testing sequence for diagnosis of HCV infection. When an HCV antibody test is reactive and no HCV RNA test is performed, testing is considered incomplete. Historically, approximately one third of patients have incomplete testing.New guidance for completion of HCV testing supports operational strategies that collect samples at a single visit, and automatic HCV RNA testing on all HCV antibody reactive samples. Use of strategies that require multiple visits to collect samples should be discontinued.Automatic HCV RNA testing on all HCV antibody reactive samples will increase the percentage of patients with current HCV infection who are linked to care and receive curative antiviral therapy.Current hepatitis C virus (HCV) testing guidance recommends a two-step testing sequence for diagnosis of HCV infection. Performing an HCV RNA test whenever an HCV antibody test is reactive (complete testing) is critical to achieve national HCV elimination goals. When an HCV antibody test is reactive and no HCV RNA test is performed, testing is considered incomplete. Historically, approximately one third of patients have incomplete testing. This update clarifies that all sites performing HCV screening should ensure single-visit sample collection. This approach allows for automatic HCV RNA testing when an HCV antibody test is reactive to avoid incomplete testing. Use of strategies that require multiple visits to collect HCV testing samples should be discontinued. Automatic HCV RNA testing on all HCV antibody reactive samples will increase the percentage of patients with current HCV infection who are linked to care and receive curative antiviral therapy. Examination of the hepatitis C care cascade in the United States reveals a substantial gap between the number of persons who have a reactive hepatitis C virus (HCV) antibody test and those who undergo nucleic acid testing (NAT) for detection of HCV RNA , a separate sample should be collected at the same visit to ensure that HCV RNA testing is completed when the HCV antibody result is reactive. If an HCV antibody is reactive and no HCV RNA test is performed, testing is considered incomplete; an HCV RNA test should be performed for all HCV antibody reactive samples to establish the diagnosis of current HCV infection. Sites performing HCV screening should ensure single-visit sample collection are used to avoid incomplete HCV testing.Complete and accurate testing is the first step in identifying persons with current HCV infection to ensure linkage to care and initiation of curative antiviral therapy. Operational strategy 1 should no longer be used because it can lead to incomplete HCV testing and gaps in the HCV care cascade. Health care facilities and laboratories should update practices to ensure operational strategy 1 is no longer used. Using a single visit to conduct both steps of the HCV testing sequence will increase complete diagnosis of current HCV infection, which will increase the percentage of patients with current HCV infection who are linked to care and receive curative antiviral therapy."} +{"text": "When this article was originally published in Psychological Medicine it omitted an acknowledgment credit. This section has been updated to include the following: \u2018The authors also grateful acknowledge research assistant Qiao Ying Leong and research fellow Dr. Alexandria Marie Remus of the N.1 Institute for Health and WisDM for their work in Prospero registration/literature review framework guidance, and literature review.\u2019.The authors apologise for this error."} +{"text": "Objectives: The past hand hygiene (HH) compliance rate has indicated the low number of opportunities for some healthcare workers (HCWs) because the infection control liaison officer (ICLO) tended to capture opportunities from nurses who were available, despite the proportional allocation of opportunities per HCW type based on the World Health Organization (WHO) HH methodology. Therefore, HH compliance rates may not have accurately represented the specific HCW type, which may have affected the overall HH compliance rate. We sought to determine an accurate baseline of HH compliance rate with consistent number of opportunities across all HCW categories. Methods: HH auditors were ICLOs trained in HH observation by the infection control nurse (ICN) according to the WHO \u201cMy Five Moments of Hand Hygiene.\u201d HH observations were conducted bimonthly with assigned areas focusing only on 1 HCW category for each session: nursing, medical, clinical support services, or environmental services. A briefing session was given on the day of observation, with the goal of collecting 20 opportunities per area with HCW focus during their peak activities. Direct feedback and positive reinforcement were given to HCWs after observations were completed. Results: A survey of 96 ICLOs indicated that observations based on HCW focus allowed them to capture more HH opportunities and concentrate on their observations. The new approach showed a significant increase in number of opportunities across all HCW categories that was more representative. We also successfully determined a new baseline for all HCW categories, with further breakdown of HCW type. Conclusions: A new methodology of HH observation with a focus on HCW category has resulted in more HH opportunities across all HCW categories and improved representation of the HH compliance rate."} +{"text": "This cross-sectional study describes the health care prices publicly posted by Humana and the price variations by geography, service, and other factors. We examined TiC price data for common services from Humana, a large national insurer, and highlighted use cases of such novel data for future research. New TiC payer data are released each month by all payers. Informed health care consumerism is a potential lever for managing costs and improving patient satisfaction.Over half of the US population receives health insurance from private insurers, and prices are negotiated rather than set administratively . This negotiation process contributes to a landscape in which private insurance prices are both higher than Medicare rates and highly variable.3 Indiana University Institutional Review Board deemed this cross-sectional study exempt from ethics review and informed consent because it was not human participant research.We obtained October 2022 TiC data from Humana\u2019s public-facing portal and downloaded data in batches (Python Software).4 We restricted analyses to in-network clinicians and facilities and used the mean posted price when the data included multiple contracted rates for the same procedure and clinician or facility within the same network.Humana rates were chosen because of its largely national coverage of clinicians and facilities and our ability to speedily parse the data files. While mostly a provider of Medicare Advantage benefits, Humana covers approximately 1 million individuals with commercial insurance.5 to match clinicians and facilities who performed these services by their National Provider Identifiers. We analyzed distributional differences in prices and coefficients of variation. Data analysis was performed using Stata 17.0 (StataCorp LLC).We focused on 7 procedures, including more shoppable codes (computed tomography [CT] scan of head or brain without contrast) and less shoppable codes (high-severity emergency department [ED] visit). A key challenge was that TiC data reported rates for clinicians and facilities regardless of whether they actually performed a given service. To identify those who performed the selected services, we used both 2019 100% Medicare fee-for-service claims data and commercial claims data from the RAND hospital price transparency projectThe The This study revealed how novel data can inform policies that improve the efficiency of the US health care system. The study was limited to a single insurer and 7 procedures; however, it opens the door to using TiC data in other, broader settings.Future work may examine the underlying causes of price variation in health care, as it is unclear whether prices are meaningfully associated with value as in nearly every market, or whether prices reflect imbalances in market power and negotiation leverage. If price variation reflects clinical or perceived quality variation, purchasers and policymakers need to find balance between receiving higher-quality care and spending financial resources elsewhere. However, if price variation is driven by consolidation or anticompetitive contracting, then regulators should design policies that ensure competitive health care markets. The factors determining price variation are likely in the middle of these 2 possibilities."} +{"text": "Scientific Reports 10.1038/s41598-022-27237-0, published online 29 December 2022Correction to: The original version of this Article contained errors.In the Material and methods section, under the subheading \u2018Patients and specimens\u2019,\u201cPatients treated with nivolumab between September 2016 and January 2021 at our hospital were enrolled in this study.\u201dnow reads:\u201cPatients treated with nivolumab between September 2017 and January 2021 at our hospital were enrolled in this study.\u201dIn addition, in the Ethical approval section,\u201cThe study protocol complied with the Declaration of Helsinki and was approved by the institutional review board of Saitama Medical University International Medical Center . All patients provided written informed consent prior to enrollment.\u201dnow reads:\u201cThe study protocol complied with the Declaration of Helsinki and was approved by the institutional review board of the Saitama Medical University International Medical Center . All patients provided written informed consent prior to enrollment.\u201dThe original Article has been corrected."} +{"text": "There was an error in the original publication . We misuA correction has been made to Methods, Paragraph 1 on the first line:\u201cThe study was systematically reviewed following the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelines [26].\u201dThe authors state that the scientific conclusions are unaffected. This correction was approved by the Academic Editor. The original publication has also been updated."} +{"text": "Urolift\u00ae is a surgical modality to treat lower urinary tract symptoms (LUTS) in patients with enlarged prostates . However We performed a video compilation with several surgical steps illustrating key aspects and critical details of the anterior bladder neck access, lateral bladder dissection from the prostate, and posterior prostate dissection to avoid ureteral and neural bundles injuries. We perform our RARP technique with our standard approach in all patients -6. Th Robotic-assisted radical prostatectomy in patients with Urolift \u00ae is challenging due to modified anatomical landmarks and intense inflammatory processes in the posterior bladder neck. When dissecting the clips placed next to the base of the prostate, it is crucial to avoid cautery because energy conduction to the other edge of the Urolift \u00ae can cause thermal damage to the ureters and neural bundles."} +{"text": "I read a recently published research study about the Mediterranean diet and its association with liver status with extraordinary interest. This research group focused on a population of overweight and obese women . AlthougHowever, it would be increasingly constructive for future research associating dieting and favorable health outcomes to use new sources for the MEDAS (14-item Mediterranean Diet Adherence Screener) validation tool other than the PREDIMED study . Althoug"} +{"text": "New sensors and modulators that interact wirelessly with medical modalities unlock uncharted avenues for in situ brain recording and stimulation. Ongoing miniaturization, material refinement, and sensitization to specific neurophysiological and neurochemical processes are spurring new capabilities that begin to transcend the constraints of traditional bulky and invasive wired probes. Here we survey current state-of-the-art agents across diverse realms of operation and evaluate possibilities depending on size, delivery, specificity and spatiotemporal resolution. We begin by describing implantable and injectable micro- and nano-scale electronic devices operating at or below the radio frequency (RF) regime with simple near field transmission, and continue with more sophisticated devices, nanoparticles and biochemical molecular conjugates acting as dynamic contrast agents in magnetic resonance imaging (MRI), ultrasound (US) transduction and other functional tomographic modalities. We assess the ability of some of these technologies to deliver stimulation and neuromodulation with emerging probes and materials that provide minimally invasive magnetic, electrical, thermal and optogenetic stimulation. These methodologies are transforming the repertoire of readily available technologies paired with compatible imaging systems and hold promise toward broadening the expanse of neurological and neuroscientific diagnostics and therapeutics. Recent progress in neural engineering has narrowed the gap between invasive precision neurotechnologies and noninvasive methods for imaging and stimulating the brain. Robust brain machine interfaces (BMIs) primarily rely on mainstay electrophysiological tools including microelectrode arrays that can be delivered by injection termed \u201cneurograins\u201d that are powered at the RF regime via a 1\u00a0GHz transcutaneous communication relay coil and capable of bidirectional recording and stimulation , epicortical recordings were demonstrated in rat motor and sensory cortices in conjunction with intracortical stimulation of nearby regions. The devices were deposited onto the cortex manually following craniotomy, but with optimized efficiency at the midfield range relying on both inductive and radiative modes for considerably smaller devices demonstrating electroneurography and electromyography with sufficient sensitivity . Further improvements to material composition were shown for optimized signal transfer in tissue empowering fUS-based wireless devices for deep brain stimulation -based agents are readily delivered by injection, and depending on their chemical properties, can cross the blood\u2013brain barrier (BBB) into target anatomical and intrasynaptic regions in the brain either naturally, through functionalization and endocytosis, or by applying osmotic, electromagnetic or ultrasonic field gradients into locally injected SPIONs resulting in the activation of heat-sensitive capsaicin receptor TRPV1 channels and modulation of neuronal activity for remote brain mechanostimulation and excess heat dissipation core coated with a piezoelectric BaTiO3 (BTO) shell were specifically simulated has wireless power transfer efficiency between one and two orders of magnitude above existing miniaturized micro-coils and a lower limit of detection between 300 and 500 pT in ex vivo neural tissue neural stimulators at 100\u2013200\u00a0Hz and piezoelectric aluminum nitride (AlN) were designed as a magnetoelectric microscale antenna sensor array loaded with complex onboard transmission and reception circuitry and theoretically able to record physiological fields with high sensitivity of up to 40 pT and 200\u00a0\u00b5m\u2009\u00d7\u200910\u00a0s to 100\u00a0s of MHz spatiotemporal resolution acting as fully biocompatible contrast agents for sensing and modulation at millisecond-scale precision and easily detectable MR signal changes in response to exogenously-infused dopamine in the brain. This strategy later developed into a toolkit for molecularly specific functional visualization of endogenous neurotransmitters across large regions of the brain in vivo ester Ca\u20134\u00a0m\u2009\u00d7\u2009100\u00a0Hz regime compared with more rapid electronic wireless neurotechnologies. Moreover, while molecular sensors can be orders of magnitude smaller than electronic devices and even nanoparticles, they are not always capable of crossing the blood brain barrier unforced due to complexities including hydrophilicity, cellular efflux and more. Future work for all tiers of agents will require the exploitation of BBB penetration schemes, sensing from nearby microvasculature using electromagnetic, chemical, or biological strategies to maneuver wireless probes into neural tissue of interest.As with MRI-based nanoparticle imaging agents, spatiotemporal resolution for most molecular probes sits at the 10In this work, we surveyed cutting-edge augmentations to wireless neural probe technologies, examined the technical challenges and ramifications of sensor size reduction and consolidation, and summarized how wireless agents for variable modalities can facilitate precision recording and stimulation of the brain and drive breakthroughs in bioelectronic medicine. To harness the exclusive capabilities of wireless micro- and nano-scale probes, their physical footprint must be aggressively minimized while maintaining recording and stimulation functionality. Because miniaturization can also lead to new forms of toxicity and adverse cellular response, excess aggregation and obstruction within the brain\u2014complementary tools must be developed for effective sensor monitoring and evacuation. We demonstrated here how devices and probes are accommodating the increasingly integrated fields of acoustic, chemical, electronic, magnetic, and optical imaging, with emphasis on cross-modality operation for creating toolkits of agents coupled to a diverse array of recording and stimulation systems\u2014rather than a single monolithic framework.We expect this approach to elevate research capabilities neuroscience and pave the way for clinical deployment."} +{"text": "With this notice, Transplant International states its awareness of concerns as raised by the authors regarding the data validity of \u201cReal-World Treatment Patterns of Antiviral Prophylaxis for Cytomegalovirus Among Adult Kidney Transplant Recipients: A Linked USRDS-Medicare Database Study\u201d published on 12 August 2022. While a revised version is currently being conducted by the authors in collaboration with the Editorial Office, this expression of concern has been posted while Transplant International awaits the revisions. It will be updated accordingly after that time."} +{"text": "Central sleep apnea represented by Cheyne\u2013Stokes Respiration (CSR) is frequently observed in heart failure (HF) patients, and its severity has been reported to be associated with morbidity and mortality in patients with HF . CSR is The study by Abulimiti et al. investigated the association between sleep disordered breathing reflecting the severity of HF and malnutrition causing frailty . They di"} +{"text": "Unconventional oil and gas (UOG) development, made possible by horizontal drilling and high\u2010volume hydraulic fracturing, has been fraught with controversy since the industry's rapid expansion in the early 2000's. Concerns about environmental contamination and public health risks persist in many rural communities that depend on groundwater resources for drinking and other daily needs. Spatial disparities in UOG risks can pose distributive environmental injustice if such risks are disproportionately borne by marginalized communities. In this paper, we analyzed groundwater vulnerability to contamination from UOG as a physically based measure of risk in conjunction with census tract level sociodemographic characteristics describing social vulnerability in the northern Appalachian Basin. We found significant associations between elevated groundwater vulnerability and lower population density, consistent with UOG development occurring in less densely populated rural areas. We also found associations between elevated groundwater vulnerability and lower income, higher proportions of elderly populations, and higher proportion of mobile homes, suggesting a disproportionate risk burden on these socially vulnerable groups. We did not find a statistically significant association between elevated groundwater vulnerability and populations of racial/ethnic minorities in our study region. Household surveys provided empirical support for a relationship between sociodemographic characteristics and capacity to assess and mitigate exposures to potentially contaminated water. Further research is needed to probe if the observed disparities translate to differences in chemical exposure and adverse health outcomes. Groundwater vulnerability to contamination from unconventional oil and gas development was assessed using a physically based modeling frameworkElevated groundwater vulnerability was associated with several sociodemographic indicators of high social vulnerabilityCo\u2010occurrence of groundwater vulnerability and social vulnerability poses distributive environmental injustice Hydraulic fracturing (\u201cfracking\u201d) involves the injection of millions of gallons of water mixed with sand and chemicals at high pressures to induce openings in the target formation and ease the flow of trapped oil and gas. UOG development has been hailed for its socioeconomic benefits, allowing the United States of America (USA) to become a net energy exporter in recent years and facilitating the transition away from coal toward lower carbon dioxide\u2010emitting natural gas is the subject of environmental justice scholarship and reflects the concept of distributive justice. In the case of UOG, the empirical evidence to date has shown some mixed indications of disproportionate placement of risks\u2014including drilling, siting of pipelines and wastewater disposal facilities, toxic emissions, water quality complaints, and negative health outcomes\u2014on marginalized communities for a 104,000\u2010sq\u00a0km region in the Appalachian Basin, covering parts of Pennsylvania, Ohio, and West Virginia Figure\u00a0. The reg2.2To assess social vulnerability, we analyzed the 15 sociodemographic characteristics comprising the Centers for Disease Control and Prevention's Social Vulnerability Index within the physically based model domain , while convergence was assessed with the Geweke diagnostic and effective sample size for each parameter in the model , in contrast to the original criterion based on the proportion of population dependent on domestic groundwater had a significantly larger proportion of populations below poverty, unemployed, aged 65 or older, and with a reported disability, as well as a larger percentage of mobile homes , with higher income households more likely to report having access to alternative sources such as bottled and municipal water. Households in the lowest income bracket were also the most likely to report having no water treatment devices installed. Respondent age was significantly associated with the frequency of well water testing , with older respondents more likely to report testing their water at least once per year. These results suggest that sociodemographic characteristics indeed capture a household's ability to assess and mitigate exposures to contaminated waters and, thus, disparities in the former can reflect disparities in the latter.While our survey results cannot be generalized to the entire study region, we can still derive notable insights from these data. Household income was significantly associated with the source of drinking water using distance\u2010based proxies alone. The ability to identify specific drinking water contamination risks is critical for communities that heavily depend on groundwater for daily use, as such risks can be potentially mitigated by household access to alternative water sources, treatment systems, and frequent well water testing. Our assessment of groundwater vulnerability to contamination and sociodemographic characteristics suggests potential environmental injustice related to a disproportionate burden of UOG risks on socially vulnerable communities. We found disproportionately elevated GWV among lower income populations, which can constitute an environmental injustice as these populations may have reduced capacity to mitigate the exposures and impacts of UOG due to lower access to healthcare, lack of alternative drinking water sources, or cumulative burdens from a multiplicity of environmental hazards (Khanassov et\u00a0al.,\u00a0Our regional tract\u2010level analyses also suggested associations between elevated GWV and higher proportions of mobile homes and persons living in group quarters. Populations in these settings have a lower likelihood of owning land or mineral rights, and thus are potentially exposed to contamination risks without accruing any direct economic benefits from UOG development. Indeed, scenarios where the largest benefits of UOG go to those who bear little to none of the environmental and public health risks have been identified by previous research in the benefit\u2010sharing environmental justice literature (Clough & Bell,\u00a04This work contributes to the mounting body of research showing disparities in the distribution of impacts and risks from UOG development. Our physically based source\u2010pathway\u2010receptor vulnerability modeling framework provides a novel, spatially explicit approach for evaluating groundwater contamination risks from UOG development. This mechanistic framework characterizes risk pathways specific to drinking water pollution, whereas previous distance\u2010based proxies only describe aggregate risks. The physically based vulnerability analysis thus provides a more focused lens with which to examine questions of environmental justice. Regional estimates of groundwater vulnerability to contamination from UOG activities were consistently elevated in communities exhibiting certain social vulnerability characteristics\u2013 lower income per capita, larger proportions of elderly populations, and larger percentages of mobile homes\u2013 suggesting the potential occurrence of distributive environmental injustice. Our household surveys provide empirical support documenting relationships between sociodemographic characteristics and a household's capacity to address domestic groundwater contamination. Additional research is needed on the social processes driving these associations and on the degree to which the observed sociodemographic disparities in groundwater vulnerability translate to differences in public health endpoints such as disease and mortality, as well as on the other dimensions of environmental justice.The authors declare no conflicts of interest relevant to this study.Supporting Information S1Click here for additional data file."} +{"text": "Normal human physiology is vitally influenced by thyroid hormones. Thyroid hormones affect almost all human tissues influencing development and growth, regulating vital body functions and metabolism . The prein vitro experiment analysing FRGs and lncRNAs in orbital fibroblasts of three TAO patients and three healthy individuals . Another manuscript found digenic variants in five individuals affected with congenital hypothyroidism (CH). In addition, seven novel genetic variants were identified in these patients . Resistance to thyroid hormone (THR) is a rare hereditary disorder caused by mutations in the thyroid hormone receptor beta (THR\u03b2) gene. Two case reports published in this Research Topic deal with the problem of this syndrome. In the first one, authors sequenced the coding region of THR\u03b2 gene and found a novel dinucleotide substitution located in codon 453 in the affected woman with coexisting autoimmune thyroid disease . In the second one, the authors reported two cases of THR\u03b2 gene mutation with coexisting papillary thyroid carcinoma (PTC). After reviewing the literature, the authors state that 17 cases of THR\u03b2 gene mutation coexisting with PTC have been described to date .Although the mentioned approaches are very important in determining the genetic function of the thyroid gland, there are other types of genetic studies that help in elucidating the function of the gland. One of the manuscripts published in this Research Topic aimed to identify ferroptosis-related genes (FRGs) that may have a diagnostic and therapeutic association with thyroid-associated ophthalmopathy (TAO). Using a high-throughput gene expression database (GEO) authors identify differentially expressed genes and differentially expressed FRGs between TAO patients and controls. The authors also applied immune cell infiltrative analysis using the CIBERSORT algorithm to prove the difference between TAO patients and controls, and finally, they also identify differentially expressed ferroptosis-related lncRNAs in the TAO group. Gained results were validated by All manuscripts published in this Research Topic contribute to the elucidation of the genetic background of thyroid gland dysfunction."} +{"text": "Acknowledgments statement. This sentence previously stated:In the published article, there was an error. A correction has been made to the \u201cWe gratefully acknowledge the Universiti Teknologi MARA and Universiti Sains Islam Malaysia and to thank all the responders who participated in this study.\u201dThe corrected sentence appears below:\u201cWe gratefully acknowledge the Universiti Teknologi MARA [600-RMC/YTR/5/3 (001/2022)] and Universiti Sains Islam Malaysia and thank all the responders who participated in this study.\u201dThe authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated."} +{"text": "Papio ursinus) that commonly ranges within urban space in the City of Cape Town, South Africa, stops using urban space after giving birth. This change of space use occurs without any significant change in daily distance traveled or social interactions that would be expected with general risk\u2010sensitive behavior after birth. Instead, we suggest this change occurs because of the specific and greater risks the baboons experience within the urban space compared to natural space, and because leaving the troop (to enter urban space) may increase infanticide risk. This case study can inform methods used to manage the baboons' urban space use in Cape Town and provides insight into how life history events alter individuals' use of anthropogenic environments.Species with slow life history strategies that invest in few offspring with extended parental care need to adapt their behavior to cope with anthropogenic changes that occur within their lifetime. Here we show that a female chacma baboon ( Papio ursinus) who commonly ranges within urban spaces in the City of Cape Town, South Africa, stops using urban space after giving birth. We suggest this change occurs because of the risks the baboons face when leaving the troop and using urban space. This case study will be important for baboon management in the region, and provides insight into how life history events alter individuals' use of anthropogenic environments.Using high\u2010resolution GPS data we show that a female chacma baboon ( Species with slow life history strategies typically have few offspring and high investment in both social relationships and parental care. Consequently species with slow life history strategies are less able to compensate for increased mortality rates are a typical slow life history species, living in cohesive mixed\u2010sex social groups with strong social relationships that ranges within and on the periphery of the Da Gama Park suburb of Cape Town, several females gave birth . At the time of our study, the troop contained two adult males with an unstable dominance relationship before and after birth .In our study system, the urban space poses specific risks in the form of residents, dogs, vehicles, electric fences, and herding behavior by designated field rangers employed to reduce baboons' urban space use ; data curation ; formal analysis (lead); investigation ; methodology ; visualization (lead); writing \u2013 original draft (lead). Charlotte Christensen: Data curation ; investigation ; writing \u2013 review and editing . M. Justin O'Riain: Project administration ; writing \u2013 review and editing . Ines F\u00fcrtbauer: Funding acquisition ; project administration ; resources ; supervision (supporting); writing \u2013 review and editing . Andrew J. King: Conceptualization ; funding acquisition ; methodology ; project administration ; resources ; supervision (lead); visualization (supporting); writing \u2013 review and editing .The authors declare no competing interests."} +{"text": "The vocal fold mucosa has a unique multilayered architecture whose layers have discrete viscoelastic properties facilitating sound production. Perturbations in these properties lead to voice loss. Currently, vocal fold pliability is inferred clinically using laryngeal videostroboscopy and no tools are available for The main objective of the present study is to evaluate viability of Brillouin microspectroscopy for differentiating vocal folds\u2019 mechanical properties against surrounding tissues.We used Brillouin microspectroscopy as an emerging optical imaging modality capable of providing information about local viscoelastic properties of tissues in noninvasive and remote manner.Brillouin measurements of the porcine larynx vocal folds were performed. Elasticity-driven Brillouin spectral shifts were recorded and analyzed. Elastic properties, as assessed by Brillouin spectroscopy, strongly correlate with those acquired using classical elasticity measurements.These results demonstrate the feasibility of Brillouin spectroscopy for vocal fold imaging. With more extensive research, this technique may provide noninvasive objective assessment of vocal fold mucosal pliability toward objective diagnoses and more targeted treatments. These fibers and collagen are interwoven with vocalis muscle, forming a bond between the two morphologically different layers. The superficial layer is connected to the deep layer loosely through intermediate layer and can move differently from the elastic conus. The superficial layer and epithelium together form the vocal fold mucosa.The porcine larynx contains two types of vocal folds, similarly to humans; however, unlike the human model, they cannot be definitely addressed as \u201ctrue\u201d vocal folds and \u201cfalse\u201d vocal folds due to lack of information regarding which vocal folds play the key role in phonation.,According to previously published research,,The vocal folds are susceptible to pathological change, which can alter their mechanical properties resulting in loss of vocal capabilities.As a gold standard, definitive diagnosis of laryngeal and vocal fold anomalies requires tissue biopsy for pathological diagnosis that can lead to unintended adverse effects on voice. We propose application of Brillouin microspectroscopy as a viable and non-invasive tool to improve noninvasive diagnostic capabilities for assessing dysphonia and the vocal folds\u2019 mechanical and morphological properties.,Structural evaluation of vocal folds and their mechanical properties can be achieved with the help of more conventional imaging modalities, such as X-ray computed tomography (CT), magnetic resonance imaging (MRI), and ultrasound (US).,,Brillouin microspectroscopy is an emerging imaging technique built around evaluation of Brillouin scattering.Complex part of the high-frequency longitudinal modulus is defined by the following equation: ,,\u2013Brillouin spectroscopic evaluation utilizes relations quantified in Eqs.\u00a0(3) and (4) allowing for non-destructive label-free determination of viscoelastic properties of various materials. In the past decade, Brillouin spectroscopy has been applied to biological systems numerous times due to its inherent non-invasive and non-destructive nature. Multiple applications in the biological field were aimed at determining mechanical characteristics of different tissue types, such as superficial cancer tissues,ex vivo studies of human larynx in the future.It was reported previously that porcine larynx shares structural similarities with human larynx, making it a suitable model for phonation pathology research.Previously published research was focused on evaluating porcine larynx structures using cyclic force application and analysis of elongation using a scientific grade ergometer.To this end, we acquired a batch of porcine larynxes, tuned the system to allow for highly scattering sample imaging, and devised the experiment to remotely measure the elastic properties of excised porcine laryngeal structures using a confocal Brillouin spectrometer.The flowchart in As shown in We targeted superior vocal fold (SVF) and inferior vocal fold (IVF) of the porcine larynx with the expectation of their mechanical properties being significantly different from surrounding tissue. The surrounding supraglottal wall (SGW) was also evaluated as comparator non-vocal fold tissue.22.1,,Data were acquired in backscattering geometry using a custom-built upright confocal Brillouin microspectrometer,Brillouin scattering was induced with 532\u00a0nm radiation, which was generated as a result of second harmonic generation in a periodically poled lithium niobate (PPLN) crystal mple (S) and collBrillouin microspectrometer axial resolution did not exceed Collected light was guided toward a confocal pinhole assembly. Radiation was focused into a precision pinhole (PH) Nationa and recoSpatially filtered radiation was transmitted through a heated iodine vapor cell (VC) spectromns (CL1) (Thorlabns (CL1) . This was followed by baseline subtraction. Baseline curve was calculated via the use of an asymmetric least-squares fit algorithm .The processed signal was then converted from pixel domain to the GHz domain via approximation of VIPA dispersion curve using a clear and homogeneous sample as a reference . Stokes and anti-Stokes peak pairs retrieved from the acetone sample along with the fit peak maxima positions and VIPA dispersion curve are shown in According to previously published results,At the time of the measurements on the porcine vocal folds, spectrometer performance was characterized in a form of systematic error measurement. It was approximated from 100 data points acquired from acetone at 20\u00b0C. The histogram and Gaussian fit are shown in As seen in 3Compiled results for studied laryngeal samples are presented in the figures listed below. Three laryngeal regions that were interrogated for their elastic properties are shown in To account for tissue heterogeneity, digital reduction of system resolution was employed. Statistical analysis of frequency shift and linewidth value data within a sampled region provided raw standard deviation values for each interrogated region. Outliers were filtered out through a combination of median filtration, statistical analysis, and smoothing of 2D value map within a sampled subregion.Total accumulated data of mean retrieved values of Brillouin frequency shift and Brillouin line width are presented as bar plots in Brillouin frequency shift and Brillouin line FWHM data follow the same trend among all interrogated samples. Compiled data are shown in All the interrogated regions retrieved Brillouin frequency shift value distributions that were significantly different stiffness,The SGW region displayed the lowest Brillouin frequency shift values and lowest Brillouin line FWHM values, suggesting that this region has the highest elasticity and lowest viscosity. According to histological analysis of porcine larynx,Vocal folds, on the other hand, have been shown to have thin epithelium, attached to collagen-rich lamina propria.Previously published results were acquired using classical evaluation techniques that utilized the entire volume of excised vocal fold. Since, from the volumetric point of view, lamina propria makes up for the larger part of the total volume of vocal folds, we can infer that mechanical properties of vocal folds are driven by it. In case of our study, this means that Brillouin frequency shift difference occurs due to high lamina propria stiffness and different morphology between IVF and SVF. This also implies that due to good arrangement with previously published data, our system is indirectly evaluating lamina propria stiffness.4The ability of Brillouin microspectroscopy to differentiate between laryngeal substructures was successfully demonstrated for the first time. The use of porcine larynx allowed for the comparison of Brillouin frequency shift values between SVF, IVF, and SGW regions of the larynx. The Brillouin frequency shift values were statistically significantly different, with SGW tissue displaying the lowest Brillouin frequency shift value, and SVF displaying the highest, in agreement with previously published results using traditional mechanical testing.While these results clearly demonstrate that porcine vocal folds can be differentiated against surrounding tissue using Brillouin frequency shift values, further study is needed. The promise of Brillouin microspectroscopy for characterizing the vocal fold mechanical properties is that it is noninvasive and can potentially be done using common endoscopic approaches for imaging the vocal folds in the office setting. If changes in the mechanical properties that correlate with vocal fold pathology can be observed with Brillouin microspectroscopy, this approach could become a powerful diagnostic tool for the laryngologist."} +{"text": "Clefts of the lip and palate (CLP), the major causes of congenital facial malformation globally, result from failure of fusion of the facial processes during embryogenesis. With a prevalence of 1 in 500\u20132500\u00a0live births, CLP causes major morbidity throughout life as a result of problems with facial appearance, feeding, speaking, obstructive apnoea, hearing and social adjustment and requires complex, multi\u2010disciplinary care at considerable cost to healthcare systems worldwide. Long\u2010term outcomes for affected individuals include increased mortality compared with their unaffected siblings. The frequent occurrence and major healthcare burden imposed by CLP highlight the importance of dissecting the molecular mechanisms driving facial development. Identification of the genetic mutations underlying syndromic forms of CLP, where CLP occurs in association with non\u2010cleft clinical features, allied to developmental studies using appropriate animal models is central to our understanding of the molecular events underlying development of the lip and palate and, ultimately, how these are disturbed in CLP. Hhat, which encodes an acyltransferase responsible for modification of Hh proteins and Ptch1 mutant filopodia are absent and the facial processes fail to fuse signalling molecules are secretory glycoproteins which signal through Frizzled (FZD) cell surface receptors. WNT signalling activates signal transduction pathways which control cell proliferation, cell polarity, cell differentiation and cell survival gene in the epithelia of the facial processes resulted in fully penetrant bilateral CLP. In contrast, deletion of Bmp4 from the same region led to isolated cleft lip or inter\u2010rugal zones on the oral side of the palate deletion of Smo, display severe early craniofacial abnormalities which precluded analysis of secondary palate development were expressed ectopically and shown to be direct targets (Sema3a and Sema3d) of Osr2. Together, these studies reveal OSR2 plays an intrinsic role in mesenchymal cell proliferation and fate, preventing premature osteogenesis and aberrant semaphorin expression. However, further studies are needed to understand the function of semaphorins during palate development is involved in many aspects of early craniofacial development signalling lies downstream of WNT signalling in other development contexts and 6Historically, palatal shelf elevation was viewed as a process of rotation is restricted to the medial nasal mesenchyme prior to palatal shelf elevation consistent with a role in osteogenesis; in contrast, Tenascin\u2010C (Tnc) is expressed throughout the medial aspect of the palatal mesenchyme clinical features as non\u2010syndromic (ns) CLP. While there is a major genetic component to nsCLP (Grosen et al., Genome\u2010wide association studies indicate that only a small proportion of disease\u2010associated SNPs result in non\u2010synonymous changes to protein\u2010coding sequences suggesting that variants in regulatory sequences contribute markedly to human disease . For nsCWhile our current knowledge of facial development and disorders is derived mainly from analysing animal models, differences in the timing of facial development, the underlying molecular architecture and the facial morphology of different species, suggests that animal models are of limited use for interpreting genetic variation in human non\u2010syndromic CLP. For example, inter\u2010species differences in gene expression patterns have been demonstrated (Fougerousse et al., To overcome these limitations, transcriptomic and epigenomic analyses of human cells and bulk tissues have been undertaken using early human neural crest cells (Rada\u2010Iglesias et al., The authors have declared that no conflict of interest exists.Nigel Lowe Hammond: Writing \u2013 original draft; Writing \u2013 review & editing. Michael Dixon: Writing \u2013 original draft; Writing \u2013 review & editing.https://publons.com/publon/10.1111/odi.14174The peer review history for this article is available at"} +{"text": "In the published article, there was an error.A correction has been made to Methods section, Paragraph 5. This section previously stated:\u201cTo quantify aspects of the racial and economic diversity of each woman's neighborhood during the day , we used a large database of daily mobility as captured from mobile phone GPS apps, as provided by Cuebiq, a data aggregation firm. Cuebiq aggregated mobile phone location data, which are collected by select smartphone apps from about 15 million anonymous users who opted in to data collection for research purposes through a GDPR and CCPA compliant framework.\u201dThe corrected sentence appears below:\u201cTo quantify aspects of the racial and economic diversity of each woman's neighborhood during the day , we used a large database of daily mobility as captured from mobile device GPS apps. Mobile device location data were collected by select apps and subsequently aggregated from about 15 million anonymous users who opted in to data collection for research purposes through a GDPR and CCPA compliant framework.\u201dThe authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated."} +{"text": "Despite lotic systems demonstrating high levels of seasonal and spatial variability, most research and biomonitoring practices do not consider seasonality when interpreting results and are typically focused at the meso\u2010scale (combined pool/riffle samples) rather than considering habitat patch dynamics. We therefore sought to determine if the sampling season influenced observed macroinvertebrate biodiversity, structure and function at the habitat unit scale (determined by substrate composition), and if this in turn influenced the assessment of fine sediment (sand and silt) pressures. We found that biodiversity supported at the habitat level was not seasonally consistent with the contribution of nestedness and turnover in structuring communities varying seasonally. Habitat differences in community composition were evident for taxonomic communities regardless of the season but were not seasonally consistent for functional communities, and, notably, season explained a greater amount of variance in functional community composition than the habitat unit. Macroinvertebrate biodiversity supported by silt habitats demonstrated strong seasonal differences and communities were functionally comparable to sand habitats in spring and to gravel habitats in autumn. Sand communities were impoverished compared to other habitats regardless of the season. Silt habitats demonstrated a strong increase in Ephemeroptera, Plecoptera and Trichoptera (EPT) taxa and functional richness from spring into autumn, while vegetation habitats displayed a peak in EPT abundance in summer. Only silt and sand habitats demonstrated temporal variability in functional evenness suggesting that these habitats are different in terms of their resource partitioning and productivity over time compared to other habitats. Gravel and vegetation habitats appeared to be more stable over time with functional richness and evenness remaining consistent. To accurately evaluate the influence of fine sediment on lotic ecosystems, it is imperative that routine biomonitoring and scientific research discriminate between sand and silt fractions, given they support different biodiversity, particularly during summer and autumn months. We sought to examine if seasonal sampling influenced observed macroinvertebrate biodiversity, structure and function at the habitat unit scale (as determined by substrate composition) and if this in turn influenced the assessment of fine sediment (sand and silt) pressures on instream ecology. Biodiversity supported at the habitat level was not seasonally consistent when considered within the wider habitat provision at the reach scale particularly for functional level. In particular, results suggest that silt habitats are perhaps not as impoverished as previously perceived when considered seasonally. We examined taxonomic and functional facets of macroinvertebrate alpha and beta diversity. Establishing this knowledge base is vital to enable scientists and managers to accurately appraise ecological health across the entire year and ensure management recommendations are effective across seasons. In particular, we tested the following hypotheses:22.1Phragmites sp.). Other individual vegetation habitats were present in the reach but have not been included herein. On each occasion 10 replicate samples were taken for each substrate habitat; only five silt samples were taken during spring due to its naturally low occurrence during this season, resulting in a total of 115 samples. Samples consisted of a 15\u2010s kick/sweep sample using a 900\u2010\u03bcm pond net following the method outlined in Armitage et al.\u00a0. Although the data herein represent a re\u2010analysis of a previously published dataset, analysis and findings presented differ substantially in their focus (fine sediment dynamics) in addition to contemporary biodiversity analyses conducted being new.One set of macroinvertebrate samples were collected from a 300\u2010m reach of the Mill stream, located in Dorset, UK over three seasons in 1992: spring (late April), summer (early July) and autumn (late September). Winter was not sampled due to high\u2010water levels typically encountered during these months. Four habitat units were visually identified in each sampling season and each represented discrete patches of substrate/vegetation as opposed to a gradient of substrate sizes or vegetation species. The four habitats examined in each season were: (a) gravel, (b) sand, (c) silt and (d) perennial vegetation to three or five (high affinity). Trait values were therefore standardised following \u2018fuzzy coding\u2019 standardisation using the Arbizu,\u00a0. Indicatbetadisper function in vegan. Statistical differences in multivariate dispersion between seasons for each habitat were tested using one\u2010way ANOVA. Where significant seasonal differences occurred for a habitat, pairwise comparisons of differences were tested via Tukey's post hoc tests. Total beta diversity was decomposed into its nestedness and turnover components to investigate the dominant processes structuring macroinvertebrate taxonomic and functional composition. Prior to functional beta analysis the Taxa\u2009\u00d7\u2009Traits matrix underwent hierarchical clustering using the unweighted pair group method with arithmetic mean were calculated.To test for potential seasonal differences in the heterogeneity of macroinvertebrate community composition (beta diversity) of each habitat classification, homogeneity of multivariate dispersions were calculated for functional and taxonomic communities using the 2.3.2dbFD function on a Gower dissimilarity matrix in the FD package. General linear models (GLM) were constructed for each response variable by dataset and were fitted with the fixed interacting effects of habitat and season using the glm function in the stats package. All models were fitted with a Gaussian error distribution with the exception of abundance, taxa richness, EPT richness and EPT abundance which were fitted with a Poisson error distribution. Significant differences associated with habitat and season were tested via post hoc pairwise comparisons of groups using estimated marginal means, and p values were adjusted for multiple comparisons via Tukey's tests within the emmeans package taxa and Pielou's evenness. Three functional metrics were calculated comprising functional richness (FRic) representing the minimum convex hull encompassing all species, functional evenness (FEve) reflecting the regularity in which species are distributed across functional space and functional divergence (FDiv) representing how abundance is distributed within the volume of functional space occupied by species in taxonomic composition, while season explained the most variation for functional composition . In both instances the interaction of habitat and season were statistically significant . Sand habitats did not support any indicator species in spring, but two chironomid genera were identified in summer and one genus in autumn regardless of the season with these species being a mixture of taxonomic orders Table\u00a0. Gravel p\u2009<\u2009.001) and vegetation (p\u2009=\u2009.001) habitats were evident with silt supporting greater functional heterogeneity . Only sand habitats displayed seasonal variations in community abundance with statistically lower values in spring compared to all other habitats . The flow regime in temperate zone rivers during autumn months are typically stable following low and base flow conditions which were recorded in high abundances in the summer in gravel habitats, and as such were identified as indicators, were absent in gravel in the autumn with only a few individuals being recorded in the vegetation. As such, studies and routine biomonitoring investigating the ecological implications of fine sediment should consider the potential influence of season in their findings and metric derivation, as we observed differing outcomes when different seasons were considered. Indeed, three species of trichopteran were determined as indicator species in silt habitats in autumn, a surprising finding given that one species, Athripsodes cinereus, was classified as moderately sensitive to fine sediment in one biomonitoring tool were identified as potential indicator species with the latter being identified in both summer and autumn. Typically, studies examining fine sediment deposition (<2\u2009mm) do not discriminate between sand and silt fractions . However, it should be noted that application of finer resolution taxonomic data does enable identification of potential indicator species that would otherwise be overlooked, given the majority of taxa inhabiting sand habitats are generalists that can tolerate a range of conditions. Two genera of chironomid . The limited number of studies which have examined the processes structuring beta diversity in fine sediment habitats have reported that the communities were shaped by nestedness and others which may display seasonal variability (silt). At present, most national biomonitoring programmes are conducted as multi\u2010habitat 3\u2010min kick samples which still enable general riverine health to be assessed. Such assessments will provide reach scale quantification of the ecological community present ; data curation (lead); formal analysis (lead); funding acquisition (lead); project administration (lead); visualization (lead); writing \u2013 original draft (lead); writing \u2013 review and editing . Patrick D. Armitage: Conceptualization (supporting); investigation ; methodology ; writing \u2013 review and editing . Matthew Hill: Writing \u2013 review and editing . Morwenna McKenzie: Writing \u2013 review and editing . Isabel Pardo: Investigation ; methodology ; writing \u2013 review and editing . Paul J. Wood: Conceptualization (supporting); writing \u2013 review and editing .The authors declare no conflicts of interest.Tables S1\u2013S8Click here for additional data file."} +{"text": "In the published article, an author contribution was incorrectly written as [Ad]. The correct initial is [AdI].In the published article, there was an error in the Funding statement. [This project was supported by Compagnia di San Paolo (Progetto Trapezio) and by Italian Miur]. The correct Funding statement appears below."} +{"text": "Neonates with hypoxic ischemic encephalopathy (HIE) are at significant risk for adverse outcomes including death and neurodevelopmental impairment. Neuromonitoring provides critical diagnostic and prognostic information for these infants. Modalities providing continuous monitoring include continuous electroencephalography (cEEG), amplitude-integrated electroencephalography (aEEG), near-infrared spectroscopy (NIRS), and heart rate variability. Serial bedside neuromonitoring techniques include cranial ultrasound and somatic and visual evoked potentials but may be limited by discrete time points of assessment. EEG, aEEG, and NIRS provide distinct and complementary information about cerebral function and oxygen utilization. Integrated use of these neuromonitoring modalities in addition to other potential techniques such as heart rate variability may best predict imaging outcomes and longer-term neurodevelopment. This review examines available bedside neuromonitoring techniques for the neonate with HIE in the context of therapeutic hypothermia. Neonatal hypoxic ischemic encephalopathy (HIE) continues to be a significant health problem leading to death and long-term disability. Randomized controlled trials have evaluated the effect of systemic hypothermia on newborns with moderate to severe HIE and the results are encouraging; however, the incidence of death or moderate to severe disability remains 44%\u201352% in infants receiving induced hypothermia . A majorThe randomized controlled trials of therapeutic hypothermia for newborns with HIE marked the beginning of active investigation into the use of continuous neuromonitoring techniques in the neonatal intensive care unit (NICU). Prior to this period, video EEG was used in the NICU, but rarely for prolonged periods of time. The primary focus has been identification of electrographic seizure activity. For prediction of outcome, EEG background pattern is the most important factor and EEGs with low voltage, persistent burst suppression, or electrocerebral inactivity are highly correlated with adverse outcome and death. Amplitude integrated electroencephalography (aEEG), a simplified, continuous EEG using a small number of electrodes provides an overall impression of cerebral activity. The cerebral function monitor (CFM) was developed by Prior and Maynard in the 1960s for use in adults and subsequently applied to newborns in the late 1970s and 1980s. Its use has become more widespread for outcome prediction as well as for seizure detection particularly in newborns with perinatal asphyxia. The terminology used in aEEG is similar to EEG allowing for communication between neonatologists and neurologists.Near infrared spectroscopy (NIRS) is a more recently utilized technology for continuous, bedside, neuromonitoring by measuring trends in cerebral oxygenation and evaluating the balance between oxygen delivery and consumption. NIRS monitors also demonstrate the relationship between oxygenation of the brain and other end organs by providing cerebral and somatic oximetry values. Heart rate variability has also emerged as a potential bedside measure which may be predictive of outcomes in the HIE population. Other bedside neuromonitoring devices used less frequently in the NICU include visual and somatosensory evoked potentials.This manuscript will discuss the evidence for the use of these neuromonitoring techniques in newborns with HIE focusing primarily on outcome prediction and seizure detection.Electroencephalography is the recording of brain waves allowing for measurement of brain function. In newborns, electrode placement is according to the International 10\u201320 System, modified for the smaller head size. In a term infant, 9\u201311 electrodes are used whereas in a preterm newborn fewer are used. Electrode placement is at specific positions on the scalp and for this reason an EEG technologist is necessary. Interpretation is performed by a neurophysiologist trained in EEG interpretation. When the EEG is for a short period such as 30\u201360\u2005min it is considered a routine or \u201cspot\u201d EEG while longer recordings are termed continuous EEG (cEEG) and record for 24\u2005h or more, frequently including video (vEEG). cEEG is a key neuromonitoring tool when caring for critically ill term and preterm neonates. Its primary roles are identification of electrographic seizure activity and prognostication by assessing the background brain activity. The American Clinical Neurophysiology Society has written guidelines for cEEG monitoring in neonates , focal, or low amplitude are not recognized as they are not visible on the compressed tracing.n\u2009=\u200931) and hypothermia treated infants (n\u2009=\u200943) . Tracing(n\u2009=\u200943) . Several(n\u2009=\u200943) . They caSeveral meta-analyses have sought to further clarify the role of aEEG in prediction of outcome for HIE. Each have had a slightly different focus. A study by Spitzmiller in 2007 was the first meta-analysis to evaluate the use of aEEG to predict outcome in HIE prior to the use of therapeutic hypothermia . In a poAs neonatal HIE may also result in hemodynamic and cerebral metabolic alterations, measurement of cerebral oxygenation (rScO2) and cerebral fractional tissue oxygen extraction (fTOE) with NIRS bedside monitoring has significant promise as a neurodiagnostic technique. A NIRS sensor placed on the left or right forehead emits near-infrared light, which penetrates through skin and bone and is differentially absorbed by oxygenated and deoxygenated hemoglobin in the underlying tissue. Residual light is then reflected back to a detector, with subsequent calculation of rScO2, a measure of tissue oxygenation. Cerebral fTOE is estimated using the combined measure of peripheral oxygen saturation (SpO2) and rScO2 (fTOE\u2009=\u2009(SpO2 \u2013 rScO2)/SpO2), indicating the balance between oxygen delivery and oxygen consumption in the interrogated tissue. Different commercial NIRS devices operate using these same principles, although with slightly different light wavelengths and algorithms, resulting in discrepancies in absolute rScO2 levels between devices . For thiIn infants with HIE, decreased cerebral blood flow after acute injury is followed by reperfusion over time. As a surrogate for cerebral blood flow, the chronologic evolution of rScO2 may be an important bedside measure and further serve as a prognostic tool . While tSeveral studies have examined cerebral NIRS measures during therapeutic hypothermia as a predictor for both adverse brain MRI outcomes and longer-term neurodevelopmental outcomes. High rScO2 and low fTOE are associated with worse neuroimaging outcomes by MRI in several small studies \u201347. A hiSimilarly, several small studies have demonstrated adverse neurodevelopmental outcomes at 18\u201330 months of age in infants with higher rScO2 and lower fTOE during cooling. These studies all were limited by relatively small sample size and differed on optimal time period to capture differences, with best predictive capabilities ranging from 12\u2005h of age, 24\u201348\u2005h of age, or after 72\u2005h and rewarming , 49\u201351.Rate of change of rScO2 may also be prognostic and associated with the severity of HIE. In a prospective, observational study, 15 non-cooled infants with mild HIE had significantly higher mean rScO2 and lower FTOE at 6\u2005h of age compared to 15 cooled infants with moderate HIE ), although differences resolved by 18\u2005h . An earlMore sophisticated NIRS techniques and analyses of NIRS data may provide additional insight for the neonatal HIE population. Several investigators have explored impaired cerebral autoregulation with failure to regulate cerebral blood flow as a mechanism for brain injury after HIE. Various measures of autoregulation using NIRS have included a higher pressure passivity index with increased coherence between mean arterial pressure and cerebral oxygen saturation, increased time domain reactivity index, and wavelet coherence analysis \u201355. RegiPerinatal oxygen deprivation with subsequent HIE may impact cortical and subcortical neuronal pathways, disrupting the integrity of the autonomic nervous system , 59. Conn\u2009=\u2009248) concluded that moderate and severe HIE was associated with a reduction in most HRV measures are typically obtained to assess the optic pathway in response to a flash of light. Most studies demonstrating association of abnormal VEPs with adverse neurodevelopmental outcome were obtained in the pre-hypothermia era, although in more recent cohorts of infants treated with therapeutic hypothermia for HIE, VEP abnormalities have been associated with impaired hearing-language scores and with abnormal glucose levels and MRI brain injury \u201378. SomaNIRS and aEEG or cEEG are the neuromonitoring modalities most utilized in NICUs, specifically in newborns with HIE. The use of both modalities has the distinct advantage of simultaneous assessment of both cerebral oxygenation and brain function. NIRS serves as a trend monitor to evaluate the balance between tissue oxygen delivery and consumption while aEEG or EEG provides a continuous monitoring of cerebral function while also alerting the medical providers to electrographic seizure activity. Stratification of hypoxic-ischemic injury may be distinguished by findings on combined neuromonitoring. In several animal models of HIE, NIRS was highly sensitive to an acute hypoxic ischemic insult, while aEEG was less sensitive, particularly for the detection of mild hypoxia ischemia , 82. HowIn clinical studies of neonates with HIE, combined use of aEEG and NIRS improved predictive value compared to either modality alone for both MRI abnormalities and neurodevelopmental outcomes at 18 months of age or 30 months of age , 51, 85.Combining neuromonitoring with NIRS and EEG may also have promise for improved seizure detection and improved assessment of hemodynamic changes during seizure evolution . Higher Simultaneous use of these neuromonitoring techniques may improve the understanding of alterations in cerebral and systemic hemodynamics and the resulting risk of cerebral injury. Some infants with HIE may also require extracorporeal life support (ECLS), and close neuromonitoring in these patients is particularly important given their inherent risk for both hemodynamic instability and abnormal brain function , 89. DesSelection of optimal neuromonitoring modalities for the infant with HIE must take into consideration sensitivity and specificity for prediction of adverse neurodevelopmental outcomes, timing of monitoring to impact clinical care, and ease of implementation and interpretation at the bedside. Neuromonitoring in neonates with HIE plays an important role for both diagnostic and prognostic purposes. As future neuroprotective strategies are developed in the era of therapeutic hypothermia, timely initiation of neuromonitoring is critical for risk stratification of neonates with HIE, early prognostication, and counseling of families. A combination of neuromonitoring techniques with HRV metrics, bedside imaging, evoked potentials, or other newer technologies in the infant with HIE requires further exploration. Multimodality neuromonitoring with aEEG/EEG and NIRS holds particular promise for continuous, bedside assessment of both electrical function and hemodynamic changes in the brain. Consideration should also be given to the necessary training of bedside staff for the implementation of neuromonitoring modalities and interpretation of results. An individualized approach to care using real-time neuromonitoring will ideally optimize neurodevelopmental outcomes in the vulnerable neonatal HIE population."} +{"text": "Cultural incompetence hinders the engagement of sexual and gender minority (SGM) youth in the HIV prevention and care continuum. Improving providers\u2019 cultural competence is critical to reducing HIV health inequities. Medical education lacks opportunity for practice and SGM perspectives. We are developing an AI training app that uses virtual patients and a framework of educational and behavior change theory to increase provider clinical and communication competence with SGM youth. SGM youth and healthcare providers have unique knowledge about provider bias and stigma barriers to care, which can inform intervention content.From Twitter's API, 42,213 Tweets were iteratively sampled by 11 SGM youth (the \u201cYAB\u201d) and analyzed thematically. These themes were combined with current clinical care guidelines for SGM youth to generate 111 stigma barriers to care agreed upon by the YAB by consensus. These themes were edited and ranked by importance by the YAB using a modified Delphi method. Using this content as a starting point, we held workshops with providers and the YAB to develop a comprehensive list of scenarios where providers noted deficient training in providing culturally competent care to SGM youth. Prioritized training scenarios selected from this list were developed into detailed scripts for AI virtual patient encounters.Using a model of social media mining informed by SGM youth and provider collaborative work, training scenarios were developed for integration into a GPT/AI provider training app. Scenarios address topics selected by SGM youth that are particularly salient to this population based on social media conversations around provider stigma and the YAB members\u2019 lived experiences. Scenarios will progress from basic to advanced topics to train providers in culturally appropriate language and foster competent care for SGM youth.SGM youth have deep insight into their care needs and lived experiences of healthcare trauma. Understanding these care needs and building provider competence is critical to reduce HIV inequities among SGM youth. Our data provide new insight into how interactions between providers and SGM youth may improve. Future research will test the app\u2019s effect on the clinical care behaviors of providers in a randomized control trial.All Authors: No reported disclosures"} +{"text": "Domestic and sexual violence and abuse (DSVA) is a global public health problem resulting in health inequalities. Community pharmacies are uniquely placed to help people affected by DSVA. We examined factors that impact pharmacists\u2019 engagement in response to DSVA when providing public health services.n\u00a0=\u00a020) were analyzed thematically, with inductive themes mapped to the Capability\u2013Opportunity\u2013Motivation Behaviour (COM-B) model.Semi-structured qualitative interviews with community pharmacists (Pharmacists were confident in providing public health services, but a lack of DSVA training meant there is a need to support their \u2018Capability\u2019 to respond to DSVA. Pharmacies were perceived as highly accessible healthcare providers on the high street, with sexual health consultations offering an ideal \u2018Opportunity\u2019 to enquire about DSVA in a private consultation room. Pharmacist\u2019s \u2018Motivation\u2019 to enquire about DSVA was driven by potential positive client outcomes and a desire to be more involved in public heath interventions, but organisation- and system-level support and remuneration is needed.Community pharmacy offers opportunities for integrating DSVA work in existing public health services. Pharmacists need training on DSVA, ongoing support, allocated funding for DSVA work, and awareness raising campaign for the public on their extended public health role. Domestic and sexual violence and abuse (DSVA) is a global public health and clinical problem rooted in gender and social inequalities.Public Health England identifies community pharmacies as an important provider of public health services, uniquely placed to reach disadvantaged, marginalized and vulnerable populations.,World Health Organisation (WHO),Our systematic review,,A few studies explored the topic of DSVA in the community pharmacy setting. One US survey found that although women considered DSVA as an important health-care issue, they did not feel that a pharmacy would be an appropriate place for addressing this problem.,In March\u2013July 2017, experienced female social science researchers conducted semi-structured interviews with community pharmacists purposefully sampled across two UK localities. We aimed to recruit a maximum variation sample for heterogeneityResearchers introduced themselves and the study and obtained written informed consent. Interviews were guided by a topic guide developed with a pharmacist and LPC managers and pilot tested in the first two interviews. Interviews were audio-recorded, professionally transcribed and imported into NVivo 10 for coding. We used a combination of inductive thematic analysisLPCs emailed study invites to 35 pharmacies ; 12 did not respond, 3 declined due to workload; 20 pharmacists consented to take part. Interviews lasted between 18 and 71\u00a0minutes (mean 33). We met our recruitment target on heterogeneity. The South West pharmacies representing independent and chain businesses were located in relatively deprived areas (mean IMD 3.6) across four local authorities. Included pharmacies served communities across both inner city and urban residential areas. Inner city pharmacies had lower IMDs, while urban residential settings were relatively less deprived. All London pharmacies were independent and located in less deprived residential areas (mean IMD 4.8) in one local authority .n\u00a0=\u00a010), supervised methadone consumption (n\u00a0=\u00a09), smoking cessation (n\u00a0=\u00a07) and flu vaccine (n\u00a0=\u00a07). All pharmacists had completed mandatory training on each public health service they provided and on safeguarding children and vulnerable adults. Only two pharmacists recalled encounters with a small number of clients who have experienced DSVA. We developed eleven themes from open coding and mapped them on the COM-B constructs. There was commonality in views across inner city and urban residential pharmacies.All pharmacies provided locally commissioned public health services other than EHC: chlamydia treatment . However, few participants from small independent pharmacies thought that even in the consultation room, some clients could feel too exposed because of the possibility of knowing and being known to the staff and people attending from the same community. One pharmacist said that despite offering consultation in a private room, some clients want conversation over the counter.Pharmacists were concerned that public and other professionals are not yet ready to use pharmacies as providers of public health services including response to DSVA. Participants thought that most people still perceive pharmacies as medicine suppliers and attend for a transaction of medicine rather than for a healthcare consultation , quote 3Pharmacists serving multicultural communities identified language and cultural barriers to engaging with patients from minority ethnic groups. Female pharmacy assistants were key for facilitating communication between women seeking EHC and male pharmacists , quote 4Pharmacists\u2019 \u2018Motivation\u2019 to enquire about DSVA was driven by potential positive client outcomes and a desire be more involved in public heath interventions, but they needed organisation- and system-level support and financial remuneration. Pharmacists believed that identification and response to DSVA could be part of their role in provision of public health services but had concerns about their confidence and competence, negative consequences and lack of incentives for engaging in DSVA work. All pharmacists saw themselves as healthcare providers with limited expertise in diagnosing and managing clinical conditions. Participants valued extended pharmacy role comprising traditional medicines supply and provision of new public health services. Some pharmacists saw DSVA work as an opportunity to enhance existing health services for vulnerable populations. Pharmacists wanted to provide holistic healthcare and suggested integrating identification and response to DSVA into the existing commissioned EHC service, supervised consumption of opioid substitution therapy and needle and syringe exchange. Some saw potential for integrating DSVA work into medicine use reviews, undergraduate pharmacy courses and public health campaigns.Participants from standalone independent pharmacies valued their social capital in the local community and believed that they could become an essential part of a multisectoral response to DSVA , quote 1One third of pharmacists said that financial incentivization would facilitate their engagement in identification and response to DSVA. They explained that community pharmacy is a private business paid per medicine or health service supplied as opposite to general practice paid per population served. Although independent pharmacists highlighted their commitment to the needs of the local community over payment, most pharmacists thought that budget for DSVA work should be allocated the same way as for other commissioned public health services , quote 3We explored barriers and facilitators to pharmacists\u2019 engagement in identification and response to DSVA and mapped them on the COM-B model to inform pharmacy-specific adaptations of the IRIS GP intervention. Qualitative interviews with community pharmacists found that they have some Capacity, Motivation and see Opportunities for integrating identification and response to DSVA into existing commissioned public health services. The main facilitators for pharmacist engagement in DSVA work were expertise and experience in providing commissioned public health services (Capability), the accessibility and inclusivity of the pharmacy setting (Opportunities), perceived relevance of DSVA work to their role and intention to get more involved in public health services (Motivation). Barriers to engaging in DSVA work included: (i) uncertainty about how to identify and respond to clients affected by DSVA in the time available, lack of training, policy/guidance, judgemental attitudes (Capability), (ii) limited resources, societal perception of pharmacy role, language and cultural norms (Opportunities), (iii) fear of negative consequences, lack of support to offload emotional burden and lack of reward (Motivation).Pharmacists suggested integrating DSVA identification and response into existing public health services, for example sexual health consultations. They saw LPCs and existing system of remuneration for commissioned public health services as vehicles for creating opportunities and motivation for engaging in DSVA work.,,One systematic review found some evidence on the acceptability, feasibility and effectiveness of pharmacy-based public health interventions for smoking cessation and weight loss.We applied the COM-B model to identify barriers and facilitators to engaging pharmacists in identification and response to DSVA during provision of commissioned public health services. Pharmacists were confident and motivated to provide public health services but lacked Capability and Motivation for engaging in identification and response to DSVA.Pharmacy setting offers Opportunities for integrating DSVA work in existing public health services. Pharmacists welcome training on DSVA, ongoing support, allocated funding for DSVA work and awareness raising campaign for the public on their extended public health role. LPC and PharmaOutcomes platform can be used to deliver the training and remunerate for DSVA work integrated into existing commissioned public health services. These findings will inform pharmacy specific adaptation of the IRIS GP programme through matching the identified barriers and facilitators with target behaviours on the Theoretical Domains Framework and using the Behaviour Change Wheel to choose behaviour change technics.,Our findings are applicable to the recent codeword schemes in community pharmacies, which have not yet been formally evaluated. In line with the previous systematic reviews,We did not interview pharmacy clients and therefore further research is required to examine their views on the acceptability of the DSVA work. This study took place before the UK Home Office introduced the codeword schemes in community pharmacies. Pharmacists\u2019 \u2018Capability\u2019 and external \u2018Opportunities\u2019 could have changed because of the training and promotional materials provided as part of these schemes.JH, GF, NVL conceived and designed the study. NVL and TS conducted interviews. NVL, TS, and JH analysed the data and produced first draft. All authors contributed to five revisions of the manuscript. All authors read and approved the final manuscript."} +{"text": "Brook trout populations have been declining throughout their native range in the east coast of the United States. Many populations are now distributed in small, isolated habitat patches where low genetic diversity and high rates of inbreeding reduce contemporary viability and long\u2010term adaptive potential. Although human\u2010assisted gene flow could theoretically improve conservation outcomes through genetic rescue, there is widespread hesitancy to use this tool to support brook trout conservation. Here, we review the major uncertainties that have limited genetic rescue from being considered as a viable conservation tool for isolated brook trout populations and compare the risks of genetic rescue with other management alternatives. Drawing on theoretical and empirical studies, we discuss methods for implementing genetic rescue in brook trout that could yield long\u2010term evolutionary benefits while avoiding negative fitness effects associated with outbreeding depression and the spread of maladapted alleles. We also highlight the potential for future collaborative efforts to accelerate our understanding of genetic rescue as a viable tool for conservation. Ultimately, while we acknowledge that genetic rescue is not without risk, we emphasize the merits that this tool offers for protecting and propagating adaptive potential and improving species' resilience to rapid environmental change. Many brook trout populations exist in small, isolated habitats where demographic stochasticity and the lack of gene flow threaten long\u2010term persistence. In this article, we review existing literature to evaluate whether genetic rescue may be a viable management goal for brook trout conservation. Although brook trout were once abundant in many mountain streams and lakes, native populations have declined over the last century due to the interactive effects of habitat loss, climate change, and competition with non\u2010native species isolated by anthropogenic activities may be particularly vulnerable to demographic and genetic collapse, as bottlenecked populations frequently experience higher genetic loads owing to inbreeding, loss of genetic diversity, and increased prevalence of deleterious mutations despite the use of an adaptively divergent source population. Likewise, Wells et al.\u00a0 and have lost substantial genetic diversity may be better candidates for genetic rescue. Ralls et al.\u00a0 suggesteSalvelinus spp., authors have been able to disentangle the relative effects of neutral and adaptive genetic processes for shaping contemporary patterns of genetic diversity, ultimately providing insights into the colonization success of introduced brook trout populations . Likewise, regional population genetic analyses are necessary to identify source populations with enough genetic diversity to enable genetic rescue and to screen for introgression with domestic hatchery lineages ; investigation (lead); visualization (lead); writing \u2013 original draft (lead); writing \u2013 review and editing (lead). Jacob M. Rash: Conceptualization ; supervision ; writing \u2013 original draft (supporting); writing \u2013 review and editing (supporting). David C. Kazyak: Conceptualization ; supervision (supporting); writing \u2013 original draft (supporting); writing \u2013 review and editing (supporting).None.The authors declare no conflicts of interest."} +{"text": "Chamaecrista fasciculata, a legume species that has previously been determined to have significant SGS. We collected genetic data from prokaryotic and fungal rhizosphere communities in association with 70 plants in an area of ~400 square meters to investigate the presence of SGS in microbial communities. Bacteria of Acidobacteria, Protobacteria, and Bacteroidetes and fungi of Basidiomycota, Ascomycota, and Mortierellomycota were dominant members of the rhizosphere. Although microbial alpha diversity did not differ significantly among plants hosts, we detected significant compositional differences among the microbial communities as well as isolation by distance. The strongest factor associated with microbial distance was genetic distance of the other microbial community, followed by geographic distance, but there was not a significant association with plant genetic distance for either microbial community. This study further demonstrates the strong potential for spatial structuring of soil microbial communities at the smallest spatial scales and provides further insight into the complexity of factors that influence microbial composition in soils and in association with host plants.Soil microbiota of the rhizosphere are an important extension of the plant phenotype because they impact the health and fitness of host plants. The composition of these communities is expected to differ among host plants due to influence by host genotype. Given that many plant populations exhibit fine\u2010scale genetic structure (SGS), associated microbial communities may also exhibit SGS. In this study, we tested this hypothesis using Chamaecrista fasciculata. Diversity of bacteria and fungi at this small scale is correlated, exhibits an isolation by distance, and is best explained by environmental factors, rather than the host plant genotype.This study characterized fine\u2010scale patterns of microbial diversity in the rhizosphere of the host plant, Given its frequent occurrence in early successional habitats, it has great potential to influence below\u2010ground community composition. C.\u2009fasciculata is symbiotic with rhizobia Rhizosphere microbial communities, encompassing bacteria and fungi, have varying composition at a local spatial scale, (2) This compositional variation exhibits genetic structuring that may be explained by (a) genetic structuring in host plants and/or (b) isolation by distance.In a previous study, we identified fine\u2010scale spatial genetic structure (SGS) for both host plants and nodulating rhizobia and found that plant genetic identity and geographic distance are significant predictors of the genetic structure of nodulating rhizobia of 22.1Pinus taeda forest . The concentration of the extracted DNA was determined using a Qubit 3 Fluorometer (ThermoFisher Scientific). By applying 319F and 806R primer pair sequences designed for the V3 and V4 region of the 16S rRNA gene was used to assess the association between genetic distance of microbial communities based on weighted UniFrac distance matrices and fungal data sets . In PCoA plots, the first two components accounted for approximately 42% of the total variance observed among bacterial communities and 30% of the total variance among fungal communities sampled among different plant hosts. Bacterial sequences from plot 1 are scattered in the PCoA plot while samples from plots 2 and 3 appear more distinct from one another in both analyses. Although significant (p\u2009<\u2009.05) predictors were identified, still relatively little of the observed genetic distance variation was explained by them and pea (Pisum arvense L.) Higher bacterial diversity in our study might be explained by higher tendency for symbiotic relationships between legumes and bacteria for nitrogen fixation and assimilation, carbon and sulfur cycling, metabolism of iron, antimicrobial and transporter properties Heynh. and suggested that host plant genotype most strongly shapes the associated microbial community when it directly affects colonization of hub taxa. Thus, while it is commonly believed that host plant traits play a role in determining the composition of their rhizosphere communities, such effects may be diluted by microbe\u2010microbe interactions that take place after initial colonization by specific \u201chub\u201d microbes. The presence of diverse symbioses common to many legumes, and discussed above, may also influence the structure of microbial communities of legumes in unique ways ; data curation ; formal analysis (lead); funding acquisition ; investigation (lead); methodology ; writing\u00a0\u2013 original draft (lead); writing \u2013 review and editing . Lisa E. Wallace: Conceptualization ; data curation ; formal analysis (supporting); funding acquisition ; investigation ; methodology ; project administration (lead); resources (lead); supervision (lead); writing \u2013 review and editing .This research was funded by the Biology Faculty Fund of Mississippi State University and the J. Robert Stiffler Endowment at Old Dominion University.The authors declare no conflict of interest."} +{"text": "Authors declare no conflict of interests for this article.To editor:Detection of lethal arrhythmia is of great importance in the diagnosis of patients with palpitations. Various noninvasive devices have been developed to monitor and detect critical arrhythmias. Kim and colleagues demonstrated that a 7\u2010day patch\u2010type continuous electrocardiogram (ECG) monitor (MEMO patch\u00ae) had acceptable feasibility and efficacy in detecting arrhythmias when compared with conventional 24\u2010h Holter ECG monitoring.Various long\u2010term ECG monitoring devices have demonstrated their efficacy so far.One of the issues of these monitoring devices is their water resistance. If the MEMO patch is not water resistant, it cannot be used during bathing, when the autonomic nervous system fluctuates dramatically and the risk of critical arrhythmias increases.The ability to detect arrhythmias depends on the duration of monitoring.It is generally challenging to automatically distinguish atrial fibrillation from sinus tachycardia by detecting R\u2010R irregularity. Was the presence of atrial fibrillation detected automatically or manually by using MEMO patch?"} +{"text": "In mental health settings, there is no place more social than where people smoke tobacco, patients and healthcare professionals alike much as many social activities in other settings even nowadays.Yet mental illness is associated with higher levels of social anxiety. Those who suffer are doing their coping and may appear to be doing better than the others but in fact may need special attention for smoking cessation because they are still smoking more than other patient populations.To reflect on tobacco smoking and social anxiety.Pubmed search using terms: tobacco and smoking and social anxiety/ social anxiety disorderIs associated with higher smoking initiation and progression to dependenceis more frequent in smokersis used as a coping mechanism for distress caused by social interactions and may alleviate negative affect and thus serve as negative reinforcementmay be associated with higher nicotine dependencehas not been definitely associated with heavier smokingmay differ in its effects according to gendermay be associated with less quit attemptsmay hinder success in quitting smoking and may be associated with higher rates of relapseSocial anxiety:Identifying and treating social anxiety may lead to better outcomes in smoking cessation in a sub-group of patients who present elevated social anxiety with or without social anxiety disorder.Patients with mental illness, especially serious mental illness, will likely present with higher levels of social anxiety which may represent a significant factor contributing to an increased difficulty in quitting tobacco smoking in this patient population.None Declared"} +{"text": "Unveiling the interactions between brain areas and determining the topology and function of the networks connecting them is central to Neuroscience as these networks do not just integrate multiple inputs but also perform complex information processing. A large repertoire of connectivity estimators has been put forward to decipher and quantify network structure and operation; they include linear/non-linear, bivariate and multivariate methods. Determining connectivity patterns faces many technical challenges which once successfully addressed, potentially offer an appropriate descriptive brain network framework amenable to further analysis through complex network theory tools. This collection of papers portrays some different approaches to achieve that.Strijbis et al.: \u201cState changes during resting state (magneto)encephalographic studies: the effect of drowsiness on spectral, connectivity, and network analyses\u201d used the phase lag index (PLI) and corrected amplitude envelope correlation (AECc) to generate a network topology that was appraised through the method of minimum spanning trees. Spectral analysis over 6 canonical frequency bands was also determined. Structural differences between open-eye, closed-eye and drowsiness states were examined. State and condition were far less distinct when seen through the proposed connectivity methods than when compared via spectral analysis. The best discrimination was achieved using spanning trees coupled with AECc, whereas only minimal delta band differences were noticed between drowsiness, closed-eye and open-eye states. Drowsiness had no impact on minimal spanning tree measures.The paper of Coelho Ramos et al.: \u201cSpectral density-based clustering algorithms for complex networks\u201d investigated functional brain network changes during anesthesia. The authors applied clustering methods including k-means graph classification and model-based approaches to compare graphs of different sizes. The networks presented by the authors have very dense structure and equal connection strengths. The paper is a methodological study of different clustering approaches for binary graph comparison.The paper of Paz-Linares et al.: \u201cMinimizing distortions in electrophysiological source imaging of the cortical oscillatory activity via Structured Sparse Bayesian Learning\u201d concerns the reconstruction of oscillating cortical sources from scalp data. The authors aimed at improving contemporary approaches by using Bayesian learning with variational approximation to the problem of setting \u201ca priori\u201d connection probabilities. The authors claim that this leads to a two order of magnitude decrease in distortion compared to other state-of-the-art methods while still being applicable to large-scale networks and not just to low-density settings as is the case of other methods.The paper by Pidnebesna et al.: \u201cTackling the challenges of group network inference from intracranial EEG data\u201d faces the problem of statistical inference and analysis of brain networks under inhomogeneous and sparse electrode placement and its differentiation across patients. The authors developed a methodological pipeline for estimating this kind of group network structure. They tested their methodology on intra-cranial data recorded for the range of visual tasks by using Directed Transfer Function (DTF)\u2014a multivariate causal measure of connectivity\u2013 that provides directed information flow and allows representing reciprocal connections and multiple loops relationships, as in The present collection of papers was motivated by the largely unsatisfactory present status of graph theoretical applications to neural connectivity following valid criticisms of ubiquitous \u201csmall word\u201d descriptions (Hilgetag and Goulas, To sum up the present Special Topic edition provided a promising panorama of approaches toward constructing brain networks whether they are binary or weighted directed graphs thereby opening perspectives for improving their analysis and systematic comparison.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication."} +{"text": "Li et\u00a0al. reports an interesting evolutionary process of EGFR alteration patterns when a stage IV lung adenocarcinoma patient is under selective pressure while on a TKI. The resistance mechanism involves enrichment of differentially mutated or abnormally amplified EGFR. The patient has relatively extended stable disease periods when a TKI is tailored based on specific EGFR alterations from biopsies at different stages of disease progression. Accumulation of novel mutations during the disease course may exhaust options of targeted therapies. Case reports in this Research Topic show off-label use of TKIs for novel EGFR mutations may have unexpected therapeutic benefits as demonstrated by other cohort studies . For example, a radiographic complete response was achieved with concurrent chemo-/radio-therapy and crizotinib for a rare locally-advanced gingival sarcomatoid squamous cell carcinoma patient with MET exon 14 skipping mutation . Interestingly, Li et\u00a0al. reported management of a intracranial lesion for an EGFR-mutated NSCLC patient with radiation followed by adaptive targeted therapies. They reported the patient had unexpectedly good clinical response with intracranial disease control of 23 months.Safety and efficacy of combined treatment of targeted therapy and radiotherapy is not well reported in clinical trials. This Research Topic showed several cases with good outcomes when concurrent or sequential treatment of radiation and targeted therapeutic agents (This series of articles in Molecular and Cellular Oncology highlights the gaps in understanding how targeting a single mutation alters the cellular microenvironment and implications of this as patients are living longer on these therapies. Further studies are needed to better understand genetic alterations in tumor response to novel targeted therapies and their interactions with chemo-/radio-therapies.SZ drafted the editorial; TT edited and revised the editorial. All authors contributed to the article and approved the submitted version."} +{"text": "PLOS Biology finds a different pattern\u201460 SNPs spread across the genome differentiate a Penstemon species pair.The integrity of hybridizing species is usually maintained by genome-wide selection or by selection on a few genomic regions. A study published in Hybridizing species are usually maintained by genome-wide selection against introgression or by selection on a few \u201cgenomic islands\u201d. This Primer explores the implications of a study which finds a new pattern \u2013 60 SNPs spread across the genome differentiate Penstemon species with different pollinators. PLoS Biology by Wessinger and colleagues [The past two decades of research on speciation genomics have shown that a simple bifurcating tree\u2014the major metaphor presented to describe the process of speciation\u2014is a biological exception rather than the rule. Speciation genomic analyses have revealed that post-divergence gene flow is common and have tended to find one of two genomic patterns of introgression, each of which involve heterogeneity in interspecific divergence across the genome. One commonly reported pattern is a negative relationship between recombination rate and introgression, and this pattern is interpreted as genome-wide selection against minor parent ancestry . The othlleagues fits neiPenstemon species is the result of repeated shifts from bee to hummingbird pollination, with the shifts in floral traits forming the basis of taxonomic species delineation. Wessinger and colleagues [P. barbatus is not monophyletic, but rather appears as two distinct subclades within the broader diversity of the bee-pollinated P. virgatus and P. neomexicanus that distinguish otherwise similar populations have long served as a model to understand the process of speciation . Shifts lleagues focus onlleagues discoverexicanus . SpecifiDespite this genome-wide pattern of low interspecific divergence relative to intraspecific diversity, Wessinger and colleagues found 60P. barbatus/P. virgatus\u2014with dozens of SNPs associated with species identity spread across the genome and colocalizing with loci underlying species differences despite isolation by distance both within and between species\u2014is potentially consistent with numerous alternative evolutionary histories . The authors are careful not to overinterpret the observed pattern, but suggest that the patterns observed are best explained by occasional local gene flow between species (despite the paucity of hybrids observed in the field) with strong assortative mating and divergent selection maintaining divergence in floral syndromes. While these patterns and analysis are exciting, it is not the only such case\u2014regions associated with coloration differences between two subspecies are spread across multiple chromosomes in hybridizing subspecies of the northern flicker, Colaptes auratus [As de novo genome assemblies and large-scale resequencing efforts continue to spread to non-model species, solving the major problem of connecting patterns of divergence across the genome to the underlying process of speciation is becom auratus . It is cThe future of speciation genomics research is quite exciting. Combining genetic mapping and genome scans (like those of Wessinger and colleagues and that"} +{"text": "The authors reply: As representatives for our entire team, we thank Vinayachandran and Balasubramanian , paternal alcohol exposure (PatExp), and dual parental alcohol exposure and measThese preliminary data reinforce our assertion that FAS craniofacial phenotypes can emerge from paternal alcohol exposures alone and that the exclusive focus on maternal exposures as a diagnostic criterion of FAS may be flawed. Moreover, they suggest that the craniofacial defects described in our E16.5 fetal samples may persist through development into adulthood, emphasizing that FAS is not merely a pediatric disorder but persists across the life course ("} +{"text": "Observational studies have found Attention Deficit Hyperactivity Disorder (ADHD) to be associated with an increased risk of adverse outcomes as well as with early risk factors; however it is not clear whether these associations reflect causal effects. Alternatives to traditional observational studies are needed to investigate causality: one such design is Mendelian randomization (MR), which uses genetic variants as instrumental variables for the exposure.In this review we summarise findings from approximately 50 studies using MR to examine potentially causal associations with ADHD as either an exposure or outcome.To\u2010date, few MR ADHD studies have investigated causal evidence with other neurodevelopmental, mental health and neurodegenerative conditions but those that have suggest a complex relationship with autism, some evidence of a causal effect on depression and limited evidence of a causal effect on neurodegenerative conditions. For substance use, MR studies provide evidence consistent with a causal effect of ADHD on smoking initiation, but findings for other smoking behaviours and cannabis use are less consistent. Studies of physical health suggest bidirectional causal effects with higher body mass index, with stronger effects for childhood obesity, as well as some evidence of causal effects on coronary artery disease and stroke in adults and limited evidence of causal effects on other physical health problems or sleep. Studies suggest bidirectional relationships between ADHD and socio\u2010economic markers and provide some evidence that low birthweight may be a causal risk factor for ADHD, while bidirectional evidence has been found for some environmental factors. Finally, there is emerging evidence of bidirectional causal links between ADHD genetic liability and biological markers of human metabolism and inflammation.While MR has advantages over traditional observational designs in addressing causality, we discuss limitations of current ADHD studies and future directions, including the need for larger genome\u2010wide association studies (and using samples of different ancestries), and for triangulation with different methods. Observational studies have found ADHD to be associated with an increased risk of adverse outcomes as well as with early risk factors; however it is not clear whether these associations reflect causal relationships. In this review we summarise findings from approximately 50 studies using Mendelian randomization (MR) to examine causality with ADHD as either an exposure or outcome. Observational studies have found ADHD to be associated with an increased risk of both adverse outcomes and early risk factors: it is not clear whether these reflect causal evidence.We summarise findings from approximately 50 studies using MR to examine causality with ADHD as either the exposure or outcome.MR studies have examined causal evidence\u00a0between ADHD and (i) neurodevelopmental, mental health, neurodegenerative conditions and personality, (ii) substance use, (iii) a range of physical health and sleep measures, (iv) cognitive ability test scores and indicators of socio\u2010economic status, (v) birthweight, gestational age and environmental exposures, and (vi) biological markers.MR has advantages over traditional observational designs in examining causal evidence in ADHD, which could have prevention and treatment implications.Attention Deficit Hyperactivity Disorder (ADHD) is a neurodevelopmental condition that typically onsets in early in development studies have identified several risk factors for ADHD. ADHD is highly heritable \u2013 twin studies estimating 70%\u201380% heritability and limited evidence for shared familial environmental contributions as instrumental variables (proxies) for the exposure: as shown in Figure\u00a0In this paper we will summarise findings from studies using MR to examine potentially causal evidence with ADHD as either an exposure or outcome. We focus on 2\u2010sample MR studies, in which data on the exposure and outcome of interest are identified from summary statistics of two different GWAS (one for the exposure and one for the outcome), although some alternative designs are briefly covered. Our search strategy for identifying MR studies of ADHD is outlined in the Supporting Information.Despite substantial phenotypic overlap, few MR studies have investigated causal evidence between ADHD and other conditions involving the brain, mainly due to the challenges of identifying causal effects with genetic instruments that display extensive pleiotropic effects across different conditions. Two studies of ADHD and autism spectrum disorder (ASD) suggested a complex relationship, with evidence of a causal effect of genetic liability to ASD on ADHD and of genetic liability to ADHD on ASD, with effects in both directions showing substantial heterogeneity inflammatory bowel disease . At the moment, there is limited evidence of ADHD genetic liability causing other physical health problems or affecting sleep.Three MR studies investigating ADHD and cognitive ability test scores using the same cognitive ability GWAS have found evidence of a causal effect of genetic liability to higher cognitive ability test scores on lower risk of ADHD, with little evidence of horizontal pleiotropy and two measures of sexual behaviour with bidirectional effect for the latter with genomics to investigate causal roles of blood biomarkers on ADHD.One 2\u2010sample MR study integrated metabolomic and genomics and used data from a GWAS of 486 metabolites have been tested for causal effects on psychiatric disorders, including ADHD, with the MR study not identifying any potential causal effects , ADHD MR study findings \u2013 whether ADHD is the exposure or outcome \u2013 have theoretical, prevention and treatment implications. Where study findings are consistent with a causal effect of ADHD on outcomes, such as have been observed for depression and smoking initiation, these suggest that effective treatment of ADHD may reduce the risk of these outcomes, although triangulation of findings is needed (see below). Multivariable MR can provide indications of potential causal pathways of these relationships, for example, findings implicating childhood obesity as a mediator of the causal link between ADHD and coronary artery disease (Leppert et\u00a0al.,\u00a0Where MR studies have investigated ADHD as the outcome, findings consistent with a casual effect on ADHD, such as have been observed for low birthweight, suggest that targeting these exposures in those at risk of ADHD may reduce risk of ADHD. However, it is not clear what causal effects of phenotypes such as low birth weight capture, maybe other underlying foetal problems. Finally, emerging evidence of bidirectional causal links between ADHD genetic liability and biological markers of human metabolism and inflammation may provide clues as to the biological underpinnings of ADHD, which could help refine treatment options.While MR studies have advantages over traditional observational designs in minimising unmeasured confounding and reverse causation, this is based on MR assumptions (Davey Smith & Hemani,\u00a0Population stratification is another source of bias in MR: many of the studies we reviewed restricted the ADHD GWAS used to European origin samples (which comprise the majority of samples included in the largest ADHD GWAS; Demontis et\u00a0al.,\u00a0Finally, triangulation between MR findings and those of other methods is required as different designs will have different sources of bias (Lawlor et al., \u00a0In conclusion, to\u2010date approximately 50 studies have been published examining potentially causal evidence with ADHD as either the exposure or outcome. Few MR studies investigated causal effects between ADHD and other conditions involving the brain, but those that have suggest a complex relationship with ASD, some evidence of a causal effect on depression and limited evidence of a causal effect on neurodegenerative conditions. Mendelian randomization studies of substance use provide evidence consistent with a causal effect of ADHD on smoking initiation, but findings for other smoking behaviours and cannabis use are less consistent. Several studies have examined causal evidence with different aspects of physical health: these suggest bidirectional causal effects with higher BMI, with stronger effects for childhood obesity, as well as some evidence of causal effects on coronary artery disease and stroke in adults, but limited evidence of causal effects on other physical health problems or sleep. Studies suggest bidirectional relationships between ADHD and socio\u2010economic markers, which could reflect an increased likelihood for ADHD symptoms being impairing in the absence of sufficient or additional resources and that ADHD can lead to barriers in attaining higher education and income. Studies have also provided some evidence that low birthweight may be a causal risk factor for ADHD, while bidirectional effects have been found for some environmental factors. Finally, there is emerging evidence of bidirectional causal links between ADHD genetic liability and biological markers of human metabolism and inflammation. While MR has advantages over traditional observational designs in addressing causality it is not without limitations and triangulation with different methods is needed to increase confidence in causal association.Lucy Riglin: Data curation, Investigation, Writing \u2013 original draft. Evie Stergiakouli: Investigation, Writing \u2013 original draft.The authors have declared that they have no competing or potential conflicts of interest.No ethics approval was required for this article as no new data were created or analyzed in this study.Supplementary MaterialClick here for additional data file."} +{"text": "OXTR methylation) affects the salience of social information in young adults. Little is known about how the oxytocinergic system ages and what effect this aging system has on social cognitive abilities throughout the lifespan.Social isolation is one of the strongest predictors of increased risk of mortality in older adulthood. The ability to form and maintain the social relationships that mitigate this risk is partially regulated by the oxytocinergic system and one\u2019s ability to attend to and process social information. We have previously shown that an epigenetic change to the DNA of the oxytocin receptor gene (OXTR DNA methylation in young (age 18\u201331) and older (age 58-81) adults. Participants underwent fMRI during a selective social attention task and provided a DNA sample for the assessment of OXTR methylation.Here we explored age-related differences in the association between neural response during selective social attention and OXTR methylation interaction on neural response when attending to social stimuli in a complex display; young adults displayed a positive association between OXTR methylation and neural activation, replicating our prior finding that young adults with presumed diminished endogenous access to oxytocin recruit regions of the attentional cortex to a greater extent. This association did not hold for older adults. Instead, perceived social support interacted with OXTR methylation to influence neural response during selective social attention. These data suggest that environmental factors like social support moderate biological processes in aging and highlight the importance of a lifespan perspective for understanding associations between individual differences in the oxytocinergic system, neural function, and social behavior.We found that older adults activated diffuse areas of visual cortex and dorsolateral prefrontal cortex during selective social attention, consistent with the dedifferentiation and compensatory neural activation commonly reported in aging. We found a significant age-by- Understanding the neurobiological factors that enable successful social functioning across the lifespan has important psychological and public health implications . The faiMultiple streams of incoming information simultaneously compete for our attention. Because our perceptual and cognitive systems have a limited processing capacity, we must selectively attend to stimuli that are relevant to the task at hand while simultaneously ignoring distracting information . SalientAdditionally, our socio-cognitive abilities and motivations change across the lifespan. While affective empathy and social competencies generally improve with age, abilities to detect cues in human emotion and accurately attribute mental states to others decrease in older adulthood . These cOXTR) is partially responsible for this variability in expression. Our group has identified a cytosine-phosphate-guanine (CpG) site within the promoter of OXTR, site \u2212934 fail to find social information intrinsically salient and therefore engage additional, compensatory neural mechanisms to attend to social information.We have previously shown in a sample of healthy young adults that differences in neural response during selective social attention is associated with ite \u2212934 . Specifiey nodes . These rOXTR methylation is associated with social signal detection across adulthood. Additionally, we explored the moderating effect of social support in older adulthood on the relationship between OXTR methylation and neural response during selective social attention.While a few studies have begun to investigate how the endogenous oxytocinergic system ages , no studM\u2009=\u200921.14) and 88 older adults aged 58\u201381\u2009years (M\u2009=\u200968.27) participated in the present study. To control for potential population stratification artifacts related to epigenetic testing, only individuals who self-identified as white and of European descent were included in analyses. Older adults were recruited as a subset from the Virginia Cognitive Aging Project (VCAP), a longitudinal aging study ongoing since 2001. VCAP participants were determined to be cognitively normal on the Mini-Mental Status Exam . Additional cognitive related data including the Trail Making Test A assessing processing speed and working memory younger adults aged 18\u201331\u2009years years provided both sample types to determine whether methylation values differ across tissue collection methods. DNA was isolated and subjected to bisulfite treatment which converts non-methylated cytosines to uracil and leaves methylated cytosines unmodified. We then amplified a 116-base pair region of OXTR containing CpG site-934 for assessment of peripheral blood mononuclear cell (PBMC) methylation (younger adults), or PAXGene Blood DNA Tubes for assessment of whole blood methylation (older adults). An independent tissue comparison sample consisting of 156 adult participants aged 16\u201364 since 2009. Average social support scores ranged from 36 to 98.5.Participants in VCAP have been completing the Social Support Questionnaire at each Imaging acquisition details for the original young adult sample are detailed in Data preprocessing was carried out using the FMRI Expert Analysis Tool (FEAT) Version 6.00, part of the FMRIB Software Library (FSL) . The folImaging analysis was conducted using FEAT. At the subject level, time-series statistical analysis was carried out using FSL\u2019s improved linear model (FILM) with local autocorrelation correction . RegressZ (Gaussianised T/F) statistic images were thresholded using clusters determined by Z\u2009>\u20092.3 (p\u2009<\u20090.01) and a corrected cluster significance threshold of p\u2009<\u20090.05 \u2009>\u20091 stage 1 with FSLp\u2009<\u20090.05 . Cluster (D)\u2009>\u20091 as criteOXTR methylation and task-specific Attend Faces > Attend Houses activation (n\u2009=\u200949). Two regressors were included in the model \u2013 group mean and mean-centered OXTR methylation \u2013 and contrasts testing for positive and negative linear associations between OXTR methylation and Attend Faces > Attend Houses BOLD activation were computed. One outlier was removed from this analysis. All subsequent analyses considered the full young adult sample (n\u2009=\u2009103).We first conducted a whole-brain analysis to determine the replicability of the association between tivation in our nWe next tested for group-level differences in task-specific Attend Faces > Attend Houses activation using a two-sample unpaired t-test with task accuracy included as a nuisance regressor to account for differences in task performance across age groups. Three outliers (2 younger adults) were removed from this analysis.OXTR methylation to determine whether the slope of the linear association between OXTR methylation and Attend Faces > Attend Houses BOLD activation varied as a function of age group. Task accuracy was included as a nuisance regressor in this analysis. Eight outliers (3 younger adults) were removed from this analysis.We next tested for an interaction between age group and OXTR methylation, social support, and neural activation during selective social attention in the older adult group. We included the mean-centered measures and the interaction terms as regressors and Task Accuracy as a nuisance regressor in the model, and computed contrasts testing for linear associations between each regressor and Attend Faces\u2009>\u2009Attend Houses activation. Two outliers were removed from this analysis. Finally, we computed the percent of significant voxels that overlapped between the analysis considering total Support Score and analyses considering each of the individual subscales to explore whether any particular factor of social support most accounted for our results.Finally, in an exploratory analysis we tested for associations between SD\u2009=\u20096.33). Whole blood methylation values averaged 46.45% (SD\u2009=\u20096.82). Methylation values were significantly correlated across tissue types , indicating methylation values in our two imaging samples can be compared.We first determined that methylation values do not differ across whole blood and PBMC tissue collection types. In the independent tissue comparison sample, PBMC methylation values averaged 46.72% (SD\u2009=\u20096.06). Whole blood methylation for the older adult sample averaged 47.09% (SD\u2009=\u200910.31). Mean methylation values did not significantly differ across age groups , although the variance in methylation values was significantly higher for older adults .PBMC methylation for the younger adult sample averaged 47.30% such that younger adults performed significantly better on the task than older adults . Performance on the Attend Faces and Attend Houses conditions was significantly correlated for both younger and older adults. Task performance as well as error type by task condition and age group are plotted in A logistic regression model predicting proportion correct from age group revealed a significant effect of age group on task performance (n\u2009=\u200948) to test for the replicability of the association between OXTR methylation and neural response during selective social attention we previously identified in young adults activated specific regions of the face perception network including left orbitofrontal cortex and posterior cingulate cortex to a greater extent than older adults than older (n\u2009=\u200985) adults within regions of the attentional control network including anterior cingulate cortex, left dorsolateral prefrontal cortex, and left superior parietal lobule . We found a significant interaction between OXTR methylation and reported social support on neural response during selective social attention. To illustrate the interaction, we plotted social support by median split and found that older adults who reported lower levels of social support displayed a positive association between OXTR methylation and neural activation within right dorsolateral prefrontal cortex, whereas this association was negative for those that reported higher levels of social support . These results were primarily driven by the Enacted Support subscale continue to exert effort and recruit regions of the attentional control network because they intrinsically find social information worthy of attention and investment of cognitive resources. Conversely, with increasing methylation (presumed less sensitivity to endogenous oxytocin), those who report a supportive social environment may not feel the need to exert extra effort to attend to the social information in this task.We therefore hypothesized that an epigene-by-environment interaction may account for individual differences in neural response during selective social attention in our older adult sample. In an exploratory analysis, we found an interaction between ionships . PerhapsOXTR DNA methylation and autism than older females (n\u2009=\u200960). Therefore, while we could not reliably assess sex differences in the older adult sample, we found a positive association between OXTR methylation and neural response within attentional control regions across sexes in our younger adult sample. The oxytocinergic system, social behavior, and their associated neural systems all show sex differences (Our sample had fewer older males (ferences ; therefoOXTR methylation and neural response during selective social attention for the theory of socioemotional selectivity, future research should present both familiar and unfamiliar stimuli to understand how age and methylation impact differential activation of attentional control regions during the perception of familiar vs. unfamiliar faces. Finally, age-related changes in functional connectivity within attention and salience networks should be explored in future work, particularly given that DLPFC activation and functional connectivity during selective social attention interacted to impact task performance in our prior young adult work (Although we controlled for task performance by including this variable as a nuisance regressor in all analyses, future research should set individual threshold levels for task performance so that the neural and epigenetic mechanisms supporting task performance are matched across cognitive demand. However, that task performance varied greatly within both age groups reflects an advantage for our study design as young adults typically score at ceiling on many social cognitive tasks. Additionally, to further probe the hypothesized mechanism of ult work .Our results highlight the importance of adopting a lifespan perspective for understanding associations between individual differences in oxytocinergic system function, neural systems supporting social cognition, and social behavior. Maintaining social relationships is critical for longevity, happiness , and redThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving humans were approved by the University of Virginia Institutional Review Board for Health Sciences Research. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.MP, JC, and JM designed and conceptualized the study. MP, ML, and MN wrote the paper. MP collected the younger adult data and performed the fMRI and epigenetic analyses. ML collected the older adult data and isolated whole blood DNA for the older adult samples. JC and JM provided edits and comments to the manuscript and funding for the work. All authors contributed to the article and approved the submitted version."} +{"text": "While therapeutic hypothermia improves outcome after moderate and severe neonatal hypoxic\u2010ischaemic encephalopathy (HIE) in high\u2010income countries, many cooled infants without cerebral palsy (CP) have cognitive impairment and memory deficits during childhood.Spencer et al.Why does hippocampal injury persist in children without CP despite rescue hypothermic neuroprotection? Excluding children with CP suggests that this cohort of cooled infants were probably at the milder end of neonatal brain injury, where hypothermia is expected to be more neuroprotective. Hippocampus is one of the first regions to be injured in preclinical models of hypoxic\u2013ischemic injury, well before deep brain nuclei injury.Although preclinical models of single acute hypoxia\u2010ischemia have had a major role in the discovery of hypothermic neuroprotection, clinical scenario is far more heterogenous than current preclinical models. More complex preclinical models, representative of clinical scenario, are required to further advance the field of neuroprotection. Another major implication of these observations relates to the therapeutic drift of cooling in mild encephalopathy. Although CP is unlikely after mild encephalopathy, many of these infants have cognitive and memory deficits during childhood. Does it mean that hypothermia may not benefit these infants? Clearly, further research is required to explore these issues."} +{"text": "Girls are more at risk of non-communicable diseases associated with insufficient levels of physical activity (PA). Maternal support plays an important role in girl\u2019s PA. Yet little is known about how mothers provide support to keep their daughters active. The aim of this study was to explore mothers\u2019 experiences of supporting their daughter to be physically active.A qualitative study with semi-structured interviews was conducted with a purposive sample of mothers (n\u2009=\u200929) of girls . Reflexive Thematic Analysis was used to analyse the data, with themes/subthemes mapped to the relevant domains of the Theoretical Domains Framework (TDF).Most mothers recognised the importance of supporting their daughter to be active. Themes highlighted how a mother\u2019s identity and confidence in supporting their daughter to be active influenced supportive behaviours. Social interactions played an important role in facilitating or inhibiting how a mother supports her daughter. The circumstances of a mother\u2019s environment and her perceptions of her daughter\u2019s attitudes towards PA influenced the type of support mothers provided.The findings advance our understanding of maternal PA support behaviours which will inform strategies to enhance maternal PA support with the ultimate goal of improving girls\u2019 participation in PA. Future research should explore the appropriate behaviour change techniques for maternal PA support and involve mothers in the co-design process to develop interventions that are more feasible, acceptable and implementable."} +{"text": "PLOS ONE article ..PLOS ONEPLOS ONE study included more details and discussion of limitations of its reported model as compared to the Diversity and Distributions study, and found that whether species\u2019 area of habitat is larger or smaller than the original range map differs across IUCN Red List categories.Both articles , 2 addre"} +{"text": "This is the authors\u2019 response to peer-review reports for \u201cEarly Experience With Neutralizing Monoclonal Antibody Therapy for COVID-19: Retrospective Cohort Survival Analysis and Descriptive Study.\u201dFormatting in paper changed Figures changedAdded suggested contextualizationFortified DiscussionAlthough we added references and discussion of the inflammatory response to cytokines, it should be recognized that these antibodies work by neutralization of the virus, not by affecting cytokines."} +{"text": "Xeroderma pigmentosum is an autosomal recessive disorder with various ocular manifestations of which bilateral limbal stem cell deficiency is a rare manifestation. Timely diagnosis and meticulous management are vital in these cases to prevent irreversible ocular sequelae.Bilateral limbal stem cell deficiency (LSCD) can be a rare manifestation in patients afflicted with xeroderma pigmentosum (XP). The authors report a rare case of a 12\u2010year\u2010old boy who presented with redness and defective vision and was diagnosed with bilateral LSCD and hyperpigmented lesion over the face and trunk suggestive of XP. Kirandeep Kaur: Conceptualization; data curation; formal analysis; investigation; methodology; project administration; resources; software; supervision; validation; visualization; writing \u2013 original draft; writing \u2013 review and editing.There are no conflicts of interest.Written informed consent was obtained from the patient to publish this report in accordance with the journal's patient consent policy.At our institute case reports, images and case series are exempted from IRB approval and the research followed the tenets of the Declaration of Helsinki."} +{"text": "Infectious diseases physicians invest significant time mentoring medical students and internal medicine residents developing research projects as well as writing case reports. The impact of research or case reports on future application to infectious diseases fellowship is unknown.All research projects and case reports published or presented at conferences from Brooke Army Medical Center between 2014-2022 with an infectious diseases senior author and a medical student or internal medicine resident first author were evaluated. The presentations and publications that resulted from each project as well as whether the trainee applied to infectious diseases were recorded.During the study period, 12 faculty mentored 51 trainees in 35 research projects and 26 case reports. Research and case reports were primarily performed by residents (88% and 96% respectively) (Table 1). Most trainees presented or published their research in multiple venues. Compared to case reports, research projects were more likely to be presented at national meetings . Individuals who completed research projects had greater rates of infectious diseases fellowship application as compared to those who completed case reports (Table 2).Summary of Research and Case Reports by Medical Students and Residents Mentored by Infectious Diseases Faculty at Brooke Army Medical Center, 2014-2022Internal Medicine Resident and Medical Student Application to Infectious Diseases Fellowship Organized by Projects Mentored by an Infectious Diseases Faculty Member at Brooke Army Medical Center, 2014-2022Internal medicine resident and medical student involvement in research mentored by an infectious disease physician was associated with a higher infectious diseases fellowship application rate as compared to those who were mentored for case reports. Despite the significant time required to mentor trainees, investment in trainee research should be considered as an important component to the infectious diseases recruiting effort.All Authors: No reported disclosures"} +{"text": "Homo sapiens population dynamics, most likely fuelled by the ability to accumulate cultural/technological innovations that actively modify their environment. We are especially interested in establishing if the demographic transitions of pre-historic populations show the same dynamic signature of the Industrial Revolution transition (a positive relationship between population growth rates and size). Our results show that population growth patterns across different pre-historic societies were similar to those observed during the Industrial Revolution in developed western societies. These features, which appear to have been operating during most of our recent demographic history from hunter\u2013gatherers to modern industrial societies, imply that the dynamics of cooperation underlay sudden population transitions in human societies.Abrupt and rapid changes in human societies are among the most exciting population phenomena. Human populations tend to show rapid expansions from low to high population density along with increased social complexity in just a few generations. Such demographic transitions appear as a remarkable feature of This article is part of the theme issue \u2018Evolution and sustainability: gathering the strands for an Anthropocene synthesis\u2019. Human populations tend to show rapid expansions from low to high population density and social complexity in relatively few generations ). 5Our results suggest that a much more flexible approach is required to understanding human population dynamics by including cooperation and competition as general principles driving demographic changes. Given the present controversy about how human population dynamics can be a cause or a consequence of socio-cultural innovations, we present support for the idea of a mutual feedback relationship between demography and cultural dynamics \u201321,23,73Over much of the Holocene, human population trajectories experienced waves of accelerated growth rates, probably driven by a positive feedback loop among population size and technological innovations, to a \u2018propensity towards unsustainability and socio-ecological crisis' , p. 1096"} +{"text": "This challenge has to be progressively resolved to discover more effective and personalized cancer therapies.The development of tailored therapies designed to specifically target driver oncogenes has initiated a revolutionary era in cancer biology. The availability of a growing number of selective inhibitors has generated novel experimental and clinical paradigms. These represent an opportunity and a challenge for researchers and clinicians to delve deeper into the intricate dynamics of cancer development and response to treatment. By directly inhibiting key driver oncogenes involved in tumor initiation and progression, scientists have an unprecedented opportunity to conduct longitudinal and clonal evolutionary studies of how cancer cells adapt, rewire, and exploit conflictive or overlapping signaling dependencies in response to treatment Personalized cancer therapies mark a groundbreaking era in cancer research. The chance to identify new vulnerabilities stemming from cancer cell adaptation, signaling rewiring, and treatment response is now a reality. In this battle with tumors, researchers and clinicians must carefully consider their moves, just like chess players, to anticipate the response of tumors and strategize the most precise and effective therapy. ALKanaplastic lymphoma kinaseEGFRepidermal growth factor receptorKRASKirsten rat sarcoma virus oncogene homologNTRKneurotrophic tyrosine receptor kinaseRETrearranged during transfection proto\u2010oncogeneIn cancer biology, the concept of \u201concogenic sweet spot\u201d refers to the optimal signaling input resulting in best cell fitness output . This opCurrent knowledge regarding drug resistance in cancer often revolves around the identification of specific genetic mutations responsible for stable, inheritable resistance states. However, there is growing recognition of adaptive and potentially reversible mechanisms of drug resistance. These plastic mechanisms represent dynamic phenotypic diversity present within tumor cell populations which is dependent on individual oncogenic sweet\u2010spot, suggesting that disrupting these states could be highly advantageous. Therapeutic efforts can be focused on shifting the drug\u2010resistant subpopulation toward a state that represents the drug sensitivity of the majority of cells, promoting greater uniformity among tumor cell subpopulations.Systematic sequencing of tumor biopsies at relapse or longitudinal sampling of circulating tumor DNA in clinical trials and real\u2010world patients have yielded vast amounts of data that have substantially broadened the understanding of the genetic basis of tumor cell adaptation to the blockade of driver oncogenic signaling. Typically, a sustained inhibition of driver oncogenes shapes the selection of clonal populations that are driven by co\u2010existing oncogenes that never occur in treatment\u2010na\u00efve tumors , 10, 11.While our understanding of these mechanisms is still incomplete, an immediate question that arises is whether this tumor adaptability can be exploited as a liability for cancer treatment when two potent and druggable oncogenes co\u2010exist. In some models, preclinical data have demonstrated that the co\u2010existence of potent driver oncogenes is detrimental for cell fitness in untreated tumor cells. In contrast, this co\u2010existence is selected upon oncogenic driver inhibition in experimental models and patients Fig.\u00a0 12]. Wo. Wo12]. One immediate approach would be a dual therapy that simultaneously targets both mutated driver oncogenes Fig.\u00a0. While rThis approach would likely alleviate the toxicity of combined therapies and could be achieved by designing treatment regimens that exhibit synergy without administering both drugs concurrently . Such a In all these treatment scenarios, the goal is to delay or prevent the emergence of drug resistance through sequential administration of targeted therapies, offering a treatment schedule to mitigate possible constraints on dosage due to cumulative toxicities between the combined agents. In other words, a complete eradication of tumor cells might be difficult to achieve with multiple drug combinations due to the barrier imposed by toxicity. However, there could possibly be alternative ways to exploit the growing arsenal of targeted therapies to prolong the fight with drug\u2010resistance cancer cells while improving the quality of life of cancer patients.CA received research fees from Revolution Medicines, Verastem, Roche and Boehringer\u2010Ingelheim. RC is the scientific founder of ALKemist Bio.RC and CA conceived and wrote the paper."} +{"text": "We recently established a website which provides people in Germany with the nearest early detection an intervention service available (https://www.psycho-check.com). However, the overall implementation rate of early detction in Germany is quite heterogenous. We will also present recent research and ongoing projects from Germany including the first evaluation of specialized inpatient services for early psychosis, first evaluations of Individual placement and support and a mindfulness based group intervention in people with early psychosis as well as a newly desigend youth mental health service called soulspace (None Declared"} +{"text": "Weight gain is most marked if ART is started with CD4 counts below 200 cells/\u03bcL.1 Women gain more weight after starting ART than men.1 The South African ADVANCE study reported more weight gain in participants randomised to dolutegravir than efavirenz, which was more marked among women.3 This observation has caused concern because women in sub-Saharan Africa have a higher prevalence of both HIV and obesity than men.4Dolutegravir and other integrase strand transfer inhibitors are associated with more weight gain in people starting antiretroviral therapy (ART) than other antiretroviral drug classes.6There are two hypotheses for the greater weight gain observed with dolutegravir than efavirenz: dolutegravir may be causing weight gain or efavirenz may be impairing weight gain. There are no credible mechanisms by which dolutegravir could cause weight gain. Integrase strand transfer inhibitors inhibit the human melanocortin 4 receptors in vitro, which plays a role in the control of appetite in the brain, but inhibition only occurs at concentrations far exceeding those that are achievable clinically. By contrast, there are biologically plausible reasons why efavirenz could impair weight gain: by impairing appetite from chronic neuropsychiatric effects or by its toxic effects on adipocytes.7 A genetic sub-study in ADVANCE participants randomised to the efavirenz arm found that efavirenz metaboliser genotype predicted weight gain to 48 weeks: extensive metabolisers gained the most weight, followed by intermediate metabolisers, then slow metabolisers, who actually experienced a slight decline from baseline in median weight.8 A key point was that weight gain was similar between extensive metabolisers in the efavirenz arm and in the dolutegravir arm with the same nucleoside reverse transcriptase inhibitor backbone (tenofovir disoproxil fumarate and emtricitabine). A cohort study reported that efavirenz slow metabolisers gained the most weight after switching from efavirenz to integrase inhibitors when virologically suppressed, providing further evidence for impaired weight gain on efavirenz-based ART in slow metabolisers.9Recent studies provide very strong evidence for the hypothesis that efavirenz impairs weight gain in genetically susceptible individuals. Efavirenz concentrations are highly variable, which is mainly due to loss-of-function mutations in genes encoding for the cytochrome P450 enzymes responsible for metabolising efavirenz, CYP2B6 (major pathway) and CYP2A6 (minor pathway). Mutations in these genes are used to categorise people into extensive, intermediate, and slow efavirenz metabolisers \u2013 intermediate metabolisers have modestly increased efavirenz concentrations while slow metabolisers (about 20% of South Africans) have markedly increased efavirenz concentrations.The belief that dolutegravir causes weight gain is widespread among people with HIV and clinicians, which can result in inappropriate antiretroviral switching, reduced adherence, and failure to manage people who are overweight and obese with appropriate interventions.5People with marked weight gain on dolutegravir-based ART should be screened for the metabolic syndrome and treated accordingly. The weight gain should be addressed by appropriate lifestyle and other interventions. Newer interventions are more effective at maintaining weight loss for people with established obesity than lifestyle measures but access to these interventions is limited in our region, especially in the public sector. Switching antiretrovirals in people who experience weight gain on dolutegravir is recommended against in the 2023 guidelines from the Southern African HIV Clinicians Society and the National Department of Health. Switching from dolutegravir to efavirenz could result in weight loss in efavirenz slow metabolisers, but they are at increased risk of severe toxicity, including drug-induced liver-injury and chronic neuropsychiatric conditions."} +{"text": "Socio-demographic and clinical variables were recollected at the baseline evaluation. Functioning was evaluated at baseline with the functioning assessment short test (FAST). The strength of the association between the lifetime number of affective episodes and FAST subscores was explored with Spearman\u2019s correlation test. Linear regression was computed using cognitive functioning as the dependent variable and a set of clinically relevant variables including the lifetime number of affective episodes as independent variables after controlling for illness duration.261 BD patients were recruited. Patients with a higher number of lifetime affective episodes showed a significant positive correlation with higher FAST global score and FAST cognitive functioning subscore . At the linear regression, a higher number of affective episodes was associated to worse cognitive functioning .Poor cognitive functioning in BD could be the result of multiple affective relapses. A timely diagnosis with subsequent effective prophylactic treatment may prevent poor functional outcomes in real-world patients with BD.None Declared"} +{"text": "In the era of personalized medicine, magnetic resonance-guided radiotherapy (MRgRT) is a new technique aiming to reduce radiation-induced toxicity while improving oncologic outcomes by facilitating dose-escalation These differences show that there can be variations in different model\u2019s performance. Therefore, each model should be externally validated before routine clinical use. Application and evolution of artificial intelligence systems and incorporation of the results in 3D systems can also assist magnetic resonance imaging-guided nerve sparing surgery and improve erectile function preservation after radical prostatectomy. The research field is open and better training is essential both for automatic models as well as for radiation oncologists.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper."} +{"text": "Chronic infection with hepatitis B virus (HBV) induces transregulation of the host cell gene expression and eventually malignant transformation. HBV X protein acts as a transactivator of cellular promoters leading to upregulation of DNA methyltransferases and alterations of DNA methylation patterns.To contribute to our understanding of DNA and chromatin-modifying mechanisms modulated by HBV, which are responsible for the transregulation of host cells for virus particle production and malignant transformation of hepatocytes. We further study the effect of lamivudine treatment or an antiviral vector-based RNAi strategy targeting HBx on the establishment and maintenance of DNA methylation patterns. Our research not only aims to uncover epigenomic plasticity modulated by HBV. We also intend to contribute to the open problem, whether therapies lead to the restoration of regular epigenomic signatures, or whether hepatocytes persist in a deregulated epigenetic \u2018memory\u2019 state of HBV infection thus still carrying the risk for malignant transformation.Therefore we analyze expression profiles by qRT PCR arrays. DNA methylation as well as chromatin signatures of genes up- or downregulated upon HBV infection are studied by MeDIP or ChIP respectively, in murine cell lines infected with HBV.We identified several transregulated genes and monitored DNA methylation and chromatin signatures at selected loci within promoters. Moreover we observed that therapeutic strategies lead to changes in the epigenomic signature of hepatocytes.Our data show that our experimental therapeutic interventions efficiently suppress HBV proliferation, whereas an increased risk of malignant transformation cannot be excluded."} +{"text": "Epidemiological studies have demonstrated the developmental neurotoxicity associated with prenatal methylmercury exposure may play a role in conjugating methylmercury with GSH main effect on mercury retention in the umbilical cord in a UK birth cohort and GCLM also seem to affect the retention of methylmercury from fish and seafood and metallothionein metabolism exposure in urine samples of children and adolescents. Similarly, an increased frequency of symptoms occurred in those who had a mutation (rs4680) in a gene that codes for catechol O-methyltransferase (COMT) may also result in a predisposition to adverse effects from elemental mercury exposure in children rs10636 showed a significant main effect on general cognitive functioning variants modified the adverse effects of cord blood mercury on neurodevelopment .A recent prospective study of 2-year old children suggested that apolipoprotein E (BDNF), Paraoxonase 1 (PON1), Transferrin (TF) and Progesterone Receptor (PGR) genes appeared to modify the methylmercury-outcome associations with cognitive deficits in children with the minor alleles (mutations).None of these studies focused on the potential environmental exposure of methylmercury during pregnancy and each study tested only a few SNPs, with the exception of our recent study . Given that the apparent increase in IQ among those who had no more than one mutation in the four genes is likely due to the benefits from maternal seafood diets, then the much lower IQs in children with minor alleles are worrisome. In some analyses, the difference between the groups suggests that children with at least four mutations lose as much as 25 IQ point more than wild type children at a 10-fold increase in prenatal methylmercury exposure. This finding suggests that current exposure limits may be too lax for a sizable fraction of the general population. One caveat must be mentioned. As some of the candidate genes may show pronounced differences in allele frequencies between ethnic groups, adjustment for potential genetic confounding must be considered. Because of the genetic background of the English and immigration during recent decades, the ALSPAC cohort can be considered an admixed population. Heterogeneity of genetic background can potentially lead to spurious associations if ancestry is related to both a candidate gene and the disease outcome of interest, i.e., genetic confounding due to population stratification , even when adjustments for beneficial dietary factors from maternal seafood intake and social class were included in the models. One might therefore assume that the developmental neurotoxicity was negligible. However, adverse associations among genetically susceptible groups were discovered in analyses that were stratified by the SNP allelic variants. While the wild type was associated with benefits from increased methylmercury exposure one or two mutations in the genes led to lower IQs at age 8 years. Furthermore, the importance of such genetic predisposition is illustrated by the fact that 21 percent of the cohort subjects had at least four minor alleles in the four SNPs identified; this subgroup showed methylmercury-associated cognitive deficits with low GSTP1 Val105 polymorphism modified effects of prenatal p,p\u2032-Dichlorodiphenyltrichloroethane exposure on cognitive functioning in preschoolers, thus suggesting oxidative stress as a potential neurotoxicity mechanism , Morales et al., observed that In conclusion, environmental neuroepidemiology studies need to include a new focus on genetically susceptible groups in order to assess a more realistic potential risk of neurotoxicant exposures at low levels. Meanwhile, the regulatory agencies and risk assessment professionals should consider a precautionary approach taking into account the likely existence of genetic predisposition to neurotoxicity."} +{"text": "Antenatal testosterone exposure influences fetal neurodevelopment and gender-role behavior in postnatal life and may contribute to differences in developmental psychopathology during childhood. We prospectively measured the associations between umbilical cord blood testosterone levels at birth and childhood behavioral development in both males and females from a large population based sample. The study comprised 430 females and 429 males from the Western Australian Pregnancy Cohort (Raine) Study where umbilical cord blood had been collected. Total testosterone concentrations were determined by mass spectrometry and bioavailable testosterone (BioT) levels were calculated. At two, five, eight and ten years of age, the participants completed the Child Behavior Checklist (CBCL). Linear regression models were used to analyse the relationship between BioT concentrations (in quartiles) and CBCL scores . Boys had higher mean CBCL T-scores than girls across all ages of follow-up. There was no significant relationship between cord blood BioT quartiles and CBCL total, internalizing and externalizing T-scores at age two or five to ten combined. In the syndrome score analyses, higher BioT quartiles were associated with significantly lower scores for attention problems for boys at age five, eight and ten, and greater withdrawal symptoms in pre-school girls (age five). We did not identify a consistent relationship between antenatal testosterone exposure and total, internalizing or externalizing behavioral difficulties in childhood. Higher umbilical cord BioT levels were associated with lower scores for attention problems in boys up to 10 years and more withdrawn behavior in 5-year-old girls; however, these findings were not consistent across ages and require further investigation in a larger sample. Both animal and human studies have reported that fetal androgen exposure during pregnancy can influence the postnatal development of sexually differentiated behaviors, with greater testosterone exposure thought to predispose to more masculine behaviors, such as aggression. In contrast, lower levels of intrauterine testosterone exposure are associated with more feminine behaviors, such as increased nurturing behavior in utero can lead to a shift in the behavior of females towards more male-typical behavior The involvement of sex hormones in developmental psychopathology is supported by a number of small studies that have shown a greater exposure to testosterone Antenatal androgens may also influence behavior in childhood. Using digit ratio as a marker of antenatal androgen exposure, higher exposure was associated with increased externalising behavior in girls and boys at mid-childhood in utero would have higher externalizing behavior scores (representing poorer behavior).The relationship between prenatal androgen exposure and childhood behavior has not previously been measured in large population based studies using biological measures of antenatal testosterone levels. The aim of this study was to use prospectively collected data from a non-selected pregnancy cohort in conjunction with umbilical cord blood testosterone measurements to measure the relationship between antenatal testosterone exposure and child behavior from age 2\u201310 years. Specifically, we hypothesised that females and males exposed to elevated testosterone levels The Western Australian Pregnancy Cohort (Raine) Study commenced in May 1989 and recruited 2900 pregnancies at an average gestational age of 18 weeks through the state maternity hospital until November 1991. The study was initially established to investigate the effects and safety of repeated ultrasound imaging in pregnancy. A full summary of the study and enrolment details has been published elsewhere We were able to recruit 90% of eligible women approached to participate in this study and the initial cohort was representative of a tertiary hospital population a priori as outcomes for additional analyses: aggression and attention problem raw scores and withdrawn and social problem raw scores The 118 item CBCL for Ages 4\u201318 (CBCL/4\u201318) was administered at the five, eight, and ten year follow-ups and completed by the primary caregiver Umbilical cord blood collected at birth was available from 861 randomly selected singleton deliveries. Cord blood samples consisted of mixed arterial\u2013venous fetal blood. Lack of contamination was confirmed by Mendelian concordance analysis of DNA from matched maternal and cord blood in 10 randomly selected sample sets from the cohort using the Affymetrix genome-wide human single nucleotide polymorphisms array 6.0. Liquid chromatography\u2013tandem mass spectrometry after solvent extraction was used to determine total testosterone concentrations, described elsewhere in detail by Keelan et al. We adjusted for a range of antenatal factors that have previously been associated with later child behavioral outcomes a priori at each follow-up. The syndrome scores included as outcomes in these models were: aggression and attention problems for males and social and withdrawn problems for females. Separate models were constructed for males and females and all models were adjusted for the covariates already noted: alcohol and smoking in pregnancy, parity, presence of labour, mother\u2019s educational level, low family income, mother\u2019s age and gestational age at delivery.BioT and total testosterone levels were highly correlated (Spearman correlation coefficient 0.93 for girls and 0.93 for boys). We analysed mean T-scores separately for males and females at age two and five-ten years, adjusted for gestational age at delivery. Linear regression was used to investigate the relationship between BioT and total, internalizing and externalizing CBCL T-scores at ages two, five, eight and ten. CBCL scores at age 2 were analysed separately from the older age groups as the questionnaires differed slightly between the CBCL/2\u20133 and the CBCL/4\u201318. The combined CBCL outcomes at ages 5, 8 and 10 were analysed using longitudinal mixed regression models with a random intercept at the subject level to account for loss of independence due to repeated measures on the same individuals. In these longitudinal models, we checked that effects were consistent across ages by ensuring that BioT quartiles and age interaction effects were not statistically significant (p<0.05). To examine the relationship between BioT quartiles and specific syndromes, we used negative binomial regression (due to the skewed distribution of data) to analyse raw scores of four behavioral syndromes selected p<0.001 in boys compared with girls in our cohort across total, internalizing and externalizing behavior at age two and five through ten years . In the We also did not find any significant relationship between cord BioT levels and aggression problems in boys or sociain utero significantly impacts upon child internalizing and externalizing behavior This is the first published study to report the relationship between cord blood BioT concentrations and male and female childhood behavior in a large population-based sample. Contrary to previous smaller studies suggesting that greater exposure to testosterone Overall, our findings indicate that fetal testosterone concentrations in late gestation do not predict an increased risk for behaviour problems in childhood in boys or girls. Previous studies have reported significantly higher rates of childhood problem externalizing and internalizing behaviors associated with prenatal testosterone exposure. However, these studies have been limited by population selection in utero, had higher withdrawal behavior problem scores at age five may reflect a similar phenomenon underpinning the difficulties in communication and social behavior for girls with CAH that have been described in previous work Our finding regarding improved attention with higher fetal testosterone levels at birth may contribute to the understanding of the mechanisms responsible for the gender differences in attention problems, where boys are less likely than girls to have the inattentive subtype of ADHD The strength of this study is the size of the cohort and the prospective ten year longitudinal follow-up of behavioral development using a valid and detailed assessment instrument A potential limitation of our study is the use of testosterone measures obtained during late gestation. The sensitive period(s) when testosterone may affect fetal brain development are not known, but sex specific changes have been documented from early gestation. Measurements during late gestation may not necessarily reflect earlier exposure. Further, testosterone effects on the brain may not relate solely to circulating levels, but may also reflect receptor sensitivity which cannot be easily measured in human studies. We acknowledge that although we did include a range of maternal sociodemographic and lifestyle factors from pregnancy in our models as potential confounders, there are potentially other factors later in childhood that remain unmeasured and unaccounted for in this study. However, despite these limitations and our inability to determine causation from this study alone, our study of the relationship between late gestation umbilical cord testosterone levels and childhood behavior adds significantly to the existing literature in this area.In summary, our findings did not confirm the hypothesis that antenatal testosterone exposure is a significant determinant of later internalizing or externalizing child behavioral problem scores. However, we did observe significantly lower attention problem scores from age five to ten in boys with higher cord blood BioT levels and higher withdrawn problem scores at age five in girls. Further research is warranted to more thoroughly understand this relationship, but these findings suggest that the links between intrauterine testosterone exposure and behavioral development may be restricted to particular behaviors."} +{"text": "Phylogenetic diversity\u2014patterns of phylogenetic relatedness among organisms in ecological communities\u2014provides important insights into the mechanisms underlying community assembly. Studies that measure phylogenetic diversity in microbial communities have primarily been limited to a single marker gene approach, using the small subunit of the rRNA gene (SSU-rRNA) to quantify phylogenetic relationships among microbial taxa. In this study, we present an approach for inferring phylogenetic relationships among microorganisms based on the random metagenomic sequencing of DNA fragments. To overcome challenges caused by the fragmentary nature of metagenomic data, we leveraged fully sequenced bacterial genomes as a scaffold to enable inference of phylogenetic relationships among metagenomic sequences from multiple phylogenetic marker gene families. The resulting metagenomic phylogeny can be used to quantify the phylogenetic diversity of microbial communities based on metagenomic data sets. We applied this method to understand patterns of microbial phylogenetic diversity and community assembly along an oceanic depth gradient, and compared our findings to previous studies of this gradient using SSU-rRNA gene and metagenomic analyses. Bacterial phylogenetic diversity was highest at intermediate depths beneath the ocean surface, whereas taxonomic diversity showed no relationship with depth. Phylogenetic diversity estimates based on the SSU-rRNA gene and the multi-gene metagenomic phylogeny were broadly concordant, suggesting that our approach will be applicable to other metagenomic data sets for which corresponding SSU-rRNA gene sequences are unavailable. Our approach opens up the possibility of using metagenomic data to study microbial diversity in a phylogenetic context. In recent years there have been significant advances in the development of phylogenetic diversity statistics to quantify the relative importance of processes such as dispersal, competition and environmental filtering in shaping community structure As sequencing costs have declined and novel technologies developed, a new field has emerged in the study of microbial communities wherein DNA isolated from environmental samples is randomly sequenced using the same shotgun approaches used to sequence the human and other genomes The decreasing cost of sequencing technologies will lead to a massive increase in the sequencing depth and overall availability of metagenomic data To study the phylogenetic diversity of microbial communities, one needs to first generate hypotheses regarding the phylogenetic relationships among the organisms in those communities. While in theory metagenomics has enormous potential for such studies, in practice making use of metagenomic data to quantify phylogenetic diversity has been challenging. A key challenge is which gene or genes to study. While previous studies have constructed phylogenetic trees for single genes based on metagenomic data To overcome these challenges, we took advantage of the rapidly increasing availability of fully sequenced bacterial genomes For our analysis, we began with a set of 31 gene families (which we refer to as \u2018marker\u2019 genes) chosen based on their universality, low copy number, phylogenetic signal, and low rates of horizontal gene transfer MPD) separating all pairs of sequences in the sample SES) of phylogenetic diversity We next evaluated the ability of our phylogenetic marker gene approach to detect patterns of diversity along the HOT ALOHA ocean depth gradient, and compared our approach to existing approaches to analyzing microbial data, including SSU-rRNA-gene-based measures of phylogenetic diversity and taxonomic composition. By analysis of phylogenetic relatedness among metagenomic sequences from the 31 marker gene families , we measMPDSES) expresses how different the observed phylogenetic diversity value is (in units of standard deviations (sd)) from the average (mean) phylogenetic diversity in the randomly generated communities. Positive values of MPDSES indicate phylogenetic evenness , while negative values indicate phylogenetic clustering (co-occurring sequences more closely related than expected by chance).The resulting standardized phylogenetic diversity measure (MPDSES<0), meaning that the sequences were more closely related than expected by chance. In contrast, samples from intermediate depths were phylogenetically even (MPDSES>0), meaning that the sequences were more distantly related than expected by chance. The deepest sample from 4000 m did not show a clear difference from the null model; it was either phylogenetically clustered or even relative to this null model depending on the method used to measure phylogenetic relatedness . Phylogenetic diversity calculated for SSU-rRNA gene sequences showed a similar unimodal pattern with highest diversity at intermediate depths, although SSU-rRNA gene phylogenetic diversity within samples was phylogenetically clustered relative to the null expectation at all depths.Standardized phylogenetic diversity peaks at intermediate oceanic depth, with the lowest phylogenetic diversity in the shallowest samples . PhylogeThe non-random phylogenetic diversity we observed along the depth gradient provides evidence for the role of different niche-based community assembly processes structuring these microbial communities. The phylogenetic clustering of sequences in the shallowest and deepest samples is consistent with the pattern expected if closely related species are ecologically similar et al.Our approach provides a phylogenetic framework that makes sense of the inconsistent results of previous studies of microbial diversity along oceanic depth gradients. Studies using fingerprinting technologies such as T-RFLP to measure microbial diversity along depth gradients have found inconsistent results ranging from increasing to decreasing diversity with depth Phylogenetic diversity measures have commonly been applied in the analysis of SSU-rRNA data, but not for metagenomic data sets. A phylogenetic approach to measuring diversity from metagenomic data enables the detection of environmental diversity patterns that could be missed by commonly used methods that bin metagenomic sequences into OTUs or other taxonomic groupings. Since taxonomic binning methods estimate diversity using the number of distinct taxa in each community, they can provide similar measures of diversity for a sample of related versus the same number of divergent taxa. In other words, when the phylogenetic relatedness of taxa varies across communities, taxonomic binning methods may fail to detect this variation and will be sensitive to the choice of threshold for identifying distinct taxa. An added benefit of the phylogenetic approach is that it avoids the issue of choosing a similarity threshold to define OTUs or other ecologically relevant taxonomic groups, which can be extremely challenging in microbial diversity studies In addition to analyses of phylogenetic diversity, we examined variation in community composition with depth based on phylotyping of sequences using a phylogenetic framework. The AMPHORA bioinformatics pipeline performs phylotyping, which uses phylogenetic placement of the metagenomic reads to identify the taxonomic groups to which they belong. This approach is conceptually similar to existing tools for taxonomic classification of metagenomic sequences . Our taxonomic diversity analyses were based on analysis of the SSU-rRNA gene sequences and our phylogenetic diversity analyses were based on analysis of both the SSU-rRNA gene sequences and metagenomic data.We analyzed a publicly available data set of DNA sequences from microbial communities in seven oceanic water samples along a depth gradient from 10 m to 4000 m depth at the HOT ALOHA site in the tropical Pacific The 419 SSU-rRNA gene sequences were aligned using the STAP rRNA gene alignment and taxonomy pipeline Phylogenetic relationships among SSU-rRNA gene sequences were inferred using FastTree version 2.0.1 P<0.001).Phylogenetic tree inference for metagenomic sequences required a different approach due to the fact that metagenomic sequences were relatively fragmentary and non-overlapping compared to the full-length SSU-rRNA gene sequences. Using aligned reference and metagenomic sequences from the 31 AMPHORA gene families, we combined reference sequences with metagenomic reads into a single large alignment . ReferenWe placed all metagenomic reads on the reference phylogeny using the single-sequence likelihood insertion heuristic implemented in RAxML version 7.2.2 There were some metagenomic sequences that were either highly divergent or poorly placed on the reference phylogeny, resulting in a relatively long branch length subtending the sequence or relatively high uncertainty in placement of the sequence on the reference phylogeny. To assess the effect of these sequences on our results, we repeated analyses with a more conservative set of sequences by dropping the sequences whose subtending branch length connecting them to the reference phylogeny was in the top 5th percentile of subtending branch lengths, as well as sequences with fewer than 50 unmasked amino acids. Using a more conservative set of sequences did not change the trends we observed.Taxonomic classification of the aligned SSU-rRNA gene sequences by the STAP rRNA alignment and taxonomy pipeline MPDSES) separating all pairs of sequences in each sample MPDSES was calculated by simulation, based on a comparison of observed phylogenetic diversity with the phylogenetic diversity in 999 random draws of the observed number of sequences in a sample from the phylogeny including all metagenomic sequences. Standardized phylogenetic diversity was calculated for each sample based on the SSU-rRNA gene phylogeny, the maximum likelihood metagenomic phylogeny, and across the 100 replicate metagenomic phylogenies inferred with a phylogenetic bootstrap.We used the Picante software package 10-transformed depth to determine how diversity varied with depth, and whether the diversity-depth relationship was linear or quadratic . While sample sizes were too small to allow formal model comparisons for taxonomic diversity and phylogenetic diversity based on the maximum likelihood tree, for the bootstrap replicate phylogenies the quadratic model of the phylogenetic diversity - depth relationship was a more parsimonious fit to the observed data than the linear model (MPDSES versus log10(depth): AIC of quadratic model: 2171.9, AIC of linear model: 2256.3).Relationships between diversity and depth were calculated for all diversity measures. We compared linear and quadratic regressions of taxonomic and phylogenetic diversity versus logTable S1Relative abundances of sequences assigned to differenet outgroups on a reference phylogenetic tree by AMPHORAfor metagenomic sequences collected along an oceanic depth gradient at the HOT ALOHA site. Outgroups represent the reference sequence most closely related to each metagenomic sequence based on a phylogenetic placement of each sequence on a phylogeny based on 31 gene families from 571 fully sequenced bacterial genomes.(PDF)Click here for additional data file.Data Set S1FASTA alignment file containing aligned AMPHORAreference sequences and metagenomic sequences from 31 gene families along an oceanic depth gradient at the HOT ALOHA site.(FASTA)Click here for additional data file.Data Set S2Newick-format phylogenetic tree linking metagenomic sequences from 31 gene families along an oceanic depth gradient at the HOT ALOHA site. The tree is the one that was identified as having the maximum likelihood by placing metagenomic reads on a reference phylogeny inferred with a WAG + G model partitioned by gene family in RAxML (NEWICK)Click here for additional data file."} +{"text": "Activation of cancer promoting genes or silencing of tumour suppressors is often due to aberrant expression of transcription factors or transcriptional regulators; interestingly, the expression of many of these transcriptional regulators is usually restricted to male germ cells and Prednisone) in combination with the monoclonal therapeutic antibody Rituximab\u2026The monoclonal antibody Rituximab (trade names Rituxan and MabThera) targets CD20, a surface protein mainly expressed in the B-cell lineage, leading to selective B-cell killing. The Rituximab\u2009+\u2009CHOP chemotherapy regime is a more efficient treatment strategy compared to chemotherapy alone in high-risk B-cell lymphoma bromodomain inhibitor JQ1 correlated with gene signatures of cancer testis antigens of the MAGE family and also matched expression profiles that are characteristic for aggressive subtypes of multiple myeloma. CYCLON knockdown also increased the sensitivity of lymphoma cells to Rituximab http://www.cancer.gov/drugdictionary?cdrid=733799). Since, however, oncogenic MYC is only one factor sensitising cancer cells to BET inhibition and considering its high expression levels in most tumours it is not likely that this oncogene will have predictive value for patient stratification. The study by Emadali et al give rise to the development of NUT midline carcinoma (NMC), an incurable uniformly fatal subtype of squamous carcinoma and in rare cases also cancers of other origins French, providinli et al makes th\u2026makes therefore an important contribution by identifying the nuclear, male germ cell specific factor, CYCLON as a downstream effector of oncogenic MYC signalling in lymphoma.The author declares that he has no conflict of interest."} +{"text": "Recent studies have revealed that proteases encoded by three very diverse RNA virus groups share structural similarity with enzymes of the Ovarian Tumor (OTU) superfamily of deubiquitinases (DUBs). The publication of the latest of these reports in quick succession prevented proper recognition and discussion of the shared features of these viral enzymes. Here we provide a brief structural and functional comparison of these virus-encoded OTU DUBs. Interestingly, although their shared structural features and substrate specificity tentatively place them within the same protease superfamily, they also show interesting differences that trigger speculation as to their origins. The covalent attachment of ubiquitin (Ub) to protein substrates, i.e., ubiquitination, plays a pivotal regulatory role in numerous cellular processes Ubiquitination often involves the formation of polyubiquitin chains The proteasomal degradation pathway is an important cellular route to dispose of viral proteins, as exemplified by the turnover of the TYMV polymerase Polyubiquitin chains can adopt a number of different configurations, depending on the type of covalent linkage present within the polymer It is important to note that many positive-strand RNA viruses, including arteriviruses and tymoviruses, encode polyproteins that are post-translationally cleaved by internal protease domains Based on mutagenesis of putative catalytic residues, arterivirus PLP2 and tymovirus PRO were initially generally classified as papain-like cysteine proteases Structural characterization of nairovirus (CCHFV and DUGV) OTU domains and EAV PLP2 in complex with Ub revealed that while these viral proteases adopt a fold that is consistent with eukaryotic OTU DUBs, they possess additional structural motifs in their S1 binding site that rotate the distal Ub relative to the binding orientation observed in eukaryotic OTU enzymes [22]\u2013[26A remarkable feature of EAV PLP2 is the incorporation within the OTU-fold of a zinc finger that is involved in the interaction with Ub , 2E. TheFinally, an interesting structural difference between TYMV PRO and other OTU proteases of known structure is the absence of a loop that generally covers the active site . BecauseThe presence of structurally similar proteases, each displaying unique features, in these highly diverse virus groups suggests that their ancestors have independently acquired their respective OTU enzymes. Although their origins remain elusive, one possible scenario is the scavenging of an OTU DUB-encoding gene that directly enabled the ancestral virus to interact with the cellular ubiquitin landscape"} +{"text": "Diabetic foot ulcers are one of the most hospitalised diabetes complications and contribute too many leg amputations. Trained diabetic foot teams and specialists managing diabetic foot ulcers have demonstrated reductions in amputations and hospitalisation by up to 90%. Few such teams exist in Australia. Thus, access is limited for all geographical populations and may somewhat explain the high rates of hospitalisation. This pilot study aims to analyse if local clinicians managing diabetic foot complications report improved access to diabetic foot specialists and outcomes with the introduction of a telehealth store-and-forward system.A store-and-forward telehealth system was implemented in six different Queensland locations between August 2009 and February 2010. Sites were offered ad hoc and/or fortnightly telehealth access to a diabetic foot speciality service. A survey was sent 6 months following commencement of the trial to the 14 eligible clinicians involved in the trial to gauge clinical perception of the telehealth system.Eight participants returned the surveys. The majority of responding clinicians reported that the telehealth system was easy to use (100%), improved their access to diabetic foot speciality services (75%), improved upskilling of local diabetes service staff (100%), and improved patient outcomes (100%).This pilot study suggests that clinician\u2019s found the use of a telehealth store-and-forward system very useful in improving access to speciality services, clinical skills and patient outcomes. This study supports the recommendation that telehealth systems should be made available for diabetic foot ulcer management."} +{"text": "Prostate cancer is the most common visceral malignancy in Western men and a major cause of cancer deaths. Increased activation of the AKT and NFkB pathways have been identified as critical steps in prostate cancer initiation and progression. GGAP2 (GTP-binding and GTPase activating protein 2) is a multidomain protein that contains an N-terminal Ras homology domain (GTPase), followed by a PH domain, a C-terminal GAP domain and an ankyrin repeat domain. GGAP2 can directly activate signaling via both the AKT and NFkB pathways and acts as a node of crosstalk between these pathways. Increased GGAP2 expression is present in three quarters of prostate cancers. Mutations of GGAP2 have been reported in cell lines from other malignancies. We therefore analyzed 84 prostate cancer tissues and 43 benign prostate tissues for somatic mutations in GGAP2 by direct sequencing of individual clones derived from the GAP and GTPase domains of normal and tumor tissue. Overall, half of cancers contained mutant GAP domain clones and in 20% of cancers, 30% or more of clones were mutant in the GAP domain. Surprisingly, the mutations were heterogeneous and nonclonal, with multiple different mutations being present in many tumors. Similar findings were observed in the analysis of the GTPase domain. Mutant GGAP2 proteins had significantly higher transcriptional activity using AP-1 responsive reporter constructs when compared to wild-type protein. Furthermore, the presence of these mutations was associated with aggressive clinical behavior. The presence of high frequency nonclonal mutations of a single gene is novel and represents a new mode of genetic alteration that can promote tumor progression. Analysis of mutations in cancer has been used to predict outcome and guide therapeutic target identification but such analysis has focused on clonal mutations. Our studies indicate that in some cases high frequency nonclonal mutations may need to be assessed as well. A variety of genetic and epigenetic alterations have been described in prostate cancer. Numerous studies have found consistent patterns of copy number alterations such as loss of 8p and 13q14 and gain of 8q24 in clinically localized and advanced prostate cancers GGAP2 is a G-protein which has a strong GTPase activity, as expected from its RAS homology domain. It also contains a GAP domain can activate the GTPase activity via either intramolecular or intermolecular interaction. GGAP2 binds to activated AKT and strongly enhances its activity and this interaction is promoted by GTP binding Hu et al have identified mutant forms of GGAP2 in sarcoma, neuroblastoma and glioblastoma cell lines To determine if GGAP2 is mutated in prostate cancer we initially focused on the GAP domain, since this region is an important negative regulator of GGAP2 activity. We analyzed cDNAs from 15 cancers and 9 benign prostate tissues from radical prostatectomy specimens. The GAP domain was amplified and individual clones were isolated and sequenced. Surprisingly we found that the missense mutations were highly heterogeneous. There were no recurrent missense mutations involving more than two tumors. In tissues with multiple mutant clones there were only two cases with two identical mutant clones. There was variability in the distribution of the missense mutations with the regions between amino acids 640\u2013660 and 700\u2013710 having relatively more frequent mutations while mutations were uncommon from amino acids 540\u2013570 but there was no statistically significant \u201chot spots\u201d. In several clones we found 2 mutations in the same clone. This is similar to the observation of Hu et al Given this surprising heterogeneity we considered the possibility that this may represent a PCR misincorporation artifact. However, we found only 3 silent mutations among 540 clones from cancer tissues (versus 90 missense or stop) while in the benign tissues we found 2 silent mutations among 304 clones (versus 3 missense mutations). The proportion of missense and stop versus silent mutation was much higher in the cancer tissue than in the benign tissue and the difference was statistically significant . This is inconsistent with a random misincorporation. To further examine this point, we systematically determined the consequences of transition mutations on amino acid sequence for all nucleotides in the GAP domain. We only examined transitions since 82% of the observed mutations were transition mutations (data not shown). Systematic transition mutation of each nucleotide in the GAP domain would yield 273 missense, 15 stop mutations and 162 silent mutations. The difference in the proportions of missense and stop versus silent mutations we observed (90 and 3) compared the predicted distribution (288 and 167) was highly statistically significant . Finally, we considered the possibility that the cancer tissues had an increase rate of mutation targeting the first and second bases of each codon resulting in random missense mutation in all genes. We therefore examined 5 cancer and 5 benign tissues for mutations in \u03b2-actin. We found no mutations in 32 clones from cancer tissue and 36 clones from benign tissues. The proportion of missense and stop clones in GGAP2 was statistically significantly higher than in \u03b2-actin . Thus the observed heterogeneous mutations in the GAP domain of GGAP2 are indeed genuine.The GTPase domain is also a key regulator of GGAP2 activity. We therefore examined cDNAs from 23 cancers and 12 benign tissues for GTPase domain mutations. The results were very similar to those observed with GAP domain . FifteenWe have shown that NF\u03baB can increase expression of FOS in prostate cancer cells and thus AP-1 activity To determine whether the presence of missense or stop mutations in the GAP domain were associated with aggressive disease we examined in proportion of such mutant clones in prostate cancers with various clinical and pathological parameters associated with aggressive disease . Early PClonal mutations in clinically localized prostate cancer are uncommon and usually involve tumor suppressor genes in such cases. This is a minimum figure since it does not include GTPase domain mutations and potential mutations in other regions of GGAP2, which have been reported Are the mutations we observed significant? The missense mutation frequency observed in the GAP domain in cancer tissues was 370\u00d710Most studies of mutations in cancer have justifiably focused on clonal mutations since it is easier to see the significance of such mutations. Heterogeneous nonclonal mutations will not be detected by many analytical methods or are not further analyzed since it is unclear whether they may be PCR artifacts or simply passenger mutations. Our findings indicate that in some cases high frequency heterogeneous nonclonal mutations can occur and may be clinically important. It remains to be determined how often this is the case with other tumor suppressor genes and oncogenes. In some cases groups have analyzed primary prostate cancers for the presence of mutation using single stranded conformation polymorphism assays followed by sequencing of abnormally migrating bands and found relatively high rates of mutation in some genes. For example, using this approach, mutations in plexin-B1 in were identified in 46% of primary prostate cancers The potential for high frequency nonclonal mutation adds another layer of complexity to the complex mutational landscape of common cancers that has been revealed by large scale sequencing Normal peripheral zone and cancer tissues were collected with written informed consent from men undergoing radical prostatectomy by the Baylor College of Medicine Prostate Cancer Program Tissue Bank and snap frozen as described previously CCGCTCCATTCCTGAACTG; Reverse: GTTGCTGCTTGCGCAAG for the GAP domain: Forward: CACAGACAGCCAAAGCGA; Reverse: CCAAAAGCAGGAGAACGGTAG. DNAs were sequenced in both directions and all base pair changes called by the machine read of the sequence were confirmed by visual examination of sequencing traces. Clones with poor quality sequencing traces were not analyzed. No novel reportable germ line variants were detected.The N-terminal GTPase domain and the C-terminal GAP domain of GGAP2 gene were amplified using PCR and cloned into the PCR 2.1 TOPO vector using TOPO TA cloning kit ( Invitrogen). PCR was performed using Platinum Taq (Invitrogen) to minimize misincorporation. Primers used for cloning were: for GTPase domain Forward: Single nucleotide mutagenesis was carried out according to the manufacturer's protocol (Stratagene). Briefly, primers with the target mutations were used in PCR to generate GGAP2 expression constructs containing 9 different missense mutations. Primers used are shown in Luciferase transcriptional reporter assays were performed as described previously To compare rates of mutation between groups chi square or Fisher exact analysis was performed. Luciferase activity of mutant clones was compared using analysis of variance (ANOVA). For all tests p<.05 was considered significant."} +{"text": "A 54-year-old man with multiple comorbidities including wheelchair use secondary to a left below knee amputation, underwent a 2-vessel coronary artery bypass graft surgery and subsequently developed a noninfected sternal nonunion Fig . SubsequWhat is the current recommendation for coverage of mediastinal wounds?When are local muscle flaps indicated, and when are they not indicated?What other options are there besides local muscle?,Myocutaneous flaps are the preferred time-honored approach for sternotomy wound closure following sternal nonunion and mediastinitis.2Perforators of the deep superior epigastric artery (DSEA) have been the basis for both myocutaneous and fasciocutaneous flaps applied in a variety of clinical settings in the past.,4Sternal wound dehiscence presents a reconstructive challenge especially in clinical situations where muscle loss becomes unfeasible. Deep superior epigastric artery-based fasciocutaneous flaps offer an alternative with several advantages including the preservation of rectus abdominis or pectoralis major muscles for future myocutaneous flaps, prevention of functional impairment associated with loss of muscle, and the benefit of shorter operative and anesthetic times in a patient population with typically high surgical risks.2For this patient, because pectoralis major muscle function is essential for transfer in and out of wheel chairs, the possibility of functional impairment associated with muscle loss was unacceptable and an alternative reconstructive option was pursued. A fasciocutaneous flap based on a single DSEA perforator was designed as a vertical ellipse and rotated 180 degrees clockwise after skeletonizing the perforator to cover the defect Fig . The len"} +{"text": "While our paper confirms the obvious assumption that caliper measurement is more accurate than clinical palpation, it also highlights that caliper measurement is statistically comparable with accurate ultrasonography measurement for clinically palpable neck lumps. We therefore emphasise the merit of this inexpensive adjunct in assessing neck lump size when more expensive tools are not immediately available.Our study highlights the use of calipers in augmenting clinical assessment at neck lump clinics. As previously discussed, we acknowledge that calipers cannot substitute ultrasonography in the assessment of lump morphology, vascular flow and anatomical origin or targeting for fine needle aspiration. We also appreciate that different nodal levels have varying acceptable sizes for normality. Suspicious lymph nodes with a minimal axial diameter greater than 10mm have a sensitivity and specificity of approximately 70% for neoplastic involvement.All data were obtained in an ear, nose and throat neck lump clinic. This is why oral and maxillofacial surgeons were not mentioned in the paper. However, we fully acknowledge that head and neck cancer management is a multidisciplinary effort to which oral and maxillofacial surgeons provide an invaluable contribution."} +{"text": "For example, if the word left enables us to remember which way to turn, preventing its activation might be expected to disrupt navigation. Manipulating the labeling process (and the engagement of language more broadly) is therefore very useful in exploring how language influences cognition. In this paper, we review two methodologies for implementing linguistic manipulations: verbal interference and transcranial direct current stimulation (tDCS), and discuss what we can learn about the role of language in cognitive processes from this line of research.Questions about the relationship between language and thought have long fascinated psychologists, philosophers, and the general public. One specific question is the extent to which verbal labels causally impact cognitive processes\u2014how does calling an object by a particular name influence the way people categorize it; how does knowing words for mental states influence our reasoning about the minds of others; how does learning and using words like \u201c[I]f linguistic processes play an active, online role in perceptual tasks, then a verbal dual task, but not a non-linguistic dual task, should diminish the goluboy/siniy [light blue / dark blue] category advantage found in Russian speakers,\u201d a more systematic comparison of linguistic perturbation methods, specifically comparing behavioral linguistic perturbations to perturbations utilizing stimulation techniques such as tDCS; (2) a more rigorous comparison of different verbal interference tasks; and (3) a theory that elucidates the mechanisms by which verbal interference actually works. Such an examination will be critical to clarifying the contributions of language to cognition, helping us answer such questions as whether the mechanisms by which language affects perception of color categories are in some broad sense similar to mechanisms by which language affects spatial cognition. Performing the cross-method comparisons we advocate will highlight possible contradictions leading to further theoretical refinement. For example, what would it mean for the underlying cognitive and neural processes if one verbal interference method affects a primary task but another does not?The extant empirical literature on effects of language on cognition and perception takes us considerably beyond the question as it is often phrased: \u201cDoes language affect thought,\u201d (Boroditsky,"} +{"text": "The Colitis Once Daily Asacol\u00ae (CODA) study assessed the efficacy and safety of once daily (OD) dosing with Mesalazine versus one 800mg tablet three times daily (TDS) over a 12 month period for patients in remission with ulcerative colitis (UC). Emerging evidence suggests OD Mesalazine is as effective as divided doses [To (i.) describe the use of the MEMS for a detailed assessment of medication adherence and (ii.) present results from the CODA sub study, comparing adherence data collected using the MEMS with the methods used in the main trial.The main CODA trial collected tablet counts at patient visits and asked questions on perceived adherence. The CODA sub study used the MEMS cap data, which electronically recorded the time and date of each cap opening. It was assumed that each cap opening represented a patient taking the correct medication from the bottle .A total of 58 patients had usable adherence data (49 with complete data (12 months or until relapse), 9 with partial data, 3 patients were withdrawn). The frequency of cap openings split by trial arm will be presented. The percentage of days adherent was significantly different between the two trial arms, with OD patients considerably more adherent than TDS patients. The impact of controlling for adherence on relapse rates will be presented. A comparison will be made between the MEMS adherence data and the adherence data obtained in the main trial. More detailed analysis of patient adherence; including (i.) weekday versus weekend adherence; (ii.) adherence around visit dates versus regular adherence and (iii.) patterns of adherence over time will be considered.Collecting adherence data electronically using products such as the MEMS provides an adequate representation of the complexities of patient adherence that may not be possible to obtain through other means (e.g. tablet counts and patient perception)."} +{"text": "We present an unusual case of low pressure headaches in a patient with Marfan\u2019s Syndrome caused by a giant anterior sacral meningocele. The patient, a lady with Marfan\u2019s Syndrome and previous cardiac and ocular complications, presented initially with recurrent postural headaches, worse on standing, occurring over a 5 year period. During these periods she would develop pulsatile tinnitus on standing and also felt the headaches improved when she breathed in. CT brain scans, MRI scans of head and neck and recurrent lumbar punctures done during this period where normal although opening pressures were not recorded. Since 2007 she has had a chronic daily headache with no associated features. Neurological examinations were normal throughout her presentations. A MRI of her lumboscaral spine was performed in 2011 with showed a massive fluid filled structure in the pelvis and a diagnosis of a giant dural ectasia or anterior sacral meningocele was made. Anterior sacral meningoceles are an uncommon congenital abnormality consisting of a spinal fluid filled sac in the pelvis communicating with the subarachnoid space. They are associated with Currarino\u2019s syndrome and Marfan\u2019s syndrome. Patients most often present with abdominal symptoms, symptoms of cauda equina or with headaches (both high and low pressure). Neurological symptoms may respond to surgical treatment of the meningocele."} +{"text": "A 12 year old Caucasian female presented with malaise, pyrexia and painful bullous hemorrhagic skin lesions on both upper and lower extremities. She was the second child of a healthy unrelated Caucasian couple and her past medical history was unremarkable. Laboratory tests showed raised inflammatory markers and chest radiograph showed a left lower lobe collapse. She was started on intravenous broad spectrum antibiotics and steroids, with a provisional diagnosis of vasculitis secondary to infection. Autoimmune tests (including ANCA) were negative and she had no evidence of systemic vasculitis. Given the extent of the cutaneous lesions, tests looking at the immune system and for an infection precipitant were performed. Fluid aspirate from the lesion was sterile and skin biopsy showed non specific inflammation. Mycoplasma serology titers were extremely high 1:5200 and the patient had complete mannose binding lectin deficiency (MBL). She was treated with macrolides and was started on chemoprophylaxis.Mannose binding lectin (MBL) is a serum protein believed to play an important role in the innate immune response. Defective MBL production is regarded as the most common immune deficiency in the general population, affecting approximately 5-7% of individuals. Severe MBL usually presents in the early infancy or in early childhood. Late presentation with severe clinical features such as this is not well documented. Patients with primary antibody deficiencies are susceptible to mycoplasma infections (mainly arthritis) due to defective function on the mannose binding lectin pathway. Conversely, mycoplasma is a common pathogen in the paediatric population whilst erythema multiforme associated with mycoplasma infection is a rare presentation. This case is first report in literature where complete MBL deficiency presented in late childhood with severe mycoplasma infection related severe cutaneous vasculitis and no previous history of recurrent infections."} +{"text": "Various diagnostic methods have been used to evaluate hypertensive patients under physical and pharmacological stress. Several studies have shown that exercise hypertension has an independent, adverse impact on outcome; however, other prognostic studies have shown that exercise hypertension is a favorable prognostic indicator and associated with good outcome. Exercise hypertension may be encountered as a warning signal of hypertension at rest and future hypertensive left ventricular hypertrophy. The results of diagnostic stress tests support that hypertensive response to exercise is frequently associated with high rate-pressure product in hypertensives. In addition to the observations on high rate-pressure product and enhanced ventricular contractility in patients with hypertension, evaluation of myocardial contractility by Doppler tissue imaging has shown hyperdynamic myocardial function under pharmacological stress. These recent quantitative data in hypertensives suggest that hyperdynamic myocardial function and high rate-pressure product response to stress may be related to exaggerated hypertension, which may have more importance than that it has been already given in clinical practice. Measurement of blood pressure at rest has been usually used in epidemiological studies . Some otIt is well documented in large prospective epidemiological trials that high blood pressure at rest results in fatal and nonfatal cardiovascular events . There aAlthough some studies suggested that systolic blood pressure rises with dynamic exercise, while diastolic blood pressure does not change or falls slightly at submaximal exercise , in anotA finding of another study was a high prevalence of masked hypertension in apparently healthy patients presenting with normal office blood pressure but high blood pressure during a clinically indicated exercise stress test . The preThe findings that an individual's risk of developing hypertension in those with high-normal blood pressure was greatly increased if they exhibit an exaggerated blood pressure response to exercise confirms an incremental contribution of exercise blood pressure response above resting blood pressure in predicting future hypertension. Therefore, exercise testing in populations at high risk for hypertension could provide important additional information concerning hypertension risk .A hypertensive response to exercise is associated with subtle systolic dysfunction, even in the absence of resting hypertension. These changes occur before the development of left ventricular hypertrophy or detectable diastolic dysfunction and likely represent early hypertensive heart disease .An exaggerated increase in systolic blood pressure prolongs myocardial relaxation and increases left ventricular chamber stiffness, resulting in an increase in left ventricular filling pressure. Left ventricular filling pressures can be estimated reliably by combining mitral inflow early diastolic velocity (E) and annulus velocity (E'). An increased E/E' ratio reflects elevated filling pressures and may be useful in assessing an abnormal increase in filling pressures for patients with diastolic dysfunction. In a study to evaluate left ventricular diastolic dysfunction leading to exercise intolerance, even in the absence of resting hypertension, (129 subjects with a preserved ejection fraction and a negative stress test) hypertensive response to exercise was evaluated at the end of a 6-min exercise test using the modified Bruce protocol. It is shown that early diastolic mitral annular velocity (E') by tissue Doppler imaging was significantly lower and the ratio of early diastolic mitral inflow velocity (E) to E' (E/E') was significantly higher in patients of the hypertensive response to exercise. Irrespective of the presence of resting hypertension, patients with hypertensive response to exercise had impaired left ventricular longitudinal diastolic function and exercise intolerance . DiastolVarious diagnostic methods have been used to evaluate hypertensive patients and there have been conflicting data relating the effectiveness of the diagnostic methods. Exercise electrocardiography has low specificity especially in patients who have electrocardiographic abnormalities at rest . DobutamFactors related to false-positive results of exercise echocardiography to diagnose coronary disease have been extensively studied and it has been suggested that the factors predictive of false-positive results during exercise echocardiography to diagnose coronary disease were female sex, higher blood pressure at peak exercise, lower risk by Duke score and lower number of abnormal segments and wall motion score index after peak exercise. Women appear to be susceptible to wall motion abnormalities caused by elevated exercise blood pressure .We have recently documented increased regional myocardial contractility of left ventricular base using exercise stress echocardiography tissue Doppler imaging in hypertensives with left ventricular hypertrophy and relation between stress-related regional tissue dynamics and left ventricular outflow tract blood flow Figure 28]. In. In28]. We have observed that peak pharmacologic stress causes high systolic velocities on basal septal tissue in the hypertensive patients with basal septal hypertrophy suggesting stress may produce an enhanced left ventricular contractility Figure 30]. It. It30]. Left ventricular end-systolic pressure-volume relationship provides a robust, relatively load-insensitive evaluation of contractility and can be assessed non-invasively during stress echocardiography. This index of global contractility is reasonably simple, does not affect the imaging time, and only minimally prolongs the off-line analysis time. It allows unmasking quite different, and heterogeneous, contractility reserve patterns underlying a given ejection fraction at rest . RecentlExaggerated hypertension is well tolerated and associated with only minor symptoms in most of the patients for the evaluation of hypertensives with chest pain . In thesSystemic hypertension and an exaggerated blood pressure response with exercise have been associated with 'false-positive' findings on stress electrocardiography and echocardiography . In a coIt has been reported that hypertensive patients who have well controlled blood pressure at rest may give exaggerated blood pressure response to exercise . HyperteIt has been suggested that exaggerated blood pressure response during exercise testing may provide a foresight for the future of sustained hypertension development . It is kIt has been documented that the patients with exercise hypertension presented significantly greater posterior wall thickness and Doppler functional data showed significant increase in the late to early flow velocity ratio. Even in the absence of hypertension, exaggerated blood pressure reactivity to physical exercise suggests greater blood pressure elevation during daily activity as well as enhanced sympathetic nervous tonus, which may be considered a risk factor promoting hypertension and these hemodynamic and neurohumoral behaviors are associated with a tendency to develop target organ abnormalities in the heart .Although the mechanism responsible for the exaggerated blood pressure response to exercise has not been revealed, there are some plausible mechanisms linking with underlying structural abnormalities in the cardiovascular system. Wilson et al. found thLauer et al. have detAl-Nasser et al. have shoSome results indicate that an angiotensin converting enzyme inhibitor provides a sustained 24 h blood pressure reduction associated with blood pressure reduction during physical exercise. These effects have been obtained without altering diurnal blood pressure profile, without excessive hypotension at the time of maximal antihypertensive effect and with a slight improvement of exercise tolerance . It has Franz et al. examinedRecent quantitative data support that exercise hypertension in hypertensive patients may be related to enhanced ventricular contractility and excessive rate-pressure product response to stress and this finding may have more importance than that it has been already given in clinical practice.The authors declare that they have no competing interests.All authors contributed to the paper and meet the criteria for authorship. All authors read and approved the final manuscript."} +{"text": "In Norway spruce, environmental conditions during the reproduction can greatly influence progeny performance. We found that the temperature during post meiotic megagametogenesis (zygotic embryogenesis) and seed maturation shift the growth cycle program of the embryos in the seeds, resulting in significant and long lasting phenotypic changes in the progeny. Traits that are affected include the timing of dehardening and bud burst in the spring; leader shoot growth cessation in the summer, and bud set and cold acclimation in the autumn. All processes are advanced or delayed in correspondence with the temperature during female reproduction. Colder reproductive environment advance bud set and cold acclimation during autumn and dehardening and bud burst during spring in their progenies. Temperature dependent difference in timing of terminal bud formation in identical clones was equivalent to a 4\u20136\u00b0 latitudinal ecotypic difference. The progeny \u201cremember\u201d the temperatures and photoperiod prevailing during zygotic embryogenesis and seed maturation and this memory, affecting the climatic adaptation in this species, is an epigenetic phenomenon.This phenomenon is not only of evolutionary significance but has clear practical implications. This memory can help the conifer to cope with the anticipated rapid change in climatic conditions. It will have importance for the deployment of seedlings produced in seed orchards containing clones that are translocated to warmer sites, and it may be used to produce seedlings that have specific adaptive properties. So, it is possible to produce distinct phenotypes (epitypes) in Norway spruce, however this type of long lasting effects is not well documented in other organisms so far.PaDCL1 and 2 and PaSGS3 as well as transposons related genes are differential expressed in the epigenetically different progenies with phenotypic differences in bud burst and bud set.The molecular mechanism behind this striking epigenetic memory phenomenon is not yet unraveled but transcriptional changes are clearly involved. In epigenetically different progenies, transcriptional analysis revealed that seedlings from full-sib families produced at different embryogenesis temperature under long and short day conditions differed. Suppressive subtracted cDNA libraries revealed significant differences in their transcriptomes. Using qRT-PCR, microRNA pathways genes MicroRNAs (miRNAs) are endogenous small RNAs that can exert epigenetic gene regulatory impacts. We have examined the possible role of miRNA in the epigenetic phenomena, and found that Norway spruce contains a set of conserved miRNAs as well as a large proportion of novel non-conserved miRNAs. From concatemerized small RNA libraries from seedlings from the same parents, originated from seeds developed in a cold and warm environment from a family with distinct epigenetic effects, contrasted to one from a family with little response, miRNAs potentially involved in this epigenetic memory was identified. Most of the miRNAs target unknown genes or genes with no known function. The expression of seven conserved and nine novel miRNAs showed significant differences in transcript levels in progenies with distinct epigenetic difference in bud set, but not in the progenies from a non-responding family, making them excellent candidate miRNAs. The differentially expression of specific miRNAs in genetically identical but epigenetically different progeny indicate their putative participation in the epigenetic regulation.Epigenetic mechanisms influence phenotype through altered regulation of gene expression that is mitotically propagated. Understanding the mechanisms involved in the initiation, maintenance, and heritability of epigenetic states is an exiting aspect of research in current biology. Epigenetic regulation may be realized through several interconnected molecular pathways including DNA methylation, histone modification and chromatin remodeling, small non-coding RNAs and transposable element regulation.Among spruce ESTs we found 64 homologs of genes described as involved in DNA methylation, histone modification and chromatin remodeling and small RNA biogenesis in other plant species. In general, known epigenetic mechanism related genes are very well represented in the spruce genome. We analyzed the transcription patterns of these genes using RT-PCR in epigenetically different zygotic embryogenic samples on different stages of development and in seedlings, originated from full-sib families clearly differed in epigenetic response. The largest difference in gene expression of selected genes was found at the earlier stages of embryogenesis while in seedlings a low number of these genes were differentially expressed. Most of the known epigenetic mechanism related genes in seedlings were steadily expressed in all studied samples independently of their epitype.P. abies during early stages of embryonic development. We selected genes which could be involved into epigenetic response by comparison warm and cold originated \u201cembryonic epitypes\u201d from the same full-sibs family somatic embryos developed in cold (18\u00b0C) and warm (30\u00b0C) environmental conditions. Additionally, for more distinct analysing of the large amount of \u201cno database hit\u201d reads we sequenced one normalised library using 454 Titanium GS FLX sequencing to get reference transcript set of expressed genes. The sequencing data is currently under processing and we are going to discuss main results here.To get a deeper analysis of epigenetic related transcriptome we used high-throughput sequencing (RNA-seq and miRNA-seq) in cooperation with GenXPro GmbH. Using MACE (massive cDNA 3\u2019end sequencing) deep mRNA sequencing on the Illumina GSII platform, we analyzed the genes differentially expressed in To proceed with our initial study of miRNAs in spruce, we used Illumina/SOLEXA sequencing to identify small RNAs expressed at the same epigenetic responsive family developed in warm and cold environment progenies following short-day treatment. The identification of novel miRNA candidates are in progress and the confirmation of conserved and novel miRNA by qRT-PCR analysis will be presented."} +{"text": "Trypanosoma sp. and Leishmania sp. are responsible for neglected diseases like Chagas\u2019 disease, African sleeping sickness or Leishmaniasis. The trypanothione synthetase (TryS) is an attractive new drug target for the development of trypanocidal and antileishmanial drugs [The protozoan parasites of the genus al drugs .In our virtual screening campaign for targeting the trypanothione synthetase (TryS) we used representative protein conformations derived from a computational analysis using molecular dynamics (MD) simulations of this key component of trypanothione biosynthesis. The publicly available crystal structure lacks a variable loop region that is known to be important for trypanothione biosynthesis. MD simulations turned out to be a good tool to model this loop region and obtain a more complete set of protein conformations for subsequent use in virtual screening .For creating a structure-based consensus pharmacophore model, Superstar was deplWe will discuss in detail this combined pharmacophore/ensemble docking approach based on MD simulations and will present the results of the compound selection for testing."} +{"text": "Salmonella persistence or transmission within a remote and isolated wild pig (Sus scrofa) population. We determined the Salmonella infection status of wild pigs. Salmonella isolates were genotyped and a range of data was collected on putative risk factors for Salmonella transmission. We a priori identified several plausible biological hypotheses for Salmonella prevalence versus transmission (molecular case series study design) and fit the data to these models. There were 543 wild pig Salmonella observations, sampled at 93 unique locations. Salmonella prevalence was 41% . The median Salmonella DICE coefficient was 52% (interquartile range [IQR]: 42\u201362%). Using the traditional cross sectional prevalence study design, the only supported model was based on the hypothesis that abundance of available ecological resources determines Salmonella prevalence in wild pigs. In the molecular study design, spatial proximity and herd membership as well as some individual risk factors determined transmission between pigs. Traditional cross sectional surveys and molecular epidemiological approaches are complementary and together can enhance understanding of disease ecology: abundance of ecological resources critical for wildlife influences Salmonella prevalence, whereas Salmonella transmission is driven by local spatial, social, density and individual factors, rather than resources. This enhanced understanding has implications for the control of diseases in wildlife populations. Attempts to manage wildlife disease using simplistic density approaches do not acknowledge the complexity of disease ecology.Infectious wildlife diseases have enormous global impacts, leading to human pandemics, global biodiversity declines and socio-economic hardship. Understanding how infection persists and is transmitted in wildlife is critical for managing diseases, but our understanding is limited. Our study aim was to better understand how infectious disease persists in wildlife populations by integrating genetics, ecology and epidemiology approaches. Specifically, we aimed to determine whether environmental or host factors were stronger drivers of Infectious diseases of wildlife have caused important global pandemics in people Cross sectional surveys \u2013 where a representative sample of a population is taken, prevalence of disease measured and a contrast made between those with and without the infection to infer risk factors \u2013 have frequently been a mainstay of wildlife epidemiological study Salmonella and a contemporary molecular epidemiological study design to assess risk factors for transmission of Salmonella in wild pigs. We use wild pigs (Sus scrofa) as our model as they are a species of global importance, being found on every continent except Antarctica SalmonellaSalmonella species, six subspecies and more than 2500 serovars Salmonella refers to serovars within Salmonella enterica subspecies enterica.Here, we use both the traditional and widely accepted cross sectional survey design to investigate risk factors for prevalence or persistence of Salmonella infection status of 543 wild pigs (Sus scrofa) by culturing mesenteric lymph nodes and faeces Salmonella isolates were genotyped using Pulsed field gel electrophoresis (PFGE) Salmonella transmission including: pig genotype (using microsatellites) We sampled Salmonella infection and risk factors.A traditional cross sectional prevalence based logistic regression analysis which modelled associations between Salmonella isolates which modelled Salmonella genetic relatedness for each pair of infected pigs against risk factors using linear regression. It was assumed that increasing similarity between Salmonella isolates was correlated with transmission.A pair-wise molecular analysis of all a priori identified several plausible biological hypotheses for both Salmonella prevalence in pigs and Salmonella transmission between pigs (molecular case series study design). Two models (paired) were implemented for each hypothesis, one for the cross sectional prevalence study design and one for the molecular case series study design. We used information theoretic approaches to select the most supported models within each study design. We compared the two study design for utility for wildlife disease investigation and inferred mechanisms for Salmonella persistence and transmission between wild pigs.We 2, lying between 18.612 to 18.037\u00b0S and 124.922 to 126.270\u00b0E. The area has a tropical monsoonal climate with mean rainfall over the last century of 484 mm per year (range 163\u2013907) and hot temperatures (mean daily maximum temperature 30\u201339\u00b0C [range 18\u201346\u00b0C]).The sampling strategy was to systematically search water features by helicopter. All pigs observed were targeted for humane destruction according to Australian standard operating procedures The study was undertaken on a 200 km length of the Fitzroy River floodplain in the west Kimberley region of north-western Australia see in OctobSalmonella isolates confirmed by serotyping were genotyped by PFGE S. Braenderup reference strain. Bands were detected automatically and verified manually. Salmonella PFGE DICE similarity coefficient was extracted for each pair-wise comparison of isolates using optimised parameters (optimisation (0.2%), tolerance (1.375%), tolerance change (0%), minimum height (9%), minimum surface (2%), uncertain bands (ignore), relaxed doublet matching (no), fuzzy logic (no), area sensitive (no) and active zones (4.3\u201388.6%)). DNA was extracted from pig ear tissues using the Machery Nagel NucleoSpin Tissue kit. Fourteen pig microsatellite markers Faeces and MLN were cultured according to the Australian standard for microbiology of food and animal feeding stuffs for the detection of Salmonella spp. Salmonella isolates . The outcome variable was the pair-wise genetic similarity (DICE) for each pair of Salmonella isolates. Covariates were the same as for the first data set except that they were the absolute difference between pair-wise covariates values, pair-wise contrasts, for example Euclidean distance between pigs, or pair-wise indicator variables (for example to indicate the same herd membership).Two data sets were assembled. The first data set consisted of a single observation for each pig that was sampled. Presence or absence of infection was determined by culture for each pig in this dataset. Covariate data . For the pair-wise data set, multivariable linear models were developed that related Salmonella DICE linearly to pair-wise covariates. Permutation based methods or transmission were made.Five biologically plausible hypotheses were developed area see . Two sepThis study was approved by the University of Sydney Animal Ethics Committee (N00/6-2010/1/5319).Salmonella observations, sampled at 93 unique locations across the study area. The mean weight of pigs was 51 kg (95% CI: 47\u201354) with a range of 2\u2013150 kg. There were 264 males (49%) and 279 females (51%). Mean male weight was slightly greater than female weight 54 (95% CI: 50\u201358) versus 49 (95% CI: 45\u201352) kg, respectively. Assuming an adult wild pig is \u226530 kg Salmonella infection was 41% (95% CI: 37\u201345%). The median Salmonella DICE coefficient Salmonella genetic similarity) was 52% . The median pig genetic dissimilarity We made 543 wild pig Salmonella infection in wild pigs . Transmission was more likely between pigs that were geographically closer with the similarity of Salmonella declining as the Euclidean distance between pigs increased. Interpretation of the remaining significant covariates from the resources and host immunity models was more complex as these covariates were the absolute differences between the value of the covariate for each pig being compared, and are thus undirected associations. There was an association between Salmonella similarity and divergent ages and densities of source population of the pigs being compared. The coefficient for condition score indicated that transmission was less likely between pigs of differing condition score.Significant coefficients in the cross sectional prevalence data resource model indicateSalmonella prevalence was higher in well conditioned (fatter) pigs and in resource rich areas across the landscape. Prevalence of an infection in a population depends on several factors, especially transmission rates, but also disease induced mortality, duration of infection and the length of time an infection has been present in a population Salmonella in pig populations. With regards to condition score as a risk factor, it is possible that those pigs that were fatter exhibited some specific behaviour that increased exposure to infection. Given their better body condition, these pigs may have been travelling further and foraging more effectively and widely for food. This may have exposed them to more infection, resulting in higher prevalence in these pigs through more effective contacts. This is consistent with prior research in human infections Salmonella infection.It is interesting to posit the reasons that Salmonella in our study area.The molecular study design demonstrated that transmission was more likely within social groups and to other pigs within close proximity. This was expected as wild pigs have been shown to be generally very sedentary 2 values associated with both the risk and resources model, indicating the majority of the variability in the Salmonella genetic relatedness was not due to direct transmission between wild pigs. Instead complex phylodynamic processes Salmonella ecology likely introduced considerable Salmonella genetic diversity that we could not model under the assumption of transmission between pigs. Phylodynamic processes include mechanisms associated with the persistence of Salmonella infections within individual pigs, leading to increased selection pressure and interplay between host immune responses and mutations 2 value of the cross sectional resources model an order of magnitude greater than the resources or risk model in the molecular study. Additionally, an assumption that transmission was associated with resources would have been made, instead of the likely reason that resources affected prevalence through other reasons such as persistence of Salmonella in pig populations in resource rich areas.An important result from the molecular study design were the low RSalmonella persistence in pig populations is associated with resource abundance, and conversely that density has little role in persistence, have implications for control of infection in pigs in Northern Australia and in wildlife more generally. It indicates that control of wildlife infections may not always be achieved through simplistic application of threshold density concepts, as indicated by prior theoretical models Our empirical findings, that Salmonella is an important human pathogen. It is also important in livestock production, both due to its effect on health and productivity and due to its role as a foodborne zoonosis. Salmonella has been isolated from the carcasses of wild pigs harvested for human consumption in Australia Salmonella infection of wild pig populations might represent a reservoir of infection for grazing livestock (sheep and cattle) or pose a direct and a foodborne zoonotic hazard Salmonella for domestic livestock apparently has not been investigated. Whether this is an ecosystem with no overt domestic animal (or human) involvement i.e. wild pigs as a reservoir, or spillover from domestic animals to wildlife populations living in proximity Salmonella for co-grazing cattle by using molecular and spatial epidemiological methods.Salmonella ecological system, strong signals were evident and greater inferences were possible than using either approach alone. Our analyses indicated that the abundance of ecological resources critical for wildlife influences Salmonella prevalence, likely through greater persistence of Salmonella in wild pig populations. Importantly the use of a molecular approach allowed differentiation between persistence and transmission of Salmonella, revealing that transmission is influenced by local spatial, social and individual factors, rather than just resources. Additionally, reliance on only cross sectional data for evaluating Salmonella transmission would have overestimated the proportion of variability of Salmonella data that could be explained, with R2 values orders of magnitude greater than with the molecular approaches. The integration of molecular and cross sectional approaches also allows nuanced inferences for control. Implementation of control zones for wildlife disease management should be structured on complex spatial, social, density and resource distribution principals that aim to reduce prevalence as well as transmission, rather than on simple host density principals outlined in previous theoretical models.We conclude that molecular epidemiological approaches and traditional cross sectional surveys are complementary and can enhance the understanding that can be achieved using either approach alone. Even in a complex hyper-endemic"} +{"text": "Neurons as Time Encoding Machines (TEMs) have been proposed to capture the information present in sensory stimuli and to encode it into spike trains . These nBy exploiting sparsity, the LowRate IAF neuron encodes the information present in the input stimulus into spike trains with average firing rate well below Nyquist rate while using the spiking information in a smart manner to improve stimulus recovery."} +{"text": "Research conducted to date, has documented hand hygiene (HH) compliance rates for medical students ranging between 8% and 52%. While compliance rates have increased in recent years for medical students, they are still well below the ideal levels. The audit data by hand hygiene Australia indicate that currently hand hygiene of medical studnets in Australia is below 70% .Our study aimed to examine current teaching and assessment practices used in Australian medical schools to teach students the concepts of HH.A cross-sectional survey was sent to medical education experts across all Australian medical schools (n= 17). The survey was made up of a mix of open and closed questions and statistical analysis was undertaken on all surveys using SPSS version 21.Sixteen medical schools indicated that concepts of hand hygiene are taught and reinforced throughout the training program. Skills stations was reported as the most common teaching method used reported by fifteen medical schools followed by case scenarios were reported by twelve medical schools. At sixteen medical schools indicated that the HH concepts are assessed at least once during the medical training and assessment is done most commonly during OSCEs and through clinical practical exams and competency checks. All medical schools rated their students hand hygiene compliance as high to very high. Teaching and learning of HH was considered adequate and was supported by good infrastructure. However half the participants did not consider HH as important as other medical concepts and role models were considered as important influence in reinforcing HH practices in a variety of clinical environments.Appropriate knowledge is a starting point for improving practice and for instilling the correct attitude to infection prevention. The frequency and method of teaching, as well as other measures aimed to enhance HH compliance amongst medical students, must be re-examined.None declared"} +{"text": "This analysis also identified transcript expression differences between serous and endometrioid cancers and tumor grade, but no apparent differences were identified as a function of depth of myometrial invasion. Four genes were validated by quantitative PCR on an independent set of cancer and normal endometrium samples. These findings indicate that unique gene expression profiles are associated with histologic type and grade, but not myometrial invasion among early stage endometrial cancers. These data provide a comprehensive perspective on the molecular alterations associated with stage I endometrial cancer, particularly those subtypes that have the worst prognosis.Endometrial cancer is the most common gynecologic malignancy in the United States but it remains poorly understood at the molecular level. This investigation was conducted to specifically assess whether gene expression changes underlie the clinical and pathologic factors traditionally used for determining treatment regimens in women with stage I endometrial cancer. These include the effect of tumor grade, depth of myometrial invasion and histotype. We utilized oligonucleotide microarrays to assess the transcript expression profile in epithelial glandular cells laser microdissected from 79 endometrioid and 12 serous stage I endometrial cancers with a heterogeneous distribution of grade and depth of myometrial invasion, along with 12 normal post-menopausal endometrial samples. Unsupervised multidimensional scaling analyses revealed that serous and endometrioid stage I cancers have similar transcript expression patterns when compared to normal controls where 900 transcripts were identified to be differentially expressed by at least fourfold (univariate PTEN tumor suppressor gene as well as defects in DNA mismatch repair resulting in microsatellite instability that enrolled on one of two different protocols: (1) a tissue and data collection protocol approved by the Institutional Review Board (IRB) at Duke University Medical Center; and (2) a tissue and data collection protocol (GOG-136) managed by the Gynecologic Oncology Group which was approved by the IRB of each institution that provided specimens to the central repository. The current project used a de-identified sample set of specimens and data from these two resources after approvals from the Duke University IRB and the GOG Protocol Committee. Second level review was provided by the Office of Human Protections at the U.S. Medical Research and Material Command. An ethics committee review was not required to review based on the proposed research efforts.Hematoxylin and eosin stained tissue specimens were evaluated by one of two board certified gynecologic pathologists (CZ and who else?) to confirm the original diagnosis. Only homogenous serous endometrial cancers were used for the analysis and mixed epithelial cancers were excluded. Normal endometrial samples obtained from hysterectomy specimens from 12 age-matched post-menopausal women were used for the comparison.For the discovery analysis, samples differed by grade and stage as follows: 9 IAG1, 14 IAG2, 7 IAG3, 14 IBG1, 12 IBG2, 12 IBG3, 7 ICG1, 10 ICG2, and 6 ICG3. There were 79 endometrioid and 12 serous cancers. Sub-stage designations were based on the 1988 version of FIGO classifications of disease that more clearly describe the extent of invasion and thus allow for comparison of non-invading versus deep invading tumors as compared to the revised 2010 criteria.Additionally, array data from six pre-menopausal endometrium samples were compared to the array data from the 91 stage I cancers to assess whether ontology of differentially expressed genes varied substantially with the menopausal status of the control endometrium.n\u2009=\u20099 proliferative phase and n\u2009=\u20099 secretory phase) samples.An independent set of cases used for validation of discovery gene expression data included 40 stage I endometrioid endometrial cancers, 18 stage I serous endometrial cancers, 7 normal post-menopausal endometrial samples, and 18 pre-menopausal endometrial for each cancer case and up to 40 serial tissue thin sections for each normal control were required to obtain sufficient RNA. Laser microdissection enabled collection of greater than 95% purity of cancer or normal epithelial cells.Approximately 50\u2009ng of total RNA was extracted from each sample and processed using the GeneChip two-cycle cDNA synthesis kit . Approximately 10\u2009\u03bcg of amplified cRNA was labeled, hybridized, washed, and scanned according to the manufacturer\u2019s specifications (Affymetrix). The Affymetrix HG-U133 plus 2.0 GeneChip system was used to analyze over 54,000 transcripts covering 28473 UniGene clusters.\u00ae Gene Expression Assays, Applied Biosystems Inc., Foster City, CA, USA). About 18S ribosomal RNA was utilized as the reference. Samples were analyzed according to manufacturer\u2019s suggested protocols in triplicate. Relative expression values for each targeted transcript were calculated for each sample using the comparative CT method. The geometric average of the mean ratios of each histologic group was calculated along with the standard error of the mean.The expression levels of transcripts chosen for validation were determined by multiplexed PCR (TaqManhttp://linus.nci.nih.gov/BRB-ArrayTools.html). Parametric p-values are reported and the false discovery rates (FDR) were calculated using the Benjamini and Hochberg method. The identification of differentially expressed transcripts was carried out using MetaCore . The raw data were deposited in GEO (GSE17025).The transcript expression data were normalized to a target intensity of 500 . Affymetrix.cel files were processed with the program GCOS to generate signal values. Multidimensional scaling (MDS) was performed using one-correlation as distance metric. Unsupervised hierarchical clustering of samples was performed with Cluster 3.0 and the dendrograms visualized with Tree View Clustering was performed with the entire gene list (unsupervised) to determine whether there are groupings of samples and the same software was utilized to generate heat maps of the most differentially expressed genes. Supervised differential gene expression between groups of samples was performed using BRB array tools from the National Cancer Institute indicating that endometrial cancer cells possess highly distinct transcript profiles compared to glandular cells from the normal epithelium . We further evaluated the expression of six of these genes using real-time quantitative PCR to validate the methodology of our array processing. Quantitative PCR results were highly consistent with the microarray analysis . Comparison of serous endometrial cancers versus controls revealed 4300 differentially expressed transcripts between the two groups . A comparison of the differential expressions between the histotypes assessed by matched sample sizes reveals common and distinct transcripts . Although an unsupervised MDS analysis indicated no distinct clustering according to grade (data not shown), a supervised differential expression analysis resulted in identification of 498 statistically significant differentially abundant transcripts .To understand if distinct transcript expression changes associated with tumor grade exist, an unsupervised MDS analysis of grade in the endometrial cancer samples was performed to identify changes in transcript expression associated with poorly differentiated tumors. When the entire sample set was evaluated, including both serous and endometrioid cancers, the grade three endometrioid cancers did not appear to segregate with the serous tumors (grade 3 by definition) Because serous cancers were shown to be different compared to endometrioid tumors we conducted a focused analysis of transcript expression levels as a function of grade in endometrioid endometrial cancer against those that were deeply invasive (n\u2009=\u200923). In 2010, FIGO updated endometrial cancer staging to reflect in part the relative lack of prognostic difference between the previous stage IA and stage IB sub stages using 1988 FIGO staging criteria. Despite these clinical data, we chose to compare only the extremes of invasion . Unsupervised MDS analysis indicated no separation of stage IA cancers versus stage IC cases (data not shown) suggesting that depth of invasion is not reflected in a global transcript expression pattern. Univariate t-tests indicated only 46 transcripts which may be found merely by random chance.To address whether depth of invasion is the result of biologic differences between cancers or simply a reflection of the temporal progression of disease, we analyzed the gene expression of endometrioid cases with no myometrial invasion , DLG7 (DLGAP5), and MELK] whose expression levels were identified as altered by hybridization-based microarray analysis in the discovery set were chosen for validation in an external sample set comprised of 58 endometrial cancer (40 endometrioid and 18 serous) and 25 normal endometrial tissue specimens utilizing by qRT-PCR versus normal post-menopausal endometrium control gene list using the MetaCore (GeneGo) pathway and ontology mining database revealed enrichment of pathways involved in cell cycle, cytoskeletal remodeling, chemokines in cell adhesion, and several signaling pathways including PTEN, Wnt, Flt, and CREB Figure . In ordeIn this study, we examined the transcript expression profiles of stage I endometrial cancers and post-menopausal epithelium to gain insight into the biology and to determine the relevance of expression profile differences to clinical and pathologic variables used to characterize this disease. Although several investigations have previously examined gene expression profiles associated with different subtypes of endometrial cancer, few have been designed specifically to identify transcripts that are differentially expressed between endometrial cancer and normal endometrium, and none have examined the effect of sub-stage on transcript expression. We selected a heterogeneous group of stage I endometrial cancers on the basis of grade and degree of myometrial invasion representing the full range of stage I sub-strata.Two previous study performed comparisons of normal post-menopausal samples with serous and endometrioid cancers and the maternal embryonic leucine zipper kinase MELK transcripts were elevated in endometrial cancer specimens compared to controls. Recently MELK has been described as over-expressed in carcinomas and in particular their stem cell niche in normal and tumor biology remains to be determined. The identification of up-regulated MELK has the potential to be exploited clinically if a tumor growth phenotype is attributed to MELK, as kinases have often been effectively targeted by small molecule therapy in cancer. Similarly the DLG7 gene product (DLG7) is associated with several malignancies has been extensively studied in the uterus and is a key signaling molecule in regulation of the uterine epithelium in preparation for implantation . For example, the The loss of cell polarity and normal cell adhesion processes are central features of cellular transformation and metastasis. We noted a distinct enrichment for dysregulated genes in the cell adhesion (tetraspanins and integrin) pathway by GeneGo analysis of all of the differentially expressed genes. Although members of the integrins and tetraspanins have been examined in endometrial cancer there has not been focused study for many of these key molecules in early disease. Our data show that signaling through deregulated integrin and tetraspanins may account for the observed increased expression of focal adhesion kinase (FAK) and ezrin in this endometrial cancer data set. FAK is a central component regulating invasion and metastasis and limited data suggest it is up-regulated at the protein level in some endometrial carcinomas , was not among the few differentially expressed genes identified.In summary, we determined that stage I endometrial cancers have unique global expression patterns based on their histologic subtype. However, this study importantly describes a large number of gene transcripts that are distinctly dysregulated regardless of histotype when these cancers are compared to normal endometrial epithelium. Most surprising was the distinct lack of changes that existed between superficially invasive stage I cancers when compared to those that are deeply invasive, which suggests that depth of invasion may have a temporal etiology and does not necessarily reflect a unique biological expression profile that develops as an endometrial cancer becomes more advanced. Future studies aimed at identifying effective prognostics for predicting stage I cancer recurrence will likely have to focus away from the traditional clinical and pathologic criteria in order to be effective. Given that many early stage endometrial cancers share significant common gene expression changes, broad-based preventive and chemotherapeutic strategies have the potential to be developed that impact both endometrioid and serous subtypes of endometrial carcinoma.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.http://www.frontiersin.org/Women\u2019s_Cancer/10.3389/fonc.2013.00139/abstractThe Supplementary Material for this article can be found online at Supplementary Figure S1Transcript expression from quantitative PCR (top row) and microarray analysis (bottom row) for six selected genes differentially expressed between endometrial carcinoma endometrioid (E), papillary serous carcinoma (PS), and normal endometrium (N).Click here for additional data file.Click here for additional data file."} +{"text": "The article raises a number of concerns.We read with interest the case report by Dobranici et al. about a 23-year-old female with dilated cardiomyopathy and left ventricular hypertrabeculation / non-compaction (LVHT) who also suffered from chronic toxoplasmosis [2].Since LVHT develops during life in single cases (acquired LVHT) and since there is uncertainty about the pathogenesis of LVHT, it cannot be postulated that LVHT is exclusively an embryonic abnormality . Did a neurologist for NMD investigate the presented patient? Did the patient suffer from a multi-system neurological disease? Was the family history positive for NMD? As mentioned by the authors, LVHT may be associated with neuromuscular disorders (NMDs) , it would be interesting to know if other family members were investigated for ECG abnormalities and LVHT and if LVHT was found in any of them?Since LVHT occurs familial [5]. Possibly, toxoplasmic cysts may mimic LVHT. We have also shown that abscesses due to candida may pretend LVHT on echocardiography [6] and reversible LVHT has been reported in a young male with Coxsackie myocarditis [7].Toxoplasma myocarditis in immunocompetent patients is not unknown as demonstrated by several case reports in the literature [8]. Which antibiotic treatment did she receive and why for such a long time, which is unusual? Which were the results of the follow-up investigations for serum IgG antibodies against toxoplasma?Based on which diagnostic procedures, was toxoplasmosis diagnosed in the presented patient? Were any signs of inflammation or myocardial oedema found on cardiac magnetic resonance imaging? Which was the HIV-status of the patient? Toxoplasma myocarditis particularly occurs in HIV-positive patients [For how long was the patient already receiving antibiotic drugs for toxoplasmosis when dilated cardiomyopathy was diagnosed? Assuming that antibiotic treatment for toxoplasmosis was effective, dilated cardiomyopathy should have already been resolved and dilated cardiomyopathy could be attributed to another, most likely, genetic cause. Why was the patient anticoagulated? Was atrial fibrillation ever recorded? Did heart failure not resolve under treatment? LVHT in the absence of severe systolic function or atrial fibrillation per se is no indication for oral anticoagulation. Why did the patient receive an ICD? Were severe ventricular arrhythmias ever recorded by long-term ECG recordings?The follow up period of the patient is unclear in the presentation. Was LVHT also present after the resolution of systolic dysfunction and heart failure? Did ECG abnormalities resolve during follow-up? Which was the long-term outcome of the patient?Overall, it would be eligible to increase the information about comorbidity in the presented patient and his relatives. Treatment of LVHT should rely on the therapy of comorbidities, such as systolic dysfunction, ventricular arrhythmias, or stroke/embolism. LVHT per se is no indication for cardiac therapy."} +{"text": "EHP that BPA exposure late in gestation alters airway cell development in rhesus macaques.Prenatal bisphenol A (BPA) exposure has been shown to alter the development of reproductive organs in animal models,Previous studies have associated BPA exposure with an experimental model of asthma in mice.BPA exposure is widespread. One report from the National Health and Nutrition Examination Survey found that more than 90% of urine samples collected from U.S. males and females over age 6 years contained detectable levels of the chemical.For the current study, pregnant rhesus macaques received BPA via subcutaneous implant for 50 days during gestational days 50\u2013100 or 100\u2013150 . Control macaques received a corn oil implant or ate corn oil\u2013treated fruit. Treatment groups included at least 6 animals, with histopathologic analyses conducted on smaller subgroups.\u201cOur goal was to model constant serum levels of BPA that have been measured in humans,\u201d says first author Laura Van Winkle, a toxicologist at the university. \u201cLung development patterns and cellular abundance in the airways of these animals match humans much more closely than rodent models.\u201dAt the end of each group\u2019s exposure period the researchers collected fetal airway tissue samples. They used only female fetuses, as the study originated from a project designed to examine the effects of BPA on female reproductive development.They found that BPA exposure in late pregnancy was associated with increased expression of secretory proteins in fetal tissue. The cells that produce these proteins mature late in gestation; the proteins themselves\u2014Clara cell secretory protein (CCSP) and the mucins MUC5AC and MUC5B\u2014are key components of airway mucous secretions.Muc5B gene was approximately six times higher at 150 days\u2019 gestation in fetuses whose mothers were exposed to BPA late in pregnancy compared with those whose mothers received no BPA exposure. Expression of the Muc5AC gene also was increased. Histological staining of the lung tissue indicated there were more mucous cells in the airway epithelium of exposed fetuses and suggested an increase in the amount of mucous production .Expression of the There were no significant changes in protein expression after exposure during mid-pregnancy, suggesting that late pregnancy may be a critical period in which BPA exposure may alter airway cell development. This critical window may also apply to human exposures to BPA in the third trimester of pregnancy due to similarities in the timing of cellular development and airway structure between rhesus macaques and humans.\u201cTaken together with earlier studies in humans, this study in monkeys is important because it is another link in the chain of evidence [potentially] connecting BPA exposure to lung disease,\u201d says Donohue.An increase in mucous cell abundance is one of the hallmarks of asthma, the authors report. However, they say, the clinical relevance of this particular study remains to be seen. The researchers examined only fetal airway tissue samples, and it is unknown whether an increase in mucous cell abundance would have resulted in airway disease after birth.MUC5B in cultured airway epithelium cells from humans,The potential effects of environmental estrogens such as BPA on lung development also are not well known, says Van Winkle. Although estrogen is known to increase the expression of ,Recent experimental studies have suggested that BPA may also disrupt fetal development through epigenetic and nonestrogenic pathways."} +{"text": "Human breast cancer is a heterogeneous disease consisting of at least five molecular subtypes. With our increasing understanding of the genetic underpinnings of human breast cancer subtypes, many genetically engineered mouse models (GEMMs) have been created to mimic these subtypes. Traditionally, these GEMMs have been analyzed individually; however, when consolidated into a single dataset, these models have an increased sensitivity for detecting significant overlap with human subtypes. These associations between GEMMs and human subtypes can provide insight into the genetic alterations associated with the human subtypes and can provide a translational resource for preclinical drug testing. We previously performed an analysis of 13 GEMMs ["} +{"text": "Rationale: The adult brain maintains the ability for reorganization or plasticity throughout life. Non\u2013 invasive techniques such as functional magnetic resonance imaging (f\u2013MRI), Transcranial Magnetic Stimulation (TMS) and magnetoencephalography could be used to show recovery of function after stroke. Objective: Explanation of neuronal plastisity and intra\u2013hemisferic reorganization with functional recovery of a case who has total extensive damage on the left hemisphere. Case Report: Twenty\u2013five years old female patient was admitted to our hospital with a right\u2013sided sequel hemiparesis and homonymous hemianopia. She had right\u2013sided paresia after an inguinal hernia operation when she was one and a half years old. On neurological examination she was speaking fluently, and cooperated on complicated comments. Motor examination revealed right\u2013sided spastic hemiparesis predominant on distal parts and right sided visual field defect. But she was continuing her activity of daily livings without help. Her detailed neuropsycological examination revealed mild cognitive dysfunction. Cranial MRI showed total left hemisphere encephalomalasia. Right hemisphere function was noticed on task\u2013related brain activation during voluntary movement of her right leg (she was not capable of performing right hand function tasks by herself ) on functional MRI. Conclusion: Cerebral lesions in the early life can be compensated with the unaffected hemisphere by the neuronal reorganisation and a patient with complete left hemisphere lesion such as our patient can speak, maintain her life without assistance but with mild cognitive decline, compared with elderly stroke patients. Nervous system has a property to reorganize its function after a lesion or environmental change . The matNeuronal plasticity has been shown by many experimental studies and clinical experiences, and non-invasive techniques such as functional magnetic resonance imaging (f\u2013MRI), transcranial magnetic stimulation (TMS) and magnetoencephalography (MEG) allow understanding of the functional adaptive changes in human beings \u20135. Most We report a patient with mild motor and cognitive deficit despite a total left hemisphere lesion and we explain the function of neuronal reorganization in early stage of life.Nervous system has a property to reorganize its function after a lesion or environmental change . The matNeuronal plasticity has been shown by many experimental studies and clinical experiences, and non\u2013invasive techniques such as functional magnetic resonance imaging (f\u2013MRI), transcranial magnetic stimulation (TMS) and magnetoencephalography (MEG) allow understanding of the functional adaptive changes in human beings \u20135. Most We report a patient with mild motor and cognitive deficit despite a total left hemisphere lesion and we explain the function of neuronal reorganization in early stage of life.When she admitted to our hospital outpatient clinic, she was complaining of her right sided homonymous hemianopia and that it worsened in two days\u2019 time. On the neurological examination she was speaking fluently, and cooperated on complicated comments. Motor examination revealed right\u2013sided hemiparesis with increased tone predominantly on distal parts, right sided hyperreflexia and Babinski sign. At rest and particularly when she walked, her right foot inverted and assumed a dystonic posture. But she continued her activity of daily living without help. Her detailed neuropsychological examination revealed normal visual and spatial functions, mild impairment of executive functions and broadening of simple attention suggesting mild cognitive dysfunction. Cranial MRI showed total left hemisphere encephalomalasia which looked like the previous cranial CT . Blood aExtensive damage to the left hemisphere, which can be caused by stroke or tumor in adults, can result in profound and chronic aphasia. But when such a lesion occurred during childhood, especially before the complete maturation of brain, rarely results in pronounced speech and language disorders . The mecSignificant changes in the topography of the cortical somatosensory and motor maps have been demonstrated using non invasive mapping techniques as multichannel EEG, evoked potential, transcranial magnetic stimulation, functional magnetic resonance imaging and positron emission tomography . PlasticNeuroimaging studies of both adults and children have indicated that right hemisphere language lateralization is more likely to be observed early rather than late. Liegeois and et al was studPerilesional reorganization has also been observed in adults who suffered a left inferior frontal stroke and showed good language outcome . The mecIn the present case, there is an extensive damage to the left hemisphere but the clinical outcome is much better than expected. Functional MRI of the patient shows the adaptive changes of the human brain and permits the patients to live with mildly motor and cognitive dysfunctions. We could not find in the literature a lesion large enough to cause mild deficit although there is a total hemispheric damage."} +{"text": "Targeted integration (TI) allows fast and reproducible genetic modification of well characterized previously tagged host cells thus generating producer cells with predictable qualities. Concurrently, timelines are cut by 50% compared to random integration (RI) based cell line development. In contrast to commonly low productivities of cell lines generated by TI, we developed a system for CHO cells leading to productivities of more than 1 g/L within weeks using the TurboCell\u2122 platform.The system is based on CHO K1 cells that have been tagged with a GFP expression cassette flanked by recombinase recognition sites. Following GFP based FACS enrichment and cloning of the tagged cells, over 4000 clones were screened for growth, productivity, GFP expression stability and integration status of the GFP expression cassette. The best clones were selected to be used as \"Master TurboCell\" (MTC) host cell lines for recombinant cell line development.A selected MTC host cell line is co-transfected using a TurboCell\u2122 expression plasmid containing the gene of interest (GOI) expression cassette flanked by matching recombinase recognition sites together with a plasmid encoding the recombinase enzyme required for RMCE. Upon transfection both plasmids enter the MTC's nucleus initiating transient expression of the recombinase which further mediates the stable exchange of the GFP expression cassette against the GOI expression cassette. Thus, the GOI is stably introduced into the tagged genomic spot shortly after the transfection. Cells are cultivated for a few days to recover from the transfection procedure and to allow GFP to fade out of RMCE positive cells. The transfected pools are thereupon sorted by FACS in order to remove the majority of GFP positive cells. The remaining producer TurboCell\u2122(PTC) pools in general comprise of 90-99% GFP negative, GOI expressing cells that are genetically identical due to the conserved locus of GOI integration. This allows the production of recombinant protein from PTC enriched pools at a very early stage of 3 weeks upon transfection. Due to their genetic homogeneity the physiological diversity of the clones within the pool is limited thus leading to only small variations in the recombinant protein produced. Therefore, material drug candidate screening prepared on the parental PTC pool level should only differ slightly from material produced from clones thereof. Following FACS sorting, the PTC pools can be cloned, if required. Due to the high degree of similarity of all clones, the screening effort to find the best clone can be limited to about 10 clones. Recombinant protein material from clones can be produced 9 weeks upon transfection.In order to prove successful RMCE reproducibly taking place without additional random integration of the remaining plasmid, genomic DNA was prepared from clonal PTC for Southern Blot analysis. The genomic DNA was digested with a restriction enzyme cutting the correctly integrated targeting vector into two pieces, one fragment only comprising internal vector sequences, as well as a second fragment also comprising CHO derived sequences of the specific integration locus. As both fragments carry sequences of the CMV promotor, both can be visualized using one single CMV promoter-specific probe. As only two bands occur in case of successful RMCE, cell lines showing more than two bands indicate clones with randomly integrated targeting vector molecules in addition to RMCE. Statistics of several cell line generation projects show that in about 90% of all analyzed clones a correct RMCE without additional random integration events takes place. This allows for a significant reduction in clone screening efforts to a level of 10 clones per project.6 cells/mL, as well as the integral of viable cells over the cultivation time, and the maximum product titer of more than 1 g/L IgG1 proved the feasibility of the TurboCell\u2122 system for the production of recombinant proteins even in larger amounts at a very early stage of a biopharmaceutical development project.To show the feasibility of the TurboCell\u2122 system for recombinant protein production in fed batch cultivations, a PTC clone producing IgG1 was cultivated in a stirred tank bioreactor. The data of this bioreactor were compared to two shake flask fed batch runs performed with the same PTC and the same media system in parallel. Figure Both amount of glycosylation and glycosylation pattern in different Turbo Cell subsets were analyzedFigure Within 3 weeks upon transfection and targeted integration, producer cell pools were FACS sorted to purities of >95%. These cells were suited for high quality recombinant protein material production in fed batch runs exceeding 1 g/L IgG1. Clones generated thereof behaved similar to the pools in terms of productivity and product quality, cell growth and metabolism. From those clones analyzed a mean of about 90% showed successful RMCE without unintended random integration. Cellular properties and productivities of the clones were as expected and variations between different clones were marginal. Thus, the TurboCell\u2122 system reduces clone screening efforts to a minimum allowing the simultaneous production of multiple recombinant proteins in stable CHO cells with optimal use of resources. This makes the TurboCell\u2122 system an interesting tool for candidate screening and early phases material production even in large scale setups."} +{"text": "Symptomatic severe aortic stenosis carries a two year survival of only 50%. However many patients are unsuitable for conventional aortic valve replacement as they are considered too high risk due to significant co-morbidities. Transcatheter Aortic Valve Implantation (TAVI) offers a viable alternative for this high risk patient group, either by the femoral or apical route. This article reports a case of a pseudoaneurysm of the left ventricle following an apical approach TAVI in an elderly lady with severe aortic stenosis. To our knowledge pseduoaneuryms of the left ventricle have been reported infrequently in the literature and has yet to be established as a recognised complication of TAVI. However many patients are unsuitable for conventional aortic valve replacement as they are considered too high risk due to significant co-morbidities. Transcatheter Aortic Valve Implantation (TAVI) offers a viable alternate for this high risk patient group, either by the femoral or apical route. The procedure was first described in 2002 by Cribier et al and seveet al .This article reports a case of a pseudoaneurysm of the left ventricle (LV) following an apical approach TAVI in an elderly lady with severe aortic stenosis. To our knowledge pseduoaneuryms of the left ventricle have been reported only infrequently in the literature and it has yet to be established as a recognised complication of TAVI ,6.2) who described worsening dyspnoea and chest discomfort on minimal exertion. Her past medical history included coronary artery bypass grafting, hypertension and diabetes mellitus type 2.We report the case of an 86 year old British Caucasian lady with severe aortic stenosis . Immediate transoephageal echocardiography and fluoroscopic imaging demonstrated excellent seating of the valve, subsequently confirmed angiographically.-1. Cardiovascular magnetic resonance (CMR) showed a discrete pseudoaneurysm with late gadolinium myocardial enhancement sequence showing pseudoaneurysm of the LV apex at the incision site.Click here for fileFirst pass gadolinium scan showing flow into the pseudoaneurysm.Click here for file"} +{"text": "Recent advances in genome wide transcriptional analysis have provided greater insights into the etiology and heterogeneity of breast cancer. Molecular signatures have been developed that stratify the conventional estrogen receptor positive or negative categories into subtypes that are associated with differing clinical outcomes. It is thought that the expression patterns of the molecular subtypes primarily reflect cell-of-origin or tumor driver mutations. In this study however, using a genetically engineered mouse mammary tumor model we demonstrate that the PAM50 subtype signature of tumors driven by a common oncogenic event can be significantly influenced by the genetic background on which the tumor arises. These results have important implications for interpretation of \u201csnapshot\u201d expression profiles, as well as suggesting that incorporation of genetic background effects may allow investigation into phenotypes not initially anticipated in individual mouse models of cancer. The past decades have seen significant advances in our understanding of and ability to model breast cancer. The advent of genome-wide expression profiling has led to the development of prognostic gene signatures In addition to cell of origin and somatic mutation events, studies over the past 10 years have demonstrated that genetic polymorphism can significantly affect gene expression. Studies in a variety of species have shown that the expression of a significant fraction of the genome can vary across genetically segregating populations The investigations in our laboratory have been based on a single mouse model of metastatic breast cancer, the MMTV-PyMT transgenic model To explore this possibility, analysis of the tumor characteristics of genetic crosses between MMTV-PyMT and NZB/B1NJ, a common laboratory strain, MOLF/Ei, a wild-derived mouse strain, and the Diversity Outcross, an outbred population based on eight founder strains + N2 females. Diversity Outbred animals used in this experiment were generated by breeding MMTV-PyMT males to DO females to generate PyMT+ F1 females. Animals were housed in groups of 3\u20135 animals in conventional or specific pathogen free conditions on shaved pine bedding. The animals were maintained on a 12 hour light/dark cycle, with food and water provided ad libidum. PyMT+ animals were aged to permit tumor development then euthanized as total tumor burden approached the 10% body weight humane endpoint. Individual animals in all of the crosses developed tumors in 6\u201310 of the mammary glands. Euthanasia was performed by cervical dislocation after Avertin anesthesia. All experiments were performed under protocols approved by the Fox Chase Cancer Center or National Cancer Center Institutional Animal Care and Use Committees. Tumor and metastatic phenotypes were collected as previously described The NZB backcross has been previously described http://cancergenome.nih.gov/).RNA from mammary tumors was isolated using Trizol, following manufacturer\u2019s recommended protocol. The NZB tumors were profiled using Affymetrix MOE430 v2 chips, as described PAM50 gene listing was from the data sets of the R package genefu The subtype prediction of the mouse samples were estimated by the sub-clustering analysis. The hierarchical clustering was performed using the complete linkage algorithm with GSE2034 and one of other 4 data sets. For each hierarchical cluster, the sub-clusters were constructed by the cut heights given the desired number from 2 to 20. The proportions of the GSE2034 subtypes were calculated for each sub-cluster if the number of GSE2034 sample in the sub-cluster is more than 4. The proportion of the GSE2034 subtypes was treated as the probabilities of the mouse and TCGA subtypes. The TCGA genefu prediction was used for algorithm performance analysis.The normalized data for the PAM50 classifier for all 5 data sets was imported into BRB Array Tools Immunohistochemistry staining was performed by the Frederick National Laboratory for Cancer Research Pathology/Histochemistry Laboratory.Previous studies using network analysis demonstrated that conserved network modules exist between human patient samples and MMTV-PyMT derived mouse tumor samples that are prognostic for distant metastasis and survival To address this hypothesis, unsupervised hierarchical clustering of the gene expression data from an NZB backcross was performed. This backcross expression data set consists of 68 mammary tumors from a genetic mapping backcross performed between NZB/B1NJ and MMTV-PyMT mice Further investigation was performed by expanding both the mouse and human data sets. In addition to the small NZB backcross two additional mouse expression data sets were generated. 134 tumors from the previously described MOLF mouse backcross \u20135\u22121.79\u00d710\u20136, FDR\u200a=\u200a0.04\u20130.0073). The different colors of the peaks indicated that multiple DO progenitor strains contributed to the overall HER2/Luminal B susceptibility in this cross.The clustering results are consistent with the presence of inherited loci that contribute to subtype gene expression signatures. If true, this suggests that it should be possible to map inherited loci that predispose tumors to assignment to specific intrinsic subtypes. Genotyping of the MOLF and DO mouse populations was therefore performed to attempt to map subtype susceptibility genes. Due to the relatively small number of samples in the NZB cross this data set was not included in this analysis. Genotyping was performed using spleen DNA on either the Illumina Mouse Medium Density Linkage Panel or high density MUGA SNP chip . The genotype data was then screened for SNPs that showed significant associations with subtype assignments based on the unsupervised hierarchical clustering analysis. A single locus on mouse chromosome 1 (53.8\u201361.9 mb; To determine whether the genetic background of the tumors influenced the expression of standard clinical immunohistochemical markers as well as PAM50-defined intrinsic subtypes staining of tumors was performed. Three representative tumors clustering with human basal or luminal A subtypes from the MOLF cross were stained for estrogen or progesterone receptor, Ki67, Her2, the basal cytokeratin marker keratin 5 and the luminal cytokeratin 8. Basal cytokeratin 5 staining was restricted to the basal cells in the normal ducts, with occasional positive cells observed within tumor masses in all tThe PAM50 classifier has been investigated as a tool to improve standard histopathological methods for subdividing breast cancer patients into classes with differing clinical outcomes. Outcome for most epithelial cancers are related to the development of metastatic disease and one might anticipate metastatic tumors would cluster in those subtypes with worst overall outcome. Outcome in human disease however is linked to many different factors, including tumor subtype, age of diagnosis, treatment selection, response to treatment etc. which could obscure potential association between subtype classification and metastatic susceptibility.To investigate this possibility the association between PAM50 subtypes and distant metastasis free survival was investigated for the GSE2034 human breast cancer data set as well as the mouse samples. The GSE2034 data set consists of lymph node negative breast cancer patients whose tumors were resected but not treated with adjuvant therapy and thus represent the natural history of the disease without systemic therapy. The mouse populations were not subject to any therapeutic intervention and therefore represent the natural course of the disease. As can be observed in Diagnostic and prognostic gene signatures have great potential to improve the ability to stratify patients into appropriate treatment regimens. Improved stratification will enable clinicians to select the most efficacious treatment as first-line therapy which likely will significantly improve patient response and long term outcome. Just as importantly, clinicians would be able to avoid those therapeutic options that would be less effective, sparing patients unnecessary morbidities and toxicities. With these significant advantages it is understandable that a great deal of effort has been spent in developing and validating molecular signatures for clinical use exs .One issue regarding these signatures however is their significance in regard to molecular and cellular origins of the transcriptional profiles. In general, these gene expression profiles are generated by supervised analysis of one set of patient samples, followed by validation in an independent set of samples. The signatures are therefore based on correlations with clinical data or outcomes rather than on molecular events. Any mechanistic basis ascribed to the generation of the signatures is therefore usually by correlation of the expression patterns to known expression patterns that provide plausible explanations.The lack of experimental validation of the molecular basis of these signatures does not in any way detract from their potential clinical value. However, comprehensive understanding of what induces these transcriptional profiles might reveal important biological insights into disease biology. In this study we have explored some of the factors that may contribute to one of these clinically relevant signatures, the PAM50 breast cancer subtype profiling tool Thus, if these results can be extrapolated into human patients, subtype assignments based on gene expression alone may reflect a combination of cell of origin, somatic genetic alteration spectrum, and an inherited predisposition to develop tumors with a particular subtype signature. The data from this study suggests that inherited polymorphism may be an additional contributing factor to the establishment of the PAM50 signatures. Moreover, the data indicate that pre-disposition for particular classes of breast cancer likely exist in human populations. Intriguingly, recent studies in human epidemiology support this interpretation, with the demonstration of the existence of loci pre-disposing women to triple negative breast cancer, a subset of basal tumors Finally, this study also has important implications for the use of animal models for cancer research. Current strategies are to generate individual models based on single or combinations of mutations with the aim to model a particular breast cancer subtype. Each of these models is generally explored on a single genetic background. The results from this study indicate that varying the genetic background gives the ability to investigate additional biological questions for a particular genetically engineered model. This is most clearly evidenced by our ability to identify susceptibility loci associated with ER\u2013 breast cancers using a model thought to most closely represent an ER+ luminal subtype. By incorporating genetic background as a variable in research strategies individual mouse models of cancer may therefore have additional utility to explore the complex biology than originally appreciated."} +{"text": "Model selection using AIC suggested strongest support for linear mixed effects models in which the variables (i) fishing effort, (ii) geographic location and (iii) demersal fish assemblage had approximately equal importance in explaining elasmobranch biomass. In the eastern Celtic Sea, sampling sites that occurred in the lowest 10% of the observed fishing effort range recorded 10 species of elasmobranch including the critically endangered Dipturus spp. The most intensely fished 10% of sites had only three elasmobranch species, with two IUCN listed as Least Concern. Our results suggest that stable spatial heterogeneity in fishing effort creates de facto refugia for elasmobranchs in the Celtic Sea. However, changes in the present fisheries management regime could impair the refuge effect by changing fisher's behaviour and displacing effort into these areas.The life history characteristics of some elasmobranchs make them particularly vulnerable to fishing mortality; about a third of all species are listed by the IUCN as Threatened or Near Threatened. Marine Protected Areas (MPAs) have been suggested as a tool for conservation of elasmobranchs, but they are likely to be effective only if such populations respond to fishing impacts at spatial-scales corresponding to MPA size. Using the example of the Celtic Sea, we modelled elasmobranch biomass (kg h An emerging requirement of the Ecosystem Approach to Fisheries Management (EAFM) is to understand the spatial scales at which the ecological impacts of fishing operate Dipturus batis was already very rare in the Irish Sea by 1981 Dipturus intermedia and D. flossada) has been listed by the IUCN as Critically Endangered Fishing-induced curtailment of fish community size-structure e.g., reflectset al. et al. Squalus acanthius. In contrast, Walker & Heessen 2) are associated with positive responses in the abundance and biomass of some fish species, although often this coincides with strong habitat association within the MPA boundary If fish community size-structure and species composition change in space with environment and fishing intensity, then heterogeneity in distribution and abundance of vulnerable elasmobranchs can be expected. Rogers D. intermedia and D. flossada. In the current paper, we combined fisheries-independent survey data and fine-scale fishing effort data from the Celtic Sea with several key environmental descriptors. The objective was to establish whether spatial heterogeneity in fishing effort can lead to a temporally stable mosaic of fished and unfished areas that would generate de facto refugia resulting in local changes in biomass and species composition of elasmobranchs. De facto refugia (sensu The Celtic Sea retains some of the largest remaining populations of many NE Atlantic elasmobranch species ia sensu are here\u22121) on biomass of elasmobranchs caught per hour of survey trawl sampling (kg h\u22121) in the Celtic Sea.In studies of spatial or temporal variation in fish abundance, standardised catch rate is often used. Standardised CPUE accounts for variation in abundance or biomass due to environmental or other factors (see The Irish Groundfish Survey (IGFS) is a standardized bottom-trawl survey that includes the Celtic Sea , and has\u03b2), where the parameters \u03b1 and \u03b2 were obtained by direct analysis (common species) or from FishBase (www.fishbase.org). Catch weight at each length class of each demersal fish species in each trawl sample (haul) was then converted to a density (kg h\u22121) by dividing by the precise trawl duration. Elasmobranch species richness and biomass density (kg h\u22121) was then calculated for each survey haul.Using IGFS survey data (2006\u20132011), catch numbers at length were converted to weight (W) at length using weight at length relationships (W\u200a=\u200a\u03b1La posteriori that were significantly (P<0.05) different www.searchmesh.net). Because of differences in the fish community between the shallower eastern area of the Celtic Sea and the deeper western shelf within a 20 km radius circle from the survey haul midpoint records (2006\u20132011) for the area of the Celtic Sea within the Irish Exclusive Economic Zone (EEZ) . VMS tramidpoint . Interna\u22121) on elasmobranch biomass (kg h\u22121) by survey haul was estimated using models that accounted for environmental variables. Model selection was conducted in an information theory context using AIC. The full starting model included: Fish assemblage region (Assemblage), Substratum, Depth (m), sampling location (Location: latitude + longitude) and interactions. A preliminary comparison indicated that a linear model had lower AIC than a (nonlinear) GAM and hence further analysis focused on linear models. Boxplots of model residuals showed variation in elasmobranch biomass by Prime Station for observation (haul) j at Prime Station i and ia is the random effect of Prime Station. Residual distributions suggested non-heterogeneity so a structure was added that allowed variance to change with location; this resulted in acceptable residuals. A spline correlogram of model residuals against location (latitude and longitude) showed no spatial autocorrelation.Where: Biomassde facto refuge developed because fishermen avoid the area for one or more of the following reasons:This statistical modelling indicated a distinct area in the NE Celtic Sea where minimal fishing effort was combined with greater biomass and species richness of elasmobranchs. We hypothesised that this The catch of target species is relatively low in this area.The relative cost of fishing this area, measured as distance from nearest port, is high.The risk of losing gear is unacceptably high due to rough or unpredictable seabed conditions.Data were not available to support a robust quantitative analysis of this question, so a qualitative approach was taken involving mapping and informal questioning of fishermen who operate around the area.Model coefficients indicated a negative effect of fishing effort on Celtic Sea elasmobranch biomass. There was also an effect on elasmobranch biomass of fish assemblage region, and an interaction between effort and assemblage region with the strongest effort effect across the \u2018East\u2019 region where grde facto elasmobranch refuge.There was a distinct area in the NE Celtic Sea where low fishing effort overlapped closely with greater elasmobranch biomass and species richness . This ar\u22122. We find that these areas have many more elasmobranch species and greater elasmobranch biomass than geographically proximate heavily-fished areas. Our results suggest that heterogeneity in effort may create de facto refugia for Celtic Sea elasmobranchs, provided this mosaic of fishing effort distribution remains stable through time.The spatial distribution of fishing effort is often very uneven de facto elasmobranch refugia may occur when low commercial fishing effort overlaps with favourable habitat.The distribution of elasmobranchs in the NE Atlantic shows broad patterns that are most likely driven by environmental parameters at regional scales (100 s km) Anecdotal information from fishers suggested that shifting sandy seabed in parts of the refuge area makes trawling difficult and hence uneconomic under the current management regime. The environment may thus impart some degree of on-going natural protection from fishing. However, some commercial fishing does occur in the area and LPUE can be quite high. This existing effort means that changes in the present fisheries management regime in the Celtic Sea could displace the distribution of effort into this area de facto refuge functions to sustain elasmobranch biomass and species richness. Protection of nursery areas has been considered important in management of shark populations Raja clavata can show strong site fidelity et al. Rajids, and also found that juveniles of the critically endangered D. Batis were only found in the Celtic Sea Further work is required to understand how this D. batis, are highly sedentary In contrast, recent evidence suggests that protection of adults may be a more effective elasmobranch conservation strategy than focusing on nursery grounds (see review in 2.The legal and management cost of establishing an MPA can be significant"} +{"text": "Despite clinical remission a substantial proportion of Juvenile Idiopathic Arthritis (JIA) patients will flare after a period of inactive disease, suggesting ongoing disease activity which cannot be detected by current assessment methods. As MRI has proven to depict subclinical inflammation in JIA, it may identify patients at risk for flaring.The purpose of this study was to use MRI to evaluate the knee for signs of subclinical inflammation in JIA patients who are in clinical remission.Prospective cohort study of 30 patients with JIA and fulfilling the PRINTO remission criteria. Contrast-enhanced MRI of the previously most involved knee was used to evaluate the degree of synovial hypertrophy .Data of 30 patients were analysed: 26 patients in clinical remission on medication (CRM) and 4 patients who achieved clinical remission off medication (CR) . Thirty percent of CRM patients and 1 of 4 CR patients had > 3 mm synovial thickening, suggestive for active synovitis.Numerous JIA patients who satisfy the PRINTO remission criteria had MRI detected synovitis in this study. Subclinical inflammation may be the reason for the high percentage of JIA patients who flare after tapering medication. MRI may be a sensitive tool to define true remission and may identify JIA patients at risk for flaring of disease."} +{"text": "Two hemodynamic parameters that have been used for this purpose is the wall shear stress (WSS), which is known to regulate endothelial cell function, and the normalized flow displacement from the vessel center, which was recently shown to correlate with increased growth rates of ascending aortic dilation ,2. Analy25 patients previously studied with 4D Flow imaging were included. Previously reported data on interval aortic growth was available for each subject [CMR velocity data from a plane perpendicular to the ascending aorta just distal to the sinotubular junction was collected independently by two blinded reviewers, and then separately segmented by two blinded observers Figure . SubsequInter-observer correlations with regards to plane selection and contour delineation are reported in Table Normalized flow displacement is a reproducible hemodynamic marker that shows good correlation with interval aortic growth. Reproducibility of contour delineation for WSS analysis was good and in line with previous reports . HoweverCovidien/Radiologic Society of North America Research Scholar Grant 2012-2014."} +{"text": "Modern surgery is faced with the emergence of newer \u201crisk factors\u201d and the challenges associated with identifying and managing these risks in the perioperative period. Obstructive sleep apnea and obesity hypoventilation syndrome pose unique challenges in the perioperative setting. Recent studies have identified some of the specific risks arising from caring for such patients in the surgical setting. While all possible postoperative complications are not yet fully established or understood, the prevention and management of these complications pose even greater challenges. Pulmonary hypertension with its changing epidemiology and novel management strategies is another new disease for the surgeon and the anesthesiologist in the noncardiac surgical setting. Traditionally most such patients were not considered surgical candidates for any required elective surgery. Our review discusses these disease entities which are often undiagnosed before elective noncardiac surgery. Medical literature abounds with wide recognition and awareness of cardiac risk factors and their effective management in modifying perioperative morbidity and mortality. Although large cohort studies have shown similar rates of postoperative pulmonary and cardiac complications in patients undergoing noncardiac surgery (NCS), there are no well-known risk indices that can help predict and manage postoperative pulmonary complications from underlying pulmonary disease \u20133. GuideThe prevalence of obesity is rising and according to NCHS data more than one third of adults 78 million) are obese as defined by a body mass index (BMI) > 30 . The obe8 millionWith such high prevalence in the presurgical population a large majority of patients with OSA remain undiagnosed at the time of surgery. The gold standard for diagnosing and treating OSA is polysomnography (PSG); it may however not be practical for use in the preoperative setting in such a large population. Trying to identify patients at risk for OSA remains a challenge and surgical preoperative centers should have policies to help identify these patients resulting in appropriate triage and perioperative management. Screening should start with questions about daytime sleepiness, heavy snoring and sudden awakenings with need to catch breath, and witnessed apnea by a partner. Physical exam may provide additional clues like BMI > 30, short, thick neck, narrow oropharynx, tonsillar hypertrophy, and retrognathia. Among the available screening tools are an 11-point scoring instrument named the Berlin Questionnaire and the STOP and STOP-BANG questionnaire which is much easier to use. Pulse oximetry is more widely available and cheap; however, its sensitivity and specificity vary widely in studies \u201315. The P = 0.004) and hospital length of stay . Wh. Wh49]. 2 rather than positive end expiratory pressure (PEEP).General anesthesia is the most commonly used especially in patients with prolonged surgical time and spinal anesthesia is usually avoided due to its rapid onset and profound sympatholytic effect. No studies have compared the use of general versus regional anesthesia in PH patients. Anesthetic agents with minimal effects on myocardial contractility and SVR like etomidate are preferred while propofol and sodium pentothal having the exact opposite effects should be avoided for induction , 51. DurPatients on PH specific therapy should continue this without interruption. If initiation is required for the first time, short acting pulmonary vasodilators like inhaled nitric oxide or prostacyclin or oral/parenteral sildenafil are recommended , 53. Lif"} +{"text": "Lower than predicted productivity gains at elevated CO2 under nitrogen or water stress may indicate shoot\u2013root signaling lacks plasticity to respond to rising atmospheric CO2 concentrations. The following presents recent research results on shoot\u2013root nitrogen and water signaling, emphasizing the influence that rising atmospheric carbon dioxide levels are having on these source\u2013sink interactions.Terrestrial higher plants are composed of roots and shoots, distinct organs that conduct complementary functions in dissimilar environments. For example, roots are responsible for acquiring water and nutrients such as inorganic nitrogen from the soil, yet shoots consume the majority of these resources. The success of such a relationship depends on excellent root\u2013shoot communications. Increased net photosynthesis and decreased shoot nitrogen and water use at elevated CO Land plants occupy highly dissimilar aboveground and belowground environments and face the basic allocation dilemma of where to invest resources . Too lit2 uptake increases with drought and salinity, induces stomatal closure, and inhibits transpirational water loss . Low rooAbscisic acid-induced stomatal closure is not solely dependent on root ABA production. Shoot vascular tissue ABA production and ABA + and Cl- are transported in xylem sap and concentrated at sites of evaporation in leaves. High leaf apoplastic Na+ and Cl-decrease water potential, prompting osmotic adjustment and, in some halophytes, stomatal closure . Critical assessment of limitations in shoot\u2013root signaling at elevated CO2 and careful genetic manipulations of N and water signaling could enhance crop response to rising atmospheric CO2 and avoid declines in plant N.Shoot\u2013root N and water signaling involve both resource and phytohormone transport from source organs to distant sink organs to achieve a functional equilibrium between roots and shoots. Rising atmospheric COThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Ecological habitats with greater structural complexity contain more species due to increased niche diversity. This is especially apparent on coral reefs where individual coral colonies aggregate to give a reef its morphology, species zonation, and three dimensionality. Structural complexity is classically measured with a reef rugosity index, which is the ratio of a straight line transect to the distance a flexible chain of equal length travels when draped over the reef substrate; yet, other techniques from visual categories to remote sensing have been used to characterize structural complexity at scales from microhabitats to reefscapes. Reef-scale methods either lack quantitative precision or are too time consuming to be routinely practical, while remotely sensed indices are mismatched to the finer scale morphology of coral colonies and reef habitats. In this communication a new digital technique, Digital Reef Rugosity (DRR) is described which utilizes a self-contained water level gauge enabling a diver to quickly and accurately characterize rugosity with non-invasive millimeter scale measurements of coral reef surface height at decimeter intervals along meter scale transects. The precise measurements require very little post-processing and are easily imported into a spreadsheet for statistical analyses and modeling. To assess its applicability we investigated the relationship between DRR and fish community structure at four coral reef sites on Menjangan Island off the northwest corner of Bali, Indonesia and one on mainland Bali to the west of Menjangan Island; our findings show a positive relationship between DRR and fish diversity. Since structural complexity drives key ecological processes on coral reefs, we consider that DRR may become a useful quantitative community-level descriptor to characterize reef complexity. Goreau's 1959 seminal paper on the zonation of West Indian corals highlighted hermatypic corals as the structural architects of the reef Risk's original chain/tape ratio analog method was universally accepted because it was simple, inexpensive, and captured the essence of rugosity in a simple ratio. But chains tangle easily with reef organisms and their substrate and the ratio is an imprecise descriptor of structural complexity across a wide range of scales. Sampling techniques that increase small scale spatial resolution are time consuming In this communication we describe a new technique to digitally parameterize coral reef structural complexity with a diver operated recording digital submersible level gauge designed to monitor groundwater water levels.Preliminary data are presented suggesting a positive relationship between digital reef rugosity (DRR) and fish diversity across a number of different habitats on shallow water Balinese coral reefs, reinforcing the conclusion of others that structural complexity is a fundamental ecological property of reef communities Acropora foliose and branching corals fitting into the r-K-s ternary classification CC\u200a=\u200a2 of Endinger and Risk for Indonesian coral reef conservation Four sites on Menjangan Island off the northwest corner of Bali, Indonesia and one on mainland Bali to the west of Menjangan Island (Kelor Point), were surveyed . The reeOur field studies were conducted in calm seas on the terrace between the reef flat and wall communities. At each site a 20\u201350 meter transect tape was set parallel to the general reef zonation (approximately perpendicular to swell direction) working along bathymetric contours within rather than across habitat zones. Transects varied in length depending on the patch size of reef substrate and depth .http://www.onsetcomp.com/products/data-loggers/u20-001-02). The ceramic pressure transducer of the instrument has a nominal operating depth range of 0\u201330 meters with a resolution of 0.41 cm and an accuracy of +/\u2212 1.5 cm over its depth range. Temperature recording enables a diver to profile water column temperature and/or assess the fine scale distribution of temperature on the reef. The instrument has the capacity to record 42,400 individual data points at intervals as fine as one second, equaling almost 4 hours of data collection for pressure, temperature, time, and battery voltage which can be extended to almost 6 hours by omitting the battery voltage recording. The instrument communicates with a computer via an optically coupled base station and proprietary software . The level gauge can be programmed to start recording at a predetermined time which alleviates the necessity of bringing a computer into the field. Recording stops when the instrument's memory capacity is reached or by software control. Thus the instrument logs data continuously for an entire dive or day in the field until its memory is full. The actual useful data are subsets from the raw file after it has been downloaded and exported to a spreadsheet. While this greatly simplifies operation underwater, the operator must keep careful track of time and develop a protocol (as described in the next paragraph) to embed metadata information such as transect measurement start and stop into the constantly streaming raw data recording.Structural complexity was parameterized with fine scale pressure measurements recorded by a digital level gauge, an instrument normally used to track groundwater or stream levels as the standard deviation describes the variation of a set of measurements. This is different from the traditional rugosity chain/tape ratio but it has been widely used in many studies that do not employ the traditional methodology STD can be calculated from raw sensor data because all the calibration factors are constants which do not affect transect variability. Fast Fourier transform (DRRFFT) was employed to explore the spatial distribution of structural complexity at horizontal scales within transects. The pressure measurements were subject to Single Series Fourier Analysis with an even number of samples based on the assumption of equally spaced sampling points at 10 cm intervals along each transect .The raw level gauge data were downloaded, exported in ASCII format to a spreadsheet for atmospheric pressure correction and conversion into units of depth (meters), and parsed into individual transects. The distant marks were notated in the data file and used to examine the rate of travel (points-per-meter) for consistency. The contour of the reef along each transect was calculated by subtracting the deepest point from all other depths (relative depth). Consecutive depth differences were calculated from these data by subtracting each point from the next point \u22121 and biomass hectare\u22121 using published length-weight relationships at four organization levels: species, genera, families and morphological groups Benthic community percent cover was estimated by pointcounting consecutive still digital photographs of each transect The study sites spanned a wide range of combined stony and soft coral cover . Coral rTen transects varying in length from 23 to 50 meters with 6 of the surveys coinciding with fish counts were completed . The samSTD varied between transects and reefs but was not significantly correlated with coral cover . Levels of DRRSTD may be characteristic of a particular reef zone and/or morphology but the sample size was too small to make any assertions.The richest of the reefs we sampled were virtually intact with bush-like branching colonies closely packed to form a dense, near-continuous canopy. Transects on the deeper forereef were more complex as the coral colonies of many different morphologies grow seaward and upwards, rather than simply upwards as in shallow water . DRRSTD The periodograms resulting from Fast Fourier Transform varied between sites and depth. Highly degraded reef displayed very little variability across scale compared to an adjacent nearly intact rich shallow reef . Richly More than 120 species of fish were observed whose abundance, biomass, and diversity varied widely across sites . These dSTD as has been seen in other studies of rugosity STD and fish species diversity (Shannon index (H\u2032\u200a=\u200a\u2212\u2211 pi Log pi) based on either fish species abundance or estimated biomass and DRRIn this communication we have presented a novel digital method to parameterize the structural complexity (rugosity) of coral reefs using a robust, off-the-shelf recording submersible pressure gauge. Initially, we have chosen to use the standard deviation of these fine scale pressure measurements as an estimate of rugosity but, with development, there may be other statistics that perform better. DRR measurements are relatively easy to carry out by a skilled SCUBA diver. The diver must swim the transect at a relatively constant swimming speed and use good buoyancy control; both are achievable with practice and concentration. Since it is a pressure measurement, waves and swell of any significant size will add information to the data. Increased wave height will add noise to the signal. Deeper transects will experience less \u201cnoise\u201d from waves and/or swells as their signal becomes proportionally smaller with depth. At our sites below 5 meters the \u201cnoise\u201d added to the variation in pressure was insignificant compared to the vertical relief of the coral reef. In some instances the use of a second stationary reference instrument may be helpful to remove wave variability in post processing, but one also has to recognize when it is too rough to work as it is far less complicated to confine data collection to calm sea conditions. Since diving conditions and substrates vary widely, each investigator will have to adapt the methodology to their specific study site conditions.std and fish diversity corroborates earlier work on fish species richness and reef structural heterogeneity analogous to the classic work of MacArthur for terrestrial forests The Menjangan Island fringing reef community provided a range of reef habitats to explore the utility of DRR at scales commensurate with the scale of many coral reef monitoring programs (10 to 50 m transects). The positive correlation between DRRstd because stony corals and soft corals generate the topographical structure. High coral cover often generates a tightly knit canopy which reduces rugosity but may impart a DRR signature that will have diagnostic importance for long term monitoring as has been suggested for monitoring tropical forest systems with remote sensing It is also somewhat perplexing that there was no significant correlation between coral cover and DRRFast Fourier analysis partitioned the variation in structural complexity as a function of horizontal transect scale within the range of 0.2 m to approximately 5 m. The one-second sampling rate approximating 10 cm of horizontal distance enabled a spatial resolution of 20 cm following Nyquist sampling theory where the smallest scale detectable is twice the sampling interval FFT reveal the distribution of rugosity as a function of scale . DRRFFT spectral density plots from two rich shallow reefs (Kelor Point and Northwest Corner) displayed strong variation at similar scales ranging from 0.5\u20132 m (FFT may improve our understanding of the contribution made by individual colonies to community structural complexity and reef morphology. Additionally, since fish and other mobile organisms tend to scale with their habitat FFT may provide further insight into the influence of habitat complexity on substrate preference and niche partitioning.The periodograms of DRR 0.5\u20132 m . These r 0.5\u20132 m and 3A. 0.5\u20132 m illustra 0.5\u20132 m . ColonieFFT analysis could be employed to characterize the relationship of reef canopies with prevailing seas or calculate hydrodynamic forces through an analysis of wave height time series measurements along depth transects using stationary instruments Digital Reef Rugosity can be applied across a wide range of spatial and temporal scales without sacrificing accuracy or precision since the length of a transect is virtually unlimited. Thus the technique provides a precise link between marine ecology and remote sensing which has eluded satellite-based remote sensing efforts because the smallest scale detectable from space is usually greater than the scale used in most ecological studies Globally, coral reef decline is embodied by the loss of coral cover, which is itself a proxy for reduced structural complexity analogous to terrestrial deforestation. Rugosity that is generated by coral colonies integrates ecological integrity, complexity, and vitality because these functions are biologically inseparable from structure on reefs. At some point in the degradation process the structural complexity of the reef ceases to support its fish community. In this time of dwindling management resources and accelerating reef degradation, a simple field technique that could help detect, or even predict, such tipping points would be an invaluable tool to help triage reefs for the most appropriate course of conservation; no-take policies might be applicable for overfished reefs but others might require rebuilding the physical structure to augment rugosity. Digital Reef Rugosity provides a modern method to more fully parameterize the fundamental community property of coral reefs so elegantly conceived by Risk over 40 years ago"} +{"text": "Epilepsy is associated with an abnormal expression of neural oscillations and their synchronization across brain regions. Oscillatory brain activation and synchronization also play an important role in cognition, perception and motor control. Childhood epilepsy is associated with a variety of cognitive and motor deficits, but the relationship between altered functional brain responses in various frequency ranges and functional impairment in these children remains poorly understood. We investigated functional magnetoencephalographic (MEG) responses from motor cortex in multiple functionally relevant frequency bands following median nerve stimulation in twelve children with epilepsy, including four children with motor impairments. We demonstrated that children with motor impairments exhibit an excessive gamma-band response from Rolandic cortex, and that the magnitude of this Rolandic gamma response is negatively associated with motor function. Abnormal responses from motor cortex were also associated with ictal desynchronization of oscillations within Rolandic cortex measured using intracranial EEG (iEEG). These results provide the evidence that ictal disruption of motor networks is associated with an altered functional response from motor cortex, which is in turn associated with motor impairment. Childhood epilepsy is associated with a host of cognitive and behavioural deficits, including problems with motor function Epilepsy is also associated with the abnormal expression of neural oscillations and their synchronization across brain regions Previously, we demonstrated that seizure-induced alterations in oscillatory synchronization involving Rolandic cortex were associated with motor impairment in a group of children with focal cortical dysplasia (FCD) who were undergoing evaluation for surgery for intractable epilepsy We investigated motor ability and functional responses from Rolandic cortex in a group of children with FCD in whom the relationship between ictal Rolandic desynchronization and motor impairment has previously been established \u00b5sec, supra-motor threshold). For each subject, 400 trials were recorded with trial length of 200 ms with a 50 ms pre-trigger interval. MEG was recorded at 2500 Hz with a band-pass of 0\u2013833 Hz, and a 60 Hz notch filter to remove line noise and 3rd order gradient noise reduction was employed using a 151 channel MISL Omega system . Data were epoched and averaged; localization of the generator of the first major neuromagnetic peak following median nerve stimulation using an equivalent current dipole (ECD) method produces an anterior-facing dipole which accurately localizes primary somatosensory cortex, and ECD localization of the second peak produces a posterior-facing dipole which accurately localizes motor cortex or analysis of covariance (ANCOVA) was performed for categorical and continuous variables respectively. Outcomes were considered statistically significant at p<0.05. Statistical analysis was performed using SAS Statistical Software 9.3 .p\u200a=\u200a0.014; p\u200a=\u200a0.036), age (p\u200a=\u200a0.020), duration of epilepsy (p\u200a=\u200a0.034), and whether or not the epileptogenic hemisphere was ipsilateral or contralateral to the dominant hand (p\u200a=\u200a0.005).The magnitude of the functional response from median nerve stimulation was obtained in each analyzed frequency from the signal amplitude (pseudo-Z) at the peak latency of the motor cortex response (second peak associated with dipole with posterior orientation) of each subject. To investigate the relation between functional responses in particular frequency ranges, the amplitudes of the filtered signals were contrasted between children with normal and abnormal motor function, as determined by neuropsychological assessment and neurological examination. Children with abnormal motor function showed greater gamma1 (36\u201344 Hz) activation in motor cortex within the epileptogenic hemisphere and ipsilateral hand . Correlation statistics were then employed to assess relations between individual gamma1 responses from motor cortex and motor ability, as indexed by standard neuropsychological assessment. This revealed that the amplitude of gamma1 motor cortex MEG response in the epileptogenic hemisphere was negatively correlated with performance on the grooved pegboard task for both the contralateral . This indicates that greater ictal desynchronization of Rolandic cortex is associated with a more abnormal functional response from motor cortex elicited during median nerve stimulation band we focused our examination of the relation between MEG and iEEG data in this frequency range. The analysis of ictal iEEG desynchronization has been previously reported mulation .We provide the first evidence that motor deficits in childhood epilepsy are related to an atypical gamma-band response from Rolandic cortex. We also uniquely show that this altered neuromagnetic response is associated with ictal Rolandic desynchronization measured using iEEG. We previously showed that ictal Rolandic desynchronization was associated with motor impairment in children with epilepsy Our previous analysis of motor network dynamics using iEEG indicated that motor impairment was most strongly associated with differences between ictal and interictal periods, but that ictal activity was much more strongly associated with motor impairment than interictal activity. This, taken together with the fact that MEG responses from sensorimotor cortex are large and robust In the present study, we demonstrate that gamma-band MEG responses from motor cortex are abnormal in children with epilepsy who suffer from motor impairment and show that these atypical responses are associated with individual differences in motor ability in this population. These effects were not attributable to age, sex, duration of epilepsy or whether the dominant hand was ipsilateral or contralateral to the epileptogenic hemisphere. This indicates that the reported findings are not attributable to potentially confounding variables which could impact the amplitude of the Rolandic gamma-band response. The findings of the present study provide new insights into how ictal disruption of functional networks impacts brain activation and function.The finding that differences between children with normal and abnormal motor function occurred in the gamma1 frequency range is consistent with numerous previous observations which have reliably implicated gamma activity in diverse cognitive, perceptual and motor processes The results presented here also conform with a growing body of evidence implicating altered brain oscillations in epilepsy and its associated impairments of function. Excessive high-frequency cortical activity is increasingly being accepted as a biomarker of epileptogenicity The present study was able to investigate relations among functional MEG responses, ictal iEEG activity and motor function in pediatric epilepsy by taking advantage of clinically collected data. One limitation of this approach is the lack of a control group, which we compensated for by comparing patients with motor impairments with patients without motor impairment. Clinically employed data collection techniques also record very short segments of data which are epoched at collection. This prevented analysis of activity relative to a pre-stimulation baseline. To facilitate comparison with previous results describing relations between ictal desynchronization and motor impairment We provide the first evidence that MEG gamma-band responses from motor cortex are associated with motor impairments and ictal desynchronization of Rolandic cortex in children with epilepsy. These results suggest that invasion of eloquent cortical regions by epileptic activity may induce long-lasting changes which negatively affect the brain\u2019s ability to recruit a normal functional response. Such disruption of specific functional brain systems accordingly may lead to impairment in the corresponding domain. These multimodal neurophysiological findings comparing invasive ictal (iEEG) network mapping with interictal noninvasive recordings (MEG) indicate that the long-lasting effects of ictal disruption of functional brain systems can be noninvasively measured using MEG. This strategy promises to provide new means to assess the health of functional brain networks during presurgical planning, to index their function following surgery, and to track the impact of seizures on the function of eloquent cortical areas and networks. The findings of the present study also provide new insights into relations among ictal network dynamics, long-term changes in functional brain activity and developmental difficulties prevalent in pediatric epilepsy."} +{"text": "C-terminus of the vimentin protein, respectively), we examined whether either of these domains would be displayed on the surface of three commonly studied prostate cancer cell lines isolated from different sites of metastases. Confocal analysis of LNCaP, PC3 and DU145 prostate cancer cell lines demonstrated that both domains of vimentin are present on the surface of these metastatic cancer cell types. In addition, flow cytometric analysis revealed that vimentin expression was readily detected along with CD44 expression but only a small subpopulation of prostate cancer cells expressed vimentin and the putative stem cell marker CD133 along with CD44. Finally, Cowpea mosaic virus (CPMV) nanoparticles that target vimentin could bind and internalize into tested prostate cancer cell lines. These results demonstrate that at least two domains of vimentin are present on the surface of metastatic prostate cancer cells and suggest that vimentin could provide a useful target for nanoparticle- or antibody- cancer therapeutic agents directed against highly invasive cancer and/or stem cells.Vimentin was originally identified as an intermediate filament protein present only as an intracellular component in many cell types. However, this protein has now been detected on the surface of a number of different cancer cell types in a punctate distribution pattern. Increased vimentin expression has been indicated as an important step in epithelial-mesenchymal transition (EMT) required for the metastasis of prostate cancer. Here, using two vimentin-specific monoclonal antibodies (SC5 and V9 directed against the coil one rod domain and the When prWe propose that surface vimentin could be such a common marker of highly metastatic cancer cells and as well possibly related to prostate cancer stem- or progenitor cells. Proteome analysis indicated vimentin expression correlated with invasion and metastases of androgen-independent prostate cancers . To achiInterestingly, EMT is a normal process that occurs in embryonic development during organogenesis allowing epithelial cells to differentiate into spindle-shaped cells with mesenchymal properties showing or possessing invasive properties. The developing embryonic organ invasion fronts resemble fronts of tumor metastases, and cells undergoing EMT could share characteristics of primitive tissue stem cells and actually represent tumor stem cell populations. A link between EMT and hypoxia may be a trigger of prostate cancer metastases in the tumor microenvironment. Vimentin expression was induced under hypoxic conditions and corresponded to increases in invasive metastatic potential of LNCaP tumor cells . Cell suThe prostate cancer cell lines DU145, LNCaP and PC3 can be passaged and used to form xenograft tumors. Prostate cancer cells have previously been reported to be heterogeneous in their tumorigenic capacity . In accoin vivo properties of CPMV are well understood; after intravenous inoculation CPMV particles are cleared rapidly from circulation with no apparent toxicity or pathological effects . We suggest the potential utility of CPMV nanoparticle to target prostate cancers via vimentin interactions, this, however, requires formal testing. An additional possible benefit for targeting through vimentin recognition is that a unique extracellular deposition for vimentin has now been shown to mark the stroma of prostate cancer lesions but not prostate hyperplasia or normal prostate epithelial tissue [With regard to prostate cancer targeting, we were able to show uptake of CPMV nanoparticles by the prostate cancer cell lines and that HMEC-1 cells (surrogate cells for human vascular endothelium) did not bind the SC5 vimentin specific mAb. This result indicates that intravenous delivery of vimentin targeting antibody constructs or nanoparticles may not be taken up non-specifically in the vasculature and preferential target metastatic tumor cells. Indeed previous studies have shown that CPMV homing to tumors observed ,34. Furtl tissue . This unversus non-metastatic breast cancer cells [We found that surface vimentin is detectable on the metastases-derived prostate cancer cells. This finding is consistent with other studies on expression of vimentin for metastatic er cells . SurfaceFinally, determining the actual topography of what domains of vimentin are expressed on the surface of prostate and other metastatic cancer cells could provide insights for the creation of novel therapeutic antibody agents, such as spectral-targeted antibody derivative constructs . These a5.We describe in this report that at least two different epitope domains of vimentin can be detected on the surface of three different prostate cancer tumor cell lines derived from different metastatic tissue sites. This commonality in expression on the metastatic lines makes surface vimentin an interesting and possibly universal target by therapeutic agents designed to treat metastases. Surface vimentin was found to be co-expressed on a subpopulation of cells along with CD44 and CD133 suggested to be markers of prostate cancer stem cells. Whether surface vimentin also marks stem- or pro-genitor cells, remains to be formally demonstrated. Finally as a proof-in-principle, we were able to demonstrate CPMV nanoparticles can target prostate cancer cells. Our findings suggest the possibility of creating novel nanoparticle or antibody derivative constructs using vimentin as a way to target prostate cancer metastases and/or stem cells therapeutically."} +{"text": "High throughput screening (HTS) is one of the most prominent techniques used in the beginning stages of a drug discovery programme to identify those few hit compounds that can be used as starting points in subsequent studies ,2. HowevThe novel workflow was evaluated under various aspects in a retrospective study using publicly available quantitative HTS (qHTS) datasets . One of Vortex, a platform for analysing chemical information using chemoinformatics methods and data visualisations tools [The workflow was integrated into Dotmatics\u2019 ns tools . This in"} +{"text": "Dear Editor,Poorly controlled diabetes mellitus can lead to blindness due to severe proliferative diabetic retinopathy. The pathophysiology of retinal involvement is important in diabetic retinopathy, with stress-induced cellular ion change and oxidation generally proposed to be the underlying mechanism \u2013 2. BasThe authors report no conflicts of interest in this work."} +{"text": "Numerous studies have revealed a role for the hippocampal dentate gyrus in behavioral discrimination between similar contexts or objects, referred to as pattern separation. Recently many behavioral studies have demonstrated a role for dentate neurogenesis in such pattern separation. While several computational studies have modeled the effect of neuronal turnover on learning in simple -3 and mo"} +{"text": "Patterns of activity in brains are commonly composed of temporal sequences of periods with steady-state firing rates lasting several hundred milliseconds separated by sharp transitions during movement , perceptWe also used the present model to control specific motor sequences in a robotic device. The population activity pattern in this modeled nervous system has similarities to that observed in primate frontal cortex during multi-segmented limb movements."} +{"text": "Proteins introduced into foods through genetic engineering must be evaluated for potential risks of food allergy (FA) or if derived from grain, potential elicitation of celiac disease (CD). The evaluations are based on Codex Alimentarius guidelines (2003). Novel food ingredients may also be evaluated. Amino acid sequence comparisons are recommended to identify proteins that should be screened further by serum IgE tests for food allergy (FA) or T cell proliferation for potential CD eliciting proteinsprior to use in foods.The www.allergenonline.org database was established in 2004 and updated annually. Criteria were defined by a panel of recognized allergy experts who also review updates annually based on published evidence the proteins are allergenic or are from an allergenic source and bind IgE specifically allergic individuals. Users enter an amino acid sequence for comparison against the database. The new celiac database was constructed by reviewing major publications of native, mutaedor deamidated peptides derived from glutens and suggested as elicitors of CD. Both are available at no cost for public use.Version 12 of the allergen database (February 2012) includes 1603 sequences from 603 taxonomic-protein groups associated with IgE mediated allergy. References are provided for each group and sequences can be searched for matches exceeding regulatory criteria. The new experimental celiac database was constructed and released in February 2012 and includes 1016 published peptide sequences from 68 glutens of wheat, barley, rye or oats that are associated with celiac disease. Publications were evaluated regarding reported T cell proliferation from PBMC or clones from MHC Class II DQ 2.5 or DQ 8 restricted CD subjects. Criteria for inclusion of sequences in both databases and appropriate uses are described online. Criteria, simple to follow sequence comparison instructions and interpretations are illustrated.The two databases provide efficient and simple tools to evaluate candidate food proteins that might pose a risk of elicitingFA symptoms or CD in affected individuals. Proteins that do not exceed criteria have a small likelihood of elicitingFA or CD in sensitized consumers. Proteins that exceed criteria should be evaluated further or not used in new food sources."} +{"text": "Efficient determination of structural similarities between protein binding pockets is an important challenge in computational chemistry. A degree of similarity in the mutual comparison is often estimated in terms of graphs and by calculating a metric such as the maximum shared common subgraph. Cavbase was deveRapid Pocket Matching using Distances (RAPMAD), a new modeling formalism for Cavbase entries which allows for highly efficient similarity calculations. Here, protein binding sites are represented by sets of distance histograms based on specific spatial reference points [In this study we propose e points in orderWe demonstrate the discriminative power and the orders of magnitude faster runtime of this novel method by carrying out several classification and retrieval experiments. Among others, datasets of protein cavities hosting specific cofactors are used for classification experiments, where RAPMAD results in a considerably higher rate of correct classifications compared to other alternative approaches while it requires only a fraction of their runtime. Moreover, a set of proteases was inve"} +{"text": "When in 1991 this company was informed by an investigator about a safety issue with piperacillin . We selected trauma surgeons as investigators who worked in large University trauma centers (\u201cknife and gun clubs\u201d). We expected cases with colonic penetration resulting in spillage of gut bacteria into the abdomen to benefit of piperacillin versus a comparator antibiotic.Perioperatively, patients received one of the two study antibiotics plus vecuronium, a non-depolarizing neuromuscular blocking agent. An investigator informed me of the adverse event of prolonged neuromuscular blockade after an abdominal operation.The investigator knew the Drug Interaction section in vecuronium\u2019s package insert includedMy medical director asked a colleague uninvolved with this trial to break the blind. We discovered the patient received our drug. Vecuronium\u2019s package insert (Adverse Reactions section) states the most frequent adverse reaction to non-depolarizing blocking agents as a class consists of an extension of the drug\u2019s pharmacological action beyond the time period needed. This may vary from skeletal muscle weakness to profound and prolonged skeletal muscle paralysis resulting in respiration insufficiency or apnea. What should my company do with the knowledge of this adverse event reported from one of its clinical trials? Lederle, a very ethical big pharmaceutical company did the right thing. We designed and conducted a small study (30 subjects) to determine the magnitude of the neuromuscular blockade of piperacillin in combination with vecuronium. We included a positive control (an antibiotic known to cause this effect). We conducted the trial in a major university hospital. An anesthesiologist took twitch monitor measurements. This machine stimulates nerve causing muscle fibers to twitch; this allows a quantitative monitoring of the blockade.The study confirmed piperacillin caused neuromuscular blockade and the control antibiotic showed the same amount of blockade as had been previously reported . We repo"} +{"text": "Ultimately, we will work towards the goal of taking any defined part of the genome and accurately quantifying the Histone Codes, detecting all the non-histone proteins that reside on these distinct pieces of chromatin, and then mapping this proteomic data back to specific genomic locations, therefore taking a proteomic snapshot of what that chromosome landscape looks like during any nuclear event. These studies in combination with biological experiments will help provide a systems biology outlook on gene expression that will lay down the basic scientific foundation to advance several applications, such as stem cell reprogramming and cancer progression.Histones are small proteins that package DNA into chromosomes, and a large number of studies have showed that several single post-translational modification sites on the histones are associated with both gene activation and silencing. Nevertheless, what type of effect distinct combinations of simultaneously occuring histone modifications (Histone Codes or patterns) have upon cellular events is poorly understood. The main reason for this lack of knowledge is that robust high-throughput methods for quantitative characterization or even qualitative identification of combinatorial Histone Codes by any standard biological, immunological or physical technique do not exist. We plan to specifically address this deficiency by developing novel mass spectrometry based proteomic methods and accompanying bioinformatics to quantitatively characterize molecular level descriptions of combinatorial Histone Codes, and apply these methods to study how these dynamic Histone Codes influence gene expression under different biological conditions. Here we present initial proteomics data that describes: (i) high-throughput comparison of histone modifications from multiple cellular states (ii) developing mass spectrometry methods for quantitative tracking of combinatorial Histone Codes (iii) monitoring"} +{"text": "Unipolar brush cells (UBCs) are excitatory glutamatergic interneurons of the cerebellar granular layer receiving both primary and secondary vestibular inputs through mossy fibers (excitatory input) and Golgi cell axon (inhibitory input). When injected with progressively increasing depolarizing currents from a negative membrane potential, the UBC generates a burst sustained by a calcium spike and then a protracted discharge with shorter latency and spike frequency adaptation. The intrinsic excitability of UBCs is determined by an H current and by Low Voltage activated and High Voltage activated calcium currents ,3. Fast"} +{"text": "Accurate prediction of neurological outcome after cardiac arrest is desirable to prevent inappropriate withdrawal of life-sustaining therapy in patients who could have a good neurological outcome, and to limit active treatment in patients whose ultimate neurological outcomes are poor. Established guidelines to predict neurological outcome after cardiac arrest were developed before the widespread use of therapeutic hypothermia. The American Association of Neurology guidelines currentlA review of existing literature was undertaken to examine whether the utility of motor scores to predict poor neurological outcome is influenced by the use of therapeutic hypothermia.Six studies were identified -7 that iMotor scores at day 3 post cardiac arrest of extension or worse do not reliably predict poor neurological outcome when therapeutic hypothermia has been used. Clinical neurological findings may not be valid predictors of poor neurological outcome after therapeutic hypothermia."} +{"text": "Black blood carotid artery wall imaging can evaluate not just atherosclerotic plaque burden but also high risk components like lipid core and intraplaque hemorrhage. A current limitation in its clinical application, however, is its susceptibility to motion artifacts caused by involuntary motions like swallowing, coughing and breathing. Due to the natural bright signal from subcutaneous fat, unsuppressed fat signal contributes to the majority of motion artifacts in the carotid artery wall region. The fat signal cannot always be properly suppressed using spectrally selective RF pulses due to magnetic field inhomogeneities. Modified Dixon (mDixon) technique, however, is less susceptible to field inhomogeneities because it can separate fat signal without relying on the absolute frequency of the fat spectral.The aim of this study is to explore the feasibility of using mDixon techniques to better separate the strong fat signal and thus reduce motion artifacts near carotid wall region.A two-point mDixon acquisition was programmed to make an automatic extra acquisition with the acquisition window shift after each regular TSE acquisition. The time shift was selected to be 1.15ms, which corresponds to exactly 180 phase separation between water and fat signal at 3T. All images were acquired using a whole body scanner with a dedicated carotid coil. 5 patients with diagnosed carotid atherosclerosis diseases were scanned with both regular spectral selective fat saturationT1 MSDE and an mDixon T1 MSDE sequences. After each mDixon acquisition, the water image was automatically calculated by the algorithm reported before (1). The image quality of the carotid artery on each image was individually evaluated according to an established method (2).The black blood mDixon image presented significantly improved fat saturation at the peripheral regions of the neck Fig. arrows omDixon water fat separation technique improved fat suppression in carotid black blood MRI and helped reduce motion artifacts from fat in carotid artery wall images. Benefited from the improved fat suppression, dramatically improved carotid wall delineation can be observed when motion artifact presents.N.A."} +{"text": "Client change talk has been proposed as a mechanism of change in motivational interviewing (MI) by mediating the link between therapist MI-consistent behaviors (MICO) and client behavioral outcomes. In the present study, we tested under what circumstances this generic MI conceptualization was supported. The study context was a Swiss clinical trial of brief MI for heavy drinking among non-treatment seeking young men (age 20). We conducted psycholinguistic coding of 174 sessions using the MI Skill Code and derived the frequency of MICO and the strength of change talk (CTS) averaged over the session. CTS was examined as a mediator of the relationship between MICO and a drinking composite score measured 3 months after baseline, controlling for the baseline drinking composite score. Finally, we tested therapist gender and MI experience and client readiness to change and alcohol problems severity as moderators of this mediation model (see Hayes 2013). CTS significantly predicted outcome in the expected direction (i.e. higher strength related to less drinking), but MICO did not significantly predicted CTS. However, CTS mediated the relationship between MICO and drinking outcomes when therapists had more experience in MI and when clients had more severe alcohol problems . Findings showed that the mechanism of change hypothesized by MI theory was operative in our brief MI model with heavy drinking young men, but only under particular conditions. Our results suggest that particular attention should be paid to therapist selection and/or training and supervision until they reach a certain level of competence, and that MI might not be appropriate for non-treatment seeking clients drinking at lower level of risk."} +{"text": "We consider an anatomical connectivity matrix (66 nodes), obtained via diffusion spectrum imaging (DSI) and white matter tractography , describCriticality has been proposed to characterize brain signals . The criRecent works have simulated brain activity implementing several dynamical on the connectome structure, retrieving in some cases correlation-based networks similar to those observed from the analysis of neuroimaging data (mainly fMRI at rest). The present work extends the analysis to dynamical networks who take into account lagged and directional influences, concentrating for this abstract on the nodes that more prominently express this disparity between incoming and outgoing information."} +{"text": "Heart failure is a significant and growing health problem. To enable a preventative health care system in heart failure, a move is required from the current, intermittent episodical treatment to continuous and ubiquitous access to medical excellence. Home telemonitoring systems that monitor vital body signs and symptoms with wearable technology, have been proposed to enhance treatment and anticipation of adverse events in heart failure patients (decompensation).We investigated whether the data recorded in a home telemonitoring system using innovative sensors had predictive value in detecting heart failure related events.In the MyHeart observational clinical trial, 148 heart failure patients were followed daily for up to one year using an innovative telemonitoring system recording symptoms questionnaires, basic parameters such as body weight and blood pressure, as well as advanced information provided by a bed sensor monitoring night heart rate and activity and a thorax bioimpedance (BIM) device, designed to monitor fluid accumulation in the lungs. The area under the receiver operator characteristic curve (AUC) was used to evaluate the predictive value for heart failure related events of single and combined telemonitoring measurements.The telemonitoring system including innovative sensors was rated positively by the patients, which is further supported by the reasonable compliance rates (>65%) measured. The most predictive variables 14 days preceding an event were thoracic bioimpedance (area under the curve of 0.70), night breathing rate and some symptoms. The initial multiparametric analysis achieved a mean accuracy of 80.6% but would require more data to confirm its generalisation ability.The measurements performed during the MyHeart trial with 148 patients and 12 months\u2019 follow-up provided evidence supporting the development of predictive algorithms of decompensation to support heart failure management at home. An innovative wearable monitor of thoracic congestion by means of bioimpedance was tested and showed the highest predictive value for heart failure decompensation among a large set of monitored vital signs and symptoms."} +{"text": "Our hypothesis is that patients who demonstrate acute pulmonary vein narrowing or stenosis secondary to edema following radiofrequency ablation (RFA) procedure for atrial fibrillation as demonstrated on immediate post procedural cardiac magnetic resonance (CMR) examination are at an increased risk for developing chronic pulmonary vein stenosis at 3 month followup CMR.From January, 2010 to September, 2011, 224 patients who underwent pulmonary vein isolation and debulking of the septal and posterior walls of the left atrium as part of RFA procedure for atrial fibrillation were imaged on a 3 Tesla MR scanner . Time interval between the conclusion of ablation procedure and placing the patient in the scanner was typically less than one hour. The MR protocol included double inversion recovery T2-weighted TSE, contrast enhanced MR angiography and 3D late gadolinium enhancement (LGE) scans.Retrospective review of records was performed to determine those patients in whom acute pulmonary vein narrowing or stenosis was identified on their immediate post RFA procedure CMR examination. Prospective followup of these identified patients was then performed to determine those patients who went on to develop chronic pulmonary vein stenosis at 3 month followup CMR imaging.Data collection is ongoing. Currently 30 patients have been identified as having findings of acute pulmonary vein narrowing or stenosis secondary to edema on their immediate post procedural CMR. Sixteen of these patients have had three month followup CMR examination. Twelve patients have pending 3 month followup imaging scheduled within the next several months. Two patients have been lost to followup to date. Of the 16 patients found to have acute pulmonary vein stenosis secondary to post procedural edema per CMR, and who have had a 3 month followup to date, 13 of the 16 patients (81%) demonstrate chronic pulmonary vein narrowing or stenosis at 3 month followup examination. Additional patients and followup data will be included in the final results. Results will include extent of immediate post procedural and chronic pulmonary vein narrowing and stenosis.Findings of acute pulmonary vein narrowing or stenosis on immediate post radiofrequency ablation procedures for history of atrial fibrillation have an increased risk of developing chronic pulmonary vein stenosis at 3 month followup CMR examination. It has previously been demonstrated that no patient with a normal examination at 3 month post RFA procedure demonstrated significant stenosis at 6 to 12 months.No grants or additional funding was obtained."} +{"text": "To advance the understanding of the role other drug use plays in alcohol brief intervention (BI), we examined the effects of baseline drug dependence on alcohol use outcomes and the effects of alcohol BI on drug use among injured patients. Hierarchical linear modeling was used to conduct a secondary analysis of data from a randomized trial of patients admitted to a Level-1 trauma center who screened positive for alcohol misuse. A series of two-level models were developed to test the interaction of drug dependence and treatment on alcohol use outcomes for Hispanic (n = 539), white (n = 667), and black (n = 287) patients and the effects of alcohol BI on drug use at 12 months. Results showed significant effects of BI on alcohol outcomes among Hispanic patients but not among white or black patients for percent days abstinent , volume per week , and maximum amount consumed per drinking occasion . Analysis for drug use as an outcome at 12 months showed no significant effects for any race/ethnicity group. In contrast to white and black patients, Hispanic patients with drug dependence who received alcohol BI were more likely to reduce drinking than those who received standard care. Alcohol BI did not appear to influence drug use at follow-up in any group. These results suggest drug use at baseline does not negatively influence drinking outcomes, and alcohol BI does not appear to influence drug use. Interventions specifically targeting drug use may be more likely to influence drug use."} +{"text": "Irisin, secreted by skeletal muscle and possibly fat, is hypothesized to play an important role in modulating energy expenditure, obesity and metabolism. Coffee consumption also increases energy expenditure and leads to positive metabolic effects, but whether these effects are mediated by irisin remains unknown. The objective of this study was to determine the association between baseline irisin levels and the metabolic profile in humans and to investigate whether consumption of caffeinated coffee alters irisin levels. To this end, a secondary analysis was performed investigating irisin levels at baseline and after eight weeks in 32 healthy, overweight coffee drinkers who were randomized to consumption of 5 cups per day of instant caffeinated coffee, decaffeinated coffee, or water. Spearman correlation and analysis of covariance analyses were performed to identify possible associations. Irisin levels were positively correlated with waist circumference , fat mass and CRP . Though there was a trend towards increased levels of irisin over time in the caffeinated coffee group (+1.8%) when compared to the placebo group (\u22124%) this did not reach statistical significance (p\u200a=\u200a0.75 for the trend). This first randomized trial failed to reveal any effects of coffee consumption on irisin levels, but a larger trial, appropriately sized on the basis of data provided by this study, is needed to conclusively investigate such a relationship.NCT00305097Clinicaltrials.gov Irisin is a novel myokine thought to play an important role in energy expenditure by mediating the exercise-induced browning of fat Coffee consumption increases energy expenditure and is thought to decrease the incidence of diabetes The purpose of this study is twofold: to identify associations between irisin and markers of metabolism in humans and to determine whether coffee consumption affects irisin levels. As caffeine increases energy expenditure and irisin levels appear to rise with increased energy expenditure, we hypothesize that irisin levels will increase with coffee consumption. To this end, we have performed a secondary analysis of the serum levels of irisin in overweight coffee drinkers who were randomly assigned to consumption of caffeinated coffee, decaffeinated coffee, or water for eight weeks. This study aims to shed further light on the possible mechanisms by which irisin and coffee consumption can lead to improved health outcomes.2) but otherwise healthy adults who were regular coffee drinkers (\u22652 cups/day) were recruited between 2006 and 2008 with inclusion and exclusion criteria that have previously been described Forty-one overweight Committee on Clinical Investigations and Institutional Review Board in accordance with the Helsinki Declaration of 1975 as revised in 1983. All participants gave written informed consent prior to participation in the study. The clinical trial registration number is NCT00305097 at clinicaltrials.gov.After a two-week long caffeine washout and an overnight fast, participants underwent baseline physical examination, blood draw and oral glucose tolerance test (OGTT). Participants were instructed to maintain their usual exercise and dietary habits and to abstain from caffeine-containing foods throughout the study. Participants returned after eight weeks for adherence questionnaires and repeat blood draws.Each day, participants in the two coffee arms of the study consumed five two-gram portions of instant coffee (caffeinated or decaffeinated Nestl\u00e9\u2019s Taster\u2019s Choice) prepared with 6 ounces of boiling water. Participants randomized to the no coffee arm drank instead five 6-ounce glasses of water. As was previously reported, the five daily cups of caffeinated coffee provided 345 mg caffeine and 302 mg chlorogenic acid while five cups of decaffeinated coffee provided 216 mg chlorogenic acid At each study visit, weight, height, and waist circumference were measured by a trained investigator according to standard definitions. Body composition was measured using Tanita single frequency bioelectrical impedance analysis 2 test for categorical variables and analysis of variance for the continuous variables with normal distribution . All data used in these analyses can be made available on request.The average age (\u00b1SD) of these 32 subjects was 41.4 (\u00b113.8) with a BMI of 30.1 (\u00b11.9). Other baseline characteristics of the study population are shown in Correlations between irisin levels and baseline anthropometric data are shown in Correlations between irisin levels and changes in other biomarkers over the study period are shown in Results of the ANCOVA model calculating percentage change in irisin levels over eight weeks by coffee group are graphically depicted in In this study, irisin levels demonstrated positive correlations with markers of adiposity such as fat mass and waist circumference and CRP, a marker of inflammation. While we had hypothesized that irisin levels would rise with increased caffeine consumption, this did not reach statistical significance.The positive associations between irisin and markers of obesity in this study support the findings of several recent studies and are consistent with our prior hypothesis that irisin levels are increased in obesity as a means of counteracting rising insulin resistance Coffee intake increases energy expenditure and has been associated with a decreased incidence of diabetes and the metabolic syndrome in multiple studies The key limitation of this study is the small and fixed sample size, limiting the power of the study to identify significant relationships and increasing the likelihood of extreme values affecting the results; however, prior to this study no data were available to determine the appropriate sample size. In terms of anthropometric data, the association between irisin and BMI might have been attenuated by the small range of BMI in this study given that all participants were overweight at baseline. The small size and relative homogeneity of this study population also limits generalizability of our findings.Strengths of the study include that it is the first randomized controlled trial that has investigated the effects of coffee intake on irisin levels. We found positive associations between irisin and several markers of obesity, lending credence to the hypothesis that irisin may be secreted by fat in addition to muscle in response to obesity-linked insulin resistance. The data reported in this rather small study can be used to perform power calculations for larger clinical trials and mechanistic studies which are needed to more firmly establish the role of irisin in metabolism and to better understand its potential association with coffee.Figure S1Scatter plot depicting irisin levels versus BMI and waist circumference.(TIF)Click here for additional data file.Figure S2Scatter plot depicting irisin levels versus waist: hip ratio and fat mass.(TIF)Click here for additional data file.Figure S3Scatter plot depicting irisin levels versus CRP and adiponectin.(TIF)Click here for additional data file.Figure S4Scatter plot depicting irisin levels versus fasting plasma glucose and HOMA-IR.(TIF)Click here for additional data file.Figure S5Scatter plot depicting irisin levels versus IL6.(TIF)Click here for additional data file.Table S1Spearman correlation coefficients between irisin levels and baseline anthropometric data.(TIF)Click here for additional data file.Table S2Spearman correlation coefficients between irisin levels and changes in anthropometric data.(TIF)Click here for additional data file.Table S3Spearman correlation coefficients between irisin levels and baseline biomarker levels.(TIF)Click here for additional data file.Table S4Spearman correlation coefficients between irisin levels and changes in biomarkers.(TIF)Click here for additional data file.Checklist S1CONSORT Checklist.(DOC)Click here for additional data file.Protocol S1Study Protocol.(DOC)Click here for additional data file."} +{"text": "But it\u2019s been difficult to translate these findings into health-protective policies because of inconsistencies across studies\u2014something the International Collaboration on Air Pollution and Pregnancy Outcomes (ICAPPO) is working to remedy. This worldwide, multicenter project used a common analysis protocol to derive combined effect estimates of maternal exposure to coarse and fine particulate matter compared with those that accounted for both spatial and temporal elements of exposure .Differences in average PMAlthough the study does not include direct measurements of personal exposure levels, it does provide a comprehensive estimate of the global effects of maternal exposure to particulate matter on birth weight. This could prove useful for shaping meaningful public health policies regarding air pollution."} +{"text": "Rhinoliths are uncommon clinical entities reported in clinical practice as unusual cause of unilateral nasal obstruction and foul smell nasal discharge. Rhinolith is calcified material found in the nasal cavity incidentally or due to patient complaint. It should be suspected when patient presents with nasal symptoms and found to have stony mass showed radiologically. We reported a 28-year-old Saudi male with left sided (LT) nasal obstruction and foul smell discharge for 5 years suspected as being due to foreign body presence since childhood due to head trauma following car accident in sandy area. Rhinoliths are calcified material around intranasal foreign body. They can be endogenous if occur around body tissues as tooth or exogenous if they occur around foreign subject as stones, cotton, or beads. They are found usually in anterior nasal cavity commonly associated with narrowing due to deviated septum, spurs, and/or turbinate hypertrophy. Endoscopic appearance is the main step in diagnosis which can be supported by radiology. Complete resolution of symptoms occurs after endoscopic surgical removal \u20133.A 28-year-old Saudi male presented to ORL HNS clinic referred from another hospital for his complaint of left (LT) nasal obstruction and foul smell discharge for five years. Symptoms were progressively noticed and disturb the patient's life in the last 3 years. He received multiple courses of antibiotics and nasal steroids with no benefit. He had no history of foreign body introduced into nasal cavity. He had history of head trauma after car accident at childhood in sandy area. Anterior rhinoscopy showed irregular hard material with crustations and thick secretions around, stuck between septal spur and inferior turbinate at LT anterior nasal cavity . Trials Bartholin first described rhinolith in 1654. Then more than 600 cases had been reported \u20135.Rhinolith was found frequently as incidental finding during rhinoscopy as irregular, hard dark mass with greenish foul smelling crustations around. Radiological investigations such as plain X-ray and CT scan can support diagnosis and direct the management. Maclntyre was the first to describe rhinolith radiographically in 1900 . Rhinoli To our knowledge this case is the first case reported here in Saudi Arabia and the most interesting is the long standing time it takes to present, and ultimately the final diagnosis was reached by clinical suspicion and endoscopic management is the standard modality of treatment."} +{"text": "The correct cementing technique for knee replacement involves maintaining the knee in extension for a protracted period of time to compress the cement effectively during polymerisation.An abdominal brace is used routinely during knee replacement as a foot support to maintain the knee in flexion. Placing a second, larger abdominal brace distally offers no negative interference to surgery . HoweverWe have used this technique in every primary knee replacement performed over the last 18 months. It has facilitated an increased ease of procedure, negating both assistant discomfort when operating on large patients and the need for an assistant during closure. It is now used as standard operative practice by the senior author."} +{"text": "Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-beta superfamily and were originally identified as proteins that induce a full cascade of endochondral bone formation in vivo and ectopically. We hypothesized that the BMP signaling pathway could also play a role in the process of ankylosis that characterizes ankylosing spondylitis and related spondyloarthritides. By over expression of noggin, a BMP antagonist, in a dedicated mouse model of joint ankylosis, we provided evidence for this hypothesis. Our current studies focus on the relationship between stromal cell activation and inflammation, a link that is essential in ankylosing spondylitis. Emerging functional and genetic evidence further corroborates the essential role of BMPs in these processes. BMPs can trigger different downstream signaling cascades, in particular Smad and p38 signaling. Inhibition of p38 signaling in vivo in the spontaneous ankylosing enthesitis model appears to accelerate ankylosis while inhibiting in vitro chondrogenesis. Genetic analysis of intercrosses between the susceptible DBA/1 strain and the H2 identical Balb/c strain point towards a region containing the BMP type Ib receptor as a factor determining genetic susceptibility. Recent clinical data suggest that levels of BMPs are higher in patients with progressive ankylosis in comparison with patients that show no structural progression of disease. Taken together, these data support our hypothesis that BMP signaling is a therapeutic target in ankylosing spondylitis."} +{"text": "The purpose of this study is to evaluate semi quantitative perfusion parameter maps generated based on a fully automated non-rigid motion correction during a first pass myocardial perfusion (FPMP) MRI in patients with suspected coronary artery disease (CAD) or coronary micro vascular disease (CMVD).FPMP MRI is commonly used to assess CAD and most recently to assess cardiac involvement in asymptomatic patients with CMVD such as systemic sclerosis and diabetes mellitus.FPMP MRI evaluation relies on visual inspection for qualitative analysis but quantitative analysis of rest and stress perfusion data is desired to improve diagnosis. One main challenge of qualitative analysis includes cardiac and respiratory motion. To minimize this challenge, a previously described inline, fully automated motion correction method generates a motion corrected dataset as well as pixel-wise upslope maps. Using the image at a time point selected for peak signal change during the first pass of contrast agent as the template, all other time points were registered into the template coordinate system. We compare qualitatively and quantitatively the original free breathing images and motion correted images with the corresponding maps pixel-wise upslope maps in patients with suspected CAD or CMVD.Seventy one patients with suspected epicardial CAD or CMVD underwent adenosine stress and rest perfusion scans on 1.5T scanner using a framework to fully automatically analyze cardiac FPMP MR.Three short axis slices were acquired during infusion of 0.075 mmol/kg of Gadolinium adenosine infusion was administrated to induce stress. Free breathing, motion-corrected images and corresponding perfusion maps were assessed by two radiologists independently using the AHA 16 model and scored using a four point Likert scale (poor to excellent) to evaluate image quality and confidence level for the presence or absence of hypo-perfusion regions. Signal intensity curves upslope index from both free breathing and motion corrected images during stress and rest were manually calculated in non ischemic and ischemic areas and compared to the corresponding pixel wise parameter map generated based on motion corrected images.All patients were successfully scanned. Segmental perfusion defects were identified in 27 of the 49 patients with suspected CAD patientsFig. and in tSemi quantitative perfusion parameter maps obtained by a fully automated non-rigid motion correction during a FPMP MRI correlated both qualitatively and quantitatively with the free breathing images in patients with epicardial CAD and CMVD.Astellas Pharma Global Development, Inc."} +{"text": "Tau protein is a microtubule-associated protein found in the axonal compartment that stabilizes neuronal microtubules under normal physiological conditions. Tau metabolism has attracted much attention because of its role in neurodegenerative disorders called tauopathies, mainly Alzheimer disease. Here, we review recent findings suggesting that axonal outgrowth in subgranular zone during adult hippocampal neurogenesis requires a dynamic microtubule network and tau protein facilitates to maintain that dynamic cytoskeleton. Those functions are carried out in part by tau isoform with only three microtubule-binding domains (without exon 10) and by presence of hyperphosphorylated tau forms. Thus, tau is a good marker and a valuable tool to study new axons in adult neurogenesis. Newborn granule cells generated in the SGZ grow dendrites into the molecular layer and send axons into the CA3 region. This process is similar to that which occurs during neuronal polarization mainly studied with rodent embryonic hippocampal neurons New neurons in the SGZ of the hippocampal formation grow dendrites into the molecular layer, and send axons into the CA3 region. Major glutamatergic synaptic activation from perforant path afferents does not occur until new neurons are two or more weeks old concurrent with appearance of spines on dendrites of newly born neurons and diverse neurodegenerative (FTDP-17 and Alzheimer disease) models.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Observational evidence suggests that improving fetal growth may improve adult health. Experimental evidence from nutritional supplementation trials undertaken amongst pregnant women in the less developed world does not show strong or consistent effects on adult disease risk and no trials from the more developed world have previously been reported. To test the hypothesis that nutritional supplementation during pregnancy influences offspring disease risk in adulthood Clinical assessment of a range of established diseases risk markers in young adult offspring of 283 South Asian mothers who participated in two trials of nutritional supplementation during pregnancy at Sorrento Maternity Hospital in Birmingham UK either unselected or selected on the basis of nutritional status. 236 (83%) offspring were traced and 118 (50%) of these were assessed in clinic. Protein/energy/vitamins supplementation amongst undernourished mothers was associated with increased infant birthweight. Nutritional supplementation showed no strong association with any one of a comprehensive range of markers of adult disease risk and no consistent pattern of association with risk across markers in offspring of either unselected or undernourished mothers. We found no evidence that nutritional supplements given to pregnant women are an important influence on adult disease risk however our study lacked power to estimate small effects. Our findings do not provide support for a policy of nutritional supplementation for pregnant women as an effective means to improve adult health in more developed societies. The nutritional \u201cfetal origins\u201d hypothesis holds that physiological adaptation to nutritional status during gestation has life-long consequences for health,2. ExperAll participants provided written informed consent. All study procedures were approved by South Birmingham Local Research Ethics Committee (LREC no. 0273). Details of participants and procedures in the original unselected and selected trials of nutritional supplementation at the Sorrento maternity hospital in Birmingham have been published elsewhere-17.BriefSupplements were delivered in batches to the mothers\u2019 home at five week intervals. Blinding of mothers to their trial allocation group was not attempted. Empty containers from the previous delivery were collected to assess apparent consumption. Maternal compliance was also checked by study midwives and dieticians and monitored through blood biochemistry. Analysis was by intention to treat.Personal identifier information for women who participated in the trial and their offspring were extracted from archived trial and maternity records. Individuals were then traced through the NHS central register.All living offspring for whom contact details were available were sent a letter describing the study and inviting them to attend for a clinical assessment at the Wellcome Trust Clinical Research Facility at the Queen Elizabeth Hospital in Birmingham. Non-responders were sent reminder letters. Home-visits and telephone calls were used to confirm contact details and receipt of the study invitation. Results of maternal tracing and offspring follow up in clinic are summarised in a modified CONSORT diagram see .Participants attended the clinic in a fasting state; a baseline blood sample was obtained prior to administration of a standard glucose tolerance test. Further blood samples were obtained at 30 and 120 minutes after the glucose load. Fasting blood samples were used to assess blood lipids, glucose, glycosylated haemoglobin, C peptide, testosterone and Sex Hormone Binding Globulin according to standard methods. Insulin and glucose were also estimated on the 30 and 120 minutes post glucose samples according to standard methods. Insulin resistance and beta cell sensitivity was estimated using the HOMA method. Sex HorInsulin like growth factors IGF1 and IGFBP3, the levels of which have been associated with increased risk of some common cancers ,21, wereParticipants were fitted with a Spacelabs 90207 ambulatory blood pressure monitor that was collected after completion of 24 hours of measurement allowing estimation of mean systolic and diastolic blood pressure over the measurement period.Participants provided a 24 hour urine sample that was tested for steroid metabolites using gas chromatography/ mass spectroscopy. Urinary cortisol:cortisone (F/E) ratio was taken as a marker of 11 B- hydroxysteroid dehydrogenase 2 activity in the kidney (with increased ratios suggesting the impaired activity associated with failure to inactivate cortisol to cortisone associated with hypertension), tetrahydrocortisol + 5 allo tetrahydrocortisol: tetrahydrocortisone (THF + 5a \u2013 THF/ THE) ratio was taken to be a marker of 11 B- hydroxysteroid dehydrogenase 1(increased ratios suggesting the impaired activity associated with adiposity). ParticiWe investigated the primary null hypothesis that nutritional supplementation during pregnancy has no influence on disease risk in adulthood. We had no strong a priori basis to expect an effect of a particular size in relation to any of the disease risk markers measured with a particular supplementation regime. In the original Sorrento studies the strongest effect on birth weight was seen amongst offspring of mothers selected on the basis of their apparent under-nutrition who received protein, carbohydrate and vitamin supplements as described above. Mean values of infant birth weight and all adult outcome variables along with their standard errors were calculated according to original trial allocation and were compared using analysis of covariance amongst participants assessed as adults. Birth weight according to trial allocation was also examined amongst the larger group of participants successfully traced as adults. The original Sorrento studies were randomised therefore an unadjusted intention to treat analysis was appropriate. In the original studies a stronger effect on birth weight amongst under-nourished mothers was seen in a per protocol analysis excluding apparently non-compliant mothers. We alsoNot all individuals assessed as adults had full information on all measures. To increase power and investigate the possibility that this might have led to bias we used multiple imputation of missing adult data using the ICE routine in Stata 10. All varOne participant in Trial I was excluded from the analyses as they had been measured on only one of the adult disease risk markers (BMI). Anonymised study data are available on request from the corresponding author. All analyses were performed using Stata 10.118 offspring of mothers participating in the original Sorrento trials were assessed in clinic between June 2002 and October 2004. Participant characteristics at follow up are given in 2,62 =0.73, p=0.48) . Amongst children of under-nourished mothers those receiving protein-energy supplements have higher birth weights however in the group of these children assessed as adults this difference is no longer apparent and any difference between groups is small and imprecisely estimated.Distribution of those assessed according to allocation in the original trial is given in Amongst young adult offspring of mothers randomised to receive one of three nutritional supplementation regimes during pregnancy either unselected or selected on the basis of evidence of their nutritional status we found no evidence that supplementation influenced adult risk markers of cardiovascular disease, diabetes or cancer. Amongst mothers with evidence of under-nutrition, protein-energy supplementation during the final pregnancy trimester was associated with increased birth weight but this increase was not associated with any strong or consistent change in any marker of adult disease risk measured. These risk markers included many of those found to be associated with birth size in observational studies such as blood pressure , markersThree other trials of the effectiveness of nutritional supplementation during pregnancy on perinatal and early childhood outcomes have now been extended to include later childhood or early adult follow up-14. ThesMany of the risk markers we assessed were not measured in these follow up studies so direct comparison is not possible. We chose measures that appeared to provide the most valid index of the risk parameter of interest in relation to all the elements and mechanisms of risk previously discussed in relation to the fetal origins hypothesis. Thus in addition to standard clinic assessments we used 24 hour ambulatory blood pressure measurement and we assessed body composition and adiposity through DEXA scanning in addition to standard measures of height and weight. Fetal corticosteroid exposure has been suggested as an alternative mechanism whereby smaller birth size could be associated with higher risk of later cardiovascular disease principally through an effect on hypertension or adiposity. BecauseThe main strength of this study is its experimental design. Also, our study was undertaken in a high income setting with facilities for assessment of a wide range of risk markers using relatively sophisticated technologies. We also provided evidence in relation to the value of targeted supplementation based on nutritional assessment compared to universal supplementation. Finally this study provided evidence in relation to a supplementation intervention feasibly delivered within a health care system typical of high-income countries. The main limitation of this study is its small size and consequently limited power and precision. Sample size was set by the original Sorrento study and was further reduced by incomplete follow up. These considerations are particularly relevant to the second Sorrento trial amongst mothers selected on the basis of their evidence of under-nutrition. Considering the age of the study population and the fact that assessments involved a clinic visit, follow up rates of 50% amongst those with valid contact details were relatively high. Amongst individuals with incomplete assessment data we used multiple imputation of missing values which increased power and did not suggest any important bias in the complete case analysis. Our participants were all of South Asian ethnicity however observational evidence supporting the \u201cfetal origins\u201d hypothesis has also been reported from South Asian populations. Given tWe found no evidence that, irrespective of other benefits it may have, improving the nutritional status of pregnant women is likely to have substantial long term effects on offspring risk of cardiovascular disease or diabetes. We also found no evidence of any substantial adverse effects on increased cancer risk. It is important to note that we lacked power to detect small effects in relation to these outcomes. Within this caveat our study suggests that rather than expanding public health policy around cardiovascular prevention to include fetal nutrition preventive policy should continue to be focused on the established adult behavioural and physiological risk factors hypertension, dyslipidaemia, diabetes, obesity, lack of exercise and smoking alongside the social disadvantage that reinforces adverse risk profiles in these. Figure S1Flow Diagrams showing maternal participation in original Sorrento trial and offspring follow up.(DOCX)Click here for additional data file."} +{"text": "An initial Delphi survey delineated key commonalities for a standard definition of clinical remission and inactive disease in jSLE. However, several additional clarifications were still required.To develop a definition and criteria of clinical remission and inactive disease in jSLE.A second international Delphi survey was conducted among pediatric rheumatologists. Consensus was set at 75%.There were 210 respondents. Consensus was achieved regarding the key definitions under consideration (Table Consensus has been reached on the definition of \u2018Clinical Remission\u2019 and \u2018Clinically Inactive Disease\u2019 in jSLE. The results of the Delphi process will be used to guide the data-driven development of provisional criteria of clinical remission and inactive disease in jSLE."} +{"text": "Populus spp.) is favored for forestry and reclamation purposes all over the northern hemisphere where they represent a commercially important resource. Poplars may become a component of programs to optimize carbon sequestration however; poplars are generally regarded as drought sensitive. The patterns of episodic drought over the last decade suggest that the development of drought tolerant poplar genotypes could be a useful tool to achieve sustained forest productivity [The cultivation of poplars were obtained from Dr. Barb Thomas (Alberta Pacific Forest Industries) and Bill Schroeder . Cuttings were established in the greenhouse and plants were obtained for the drought stress trial. At the start of the trial, water was withheld from plants used for drought experiment whereas control plants were watered regularly. During the drought stress trial, a plastochron index was established that helped to collect the data from similar leaves of all drought stressed and control plants. Data were collected from 9 different poplar hybrids at 3 time points . Split plot design was used to collect the data from drought stressed and control plants using genotypes as main plots (9) and time points as sub-plots (3). Physiological data were collected for height growth, new leaf formation, water potential (\u03c8) and relative water content (RWC) from drought stressed and control plants. Young leaves were also collected for gene expression analysis from both stressed and control plants. Quantitative polymerase chain reaction (Q-PCR) was used to analyze the expression of several candidate drought responsive genes.Drought significantly affected the growth of the trees . Data from 3 drought stress trials in the greenhouse showed that different hybrids responded drought stress differently. Some hybrids behaved as tolerant whereas others as sensitive and some hybrids showed intermediate response to drought. By statistical analysis of physiological data, we ranked these 9 poplar hybrids according to their drought tolerance ability. Statistical analysis showed that Walker maintained significantly better height growth, leaf formation, \u03c8 and RWC during mild, severe stress and recovery as compared to all other hybrids; therefore Walker was identified as the most drought tolerant hybrid genotype. On contrary, Green Giant showed poor maintenance of physiological parameters at all time points and hence was regarded as the least drought tolerant hybrid genotype. An intermediate response of all other hybrids was recorded during 3 drought stress trials in the greenhouse. Further, we focused only on 2 genotypes (the most and the least drought tolerant) to analyze the expression of several candidate drought responsive genes, which were obtained from microarray studies in poplar. Q-PCR results revealed that selected 2 genotypes had no significant effect on the stress response of some genes, however, other genes showed marked and significant differences in expression between the two genotypes.\u2022 Three trials in the greenhouse showed that physiological response to drought varies among different poplar hybrids.\u2022 Walker was identified as the most whereas Green Giant as the least drought tolerant genotype among a group of poplar hybrids.\u2022 Q-PCR analysis showed that among a set of candidate drought responsive genes, some genes showed differential expression between the most and the least (Green Giant) drought tolerant genotypes.\u2022 The genes showing differential expression in the most and the least drought tolerant genotypes might be playing an important role in drought tolerance of poplar..Populus genes those expression correlate with drought tolerance, providing candidate genes for drought tolerance breeding.\u2022 We have identified Populus.\u2022 These genes may also be used as molecular markers for drought tolerance in"} +{"text": "The aim of this study is to establish whether different types of sepsis have an impact on selenium levels. Selenium is an essential trace element involved in antioxidant and immunological reactions. Selenium levels have been shown to be low in patients with systemic inflammatory response syndrome and sepsis. Selenium replacement has been recommended in patients with sepsis ,2. GreatThis is a prospective survey where selenium levels were collected from patients admitted with septic shock to a tertiary ICU, for 6 months from October 2010 to March 2011.Selenium levels were measured in 31 patients with septic shock. Abdominal and chest sepsis were the main sources of infection. Those with an abdominal source of sepsis had the lowest levels, as shown in Table All patients admitted with early septic shock had subnormal selenium levels. Patients with an abdominal source of septic shock had the lowest levels. This survey supports previous studies indicating early supplementation may be beneficial in septic shock patients."} +{"text": "The last few years have witnessed rapid scaling-up of key malaria interventions in several African countries following increases in development assistance. However, there is only limited country-specific information on the health impact of expanded coverage of these interventions.Paediatric admission data were assembled from 4 hospitals in Malawi reflecting different malaria ecologies. Trends in monthly clinical malaria admissions between January 2000 and December 2010 were analysed using time-series models controlling for covariates related to climate and service use to establish whether changes in admissions can be related to expanded coverage of interventions aimed at reducing malaria infection.In 3 of 4 sites there was an increase in clinical malaria admission rates. Results from time series models indicate a significant month-to-month increase in the mean clinical malaria admission rates at two hospitals (trend P<0.05). At these hospitals clinical malaria admissions had increased from 2000 by 41% to 100%. Comparison of changes in malaria risk and ITN coverage appear to correspond to a lack of disease declines over the period. Changes in intervention coverage within hospital catchments showed minimal increases in ITN coverage from <6% across all sites in 2000 to maximum of 33% at one hospital site by 2010. Additionally, malaria transmission intensity remained unchanged between 2000\u20132010 across all sites.Despite modest increases in coverage of measures to reduce infection there has been minimal changes in paediatric clinical malaria cases in four hospitals in Malawi. Studies across Africa are increasingly showing a mixed set of impact results and it is important to assemble more data from more sites to understand the wider implications of malaria funding investment. We also caution that impact surveillance should continue in areas where intervention coverage is increasing with time, for example Malawi, as decline may become evident within a period when coverage reaches optimal levels. Malaria poses a major public health challenge in many part of sub-Saharan Africa and is recognized as part of the global health agenda as a significant barrier to achieving the Millennium Development Goal of improving child survival by 2015 There is a growing body of evidence of the health impact associated with increasing flows of malaria ODA Malawi is a high mortality burden In comparison to other countries in sub-Saharan Africa, Malawi was slower to realize adequate donor support before 2008 In 2000 less than 4% of Malawian children were protected by an ITN Initially IRS activities were limited to a single pilot district, Nkhotakota in 2007 covering 27,000 houses in the northern section and were subsequently repeated in 2008 and 2009. In 2010 the President\u2019s Malaria Initiative (PMI) expanded IRS activities to the whole district this time including neighbouring Salima District. This has now been scaled-up covering approximately 500,000 households in five additional highly endemic districts in December 2010: Karonga, Nkhata Bay, Mangochi, Chikwawa, and Nsanje Districts Between 2000 and 2007 sulphadoxine-pyrimethamine (SP) was the only drug available in most government clinics Thus within the context of financing, intervention delivery and coverage, between 2000 and 2006, there was a definite lack of significant progress toward achieving internationally agreed and nationally adopted coverage targets of malaria interventions. However, starting in 2007 there was an increase in external funding from the Global Fund (GF) and PMI, implementation of AL for treatment and a revised ITN policy to rapidly scale up ITN coverage including the use of mass campaigns.Malawi is divided into three regions: Central, Northern and Southern with a total of 28 districts. Within this there are 4 central hospitals, 22 designated district general hospitals and 46 rural hospitals managed by either the government or mission sectors. District hospitals provide secondary level health care services and serve to support peripheral clinical services by providing in-patient care and other specialized services. Despite recent efforts at improvement, traditionally routine health management information system (HMIS) reporting from all levels of service provision has been largely incomplete over long time periods and often inappropriately summarized by cause or age. To overcome the perennial inadequacies of routine HMIS data we selected a sub-set of hospitals for a more detailed inpatient review where each hospital had records available for investigation and represented the malaria ecologies typical of the country. The four hospitals selected for review are shown in We computed catchment populations to standardise the admission data across the four hospitals. Previously we have attempted to define catchment areas using addresses of admissions in Kenya Using the lowest level of administrative units available, Traditional Authorities, all units that intersected the boundaries of the Thiessen polygon of the study hospital were included and used as the first step in defining the hospital\u2019s catchment. In the second step, a provisional catchment was defined for each hospital using a radial distance of 30 km which had been found to capture over 90% of admissions in Kenya At each selected hospital, paediatric ward in-patient registers were identified for most months from January 2000 to December 2010. Each admission entry in the registers was recorded on a tally sheet indicating the month of admission and whether a primary working diagnosis of malaria had been defined for the child or whether the admission diagnosis did not include an indication of malaria. We have restricted the data assembly to records of children less than five years because they bear the burden of severe malaria in most highly endemic African countries. Individual register entries were not reconciled with patient notes and we have assumed that the admission diagnosis remained the diagnosis upon which each admission was managed clinically. Slide confirmed malaria diagnoses at admission were not universally available within, nor between hospital sites, however we have assumed that most admissions were likely to have a blood film prepared pre-admission. We remain uncertain about how, when available, parasitological results were used to guide diagnosis given the vagaries of presumed and parasitological diagnosis for in-patient paediatric case-management This was a routine audit from registers and something undertaken as part of routine activities. The data were assembled as anonymised counts, all the data were de-identified monthly tallies of hospital admissions therefore no patient level details were included.PfPR2\u201310) is a common metric of transmission intensity that is used universally and scales with paediatric malaria disease risks PfPR2\u201310) sample surveys undertaken during this period using a space-time Model Based Geostatistical framework with Bayesian inference and implemented using the Markov Chain Monte Carlo algorithm. Posterior distributions of the predicted mean PfPR2\u201310 were generated at every unsampled 5\u00d75 km grid in Malawi for the years 2000, 2005 and 2010 We have used parasite prevalence as a measure of transmission intensity at each hospital site selected for investigation. Parasite prevalence in children aged 2\u201310 years from the national household sample Demographic Health Surveys (DHS) undertaken in 2000, 2004 and 2010 admission months where 98,773 (38%) had a primary working admission diagnosis of malaria. The total admissions, clinically diagnosed malaria admissions and proportion of admissions that are clinical malaria cases are shown in To examine the long-term temporal signals within the monthly time-series several model forms were explored. The predictions from the best fitting model that adjusted for non-malaria admission rates and rainfall at different lags while controlling for auto-regressive and moving average effects within the data are shown in PfPR2\u201310 from within the defined catchment areas of each hospital mean PfPR2\u201310 was 41%, however it was predicted to be lower in Zomba during this period mean PfPR2\u201310 had increased marginally or remained unchanged in all the four sites households in the vicinity of Salima District Hospital had been sprayed within the preceding 12 months.The proportions of reported paediatric fevers in the last 14 days recorded during the most recent household surveys that were treated with an ACT remains low. Between 2007 and 2012, there have been two surveys that sought to evaluate malaria case management practices for febrile children below 5 years of age Since the launch of RBM in 2000 there has been a slow increase in external donor support and its translation into effective disease control and prevention coverage in Malawi, until important changes in access to malaria ODA and support for drug policy change and ITN distribution after 2007. Paediatric clinical malaria admissions at four hospitals across the country were analysed over 11 years, 2000 to 2010, to explore whether annualized, catchment population standardized admission rates had declined following the launch of the first NMS in 2001 and following significant changes in intervention coverage.At three of the four hospital sites investigated, malaria admission rates have increased or remained unchanged since the period of pre-scaled intervention and funding (2000\u20132006) through to a period when Malawi had received improved financial support and made progress towards malaria control (2007\u20132010). Conversely, with the exception of Salima, non-malaria pediatric admissions remained relatively constant. Analysis of monthly time-series malaria admission rates using autoregressive models adjusting for external factors shows a significant increase in paediatric admissions from January 2000 through to December 2010 within five years than when starting endemicity is higher Trends reported here are similar to reports from other high transmission sites in Uganda and Kenya One major caveat to our retrospective analysis of hospital-based data on clinically diagnosed paediatric malaria admissions is that we cannot be certain that each recorded case of \u201cmalaria\u201d was in fact malaria. Parasitological confirmed cases of malaria are more common among patients admitted to hospital than those treated as out-patients, and therefore represent a more specific clinical temporal series but testing is never universal; laboratory records were incomplete and not linked to ward registers; and often incorrect where technologists err on the side of caution and record \u201cscanty\u201d for slides without parasites. This represents a perennial problem for all long-term hospital data on malaria in Africa and applies to all analyzes of hospital malaria trends reported to-date Malawi has only enjoyed reasonable levels of ITN coverage for a few years and the impact on transmission and subsequently disease may take several more years to become evident. What is clear is that current coverage has not had a dramatic impact and that additional measures, as proposed in Malawi, like IRS combined with higher levels of ITN are necessary to meet the ambitions of the latest national malaria strategy to reduce by 50% the 2010 malaria burden. This will require an increase in malaria ODA and a more targeted plan of action and in order to document future epidemiological changes a result of increased ODA, better surveillance and better data.Figure S1Map of clusters from the Malawian DHS dates of 2000, 2004 and 2010 within a 40 km radius of each hospital extracted in ARCGIS 9.3 to provide data with which to define ITN/IRS coverage within hospital catchments.(DOCX)Click here for additional data file.Table S1Temporally aggregated paediatric admission data for malaria admissions data and for all cause admissions in each of the 4 hospitals between 2000\u20132006 and 2007\u20132010 expressed per 1000 children aged 0\u20134 years per annum and 95% confidence intervals computed using a Poisson distribution. Percentage change is the change in average annual malaria admission rates or average annual all-cause admission rates between the first (2000\u20132006) and the second (2007\u20132010) period.(DOCX)Click here for additional data file."} +{"text": "Many cognitive and motor functions are enabled by the temporal representation and processing of stimuli but it remains an open issue how neuronal circuits could reliably encode such sequences of information. We consider the task of generating and learning spatiotemporal spike patterns in the context of an attractor memory network, in which each memory is stored in a distributed fashion represented by increased firing in pools of excitatory neurons. Excitatory activity is locally modulated by inhibitory neurons representing lateral inhibition that generates a type of winner-take-all dynamics. Networks of this type have previously been shown to exhibit switching between a non-coding ground state and low-rate memory state activations displaying gamma oscillations ; howeverAssuming a probabilistic framework in which local neuron populations discretely encode uncertainty about an attribute in the external world , we model inter-module synapses using the Bayesian Confidence Propagation Neural Network (BCPNN) plasticity rule . We use Introducing plastic BCPNN synaptic projections into the attractor network model allows for stable associations between distinct network states. Associations are mediated by different synaptic timescales with fas"} +{"text": "It is possible to infer the past of populations by comparing genomes between individuals. In general, older populations have more genomic diversity than younger populations. The force of selection can also be inferred from population diversity. If selection is strong and frequently eliminates less fit variants, diversity will be limited because new, initially homogeneous populations constantly emerge.Here we translate a population genetics approach to human somatic cancer cell populations by measuring genomic diversity within and between small colorectal cancer (CRC) glands. Control tissue culture and xenograft experiments demonstrate that the population diversity of certain passenger DNA methylation patterns is reduced after cloning but subsequently increases with time. When measured in CRC gland populations, passenger methylation diversity from different parts of nine CRCs was relatively high and uniform, consistent with older, stable lineages rather than mixtures of younger homogeneous populations arising from frequent cycles of selection. The diversity of six metastases was also high, suggesting dissemination early after transformation. Diversity was lower in DNA mismatch repair deficient CRC glands, possibly suggesting more selection and the elimination of less fit variants when mutation rates are elevated.The many hitchhiking passenger variants observed in primary and metastatic CRC cell populations are consistent with relatively old populations, suggesting that clonal evolution leading to selective sweeps may be rare after transformation. Selection in human cancers appears to be a weaker than presumed force after transformation, consistent with the observed rarity of driver mutations in cancer genomes. Phenotypic plasticity rather than the stepwise acquisition of new driver mutations may better account for the many different phenotypes within human tumors. A barrier to a better understanding of human cancer is the inability to directly observe how cancers evolve. Progression is clinically important, with localized cancers more easily treated than deeply invasive or metastatic cancers. A logical presumption is that progression occurs stepwise after transformation with theAlternatively, the ability to invade or metastasize may already be present at the time of transformation Tumor heterogeneity measurements are complicated because many mechanisms can contribute to diversity Colorectal adenocarcinomas have neoplastic glands that partition tumor cells into distinct small neighborhoods. The extent of hitchhiking diversity within and between glands depends on the timing of the last clonal expansion or selective sweep . A selecBecause somatic mutations are relatively rare in human CRCs , distant cells are almost related as adjacent cells because its different parts are essentially created at the same time. Therefore, PWDs will be independent of physical distance or location. By contrast, if tumors are created by stepwise selection and clonal evolution, PWDs depend on the physical location of the cells. Older regions should be more diverse than younger regions, and PWDs should increase when comparisons are made between younger and older tumor regions .The xenografts simulate these different progression scenarios. A single recent clonal expansion is represented by xenografts initiated from the same single cell progenitor. Stepwise clonal evolution is represented by comparisons between the polyclonal and clonal xenografts. Intergland PWDs were lower in the clonal xenografts, greater in the polyclonal xenografts, and greater between the clonal xenografts and their parental polyclonal xenografts or the other independent clonal xenograft . These eThe cloning experiments are potentially complicated by unseen bottlenecks caused by natural selection . The \u201cpoIf significant bottlenecks occur during tumorigenesis, xenograft diversities will be similarly limited whether the initial inoculate was clonal or polyclonal. BGN tag diversity was significant less when xenografts were initiated with the single cell clones compared with a polyclonal inoculate , indicating that selective bottlenecks do not occur with the Lovo cell line during xenograft tumorigenesis . LOC tagAnother way to detect localized selection is to compare the diversity within glands with the diversity between glands. In the absence of selection, older more diverse tumors should have older more diverse glands. If selection occurs within glands, diversity is reduced as variant cells are eliminated , and intIn the above studies, only eight tags were sampled per specimen. To confirm the diversity in the small fragments, 24 tags were sampled . AdditioA prior study inferred that small human CRC glands are diverse populations Human CRC gland diversities were high indicating that neighboring cells are not closely related . For thrLow cancer gland diversity does not directly reflect selection because even without selection a new clonal expansion would initially be homogeneous. To correct for tumor age, as a rough approximation, the age of a tumor is the time or numbers of divisions between cells from opposite tumor sides, because these cells last shared a common ancestor around the time of transformation If selection depends on mutations, a simple prediction is that selection should occur more often when mutation rates are elevated. Two of the five CRCs were deficient in DNA mismatch repair (MMR), which is associated with 100- to 1000-fold higher mutation rates A quantitative way to describe selection is to estimate the numbers of long-lived lineages per cancer gland, which are effectively CSCs A previous analysis of data with eight tags per gland estimated relatively high CSC frequencies The EDTA gland isolation method may bias sampling to superficial tumor regions, which may be the oldest part of a tumor that progresses via clonal evolution . Laser cWith sequential stepwise progression, there should be a diversity gradient , whereas all regions should be similarly diverse if a tumor is essentially a single clonal expansion. Nine CRCs were examined . DiversiIntergland PWDs were similar between superficial and invasive portions of three Stage II cancers . For two of the six metastatic cancers (D and F), superficial, invasive, and metastatic regions were similarly diverse, including three different lymph node metastases of Cancer F. Significant diversity differences were present between four primary cancers and their metastases. Both the invasive and metastatic regions of Cancers H and I were significantly less diverse than their superficial cancer regions. For Cancer G, only the metastatic lesion was significantly less diverse. The metastasis of Cancer A was significantly more diverse than its primary.To search for regional clonal evolution, intergland PWDs were compared with physical distances . The supTumor cells encounter and colonize many different microenvironments during tumorigenesis, and conceptually selection efficiently maximizes fitness to drive this progression. Clonal evolution depends on new driver mutations, which should be readily generated by the genomic \u201cinstability\u201d thought to be present in many cancers Without a measure of selection it is difficult to judge the roles of the numerous mutations and epigenetic changes found in cancer genomes. Selection efficiently optimizes fitness whenever and wherever opportunities arise, but a practical question is how much cells differ before selection intervenes. Although selection is difficult to measure, a selective sweep produces a bottleneck and loss of cellular diversity. Therefore, the diversity of hitchhiking passenger changes within a population is a meaIf selection is weak and a stepwise acquisition of new capabilities occurs infrequently, the first transformed cell may already produce well-adapted and versatile progeny with abilities to invade or metastasize Without clonal evolution, present day tumor cells would form a single population with uncomplicated star-shaped ancestries and frequent long-lived lineages. The relative diversities within glands versus between glands (intragland to intergland PWD ratios) indicate how much remodeling or extinction occurs within glands. Simulations of cancer gland diversity suggested limited cancer cell extinction and were consistent with stem cell hierarchies with multiple long-lived CSC lineages per gland rather than extremely rare CSCs. CRCs are often resistant to chemotherapy Tumor evolution is commonly thought to increase fitness, but if the first transformed cell is already optimally \u201cfit\u201d, its progeny may suffer from progressive declines in fitness, an asexual reproduction phenomenon called Muller's Ratchet Exactly how individual human tumors progress is uncertain because serial observations are impractical and unethical. Translation of traditional molecular phylogeny approaches to somatic cell populations can potentially reconstruct the pasts of individual human tumors CRC samples were obtained in the course of routine clinical care from the Norris Comprehensive Cancer Center, with approval from our institutional review board . The mouse xenograft studies were approved by our institutional review board .Limiting dilution was used for single cell cloning of the Lovo CRC cell line CRC samples were obtained in the course of routine clinical care from the Norris Comprehensive Cancer Center, with approval from our institutional review board. The 24 tags per gland sampling was performed on previously analyzed tumors . LCM was performed as previously described Figure S1A. Sequences of the LOC and BGN X-chromosomal tags. Primers are underlined and CpG sites are highlighted in red. B. Sample data, with 8 epialleles sampled from each specimen. The polyclonal specimens are more diverse (higher average PWDs) compared to the clonal culture or xenograft. Filled circles represent methylated CpG sites.Methylation tags. (PDF)Click here for additional data file.Figure S2Intergland PWDs were similar with EDTA- (black) or LCM-sampling (red) for Cancers A\u2013E, indicating the sampling approaches are equivalent. Only for Cancer B were the values significantly different .(PDF)Click here for additional data file."} +{"text": "Transcranial alternating current stimulation (tACS) seems likely to open a new era of the field of noninvasive electrical stimulation of the human brain by directly interfering with cortical rhythms. It is expected to synchronize (by one single resonance frequency) or desynchronize cortical oscillations. If applied long enough it may cause neuroplastic effects. In the theta range it may improve cognition when applied in phase. Alpha rhythms could improve motor performance, whereas beta intrusion may deteriorate them. TACS with both alpha and beta frequencies has a high likelihood to induce retinal phosphenes. Gamma intrusion can possibly interfere with attention. Stimulation in the \u201cripple\u201d range induces intensity dependent inhibition or excitation in the motor cortex (M1) most likely by entrainment of neuronal networks, whereas stimulation in the low kHz range induces excitation by neuronal membrane interference. TACS in the 200 kHz range may have a potential in oncology. Transcranial alternating current stimulation (tACS)\u2014the external application of oscillating electrical currents\u2014is able to influence cortical excitability and activity and in the so called \u201cripple\u201d range was observed induced an entrainment of the applied oscillatory activity increases excitability in a similar way than anodal tDCS, when 1 mA intensity is used it is clearly cheaper due to the small and compact equipment; (2) it can be more easily combined with online cognitive projects; (3) it produces no acoustic noise and muscle twitching of cranial muscles and it causes much less or no perceptual skin sensations (Ambrus et al., tACS is only in its beginnings. A seemly indefinitely number of stimulation paradigms will have to be condensed to those with highest physiological relevance. As a prerequisite, this requires a clearer picture of the neuronal mechanisms involved in tACS-induced entrainment. Knowledge of their dynamics over time would enable to formulate optimized protocols for future tACS studies.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Existing infection control(IC) programs in hospitals are not effective due to lack of coordinated educational modules for stake holders in health care environment. We highlight our experience on a new knowledge management program which aims to educate and update information regarding IC practices to all Health Care Practitioners (HCP).A working core group was formed comprising of Heads of all clinical departments and Microbiology. Three members of the core group initiated a program at departmental level to identify their needs & issues, and to find out areas of constraints & possible remedial measures. The self generated team (BLUE BOARD) interacted through electronic media. A common platform was created so that different units could revise existing policies considering guidelines & feasibility of implementing the revised ones. Achievements were assessed by indicators viz., infection rate and antimicrobial usage. Data was analysed by simple descriptive statistics.They met at regular intervals & utilized learning materials, guidelines & other scientific data from authorized resources. They contributed to revise the program based on feedback from members. This continuous on-going education cum training programme blended with computer technology, interactive discussions & role plays brought down infection rates (25%) and reduction in the use of antimicrobials (17%).\u201cBlue Board\u201d has streamlined IC practices, improved the attitude of HCPs. Based on the confidence gained, BLUE BOARD system extended their services to other hospitals within the locality, disseminated knowledge gained and maintained uniformity in IC practices.None declared."} +{"text": "Sir,Central venous (CV) catheterisation is a routine procedure in intensive care units (ICU), with an overall complication rate of 12%. Loss of a complete guide wire into the circulation is one of its rare and preventable complications.[Migration of a guide wire into the circulation can occur from any of the usual CV catheter insertion sites.\u20133 A compThe usual attributes for a guide wire loss are operator inexperience, inattention and inadequate supervision during catheterisation. In our p"} +{"text": "Electrophysiological recordings suggest that cortical circuits operate in a regime where the excitatory and inhibitory currents received by individual neurons are highly correlated in both time and stimulus selectivity. For such balanced input, neurons are activated by fluctuations in the input and tend to fire asynchronously and at irregular time intervals, a regime known as asynchronous irregular state.rand. The phase sequence as originally proposed by Donald Hebb [rc>prand while neurons in subsequent assemblies are connected with feed-forward probability pff>prand. Each assembly has a corresponding inhibitory subpopulation to which it is recurrently connected with probability prc , the network resembles a synfire chain: a feed-forward network with convergent-divergent connections between subsequent groups of neurons [However, transient synchronization and precise sequential firing has been observed during certain perceptual tasks or behavioral states. The neural mechanism behind these activity patterns has not yet been resolved. Here we address the problem by modeling a phase sequence embedded into a random recurrent network with sparse connectivity pald Hebb is a ser neurons .ff that is required for the propagation of activity (Figure In contrast to synfire chain models, the balanced recurrent network dramatically reduces the connection probability py Figure . Simulat"} +{"text": "Background: Although the effects of opioids on intracranial pressure (ICP) have long been a subject of controversy, they are frequently administered to patients with severe head trauma. We present a patient with an uncommon paradoxical response to opioids.Case Report: A patient with refractory intracranial hypertension after closed head injury was managed with standard medical therapy with only transient decreases in the ICP. Only after discontinuation of opiates did the ICP become manageable without metabolic suppression and rescue osmotic therapy, implicating opiates as the etiology of refractory intracranial hypertension in this patient.Conclusion: Clinicians should consider opioids as a contributing factor in malignant intracranial hypertension when findings on neuroimaging do not explain persistent and refractory intracranial hypertension. Although opioids frequently are administered to patients with severe head trauma, the effects of such drugs on intracranial pressure (ICP) are controversial using the Arctic Sun device as an adjuvant to osmotic therapy with pharmacological neuromuscular paralysis to control the shivering response. Several attempts at reducing the degree of metabolic suppression by slowly increasing the core body temperature and lightening the degree of neuromuscular paralysis and propofol requirement failed. She consistently required a temperature under 35\u00b0C and a dose of Propofol greater than 40\u2009mcg/kg/min to maintain control of her ICP. A repeat CT scan on hospital day 11 (Figure Morphine and fentanyl are the most commonly used opioids for analgesia in patients suffering head trauma (Shapiro et al., Another possible mechanism is a direct vasodilatory effect associated with opioid administration Wahl, . The facAn alternative mechanism which could induce increased ICP indirectly is opioid-induced histamine release by a vasodilatory effect and resultant increased CBF. Our patient consistently responded to osmotic agents but always briefly. This was likely due to a rheologic reduction in cerebral blood volume rather than to osmotic diuresis as she did not have evidence of significant cerebral edema on imaging. While this may explain the sudden increase in ICP following morphine administration in our patient, it does not fully explain the reduction in ICP after discontinuation of fentanyl infusion as fentanyl does not typically trigger histamine release (Flacke et al., Although the mechanism refractory intracranial hypertension in our patient is unclear, the sudden surge in ICP following morphine administration in addition to the resolution of refractory intracranial hypertension following discontinuation of fentanyl infusion strongly argues for a causal relationship. The mechanism of the increased ICP in our patient is unclear, however, it is reasonable to assume that it could be related to intrinsic properties of opioids such as direct cerebral vasodilation combined with some predisposition in our patient. Despite widespread use of opiates in patients with head injury, we encourage the clinician to consider opiates as a possible cause for refractory intracranial hypertension in patients at risk for decreased intracranial compliance, particularly if a sufficient explanation for persistent intracranial hypertension is lacking.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Dietary iodine status is routinely assessed by measuring urinary iodine excretion (UI). In most European countries iodine intake is maintained at WHO recommended levels by iodisation of table salt . ExceptiSubjects were not selected on a systematic basis but were a combination of available findings from different Irish populations studied over the years specified. Although most study groups were comprised of adult females, where these were not available, findings from female schoolchildren were assessed. Median values for UI in study populations ranged from approximately 50-140 \u00b5g/l. In the absence of iodised salt availability, milk and dairy products constitute a major iodine source but their content shows seasonal variation with a higher iodine content when cattle in winter housing are fed dietary supplements including iodine ,10. ThusIn the absence of iodine supplementation of table salt, dietary iodine intake is entirely opportunistic. Consumption of milk and dairy products obviously plays a part but this communication reports on the investigation of possible other modes of iodine intake aimed at establishing if living near the sea in a seaweed abundant environment, and therefore exposed to gaseous I2 ingestion by respiration, may confer advantages in terms of iodine intake. Also, as adequate iodine nutrition for the foetus depends not only on maternal iodine supply, but also on the ability of the placenta to successfully transport iodide to the foetal thyroid for use in thyroid hormone biosynthesis, it is proposed to report on placental uptake and possible storage of iodine as a means of maintaining adequate iodine intake in utero.Ireland has traditionally been regarded as an area of borderline iodine deficiency which might not be expected on an island where few live more than 200Km from the sea. However as has become apparent in recent times, availability of iodine in the diet is dependent on many factors, of which consumption of seafoods is but one. Seaweed is the major source of iodine and for this study we utilised for iodine intake studies measurements whose purpose was to determine the concentration of gaseous I2 released by seaweeds and its effect on cloud formation and changes in weather ,13. To dThe other area of study presented is the role of the placenta in iodide transport/storage. How is iodide transported into the placenta? Is iodide transport concentration dependent? Is such transport hormone dependent? Both fresh placental tissue obtained from women undergoing elective prelabour caesarean section at term and primary cultures of trophoblastic cells accumulate radioactive 125I which could be partially blocked by perchlorate inhibition [unpublished observations]. RNA was isolated from placental trophoblasts and real time RT-PCR performed using sodium iodide symporter (NIS) and pendrin (PDS) probes. Trophoblastic cells expressed both NIS and PDS. 125I uptake in primary cultures from placental tissues was enhanced by individual pregnancy related hormones, particularly hCG and oxytocin, with synergism between hormone combinations. These incremental responses were mirrored by increased expression of NIS but not PDS when measured by Real time PCR suggesting that the increased iodide uptake was solely due to increased NIS expression. Measurement of placental tissue iodine content (mean 40ng/g tissue), while not approaching thyroid levels , is significantly greater than that of other non-thyroidal tissues and appears to be directly proportional to iodine intake as determined by UI ,15.The above findings demonstrate that in the absence of an iodine supplementation through iodisation of salt or otherwise, the study population appears to be only borderline iodine deficient. Even this apparent deficiency depends on the applicability to the definition of iodine deficiency of the WHO recommended UI cut off point (Median UI 100 \u00b5g/l) as it has been suggested that using the Recommended Daily Allowance (RDA) of the Institute of Medicine (95 \u00b5g) would lower this cut off point from 100 \u00b5g/l to 63 \u00b5g/l .Investigating alternative pathways of iodine intake such as gaseous I2 ingestion or placental iodide transport in utero may help to explain maintenance of adequate iodine intake. However calculation of the potential exposure to gaseous I2 ingestion in children residing in a seaweed abundant coastal area is difficult to reconcile with the relatively high UI values observed. On the basis of measured atmospheric gaseous I2, extremely preferential I2 absorption would be required to make a significant contribution to daily iodine intake. Although living near the sea may not in itself be sufficient to maintain satisfactory iodine intake, findings in this study do not exclude the possibility of a significant role for iodine inhalation in influencing iodine status.Iodine (125I) uptake in primary cultures from placental tissues was enhanced by individual pregnancy related hormones, particularly hCG and oxytocin with synergism between hormone combinations. These incremental responses were mirrored by increased expression of placental NIS. As only up to 30% of 125I uptake by placental cells was blocked by perchlorate, it is likely that passive diffusion as well as active transport is involved. However iodide uptake does appear to involve placental storage as placental tissue had a significantly higher iodide content than other non-thyroid tissues. Also it has been demonstrated that maternal urinary iodine (UI) excretion in the immediate antenatal and early postpartum periods showed a precipitous fall in median values from 93 \u00b5g/l antenatally to 36 \u00b5g/l at delivery which could be possibly explained by placental loss .It therefore appears that iodide uptake and transport from other than conventional dietary sources may assist in maintaining normal thyroid function even dietary intake is apparently deficient."} +{"text": "The benefits of pulmonary rehabilitation (PR) programs in patients with chronic obstructive pulmonary disease (COPD) are well established today . PR progultidisciplinary Respiratory Medicine Ki-song Kim & coll [In the present issue of Mm & coll present It is well known that posture during spontaneous quiet breathing influences not only thoraco-abdominal kinematics but also gas exchange and the cardiovascular system. Hence, in recent years functional studies and respiratory rehabilitation procedures have taken posture into account as a strategic way to understand and treat cardiorespiratory disability.PR in the past was performed with good results for patients but often subjects were studied in seated position only, with spirometry measuring forced expiratory volume in 1\u2009s. In this way the evidence of cardiorespiratory function improvements after treatment was lacking. Moreover PR was considered like a flying bumble-bee (accordiAs an example of real life, very recently respirataO2 is frequently measured in blood samples taken from supine subjects, and compared to normal values obtained in seated subjects. Such comparisons are not valid because in the supine position the values of PaO2 tend to be lower than those in seated position, particularly in elderly subjects. The same applies to estimates of arterial oxygen saturation.Another example commonly present in real life measurement can be considered. In normal elderly subjects the arterial oxygen pressure is lower in supine than in sitting position, because closing capacity exceeds functional residual capacity with increased inhomogeneity of ventilation-to-perfusion due to airway opening and closure during quiet breathing . This siSo any attempt to carefully understand functional respiratory mechanisms and respiratory muscle involvement in the real life procedures during PR maneuvers is important, also for the purposes of clarifying if the addition of inspiratory muscle training to general exercise training results in greater benefits than exercise training alone in COPD patients."} +{"text": "We present two children with a diagnosis of upper limb arthrogryposis and report on findings about brachial plexus exploration and a nerve transfer procedure to reanimate elbow flexion. Although the etiology of arthrogryposis multiplex congenita remains unknown and multifactorial, it can be worthful to explore the brachial plexus in the affected upper limb and to perform selective motor nerve transfers on morphologically well developed but not sufficiently innervated target muscles, like the biceps brachialis, brachialis, deltoid and supra-/infraspinatus muscles. This strategy may reduce the necessity of later muscle transfers and improves the overall functional status of the affected limb(s). Arthrogryposis multiplex congenita (AMC) is a well known clinical entity of unknown, certainly multifactorial etiology ,2. AkineAMC is characterized by a variable functional impairment of upper and/or lower limbs due to muscular hypotrophy and imbalance and joint ankylosis since birth. The treatment is orthopaedic and surgical according to the severity. In upper limb impairment, some authors claim an early surgical correction by joint releases and muscle transfers, to reanimate essential motor functions like the elbow flexion . Little We present two clinical observations allowing insights in nerve variations and a possible strategy for an early functional improvement. Further studies and observations should strengthen the hypothesis of treatable proximal motor nerve alterations in some cases of upper limb AMC.Girl patient 1 was born as the first child of a mother with a known uterus malformation (septum) by caesarian section. Immediately after birth, hypotrophy of the partially paralyzed left upper limb was observed. Due to the lack of active shoulder and elbow movements in a medially rotated arm, the diagnosis of severe upper obstetric brachial plexus palsy was hypothesized and the child was presented at our consultation.As the palsy was severe and did not show any clinical improvement at three months, a surgical exploration of the left brachial plexus was performed when she was aged four\u00a0months Figure\u00a0 under thThe brachial plexus showed to be hypoplastic, with thin roots and trunks Figure\u00a0a and b. right upper arm to verify the presence of the biceps muscle and to try to functionally reanimate the elbow flexors by a fascicular ulnar nerve transfer [Boy patient 2 was born with typical AMC affecting all four limbs. No active elbow flexion was present at 15\u00a0months and we decided together with the parents to explore the transfer , targetiExploration was performed when he was 21 months old using an anterior approach of the upper arm, showing a good muscle bulk corresponding to the biceps brachii muscle and the presence of a rather thin musculocutaneous nerve, silent on direct electrical stimulation. We identified the motor branch directed to the biceps brachii muscle and performed a typical nerve transfer according to the technique described by Oberlin , using mThese two cases illustrate that upper limb AMC may be associated with brachial plexus root hypoplasia, like seen in traumatic partial root avulsions. All five roots in patient 1 were thin and did only partially and weakly respond to direct electrical stimulation. This pattern has been observed in children suffering from severe upper obstetric brachial plexus palsy after a breech delivery with proven partial or total root avulsions. True hypoplastic malformations or congenital abnormalities of the brachial plexus are not described in the literature; but are reported by surgeons with longlasting experience: Gilbert described 3 cases of brachial plexus malformation out of 1000 operated children .In our clinic, after these first two cases with specific upper limb involvement, presence of a good muscle mass with absent or poor motor innervation has since been verified in three other children Figure\u00a0. Only inSelective motor nerve transfers thus might be helpful in these children, if enough muscle mass is present at the shoulder or arm level and if dispensable motor nerve donors are available, without compromising the existing and sometimes weaker than normal motor functions. Obviously, the challenge in patient 2 was to avoid downgrading the overall good hand function, which did not appear on global hand function assessment postoperatively.Nerve transfers prior to muscle transfers could change the prognosis and functional outcome in selected AMC children, as morphologically developed target muscles even with poor motor innervation could be salvaged and functionally upgraded. Muscle transfer options still remain possible, even at an early moment.Written informed consent was obtained from the patients\u2019 parents for publication of both case reports and any accompanying images and videos. A copy of the written consent for each case report is available for review by the editorial office.The author declares he has no competing interests.Video. Patient 2 right upper limb function after surgery.Click here for file"} +{"text": "Many lower extremity, pelvic and acetabular fractures require traction as first aid management prior to definitive fixation. While skin traction and Thomas splints are generally available, weights to provide countertraction are often missing or in parts of the hospital remote to the emergency department. A sharps bin filled two-thirds with tap water and tied via its bucket handle to skin traction provides"} +{"text": "Sepsis remains a major and increasing healthcare problem with a mortality exceeding 25%. The early detection of infection is important in treating sepsis. Nucleic acid amplification methods have the potential to improve the timeliness, sensitivity, and accuracy of the tests used to detect respiratory pathogens. We used a quantitative real-time PCR (rt-PCR) to detect the enteric bacterial counts in blood from patients in the emergency room.EDTA samples were collected from patients with systemic inflammatory response syndrome (SIRS) presenting to the emergency room after obtaining informed consent. Enteric bacterial loads in blood samples were assayed by rt-PCR to quantitate the bacterial 23S rDNA and EB rDNA loads. Descriptive and clinical data were collected from the medical records and compared with 23S and EB rDNA results.From January 2011 to April 2011, 39 patients were enrolled in the study. There was no correlation between serum lactate and enteric bacterial load in patients with SIRS. However, in a subgroup comprising patients presenting with respiratory distress and abnormal blood white cell count, the enteric bacterial rDNA load was higher and showed correlation with serum lactate level. The serum enteric bacterial rDNA loads were significantly higher in patients with positive cultures and in patients presenting with higher serum lactate. Correlations between serum lactate and enteric bacterial rDNA load were also significant in the patients with positive culture results.The quantitative assay for enteric bacterial rDNA could be a useful tool to detect early enteric bacterial translocation in patients presenting to the emergency room with elevated serum lactate level or with respiratory distress and abnormal white blood cell counts."} +{"text": "Shear wave elastography is a new ultrasound technique which shows promise in the differential diagnosis of solid breast masses. This study compares the mean tumour stiffness with mammographic findings and mammographic density in women with invasive breast cancer.Mammographic morphological features and BIRADS density assessment of the contralateral breast were documented in 96 consecutive patients with operable invasive breast cancer who had undergone shear wave elastography. Mammographic assessment was performed blinded to the elastography and pathological findings. Mean stiffness values of individual tumours were classified as either above or below the average mean stiffness of the cancers. Chi-squared tests for trend were used to compare mean stiffness with mammographic features and density.There was no statistically significant difference in the mean stiffness of tumours according to their mammographic features. Mean tumoural stiffness increased with breast density , with a statistically significant trend of stiffness being above average for a cancer in a denser breast. No statistically significant correlation was shown between tumour size or grade (factors known to be associated with increased stiffness) and mammographic density.Tissue stiffness of breast cancers is greater in women with mammographically denser breasts. Assuming elastographic stiffness reflects characteristics of stroma and tumour, our findings suggest that tumour-stroma interactions may vary with mammographic density."} +{"text": "Cases of percutaneous transluminal renal angioplasty for renal artery stenosis are increasing. However, percutaneous transluminal renal angioplasty with stenting for stenotic venous bypass grafts has never been reported. Herein, the authors describe two cases of percutaneous transluminal renal angioplasty with stenting for a stenotic venous bypass graft. The patients in both cases had undergone bypass grafting using autologous saphenous veins, which were anastomosed directly to their abdominal aortas. We successfully conducted percutaneous transluminal renal angioplasty with stenting. One of the keys for technical success is an appropriate selection of guiding catheter compatible with postoperative nonanatomical vasculature, and the other is relatively high pressure dilation for venous stenosis. Percutaneous transluminal renal angioplasty (PTRA) was first described in 1978 for stenotic venous bypass grafts is generally conducted as one of the way of salvage which shape of the tip was relatively straight (Multipurpose D). Two sites of stenosis were detected at the left renal bypass graft Figure\u00a0a. We pasThe second case is very similar with the first case mentioned above. A 59-year-old man was referred to our department for PTRA. He underwent right nephrectomy and patch plasty of left renal artery using an autologous saphenous vein for fibromuscular dysplasia and induced right renal complete dysfunction in his twenties. Around 30\u00a0years after the operation, a left renal artery aneurysm was detected. Left renal artery bypass grafting was performed using autologous saphenous vein grafts which shape of the tip was straight. Stenosis was detected at the proximal region of the posterior branch of the left renal bypass graft Figure\u00a0a. We pasAlthough there are many reports about PCI for stenotic venous bypass grafts (Hernandez-Antolin et al. In each of our two cases, PTRA was performed for the stenotic venous bypass graft, which had been grafted for the treatment of renal artery aneurysm and was anastomosed directly to the abdominal aorta distal to the renal artery branches nonanatomically Figures\u00a0 and 3. ACompared with atherosclerotic arterial stenosis, venous stenosis requires high pressure dilation (Trerotola et al. Our report of PTRA for stenotic venous bypass grafts represents just two cases and follow-up period after PTRA is short. Therefore, long-term prognosis such as risk of restenosis or course of renal function is not clear. Protection from distal embolism is discussed in the field of PCI for stenotic venous bypass grafts, but was not preformed in our procedures. We should consider long-term follow-up and protection from distal embolism in the future.Our findings indicate that an appropriate selection of guiding catheter compatible with postoperative nonanatomical vasculature and relatively high pressure dilation for venous stenosis are the keys for technical success in PTRA with stenting for stenotic venous bypass grafts.Written informed consent was obtained from the patient for the publication of this report and any accompanying images."} +{"text": "SACS gene were subsequently identified in these families in keeping with a mutational founder event in a geographically isolated population.SACS mutations, the identification of ancillary features linked with these genetic defects could prove particularly useful in prioritising the most appropriate lines of investigations when confronted with a suspected case of ARSACS. Although prominent retinal hypermyelination is thought to be a characteristic manifestation of classical ARSACS among Qu\u00e9bec patients, this ophthalmological finding has only been described infrequently in patients from Europe, Asia and the Middle East.Autosomal-recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) was first described among French Canadian patients from Qu\u00e9bec presenting with a stereotypical triad of early-onset cerebellar ataxia, spastic paraplegia and peripheral neuropathy. Two recurring pathogenic mutations in the To more accurately define the nature of the retinal findings in ARSACS and its possible practical relevance as a screening tool in routine clinical practice, we carried out a comprehensive neuro-ophthalmological examination of five patients from the North of England with molecularly confirmed ARSACS .45 TopoNo patient had evidence of retinal hypermyelination. Retinal striations were observed around the optic discs in four patients and OCT measurements showed significant generalised peripapillary RNFL thickening (see online supplementary figures S1\u2013S4). Patient E had no fundus abnormalities, and the peripapillary RNFL thickness was within the normal range for healthy controls (see online supplementary figure S5).SACS mutations. Significant peripapillary RNFL thickening has also been previously reported in eight patients harbouring confirmed pathogenic SACS mutations with different OCT imaging platforms to the one used in our study.Our case series has provided convincing evidence that abnormal retinal thickening is a common, although not universal, feature among patients with ARSACS-related phenotypes secondary to pathogenic SPG4 mutations, which account for \u223c40% of all autosomal-dominant cases of hereditary spastic paraplegia.SACS genetic screening should be considered.The investigation of a large multicentre cohort of patients with ARSACS will be needed to determine whether the degree of RNFL thickening correlates with disease severity and progression or whether there are any specific genotype\u2013OCT correlations. The molecular genetic basis of inherited ataxia and spastic paraplegia syndromes is highly heterogeneous\u2014a situation that poses a number of diagnostic challenges in neurology clinics. In Friedreich's ataxia, which is the most common form of autosomal recessive ataxia, variable reduction in RNFL thickness has been reported among visually asymptomatic patients."} +{"text": "The ability to grow new capillaries, adapt their caliber to higher flow and develop collaterals from obstructed arteries is essential to maintain or restore tissue perfusion. Better knowledge of the molecular and cellular details of postnatal blood vessel growth and remodelling is necessary to develop improved vascular regenerative therapies.Despite the crucial roles of myeloid cells in immune defense and angiogenesis, we still lack a thorough understanding of their identity and functional regulation.ex vivo priming with Ang-1 improves the vasculogenic potential of peripheral blood (PB) stem cells and increases their engraftment and therapeutic angiogenesis response after transplantation in a mouse model of LI and Tie2 receptor expressing monocytes/macrophages (TEM) team up to promote reparative vascularization in limb ischemia in subendocardial medium\u2013sized vessels skews macrophages to M2 type, increases M2 macrophage engraftment and their release of arteriogenic factors in limb muscles, thus leading to enhanced VSMCs recruitment and growth and arterial collateralization under non-ischemic conditions in mice. Consequently, Phd2+/\u2212 mice are \u2018preconditioned\u2019 to better respond once LI is experimentally induced , which share enzymatic properties but differ by their expression profiles. PHD1 inhibition induces hypoxia tolerance in ischemic muscles by reprogramming their glucose metabolism from oxidative to more anaerobic ATP production, thus conferring protection of myofibers (Aragones et al, In conclusion, TEM, Ang-1 and PHDs should be regarded as important cellular and molecular targets able to promote therapeutic neovascularization in CLI and other ischemic diseases. Further investigations allowing translation to interventional clinical trials should be developed."} +{"text": "Ovarian cancer is the most deadly gynecological cancer. The high rate of mortality is due to the large tumor burden with extensive metastatic lesion of the abdominal cavity. Despite initial chemosensitivity and improved surgical procedures, abdominal recurrence remains an issue and results in patients' poor prognosis. Transcriptomic and genetic studies have revealed significant genome pathologies in the primary tumors and yielded important information regarding carcinogenesis. There are, however, few studies on genetic alterations and their consequences in peritoneal metastatic tumors when compared to their matched ovarian primary tumors. We used high-density SNP arrays to investigate copy number variations in matched primary and metastatic ovarian cancer from 9 patients. Here we show that copy number variations acquired by ovarian tumors are significantly different between matched primary and metastatic tumors and these are likely due to different functional requirements. We show that these copy number variations clearly differentially affect specific pathways including the JAK/STAT and cytokine signaling pathways. While many have shown complex involvement of cytokines in the ovarian cancer environment we provide evidence that ovarian tumors have specific copy number variation differences in many of these genes. Epithelial Ovarian carcinoma (EOC) is the sixth most common malignancy in woman and the leading cause of death from gynecological cancer in the world Efforts have been made to delineate gene expression signatures for prognostic predictions as well as chemotherapeutic responses The complex cytogenetic alterations of ovarian carcinoma and the lack of high-resolution technologies have hindered the identification of specific genes involved in the metastatic process. Using low-resolution platforms, wide-spread copy number changes of 7 amplicons in high-grade tumors were identified while a relatively flat and quiet chromosomal landscape was found in low-grade tumors Therefore we hypothesize that a prospective collection of homogenous primary and metastatic lesions from patients with advanced ovarian carcinoma would allow a comprehensive view of genetic modification and have the potential to define important pathways for the occurrence of peritoneal metastasis in serous papillary ovarian carcinoma.We identified 9 patients with matched ovarian and peritoneal metastatic tumors . All priWe used the Affymetrix SNP 6.0 chip to detect regions with significant copy number variations (CNV) with respect to either a HapMap control set or the matched primary tumors. For validation, we selected 14 regions for quantitative-PCR validation of peritoneal metastasis versus primary tumor copy number. The regions included 3 controls shown to not be within CNVs in the patient's studied here, and an additional 11 regions within 5We first compared genomic DNA from primary and metastatic lesions with a dataset of normal tissues provided by the HapMap project. This should yield cancer specific amplifications and deletions when compared to normal tissue . Only reWhile our data agrees well with previous ovarian CNV studies et al., in preparation) for genes differentially expressed between primary and metastatic tumors. Functional analysis of gene expression data also revealed enrichment of differentially expressed genes in the cytokine/receptor interaction pathway and in the JAK-STAT signaling pathway. These findings are not unexpected as the underlying CNVs likely affect gene expression.For corroboration of pathways identified in the CNV analysis, we searched gene expression data of the same samples were mapped to the JAK/STAT signaling pathway . It is cWe documented all cytokine/chemokine signaling pathway related genes in CNVs and observed trends specific to primary and metastatic tumors . SpecifiLikewise, large numbers of the CXC chemokine subfamily were amplified in primary but not metastatic lesions , howeveret al., in preparation) for genes contained within a CNV and that had significant gene expression differences between primary and metastatic tumors. Of 9 differentially expressed chemokine pathway genes, 6 had the expected gene expression trend based on the CNV. Specifically, CXCR6, CCR2, CCR4, IL2Ra were both deleted and had lower gene expression in the primary versus metastatic tumors. Conversely, CCL28 and VEGF-A were both amplified and had higher gene expression in the primary versus metastatic tumors.We further searched gene expression data of the same samples in all but one patient (OV08-2) suggesting this pathway is altered with high frequency. While we realize the number of cases is too small to draw firm conclusions, the relative homogeneity of ovarian cancer CNVs (our data as well as Bowtell group) might advocate for the possibility of only a few pathways being required in the change toward metastasis. Genome-wide CNV analysis on a greater number of patients will allow us to determine if indeed the pathways to metastasis are few and therefore consistent. Interestingly, patient OV08-2 was the only patient with refractory cancer in our cohort. Whether the complete absence of CC chemokine deletions in the primary tumor is related to this refractory state will require further investigation of a higher number of such patients. While the approach of using matched primary and metastatic lesions to study ovarian cancer genome-wide CNVs is unique in this study, a similar study in breast cancer has been reported It is clear from our study that metastatic tumors are different from their ovarian primary source. Microenvironment pressures as well as the requirement for migration may select for copy number variations in these different pathways. The most frequently differentiating pathway we observed among primary and metastatic tumors was in the cytokine family of genes. This finding was corroborated by similar trends in gene expression data. The frequent involvement of cytokines in immune response and migration in cancer makes this an interesting finding. Indeed the role of immune infiltration has been recently described in different tumors including colon cancer and ovarian cancer Recently the TCGA group published the results of their comprehensive analysis of 489 patients with high-grade serous ovarian adenocarcinoma Our study highlights the benefit and importance of performing paired analysis of primary tumors and their metastatic lesions in ovarian cancer. While comparison of primary and metastasis as groups provided insight into cancer development, the matched analysis allowed more specific detection of consistent differences. Indeed advanced disease allows access to not only the primary but also the different metastatic sites. It has been clearly demonstrated that the patients' prognosis relies on tumor residue; therefore it is critical to understand the biology of the metastatic lesions in order to design appropriate new therapeutic approaches. The results presented here should be a step in that direction.All the samples were collected in the department of Gynecologic Oncology at the institut Claudius Regaud . The project was reviewed and approved by the institution's Human research Ethics Committee. All patients included in the study gave informed written consent prior to surgery. 9 patients with advance Stage III or IV papillary serous ovarian adenocarcinoma were prospectively enrolled in this study at the time of primary surgery before any treatment was given. The patients had a biopsy of the primary lesion as well as a peritoneal metastasis outside of the pelvis. In order to ensure very little contamination by the stromal components the biopsies specifically took the tumoral nodules without the underlying peritoneal elements. All biopsies were immediately liquid nitrogen snap frozen. A representative haematoxylin and eosin stained section was assessed and samples with 80% epithelial cells and less than 20% of necrosis no greater than 20%.Gene lists from both the gene expression and copy number variation analysis were entered into DAVID Table S1Primers used for qPCR validation of CNVs. Primers including controls, their product coordinates on hg19 and their sequence are provided.(XLSX)Click here for additional data file.Table S2Quantitative-PCR validation results of CNVs. 11 regions in 5 genes were used to determine copy number in regions identified by arrays as deviating from 2 copies. Non-concordant results are highlighted in red.(XLSX)Click here for additional data file.Table S3Primary ovarian tumor copy number variation regions identified in 9 patients. Segments of variation including chromosomal location, which patients are amplified and deleted, and genes within the region are listed.(XLSX)Click here for additional data file.Table S4Peritoneal metastasis tumor copy number variation regions identified in 9 patients. Segments of variation including chromosomal location, which patients are amplified and deleted, and genes within the region are listed.(XLSX)Click here for additional data file.Table S5Genes identified in copy number variation regions both shared by primary and metastatic tumors and those specific to each tumor type. Both official gene symbols and Refseq IDs are provided. Comparisons include using a HapMap provided baseline or comparing the Peritoneal metastasis to the primary tumor baseline (Peri V Primary).(XLSX)Click here for additional data file.Table S6Cytokine-Cytokine Receptor gene Copy Number Variations. Cytokine/Receptor genes were noted for presence in CNVs for all tumor comparisons conducted. Amplifications are colored in red and deletions in blue. The CC subfamily is especially deleted in primary but not metastatic tumors.(XLSX)Click here for additional data file.Table S7Peritoneal metastasis tumor copy number variation regions when compared to matched primary ovarian tumors identified in 9 patients. Segments of variation including chromosomal location, which patients are amplified and deleted, and genes within the region are listed.(XLSX)Click here for additional data file."} +{"text": "Wrong level spine surgery dominates malpractice claims.Prior to skin incision, lateral fluoroscopy is performed with the patient prone. A radiopaque pointer is used to identify the level of the disc being approached and thisIn 2010 a retrospective study by Irace and Corona reported the use of a pre-incision wire marker inserted to the spinous process using radiography for patients undergoing microlumbar discectomies."} +{"text": "The purpose of this literature review is to look at the potential of cerebral oxygen monitoring in the intensive care setting and how this monitoring modality will impact our current practice.A PubMed literature search was conducted using the search items 'cerebral, oxygenation, and monitoring'. The search was limited to adults and the search items limited to the title or abstract. Articles selected were those that demonstrated a positive or negative benefit of cerebral oxygen monitoring on neurological outcome after surgery or intensive care.The search revealed a total of 449 possible articles when conducted in December 2010. This was narrowed down to 18 articles related to monitoring cerebral oxygen. Patient outcomes: cerebral oxygen monitoring and the aggressive treatment of cerebral hypoxia reduced mortality and improved long-term outcomes after traumatic brain injury and coronary artery bypass surgery. Near-infrared spectroscopic cerebral oxygen monitoring is capable of detecting ischaemic cerebral perfusion deficits and may be more sensitive than transcranial Doppler in assessing blood flow and detecting delayed ischaemic deficits in subarachnoid haemorrhage. Cerebral hypoxia can persist despite good cerebral perfusion and normal intracranial pressure. Cerebral oxygenation monitoring can prevent iatrogenically driven hyperoxia and hyperperfusion, and can detect cerebral hypoxia before drops in standard pulse oximetry monitoring.The authors believe evidence is gathering suggesting that cerebral oxygen monitoring may play an important role in neurointensive and adult intensive care centres. Cerebral hypoxia worsens long-term neurological outcomes, and this modality has potential to help reduce morbidity."} +{"text": "In this \"featured arrhythmia\" article we present a set of unusual intracardiac electrode tracings that were recorded in a patient with typical clockwise flutter but a very dilated right atrium. The potential mechanism underlying this phenomenon is discussed with reference to the current literature. The 12-lead electrocardiogram was consistent with typical isthmus-dependent counter-clockwise (CCW) flutter. Under fluoroscopic guidance in the LAO projection, a steerable decapole catheter was placed in the coronary sinus, an adjustable duodecapole catheter placed around the tricuspid valve annulus and an ablation catheter placed on the cavotricuspid isthmus at the 6 o'clock position during typical atrial flutter has been well documented . In thesPersistent conduction delay has been demonstrated in fixed anatomical conduction block, such as that observed in the canine right atrial crush model of atrial flutter or in in"} +{"text": "Biobanking represents a critical resource for personalized translational medicine discovery. It is a great challenge to establish a high quality and comprehensive tissue biobank for cancer research. Many hospitals and clinical centers have large archival existing formalin fixed paraffin embedded (FFPE) tissue depository in the pathology department along with rich history of clinical diagnosis and long term follow up information. This is an existing gold mine for biomarker discovery. Our laboratory has first systematically demonstrated that unlike coding mRNA, a class of non-coding RNA, microRNA (miRNA), is rather stable in FFPE specimens . This di"} +{"text": "Adipose tissue displays characteristics of an endocrine organ releasing a number of adipocyte\u2013specific factors known as adipocytokines.It has been recently suggested that adipocytokines may play a role in pathogenesis and progression of certain cancers, in particular in colorectal cancer. The aim of this study was to investigate the association between several blood adipocytokine levels and clinicopathological characteristics of colon cancer patients undergoing surgery. The study group comprised of 29 patients who underwent surgical resection for colon cancer at Emergency University Hospital Bucharest and 27 healthy volunteers. The serum levels of adipocytokines were measured using multianalyte xMap profiling technology (Luminex). Resistin levels were significantly higher in colon cancer patients while leptin serum levels were significantly lower as compared to controls. Leptin levels decreased gradually with tumor stage and aggressiveness. Taken together, these results of this study suggest that adipokines, in particular resistin and leptin may be involved in development and progression of colon cancer. In the past decade it has become increasingly clear that adipose tissue displays characteristics of an endocrine organ releasing a number of adipocyte\u2013pecific factors known as adipocytokines ,2. It haAlthough a strong association between the prevalence of colorectal adenomas and increased levels of proinflammatory cytokines such as IL\u20136 and TNF\u2013alpha has been documented there arThe aim of this study was to investigate the association between several blood adipocytokine levels and clinicopathological characteristics of colon cancer patients undergoing surgery. The study group comprised of 29 patients who underwent surgical resection for colon cancer at Emergency University Hospital Bucharest and 27 healthy volunteers. All subjects (patients and controls) provided informed consent prior to the collection and analysis of blood samples and the study was approved by local Ethics Committee.Blood samples were collected from colon cancer patients before and after surgery. Whole blood was allowed to clot for 1 hour at 37 degrees C and spun to serum (quick run). Serum was aliquoted and stored at \u201320 degrees C until analyzed. The following adipocykines were tested in serum samples: adiponectin, leptin, resistin and serpin/type\u20131 plasminogen activator inhibitor (PAI\u20131) using Human Obesity Multianalyte Profiling Base Kit and Fluorokine MAP Human microbeads for each of the above mentioned parameters following the protocol described in detail elsewhere . Plate rStatistical analysis was performed using R software . Serum lThe adipokines concentration were measured in patients' serum at two time points: 3\u20134 days before surgery (time point 1: \u2018preop\u2019), and 5\u20137 days after surgery (time point 2: \u2018postop\u2019). The concentration of adiponectin and PAI\u20131 in undiluted serum was above the upper limit of linearity of the test for both patients and controls. Therefore, we decided to examine comparatively only the concentration of resistin and leptin which did not exceed the measurable range of the test in undiluted sera of patients and controls. As can be seen in , serum lInterestingly, for both mediators, we noticed a statistically significant positive correlation between preoperative and postoperative values . Next, we looked for possible association between measured adipokines serum levels and clinicopathological features such as tumor stage, histological grade of differentiation or localization. There was no statistically significant difference in serum concentration of resistin or leptin between A+B vs. C+D Dukes' stage . As can bee seen, for both subgroups of patients, serum resistin levels were significantly higher as compared to controls (P<0.01 for Dukes' B1+B2 stages and P<0.001 for Dukes' C+D stages). Leptin serum concentration was significantly decreased than those in controls only in patients in more advanced stages (P<0.05 as compared to controls). Moreover, a significant negative correlation was present between leptin and T stage ; however no correlation was noticed between leptin concentration and Dukes' stage. Disease stage and resistin displayed no correlation. Although a trend toward higher values was noticed for both tested mediators in patients with well and moderately differentiated tumors versus poorly differentiated or mucinous colon adenocarcinoma, the differences did not reach the statistical significance . When coNo significant differences in adipokines serum concentration according to tumor localization were noticed (P>0.05). The aim of this study was to determine the correlation, if any, between clinicopathological features of patients with colon cancer undergoing surgery and the serum level of adipokines. We observed that circulating resistin levels were significantly increased in colon cancer patients as compared to controls. These results are in agreement with other studies which found higher levels of resistin in patients with colorectal ,11, breaIt has been suggested that high resistin levels are related to cancer associated chronic inflammation. Recent data indicate that stimulation of macrophages in vitro with endotoxin or proinflammatory cytokines leads to a marked increase in resistin production and vice versa, resistin strongly up\u2013regulate IL\u20136 and TNF\u2013alpha production . Thus reThe association between leptin levels and the risk of colorectal cancer or adenoma has remained controversial. Although it has been suggested that in men, leptin may be associated with risk of colorectal adenomas ,19 otherOur study has some limitations. We had no information regarding body weight changes in the patients and controls before the sampling, and thus it was not possible to determine whether the changes in adipokines levels in the patients was caused by obesity before the sampling. Notably, several studies made the observations that adipokines concentration was not correlated with anthropometric measures of adiposity or weight loss in both control subject and cancer patients ,11. Howe"} +{"text": "En bloc radical vulvectomy with bilateral inguinofemoral lymphadenectomy has now been replaced by radical wide excision and selective inguinal lymphadenectomy based on the stage and location of invasive vulvar cancer. Early stage lateral cancers can be effectively treated by radical wide excision and ipsilateral superficial inguinal lymphadenectomy. Lymph node mapping using perilesional injection of radiocolloid and blue dye may identify sentinel lymph nodes which can be removed, thereby avoiding the morbidity of full inguinal lymphadenectomy in selected patients with early stage disease. Although squamous cell carcinoma of the vulva is not common, it is occurring with increasing frequency in younger women, particularly in those exposed to human papilloma virus (HPV). With efforts at education, many patients are presenting with early stage disease which is amenable to surgery. This year, there will be approximately 3500 new cases of vulvar cancer in the United States, representing 5% of all gynecologic cancers . The surThe lymphatic drainage of the vulva has been studied extensively and described in numerous publications , 3. Gene1 or T2 vulvar cancers when a margin of at least 8\u2009mm normal skin was excised with the primary lesion. In contrast, patients with a margin of <8\u2009mm had a local recurrence rate of 22%. At present, it is recommended that a margin of at least 1\u2009cm of normal skin around the circumference of the primary vulvar lesion be included in the surgical specimen and that the underlying subcutaneous tissue be removed to the level of the perineal fascia. Inguinal lymphadenectomy is then performed through separate incisions.The Basset-Way operation which was the standard of care in the operative management of patients with vulvar cancer included the en bloc resection of the primary lesion and surrounding vulvar skin as well as the skin over both groins 4]. Alt. Alt4]. Sentinel lymph node excision is now being recommended in selected patients with early stage squamous cell carcinoma as a means to avoid the operative morbidity associated with inguinofemoral lymphadenectomy \u201310. It iA persisting concern about sentinel lymph node mapping in vulvar cancer is the frequency of groin recurrences in patients with negative sentinel lymph nodes. In a multi-institutional observational study, Van Der Zee and coworkers performeThere is a definite learning curve in the performance and interpretation of lymph node mapping, and it is recommended that a multidisciplinary group within each institution perform sentinel node mapping in at least 10\u201320 cases before it becomes an accepted procedure . If sentPatients with a positive sentinel node should undergo a full inguinofemoral lymphadenectomy followed by postoperative radiation therapy to the involved groin and pelvis. However, if the sentinel lymph nodes identified by mapping are histologically negative after review by an experienced multidisciplinary team, no further treatment is indicated. 1 or T2 unifocal vulvar cancers <4\u2009cm diameter with nonpalpable groin nodes [It should be emphasized that optimal candidates for sentinel lymph node mapping are patients who have lateral Tin nodes . This prin nodes . 1 vulvar cancers <1\u2009cm diameter with stromal invasion >5\u2009mm who underwent radical wide excision and superficial inguinal lymphadenectomy. Women with positive superficial inguinal nodes underwent deep inguinal lymphadenectomy and radiation, whereas patients with negative superficial inguinal nodes received no further treatment. There were no isolated groin recurrences noted during a follow-up period of 36 months. Importantly, only 1 patient died of recurrent cancer, and wound complications were observed in only 12% of patients. These authors concluded that radical wide excision of the primary lesion and superficial inguinal lymphadenectomy was the treatment of choice for most women with early stage vulvar cancer and no evidence of enlarged inguinal lymph nodes on clinical examination. A persisting concern of this approach has been a significant but low incidence of groin recurrence in patients with negative superficial inguinal lymph nodes at the time of lymphadenectomy. Two investigators have reported a 4\u20137% incidence of subsequent ipsilateral groin failure after negative primary superficial groin dissection [\u223c3 per groin). Therefore, these authors recommend superficial inguinal lymphadenectomy as the initial surgical approach in patients with lateral Stage I or Stage II vulvar cancers provided that an adequate number of superficial inguinal lymph nodes (8\u201310) are removed [1 and T2 vulvar cancers having >1\u2009mm stromal invasion and no clinical evidence of enlarged groin nodes.Many surgeons concerned about the accuracy of sentinel lymph mapping have elected to perform superficial inguinal lymphadenectomy as the treatment of choice in patients with early stage vulvar cancer, believing that the superficial inguinal nodes are themselves \u201csentinel nodes.\u201d Specifically, all inguinal lymph nodes above the cribriform fascia are removed en bloc . Approxissection , 22. Thissection the anatAnatomic studies have indicated that some superficial inguinal nodes may be located within the interstices of the cribriform fascia and could be missed by purely superficial inguinal lymphadenectomy . TherefoAt one time, patients with positive superficial or deep inguinal nodes were treated by pelvic lymphadenectomy. However, a prospective trial conducted by the Gynecologic Oncology Group showed that patients with inguinal lymph node metastasis at the time of groin dissection who were treated by postoperative inguinal and pelvic radiation had a significant survival advantage when compared to similar patients treated by pelvic lymphadenectomy . Patient1 or T2 vulvar cancers by radical vulvectomy (N = 60) or radical wide excision (N = 62) and bilateral inguinal lymphadenectomy. Twenty-six patients (21%) had ipsilateral inguinal lymph node metastases, but there were no cases of spread to the contralateral inguinal lymph nodes. Patients with positive inguinal nodes were treated by postoperative radiation therapy to the involved groin and pelvis. All patients in the study were followed periodically by clinical examinations 10\u2013195 months (mean 59 months) after treatment. Eighteen patients (15%) developed recurrent vulvar cancer\u201413 to the ipsilateral vulvar skin, 2 to the ipsilateral groin, and 3 to the lung. There were no recurrences to the contralateral vulvar skin or groin. Likewise, there was no difference in the local recurrence rate of patients treated by radical vulvectomy versus those treated by radical wide excision. The 5-year disease-free survival was 98% for patients with T1 lesions and 93% for patients with T2 lesions. These authors concluded that women with lateral T1 and T2 vulvar cancers could be treated effectively by radical wide excision and ipsilateral inguinal lymphadenectomy.Anatomic studies have confirmed that efferent lymphatics from the lateral vulva drain initially to the ipsilateral superficial inguinal lymph nodes , 27. LikThere is still some controversy concerning the performance of routine contralateral inguinofemoral lymphadenectomy in patients with lateral vulvar cancers and positive ipsilateral inguinal nodes. Isolated reports have indicated that contralateral inguinal lymph node metastases can occur in these patients. However, the frequency of contralateral spread in these patients is extremely low, and the risk of contralateral inguinal lymphadenectomy may outweigh its benefits, particularly if only a small number of ipsilateral nodes are positive. All vulvar cancers located within 1\u2009cm of midline structures have the potential to spread to both groins, and should be treated by radical wide excision and bilateral inguinofemoral lymphadenectomy. The Bassett-Way operation, which emphasized en bloc resection of the vulva and both groins, has been replaced by radical wide excision and selective inguinal lymphadenectomy through separate groin incisions. The specific type of inguinal lymphadenectomy indicated depends on the stage and location of each cancer. Early stage lateral vulvar cancers can be treated safely by radical wide excision and ipsilateral superficial inguinal lymphadenectomy, whereas central vulvar cancers require bilateral inguinal lymphadenectomy. Deep inguinal lymphadenectomy, which involves surgical removal of the deep femoral nodes, is indicated in patients with centrally located early stage vulvar cancers, advanced stage vulvar cancers, and in patients with positive superficial inguinal lymph nodes. The deep femoral lymph nodes are always located medial to the femoral vein and can be removed without incising the deep fascia or dissecting the femoral vessels. Sentinel lymph node mapping should be offered to select patients with early stage lateral vulvar cancers as a means to avoid the postoperative morbidity associated with full inguinofemoral lymphadenectomy."} +{"text": "A cursory review of the current socket preservation literatures well depicts the necessity of further esthetic considerations through the corrective procedures of the alveolar ridge upon and post extraction. A new technique has been described here is a rotational pedicle combined epithelialized and connective tissue graft (RPC graft) adjunct with immediate guided tissue regeneration (GBR) procedure.We reviewed this technique through a case report and discuss it\u2019s benefit in compare to other socket preservation procedures.The main advantages of RPC graft would be summarized as follows: stable primary closure during bone remodeling, saving or crating sufficient vestibular depth, making adequate keratinized gingiva on the buccal surface, and being esthetically pleasant. Several studies have concerned the morphological alterations occurred in alveolar process as a consequence of tooth extraction, both vertically and in the width of the residual bone ,2. The rOn the other hand, various techniques have been discussed to achieve proper soft tissue closure at immediate implant sites. Pedicle flap technique and coronally displaced flap are two of the most important relevant corrective approaches . Also, tIt seems that if graft is pedicle and self-supplied with sufficient blood, a significantly higher survival rate would be expected. Then, it is necessary to seek a new soft tissue graft approach which will meet both keratinized tissue augmentation and blood supply demands. A review of the current socket preservation literatures depicts the necessity of further esthetic considerations through the corrective procedures of the alveolar ridge upon and post extraction.A 35\u2009years old female with congenital missing right maxillary first premolar and tilting of the adjacent teeth towards the edentulous area was referred to our clinic. The right maxillary second premolar was crown-less and was endodontically hopeless. Based on the wax up analysis, the best potential implant site was determined to be mesial to the remained root Figure -a. It waThe remained root was extracted with minor violation to the structures in vicinity (atraumatic conservative approach). A mucoperiosteal flap was raised on the buccal side of the edentulous site Figure -b. No reThe patient was an implant supported prosthesis candidate. Reevaluation six months later showed sufficient bone volume and soft tissue maturation ready for implant placement. Bone width in the implantation site was 6\u2009mm and the height was 15\u2009mm. A punch whole was made in the potential implant site and fixture was placed without flap reflection Figure -a and b.Socket preservation techniques have mostly concerned hard tissue augmentation and prevention of ridge collapse. Van der Weijden et al had a review of data about alveolar bone dimensional changes of extraction sockets in human. They calculated the data from 12 publications and reported an average 3.87\u2009mm reduction in width and 1.67\u2009mm bone loss in height after normal remodeling . Soft tiadvantages of above mentioned technique could be summarized as:The main \u00b7 The epithelialized part of the pedicle graft covers the socket orifice and the de-epithelialized part is placed under the buccal flap. This not only ensures the proper closure of the socket, but also enhances the contour on the buccal side and contributes to the blood supply of the graft site. Underlying palatal graft will enhance the quality of the covering attached gingiva.\u00b7 The healing process then occurs through first intention on the socket orifice. It must be mentioned that one early clinical concern in all kinds of socket preservation procedures was wound premature opening .\u00b7 There is no need for releasing incisions since buccal flap is aimed to form a pocket.\u00b7 There is no need to coronally position the buccal flap. The mucogingival line level is then preserved as normal and buccal flap may even be positioned apically in an attempt to correct inadequate vestibular depth.\u00b7 Placing the non-epithelialized part of the pedicle graft under the buccal pocket beyond the mucogingival junction will make amends for the probable future buccal collapse.\u00b7 Due to adequate blood supply within the pedicle graft, socket inclusions will be nourished not only through socket walls but also from the flap. This will increase the chance of graft survival and enhance the future osseointegration in the potential implant site.\u00b7 This technique will not only provide sufficient functional masticatory mucosa but also will provide maximum buccal soft tissue augmentation Figure .\u00b7 Future buccal depression results from the tissue collapse (which is an inevitable consequence of remodeling) will be prevented due to the overbuilding of soft tissue in the area.\u00b7 This technique along with the harvest of a thick graft in the mesial and distal donor pedicle, papillae generation could be achieved to some extent in patients where gingival papillae have become flattened.\u00b7 This technique is associated with esthetically pleasant outcomes since the connective tissue graft have the advantage of color matching to the overlying tissue Figure .\u00b7 Adequately extended incisions along with the application of cut-back incisions will allow free rotation of the flap and its passive placement on the expected area. There is then no need to suture the graft in place. When needed, the flap end may be sutured using resorbable material to the underlying connective tissue.\u00b7 This technique usually does not need a coronal repositioning of the buccal flap and thus no mucogingival junction displacements would be expected . The premodifications of this technique are as follows:The possible \u00b7 Full thickness flap is suggested in cases where buccal marginal bone needs overbuilding and partial thickness flap is recommended where adequate intact (minimum of 2\u2009mm) buccal table is present post-extraction .\u00b7 In the cases where the buccal table needs overbuilding, the socket width will be covered with a resorbable collagen membrane after bone graft is placed. However, if the buccal plate is intact, there is no need for buccal table overbuilding or membrane application.\u00b7 The socket may be filled with resorbable bone substitutes like DFDBA or semi-resorbable bone substitutes like nano-bone.\u00b7 There is then no need to suture the graft in place. When needed, the flap end may be sutured using resorbable material to the underlying connective tissue.Based on the type of bone substitute used, a 3 to 6\u2009month bone healing period should be considered prior to implantation . Also, tSocket preservation procedures used widely to manage the tissue dimensional alterations after tooth removal. These techniques considered as predictable procedures to reduce the need for extensive bone augmentation operations in implant dentistry. There are different socket/ridge preservation techniques with different outcomes. Also, there is no evidence to support the superiority of one specific technique over another. Our recommended technique named RPC graft would be useful in high esthetic demand cases due to its ability to reconstruct hard and soft tissue, simultaneously.The authors declare that they have no competing interests."} +{"text": "Many mental health interventions constitute complex interventions since they are characterised by multiple components which when given in combination are thought to improve outcome as well as the components changing distal clinical outcome indirectly by inducing change in intermediate process variables (mediation). Ideally the evaluation of a complex intervention should be both pragmatic (assess effectiveness) as well as explanatory (measure the efficacy of the intervention components and separate their direct and indirect effects). Ordinary least squares (OLS) estimates of parameters of interest in explanatory analyses will suffer bias despite random treatment allocation when covariates are endogenous. In mental health trials such endogeneity is likely to arise as the result of non-compliance with randomised treatments, hidden confounding or measurement error in the treatment components received or process variables.In this talk I discuss ways of designing a complex intervention trial so that all causal parameters of interest can be estimated consistently using instrumental variable (IV) methods.Pearl\u2019s graphical checks of identifiability are applied to establish the most general causal model for mental health complex intervention trials under which parameters of interest remain identified.Having determined this model, experimental actions including the generation of relevant IVs, that can ensure model assumptions hold, are reviewed.Monte Carlo simulations are carried out to compare the performance of na\u00efve OLS estimators with that of IV estimators.Allowing for non-compliance with randomised treatment offers, hidden confounding or measurement errors requires independent randomisation of constituent treatment components and the availability of further instruments for hypothesised process variables. A number of avenues for generating the latter exist.Simulations confirm that the biases suffered by OLS estimators are corrected by IV methods. The variance inflation of IV treatment component effect estimators is acceptable in standard sized mental health trials. However, instruments for process variables may be less strong raising the possibility that the variance inflation of mediation effect estimators renders them impractical for use.Investigators planning to carry out explanatory analyses of complex interventions should consider the generation of relevant IVs at the design stage.The unbiased estimation of effects of the treatment components actually received (efficacy) is feasible under non-compliance or treatment misclassification.The generation of strong IVs for process variables may be more problematic. In mental health research mediation analyses allowing for hidden confounding and measurement error may only be feasible for large trials or after combining trials."} +{"text": "A prominent feature in the ascending auditory pathway is that of broadband onset inhibition, and there are many hypotheses regarding its functional role -4. Here,Onset inhibition in the auditory brainstem can improve harmonic signal strength of speech sounds by suppressing the inharmonic onset noise associated with glottal pulses."} +{"text": "Since far genes , a singlar genes . In thisde novo CNVs identified in patients with developmental disorders from the DatabasE of Chromosomal Imbalances and Phenotype in Humans using Ensemble Resources (DECIPHER). Employing a novel integrated functional linkage network, we examined clustering and importance of the genes affected by these de novo CNVs. A fast neighbor joining clustering algorithm was used to identify gene groups within each CNV defined as those linked by the top 1 % of shortest paths within the functional linkage network. To ensure functional clusters were not simply the result of tandem duplications, we collapsed paralogous genes into a single copy within the genome. Significant enrichments in clustering and central network position were used to build a predictive model able to scan the genome and identify similar regions whose copy number change may also predispose to disease.We obtained a set of 626 de novo CNVs were significantly enriched for large functional clusters with low within-cluster similarity compared with gene-number matched randomizations. Functional clusters were even more significantly enriched when paralogous genes were collapsed. Clusters were present in 357 of 626 CNVs, with an average size of four genes. In addition all measures of network centrality were significantly high, with average maximum betweenness (bottlenecks) the most significant. Bottleneck genes tended to be haplo-insufficient and highly pleiotropic when knocked out in mice. Functional clusters frequently contained bottleneck genes and these regions were frequently affected by CNVs in more than one patient.DECIPHER de novo CNVs identify putative epistatic hotspots, which are clusters of functionally related genes whose disruptions are associated with developmental disorders. In addition, these hotspots are enriched in bottleneck genes that may play a role in the diversity of phenotypes observed for these patients.DECIPHER"} +{"text": "Axonal injury and swelling is a common outcome of brain trauma and/or concussion. Recent experimentally observed axonal deformations induce irregularities in the axonal morphology and geometry ,2. Such We develop a computational framework to evaluate and distinguish between axonal injuries that lead to geometrical enlargements responsible for producing minor changes in propagation from those that result in critical phenomenon such as reflection or blockage of the original traveling spike train. We use a few geometrical parameters to model a prototypical axon enlargement and explore numerically the parameter space characterizing all possible propagation regimes and dynamics in an unmylienated action potential model. Figure Contrary to earlier notions that large diameter increases mostly lead to blocking, we demonstrate transmission is stable provided the geometrical changes occur in a slow (adiabatic) manner. As a consequence, swellings of lesser volumes but with abrupt increase in diameter can be more dangerous than those of larger volumes with adiabatic changes in geometry. Our method also identifies a narrow range of parameters leading to a reflection regime. The distinction between these three regimes can be evaluated by a simple analytical function of the geometrical parameters inferred through numerical simulations. Additionally, the effect on information encoding can be evaluated in such injured axons by performing a spike metric analysis . Given t"} +{"text": "Risk factors for breast cancer can be allocated to one of four groups:1 Family History/genetic2 Reproductive/hormonal3 Proliferative benign breast disease4 Mammographic densityThese four factors have now been thoroughly studied and accurate quantitative estimates for the risk are now available for many of them. The most useful summary comes from the Oxford collaboration, which has now produced a series of papers estimating risk for individual factors. Less is known about the possible interaction between these factors and virtually nothing is known about how different factors influence the risk of different types of breast cancer e.g. oestrogen receptor positive versus negative tumours. Risk factors appear to be largely independent and this facilitates building a model to predict risk for individuals. Previous models have focused on either non-genetic factors where imHere we briefly review the main risk factors for breast cancer and describe our own model and a co"} +{"text": "The Global Malaria Action Plan places scaled-up effective malaria control as a necessary prerequisite for malaria elimination. Effective and sustained malaria control depends on vastly strengthened health systems in malaria endemic countries. However definitions of health system strengthening, and the means to achieve it are still lacking. Emerging approaches of system-wide thinking that applies systems thinking tools in implementation science can open new possibilities to accelerate system strengthening with a particular focus on the systems effectiveness of malaria surveillance and control interventions. This presentation will discuss how tipping point revolutions in health systems could be harnessed to strengthen health systems to become malaria elimination ready. These include social network analysis to better manage stakeholder partnerships that bring malaria control and implementation research closer together; new concepts in governance of health systems to improve both system design and systems management; new approaches to diagnose system effectiveness bottlenecks and target system-level interventions; and modern mHealth strategies to improve procurement and supply chains as well as surveillance as an intervention in real time. For rapid progress across these dimensions, closer and better managed partnership among health system managers, health systems researchers and implementation scientists is essential."} +{"text": "Periodization of resistance training or planned changes in training volume and intensity are used to maximize strength and fitness gains. Several types of periodized resistance training plans have been developed. The most common of these plans is linear also termed classic or strength/power periodization and nonlinear periodization. The biggest difference between these two types of training plans is with nonlinear periodization changes in training volume and intensity are made more frequently. The most common type of nonlinear periodization is daily nonlinear periodization where substantial changes in training intensity and volume are made from one training session to the next training session. Periodized resistance training does result in greater strength gains than non-periodized programs. While both linear and nonlinear periodization plans result in significant strength and fitness gains some research indicates greater strength gains with daily nonlinear periodization. Training periodization has long been used by athletes and coaches in an attempt to maximize fitness gains and physical performance. More recently fitness enthusiasts and personal trainers have also begun to utilize periodized training plans. Although all types of training can be periodized here only periodization of resistance or weight training will be considered. Periodization of resistance training refers to planned changes in the acute training program variables of exercise order, exercise choice, number of sets, number of repetitions per set, rest periods between sets and exercises, training intensity, training volume, and number of training sessions per day in an attempt to bring about continued and optimal fitness gains. The main goals of periodized training are optimizing training adaptations during both short periods of time and long periods of time . Some periodized plans also have as a goal to peak physical performance at a particular point in time, such as for a major competition. Another goal of periodized resistance training is to avoid training plateaus.A large number of studies and a meta-analysis indicate periodized resistance training programs result in greater strength increases in both sexes, untrained individuals and trained individuals compared to non-varied programs or performing the same number of sets and repetitions per set for the entire training program . ChangesLong-term programs of classic, linear or traditional strength/power periodization programs begin with high volume-low high-intensity training and progress towards low-volume high-intensity training. Normally the entire training plan takes several months to complete. If training is continued the entire plan beginning with high-volume low-intensity training is repeated. Typically with linear periodization different training phases of different volumes and intensities last approximately 4\u20136 weeks. Thus 4\u20136 weeks of training may be spent predominantly training with a particular training zone, such as 8\u201310 or 1\u20133 repetitions per set. Different training phases in many linear plans have a specific training goal and name, such as hypertrophy, strength, strength/power and power. One goal of many linear periodization programs is to peak or maximize strength/power after the last training phase typically termed a power phase.While with nonlinear periodization training intensity and volume are changed much more frequently . In factNo matter which type of periodized resistance training is used other changes than the number of repetitions per set can also be made. Number of sets per exercise can be increased or decreased to change total training volume. Different exercises can be performed as the training progresses. Typically this change involves removing single joint exercises while keeping multi-joint exercises as training progresses. In many training plans other changes in the type of resistance exercises are also made, such as variations of the Olympic lifts, are emphasized when a training phase emphasizes power. In many periodized plans changes in intensity and volume are utilized mainly for multi-joint or multi-muscle group exercises. Thus in many periodized plant single joint or single muscle group exercises are not periodized. Although all of these changes are used in periodized plans research has predominantly examined the effect of different numbers of repetitions per set, which is a change in intensity and volume, when comparing periodized training plans.Most research comparisons of periodization models are between daily nonlinear and linear periodization with training durations of between 9\u201315 weeks. Some comparisons show significantly greater strength gains with daily nonlinear peridoization in college-age males . While oA limited number of studies indicate motor performance and power increases are not significantly different between daily nonlinear and linear periodization . AdditioComparisons of weekly and biweekly nonlinear to linear periodization also show little difference in fitness gains between periodization plans. For example, a comparison of biweekly nonlinear periodization, linear periodization and a non-varied training program (3 sets of 6 repetitions) has been performed. The results showed all training types significantly increased maximal strength, vertical jump and fat-free mass, with no significant differences between training types . CollectFlexible nonlinear periodization is a relatively new type of periodization. Flexible nonlinear periodization uses the nonlinear training model but allows changes in training based upon the readiness of a trainee to perform a specific training zone. The decision to change the planned training zone for a specific training session is made using several pieces of information. A test, such as a maximal vertical jump, standing long jump or medicine ball throw, can be performed immediately prior to a training session to help determine the readiness of a trainee to perform a specific training zone. The beginning sets of the first few exercises in a training session can also be monitored to help determine the physical readiness of a trainee to perform a specific training session.For example, if a standing long jump is performed immediately prior to a training session and the trainee cannot achieve at least 90% of their previous maximal standing long jump, the trainee may be fatigued. Similarly fatigue is indicated if an individual could previously perform 10 repetitions of an exercise with a specific resistance and at the start of a training session can only perform seven repetitions with this resistance. The fatigue or other physiological factor, such as delayed onset muscle soreness, could be due to previous resistance training sessions or other types of training being performed as part of the total training program. Psychological stress due to work or any other factor could also prevent performing up to previously demonstrated abilities. No matter what the reason in this example if a moderate-intensity moderate-volume (4 sets of 10\u201312 repetitions) training zone was scheduled to be performed the training zone would be changed to a different zone (3 sets of 12\u201315 repetitions).It is also possible to change from a low-intensity high-volume training zone to a higher intensity and lower volume zone. For example, a standing long jump is performed and 100% of the best standing long jump is achieved or sets of 8\u201310 repetitions are planned, but the trainee achieves 12 repetitions per set in the first exercise of a training session. In this case rather than continuing with a training zone of 8\u201310 repetitions a higher intensity zone (4\u20136 repetitions) may be performed because fatigue is not indicated and it appears the trainee is ready to train at a high intensity. Flexible daily nonlinear periodization and training zone changes have been previously extensively discussed .To date, little research has been performed concerning flexible nonlinear periodization. A variation of this type of periodization has been employed to maintain and increase physiological markers in collegiate Division I soccer players throughout a 16-week season . ResistaA comparison of a flexible daily nonlinear to nonlinear periodization indicates flexible nonlinear periodization offers some advantages . StudentPre- to post-training one repetition maximal (1 RM) chest press ability and maximal standing long jump ability significantly increased with both training plans with no significant difference shown between plans. However, 1 RM leg press ability increased significantly more with the flexible nonlinear program. These results indicate the flexible nonlinear periodization plan did not result in a significant greater increase in upper body strength, but did significantly increase lower body strength to a significantly greater degree. Although little research has been performed on flexible nonlinear periodization this type of training plan appears promising.Periodized resistance training does result in greater fitness increases than non-periodized programs. Nonlinear periodization results in similar fitness gains or possibly even greater fitness gains than linear periodization. While flexible nonlinear periodization has received little study by the sports science community it appears to be a promising periodized type of training. Thus coaches and fitness enthusiasts can use nonlinear and flexible nonlinear periodization plans with confidence that these types of periodization will result in significant fitness gains."} +{"text": "In patients with HIV and hepatitis B or C co-infection initiation of early HAART therapy is clearly recommended by the international guidelines reflecting the overall survival benefit with regard not only to HIV but also clinical endpoints of liver disease in the co-infected patient population. In patients with hepatitis C co-infection administration of successful HAART is associated with less inflammation in liver biopsies and hence, over time less fibrosis generation. Obviously, drugs which are dually active against HIV and HBV do not only prevent HIV disease progression, but also decrease the progression of chronic hepatitis B related liver disease. Nevertheless, recent reports on the emergence of non cirrhotic portal hypertension as a rare cause of upper gastrointestinal bleeding in HIV patients most likely following prolonged didanosin exposure have raised questions with regard to potential drug toxicity as sequelae of HAART causing possible damage to the liver and the portal vascular system. Under consideration of metabolic changes including dislipidemia and insulin resistance caused by various components of different drug classes the question also remains in how far risk for fatty liver disease may rise after decades of HAART administration. Therefore, close surveillance programs and monitoring are required to answer the long term safety questions around HAART administration and liver safety. Nevertheless, the clear demonstration of overall increased survival under HAART in patients with concomitant hepatitis and HIV clearly underlines that the benefits of HAART outweighs potential toxicity risks. Also with the knowledge obtained around mitochondrial toxicity observed under D-nucleoside therapy treatment algorithms for co-infected patients now specifically refrain from using these drugs in particular. With the advent of new drug classes with low hepatotoxicity profiles new combinations arise which potentially improved liver safety profile over time. But again, long term data will be needed to eventually decide what are the best HIV treatment options in patients with concomitant liver disease."} +{"text": "Most studies exploring cancer progression have focused on the influence of individual genes, and few efforts have investigated the effects of interactions between genes within the genome. Our hypothesis is that cancer cells thrive by exploiting combinations of genes, in fact by exploiting networks of genes that both protect the cell against destruction and enhance its survival. We believe that these networks involve genes that tend to be coordinated in their copy number alterations, even when they are located at a distance in the genome. Radiation hybrid (RH) cells have a random assortment of genes as triploid rather than diploid. Our recent work studying genetic networks in libraries of RH cells has elucidated key survival-enhancing interactions with high specificity . BecauseOur work with the RH data provided the rationale for an investigation of cancer survival networks, in particular for glioblastoma multiforme, a formidable brain cancer for which extensive datasets are available but few treatment options. We investigated correlated patterns of copy number alterations for distant genes in glioblastoma multiforme tumors using the same method we employed to construct the RH survival network. Public data were analyzed from 301 glioblastomas that had been assessed for copy number alterations using array comparative genomic hybridization .P = 3.7 \u00d7 10\u201331). We therefore exploited the high-resolution mapping of the RH data to obtain single-gene specificity in the glioblastoma network. The combined network features 5,439 genes and 13,846 interactions <5%) and suggests novel approaches to therapy for glioblastoma. For example, although the epidermal growth-factor receptor (EGFR) oncogene is frequently activated in glioblastoma, EGFR inhibitors have limited therapeutic efficacy [The glioblastoma and RH survival networks overlapped significantly (By elucidating a genetic survival network for glioblastoma, we gained insight into the mechanisms of proliferation of this cancer and opened up new avenues for therapeutic intervention."} +{"text": "For virtual screening and similarity searching numerous descriptors can be employed to represent molecular structures and properties. Concurrently these descriptors always create a chemical property space. Typically, we lack information on how these spaces are structured and organized due to their high dimensionality. We present a projection method that allows for the visualization of such property spaces of large databases while maintaining the high-dimensional spatial structures and neighbourhood behaviour Figure . The pro"} +{"text": "Modifier genes are an integral part of the genetic landscape in both humans and experimental organisms, but have been less well explored in mammals than other systems. A growing number of modifier genes in mouse models of disease nonetheless illustrate the potential for novel findings, while new technical advances promise many more to come. Modifier genes in mouse models include induced mutations and spontaneous or wild-derived variations captured in inbred strains. Identification of modifiers among wild-derived variants in particular should detect disease modifiers that have been shaped by selection and might therefore be compatible with high fitness and function. Here we review selected examples and argue that modifier genes derived from natural variation may provide a bias for nodes in genetic networks that have greater intrinsic plasticity and whose therapeutic manipulation may therefore be more resilient to side effects than conventional targets. Beaded and truncate wings in fruit flies and pigmentation in hooded rats. In each of these cases, the phenotype varied widely among offspring of parents with established genotypes. Mutant frequencies did not follow Mendelian ratios and\u2014more troubling still\u2014varied among derived lines carrying the same mutation. This led some workers to question whether genes were constant physical units or changed in properties during transmission truncate flies Beaded flies The concept of modifier genes originated with the solution to an early genetic mystery. As early workers tested Mendel's segregation ratios with observations on a wide range of traits and species, several cases of \u201cinconstant inheritance\u201d caused some to question the Mendelian basis of factors underlying these traits The term has been applied to several classes of genetic activities, some of which overlap. Genetic variations that alter the activity of a protein encoded by a second locus are one class. To the extent that the modifying effect is independent of allelic variation in the gene being modified, this usage is conceptually equivalent to any primary mutation whose phenotypic consequences include loss of interaction with its normal targets . Interactions between mutations that were each previously recognized by their independent phenotypes are another class. While these genetic interactions can be revelatory, one need not invoke the term \u201cmodifier\u201d to fully describe both individual and interaction effects, as each locus is identifiable without the other. Quantitative trait loci (QTL) are sometimes referred to as modifiers in the context of a major effect locus, which can be nearer the original meaning, depending on the structure of interactions in the QTL model. For the purposes of this review, we will use \u201cmodifier\u201d to mean a genetic variant that is best recognized by its ability to change the phenotypic outcome of an independent \u201cconditioning\u201d variant at another locus. Modifier genes in this usage have no obvious phenotype of their own prior to discovery and are effectively silent\u2014or at least quiet\u2014with respect to the phenotype under study, in the absence of the conditioning mutation.Early workers quickly appreciated that modifier genes are pervasive across experimental systems and organisms. Modifier genes need not be subtle and can have phenotypic effects as large as the initial conditioning mutation. These early observations contributed to the conceptualization of genetic pathways, prior to knowing the molecular identities of any components. This formal concept remains useful in analyzing genetic networks for sensitive nodes (genes) through which to manipulate mutant phenotypes in genetic disease or experimental biology.Modifier genes are also frequent in human disease and often invoked to explain divergent outcomes in genetic disorders with apparently equivalent cause. Among the best examples is cystic fibrosis (CF). CF patients homozygous for the \u0394508 allele present with a broad range of organ involvement and clinical severity, much of which is controlled by modifier genes Identification of modifier genes in mouse models offers an opportunity to understand forms of plasticity in mammalian genetic architectures that could be exploited as a preclinical knowledge base in designing therapeutic strategies The examples below argue that modifier genes are a field ripe for harvest, particularly with the recent arrival of several new community resources that improve experimental access to genetic variations among common strains. The situation is reminiscent of early days of positional cloning in many respects. Large numbers of loci have been reported and mapped , but fewMany of the genetic interactions known in mice come from direct tests of candidate interactions. Testing interactions first observed in other species can identify physiological context for gene pairs or networks that are conserved more deeply than the physiology or organ system for which they are most relevant to human disease. For observed binding partners, genetic interactions can test the biological relevance of likely physical contacts. Proposed interactions between mutations with similar phenotypes can also clarify points of convergence between previously separate pathways.Candidate interactions are often based on homology to interacting genes in other species, including components of developmental signaling pathways, homeotic regulators, and transcription factor cascades in development. Some of these interactions may confirm modifier genes in the strict sense of Prkdc, which encodes a DNA-activated protein kinase, as the sensitizing variant and asked whether this might be relevant to other mtDNA dysfunctions Mpv17, which encodes an inner mitochondrial membrane protein, is mutated in mtDNA depletion syndromes Prkdc potentiates mtDNA loss in Mpv17-mutant mice: double mutant mice (but neither single mutant) suffer mtDNA loss and other features of adriamycin-induced nephropathy without adriamycin exposure, providing parallel gene\u00d7gene and gene\u00d7environment models.Synthetic interactions between environmentally sensitizing alleles are another kind of candidate gene approach. For example, BALB/c and 129S1 mice are sensitive to adriamycin-induced nephropathy and show mitochondrial DNA (mtDNA) depletion in vulnerable organs after adriamycin treatment. Gharavi and colleagues identified a single amino acid substitution in the shared BALB/c and 129S1 allele of The many successes in modeling the phenotypic consequences of predicted candidate gene interactions in an intact mammal should encourage us to consider what value might be obtained from screens that are less constrained by prior predictions\u2014and therefore capable of identifying novel and unexpected interactions that might catalyze more rapid progress in the often complex genetic architectures relevant to disease.dilute suppressor (dsuMreg). This modifier arose spontaneously in a non-agouti, dilute stock, suppressing the coat color dilution but with no obvious phenotype of its own dsu similarly suppressed pigmentation phenotypes in mutations at five of 11 classical coat color loci tested MelanoregulinMvb1Nxf1, has been effective on a larger number.An early success in using modifier genes to understand a genetic network in mice came from the Sodium channel modifier 1 (Scnm1) locus was identified as a strain-dependent modifier of med-JScn8a, a mutation in a neuronal sodium channel gene responsible for a range of neurological phenotypes Scn8a mutations, the modifier is specific for the Jmed allele, an intronic single nucleotide variant that alters 5\u2032 splice site usage. Positional cloning of the modifier identified a novel gene whose protein plays a direct and previously unsuspected role in pre-mRNA splicing Scnm1 and med-JScn8a appears highly specific, as Scnm1 does not alter general RNA patterns in brain nor modify other tested mutations with similar defects Scnm1 allele is only found in C57 and C58 strains, but not in other strains with otherwise similar Scnm1 haplotypes, it is proposed to have arisen as a spontaneous mutation in a progenitor stock rather than as a wild population variant dsu and Scnm1 spontaneous modifiers should encourage further explorations of modifier genes.Spontaneous mutations can also contribute to nominally wild-type inbred backgrounds. The Random mutagenesis to introduce and screen new variants has several advantages for identifying genetic interactions and trait architectures. Ethylnitrosourea (ENU) is especially efficient in mice Agouti alleles and green fluorescent protein (GFP) transgenes that show variegated expression as phenotype reporters, Whitelaw and coworkers have recovered a substantial collection of ENU-induced Modifier of murine metastable epialleles dominant (MommeD) mutations. This approach creates an \u201coutside-in\u201d interaction network module, with multiple edges (interactions) converging on a single node (conditioning mutation or reporter gene) . At leaslacZSox10/+ model of Waardenburg syndrome Mos1,2,3). Remarkably, these three induced mutations mapped to locations distinct from several previously identified modifiers of Sox10 and from each other, suggesting that Sox10 phenotypes are sensitive to perturbations at many positions in an extended genetic network. A collateral phenotype and map position strongly suggested Gli3 as the Mos1 gene, which sequence and complementation analysis rapidly confirmed. Mos2 was subsequently identified as a null allele of the RNA regulator Magoh, with severe haploinsufficiency phenotypes in brain development Another instructive example comes from an ENU screen for dominant enhancers of dominant white spotting in a As illustrated in these examples, chemical mutagenesis allows efficient exploration of genetic networks unconstrained by prior hypothesis. Networks ascertained by this approach, in both examples, comprise modifier alleles that typically have independent deleterious phenotypes. Whether this is more often true of induced mutations or is a property of the specific networks tested, and whether milder alleles at the same genes might retain modifier effects without collateral phenotypes remains to be seen. Alternatively, networks based on variants in natural populations\u2014or nominally wild-type strains\u2014might highlight different genes and network connections, depending on biological trade-offs between modifier effect and collateral phenotypes in any gene's potential allele spectrum and the forces of selection through which a given variant has passed prior to discovery.While inbred mouse strains are often referred to as nominally \u201cwild type,\u201d each represents a different sampling of wild (and a few laboratory-derived) alleles across the genome. Inbred strains in aggregate represent an abundant source of captive variation MinApc-dependent tumor phenotypes in a genetic cancer model Modifier of Min-1 (Mom1) is the most well studied, beginning with its discovery by linkage analysis for intestinal tumor number nearly two decades ago Pla2g2a was proposed as a positional and functionally variant candidate gene, with an apparent null allele caused by a single base insertion in MinApc sensitive strains Mom1 is semidominant and analysis of tumor DNA suggested that its effects are not cell autonomous, consistent with its identification as a secreted enzyme. Complementation studies confirmed that Pla2g2a accounted for a large fraction of the variance in tumor number Mom6, was inferred to account for the full effect of the initial linkage PLA2G2A was subsequently associated with survival in gastric cancer patients Mom1 alleles in mice are widely distributed across inbred strains, suggesting an early origin. Re-sequencing Mom1 single base insertion is a derived allele that arose on a unique haplotype and that at least 2 Mb of this interval is shared by all characterized SMom1-allele strains. may impact several themes raised above, including selection, specificity, and epigenetic regulation. The functionally tested alleles are both well dispersed across inbred strains Dac alleles of Fbxw4, caused by independent insertions of MusD-family retrovirus elements at different sites Mdac alleles alter both DNA methylation and the accumulation of inhibitory histone modifications on the Dac MusD elements Among modifiers not yet identified molecularly, the Identifying modifiers on the basis of natural variants may require more resources than de novo mutations (once generated), but have the added value of generally highlighting alleles, genes, or networks whose manipulation appears well tolerated by the organism and largely compatible with normal function, having been vetted by selective pressures. In the ultimate goal of finding pressure points through which to modulate disease networks, this may prove advantageous.http://bacpac.chori.org/, among others) for transgenic studies, larger-scale tools for engineering ES cells How readily can we use modifier genes to predict sensitive pathways and nodes for therapeutic interventions? New tools and resources in mouse genetics should help to unclog the pipeline of modifier genes that have been detected but not molecularly defined. Maps of copy number variation Table S1A curated list of mouse modifier genes. The attached spreadsheet collects several examples of modifiers and a few exemplar interactions that may strain the strict definition of the term. Entries were manually curated from entries in the Mouse Genome Database (XLSX)Click here for additional data file."} +{"text": "BRCA2) or because the gene is not routinely tested in the context of breast cancer without additional clinical manifestations (PTEN). Among the remaining families candidate genes are currently being assessed for segregation among family members and for prevalence among an addition 800+ unexplained breast cancer families. In particular, we found truncating mutations in two genes that are involved in well-recognised DNA repair pathways but have not previously been associated with an increased risk of breast cancer. In summary, whole exome sequencing of multiple individuals from within each cancer family is proving to be an efficient strategy for rapidly identifying novel familial predisposing mutations.Recent advances in technology have opened up the possibility of using next generation sequencing to efficiently uncover predisposing mutations in individuals with inherited cancer in an unbiased manner. We are conducting whole exome sequence analysis of germline DNA from multiple affected relatives from breast cancer families with the aim of identifying rare protein truncating and non-synonymous variants that are likely to include novel cancer predisposing mutations. Data from >70 exomes show that on average each individual only carries 30-50 protein truncating mutations and 300-400 rare non-synonymous missense variants. By considering only those variants shared by multiple affected relatives the number of candidate predisposing mutations can be dramatically reduced to just 3-5 truncating mutations and 10-20 nonsynonymous variants per family. Among the first 10 breast cancer families studied in detail, two harbour mutations in known breast cancer genes that were missed by clinical genetic testing either because the index case was a phenocopy who did not carry the mutation ("} +{"text": "Aldehyde dehydrogenase-2 (ALDH2) was characterized as the main enzyme responsible for bioactivation of the antianginal drug nitroglycerin (GTN). We have recently shown that ALDH2 is mainly cytosolic in murine vascular tissue, challenging the general assumption that GTN bioactivation takes place in the mitochondrial matrix of vascular smooth muscle cells.In the present study we investigated whether bioactivation of GTN is affected by the subcellular localization of ALDH2 using immortalized ALDH2-deficient aortic smooth muscle cells with selective overexpression of the enzyme in either cytosol or mitochondria. Furthermore we investigated a potential correlation between the relaxation potency of GTN and the distribution of ALDH2 in arterial blood vessels from different species as well as the subcellular distribution of the enzyme in several murine organs.Radio thin layer chromatography analysis showed that cytosolic overexpression of ALDH2 led to denitration rates up to 4 times higher than mitochondrial overexpression, suggesting a more efficient bioactivation by cytosolic ALDH2. Interestingly, denitration rates of smooth muscle cells were even higher in cells without functional mitochondria (Rho0 cells), suggesting possible adverse effects of mitochondria on the bioactivity of GTN. Quantitative immunoblotting revealed that ALDH2 is mainly cytosolic in murine, rat, guinea-pig and rabbit aortas as well as in porcine, bovine and human coronary arteries. A similar expression pattern was found in several murine organs, except liver. Cumulative concentration-response curves to GTN established by vasorelaxation studies were biphasic for aortas with more than 10% ALDH2 in mitochondria (mouse and rabbit), strengthening the hypothesis that mitochondrial GTN metabolism counteracts cytosolic bioactivation of the drug.The data indicate that cytosolic expression is essential for GTN bioactivation in arterial blood vessels and aortic smooth muscle cells, presumably due to limited access of GTN to the mitochondrial matrix."} +{"text": "Rangifer tarandus) populations are declining worldwide in part due to disturbance from human development. Prior to human development, important areas of habitat should be identified to help managers minimize adverse effects. Resource selection functions can help identify these areas by providing a link between space use and landscape attributes. We estimated resource selection during five summer periods at two spatial scales for the Teshekpuk Caribou Herd in northern Alaska prior to industrial development to identify areas of high predicted use for the herd. Additionally, given the strong influence parturition and insect harassment have on space use, we determined how selection differed between parturient and non-parturient females, and between periods with and without insect harassment. We used location data acquired between 2004\u20132010 for 41 female caribou to estimate resource selection functions. Patterns of selection varied through summer but caribou consistently avoided patches of flooded vegetation and selected areas with a high density of sedge-grass meadow. Predicted use by parturient females during calving was almost entirely restricted to the area surrounding Teshekpuk Lake presumably due to high concentration of sedge-grass meadows, whereas selection for this area by non-parturient females was less strong. When insect harassment was low, caribou primarily selected the areas around Teshekpuk Lake but when it was high, caribou used areas having climates where insect abundance would be lower . Areas with a high probability of use were predominately restricted to the area surrounding Teshekpuk Lake except during late summer when high use areas were less aggregated because of more general patterns of resource selection. Planning is currently underway for establishing where oil and gas development can occur in the herd\u2019s range, so our results provide land managers with information that can help predict and minimize impacts of development on the herd.Many caribou ( Rangifer tarandus) populations are exhibiting synchronous declines Across the circumpolar north, many caribou and reindeer on the landscape. Resource selection functions RSFs; are a vaThe Teshekpuk Caribou Herd resides in northern Alaska with nearly all of its annual range overlapping with the National Petroleum Reserve-Alaska NPRA; . LimitedCervus elaphus) select areas with low predator density when choosing seasonal ranges, but select for forage quality at finer spatial scales By estimating resource selection functions at multiple spatial scales through summer we can also better understand the choices caribou make in selecting their seasonal ranges. Considering multiple scales of selection is particularly important for migratory species, such as caribou, where individuals may select certain features of the landscape for placement of seasonal ranges , but show different patterns of selection within seasonal ranges as they search for forage .2 to >800 km2Eriophorum spp.) and Carex aquatilis. The study area has a short snow-free growing period that lasts from June through September.Our study area was restricted to the summer range of the TCH which lies almost entirely within the NPRA . The TCHCanis lupus), brown bears (Ursus arctos), and Golden Eagles (Aquila chrysaetos) prey on the TCH, but are relatively rare on the coastal plain The TCH typically calves near Teshekpuk Lake, uses the area between Teshekpuk Lake and the Beaufort Sea as mosquito-relief habitat, then disperses further inland during late summer 2\u200a=\u200a2.02, P\u200a=\u200a0.98). We also excluded locations obtained while caribou were assumed to have joined another herd. We assumed a caribou switched herds if it was in the calving area of another herd during a subsequent calving season.Between 2004 and 2010 we captured 41 adult female caribou from the TCH with a net gun fired from a helicopter and collared them with GPS collars . The number of individuals monitored differed annually , with most individuals monitored for >1 year (n\u200a=\u200a35). We obtained locations every 3 (2004\u20132005) or 2 hours (2006\u20132010) and removed erroneous points and those with 2D fixes or a positional dilution of precision >10. These cut-offs provide the greatest improvement in mean location accuracy Culex spp.) harassment (1\u201315 Jul), oestrid fly (Hypoderma spp. and Cephenemyia spp.) harassment , and late summer (8 Aug \u201315 Sep). While there is likely annual variation of when these periods occur, we feel these dates adequately capture the majority of each biological period annually and the relevant selective forces acting that we hoped to estimate with the RSFs. Additionally, we divided each of the calving, mosquito harassment, and oestrid fly harassment seasons\u2019 data into two distinct groups. For calving, we partitioned the data into parturient and non-parturient females (n\u200a=\u200a23). We divided mosquito and oestrid seasons into periods when mosquitoes or oestrid flies were predicted to be highly active and periods when they were not predicted to be highly active.We divided each summer into five periods based on previously described life history traits for the herd (We obtained an index of mosquito and oestrid fly activity based on hourly weather observations at Inigok (69 59.377 N 153 05.630 W) and Fish Creek weather stations . Weather stations were located inland, southeast of Teshekpuk Lake in areas used by caribou during midsummer. Based on air temperature and wind speed we calculated mosquito and oestrid fly activity indices using the formulas of Russell et al. Previous studies on resource selection by caribou in the Arctic have highlighted the importance of vegetation type and indices of quality at multiple scales http://free.vgt.vito.be/home.php, accessed 9 Aug 2011). SPOT imagery is provided as 10 day composite images of NDVI and thus minimizes the potential for large areas to be masked by cloud cover. Conversely, it provides a lower temporal resolution of plant phenology, thus it is only serves as a general metric of phenology across the landscape. We defined date of green-up for each pixel as the day that the NDVI value exceeded background values of no-growth from SPOT satellite imagery . We used the digital elevation model to calculate terrain ruggedness using the vector ruggedness measure with an 8 pixel window following the methods of Sappington et al. http://www.snap.uaf.edu/downloads/alaska-climate-datasets, accessed 9 Aug 2011). PRISM data had a resolution of 2 km.We used a 60 m Digital Elevation Model to obtain elevation data with the \u2018corvif\u2019 function found in the AED library in R We estimated resource selection at two separate scales. At the landscape scale, we estimated selection within the herd\u2019s range for a given period. We produced range maps for each period of summer in the study by estimating kernel density estimates To estimate patch-scale selection we used conditional logistic regression following the methods of Forester et al. The resolution of explanatory variables varied such that many variables had grain sizes too coarse to be meaningful in the patch-scale RSFs. Thus, for these models we ran models with all possible combinations of vegetation type, elevation, distance to coast, and terrain ruggedness. We selected the best model in our set of candidate models based on the lowest Akaike\u2019s Information Criterion (AIC) score w(x) is the relative probability of a pixel being selected, \u03b20 is the estimated intercept, and \u03b2i is the coefficient estimate for variable xi.Finally, we created maps to visualize the results of each RSF by using the logistic regression equation to convert each model\u2019s results into values ranging from 0 to 1 C. aquatilis) throughout summer which forage on similar plant types as caribou during winter Caribou selection for patches of different vegetation types generally did not vary between the two spatial scales. One explanation for this lack of pattern is the strong latitudinal gradient in vegetation types in northern Alaska leading to low environmental heterogeneity in the distribution of vegetation types. This same pattern of scale-invariant selection for vegetation types has been shown for muskoxen in the arctic (Eriophorum sp.) and C. aquatilis which have been shown to provide parturient caribou with highly digestible and high nitrogen content forage during lactation in northern Alaska The two scales of analysis were particularly informative during the calving period. Although parturient and non-parturient females did not show vastly different resource selection patterns at the patch-scale, their landscape-level resource selection differed. Parturient females showed substantially higher selection for areas with high densities of sedge-grass meadows than non-parturient females. Sedge-grass meadows at our study site are dominated by cotton grass (The higher energetic requirements and intestinal changes of parturient females are predicted to lead to different space use patterns compared to non-parturient female ungulates During periods with insects, our landscape-scale RSFs revealed that caribou selected areas on the landscape that have climatic conditions associated with lower rates of insect harassment such as near the coastline or along river terraces where wind and cooler temperatures reduce insect activity Similar to Witter et al. Our conclusions about the effects of insect activity on selection by the herd should be viewed cautiously as we relied on a coarse index of insect activity without any associated monitoring of how the index related to actual measures of insect abundance. When Russell et al. Our study highlights the importance of maintaining existing calving areas for the herd because there are limited areas across the NPRA that share features the herd currently selects during calving and post-calving seasons. Additionally, given the importance of the coastline for mosquito relief, care must be taken to avoid hindering movement through the narrow corridors on either side of Teshekpuk Lake that much of the herd uses to reach the coastline"} +{"text": "Bacillus thuringiensis are use worldwide in transgenic crops for efficient pest control. Among the family of Cry toxins, the three domain Cry family is the better characterized regarding their natural evolution leading to a large number of Cry proteins with similar structure, mode of action but different insect specificity. Also, this group is the better characterized regarding the study of their mode of action and the molecular basis of insect specificity. In this review we discuss how Cry toxins have evolved insect specificity in nature and analyse several cases of improvement of Cry toxin action by genetic engineering, some of these examples are currently used in transgenic crops. We believe that the success in the improvement of insecticidal activity by genetic evolution of Cry toxins will depend on the knowledge of the rate-limiting steps of Cry toxicity in different insect pests, the mapping of the specificity binding regions in the Cry toxins, as well as the improvement of mutagenesis strategies and selection procedures.Insecticidal Cry proteins produced by Bacillus thuringiensis (Bt) is a Gram-positive bacterium that produces insecticidal proteins as crystal inclusions during its sporulation phase of growth, known as Cry or Cyt toxins, which have been proven to be effective against important crop pests and also against mosquitoes that are vectors of human diseases such as dengue and malaria (Bombyx mori) was isolated. Few years later Bt was rediscovered in Germany by Ernst Berliner from a Mediterranean fluor moth (Ephestia kuehniella) where toxins belonging to each Cry group share less than 40% amino acid identity with proteins from other groups or aminopeptidase-N (APN) and cadherin-like protein resulting in the formation of a pre-pore oligomeric structure that is proficient in membrane insertion and pore formation (Manduca sexta) larvae. It is proposed that Cry1Ab binds with low affinity (Kd 100\u2013200 nM) to glycosyl-phosphatidyl-inositol anchored ALP or APN receptors. This binding step is believed to concentrate the monomeric toxin in the surface of the brush border membrane. Following this binding step Cry1Ab binds with high affinity (Kd 1 nM) to cadherin; this binding step facilitates the proteolytic removal of helix \u03b11 of domain I, inducing toxin oligomerization. Cry1Ab toxin oligomers gain binding affinity to both ALP and APN (Kd 0.6 nM) and this final binding step facilitates oligomer membrane insertion and pore formation (reviewed in The mode of action of Cry1Ab toxin has been described to some detail in the tobacco hornworm (Leptinotarsa decemlineata) it shows very low toxicity against Western corn rootworm (Diabrotica virgifera virgifera). The low toxicity of Cry3Aa against Western corn rootworm was proposed to be due to the low solubility of the protease activated Cry3Aa that yield a 67 kDa fragment. However, activation with chimiotrypsin was shown to increase the yield of a fully processed 55 kDa Cry3A fragment that showed increased solubility and toxicity Cry1Ac resistant colony linked to mutations in the cadherin gene (Heliothis virescens) Cry1Ac resistant colony whose resistance was recently shown to be genetically linked to a mutant allele of an ABC transporter (ABCC2) (Plutella xylostella) and the cabbage looper (Trichoplusia ni) that were recently shown to be also linked to mutations in the ABCC2 transporter (Appearance of insect resistance threatens the use of Cry toxins in transgenic plants . CadheriIn vitro domain III swapping among different Cry toxins have resulted in some cases in hybrid toxins with improved toxicities against certain insect species (Spodoptera exigua) compared with Cry1C (Tenebrio molitor) , cotton bollworm (Helicoverpa armigera) and black cutworm (Agrotis ipsilon) (in silico (Anoplophora glabripennis) was achieved by fusion of an eight amino acid residue peptide that was shown to specifically bound longhorn beetle midgut Cx-cellulase cadherin fragment. After five rounds of selection using cadherin coated magnetic beads, a domain II loop 2 mutant with apparent similar binding affinity to cadherin, and a fourfold higher insecticidal activity against silkworm larvae than Cry1Aa was selected (Telchin licus licus) is not susceptible to the known Cry toxins including Cry1Ia that was shown to be toxic to fall armyworm (Spodoptera frugiperda) (Anthonomus grandis). Cry8Ka gene was identified as a toxin showing moderate toxicity against cotton boll weevil. Gene shuffling and the selection of improved Cry8Ka binders against cotton boll weevil BBMV resulted in a mutant (Cry8Ka5) with threefold higher toxicity (Despite the problems mentioned above on the efficiency to display certain Cry toxins in phage particles, at least three successful examples of selection of improved variants of Cry toxins selected by biopanning of phage display libraries have been described (in vitro evolution of toxicity of Cry toxins. The rational behind this strategy is that Cry toxin mutants with improved binding affinities to either BBMV from the target insect or isolated insect Cry-binding proteins, will provide mutants that are likely to show enhanced insecticidal activity. The first step is the construction of cry gene library of variants that could then be screened for binding to BBMV or toxin-receptors from the target insect. Several methods for creating variability could be exploited depending on the binding selection procedure. Using general mutagenesis strategies as gene shuffling or prone PCR can explore the whole gene cry sequence including domain I. As discussed earlier, there are examples of domain I mutations that enhance Cry toxicity presumably by enhancing membrane partioning into the membrane (cry gene libraries are cloned into phagemid vectors for the display of Cry mutants in the phage particles. The third step is the screening of libraries by biopanning against BBMV or pure receptor molecules. The fourth step is the selection of Cry mutants with enhanced binding to BBMV or receptors. Finally, the fifth step is the determination of toxicity of Cry toxins with improved binding against the target insect. Improved variants could be the substrate for additional mutagenesis, binding selection and bioassays.We have revised several examples of evolved Cry toxins with improved performance in controlling different insect pests. Some of these Cry mutants show novel insecticidal activity, others improved toxicity against a specific target and others were shown to be active against resistant insects to Cry toxins. Nevertheless, as pointed before, most of these Cry mutants were the result of analysing few mutants in different insect species but not from a high through output system that could detect improved mutants from a large number of variants. We believe that the evolution of Cry toxicity using high through output systems is likely to provide toxins that will perform better in controlling insect pests. In"} +{"text": "Endogenous retroviruses (ERVs) are genetic remnants of exogenous retroviral infections that have endured selective processes and made their way into ancestral host genomes. While many of them have deteriorated beyond the ability to code for detectable proteins, some still retain activity or the potential for coding activity -3. A fewItrharvest[gag, pol or env by a BlastX [The genomes of twelve vertebrates were acquired from UCSC Genome Browser and analyzed with an in-house method based on the a BlastX against Pol genes appear to possess the highest number of conserved ORF and a high number of recent integrations with full conservation has been found in the mouse genome.Our study reveals the existence of a few conserved full length open reading frames in loci whose LTRs present a similarity lower than 70% and plenty of open reading frames occur across all genomes in loci with LTR similarity below 80%. Current method limitations may cause this number to be an underestimation but nonetheless reveals the existence of old retroviral infections that have, to this date, kept some coding potential. In the studied primate genomes, a select number of loci still retain long to full length open reading frames on all three major genes even on loci with LTR divergence under 95% Table . Pol gen"} +{"text": "Hepatitis B is a potentially life threatening liver infection which leads to millions of deaths annually. Serological and molecular assays for hepatitis B virus are the major diagnostic tools. Dried blood spot (DBS), a minimally invasive procedure, is an alternative to serum and can be used for field based studies for molecular detection. The present aim of this study is to diagnose HBV infection by combination of serological and molecular methods from serum and dried blood spot.Blood was collected from suspected cases of liver diseases attending JIPMER Hospital, during September 2010 to October 2011. The study group is divided into two each having 30 cases of hepatitis B surface marker positive and negative profile respectively. Samples were analyzed for complete serological tests (surface and core antigen) and PCR were performed on serum samples and DBS.Out of 30 HBsAg positive cases screened by ELISA, 22 samples were found positive of HBV DNA by PCR method from serum, which includes 2 samples with only surface (HBsAg) and antibody to core antigen (IgM anti HBc) positive. All these 22 positive cases were also been detected from DBS after storing the sample at 25\u00b0C for 4 and 7 days.It is important to detect both serological and molecular markers to diagnose hepatitis B for appropriate management of disease Since dried blood spot yields comparable results to serum in detecting HBV DNA it can be used as convenient method of collecting samples than venous blood, particularly in resource limited settings."} +{"text": "Objective: This article serves to review latissimus dorsi myocutaneous flap as an option for breast reconstruction postmastectomy. Since the introduction of the latissimus dorsi myocutaneous flap in the late 1970s, its use has always been as a secondary technique, particularly after the development of the transverse rectus abdominus myocutaneous flap in the 1980s. Methods: A literature review of the history of latissimus dorsi myocutaneous flap utilized for breast reconstruction as well as a review of our institution's experience with latissimus dorsi myocutaneous flap and tissue expander placement was performed. Results: There remains a paucity of published studies investigating latissimus dorsi myocutaneous flap for breast reconstruction. Most studies have small numbers and do not utilize tissue expanders. More recently several small studies have been published that show acceptably low complication rates with aesthetically pleasing outcomes when latissimus dorsi myocutaneous flap is employed with a tissue expander. At our institution, we have employed latissimus dorsi myocutaneous flap with tissue expander placement for both delayed and immediate reconstruction with subsequent replacement with a permanent implant with a capsular contraction rate of 10.5%. Our data and others more recently published demonstrate very acceptable capsular contracture rates and aesthetic outcomes, particularly when an expander is utilized. Conclusion: The latissimus dorsi myocutaneous flap remains an excellent choice for breast reconstruction with a low risk of complications. Over 202,000 women were diagnosed with breast cancer in 2007.A literature review of the history of LDMF utilized for breast reconstruction as well as a review of our institution's experience with LDMF and tissue expander placement was performed.,10The LDMF was first described in the late 1800s by Italian surgeon Tanzini7-In early series, LDMF was heavily criticized for high capsular contracture rates and other complications such as seroma formation.11-12-As stated, the most significant criticism has been the LDMF's reported incidence of capsular contracture (7.4%-75%).11McCraw and MaxwellMore recently, however, Venus and PrinslooThere continues to be a dearth of literature investigating LDMF breast reconstruction in conjunction with initial placement of tissue expanders rather than an implant. Abdalla et al19There is variation in published complication rates in LDMF in irradiated breasts as well. Garusi et alAt our institution, we have employed LDMF with tissue expander placement for both delayed and immediate reconstruction with subsequent replacement with a permanent implant Fig . We publ13-28Aesthetically, a pleasing outcome is obtainable with LDMF reconstruction when tissue expanders are employed Figs and 3. T25In summary, LDMF for breast reconstruction after mastectomy is a procedure that has been erroneously maligned over the years for high capsular contracture complications. In reality, a lot of the data regarding capsular contracture rates in LDMF are outdated and skewed. Proper study of LDMF most likely was hindered by the rapid rise in the popularity of the TRAM flap in the early 1980s. Our data and others more recently published demonstrate very acceptable capsular contracture rates and aesthetic outcomes, particularly when an expander is utilized. We believe that LDMF should be utilized more often, as it is a technically straightforward procedure that gives acceptable cosmetic outcomes with few complications."} +{"text": "VEGFA gene results in the formation of various VEGFA isoforms. Each isoform has unique properties and is identified by the number of amino acids within the mature protein. Proangiogenic isoforms (VEGFA_XXX) are encoded by exon 8a, whereas a sister set of isoforms (VEGFA_XXXB) with antiangiogenic properties is encoded by exon 8b. The antiangiogenic VEGFA_XXXB isoforms comprise the majority of VEGFA expressed in most tissues, whereas expression of the proangiogenic VEGFA isoforms is upregulated in tissues undergoing active angiogenesis. Although proangiogenic and antiangiogenic isoforms can now be distinguished from one another, many studies evaluating VEGFA in ovarian and follicular development up to now have not differentiated proangiogenic VEGFA from antiangiogenic VEGFA. Experiments from our laboratory indicate that proangiogenic VEGFA promotes follicle recruitment and early follicular development and antiangiogenic VEGFA inhibits these processes. The balance of proangiogenic versus antiangiognic VEGFA isoforms is thus of importance during follicle development. Further studies are warranted to elucidate the way that this balance regulates follicular formation and progression.Vascular endothelial growth factor A (VEGFA) has been extensively studied because of its role in follicular development and is a principal angiogenic factor essential for angiogenesis. Since vascularization of the theca layer increases as follicles progress in size through preantral and antral stages, VEGFA might influence follicle growth via the regulation of angiogenesis. However, VEGFA might also influence follicular development through nonangiogenic mechanisms, since its expression has been localized in nonvascular follicles and cells. Alternative mRNA splicing of eight exons from the Angiogenesis is the term used to describe the formation of new vessels from the remodeling and expansion of the existing vascular network. This process involves both proliferation and migration of endothelial cells and can lead to the vascularization of previously avascular tissues Patan . OvarianFollicle assembly and initial recruitment of primordial follicles begins near the corticomedullary border and progresses outward to the periphery , fibroblast growth factor 2, members of the platelet-derived growth factor family and angiopoietins has been shown to promote migration, proliferation and tube formation in endothelial cells Patan and is aTwo tyrosine kinase receptors bind VEGFA with high affinity: FLT1 and KDR . Both of these receptors have seven extracellular immunoglobulin-like domains, a single transmembrane region and an intracellular tyrosine kinase sequence with a kinase insert domain Ferrara . FLT1 waAlthough FLT1 has a higher affinity for VEGFA than KDR, VEGFA binding to KDR induces stronger tyrosine phosphorylation die between 10.5-12.5 dpc with multiple vascular defects and mutant mice that overexpress NRP1 die at 17.5 dpc with excessive and dilated vasculature from bovine granulosa cells. Based on the predicted amino acid sequence, the antiangiogenic isoforms appear to have the same number of amino acids as the proangiogenic isoforms in the bovine. However, we have also sequenced the mRNA for Vegfa_165b from rat ovaries and, based on the predicted amino acid sequence, the antiangiogenic isoforms appear to have an additional amino acid compared with their respective proangiogenic isoforms in rats but the corresponding isoform in mice, rats and cattle consists in only 164 amino acids administration of adult mice with an antibody designed to neutralize VEGFA reduces the number of primordial follicles by approximately 50% without having an effect on primary or secondary follicles. Similar results are seen with intrabursal administration of the same antibody to prepubertal (6- to 8-weeks-old) mice. Intrabursal administration of a KDR antibody also reduces primordial follicle numbers in prepubertal mice but injection with a FLT1 antibody has no effect. Although these differences in primordial follicles numbers are lost between 30\u00a0days (immature mice) or 6\u00a0months (adults) after treatment significantly reduces vascular density, increases the percentage of primordial follicles and decreases the percentage of developing follicles compared with control ovaries Fig.\u00a0. In contFurther experiments with the same P3/4 rat ovary culture system demonstrated that treatment with recombinant VEGFA_165 or a VEGFA_XXXB antibody resulted in an increase in vascular density. In addition, treated ovaries had a decreased percentage of primordial follicles and an increased percentage of developing follicles compared with controls Fig.\u00a0. Both ex2) and progesterone (P4) in initial follicle recruitment. Approximately half the number of primordial follicles transitioned to primary follicles in PO rat ovary cultures that were treated with E2 or P4 compared with control ovary cultures in prepubertal (P21) rat pups significantly increases the number of both primary and small secondary follicles in the treated ovary compared with the contralateral ovary. Intrabursal injection with recombinant VEGFA has similar effects on follicle dynamics. In the same study, both systemic and intrabursal administration of E2 is shown to result in an increase in ovarian VEGFA protein expression receptors during the preantral stage and theca cells express luteinizing hormone (LH) receptors as soon as they form (Roy et al. In postnatal rat ovaries, VEGFA, FLT1 and KDR have been localized to granulosa cells, theca cells and the cytoplasm of oocytes of preantral follicles (McFee et al. VEGFA_121, VEGFA_165, VEGFA_189, FLT1 and KDR has also been demonstrated in fetal bovine ovaries and levels of VEGFA_121 and VEGFA_189 increase as development proceeds. Messenger RNA levels for VEGFA are consistent across follicle development in theca cells, whereas the expression of KDR and FLT1 is weak in granulosa cells but strong in theca cells (Yang and Fortune VEGFA_165 and VEGFA_121 isoforms and the expression of VEGFA increases as antral follicles increase in size (Berisha et al. VEGFA_120 and VEGFA_164 isoforms but also weakly express mRNA for VEGFA_189. These cultured cells additionally express mRNA for KDR (Greenaway et al. VEGFA is expressed in both granulosa and theca cells of secondary and tertiary follicles, whereas FLT1 and KDR are expressed by the endothelial cells within the theca layer in marmoset monkeys. The mRNA levels for VEGFA in granulosa cells increase from the secondary follicle stage to the tertiary stage (Wulff et al. Expression of mRNA for 2 and wider vascular networks within the follicular wall than medium follicles with low VEGFA levels (Mattioli et al. VEGFA protein in follicular fluid and granulosa cells has been demonstrated to increase in both bovine and porcine follicles as they increase in size from small to large antral follicles (Berisha et al. VEGFA gene fragments into the ovaries of miniature gilts results in increased numbers of large antral follicles, increased mRNA expression of the VEGFA_165 and VEGFA_121 isoforms in granulosa cells and increased VEGFA protein levels in follicular fluid. In addition, the capillary density within the theca interna is increased in follicles from VEGFA-injected gilts compared with control animals (Shimizu VEGFA mRNA levels in granulosa cells of follicles larger than 4\u00a0mm in diameter (Barboni et al. Based upon mRNA and protein expression alone, VEGFA appears to be involved in the growth of follicles from the preantral and early antral stages to later antral stages. Numerous other studies have added support for the role of VEGFA in follicle development. Culturing pieces of bovine fetal ovarian cortex in the presence of VEGFA has no effect on the number of primordial or primary follicles but does increase the number of secondary follicles (Yang and Fortune Shimizu . IntramuVEGFA regulation of preantral and antral follicle growth appears to be mediated through FLT1 and KDR signaling. Inhibition of VEGFA with intrabursal injection of a soluble FLT1/Fc chimera Trap does not alter the number of preantral or early antral follicles in prepubertal rats treated with eCG. However, the number of atretic follicles increases compared with control rats, together with increased BAX and decreased BCL2 protein levels in follicular cells (Abramovich et al. A strong body of evidence supports a role for VEGFA in initial follicle recruitment and development. Even though VEGFA is considered a prominent proangiogenic factor, VEGFA has been localized to nonvascular follicles and cells and might influence follicular development via nonvascular mechanisms. In addition, VEGFA isoforms have now been identified that have antiangiogenic properties (Harper and Bates"} +{"text": "Togaviridae) infection in mosquitoes require the use of recombinant viruses engineered to express a visibly detectable reporter protein. These altered viruses expressing fluorescent proteins, usually from a duplicated viral subgenomic reporter, are effective at marking infection but tend to be attenuated due to the modification of the genome. Additionally, field strains of viruses cannot be visualized using this approach unless infectious clones can be developed to insert a reporter protein. To circumvent these issues, we have developed an insect cell-based system for detecting wild-type sindbis virus infection that uses a virus inducible promoter to express a fluorescent reporter gene only upon active virus infection. We have developed an insect expression system that produces sindbis virus minigenomes containing a subgenomic promoter sequence, which produces a translatable RNA species only when infectious virus is present and providing viral replication proteins. This subgenomic reporter RNA system is able to detect wild-type Sindbis infection in cultured mosquito cells. The detection system is relatively species specific and only detects closely related viruses, but can detect low levels of alphavirus specific replication early during infection. A chikungunya virus detection system was also developed that specifically detects chikungunya virus infection. Transgenic Aedes aegypti mosquito families were established that constitutively express the sindbis virus reporter RNA and were found to only express fluorescent proteins during virus infection. This virus inducible reporter system demonstrates a novel approach for detecting non-recombinant virus infection in mosquito cell culture and in live transgenic mosquitoes. Current methods for detecting real-time alphavirus (Family Alphaviruses are mosquito-borne pathogens that can cause severe human and veterinary disease, several of which are considered potential biological weapons ,3. AlphaDefining how alphaviruses infect the mosquito vector and transmit to mammalian hosts is an active area of study, but the tools for monitoring alphavirus infection in mosquitoes have largely relied on postmortem analysis or using recombinant viruses engineered to express fluorescent or luminescent proteins from duplicated subgenomic promoters (SGP) . AlthougTogaviridae, genus alphavirus) are positive strand RNA viruses with a genome size of ~12Kb containing a 5\u2019 RNA cap and a 3\u2019 polyadenylated tail ) resulting in pBG471 . Th. ThAe. apromoter to optimThe reporter RNAs used in this study express fluorescent proteins during infection and allow visual confirmation of Sindbis virus infection. However, a system similar to this could be engineered to express other proteins only during active virus replication. An interesting application for this technology would be to engineer reporter RNAs that encode a cytotoxic or mosquitocidal gene, which would only be expressed concurrent with virus infection. For example, expression of the Saporin ribosomal toxin gene during iThe subgenomic reporter RNA system we present can detect alphavirus infection in cell culture systems and in transgenic mosquitoes. Once optimized, this novel system will provide a valuable method to visually monitor dissemination of wild-type alphaviruses throughout insect cells and be useful for monitoring the transmission of alphaviruses from mosquitoes to mammalian hosts, increasing our understanding of viral transmission cycle."} +{"text": "Our study aimed to determine the prevalence of functional SNPs of TLR4 receptors, in healthy volunteers and septic patients in a Brazilian population and to correlate the presence of these polymorphisms in septic patients with clinical outcome.NcoI for SNP 299 and HinfI for SNP399 followed by electrophoresis for identification of alleles.We verified the presence of polymorphisms ASP299GLY, THR399 ILE by PCR-restriction fragment length polymorphism followed by digestion with enzymes P = 0.001). We also observed a statistically significant difference between genotype groups formed by the Asp299Gly and Thr399Ile polymorphisms and respiratory dysfunction more often featuring group 299Selv/399Selv grupo299Het/399Het and less frequently in individuals with respiratory dysfunction than those without this condition (P = 0.003).We observed a statistically significant difference between the genotypes of the Thr399Ile polymorphism and respiratory dysfunction, indicating a higher frequency than wild-type genotype in subjects with respiratory dysfunction than those without this condition (Our study shows for the first time an assessment of the prevalence of polymorphisms of TLR4 Asp299Gly and Thr399Ile considering its cosegregation in healthy individuals and septic patients. And that septic patients who develop respiratory dysfunction have more presence and genotypes 399Selv 299Selv/399Selv and less the presence of genotype 299Het/399Het, featuring a protective effect of the polymorphism Thr399Ile."} +{"text": "Matched molecular pairs (MMPs) are commonly used to assess the importance of chemical modifications on small molecules versus a particular property . One majHere we present a novel methodology for providing robust statistics for fuzzy context specific MMPs even on medium sized data-sets. Molecules are transformed to a reduced graph using the Discngine Pharmacophore Graph methodology , a molecValidation of the fuzzy context specific MMP (fcsMMP) is presented and outcome is compared to classical MMP analysis. Last the Discngine Network framework is used to organize the derived design rules for efficient large scale mining and results extraction in a real world Med Chem context and applications are shown."} +{"text": "Most patients with thoracic ossification of the posterior longitudinal ligament (OPLL) exhibit delayed recovery of gait dysfunction after spinal injury. The hybrid assistive limb (HAL) is a new robot suit controlling knee and hip joint motion by detecting very weak bioelectric signals on the surface of the skin. This study is to report the feasibility and benefits of patient-assistive HAL walking rehabilitation for facilitating locomotor function after spinal surgery. The patient was a 60-year-old woman with thoracic OPLL, and her motor and sensory paralyses did not improve after spinal surgery, indicating severe impairment in the paretic legs. The subject underwent 6 HAL sessions per week for 8 weeks, consisting of a standing and sitting exercise and walking on the ground with HAL. Clinical outcomes were evaluated before and after HAL training and 1 year after surgery. The subject improved considerably as a result of HAL training. Subsequently, her walking ability recovered rapidly, and she was able to walk unaided six months after surgery. This case study suggests that HAL training is a feasible and effective option to facilitating locomotor function and the early HAL training with physiotherapy may enhance motor recovery of patients with residual paralysis after surgery. Decompression is the primary treatment for patients with compressive myelopathy due to thoracic ossification of the posterior longitudinal ligament (OPLL) and ossification of the ligamentum flavum (OLF), but surgical outcomes vary. Studies of postoperative clinical outcomes of thoracic OPLL indicate that most patients exhibit delayed recovery of motor weakness in the lower limbs and gait dysfunction after surgery , 2. GaitRobotic therapy is becoming increasingly common for gait rehabilitation after stroke or spinal cord injury, using an exoskeleton robotic device or a robotic device with foot-driven plates \u20136. The rThis case was markedly improved locomotor function by training with HAL, although recovery did not start until 7 weeks after spinal decompression of thoracic OPLL. Therefore, we report a case of patient-assistive HAL walking rehabilitation from an early stage for facilitating locomotor functions for patients with severe residual paralysis.2) presented with onset of pain and numbness in her right lower limb and gait disturbance. The diagnosis was cervico-thoracic OPLL. After 15 months, her symptoms had gradually progressed, showing motor and sensory paresis of the lower limb and urinary disturbance. Magnetic resonance imaging showed areas of OPLL extending from T2 to T8 and T9/T10 OYL and 8 weeks and 8 months after HAL intervention (15 weeks and 1 year after surgery, resp., Locomotor functions of the patient improved considerably by the intervention of HAL training. Subsequently, her walking ability recovered rapidly and she was able to walk independently six months after surgery. This case report describes the feasibility of facilitating locomotor functions with HAL training for patients with residual paralysis after spinal surgery. Matsumoto et al. reportedHAL is a robotic device with potential rehabilitation applications that are dependent on the physical support it can provide . A patieThis study had a clear limitation in that the HAL training was started relatively soon after surgery. However, even if this patient was still in the recovery period, her locomotor function markedly improved by combining training with HAL. HAL training at an early stage may be necessary to prevent disuse syndrome such as muscle weakness in the lower limbs or joint contracture. The subject may also have experienced improved motivation for rehabilitation by HAL training use from an early stage, because she had been bedridden for 7 weeks after surgery. The findings from this case report suggest that HAL training for voluntary control of leg joint motion from an early phase is a safe and effective option for restoring locomotor functions in patients with residual paralysis after spinal surgery.We concluded that for patients of thoracic OPLL, the early HAL training with physiotherapy may enhance motor recovery after surgery. Early mobilization using HAL may be also advocated to prevent post surgery complications, such as contractures and deep vein thrombosis."} +{"text": "Beta cell dysfunction and insulin resistance are inherently complex with their interrelation for triggering the pathogenesis of diabetes also somewhat undefined. Both pathogenic states induce hyperglycemia and therefore increase insulin demand. Beta cell dysfunction results from inadequate glucose sensing to stimulate insulin secretion therefore elevated glucose concentrations prevail. Persistently elevated glucose concentrations above the physiological range result in the manifestation of hyperglycemia. With systemic insulin resistance, insulin signaling within glucose recipient tissues is defective therefore hyperglycemia perseveres. Beta cell dysfunction supersedes insulin resistance in inducing diabetes. Both pathological states influence each other and presumably synergistically exacerbate diabetes. Preserving beta cell function and insulin signaling in beta cells and insulin signaling in the glucose recipient tissues will maintain glucose homeostasis. A progressive decline of beta cell function leading to beta cell exhaustion precedes beta cell demise and reactive nitrogen species (RNS) are formed during both cytokine-mediated proinflammatory beta cell aggression in type 1 diabetes and glucolipotoxicity-mediated beta cell dysfunction in type 2 diabetes may potentiate the effect of genetic variants on insulin resistance pathways, an effect that could be attributed to tissue-specific responses to the obesogenic environment in type 2 diabetes is attributed to nutrient overload leading to metabolic exhaustion in beta cells whereas beta cell hyperplasia occurs by beta cell replication or beta cell neogenesis from non-beta cells. Both beta cell replication and neogenesis contribute to the expansion of beta cell mass and require external stimuli such as hormones and growth factors in response to fluctuations in the demand for insulin. In states of increased insulin demand such as hyperglycemia, beta cells compensate to restore glucose homeostasis (maintain normoglycemia) via beta cell hypertrophy, and hyperplasia (to collectively increase beta cell mass) Cerf, also incBeta cell mass is dynamic and can respond to environmental cues such as glucose and insulin are highly variable from 2% and ultimately contribute to beta cell preservation. Beta cell preservation refers to the maintenance of both beta cell structure and function (beta cell physiology).Serum response factor (SRF), an essential regulator of myogenic and neurogenic genes, is highly enriched in beta cells and the insulin gene is a target of SRF is a target of HNF4\u03b1 in beta cells does not contribute to mitochondrial uncoupling in beta cells in intact islets , the growth inhibitory peptide or glucose-dependent insulinotropic polypeptide (GIP), the transcription factor paired box gene (Pax4), and the orphan nuclear liver receptor homolog 1 (LRH1) are factors that exert several beneficial actions on beta cells simultaneously such as conferring beta cell protection and enhancing proliferation rats agonist, enhances beta cell function and survival during culture conditions used in clinical islet transplantation which presents a feasible approach to preserve beta cells during pre-transplant islet culture activation and targets, cyclin D2, and cyclin R (Lopez-Acosta et al., Insulin secretion in the presence of resveratrol is increased in various pancreatic beta cell cultures (Chen et al., A healthy normocaloric diet that is well balanced, limited in saturated fat content, and meets the recommended daily allowances of key nutrients protects beta cells and enhances longevity in healthy individuals. Reduced energy intake combined with exercise improves insulin sensitivity (Weickert, Regular exercise, although it may not necessarily reduce weight, enhances insulin sensitivity. Importantly, exercise concomitant with weight loss improves beta cell function (Dela et al., Cytokine-mediated oxidative stress and inflammation, inherent in obesity and insulin resistance, induces beta cell death therefore the beta cell population declines contributing to the manifestation of beta cell dysfunction Figure . SimilarMetabolites, hormones, and transcription factors stimulate beta cell proliferation to sustain beta cell compensation Figure . Beta ceProliferative agents, novel compounds, and healthy lifestyles help to preserve beta cells by maintaining beta cell integrity and function Figure . TherefoBeta cell dysfunction signals an advanced state of diabetes as insufficient insulin is secreted to meet demand. Insulin resistance precedes the pathogenesis for several modern diseases (Samuel and Shulman, The relationship between insulin resistance and beta cell dysfunction is dynamic and largely dependent on the metabolic state that is primarily determined by glycemic status and consequently insulinemic status. Both a high fat diet and obesity trigger insulin resistance independently, with a high fat diet contributing to overweight and obesity Figure . In the Beta cell physiology should be preserved throughout life but is adversely impacted with aging and altered metabolic states such as obesity that requires a sustained increase in insulin. Insulin resistance promotes beta cell demise and inhibits beta cell compensation which thereby promotes beta cell dysfunction. Replenishing or preserving beta cells maintains beta cell physiology and allows for beta cell compensation which combats beta cell demise and beta cell dysfunction. The preservation of beta cells by replenishment to mitigate against insults and maintain beta cell physiology will improve the metabolic outcomes associated with diabetes.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Schizophrenia provides a striking example of a disorder in which complex genetic and environmental factors combine to produce abnormal brain development and function. In order to fully understand the disorder, and develop more effective and targeted treatments, more accurate and sophisticated animal models are required, which incorporate genetic and environmental variables and their associated gene-environment interactions. We discuss key considerations in modeling gene-environment interactions, with a focus on the recent proposal that schizophrenia involves \u201cdecanalization,\u201d whereby \u201cexperience-expectant\u201d brain development can have its trajectory derailed when particular genotypes (and associated cryptic genetic variants) are exposed to \u201cunexpected\u201d environmental conditions. This has broader implications for the modeling of schizophrenia and other brain disorders involving neurodevelopmental etiology, including autism spectrum disorders (ASDs). We propose that it is insufficient to examine animal models expressing particular genetic variants or mutations only in the single environmental context of \u201cstandard\u201d housing conditions. The exploration of disease-associated polymorphisms and mutations under housing conditions in which environmental factors of clinical relevance are systematically manipulated will facilitate the testing of specific hypotheses associated with pathogenic gene-environment interactions and decanalized development.Over the past several decades, significant advances in genetics and neuroscience have transformed our understanding of how the brain produces adaptive behavior and the ways in which normal functioning becomes disrupted in neurodevelopmental disorders, such as schizophrenia and ASD. Nevertheless, we have only begun to comprehend how particular combinations of genomes and environmental histories combine to produce a given set of clinical symptoms.A major challenge for translating these findings to specific, effective treatments has been the heterogeneity that exists both in clinical presentation and in the genetic associations that have been uncovered. Both schizophrenia and ASD exhibit high heritability and significant research efforts have been geared toward uncovering genetic variation in a bid to explain cause. Genome-wide association studies (GWAS) have identified hundreds of common variants associated with complex diseases, however, the overall genetic risk explained by these loci remains modest (Manolio et al., The problem of missing heritability may be at least partly addressed by improving the discovery rate of genetic variants via statistically well-powered cohorts of individuals that are better characterized for disease phenotypes, genetic background, and environmental exposure (Manolio et al., Humans are far from being at genetic equilibrium, owing to marked changes in population size, admixture, and environment in the past few thousand years. Principle among these environmental changes are dietary shifts, modern hygiene, altered pathogen exposure, urban living, stress, and departure from natural circadian rhythm through artificial lighting. These, and other, environmental perturbations may alter the genetic contributions to phenotype by revealing cryptic genetic variation, especially among individuals with existing genetic vulnerability Gibson, .The capacity of a population to produce the same phenotype, regardless of variability in environment or genotype has been termed canalization (Waddington, Homo sapiens lineage (McGrath et al., Human brain development may be particularly susceptible to decanalization due to rapid evolution of brain structure and function in the relatively recent This conceptual framework also has implications for the way in which preclinical trials in animal models are designed and implemented. We have recently proposed that environmentally enriched conditions could be used as a \u201csecondary screen\u201d for those preclinical studies initially conducted under standard housing conditions (Burrows et al., An additional implication of this theoretical framework is that brain development may be canalized in appropriately stimulating enriched environments, but may become decanalized under conditions of sensorimotor deprivation or environmental stress. Thus, the analysis of animal models in a single \u201cstandard\u201d housing environment has limited heuristic value (Richter et al., The potential importance of decanalization as a mechanism mediating G \u00d7 E interactions in schizophrenia is supported both by epidemiological evidence and animal models (McGrath et al., in utero or in mothers prior to conception could be particularly important in precipitating such decanalized brain development.ASDs involve abnormal brain development and contributions from both environmental and genetic factors. Furthermore, there is evidence of overlapping genetic etiologies amongst neurodevelopmental disorders such as schizophrenia and ASD (Cristino et al., Applying the decanalization framework to clinical and preclinical research may help to identify clusters of neuropsychiatric disorders that emerge from shared decanalization events. This approach has been partially utilized in the human genetics field with a recent genome-wide analysis leading to the identification of risk loci with shared effects on five major psychiatric disorders (Smoller et al., A major limitation in applying this framework is the feasibility of incorporating the staggering complexity that underlies psychiatric disorders into research designs. Furthermore, simple models are not likely to be sufficient to unravel this complexity. There has been a realization that disease entities that appear to be a single disorder actually have separate developmental trajectories arising from distinct genetic vectors that influence the epigenetic landscape (McGrath et al., Understanding how evolved genetic programs and biological systems are dynamically sculpted by G \u00d7 E interactions is the next frontier in the analysis of complex traits and in understanding the origin of neurodevelopmental disorders such as schizophrenia and ASD. In the past, G \u00d7 E interactions driving variation in complex traits have been regarded by some as a nuisance, leading to difficulty in replicating results across cohorts and to the rejection of interesting genetic effects that are significant in specific environments but exhibit diminishing significance when averaged across constellations of differing environments. The realization that G \u00d7 E interactions via decanalization may be integral to the development of major disorders such as schizophrenia will motivate research aimed at elucidating mechanisms as well as identifying and modifying key environmental risk factors. Furthermore, incorporation of G \u00d7 E interactions into our models of neuropsychiatric disorders will provide us with the powerful tools to understand how the decanalized brain produces suboptimal phenotypes and to develop more effective therapeutic approaches."} +{"text": "The proposed method successfully tracks the time-varying causal network in simulated data, and when applied to real neural data recorded in the rat insular cortex, it identifies the change of causal relationships between neurons to a relevant behavioral stimulus (see Figure Neurons in many brain regions change their spiking responses and interactions among them to relevant stimuli. Tracking the dynamics of neural system is crucial for understanding how neural systems adapt their responses to relevant biological information. Granger causality has beenThe time-varying causal connectivity between neurons was assessed based on the instantaneous point process LLR calculated using the sequential probability assignment method. The proposed framework can be easily extended to different modalities of neural signals using general exponential family of distributions."} +{"text": "Toxin-antitoxin (TA) systems are small genetic elements ubiquitous in prokaryotic genomes that encode toxic proteins targeting various vital cellular functions. Typically, toxin activity is controlled by adjacently encoded protein or RNA antitoxins and unleashed as a consequence of genetic fluctuations or stressful conditions. Whereas some TA systems interfere with replication or cell wall synthesis, most of them influence transcriptional and post-transcriptional gene regulation. Antitoxin proteins often act as DNA binding transcriptional regulators and many TA toxins exhibit endoribonuclease activity to selectively degrade different RNA species and thus alter gene expression patterns. Some TA RNases cleave tRNA, tmRNAs or rRNAs, whereas most commonly mRNAs either in association with the ribosome or as free transcripts, are targeted. Examples are provided on how TA toxins differentially shape gene expression in bacterial pathogens by creating specialized ribosomes or by altering the transcriptome and how this may be tied in the control of pathogenicity factors. E. coli episomes. There, cells which suffered stochastic plasmid loss in the course of cell division were found to be eradicated by a stable TA toxin whose less stable cognate antitoxin was no longer produced. For example, the hok-sok system ensures faithful partitioning of the plasmid R1 via the membrane pore forming Hok toxin systems are small and frequently bicistronic elements that are widely distributed throughout bacterial and archaeal genomes. They generally encode a low molecular weight protein which interferes with vital cellular functions and another protein or RNA molecule that keeps the toxin activity in check. The first TA loci have been characterized as plasmid addiction systems on Generally, TA toxin activity is unleashed by an imbalance in favor of free toxin molecules, generated either by stochastic fluctuations in the TA systems' gene expression or cellular stress homolog of M. tuberculosis in vitro and in E. coli have shown cleavage of the 23S ribosomal RNA between G and A in a prominent loop region , the concomitant stimulation of translation initiation at elongator codons in lieu of canonical start codons may globally affect the cellular translation program sequence of the 16S rRNA is removed, yielding so called \u201cstress ribosomes\u201d. These are capable of translating a set of leaderless mRNAs with clipped SD sequences as another result of sequence specific MazF activity. This orthogonal translation system produces about 50 proteins which are associated with population heterogeneity in E. coli cultures that leaves only few survivors behind , which inhibits nucleic acid cleavage activity of both enzymes. This interaction exemplifies an additional regulatory function of a TA interferase beyond ribonuclease activity , acpP (acyl carrier protein) and hns (DNA condensing and supercoiling protein) at AAG, GAA, and ACA was demonstrated mRNA in vivo, three nucleotides downstream of the start codon , the RatA toxin, encoded by the The PhD/Doc system blocks general translation by binding to the 30S ribosome subunit and thus inhibiting translation elongation affected by TA RNases throughout different bacteria and virulence in a mouse model (De la Cruz et al., Insights into extended roles of type II antitoxins are emerging. Besides controlling toxin activity by protein-protein interaction and transcriptional autoregulation, some antitoxins have been proven to additionally act as transcription factors controlling other regulons (Kim et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Cardiac retransplantation in the setting of primary graft failure has been traditionally associated with dismal results. We report a successful outcome of a patient who suffered acute graft failure and was initially rescued with short term mechanical assist support and subsequently retransplanted.Case report of 56 year old male with end stage heart disease on the background of ischemic cardiomyopathy bridged to transplantation witha continuous-flow mechanical assist device. After 125 days of support with HeartMate II he received a cardiac allograft from 53 year old male who died from intracranial hemorrhage. After unremarkable procedure with bicaval technique with a total 83 minutes of cold ischemia we observed poor contractility during initial attempt at weaning from CPB. Severe acute predominantly left ventricular failure was confirmed on transesophageal echocardiography. After sixty minutes of reperfusion together with inotropic, vasodilator and inhaled nitric oxide we made the second unsuccessful attempt at weaning and finally resolved to rescue the patient with short-term LVAD Levitronix CentriMag. After stabilization another donor heart became available 16 days after the first transplantation. The second graft came from a 35 year old female who succumbed to embolic stroke. The retransplantation (ischemic time 82 minutes) went uneventfully and the patient was eventually discharged. He is now four years out of surgery in clinically good condition with good graft function.Primary graft failure after the heart transplantation is a catastrophic complication with gloomy prognosis. Growing experience with mechanical circulatory support devices and improvements in peritransplant care has made the salvage of even these critically ill patients possible. Given the huge financial and human cost involved as well as scarcity of donor organs the decision to proceed with retransplantation has to be based on careful patient selection and subject to vigorous multidisciplinary validation."} +{"text": "Nearly 50% of human genetic diseases are an outcome of splicing errors and over 95% of our genes undergo alternative splicing ,2. HenceWe present a minigene system that exhibits a shift in splicing pattern in cells as a function of time following transfection, we evaluated whether the changes in splicing pattern were reflected by changes in nucleosome occupancy; we mutated the plasmid, used different drugs that affect chromatin structure, analyzed RNA polymerase II processivity. For all experiments MNase treatment and chromatin immunoprecipitation assay were used following QPCR analysis.Chromatin organization affects alternative splicing and previous studies have shown that exons have increased nucleosome occupancy compared with their flanking introns . To deteOur data indicate that there is a delicate bi-directional interplay between chromatin organization and regulation of mRNA splicing. Regulation of alternative splicing at the RNA level influences an upstream process \u2013 the organization of the chromatin. U1 snRNA binding to the 5\u2019ss provides the necessary signal from the splicing reaction back to chromatin organization."} +{"text": "Hemodynamic changes during a deep inspiration maneuver predict fluid responsiveness in spontaneously breathing patients\u201d with great interest. Based on the fundamental ideas of dynamic hemodynamic monitoring, inducing cardiac preload variations and monitoring corresponding pulse pressure variations [Sir, we have read the paper by S. Preau et al. \u201criations , the papConcerning compliance and applicability in terms of intention-to-treat: it is not clear which proportion of SB patients with acute circulatory failure was able to comply with the DIM. Can compliance be expected to be as high as 87% (26 of 30) for this generally defined patient group? Concerning the calculations: DIM-induced changes are defined by the authors as.\u2009 \u2009DIM-induced changes = //2).minimal value during quiet SB prior to DIM? Did the time window vary or was it fixed?How large was the time window when estimating the maximal value during DIM was found in either phase 2 or phase 4 of the DIM. In which phase was the maximal value typically found? Were the maximal values during the two phases generally of the same magnitude?The Instructing the patients to perform the DIM: this is important should the technique be validated by other study groups and in turn gain widespread use.slow continuous inspiration strain was associated with a more or less constant inspiratory flow or was most air generally inspired in the first phase of inspiration, perhaps amplifying maximal value during DIM phase 2? How were instructions given on inspiratory flow rate?Did the authors in any way evaluate if the maximal or convenient lung volume?Did you make any instructions to the patients (directly or indirectly) what tidal volume should be during DIM? Did they inspire to, for example, Concerning the physiological mechanisms: did you experience any patients with significant heart rate variability (HRV)? The respiratory part of HRV (with its subsequent varying passive filling time of the ventricles) could be another mechanism inducing preload variations and as such it could confound results obtained with the DIM technique. On the other hand, HRV is known to be significantly reduced during sepsis .We also have a few comments and suggestions to the proposed physiological mechanisms.The decrease in left ventricular stroke volume during DIM phase 1 is suggested to be induced by increasing ventricular afterload. However, we think that another and perhaps more important mechanism is the reduced intrathoracic pressure reducing left ventricular preload during the initial phase of inspiration\u2014until the increasing right ventricular stroke volume with the delay of pulmonary transit time again increases left ventricular preload substantially. In line with this suggestion, but as a \u201creversed mechanism,\u201d we also believe that the increasing intrathoracic pressure during passive expiration (DIM phase 4) contributes somewhat to the increasing left ventricular (preload and) stroke volume in DIM phase 4 in addition to the suggested overall increase in intrathoracic blood volume.As a last comment, we would like to suggest that the DIM phases 2 and 4 could potentially disclose right ventricular failure: a large maximal value during DIM phase 2 relies on biventricular preload responsiveness whereas a large value in DIM phase 4 relies mostly, if not fully, on left ventricular preload responsiveness. Even though the authors did not encounter any cases of preload unresponsiveness associated with high \u0394PPdim (specificity was 100%) low phase 2 values associated with high phase 4 values could theoretically disclose right ventricular failure\u2014even when taking interventricular dependence into account."} +{"text": "The DNA sequence 5\u2019CG (CpG) is self-complementary and can exist in three major chemical forms depending on the modification status of its cytosine moiety. To understand the functional significance of the CpG dinucleotide, we study proteins that bind either its methylated or unmethylated form. These proteins are likely mediators of CpG signalling that influence chromatin modification and thereby genome activity. The local density of CpG varies dramatically within genomic DNA. In the bulk genome CpG is rare and highly methylated, but in so-called \u201cCpG islands\u201d (CGIs) it is dense and usually non-methylated. A signature histone mark at non-methylated CGIs and also at transcriptionally active genes is trimethylation of histone H3 lysine 4. We are exploring the mechanisms by which DNA sequence features that are shared by all CGIs influence this and other epigenetic marks. In contrast, proteins that interact with methyl-CpG are thought to promote gene silencing by recruiting transcriptional corepressors. In particular mutations in the gene for the methyl-CpG binding protein MeCP2 cause the autism spectrum disorder Rett Syndrome. By studying MeCP2 we are learning about its partner proteins that mediate effects on chromatin. These findings allow us to evaluate competing models for MeCP2 function and they illuminate the biology of DNA methylation and the molecular basis of this neurological condition."} +{"text": "The fibrinolytic activity of plasmin plays a fundamental role in resolution of blood clots and clearance of extravascular deposited fibrin in damaged tissues. These vital functions of plasmin are exploited by malignant cells to accelerate tumor growth and facilitate metastases. Mice lacking functional plasmin thus display decreased tumor growth in a variety of cancer models. Interestingly, this role of plasmin has, in regard to skin cancer, been shown to be restricted to male mice. It remains to be clarified whether gender also affects other phenotypic characteristics of plasmin deficiency or if this gender effect is restricted to skin cancer. To investigate this, we tested the effect of gender on plasmin dependent immune cell migration, accumulation of hepatic fibrin depositions, skin composition, and skin wound healing. Gender did not affect immune cell migration or hepatic fibrin accumulation in neither wildtype nor plasmin deficient mice, and the existing differences in skin composition between males and females were unaffected by plasmin deficiency. In contrast, gender had a marked effect on the ability of plasmin deficient mice to heal skin wounds, which was seen as an accelerated wound closure in female versus male plasmin deficient mice. Further studies showed that this gender effect could not be reversed by ovariectomy, suggesting that female sex-hormones did not mediate the accelerated skin wound healing in plasmin deficient female mice. Histological examination of healed wounds revealed larger amounts of fibrotic scars in the provisional matrix of plasmin deficient male mice compared to female mice. These fibrotic scars correlated to an obstruction of cell infiltration of the granulation tissue, which is a prerequisite for wound healing. In conclusion, the presented data show that the gender dependent effect of plasmin deficiency is tissue specific and may be secondary to already established differences between genders, such as skin thickness and composition. In humans, loss of plasmin dependent fibrinolysis leads to abnormal wound healing and development of pseudo membranes on mucosal tissues throughout the body. The gastrointestinal tract, respiratory system, and genitourinary tract are often strongly affected, which may ultimately lead to organ failure and increased morbidity. However, the most noticeable clinical syndrome in these patients is ligneous conjunctivitis represented by fibrin rich lesions on the eyelids, which may be resolved by local treatment with Plg \u2212/\u2212 female mice are not prone to the same decree of tumor thrombosis or can resolve fibrin clots by expressing fibrinolytic protease(s) besides plasmin. Alternatively, the endogenous differences in skin composition between genders could be responsible for the different effects of plasmin deficiency on skin tumor growth in male and female mice.Besides the important functions of plasmin in maintaining homeostasis and tissue integrity, plasmin and the Plg activation (PA) system also mediate pathological processes, such as tumor growth and cancer metastasis We here describe a systematic investigation of the phenotype of Plg deficiency in male and female mice, and reveal an effect of gender that is restricted to the skin. This gender effect could not be resolved by ovariectomy (OVX) thus suggesting that it is likely linked to variations in skin composition and less likely to a hormone dependent expression of a fibrinolytic protease.All experimental animals were monitored daily by trained animal caretakers. Animals showing signs of distress were euthanized by cervical dislocation. All experiments were conducted according to institutional and national guidelines. The experiments performed in the current project were specifically approved by the Danish Animal Experiments Inspectorate (permit number 2010-561-1778). Following the experiments, animals were euthanized by anaesthetizing using hypnorm/dormicum followed by perfusion, as described below.\u2212/\u2212 mice and their littermate controls were generated by breeding heterozygous mice (Plg\u2212/+), that had been backcrossed into an FVB/n background for at least 30 generations . C57Bl/6 Plg\u2212/+ mice were likewise backcrossed for at least 30 generations into the C57Bl/6 background. All mice were bred in the SPF facility at University of Copenhagen and during experimentation they were housed individually. Genotyping was performed as previously described Both FVB/n and C57Bl/6 mice were used for wound healing studies. Tissue libraries were derived from FVB/n mice Mice of approximately five weeks of age were anaesthetized using hypnorm/dormicum and the ovaries were excised as previously described Six weeks old mice were used for incisional wound healing studies as described previously Six weeks old mice were injected intraperitoneally (IP) with 1 ml of a 3.85% TG brew , which had been incubated for at least four weeks at 4\u00b0C. After three days, the mice were anaesthetized with hypnorm/dormicum, the abdominal skin removed leaving behind the abdominal muscle tissue, and the mice were then injected IP with 4 ml PBS and gently shaken on a lab vibrator for 1 min. The lavage fluid was harvested and the cell concentration was defined using an automated cell counter .\u2212/\u2212 mice were derived from a tissue collection containing samples isolated from mice with an age of eight, 12 and 26 weeks Liver and skin samples from na\u00efve wildtype (Wt) and Plg+ system in combination with NovaRED HRP substrate . Stained sections were scanned using a motorized Olympus BX51 microscope with a 20X objective controlled by Visiopharm software or by a NanoZoomer-2.0HT using a 20X or 40X objective. In wound tissue, cell counting was performed in randomly selected areas either containing or devoid of fibrin. The average size of these regions of interest (ROIs) was 0.035 mm2. The average thickness of the epidermis was derived from the area of the hyperplastic epidermis divided by the length. The combined area of fibrin rich lesions in the provisional matrix was determined using the staining analysis software VisiomorphDP, which is part of the Visiopharm software package . In the liver, the degree of fibrin deposition was determined on one whole scanned tissue slide from each liver using VisiomorphDP . Likewise, was the area of CD34 positive staining quantitated by use of VisiomorphDP on whole tissue slides scanned at 40X.Paraffin embedded tissues were sectioned, rehydrated and stained using a standard H&E staining protocol. For immunohistochemical detection of fibrin(ogen) and CD34, the following antibodies were used: rabbit-anti-mouse fibrin(ogen) Statistical analyses were performed using Two-way ANOVA, One-way ANOVA with a Newman-Keuls post test or with student\u2019s t-tests.\u2212/\u2212 mice, we conducted a histological analysis of livers derived from a cohort of Plg\u2212/\u2212 mice. Immunohistochemical staining revealed no signs of fibrin rich lesions in livers from Wt mice regardless of age (up to 26 weeks). However, in Plg\u2212/\u2212 mice, fibrin rich lesions were evident already at an age of eight weeks. Computerized staining analysis revealed that the number, size and combined area of these lesions increased significantly up to the age of 26 weeks. However, when comparing livers from male and female Plg\u2212/\u2212 mice it was evident that there were no differences between genders is known to depend on Plg \u2212/\u2212 mice (C57Bl/6 backcrossed for at least 30 generations) of both genders totaling 26 mice. Three days after TG injection, Wt mice presented an almost tenfold increase in the concentration of cells in the peritoneal cavity. This response to TG was independent of gender. When comparing the response to TG in Wt and Plg\u2212/\u2212 mice, a striking difference was observed, as the latter mice seemed completely unaffected by the administration of TG. This, however, was regardless of gender (p\u200a=\u200a0.8885 for the difference between genders) . The lac\u2212/\u2212 mice, we speculated that gender only affected the phenotype of Plg\u2212/\u2212 mice in terms of skin pathologies. As it has been thoroughly documented that skin wound healing is highly dependent on plasmin driven fibrinolysis \u2212/\u2212 FVB mice of both genders to heal 20 mm long incisional skin wounds on the back. No difference in the time to complete skin wound healing was observed between Wt male and female mice , while the time needed to complete wound healing in Plg\u2212/\u2212 mice of both genders was markedly delayed compared to the Wt mice no differences between the genders could be seen , while there were no differences in terms of average time to complete wound closure (p\u200a=\u200a0.2023). These data suggested that it is not only skin tumor growth in Plg\u2212/\u2212 mice that is affected by gender but also other types of tissue remodelling processes that takes place in the skin.It has previously been demonstrated that Plg deficiency affects chemically induced skin tumor growth differently in male and female mice. Whereas Plg deficiency decreased tumor burden by 52% in male mice, biallelic Plg disruption did not affect the skin tumor burden in female mice compared to littermate control mice Wt mice . However be seen . The hea\u2212/\u2212 mice. Indeed, the effect of gender was more pronounced in C57Bl/6 mice than in the corresponding FVB mice . Though C57Bl/6 mice with a dysfunctional PA-system also display fibrin depositions in the liver \u2212/\u2212 FVB and C57Bl/6 mice has never been performed. Thus, the phenotype of Plg deficiency may very well be partly strain dependent.To further substantiate the concept of a gender dependent effect of Plg deficiency in terms of wound healing, a wound healing study similar to the one performed in FVB mice was performed in fully backcrossed C57Bl/6 mice (backcrossed for at least 30 generations). This experiment was carried out by independent research technicians that were unaware of the gender dependent phenotype observed in FVB mice. This experiment confirmed the existence of a gender dependent phenotype in relation to skin wound healing in PlgFVB mice . Notewor\u2212/\u2212 mice to execute tissue remodelling processes in the skin is affected by gender, prompted us to investigate if the known differences between the genders in terms of thickness of the dermal and hypodermal compartments were affected by Plg deficiency. For this purpose we analyzed normal skin samples from the previous mentioned cohort tissue collection \u2212/\u2212 mice.The obtained data, suggesting that the ability of Plg\u2212/\u2212 mice.Expression of distinct extracellular matrix proteases with a potential fibrinolytic activity may to some extent be regulated by sex-hormones. It can thus not be ruled out that female mice, during the process of skin wound healing, have the capacity to express fibrinolytic proteases, which can compensate for the lack of plasmin. To test this hypothesis we analyzed the effect of OVX on skin wound healing in Wt and Plg\u2212/\u2212 mice, the gender differences in wound healing were still clearly evident despite sham and OVX surgeries. However, in contrast to Wt mice, OVX in Plg\u2212/\u2212 mice led to a subtle delay in the initial phase of the wound healing process compared to both sham Plg\u2212/\u2212 male and female mice. However, around day 30 post wounding, it was no longer possible to distinguish between sham and OVX Plg\u2212/\u2212 female mice . The wounds all displayed a hyperplastic epidermis, which covered a dermal wound gap containing yet unresolved granulation tissue. The lengths of the hyperplastic epidermis and wound gap were similar in Wt male and female mice, while the thickness of the hyperplastic epidermis was increased in Wt females. Interestingly, the lengths of the hyperplastic epidermis and dermal wound gap were significantly longer in Plg\u2212/\u2212 mice than in Wt mice, and furthermore, the lengths were 48% longer in Plg\u2212/\u2212 male than in Plg\u2212/\u2212 female mice . Furthermore, the concentration of cells in the fibrin rich areas was decreased by almost 80% in comparison with areas not containing fibrin. In addition, the granulation tissue in Plg\u2212/\u2212 female mice contained fewer cells than in Plg\u2212/\u2212 male mice, both within and outside the fibrin rich areas was compared. CD34 positive vessels were mainly observed within close distance to the epidermis in both Wt and Plg\u2212/\u2212 mice of both genders and no overt differences were observed in terms of size or shape between these groups of mice (\u2212/\u2212 mice having decreased amounts of CD34 compared to Wt mice (Wound healing is intimately associated with the generation of a vascular network throughout the granulation tissue. This process depends on the interaction between endothelial and matrix proteins as well as proteolytic matrix reorginization by MMPs and the PA system of mice . The qua Wt mice .\u2212/\u2212 mice was affected by gender. Furthermore, the gender specific differences in na\u00efve skin described in Wt mice were unaffected by Plg deficiency. Histological examination of healed wounds revealed that the combined area of fibrin rich lesions in the provisional matrix was larger in male Plg\u2212/\u2212 mice than in female Plg\u2212/\u2212 mice, and that these fibrin rich lesions contained far less cells than the surrounding wound matrix in both male and female mice. The difference in the area of fibrin rich lesions between genders correlated well to the differences in the length of the hyperplastic epidermis and amount of granulation tissue in healed wounds as well as to the measured difference in time to complete wound reepithelialization.In the present study the effect of gender on the phenotype of Plg deficiency was illuminated. Three well described characteristics were assessed; spontaneous accumulation of fibrin in the liver, Plg dependent cell recruitment to the peritoneal cavity, and incisional skin wound healing. Among these, only skin wound healing in Plg\u2212/\u2212 mice is the same in males and females, though subtle differences may be observed \u2212/\u2212 phenotype. However, as spontaneous in vivo clot lysis, determined by intra-venous injections of radiolabeled fibrin clots, is remarkably reduced in Plg deficient mice compared to Wt mice \u2212/\u2212 mice. Yet, we find this questionable as identical levels of fibrin depositions in the liver of male and female Plg\u2212/\u2212 mice were observed, which not only suggests that the livers in male and female Plg\u2212/\u2212 mice are equally degenerated over time, but also that a putative but insufficient extravascular fibrin clearance occurs at the same rate in both genders. Furthermore, the evident lack of a gender effect on the recruitment of immune cells to the peritoneal cavity in response to TG injections, suggests that males and females depend equally on plasmin activity to execute an inflammatory response. In accordance with these observations, Plg deficiency has not previously been reported to show a gender effect in macrophage recruitment \u2212/\u2212 mice challenged with a proinflammatory agent, inflammatory reactions seem to be fully executed in some chronic pathological settings, such as in the development of inflammatory colon lesions and skin tumors It is evident that the general phenotype of unchallenged Plg\u2212/\u2212 mice was highly dependent on gender. The intricate process of wound healing involves fibrin clearance, which likely facilitates the recruitment of cells into the granulation tissue and/or keratinocyte migration \u2212/\u2212 mice but not to the extent observed when treating Plg\u2212/\u2212 mice with the broad spectrum MMP inhibitor Galardin \u2212/\u2212 mice may very well be directly linked to obstructed cell recruitment or keratinocyte migration. In the current study, we found, that reepithelialized skin wounds in Plg\u2212/\u2212 mice still contained large dermal wound gaps, which was significantly larger than that observed in Wt mice. This observation suggests that epidermal wound closure may be less affected by Plg deficiency than reconstruction of the underlying dermal compartment.In contrast to fibrin deposition in the liver and immune cell recruitment, we found that incisional skin wound healing in Plg\u2212/\u2212 mice induced a subtle but statistically significant delay in wound closure compared to sham operated female Plg\u2212/\u2212 mice. We speculate, that the enhanced effect of OVX on wound healing in Plg\u2212/\u2212 mice compared to Wt mice may relate to specific events during the healing process. Skin wound healing can be divided into several and partially overlapping phases. Following the initial event of coagulation and haemostasis, neutrophils are recruited to the wound site followed by macrophages, which phagocytose remaining neutrophils and secrete tissue growth factors that activate keratinocytes and fibroblasts \u2212/\u2212 mice in the current study could be related to an additive or synergistic effect of Plg deficiency and depletion of female sex hormones on macrophage migration and activation \u2212/\u2212 mice Concerning the role of female sex hormones on cutaneous wound healing, it was noted that during the first four weeks post wounding, OVX of Plg\u2212/\u2212 C57Bl/6 healed faster than the corresponding FVB mice, thus indicating major differences in either skin composition per se or in one or more of the distinct phases of the wound healing process.Of notice is that we did not observe any gender dependent difference between sham and OVX operated Wt mice with regard to wound healing. This observation is contradicted by previous reports stating a role for estrogen/estrogen receptor \u03b2 interaction in cutaneous wound healing \u2212/\u2212 mice. In male mice we observed larger dermal thickness compared to female mice, a parameter that has been reported to be under the influence of male hormones Additional explicatory factors may relate to endogenous differences between male and female mice in skin properties, such as skin strength and mechanical features. In the current study we therefore performed analyses of unchallenged skin biopsies from Wt and Plg deficient male and female mice. In line with other reports, we demonstrated inherent gender differences in normal skin histology. Although these differences were not affected by Plg deficiency, they might affect wound healing in Plg\u2212/\u2212 female mice compared to Plg\u2212/\u2212 male mice could simply be a reflection of the fact that the overall thickness of the skin is smaller in females \u2212/\u2212 female mice is smaller than in the corresponding males. However, it is conceivable that the decreased level of fibrin deposition in Plg\u2212/\u2212 female mice is secondary to an accelerated fibrinolytic process in comparison with male mice. Interestingly, the differences in dermal fibrin levels between male and female Plg\u2212/\u2212 mice did not seem to result in differences within vessel density of the fully healed wounds. However, disregarding gender Plg\u2212/\u2212 mice did tend to have fewer CD34 positive vessels underneath the hyperplastic epidermis compared to Wt mice. As extracellular proteases mediate angiogenic processes and that angiogenesis is important for skin wound healing \u2212/\u2212 mice may indeed be partly related to hampered angiogenesis.Our data showing that the combined area of fibrin rich lesions in the provisional matrix was significantly lower in Plg\u2212/\u2212 mice is not solely related to sex-hormones, though the lack of female sex-hormones, induced by OVX, affected wound healing in female Plg\u2212/\u2212 mice to some extent. It is possible that the endogenous differences in skin histology between genders are responsible for the varying effect of Plg deficiency on wound healing, which would explain why the gender effect is restricted to the skin.In conclusion, the presented data show that the effect of Plg deficiency is both gender and tissue specific. The results further suggest that the observed differences in wound healing between male and female Plg"} +{"text": "The NIHR HTA funded IQuaD Trial is a 5 year multi-centre, cluster randomised, open trial with blinded outcome evaluation based in dental primary care in Scotland and the North East of England. It compares scale and polish intervals and oral hygiene advice delivery for the maintenance of oral health. We aim to recruit 1860 adults across 60 dental practices. Trials of this size have not previously been carried out in primary dental care, reflecting these uncertainties Outcome Assessors are employed by the trial to go into dental Practices to consent eligible patients as well as collecting baseline measurements.Novel strategies used include:\u2022 1. Trial staff arranging and managing clinical recruitment sessions with Practice staff in advance\u2022 2. Inviting patients to attend recruitment sessions in line with each participating Practices' routine methods of contacting patients\u2022 3. Sending information about the Trial along with an invitation letter and baseline questionnaire to patients in advance of clinical recruitment sessions\u2022 4. Organising flexible appointment systems with Practices allowing patients to opt out of the Trial in advance of their dental appointment or following discussion with trial and dental staff at recruitment sessionsBased on our experiences in IQuaD, we will discuss the challenges of incorporating trial recruitment processes into general dental primary care and how these strategies enabled us to accurately predict and maximise recruitment to meet targets."} +{"text": "The published literature suggests uncertainty about whether operative or nonoperative treatments are best for femoroacetabular impingement (FAI). Without the same level of uncertainty (equipoise) amongst surgeons, a RCT will be challenging. A qualitative study was conducted to explore the level of equipoise amongst arthroscopic FAI surgeons. In phase 1, 14 hip arthroscopy surgeons were interviewed and asked to make treatment decisions based on real life cases that included actively recruiting patients to a theoretical RCT. In phase 2, 9 hip arthroscopy hip surgeons participating in a pilot RCT were interviewed about their experiences so far of taking part in a pilot RCT. Five surgeons took part in both phase 1 and 2. Sixteen (89%) surgeons believed that they were in equipoise and that a RCT was required to generate superior scientific evidence and guidelines for the care. Despite this 5 (36%) surgeons showed a lack of active clinical equipoise when faced with real life case scenarios or discussing involvement with a pilot RCT. Some of the reasons behind surgeons\u2019 lack of equipoise, ranged from lack of belief in the FAI pathology, to personal enthusiasm and gut instinct about the efficacy of surgery on one hand; but conservatism on the other. Although many would like a RCT to guide care, there may be particular challenges amongst this same population when actively recruiting patients to a RCT. Qualitative methodology can be used to help design surgical RCTs and address any subsequent difficulties with recruitment."} +{"text": "Bombus) across spatial scales comparing: local communities to regional pools, regional communities to continental pools and the continental community to a global species pool. Species composition and data on tongue lengths, a key foraging trait, were used to test patterns of relatedness and trait similarity across scales. Although expected to exhibit limiting similarity, local communities were clustered both phenotypically and phylogenetically. Larger spatial scales were also found to have more phylogenetic clustering but less trait clustering. While patterns of relatedness in mobile species have previously been suggested to exhibit less structure in local communities and to be less clustered than immobile species, we suggest that mobility may actually allow communities to have more similar species that can simply limit direct competition through mobility.Despite the expansion of phylogenetic community analysis to understand community assembly, few studies have used these methods on mobile organisms and it has been suggested the local scales that are typically considered may be too small to represent the community as perceived by organisms with high mobility. Mobility is believed to allow species to mediate competitive interactions quickly and thus highly mobile species may appear randomly assembled in local communities. At larger scales, however, biogeographical processes could cause communities to be either phylogenetically clustered or even. Using phylogenetic community analysis we examined patterns of relatedness and trait similarity in communities of bumble bees ( Understanding patterns of species diversity and assembly is a major objective of research in ecology, evolution and biogeography. The recent development of methods to integrate phylogenetics into community ecology\u2013\u201cphylogenetic community ecology\u201d\u2013makes it possible to simultaneously address spatial and temporal questions about how species assemble and what processes impact assemblage membership Phylogenetic methods are commonly used to determine the phylogenetic clustering vs. evenness (i.e. the degree of relatedness), and the degree of phenotypic similarity or differentiation of community members in local communities, compared to null communities drawn from a larger, regional species pool Increasing the scale of analysis used for phylogenetic community analysis could also help expand studies to mobile taxa for which patterns are believed to arise at scales larger than those normally considered by community ecology Bombus spp.) are likely to disproportionately provide pollination service to many crops and wildflowers Bombus could provide both vital information for pollination service in sensitive areas and insight into local assemblage and biogeographical patterns of highly mobile species.Bumble bees offer an excellent model group to test the impacts of spatial scale on patterns of community assembly. As generalist, large bodied pollinators, bumble bees Does tongue length show significant patterns of phylogenetic conservatism? 2) Are there non-random patterns of tongue length or relatedness among co-occurring Bombus species? 3) Do trait and phylogenetic patterns vary with spatial scale? As mobility may allow species to limit direct competition and assemble freely in local communities As a large, native eusocial bee, bumble bees are assumed to exhibit strong intra-generic competition, due to the high resource demand to support colonies. If closely related species or species with similar tongue lengths compete more strongly, we would expect communities to be evenly dispersed in terms of relatedness or trait distributions, respectively. Using the Data on local assemblages were shared by researchers with the full knowledge that it would be used to analyze patterns of relatedness.Bombus tongue length data were collected through literature searches in ISI Web of Science during the spring of 2009 using search terms: (Bombus or bumble*) and (proboscis or tongue). Additional sources were acquired by searching cited literature. Only data for the worker caste that was directly measured as the sum of prementum and glossa were used Psythirus were removed from analysis because they do not have a worker caste and their existence in a community is dependent on an appropriate host.Bombus to acquire original databases on bumble bee species presence in the Nearctic Ecozone. Original data were required because published data was typically pooled spatially or temporally. Only data that was collected from sites greater than one km apart and in which sampling was conducted across all plant species were used to ensure sites were distinct and no species were excluded by sampling a single plant species Bombus or pollinator diversity so although they varied in size and sampling intensity . Using DiscoverLife.org, a freely available database that pools occurrence and location records from museums and databases of global species occurrence, we determined the species that occurred in each grid cell within Nearctic regions of North America (hereafter Nearctic) and compared these to published records of species occurrence when possible. Only data points that had been verified by a taxonomist and georeferenced were used from the Discover Life database. Using predefined regional areas helps limit the variability in species pool size and definition across studies, which can significantly impact power of analysis To test for non-random patterns in observed communities, regional species pools were used to generate null communities for comparison with observed community phylogenetic distance and trait distributions. Regional species pools were defined based on equal area grid cells, following Williams To determine if species trait and phylogenetic structure appear at spatial scales larger than local communities, regional species pools were then compared to the entire Nearctic pool and the continental pool was compared to the global species pool.R 2.10.1 using the picante package All analyses were conducted in To determine levels of trait conservatism, we calculated Blomberg's K value, a metric for describing the distribution of phenotypic variation across the tips of a phylogeny We used the ultrametric, gap-coded, phylogenetic tree published by Hines picantePhylogenetic community analysis can identify patterns of relatedness in assemblages compared to null assemblages via several metrics. Here we use: 1) Mean Nearest Neighbor Distance (MNND) and 2) Mean Phylogenetic Distance (MPD) as defined and implemented in picante). The observed value is then ranked compared to the null values and the p-value is the rank/1000. From MNND and MPD corresponding z- score standardizations referred to as the Nearest Taxon Index (NTI) and Net Relatedness Index (NRI) using the mean and standard deviation are typically calculated to allow comparison across groups. Positive values of NTI and NRI indicate clustering of species in an assemblage compared to the nulls. Multiplying by negative one allows the indices to have more intuitive meaning with negative values indicating phylogenetic evenness and positive values indicating clustering. However, as highlighted by Cooper et al. For comparison, mean null values of MNND and MPD are calculated from 999 randomly generated assemblages with species richness equal to each of the observed assemblages and species selected at random from the regional species pool of the observed community and Mean Trait Distance (MTD). MNTD and MTD are equivalent to MNND and MPD, respectively. Using the tongue length data for Nearctic species we created a phenotypic distance matrix of all species and calculated MNTD and MTD using the same method as above. Just as with phylogenetic distance, observed scores that are larger than nulls indicate limiting trait similarity in an assemblage. Positive Z-scores of MNTD and MTD indicate trait filtering.r) To determine if tongue length was consistently spaced along a trait axis, potentially limiting competition within a site, we calculated the standard deviation of the successive neighbor distance when divided by the trait range within the assemblage , we calculated the MNND, MPD, MNTD, MTD, SDNNr and range using trait and phylogenetic distance matrices for each of the 45 regional assemblages, using the entire Nearctic as the species pool for comparison.We also calculated MNND, MPD, MNTD, MTD, SDNNr and range for the Nearctic assemblage compared to the global species pool to compare patterns of phylogenetic and trait distance within the continent compared to the global species pool. We were able to use the Z-score and p-value of the observed community compared to the null distribution because only one assemblage (the Nearctic) was examined.We found a total of 18 published studies and 1 unpublished Master's thesis with measured worker tongue length spanning 79 species globally and 34 species in the Nearctic see Figure 1We identified 110 assemblages in 8 of the 45 grid cells in Nearctic areas to analyze tongue length and relatedness across co-occurring species. Richness ranged from 2 to 8 species across assemblages and regional species pools for these assemblages ranged from 11 to 20 species see . When teFor the same 110 observed communities above, the trait analysis revealed that tongue length had significantly lower nearest trait distance (MNTD) and significantly more similar overall tongue lengths (MTD) in observed assemblages compared to nulls but was not significant for any trait measure.Using the results above we can look at trends across the 3 spatial scales by plotting the Z-scores compared to null communities. Results for MNND reveal increasing phylogenetic clustering across spatial scales but metrics were similar at local and regional scales (see Bombus had significant clustering of mean phylogenetic distance (MPD) but not of mean nearest neighbor distance which suggests that local assemblages are overall closely related but not simply made up of sister taxa. Traits in local assemblages were also clustered for mean nearest trait distance (MNTD), mean trait distance (MTD) and range which suggests that local assemblages have more similar tongue lengths than expected by chance. As tongue length has strong phylogenetic signal and is prone to convergence on both the global and Nearctic phylogeny, the trait clustering is consistent with phylogenetic clustering. This pattern arises despite high levels of variability in worker size in Bombus nests Under the competition-relatedness hypothesis Bombus communities. Competitive interactions, depending on their strength, can cause assemblages to be clustered or even Similarity in trait values and higher than expected relatedness among co-occurring species may suggest that other biotic and abiotic features are more important in structuring local At regional and continental scales no significant pattern was found for trait metrics but MNND was clustered at both scales and MPD was clustered continentally. The lack of significant clustering at the regional scale is supported by significant SDNNr suggesting even spacing of traits. Clustering of MNND and MPD at the continental scale may be a reflection of geographic barriers to bumble bees reaching the New World while the significant clustering of MNND and lack of clustering of trait metrics in regional areas may suggest radiation to fill the various niches. These regional radiations may also explain why tongue length is less conserved along the phylogeny than expected under Brownian motion. So although it has been suggested that assemblages of mobile species should have less signal of phylogeny over large spatial scales Recent studies in the United Kingdom reported that the observed declines in bumble bee diversity disproportionately affect longer tongued species compared to co-occurring shorter tongued species Table S1Tongue lengths of all Bombus species recorded during study and source. Note: A weighted average was used for species with multiple published measurements. Species are in the same order as in (DOCX)Click here for additional data file."} +{"text": "For patients with partial epilepsy, one or several brain regions generate seizures ('epileptogenic zone' ), which Intracranial EEG times series show that brain regions and seizures may display a large variability in dynamics: (i) some seizures recruit more brain regions than others, (ii) the delays between the onset of the seizure in the epileptogenic zone and other areas can change on a time scale of several seconds, (iii) recruited areas exhibit different levels of coherence with the epileptogenic zone according to their position.Here we propose a model able to reproduce these 3 features . Our"} +{"text": "Cimex lectularius) have been increasing worldwide over the last few decades Bed bug infestations are maintained in nature by zoonotic transmission cycles involving an arthropod and nonhuman vertebrate host. Epidemics occur when transmission spills over to unintended hosts or the pathogen jumps into a new vector species Togaviridae, genus Alphavirus) was repeatedly isolated from cliff swallows, house sparrows, and cliff swallow bugs (Oeciacus vicarius) Alphavirus genus most closely related to FMV are usually transmitted in zoonotic cycles involving mosquitoes and birds , was also repeatedly isolated from swallow bugs during the years 1974\u20131976 From these examples there is reason to suggest that other alphaviruses, such as eastern equine encephalitis virus (EEEV) or other members of the VEEV or WEEV complexes may have already adapted to cimicids, or could do so in the future. Despite a principle transmission cycle involving mosquitoes, EEEV has been isolated from naturally infected chicken mites Bunyaviridiae, genus Orthobunyavirus) was repeatedly isolated from bat bugs (Cimex insuetus and Stricticimex parvus) collected from several caves in Thailand Ornithodorus hermsi) also present in the caves, suggesting a true biological role for bat bugs in the transmission of the virus. The bat bugs aggressively fed on humans who entered the caves to mine bat guano; many such workers complained of illness after entering the caves for the first time and were found to be seropositive for KKV, suggesting true epizootic transmission was occurring In 1970\u20131971, Kaeng Khoi virus may well be supporting undiscovered enzootic transmission cycles. If this proves to be the case, the probability for spillover into human populations will increase as cimicid (including C. lectularius and C. hemipterus) populations continue to increase worldwide. Human populations most at risk would be those coexisting with long-term bed bug infestations such as those living refugee camps Concluding that bed bugs do not transmit infectious agents may be premature, as most published studies that have investigated this question have focused on pathogens unlikely to be vectored by any bloodfeeding arthropod. While the ecological conditions associated with single-dwelling infestations are extremely unlikely to maintain arbovirus transmission cycles, the large and diverse number of cimicid species associated with highly gregarious bat or bird populations around the world (including wild"} +{"text": "Maternal diabetes, genetic predisposition and vascular hypoperfusion have been suggested as possible causative factors but no true causative factor has been determined. Associated organ system dysfunction depends on the severity of the disease. Prenatal ultrasonographic diagnosis of this syndrome is possible at 22 weeks of gestation. We report a case of a four months old male newborn to a known diabetic mother.Caudal regression is a rare syndrome which has a spectrum of congenital malformations ranging from simple anal atresia to absence of sacral, lumbar and possibly lower thoracic vertebrae. It results from a disturbance in the fetal mesoderm in early pregnancy (< 4"} +{"text": "To differentiate pooled external stimuli the sensory center requires distinct neural representations. It is generally considered that one major function of the antennal lobe in Drosophila is to separate the inputs from olfactory sensory neurons (SNs) and encode them into only weakly uncorrelated neural representations at the level of projection neurons (PNs). Several reports ,2 demonsHere, we show that the correlation between odor representations can be either decreased or increased in a model of the antennal lobe given the identical heterogeneous glomerular innervation of local neurons (LNs). The investigation of glomerular innervation of LNs confirmed that whereas some LNs innervate to only a few glomeruli, others innervate many and yet others to almost all glomeruli . Our sim"} +{"text": "Puccinia graminis f. sp. tritici (Pgt TTKSK or Ug99), was discovered in Uganda. Most of the wheat and barley cultivars grown currently worldwide are susceptible to this new race. Pgt TTKSK has already spread northward into Iran and will likely spread eastward throughout the Indian subcontinent in the near future. This scenario is not unique to stem rust; new races of leaf rust (Puccinia triticina) and stripe rust (Puccinia striiformis) have also emerged recently. One strategy for countering the persistent adaptability of these pathogens is to stack complete- and partial-resistance genes, which requires significant breeding efforts in order to reduce deleterious effects of linkage drag. These varied resistance combinations are typically more difficult for the pathogen to defeat, since they would be predicted to apply lower selection pressure. Genetical genomics or expression Quantitative Trait Locus (eQTL) analysis enables the identification of regulatory loci that control the expression of many to hundreds of genes. Integrated deployment of these technologies coupled with efficient phenotyping offers significant potential to elucidate the regulatory nodes in genetic networks that orchestrate host defense responses. The focus of this review will be to present advances in genetical genomic experimental designs and analysis, particularly as they apply to the prospects for discovering partial disease resistance alleles in cereals.Rusts are one of the most severe threats to cereal crops because new pathogen races emerge regularly, resulting in infestations that lead to large yield losses. In 1999, a new race of stem rust, Puccinia species ], wheat leaf rust (Puccinia triticina), stripe rust (Puccinia striiformis), barley leaf rust (Puccinia hordei), and oat crown rust (Puccinia coronata). All of these species can infect a large range of cereal hosts: 365 grass species for Puccinia graminis alone.The heteroecious rust fungi are some of the most important pathogens of cereal crops. These comprise 3000 R) alleles bred into elite varieties . A new threat to wheat and barley production is the discovery of a novel race (Pgt TTKSK) of wheat stem rust in East Africa analyses aimed at identifying partial resistance loci require large population sizes in order to detect these less obvious effects , intermated recombinant inbred lines (iRIL), double haploids (DHs), and back cross (BC). Based on simulation studies, a well-developed RIL population appears to be the most efficient for accurate QTL mapping \u22121, where r is the recombination frequency in the corresponding F2 is much more difficult to dissect than one whose inheritance architecture is oligogenic of a partial resistance allele epistatically depends on the presence of the R gene allele that confers resistance or elsewhere (trans). This level of characterization helps define the putative function of the eQTL, promoter difference vs. transcription factor difference, for example. Also, when searching for genes that act as capacitors of a significant process, it is helpful to know if many genes involved in a process are regulated by a locus where few or none of them reside. Such loci are termed eQTL hotspots and can regulate more than 1000 genes in some cases facilitates these goals.Epistasis has been shown to have an impact on numerous major phenotypic QTL and will likely explain significant variance components of plant gene expression high resolution population(s) that harbor genetic variation for resistance to the pathogen\u2014intermated RILs should provide the resolution, while simultaneously providing a reasonable number of individuals for downstream molecular work; (2) all-genes expression-profiling platforms for the hosts and pathogens in question\u2014with NextGen sequencing technologies, these are becoming possible at a reasonable cost; (3) high-throughput genotyping\u2014several platforms offer the possibility of genotyping individuals with multiplex capability (Poland et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "It has been reported that luteinizing hormone (LH) had thyropropic effect on rat and human thyroid membrane. It has been known that patients with PCOS have elevated LH levels in comparison to healthy controls.The goiter prevalence is more common in women than in men regardless of population. The higher incidence of thyroid diseases in women has been previously attributed to higher estradiol levels. Estradiol has been shown to enhance proliferative and mitogenic activities of thyroid cells. However, in recent years chronic estradiol treatment has been shown to reduce volume densities of thyroid follicles, follicular epithelium and thyroid gland volume. It is thought to be due to LH suppression.Therefore we suggested that increased LH levels might provide a stimulus for growth on thyroid and alter thyroid function. Therefore patients with PCOS who had elevated LH levels should be treated by combined estradiol pills such as estrogen-progestin contraceptives for suppression of LH secretion. Further studies are needed to evaluate the association between LH, LH suppression and thyroid volume in patients with PCOS. Human chorionic gonadotropin (hCG) secreting placental tumors such as hydatidiform moles and choriocarcinomas have been found to be associated with hyperthyroidism and HCG Luteinizing hormone is a glycoprotein hormone like TSH and has similar alpha subunit. In Carayon et al. study LH has been identified to increase thyroid adenylate cylase activity 65 times more potently than HCG in human thyroid membranes [Our hypotheses is increased LH levels might have a growth effect on thyroid gland in patients with polycystic ovary syndrome (PCOS). Polycystic ovary syndrome is a common endocrine disorder affecting at least five to 10% of women of reproductive age .The goiter prevalence is more common in women than in men regardless of population . HoweverIt has been known that patients with PCOS have elevated LH levels ,14 and aIn the recent hypotheses increased LH levels might provide a stimulus for growth on thyroid and alter thyroid function. Therefore patients with PCOS who had elevated LH levels should be treated by combined estradiol pills such as estrogen-progestin contraceptives for suppression of LH secretion. Further studies are needed to evaluate the association between LH, LH suppression and thyroid volume in patients with PCOS."} +{"text": "The multimodal nature of perception has generated several questions of importance pertaining to the encoding, learning, and retrieval of linguistic representations , our percepts appear stable due to dimensionality reduction. These questions become even more complex in multisensory speech perception since we are now dealing with the issue of how visual speech gestures coalesce with the auditory signal as the respective signals unfold at different rates and reach cortical areas at different times. In fact, these signals must co-occur within an optimal spatio-temporal window to have a significant probability of undergoing integration Temporally congruent auditory and visual inputs will be processed by cortical integration circuitry, (2), internal representations (\u201cfixed Bayesian priors\u201d) are compared and matched against the inputs, and (3) hypotheses about the intended utterance are actively generated. van Wassenhove et al.'s (The et al.'s EEG studet al.'s . Predictet al.'s .Hypothetically, visual cues can provide predictive information so long as they precede the auditory stimulus and provide reliable cues (see Nahorna et al., While these studies suggest some rigidity of priors, I would emphasize that prior probabilities or \u201cinternal rules\u201d remain malleable into adulthood. This adaptive perspective finds support among Bayesian theorists who argue that priors are continually updated in light of new evidence. Research indicates that differences in the ability to detect subtle auditory-visual asynchronies changes even into early adulthood (Hillock et al., Such findings appear consistent with a unified parallel framework where visual information influences auditory processing and where visual predictability can be reweighted through learning. Figure"} +{"text": "While many neural coding models have been proposed for V1, it remains an open question as to which model best describes the diversity of observed response properties. For instance, the canonical linear-nonlinear model (LN) partially explains some fundamental mechanistic and phenomenological properties of V1, but is unable to explain many nonlinear response properties that are likely associated with the keys to efficient and robust human vision.For example, surround suppression is one such nonlinear response property in which visual stimuli extending beyond the classical receptive field (CRF) selectively diminish neural responses. This property has been studied through electrophysiology experiments with synthetic stimuli . Surprisingly, high level sparse coding models implemented in a biologically plausible dynamical system have been shown to produce surround suppression effects that match individual and population observed responses . More re"} +{"text": "In the last years severe efforts turned into extensive research on development of novel antimicrobial compounds. Among them, antimicrobial peptides, commonly isolated from several organisms, have been considered part of innate immune system and also as potential antimicrobial drugs. AMPs have variable amino acid composition and size (ranging from less than 5\u2013100 amino acid residues), commonly showing cationic and amphipathic properties. Nowadays about 2300 AMPs have been reported in the Antimicrobial Peptide Database (AMP database). Such research endeavor resulted in more than 100 peptide-based drugs available in the market with approximately 500\u2013600 candidates in pre-clinical test development and host defense peptides (HDP) they also represent important candidates as novel drugs and have been carefully addressed in the current issue. For instance, cationic peptides have been considered as anticancer agents for presenting numerous advantages over chemical agents such as higher specific cytotoxicity to tumor cells, lower side effects and easier absorption as recently listed (Roy et al., Despite the great number of peptides available in databases, isolation of new molecules using classic purification techniques is crucial to identify novel molecules with diverse activities. Indeed a proteinase inhibitor isolated from in vitro and in vivo validation. In this sense it is also necessary to develop rapid assays that can be performed concomitant to million candidates. A recent method enabled the selection of AMPs directly on peptide microarrays allowing identification of AMPs that bind and are bactericide among those that bind but do not kill bacteria (Wimley, Lately not only nature has become a source of AMPs. Besides isolation of natural organisms, antimicrobial peptides might be improved or created using computational tools. This opens even more this so amazing field by creating infinite novel and remarkable possibilities. Recently a study screened the distribution of known motifs in prokaryotic extracellular and virulence proteins across a range of bacterial species in order to identify novel motifs in virulence proteins (Ruhanen et al., Wimley, . In anot Wimley, . Thus ad Wimley, .in silico models, several systemic strategies such as transcriptome and proteome have been utilized for understanding peptide interaction with its target, which may allow improvement or design new molecule properties (Tavares et al., In alignment with Overall the current issue highlights the relevance of such research topic with perspectives to develop entirely new molecules with vast application within health and agricultural field with c higher affinity for its target with concomitant reduction of side effects.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Rearfoot pronation is one of the factors having been linked to overuse injuries in running (1). Many studies have used shoe eversion as a measure for movement about the subtalar joint axis. Others used foot movement expressed as rotations about the anatomical main axes (2). If joint coupling and with that muscle loading is to be investigated it may be beneficial to assess rotations about the anatomical axes, namely the talocrural and subtalar joints.The aim of this study was to describe how the relationship of the two ankle joint movements is affected by moderate geometric changes of the midsole of a running shoe. Secondly, the relationship of total ankle movement in main anatomical planes and the two anatomical ankle joints was to be explored.Eleven experienced runners were asked to run in a running shoe (Nike Pegasus) with neutral (NE), +4\u00b0 varus (VR) and \u20134\u00b0 valgus (VG) midsole. Subjects were given 1 \u2013 2 km of running to accommodate. Three-dimensional kinematics were recorded using an eight-camera system . Ground reaction forces were sampled at 1200 Hz from 2 force plates (Bertec).A lower extremity model was scaled to each individual and inverse dynamics analysis carried out in the Any Body Modelling System (3). Subtalar and talocrural joint kinematics, joint kinetics and muscle activations were extracted after optimisation. Additionally, foot rotations with respect to the leg segment in the cardinal anatomical planes were extracted for comparison. Regression techniques were employed to test for relationships of kinematics and muscle activations (p<.05).Sagittal plane ankle movement consistently showed significant correlations with talocrural joint excursions while movement about the subtalar joint showed moderate to high correlations to movement in the frontal and transverse planes. However, for some subjects these relationships were inverted for non-neutral shoe conditions. Muscle forces were generally closer related to rotations about the anatomical joint axes than projected to the cardinal planes.When addressing the relationship between soft tissue loading and joint kinematics anatomical joint axes orientations should be preferred. It remains, however, open to verification how such loading distribution is affected by anatomical variations in individuals."} +{"text": "Intermittent preventive treatment during pregnancy (IPTp) at routine antenatal care (ANC) clinics is an important and efficacious intervention to reduce adverse health outcomes of malaria infections during pregnancy. However, coverage for the recommended two IPTp doses is still far below the 80% target in Tanzania. This paper investigates the combined impact of pregnant women\u2019s timing of ANC attendance, health workers\u2019 IPTp delivery and different delivery schedules of national IPTp guidelines on IPTp coverage.Data on pregnant women\u2019s ANC attendance and health workers\u2019 IPTp delivery were collected from ANC card records during structured exit interviews with ANC attendees and through semi-structured interviews with health workers in south-eastern Tanzania. Women\u2019s timing of ANC visits and health worker\u2019s timing of IPTp delivery were analyzed in relation to the different national IPTp schedules and the outcome on IPTp coverage was modelled.Among all women eligible for IPTp, 79% received a first dose of IPTp and 27% were given a second dose. Although pregnant women initiated ANC attendance late, their timing was in line with the national guidelines recommending IPTp delivery between 20-24 weeks and 28-32 weeks of gestation. Only 15% of the women delayed to the extent of being too late to be eligible for a first dose of IPTp. Less than 1% of women started ANC attendance after 32 weeks of gestation. During the second IPTp delivery period health workers delivered IPTp to significantly less women than during the first one (55% vs. 73%) contributing to low second dose coverage. Simplified IPTp guidelines for front-line health workers as recommended by WHO could lead to a 20 percentage point increase in IPTp coverage.This study suggests that facility and policy factors are greater barriers to IPTp coverage than women\u2019s timing of ANC attendance. To maximize the benefit of the IPTp intervention, revision of existing guidelines is needed. Training on simplified IPTp messages should be consolidated as part of the extended antenatal care training to change health workers\u2019 delivery practices and increase IPTp coverage. Pregnant women\u2019s knowledge about IPTp and the risks of malaria during pregnancy should be enhanced as well as their ability and power to demand IPTp and other ANC services."} +{"text": "Corticosteroids are drugs that can reduce the inflammation caused by infection, used as an adjunct to antimicrobial drugs to improve the outcome in various infectious diseases, but their role is controversial. Research on the use of corticosteroids in addition to antimicrobials has had conflicting results. Corticosteroids are also known to have certain adverse effects.We have studied the Cochrane systematic reviews addressing the systemic use of corticosteroids in:\u2022 acute bacterial meningitis ; 24 randomized controlled trials (RCTs) enrolling 4041 patients;\u2022 tuberculous meningitis ; 7 RCTs involving 1140 people;\u2022 infectious mononucleosis (to assess efficacy and safety of steroids for symptom control); 4 trials that compared the effectiveness of a steroid for short-term symptom control to a placebo and 3 other trials that used a combination of steroid and an antiviral drug, involving 362 participants;\u2022 acute sinusitis (to assess the effectiveness of systemic corticosteroids in relieving symptoms); 4 RCTs with a total of 1008 adult participants;\u2022 acute pharyngitis 8 trials involving 743 participants (369 children and 374 adults);\u2022 acute pneumonia (to assess efficacy and safety of corticosteroids), 6 studies including 437 participants;\u2022 croup in children (to determine the effect of glucocorticoids), 38 studies were included, enrolling 4299 patients;\u2022 acute viral bronchiolitis in children , 17 trials with 2596 participants;\u2022 severe sepsis and septic shock (to examine the effects of corticosteroids on death at one month in sepsis), 20 studies involving 2384 patients;\u2022 tuberculous pleural effusion , 6 trials with 633 participants.The systematic review and meta-analysis facilitates an interpretation of variable results from different RCTs and is the first step in making evidence based medicine recommendations regarding corticosteroid use."} +{"text": "Dramatic increases in obesity and sugar-sweetened beverage consumption over the past several decades have become major public health and clinical concerns. Obesity rates tripled in 30 years, and sugar-sweetened beverage consumption among children more than doubled in the last 2 decades of the twentieth century . Many chThese trends and the evidence that modest but persistent reductions in calories (approximately 1 can of sugar-sweetened beverage per day) could halt the obesity epidemic for 90% of the population or more has focuAlthough U.S. states have taxed carbonated soft drinks for nearly 100 years as a means of raising revenue, only recently has this policy been evaluated for its potential effect on reducing obesity rates. Evidence on this issue is available primarily in 2 varieties. A host of studies have used data on household consumption and grocery store prices to show that households that encounter higher prices reduce carbonated soft drink purchases . These eThese studies do not fully address several complex behavioral and biological responses, however. These responses include the possibility that consumers would substitute other caloric beverages or foods for sugar-sweetened beverages if only the latter are taxed. Another possibility is that manufacturers would respond to higher taxes by lowering prices (\u201cstrategic pricing\u201d). A third possibility is a dynamic response of weight to a change in the rate of sugar-sweetened beverage consumption. A second variety of studies addresses these issues by directly linking existing state-level carbonated soft drink tax rates to information about both daily beverage consumption and measured weight to estimate actual tax effects. These studies have found little or no evidence that people faced with higher carbonated soft drink taxes have lower weight .How could these 2 sets of studies come to such different conclusions on the effects of carbonated soft drink taxes on weight? A key distinction between these similar approaches is in how substitution patterns are incorporated \u2014 that is, if consumers drink fewer carbonated soft drinks, what, if anything, do they drink instead? Studies of the first variety typically assume that consumers respond only partially to the reduction in carbonated soft drink calories by increasing intake of other caloric beverages such as juice or whole milk. For example, some researchers assume oOn the basis of substantial increases in both obesity and sugar-sweetened beverage consumption in the past decades and the links between them, the effectiveness of sugar-sweetened beverage taxation in reducing rates of obesity is essential to consider in any discussion of public health measures available to reduce obesity rates. Evidence suggests caution in enacting sugar-sweetened beverage taxation legislation with a core purpose of obesity reduction. \u201cBig taxes\u201d may have different effects than the small taxes currently in place, but these big taxes would need to dramatically shift the substitution patterns we see when small taxes are implemented. Big taxes would need to effectively shift people toward water and other low-calorie drinks in a way that small taxes do not; taxes alone would not likely cause such a shift.However, what if sugar-sweetened beverage taxes are ineffective in reducing obesity? Does this mean that we should not consider implementing them as public health measures? Not necessarily \u2014 the broader benefits of tax increases on sugar-sweetened beverages must be considered. Most studies suggest that such taxes will likely reduce consumption of sugar-sweetened beverages, and researchers find thaIn addition to encouraging other potential health benefits, sugar-sweetened beverage taxes may be helpful in reducing obesity rates if they are used as one aspect of a larger, more comprehensive policy approach that aims to redirect consumers away from caloric sweeteners and toward more healthful alternatives such as water or food without added sweeteners. For example, a tax combined with a subsidy for water could be more effective than a tax in isolation. Additionally, a recently proposed extension of a sugar-sweetened beverage tax that instead taxes all caloric sweeteners at the manufacturer level would limit consumers\u2019 ability to easily switch to foods with caloric sweeteners or other unhealthful beverages . A key f"} +{"text": "OpenWorm is an international collaboration with the aim of producing an integrative computational model of Caenorhabditis elegans to further the understanding of how macroscopic behaviour of the organism emerges from aggregated biophysical processes. A core component of the project involves the integration of electrophysiological modelling and predictive-corrective incompressible smoothed particle hydrodynamics (PCISPH) to model how neuronal and muscle dynamics effect the nematode's behaviour. Several tools are being utilised and developed in the course of the project:\u2022 Electrophysiological model parameters are constrained to reproduce experimental measurements using the Optimal Neuron toolkit \u2022 A PCISPH solver is under development - a comb\u2022 A generic model integration framework (Gepetto ) will be\u2022 All electrophysiological models are NeuroML-compatible .\u2022 The Open Worm Browser provides a powerful way to visualise C. Elegans anatomy All of the above mentioned applications are open source, freely available and can be used for modelling other neuronal systems."} +{"text": "Recent studies have demonstrated that frequent laboratory testing does not necessarily relate to better outcomes. Our aim is to reduce unnecessary blood draws for ICU laboratory tests by predicting which tests are likely to return as normal or abnormal and therefore influence clinical management around gastrointestinal (GI) bleeding.An artificial intelligence tool, namely fuzzy systems, was applied to 1,092 GI bleed patients extracted from a large ICU database with over 32,000 patients. A classification approach for laboratory test outcome was utilized for a total of seven outcome variables shown in Table Classification accuracy of greater than 80% was achieved for all of the seven outcome variables Table . SensitiReducing frequent laboratory testing, and potential phlebotomy complications, is a major concern in critical care medicine. If one could predict in advance whether a laboratory test would be normal or abnormal then that particular laboratory test may not be ordered, and thereby reducing potential complications and costs. In this work we present an artificial intelligence method for the classifying the likelihood of a blood test being normal or abnormal. Our results show acceptable classification accuracy both in terms of sensitivity and specificity."} +{"text": "Observational studies suggest that HIV-focused pharmacies can improve antiretroviral therapy (ART) refill adherence, but there is a lack of clear documentation about the kind and variability of adherence interventions that are conducted.To use qualitative research methods to obtain an in-depth understanding of how ART adherence support and counseling is provided in human immunodeficiency virus (HIV)-focused community pharmacies. To determine relevant facilitators and barriers around adherence support from both patient\u2019s and pharmacist\u2019s perspectives.A qualitative research study of patients who patronized and pharmacists who were employed at HIV-focused pharmacies in the San Francisco Bay Area was conducted. Participants were recruited using flyers at HIV clinics and community-based organizations and using blurbs in newsletters. Transcripts were analyzed using grounded theory methods to determine emergent themes in the data.19 eligible patients with a self-reported diagnosis of HIV, who were taking their current ART regimen for at least 3 months, and who obtained their ART from a community pharmacy in the San Francisco Bay Area were included; 9 pharmacists who were employed at 9 different pharmacy locations frequented by participants were interviewed. Emergent themes included descriptions of pharmacy adherence counseling and support, roles and responsibilities regarding medication adherence, barriers to providing adherence support, and feeling connected as a facilitator to adherence support relationships.Pharmacists provide diverse types of ART adherence support and are uniquely positioned to help clients manage their medications. Additional training on developing relationships with patients and advertising their adherence services may further the role of community pharmacists in supporting antiretroviral adherence."} +{"text": "The health and weight control benefits of low carbohydrate diets are well established. Likewise, nutrient timing has been shown to effectively enhance exercise performance. However, there exists an apparent conflict between these two dietary strategies. In fact, many authorities consider high glycemic carbohydrates to be a necessary component of nutrient timing and there is no place in athletic training or competition for low carbohydrate diets.Various low carbohydrate diets have been shown to provide beneficial changes in body mass, lipid profiles and other health risk factors. Recent evidence indicates that diets with lower glycemic index carbohydrates and increased protein provide greater weight loss and maintenance of the reduced weight as compared with high glycemic and low protein diets. Insulin release is lower with lower blood glucose levels, thereby reducing fatty acid metabolism and storage. High intake of high glycemic response carbohydrates is associated with increased levels of insulin resistance which appears to be the common link between obesity and metabolic syndrome.Nutrient timing is generally regarded as a nutritional strategy in which precise amounts of particular nutrients are delivered at precise time points, relative to exercise, in order to enhance performance or training effects. This somewhat general definition has been operationally limited by many to diets that utilize high glycemic carbohydrates prior to, during, and/or following exercise. These carbohydrates are considered vital as they provide an energy source as well as inducing increased insulin levels. As insulin directly influences the production of nitric oxide, vascular musculature is relaxed and circulation into the capillary beds of exercising muscles is increased. Carbohydrates, in particular higher glycemic carbohydrates, supply these critical benefits.The basic model of low carbohydrate nutrient timing applies specific proven micronutrients for enhanced exercise performance rather than relying on the ingestion of sugar and the subsequent insulin responses. First, reduced carbohydrate intake produces reduced insulin responses which shifts the metabolism to fatty acid utilization. Secondly, various nutritional components can provide additional energy sources and/or produce increased nitric oxide production with subsequent vasodilation. Items such as creatine and beta alanine can influence energy levels by affecting energy replenishment and acting as an anaerobic buffer. Branched chain amino acids provide a third energy source without which muscle tissue may be consumed with intense exercise. Various micronutrients can increase muscle blood flow to some degree. In particular, glycine proprionyl l-carnitine (GPLC) has been shown to dramatically increase nitric oxide synthesis in response to exercise stresses and to significantly increase exercise performance with reduced production of lactate.The limited research in the area suggests that some athletes can train and compete in certain settings successfully with relatively low intake of dietary carbohydrates. It has been shown that pre-workout supplements containing common ingredients such as creatine, beta alanine, branched chain amino acids can substantially enhance exercise performance without ingestion of additional carbohydrates. Controlled clinical trials are needed to examine the effectiveness of nutrient timing with a low carbohydrate diet in various sports settings."} +{"text": "With this study we wanted to test the hypothesis that individual like and dislike as occurring in relation to brand attitude can be objectively assessed. First, individuals rated common brands with respect to subjective preference. Then, they volunteered in an experiment during which their most liked and disliked brand names were visually presented while three different objective measures were taken. Participant's eye blinks as responses to acoustic startle probes were registered with electromyography (EMG) (i) and their skin conductance (ii) and their heart rate (iii) were recorded. We found significantly reduced eye blink amplitudes related to liked brand names compared to disliked brand names. This finding suggests that visual perception of liked brand names elicits higher degrees of pleasantness, more positive emotion and approach-oriented motivation than visual perception of disliked brand names. Also, skin conductance and heart rate were both reduced in case of liked versus disliked brand names. We conclude that all our physiological measures highlight emotion-related differences depending on the like and dislike toward individual brands. We suggest that objective measures should be used more frequently to quantify emotion-related aspects of brand attitude. In particular, there might be potential interest to introduce startle reflex modulation to measure emotion-related impact during product development, product design and various further fields relevant to marketing. Our findings are discussed in relation to the idea that self reported measures are most often cognitively polluted. These days, our environment not only provides us with stimuli that have to be evaluated to directly protect the conservation of oneself in order to survive. Instead, a lot of stimuli out there are not critically important, but still do play a certain role in our everyday life. Brand names are good examples for such stimuli. They stand for products sold under their names. They are associated with knowledge about respective products, with experience related to companies producing and/or selling them and supposedly they are also associated with emotions altogether forming the basis for individual brand attitude. From a company's perspective, creating a positive brand attitude is of utmost importance. The rationale for this circumstance is twofold. First, the attitude towards an object affects individual object-related behaviour However, given the vast amount of research including brand attitude to explain and predict other phenomena, there is a surprisingly small amount of conceptual and empirical discussions on the concept of brand attitude itself. Brand attitude comprises cognitive aspects in terms of brand associations Although largely viewed as separate functions, emotion and cognition are more and more considered interdependent. More precisely, it is believed that they influence each other while their merged output drives decision making and finally behaviour Applied Neuroscience offers a number of ways to focus on emotion-related processing in the human brain. Besides common brain imaging methods such as electroencephalography (EEG) Besides the objective nature of this method (no language-related explicit responses have to be given by study participants) the crucial advantage of startle reflex modulation is its independence from cognitive information processing and from arousal. It was demonstrated in 1988 The fact that startle responses quickly adapt to changing emotion context together with its independency from cognitive influence makes it an ideal tool to quantify emotion on a fine graded scale.We therefore formed the hypothesis that ongoing visual perception of strongly liked brand names elicits less pronounced startle responses compared to ongoing visual perception of strongly disliked brand names. According to the above mentioned features of startle responses we further link any such significant findings with differences in emotion-related information processing initiated during visual perception of known brand names.Besides startle reflex modulation, two traditional measures in psychophysiology, skin conductance and heart rate, are also known to be sensitive to certain aspects of emotion The hypothesis here is that both heart rate and skin conductance differ significantly while being recorded during ongoing visual perception of written brand names as a function of prior stated subjective preference.This study was conducted by using non-invasive methods, thus no local ethics committee was needed to carry it out at our institution. However, it was ethically approved by the head and by the deputy head of the Biological Psychology Unit of the Faculty of Psychology. In addition, written informed consent was given by all participants who could withdraw at any time during the experiment without any negative consequences.Bank Austria (see acknowledgements). They all gave their written informed consent.29 young and healthy study participants volunteered in the present study. 8 participants had to be excluded from the analysis because of unwanted muscle-artefacts, no clear startle responses or too many missing values. The mean age of the remaining 21 participants was 28.14 (SD\u200a=\u200a6.29). They were all right handed and with normal or corrected to normal vision. All volunteers were paid for participation which was possible due to a generous financial support by the Austrian Bank www.globalpark.com). This online survey allowed study participants to rate all brand names according to individual attitude (like or dislike). For this purpose each brand name was simultaneously presented with a slider scale ranging from 1 to 21 . A bar could be placed at one of the fine grades between the extremes by using a computer mouse. The left side was meant to rate for dislike-related intensity while the right side was meant to rate for like-related intensity. As a consequence of this pre-evaluation we were able to identify the 10 most liked and the 10 most disliked brand names out of the entire list for every participant . These individual sets of 20 brand names were then introduced as independent variables (target brands) in a following startle reflex modulation experiment which also included measures of skin conductance and heart rate. For every participant a separate list was created including individual target brand names mixed up with 180 non-target brand names (filler items). The order of target and non-target brand names was randomised between participants.300 common brand names (common in German speaking countries) were put together on a list on the basis of subjective selection by the experimenters. These brand names were all presented through an online survey using the EFS Survey solution offered by Globalpark method to transform EMG signals into amplitudes. The resulting amplitudes were then subject to statistical analysis.Eye blinks as startle responses were elicited by a 50 ms burst of acoustic white noise at 105 dB sound pressure level delivered through professional headphones (AKG) fully covering both ears. Sound pressure level was measured with a mobile measuring device (produced by Voltcraft). To achieve the respective loudness a commercial headphone pre-amplifier was used . With bipolar electromyography (EMG) carried out with a Nexus-10 mobile recording system (by Mind Media BV) muscle potential changes of the Skin conductance was recorded at a rate of 32 samples/s with a Nexus-10-SC/GSR sensor (Two finger sensor) connected to the Nexus-10 recording system with a 24 bit resolution which is able to register changes of less than 0.0001 microsiemens. We attached one sensor to the middle finger and the other sensor to the ring finger of the left hand.Heart rate was calculated from the raw blood volume pulse signal recorded at a rate of 32samples/s with a Nexus-10-Blood Volume Pulse sensor. This sensor works with near infrared light, a method known as photoplethysmography. It was also connected to the Nexus-10 recording system and attached to the left index finger.After arriving at the lab every participant was seated on a comfortable chair viewing a computer monitor which was placed in front of them on a desk. It was all set up in a specially designed experiment room with a computer monitor on a table and one chair. A door could be closed to leave each study participant alone to fully concentrate on the task. All stimulus presentation and physiological signal recording could be controlled from outside. The procedure was explained to the participants while all sensors were attached, the cables connected to the recording system and all computers and monitors turned on. To visually present the individually prepared stimulus lists we used the software Biotrace+ (Mind Media BV). Each brand name (white letters on black background) was presented for 5 s with an inter stimulus interval of 2 s. Between stimuli a black screen with a fixation cross in the centre was shown. As mentioned above, stimulus lists were created individually based on each subject's online rating. Thus, lists differed between subjects. The 180 filler brand names were randomly mixed with the 20 individual brand names for every subject. All 20 target brand names were associated with a startle probe at 4.2 s after their onset. Inter stimulus interval between startle probes was kept between 40 s and 70 s to avoid unwanted startle habituation effects. This was done although it was demonstrated that the modulatory influence of the startle reflex is much less subject to habituation than is the obligatory startle pathway The instruction given to study participants was to read each brand name and to rate their buying intention on a five-graded scale. This instruction was simply used to keep our participant's conscious attention focused on the brand names. All participants were naive to the nature of the experiment before being contacted, but they were fully informed shortly before actual testing.BioTrace+ was also used to manage data registration and following data pre processing before statistical analysis.All raw EMG signals following startle stimulations (20 per participant) were recalculated into amplitude signals by using the root mean square method (RMS) for all study participants (as implemented in the software Biotrace+) . For comMean skin conductance values (in microsiemens) as well as mean heart rate values (in beats per minute) were calculated for time windows starting from the onset of brand name presentation until 4 s after the onset for all 20 target brands. No missing values occurred. The final data sets each included 10 single skin conductance or heart rate values for liked brand names and further 10 single skin conductance or heart rate values for disliked brand names. These two separate data sets were then subject to statistical analysis in terms of repeated measures ANOVA to calculate condition main effect significance. Effect sizes were also calculated to better interpret statistical findings.2\u200a=\u200a.203). The mean eye blink amplitude during visual presentations of liked brand names across all study participants was 31.98 \u00b5V RMS (SD\u200a=\u200a22.4). The mean eye blink amplitude related to visual presentations of disliked brand names across all study participants was 34.94 \u00b5V RMS (SD\u200a=\u200a20.94). Repeated measures ANOVA revealed a significant condition main effect on eye blink amplitudes . Despite relatively high standard deviations because of inter individual differences, within-subject analysis revealed a significant condition main effect. We found a significantly reduced skin conductance value associated with visual presentations of liked brand names compared to disliked brand names Repeated measures ANOVA revealed a significant condition main effect . We found a strong trend towards a significantly reduced heart rate associated with visual presentations of liked brand names compared to disliked brand names . Repeated measures ANOVA revealed an almost significant condition main effect are most likely influencing eyeblink amplitudes as responses to acoustic startle stimulation. There are reports about various other influences as well, but these seem to depend on specific circumstances related to stimulation procedures and instructions given to participants. According to a review about the psychological significance of human startle eyeblink modification by The startle reflex modulation findings from the present study can be interpreted in two directions. First of all, they are suggested to provide empirical evidence that brand attitudes indeed do contain emotion-related aspects with a specific focus on valence. This first claim is based on numerous previous investigations using startle reflex modulation. By using all sorts of emotional foreground stimuli such as facial expressions, sounds The skin conductance data from the present study also demonstrate a clear sensitivity related to subjective brand preference. Skin conductance was markedly reduced in case of visual perception of liked brand names versus disliked brand names. Skin conductance is usually suggested to measure levels of arousal rather than emotion valence In the present study, skin conductance was significantly reduced in case of viewing liked brand names compared to viewing disliked brand names. It can therefore be concluded that visual perception of liked brand names elicited a more relaxing state compared to disliked brand names. Whereas our startle reflex modulation data reflect more positive emotion in association with liked brand names our skin conductance data add that liked brand names elicit a more relaxing state than disliked brand names.It is known that heart rate in humans varies over time even in the absence of any changes in physical activity. Besides respiration rate having an influence on heart rate In particular, the significance of startle reflex modulation could be successfully demonstrated. Attitudes towards brands contain describable emotion aspects. Startle reflex modulation offers the opportunity to get insight into emotion-related aspects of brand attitude without demanding explicit responses. It overcomes the weaknesses traditional self-report measures of brand attitude have to deal with. It overcomes the issue that many responses to attitude questions may be measurement artefacts created simply because the question was asked"} +{"text": "Mammalian target of rapamycin (mTOR) kinases are emerging as master regulators of cellular metabolism During infections, proinflammatory and anti-inflammatory immune responses are cross regulated to achieve host protection while limiting pathologic insult ENV-mediated signal transduction, which impedes autophagy in mucosal DCs, resulting in reduced autophagosome degradation, less processing and presentation of HIV-1 antigens, and enhanced viral transfer from DC to CD4+ T cells are known to activate the mTOR complexes in DCs, triggering the production of the anti-inflammatory cytokine IL-10 and thereby promoting their survival in the host Leishmania donovani and Leishmania major. Toxoplasma gondii targeting of mTORc2 may limit the mobility of the host cell and may prevent the spreading of the infection mTOR is a serine/threonine kinase that performs fundamental roles in integrating cell growth and metabolism Leishmania infection in macrophages have shown that virulence factors directly modulate mTOR stability Studies of Leishmania infection We speculate that many potential pathogen targets exist which could be influenced for the benefit of the host by modulating mTOR signaling. Robust generation of the type I interferons (IFN-\u03b1 and IFN-\u03b2) from DCs is one of the crucial early innate antiviral immune responses. It has been demonstrated that blocking mTOR or its downstream signaling molecules impairs the production of type I interferon from plasmacytoid DCs; thus, preemptive activation of mTOR signaling in DCs could stop many infections before they get a chance to establish a foothold. However, blocking mTOR signaling could be beneficial for the replication of therapeutic oncolytic viruses, which are sensitive to type I interferon + T cell fate mTOR complexes are also implicated in the generation of durable and functional antimicrobial memory T cell responses. Rapamycin administration leads to an enhancement of the quality and quantity of memory T cell responses in the lymphocytic choriomeningitis virus (LCMV) model In the context of the apparently contrasting immunological outcomes of mTOR's function in T cells and APCs Leishmania would activate mTOR in macrophages and effector T cells while suppressing mTOR in T cells destined to become memory cells. This would limit pathogen replication, promote immunity, and generate protective memory. This therapeutic modality of reversion of propathogenic metabolic phenotype may lead to host protection that could rightly be called \u201cimmuno-metabolic therapy.\u201d Thus, the differential host and pathogen, immune cell type-specific and disease phase\u2013specific functions of mTOR represent a conundrum that must be carefully considered.Pathogens are armed with virulence factors that target mTOR to manipulate the host towards a unique metabolic state with immunological outcomes that favor pathogen survival. In contrast, there are few effective antibiotics that alter the host metabolism to favor pathogen clearance. One reason for this is that systematic delivery of metabolism-altering agents such as rapamycin often has opposing actions: they limit pathogen replication but also alter the immune response to limit pathogen clearance. We propose the design of drugs that promote distinct and likely contradictory immunological changes in the APC and T cells. For instance, the optimal therapy to combat"} +{"text": "Taxonomically exhaustive and continent wide patterns of genetic divergence within and between species have rarely been described and the underlying evolutionary causes shaping biodiversity distribution remain contentious. Here, we show that geographic patterns of intraspecific and interspecific genetic divergence among nearly all of the North American freshwater fish species (>750 species) support a dual role involving both the late Pliocene-Pleistocene climatic fluctuations and metabolic rate in determining latitudinal gradients of genetic divergence and very likely influencing speciation rates. Results indicate that the recurrent glacial cycles caused global reduction in intraspecific diversity, interspecific genetic divergence, and species richness at higher latitudes. At the opposite, longer geographic isolation, higher metabolic rate increasing substitution rate and possibly the rapid accumulation of genetic incompatibilities, led to an increasing biodiversity towards lower latitudes. This indicates that both intrinsic and extrinsic factors similarly affect micro and macro evolutionary processes shaping global patterns of biodiversity distribution. These results also indicate that factors favouring allopatric speciation are the main drivers underlying the diversification of North American freshwater fishes. Levels of genetic divergence and mutation rates vary between and among species as well as between geographic regions Many factors have been hypothesised to influence global patterns of biodiversity distribution among taxonomic groups and geographic regions. A first general hypothesis suggests that climatic fluctuation of late Pliocene - Pleistocene glacial cycles has played a major role in shaping a latitudinal biodiversity gradient A second general hypothesis suggests that metabolic rate influence the pace of evolution in acting on mutation rate A full understanding of the relative role of climatic fluctuation and metabolic rate in shaping global patterns of diversity requires not only investigating geographic patterns of species richness but also those of genetic divergence at both the intraspecific and interspecific levels North American freshwater fish fauna represent an exceptional model to investigate the determinants of global pattern of biodiversity. First, it is species rich with 903 formally recognised species in the U.S. and Canada, and yet most species are endemic to the continent In this study, we test the two general hypotheses that temporal habitat stability associated with the late Pliocene - Pleistocene glacial cycles, as well as metabolic rate associated with thermal regime and size, have dually contributed in shaping global patterns of intraspecific and interspecific genetic divergence. More specifically, we predict 1.a) a negative relationship between genetic divergence and latitude, as well as 1.b) a breakpoint in this relationship located at the known southern limits of Wisconsinan glaciers (46\u00b0), if glacial cycles have importantly influenced current patterns of genetic divergence. Under the hypothesis that metabolic rates largely influence genetic divergence and mutation rates, we predict 2.a) a positive relationship between genetic divergence and metabolic rate, 2.b) that the explanatory power of metabolic rate should be higher than the ones of body size and temperature, as well as 2.c) a tendency for mutation rates to increase with metabolic rate. To test those hypotheses, we measured the extent of mtDNA phylogroup divergence within species and the extent of genetic divergence between closely related species from the majority of North American freshwater fish species. We then use generalized linear mixed models to assess the relationship of genetic divergence with latitude, metabolic rates, body size and temperature. We verify the effect of metabolic rate on mutation rate by using ratio of branch length at node between sister species.www.boldsystems.org).We first obtained two datasets, one at the intraspecific level and the other at the interspecific level, including only native North American species that spend their entire life in freshwater, thus excluding those that are obligatory diadromous or mainly occurring in saltwater. These two datasets were obtained from an initial dataset including 5674 specimens from 752 species oM \u22121/4 e\u2212E/kT, where B is the metabolic rate, bo is a normalization coefficient independent of mass and temperature, M is body mass, e\u2212E/kT is the Boltzmann factor, which underlies the temperature dependence of metabolic rate . To estimate body mass, we first obtained maximum total length from literature for each species 2\u200a=\u200a0.48, For each species included in the analysis, we first used extent literature We tested the relationship between the genetic divergence parameters and the hypothesised explanatory variables using generalized linear mixed models (GLMM). Such models are particularly well suited for dealing accurately with non-normal data and random variables We performed an additional test in order to further assess the association between genetic divergence and the late Pliocene\u2013Pleistocene glaciations events. We predicted a breakpoint in the relationship between sequence divergences and midpoint latitude located at the known southern limits of Wisconsinan glaciers (46\u00b0), if glaciations had a significant impact on geographical patterns of genetic divergence. We tested this prediction using piecewise generalized linear mixed models, involving or not a breakpoint at 46\u00b0 of latitude, between both phylogroup and nearest neighbour species sequence divergences and midpoint latitude. Lower AIC values We also performed an additional test in order to further investigate the effect of mass specific metabolic rate on patterns of sequence divergence and directly tested if mutation rates increase with mass specific metabolic rate. Therefore, we measured relative mutation rate by using the ratio of branch length at node between sister species under the following rationale. For any given pair of sister species, the sister is each other\u2019s closest relative and thus form a monophyletic group. Their time of divergence from the closest common ancestor is thus the same and differences in the number of mutations between the node and the tips of two sister species is theoretically predicted to reflect differences in mutation rates We also verified if there is a correlation between genetic divergence at both mitochondrial and nuclear DNA levels using published data Our results show a highly significant effect of both midpoint latitude and mass specific metabolic rate on the extent of phylogroup genetic divergence whereby phylogroup sequence divergence decreases with midpoint latitude and increases with mass specific metabolic rate . The besResults of the generalized linear mixed model analysis using different combination of explanatory variables support the results of the generalized linear mixed model conducted using both variables independently. We only present the model having the lowest AIC value, considered to be the best model, in The random effect order had a highly significant effect in all models , meaning that some orders were characterized by higher levels of intraspecific or interspecies divergence than others. For example, levels of phylogroup divergence are the highest in Siluriformes and particularly low in Esociformes . For nearest neighbour species divergence, the extremes were observed in Siluriformes and Scorpaeniformes .We observed that the piecewise regression involving a breakpoint at glacier margin (46\u00b0) had a better fit (lower AIC) than the simple midpoint latitude model in explaining patterns of genetic divergence between nearest neighbour species, but not for phylogroup divergence within species . HoweverWe found a significant relationship between mutation rate and mass specific metabolic rate. Results of non parametric 2-tailed sign test and parametric linear regression support the hypothesis that species with higher mass specific metabolic rate had significantly higher mutation rates. We found no relationship between mutation rate and body size . Based on our analyses of a published North American cyprinid phylogeny that used both mitochondrial and nuclear DNA data sequences Our results globally support the hypothesis that late Pliocene \u2013 Pleistocene climatic fluctuations that caused glacial cycles had a generalised effect on both the patterns of intraspecific (phylogroup) and interspecific (nearest neighbour species) genetic divergence over the whole North American freshwater fish fauna. We observed a significant and negative relationship between latitude and genetic divergence as well as a breakpoint in the relationship that corresponds to the southern extends of the Wisconsinan ice sheet in the case of interspecific divergence . Even usCoregonus sp. Gasterosteus sp. Oncorhynchus sp. Gasterosteus sp.) in North America are limited to a few lakes of the north-western Pacific coast whereas the species is ubiquitous to coastal areas all over North America The generally lower level of sequence divergence between nearest neighbour species at higher latitudes may suggest younger burst of ecological speciation influenced by climatic fluctuation 2\u200a=\u200a0.475, p-value\u200a=\u200a0.0296), indicating that more lineages might be near the final stage of allopatric speciation in southern latitudes relative to northern.The potential for genetic divergence and allopatric speciation might have been greater at southern latitudes because phylogroups and species occurring in these temporally more stable environments could survive long periods of time in geographic isolation (that is lower extinction rate associated with habitat loss), and thus accumulate more pronounced genetic differences. Here, we reiterate that the majority of analyzed intraspecific phylogroups are geographically partitioned (88%) Our results also suggest that metabolic rates significantly impact the continental wide pattern of intraspecific and interspecific sequence divergence of the North American freshwater fish fauna. This was supported by the positive and significant relationship between metabolic rates and genetic divergence, by a better model fit for this factor compared to midpoint latitude, body size and temperature , as wellHigher mutation rates may induce faster divergence and speciation rates Although it often remains challenging to tease apart the effect of correlated characteristics, our results suggest that mass specific metabolic rate have a prime effect on genetic divergence and molecular evolution. Indeed, mass specific metabolic rate is correlated with the thermal environment, particularly so in poikilotherms. Thus, mass specific metabolic rate is also expected to covariate with latitude, which likely explains the similar patterns observed for both factors. However, the fact that the model considering mass specific metabolic rate had a better fit and explanatory power than the midpoint latitude and temperature models and thatWe thus propose that across the whole North American freshwater fish biota, genetic divergence and speciation rates has been particularly high in southern latitude because of more stable environments, allowing longer geographic isolation, coupled with a faster pace of molecular evolution causing more rapid accumulations of genetic differences and incompatibilities. This coupled, in conjunction with a lower probability of extinction relative to northern latitudes, may be largely responsible for the latitudinal gradient of species richness observed among North American freshwater fishes By simultaneously considering the dual roles of two very distinct mechanisms generating a spatial variation in intraspecific and interspecific genetic divergence, this study sheds new light on some longstanding questions in ecology and evolution. First, while some previous studies have suggested that current latitudinal gradient of diversity is primary the results of extrinsic factors www.boldsystems.org).Sequence data are available on GenBank . DNA sequences and sampling information for all species are available on the BOLD website in the project \u201cNorth American freshwater fish\u201d . This dataset is described in detail by April and collaborators (2011).(DOCX)Click here for additional data file.Table S1Results of generalized linear mixed models using a dataset that includes species sharing haplotypes. For the model testing the presence of a breakpoint at glacier margin, we conducted a piece-wise regression involving a break at 46\u00b0 of latitude which corresponds to the maximal extent of Pleistocene glaciations events.(DOCX)Click here for additional data file.Table S2Pearson correlations between explanatory variables.(DOCX)Click here for additional data file.Text S1List of species included in the intraspecific divergence analyses.(DOCX)Click here for additional data file.Text S2List of species included in the interspecific divergence analyses.(DOCX)Click here for additional data file.Text S3List of species included in the analyses testing the correlation between mass specific metabolic rate and mutation rate.(DOCX)Click here for additional data file.Text S4List of species included in the analyses testing the correlation between mtDNA divergence and nucDNA divergence Data from: Sch\u00f6nhuth S, Mayden RL (2010) Phylogenetic relationships in the genus Cyprinella (Actinopterygii: Cyprinidae) based on mitochondrial and nuclear gene sequences. Mol Phylo Evol 55\u223677\u201398.(DOCX)Click here for additional data file."} +{"text": "Diabetic patients are considered as a high-risk population for the development of pulmonary tuberculosis (PTB). Usually, PTB is found predominantly in the upper lobes. Lower lung field tuberculosis occurs but is often misdiagnosed as pneumonia, carcinoma, or lung abscesses. In a number of published comparative studies, chest X-ray images from patients having PTB with diabetes mellitus (DM) have been described as \u2018atypical\u2019, mainly because they frequently involve the lower lung fields, often with cavities.2 A higheWe studied 50 patients with pulmonary tuberculosis with or without extra pulmonary tuberculosis having DM. All other forms of extra pulmonary tuberculosis, and HIV seropositive patients were excluded from the study to allow better data comparison. All were subjected to sputum smear for AFB examination, X-ray chest PA view and hematological investigations. Bronchoalveolar lavage and mantoux test were performed only in selected patients. Patients were considered to have a diagnosis of diabetes mellitus if they were receiving insulin or an oral hypoglycemic agent at the time of hospital admission or were found to have two or more fasting blood glucose levels greater than 140 mg%. Upper lung field tuberculosis was defined as tuberculosis involving upper zone. Lower lung field tuberculosis was defined as tuberculosis involving the middle zone and/or lower zone. Cavitation was considered to be present only when its diameter was more than 2 cm.We found that there was a higher involvement of lower lung field (84%) as compared to upper lung field (16%). Bilateral involvement was present in 32% while unilateral involvement was present in 68%. Ten patients out of the 50 had cavitary disease. Cavitary lesions were more frequently confined to lower lung field (80%). Nodular lesions were found in 36%, exudative lesions were found in 22% and mixed lesions were found in 22%.There had been much debate concerning the atypical radiographic findings of TB. Some authors have repWe conclude that the patients with tuberculosis and DM are more likely to present with atypical radiological images. Among diabetic patients presenting with lower lung field lesions, possibility of TB should always be considered for prompt diagnosis and management."} +{"text": "Adenoma or florid papillomatosis of the nipple (FPN) is a rare benign disease which has histopathological features similar to those of a mammary papillary carcinoma. Here, we report a rare case of bilateral florid papillomatosis of the nipple and breast cancer, with a literature review. Adenoma or florid papillomatosis of the nipple (FPN) is a rare benign disease which clinically resembles Paget's disease of the nipple and has histopathological features similar to those of a mammary papillary carcinoma . In thisin situ with apocrine features and microinvasive changes. Twelve years prior to that, she had a benign cyst removed from her left breast. Her mother died of ovarian cancer.A 63-year-old Caucasian woman presented to our breast clinic with a cracked right nipple and a chronic yellow discharge for 1 year. She had a left side mastectomy with Latissimus Dorsi flap reconstruction 2 years ago for breast cancer. Histopathological examination of the left mastectomy specimen reported features of left nipple florid papillomatosis along with multifocal ductal carcinoma Findings on clinical examination were those of eczematous-like changes of her right nipple with crusting and nipple inversion. A yellow discharge could be expressed on examination and no palpable lumps were found in her right breast or either axillae. Her right breast mammogram showed heterogeneous glandular parenchyma which was unchanged compared to previous examinations. No new suspicious mammographic features were identified. The patient has subsequently undergone a major duct excision with a specimen size of 30 \u00d7 27 \u00d7 17\u2009mm.Histology of the breast tissue included major nipple ducts in the breast tissue, several of which showed florid epithelial hyperplasia with papillary hyperplasia in some areas . Ducts eThis patient's case was discussed at our multidisciplinary meeting and it was decided that close followup is the best option to detect any future changes in her right breast.in situ).The term \u201cpapillomatous breast lesions\u201d refers to benign proliferative epithelial breast lesions with underlying papillary architecture. The papillary layers are composed of glandular epithelial cells, basal myoepithelial layers, and fibrovascular cores. Basement membranes enclose these layers in a \u201cpapillary pattern.\u201d Such lesions can either be central or peripheral . In a significant proportion of mammary papillomatous lesions, atypical changes can occur which constitute atypical proliferative tissue of the ductal type , revision of previous literature series indicated the presence of a risk for developing malignant changes in up to 14%\u201317% of patients with FPN, even in the absence of cellular atypia at the time of FPN biopsy. Furthermore, there is also the low risk of developing subsequent carcinoma following excision of FPN which warrants clinical followup.To the best of our knowledge, this is the first reported case of bilateral florid papillomatosis of the nipple (FPN) with an associated metachronous cancer. FPN is a rare benign disease entity which is proved to be associated with the risk of development of intraductal and invasive mammary carcinomas, either at the time of diagnosis or even years following the benign disease excision. Disease excision (in contrast to mastectomy) and close followup are the best management option."} +{"text": "Professionals promoting hand hygiene (HH) with multimodal strategies featuring the introduction of alcohol-based handrub (ABHR) are confronted by a common set of impeding opinions regarding the indications, safety and efficacy of ABHR. Some of these could be referred to as HH myths.Clostridium difficile disease; and 5) ABHR as a fire hazard.We undertook a review of the literature to assess the currently available evidence regarding five common barriers to the successful implementation ABHR: 1) poor HH before patient contact; 2) the risk of systemic absorption of alcohol; 3) adverse dermatologic effects; 4) risk of Clostridium difficile infection incidence. Fire events related to ABHR are extremely rare and almost exclusively associated with inappropriate use.Hand hygiene compliance is usually better after patient contact than before, despite a lack of evidence to suggest this is an effective means to prevent transmission of pathogens. Blood levels of ethanol and acetone after even supra-normal exposure are undetectable or insignificant. Appropriately formulated ABHR products are less likely to result in dermatitis than washing with soap and water. Appropriate implementation of hand hygiene guidelines does not result in Like any medication, ABHRs do have potential adverse effects, but these can be minimised by appropriate usage. Rare or even mythic complications should be weighed realistically against the potential of ABHR to prevent countless healthcare-associated infections each year.None declared."} +{"text": "Motivated by the challenges in assessing physician-level cancer screening performance and the negative impact of misclassification, we propose a method (using mammography as an example) that enables confident assertion of adequate or inadequate performance or alternatively recognizes when more data is required.Using established metrics for mammography screening performance\u2013cancer detection rate (CDR) and recall rate (RR)\u2013and observed benchmarks from the Breast Cancer Surveillance Consortium (BCSC), we calculate the minimum volume required to be 95% confident that a physician is performing at or above benchmark thresholds. We graphically display the minimum observed CDR and RR values required to confidently assert adequate performance over a range of interpretive volumes. We use a prospectively collected database of consecutive mammograms from a clinical screening program outside the BCSC to illustrate how this method classifies individual physician performance as volume accrues.Our analysis reveals that an annual interpretive volume of 2770 screening mammograms, above the United States\u2019 (US) mandatory (480) and average (1777) annual volumes but below England\u2019s mandatory (5000) annual volume is necessary to confidently assert that a physician performed adequately. In our analyzed US practice, a single year of data uniformly allowed confident assertion of adequate performance in terms of RR but not CDR, which required aggregation of data across more than one year.For individual physician quality assessment in cancer screening programs that target low incidence populations, considering imprecision in observed performance metrics due to small numbers of patients with cancer is important. Metrics used to evaluate the quality of a cancer screening program often parallel performance characteristics of randomized controlled trials (RCTs) that have demonstrated a mortality benefit and have thereby established the efficacy of the test\u2013typically detection rates and false positives Mammography screening may be the best studied screening test, perhaps due to rigorous performance of RCTs, development of large, high-quality, population-based data sets and subsequent quality legislation. For these reasons, we use mammography as our example. The Mammography Quality Standards Act (MQSA), established in 1992 in the US, requires each mammography facility to have a medical audit system for follow-up and outcome analysis but stops short of requiring that physicians meet specific performance criteria Observed performance values for many physicians in the BCSC were based on a small number of mammograms, especially those performed on women with cancer, possibly leading to misclassification of some physicians based on imprecise estimates. The volume of interpreted mammograms directly influences the size of the confidence interval around observed performance values and these confidence intervals should be considered in the evaluation of individual physicians. Although interpretative volume has been recognized as a source of inaccuracy when assessing performance benchmarks historically Our Institutional Review Board did not require that this HIPAA-compliant, retrospective quality-assurance project involve informed consent. We define CDR and RR benchmarks based on the BCSC reference distribution derived from seven mammography registries in the US benchmark threshold as a limit that the confidence interval of an individual physician\u2019s performance value must not overlap in order to be deemed adequate. For CDR, we define the benchmark threshold as the 10th percentile of the BCSC reference distribution, which is 2.4/1000.Cancer detection rate (CDR) is the number of true positive screening mammograms divided by the total number of screening mammograms performed th percentile of the BCSC reference distribution, which is 16.8%.Recall rate is the number of positive screening mammograms divided by the total number of screening examinations interpreted adequate performance) meaning all the values in the confidence interval for the individual performance value meet or exceed the benchmark threshold, 2) uncertain performance meaning the 95% confidence interval overlaps the benchmark threshold, and 3) did not meet benchmark \u201cwith confidence\u201d (inadequate performance) meaning that all the values in the confidence interval for the individual performance value fall short of the benchmark threshold.We divide screening interpretive performance into 3 categories: 1) met benchmark \u201cwith confidence\u201d from 1/1/2006 to 12/31/2008. All mammographic findings were prospectively described and recorded (at the time of mammography interpretation) by the interpreting physician using the Breast Imaging Reporting and Data System (BI-RADS) assessment categories\u2013Since demographic factors like age, family history of breast cancer, personal history of breast cancer, breast density, and comparison with prior mammography We calculated cancer detection rate and recall rate as per BI-RADS methodology on an individual physician level We propose a graphical method to illustrate the classification of performance into three categories based observed performance values and interpretive volume, for a given benchmark threshold. Performance categories are defined by first calculating a 95% confidence interval (CI) for the observed performance and then assessing whether the benchmark threshold lies above, within, or under the 95% CI. We used the Wilson score confidence interval method with continuity correction Graphical representations of the observed performance values required to provide 95% confidence of adequate or inadequate performance given our selected benchmark threshold and a range of volumes are shown in During the 3 year time period we analyzed clinical data (from outside the BCSC), 30,363 screening mammograms were performed for 18,069 women. We compare our study population to the BCSC population in Based on this clinical data we analyzed both CDR and RR over three consecutive years. The average yearly volume for the four included physicians was 1918 screening mammograms per year per physician. Plotting observed performance values as volume increases demonstrAnalysis of clinical data from one non-BCSC practice demonstrates that physicians often appear to be underperforming if a single year is viewed in isolation. Out of 12 annual measures of CDR (three for each physician), only 5 demonstrated adequate performance and 7 were in the uncertain range . FurtherA physician performing a cancer screening test is expected to have a high detection rate while simultaneously maintaining a low false positive rate in order to reap the mortality benefits of early detection while simultaneously minimizing harms. Variability of practice diminishes screening program efficacy We use mammography with associated national benchmark data (the BCSC reference distribution) as an example to establish combinations of volume and performance that are adequate with certainty, inadequate with certainty, or uncertain and thus require more data. We found that much larger volumes are required to confidently classify physicians based on CDR vs. RR; given cancer detection is a much rarer event than recall. For physicians with observed performance values at the benchmark median, volumes of 2770 screening mammograms for CDR compared to only 120 screening for RR are required to confidently assert their performance is adequate. Importantly, below this volume, physicians must have observed performance values above the benchmark median to confidently assert adequate performance. The average annual screening interpretive volume for a large sample of physicians in the US was 1777 mammograms By applying our method to physicians outside the BCSC, we find that assessing annual observed performance values to judge CDR for screening mammography without considering volume (i.e. variability) is perilous, because observed measures for individual physicians may fall below the benchmark threshold by chance in a given year. In fact, this occurred for two out of four physicians when annual performance values were viewed without considering their confidence intervals, despite adequate performance when larger volumes for the same physicians were aggregated . Based on established benchmark thresholds in the literature, and confidence level (95%) is somewhat ad-hoc. We do not contend that this choice is \u201ccorrect,\u201d just reasonable and useful for illustrative purposes. Our choices might optimally be more or less strict depending on the values, financial resources, and workforce considerations of the health system or population. Perhaps a screening program might rather use a 99% (wider) confidence interval for observed performance values thereby creating a stricter standard for classifying someone as adequate or inadequate. This would result in more physicians being in the uncertain zone, which would require more data or some other type of review to determine if performance is adequate. Using an 80% (narrower) confidence interval for observed performance values would more easily classify someone as adequate or inadequate, with fewer physicians in the uncertain zone. The exact values prescribed are not the point of our manuscript. Our methodology is intended to support any reference distribution, selected benchmark threshold (or consensus-developed performance range), and confidence interval considered appropriate for a given screening program We recognize that our choice of benchmark threshold values Click here for additional data file.Figure S2RR performance estimates create a sawtooth appearance but it becomes smoother sooner due to higher event rate. The continuity correction can be safely ignored even for low N, because the recall rate (RR) is higher than the cancer detection rate (CDR). Curves derived using the Poisson distribution illustrating the effect of the continuity correction.(EPS)Click here for additional data file.Materials S1Online Data Supplement: Statistical methodology.(DOCX)Click here for additional data file."} +{"text": "Despite increasing reports of successful pregnancies whilst using Efavirenz (EFZ), the drug remains Category C during pregnancy due to concerns around teratogenicity . AdditioCase-note review of all women taking EFZ in Jan 2008 and again in Feb 2010. Current contraception used, advice on teratogenicity, and advice on efficacy documentation was recorded. Women over 50, with documented menopause or hysterectomy were excluded.In 2008 we identified 31 females using EFZ in our cohort of 912 patients. Contraceptive choices are shown in Figure Simple changes such as adding contraception to a clinic proforma can help improve sexual and reproductive health outcomes in HIV positive women. However, there are still improvements to be made in documentation of advice given, particularly when using a Category C drug in women who may become pregnant. Additionally, women should be made aware of the potential interaction between antiretrovirals and hormonal contraceptives at the HIV clinic \u2014 particularly as some may not disclose their status to Family Planning or GP services and therefore we cannot assume that this advice is being given elsewhere."} +{"text": "Tamara T., Sflomos G et al., Science Translational Medicine, 2013, Vol. 5, Issue 182, p. 182) presents and validates a novel ex vivo culture model based on tissue microstructures from fresh human reduction mammoplasty specimens. For the first time, physiological hormone action can be studied in a human model and new opportunities to unravel hormone action open up. The work revealed Progesterone/RANKL axis as a major driver of cell proliferation in the adult human breast with potential clinical implications for breast cancer patients. Current knowledge about the mechanisms by which female reproductive hormones control mammary gland development and breast carcinogenesis stem mainly from animal studies. In particular, the mouse model, in which genetic tools can be combined with powerful tissue recombination techniques, has been instrumental in revealing that epithelial estrogen receptor (ER\u03b1) signaling controls pubertal gland development whereas progesterone receptor (PR) signaling is the major proliferative stimulus in the adult mouse mammary gland. Across species, 30-50% of the breast epithelial cells express the ER\u03b1 and PR. The mouse model revealed that at the cellular level, hormone action relies largely on paracrine signaling. In particular, receptor activator of nuclear factor kappaB ligand (RANKL) is a central mediator of PR-induced cell proliferation. RANKL, a transmembrane type II protein is a member of the tumor necrosis factor superfamily and signals through its receptor RANK and progesterone (P) are major determinants of postnatal mammary gland development and are thought to promote breast carcinogenesis. How do they impact on the human breast? The most widely used models to study E and P signaling are hormone receptor positive breast cancer cell lines. However, it is unclear to what extent they provide insights into the breast tissue molecular pathophysiology. The study are grown on tissue culture plastic they lose hormone receptor expression and cannot be used to study hormone action. Hormone receptor positive breast cancer cell lines such as MCF-7 and T47D express high levels of hormone receptors ex vivo approach, breast cells retain hormone receptor expression and remain hormone responsive. Interestingly, estrogens failed to elicit cell proliferation in a consistent fashion whereas progesterone signaling induced proliferation in most samples. Activation of progesterone receptor signaling did not affect RANKL expression in MCF-7 and T47D cell lines but induced it in the tissue microstructures. RANKL induction by progesterone receptor signaling is mediated mainly by molecular mechanisms controlling RANKL's mRNA maturation and stability, leading to elevated amount of this protein in the cell have been developed for gynecological disorders and have fewer side effects compared to previous generations. If the paracrine axis persists in patients bearing PR+ breast cancers then these patients may benefit from these drugs.ex vivo system can be useful to address whether progesterone and progestins differ in their ability to induce RANKL and/or other targets and promote cell proliferation not only from microstructures derived from reduction mammoplasties but also from tumor samples.Large studies of women on hormone replacement therapy have revealed puzzling results. The intake of combined estrogen and progestin preparations increased breast cancer risk whereas therapy with estrogen and natural progesterone did not. The Over decades, the approval of oral contraceptives has substantially improved birth control and prevented unwanted pregnancies. However, many studies argue that contraceptive pills, most of which include progestins, can increase breast cancer incidence for reasons that are not yet fully understood. Additional bench work using the human microstructure model will help us to identify the molecular underpinnings of these differences and to determine individual risk factors related to new formulations of contraceptives and to a woman's age or reproductive status. This new experimental model holds promise to help us gain newinsights into hormone action in the human breast that is ever so important to tumor development in this organ."} +{"text": "Two major breast cancer sub-types are defined by the expression of estrogen receptors on tumour cells. Cancers with large numbers of receptors are termed estrogen receptor positive and those with few are estrogen receptor negative. Using genome-wide single nucleotide polymorphism genotype data for a sample of early-onset breast cancer patients we developed a Support Vector Machine (SVM) classifier from 200 germline variants associated with estrogen receptor status (p<0.0005). Using a linear kernel Support Vector Machine, we achieved classification accuracy exceeding 93%. The model indicates that polygenic variation in more than 100 genes is likely to underlie the estrogen receptor phenotype in early-onset breast cancer. Functional classification of the genes involved identifies enrichment of functions linked to the immune system, which is consistent with the current understanding of the biological role of estrogen receptors in breast cancer. Breast cancer sub-types may be classified according to the number of estrogen receptors present on the tumour. Tumours expressing large numbers of receptors are termed estrogen receptor positive (ER+) and, conversely, estrogen receptor negative (ER\u2212) for few or no receptors. ER status is extremely important since ER+ cancers grow under the influence of estrogen, and may therefore respond well to hormone suppression treatments, while the proliferation of ER\u2212 cancers is not driven by estrogen and does not respond to estrogen modulation. Deroo and Korach Two hypotheses seek to explain the relationship between estrogen and breast cancer. The first considers the proliferation of mammary cells stimulated by the binding of estrogen to the ER leading to an increase in the number of target cells and associated elevated risk for replication errors and acquisition of deleterious mutations during cell division and DNA replication. A second hypothesis identifies genotoxic by-products of estrogen metabolism which may lead to DNA damage and, subsequently, cancer. Evidence exists to support both hypotheses as mechanisms to initiate and promote tumour development ESR1 and ESR2 genes respectively. The two forms have distinct roles in breast tissue; ER\u03b1 promotes cell proliferation in response to estrogen while ER\u03b2 inhibits proliferation and tumour formation ESR1 gene have been associated with increased susceptibility to breast cancer, however they are fairly rare ESR2 gene may also be important in disease susceptibility however, no SNPs demonstrating a strong association with breast cancer risk have been identified The estrogen receptor has two forms, \u03b1 and \u03b2, which are encoded by the We hypothesized that patients who develop ER+ and ER\u2212 tumours would show distinct constitutional genetic profiles the exploration of which could yield new insights into the biological effect of the host genomic environment on the emergence of these forms of breast cancer. We developed machine learning (ML) classifiers to explore the distinction between profiles in well characterised breast cancer cases. ML is used extensively in many scientific fields for classification purposes. ML methods have been used in genetic studies to explore the underlying genetic profile of disease and build models capable of (i) detecting gene-gene interactions; (ii) predicting disease susceptibility; (iii) predicting cancer recurrence; and (iv) predicting cancer survivability The overall classification accuracy of a ML classifier is a measure of how successful the method is at assigning samples to the correct class. In this study the highest classification accuracy was achieved using 200 SNPs fully genotyped in all 542 study samples and indiClassifier performance was further evaluated using the receiver operating characteristic (ROC) area under curve (AUC) values which indicate these models have excellent accuracy: all exceed 0.9 . ROC curSVM classifiers were produced for two additional subsets of SNP features to further investigate classification accuracy. A set of 200 SNPs showing no individual association for the ER+/ER\u2212 distinction and a subset of 200 randomly selected SNPs were used to produce classification models . AccuracTo identify biological terms and pathways that are particularly enriched for genes represented in the classifier we used DAVID analysis was also performed for the 100 SNPs with the highest absolute classifier weights from theMachine learning techniques have an important role to play in disease classification and the discovery of underlying disease mechanisms, including gene-gene interactions or signalling pathway enrichment which influences disease. Support vector machines in particular are state-of-the-art classifiers Although SVM classification accuracy is an important indicator of success it can be misleading, particularly in the case of an unbalanced data set , as in this study Classifier performance can be further evaluated using receiver operator characteristic (ROC) curves which are based on the true positive and true negative rates at several different thresholds. One of the major advantages of the ROC curve is that it is unaffected by unbalanced datasets capacity describing how complex a model can be: learning machine functions with high capacity may generate lower training error rates but require larger training sets than simpler, low capacity models. The best theoretical performance guarantee is achieved through the right balance between the accuracy attained for a given training set and the model capacity. Because analysis of genomic disease data considers potentially very large number of features (SNPs) evaluated on relatively small numbers of samples (genomes) feature selection strategies aim to reduce overfitting. Alternatives to the approach to reduce feature complexity adopted here include Recursive Feature Elimination (RFE) applied to linear SVMs using the ranked SVM weights to recursively eliminate features Feature selection is an important component of building a ML classifier. Much of the SNP data in these samples will not be useful for building an accurate model so it waThe underlying biological nature of the genes identified as discriminators of ER+/ER\u2212 breast cancer was of particular interest in this study. To identify gene enrichment in gene groups and pathways we used the DAVID toolset. Analysis of the 139 genes that the classifier SNPs reside in, or are closest to, identified gene groups, pathways and annotation terms that were particularly enriched and 3. OThe role of estrogen and estrogen receptors as regulators of proliferation and differentiation in breast tissue is well-established and is crucially important for disease progression in many cases + T lymphocytes present at the tumour site influences patient survival, with higher numbers being associated with better survival rates. This effect is more evident in patients presenting with ER\u2212 tumours compared to ER+ tumours The estrogen receptor status of breast cancer patients has long been recognised as a strong prognostic factor that influences patient treatment options and survival. Patients with ER\u2212 forms of the disease tend to show decreased survival rates in the first few years after diagnosis and present with more aggressive tumours DAVID analysis of the gene set also identified five genes implicated in the \u2018axon guidance\u2019 pathway . Axon guGenome wide association studies have identified risk-related SNPs for many diseases. Thirty-five SNPs, which lie in or near to 36 genes, are identified as breast cancer risk SNPs in the Catalog of Published Genome-Wide Association Studies Prospective study of Outcome in Sporadic versus Hereditary breast cancer\u2019 (POSH) cohort 542 early-onset breast cancer patients were selected from the \u2018Genotyping of the breast cancer samples was conducted using the Illumina 660-Quad SNP array. Genotyping was conducted at the Mayo Clinic, Rochester, Minnesota, USA (261 samples), and the Genome Institute of Singapore, National University of Singapore (281 samples) SNPs showing significant association with ER\u2212 cases were identified from the additive chi-squared association test implemented in the PLINK toolset in which ER+ samples were labelled as \u2018controls\u2019 and ER\u2212 samples were labelled as \u2018cases\u2019. Based on results from the chi-squared test all SNPs were ranked in terms of association with the ER+/\u2212 classification. Subsets of SNPs were selected as features for SVM models from the ranked list of SNPs and models were produced from subsets of 50, 100 and 200 SNPs to test utility as discriminatory factors for ER+/ER\u2212 breast cancer.The three genotypes at each SNP were converted into numeric values following n equally sized sets testing n models. We used 10-fold cross-validation: data were divided into 10 approximately equal-sized sets and a classifier built based on the data in 9/10 of these sets. The remaining 10% of data was used as a test set to determine the accuracy of the classifier. This process was repeated 10 times with each set representing the test data once and average classification accuracy determined. We further explored 10-fold cross-validation using 100 replicates and mean accuracy from 1000 resultant models was obtained for alternative kernel models.Support vector machines are supervised machine learning algorithms which build models based on \u2018training\u2019 data and search for similar patterns in \u2018test\u2019 data The SVM classification model was produced using the Weka data mining software Annotation of sets of SNPs used in the classification models was undertaken using the ANNOVAR software Functional gene annotation clusters were identified using the \u2018Gene Functional Classification\u2019 tool in DAVID Table S1200 SNPs which most strongly discriminate ER+ and ER\u2212 breast cancers used in the classification models. The weights are taken from a linear model built using one iteration of 10-fold cross-validation in the WEKA Explorer. Classification accuracy for this model was 92.4%. The magnitude of the absolute values of the SNP weights indicates importance of the SNP for classifying cases. Positive SNP weights relate to classifying ER+ cases while negative SNP weights relate to classifying ER\u2212 cases. For those SNPs that are not located within a gene the nearest gene is given and the distance of the SNP from this gene is indicated by dist\u200a=\u200a.(DOCX)Click here for additional data file.Table S2Weka kernels and classification results for 100 and 50 SNPs with highest chi-squares. Comparison of classifiers built with 100 and 50 highest ranked SNPs from PLINK chi-square test.(DOCX)Click here for additional data file.Table S3Weka kernels and classification results for bottom 200 and random 200 SNPs. Comparison of classifiers built with 200 random SNPs and the lowest ranked by PLINK chi-square test.(DOCX)Click here for additional data file."} +{"text": "Objective: Test the hypothesis that right hemisphere stroke can cause extinction of left hand movements or movements of either hand held in left space, when both are used simultaneously, possibly depending on lesion site.Methods: 93 non-hemiplegic patients with acute right hemisphere stroke were tested for motor extinction by pressing a counter rapidly for one minute with the right hand, left hand, or both simultaneously with their hands held at their sides, or crossed over midline.Results: We identified two distinct types of motor extinction in separate patients; 20 patients extinguished left hand movements held in left or right space ; the most significantly associated voxel cluster of ischemic tissue was in the right temporal white matter. Seven patients extinguished either hand held in left space (left space extinction), and the most significantly associated voxel cluster of ischemic tissue was in right parietal white matter.Conclusions: There was a double dissociation between left canonical body extinction and left space motor extinction. Left canonical body extinction seems to be associated with more dorsal ischemia, and left canonical body extinction seems to be associated with more ventral ischemia."} +{"text": "However, previously tested specimens might not provide representative mechanical properties as prior testing may have altered the tissue. The purpose of this study was to test the effect of prior compression testing on the plantar soft tissue shear and compressive properties using paired specimens in a two-part study.Changes in the shear plantar soft tissue properties with diabetes likely play a role in plantar ulceration, yet little is known about these characteristics. We recently conducted Four pairs of cylindrical specimens (n=8) were isolated per previous methods from theThe results Table of the fThis study demonstrates that prior compression testing of the plantar soft tissue may alter the compressive properties. However, since the shear parameters were not affected by prior testing in compression, shear tests using previously compression tested specimens should provided representative properties."} +{"text": "It is not known why continuing instability exists after ankle sprain. The most common hypotheses include impairments in proprioception, muscle power or postural control but a reSixty three participants with history of either no ankle sprain or only one ankle sprain were recruited. Functional ankle instability was measured using the Cumberland Ankle Instability Tool (CAIT), a highly reliable measure of functional ankle instability . InvertoNo correlation was found between the CAIT scores and the three measured variables. The strongest correlation was between CAIT score and inversion peak power at 30\u00b0/s .Based on our findings, functional ankle instability (as measured by CAIT) is not related to inversion/eversion peak power, or with joint movement detection or position sense at the ankle. These findings are consistent with the results of previous studies investigating the relationship between CAIT score and other functional tests . The lac"} +{"text": "Recent advances in neural recording techniques open exciting possibilities of better understanding whole populations of neurons. Devices such as APS MEA (Active Pixel Sensor Microelectrode Array) ,2 allow Pairwise maximum entropy model , when fit to the data, yields a minimally structured probability distribution of network states that respects first and second order interactions. It is a convex, parsimonious and readily interpretable model that has been shown to characterize spiking patterns surprisingly robustly in many cases ,4. AdditIn present work we examine the results and performance of the MaxEnt model fitting in different preparation types and parameter regimes; owing to high resolution recordings we can specifically focus on varying spatial scales. As can be seen in Fig."} +{"text": "Molothrus ater), which is positively associated with increasing landscape forest cover in the midwestern United States. However, cowbirds are also experiencing long-term population declines, which should reduce parasitism pressure and thus increase productivity of host species. We used 20 years of nest monitoring data from five sites in Missouri across a gradient of landscape forest cover to assess temporal trends in the rate and intensity of brood parasitism for Acadian Flycatchers (Empidonax virescens), Indigo Buntings (Passerina cyanea), and Northern Cardinals . We evaluated whether there were concomitant changes in fledging brood size, nest survival, a combination of the two metrics , and whether such changes were more substantial with decreasing landscape forest cover. Parasitism rates and intensities declined substantially during 1991\u20132010. Fledging brood size and nest survival rates were positively associated with landscape forest cover, confirming the fragmentation hypothesis for Midwest forest birds. Declining parasitism rates were associated with increased fledging brood sizes, with more pronounced increases as landscape forest cover decreased. Nest survival increased insubstantially across time during laying and incubation, but not during the nestling stage. The best predictor of nest survival was parasitism status, with parasitized nests surviving at lower rates than unparasitized nests. Overall, productivity increased during 1991\u20132010, with more pronounced increases associated with lower levels of landscape forest cover. The negative effects of cowbirds on nest survival in addition to fledging brood size in less forested landscapes suggest that cowbirds may be a primary cause of forest fragmentation effects on songbird productivity in the Midwest. Our results underscore the dynamic nature of demographic parameters, which should be accounted for in predictive models of wildlife responses to future environmental conditions.Many songbird species have experienced significant population declines, partly because of brood parasitism by the Brown-headed Cowbird ( Molothrus ater; hereafter cowbird), which exhibit increased abundances that result in increased rates of brood parasitism with decreasing regional forest cover in the midwestern United States Many species of Neotropical migrant songbirds have experienced significant long-term population declines Spiza americana) compared to an earlier study at the same location Like many other passerines, cowbirds have exhibited long-term declines in population abundances successful nest), nest survival rates, and an overall productivity metric that combined fledging brood size and nest survival for three species of forest songbirds across a gradient of forest cover in Missouri, USA, landscapes that have remained largely unchanged during the past 20 years. We predicted that a statewide reduction in cowbird abundances builds nests in shrubs within old fields, along forest edges, and in dense forest understory vegetation. The Acadian Flycatcher is a forest-interior species that typically nests in the branches of sub-canopy trees. The Northern Cardinal is a habitat generalist that nests in shrubs and trees at a variety of heights in old fields and wooded habitats. Researchers used systematic search or behavioral cues to find nests We compiled nest monitoring data from multiple studies that occurred on public lands in Missouri, USA, during 1991\u20132010 . The Ripn\u200a=\u200a1,186) or from nest locations marked on gridded maps in a GIS (n\u200a=\u200a959). One study at Baskett did not have exact nest locations mapped but did record subplot locations ; we defined the center of the subplot as the location for these nests (n\u200a=\u200a718). The remaining nests were assigned locations based on the center of a breeding territory (n\u200a=\u200a45), or an estimated location based on written descriptions (n\u200a=\u200a4).We obtained geographic coordinates recorded from handheld GPS units (sensuhttp://www.fia.fs.fed.us/) for all the counties that fell within a 10-km radius of our nests and assessed forest cover change from 1989 (data were not available for 1990\u20131991) to 2010. Second, we downloaded two NLCD land cover change databases that correct for compatibility issues between releases and identify pixels that have changed between releases. We used the pixels within the 10-km buffers surrounding nests that changed to or from forest to calculate the percent change in forest cover.We used landscape forest cover as a metric of habitat fragmentation sensu. We asseWe concluded it was appropriate to use the 2001 database to calculate landscape forest cover for all nests to avoid compatibility issues between different NLCD releases (see results). We used ArcMap 9.3 Our overall approach was to evaluate sets of candidate models explaining variation in nest parasitism rates, parasitism intensity, fledging brood size, and nest survival within an information-theoretic approach iw) and evaluate the relative support of each model in the candidate set. All models included study site as a random effect to acknowledge the potential for correlated fates within sites and to account for site-specific variation in parasitism rates. All models except the null also included host species and nest site habitat type, both of which can influence parasitism rates We modeled parasitism rates using logistic regression with a binary response variable (parasitized versus unparasitized) with the GLIMMIX procedure in SAS Because interpretation of model-averaged coefficients is problematic when some parameters occur as both additive and interactive terms in various models We used nests for which exact counts of host young were known to model fledging brood size as a function of forest cover and year. We used the same approach and model set as described for parasitism rates and intensity , but becWe used the logistic exposure method To assess temporal changes in productivity, we combined fledging brood size and nest survival estimates. To do this, we used a parametric bootstrapping approach that allowed us to incorporate the error associated with the fledging brood size and nest survival estimates th percentile of observed forest cover values) versus 3% (95% CI: 2\u20134%) for a population of nests at 94% forest cover (the 95th percentile). A parameter for year was in the two best-supported parasitism rate models, which combined for 100% of the overall weight of evidence between 1989 and 2010 according to FIA data. In contrast, NLCD data from a 10-km radius surrounding each nest suggested that forest cover declined by 1.8% from 1992 to 2001, and by 0.4% from 2001 to 2006. Of the nests monitored wherein contents were reliably observed, 423 of 1,524 (28%) bunting nests, 76 of 1,021 (7%) flycatcher nests, and 79 of 367 (22%) cardinal nests were parasitized. All three species were well represented across the gradient of forest cover are frequent predators during incubation Buteo platypterus) and Barred Owls (Strix asio), frequent predators that depredate nests almost exclusively during the nestling stage Because parasitism rates increase and nest survival rates decrease with increasing forest fragmentation in the Midwest Lower nest survival and reduced fledging brood sizes associated with low landscape forest cover led to a substantial negative correlation between forest cover and our combined productivity metric . With predicted productivity values \u226550% lower at 23% forest cover compared to 94% forest cover regardless of year , our datsensuDespite the substantial decline in predicted parasitism rates during 1991\u20132010, the concomitant increase in productivity was comparatively modest because the strongly negative impact of parasitism on the productivity of a single nest is muted across a population of nests wherein most are not parasitized and many parasitized nests are depredated. Nevertheless, lower landscape forest cover was associated with a greater increase in predicted productivity, which provides support for our hypothesis that temporal trends in productivity should be landscape-specific. It also suggests that some habitat patches that were formerly population sinks sensu may now We stress that the patterns we observed in Missouri almost assuredly do not apply throughout the extensive range of the Brown-headed Cowbird. The BBS data suggest that temporal trends in cowbird abundances are not uniform; 19 U.S. states have seen substantial cowbird declines between 1966\u20132010, but 12 states have had significant increases in cowbird abundances during the same timespan Predictive models designed to assess the effects of climate change on wildlife distributions and abundances into the next century are increasingly common in the literature. Such models will be most useful when they incorporate important biotic interactions"} +{"text": "High risk) develop faster than those from permanent ponds (Low risk) and/or show increased developmental plasticity in response to drying conditions. Our analyses support shorter developmental times in High risk, primarily by decreasing body mass at metamorphosis. Plasticity in developmental times was small and did not differ between groups. However, accelerated development in High risk species generally resulted in reduced sizes at metamorphosis, while some Low risk species were able compensate this effect by increasing mean growth rates. Taken together, our results suggest that plastic responses in species breeding in ephemeral ponds are constrained by a general trade-off between development and growth rates.Anurans breed in a variety of aquatic habitats with contrasting levels of desiccation risk, which may result in selection for faster development during larval stages. Previous studies suggest that species in ephemeral ponds reduce their developmental times to minimize desiccation risks, although it is not clear how variation in desiccation risk affects developmental strategies in different species. Employing a comparative phylogenetic approach including data from published and unpublished studies encompassing 62 observations across 30 species, we tested if species breeding in ephemeral ponds ( One of the primary goals of life history theory is to explain the variation in age and size of organisms at ontogenetic niche transitions , and how potential trade-offs may constrain its plasticity and evolution . For insLarval amphibians occupy aquatic environments along a wide permanency gradient , and theEnvironmental variability adds another level of complexity because different strategies may be favored depending on the conditions encountered during development. Smaller temporal ponds are expected to appear and disappear within a relatively short period of time, subjecting those species breeding in these habitats to a higher risk of desiccation. Evolutionary responses to increased desiccation risk may involve an overall shift toward faster development, the evolution of plasticity triggered only when ponds are drying , or bothIn the present study, we determined whether amphibian anuran species facing higher risks of desiccation have evolved faster developmental rates and increased plasticity than species living in more stable pond environments. We tested these hypotheses with a comparative approach and a large dataset that includes several anuran species belonging to distinct taxonomic groups. Importantly, several comparative studies have shown that evolutionary inferences may change when phylogenetic relations among species are taken into account , employing the key words \u201cpond desiccation,\u201d\u201chydroperiod,\u201d\u201cmetamorphosis,\u201d\u201clife history,\u201d and \u201clarval period,\u201d and the references of all papers obtained with this search were subsequently reviewed. Data from individual studies were included only if they met the following criteria. First, the study experimentally manipulated water level or water permanency directly by comparing developmental time and size at metamorphosis in constant water-level treatment versus desiccation treatment in the laboratory or mesocosm, or indirectly by comparing permanent versus temporary ponds in the field. When more than one level was available, the extremes were selected to maximize the difference in desiccation risk between treatments. Second, we excluded studies that combined pond drying effects with another variable to avoid confounding effects . Third, studies that utilized snout vent length as an estimate of metamorphic size were not included, but we did include data from one study that reported body volume because Rana sphenocephala and Rana blairi and the hybrid of Pelophylax lessonae and Pelophylax ridibunda (Pelophylax esculenta), that were considered as \u2018ecological\u2019 species , from 25 studies in the literature, published between 1989 and 2006, and three unpublished datasets (Table S1). Developmental rates were calculated as 1/developmental period and mean growth rates as body mass/developmental period. This later estimate of mean growth rate was employed for simplicity as a general descriptor of growth rates Low risk. Species exposed to a lower risk of pond desiccation, basically permanent ponds holding water year-round in most years with rare events of desiccation. Desiccation risk varies more within and between years in ephemeral and temporary ponds than in permanent ponds (http://amphibiaweb.org/) or Global Amphibians Assessments Project database and We operationally assigned species into two categories, according to the typical habitat they employed for breeding, to analyze the evolutionary consequences of developing under contrasting variation of desiccation risks: (1) nt ponds . In mostape package available in R (http://cran.r-project.org/). Because we did not detect any significant effects of different experimental venues in preliminary analyses, values obtained from laboratory, mesocosm, and field experiment were combined in subsequent analyses. Comparisons of developmental strategies between High and Low risk species were performed employing values measured under constant water-level conditions, because this minimizes the potentially confounding effects of plasticity. We compared developmental rates, mean growth, and body mass with a linear model including desiccation risk as a categorical factor (High risk vs. Low risk). In these analyses, all variables were log10-transformed to meet the assumption of normality. A similar model including developmental time as a covariable was subsequently employed to compare mean growth rates and body mass at metamorphosis controlling for differences in developmental time.Analyses were performed employing phylogentic generalized linear models (see dmass\u2212cmass)/cmass where c and d correspond to constant and drying conditions, respectively). To ensure that none of the analyses were affected by variability within treatments, we also estimated plasticity as standardized effect sizes to estimate the relative weight of the evidence in favor of each model . The AICch model , which cAIC and wAIC show that phylogenetic models comparing developmental rates, mean growth rates, and body mass at metamorphosis had a substantially better fit than conventional analyses (High risk group exhibit significantly lower body mass at metamorphosis and mean growth rates (P < 0.01 in both cases), supporting the prediction that these species accelerate their development at the expense of growth . In other words, the reduction in developmental time due to plasticity is a relative constant fraction of developmental times under constant \u201coptimal\u201d conditions, regardless of whether a species takes 20 d or 170 d to develop. Conversely, the relative reduction in body mass at metamorphosis was significantly higher in larger species . Thus, larger tadpoles were able to metamorphose at a substantially lower fraction of their mass under constant conditions.Does the variation in mean estimates measured under constant conditions correlate with plasticity estimates? Phylogenetic generalized linear models suggest that plastic responses in developmental times are relatively constant across species and independent of the absolute length of the development period , which fits the data substantially better (AIC = 537.17 and wAIC = 0.94) than the phylogenetic model (AIC = 543.58 and AICw = 0.06). Interestingly, the magnitude of the plastic response in developmental times was not significantly related to variation in either body mass at metamorphosis and mean growth rates , hence the reduction in developmental times seems to be relatively constant across species regardless of their size at metamorphosis and mean growth rates.A similar pattern was observed for plasticity in mean growth rates: species growing on average faster showed larger reductions in mean growth rates when exposed to decreasing water levels. This trend was significant according to a conventional linear model Species that typically breed in temporary ponds have faster development rates than do species that typically breed in permanent ponds. This evolutionary response is associated with a reduced body size at metamorphosis and lower mean growth rates . (2) TheHigh risk species have evolved lower threshold sizes to reduce developmental time. Previous studies have suggested that anuran larvae that exhibit shorter mean developmental times in ephemeral ponds with increased risk of desiccation also show reduced body mass at metamorphosis . Our anaIn addition, analyses controlling for differences in developmental rates suggest that species breeding in ephemeral ponds actually grow slower than their counterparts breeding in permanent ponds. This is counterintuitive if one assumes that a critical size is necessary to trigger metamorphosis into the adult form, given that lower overall growth rates would delay metamorphosis and may potentially have a negative impact in fitness . Taken tAt the level of developmental plasticity, comparisons between species breeding in ephemeral and permanent ponds showed that both groups showed a similar reduction in developmental period in response to drying conditions. However, the nature of plastic responses actually depended on the breeding habitat: whereas species breeding in ephemeral ponds showed pronounced reductions in size at metamorphosis in response to drying conditions, this was generally not observed in species inhabiting permanent ponds . Even thDevelopment and growth involve two very distinct physiological processes, where the former is essentially associated with cell differentiation while the second is primarily determined by cell proliferation and growth . Althougn = 16 cases) provided a similar picture, an average (\u00b1SE) increase of 3.6 \u00b1 0.6% in species from ephemeral ponds versus a 29.1 \u00b1 8.3% increase in their counterparts from permanent ponds . This provides compelling evidence that anuran species breeding in ephemeral ponds are maximizing mean growth rates and consequently show little plasticity in this trait , considerable variation in the nature and the magnitude of plastic responses was observed across species breeding in permanent ponds. Many species did not increase their developmental rates in response to drying conditions, even though some of these species exhibited a reduction in body size at metamorphosis of almost 40% . ConversAbsolute limits to performance certainly exist, but this does not account for the interspecific differences observed in developmental strategies. Although differences may be partly due to the stressful physical and biotic conditions of desiccating ponds, our results suggest that an important fraction of the variation can be attributed to phylogenetic history. For example, bufonids in general metamorphose at very small body sizes, whereas the opposite pattern is true for scaphiopodids . It is pUnderstanding which factors ultimately explain these differences is crucial to determine if and how anuran species may respond to increasing risks of desiccation. Even though comparative studies are strictly correlational and may provide limited information on the mechanisms underlying species developmental differences, phylogenetic information can be valuable for predictive purposes, because closely related taxa seem to employ similar developmental strategies and may also potentially share the same physiological limitations."} +{"text": "MicroRNAs (miRNAs) are a group of small non-coding RNAs that play important regulatory roles at the post-transcriptional level. Although several computational methods have been developed to compare miRNAs, it is still a challenging and a badly needed task with the availability of various biological data resources. In this study, we proposed a novel graph theoretic property based computational framework and method, called miRFunSim, for quantifying the associations between miRNAs based on miRNAs targeting propensity and proteins connectivity in the integrated protein-protein interaction network. To evaluate the performance of our method, we applied the miRFunSim method to compute functional similarity scores of miRNA pairs between 100 miRNAs whose target genes have been experimentally supported and found that the functional similarity scores of miRNAs in the same family or in the same cluster are significantly higher compared with other miRNAs which are consistent with prior knowledge. Further validation analysis on experimentally verified miRNA-disease associations suggested that miRFunSim can effectively recover the known miRNA pairs associated with the same disease and achieve a higher AUC of 83.1%. In comparison with similar methods, our miRFunSim method can achieve more effective and more reliable performance for measuring the associations of miRNAs. We also conducted the case study examining liver cancer based on our method, and succeeded in uncovering the candidate liver cancer related miRNAs such as miR-34 which also has been proven in the latest study. We applied the miRFunSim method to compare 100 miRNAs whose target genes have been experimentally supported from TarBase Figure S1The robustness analysis results for measuring the relationship of miRNAs using miRFunSim. (A) A comparison of functional similarity scores between intrafamily and interfamily miRNA pairs, and between intracluster and intercluster miRNA pairs using predicted miRNA targets. (B) A comparison of functional similarity scores between intrafamily and interfamily miRNA pairs, and between intracluster and intercluster miRNA pairs by the removal of 5% network nodes in the protein interaction network randomly. (C) A comparison of functional similarity scores between intrafamily and interfamily miRNA pairs, and between intracluster and intercluster miRNA pairs by the removal of 10% network nodes in the protein interaction network randomly.(DOC)Click here for additional data file.File S1Information of experimentally verified miRNA targets from TarBase.(TXT)Click here for additional data file.File S2Information of predicted miRNA targets from starBase by at least three prediction algorithms with readNum>\u200a=\u200a1 and biological complexity >\u200a=\u200a1.(TXT)Click here for additional data file."} +{"text": "As a rule of thumb, the following people are likely to need low vision services and must be referred wherever possible:All children who have undergone bilateral cataract operations, both those with pseudophakia and those with aphakiaPeople with diabetic macular oedema whose vision remains poor despite laser treatmentPeople with age-related macular degenerationChildren with oculocutaneous albinismPeople with optic atrophy, whatever the causeAny person who still has difficulty performing their daily activities because of their vision, even after treatment and refraction.People with low vision are affected in different ways. They may suffer from some or all of the following:Severely reduced visual acuityBlurred visionVisual field loss: central or peripheralLoss of contrast sensitivityIncreased light sensitivity.Many people with low vision suffer from blurred vision Figure , for exaPeople with optic atrophy or age-related macular degeneration will have loss of central visual acuity Figure , which mSomeone with glaucoma or retinitis pigmentosa will have constricted visual fields, i.e. loss of peripheral vision Figure . This maLoss of contrast sensitivity Figure can haveIncreased light sensitivity makes it very difficult for people to see detail or make sense of what they see if they are in bright light, or glare Figure ."} +{"text": "According to recent clinical findings epileptiform activity in temporolimbic structures may cause depressive and other psychiatric symptoms that may occur independently of any seizure in patient's history. In addition in these patients subclinical seizure-like activity with indirect clinical manifestations likely may occur in a form of various forms of cognitive, affective, memory, sensory, behavioral and somatic symptoms . A typical characteristic of epileptiform changes is increased neural synchrony related to spreading of epileptiform activity between hemispheres even in subclinical conditions i.e. without seizures. These findings suggest a hypothesis that measures reflecting a level of synchronization and information transfer between hemispheres could reflect spreading of epileptiform activity and might be related to complex partial seizure-like symptoms.Suitable data for such analysis may provide various physiological signals reflecting brain laterality, as for example bilateral electrodermal activity (EDA) that is closely related to limbic modulation influences. With this purpose we have performed measurement and analysis of bilateral EDA and compared the results with psychometric measures of complex partial seizure-like symptoms, depression and actually experienced stress in 44 patients with unipolar depression and 35 healthy controls. The results in unipolar depressive patients show that during rest conditions the patients with higher level of complex partial seizure like symptoms (CPSI) display increased level of EDA transinformation (PTI) calculated between left and right EDA records .The result may present potentially useful clinical finding suggesting that increased EDA transinformation (PTI) could indirectly indicate increased neural synchrony as a possible indicator of epileptiform activity in unipolar depressive patients treated by serotoninergic antidepresants. Recent clinical findings indicate that epileptiform activity in temporolimbic structures may cause depressive and other psychiatric symptoms that may occur independently of any seizure in patient's history These findings are in agreement with the kindling hypothesis proposed by Post Together these findings suggest that in many cases depression and its pathogenesis may be closely related to an epileptiform process that may occur in temporolimbic structures frequently without any evidence on scalp EEG. Additionally there is evidence that this epileptic-like process may emerge in the form of symptoms similar to several symptoms of temporal lobe epilepsy that occur as cognitive, affective, memory, sensory, behavioral and somatic symptoms Although these symptoms typically occur in temporal epileptic patients and psychometric measures of the complex partial seizure-like symptoms were designed for epileptic patients, there is clinical evidence that clinically significant level of the same symptoms occur in non-epileptic patients as for example in patients with depression, schizophrenia, PTSD or in patients with traumatic brain injury Although seizures in temporal lobe structures may occur only locally and unilaterally, frequently the seizure activity tend to increase neural synchronization and extend to the other hemisphere which significantly influences interhemispheric information transfer that may reflect temporal lobe \u201cepileptogenicity\u201d Because depressive patients predominantly have normal EEG and conventional scalp EEG is not able to provide information on subcortical structures, it is likely that direct measurement of limbic activity or its manifestations using EEG likely is not possible. Nevertheless there is evidence that epileptiform activity may manifest in the autonomic nervous system With respect to this possibility recent evidence indicates that sensitive measure of autonomic changes reflecting brain functions presents bilateral electrodermal activity (EDA). The evidence shows that EDA is governed by ipsilateral limbic modulation influences and correlates with amygdala activity, although also other structures, such as the ventromedial and dorsolateral prefrontal cortices, anterior cingulate gyrus, parietal lobe, insula, and hippocampus are also involved in EDA modulation Together these findings suggest a hypothesis that EDA could reflect epileptic-like conditions that are not directly presented on scalp EEG. In this context, it is possible to suppose that bilateral EDA could reflect increased synchrony and interhemispheric information transfer (transinformation) related to extension of epileptiform activity between left and right temporal lobe structures during resting conditions detected in EDA baseline activity that is not disturbed by outside stimuli.For empirical examination of suggested hypothesis the methods of psychometric assessment and EDA measurement were used in 44 outpatients of the Charles university hospital predominantly with high school education (mean of education 13.54 years). The patients had diagnosis of unipolar depressive disorder (26 patients with depressive episode and 18 patients with recurrent depression with mean period of depression 3.5 years), confirmed by clinical interview according to DSM IV criteria With exception of age range (20\u201350), diagnosis (unipolar depression) and medication (serotoninergic antidepressants) and with exception of below mentioned exclusion criteria, there were not applied any selection criteria and the patients were assessed consecutively in order of their usual visits of the outpatient center.Exclusion criteria for the patients were organic illnesses involving the central nervous system, sensory disorders, heart diseases, gastrointestinal disorders and other internal diseases, any form of epilepsy or epileptiform abnormalities on scalp EEG and mental retardation [IQ Raven higher than 90], psychotic disorders, electroconvulsive therapy, bipolar disorder, alcohol and drug abuse. Because high numbers of outpatients have unipolar depression and are treated by serotoninergic antidepressants we used this criterion for sample homogeneity. At this point the sample homogeneity was also criterion why we did not include unmedicated patients and patients with reactive depression, who may be momentarily influenced by stress factors.With a purpose to compare the results from unipolar depresive patients we have included also 35 healthy controls . The control group involved 15 men and 20 women both predominantly with highschool education. The healthy controls were selected from general population that included hospital and university stuff members (N\u200a=\u200a21) and university students (N\u200a=\u200a14). All the controls were psychiatrically healthy according to M.I.N.I.All the patients and controls gave written informed consent and the clinical study was approved by the university ethical committee.For the assessment of depressive symptoms Beck depression inventory- BDI-II Complex partial seizure-like symptoms were assessed using complex partial seizure-like symptoms inventory\u2013 CPSI Symptoms of momentarily experienced stress were measured using Impact of Event Scale- IES EDA was recorded bilaterally using a two-channel SAM unit and Psylab software (Contact Precision Instruments) connected to a personal computer with sampling frequency 1000 Hz. The measurements were performed in a quiet room with a room temperature of about 23\u00b0C. During the measurement the participant was in resting state and sat in a comfortable chair. The measurement was performed using two pairs of Ag/AgCl electrodes (8 mm diameter active area) filled with electroconductive paste that were attached to the medial phalanges of the index and middle finger of each hand.Practical approaches to studying complex dynamical systems, such as the human brain, present methods of time-series analysis This method of nonlinear data analysis was applied to 100 seconds long left- and right- EDA time series during rest using algorithm for pointwise transinformation which is included in software package Dataplore. The algorithm is performed through calculation of transinformation between EDA time series x1 (left) and x2 (right). The pointwise transinformation is a time-resolving variant of the transinformation, returning an estimation of the transinformation for each sample (point in time).The defining formula for the i-th time step is:Statistical evaluation of PTI values (in bits), CPSI, BDI II and IES values was performed using software package Statistica version 8.0 and included descriptive statistics, Spearman correlations and nonparametric Mann-Whitney test for independent samples.The results show that during rest conditions complex partial seizure-like symptoms (CPSI) are significantly correlated with the information transfer (PTI) . Other cAdditionally, we have used Mann\u2013Whitney test for independent samples as a confirmation method for correlation analysis which indicate that the patients with higher level of complex partial seizure like symptoms (CPSI) display increased level of interhemispheric information transmission measured by PTI calculated between left and right EDA records in comparison to patients who have lower CPSI score . Resultsf .To distinguish the effect of BDI-II, IES and PTI on CPSI, we have used a multiple linear regression because it may be useful to know whether depression and PTI in their specific interactions are linked to increased levels of CPSI and whether BDI-II and PTI are tightly linked together. The result shows that multiple R\u200a=\u200a0.76 is statistically significant which enables to define CPSI as a linear function of three variables CPSI\u200a=\u200aIn further evaluation we have also performed correlational analysis of the psychometric measures with EDA activity (skin conductance level) for each patient on both hands. The results indicate that there is no direct association between EDA and psychometric measures and also we have not found any association between EDA changes and disease conditions such as the absence of differences between EDA in relapse and in remissions, as well as between pharmacotherapy response and EDA. In addition worthy of attention are also correlations between BDI-II and CPSI and between IES and CPSI that likely also may influence the PTI.The same correlational analysis of the psychometric measures with PTI and EDA activity (skin conductance level) for each participant on both hands we have also performed in the healthy control group. The results indicate that there is no direct association between PTI, EDA and psychometric measures. Similarly as in depressive patients we have found statistically significant correlations between BDI-II and CPSI but not between IES and CPSI.The results indicate that information flow between two EDA channels is related to complex partial seizure-like symptoms but not to depression or actual stress symptoms. With respect to current findings this result provides the first evidence on the relationship of EDA transinformation with complex partial seizure-like symptoms presented as cognitive, affective, memory, sensory, behavioral and somatic symptoms in depressive patients.Findings of the present study are also in agreement with clinical data reported by several studies. For example, Silberman et al. Similar result reflecting relationship between EDA transinformation (PTI) and psychosensory symptoms of temporal epilepsy we have found also in our previous study in alcohol dependent patients who during withdrawal and also in abstinent period in many cases display reduced inhibitory functions and kindling that likely may appear also in the form of psychosensory symptoms similar to temporal lobe epilepsy frequently in conditions of normal EEG and without seizures In this context, increased EDA transinformation could be explained by increased synchrony that may be caused by covered epileptic-like process, although this pathological process in principle does not mean that these patients have underlying neurological disorder, in fact epilepsy. With respect to close association of CPSI symtoms with stress (IES) and also with depression (BDI-II) and PTI it is likely that some symptoms of CPSI could reflect the increased emotional tension and stress at least in a subgroup of depressed patients. This assumption is in agreement with reported studies that repeated stressful stimuli may lead to increased vulnerability to stressors and influence sensitization with kindling-like progression that may result to limbic epileptiform activity Taken together these clinical findings are in agreement with evidence that focal seizures restricted to the hippocampus may progress without neurological clinical symptoms that are manifested when the focal hippocampal seizures spread to other structures such as the parahippocampal cortices and amygdala Partial limitation of this study is that we had not sufficiently large group of unmedicated unipolar depressive patients who could be included to the study. Inclusion of unmedicated patients in principle would be very useful for comparison of the results because SSRIs may cause various side effects that in principle could influence complex partial seizure-like symptoms and EDA transinformation. It is also possible that relationship between EDA transinformation and complex partial seizure-like symptoms could be stronger in unmedicated patients.The relationship between EDA transinformation and complex partial seizure-like symptoms is also in agreement with findings suggesting that an important factor that implicates increase in neural synchrony and a possible transition of latent epileptiform process to clinical seizure is the speed of interhemispheric propagation of established epileptiform activity"} +{"text": "Since sulfotransferases exhibit low substrate specificities caused by their high degree of conformational freedom [As metabolism is considered a main cause for adverse drug reactions and failures of new drug candidates, our goal is to establish an freedom , activitWe therefore established a workflow based on molecular dynamics (MD) simulations to cover the whole spectrum of structural flexibility and incorporated it into multiple pharmacophores that represent specific modes of action. Using an ensemble of pharmacophores for virtual screening ensures accurate categorization of potential SULT ligands . Recent advances in MD technology allowed"} +{"text": "Individuals with osteoporosis are predisposed to hip fracture during trips, stumbles or falls, but half of all hip fractures occur in those without generalised osteoporosis. By analysing ordinary clinical CT scans using a novel cortical thickness mapping technique, we discovered patches of markedly thinner bone at fracture-prone regions in the femurs of women with acute hip fracture compared with controls.We analysed CT scans from 75 female volunteers with acute fracture and 75 age- and sex-matched controls. We classified the fracture location as femoral neck or trochanteric before creating bone thickness maps of the outer \u2018cortical\u2019 shell of the intact contra-lateral hip. After registration of each bone to an average femur shape and statistical parametric mapping, we were able to visualise and quantify statistically significant foci of thinner cortical bone associated with each fracture type, assuming good symmetry of bone structure between the intact and fractured hip. The technique allowed us to pinpoint systematic differences and display the results on a 3D average femur shape model.The cortex was generally thinner in femoral neck fracture cases than controls. More striking were several discrete patches of statistically significant thinner bone of up to 30%, which coincided with common sites of fracture initiation .Femoral neck fracture patients had a thumbnail-sized patch of focal osteoporosis at the upper head-neck junction. This region coincided with a weak part of the femur, prone to both spontaneous \u2018tensile\u2019 fractures of the femoral neck, and as a site of crack initiation when falling sideways. Current hip fracture prevention strategies are based on case finding: they involve clinical risk factor estimation to determine the need for single-plane bone density measurement within a standard region of interest (ROI) of the femoral neck. The precise sites of focal osteoporosis that we have identified are overlooked by current 2D bone densitometry methods. The annual incidence of hip fractures is projected to rise fourfold to 6.3 million worldwide by 2050, because of the exponentially increasing risk of fracture as people live longer. Studying femoral neck and trochanteric fractures is therefore a health priority A new CT image processing technique From 2006 to 2009, women admitted to Bulovka University Hospital, Prague with an acute hip fracture were consented to the pragmatic \u2018Surgical treatment of the hip joint in trauma\u2019 study (PI Professor P Dungl), part of which involved a clinical CT scan of both hips before surgical fixation The analysis method is illustrated in left upper panel and trochanteric fractures vs controls; right upper panel). Similar maps were created to visualise the statistical significance of differences (lower panels). Several distinct patches of up to 30% thinner cortical bone were identified in fracture cases which coincided with typical sites of hip fracture. No regions of statistically significant thicker bone were seen in fracture cases. WHO-defined osteoporosis was present in less than half of hip fracture patients and 9/75 (12%) controls. The age, height and weight adjusted values for the clusters of thinner bone associated with each fracture type are shown in Percentage differences in cortical thickness between each hip fracture group and the control group were displayed on an average right femur surface map using a colour scale. Views from several anatomical planes were chosen to illustrate the differences that controls could have substantial thickening of bone at various sites through unknown mechanisms. Finally, although studying the cortex is important in determining bone strength, alternative methods such as finite element (FE) models use whole bone biomechanics, and can therefore be informative in determining how and why individuals fracture their hips (as reviewed recently by Cristofolini et al In related work, Li et al applied SPM to 3D density maps of femurs and discovered focal regions where clustered voxels of bone density differed significantly between hip fracture cases and controls"} +{"text": "Surgery can only offer palliation in an attempt to slow the progression of malignant pleural effusion (MPE). We want to assess the effectiveness and safety of decortication in selected patient with trapped lung compare with control group with talk pleurodesis only.We reviewed our VATS decortication results in 26 patients with MPE over a 6 year period, compare with 40 patient treated with talk pleurodesis and drainage only. Major symptoms were chest pain, cough and dyspnea, and radiographic findings of pleural fluid. Trapped lung was demonstrated after MPE evacuation in all patients preoperatively. Patient with malignant mesothelioma end bronchial carcinoma and atelectasis was excluded from this study.The patients underwent subtotal (80%) or total decortication (20%). No surgical mortality and the morbidity rate was 3%. Morbidity included prolonged air leak (n = 2), reaccumulation of pleural fluid (n =1). Palliative results included dyspnea and cough relief in all patients with VATS decortication, chest relief in (85%) and pleural fluid control in 23 (96%) patients. Median survival was 18 months in VATS patients compare to 6 mounts in patient with talk pleurodesis only. Chi test was used to compere groups and Kaplan-Mayer method for estimating the survival analysis.We conclude that VATS decortication and talk pleurodesis safely provides effective treatment of pleural effusion and symptoms and therefore excellent palliation in selected patients with malignant pleural effusion and trapped lung."} +{"text": "Our objective is to develop viral vaccine vectors that will elicit neutralizing antibodies that are specific for the functional attachment protein on the HIV particle. To achieve this goal, we are developing vectors that express membrane-anchored Env trimers that closely mimic authentic functional glycoprotein spikes.We are using vesicular stomatitis virus (VSV) as a vector platform for delivery of Env immunogens as transmembrane glycoproteins. We have investigated a variety of vector designs and Env modifications to identify combinations that balance the practical requirement for vector genetic stability with factors influencing antibody responses including immunogen abundance, efficient post-translational processing, and presentation of antigenic determinants representative of a functional trimeric spike.Substituting domains in Env with analogous regions from VSV G, we have developed a number of immunogens that are efficiently expressed and incorporated in the infected cell plasma membrane, and in most cases, progeny virus particles. Antigenicity was evaluated using a panel of monoclonal antibodies specific for various Env epitopes.We identified modified Env immunogens that contain determinants for most classes of known broadly neutralizing monoclonal antibodies including those with specificity for the CD4 binding site , V3 and carbohydrate (PGT126), the MPER (2F5 and 4E10), the glycan shield (2G12), and structures formed by V1/V2 and carbohydrate . Results from ongoing immunogenicity studies with vectors encoding SIV or HIV Env immunogens indicate that the modified trimers elicit antibody responses in small animals and nonhuman primates, and that some live vectors induce mucosal antibodies. Study sera are being analyzed for virus neutralization activity and fine specificity."} +{"text": "Generalised excessive or prolonged foot pronation has been implemented in numerous functional changes to the lower limb resulting in overuse injuries affecting the lower back hip, knee, lower leg, ankle and foot. Motion control components incorporated in midsole of shoes have been based on biomechanical reasoning which assumes excessive motion can be controlled via mechanisms of restraint however this has not be clearly demonstrated in the literature. The aim of this project was to determine the effect of dual density midsoles and neutral midsoles compared with a barefoot condition on the rearfoot kinematics and kinetics in walking gait.Sixteen participants with a Foot Posture Index of 0 to +5 indicating a normal foot type were recruited for this study. Each participant performed walking trials in barefoot, neutral shoe and dual density shoe conditions. A nine-camera, three -dimensional motion analysis system was used to measure frontal plane rearfoot motion and tibial rotation for each condition during the stance phase of gait. Frontal plane rearfoot moments were calculated for each condition.There was no significant difference in peak rearfoot frontal plane motion or peak tibial rotation between the barefoot and shoes conditions. The dual density shoe demonstrated a trend towards reduced maximum eversion compared with the barefoot condition (p=0.06). There was a significant increase in peak inversion moment between the barefoot condition and the dual density shoe in walking and running gait (p<0.05). No significant difference in peak rearfoot eversion or peak inversion moment was found between footwear conditions however the neutral shoe was associated with a non-significant increase in peak inversion moment. Time series data demonstrated earlier onset and longer duration of inversion moment in both walking and running gait in the barefoot condition.The results of this study indicate that dual density and neutral sports shoes do not have a statistically significant effect on kinematics of the tibia or rearfoot, however there is evidence to suggest a dual density shoe may reduce peak rearfoot eversion. The reduction in inversion moment associated with both types of footwear suggests there may be less demand on anti-pronatory muscles associated with footwear use."} +{"text": "Since the development of the living / controlled radical polymerization method ATRP in 1995 [Guanidine copper complexes display high activity in ATRP of styrene but the factors imposed on the activator/deactivator equilibrium are multifaceted . Herein"} +{"text": "International depression screening guidelines in heart failure (HF) are partly based on depression treatment efficacy from randomized controlled trials (RCTs). Our aim was to test the external validity of depression RCT criteria in a sample of real-world HF patients.HF patients admitted to 3 hospitals in South Australia were referred to a HF psychologist if not already receiving current psychiatric management by psychologist or psychiatrist elsewhere. Screening and referral protocol consisted of the following; (a). Patient Health Questionnaire \u226510; (b). Generalized Anxiety Disorder Questionnaire \u22657); (c). positive response to 1 item panic attack screener; (d). evidence of suicidality. Patients were evaluated against the most common RCT exclusion criteria personality disorder, high suicide risk, cognitive impairment, psychosis, alcohol or substance abuse or dependency, bi-polar depression.Total 81 HF patients were referred from 404 HF admissions, and 73 were assessed . Nearly half (47%) met at least 1 RCT exclusion criterion, most commonly personality disorder (28.5%), alcohol/substance abuse (17.8%) and high suicide risk (11.0%). RCT ineligibility criteria was more frequent among patients with major depression and dysthymia but not significantly associated with anxiety disorders. RCT ineligible patients reported greater severity of depression and were higher consumers of HF psychotherapy services .In this real-world sample comparable in size to recent RCT intervention arms, patients with depression disorders presented with complex psychiatric needs including comorbid personality disorders, alcohol/substance use and suicide risk. These findings suggest external validity of depression screening and RCTs could serve as a basis for level A guideline recommendations in cardiovascular diseases. Depression has gained widespread research attention with respect to prognosis of heart diseases including heart failure (HF) Though a number of studies have applied routine depression screening protocols to improve recognition of depression The topical nature of routine depression and anxiety screening To what extent are real-word HF-patients with depression covered by the inclusion and exclusion criteria of RCTs on depression in HF patients?Do RCT ineligible patients differ from RCT eligible patients with respect to demographic and clinical characteristics?What are the prevalence rates of various depression and anxiety disorders among HF patients referred for integrated mental health management after routine depression and anxiety screening?This study received ethics approval and all participants provided written and informed consent prior to assessment . Between April 2011 and June 2012 patients with verified HF admission were managed by specialist HF nurses in a HF self-management program (HFSMP) Referred patients were contacted by telephone to schedule the initial mental health assessment and all facets of HFSMP care was provided at no cost. The HFSMP was community based, delivered flexibly at home visit, hospital site, or prior to weekly HFSMP exercise classes at Hampstead Rehabilitation Hospital. Ineligibility criteria for psychologist referral was not having cardiologist verified HF or currently receiving psychology and/or psychiatrist support elsewhere.Patients who consented to standard HFSMP psychology assessment were free to refuse treatment at any time in accordance with ethical guidelines and government primary health care protocols. Patients not desiring the HFSMP psychology assessment (n\u200a=\u200a5) were provided with alternative counselling arrangements including psychiatrist referral, local psychologist support and tele-counselling. Refusal did not impinge on standard cardiology care. HFSMP psychology was withdrawn in cases when patients transitioned to a palliative care team and the associated mental health supports. Patients requiring acute psychiatric care were managed by the treating psychologist in collaboration with the 24 hour South Australian Mental Health Emergency Triage Service for Community and Older Persons (Acute Crisis Intervention Service).Referred patients repeated the depression and anxiety questionnaire at the psychologist intake assessment and again before each subsequent psychologist appointment to verify symptom response to treatment. The PHQ-9 In the last 4 weeks, have you had an anxiety attack \u2013 suddenly feeling fear or panic?\u201d) Patients also completed an 8 item questionnaire regarding anxiety ; and a one-item panic screener \u201cPatients were assessed with the Structured Clinical Interview for DSM-IV Axis-I and AXIS-II disorders Comparison of the present community treatment sample against RCT exclusion criteria focussed on depression interventions as there are no known anxiety disorder interventions in HF patients. Ineligibility for RCT was determined from the recent systematic review of depression interventions in HF reported by Woltz and colleagues personality disorder suicide risk cognitive impairment current or past psychosis active alcohol/substance abuse or dependency current or past bi-polar Data analysis was performed with SPSS\u00ae 19.0 . Descriptive comparisons between RCT eligible and ineligible groups employed the independent samples t-test, and the chi-square statistic with Fisher\u2019s exact test as appropriate. All statistical tests were two-tailed, an alpha value p<.05 was considered statistically significant. This exploratory study pertains to RCT criteria validation and we have therefore not adjusted for multiple comparisons During the study period 81 patients were referred to HF mental health care, 8 were not included (did not want support (n\u200a=\u200a5), HF death prior to mental health assessment (n\u200a=\u200a2), receiving psychology treatment elsewhere (n\u200a=\u200a1). This left a sample of 73 patients whom underwent mental health assessment and psychotherapy as appropriate of assessed patients would be excluded from RCTs according to the six standard exclusion criteria . The mosComparison of the RCT eligible and ineligible patients with respect to demographics and comorbidities is shown in Comparison of the RCT eligible and ineligible patients with respect to clinical psychiatric factors is shown in The prevalence of depression and anxiety disorders is shown in This study reports the mental health status subsequent to depression and anxiety screening among HF patients. Psychological assessment suggested that patients commonly presented with emotional disorders other than depression including GAD and panic disorder, consistent with other research A number of effective treatments for depression have been reported The current findings should not detract from the importance of prior RCT studies The current findings should thus serve to raise awareness regarding psychiatric illness complexity and comorbidity, particularly as treatment-resistant depression increases cardiovascular risk The strength of this study was comprehensive psychological assessment after a routine depression and anxiety screening initiative in ambulatory HF patients thus facilitating mental health care tailored to individual patient needs. This study is presented with several limitations that temper the generalizability of these findings. Firstly, the use of anxiety questionnaires may have elicited more referrals for patients with comorbid anxiety-depression such as GAD and panic disorder In conclusion, implementation of routine depression screening protocols in cardiology settings may underestimate the severity and complexity of psychiatric needs in HF such as comorbid personality disorders, alcohol/substance use, suicide risk and anxiety disorders. Application of six standard exclusion criteria suggested that the extant RCT evidence may not apply to half of HF patients referred for psychiatric care. Further investigation into external validity of depression RCTs in cardiology settings is recommended to better reflect typical HF patient needs"} +{"text": "Stress hyperglycemia in the critically ill is a complex process in which insulin signaling is systematically hijacked to provide energy substrate for metabolic priorities such as cell healing or infection containment. Fluctuating levels of plasma glucose are associated with increased mortality in the ICU . We deveInsulin resistance following insult has been shown to be driven primarily by the immune response via the cytokine IL-6 . We creaInhibitory dynamics driven by IL-6 were incorporated into the cellular model to attenuate an insulin signaling intermediate (insulin receptor substrate 1) according to the proposed biological mechanisms. The percentage reduction in glucose uptake as a function of IL-6 concentration was fit to data from patients who underwent elective abdominal surgery , shown iA multiscale model has been developed to describe the inhibitory effects of IL-6 on insulin-mediated glucose uptake. Cellular inhibitory dynamics were shown to capture reduced insulin sensitivity on the macroscale, which could then be used to characterize insulin sensitivity and to provide insulin treatment advice to reduce glucose variability."} +{"text": "Acute pain is one of the most common problems faced in prehospital emergency medicine. Sufficient prehospital pain therapy reduces psychological and emotional stress. Other clinical benefits include optimized conditions for patient transport, increased patient satisfaction and a better chance of timely and proper analgesia at the emergency department. Unfortunately, undertreatment of acute pain is common. Oligoanalgesia is associated with following factors:\u2022 Variable clinical experience\u2022 Concern for masking illness or injury\u2022 Focus on other clinical symptoms\u2022 Poor education in pain management and insufficient compliance with pain management protocols\u2022 Fear of inducing adverse effects\u2022 Lack of follow-up after initial pain therapy administrationOne approach to minimize oligoanalgesia is to increase the use of fentanyl in the prehospital environment. Fentanyl is an opioid with rapid-acting properties and short time of action allowing safe titration and few side effects.In order to optimize prehospital pain management all ambulance personnel in Central Denmark Region have been taught how to administer fentanyl in specific clinical situations and under certain circumstances. We wish to present the preliminary results from a 3-month period in which rescuers working for one of the two ambulance companies operating in the region, Responce and Falck, were allowed to administer fentanyl.A total of 204 patients were treated with fentanyl by ambulance personnel over a period of 3 month. About one half had some kind of injury (n=114) and the remainder experienced pain due to acute coronary syndrome (n=52), abdominal pain (n=18) or other clinical conditions (n=20). None of the patients experienced side effects. Antidote was not required in any of the cases.The administration of fentanyl by ambulance personnel seems to be safe. Future studies will further evaluate pain and the safety and effectiveness of fentanyl administered by ambulance personnel in Central Denmark Region."} +{"text": "Gastrointestinal bleeding appears to be a common adverse event associated with dasatinib therapy. Here we present a case of a 59-year-old man with chronic myeloid leukaemia (CML) developing the rarest complication of haemorrhagic colitis with dasatinib therapy which resolved rapidly after treatment withdrawal. Dasatinib is a widely used therapy for chronic myeloid leukaemia especially when resistance has occurred to first line therapies such as Imatinib . Althoug9/L, the total and differential white cell count and prothrombin time were within normal limits. Serum albumin was significantly reduced at 24\u2009g/L and serum CRP was significantly raised at 99\u2009mg/L. Stool cultures were negative for Clostridium difficile and other pathogenic enteric bacteria. Endoscopic examination of the lower gastrointestinal tract revealed a granular and congested mucosa in the rectum along with large ulcers between the descending colon and splenic flexure (see A 59-year-old man with chronic-phase chronic myeloid leukaemia (CML), first diagnosed in 2004, was initially treated with Imatinib 400\u2013800\u2009mg once a day. He developed cytogenic resistance to Imatinib and was switched to dasatinib 70\u2009mg twice a day, achieving a good haematological response. Mutation analysis revealed an E450Q mutation in the ABL kinase domain of the BCR ABL fusion gene which has been shown to be associated with Imatinib resistance . Three yxure see . The posHistology revealed an acute colitis. The crypt architecture was well preserved and this argued against a diagnosis of inflammatory bowel disease. There was no leukaemic involvement and given the clinical presentation a drug aetiology was raised , duration of CML, and advanced disease (bleeding noted more commonly in the accelerated and blast phase of CML as opposed to the chronic phase of CML) [The variables thought to increase the risk of bleeding whilst on dasatinib therapy include thrombocytopenia (platelets <30 \u00d7 10 of CML) . Our patin vitro platelet activation by collagen was reduced and tail bleeding in mice was increased following dasatinib exposure [in utero angiogenesis and capillary wall development leading to microaneurysm formation and haemorrhage [The causes of bleeding secondary to dasatinib are likely to be multifactorial and the underlying pathophysiology remains poorly understood. Dasatinib has been shown to significantly reduce platelet aggregation in CML patients when compared to the use of other tyrosine kinase inhibitors . A furthexposure . This stexposure . PDGFR nmorrhage . Mustjoki et al. described an association between lymphocytosis secondary to significant expansion of clonal large granular lymphocytes (LGL) and patients receiving dasatinib . DasatinA more recent report looking at bleeding diathesis in CML patients receiving dasatinib therapy noted that 81% of all bleeding episodes were confined to the gastrointestinal tract . GastroiHere we describe a case of acute haemorrhagic colitis secondary to dasatinib therapy. Current literature describing this association is scarce with only four cases being previously reported. There first case is a paediatric patient with Philadelphia acute lymphoblastic leukaemia who similarly developed haemorrhagic colitis during dasatinib therapy which responded to steroids and discontinuation of the drug . In thisIn summary, although gastrointestinal bleeding is a recognised complication of dasatinib therapy in CML, haemorrhagic colitis is much less common but responds well to discontinuation of therapy. Physicians are advised to be astute to this particular presentation especially in patients with thrombocytopenia and advanced disease. Further studies are needed to understand the exact pathophysiology of this complication."} +{"text": "Carotid cavernous fistulas (CCF) are a dural arteriovenous fistulas which include pathological communications between the arterial system and the venous cavernous sinus situated at the wall of the cavernous sinus (CS). It can be demonstrated by wide range clinical presentations, like ophthalmic signs and symptoms , cranial nerve pareses, bleeding from the cranial structures and intracranial hemorrhage. We report a case of a patient which has diagnosed with CCF initially presented with persistent daily headache followed with promptly neurological progression. Case report: Twenty eight years old women has presented with 3 months history of daily left side headache, predominantly localized in left eye profound, without free after analgotherapy. Parallel with headache development she described sensation as a heart sound in pain area. Her mother died since some brain AV malformations. On examination, she had no ophthalmoplegia, proptosis and chemosis with normal neurological status. Auscultation under her left eye has found a whir. Digital subtraction angiography was performed and revealed a left side ophthalmic and facial venous dilatation with CS dilatation during all the time beginning from early arterial phase. As a result of panangiography detected a direct fast flow CCF, without presentation of left ICA intracranial parts with promptly retrograde venous swelling into the ophthalmic and facial venous and petrous sinus. During three days in neurological examination have occurred fully left side ophtalmoplegia with diplopia, left side ptosis and hypestesis in V1 area. The patient was scheduled for endovascular therapy (performed stent assisted coil embolization with complete occlusion of the fistula).In the small number of cases CCF can be presented by minimal symptoms such as persistent daily headache. This condition must be diagnosed and treated promptly since it has a high risk of clinical progression."} +{"text": "Cardiac MRI is the accepted gold standard for the assessment of left ventricular systolic function. However, no standards exist for the MRI assessment of left ventricular diastolic function. We hypothesize that the superior morphologic assessment enabled by ultrafast ECG gated cine TrueFISP imaging will enable assessment of left ventricular diastolic function.The purpose of this study is to determine if morphologic assessment of the left atrial size coupled with assessment of left ventricular filling on ultrafast cine TrueFISP imaging is able to differentiate subjects with normal and abnormal left ventricular diastolic function.18 self-reported healthy volunteers and 6 patients with diastolic dysfunction underwent cardiac MRI on an investigational 1.5T cardiovascular magnet . All subjects underwent ultrafast cine MR imaging in four and two chamber orientations using an ECG-gated parallel imaging accelerated steady state pulse sequence, optimized to minimize the temporal resolution. Atrial volumes were calculated using the area-length method during left ventricular isovolumetric relaxation. Myocardial relaxation was determined visually off four chamber cine images. Diastolic dysfunction was defined as present when myocardial relaxation was abnormal or left atrial size was increased in the absence of overt mitral valvular disease or fluid overloaded status. A single reviewer, blinded to subject identity reviewed all images and performed the above measurements. All healthy volunteers were assumed to have normal diastolic function; echocardiography with tissue Doppler was the gold standard in all patients.The left ventricular diastolic function of 15 of 18 healthy volunteers (83%) and 5 of 6 patients (83%) was correctly classified at MRI. Three volunteers demonstrated borderline left atrial volume with sluggish chamber filling suggestive of diastolic dysfunction. There was a statistically significant difference in age between these three volunteers and the rest of the volunteer cohort (p<0.05). The average left atrial volume of healthy volunteers was not significantly different from that in patients with diastolic dysfunction (p>0.05). One patient with echocardiographic evidence of diastolic dysfunction was indeterminate by MRI secondary to moderate mitral regurgitation.Gated ultrafast cine Cardiac MRI may be able to distinguish subjects with diastolic dysfunction. Combined with phase contrast imaging, morphologic assessment at MRI may enable grading of left ventricular diastolic function. Further work is ongoing to quantify relaxation on cine imaging using an automated algorithm."} +{"text": "Unilateral lung agenesis is extremely rare. These patients usually present in infancy or childhood with recurrent respiratory infection and cardiopulmonary insufficiency. Here, we report a rare case of bronchial asthma and allergic rhinitis whose investigation showed complete right lung agenesis.A 26 year-old lady presented with episodic breathlessness, chest tightness, recurrent nasal obstruction and excessive sneezing, mainly during change of season along with opacity of the right hemithorax on chest x-ray. Further detailed work-up including spirometry, high resolution CT scan of the thorax and fibreoptic bronchoscopy confirmed complete right lung agenesis in patients with bronchial asthma and allergic rhinitis.Chest x-ray PA view showed homogeneous opacity with signs of volume loss on the right side. After that we confirmed by CT images of the thorax that revealed complete absence of right lung with hyperinflation and herniation of the left lung to the right side. Further, fiberoptic bronchoscopy showed completely absent right main bronchus and trachea directly leading to left main bronchus. Spirometry was also performed which revealed obstructive airway disorder with 13% post bronchodilator reversibility confirming the diagnosis of bronchial asthma. Complete control of symptoms was achieved with formoterol 6 \u03bcg and mometasone 200 \u03bcg and intranasal fluticasone 50 \u03bcg 2 puffs twice daily and oral montelukast 10 mg with levocetirizine 5 mg once daily. The patient was completely asymptomatic when reviewed after 1 month follow up and she was advised to stop intranasal fluticasone and to continue formeterol and mometasone inhaler with oral montelukast and levocetirizine preparation.This rare case illustrates that a high index of suspicion is necessary to diagnose the condition. Simple and regular asthma medications may good enough to relieve symptoms even in patients with unilateral lung agenesis for control of symptoms."} +{"text": "To measure the changes in post opertive wound infections before and after introduction of hand hygiene and safe surgery check list.To see the compliance of health workers on hand hygiene and on using the safe surgery check list.Baseline assemnet made on wound infection rate of 100 patients before the impelemenation of hand hygiene using the WHO accreditted alcohol based hand rub technique. Six monthe after, surgical site wound infection in 100 pateints of similar profile is reaudited. Wound infection is declared if there are clinical signs of wound infection from day 3 on post operative course. Wounds already infected during or befoe surgery were excluded. Study was conducted in two wards namely general surgical and Obstetric wards.Post operative wound infection rate in surgical wards was reduced by 45% while surgical site infection in obstetric wards went down by 33% after health workers used the hand hygiene in and check list in 50% of the cases.the implementation of hand hygiene and safe surgery check list was only 50 % while it brought about significant changes surgical site infection in both wards. the impact on paient safety in genreal and surgical site infection rates will go down by fully implementing the hand hygiene and safe srgery check list.None declared."} +{"text": "Many foot posture measurement approaches are not suitable for routine use as they are time-consuming or require specialised equipment and/or clinical expertise. The objective of this study was to develop and evaluate a simple visual assessment tool for foot posture assessment based on the Arch Index (AI) .Fully weightbearing footprints from 602 people aged 62 to 96 years were obtained using a carbon paper imprint material, and cut-off AI scores dividing participants into three categories were determined. A visual tool was created using representative examples for the boundaries of each category Figure . Two exaInter- and intra-tester reliability of the examiners was almost perfect . Examiner\u2019s scores were strongly correlated with actual AI values and AI categories . There was a slight tendency for examiners to categorise participants as having higher arches than their AI scores indicated.Foot posture can be quickly and reliably categorised as high, normal or low in older people using a simplified visual categorisation tool based on the AI."} +{"text": "Biochemical markers of bone turnover have been used in research for a long time and are now being recognised as helpful tools in the clinical management of bone disease. It is important to establish robust reference values for the interpretation of these markers. In addition to standardising pre-analytical variability it is unclear whether manufacturer\u2019s ranges take into account global differences between subjects and so each laboratory should investigate the transferability of the expected values to its own patient population and if necessary determine its own reference ranges.Serum samples from 70 healthy volunteers were analysed for biochemical markers of bone formation and bone resorption (beta carboxyterminal cross-linking telopeptide of bone collagen [\u03b2CTX] on the Roche Elecsys 2010. The results were compared to the manufacturer\u2019s reference range and to 46 samples from patients with severe refractory rheumatoid arthritis prior to treatment with rituximab.We found substantial inter-person variability in all of the biomarkers and differences between the manufacturer and healthy control ranges Table .These results demonstrate the large between-person variability in serum bone markers and the differences between defined \u2018healthy control\u2019 ranges, this can be critical when assessing patients with bone disease. We suggest that it is therefore important for each laboratory to investigate the transferability of the quoted reference range to its own patient population based on equivalent standardised collection conditions, and where necessary determine its own ranges. We recognize that it is often difficult to recruit sufficient healthy volunteers but the widespread availability of automated bone marker assays now means that harmonisation of methods and specific reference ranges may be possible using well-characterised populations in larger cohorts."} +{"text": "Tadarida brasiliensis) form dense aggregations in cave roosts in Texas. These bats emerge from caves daily to forage at high altitudes, which makes them detectable with Doppler weather radars. Timing of emergence in bats is often viewed as an adaptive trade-off between emerging early and risking predation or increased competition and emerging late which restricts foraging opportunities. We used timing of emergence from five maternity colonies of Brazilian free-tailed bats in south-central Texas during the peak lactation period (15 June\u201315 July) to determine whether emergence behavior was associated with summer drought conditions and daily temperatures. Bats emerged significantly earlier during years with extreme drought conditions than during moist years. Bats emerged later on days with high surface temperatures in both dry and moist years, but there was no relationship between surface temperatures and timing of emergence in summers with normal moisture levels. We conclude that emergence behavior is a flexible animal response to climate and weather conditions and may be a useful indicator for monitoring animal response to long-term shifts in climate.Interest in forecasting impacts of climate change have heightened attention in recent decades to how animals respond to variation in climate and weather patterns. One difficulty in determining animal response to climate variation is lack of long-term datasets that record animal behaviors over decadal scales. We used radar observations from the national NEXRAD network of Doppler weather radars to measure how group behavior in a colonially-roosting bat species responded to annual variation in climate and daily variation in weather over the past 11 years. Brazilian free-tailed bats ( Changes in climate can affect animal and plant populations in numerous ways One limitation to understanding how climate affects animal behavior is lack of long-term datasets that adequately measure behavioral response at the time scales necessary to detect responses to shifts in climate. Use of remote sensing data to measure changes in primary productivity provide a means to assess changes in vegetation communities The data archive maintained by the National Climatic Data Center (NCDC) of national networked weather radars (collectively known as NEXRAD) contains signals of animals aloft in the aerosphere going back to the early 1990s Timing of emergence to forage by bats is an adaptive behavior that has important fitness consequences in terms of trade-offs between increased risk of predation or competition with diurnal aerial insectivores and forfeiting foraging opportunities during peak prey availability Tadarida brasiliensis) during the maternity season is associated with variation in summer drought conditions over the past 11 years. Drought causes physiological stress for many bat species, particularly in summer months when bats are reproductively active Here, we test whether emergence behavior of Brazilian free-tailed bats (www.ncdc.noaa.gov), including all three conventional radar products: radar reflectivity factor (Z), radial velocity (vr), and spectrum width (\u03c3w). The measure of backscattered intensity, radar reflectivity factor (Z), can be directly related to the number of aerial organisms occupying the aerosphere To compare bat emergence behavior with daily and seasonal meteorological conditions, it was first necessary to establish a record of the time of emergence for a selection of bat colonies. Brazilian free-tailed bats disperse nightly in dense columns from cave and bridge roosts and forage at high altitudes (300\u20132500 m AGL) over large spatial extents that are regularly detected by the NEXRAD network of weather surveillance radars We chose five maternity colonies of Brazilian free-tailed bats in south-central Texas, which are regularly detected by radar . Becausehttp://soar.ou.edu) web portal. Data generated for this analysis were processed by special request by the National Severe Storms Laboratory that hosts SOAR. We chose to focus on data from the period of June 15 through July 15, corresponding to peak lactation period for Brazilian free-tailed bats NCDC stores NEXRAD radar products from individual radars in polar coordinates. To provide the best spatial coverage of the selected caves, we chose four of the surrounding NEXRAD installations for our analysis . Using aTo determine emergence time for each colony on each day, we defined a 40 by 40 pixel (20 km by 20 km) spatial domain centered on each of the five cave locations . A broadWe observed emergence of Brazilian free-tailed bats from Frio Cave on 10 nights from 22 June\u20131 July in 2011 to confirm radar observations. Visual estimates of timing of emergence were similar to those derived from radar. Radar reflectivity factor values can be assumed to derive primarily from Brazilian free-tailed bats given that other bat species that may use these caves occur in much lower densities, fly at lower altitudes, and do not emerge in dense columns.http://www.cpc.ncep.noaa.gov/products/monitoring_and_data/drought.shtml) . We reprt.shtml) . We averWe compared five a priori linear regression models using generalized least squares to determine how daily weather conditions influenced timing of emergence given yearly drought conditions. For this analysis, we calculated daily averages of emergence time offsets by averaging values for each of the five maternity colonies on each of the 30 days in 2001\u20132011 for the response variable. The five a priori models included a null model (emergence time constant), a main effects model with daily surface temperature as a predictor, a main effects model with a categorical variable of years classified as dry (PDSI score<\u22121), normal (PDSI score\u200a=\u200a\u22121 to 1), or wet (PDSI score>1), and parallel and varying slopes models with daily temperature as a continuous predictor and type of year as categorical predictor . We usedVisual inspection of residuals using the auto-correlation function (acf) in Program R Analyses were conducted in R.12. Brazilian free-tailed bats emerged to forage significantly earlier in the evening during drought events than in years with normal to unusually moist conditions (p<0.01) . The estThe varying slopes model after accounting for significant temporal auto-correlation in model residuals was the best fit according to AIC (AIC weight\u200a=\u200a0.98) and indicates that timing of emergence was significantly different in dry, normal, and wet summers and that the relationship between daily timing of emergence and temperature depends on summer climate type ; Table 3Our results demonstrate a strong association between climatic conditions and emergence behavior in Brazilian free-tailed bats. Bats emerged earlier in years that were characterized by severe drought conditions and later in years with moist conditions . This paDaily weather also influenced timing of emergence such that bats emerged later on hotter days in both dry and moist years . ForaginPhenotypic plasticity in response to climate can be an adaptive response that mediates impacts of changing climate on wild populations t+1\u226aNt). We are currently working on estimating aerial densities of bats directly from radar products One way to determine if emergence behavior in response to climate conditions results in changes in population growth would be to estimate population sizes of bat colonies over the same time frame in order to test whether years following severe drought were associated with significant population declines (i.e. NPast studies have investigated the functional significance of timing of emergence by assessing adaptive trade-offs, comparing foraging habits, and determining differences in age and reproductive conditions Brazilian free-tailed bats in our study emerged substantially earlier than reported emergence times of other bats. In a review comparing emergence times of bats, Jones and Rydell Our results were similar to emergence times reported for Brazilian free-tailed bats from Frio Cave in 1996 and 1997 Our study is the first to use a sufficiently long yearly time series to assess how annual variation in climate conditions influences emergence behavior in bats. Annual variation in emergence times demonstrates that plasticity in emergence behavior of bats is a response to environmental cues by which bats can alter foraging strategies to meet energy needs. Our data suggest that bats respond to both daily and seasonal conditions and that drought conditions are associated with riskier behaviors of emerging earlier. Emergence timing may be a useful long-term indicator of response to climate change by bats, particularly in arid environments. Future studies should aim to link the fitness consequences of emergence behavior response to climate and weather patterns.We used remote-sensing technology and freely available climatic indices to associate animal behavior with annual variation in climate and daily weather conditions. Numerous studies have investigated timing of emergence in bats, as it is an easily measured behavioral signal"} +{"text": "Glandular tumors involving the middle ear are rare and distinguishing between adenoma and adenocarcinoma remains difficult. A distinct subclass of these tumors demonstrates microscopic papillary architecture and has a propensity to erode the petrous bone and extend intracranially. The term \u201caggressive papillary middle ear tumor\u201d has recently been proposed to describe this more invasive type of middle ear tumor. These tumors cause symptoms even when microscopic in size. Although histologically benign, they have been locally destructive with frequent intracranial extension and patients may die of uncontrolled local disease. These tumors do not metastasize but there is single case report of drop metastasis to the spine in the literature. Hence this tumor must be distinguished from other benign tumors of the middle ear. These rare neoplasms constitute a distinct pathological entity and deserve wider recognition. Endolymphatic sac tumors are rare, and their true origin is not clear . Althoug63-year-old male patient came with the history of otalgia, hearing loss, lower cranial nerve palsy, and recurrent bleeding in the left ear since 6 years. On examination a reddish blue mass bulging from the tympanic membrane was seen in the left ear. MRI demonstrated , arrow aTumors with papillary architecture arising from the epithelium of the middle ear and invading into the adjacent bony structures has been called as \u201cPrimary aggressive papillary tumor of the middle ear\u201d by Gaffey et al. . HeffnerEndolymphatic sac tumors are rare skull base tumor originates from endolymphatic epithelium within the vestibular aqueduct, characterized clinically by slow growth with local invasion and bone destruction. Metastasis is reported in only one case. Histologically it has bland cytologic features and a papillary growth pattern. Surgical exploration of the tumor and sac is the treatment of choice but recurrence may occur due to subtotal resection."} +{"text": "DNAH5 is the gene most frequently mutated in PCD. It encodes a large 500kDa structural protein with ATPase activity that powers ciliary movement. PCD rarity, the shortage of genotyped patient samples and lack of suitable animal models means that better model systems are needed in which to study gene therapies. A further challenge is that gene transfer of DNAH5 will require an efficient non-viral delivery vector capable of packaging this gene, which is too large for commonly used viral vectors. To model PCD we have used RNA interference to silence DNAH5 in normal human bronchial epithelial cells grown in air-liquid interface cultures. Cells were transduced with a lentivirus expressing an shRNA for DNAH5. Silencing of DNAH5 expression was demonstrated and preliminary evidence that the cilia were immotile. We have cloned DNAH5 from mRNA of human ciliated cells into a mammalian expression vector and sequenced it. The DNAH5 clone expressed both mRNA and protein in transfected cells. Transfections were performed with a nanocomplex formulation comprising liposomes and targeting peptides that we have developed for DNAH5 gene transfer to the respiratory epithelium. We are now able to investigate PCD gene therapy using these models, DNAH5 constructs and vector delivery system.Primary ciliary dyskinesia (PCD) describes a family of rare genetic disorders affecting ciliary motility in several organ systems. The respiratory defects that can lead to lung failure, however, are most concerning. New treatments for PCD are needed that prevent progressive lung damage and we aim to develop gene therapy to achieve this."} +{"text": "We use historical population modelling to demonstrate that brown bears are highly unlikely to have survived through the LGM in Ireland under any combination of life-history parameters shown by living bear populations, but instead would have rapidly become extinct following advance of the British\u2013Irish ice sheet, and probably recolonized Ireland during the end-Pleistocene Woodgrange Interstadial from a closely related nearby source population. The time available for brown bear\u2013polar bear hybridization was therefore restricted to narrow periods at the beginning or end of the LGM. Brown bears would have been extremely vulnerable to extinction in Quaternary habitat refugia and required areas substantially larger than southwestern Ireland to survive adverse glacial conditions.Brown bears are recorded from Ireland during both the Late Pleistocene and early\u2013mid Holocene. Although most of the Irish landmass was covered by an ice sheet during the Last Glacial Maximum (LGM), Irish brown bears are known to have hybridized with polar bears during the Late Pleistocene, and it is suggested that the Irish brown bear population did not become extinct but instead persisted This alternative hypothesis has received support from genetic analyses of some Irish small mammals [The origin of Ireland's modern terrestrial vertebrate fauna is the subject of ongoing debate \u20133. Late Holocene . HoweverHolocene \u20137. Many Holocene . HoweverHolocene , suggest mammals and amph mammals .Ursus maritimus) also falls within the genetic diversity of Irish brown bears, indicating that this brown bear population hybridized with polar bears; it is suggested that hybridization may have occurred when the British\u2013Irish ice sheet reached its maximum extent 22\u201320 kya and provided suitable polar bear habitat in Ireland [Although no Irish brown bear fossils are known from the LGM (they are absent from the Irish fossil record between 26 340 \u00b1 320 and 12 143 \u00b1 46 year BP ), it has Ireland .in situ Irish brown bear survival was possible for the duration of the LGM.Spatial patterns of species range change in response to natural or human-mediated environmental change remain incompletely understood. Broad taxon-focus and species-specific analytical studies of \u2018dynamic biogeography\u2019 have demonstrated that many species persist in peripheral subpopulations rather than core areas of their geographical range ,12, alth2.ortex [2 in ArcView v. 9.3 using Ehlers & Gibbard [2, and with LGM sea levels 90 m lower along southeastern and western coasts and 100 m lower along the southwestern coast for the general model, and 149 years for the barren-ground model. Sensitivity analysis showed that interbirth interval and female mortality rate have greatest impact on population persistence ; there is little difference in these parameters between barren-ground and coastal brown bear populations, due to higher levels of intraspecific competition in more productive environments , suggest4.in situ, and are likely to have recolonized Ireland during the Woodgrange Interstadial from a closely related source population (probably on unglaciated southern Britain). Although the area of Irish ice cover varied between 27 and 15 kya, with a possible retreat 24\u201323 kya followed by re-advance [PVA is an analytical tool providing a modelled outcome from available parameter values, in this case modern-day populations ,25. As sOur findings reveal that brown bears would have been extremely vulnerable to extinction when their populations were periodically restricted to habitat refugia during Quaternary climatic fluctuations and would have required areas substantially larger than southwestern Ireland for long-term survival through adverse glacial environmental conditions. Conversely, although we demonstrate that remnant brown bear populations are very unlikely to persist in restricted geographical refugia through long periods of Quaternary climate change, they may still be able to survive for several hundred years or more in the absence of any population connectivity. Although brown bear populations in northern Eurasia and North America are still large and contiguous, the species has been extirpated from much of the southern portion of its former range, and most surviving populations in western and central Europe, central Asia and the United States are now small and fragmented due to historical-era anthropogenic pressures , in mos"} +{"text": "The emotional impact of genetic testing has been extensively studied in individuals at high risk for Hereditary Breast and Ovarian Cancer Syndrome (HBOS). Studies on the impact of genetic testing for Lynch Syndrome (LS) are not as prevalent and few studies have compared the two cohorts.To compare the psychological impact of genetic testing in participants at high risk for LS and HBOS and identify variables associated with genetic testing distress, anxiety and depression at one week and one year post disclosure of genetic test results.140 individuals undergoing genetic testing for LS and 133 undergoing genetic testing for HBOS at the Dana-Farber Cancer Institute were enrolled in a longitudinal questionnaire study. We assessed levels of genetic testing distress, anxiety and depression using The Multidimensional Impact of Cancer Risk Assessment (MICRA) and the Hospital Anxiety and Depression Scale (HADS). Multivariable regression analysis was used to identify factors associated with psychological distress in the LS and HBOS cohorts. One year follow-up data is based on 96 LS and 94 HBOS tested individuals that completed the one year questionnaire.For both cohorts, individuals who had the expectation for carrying a deleterious gene pre-test and/or those who received a positive or variant of uncertain significance (VUCS) test result had significantly higher genetic testing distress at one week post-disclosure (p \u2264 0.05). Additional independent predictors of higher genetic testing distress in the LS cohort included not being married and having a personal history of cancer. In the HBOS cohort, predictors included having a higher HADS anxiety score at baseline and having an annual household income of \u2264 $50,000.At one year post-disclosure, individuals that tested positive or VUCS and/or had the expectation of carrying a deleterious gene pre-test continued to have significantly higher genetic testing distress (p \u2264 0.01). While there were no differences between the cohorts in baseline or post-disclosure HADS anxiety or depression scores, significantly more individuals in the LS cohort had HADS depression scores \u2265 8 one year after testing .Individuals at risk for LS and HBOS whose genetic tests reveal a pathogenic mutation or VUCS can continue to demonstrate genetic testing distress one year after receiving genetic test results. These individuals may benefit from counseling beyond the immediate disclosure period."} +{"text": "Molecular chaperone or heat shock proteins (HSP) are vital proteins that increase cell survival by allowing it to combat stress caused by injurious stimuli through certain cyto-protective mechanisms . These cStabilization of the structure of important agents in malignant transformation, such as kinases (Src and Met tyrosine kinases) and transcription factors allows molecular chaperones to stimulate angiogenesis by promoting endothelial cell proliferation and permitting growth of cancer beyond the oxygen capacity of tissue diffusion . MoleculThe new therapeutic agents or Heat Shock Protein inhibitors function by blocking the intrinsic ATPase activity of molecular chaperones allowing oncogenic proteins to be taDespite effective results in phase 1 of clinical trials , HSP inhAlthough still in phase 2 of clinical trial, the development of HSP inhibitors provides an exciting alternative for molecular-based therapy in cancer . HSP inh"} +{"text": "Severe malaria is a potentially life-threatening infectious disease. It has been conclusively shown that artesunate compared to quinine is superior in antiparasitic efficacy and in lowering mortality showing a better short-term safety profile. Regarding longer-term effects, reports of delayed haemolysis after parenteral artesunate for severe malaria in returning travellers have been published recently. So far, delayed haemolysis has not been described after the use of parenteral quinine.In this retrospective study, all patients treated for severe malaria at the University Medical Centre Hamburg-Eppendorf were included between 2006 and 2012. The primary endpoint was the proportion of delayed haemolysis in patients treated with quinine versus those who received artesunate. As secondary endpoint, the proportion of any adverse event was assessed.A total of 36 patients with severe malaria were included in the analysis. Of these, 16 patients contributed sufficient data to assess the endpoint delayed haemolysis. Twelve were treated primarily with intravenous quinine \u2013 with four patients having received intrarectal artesunate as an adjunct treatment \u2013 and five patients were treated primarily with artesunate. Five cases of delayed haemolysis could be detected \u2013 two in patients treated with quinine and intrarectal artesunate and three in patients treated with artesunate. No case of delayed haemolysis was detected in patients treated with quinine alone.While adverse events observed in patients treated with artesunate were limited to delayed haemolysis and temporary deterioration in renal function , patients treated with quinine showed a more diverse picture of side effects with 22 patients (71%) experiencing at least one adverse event. The most common adverse events after quinine were hearing disturbances , hypoglycaemia and cardiotoxicity .This study provides further evidence on delayed haemolysis after artesunate and underlines the importance of a standardized follow-up of patients treated with artesunate for severe malaria. Plasmodium falciparum malaria are notified per year in the WHO European region with around 25 deaths..Case desClick here for fileCase description \u2013 patient 2 [-[.atient 2 -33]..Case desClick here for file"} +{"text": "Dear Sir:Giardia duodenalis, present ongoing challenges to military personnel serving on deployments in Djibouti, and elsewhere.We thank Dr. Coton for his kind letterGiardia in 3 of 113 specimens (3%) collected from service members presenting with diarrhea or with high fever.Although published information is lacking on the incidence of giardiasis specific to deployed French Forces, a research study conducted between 1992 and 1993 during a previous United States military operation in neighboring Somalia found Giardia infection, presumptive treatment with either metronidazole or albendazole are both equally effective and each are fairly well tolerated among adults.3For cases of persistent diarrhea thought caused by Giardia infection should remain a priority. The wider use of rapid assays for diagnosis may help to overcome many of the limitations found in certain deployed settings where adequate laboratory facilities are often lacking, and thus aid in improving surveillance in settings where this infection may be a significant and unappreciated cause of prolonged gastrointestinal illness.Despite the availability of such therapies, improving opportunities for definitive diagnosis of"} +{"text": "Panthera leo. We conducted two 96 hour, continuous follows of lions in Addo Elephant National Park seasonally from December 2003 until November 2005 (16 follows), and compared prey encounter rate with prey abundance, hunt rate with prey encounter rate, and kill rate with prey hunt rate for the major prey species in Addo using Jacobs' electivity index. We found that lions encountered preferred prey species far more frequently than expected based on their abundance, and they hunted these species more frequently than expected based on this higher encounter rate. Lions responded variably to non-preferred and avoided prey species throughout the predatory sequence, although they hunted avoided prey far less frequently than expected based on the number of encounters of them. We conclude that actions of lions throughout the predatory behavioural sequence, but particularly early on, drive the prey preferences that have been documented for this species. Once a hunt is initiated, evolutionary adaptations to the predator-prey interactions drive hunting success.Research on coursing predators has revealed that actions throughout the predatory behavioral sequence drive observed prey preferences. We tested whether similar actions drive the observed prey preferences of a stalking predator, the African lion Panthera leo kill blue wildebeest Connochaetes taurinus, Cape buffalo Syncerus caffer, gemsbok Oryx gazella, giraffe Giraffa camelopardalis, and plain's zebra Equus burchelli significantly more than expected based on their composition of the prey community at a site Lycaon pictus, cheetah Acinonyx jubatus, and leopard Panthera pardus also kill a small number of prey species significantly more frequently than expected, based on their relative abundance in the prey community African lions Despite this overwhelming evidence of predatory specialization via preferential predation, the question arises whether the predator behaviour drives selection for certain prey species or whether the preferential predation findings arise simply from increased hunting success for those species that the predator has evolved morphological specializations to successfully hunt. Cheetahs do not capture larger prey in more densely vegetated sites, leading to the conclusion that the prey they kill is limited by morphological factors rather than kleptoparasitism avoidance We aimed to test which actions of a stalking predator \u2013 the African lion \u2013 influenced whether or not it pursues a particular prey item and whether it captures it. We did this by determining whether lions improved their likelihood of a successful hunt at three distinct phases of the predatory behavioural sequence. Firstly, we determined whether lions encountered preferred prey species more frequently than expected based on the abundance of prey in the community. Secondly, we determined whether lions elected to hunt preferred prey species more frequently than expected based on the encounter rate of prey. Finally, we determined whether lions kill preferred prey more frequently than expected based on the number of attempted hunts. We predicted that lions would 1) encounter preferred prey more frequently than expected in a random encounter rate with prey; would 2) hunt preferred prey more frequently than expected based on random hunts of the encountered prey; and would 3) have a higher hunting success rate on preferred than non-preferred prey species. If these predictions were supported, we believe this would show that lions were making decisions throughout the predator behavioural sequence to optimize their foraging success.The buffalo population in Addo exceeded 300 individuals, yet these were encountered by lions far less frequently than expected based on their relative abundance . This isThe small zebra population in Addo was encountered by lions far more frequently than expected, based on their relative abundance, and hunts were conducted as frequently as expected based on this high encounter rate . There wKudu was the dominant herbivore in Addo, with roughly 1000 present in the park. Lions encountered kudu in accordance with their abundance, they hunted kudu in accordance with their encounter rate, and they killed kudu in accordance with the number of hunts they conducted . Lions wThe warthog population in Addo increased from 298 in 2004 to over 600 in 2005. Lions encountered warthogs more frequently than expected based on their abundance, and they elected to hunt the encountered warthogs as expected based on the encounter rate . Lions cThe eland population in Addo averaged around 100 individuals and these were encountered by lions as frequently as expected based on their total relative abundance . Like buThe 280 hartebeest in Addo were encountered by lions far more frequently than expected based on their relative abundance . Lions hThe ostrich population of Addo varied between 180 and 260 over the study period. These individuals were encountered as frequently as expected, however they were hunted far less frequently than expected based on the number of encounters, even though the number of kills were far more frequent than expected based on the hunting effort . There wAddo's elephant population exceeded 300 individuals made up of eight herds and dozens of male groups Lions encounter preferred prey more frequently than expected . ConversBoth buffalo and zebra were preferentially preyed upon in Addo. This preferential predation arose through a high encounter rate and a high hunting rate . Hence, preferential predation appears to be initiated early in the predatory behavioural sequence in a stalking predator like lions, as it is in coursing African wild dogs Hunting success is not highest for the preferred prey species suggestiOur results agree with data on African wild dogs, which showed that their prey preferences were the result of a series of preferential decisions throughout the predatory behavioural sequence Lions had a higher than expected encounter rate with warthog and the highest hunting success rate for this species & Fig. 2Canis lupus reintroduction Clethrionomys glareolus where anti-predator behaviour appears innate Canis lupusThe lions' hunting success rate improved from the first year to the second year of the study for all species except buffalo . The preIt could be argued that small sample size may limit the broader conclusions of this study, however these results came from observations of six unrelated lions living in two to five groups reintroduced to a novel environment with novel prey, so their actions are likely to reflect characteristics of the species as a whole. Furthermore, it is highly unusual to be able to monitor individual lions at such a high level of detail and few other studies have been able to achieve this There are other factors that may influence the decision making process in stalking predators. Hunger level may influence the decision whether to attempt a hunt, however there was no relation between hunger and hunting effort in Addo's lions, although this may have been due to methodological problems The lions reintroduced to Addo's dense thicket environment came from the open, arid Kalahari ecosystem after an absence of over 100 years. Both predators and prey rapidly altered their behaviour to contend with these new challenges and there is little evidence to date that the reintroduction programme has caused a predator-prey imbalance.2 Main Camp section of South Africa's Addo Elephant National Park (Addo) where six radio collared lions were reintroduced by the South African National Parks in 2003 after an absence of over 100 years We studied lion predatory behaviour in the 134 kmPortulacaria afra as part of the densely vegetated Thicket Biome Addo is situated in the Eastern Cape Province and is approximately 70 km north of Port Elizabeth. The original vegetation of Addo was dominated by spekboom Phacochoerus africanus and kudu Tragelaphus strepsiceros are also present and are generally killed more frequently than expected in the thicket biome Tragelaphus oryx and hartebeest Alcephalus busephalus are killed in accordance with their availability Struthio camelus and elephant Loxodonta africana are universally avoided by lions Prey species abundances at Addo have been published previously Data on lion hunting behaviour was collected during 16 continuous follows of focal animals that occurred twice per season between summer (December) 2003 and spring (November) 2005. These continuous follows involved following the focal animal continuously for periods of up to 96 hours We recorded an encounter with prey if the prey individual or group was detectable to us and if there were no obvious impediments to detection by the focal lion. We counted the number of encounters with groups rather than the number of individuals in each group encountered because we were unable to accurately count group sizes in the dense, thicket vegetation. We defined hunts as characteristic behaviours that involved focused attention on a potential prey item, stalking toward the prey individual, and ultimately charging Annual estimates of the prey community were made by standardized aerial censuses using helicopters and several observers. These data have been published elsewhere ip is the relative proportion of the previous predatory behavioural phase and ir is the relative proportion of the latter predatory phase . We analysed buffalo and eland abundance based both on individual counts and our estimate of number of mixed sex and bachelor herds. This was not conducted for other species, as buffaloes and eland exist in large herds of up to 100 individuals and so the discrepancy between the number of individuals and herds is greatest for them. We considered Jacobs' index values >0.2 as implying preference and <\u22120.2 as implying avoidance, with values between these two figures indicating a behaviour is performed as frequently as expected.We used Jacobs' selectivity index"} +{"text": "Three dimensional pharmacophores and pharmacophore searches are well established in virtual screening and have been applied successfully in many prospective and retrospective drug discovery campaigns . While tRecently, Silicos NV provided their freely available ligand centric pharmacophore method Pharao . Pharao Cavity Knowledge Acceleration), our own in-house strategy for structure based pharmacophore generation. CavKA interprets ligand-receptor complexes and detects interaction between ligand and binding site in order derive pharmacophore models automatically. In addition Grid [Here we present a hybrid model that utilizes the Gaussian pharmacophore representation of Pharao to be adapted and used in CavKA (ion Grid MoleculaHYBRID is compared to the purely ligand centric approach Pharao in a retrospective virtual screening on the Field Screen [HYBRID is analysed.The performance of CavKAd Screen dataset."} +{"text": "First, we found that glucose uptake rate in the dorsal caudate nucleus was higher in obese than in normal-weight subjects. Second, obese subjects showed increased hemodynamic responses in the caudate nucleus while viewing appetizing versus bland foods in fMRI. The caudate also showed elevated task-related functional connectivity with amygdala and insula in the obese versus normal-weight subjects. Finally, obese subjects had smaller responses to appetizing versus bland foods in the dorsolateral and orbitofrontal cortices than did normal-weight subjects, and failure to activate the dorsolateral prefrontal cortex was correlated with high glucose metabolism in the dorsal caudate nucleus. These findings suggest that enhanced sensitivity to external food cues in obesity may involve abnormal stimulus-response learning and incentive motivation subserved by the dorsal caudate nucleus, which in turn may be due to abnormally high input from the amygdala and insula and dysfunctional inhibitory control by the frontal cortical regions. These functional changes in the responsiveness and interconnectivity of the reward circuit could be a critical mechanism to explain overeating in obesity.Obesity is characterized by an imbalance in the brain circuits promoting reward seeking and those governing cognitive control. Here we show that the dorsal caudate nucleus and its connections with amygdala, insula and prefrontal cortex contribute to abnormal reward processing in obesity. We measured regional brain glucose uptake in morbidly obese ( Similar to drugs of abuse, food consumption is associated with dopamine release in the dorsal striatum in healthy subjects, and the amount of dopamine released is correlated positively with ratings of food pleasantness 2R density, which is directionally proportional to BMI Palatable foods carry strong motivational power. Mere sight of a delicious cake or the smell of our favourite food may elicit a strong urge for eating right now, and exposure to such cues may override physiological satiety signals and trigger food consumption 2 receptor density - a hallmark of dysregulated reward circuit - in obese subjects connectivity of the reward circuit. While a recent imaging study in healthy humans demonstrated that connectivity within the human reward circuit is dependent on individual sensitivity to external food cues Although altered sensitivity of the reward circuit may be a critical factor explaining obesity, it remains elusive how exactly the reward circuitry contributes to food-related anticipatory reward functions in obese individuals. First, previous demonstrations of elevated reward circuit responses to foods in normal-weight and obese subjects 18F]FDG PET with an fMRI experiment involving anticipatory reward induced by presentation of appetizing and bland food pictures. Note that although no rewards were actually delivered to the participants, we use the term \u2018anticipatory reward\u2019 for the sake of conciseness, as seeing highly rewarding targets such as foods reliably induces reward anticipation responses in the ventral striatum, even when no rewards are actually delivered In this study we applied multimodal brain imaging by combining FDG, and another into the opposite warmed arm for sampling of arterialized blood. The euglycemic hyperinsulinemic clamp technique was used as previously described \u22121 \u00b7 min\u22121 . During hyperinsulinemia, euglycemia was maintained by infusing 20% glucose intravenously. The rate of glucose infusion was adjusted according to plasma glucose concentrations measured every 5\u201310 min from arterialized blood. At the time point 100+\u221210 minutes of euglycemic hyperinsulinemic clamp, [18F]FDG (189\u00b19 MBq) was injected intravenously over 40 second and the dynamic brain scan for 40 min started. During the scan arterial blood samples were drawn for radioactivity analysis. A GE Advance PET scanner with resolution of 4.25 mm was used for PET studies as previously described 18F]FDG was synthesized as previously described The studies were performed after 12 hours fasting. Subjects refrained from caffeine-containing drinks and from smoking 24 hours before PET studies. Any kind of strenuous physical activity was prohibited from the preceding evening. Two catheters were inserted into antecubital veins, one for saline, insulin and glucose infusions and injection of radiotracer FDG PET scan would predict abnormal anticipatory reward on fMRI, we first extracted subject-wise GMR values in the caudate nucleus from the parametric GMR images. Next, we used these values as a regressor in a second-level model comparing the BOLD responses to appetizing versus bland food in fMRI. This analysis showed that increased glucose metabolism in the caudate nucleus predicted smaller responses to appetizing versus bland food specifically in the right lateral frontal cortex , primary somatosensory cortex and posterior insula than normal-weight subjects density in the striatum, and blunted dopamine release following the administration of the drug of abuse 2R density, and it has been proposed that this may reflect downregulation which compensates frequent transient dopamine increases due to perpetual overestimulation of the reward circuit by drug use or eating Dorsal caudate nucleus has been implicated in habitual stimulus-response learning, motivation and conditioning, and imaging studies in humans suggest that it contributes to variety of functions related to reward signaling and addictions. Patients with drug addiction show lower baseline DBy using the hyperinsulinemic clamp, we simulated a situation where the body is in a satiated state in terms of insulin signaling. Although this approach does not completely simulate physiological satiety due to a lack of orosensory stimulation and release of hormones from the gut, placebo-controlled intravenous glucose has been shown to increase hormonal markers of satiety 2R availability in striatum is negatively associated with frontocortical glucose metabolism elevated frontal responses to food pictures in obese versus normal-weight individuals. It is likely that these discrepancies across studies reflect task-dependent engagement of the frontal cortex: whereas our study involved implicit processing of briefly presented food cues, Rothemund and colleagues employed relatively long stimulus presentation with a memory task. It is thus possible that the obese individuals may fail to activate the cognitive control circuits particularly when they are not explicitly processing the food items they are viewing. Accordingly, this suggests that even \u2018unseen\u2019 or unattended food pictures in various advertisements could trigger powerful urges for eating in obese individuals.It has been established that in obese subjects, DThe amygdala is involved in early stages of reward processing The interpretation of a significant PPI is that there is differential engagement of anatomical connections as a function of psychological context. Although the PPI cannot be used to reveal whether or not such connections exist, it is likely that the PPIs we observed reflect changes in the engagement of direct anatomical connections between the seed and target regions because such direct anatomical connections between the striatum and amygdala are supported by tracing studies in other primates Amygdala neurons facilitate reward seeking via their projections to the striatum anterior insula were smaller in the obese subjects. The anterior insula integrates autonomic and visceral signals into motivational and emotional functions, whereas the posterior insula is thought to underlie somatosensory, vestibular and motor integration as well as monitoring bodily states elevated anterior insular responses to expected and consummatory food-related rewards in obese rather than in lean individuals The PPI analyses revealed that the interconnectivity between dorsal striatum and posterior insula was elevated in the obese versus normal-weight subjects, whereas regional responses to appetizing versus bland foods in the n\u200a=\u200a35) the between-group comparisons for fMRI data were not significant when corrected for multiple comparisons. Although the between-group differences were observed in predicted regions, some caution should be warranted when interpreting the findings. Furthermore, it must be stressed that we were not able to fully delineate the exact psychological mechanism that results in elevated brain responses to food pictures in obese individuals. Although we acquired ratings of the perceived pleasantness (\u2018liking\u2019) of the foods, these were similar across obese and normal-weight individuals. Accordingly, elevated liking of appetizing foods in obesity is unlikely to contribute to the differences in brain responses. However, it could be speculated that food craving rather than liking could be the key factor that modulates brain responses to food pictures in obesity. In support of this hypothesis, it has been shown that although obese and normal-weight individuals \u2018like\u2019 foods similarly, stress-induced food craving is much higher in obese individuals One obvious limitation of the present study was that despite a large sample size (We show that obesity is associated with elevated glucose metabolism of the caudate nucleus, as well as modified regional responses and altered connectivity of the reward circuit when seeing appetizing versus bland foods. These data parallel with the findings on altered brain functioning in addictive disorders, and support the view that obesity may share a common neural substrate with addictions"} +{"text": "Mesenchymal stromal cells (MSCs) are under investigation for clinical application. Despite approval by the United States Food and Drug Administration for MSC use in pediatric steroid-refractory acute GvHD after allogeneic hematopoietic stem cell transplantation , were infused via tail vein injection. MSCs exhibited perivascular homing remote to the lungs and liver as well as paracrine expression of growth factors mediating vascular regeneration in specific sites. We were able to visualize MSCs in vivo by intravital fluorescence microscopy and laser scanning confocal microscopy and post mortem histologically in the peripheral tissue after transplantation of fluorescent allogeneic MSCs. Freshly isolated LinCultured MSCs may not be phenotypically distinguishable from fibroblasts and may even share similar surface antigens or differentiation potential Hematti, . With reIn the past, it has been shown that the duration and degree of cell expansion and culture has a clear impact on MSC morphology, differentiation, viability, and migratory properties If the degree of this phenomenon varies with the size of MSCs infused (based on passage cycle or culture denominators); (2) if there are long-term effects on lung function due to the entrapped cells and; (3) if the immunological efficacy of MSCs could be improved through direct arterial delivery to the graft or specific end organs. There is some evidence that the loss of cells through a first pass effect is indeed lower with freshly isolated MSCs indicating a link to smaller cell size or possibly related to enhanced viability and homing capacity.Taken together, studies comparing effects of fresh isolated MSCs delivered intra-arterially to the graft or in proximity to the end organ to those secondary to passaged MSCs delivered via a peripheral intravenous route may be important to define if indeed this is a technical or procedural consideration essential for incorporation into pre-clinical protocols to optimize overall outcomes."} +{"text": "To the Editor: Bacchetti are likely transported from the gut to lymphoid tissue by cells such as migrating intestinal dendritic cells (Res appears to be amplified by follicular dendritic cells (,In 1979, Dickinson and Outram (Res to follicular dendritic cells.Both in vitro and in vivo animal and human studies demonstrate age-related declines in both humeral and cellular components of the immune system (Res challenge of young versus old animals. Because the intracerebral challenge bypasses the immune system portal, old, peripherally challenged animals should show a disproportionate reduction in disease risk if immune system senescence is important in regulating pathogenesis.This hypothesis could be readily tested by intracerebral versus peripheral PrP"} +{"text": "Xerus inauris, a ground squirrel found throughout southern Africa, is extremely social and promiscuous with one of the highest male reproductive investments among rodents. These life-history attributes suggest males and females should demonstrate a large dichotomy in immunity. Contrary to our prediction, we found no difference in spleen mass between the sexes. However, we did find significant biases in leukocyte types and red blood cell counts, possibly reflecting responses to parasite types. Among males, we predicted greater investments in spermatogenesis would result in reduced immunological investments. We found a negative association between testes and spleen size and a positive relationship between testes and number of lice suggesting trade-offs in reproductive investment possibly due to the costs associated with spermatogenesis and immunity. We suggest when measuring sexual differences in immunity it is important to consider the effects of reproductive pressures, parasite types, and life history costs.Differences in how males and females respond to foreign antigens are common across taxa. Such sexual differences in the immune system are predicted to be greater in species with high promiscuity and sociality as these factors increase the likelihood of disease transmission. Intense sperm competition is thought to further this sexual dichotomy as increased investment in spermatogenesis likely incurs additional immunological costs. Sexual selection imposes different selective pressures on males and females resulting in a dichotomy in fitness strategies Male vertebrates typically show increased susceptibility to disease and higher rates of parasitic infections compared to females The ICHH predicts species with high investment in spermatogenesis are more likely to demonstrate a trade-off between reproductive and immunological investment Xerus inauris), a species that is both highly social and promiscuous We addressed sex differences in immunity in the Cape ground squirrel or spleen size after controlling for body size. Despite finding no significant difference between body condition of males and females , males did have significantly more ectoparasites per individual averaging 71.3\u00b118.3 compared to females . The majority of those ectoparasites for both sexes were lice . We took blood from live animals in addition to those we euthanized such that we had 7 additional male samples and 22 additional females, although red blood cell counts were not performed on all additional animals. We found the percentage of red blood cells to be significantly lower in males than females and lymphocytes and females having higher percentages of neutrophils . Testes =\u200a0.020) . Despite=\u200a0.020) .X. inauris have been shown to carry high parasite loads with males having significantly higher ectoparasite loads and females having significantly higher endoparasite loads X. inauris while ectoparasites are suspected to be under hormonal control Sex biases in parasitism often are attributed to sex-specific life-history strategies that affect parasite susceptibility and exposure such that differences in reproductive strategies should determine male and female immunocompetence The field of ecological immunity is becoming increasingly complex as variables such as parasite type X. inauris, however, are year-round, asynchronous breeders, requiring both sexes to continuously invest in reproduction X. inauris significantly increases reproductive success suggesting females also are immunologically challenged Previous studies of immunosuppression imply selection only imposes stress on males during the energetically expensive breeding season Sceloporus jarrovi) demonstrated a positive association between plasma testosterone levels and mites but a negative association with endoparasites Significant increases in ectoparasite loads do not arise in males until they reach reproductive maturity Our findings support the ICHH and the male\u2019s response to testosterone but also suggest a larger issue rarely addressed in the literature. Different parasite types trigger different immune responses. In this system, both males and females have high energetic constraints but significant differences in parasite loads We handled and euthanized animals in accordance with the American Mammal Association guidelines X. inauris from May - June 2007 at S.A. Lombard Nature Reserve near Bloemhof, South Africa . We trapped squirrels using live traps , targeting males for use in multiple studies, and euthanized a subset of adult animals on site with a halothane overdose. While this subset was chosen opportunistically, we are confident that we did not introduce unintentional bias as previous trapping history has no indication of biases in the condition of the animals trapped We sampled To assess immunity, we measured spleen size in euthanized animals and percentage of red and white blood cells in all animals. Although such measures are proxies for immunity, they are frequently used to assess immunocompetence To measure male reproductive investment, we measured mass (\u00b10.01 g) of each testis. To remove effects of body size, we calculated the residuals of a least squares regression of total testes on body mass. We removed outliers if the value fell two standard errors outside of the mean. We then compared relationships between residuals using a least squares regression"} +{"text": "We report a rare case of papillary thyroid carcinoma incidentally found within a branchial cleft cyst. Only four other cases have been described in the literature. A total thyroidectomy and selective neck dissection was performed, and no evidence of occult primary disease was found after review of fine sections. Branchial cleft cysts are the most common lateral neck masses. Ectopic thyroid tissue within a branchial cleft cyst is an unusual phenomenon, and papillary thyroid carcinoma arising from this tissue is extremely rare. Clinicians are left with a diagnostic dilemma when presented with thyroid tissue neoplasm within a neck cyst in the absence of a thyroid primary\u2014is this a case of metastatic disease with a missed primary or rather carcinoma arising in ectopic thyroid tissue? A thorough discussion of the etiologies of these lateral neck masses is reviewed including the embryogenesis of thyroid tissue in a branchial cleft cyst. The prognosis of patients with papillary thyroid carcinoma in lateral neck cysts without a primary site identified appears to be good following excision of the cyst and total thyroidectomy. Other management recommendations regarding these unique lateral neck malignancies are also presented. Branchial cleft cysts are the most common lateral cystic neck masses. Ectopic thyroid tissue within a branchial cleft cyst is a rare phenomenon, and papillary thyroid carcinoma (PTC) arising from this tissue is extremely rare , 2.We report a case of PTC incidentally found in a branchial cleft cyst. A total thyroidectomy and selective neck dissection was performed, and no evidence of occult primary disease was found after review of fine sections. A brief discussion of the etiologies of these lateral neck masses is reviewed including how ectopic thyroid tissue may be present in a lateral neck mass. Management recommendations regarding lateral neck malignancy without a primary are also presented. This paper was approved by the Tripler Institutional Review Board.A 20-year-old euthyroid man presented with a history of painless right-sided neck swelling. Physical examination revealed a 6\u2009cm mass in his right neck without a cutaneous fistula. His referring provider ordered a contrast-enhanced CT scan of the neck that showed a cystic lesion in the lateral neck without cervical lymphadenopathy Figures and 2 coPresuming metastatic spread from a thyroid primary, a total thyroidectomy and selective neck dissection was performed. Serial thin sections of the complete thyroid gland and analysis of lymph nodes did not detect carcinoma. Since the tumor was 1\u2009cm without evidence of other neck diseases, radioactive iodine-131 was not given.To our knowledge, only four cases have reported PTC arising in a branchial cyst without a primary in the thyroid \u20133, 9. AtBranchial cleft cysts are the most common lateral cystic neck lesion and typically present in the fourth decade of life. However, despite considerable study of branchial cysts, their etiology remains unclear. Previous theories describe branchial cleft cysts as congenital malformations resulting from failure of the branchial pouch apparatus. Recent theories, however, suggest that some of these lateral neck masses are the result of cystic degeneration triggered by epithelial inclusions that migrate into lymph nodes. Proponents of this acquired \u201cinclusion theory\u201d for branchial cyst formation suggest that epithelium from upper aerodigestive tract or glandular tissue enters a cervical lymph node via lymphatics and stimulates degeneration into a lateral cervical cyst . ExaminaAnother acquired theory proposes that the lateral anlage of the thyroid develops from the fourth-fifth branchial pouch and can aberrantly entrap normal thyroid tissue . ThyroidRegardless of etiology, the prognosis of patients with carcinoma in lateral neck cysts without a primary site identified after total thyroidectomy appears to be good \u20133, 9. Th Although few examples exist of thyroid carcinomas presenting in a lateral neck cyst without a primary in the thyroid, management recommendations are similar in the literature. Imaging and fine needle aspiration cytology (FNAC) do not take the place of tissue diagnosis, and excisional biopsy should be performed. When the clinician is presented with a thyroid carcinoma in a lateral neck cyst a thorough examination of the neck is necessary. Imaging such as ultrasound and CT scan of the neck plays a role to help distinguish the presence of metastatic nodes from branchial cysts as they can appear similar. Total thyroidectomy is strongly recommended \u20133, 9, 10"} +{"text": "To assess the role of CMR in the treatment of true chronic total occlusions (CTO) with percutaneous coronary intervention (PCI) and drug eluting stent implantation.Successful PCI for CTO may confer an improved prognosis and a reduction in major adverse cardiac events. However most trials have included occlusions of short duration (less than 4 weeks). In this study we assessed the impact of PCI on LV function in patients with true CTOs (TIMI flow grade 0 and greater than 12 weeks duration) using serial CMR imaging as well as the predictive value of late gadolinium enhancement when performed prior to revascularization.Thirty patents referred for PCI to a single vessel CTO underwent CMR examination prior to and six months after PCI. Technical success was defined as recanalization of the occluded vessel and DES implantation with a final residual diameter stenosis <30%. LV function and infarct size were assessed using a 1.5T GE MRI system. Segmental wall thickening (SWT) was measured within the perfusion territory of the CTO using the 16-segment model and segments were dysfunctional if the SWT was \u226445%. The transmural extent of infarction (TEI) was calculated by dividing the hyperenhanced area by the total area x 100; a score of \u226425% were considered viable.Technical success was achieved in 19 of the 30 patients (63%). CTO duration was greater in patients with failed revascularization but other baseline demographics were well matched between groups Table . There wPCI-CTO success of true CTOs can improve global LV function. The TEI, assessed with CMR, can be used to help predict improvements in regional wall function. Failed PCI was not associated with increased MACE at medium-term follow up."} +{"text": "Traumatic brain injury studies predominantly use an assessment of neurological function some time after hospital discharge as the primary endpoint. Recent studies have followed up patients at 6 months after injury with very variable loss to follow up ,2. We haThe DECRA trial is a prospective randomised trial of 155 patients from Australia, New Zealand, and Saudi Arabia. Patients with severe traumatic brain injury and refractory intracranial hypertension were randomly assigned to receive either a decompressive craniectomy or to continue with standard medical management. The primary outcome was patient's neurological function using the Extended Glasgow Outcomes Scale (GOSE) at 6 months after injury. Patients were tracked following hospital discharge by the Research Coordinators at each participating site. The GOSE assessments were conducted by three blinded assessors using structured telephone questionnaires. The assessment team was led by an experienced assessor. Two assessors were located in Australia and one assessor in Saudi Arabia. Assessors were trained using a prepared training package of examples and self-testing exercises. The chief assessor reviewed the outcome assessments performed by the other assessors. Any complex assessments were referred to an assessment panel for a consensus decision.DECRA commenced recruitment in 2003 and the last patient was enrolled in April 2010. Research coordinators successfully tracked all surviving patients, which resulted in a 100% follow-up rate for the primary study outcome measure.We have successfully completed a prospective randomised controlled trial with zero loss to follow up for the primary outcome measure of GOSE at 6 months. Assessments were reviewed by the chief assessor and a consensus panel if required to ensure consistency of the assessment."} +{"text": "Neurons in the medial superior olive (MSO) and lateral superior olive (LSO) of the auditory brainstem code for sound source location in the horizontal plane by extracting interaural time differences (ITD) from the fine structure or envelope of sound stimuli. Both cell types are tuned to frequency and are organized along a tonotopic axis.The statistics of natural sounds vary with frequency, e.g., the signal to noise ratio of combined behaviorally relevant and background noise stimuli typically decrease with increasing frequency . Also, ain vitro whole-cell recordings we characterized the membrane filters of cells in the guinea pig MSO and LSO with ZAP current injections. All MSO cells and some of the LSO cells showed membrane potential resonances with peak frequencies between 80 and 400 Hz. The experiments suggest that the peak resonant frequencies decrease along the tonotopic axis (with increasing CF). Using a modeling approach we first assessed what membrane currents could underlie the resonance. Linear models fitted to the data predict that a gradient in both the density and activation time constant of a low threshold potassium current (IKLT) is probably underlying the resonant frequency gradient. We subsequently examined how the filter gradient affects ITD sensitivity to natural sounds. Simulations where we fed guinea pig vocalizations to the neural filters via an auditory nerve model show that ITD sensitivity increases with the cell\u2019s peak resonant frequency, also in noisy environments. Hence, our results suggest that ITD sensitivity decreases along the tonotopic axis. This finding could underlie the decreasing performance in ITD discrimination with increasing CF and amplitude modulation frequency found in psychophysics [We studied whether the intrinsic properties of MSO and LSO cells vary along the tonotopic axis in order to optimize ITD sensitivity to natural sounds. Using ophysics ."} +{"text": "RCTs are critical to evidence-based medicine, but recruitment is routinely problematic. Robust tests of recruitment methods, where alternative approaches are compared within RCTs, are rare. The MRC funded START programme aims to test the feasibility of routinely nesting recruitment interventions in host RCTs. This will rapidly extend the evidence base, allow promising recruitment interventions to be tested across multiple trials, and improve our understanding of the impact of contextual factors on recruitment.Newly commissioned or recruiting trials were identified via PCRN and HTA portfolios.A total of 225 PIs were sent a flyer describing the MRC START programme with a covering email (from PCRN or HTA) inviting them to contact the team if they would like to know more about the study. Preliminary discussions were held with interested trials to screen them for suitability. Formal agreements were then put in place and trial specific intervention content was developed.Nearly a third (32%) of PIs approached expressed an interest in the study. Just over half (52%) were excluded, reasons included incompatible recruitment strategies, timetable issues or trial size. Reaction to the additional burden placed on trials has so far been positive. Documentation has been found fit for purpose and the process of implementation supported by a \u2018can do' culture and complex research skills in trial managers.This presentation will:\u2022 (a) describe START and report on progress to date,\u2022 (b) discuss early findings in relation to feasibility and host trial experience,\u2022 (c) present materials to support the recruitment of host trials."} +{"text": "There is increasing recognition of the utility of focused echocardiography in critically ill patients and a need for suitable training programmes to be developed to meet the specific needs of critical care. Critical care communities across Europe have struggled to implement focused echocardiography into everyday clinical practice. We aim to determine whether a training programme could be implemented during a year of advanced intensive care training in a region where none of the critical care consultant body had accreditation in echocardiography, and to establish the perceived training requirements in critical care echocardiography in our region and to evaluate what information clinicians wished to obtain from a focused echocardiography examination.Trainees attended a course designed for echocardiography in a peri-arrest situation. Local cardiac anaesthetists with experience in transthoracic echocardiography were recruited as mentors. Data archiving protocols were established. Trainees performed an initial 10 scans directly supervised on the cardiac ICU. A further 40 scans were completed independently on the general ICU. A logbook was maintained and the scans reviewed with a mentor prior to final sign off. This process was supported by a regional educational meeting where personnel interested in echocardiography reviewed the types of training provided and how this matched local needs and resources. This included trainees and trainers in intensive care medicine, anaesthesia and acute medicine.Although 91% of doctors wished to incorporate focused echocardiography into their clinical practice, only 36% had undergone any focused echocardiography training and only 5% had focused echocardiography accreditation. The majority of respondents wished only to incorporate eyeball assessments of ventricular function but did not wish to perform more complex examinations such as Doppler assessment.It is possible to implement a simple training programme in echocardiography in an intensive care medicine department with no prior experience in critical care echocardiography. Within our region there is strong demand for simple training in focused echocardiography rather than a higher level of accreditation currently offered by many courses."} +{"text": "Clinical trials engrafting human fetal ventral mesencephalic tissue have demonstrated, in principle, that cell replacement therapy provides substantial long-lasting improvement of motor impairments generated by Parkinson's Disease (PD). The use of fetal tissue is not practical for widespread clinical implementation of this therapy, but stem cells are a promising alternative source for obtaining replacement cells. The ideal stem cell source has yet to be established and, in this review, we discuss the potential of neural stem cells in the adult subventricular zone (SVZ) as an autologous source of replacement cells. We identify three key challenges for further developing this potential source of replacement cells: (1) improving survival of transplanted cells, (2) suppressing glial progenitor proliferation and survival, and (3) developing methods to efficiently produce dopaminergic neurons. Subventricular neural stem cells naturally produce a dopaminergic interneuron phenotype that has an apparent lack of vulnerability to PD-mediated degeneration. We also discuss whether olfactory bulb dopaminergic neurons derived from adult SVZ neural stem cells are a suitable source for cell replacement strategies. Parkinson's Disease (PD) is one of the most prevalent neurodegenerative disorders and afflicts ~1% of the population over 60 years of age in industrialized nations of the lateral ventricles of neuronal precursors in SVZ cultures prior to transplantation (Deleyrolle and Reynolds, in vitro culture conditions with Noggin may also enhance suppression of glial phenotypes. SVZ neural stem cells and neural progenitors express BMP receptors and antagonism of these receptors by Noggin inhibits glial differentiation (Lim et al., A second challenge is the suppression of glial progenitor proliferation and survival. SVZ neural stem cells prodigiously generate neuronal progenitors in their endogenous niche, but most of the progenitors generated by these cells either do not to survive or they adopt alternative fates when cultured Nurr1 and Mash1, avoids the concerns of genetic reprogramming, but retroviruses are questionable for clinical application since they can increase the risk of tumor formation by disrupting endogenous gene expression upon their insertion into the host genome (Yi et al., A third challenge is developing efficient methods to produce dopaminergic neurons. Treatment either with growth factors, morphogens, signaling molecules, or chromatin modifying agents does not direct SVZ-derived progenitors toward a dopaminergic fate. By contrast, significant dopaminergic neuron production has been only achieved through genetic modification of SVZ-derived progenitors (Shim et al., Nurr1 and Mash1 has provided the best yield of SVZ-derived dopaminergic neurons to date, only a subset of transduced cells adopted a dopaminergic phenotype. This partial induction may reflect the heterogeneity within the SVZ stem cell niche. Specific subsets of olfactory bulb interneurons are generated from the neural stem cells that are spatially localized in distinct regions of the adult SVZ (Kelsch et al., in vitro. In addition to transcription factors, heterogeneous expression of microRNAs may also be instrumental in shaping the in vitro potential of adult SVZ neural stem cells to generate dopaminergic neurons (De Chevigny et al., Even though over-expression of The adult SVZ is a promising source of neural stem cells to generate dopaminergic neuronal progenitors for cell transplant strategies in PD. Since these stem cells can be endogenously harvested from patients, they are a potential autologous source of replacement cells. As discussed in this review, however, there are several important technical issues to be addressed in order for adult SVZ neural stem cells to be competitive source for clinically suitable dopaminergic neurons.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "The management of medicinal clinical trials commonly presents challenges to trial teams, particularly when seeking practical and affordable systems for managing the trial randomisation process and associated Investigational Medicinal Product (IMP) distribution.In blinded IMP trials, these challenges include:\u2022 1) how to manage IMP supply when the preferred randomisation algorithm does not guarantee balance between trial arms in each centre\u2022 2) tracking of IMP expiry dates and stock levels to ensure continuous supply of both trial arms at site\u2022 3) tracking availability of central stock of manufactured IMP to facilitate planning of subsequent manufacturing runsTo provide an efficient and robust solution to these challenges, a web-based randomisation system with integrated IMP management, is presented.Designed within the King's Clinical Trials Unit, the system accommodates simple randomisation, block randomisation, stratified block randomisation and minimisation, enabling trial teams to manage blinded IMP distribution to sites while remaining fully blinded to treatment allocation. Blinding is maintained even where there is gross imbalance between trial arms of patients within a study site.This system avoids the need for IMP manufacturing companies to take on this role. Further, it avoids the need for individuals within the study management team to be unblinded while managing the IMP supply during the conduct phase. It has also been designed to accommodate many of the common events that occur in clinical trials, such as patients losing packs of medication or IMP expiry dates being extended mid-study."} +{"text": "Most available tools for conformer generation, like OMEGA , ROTATE For our systematic search for pairwise dependent torsion angles, we assembled a set of about 200 chemical patterns, each describing a torsion angle and part of its environment. The patterns range from very general descriptions like 'rotatable bond between two sp3 hybridized atoms' to patterns describing a more specific molecular environment.As a first approach we tried to replicate the examples given by Bramelt et al. using suitable chemical patterns and a CSD subset oUsing our systematic search approach we found many additional examples for dependent torsion angles, confirming the findings of Bramelt et al. and supporting their advice to search for pairs of mutually dependent conformation variables."} +{"text": "By simulating natural food shortage in mice that work for food we show that reduced food intake alone shifts the activity phase from the night into the day and eventually causes nocturnal torpor . Release into continuous darkness with ad libitum food, elicits immediate reversal of activity to the previous nocturnal phase, indicating that the classical circadian pacemaker maintained its phase to the light-dark cycle. This flexibility in behavioral timing would allow mice to exploit the diurnal temporal niche while minimizing energy expenditure under poor feeding conditions in nature. This study reveals an intimate link between metabolism and mammalian circadian organization.Nocturnal rodents show diurnal food anticipatory activity when food access is restricted to a few hours in daytime. Timed food access also results in reduced food intake, but the role of food intake in circadian organization In such restricted schedules the food received is limited both in time and in quantity . This procedure induces food anticipatory activity (FAA) a few hours before the food is delivered i.e. spend time and energy) to obtain food. To study the effect of reduced food intake on circadian organization, we simulated natural food scarcity by having mice work for their food at increasing levels of workload under a 12 h light - 12 h dark (LD) cycle, without restricting food access to a specific time of day.In nature, food availability is hardly periodic to most rodents, and diurnal activity has often been observed in nocturnal animals ad libitum food; ad libitum food access throughout the experiment did not show advanced activity onsets in LD but rather delayed offsets advanced for the onset of activity and 2.4 h advanced for the offset of activity (green vs. grey bar) when compared to the ad libitum control mice. Taken together, these data indicate that reduced food intake shifts activity from the dark phase into the light phase. Upon release into DD with food available ad libitum, activity patterns instantaneously reverted to the previous dark phase reduced daily food intake as descrrk phase . This maperiment .b) were associated with activity both in mice that work for their food and in ad libitum fed mice , which seems unaffected by the work for food protocol, and a less intense activity component (Actshifted) that followed the main activity component under high reward ratio and ad libitum conditions. Actshifted component shifted and preceded the main activity component under medium and low reward ratio conditions activity when feeding is restricted during a few hours in the light phase. This restricted diurnal meal timing is thought to uncouple activity rhythms from the circadian pacemaker (SCN) signal All animal experiments were approved by the University of Groningen ethical committee and carried out following (inter-) national animal welfare regulations.ad libitum food condition, because excess pellets were observed in each individual kept under these conditions. During the reward reduction phases of the experiment, workload was increased daily by an extra 10\u201320 revolutions per pellet up to 200 revolutions per pellet (medium reward schedule) or 300\u2013400 revolutions per pellet (low reward schedule). Throughout the experiment body mass was closely monitored at least every 3rd day at different times of day and high workload levels were individually titrated to keep the mice above 18 grams and 75% of their initial body mass.Adult male mice were kept under standard laboratory conditions see . Sample Body temperature was measured by customised temperature loggers implanted in the abdominal cavity of the mice and set to a sampling rate of once every 20 minutes see .Text S1Supporting Information for \u2018Working for food shifts nocturnal mouse activity into the day\u2019.(DOC)Click here for additional data file.Figure S1Reduced food reward affects body temperature patterns. Body temperature patterns of two representative animals with gradually reduced food reward and an ad libitum control animal (C). Body temperature occurring during the rest phase is reduced, eventually leading to torpor. Data are from the same animals as presented in (TIF)Click here for additional data file.Figure S2Reduced food reward shifts activity rhythms before torpor occurs. This example shows that some animals in the experiment presented in (TIF)Click here for additional data file."} +{"text": "Formulas based on protein hydrolysates or free amino acids are used to manage food allergies in infants and young children. Three formula categories are available: Products containing partially hydrolyzed proteins which show some decrease in clinical reactivity but are not recommended for food-allergic infants, formulas based on extensively hydrolyzed proteins that are useful in clinical management of food allergies in >90% of patients, and free amino acid-based formulas used for the most strongly allergic patients.Formula hypoallergenic reactivity is only established by clinical trials, but these studies should be preceded by evaluation in appropriate animal models. For partially hydrolyzed formulas the goal of the model is to demonstrate some reduction in immunologic reactivity that justifies their \"Hypoallergenic, not for use in food-allergic infants\" labeling. For formulas based on extensively hydrolyzed proteins or amino acids, results of the animal models must predict the accepted standard for hypoallergenic clinical performance .Guinea pig oral sensitization is used to show decreased immunological reactivity of partially hydrolyzed formulas. Model sensitivity can be controlled by varying feeding duration, with positive controls (intact cow milk formula) reacting after a 15 day feeding. However, some partially hydrolyzed formulae that give negative results with 15 day feedings are sensitizing if fed for 35 days. Extensively hydrolyzed and amino acid formulas fed 35 days are not sensitizing. Symptom standards for evaluating reactions will be presented along with formula type and feeding duration comparisons.The laboratory animal hyperimmunization model is sensitive at the lower end of the immunological reactivity scale and can be used to differentiate extensively hydrolyzed and amino acid formulas which meet hypoallergenic clinical performance standards from partially hydrolyzed formulas not recommended for allergic patients. Analytical considerations and formula type comparisons will be presented."} +{"text": "Postoperative complications from the Central Nervous System after Coronary Artery Bypass Grafting Operations (CABG) can range from transient neurologic or psychological changes to massive brain infarction and irreversible coma. The risk of stoke after CABG is approximately 2%. Multiple mechanisms such as brain hypoperfusion during or after the operation, carotid stenosis and embolization can lead to these complications.635 patients with risk factors for Cerebral Vascular Accident (CVA) such as history of stroke, peripheral vascular disease (PVD) and age over 65 years old compared to 331 patients <65 years old without any of these risk factors. All patients underwent CABG procedure and carotid endarterectomy at the same time when was necessary. Other cardiac operations excluded. The following risk factors for CVA were considered: age>65 years old, history of CVA, history of PVD. All patients had a preoperative carotid triplex. For practical reasons the Carotid Artery Stenosis was defund as significant> 50-75% and severe > 75%. EuroSCORE I calculated as well for all patients in order to calculate the postoperative mortality score.From the low risk group (311 patients), 33 (10%) were found to have significant carotid stenosis and 5 had severe stenosis and underwent Carotid endarterectomy and CABG at the same time. None of them suffered from postoperative stroke and one patient died due to chest infection. From the high-risk group (635 patients) 15 developed transient neurologic symptoms and 6 developed severe postoperative stroke and 2 died.Because of the catastrophic neurological complications in patients undergo Coronary Artery Bypass operations even if they are low risk patients for CVA we suggest to have a preoperative carotid triplex in order to find asymptomatic lesions and take all the perioperative precautions. That will improve the outcome and will reduce more the morbidity and mortality."} +{"text": "Myasthenia gravis is an autoimmune neuromuscular disease affecting the postsynaptic acetylcholine receptors that are blocked by specific antibodies, with resultant muscle weakness and fatigue. Anesthesia for thymectomy can be classified into: muscle relaxant or nonmuscle relaxant techniques. Our description of the nonmuscle relaxant technique retuned back to 1994, when it was first described.3 In this results."} +{"text": "This presentation addressed cultural considerations and sensitivities particularly relevant to the southern US when providing alcohol screening and brief intervention (SBI) to individuals demonstrating risky drinking behavior. Broader implications for education and training were also reviewed. The literature has begun to identify key issues pertaining to cultural sensitivities and considerations for successful change work with individuals. Key lessons learned about cultural considerations pertaining to alcohol SBI in one trauma center in the southern US were presented. Information included in the presentation was based on three years of alcohol SBI provided by counselors and doctoral and masters level student interns in a level-I medical trauma center. Implications regarding training SBI providers in cultural sensitivity were also discussed. To maximize an individual's acceptance of alcohol SBI, key cultural considerations may be implemented. In addition, educating and training SBI providers to enhance their cultural sensitivity is recommended."} +{"text": "The analysis of randomised controlled trials by subgroups of individuals (e.g. according to age or gender) remains controversial and often misinterpreted. It is recognised that such analyses should be limited to a few pre-specified key baseline factors. In addition an appropriate analysis typically calculates effect estimates and confidence intervals within such subgroups together with an overall interaction test rather than calculating separate p-values within each category of the subgroup.However, patients vary considerably often presenting with multiple risk factors for the outcome of interest. Hence an alternative approach to analysing subgroups is proposed whereby patients are analysed according to their underlying risk rather than individual characteristics and a single interaction test performed between underlying risk and treatment. Patients can be categorised into several ordered risk groups and the absolute and relative risk reduction in each group presented. This helps the decision making process for an individual patient when considering appropriate treatment strategies. For instance, even if a beneficial relative risk is steady across groups, the absolute benefits in the low-risk group may be too small to justify the new treatment\u2019s use.These issues will be illustrated using data from trials in acute coronary syndrome. The FIR collaboration combined data from three trials interventional and conservative strategies. The ACUITY trial assessed the impact of three anti-thrombotic therapies for reducing clinical outcomes and major bleeding.We recommend that risk stratified subgroup analyses be routinely reported in trials that claim a treatment benefit."} +{"text": "Tens of thousands of healthcare workers worldwide can only wear a plain wedding ring at work, if any at all. This arose from policies citing early laboratory evidence that rings can carry clinically relevant bacteria, but with little supporting clinical data. Policies that are both invasive and perceived as lacking evidence create a broader scepticism of infection control guidelines: it is therefore important to regularly review the evidence for such guidance.A systematic literature review was performed of studies investigating the infection risk of ring wearing by healthcare workers. PubMed, Cochrane Library and clinical trials registries were searched. Data was extracted on study design and quality, and the following outcomes: hospital acquired infection (HAI) rates, bacterial transmission, and bacterial contamination of healthcare workers\u2019 hands.Two interventional randomised controlled trials (RCTs) and ten observational studies were identified. No study investigated an association between ring wearing and HAI rates. The RCTs were very small and used hand colonization as the primary outcome. One RCT found higher colonization of hands of healthcare wokers randomised to wear rings than those not wearing rings, whereas the other RCT found no difference. One observational study assessed bacterial transmission through handshaking and found the presence of a ring did not result in higher transmission. Three observational studies found higher bacterial contamination of hands with rings, and five studies found no difference. The presence of rings did not result in higher contamination after handwashing in most studies. No study identified a significant increase in hand contamination with multiple rings compared with one ring, nor between different types of ring.No direct evidence was found that healthcare workers wearing rings results in higher HAI or bacterial transmission rates. Most studies did not identify higher contamination associated with ring wearing; furthermore, the clinical significance of a statistical difference in the number of colony forming units is unclear. Guidelines could benefit from reconsidering ring wearing guidance, and focussing on interventions with a more defined evidence base; fewer intrusions into healthcare workers\u2019 personal autonomy may increase willingness to participate in other important interventions.None declared"} +{"text": "We describe a novel method to develop energetically optimized, structure-based pharmacophores for use in rapid in silico screening. The method combines pharmacophore perception and database screening with protein ligand energetic terms computed by the Glide XP scoring function to rank the importance of pharmacophore features. We derive energy-optimized pharmacophore hypotheses for 30 pharmaceutically relevant crystal structures and screen a database to assess the enrichment of active compounds. The method is compared to three other approaches: (1) pharmacophore hypotheses derived from a systematic assessment of receptor ligand contacts, (2) Glide SP docking, and (3) 2D ligand fingerprint similarity. The method developed here shows better enrichments than the other three methods and yields a greater diversity of actives than the contact-based pharmacophores or the 2D ligand similarity. We then apply this method on fragment docking using fragment-specific settings aimed to generate poses that explore every pocket of a binding site while maintaining the docking accuracy of the top scoring pose. Next, we describe how the energy terms from the Glide XP scoring function are mapped onto pharmacophore sites from the docked fragments in order to rank their importance for binding. We show that the most energetically favourable pharmacophore sites are consistent with features from known tight binding compounds. The derived pharmacophore models are able to recover known active compounds from a database screen and retrieving diverse compounds that are not biased by the co-crystallized ligand."} +{"text": "Research has tended to focus on the information that researchers and ethicists deem important for informed consent to clinical trial participation and on the deficits in patients\u2019 understanding of this information. Drawing on an alternative \u2018capabilities\u2019 approach we explored what information parents prioritised when making a decision about their child\u2019s participation in a clinical trial, parents\u2019 comprehension of these issues and how far they voiced their priorities during discussions with recruiters.Qualitative interview and observational study examining recruitment in four placebo-controlled, double-blind randomised clinical trials of medicines for children. We compared audio-recorded routine parent-recruiter discussions (N=41) about the clinical trials with subsequent semi-structured interviews with parents about their experiences of recruitment.When making a decision about trial entry, parents\u2019 main priorities were: what clinical benefit the trial might offer their child, their child\u2019s safety; practicalities of participation; research for the common good: access to medication and randomisation. Parents had specific misunderstandings linked to these priorities which had the potential to influence their decisions. While parents had many questions and concerns about trial participation which influenced their decision-making, they rarely voiced these during discussions with recruiters.Knowing what information patients and carers prioritise when deliberating about entry to a trial can help recruiters when approaching potential participants. Tailoring discussion around these issues and helping patients to voice their particular priorities may enhance recruitment conduct."} +{"text": "Consider the anger that arises in a heated argument with your romantic partner, or the dreadful anxious anticipation in the dentist's waiting room prior to a root canal procedure. Our daily lives are densely populated with events that make us emotional. Luckily, however, we developed numerous ways to control or regulate our emotions in order to adapt major determinants and (2) underlying mechanisms of emotion regulation choice . A classic early selection strategy is distraction, which involves producing neutral thoughts that are independent from and not in conflict with emotional information and a second regulatory option was cognitively effortful yet highly disengaging from emotional processing (subtract 7s). Findings supported the centrality of the engagement/disengagement factor with an increased preference to use the more disengaging subtract 7s distraction as negative emotional intensity increased. These findings suggest that individuals are willing to exert substantial cognitive effort in order to obtain adequate levels of disengagement. Nevertheless, future studies should parametrically manipulate varying levels of engagement/disengagement and cognitive effort in order to better understand the relationship between them. In a complementary study we showed that the engagement/disengagement dimension is central within the reappraisal category. Specifically, we found that under high negative emotional intensity participants choose to use \u201creality challenge\u201d reappraisals which involves disengaging by not considering emotional consequences of events and one late selection strategy . It is clear that people typically use many other strategies and that their regulatory choice patterns may have important consequences for well-being and psychopathology. Consider avoidance which disengages from emotional processing at an early selection stage, and rumination which involves magnifying emotional information at an early attentional stage and elaborating it in a late selection phase. Our account suggests that deviations from the preference to disengage from high emotional intensity by overly engaging via rumination may be related to depression (Nolen-Hoeksema et al., Second, we concentrated on deliberate regulatory choices among explicit regulation strategies. While the vast majority of studies in the field concentrated on explicit forms of regulation (Gross and Thompson,"} +{"text": "Metals can accumulate in flowering plants grown in contaminated soils and have been found in honey samples collected near polluted sites. Investigators at the University of Pittsburgh now report that metal contamination of flowers may affect bumblebees\u2019 foraging behavior and potentially harm pollinator health.A survey of carbon dioxide tissue culture incubators found that ambient static and time-varying magnetic fields in six popular models differed by orders of magnitude between incubators as well as between locations centimeters apart within single units.The U.S. EPA is accepting applications until 13 May 2013 for a new program designed to discover innovative ways to utilize Toxics Release Inventory (TRI) data.GSTM gene, which is involved in detoxification of PAHs, appeared particularly vulnerable. The findings suggest that reducing exposures to either allergens or combustion sources of PAHs could reduce asthma risk in urban communities.Cockroach allergens are a major contributor to asthma in children, especially in lower-income urban environments. Investigators now report that children with higher prenatal exposure to both cockroach allergens and polycyclic aromatic hydrocarbons (PAHs) had an increased risk of developing cockroach allergy at ages 5 to 7 years.In March 2013 the Washington State Legislature passed a bill requiring that all asbestos-containing building materials sold in the state be clearly labeled."} +{"text": "The NPWT is becoming an important tool in the treatment of both acute and chronic wounds. The authors describe their initial experience using NPWT to fix a dermal substitute for preserving maximal foot length after surgical debridement in diabetic patients with foot lesions that were assessed for sensory-motor neuropathy and infection. The application of dressings to fix dermal templates can reduce shearing forces, restrict seroma and haematoma formation, simplify wound care and improve patient tolerance.INTEGRA\u00aeDermal Regeneration Template, SIAD) fixed by NPWT was performed. Later the wounds were closed with thin skin autografts.Two male patients, respectively 68 and 70 years old, with diabetes and peripheral neuropathy but without vascular dysfunctions were observed in our outpatient service in consequence of traumatic foot wounds with exposed bones and tendons. Antibiotic drugs were provided and NPWT was applied for three weeks after urgent surgical debridement. During this period, an increase of granulation tissue and decrease of nonviable tissue were observed. After three weeks in both patients minor amputations were performed because of bone necrosis. The patients continued NPWT (V1STA\u00ae Smith & Nephew) for 1 week until limited granulation of the wound bed was obtained. Subsequently an artificial graft with a dermal substitute (In our initial experience, the use of NPWT to fix dermal templates reduced shearing forces and restricted seroma and haematoma formation. Moreover this practice achieved a faster granulating wound bed in order to prepare it for surgical closure techniques.The use of NPWT and dermal substitute make wound healing during the treatment of diabetic foot ulcers faster, decreases hospital stay and prevents major amputations. Moreover the application of NPWT dressings to fix dermal templates can simplify wound care and improve patient tolerance."} +{"text": "Integrative medicine is a relatively new field which seeks to combine a wide range of conventional and nonconventional approaches to patient care. Many academic health centers have now established integrative medicine clinics, yet little is known about the clinicians who practice at them.We conducted a nationwide survey of clinicians who practice at integrative medicine clinics which are affiliated with academic health centers, seeking to characterize their educational backgrounds, clinical practices, and participation in research and education activities.We received completed surveys from 136 of 162 clinicians (84% response rate). The integrative therapies that clinicians most often reported providing themselves were breathing exercises (66%), herbal medicine prescribing (61%), meditation (44%), and functional medicine (34%). The integrative therapies that clinicians most often referred their patients for were acupuncture (96%), massage (92%), yoga (85%), and meditation (79%). Respondents reported spending a mean of 20% of their time training medical students, and 63% had participated in research in the past year.This survey provides the first national assessment of clinicians practicing integrative medicine at academic health centers. These clinicians employ a wide variety of complementary and alternative therapies and appear involved in the research and education missions of their academic health centers."} +{"text": "Podiatry services for patients attending haemodialysis at Caulfield Hospital (CH) began in June 2009 due to emerging evidence indicating people with chronic kidney disease and end stage renal failure are at an increased risk of developing lower limb complications such as ulceration and amputation. Risk factors become more frequent with progression of the disease, some research also suggests 95% of people with diabetes who receive haemodialysis treatment have at least one risk factor for foot ulceration.Preliminary patient surveys were undertaken to establish patient interest in receiving podiatry treatment. Prior to receiving treatment all patients underwent an initial podiatry assessment establishing their baseline vascular and neurological status and other information to help determine their level of risk. Following a 12 month pilot further process evaluation data was collected.An overwhelmingly positive response was received from patients and haemodialysis nursing staff in requesting access to podiatry services. At the time of the initial survey only 43% of haemodialysis patients were accessing podiatry. A haemodialysis podiatry ward round has been established on alternating days and weeks with an 8 weekly review period to ensure easy access for all patients. Treatment is provided at no cost to the patients and takes place during haemodialysis treatment hours; making it more accessible and convenient for patients. Additional funding was not obtained for this service and was redistributed from existing funding streams. A 12 month service review was conducted in July 2010 surveying podiatry staff, haemodialysis nursing staff and patients. The feedback from all parties was positive and encouraging and it was decided the haemodialysis ward round would continue with some minor changes. The survey revealed the percentage of people accessing podiatry services had increased to 73% (from 43%) within one year; 91% of these patients reported receiving podiatry treatment whilst attending haemodialysis at CH. Most recently funding has been procured to implement a limited podiatry service at Sandringham Hospital. The service will include access for patients receiving haemodialysis and will utilise the model previously piloted at CH.The future aim is to obtain funding for podiatry services for haemodialysis patients across all Alfred Health sites and encourage more patients to access the service by ensuring relevant information and education is provided. Ethics approval has been sought to further investigate the effects of haemodialysis on the foot. The scope of this would include increasing awareness amongst the greater community of foot complications in haemodialysis patients and assist in establishing better access to podiatry services for patients undergoing haemodialysis."} +{"text": "Fragment-based approaches have become popular tool in drug design due to their ability to screen large portions of chemical space with comparatively small libraries. However fragments can exhibit unspecific binding and even if they bind to a specific binding site in some cases more than one binding mode is observed . For com1H and 15N CSP are easily obtainable from 15N HSQC spectra and can be used as probe for ligand orientation but also include information about conformational changes on the protein side. CSP data has been used to orientated drug like molecules into protein binding sites [The sensitivity of a nuclei's NMR chemical shift to changes in its chemical environment can be used to measure chemical shift perturbations (CSP) of protein atoms upon ligand binding. Especially ng sites ,3 and cang sites .Here we show how CSP can be used to quickly validate docking poses of smaller fragments by filtering them for their agreement between experimental CSP and simulated CSP for the docked poses. Additionally a more detailed analysis of the differences between the experimental and the simulated CSP profiles can be used to highlight protein regions and even single residues which undergo structural changes upon fragment binding."} +{"text": "Sensory input arrives in the cortex in the form of dynamic synaptic currents to populations of neurons. How cortical neurons encode and transmit these inputs ultimately determines the cognitive and behavioral response of the animal. Therefore, a number of theoretical studies have attempted to explain the cortical population response in model neuronal networks . Yet, thin vivo [Optogenetic techniques in dissociated cultured networks (DCNs) that are grown on multielectrode arrays (MEAs) offer an opportunity to test key aspects of network response dynamics. However, controlling the spontaneous activity of DCNs and imposing a specific irregular activity pattern is challenging . Here, win vivo ; (2) theThese results indicate that amplitude modulated optogenetic stimulation with LEDs is an efficient tool for providing correlated input currents to thousands of pyramidal cells embedded in DCNs while recording spiking activity in hundreds of individual neurons using an MEA. Because DCNs can be created with specified proportions of different cell types and allow easy pharmacological access, this technique opens a new level of control over living neuronal populations for testing theories of cortical response properties and small signal representation under different network parameter regimes."} +{"text": "Bioisosteres are defined as structurally different molecules or substructures that can form similar intermolecular interactions, and therefore fragments that bind to similar protein pocket can be considered to have exhibited a degree of bioisosterism ,2. In thThe utility of the approach is demonstrated by (i) separating known similar binding site pairs from random binding site pairs and (ii) a bioisosteric replacement study for fragments binding to subpockets of different proteins."} +{"text": "Electronic structure theory under the influence of apolar solvents suffers from substantial methodical difficulties since in this case the solvent-induced solute polarization originates mainly from specific directional interactions and higher electric multipoles. Continuum solvation models based on the dielectric solvent response such as the PCM approach ignore such interactions and can therefore not adequately model solvation effects in nonaqueous environments.Ka shifts [The \u201cembedded cluster reference interaction site model\u201d (EC-RISM) retains a shifts and tauta shifts .Here we outline the strength of the integral equation model by studying benzene and hexafluorobenzene solutions. In particular, the thermodynamics of differential solvation is quantified for organic compounds dissolved in these media. Moreover, it is shown that the respective solvent structures around particular solutes differ strongly, possibly leading to changes in the thermodynamic stability scale of various isomers which are not reproduced by the PCM model."} +{"text": "Polycomb Group (PcG)-mediated repression is critical for cell fate determination and maintenance of gene expression during embryonic development. However, the mechanisms underlying PcG recruitment in mammals remain unclear since few regulatory sites have been identified. We have identified and characterized two new potential PcG-dependent regulatory elements within the human HOXB and HOXC clusters. Their repressive activities are similar to a previously identified element in the HOXD cluster. The PcG proteins BMI1 and SUZ12 are recruited to a reporter construct in mesenchymal stem cells and confer repression that was dependent upon PcG expression. In addition, JARID2 was observed to localize to these three elements. Interestingly, the requirement for JARID2 is variable at the different regions despite its localization. We conclude that distinct regions of the mammalian HOX clusters can recruit components of the PcG complexes and confer repression and that JARID2 plays a role in the recruitment of PRC2."} +{"text": "Many molecular systems involved in controlling development are also implicated in malignant transformation and are associated with poorer survival outcomes in cancer patients. In fact, lung tumours with similar gene expression profiles to fetal lung cells are associated with aggressive phenotypes of the disease. As targeting fetal developmental pathways selectively reactivated in cancer cells would presumably spare normal adult cells from toxicity, these pathways are considered ideal therapeutic targets for cancer treatment. Therefore, a better understanding of the interplay between fetal lung development and lung cancer on the level of individual miRNAs will provide new insights for translational research. In this study we applied sequencing technology to identify miRNAs with similar expression patterns between fetal lung and lung tumours.Eighty-nine pairs of tumour and adjacent non-malignant lung and five fetal lung tissue samples were profiled by miRNA-sequencing. Mann\u2013Whitney U tests were performed with Benjamini-Hochberg multiple testing correction to identify common expression patterns.We describe, for the first time, a comprehensive miRNA expression profile of human fetal lung. We have not only detected miRNAs that exhibit similar expression levels between fetal lung and non-malignant lung tissue which may play roles in lung tissue maintenance, but also identified multiple miRNAs with similar expression patterns between fetal lung and lung tumours. Some of these miRNAs have been previously implicated in lung cancer, while others have not yet been described to play a role in this disease. Pathways known to be targeted by miRNAs commonly expressed between fetal lung and lung tumours are involved in cell proliferation and angiogenesis. Further studies will elucidate the specific roles of these miRNAs in lung tumourigenesis.We identified several miRNAs with similar expression profiles between fetal lung and lung tumours which potentially play critical roles in lung cancer and might lead to the identification of promising therapeutic targets.DDBS is supported by University of British Columbia 4YF scholarship, KLT by Vanier Canada Graduate Scholarship. This work was supported by grants from the Canadian Institutes for Health Research."} +{"text": "A 70-year-old woman with short stature and hypermastia underwent ascending aortic aneurysm repair with a prosthetic graft, complicated by postoperative mediastinitis and sternal dehiscence. She was treated with aggressive sternal debridement and negative pressure wound therapy for 1 week prior toWhat are the requirements for debriding sternal wounds complicated by mediastinitis?What is the role of negative pressure wound therapy in postoperative mediastinitis?What are the options for reconstruction of sternal wounds in cardiothoracic surgery patients?Why was the omental flap the best option for this particular patient?,Mediastinitis, or deep sternal wound infection, is a feared life-threatening complication of cardiac surgery involving a midline sternotomy, with an in-hospital mortality rate as high as 31%.1Negative pressure wound therapy is a highly effective treatment strategy for infected sternal wounds as a temporary dressing between debridements until the wound is clean enough for definitive closure.3,Options for reconstruction of sternal wounds include a unilateral pectoralis major muscle \u201cturnover\u201d flap, unilateral or bilateral pectoralis major muscle or myocutaneous advancement flap, rectus abdominis muscle or myocutaneous flap, latissimus dorsi muscle or myocutaneous pedicled or microvascular flap, omental flap, or a combination of these.5This particular case involved an exposed prosthetic aortic root graft and suture line lying deep within the mediastinum. A pectoralis or other muscle flap would have not been adequate to reach and envelop the graft suture line in the posterior mediastinum. Omentum is more malleable than muscle and has a longer pedicle. It also has a minimal raw surface and donor site, which is helpful in reducing hematoma risk in anticoagulated patients. Consequently, the omentum was the best choice to mold into and fill this deep mediastinal defect."} +{"text": "The prevalence of metabolic syndrome worldwide is high and rising . According to all definitions visceral obesity, increased triglycerides, decreased HDL cholesterol, impaired glucose tolerance and increased blood pressure are part of the definition. Thyroid hormones impact on all these factors. In a large Chinese case control study all components of the metabolic syndrome were associated with systematically higher TSH levels . Recent data on TH replacement following thyroidectomy further strengthen the close relation between TH status and body weight. Total thyroidectomy despite full replacement with T4 increases body weight by a mean of 2 kg above controls within 1 year (Jonklaas Thyroid 2011). When patients are treated with T3 instead of T4 to an identical 24 h mean of TSH body weight decreases by more than 2% within 1 year . The substantial effects of thyroid hormones on lipid components have been frequently reviewed (Duntas Med Clin North Am. 2012). Thyroid hormones are positively associated with insulin resistance not only in clinically diagnosed DM but also in subjects with a normal glucose tolerance. Indices of insulin resistance as judged by HOMA are closely linked to TH status. They modulate gluconeogenesis and peripheral glucose uptake . Finally, recent data from children and adolescents show a major impact of TH on the regulation of blood pressure. As other components of blood pressure dysregulation are less prominent at this age the small positive association of thyroid hormones with systolic and diastolic blood pressure support previous studies on the impact of TH status on blood pressure regulation . Thus, all components of the metabolic syndrome are altered by thyroid hormone status. Measurement of thyroid hormone status ought thus to be considered in any patient with metabolic syndrome or diabetes mellitus to rule out an easily manageable cofactor of the disease."} +{"text": "Epigenetic signatures are highly cell type specific. Separation of distinct cell populations is therefore a prerequisite for all epigenetic studies. To date little information is available whether separation protocols might influence epigenetic and/or geneexpression signatures. We therefore investigated the influence of two frequently used protocols to isolate intestinal epithelium cells (lECs) \u2013 EDTA/DTT and enzymatic release \u2013on gene expression and DNA methylation patterns of lECs obtained from 6 healthy individuals. While cell purity was >95% using both approaches, gene expression analysis revealed significantly higher mRNA levels of several inflammatory genes in EDTA/DTT when compared to enzymatically released cells. In contrast, DNA methylation of selected genes was less variable but tended to be lower in inflammatory and higher in maintenance genes in EDTA/DTT released cells. Taken together, this highlights the importance of considering the effect of cell separation procedures particularly when it comes to correlating epigenetic changes with gene expression."} +{"text": "Astronomers and physicists noticed centuries ago that visual spatial resolution is higher for dark than light stimuli ,2, but tWe hypothesized that a nonlinear encoding of luminance increments and decrements could explain these differences. We found that OFF cells in LGN increase their responses roughly linearly Figure , right wConsistent with our hypothesis, we show that a simple computational model based on the ON-channel nonlinearity can explain the larger receptive fields and lower spatial resolution of ON cells, the differences in human spatial resolution between darks than lights, the differences in the size of ON and OFF dendritic fields from retinal ganglion cells and the recently demonstrated OFF dominance in visual cortex . These results demonstrate a fundamental difference in processing between ON and OFF channels, which could have major implications in visual perception and cortical development."} +{"text": "In nature, the root systems of most plants develop intimate symbioses with glomeromycotan fungi that assist in the acquisition of mineral nutrients and water through uptake from the soil and direct delivery into the root cortex. Root systems are endowed with a strong, environment-responsive architectural plasticity that also manifests itself during the establishment of arbuscular mycorrhizal (AM) symbioses, predominantly in lateral root proliferation. In this review, we collect evidence for the idea that AM-induced root system remodeling is regulated at several levels: by AM fungal signaling molecules and by changes in plant nutrient status and distribution within the root system. When plants made the transition from freshwater to terrestrial environments more than 400 million years ago, fundamental morphological changes were needed for the acquisition of mineral nutrients from the soil instead of from the aqueous substratum. Preceding the development of complex root systems the alliance with symbiotic fungi such as the Glomeromycetes and Mucoromycotina is believed to have greatly assisted this transition , that suRoot systems consist of individual modules with different function: the shoot-born dicotyledon tap roots and monocotyledon crown roots (CRs) are mainly involved in anchorage and support whereas lateral roots mediate nutrient uptake . Root syFigure 1; Figure 1). It has been shown in maize that phosphate starvation stress leads to an increased transcription of genes involved in secondary cell wall biosynthesis and the fine lateral roots (FLRs), which lack cortex tissue , and arela roots . Althougot zones . This is surface . These p surface . They in surface . PDR1 (p surface . This miLotus japonicus hairy root cultures of the putative transcription factor gene meristem and arbuscular mycorrhiza induced (LjMAMI) that lacks embryonic lateral roots (Numerous studies report root system changes in response to arbuscular mycorrhiza leading to an increased root branching and root system volume (reviewed in (LjMAMI) . Here, cal roots . Inoculaal roots . Taken tRoot system architectural changes in response to AM colonization are regulated on at least two levels as evidenced by their induction prior to or after establishment of AM colonization .M. truncatula, germinating AM fungal spores that were separated from the root by a semipermeable membrane induced lateral root formation, indicating that diffusible signals released by these spores activate the lateral root developmental program and DMI2 (SYMRK), two genes that act upstream of Ca2+-spiking as part of the common SYM pathway (DMI3 (CCamK), that acts downstream of Ca2+-spiking , and DMI3 (CCAMK) for lateral root induction by GSEs (Lotus japonicus mutant hypernodulation aberrant root formation 1 (har1), that hypernodulates and is hypercolonized by AM fungi, constitutively forms supernumerary lateral roots , and this might for example explain SYM pathway-independent lateral root induction in rice, while in nodulating legumes common SYM-mediated lateral root induction might be epistatic to auxin signaling.Lateral root formation is regulated by auxin in conjunction with other phytohormone signaling pathways . Impairm outcome . Ectomycompounds . LikewisAllium porrum and Prunus cerasifera uptake pathway dominates and involves suppression of the transporter genes involved in epidermal direct uptake . It is aLateral root formation can be triggered by carbon supply in the growth medium, suggesting its dependence on sufficient carbon . There iPlant productivity strongly depends on an appropriately adapted root system architecture for the uptake of nutrients and water under adverse soil conditions. Thus modulation of the root system architecture in response to environmental conditions is considered an important target for genetic crop improvement . AM fungThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Several recent studies reveal a close interplay of neurons and astrocytes in information processing . AstrocyWe implemented both mechanisms in a highly recurrent single layer 2d map model based on . NeuronsWe observed that a selective increase in modalities towards inhibitory neurons even leads to a sharpening of orientation tuning. While selective enhancement in the modalities towards excitatory neurons leads first to a drop in orientation tuning and very fast for further increase to pathological firing rates. For both mechanisms the closest fit in orientation tuning (HWHM) to the experimental observation with TBOA was found for a stronger effect on excitatory neurons along with a simultaneous but weaker effect on the inhibitory population. While both models can explain the current data, they, however, provide different predictions for sub-threshold properties and for neurons close to pinwheels or domain centers."} +{"text": "To the Editor: Fox et al. used computer-assisted telephone interview (CATI) techniques in an outbreak of cryptosporidiosis .A \"control bank\" allows investigators to rapidly obtain contact details for appropriately matched controls because age and sex of all household members are recorded in a database. Using control banks with CATI allows completion of studies quicker than CATI or traditional methods alone of 6,087 households agreed to be in a control bank (Investigators may find CATI useful, although it can be costly and introduce biases ("} +{"text": "From one year of liver's transplantation and cyclosporin (CSA) maintenance treatment(100 mg a day), a 43 years old women presented with abdominal pain and diarrhea immediately after eating milk and egg. She underwent the transplantation after an acute liver toxicity caused by isoniazid during tuberculosis treatment. Increasing hyperosinophilia (40%) and eosinophilic oesophagitis (EE) presented with food allergy. Total IgE were low (45UI).Commercial skin prick test and prick by prick as specific IgEs detection (IMMUNOCAP) were positive for milk's proteins, egg, rice and wheat flour. Hypereosinophilia persisted inspite of an elimination diet for the culprit allergens while the clinical symptoms of food allergy improved.Other causes of hypereosinophilia were excluded.Interestingly only an inhaled fluticasone propionate treatment for three months was followed by an outstanding reduction of EE and hypereosinophilia (less than 1000/mm3 and <25%) without changing CSA dosage. Food allergy and hypereosinophilia has been increasingly reported in children in the setting of liver transplantation during tacrolimus treatment.On the contrast reports in adults are very rare expecially during CSA treatment. In our patient elimination diet wasn't followed by a reduction of hypereosinofilia as generally occurs in pediatric cases. Our patient didn't present any allergy before the transpantation.No information was available on donor's known allergy.Different meccanisms are supposed underlying the new onset of food allergy and hypereosinophilia in liver's transplantation:\u2022 An imbalance between Th1/Th2 cells or an increased enteric permeability.\u2022 Immune effects of viral infections associated with the immunosoppressive state.\u2022 Acquired food allergy and hypereosinophilia due to a transfer of hepatic hematopoietic stam cells or active IGE from the donor's liver.More studied are needed in a controlled setting to identify similar findings among liver transplants.Moreover in the pretransplant investigation should be inlcluded the allergic status both of the donor as the recipient."} +{"text": "Predation shapes many fundamental aspects of ecology. Uncertainty remains, however, about whether predators can influence patterns of temporal niche construction at ecologically relevant timescales. Partitioning of time is an important mechanism by which prey avoid interactions with predators. However, the traits that control a prey organism's capacity to operate during a particular portion of the diel cycle are diverse and complex. Thus, diel prey niches are often assumed to be relatively unlikely to respond to changes in predation risk at short timescales. Here we present evidence to the contrary. We report results that suggest that the anthropogenic depletion of daytime active predators in a coral reef ecosystem is associated with rapid temporal niche expansions in a multi-species assemblage of nocturnal prey fishes. Diurnal comparisons of nocturnal prey fish abundance in predator rich and predator depleted reefs at two atolls revealed that nocturnal fish were approximately six (biomass) and eight (density) times more common during the day on predator depleted reefs. Amongst these, the prey species that likely were the most specialized for nocturnal living, and thus the most vulnerable to predation (i.e. those with greatest eye size to body length ratio), showed the strongest diurnal increases at sites where daytime active predators were rare. While we were unable to determine whether these observed increases in diurnal abundance by nocturnal prey were the result of a numerical or behavioral response, either effect could be ecologically significant. These results raise the possibility that predation may play an important role in regulating the partitioning of time by prey and that anthropogenic depletions of predators may be capable of causing rapid changes to key properties of temporal community architecture. Predation is believed to have played an important role in shaping the long-term evolution of diel cycles and temporal niche partitioning in animals. For example, a major factor that contributed to the nocturnal disposition of multiple species appears to have been the capacity to avoid daytime active predators Generally there is much interest amongst ecologists in understanding how key properties of community organization and function, such as diel partitioning, change when the drivers that brought them about are relaxed. In the case of predation there is considerable field and experimental evidence demonstrating that a variety of basic life history traits (e.g. growth and reproduction) can evolve rapidly when predation pressure is reduced Here we engage these issues by considering whether nocturnal animals are capable of behaviorally or numerically responding to reductions in predation risk from daytime active predators. To do this we compared the relative abundance of nocturnal prey fish communities observed during the day at a coral reef ecosystem where large daytime active predators were abundant to those on physically similar reefs where these predators have been fished to low levels in the last three decades. Our results suggest that predators may indeed play an important role in enforcing the boundaries of nocturnal prey niches. Such observations challenge us to more broadly consider the controls that predators may have upon fundamental aspects of niche construction and inform our understanding of the full extent to which anthropogenic change is capable of impacting animal ecology.This work was carried out under permission from USFWS SUP # 12533-06032 and from the Republic of Kiribati Environment and Conservation Division. This research was conducted at Palmyra and Tabuaeran Atolls. Palmyra is a US National Wildlife Refuge and prohibits the take of all reef fish. The reefs of Palmyra are among the least disturbed in the world and host especially high densities of large predatory fish We used SCUBA belt transect surveys to inventory fish assemblages during full light conditions (1000 \u2013 1600 h) on the forereefs (ocean-side of reef crest) of both Palmyra and Tabuaeran. A complete fish survey consisted of four belt transects, the dimensions of which were matched to size of individual fish being inventoried: \u226550 cm TL fish \u221250\u00d78 m transect; 30\u201349 cm TL fish \u221250\u00d74 m transect; 15\u201329 cm TL fish \u221250\u00d74 m transect; <15 cm TL fish \u221225\u00d72 m transect Fish within each of these three categories differ in their degree of conformity and specialization to the specified diel modes. In the case of nocturnal fishes, the ratio of eye diameter to fish standard length (SL) serves as a convenient proxy for picking out gradients in adherence to nocturnal living Differences in the density and biomass of diurnal, cathemeral, and nocturnal fish species were compared between Palmyra and Tabuaeran using effect size measurements .The significance of these effect size comparisons was evaluated using Kruskal-Wallis and Wilcoxon rank sum tests \u2013 as data could not be transformed to meet parametric assumptions. Post-hoc Holm's sequential Bonferroni corrections P\u200a=\u200a0.04). This finding parallels observations made by other researchers that have reported pronounced anthropogenic depletions of large piscivores at Tabuaeran Of the 185 species that were shared between Palmyra and Tabuaeran we classified 141 as diurnal species, 26 as cathemeral species, and 18 as nocturnal species (Supporting dataset 1). Nocturnal fish included representative species from 6 families: Holocentridae, Priacanthidae, Pempheridae, Lethrinidae, Mullidae, and Serranidae. No nocturnal fish in this assemblage achieved a SL >50 cm , generally qualifying all as potential prey for large piscivorous reef fish. Data from our fish surveys indicated that large daytime active predators were abundant on the reefs of Palmyra, but were considerably depleted at Tabuaeran but only significantly different (post-correction) from cathemeral species in the case of biomass . Abundance increases in cathemeral species between Tabuaeran and Palmyra were intermediate to nocturnal and diurnal species, and always significantly higher than the diurnal increase . Presence/absence data summarized from these fish surveys conveyed a similar conclusion. Nocturnal fish were sighted in considerably more surveys at Tabuaeran than Palmyra (P<0.001). Cathemeral species were also sighted at higher frequencies at Tabuaeran but diurnal species were not different between atolls (W\u200a=\u200a10196 P\u200a=\u200a0.87).Analysis of the effect size patterns of diurnal, cathemeral, and nocturnal fish species indicated that nocturnal fish showed by far the strongest increases in both density and biomass during the day at Tabuaeran relative to Palmyra . These i2\u200a=\u200aR0.12, P<0.0001).We observed a significant positive relationship between eye diameter: SL ratios and the density effect size response of nocturnal reef fish species. This observed relationship indicates that the most dark adapted species, which are expected to be more vulnerable to daytime active predators, showed the strongest increases in abundance at Tabuaeran . No suchOur observations suggest that reductions in predator density in coral reef ecosystems can facilitate the temporal niche expansion of prey species. While these conclusions are drawn from the study of only two systems, the resultant observations are quite compelling. At our Tabuaeran Atoll study sites where fishers have depleted large diurnal and cathemeral predatory fish, we observed strong increases in the daytime density, biomass, and sighting frequencies of nocturnal prey fish compared to our sites at Palmyra Atoll where daytime predators remained naturally abundant and 3. DWithout data on the abundance of nocturnal prey fish at Tabuaeran during the periods prior to the depletion of its large predators, or the capacity to engineer exclosure manipulations large enough to meaningfully capture large scale interactions between reef predators and nocturnal prey fish, we cannot directly identify the mechanisms that have caused the stark differences in nocturnal prey fish abundance on Tabuaeran's predator depauperate reefs. However, a predatory release explanation for these shifts is well supported by the relationships observed between degree of specialization for nocturnal living by prey fish and the effect sizes of their abundance responses to predator reductions. If risk of predation from daytime predators is indeed playing an important role in constricting the diel niches of nocturnal fishes at these sites, we would expect the nocturnal species that are most vulnerable to daytime predators to show the strongest responses when these predators are reduced in number. This was indeed what was observed: nocturnal prey fish species that were highly adapted to functioning at night and presumably most at risk to predation during the daytime showed the strongest positive responses to the predator reductions at Tabuaeran . Other aThere is no reason to believe that nocturnal fishes are the only taxa whose temporal niche space would be affected by the removal of reef predators at Tabuaeran. Observations made in this same archipelago have demonstrated that diurnal prey fish undergo dramatic shifts in behavior (i.e. excursion distance) in the environments where predation risk is minimized It is not possible to determine conclusively whether the changes we observed in the nocturnal fish communities of Tabuaeran were principally the result of alterations in their abundance or their behavior. Nocturnal fish cannot be counted effectively using non-destructive methods while in diurnal refuges Determining the ecological consequences of these diurnal increases in the abundance of nocturnal predators will require gathering more data on the foraging efficiency of these nighttime feeders. The fish species with large eye: SL ratios, which showed the strongest increases in abundance on predator depleted reefs, are also likely to be the poorest daytime foragers given the physiology of their vision. These taxa may be weak competitors with more light-adapted diurnal species feeding on similar prey. Future research will help resolve how the dynamics of both competition and resource availability in this reef system may be altered by these increases in the abundance of nocturnal consumers.Rapid changes in patterns of diel partitioning following disruptions in predator regimes have been observed in mammals The possibility that changes to predation regimes can alter well-established patterns of temporal partitioning raises many interesting questions. Do prey and competitors respond to these rapid shifts in temporal niche space, causing evolutionarily significant temporal cascades in ecosystems Future research on the effects of predators on the temporal ecology of prey will be necessary to examine the geographic and taxonomic ubiquity of the trends we report herein. Developing this line of research is critically important given the rapidity and severity by which humans are depleting predator populations in both marine and terrestrial environments Dataset S1Temporal niche classifications of the fishes at predator rich Palmyra and predator depauperate Tabuaeran Atolls. Comparisons of the density and biomass of these fish species, as conducted using effect size measurements, are also reported.(DOC)Click here for additional data file.Text S1Mechanics of Cohen's D effect size measurements used to compare the density and biomass of fish species on the reefs of Palmyra and Tabuaeran.(DOCX)Click here for additional data file."} +{"text": "To the Editor: Dengue is one of the most widespread infectious diseases globally; transmission now occurs in 128 countries. Although dengue virus (DENV) control strategies have targeted vector control and disease surveillance, the development of an effective vaccine is the holy grail of prevention.Dengue vaccine development has spanned many decades. A candidate vaccine containing all 4 DENV serotypes is in advanced clinical testing. However, when given to school children in Thailand, this live-attenuated, tetravalent, dengue\u2013yellow fever 17D chimeric virus vaccine showed major but incomplete efficacy against 3 of the 4 DENV serotypes in the intention-to-treat group but no protection against DENV 2, the most pathogenic of the DENV serotypes doses 2 and 3 of the Sanofi Pasteur vaccine given to children over a 1-year period failed to improve efficacy outcomes. These results were obtained even though 91% of the children had circulating dengue or Japanese encephalitis antibodies before vaccination, neutralizing antibodies developed to all 4 DENVs, and neutralizing antibody titers increased 2\u20133 fold after 3 doses of vaccine in 80%\u201390% of vaccinated children.The inability of a mixture of 4 dengue chimeric viruses to elicit an initial primary neutralizing antibody response in nonhuman primates and susceptible humans was recognized during preclinical testing and explained by the phenomenon of interference (Infections with 2 different DENVs can protect against severe disease during subsequent infections (We believe that the unanticipated results of the dengue vaccine efficacy trial in Thailand call for new methods of assessing dengue immunity. Myeloid cells are major targets of dengue infection in humans. We and others have described the unique biologic responses when dengue virus\u2013antibody complexes are presented to myeloid cells (To our knowledge, only once has an in vitro test correctly predicted which children would be susceptible or have silent infections accompanying a second heterotypic dengue infection (Although human Fc-receptor cell lines may be convenient for assaying DENV antibodies, decisions regarding their use should be deferred until they are shown to model primary myeloid cells. Because antibody titers often wane after vaccination, the ability of serum samples from vaccinees to protect against infection of myeloid cells with the 4 DENVs should be studied over many years. Changes to in vitro systems for measuring immune responses after dengue vaccination may provide a better surrogate of protection by realigning antibody measurement systems to our contemporary understanding of the pathogenesis of this complex disease."} +{"text": "Patients with amnestic mild cognitive impairment are at high risk for developing Alzheimer's disease. Besides episodic memory dysfunction they show deficits in accessing contextual knowledge that further specifies a general concept or helps to identify an object or a person.Using functional magnetic resonance imaging, we investigated the neural networks associated with the perception of personal familiar faces and places in patients with amnestic mild cognitive impairment and healthy control subjects. Irrespective of stimulus type, patients compared to control subjects showed lower activity in right prefrontal brain regions when perceiving personally familiar versus unfamiliar faces and places. Both groups did not show different neural activity when perceiving faces or places irrespective of familiarity.Our data highlight changes in a frontal cortical network associated with knowledge-based personal familiarity among patients with amnestic mild cognitive impairment. These changes could contribute to deficits in social cognition and may reduce the patients' ability to transition from basic to complex situations and tasks. For example, unexpectedly encountering a person we already met before, could elicit a feeling of familiarity although we may not be able to remember any specific details about this person The potential influence of personal familiarity on object use and person recognition plays a substantial role in treatment and care of patients with pathological cognitive decline. For example, Giovannetti et al. personal familiarity in aMCI patients. Recent behavioral data suggest that aMCI patients may have difficulties in accessing episodic memory details In this study we investigated patients with amnestic mild cognitive impairment (aMCI). Although these patients are not demented, they are at high risk for developing Alzheimer's Disease (AD), with annual conversion rates of 10\u201312% personal (knowledge-based) familiarity revealed additional activity in medial prefrontal, anterior cingulate and posterior temporal areas Studies aimed at investigating the neural correlates underlying familiarity are rare, and they usually focus on young, healthy people. In functional magnetic resonance imaging (fMRI) studies, familiar stimuli have been shown to activate medial posterior brain regions across various stimulus modalities, such as faces, places or voices Recent structural and functional imaging studies investigating MCI patients suggest complex anatomical and functional changes in brain regions that are associated with familiarity To reveal the neural networks associated with personal familiarity, in this study, photographs of personally familiar faces (spouse or children) and places (from the participants' own homes), as well as unfamiliar faces and places, were presented to aMCI patients and healthy elderly control participants during fMRI scanning. We predicted that in line with the ability to recognize the visual stimulus as familiar, both participant groups would engage the posterior cingulate/precuneus region irrespective of stimulus type (face/place), when perceiving familiar versus novel context. We further hypothesized that associated with impairment in accessing rich contextual details for the familiar stimulus, aMCI patients would exhibit reduced activity in prefrontal cortical areas.All aMCI subjects were recruited through the University's Memory Clinic. Control participants responded to public advertisements. The experiments were done in accord with the Helsinki Declaration of 1975. The Ethics Committee of Dresden University's Medical Faculty \u2018Carl Gustav Carus\u2019, Dresden, Germany approved the study and written informed consent was obtained. Twelve aMCI subjects meeting Petersen et al. Twelve cognitively healthy subjects participated as control subjects. These subjects performed within the normal range in all neuropsychological tests. The healthy subjects also served as participants in a previous study investigating familiarity effects in normal aging For familiar faces, we obtained photographs of each participant's close relatives (spouse or children) with a digital camera. Each relative was photographed from five different angles . Images were digitally manipulated to ensure similar head size, luminance, and background. Pictures of unfamiliar faces were obtained from family members of the clinical staff. Familiar and unfamiliar face stimuli were matched for gender and approximate age. Images of familiar places were taken of the participants' homes. We obtained photographs of rooms rather than of single furniture. Pictures of unfamiliar places were obtained from the homes of clinical staff members and their relatives.3.In order to investigate the neural activity associated with different stimulus modality and personal familiarity, we used a blocked factorial design, presenting images of personally familiar faces and places, and unfamiliar faces and places during an fMRI experiment. We utilized the same experimental procedure as in our previous study investigating familiarity effects in healthy aging Image processing and statistical calculations were performed using MATLAB and statistical parametric mapping software . The first five EPI images were discarded to allow the MRI signal to reach a steady state. To correct for head movement we spatially realigned individual data to the first volume. We used a standard EPI template (MNI brain) for normalization. After resampling to achieve 3\u00d73\u00d73 mm isotropic voxels we smoothed the functional data using an isotropic Gaussian kernel of 10 mm FWHM. At the single subject level, we modelled all four conditions of the paradigm in the context of a general linear model (GLM). We also modelled the question stimulus, the subjects' response (button presses) and feedback separately from the rest condition (focusing on a fixation cross). We used a flexible factorial modelling procedure for second level analyses in a 2*2*2 factorial design, investigating the factors stimulus type (face/place), familiarity (familiar/unfamiliar), and group (control/aMCI). After examining the factors' main effects, we investigated all two-way interactions . In case of significant interactions we additionally calculated the respective simple main effects (e.g. effect of familiarity in both groups). Although our groups did not differ in mean age or education status, we additionally investigated whether modelling age and education as covariates would change our findings. Voxels in MNI-space were considered statistically significant at a threshold of p<0.05 (corrected at cluster level) using a height threshold of p<0.001 uncorrected, corresponding to T\u200a=\u200a3.28 and a cluster size of at least 30 activated voxels. Sociodemographic data and neuropsychological scores were compared using two-tailed t-tests., , Given our hypotheses, we were specifically interested in examining a possible interaction between the factors group and familiarity. We detected significant main effects for familiarity but not for group Table 2,The factor stimulus type was of no primary interest for our main hypothesis. However, investigating main effects Table 2 In a post-scanning debriefing, individual stimuli used during the scan were again presented on a computer screen. Both participant groups did not significantly differ in their ability to correctly categorize familiar and unfamiliar stimuli.In this study we demonstrated that aMCI patients compared to healthy elderly subjects showed lower activity in right prefrontal brain regions when perceiving personally familiar faces and places. Within-group comparison revealed that control participants activated a large neural network including frontal, posterior cingulate and temporal cortices for personally familiar versus unfamiliar stimuli, whereas aMCI patients showed activity in the bilateral precuneus and right posterior cingulate cortex only. These differences in neural activity occurred irrespective of visual stimulus modality (face/place), and despite the fact that both groups did not show neural activity differences when perceiving faces or places per se, irrespective of familiarity.Personal familiarity associated with close family members and one's own home arises from years of interaction and exposure. The recollection of specific knowledge and experiences associated with a familiar stimulus has been shown to recruit brain regions involved in social cognition and episodic memory Throughout the literature there is a main focus on the medial temporal lobe with respect to patients suffering from pathologic cognitive decline. Our data contribute to the emerging evidence that changes in frontal cortical functioning are also involved relatively early in the course of cognitive impairment. Due to its late myelination in brain development, the frontal cortex is susceptible to myelin damage personally familiar environment is particularly helpful in dementia care and therapy In contrast to prefrontal cortical activity, we did not detect a group difference in the posterior cingulate cortex when the subjects perceived personally familiar stimuli. The posterior cingulate/precuneus region is closely associated with perceptual familiarity, irrespective of whether or not there is any knowledge available that would further individuate the perceptually familiar stimulus With respect to activation laterality, it should be highlighted that our group difference associated with personal familiarity was detected in the right prefrontal cortex. Whereas left more than right prefrontal regions are involved in episodic memory encoding, the opposite pattern has been described for episodic memory retrieval It is a limitation of this study that we were not able to directly investigate the participants' performance of retrieving detail-rich contextual information on the behavioral level. However, we previously showed that aMCI patients retrieve autobiographical events with fewer details when compared to healthy subjects"} +{"text": "There is clear evidence documenting the morbidity and mortality benefit of regular colonoscopy surveillance in reducing the high risk of primary and metachronous colorectal cancers associated with Lynch Syndrome (LS) . The Australian NHMRC guidelines (2005) recommend LS patients undergo a colonoscopy every 1-2 years (NHMRC 2005). At Peter Mac yearly colonoscopy screening is usually advised for LS patients.The long term adherence rate to colonoscopy in LS patients as reported in the literature is between 60-88% . Some factors have been associated with inadequate LS patient screening adherence including lack of sedation, inappropriate advice from managing doctors, financial costs, embarrassment and lack of patient time . Whilst not studied, some of these factors may also contribute to a phenomenon known as \u2018screening fatigue\u2019, whereby after one or more normal screening procedures patients begin to attend less frequently for the recommended screening procedures.At the Peter Mac patients and their managing doctors are given GI screening recommendations by the FCC when the patient receives their gene mutation results. However, it is unclear whether LS patients continue to follow the GI screening advice set out by an FCC over time and whether there is any evidence that \u2018screening fatigue\u2019 exists. Furthermore it is unknown whether differences regarding the frequency of colonoscopy offered/performed, adherence to colonoscopy or quality of endoscopy exist for patients who participate in a high risk GI management clinic versus those who are screened by endoscopists in generalist community based settings.The FCC at Peter Mac undertook an audit of the GI screening practices of 74 confirmed Lynch Syndrome gene mutation carrier patients from 2006-2010. Of these patients 27 participate in a high risk GI management clinic where they receive their GI screening. The additional 47 patients have GI screening conducted through either private endosocopy clinics or at public hospitals.This work will;\u2756 Provide a clinical description of our population of LS patients\u2756 Describe the rate of adherence to recommended GI screening overtime. This is assessed by comparing the screening practices undertaken with the recommendations provided by the FCC at the time of their gene test result. We will also present data about whether updates of screening recommendations by our FCC were acted upon by patients/their managing doctors.\u2756 Assess for evidence of \u2018screening fatigue\u2019\u2756 Compare rates of adherence to GI screening practices between patients attending GI high risk management clinics versus patients attending for GI screening in community settings\u2756 Describe the overall colorectal polyp and GI cancer detection rate\u2756 Use colorectal polyp detection also a measure of quality of colonoscopyWe will make some suggestions for further research particularly into interventions that may improve adherence to GI screening advice for a population of LS patients."} +{"text": "The highly variable nature of acupuncture needling creates challenges to systematic research. The goal of this study was to test the feasibility of quantifying acupuncture needle manipulation using motion and force measurements. We hypothesized that distinct needling styles and techniques would produce different needle motion and force patterns that could be quantified and differentiated from each other.A new needling sensor tool (Acusensor) was used to record needling in real time as performed by six New England School of Acupuncture (NESA) faculty from the \u201cChinese Acupuncture\u201d (Style 1) and \u201cJapanese Acupuncture\u201d (Style 2) programs (three from each). Each faculty expert needled twelve points in twelve healthy human subjects using both tonification (Technique 1) and dispersal (Technique 2). Parameters calculated from the raw needling data were displacement amplitude, displacement frequency, rotation amplitude, rotation frequency, force amplitude, and torque amplitude.Data analysis revealed significant differences in the amplitude of both displacement and rotation between needling performed by faculty from two different acupuncture styles. We also found significant overall differences in the frequency of displacement between tonification and dispersal that were not dependent of the style of acupuncture being performed. The relationships between displacement and rotation frequencies, as well as between displacement and force amplitudes, showed considerable variability across individual acupuncturists and subjects.Needling motion and force parameters can be quantified in a treatment-like setting. Needling data can subsequently be analyzed, providing an objective method for characterizing needling in acupuncture basic and clinical research."} +{"text": "Taking folic acid supplements before pregnancy to reduce the risk of a neural tube defect (NTD) is especially important in countries without universal folic acid fortification. The extent of folic acid supplementation among women who had antenatal screening for Down\u2019s syndrome and NTDs at the Wolfson Institute of Preventive Medicine, London between 1999 and 2012 was assessed.466,860 women screened provided details on folic acid supplementation. The proportion of women who took folic acid supplements before pregnancy was determined according to year and characteristics of the women. The proportion of women taking folic acid supplements before pregnancy declined from 35% (95% CI 34%\u201335%) in 1999\u20132001 to 31% (30%\u201331%) in 2011\u20132012. 6% (5%\u20136%) of women aged under 20 took folic acid supplements before pregnancy compared with 40% of women aged between 35 and 39. Non-Caucasian women were less likely to take folic acid supplements before pregnancy than Caucasian women; Afro-Caribbean 17% (16%\u201317%), Oriental 25% (24%\u201325%) and South Asian 20% (20%\u201321%) compared with 35% (35%\u201335%) for Caucasian women. 51% (48%\u201355%) of women who previously had an NTD pregnancy took folic acid supplements before the current pregnancy.The policy of folic acid supplementation is failing and has led to health inequalities. This study demonstrates the need to fortify flour and other cereal grain with folic acid in all countries of the world. In 1991 the results of the Medical Research Council Vitamin Study were published. This randomised controlled trial showed that taking folic acid before conception reduced the risk of a neural tube defect (NTD) pregnancy by an estimated 72% The results in the paper arose out of an audit of our screening service for which no explicit consent was sought but women screened were informed that data arising from the screening programme might be used to make improvements to the services provided. All the data were anonymous and no enquiries or procedures were performed other than those that formed part of the routine service so ethical approval was not required.In 1998 the antenatal Down\u2019s syndrome and NTD screening service at the Wolfson Institute of Preventive Medicine, London introduced a question on screening request forms asking whether women had (i) started taking folic acid supplements before pregnancy, (ii) started once pregnancy was confirmed or (iii) not taken folic acid supplements. Between January 1999 and December 2012 520,570 pregnant women were routinely screened at the Wolfson Institute for Down\u2019s syndrome using the late first trimester (11\u201313 weeks\u2019 gestation) Combined test or the early second trimester (15\u201320 weeks\u2019 gestation) Quadruple test . Women screened with the Quadruple test were also screened for NTDs using maternal serum alphafetoprotein, collected as part of the Quadruple test, and a further 867 women were screened for NTDs only. A total of 466,860 provided data on folic acid supplement status. The proportion of women who started taking folic acid supplements before pregnancy was determined for each calendar year. Data on the following specified factors that are routinely collected as part of the screening service were used to determine the independent effects of each: previous NTD pregnancy (yes/no), previous Down\u2019s syndrome pregnancy (yes/no), ethnicity , insulin dependent diabetes mellitus (yes/no), in-vitro fertilisation , smoker (yes/no), maternal weight (five 10 kg categories) and maternal age (seven 5-year categories), region of residence , and time of screening (first or second trimester). The relative use of taking folic acid supplements before pregnancy versus not taking folic acid supplements before pregnancy was calculated according to these factors and categories. Adjusted relative use estimates were calculated using multivariate Poisson regression with robust variances All factors were statistically significantly associated with taking folic acid supplements before pregnancy and each factor, adjusting for all other factors, remained statistically significant despite the unadjusted percentages being the same (40%). The main contributor to the lower adjusted percentage for women aged 45 or older compared with women aged 40\u201344 is the proportion of women who had undergone IVF treatment (who were most likely to take folic acid before pregnancy) in the 45 or older age group; 34% compared with 7% of women aged 40\u201344.During the study period the proportion of women who started taking folic acid supplements after pregnancy had been confirmed increased from 45% to 62% see . HoweverA strength of this study is the large sample size; almost half a million pregnancies resulting in very narrow confidence intervals of our estimates and even small differences are highly statistically significant. The results show that the policy of folic acid supplementation has failed. The policy exacerbates health inequalities among ethnic minorities and younger women. Our study is based on women from England and the Isle of Man, but the results are similar to other countries; a review of 34 studies conducted in 8 countries between 1992 and 2001 showed that nowhere was the proportion of women taking folic acid supplements before pregnancy greater than 50% and that overall it was about 25% The global number of spina bifida and anencephaly births has been estimated at 300,000 to 400,000 per year It is widely judged that folic acid fortification and supplementation is safe. Concerns over the possible increased risk of cancer being associated with folic acid fortification are not justified The prevention of neural tube defects is a global problem. Our results show that public health policy cannot rely on pre-pregnancy folic acid supplementation alone. Improved education in communities with particularly low rates of pre-pregnancy folic acid supplementation may increase the rates but for all women to benefit countries that have not introduced folic acid fortification should do so to avert a preventable serious birth defect responsible for still births, severe physical disability and needless therapeutic abortions.Table S1Percentage of women taking folic acid before pregnancy, once pregnancy confirmed or not at all with unadjusted and adjusted relative use compared with reference categories.(DOCX)Click here for additional data file."} +{"text": "In situ observations of larval swimming are few given the constraints of field sampling. Active behaviour is therefore often inferred from spatial patterns in the field, laboratory studies, or hydrodynamic theory, but rarely are these approaches considered in concert. Ichthyoplankton survey data collected during 2004 and 2006 from coastal Newfoundland show that changes in spatial heterogeneity for multiple species do not conform to predictions based on passive transport. We evaluated the interaction of individual larvae with their environment by calculating Reynolds number as a function of ontogeny. Typically, larvae hatch into a viscous environment in which swimming is inefficient, and later grow into more efficient intermediate and inertial swimming environments. Swimming is therefore closely related to length, not only because of swimming capacity but also in how larvae experience viscosity. Six of eight species sampled demonstrated consistent changes in spatial patchiness and concomitant increases in spatial heterogeneity as they transitioned into more favourable hydrodynamic swimming environments, suggesting an active behavioural element to dispersal. We propose the tandem assessment of spatial heterogeneity and hydrodynamic environment as a potential approach to understand and predict the onset of ecologically significant swimming behaviour of larval fishes in the field.During the pelagic larval phase, fish dispersal may be influenced passively by surface currents or actively determined by swimming behaviour. The eggs and larvae of many marine organisms are transported during a pelagic dispersive stage through interaction between passive oceanographic processes Gadus morhua) Multiple studies hypothesize that larval swimming ability is critical to success or failure in dispersal Behavioural influences on spatial distributions of larvae are difficult to demonstrate without direct observations. Previous studies of coral reef systems Spatial distributions of marine organisms are rarely uniform and often patchy. Spatial patchiness of fish larvae is determined by passive oceanographic mechanisms Previous patchiness studies do not specifically address the role of the hydrodynamic environment and how it might provide another framework in which to consider swimming as a quantifiable influence on spatial heterogeneity during the larval period. Researchers have used functional morphology to explain allometric growth trajectories in relation to hydrodynamic environments Recent laboratory Scorpaeniformes, Plueronectiformes. Perciformes and Osmeriformes. This mix of taxa provides additional opportunities to test potential swimming contributions to spatial heterogeneity. We compare changes in spatial patterns of multiple species to passive flow conditions while simultaneously considering the hydrodynamic swimming environment and biological environment to which these larvae occupy. Specifically, we evaluate changes in spatial pattern related to swimming capability in order to test our hypothesis that increases in spatial patchiness coincide with critical changes in the hydrodynamic swimming environment of sampled larvae, thus helping to explain natural patterns in ichthyoplankton catch data.Smith Sound, Trinity Bay supports a large, persistent inshore spawning aggregation of Atlantic cod 3) collected by the Tucker trawl is designed to minimize such biases. We preserved samples in a buffered seawater and 4% formalin solution, and later identified all fish larvae to species using published keys We collected larval data from ichthyoplankton surveys of Trinity Bay, Newfoundland during the spring of 2004 and 2006, and the summer of 2004, on board the Canadian Coast Guard Ship (CCGS) Shamook. At each survey station tows were carried out using a 2.0 m by 2.0 m Tucker trawl fitted with decreasing mesh sizes of 1000, 570, and 333 m progressing from the front of the net to the cod end. We sampled twenty ichthyoplankton stations in a \u201cbullseye\u201d pattern radiating out from Smith Sound on the western side of the bay in obliq\u22121 calibration using ImageJ\u00ae image analysis software, and grouped larvae into 1 mm size bins. Standard length was defined as the measurement from the anterior tip of the body to the midlateral posterior edge of the hypural plate.Larvae were imaged at the Ocean Science Centre's Image Data Analysis Facility, where we measured lengths of a maximum of 100 individuals for each species for each station. Samples with more than 100 individuals of a single species were sub-sampled using a Motoda plankton splitter. We calculated length from the images to the nearest 0.1 mm with a pixel mmLinear kriging in Surfer\u00ae 8 was used to increase spatial resolution of surveys by using all stations sampled We used Lloyd's index of mean crowding k) into Lloyd's index of patchiness P is an estimate of patchiness and k is the dispersion parameter estimated with the maximum likelihood approach P greater than 1 denote how many more times crowded an individual is than a random distribution The calculation of Lloyd's index requires an estimate of population variance where the use of sample variance may be inappropriate. Several authors estimated population variance by applying a negative binomial distribution to ichthyoplankton data Re) following Brett critU\u200a=\u200acritical swim speed (m s\u22121), L is the length scale and \u03bd is the kinematic viscosity of seawater (m2 s\u22121). We used total length (L), defined as the mean size within a 1 mm size bin and applied this mean length to the swimming parameter (Ucrit) of the Reynolds number equation. Critical swim speeds for Atlantic cod were estimated according to length swimming relationships derived from an image database and corresponding swim data reported in Guan et al. 2\u200a=\u200a0.90) presented by Guan et al. 2008. The consistent functional relationship between swimming capacity, beat frequency, and length in larval fish suggests some generality among temperate species at comparable ontogenetic stages For each larval fish measured we calculated the Reynolds number (We obtained measurements of average mixed layer (upper 40 m) temperature, salinity, and fluorometry from vertical conductivity-temperature-depth (CTD) profiles taken concurrently with each ichthyoplankton sample. Kriging was used to analyze spatial patterns of environmental variables which we then compared with spatial patterns of larvae produced the same way. Kinematic viscosity (\u03bd) was calculated from mean survey temperature data collected from CTD casts. Kinematic viscosity also changes as a function of salinity, however, average salinity differences among survey seasons were negligible (maximum difference of 0.7), and we therefore did not consider them.Re, varies among studies. We used a Reynolds number of 300 as a critical point marking the shift between a clearly viscous swimming environment to one which is intermediate We estimated Reynolds number for a specific size range of larvae based on swimming velocities from the morphometric kinematic relationships and average kinematic viscosities. Estimates of the transitional point between swimming environments, defined by We estimated both Lloyd's index and Reynolds numbers for larvae collected in the field using length as the linking factor in all calculations. Information on the hydrodynamic environment, defined by Reynolds number boundaries, was then considered with patchiness data to illustrate linkages between hydrodynamics and spatial heterogeneity in the field.\u22121 using a Motoda plankton splitter. We then mapped and compared zooplankton abundance following the same methodology outlined for larval fish lengths and Atlantic seasnail (Liparus atlanticus) to 3.2 in Atlantic seasnail. The effect of zero counts (stations where no larvae of a given species at any size were caught) differed among species, exaggerating patchiness estimates for species with abundant larvae (e.g. capelin) or reducing estimates for species with few larvae (e.g. redfish). Despite this variability, the overall pattern with size was similar irrespective of whether we included zero values. To be consistent with other authors who used this method We limited our spatial analysis of Lloyd's index as a function of the full range of ontogenetic egg development to Atlantic cod, which was the only species in our samples with sufficiently abundant developmental stages to include in patchiness estimates. Nonetheless, comparisons of larval patterns for multiple species indicated consistent patterns of patchiness through larval ontogeny ,4. For Aanticus) . The staSebastes spp.), radiated shanny, and witch flounder were all significantly more abundant on the eastern side of Trinity Bay throughout larval ontogeny. Arctic shanny (Stichaeus puntatus.) and Atlantic seasnail shifted between the east and west divide during the latest larval stage. The remaining species were significantly more abundant on the western side of Trinity Bay throughout ontogeny. Larger individuals of Atlantic cod, American sandlance (Ammodytes americanus), capelin , redfish, and Atlantic seasnail were more common on the western side of Trinity Bay near Bonaventure Head.Spatial heterogeneity as a function of larval ontogeny for larvae of 8 species suggested that patterns in early and late larval stages are more defined than in intermediate stages ,6, as inKriging smoothed out bias in the sampling grid, such as our highly clustered stations near the presumed natal source of cod larvae in Smith Sound. This procedure provides a reasonable estimate of mean concentration but an unreliable estimate of variance for each side of the bay because variance estimates for the east and west side of the bay are not independent. To determine if this statistical violation impacted spatial interpretation, we divided the bay into western and eastern regions, and individually kriged data on Atlantic cod and American sandlance . With thcrit) for Atlantic cod larvae of a given total length (TL) derived from:For Atlantic cod, of all morphometric variables analysed, total length was the best predictor of kinematic potential \u22126 m2 s\u22121 .We calculated kinematic viscosity from the mean temperature for the mixed layer (top 40 m) at all stations during all surveys. Although this method ignores spatial variability in viscosity, extreme high and low field temperatures change the estimate of Re by less than 3.5%, and therefore have little effect on data interpretation. Mean temperature among surveys yielded an average kinematic viscosity of 1.714\u00d710We found similar patterns of patchiness in the context of hydrodynamic transitions among species by calculating the mean change between the size classes for each species immediately before and after the proposed viscous transition (Re\u200a=\u200a300). For all species except Atlantic seasnail (decrease 10%), an increase in patchiness coincided with the lead up to and transition out of the viscous environment with the western side of the bay near the cold upwelling as the dominant predators of larval fish The results presented above indicate that passive circulation alone cannot explain spatial distribution pattern, leaving two possible explanations for our results other than active swimming - predation or food availability. Size-dependent predation in particular could influence spatial patterns in ichthyoplankton. Several studies of larval distribution in Newfoundland embayments identified juvenile or adult capelin (Food availability can also concentrate organisms into patches Using behaviour to explain spatial pattern necessitates the resolution of bearing or frame of reference Data from our study do not permit distinction of whether this behaviour is a response to single or multiple cues. However, our study does suggest that multiple species conform to a pattern fitting predictions of behaviour and not one predicted by a purely passive model. Increased swimming abilities both horizontally and vertically will enable larvae to utilize and orient towards a greater diversity of cues Spatial analysis using total length as a proxy for ontological development revealed higher patchiness estimates for late stage and in some cases early stage larvae than at intermediate stages, confirming patterns found in other temperate species"} +{"text": "Physiological instability is a common clinical problem in the critically ill. Physiological adaptation can be regarded as a dynamic process, with stability being conferred by a number of apparently complex, fluctuating homeokinetic processes . Many nam) and multifractal spectrum width-at-half-height (WHH). We investigated how these parameters changed with pharmacological interventions such as vasoconstriction.We employ the wavelet modulus maxima technique to characterize the multifractal properties of physiological time series such as heart rate (HR) and mean arterial pressure (MAP) under conditions of clinical physiological instability. We calculated point estimates for the dominant H\u00f6lder exponent (hm for MAP, consistent with a more highly fluctuating, antipersistent and complex behavior. Blood pressure support with pharmacological vasoconstriction led to a transient increase in hm for MAP (Figure m as estimated for HR. On the other hand, supporting the heart rate with atropine had no effect on hm for MAP, but did tend to increase hm for HR.Hypotensive patients showed lower values of hP Figure revealinWe demonstrate increasing signal complexity under physiological challenge consistent with the activation of homeokinetic processes. Differential fractal behavior for HR and MAP suggests that the homeokinetic systems are recruited in a targeted way depending on the physiological challenge. Pharmacological restoration of homeostasis leads to system decomplexification suggesting that homeokinetic mechanisms are derecruited as physiology is restored. We suggest fractal geometry provides a method for characterizing physiological instability and measuring the homeokinetic stress response during physiological challenges."} +{"text": "Severe therapy resistant asthma is a relatively rare entity. This has been variously defined based on clinical and spirometric criteria. It has always been known that the correlation between clinical and physiological parameters is not very consistent and hence the difficulty in defining this entity. We have looked at our cohort of consecutive patients with severe and moderate asthma in trying to establish a correlation between clinical and spirometric parameters.We looked at 850 consecutive patients with diagnosis of asthma based on Spirometric criteria (GINA guidelines). These were patients were compliance to medications was ensured and technique was optimised by trained healthcare personnel. We then selected the ones who had \u201cSevere\u201d & \u201cModerate\u201d asthma basedon Spirometry. We now looked at these patients and correlated their Spirometry with thesymptomatology and exacerbation rates .Only 79 (9.29%) patients out of 850 had severe asthma & 352 (41.41%) patients had moderate asthma based on Spirometric criteria. We found that Spirometric findings correlated with clinical parameters in 69.4% patients with Severe Asthma & 86% patients with Moderate Asthma.Only a small but significant percentage of patients in our population satisfy the Spirometric criteria of Severe Asthma (less than 10%). Even in this population there is lack of homogeneity between clinical parameters (symptoms & exacerbations) and physiological parameters (Spirometry). This correlation was much more consistent in patients with Moderate Asthma. Interestingly, we found more severe the asthma based on Spirometry, less was the correlation with symptomatology and exacerbations. This phenomenon and its causation needs to be studied in greater detail in longitudinal studies."} +{"text": "Profound synapse loss is one of the major pathological hallmarks associated with Alzheimer\u2019s disease (AD) and might underlie memory impairment. The homeostasis of metal ions plays an important role in health and neurodegenerative disease by influencing cellular biochemical pathways. The disturbance of some metal ions may have cytotoxic effects, which may cause cell death leading to neurodegenerative disorders such as AD. The aim of the present study was to investigate metal concentrations in whole blood from Chinese AD patients with APOE \u03b54 allele carrier. Concentrations of metals were determined in whole blood by inductively coupled plasma mass spectrometry (ICP-MS) in 40 Chinese people with different Mini-mental state examination (MMSE) score. Normal APP processing could be restored when magnesium was adjusted back to physiological concentration. Our findings suggest that supplementation of magnesium has a therapeutic potential for preventing AD. We observed that Plasma Mg, Zn and Se levels were found to be significantly lower in patients with AD compared with controls. Furthermore, there is a significant negative correlation between manganese and MMSE score. Whereas other metal ions have no association with MMSE score. These result suggests that dishomeostasis of metal ions may involve in the progress of AD pathology, and elevation of brain magnesium exerts substantial synaptoprotective effects in a mouse model of AD and may have therapeutic potential for treating AD in humans."} +{"text": "While an outright cure or a preventive vaccine for HIV/AIDS remain elusive, remarkable advances in HIV treatment have been achieved over the past two decades. Most significant among these advances is the development of highly active antiretroviral therapy (HAART).Available evidence increasingly supports the notion that the viral load suppression achieved by HAART has a preventive impact on the transmission of HIV. Hence, expanding HAART coverage to all those in medical need, represents a key strategy to not only decrease HIV and AIDS related morbidity and mortality but also to dramatically reduce HIV transmission by all routes.In British Columbia, the expansion of HAART coverage has been associated with a substantial reduction in HIV new diagnoses . The role of treatment as prevention has now been formally incorporated within the UNAIDS global AIDS control strategy, under the \"Treatment 2.0\" initiative."} +{"text": "Prevention of unintended pregnancy, especially among HIV positive couples and in settings where both HIV and total fertility rates are high, is a critical public health initiative.A factorial randomized controlled trial evaluated the effect on incident pregnancy of two interventions to promote long-term contraceptive use among HIV serodiscordant and concordant positive couples (N = 1060) identified from CVCT clinics in Lusaka, Zambia.Couple baseline serostatus and contraception usage were both individual effect measure modifiers (p<0.0001). Among couples in which the woman was not using a contraceptive method at baseline (N=782), there was no significant effect of the interventions overall or when stratifying by couple serostatus on incident pregnancy. Among couples in which the woman was using a contraceptive method at baseline, concordant positive couples , and couples in which the woman was HIV positive at baseline who received \u201cMethods + Both\u201d interventions - which combined information on contraceptive methods and motivational messages for future planning behaviors - were at significantly decreased hazard for pregnancy relative to those receiving \u201cMotivational + Control\u201d interventions -- which provided motivational messages for future planning but not information on contraceptive methods.An educational intervention promoting long-term contraceptive method uptake among HIV positive couples is successful at decreasing time to pregnancy in the context of couples\u2019 HIV testing, particularly among women who are HIV+ and already using a contraceptive method. A combination of motivational messages for future planning behaviors and information on long-term contraceptive methods appears to be the best intervention for reducing incident pregnancy among concordant positive and serodiscordant couples. Further work is needed to understand the interventions appropriate for women who are currently not contraceptive users."} +{"text": "Strix occidentalis caurina) habitat are largely initiated by establishing habitat occupancy. Northern spotted owl occupancy is typically assessed by eliciting their response to simulated conspecific vocalizations. However, proximity of barred owls (Strix varia)\u2013a significant threat to northern spotted owls\u2013can suppress northern spotted owl responsiveness to vocalization surveys and hence their probability of detection. We developed a survey method to simultaneously detect both species that does not require vocalization. Detection dogs (Canis familiaris) located owl pellets accumulated under roost sites, within search areas selected using habitat association maps. We compared success of detection dog surveys to vocalization surveys slightly modified from the U.S. Fish and Wildlife Service\u2019s Draft 2010 Survey Protocol. Seventeen 2 km \u00d72 km polygons were each surveyed multiple times in an area where northern spotted owls were known to nest prior to 1997 and barred owl density was thought to be low. Mitochondrial DNA was used to confirm species from pellets detected by dogs. Spotted owl and barred owl detection probabilities were significantly higher for dog than vocalization surveys. For spotted owls, this difference increased with number of site visits. Cumulative detection probabilities of northern spotted owls were 29% after session 1, 62% after session 2, and 87% after session 3 for dog surveys, compared to 25% after session 1, increasing to 59% by session 6 for vocalization surveys. Mean detection probability for barred owls was 20.1% for dog surveys and 7.3% for vocal surveys. Results suggest that detection dog surveys can complement vocalization surveys by providing a reliable method for establishing occupancy of both northern spotted and barred owl without requiring owl vocalization. This helps meet objectives of Recovery Actions 24 and 25 of the Revised Recovery Plan for the Northern Spotted Owl.State and federal actions to conserve northern spotted owl ( Establishment of occupancy is often critical for initiating management practices aimed at conserving endangered species. However, reliable assessment of occupancy requires a methodology that provides reasonable probability of detecting the species when present Many conservation actions for northern spotted owls are enacted only when their occupancy is established. Northern spotted owl presence is typically confirmed by vocal response to simulated calls on potentially occupied habitat Unfortunately, the presence of invading barred owl competitors can suppress spotted owl responsiveness to vocalization surveys Canis familiaris) to locate DNA-confirmable wildlife sign can provide a useful complementary survey strategy that is largely independent of the target species\u2019 behavioral response or physiological status. Dogs are selected for an extreme drive to play with a toy, generally a ball. Once the dogs are trained to associate detection of the target scent with their play toy reward, sample detection becomes driven solely by the dogs\u2019 obsession to obtain their reward. Sample detection thus becomes detached from the target species\u2019 sex, life history stage, responsiveness to vocalization or other characteristics that might cause detection bias Use of detection dogs analysis of mtDNA extracted from the swabs of each pellet (see below).The present study examines the use of detection dogs to simultaneously document occupancy of the federally threatened northern spotted owl and its closely related competitor, the barred owl. In spring 2010, we conducted a study comparing the cumulative detection probabilities of northern spotted and barred owls from dog surveys and vocalization surveys using the U.S. Fish and Wildlife Service (USFWS) Draft Northern Spotted Owl Survey Protocol An important objective of this paper is to determine whether dog and vocal survey methods differ in detection probabilities for both northern spotted owl and barred owl, and whether these survey method differences are impacted by number of sampling sessions conducted in each polygon and by habitat. For northern spotted owl, we also wanted to know if barred owl presence impacted northern spotted owl probabilities of occupancy and detection and whether the latter varied by survey method. If barred owl inhibit northern spotted owl responsiveness to playbacks, barred owl presence should reduce northern spotted owl detection probability in vocal surveys but have no impact on dog survey detection probability. Finally, we wanted to know whether there were differences between teams conducting the same survey method.We used an occupancy model approach Sample collection methods were approved by the University of Washington\u2019s Institutional Animal Care and Use Committee (IACUC) under permit numbers 2850-04 and 2850-08.Pseudotsuga menziesii), Ponderosa pine (Pinus ponderosa) and oak (Quercus spp.). Steep topography is typical in the study area. Barred owls were thought to be relatively uncommon in the Shasta-Trinity National Forest at the time of the study . The study area had not been completely surveyed since at least 1997. Thus, most owls had little or no experience with mouse offerings that could increase their responsiveness to vocalization surveys.Our study was conducted in the South Fork Management Unit of Shasta-Trinity National Forest in northern California. The forest consists of mixed coniferous and deciduous trees, comprised primarily of Douglas fir to locate northern spotted and barred owl pellets and feces by scent, using methods described in Wasser et al. We acquired pellets from captive barred owls at the Woodland Park Zoo in Seattle and from wild spotted owls collected during an independent study in Shasta-Trinity National Forest Once on site, we spent the first week acclimating the dogs to the study area and facilitating their detection of pellets that naturally occurred in the field. This was accomplished by teams visiting previously known spotted owl sites located outside our study area, eliciting a vocal response from the owl and then hiking in with the dog to search for pellets. Dogs were worked off leash and rewarded upon sample detection. Supplemental exercises also occurred at the base-camp 2\u20133 times per day during that week, using previously acquired northern spotted and barred owl samples.Twenty historic northern spotted owl territories documented between 1987 and 1997 were selected for survey by both dog teams and vocalization teams between 11 May and 4 July 2010. However, three polygons were excluded after the first session due to signs of marijuana cultivation and the inherent danger to field crews. The study area was part of a late successional reserve, as identified by the Northwest Forest Plan. All polygons were delineated and consecutively numbered prior to our arrival in California. However, not all of the polygons could be searched . Those sites were abandoned, but all original polygon numbers were retained.2 search polygon that encompassed as much northern spotted owl nesting/roosting habitat as possible \u22641 km from each historic nest site. Nesting/roosting habitat is generally characterized by moderate to high canopy closure (60\u201390 percent) and a multi-layered, multi-species canopy with large (mean diameter at breast height (DBH) \u226530 inches) overstory trees 2 because our previous studies in this area Two separate northern spotted owl habitat quality models While all crews had at least one member familiar with the Shasta Trinity National Forest, no one had specific data on any of the sites being surveyed in 2010; unintentionally, crew leaders did have some familiarity with 2\u20133 sites from previous years. Dog and vocalization teams surveyed each polygon independently of the other. With few exceptions (see below), vocalization crews began their surveys at roadside call points and only hiked in when an owl responded. Dog crews started and ended at a different location each survey, never covering the same area twice. Thus, there were virtually no opportunities for a dog to follow the trail of another dog or human surveyor. We also made every effort to maintain a \u201cfirewall\u201d between vocalization and dog teams; each team had separate field supervisors, used independent vehicles and equipment, and was prohibited from sharing survey results. Because of illegal marijuana farming within the study area, crews also worked closely with Shasta Trinity National Forest law enforcement personnel.Each dog team consisted of a detection dog, a handler, and an orienteer that processed samples and kept the team within the designated survey area using a hand-held Global Positioning System (GPS) device. Dog teams searched each 2 km \u00d72 km polygon a total of three times (sessions 1\u20133). The same dog searched a given polygon on the first and third session, with the other dog searching during the second session. In no cases was the same route searched twice within a polygon.For any given session, each dog team walked a \u223c5 km (or 6 hr) loop, taking intermittent rests (\u223c10 min) throughout the continuous 6 hr period at a frequency that depended on ambient temperature and steepness of terrain. Whenever a dog located a spotted or barred owl pellet, it sat at the sample to indicate detection. The handler then checked the sample and immediately rewarded the dog. The dog also had a rest period while each sample was being processed.Habitat selection models Latex gloves were worn whenever preparing swabs and collecting specimens. The outer surface of each pellet was swabbed twice, using buccal swabs saturated with 1X PBS buffer. The entire surface of the pellet was lightly swabbed for surface mucosal cells while rotating the swab to maximize the surface area covered Each swabbed pellet was then placed in a paper bag labeled with the pellet ID, date, and UTM location. The paper bag was placed inside an identically-labeled freezer-safe plastic bag and stored in the freezer. At the end of the study, swabs and pellets were transported on dry ice to our laboratory at the University of Washington.Each owl pellet swab was extracted using a modified version of Qiagen\u2019s DNeasy Tissue DNA extraction protocol and eluted in 200 uL AE buffer. Negative controls were included in every extraction to control for any laboratory contamination, and all extractions were performed in a room that was free of PCR products.ACAGCTAAACTTGGGA, which together amplify a 358 bp fragment.We developed a PCR-RFLP assay for species identification using mitochondrial DNA variation. We obtained numerous sequences of the control region in northern spotted owls (n\u200a=\u200a18) and barred owls (n\u200a=\u200a45) from the USFS and GenBank. Conserved regions in both species were identified for primer development by sequence alignment using CLC DNA Workbench. The forward primer, NSO3, has the sequence CACYCTAATYCATGACA and the reverse primer, NSO2, has the sequence Sequence alignment also revealed an AvrII restriction enzyme cut site present in all 45 barred owl sequences and absent in all 18 northern spotted owl sequences, which cuts a 134 bp fragment from the 358 bp fragment for barred owls only. Positive control tissue samples of northern spotted owl and barred owl used for assay validations had 100% consistency with expected results described above, and were included in every PCR run. All samples were analyzed on an ABI3100 Genetic Analyzer using Genescan and Genotyper software (Life Technologies Applied Biosystems), with a 5\u2032 6-FAM label attached to the forward primer NSO3.Two two-person northern spotted owl vocalization teams surveyed each 2 km \u00d72 km polygon six times (sessions 1\u20136). All vocalization surveys were conducted in spring 2010, coincident with dates of the dog surveys. Crew members were trained by senior owl surveyors from the USFWS office and both survey teams had a crew leader with at least two years of experience conducting northern spotted owl vocalization surveys.All visits complied with the U.S. Fish and Wildlife Service\u2019s 2010 Draft Protocol for Surveying Proposed Management Activities That May Impact Northern Spotted Owls Vocalization crews generally arrived on site at 8:30 pm and surveyed until 1 or 2 am (\u223csix to eight hours in the night). All owl responses were followed by a search at sunrise, typically requiring an additional five hours of effort spent hiking and calling.Consistent with the protocol, northern spotted owl calls were generated using high quality digital wildlife callers. Historical information, topographical maps, and aerial data were used to determine call points prior to beginning the survey period. As directed by the 2010 Draft Protocol, sites with recent owl activity from a previous season would receive a daytime initial site visit prior to the night survey. Thus, per protocol, vocalization survey teams conducted historical stand searches before night surveying of polygons 7, 22 and 24 since northern spotted owls had previously been detected there.In a few cases, some call points were placed outside the polygon if more geographically logical. If predetermined call points along roads did not cover all suitable habitat, continuous walking surveys were conducted directly following an unsuccessful pre-dawn vocalization survey for \u223c4 hrs immediately after sunrise. Surveys continued until all suitable habitat that could not be covered by road call points had been searched and called. In such instances, calling occurred within the polygon, off the road and in nesting, roosting, or foraging northern spotted owl habitat. All but two polygons had excellent coverage from night call point locations. Each polygon had a different number of call points depending on road access and suitable habitat, ranging from 3 to 7 points per polygon, spaced 0.25 to 0.5 mi apart depending on acoustic conditions.Call times were increased from 10 minutes to 12 minutes on sites that had no response after four visits to improve the chances of owl response. No surveys were conducted in heavy wind or rain that might hinder auditory detection. Unlike the dog surveys, in most cases the same team conducted all 6 sessions per polygon because their experience from previous sessions made it easier to navigate the area. However, if a team was unsuccessful at locating owls on several visits, the other team would often survey that polygon.All owl pellets located by detection dogs had to be DNA confirmed to owl species by RFLP analysis of mtDNA. The same species-specific DNA fragment had to be observed at least twice from the same sample to be listed as a species confirmation. Reproductive status required a visual identification of the northern spotted or barred owl(s), typically accompanied by presentation of a live mouse.Occupancy models 2 site based on the Zabel et al. a priori, it was included in the model selection concurrently with the main effect.Predictor variables examined included mean and standard deviation in habitat quality per 4 kmStrix owl pellets on all 20 of the 2 km \u00d72 km polygons searched. Three of these sites were subsequently determined to be too dangerous for further searching due to evidence of illegal marijuana farming, although dogs found pellets during the first session in all three cases. A fourth site had to be similarly abandoned after the third dog team visit and thus was only partially included in the vocalization surveys ; three of those sites had both northern spotted owl and barred owl in the same polygon for dry, intact pellets. However, our protocol of collecting numerous pellets per site generally resulted in at least one DNA confirmation at any given polygon . We beliStrix species in all but three of the 17 polygons they surveyed . Spotted owl occupancy was uniformly high across our study area, as was habitat quality based on the Zabel et al. The best predictors of spotted owl detection probability were survey type , session number and their statistical interaction. Dog surveys had significantly higher detection probabilities for northern spotted owls than did vocalization surveys, and this difference increased with the number of surveys conducted per polygon. Dog surveys had cumulative detection probabilities of DNA confirmed northern spotted owls of 29% after session 1, 62% after session 2, and 87% after session 3. Cumulative detection probability of northern spotted owls by vocalization surveys was 25% after session 1, and increased to 59% by session 6 .Barred owl occupancy was comparatively low across the study area. The best barred owl model (according to AIC) included habitat quality as a predictor or occupancy probability . However, the habitat quality covariate was not significant in the model output for probability of occupancy .The best predictors of barred owl detection probability were habitat quality and survey type . Mean deWe found no impact of barred owls on spotted owl occupancy or detection probabilities using either survey method, although this may have been a function of the small size of our study area combined with low number of barred owls found in the area.Team was not a significant predictor of detection probabilities for either species, indicating that dog teams were not significantly different from one another, nor were vocalization teams. Both dogs also detected comparable numbers of DNA-confirmed owls over the study period (13 for dog 1 and 17 for dog 2). However, the dog without prior owl experience (dog 2) showed marked improvement in spotted owl detections between sessions 1 and 2.This study aimed to directly compare detection probabilities of surveys conducted by detection dogs with those of vocalization crews employing the latest draft USFWS survey protocol. Use of the draft USFWS survey protocol provided more detailed information upon locating individual owls . However, by the third visit per polygon, the DNA-confirmed cumulative detection probability of dog surveys was 28% higher than the cumulative detection probability achieved by vocalization surveys after six visits for spotted owls . OverallAlthough spotted owl detection probabilities from our vocalization surveys may seem low in comparison to past studies Spotted owl detection probabilities also declined with sampling session for vocalization surveys but increased for scat dog surveys. The drop-off in vocalization survey detection probabilities with sampling session most likely occurred because of the high likelihood that owls within hearing distance of surveyors, and a propensity to respond, will do so on the first attempt. In perfect environmental conditions, vocalization surveys can detect owls at distances greater than a half mile radius from a call point. Moreover, call points are located so that complete coverage of the polygon occurs. Although rare, some abiotic or biotic factors can still inhibit or prevent a response. For example, the topography in our study area consists of steep mountains with deep and numerous ravines and drainages, creating circumstances where the location of the owl(s) during the first attempt would preclude the owl or the vocalization surveyors from hearing each other. Detection might subsequently become possible if the owl(s) changed their location in later visits, making their response audible to surveyors. Detection probability declines with session may also occur because further surveys are not conducted within hearing distance of the animal(s) once a northern spotted owl has been located and nesting status confirmed. To minimize disturbance, only those portions of the polygon where owls have not been documented are subsequently surveyed under those circumstances.Unlike vocalization surveys, canine transects covered completely new survey areas within the polygon on subsequent visits. Changing locations within a polygon each session likely increases overall detection rates by increasing polygon coverage, as also reported in mark recapture studies In only one case did the vocalization surveys detect an owl species that was not detected by DNA-confirmed dog surveys in the same polygon. By contrast, several northern spotted owls and barred owls were detected by dog surveys but not by vocalization surveys. This occurred in three instances where barred owls occurred on sites already occupied by northern spotted owls and one case where a northern spotted owl was present at a site occupied by a nesting barred owl pair . This suWhere noninvasive survey techniques are desired or where both northern spotted owls and barred owls are present, detection dogs can provide an alternative or complement to vocalization surveys that does not rely on a behavioral response from either species. Combining detection dog and vocalization survey methods, including offering mice to confirm owl reproductive status, may provide additional biological and ecological insights into the consequences of competitive interaction between these two owl species. For example, the three northern spotted owl pairs found in polygons that were sympatric with barred owls were non-reproductive and no barred owls were documented in the three polygons where northern spotted owl pairs were nesting . These oWhile the dog\u2019s presence on territories could be a source of disturbance to owls, dogs were trained not to chase or otherwise harass wildlife. Future studies could evaluate these impacts by comparing glucocorticoid levels Vocalization surveys can cover a large, three-dimensional area in minutes. This differs from the two-dimensional dog surveys described here. Dogs searched for owl pellets along a somewhat pre-defined transect focused on the habitat with the highest probability of owl occupancy. Since pellets must subsequently be DNA-amplified to confirm the species, low amplification success of DNA from owl pellets is a potential drawback of the detection dog method. However, amplification success could probably be improved by identifying a species-specific mtDNA fragment smaller than the 358 bp DNA fragment we used in this study. Pellet detection could also be combined with visual confirmation on occasion to increase the likelihood of confirming owl presence as well as opportunities to establish reproductive condition by offering mice Detection dogs provide an effective noninvasive method for determining presence of both northern spotted owls and barred owls, independent of owl responsiveness. This method can provide a valuable complement to vocalization surveys, facilitating more effective northern spotted owl conservation actions in the face of the species\u2019 continued decline Vocalization surveys that include \u201cmousing\u201d techniques remain the best method for determining reproductive status of northern spotted owls Figure S1Northern spotted owl occupancy plotted as a function of habitat quality. Habitat quality is based on amount of old growth and mature forest (see Carroll and Johnson 2008). Dotted lines are 95% confidence intervals.(DOCX)Click here for additional data file.Table S1Northern spotted owl (NSO) and barred owl (BO) roosts located by detection dog versus vocalization surveys.(DOCX)Click here for additional data file.Table S2Northern Spotted Owl Occupancy Model Using Forward Model Selection.(DOCX)Click here for additional data file.Table S3Barred Owl Occupancy Model Using Forward Model Selection.(DOCX)Click here for additional data file."} +{"text": "The aim of the workshop is to conduct a biomechanical foot assessment of the rheumatoid foot. Participants will be involved in an interactive workshop that includes patients with rheumatoid arthritis. We will examine foot-related outcome tools designed to evaluate foot function, pain and disability. Delegates will be divided into small groups to analyse and interpret temporal and spatial gait parameters and plantar pressure measurements.Upon completion of this workshop, participants should be able to:\u2022 Describe key components of a foot and ankle examination for the rheumatology patient\u2022 Discuss how current technology pertains to conceptualizing and treating patients with rheumatic disease\u2022 Describe the clinical and research applications of validated foot outcome assessment instruments\u2022 Formulate evidence-based non-pharmacological treatment strategies based on pain mechanisms, unique clinical features and biomechanical characteristics\u2022 Demonstrate how biomechanical concepts and related treatment strategies can be used to address complex cases of rheumatological patients with foot pain, impairments and disability"} +{"text": "The authors have justified to the Editor the contribution made by each author. All authors reviewed and approved the final version.The left internal mammary artery (LIMA) to left anterior descending coronary artery (LAD) graft is the most important graft in coronary artery bypass graft surgery.\u00ae suture using a 2/0 Ethibond NJ, US) . This maWe have used this technique successfully in a few cases where we have found the LIMA to be under likely tension or were concerned with emphysematous lungs stretching the LIMA. This simple and easily reproducible technique, which has also been used by others, is a helpful solution to an occasional problem."} +{"text": "The activity of dopaminergic (DA) neurons has been hypothesized to encode a reward prediction error (RPE) which coRecent recordings of DA neurons during multi-choice tasks investigated this issue and raised contradictory interpretations on whether DA's RPE signal is action dependent or not . While tBy quantitatively comparing the ability of the different TD learning algorithms, this work shows the limitation of these algorithms to fit both the behavior and the DA activity observed in a multi-choice task, when interpreting DA activity as a RPE only. Unexpectedly we show that a value function better fits DA activity suggesting that DA neurons recorded in this task may encode multiple information."} +{"text": "Solid tumors including prostate cancer activate angiogenic signals to ensure an adequate blood supply. In parallel, amino acid transporters on the cell surface are also increased so as to provide nutrients for the higher metabolic and growth demands of cancers. We are studying the L-type amino acid transporters (LAT1 and LAT3) that mediate uptake of essential amino acids including leucine. Leucine has recently been shown to be critical for the activity of mTORC1, which regulates protein translation and cell growth. Therefore, increased amino acid transport in prostate cancer cells may drive the mTORC1 signaling pathway to promote unrestrained cellular proliferation.in vitro cell growth, cell cycle and signaling pathway analysis as well as in vivo bioluminescent tumor growth assays and clinical data correlations with experimental data from primary human cancers.We have used the androgen dependent (LNCaP) and androgen independent (PC-3) prostate cancer cell lines to test the role of amino acid transport in cancer. We have used both amino acid uptake inhibitors (BCH) and shRNA knockdown to test the effects on in vivo.Our results have demonstrated that prostate cancer cells coordinate the expression of LAT1 and LAT3, thereby increasing leucine uptake to promote mTORC1 signaling and cell growth [These data show that prostate cancer cells respond to the demand for increased amino acids through an integrated pathway, leading to increased amino acid transporter expression and cell growth. Furthermore, LAT3 and LAT1 may provide novel therapeutic targets in early and late stage prostate cancer respectively."} +{"text": "We present an interesting image showing sequential loss of anterograde, and subsequently, retrograde conduction during radiofrequency ablation of an accessory pathway. We discuss the possible mechanisms and prior literature concerning this interesting finding. A young male with a history of recurrent palpitations and preexcitation in the electrocardiogram underwent an electrophysiology study. A right free wall accessory pathway with anterograde and retrograde conduction and inducible orthodromic atrioventricular reentrant tachycardia were demonstrated. Mapping was performed with a 4 mm tip ablation catheter during atrial pacing and showed early ventricular activation at 10'O clock on the tricuspid annulus. At the best site, local bipolar ventricular activation was about 50 ms ahead of surface delta wave onset, a sharp pathway potential was seen and unipolar electrogram showed a qS pattern , panel ALoss of preexcitation was seen two seconds after radiofrequency ablation was started , panel BDifferential effects on anterograde and retrograde accessory pathway conduction by radiofrequency ablation -3 and cr"} +{"text": "Tetralogy of fallot (TOF) is the most common cyanotic congenital heart disease. Anesthesia induction is a challenging issue in these patients due to the risk of worsening hypoxemia following decrease in pulmonary blood flow. We evaluated the effect of three anesthetic induction regimens on the arterial oxygen saturation (SaO2%) in children with TOF.Seventy six children aged 50 days to 15 years old with TOF, scheduled in Nemazee and Faghihi hospitals to undergo elective cardiac surgery during 1385-1388 were randomly divided into 3 groups to receive three anesthetic induction agents including ketamine , ketamine and halothane for gas induction. SaO2% and heart rate were recorded before induction and thereafter every 1 minute during induction of anesthesia till 10 min post-induction.There were not significant differences between three groups regarding pattern of changes in SaO2% during 10 min post-induction. All three groups showed an increase in SaO2% committed over 6th minute but this pattern was not seen after that time. In addition, there were not significant differences among groups according to heart rate in the study period.It seems that anesthesia induction in TOF patients with ketamine IV and IM and halothane did not have significant adverse effects on SaO2%. Indeed, oxygenation during induction may offset other possible adverse effects of induction drugs on SaO2%. Tetralogy of fallot (TOF) is the most common cyanotic congenital heart disease (CCHD) which characterized by a large single ventricular septal defect (VSD), an overridden aorta, obstruction to right ventricular out flow and right ventricular hypertrophy. Hypercyanotic attacks in TOF patients are common and characterized by sudden spells of hypoxemia due to a sudden decrease in pulmonary blood flow (PBF). Surgical interventions in these patients include total correction and some palliative procedures to increase PBF transiently.Anesthesia management in these patients require understanding of the events or drugs that influence the magnitude of right to left (Rt to Lt) shunting of blood, right ventricular outflow obstruction and resulting PBF and arterial oxygen saturation SaO2%). Both intravenous (IV) and inhalational anesthetic regimens have been recommended for induction of anesthesia in patients with TOF. Ketamine is the suggested anesthetic drug for induction due to its beneficial cardiovascular effect on increasing systemic vascular resistance (SVR) and resulting decreased Rt to Lt shunting via VSD and improved oxygenation due to increased PBF. On the other hand, ketamine can cause infundibular cardiac muscle spasm via its positive inotropic effect. Anesthetic induction with volatile agents such as sevoflurane and halothane are acceptable but have some adverse and/or beneficial effects on PBF and SaO2%. A sma A sma4] The adverse effect of ketamine on PBF (increased PR) may be opposed by its increasing effect on SVR and decreased magnitude of Rt to Lt shunt. Animal investigations suggest that the effects of ketamine on PR are complex. It increased concentrations of epinephrine and norepinephrine in plasma and increased PR in isolated rat lungs.11] Howe Howe11] In contrast, positive inotropic effect of ketamine on myocardium may cause infundibular spasm of cardiac muscle and following increasing Rt to Lt shunt magnitude, decreased PBF and SaO2%. Intravenous and intramuscular injections are acceptable routes of administration of ketamine in anesthesia induction. It seems that adverse or beneficial hemodynamic effects of IM ketamine appear more gradual than IV ketamine. We compare these two routes of administration of ketamine in TOF patients regarding effect on SaO2% and HR and observed that both groups of ketamine (IV and IM) had similar increasing course in SaO2% in comparison to awaken values during the first 10 min after induction of anesthesia.Gas induction with volatile agents is another acceptable anesthetic induction regimen in TOF patients. We found the similar increasing course of SaO2% in TOF children anesthetized with halothane as gas induction in comparison to ketamine groups despite its significant vasodilatory effect. A large decrease in SVR would be expected to result in a net increase in Rt to Lt shunting and aggravated hypoxemia. However, decrease in SaO2% was not noticed in any of the patients in halothane group. This finding was also reported by William and co-workers when compared the effect of halothane and IM ketamine on SaO2% in children with cyanotic congenital heart disease.There are some explanations for this observation. In our patients, tissue oxygen utilization decreased as the children progressed from awaken premedicated state to the anesthetized situation. This resulted in both a decrease in PBF requirement and an increase in mixed venous oxygenation. Keeping the PBF constant, a rise in mixed venous oxygenation has been shown to increase SaO2% in cyanotic congenital heart disease. Another SaO2% is affected by several factors in patients with cyanotic congenital heart disease: (i) The inspired fraction of O2 (FIO2); (ii) The extent of Rt to Lt shunting; (iii) O2 consumption; (iv) Cardiac output; and (v) The mixed venous oxygen tension. If the mThere are some limitations and defects in our study including unrecorded blood pressure of patients during study period and possible inaccuracies or errors in SaO2% values. Some events such as hypothermia, low cardiac output, drug induced vasoconstriction, motion artifact (more common with finger probe than ear or forehead probes monitoring) and sever hypoxia may cause bias or imprecision in SaO2% values. We used finger probe pulse oximetry while movement of patients was inevitable in children especially at the beginning of anesthesia induction.In conclusion, our study indicated that the use of the IV or IM ketamine and halothane as anesthetic induction agents did not significantly affect SaO2% and all were hinge to especial attention regarding hemodynamic stability."} +{"text": "Surgery for acute aortic dissection is always challenging, especially in the case of cerebral malperfusion. Question remains open, whether to perform only aortic repair or to reconstruct arch vessels if there flow is severely impaired by disease process.This is a case of acute aortic dissection with multiple tears, occluding innominate artery and causing brain and right hand malperfusion. Patient underwent successful emergent surgery in deep hypothermic circulatory arrest with antegrade cerebral protection for complete replacement of innominate artery and hemiarch. Complete innominate artery was replaced during cooling period on 22\u00b0C.There is still no consensus about arch vessel repair in the case of complicated aortic dissection. This technique is promising as it do not increase circulatory arrest time and it is safe and reproducible for patients with cerebral malperfusion."} +{"text": "Amazona auropalliata) by moving individuals within and across dialect boundaries in Costa Rica. One juvenile translocated across dialect boundaries altered its contact call to imitate the acoustic form of the local call six weeks post-release. In contrast, four adults translocated across dialect boundaries returned to their original capture site within 120 days, while five cross-dialect translocated adults who remained at the release site did not alter their contact calls. Translocated individuals were observed to show some segregation from resident flocks. The observation of vocal imitation by the juvenile bird supports the vocal imitation, whereas the behavior of adults is more consistent with the reduced dispersal hypotheses. Taken together, our results suggest that both post-dispersal learning by juveniles and high philopatry in adults could explain the stability of vocal dialects in the face of immigration and gene flow.Studies of avian vocal dialects commonly find evidence of geographic and acoustic stability in the face of substantial gene flow between dialects. The vocal imitation and reduced dispersal hypotheses are alternatives to explain this mismatch between vocal and genetic variation. We experimentally simulated dispersal in the yellow-naped amazon ( Zonotrichia leucophrys nutalliVocal dialects are a common manifestation of vocal learning, in which variation in calls or songs is much lower within than between geographic regions. Vocal dialects in birds were first described in white-crowned sparrows, Two main hypotheses have been proposed for the long-term maintenance of vocal dialects. The reduced dispersal hypothesis proposes that individuals rarely immigrate across dialect lines because they encounter fitness costs when associating with individuals using different acoustic signals. A reduction in fitness could result from assortative mating or social association by call type by the immigrants in the new dialect Current evidence provides mixed support for the two hypotheses. Studies of mating preferences using individuals from different dialects typically find a stronger positive response between same dialect individuals Amazona auropalliata) is a medium size parrot that exhibits three distinct vocal dialects in Costa Rica with well defined boundaries The yellow-naped amazon and with permission from the land owners of our study sites.We captured parrots in May to August from 2006\u20132008 and from January to May in 2009 using canopy mist-nets in trees adjacent to four roosts in privately owned lands . IndividWe located radio-collared individuals using directional antennas and approached them within 50 m for observations. We attempted to obtain behavioral observations and vocal recordings from radio-collared individuals at least four times a week after release. Due to the wide-ranging movements of this species, not every individual was located during all periods. When a radio-collared individual was located, we alternated five minute focal observations on the radio-collared individual and on an unmarked individual in the same flock. We recorded every instance of aggressive acts (displacement and attack) or affiliative acts directed towards all translocated birds. To obtain an estimate of each individual\u2019s original call structure, we recorded radio-collared birds\u2019 contact calls immediately after release while individuals were flying, foraging or socializing with a Sennheiser ME67 shotgun microphone on a Marantz PMD670 or PMD660 solid state recorder. We continued to obtain vocal recordings throughout the study period to quantify change in vocal structure over time by recording individuals whenever located.a priori into a first phase (<6 weeks post-release) and a second phase (>6 weeks post-release). Six of the cross-dialect translocated individuals from the Southern dialect fulfilled the minimum requirements of being tracked for >6 weeks and having at least one good quality recording bout per phase ; this pattern was observed regardless of whether or not they imitated local calls or given . No differences were observed between translocated and resident birds in either receiving or giving affiliative behaviors .We conducted 98 (n\u200a=\u200a2 birds) and 135 (n\u200a=\u200a10 birds) focal observational periods for resident and translocated birds respectively. Few direct social interactions were observed, with social behaviors recorded in only 27% of the observational periods. Aggressive acts were observed in only 3% of the observational periods; across-dialect translocated and resident birds did not differ in the number of aggressive acts received . Some of these translocated individuals returned to the capture site 30 km away in their original dialect, a significant distance given observed foraging distances in the species. These patterns suggests that adult yellow-naped amazons are highly philopatric and either unable or unwilling to learn the calls of a new dialect. It is also consistent with the idea that individuals that disperse across dialect boundaries might suffer from reduced fitness due to the lack of call sharing with local individuals; however, it is important to note we did not directly measure the relative fitness of translocated birds. A high degree of philopatry suggests that movement by adults across dialects is limited, as predicted by the reduced dispersal hypothesis. Such philopatry may be driven by natal habitats preference Recent population level processes in the yellow-naped amazon could be affecting the relative contribution of each of the mechanisms in maintaining vocal dialects in this species. Although indirect estimations of dispersal in the yellow-naped amazon point to considerable flow of individuals across dialect boundaries Melopsittacus undulatusThe adaptive value of learning local calls by immigrant birds is uncertain. The password hypothesis, which suggests that imitation of a group\u2019s call is an honest signal of local experience that allows an immigrant to gain group benefits Our results suggest that the occurrence of vocal imitation may be associated with age-specific constraints or costs. The fact that the only vocal-imitating individual in our study was a juvenile suggests that the motivation for vocal learning may be related to the strength of previous social ties and individual\u2019s age. Dispersal typically occurs in juvenile animals Although parrots are known for being open-ended learners, data collected from adults in this study would suggest that learning capabilities are reduced or lacking in adults, as found in other species previously thought to be true open-ended learners Sound Example S1Sound file: Example of Northern dialect vocalizations at the recipient site.(AIF)Click here for additional data file.Sound Example S2Sound file: Example of Southern dialect vocalizations at the source site of translocated birds.(AIF)Click here for additional data file.Sound Example S3Sound file: Example of original Southern dialect vocalization of the translocated juvenile in the 1st phase prior to altering vocalization.(AIF)Click here for additional data file.Sound Example S4Sound file: Example of Northern dialect vocal imitation by the translocated juvenile in phase two after altering vocalization.(AIF)Click here for additional data file."} +{"text": "We regret any confusion or inconvenience this error may have caused.In the article \"Trends in Selected Chronic Conditions and Behavioral Risk Factors Among Women of Reproductive Age, Behavioral Risk Factor Surveillance System, 2001-2009,\" the title and some column heads for Table 3 were incorrect. The title should have read \"Prevalence Ratios for Risk Factors and Chronic Conditions Among Adult Women of Reproductive Age, BRFSS, 2001-2009,\" and the column heads should have read \"Crude Ratio,\" \"Adjusted Ratio,\" and \"Adjusted Ratio P Value.\" The correction was made to our website on October 24, 2011 and appears online at"} +{"text": "Despite scientific and technological advances, Legionella continues to be a major threat to patients in healthcare settings. To be effective the control of Legionella need a competent attention based on a correct risk assessment and on consequent operative choices.A tool called Legionella Priority Control [LPC], was designed to be comprehensive, easily implementable and able to define intervention priorities in the healthcare setting.LPC was developed within a project funded by the Italian Ministry of University through the following stages:- Review of literature to identify all the possible critical points in Legionella control;- Classification of all selected critical points in three independent areas of interest: management, water distribution system and patient susceptibility;- Identification of several critical points for each area;- Split of each critical point into items to be answered in form of questionnaire;- Classification of items through a multidisciplinary consensus into 4 risk categories /medium risk, 3 - probable Legionella presence (evidence based)/high risk, 4 - demonstrated Legionella presence/very high risk);- Analysis of the 3 areas scores in order to define a general risk score and a structure related risk score;- Use of a risk matrix to define final priorities.LPC includes:- A check list of items which, once filled in, allows the definition of existing problems ;- A risk matrix which allows definition of an initial list of priorities;- A subjective feasibility score which allows definition of priorities tailored to the local setting.LPC offers to the infection control practitioner an easier yet comprehensive way to define priorities for Legionella control tailored to the local healthcare setting.None declared"} +{"text": "We have also used this approach to show that nucleosomes shift during mitosis to cover the transcription start sites of genes. This may be a mechanism to bookmark genes for expression in the following G1 phase of the cell cycle.DNA methylation and nucleosome positioning work together to generate chromatin structures that regulate gene expression. Nucleosomes are typically mapped using nuclease digestion but we have developed a method (NOMe-Seq) that uses a GpC methyltransferase (M.CviP1) and next generation sequencing to generate high resolution footprints of nucleosome positioning genome wide using less than 1 million cells while retaining endogenous DNA methylation information from the same strand. We have developed a novel bioinformatics pipeline that shows a striking anti-correlation between nucleosome occupancy and DNA methylation at CTCF regions that is not present at promoters. The extent of nucleosome depletion of promoters is directly correlated with expression level and can accommodate multiple nucleosomes. Genome wide evidence also shows that expressed non-CpG island promoters are nucleosome depleted. Because the method obtains DNA methylation and nucleosome positioning information from the same DNA molecule we can also use this method to track the relationship between nucleosome occupancy and DNA methylation during differentiation and mitosis. These experiments have shown that nucleosome occupancy precedes DNA methylation in the OCT4 promoter which is consistent with the idea that nucleosomal DNA is the preferred substrate for"} +{"text": "Although a small subset of patients with co-inherited cystic fibrosis and other hemoglobinopathies have been reported, this patient developed early hematologic and pulmonary complications that were more severe than the previous cases. To assess pulmonary co-morbidities, we used infant pulmonary function testing through the raised volume rapid thoracoabdominal compression technique as both an established study of early cystic fibrosis and also as a newer study of mechanism for early sickle cell lung disease. This further serves as the first report of the raised volume rapid thoracoabdominal compression technique to determine raised volume forced expiratory flows and fractional lung volumes in a patient with a hemoglobinopathy.This is the first published report of a young girl with co-inherited sickle cell-\u03b2+ thalassemia developed severe hematologic complications during periods of cystic fibrosis pulmonary exacerbations and weight loss. Because cystic fibrosis and sickle cell-\u03b2+ thalassemia both confer distinct patterns of pulmonary disease, infant pulmonary function testing with the raised volume rapid thoracoabdominal compression technique was used to define respiratory pathophysiology and guide treatment options. Infant pulmonary function testing data demonstrated moderate-to-severe lower airways obstruction, moderate air trapping, and no evidence of restrictive lung disease.A 2-year-old African-American girl with co-inherited cystic fibrosis and sickle cell-\u03b2+ thalassemia. Although this is an original case report on a unique patient, this case highlights the need to evaluate early respiratory pathophysiology in a broader population of young patients with hemoglobinopathies and screen those at risk for early pulmonary co-morbidities.Infant pulmonary function testing with the raised volume rapid thoracoabdominal compression technique guided therapy in this patient with cystic fibrosis and sickle cell-\u03b2 Multiple early interventions have been instituted in an attempt to reduce the incidence and severity of respiratory complications associated with SCLD and CF that often start in infancy. These have included newborn screening programs for CF and hemoglobinopathies along with referral to subspecialty physicians and established care programs focused on minimizing anticipated complications. In recent years, infant pulmonary function testing (IPFT) using the raised volume rapid thoracoabdominal compression (RVRTC) technique has been applied as a clinical tool and research aid to assess early airway obstruction and air trapping in infants with CF . -12. thalin vitro assessment of airway secretions exposed to different therapeutic modalities may help guide therapy. DNase has been shown in both CF and ACS to aid in airway clearance [The rationale for concurrent pulmonary and hematologic complications is probably multifactorial and may be explained due to a combination of pathophysiologic effects. Intraluminal airway secretions are known to have different cellularity in CF bacteriologic bronchitis compared to plastic bronchitis associated with ACS. If airway secretions are available from sputum collection or bronchoalveolar lavage, formal pathologic assessment may guide therapeutic options based upon cellular and/or fibrinous appearance. In addition, learance -15. Addilearance ,16, alonlearance althoughAnother complicating factor was the need for high dose opioid narcotics during pain crises. Opioid narcotics may further contribute to generalized histamine release that may contribute to airway hyperresponsiveness or increased airway edema. Use of aggressive narcotics for pain to allow airway clearance was favored over the possibility of worsened lower airways obstruction due to a direct opioid effect. Analgesic alternatives including intravenous ketorolac and transdermal lidocaine were utilized in the interest of minimizing opioid dosing.The approach to managing an acute respiratory decline proved difficult in this patient due to the multiple disease processes involved. IPFT was utilized to help delineate various possible pathophysiologic mechanisms: lower airways obstruction, regional air trapping, and/or restrictive lung disease. Children with SCD are more likely to have wheezing and symptoms of airway hyperresponsiveness during episodes of ACS and have a higher incidence of asthma than the general population. Data supports an association between asthma and increased frequency of ACS in older patients with SCD however the temporal relationship has not been defined . As thesAlthough the co-inheritance of CF and a hemoglobinopathy is relatively rare, the use of infant lung function testing as detailed in this report may be of benefit to a broader population of young children with SCD. With the advanced technology allowed by the RVRTC technique, young patients with SCD may have their lung function followed longitudinally. Should this population have evidence of early lung disease, IPFT could become an additional screening tool to allow for earlier interventions that could minimize the progression of chronic lung disease in SCD.Since the patient described in this report is a minor, the authors have obtained written informed consent from the patient\u2019s legal guardian for publication of this case report and accompanying images. A copy of this written consent is available for review by the Editor-in-Chief of this journal.+ thalassemia; SCD: Sickle cell disease; SCLD: Sickle chronic lung disease; VOE: Vaso-occlusive events.ACS: Acute chest syndrome; CF: Cystic fibrosis; IPFT: Infant pulmonary function testing; RVRTC: Raised volume rapid thoracoabdominal compression; S-\u03b2thal: Sickle cell-\u03b2The authors declare that they have no competing interests.KS collected and analyzed infant pulmonary function case data and was the major contributor in writing the manuscript. CT provided critical manuscript revision and insight pertaining to the hematologic manifestations of this case. JV was involved in intellectual critique of the clinical pulmonary aspects of this case. SD provided critical manuscript revision pertaining to IPFT performed on this patient. SPC collected and analyzed infant pulmonary function case data, contributed to certain aspects of the written manuscript, and offered critical revision pertaining to pulmonary aspects of this case, in particular as it related to IPFT. All authors read and approved the final manuscript."} +{"text": "Autism is well known as a complex developmental disorder with a seemingly confusing and uncertain pathogenesis. The definitive mechanisms that promote autism are poorly understood and mostly unknown, yet available theories do appear to focus on the disruption of normal cerebral development and its subsequent implications on the functional brain unit. This mini-review aims solely to discuss and evaluate the most prominent current theories regarding the pathogenesis of autism. The main conclusion is that although there is not a clear pathway of mechanisms directed towards a simple pathogenesis and an established link to autism on the symptomatic level; there are however several important theories which appear to offer an explanation to how autism develops. It seems probable that autism\u2019s neurodevelopmental defect is \u2018multi-domain\u2019 in origin and is hence distributed across numerous levels of study . A more definitive understanding of the pathogenesis could facilitate the development of better treatments for this complex psychiatric disorder. Autism is one of the pervasive developmental disorders (or autism spectrum disorders) and a commonly diagnosed condition in child and adolescent psychiatry, which was initially described by the psychiatrist Leo The aetiology of autism is complex and is likely to be of multifactorial descent encompassing both genetic predisposition and environmental factors . PotentiThe mechanisms that lead to autism are at best poorly understood, however they do centre around the disruption of normal cerebral development and its subsequent implications on the functional brain unit . Numerous neuropsychiatry papers attribute the pathogenesis of autism specifically to \u2018localised\u2019 anomalies , which have the potential to detrimentally effect CNS structure and function . The folOne specific theory emphasises that early brain overgrowth and neural overconnectivity are key in the pathogenesis. It is speculated that excess neuron numbers may promote defects in neural patterning and wiring, with subsequent over energetic short-distance cortical interactions hindering long-distance interactions that communicate between critical brain regions. Suggesting a more diffuse pathology across a multitude of cortical areas rather than just a single confined defect. This neuroanatomical anomaly has the potential to underlie the deficits in socio-emotional and communicational function observed in autism .Conversely another related theory postulates that the neuropathological basis of disrupted cognition in autism is linked to reduced intracortical connectivity which subsequently promotes a lower degree of information integration across multiple cortical regions . WhetherCerebral cortical malformations observed in autism may result from defective neural migration to the cerebral cortex during the first 6 months of gestation. The subsequent cortical dysgenesis seen in autism patients with disturbed neural migration includes among others; thickened cortex, high neuronal density, poor grey-white matter boundaries and ectopic grey matter . This hyThe theory of unbalanced excitability-inhibitory networks in autism also carries certain credibility. Available research concludes that chromosomal rearrangements involving GABA receptor gene clusters are heavily implicated, promoting abnormal CNS system excitability and function. The additional role of glutamate receptors in synaptic maintenance also shows potential relevance to autism\u2019s pathophysiology . Yet it The abnormal assembly of synapses and dendritic spines may also be a contributing factor in autism\u2019s pathogenesis. Notably, it has been found that autistic brains feature increased numbers of long, thin, dendritic spines . CrucialThe topic of neuroimmune disturbance and its impact on the pathogenesis of autism has also been widely researched. Immune disorders noted in autistic patients include among others: abnormal T-helper cell type 1/2 responses, general suppression of cell-mediated immunity, subnormal levels of CD4+ lymphocytes, imbalance of antibody levels and reduced natural killer cell functions . AutoimmCalcium signalling may also contribute to autism via the important nature of activity-dependant calcium influx into neurons which subsequently regulates (via transcription) numerous cortical excitatory synapses . AlteredThe favourable mirror neurone system theory links neuropathology to autism on a more symptomatic level. The mirror neurons themselves are pre-motor and parietal cells of the cerebral cortex that fire action potentials not just when we are in action or motion, but when we observe others performing similar actions. These neurons essentially \u2018mirror\u2019 the behaviour of others and provide a suitable physiological mechanism for the great scope of social behaviours and skills that humans implement . Thus, mirror neurone dysfunction could be the central deficit observed in autism leading to the profound social and communication difficulties experienced . AlthougAside from the above theories there are several other lines of research into the pathogenesis of autism. One such theory involves decreased levels of apoptosis (programmed cell death) since subnormal levels of Bcl-2 and p53 protein have been documented in the cortices of autistic brains . WhetherIt is clear from the above mentioned theories that there is not a direct pathway of mechanisms pointing towards a simple pathogenesis and an explanation of autism\u2019s rather distinctive set of symptoms. It seems much more favourable to summarise autism\u2019s pathogenesis as being multidimensional and uncertain but yet promising with regard to some of the aforementioned theories. These include: disturbed neural connectivity; defective neural migration; unbalanced excitatory-inhibitory networks; abnormal dendritic morphology; neuroimmune disturbances; calcium signalling and the mirror neurone system theory. Further research in the field of autism pathogenesis should focus on elucidating whether exactly the excitation abnormality is due to GABA receptor activity suppression or glutamate receptor activity elevation or defects in calcium signalling?; whether the neuroimmune pathogenic element is due to suppression of immune responses or more related to autoimmune insults?; why the mirror neurone theory cannot explain the great array of behavioural and psychosocial symptoms seen in autism?; what specific relevance disturbed neural connectivity has to autism, and whether there is a valid anti-apoptotic link? and finally how all the mentioned pathogenic factors interact together to produce a deficit in the functional brain unit?Overall it seems most likely that the anomaly encountered in neurodevelopment that leads to autism is not merely \u2018localised\u2019 to a single functional neural defect as many theories have suggested, but more distributed across numerous levels of study becoming a \u2018multi-domain\u2019 disorder . Further"} +{"text": "APACHE II scores and Glas\u00ae) and scores (APACHE II and GAS) were calculated for each of these patients. The mortality rates were compared with the national average [This retrospective audit included patients who underwent emergency open AAA repair and were admitted to our ICU, over a period of 1 year (November 2008 to November 2009). These patients were identified from our local ICU database had undergone emergency (ruptured) repair. Seven patients (20%), including two females, died in the ICU. There is an increase in mortality with increasing APACHE II scores Figure . The samAPACHE II scores seem to be more predictive of our unit AAA mortality rates than GAS scores. We aim to apply these scores to a larger dataset and also determine possible reasons for improved survival."} +{"text": "The Cambridge Structural Database ( CSD ) contains a large amount of molecular structure data Much of this data has previously been extracted in histogram form and provided in the Mogul program. Histograms however have several disadvantages e.g. they are not smooth, they depend on bin widths and bin end points.Kernel density estimators do not bin data and have no end points but centre a kernel function at each data point and smooth kernel functions will generate smooth density estimates . A diffiIn this work kernel density estimation is used to generate probability density functions ( pdfs ) for bond length, bond angle and torsion angle histograms derived from the CSD. Gaussian kernels are used for bond length and bond angle data and a von Mises kernel is used for the torsion angle data . The res"} +{"text": "The diagnosis of septic arthritis in children remains challenging despite reasonable evidence for the use of laboratory tests in diagnosis. There is also limited data on the diagnostic utility of gram stain microscopy in diagnosis. We therefore aim to establish the diagnostic utility of gram stain and predictive clinical and laboratory features of paediatric septic arthritis.We conducted a retrospective review of all patients of 16 years and under that underwent aspiration with or without washout of suspected septic joint from January 2005 to March 2011. Cases were defined as any patient with an organism identified on microbiology culture. The association between clinical features, laboratory results, operative findings and gram stain examination were compared against final culture results with chi-square test and Mann Whitney test (for non-parametric data).Twenty three paediatric patients were identified during the time period, of which 9 (39%) had positive culture. There was no statistically significant data to show that clinical features or operative findings were predictive of final results. Of the blood test results found, CRP has statistical significant rise (p=0.01) in culture positive septic joints compared to culture negative with CRP >65 rendering sensitivity 100% and specificity 78%. Gram stain microscopy showed 33.3% sensitivity and 100% specificity.Presenting features, operative findings and most laboratory tests are unhelpful in predicting diagnosis for septic arthritis. However a high CRP (>65) may be useful diagnostic tool proven by high sensitivity. Positive gram stain is strongly predictive of culture positive septic arthritis although diagnosis cannot be excluded on the basis of negative gram stain. Further research should be conducted using CRP and gram stain alongside each other as diagnostic utility for paediatric septic arthritis as demonstrated by the research."} +{"text": "Xie et al. in their article entitled \u201csleep drives metabolite clearance from the adult brain\u201d in Science 342: 373\u2013377) , provide: 373\u2013377Key to understanding their paradigm is an exploration of the differences between physiological clearance of cellular waste from the central nervous system and peripheral tissues. Systemic waste interstitial proteins are transported via lymph vessels and the systemic circulation to the liver for degradation . The brain vivo using two-photon imaging and electromyography (EMG) determined sleep-wake status, with a sleep state practically defined as an increased power of slow waves and an awake state associated with a relative reduction in slow waves and increase in power of fast waves .On entering sleep there was a \u201crobust influx\u201d of fluorescent CSF tracer along the periarterial spaces, subpial region, and into the brain parenchyma . Sleep wOn awakening, CSF tracer influx reduced dramatically by 95% . The staThese results were replicated in a cohort of mice, examined firstly whilst awake. The intracisternal infusion of fluorescent CSF tracer was associated with only minimal influx into the brain. Then on induction of anesthesia there was a rapid influx of CSF tracer along the periarterial spaces into the brain parenchyma, similar to naturally sleeping mice .They postulate that CSF influx into brain parenchyma is not simply a function of arterial pulsation and blood pressure but rather the interstitial space volume is dynamic and contracts during wakefulness and expands during sleep .+) method whereby TMA+ was delivered via an iontophoresis microelectrode in the vicinity of a TMA+-sensitive microelectrode stationed approximately 150\u2009\u03bcm away in the interstitial space. In the paradigm a small interstitial space results in reduced TMA+ dilution after delivery and thus higher levels of residual TMA+ are detected by the sensing electrode sleep disorder merely a manifestation of alpha synucleinopathies or could it be implicated in pathogenesis? Why do some patients with Parkinson\u2019s disease derive sleep benefit? What causes fluctuating arousal in dementia with Lewy bodies (DLB)? Does sleep-wake state influence volumetric analysis in MRI? Given the invasive nature of the experimental methodologies used they could not be reproduced in humans in their current format, however they could provide a platform for the investigation of clearance of other neurotoxic proteins involved in neurodegeneration, using a Parkinson\u2019s disease mouse model for example. Furthermore, they may be useful for investigation of potential drugs to promote the clearance of neurotoxic proteins, whether they be currently used adrenergic antagonist drugs or novel agents. When we try to encourage a good nights sleep for our patients perhaps we are doing them more good than we thought."} +{"text": "The recent article by Williams and Kriegsfeld was highFor example, patients with obstructive sleep apnea (OSA) usually demonstrate low levels of free testosterone. These decreased levels in OSA patients are remarkably independent of the body mass index. For instance, the mean free testosterone level in patients with severe OSA is about 69\u2009pg/ml in comparison to 75\u2009pg/ml in those without any OSA therapy. Similar improvements in erectile dysfunction can be seen following successful treatment of OSA. A significant decline in prolactin levels is also noticed following the institution of CPAP therapy thus having a further positive impact on male fertility.Sleep apnea may also be seen with decreased reproductive function and adjunct obesity in females. Such patients may have underlying polycystic ovarian syndrome (PCOS; Randeva et al., Individuals with acromegaly may have coexistent OSA and decreased reproductive function (Giustina et al., Females with irregular menstrual cycles have almost twice the incidence of sleep abnormalities such as \u201clight sleep stages\u201d in comparison to those with regular menstrual cycles (Hachul et al., The above examples clearly illustrate a higher incidence of reproductive dysfunction and sleep disorders in patients with the above conditions and the need to regularly screen such patients for sleep disorders."} +{"text": "The design of an effective T cell based vaccine relies on determining the most highly constrained regions of the HIV proteome.Using publicly available crystal structures from the Protein Databank (PDB), we performed a systematic analysis of the local microenvironment of every amino acid within the HIV Proteome for which structural data was available (65.8%). Structural constraint parameters included involvement in protein secondary structure, relative solvent accessibility, and involvement of amino acid side chains in intermolecular interactions . Calculations of these constraints were carried out using validated methods of protein structure analysis and distance geometry, with weighted values cumulatively summed to provide a constraint score for every amino acid.Amino acids with a higher constraint score were observed to strongly correlate with low values of entropy within viral sequences from every clade of HIV. Analysis of constraint score variation across the HIV proteome reveals that the p24 capsid protein to be the most highly interconnected and constrained. Evaluation of amino acids within known HLA-restricted epitopes further elucidated a preference of controller alleles for buried and interconnected amino acids, while progressor alleles predominantly targeted exposed and non-connected amino acids. Thermodynamic stability analysis further demonstrated a strong correlation with amino acid constraint and change in predicted Gibbs' Free Energy.Our analyses reveal that evaluation of local amino acid microenvironments represents a novel method for the determination of constraint within the HIV proteome, setting the stage for more robust targeting of these constrained regions and enhanced immunogen design."} +{"text": "The gain in computational efficiency of CG methods often comes at the expense of non-protein like local conformational features. This could cause problems when transitioning to full atom models in a hierarchical framework. Here, a CG potential energy function was validated by applying it to the problem of loop prediction. A novel method to sample the conformational space of backbone atoms was benchmarked using a standard test set consisting of 351 distinct loops. This method used a sequence-independent CG potential energy function representing the protein using The prediction of protein structure to atomic level resolution and the design of de novo proteins with large scale backbone sampling are largely unsolved problems although there has been a great deal of progress in recent years. Both problems require the ability to rapidly sample a large number of backbone conformations. Sampling protein conformational space using full atom models can be prohibitively computationally expensive so a variety of different approaches have been developed to reduce the search space. This can be achieved by using coarse-grained (CG) protein models, by assembling backbone models from short fragments taken from known protein structures or by a combination of both of these methods.Coarse-grained models have been increasingly used for modelling large biomolecules over long time scales due to the computational efficiency provided by these methods While CG models in the past were mostly used as toy models to study the general principles of protein folding The accuracy of full-atom reconstruction depends on the level of coarse-graining de novo methods. Loop modelling is also important for computational protein design applications where the backbone structure needs to be redesigned in order to satify some functional constraints de novo structure prediction methods due to their high degree of structural variability and a weaker sequence-structure relationship compared to secondary structure elements. While many loop prediction methods have been previously described For most protein sequences, experimentally determined structures of homologous sequences are available and can be used as templates for accurate modelling In this paper we validate a previously developed sequence-independent CG potential energy function The method presented here was compared to RAPPER et alet alThe loop prediction benchmark test proposed by Fiser et alThe RAPPER Sidechains were added to the generated backbone loops using the default Rosetta simulated annealing repacking algorithm and the whole loop (including the backbone) was then gradient minimised in a iterative manner as described in the Methods. The lowest energy loop was taken as the prediction. The results were comparable to existing methods and in some cases better . OverallAs a measure of backbone structure quality, the Ramachandran distribution was calculated for all generated loop decoys . Most fePredictions were carried out on a new loop set taken from recently desposited structures with low sequence or structural similarity to solved structures deposited in the PDB before April 2012 , bond moves where two A backbone potential energy function was used for conjugate gradient minimisation after rough backbone atom positions were added to Initial loop Sidechains were added using the default Rosetta simulated annealing repacking algorithm and the loop atoms gradient minimised in the Rosetta all atom potential energy function using a PyRosetta \u00c5), contained chain breaks/missing residues, or had no loops in the range 8\u201312 were removed leaving a final set of 4 structures and 7 loops were BLASTed 7 loops . Loops wFile S1Supporting figures and tables.(PDF)Click here for additional data file."} +{"text": "AbstractCalosoma (Coleoptera: Carabidae) is a group of large, sometimes ornate beetles, which often voraciously attack caterpillars. Many studies have reported Calosoma beetles being highly conspicuous during defoliator outbreaks. Based on observations of individual beetle behavior, patterns of activity density and phenology we provide a hypothesis on how environmental cues may synchronize Calosoma activity with periods of high defoliation. We have observed that adults of Calosoma frigidum construct underground burrows similar to those reported to be created by larvae for pupation. We propose that small increases in soil surface temperature caused either by defoliation events or decreased albedo of blackened, burned soil causes beetles to leave their underground burrows and begin foraging. Indirect support for this hypothesis comes from high levels of adult Calosoma frigidum collected in relatively small patches of burned forest (<200m2) relative to the surrounding mosaic of unburned forest shortly after a prescribed surface burn.The genus Calosoma frigidum Kirby, also known as \u201cThe Cold Country Caterpillar Hunter\u201d , was positively impacted by burning, salvage harvesting and a herbicide treatment applied after harvesting, however catches of both these species in control stands were extremely low . The 10 ha stand chosen for the study was dominated by trembling aspen (Populus tremuloides Michx.) and balsam poplar with the understory dominated by green alder (Alnus crispa (Aiton))and river alder.\u00a0 Further information on the EMEND site and ground beetles responses can be found in This study took place at the Ecosystem Management by Emulating Natural Disturbance (EMEND) research site in northwestern Alberta, Canada in which burning was clearly evident and four additional areas with no evidence of burning, and installed two pitfall traps per area on June 5th, 2000. Our pitfall traps consisted of an outer permanent (1L) cup, acting as a sleeve to maintain the pit and minimize disturbance at checks, and a removable (500ml) inner cup using the aov function. Tukey HSD post-hoc tests were carried out using the HSD.test function in the agricolae library of R than in the unburned patches or anywhere in the stand the following year . The highest catch rates were recorded in the July 11th collection in 2000 and for the June 21 in 2001 . This would also explain why the highest levels of capture were recorded at 50% green tree retention the 2 years following harvest at EMEND . Calosoma populations could initially build-up during an outbreak through a combination of both localized immigration from neighboring stands and emergence of resident adults from underground burrows within the stand. If defoliator populations remain high, both immigrants and residents could conceivably accumulate in the stand in adult burrows in the soil. When an outbreak collapses and there are no signals to emerge and run, these adults could lay dormant for several years. As the next outbreak unfolds, increased soil temperatures would effectively synchronize adult beetles with an increasing source of prey as well with potential viable congeners to facilitate reproduction. The proportions of these individuals that emigrate following the collapse or remain in adult burrows as a \u2018sit and wait\u2019 strategy should vary with outbreak frequency, but such data remain to be collected.PageBreakCalosoma beetles, using specific evidence from Calosoma frigidum in NW Alberta, Canada. Increases in the catch rate and activity patterns of Calosoma beetles have been recorded during defoliation events, herbicide treatments, mechanical canopy thinning and soil charcoal deposits following fire. All these events have the potential to warm the soil surface in a sustained way, and we suggest that such warming could cue beetles to emerge from their underground burrows and begin foraging. Although we acknowledge that this evidence is somewhat circumstantial, we hope that it offers useful insight to inspire further work about what triggers of foraging behavior in this very interesting beetle.We have offered a possible mechanism for initiating the foraging response in dormant"} +{"text": "Independent component analysis (ICA) is a signal processing technique using higher-order statistics to extract signals by unmixing signal mixtures. McKeown et al. introducSICA has become widely used for fMRI analysis since its original application to fMRI 15 year ago approach, which assesses FN overlap by detecting the involvement of the same functional clusters in different FNs, to assess FN overlap from fMRI acquired at resting condition (Yan et al., The knowledge of FN overlap and their task-related modulation has important theoretical implications in understanding brain functional organization, and can help reconcile inconsistent data existing in the literature. For example, fMRI studies often show task-induced deactivation in the medial PFC and precuneus/PCC during tasks demanding working memory or cognitive control. It has been hypothesized that these brain regions are the core of DMN and associated with intrinsically generated, task-independent mental activity, e.g., task-unrelated thoughts (Fox et al., In addition to the theoretical significance, the knowledge of FN overlap has important practical implications in how to assess brain functional organization. For example, seed-based approach is another popular technique for assessing FNs. This approach generates FNs by extracting the timecourse of fMRI signal from a selected voxel or ROI and then correlating this timecourse with the timecourses of other voxels or ROIs. Therefore, it does not consider different timecourses of different source signals from the same voxel or ROI, and thus may generate findings different from those by sICA, because some source signals from different ROIs may show significant correlations while the signal mixtures from these ROIs may show no significant correlations. Another example is that fMRI studies using GLM based analysis often report different changes in fMRI signal at some brain regions during task performance between patients and healthy controls. This difference in signal mixture could be due to changes in one or more source signals. The knowledge of changes in a specific source signal may provide further insight into etiology and neuropathology of the disease under investigation relative to the knowledge of changed signal mixture.In summary, sICA can separate source signals from the same brain regions, and this specific capacity has not been fully exploited in most published fMRI studies. Two recent studies explicitly employed this capacity and revealed new insight into functional activity hidden from a GLM based analysis. These new insights have significant theoretical and practical implications in understanding brain functional organization. Thus, we recommend that investigators should use sICA or some other approach which can account for overlap cancelation to perform secondary analysis of published fMRI studies analyzed by GLM alone, and use both GLM and sICA in future fMRI studies for a more complete understanding brain functional organization."} +{"text": "Diabetic foot complications are acknowledged as the leading cause of amputation and diabetes-related hospitalisation. Podiatry-led multi-disciplinary clinics are recognised as important strategies to improve diabetic foot outcomes. Data suggests diabetic foot complications are increasing at a faster rate than diabetes is being diagnosed. A reliable, competent workforce is urgently required to adequately manage patients with diabetes foot complications. Clinical training is known to have a beneficial impact on diabetic foot ulcer outcomes. As the basis for podiatry clinical training occurs at the undergraduate level in Australia, the development of students equipped with best practice skills to manage the growing population of diabetic foot complications is essential. The aim of this paper is to develop, implement and evaluate a student led \u201chigh risk foot\u201d clinic and evaluate its impact on undergraduate learning and diabetic foot outcomes.In January 2011 a designated Queensland University of Technology (QUT)/ Queensland Health (QH) \u201chigh risk foot\u201d podiatry led student clinic will commence on the QUT campus with support from the QUT/QH clinical educator and other senior podiatrists. To complement the student clinical experience, placements in QH \u201chigh risk\u201d foot services and exposure to simulation training in high risk foot management will be fostered. Primary evaluation will be undertaken quarterly and involve addressing satisfaction and clinical competencies. Surveys will be obtained from students, clinical supervisors and placement supervisors. Further evaluation of students\u2019 diabetic foot clinical indicators and patient outcomes will also be evaluated.The presentation will review the initial planning and implementation of the \u201chigh risk foot\u201d student clinic at QUT. The first three month evaluation will be presented at the conference.The explosion of diabetic foot complications requires a well skilled workforce. Undergraduate education remains at the core of podiatry learning in Australia. The development of student \u201chigh risk foot\u201d clinics may be an innovative and effective strategy to meet the needs of Australia\u2019s diabetic foot complications. It is envisaged two years worth of data will be analysed and presented to the 2013 Australasian Podiatry Conference."} +{"text": "The DNA sequence 5\u2019CG (CpG) is self-complementary and can exist in three major chemical forms depending on the modification status of its cytosine moiety. To understand the functional significance of the CpG dinucleotide, we study proteins that bind either its methylated or unmethylated form. These proteins are likely mediators of CpG signalling that influence chromatin modification and thereby genome activity. The local density of CpG varies dramatically within genomic DNA. In the bulk genome CpG is rare and highly methylated, but in so-called \u201cCpG islands\u201d (CGIs) it is dense and usually non-methylated. A signature histone mark at non-methylated CGIs and also at transcriptionally active genes is trimethylation of histone H3 lysine 4. We are exploring the mechanisms by which DNA sequence features that are shared by all CGIs influence this and other epigenetic marks. In contrast, proteins that interact with methyl-CpG are thought to promote gene silencing by recruiting transcriptional corepressors. In particular mutations in the gene for the methyl-CpG binding protein MeCP2 cause the autism spectrum disorder Rett Syndrome. By studying MeCP2 we are learning about its partner proteins that mediate effects on chromatin. These findings allow us to evaluate competing models for MeCP2 function and they illuminate the biology of DNA methylation and the molecular basis of this neurological condition."} +{"text": "Aspirin reduces the absolute risk of death or dependence following an acute ischaemic stroke. An associated increase in risk of haemorrhage may cause considerable harm. We hypothesised that patients at a high predicted risk of further thrombosis or a low risk of haemorrhage would experience greater absolute benefit from aspirin. In addition we explored the assumption that absolute benefit increases with baseline risk.We applied formal prediction methods to the three largest randomised trials of aspirin in patients with acute ischaemic stroke. We developed new prediction models for early events (14 day thrombosis and haemorrhage) and for long term functional outcome (six month death or dependence) and internally evaluated their performance. We calculated the absolute risk reduction of death or dependence with aspirin within quarters of predicted patient risk from early events across trials and pooled the results using random effects meta-analysis.Simple prediction models discriminated early events poorly (AUROCC 0.56 and 0.60) but were moderate at discriminating long term death or dependence (AUROCC 0.77). There was no evidence of greater benefit or of harm from aspirin across the sixteen defined subgroups of predicted risk nor was there any evidence that absolute benefit increased linearly with baseline risk. The best estimate of the effect of aspirin was the overall absolute risk reduction of death or dependence of 1% (95%CI: 0% to 2%) in all risk groups.We found no evidence to support targeting aspirin to acute ischaemic stroke patients with a high predicted risk of thrombosis or a low predicted risk of haemorrhage. The modest absolute benefit of aspirin was similar across predicted patient risk of death or dependence."} +{"text": "Heart hand syndromes are characterized by radial abnormalities and associated defects in the heart. We here describe an extremely rare heart hand syndrome known as Baller-Gerold syndrome. Baller-Gerold syndrome characterized by a combination of preaxial upper limb reduction defects and craniosynostosis described separately by Baller in 1950 was nameA five-month-old child, born at term, a product of consanguineous marriage was referred for echocardiographic evaluation following detection of grade II/VI ejection systolic murmur in the left upper parasternal area along with a wide split second heart sound. Echocardiographic evaluation revealed situs slitus, levocardia, and atrioventricular and ventriculoarterial concordance with intact ventricular septum and a 15\u2009mm ostium secundum atrial septal defect with lefThough there are many well-described heart hand syndromes characterized by deformities of the radius bone and congenital heart defects like thrombocytopenia absent radius syndrome and HoltIn an infant presenting with upper limb skeletal abnormalities and a cardiac defect, one should always look for craniosynostosis resulting in prominent and palpable cranial sutures to avoid missing the diagnosis of this rare syndrome reported to be associated with the syndrome."} +{"text": "There is an ongoing debate about when to use efavirenz (EFV) or nevirapine (NVP) for first line antiretroviral treatment (ART) in developing countries, fuelled by EFV's link with teratogenicity in rats and NVP's risk of hepatotoxicity with increasing CD4 levels and it's interactions with Rifampicin. This paper compares the effectiveness of these two drugs in a multicentre adult cohort of ART patients attending public health facilities in South Africa.A retrospective cohort analysis of routine data on 27350 ART na\u00efve adults initiated between March 2004 and March 2007 at 56 public health sites across 4 provinces was completed. Stata 9 was used to conduct analyses which included Kaplan Meir survival analysis, logistic regression, generalised estimating equations and Cox proportional hazard models.Median follow up time was 9.3 months and median baseline CD4 count 113 cells/mL (IQR= 57-165). Multivariate analyses showed patients receiving first line regimens containing EFV to have been more likely to suppress virologically both at 6 months and at any time between 6 and 36 months , less likely to change regimen and more likely to die . A subset analysis of 18527 patients with pregnancy and tuberculosis status reported showed no difference in hazard rates for death between the two groups .This data shows superior results for patients on EFV with respect to all outcomes except death. Retrieval of missing data about pregnancy and tuberculosis status may push this last association toward the null. Protease inhibitors are an alternative to non-nucleoside reverse transcriptase inhibitors (NNRTIs) for first line use; but are currently too costly. There is an urgent need for further research into currently available NNRTIs; as well as the development of new antiretrovirals for resource depleted settings. In the interim the developing world needs to increase access and bring down the cost of existing drugs and implement more efficient treatment strategies."} +{"text": "Recent research has demonstrated that spontaneous brain activity is not random. At the level of large-scale brain systems, the ongoing activity measured with functional MRI reflects the organization of many highly coherent functional networks . SynchroSynchronization likelihood could prove helpful for detection and estimation of functional relations between electrodes within and without brain regions."} +{"text": "Parental interviews highlighted the array of circumstances which can exist for parents of multiples enrolled in trials. Issues discussed with professionals included:Neonatal trials which include preterm babies often recruit multiples (twins or higher order births). For such trials, methodological decisions must be made regarding recruitment and randomisation of multiples. Enrolment can take place in complicated and challenging situations which are compounded if one or more babies die. In the BRACELET Study \u2022 o \u2018Group' randomisation \u2022 o Randomisation time-points (siblings may become eligible at the same or different times)\u2022 Analysis of outcomes for multiples \u2022 Policies on feedback of trial results to parents Including multiples in neonatal trials is important, but interviews from the BRACELET Study show the need to consider the complexity of the issues raised in the conduct of trials on both scientific and compassionate grounds."} +{"text": "Our recently published review article containe\"Results of a randomized trial investigating mortality in 3,141 children with severe febrile illness and impaired perfusion in sub-Saharan Africa surprisingly showed higher mortality at 48 hours and at 4 weeks in the group that did not receive any fluid boluses compared with two groups resuscitated with albumin or saline 38.\"should have instead read:lower mortality at 48 hours and at 4 weeks in the group that did not receive any fluid boluses compared with two groups resuscitated with albumin or saline 38.\"\"Results of a randomized trial investigating mortality in 3,141 children with severe febrile illness and impaired perfusion in sub-Saharan Africa surprisingly showed The authors sincerely regret this error.TJG has received research funding from Fresenius Kabi and honoraria from Hospira and Baxter. KB and RHT declare that they have no competing interests."} +{"text": "This systematic review and meta-analysis was conducted to provide a clear guide for the best evidence based approach to conservatively manage Achilles tendinopathy. A recent systematic review provided a descriptive summary of the evidence with the absence of a quality assessment of the study methodologies.A systematic literature search was conducted to identify randomised, controlled studies using pain or function outcome measures which were published in English. Quality assessments were performed using the Modified PEDro rating scale. Standardised mean differences for each intervention and pooled data were calculated using Review Manager software.The search strategy yielded 22 studies whose methodological quality rated between 2 and 12 out of 14 on a Modified PEDro rating scale. Studies were grouped by their primary intervention . Follow up times were predominantly within three months. A meta-analysis was able to be performed for two intervention comparisons; shock wave therapy (SWT) versus eccentric exercise (EE) and laser therapy versus a sham laser therapy, where both groups received an EE program. The pooled data found a moderate significant effect favouring SWT and small significant effect favouring laser therapy. Of the eleven studies evaluating EE, six reported that EE had superior results to the control intervention.This systematic review emphasises the need for a consistent use of valid and reliable outcome measures, larger subject numbers and longer follow-up times to build a larger body of high quality evidence and a greater opportunity to perform meta-analyses using studies that examine conservative interventions for Achilles tendinopathy. The current evidence supports the use of EE, SWT and laser therapy for the management of Achilles tendinopathy."} +{"text": "Arhgef1\u2212/\u2212) that display constitutive pulmonary inflammation and decreased lung elastance reminiscent of emphysema. The results from this study show that sub-chronic second hand smoke exposure leads to significantly increased numbers of airspace leukocytes in both healthy and mutant animals. While sub-chronic cigarette smoke exposure is not sufficient to induce changes in lung architecture as measured by mean linear intercept, both groups exhibit a significant decrease in lung elastance. Together these data demonstrate that even sub-chronic exposure to second hand smoke is sufficient to induce pulmonary inflammation and decrease lung elastance in both healthy and diseased animals and in the absence of tissue destruction.Exposure to second hand tobacco smoke is associated with the development and/or exacerbation of several different pulmonary diseases in humans. To better understand the possible effects of second hand smoke exposure in humans, we sub-chronically (4 weeks) exposed mice to a mixture of mainstream and sidestream tobacco smoke at concentrations similar to second hand smoke exposure in humans. The inflammatory response to smoke exposures was assessed at the end of this time by enumeration of pulmonary leukocyte infiltration together with measurements of lung elastance and pathology. This response was measured in both healthy wild type (C57BL/6) mice as well as mouse mutants deficient in the expression of Arhgef1 ( Second hand smoke exposure has been associated with a variety of negative health outcomes in a number of epidemiological studies exposure to a mixture of mainstream and sidestream tobacco smoke. We evaluated this exposure in both wild type (C57BL/6) and 3 and 190 ppm, respectively as previously described as previously described and exposure [filtered air (FA) vs. second hand smoke (SHS)] a Two-Way ANOVA was performed on all appropriate data sets.JMP\u00ae was used for all statistical analysis. For data sets including four groups a One-Way ANOVA was performed. If significance was detected with the One-Way ANOVA, a Arhgef1\u2212/\u2212 mutant mice were exposed to either FA or SHS for 4 weeks after which time the number of pulmonary leukocytes in lung tissue and airspace were enumerated and characterized.To explore the effects of second hand tobacco smoke exposure on the pulmonary compartment we exposed mice to a mixture of sidestream and mainstream smoke at concentrations similar to what an individual would experience through second hand smoke exposure were also increased in the BAL of SHS exposed animals compared to FA controls for both genotypes, although this increase was only significant in Arhgef1\u2212/\u2212 cohort and exposure (FA vs. SHS). Because of the increase in leukocytes recovered from SHS exposed animals, we were able to further identify the lymphocyte populations present in these samples from both C57BL/6 and S Figure . This inS Figure . Neutropt Figure . We perfs Figure . These fs Figure .Arhgef1\u2212/\u2212 lungs exhibit significant airspace enlargement compared to the C57BL/6 lungs consistent with our previous report and exposure (FA vs. SHS). Therefore, we conclude that the pathways leading to decreased lung elastance in the Arhgef1\u2212/\u2212 mice are independent of the pathways leading to decreased lung elastance after SHS exposure. The results from these experiments demonstrate that sub-chronic SHS exposure is sufficient to significantly decrease lung elastance in both mouse strains examined exposure to SHS is sufficient to induce significant increases in the number of airspace leukocytes present in either healthy or mutant lungs.This study documents that 4 week sub-chronic exposure to second hand cigarette smoke does not lead to measureable leukocyte infiltration within lung tissue but does result in airspace inflammation and decreased lung elastance. The inability of sub-chronic smoke exposure to promote lung tissue inflammation or changes in airspace structure is consistent with previous reports . Initially we were surprised to observe a change in lung mechanics with a relative short smoke exposure protocol. A previous report failed to observe any changes in the lung mechanics of C57BL/6 mice exposed to smoke for 6 months . A recent review included both of these measurements performed in the pallid mouse strains lungs may be more sensitive to detecting modest changes in lung elastance than measurement performed at lower pressures.Significant decreases in lung elastance are evident in the C57BL/6 and Arhgef1\u2212/\u2212 mice and C57BL/6 mice across these ages reveal the most pronounced differences between strains occur at 6 months of age (Hartney et al., In addition to the differences in methods of measurements it is also worth noting the difference in duration of smoke exposure protocols between the studies, 4 weeks versus 6 months (Guerassimov et al., Comparing the lung mechanics and airspace architecture between all four groups suggests that changes in murine lung mechanics can occur in the presence or absence of changes in lung architecture. Note the SHS exposed C57BL/6 mice have lung elastance values lower than the na\u00efve Arhgef1-deficient mice despite the lack of alteration in airspace structure, as measured by mean linear intercept Figures and 5. TArhgef1\u2212/\u2212 and wild type mice to SHS exposure. Using a Two-Way ANOVA, no interaction is found between genotype and response to cigarette smoke exposure in any of our data sets. Thus, we conclude that these pathways, Arhgef1 and cigarette smoke exposure induced responses appear to occur independently of each other.We have previously described a signaling pathway that operates in pulmonary myeloid cells that leads to the production of pro-inflammatory mediators and is normally inhibited by Arhgef1 (Hartney et al., In conclusion the data presented here demonstrate that sub-chronic SHS exposure is sufficient to induce a significant increase in airspace leukocytes and decrease in lung elastance in both healthy animals and a mutant mouse strain with pre-existing pulmonary inflammation and pathology. This change in lung mechanics appears to occur as a result of processes that can be independent of changes in airspace structure. Further examination of the pathways responsible for SHS induced changes in lung mechanics may identify novel targets for restoring or retaining lung elastance in human subjects exposed to second hand smoke.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "A review of 100 patients between January 2009 - June 2010 undergoing foot surgery requiring an ankle or popliteal block were assessed on their personal perceptions of the injection procedure in terms of expectations of pain and intra-surgical experience. Comparative results are made with those who had anaesthetist led sedation and an ankle block only. Preliminary results indicate those patients selected/elected for the local anaesthetic had a favourable experience, the majority would be happy to repeat their procedure under similar conditions. Sedation patients had a very similar patient experience profile. This small study supports the view that good practice for Podiatric Surgical patients is to offer an anaesthetist led sedation or general anaesthesia option but it is not essential or desirable for all patients."} +{"text": "Drosophila provide an excellent opportunity to study cilia formation and function as the sensory neurons are the only ciliated somatic cells, and so cilium mutations are recognisable by sensory behavioural defects. A reverse genetic screen was conducted to implicate candidate genes in ciliary processes. Candidates were selected from a chordotonal sensory neuron transcriptome analysis and refined by consideration of gene ontology, existence of human homologues, and participation in protein-protein interaction networks . P element insertions and Gal4/UAS induced RNAi were used to disrupt gene function. Candidate genes were screened using a climbing assay to select for proprioceptive and gravitactic deficiency, indicating disrupted chordotonal neuron function. Positive hits from the screen are being investigated by observing the ciliary dendrite structure of pupal antennae and larval chordotonal neurons. Positive candidates are also tested for fertility defects due to disrupted flagellum formation during spermiogenesis. Our analysis has identified several novel conserved ciliary genes. Particular attention has been given to genes regulated by the transcription factor fd3F, which is required for the transcription of several genes involved in cilium motility."} +{"text": "The proper management of infectious conditions often requires optimized clinical decisions based on a complex decision making process. Computers are able to aid this process only if the medical knowledge used is machine readable, which requires scrupulous preparation and modeling. Here we describe and evaluate a dynamic model framework for Urinary Tract Infection (UTI).We used Bayesian Belief Network (BBN), a probabilistic model that can represent conditional dependencies between variables. Its dynamic characteristics allow modeling complex relationships between medical processes and symptoms. For the medical information we turned to clinical practice guidelines for UTI and abstracted as well as modeled their content with standard ontological concepts. Alltogether we extracted several hundreds of rules describing diagnostic and therapeutic relationships. Finally we validated these rules against the original guidelines using an open-source reasoner (Euler) and a battery of test cases.We found that the results proposed by the reasoner coincided with that of the clinical guideline in 97% while allowing a much higher complexity with the possibility of freely adding and combining diagnostic and therapeutic parameters.We conclude the BBN models in infectious conditions deliver not only accurate decisions but their application may also be warranted to effectively deal with large number of combinations of conditions when making complex decision. Similarly it also allows dynamic addition of non-medical parameters for a better optimized decision making process.None declared."} +{"text": "Motivational support is crucial for the success of smoking cessation. Significant others are a proven source of that support ,2. As faThis was a randomized population-based intervention study at the smoking cessation clinic of Evaggelismos hospital. We monitored for people that are related in the initial screening stage. Couples included life partners, family members or very close friends. Smokers were in all motivational stages. All participants underwent the same intervention with motivational and behavioural components in the smoking cessation groups and received medical consultation and pharmacotherapy (Varenicline). We compared so far the smoking cessation rates of 25 \"couples\" and 50 randomized smokers that followed our smoking cessation program.We found that participants that joint the COSO quit smoking in a higher rate (58%) than of smokers (38%). Within the dyad the person more motivated to quit smoking was usually the first to quit. Among couples that quit smoking, men were more successful (63%) than women (49%).We conclude that higher smoking cessation rates were obtained in COSO joining our smoking cessation program."} +{"text": "The eating disorders (EDs), anorexia nervosa (AN), and bulimia nervosa (BN) are severe psychiatric disorders of unknown etiology. EDs usually begin during adolescence and occur most commonly in females or excessive food intake , which may interact with dopamine circuits that are vulnerable to the effects of high or low food intake. Another important factor in triggering dopamine system alterations is puberty. Estrogen has a variety effects on eating behavior via neurotransmitter function and may play an important role in ED risk .The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Inactivation of the cGKI gene is associated with anemia in mice . It has cGKI-/- and cGKI rescue mice have significant lower erythrocyte levels than their control littermates, lower hematocrit and hemoglobin concentrations and elevated reticulocyte numbers. The spleens of these mice are massively enlarged. cGKI mutant mice clearly suffer from iron deficiency due to decreased plasma iron levels and the lack of iron in the spleen and liver. To maximize intestinal iron absorption in cGKI -/- mice hepcidin mRNA levels are down regulated in the liver. The iron regulatory hormone hepcidin binds to the only known cellular iron exporter ferroportin which leads to its degradation and therefore inhibits the iron transport via ferroportin ,4. IL-6 Feeding the proton pump inhibitor (PPI) esomeprazol (18 mg/kg chow) did stop the intestinal bleeding as shown by Haemoccult test and prolong the survival of cGKI-/- mice. Perls Prussian blue tissue staining for ferric iron showed that PPI treatment rescues the iron deficiency in the spleen of cGKI -/- mice and normalized serum iron levels. These results were confirmed by western blot analysis for the iron storage protein ferritin light chain. In the liver ferritin levels and hepcidin mRNA levels are increased. Treatment with PPI normalizes red blood cell counts, hematocrit and hemoglobin levels as well as reticulocyte numbers and prevents splenomegaly. Serum IL-6 concentration was not affected by PPI treatment of cGKI mutants.These data indicate that anemia and splengomegaly in cGKI-/- and cGKI rescue mice is caused by iron deficiency anemia due to intestinal blood loss and can be rescued by treatment with esomeprazol."} +{"text": "Temporal lobe epilepsy remains a significant cause of disabling morbidity. Patients that are refractory to medical treatment are often considered for invasive surgery to remove the epileptogenic focus originating from the structures of the medial temporal lobe. A range of surgical options now exist with varying degrees of efficacy. The goal of any surgical intervention is to minimize disruption of adjacent normal cortical tissue while removing the offending structures. MRgFUS provides the opportunity to perform this task in an elegant fashion. We tested the feasibility of this approach in a cadaveric model.Cadaveric skulls were filled with custom fitted thermo reactive gels and thermocouples placed along anatomical areas of interest along the skull base to assess temperature rises during the sonication process using the InSightec ExAblate Neuro System (650 Khz). The volume of temporal lobe structures typically removed by standard surgical excision was mapped on 3D volumetric MRI scans and overlaid onto the thermal gels to provide an appropriate target for ablation. Temperature maps in the treatment volume were created for various sonication parameters and temperature effects at key skull base structures were monitored with thermocouples.By adjusting sonication parameters it was possible to create a lesion of therapeutic significance in the required temporal lobe treatment volume using thermoreactive gels in cadaveric skulls. Temperature rises seen at the periphery of the target volume did decrease compared to more medial structures. Treatment of temporal lobe epilepsy with MRgFUS appears feasible based on this laboratory model. Thoughtful planning using a disconnection strategy or highly focused sonications on key anatomical epileptogenic foci may be required due to the large volume required to ablate an equivalent volume for an anatomic temporal lobectomy. Blocking of distant FUS beams may be required to circumvent temperature rises in the anterolateral temporal fossa."} +{"text": "Invasive endomyocardial biopsy is an important tool to diagnose cardiomyopathy. But the diagnostic yield is low because the procedure is performed \u2018blind' using, particularly in processes characterized by heterogeneous myocardial involvement. MRI tissue characterization techniques, using late gadolinium enhancement or T1 mapping for example, can identify affected regions and guide biopsy. We performed realtime MR guided transcatheter endomyocardial biopsy using a novel active visualization MR conditional bioptome in swine.An active visualization MR conditional bioptome was designed and built for transcatheter endomyocardial biopsy. All materials were chosen to minimize MR imaging artifacts and to minimize device heating during use in the MR environment. Active visualization of the bioptome shaft was achieved by embedding a loopless dipole RF antenna. The bioptome was tested in a phantom and in vivo in swine to perform transcatheter right and left ventricular endomyocardial biopsy under realtime MR guidance.The bioptome shaft was actively visualized under realtime MR imaging. The jaws appeared as a distinct signal void in the phantom and in vivo and by a SBIR contract to MRI Interventions."} +{"text": "Migraine with typical aura (MTA) and migraine without aura (MO) are common neurological disorders with complex inheritance. Recent efforts have identified 12 independent loci at which single nucleotide polymorphisms (SNPs) have shown to confer risk of migraine (Antilla V. Nat.Genet 2013). The objective of this study was to investigate whether these SNPs could be replicated in a Danish clinic based migraine sample and to test if the risk-alleles are associated with severe migraine traits.Semi-structured migraine interviews based on a validated questionnaire, blood samples and genotyping were performed on 1806 unrelated migraineurs from the Danish Headache Center, Glostrup Hospital. The control group consisted of 6415 individuals with no history of migraine. Association analyses were carried out using logistic regression. The primary endpoints were regarded as a proxy for severe migraine traits and tested against the 12 SNPs and a combined genetic risk score.Five out the 12 previously reported loci were replicated in our sample. The association was significant only for those with migraine without aura. Following correction for statistical testing, five SNPs showed nominal association with the severe migraine traits:\u2018early onset of migraine\u2019,\u2018prolonged migraine attacks\u2019 and \u2018many lifetime attacks\u2019.Our study confirms previous findings on the association of several SNPs with migraine. The association results with severe features, albeit nominal, suggests that the previously reported migraine risk alleles may be implicated in the development of severe migraine characteristics.No conflict of interest."} +{"text": "Left ventricular non compaction is a relatively rare congenital disorder characterized by prominent trabeculations and intertrabecular recesses with the potential for thromboembolism, arrhythmias, and sudden cardiac death as adverse effects. Echocardiography has traditionally been employed as the primary mode of imaging; however, with the advent of cardiac magnetic resonance as a more precise imaging technique, the disorder known as left ventricle non compaction is becoming more broadly defined with increasing recognition of right ventricle (RV) involvement.This report describes a 52-year-old Caucasian female with new onset atrial fibrillation with an unusual finding of left ventricular non compaction and right ventricular dysfunction on transthoracic echocardiogram with preserved left ventricular ejection fraction. Cardiac magnetic resonance imaging demonstrated a disproportionately affected right ventricle, with apical free wall dyskinesis.This case illustrates the unique occurrence of left ventricular non compaction with preserved ejection fraction alongside RV free wall dyskinesis and RV systolic dysfunction. The significance of this is yet unknown given the paucity of existing literature. This report serves to highlight the vast heterogeneity within left ventricular non compaction as we are better able to delineate this disorder using increasingly sophisticated imaging techniques. The estelopment . LVNC iselopment . CMR is A 52-year-old Caucasian female with new onset atrial fibrillation and a family history of premature sudden death underwent transthoracic echocardiography (TTE) to evaluate the presence of structural heart disease. A 12 lead baseline EKG demonstrated no evidence of an Epsilon wave nor repolarization abnormalities and there was no evidence of premature ventricular contractions nor ventricular tachycardia on 48\u00a0hour Holtor monitoring. TTE demonstrated abnormal thickening of the left ventricular (LV) apex with trabeculations and recesses, consistent with LVNC (Figure\u00a0Leung et al. demonstrated RVEF <35% in half of patients, and these similarly had lower LV ejection fractions [Stacey et al. noted that those patients with reduced RV ejection fractions were at higher risk for adverse outcomes, including congestive heart failure, thromboembolism, and arrhythmias, even after having adjusted for LVEF [This case illustrates the unique occurrence of LVNC with preserved ejection fraction alongside RV free wall dyskinesis and RV systolic dysfunction. Although it is plausible that the RV abnormalities identified on CMR may be associated with another underlying diagnosis of arrythmogenic RV dysplasia, there were no other clinical or electrocardiographic features supportive of this diagnosis. While the significance of RV involvement in LVNC is yet unknown given the paucity of existing literature, this report serves to highlight the vast heterogeneity within LVNC as we are better able to delineate this disorder using increasingly sophisticated imaging techniques. Several recent studies have demonstrated RV involvement, though focus has been on concomitant LV systolic dysfunction: a study of 56 patients looked at right ventricular involvement in LVNC, and focused specifically on the presence of RV systolic dysfunction, RV non-compaction, and myocardial fibrosis. It was found that RV systolic dysfunction was present in 16% of patients; however, all had associated LV systolic dysfunction [for LVEF . A studyfor LVEF . These fThis report serves to highlight the vast heterogeneity that seems to be expanding as we continue to increase our understanding of LVNC in an era of more sophisticated imaging techniques including CMR.Written informed consent was obtained from the patient for publication of this Case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal."} +{"text": "Hospital outbreaks of epidemiologically important pathogens are usually caused by lapses in infection control measures and result in increased morbidity, mortality and cost. However, there is no benchmark to compare the occurrence of hospital outbreaks across hospitals.It is worth considering setting up such surveillance data for hospital outbreaks, similar to surgical site and device related infections as found in the National Healthcare Safety Network (NHSN) report and International Nosocomial Infection Control Consortium (INICC) respectively.We implemented proactive infection control measures with an emphasis on timely education of healthcare workers and hospitalized patients about directly observed hand hygiene on outbreak prevention in Queen Mary Hospital (QMH), a teaching hospital. Our benchmarked performance (outbreak episodes per 1 million patient discharges and 1 million patient days) was compared with those of other regional public hospitals without these measures between 2010 and 2014.During the study period, QMH only had 1 hospital outbreak, resulting in 1.48 and 0.45 outbreak episodes per 1 million patient discharges and patient days respectively, which were significantly lower than the corresponding overall rates in 7 acute regional hospitals and that of all 42 public hospitals in Hong Kong .This large study on benchmarked rate of hospital outbreaks per patient discharges or patient days suggested that proactive infection control interventions may minimize the risk of hospital outbreaks.None declared."} +{"text": "Premature ovarian insufficiency (POI) is defined as a primary ovarian defect characterized by absent menarche (primary amenorrhea) or premature depletion of ovarian follicles before the age of 40 (secondary amenorrhea) with hypergonadotropism and hypoestrogenism. POI results in infertility and lifelong steroid deficiency, and is potentially associated with accelerated health risks such as cardiovascular and neurodegenerative disorders and osteoporosis.A large consanguineous Saudi family with three female affected with POI was investigated. All samples including 3 affected and 6 unaffected underwent whole genome SNP genotyping using Affymetric 250K array. Linkage analysis was carried out using HomozygosityMapper and Allegro software. Candidate gene sequencing was performed using ABI3500 genetic analyzer. Whole exome was sequenced in three affected and one normal individual using life technologies Ion Proton sequencer.Linkage analysis mapped the disease phenotype to long arm of chromosome 20. Sequence data analysis of potential candidate genes failed to detect any pathogenic variant. Exome sequencing data analysis identified a deletion mutation in gene \u2018X\u2019 on long arm of chromosome 20. This mutation is perfectly segregating with the disease phenotype in pedigree.We identified a novel gene responsible for POI in Saudi Arabian family. Our findings extend the body of evidence that supports the importance of gene \u2018X\u2019 in the development of ovary and ovarian reserves."} +{"text": "Impact ejected rocks are targets for life detection missions to Mars. The Martian subsurface is more favourable to organic preservation than the surface owing to an attenuation of radiation and physical separation from oxidising materials with increasing depth. Impact events bring materials to the surface where they may be accessed without complicated drilling procedures. On Earth, different assemblages of organic matter types are derived from varying depositional environments. Here we assess whether these different types of organic materials can survive impact events without corruption. We subjected four terrestrial organic matter types to elevated pressures and temperatures in piston-cylinder experiments followed by chemical characterisation using whole-rock pyrolysis-gas chromatography-mass spectrometry. Our data reveal that long chain hydrocarbon-dominated organic matter are unresistant to pressure whereas aromatic hydrocarbon-dominated organic matter types are relatively resistant. This suggests that the impact excavated record of potential biology on Mars will be unavoidably biased, with microbial organic matter underrepresented while metamorphosed, degraded or abiotic meteoritic organic matter types will be selectively preserved. Indigenous, unaltered organic matter has not been conclusively recognised in Martian surface materials despite a number of in situ mission attempts at its detection125With surface reactions proposed as sources of oxidising chemicals on Mars, the importance of sampling depth for effective life detection is recognised7Yet drilling is not the only way to access the Martian subsurface. Natural excavation of the Martian crust by recent impact events has been proposed as a mechanism for obtaining subsurface materials, with depth of sampling correlating with crater size and complexity; small simple craters can access depths of hundreds of meters while large complex craters can sample kilometre depths16If detected, organic matter from once-living organisms is unlikely to represent a complete high fidelity record. Most organic records represent portions of life (e.g. selected diagnostic remnants) that can act as proxies for the complete organisms. Hence, recognising how any selective preservation mechanisms operate is essential for accurate interpretations. The most common example of selective preservation is the persistence of hydrocarbon-based organic materials relative to other structures such as nucleic acids, proteins and carbohydrates during diagenesis in terrestrial materials, and similar processes can be expected to occur on Mars.Although the process of converting biomolecules to geomolecules (organic structures which are stable under geologic conditions) under a range of thermal conditions and timescales is relatively well understood, the possibility of selective destruction of organic structures during impact excavation is less well explored. Cratering events that transport materials from subsurface to surface are associated with great energies and organic structures are known to respond to such influences192324A number of studies have shown that organic matter can be preserved following impact events. Most common are studies intended to investigate the possibility of panspermia, the transfer of life between planetary bodies, which also provide a guide to whether this life would survive planetary bombardment262728et al.et al.Another approach commonly taken is to investigate specific organic compounds under impact-generated pressures in the laboratory. Compounds are typically chosen because they either have been observed in the relevant extraterrestrial environments data for both untreated and treated organic matter. The molecular products of pyrolysis access both the soluble and insoluble organic matter contained within the whole rock. We compared the pre- and post- pressurization state of the various organic matter types to reveal how each type responds to the pressures associated with impact excavation. It was our aim to document those classes of structures which selectively respond to pressure and which are therefore most likely to be involved in sampling bias in impact-ejected rocks. Hence, our focus is on the obvious survival and destruction preferences of general compound classes.Following online pyrolysis, types I and II organic matter produce numerous alkene/alkane doublets that reflect highly aliphatic biopolymers . The pre3839The online pyrolysis of types III and IV organic materials liberates numerous aromatic and phenolic units . AromatiThe relative survivability of aromatic and polar molecules is consistent with reports of fossil biomarkers in terrestrial impacts such as the Haughton and the Ries craters32Our results are in harmony with other published works that indicate the increased resistance of aromatic units to temperature and pressure: aromatic hydrocarbons in the form of polycyclic aromatic hydrocarbons have been extensively studied and found to be stable to pressures and temperatures in excess of those encountered in our studyOur work shows organic assemblages in impact-ejected rocks on Mars could suffer from two stages of preservation bias. During initial burial the more easily degradable components such as nucleic acids, proteins and carbohydrates and their monomers would be progressively lost, leaving a hydrocarbon-rich residue that may contain aliphatic and aromatic units. During impact ejection from the subsurface a second preservation bias could selectively concentrate aromatic and polar molecules relative to their aliphatic counterparts, which are either volatilized or transformed into analytically unamenable macromolecular material. These effects of pressures associated with impact ejection will make detection of organic remnants of microbes more difficult unless their organic matter has been aromatised under the influence of post burial maturation. In the context of Mars, where both biotic and abiotic organic inputs may be relevant, the consequences of such a preservation bias in impact ejected rocks may be to selectively preserve degraded and unrecognizable fossil organic matter or highly aromatic meteorite macromolecules. Examination, therefore, may superficially and incorrectly imply a world dominated by non-biological organic chemistry from meteorite sources.The relationship between the high pressures experienced by impact ejecta and our predicted observation of dominantly aromatic organic matter can be tested by reference to studies of the organic matter in Martian meteorites. All Martian meteorites are impact-ejected fragments of the subsurface. Online pyrolysis of the curated Martian meteorites would be expected to produce aromatic units. Published organic geochemical studies indicate that there is organic matter present in Martian meteorites and that it is dominantly aromatic in nature44The Martian subsurface is more likely to have preserved organic matter than the surface owing to the latter environment being subjected to radiation and oxidation. Impact excavation provides access to the subsurface without costly and difficult drilling. However, the pressure associated with impacts can selectively hide particular types of organic structure from detection using online pyrolysis, with aliphatic hydrocarbons, the most likely indicators of primitive biological organic chemistry, most strongly affected. Impact events which excavate organic records from depth will, therefore, produce a sampling bias: microbial organic matter may be rendered undetectable by impact-associated pressure, while thermally metamorphosed, degraded or meteoritic organic matter could be preferentially preserved. The effects of pressure on the fidelity of organic records of potential past Martian biology must be appreciated during future life detection missions to the red planet.Four organic matter types were selected for the pressure experiments 1455\u22121 vs 35\u2009\u00b0C m\u22121, respectively) and environmental conditions (~1013 and ~25\u2009mbar He respectively) between laboratory- and rover- based pyrolysis instrumentation, the techniques are the same and have similar effects on organic material; the difference in heating rates could affect the distribution of pyrolysis products but this does not change our interpretation\u22121. The pyrolysis products were introduced to an Agilent 6890 gas chromatograph using split injection at a 50:1 split ratio and an inlet temperature of 250\u2009\u00b0C. Separation was performed on a 30\u2009m J&W Scientific DB-5MS Ultra Inert column. The oven temperature program comprised a start temperature of 50\u2009\u00b0C held for 1\u2009minute, followed by a ramp of 4\u2009\u00b0C min\u22121 to 310\u2009\u00b0C where the temperature was held for 20\u2009min. Helium column flow was 1.1\u2009ml min\u22121. Post-separation compound identification took place using an Agilent 5973 inert Mass Selective Detector, which collected data over a scan range of m/z\u2009=\u200950 to 550. Assignments are made by considering the elution order against published work and comparing mass spectra against a standard library (NIST08).Py-GC-MS is a common technique used for organic detection on MarsHow to cite this article: Montgomery, W. et al. The nature of organic records in impact excavated rocks on Mars. Sci. Rep.6, 30947; doi: 10.1038/srep30947 (2016)."} +{"text": "The Standardized Uptake Value (SUV) from FDG PET-CT is a simplified quantitative measure used for characterization of tissues suspected for malignancy. Its rationale is based in augmented glucose consumption of oncologic cells although, many other benign pathologies, could show elevated values leading to error.The purpose of this presentation is to demonstrate that SUV value continues elevating in malignant pathology while in benign disease will decrease over time.We prospectively acquired delayed images in patients with focal increase of metabolic activity without clear morphologic pathology, or failing to show increase metabolic activity in sites with morphological findings of probable oncologic origin. We have acquired delayed evaluation in 45 patients with suspicious lesions.The most prominent findings were: 11 patients with focal hypermetabolic sites in gastrointestinal tract who demonstrate significant SUV decline on delayed images, avoiding unnecessary colonoscopies, while another 17 patients have significantly increased SUV on delayed images, correlated with polypoid lesions at colonoscopy. 7 patients with falling SUV on delayed images in adnexal hypermetabolic foci proved to be of functional origin at ultrasound follow-up, 5 patients with descending SUV at delayed images in hypermetabolic foci in the neck proved to be inflammatory on follow-up and 5 patients that SUV value continues elevating on delayed images, secondary or primary origin was confirmed.Dual time point PET-CT has proven to be an important tool for proper characterization of questionable lesions that would lead to costly mistakes or unnecessary diagnostic studies."} +{"text": "The role of resting state functional networks in epilepsy is incompletely understood. While some pathologic diagnoses have been shown to have maintained but altered resting state connectivity, others have implicated resting state connectivity in disease progression. However little is known about how these resting state networks influence the behavior of a focal neocortical seizure.Using data taken from invasively monitored patients with intractable focal neocortical epilepsy, we evaluated network connectivity ) as it relates to neocortical seizure foci both in the interictal and ictal states.Similar to what has been shown in the past for sleep and anesthesia, electophysiologic resting state networks that are defined by this slow cortical potential covariance maintain their topographic correlation structure throughout an ictal event. Moreover, in the context of focal epilepsy in which the seizure has a specific site of onset, seizure propagation is not chaotic or random. Rather, the seizure (reflected by an elevation of high frequency power) preferentially propagates along the network that contains the seizure onset zone.Taken together, these findings further undergird the fundamental role of resting state networks, provide novel insights into the network-influenced behavior of seizures, and potentially identify additional targets for surgical disconnection including informing the location for the completion of multiple subpial transections (MSPTs). The brain is becoming increasingly understood as a dynamically interconnected compilation of functional networks. These networks possess both resting state and active state profiles which can be recognized by a number of signal detection modalities including electrocorticography (ECoG) and functional magnetic resonance imaging (fMRI) There are reasons to suspect a relationship between resting state networks and epilepsy. First, pyramidal cells are the majority of cells in the neocortex To better understand this relationship between resting state networks and dynamic pathologic processes, we studied patients with intractable neocortical epilepsy requiring intracranial electrocorticographic (ECoG) recordings. Distinct from the structural disease process associated with AD, investigating epilepsy provides a unique model of how network connectivity is impacted by an acute event such as a seizure while also providing insight into how those networks influence the evolution of that pathologic process. This is due to the high level of both spatial and temporal resolution available through invasive recordings. The one-centimeter spatial distribution of the electrode arrays placed on the surface of the brain allows for adequate assessment of the spatial organization of network topographies. Moreover, the broad frequency scale accessible through local field potentials enables one to monitor multiple distinct physiologies. Specifically, the infraslow rhythms of less than 0.5 Hz, known as slow cortical potentials (SCPs) directly correlate with the resting state networks identified with functional MRI and the higher frequency rhythms, greater than 70 Hz, described as \u201cfast ripples\u201d have been associated with seizure propagation The study protocol was approved by the Human Research and Protection Organization at Washington University School of Medicine. All patients provided written informed consent for study participation.Three patients undergoing invasive monitoring for characterization of their seizure foci were included in this study. Exclusion criteria included the absence of drug-resistant, focal neocortical epilepsy Following a period of 24\u201348 hours for immediate post-surgical recovery in the intensive care unit, patients were transferred to the Epilepsy Monitoring Unit for seizure monitoring. All anti-epileptic medications were discontinued during this period. Intracranial electrode density and locations were chosen solely based on clinical indication. Physiologic recording in combination with video monitoring ensued uninterrupted for a period of 5\u20137 days to allow optimal characterization of patients' seizure foci.ECoG signals were acquired from the implanted electrode array at a sampling frequency of 512 Hz using a differential amplifier . This amplifier was equipped with a 0.5 Hz hardware filter for clinical recording purposes. Prior to analysis, a preliminary assessment of these clinically acquired signals was performed to compare them to signals obtained via an amplifier used exclusively for research purposes at a sampling frequency of 1200 Hz that did not include any hardware or software filters. The covariance patterns were similar between the two data sources see thereforTwenty minute time epochs were selected for analysis and were identified as either interictal or ictal epochs. See The seizure focus electrode was selected as the seed for the \u201cseizure network.\u201d This electrode was defined by the treating neurologist as the electrode first to demonstrate ictal discharges that ultimately manifest into seizure activity. When multiple contiguous electrodes were identified as equally contributing to seizure onset, a central electrode within the seizure focus mass was selected as the seed for further network analysis. For purposes of uniform comparison, the electrode found to be most negatively correlated with the seizure onset electrode over the entire 20 minute epoch was selected for the seed electrode in the \u201canti-correlated network\u201d. Simultaneous analysis of this and the epilepsy network serves as a negative control within the same dataset. Networked interactions between these two seed electrodes and all other electrodes were then completed as described below.The electrodes were co-registered between a pre-operatively acquired T1 MPRAGE anatomic MRI sequence and a post-operatively acquired computed tomography (CT) scan as described by Hermes, et al. Signals from every electrode were visually inspected and those electrodes identified as having poor signal to noise characteristics (amplitude greater than 10x that of the majority of electrodes in the array) were excluded from further analysis . Remaining signals were re-referenced to the common mean for further analysis.rd order digital low-pass Butterworth filter (cutoff\u200a=\u200a0.5 Hz) to obtain the SCP. The 20 minute blocks were analyzed as single time epochs across patients. Correlation of the SCP over the 20 minute epochs was then calculated for all electrode pairs over the entire 20 minute epoch. This was completed in the following manner: The covariance between each electrode pair was calculated using Networked interactions were evaluated as depicted in ith of N values of the SCP signal from the mth and nth electrodes, respectively. The correlation coefficient between each electrode pair was then obtained by Here, m\u2019 represents the seed electrode for a given network and it is compared against all other \u2018n\u2019 electrodes. Network connectivity was defined as all electrodes found to be positively statistically significantly covariant with the seed electrode as determined by a two-sample KS test of correlation coefficients were also evaluated Each subject's clinical ECoG signal had previously been interpreted by an epileptologist for mapping of their seizure focus and seizure propogation patterns during the time of grid implantation for invasive monitoring. Seizure onset electrodes and electrodes to which the seizures immediately propagated had previously been identified via this clinical interpretation. These clinically-defined electrodes were compared against the electrodes found to be included in the resting state seizure network by covariance analysis as a percent of overlap between the two groups of electrodes as depicted in All data can be made available upon request to the corresponding author.The seizure focus identified from the ictal onset, when used as a seed for SCP defined networks from the interictal periods, demonstrated statistically significant connectivity with distinct regions of brain. These regions of significant correlation included both near and remote cortex see . When coGlobal power in the epilepsy network was then compared against global power in the anti-correlated network during periods of interictal and ictal cortical activity. As depicted in Studies have identified broadband phenomena in the electric potentials produced by the brain that follow a power-law scaling in these signals Post-hoc comparisons showed significant differences in percent of activated electrodes between the epilepsy and anti-correlated networks continuing until 90 ms following seizure onset.Following seizure onset, the seizure network displayed an increasing percentage of all active electrodes . Conversely, the anti-correlated network did not show this same progression. Temporal power analysis revealed preferential power activations within the epilepsy network \u200a=\u200a 37.8) when compared to the anti-correlated network \u200a=\u200a 10.96) over time, relative to seizure onset, as shown graphically in Using data taken from invasively monitored patients with neocortical intractable epilepsy, this study demonstrates the important relationship that exists between resting state networks and the physiology associated with a seizure. Similar to what has been shown in the past with sleep and anesthesia for electophysiologic resting state networks that are defined by slow cortical potentials In prior studies, the durable nature of resting state networks has been shown in states associated with reduced or suppressed cognitive activity, including sleep and anesthesia The maintenance of resting state connectivity between seizure foci and remote cortex as determined by SCP correlation during both interictal and ictal epochs indicates that seizure foci are functionally coupled with remote, non-epileptogenic cortex at rest. This notion is not novel. The concept of epilepsy networks was first postulated by Spencer in 2002 The pathologic nature of these connections are highlighted by the differential behavior of the seizure with anatomic sites that show significant SCP correlation with the onset zone as compared to those areas that are anti-correlated. There was relatively increased global power amplification within the \u201cseizure network\u201d when compared to the \u201canti-correlated network.\u201d This relative amplification in overall power across frequency bands suggests several possibilities. First, because these different rhythms have been associated with different underlying neural circuits Beyond a general topographic difference in pattern of abnormal seizure activity, it appears that the focal cortical activation (as measured by high gamma power) emanating from the seizure focus moves preferentially along the interictally defined resting state network. Conversely, the areas shown to be anti-correlated with the seizure onset zone did not show a similar progression. In the setting of Alzheimer's Disease, previous work by Sheline et al. has demonstrated a preferential deposition of beta-amyloid plaques along anatomic regions defined as resting state networks. The group refers to this functional network-defined ordering of structural abnormalities as a \u201cstructural-functional\u201d connection between functional connectivity and disease progression in AD Medically refractory epilepsy affects nearly one million Americans annually Enhanced understanding of network connectivity related to seizure foci may improve this surgical cure rate by a number of mechanisms. First, for those patients found to harbor unresectable seizure foci, the seizure network may represent a more informed manner in which MSPTs can be applied over the cortical surface in and around the seizure focus. Alternatively, given the ictal propagation dynamics along the seizure network, completing MSPTs along this network may enhance seizure control even following resection of patients' seizure foci. Taken together, these findings may thus optimize clinical outcomes for focal neocortical epilepsy by providing cortical transection therapies when frank resection is not possible or after surgical resection of a seizure focus (i.e. making an effective therapy even better).There are several limitations to this study that deserve attention. These include a relatively small sample size of three patients as well as limited electrode grid coverage. Each patient's cortical coverage was dictated exclusively by clinical circumstances therefore our findings should only be interpreted in the context of this limited coverage. It is certainly possible that this limited cortical coverage impacts the implications of our findings, particularly if the seizure onset focus was in fact not included in the cortical region from which ECoG was recorded. While this is possible, the relatively favorable seizure outcomes across all three patients suggest that the seizure focus was included in analysis and then subsequently resected. There are however other potential implications of this limited electrode coverage which remain, including more extensive resting state connectivity that has not been appreciated or additional physiologic phenomena that have not been recognized due to the small area of cortex sampled for this clinical purpose. A third limitation is the possibility that the relationship between resting state connectivity and seizure propagation described herein is not necessary for seizure propagation but rather coincident with seizure pathophysiology. Studies investigating a relationship of necessity versus coincidence would need to be completed in the future to investigate this further. A fourth limitation is that by understanding the resting state connectivity and targeting it with MSPTs at the time of surgery, it is possible that new connectivity will develop and produce new propagation and resting state connectivity patterns. Further studies will need to be designed to investigate this possibility as well. Finally, our findings are applicable exclusively to focal neocortical epilepsy. As such, the cortical physiologic phenomena related to other patterns of epilepsy still remain unknown.In the setting of focal neocortical epilepsy, the pathologic site of seizure onset demonstrates networked interactions with broad anatomic regions on the cortical surface at rest and during a seizure. These seizure-related networks, which are present during interictal recording, are preferentially involved with the propagation and anatomic distribution of the ictal physiologic event. These findings support a more ordered, network driven phenomenon that could have implications for the surgical management of the disease.Figure S1Comparison of clinical and research covariance topographies. Exemplar cortical surface topography for a 20 minute interictal epoch aquired for clinical purposes (A) using a Natus differential amplifier which includes a 0.5 Hz hardware filter and a 20 minute interictal epoch acquired for research purposes (B) using a g.USBamp amplifier which includes no hardware or software filter.(TIF)Click here for additional data file."} +{"text": "Ex vivo machine perfused porcine livers are an excellent platform for investigating the sonoporation parameters and specifically the interaction of ultrasound driven microbubbles with the capillaries. Our objective was to identify the ultrasound parameters that are capable of causing detectable perfusion changes in the sonoporation area. Three types of perfusion changes were considered: large mechanical damage void of perfusion, reduced perfusion due to capillary destruction, and unaltered perfusion.Sonoporation is the transient and reversible cell membrane permeability change induced with ultrasound and microbubbles. It allows for the uptake of normally impermeable macromolecules and has been suggested for improving drug delivery. The exact sonoporation mechanism and the optimal ultrasound parameters are still under investigation. Porcine livers were collected from a local slaughterhouse and connected to a machine perfusion system Fig. . Injectiex vivo machine perfused liver with a combined therapy-imaging system. The use of unfocused therapy transducers led to a much larger treatment area and was easier to identify and measure perfusion changes with DCEUS. Focused therapy transducers produced higher acoustic pressures but at smaller areas that were difficult to identify with DCEUS unless the focused transducer was mechanically scanned to treat a larger area. Sonoporated areas with ultrasound pressure above 1 MPa showed a detectable perfusion change. Complete mechanical damage was present at much larger acoustic pressures (~10 MPa). Shorter acoustic pulses (50 cycles) produced less perfusion changes than longer pulses (500 cycles) for the same duty cycle.Perfusion change caused by image-guided sonoporation [Fig."} +{"text": "Occipital neuralgia(ON) is a rare presentation and might be due to multiple causes including occipital nerve pathologies and limit daily function of patients and have a severe impact on their quality of life (QoL). The clinical cahrecteristics of ON include paroximal painful attacks of electric shock- like sensation, occuring spontaneously or evoked by stimuli in specific tigger areas. Cervical cord demyelination might rareley cause ON. In this case report we would like to report a clinically definite relapsing-remitting multiple sclerosis (MS) patient who experienced occipital neuralgiform pain with a contrast enhancing demyelinating lesion on C2 cervical cord. We would like to discuss a rare case of occipital neuralgia presenting as an MS relapse. Although patients with multiple sclerosis have an increased incidence of headaches, new onset headaches might be related to a new active lesion and patient sholud be evaluated considering this possibility.No conflict of interest."} +{"text": "Populus tremula L.) genotypes grown in a randomized experimental array at two contrasting sites spanning the environmental conditions from which the aspen genotypes were collected. We found that variation in aspen genotype significantly influenced bark epiphyte community composition, and to the same degree as environmental variation between the test sites. We conclude that maintaining genotypic diversity of foundation species may be crucial for conservation of associated biodiversity.Community genetics hypothesizes that within a foundation species, the genotype of an individual significantly influences the assemblage of dependent organisms. To assess whether these intra-specific genetic effects are ecologically important, it is required to compare their impact on dependent organisms with that attributable to environmental variation experienced over relevant spatial scales. We assessed bark epiphytes on 27 aspen ( These cPopulus tremula L.) to establish randomized and replicated trials of naturally occurring genotypes at two sites with strongly contrasting climatic characteristics. Aspen has known high levels of associated diversity, including conservation priority species which are specialists [Here, we use a tree species which is readily cloned P. tremula L. was collected from each of 27 widely separated locations across Scotland [A root cutting from a single genotype (clone) of Scotland ,13 (figuScotland . Replica(b)An established standard method was used(c)Ordination of the epiphyte community composition for sampled ramets was performed using detrended correspondence analysis (DCA) . Frequen3.a). Epiphyte community composition also varied among the aspen genotypes, whose mean DCA scores along both axes one and two are illustrated in figure 2b.A total of 26 epiphytic taxa were recorded on the aspen ramets assessed at the two sites . DCA axes one and two explained 22.5% and 11.1%, respectively, of the variation in epiphyte community composition among ramets, with environmentally determined differences between the two sites clearly evident a. Epiphyp < 0.05) of both aspen genotype and experimental site and liverworts (Frullania dilatata) had optima more than 4 and were associated with aspen ramets from the oceanic Kilmichael site.Comparisons of individual species scores clarified environmental effects on the distribution of taxa: lichen species adapted to a more continental climate, such as pulicola , had opt4.The experimental design used here allowed us to directly compare the amount of variation in epiphyte community composition explained by intra-specific genetic effects sampled over corresponding environmental space. Previous attempts to demonstrate that genetic variation of foundation species determines the community composition of associated species have been criticized on the grounds that genetic variants have been sampled over a large geographical area, and the differences between them have been tested in a single site \u201310. EnviIt is important that our two contrasting experimental sites approximate the outer bounds in a bioclimatic envelope from the hyper-oceanic west of Scotland to the relatively more continental northeast and are The magnitude of the host genotype effect on associated epiphyte community composition has potentially widespread implications for conservation. Forest stands with mixed aspen genotypes may generate higher levels of accumulated diversity because of contrasting species composition among clones. Our findings also indicate that a reduction in genetic diversity of a foundation species such as aspen is likely to lead to a decline in the diversity of the associated epiphyte communities, as suggested by studies conducted in natural systems over small scales . This wi"} +{"text": "Oral Squamous Cell Carcinoma (OSCC) is the most common tumor of the oral cavity. Approximately 500.000 new cases of OSCC are diagnosed worldwide each year with the incidence seemingly increasing. Chemo- and radiotherapy are potential curative procedures although destructive surgery is often necessary if early diagnosis is not achieved. Such procedures impact heavily on human and social costs. Infection of oral mucosa by oncogenic HPV is strongly associated with OSCC among subjects with established risk factors such as tobacco and alcohol abuse. The development of OSCC is a multistep process in which accumulated genetic and epigenetic events lead to cell cycle deregulation and chromosomal abnormalities. Gain of chromosome 3 appears to be an important selective advantage in the progression of cervical intraepithelial neoplasia to invasive cancer as well"} +{"text": "Varicella infection is a highly contagious disease which can have a complicated course especially in immunocompromised children and children receiving immunomodulatory therapy.to evaluate safety and efficacy of varicella vaccination in children with JIA treated also with biologic therapy.We designed a prospective study on a long term follow up. Five patients with JIA , treated also with biologic therapy , received 2 doses of varicella vaccine. One patient treated with etanercept received the first dose before starting etanercept, others received both doses on biologic therapy. Before vaccination JIA was stable on therapy and lymphocytic populations were normal in all vaccinated children. All had negative varicella serology. Parents were asked for written informed consent before vaccination. After vaccination children were followed for disease activity, infections and protective antibodies (pAb) against varicella virus on a long term.There were no serious side effects after vaccination and no clinical varicella infection in a period of 3 months after vaccination. One patient had mild local reaction after the first dose. One patient treated with etanercept got adenovirus infection 3 days after the second dose of vaccine. Disease activity remained stable in all patients in a period of three months after the second dose. Four patients (75%) had pAb against varicella virus 6 weeks after the second dose. One patient treated with infliximab and methotrexate didn\u2019t develop pAb after the second dose. Two patients treated with etanercept developed low levels of pAb after the second dose. Two patients treated with tocilizumab developed low levels of pAb even after the first dose and very high levels of pAb after the second dose. One patient treated with etanercept got varicella infection 4 months after the second dose despite the protective, however, low levels of pAb. Varicella infection was mild. The other patient treated with etanercept lost his pAb 22 months after the second dose. One patient treated with tocilizumab still has low pAb 27 months after the second dose and one patient treated with tocilizumab has very high pAb 3 month after the second dose.Varicella vaccination appears to be safe in JIA patients treated also with biologic therapy. However, it is possible that protection is only short term and does not always protect against infection. Follow up of pAb is recommended and the need for revaccination should probably be carefully considered in cases with high risk of varicella infection. Larger cohort studies are needed to obtain more reliable data on varicella vaccination in JIA patient treated with biologic therapy.None declared."} +{"text": "In 1993, the cardiac surgeon Bakker et al. \u20136. Becau2\u2009+ handling, \u03b2-adrenergic receptors, contractile proteins, and myocardial natriuretic peptide pre- and post-CRT. It remains unclear whether these changes in gene expression are truly induced by CRT itself or whether they are the result of improvement in LV function. Because of the complexity of the phenotype \u2018CRT responder\u2019 and the intricate underlying genetic architecture, large studies will be necessary to identify genetic factors associated with volumetric CRT response.This special issue of the Netherlands Heart Journal elucidates current aspects of cardiac resynchronisation therapy. Wiegerinck et al. describeVersteeg et al. showed iOne important, underestimated factor that influences CRT response is LV lead placement. Imaging techniques such as cardiac MRI tagging or deformation echocardiography can detect the segment of latest mechanical activation; however prior to CRT implantation the exact coronary venous anatomy is usually not known. So why don\u2019t we measure the electrical delay in the side branches of the coronary sinus where the LV pacing lead will be implanted? Such an optimal lead position at the free wall of the LV may be more important in patients with ischaemic cardiomyopathy and non-typical LBBB. Van Stipdonk et al. describeSeveral current aspects of cardiac resynchronisation therapy are highlighted in this special CRT issue. These reviews and original articles can give us new insights for daily clinical practice and provide inspiration for new research projects to further improve heart failure therapy with implantable cardiac devices for our patients."} +{"text": "To the Editor: Health authorities from Cambodia and European Union member states recently described a pronounced increase in Salmonella enterica serotype Paratyphi A infections in Cambodia resulting from an ongoing outbreak often are resistant to the quinolone nalidixic acid or are multidrug resistant (The Paratyphi A outbreak in Cambodia appears to be large and ongoing. To our knowledge, information about possible sources and risk factors that could help inform prevention activities is not yet available. This outbreak highlights the urgent need for a paratyphoid fever vaccine; although typhoid fever vaccines exist, persons living in and visiting regions of active Paratyphi A transmission have no alternative to relying exclusively on close attention to food and water safety to mitigate risk ("} +{"text": "MAPT and C9ORF72 mutations. Comparing diffusivity metrics, optimal overall separation of the bvFTD group from the healthy control group was signalled using radial diffusivity, whereas optimal overall separation of the bvFTD group from the Alzheimer's disease group was signalled using fractional anisotropy. Comparing white matter changes with regional grey matter atrophy (delineated using voxel based morphometry) in the bvFTD cohort revealed co\u2010localisation between modalities particularly in the anterior temporal lobe, however white matter changes extended widely beyond the zones of grey matter atrophy. Our findings demonstrate a distributed signature of white matter alterations that is likely to be core to the pathophysiology of bvFTD and further suggest that this signature is modulated by underlying molecular pathologies. Hum Brain Mapp 35:4163\u20134179, 2014. \u00a9 2014 The Authors. Human Brain Mapping Published by Wiley Periodicals, Inc.Despite considerable interest in improving clinical and neurobiological characterisation of frontotemporal dementia and in defining the role of brain network disintegration in its pathogenesis, information about white matter pathway alterations in frontotemporal dementia remains limited. Here we investigated white matter tract damage using an unbiased, template\u2010based diffusion tensor imaging (DTI) protocol in a cohort of 27 patients with the behavioral variant of frontotemporal dementia (bvFTD) representing both major genetic and sporadic forms, in relation both to healthy individuals and to patients with Alzheimer's disease. Widespread white matter tract pathology was identified in the bvFTD group compared with both healthy controls and Alzheimer's disease group, with prominent involvement of uncinate fasciculus, cingulum bundle and corpus callosum. Relatively discrete and distinctive white matter profiles were associated with genetic subgroups of bvFTD associated with The behavioral variant of frontotemporal dementia (bvFTD) is a major neurodegenerative syndrome of younger life . DTI volumes were then combined for tensor fitting using Camino \u201crandomise\u201d tool within FSL with 10,000 permutations for each test. A significance threshold (P < 0.05) was applied after correction for multiple comparisons using family\u2010wise error (FWE) correction with threshold\u2010free cluster enhancement (TFCE) [Smith and Nichols, fslstats\u201d). The proportion of significantly affected voxels within each tract was calculated by dividing the number of significant voxels identified by the total number of voxels within each tract limited to within the skeleton. In addition, to assess the anatomical reliability of results obtained from the group skeleton, result maps were de\u2010projected and overlaid on each individual participant's FA image, which confirmed good anatomical alignment of the results with white matter tracts.To assess disease effects on white matter structures a general linear model was created with disease group membership as the factor of interest and age, gender, and disease duration (as a surrogate of disease severity and stage) included as nuisance covariates. Each metric (AX/RD/TR/FA) was analyzed with the above model, and additionally with a model that included the overall mean of the metric under study as an additional covariate. The latter model reveals areas that are specifically affected after adjusting for widespread global differences, analogous to controlling for total grey matter volume in voxel\u2010based morphometry [Mechelli et al., fslmeants\u201d command. ROC curves and AUC values were calculated for both global and individual tract diffusivity data.To determine the sensitivity and specificity of different diffusivity metrics in classifying individual participants into separate groups receiver operating characteristic (ROC) curves were constructed and areas\u2010under\u2010curve (AUC) were calculated. Two approaches were used for the classification, assessing respectively the mean of each individual metric across the whole white matter skeleton and the mean values within individual tracts identified as prominently involved in the initial DTI analysis. Mean values were extracted using the \u201chttp://www.fil.ion.ucl.ac.uk/spm) under Matlab 7.14 (http://www.mathworks.com). Segmentation, normalization, and modulation were performed using default parameter settings. Smoothing of grey matter images used a 6 mm kernel size. Final DARTEL transformations were combined with affine transformations to MNI space. To maintain consistency with the white matter analysis, grey matter was assessed using the same statistical methodology. Nonparametric permutation testing was used to examine differences in regional grey matter volume between groups and included age, gender, total intracranial volume and disease duration as nuisance covariates. Maps of grey matter atrophy were overlaid onto an MNI152 standard brain after FWE correction (P < 0.05) with TFCE. To provide information on the anatomical relationship of the grey and white matter changes, an intersection map of all four DTI metrics were overlaid on the map of grey matter atrophy.In order to compare white with grey matter changes in the bvFTD cohort, profiles of grey matter atrophy were first derived using voxel\u2010based morphometry (VBM) with the New Segment [Weiskopf et al., MAPT gene ), four had mutations in C9ORF72 and one patient who subsequently died was found to have Pick's disease pathology at postmortem. In addition to meeting diagnostic criteria for bvFTD, two patients had symptoms compatible with Progressive Supranucelar Palsy (both having sporadic bvFTD), two had symptoms compatible with motor neuron disease and one had symptoms compatible with corticobasal syndrome (being sporadic). All AD patients had a typical memory\u2010led presentation. Demographic, clinical and neuropsychological data for all participant groups are summarised in Table Of the 27 patients with bvFTD identified 14 met criteria for a diagnosis of definite bvFTD and 13 met criteria for a diagnosis of probable bvFTD [Rascovsky et al., Widespread white matter tract pathology was identified in the bvFTD group compared with both the healthy control group and the AD group Figs. , 2, 3, 4Compared with the healthy control group after global mean value correction reduced grey matter predominantly distributed in frontal and temporal cortices in both cerebral hemispheres while certain key bvFTD phenotypes were either underrepresented or not represented at all. The likely variability of pathologies within the sporadic bvFTD group may also have limited the ability to detect white matter changes specific to this group. Taking these caveats into account, the present findings provide further evidence that the profile of regional brain network disintegration in bvFTD may be modulated by underlying molecular pathologies [Warren et al., The present work further suggests that DTI can delineate molecularly defined profiles of white matter pathology within the broader bvFTD spectrum. Most striking were alterations within the uncinate fasciculus in those with The compatibility of different diffusivity metrics is an issue of considerable clinical and neurobiological relevance to the application of DTI in neurodegenerative disease. While our findings suggest partial convergence of metrics for particular white matter tracts, we found additional substantial divergence in the white matter profiles generated using different diffusivity metrics across both sporadic and genetic forms of bvFTD and putative bvFTD subtypes. Ultimately, the solution may lie in the use of multivariate or multimodal indices and composite profiles of tract alteration that are informed by an improved understanding of the pathobiology of white matter diffusion and the natural history of particular bvFTD pathologies [Avants et al., The authors thank all patients and healthy volunteers for their participation. The Dementia Research Centre is an Alzheimer's Research UK Co\u2010ordinating Centre.Additional Supporting Information may be found in the online version of this articleSupporting InformationClick here for additional data file."} +{"text": "ErratumUnfortunately, the original version of this article containeThe Abstract results should report that:\u201cIn female students with orthorexia nervosa multivariable linear regression analysis found low body areas (parts) satisfaction, high fitness orientation, high overweight preoccupation and high appearance orientation were independent predictors of greater fixation on eating healthy food.\u201dAnd conclusion that:\u201cA strong preoccupation with healthy and proper food was associated with an unhealthy body-self relationship among Polish female student with orthorexia nervosa.\u201dThe reporting of results on page 5 is correct. Because the correlations were negative the interpretation in the discussion should be corrected to indicate that in female students, a strong preoccupation with healthy eating was associated with lower appearance evaluation and body areas satisfaction and women with orthorexia nervosa were more likely to: (1) regularly incorporate exercise activities into their lifestyle , (2) concentrate on dieting, eating restraint and weight vigilance , (3) pay attention to their appearance and (4) lead a physically healthy lifestyle . These findings are not counterintuitive, but support the hypothesis that there is an association between orthorexia and features of disordered eating and body image concern."} +{"text": "The ability to effectively ablate prostate cancer with transrectal HIFU has been demonstrated by numerous publications to date. Early results from focal HIFU series suggest that side effect profiles such as stricture, erectile dysfunction, and incontinence can be greatly reduced as compared to total gland ablative HIFU treatment.Multiparametric MRI combined with both systematic and fusion biopsy is being utilized more frequently worldwide to select patients for focal therapy.The ability to effectively ablate prostate cancer with transrectal HIFU has been demonstrated by numerous publications to date. Early results from focal HIFU series suggest that side effect profiles such as stricture, erectile dysfunction, and incontinence can be greatly reduced as compared to total gland ablative HIFU treatment. Multiparametric MRI combined with both systematic and fusion biopsy is being utilized more frequently worldwide to select patients for focal therapy. Commercially available, FDA approved MRI to ultrasound fusion platforms are now readily available for use in the urology clinic setting, however, these have not yet been widely used to guide focal HIFU treatment. The multistep workflow requires:1. Creation of a 3D MRI model using MRI images of the prostate that contain a lesion proven by targeted biopsy to be malignant;2. Creation of a 3D model of the prostate using ultrasound images, rigid and elastic image fusion; and3. Use of the fused images to guide treatment.This process will be demonstrated using clinical examples and progress on the integration of an MRI to ultrasound fusion system into the Sonablate device will be described."} +{"text": "Achilles tendinopathy is a common pathology with limited treatment options. This case study displays a novel approach using autologous tendon stem cells (tenocytes) injected into a chronically affected Achilles tendon following a failed repair of an Achilles tendon rupture.Tenocytes were harvested from the patient's patella tendon and prepared using Orthocell regeneration technology. The tenocytes were multiplied and placed into an injectable scaffold, then injected under ultrasound guidance into the diseased tendon.The patient ruptured the Achilles tendon working as a nurse and underwent an orthopaedic surgery primary repair. At 6 months post operatively the patient had developed chronic tendinopathy affecting over 80% of the Achilles tendon. The patient was referred for opinion and had been off work for over 6 months with limitation in activities of daily living.The patient underwent a surgical excision of a posterior calcaneal exostosis and debridement of the diseased Achilles tendon and harvest of tenocytes. 13 weeks post-surgery the tenocytes were injected into the Achilles tendon. The patient returned to full time work and normal activities of daily living at 8 weeks post injection. MRI studies were performed pre and post injection to assess diseased state of the tendon.Autologous tenocyte injection provides the clinician with a tool for treatment of chronic tendinopathy, where other treatment options have failed.Mr Peter Manuel has no financial relationship with Orthocell nor received any funding for this case study."} +{"text": "The results are consistent with the existence of multi-modal representations of stimulus-response (SR) rules within the frontal cerebral cortex.We report a functional magnetic resonance imaging (fMRI) study which investigated whether brain areas involved in updating task rules within the frontal lobe of the cerebral cortex show activity related to the modality of motor response used in the task. Participants performed a rule switching task using different effector modalities. In some blocks participants responded with left/right button presses, whilst in other blocks left/right saccades were required. The color of a Cue event instructed a left or right response based upon a rule, followed by a Feedback which indicated whether the rule was to stay the same or \u201cFlip\u201d on the next trial. The findings revealed variation in the locus of activity within the ventrolateral frontal cortex dependent upon effector modality. Other frontal areas showed no significant difference in activity between response epochs but changed their pattern of connectivity with posterior cortical areas dependent upon response. Multivariate analysis revealed that the pattern of activity evoked by Flip rule Feedbacks within an apparently If this were the case then activity should be independent of response modality.Performing many complex tasks involves the execution of actions based upon arbitrary mappings between stimulus and response. Previous patient and neuroimaging investigations have suggested that the lateral and medial frontal regions of the human cerebral cortex might be important in maintaining arbitrary stimulus-response (SR) rules and resolving response conflict in rule based tasks Milner, . A key mA common assumption is that cognitive representations of task rules are maintained within the frontal cerebral cortex which modifies specific SR associations via top-down influences on sub-cortical and posterior cortical structures . Subjects made a left or right behavioral response dependent upon the color of the cue and the current rule linking color with direction. For \u201cEye\u201d (saccade) blocks participants were instructed to fixate one of two peripheral response locations . When the cue stimulus was extinguished they then had to make a return saccade to fixate the central location. The word \u201chold\u201d or \u201cflip\u201d was then presented at fixation to indicate to the participant whether to hold or reverse the current rule on the following trial (Feedback event). In order to resolve the fMRI response to these discrete events within a trial i.e., Cue and Feedback, the period between each event and trial was varied randomly between 2200, 4400 and 10200 ms such that inter-event intervals did not correspond to multiples of the TR period. Varying the period between individual events in this manner within as well as between trials ensures that discrete events can be resolved within a trial and two blocks required a saccadic response to be made to a response box located to the left or right of the central location (Eye blocks). Eye and hand blocks were presented alternately with half the subjects completing the task in the order Eye-Hand-Eye-Hand and the other group of subjects in the order Hand-Eye-Hand-Eye. Each task block was preceded by an instruction screen which told subjects to either \u201cRespond with Eyes\u201d or \u201cRespond with Hands\u201d as well as showing the rule to be used on the first trial which was displayed for 10 s, followed by a variable delay period before presentation of the first colored cue stimulus. Participants completed one practice block of each version of the task outside the scanner just prior to the scanning session.Each block consisted of 21 trials and each subject completed four blocks in one continuous scanning run, with two blocks requiring responses to be made with a manual press button response . Four hundred and sixty five image volumes were acquired in one continuous scanning run. An additional five \u201cdummy\u201d scans were performed at the start of each block prior to the start of the stimulus sequence.Scanning was performed on a 1.5 T Philips Gyroscan magnet at the Peninsula MR Research centre, University of Exeter, UK. A T2task and procedure above), This design allowed unique regressors to be derived for each event type by convolving the event onset times with a canonical hemodynamic response function. The eight different event types modeled at the individual subject level were:Data were analyzed using SPM8 software. The images were realigned, unwarped to remove variance caused by movement-by-field-inhomogeneity interactions, normalized to a standard EPI template, and smoothed with a Gaussian kernel of 6 mm full-width at half maximum. Inter-event periods were selected such that the response Cue and Feedback event sequences were temporally de-correlated with each other and the TR period ; Trial type (Hold vs. Flip rule) and Response type (Hand vs. Eye). All factors and levels were assumed to be non-independent (repeated measures) and the statistics and degrees of freedom used for inference were calculated accordingly. Two approaches were taken in the comparison of activation by response. Direct statistical contrasts between conditions of interest were first carried out . In addition, an exclusive masking approach was also taken to explore response mode specific activity in which activity for one response type was masked with the equivalent contrast for the other response using a very low statistical threshold (p < 0.05 uncorrected) to derive the exclusive mask factorial statistical model was applied to the one sample a priori hypotheses arising from previous fMRI studies analyses derived from other relevant contrasts or The conjunction analyses across Eye and Hand epochs tested the conjunction null hypothesis corrected for multiple comparisons are reported below and the results of all other statistical contrasts carried out at the individual or group level revealed no significantly activated voxels based upon this criteria. All coordinates given in tables are Montreal Neurological Institute (MNI) coordinates. Anatomical labels and estimated Brodmann area numbers were generated by converting MNI coordinates to Talaraich and interrogation of the Talaraich Daemon database tool . Directional errors in eye and hand responding were also very rare. A total of five such errors were made in Eye response blocks across all subjects and six manual response errors equating to <4% errors overall. For saccadic, but not manual responding, \u201ccorrected\u201d errors were observed on some trials in which a saccade was initially directed in the wrong direction but the movement was quickly corrected such that the eye was directed to the correct response box. A total of only 10 such corrected errors were observed across all participants. Means and standard errors of response times across participants for eye and hand blocks were 865 \u00b1 41 ms and 774 \u00b1 46 ms respectively. A two-way ANOVA with response type and trial type (Hold/Flip rule), revealed no significant effect of response type, trial type or interaction between response and trial type on reaction times.No voxels were significantly activated for the comparison of Trial Type (Hold > Flip or Flip > Hold) for Cue events. The comparison of Flip compared to Hold rule trial Feedback events highlighted superior bilateral parietal cortex, the anterior cingulate, left middle frontal gyrus, left inferior frontal gyrus and bi-lateral cerebellum Table . The revAreas which showed more or less activity during the two response mode epochs (Eye/Hand) were compared via both direct statistical contrasts and the exclusive masking technique for both Cue and Feedback elicited event activations.Direct statistical contrasts for Feedback and Cue evoked activity revealed no differences between Eye and Hand blocks. Cue locked activity in Eye blocks masked by Hand blocks showed activity in the left frontal eye fields (FEF), medial parietal and occipital cortex along with the Middle temporal and parahippocampal gyrus Table . The revA key motivating question for the study was whether the location of regions showing an enhanced response to Flip relative to Hold rule Feedback events varied dependent upon response type. Of particular interest is whether the increase in activity observed in some areas during Flip relative to Hold Feedback events was modulated by motor response . Activations of this type would be indicative of rule updating and cognitive control processes which were specific to particular modalities of response.Cue event) locked activity, a number of regions were found to be significantly activated suggesting that these response execution related regions were also activate during rule updating were examined for each relevant event type contrast using the conjunction null hypothesis test option in SPM8.This analysis for Cue locked activity alone revealed predominantly left superior parietal cortex activity, bilateral cerebellar and dorsolateral frontal (BA6) activity in the region of the FEF for Hand and Eye epochs showed activity in the medial frontal cortex including activation peaks within the dorsal anterior cingulate gyrus, superior frontal gyrus, left inferior frontal cortex/frontal operculum (BA47), bilateral inferior parietal cortex and left dorsolateral frontal cortex . In particular, we wanted to apply this technique to regions which the univariate analysis indicated were supra-modal in nature (such that no voxels showed significant variation in activity dependent upon response epoch) but which nevertheless showed evidence for changes in connectivity with posterior regions during rule updating.A binary support vector machine classifier was applied to Flip feedback event related activity in voxels extracted from the same left dorsolateral frontal ROI used as a seed region in the PPI analysis (see above). Using this approach it was found that the machine classifier was able to correctly identify whether activity evoked by Feedback events occurred in Hand or Eye epochs. This was found to be the case for all participants tested, with an overall mean accuracy across subjects of 85% (ranging from 75\u201398 between participants) for Hand and 84% (62\u201398) for Eye response epochs Figure .A key motivation of this study was to examine whether regions of the frontal cerebral cortex previously shown to be active in a SR rule switching task might show regional variation in activation patterns dependent upon effector modality . As would be expected, activity dependent upon the type of response (Eye/Hand) to be executed time-locked to response Cue events was found within the inferior parietal cortex, lateral occipital cortex, post-central gyrus and FEF Table . Interesdoes have an underlying motoric organization and function.Our results support previous findings which suggest that sub-regions of the ventrolateral frontal cortex support cognitive operations for particular motor domains specifically the inferior frontal gyrus pars triangularis and orbitalis. A more dorsal and anterior locus of activity was found for saccade responses, whilst manual response trials recruited ventral and posterior parts of the inferior frontal gyrus bilaterally. The inferior frontal cortex has been suggested as an important substrate of cognitive inhibitory control across different modalities of response was executed . Other neuroimaging studies which have studied cognitive task execution with hand and eye movements have also reported FEF activity during manual as well as saccade response blocks , even during events that signaled a change in rule rather than being associated with an actual motor response to be executed. Closer examination of fMRI data shows that even within frontal areas which univariate analysis suggested were equally activated during rule switching with manual or saccade responses, differences were apparent between Hand and Eye response epochs in terms of their connectivity with posterior areas as well as the overall pattern of voxels activated within the region. We suggest that higher order representations of task rules are underpinned by neural representations which represent effector modality. Such a distributed form of representation is best termed multi modal rather than supra-modal and may not always be apparent when standard univariate statistical analysis techniques are applied to fMRI data.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "To determine whether the growth of tumors is sustained by the whole cancer cell population or it is maintained only by small fraction of cancer initiating cells, has crucial impact on the design of suitable therapies. That is why the research on defining the subpopulation of cancer initiating cancer stem cells (CSC) in solid tumors has come into highlights.A complex panel assay at cellular and molecular levels has been performed on primary and metastatic cancerous tissue biopsies and peripheral blood of patients with malignant melanomas (n = 153) .Cell cultures grew out of the great majority of the starter metastatic tissue specimen and cancer initiating cells could be sorted (BD FACSAvia Sorter) by colocalized unique sialilated glycosphingolipids and anti CD20 binding capacity. Characteristic growth pattern, spheroid forming, CSC markers like CD133, Nestin, ABCB5, CD20 and unique GD3 gangliosides were found. Patients\u2032 sera and selected patients\u2032 Epstein Barr Virus transformed cell supernatants in bulk or after limiting dilution cloning were tested for cancer binding by ELISA and immunofluorescence. Our novel tumor infiltrating B cell antibody phage display technique and DNA sequence cluster analysis resulted in some antibody fragments, belonging to representative tumor binding antibody variable region subgroups, with defined silalilated glycosphingolipid specificity.Our strategy enables the detection and characterization of cancer stem cells in metastatic melanomas, with potential diagnostic importance. The novel peripheral blood and tumor infiltrating B cell antibody profile analysis proved to be a useful asset to reveal anti tumor humoral immune responsiveness and harness it by antibody engineering technique for further diagnostic and therapeutic usage."} +{"text": "Vitals at admission were stable and physical examination showed clear cut cool bilateral lower extremities below both ankle joints with all the 10 toes being black suggesting gangrene. Bilateral dorsalis pedis and Posterior tibial pulses were not felt and could not be detected with dopplers. A transthoracic Echocardiogram revealed that the patient had an Ejection fraction of 10-15% with diffuse hypokinesis, also were noted multiple biventricular thrombi with the largest in the left ventricle measuring 50\u00d730mm in size extending from anterolateral papillary muscle upto the septal myocardium. Evaluation with a cardiac and aortic catheterization revealed non obstructive coronaries, complete occlusion of the bilateral anterior tibial, posterior tibial and peroneal arteries at the ankle level with zero flow below bilateral ankle joints. No intervention could be performed and hence the patient was at first anti coagulated with heparin and then bridged to Coumadin. Patient was discharged to follow up with orthopedic surgery for bilateral amputations. Biventricular thrombi are generally seen in patients with a pro thrombotic state like anti phospholipid antibody syndrome, heparin induced thrombocytopenia induced thrombosis, hypereosinophilic syndrome. Cases have been reported in patients with viral myocarditis and libman sacks endocarditis. It is generally very rare to see multiple biventricular thrombi in patients with low Ejection Fraction."} +{"text": "For a range of nervous system disorders current treatment options remain limited. Focusing on Parkinson\u2019s disease as a neurodegenerative entity that affects an increasing quantity of people in our aging societies, we briefly discuss remaining challenges and opportunities that neural stem cell therapy might be able to offer. Providing a snapshot of neural transplantation paradigms, we contemplate possible imminent translational scenarios and discuss critical requirements to be considered before clinical implementation. Repairing the central nervous system may appear daunting in light of how little we still understand about its intricate molecular and cellular structure. Nevertheless, concepts of neural cell transplantation were explored early on, and since the 1980s there have been more concentrated systematic efforts in the area of Parkinson\u2019s disease, specifically, which is used here as a salient example .Figucan survive long-term, restore function and alleviate neurological disease by means of \u201creplacement\u201d was provided by these studies ).Adherence to such principles ensures lasting trust toward this field and may avert unwarranted hype, while maintaining the justifiable enthusiasm and motivation that clinicians, physician-scientists and basic researchers working in the fields of regenerative medicine and applied stem cell biology share in terms of bringing the joint scientific efforts to clinical fruition.As optimized protocols generate increasingly authentic and safer neural cell preparations, future efforts may focus on refining parameters of patient selection and cell delivery. Ultimately, enhanced interdisciplinary dialogue and applying the highest scientific standards on all levels from basic stem cell biology to clinical protocol development may make neural cell restoration via cell therapy a clinical reality."} +{"text": "Cx3cr1). By using confocal ophthalmoscopy, the authors imaged microglia in young mice and correlated these data with the severity of optic nerve pathology in the same animals 5 to 10 months later. Interestingly, the authors found that early microglial activation and microgliosis significantly correlate with late retinal neurodegeneration. These changes might thus predict disease severity and could be visually monitored to guide therapeutic interventions. 443Page Glaucoma is a progressive neurodegenerative disorder that affects the retinal ganglion cells and their axons. Its symptoms become manifested only at late disease stages, when vison loss is irreversible. Thus, identifying early biomarkers of degeneration is central to make therapeutic interventions possible. Alterations of microglia are detectable early during glaucoma progression; however, whether these early changes can predict the severity of late neurodegeneration in the retina is unknown. To address this, Alejandra Bosco and colleagues used a mouse model of inherited glaucoma in which microglial cells were genetically labelled with green fluorescent protein (GFP) expressed under the control of the fractalkine receptor locus ("} +{"text": "Social environment plays a critical role in the initiation of cigarette smoking among adolescents. It has been established that nicotine, the principal psychoactive ingredient of tobacco products, has both aversive and rewarding properties. Using olfactogustatory stimuli as the sensory cue for intravenous nicotine self-administration, we have shown that social learning of nicotine contingent odor cue prevented rats from developing conditioned taste aversion (CTA) and allowed them to instead establish stable nicotine self-administration. We also have shown that infralimbic cortex is a critical brain region for the effect of social learning in reversing nicotine conditioned aversion. We hypothesized that gene expression changes related to synaptic plasticity in infralimbic cortex underlies this effect of social learning.We trained rats to self-administer intravenous nicotine in either an inducing or a neutral social environment, with contingent olfactogustatory cues for three days. Rats were then tested using a standard CTA protocol on day four. Rats were killed immediately after the CTA test and infralimbic cortex was dissected. RNA was then extracted for transcriptome sequencing using the Ion Proton instrument. Reads were mapped to the reference genome (rn6). Gene expression levels were estimated using HTSeq. The RefSeq database was used as the reference. DESeq2 was then used to normalize the expression levels and to identify statistically significant differences between the groups placed in the inducing and neutral social environments.Gene set enrichment analysis (GSEA) supported our hypothesis that genes related to synaptic plasticity are preferentially regulated by the social environment."} +{"text": "Mustela nigripes), a prairie dog (Cynomys spp.) specialist, and the American badger (Taxidea taxus), a larger generalist carnivore that competes for prairie dogs and is known to kill ferrets. We expected that ferrets would spatially avoid badgers because of the risk of predation, that these patterns of avoidance might differ between sexes and age classes, and that the availability of food and space might influence these relationships. We used location data from 60 ferrets and 15 badgers to model the influence of extrinsic factors (prairie dog density and colony size) and intrinsic factors on patterns of space use by ferrets in relation to space use by different sex and age categories of badgers. We documented asymmetric patterns of avoidance of badgers by ferrets based on the sex of both species. Female ferrets avoided adult female badgers, but not male badgers, and male ferrets exhibited less avoidance than female ferrets. Additionally, avoidance decreased with increasing densities of prairie dogs. We suggest that intersexual differences in space use by badgers create varying distributions of predation risk that are perceived by the smaller carnivore (ferrets) and that females respond more sensitively than males to that risk. This work advances understanding about how competing species coexist and suggests that including information on both intrinsic and extrinsic factors might improve our understanding of behavioral interactions between sympatric species.Interactions between intraguild species that act as both competitors and predator\u2013prey can be especially complex. We studied patterns of space use by the black-footed ferret ( Interactions between sympatric taxa help shape the structure of ecological communities because they can influence the demography, distribution, and behavior of species within communities (Holt and Polis Vulpes vulpes) avoid coyotes (Canis latrans) by establishing home ranges outside of the areas used by coyotes (Major and Sherburne Lynx rufus) also avoid interactions with coyotes at fine spatial scales by establishing core use\u00a0areas that do not overlap with coyote core areas territories or in the matrix between territories of neighboring wolf packs and river otters (Lutra canadensis) persist in coastal marine environments by partitioning diets and also habitat by the amount of wave action and over-story cover coexisting in riverine environments reduce the use of their preferred habitat when occupied by long-tailed weasels (M.\u00a0frenata), a dominant guild member that has been documented to kill and eat short-tailed weasels and short-tailed weasel coexist because the subordinate least weasel can avoid confrontations with the dominant generalist using the tunnels and runways of voles (Microtus spp.), its primary prey coexist in prairie dog (Cynomys spp.) ecosystems in North America. Ferrets are obligate specialists that are dependent on prairie dogs for food and reside within burrows created by prairie dogs and extrinsic factors (prairie dog density and prairie dog colony size) on space use by ferrets in relation to space use by badgers. We hypothesized that (1) ferrets would reduce the potential for intraguild predation by spatially avoiding badgers, and we expected that; (2) adult female badgers would have the greatest influence on space use by ferrets because female badgers are more attuned to the distribution of prey resources and have smaller territories than males , green needlegrass (Nassella viridula), buffalo grass (Bouteloua dactyloides), and needle-and-thread (Hesperostipa comata). The area is primarily used for livestock grazing, but cultivated agriculture also is common. In 2007, the Lower Brule study site contained 25 prairie dog colonies totaling 642.5\u00a0ha located in a fragmented mixed-grass prairie dominated by western wheatgrass (Bouteloua gracilis), and prickly pear cactus (Opuntia polyacantha), and it is nearly devoid of tree and shrub species . The site is a mixed-grass prairie dominated by western wheatgrass, buffalograss, blue grama at the entrance of occupied badger burrows. Trapped individuals were chemically immobilized with an intramuscular injection of ketamine hydrochloride and xylazine hydrochloride while driving an ATV or walking along the perimeters of the colonies. Edges of colonies were distinguished by the distinct difference in vegetation height caused by foraging and vegetation clipping behavior of black-tailed prairie dogs Koford . We impoWe captured and marked prairie dogs to estimate relative densities at the colony level. We conducted trapping within randomly selected 0.5-ha plots during June\u2013July 2008 at the Lower Brule site, June 2009 at both study sites, and also during June 2010 at the Lower Brule site. At Lower Brule, we established four trapping plots on each of six prairie dog colonies, which resulted in sampling 1\u201322% of the area of each colony. At Buffalo Gap, we established 12 plots on the smaller colony (250.3\u00a0ha) and 16 on the larger colony (284.6\u00a0ha), which resulted in sampling 2\u20133% of each colony. Each plot contained 61 wire box traps baited with grain and molasses pellets spaced 10\u00a0m apart in a 7\u00a0\u00d7\u00a07 design with 12 additional traps placed in areas with high densities of burrow openings to minimize trap saturation. We prebaited plots for 3\u00a0days prior to trapping, and we set and checked traps twice each day for four consecutive days and checked them once on the 5th day . We marked prairie dogs on initial capture with hair dye in 2008, by clipping a toe-nail in 2009, and by clipping a toe-nail and applying individually numbered ear tags in 2010. An index of relative density for each colony was obtained each year by averaging the total number of individuals marked per plot across all plots. We attempted to improve these estimates using accumulation curves and modifications to Peterson\u2013Lincoln estimation methods Schnabel , but theOur study design followed a second-order selection analysis of habitat use versus availability Johnson . We buffWe generated utilization distributions (UDs) for each badger to estimate the relative intensity of use across space . We used sex and age of each ferret as intrinsic predictor variables and colony-level attributes (colony size and relative density of prairie dogs) as extrinsic predictor variables. We included the effect of study site in some models because features unique to each study site that we did not measure also might influence spatial interactions between ferrets and badgers.Our primary focus was the evaluation of spatial interactions, and consequently, we modeled the probability of use by ferrets in relation to the space use by badgers. Our candidate models included the intrinsic and extrinsic predictor variables and their 2-way interactions with the variable that represented intensity of badger use. Interactions between the intensity of badger use and colony-level attributes (colony size and relative density of prairie dogs) would be a consequence of how badgers use prairie dog colonies, which would vary spatially across colonies. To determine whether sex or age categories of badgers influenced ferrets differently, we developed a set of models for adult female badgers, adult male badgers, and all badgers. Each set contained 12 candidate models that included the effects of study site, sex and age of the ferret, relative density of prairie dogs, size of prairie dog colonies, and the variable for relative intensity of badger use. Each set of models only differed by the variable that represented the intensity of badger use . Because of small sample sizes, we did not evaluate models that contained an age*sex covariate. The effect of badger presence was modeled as interactions with the other variables, and consequently, must be interpreted in relation to the other variables. We compared models using quasilikelihood under the independence model criterion QIC; Pan to identn\u00a0=\u00a08, n\u00a0=\u00a017 colony-years, We recorded 707 locations from 15 badgers , and both included intensity of use by adult female badgers rather than adult males or all badgers are considered to have an organizing force on black kites (Milvus migrans), which minimize their predation risk by establishing territories outside of the influence of owl pairs , even at low densities, influenced the spatial patterns of bottlenose dolphins (Tursiops aduncus), such that dolphins avoid using areas that were used by tiger sharks even though food was abundant (Heithus and Dill Odocoileus virginianus; Mech Cervus elaphus; Fortin et\u00a0al. One explanation for the difference in apparent influence of female and male badgers on space use by ferrets is that intersexual differences in space use result in spatially concentrated versus dispersed predation risk. Badgers exhibit distinct intersexual differences in social behavior and space use: Female spatial organization is shaped largely by the distribution of food resources while male spatial patterns are determined primarily by the distribution of females, which results in the territories of females being smaller than those of males (Messick and Hornocker Alces alces, Oehlers et\u00a0al. Ovis canadensis, Berger Acinonyx jubatas, Durant Spatial responses of intraguild prey in our study differed between the sexes, with female ferrets exhibiting greater avoidance of badgers than males. Female ferrets are potentially more vulnerable to predation than male ferrets because they often need to minimize the risk of predation on neonates; dens of maternal females frequently contain prey provisioned for offspring, and badgers are attracted to burrows containing dead prairie dogs Biggins . SimilarAvailability of food resources also influenced spatial interactions between intraguild predators and prey in our study system. We expected that avoidance of badgers by ferrets would decrease as densities of prairie dogs declined because ferrets would accept higher risks of predation in exchange for access to adequate food resources (e.g., Lima and Dill Greater density of prairie dogs not only increases the amount of available food for ferrets, but the concomitant increase in prairie dog burrows also would lower risk of predation by badgers because of increased availability of refuges. Prey typically responds to predator presence by increasing use of refuges (Sih et\u00a0al. Contrary to our expectations, age did not strongly influence models of space use by intraguild prey. We expected adult ferrets to exhibit greater avoidance of badgers than juveniles because other research has suggested a social hierarchy in this species (Forrest et\u00a0al. We also expected that ferrets occupying smaller colonies would exhibit less spatial avoidance of badgers than ferrets on larger colonies because increased availability of space often is associated with lower predation risk in traditional and intraguild predator\u2013prey systems Suhonen . Our resOur results rely on a couple of important assumptions. One assumption is that we accurately characterized areas used by badgers. The number of locations used to estimate UDs for some badgers is slightly lower than 30, the number typically suggested to estimate the home range of an organism (Seaman et\u00a0al. Our alternative hypothesis \u2013 that space use patterns of ferrets and badgers would overlap at fine spatial scales \u2013 was only supported when interactions between adult female badgers and ferrets occurred at relatively high density of prairie dogs. These results might appear to contradict recent research suggesting that badgers concentrate their activities in areas frequently used by ferrets and that they actively pursue ferrets (Eads et\u00a0al. Our study has several implications for understanding spatial interactions between other sympatric species. First, we documented complex patterns of avoidance in an intraguild predator\u2013prey system, but these patterns would likely have been undetectable if the sex of both predators had not been considered explicitly. Second, intersexual differences in patterns of space use also likely shaped spatial interactions between the species by creating differences in distribution of predation risk. Because reproductive strategies of males and females often result in sex-specific patterns of space use, this factor is likely to shape spatial interactions in many ecological systems. Third, density of food resources influenced observed spatial interactions between the two intraguild competitors, which suggests that the strength and direction of interactions will vary over space and time with extrinsic factors. These results support the need for interpreting spatial relationships in the context of both intrinsic and extrinsic influences on sympatric species."} +{"text": "We sincerely appreciate the studies performed about mobilization in extracorporeal membrane oxygenation (ECMO) patients . An incrECMO is a life-saving tool although it comes with many different complications, including cannulation-related bleeding, ischemia of ipsilateral extremity, vascular injury and infection . Can theAbrams and colleagues suggest subclavian artery cannulation for easier mobilization, although this also sometimes comes with unexpected complications. We would like to report a patient treated with ECMO in our clinic who had some complications never reported previously in the literature. ECMO was established through the subclavian artery with a flow of 3,500\u00a0ml/minute but the patient had cerebral and pupil edema, so the flow was decreased to 2,000\u00a0ml/minute and the right arm was elevated \u2013 the symptoms regressed spontaneously. The patient was weaned from ECMO 8\u00a0days later and decannulated.Cannulation sides may have advantages over one another although this does not suggest that we should go for a routine subclavian cannulation as indicated by Abrams and colleagues. We would like to hear opinions and experiences about similar complications regarding subclavian cannulation.Darryl Abrams, Matthew Bacchetta and Daniel BrodieWe thank Gokalp and colleagues for raising the issue of the optimal cannulation configuration for patients requiring venoarterial ECMO in whom mobilization is anticipated, and for highlighting potential issues with subclavian arterial reinfusion . AlthougECMO: Extracorporeal membrane oxygenation.DB reports receiving research support from Maquet Cardiovascular including travel expenses for research meetings and anticipated support for upcoming studies and compensation paid to Columbia University for research consulting; he receives no direct compensation from Maquet. DB is also a member of the Medical Advisory Board for ALung Technologies and compensation is paid to Columbia University; he receives no direct compensation from ALung Technologies. MB reports receiving research support from Maquet Cardiovascular including travel expenses for research meetings and anticipated support for upcoming studies and compensation paid to Columbia University for research consulting; he receives no direct compensation from Maquet. The remaining authors declare that they have no competing interests."} +{"text": "Memory formation in the brain is thought to rely on the remodeling of synaptic connections which eventually results in neural network rewiring. This remodeling is likely to involve ultrathin astroglial protrusions which often occur in the immediate vicinity of excitatory synapses. The phenomenology, cellular mechanisms, and causal relationships of such astroglial restructuring remain, however, poorly understood. This is in large part because monitoring and probing of the underpinning molecular machinery on the scale of nanoscopic astroglial compartments remains a challenge. Here we briefly summarize the current knowledge regarding the cellular organisation of astroglia in the synaptic microenvironment and discuss molecular mechanisms potentially involved in use\u2010dependent astroglial morphogenesis. We also discuss recent observations concerning morphological astroglial plasticity, the respective monitoring methods, and some of the newly emerging techniques that might help with conceptual advances in the area. GLIA 2015;63:2133\u20132151 Much to the surprise of many classically trained neurophysiologists, over the past few decades experimental evidence has emerged implicating electrically passive astroglia in rapid information transfer and possibly memory trace formation in the brain . Their leaf\u2010like, ultrathin shapes correspond to a large surface\u2010to\u2010volume ratio thus suggesting the propensity to local chemical compartmentalization Freeman, . During In rats, astrocytes start extending fine filopodia for up to postnatal day 14 (PND14). By PND21, astroglia in the brain develop highly ramified processes subunits also change their expression levels even though NMDAR appears to dominate ionotropic glutamate receptor types in adult mouse astroglia. Metabotropic glutamate receptor mGluR5 is widely present in young animals but its expression declines with age while the mGluR3 subtype becomes dominant in adult mice and in humans to high\u2010affinity uptake by astrocytes potassium buffering. Aquaporin 4 appears after PND20 and its levels increase afterwards, according to single\u2010cell gene expression data when in fact it simply transforms into a larger and thinner leaf\u2010like structure: the latter might appear as shrinkage simply because fluorescence generated by the ultrathin transformed structure may fall below the detection threshold. Finally, it might not always be possible to separate molecular re\u2010arrangement or movement of genetically encoded fluorescent markers (such as membrane\u2010bound proteins) within astroglial compartments from genuine morphological alterations. Therefore, caution should be exercised in optical monitoring studies to avoid registration of spurious morphological changes on the nanoscale.in vivo also leads to PAP structural plasticity in the dentate gyrus of the hippocampus IP3\u2010dependent has been identified as a factor mediating morphological changes in astrocytes, by initiating outgrowth of highly ramified processes into the neuropil in in situ remains to be established. It appears that advances in super\u2010resolution microscopy will play a major role (see below) in our understanding of the exact molecular make\u2010up of PAPs and its dynamic changes.Cell adhesion molecules appear to be another logical candidate device to mechanistically explain the underpinning of structural astroglial plasticity. One of the most studied adhesion molecules that could be involved in direct signaling between astrocytes and neurons has been EphA4 around axonal terminals, dendrites, and glial membranes is shaped nonuniformly featuring various tunnels and sheets and Cx30 is beyond resolution of conventional optical microscopy (200\u2013300 nm). Furthermore, so far no reliable marker exists to enable distinguishing astrocyte processes that contact synapses from those that do not . Instead, here, we will briefly discuss respective applications that could be relevant to studying astroglia.The progress in astroglial research has been somewhat hampered by our current inability to monitor and probe, in real time, the organization and function of ultrathin processes that represent the bulk of astroglial morphology Stimulated\u2010emission depletion (STED) microscopy uses conventional excitation which is partly quenched (depleted) at its periphery by the doughnut\u2010shaped focal illumination: this reduces the emission spot thus boosting resolution beyond the diffraction limit or stochastic optical reconstruction microscopy (STORM), which are based on reconstructing the point source of fluorescence (often one molecule) from its detected image which is \u201cblurred\u201d by diffraction (Betzig et al., A general super\u2010resolution approach conceptually related to PALM enables tracking of individual molecules conjugated to bright nanoparticles, such as quantum dots, in live cells (reviewed in (Medintz et al., 2+\u2010dependent manner (Henneberger et al., 2+ within a certain dynamic range by triggering certain cellular cascades should reproduce such effects (Agulhon et al., 2+ rises in astrocytes could also boost the expression level of glutamate transporters (Devaraju et al., 2+ signals could be required for morphological changes in PAPs (Molotkov et al., A growing body of experimental evidence suggests that astrocyte signaling can, at least in certain conditions, influence excitatory synaptic transmission and its use\u2010dependent plasticity in a Cain situ. Clearly, further implementation of super\u2010resolution techniques in organised brain tissue should help to bridge the knowledge gap in our understanding of the molecular micro\u2010physiology of astroglia and its role in information processing of brain networks.The PAP remodeling, with or without local rearrangement of glutamate transporter proteins, is capable of altering the efficiency of extrasynaptic glutamate clearance or escape (Benediktsson et al.,"} +{"text": "SD]) failed to report sample sizes. The problem affects the ability to verify the accuracy of any analysis, to repeat methods used, and to assimilate the study findings into powerful and useful meta\u2010analyses. The problem is common in a variety of ecological topics examined to date, and despite previous calls for improved reporting and metadata archiving, which could indirectly alleviate the problem, there is no indication of an improvement in reporting standards over time. Here, we call on authors, editors, and peer reviewers to consider repeatability as a top priority when evaluating research manuscripts, bearing in mind that legacy and integration into the evidence base can drastically improve the impact of individual research reports.Despite the scientific method's central tenets of reproducibility (the ability to obtain similar results when repeated) and repeatability (the ability to replicate an experiment based on methods described), published ecological research continues to fail to provide sufficient methodological detail to allow either repeatability of verification. Recent systematic reviews highlight the problem, with one example demonstrating that an average of 13% of studies per year (\u00b18.0 [ A central tenet in scientific research is that theories should be testable and refutable Popper and expesystematic reviews; robust approaches to reviewing existing research evidence using strict methods set out by review coordinating bodies, such as the Cochrane Collaboration (www.cochrane.org) or the Collaboration for Environmental Evidence . The opportunities for reaching new overall conclusions on pressing fundamental and applied research questions have grown considerably with the availability of new statistical approaches for meta\u2010analysis. However, through our experience of critical appraisal of large bodies of evidence, we commonly find published academic research articles that do not provide sufficient methodological detail for studies to be repeated. For example, a systematic map of the environmental and socioeconomic impacts of high altitude land abandonment identified 111 of 190 studies as being described with low methodological detail prevent reliable studies from being included in statistical meta\u2010analysis Haddaway , missingSeveral solutions exist where primary research is missing methodological information:contact corresponding authors with requests for informationcheck whether related manuscripts have been published for the same experiment and extract methodological details where methods can be reliably assumed to be the sameperform sensitivity analysis in meta\u2010analysis to examine the influence of studies missing vital methodological information http://srdr.ahrq.gov] or postpublication platform such as PubPeer (https://pubpeer.com) making it easier for future readers to findonce found, publish missing information in a dedicated database , influential climatic conditionslaboratory studies: controlled conditions experimental setting2study date(s) and duration3selection procedures for sample selection and treatment allocation 4level of true replication5level of subsampling (number and repeat or within\u2010replicate sampling)6sampling precision study spatial scale 9outcome measurement methods and equipment10description of any data manipulation, modeling, or statistical analysis undertakenThese are not onerous requirements, and despite being the subject of previous calls to adequately document archived data, we must reiterate the need for this information again to ensure legacy of primary research is maximized. We also advocate calls for better reporting of summary data that permit meta\u2010analysis (e.g., Haddaway None declared."} +{"text": "COMT Val158Met locus, which had been tied to frontal cognitive function, trait anxiety and brain metabolic responses to emotional stimuli, was linked to the ability of acute pain to release endomorphin and displace 11C carfentanil binding following a pain challenge [COMT haplotype linkage is better understood via the epistatic interaction of alleles to alter translatability of COMT mRNA. GTP cyclohydrolase (GCH1) represents a second gene influencing pain . The gene was identified as a candidate via array expression in the rat neurotomy model. In humans, a functional haplotype predicting GCH1 mRNA expression in lymphoblastoid cell lines. Consistent with the rat data, the high expression diplotype was linked to both clinical post lumbar surgery leg pain and to experimental pain in a large population of controls. Continuing the theme that genes that alter emotion can also alter pain responses, a functional polymorphism in the NPY (neuropeptide Y) propmoter alters both amygdala and hippocampal emotional responses as well as predicting 11C carfentanil displacement after a pain challenge [Pain is a pervasive stressor often experienced chronically, and strongly modulated by the brain\u2019s emotional circuitry. In recent years functional polymorphisms at several genes have been linked to pain response, encouraging the idea that both pain response and other aspects of emotionality can be better understood by genetic studies of acute and chronic pain, as illustrated by studies performed with several genes, two of which also alter anxiety and emotional responses. The functional hallenge ."} +{"text": "Human and environmental genotoxicity biomonitoring studies involving exposure to glyphosate-based formulations (GBFs) were reviewed to complement an earlier review of experimental genotoxicity studies of glyphosate and GBFs. The environmental and most of the human biomonitoring studies were not informative because there was either a very low frequency of GBF exposure or exposure to a large number of pesticides without analysis of specific pesticide effects. One pesticide sprayer biomonitoring study indicated there was not a statistically significant relationship between frequency of GBF exposure reported for the last spraying season and oxidative DNA damage. There were three studies of human populations in regions of GBF aerial spraying. One study found increases for the cytokinesis-block micronucleus endpoint but these increases did not show statistically significant associations with self-reported spray exposure and were not consistent with application rates. A second study found increases for the blood cell comet endpoint at high exposures causing toxicity. However, a follow-up to this study 2 years after spraying did not indicate chromosomal effects. The results of the biomonitoring studies do not contradict an earlier conclusion derived from experimental genotoxicity studies that typical GBFs do not appear to present significant genotoxic risk under normal conditions of human or environmental exposures. BCblood cellsBMblood monocyte cellsBNMNbinucleated cells with micronucleiCBMNcytokinesis-block micronucleusCAchromosomal aberrationsGBFglyphosate-based formulationMNmicronucleusMOMNmononuclear cells with micronucleiSCEsister chromatid exchange8-OHdG8-hydroxydeoxyguanosinein vivo mammalian micronucleus and chromosomal aberration assays. Although some positive results for glyphosate and GBFs were reported in DNA damage assays and for the micronucleus endpoint for GBFs in non-mammalian studies, the positive results were associated with high dose levels and/or toxic effects. The preponderance of negative results in core assays supports the conclusion that reports of DNA damage or non-mammalian micronucleus effects are likely to be secondary to cytotoxicity rather than indicative of DNA-reactive mechanisms. This conclusion is consistent with and supported by a recent review of 14 experimental rodent carcinogenicity studies of glyphosate that indicated a weight of evidence that there was no carcinogenic effect related to glyphosate treatment have been extensively studied for their toxic properties. One of these toxic properties is genotoxicity and there has been a recent extensive review of glyphosate and GBF experimental genotoxicity studies . This rereatment .The earlier \u2122 formulation) that also contained searchable terms which indicated that human or environmental genotoxicity studies were performed or micronucleus endpoints). Emphasis was placed on publications in peer-reviewed journals. Abstracts or other sources with incomplete information were not considered. Reviews without original data were not considered for evaluation; however, these reviews were examined to determine if there were any cited publications that had not been detected in the literature searches.The published studies for review consideration were identified by literature searches for published reports containing references to glyphosate or GBFs , most of the studies did not present comprehensive detailed data on these confounding factors. Some of the studies had moderate to fairly large differences in gender distribution . One facHuman exposures were usually characterized by self-reporting of the types of pesticides used as determined by survey of the exposed population or by more general use information. Additionally, the use of personal protective equipment may have been indicated. In most cases pesticides were characterized only by the active ingredient and not as a specific formulation. In some cases the extent of individual pesticide use was described as a frequency of use and/or amount of use but in most cases there were exposures to multiple pesticides. There are only a few biomonitoring studies where some assessment of the specific effects of exposures to GBFs can be inferred from the circumstances or exposure data presented. The identified studies only rarely attempted to estimate actual amount of exposure to specific pesticides or to evaluate exposure by chemical monitoring. No cases of chemical monitoring of exposure to glyphosate or GBFs were encountered in the genotoxicity biomonitoring studies. Uncertainty in extent and amount of exposure and dose is a major limitation in interpretation of the genotoxicity biomonitoring studies of pesticide exposure.The most common endpoints employed in the biomonitoring studies were the CBMN assay on cultured lymphocytes (six human studies), the micronucleus assay on buccal cells (six human studies) and the comet assay on blood cells . Other endpoints included measurement of sister chromatid exchange (SCE) in cultured lymphocytes (three human studies), chromosomal aberration in cultured lymphocytes (three human studies), erythrocyte micronucleus assays , and bacterial reversion (Ames test strains) on urine (one human study). Two human studies measured DNA alterations (bulky adducts and oxidative DNA damage).The CBMN assays generally used similar standardized methodologies for culture, including addition of cytochalasin B at 44 h after phytohemagglutinin stimulation. The studies used whole blood rather than isolated leukocytes for culture and scored 1000 or 2000 binucleated cells per subject for micronuclei. Referent population frequencies of binucleated cells with micronuclei (BNMN) ranged from about 1.8 to 9 per 1000 which seems reasonably close to a mean of 6.5 per thousand with an inter-quartile range of 3\u201312 per thousand observed for a large number of normal subjects from many laboratories .The buccal micronucleus assays generally followed recommendations for number of cells scored with 1000\u20133000 cells scored per subject. There is a recommendation for the use of DNA-specific staining for this assay such as Feulgen-Fast Green . Two of The comet studies generally used similar standard methodology for cell lysis, alkaline treatment, and staining of DNA. One study used isolated leukocytes but the Most of the endpoints employed in the biomonitoring studies involve visual scoring for endpoints or visual selection of comets for image analysis. There are consistent and numerous recommendations that slides for scoring for these endpoints should be coded so that the scorer is not aware of the treatment conditions, individual or groups to which the slides belong e.g., . HoweverA number of human monitoring studies in Another set of human biomonitoring studies involved exposures to multiple pesticides but indicated frequency of exposure to specific pesticides that included a significant proportion of the exposed population using GBF . One of Three other studies with multi-pesticide exposure including significant frequency of GBF use in the exposed populations reported positive genotoxic effects for the lymphocyte SCE endpoint , the CBMAs indicated in One of these studies reported induction of blood cell comet effects on a Northern Ecuadorian population living within 3 km of areas sprayed with GBF for illicit crop eradication . The sprThe Subsequent to the original Another publication reportedThe human lymphocyte culture and scoring methodology employed in the The publication reported a small statistically significant increase in the frequency of BNMN in samples collected from people living in three regions within 5 days after spraying of GBFs compared with values for samples collected just before spraying. The publication also indicated a statistically significant increase of micronucleated mononuclear cells (MOMN) in the immediate post-spraying samples for two regions . In the samples taken 4 months after spraying, a statistically significant decrease in BNMN frequency compared to immediate post-spraying frequency was observed for one of the spraying regions (Nari\u00f1o) but the other sprayed regions did not exhibit a statistically significant difference in BNMN frequency between the immediate post-spraying and 4-month samples.Although the increases in BNMN frequencies in the post-spraying samples of the three regions suggest an effect from GBF exposure, more detailed consideration of exposure factors raises significant questions about this conclusion. The populations in each of the sprayed regions self-reported exposure to the spray . For all three sprayed regions, there was no statistically significant difference in BNMN frequency between those self-reporting spraying exposure and those self-reporting no spraying exposure. The largest percentage post-spraying increase in BNMN frequency was reported for Valle del Cauca but only 1 of 26 people from this population self-reported spray exposure. Also, it was noted that GBF spraying in Valle del Cauca was at a rate significantly lower than that in Nari\u00f1o and Putumayo . The lack of clear correlation between self-reported exposure and BNMN increases after regional GBF spraying led to some caution in interpretation by the authors. The The human genotoxicity biomonitoring studies that specifically address GBF effects appear to have some evidence for lack of persistent genotoxic effects, especially under normal conditions of exposure. One study suggests lack of DNA oxidation effects with GBF application and a study employing CBMN does not show statistically significant effects correlating with self-reported exposure to GBF spraying. One study reported effects on the blood cell comet endpoint following exposures to very high levels of GBF spraying which apparently were sufficient to elicit significant clinical signs of toxicity. However, a subsequent study conducted 2 years after GBF spraying using much larger populations did not detect chromosomal alterations or an increase in chromosomal fragility indicating that the comet effects did not appear to be manifested as persistent genotoxic effects. It should be noted that there is growing appreciation that comet endpoint effects in biomonitoring studies may result from indirect mechanisms such as inhibition of DNA repair, perturbation of cytokinesis, and oxidative stress . It seem\u00ae fungicide.There are two publications related to environmental biomonitoring for genotoxic endpoints. One study using blood cell comet and erythrocyte MN endpoints was conducted on samples from meadow voles living on or near golf courses where pesticides had been applied . DiffereA second publication reported results for the erythrocyte MN assay applied to fish collected from several dams in Brazil . GBF wasTwo environmental genotoxicity biomonitoring studies conducted on a mammalian species and fish species were not informative about possible environmental genotoxic effects of GBFs. Both studies involved exposures or potential exposures to multiple pesticides without characterizing the relative extent of GBF exposure.There have been a fairly large number of human genotoxicity biomonitoring studies where some exposure to GBFs was reported. Several of these studies were not informative about effects of GBF exposure because there was exposure to multiple pesticides and reported GBF exposure frequencies were low or very low. Another set of human biomonitoring studies were also not informative about possible genotoxic effects of GBF exposure because these studies listed exposure to large numbers of pesticides (10 to more than 30) in the exposed population without indicating the frequency or extent of exposure to any of the pesticides. Although positive genotoxic endpoint effects were observed in most of these studies no conclusions can be made regarding which pesticide exposures were responsible for the effects.A third set of human genotoxicity biomonitoring studies involved exposures to multiple pesticides but did indicate significant frequency of GBF exposure in the populations. One of these studies did not find statistically significant effects for the lymphocyte CBMN endpoint in the exposed population compared to a referent population. This study offers some limited evidence for lack of significant, detectable effects on this endpoint for human exposure to any of the pesticides with significant exposure frequencies, including GBF, but the population sizes exposed were low. Three other studies reported positive genotoxic endpoint effects but the exposure data and endpoint data presented did not permit attribution of these effects to any specific pesticide exposure.Finally, there are data from four human genotoxicity biomonitoring studies that provide information on GBF exposure effects. A study of oxidative effects on blood DNA indicated that observed increases in oxidative DNA damage did not statistically correlate with last season frequency of GBF application. These results provide limited evidence for this indirect genotoxic mechanism not operating at a significant level in humans using GBFs. Three studies involved measurement of genotoxic endpoints in human populations living in regions where GBFs were applied by aerial spraying. One study used a longitudinal design involving populations in regions of aerial GBF applications where samples were taken before, within 5 days and 4 months after GBF spraying. Statistically significant post-spraying increases for the CBMN endpoint were observed in these populations. However, the increases were not significantly correlated with self-reported exposure to the sprays or with the spraying application rate. Application of well-respected criteria for relating epidemiology cause and effect to theseThe overall conclusion from the human biomonitoring studies is that none of the reported positive results for studies involving exposure to multiple pesticides present evidence specifically relating GBF exposure to these results. There is some limited evidence for lack of oxidative DNA damage from normal human GBF exposure. The studies of populations in regions where GBF spraying occurred do not provide clear evidence correlating exposure to chromosomal effects such as aberrations or induction of micronuclei. The single study result of DNA damage comet effects in a population presumably exposed to GBF aerial spraying might well have been due to abnormally high toxic exposures to the GBFs rather than a DNA-reactive mechanism and does not indicate genotoxic risk to humans under normal exposure conditions.An earlier review of a very extensive number of experimental genotoxicity studies of glyphosate and GBFs concluded that there is a convincing weight of evidence supporting the lack of genotoxic potential for both glyphosate and typical GBFs in core gene mutation and chromosomal effect endpoints and that observations of DNA damage effects were likely to be secondary to toxicity . This ea"} +{"text": "The pattern of craniomaxillofacial fractures seen in childrenand adolescents varies with evolving skeletal anatomy andsocioenvironmental factors. The general principles of treatingmandibular fractures are the same in children and adults:Anatomic reduction is combined with stabilization adequateto maintain it until bone union has occurred. But recognitionof some of the differences between children and their adultcounterparts is important in long-term esthetic and functionalfacial rehabilitation as effect of injury, treatment providedhas a great influence on their ensuing growth. In childhood a generally impetuous nature and adventurousspirit combine to encourage participation in physicalactivities with little thought to immediate consequences, stillparadoxically facial injuries in children are much lesscommon than adults. Above all the immense capacity forhealing in children within the shortest possible time withminimum of complications, the assistance that growth cangive, and the inherent ability to adapt to new situation arequite different from what we see in adults. The principlesinvolved in treatment of facial injuries are same irrespectiveof the age of patient. However the techniques in childrenare necessarily modified by certain anatomical, physiologicaland psychological factors .This article aims to cover comprehensively the reviewof these modifications and preferable options for themanagement of mandibular fractures in these children.Mandibular fractures are the most common facial skeletalinjury in pediatric trauma patients.4Young bone possesses unique physical properties thatcoupled with space occupying developing dentition give riseto patterns of fracture not seen in adults. Bone fragments inchildren may become partially united as early as 4 days andfractures become difficult to reduce by seventh day.Growth abnormalities may occur as result of fracturedislocation of condyle due to elimination of \u2018functionalmatrix\u2019 of lateral pterygoid function, trismus or ankylosis.Methods of dentoalveolar stabilization also require somereforms. Between 2-4 years sufficient number of fullyformed deciduous teeth are present facilitating applicationof arch bars or eyelet wires. 5 to 8 years age old group maypresent with some difficulty owing to loss or loosening ofdeciduous teeth. The shape and shortness of deciduouscrowns may make the placement of circumdental wires andarch bar slightly more difficult in children. However thenarrow cervix of tooth in relation to crown and rootsprovides better retention of wires as in Ivy loops or stoutwires. Mandibular cortex is thinner in children so care mustbe taken to avoid pulling a wire through the mandible whenplacing circummandibular wiring for splints .While doing open reduction and fixation presence oftooth buds throughout the body of mandible must be aconsideration as trauma to developing tooth buds may resultin failure of eruption of permanent teeth and hence narrowalveolar ridge. However according to Koenig et al 82% oftooth buds in line of fracture erupted normally regardless ifmethod of treatment was open reduction with rigid fixationor closed reduction.The emergency management of facial trauma in pediatricpopulation also needs extra-consideration. Clinical signs ofshock may occur with even insignificant amounts of rapidblood loss due to small blood volume. Because of smallsize of airway laryngeal edema or retroposition of base oftongue may produce sudden obstruction. Tracheostomy ifrequired should be done using vertical incision avoidingfirst tracheal ring and high lying left innominate vein.Majority of body and symphysis fractures in children areundisplaced because of elasticity of mandible and embeddedtooth buds that hold the fragments together \u2018like glue.\u2019Bilateral fractures of anterior mandible occur with muchgreater frequency in children than in adults. A commonfracture pattern not seen in adults run from upper borderbeside the last tooth anteroinferiorly to the lower border inregion of canine. These fractures are generally greenstickand require no active treatment.Slight occlusal discrepancies resulting from lack ofperfect reduction correct spontaneously as permanent teetherupt and bone undergoes remodeling with function.Nondisplaced body or symphysis fractures without malocclusioncan be treated by close observation, blenderized dietand avoidance of physical activity. If displaced closedreduction and immobilization is performed. Exact methodof immobilization depends on child\u2019s chronologic age andstate of dental development. In under 2 years age very littl anchorage can be taken from teeth as most are unerupted orincompletely formed. In mixed dentition only 6 years molarsare adequate for circumdental wires. If possible arch barsare placed and elastic immobilization is done. If teeth areinadequate then fracture site is immobilized with gunningsplint or lingual splint. Intermaxillary fixation is used if splintstabilization is not enough as in fracture of posterior bodybeyond point of extension of splint. Appliance should befixed in place using circummandibular wires one on eitherside of fracture and two wires to add stability to the splint.If IMF is also required then wires can be added fromcircummandibular wires to wires at piriform region orzygoma. Splint should be left in place for three weeks.Alternatively if possible monocortical plate at inferior bordercan be placed. Short (4 mm) and broader screws 2 mmshould be used as they are more retentive in pediatric bone.The common occurrence of a combined parasymphysealand condylar fracture will warrant a more stable form ofparasymphyseal fracture fixation (miniplates and screws)so that early active mandibular range of motion with TMJfunction can occur.Fractures at angle proximal to tooth bearing area are notsufficiently immobilized with splint alone so closedreduction and intermaxillary fixation for 3 weeks arerequired. When tightening splints sawing of wire throughthe mandible has to be avoided.When a mandibular angle fracture occurs in the presenceof a condyle fracture, the combined forces may be significantenough to cause displacement unless ORIF at the anglefracture is carried out. Plating at the tension-band zone isnot recommended in the mixed dentition. In open reductionfor less than 5 years it is possible to injure tooth buds nearangle when placing intraosseous wire or screws whichrequires caution.Trauma to chin producing temporomandibular joint injuryis frequent occurrence in childhood. Mandibular condylein children is short, stout and highly vascular with thincortical plate. The impact displaces condyle posterosuperiorlyagainst skull base thus leading to range of injuryfrom capsular tear, hemarthrosis to fracture of condylar heador neck. Occasionally a crush injury to condyle can producecomminuted fracture. Children less than 3 years of age with trauma to condyle are at greatest potential for growth disturbanceespecially due to ankylosis. Inadequate or overtreatmentmay lead to growth retardation or excess while excessiveimmobilization may lead to mandibular hypomobility.Unlike adults a child with fracture condyle frequentlypresents with midline deviation away from rather thantoward the injury owing to swelling or hematoma withinthe joint. The location and degree of displacement ofcondylar fractures in children in primary and mixed dentitionstage is not that useful variable for developing treatmentplan.1In intracapsular injuries especially in less than 3 year ofage as chances of ankylosis are high mandibular exercisesand jaw stretching should be started early to avoid suchcomplications.In children in primary and mixed dentition stage withunilateral condylar fractures analgesics and blenderized dietfor 5-7 days is usually adequate treatment. Minormalocclusions will correct spontaneously. Deviation onopening is treated with midline opening exercises.In permanent dentition stage with unilateral or bilateralcondylar fractures especially if dislocation present withpersistent malocclusion after 7-10 days of intermaxillaryfixation open reduction to restore ramus length and toprevent progressive deformity must be considered as in olderchildren there is less capacity for bone to adapt and remodel.Restoration of normal symmetric jaw function providesbest chance for normal growth.Open reduction of a condyle fracture may be warrantedin a child in some instances. Indications may include thefollowing:Displacement into the middle cranial fossa.Unacceptable occlusion after a closed technique trialhas failed or mechanical obstruction present.Avulsion of the condyle from the capsule.Bilateral fractures of the condyles with comminutedmidface fractures.Penetrating wound is present.In all other cases conservative nonoperative managementproduces equally acceptable result with minimalcomplication.Decreased vertical height of mandibular body and alveolusmay occur after fracture of horizontal ramus of mandible ifteeth are lost due to injury or hardware through tooth buds.Contour defects may occur due to severely comminuted orcompound fractures when bone undergoes resorption duringremodeling. In general however mandibular body fracturespresent little risk for long-term growth abnormalities.Unilateral and bilateral condylar fractures may howevercause mandibular asymmetry and retrognathism with openbite respectively. Leake et alLund 6 carried out a prospective study of 38 patientswith subcondylar fractures to study the effect of injury onmandibular growth and the extent of remodeling that tookplace. Of 38 patients 32 were 12 years or less and 6 were13-17 years. There were 11 bilateral fractures and 27 unilateral fractures. 35 patients were treated with closeobservation alone or in combination with intermaxillaryfixation. Three patients had open reduction and fixation withK-Rod (1) and condylectomy (2). In Lund\u2019s study mandibulargrowth was generally greater on fractured side thannonfractured side so that the fractured ramus which wasinitially shorter had greater incremental growth rate so thatpossible disproportion between two sides reduced with time.This was evident when measuring distance between chinpoint to condyle.Three types of mandibular growth patterns were noted:Compensatory growth without overgrowth. Fracturedside grows more but in end remain somewhat shorterthan normal (13 of 27 patients).Compensatory growth with overgrowth. Fractured sitegrows longer than normal (8 of 27 patients).Dysplastic growth. Fractured site grows less sodifference is accentuated with time.Compensatory growth mainly occurred in patientsgrowing at time of injury.Lund also defined two groups on basis of pattern ofremodeling.Incomplete remodeling in which condyle was irregularor displacement remained at fracture site (12 of 49condyles).Complete remodeling (37 out of 49 condyles).He concluded that patients with displaced condyle hadgreater chance of incomplete remodeling. Successiveradiographs showed that remodeling consisted of resorptionand apposition. The process began at time of injury andcontinued for period of 5 to 49 months. When remodelingoccurred completely it consisted of resorption of proximalcondylar stump and outgrowth of bony process on ramusresembling normal condyle.Earlier most of the pediatric cases were treated withconservative measures or closed reduction techniques. Onlyrecently have the distinct advantages of accurate primaryrepair and the stable fixation of facial fractures been appliedto the rehabilitation of injuries in children too.Also, resorbable materials have been made available as afixation option for pediatric craniomaxillofacial fracturemanagement. According to Peterson with the exception ofmandibular condyle fractures judicious use of ORIF ispreferable to the closed reduction and immobilizationtechniques with splints when treating fractures in thedeciduous and mixed dentition.Mandibular fractures in children most commonly occur incondylar region, followed by parasymphysis and angle. Thefractures tend to be minimally displaced and in majority ofcases can be treated conservatively. Significantly displacedmandibular fractures are reduced and immobilized usingrigid internal fixation according to principles used in adults.Fractures in condylar region usually are treated usingnonoperative therapies as in most cases fracture heals andcondyle is remodeled with successful anatomic andfunctional results."} +{"text": "Venous anomalies of the thorax can occur in isolation or in association with complex congenital heart disease. The incidence of an absent right superior vena cava in the setting of a persistent left superior vena cava is very rare in the general population with only a dozen cases documented in the medical literature. Such venous anomalies can make for very challenging electronic cardiac device implantation. We report our challenging dual chamber pacemaker implant in a patient with such complex anatomy and focus on our implantation technique that helped achieve adequate lead positioning.A 73-year-old Caucasian female with degenerative complete heart block presented for dual chamber permanent pacemaker implant. Lead implantation was very challenging due to abnormal and rare vena cava anatomy; a persistent left superior vena cava drained directly into the coronary sinus and the right brachiocephalic vein drained directly into the left persistent superior vena cava as the patient had an absent right superior vena cava . Adequate right ventricular lead positioning was achieved following numerous lead-stylet manipulations and careful looping in the atria to redirect its trajectory to the ventricular apex.Abnormal superior vena cava development is uncommon and can lead to technical challenges when venous access is required during various interventional procedures. Pre-operative imaging can help identify such challenging anatomy allowing appropriate operative planning; careful patient selection is warranted for venography given the risk of contrast nephrotoxicity. Venous anomalies of the thorax can occur in isolation or in association with complex congenital heart disease. The incidence of an absent right superior vena cava (SVC) in the setting of a persistent left SVC is very rare in the general population with only a dozen cases documented in the medical literature. We report our challenging dual chamber pacemaker implant in a patient with such complex anatomy and focus on our implantation technique that helped achieve adequate lead positioning. We also review the medical literature on this topic.A previously healthy 73-year-old Caucasian female presents to the clinic with a history of progressive fatigue and dyspnea on exertion over the past couple of months. She denied angina, palpitations, syncope or any other associated symptoms and did not have any cardiovascular disease risk factors. On examination, she was stable and in no distress. Her blood pressure was 148/66\u00a0mmHg with a regular pulse of 48 beats per minute. She had no clinical evidence of heart failure on cardiovascular examination but was found to have an S4 on auscultation and cannon A waves on assessment of her jugular venous pressure (JVP). Her exam was otherwise unremarkable. Twelve-lead electrocardiogram during the clinic visit revealed complete heart block with a junctional escape rhythm at 49 beats per minute with right bundle branch block morphology; her 12-lead electrocardiogram 3\u00a0months earlier also revealed evidence of underlying conduction disease with a prolonged PR interval and right bundle branch block while in sinus rhythm. Trans-thoracic echocardiography displayed normal left ventricular size and function with mild degenerative changes of the mitral and aortic valves consistent with age related changes. There was no laboratory evidence of any metabolic or ischemic etiologies for the conduction disease. Given her age, clinical presentation, underlying conduction disease in the setting of no significant structural abnormalities on echocardiography, and lack of significant comorbidities, the patient likely had age related degenerative conduction disease. Significant coronary artery disease could not be excluded but was very unlikely given the lack of ischemic symptoms and cardiovascular disease risk factors along with normal cardiac size and function. She was arranged to have a permanent pacemaker implant for the symptomatic complete heart block and will have further risk stratification for possible coronary artery disease following the pacemaker implantation.The patient was brought to the operating theater following informed consent. Initial venous access and lead implantation was attempted across the left cephalic vein but aborted due to difficulty in delivering the pacing lead into the right ventricular (RV) cavity; the venous trajectory was that of a persistent left superior vena cava (SVC) draining directly into the coronary sinus. Right cephalic venous access was subsequently obtained and to our surprise there was an anomalous venous trajectory on the right implantation side as well . A persistent left SVC Figure\u00a0 can be iAnomalies of the right SVC can include drainage to the left atrium, low right atrial insertion, aneurysmal dilatation, and anomalous left brachiocephalic vein drainage to the right SVC . PersistLong-term prognosis in abnormal SVC development is good in the absence associated CHD . Venous The pacemaker lead implantation techniques we described above to overcome the challenges of this particular anomalous venous drainage case may not apply or be effective in all patients with similar or differing anatomy. It is therefore essential to evaluate the underlying venous anatomy at hand objectively using intra-operative venography or pre-operative imaging if possible when an anomalous anatomy is suspected. Once the anatomy is clarified, the technique that is the safest and least invasive for a particular scenario should be attempted first. Failure to implant pacing leads intravenously may warrant consideration for surgical epicardial lead implantation. In the setting of failed endocardial defibrillator lead implantation attempts, subcutaneous defibrillators can also be considered.Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.Abnormal SVC development is uncommon and can lead to technical challenges when venous access is required during various interventional procedures. Pre-operative imaging can help identify such challenging anatomy allowing appropriate operative planning; careful patient selection is warranted for venography given the risk of contrast nephrotoxicity.CHD: Congenital heart disease; CS: Coronary sinus; JVP: Jugular venous pressure; RA: Right atrium; RV: Right ventricle; SVC: Superior vena cava.The authors declare that they have no competing interestsJR, JB, and VK were equally involved in the care of the patient and case report composition. JR was the artist and creator of Figure"} +{"text": "Rae and Lonborg's findingsRae and Lonborg's finding that friendship motives had moderating effects in improving PWB among heavier FB users while connection motives had moderating effects in worsening PWB among heavier FB users illustrates that some social motives for use can be beneficial and others harmful. Alcohol research has repeatedly shown that while social-affiliative drinking motives are associated with lighter, non-problematic alcohol use, social-conformity motives are associated with more problematic alcohol use or decreasing negative emotions (coping or escape motives). One can imagine these two drinking motives having parallels in reasons for using FB. Moreover, such internal motives for FB use might be associated with elevations in psychopathological outcomes as has been shown in the substance abuse area , connection motives mainly operated as moderators for negative aspects of PWB . This suggests that positive reinforcement social motives may exert their moderating influences mainly in enhancing positive aspects of PWB whereas negative reinforcement social motives may exert their moderating influences mainly in intensifying negative aspects of PWB. Second, while friendship motives tended to interact with number of friends in predicting PWB outcomes, connection motives tended to interact with time spent using FB in predicting PWB outcomes. This suggests that positive and negative reinforcement social motives for FB use may exert their moderating effects on different aspects of FB use intensity in impacting PWB. These possibilities could be investigated more rigorously using structural equation modeling where all variables are entered into a single model.Rae and Lonborg's supplementary analyses at the level of individual PWB scales revealed some noteworthy patterns. First, while friendship motives mainly operated as moderators of FB intensity effects on While the Rae and Lonborg paper examined moderation, it is also possible to consider the potential role of FB use motives as mediator variables. In Cooper's motivatiAdditional work could be conducted on other potential moderator models . Kardefelt-Winther showed Pwhom to focus on for intervention delivery . Mediator studies identify what to focus on .Both moderator and mediator findings are important in directing intervention work. Moderator studies identify The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Plant evo-devo research aims to identify the nature of genetic change underpinning the evolution of diverse plant forms. A transcriptomic study comparing gene expression profiles in the meristematic shoot tips of three distantly related vascular plants suggests that different genes were recruited to regulate similar meristematic processes during evolution. The conquest of land by plants was one of the most significant events in our planet's history, and the radiation of diverse plant forms was underpinned by a series of ancient innovations in sporophytic shoot architecture. Whilst living bryophyte representatives of the earliest land plants have a single sporophytic axis that terminates growth by forming a reproductive sporangium A, todaysThe elaboration of these basic organ systems in vascular plants began around 400\u2013450 million years ago Partitatheca has a branching sporophytic axis that terminates in the formation of sporangia, and the earliest cooksonioid vascular plant fossils reiterate this basic construction For instance, the non-vascular fossil Vascular plant leaves have been classified into two types on the basis of morphological distinctions The architectural innovations underpinning the radiation of vascular plant forms reflect differences in the structure and activities of meristems at the growing shoot tips. Whilst flowering plants have meristems that are multicellular with zones and layers with well characterised and specialised functions, monilophytes have meristems that comprise a single stem cell capping a more rapidly proliferative region C [1]. LyWith the exception of rhyniophytes, there is little fossil evidence of meristem structure at the bryophyte\u2013vascular plant divergence et al. et al. have used a wider transcriptomic approach The work reported by Frank Zea mays) was selected to represent seed plants, and Equisetum and Selaginella were selected to represent monilophytes and lycophyts, respectively. Equisetum and Selaginella both have meristems with apical cell(s) capping a proliferative zone. Although both have microphyllous leaves, they acquired leaves by convergence, and most monilophytes have megaphylls that evolved by convergence with seed plant leaves , the meristem \u2018core\u2019\u2013 a proliferative zone subtending the apical cell(s)\u2013 and incipient leaves.The paper finds that in the meristems of all three species there are distinct gene expression profiles in each apical domain sampled. This supports a model whereby, as in flowering plants, the meristems of Selaginella and Equisetum, supporting a model whereby the gene networks regulating meristem function by and large followed independent evolutionary trajectories in each lineage (Equisetum expression profile shares some overlap with maize but not Selaginella, indicating potential homologies in meristem function that evolved after the lycophyte\u2013euphyllophyte divergence.The developmental gene families expressed in each domain are largely distinct between lineage D. The EqSelaginella and maize, but not shared by Equisetum, could indicate either that Selaginella independently recruited similar genetic networks to regulate meristem function or that the networks regulating meristem function were originally shared between vascular plants, but then significantly modified in Equisetum.A smaller overlap between the expression profiles of PINs, DEK1, and LOG1, which regulate auxin distributions PIN function in moss sporophytes can reproduce an architecture similar to Partitatheca fossils PINs in driving meristem function may be conserved to bryophytes (The data presented are consistent with distinct patterns of evolution in each extant vascular plant lineage and the wide divergence time between lineages. Exceptions indicate potential genetic homologies in vascular plant meristem function and include yophytes .et al. per se is constrained. A preliminary indicator is given by the necessity for PINs, DEK1, and cytokinin in driving gametophytic meristem functions in a moss (citations in This spotlight positions the Frank et al. The broad nature of the Frank"} +{"text": "Bilateral and isolated abducens nerve palsy is a rare initial presentation after aneurysms rupture. Several possible mechanisms including intracranial hypertension have been purposed. To date, there have been no reports with objective measurements to demonstrate the relationship between intracranial pressure and isolated abducens palsy in the setting of acute subarachnoid hemorrhage due to aneurysm rupture.A 50 year-old female presented with severe headache and bilaterally isolated abducens nerve palsy. A series of image studies showed a ruptured aneurysm over right internal carotid artery and posterior communicating artery bifurcation with minimal subarachnoid hemorrhage. Surgery of aneurysm clipping was performed and intracranial pressure monitoring was applied. Postoperatively no new neurological deficit developed but persistent headache and increased intracranial pressure measured by a fiber-optic device had been observed. The intracranial hypertension then decreased gradually with rapid recovery from the bilateral abducens palsy 7 days after the surgery. The relationship between postoperative intracranial pressure, subarachnoid hematoma and isolated abducens palsy are illustrated.The report demonstrated the clinical presentation of bilaterally isolated abducens palsy after an intracranial aneurysm rupture is related with the increased intracranial pressure level, rather than the hematoma compression to the nerve or vasospasm of pontine branches of basilar artery. A 50\u00a0year-old female visited our emergency department with sudden onset of severe headache, dizziness, vomiting and diplopia. Recent trauma history was denied. On examination, she was conscious with mild neck stiffness and significantly bilateral abducens palsy. There was no oculomotor neuropalsy. The brain computed tomography (CT) revealed vague hyperdensity along with left Sylvian fissure and bilateral frontal subdural space Fig.\u00a0a. MinimaThere was no other new neurological deficit postoperatively. Persistent headache with increased intracranial pressure (IICP), around 30\u00a0mmHg, had been noted Fig.\u00a0c. The fiTo the best of our knowledge, this is the first objective measurement report to demonstrate the relationship between IICP and abducens palsy after an intracranial aneurysm rupture.Ophthamoplegia is one of the clinical presentations of cerebral aneurysms but bilaterally isolated abducens nerve palsy is a rare initial presentation after aneurysms rupture (Koskela et al. Severe intracranial hypertension immediately after SAH causes inadequate cerebral perfusion and then leads to transient diffuse ischemic encephalopathy (Grote and Hassler For our case, direct aneurysmal mass effect, vasosopastic pontine branch of basilar artery and accumulated hematoma in the cisternal space were all not observed in a series of image studies or intraoperative findings. The high-pressure flow of CSF and brain swelling observed during the surgery revealed intracranial hypertension. Moreover, the postoperative observation showed the trend of IICP decrease and absence of delayed brain edema were compatible with that of abducens palsy recovery Fig.\u00a0d. The abIn this case report the CTA failed to show the aneurysm later recognized by MRI and DSA. The possible explanations were technical problems during the procedure or image reconstruction, thrombosed aneurysms and potential vasospasm. Other possible causes of a negative CTA study after diffuse SAH include an extremely small aneurysm, dissection or rupture of an atherosclerotic vessel wall (Agid et al. In conclusions, we are convinced that the bilaterally isolated abducens palsy after an intracranial aneurysm rupture in this case is related with the increased intracranial pressure level, rather than the hematoma compression to the nerve or vasospasm of pontine branches of basilar artery.The consent to publish has been obtained from the participant to report individual patient data."} +{"text": "Isolated in a transverse slice preparation, the preB\u00f6tC continues to generate under normal conditions \"fictive eupneic activity\" and at a lower frequency also \"fictive sighs\". The mechanisms generating the periodicity of fictive sighs as well as its intrinsic nature are largely unknown. Based on the experimental observations such as 1) sigh has the biphasic shape characterized as eupneic breath interrupted by augmentation with a second peak of activity, 2) the biphasic shape persists after blockage of inhibitory synapses, 3) the network is heterogeneous, we model PreB\u00f6tC network that reproduces both types of activities. The model contains three types of neurons: intrinsically tonic spiking, intrinsic bursting neurons and intrinsic quiescent. Neurons within the network are sparsely connected through randomly distributed excitatory synapses. In this model we propose that the mechanism of sigh generation is based on slow oscillations generated by glia. Moreover, the model predicts that depending on the modularity state of the system, the network exhibits sighs, does not exhibit sighs and shows only eupneic activity, or shows bi-stable activity meaning that sighs can be triggered between \"on\" and \"off\" states by short perturbation. The bistability depends on slow calcium oscillations. The network model predicts that the presence of IP3-like channels is necessary to produce \"sigh\" behavior and to mimic transitions between activity states caused by neuromodulators. We analyze the model dynamics and describe dose-response curve to neuromodulator such as norepinephrine. Theoretical data are similar to experimental data from"} +{"text": "Multiple other signals also initiate signaling pathways that streamline and optimize signaling pathways inside hepatocytes so that hepatocytes enter into cell cycle and proceed with DNA synthesis. Hepatocytes in the cell cycle generate paracrine signals to other hepatic cell types, producing GM-CSF (affecting Kupffer cells), VEGF-A and Angiopoietins 1 and 2 , TGF\u03b1 and FGF1 (affecting a variety of cell types including hepatocytes themselves). Non-parenchymal cells in the liver also produce an array of mitogenic signaling molecules, including HGF , IL6 and TNF (from Kupffer cells). TGF\u03b21, considered as being produced primarily by stellate cells, does not appear to exert mito-inhibitory effects on regenerating hepatocytes, primarily due to down-regulation of TGF\u03b2 receptors during regeneration. Functions served by TGF\u03b2 during regeneration are more probably related to control of synthesis of hepatic biomatrix, which is substantially downgraded and remodeled during the early stages of regeneration and is resynthesized towards the end of the process [The recent three decades emphasized research on liver regeneration, which focused on signals leading hepatocytes into proliferation and liver into regeneration. Mitogenic growth factors (ligands of EGFR and HGF) were identified as direct mitogens for both hepatocyte cultures as well as whole animals Many studies with a variety of epithelial cell types have shown elimination of growth suppressing signals in neoplastic development. Cancer tissues proceed with continual proliferation, due to elimination of internal checkpoints and cell cycle control systems.In this study, we concentrated on several systems apparently associated with termination of liver regeneration and demonstrated that such systems are either non-functional or eliminated in hepatocellular carcinomas (HCC). Conversely, by applying detailed genomic analysis, we also found specific genes deleted in the majority or in many hepatocellular carcinomas whose function, not apparent at first, appears to relate with growth suppressor signals associated with termination or negative control of liver regeneration. Such signals related to the present study are as follows:This protein is massively upregulated in HCC, to the point that it is used as a diagnostic tool. While it should be intuitively considered as enhancing cell growth, loss of function of GPC3 is associated with organ overgrowth (human Simpson Golabi Behmel syndrome). Transgenic overexpression of GPC3 in hepatocytes leads to deficient liver regeneration and smaller livers, with lower nuclear levels of protein Yap . GPC3 isWe identified this protein as having the highest number of gene deletions in a high detail copy number variation (CNV) analysis of human HCC . There wILK binds to \u03b21 integrin chain and transmits a variety of growth suppressing signals in hepatocytes and many other epithelial cells types. We and others have shown that extracellular matrix (being remodeled and degraded at the early stages of liver regeneration) has growth-inhibitory effects on hepatocytes. Genetic elimination of ILK specifically from hepatocytes leads to enhanced spontaneous hepatocyte proliferation, enhanced deposition of extracellular matrix and larger livers . ILK genetic elimination from hepatocytes is also associated with abnormal enhanced liver weight at the end of regeneration after partial hepatectomy . ILK binRegenerative responses to liver injury have become a good source of identifying extracellular and intracellular signaling pathways that have relevance to growth regulation of most other normal and neoplastic cells."} +{"text": "The diversity of plant species and their distribution in space are both thought to have important effects on the function of wetland ecosystems. However, knowledge of the relationships between plant species and spatial diversity remains incomplete. In this study, we investigated relationships between spatial pattern and plant species diversity over a five year period following the initial restoration of experimental wetland ecosystems. In 2003, six identical and hydrologically-isolated 0.18 ha wetland \u201ccells\u201d were constructed in former farmland in northeast Ohio. The systems were subjected to planting treatments that resulted in different levels of vascular plant species diversity among cells. Plant species diversity was assessed through annual inventories. Plant spatial pattern was assessed by digitizing low-altitude aerial photographs taken at the same time as the inventories. Diversity metrics derived from the inventories were significantly related to certain spatial metrics derived from the photographs, including cover type diversity and contagion. We found that wetlands with high cover type diversity harbor higher plant species diversity than wetlands with fewer types of patches. We also found significant relationships between plant species diversity and spatial patterning of patch types, but the direction of the effect differed depending on the diversity metric used. Links between diversity and spatial pattern observed in this study suggest that high-resolution aerial imagery may provide wetland scientists with a useful tool for assessing plant diversity. Plant species diversity is valued, in part, for its potential effects on ecosystem functions, such as primary productivity and nutrient cycling e.g., \u20133). Spat. Spat3])Although ecologists understand that pattern and process are related at all scales , most sImproving our understanding of the relationships between plant species diversity and spatial pattern may be particularly important and useful in wetlands, where individual species reflect important differences in microhabitat and may exhibit distinct functional attributes. Achieving high native species diversity is also often an explicit goal of wetland restoration , 12. ImpOur goal in this study was to quantify relationships between species diversity and spatial heterogeneity of wetland plants under controlled conditions. Over a five year period following planting, we assessed spatial patterning of vegetation and plant species diversity in six constructed wetland ecosystems that had been managed to achieve different levels of diversity. We expected that measures of plant species diversity in individual wetlands would be related to measures of spatial diversity, such as cover type diversity and aggregation.Fagus grandifolia Ehrh.-Acer sp. Marshall) forests and swamp forests dominated this region ).S1 Fig(TIF)Click here for additional data file."} +{"text": "The bioproduction of therapeutic proteins such as monoclonal antibodies (mAb) continues to be a fast growing sector of advanced manufacturing. The ever increasing repertoire of therapeutic proteins coupled with the emergence of biosimilars led to increasing global demand for higher, faster, more cost-effective manufacturing process. Central to any good production platform is the capacity of the production cell line. A suitable production cell line exhibit physiological traits such as high specific productivity (qp), rapid doubling time, high peak cell density and efficient metabolism. The emergence of a suitable production cell with all the aforementioned traits is an extremely rare event, as it requires all facets of cellular transcription, translation, secretion and metabolic efficiency are individually optimized and collectively synchronized into a system capable of high level protein expression. One of the major cellular bottlenecks thought to limit protein production in mammalian cells lies in the cellular translation and secretion capacity. The over-expression of complex recombinant proteins such as mAbs driven by strong viral promoters exert considerable burden in the Endoplasmic Reticulum (ER) and Golgi apparatus. The increase in ER stress triggers the Unfolded Protein Response (UPR) which can result in cell apoptosis and possibly eliminating cells with high uptake of the Gene of Interest (GOI). We adopted host cell engineering approach using XBP1 spice ratio to expand cellular translation and secretion capacity to increase qp and improve the probability of isolating suitable high producers.We screened a panel of mAb producing clonal cell lines using qPCR to identify XBP1 mRNA splice ratio as a suitable target to augment host cell translation and secretion machinery. We then employed FACS to isolate cell population with high ratio of spliced over unspliced XBP1 in order to overcome the negative regulatory effect of unspliced XBP1. We created a host cell line with high XBP1 splice ratio, exhibiting increased expression of chaperones, secretary vesicle proteins and quality assurance proteins without increasing UPR associated apoptotic markers as identified using qPCR. This host cell line was then used for in comparative mAb transient bioproduction studies with two different mAbs. Transient studies were performed using PEI-mediated transfection in industrial relevant chemically defined medium and feeding regime.The XBP1 cell line demonstrated 7.5 fold increase in qp over the control cell line and more than 4 fold increase in final volumetric productivity (Figure In conclusion, XBP1 splice ratio engineered host cell have expanded translation and secretion capacity to alleviate the ER stress experienced from during mAb bioproduction. The augmented capacity allowed for multi-fold improvements in qp and volumetric productivity and have the potential to increase the probability for isolating high producers. The engineered host cell line showed great promise to become a commercially suitable production platform cell line to significantly reduce the time and resources associated with cell line development."} +{"text": "The aim of this study was to determine the mostly needed communication topics and define communicational needs of cardiac surgery patients during their wake up process.This was a descriptive and observational study implemented in a training and research hospital between 1 March and 1 August, 2014, among 132 volunteering patients. The descriptive data of patients were obtained from patient records and the patients themselves. The preferred communication method and topics of intubated patients were decided after the observations taken place during their wake up process. Beginning after two hours after their wake up, the patients who were capable to speak were questioned face to face interview about the sufficiency of the communication during the wake up process.Seventy eight percent of volunteering patients had undergone coroner bypass surgery and sixty-eight percent were male. The communication method preferences of intubated patients during their wake up process were as follows; confirmation (48%), signing (35%), touching (09%), writing (05%), using communication cards (02%). The patients stated the needed topics during the process as; \"I can't breathe, and take the tube out\" (59%), \"Did the operation finish?\u201d Is it day or night? which day is today? What time is it?\" (52%), \"I have pain\" (48%), \"I am thirsty, I want water\" (44%). Having been asked two hours after intubation about the sufficiency of communication during the process, patients stated that they experienced difficulties in building communication (41%), couldn't communicate because of weakness, tiredness and pain (%32), couldn't remember the process (21%), and didn't experience any problem in communication.The most frequent communication method patients used was approval, whereas the most frequent topics were about physiologic needs and information related to the surgery. Patients experienced difficulty in or no communication. As the result of this study it was considered that nurses should properly observe their patients, pay attention to their communication requests know the most frequent topics and improve their communicational skills.We have no financial or other support for this study."} +{"text": "Observational research suggests that in children with cerebral palsy, the altered arm swing is linked to instability during walking. Therefore, the current study investigates whether children with cerebral palsy use their arms more than typically developing children, to enhance gait stability. Evidence also suggests an influence of walking speed on gait stability. Moreover, previous research highlighted a link between walking speed and arm swing. Hence, the experiment aimed to explore differences between typically developing children and children with cerebral palsy taking into account the combined influence of restricting arm swing and increasing walking speed on gait stability. Spatiotemporal gait characteristics, trunk movement parameters and margins of stability were obtained using three dimensional gait analysis to assess gait stability of 26 children with cerebral palsy and 24 typically developing children. Four walking conditions were evaluated: (i) free arm swing and preferred walking speed; (ii) restricted arm swing and preferred walking speed; (iii) free arm swing and high walking speed; and (iv) restricted arm swing and high walking speed. Double support time and trunk acceleration variability increased more when arm swing was restricted in children with bilateral cerebral palsy compared to typically developing children and children with unilateral cerebral palsy. Trunk sway velocity increased more when walking speed was increased in children with unilateral cerebral palsy compared to children with bilateral cerebral palsy and typically developing children and in children with bilateral cerebral palsy compared to typically developing children. Trunk sway velocity increased more when both arm swing was restricted and walking speed was increased in children with bilateral cerebral palsy compared to typically developing children. It is proposed that facilitating arm swing during gait rehabilitation can improve gait stability and decrease trunk movements in children with cerebral palsy. The current results thereby partly support the suggestion that facilitating arm swing in specific situations possibly enhances safety and reduces the risk of falling in children with cerebral palsy. The forelimbs have a clear locomotor function in quadrupedal walking. In human walking, this function most likely changed as the upper limbs do not make contact to the ground during upright walking. Irrespective of its quadrupedal neural base Jackson, , researcIn pathological populations, the arm swing pattern can be affected or altered during gait, which could result in changes in the function of the arm swing. Altered arm swing patterns have been reported in children with cerebral palsy. Cerebral palsy is a group of permanent disorders of the development of movement and posture, causing activity limitation, that are attributed to non-progressive disturbances that occur in the developing fetal or infant brain and 24 typically developing children (age range 5\u201312 years) were included in the study. The cerebral palsy group consisted of 11 children with unilateral cerebral palsy and 15 children with bilateral cerebral palsy, recruited from the Clinical Motion Analysis Laboratory of the U.Z. Leuven (Pellenberg). The children with cerebral palsy were only included in the study if they were diagnosed with the predominantly spastic type of cerebral palsy. Diagnosis and type of cerebral palsy were determined by a multidisciplinary team of neuropediatricians, pediatric orthopedicians, and rehabilitation physicians after neurological examination (including magnetic resonance imaging). The participants had to be able to walk without assistive devices and were only allowed to participate if they showed enough cooperation to follow the instructions concerning the walking trials. The children were excluded if they underwent Botulinum Toxin A treatment within the past 6 months or if they previously underwent lower limb surgery. The local ethical committee (Commissie Medische Ethiek KU Leuven) approved all experiments . In accordance with the Declaration of Helsinki, written informed consent was obtained of the participants' parents.Three-dimensional total-body kinematic data (100 Hz) were captured by an eight camera Vicon system to detect the reflective markers placed on the participant's skin. Similarly to Romkes et al. , 34 reflThe marker coordinates were filtered and smoothed using Woltring's quintic spline routine with a predicted mean-squared error of 15. Further processing in Workstation and Polygon consisted of defining gait cycles and calculating spatiotemporal gait parameters. In children with cerebral palsy, the most affected side was defined as the side on which the highest median spasticity score was obtained in the lower limb. In typically developing children, the least affected side was defined as their dominant side.Various outcome measures were assessed to determine the children's stability during walking. In accordance with recent literature concerning stability measures in experimental situations, several spatiotemporal parameters were calculated and both arm swing condition (free arm swing or restricted arm swing) and walking speed condition were included as repeated measures factors (within-subjects). Moreover, a Mann-Whitney U Test was used to compare children with unilateral cerebral palsy and children with bilateral cerebral palsy for differences regarding the Gross Motor Function Classification Scale-levels and the Modified Ashworth Scale-grades (on the most affected side).* subject group interactions of the performed general linear models were analyzed. Walking speed condition * subject group interactions were analyzed to explore group differences regarding the influence of increasing walking speed on gait stability. Similarly, arm swing condition * walking speed condition * subject group interactions were analyzed to explore group differences regarding the combined influence of restricting arm swing and increasing walking speed on gait stability. Following the general linear model, Tukey's Honestly Significant Difference-tests were used to perform pairwise comparisons. Moreover, Partial Eta Squared-tests were performed to compute effect sizes for all interactions revealed by the general linear models. Cohen's D-test were performed to compute effect sizes for the pairwise comparisons revealed by the Tukey's Honestly Significant Difference-test.A general linear model was performed to determine the influence of restricting arm swing and increasing walking speed on the outcome parameters, i.e., mean values and the variability of the previously described (1) spatiotemporal parameters; (2) kinematic parameters of trunk movement; (3) margin of stability. The general linear model included subject group as a factor (between-subjects) and arm swing condition (free arm swing or restricted arm swing) and walking speed condition as repeated measures factors (within-subjects). To explore group differences regarding the influence of restricting arm swing on gait stability, the arm swing condition Statistical analyses were performed using Statistica 8.0 . The level of significance was set at 0.05 for all tests.F = 3.690, p = 0.032). Children with unilateral cerebral palsy were significantly smaller than typically developing children (p = 0.025). No differences between children with bilateral cerebral palsy and children with unilateral cerebral palsy were found regarding Gross Motor Function Classification System-levels. Children with bilateral cerebral palsy had higher overall median Modified Ashworth Scale-grades on the most affected side compared to children with unilateral cerebral palsy .Group comparisons revealed no differences regarding age and weight Table . However* subject group interaction for walking speed and at high walking speed . Furthermore, walking speed increased more in typically developing children compared to children with unilateral cerebral palsy. This resulted from increased walking speed in typically developing children compared to children with unilateral cerebral palsy at high walking speed , while walking speed was similar at preferred walking speed. This is confirmed by the analysis of the effect sizes: the effect size regarding the increase from the preferred walking speed conditions to the high walking speed conditions is higher for typically developing children compared to both children with bilateral cerebral palsy and children with unilateral cerebral palsy .Statistical analysis revealed a significant walking speed condition * subject group interaction for double support time and at high walking speed . Furthermore, stride length increased more in children with unilateral cerebral palsy compared to children with bilateral cerebral palsy. This resulted from larger stride lengths in children with unilateral cerebral palsy compared to children with bilateral cerebral palsy at both preferred walking speed and at high walking speed .A significant walking speed condition * subject group interaction was found for trunk sway velocity compared to children with bilateral cerebral palsy .A significant walking speed condition No significant group differences were revealed regarding the influence of increasing walking speed on the margin of stability Tables , 6.No significant group differences were found regarding the spatiotemporal parameters.* walking speed condition * subject group interaction was observed for trunk sway velocity disruptions during walking by the involved leg in children with unilateral cerebral palsy . This possibly suggests that gait stability is very mildly affected. On the other hand, it is possible that restricting arm swing affects gait stability in a specific direction. Previous research indicated that children with unilateral cerebral palsy show gait instability in both the medio-lateral and the antero-posterior direction . Furthermore, the hypothesis that restricting arm swing would decrease gait stability more in children with bilateral cerebral palsy compared to children with unilateral cerebral palsy seems to be confirmed.Since we aimed to evaluate the combined effect of increasing walking speed and restricting arm swing, the isolated influence of walking speed on the measures of stability is described first.Typically developing children increased step length and stride length more compared to children with bilateral cerebral palsy when walking speed was increased. Children with cerebral palsy face muscle shortening, muscle contractures and/or spasticity. Since spasticity is dependent of muscle lengthening velocity, this could influence step length more when increasing walking speed. Children with unilateral cerebral palsy increased step length more compared to children with bilateral cerebral palsy when walking speed was increased. This difference is likely to be explained by the lower spasticity values in children with unilateral cerebral palsy and bilateral involvement in children with bilateral cerebral palsy .The reported group differences regarding the increase in trunk sway velocity when walking speed was increased could also be explained by compensations for differences regarding the increase in step length and stride length. However, step (stride) length increased more in children with unilateral cerebral palsy compared to children with bilateral cerebral palsy. If step (stride) length primarily caused the reported group differences regarding trunk sway velocity, one would expect a smaller increase in trunk sway velocity in children with bilateral cerebral palsy compared to children with unilateral cerebral palsy. Clearly, this is not supported by the results. Moreover, previous research indicated that altered trunk movements in children with cerebral palsy are not likely to be compensations due to lower limb impairments . In contrast to the research hypothesis, gait stability did not decrease more in children with cerebral palsy compared to typically developing children when walking speed was increased.An important group difference regarding the combined influence of restricting arm swing and increasing walking speed was found . A stronger increase in trunk sway velocity has been observed in children with bilateral cerebral palsy compared to typically developing children when subjects walking with restricted arm swing were asked to increase walking speed. This possibly suggests that increasing walking speed combined with restricting arm swing decreases gait stability more in children with bilateral cerebral palsy compared to typically developing children. However, these findings should certainly be interpreted with care because other factors may interfere with the combined influence of arm swing and walking speed on gait stability (as mentioned above).Furthermore, typically developing children showed a specific reaction when arm swing was restricted at preferred walking speed . Both trunk sway and trunk rotation decreased compared to walking with the arms free at the preferred walking speed. This \u201cen bloc\u201d strategy was not found in either group of children with cerebral palsy. Therefore, it is assumed that the trunk is required to move in children with cerebral palsy when arm swing is restricted.In conclusion, children with bilateral cerebral palsy increased trunk sway velocity more compared to typically developing children when subjects walking with restricted arm swing were asked to increase walking speed. Overall, evidence is insufficient to conclude that restricting arm swing combined with increasing walking speed induced larger group differences regarding gait stability compared to their isolated effects. Thereby, the experimental data could not confirm the postulated research hypotheses regarding the influence of both restricted arm swing and increased walking speed on gait stability. However, the results showed that children with cerebral palsy adopted different responses to arm swing restriction compared to typically developing children regarding trunk kinematics.When interpreting the results of the current study, certain methodological issues should be taken into account. It is possible that the study sample consisting of 24 typically developing children and 26 children with cerebral palsy was too small and/or heterogeneous. This could have caused some marginally non-significant changes that were reported. Additionally, more children with bilateral cerebral palsy had a GMFCS level II than children with unilateral cerebral palsy. These differences certainly need to be taken into account when comparing these two groups. Most of the subjects included in the group of children with cerebral palsy had a GMFCS level I. This mildly involved population could have caused an underestimation of the actual differences between typically developing children and children with cerebral palsy.Second, Bruijn et al. reportedFinally, trunk movements were not described using joint angles. However, elevation angles were used . As such, a simplified kinematic method was used. In previous research, this approach was found to be adequate to detect meaningful changes in the kinematics during walking in this population, in agreement with literature using joint angles for affected stability in children with bilateral cerebral palsy. Results were less clear for children with unilateral cerebral palsy. In contrast to the research hypotheses, increasing walking speed did not affect gait stability more in children with cerebral palsy compared to typically developing children . However, the current results indicate that increasing walking speed increased trunk compensations for altered angular momentum around a vertical axis more in children with cerebral palsy compared to typically developing children. The effects were larger in children with unilateral cerebral palsy compared to children with bilateral cerebral palsy because more trunk movements were already observed in children with bilateral cerebral palsy at preferred walking speed. In contrast to the research hypotheses, restricting arm swing combined with increasing walking speed did not induce larger group differences regarding gait stability compared to their isolated effects. Overall, it is proposed that facilitating arm swing during gait rehabilitation can improve gait stability and decrease trunk movements in children with cerebral palsy. The current results partly support the suggestion that facilitating arm swing in specific situations possibly enhances safety and reduce the risk of falling in children with cerebral palsy. Other authors already suggested a similar approach for other pathologies in previous research and from the Research Foundation Flanders . PM is supported by the European Commission (Horizon 2020) as a Marie Sk\u0142odowska-Curie fellow . The funding agencies had no role in the present study.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Dear Sir,Hypothermia in surgical neonates is associated with serious implications and during surgery may lead to the damage to various organs of body and therefore affects the ultimate outcome. Neonatal hypothermia affects the vital organ\u2019s circulation such as cerebral, myocardial and renal and these organs are more sensitive to resultant ischemia [1].The important factors which lead to hypothermia in operation room are the type and duration of surgery, and operating room temperature as neonate have poorly developed thermoregulatory mechanism and minor changes related with operating room environment, equipments and instruments that used to contact with neonatal body may produce severe hypothermia [1].In different setups different precautions are taken to prevent the hypothermia during neonatal surgery for example automatic warming system.Illumination .Warming sources (Two glass rod elements of 750 watts).Warming control system (A sensor with a servo mode).Alarm system.Special Bed for neonate.Unfortunately in majority of setups proper warming system in neonatal/pediatric surgical units have not been available. This aspect of neonatal surgical care was discussed in the Neonatal Surgery research Group (NSRG) which is a discussion group on face book. Many learned members shared their experiences and advocated recommendations for optimal management and prevention of hypothermia while operation in settings that lack proper thermoregulatory equipments [2]. These recommendations are summarized as under;Infusion of warm intravenous fluids during surgery.Cotton roll/aluminum foils/woolen cap and socks cover over head and limbs.Operating room air conditioner must be off.Use of warm sterilized towels, pyodine solution and surgical instruments or at least they should not be cold.There should not be frequent opening of the doors while operation.Warming pads around baby and warming pad under him may be used.Overhead warmer can be used to control the cold environment especially in winter.These measures are easy to observe and do not require special instrument/techniques to be employed. Fan heaters or electric heaters must not be used near the neonates as they can directly heat them and may burn the delicate body of neonates. These measures are simple and can avoid hypothermia while operation and thus benefit the outcome of surgical neonates.Source of Support: NilConflict of Interest: None declared"} +{"text": "REDCap (Research Enterprise Data CAPture) software may provide a feasible platform for CMR Centers to: a) capture clinical throughput securely for research purposes, and 2) collaborate using a common platform for either distributed or centralized data storage. REDCap may facilitate CMR Centers' participation in the research enterprise, especially those with limited resources. REDCap may catalyze multicenter studies with \"distributed data collection\" where CMR sites can clone shared data dictionaries across sites for subsequent compilation into a singular master data file.Investigators without prior REDCap training created a REDCap database hosted by the University of Pittsburgh with software developed by Vanderbilt University. In an IRB approved protocol. A full time research nurse consented patients referred for clinical CMR scans and abstracted patients' clinical data into REDCap during CMR scanning. Data elements included: key summary findings from CMR and all prior cardiac imaging data including full (unstructured) reports pasted into text fields or uploaded pdf attachments; blood testing acquired during IV placement; demographics; comorbidity; and medications. Front end quality assurance measures included data formats, data ranges, and redundant identifiers. We maximized data security by configuring users' rights, limiting data exports to the only the principal investigator, and logging every time stamped manipulation to the database. Adverse event queries occurred biannually. Data were entered via a web browser and stored on encrypted servers behind firewalls. Data were exported to statistical software packages for analysis.http://www.project-redcap.org) to demonstrate the potential for global multicenter data collection from the University of Minnesota and the University College of London via email exchange of a .csv file. We cloned the databases rendering them operational in <5 minutes similar to a new collaborative project site. We also queried the world map of REDCap capable sites (n Figure .REDCap is widely available and provides a robust platform for clinical CMR research. REDCap provides sites with limited resources a powerful means for rich data collection for clinical CMR research. Scarce resources can then be directed to the burden of rich data collection necessary for robust risk adjustment and multiple hypothesis testing. Shared REDCap data dictionaries are feasible and have the potential to enhance collaboration. REDCap has the potential to accelerate clinical CMR research.American Heart Association The Pittsburgh Foundation."} +{"text": "Rowing produces marked changes in oxygen uptake, pulmonary ventilation, cardiac output and lactate with extreme levels for blood acid-base status and pronounced concentration of catecholamines in blood that could affect coagulation. With development of potassiaemia arrhythmia may even be developed that most often may be of supraventricular origin but sudden cardiac death is reported in rowers. Structural myocardial adaptations to intense rowing training that demands the heart to work against high pressure during the stroke need to be considered. Rare cardiac diseases such as the Brugada syndrome and arrhythmogenic right ventricular cardiomyopathy may also provoke cardiac arrest during exercise. The latter is related to genetic disorders but myocarditis could be involved and following rowing the immune system is suppressed. In the subject with vulnerable myocardium abnormal tachycardia may arise during rowing but also bradycardia in the resting period. Furthermore, it is speculated whether silent arrhythmia in combination with dehydration and coagulation disorder could provoke blood clots and even stroke that recently occurred in two Danish athletes. These cases are presented along with parameters indicative of extreme physiology during rowing."} +{"text": "Estimating the rate of exchange of individuals among populations is a central concern to evolutionary ecology and its applications to conservation and management. For instance, the efficiency of protected areas in sustaining locally endangered populations and ecosystems depends on reserve network connectivity. The population genetics theory offers a powerful framework for estimating dispersal distances and migration rates from molecular data. In the marine realm, however, decades of molecular studies have met limited success in inferring genetic connectivity, due to the frequent lack of spatial genetic structure in species exhibiting high fecundity and dispersal capabilities. This is especially true within biogeographic regions bounded by well-known hotspots of genetic differentiation. Here, we provide an overview of the current methods for estimating genetic connectivity using molecular markers and propose several directions for improving existing approaches using large population genomic datasets. We highlight several issues that limit the effectiveness of methods based on neutral markers when there is virtually no genetic differentiation among samples. We then focus on alternative methods based on markers influenced by selection. Although some of these methodologies are still underexplored, our aim was to stimulate new research to test how broadly they are applicable to nonmodel marine species. We argue that the increased ability to apply the concepts of cline analyses will improve dispersal inferences across physical and ecological barriers that reduce connectivity locally. We finally present how neutral markers hitchhiking with selected loci can also provide information about connectivity patterns within apparently well-mixed biogeographic regions. We contend that one of the most promising applications of population genomics is the use of outlier loci to delineate relevant conservation units and related eco-geographic features across which connectivity can be measured. Inferring population connectivity from molecular data within a population genetic framework can shed light on the evolutionary and ecological processes that shape patterns of genetic diversity that produce weak patterns of genetic differentiation or even no differentiation at all is not justified in such species. On the contrary, m \u226b \u03bc would imply that the variation detected in one deme is mostly replenished by migration from the metapopulation rather than by locally arisen mutations. Under this assumption, the small differentiation generally observed in most marine species would not be an artifact of using multi-allelic markers, but the consequence of high migration. This has two consequences for our interpretation of genetic variation in high gene flow species: (i) homoplasy is likely to play a very limited role because homoplastic alleles will be almost equally distributed throughout the metapopulation by migration, (ii) high heterozygosity values reflect large metapopulation effective size rather than locally high population size, a pattern that remains true even with relatively low migration between populations.Since the advent of multi-allelic loci in population genetics, it has been pointed out that the maximum value taken by Wright's \u03c3). The mean dispersal distance (\u03bc) can be obtained from \u03c3 by assuming a normal distribution of dispersal distance (blue line). (B) The three sampling strategies commonly used in marine population genetic studies: 1. A geographically subdivided species range is discretely sampled, but some populations are not sampled (gray dotted circle); 2. A continuously distributed species is sampled discretely, and the geographic distance between samples is of the same order as \u03c3; 3. Continuous sampling of individuals separated by distances of the same order as \u03c3. Colored points are sampled individuals, gray points indicate nonsampled individuals. (C) The information about dispersal that can be obtained from indirect and direct methods for each sampling strategy.(A) The distribution of dispersal distances can be estimated in two ways: through the direct detection of discrete dispersal events (blue bars), or the indirect estimation of dispersal parameters like the standard deviation of parent\u2013offspring dispersal distances (Nem) from FST in the island model (Wright Nem or m under various extensions of the island model or other more refined population structures (Broquet and Petit FST does not necessarily mean that migration is strong as genetic differentiation is influenced by both effective size (Ne) and migration rate (m) or moments of the distribution of dispersal distances Fig. . For ins(m) Fig. . Second,(m) Fig. . The mai\u03c3 have been reported for a variety of species . In the case of parentage analysis, an additional constraint stems from the necessity to sample a large fraction of the potential parents. Assignment methods specifically applied to detect immigrants without identifying their origin require less extensive sampling, but their efficiency reduces quickly with decreasing genetic differentiation if the selection coefficient (s) can be measured, which actually represents a serious challenge to most case studies. However, a measure of selection can sometimes be obtained using experimental populations or genotype frequency comparisons between larvae and adults sampled from the same cohort. By contrast, inferring dispersal from a neutral hitchhiker locus only requires the recombination rate with the selected locus along the chromosome is measured between spatial coordinates \u2212500 and 500. (A) A local adaptation cline lying at the frontier between two environments where selection acts in opposite directions . The cline width parameter (w) is defined as the inverse of the maximum slope at the cline center, and k is a coefficient that depends on the selection regime (Slatkin r with the selected locus makes a shift (\u0394p) in the central region of the cline, and an external gradient of allele frequency (\u2202p/\u2202x) directly outside the cline (Barton s = 0.5). The amount of linkage disequilibrium (D) between selected loci is measured after dispersal at the center of the overlapping clines. (C) A tail of introgression produced by the inflow of foreign alleles entering a subdivided population appears on the left side of a chain of demes and then propagates to the right side from deme to deme (m = 0.01), leaving behind a complex allele frequency pattern at a neutral hitchhiking locus (r = 0.001). Local connectivity between adjacent demes is transiently revealed by the structure of the neutral hitchhiking locus, as long as gene flow re-homogenizes allele frequencies. The chromosomal signatures of selection can take the form of narrow regions of differentiation (A), large genomic islands (B), or shoulders of differentiation (C and D) centered on the selected loci.Plots show the chromosomal and geographic signatures of selection under four different selective processes. Selected and neutral loci are colored in red and green, respectively. Genetic differentiation ( Slatkin . A neutre Barton . (B) HybDrosophila melanogaster revealed the existence of several latitudinal clines takes advantage of the substantial genetic differences existing between populations or species that are on both sides of the hybrid zone. Minimal dispersal distances can be obtained through the identification of parental genotypes that crossed a hybrid zone and successfully settled in a foreign parental population or species. An even more precise estimation of larval dispersal distance can be made when the source of dispersing larvae is known, as for first-generation hybrids dispersing outside a hybrid zone. Using this strategy, patterns of larval movement among neighboring patches of blue mussels have been examined by measuring realized larval dispersal based on the genetic identification of recently settled juveniles , other adjacent demes being otherwise highly connected in the standard linear stepping-stone model. During a few thousands of generations postcontact, introgression generates a step in allele frequency between the two populations of the introgressed species, and the step then disappears as allele frequencies equilibrate between species . Importantly, the gradient only appears if dispersal is spatially limited, otherwise spatial homogenization occurs immediately as foreign alleles enter the introgressed population. In order to illustrate how this mechanism can be used to detect a local barrier to dispersal, we simulated a contact zone between two partially reproductively isolated species and the introgression of foreign alleles within one of the two species which is geographically subdivided Fig. . The extTails of introgression may be also influenced by selection acting outside the tension zone. In this case, the gradient of allele frequency within the range of the introgressed species may be steepened by a gradient of selection . Because secondary contact zones commonly coincide with environmental gradients , as well as the position of the selected locus (\u22123 kb 5\u2032 of the start codon of the EF1\u03b1 gene), the selection coefficient (s = 0.01), and incidentally the local recombination rate of the chromosomal region (\u03c1 = 1.7 cM/Mb, Bierne Another scenario that generates outlier loci happens during the spread of an unconditionally favorable allele in a spatially subdivided population. This process leaves a transient footprint at neutral markers in the chromosomal vicinity of the sweeping allele. When the overall genomic differentiation is low, as it is typically the case in marine species, this process generates an elevated level of differentiation on both sides of the selected locus Bierne , which cSubstantial progresses in our understanding of connectivity in nonmodel organism can be achieved with large population genomic datasets. High-density genome scans have reached the power to detect outlying patterns of genetic differentiation at different spatial scales, enabling conservation geneticists to identify genetic differences reflecting restriction to gene flow where classical neutral markers were hitherto most often largely uninformative, as in high gene flow species. The scope of the applications of outlier loci for assessing connectivity patterns in marine species needs further investigations, in particular through gathering a larger set of empirical data. Some of the methodologies that were proposed in this review are still underexplored, and we hope that our work will stimulate new research to test how broadly they are applicable to nonmodel marine species. Although spatially explicit methods are directly applicable to continuously distributed sessile species, selected and hitchhiker loci also have the potential to reveal cryptic genetic structure in migratory species with natal homing (Gagnaire et al."} +{"text": "Capturing symptom data for large studies (\u226560 participants) in \u226430 minutes is hard to achieve using manual data entry without substantial costs and resources. This challenge is highlighted if the data are needed to make real-time clinical decisions. The 140 participant capacity of the Environmental Exposure Unit (EEU) requires an advanced method to process symptom data.Advanced scanning technologies are used with a customized two-step quality assurance data collection process. Optical Mark Recognition (OMR) and Optical Character Recognition (OCR) capture data from paper symptom diary cards into the EEU\u2019s clinical data management system (CDMS). A template is configured to read the static diary card format and assign zones where the specific diary card data are located. The user configures field requirements within the zones to validate data captured. Cards that do not meet a predefined confidence level for any particular zone will be flagged for a quality check. The quality checking process involves one user visually confirming all data captured and a second user inputting all values from the card to ensure accuracy. Invalid data are rejected from the batch and returned to the participant for correction. Corrected cards are scanned again and all valid data are transferred into the CDMS.Capturing data using the advanced scanning system allows a team of 3 to process 120 symptom diary cards containing 9 symptoms and 3 peak nasal inspiratory flow (PNIF) scores, with 99.9% accuracy in <15 minutes. In comparison, manual data entry would require a team of 8 to achieve similar results.For large studies with short assessment periods, the scanning system utilized in the EEU is significantly more efficient in all aspects of data acquisition than manual entry. This ability to accommodate large studies in an accurate, efficient manner leads to an ideal setting for the conduct of time-sensitive clinical trials."} +{"text": "Plasmodium falciparum erythrocyte membrane protein 1 antigens thatare inserted onto the surface of P. falciparum infected erythrocytes playa key role both in the pathology of severe malaria and as targets of naturally acquiredimmunity. They might be considered unlikely vaccine targets because they are extremelydiverse. However, several lines of evidence suggest that underneath this moleculardiversity there are a restricted set of epitopes which may act as effective targets for avaccine against severe malaria. Here we review some of the recent developments in thisarea of research, focusing on work that has assessed the potential of these molecules aspossible vaccine targets.The Plasmodium falciparum infect human erythrocytes, they insert into theerythrocyte surface parasite antigens that profoundly alter the antigenic properties of thecells family of VSA astargets of naturally acquired immunity and review their potential as vaccine targets. Therehave been extremely encouraging developments in this area of research over the last 3 years.However, as our knowledge grows, so does our appreciation of the complexity of theparasite's strategy of evading our immune systems. There are no shortage of recent reviewsthat together give a comprehensive summary of variants surface antigens both in immunity andcytoadhesion . (2) Both CIDR and DBL contain blocks ofsequence (homology blocks) that are relatively conserved. These homology blocks can beused to divide each domain type into various classes and subclasses the molecules are constructed from only twobroad classes of domain . The most promisingpeptides are both located within homology block 4 it is still unclear whether EPCR is a commonreceptor for parasites since only a relatively small number of clinical parasite isolateshave so far been tested. A recent paper by Esser et al. (Despite the plausible mechanism that would explain the pathology of cerebral malaria(Taylor r et al. suggestsvar expression may beunder some level of global control (Merrick et al.et al.et al.et al.Two recent reports suggest that on the one hand var have an important effect on theantigenic properties of laboratory isolates (Chan et al.in vivo. Warimwe et al. (var genes, the observed association wasindependent of group A-like var gene expression or any other subgroup ofvar genes that could be identified. One possible interpretation of thisis that the association between rosetting and respiratory distress was driven by a subsetof parasites for which rosetting is mediated through RIFINs or STEVOR. This highlights theneed for full sequence information of VSA expressed under different levels of naturallyacquired immunity.The important question here is whether the level of sequestration supported by STEVOR andRIFINs in the absence of PfEMP1 could support parasite loads associated with severemalaria. Though knockdowns of e et al. observedSM\u2019, \u2018group A PfEMP1\u2019, \u2018DC8\u2019, in the same way asclinical definitions such as \u2018cerebral malaria\u2019 (Taylor et al.Future research on developing interventions based on PfEMP1 needs to continue to refinedefinitions of different cytoadhesive phenotypes at the molecular level. Various termssuch as \u2018rosetting\u2019, \u2018VSA"} +{"text": "Increase in complexity of neuronal network models escalated the efforts to make NEURON simulation environment efficient. The computational neuroscientists divided the equations into subnets amongst multiple processors for achieving better hardware performance. On parallel machines for neuronal networks, interprocessor spikes exchange consumes large section of overall simulation time. In NEURON for communication between processors Message Passing Interface (MPI) is used. MPI_Allgather collective is exercised for spikes exchange after each interval across distributed memory systems. The increase in number of processors though results in achieving concurrency and better performance but it inversely affects MPI_Allgather which increases communication time between processors. This necessitates improving communication methodology to decrease the spikes exchange time over distributed memory systems. This work has improved MPI_Allgather method using Remote Memory Access (RMA) by moving two-sided communication to one-sided communication, and use of recursive doubling mechanism facilitates achieving efficient communication between the processors in precise steps. This approach enhanced communication concurrency and has improved overall runtime making NEURON more efficient for simulation of large neuronal network models. The brains complex computational behavior necessitated developing large neuronal computational models. Huge amount of data is integrated by models which work on simulation tools to study the information regarding brain computational processing. This enables neuroscientists to practically observe the computational behavior similar to brain and to carry out experiments along with fluctuating processes on simulating environment. As a consequence better understanding of brain functionality can be attained and diseases like epilepsy, Parkinson's disease, and so forth can be diagnosed and cured. There is wide range of simulators that have been developed for simulating neuronal behavior. NEST, NEOSIM, SPLIT, and NEURON are few of important simulators in practice today \u20135. The ahttp://senselab.med.yale.edu/) and used parallel models from Netmod [NEURON has become a widely adopted simulation tool for building and analyzing neuronal models, using them for solving multifaceted neuronal computations . The perm Netmod . In thisThis paper is organized as follows: we begin with discussion on related work in Many studies exemplify that distributing network architecture over multiple processors has features of fast processing of data. For example, the scaffold functioning in NEURON for parallel simulations and performance scaling can be obtained by testing the model . As far A standard Message Passing Interface (MPI) is widely adopted by most of simulators and functions on use of the nonblocking point-to-point message passing utility. NEURON selects basic spike distribution method, which functions to distribute spikes among all processors . The \u201cAlIn the NBC library nonblocking extensions of collective calls have been developed, which was presented in MPI-2.1 . It provNEURON is a powerful simulation environment for performing experiments on models of neurons or network of neurons . It is aIn multiprocessing environment processors either perform multiple tasks simultaneously or distribute the same task across multiple processors for achieving the adequate level of concurrency. Message Passing Interface is used for communication between parallel processors running processes on distributed systems. MPI is an application programmer interface for inscription of message passing parallel programs which functions to cover the details of underlying system architecture. MPI is implemented as library since it enables convenient program that can manage to run similar program on parallel processors and has gone through enhancements in various versions \u201327. CommMPI two-sided communication gives semantic assurance implied by the standard and its implementation is subject to various practical restraints. The communication pattern of MPI in two-sided communication is based on two subdivisions MPI_Send and MPI_Recv. MPI_Send routines are used for sending messages from source process to destination process and MPI_Recv routines for getting messages on target process sent by source process as shown in All processes of MPI communication in MPI one-sided communication are carried out in framework of a window. Remote Memory Access unlike two-sided communication decouples data transfer from the synchronization of systems. Window is based on compilation of elements defined at the time of conception of window and adjacent area of memory at each process. One-sided communication can be used to send and receive data where one process can directly access memory address space of another process without intervention of other processes. Each processor declares a specified area of memory for remote access by MPI_Win_Create. The use of MPI-3 standard is based on three types of one-sided communication functions: MPI_Put, MPI_Get, and MPI_Accumulate. Sending data from source to destination on remote window is accomplished by using MPI_Put operation . On the n on a parallel computer with p processors using O(log\u2061p) parallel arithmetic steps [p processors requiring only O(log\u2061p) number of steps. In each step processors communicate with other processors and distance among processors increases by power of 2 and size of message in each coming step doubles as compared to the previous step. Initially data exchange is carried out by the processes which are distance 1 apart from each other. After this, the processes which are distance 2 apart share the data received from previous step and their own data as shown in The recursive doubling algorithm initially was developed to solve tridiagonal linear system of size ic steps . RecursiThe collective communication in MPI resolves around the participation of all the processes in the communication group known as communication world. The synchronization of processes is mandatory; this means that all the processes in communication group reach the point of synchronization so they can continue execution. Spikes communication in NEURON is handled by MPI_Allgather by the collective process based on two-sided communication routine. MPI_Allgather is just a wrapper above MPI_Gather and MPI_Bcast, which in depth are another cover over MPI_Send and MPI_Recv for sending and receiving messages in the communication group.Collective communication is the procedure of sending and receiving data amongst all the processors of MPI communication world. A processor in the MPI_Allgather communicator's world gathers data from every other process and distributes its own data amongst the communication group. In NEURON MPI_Allgather is used for communication between processors after each designated interval. The total runtime for simulation of models decreases when numbers of processors are increased.On the other hand for communication between processors MPI_Allgather time keeps increasing, thus becoming bottleneck when moved to large machines; even communication time may exceed computation time for simulating large neuronal network models. Limitation on 2\u201332 processors is depicted in O(log\u2061p) steps, where n is number of processors. This paper is based on limited processor version of the recursive doubling algorithm for points of multifaceted sharing between multiple processors using time interval for one-sided communication. The following are algorithms for RMA_Allgather and target calculation that were implemented and tested in NEURON.Collective communication is significant and is adopted in NEURON for communication between processors, but two-sided communication using MPI_Allgather makes implementation for optimization possible. MPI_Allgather is enhanced to RMA_Allgather one-sided communication using recursive doubling for efficient spikes exchange between processors. One-sided communication requires only one processor for communication, thus ensuring that both sender and receiver are not bounded to each other during whole communication process, enabling efficient communication. To minimize the number of steps for efficient spikes exchange recursive doubling mechanism was implemented which reduced the number of steps for exchange, thus ensuring message exchange in Calculation of target by every processor in each step lays foundation for appropriate communication. In the first step every processor communicates with other processors which is distance 1 apart and as the step increases their distance doubles as in p processors in O(log\u2061p) parallel arithmetic steps. In order to allow other processors to write remotely, the processor exposes its memory, and RMA enables the processors to access data and communicate without requiring other processors to be part of communication process. This enables processors to concurrently carry out the communication process.The recursive doubling algorithm is adopted to resolve communication bottleneck on parallel machines with O(log\u2061p) steps where n is number of processors. RMA_Allgather is carried out instead of MPI_Allgather and thus decouples data communication from system synchronization. It is nonblocking approach which allows processes to communicate concurrently without waiting for other processors to synchronize. The whole process is completed efficiently in This section will demonstrate the impact of proposed RMA_Allgather method through several experiments performed after implementation of the algorithms in NEURON on a 4-node HP BL460c cluster placed at Kadir Has University. The SMP cluster has 2 \u00d7 2.66\u2009GHz Intel Xeon Quad Core CPUs and RAM of 24\u2009GB and has 8 processing cores per node running Linux 2.6.18 connected with 20\u2009Gbps InfiniBand. The experiment is performed 5 times for obtaining each result and the results are average of 5 runs as shown in Tables Simulation tests were conducted on two published neuronal network models, Parscalebush and Parbulbnet , 8, exhiIt was observed that along with gradual increase in number of processors the size of subnet on single processor becomes smaller and MPI_Allgather becomes source of communication overhead on large number of processors. RMA_Allgather when applied to NEURON provides much better results than existing communication mechanism. Tables Speedup from parallelizing large network models in NEURON is found nearly proportional to number of processors, but spikes exchange time was found inversely affecting runtime along with increasing number of processors. In this paper, an optimization method RMA_Allgather using recursive doubling is applied for exchange of spikes in NEURON simulator that reduces spike exchange time almost 10% as compared to the existing MPI_Allgather method. RMA facilitates advantage of direct memory access to data of remote processor and reduces the synchronization overhead whereas recursive doubling limits the overall communication steps, thus benefiting the performance of NEURON for simulating large neuronal network models. In future we plan to improve remote direct memory access in NEURON by exchanging only the updated spikes to optimize the communication process."} +{"text": "Next generation sequencing (NGS), using massive parallel sequencing, is increasingly being used in the clinical setting. It is a high throughput technique that offers a fast and cost-effective testing solution for genetic disorders with a number of candidate genes.In this study we have employed NGS to sequence 48 samples that had been referred to the Molecular Genetics Department for the investigation of Monogenic Disorders of Insulin Secretion (MDOIS), Disorders of Sexual Development (DSD) and Noonan Syndrome (NS). All samples had previously been genotyped using Sanger sequencing.Next Generation Sequencing was performed on an Illumina MiSeq using an Illumina Nextera Rapid Capture Custom Enrichment Kit. This custom assay contained capture probes for the coding regions (including +/- 5 bases of intronic sequence) for 66 genes: 34 for DSD; 19 for MDOIS; and 13 for NS. Sequence data generated by the MiSeq was aligned to hg19 and variants detected using CLC Genomics Workbench. Genetic variants were annotated using Cartagenia BENCHlab NGS.Thirty mutations associated with DSD , MDOIS and NS were detected. This was concordant with genotypes detected by Sanger sequencing.Next generation sequencing has proven to be an accurate and efficient method of genotyping monogenic disorders with multiple candidate genes, and is an ideal technique for the clinical laboratory."} +{"text": "Severe combined immunodeficiency (SCID) is characterized by important impairment in differentiation of T and/or B lymphocytes and occasionally Natural Killer cells, representing a pediatric emergency. A case of immunodeficiency is described emphasizing symptoms, diagnosis and answer after bone marrow transplantation.Case report of a 2 years old male patient with severe combined immunodeficiency (SCID), diagnosed at 9 months after hospitalization due to failure to thrive, chronic diarrhea and pneumonia. Evolved with recurrent respiratory and gastrointestinal infections although using prophylaxis and immunoglobulin infusion. Alogenic, haploidentical transplantation was carried out with positive selection of CD34+, at 18 months of age due no compatible donor been found.Satisfactory answer after transplantation keeping infusion of IV immunoglobulin with clinical and laboratorial favorable evolution.Bone marrow transplantation when succeeds is supposed to restore lymphocyte system diminishing risks of severe and fatal infections.Written informed consent was obtained from the patient for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal."} +{"text": "Campylobacter in bulk tank and retail milk samples from the dairy. Available isolates from patient stool (n = 1), bulk tank milk (n = 1), and retail milk (n = 1) were identified by CDC as Campylobacter jejuni and were indistinguishable by pulsed-field gel electrophoresis (PFGE).During May 2013, the Pennsylvania Department of Health investigated an outbreak of campylobacteriosis among consumers of raw (unpasteurized) milk from a dairy certified by the Pennsylvania Department of Agriculture (PDA) to sell raw milk onsite, at retail stores, and at off-farm pick-up sites. Investigation by the Pennsylvania Department of Health and PDA identified six confirmed and two probable cases of campylobacteriosis associated with raw milk from the dairy. A confirmed case was defined as laboratory-confirmed campylobacteriosis in a person who drank the dairy\u2019s raw milk. A probable case was defined as diarrheal illness without laboratory confirmation in a person who had consumed the dairy\u2019s raw milk and was linked to a confirmed case. Four cases involved children aged \u226418 years. PDA identified C. jejuni strains isolated during the 2012 and 2013 outbreaks differed, consistent with the diversity of C. jejuni isolated from cattle on dairy farms (Campylobacter in bulk tank milk obtained from the dairy during January 2011; no associated human infections were reported.Although the dairy has consistently adhered to PDA requirements for raw milk dairies and conducted milk coliform and somatic cell testing more frequently than required, this was not the first outbreak associated with this dairy. During January\u2013February 2012, the dairy was identified as the source of a multistate outbreak of campylobacteriosis (Campylobacter. During 2005\u20132013, Pennsylvania experienced 17 salmonellosis and campylobacteriosis outbreaks associated with retail raw milk. Five producers had more than one outbreak during that period. Bacterial contamination of raw milk can occur even under optimal conditions; seasonal changes in bovine bacterial shedding or inadequate quality control during milk collection might contribute to outbreak recurrence (Repeat outbreaks from raw milk producers are not uncommon and not limited to"} +{"text": "Otitis media with effusion is common in infants with an unrepaired cleft palate. Although its prevalence is reduced after cleft surgery, many children continue to suffer from middle ear problems during childhood. While the tensor veli palatini muscle is thought to be involved in middle ear ventilation, evidence about its exact anatomy, function, and role in cleft palate surgery is limited.This study aimed to perform a thorough review of the literature on (1) the role of the tensor veli palatini muscle in the Eustachian tube opening and middle ear ventilation, (2) anatomical anomalies in cleft palate infants related to middle ear disease, and (3) their implications for surgical techniques used in cleft palate repair.A literature search on the MEDLINE database was performed using a combination of the keywords \u201ctensor veli palatini muscle,\u201d \u201cEustachian tube,\u201d \u201cotitis media with effusion,\u201d and \u201ccleft palate.\u201dSeveral studies confirm the important role of the tensor veli palatini muscle in the Eustachian tube opening mechanism. Maintaining the integrity of the tensor veli palatini muscle during cleft palate surgery seems to improve long-term otological outcome. However, anatomical variations in cleft palate children may alter the effect of the tensor veli palatini muscle on the Eustachian tube\u2019s dilatation mechanism.More research is warranted to clarify the role of the tensor veli palatini muscle in cleft palate-associated Eustachian tube dysfunction and development of middle ear problems.Optimized surgical management of cleft palate could potentially reduce associated middle ear problems. Otitis media with effusion is very common in infants with an unrepaired cleft palate under the age of 2\u00a0years. At the time of cleft palate repair, more than 90\u00a0% of the middle ears contain mucoid material (\u201cglue ear\u201d) , 2. AlthOptimal otological management in cleft palate patients has historically been a point of discussion, with ventilation tubes often being inserted preventively at the time of cleft palate repair. Although routine ventilation tube insertion leads to short-term hearing gain, the long-term otological outcome of these patients does not appear to be superior to outcomes of patients receiving ventilation tubes merely on indication , 9. TherThe high incidence of middle ear problem in cleft palate children is thought to be caused by Eustachian tube dysfunction, that is the result of aberrations of the paratubal muscles that are normally responsible for Eustachian tube opening \u201312. The Current surgical techniques used in cleft palate repair are primarily focused on restoring the barrier between the oral and nasal cavity and constructing a levator veli palatini muscle sling. By maximizing the levator veli palatini muscle sling function in velopharyngeal closure, the possibility of developing hypernasal speech is reduced . The adjAn electronic literature search of the MEDLINE database was conducted using a combination of the following search terms: \u201ccleft palate and otitis media,\u201d \u201ccleft palate and ventilation tube,\u201d \u201ccleft palate and tensor veli palatini,\u201d and \u201ctensor veli palatini and Eustachian tube.\u201d Our search yielded 53 relevant studies, including 6 experimental animal studies, 6 experimental studies involving humans, 8 histological studies, and 5 clinical studies that are presented in Tables\u00a0Before the most relevant studies retrieved from our search will be discussed, a short overview of the related anatomy and opening mechanism of the Eustachian tube is given.The course of the Eustachian tube\u2014from the middle ear towards nasopharynx\u2014is anterior, inferior, and medial Fig.\u00a0. The tubThe tensor veli palatini muscle originates from the cranial base and the lateral side of the auditory tube \u201345 Fig.. These tThe Eustachian tube is closed at rest as a result of the elastic connective tissue compliance in the tubal walls, extramural pressure of adjacent structures, and the tonus of the paratubal musculature , 51. TheIn the recent literature, there seems to be a general consensus that the tensor veli palatini muscle is the primary opener of the Eustachian tube and thereby has a key role in middle ear ventilation. The first purpose of this literature review was to investigate the tensor veli palatini muscle\u2019s role in Eustachian tube opening and middle ear ventilation. Tables\u00a0The first experimental study investigating the muscles responsible for Eustachian tube function was conducted by Honjo et al. . The autTo examine a possible synergistic action of the tensor veli palatini muscle and levator veli palatini muscle and to visualize the opening process of the tube, Honjo et al. injectedThe significance of a well-functioning tensor veli palatini muscle is underlined in three studies in which a functional Eustachian tube obstruction model was created by affecting the muscle through surgical manipulation or injection of paralyzing agents \u201327. CantAll the abovementioned experimental animal studies \u201327 conclThe abovementioned studies \u201333 proviOur second purpose was to examine to what extent anomalies in the tensor veli palatini muscle, Eustachian tube, or surrounding structures contribute to the development of Eustachian tube dysfunction and middle ear problems in cleft palate patients. In the early 1960s, several factors were proposed to play a causal role in Eustachian tube dysfunction and frequent middle ear pathology in cleft palate patients, including poor tensor veli palatini muscle development and the absence of a firm attachment of the tensor veli palatini muscle to the Eustachian tube . In lateIn Table In another study, Matsune et al. investigAdditionally, Matsune et al. found thShibahara and Sando also perAlthough cartilage cell density in cleft palate individuals is reported to be higher than normal , a studySheer et al. used comBased on the earlier mentioned studies, we can conclude that anatomical abnormalities of the tensor veli palatini muscle and Eustachian tube are considered to play a causal role in functional Eustachian tube obstruction in cleft palate patients. Morphological variations in the surrounding structures however may also play a causal role in Eustachian tube dysfunction. Rajion et al. performeThe final goal of this study was to review literature regarding the implications of surgical techniques used in cleft palate repair on the development of post-treatment middle ear pathology. Table Sehhati-Chafai-Leuwer et al. used MRISeveral surgical techniques have been proposed in an attempt to improve post-treatment tubal function in cleft palate patients in addition to the commonly performed cleft palate repair with levator sling construction. The tensor tenopexy technique consists of (1) identification of the tensor veli palatini muscle, (2) dissection from its aberrant attachment to the posterior edge of the hard palate, and (3) medial displacement of the tensor veli palatini tendon and suturing it under tension to the hamulus. After tensor tenopexy, the tendon is cut medial to the hamulus and the levator sling construction is performed. Medial displacement of the tensor veli palatini muscle tendon theoretically results in a more open configuration (flexed position) of the Eustachian tube lumen and improved middle ear ventilation. Some surgeons consider tensor tenopexy in cleft repair unwarranted as the tensor veli palatini muscle tendon has fibrous attachments to the hamulus and tensor transection will therefore not have negative effect on Eustachian tube opening . In spitTiwari et al. also invBeneficial effects from exerting an immediate tension on the tensor veli palatini muscle were reported by B\u00fctow et al. This wasDissection or dislocation of the tensor veli palatini muscle is often performed to reduce the incidence of post-operative fistula formation and oral mucosa dehiscence or to make the dissection of the levator muscle easier. However, no studies have provided empirical proof of the positive effects of this technique. Kane et al. examined the effects of hamular fracturing on the outcome of palatoplasty and found no differences in oral mucosa dehiscence rate or fistula occurrence rate. Although the authors also found no significant differences in speech results or otological outcomes, follow-ups were only performed up to the age of 3\u00a0years .Based on the present literature, preservation of the tensor veli palatini muscle integrity during cleft palate repair seems to improve the long-term otologic outcome of cleft palate patients , 45, 56.Most studies assessing the role of the tensor veli palatini muscle in Eustachian tube opening are performed on healthy patients and might not be extrapolated to cleft palate patients due to variations in anatomy. More studies are mandatory to elucidate the anatomical abnormalities found in cleft palate patients and how these influence the function of the tensor veli palatini muscle and Eustachian tube.Studies discussed in this review underline the important role of the tensor veli palatini muscle in opening of the Eustachian tube. The correlation between tensor veli palatini muscle integrity and long-term middle ear status makes it advisable not to release the tendon from the hamulus during cleft palate repair. Anatomical variations in cleft palate children may alter the effect of tensor veli palatini muscle on the dilatory mechanisms of the Eustachian tube and might contribute to recurrent otitis media with effusion as well. This review demonstrates that more research is warranted to clarify the role of the tensor veli palatini muscle within the cleft palate population in relation to the development of middle ear disease."} +{"text": "We previously showed that leg lean mass Z-score (LLMZ) correlates with metabolic exercise performance in Fontan patients. However, the mechanism by which leg lean mass influences exercise is not clear since LLMZ does not correlate with ventricular function or cardiac output at rest. We hypothesized that LLMZ would correlate with cardiac output at exercise and the change in cardiac output from rest to exercise.Thirteen patients had leg lean mass measured by dual energy x-ray absorptiometry within mean of 40 (range 0-258) days of completing an exercise cardiac magnetic resonance (CMR) protocol. LLMZs were generated from healthy reference data. Ventricular volumes and phase contrast flow measurements of the ascending (Ao) and descending (DAo) aorta, and superior vena cava (SVC) were obtained by CMR at rest and just after supine ergometer exercise to a heart rate associated with anaerobic threshold (determined by previous metabolic exercise test). Change in systemic flow (Qs = SVC + DAo) and indexed ventricular output (CI) during exercise, as well as baseline and peak exercise measures of Qs and CI were compared to LLMZ by linear regression.There was no correlation between LLMZ and resting flows. However, there was a strong linear relationship between LLMZ and change in both CI and Qs from rest to exercise (see figure). There was also a significant correlation between LLMZ and Qs at exercise . The correlation between LLMZ and CI at exercise did not reach significance .In our cohort, there was a strong correlation between LLMZ and change in both CI and Qs from rest to exercise. This suggests Fontan patients with higher LLMZ are better able to augment CI during exercise, improving performance. While it is tempting to attribute these differences to the effects of a peripheral muscle pump, cardiac output was not measured during active exercise but immediately after exercise. A prospective study will be needed to determine the effect of lower extremity strength training on cardiac output at exercise and exercise performance.This research was supported by NIH National Heart, Lung, and Blood Institute grants K23 HL089647 (KKW) and R01 HL67622."} +{"text": "CMR can provide details on ventricular pumping by sub-dividing the contribution to stroke volume (SV) into longitudinal shortening and radial inward motion of the ventricular borders. Previous studies have shown that patients with volume loaded dilated right ventricles (RV) due to pulmonary regurgitation (PR) have decreased longitudinal contribution to RVSV. Patients with atrial septal defects (ASD) also have volume loaded dilated RVs but it is not known if this affects longitudinal shortening. The purpose of the study was to determine the contribution of longitudinal shortening and radial motion to SV during dobutamine stress and rest in patients with ASD, and to study the early effects of ASD closure on ventricular pumping.Eighteen patients 13 females) with ASD and 16 healthy volunteers were imaged with CMR at rest and during a dobutamine/atropine stress protocol. Cine SSFP images were used for LV and RV volumes. Contribution of longitudinal shortening and radial motion to left ventricular (LV) SV and RVSV was measured using manual contouring of short axis images and measurement of longitudinal shortening in three long axis images in patients before closure was 2.2\u00b10.8. There was no difference in the contribution of longitudinal shortening to SV in patients and controls Table . During Ventricular pumping in patients with RV dilatation caused by ASD occurs with preserved longitudinal contribution to stroke volume. This differs from previous studies of RV dilatation in patients with pulmonary regurgitation. Dobutamine stress causes similar changes in ventricular pumping in patients with ASD and controls. ASD-closure results in altered ventricular pumping mechanics as early as on day one after treatment.The Swedish Heart-Lung foundation."} +{"text": "The bronchial dilations also called bronchiectasis are permanent and irreversible increase in the bronchial tubes. They can be extended or localized especially in pulmonary tuberculosis sequelae. This affection is serious, because it is at the origin of an embarrassing obstructive pulmonary disease, leading to social discomfort and preferentially in C\u00f4te d'Ivoire, it affects young subjects between 30 and 40 years old and former tuberculous. The place of surgery is still debated. Mr Coulibaly is 30 years old, hospitalized in the Thoracic Surgery Department of BouakeTeaching Hospital for pulmonary tuberculosis sequelae type symptomatic left lower bronchiectasis lobe and well localized (A). After a satisfactory preoperative evaluation, we performed a left lower lobectomy on this patient. Transection in the third world of the bronchial lower lobe resected reveals multiple tubular dilations with thickened wall containing purulent secretions (B). The specimen was sent to the pathology laboratory for confirmation of tuberculosis sequelae."} +{"text": "The past decade has seen notable progress in confronting the HIV epidemic. By the end of 2013, almost 13 million persons living with HIV had access to antiretroviral therapy (ART) in low- and middle-income countries (LMIC). In addition, progress has also been achieved in terms of HIV prevention with 26 LMIC achieving a 50% decrease in number of new infections including 15 countries in sub-Saharan Africa. New research findings also offer new efficacious prevention interventions that offer new opportunities for further mitigation of new HIV infections. Such progress has inspired ambitious pronouncements such as achieving \u201cThe End of AIDS\u201d or \u201cAn AIDS Free Generation.\u201d This progress has also motivated the development of new guidelines aimed to achieving these goals. Implementation of guideline recommendations, however, requires translation of these guidelines into actionable steps and then their successful delivery within established health services. The opportunities offered by new guidelines frequently meet the realities of the frailties of the health system. Achieving current ambitious global targets must confront the specific deficits in the health system that inhibit access and acceptability of programmes as well as hinder achievement of high quality or desired coverage."} +{"text": "Unfortunately, the original version of this article containeThe original paragraph read:\u201cThis study demonstrates that the children with evidence of allergy were equally likely as children without evidence of allergy to demonstrate evidence of bacterial infection in PCR tested MEE specimens. Although this study did not demonstrate a positive relationship between allergy and the bacterial infection in the pathogenesis of OME. However, numerous studies have indicated that allergic subjects are more susceptible to OME than non-allergic controls.\u201dHowever this sentence should read:\u201cThis study demonstrates that the MEE specimens from children with evidence of allergy were equally likely as those from children without evidence of allergy to demonstrate evidence of bacterial infection by PCR. Although this study did not demonstrate a positive relationship between allergy and the bacterial infection in the pathogenesis of OME, numerous studies have indicated that allergic subjects are more susceptible to OME than non-allergic controls.\u201d"} +{"text": "Autism spectrum disorders (ASDs) are genetically and clinically heterogeneous and lack effective medications to treat their core symptoms. Studies of syndromic ASDs caused by single gene mutations have provided insights into the pathophysiology of autism. Fragile X and Rett syndromes belong to the syndromic ASDs in which preclinical studies have identified rational targets for drug therapies focused on correcting underlying neural dysfunction. These preclinical discoveries are increasingly translating into exciting human clinical trials. Since there are significant molecular and neurobiological overlaps among ASDs, targeted treatments developed for fragile X and Rett syndromes may be helpful for autism of different etiologies. Here, we review the targeted pharmacological treatment of fragile X and Rett syndromes and discuss related issues in both preclinical studies and clinical trials of potential therapies for the diseases. Autism spectrum disorders (ASDs) encompass a group of neurodevelopmental disorders which are of different etiologies and characterized by impairments in socialization and communication, abnormalities in language development, restricted interests, and repetitive and stereotyped behaviors gene, which eventually leads to the absence of its protein product, fragile X mental retardation protein (FMRP) play important roles in synaptic plasticity, learning and memory -pyridine (MPEP) stabilized hippocampal protein synthesis, increased the density or rescued the morphology of hippocampal dendritic spines, corrected altered brain network function, reduced audiogenic seizures and repetitive and/or perseverative behaviors (marble burying), rescued the deficits in prepulse inhibition of startle response, and improved the maze and motor learning decreased repetitive and/or perseverative behaviors corrected neuronal hyperexcitability in the amygdala is the main inhibitory neurotransmitter in brain and signals through GABAB receptor agonists have potential to improve social function and behaviors in fragile X patients in blood was observed with treatment and might become a useful biomarker for future clinical studies system is involved in various brain functions, including synaptic plasticity, learning and memory activate cannabinoid receptors (CB1R and CB2R), and modulate synaptic plasticity and cognitive function ) mice, whereas CB2R antagonism with AM630 normalized anxiolytic-like behaviors in those mice with JZL184, normalized this synaptic defect and corrected behavioral abnormalities in fragile X mice are widely expressed in the central nervous system and mediate the metabotropic actions of acetylcholine and reduced susceptibility to audiogenic seizures in Oxytocin acts as a neuromodulator through its receptors in various brain areas and regulates social cognition and behaviors Neumann, . It is eThe clinical actions of oxytocin have been validated in ASD patients, providing preliminary evidence that oxytocin is able to enhance brain function and improve social behaviors in autistic patients is impaired in Fmr1 knockout mice suggested that memantine may exert therapeutic capacity for fragile X syndrome through a stimulatory effect on dendritic spine maturation and excitatory synapse formation , PI3K, mTOR and glycogen synthase kinase-3 (GSK3)] which could be downstream of neurotransmitter receptors such as glutamate, GABA, endocannabinoid, 5-HT, dopamine or mACh receptors. These signaling pathways may serve as therapeutic targets for fragile X syndrome Figure , 2.Fmr1 knockout mice and regulates many cellular processes through phosphorylation of their substrates. The activity of GSK3 itself is controlled by inhibitory serine phosphorylation induced by various intracellular pathways including PI3K/Akt and MEK/ERK that converge on GSK3 are a family of extracellular proteases that are involved in synaptogenesis, neurotransmission and synaptic plasticity are critical for regulation of actin polymerization, dendritic spine morphogenesis and synaptic plasticity is a transmembrane protein that plays roles in synaptogenesis and synaptic plasticity are responsible for RTT , both FDA-aproved drugs for the treatment of multiple sclerosis. Fingolimod is a modulator of the sphingosine-1 phosphate receptor, which leads to an increase in BDNF expression and activation of TrkB downstream signaling pathways is a growth factor that, by binding to the IGF-1 receptor, activates similar intracellular signaling cascades to those triggered by BDNF activation of TrkB receptors. Indeed, IGF-1 modulates synaptic plasticity and neuronal maturation through a tyrosine kinase signaling pathway that includes PI3K-Akt and MAPK in autopsy RTT brains and in Mecp2 knockout mice leads to the premature termination of transcripton due to a premature STOP codon (Schanen et al., MECP2 mutations that result in single amino acid substitutions. Aminoglycoside antibiotics like gentamycin are so-called \u201cread-through\u201d compounds because they allow ribosomal read-through of the premature STOP codon during translation, yielding a full-length functional protein. Aminoglycoside and non-aminoglycoside \u201cread-through\u201d compounds have been tested for therapeutic efficacy in Duchenne muscular dystrophy and cystic fibrosis (Zingman et al., In approximately one-third of RTT individuals, a nonsense mutation in Although many targeted treatments have shown efficacy across multiple aspects in ASD animal models, none has thus far demonstrated the same effectiveness in patients (Katz et al., One of the key issues in preclinical studies is that almost every study has applied its own animal behavioral experiment battery to evaluate the effects of genetic or pharmacological manipulations, making comparison of efficacy among different treatments a challenge. This situation will be greatly improved if animal behavioral test batteries for identifying potential therapy could be standardized. Despite the discrepancies in test batteries from different laboratories, many of the targeted treatments achieve predicted effect and rescue at least part of phenotypic features of the disorders in animal models (Braat and Kooy, Despite progress made in clinical trials, one of the major concerns for these studies is the lack of appropriate outcome measures for an objective assessment of patients\u2019 daily performance (Berry-Kravis et al., Fmr1 promoter might help identify a subgroup of fragile X patients that will respond well to this treatment (Jacquemont et al., Differential responses to one specific treatment have been observed in individuals with ASDs. Some subgroups within the patient cohorts respond to the therapy where others do not, as evident from clinical trials in fragile X syndrome (Jacquemont et al., Interference with the molecular pathways disturbed in ASDs has led to the initiation of clinical trials. The fragile X and Rett syndromes are the prototypes of neurodevelopmental disorders for which targeted treatments are becoming realities (Samaco and Neul, It is generally believed that earlier interventions in developmental disorders will have better outcomes. Abnormalities occurring during early development have usually been considered irreversible in adulthood. However, studies in mouse models of neurodevelopmental disorders, including fragile X and Rett syndromes, suggest that many pathophysiological aspects associated with the disorders can be reversed by genetic or pharmacological manipulations performed during adulthood (Castr\u00e9n et al., The increasing need for effective treatments of fragile X and Rett syndromes, coupled with the availability of animal models and iPSC-derived neurons from human individuals with these disorders (Marchetto et al., The rational therapeutics for ASDs requires the knowledge of an entire spectrum of symptoms that relate to each specific disorder. The complicated pathophysiology of fragile X and Rett syndromes should be taken fully into account in designing preclinical and clinical studies. It should be recognized that loss of FMRP or MeCP2 will affect a number of downstream targets which exist not only on neurons, but on glial cells and other tissues (McCauley et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "The anteroposterior compartments of the developing hindbrain (rhombomeres [r]) are normally patterned by the combinatorial action of distinct Hox genes. Using Affymetrix GeneChips to define the repertoire of genes regulated in each rhombomere, we have performed a systematic survey of the transcriptional status of individual segments of the developing chick hindbrain (r1-5) at a key stage of early development (HH11) and identified hundreds of previously un-described genes expressed in this region. For comparative purposes, we have also included the adjacent region of the embryonic midbrain (m) in our dataset. In summary, six different embryonic brain regions are represented by biological duplicates to give a raw dataset comprised of 12 individual Affymetrix GeneChip Cel and CHP files. These data give an opportunity to assess the genome-wide complexity of gene expression during patterning of the chick developing midbrain and hindbrain, and may be relevant to extending our understanding of the genes regulated by Hox family transcription factors. However, despite this being a well-recognised and investigated paradigm, we still remain relatively ignorant of the repertoire of subsequent molecular \u2018effectors\u2019 that then impart specific facets of neuronal phenotype, behaviour and connectivity. The developing vertebrate hindbrain and the adjacent mid-hindbrain boundary (MHB) signalling centre provide the ideal accessible systems in which to examine the molecular controls underpinning the emergence of neuronal diversity4\u20137.The vertebrate central nervous system (CNS) consists of thousands of distinct neuronal cells, each with unique molecular genetic profiles, organised into complex patterns of circuitry that ensure appropriate functional output. A key goal of modern developmental neuroscience is to unravel the molecular logic that first establishes the diversity in neuronal cell type and subsequently dictates their development into integrated higher order networks. Recent years have seen considerable progress in establishing the initial framework that underpins the acquisition of neuronal identity. Several lines of evidence have shown that cells are specified in discrete steps within the neuroepithelium influenced by the anteroposterior (AP) and dorsoventral (DV) position of the precursor cell. In response to diffusible molecular cues released by discrete cell groups known as local organisers a series of high level \u2018selector\u2019 genes establish the first molecular co-ordinates that imprint progenitor cells with an outline fate9. However, despite their well-defined roles in the establishment of anteroposterior (AP) pattern and considerable research into their mechanism of action, relatively few target genes have been identified in the downstream regulatory network and the caudal portion of the midbrain. Our dataset comprises systematic surveys of the transcriptional status of individual segments of the developing chick hindbrain (r1-5) and the adjacent region of the embryonic midbrain (m) during the HH11 stage of chick development. In summary, six different embryonic brain regions are represented by biological duplicates to give a raw dataset comprised of 12 individual Affymetrix GeneChip Cel and CHP files.These data can be exploited to investigate how neuronal specificity is achieved by the recruitment of effector genes and will afford insights into 3 key areas;What and how are the developmental genetic factors used in response the patterning influences of the mid-hindbrain signalling centre?For the understanding of how Hox genes and signalling molecules drive downstream networks of effector gene activityDriving the differentiation of embryonic-derived stem cells towards specific fatesA more complete understanding of the molecular logic of neuronal specification and how patterning instructions are relayed to a defined set of cellular cues is a prerequisite to expanding out knowledge of nervous system function in health and disease and for developing rational approaches to progressing stem cells towards a defined fate for use in restorative medicine.4\u20136. These mouse and chick parallel datasets can be used to define the underlying conserved genetic modules controlling the equivalent patterning processes in the midbrain and hindbrain as well as serving to cross validate each other. Similarly, these data will offer insights into the molecular mechanism that underpin the fundamental patterning differences between mouse and chick. For example, the migration of cell bodies of the r4-derived facial motor nucleus differs between the two species and the molecular genetic control of this is likely to be reflected in the array data sets.This study complements our previous approaches to address similar questions in the chick and mouse developing midbrain and hindbrainNeural tubes from stage-matched embryonic day (E) 3 [HH11] chick embryos were isolated from surrounding ectoderm and mesoderm as follows; embryos were removed from the egg, washed in phosphate buffered saline (PBS) for 5\u2009min and then transferred to 1\u2009mg/ml Dispase for 5\u2009min. Mechanical dissection with sharpened tungsten needles was used to isolate the entire neural tube free of mesoderm and ectoderm. The characteristic neuromeric landmarks of the developing neural tube were used to guide the dissection . Neural Total RNA was isolated from each pool using the Absolutely RNA microprep kit in conjunction with DNase-treatment (Agilent Technologies). Tissue samples collected by the above criteria but on different occasions by the same operator were designated as biological replicates. Thus, each region of the neural tube is represented by duplicate pools (denoted as sets 1\u20132).Labelled extracts, for processing on microarrays, were generated from 10\u2009ng of total RNA by the NuGen Ovation V2 protocol (NuGEN Technologies Inc). Labelled extracts (7\u2009\u03bcg SPIA-generated cDNA) for each sample were hybridised to Affymetrix Chicken GeneChips at 45\u2009\u00b0C for 20\u2009h before being washed, stained (GeneChip\u00ae Fluidics Station 450) and scanned (GeneChip Scanner 3000 7G) as per manufacturer\u2019s instructions (NuGen Technologies Inc & Affymetrix) to produce DAT files, and as described in The DAT file contains generated from the Affymetrix scanner contain multiple pixel intensity values for each probe on the GeneChip. The DAT files are processed by the Affymetrix image analysis package into a Cel file format that represents a single intensity value for each probe on the array . For thiFor the Affymetrix Chicken GeneChip used here, each gene sequence is represented by 11 independent probes on an array. The final expression value for a transcript is derived from the Cel file by summarisation. This is a process where the 11 probe set intensity values are transformed into a single value that is used to represent the expression level. The data files described here have been summarised by the Affymetrix MAS5 algorithm and represented in the CHP (txt) files. These txt files therefore contain information on each probe set identity, a single expression value and some biological descriptors of the gene identity and function. In addition, Affymetrix associate a \u2018flag\u2019 designation for each gene expression value that represents measure of the signal reliability; P (Present). M & A (Absent). Thus, the MAS5 processed expression values have been adjusted for the signals detected by the MM probes . HoweverA raw dataset, comprised of 12 individual Affymetrix GeneChip Cel and CHP files, representing biological duplicates of six different embryonic brain regions is deposited in the Gene Expression Omnibus (Data Citation 1).via mRNA Pico chips. Total RNA populations derived from each neural tube segment each recorded RNA Integrity numbers greater than 7 in midbrain microarray samples only which is concordant with its known restricted expression to this tissue in the developing embryo relative to the other samples in this dataset . Similarly, both Fgf8 and Hoxb1 are significantly up regulated in discrete microarray samples (m/r1 & r4 respectively) in line with the known restricted domains of mRNA expression in an equivalent-stage chick embryo was investigated using PCA. \u2018Following normalisation and the removal of non expressed genes and Affymetrix control signal (AFFX controls), the overall distribution of gene expression levels for each sample was examined with PCA or combinations of tissues . The dataset described here is represented by biological duplicates for each condition. Consequently, this may limit any potential application of this dataset when compared to those with statistical power .http://www.genomics.agilent.com]) or freeware packages designed for the statistical interrogation and visualisation of microarray-based experiments.The data files described here can be analysed with either proprietary or other statistical tests. The application of a multiple testing correction is optional. Statistically different genes can be functionally classified using a combination of Gene Ontology (GO) criteria and other molecular descriptions derived from UniGene, GenBank and Entrez Gene databases. Genes may also be clustered into potentially co-regulated groups using both unsupervised and supervised approaches including self-organising maps and quality threshold clustering. A more detailed biological interpretation of the set of differentially expressed genes can be derived from the use of gene network analysis packages .Data can be analysed using the GeneSpring package . Briefly, the suitability of the expression data sets for inclusion in the analysis and the overall relationship between and within the biological replicates is first assessed using quantile plots, principle components analysis (PCA), hierarchical clustering and assessment of internal control performance. Differential gene expression between samples maybe determined by the application of a multi-step process; briefly, samples are first \u2018globally\u2019 normalised across the expression dataset to compensate for potential technical variation in the levels of hybridisation of the arrays, and then each gene expression value is scaled to a user defined value . Prior to statistical analysis to determine significant differential gene expression between samples, the set of genes to be analyzed can be pre filtered. For example, genes classed as being not expressed or not varying their expression above a user defined level can be removed. Similarly, genes with low \u2018raw\u2019 values across all of the samples can also be removed prior to statistical analysis. From the remaining set of genes, genes whose expression levels differ significantly between each sample can be determined by one-way analysis of variance ."} +{"text": "Panicum virgatum) monocultures and species-rich prairie plantings on private farm fields that were managed similarly to bioenergy plantings. The other study was an experiment where switchgrass was planted in monoculture and in combination with increasingly species-rich native prairie mixtures. Overall, we found that bioenergy plantings with higher species richness did not produce more biomass than switchgrass monocultures. The lack of a positive relationship between planted species richness and production in our studies may be due to several factors. Non-planted species (weeds) were not removed from our studies and these non-planted species may have competed with planted species and also prevented realized species richness from equaling planted species richness. Also, we found that low seeding density of individual species limited the biomass production of these individual species. Production in future bioenergy plantings with high species richness may be increased by using a high density of inexpensive seed from switchgrass and other highly productive species, and future efforts to translate the results of biodiversity experiments to bioenergy plantings should consider the role of seeding density.Biodiversity experiments show that increases in plant diversity can lead to greater biomass production, and some researchers suggest that high diversity plantings should be used for bioenergy production. However, many methods used in past biodiversity experiments are impractical for bioenergy plantings. For example, biodiversity experiments often use intensive management such as hand weeding to maintain low diversity plantings and exclude unplanted species, but this would not be done for bioenergy plantings. Also, biodiversity experiments generally use high seeding densities that would be too expensive for bioenergy plantings. Here we report the effects of biodiversity on biomass production from two studies of more realistic bioenergy crop plantings in southern Michigan, USA. One study involved comparing production between switchgrass ( Biofuel production has increased rapidly in the USA , partialA number of experimental studies in grasslands show that high diversity plantings produce more biomass than monocultures . This haExperimental biodiversity studies prevent invasion of non-planted species by hand-weeding, whereas agricultural bioenergy plantings would allow low abundances of weeds or use herbicides or tillage to control weeds. The absence of hand-weeding will reduce differences between species richness in monoculture and high diversity plantings because non-planted species can (and do) invade these systems. Roscher et al. show thaIn addition, bioenergy plantings are likely to only use highly productive species in monoculture whereas, for experimental rigor, biodiversity studies have used both productive and unproductive species in monocultures and mixtures. Thus, some of the increase in biomass production between monocultures and more diverse mixtures in biodiversity experiments have been attributed to the sampling effect, in which more diverse mixtures are more likely to include highly productive species and, consequently, higher community-level biomass yields . HoweverPanicum virgatum), a native C4 grass that has high potential as a bioenergy crop because of its high productivity and broad geographic range in North America , where ps is the percent of total biomass made up of each species harvested from the plot and S is the number of species harvested from the plot.We completed several analyses from the LTER experiment hand harvest data. We analyzed the relationship between biomass production and planted species richness with an ANCOVA using seeding density as the covariate, planted species richness as a main plot treatment, and species identity as a nested treatment. Only the 6 grass species planted into both the 6-species and 18-species richness treatments were included in this ANCOVA because only these 6 species were present in both treatments. We calculated species\u2019 expected and observed percent of total biomass based on the initial seeding density or observed biomass production, respectively. We calculated Simpson\u2019s evenness from observed biomass production using thThe data in the analyses were normally distributed and no transformations were necessary, except for 2012 individual species biomass in the LTER experiment (relationship between seeding density and biomass production), where the data were log transformed. The SAS code for all analyses is included in Panicum virgatum) and higher species richness prairie plantings (Andropogon gerardii) and other native C4 grasses monocultures and diverse prairie mixtures. This is one of the first tests of the potential role of biodiversity to produce more biomass in bioenergy plantings using species mixes and seeding densities more realistic to agriculture [see also 18]. The lack of a relationship between plant richness and biomass production in our field surveys and experiment challenges the presumption that results of biodiversity-productivity studies can be directly translated to agriculturally realistic planting designs and management [In our studies, we did not find a persistent positive relationship between species richness and biomass production. In the LTER experiment there was only a positive relationship between planted species richness and biomass production in the first year of sampling (2010). In subsequent years, this relationship was not significant or was negative. In the GLBRC field surveys, there was no significant difference in production between the fields planted to switchgrass were not removed from low richness treatments in our GLBRC field surveys and LTER experiment, whereas non-planted species were removed (by hand weeding) to maintain species richness treatments in past biodiversity studies. In our GLBRC field surveys and LTER experiment, non-planted species accounted for an average of 20% and 13%, respectively, of the total biomass in the switchgrass monoculture plantings, and non-planted invasion led to little difference in observed richness between switchgrass monocultures and more diverse plantings see Figs . The mosuctivity ,30 and ructivity ). This auctivity and ther-1 in a grassland species richness experiment established at the Cedar Creek Ecosystem Science Reserve in Minnesota, USA, whereas our LTER experiment used a total seeding density of 7.2 kg ha-1 in the highest richness treatment can limit production during the first year of establishment [-1 may also increase biomass production. Future research should seek to create designer planting mixes that maximize complementarity. We cannot directly test the role of complementarity in our study because we have not grown each species in monoculture, but we did not observe that seeding more functional groups than just switchgrass (a C4 grass) increased biomass production. However, legumes and non-planted species were present even where only switchgrass was seeded seed such as switchgrass and other highly productive species are not reduced in more diverse plantings. This should increase total production. It may be difficult to find seeding densities that allow both high biomass production and high realized plant diversity ,42, but S1 Fig(PDF)Click here for additional data file.S1 TableTotal biomass from each year of sampling and further information about environmental conditions of sites.(XLSX)Click here for additional data file.S2 TableSpecies used in the Cedar Creek and our (XLSX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file."} +{"text": "Electrophysiological recording and morphological reconstruction of pyramidal neurons from layer 3 prefrontal cortex of young and aged adult rhesus monkeys reveals higher firing rates and structural differences in aged neurons relative to young . Prior cWe fit ion channel parameters in a neuron model using a rapid, multi-stage, semi-automated approach based on our previous optimization protocol , resultiWe have used this method to generate populations of models for 1 aged and 3 young neurons. Linear discriminant analysis reveals that these four populations are readily distinguished within the parameter space. Models of the three young neurons have different ion channel parameters despite similarities in electrophysiological responses. Principal component analysis (PCA) reveals that all models of the aged neuron can be separated from models of the young neurons along the first principal component. Preliminary differences between parameters fitting these four neurons reveal, for example, that in the aged neuron the L-type calcium channel activates more slowly and has greater maximal conductance, and voltage dependence of the persistent sodium channel is lower. Generation of populations of models for other empirically characterized young and aged neurons is underway with our optimization protocol. Analytical techniques such as PCA will help generate predictions about intracellular changes during normal aging."} +{"text": "Opioids are among the most effective drugs to treat severe pain. They produce their analgesic actions by specifically activating opioid receptors located along the pain perception pathway where they inhibit the flow of nociceptive information. This inhibition is partly accomplished by activation of hyperpolarizing G protein-coupled inwardly-rectifying potassium (GIRK or Kir3) channels. Kir3 channels control cellular excitability in the central nervous system and in the heart and, because of their ubiquitous distribution, they mediate the effects of a large range of hormones and neurotransmitters which, upon activation of corresponding G protein-coupled receptors (GPCRs) lead to channel opening. Here we analyze GPCR signaling via these effectors in reference to precoupling and collision models. Existing knowledge on signaling bias is discussed in relation to these models as a means of developing strategies to produce novel opioid analgesics with an improved side effects profile. The two channel effectors prominently mediate analgesic effects of opioids. For example, studies in rodents have shown that activation of MORs on small, unmyelinated nociceptors , \u03b4 (DOR), and \u03ba (KOR) receptors . Although all four of them are expressed in peripheral influence opioid dose requirements for both acute management of postoperative pain variations showed no effect -mediated signaling by MOR agonists molecules and four Na+ ions bound to corresponding regulatory sites on the channel and bioluminescence resonance energy transfer (BRET)-based approaches have both revealed spontaneous energy transfer between channel subunits and G proteins , would be of particular interest since they could selectively enhance Kir3 signaling by DORs and no other receptors that modulate this effector and effectors (Kir3 channels) were provided.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Among methods for preventing pneumonia and possibly also bacteremia in ICU patients, selective digestive decontamination appears most effective within randomized concurrent controlled trials (RCCTs) . HoweverBacteremia incidence proportion data were extracted from component (control and intervention) groups from studies investigating antibiotic (SDD) or nonantibiotic methods of VAP prevention. Both the counterfactual and the contextual effects of SDD factorized as topical or protocolized parenteral exposures were estimated using random-effects meta-analysis of study and group level data. Studies without any prevention methods under study constituted the reference category (benchmark groups).n = 18 studies). As a contextual however, the mean bacteremia incidence among 27 control groups and 12 intervention groups receiving topical antibiotic alone from SDD RCCTs is double that of 36 benchmark groups and 19 control groups from studies of nonantibiotic methods . The upward dispersion in bacteremia incidence among component groups from SDD RCCTs away from this benchmark is striking with all but two of the 27 control groups and all but two of 12 SDD intervention groups that did not receive PPAP being above this benchmark.As a counterfactual within RCCTs, SDD when given as combined topical and parenteral antibiotic appears to halve the bacteremia incidence (odds ratio (OR) 0.59; 0.48 to 0.73; The major contextual hazard of SDD toward bacteremia among ICU patients is inapparent within individual studies. The apparent protection in SDD RCCTs is spurious as the SDD counterfactual is conflated by the strong contextual effect with partial mitigation by SDD protocolized parenteral antibiotic. Not only is the safety of SDD within the ICU environment unclear, but this SDD contextual effect may conflate the apparent SDD counterfactual effect on the incidence of bacteremia, as with VAP."} +{"text": "Global policy supports service integration to ensure that the contraceptive needs of women using HIV/AIDS services are met. We conducted an intervention study in 5 public sector health facilities in South Africa to test a strategy for serving the reproductive health (RH) needs of postpartum PMTCT clients. We conducted another study in 8 public sector health facilities in Uganda to test an intervention for increasing dual method use (condom + another contraceptive) among clients receiving antiretroviral therapy. Both studies failed to demonstrate increases in contraceptive prevalence. We examined process data to understand why RH-HIV service integration was not successful when tested in routine practice.To complement the two intervention trials, we conducted process evaluations to evaluate intervention implementation. Using a tracking tool, we systematically documented how intervention components were implemented, noting how actual implementation compared to the original design. The tool documented problems encountered during implementation. It provided space for the study team to make recommendations on adjustments to be made for future implementation.Process data from both studies revealed problems with intervention implementation that were rooted in health system constraints. These included inadequate human resource management, weaknesses in commodity management, poor coordination to support referrals, and inadequate reporting mechanisms. The process evaluation revealed that for RH-HIV service integration to succeed in routine service delivery contexts, attention must be directed toward reinforcing the health system components undergirding service delivery. These two research experiences highlighted the importance of process evaluation to know why interventions fail.Results on the implementation process and the influence of context where interventions are introduced should be systematically reported so that trials results can be interpreted accurately and health service managers can confront systemic problems that limit health service innovations.Funding for both studies was provided by the US Agency for International Development."} +{"text": "Idiopathic Intracranial Hypertension (IIH) is characterised by an increased intracranial pressure (ICP) in the absence of any central nervous system disease or structural abnormality, and normal CSF composition. Management becomes complicated once surgical intervention is required. Venous sinus stenosis has been suggested as a possible aetiology for IIH. Venous sinus stenting has emerged as a possible interventional option. Evidence for venous sinus stenting is based on elimination of the venous pressure gradient and clinical response. There have been no studies demonstrating the immediate effect of venous stenting on ICP.Patients with a potential or already known diagnosis of IIH were investigated according to departmental protocol. ICP monitoring was performed for 24 hours. When high pressures were confirmed, CT venogram and catheter venography were performed to look for venous stenosis to demonstrate a pressure gradient. If positive, venous stenting would be performed and ICP monitoring would continue for a further 24 hours after deployment of the venous stent.Ten patients underwent venous sinus stenting with concomitant ICP monitoring. Nine out of ten patients displayed an immediate reduction in their ICP that was maintained at 24 hours. The average reduction in mean ICP and pulsatility was significant (p=0.003). Six out of ten patients reported a symptomatic improvement within the first 2 weeks.Venous sinus stenting results in an immediate reduction in ICP. This physiological response to venous stenting has not previously been reported. Venous stenting could offer an alternative treatment option in correctly selected patients with IIH."} +{"text": "Driving a taxicab remains one of the most dangerous occupations in the United States, with leading homicide rates. Although safety equipment designed to reduce robberies exists, it is not clear what effect it has on reducing taxicab driver homicides.Taxicab driver homicide crime reports for 1996 through 2010 were collected from 20 of the largest cities in the United States: 7 cities with cameras installed in cabs, 6 cities with partitions installed, and 7 cities with neither cameras nor partitions. Poisson regression modeling using generalized estimating equations provided city taxicab driver homicide rates while accounting for serial correlation and clustering of data within cities. Two separate models were constructed to compare (1) cities with cameras installed in taxicabs versus cities with neither cameras nor partitions and (2) cities with partitions installed in taxicabs versus cities with neither cameras nor partitions. Cities with cameras installed in cabs experienced a significant reduction in homicides after cameras were installed and compared to cities with neither cameras nor partitions . Cities with partitions installed in taxicabs experienced a reduction in homicides compared to cities with neither cameras nor partitions, but it was not statistically significant.The findings suggest cameras installed in taxicabs are highly effective in reducing homicides among taxicab drivers. Although not statistically significant, the findings suggest partitions installed in taxicabs may be effective. Taxicab drivers work in one of the most violent occupations in the United States . Two covariates were included in the statistical analysis: the concurrent decline in homicide rates in the United States since 1990 and the number of licensed taxicabs from municipal transportation regulators (denominator). A standardized search strategy tailored for municipal police crime departments was used to locate taxicab driver homicides using any one of the following criteria: (1) crime premise designated as \u2018vehicle\u2019, (2) name of licensed taxicab companies, (3) keywords \u2018cab\u2019, \u2018taxi\u2019 or \u2018driver\u2019 in the crime report or (4) news clippings reporting taxicab driver homicides. Each crime report provided by each municipal police department was reviewed for relevance by the first author. Information on installation year of camera or partition (or neither) was obtained directly from city transportation regulators. A city was designated annually as a \u201ccamera city\u201d or \u201cpartition city\u201d if >70% of the cabs had cameras or partitions during the study period; a cut point of 70% was decided Generalized estimating equations accounted for serial correlation and clustering of data within cities. Annual taxicab driver homicide rates were modeled on installation status (camera or partition) compared to control cities in two separate models adjusting for covariates. A separate model restricted to camera cities only compared post-installation versus pre-installation homicide rates. The Wald test statistic determined the significance of installation status. The natural logarithm of the number of licensed taxicabs by city per year was used as an offset variable. The taxicab driver homicide counts were assumed to follow a Poisson distribution; the offset variable provided the denominator used to calculate the homicide rates. The data were tested for dispersion and found to be slightly under-dispersed . Therefore, all confidence intervals were considered conservative in their range.Police crime report data were analyzed for 7 cities where cameras were installed in taxicabs, 6 cities where partitions were installed in taxicabs and 7 cities where neither partitions nor cameras were installed in taxicabs. From 1996 through 2010, 95 taxicab driver homicides were investigated by law enforcement authorities after controlling for pre-existing annual changes in city taxicab driver homicide rates (\u201cyear\u201d) and the background city homicide rate. A sub-analysis exploring changes in taxicab driver homicide rates among cities with only a company policy found a statistically significant reduction in rate that persisted after covariate adjustment. Modeling annual citywide taxicab driver homicide rates on camera installation in camera cities compared with control cities post-installation versus pre-installation revealed a statistically significant reduction in homicide rate (Model 2) that persisted after controlling for covariates. A sub-analysis exploring changes in taxicab driver homicide rates in cities where camera installation is not citywide but due to a company policy found a reduced rate compared to control cities in both unadjusted and adjusted models . Modeling annual citywide taxicab driver homicide rates on partition installation in partition cities compared with control cities revealed a slightly higher rate which, after controlling for declining homicide rates and background crime rates, presented a reduction in taxicab driver homicide rate that was not statistically significant .Modeling annual citywide taxicab driver homicide rates among camera cities post-installation compared with pre-installation revealed a statistically significantly reduced homicide rate taxicab company implemented a policy of camera-equipped taxicabs. Findings suggest that cities with partition-equipped taxicabs may experience reduced taxicab driver homicide rates compared to nonpartition-equipped other cities.The observance of a significant reduction in taxicab driver homicides in cities where taxicabs are equipped with cameras, and the more pronounced effect in cities where taxicabs are equipped with cameras as mandated by a city ordinance, was an encouraging one. Based on anecdotal evidence from transportation regulators in cities already mandating cameras by ordinance, transportation regulators are currently considering promulgating city ordinances for their cities that will mandate the use of security cameras in taxicabs. Our findings suggest cities where taxicabs are equipped with cameras could result in fewer taxicab driver homicides particularly in cities with an ordinance mandating taxicabs are equipped with cameras. Furthermore, these findings are consistent with a previous analysis examining news clippings to identify homicides we could not estimate individual risk of homicide for taxicab drivers across safety equipment types and (2) the lack of uniform data in the crime reports on homicide circumstances precluded more detailed analyses. Significant strengths of this study are the 15-year time span, systematic collection of data in 20 major US cities, pre-post design that includes use of comparison cities with neither camera-equipped nor partition-equipped taxicabs, and statistical analysis that accounts for serial correlation of data that adjusts for two crucial covariates. Additionally, in working with city regulators and counting only licensed drivers, we were able to assume the drivers were in taxicabs installed with required safety equipment. The study and its findings make a significant contribution to understanding the possible effect of taxicab safety equipment on taxicab driver homicides.Taxicab driver personal safety in Seattle and King County, Final report and recommendations. The report of the Taxicab Advisory Group Committee on Driver Safety to the Director of the Department of Executive Administration for the city of Seattle. June 18, 2004."} +{"text": "Schwarzer et al. showed dIn our opinion, the key message of their study is that commercially available BGAs are not accurate when measuring [iCa] outside the reference range and therefore clinicians should avoid using multiple BGAs to guide RCA in individual patients. However, there is no indication to change a RCA protocol which has been proven safe and effective in >10 studies including >2000 patients from different countries regardless of the BGA used .Schwarzer et al. also raise concern about the potential risk of life-threatening citrate intoxication. Whether raised citrate levels are toxic or merely indicative of impaired cellular metabolism remains unclear, but excess citrate can cause metabolic alkalosis. The Fresenius RCA protocol and the technical specifications of the multifiltrate machine both include safety mechanisms to detect potential citrate accumulation early. The risk of citrate toxicity is low <3\u00a0%), even in high-risk patients with liver failure \u00a0%, even .Given the proven advantages of RCA and the Kidney Disease Improving Global Outcomes (KDIGO) recommendation to use citrate as the first-line anticoagulant during continuous renal replacement therapy, the accuracy of commercially available BGA devices should be improved. In our opinion, there is no need to change a safe and effective protocol."} +{"text": "Silicon based photon detectors make it nowadays possible to build highly integrated PET detectors for simultaneous PET/MRI. Although the operating principle of silicon photomultipliers is believed to be insensitive to the strong magnetic fields inside MRI machines, the construction of an MRI compatible detector has to cover considerably more aspects of MRI compatibility. In this paper we report on our development of an MRI compatible PET detector stack based on digital SiPMs for the Hyperion IID scanner.We developed an MRI compatible PET detector stack composed of two main assembly groups, called sensor tile and interface board. The sensor tile contains an array of 64 digital SiPM and is connected via two connector to the interface board. The interface board contains a Xilinx Spartan 6 FPGA for data collection, configuration and voltage control.We investigated the PET performance and MRI compatibility of the detector stack in combination with pixilated LYSO scintillator arrays with 1 mm pitch and 4 mm pitch inside a 3 Tesla Philips Achieva MRI system. We used MRI sequences with intensive gradient switching, field homogeneity and spurious noise scans to investigate PET/MRI interference effects. Additionally we build up laboratory setups with electromagnets and strong permanent magnets to study gradient induced effects and B0 dependencies in more detail.The detector stack is fully functional inside the B0 field and position histograms are undistorted. Energy histograms showed on average a 1 % upward shift of the 511 keV photopeak position caused by a shift of bias voltage supply. Initially observed effects of gradient switching on energy resolution and stability could be traced back to noise pickup of the voltage controllers.We couldn't observe any direct effect of the MRI environment on the digital SiPM itself. In fact MRI compatibility in practice is determined by proper design of the entire system."} +{"text": "Two popular protocols for inducing LTP in CA1 pyramidal cells are high-frequency stimulation (HFS), typically continuous 100 Hz tetanization for 1 second, and burst stimulation with 4 pulses at 100 Hz repeated at 200 ms intervals. NMDA blockers prevent LTP with burst stimulation, but some NMDA-independent LTP remains with HFS. Conversely, the L-channel antagonist nifedipine strongly inhibits LTP following HFS, but has a relatively small effect on LTP induced by burst stimulation [With continuous tetanization it has been observed that EPSPs sum and produce a large depolarization that triggers action potentials early in the train, but later in the train EPSPs are smaller and fail to produce action potentials even though voltage remains elevated . To accoWe found that total calcium influx at the soma was considerably larger with burst stimulation than with a continuous tetanus and was directly related to the number of action potentials produced by each protocol. However calcium influx with continuous tetanization was larger early in the train causing the average soma calcium concentration change for the first few hundred milliseconds to be up to 2-fold larger than with burst stimulation. At spines calcium influx was also larger at early times with continuous tetanization, but again total calcium influx was considerably larger with burst stimulation. Curiously, we occasionally saw spikelets of 10s of mV generated in the dendrites with continuous tetanization, but these did not propagate successfully to affect soma voltage. Initial spikes or spikes generated after a quiet period tended to be generated at the initial segment, but the site of action potential generation of subsequent spikes moved into the dendrites.The larger total calcium influx seen with burst stimulation is consistent with the observation that this is a more effective protocol than continuous tetanization for inducing LTP. How the differences in calcium signals observed at spines and the soma with these two different protocols affect LTP induction or explain the different actions of L-channel and NMDA blockers on LTP induction with these protocols awaits further study."} +{"text": "Mitochondrial homoplasmy signifies the existence of identical copies of mitochondrial DNA (mtDNA) and is essential for normal development, as heteroplasmy causes abnormal development and diseases in human. Homoplasmy in many organisms is ensured by maternal mtDNA inheritance through either absence of paternal mtDNA delivery or early elimination of paternal mtDNA. However, whether paternal mtDNA is transcribed has remained unknown. Here we report that paternal mtDNA shows late elimination and transcriptional quiescence in cyprinid fishes. Paternal mtDNA was present in zygotes but absent in larvae and adult organs of goldfish and blunt-snout bream, demonstrating paternal mtDNA delivery and elimination for maternal mtDNA inheritance. Surprisingly, paternal mtDNA remained detectable up to the heartbeat stage, suggesting its late elimination leading to embryonic heteroplasmy up to advanced embryogenesis. Most importantly, we never detected the cytb RNA of paternal mtDNA at all stages when paternal mtDNA was easily detectable, which reveals that paternal mtDNA is transcriptionally quiescent and thus excludes its effect on the development of heteroplasmic embryos. Therefore, paternal mtDNA in cyprinids shows late elimination and transcriptional quiescence. Clearly, transcriptional quiescence of paternal mtDNA represents a new mechanism for maternal mtDNA inheritance and provides implications for treating mitochondrion-associated diseases by mitochondrial transfer or replacement. Musculista senhousia) has mtDNA that show differences in size and gene number between male and female origins891012The mitochondrion (MT) is a membraned organelle present in all eukaryotic organisms. MT converts the energy of food molecules into ATP to support cellular and organismal metabolism, and is involved also in regulating diverse processes such as apoptosis and innate immunity14Many organisms are homoplasmic, because their cells possess a pool of homogeneous mtDNA molecules. Homoplasmy is very important for normal development, because heteroplasmy\u2013mixing of even two different normal mtDNAs\u2013may lead to genetic instability in mice31819Different degrees of paternal inheritance or leakage of mtDNA may occur even in organisms with demonstrated MUI such as Drosophila21219Carassius auratus red var.) and blunt snout bream as a model of cyprinid fishes. We show that MUI of mtDNA operates in both species by the elimination of paternal mtDNA during embryogenesis. Interestingly, we demonstrate that paternal mtDNA can persist to fairly advanced stages of embryogenesis and remains transcriptionally quiescent, excluding its phenotypic contribution to the developing embryos.This study was aimed at investigation of the fate and behavior of paternal mtDNA in reciprocal hybrids between goldfish Oryzias latipes)Two major modes operate to ensure MUI of mtDNA. One is paternal mtDNA exclusion, where sperm mitochondria do not enter into the egg but remain outside, and are thus prevented from mtDNA inheritance. This mode has been thought as exceptional because it has so far been limited to the Chinese hamster . Goldfish and blunt-snout bream were maintained at the National Education Ministry Breeding Center of Polyploidy Fish, Hunan Normal University as describedFish work was performed in strict accordance with the recommendations in the Guidelines for the Care and Use of Laboratory Animals of the National Advisory Committee for Laboratory Animal Research in China and approved by the Animal Care Committee of Hunan Normal University Sequences were analyzed by using Blast search and aligned by using Vector NT.DNA was extracted from freshly dissected organs of adult fish or 20 pooled embryos at each stage by using the TaKaRa MiniBEST Universal Genomic DNA Extraction Kit as describedTM RT reagent Kit with gDNA Eraser (TaKaRa), and PCR was run for 35 cycles in a 25-\u03bcl volume containing 10\u2009ng of template cDNA and appropriate primers for cytb and tfam, or for 30 cycles for \u03b2-actin as a loading control. Primers used are listed in Genomic DNA PCR was run for 35 cycles in a 25-\u03bcl volume containing 50\u2009ng of template DNA and appropriate primers for cytb, tfam and \u03b2-actin as describedHow to cite this article: Wen, M. et al. Transcriptional quiescence of paternal mtDNA in cyprinid fish embryos. Sci. Rep.6, 28571; doi: 10.1038/srep28571 (2016)."} +{"text": "There is increasing interest in neurosurgical interventions for hypertonicity in children and young people (CAYP), which often presents with a mixture of dystonia and spasticity. Significant spasticity would usually be considered a contraindication for deep brain stimulation (DBS) and more suitably treated with intrathecal baclofen (ITB). We aimed to explore whether white matter microstructure, as measured by Fractional Anisotropy (FA), differed between CAYP selected for DBS compared to ITB surgery.We retrospectively analysed Diffusion Tensor Imaging for 31 CAYP selected for DBS surgery and 10 CAYP selected for ITB surgery. A voxel-wise comparison of FA values was performed using tract-based spatial statistics, comparing primary and secondary dystonia groups to the ITB group, and the two dystonia groups.Widespread areas of reduced FA were demonstrated in ITB compared to either DBS group and in CAYP with secondary compared to primary dystonia. These changes were not restricted to motor pathways. Region of interest (ROI) analysis from the corticospinal tract (CST) demonstrated groupwise differences but overlapping values at the individual level.DTI measures may contribute to decision making for CAYP selection for movement disorder surgery. Significant differences in CAYP with secondary dystonia selected for DBS surgery compared to CAYP selected for ITB pump implants, suggesting that more extensive white matter injury may be a feature of the spastic motor phenotype. Altered white matter microstructure could potentially explain the reduced responsiveness to interventions such as DBS in secondary compared to primary dystonia. Hypertonicity in childhood has been variably described by the terms \u201cspasticity\u201d, \u201cdystonia\u201d and \u201crigidity\u201d. Spasticity can be defined as \u201cresistance to externally imposed movement increases with increasing speed of stretch and varies with the direction of joint movement,\u201d whilst dystonia is defined as \u201ca movement disorder in which involuntary sustained or intermittent muscle contractions cause twisting and repetitive movements, abnormal postures, or both\u201d . In chilA number of neurosurgical interventions are available in hypertonicity management, with intrathecal baclofen (ITB) and deep brain stimulation (DBS) used most widely. DBS of the globus pallidus interna (GPi) has been widely adopted in the management of primary dystonia in adulthood, with sustained beneficial effects seen up to several years following initial implantation , 4. DBS In primary dystonia, DBS, where available, is universally offered rather than ITB. For secondary dystonia, the choice between DBS and ITB is strongly influenced by the degree of spasticity and corticospinal impairment. Significant impairment of the corticospinal tract (CST), as indicated by abnormal Central Motor Conduction Time (CMCT) and other measures , would bDiffusion Tensor Imaging (DTI) is an MRI technique which provides information about white matter microstructure and is derived from the differential diffusion of water molecules . In whitDirect comparisons of DTI metrics between spastic and other subtypes of CP are currently limited. Two studies from one group, one using a region-of-interest (ROI) and one using an atlas-based approach, have compared children with spastic and dyskinetic CP , 13. TheThe potential application of diffusion tensor imaging-based techniques to the field of movement disorder surgery is an area of active research. This includes improving target selection, improving the understanding of the distributed network activated by DBS and also monitoring the effects of the intervention. We aimed to determine if quantitative white matter differences could be found between children and young people (CAYP) undergoing DBS and those undergoing ITB at our centre using tract-based spatial statistics (TBSS) analysis. This retrospective analysis was intended to explore the possible utility of DTI-derived metrics as part of the clinical assessment of hypertonic children proposed as candidates for movement disorder surgery and also to explore differences in white matter microstructure between the predominantly dystonic and predominantly spastic motor phenotypes.p\u2009<\u20090.05). Gross Motor Function Classification scores (GMFCS) [p\u2009<\u20090.05) but did not differ between the secondary dystonia DBS compared to ITB group.The case notes and imaging records of all CAYP referred to our service between July 2008 and January 2012 for evaluation prior to possible neurosurgical intervention were reviewed. CAYP who had undergone suitable DWI imaging during their clinical assessment where identified for further processing. Suitable imaging sequences were available for 31 CAYP put forward for DBS surgery and 10 CAYP put forward for ITB surgery . In all cases, MRI was performed under general anaesthesia as the severity of movement disorder precluded acquisition in the awake state.DWI sequences were obtained as part of the routine clinical work up of CAYP at our centre under evaluation for possible DBS or ITB surgery. All images were acquired on a 1.5T MRI scanner using an eight-channel phased array head coil. Diffusion-weighted single shot EPI sequences . TBSS projects all subjects\u2019 FA data onto a mean FA tract skeleton, before applying voxel-wise cross-subject statistics. Firstly, diffusion-weighted images were registered to a non-diffusion weighted reference volume for correction of head motion and eddy currents. A mask was created to remove non-brain matter using the FMRIB Brain Extraction Tool (BET) [Voxel-wise statistical analysis of the fractional anisotropy (FA) data was carried out using Tract-Based Spatial Statistics (TBSS part of ol (BET) . FA imagol (BET) .n\u2009=\u200914) were compared to those selected for ITB surgery (n\u2009=\u200910).CAYP selected for DBS surgery with primary dystonia (n\u2009=\u200917) were compared to patients selected for ITB surgery (n\u2009=\u200910).CAYP with a diagnosis of secondary dystonia selected for DBS surgery (n\u2009=\u200914) were compared to those with a diagnosis of secondary dystonia selected for DBS surgery (n\u2009=\u200917).CAYP with a diagnosis of primary dystonia selected for DBS surgery white matter tractography atlas . To examEthical approval was waived by Westminster Ethics Committee. Informed consent for all investigations was obtained from CAYP and/or their legal next of kin.Results of the group comparison TBSS are shown in Fig.\u00a0FA values extracted from voxels within the left and right CST are shown in Fig.\u00a0TBSS demonstrated extensive differences in white matter microstructure between children and young people selected for ITB and those selected for DBS surgeries. Differences in FA values were found in all major white matter pathways. Lower regions of FA were found in candidates for ITB surgery compared to candidates for DBS surgery (both as a combined group and when separated into either primary or secondary dystonia groups). Comparing the cohorts of candidates for DBS surgery, significant differences were seen across the major white matter pathways. Lower FA values were seen in the secondary compared to primary dystonia group. ROI analysis from the PLIC would suggest that the magnitude of this difference was smaller.Dystonia and spasticity arise from distinct pathophysiological processes. Previously considered a disease of the basal ganglia, increasing evidence suggests that dystonia may arise from perturbations at many points of the distributed motor network . PathophNeuroimaging correlates of motor phenotype in childhood have been most extensively studied in children with CP. Systematic reviews of neuroimaging findings in this population have demonstrated an imperfect relationship between motor phenotype and conventional imaging findings . In chilWhite matter abnormalities have been more variably reported outside of the CST, with the superior and posterior thalamic radiations and corpus callosum most frequently examined . Most stOur findings are in contrast to the previous observation of more severe white matter disruption in dyskinetic compared to spastic CP , 13, insOne interpretation of our findings is that greater white matter integrity is required to express dystonia. This would be consistent with the observation that white matter injury is most commonly seen in CAYP with spastic CP, and also our previously reported findings of normal CMCT in the majority of children with dystonia and abnormal neuroimaging . AbnormaWhilst group-wise differences in FA between ITB and DBS groups were found, an overlap in value within the CST could be seen Fig.\u00a0, suggestReduced white matter integrity was shown in the secondary dystonia compared to primary dystonia group. We hypothesise that this reduced white matter connectivity may account for differences to response to DBS seen comparing primary and secondary dystonia. Differences in FA values were seen very broadly across the major white matter tracts, reflective of the global injury to the brain arising from an insult such as HIE.A number of limitations to our study must be acknowledged. DWI images were obtained during the routine clinical assessment of CAYP referred to our service. Sequence length was consequently constrained, restricting the number of directions to 32. Numbers of CAYP within each sub-group were relatively small, with a range of aetiologies in each group. This included a range of focal radiological abnormalities consistent with diagnosis shown in Table In a cohort of children and young people with hypertonicity of mixed origins undergoing evaluation for neurosurgical intervention, extensive areas of statistically significant lower FA were found comparing candidates for ITB and candidates for DBS surgery. DTI measures may therefore contribute to decision making for patient selection for movement disorder surgery. Significant differences were found in CAYP with secondary dystonia selected for DBS surgery compared to CAYP selected for ITB pump implants, suggesting that more extensive white matter injury is a feature of the spastic motor phenotype. Altered white matter microstructure was found across almost all of the major white matter pathways of the brain, which could possibly explain the reduced responsiveness to interventions such as DBS in this group."} +{"text": "Respiratory failure is a well-known complication of aortic aneurysm surgery. We describe the impact of a protocol, using CPAP after elective surgery to reduce the need for unplanned invasive ventilation.In 2012 we introduced a CPAP protocol for patients undergoing elective aortic aneurysm surgery, either open (AAA) or as an endovascular repair (EVAR). According to pre-existing risk factors (see Table Results are reported as the split between open surgery and endovascular repair. Table We compare patient cohorts in the years 2010 and 2011 (pre protocol) with 2013 and 2014 (post protocol). There is a clear reduction in the need for unplanned IPPV in both patient groups. An audit in 2013 showed incomplete protocol adherence in the ITU, therefore benefits may be underestimated."} +{"text": "Polio is still crippling disease in Pakistan around 306 cases reported by year of 2014, this is alarming situation regarding polio virus around globe too, therefore to strengthen routine immunization eradication of polio is a goal to achieve health standard better regarding morbidity and mortality.The innovative stamp campaign for psychological, emotionally and healthy activity of stamps on childrens hand about polio to develop interest and impact for polio awareness /education for eradication.To eradicate polio free world, and object is educate, aware children about importance of polio drops and routine immunization.Descriptive study by questioner among the school children to enhance capacity about polio awareness, therefore we put polio stamps on children hands and educate them about polio after that we analysis the data by asking question about polio and its importance on spss version 11.Sample size: 500 hundred children were stamping regarding polio.The outcomes of the properly given answers:What is polio? Answers were: 353 (70.6%), what happen in polio? Answers were: 293(58.6%)How many drops given? Answers were: 343 (68.6%), at what age groups taken polio drops? Answers were: 281 (56.2%), Did you like the polio stamp campaign? Answers were: 427 (85.4%)Polio is challenging issue by Innovation of stamp campaign play most important role in development according to growth of brain (age) here we are still facing crawling love disease (polio), therefore to regret the wrong myths create awareness engage community and children like polio stamp campaign the outcomes and impact of such campaign gives meaning full evaluation results regarding awareness that will bring change to eradicate polio from globe.None declared."} +{"text": "The data presented here are associated with the article \u201cThe blister fluid proteome of paediatric burns\u201d [1]. Bur Specifications Table\u2022First and most comprehensive proteome of burn blister fluid that can be used to compare against other disease states/patient populations.\u2022These data provide a reference list of known, observed proteins within paediatric burn blister fluid, which will be of interest for the burn wound research community and clinicians.\u2022Qualitative evaluation of the biochemical differences between burns of different depths will enable future targeted quantitative analyses of protein abundance based on burn depth.\u2022The dataset allows for extensive Gene Ontology (GO) term analysis of the burn blister fluid proteome and the interaction/interrogation of this proteome through the Cytoscape data file provided herein.wound healing\u2019 and \u2018response to stress\u2019 are shown in Presented in this publication is an inventory of proteins identified in paediatric burn blister fluid (1% FDR corrected), and the depths at which they were detected , molecular functions (MF), and cellular components (CC) were determined through Gene Ontology (GO) enrichment analysis of the whole blister fluid proteome using the BiNGO app within Cytoscape ("} +{"text": "Accurate chromosome segregation during mitosis is ensured by a sophisticated surveillance mechanism, the spindle assembly checkpoint (SAC) that only allows sister chromatid separation once all kinetochores have bound to microtubules. A major unresolved question has been how the SAC senses microtubule binding to kinetochores and how this couples with turning the checkpoint off. Two papers now show that the kinetochore binding site for microtubules and the checkpoint kinase Mps1 overlap providing an elegant answer to this question.Kinetochores are large protein assemblies at the constriction point of chromatids that perform two key functions during mitosis. First, kinetochores can bind directly to microtubules of the mitotic spindle, a requirement for sister chromatid separation. Second, unattached kinetochores activate the SAC by recruiting checkpoint proteins and their localization to this structure results in the generation of a diffusible \u201cwait anaphase\u201d signal. The KMN network is an outer kinetochore complex composed of KNL1, the Mis12 complex and the Ndc80 complex that executes and integrates these two important functions of kinetochore biology repeats in KNL1 that creates binding sites for the Bub1-Bub3 checkpoint complex [in vitro and in cells prevents Mps1 binding showing direct competition brought about by changes in kinetochore architecture due to microtubule binding contributes to checkpoint silencing . By careWe clearly now have a much better understanding of how microtubule binding to kinetochores turns off SAC signaling by hampering Mps1 phosphorylation of KNL1. Whether microtubule binding affects other aspects of SAC signaling remains to be explored."} +{"text": "Seed priming plus foliar spray of MLE and kinetin significantly improved stand establishment especially under early sown crop as indicated by reduced mean emergence time (MET), improved emergence index (EI) and final emergence percentage (FEP). Similarly increased chlorophyll contents, crop growth rate, leaf area index, photosynthetic rate, transpiration rate, relative water content and decreased membrane permeability were recorded in both early and optimum sowing conditions in MLE priming plus foliar spray treatment. All these improvements were harvested in the form of increased yield and harvest index compared with control treatment. Overall crop sown at optimum time performed best but exogenous application of MLE through seed priming and foliar spray maximally improved the performance of early sown maize crop which is attributed more likely due to improved stand establishment, chlorophyll and phenolic contents, increased leaf area duration and grain filling period. It can be concluded that seed priming with MLE along with its foliar spray could increase production of maize under temperature extremes.Low temperature at stand establishment and high temperature at reproductive stage are involved in reduction of grain yield of spring maize. A field study was therefore conducted to evaluate different physiological strategies for improving performance of spring maize under temperature extremes. Seed priming and foliar spray with 3% moringa leaf extract (MLE) and 100 mg L Out of 1.1 billion tons of coarse grains produced in the world during 2010 for food, feed and industrial purposes, maize accounts for 74% of aggregate output . Early pMoringa oleifera could be an alternative source of plant hormones as moringa leaves are rich source of antioxidants , vitamins like , different essential minerals , proteins and zeatin . F. F16]. F machine .-1 kinetin solution was also applied using same procedure and equipment as described for MLE spray.Fresh moringa leaf extract was diluted up to 3% and was applied with a hand sprayer at knee height, tasseling and grain filling stages to field-grown maize plot. Foliar spray of 100 mg LNumbers of emerged seedlings were recorded daily according to the seedling evaluation handbook of the Association of Official Seed Analysis . Mean emWhere n is the number of seedlings emerged on day D, and D is the number of days counted from the beginning of emergence. Final emergence percentage was calculated by counting final number of seedling emerged in all days and divided it by total number of seeds sown and then multiplied it by 100.CGR = Crop growth rate1 = Total dry matter at the first harvestW2 = Total dry matter at the second harvestW1 = Date of observation of first dry mattert2 = Date of observation of second dry mattertLeaf area was measured using leaf area meter (Model: CI 203) and leaf area index was calculated using equation;One square meter area containing four plants was harvested from each replicate at fortnight interval and fresh weight of harvested samples was recorded. 50 g sample comprised of stem, leaves and cobs in equal ratio was dried in oven for determination of dry weight. Crop growth rate (CGR) was measured using equation .CGR\u00a0=\u00a0W2 area in the central part of the leaf blade that did not include midrib. These measurements were made from 10.00 a.m. to 3.00 p.m in bright sunny day.Gas exchange measurements for photosynthic rate and transpiration rate were taken from ear leaf at silking stage. For this purpose a portable photosynthesis system was used. Three plants were tagged from each plot and measurements were taken on 6.25cmf) were rinsed in water until the weight of the leaves was constant. The saturated leaves were weighed (Ws) and then dried for 24 h at 80\u00b0C for determinations of the dry weigh (Wd). Relative water content (RWC) was calculated by using formula [Ear leaves were collected at silking stage for biochemical analysis as major contribution towards final yield is made by the ear leaf. Membrane stability was determined in terms of electrolyte leakage in leaf samples which were cut into six equal size segments . Fresh l formula :RWC\u00a0=\u00a0WPlants from two rows comprising an area of 6.0 m \u00d7 3.0 m were harvested at when grains became blackish at the point of attachment with cob, giving a sign of harvest maturity. Data of agronomic traits and yield components including number of grains per row, number of grains per cob, 1000-grain weight, grain yield, biological yield and harvest index were recorded following standard procedures. Plant samples were sun-dried for determination of biological yield and harvest index .Soxhlet fat extraction method was used for measurement of oil content in grain. Grain protein content was determined by using Micro Kjeldahl Method .The daily fluctuation in average temperature during whole crop growth period for early and optimal sowing of spring maize under climatic conditions of Faisalabad was as given in Benefit to cost ratio for each treatment was calculated by dividing the gross income with total expenditures. Total expenditures for each treatment were calculated by the addition of total fixed cost and total variable cost (different for each treatment including cost of exogenously applied agents). There was no cost of MLE and water spray except labor charges for exogenous application. The gross income was estimated using the prevailing average market prices of grain and stover in Pakistan.Data were analyzed statistically by using Fisher\u2019s Analysis of Variance Technique and treatment means were compared by using Least Significantly Difference (LSD) test at 5% probability level. Emergence data were presented graphically in term of final emergence percentage using Microsoft Office Excel.-1 under optimum sowing conditions and increased emergence index (EI) and final emergence (FEP) under both early and optimum sowing conditions. Significant interaction was observed between treatments and sowing dates. Maximum final emergence percentage and emergence index while lower mean emergence time was observed in seeds primed with MLE and kinetin 100 mg Lnditions .Likewise seed priming and foliar spray strategies significantly improved yield related traits and interaction between different strategies and sowing dates was found significant . Among vSeed priming and foliar application resulted in significantly higher 1000-grain weight, grain and biological yields and harvest index as compared to control. Seed priming plus foliar spray of MLE showed best results both in early and optimum sown maize crop followed by seed priming and foliar application of kinetin . As for All priming and foliar treatments increased chlorophyll a and b contents in both Crop growth rate recorded at 45 and 60 days after sowing was higher in response to application of MLE through seed priming and foliar spray . LikewisUniform emergence with higher final emergence count is the key foundation, which ensures the improvement of seedling performance and overall growth of crop. This study shows that seed priming treatments not only improved the stand establishment but also provided an energetic start to maize seedling under low temperature conditions . A signiChilling stress in maize plants leads to the generation of ROS, which causes oxidative damage and impairing the optimal cellular functions by reacting with important macromolecules . Seed prSeed priming resulted in rapid and uniform germination which leads to the production of vigorous seedlings with high chlorophyll contents in their leaves . In presAll the improvements in stand establishment, crop growth and phenology were harvested in the form of increased 1000-grain weight, better grain and biological yield, and higher values of harvest index . Foliar Different nutrients, vitamins, minerals and growth promoting substances present in MLE boosted the crop growth rate and photosynthetic rate and ultimately more photo assimilates were translocated towards grain increasing its oil contents. The lower protein contents in the grains from MLE priming plus foliar spray treatment might be due to the fact that in developing seed more photosynthates were translocated towards lipid biosynthesis. Similar inverse relation for oil and protein contents has been reported .Of various physiological strategies, priming of maize seed with 3% MLE along with its foliar spray at knee height, tasseling and grain filling stage was most effective in improving stand establishment, growth, biological yield, grain yield and grain quality under both early and optimum sowing conditions. Furthermore among the various growth promoting substances used for priming and foliar spray MLE seems to be more practical being least expensive , non tox"} +{"text": "Taylor\u2019s spatial frame (TSF) and Ilizarov external fixators(IEF) are two circular external fixator commonly used toaddress complex deformity and fractures. There is currentlyno data available comparing the biomechanical propertiesof these two external fixators. This study looks into themechanical characteristics of each system. TSF rings with6 oblique struts, 4 tube connectors, 4 threaded rods, and6 threaded rods were compared to a standard IEF ringswith 4 threaded rods. Compression and torsional loadingwas performed to the frame as well as construct withPolyvinylchloride tubes. TSF rings with 4 tube connectorshad the highest stiffness (3288 N/mm) while TSF ringswith 6 struts was the least stiff. The situation was reversedfor torsion where TSF rings with 6 oblique struts had thehighest torsional stiffness (82.01 Nm/Degree) and frameIlizarov rings with 4 threaded rods the least. Standard TSFconstruct of two ring with 6 oblique struts have bettertorsional stiffness and lower axial stiffness compared tothe standard IEF.Taylor\u2019s Spatial Frame, Ilizarov External Fixator,Biomechanical properties Accurate and purposeful positioning of hinges and distractors will also allow gradual correctionof complex deformities.Conventional Ilizarov external fixator (IEF) is composedof stainless steel rings connected with threaded rods thatcan be configured in various ways to Manage differentindications. The frame is fixed to the bone using eitherstainless steel wires under tension or rigid Schanz pins thatcauses minimally disruption of the soft tissue. These basicIlizarov external fixator construct and method of fixationprovides favorable mechanical and biological environmentfor bone healing Planning and application of Ilizarov external fixtor requireconsiderable experience, and the need for post-operativeframe re-adjustment is not infrequent. Newer generationof external fixators make use of hexapod system toperform gradual multiaxial correction in six degree offreedom without changing the position and orientation orconnecting elements. In this type of fixator, six obliquelyplaced adjustable struts are connected to the proximal anddistal rings. To achieve desirable correction of the bonesegments, gradual adjustment of individual strut lengthswill be guided by computer software.3-5. We would expect multiaxialexternal fixator frame to have additional free play thatarises from the universal hinges on both sides of theoblique struts. To our knowledge, there has been no studycomparing the mechanical properties of the conventionalIEF with multiaxial frame. Information on the mechanicalproperties may allow the user to modify the construct ofthese frames to provide optimum environment for fractureunion or bone healing.Stiffness and fixation stability of conventional IEF havebeen reported in literature We therefore conducted this study to compare themechanical properties of IEF and multiaxial externalfixator.Four pairs of 155mm Taylor Spatial Frame full rings and one pair of150mm IEF rings were used in this study. Five frame configurations wereconstructed 6,7. Loading was performed along the midaxialplane as well as offset axial loading These frames were loaded on an Instron 3365 under displacement control with constant ram speed of0.5mm/sec up to a maximum load of 700 N. The loadof 700N was chosen as this is the average weight ofadult patients 8. Theabove measurement was repeated six times to obtain anaverage value. Torque vs angular displacement curveswere plotted to calculate the torsional stiffness.Torsion load was applied manually using a torque wrenchand measuring the angular displacement between theproximal and distal rings. An incremental load of 5Nmup to a maximum of 30 Nm of torque was applied The same construct were later mounted withpolyvinylchloride (PVC) tubes (inner diameter of 42mmand outer diameter of 32mm) to represent bone innerdiameter of 42 mm and outer diameter of 32 mm PVCwas used to standardize the material property as the aimof this study is to compare the stiffness of the frameconstruct and not the holding strength to the bone. Theother consideration is that the PVC tubes are readilyavailable and cheap compared to cadaveric bone. Therehas been studies in the past utilizing PVC and wood tosimulate bone 9,10. The tubes were fixed to the frameseccentric to center of the ring to simulate tibia bonefixation in clinical practice. One surface of the tube willbe placed 20mm from the inner border of the ring torepresent the anterior medial cortex of tibia bone The first author performed all the mechanical testing andits measurement. Analyses of the results were performedwith SPSS ver 17.0 and Microsoft Excel 2010, usinganalysis of variance (ANOVA). P value of 7lt; 0.05 wasconsidered significant.When we compare the frame stiffness, true axial loadingshowed higher stiffness values compared to offset axialloading When we compare the loading stiffness through the bonesubstitute, the overall pattern is similar to the loading ofthe frame with Frame B (TSF rings with hollow rods)being the stiffest in axial loading while the Frame A(TSF rings with 6 struts) was stiffest in torsional loadingAxial stiffness reduces markedly when we comparedtesting of the frames and testing of the tubes. However,for torsional stiffness, the differences between frameand tube testing were less obvious 12. Ilizarov in his experimentswith canines reported a direct correlation betweenframe stiffness and bone regeneration13. External fixatornaturally would not be as stiff as internal fixation due tothe long lever arm of the fixation wires and pins. This wasalso evident from our study where stiffness of the framewas much higher compared to that of the bone substitutesespecially on axial loading Bone is a living and dynamic entity and bone healingprocess is influenced by both biology as well as mechanicalenvironment. Preservation of blood supply to the boneprovides a favorable biological condition for healing tooccur. At the same time, adequate stability of bone endsis also essential to prevent excessive movement that maylead to nonunion. It has been reported that interfragmentarystrain of less than 10% between bone ends was necessaryfor desirable fracture union When we analyze the stiffness on axial loading, our resultsshowed that TSF rings are stiffer than Ilizarov rings asevidenced by the higher stiffness of Frame C comparedto Frame E, although the difference was not statisticallysignificant. TSF rings arethicker compared to IEF rings , and this may be the main contributingfactor. However, when we compared the standard TSFconfiguration with 6 oblique struts to the standard IEFconfiguration with 4 threaded rods, we noted that IEF(Frame E) was significantly stiffer than TSF frame(Frame A) on axial loading. Lower stiffness recorded onTSF frame was mainly contributed by the design andconfiguration of the connecting struts. As expected,stiffness of PVC tubes fixed with both types of fixatorframes will be lower than stiffness of correspondingfixator frames alone, due to the long lever arm of wiresand pins used to secure the bone substitutes on to theframes. Degree of stiffness on the PVC tubes fixedwith TSF frame remained lower than that of IEF frame,but the difference was not statistically significant.10. Oblique struts of TSF accentuates thepliability in the wire and pin fixation to provide axialcompression to stimulate new bone formation, and at thesame time resist torsional and translational motions thatis detrimental to bone healing. Our findings showed thatstandard configuration of TSF with 6 obliquely placedstruts are able to provide favorable mechanical propertiesfor bone healing.When we compared torsional stiffness of the frames, TSFframe (Frame A) stands out as the frame with higheststiffness compared to other types of frames . In fact differences in torsional stiffness betweenthe other frames were not statistically significant. Comparedto connecting elements that were placed perpendicular tothe fixator rings, TSF struts placed in an oblique positionwould provide additional resistance against translationalforce along the axis of its body. With six struts distributedin a circular manner, the configuration would make theframe stiff on horizontal plain against loading from anydirection. Advantage of conventional IEF over otherfixator designs was based on its ability to allow axialmotion and resist torsional or translational motions overthe bone ends Stiffness of an external fixator can be improved byapplication of additional elements to the fixator frame.However, this may provide little benefit to the overalltreatment because this may be offset by the addedweight, bulk and cost of the device. Improvement in thebasic design and configuration of external fixator framewould be more effective to achieve better outcome, andthe additional stability provided by obliquely placedconnecting elements between fixator rings will have thepotential to increase the quality and final outcome in themanagement of nonunion, bone lengthening and deformitycorrection. A comparative study on animal and humansubjects would be necessary to provide clinical evidenceto support these findings.14.There are some limitations is our study. Firstly althoughthe PVC tube offers a standardized material for testing,it may not represent the property of bone in clinicalpractice. Torsional load was measured using a manualmethod that may have introduced errors. The free playin the TSF frame was not taken into consideration duringtesting as very little loads are required to take up the freeplay and thus making testing difficult. A larger samplesize utilizing composite resin bone models or cadavericmodels may provide more representative values for use inclinical practice. This study also did not consider varyingangles of the TSF strut as it has been reported that the TSFframe has some inherent instabilities when the strut anglesare less than 30\u00b0 Standard TSF with 6 oblique struts fixed on to bonemodel can provide comparable stiffness on axial loadingand better stiffness on torsional loading to conventionalIEF with 4 threaded rods. The mechanical properties aretheoretically favorable for both fracture healing and newbone formation. Changing to stronger hollow connectingbars or increasing the number of threaded rods did notsignificantly increase the stability against torsional forces.Our findings suggest that TSF may provide a betteralternative to conventional IEF as far as mechanicalproperty is concerned."} +{"text": "Epidural venous plexus engorgement may occur due to several conditions that prevent the normal venous circulation. Inferior vena cava agenesis is a very rare cause of epidural venous enlargement. We present a case with a very thin inferior vena cava and left iliac vein agenesis who presented with back pain due to epidural vein engorgement and lacked other venous problems such as deep vein thrombosis. Back pain and nerve root compression symptoms are very frequent complaints of patients with herniated discs which are demonstrated on lumbar MRI. However, herniated discs may not be the only pathology and MRI may also reveal epidural venous engorgements which cause nerve root compression. In that case, underlying etiological factors must be thought in the differential diagnosis such as absence of inferior vena cava, intracranial hypotension, and Marfan syndrome . EngorgeHerein we present a young patient with back pain who had epidural venous engorgement on lumbar MRI. Subsequent abdominal CT revealed a very thin inferior vena cava and absence of left common iliac vein.A 24-year-old woman with back and left leg pain was admitted to the department of neurosurgery. She mentioned that the back pain was refractory to medical treatment. She had no prior medical problems except for polycystic ovaries and irregular menstruation periods. Physical examination revealed the Lasegue positivity at 45 degrees. A lumbar MRI with the prediagnosis of disc herniation was requested. On lumbar MRI, no herniation was evident; however there were venous engorgement of the epidural veins and obliteration of the anterior epidural space on axial and sagittal T2-weighted images Figures . The patEpidural venous engorgement may be seen in blocked venous system due to various pathologies such as portal hypertension, Budd-Chiari syndrome, intracranial hypotension, superior or inferior vena cava thrombosis, and abdominal malignancy or in a physiologic state such as pregnancy. It may be asymptomatic, or in case of nerve root compression, there may be radicular symptoms , 4. The Thrombosis of inferior vena cava due to several diseases, some of which are mentioned above, is more common. Absent inferior vena cava is a very rare cause of this disorder. Normally developed inferior vena cava is formed by anastomosis of several embryological segments. Absence of suprarenal segment results in continuation of venous flow into the azygos system Congenital aplasia of the infrarenal segment of IVC itself is extremely rare , 7. EvenRadiologically it is important to be aware of other structures rather than disc pathology which may cause radicular symptoms. In a case report by Dudeck et al., the dilated veins were thought to be retroperitoneal enlarged lymph nodes on MRI and ultrasound revealed multinodular left-sided retroperitoneal mass which later turned out to be thrombosed collateral veins . RadioloGenerally published cases are presented with findings of thrombosis, and inferior vena cava agenesis without thrombosis is reported in an article by Kamerath and Morgan which became symptomatic during exercise . Our patThe cases in the literature took place in clinical journals rather than radiology journals; however, we think that radiologists should be aware of this phenomenon and in such cases immediate abdominal workup for any vascular pathology and also doppler sonography should be performed to rule out possible deep vein thrombosis. Diagnosis of inferior vena cava or iliac vein agenesis or thrombosis will also help to lead the antithrombotic treatment and also knowing any Factor 5 Leiden mutation is also important in young women who may be pregnant and have the possibility of thrombosis-related problems in the future. This problem is not confined to the lumbar region and ascending lumbar vein and epidural venous plexus enlargement should be sought in the cervical or thoracal regions."} +{"text": "Sepsis is defined as the presence of infection with systemic signs of infection, and severe sepsis as sepsis plus sepsis-induced organ dysfunction or tissue hypoperfusion . Since tAll referrals to our critical care response team with a diagnosis of sepsis over a 3-month period (September to November 2014) were investigated to determine how many had an EPR sepsis alert comprising a prompt for blood cultures, serum lactate measurement, fluid challenge if hypotensive, and antibiotics within 1 hour.Only 25/174 (14%) patients with a diagnosis of sepsis had an EPR sepsis alert. There was no significant difference between acute and nonacute ward areas in their likelihood of using the screening tool or alert, in contrast to previous audits of the alerted population which showed that acute areas such as A&E and medical acute admission wards had higher utilisation and bundle completion rates.Despite these interventions, most patients still do not receive the full recommended treatment bundle. These findings have prompted a point prevalence audit at ward level, which will examine all patients' notes for the preceding 24 hours to ascertain if sepsis is truly unrecognised or whether it is simply that our current tool is not a helpful adjunct to care. With national guidelines expected within the year, we will redesign and re-launch our screening tools and education programme to improve awareness and management of this common medical emergency."} +{"text": "The biologic dose response curves of thermal dose and absorbed radiation dose have not been compared to each other even though they have both been extensively investigated separately and combined. Although heat and radiation produce cell kill by different biological mechanisms a comparison of dose response curves is possible using the endpoint of cell survival.Survival curves for both thermal and radiation doses were extracted for three different types of cells from previously published data. Using models based on the beam shapes of the current clinical systems for the dose profile, the survival curves were generated and the survival profiles were compared for both modalities, Focused Ultrasound (FUS) and Gamma Knife (GK), for a thalamotomy. The thermal dose profile was calculated according to Dewey (1994), from temperature maps simulated with a 3D finite differences time domain code solving the bio-heat equation with a heat deposition term dependent on the pressure field. Radiosurgery dose distributions were exported from the Gamma Knife treatment planning software with the smallest target as an input.The comparison showed that focused ultrasound exhibits a steeper dose and survival profile than gamma knife. As shown in Figure"} +{"text": "On a backdrop of increasingly distressing opioid misuse in our communities, and safety concerns expressed by The Joint Commission and others, emergency physicians are further increasing their utilization of these important agents in our patients.The timely report by Sutter et al.5The Patient Safety Movement Foundation recommends that \u201call patients receiving IV opioids have continuous \u2026 pulse oximetry\u201d and those patients receiving supplemental oxygen have continuous respiratory rate monitoring. At a time when alarm fatigue and ED-crowding are factors which may distract providers from adequate monitoring, excessive monitoring may add additional burdens which may not ultimately benefit overall ED patient safety. Clinical personnel close to the bedside during the known peak action of the drug administered can provide both thoughtful monitoring and the immediate ability to respond.. The authors also point out that naloxone reversal was used ~73% of the time for patients with an increased likelihood of respiratory depression. This indicates that patient selection is important in avoiding adverse effects of opioids. Clearly patients with intoxication, sedation and other medications sedative-hypnotic agents on-board should be vigorously monitored and opioid dosing constrained.7It has been well documented that we are using opioids to effectively reduce patient suffering, in many differing clinical conditions.13Increasingly states have adopted approaches to monitor and provide practitioner feedback opportunities to monitor prescription drug prescribing. Reducing over-consumption, diversion and other misuses of prescription medications is an important goal shared by emergency medicine practitioners.14Providing for life-saving and innovative interventions in the ED and our communities should be strongly considered. Mechanisms and policy for advancing the use of community based reversal of opioid over-doses is life-saving.16So it seems we have effective analgesic agents with a good safety margin when used appropriately with safe monitoring. Our ability to utilize them appropriately appears to depend upon early administration, dosage adjustment for patients at risk of adverse effects, appropriate monitoring, and advocating for innovations which will assist in reducing the risk of substance abuse and treatments."} +{"text": "Electrical microstimulation can be used to drive neural responses to match meaningful spiking patterns corresponding to natural sensory stimuli or motor behaviors. Optimizing microstimulation sequences requires repeatedly stimulating the neural system to obtain sufficient probing data to construct an inverse model. This is challenging in the real brain where probing time may be limited and plasticity may be induced. Biologically realistic models allow the system to be repeatedly probed and reset, providing a unique test bed for understanding the dynamic interaction between ongoing neural activity and artificially applied stimulation. Here, we employ a biomimetic spiking model (BMM) of sensorimotor cortex which controls a realistic virtual musculoskeletal arm that performs reaching movements .After training the BMM and virtual arm to reach a target, a set of 1536 microstimulation probing sequences were applied to the target sensory population. The output motor population activity and the virtual arm trajectories were recorded (Figure This work demonstrates the advantages of employing in silico brain simulations and realistic limb models as a test bed for microstimlation-based neural controllers. The proposed system, which has been previously interfaced with a neural data acquisition system and a robotic arm in real time , paves t"} +{"text": "Dear Editor,I read the article entitled \u201cThe Maternal and Neonatal Effects of Adding Tramadol to 2% Lidocaine in Epidural Anesthesia for Cesarean Section\u201d . The autOther important comments when reflecting about the results section, I was wondering the following points could have been provided to allow fully result analysis:The mean dose of lidocaine and sufentanil request intraoperative were presented, but it was not showed the number of patients that demanded complementation analgesia and when;Around 30% of patients present some complications but they did not mention what kind of complications and these numbers is relatively bigger than other found on literature andThey also comment that general anesthesia is an option when maternal apnea lasts longer than 20 seconds or enable to speech or if the mother lost consciousness or did not respond to stimuli but they did not comment if some patients need it, considering the risks of general anesthesia, it is an important date.Finally, the article core idea is very interesting, since epidural tramadol has comparable analgesic efficacy with lower side effects than epidural morphine, specifically concerning about respiratory depression . Althoug"} +{"text": "Whereas past wolf management in the United States was restricted to recovery, managers must now contend with publicly contentious post-recovery issues including regulated hunting seasons. Understanding stakeholder concerns associated with hunting can inform stakeholder engagement, communication, and policy development and evaluation. Social identity theory (SIT) has been used to understand how groups interact, why they conflict, and how collaboration may be achieved. Applying SIT to stakeholder conflicts about wolf hunting may help delineate groups according to their concern about, support for or opposition to the policy choice of hunting wolves. Our objective was to assess concerns about hunting as a tool to resolve conflict in Michigan, using SIT as a framework. We used a mixed-modal sampling approach with wolf hunting-related public meeting participants in March 2013. Survey questions focused on 12 concerns previously identified as associated with hunting as a management tool to resolve conflict. Respondents (n \u200a=\u200a 666) cared greatly about wolves but were divided over hunting wolves. Wolf conflicts, use of science in policy decisions, and maintaining a wolf population were the highest ranked concerns. Principle components analysis reduced concerns into three factors that explained 50.7% of total variance; concerns crystallized over justifications for hunting. General linear models revealed a lack of geographic influence on care, fear and support for hunting related to wolves. These findings challenge assumptions about regional differences and suggest a strong role for social identity in driving dichotomized public perceptions in wildlife management. Effective decision-making in wildlife management may be inhibited by conflict between and among stakeholders, especially when management decisions or actions are controversial One dominant paradigm for reducing conflicts among stakeholders over HWC is stakeholder engagement Psychology's social identity theory (SIT) posits that perceptions of unequal power help drive intergroup competition and bias individuals against competing groups with different ideologies ingroups consisting of like-minded individuals. The individual views himself as a representative of that group and acts according to group expectations and norms ingroup bias, whereby individuals seek to increase positive ingroup characteristics and negative aspects of outgroups SIT explains how individuals view themselves through their group memberships and the value and meaning attached to that membership Incorporating SIT into wildlife-related decision-making may advance stakeholder engagement beyond stereotypes, offer another way to understand underlying stakeholder concerns about hunting wolves, and potentially bears implications for resolving human conflict over HWC. Because people tend to underestimate attitudinal heterogeneity within a group and socio-demographics alone may not explain group interactions Gray wolves were eradicated from the Western Great Lakes region except in Northern Minnesota by the 1930s, listed as endangered under the Endangered Species Act in 1973 and naturally emigrated back to Michigan's Upper Peninsula over the past two decades In Michigan, some groups have publically taken various positions about hunting wolves and aligned themselves with similarly positioned groups. Although these identity groups may publically present dichotomized pro- or anti-hunting policy positions, their underlying justifications for policy preferences are not necessarily the same. For example, nuanced but important differences of opinion can exist between hunters who hunt deer versus those who hunt bear. Treating identity groups with different concerns about hunting wolves as a single stakeholder group based solely on policy position may lead individuals to assume that the opposition holds homogenous attitudes in direct conflict with the individual and his/her identity group Michigan State University's Committee on Research Involving Human Subjects (IRB# x11-1144e) reviewed and approved methods used in this research. Committee-approved informed consent was obtained in written form. Respondents had to first read the informed consent statement, continuing on to the survey was consent to participate in the study.http://datadryad.org/[DOI will be added after acceptance].Data are archived at: In March 2013, we used a snowball sampling technique that included two modes, whereby paper and online version of the questionnaire were made available Paper and online versions of the survey used identical measures and formats. Five-point Likert-style questions measured twelve concerns regarding hunting wolves as a tool to address conflict. The twelve concerns were originally identified by the Michigan Wolf Advisory Council, a MDNR-initiated group of stakeholder representatives Paper and web-based survey responses were pooled into a single dataset and duplicate responses (subsequently dated IP addresses) were deleted A total of 676 respondents completed our survey and 10 duplicate electronic responses were deleted for a usable sample of 666. Michigan residents comprised the majority of respondents. Upper Peninsula residents were overrepresented in the sample compared to their relative proportion in the statewide population Of the 12 concerns presented, the top-ranked three concerns associated with hunting as a tool to manage wolves were about: (1) managing conflicts with wolves, (2) use of science in decision-making, (3) and maintaining a wolf population . Support2\u200a=\u200a662.394, df\u200a=\u200a66, p<0.01) that all 12 concerns loaded significantly (\u22670.45) on three factors that explained 50.7% of total variance ; more fearful respondents were more likely to disagree with concerns. Among respondents, region of residence was not significantly related to concerns .General linear model analysis revealed care was significantly and positively correlated with concerns about hunting wolves or negative (e.g. fear) attitudes toward wolves, not region, may be important factors driving public perceptions about wolf hunting Our results reaffirm literature suggesting social identities among stakeholders highly involved in wolf management are polarized and concerns about hunting wolves are easily dichotomized (into the two predominant Factors 1 and 2)Although positions and interests related to wildlife management can be nuanced and diverse, the dichotomy among study participants vis-\u00e0-vis current wolf management may be the result of group reactions to perceived status threats SIT provides insight about how to cope with the dichotomies for communication within groups and cooperation among groups such as those found in among our study participants. First, communication geared toward each identity group that addresses their specific concerns may be more persuasive and effective than messages targeting stakeholders generally. Stakeholder engagement that fails to address the unique concerns of a particular stakeholder may result in magnified negative effects such as lawsuits and noncompliance Second, SIT tells us simultaneously encouraging \u201ccare\u201d for wolves while decreasing \u201cfear\u201d as measured by our survey instrument may help usher identity groups toward common understanding and greater agreement about management strategies. Because our results suggest that fear influences management acceptability, mitigating perceived risk may be an especially effective tool to address conflict over HWC. Strategic risk communication may help decrease \u201cfear of wolves\u201d by emphasizing wolf-related benefits The salience of a broader, inclusive group identification can also be increased by emphasizing the distinctive qualities of that group and areas where group standing can be improved Ursus arctos arctos), return to historic ranges How the case of wolf management evolves in Michigan has implications for large carnivore management in diverse contexts and other regions"} +{"text": "Electrical impedance tomography (EIT) is a functional imaging technology allowing one to regionally monitor aeration of the lungs. We used EIT with increased signal quality and spatial resolution to describe and quantify the regional changes in aeration caused by body position, both during spontaneous breathing and mechanical ventilation in pulmonary healthy patients undergoing laparoscopic prostatectomy.In 40 patients we performed EIT measurements at five points of time Table with theP < 0.05).Perioperative changes of NSS and DSS are shown in Table We describe for the first time the mapping of Silent Spaces during spontaneous breathing and changing ventilation conditions and body positions in patients with healthy lungs using EIT. This mapping of Silent Spaces might prove useful for developing perioperative protective ventilation strategies."} +{"text": "Atrial fibrillation (AF) occurs frequently after cardiac surgery and is mostly self-limiting. It relates to direct surgical effects on the atria, atrial ischaemia during surgery, adrenergic activation and a general state of inflammation. Although most postoperative AF is transient, it may lead to a prolonged hospital stay and increased mortality . TreatmeNetherlands Heart Journal, Jacob et al. [In this edition of the b et al. report ab et al. , which aThe authors have put much effort into the sophisticated analysis of transoesophageal echocardiography recordings (TEE). We commend the investigators for their focus on atrial function rather than atrial size only, especially since atrial function may better reflect atrial pathophysiology than merely looking at atrial anatomy. Their methodology should be followed when evaluating atrial performance in order to understand atrial remodelling processes much more efficiently.In the original DECS study , dexametWe again commend the investigators for having performed such extensive TEE analyses. We understand that collecting data immediately after surgery was most appropriate considering the complex logistics and limited acceptance of TEE in the awake patient. On the other hand, atrial inflammation may not be at its peak right after surgery and therefore the investigators may have missed the beneficial effects of dexamethasone on atrial function. This notion is fed by the fact that postoperative AF reaches its highest incidence on day two after cardiac surgery .Comparing preoperative and postoperative left atrial (LA) function within the dexamethasone and placebo groups (rather than the presented comparison between dexamethasone and placebo before and after surgery) suggests that the LA total emptying fraction is stable in the placebo group, while it seems to decrease in the dexamethasone group. Whilst correction of the LA function parameters for fluid status may appear explanatory one cannot exclude an early unexpected deleterious effect of dexamethasone potentially precluding a net positive effect during the next few days after surgery. Future studies on postoperative atrial inflammation may shed more light on these notions.The present study shows the feasibility of assessing perioperative atrial function using TEE. However, it also illustrates the limitations of fitting methodology to standard care which precluded observing atrial inflammation when it\u2014expectedly\u2014was at its peak. To paraphrase the present Dean and Vice Chairman of the Board of the University Medical Centre Utrecht, it is time for a \u2018transition in science\u2019 . Among oNone.None declared."} +{"text": "This is the official guideline endorsed by the specialty associations involved in the care of head and neck cancer patients in the UK. It provides recommendations on the assessment and interventions for the psychological management in this patient group.\u2022 Audit of information supplied to patients and carers should be conducted on an annual basis to update and review content and media presentation. (G)\u2022 Patients and carers should be invited to discuss treatment options and relate possible outcomes to functional retention or loss to provide a patient-centred approach. (G)\u2022 Clinical staff should inspect their systems of assessment to make them sensitive enough to identify patients with psychological difficulties. (G)\u2022 Flexibility, rather than rigid formulation is required to assess patients frequently, and to allow for change in circumstances to be noted. (G)\u2022 Multidisciplinary teams should determine the supportive care services available and commission extra assistance to provide patients and carers with timely information, education or brief supportive advice. (G)\u2022 Multidisciplinary teams need to inspect specialist services for mental health interventions at structured and complex levels for the small proportion of patients with more serious, but rarer, psychological difficulties. (G)\u2022 Clinical staff at all levels should receive communication skills training to raise and maintain consultation expertise with difficult patient and/or carer interactions. (G) The head and neck cancer patient and their carers have considerable challenges to overcome.,,Evidence from other areas of treating cancer at other sites has demonstrated clearly that the way in which the diagnosis is presented to the patient is important to their psychological response to the disease and treatment.,\u2022Audit of information supplied to patients and carers should be conducted on an annual basis to update and review content and media presentation (G)\u2022Patients and carers should be invited to discuss treatment options and relate possible outcomes to functional retention or loss to provide a patient-centred approach (G)Considerable efforts have been expended to determine the information needs of head and neck cancer patients.The use of routine assessments for psychological distress such as the Distress Thermometer and the Hospital Anxiety and Depression Scale are being considered as a means to identify those patients who may suffer during the process of treatment preparation, the treatment itself, initial stages of recovery and follow-up out-patient appointments.,The types of psychological distress require attention and definition. The classical typology of mental distress includes anxiety and depression. In addition, assessments of recurrence fears (the most frequent reported concern of head and neck cancer patients), facial disfigurement, body image, loneliness and sexual dysfunction may also be compiled within an MDT assessment profile library for occasional use when required.\u2022Clinical staff should inspect their systems of assessment to make them sensitive enough to identify patients with psychological difficulties (G)\u2022Flexibility, rather than rigid formulation is required to assess patients frequently, and to allow for change in circumstances to be noted (G)\u2022Multidisciplinary teams should determine the supportive care services available and commission extra assistance to provide patients and carers with timely information, education or brief supportive advice (G)\u2022Multidisciplinary teams need to inspect specialist services for mental health interventions at structured and complex levels for the small proportion of patients with more serious, but rarer, psychological difficulties (G)The profile of staff expertise and skills needs close inspection to enable a flexible and tailored matching of need to professional training of support or specialist staff. Multidisciplinary teams need to plan their services to provide escalating level of care according to the specific need of psychological difficulty presented by the patient. The newly developing Map of Medicine describes in detail the levels of intervention (1\u20134). Timely support and educational approaches are conducted at levels 1 and 2. Structured interventions are provided at level 3 by staff with a mental health qualification. Level 4 interventions consisting of complex psychotherapeutic approaches are delivered by clinical psychologists, counselling psychotherapists and liaison psychiatrists.It is important for the MDT to raise survivorship issues with patients.\u2022Clinical staff at all levels should receive communication skills training to raise and maintain consultation expertise with difficult patient and/or carer interactions (G)Communication with the patient assumes even greater importance when curative treatment options are not available and care focuses towards a palliative approach.\u2022Develop information services for patients and carers. Consider introducing new technology to collect routine patient self-report data on health behaviour, psychological responses to care received, outlining of key messages and outcome assessments\u2022Develop decision-making tools (such as explanatory tablet applications) for the aid of patients to enter into discussion with multidisciplinary team to agree on treatment plan\u2022Collect routine psychological assessments at key points during course of care. These indicators must be supported with dedicated and tailored interventions to prevent neglect of identified psychological distress or depression\u2022Focus on level of support and intervention that current team can realistically provide with current level of resource. Remain cautious when introducing change, but strengthen and build upon supports already available\u2022Develop more comprehensive support services by improving generic communication skills training for current staff and ensure consistency of message giving to patients and/or carers across the multidisciplinary team\u2022Introduce staff training to assist with management of potential burnout in multidisciplinary team staff. Consider flexible responses including secondments, study breaks and peer-support programmes\u2022Audit current psychological services applied in the head and neck cancer service. Identify current usage, gaps in service and develop forward plans to address these gaps\u2022Assess current capability of specialist clinical nurse skills to support head and neck cancer patients psychologically, and introduce dedicated training and supervision programmes\u2022Actively search for clinical psychology service input and negotiate improved access and response time. Estimate likely demand of service\u2022Consider appointing sessional input to cancer network of a clinical or counselling psychologist or psychotherapist\u2022Identify liaison psychiatry service and negotiate referral pathway and response time."} +{"text": "Nonsubspecialized pathologists frequently request expert consultation in challenging dermatopathology cases. Traditional consultation practice utilizing shipment of glass slides is costly, slow, and of limited educational benefit to the referring physician. Whole slide imaging (WSI) has been suggested as a potential method of overcoming these limitations in the current glass slide consultation practice, but there have been concerns regarding the adequacy of image quality for interpretation of challenging dermatopathology cases. We aimed to investigate the performance of WSI in challenging dermatopathology consult cases.52 consecutive clinical consultation dermatopathology cases sent from a community hospital to an academic medical center were sampled and diagnosed by traditional microscopic examination and by whole slide image examination. Matched pairs of diagnoses were evaluated for diagnostic accuracy rates via a masked adjudication process.Two of 52 cases (3.8%) had major discrepancies. After adjudication the WSI diagnosis was preferred in one case and the glass slide diagnosis was preferred in the other. 13 of 52 cases (25%) had minor discrepancies, with the WSI diagnosis preferred in 6 cases and the glass slide diagnosis preferred in 4 cases and with no preference in 3 cases. Differences in diagnosis were primarily due to interobserver variability and thresholding inherent in challenging dermatopathology consult cases and not due to image quality.Overall, the sampled accuracy rates of both WSI and glass slide techniques were equivalent. These results suggest that WSI may be feasible for even challenging dermatopathology consultation cases."} +{"text": "Lateral wedge insoles have consistently shown to reduce the external knee adduction moment (EKAM) in medial knee osteoarthritis (OA) patients; although there is evidence that certain patients have a paradoxical increase in EKAM. This may be a key factor in determining clinical response and thus identifying and understanding why these patients increase EKAM is critical for prescribing the correct treatment for these patients. Previous evidence has suggested that foot and ankle biomechanics play a role in reducing EKAM by shifting the centre of foot pressure (COFP) laterally and increasing the valgus orientation of the calcaneus, which shortens the lever arm in respect of the knee, thus reducing the EKAM. To date, patients have been studied irrespective of biomechanical response to lateral wedge insoles. In this study we investigated whether dynamic ankle biomechanics can assist in identifying and explaining why some patients increase EKAM and other decrease EKAM when wearing a lateral wedge.both lateral wedge conditions compared to the control shoe. We defined biomechanical non-responders (non-responder) as those whose EKAM increased when wearing both lateral wedges compared to the control shoe. Fixed-effects multiple linear regressions were used to test for effects of the lateral wedge on coronal ankle variables, in both wedge conditions and subsequently when dichotomising individuals into biomechanical responder and non-responders. Finally, logistic regression was performed to see which coronal ankle variables, measured in the control condition only, could predict response to EKAM.Participants diagnosed with medial knee OA were recruited to the study. Each participant underwent a 3D kinematic and kinetic analysis whilst walking in a control shoe and the two different lateral wedge insoles which were inserted bilaterally into the control shoe. The order of testing was randomised. We classified participants as biomechanical responders (responder) if participants decreased EKAM under p = 0.036) or a higher eversion angle at peak EKAM during the control condition were more likely to classified as a biomechanical responder to the lateral wedges.Of the 70 participants studied , 20% increased their EKAM and 54% decreased their EKAM. Both pairs of lateral wedge insoles caused the foot to be in a significantly more everted position compared to the control condition with one insole greater. Change in ankle angle excursion significantly predicted EKAM change with lateral wedge insoles. Additionally, individuals with a higher peak ankle eversion angle (OR 1.31; 95% CI 1.019 to 1.703; In conclusion, we have demonstrated for the first time that coronal plane foot and ankle biomechanical measures are key mechanisms for the reduction of EKAM when wearing lateral wedge insoles. Furthermore, our findings also demonstrate that coronal plane ankle biomechanical measures under the control condition predict if an individual is likely to decrease their EKAM when wearing lateral wedge insoles. These findings may provide future insights into determining who will respond to lateral wedge insoles.ISRCTN: 83706683."} +{"text": "Alcoholic beverages are traditional to the Balkans region since the Antiquity. Consumption of domestic honey-made spirits and wine was wide spread among the Serbs even in the early medieval history before spreading of Christianity . Today, Likewise, elsewhere in the European community, traffic accidents caused by alcohol consumption posed a substantial challenge to the road safety . UndispuAmong direct alcohol effects on mortality are classified as mental disorders with suicidal tendency as well According to the criminal justice procedure in Serbia, there are some 106 beds of Special Prison Hospital in Belgrade reserved for mandatory treatment of offenders who committed crimes in alcoholic condition. Such treatments are either been conducted in the penitentiary institution itself or elsewhere, in a specialized medical facility.Quite a peculiar issue is non-fatal alcohol overdoses whose treatment lies within responsibility of the National Poison Control Center of the Military Medical Academy being the reference institution in the country for diagnostics and treatment of intoxications. This facility reported 2078 patients admitted because of acute ethylalcohol intoxications or almost 50% of all cases in 2012 . Broad pHealth economics evidence is still not being deployed as mandatory tool in a routine policy making across the Balkan region . GradualLesch Alcoholism Typology (LAT) as an interactive software containing treatment guidelines has been introduced in seven major regional addiction centers throughout Serbia. It distinguishes among four major subtypes of alcohol-dependent patients. The first one is \u201callergy model\u201d ; the second one \u201cconflict resolution and anxiety model\u201d (craving caused by stress); the third \u201cdepressive model\u201d (craving caused by mood); and the fourth is known as the \u201cconditioning model\u201d (craving caused by compulsion). An important feature of alcohol-dependent patients in Serbia is significantly higher anxiety levels and clear clinical domination of LAT type III. Major underlying reasons for this phenomenon might be the post-traumatic stress syndrome among almost one million refugees and internally displaced persons suffering from consequences of civil wars in former Yugoslavia back in 1990s. Another key issue is increased poverty levels due to global recession. The increased anxiety presence among the addictive patient population should be regarded as an important local feature of clinical presentation uncommon to many high-income EU communities. Such morbidity structure is inevitably reflected to demand for medical services including psychotherapy and pharmaceuticals . An esseRegardless of quite strong legacy of alcohol abuse and dependence, national capacities for necessary medical care remain insufficient. Long-term-based residential alcoholism rehabilitation covers <10% . AlthougSerbia\u2019s health system has passed a long way from former municipally owned health care funded through mixed-Bismarck model of former Yugoslavia. It should not be forgotten that the largest market of Western Balkans, due to historical circumstances, actually entered socioeconomic transition with one decade long delay compared to most of Eastern Europe . Its achOne of the promising trends observed in time series of cross sectional data on the region reported by the national authorities to European Health for All Database (HFA-DB) is steadAnother intriguing local developments were related to the official implementation and staff training in LAT software and its clinical guidelines in seven major regional addiction centers in Serbia since 2011 . After 2At contemporary momentum, it appears that accessibility and affordability of alcohol dependency medical care remain far from satisfactory levels in Serbia. Efficient new financing instruments will be required to fund existing network of specialty addiction centers as well as to provide reimbursement of anti-craving medicines. There is a long way ahead. Current support from ground professional associations such as ESBRA and EU iNo conflict of interest with regards to the manuscript disclosed by Mihajlo B. Jakovljevic, Mirjana Jovanovic, and Otto Michael Lesch."} +{"text": "The effectiveness of conservation organizations is determined in part by how they adapt to changing conditions. Over the previous decade, economic conditions in the United States (US) showed marked variation including a period of rapid growth followed by a major recession. We examine how biodiversity conservation nonprofits in the US responded to these changes through their financial behaviors, focusing on a sample of 90 biodiversity conservation nonprofits and the largest individual organization . For the 90 sampled organizations, an analysis of financial ratios derived from tax return data revealed little response to economic conditions. Similarly, more detailed examination of conservation expenditures and land acquisition practices of TNC revealed only one significant relationship with economic conditions: TNC accepted a greater proportion of conservation easements as donated in more difficult economic conditions. Our results suggest that the financial behaviors of US biodiversity conservation nonprofits are unresponsive to economic conditions. The decade of the 2000s was characterized by highly variable economic conditions globally and within the United States (US), including a period of rapid growth followed by the largest recession since the Great Depression Poole ; Fig.1. ; Kenward et\u00a0al. These divergent predictions regarding the impact of changing economic conditions on conservation may hinge on conservation organizations' responsiveness or ability to adapt to change. The ability of conservation organizations to adapt to change has been suggested as a key driver of their overall effectiveness . We use financial data from the US Internal Revenue Service (IRS) tax returns for a random sample of biodiversity conservation nonprofits to calculate \u201cfinancial ratios\u201d indicative of organization behavior. Originating from for-profit applications like predicting bankruptcy risk . We standardized all monetary amounts to 2010 US dollars ($) using the Consumer Price Index (http://www.bls.gov/CPI/). A minority of our nonprofits (35 of 90) reported in fiscal years different from the calendar year; for these organizations, we standardized fiscal years by calculating averaged monthly values and summing these into corrected calendar years.Our cross-sectoral analyses use a stratified random sample of 90 biodiversity conservation nonprofits drawn from an existing dataset of over 1700 such organizations . We included only organizations that filed IRS 990 forms every year between 2000 and 2009, excluding apparent exits (failures) for two reasons. Prior to 2008, small nonprofits with gross revenues below $25,000 were not required to file IRS 990 forms, and consequently true exits were difficult to parse from the more common incidence of organizations failing to file taxes for several consecutive years liquid funds interval; (2) revenue concentration; (3) ratio of personnel costs to total expenditures; and (4) ratio of total liabilities to total assets to another revenue source of equal magnitude would go undetected (see Appendix\u00a04). For this reason, we also sought to complement our use of coarse financial ratios with a more resolved analysis focusing on land acquisition practices of one major organization. However, we recognize that focused interviews of organizational leaders might be better suited for some aspects of more fine-grained responses and using conservation easements . For each transaction, we considered the total area (hectares), total cost , the proportion of costs that were donated relative to fair market values estimated from independent appraisals of property values, the price per hectare of acquisitions, and finally the ratio of conservation easements to fee simple acquisitions. We aggregated these fields across deals done in each financial quarter in 2000 through 2009. All TNC responses were log10 transformed for analyses, with additional detail on sources and management of TNC data given by Fishburn et\u00a0al. . We also included a set of models that incorporated an interaction term between biodiversity conservation nonprofit size and economic conditions (time-detrended GDP). Predictions of the role of organization size on financial ratio responses are given by TableR2 values were calculated for cross-sectoral mixed models as the relationship of model fitted to observed response values.We sought to relate our indicators of nonprofit behavior (above) to changing economic conditions. We used log10 $ Fig. because Three of the four financial ratios considered in cross-sectoral analyses were affected by biodiversity conservation nonprofit size Table. SpecifiR2 values suggest that biodiversity conservation nonprofits are not particularly responsive to changing economic conditions responded as predicted Table to econoOur analysis of TNC's land acquisition behavior provides an opportunity to test for behavioral responses to economic conditions at a much more resolved, if organization specific, level. However, again we detected little discernable response in behavior. Only one TNC land acquisition behavior was significantly affected by GDP and explained a meaningful proportion of the variance Table. The pro, Woodward Scientists regularly express in popular media , suggests TNC maintains their pace of conservation activity under poor economic conditions in part by taking as donations lands they might not prefer under more favorable conditions. This behavior also likely displaces some of the cost of conservation onto state and federal governments via land owner tax deductions for easement donations at times (economic recessions) when government budgets are already stressed by decreases in revenue.Surveys and interviews of employees or board members might be used to test our conclusion of little responsiveness by biodiversity conservation nonprofits to economic conditions, and also to further characterize how such responsiveness relates to meeting organization objectives and conservation goals in some kind of consistent \u201cfiscal homeostasis.\u201d Related, Zietlow in a stuWe conclude by emphasizing that efforts to characterize effectiveness of conservation activity for the sector in aggregate remain in their infancy (Gaston et\u00a0al."} +{"text": "AlthougSome scientists reported that paradoxal responses are defined as worsening of existing symptoms or the appearance of new lesions in patients who initially responded well to antituberculous therapy , 3.Triple-drug antituberculous chemotherapy can play a main role in treating tuberculosis if the lCurrently, there are few widely accepted classification systems based on objective data that can provide guidance on selecting the proper treatment approach for patients with spinal tuberculosis. In 2008, Oguz et al. developeWe believe that this classification system should be considered as a practical guide for spinal tuberculosis treatment planning in all countries."} +{"text": "Headache is a rare presenting demyelinating feature in people who go on to develop multiple sclerosis (MS). It has been suggested that approximately 2% of MS patients present with headache. There has been a debate whether MS patient group are at higher risk of developing headaches. We are not aware of any case reports where an occipital headache heralds first or recurrent high cervical cord demyelinating lesion.To report headache characteristics in patients whose headache was the main presenting symptom of MSWe prospectively collected data from patients who were referred to the MS clinic with new onset headaches who were found to have first demyelinating episode or MS relapse over the last 12 months. Magnetic Resonance Imaging (MRI) scans were reviewed for relevant demyelinating lesions.We identified four patients with evidence of demyelination whose main symptom was new onset-headache and were found to have relevant abnormal physical examination.Of these four patients, three had demylinating plaque at C1-C2 segments. In 2 out of 4 cases, headache represented the first demyelinating episode.Cervical spinal cord is a heavily myelinated structure, demyelinating plaques could induce nociceptive inflow by affecting the trigemino-cervical complex.It is the traditional thinking that headaches are an unlikely presentation of a demyelinating episode, however detailed headache history coupled with appropriate examination and investigations could potentially reveal conditions which requires totally different therapeutic approach from the common primary headache disorders.No conflict of interest."} +{"text": "Computain vitro . It is nDifferential Evolution (DE) is an evolutionary optimization method capable of identifying a population of candidate models throughout parameter space that closely match empirically observed firing patterns. The quality of fit achieved by an optimization is reliant on the \u2018fitness functions\u2019 used to measure the accuracy of the model. Previous neuronal compartment modeling studies using parameter optimization have introduced multiple types of fitness measurement , but havWe demonstrate the method with a compartment model comprising a simplified pyramidal neuron morphology and three ion channels, optimized to data from representative young and aged neurons. Compared to a manual approach involving iterative generation of fitness functions, our novel method produces better fitting models using a tenth of the computation time. Future work will extend the automatic protocol generation to prioritize which parameters to optimize, a critical step as more ion channels are added to the model to improve fitness. This method will be used to generate morphologically detailed models of 20+ young and aged PFC neurons, predicting which ionic mechanisms underlie age-related physiological changes."} +{"text": "Additionally they assumed by earlier radiographic diagnoses of FAI and AD would lead to earlier hip preservation and reduce early osteoarthritis (OA). They hypothesize \u2018FAI and AD often have overlapping symptoms, making an accurate diagnosis challenging. Therefore, radiographs remain the cornerstone in the evaluation, diagnosis and management of patients with these pre-arthritic hip condition,\u2019 and \u2018the ability to accurately and consistently interpret the radiographs in this particular patient population is of great importance.\u2019 They used 14 parameters (6 objectives and 8 subjectives) to evaluate 55\u2009hips hip radiographs with on follow-up 6 weeks later on 20\u2009hips for a reliability test, with blinded radiographic reviews of all 55 patients by an attending orthopaedic hip surgeon, an attending musculoskeletal radiologist, an orthopaedic sports fellow and a 3-year orthopaedic surgery resident. They found \u2018a relatively low level of interobserver and intraobserver reliability between readers, especially for subjective parameters. Thus, many of the standard radiographic measurements on anterior posterior (AP) pelvis and lateral hip views to diagnose FAI or AD were observed as not being reproducible,\u2019 and found interobserver reliability to be highest among objective parameters such as the lateral center edge angle and alpha angle. Poor interobserver reliability values were observed with predominately subjective measures such as joint congruency, T\u00f6nnis grade and detection of herniation pits. The unique aspect of their study was that the amount of surgical hip experience increased the likelihood of radiographically making a pathologic diagnosis with their senior hip surgeon demonstrating the highest diagnostic sensitivity among all readers; however, he also demonstrated the lowest degree of diagnostic specificity with over reading normal radiograph as abnormal. They concluded a tendency to over diagnose radiographs can explain the increasing incidence of hip arthroscopy procedures. While this study uses 14 parameters to radiographically diagnose FAI and AD, most astute clinicians base their diagnosis on a sound history and an accurate physical exam the radiographs as an adjunct to their clinical impression. We know from other studies that radiographic FAI may not necessarily be symptomatic [Patrick C. Schottel"} +{"text": "Optic neuritis is a common presentation of demyelinating disorders such as multiple sclerosis. It typically presents with acute painful monocular vision loss, whereas chronic optic neuropathy can be caused by compressive lesions along the anterior visual pathway, genetic, toxic, or nutritional causes. We report an unusual presentation mimicking optic neuritis, which was subsequently diagnosed as optic nerve sheath meningioma (ONSM). Misinterpretation of white matter lesions on MRI of brain and the failure to image the optic nerves at the time of acute loss of vision led to the misdiagnosis of optic neuritis in this case. A comprehensive accurate history and ordering the appropriate imaging modality remain paramount in diagnosing progressive visual deterioration. Optic nerve sheath meningioma (ONSM) usually arises from the intraorbital part of the optic nerve sheath and accounts for approximately 2% of all orbital tumor . TypicalA 47-year-old woman presented with history of acute decrease in vision of the right eye 4 years ago. She has reported that the onset was \u201csudden\u201d yet with rapid progression within 1 week and was associated with \u201cpain with eye movement.\u201d She denied any previous history of weakness, paresthesia, or bulbar or bowl/bladder symptoms. Review of her records showed that initial neuro-ophthalmological examination four years ago revealed that her visual acuity was hand motion recognition in the right eye and 20/20 in the left eye. She had a large right relative pupillary afferent defect (RAPD). Anterior segment examination was within normal limits and her ocular motility was full with no ocular misalignment or limitation of eye movements. Fundoscopic examination showed normal optic discs and maculae in both eyes. Humphrey visual fields 30-2 showed a large right central scotoma in the right eye and was normal in the left eye. Neurological examination was unremarkable. The initial MRI of the brain reportedly showed multiple nonenhancing T2/FLAIR hyperintense lesions within the periventricular, deep, and subcortical white matter in a nonspecific distribution. Orbital cuts were not performed in the original MRI. The white matter lesions were seen in the subsequent MRI obtained when the patient presented to us Figures . There wOur case highlighted the difficulty in diagnosing ONSM when presented with acute visual loss in association with painful eye movements mimicking optic neuritis. The unusual presentation can be attributed to the \u201csudden awareness\u201d rather than the sudden onset in a patient who was not sufficiently cognisant to give an accurate history. The normal fundoscopic examination was also supportive of acute retrobulbar optic neuritis. It was obvious that the MRI findings of white matter lesion had a significant impact on directing the provisional diagnosis toward a demyelinating process, specifically multiple sclerosis. Misinterpretation of the MRI resulted in a delay of the diagnosis and possibly missing the therapeutic window in this patient. In addition, the omission of high-spatial-resolution contrast-enhanced MRI of the orbit with thin sections (less than 3\u2009mm) particularly with progressive loss of vision was also an important factor in the delay of diagnosis. Progressive visual loss and unresponsiveness to high-dose steroids are usual red flags in optic neuritis secondary to multiple sclerosis and they usually point toward more serious diagnoses such as neuromyelitis optica or orbital pathology. Although OSNM typically presents with slowly progressive visual loss, atypical acute presentation leading to an initial diagnosis of \u201coptic neuritis\u201d has been reported \u201310. JackIn summary, ONSM may present with atypical features such as acute painful visual loss in association with normal fundoscopic examination. Lack of recovery or progressive loss of vision should prompt the clinician to explore alternative diagnoses other than typical demyelinating optic neuritis. Finally selecting the appropriate imaging protocol in the setting of progressive loss of vision, particularly high resolution contrast MRI of the orbit with fat-suppression sequences, can be crucial in making the correct diagnosis."} +{"text": "Plants select plant growth promoting rhizobacteria (PGPR) that are competitively fit to occupy compatible niches without causing pathological stress on them. However, when screening bacteria for plant growth promoting (PGP) agents, it is better to select bacteria for achieving the most promising isolates having suitable colonization and PGP traits. In most researches, it has been seen that following incubation, bacterial flora are taken at random from petri dishes for further study. However, this type of selection may remove some superior bacteria in terms of PGP traits and high colonization ability. Therefore, it is essential to study all the isolated bacteria in an economic way and select the best bacteria in terms of PGP traits and high colonization rate. A simple screening method to detect endophytic and rhizosphere bacteria, isolated from the plants in rotation with rice, for rice PGP agents based on a root colonization bioassay and a PGP trait is characterized.\u2022Selected bacterial isolates based on their IAA producing trait have the potential for more PGP and colonization of rice plant.\u2022IAA may be the first PGP trait for screening bacteria isolated from plant rotated with rice for rice PGP agents.\u2022The screening procedure appears to be very effective and less time consuming. Find isolates without any antagonistic effect with each other by an Note: This assay must be performed on four groups of plant growth promoting bacteria (PGPR) with different PGP traits namely IAA producing isolates, ACC deaminase producing isolates, siderophore producing isolates and phosphate solubilizing isolates separately to find isolates without any antagonistic effect with each other and having the same PGP traits.5.4)2SO4 under in vitro conditions as described by Etesami et al. Inoculate each of IAA, siderophore and ACC deaminase producing isolates and phosphate solubilizing isolates as mixtures of strains each (isolates without antagonistic effect with each other and having the same PGP trait as batch inoculations) on rice plant seedlings in sterile Hoagland\u2019s medium containing 8\u00a0mM , and examine their endophytic competence (infection and persistence characteristics).6.Isolate endophytic and rhizosplane bacterial isolates inoculated on rice seedlings after 20 days.7.Screen all endophytic and rhizoplane strains isolated from rice seedlings for the same plant growth promoting (PGP) traits as mentioned above.Note: this step was performed for the first time to determine whether bacteria reisolated from rice seedlings can have other PGP traits. For example, whether ACC deaminase producing isolates can also produce IAA or siderophore or solubilize phosphate. In our studies 8.4)2SO4 under in vitro conditions as described by Etesami et al. Inoculate each of IAA, siderophore and ACC deaminase producing isolates and phosphate solubilizing isolates as single-strain inoculants on rice plant seedlings in sterile Hoagland\u2019s medium containing 8\u00a0mM , were able to colonize within roots and promote plant growth. According to the results obtained, 7 out of 10 isolates tested (70%) behaved as potentially good PGP and colonizing agents. Seedlings inoculated with IAA producing isolates yielded more shoot biomass (stem plus leaves), root length and colonization than the control plants inoculated with IAA non-producing strains and plants inoculated with PGPR producing other PGP traits To confirming the efficiency of the bioassay, ten isolates isolated from berseem clover or canola plants grown at field, which had been screened only based on the production of IAA, were retested, using the same procedures. As additional negative controls, two IAA non-producing isolates were also included. Screening to detect isolates with good potential as rice growth-promoting agents indicated that seven out of ten endophytes, inoculated on five rice cultivars , it is possible that IAA producing bacteria by increased root system can colonize plant roots better than other bacteria. In addition, IAA levels weaken plant defence mechanisms making colonization easier \u2022Bacterial IAA can loosen plant cell walls and as a result promotes an increasing amount of root exudation that provides additional nutrients to support the growth of rhizosphere bacteria \u2022It is known that bacterial IAA can loosen plant cell walls and as a result promotes an increasing amount of root exudation that provides additional nutrients to support the growth of rhizosphere bacteria \u2022IAA stimulates overproduction of root hairs and lateral roots in plants and release of saccharides from plant cell walls during the elongation \u2022Bacterial IAA increase root surface area and length, and thereby provides the plant greater access to soil nutrients and water uptake \u2022In view of function of bacterial IAA in increased root system, it is may proposed that IAA producing bacteria can provides more number of active sites and access to colonization for other PGPRs. For example, the presence of PGPRs in the root vicinity could improve ability of rhizobia to compete with indigenous populations for nodulation \u2022It is hypothesized that the secretion of IAA may modify the microhabitat of epiphytic bacteria by increasing nutrient leakage from plant cells; enhanced nutrient availability may better enable IAA-producing bacteria to colonize the phyllosphere and may contribute to their epiphytic fitness \u2022Bacterial IAA can obviate to a certain extent the function of ACC deaminase and siderophore producing bacteria and phosphate solubilizing bacteria There are many evidences that IAA may be the first PGP trait compared to ACC deaminase activity, siderophore production and phosphate solubilization traits for screening rhizosphere and endophytic bacteria for rice plant PGP agents below:in vitro potential of IAA production could provide a reliable base for selection of effective PGP bacteria. Many studies have shown that the interaction of IAA-producing bacteria with plants might posit that since IAA activates the transcription of ACC synthease, these bacteria should all ultimately result in the production of relatively high concentrations of ACC and subsequently inhibitory levels of ethylene. Thus, in the absence of some other mechanism, IAA-producing bacteria might all be expected to ultimately be inhibitory to plant growth. However, according to Glick It may be suggested that plants select endophytic and rhizosphere bacteria with these traits or that these bacteria harbor other traits that allow them to more effectively reach and establish themselves in rhizoplane and the inner plant tissue Therefore, the screening procedure appears to be very effective and less time consuming . This sc8 bacteria g14+ and phosphorus were available to rice seedlings in sterile Hoagland\u2019s medium and the same in rice fields due to anaerobic conditions (high availability of Fe +2 and phosphorus), it is seemed rice seedlings did not have more need to attract isolates helping the increase of solubility of these nutrients. However, due to possibility of production of ethylene under constant flooding conditions, ACC deaminase producing isolates could be next option to be attracted by rice seedlings.PGPRs may use different mechanisms to enhance plant growth as experimental evidence suggests that the plant growth stimulation is the net result of multiple mechanisms that may be activated simultaneously"} +{"text": "The NEURON simulator software is widely used by the computational neuroscience community for neuronal electrophysiology modeling and we have recently extended it to support reaction-diffusion dynamics . This clSBML is an XMWe have added the capability to import and export models in SBML format to NEURON\u2019s reaction-diffusion module using the libSBML library . This al\u2022 Electrophysiology model is loaded from ModelDB or constructed de novo.\u2022 NEURON loads SBML data and instantiates appropriate reaction diffusion objects: rxd.Region, rxd.Species, rxd.Reaction. User must interactively or preemptively match state-variable names across combined models to combine the two models into one.\u2022 SBML models do not include diffusion so diffusion constants can be added to provide the spatial element of the combined model.\u2022 User can adjust the parameters and make simulation runs of the model.We have used these new features to study the interaction of a calcium induced calcium release model from BioModels with electrical activity in a detailed model of a hippocampal CA1 pyramidal neuron."} +{"text": "A modifiable spreadsheet tool will enable health officials to plan for thesebirths. The marked increase in infants born with microcephaly in Brazil after a 2015 outbreakof Zika virus (Zika virus) disease suggests an association between maternal Zikavirus infection and congenital microcephaly. To project the timing of delivery ofinfants born to mothers infected during early pregnancy in 1 city in Bahia State,Brazil, we incorporated data on reported Zika virus disease cases and microcephalycases into a graphical schematic of weekly birth cohorts. We projected that thesebirths would occur through February 2016. Applying similar projections to ahypothetical location at which Zika virus transmission started in November, weprojected that full-term infants at risk for Zika virus infection would be bornduring April\u2013September 2016. We also developed a modifiable spreadsheet toolthat public health officials and researchers can use for their countries to plan fordeliveries of infants to women who were infected with Zika virus during differentpregnancy trimesters. Aedes aegyptimosquitoes; maternal\u2013fetal transmission of Zika virus has been reported reported an outbreak of Zika virus (Zikavirus) disease in Brazil , we created figures demonstrating cohorts of pregnant women by week ofdelivery and then extrapolated to the beginning of pregnancy. Live-birth data fromBrazil showed small differences in the proportions of infants born at full term(37\u201341 weeks) with microcephaly (76.7%) compared with those born at full termwithout birth defects (83.6%) . We alsoIn Bahia, \u22484,000 infants are born each week . DuThe increased number of reported cases of microcephaly in Bahia State began with Octoberbirths; reported cases rose sharply during November 2015\u2013January 2016. For thecity of Salvador, these November 2015\u2013January 2016 births corresponded to thehighest likelihood of infection occurring in the first trimester or early in the secondtrimester of pregnancy, assuming that the date of report approximates the date of birth.There are no reports to indicate whether the city of Salvador experienced the Zika virusdisease outbreak earlier or later than the rest of Bahia State.In Country A , for theTo enable readers to project months when births with exposure in different trimesterscan be expected, we developed a modifiable spreadsheet tool . Users mOur projections, based on ecologic data, indicate that in Bahia State, Brazil, Zikavirus infection during the first trimester or early in the second trimester of pregnancyis temporally associated with the observed increase in infants born with microcephaly;this projection is consistent with the observed reported decline for January andFebruary 2016. This finding adds to pathologic findings documenting Zika virus infectionin several infants with microcephaly and loca,Understanding the timing of Zika virus infection of pregnant women is key because theeffects of infection on pregnancy and fetal and infant outcomes is likely to vary bygestational timing, as has been demonstrated for other congenital infections such asrubella and cytomegalovirus; transmission risk may also vary according to gestationaltiming (Some of the reported cases of microcephaly included in the graph are still beingassessed, and some might not meet the final case definition for microcephaly in Brazil(i.e., head circumference We assumed that the birth rates in these models remain constant throughout the year,which is not true for all locations. The data for Zika virus infection and infants withmicrocephaly are based on dates of report, which are probably later than actualoccurrence.Despite these limitations, our assessments provide some indication that the period ofhighest risk might be during the first trimester or early in the second trimester ofpregnancy. This assessment can help inform public health officials about risks formicrocephaly and help them plan for deliveries in areas where Zika virus diseaseoutbreaks occur. Conducting surveillance for microcephaly but also other pregnancyoutcomes such as pregnancy loss and other birth defects will enable continued evaluationof any effects of Zika virus disease might have on pregnancy. These data also emphasizethe role of arboviral disease\u2013tracking activities for informing public healthplanning. The US Centers for Disease Control and Prevention has prepared interimguidelines for US healthcare providers who care for women who are pregnant during a Zikaoutbreak (The consequences of Zika virus infection during pregnancy are not fully understood.Given the growing evidence of an association with microcephaly (Modifiable spreadsheet for projecting periods of delivery of at-risk infants afterZika virus disease outbreaks."} +{"text": "Gs\u03b1 plays a crucial role in intracellular signal transduction of peptides, hormones and neurotransmitter receptors in multiple tissues. Key features of PHP include Albright Hereditary Osteodystrophy (AHO) and biochemical evidence of multiple hormone resistances. There are several conflicting mechanisms for its heterogeneity; a possible explanation is a tissue-specific differential imprinting of Gs\u03b1 protein. PHP type1a has AHO with multiple hormone resistance and is inherited as maternal imprinting defect. PHP type 1b presents with hormone resistance but no AHO features and is probably due to epigenetic methylation defects. Peudopseudohypoparathyroidism is another subtype with AHO but absence of hormone resistance and is inherited as paternal inactivating mutation. We describe a family with female members having PHP with variable clinical and biochemical features.Pseudohypoparathyroidism (PHP) encompasses a heterogeneous group of disorders due to an inactivating mutation in the GNAS gene which encodes the \u03b1 subunit of GTwo female siblings from non-consanguineous parents, patient A 15 year) & patient B (13 year) presented at birth with raised TSH levels and were diagnosed to have congenital hypothyroidism and treated with thyroxine replacement. Patient A was noted to have infantile obesity with short stature and evolving phenotypic features consistent with AHO. Further history revealed that her sister patient B has similar phenotypic appearance and was confirmed on clinical exam. Biochemical evaluation for Patient A showed a borderline low calcium and normal phosphate level with elevated PTH while Patient B had normal calcium and phosphate levels with high PTH. Both patients entered puberty at 11 years and patient A progressed to breast tanner 4 but has no menarche at 15 years despite pubertal gonadotropin levels (Table 5 year & The two patients with PHP described above had an interesting presentation as congenital hypothyroidism with features of AHO evolving later in life. They exhibit clinical evidence of maternal transmission with end-organ resistance. This family illustrates heterogeneity of presentation of GNAS mutation.Written informed consent was obtained from the patients for publication of this abstract. A copy of the written consent is available for review by the Editor of this journal."} +{"text": "Delivery of infants who are physiologically mature and capable of successful transition to the extrauterine environment is an important priority for obstetric practitioners. During the past 15 years in the United States the percentage of infants born before 40 weeks\u2019 gestation has dramatically increased and the percentage of infants born after 40 weeks\u2019 gestation has decreased. In this shift several factors have been implicated: increased medical surveillance and interventions, increased multifetal pregnancies, maternal obesity , maternal autonomy, route and timing of delivery.+0 to 36+6 weeks\u2019 gestation). Age stratified cohort studies confirms that adverse neonatal outcome decrease with increasing gestational age independent of delivery mode.Birth before fetal maturity contributes to short-term and long-term morbidity and mortality in late preterm are based on the balance between maternal and newborn risks of early delivery with the risk of further continuation of pregnancy.To decrease the mortality and morbidity associated with late preterm births, prevention is one of the key components. The ACOG does not recommend induced vaginal or planned cesarean delivery prior to 39 weeks gestation unless medically indicate. If elective induction is undertaken for nonmedical reasons, it should only take place if the preinduction assessment ensures the gestational age is at least 39 weeks.In addition, further research is needed to refine the management of late preterm gestation :\u2022 Assess the risk/benefit ratio for indications for late preterm delivery, such as more accurate estimation of fetal outcome in presence of maternal diseases.\u2022 Identify management strategies to improve outcomes in late preterm infant \u2022 Improve the precision of determining gestational age."} +{"text": "Neurons are fundamental components of neural systems. Organized neural activity is key for proper function at many scales, from small central pattern generating circuits to large populations of neurons. Neurons can show considerable variability in their spiking regularity; this variability is attributed to a number of different sources, including probabilistic synaptic processes and stochastic ion channel activity . The rolAplysia californica invertebrate neurons for 2 or more hours in a temperature-controlled preparation to determine the regularity of interspike intervals. We determined that sequential ISIs show strong positive correlation with prior ISIs and this correlation remains high for many lag values, indicating that the ISI sequence shows history. In contrast, sequential values of the change in ISI (delta ISI) show a single prominent negative autocorrelation coefficient; this indicates that delta ISI correlates strongest with one previous delta ISI, and the negative sign suggests a tendency for delta ISI values to remain within some range. We find that ISI sequences from intrinsically spiking neurons in this system can be represented using autoregressive integrated moving average (ARIMA) models. In addition, delta ISI distributions are symmetric and Gaussian-like with heavy tails. Recent literature reports that heavy-tailed ISI distributions that show positive serial correlation values may result from stochastic activity in populations of adapting ion channels [In this work, we characterized the variability of intrinsically active neurons over time and found that all cells studied shared certain features. We measured intracellularly from synaptically-isolated, endogenously spiking channels ,5; we arWe present here an ISI variability model that captures statistical features of experimental data, which are essentially history and randomness. Existing explanations for neural spiking variability include properties of synaptic conductances , neural This work was funded by NIH NS 54281."} +{"text": "Sensory modulation utilises sensory input from external senses and internal senses , along with abdominal breathing. It has been shown to help with stress reduction, relaxation, emotional regulation and symptom management of adolescent consumers and elderly consumers. An exploratory method was used to evaluate if Sensory Modulation had an impact on reducing post meal distress in adult consumers suffering from eating disorders. The data was collected from inpatient eating disorder consumers. There ranged between five and eight consumers per group per week. The Sensory Modulation Group ran once a week for forty five minutes over a ten week period. The Sensory Modulation Consumer Self Rating Tool was used to rate the consumer mood before and after Sensory Modulation was implemented. It was found that Sensory Modulation utilised after a meal reduced post meal distress by 10%-20%. With increased implementation on the inpatient unit, Sensory Modulation allows inpatient consumers to find a sensory modality that works for them that they can utilise in the community to manage post meal distress."} +{"text": "The percent identity matrices of two sequence multiple alignments between linker histones from chicken and mammalian species are described. Linker histone protein sequences for chicken, mouse, rat and humans, available on public databases were used. This information is related to the research article entitled \u201cIdentification of novel post-translational modifications in linker histones from chicken erythrocytes\u201dpublished in the Journal of Proteomics The fir"} +{"text": "Orthodontists and maxillofacial surgeons have always been interested to quantify the influence of orthodontic and/or orthognathic surgery treatment on craniofacial morphology.Historically, superimpositions of cephalometric radiographs have been used to study growth and treatment effects. In recent years advances in 3D imaging techniques have led to their widespread use for setting treatment goals, choosing treatment modalities, predicting treatment results and evaluating treatment and/or growth changes.Various different techniques have been reported for superimposition of 3D datasets derived from either conventional computed tomography (CT) or lower radiation cone beam CT (CBCT) images.This lecture presents the main 3D superimposition techniques that are currently available and discuss their unique specifications and inherent limitations."} +{"text": "Cardiovascular autonomic nervous dysfunction has been demonstrated to severely debilitate HIV infected patients, namely by postural hypotension and syncopes. It has important implication in health care of HIV patients. Presence of autonomic neuropathy signals the need for added precautions when invasive procedures are performed on HIV patients.Fifty patients (25 HIV +ve without AIDS and 25 HIV+ with AIDS) who fulfilled the inclusion and exclusion criteria and 50 healthy matched controls were enrolled in the study. All HIV positive/AIDS patients were evaluated according to a detailed proforma with elicitation of history, symptoms, signs and routine and specialized investigations.In the present study, 16% of HIV +ve with the AIDS had abnormal autonomic dysfunction and 4% of HIV positive without AIDS had abnormal autonomic dysfunction. Reduced heart rate variability is the commonest manifestation of autonomic dysfunction noted in both HIV positive without AIDS and HIV positive with AIDS groups. Diastolic BP response to sustained handgrip has a limited role in discriminating autonomic function in HIV infected patients. There is no statistically significant correlation with the CD4 level and the presence of autonomic nervous system dysfunction in both the groups.Cardiac autonomic nervous dysfunction is a common and relevant clinical problem affecting both HIV positive without AIDS and HIV positive with AIDS groups. It may provide an alternative explanation for symptoms commonly observed in HIV infected individuals such as bowel and bladder dysfunction, impotence, syncope and sweating abnormalities."} +{"text": "AlthouThe liver is known for its outstanding capacity to regenerate after toxic damage. Within a relatively short period of time millions of cells find their new position to restore functional tissue architecture. Until recently, little was known which mechanisms orchestrate this process . In ). In 7])Recently, a Systems Toxicology approach with a metabolic model of ammonia metabolism predicted that under specific conditions of hyperammonemia, glutamate dehydrogenase (GDH) may switch its direction from ammonia production to ammonia consumption . This iUnderstanding the mechanisms of organ toxicity has always been a major goal in toxicology (Campos et al., 2014; Hammad,"} +{"text": "There are significant challenges to restoring binaural hearing to children who have been deaf from an early age. The uncoordinated and poor temporal information available from cochlear implants distorts perception of interaural timing differences normally important for sound localization and listening in noise. Moreover, binaural development can be compromised by bilateral and unilateral auditory deprivation. Here, we studied perception of both interaural level and timing differences in 79 children/adolescents using bilateral cochlear implants and 16 peers with normal hearing. They were asked on which side of their head they heard unilaterally or bilaterally presented click- or electrical pulse- trains. Interaural level cues were identified by most participants including adolescents with long periods of unilateral cochlear implant use and little bilateral implant experience. Interaural timing cues were not detected by new bilateral adolescent users, consistent with previous evidence. Evidence of binaural timing detection was, for the first time, found in children who had much longer implant experience but it was marked by poorer than normal sensitivity and abnormally strong dependence on current level differences between implants. In addition, children with prior unilateral implant use showed a higher proportion of responses to their first implanted sides than children implanted simultaneously. These data indicate that there are functional repercussions of developing binaural hearing through bilateral cochlear implants, particularly when provided sequentially; nonetheless, children have an opportunity to use these devices to hear better in noise and gain spatial hearing. This is important for survival and communication. Directional hearing helps us to listen to one voice amongst many by comparing sounds reaching our two ears. Sounds sources in space hit the outer ears at different places and angles producing unique and recognizable spectral patterns The cochlear implant is surgically placed in each inner ear (cochlea) to bypass the impaired sensory system and electrically stimulate the auditory nerve on each side Reduced sensitivity to interaural timing cues in adults occurs despite efforts to match place of stimulation on the two sides Given such challenges inherent to cochlear implants, can children who are deaf develop binaural hearing with these devices? Early findings suggest that children with bilateral implants have considerably poorer sound localization and perception of binaural cues than adult users who lost their hearing later in life Although it is clear that cochlear implantation should proceed as early as possible for oral speech and language development Importantly, the asymmetric development promoted by unilateral implant use did not eliminate integration of binaural input in the brainstem The study protocol and consent procedure were approved by the Hospital for Sick Children's Research Ethics Board (#1000002954). A total of 95 adolescents/children consented to participate in this study. Written consent was obtained from caretakers or guardians on behalf of participants who were under 18 years of age at the time of enrollment and written assent was obtained in those children who were able to provide it. Three groups used bilateral cochlear implants and one group had normal hearing. The demographic details are shown in The normal hearing group listened to 500 ms trains of clicks presented at 250 clicks/s and bilateral cochlear implant users listened to 500 ms trains of biphasic electrical pulses presented at 250 pulses/s. Interaural level differences (ILD) \u200a=\u200a0 approximated levels which were most likely to be perceived as balanced. For normal hearing children, we measured behavioral thresholds to the click stimulus and presented stimuli in each ear at 40 dB above threshold (sensation level (SL). By contrast, it was not possible to assume a constant range of current between threshold and comfortable listening levels in cochlear implant users. We therefore used electrophysiological measures of brainstem activity evoked by each implant to determine approximately balanced current levels at the upper part of the dynamic range Differences between levels were introduced while holding the overall current delivered constant; current increases in one device occurred with equal decreases in current in the other. The manufacture defined Clinical Units (CU) were used during testing because these are logarithmic values and provide linear increases/decreases but the conversion to standard current (amperes) varies slightly with device. All children were asked to indicate on which side of their head they heard the sound (left or right) by pointing or speaking. The first 23 of the 34 adolescents tested in the first months of implant use were provided with 2 additional choices (middle and both) as in a previous study Repeated measures ANOVAs were used to assess change in response proportions with ILD and ITD conditions. Binary logit regression was used to fit responses across ILD and ITD conditions in each participant. ILD and ITD slopes were calculated using a bias-reduction general linear model; a t-test was used to compare slopes between bilateral groups. Linear regression was used to assess time-intensity trading by fitting predicted ITD for a centered perception against ILDs at predicted balance. Two way repeated measures ANOVAs were used to test for effects of ITD, group and ITD*group interactions. Significance was considered at p<0.05. Bonferroni and Dunnett T3 adjustments were used for post-hoc ANOVA comparisons.Although children using bilateral cochlear implants were previously found to detect changes in inter-implant current levels (measured in dB re: 100 \u00b5A), it is not clear whether these skills required bilateral implant experience to develop nor whether this ability would be present in adolescents who, by virtue of their age and unilateral CI use, were questionable candidates for bilateral implantation Because most responses were to the right or left side of the head, binary logit regression analyses were used to assess whether the increase in responses to the side of first implant increased as ILD became increasingly weighted to that side for each child as shown in Given that perception of binaural level cues appeared so early in bilateral implant use and even in children who had very long delays between implantations, we asked whether there was an effect of the timing of implantation on this perception. In this part of the study, we measured proportion of responses to the left and right sides of the head in response to bilateral input in 3 groups of children matched for bilateral hearing age: 1) 29 children receiving bilateral implants simultaneously ; 2) 16 children receiving bilateral implants sequentially ; and 3) 16 children with normal hearing . Mean (SE) responses in the 3 groups are plotted in In contrast to the encouraging findings with respect to detection of binaural level cues, the perception of interaural timing differences was not evident in the cohort of adolescents studied during the first year of bilateral implant use. Mean \u00b1 SE proportion of responses in the group tested after 1.66\u00b10.80 months of bilateral implant experience are shown in Based on the lack of ITD perception in the adolescent cohort, we asked whether these skills might develop in the best conditions, when bilateral cochlear implants are provided without delay in young children, and with long periods of bilateral implant use. Data in As mentioned above, the proportion of right responses to bilateral input presented without level differences (ILD\u200a=\u200a0) or timing differences (ITD\u200a=\u200a0) were not significantly different than the proportion of left responses in children implanted bilaterally in simultaneous or sequential procedures. Nonetheless, as shown in In Additional group differences are shown in the mean (SE) proportion of right responses with changes in ITD plotted in In the present set of experiments, we took an optimistic view and hypothesized that perception of binaural cues could be established in children who are deaf in both ears. In support, detection of interaural level differences was found to be present at very early stages of bilateral implant use even in adolescents who had developed hearing from an implant in one ear for most of their lives. Moreover, children with longer term bilateral implant use responded to changes in interaural level differences similarly to their normal hearing peers with no apparent effects of unilateral hearing before bilateral implant use. Even more encouraging, we provide the first evidence that these children were also able to detect interaural timing cues. These skills took many years to develop as they were not evident in the present cohort of adolescents who recently received bilateral implants nor in a younger group of children tested after 2.2\u00b11.1 years of bilateral implant use as previously reported Data presented in Further evidence that bilateral and unilateral deprivation in development did not eliminate ILD perception is shown in The rapid ability by children who are deaf to detect binaural level cues is clinically important because cochlear implant levels must be customized for each child. The behavioral lateralization task used in the present study could help to establish bilaterally balanced perception even in early stages of device use. Although electrophysiological measures can be used to help approximate balanced bilateral levels, this must be confirmed behaviorally Unlike the rapid restoration of binaural level perception, adolescents receiving bilateral cochlear implants were unable to detect even very large differences in binaural timing during the first year of bilateral implant use as shown in The first evidence to our knowledge that children who are deaf can develop ITD perception through long term bilateral cochlear implant use is shown in Although children using bilateral implants develop perception of binaural cues after long term bilateral implant use, differences from normal remain. As shown in It is not entirely surprising that children with bilateral cochlear implants acquire abnormal ITD sensitivity. Their abilities were in line with that shown in adult bilateral implant users who had normal hearing in both ears before the onset of deafness We have shown in the present study that ITD detection in children using cochlear implants is an emerging skill which leads us to ask whether these skills might further develop with time. It is true that the children with normal hearing had longer binaural hearing on average than the bilateral cochlear implant users . Yet, even the youngest children with normal hearing, aged 5.55 years and 6.33 years, detected the ITD cues presented highly accurately and it is not clear whether children with bilateral cochlear implants, even those receiving bilateral implants simultaneously, would outperform adult CI users. Methods which seek to better match the place and level of stimulation between the devices, as discussed above, could help both adults and children using CIs along with better coordination of temporal cues between the devices to help ensure binaural fusion of the input. In addition, behavioral therapy might help bilateral implant users increase their awareness of these important binaural cues and translate them into useable improvements in sound localization and speech recognition in noise.Despite bilateral deafness in early childhood, interaural level cues were available almost immediately to most bilateral implant users. The emerging detection of timing cues after many years of bilateral cochlear implant stimulation reflects the plasticity of the developing system whilst the findings of preferred responses toward a unilaterally stimulated ear during development demonstrate that limitations to auditory development remain. Nonetheless, the restoration of binaural cues through bilateral cochlear implantation is a marked endorsement of this clinical intervention and highlights the potential for children to recover, at least to some extent, a missing sensory ability."} +{"text": "Stereoelectroencephalographic (SEEG) depth electrodes have the potential to record neural activity from deep brain structures not easily reached with other intracranial recording technologies. SEEG electrodes were placed through deep cortical structures including central sulcus and insular cortex. In order to observe changes in frequency band modulation, participants performed force matching trials at three distinct force levels using two different grasp configurations: a power grasp and a lateral pinch. Signals from these deeper structures were found to contain information useful for distinguishing force from rest trials as well as different force levels in some participants. High frequency components along with alpha and beta bands recorded from electrodes located near the primary motor cortex wall of central sulcus and electrodes passing through sensory cortex were found to be the most useful for classification of force versus rest although one participant did have significant modulation in the insular cortex. This study electrophysiologically corroborates with previous imaging studies that show force-related modulation occurs inside of central sulcus and insular cortex. The results of this work suggest that depth electrodes could be useful tools for investigating the functions of deeper brain structures as well as showing that central sulcus and insular cortex may contain neural signals that could be used for control of a grasp force BMI. Intracranial electrodes , placed for clinical localization of seizures in patients with pharmacologically resistant epilepsy, offer a platform for understanding fundamental human neurophysiology, as well as investigating novel cortical areas for implanting electrodes for use in brain-machine interfaces (BMIs). In the present study, we use intracranial SEEG to first investigate the efficacy of using field potentials recorded from insular cortex and the intrasulcal wall of motor cortex to discriminate forces of hand grasping for controlling a BMI, and secondly to compare the modulations of the high (gamma) and low band activities from motor and insular cortices during grasp force production.In contrast to subdural electrocorticography (ECoG) electrodes which record from the cortical surface, and intracortical microelectrodes which typically record from 1\u20131.5 mm deep into cortical tissue, SEEG depth electrodes can record neural activity from deeper cortical structures (such as insular cortex) that are not typically accessible by ECoG or intracortical arrays, and yet may exhibit movement-related modulation. Functional magnetic resonance imaging (fMRI) investigations have elucidated spatial patterns of activation during hand and finger movements that include activity in the central sulcus wall of motor cortex (MC) and the insular cortex (IC). Specifically, increased blood-oxygen-level-dependent (BOLD) signals have been observed inside the walls of central sulcus during hand and finger movements and isomAt a level deeper than discrimination of hand states, we sought to understand the temporal and spatial relationships between low and high (gamma) band activities in insular and motor cortices during hand grasp force production. Event-related synchronization (ERS) of the gamma band (30+ Hz) and event-related desynchronization (ERD) of the beta band (12-30Hz) have long been shown to be hallmarks of cortical sensorimotor activity during motor execution and even imagery. Yet it is unclear whether these same characteristic patterns of cortical modulation are evident in insular cortex, despite the reported contribution of insular cortex during movement generation. Beta band desynchronization occurs at the beginning of, and sometimes prior to, movement activity and is believed to be related to asynchronous activity of neurons within the cortical network as the neurons increase their firing rate. Beta desynchronization is also accompanied by synchronization in higher frequencies as more of the neurons start to fire at different rates, resulting in increased power in the high gamma frequencies . Once thSeveral lower frequencies have been shown to have task related modulation as well. Modulation in the alpha frequency band (6\u201312 Hz) has been reported in EEG to respond to executed and imagined movement tasks and isomThe present study reports comparative electrophysiological analysis of the relationships of neural activity from insular cortex and the intrasulcal wall of MC to production of hand grasp force. The findings show that insular cortex (at least in one participant) exhibits movement related cortical modulation, with focus in the alpha (6\u201312 Hz) frequency bands, and that this modulation alone is sufficient for discrimination of force execution vs non-force. Higher gamma frequency modulation (> 30 Hz) was not present in insular electrodes, in contrast to the clear gamma modulation observed in the intrasulcal wall of motor cortex. The present findings suggest that the motor contributions of insular cortex is fundamentally different than that of motor cortex, predominantly focusing modulation in the lower frequency band, rather than the higher frequency bands, and the observed modulations allows for motor discrimination during a hand grasp force task.Four human participants were enrolled into the study after providing written consent. The study was reviewed and approved by the Institutional Review Boards of Louis Stokes Cleveland VA Medical Center (IRB# 11068-H14) and University Hospitals Case Medical Center (IRB# 07-12-33). All study participants were patients at University Hospitals Case Medical Center admitted for epilepsy monitoring prior to resection surgery. Participants were implanted with SEEG electrodes from either Integra LifeSciences Corporation or PMT Corporation . Each Integra electrode consisted of 12 platinum-iridium macro-contacts measuring 1.1 mm in diameter and 2.3 mm in length, evenly spaced at 5 mm intervals. One participant had two PMT combination electrodes implanted consisting of 4 SEEG macro contacts with 4x6 circumferentially spaced microwires between the macro contacts (1.4mm macro contacts with 7mm center spacing). All participants underwent implantation of electrodes into the target structures exclusively for clinical purposes . Only electrodes passing directly through sensorimotor areas were used for this study. Study procedures were performed during non-epileptic episodes.http://surfer.nmr.mgh.harvard.edu/) [http://fsl.fmrib.ox.ac.uk/fsl/fslwiki/) [https://github.com/mcintyrelab).Each electrode contact was localized in 3-dimensional anatomical space in a custom Python-based visualization environment using the electrode contact artifacts in post-operative volumetric CT scans co-registered with the pre-operative MRI scans. Since the SEEG electrodes are fabricated from polyurethane tubing and the electrode contacts span 60 mm, the shaft of the electrodes can follow a curved course between superficial and deep targets. Thus, each contact was individually represented as a sphere and labeled by color according to the nearest neural structure. Using the T1-weighted image, FreeSurfer (rd.edu/) parcellard.edu/) and to cslwiki/) . We defislwiki/) . For morEach participant performed a series of force matching tasks with the arm contralateral to electrode locations resting on a pillow or tabletop. An instrumented pinch gauge and power grasp dynamometer were used to record exerted grasp forces . Hand orTwo participants (A and B) performed blocks where they only imagined making the desired force level without exerting any force on the sensor. These blocks were interspersed with the executed blocks to help the participants better imagine performing the desired forces. Visual cues were still given during these trials and the force sensor readings were monitored to make sure no forces were actually being applied.SEEG signals were split between the clinical Nihon Kohden system and the research recording system using two 64-channel touch-proof connector splitter boxes . The experimental setup used a Neuroport data acquisition system (Blackrock Microsystems) to record signals at 2 kHz from up to 128 channels depending on the participant. Electrodes were hardware referenced to a depth electrode contact in a different anatomical region of the brain that was not related to seizure generation. SEEG signals were also common average referenced by grouping contacts passing through similar tissue structures and on the same electrode to reduce the large amounts of ambient noise from the hospital equipment. Particularly noisy channels were excluded from the common average.The powers in six frequency features were extracted from each channel after preprocessing using a short-time Fourier transform in 1 Hz frequency bins with a temporal window size of 512 ms and an update of 50 ms. For each 1 Hz frequency band, power was converted to a z-score for normalization. The local motor potential (LMP)/Low-frequency content(LFC) was also calculated as a feature for comparison with frequency power features. The LMP was computed in this study using a 2nd order Savitzky-Golay filter with a 250 ms window similar to Pistohl et al. (2012). The aveTo determine statistically significant event related synchronization and desynchronization modulation peaks of each frequency feature, the maximum modulation/demodulation from baseline was found during a time window of -1 seconds to force onset. The maximum change was then compared against a baseline period of -3 to -1 seconds prior to force onset using a two-sample t-test with a very conservative p value of 0.002.p-values for multiple comparisons using the false discovery rate proposed by Benjamini and Hochberg [Cortical discrimination of grasp force states was assessed using a two-stage, cross-validated support vector machine (SVM) implemented with the libSVM package for Matlab . A time Hochberg . The falSeveral SEEG channels showed obvious modulation during force trials . Fig 3A We examined the amount of information the modulation of each frequency band in insular and motor electrodes conveyed for discrimination of force grasp states, through a support vector machine. For each grasp force trial, activity in each band was represented by the average power in a -400 ms to 400 ms window centered around force onset. This time window was chosen as it contained areas of greatest modulation in band powers as seen in We further investigated whether multiple grasp force levels could be discriminated beyond simply rest vs force. SVM classification of cortical modulation using sequential feature selection was not able to discriminate beyond chance between 10%, 20%, and 40% MVC for any participant or grasp configurations. Upon closer inspection of each participant\u2019s ability to correctly modulate their force level to the desired target, it was observed that when participants were instructed to execute 30% MVC grasp force the recorded force values often overlapped the 20% or 40% MVC levels. Thus we attempted state classification of just the light and hard force levels (20 and 40% MVC which had more separation) plus rest. We also examined to what extent insular and motor cortical bands modulated their activities during tasks of purely imagined grasp force production in two participants (A and B). The modulation during imagined force trials was significant enough to give above-chance classification of imagined move versus rest using sequential feature selection as can be seen in The goal of this work was to investigate whether SEEG electrodes could record neural activity from sulci and deep cortical structures related to hand grasp force. The results show that SEEG electrodes are able to record signals from the insular cortex and inside of central sulcus that correspond to motor activity. These structures are usually inaccessible to other invasive brain recording electrode technologies such as ECoG grids or standard 1\u20131.5mm penetrating microelectrodes. This is the first study to our knowledge to investigate whether SEEG signals taken from the sulci and insular cortex could be used to discriminate executed and imagined grasp force levels.Several cortical locations showed modulation when comparing force generation trials to resting trials. These structures included cortical tissue of the motor wall of central sulcus from the fundus all the way up to the top of the gyri, as well as, different portions of insular, premotor and sensory cortices. While there still is some debate about the relationship between electrophysiological and BOLD recordings from inside of sulci due to the presence of blood vessels, our results do correspond with several fMRI imaging studies which show that much of the BOLD signal increases for hand and finger movements occur inside of the central sulcus ,2. OtherThe modulation observed in the insular cortex of participant B was found predominantly in the alpha band for both pinch and power grasp configurations. This is in contrast to the motor contacts which showed modulation in almost all of the bands , the regSome channels that appear to be in white matter tracts recorded movement-dependent modulation. There is some level of inaccuracy when coregistering pre-op MRI and post-OP CT scans as well as automated grey matter parcellations so it is possible that these electrodes might actually be close enough to be recording from surrounding grey matter. Without fully knowing the recording distance of these SEEG contacts it iThe frequency features found to deterministically modulate during force versus rest trials were the high gamma bands and the beta band (12-30Hz). Participants B and D also exhibited modulated activity in the alpha band. When looking at force discrimination tasks and excluding sensory cortex from participant D, however, only high gamma modulation resulted in classification accuracy greater than chance. This is consistent with other published literature showing that high gamma bands carry movement specific information while beta band modulation is predominantly useful for discrimination of rest versus movement activity \u201346. The The peak in modulation can easily be seen in participant B\u2019s spectral traces in Both participants still showed significant levels of modulation in both motor and insular cortices during imagined force execution. During the imagined force trials, the magnitudes of modulation were much lower than during the executed trials. This decrease in modulation amplitude is consistent with surface ECoG results seen in Miller et al. (2010).Interestingly, the LMP was not able to achieve statistical significance of force versus rest classification. This is in contrast to several recent ECoG papers that showed LMP to be their most useful feature for classification of grasp forces ,27. PerhUsing only a single feature, several frequency bands across multiple contacts could be used to achieve classification accuracies well above chance for rest versus force classifications. It was not possible to achieve statistically significant classification of 3 force levels even using multiple features, however. Also, even though Figs The force targets chosen may have been too close to be differentiable using the field potentials and a larger gap in percentage of MVC might be necessary to achieve higher success in discrimination of force state. The force levels generated by the participants often overlapped with other force target levels especially when a participant would overshoot and undershoot before settling close to the target force level. There was also a lot of variability in the signals even during the same force tasks. This is most likely due to the fact that the magnitude of the signal modulations can vary considerably depending on the level of focus or attention given to the task as well A potential limitation of the current macro-contact depth electrodes is that the contact size may simply be too large to be able to distinguish between higher resolutions of motor control associated with smaller cortical networks. Kent and Grill (2014) modeled Even though the electrode locations varied between participants in this study due to clinical necessity, there was a large overlap in cortical regions that were sampled. All four participants had contacts placed in motor cortex, although participants A, B and C had electrodes passing through central sulcus and D had two electrodes passing through the gyrus, one in arm and hand area and the other in leg area. The fact that participant D\u2019s motor contacts passed through the gyrus rather than by the central sulcus might account for the reduced classification ability from these electrode contacts. Participants A, B and C all had electrode contacts passing through the posterior and anterior insular cortex as well. Participants C and D also had electrode contacts that passed through sensory cortical tissue. Looking at the power spectral density plots in The modulation shown on all participants in motor cortex and sensory cortex for participants C and D is consistent with numerous other studies. The recording of force-onset related signals from the insular cortex in participant B, however, have not been reported in the literature to our knowledge. Imaging studies have implied its involvement in hand movement and force tasks but since it is hard to reach with other intracranial electrodes, recordings from it have not been demonstrated previously. The desynchronization of activity in this region in the alpha frequency band could mean that the insular cortex is involved with releasing other networks from inhibition. The activity in different regions of the insular cortex could be related to different networks as well. It has been proposed that the posterior insular cortex is largely related to viscerosensory and somatosensory information while thDepth electrodes have the potential to be a new technology for investigation of neural activity from deeper brain structures in awake human participants. These electrodes allow for recording from deeper cortical structures such as the insular cortex and inside of the sulci which are difficult to approach using other invasive recording technologies. Our results corroborate with imaging studies that show force related modulation within these deeper structures, and specifically insular cortex. The current gold standard technology of silicon penetrating microelectrodes achieve fairly high levels of performance by recording individual neuron signals but only penetrate 1\u20131.5mm into the surface of the brain which would not be able to reach these deeper structures. While there are some reports of ECoG contacts implanted inside sulci ,54,55, iThe penetrating microelectrodes also have been shown to be prone to signal degradation over time \u201359. SEEGSubdural ECoG grids and penetrating microelectrode arrays both currently involve surgeries requiring craniotomies. SEEG placement is minimally invasive and requires only a minor stab incision and a small burr hole to access the intracranial space. Several studies have shown that SEEG implantation is at least as safe craniotomy for implantation of electrode arrays \u201369. CompFurther testing is necessary to determine whether more discriminable force signals can be attained using depth SEEG electrodes with smaller contacts with more specific targeting of cortical structures. Smaller contacts may provide better accuracies than shown here and in the ECoG studies. New combination macrocontact/microwire depth electrodes could potentially provide the best of both worlds: high information throughput from single-unit recordings but also the more stable recordings of population dynamics from the field potentials. Placements of electrodes in this study were limited to anatomic structures that required clinical monitoring. By using fMRI to pinpoint exact cortical regions that modulate with desired tasks prior to surgery, performance might be increased. Deep brain stimulation electrodes appear to be tolerated by neural tissue well enough for years of implantation and depth electrodes are similar in size and material. It will be necessary to show the long-term viability of depth electrodes for chronic implants for long-term neurophysiology studies or brain-machine interfaces (BMI) but other field potentials have been shown to be stable for long periods of time. This study also corroborates with imaging studies that show force-related activity occurs inside the central sulcus and insular cortex. SEEG electrodes can be used to record activity from these brain regions which are difficult to reach with any other intracranial electrode technology. While not able to provide significant discrimination of different force levels with the electrode contact setup used in this study, SEEG electrodes were able to show significant modulation in the central sulcus and insular cortex between force and movement. The possibility of SEEG electrodes being able to record from white matter tracts should also be investigated further. These regions could prove useful targets for future BMI hand grasp systems if force level discrimination could be shown in future studies. The ability to record signals related to force activities when force is not actually being applied is also important for BMI in paralyzed patients. Though this study was not able to discriminate different levels of force during imagery, it was able to show discrimination of force imagery versus rest. This is important to the field because these signals were recorded from intrasulcal motor cortex during motor imagery which, to our knowledge, has not been shown before. It also lends support to the idea that force related activity can be found in motor cortex and that the changes in modulation are not just artifacts of sensory information changes. In conclusion, SEEG electrodes already being placed clinically for epilepsy monitoring also provide a unique platform for advanced electrophysiological study of deeper brain tissues in a wide assortment of tasks beyond just grasp force generation, such as decision making, and should be utilized to advance our understanding of these deeper structures.S1 Text(DOCX)Click here for additional data file.S2 Text(DOCX)Click here for additional data file.S1 FigA/H = primary motor in arm/hand area, ML = primary motor in leg area, SA/H = sensory cortex in arm/hand area and SL = sensory cortex in leg area). Square electrode contacts are color coded to match the cortical structures they pass by as shown in Electrodes are organized by clinical location they targeted Click here for additional data file.S2 FigThis figure shows the rendered insular cortex for Participants A-C. Electrode contact locations are shown as spheres color-coded to the brain regions they are passing through . The insular cortex renderings are shown in yellow in order to make the locations of the purple insular contacts more apparent. The fMRI region of interest shown in Mutschler et al. 2009 with the(TIF)Click here for additional data file.S3 FigHere is an example from Participant A showing the common average referenced power spectral density for single contacts in 3 different brain regions . The top row shows the raw PSDs while the bottom row shows the z-score normalized change in PSDs. Red traces show average resting values while blue shows the average force trial values -400ms to +400ms centered on force onset.(TIF)Click here for additional data file.S4 FigHere are example spectrograms from participant B with frequency on the y-axis and time on the x-axis showing average trial modulation for three different contacts located in different brain regions . Spectrograms are the z-score normalized log powers across all force trials for each contact and the ranges are cutoff at a maximum of \u00b1 0.5. It can be seen that the largest modulation occurs at the beginning of the force trial during force onset (dotted black line) for motor cortex but last longer and are contained in lower frequency bands for the insula and white matter contacts.(TIF)Click here for additional data file."} +{"text": "Aspergillus EBFI not to result in respiratory failure.Endobronchial fungal infection (EBFI) is notoriously difficult to diagnose early since it may present few systemic features and does not cause characteristic parenchymal lesions on lung CT scanning. We report a 9-year-old girl who suffered extended neutropenia following graft failure after haematopoietic stem cell transplantation (HSCT) for severe aplastic anaemia. CT scan prior to retransplantation was normal despite persistent cough but lobar collapse was shown on repeat scan 16 days later. The probable diagnosis of EBFI (later proven on bronchoscopy) was only suspected when subsequent chest X-ray (CXR) demonstrated lack of an air bronchogram in the partially collapsed lung. Early radiological suspicion resulted in multiagent antifungal therapy followed by delayed lobectomy, and led to this being the first reported case of Aspergillus fumigatus infection through bronchoscopy, and definitive curative treatment with lobectomy. Early radiological investigations had been normal in this symptomatic patient. We discuss the value of repeat imaging and a high index of diagnostic suspicion in this underrecognized and underreported disease entity [Endobronchial fungal infection (EBFI) is a rare form of fungal invasion that is initially localized to the bronchial tree . There ae entity .A 9-year-old girl was admitted to the Bone Marrow Transplant Unit for a second haematopoietic stem cell transplantation (HSCT) procedure for SAA from her HLA-matched sister. Her blood count had returned to normal after her first transplant but she became pancytopenic again following a primary parvovirus infection 18 months after initial transplantation. This resulted in severe neutropenia for six months prior to admission whilst awaiting viral clearance. She was maintained on prophylactic cotrimoxazole and itraconazole throughout this period.Prior to admission the patient had been nonspecifically unwell for a number of weeks, with a persistent cough, but was afebrile with a CRP of less than 10\u2009mg/dL. Due to these symptoms she was thoroughly investigated on admission. Echocardiogram, lung function tests, and CT scan were all normal (the latter shown in She remained well in herself (apart from her mild cough) until day 5 after transplant when she became febrile. Antibiotics were administered but pyrexia worsened and CRP rose to 133\u2009mg/dL on day 7 after transplant was added to caspofungin and an increased dose of liposomal amphotericin (5\u2009mg/kg) was used.Dyspnoea progressed and a CXR on day 11 after HSCT identified progressive lung collapse with lac Aspergillus fumigatus was grown in culture. The patient remained afebrile but with a persistent CRP between 20 and 40 and was therefore commenced on additional nebulized amphotericin on day 38. Her CRP fell to 12 on this regime by day 48.The patient initially exhibited a good response to triple antifungal therapy (pyrexia and dyspnoea resolved), supported by a rising neutrophil count . However Aspergillus infection five years later.Whilst left lower lobe atelectasis resolved, the upper lobe collapse remained and lobectomy was required subsequently. This entailed a complex, eight-hour procedure which involved extensive bronchovascular dissection because of adherence of nodes to the hilar structures including opening of the pericardium and the left main bronchus. The patient remained well and free of Aspergillus infection which is the leading cause of EBFI, accounting for 53% of cases in a 238-case review [EBFI predominantly affects severely immunocompromised patients such as those undergoing solid organ transplantation or HSCT or receiving cytotoxic chemotherapy , 5, 6. Te review . This pre review .The potential for EB aspergillosis to progress rapidly to respiratory failure is depicted by previously reported cases \u20134. FurthIn EBFI the classic \u201chalo\u201d and \u201ccrescent\u201d signs of invasive parenchymal aspergillosis will not show on CT scans due to the intraluminal nature of the infection . HoweverAnecdotally, during our discussions of this case with members of the HSCT community, we have found that many senior transplant professionals are unaware of the existence of occlusive EBFI and its difficulties of detection on CT scanning. This is crucial when CT has become the gold standard investigation for fungal infection in HSCT patients. Bronchoscopy is now much less widely used than previously in HSCT patients because of the advent of PCR viral detection systems for throat swabs/sputum analysis and because of the ease and efficacy of high-resolution CT scanning. This case demonstrates that it is still crucial to consider bronchoscopy if these investigations are negative but the patient is persistently febrile and/or develops respiratory signs/symptoms.This report illustrates the nonspecific clinical presentation of EBFI and therefore the high index of suspicion required to diagnose these patients. Radiological suspicion played a pivotal role in rapid escalation of antifungal therapy; it was almost certainly this which prevented progression to respiratory failure and ventilation and enabled a curative outcome for this patient ."} +{"text": "Systematic investigations into the role of semantics in the speech production process have remained elusive. This special issue aims at moving forward toward a more detailed account of how precisely conceptual information is used to access the lexicon in speaking and what corresponding format of conceptual representations needs to be assumed. The studies presented in this volume investigated effects of conceptual processing on different processing stages of language production, including sentence formulation, lemma selection, and word form access.Ganushchak et al. show that contextually new referents are fixated with priority over contextually old referents. The time course of the contextual effects on gaze patterns suggests that contextual information might well be taken into account during sentence formulation. Hsiao et al. present data from a sentence production task and a corpus study that show that speakers of Mandarin Chinese are more prone to omitting subject pronouns in their utterances when the subject and object of the sentence are conceptually similar than when they are conceptually dissimilar.Using an eye-tracking paradigm in which participants are prompted to describe pictures of two-character transitive events, Harvey and Schnur investigated semantic interference in picture naming and word\u2013picture matching. Using a blocked-cyclical paradigm they show that semantic interference in naming generalizes to novel objects, but semantic interference in word\u2013picture matching does not. This is taken as evidence that semantic interference effects in naming and word\u2013picture matching arise at different processing stages. Naming novel items that corresponded to semantic categories that had been previously encountered in word\u2013picture matching induced semantic interference. The latter result suggests a common origin of semantic interference across tasks.The majority of studies aimed at gaining further insights into classic distractor effects. B\u00f6lte et al. investigated the origin of semantic interference effects in the picture\u2013picture paradigm. Participants named pictures of German compound words which were accompanied by categorically or associatively related distractor objects. Categorically related distractors facilitated naming at SOAs at which semantic processing is expected (in this case +200). The authors argue that the absence of semantic interference means that such distractors activate their conceptual-semantic information but do not activate the corresponding lemma.Vieth et al. investigated semantic interference from distinctive features. Their first experiment showed no evidence that distractors that differed from target items on a distinctive feature were processed differently from semantically matched distractors with no distinctive feature differences . Further experiments showed that distractors denoting visible parts of target objects that are also found in other objects slowed down naming of target items. The authors argue that this reflects competition from semantically related items .Damian and Spalek used a picture\u2013word-interference paradigm with distractors that were either unrelated, categorically related, associatively related, or both categorically and associatively related. In addition the authors manipulated the visibility of distractors by presenting them in between forward and backward masks. Results replicate earlier or competitive accounts .Von Holzen and Mani show that bilinguals implicitly generate labels for pictures simultaneously in their first and second languages. Targets preceded by phonologically related pictures showed lower N400 effects irrespective of whether the phonological relationship was within or between languages. This implies that the non-selected (non-target language) lemma can send activation cascading forward to the phonological level. Correia et al. studied the reverse flow of activation. Using multivariate pattern analysis of EEG data, they show that in bilingual listeners language invariant semantic representations can be decoded around 550 ms following the onset of a spoken word.Two studies investigated relationships between conceptual and word form activation in bilingual speakers. Mulatti et al. show that white noise interferes with naming pictures of objects with typical sounds but not with objects without typical sounds. This suggests that an object's sound attribute is used during lemma retrieval. Lloyd-Jones and Nakabayashi examined the retrieval of object color information using a picture naming and semantic matching task. Their results suggest differential retrieval of color information for object names and object shapes.Finally, two studies investigated the role of attribute retrieval in naming. It becomes clear in this volume that effects of conceptual processing extend beyond the conceptual level and can affect many levels of processing. The range of conceptual relationships that are explored is just beginning to be expanded beyond categorical and associative relationships.All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.This research was funded by the Deutsche Forschungsgemeinschaft (DFG) Collaborative Research Centre (CRC) 991.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Neonates with out-of-hospital cardiovascular emergencies get to Emergency Department or Neonatal Intensive Care Unit in a state of shock, which is a complex clinical syndrome characterized by an acute failure of the circulatory system with inadequate tissue and organ perfusion.It has been demonstrated that early recognition and time-sensitive aggressive resuscitation of neonatal shock significantly reduce mortality and improve outcomes .2[The update algorithm for goal-directed treatment of neonatal shock emphasizes early use of therapies directed to restore threshold heart rates, normalize blood pressure and capillary refill \u22642 seconds and subsequent intensive hemodynamic support aimed to goals of central venous oxygen saturation (ScvO2) >70% and cardiac index (CI) >3.3 L/min/m2.Rapid attainment of a vascular access is pivotal for fluid resuscitation and inotrope therapy and an intraosseous needle must be early inserted especially in the case of cardiac arrest or decompensated shock .Rapid crystalloid boluses of 10-20 ml/kg (over 5-10 minutes) up to 60 ml/kg in the first hour must be administered paying attention to signs of fluid overload because overaggressive therapy can be detrimental in case of cardiogenic shock. An echocardiographic analysis can help clinicians to assess the inferior vena cava diameter or collapsibility as an index of preload status and to eEmpiric antibiotics should be administered within one hour if a sepsis is suspected .Neonates with fluid refractory shock need to start and titrate a peripheral inotropic support and progressively to be added vasopressors according to the hemodynamic state.Many methods are available for the hemodynamic monitoring after the first hour of stabilization of the neonate. A safer ultrasound-guided central venous access can easily be obtained and ScvOMany aspects of the intensive hemodynamic support of neonatal shock resemble those of paediatric critical care medicine (PCCM) usually provided by anaesthesiologists.We believe that neonatologists need a specific PCCM training before dealing with critically ill neonates and for this purpose our team has devised some courses Figure to meet"} +{"text": "The incidence of human immunodeficiency virus (HIV) infection has significantly increased among black men who have sex with men (MSM) in the United States, and young black MSM have been disproportionately affected . HIV-infPartner services include a broad array of medical , prevention , and psychosocial services provided to persons diagnosed with HIV infection and their partners. One critical function of partner services is partner notification, a process routinely used in the prevention and control of sexually transmitted diseases (STDs), including HIV. Persons infected with HIV are interviewed to elicit information about their partners and social contacts who can then be confidentially notified of their possible exposure to or potential risk for HIV infection . PartnerScreening Targeted Populations to Interrupt Ongoing Chains of HIV Transmission with Enhanced Partner Notification (STOP) is a prospective study evaluating acute HIV infection diagnosis linked to partner services at 12 HIV testing sites in North Carolina; New York, New York; and San Francisco, California . Participol) gene sequences of newly diagnosed HIV-infected persons were analyzed with standard phylogenetic techniques and there was high statistical support in phylogenetic analyses (bootstrapping >99% and posterior probabilities =1.0) to suggest the sequences were highly related compared with local controls. This analysis was limited to a subset of black MSM tested in the STOP study in North Carolina for whom HIV-1 pol sequences were available.For partner services, HIV-infected participants (index patients) were offered notification services. Contact information was elicited for sex partners from the previous 3 months for index patients with acute HIV infection and the previous 12 months for index patients with established or previously diagnosed HIV infection. In addition, contact information was elicited for social contacts considered by the index patient to be at high risk for HIV infection . Health department personnel trained as disease intervention specialists contacted sex partners and social contacts and used Internet-based communication and text messaging when available. Sex partners and social contacts were offered HIV testing. HIV status was defined as 1) previously diagnosed HIV infection, 2) newly diagnosed HIV infection, 3) HIV-negative (HIV testing during partner services was negative), or 4) HIV status unknown (could not be located or refused testing). HIV polymerase . The 30 index patients named 95 persons , of whom 39 (41%) previously had been diagnosed with HIV infection, including 14 who had been diagnosed within the most recent year and 17 who were aged <25 years. An additional 29 (31%) of the 95 named sex partners and social contacts accepted an HIV test, and two sex partners were newly diagnosed with HIV infection. Of the remaining sex partners and social contacts, eight refused HIV testing, eight refused any partner services counseling, and 11 could not be located. Most sex partners and social contacts were male (98%) and black (81%), with a median age of 26 years who had a reactive HIV test result and an available HIV-1 26 years . Sex parpol gene sequences were available for the 30 index patients, but not for their 95 sex partners and social contacts. Although none of the 30 index patients named another index patient as a sex partner, phylogenetic analyses identified four highly supported clusters involving eight (27%) index patients (two men per cluster). The sexual network and molecular phylogenetic data were combined for each of these four clusters. Based on data collected during April 2012\u2013April 2013, the largest of the resulting networks included 23 black MSM connected by 20 sexual relationships, one social contact, and one molecular phylogenetic link infection has significantly increased among black men who have sex with men (MSM) in the United States, and young black MSM have been disproportionately affected. Previous studies have demonstrated that black MSM have risk behaviors similar to MSM of other racial and ethnic groups but are more likely to have an HIV exposure within their sexual network.What is added by this report?Among black MSM who received partner services in North Carolina, a high proportion (41%) of sex partners and social contacts had been previously diagnosed with HIV infection, whereas only 2% of partners were newly diagnosed with HIV infection. Based on sexual and social network and molecular phylogenetic data, a representative partner network demonstrated that HIV-infected and HIV-negative partners were frequently in the same network and that the majority of HIV-infected partners were already aware of their diagnosis but had not achieved viral suppression.What are the implications for public health practice?Diagnosing persons unaware of their HIV status provides a potential opportunity to reengage HIV-infected partners already aware of their status in medical care. This public health intervention might be particularly important among young black MSM in an HIV transmission network, who are disproportionately affected by new HIV infections and less likely to maintain sustained access to HIV medical care.Partner notification is an important opportunity to diagnose persons who are unaware of their HIV infection. This report illustrates that partner notification can also be an important opportunity to identify and link to care HIV-infected partners who are aware of their diagnosis but have not achieved viral suppression. This public health intervention might be particularly important among young black MSM, who are disproportionately affected by new HIV infections and less likely to maintain sustained access to HIV medical care . Among bpol sequence that clustered by molecular phylogenetic analysis with a person who was not reported as a sex partner. This finding is consistent with the named sex partner and social contact characteristics, which were relatively homogeneous with respect to age (predominantly young), race/ethnicity (81% black), and geography (91% from North Carolina). This degree of clustering and homogeneity suggests that sexual networks among black MSM in North Carolina are highly connected, and that HIV prevention efforts targeting persons in a central sexual network location might result in substantial decreases in HIV transmission.Previous studies have demonstrated that black MSM have risk behaviors similar to MSM of other racial and ethnic groups but are pol sequence were excluded, which might exclude men who are less likely to link to medical care. However, the results are consistent with national statistics demonstrating high rates of incident HIV infections among black MSM within these HIV transmission networks engage in HIV medical care. Interventions for HIV-infected and HIV-negative partners could have a substantial impact on transmission within these networks, improving the HIV continuum of care among black MSM and reducing the number of new infections."} +{"text": "Juvenile Dermatomyositis (JDM) is the most common idiopathic inflammatory myopathy in childhood, a systemic vasculopathy that usually affects skin and skeletal muscle but also can affect gastrointestinal tract and other organs. Diagnosis is based on Bohan and Peter's criteria and the goal of treatment includes control of skin and muscle symptoms and prevent disease complications. Stepwise aggressive treatment decreases JDM activity and improves long-term outcome.Role of magnetic resonance imaging (MRI) as a non invasive tool to asses muscle edema have increased last years with a diagnostic sensitivity being 76%, compared with 64% for creatine kinase and ability to distinguish between active JDM patients and inactive healthy children. Attempt to establish MRI scores have been done whether evaluating thigh muscle groups or whole body MRI.To evaluate muscle edema on arm and tight MRI of our JDM patients after and before treatment.We made a descriptive retrospective review of our JDM patients with muscle MRI before and after treatment. Muscle edema was assessed independently by two radiologists on four arm and thigh muscles of both sides on a 4-point-score before and after treatment. We also scored the presence of fascia and soft-tissue edema .8 patients were included. All have pathological MRI before treatment. We did not find differences between each side. The mean scores for each muscle, arm and tight before and after treatment are given in table 1 and 2 respectively. We did not find differences between arm and tight muscle edema if we compared whole means but individually supraspinatus, infraspinatus and gluteals were the most affected muscles. Almost all muscle return to normal signal on MRI after treatment and the ones which still remained slightly pathological were the most affected before treatment. Fascia and soft tissue edema disappeared after treatment.MRI is an important not invasive tool to evaluate muscle edema in JDM patients. Our results, as others have suggested in the past, shown that tight MRI could be enough for studying JDM patients at diagnosis. Whole body MRI seems promising not only for diagnose but also for follow up and as an outcome tool in these patients.None declared."} +{"text": "Obesity is associated with increased risk of death that may be related to underlying structural and functional changes in the heart. Cardiac MR (CMR) can provide detailed structural and functional information about the heart and vasculature that may improve our understanding of obesity related cardiac morbidity and mortality.A cohort of 77 obese patients referred for stress CMR for evaluation of dyspnea prior to weight-loss surgery (WLS) were identified from our research registry. All patients underwent standard vasodilator stress perfusion protocol with late gadolinium enhancement (LGE) imaging on a 1.5T MR system. We collected demographic and CMR data including ventricular volumes, global and regional functional indices, and results of stress and LGE imaging. Comparison of CMR was made to previously derived and universally accepted normative data. The goal will be to assess these parameters 1 year post WLS and analyze the structural, functional and microvascular changes.Please see table Multiple cardiovascular abnormalities are noted in severely obese patients including LV and RV dilation and microvascular ischemia that may contribute to the increased cardiovascular morbidity and mortality observed in this population.Empire Clinical Research Investigator Program (ECRIP)."} +{"text": "The prevalence of Coronary artery disease (CAD) in patients with peripheral vascular disease (PVD) varies widely from 28% to 94% in published reports.We share our experience with a concomitant procedure for coronary artery disease and peripheral vascular disease using an artificial conduit from ascending aorta to peripheral vessel in a single sitting.Records of 41 patients who underwent cardiac and peripheral vessel revascularisation between January 2009 and January 2014 were retrospectively analysed. All patients had diseased abdominal aorta with claudication pain and non-healing ulcer over the foot and a coronary angiogram showing either a triple vessel disease (27) or a double vessel disease (14). All patients underwent coronary artery bypass grafting and aorto-bifemoral grafting in a single sitting.Post-operative Doppler study showed good peripheral blood flow in all patients. Patients were relieved from rest pain and non-healing ulcers were converted to healing ulcers and limb salvage was possible in all cases. Four patients had pericardial effusion due to weeping of graft, which was drained with the help of pig tail catheter. Two patients had serous collection at the inguinal site which required drainage. These complications did not compromise the hemodynamics of the patient in the form of cardiac tamponade or limb ischemia.Single sitting for CAD and PVD revascularization has reduced morbidity, is easy to perform, cost effective and reduced hospital stay."} +{"text": "An important discussion at colleges is centered on determining more effective models for teaching undergraduates. As personalized genomics has become more common, we hypothesized it could be a valuable tool to make science education more hands on, personal, and engaging for college undergraduates. We hypothesized that providing students with personal genome testing kits would enhance the learning experience of students in two undergraduate courses at Brigham Young University: Advanced Molecular Biology and Genomics. These courses have an emphasis on personal genomics the last two weeks of the semester. Students taking these courses were given the option to receive personal genomics kits in 2014, whereas in 2015 they were not. Students sent their personal genomics samples in on their own and received the data after the course ended. We surveyed students in these courses before and after the two-week emphasis on personal genomics to collect data on whether anticipation of obtaining their own personal genomic data impacted undergraduate student learning. We also tested to see if specific personal genomic assignments improved the learning experience by analyzing the data from the undergraduate students who completed both the pre- and post-course surveys. Anticipation of personal genomic data significantly enhanced student interest and the learning environment based on the time students spent researching personal genomic material and their self-reported attitudes compared to those who did not anticipate getting their own data. Personal genomics homework assignments significantly enhanced the undergraduate student interest and learning based on the same criteria and a personal genomics quiz. We found that for the undergraduate students in both molecular biology and genomics courses, incorporation of personal genomic testing can be an effective educational tool in undergraduate science education. Finding promising teaching practices for improving undergraduate science education is an important issue and evidence suggests a beneficial way to do this is to increase hands on experiential and active learning by working on real world issues and technologies \u20133. StudeResearchers recently found that incorporating personal genome testing in the medical school classroom enhanced the learning experience of students on multiple levels. They concluded personal genome testing and using personal genotype data in the classroom enhanced both students\u2019 self-reported and personal genomics based quiz scores . We hypoWe determine to test the hypothesis that anticipation of getting personal genome data increases student motivation in the subject and student learning by making the topic more relevant and personal. This was tested in two senior-level science courses at Brigham Young University [Advanced Molecular Biology (MMBIO 441) and Genomics (MMBIO 468)]. Of particular note, we found that student anticipation of obtaining their personal genomic data results in 1) increased self-reported motivation and learning based on survey responses, and 2) increased reported time working on personal genomics products.https://www.23andme.com/academic/) at an academic price using funds from a Brigham Young University Teaching enhancement grant. In the winter semester of 2014 there were a total of 84 students who completed both the pre and post surveys and received personal genomics kits (42 students in Advanced Molecular Biology and 42 students in Genomics) and in the winter semester of 2015 there were 71 students who completed both the pre and post surveys and did not receive personal genomics kits (40 students Advanced Molecular Biology and 31 students in Genomics).Subjects were undergraduate students enrolled in two senior-level undergraduate science courses at Brigham Young University [Advanced Molecular Biology (MMBIO 441) and Genomics (MMBIO 468)] offered in the Winter 2014 and 2015 semesters. The classes had approximately 40\u201350 students each and met three times per week for 50 minute time periods. All of the students enrolled in these two courses had previously completed an introductory molecular biology course (MMBIO 240). Students in the courses taught during the 2014 semester were given access to demo data sets and the option to receive personal genomics kits, whereas students in the 2015 semester were given access to demo data sets, but were not given kits to measure their own genomic data. Any students who had previously undergone personal genomics testing or did not complete both the pre and post surveys were not included in the data analysis. The personal genomics kits were purchased from 23andMe described how the testing assays work and what can and cannot be concluded from this type of data. The instructors also demonstrated how demo genomic data could be accessed using an online demo data set provided by 23andMe. This demo data included genomic data for an anonymous family (Mendel family) as well as 12 diverse anonymous individuals. Students were given a packet with instructions on how to obtain access to these demo data sets as well as a step-by-step packet on analysis of Alzheimer\u2019s disease. In the Advanced Molecular Biology course, completing this packet was optional for students, whereas in Genomics completion of this packet was coupled with a required homework assignment.At the start and conclusion of the two-week personal genomics emphasis, we administered a survey to measure the student attitudes and understanding of personal genomics and personalized medicine. These pre and post surveys were adapted from a survey used in the Stanford Medical Student study and assessed attitudes and knowledge about personal genomics testing and the classroom learning experience of the students during this two-week personal genomics emphasis in the courses . AssessmWe analyzed responses from students who completed both the pre and post surveys and had not had their personal genome analysis done previously. Students who were enrolled in both courses and took the pre and post surveys had their responses counted only once. Student attitudes were assessed on a 5-point Likert scale and data comparisons between kit and no kit for Likert questions were performed using a Mann-Whitney U-test. Comparisons of student binary survey data and change in student hours were analyzed comparing kit and no kit responses using an independent-samples t-test. Comparisons of pre- and post-survey data were evaluated using a paired t-test. The difference in improvement between assignment and no-assignment students on a personal genomics quiz was assessed by an independent-samples t-test. All statistics were performed using Prism 6 software (GraphPad).Eighty-four students completed both the pre and post surveys and received personal genomics kits (42 students in 2014 Advanced Molecular Biology course and 42 students in the 2014 Genomics course) and 71 students completed both the pre and post surveys and did not receive personal genomics kits (40 students in the 2015 Advanced Molecular Biology course and 31 students in the 2015 Genomics course). All of these students were undergraduates taking the Advanced Molecular Biology or Genomics courses at Brigham Young University during winter semester of 2014 or winter semester of 2015.As part of our study we surveyed student attitudes towards personal genome testing after our two-week focus on personal genomics. We asked students (Y/N) if they were to undergo personal genomics testing would they ask a health care provider for help in interpreting the results. We found that those who had submitted their personal genome kits and were anticipating receiving their data were significantly less likely to answer that they would ask a health care provider for help interpreting their results .Students were asked to rate their confidence on a number of topics regarding their attitudes towards personal genotyping analysis and regulation using a 5-point Likert scale . Those sStudents from both groups were asked about the ability of others to interpret personal genomics results and had similar levels of confidence in the abilities of physicians, other people, and personal genomic companies . We then calculated the change in hours (increase) between the pre and post surveys and compared the increase in hours of students who did not have the kit with those who did. When we did this we found that those that were given the kits spent significantly more time using personal genomics products, almost 2 hours more, than students who were not anticipating getting their own personal genomics data ; p<.01. As part of our study, we had one course (Genomics) that gave a specific personal-genomics assignment using the online, personal-genomics demo data for students to complete; whereas the other course (Advanced Molecular Biology) gave students an optional packet to guide them through how to access the online personal genomics demo profiles. This allowed us to evaluate the individual impact of kits and homework on the number of hours student spent researching personal genomics. When looking at the hours spent using personal genomics products, those that did not receive a kit or a graded assignment did not have significant differences in hours between pre and post surveys for each of these groups and found that having an assignment did not significantly change the number of hours spent for those who did not receive a kit, but did significantly increase the hours spent by those that received a kit ; p<.05. As mentioned above, the Genomics course had a required online personal-genomics homework assignment and the Advanced Molecular Biology had an optional packet outlining how to analyze online demo genomic data. This allowed us to evaluate the individual impact of kits and homework on student scores on the personal genomics quiz given during the pre and post surveys. We found that when students did not have the required assignment (with or without a kit), student scores significantly increased from the pre to post survey ; p<.01. We report here the first study, to our knowledge, to evaluate the benefits of incorporating personal genomics kits into an undergraduate science course and testing the effect of anticipation of obtaining personal genomic data on student interest and learning environment. Our results suggest that integration of personal genomics into undergraduate classrooms can be a valuable means of improving student interest and the learning environment when teaching the concepts of molecular biology and genomics. At the end of the course our students reported that anticipation of getting their personal genomics data had enhanced their learning experience, time spent learning about personal genomics, and overall attitude about the course in an undergraduate classroom.In this study we tested the prediction that student anticipation of obtaining their personal genomics data enhances student interest and learning experience. Analysis of our data revealed that student anticipation of getting their own personal genomic data did increase student interest and the learning environment based on student reports and increased their reported time spent working on personal genomics. We also found anticipation of analyzing their own personal genomics data coupled with homework assignments resulted in even greater interest compared to students without homework.While incorporating personalized genomics into the classroom can be a valuable educational tool, it also comes with a number of potential ethical issues that can be problematic. For example, the highest profile problem with personalized genomics and undergraduate education occurred in 2010 when U.C. Berkley started a \u201cBring your Genes to Cal\u201d program for incoming freshman and transfer students that gave them the opportunity to submit their DNA for genetic analysis . InitialAn important part of our study is that our students submitted their own samples and did not actually receive their personal genomic data until the course had ended. No one besides the student had access to his or her personal genomic data. While integrating analysis of personal genomics data as a part of the course could be a valuable way to enhance learning, it also produces additional classroom privacy issues that would need to be addressed . ImportaUpon completion of the two-week personal genomics focus, we found that students who were anticipating analyzing their data had spent more time using personal genomics products. This likely played a role in the increased confidence that these students had in their ability to interpret their personal genome results. We also found that these same students reported less support for health care providers having their data and less agreement that personal genome testing companies should be regulated. Our students also self reported being more interested in the class topics, spending more time studying and learning, feeling like the course was more applicable, and enjoying the learning experience more. We also examined the effects of homework with and without the students receiving the kit. A required homework assignment increased the hours spent using personal genomics products as well as learning based on scores on the personal genomics quiz Figs and 6C, This study has a large sample size and students from multiple classes, but does have several limitations when broadly generalizing these findings. All of this work was done at a single institution and it will be interesting to see other institutions examine student anticipation of personal genomics in enhancing learning and interest in molecular biology and genomics. These limitations notwithstanding, our study represents the first evidence that undergraduate student anticipation of receiving personal genomics data can enhance learning and interest. As personal genomics is more routinely incorporated into classroom education, additional randomized studies at multiple institutions can further enhance our understanding of what effect personal-genomics testing has upon undergraduate student learning.We believe it is critical for undergraduate science educators to find practical and engaging ways to improve science education and personal genomics represents a current and powerful means of doing this. Personal genomics provides a valuable method of incorporating discussions and critical thought about rapidly evolving medical, ethical, and privacy issues that currently confront society and will help prepare our students to contribute to future policy discussions. Although further analysis of the value of using personal genomics in undergraduate education is necessary, we believe it is a powerful educational resource that should be thoughtfully incorporated into molecular biology and genomics education.S1 FigStudent responses to Question #2 regarding whether they would share their results with a physician. There were four answer options and percent response for each option is displayed. .(EPS)Click here for additional data file.S2 Fig(PDF)Click here for additional data file."} +{"text": "Improving medication adherence across the health care system is a vital component to improving patient outcomes and reducing downstream health care costs. Research suggests that pharmacists can be a highly effective tool for improving medication adherence when equipped with knowledge and skills on conducting adherence screenings and interventions.The objective of this large scale pharmacy demonstration study is to evaluate the impact of a pharmacy-based intervention on adherence to five chronic medication classes.283 pharmacists from a national community pharmacy chain were assigned to the intervention group. Collectively, they screened 29,042 patients for poor adherence risk and provided brief interventions to individuals with an elevated risk. Results from these screenings and interventions were compared to those of a control group consisting of 295 pharmacists who screened 30,454 but did not provide any brief interventions.Patients in the intervention group significantly improved adherence for all medication classes. Adherence for oral diabetes medications improved 4.8 percent. Adherence for beta-blockers improved 3.1 percent. Additionally there was a significant reduction in per patient annual health care spending for patients taking statins ($241) and oral diabetes medications ($341).This study demonstrated that interventions are a cost-effective tool that can be applied in health care sites across the country. Furthermore, pharmacies provide a yet untapped source of health care professionals who can perform screening and brief interventions with patients."} +{"text": "The cardiac natriuretic peptides ANP and BNP stimulate adipocyte metabolism to increase lipolysis of triglycerides to free fatty acids, as well promote brown adipocyte energy expenditure through uncoupled mitochondrial respiration. Energy expenditure in brown adipocytes holds potential for improving cardiometabolic disease by consuming glucose and fatty acids and thereby improving insulin sensitivity. This presentation will describe studies in mouse models lacking the natriuretic peptide \u2018clearance receptor\u2019 Nprc (gene = Npr3) in adipose and other tissues, as well as more clinically oriented studies in human subjects examining a role for the natriuretic peptide system in insulin sensitivity and energy expenditure. We will also describe novel signal transduction mechanisms downstream of PKA and PKG that promote the increase in brown adipocytes within white adipose tissue and their metabolic activation."} +{"text": "A 42-year-old male with a history of multiple shoulder dislocations presented to the emergency department via emergency medical services with both arms locked above his head, stating that he had been jumped at a bar and had since been unable to move his arms. A single anteroposterior chest radiograph demonstrInferior shoulder dislocations are the rarest of all glenohumeral joint dislocations, accounting for less than 1%; and bilateral inferior dislocations are therefore extremely infrequent. The classic presentation is the patient whose arm is locked above his head in a hyper-abducted fashion. The inferior dislocation must first be converted to an anterior dislocation by slowly adducting the arm. The anterior dislocation can then be reduced using routine maneuvers.In our case the patient underwent reduction with propofol sedation and was placed in bilateral shoulder slings. He was observed overnight by the orthopedic service for ethanol metabolism and was safely discharged home the next day in bilateral arm slings."} +{"text": "The utrophin-dystrophin deficient (DKO) mouse model has been widely used to understand the progression of Duchenne muscular dystrophy (DMD). However, it is unclear as to what extent muscle pathology affects metabolism. Therefore, the present study was focused on understanding energy expenditure in the whole animal and in isolated extensor digitorum longus (EDL) muscle and to determine changes in metabolic enzymes. Our results show that the 8 week-old DKO mice consume higher oxygen relative to activity levels. Interestingly the EDL muscle from DKO mouse consumes higher oxygen per unit integral force, generates less force and performs better in the presence of pyruvate thus mimicking a slow twitch muscle. We also found that the expression of hexokinase 1 and pyruvate kinase M2 was upregulated several fold suggesting increased glycolytic flux. Additionally, there is a dramatic increase in dynamin-related protein 1 (Drp 1) and mitofusin 2 protein levels suggesting increased mitochondrial fission and fusion, a feature associated with increased energy demand and altered mitochondrial dynamics. Collectively our studies point out that the dystrophic disease has caused significant changes in muscle metabolism. To meet the increased energetic demand, upregulation of metabolic enzymes and regulators of mitochondrial fusion and fission is observed in the dystrophic muscle. A better understanding of the metabolic demands and the accompanied alterations in the dystrophic muscle can help us design improved intervention therapies along with existing drug treatments for the DMD patients. Ho. Ho7]. HIn comparison to glucose as a substrate, the DKO EDL exhibits greater potentiation of force when pyruvate is used as a substrate. This preference in substrate utilization is intriguing since CLAMS data indicate that the DKO and WT mice have similar RER suggesting no alteration in terms of whole body fuel utilization. Previously this potentiation in response to pyruvate has been observed in soleus but not in a fast twitch glycolytic muscle like EDL . It has mdx mice that there is an inefficiency in energy production by mitochondria [mdx mice suggest that the higher metabolic needs of DKO mice as indicated by increased oxygen consumption relative to work done, is most likely due to a combination of inefficiency in energy production and altered distribution of mitochondria [An exciting finding of this study is that proteins involved in fusion and fission of mitochondria were upregulated several fold in DKO muscles. Expression of mitochondrial fusion regulator, Mfn 2, is often associated with states of increased energy expenditure like exercise and cold induced thermogenesis , 51 and chondria . Hence uchondria . High lechondria \u201355. An ichondria and causchondria . The dyschondria , 58\u201359 achondria .The inefficiency in oxidative phosphorylation along with the constant damage and repair that is seen in DKO muscle places a tremendous metabolic stress on these muscles. In such a situation the diseased muscle would have to rely on increased glycolysis to meet its energy demands. In this regard earlier studies in DMD patients show alterations in HK 2 levels \u201361; an iIn summary this study shows for the first time the impact of muscular dystrophy on whole body energy expenditure and its effect on muscle performance, especially on fast twitch glycolytic muscle. Collectively our data and published studies , 9, 42 s"} +{"text": "Recently, we provided human validation for a cooperating oncogenic role for cAMP in brain tumorigenesis when we found that SNPs in ADCY8 were correlated with glioma (brain tumor) risk in individuals with Neurofibromatosis type 1 (NF1). Together, these studies provide a strong rationale for targeting cAMP in brain tumor therapy. However, the cAMP pathway is well-known to be sexually dimorphic, and SNPs in ADCY8 affected glioma risk in a sex-specific fashion, elevating the risk for females while protecting males. The cAMP pathway can be targeted at multiple levels in the regulation of its synthesis and degradation. Sex differences in response to drugs that target cAMP regulators indicate that successful targeting of the cAMP pathway for brain tumor patients is likely to require matching specific mechanisms of drug action with patient sex.A relationship between cyclic adenosine 3\u2032, 5\u2032-monophosphate (cAMP) levels and brain tumor biology has been evident for nearly as long as cAMP and its synthetase, adenylate cyclase (ADCY) have been known. The importance of the pathway in brain tumorigenesis has been demonstrated Soon after its discovery, it became clear that cAMP was a ubiquitous second messenger and that normal physiology was dependent upon precise regulation of its synthesis and degradation. As a corollary, investigations rapidly ensued to determine whether pathology was associated with dysregulation of cAMP levels. In 1971, multiple studies were published that associated differences in cAMP levels with differences between normal and cancer cells pathway , and the PAC1 ligand, Pituitary Adenylate Cyclase Activating Peptide (PACAP), have increased incidence of medulloblastoma compared to mice with Ptc deficiency alone and is negatively correlated with survival and this mechanism has been correlated with tumor biology and therapeutic responses. In diffuse large B cell lymphoma, decreased mir-124 expression led to increased PDE4B expression and subsequent insensitivity to glucocorticoid treatment exhibit significantly different levels of GTP loading of G\u03b1i despite equal expression of CXCR4 . When treated with CRF, female neurons exhibit both greater cAMP elevation and greater PKA activation compared to male neurons. This has been proposed to be the consequence of greater coupling between G\u03b1s and the CRF receptor in female neurons increase glioma risk in female patients with NF1, but are protective against glioma in male patients in alcohol dependence array analysis of polymorphisms in the cAMP pathway with DNA from individuals with NF1 with and without optic pathway gliomas revealed that polymorphisms in Nf1\u2212\u2215\u2212 astrocytes would render male and female Nf1\u2212\u2215\u2212 astrocytes differentially sensitive to Rolipram. Male Nf1\u2212\u2215\u2212 astrocytes exhibit lower basal levels of cAMP and are insensitive to Rolipram (Figure Nf1\u2212\u2215\u2212 astrocytes exhibit higher baseline levels of cAMP and these levels further increase with Rolipram treatment. Thus, female brain tumor patients may be more responsive to Rolipram treatment than male brain tumor patients.We would propose that therapeutic cAMP elevation is an ideal setting to test the hypothesis that sexual dimorphism in the cAMP pathway will render males and females differentially sensitive to specific cAMP modulating agents. We previously reported that PDE4 inhibition with Rolipram had significant anti-brain tumor effect in multiple brain tumor models (Yang et al., m Figure . In contThe multiplicity of agents that can target different levels of the cAMP regulatory pathways from GPCR through AC and PDE should allow for exhaustive determination of how significant the effects of sex are on cAMP regulation and therapeutic responses to cAMP modulation. Only with this kind of knowledge can we hope to harness the full power of cAMP elevating agents to treat cancer.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Contemporaneous work from Dr. Anurag Agrawal\u2019s lab reveals the molecular regulation of mitochondrial movement during donation and shows how this can be engineered to increase therapeutic efficacy of MSC. They find that Miro1, a calcium sensitive mitochondrial Rho-GTPase that attaches mitochondria to KIF5 motor proteins on microtubules and regulates neuronal mitochondrial trafficking, is necessary for mitochondrial transfer by MSC. Overexpressing Miro1 in MSC (MSCmiroHi) led to increased mitochondrial transfer and therapeutic efficacy in mouse models of lung injury as well as asthma. This is a significant addition to the field of mitochondrial biology and stem cell therapeutics. It is also of general interest to physicians and members of public with interest in regenerative medicine, because this is highly translatable into more effective therapies.There is increasing interest in whether mesenchymal stem cells (MSC) act as mitochondrial donors for rescuing injured cells. Islam"} +{"text": "Ecosystem service\u2010based management requires an accurate understanding of how human modification influences ecosystem processes and these relationships are most accurate when based on functional traits. Although trait variation is typically sampled at local scales, remote sensing methods can facilitate scaling up trait variation to regional scales needed for ecosystem service management. We review concepts and methods for scaling up plant and animal functional traits from local to regional spatial scales with the goal of assessing impacts of human modification on ecosystem processes and services. We focus our objectives on considerations and approaches for (1) conducting local plot\u2010level sampling of trait variation and (2) scaling up trait variation to regional spatial scales using remotely sensed data. We show that sampling methods for scaling up traits need to account for the modification of trait variation due to land cover change and species introductions. Sampling intraspecific variation, stratification by land cover type or landscape context, or inference of traits from published sources may be necessary depending on the traits of interest. Passive and active remote sensing are useful for mapping plant phenological, chemical, and structural traits. Combining these methods can significantly improve their capacity for mapping plant trait variation. These methods can also be used to map landscape and vegetation structure in order to infer animal trait variation. Due to high context dependency, relationships between trait variation and remotely sensed data are not directly transferable across regions. We end our review with a brief synthesis of issues to consider and outlook for the development of these approaches. Research that relates typical functional trait metrics, such as the community\u2010weighted mean, with remote sensing data and that relates variation in traits that cannot be remotely sensed to other proxies is needed. Our review narrows the gap between functional trait and remote sensing methods for ecosystem service management. Evaluation of ecosystem service policy and management requires understanding the consequences of human modification on ecosystem processes and dependent ecosystem services at regional scales or functional diversity indices, rather than vegetation types Grime . These mA promising alternative approach is the fine\u2010resolution regional mapping of functional traits using remote sensing Fig.\u00a0. CurrentTo better understand the application of functional traits to ecosystem service assessments in human\u2010modified regions, our objectives are to review (1) the current conceptual understanding of trait\u2010based approaches for sampling trait variation across spatial scales and (2) existing remote sensing\u2010based methods that can be used to scale up trait variation from plot to regional scales. We begin our first objective by reviewing the sources of functional trait variation found across ecological levels of organization that span individuals, species, communities, and landscapes. We then synthesize how these sources combine to structure functional trait variation across space in light of human modification, and the implications that the resulting spatial trait variation has for the design of local plot\u2010scale sampling methods. We structure our second objective around groups of remotely sensible traits that correspond to different sets of available remote sensing methods. In general, phenological and chemical plant traits can be sampled by optical\u2010based remote sensing while plant structural traits can be sampled by active laser\u2010based remote sensing. Animal traits may be inferred by combining remote sensing of plant and vegetation structural traits with landscape structure. We illustrate the applicability of some of these methods by citing examples of how variation in plant and animal traits has been sampled and scaled up to regional scales. We end our review by providing a brief synthesis of results, identified knowledge gaps, and outlook for further development of these methods to improve ecosystem service assessments.Functional traits can vary across individuals, species, communities, and landscapes. A better understanding of the sources and spatial scales in which most of the effect trait variation is found will allow for better allocation of sampling effort in trait\u2010based approaches. Although we cannot logistically measure all trait values across all ecological levels, understanding the magnitude of trait variation sources will reduce uncertainty when scaling from local plot\u2010scale estimates of trait values to broader spatial scales.Intraspecific variation in functional traits arises from microsite environmental variability and gradients occurring across the geographical range of plant and animal populations due to founder effects and successional processes per site per community type (\u22653 sites per community type) at selected points across the prevailing environmental gradients can be enough to capture the necessary effect trait variation into CWM or trait diversity indices , can be mapped using satellite\u2010based passive multi\u2010 to hyperspectral remote sensing Fig.\u00a0. PassiveEmpirical models use regression analysis to establish statistical relationships between field measurements of traits and passive remote sensing data. A limitation of empirical models has been that most multispectral sensors sample few portions of the electromagnetic spectrum at bandwidths too wide to capture important features for the discrimination of canopy traits Ollinger . This liPhysical models of radiative transfer account for light absorption and scattering processes to simulate leaf to canopy reflected or emitted optical spectral properties based on multi\u2010 or hyperspectral data habitat structure across landscapes. Some useful LiDAR\u2010derived structural variables include understory vegetation density, LAI, canopy architecture, snag size and density, and tree biomass and basal area Fig.\u00a0. In situOur review found and synthesized various issues to consider and corresponding viable approaches for scaling up plant and animal traits from plot to regional scales using remote sensing Table\u00a0. DecidinOur review focused on the objectives of conducting field sampling of trait variation and scaling up trait variation using remotely sensed data with the ultimate goal of improving ecosystem service assessments. An important knowledge gap implicitly found by our review is the lack of research directed toward linking the functional trait metrics that are typically related to ecosystem processes and services, the CWM and functional diversity indices, with remotely sensed data. We could not find any papers on this topic, and this remains a next step to improve the utility of functional traits for ecosystem service management. In addition, most of our review of methods for scaling up traits applied to traits that can be remotely sensed. Other plant traits that may be useful for ecosystem service assessments, such as wood density or belowground biomass, still would need to be inferred based on their relationship to environmental variables or to other traits via modeling approaches akin to the inference of animal traits as illustrated by our review. Nevertheless, fusion of passive and active remote sensing along with technological developments that increase sensor spectral, spatial, and temporal resolutions can improve the mapping of sensible plant functional traits and animal traits related to landscape and vegetation structure. At present, remote sensing is a powerful tool for capturing variation in functional traits at multiple spatial scales and, to improve their accuracy, ecosystem service assessments should take advantage of traits that can be remotely sensed.All data are included in the manuscript.None declared."} +{"text": "GRN mutation causes neuronal ceroid lipofuscinosis, also known as Batten's disease\u2014a progressive neurodegenerative condition belonging to a class of disorders called lysosomal storage diseases . This was discovered when Baker et al. . Homozygous or compound heterozygous loss of function mutations in this gene cause a lysosomal disorder called Gaucher disease. Like Batten's disease caused by Grn knockout in mice, Gba knockout produces similar neuropathology; both mouse models display conspicuous loss of neurons in the VPM/VPL that is accompanied by marked gliosis (Farfel-Becker et al., GBA is over-represented in Parkinson's disease patients (Gan-Or et al., GBA and Parkinson's disease has since been corroborated by numerous genetic studies.Like GBA and GRN haplo-insufficiency raise immediate questions about the nature of pathogenesis in later onset neurodegenerative diseases such as FTD, Alzheimer's disease and Parkinson's disease. Is risk for late onset neurodegenerative disease from mutation in GBA or GRN related to gene-product specific mechanisms? Or is it general inefficiency in lysosomal flux that causes or contributes to late onset neurodegenerative disease? Surely, the fact that mutations in two very different LSD causing genes that also contribute to late onset neurodegenerative disease suggests variation in lysosomal flux is important. This idea has been further corroborated by a study that showed FTD associated with mutation in the lysosomal network gene, CHMP2B, was accompanied by neuronal lysosomal storage material (Clayton et al., The obvious parallels between With this in mind, are mutations in other lysosomal proteins risk factors for late onset neurodegenerative disorders? With over 50 genes that cause lysosomal storage disease (Cox and Cach\u00f3n-Gonz\u00e1lez, GRN mutation in late onset neurodegenerative disorders appears similar to the relationship between GBA, another lysosomal gene, and Parkinson's disease. This suggests generalized reduction in lysosomal network flux may be a key driver of pathogenesis in late onset neurodegenerative disease.In conclusion, the study by Tanaka and colleagues directly links homozygous loss of progranulin to other models of LSD. Lysosomal storage of un-degraded material, along with regionally specific neuronal cell death consistent with other diverse lysosomal disease models is strong evidence that progranulin deficiency causes lysosomal storage disease. Further to this, the role of heterozygous The author confirms being the sole contributor of this work and approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Flash pulmonary edema is characteristically sudden in onset with rapid resolution once appropriate therapy has been instituted . Acute increase of left ventricular (LV) end diastolic pressure is the usual cause of sudden decompensated cardiac failure in this patient population. Presence of bilateral renal artery stenosis or unilateral stenosis in combination with a single functional kidney in the susceptible cohort is usually blamed for this condition. We describe a patient who presented with flash pulmonary edema in the setting of normal coronary arteries. Our case is distinct as our patient developed flash pulmonary edema secondary to unilateral renal artery stenosis in the presence of bilateral functioning kidneys. Percutaneous stent implantation in the affected renal artery resulted in rapid resolution of pulmonary edema. Flash pulmonary edema commonly presents with sudden onset symptoms which typically resolves rapidly . Acute iOur patient is a 76-year-old woman who presented to a peripheral hospital with sudden onset dyspnea, nausea, orthopnea, chest pain, and vague abdominal discomfort. She had a history of vague headaches and a family history of hypertension and ischemic heart disease but did not have a formal diagnosis of hypertension herself. Initial examination and investigations revealed a normal hemoglobin level (12.1\u2009mg/dL), minimally raised neutrophil count, and a normal renal profile. Chest X-ray revealed severe congestive cardiac failure with markedly raised blood pressure (230/130). ECG showed normal sinus rhythm with left ventricular hypertrophic changes which were incorrectly interpreted as left bundle branch block (LBBB). A quick bedside echo revealed moderate left ventricular hypertrophy with a normal left ventricular ejection fraction (LVEF) and no significant valvular lesions which could have accounted for her clinical deterioration. She was commenced on an intravenous nitrate infusion and transferred to the cardiac catheterization laboratory in our tertiary care facility for a presumed acute coronary event leading to decompensated cardiac failure.On arrival to our facility, the patient was extremely moribund and complained of severe dyspnea and chest discomfort. Her blood pressure was still unchanged at 230/130\u2009mm\u2009Hg. Additional antihypertensive pharmacotherapy was considered; however, in view of her unstable clinical status, we decided to shift her to the cardiac catheterization laboratory.Coronary angiogram revealed nonobstructive coronary artery disease with severely raised end diastolic pressure. In view of the nature of her presentation, renal angiography was performed which showed a normal right renal artery while an ostial occlusion of the left renal artery was noted Figures and 2. BBlood pressure rapidly normalized and the clinical status of the patient improved after intervention. Patient had a detailed echo the following morning, which confirmed a normal left ventricular ejection fraction, left ventricular hypertrophy with grade 1 diastolic dysfunction, trace to mild MR, left atrial dilation, trace aortic regurgitation, and mild tricuspid regurgitation. An abdominal ultrasound revealed no significant renal abnormalities. Telemetry revealed frequent premature atrial complexes (PACs) and premature ventricular complexes (PVCs) overnight which settled over the following 48 hours. No evidence of atrial fibrillation was noted.She has been followed up on half yearly basis since and her blood pressure and heart failure control have remained satisfactory. She was prescribed dual antiplatelet therapy for 18 months. A CT aortogram was not performed in her case as atherosclerosis is the commonest cause of renal artery stenosis in people aged >45 years and we felt that further radiation and dye exposure was not warranted.Atherosclerotic renal artery stenosis is a progressive disease. Sudden progression to complete occlusion of bilateral renal arteries is associated with acute renal failure and sudden onset fluid overload with resultant flash pulmonary edema. Unilateral renal artery stenosis rarely presents with flash pulmonary edema. The exact mechanism of this presentation is not well understood.Acute renal infarction primarily occurs in patients with other comorbid conditions such as diffuse atherosclerotic disease and atrial fibrillation. These patients typically complain of acute onset of flank or generalized abdominal pain, frequently accompanied by nausea and vomiting. These findings are usually accompanied by an acute elevation in blood pressure that is presumably mediated by increased renin release. Laboratory findings include deterioration in renal indices (creatinine and eGFR), hematuria, and increased LDH . These cSeveral clinical trials have demonstrated significant improvement in overall renal function in patients with unilateral renal stenosis, following reinstitution of blood flow to the stenotic kidney , 4. One La Batide-Alanore et al., showed that bilateral renal artery disease or comparable conditions like unilateral renal artery stenosis with a single functioning kidney, differed from unilateral stenosis with bilateral functioning kidneys in the mechanism by which fluid overload precipitates . BilaterPotential pathophysiological mechanisms involved in our patient's presentation are complex and probably do involve the RAS system. We agree with Noh et al. who havePossible treatment strategies in patients with acute renal artery stenosis include medical, surgical, and percutaneous options . PercutaIn our patient, the severity of situation and resource availability prompted us towards the percutaneous intervention route which proved successful.Flash pulmonary edema is a rare manifestation of renal artery stenosis. In our patient, it occurred secondary to unilateral renal artery stenosis with bilateral functioning kidneys. Our case demonstrated that unexplained flash pulmonary edema with hypertension should be pursued with a renal angiogram once the coronary artery disease has been outruled. Revascularization in this patient proved that therapeutic intervention with stenting in symptomatic individuals should be performed to reduce morbidity and mortality."} +{"text": "Chlamydomonas flagella we identified two types of IFT trains we named long and short trains, each characterized by a specific ultrastructure and a definite internal repeat, and proposed that long, less compact trains could represent anterograde IFT while the short, more compact trains could be retrograde. To challenge such model, we monitored by transmission electron microscopy the IFT trains expressed both in wt regenerating flagella and during flagellar reabsorption induced in the ts mutant pf1-fla10. We also progressed in our electron tomographic 3D modeling of short IFT trains. Our data suggest that long IFT trains are not the only anterograde IFT component. Rather, anterograde IFT is contributed also by a subclass of short trains that is expressed in a flagellar length-dependent fashion.Intraflagellar Transport (IFT) is the molecular process responsible for the active bidirectional trafficking of structural and functional components that occurs in the flagellar compartment of eukaryotic cells. Flagellar components undergo a constant turnover at flagellar tip and multiple evidences indicate that flagellar elongation, maintenance and reabsorption depend on the correct balance between anterograde and retrograde trafficking. IFT particles are formed by >22 polypeptides assembled into two subcomplexes, A and B, and are moved bidirectionally along the outer surface of axonemal doublets as linear rows of IFT particles, for which we proposed the term \"train\". Anterograde IFT trains are moved by kinesin II and carry to flagellar tip the retrograde motor cytoplasmic dynein 1b, responsible for retrograde IFT. In a previous study carried out on"} +{"text": "The aims of this study were to investigate the relationship between attachment styles and maladaptive eating practices, and to determine whether the relationship was mediated by perfectionism. Attachment style, perfectionism and body image dissatisfaction have been examined separately in previous studies but the relationship with maladaptive eating practices has not been explored in community samples. The current study investigated whether perfectionistic tendencies mediated the relationship between attachment style and maladaptive eating practices. A total of 131 community individuals completed the following scales: Experiences in Close Relationships-Revised (ECR-R), Frost's Multidimensional Perfectionism Scale (FMPS), and the Maladaptive Eating Practices Questionnaire (MEPQ). Consistent with the research hypotheses, hierarchical regression established that maladaptive perfectionism mediated the relationship between attachment avoidance and maladaptive eating practices. In addition, maladaptive perfectionism mediated the relationship between attachment anxiety and maladaptive eating practices. Adaptive perfectionism also mediated the relationship between attachment anxiety and maladaptive eating practices but not between attachment avoidance and maladaptive eating practices. These findings suggest that perfectionistic tendencies whether \u2018maladaptive\u2019 or \u2018adaptive\u2019 act as an explanatory mechanism linking attachment style to maladaptive eating practices. Recommendations for professional practice and future research are suggested."} +{"text": "Breast cancer is the second leading cause of cancer deaths today after lung cancer and is the most common cancer among women. The primary drug tamoxifen is used treat breast cancer has several problems including poor oral bioavailability. Bile acids/salts are known to be components of endogenous molecular pool that solubilizes, absorbs dietary fat/lipid molecules in the form of micelles. Bile acids are interesting chemical scaffold for drug conjugation due to presence of different number of free hydroxyl groups and a free carboxylic acid. We have been exploring bile acids as drug carriers for cancer therapy. We used three bile acids: lithocholic acid (LCA), deoxycholic acid (DCA) and cholic acid (CA) to engineer bile acid tamoxifen conjugates with free amine and acid functionalities. In this talk, I would present the interactions of these new drug carriers and their therapeutic potential for breast cancer therapy."} +{"text": "It is more ideal to estimate neurological recovery after SCI to match the injury level.Spinal cord injury (SCI) is a major public health problem and a devastating event for individuals. Because the central nervous system has a limited capacity for endogenous regeneration and repair, it is necessary to identify factors that exacerbate SCI to prevent any further deterioration of neurological function and improve the outcomes of injuries. We prevTo clarify the effects of hyperglycemia on the functional outcomes after SCI.To assess whether hyperglycemia purely affects neurologic outcomes of the patients with the same injury level, in this study, we selected acute C3-C4 cervical cord injury patients without any bony damages, which is most popular SCI in Japan. We also eliminated the patients with surgical treatments to exclude the surgical effect on neurological recovery. We retrospectively identified 48 SCI patients admitted within 24 hours after injury to the Spinal Injuries Center between June 2005 and May 2011. The blood samples of the patients were corrected immediately after being transported to our hospital (within 24 hours after injury), and the blood glucose concentration was measured in each sample. We examined the relationships between the admission blood glucose concentration values and other functional/clinical measurements, including American Spinal Injury Association (ASIA) Impairment Scale (AIS) grade, and the total spinal cord independence measure (SCIM) scores (for ADLs) at the final follow-up.2 analysis of data for 48 patients with SCI indicated that hyperglycemia on admission (glucose concentration \u2267 126 mg/dl) was a significant risk predictor of poor functional outcome.Pearson xWe showed that hyperglycemia during acute SCI may be a useful prognostic factor with a negative impact on motor function, highlighting the importance of achieving tight glycemic control after central nervous system injury.This work was supported by JSPS KAKENHI Grant Number 26861198."} +{"text": "Data for calibration and out-of-sample error testing of option pricing models are provided alongside data obtained from optimization procedures in \"On calibration of stochastic and fractional stochastic volatility models\" Specifications TableValue of the data\u2022The provided data might help to improve methods described in [1].\u2022New approaches might be compared to the ones used.\u2022Methodology of the calibration testing might be of interest to both practitioners and academic researchers in the field.1We describe in detail data sets used for calibration trials in The data obtained from optimization routines is depicted by figures in 2In Further data were obtained from specific calibration routines, models and problem formulation. The formulation differed only with respect to weights, all employed weight sets are included in the supplementary spreadsheets. Considered models were the following: the popular Heston stochastic volatility model and the newly introduced approximative fractional volatility model (FSV). Three global optimizers were considered for the calibration task, Genetic Algorithm (GA), Simulated Annealing (SA) and Adaptive Simulated Annealing (ASA). As a local search method a trust region reflective method (LSQ) was used. The most interesting approach in"} +{"text": "Mathematical modelling provides an effective way to challenge conventional wisdom aboutparasite evolution and investigate why parasites \u2018do what they do\u2019 within the host. Modelscan reveal when intuition cannot explain observed patterns, when more complicated biologymust be considered, and when experimental and statistical methods are likely to mislead.We describe how models of within-host infection dynamics can refine experimental design,and focus on the case study of malaria to highlight how integration between models anddata can guide understanding of parasite fitness in three areas: (1) the adaptivesignificance of chronic infections; (2) the potential for tradeoffs between virulence andtransmission; and (3) the implications of within-vector dynamics. We emphasize that modelsare often useful when they highlight unexpected patterns in parasite evolution, revealinginstead why intuition yields the wrong answer and what combination of theory and data areneeded to advance understanding. To simplify calculations, models commonly useconstants to represent transmission to and from the vector, including efforts to estimatedisease risk from relapses; transient increases in parasite biomass appear advantageousin the short-term, since they coincide with enhanced transmission rates (Cornet etal.Distinguishing between these possibilities is essential to predicting the consequences ofselectively removing older mosquitoes Models have been immensely useful for refining intuition and improving methods forinferring what cannot be observed directly. Recent work \u2013 theoretical and experimental \u2013 hashighlighted the rich detail of within-host ecology, necessary for placing parasite lifecycles into a robust evolutionary framework. These details are likely to matter as diseaseintervention efforts become more sophisticated, and integration between models and data canlocate the critical gaps in knowledge and augment efforts to unravel the complex patterns ofparasite evolution."} +{"text": "Polycarbonate (PC) is one of the most widely used engineering polymers due to its unusual combination of optical clarity, heat resistance, high impact strength and dimensional stability over wide thermal range . Low watof research was to develop novel low toxic biocides for PC having sufficient thermal stability for the joint melt processing with PC resin.Pseudomonas fluorescens SBW25 with a help of classical microbiological methods for bacterial suspension as well as a live-dead assay for biofilms [Two kinds of potential antimicrobial additives for PC have been synthesized: imidazolium and guanidinium ionenes, as well as imidazolium based ionic liquids. PC films containing from 1 to 10 wt% of biocides were prepared by solvent casting or by compression molding methods. The thermal stability of novel biocides as well as modified PC was investigated by using thermogravimetric analysis (TGA). Antimicrobial testing of PC composites were performed using a model bacteria biofilms .oC that is quite sufficient for their melt processing. The results of microbiological investigations showed pronounced antimicrobial efficacy of PC films containing 3-4 wt% of ionic liquids or 5-8 wt% of ionene polymers.According to TGA data, the thermal stability of PC samples containing biocidal additives was found to be in the range of 350-425 Imidazolium ionic liquids and ionene polymers are promising antimicrobial additives for PC resins since they combine high thermal stability, low toxicity and broad spectrum of antimicrobial activity.According to the obtained results we recommend to expand the application of polimers to design polycarbonate based medical devices for prevention of postoperative infectious complications in orthopedics after performing relevant biosafety and preclinical tests."} +{"text": "The UK faces a public health challenge arising from unhealthy behaviours. Some health care workers engage in the same unhealthy behaviours as the general population. This paper explores the issues arising from some primary care staff adopting unhealthy behaviours upon healthcare organisations, professional practice and patient perceptions in terms of the promotion of health in the primary care setting. The primary care workforce is at the frontline for promoting healthy behaviours but the social judgements of their patients may undermine the credibility of health professionals as health promoters.\u2022 The health care workforce exhibits the same health behaviours as the general population.\u2022 Unhealthy behaviours increase ill health and unplanned staff absences which impact negatively upon care delivery.\u2022 Personal health behaviours of health professionals may influence their clinical practice.\u2022 Health professionals with unhealthy behaviours may be less credible as health promoters.\u2022 Primary care staff are the face of the NHS and can be role models for their patients.The Chief Medical Officer\u2019s (CMOs) rThe concerns raised by the CMOs report have been reiterated in the future strategy for the NHS which caThe evidence suggests that the health care workforce exhibits the same health behaviours as the general population. Thus theNonetheless primary care staff, like all healthcare staff, are expected to exhibit professional behaviours at all times regardless of their personal health behaviours. This means that every opportunity should be taken to promote health and optiDo the health behaviours of health professionals matter?At the organisational level, services depend upon motivated staff capable of delivering high-quality care to their patients and this is undermined when there are staff absences which reduce care capacity and disrupt continuity of care. Unplannen\u00a0=\u00a0355) and a third of qualified nurses (n\u00a0=\u00a0409) in their sample misperceived their weight status in favour of a lower BMI which may impact upon their assessment of patients in need of weight management support. Further, although the nurses who worked in the community reported more weight management practices, Zhu et al.\u2019s [n\u00a0=\u00a0420) directly and positively predicted their weight management practices; that is, the nurse\u2019s belief in their ability to deliver weight management practices is important to understand their professional practice. This suggests that the enhancement of nurses\u2019 self-efficacy may be an effective strategy to improve their weight management practices.There is a growing body of evidence that the personal health behaviours of health professionals may influence how they practice clinically. Two systematic reviews suggest that the personal body weight of doctors and nurses are related to: (1) their attitudes towards weight management ; and (2)et al.\u2019s path anaet al.\u2019s of the qn\u00a0=\u00a0623) has reported that many nurses are not achieving the recommended levels of physical activity. Further, 25% of the sample were at risk of hazardous drinking or had an active alcohol disorder and 11% were current smokers and 17% were past smokers. This study confirmed a relationship between the nurses\u2019 personal health behaviours and their physical activity health promotion practices. Two other systematic reviews suggest that personal tobacco use [Similarly, personal engagement in physical activity appears to be related to levels of physical activity promotion although the self-report measurement of physical activity is problematic. A study acco use and alcoacco use are alsoThe personal health behaviours of health professionals may influence how patients view their credibility as a health promoter. Indeed, negative attitudes towards obese people are widespread across society with simThe health care workforce, including those working in primary care, is likely to exhibit the same health behaviour trends as the general population. This may have important implications for health care delivery both in terms of staff absences and suboptimal health promotion. Perhaps we need to consider what we can reasonably expect of primary healthcare staff as role models and as the face of the NHS. A good first step may be to implement NICE guidelines relating to the promotion of health and well-being within primary care workplace settings thereby supporting staff in their adoption of healthy lifestyles. The relevant NICE guidelines include those relating to promoting physical activity, mental wNone required"} +{"text": "In recent years the number of extremely premature born (very low birth weight (VLBW<1500 g) children has increased as a consequence of increased survival rate. Those preterm neonates who survive have an increased risk of long-term neurological disabilities and chronic pulmonary disease. In addition, in the last two decades alterations in body composition and increased metabolic risk have been added to the list of consequences. In order to evaluate whether early patterns of infancy anthropometry and nutrition have an impact in metabolic hormonal profile and body composition we have prospectively assessed two cohorts of preterm infants and results will be presented."} +{"text": "To identify the therapeutic effect of the talus control foot orthosis(TCFO) in children with severe flexible flat foot.TCFO is foot orthosis which combines inverted rigid foot orthosis(RFO) with broad upright portion that covers and protects the talonavicular joint, rising well above the navicular.Fourty children were participated in this study who had been diagnosed as flexible pes planus, and had more than two consecutive radiologic studies. The anteroposterior talocalcaneal angles (APTCA) and lateral talocalcaneal angles (LTTCA), the lateral talometatarsal angles (LTTMA) and the calcaneal pitch (CP) of both feet were measured to evaluate foot alignment. Severe flexible pes planus was diagnosed when Beighton hypermobility score was greater than 4 points and when either of the feet had greater than 10 degrees valgus of resting calcaneal stance position angle and indicators showed greater than 55 degrees in APTCA and LTTCA, greater than 10 degrees in LTTMA, lesser than 10 degrees of CP.Twenty children with flexible flat foot were fitted with a pair of RFO and another 20 children were fitted with TCFO. They were recommended to put on orthosis more than 8 hrs a day, to walk with heel strike at initial contact and reciprocal arm swing to normalize gait pattern. The follow up clinical evaluation with radiologic study was done after 12 months.With TCFO, all radiologic indicators changed toward corrective direction than with RFO. There were statistically significant improvements in CP and RCSP in both groups. (p < 0.05) In TCFO group, APTCA, RCSP improved significantly compared with RFO group.This study showed that TCFO is effective in the treatment of children with severe flexible pes planus than RFO. The evaluation with long term follow up of radiographic study would be necessary to confirm the therapeutic effect of TCFO in children with pes planus."} +{"text": "After decades of slight to modest changes in outcomes, survival for patients with glioblastoma has shown consistent and sustained improvement. Whether this improvement is due to enhanced imaging technologies, increased diagnostic accuracy, earlier detection, advanced microsurgical techniques, functional tissue preservation, postoperative critical care, targeted radiation, or novel adjuvant therapies is uncertain. What is known is that the field is moving in a more promising direction. In this issue we have selected novel and exciting contributions that represent our current landscape and illustrate new directions, highlighting their opportunities as well as their limitations. The goal of this special issue is to stimulate new understanding and encourage cross-discipline collaborations that will assist in changing the outlook for this disease.Fluorescence-guided surgery and biopsy in gliomas with an exoscope system\u201d and combine intraoperative imaging with tissue characterization and biomarker utilization such as \u201cIntraoperative cerebral glioma characterization with contrast enhanced ultrasound.\u201d Original contributions presenting improved immunotherapy strategies such as \u201cInterleukin-13 receptor alpha 2-targeted glioblastoma immunotherapy\u201d and alternative gene therapy approaches were also included such as \u201cNewcastle disease virus interaction in targeted therapy against proliferation and invasion pathways of glioblastoma multiforme.\u201d The role of cancer stem cells in GBM malignancy and the progress made in its diagnostic and therapeutic implications are nicely reviewed as in \u201cStem cell niches in glioblastoma: A neuropathological view.\u201d The impact of the intracranial tumor microenvironment and its pathophysiological implications are also discussed in \u201cProgesterone induces the growth and infiltration of human astrocytoma cells implanted in the cerebral cortex of the rat.\u201dWe have invited investigators to contribute original research articles as well as topical reviews that provide updated perspective and present exciting contributions. We have included articles that explore intraoperative technological advances like \u201cIt is our intention that this issue will promote discussion of new topics of interest and stimulate interdisciplinary collaborative efforts to improve outcomes for patients with glioblastoma. New modalities of treatment, characterization of the tumor's microenvironment, developing intraoperative innovative techniques, and targeting tumor-specific receptors are all disciplines on the cutting edge of glioblastoma research, the results of which will hopefully lead to marked improvements in outcome.Betty TylerFrancesco DiMeco Rachel GrossmanGustavo Pradilla"} +{"text": "While contrast reactions are rare we have anecdotally observed that patients of certain racial backgrounds appear more likely to experience reactions of nausea or vomiting after Multihance\u00ae infusion and sought to formally examine this possible relationship.Gadolinium based contrast agents are frequently given to patients who undergo a Cardiac MRI examination to maximize contrast between normal and abnormal tissues in order to demonstrate pathologies such as scar or fibrosis in the heart and for MR angiography. Multihance\u00ae from our CMR clinical database. These included 161 patients of self-described African descent. The remaining 1612 individuals were of other races, predominately Caucasian with a small minority of Asian , Hispanic and other populations. An additional 240 individuals elected not to report ethnicity Patients were told that no preparation was required for their examination; therefore the time since their last meal was unknown. All individuals were monitored in the same way during their study including ECG, non-invasive blood pressure and respiratory monitoring. Verbal communication was maintained with all individuals before, during and after the administration of the contrast agent.We retrospectively identified 1773 consecutive CMR examinations over a 4 year period (2010-2014) utilizing Multihance\u00ae infusion including 12 African descent, 17 Caucasian, 1 Hispanic and 1 of unknown race/ethnicity reactions involving nausea and vomiting immediately after Multihance\u00ae brand of gadolinium based contrast agent in individuals of African descent as compared to individuals who identify themselves as of non-African descent. Further prospective studies are needed to determine if this finding remains consistent and if ethnicity should be considered in the choice of CMR contrast agent.In this consecutive, retrospective study of patients referred for clinical CMR study, we found a much higher incidence of nausea/vomiting reactions to the MultihanceN/A."} +{"text": "In this data article, we evaluated the daily fluoride contents in 20 household desalinators working by reverse osmosis (RO) Specifications TableValue of the data\u2022Data can be used as a base-line data for the fluoride content in drinking water prepared by household desalinators.\u2022Data shown here will be informative in computing fluoride daily intake by drinking water as well as food consumption.\u2022Data shown here can be useful for health policy makers by assigning prevention measures against adverse health effects of fluoride with considering fluoride intake by different sources.1In the data, as shown in 2This cross-sectional descriptive study was carried out using random sampling (In different areas of Bushehr). Samples were taken from inlet and outlet waters of household desalinators working by RO process between February and March 2016. A total number of 40 samples (Inlet and outlet waters) were taken from 20 household desalinators and analyzed for fluoride contents. For sampling, we used plastic containers. All bottles were stored in a dark place at room temperature until the fluoride analysis was made by the standard SPADNS method"} +{"text": "Despite prompt reperfusion by primary percutaneous coronary intervention (PPCI), the mortality and morbidity of patients presenting with an acute ST-segment elevation myocardial infarction (STEMI) remain significant with 9% death and 10% heart failure at 1\u2005year. In these patients, one important neglected therapeutic target is \u2018myocardial reperfusion injury\u2019, a term given to the cardiomyocyte death and microvascular dysfunction which occurs on reperfusing ischaemic myocardium. A number of cardioprotective therapies , which are known to target myocardial reperfusion injury, have been shown to reduce myocardial infarct (MI) size in small proof-of-concept clinical studies\u2014however, being able to demonstrate improved clinical outcomes has been elusive. In this article, we review the challenges facing clinical cardioprotection research, and highlight future therapies for reducing MI size and preventing heart failure in patients presenting with STEMI at risk of myocardial reperfusion injury. Although, myocardial reperfusion is a pre-requisite to salvaging viable myocardium, the process of restoring coronary blood flow can paradoxically induce myocardial injury and cardiomyocyte death, thereby mitigating the full benefits of reperfusion in terms of MI size reduction\u2014a phenomenon which has been termed \u2018myocardial reperfusion injury\u2019.In this article, we review the challenges facing clinical cardioprotection research, and highlight future opportunities for reducing MI size and improving clinical outcomes in patients presenting with STEMI at risk of myocardial reperfusion injury.There are a number of challenges facing clinical cardioprotection research. First, the translation of novel cardioprotective therapies into the clinical setting for patient benefit has been extremely difficult. Extensive literature exists on the complex array of prosurvival signalling pathways which underlie cardioprotection, and the readers are referred to a number of recent reviewsThe reasons for this failure to translate cardioprotection into the clinical setting have been attributed to a number of factors including the use of inappropriate animal MI models and poorly designed clinical studies\u2014a topic which has been discussed extensively in the literature and is highlighted in a following section.17A second challenge facing clinical cardioprotection research is that clinical outcomes of patients presenting with STEMI following PPCI continue to improve, making it increasingly difficult to demonstrate a reduction in MI size and improvement in clinical outcomes with a novel cardioprotective therapy. However, although mortality following STEMI is in decline, the number of patients surviving STEMI and going on to develop heart failure is increasing. There remains, therefore, an unmet need to discover novel therapeutic strategies capable of preventing myocardial reperfusion injury and reducing MI size, so as to preserve LV systolic function and prevent the onset of heart failure in patients presenting with reperfused STEMI. In the next section and in et al,31Interrupting myocardial reperfusion with several short-lived episodes of myocardial ischaemia has been demonstrated in experimental animal studies to prevent myocardial reperfusion injury and reduce MI size\u2014a phenomenon which has been termed \u2018ischaemic postconditioning\u2019 (IPost).et alExperimental animal studies have found that administering atrial natriuretic peptide (ANP) prior to myocardial reperfusion can reduce MI size through the activation of known prosurvival signalling pathways.et alThe opening of the mitochondrial permeability transition pore (MPTP) in the first minutes of reperfusion is a critical mediator of reperfusion-induced cardiomyocyte death, and preventing its opening using pharmacological inhibitors of the MPTP such as ciclosporin-A (CsA) has been reported in experimental studies to limit MI size.Exenatide (Byetta), a synthetic version of exendin-4 , is a long-acting analogue of glucagon-like peptide-1 (GLP-1), a hormone which lowers blood glucose by stimulating insulin secretion.45et alet alLonborg Inducing brief non-lethal episodes of ischaemia and reperfusion in the arm or leg, using serial inflations and deflations (three to four 5\u2005min cycles) of a standard blood pressure cuff placed on the upper arm or thigh, has been shown to protect the heart against acute ischaemia-reperfusion injury (IRI)\u2014a phenomenon known as \u2018remote ischaemic conditioning\u2019 (RIC).48A number of proof-of-concept clinical studies have demonstrated MI size reduction using limb RIC in patients presenting with STEMI treated by either PPCIet alRepeated RIC post MI has attracted attention as a potential chronic cardioprotective therapy. Wei et alWhether early \u03b2-blocker therapy is beneficial in patients presenting with reperfused STEMI is controversial and had not previously been investigated in the PPCI era. Using a porcine mode of acute myocardial IRI, Ibanez et alPrior attempts to reduce MI size in patients presenting with STEMI have relied on targeting one single component of myocardial reperfusion injury with a single agent. Whether, using combination reperfusion therapy can provide more effective cardioprotection against myocardial reperfusion injury remains to be investigated. In this regard, Alburquerque-Bejar Patient selection\u2014where possible, consider selecting those patients presenting with STEMI who are most likely to benefit from the novel cardioprotective therapy\u2014those presenting: early and concomitant medication may interfere with the efficacy of the novel cardioprotective therapy.Timing of the cardioprotective therapy\u2014as myocardial reperfusion injury manifests in the first minutes of reperfusion, it is essential that the novel cardioprotective therapy is administered prior to myocardial reperfusion if it is to be effective. A number of neutral clinical studies were due to administering the cardioprotective therapy after reperfusion had already taken place.Study end points\u2014consider selecting study end points which are most likely to be affected by the novel cardioprotective therapy\u2014these include surrogate end points such as MI size , myocardial salvage index (which is more sensitive than MI size reduction but requires the assessment of the AAR); microvascular obstruction , LV size and function and hard clinical end point such as heart failure hospitalisation, and cardiac death.There are number of factors which need to be taken into consideration when designing clinical cardioprotection studies for reducing MI size in patients presenting with reperfused STEMI, as this may help facilitate the translation of novel cardioprotective therapies into the clinical setting for patient benefit.Clinical cardioprotection research remains a challenge\u2014mortality rates following a PPCI-treated STEMI are in decline, which makes demonstrating a further reduction in MI size and improved clinical outcomes increasingly more difficult. However, an increasing number of patients are developing heart failure\u2014as such, novel cardioprotective therapies which target myocardial reperfusion injury and reduce MI size provide the opportunity to preserve LV systolic function and prevent the onset of heart failure. The failure to translate a large number of infarct-limiting therapies into the clinical setting should not be taken as evidence that myocardial reperfusion injury does not exist in humans. Rather, it should provide the impetus to optimise the design of our experimental animal and clinical studies, and improve how we select which novel cardioprotective therapy to test in the clinical setting\u2014this may hopefully facilitate the discovery of new effective therapies for reducing MI size and preventing heart failure in patients presenting with reperfused STEMI. Initial data suggest that using a combination of therapies to target myocardial reperfusion injury may be more beneficial than using a single therapy approach, and using this approach may result in improved clinical outcomes in this patient group."} +{"text": "In an era increasingly focused on quality improvement and cost containment, more emphasis is being placed on wiser utilization of medical care resources. One underutilized resource in early neonatal care is umbilical cord blood.Umbilical cord blood can be utilized for admission laboratory studies in neonates thereby avoiding a significant phlebotomy event in the first minutes to hours of life. Additionally, umbilical cord blood can also be safely \u201ctransfused\u201d into the neonate via delayed cord clamping or milking of the umbilical cord. This has been demonstrated to be particularly beneficial in premature infants by decreasing the rate of intraventricular hemorrhage. Delayed cord clamping has been formally endorsed by a number of medical societies, however it has not yet been universally adopted by obstetricians and neonatologists.Both uses of umbilical cord blood for neonatal admission laboratory testing and delayed cord clamping/milking of the umbilical cord have resulted in decreased transfusion rates as well as other outcomes reviewed herein. In an era increasingly focused on quality improvement and cost containment, more emphasis is being placed on wiser utilization of medical care resources. Emphasis has included aspects such as consistency in the use of medical interventions, creating evidence-based \u201cbundles\u201d of care, and process standardization. One underutilized resource in early neonatal care is the use of umbilical cord blood. This review will present proposed uses of this resource in the minutes immediately following delivery. Specific uses will include utilizing otherwise discarded umbilical cord blood for all admission laboratory blood studies, and delayed cord clamping (DCC) or milking of the umbilical cord (MUC). We will not review the use of umbilical cord blood for autologous transfusion, umbilical cord blood banking, or cord blood mesenchymal stem cells for neonatal therapy in this manuscript.Blood tests are required to care for critically ill neonatal intensive care unit (NICU) patients, including VLBW infants. VLBW infants typically have greater phlebotomy blood loss, per kilogram body weight, on the first day of life than any other day during their hospitalization. In one study the mean phlebotomy loss on the first day of life was >10\u00a0mL/kg [In paired-samples of cord blood versus blood drawn at birth from the neonate, CBC and leukocyte differentials are clinically equivalent. This has been demonstrated in both term and preterm infants \u20136. In onSeveral studies have contrasted blood culture results when blood is drawn directly from the neonate versus otherwise discarded blood from the umbilical cord or fetal vessels on the placenta , 4\u20138. ItDetermination of the neonatal blood type is frequently done from umbilical cord blood. Both pediatric and obstMany states recommend drawing the newborn metabolic screening tests of VLBW infants before antibiotics are started, before blood products are transfused, and before amino acid-containing hyperalimentation solutions are administered. The Clinical and Laboratory Standards Institute recommends obtaining the first newborn metabolic screen upon admission for premature infants . ObtainiTo date one pilot study and one Studies over 45\u00a0years ago demonstrated a higher fetal blood volume and a proportionately lower placental blood volume in term infants as a result of DCC for up to 180\u00a0s . At termFor DCC to effectively transfuse placental blood to the newly delivered neonate fetal blood in the placenta must continue to transfer into the neonate for a period of time, i.e., potentially until the umbilical cord is clamped or cord pulsations cease. If the umbilical vein closes before the umbilical arteries, the neonate could \u201chemorrhage\u201d back into the placenta. To study this, the direction of blood flow during DCC was evaluated using doppler ultrasonography in term infants after vaginal delivery . The durMilking of the umbilical cord is another method of transferring fetal blood from the placenta into the fetus before clamping and cutting the umbilical cord. This method offers the potential advantage of being completed more quickly than DCC. It may also be more enthusiastically adopted by obstetricians if they are uncomfortable holding an extremely premature infant during DCC without any perceived intervention. Several studies have used sonography to assess the umbilical vessel sizes at various gestational ages \u201325. We uUntil recently, current practice was ICC following delivery of a premature infant. Over the past several years a number of organizations including the World Health Organization, Royal College of Obstetricians & Gynaecologists, and American College of Obstetricians & Gynecologists have formally endorsed DCC as standard of care \u201329. The Premature infant brains are sensitive to fluctuations in blood pressure, particularly hypotension due to poor cerebral autoregulation . MaintaiRecently benefits of DCC versus ICC have been assessed in premature infants. By 2004 at least seven randomized controlled trials compared ICC with DCC of preterm infants. Although the definition for DCC varied from 30 to 120\u00a0s, DCC was associated with higher hematocrits, fewer transfusions, and less intraventricular hemorrhage . While sUmbilical cord blood may be the most valuable underutilized resource in the care of premature neonates. Utilization of umbilical cord blood for admission laboratory testing of neonates is a promising new practice that has been shown to improve neonatal outcomes. While DCC and MUC have been formally endorsed by a number of professional organizations they have not yet been universally adopted by obstetricians and neonatologists. Full implementation of this practice is therefore an important step in better utilization of umbilical cord blood in improving the outcomes of premature neonates."} +{"text": "How variable and noisy is the neural code arising from the joint activity of recurrently connected cells? Isolated neurons are known to respond to fluctuating input currents with reliable spike patterns ,2, but vWe focus on spiking model networks with sparse, random connectivity and balanced excitation and inhibition that reproduce the irregular firing that typifies cortical activity. In such models, activity is known to be chaotic, with extremely strong sensitivity of spike outputs on tiny changes in a network\u2019s initial conditions -6. NeverWe derive a bound for the entropy of joint spike pattern distributions in large spiking model networks in response to a fluctuating temporal signal. The analysis is based on results from random dynamical systems theory and complimented by detailed numerical simulations. We find that despite very weak conditional correlations between neurons, the resulting joint variability of network responses is surprisingly lower than what would be expected by considering only limited statistical neural interactions. Moreover, joint spiking variability is strongly constrained by the level of temporal features of input stimuli."} +{"text": "LMNA) defect include different phenotypes that can be classified as i) severe phenotype with generalized muscular weakness and contractures by birth, ii) \u2018dropped head\u2019 phenotype with prominent involvement of axial muscles that generally evolves to rigid spine phenotype and iii) early Emery-Dreifuss phenotype. All these condition generally lead in the first 2 decades to cardiac disturbances, respiratory insufficiency, orthopedic complication and metabolic disorders. The clinical management requires a multidisciplinary and rigorous approach focused on early medical and rehabilitative interventions with the main aims to prevent \u2018fatal heart event\u2019, to cure co-morbidities and to improve the quality of life of these children.Congenital Muscular Dystrophies are a heterogeneous group of muscular disorders defined as early onset muscle weakness and progressive course associated to dystrophic features at muscle biopsy. CMDs related to lamina A/C gene ("} +{"text": "Philosophy, Ethics, and Humanities in Medicine would like to thank all our reviewers who have contributed to the journal in Volume 10 (2015).The editors of A PascalevBulgariaRoland BenedikterItalyJohn ShookUSASilke SchicktanzGermanyDan SteinSouth AfricaPatrick BrownUKDavid MillerUSAMichael SchwartzUSARay GreekUSAJeff CollmannUSAElizabeth DavenportUSAGuillermo PalchikUSAJoan EngebretsonUSA"} +{"text": "Mechanisms involved in regulating metabolic reprogramming in cancer cells are not fully understood. Acetylation is emerging as a major regulator of mitochondrial metabolism and may contribute to metabolic derangements that occur in cancer cells. Sirtuin-3, (SIRT3), is the main mitochondrial deacetylase and it serves to maintain mitochondrial energy homeostasis by deacetylating and activating mitochondrial proteins. Loss of SIRT3 leads to altered cellular metabolism including reduced ATP production and decreased fatty acid oxidation . RemarkaStable cell lines expressing shScramble or shSIRT3 were made by lentiviral transduction in HepG2 and 293T cells. Metabolomic analysis of stable cell lines were done by GC/MS and MS/MS. Glutamine oxidation was measured by treating cells with 14C glutamine and capturing radiolabeled CO2.Protein acetylation is particularly sensitive to nutrient status, such as high fat diet feeding . Our datThese data illustrate an anabolic shift in cell metabolism that is required to supply biosynthetic precursors necessary for rapid cell growth. We believe that loss of SIRT3 and subsequent hyperacetylation of mitochondrial proteins leads to a rerouting of energetic substrates supporting oncogenically driven tumor growth."} +{"text": "Neural output from the locomotor system for each arm and leg influences the spinal motoneuronal pools directly and indirectly through interneuronal (IN) reflex networks. While well documented in other species, less is known about the functions and features of convergence in common IN reflex system from cutaneous afferents innervating different foot regions during remote arm and leg movement in humans. The purpose of the present study was to use spatial facilitation to examine possible convergence in common reflex pathways during rhythmic locomotor limb movements. Cutaneous reflexes were evoked in ipsilateral tibialis anterior muscle by stimulating (in random order) the sural nerve (SUR), the distal tibial nerve (TIB), and combined simultaneous stimulation of both nerves (TIB&SUR). Reflexes were evoked while participants performed rhythmic stepping and arm swinging movement with both arms and the leg contralateral to stimulation (ARM&LEG), with just arm movement (ARM) and with just contralateral leg movement (LEG). Stimulation intensities were just below threshold for evoking early latency (<80 ms to peak) reflexes. For each stimulus condition, rectified EMG signals were averaged while participants held static contractions in the stationary (stimulated) leg. During ARM&LEG movement, amplitudes of cutaneous reflexes evoked by combined TIB&SUR stimulation were significantly larger than simple mathematical summation of the amplitudes evoked by SUR or TIB alone. Interestingly, this extra facilitation seen during combined nerve stimulation was significantly reduced when performing ARM or LEG compared to ARM&LEG. We conclude that locomotor rhythmic limb movement induces excitation of common IN reflex pathways from cutaneous afferents innervating different foot regions. Importantly, activity in this pathway is most facilitated during ARM&LEG movement. These results suggest that transmission in IN reflex pathways is weighted according to the number of limbs directly engaged in human locomotor activity and underscores the importance of arm swing to support neuronal excitability in leg muscles. Progress in understanding human movement indicates that the neuronal coordination for fore and hind limbs observed in quadrupedal locomotor systems is preserved in human locomotion Nerve or location-specificity of reflex amplitudes has been described in leg muscles during walking and leg cycling after activation of cutaneous nerves in the foot Using spatial facilitation in a feline model, Labella and McCrea (1990) reported that cutaneous afferents from two different nerves converge onto common spinal interneurons (IN) to produce excitation and inhibition in functionally related groups of motoneurons Although neural output from the locomotor generating systems for each arm and leg projects directly to each motoneuronal pool and indirectly through IN reflex networks Previous studies reported that during the rhythmic movement of \u201call four limbs\u201d, the influence of the arms on reflex expression in the legs was superimposed on the dominant effect of the legs The purpose of the present study was to examine convergence in reflex effects following cutaneous stimulation of two different nerves of the foot during arm and leg three-limb stepping by using spatial facilitation. We tested the hypothesis that reflex pathways arising from two different cutaneous nerves converge on putative common INs in a spinal cord pathway for leg muscles during \u201creduced\u201d locomotion. Furthermore, we hypothesized that the effect during combined stimulation of two nerves would be modulated according to the number of limbs engaged in arm and leg movement.Twelve subjects between the ages of 22 and 49 years participated with informed, written consent in a protocol approved by the Human Research Ethic Board at the University of Victoria and conforming to the Declaration of Helsinki (1964).Participants used a recumbent arm and leg stepping ergometer to perform the following rhythmic movement tasks: (1) bilateral arm and contralateral leg ARM&LEG, ; (2) bilCutaneous reflexes in TA were evoked with trains (5 pulses\u00d71.0 ms at 300 Hz) delivered by an isolated constant current stimulation . Stimulation was applied to the distal tibial , Sural and combined simultaneous stimulation of both nerves (TIB&SUR) see . StimulaInitially, subjects performed the ARM&LEG task in 3 different randomly ordered nerve stimulation conditions: 1) TIB, 2) SUR and 3) TIB&SUR. Stimuli were applied at near the start of the recovery phase of the contralateral leg by using the elbow or knee goniometer as a trigger signal . After tStimulation intensities of SUR (\u223c1.4\u20132.2\u00d7RT) and TIB (\u223c0.8\u20132.2\u00d7RT) was adjusted to just below the threshold [just below 2 standard deviations (SDs) of the mean background TA EMG (BG EMG)] that produced an early latency (ELR latency: 45\u201380 ms after onset of stimulation) facilitatory reflex during the ARM&LEG task. These trains of stimuli were delivered every 2\u20134 step cycles for a total of 20 times in each trial and with a trial duration of \u223c60 s. Furthermore, resting duration between trials was given at 3\u20135 min. To investigate convergence effects on ELR amplitude, simultaneous nerve stimulation (TIB&SUR) was applied using the identified subthreshold intensities.After abrasion and cleaning of the skin with alcohol, disposable surface EMG electrodes were applied in a bipolar configuration with a 2 cm interelectrode distance over 8 muscles in the arms and legs. The muscles were anterior deltoid (AD), vastus lateralis (VL), medial gastrocnemius (MG) and tibialis anterior (TA). EMG signals were amplified (\u00d75000) and bandpass filtered at 100\u2013300 Hz .Data were sampled at 1000 Hz with a 12 bit A/D converter connected to a microcomputer running LabView Software . Evoked EMG in ipsilateral TA was analyzed for phasic reflex amplitudes and latencies. During offline digital processing using custom written MATLAB routines, the stimulus artifact was removed and the sweeps were then filtered with a dual pass Butterworth at 100 Hz. The peak response within the ELR window was determined and a 5 ms average centered around this peak was calculated. Reflex amplitudes were normalized as a percentage of the maximal EMG values while performing maximal voluntary contraction. These data from each participant were then averaged across all participants to obtain the group data.To determine whether ELR amplitudes from TIB, SUR, simultaneous TIB&SUR stimulation and algebraic summation (TIB+SUR) were significantly modulated during ARM&LEG, one-way repeated measures analysis of variance (ANOVA) was performed. Also, comparison of reflex amplitudes and BG EMG activity following TIB&SUR stimulation across ARM&LEG, ARM, and LEG tasks were analyzed by using one-way ANOVA. Before calculating one-way ANOVA, Mauchly's sphericity test was performed as a test of the homoscedasticity of our samples If the results of ANOVA were statistically significant, multiple comparisons were performed with the Bonferroni post-hoc test All data are expressed as means and +/\u2212 SD. All repeated one-way ANOVA and post-hoc tests were performed using SPSS software Ver.11 . Significant differences were recognized at the p<0.05 level in all cases.To investigate whether the number of moving limbs modulates facilitation of ELR following simultaneous stimulation, data were compared across the ARM&LEG, ARM and LEG tasks .The present investigation demonstrated a non-linear facilitation of ELR amplitudes evoked by stimulating TIB and SUR nerves when activated simultaneously during rhythmic locomotor movement. During ARM&LEG movement, facilitation of ELR in TA following combined TIB&SUR stimulation was significantly larger than the amplitude anticipated by mathematical summation of amplitudes for individual nerve stimulation conditions (i.e. TIB+SUR). Interestingly, this extra facilitation seen during combined TIB&SUR was significantly reduced if subjects performed ARM or LEG tasks compared to the ARM&LEG task. An important advance of the present study was our ability to determine the incremental effect the number of rhythmically active limbs has on the excitability of common reflex pathways from sensory pathways innervating different foot regions.Stimulus intensities for individual nerve (TIB and SUR) were set to just below threshold for evoking significant ELR during ARM&LEG. Despite that, we found significant facilitation of ELR in TA following combined TIB&SUR stimulation. Moreover, this facilitation was larger than predicted by mathematical summation of responses following individual nerve stimulation. Thus, it is unlikely that our \u201cextra facilitation\u201d effect can be explained by the simple summation effects arising from excitability in \u201cprivate reflex pathway\u201d from inputs evoked by SUR and TIB stimulation The simplest interpretation of our results is that they are consistent with the concept of convergence onto shared interneurons within the polysynaptic reflex pathways arising from two different inputs described by Anders Lundberg and his co-workers Although comparatively little is known about shared reflex pathways following low-threshold cutaneous nerves stimulation in humans, we found spatial facilitation in reflex pathways from cutaneous afferents innervating different foot regions during ARM&LEG movement Interestingly, the range of ELR investigated in the present study is consistent with early latency or P1 components in the TA described by previous studies in the cat Taken in sum, our findings suggest that locomotor commands and afferent feedback during ARM&LEG movement produce the neural substrate that facilitates transmission of ELR pathways during TIB&SUR nerve stimulation. We suggest that signals for rhythmic limb movement converge onto common reflex pathways coming from cutaneous afferents of different nerves innervating the foot.We demonstrated that ELR amplitudes following TIB&SUR stimulation were strongly dependent on the task performed. That is, the amplitude of the ELR was significantly reduced when subjects performed ARM alone and LEG alone compared to ARM&LEG movement. This general feature of task dependency is in line with many previous reports obtained from individual nerve stimulation To provide a context for our findings, we propose a schematic representation of the possible neural mechanisms as shown in c.f.We found that rhythmic locomotor movement activates presumed common reflex pathways from sensory inputs innervating different foot regions. In addition, our results suggest that transmission in these reflex pathways is weighted according to the number of limbs directly engaged in human locomotor activity. Sensory information modulates motor output in locomotor generator systems in the spinal cord"} +{"text": "The severely limited ability of the heart to repair itself following infarction has led to the development of a number of novel cell therapies aimed at stimulating myocardial regeneration. One promising technique is cellular monolayers consisting of fibroblasts and cardiomyocytes applied directly to the infarct region to promote regeneration. The study aims to assess the success of this patch treatment in rats using a combination of magnetic resonance imaging techniques to perform regional strain analysis and assess myofibre remodelling and use optical imaging techniques for tracking therapeutic cell retention.Monolayers consisting of 50/50 neonatal rat cardiomyocytes and fibroblasts were cultured on temperature sensitive dishes and labelled with fluorescent cell tracker DiI. These patches were lifted and layered over the infarct region during permanent suture ligation of the coronary artery (n=2). Animals were scanned using a 9.4T MRI system 7 days post-infarction using a DANTE tagging sequence Fig. Hearts Regional function was analysed in 8 sectors of myocardium using inTag\u00a9 Fig . The conThis work is the first to combine advanced MRI tissue tagging and DTI with optical projection tomography to evaluate cardiac regenerative therapy. Initial data shows that treated hearts have reduced loss of contractility when compared to untreated infarctions. Myofibre remodelling in infarcted tissues is vital to functional recovery for infarcted hearts and DT-MRI can be used to monitor myofibre orientation and tissue anisotropy.Medical Research Council, UK."} +{"text": "Divergent selection and adaptive divergence can increase phenotypic diversification amongst populations and lineages. Yet adaptive divergence between different environments, habitats or niches does not occur in all lineages. For example, the colonization of freshwater environments by ancestral marine species has triggered adaptive radiation and phenotypic diversification in some taxa but not in others. Studying closely related lineages differing in their ability to diversify is an excellent means of understanding the factors promoting and constraining adaptive evolution. A well-known example of the evolution of increased phenotypic diversification following freshwater colonization is the three-spined stickleback. Two closely related stickleback lineages, the Pacific Ocean and the Japan Sea occur in Japan. However, Japanese freshwater stickleback populations are derived from the Pacific Ocean lineage only, suggesting the Japan Sea lineage is unable to colonize freshwater. Using stable isotope data and trophic morphology, we first show higher rates of phenotypic and ecological diversification between marine and freshwater populations within the Pacific Ocean lineage, confirming adaptive divergence has occurred between the two lineages and within the Pacific Ocean lineage but not in the Japan Sea lineage. We further identified consistent divergence in diet and foraging behaviour between marine forms from each lineage, confirming Pacific Ocean marine sticklebacks, from which all Japanese freshwater populations are derived, are better adapted to freshwater environments than Japan Sea sticklebacks. We suggest adaptive divergence between ancestral marine populations may have played a role in constraining phenotypic diversification and adaptive evolution in Japanese sticklebacks. Yet adaptive divergence and diversification is not ubiquitous; for example Darwin's finches have evolved remarkable phenotypic diversity in foraging behaviour and morphology, while Gal\u00e1pagos' mockingbirds have not Colonisation of new environments can lead to adaptive divergence, the evolution of reproductive isolation and progression towards speciation within evolutionary lineages de novo mutations e.g. the chance formation of novel island and lake environments and in turn, chance colonisation Many potential factors may constrain phenotypic diversification and adaptive divergence Gasterosteus aculeatus) offer an excellent example with repeated colonisation of freshwater environments throughout the Pleistocene resulting in parallel phenotypic adaptation and genomic divergence The formation of freshwater lakes and rivers by glacial and interglacial cycles has repeatedly created novel unoccupied niche space during the late Quaternary Despite freshwater colonization being characteristic of three-spined sticklebacks i.e. where only a single form occurs).Both lineages have extant marine forms that breed in brackish waters and rivers, the Pacific Ocean and Japan Sea anadromous forms (PA and JA hereafter). These lineages are reproductively isolated from one another due to hybrid male sterility i.e. gill raker morphology and resource use - has occurred between marine and freshwater populations in the Pacific Ocean lineage. In contrast, we expected the Japan Sea lineage to lack phenotypic diversification because of a lack of adaptive divergence and our results strongly indicate this is the case.Focusing on the Japan Sea and Pacific Ocean stickleback lineages we first asked whether phenotypic and ecological diversification rates are higher in the Pacific Ocean lineage compared to the Japan Sea lineage. Using phylogenetic comparative methods we quantified diversification in trophic traits, including gill raker number and resource use in both anadromous and freshwater populations. We expected that divergence in trophic ecology would be consistent with foraging trait divergence between lineages. This first part of our study aimed to explicitly test the hypothesis that significant adaptive divergence in freshwater foraging traits \u2013 Second, we asked how anadromous forms of the two lineages differ in trophic morphology, ecology and feeding behaviour, specifically focusing on sympatric (i.e. both forms co-occurring) and allopatric (i.e. only one form present) populations of JA and PA fish. Forms from both lineages migrate to coastal regions to spawn and previous research has suggested PA migrate further upstream than JA Animal use protocols were approved by the Institutional Animal Care and Use Committee of the National Institute of Genetics (23-15). Fish sampling in Hokkaido was conducted under a permit issued by Hokkaido Prefecture.Anadromous and freshwater stickleback populations (PF) were sampled across Northern Japan using minnow traps and seine nets between June 2006 and May 2012 . JA and Benthic macroinvertebrates were collected clinally from the lake in the Bekanbeushi and Shiomi river systems and marine invertebrates from Akkeshi Bay . SamplesDietary inference is an important means of determining habitat and resource use between divergent populations 13C and \u03b415N values from fish muscle tissue typically reflect dietary assimilation during spring/summer growth as during winter nutrients are used to sustain basal metabolic processes 13C and \u03b415N values may indicate habitat divergence throughout life history. Three-spined sticklebacks from each of the sample sites were processed for stable isotope analysis (n\u200a=\u200a314). Dorsal muscle was dissected from each fish, dried for 48 hours at 60\u00b0C, ground and weighed. Benthic macroinvertebrates (n\u200a=\u200a66) were similarly processed. Samples were analysed for \u03b413C, \u03b415N, % C and % N on a Carlo Erba Elemental Analyser and a Thermo Finnigan Delta Plus XL mass spectrometer at the Duke Environmental Isotope Analysis laboratory (DEVIL) at Duke University, North Carolina, USA and at UC Santa Cruz Stable Isotope Laboratory, California, USA. Prior to analysis, fish muscle \u03b413C values were lipid-normalised The long-term resource use signal from stable isotope analysis is informative for anadromous species making large distance migrations across salinity gradients. As ectotherms, \u03b4C) and Bayesian estimated standard ellipse area (SEAB).Carbon and nitrogen isotopes can also be used to quantify the isotopic niche, a proxy for ecological niche We additionally counted the number of gill rakers, a functionally important trait closely correlated with trophic ecology D and 2\u03b4\u03bcStn170, Stn233, Stn64, Stn159, Stn46, Stn90, Stn120, Stn278, Stn332 and Stn384) located on different three-spined stickleback linkage groups not linked to sex D performs best when divergence time is relatively recent whereas 2\u03b4\u03bc performs better when divergence is older ape so that only populations with ecological data remained Two phylogenetic trees were estimated from microsatellite data in anadromous and freshwater populations from the Japan Sea and Pacific Ocean lineages using both Nei's 13C and \u03b415N) we first used the method developed by O'Meara et al. (2006). This allows phenotypic traits to evolve along a phylogeny under Brownian motion (BM) and estimates the likelihood of two models; a single rate only and separate rates (\u03c32) for the Japan Sea (JS) and Pacific Ocean (PO) lineages. Lineage was mapped onto each tree and nested Brownian motion models were fitted using the brownie.lite function in phytoolsTo test for lineage specific rates of diversification for gill raker number and niche use using Post's Baseline-corrected \u03b4Isotope mixing models can estimate source proportions, providing a time-averaged indication of dietary preference i) Correlation between dietary preferences and ecomorphological traits is an proxy for detecting divergent natural selection between environments We also conducted benthic foraging experiments to test whether freshwater foraging efficiency was greater in PA. Fish used were captured from Bekanbeushi system, returned to the laboratory and kept for one month. Experiments were conducted in a 63-litre clear glass tank filled with 10% seawater. The tank was placed on the floor of a well-lit room with an ambient of temperature of 16\u00b0C. All sides apart from the front were covered to prevent startling the fish. Substrate consisted of fine sand and gravel, spread thinly to prevent benthic prey items from burying themselves beyond reach. Before the first trial on a given day, 60 live chironomid larvae were added to the test arena and spread at random across the substrate. Trials were then filmed using a SONY HVR HD 1000 placed at 1.5 m from the test tank. Trials were observed remotely and could be initiated, monitored and recorded without disturbing or startling the fish. A total of 42 trials were conducted over four days in June 2012 and the test arena was cleared, cleaned and refilled on each day.Artemia and frozen chironomids for one week and were then starved for 24 hours before a trial to ensure feeding. Trials were conducted using the following protocol; a fish was chosen at random from a holding tank and then placed in the test arena containing food items. Once a fish made a vertical strike at a prey item, recording was started and a ten-minute foraging trial initiated. If no strike was made within 10 min after introduction, the trial was ended and the fish removed. Following trial completion, successful or not, fish were removed and their standard length recorded. Following each trial approximately five chironomid larvae were added to the test tank and the substrate raked to ensure prey visibility.Fish were fed a diet consisting of live Measures of foraging efficiency were recorded from video footage. All vertical and horizontal strikes were counted; vertical strikes were defined as strikes made at a prey item resting on the substrate; horizontal strikes were defined as strikes made at prey suspended in the water column. The former captures the number of attempts to feed on new prey while the latter captures the number of strikes required to handle prey. Videos were reviewed again to calculate the number of chironomid larvae consumed per trial. The number of chironomids handled or abandoned was recorded and the difference equalled the number of chironomids consumed.preyT is total number of prey items consumed and VS and HS are vertical and horizontal strikes. This measure ranges from 0 to 1, with larger values indicating lower numbers of strikes per prey item consumed. We further calculated the number of vertical strikes per second to obtain a measure of foraging rate.We calculated ratios of the number of vertical and horizontal strikes as well as the number of abandoned prey items to the number of chironomids handled in order to give an indication of handling efficiency. We additionally calculated a measure of foraging efficacy.10 transformed prior to analysis; all other foraging efficiency measures were square root transformed. Foraging efficiency measures were tested using GLMs with species as a factor and standard length as a covariate. All statistical analysis was conducted using R 2.15.1 For SIA data, generalised linear mixed models (GLMMs) were used with species and site set as fixed or random factors depending on the test for all three of these trophic traits were significantly higher for the Pacific Ocean lineage than the Japan Sea lineage was found, indicating a functional link to trophic ecology, although there was no \u03b413C correlation (P\u200a=\u200a0.17).Mean gill raker number was lower in Pacific Ocean freshwater populations than in Pacific Ocean and Japan Sea anadromous forms . PA fish varied spatially for both \u03b413C and \u03b415N . This was largely driven by \u03b413C variation between fish captured at the bay site and those in the river (P<0.05 in both cases). Focusing on the bay and midstream sites where both species co-occur, \u03b413C differences occurred between sites and forms (P<0.0001) but no significant interaction could be detected (P\u200a=\u200a0.48), suggesting the difference between species did not vary between sites.Spatial variation in stable isotope values was also apparent within forms . JA sticP\u200a=\u200a0.002) while freshwater benthic prey was a more important prey resource for PA fish , consistent with a greater marine contribution to diet.Bayesian source estimation revealed a greater contribution of marine benthic sources to JA fish so size was included as a factor in the analysis to test for size specific effects. PA fish consumed a greater number of chironomids per trial than JA fish , a significant interaction indicated a size effect on wild fish captured from the Bekanbeushi system of which13C values for PA were lower than JA populations at both sympatric and allopatric sites, indicating greater freshwater foraging in the former and a significant species * distribution interaction indicated that JA fish occurring in sympatry with PA fish had a more marine \u03b413C signal than allopatric JA populations; no difference occurred between allopatric and sympatric PA populations and sympatric (i.e. both lineages are present) JA and PA populations differed in trophic ecology. Distribution did account for \u03b4ulations . IsotopiThe results of our comparative phylogenetic analyses show that rates of phenotypic and ecological diversification are higher in the Pacific Ocean stickleback lineage compared to the closely related Japan Sea lineage. Since marine-freshwater adaptive divergence has only occurred in the former, our findings support the hypothesis that colonization of freshwater environments facilitates the evolution of increased diversity in stickleback foraging traits between marine-freshwater populations within lineages. Different mean optimal gill raker numbers between JA and PA populations indicate divergent ecological selection also occurs between these anadromous forms. To further investigate differences in optimal trophic trait values, we quantified divergence in resource use, foraging morphology and behaviour between the anadromous forms (PA and JA) in these two lineages. PA exploited more benthic freshwater resources, consistent with fewer gill rakers and improved prey handling on benthic macroinvertebrates compared to JA in both sympatry and allopatry. Stable isotope analysis also suggested allopatric JA populations exploit more freshwater resources when PA are not present, suggesting the possibility that competition may occur between the two anadromous forms. Together, our results confirm that only the Pacific Ocean lineage has undergone extensive diversification in foraging behaviour, ecology and morphology as a result of marine-freshwater adaptation and that substantial ecological divergence has occurred between Pacific Ocean and Japan Sea anadromous forms.Sticklebacks have been extensively studied as model organisms for studies of adaptive divergence and evolution Selection between different habitats, resources and niches is the major determinant of adaptive divergence between populations In addition to our comparative phylogenetic results, our other analyses and previous research provides a strong case for adaptive divergence between anadromous and freshwater resident populations of the Pacific Ocean lineage. Our stable isotope data showed clear structuring along a marine-freshwater axis, with obligate freshwater PF populations at one end and JA populations at the other . While PEvolutionary diversification is closely linked to the colonization of new environments and establishment success often depends on the similarity of these environments to the source habitat as colonisers may already possess suitable adaptations Our stable isotope and stomach content analyses demonstrated divergent resource use between anadromous forms from the two lineages. PA stickleback exploited a greater proportion of freshwater resources than JA across their distribution with mean isotopic values more similar to freshwater resident populations (PF). Spatial isolation of spawning sites following migration upstream between the PA and JA forms at sites where both forms co-occur is supported by our present stable isotope data and also by previous longitudinal demographic studies . Habitat isolation arising as a by-product of divergent natural selection occurs quite readily between stickleback species pairs Our comparative OU analysis supported a three optimum model for gill raker morphology \u2013 i.e. Japan Sea fish have a higher mean number of gill rakers than Pacific Ocean anadromous and freshwater populations. Support for an additional optimum in Pacific freshwater populations suggests freshwater colonization is characterised by a reduction in gill raker number and a shift towards a new adaptive peak 15N values and higher trophic level in Japan Sea anadromous fish indicate increased pelagic diet. Correlations between morphology and behaviour are compelling but further work is necessary to test whether gill raker morphology directly increases foraging efficiency in the Japanese stickleback system.Our behavioural experiment supports a functional link between gill raker morphology and foraging efficiency in the sympatric Bekanbeushi population. PA fish consumed more benthic prey and demonstrated improved prey handling. As variation in gill raker number was not included in our experimental design, we cannot conclude that lower efficiency with benthic prey in JA fish is as a result of morphological adaptation to feeding on pelagic prey. However gill raker number has been shown to correlate closely with foraging efficiency in stickleback and other fish species Body size can also influence prey capture success in fishes and may be more important in determining benthic foraging success than trophic morphology Although our present study has focused on ecological factors arising from differences in foraging and morphology, physiological constraint may have also played a role in preventing marine-freshwater adaptive divergence in the Japan Sea lineage. \u2018Key innovations\u2019 are adaptive traits which allow new niches to be exploited and can increase diversification, potentially leading to adaptive radiations when they arise 13C values in allopatric JA populations suggest that the JA form is able to make greater use of freshwater resources when PA fish are absent. Competition for resources can also have a negative effect on population densities during colonization Ecological interactions, such as competition for resource use, may also play an important role in shaping colonization of and adaptation to novel environments Priority effects may also explain the difference in the ability of the two lineages to adapt to freshwater. Priority effects are species-specific interactions that influence establishment and fitness Pungitius spp.) could also act as a competitor, having adapted to freshwater environments connected to the Sea of Japan Competition may play some role in determining differences in resource use when the two lineages occur in sympatry, however it seems unlikely this is the sole explanation for the failure of the Japan Sea lineage to diversify by colonizing freshwater environments, Resource competition would only prevent colonisation where the two forms overlap and therInternal and external constraints on adaptation and phenotypic diversification should not be considered in isolation as the interaction between both classes of constraint is often more informative Genomic constraints on phenotypic diversification remain uncertain, although the loss of allelic variants underlying traits may constrain adaptive divergence Understanding factors that facilitate or constrain adaptive divergence, phenotypic diversification and adaptive radiation is a fundamental question for evolutionary biologists. Both phylogenetic and experimental data have demonstrated that ecology of founders can influence the patterns of adaptive radiation Ecological opportunity is thought to be key for phenotypic diversification File S1Supplementary text, tables and figures.(DOCX)Click here for additional data file."} +{"text": "In-vitro differentiated islets from various adult stem cells sources were assessed at morphological, molecular, immunological and functional level and further evaluated for glucose lowering effect after transplantation in Streptozotocin (STZ) induced diabetic balb/c mice.In the area of regenerative medicine, researchers have been exploring the potential of Stem Cells in preclinical and clinical studies to improve therapies that can resolve injuries by enhancing endogenous repair, opening a new paradigm in cell therapy.Stem cells may it be embryonic stem cells (ESC), induced pluripotent stem cells (iPSC) or adult stem cells have been used as potential sources for beta cell replacement therapy across the globe. Numerous studies have shown the differentiation potential of ESCs and iPSCs towards producing pancreatic progenitors but after terminal differentiation into insulin producing cells these lacked various important pancreatic markers and have low index of glucose stimulated insulin secretion (GSIS). This along with other ethical conundrums makes it difficult to use ESCs as a source for cell therapy. Adult stem cells provide clinically and ethically accepted option over embryonic or induced pluripotent stem cells, as they can be used as autologous source for cell transplant in treatment of diabetes. Widely used option of cadaveric islet therapy has its own problems due to insufficient amount of islets available for transplantation. Hence, our research is focused on increasing islet mass from various adult stem cells using neutraceutic bioactive compounds isolated from a plant demonstrating efficient anti-diabetic activity and further explored them as potent differentiating agent under"} +{"text": "However, the wide dynamic range of velocities in the left ventricle can create difficulties when optimizing acquisition protocols, especially when attempting to measure complex flow features during filling and diastasis. Additionally, there is no reference standard method to validate velocity flow and wall shear stress measurements. Here we describe our experience with a custom-built MRI compatible physiologic left heart flow phantom which can be used to optimize acquisition parameters, and can be used in the lab to make velocity measurements with particle image velocimetry (PIV).The left heart flow phantom Figure consistsThe 4D PCMR images captured the 3D velocity field within the LV throughout the cardiac cycle. The spatial and temporal resolution chosen was sufficient enough to extract and reconstruct the temporal motion of the ventricular wall Figure . The anaA 4D PCMR scan was performed on the left heart flow phantom developed for this study. The scan parameters used here found that 3D blood flow and wall motion information were extracted; nonetheless, further optimization such as k-t undersampling may allow for higher resolution and shorter scan times. The left heart flow phantom can be used to identify (or optimize) alternative MRI sequences to research valvular and ventricular pathologies.NHLBI (RO1HL07262)."} +{"text": "A tremendous leap in the field occurred using mouse ES and iPS cells wherein they were first differentiated into epiblast-like cells and then primordial germ cell-like cells. These on further development produced sperm, oocytes and live offspring (had associated genetic problems). Evidently differentiating pluripotent stem cells into primordial germ cells (PGCs) remains a major bottleneck. Against this backdrop, we propose that a novel population of pluripotent stem cells termed very small embryonic-like stem cells (VSELs) may serve as an alternative, potential source of autologus gametes, keeping in mind that they are indeed PGCs surviving in adult mammalian ovaries and testes. Both VSELs and PGCs are pluripotent, relatively quiescent because of epigenetic modifications of parentally imprinted genes loci like Igf2-H19 and KCNQ1p57, share several markers like Stella, Fragilis, Mvh, Dppa2, Dppa4, Sall4, Blimp1 and functional receptors. VSELs are localized in the basement membrane of seminiferous tubules in testis and in the ovary surface epithelium. Ovarian stem cells from mouse, rabbit, sheep, marmoset and humans spontaneously differentiate into oocyte-like structures in vitro with no additional requirement of growth factors. Thus a more pragmatic option to obtain autologus gametes may be the pluripotent VSELs and if we could manipulate them in vivo \u2013 existing ethical and epigenetic/genetic concerns associated with in vitro culture may also be minimized. The field of oncofertility may undergo a sea-change and existing strategies of cryopreservation of gametes and gonadal tissue for fertility preservation in cancer patients will necessitate a revision. However, first the scientific community needs to arrive at a consensus about VSELs in the gonads and then work towards exploiting their potential.The urge to have one\u2019s own biological child supersedes any desire in life. Several options have been used to obtain gametes including pluripotent stem cells (embryonic ES and induced pluripotent iPS stem cells); gonadal stem cells , bone marrow, mesenchymal cells and fetal skin. However, the field poses a huge challenge including inefficient existing protocols for differentiation, epigenetic and genetic changes associated with extensive Premature ovarian failure (POF) is a heterogeneous disorder that occurs at the frequency of less than 1% in women less than 40\u00a0years of age. Besides genetic basis and autoimmune etiologies, POF is caused by surgical removal of ovaries for conditions such as severe endometriosis, cancer and also as a side effect of oncotherapy for various non-gynecological malignancies. Similarly, besides a genetic basis, azoospermia in men occurs as a side effect of oncotherapy or infections. The option to preserve fertility prior to oncotherapy by way of cryopreservation of gametes or embryos is not yet widely available in several countries and also not useful to young pre-pubertal cancer patients due to non-availability of gametes. Women willingly go through 6\u20137 failed IVF cycles with a hope to become pregnant. However, assisted reproductive technologies of IVF and ICSI fail to benefit 30% of couples diagnosed with unexplained infertility and in cases where patients are entirely devoid of viable gametes. Donor gametes or adoption are available options however, the urge to have one\u2019s own biological child supersedes any other desire in life. Recent advances in the field of reproductive medicine are focused on exploiting pluripotent stem cells to differentiate into gametes with a hope to deal with infertility.Gametes derived from pluripotent stem cells may provide potential reproductive options to individuals who are rendered infertile due to injuries, exposure to toxicants or immune-suppressive treatments, in cases with gonadal insufficiency due to premature ovarian failure or azoospermia, reproductive aging and idiopathic cases of poor gametes quality and IVF failure. These artificial gametes derived from stem cells may also serve as an invaluable model system to study both genetic and epigenetic programming of germ cells development First human pluripotent embryonic stem (hES) cell lines were reported more than 15\u00a0years ago . We encoCell that it is possible to obtain live pups from sperm derived from pluripotent stem cells (ES or iPS cells) [Science that following a similar strategy, offspring are obtained from oocytes derived from ES or iPS cells [in vitro what happens in vivo during early embryo development. Two main strategies that have been used in the past to induce germ cells from pluripotent stem cells (PSCs) include (i) spontaneous differentiation of PSCs to make embryoid bodies (EBs), isolate cells expressing germ cell markers for further manipulation and (ii) to use mouse epiblast stem cell lines to obtain germ cells. Both these approaches, although provide proof of concept that it may be possible to differentiate PSCs into germ cells, remain highly inefficient. Primordial germ cells (PGCs) are available in very few numbers and are relatively quiescent and thus the embryonic germ cell lines derived from them [in vitro culture and parallel studies in humans remain a distant dream [It is intriguing to note that offspring born when starting with PGCs are normal compared to when starting with ES/iPS cells. Hayashi et al. [A careful review of published literature shows that a group from Japan, including Prof. Hayashi and Prof. Saitou has achieved major progress in the field of generating gametes from mouse pluripotent stem cells (mES/iPS cells). In 2011 they published in S cells) . In 2012PS cells . In 2013PS cells . Basic rrom them , 9 have nt dream transplant dream reportednt dream and Hashnt dream reportedi et al. reviewedin vitro in mice [in vitro growth and in vitro maturation however challenges remain to be overcome and to develop a perfect culture to obtain a healthy oocyte from primordial follicle [Work on spermatogonial stem cells (SSCs) has progressed and recent reports suggest that it may be possible to expand SSCs in mice and also in mice . However in mice who obta in mice , 22. Til in mice . OSCs ar in mice . Recentl in mice . Severalfollicle .bone marrow[as well as MSCs[have a sub-group of pluripotent very small embryonic-like stem cells (VSELs) which could possibly be responsible for the observations made by various groups . During the ageing process, proliferation-repressive epigenetic marks progressively disappear, resulting in the increased sensitivity to Ins/Igf signaling and thus depletion of VSELs [a common population of VSELs exists in adults that undergoes hematopoiesis in bone marrow and gametogenesis in the gonads. It is time to think beyond the existing paradigm that PGCs migrate only to the gonadal ridge and give rise to germ cells \u2013 rather they possibly migrate and settle in various adult organs and survive throughout life serving as a backup pool for tissue committed stem cells.After gastrulation, majority of epiblast stem cells lose expression of pluripotency transcription factors and further develop into somatic organs whereas the pluripotency markers are selectively expressed in the PGCs Figure\u00a0. Variousof VSELs . A direcof VSELs . A lot oVSELs (PGCs) have been reported in adult human and mousadult peri-menopausal ovarian VSELs express Stella and Fragilis (specific markers for PGCS) suggesting that the VSELs are indeed the PGCs that survive into adulthood.Thus of the two models proposed by Felici and Barrios , we agrein vitro. Anand et al. [We have further observed that VSELs in the mouse ovary and testis survive chemotherapy in agreement with earlier report in mouse bone marrow after total body irradiation . VSELs ed et al. recentlyThis distinct expression profile of VSELs isolated from adult human ovary shows that they are more related to PGCs than ES cells.Table\u00a0Using adult mice, our group has recently documented the effect of ovarian stimulation on the stem cells (VSELs and OSCs) localized in the OSE . Ovariesin vitro. Oocyte-like structures were obtained in vitro using samples collected from menopausal women as well as those who had premature ovarian failure unlike the conventional IVF procedure where maternal age more than 35\u00a0years is considered as a high risk due to the genetic abnormalities. Later Virant-Klun and her group [in vitro oogenesis in adult human OSE cultures along with characteristic expression of stem/germ cell/oocyte markers [Bukovsky et al. , 75 firser group \u201378 reporer group . We also markers . Time la markers , 79.No additional growth factors are added to the medium to induce differentiation of oocyte-like structures. It appears that the VSELs in the OSE scrapings are pre-programmed to differentiate into oocytes. This is indeed facilitated by the epithelial cells which form a bed of fibroblasts and were present in close association with the differentiating stem cells. Similarly the isolated OSCs also undergo spontaneous differentiation into oocytes in culture [The striking fact is the spontaneous nature of this kind of differentiation of VSELs into oocyte-like structures. culture , 43, 80. culture have shoin vitro during OSE culture. Similar in vitro culture studies are ongoing in our lab using testicular VSELs. More studies are required to further substantiate the potential of VSELs and their ability to differentiate into gametes. We propose that rather than the existing concept of in vitro differentiation of stem cells into oocytes and sperm for assisted reproduction, it would be ideal to manipulate VSELs that survive oncotherapy in vivo to achieve restoration of gonadal function .It may be possible to obtain human gametes provided efficient and directed differentiation of ES or iPS cells into PGCs is achieved. But this may not be mandatory since emerging literature suggests that PGCs persist as a sub-population of VSELs along with SSCs in testis and OSCs in ovary. Similar to the PGCs, VSELs are quiescent in nature, do not expand in culture like ES or iPS cells and throughout life serve as a backup pool and give rise to SSCs/OSCs which undergo clonal expansion, meiosis and further differentiation to produce haploid gametes. Ovarian VSELs respond to FSH via FSHR3 and spontaneously differentiate into oocyte-like structures In contrast to genetically affected offspring born from ES/iPS derived gametes, healthy offspring born starting with OSCs and the oocytes formed after in vitro spontaneous differentiation of ovarian stem cells show normal ploidy status. This is evidently because of the similar epigenetic status of PGCs and VSELs which is possibly difficult to be replicated in vitro while differentiating ES/iPS cells into PGCs . Scientific community needs to slow down, re-think and make efforts to exploit clinical potential of pluripotent stem cells (VSELs) and progenitors (SSCs and OSCs) which exist in the adult gonads as an alternate option to ES/iPS cells!\u2009Current status of making gametes from pluripotent stem cells (ES and iPS) to help infertile couples is highly inefficient and still remains a distant dreamin vitro. PGCs are pre-programmed and hence easily and spontaneously differentiate into gametes\u2009Major obstacle in the field is apparently to establish protocols to obtain primordial germ cells (PGCs) from the pluripotent stem cells (ES and iPS) \u2009Published literature is reviewed suggesting that this challenge of making gametes can be easily overcome since PGCs indeed survive in adult human ovaries and testes as very small embryonic-like stem cells (VSELs)\u2009VSELs are pluripotent stem cells (surviving PGCs) which exist as a sub-population localized in the adult ovary surface epithelium and in the basement membrane of seminiferous tubules in the testes. They are present in normal adult and aged testes and ovaries . Moreover VSELs survive oncotherapy because of their quiescent nature.\u2009Three weeks culture (simple culture medium with no added growth factors) of ovary surface epithelial cells enriched with VSELs and ovary stem cells (OSCs) spontaneously differentiate into oocyte-like structures - because the gonadal VSELs (PGCs) and OSCs (arise from the VSELs) are pre-programmed to develop into gametesin vitro, a better approach will be to manipulate them in vivo to give rise to functional gametes. This approach will give rise to autologus gametes, with no associated ethical/regulatory constraints and epigenetic/genetic issues may not exist by avoiding in vitro culture.\u2009We propose that rather than manipulating gonadal VSELs (PGCs) DB is working on pluripotent stem cells for almost 11\u00a0years. IH is leading IVF expert and well understands the need of synthetic gametes by infertile couples. HP is a PhD student at NIRRH and RB works at Hinduja Hospital."} +{"text": "Administration of trials involving large numbers of participants, coordinating multi-centred sites or drug management can all result in major logistical challenges for any trial. The enormity of these challenges can be drastically reduced through the adoption of healthcare informatics.ECLS (Early Cancer detection test - Lung cancer Scotland) is a high impact study with the aim of detecting often-deadly lung cancer at an earlier and more treatable stage. This project, a collaboration between researchers at the Universities of Dundee, Glasgow and Nottingham, NHS Scotland, the Tayside Clinical Trials Unit (TCTU) and supported by the Health Informatics Services Centre (HIC), provides a prime example of how healthcare informatics can be successfully applied through the full life cycle of a trial:\u2022 A web based Patient Recruitment system is being utilised to invite ~50,000 potential participants to take part in this study. This provides the facility to track participant progress, generate online reports and record participant appointments.\u2022 A Letter App supports the generation of letters as required by the study (e.g. appointment/result letters).\u2022 The Tayside Randomisation System (TRuST) balances the randomisation allocation for this study based on a minimisation with stratification algorithm. TRuST can also be utilised for allocation clustering preventing potential contamination and drug management offering full accountability.\u2022 On completion of recruitment the study will employ the services of the HIC Data Linkage Service to follow up participants and link to their routinely collected electronic health records.\u2022 HIC Services further supports clinical trials through the creation of study websites and data collection tools."} +{"text": "Massive pulmonary embolism (PE) is frequent lethal, but rapid diagnosis and aggressive therapy with endovascular or surgical thromboembolectomy and extracorporeal membrane oxygenation (ECMO) may be lifesaving. We reviewed our current cases to find that whether the aggressive surgical approaches is the best alternative or not.Five female patients were diagnosed as massive pulmonary embolism with either acute irreversible pulmonary failure or cardiac collapse (2 CPR) by Chest CT or pulmonary angiography. All patients required ECMO support in addition to 2 endovascular Angiojet aspiration or 1 surgical pulmonary thromboembolectomy. The duration for ECMO survivors were 1, 2 and 7 days, respectively. All patients required anticoagulation to resolve the residual emboli.One died from ECMO cannula insertion complication of massive retroperitoneal hematoma and bleeding and the other one died of multiorgan failure (MOF). Three were weaned from ECMO and was discharged in good condition at follow-up.Aggressive endovascular or surgical pulmonary thromboembolectomy in combination with ECMO appears to have beneficial effects for massive pulmonary embolism with acute cardiopulmonary failure."} +{"text": "Invasive right heart catheterization plays a central role in the investigation of patients with cardiac and pulmonary vascular disease. Physiological provocations during invasive heart catheterization augment the diagnostic yield and can provide useful prognostic information. MRI catheterization combines invasive hemodynamic measurements with MRI structural and functional evaluations - thus providing superior diagnostic information than either test alone. An additional benefit to both patient and operator is no ionizing radiation, which is of particular value in pediatric patients.We propose a diagnostic algorithm for MRI catheterization integrating serial physiological provocations designed to unmask latent pathology and aid prognostication. The algorithm allows each MRI catheterization to be tailored to the individual patient. We have developed a concise MRI examination protocol to be performed alongside invasive hemodynamic measurements during each provocation. This protocol provides cardiac chamber volumes, pulmonary and systemic blood flow measurements with each provocation. The MRI examination is performed free breathing, which allows the study to be performed in patients with dyspnea or under moderate sedation.Real-time MRI provides excellent anatomical imaging for catheter navigation. Challenging procedural steps using X-ray guidance, such as navigating into the SVC Figure or left Provocative MRI catheterization can uncover diagnoses not apparent at rest and can provide incremental structural and functional information compared with invasive heart catheterization or MRI alone.This work is supported by the Division of Intramural Research , National Heart Lung and Blood Institute, National Institutes of Health, USA."} +{"text": "AbstractPrimary torsion of the omentus is an extremely unusual cause of acute abdomen in the pediatric population. This condition occurs from twist of the pedicle of the omental apron around its longer axis, leading to edema, ischaemia and necrosis. Here we present a rare case of a 9 year old girl referred by her general practitioner due to severe right lower quadrant abdominal pain with a presumed diagnosis of acute appendiceal inflammation. Surgical operation disclosed primary omental torsion. The infarcted segment was resected and the girl\u2019s clinical recovery was uneventful without any complication. The condition may mimic a variety of other causes of acute abdominal symptoms. In this case report, a presumed diagnosis of acute appendicitis urgently induced the decision of a surgical approach. Physicians involved in the acute pediatric care have to include this rare condition in the differential diagnosis of acute onset right-sided abdominal pain. It is remarkable that Kimber et al, in a 20 years retrospective study among a paediatric population reported that the ratio of primary omental torsion to appendicitis was found less than 4/1000 respectively [3]. In a similar 10 year study in Greece this ratio was found 1/587 with overweight males to be mainly affected [2]. Primary omental torsion represents an extremely rare etiology of acute abdomen . Ultrasound (US) examination showed a small amount of free fluid in the pouch of Douglas. Visualization of the appendix was not feasible, while ovaries were found normal. Due to the increasing character of the abdominal pain, the girl was transferred to the operation room. Intraoperatively, during exploration of the peritoneal cavity was detected in the sero-sanguineous fluid. Appendix was found normal. Further exploration for Meckel\u2019s diverticulum was negative. Torsion of the right omental part around its long axis (4]. In regard to pathogenesis, torsion of the omentum around a pivotal point impairs its vascular perfusion resulting to severe congestion and oedema . Spontaneous derotation may occur at this stage [6]. However, if the torsion continues, edema progresses to acute hemorrhagic infarction and omental necrosis [7]. Extravasation of a sero-sanguineous liquid into the peritoneal cavity is often detected during surgery [5]. Omental torsion was first described in 1899 by Eitel [1]. Secondary omental torsion occurs more frequently than the primary form and is associated with intra-abdominal inflammatory situations, tumours or cysts [8]. Morris et al reported that the majority of secondary omental torsion cases are presented in patients suffering from inguinal hernia [9]. Regarding the primary or idiopathic form, it occurs in the absence of any intra-abdominal disorder [5]. The condition according to its aetiology is further classified as primary or secondary to certain predisposing risk factors [1]. Intraoperatively, twist of the greater omentum around the right epiploic artery is often observed [10]. Increased mobility and length of the right side of the omentum may explain the increased prevalence of right omental infarction [11]. Factors that predispose a patient to torsion include congenital pathological variations of the omentum. Among these are reported bifid omentum which is an accessory omentum derived from a narrow route, abnormal embryological position of the right omental part with secondary fragile blood vessels [12] and irregular accumulation of omental fat [1]. Precipitating factors are those causing sudden omental displacement and include trauma, coughing, violent exercise, hyperperistalsis and compression between abdominal wall and the liver [1]. A certain causative mechanism for primary omental torsion has not yet been established [1]. Acute appendicitis, acute cholecystitis [5] and diverticulitis are conditions included in the differential diagnosis [1]. Right sided acute abdominal pain, fever, nausea and vomiting are the most frequently encountered symptoms at onset [13]. Physical examination reveals signs of peritoneal irritation with guarding and rebound abdominal tenderness [14]. A palpable abdominal mass has been also reported in rare cases [13]. Remarkably, patients with omental torsion share a more prolonged clinical course with less systematic manifestations compared to acute appendicitis [15]. Patients with omental torsion may present with a variety of non-specific symptoms and can mimic various aetiologies of acute abdomen [14], the pathology is rarely diagnosed preoperatively, making exploratory laparotomy the most optimal diagnostic and therapeutic modality [5]. Surgical management of the primary omental torsion includes resection of the affected omental part with or without appendectomy [16]. Laparoscopic approach has been reported to be a safe and effective option [14]. Although conservative management has been described in some reports, surgery has been recommended as the treatment of choice in order to prevent severe complications related to conservative therapy such as sepsis and intra-abdominal abscess formation [14]. Although, US and Computed Tomography imaging are useful tools that assist the diagnosis by excluding acute appendicitis, cholecystitis and diverticulitis [ In this case report, a presumed diagnosis of acute appendicitis urgently induced the decision of a surgical management. The intraoperative diagnosis was this of omental torsion. Describing, in detail, this episode of care which occurred in a 9 year old child, it is crucial to properly rule out causes of abdominal symptoms.We presented an unusual case of primary omental torsion in a non-obese 9 years old girl. Although accurate preoperative diagnosis is seldom feasible, physicians involved in the pediatric care have to consider this condition in cases of acute onset right-sided abdominal pain."} +{"text": "To provide knowledge about the clinical presentation of eating disorders in young males (< 18 years).The sample comprised young males with eating disorders (n = 53) and females with eating disorders (n = 704). The data source was the Helping to Outline Paediatric Eating Disorders (HOPE) Project registry (N ~ 1000), a prospective and ongoing registry study comprising consecutive paediatric tertiary eating disorder referrals.Young males with eating disorders more commonly presented with unspecified eating disorders (40%). In comparison to young females with eating disorders young males were less likely to report self-induced purging, endorsed lower weight concern, and presented with an earlier age of onset. Young males and females presented with a similar duration of untreated illness.Young males with eating disorders are an understudied group who are systematically different from young females with eating disorders. Diagnostic classification, assessment instruments, conceptualisation and treatment methods need to be refined to improve application to young males.Service Initiatives: Child and Adolescent stream of the 2014 ANZAED Conference.This abstract was presented in the"} +{"text": "Most cancers have intact mitochondria function and we previously showed that mitochondria metabolism and ROS is essential for tumorigenesis. However, there are a subset of cancers which arise from mutations which abolish activity of TCA cycle enzymes, including Fumarate Hydratase (FH) deficient renal cancer cells. While oxygen consumption is severely reduced in FH deficient human cancer cells, we uncovered that they are still dependent on mitochondrial metabolism and ROS for cell proliferation. Based on these results we propose that mitochondrial metabolism and/or ROS could be an attractive target for therapy. We will discuss the role of mitochondria in regulating cancer and how metformin might exert its anti-tumorigenic properties by inhibiting certain aspects of mitochondrial function."} +{"text": "Foot muscle weakness has been linked with painful foot pathologies. This systematic review evaluated the relationship between foot muscle weakness and foot pain in adults.Electronic databases and reference lists were searched for all years up to March 2013. Two independent reviewers rated all included papers for methodological quality using a modified checklist from the Quality Index Tool. Due to the heterogeneity of studies, no data were pooled for meta-analysis.Seven studies evaluated the relationship between foot muscle weakness and foot pain. Methodological quality varied from poor (40%) to very good (89%). Four studies reported a significant relationship between foot muscle weakness and foot pain. Participants with plantar fasciitis were reported to have significant foot pain associated with a decrease in the cross-sectional area of the forefoot musculature and reduced toe flexor force. A study considering non-specific foot pain found a significant difference in dynamic toe flexor force between participants with disabling foot pain versus no pain on some day(s) and on most/every day. Finally, a clinical trial evaluating hallux limitus reported a significant improvement in pain and hallux plantar muscle strength after treatment. Of the three studies reporting no association, two reported only on hind foot muscles and one had a restricted sample. Summary of data extracted and quality index scores is shown in Table Despite some conflicting data encountered in this systematic review, there is evidence of a significant association between foot pain and muscle weakness, primarily related to toe flexion and foot pain, when the pain is of frequent disabling intensity."} +{"text": "Clear guidelines have been published by ESPGHAN and RCPCWe have observed 5 infants (over 18 months) where there has been emergence of IgE mediated allergic symptoms on reintroduction of cow\u2019s milk following an exclusion diet for intolerance symptoms such as GORD. All infants had been sensitized to cow\u2019s milk, and had been able to tolerate it without immediate hypersensitivity prior to the exclusion diet. Not all had allergy testing prior to challenge.This has not been described in the literature following exclusion for GORD, but there have been reports of acute allergic reaction following exclusion diet in the management of severe eczema. Flinterman el al. describeThis finding has led us to consider changing our practice. We will now perform allergy testing of all children (SPT +/-RAST) who have been following an exclusion diet for intolerance symptoms before proceeding to formal milk challenge. This is because of the chance of developing acute allergic reactions. Parents should be counselled of this risk."} +{"text": "Identifying populations within tree species potentially adapted to future climatic conditions is an important requirement for reforestation and assisted migration programmes. Such populations can be identified either by empirical response functions based on correlations of quantitative traits with climate variables or by climate envelope models that compare the climate of seed sources and potential growing areas. In the present study, we analyzed the intraspecific variation in climate growth response of Douglas-fir planted within the non-analogous climate conditions of Central and continental Europe. With data from 50 common garden trials, we developed Universal Response Functions (URF) for tree height and mean basal area and compared the growth performance of the selected best performing populations with that of populations identified through a climate envelope approach. Climate variables of the trial location were found to be stronger predictors of growth performance than climate variables of the population origin. Although the precipitation regime of the population sources varied strongly none of the precipitation related climate variables of population origin was found to be significant within the models. Overall, the URFs explained more than 88% of variation in growth performance. Populations identified by the URF models originate from western Cascades and coastal areas of Washington and Oregon and show significantly higher growth performance than populations identified by the climate envelope approach under both current and climate change scenarios. The URFs predict decreasing growth performance at low and middle elevations of the case study area, but increasing growth performance on high elevation sites. Our analysis suggests that population recommendations based on empirical approaches should be preferred and population selections by climate envelope models without considering climatic constrains of growth performance should be carefully appraised before transferring populations to planting locations with novel or dissimilar climate. Climate change poses serious threats to the ability of forests to provide multiple ecosystem services . In manyPseudotsuga menziesii [Mirbel] Franco). Due to its superior growth, wood quality and market value and x refers to DBH [cm]. RMSE refers to root mean squared error.The quadratic forms are y = a + bx(DOCX)Click here for additional data file.S2 Tables).Here: x = MATp (MAT of population origin);Y = H24 [m] andY1 = BA24 [m2ha-1].Column 1 shows the trial sites from which anchor points were developed. Figures within parentheses refer to mean annual temperature (MAT) of the respective trial sites (MAT(DOCX)Click here for additional data file.S3 TableFor definition of the acronyms see (DOCX)Click here for additional data file.S4 TableThe table gives the mean, median, maximal and minimal correlation coefficients of the individual trials as well as correlation across all trial sites, demonstrating the equal contributions of tree density and DBH to basal area.(DOCX)Click here for additional data file."} +{"text": "Studies designed to promote unbiased research increasingly show that human preferences exert a major influence on randomised controlled trials (RCTs) -3. More We collected 20 unselected and consecutive RCTs published in a high impact paediatric journal from July to November 2013. Two experienced reviewers identified the following five domains and a grading method to score discrepancy between what author\u2019s state in clinical trial registries (CTRs) and report in published RCTs: reported funding (1 point), study hypotheses, information on patients enrolled and study conduction (3 points); primary and secondary outcomes, early study completion, and upgrading or downgrading outcome results (5 points). Higher scores implied marked discrepancy. Two reviewers then independently applied the method on the RCT sample by mapping and coding information for the domains identified and reported discrepancies by comparing CTRs and RCTs and 4 had discrepancy in declaring funding. In 12 studies researchers completed the study early and in 8 studies they downgraded or upgraded outcomes. Only 5 CTRs were updated but they neglected to include published RCT results. Only in 5 CTRs, dataset supervisors indicated the RCT URL. None of the 20 RCTs allowed us to assess patients\u2019 preferences . No difference was found in discrepancy scores among the five CTR databases.The high discrepancy scores obtained by comparing what researchers stated in CTRs and published in RCTs suggest possible misconduct. Patients\u2019 preferences during RCT enrolment and conduction remain undetectable owing to the lack of targeted protocols to elicit this issue. These results, if confirmed in further studies, should prompt international regulation developers to encouClick here for file"} +{"text": "High field homogeneity is needed for a good MRI acquisition in general as well as in certain applications. Typically, active shimming using dedicated coils is applied to improve the field homogeneity. However, these techniques are limited especially for localized distortion profiles with higher-order characteristics caused by PET/MRI inserts. As a consequence, we are exploring the potential application of shimming on PET detector level (for the Hyperion-IID PET/MRI insert), meaning that the distortion profile caused by PET modules is compensated using additional magnetic materials (passive shimming) and DC coils (active shimming). To explore the technique, B0 field measurements have been performed using a whole-body phantom in combination with the MRI body coil. An FFE sequence was used to measure distortion maps of DC loops and small magnetic objects . These distortion maps served as input for a software framework which has been written to perform the field optimization. The implementation was verified by measurements and fits were performed to extract characteristic parameters of the tested objects. Finally, the implemented software framework was used to homogenize a measured distortion map produced by a single PET module by superimposing distortion corrections from additional simulated materials. The resulting superimposed distortion map showed a significantly improved B0 field map quality . The simulated susceptibility distribution will be applied on PET module level and tested in experiments. Results and details about this study will be presented at the conference.Combining PET and MRI into a hybrid device is challenging since both systems might influence each other. A typical interference problem of such a combined device is the distortion of the MRI\u2019s B"} +{"text": "The multi-step treatment of severe dentofacial deformities starts after the termination of skeletal bone growth. In cases of additional transversal discrepancies the SARPE procedure has to be executed at first, followed by the orthodontic alignment of the dental arches. Then the at least three step treatment starts: it consists of the pre- and postoperative orthodontic treatment and the treatment in between surgical correction. This combined treatment may last up to three years and longer.For economic reasons as well as for an increasing motivation of the patient the \u2018Surgery First\u2019 concept was developed to reduce treatment time. We take advantage of the Regional Acceleration Procedure (RAP), which is known since K\u00f6le published the interdental corticotomy in the crowded lower front to accelerate the orthodontic alignment in the 1950s.Instead of hyrax screws for transversal widening we prefer he application of bone borne devices for SARPE procedures. Fixed appliances treatment starts earlier using RAP effect.Instead of preoperative orthodontic treatment we start with the operation and align the dental arches postoperatively within 6-8 months.Advantages of \u2018Surgery First\u2019:\u2022 The patient sees the operative results immediately.\u2022 Orthodontic treatment is accelerated by RAP effect.\u2022 Treatment is completed after 6-8 months, sometimes after 10 months.Disadvantages of \u2018Surgery First\u2019:\u2022 Orthodontists must be experienced and needs to plan the treatment in advance.\u2022 More close cooperation between surgeon and orthodontist is needed.\u2022 Orthodontic treatment starts 1-2 weeks after the operation with intervals of two weeks.\u2022 RAP effect takes three to four months.The \u2018Surgery First\u2019 concept can be performed easily with stable results in Angle Class II and III cases with crowding, frontal compensation and pronounced Spee curve. (Segmented) Bimaxillary osteotomies with or without CWR or CCWR can be performed with additional procedures as chin correction or jaw bone augmentation.The \u2018Surgery First\u2019 concept delivers safe results also in open bite cases, elongated front regions, minor transversal discrepancies and asymmetries. In cases of severe facial deformities and transversal discrepancies \u2018Surgery First\u2019 cannot be recommended yet."} +{"text": "Systemic lupus erythematous (SLE) is a complex disease which is rare in childhood and can present insidiously. Libman-Sacks endocarditis (LSE) is a recognised cardiac manifestation of SLE with valvular abnormalities that are clinically silent prior to significant valve dysfunction. Multiple reports have associated isolated mitral regurgitation with SLE and the presence of antiphospholipid antibodies in SLE patients increases the prevalence of mitral valve regurgitation by three fold.To highlight awareness of Libman-Sacks endocarditis as a presentation of juvenile SLECase reportWe present a case of a 17 year old boy with phenotypic Noonans (SHOC2 mutation) who presented aged 14 with a pericardial effusion, verging on tamponade and requiring surgical drainage and diuretic therapy. A previous cardiac ultrasound aged 12 had shown no significant abnormality. One year later he developed mitral regurgitation, deteriorating over the following 18 months with the development of increasingly severe congestive cardiac failure (WHO class IV). Further history revealed new onset headaches and difficulty in concentration as well as a history of intermittent arthralgia. There were no rash, fever or mouth ulcers.Investigations demonstrated lymphopenia, prolonged APTT with positive lupus anticoagulant and anticardiolipin antibodies, raised immunoglobulin levels, low C4, persistently raised ESR but normal CRP as well as strongly positive ANA(1:2560) and anti-DNA antibody(86 IU/ml). Blood cultures, throat swabs and viral serology were negative. A diagnosis of SLE with Libman-Sacks endocarditis was made. There was no evidence of renal involvement. A course of intravenous methylprednisolone followed by oral steroids was given to minimise active inflammation prior to mitral valve replacement with a mechanical valve. Histopathology of the damaged mitral valve demonstrated fibrinous deposits with neovascularisation and myxoid degenerative changes, consistent with LSE. He made a good recovery following surgery with resolution of his dyspnoea. He has been anticoagulated with warfarin and commenced on hydroxychloroquine and mycophenolate mofetil for maintenance immunosuppression.LSE is rare in childhood with only six previous cases described in the literature. In adults with SLE the prevalence of progressive valvular abnormalities is higher when SLE is associated with antiphospholipid antibodies. Interestingly, SHOC2 mutation is associated with congenital mitral valve defects but acquired mitral valve disease has not been reported.This case highlights the difficulties and potential delays in diagnosing SLE due to its varied and often insiduous presentation and demonstrates that LSE can occur in children with lupus. It also reaffirms the importance of considering autoimmune inflammatory conditions in cases of pericarditis with no evidence of an infectious cause.None declared."} +{"text": "Clinicians need to have intimate knowledge and thorough understanding of both pulp chamber and root canal anatomy. They should be aware of possibility of anatomical variations in the root canal system during endodontic treatment. Maxillary canines usually have single root and root canal but rarely may have single root with two root canals. This case describes a lengthier maxillary canine with two root canals. Thorough knowledge and understanding of pulp chamber and root canal system anatomy are essential for successful root canal therapy. Familiarity with variations in tooth anatomy and characteristic features in various racial groups can aid location and negotiation of canals . Missed Pulp canal system is complex with branching and divisions throughout the root length. Vertucci (1984) classified the root canal configurations of human permanent teeth into various types ranging from single to three separate distinct canals .Permanent maxillary canines are more commonly single rooted, single canal teeth. Presence of two root canals is a rarity . MajoritThis case report presents a permanent maxillary canine having two root canals exiting as single foramen.A 35-year-old female patient reported to dental clinic with a chief complaint of spontaneous pain from four days in maxillary left anterior region. Subjective symptoms include sharp, severe, continuous, throbbing pain and are aggravated by taking hot foods and relieved by medication. Past medical history was noncontributory.Oral examination revealed a deep carious lesion involving maxillary left canine . The teeth was asymptomatic to palpation and tested negative using electrical pulp tester and gave an exaggerated severe response to cold test. No mobility was seen and tested negative for percussion. Radiographic examination revealed an abnormal root canal morphology. A diagnosis of acute irreversible pulpitis of 23 was established and endodontic treatment was planned.Following local anesthesia with 2% lidocaine, the tooth was isolated with rubber dam , and an endodontic access was made on palatal side with #1014 round bur and endo-Z carbide bur. The vital pulp tissue was extirpated and initially two canal orifices were located. Working length of 31\u2009mm was measured by using K flex files #15 of length 31\u2009mm . The palDebridement of root canal to remove pulpal remnants, bacteria, and their byproducts before obturation is primary requisite for successful endodontic treatment. Being unable to locate and fill a canal results in failure of root canal therapy. Therefore, it is imperative to have knowledge of anatomic variations as endodontic success is related to canal debridement .The diagnostic difficulty and possible canal superimposition on radiographic examination should be kept in mind when examining such cases. When locating extra canals, identification of periodontal ligament space that often projects onto root surface resembling a canal should be differentiated. Vertucci (1984) classified root canals according to number of canals present and their configuration into eight types .Anatomic anomaly observed at first appointment should be checked for similar anomaly of tooth on the other side . In the \u00c7ali\u015fkan et al. studied In the present case, the maxillary canines had an unusual root length of 31\u2009mm which necessitated use of lengthier k flex files of 31\u2009mm removing silicone directional stopper before use. They showed canal configuration of type II similar to that reported by Alapati et al. and Onay and Ungor. Two distinct canal orifices were located in labial/palatal direction which joined in apical third, forming a type II configuration.Teeth with type II configuration during treatment may pose problems. The canal that is in line with the main passage is usually amenable to adequate enlarging and obturation procedures; the preparation and filling of the other canal are often extremely difficult .A thorough knowledge of root canal anatomy and operator skill are essential for endodontic success. Careful clinical examination with radiographs from several different angles may lead to suspicion or identification of additional canals and leads to higher possible success .Several variations exist in the root canal system and clinicians should be aware of the variations for complete infection removal and prevention of reinfection. Special care with careful endodontic exploration, different angle radiographs, and magnification with surgical microscope aids in detection and treatment of extra canals."} +{"text": "Aerial dispersion of bioaerosols and subsequent contamination of surfaces is recognised as a potential transmission route for some HCAI infections. Pathogens have been shown to accrue on health-care workers\u2019 (HCW) hands as they touch surfaces and hencTo link design of current hospital rooms with infection risk from surface contamination and HCW contacts.Computational fluid dynamics simulations, validated through bioaerosol experiments were used to accurately predict spatial distributions of bioaerosol deposition in a single and multi-bed hospital rooms. A Markov chain Monte-Carlo model was developed using the deposition patterns in conjunction with clinical observation of HCW surface contact sequences, to predict the contamination levels of bacteria on HCWs\u2019 hands as they perform routine patient care in the two rooms types.Hand colonisation depends on care type, room layout, the number of surface contacts and in particular on the spatial distribution of pathogens between surfaces, which is influenced by ventilation strategy. Contamination on the HCWs' hands decreases monotonically after patient care in a single room due to hand hygiene. During care within multi-bed rooms colonisation levels increase throughout due to the spatial spread of microorganisms contaminating multiple patient surfaces caused by the ventilation strategy. Positioning infectious patients within an unobstructed path between the inlet and outlet diffuser significantly reduces cross contamination to other patients surfaces.Results indicate that colonisation levels of HCWs\u2019 hands are likely to be significantly lower after care in single patient rooms than after care in a multi-bed ward and that patient and ventilation design is vitally important in helping curtail the risk of bioaerosol spread.None declared."} +{"text": "Foodborne illness is prevented by inspection and surveillance conducted by health departments across America. Appropriate restaurant behavior is enforced and monitored via public health inspections. However, surveillance coverage provided by state and local health departments is insufficient in preventing the rising number of foodborne illness outbreaks. To address this need for improved surveillance coverage we conducted a supplementary form of public health surveillance using social media data: Yelp.com restaurant reviews in the city of San Francisco. Yelp is a social media site where users post reviews and rate restaurants they have personally visited. Presence of keywords related to health code regulations and foodborne illness symptoms, number of restaurant reviews, number of Yelp stars, and restaurant price range were included in a model predicting a restaurant\u2019s likelihood of health code violation measured by the assigned San Francisco public health code rating. For a list of major health code violations see . We buil Food pathogens cause 9.4 million pathogen confirmed foodborne illnesses in the United States each year . Each yeAnnual health inspections only capture a small window of time and may not accurately reflect the true practices of an establishment. The coverage provided by health inspection covers less than 1% of a restaurant\u2019s annual operation time. At most, the SFDPH inspects each restaurant two to three times per year. Current risk ranking dictates that restaurants that receive a favorable health code rating (> 80 out of 100 points) will not be inspected again for the rest of the year. Restaurants with more severe findings (Major Violations) will have a greater number of points deducted from their score. Through this approach, those restaurants that have the highest risk violations will be re-inspected first. Additionally, while restaurants receiving suboptimal health department scores are more likely to be connected to foodborne illness outbreaks, suboptimal scores currently predict a minority of restaurant related outbreaks . Foodbornow-casts\u2014but also forecasts of its future state\u201d . In . In 29].Mining publicly-available, crowdsourced data to develop a surveillance method for tracking foodborne illness risk factors gives health inspectors an improved ability to identify restaurants with greater odds of low health code ratings and violations outside of the normal inspection window. Our approach uses available data and open source software. Our code for data extraction and analysis is available in public repositories and may be built upon to increase the breadth and transferability of our model. Additionally, tracking clusters of food safety compliance and foodborne illness-related keywords in large, crowdsourced data sets improves traditional surveillance methods without substantially increasing costs. This study serves as a step forward in illustrating how social media data may be used for the benefit of public health.S1 Fig(TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file."} +{"text": "Increases in skeletal muscle mass arises as a result of positive net protein balance, such that muscle protein synthesis (MPS) exceeds muscle protein breakdown (MPB). Increased mechanical loading and provision of high quality amino acids are potent and independent stimulators of MPS through activation of key cell signaling pathways involving the mTOR-p70S6K signaling axis. Importantly, the combination of resistance exercise and dietary protein intake elevate MPS over and above their independent effects, thus highlighting that a well formulated resistance training programme (incorporating multiple sets to failure) and increased dietary protein intake (spread evenly throughout the day) should form the basis of training and nutritional strategies. However, given that many elite athletes (such as rowers) may simultaneously be training to develop endurance as well as strength, it is also important to consider the periodization of endurance type training sessions alongside high volume resistance sessions. In this regard the signaling pathway known to regulate the endurance phenotype (i.e. the AMPK-PGC-1 axis) may potentially attenuate the activation of growth related pathways thereby mediating a training interference effect by which lean mass growth is negated. As such, careful consideration should also be given to the training structure and nutrient availability both within and between days so as to maximize training adaptation and recovery. In this presentation, the author presents data from both his own laboratory and others that briefly outlines the molecular regulation of muscle mass growth and endurance adaptations before providing nutritional and training periodization strategies such that both aspects of training adaptation may be developed simultaneously."} +{"text": "States of arousal, or consciousness with the brain are regulated largely by the neurotransmitter acetylcholine (ACh). Specifically, ACh is likely responsible for the transition between slow wave sleep and rapid eye movement sleep or waking states (where ACh is high). Patterns of neural activity within the cerebral cortex corresponding to these states are markedly different. During SWS there are traveling waves of intense activity in the cortex while in other states locally organized stationary patterns occur . From a"} +{"text": "This section on 'EP Update - Clinical' summarizes the recent literature on ventricular tachyarrhythmia and related sudden cardiac death from leading journals. The articles have been hand picked and reviewed by the editors of IPEJ for the benefit of readers.Goldberger JJ and colleagues have provided a meta analysis on risk stratification for arrhythmic events in nonischemic dilated cardiomyopathy . They idSudden arrhythmic deaths occur in the range of about one in a hundred thousand population. Identifying inherited cardiac conditions in this group will be of benefit to the family members in prevention of future catastrophes. Wong CH et al looked aIt is well known that highest percentage of biventricular pacing is optimal for patients on cardiac resynchronization therapy (CRT) for heart failure. Ruwal MH and associates have stuVentricular tachycardia (VT) associated with structural heart disease is often difficult to treat by catheter ablation. Recurrence of arrhythmia after ablation is associated with increased mortality. Nagashima K et al found thSpontaneous type 1 Brugada ECG with history of syncope likely to be due to ventricular tachyarrhytmia is a Class IIa indication for the implantation of an ICD as per HRS/EHRA/APHRS Expert Consensus Statement 2013. Class IIb recommendation for ICD implantation was given for those with either a spontaneous or drug induced type 1 Brugada ECG and inducible ventricular fibrillation (VF) on programmed electrical stimulation. A Japanese multicenter study has furt"} +{"text": "Surgery first concept in orthognathic surgery has been introduced less than a decade ago. The advantages of this approach are claimed to be:\u2022 Shorter entire treatment period.\u2022 Better satisfaction because of the patients\u2019 main complaint, facial aesthetics, is achieved and improved at the beginning of the treatment.\u2022 Postoperative orthodontic tooth movement is accelerated because of favourable metabolic bony changes due to surgery.Evidence to support these claims is, however, weak. One may further question if surgical stability is compromised without presurgical orthodontics, which in conventional orthognathic surgery is thought to maximize stable postoperative occlusion and reduce relapse tendency. Evident longer postoperative orthodontics treatment duration in the surgery first concept may also cause problems, since it has been reported, that longer than nine months postoperative treatment is no longer well tolerated by the patients.From the orthodontic point of view reasons for surgery first include:\u2022 Early surgery in patients with severe sleep apnea.\u2022 Deep bite and scissors bite cases where presurgical orthodontic treatment is not possible.\u2022 Class III patients in whom presurgical treatment would lead to significant worsening of the facial outlook.Surgery first concept requires close collaboration with the surgeon and orthodontist to plan the surgery, since in many cases surgical overcorrection has to be considered to compensate for the space required for final tooth alignment. Postsurgical orthodontic mechanotherapy is also demanding, but modern skeletal anchorage systems offer here a valuable tool.Without proper scientific evidence surgery first has to be considered as an experimental concept with a need of much further investigation."} +{"text": "Due to the helical structure of DNA the process of DNA replication is topologically complex. Freshly replicated DNA molecules are catenated with each other and are frequently knotted. For proper functioning of DNA it is necessary to remove all of these entanglements. This is done by DNA topoisomerases that pass DNA segments through each other. However, it has been a riddle how DNA topoisomerases select the sites of their action. In highly crowded DNA in living cells random passages between contacting segments would only increase the extent of entanglement. Using molecular dynamics simulations we observed that in actively supercoiled DNA molecules the entanglements resulting from DNA knotting or catenation spontaneously approach sites of nicks and gaps in the DNA. Type I topoisomerases, that preferentially act at sites of nick and gaps, are thus naturally provided with DNA\u2013DNA juxtapositions where a passage results in an error-free DNA unknotting or DNA decatenation. We modir represents center-to-center distance between two beads, r \u2264 8q = \u22122 as that closely corresponds to the remaining fraction of non-neutralized charges per 8 bp fragment in physiological solutions containing mono- and divalent counterions (Bl) for aqueous solutions was set to 0.71 nm = 0.28\u03b8 is the angle formed by two consecutive principal bonds of the chain. The bending constant Kb was adjusted so that the chain stiffening resulting from the cumulative action of excluded volume, electrostatic repulsion and bending rigidity produced the chain with the persistence length of ca 50 nm. In order to introduce torsional constraint and also to mimic the rotational drag acting on DNA molecules in solution, we modified the classical beaded chain model by introducing periaxial beads (P) with no excluded volume but experiencing hydrodynamic drag . The periaxial beads (one per bond connecting main chain beads) were placed at distance of m. This positioning of periaxial beads ensures that the dihedral angle between consecutive bonds connecting periaxial beads with the skeletal bonds of the chain is defined even for 90\u00b0 bending between consecutive main bonds of the chain. The torsional constraint was introduced by dihedral angle potential acting on consecutive bonds connecting auxiliary periaxial beads (P) with auxiliary axial beads (A). The dihedral angle potential had the form V(\u03d5) = KD[1 \u2212 cos (\u03d5 - p)], with the constant KD = 20\u03d5 and the phase p. For generic regions of modelled DNA the phase p was set to 0.The chain construction is presented in Supplementary Figure S1. Consecutive main chain beads (M) were bonded by a finitely extensible non-linear elastic potential (FENE) with the equilibrium bond length set to 1otential , so thattherwise , \\documeximation , \\documeer study . The masp was initially set to 0 but then was gradually changed during the simulation. To introduce a passive swivel mimicking the effect of a nick, the dihedral lock was interrupted between two consecutive beads. To mimic the effect of a short single-stranded gap, in addition to removing dihedral lock over a distance of three beads we also decreased by half the electrostatic charge per bead (since one bead represents then 8 bases and not 8 base pairs) and decreased the persistence length, since ssDNA is much more flexible than dsDNA . This corresponds to the situation of freshly replicated plasmids that contain nicks and gaps in the terminator region and whic18 is pushed by arising supercoiling towards nicks or gaps (inset in the figure presents a standard tabular representation of the knot 818).Pushing of DNA entanglements towards nicks or gaps is not limited to cases where the sites of gyrase action are maximally distant from the sites where the supercoiling is released i.e. nicks or gaps. Practically the same phenomenon was observed when the active and passive swivel sites were present in the same quarter of modelled 3 kb plasmid (see Figure It is known that bacterial DNA topoisomerase III promotes decatenation and unknotting in DNA molecules by mediating passages of duplex regions through short single stranded gaps ,19. We tThe presented results suggest a possible model of efficient mechanism of DNA unknotting and DNA decatenation. Figure DNA unknotting is important for cells, however, of much more fundamental importance is very efficient decatenation of freshly replicated circular DNA molecules that end the replication forming torus type of catenanes with several interlinks . Since sThe confinement of interlinked region by supercoiling should also facilitate the main decatenation pathway involving the action of topoisomerase IV. Strong confinement is likely to induce formation of higher order supercoiling with favourable geometry of dsDNA\u2013dsDNA juxtaposition for topoIV action .It should be mentioned here that earlier simulation studies revealed that in a \u2018static\u2019 situation, where knotted molecules were already supercoiled and where gyrase does not introduce additional supercoils, knotted portions of molecules get more confined than in non-supercoiled DNA . HoweverIt is good to add here that the linking number change introduced by DNA gyrase is redistributed into the change of twist and the change of writhe. Only the writhing and more specifically formation of plectonemes is responsible for pushing the knots towards nicks and gaps.In vivo the rotational drag of DNA results not only from relatively large viscosity of cellular milieu but also from the fact that DNA is bound by various proteins that effectively increase the diameter of formed nucleoprotein complexes and hence increase the drag. In vivo experiments demonstrated that transcribing RNA polymerase, inducing ca 5 axial DNA rotations per second, is sufficient to induce formation of supercoils when another RNA polymerase is simply bound to DNA stabilizing there a DNA bend that provides a strong rotational drag (As mentioned in the Material and Methods section, our simulations were performed under conditions corresponding to an upper range of physiologically observed viscosities . Under tnal drag . In our nal drag .in vivo to move knots and catenane crossings towards nicks and gaps for unknotting and decatenation, respectively. In simulated 3 kb large plasmids ca 100 rotations of DNA were usually needed to push the knot towards a nick. With DNA gyrase action resulting in ca 5 rotations per second (It is interesting to estimate how much time it may take r second it will Escherichia coli cells devoid of gyrase activity accumulated knotted DNA plasmids suggesting that gyrase activity is needed for efficient DNA unknotting (E. coli cells without TopoI but having TopoIII as the only type I DNA topoisomerase require supercoiling activity of gyrase for chromosome segregation (Proposed by us models of supercoiling-assisted topoIII-mediated unknotting and decatenation seem to be attractive. Is there however an experimental evidence supporting these models? Shishido et al., showed that knotting . Separatregation . The intregation ."} +{"text": "Focused echocardiography is increasingly used by clinicians in the management of critically ill patients and has been adopted by the Defence Medical Services as a tool to guide flow assessment and resuscitation in deployed critical care.We aimed to explore whether two focused echo techniques; Inferior Vena Cava (IVC) and Left Ventricular Outflow Tract (LVOT) Velocity Time Integer (VTi) variability could be taught to a group of critical care nurse who had no previous exposure to ultrasound imaging.Ethical approval was waived for this service improvement study. After a five week program of training validation was carried out on healthy volunteers. The mentor, an accredited focused echo trainer, and six nurses performed a total of 48 scans on 11 volunteers. The mentor and students acquired subcostal long axis and apical five chamber windows using a high frequency linear ultrasound probe . Mean values from three measurements were obtained for IVC diameter and LVOT VTi. Minimum and maximum values were recorded for both variables across a full respiratory cycle. Echo images were saved and at least two images for each student were reviewed offline by an accredited echo training supervisor.In all cases students were able to obtain adequate echo windows. There was good correlation between values recorded by the mentor and students for both IVC diameter and LVOT VTi . Bland Altman analysis showed good correlation with minimal bias for VTi measurements. There was, however, some increase in bias for IVC measurements below 1.2 cm.We demonstrated that two focused echo techniques for assessing intravascular volume status could be acquired by specialist nurses, with no previous experience, in a relatively short time and that results were comparable to those produced by an experienced practitioner. These results will need to be replicated in a clinical setting before being adopted into practice."} +{"text": "Mycobacterium tuberculosis (MTB) from non tuberculous mycobacteria, are important issues which impinge on the WHO\u2019s millennium goal of disease reduction by 2015. Traditional methods of TB detection and anti tB drug susceptibility assays are time consuming and with a steep rise in number of MDR tB cases the need of the hour is rapid diagnostic methods like direct drug susceptibility testing in liquid medium, molecular hybridization methods and RealTime PCR.Tuberculosis is the second leading cause of death due to infectious disease worldwide. Delay in bacilli isolation in culture, cumbersome susceptibility testing methods, lack of methods for differentiation of This study was conducted to assess the efficacy of line probe assay in detecting MTB from smear negative culture negative samples in a tertiary care centre catering to the need of a high burden TB population of Uttarakhand.Between January 2013 and November 2013 a 22.7%positivity rate of sputum samples by fluorescent staining was reported. Culture by MGIT (Mycobacterium growth indicator tube) of 162 smear negative samples from patients clinically or radiologically pulmonary TB suspects was performed and 78(48.1%) samples grew MTB. Further, 50 samples which were smear negative and MGIT negative from patients with strong suspicion of pulmonary tuberculosis were tested by line probe assay and 34(68%) samples turned out to be positive.Newer methods offer definite improvements in the ability to detect MTB. However, there is a strong need to deploy these tests in areas where MTB burden is highest."} +{"text": "Central Line Associated blood stream infections (CLABSI) is High Risk and High Volume problem, it prolongs hospitalization by mean of 7 days. Attribute cost per blood stream infection are estimated to be between 3700 $ and 29000$. Central line (CL) bundle is evidence-based practices which when implemented has demonstrated striking reduction in the rate of both central line related infections.According 2012 Risk Assessment at king Saud Medical City, reduction of Central Line Associated Blood Stream Infection Rate in Adult ICU was a prioritized goal and planned to be achieved through implementation of central line bundle of care.Reduce CLABSI rate at ICU by 20% annually staring in 2013Achieve 90% CL Bundle compliance by end of 2014FOCUS PDCA Performance Improvement Project implemented. Team formulated of Infection control staff, ICU nurses and physicians and ICU director and the current practice of insertion and maintenance of Central Line catheter has been discussedImprovment strategies include:\u2022Education about Central Line Bundle of Care for ICU staff\u2022Make the supply available in ICU to maintain bundle of care for central line .\u2022Checklist developed for bundle variables and placed at patient Medical Record\u2022Supervised central line insertions by secret observer\u2022Nurses are empowered to stop any central line insertion in case of any breach of infection control guidelines and practices.\u2022Replaced povidone iodine with CHG which is superior.\u2022Encourge strict adherence to hand hygiene practices.\u2022Discouraged the use of suboptimal site such as the femoral by means of email alerts\u2022Communication with emergency room team to reduce the femoral central lines coming from E.R.\u2022Replacing CVL inserted under emergent situation within 48 hours\u2022Audit sheet used to calculate bundle compliance\u2022Doing daily bathing with CHG for the patients.40% reduction of CLABSI occured within 2 years (from 11.3 in 2012 to 6.8 in 2014)Bundle compliance ranges between 84% to 97% during 2014Femoral insertions declines from 10/month to 1-3/month in 2014Involving process owner champions, consistent availability and accessibility of required supply for Central Line Bundle and continuous Staff education and monitoring with feedback for caregivers are effective strategies to reduce CLABSINone declared."} +{"text": "There is increasing evidence that environmental factors in early life predict later health. The early adiposity rebound recorded in most obese subjects suggests that factors promoting body fat development have operated in the first years of life. Birth weight, growth velocity and body mass index (BMI) trajectories seem to be highly sensitive to the environmental conditions present during pregnancy and in early life (\u201cThe first 1000 days\u201d). Particularly, nutritional exposure can have a long-term effect on health in adulthood. The high protein-low fat diet often recorded in young children may have contributed to the rapid rise of childhood obesity prevalence during the last decades. Metabolic programming by early nutrition could explain the development of later obesity and adult diseases. There is now clear evidence that nutritional and metabolic exposure during critical periods of early human development (\u201cthe first 1000 days\u201d) can have a long-term effect on health in adulthood. Early environmental factors can permanently change the structure and function of the body, a phenomenon known as \u201cprogramming\u201d . SeveralAssessment of nutritional status and secuDecreasing consumption of fat and increasing consumption of proteins was the consequence of replacing whole milk by low fat milk . In 1973The main explanation usually proposed to account for the trend of decreasing energy intake is decreasing energy expenditure. Low physical activity is indeed an important factor, however, this hypothesis seems less convincing in very young children. Other factors may explain the rising trend of obesity.Early nutrition is associated with metabolic risks through its impact on growth processes and hormonal status .An association between high protein intake in early life and increased body fat in later development was reported two decades ago in the Etude Longitudinale Alimentation Nutrition et Croissance des Enfants (ELANCE) study . High prMany studies have confirmed the association between early protein intake and later risk of obesity ,8. A sysMore recently, the ELANCE study investigating the association between early nutrition and adult measurements showed that early low fat intakes were associated with adults being overweight and having high serum leptin ,11, but The impact of nutritional conditions on BMI trajectories can be suggested by examining the trends in BMI growth curves. Data from the Fels study have shown that BMI curves of children born during the obesity epidemic were characterized by lower BMI values before the adiposity rebound and by rapid subsequent BMI gain . This trThe association between high protein intake in early life and later obesity may occur through increased growth factors stimulating growth and promThe adverse effect of reducing fat intake is consistent with the results of studies conducted in different contexts. Low birth weight, which is associated with low leptin concentration or earlyThere are many factors which could account for the protective effect of breast feeding. The nutrient composition of breast milk could explain the association between breast feeding and reduced risk of becoming overweight . The latThe positive association recorded between early protein intake and later body fat, and the negative association recorded between early fat intake and later body fat have highlighted the inadequate nutrient balance of the infant diet in industrialized countries. Protein intake represents about 3\u20134 times the protein needs at this age and fat intake is remarkably low in many countries ,11. ThisParadoxically, fat intake increases with age, while it should be high in infancy and decrease thereafter . In the Evidence suggests that an imbalanced diet in early life can have deleterious consequences on body composition and health. The high protein-low fat diet recorded in young children may have contributed to the obesity epidemic. The consequences of inadequate nutrition at different ages stress the importance of providing nutritional intakes adapted to the child\u2019s metabolic needs at the various stages of growth."} +{"text": "We found that all species underwent more or less intensive changes in population size but we also found consistent differences between demographic histories of pelagic and benthic species. Contemporary pelagic populations are significantly more genetically diverse and bear traces of older demographic expansions than less diverse benthic species that show evidence of more recent population expansions. Our findings suggest that the lifestyles of different species have strong influences on their responses to the same environmental events. Our data, in conjunction with previous studies showing a constant diversification tempo of these species during the Pleistocene, support the hypothesis that Pleistocene glaciations had a smaller effect on pelagic species than on benthic species whose survival may have relied upon ephemeral refugia in shallow shelf waters. These findings suggest that the interaction between lifestyle and environmental changes should be considered in genetic analyses.Major climatic changes in the Pleistocene had significant effects on marine organisms and the environments in which they lived. The presence of divergent patterns of demographic history even among phylogenetically closely-related species sharing climatic changes raises questions as to the respective influence of species-specific traits on population structure. In this work we tested whether the lifestyle of Antarctic notothenioid benthic and pelagic fish species from the Southern Ocean influenced the concerted population response to Pleistocene climatic fluctuations. This was done by a comparative analysis of sequence variation at the cyt Rubus chamaemorus) , Re, Reb areerences: .(DOCX)Click here for additional data file.S3 Table(TXT)Click here for additional data file."} +{"text": "Poor retention of participants in randomised controlled trials (RCTs) is a commonly recognised issue. Attrition rates in studies across different disciplines have been reported to vary from 5% to 70%. There is no systematic evidence reporting attrition rates specifically in trials involving people with severe and fluctuating mental health conditions.To purpose of this review was to synthesise the reported retention rates in RCTs evaluating non-pharmacological interventions for psychosis and identify study characteristics associated with high dropout.Five key electronic databases and key journals were systematically searched to identify studies published between 1996 and 2015. The search included large scale RCTs (n\u2265100 at baseline) including adults with psychosis receiving non-pharmacological interventions. Data extraction included participant flow information reported in the Consolidated Standards of Reporting Trials (CONSORT) statement or flow diagram. Random effects logistic regression analysis was conducted to examine effects of key study characteristics on participation rates.62 papers were included in the review. This paper will discuss the participation rates reported in RCTs, the quality of reporting in publications, and the study characteristics that predict participation rates."} +{"text": "Several countries have set up early awareness and alert systems (EAAS) to inform their customers about technologies that may have a significant impact on the health care system .The Austrian Ludwig Boltzmann Institute for Health Technology Assessment (LBI-HTA) implemented an EAAS specifically for oncologic drugs in 2009 [In order to identify potential ways of collaboration a workshop with 12 agencies from nine countries all involved in assessing new oncologic drugs was held in 2010. Following this workshop, the LBI-HTA started to send out \"calls for collaboration\" to identify partners interested in jointly conducting reports. Up until now, 11 calls have been sent out resulting in 15 collaborations with a total of six institutes. Collaboration initially led to some delays in report production, but since the same agencies repeatedly indicated interest in collaboration, familiarity with processes and development of trust was eventually achieved ultimately leading to efficiency gains.Besides the active production of joint reports, rapid relative effectiveness assessments produced in international collaboration within the European HTA Network EUnetHTA can serve as basis for local reports . So far,Increasing financial pressure on health care systems and limited research resources necessitate exploring ways of sharing and reusing research findings. Local initiatives driven by individual agencies but also European-wide developments offer the opportunity to produce assessments more efficiently and to reduce redundancies."} +{"text": "Small renal masses are increasingly diagnosed incidentally. This results in management dilemma because at histopathology significant numbers of small renal masses are either benign tumors such as angiomyolipoma (AML) or oncocytoma, or are neoplasms with relatively indolent behavior . SurgicaWithin this paradigm, the role of radiologist and imaging is evolving from traditional role of identifying renal lesion and detecting enhancement, to predicting aggressiveness and biology of the tumor as well as providing operative guidance. MR imaging can play a very important role not only as a problem solving tool in traditional sense by detecting subtle enhancement and macroscopic and microscopic fat, but can provide deeper insight into tumor biology. Number of key observations highlighting the role of MR in evaluation of renal masses is as listed below:- There is some controversy regarding the role of signal loss on opposed phase chemical shift imaging in discriminating AML from RCC ,4.- Lipid poor AML tend to have uniform low T2 signal and uniform enhancement without evidence for necrosis ,6.- There is overlap in the morphologic features of Oncocytoma and RCC on conventional imaging . Further- Papillary subtype of RCC usually have low T2 signal and are hypovascular when compared to clear cell RCC. Furthermore, clear cell subtype have heterogeneous T2 signal and demonstrate heterogeneous hypervascularity .- Chromophobe subtype is difficult to differentiate from clear cell RCC on the basis of enhancement. However, advance diffusion and perfusion MR techniques have shown some promise .- Cystic RCC with less than 25% solid enhancing component tend to be less aggressive than solid RCC .- High stage clear cell RCC tend to me more heterogeneous with different texture compared to low stage RCC on Apparent diffusion coefficient (ADC) map .- High grade clear cell RCC tend to have lower ADC compared to low grade clear cell RCC ."} +{"text": "In summary, our data demonstrate a previously unappreciated role for acetate as a nutritional source for the growth and survival of breast and prostate cancer cells under metabolic stress.During tumour formation and expansion, cancer cells encounter constantly changing environmental conditions in which nutrient and oxygen availability may be severely compromised. The metabolic transformation of cancer cells is characterised by distinct changes in metabolic activity that satisfy the exigencies of energy and biomass production imposed by continued cell proliferation. These metabolic adaptations often involve increased consumption and metabolism of extracellular nutrients, mainly glucose, amino acids and lipids. During periods of nutrient or oxygen deprivation, cancer cells can also modify their metabolism to adapt to these specific challenges. Here we report the results of a functional genomics study that revealed that the activity of acetyl-coA synthetase 2, an enzyme that converts acetate into acetyl-coA, contributes to cellular growth under oxygen and nutrient stressed conditions. ACSS2 was required to provide acetyl groups for lipid biosynthesis. Moreover, ACSS2 was essential for cancer cell growth and survival under physiologically relevant growth conditions and its depletion blocked tumour growth"} +{"text": "We observe negative correlations between basal ganglia network activity and performance in Semantic Fluency test and Part A of the Trail Making Test for patients with poor cognitive recovery. A reverse pattern was observed between frontal network activity and the abovementioned tests for the same group. Hum Brain Mapp 35:3819\u20133831, 2014. \u00a9 2014 The Authors. Human Brain Mapping published by Wiley Periodicals, Inc.Resting\u2010state studies conducted with stroke patients are scarce. First objective was to explore whether patients with good cognitive recovery showed differences in resting\u2010state functional patterns of brain activity when compared to patients with poor cognitive recovery. Second objective was to determine whether such patterns were correlated with cognitive performance. Third objective was to assess the existence of prognostic factors for cognitive recovery. Eighteen right\u2010handed stroke patients and eighteen healthy controls were included in the study. Stroke patients were divided into two groups according to their cognitive improvement observed at three months after stroke. Probabilistic independent component analysis was used to identify resting\u2010state brain activity patterns. The analysis identified six networks: frontal, fronto\u2010temporal, default mode network, secondary visual, parietal, and basal ganglia. Stroke patients showed significant decrease in brain activity in parietal and basal ganglia networks and a widespread increase in brain activity in the remaining ones when compared with healthy controls. When analyzed separately, patients with poor cognitive recovery ( Acute ischemic stroke is the second most common cause of death worldwide and a major cause of disability in the elder population .Demographic and clinical data are given in Table Stroke group in general demonstrated a significant acute\u2010to\u2010subacute improvement in the following cognitive tests: MMSE, SFT , Boston Naming Test, TMTA, and the GPT Table . We haveP\u2009=\u20090.053) and the number of omissions in the attention subtest (P\u2009=\u20090.052) than patients with poor cognitive recovery (data not shown).There was no significant difference between the two stroke groups in any cognitive test evaluated in the acute phase. In the subacute phase however, patients with good cognitive recovery performed significantly better in the TMTA frontal network; 2) fronto\u2010temporal network; (3) DMN, (4) secondary network, and decreased brain activity in (5) Basal Ganglia network, and (6) parietal network a lower correlation between Basal Ganglia activity change and SFT score and a higher correlation between Basal Ganglia activity and TMTA time Table , (b) a hThis study aims to identify resting\u2010state functional connectivity patterns characterizing ischemic stroke in subacute phase and their relations with cognitive recovery. Our pICA analysis identified eighteen relevant components matching the standard RSN reported in healthy subjects [Beckmann et al., Resting\u2010state studies have already been carried out in stroke populations in relation to motor recovery investigating interhemispheric resting activity as a measure of normal function [Carter et al., There is an on\u2010going debate in the literature regarding the role of the contralesional hemisphere activity in stroke recovery. Considering the motor function, stroke patients typically show pathologically enhanced neural activity in a number of areas both in the lesioned and in the contralesional hemisphere [Grefkes et al., The brain areas where we found increased activity at rest are related to cognitive functions impaired in our stroke patients, such as executive, attentional, and motor functions : the paracingulate cortex involved in top\u2010down and bottom\u2010up control to other areas [Allman et al., RSN changes by themselves could be interpreted as brain disturbances due to stroke. The fact that they were stronger in stroke patients with poor cognitive recovery also supports this hypothesis. However, a larger portion of brain activity at rest was in the left hemisphere . This pattern of activity is in agreement with results obtained from stroke recovery research both in animal models and clinical patients showing that widespread changes in activity patterns can even extend to the unaffected hemisphere [Carmichael and Chesselet, Most importantly, the magnitude of these changes correlated well with cognitive performance: increased Frontal activity having a positive correlation with cognitive tests, and decreased Basal Ganglia activity having a negative correlation with cognitive tests. The (not significantly) weaker correlations in patients with good cognitive recovery, and the significantly weaker correlation or reverse correlation in healthy controls also support their compensatory nature. In stroke patients with poor cognitive recovery, they seem to have a negative effect on performance probably due to disruption of the interplay between the brain areas. When some of those brain areas are damaged in stroke patients, they compensate these damages via shifting the functional connectivity to favor unaffected brain areas. Therefore, patients demonstrating a larger shift in functional connectivity provide a better cognitive performance.However, these changes seem to play no role in recovery; they actually diminish to allow coming back to \u201cnormal\u201d brain activity. That explains their weaker presence in patients with good cognitive recovery. This is in agreement with other studies linking improved recovery with regaining the\u201cnormal\u201d brain activity [Dijkhuizen et al., Rs\u2010fMRI is becoming an excellent tool for clinical studies, because it does not impose attentional demands or cognitive burdens on the patient. Although rs\u2010fMRI has already been employed in other stroke studies, they do not take into account the whole range of brain networks as this study. Moreover, we not only employed a detailed neuropsychological evaluation covering the whole cognitive spectrum in acute stroke; but also investigated how they are associated with recovery. Finally, the study design allows examining how resting\u2010state brain activity relates to recovery, and whether rs\u2010fMRI has any predictive value regarding clinically relevant outcome. However, our sample is small due to our strict criteria. This may also decrease the sensitivity, and restrict the generalizability of our preliminary results.Finally, although we did not reported statistical significant differences regarding the volume of ischemic lesions, their size was heterogeneous. This is a limitation because whereas recovery after a small ischemic lesion may involve preserved peri\u2010infarct tissue with function similar to the infarcted tissue [Brown et al., Summarizing, our results confirm our hypotheses and may expand our understanding of brain changes occurring after stroke, as well as stimulate new researches on lesion\u2010induced network plasticity changes and fMRI biomarkers of recovery/progression not only in stroke but also in vascular cognitive impairment and vascular dementia.Brain connectivity changes is stroke patients have been already described in task related fMRI studies and in a few resting\u2010state functional connectivity studies focusing on specific networks. Our less restricted study also demonstrated that these changes affect several brain networks, which not only explains the multiplicity of the deficits following a focal lesion but may also indicate compensatory brain plasticity. As a consequence, they are more pronounced in patients with poor cognitive recovery, whereas patients with good cognitive recovery show \u201cnormalization\u201d of these compensatory changes. More importantly, there are strong correlations between functional connectivity changes and cognitive recovery further supporting the relevance of the study of resting\u2010state functional data.Our results suggest that resting\u2010state fMRI provides information for cognitive recovery prognosis and could be a potential biomarker in stroke patients detecting early neural dysfunction and compensatory mechanisms prior to brain atrophy."} +{"text": "Associations of genetic changes and aneuploidy with tumor growth are traditionally attributed to alterations in DNA sequence manifested as mutations, deletions and amplifications. Inactive tumor suppressor genes could serve as drivers of tumor progression due to not only altered or lack of protein function but may also contribute to phenotypic changes that may provide distinct growth advantage in a hostile environment in the host. Human variation is also due to epigenetic alterations and heritable change that leads to altered gene expression; the functional consequence of which may contribute to definitive trait. A number of key regulatory genes associated with epigenetic silencing due to DNA methylation in cervical cancer have been reported. Elucidation of differentially methylated genes may identify new targets and further strengthen our understanding of molecular mechanism governing pathogenesis of cervical cancer. Thus, to identify DNA methylation regulated genes in cervical cancer, we have employed DMH based microarray experiments in pre-malignant and malignant cervical sample. Microarray data analysis and validation using bisulfite genomic sequencing lead to the identification of several CpG island as altered during cervical carcinogenesis and showed the potential for early screening of cervical cancer. One of the candidate gene identified was Double C2 like Domain beta (DOC2B), a key calcium regulator protein whose alteration has never been linked to cancer. We provide evidence that DOC2B is depressed in cervical cancer due to promoter hypermethylation and act as a novel tumor suppressor gene by regulating multiple pathways in cervical cancer."} +{"text": "The introduction of the implantable cardioverter-defibrillator (ICD) for primary prevention of sudden cardiac death has led to an important reduction in mortality in patients with impaired left ventricular ejection fraction (LVEF) , 2. ConsThe study performed by De Haan et al. published in the current issue of the Netherlands Heart Journal included 152 patients who were referred for primary prevention ICD implantation [The lack of uniformity between imaging modalities to assess LVEF has been well documented. Although comparative studies have shown some discrepancies, most of the data suggest that LVEF tends to be highest when utilising 2D echocardiography and subsequently gradually declines with CMR and nuclear ventriculography, respectively . The quathe most clinically accurate and appropriate in their institution\u2019 [So what should our practice be in the meantime? As current clinical guidelines for ICD implantation are predominantly based on studies using 2D echocardiography, it could be argued that this type of imaging should be preferred to practice evidence-based medicine. Indeed, the ACC/AHA/ESC 2006 guidelines for ICD implantation actually recommend echocardiography for the assessment of left ventricular function . It is oitution\u2019 . The cur"} +{"text": "Arenaviruses cause up to 500,000 zoonotic infections per year in endemic areas of Africa and South America and can lead to severe and lethal hemorrhagic fever (HF) symptoms . CurrentOur laboratory has been involved in characterizing the molecular mechanisms behind innate immune suppression mediated by arenaviral nucleoprotein (NP). Our high-resolution structure of the Lassa virus (LASV) NP reveals a conserved DEDDh exonuclease in its C terminal domain , which cin vivo.Our findings are supported by recent studies with recombinant LASV carrying the NP catalytic double mutations (D389T/G392A and D389A/G392A), which lose the ability to suppress the type I IFN-induction pathway in dendritic cells or macrophages, which are thought to be primary target cells for virus infection , 7. The Taken together, studies conducted in our laboratory as well as by other laboratories have provided strong evidence to support the role of the arenaviral NP exoribonuclease function in mediating immune suppression upon viral infections. It is important to highlight the fact that this is the first time that a viral exoribonuclease function has been attributed to mediating host immune evasion. Because there is currently no specific antiviral therapy available against arenaviruses, a better level of understanding of how arenaviruses are able to evade host immunity is warranted as it may offer new ways to evaluate future treatment options and to design and test vaccine candidates against these deadly human viral pathogens."} +{"text": "A. leptorhynchus transforms the firing rate, yielding a code for stimulus intensity (distance) that is invariant to the rate of change of intensity with respect to time (speed). As such, estimates of both object distance and approach speed are effectively parsed into distinct measures of an electroreceptor afferent\u2019s firing rate [Object saliency is based on the relative local-to-background contrast in the physical signals that underlie perceptual experience. Extracting meaningful stimulus representations from these contrast patterns constitutes a serious inverse problem for neural coding, where many different stimulus features map into a scalar firing rate. This decoding problem is even more acute for neurons whose dynamics are characterized by fixed timescales, which produce distinct firing rate responses to different temporal parameterizations of a stimulus . By studing rate .Despite its important role in neural coding, adaptation suffers a serious caveat for encoding natural inputs: it generates skewed responses when signal intensity changes from increasing to decreasing (or vice versa). As predicted by generic models of spike-frequency adaptation, we report a skew in the electroreceptor afferent firing rate upon motion reversal, introducing further ambiguity into the estimation of object distance from electrosensory contrast . The ele"} +{"text": "Delayed puberty, while not uncommonly a variant of normal pubertal physiology, could be an indicator of a wide range of less common but important clinical disorders. Congenital pathologies may involve defective developments of the gonads, pituitary gonadotrophs or hypothalamic GnRH neurons. Acquired pathologies could likewise be acting at each level. Clinical diagnostic evaluation to delineate a good range of these disorders and the respective managements will be covered in this session."} +{"text": "Pulmonary hemosiderosis is a disorder with unknown cause and characterized by hemosiderin appreciation in alveolar interstitium from decomposed hemoglobin following alveolar capillary bleeding, which finally leads to pulmonary fibrosis. It can be divided into primary and secondary types in terms of its etiology. While primary types are related to autoimmunity, secondary types can be associated with cardiovascular and pulmonary causes such as mitral stenosis leading to pulmonary congestion. We report a case of cor triatriatum sinister in a child who presented with hemoptysis as a main clinical manifestation and had been previously diagnosed with idiopathic pulmonary hemosiderosis. Based on clinical signs and imaging examinations, we considered the hemoptysis was most likely due to cor triatriatum. The child underwent corrective surgery with uneventful recovery. The hemoptysis has not recurred any more after operation. Cardiovascular disease including cor triatriatum should be considered with regards to the etiology of pulmonary hemosiderosis. Pulmonary hemosiderosis is a disorder characterized by precipitation of hemosiderin from decomposed hemoglobin following alveolar capillary bleeding in alveolar interstitium were found under microscopy Fig.\u00a0. She wasCor triatriatum is a rare congenital cardiac malformation, the incidence of which has been reported as 0.1\u20130.4\u00a0% of all congenital heart diseases (Chen et al. Pulmonary hemosiderosis can be divided into primary and secondary types (Ioachimescu et al. For idiopathic pulmonary hemosiderosis, systemic glucocorticoids can reduce the morbidity and mortality from acute episodes of alveolar bleeding and control progression to pulmonary fibrosis (Green et al. We report a very rare combination of pulmonary hemosiderosis with cor triatriatum sinister. When primary pulmonary hemosiderosis is considered, precautions should be given to exclude all possible underlying causes before a diagnosis is achieved. In such a case as presented here, hemosiderosis can be well managed by correction of the causative factor."} +{"text": "Mesenchymal stromal cells (MSC) are multipotent cells with an immune suppressive capacity. In the last decade the therapeutic application of these cells has been tested in inflammatory diseases like graft versus host disease and rheumatoid arthritis. Injection of MSC can be a potential therapy for juvenile idiopathic arthritis patients refractory to conventional therapies.We tested the mechanisms of MSC immune modulation with a specific focus on the suppression of synovial fluid derived immune cells.in vitro culture system to test the suppressive capacity of MSC on both peripheral blood cells (PBMC) and synovial fluid cells (SFMC). We tested T cell proliferation and cytokine production of healthy controls and JIA patients. Furthermore, we investigated the induction or suppression of regulatory T cells (Treg) and Th17 cells as these cells have an important role in JIA pathogenesis and tested the effect of inflammatory cytokines and monocytes on MSC suppression.We setup an MSC dose dependently suppressed both PBMC and SFMC, but SF T cells were less receptive towards MSC suppression. Tnfa and ifng were suppressed in both PBMC and SFMC, but IL-17 was not affected. In PBMC, but not SFMC, Th17 cell numbers were reduced by MSC and Treg numbers increased. We found no clear role for monocytes or inflammatory cytokines tnfa and ifng in activating the immune suppressive activity of MSC as published before.We conclude that MSC can suppress T cells derived from JIA synovial fluid. However, this suppression is reduced as compared to T cells derived from peripheral blood.None declared."} +{"text": "Juvenile Dermatomyositis (JDM) is a rare life threatening disease affecting children. Symptoms include severe proximal muscle weakness and characteristic skin rashes. Vascular pathology is often a key finding in JDM including typical features of capillary drop out and abnormal blood vessel endothelial cells. We have observed that another common finding in JDM biopsies is the presence of tubuloreticular inclusions (TRI) detected by electron microscopy (EM) in blood vessel endothelial cells in muscle and the overlying skin. These are tubule-like structures within cisternae of endoplasmic reticulum.The aim of this study was to determine the frequency and specificity of TRIs in JDM biopsies compared to muscle biopsies investigated for other pathologiesThe UK JDM Biomarker and Cohort Study (JDBCS) provides access to a large JDM cohort, with linked samples and biopsies, . We examined pathology by EM of 41 JDM biopsies and recorded reports of TRIs in blood vessel endothelial cells.Tubuloreticular inclusions were demonstrated in blood vessel endothelial cells in 80% of JDM muscle biopsies (33/41) and in the overlying clinically unaffected skin in 78% of cases (26/33) where this tissue was available. In contrast no TRIs in vessel endothelial cells had previously been reported in muscle biopsies investigated for other pathologies in children (n>500).The high number of JDM muscle biopsies with identified TRIs compared to control biopsies investigated for other diseases suggests that TRI\u2019s are a specific marker of JDM pathology. TRIs have also been found in other diseases such as glomerulonephritis (GN), lupus nephritis (LN), HIV and Degos disease and are suggested to be a biomarker of type1 interferon (IFN) exposure. Type 1 IFN gene and chemokine signature is thought to be associated with JDM pathology and clinical features. The high specificity of TRIs in vessel endothelium in JDM biopsies compared to non-JDM biopsies suggests that their detection is useful in supporting a diagnosis of JDM in patients.None declared."} +{"text": "Until recently tactical analysis in elite soccer were based on observational data using variables which discard most contextual information. Analyses of team tactics require however detailed data from various sources including technical skill, individual physiological performance, and team formations among others to represent the complex processes underlying team tactical behavior. Accordingly, little is known about how these different factors influence team tactical behavior in elite soccer. In parts, this has also been due to the lack of available data. Increasingly however, detailed game logs obtained through next-generation tracking technologies in addition to physiological training data collected through novel miniature sensor technologies have become available for research. This leads however to the opposite problem where the shear amount of data becomes an obstacle in itself as methodological guidelines as well as theoretical modelling of tactical decision making in team sports is lacking. The present paper discusses how big data and modern machine learning technologies may help to address these issues and aid in developing a theoretical model for tactical decision making in team sports. As experience from medical applications show, significant organizational obstacles regarding data governance and access to technologies must be overcome first. The present work discusses these issues with respect to tactical analyses in elite soccer and propose a technological stack which aims to introduce big data technologies into elite soccer research. The proposed approach could also serve as a guideline for other sports science domains as increasing data size is becoming a wide-spread phenomenon. Tactics are a central component for success in modern elite soccer. Yet until recently, there have been few detailed scientific investigations of team tactics. One reason in this regard has been the lack of available, relevant data. With the development of advanced tracking technologies this situation has changed recently. Instead, now the amount of available data is becoming increasingly difficult to manage. In the present article we discuss how recent developments of big data technologies from industrial data analytics domains address these problems. Further, the present work provide an overview how big data technologies may provide new opportunities to study tactical behavior in elite soccer and what future challengers lie ahead.According to the Oxford dictionary, tactics describe \u201can action or strategy carefully planned to achieve a specific end\u201d. Regarding competitive soccer, naturally the aim the end of the activity is to win the game. Choosing an appropriate tactic is therefore crucial for every pre-game preparation algorithms based on game position data (Bialkowski et al. A second group of ML approaches featuring prominent in the soccer literature uses neural network modeling (compare Dutt-Mazumder et al. This short overview shows that although many interesting analyses are available what is lacking is a conceptual connection between them. Accordingly, it appears that the main obstacle to study team tactics stems from the lack of a theoretical model Garganta . One modThe lack of a higher-order description about soccer team dynamics also prevents the current analytical approaches from making a real impact with practitioners (Carling et al. A potential solution with respect to model building and the combination various data sources might present itself through the recent rise of big data technologies which has been already suggested as shaping the future of performance analysis in elite soccer (Cassimally A candidate big data soccer technological stack for soccer tactics analyses should be organized along several levels (compare Fig.\u00a0Yet, what immediately becomes clear from Fig.\u00a0As computational approaches increasingly become more complex reproducibility issue will also become more important as the development of novel algorithmic approach will become the focus of future publication results Mesirov . In this In conclusion, exciting times are emerging for team sports performance analysis as more and more data is going to become available allowing more refined investigations. The adaption of big data technologies for soccer research may therefore provide solutions to some of the key issues outline above. Thus, by providing novel methods to analyze the data and a more comprehensive theoretical model and understanding of tactical team performance in elite soccer may be within reach. This implies however, that future soccer research will have to embrace a stronger multi-disciplinary approach. Performance analysts, exercise scientists, biomechanists as well as practitioners will have to work together to make sense of these complex data sets. As has been pointed out, most of the machine learning approaches presented were performed by computer science research groups. Accordingly, future collaborations between computer and sports scientists may hold the key to apply these complex approaches in a more relevant manner. In turn, relying increasingly on more complex data analysis techniques will also pose new challenges for future sports scientists. Therefore, university curricula will have be augmented to ensure that future students receive the required background training to be able to not only use these techniques but to have at least some understanding of their theoretical and computational underpinnings. The introduction of big data technologies will also require a discussions within the research community of how to share data and techniques across research teams. To make the new insights relevant for practice a tight interchange with practitioners is required. Finally, taking a broader view on the issue of big data and sports science the proposed model for tactical analyses in elite soccer might also prove beneficial for other sports science domains where data sizes are bound to increase as well and accordingly similar problems will surface."} +{"text": "As our population ages, there has been an increase in the prevalence of cancer and heart disease . Modern In 2005 trastuzumab in combination with chemotherapy was shown to significantly improve disease-free and overall survival in women with early stage HER2 positive breast cancer , 7. WhilThe last several years have seen the development and approval of a plethora of cancer drugs, many of which may negatively impact the heart and cardiovascular system. Tyrosine kinase inhibitors can cause or exacerbate preexisting hypertension and BCR-ABL inhibitors can cause Q-T prolongation. In the modern era of cancer therapy it is imperative that oncologists work closely with cardiologists in order to provide the best possible cancer care without compromising cardiac health . This isIn this special issue we gain insight into the challenges that health care providers face when treating this unique population of patients. While our understanding of how modern cancer therapies impact the heart continues to evolve, many knowledge gaps persist.How do we identify cancer patients at high risk of cardiotoxicity? In this issue, M. Davis and colleagues highlight the importance of cardiovascular risk assessment in cancer patients prior to commencing therapy. In a cohort of prostate cancer patients, they identified a high prevalence of baseline cardiovascular risk factors and cardiovascular disease (25%) prior to initiation of cancer therapy. A standardized approach of cardiovascular risk assessment prior to initiation of treatment is needed for all cancer patients in order to optimize cardiovascular health prior to, during, and after treatment.What are the best modalities to detect cardiotoxicity? Two-dimensional (2D) echocardiography and MUGA scans are the most widely used modalities for monitoring cardiac function in chemotherapy treated patients\u2014but is this the best strategy? F. Pizzino and colleagues discuss newer imaging modalities, including 2DE tissue Doppler imaging (TDI), cardiac magnetic resonance imaging (CMR), and 2D and 3D speckle tracking echocardiography. Left ventricular ejection fraction (LVEF) has been the \u201cgold standard\u201d used to detect cardiotoxicity\u2014but it is clear that this is not the best method . A. CallHow do we manage cardiotoxicity in this patient population? In this issue, J. Sulpher and colleagues clearly identify knowledge gaps between cardiologists and oncologists in the appropriate clinical management of cancer patients who develop cardiotoxicity secondary to their cancer treatment, underscoring the need for collaboration between oncologists and cardiologists. In order to facilitate this collaboration, a number of dedicated cardiac oncology clinics have been established (mainly in academic centers) but are these specialized clinics impacting patient care? J. Sulpher and colleagues describe the clinical outcomes of cancer patients referred to a dedicated cardiac oncology clinic. While their conclusions are limited by the observational nature of their study, their results are encouraging (majority of cancer patients completed treatment) and support ongoing collaboration and research in this area.And finally how do we manage those patients who develop end stage heart disease due to cancer therapy? N. Ghosh and colleagues describe the unique challenges and clinical outcomes of cancer patients with end stage heart failure who require advanced therapies such as inotropic support, orthotopic heart transplantation, or left ventricular assist devices.Modern cancer therapies have led to more individuals surviving a diagnosis of cancer. There is an increasing appreciation, by health care providers, of the potential negative impact of cancer therapies on cardiovascular health. This special issue adds to our current knowledge in the discipline of cardiac oncology and we look forward to future research that will help guide best practices.Susan DentSusan DentPeter LiuPeter LiuChristine Brezden-MasleyChristine Brezden-MasleyDaniel LenihanDaniel Lenihan"} +{"text": "ABSTRACT. The molecular basis by which fungal and mammalian prions arise spontaneously is poorly understood. A number of different environmental stress conditions are known to increase the frequency of yeast [PSI+] prion formation in agreement with the idea that conditions which cause protein misfolding may promote the conversion of normally soluble proteins to their amyloid forms. A recent study from our laboratory has shown that the de novo formation of the [PSI+] prion is significantly increased in yeast mutants lacking key antioxidants suggesting that endogenous reactive oxygen species are sufficient to promote prion formation. Our findings strongly implicate oxidative damage of Sup35 as an important trigger for the formation of the heritable [PSI+] prion in yeast. This review discusses the mechanisms by which the direct oxidation of Sup35 might lead to structural transitions favoring conversion to the transmissible amyloid-like form. This is analogous to various environmental factors which have been proposed to trigger misfolding of the mammalian prion protein (PrPC) into the aggregated scrapie form (PrPSc). Sc) of the cellular PrPC protein.Sc \u2018seeds\u2019. The mechanism(s) underlying this switch from a normally soluble protein to a misfolded, infectious form are poorly understood. It is important that these mechanism(s) are established if we are to develop effective preventative measures for human and animal amyloidoses.Most cases of prion diseases are sporadic, that is they form spontaneously without any underlying genetic change. The emergence of disease correlates with a novel conformational form , which only modestly affect wild-type yeast growth, induce de novo formation of [PSI+] from wild-type Sup35.PSI+] prion formation is also observed in response to oxidative stress caused by the superoxide anion.Increasing evidence suggests that protein oxidative damage can trigger misfolding events resulting in prion formation. Oxidative stress induced by exposure to hydrogen peroxide was found to significantly increase [PSI+] induction did not identify any antioxidants.PSI+] formation from nuclear gene mutations by their irreversible elimination in guanidine hydrochloride (GdnHCl). Reversible curability is a well-established genetic criterion for yeast prionsPSI+] formation also exhibit significantly increased rates of nuclear mutations.TSA1 results in an increased rate of spontaneous mutations which arise as a result of endogenous ROS production.PSI+] prion formation in antioxidant mutants.An unbiased genome-wide screen for factors which increase prion formation in a [PIN+][psi\u2212] strain.PSI+] prion formation. Increased MetO levels were detected in Sup35 from antioxidant mutants grown under normal non-stress conditions indicating that endogenous ROS levels are sufficient to promote Sup35 oxidation. Increased methionine oxidation is commonly detected in a number of disease states as well as during the aging process. For example, MetO antibodies have been used to detect increased methionine oxidation in aged mouse samples and samples from patients with Alzheimer's disease.We have used an anti-MetO antibody to detect methionine oxidation of Sup35. Oxidative stress conditions induced by exposure to hydrogen peroxide or the superoxide anion significantly increased MetO levels and increased the frequency of [PSI+] prion formation. Sup35 has 3 functional regions: an N-terminal region (residues 1\u2013123) which is essential for prion formation, a highly charged middle (M) region (residues 124\u2013253) and a C-terminal region (residues 254\u2013685) which is required for translation termination activity.PSI+] prion in the absence of the NM region, oxidation of Met residues in the C domain might be expected to affect the overall protein structure/folding of Sup35 which in turn might promote the formation of misfolded structures that have a higher propensity to switch to the prion conformation. Alternatively Met mutations may affect specific Sup35-chaperone interactions. For example, the M region interacts with Hsp104 and is required for prion curing but not propagation.de novo [PSI+] prion formation, although the molecular details are not well defined.It is not know which methionine residues in Sup35 are important for [PSI+] formation observed in response to Sup35 overexpression requires the presence of another prion, the best studied example of which is the [PIN+] prion form of Rnq1.PIN+] is still required for the elevated frequency of [PSI+] prion formation observed in tsa1 tsa2 mutants, indicating that oxidative stress does not overcome the requirement of cells being [PIN+] to form the [PSI+] prion.PIN+] prion aggregates are thought to provide imperfect templates which seed the polymerization of Sup35, facilitating the formation of its transmissible prion form.PIN+]-dependent manner.The elevated frequency of [PSI+] prion formation, confirming that Sup35 oxidation can cause the switch from a soluble to an aggregated prion-form of Sup35 in antioxidant mutants.PSI+] prion formation as shown in PIN+] aggregates promote de novo formation of the [PSI+] prion. Sup35 oxidation may cause misfolding by altering the conformation of its polypeptide backbone and decreasing thermal stability, or alternatively, it may disrupt normal Sup35-chaperone interactions. It is unknown whether Sup35 oxidation occurs on the nascent polypeptide chain or in pre-existing Sup35 molecules .PSI+] prion form. Methionine oxidation does not appear to account for the formation of all yeast prions since we were unable to detect methionine oxidation of Rnq1 in a tsa1 tsa2 mutant.PIN+] formation is elevated in tsa1 tsa2 and sod1 mutants suggesting that prion formation may be a common phenomenon in antioxidant mutants.PIN+] prion formation is significantly higher than [PSI+] prion formationPIN+] prion form. Alternatively, other forms of protein oxidation may underlie the switch from soluble Rnq1 to its amyloid form.Under normal non-stress growth conditions, MetO formation is not detected in Sup35 suggesting that cellular antioxidants are sufficient to protect against endogenous ROS exposure and MSR enzymes function to reduce any MetO that might be formed. The addition of an exogenous oxidant, which can overwhelm the cellular antioxidant defense systems, results in Sup35 oxidation and misfolding, prior to targeting to the IPOD where it is converted to the heritable [Sc purified from brain samples; although it was unclear whether oxidation actually occurred in the brain or during the protein purification and analysis protocol.C to an oxidant has been shown to result in methionine oxidation,in vitro studies suggested that methionine oxidation of recombinant hamster and mouse PrPC interferes with its conversion into amyloid fibrils.in vivo studies showed that MetO (particularly Met213 in humans) could be detected in PrPSc isolated from brain samples, but not in PrPC, leading to the suggestion that MetO is a specific marker for PrPSc.C and PrPSC isolated from a hamster-adapted scrapie model found comparable low levels of methionine oxidation suggesting that MetO213 should not be regarded as a prion-specific covalent signature.in vivo, it raise the question as to whether methionine oxidation underlies the structural switch that converts PrPC to PrPSc, or is simply a biomarker for PrPSC formation. Specificity for PrPSc might arise if methionine oxidation requires partial denaturation of PrPC before methionine residues can become oxidized, or else they may be oxidized only after PrPSC forms.Conflicting data has been reported regarding the potential role of methionine oxidation in the aggregation and pathogenicity of mammalian prions. Mass spectrometry was originally used to detect MetO in PrPin vivo studies are able address the importance of methionine oxidation in sporadic PrPSC formation. In vitro studies have attempted to examine the importance of methionine oxidation as a destabilizing event that triggers PrPC misfolding leading to spontaneous prion formation. For example, methionine analogs have been used to correlate the degree of PrPC methionine oxidation with the structural transitions that underlie its conversion to the infectious PrPSC form.C has been shown to reduce its thermal stability, promoting generation of a molten globule fold which is thought to be a key step in the misfolding pathway.C which can trigger its conversion to the pathogenic PrPSC form.C structure resulting in destabilization and misfolding.C was recently found to be more susceptible to methionine oxidation.C decreased its structural stability, enhanced its aggregation and increased neurotoxicity. This suggests that methionine oxidation can have synergistic effects with other pathogenic mutations in PrPC which cause prion diseases.None of the existing C destabilization or PrPSC stabilization such as inhibiting clearance of misfolded forms and favoring misfolding pathways which result in amyloid formation. This will however, be experimentally difficult without the tractability of the yeast model system.Further work will be required to establish a causal role for methionine oxidation in PrP misfolding and the formation of toxic oligomers and amyloid plaques. This may occur via PrPNo potential conflicts of interest were disclosed."} +{"text": "IgA nephropathy (IgAN) is a clinically and pathologically heterogeneous disease. Endocapillary proliferation is associated with higher risk of progressive disease, and clinical studies suggest that corticosteroids mitigate this risk. However, corticosteroids are associated with protean cellular effects and significant toxicity. Furthermore the precise mechanism by which they modulate kidney injury in IgAN is not well delineated. To better understand molecular pathways involved in the development of endocapillary proliferation and to identify novel specific therapeutic targets, we evaluated the glomerular transcriptome of microdissected kidney biopsies from 22 patients with IgAN. Endocapillary proliferation was defined according to the Oxford scoring system independently by 3 nephropathologists. We analyzed mRNA expression using microarrays and identified transcripts differentially expressed in patients with endocapillary proliferation compared to IgAN without endocapillary lesions. Next, we employed both transcription factor analysis and in silico drug screening and confirmed that the endocapillary proliferation transcriptome is significantly enriched with pathways that can be impacted by corticosteroids. With this approach we also identified novel therapeutic targets and bioactive small molecules that may be considered for therapeutic trials for the treatment of IgAN, including resveratrol and hydroquinine. In summary, we have defined the distinct molecular profile of a pathologic phenotype associated with progressive renal insufficiency in IgAN. Exploration of the pathways associated with endocapillary proliferation confirms a molecular basis for the clinical effectiveness of corticosteroids in this subgroup of IgAN, and elucidates new therapeutic strategies for IgAN. IgA nephropathy (IgAN) is the most common cause of primary kidney disease globally and up to one third of patients with IgAN will progress to kidney failure by 10 years following diagnosis A new pathologic classification system for IgA nephropathy has recently been proposed. Through a rigorous international collaborative effort, reproducible morphologic features in IgAN independently correlated with long-term renal disease outcome were identified Corticosteroids modulate activity of several transcription factors, and their main effects on immune responses are ascribed to inhibition of the activity of nuclear factor \u03ba-B (NF\u03baB) To determine the molecular pathways involved in the development of endocapillary proliferation in patients with IgAN, and to identify novel therapeutic targets, we evaluated the glomerular transcriptome of microdissected kidney biopsies from patients with IgAN. We compared the mRNA expression profile of biopsies with glomerular endocapillary proliferation (E1) to biopsies without endocapillary proliferation (E0), based upon interpretation by three independent nephropathologists. We then identified the mRNA transcriptomic profile associated with the pathologic finding of endocapillary proliferation in IgAN. Next, we employed both transcription factor analysis and in silico drug screening and confirmed that the endocapillary proliferation transcriptome is enriched with pathways modulated by corticosteroid exposure. With this approach we also identified new therapeutic targets that may be responsible for the pathogenesis of this lesion, and a panel of small molecules that may be candidates for modulation of the pathways responsible for development of endocapillary proliferation. Taken together, our findings are proof of principle that evaluation of the human tissue molecular phenotype associated with a distinct, clinically important and carefully annotated pathologic phenotype can yield insights regarding novel therapeutic strategies for the treatment of IgAN.Biopsy material was obtained from the European Renal cDNA bank (ERCB) For both cohorts, the RNA was extracted from the microdissected glomerular compartment of kidney biopsies using identical protocols as previously described A single PAS-stained section from each case was reviewed by four nephropathologists blinded to the reported diagnosis, and the glomeruli were scored according to the Oxford MEST scoring system http://brainarray.mbni.med.umich.edu/Brainarray/default.asp). Microarray gene expression data of the ERCB and Toronto cohorts were combined and batch-corrected using the Combat method implemented in the GenePattern pipeline (http://www.GenePattern.org). Principal component analysis using ArrayTrack software (http://www.fda.gov/ArrayTrack) supported the use of the merged ERCB and Toronto mRNA expression profiles for further analysis.Microarray data normalization and filtering were performed as previously described Differential mRNA expression between the E1 and E0 biopsies was evaluated using the Significance Analysis of Microarrays (SAM) method implemented in the TIGR MultiExperiment Viewer (TMEV) application www.genomatix.de). Canonical pathways were analyzed using Ingenuity Pathway Analysis software (IPA) (www.ingenuity.com). Gene expression datasets are available on Gene Expression Omnibus (GEO) under the accession number GSE50469 (http://www.ncbi.nlm.nih.gov/geo/).The functional context of significantly regulated genes was studied by creating biological literature-based networks using Genomatix Pathway System software (GePS) (www.broadinstitute.org/cmap) http://www.maayanlab.net/DPS). This database provides access to data regarding in vitro cellular responses of human cell lines to approximately 1300 bioactive compounds. This allows users to identify which compounds would be expected to revert disease-associated gene expression signatures derived from user-provided datasets. For this CMAP analysis the imported query of differentially expressed transcripts was compared with predefined gene expression signatures of therapeutic compounds and ranked according to a connectivity score (+1 to \u22121) representing relative similarity in regulation to the imported gene list. A filtering step for the \u201cmost representative\u201d gene expression signature was employed to manage the large amount of data in the CMAP database. Compounds with negative connectivity scores, representing genes discordantly expressed to the imported query, are predicted to exert biologic effects that revert disease signatures in the renal tissue. Drug Pair Seeker was used to predict and prioritized which drug pairs from CMAP data could be associated together to reverse the direction of gene expression.Differentially expressed genes with a fold change \u22651.5 for the up-regulated genes and \u22640.6 for the downregulated genes were converted into probeset signatures, imported, and analyzed by Connectivity Map (CMAP) and 15 biopsies without endocapillary proliferation (E0). The clinical characteristics of the patients at the time of kidney biopsy are described in We identified 424 genes that are differentially expressed in microdissected glomeruli with endocapillary proliferation versus glomeruli without endocapillary proliferation at a conservative false-discovery-adjusted q-value of <0.05 . The expTo identify relevant biological pathways represented in these 424 differentially expressed genes, we performed canonical pathway analysis . The top enriched canonical pathways differentially expressed in biopsies with endocapillary proliferation are provided in The only glomerular pathologic phenotype as defined by the Oxford classification associated with a distinct molecular signature was endocapillary hypercellularity. Only 9 and 4 genes were significantly regulated in biopsies with versus without mesangial hypercellularity or segmental glomerulosclerosis, respectively.Therapy with corticosteroid is associated with complete regression of endocapillary proliferation in serial biopsies of patients with IgAN. Given that the primary mechanism of action underlying the immunosuppressive action of prednisone is interruption of transcription of nuclear factor kappa-B-(NF\u03baB) regulated genes, we performed transcription factor analysis to evaluate whether there was an overrepresentation of genes with an upstream NF\u03baB consensus binding unit. We found that one quarter (144 of 424) of the differentially expressed genes contained upstream NF\u03baB promoter sites representing significant enrichment of pathways that may be modulated by inhibition of corticosteroids . Using ain vitro models of cellular responses to a panel of bioactive molecules To identify bioactive compounds that modulate expression of the transcripts represented in our endocapillary proliferation signature, we used the Connectivity Map (CMAP) approach. With this approach we compared our gene signatures to expression profiles in experimental We have employed a translational approach to delineate molecular mechanisms underlying the development of endocapillary proliferation in human subjects with IgAN, and to identify potential therapeutic targets to modulate this disease. Our approach to identification of clinically and biologically relevant processes involved in progressive IgAN was based upon two important clinical observations. First, the pathologic lesion of endocapillary proliferation is associated with a higher risk of progressive loss of renal function, and second, it is potentially modifiable by corticosteroid therapy.Large observational studies suggest that corticosteroids may be effective in preventing progression of IgAN Our first finding is that in the isolated microdissected glomeruli of human diagnostic kidney biopsies, endocapillary proliferation is associated with a distinct signature of differentially expressed mRNA transcripts. We identified the biologic processes and pathways represented in this signature, using canonical pathway analysis. Given that endocapillary proliferation may be amenable to therapy with corticosteroids and that the prime mechanism of action of this drug is interruption of NF\u03baB-mediated gene transcription, we verified that our signature includes transcripts regulated by this transcription factor. Indeed one-third of the transcripts contain upstream promoter binding sites. Finally, we employed in silico drug screening and confirmed that the endocapillary proliferation transcriptome is significantly enriched with pathways modulated by corticosteroids, which is supportive of clinical observations of efficacy of corticosteroids in patients with this lesion. With this approach we also identified novel therapeutic targets and bioactive small molecules that may be considered for treatment of IgAN.The glomerular mRNA expression profile associated with endocapillary proliferation includes a significant number of transcripts encoding proteins involved in the innate immune response, such as several toll-like receptors (TLRs). One of the potential mechanisms hypothesized to be responsible for development of IgAN is a dysregulated immunologic response to a mucosal microbial challenge The activation of TLRs and binding of intracellular adapter proteins results in increased activity of transcription factors interferon transcription factor 3 and NF\u03baB www.Nephropmine.org). The Connectivity Map datasets have been used to predict and prioritize pairs of drugs that reverse or aggravate the direction of differential gene expression (\u201cDrug Pair Seeker\u201d available at http://www.maayanlab.net/DPS/). With this approach we identified several compounds predicted to reverse the gene expression changes associated with endocapillary proliferation. Resveretrol emerged as a potential novel compound with this potential therapeutic effect, and interestingly in our analysis both methylprednisolone and corticosterone were predicted to reverse the E1 transcriptional responses when used in combination with resveratrol. This compound, a polyphenol naturally occurring in grapes, has been shown to have attenuate kidney injury induced in the ischemia-reperfusion rat model To evaluate functional connections between corticosteroids and our differentially expressed transcripts we used CMAP analysis. This tool is a reference catalogue of gene expression data derived from cultured cells exposed to a spectrum of bioactive compounds In summary, we have used a novel approach to identify a distinct glomerular molecular signature associated with endocapillary proliferation. Our transcriptional and pathway analyses support the clinical observation that this lesion may be modifiable with use of corticosteroids. We have also identified therapeutic targets and agents for future study in treatment of IgAN.Figure S1Analytic approach.(DOCX)Click here for additional data file.Table S1Oxford MEST scoring.(DOCX)Click here for additional data file.Table S2List of the 424 genes significantly differentially regulated in E1 vs. E0 IgAN biopsies , sorted by q-value. The 144 genes in bold type also possess an upstream binding site for NF\u03baB1 in their promoter region.(DOCX)Click here for additional data file.Table S3Considering endocapillary proliferation as a continuous variable. As illustrated in (DOCX)Click here for additional data file.Table S4Canonical pathways significantly regulated from the 424 genes regulated in E1 vs. E0 biopsies, as assessed by IPA .(DOCX)Click here for additional data file.Table S5144 genes of the 424 regulated in E1 vs E0 have a binding site for NFKB1 in their promoter.(DOCX)Click here for additional data file.Table S6Transcription factor analysis results. This list includes TFs able to bind to the promoter region of at least 30 different genes from the 424 genes differentially regulated in E1 vs E0.(DOCX)Click here for additional data file.Table S7Compounds from Connectivity map analysis results . These compounds are predicted to have favourable effects reversing the majority of differential mRNA expression changes associated with endocapillary proliferation. Compounds of interest are highlighted in grey.(DOCX)Click here for additional data file.Table S8Drug pair seeker analysis. Top computationally-predicted drugs that would enhance the reversal of gene expression changes associated with endocapillary proliferation when combined with methylprednisolone or corticosterone. Coverage\u200a=\u200anumber of desirable targets that the drug affects, meaning the transcripts described in endocapillary proliferation that the drug would reverse. Conflict\u200a=\u200athe number of genes the drug is potentially changing in an undesired direction in endocapillary proliferation.(DOCX)Click here for additional data file."} +{"text": "Early mobilization (EM) of patients on mechanical ventilation (MV) is shown to improve outcomes after critical illness. Little is known regarding clinician knowledge of EM or multi-disciplinary barriers to use of EM in the intensive care unit (ICU). The goal of this study was to assess clinician knowledge regarding EM and identify barriers to its provision.Simultaneous cross-sectional surveys of medical ICU (MICU) nurses (RN)/physical therapists (PT) respondents and physician (MD) respondents in a single MICU at an academic hospital in Seattle, WA in 2010\u20132011. Responses were indicated on a 5 point Likert scale and reported as proportion of respondents agreeing or disagreeing. Chi-square testing and Fisher\u2019s exact testing was performed to determine whether responses differed by duration of employment or prior EM experience.A total of 120 clinicians responded to the survey (91 MDs (response rate 82% (91/111)), 17 RNs ), and 12 PTs ), overall response rate 86%). Most clinicians indicated knowledge regarding benefits of EM. More attending physicians reported knowledge of EM benefits, but also that risks of EM outweigh the benefits compared to trainees (p\u2009=\u20090.02 and 0.01). Clinicians across disciplines reported near universal agreement to use of EM for patients on MV, while the minority reported agreement to EM for patients on vasoactive agents. The most frequently reported cross-disciplinary barriers to EM were staffing and time. Risk of self-injury and excess work stress were indicated as barriers by RN and PT respondents.MICU clinicians, at our institution, reported knowledge of EM in the ICU. Staffing and clinician time were frequently identified cross-disciplinary barriers. Risk of self-injury and excess work stress were frequently reported RN and PT barriers.The online version of this article (doi:10.1186/1471-2253-14-84) contains supplementary material, which is available to authorized users. Critically ill patients, particularly those requiring mechanical ventilation (MV), are prone to impairments in physical function associated with immobility and intensive care unit (ICU) acquired weakness. Functional impairments acquired during ICU hospitalization result in increased need for long term nursing care, greater risk of readmissions and reductions in health-related quality of life for ICU survivors \u20136. ThereThese studies show that EM is safe, feasible, and results in significant improvements in delirium and function at time of hospital discharge \u201316. DespQuality improvement projects have attempted to understand whether clinician attitudes and education around EM serve as barriers to its delivery , 14, 19.Therefore, the aim of this study was to investigate whether clinicians in the medical intensive care unit at our institution are knowledgeable regarding the benefits of early mobilization and to identify perceived barriers to delivery of mobility in the ICU.We performed two simultaneous cross-sectional surveys of clinicians providing care in the medical intensive care unit (MICU) at our institution between July 2010 and July 2011. Our institution is a large, urban academic medical center in Seattle, WA. Physician subjects were identified during clinical rotations through the medical intensive unit. All rotating residents, fellows and attending physicians were invited to participate. Registered nurses and physical therapists working in the MICU were identified by the MICU nurse manager and the head of the physical therapy department. Participation was voluntary and each participant contributed one, distinct survey to the study. Physician participants were invited to participate at the start of clinical rotations through the MICU. Nursing and physical therapy participants were invited to participate during a regularly scheduled staff meeting. Potential participants were only contacted for potential participation on one occasion. Surveys were administered by an individual not involved in data analysis or primary study design with physician surveys administered on paper and nursing/physical therapist administered electronically. All surveys were anonymous and were collected in an anonymous manner.Surveys were conducted prior to implementation of an ICU mobility program at our institution. At the time of survey administration, activity orders for critically ill patients required a physician orders with all activity performed by either the bedside nurse and/or a physical or occupational therapist who shared acute care floor and ICU duties. Physician surveys differed in wording from nursing/physical therapy surveys occurring within 48 hours of initiation of mechanical ventilation for our study purposes. Prior experience with early mobilization was defined as a respondent responding \u201cyes\u201d to the question, \u201cHave you ever trained and/or worked at an institution that actively mobilizes patients receiving MV?\u201d \u201cCorrect\u201d answers for the knowledge questions were identified prior to survey administration. Reponses of strongly disagree or disagree were considered \u201ccorrect\u201d answers for the questionnaire item assessing whether range of motion was sufficient to maintain muscle strength. This was identified as a correct answer. Reponses of agree or strongly agree were considered \u201ccorrect\u201d answers to the questionnaire item assessing whether the use of early mobilization was associated with a decrease in duration of mechanical ventilation. This was chosen as a \u201ccorrect\u201d based on the results of the single randomized controlled trial using early mobilization as an intervention to date demonstrating a shorter duration of mechanical ventilation in patients receiving early ICU mobility . For allDescriptive statistics were used to describe respondents. Likert scale responses were reported as frequencies and proportions. Chi-square testing was performed to test whether responses differed significantly among physician providers differing by level of training (faculty vs. trainee physicians) and differing by prior experience with early mobilization. Fisher\u2019s exact testing was performed to test whether responses differed significantly among nursing and physical therapist providers differing by levels of experience (\u22655 years vs. < 5 years) and differing by prior experience with early mobilization. A two-sided p-value of <0.05 was considered statistically significant for all analyses. Statistical calculations were performed using STATA 12.0 .This study was conducted as part of an educational quality improvement initiative aimed at understanding institutional use of EM. The study was reviewed and considered non-Human Subjects research by the University of Washington Institutional Review Board. This study adheres to the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelines ). Most physicians were Internal Medicine or Pulmonary and Critical Care Medicine trainees (Table\u00a0/111). MoMost physicians surveyed indicated that they would allow EM for patients on MV and most indicated they would be willing to alter their ventilator strategy n\u2009=\u200964, 71%) to allow for EM. Forty-three percent (n\u2009=\u200939) of physicians indicated that they disagreed with EM for patients on vasopressor agents. Prior experience with EM was not associated with reported agreement with EM for patients on vasopressors . Eighty percent (n\u2009=\u200973) of physicians surveyed indicated that EM should occur automatically via nursing and PT protocols unless the physician specifically orders otherwise. Staffing, excessive sedation, delirium and patient safety were identified most frequently as barriers to EM . Most nurse respondents reported greater than 5 years of employment in their current medical ICU. Most MICU nurses n\u2009=\u200913, 76%) indicated that range of motion was insufficient to maintain muscle strength in critically ill patients and that use of EM reduces duration of MV . Responses did not differ based on years of experience nursing . Nurses who reported a history of prior experience with EM were more likely to report that reduction in duration of MV as a potential benefit (p\u2009=\u20090.04). Most MICU nurses surveyed agreed that it was possible to mobilize patients on MV while less than half agreed that it was possible to mobilize patients on vasopressor agents. Three nurses (18%) surveyed agreed that the risks of EM to patients outweighed the potential benefits completed a single survey regarding EM . Most PT respondents reported greater than 5 years of employment in their current medical ICU. Most physical therapists indicated that range of motion was insufficient to maintain muscle strength in critically ill patients and most indicated that EM reduces duration of MV. Responses to the knowledge questions did not differ by years of experience as a physical therapist or by prior experience with EM . All of the MICU PTs surveyed agreed that it was possible to mobilize patients on MV while fifty-eight percent (n\u2009=\u20097) agreed that it was possible to mobilize patients on vasopressor agents regarding knowledge and perceived barriers towards EM in critically ill patients. We targeted clinicians involved in patient care in the MICU given that the largest body of literature around EM to date exist for medical ICU patients, particularly patients diagnosed with respiratory failure , 11, 15.There were a number of shared barriers reported across all types of MICU clinicians including staffing and time. Finding the time and personnel necessary to mobilize can be a deterrent to implementation of mobility programs and an often reported concern in quality improvement studies targeting broader acceptance of mobility , 22, 25.Nursing and physical therapy staff highlighted a number of previously unreported barriers to EM that may be key to implementation of EM including risk of self-injury, perceived work stress and concerns over delay of usual work. Although studies demonstrate that EM is safe and feasible for patients, missed work related to staff musculoskeletal injury is under explored and may represent an important occupational health barrier to delivery of EM , 29. ConDisconnect between physicians and nursing on patient-nurse safety risk may hinder efforts to optimize mobility if nursing staff perceive personal safety risks around mobilizing certain patient populations that are not conveyed to physicians. Targeted focus on mobility readiness that incorporate all members of the care team, including physicians, are needed in order to develop shared mental models with the patient and the mobilizing team . EnhanciRespondents in our study again identified sedation and delirium as barriers to EM. Prior mobility studies demonstrate a link between reducing levels of sedation in critically ill patients and subsequent increase in ICU mobility , 17\u201319. Surprisingly, attending physicians were significantly more likely than trainees to report excess risk to early mobilization, potentially reflecting practice biases and reluctance to adopt new interventions too early. This sense of \u201cclinical inertia\u201d is reported in other care interventions and may serve as a safeguard given the uncertain nature of medicine , 35. TheInstitution of checklists with targeted user feedback regarding performance adherence may aid in overcoming clinical inertia. Clinicians surveyed in our study appeared amenable to such interventions with 80% of physicians reporting willingness to relinquish decision making regarding early mobilization to multidisciplinary protocols. Further studies are needed to better define ideal care team models and care delivery systems for EM. Better definitions of care roles may allow for more targeted surveys of attitudes and behaviors that may influence implementation and adherence to mobility programs , 37. CreThis study has a number of important limitations. First, the surveyed sample represents a small sample of ICU care providers from a single institution. Our results are subject to selection bias regarding clinicians who opted to participate in the survey, particularly around the nursing respondents given the low response rate. While focus groups and pilot testing were utilized for survey development, our surveys were limited in length and scope by timing of administration and feasibility. Potential interactions may exist between survey questions that may introduce response bias. Additionally, answers to \u201cknowledge\u201d questions may be influenced by the limited early mobilization literature and potential reduced generalizability from EM clinical trials. Our results represent a baseline for our institution, but cannot fully address all potential attitudes and barriers towards early mobilization. Larger survey-based studies are needed to understand how pervasive these attitudes are across the broader ICU mobility. Finally, our survey did not survey administrative leaders. Understanding administrator attitudes towards EM is necessary when addressing hospital-level barriers including resource allocation and staffing. The strength of our study is that it is the first to broadly survey all MICU care clinicians within an institution to better understand cross-disciplinary care concerns around EM. As our study was focused at a single institution, it is possible that the results reflect local culture limiting generalizability and the low response rate by nursing providers limits generalizability of the nursing results. However, selection of an average MICU in a large academic hospital mirrors the care settings represented in most EM literature to date.Medical intensive care unit clinicians at our institution, overall, were knowledgeable regarding the potential benefits of early mobilization for critically ill patients. Clinicians across MICU care disciplines agreed to EM of patients on MV, but disagreed regarding EM in patients on vasoactive agents. Attending physicians were more likely to report the risks of EM outweighed the benefits compared to trainees, despite being more likely to identify potential benefits to EM. Staffing and clinician time were the most frequently reported barriers to EM across disciplines. Risks of self-injury, excess work stress and delay of usual care were identified as nursing and PT specific barriers to EM. Additional research on appropriate patient selection for EM and risks of EM to staff combined with enhanced education, may be necessary for broader implementation of EM programs across medical intensive care units.\u2002Medical intensive care unit nurses, physical therapists and physicians are knowledgeable regarding potential benefits to early mobilization including reductions in duration of mechanical ventilation and maintenance of muscle strength.\u2002Attending physicians were significantly more likely to report knowledge of benefits to early mobilization and to report that the risks of mobilization outweigh the potential benefits compared to trainees.\u2002Physical therapy and nursing staffing and time were the most frequent physician reported barriers to early mobilization.\u2002Excess work stress and risk of self-injury were commonly reported nursing and physical therapy reported barriers to early mobilization.Clinician attitudes survey supplement 1.(DOCX 15 KB)Additional file 1:STROBE Statement\u2014Checklist of items that should be included in reports ofcross-sectional studies.(DOC 79 KB)Additional file 2:"} +{"text": "Curing HIV is a new strategic priority for several major AIDS organizations. In step with this new priority, HIV cure research and related programs are advancing in low, middle, and high-income country settings. This HIV cure momentum may influence existing HIV programs and research priorities.Despite the early stage of ongoing HIV cure efforts, these changes have directly influenced HIV research funding priorities, pilot programs, and HIV messaging. The building momentum to cure HIV infection may synergize with strategic priorities to better identify adults and infants with very early HIV infection. Although HIV cure represents a new goal, many existing programs and research techniques can be repurposed towards an HIV cure. HIV messages focused on engaging communities towards an HIV cure need to be careful to promote ARV adherence and retention within the HIV continuum of care.An increased emphasis within the AIDS field on finding an HIV cure has several important implications. Strengthening connections between HIV cure research and other areas of HIV research may help to catalyze research and facilitate implementation in the future. HIV cure research and related programs are accelerating around the world. This new priority developed over the last five years because of an increased understanding of the long-term side effects of lifelong antiretroviral therapy (ART), the public health challenges of current strategies, and basic science advances regarding HIV latency . The goaAlthough the field of HIV cure is still in its infancy and many decisions about cure efforts or pilot programs have not been centrally organized, there are already important implications of expanding HIV cure momentum . The purThe most substantial implication of the increased emphasis of the AIDS field on finding a cure for HIV is changing HIV research priorities. The International AIDS Society HIV cure resources tracking group estimated that 157.9 million USD was invested in HIV cure research in 2014 , the MedThe new emphasis on HIV cure within broader HIV funding priorities raises an important question about the relationship between HIV cure and other HIV research priorities. The International AIDS Society \u201cTowards a Cure\u201d initiative has explicitly stated that the global HIV response should not divert research funding from other areas of HIV research towards cure, instead arguing for greater HIV cure resources . In addiAnother important policy implication of the HIV cure momentum is enhanced detection of adults and adolescents with acute or very early HIV infection. The first few weeks or months after infection, but prior to seroconversion, constitute the period of acute HIV infection . We defiNew HIV cure momentum also provides an opportunity to synergize and extend existing programs focused on identifying very early HIV infected individuals. Programs seeking to increase or improve diagnosis and treatment during very early infection are driven by both public health imperatives and concerns for long-term patient well-being. Public health programs focused on identifying very early HIV infection have been established because of the increased risk of onward transmission during very early infection . AdditioAlthough the additional momentum created by HIV cure research is unlikely to overcome logistical problems, HIV cure may provide another strong reason to enhance detection of very early HIV infected adults within clinical trial sites. HIV cure related interest in very early infection may help to raise awareness about acute HIV infection, its symptoms, and why early detection and suppression are important. This would be useful given that many patients and primIn addition to enhanced detection of very early infected adults and adolescents, HIV cure momentum may increase the number of neonates discovered with very early HIV infections. Neonates who acquire HIV infection from their mothers are another key group for HIV cure research efforts as demonstrated by the extended remission of the Mississippi child case . AnalogoFinally, HIV cure has progressively changed how we talk about HIV in public health messages. Although the resources necessary for implementing HIV control strategies are substantial, available HIV resources have been stable or decreasing in many regions , 31. TheGiven the opportunity presented by a growing HIV cure momentum around the world, we have several recommendations for enabling these policy-program synergies. First, engaging a wide range of global HIV stakeholders, especially in low and middle income settings, will be important for realizing the programmatic synergies articulated above, just as it was in the development of previous interventions . PolicymHIV cure momentum is building in a range of global settings. While this is a new priority, there are several ways that research towards an HIV cure can strengthen existing HIV policies and practices."} +{"text": "A quality improvement collaborative uses multiple approaches, including coaching, to teach the necessary skills to the change leader in an effort to teach them quality improvement skills and to enhance their knowledge acquisition in order to implement effective organizational changes. However, little is known about the individual teaching and learning styles of the coach or change leader and how a particular style or a match between teaching and learning style influence quality improvement outcomes. This study builds on the NIATx200 results. It seeks to answer two research questions: 1) What are the learning and teaching style typology in a quality improvement collaborative? (2) How do levels of convergence and divergence between staff learning style and coach teaching style influence the outcomes in a quality improvement collaborative?The Grasha-Riechmann Student Learning Style survey and Teaching Style Inventory, developed and validated within the field of educational research, were modified to identify the individual teaching and learning styles of participants in a quality improvement collaborative. Change leaders, executive sponsor, and coaches were invited to complete the surveys. Using NIATx200 results, each outcome was classified as improved or not improved for each site. A pooled factor effect backwards stepwise elimination regression model explored the relationship between the different styles and the NIATx200 outcomes.Coaches (n = 17) in a QIC exhibit similar teaching styles identified in an educational setting. The learning style of change leaders (n = 77) in a quality improvement collaborative differs from how students learn in an education setting. Results indicate the presence of 10 different learning styles. The regression results indicate that higher competitive leader learning styles scores is associated with lower wait-time improvements for providers in the learning session intervention. Higher expert and personal model teaching styles are associated with wait time improvements for the coaching and combination arm . Coaches with higher expert teaching style scores showed greater admission improvements in the coaching intervention arm .The preliminary results suggest that certain individual learning and teaching styles influence organizational outcomes for certain interventions within a quality improvement collaborative. Further research is required to understand how teaching and learning styles interact to influence outcome improvement. The findings could suggest how to tailor a quality improvement collaborative to improve outcomes."} +{"text": "Homeostasis in electrical activity is a guiding principle for network formation and reorganization throughout life. Traditionally homeostatic plasticity is thought to adapt the weight of existing synapses so that postsynaptic firing rates are regulated towards or maintained at a desired set-point. However, neither during development nor in adulthood the connectivity structure of sparsely connected brain networks is fixed but continuously rewires. Here, we summarize our most recent modeling results showing that homeostasis in electrical activity can drive network formation during development as well as network reorganization after focal retinal lesions.For both studies, we applied our model of structural plasticity (MSP) ,2 in comInterestingly, during network formation neurons started to form longer connections than expected from the kernel function as soon as they approached a homeostatic equilibrium in electrical activity making networks more efficiently connected as networks formed without a homeostatic rule for synapse formation . Our theWe also applied MSP to restore neuronal activities after a focal loss of input . AlthougThis work broadens our understanding of brain plasticity as observes forms of plasticity that go beyond mere Hebbian synaptic weight plasticity. Especially structural plasticity acts on long timescales from days to weeks and months that are crucial for brain development and network repair after brain lesions and so far hardly respected by computational neuroscience."} +{"text": "Melanoma is a highly malignant melanocyte-derived tumor and its incidence is increasing at outstanding rate. Despite different types of immunotherapy became available for the treatment of melanoma, including adoptive T cell transfer, immune checkpoint blockade, and vaccines such as dendritic cell vaccines, when applied to treat metastatic melanoma, only 10 to 40% of the patients have long term benefit. Furthermore, given the toxicity and high costs associated with immunotherapy there is a stringent need to identify biomarkers that may predict its potential efficacy. We have investigated whether the presence and distribution of T cells within the primary tumor of melanoma patients correlates with survival when treated for metastatic disease with dendritic cell based cancer vaccines. Quantitative multispectral imaging has been used to compare the tumor microenvironment of responding patients that survived longer than 20 months after immunotherapy, and non-responding patients that survived less than 12 months after immunotherapy. T cell infiltrates have been initially assessed in primary melanomas of 19 metastatic melanoma patients treated with dendritic cell based immunotherapy (discovery set) and subsequently in an independent cohort of 39 patients . In the discovery cohort we observed a very high correlation between the ratio of peritumoral over intratumoral T cells in the primary tumor and overall survival. Lower peri/intratumoral T cell ratios in primary tumors were associated with improved clinical outcome. ROC curve and multivariate analysis were exploited to evaluate the predictive power of the T cell ratio and established prognostic markers. Statistical analyses in the validation cohort confirmed our findings showing that the peri/intratumoral T cell ratio correctly predicted long term survival after DC vaccination in 90% of the cases and was the strongest predictor of survival in the multivariate analysis. This study indicates that the ratio between CD3 positive peri/intra-tumoral T cells is a very strong predictor of patient outcome and response to dendritic cells immunotherapy in melanoma patients."} +{"text": "Extracellular electrical potential oscillations recorded as local field potentials (LFPs) in the hippocampal formation are thought to be involved in cognitive processes such as working memory retention, memory consolidation and spatial navigation. Recent studies have shown that combining spikes with LFP phase can increase the information content of spikes ,2 and th"} +{"text": "The abscopal effect is a phenomenon that occurs when radiation of disease at one-site leads to regression of distant, untreated tumors. Historically, the abscopal effect caused by conventional radiation therapy has been rare. However, the combination of radiation and immunotherapy has increased the reported incidence of this effect, which is thought to be mediated by an adaptive immune response. We have demonstrated that radiation alone (RT) is weakly effective at generating anti-tumor adaptive immune responses despite release of tumor-associated antigen. We hypothesized that providing a potent adjuvant to the tumor would improve the adaptive immune response increasing immune control of local and distant disease The STimulator of INterferon Genes (STING) innate signaling pathway is being investigated for its ability to propagate an adaptive immune response that is initiated in the tumor microenvironment (TME). STING is a cytosolic receptor that senses either exogenous or endogenous cyclic dinucleotides (CDNs), produced by bacteria or by host cell cyclic GMP-AMP synthetase in response to binding dsDNA, respectively. In the current work, we evaluated synthetic CDN derivatives modified to be resistant to degradation by phosphodiesterases and to activate all known human STING allele variants. To test our hypothesis, we established bilateral Panc02 flank tumors in immune competent C57BL/6 mice. 14 days post tumor challenge mice were randomized to receive a suboptimal dose of 10 Gray of RT to one tumor, and intratumoral injection of CDN or vehicle to that same tumor immediately following radiation and again at 24 hours. CDN injection resulted in rapid local and systemic induction of inflammatory mediators including IFN\u03b3 and TNF\u03b1 , and vascular damage that spread through the injected tumor without causing detectable damage to normal tissues. CDN injection caused rapid innate-mediated regression of injected tumors , and when combined with local radiation therapy also caused adaptive-immune mediated control of distant tumors . We conclude that these CDNs are potent activators of innate immunity that in this model combine effectively with radiation therapy to promote adaptive immune activation and systemic immune surveillance."} +{"text": "DNA methylation is an essential epigenetic modification for mammalian development and is crucial for the establishment and maintenance of cellular identity. Traditionally, DNA methylation has been considered as a permanent repressive epigenetic mark. However, the application of genome-wide approaches has allowed the analysis of DNA methylation in different genomic contexts revealing a more dynamic regulation than originally thought, since active DNA methylation and demethylation occur during cellular differentiation and tissue specification. Satellite cells are the primary stem cells in adult skeletal muscle and are responsible for postnatal muscle growth, hypertrophy, and muscle regeneration. This review outlines the published data regarding DNA methylation changes along the skeletal muscle program, in both physiological and pathological conditions, to better understand the epigenetic mechanisms that control myogenesis. DNA methylation was the first discovered epigenetic modification are pluripotent cells showing very low methylation levels at CpG-rich sequences and differentiated myotubes (MTs), using the non-promoter-oriented RRBS method, showed no significant methylation changes during myogenic terminal differentiation when he realized that treated cells turned into a huge syncytium of multinucleated cells and proliferating MBs showed no gene expression is a key indicator of myofibroblast differentiation into fibroblast, and its accumulation in tissue remodeling and fibrosis leads to a deleterious excess of extracellular matrix components had the capacity to convert 5mC into 5-hydroxymethylcytosine (5hmC) raised the possibility that 5hmC might constitute a distinct epigenetic state contributing to dynamic changes in DNA methylation and \u201caxon guidance signaling\u201d (P = 6.17E\u201310), suggesting that the dmCpG sites identified within the aged group were relevant to muscle tissue functions and neuromuscular junctions and old (68\u201389 years of age) males showed a predominant pattern of hypermethylation in the DNA of aging skeletal muscle is mutated gene is a severe chronic childhood autoimmune disease showing the affected children muscle weakness caused by chronic muscle damage showed a small but very rapid global DNA methylation decrease in young muscles (Barr\u00e8s et al., soleus muscles 45 min after ex vivo contraction correlating with increased gene expression (Barr\u00e8s et al., Barr\u00e8s et al. analyzed DNA methylation levels of the Altogether, these results suggest that physical exercise may modify DNA methylation patterns, although future studies will be needed to deeper understand the involvement of epigenetic mechanisms in the beneficial effects of regular exercise on human health.DNA methylation plays an important role in mammalian development, as illustrated during skeletal muscle cell fate commitment and differentiation. During development muscle stem cells acquire an unique DNA methylation signature associated with its specialized functions, and specific-myogenic factors are activated in a demethylation-dependent manner. DNA methylation patterns are not fixed but dynamic, and can be modulated by external influences. We have highlighted the recent data regarding how skeletal muscle methylome changes in response to physical exercise, aging and in muscle-related pathologies including cancer (summarized in Figure EC and MS wrote the manuscript. Both authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Unhealthy drinking and other drug use are common comorbid problems among individuals receiving treatment in psychiatric Emergency Departments (ED). Several studies have shown that systematic screening and motivational brief interventions (SBIRT) are promising approaches for reducing alcohol and drug use that can be implemented in psychiatric ED settings. Recent digital screening and brief intervention trials have shown reductions in alcohol and other drug use among general (non-psychiatric) ED patients. These electronic approaches have yet to be tested for those receiving care in psychiatric ED settings. This presentation will provide an overview and framework for considering how SBIRT might be delivered electronically among psychiatric ED patients and how digital devices might be used to extend the impact of SBIRT beyond the psychiatric ED.Systematic literature review of published literature on digital behavioral interventions for individuals with major mental disorders. Review of existing literature on electronic SBIRT approaches in the ED setting.general ED and other healthcare settings. However, the potential use of digital interventions for alcohol and other drug use among psychiatric ED patients have been inadequately explored.Several studies have shown promising results for the use of tablet computers, wearable tracking devices and smartphone apps for individuals with mental disorders. The targeted behavioral interventions have typically focused on general fitness and weight loss. Several published studies have shown promise for digital SBIRT and computer-assisted SBIRT approaches (computer decision support) addressing alcohol and other drug use in Integrating digital SBIRT interventions initiated in psychiatric ED settings, with potential continuing care using mobile devices, could both improve patient access to SBIRT and provide alternative enhancements to psychiatric care to improve outcomes for these vulnerable patients. Electronic delivery of SBIRT, combined with the use of other digital technologies for patients with major mental disorders and concurrent substance misuse, should be explored."} +{"text": "Current hypotheses about the pathophysiology of schizophrenia suggest that the disease results from disordered connectivity in the brain. Human functional imaging studies have lent support to this idea by demonstrating reduced temporal correlations between cortical areas in schizophrenic patients ; howeverTo characterize changes in synaptic communication between neurons in the disease state, we evaluated timing relationships in spike trains of simultaneously recorded neurons. We employed a Generalized Linear Model (GLM) approach to infer"} +{"text": "Tremor associated with Parkinson\u2019s disease can be medication resistant and disabling. A clinical trial of stereotactic focused ultrasound thalamotomy is underway.This pilot study is being conducted in two centers with a randomized, controlled study design using blinded and validated rater assessments and a sham procedure. The primary outcome variable is hand tremor and the Unified Parkinson\u2019s disease rating scale in the medicated state at three months. Comprehensive cognitive assessments before and after the procedure are being conducted for additional safety. A total of thirty patients will be enrolled."} +{"text": "Central nervous system tumors are marked with high intra- and inter-tumoral heterogeneity. For instance, an integrative large scale gene expression study performed in 2010 revealed 4 subtypes glioblastoma multiforme (GBM) with differential responses to treatment and prognosis [Two, not mutually exclusive, general models have been proposed to explain tumor heterogeneity . The gen2 to have an animal model in which both the cell of origin and genetic insult can be conveniently and independently manipulated. To achieve this, we have recently developed a central nervous system tumor model in the rat in which multiple oncogenes can be expressed in selected cell populations at different times in brain development [piggyBac transposon system [in utero electroporation (IUE). Using this model, we evaluated the contribution of cell of origin and genetic mutation in tumor heterogeneity.To fully explore the causes of tumor diversity, it is desirableelopment . In thisn system to stablTo test whether the same oncogenic event in different, but closely related, cell population gives rise to same or different tumors, we directed HRasV12/AKT expression in disparate cell populations in the radial glia lineage with promoters that are ubiquitously active (CAG promoter), astrocyte selective GFAP promoter or oligodendrocytes selective MBP (myelin basic protein) promoter. We showed that HRasV12/AKT expression under CAG or GFAP promoter induced similar tumors, glioblastoma multiforme (WHO grade 4). However, HRasV12/AKT expression controlled by MBP promoter induced anaplastic oligoastrocytoma (WHO grade 3). We further showed that these induced induced anaplastic oligoastrocytoma differed from glioblastoma multiforme both in histology and molecular signature. These results indicate that oncogenic events occurring in different cell types in the same cellular lineage can lead to different tumor types.We next investigated whether tumor phenotype could be modified by expression of neurogenic bHLH family protein Neurogenin2 (Ngn2) or Neural differentiation 1 (NeuroD1). Members of the bHLH gene family are well known to have important roles is cell-type determination in normal development. Expression of either Ngn2 or NeuroD1 along with HRasV12/AKT resulted in atypical teratoid rhabdoid tumor like (ATRT like) tumor, a tumor type not previously observed after expression of HRasV12/AKT oncogenes alone. We further tested whether this phenotypic transformation from GBM to ATRT like tumor was due to transient expression of bHLH factors in radial glia or due to expression in tumor cells. Our data showed that transient expression of Ngn2 in radial glia, prior to transformation by HRasV12/AKT was able to induce ATRT like tumor formation. These results may indicate that the same oncogenic events occurring in similar cell types expressing different levels of individual bHLH transcription factors can lead to very different tumor types.piggyBac IUE method is that it allows for introduction of multiple transgenes controlled by independent promoters. The high co-expression rates allowed us to direct expression in different subpopulations in sequence. In the same system, we also demonstrated use of multi-color fluorescent protein expression to produce a clonal readout of tumor growth and invasion. There are several additional features of the piggyBac system that make it useful for other applications in investigating tumor biology. For example, the piggyBac- IUE approach could be applied to other species other than rodent extending CNS tumor biology to other species, such as ferret. Combination of piggyBac-IUE with existing transgenic mouse models could potentially broaden the utility of both approaches. Also multicolor clonal labeling of tumor cells could facilitate in vivo imaging of clonally related tumor cells. These functionalities should make this approach a useful platform for screening potential modifiers of tumor development and for studying further how genetic modifiers and cell or origin are related to tumor development and heterogeneity.The main advantage of"} +{"text": "Hum Brain Mapp 36:1637\u20131647, 2015. \u00a9 2015 The Authors Human Brain Mapping Published by Wiley Periodicals, Inc.Refractory mesial temporal lobe epilepsy (mTLE) is a debilitating condition potentially amenable to resective surgery. However, between 40 and 50% patients continue to experience postoperative seizures. The development of imaging prognostic markers of postoperative seizure outcome is a crucial objective for epilepsy research. In the present study, we performed analyses of preoperative cortical thickness and subcortical surface shape on MRI in 115 of patients with mTLE and radiologically defined hippocampal sclerosis being considered for surgery, and 80 healthy controls. Patients with excellent I) and suboptimal (ILAE II\u2013VI) postoperative outcomes had a comparable distribution of preoperative atrophy across the cortex, basal ganglia, and amygdala. Conventional volumetry of whole hippocampal and extrahippocampal subcortical structures, and of global gray and white matter, could not differentiate between patient outcome groups. However, surface shape analysis revealed localized atrophy of the thalamus bilaterally and of the posterior/lateral hippocampus contralateral to intended resection in patients with persistent postoperative seizures relative to those rendered seizure free. Data uncorrected for multiple comparisons also revealed focal atrophy of the ipsilateral hippocampus posterior to the margins of resection in patients with persistent seizures. This data indicates that persistent postoperative seizures after temporal lobe surgery are related to localized preoperative shape alterations of the thalamus bilaterally and the hippocampus contralateral to intended resection. Imaging techniques that have the potential to unlock prognostic markers of postoperative outcome in individual patients should focus assessment on a bihemispheric thalamohippocampal network in prospective patients with refractory mTLE being considered for temporal lobe surgery. Mesial temporal lobe epilepsy (mTLE) due to hippocampal sclerosis (HS) is the most common and most frequently operated medically intractable epilepsy disorder [Janszky et al., Despite that resective surgery will significantly reduce the number of debilitating seizures for the majority of patients, up to 40% of patients with mTLE and HS will continue to experience postoperative seizures at 2\u2010year follow\u2010up [Berkovic et al., There is inconsistent evidence for the relationship between hippocampal structural alterations on MRI and postoperative outcome in mTLE, with some studies reporting increased hippocampal atrophy in patients with persistent postoperative seizures relative to those rendered seizure free [Keller et al., We studied 115 patients with refractory mTLE and neuroradiologically defined ipsilateral HS who were being considered for temporal lobe resection at University Hospital Bonn, Germany. As per standardised protocol, each patient had a detailed clinical assessment to ascertain seizure semiology, interictal electroencephalography (EEG), long\u2010term video EEG monitoring, if clinically necessary additional intracranial electrode recording, MRI images), and neuropsychological assessment [Kral et al., 3, flip angle 10\u00b0).All study participants underwent MRI at the Life & Brain Center in Bonn on a 3 Tesla scanner . An eight\u2010channel head coil was used for signal reception. Morphometric analyses in this study were performed on 3D T1\u2010weighted MPRAGE images \u2009=\u20091300 ms, inversion time (TI)\u2009=\u2009650 ms, echo time (TE)\u2009=\u20093.97 ms, resolution 1.0 \u00d7 1.0 \u00d7 1.0 mmhttp://surfer.nmr.mgh.harvard.edu/). This approach has been previously described in detail [Dale et al., P\u2009<\u20090.05, False Discovery Rate).We analysed differences in regional cortical thickness between participant groups using Freesurfer software for the automated segmentation, volume calculation and surface shape analysis of the subcortical structures of interest: the left and right hippocampus, amygdala, thalamus, and the major structures of the basal ganglia . As described in detail elsewhere [Kim et al., P\u2009<\u20090.05, corrected for multiple comparisons. Results from surface analysis were visualized using Freesurfer's Freeview software. To compare global parenchyma volumes between groups, and to determine intracranial volume (ICV) for normalisation of subcortical volumes, each image was brain extracted using FSL's brain extraction tool [Smith, We used FSL\u2010integrated registration and segmentation toolbox (FSL\u2010FIRST) software, version 5.0 , patients with left mTLE (n\u2009=\u200975) and those with right mTLE (n\u2009=\u200940). We subsequently compared cortical and subcortical morphology between patients who were surgically rendered seizure free and patients who continued to experience persistent postoperative seizures. To increase sample size and thus the sensitivity in detecting pathological alterations in one patient postoperative outcome group relative to another, we side\u2010flipped the images of patients with right mTLE so that all patients could be analysed together and abnormalities treated as ipsilateral and contralateral to seizure onset/intended surgery only when analysing MRI relationships with postoperative outcome, as undertaken previously [Bernhardt et al., Comparisons were initially made between healthy controls .Patients who were rendered seizure free and those who continued to experience postoperative seizures showed very similar patterns of reduced cortical thickness relative to controls Fig. . A reducF\u2009=\u2009119.3, P\u2009<\u20090.001), amygdala , thalamus , and putamen , the right amygdala , thalamus , and accumbens area , and whole gray and white matter. Significant volume reduction in patients with right mTLE was restricted to the right hippocampus and accumbens area relative to controls. Whole structural volumes were not significantly different between patients who were rendered seizure free and those who experienced persistent postoperative seizures .Relative to controls, patients with left mTLE had significantly reduced global volume of the left hippocampus , and hippocampal alterations in posterior lateral/dorsal areas . When we explored results without stringent correction for multiple comparisons, degenerative changes in the posterior ipsilateral hippocampus was observed in patients with persistent postoperative seizures relative to those rendered seizure free . There were no significant differences in the surface shape of basal ganglia structures or the amygdala between patient outcome groups at corrected or uncorrected statistical thresholds. We furthermore determined the relative shape change for each significant cluster from each comparison in every patient and investigated correlations with clinical variables. There were no significant correlations between age of onset of mTLE or preoperative frequency of seizures and subcortical shape changes . There was a borderline significant correlation between increasing duration of preoperative mTLE and clusters of maximum shape change in the ipsilateral and contralateral thalami across all patients, but not when corrected for patient age . No other significant effects were observed.Patients who continued to experience postoperative seizures had significant regional inward surface deflation of the thalamus bilaterally and of the hippocampus contralateral to the side of intended surgery relative to patients who were rendered seizure free Fig. . ThalamiRefractory mTLE is associated with widespread cortical and subcortical brain atrophy, but it is preoperative focal alterations of the thalamus in both cerebral hemispheres and of the hippocampus contralateral to intended resection that are associated with persistent postoperative seizures. Our patient outcome groups did not differ in cortical thickness, subcortical volume, global gray and white matter, or surface shape of the basal ganglia and amygdala. These findings suggest that persistent postoperative seizures are not simply due to a more widespread distribution of brain damage, but potentially a localized alteration in a bilateral thalamohippocampal network.Abnormalities of the thalamus are well established in imaging studies of human mTLE [Barron et al., Our thalamic structural findings are theoretically compatible with an invasive stereoelectroencephalography study on ictal mesial temporal\u2014thalamic coupling in patients with mTLE [Guye et al., Some studies that have investigated the volume of the whole hippocampus on MRI have reported no significant association between postoperative outcome and the nonoperated hippocampus in patients with mTLE [Jack et al., We also found evidence of subtle hippocampal atrophy in the posterior ipsilateral hippocampus in patients with postoperative seizures when statistics were uncorrected for multiple comparisons. We reported this finding as preliminary data in an earlier study in a small group of patients with left mTLE [Keller et al., Correlations between an increasing duration of mTLE and decreasing brain volume have been used in some studies to infer progressive brain degeneration due to uncontrolled seizures. Ultimately, only longitudinal imaging can resolve whether recurrent seizures cause progressive brain damage, and only when patient brain changes are considered alongside normal maturational changes observed in neurologically healthy people. An increasing duration of mTLE has been related to hippocampal [Bernasconi et al., Our results may provide some insight into the underlying biological mechanisms of seizure severity in mTLE. Consistent with other work [Janszky et al., It is important to consider that there may be discrete pathological alterations of cortex underlying postoperative seizures that are spatially discordant between patients, and will therefore be obscured in group analyses such as the present study. One electrophysiological study reported that 55% of patients with persistent postoperative seizures were considered to have seizure onset in the ipsilateral temporal neocortex [Hennessy et al., The quantitative assessment of subcortical surface shape is a novel way of determining regional atrophy of subcortical structures on MR images. The approach we have used has previously been shown to reveal regional thalamic alterations that are consistent with focal alterations in thalamocortical connectivity [Hughes et al., both patients with left and right mTLE. Finally, it should be noted that the apparent large difference in number of left (n\u2009=\u200975) and right (n\u2009=\u200940) sided mTLE patients recruited into this study were consecutively recruited. Left mTLE is significantly more prevalent than right mTLE [Janszky et al., Side\u2010flipping MRI data in patients with unilateral neurological disorders/lesions is frequently performed in quantitative studies so that data can be pooled and analysed with respect to a primary factor. This was done in the present study, with the primary factor being postoperative outcome, which is an approach taken in previous MRI studies of outcome in mTLE [Bernhardt et al., mTLE due to HS is a systems seizure disorder without a circumscribed brain abnormality. There are networked structural alterations in mTLE that support seizure initiation, propagation and modulation, which include the mesial temporal lobe, thalamus, basal ganglia, and cortex. Our data suggest that new imaging techniques that have the potential to unlock prognostic markers of postoperative outcome in individual patients should focus assessment on a bihemispheric thalamohippocampal network in prospective patients with refractory mTLE being considered for temporal lobe surgery. Damage to this network may prognosticate seizure resistance to conventional temporal lobe surgery.Supplementary InformationClick here for additional data file."} +{"text": "One has to use antimalarial drugs differently for elimination than ordinary treatment. When treating sick patients, the objective is to kill many parasites in a few sick people whereas malaria elimination involves killing a few parasites in many well people. There are standard public health means to reach very low levels of malaria infection; what is difficult is moving from low to no malaria. Although mass drug administration has been used successfully in China, it is not entirely clear how such methods might be adapted for other populations especially those with more persons having G6PD deficiency. Primaquine and methylene blue are among our oldest antimalarial drugs and are established means of killing the gametocyte transmission stages. Adverse events and long dosage regimens make both primaquine and methylene blue far from the mass drug administration model used during antifilarial drug campaigns. A long acting primaquine analog, tafenoquine, is in late clinical trials and may have some role in eliminating relapsing malaria hypnozoites from a population. Other new chemical compounds such as the imidazole-piperazine KA156 are in the development pipeline which have anti-transmission potential although this has not be demonstrated in field trials to date. Eliminating malaria will require a multi-faceted approach that will necessarily involve new means of using drugs to kill small residual parasite populations long after the major public health effects of malarial disease has disappeared."} +{"text": "This is the official guideline endorsed by the specialty associations involved in the care of head and neck cancer patients in the UK and provides recommendations on the pre-treatment oral and dental assessment, during and after treatment and oral rehabilitation. Restorative dentists are core members of the multidisciplinary team treating head and neck cancer patients, involved from the treatment planning phase through to long-term rehabilitation.\u2022 Preventative oral care must be delivered to patients whose cancer treatment will affect the oral cavity, jaws, salivary glands and oral accessibility. (G)\u2022 Close working and communication between the surgeons, oncologists and restorative dental specialists is important in ensuring optimal oral health outcomes. (G)\u2022 Intensity-modulated radiotherapy has been shown to reduce long-term xerostomia and should be offered to all appropriate patients. (R)\u2022 If patients are deemed at risk of trismus they should be warned and its progressive and potentially irreversible nature explained. (G)\u2022 Where it is known that adjuvant radiotherapy will be given, extractions should take place at primary surgery to maximise the time for healing and minimise the number of surgical events for patients. (G)\u2022 Osseointegrated implants should be considered for all patients having resection for head and neck cancer. (G) The consultant in restorative dentistry and oral rehabilitation is a core member within the head and neck cancer team as many patients face complex oral rehabilitation and dental health issues during and after their treatment. This section addresses the issues relating to pre-treatment oral and dental assessment, preventative advice, during and after treatment and oral rehabilitation.Patients whose oral cavity, teeth, salivary glands and jaws will be affected should have assessment and appropriate management as early as possible to allow time for any necessary dental treatment.\u2022Avoidance of unscheduled interruptions to primary treatment as a result of dental problems\u2022Pre-prosthetic planning and treatment, e.g. planning for primary implants and/or impressions for obturator\u2022Planning for extraction of teeth which are of doubtful prognosis or are at risk of dental disease in the future and are in an area where there would be risk of osteoradionecrosis.\u2022Planning for restoration of remaining teeth as required\u2022Preventive advice and treatment\u2022Assess potential for post-treatment access difficulties, e.g. trismus, microstomia.The aims of pre-treatment assessment are:Treatment for head and neck cancer may involve surgery, chemotherapy and radiotherapy which can cause adverse short- and long-term oral side effects as follows:\u2022Mucositis: inflammation and ulceration of the mucosal lining of the oral cavity\u2022Infection: chemotherapy-induced neutropenia makes the patient susceptible to bacterial, viral and fungal infections. Oral candidal infections are extremely common following chemotherapy or radiotherapy\u2022Xerostomia: dry mouth resulting from a decrease in the production of saliva as a result of radiotherapy.Short term:\u2022Altered anatomy: surgical ablation and reconstruction can cause permanent changes in oral anatomy making prosthetic rehabilitation difficult\u2022,Rampant dental caries: radiogenic dental caries\u2022Trismus: may be caused by surgical scarring or by radiotherapy induced fibrosis of the masticatory muscles\u2022Mastication difficulties: if a significant number of opposing pairs of teeth are lost\u2022Osteoradionecrosis: hypovascularity and necrosis of bone followed by trauma-induced or spontaneous mucosal breakdown, leading to a non-healing wound\u2022Xerostomia: intensity-modulated radiotherapy (IMRT) reduces the risk of xerostomia after treatment and possibly osteoradionecrosis.Long term:\u2022Maintenance of good oral hygiene by effective tooth brushing; flossing daily\u2022Dietary advice with regard to caries prevention\u2022Daily topical fluoride application (5000\u00a0ppm fluoride toothpaste) in custom-made trays or brush-on.\u2022\u2122 containing free calcium or other remineralising agentDaily use of GC Tooth Mousse\u2022Saliva replacement therapy and use of frequent saline rinses\u2022Jaw exercises to reduce trismus.Treatments include Chinese medicines, hydrolytic enzymes, ice chips, benzydamine, calcium phosphate, etoposide bolus, manuka honey, iseganan and zinc sulphate.There is strong evidence that some antifungal drugs prevent oral candidiasis caused by cancer treatment, but nystatin does not appear to work. Chlorhexidine gluconate has antifungal and antibacterial properties in addition to antiplaque effects; however, its value is still unconfirmed. Its tendency to stain teeth and its alcohol content, which can irritate inflamed tissues, are potential drawbacks.\u2122 should not be used by dentate patients as their pH is below the critical pH of 5.5. Pilocarpine (5\u201310\u00a0mg/day) may improve radiation induced xerostomia in patients with evidence of some intact salivary function.This can be managed by sipping sugarless fluids frequently, chewing sugarless gum or lozenges, and using a carboxymethyl cellulose saliva substitute as a mouthwash. Oral balance gel may be best accepted by patients because of its extended duration of effect. Acidic salivary stimulants such as GlandosaneProstheses may be required to replace missing oral and facial tissues. These may be implant supported.Management must be individualised, and patients must be assessed at regular intervals to determine the caries risk and caries activity to provide guidance for maintenance of the dentition.This can be minimised by maintenance of the dentition and use of well-made prostheses.\u2122 prior to and during radiotherapy may limit the severity of trismus, but they will not mobilise fibrosis once it has occurred. They may help surgically induced trismus (as may coronoidectomy). Dental work that was deferred during radiotherapy should be completed. Frequent dental follow-up appointments (3\u20134 monthly), either with local general or community dental practitioner is warranted for these patients.Jaw exercises and the use of devices such as the Therabite,\u2022Preventive oral care must be delivered to patients whose cancer treatment will affect the oral cavity, jaws, salivary glands and oral accessibility (G)\u2022Close working and communication between the surgeons, oncologists and restorative dental specialists is important in ensuring optimal oral health outcomes (G)\u2022IMRT has been shown to reduce long-term xerostomia and should be offered to all appropriate patients (R)\u2022If patients are deemed at risk of trismus they should be warned and its progressive and potentially irreversible nature explained (G)\u2022Where it is known that adjuvant radiotherapy will be given, extractions should take place at primary surgery to maximise the time for healing and minimise the number of surgical events for patients (G)\u2022Osseointegrated implants should be considered for all patients having resection for head and neck cancer (G)Osseointegrated implants allow effective oral and facial rehabilitation following cancer treatment including radiotherapy.,The placement of intra-oral implants at the same time as tumour resection may be beneficial for carefully selected patients and where there is continuity of the mandible or in patients who require the prosthetic obturation of significant maxillary defects where retention of the obturator is likely to be compromised or in patients undergoing rhinectomy or orbital exenteration.For many patients, the placement of osseointegrated implants will be considered following cancer treatment in response to ongoing problems with oral function. A secondary approach allows a detailed assessment of the patient's overall prognosis, their individual risk factors as well as their anatomical factors such as the presence of reconstructive hard and soft tissue grafts, metal hardware, tongue function and mouth opening. Comprehensive prosthodontic planning should be undertaken prior to surgery and the use of computerised planning and surgical guide stent technology is useful.\u2022Consultants in Restorative Dentistry are core members of the multidisciplinary team dealing with head and neck cancer patients\u2022Patients whose oral cavity, teeth, salivary glands and jaws will be affected by their treatment should have a dental assessment and appropriate management as early as possible to allow time for any necessary dental treatment\u2022Patients requiring maxillary obturation should be carefully prepared for treatment by a Restorative specialist who should ideally be present during surgery\u2022Consideration should be given to the placement of osseointegrated titanium implants at the time of primary resective surgery in selected patients in order to support dental and facial prostheses\u2022Liaison with the patient's general dental practitioner is important for ongoing dental care with support from the Restorative specialist where advice is required."} +{"text": "The International Federation of Gynecologists and Obstetricians (FIGO) staging system for ovarian cancer is surgically based. It does not formally include imaging but the FIGO committee encourages the use of imaging techniques if available to assess the important prognostic factors such as disease resectability and lymph node status. FIGO has recently revised the staging of ovarian cancer . It inclThe standard of care for patients with newly diagnosed advanced ovarian cancer has been comprehensive staging laparotomy and primary optimal surgical cytoreduction followed by adjuvant chemotherapy. However, the use of neoadjuvant chemotherapy followed by interval debulking surgery (IDS) as a suitable alternative is supported by multicenter randomized controlled trials . ImagingCT is the primary imaging modality used to stage ovarian cancer. It is complimentary to surgical staging identifying possible sites of unsuspected disease such as pelvic peritoneum, paraaortic nodes, diaphragm and chest . The tho"} +{"text": "A major challenge in rare diseases is conducting clinical trials with sufficient power to inform best clinical practice when anticipated sample sizes are small. Historically, this has been a major barrier in rare paediatric autoimmune diseases. Bayesian methodology can be used to augment the sparse therapeutic data obtained from clinical trials in these circumstances.We elicited expert prior opinion for a future Bayesian randomised controlled trial for a rare inflammatory paediatric disease, polyarteritis nodosa .A Bayesian prior elicitation meeting was convened. Participating experts were drawn from across the EU and Turkey. Opinion was sought on the probability that a patient in the MYPAN trial treated with cyclophosphamide would achieve disease remission within 6-months, and on the relative efficacies of mycophenolate mofetil and cyclophosphamide. Expert opinion was combined with previously unseen data from a recently completed randomised controlled trial of mycophenolate mofetil versus cyclophosphamide in anti-neutrophil cytoplasmic antibody associated vasculitis.A pan-European group of fifteen experts participated in the elicitation meeting. Consensus expert prior opinion was that the most likely rates of disease remission within 6 months on cyclophosphamide or mycophenolate mofetil were 74% and 71% respectively. This prior opinion will now be taken in to account and will be modified to formulate a Bayesian posterior opinion when data from 40 patients completing the trial randomised at a 1:1 ratio to either receive cyclophosphamide or mycophenolate mofetil are available.We suggest that this methodological template could be applied to trial design for other rare diseases, and is of particular relevance to rare autoimmune conditions that currently lack a good evidence base for treatment.None declared."} +{"text": "This study aimed to assess current education and practices of emergency medicine (EM) residents as perceived by EM program directors to determine if there are deficits in resident discharge handoff training. This survey study was guided by the Kern model for medical curriculum development. A six-member Council of EM Residency Directors (CORD) Transitions of Care task force of EM physicians performed these steps and constructed a survey. The survey was distributed to program residency directors via the CORD listserve and/or direct contact. There were 119 responses to the survey, which were collected using an online survey tool. Over 71% of the 167 American College of Graduate Medical Education (ACGME) accredited EM residency programs were represented. Of those responding, 42.9% of programs reported formal training regarding discharges during initial orientation and 5.9% reported structured curriculum outside of orientation. A majority (73.9%) of programs reported that EM residents were not routinely evaluated on their discharge proficiency. Despite the ACGME requirements requiring formal handoff curriculum and evaluation, many programs do not provide formal curriculum on the discharge transition of care or evaluate EM residents on their discharge proficiency. Millions of patients are seen in the emergency department (ED) with approximately 86% rate of discharge.8Evidence on how to ensure ideal handoffs has been limited, but multiple sources have identified process standardization as an opportunity for quality improvement.12Although there are clear mandates to ensure that handoffs are standardized, and formal handoff curriculum and evaluation are provided to emergency medicine (EM) residents, there is no information available to identify the current practices of EM training programs on the discharge transition of care.The objectives in this study were to (1) assess the current scope of discharge training among EM residency programs by surveying their residency leadership, (2) assess current educational and evaluation practices related to discharge training, and (3) identify whether additional training is necessary based on the current practices and perceived competencies.This survey study was guided by the six-step Kern model for medical curriculum development.The institutional review board at Alameda County Medical Center granted exempt approval for this study. Members of CORD were invited to complete the survey electronically. The CORD e-mail listserve is exclusive to educators in EM residency programs and includes associate, assistant and primary residency program directors from the 167 ACGME-accredited EM residency programs. Program leaders were recruited via the CORD listserve from March to April 2014. The survey was opened for six weeks in which 87 identified programs responded. Duplicate responses were reviewed and clarified with program directors directly in April 2015 (22 programs). Two programs requested their duplicate responses be deleted and completed new surveys to accurately represent their current practice. All other programs selected their most accurate survey responses. Direct emails were then sent to program directors with links to the survey in April 2015; 32 additional programs completed the survey and the survey was closed on April 23, 2015. Only programs that identified their program name were included in the study to ensure that all surveyed programs represented ACGME-accredited programs and to avoid potential duplicative responses.A total of 119 programs were surveyed, making the overall response rate 71.2% of the 167 currently accredited ACGME EM residency training programs. A majority of programs indicated that residents are given informal education regarding discharge training by senior residents and faculty (87.4%); just under half provide formal curriculum at orientation (42.9%) and/or outside of orientation (5.9%). A small percentage of programs offer no training (6.7%) . Over haMost residency programs reported using a structured discharge system in the ED , while a small minority report using none or being unsure if this is provided in their main ED . For those with a structured process of discharge most reported that this is being performed most of the time or always . A majority of programs reported providing structured written modifiable written discharge instructions and bidirectional conversations with patients . A majorAll programs reported a variety of tools to assist in the discharge process with only 34, or 43%, of the respondents being satisfied or extremely satisfied with these tools . Over twOver three-quarters of program leadership reported that junior level residents are \u201csomewhat competent\u201d in their discharge competency. Almost a quarter felt that their junior residents were \u201ccompetent\u201d . One program (0.8%) reported their junior residents were extremely competent . For senior level residents, described as residents with over eight months experience, their program leadership identified them as competent or extremely competent . A minority of programs reported their senior level residents as \u201csomewhat competent\u201d in their discharge skills . None of the respondents reported their junior or senior level residents to be incompetent in their discharge abilities.These results provide insight into the discharge educational practices and clinical training experience surrounding discharge of EM residents as reported by their program leadership. Standardized formal training and evaluation is not the current norm at most programs. Most formal training provided to EM residents is at their orientation, and few programs offer formal educational opportunities beyond the first few weeks of training. Since a majority of program leaders indicated that ideal educational practices would include formal training at orientation and/or workshops or classes outside of orientation, programs may value structured education of discharge competencies but may be constrained by other limitations such as faculty time, didactic scheduling or curriculum availability. The same gap was seen between current training and ideal training for evaluation processes. While most programs do not perform formal evaluations on their residents\u2019 discharge competency, a majority of programs identify that this would be an ideal practice. This implies that EM program leadership values formal evaluation of their residents\u2019 discharge competency but they may be constrained by limitations such as a recognized evaluation tool and/or faculty time. These data also suggest that while program leadership values discharge competency training and evaluations, this education may not be valued as a high priority since most programs perceive their senior level residents to be competent. Although it is difficult to fully endorse competency without a standardized evaluation process, there is support that informal evaluation may be valid in identifying residents\u2019 clinical competencies.16The fact that this study relies on perceptions of program leadership is a major limitation as there is no gold standard to formally measure discharge competency even for programs providing formal evaluation of their residents\u2019 discharge competency. While most program leadership feel that their current process of discharge is \u201csafe and effective,\u201d this perception may be limited. Given that each respondent based their program results on his or her perceptions of the discharge education and performance of residents within the clinical environment, construct underrepresentation and irrelevant variance represent threats to the validity of clinical performance ratings in this study.Program leadership relies on general gestalt that their residents are competent in their discharge proficiency since most are not evaluating this competency. Program leadership may not identify any limitations in senior resident discharge competency because there may be larger, more systematic failures of discharge communication with patients and/or caregivers. In this scenario, resident performance of discharge may be at an acceptable level at the departmental level but departmental expectations of patient discharge competency may not be meeting the patient needs to create safe and effective discharges. Standardizing the process of handoffs between providers in the hospital environment has demonstrated improvement in the quality of communication and increased patient safety in the clinical arena.The conflation of the concept of \u201csafety\u201d and \u201ceffectiveness\u201d may be another limitation of this survey. Programs were asked about safe and effective discharges in a single question. These two concepts may be inappropriately linked together and it may be that safety may exist without being effective and vice versa. This may represent a construct error in the survey design and affected results of this survey.Lastly, a major limitation of this study was the gap of approximately one year between the surveys of the first cohort of surveyed programs (87) and the second (32). Although it is unlikely that most programs changed their educational practices dramatically within that time period, it is possible.Further work should focus on the more structured assessment of resident discharge competency through direct observation and evaluation of resident performance to corroborate program leaders\u2019 assessment of resident competence. These evaluation tools should then be validated and studied in a clinical setting with specific process measures and patient outcomes. Following Kern\u2019s six-step model for curriculum development, the next step would be to create specific goals and objectives of the discharge curriculum and develop educational interventions aligned with these goals. Curricular tool suggestions that have been made to structure education and evaluation for provider-to-provider handoffs might be used directly or modified to educate residents in the discharge transitions of care.18The results of this targeted needs assessment indicate a lack of structured training and assessment of resident discharge competency despite current guidelines for formalized training in all handoffs. Although most programs reported senior residents are competent in discharge proficiency, the residents\u2019 training is primarily informal which may lead to significant variability in resident experience and performance. Further research should be aimed at assessing proficiency of resident discharge performance through objective observation with validated evaluation tools. Structured training and assessment recommendations should follow from this research with increased attention to implementing a standard curricular model or toolbox, objective, valid evaluation methods, and identification and management of high-risk discharges."} +{"text": "Fifteen years ago in Health Services Research 1999) qualitative research methods were argued to be useful and valid. Since that time qualitative research methods have gained increasing legitimacy however qualitative research papers remain underrepresented in high impact health journals [999 qualiNarrative inquiry is based on the premise that by listening to the stories of others we can make sense of their experience and understand how they construct meaning within a broader social context. Data is usually collected through interview. Data analysis includes narrative analysis and/or paradigmatic analysis of narratives . NarratiWe used narrative inquiry to explore health service experiences of 18 young adults with cerebral palsy. It is important to select a scholarly framework to guide but not constrain the inquiry. We selected Polkinghorne . We collThe techniques and processes used in this study can be replicated and transparently reported thus demonstrating the rigour required for narrative inquiry in health services research."} +{"text": "Polymeric filament like type IV Pilus (TFP) can transfer forces in excess of 100 pN during their retraction before stalling, powering surface translocation(twitching). Single TFP level experiments have shown remarkable nonlinearity in the retraction behavior influenced by the external load as well as levels of PilT molecular motor protein. This includes reversal of motion near stall forces when the concentration of the PilT protein is loweblack significantly. In order to explain this behavior, we analyze the coupling of TFP elasticity and interfacial behavior with PilT kinetics. We model retraction as reaction controlled and elongation as transport controlled process. The reaction rates vary with TFP deformation which is modeled as a compound elastic body consisting of multiple helical strands under axial load. Elongation is controlled by monomer transport which suffer entrapment due to excess PilT in the cell periplasm. Our analysis shows excellent agreement with a host of experimental observations and we present a possible biophysical relevance of model parameters through a mechano-chemical stall force map. Pseudomonas aerginosaNeisseria gonorrhoeaein vivo TFP retraction force-velocity characteristic of N. gonorrhoeae loaded using laser trapped micro bead showed constant retraction velocity at lower forces which then decayed to a stable indefinite stall as load was increased N. gonorrhoeae using similar set up showed that TFP retraction may even be reversed at stall fairly quickly into elongation for mutants with low concentration of PilT N. gonorrhoeae have shown an yet undiscovered higher retraction velocity at lower forces for high PilT concentration mutants coevolution whose strong evidence for N. gonorrhoeae has been reported in recent experiments Elongation, adhesion and retraction of long polymeric nano-fiber called type-IV pilus (TFP) results in a form of bacterial surface translocation called twitching motility which causes complex colonization events such as virulence, biofilm formation and fruiting bodies N. gonorrhoeae, PilD which is a preplin peptidase Neisseria meningitidisN. meningitidis and N. gonorrhoeae have been well known We first simplify the cell wall portion of TFP bio-system illustrated in polar complex(PC) which propels pilin recruitment through the charged end of growing TFP during elongation The TFP base may host a A free body diagram of the pilus depicting all the forces is now shown Neglecting inertia and using the balance of moments about A in the tion see we get: for the filaments together with Euler-Bernoulli kinematics, the moment equations can be written as:Where The normalized adhesive traction N. gonorrhoeae TFP, we have Taking the geometrical properties of a typical This is a characteristic of our model where the elongation can be significantly attenuated by increasing levels of PilT in the inner membrane due to increased pilin entrapment by PilT during transport. In the case where elongation is no longer possible due to a precipitous drop in pilin transport, the stall would represent a stable equilibrium. Although, purely concentration based diffusive transport has been ruled out since retraction rate was found to be indifferent to either the length of the retracted TFP or levels of pilin Interestingly this transport step which involves material transportation is slower than reaction and thus elongation process will exhibit pauses to allow for pilin buildup at TFP base, another observed hallmark mechano-chemical region where there is a complex interplay of the cohesive and the binding energy making it possible to arrive at a stall force through a relatively small variation of properties of both TFP interface and molecular motor. Since higher levels of PilT can produce additional weaker interfaces as well, this region provides maximum gains through PilT concentration changes. More specifically, in this region, poor alignment of PilT units due to excessive crowding which can otherwise reduce binding free energy and thus stall force may be mitigated automatically through additional cohesive energy. Thus the stall force which is an important parameter for survival and replication of these bacteria including biofilm formation and virulence N. gonorrhoeaecoevolution of both TFP properties as the underlying molecular motors, reported recently We now generate a mechano-chemical stall plot in N. gonorrhoeae, TFP processing system is known to be extremely primitive and thus shows similar properties across a wide gamut of bacterial species thriving in widely different environmental landscape To conclude we have developed a simplified but biophysically consistent model to understand the behavior of the TFP retraction behavior which includes the pilus deformation. We discover that inclusion of TFP deformation along with an interplay between its surface-interfacial and end-binding behavior plays a key role in explaining a host of yet unexplained experimental behaviors. This includes the excellent quantitative reproduction of the experimentally observed force-velocity curves, force induced switching of retraction to elongation only at depressed levels of PilT, the instantaneous reversion to retraction when optical trap is turned-off, the apparent asymmetry between retraction and elongation in the velocity profile, the relative independence of retraction and elongation behavior on PilU or PilE (pilin) levels and a possible reason for an elusive bi-modal retraction velocity profile. Furthermore, this deformation based model which is used to construct an energy phase diagram mapping the experimental locus on a interfacial-binding energy axis. This phase map was shown to provide a possible explanation for the observed co-evolution between the molecular motors and the TFP itself. Note that although the experiments yielding the parameters were conducted on"} +{"text": "The enteric nervous system (ENS) autonomously controls gut muscle activity. Mechanosensitive enteric neurons (MEN) initiate reflex activity by responding to mechanical deformation of the gastrointestinal wall. MEN throughout the gut primarily respond to compression or stretch rather than to shear force. Some MEN are multimodal as they respond to compression and stretch. Depending on the region up to 60% of the entire ENS population responds to mechanical stress. MEN fire action potentials after mechanical stimulation of processes or soma although they are more sensitive to process deformation. There are at least two populations of MEN based on their sensitivity to different modalities of mechanical stress and on their firing pattern. (1) Rapidly, slowly and ultra-slowly adapting neurons which encode compressive forces. (2) Ultra-slowly adapting stretch-sensitive neurons encoding tensile forces. Rapid adaptation of firing is typically observed after compressive force while slow adaptation or ongoing spike discharge occurs often during tensile stress (stretch). All MEN have some common properties: they receive synaptic input, are low fidelity mechanoreceptors and are multifunctional in that some serve interneuronal others even motor functions. Consequently, MEN possess processes with mechanosensitive as well as efferent functions. This raises the intriguing hypothesis that MEN sense and control muscle activity at the same time as servo-feedback loop. The mechanosensitive channel(s) or receptor(s) expressed by the different MEN populations are unknown. Future concepts have to incorporate compressive and tensile-sensitive MEN into neural circuits that controls muscle activity. They may interact to control various forms of a particular motor pattern or regulate different motor patterns independently from each other. Neurons controlling gastrointestinal functions are located in a continuous ganglionated network within the gut wall. This autonomous system is referred to as the enteric nervous system (ENS). The name was coined by Langley in 1921 to acknowledge the ENS as a third division of the autonomic nervous system and to emphasize its functional independency from the central nervous system Langley, . The ENSNeurons residing in the myenteric plexus are constantly deformed during muscle contraction and relaxation. This becomes obvious when viewing the deformation of a myenteric ganglion during muscle movements . These neurons, according to our previous findings, were defined RAMEN . In order to better define adaptation we calculated an adaptation index fired at a very low frequency even at the highest flow rate. In other studies we used intraganglionic injection of a small volume of Krebs solution which exerted compressive and shear forces. We can exclude a major role of shear stress based on the following findings. While increasing compressive forces by longer duration of intraganglionic injection changed the response pattern of MEN, there was no change if the flow rate and thereby the shear forces were increased.The first studies showing that some myenteric neurons responded to compression were performed in the \u201870s . IPANs were mostly multipolar neurons with a slow after-spike hyperpolarization mechanical stress . This conclusion is based on experiments where we recorded from MEN after distension of the ganglionic area and after intraganglionic volume injection. Measurements of the changes in ganglionic area during deformation indicated that the former stimulus would primarily mimic tensile forces while the latter one induces compressive forces. Fifty-three percent of MEN responded to both stimuli suggesting that the same neuron is able to sense different modes of mechanical stress. This finding was also supported by the fact that 46% of primary cultured MEN responded to tensile stress (pulling an elastic matrix) and compression (von Frey hair) (own unpublished results). Increase in tension caused slowly adapting firing while compression in the same neuron was associated with rapidly adapting firing. It remains to be studied whether MEN express structures specifically encoding tensile and compressive forces or whether the same mechanosensitive element is activated differently by compression and tension. It seems to be a general feature of MEN that responses to compressive stress adapt rapidly whereas, responses to tensile stress adapt slowly.Intestinofugal neurons in the guinea pig colon responded to von Frey hair compression and circumferential tissue stretch because their properties are similar nor their firing pattern has been considered in enteric reflex circuits. One of the biggest challenges is to incorporate compressive and tension-sensitive MEN into a neural pathway that controls muscle activity Figure . MEN mayGrammostola mechanotoxin 4, efficiently attenuated the swelling induced Ca2+ increase in cultured enteric neurons from rat esophagus or receptor(s). The question to address then is whether compression and tension-sensitive MEN express different channels and how targeted pharmacological interventions affect motor patterns. There are too few studies to make firm conclusions but they produced some promising results. One possible mechanosensitive structure is the large\u2013conductance (BK) potassium channels. Mechanical deformation by increasing intraelectrode pressure increased open probability of BK channels and the Technische Universit\u00e4t M\u00fcnchen within the funding program Open Access Publishing.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "More than half of those newly infected with HIV are young people yet youths have lower medication adherence than adults. Short message service (SMS) based interventions have demonstrated their effectiveness in improving adherence among adults in resource limited settings, but their efficacy with youths has not been studied. This study is one of the first qualitative studies to explore youths view on strategies for best implementing SMS based interventions.Six focus groups were conducted with 20 male and 19 female HIV-positive youths in Kampala, Uganda. Participants discussed barriers to adherence, availability and acceptability of SMS messages and strategies for implementation. Verbatim transcripts were managed in ATLAS.ti and analyzed using a framework approach.We found that major barriers to adherence were forgetfulness, stigma, poor social support, inadequate food supply and stressful working conditions. Participants reasoned that SMS based interventions would improve adherence by providing them with much needed reminders and social support. However, youths noted that the success of such interventions hinged on: i) ensuring HIV status anonymity given that text messages are vulnerable to security threats ii) developing effective content and having appropriate delivery times so that youth remain engaged iii) increasing accessibility to include youth without phones iv) addressing challenges related to phone sharing and restrictions on the use of phones by younger adolescents.SMS based interventions have the potential to promote adherence among youths. The primary recommendation of this study is to ensure confidentiality and increase accessibility."} +{"text": "Grb10 and Dlk1, on body composition in mice. These findings pose the question whether there is an evolutionary conflict between genes of maternal and paternal origin over the optimal proportions of body fat and lean muscle mass.Variation in body composition is a popular obsession. The culturally \u2018ideal\u2019 body type is light on fat and heavy on muscle but the human population is collectively laying on fat. A new study finds antagonistic effects of two imprinted genes, http://www.biomedcentral.com/1741-7007/12/99See research article: Grb10 and Dlk1 appear to modulate this trade-off between productive investment and precautionary savings [Grb10 is expressed from the allele a mouse inherits from its mother, but not the allele it inherits from its father, whereas Dlk1 is expressed when inherited from fathers but not mothers.Organisms evolve to maximize their genes\u2019 chances of leaving descendant copies. In pursuit of this goal, some species live fast and die young while others adopt a more sedate pace of life. Life-history theory attempts to understand ecological factors that shape strategic allocations between size versus number of offspring, reproductive effort versus body maintenance, early versus delayed reproduction, and making the best of current opportunities versus preparing for an uncertain future. A major strategic decision involves the relative proliferation of cells contributing to muscle mass (conferring superior earning capacity but increased maintenance costs) and fat mass (a load to be carried but insurance against hard times). Antagonistic effects of Grb10 are born large and develop into lean adults whereas mice with inactivation of the expressed paternal copy of Dlk1 are born small and develop into obese adults should benefit the individual in which they are expressed at a cost to patrilineal kin or impose individual costs for the benefit of matrilineal kin (including mothers). Conversely, paternally expressed genes (PEGs) are predicted to benefit an individual at a cost to matrilineal kin or impose individual costs for the benefit of patrilineal kin . The heaGrb10 and Dlk1 on muscle and fat hint at evolutionary conflict between MEGs and PEGs over body composition, with MEGs favoring more fat and PEGs favoring more muscle. Consistent with this pattern, Prader-Willi syndrome, which is caused by the failure to express one or more PEGs on human chromosome 15, is associated with morbid obesity and low muscle mass. Although Grb10 behaves as a MEG in muscle and fat, it behaves as a neuronal PEG. This switch from maternal-specific to paternal-specific expression in the central nervous system is recapitulated in culture as mouse embryonic stem cells differentiate into neurons [Contrasting effects of neurons .Evolutionary scenarios that invoke differential consequences of body composition for matrilineal and patrilineal kin can be constructed to explain why MEGs and PEGs might favor different allocations between muscle and fat or between brain and brawn. As an example, if (i) mice occupy territories with matrilineal kin and (ii) groups of smaller, plumper mice better survive periods of famine but (iii) greater muscle mass confers an advantage in competition for food within groups, then MEGs should favor more fat and less muscle than PEGs. However, we need to know much more about patterns of cooperation and competition among kin in wild mice before such scenarios can be as compelling as the simple story of maternal-paternal conflict over fetal growth.Imprinted genes influence not only the balance of tissues within bodies but also basic metabolic parameters. Choices of fuel - whether to burn or stored fat and whether to use amino acids for gluconeogenesis or to build proteins - are implicated in major life-history trade-offs, including those between investment in present reproduction and precautionary savings. One aspect of murine metabolism in which imprinted genes play a significant role is the control of thermogenesis (heat production) by brown adipose tissue (BAT).Grb10 (a MEG) promotes thermogenesis [Dlk1 (a PEG) inhibits thermogenesis and reduces BAT mass at weaning [Dlk1, however, is associated with increased BAT in the immediate postnatal period [Dlk1 on early and late BAT reflect a reversal in the ratio of individual to communal benefits of heat production.Huddling for warmth is a simple cooperative behavior in which the costs of heat production are borne by individuals but benefits are shared. MEGs are predicted to favor greater contributions to the collective good and PEGs to favor free-riding under the assumption that mice preferentially huddle with kin, such as sibs from multiple paternity litters, who are closer relatives on the maternal side than on the paternal side . Thus, Gogenesis whereas weaning . Overexpl period . The bodGrb10 exhibit increased glucose uptake by skeletal muscle and rapid clearance of a glucose load [Grb10 are obese and glucose intolerant [GRB10 in a meta-analysis of genome-wide association studies of insulin response to an oral-glucose tolerance test. One SNP (rs933360-A) was associated with lower fasting glucose and enhanced insulin sensitivity when maternally inherited but higher fasting glucose and reduced insulin sensitivity when paternally inherited [GRB10 and DLK1 are also implicated in the regulation of blood glucose levels. Lean mice with an inactivated maternal copy of tolerant . This suOrganisms must prudently manage their portfolio of investments over the course of a lifetime. Strategic choices at the organismal level must emerge from information-processing within cells. Each cell must not only manage its own metabolism, second by second and minute by minute, but also coordinate its activities over much longer periods with many other cells of the same and different types. Hormones and other circulating factors provide cues that allow cellular behavior to be matched to organismal goals. Growth hormone, for example, modulates fuel choice between fats and protein (via gluconeogenesis). Increased reliance on lipids to conserve protein would be favored both during rapid linear growth and during malnutrition but the adaptive tissue-specific responses in these two situations should differ markedly as elevated growth hormone is integrated with other cues.A model of organisms as well-designed machines optimizing well-defined fitness functions is implicit in much of traditional physiology and modern systems biology (as well as in the preceding paragraph). But antagonistic effects of MEGs and PEGs suggest that genes of maternal and paternal origin have had different fitness functions with respect to body composition and other metabolic parameters. Such divergence of fitness functions occurs when an organismal phenotype mediates fitness trade-offs among kin . A chall"} +{"text": "The aim of this study was to identify (a) nurses\u2019 knowledge towards patients\u2019 smoking habits (b) nurses\u2019 beliefs towards psychiatric patients\u2019 smoking practices and (c) nurses\u2019 attitudes and practices.A questionnaire based study was contacted among psychiatric nurses working on two major psychiatric hospitals. The total sample consisted of 125 psychiatric nurses which represents the 48% of licensed nurses working full-time.Various practices were noted among nurses concerning the assessment of patients\u2019 smoking history, passive smoking, smoking habits and cessation plans. The majority of nurses noted that psychiatric patient should be handled differently. They stated that smoking cessation may exacerbate psychiatric symptoms and may lead to an illness relapse . Nurses had some knowledge about the health effects of smoking and they feel responsible to help patient quit smoking.To our knowledge this is the first attempt to describe tobacco-related knowledge and practices among psychiatric nurses in Greece. The findings indicated that half of psychiatric nurses smoke in their work environment and are against the application of the anti-smoking law in psychiatric hospitals. They believe that psychiatric patients should be handled different from other patients even though they are aware of the dangers of smoking."} +{"text": "Primary arteriovenous fistula (AVF), failure remains a major problem for hemodialysis patients. Vascular access thrombosis prophylaxis needs to start early in the end-stage renal disease patient. Ticlopidine seems to be effective and safe for prevention of primary AVF failure in hemodialysis patients.et al. showed a negative association between antiplatelet therapy and access patency have been completely observed by the author.This work was financially supported by a grant from vice chancellor for research of Ahvaz Jundishapur University of Medical Sciences."} +{"text": "Recognition of nonverbal sounds in semantic dementia and other syndromes of anterior temporal lobe degeneration may determine clinical symptoms and help to define phenotypic profiles. However, nonverbal auditory semantic function has not been widely studied in these syndromes. Here we investigated semantic processing in two key nonverbal auditory domains \u2013 environmental sounds and melodies \u2013 in patients with semantic dementia and in patients with anterior temporal lobe atrophy presenting with behavioural decline in relation to healthy older controls (n\u00a0=\u00a020). We assessed auditory semantic performance in each domain using novel, uniform within-modality neuropsychological procedures that determined sound identification based on semantic classification of sound pairs. Both the SD and TL groups showed comparable overall impairments of environmental sound and melody identification; individual patients generally showed superior identification of environmental sounds than melodies, however relative sparing of melody over environmental sound identification also occurred in both groups. Our findings suggest that nonverbal auditory semantic impairment is a common feature of neurodegenerative syndromes with anterior temporal lobe atrophy. However, the profile of auditory domain involvement varies substantially between individuals. \u2022Nonverbal auditory processing is an understudied area of semantic memory.\u2022We assessed novel auditory semantic tasks in patients with temporal lobe atrophy.\u2022Environmental sound and music processing were comparably impaired across the cohort.\u2022Individual patients may show relative sparing of melody processing.\u2022Nonverbal auditory deficits integrally accompany temporal lobe degeneration. The nonverbal auditory domain encompasses both environmental sound sources and events and music who also had selective temporal lobe atrophy on MRI. Our objectives were to assess in detail clinically-relevant domains of nonverbal auditory semantic memory in SD; and to compare the auditory semantic profile in SD with another syndrome (bvFTD) associated with anterior temporal lobe degeneration. Identification of environmental sounds and melodies was compared using a novel, uniform procedure based on within-modality stimulus matching, thereby obviating the need for cross-modal labelling. Based on previous neuropsychological and clinical evidence 22.1Nine consecutive patients fulfilling consensus criteria for typical SD 2.2We adapted a previously described paradigm 2.3s) tests over the combined patient cohort. In addition, we performed a receiver-operating-characteristic (ROC) analysis to assess the potential diagnostic value of the novel nonverbal auditory semantic tests in discriminating patients from healthy participants. For all group comparisons, p\u00a0<\u00a00.05 was taken as the threshold criterion for statistical significance.All experimental data were analysed in linear regression models using STATA12\u00ae with independent variables of group membership and test type: this design allowed assessment of interaction effects between tests and experimental groups, considering the combined patient cohort and the SD and TL groups separately versus the healthy control group. Age and years of musical training were included as nuisance covariates. Non-normally distributed neuropsychological data were analysed using Wilcoxon's rank sum test. Relations between experimental nonverbal auditory semantic tasks and standardised semantic and general cognitive measures (Tables S1 and S5) were assessed using Spearman's rho . The SD and TL groups displayed similar general neuropsychological profiles in line with their clinical syndromes, including significantly (p\u00a0<\u00a00.05) more severe impairment of verbal semantic functions in the SD group than the TL group. Participant performance profiles on the experimental auditory semantic tests are summarised in s\u00a0=\u00a00.55, p\u00a0<\u00a00.01), though not within the patient cohort ; within the patient cohort, auditory nonverbal semantic performance was significantly positively correlated with performance on a standardised semantic memory measure but not with general cognitive capacity . Auditory apperceptive performance (indexed by PALPA-3) was intact in the TL group and did not correlate with environmental sound identification, suggesting that perceptual factors did not substantially confound the results.Scores on experimental auditory semantic tests were significantly positively correlated across the combined participant cohort revealed that patients generally performed better on the environmental sound identification test than the melody identification test. However, three patients but only one (5%) of the healthy control participants performed substantially better for melodies. Two of these three patients had little or no musical training, arguing against an idiosyncratic effect of prior expertise. One patient exhibited musicophilia. All three patients showed substantial bilateral temporal lobe atrophy, with somewhat more marked involvement of the nondominant temporal lobe in one case .4Here we have demonstrated that patients with clinically significant anterior temporal lobe atrophy have deficits of semantic processing in two key nonverbal auditory domains, identification of environmental sounds and melodies. The findings support earlier work While auditory semantic measures were correlated with general semantic competence in our patient cohort, individual patient performance dissociations and the lack of correlation between environmental sound and melody identification performance (Table S5) suggest that semantic profiles for music versus other sounds may be separable in these syndromes. These data are consistent with the hypothesis that mechanisms subserving semantic knowledge across domains may reside in a distributed anterior temporal lobe network This study has several limitations that suggest additional directions for future work. Group sizes here were small, limiting power to detect effects: there is a need for larger cohort studies representing the wider spectrum of temporal lobe diseases and pooling data on genetic and other uncommon syndromes across specialist centres. The small TL group here encompassed substantial syndromic and pathological heterogeneity . The patients in this study might in effect be regarded as a series of single cases, each potentially informative in their own right. Analysis of larger cohorts might provide more fine grained auditory semantic signatures of particular disease groups, and group studies should be supplemented by detailed experimental investigation of individual patients. Such work will be required to delineate critical relations between auditory and other semantic modalities and between auditory semantic and perceptual mechanisms. To define fully the brain substrates that mediate these cognitive operations is likely to require functional neuroimaging techniques, correlated with subsequent structural brain damage. Beyond elucidating the organisation of the human nonverbal semantic system, there is a clinical imperative to assess the diagnostic value of nonverbal auditory tests in relation to standard tests of semantic memory; and to determine how nonverbal deficits relate to daily life disability and symptoms that patients and their caregivers report. Longitudinal tracking of syndromes including the presymptomatic phase of genetically mediated diseases and ultimately, histopathological correlation will be essential to establish how deficits relate to one another and whether certain nonverbal semantic profiles might predict particular neurodegenerative pathologies or anticipate clinical evolution. Taking these caveats into account, our findings suggest that nonverbal auditory semantic impairment is a common feature of neurodegenerative syndromes with anterior temporal lobe atrophy, modulated by individual variation in the profile of auditory domains involved. The findings should motivate further study of this issue and more detailed exploration of its clinical significance.The authors have no conflicts of interest to declare."} +{"text": "Compulsive production of verse is an unusual form of hypergraphia that has been reported mainly in patients with right temporal lobe seizures. We present a patient with transient epileptic amnesia and a left temporal seizure focus, who developed isolated compulsive versifying, producing multiple rhyming poems, following seizure cessation induced by lamotrigine. Functional neuroimaging studies in the healthy brain implicate left frontotemporal areas in generating novel verbal output and rhyme, while dysregulation of neocortical and limbic regions occurs in temporal lobe epilepsy. This case complements previous observations of emergence of altered behavior with reduced seizure frequency in patients with temporal lobe epilepsy. Such cases suggest that reduced seizure frequency has the potential not only to stabilize or improve memory function, but also to trigger complex, specific behavioral alterations. The neural substrates of complex behaviors remain largely unknown and this is especially true of creative activities such as music and poetry is a distinctive syndrome of temporal lobe epilepsy often accompanied by a persistent interictal disturbance of memory , losing points for orientation and recall; she had a well-preserved social fa\u00e7ade but evidence of impaired recall and recognition and word finding difficulty. This was corroborated by detailed neuropsychological assessment (summarized in The patient underwent a number of investigations. Routine blood screens were unremarkable. Brain CT (MRI contraindicated) showed mild diffuse cerebral volume loss without disproportionate temporal lobe atrophy, and mild white matter ischemic changes . RoutineSeveral months after starting lamotrigine, the patient suddenly began to write original verse. Whereas poetry had never previously been among her pastimes, she now produced copious short poems (around 10\u201315 each day) on quotidian topics such as housework or about the act of versifying itself and sometimes expressing her opinions or regret about past events. These poems often had a wistful or pessimistic nature but did not have a moral or religious focus. Her husband characterized them as \u201cdoggerel\u201d because they were generally rhyming and often featured puns and other wordplay (examples in This case illustrates an unusual behavioral alteration in an unusual clinical context. Although hypergraphia is well described in the setting of epilepsy and other disorders affecting the temporal lobes and non-dominant hemisphere (Flaherty, Hypergraphia has been associated with right temporal lobe seizure foci, putatively due to disinhibition of left temporal lobe language centers, and generally arises interictally in the context of ongoing seizures (Flaherty, The syndromic diagnosis in this case was not entirely straightforward. On the one hand, our patient exhibited a number of features in line with previous descriptions of TEA. These included the phenomenology of her attacks and ensuing amnesic periods, sparing of cognitive domains such as executive function and language, EEG findings and compelling response to low dose anticonvulsant monotherapy (Butler et al, A further unresolved dimension in this case is the role played by anticonvulsant therapy. Our patient was taking a number of medications when compulsive versifying emerged. Hypomania has been described previously in association with lamotrigine (Villari, Rocca, Frieri, & Bogetto, The nature of our patient's behavioral change raises a broader issue concerning the brain mechanisms that sustain \u201ccreativity.\u201d Novel idea generation has been modelled as a reciprocal interaction of fronto-temporo-limbic networks modulated by dopaminergic and other neurotransmitter pathways (Flaherty, JDW receives salary support from the Wellcome Trust. The remaining authors have no competing interests."} +{"text": "Explaining the disparity of species richness across the tree of life is one of the great challenges in evolutionary biology. Some lineages are exceptionally species rich, while others are relatively species poor. One explanation for heterogeneity among clade richness is that older clades are more species rich because they have had more time to accrue diversity than younger clades. Alternatively, disparity in species richness may be due to among-lineage diversification rate variation. Here we investigate diversification in water scavenger beetles (Hydrophilidae), which vary in species richness among major lineages by as much as 20 fold. Using a time-calibrated phylogeny and comparative methods, we test for a relationship between clade age and species richness and for shifts in diversification rate in hydrophilids. We detected a single diversification rate increase in Megasternini, a relatively young and species rich clade whose diversity might be explained by the stunning diversity of ecological niches occupied by this clade. We find that Amphiopini, an old clade, is significantly more species poor than expected, possibly due to its restricted geographic range. The remaining lineages show a correlation between species richness and clade age, suggesting that both clade age and variation in diversification rates explain the disparity in species richness in hydrophilids. We find little evidence that transitions between aquatic, semiaquatic, and terrestrial habitats are linked to shifts in diversification rates. One of the most remarkable and pervasive patterns on Earth is the uneven distribution of species richness among clades. Indeed, some clades such as beetles are astoundingly species rich, while others such as monotremes are species poor. While there has long been interest in the disparity of species richness across the tree of life One intuitive explanation for disparity in species richness is that species rich clades are older, and thus have had more time to accumulate diversity than younger clades Studies investigating the disparity of species richness among clades have been met with mixed results Here we investigate clade age and diversification rate in water scavenger beetles (Hydrophilidae). Hydrophilids are an excellent group for investigating the processes that determine species richness patterns because they are a diverse group with over 3000 described species and a nearly global distribution Habitat has previously been show to influence diversification rates in aquatic beetles, although most studies have focused on microhabitat differences such as lotic and lentic environments The absence of a comprehensive time-calibrated phylogeny for hydrophilids has precluded efforts to disentangle the evolutionary processes that explain species richness patterns in this group. Here we use an extensive set of recently revised fossil taxa and relaxed molecular clock analyses to estimate diversification times for water scavenger beetles. We integrate our phylogeny with data on species diversity based on detailed taxonomic expertise of the group and data on habitat preferences of particular taxa largely based on our direct observations in the field. We use comparative methods to investigate the roles of clade age and among-lineage diversification rate variation in determining patterns of species richness in water scavenger beetles, and explore the influence of transitions between aquatic and terrestrial habitats in driving diversification rates.Protochares brevipalpis and Baissalarva hydrobioides Hydrobius titan ; Limnoxenus olenus Anacaena paleodominica from Dominican amber Helochares (Hydrobaticus) sp. and Cercyon sp. from Baltic amber th generation. We verified convergence of parameter estimates and that effective sample sizes were >200 for all parameters using Tracer 1.5 To determine divergence times for hydrophilids we used a six-gene molecular data set of 151 species that included all major lineages of Hydrophilidae To identify shifts in diversification rate we used MEDUSA We determined the expected species richness of a clade given a net diversification rate (using background rate from MEDUSA), a relative extinction rate, and clade age We used phylogenetic generalized least-squares regression and standard linear regression to test for a relationship between clade age and log-transformed species richness values using the stem clade ages (some clades were represented by a single representative preventing the use of crown ages) from our hydrophilid time tree and current figures for species richness for each major lineage compiled from literature. It is well known that species-rich lineages of insects harbor much greater diversity than is presently described To explore the relative number of transitions between aquatic, semiaquatic and terrestrial habitats we conducted ancestral character reconstruction. Our taxon sampling does not allow us to determine the absolute number of transitions between these macrohabitats, but we can assess where across the entire hydrophilid tree transitions have occurred, and couple this with MEDUSA determined diversification rate shifts to explore a possible relationship between habitat type and diversification rates. We coded aquatic, semiaquatic, and terrestrial habitats as discrete, unordered character states. All character reconstructions were conducted on the maximum clade credibility tree from our BEAST analyses. We used maximum likelihood (ML) in Mesquite v2.6 We recovered a topology for Hydrophilidae in our relaxed clock analysis that is consistent with Short & Fik\u00e1\u010dek Our MEDUSA analyses selected a Yule (pure birth) model as the best-fit diversification model. We found a single net diversification rate increase that occurred within the exceptionally diverse Megasternini (r\u200a=\u200a0.053) relative to the background rate for hydrophilids (r\u200a=\u200a0.032) . We did Using phylogenetic least squares regression and standard linear regression we found no relationship between clade age and (estimated) species richness across the full hydrophilid tree . We did not recover any instances where intermediate habitats were a transitional step between aquatic and terrestrial environments.Our ancestral character reconstructions indicate that hydrophilids were ancestrally aquatic . We infeOur results show that the origin of Hydrophilidae (214 Ma) considerably predates the estimate found by Hunt et al. Our study suggests that both clade age and among lineage diversification rate differences explain the disparity of species richness among hydrophilid clades. Our MEDUSA analyses revealed a single increase in diversification rate that occurred in Megasternini. We also found that Amphiopini has fewer species that expected (95% CI) under both high and low extinction rate models given a constant diversification rate , suggestIt is not clear whether net diversification rate or clade age is the predominate factor in determining the pervasive disparity of species richness across the tree of life Our results support the hypothesis that water scavenger beetles were ancestrally aquatic and have repeatedly shifted between aquatic and terrestrial habitats We did not detect a diversification rate shift directly associated with any transition between major habitat types & 5. InsWith over 540 described species and an estimated 870 species, the Megasternini is remarkably diverse compared to other major hydrophilid clades. This diversity is ultimately the result of an exceptional increase in net diversification rates compared to other hydrophilids . One intHeliconia inflorescences, leaf litter, ant nests, and mammal dung. For example, the tribe is the only lineage to have significant radiations of myrmecophilous or beach wrack taxa Though our results do not implicate a transition from aquatic to terrestrial environments as a cause for explosive diversification within the family, the exceptional species diversity of Megasternini may have an ecological explanation. Megasternini occupies a remarkable diversity of niches within the terrestrial environment and are found in a broader array of habitats than most other hydrophilid lineages & 5, sucDespite its early Jurassic origin, the Amphiopini is significantly more species-poor than expected under both high and low extinction scenarios given a constant rate . Our MEDAnother factor contributing to low species richness in Amphiopini may be its relatively restricted geographic range. Amphiopini is notable in being one of only two major lineages of Hydrophilidae to be absent from the New World. Interestingly, the only tribe that is less diverse (though younger) than the Amphiopini\u2013the Protosternini\u2013also has a relatively restricted geographic range compared to other hydrophilid lineages. It may be that while the diversification rate of Amphiopini has remained similar to the rest of the family, the smaller geographical scale over which it has diversified has limited its absolute species richness.Figure S1Hydrophilidae time tree uniform priors.(PDF)Click here for additional data file.Figure S2Hydrophilidae time tree exponential priors.(PDF)Click here for additional data file.Figure S3Hydrophilidae time tree with posterior support values.(PDF)Click here for additional data file.File S1Supplementary materials.(DOCX)Click here for additional data file.File S2Genbank vouchers.(DOCX)Click here for additional data file."} +{"text": "Arabidopsis, have significantly enhanced our understanding of plant innate immune perception and signaling. For example, the identification of classical plant resistant genes in Arabidopsis and other model dicots facilitated the successful cloning of multiple wheat rust resistant genes across plant species will significantly enhance scientist's ability to develop novel disease control strategies , narrow host range (Puccinia triticina) or a continuum between the two (Bettgenhaeuser et al., This Research Topic encompasses a collection of reviews, opinions, perspectives, as well as primary research articles investigating the interaction between crop plants and a variety of pathogens including fungi, bacteria, viruses and aphids (Ellis et al., Cuscuta is hypothesized to employ the same classes of immune receptors yet knowledge of the molecular mechanisms involved during the infection process and the ensuing immune response are still sparse (Kaiser et al., Parasitic plants, viruses as well as insects significantly impact agricultural production. Aphids cause significant crop losses by acting as a vector for plant viruses as well as causing damage due to feeding (Jaouannet et al., NLR gene. This knowledge of the molecular identities will enable the deployment of cultivars recognizing conserved pathogen effectors and monitoring their allelic variation in the field (Huet, NLR /effector pairs are still a major bottleneck in plant microbe research, because they are time consuming and labor intensive. The development of accurate, high throughput phenotyping platforms will significantly impact our ability to identify promising phenotypes and facilitate hypothesis testing (Mutka and Bart, We have entered an exciting era of research on plant-pathogen-microbe interactions. Large-scale genome sequencing of both plant and pathogen populations is now feasible and can help formulate multiple testable hypotheses. The \u201cIntegrated Decoy Hypothesis\u201d posed by Dodds and colleagues was developed from a combination of wet lab experiments coupled with genome analyses to identify genetically linked NLR pairs, where one receptor has an additional \u201csensing\u201d domain targeted by effectors (Cesari et al., ld Huet, . To dateThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Impaired cerebral venous sinus outflow leading to cerebral venous hypertension has been implicated as a potential final common pathway in the pathophysiology of idiopathic intracranial hypertension (IIH). The aim of this study is to assess the role of endovascular management strategies in the form of either primary venous sinus angioplasty or venous stenting for refractory IIH.Retrospective study of 37 consecutive patients with refractory IIH and imaging evidence of cerebral venous sinus outflow impairment. Primary venous angioplasty or secondary stenting were performed and clinical outcomes were documented on a standardised proforma.20 out of the 37 cases showed positive pressure gradients and had endovascular management where there was variable reduction of the pressure gradient. 17 (of whom 12 had only sinus venoplasty and 5 had venoplasty followed by sinus stenting) out of 20 showed clinical improvement or resolution of symptoms. 3 patients were refractory to endovascular management and stabilised after ventriculo-peritoneal shunting.The pathophysiology of IIH from venous hypertension secondary to venous outflow impairment is controversial. A selected group of patients with IIH and cerebral venous outflow impairment can benefit from endovascular treatment. In our experience 60% of patients showed clinical improvement with primary sinus venous angioplasty alone. This is a potential alternative to CSF shunting or primary stenting of venous sinus. After additional venous sinus stenting of refractory cases 85% of our patients in our cohort improved clinically."} +{"text": "To better understand the critical characteristics responsible for the clinical effectiveness of cellular therapies we and others have been studying the potency of cellular therapies. Specific phenotypes have been associated with better clinical outcomes. Animal models suggest that T memory stem cells have greater proliferative potential and persistence than effector T cells and are more effective for adoptive cellular therapy. We have found that high throughput molecular assays are useful at identifying markers that are useful at measuring cell potency. In conclusion, in order to improve the clinical effectiveness of cellular therapies it\u2019s important to better understand the biological functions that contribute to product potency and develop potency markers and assays to ensure that the highest quality cells are consistently produced.Adoptive cellular therapy is becoming an important cancer therapy. Tumor infiltrating T cells and peripheral blood T cells engineered to express high affinity T cell receptors specific to tumor antigens have been effective in treating melanoma. T cells engineered to express chimeric antigen receptors are being used to treat cancers and hematologic malignancies. Clinical studies have found that the characteristics of cellular therapies effects clinical outcomes. There is considerable variability between production lots of adoptive cellular therapies. This variability affects product characteristics and clinical outcomes. Production variability is due to both donor and manufacturing factors. Donor genetics as well as donor age, gender, race and prior therapies may affect product characteristics and function. The complexity involved with manufacturing cellular therapies also contributes to variability in the final product. Manufacturing adoptive cell therapies may involve stimulation with antibodies, cytokines, growth factors or allogeneic cells; prolonged culture and expansion; and genetic engineering. Product characteristics can affect clinical outcomes. Several studies have found that adoptive cellular therapies that have greater"} +{"text": "The vascular system performs the critical functions of supplying tissues with nutrients and clearing waste products. Vascular leakage, caused by acute or chronic exposure to vascular endothelial growth factor (VEGF), is unique amongst angiogenic factors since it primarily results in endothelial dysfunction/disruption . VEGF wRecent research advancements have brought new insights into the regulation of these proteins by various agents including exogenous expression of FVIIa or silver nanoparticles in order to decrease VEGF-induced vascular leakage have been known to inhibit angiogenesis or tumor neovasculature formation, but the underlying mechanisms have not been well studied. Resveratrol, a polyphenolic compound found in grapes and other fruits has been reported to inhibit VEGF-induced vascular leakage in endothelial cells through the Src pathway (Lin et al., 2003[The author declares that he has no conflict of interest."} +{"text": "Data from three critically brain-injured patients exhibiting a change in CVPR were investigated. An optimal value for k_aut was determined to minimise the difference between measured and simulated outputs. Optimal values for k_aut appropriately tracked changes in CVPR under most circumstances. Further development of this technique could be used to track CVPR providing targets for individualised management of patients with altered vascular reactivity, minimising secondary neurological insults.Understanding changes in cerebral oxygenation, haemodynamics and metabolism holds the key to individualised, optimised therapy after acute brain injury. Near-infrared spectroscopy (NIRS) offers the potential for non-invasive, continuous bedside measurement of surrogates for these processes. Interest has grown in applying this technique to interpret cerebrovascular pressure reactivity (CVPR), a surrogate of the brain\u2019s ability to autoregulate blood flow. We describe a physiological model-based approach to NIRS interpretation which predicts autoregulatory efficiency from a model parameter Cerebral blood flow (CBF) is tightly regulated by cerebral autoregulation (CA), forming a critical link between oxygen supply and demand. Myogenic, metabolic and neurological mechanisms lead to a complex pattern of vascular reactivity over different time scales combining to maintain constant perfusion across a wide range of perfusion pressure. Following brain injury, acute disturbances of CA may lead to hyper or hypo-perfusion and secondary neurological insults; maintaining cerebral perfusion is thus a core goal during the neurointensive care treatment of brain injury. However, delivering this is not straightforward as there is no convenient means of monitoring CBF or CA continuously at the bedside.Measures of vascular reactivity, derived using surrogates of cerebral blood volume (CBV) or CBF, may be compared with arterial blood pressure (ABP) to investigate efficiency of cerebrovascular pressure reactivity (CVPR) and CA . Recentl2 or O2 tension, cerebral metabolic rate (CMRO2) and arterial to venous volume ratio.While these modes of analysis are simple and easily performed at the bedside, they do not account for the non-stationary and non-linear complexity within the range of measured signals. A model-based approach might make best use of the available data combining a priori knowledge of complex cerebral physiology with multiple measured variables to establish fully informed physiological predictions. This might account for additional important contributions to our interpretation of NIRS measured signals such as changes in COk_aut has been designed to represent changes in the efficiency of CA ranging from 0 with an absence of CA to 1 where it is completely intact. This work translates our model ) and deoxyhaemoglobin (\u2206[HHb]) were determined by the modified Beer\u2013Lambert method. Invasive ABP from a radial artery catheter, end tidal CO2 (ETCO2) and pulse oximetry (SpO2) were gathered through an Intellivue monitor . Signals were synchronised, downsampled to 1 Hz and filtered with a lowpass 0.1 Hz fifth-order Butterworth filter to remove high frequency noise and respiratory influences. Of these measured signals ABP, ETCO2 (approximating PaCO2), SpO2 and ICP were used as model inputs. These produced simulated outputs for CBF, total haemoglobin ([HbT]), [HbO2], [HHb] and TOS which were compared with their measured counterparts Vmca and NIRS .For each patient dataset, CVPR was initially characterised using the pressure reactivity index (PRx) and mean velocity index (Mx) . Two 30-k_aut (representing CA) and an additional parameter u reflecting cerebral energy demand. Reduction of this additional parameter below its basal level simulating a reduction in cerebral metabolism was required to adequately fit the measured NIRS signals. This seems physiologically plausible because all patients were deeply sedated at the time of study. k_aut values produced by these different optimisation strategies were compared to index-based predictions of CVPR for consistency. The difference between simulated outputs and measured signals is expressed as the mean absolute difference between the two. The improvement in the fit following optimisation is given as the percentage difference between measured signals and simulated outputs at basal parameter settings and optimised parameter settings divided by the basal value.Optimisation was performed by minimising the difference between measured signals and simulated outputs for Vmca and NIRS finding optimal values for parameter k_aut was associated with intact CVPR and a low value disturbed CVPR in all simulations excluding those optimised on the basis of TOI. An example dataset is shown in Fig. k_aut (0.3), reflecting dramatically impaired autoregulation, simulates Vmca and nTHI most accurately.A high k_aut varied in the relationship between k_aut and predicted CVPR. When k_aut is optimised by minimising the difference between measured Vmca and simulated CBF alone in all epochs , suggesting that k_aut appropriately reflects the level of CA. When measured NIRS signals are included in this strategy in those with reduced CVPR. However, to achieve the best fit requires a value for u that is unphysiologically low.Model simulations using different measured signals for optimisation of hs Table , there igy Table it is pok_aut do not reflect the level of predicted CA in this final approach. The behaviour of measured TOI differs significantly from simulated outputs of TOS; large simulated changes in TOS result from large changes in CBF which are not present in the measured TOI (Fig. Inclusion of TOI within the optimisation strategy is problematic and it is not possible to fit TOI well in combination with other measured signals (Table TOI Fig. .Fig. 13.k_aut which simulates changes in CA and improves prediction of NIRS signals in brain injury. The optimal value of k_aut may thus represent a composite biomarker of cerebral autoregulatory function informed from multiple NIRS inputs. This approach aims to form cohesive physiological predictions based on prior knowledge of physiology, maximising the potential of the available data.We have identified a model parameter 2], \u2206[HHb] and TOS encode metabolic components and differential effects of arterial and venous components become more influential. It was impossible to achieve an adequate fit for TOS by varying only the model parameters k_aut and u. Despite qualitative agreement, the magnitude of variation and baseline saturation showed large discrepancies. It is unlikely that further optimisation within physiological plausibility could explain the lack of variability despite the large changes in CBF observed. However, a differing baseline is more easily explained. Similar observations were made during studies in healthy volunteers [Fitting nTHI was least problematic probably because fewer physiological processes influence this signal. In comparison \u2206[HbOlunteers , but in This study was of limited power including only six epochs from three patients. However these datasets demonstrate an extreme of physiological dysfunction with large changes in ABP and CBF, representing an excellent challenge for our model. Further work must include large numbers of patients undergoing a range of physiological challenges to increase the quality of measured signals in patients with lesser degrees of impaired CA. Although this approach has not necessarily been followed for many established indices of CVPR it should be viewed as a prerequisite to translation into the clinic.k_aut as a biomarker of CA efficiency could inform pathophysiology and potentially provide a target for physiological optimisations to improve outcome.With further investigation, model-informed interpretation of NIRS signals might offer enhanced prediction of CA across widely varying physiological and pathophysiological contexts. Prior knowledge of population characteristics and further model simplification should improve computational efficiency and move toward bedside implementation. This form of interpretation progresses beyond simple correlation analyses by combining information from multiple NIRS and systemic measures with a priori knowledge of physiology to provide cohesive predictions of cerebral well-being. Thus, use of"} +{"text": "Dromedary camels are a putative source for human infections with Middle East respiratory syndrome coronavirus. We showed that camels sampled in different regions in Kenya during 1992\u20132013 have antibodies against this virus. High densities of camel populations correlated with increased seropositivity and might be a factor in predicting long-term virus maintenance. Betacoronavirus clade C were attributed to insectivorous bats in Europe and Africa was discovered in a patient from Saudi Arabia in 2012 and has since caused \u2265250 human infections and 93 deaths and 7 counties during 1992\u20132013 . Blood s,All serum samples were tested for MERS-CoV antibodies by using a recombinant MERS-CoV spike protein subunit 1\u2013based ELISA (rELISA) as described ,. All 228As a final step, antibody specificity was confirmed by using a highly specific MERS-CoV microneutralization assay as described but at 2 locations . Antibody levels of nomadic dromedary camels from the Eastern region were significantly higher than those for farmed animals from the Rift Valley (corrected \u03c7p<0.005) . Adult a2) than in the Rift Valley region .,,,,The present study showed that dromedary camels from Kenya have antibodies against MERS-CoV, which complements the current finding that MERS-CoV is a common pathogen in dromedary camel populations (,To project and potentially control virus spread, the public health community must understand factors determining virus maintenance. Our group and others have demonstrated that young dromedary camels have lower seroprevalences and are more likely to carry infectious virus (We also demonstrated that dromedary camel population density shows a positive correlation with MERS-CoV seropositivity, which suggests efficient MERS-CoV maintenance or spread if herd density is high. Different types of animal husbandry in the Northeastern and Eastern regions of Kenya might be better predictors of virus transmission among camels. Dromedary camels in this area are often nomadic following rainfall patterns, and are taken across borders into neighboring countries, such as Ethiopia, for trade purposes (Conversely, dromedary camels that originated in the Northeastern region but had been held in isolation since 1998 were negative for MERS-CoV antibodies, which is consistent with absence of antibodies in dromedary camels bred in isolation in Dubai (Because exportation of dromedary camels is largely unidirectional from eastern Africa into the Arabian Peninsula (Dromedary camel population densities in 3 regions in Kenya during 2 periods and average numbers of dromedary camels in Kenya, 1992\u20132013."} +{"text": "The study found that more than one third ofpatients in the ICUs received resuscitation fluids on the study day and that thechoice of fluid administered was determined by local practice and habit rather thanby any identifiable patient characteristic. Given that many thousands of patientsreceive resuscitation fluid each day, it is an intervention that has the potentialto result in either great benefit or great harm. Although recently published trialsby clinician investigators have dramatically increased the evidence base in thisarea,-9 some questions remain unanswered.In 2010 the SAFE-TRIPS investigators reported on resuscitation fluid administrationin 391 intensive care units (ICUs) in 25 countries. The SAFEstudy compared the safety and efficacy of 0.9% saline and 4% albumin forresuscitation in 6997 adult general intensive care patients and established thatoverall use of the two fluids resulted in almost identical mortality rates and nosignificant difference in other outcomes. However, in the subgroup of patients withtraumatic brain injury, albumin administration increased mortality. Additionally, the SAFE studysupported the hypothesis that albumin might decrease mortality in patients withsevere sepsis; this observationled to further trials of albumin in patients with severe sepsis and septic shock,unfortunately these trials have not provided a definitive answer.Published in 2004, the Saline versus Albumin Fluid Evaluation (SAFE) Study was thefirst ICU \"mega-trial\".,5,6 These RCTs have provided convincingevidence that both higher molecular weight preparations and the newer low molecularweight starches cause harm.,5,6 In high quality trials HES administration has consistentlyincreased mortality and the incidence of acute kidney injury and its use results inmore patients being treated with renal replacement therapy; these adverse effectsare observed both in patients with severe sepsis and in the general ICU population. Other colloids, specificallydextrans and gelatins have not been extensively studied. The Cochrane Collaborationregularly reviews the totality of evidence regarding fluid choice and concludes thatcolloids offer no demonstrable clinical benefit over crystalloids; colloids areassociated with no improvement in survival and are more costly, making their use inclinical practice hard to justify.Hydroxyethyl starches (HES) is the other colloid that has been extensivelyinvestigated in randomised controlled trials (RCTs). despiteconcerns that its high chloride content is associated with worse patientoutcomes. Inobservational studies, the use of fluids with lower chloride concentrations, such asbalanced (buffered) salt solutions, has been associated with reductions in majorsurgical complications, in the incidence of acute kidney injury, and reductions inhospital mortality. Theseobservational data appear to be influencing clinical practice with increasing use ofbalanced salt solutions in some regions. Large scale RCTs comparing outcomes in patients assignedto receive either normal saline or balanced salt solutions are currently beingplanned (NHMRC APP1101765).Normal (0.9%) saline has been the most commonly used resuscitation fluidworldwide Although thisis likely confounded by severity of illness with sicker patients being more likelyto have a positive fluid balance, it begs the question of whether we shouldre-evaluate the liberal use of resuscitation fluids in ICUs. Recent trials suggestthat adopting a more restrictive fluid strategy in patients with lung injury andfollowing major abdominal surgery may produce better short term outcomes.,15 In another context, the Fluid Expansion as SupportiveTherapy (FEAST) trial, reportedthat African children with severe infections who received fluid boluses had increased mortality compared with those who did not receive fluidboluses. While the applicability of these results to other healthcare settings isunclear, the impact of fluid boluses and liberal resuscitation strategies shouldboth be studied in large high quality RCTs. Such studies should examine both shortand long term outcomes as later cognitive impairment may be more common in patientsassigned to a restrictive fluid strategy.Another remaining challenge is deciding whether a liberal or restrictive fluidpractice is best for critically ill patients. A positive fluid balance is associatedwith adverse patient outcomes in patients with sepsis and in patients with renalfailure.It is clear that \"crystalloid or colloid\" is the wrong question with irrefutableevidence that different colloid solutions have different effects and the effects arealso different in different populations. The same may be true of crystalloids butoverall, the existing evidence favours the use of crystalloids as first lineresuscitation solutions.Currently, the two outstanding questions to be addressed are whether chloriderestriction through the use of balanced salt solutions and separately fluidrestrictive strategies are beneficial to critically ill patients or not. Given thewidespread use of resuscitation fluids both these testable hypotheses should beaddressed as a matter of public health priority and ultimately for the good of ourpatients."} +{"text": "Populations occurring at species' range edges can be locally adapted to unique environmental conditions. From a species' perspective, range\u2010edge environments generally have higher severity and frequency of extreme climatic events relative to the range core. Under future climates, extreme climatic events are predicted to become increasingly important in defining species' distributions. Therefore, range\u2010edge genotypes that are better adapted to extreme climates relative to core populations may be essential to species' persistence during periods of rapid climate change. We use relatively simple conceptual models to highlight the importance of locally adapted range\u2010edge populations (leading and trailing edges) for determining the ability of species to persist under future climates. Using trees as an example, we show how locally adapted populations at species' range edges may expand under future climate change and become more common relative to range\u2010core populations. We also highlight how large\u2010scale habitat destruction occurring in some geographic areas where many species range edge converge, such as biome boundaries and ecotones , can have major implications for global biodiversity. As climate changes, range\u2010edge populations will play key roles in helping species to maintain or expand their geographic distributions. The loss of these locally adapted range\u2010edge populations through anthropogenic disturbance is therefore hypothesized to reduce the ability of species to persist in the face of rapid future climate change. Anthropogenic climate change represents a major threat to global biodiversity Urban and is cBy their very nature, species range shifts take place at range edges, and therefore, range\u2010edge populations play a critical role during climate\u2010driven range shifts. At the leading range edge , populations can act as important stepping stones by serving as dispersal\u2010foci or by maintaining unique genetic adaptations that promote colonization of newly suitable areas within the Amazon. MCWD is an integrative measure of the accumulative water stress experienced by plants in an area over the course of a year and has previously been found to be a strong predictor of species distributions as well as the location of humid tropical forests and other tropical biomes . Using the future climate (TAP and MCWD) predictions, we then identified and tallied the areas within the lowland Amazon forest that have analogous current climates. We then calculated the relative reduction (% decrease) in the number of current climate analogs corresponding to each cell due to the loss of area under current (2002) and predicted future (2050 BAU) deforestation.We found that current deforestation reduces the extent of climate analogs by an average of 16% Fig.\u00a0A and thaOur analyses are meant to highlight the potential importance of range edge populations in trees and hence the danger of losing these populations through deforestation. The actual effects of deforestation on species persistence under climate change will be highly species\u2010specific and will depend on many different factors including the degree of local adaptation, the overlap between the species' geographic ranges and deforestation, and the species' dispersal ability, including the ability to cross\u2010disturbed/modified habitats . Identifying the responsible genetic controls of these traits will aid in conservation efforts and advance our ability to accurately forecast where and when locally\u2010adapted range\u2010edge populations will be important determinants of species persistence. Even in well\u2010studied traits, such as cold temperature adaptation in northern hemisphere trees, we still have a relatively basic understanding of how specific genes control phenotypic expression and how genes are linked or interact (Howe et\u00a0al. The recent and rapid advances in molecular techniques may allow for the disentangling of genetic controls on beneficial traits and allow researchers to weigh the importance of range shifts versus rapid evolutionary adaptation in a timeframe relevant for contemporary climate change. Work of this nature should focus on intraspecific population genetics across a wide variety of taxa and functional groups that exhibit different life history strategies. Efforts such as these will greatly increase our ability to predict species' range shifts and extinctions and therefore help to most effectively focus the limited resources available for conservation.None declared.Data S1. Methods.Click here for additional data file.Figure\u00a0S1. The percent reduction in number of potential future climate analog source populations in Amazonia under 2070 climate projections accounting for climate analogs lost due to deforestation as of 2002 and under future BAU deforestation for 2050. Black represents a 100% reduction in available climate analogs based on losses from deforestation and the introduction of novel climates.Click here for additional data file."} +{"text": "For stroke and traumatic brain injury (TBI) patients minimizing the time from stroke onset/accident to treatment is fundamental to increase the chances of achieving good clinical outcome. For patients with ischemic stroke thrombolytic treatment may be effective, but only 1\u20138% receive this treatment due to delays in seeking medical attention and late diagnosis. TBI patients with severe injury require immediate transportation to a trauma center. Microwave technology (MWT) has potential to be used for prehospital diagnosis of stroke and TBI patients by detecting intracranial bleedings and thereby make prehospital thrombolysis for stroke patients possible and increase triage accuracy for TBI patients.Two clinical trials enrolling 20 + 25 stroke patients performed with research prototype systems have been completed. Two further studies are ongoing.Regarding TBI laboratory experiments using a human cranium phantom and numerical simulations of subdural hematoma (SDH) have been performed. The first clinical study assessing the potential for MWT to detect SDH has recently started.The microwave-based systems use 8\u201312 transmitting and receiving antennas. The classification algorithm is trained on measurements on patients with confirmed diagnosis, using a leave-one-out procedure.For the clinical studies on stroke patients all cases of hemorrhagic stroke could be detected while correctly classifying most cases of ischemic stroke . For theMWT has potential to improve the acute care for stroke and trauma patients by making a prehospital diagnosis. This would lead to decreased human suffering and large societal economic savings."} +{"text": "The ECG on admission shows ectopic atrial tachycardia with multilevel block: variable Mobitz type II exit block to the atrium and Wenckebach phenomena in the atrioventricular node . Chest computed tomography scan also showed mediastinal adenopathy (not shown). This combination of findings narrowed the differential diagnosis to atrial infiltration due to sarcoidosis or lymphoma/tumours. Biopsies collected during minimal thoracotomy established the diagnosis of sarcoidosis (not shown).In cardiac sarcoidosis, conduction disturbances are related to granulomatous infiltration of the conduction system . Their eThis case underlines that when conduction disturbances are seen in younger patients, a search for cardiac structural abnormalities is warranted and sarcoidosis should be suspected ."} +{"text": "Iron restriction is the body\u2019s removal of freely available iron from blood and extracellular fluid, as part of the systemic inflammatory response . This prIron is essential for the growth and proliferation of almost all microorganisms including bacteria, viruses and protozoa . Many paIn support of this hypothesis, iron overload is associated with poor clinical outcomes in TB and HIV n = 24 000) showed that iron and folic acid supplements increase the risk of malarial infection and mortality in children with normal iron status, but not in iron-deficient children [In certain situations iron supplementation may be harmful. A randomized controlled trial (children . This coAn evolutionary approach to iron raises further questions. Should we suspend iron therapy when treating infection? Does dietary iron intake alter susceptibility to infection? Could iron chelating agents yield a"} +{"text": "Spiral phase velocity mapping (PVM) has been used to assess both blood flow and myocEach spiral path is the same except for a rotation around the centre of k-space and hence each contributes equally to the final image. The final velocity-time curve reconstructed from a multi-spiral trajectory can therefore be estimated as the average of the velocity-time curves which would be generated by each spiral individually. This simulation study assumes data acquisition over 13 spiral interleaves, with one interleaf acquired per heartbeat. The input velocity-time curve used Figure is typicResults are shown in Figure These simulations show that for this application retro allows more accurate measurements of peak velocities in the presence of heart rate variation than pro: at 15% RR variation, D and AS are both underestimated by more than 20% with pro but by less than 10% with retro. This is because during the normalisation step retro effectively stretches each heartbeat linearly before combining to produce an image while pro makes no attempt to deal with variation. Pro therefore averages velocities from different parts of the cardiac cycle in the presence of RR variation, while the linear stretching in retro goes some way towards reducing the effect. These results show that incorporation of more physiological stretching algorithms could further improve future retro algorithms.CBRU Royal Brompton Hospital. HRUK Grant number RG2584."} +{"text": "Given evidences that diverse ecosystems provide more services than depauperate ones, much attention has now turned toward finding meaningful and operational diversity indices. We ask two questions: (1) Does phylogenetic diversity contain additional information not explained by functional traits? And (2) What are the strength and nature of the correlation between phylogeny and functional traits according to the evolutionary scale considered? We used data from permanent forest plots of northeastern Canada for which these links have been demonstrated and important functional traits identified. We show that the nature of the relationship between traits and phylogeny varies dramatically among traits, but also according to the evolutionary distance considered. The demonstration that different characters show phylogenetic autocorrelation at different evolutionary depths suggests that phylogenetic content of traits may be too crude to determine whether phylogenies contain relevant information. However, our study provides support for the use of phylogenies to assess ecosystem functioning when key functional traits are unavailable. We also highlight a potentially important contribution of phylogenetics for conservation and the study of the impact of biodiversity loss on ecosystem functioning and the provision of services, given the accumulating evidence that mechanisms promoting diversity effects shift over time to involve different traits. The importance of biodiversity for ecosystem functioning and for the provisioning of services to humanity is well established as a proxy for FD, imply the existence of a relationship between the phylogeny and traits of tree productivity .Tree productivity, specifically total annual aboveground biomass increments, calculated from biomass estimated using allometric equations.We used the permanent forest survey dataset from the province of Qu\u00e9bec (eastern Canada) to identify the different biodiversity elements that impact on tree productivity (Paquette and Messier www.ncbi.nlm.nih.gov) and BOLD was computed using the phylogenetic species variability index (PSV) using parametric bootstrapping (1000 simulations) to the total (Table\u00a0A1) , it was plotted beside the phylogeny as it is a key ecological characteristic of species often used in explaining forest dynamics. Shade tolerance also illustrates parallel adaptation with both angiosperms and gymnosperms containing shade-tolerant and shade-intolerant species. FigureA simple graphical representation of trait values into the phylogeny of species clearly illustrates that species functional traits are linked to the evolutionary history of species Fig.. Functio\u03bb. Whereas maximum height had a very low \u03bb, which suggests independence from the phylogeny, all other traits were found to have relatively important phylogenetic content . But phylogenetic information may still be used advantageously in cases where data on traits are scarce or incomplete to accelerate the investigation of B-EF relationships in undocumented ecosystems, or at wide continental scales. The exact relationships are probably context specific, but a relationship between PD and FD should exist wherever the functional traits of importance have a genetic basis. Also, as more studies report how each species\u2019 contributions to B-EF become increasingly singular with time , following the insurance hypothesis, evolutionary distance approaches may become even more relevant and powerful, being more inclusive of each species\u2019 past, present and future contributions (Allan et\u00a0al. We also note that whereas almost all of phylogenetic diversity effect on ecosystem function could be captured by functional diversity, the opposite was not true. The fact that functional diversity contained some unique information is important and points out that good quality and most importantly relevant functional traits are still stronger predictors of EF than is phylogenetic information alone, as noted elsewhere (Pavoine et\u00a0al. There is clearly renewed interest for phylogenetic comparative methods as ecologists delve into the past to better understand how it shaped the present state of ecosystems, such as community composition or selection on traits in communities (Davies et\u00a0al."} +{"text": "There is a need for patient involvement when selecting trial outcomes, since their priorities may differ from healthcare professionals. Qualitative research can be used to identify outcomes that matter to patients in the development of core outcome sets (COS) and in trial feasibility studies that aim to feed patient perspectives into outcome domain selection for the definitive trial. For example, the COMET database currently includes 24 published (2-3/year) and 33 ongoing COS studies utilising interviews or focus groups with patients, carers and their representatives. However, it is unclear whether direct approaches to eliciting outcomes or indirect approaches focusing on the disease and treatment are most useful in informing trial design and outcome selection.This presentation will consider the relative value of qualitative data collection approaches that (a) explore patients' experiential accounts of illness and treatment as a means to infer priorities for outcome domains and (b) involve explicit discussion with patients of outcome domains and preferences. Drawing on lessons from ongoing qualitative work within COS and trial feasibility studies, the presentation will consider how outcome focused discussions resonate with patients; what can be inferred about patients' outcome preferences from more expansive experiential accounts of illness and treatment; the role of researchers in interpreting these accounts and translating them to outcome domains; and the results of studies that collect both \u2018types\u2019 of data. Potential implications for COS development will be outlined."} +{"text": "Modern agriculture has created a demand for plant biotechnology products that provide durable resistance to insect pests, tolerance of herbicide applications for weed control, and agronomic traits tailored for specific geographies. These transgenic trait products require a modular and sequential multigene stacking platform that is supported by precise genome engineering technology. Designed nucleases have emerged as potent tools for creating targeted DNA double strand breaks (DSBs). Exogenously supplied donor DNA can repair the targeted DSB by a process known as gene targeting (GT), resulting in a desired modification of the target genome. The potential of GT technology has not been fully realized for trait deployment in agriculture, mainly because of inefficient transformation and plant regeneration systems in a majority of crop plants and genotypes. This challenge of transgene stacking in plants could be overcome by Intra-Genomic Homologous Recombination (IGHR) that converts independently segregating unlinked donor and target transgenic loci into a genetically linked molecular stack. The method requires stable integration of the donor DNA into the plant genome followed by intra-genomic mobilization. IGHR complements conventional breeding with genetic transformation and designed nucleases to provide a flexible transgene stacking and trait deployment platform. The Green Revolution in the 1960s combined advances in breeding and agricultural practice, and provided food security to millions of people . In addiGenetically modified (GM) trait technology in the mid-1990s made a major impact in meeting the world food demand and there has been a rapid adoption of the technology. These first generation trait products involved simple herbicide and insect traits that required introduction of a single gene. Control of the broad range of insect pests and weeds desired today requires multiple insect and herbicide tolerance genes . In addiDesigned nucleases have become a powerful gene targeting (GT) tool to create targeted DNA double strand breaks (DSBs) at specified genomic locations, which stimulate the cell\u2019s DNA repair machinery leading to integration of exogenously supplied transgenes into a specified genomic site. While designed nuclease-mediated targeted mutagenesis is becoming routine in plants \u20139, site-DSBs can arise spontaneously, may be induced by ionizing radiation and chemicals, or recently by designed nucleases . GenTargeted DSB-induced NHEJ has been previously described for mutagenesis, deletions or imprecise insertions , 19, 20.Targeted DSBs stimulate the cell\u2019s DNA repair machinery making the DSB site accessible to a donor transgene for site-specific integration. The DSBs however do not preclude the ectopic integration of a donor transgene elsewhere in the genome. In addition, the GT process requires efficient delivery of the donor molecule to the DSB site and the ability to regenerate whole plants from the cells with a precisely repaired targeted genomic site. Random integration of the donor transgene and an inefficient transformation method for the donor delivery therefore are two major challenges for the routine deployment of GT technology in crop plants. Positive selection for GT such that precise insertion of the donor complements the non-functional selectable marker in the target locus has been used to avoid random integration of the donor , 23, 24 Agrobacterium wou wou27] wve cells \u201359 can cFuture biotech crops are projected to require multiple transgenes to confer resistance to a broad spectrum of insect pests and provide herbicide tolerance with different modes of action. Insects and weeds will eventually develop resistance, new target pests will emerge and new traits will inevitably be needed and desired, so designing those future products to be further modified and developing capabilities to accomplish the modifications are wise investments. It is clear that producing and modifying transgenic events through GT has many advantages over random integration, and technology continues to develop to make GT increasingly efficient and flexible. Intra genomic homologous recombination using designed nucleases has good potential to overcome limitations in plant transformation and breeding to achieve targeted and highly complex stacked trait crops."} +{"text": "Talcoma is a pleural mass which may develop as a rare complication following talc pleurodesis. Talc pleurodesis is performed to obliterate the pleural space to prevent recurrent pleural effusions or persistent pneumothoraces. The present report describes a case of a patient who developed enlarging pleural mass following talc pleurodesis. Talcoma is a pleural mass which may rarely develop as a complication to talc pleurodesis. Talc pleurodesis is a procedure performed to obliterate the pleural space to prevent recurrent pleural effusions or persistent pneumothoraces. Pleurodesis is commonly performed by draining the pleural fluid, followed by either a mechanical procedure or instillation of a chemical irritant into the pleural space. The resulting inflammation and fibrosis prevent further accumulation of air and fluid within the potential pleural space. Talc is the most effective sclerosant available for chemical pleurodesis involving malignant pleural effusions . When coThe present report describes a case of a patient who developed a slowly enlarging pleural mass over the course of 16 years following talc pleurodesis for recurrent pneumothoraces.A 28-year-old male with history of immunodeficiency secondary to Hyperimmunoglobulin E syndrome (Job syndrome) had a complex surgical history including bilateral video assisted thoracic surgery (VATS) with bullae resections secondary to multiple pulmonary infections and recurrent pneumothoraces at an outside hospital. The left hemithorax bulla resection was complicated by lack of reexpansion requiring talc pleurodesis.He presented to us approximately 8 years after talc pleurodesis with a chest CT to be evaluated for pneumonia. His chest CT demonstrated postoperative changes from prior bullectomy with recurrent large bullae occupying the bilateral lung apices. A well-circumscribed heterogeneous soft tissue density mass was noted at the left cardiophrenic angle measuring 2.4 \u00d7 1.8\u2009cm . As therSurgical resection was advocated as the mass demonstrated considerable growth over the years. A left-sided thoracotomy was performed for excision of this pleural mass, during which significant pleural adhesions were noted from prior pleurodesis. The mass was dissected free from the adherent pericardium. Pathology demonstrated chronic pleuritis, calcifications, and a foreign body giant cell reaction secondary to talc pleurodesis .Talc pleurodesis is a widely used procedure in the treatment of recurrent pneumothoraces and pleural effusions with a low recurrence rate. Talc pleurodesis has also been shown to be effective in treating recurrent benign and malignant pleural effusions, thus allowing the rare diagnosis of talcoma to be seen in multiple patient populations.Ahmed and Shrager reported a young woman with a large, calcified anterior mediastinal mass discovered 18 months following a left talc pleurodesis. The lesion was evaluated and treated as the thymoma or teratoma with excision by a transcervical approach. Pathologic examination revealed a giant talc granuloma . In our When talc particles are infused into the pleural space, they are unable to enter the alveoli or systemic circulation . These pThe diagnostic challenge of evaluating a talcoma arises from its nonspecific imaging characteristics. Calcified pleural thickening and obliteration of the costophrenic angle with increased FDG avidity on PET-CT may be the only diagnostic clues. Although it has only been rarely observed after talc pleurodesis, the possibility of talcoma should still be considered in the differential diagnosis of pleural masses with patients that have a remote history of talc pleurodesis."} +{"text": "Serum specific igE levels, serum total igE levels and serum total eosinophil counts in asthma bronchiale patients were compared with healthy controls and the relation between serum specific igE levels, serum total igE levels and serum total eosinophil counts were evaluated.5 specific igE level measurements and serum total igE levels, serum total eosinophil counts of a total 60 patients with Asthma aged 19 to 52 and 34 healthy persons were compared.60 percent of patient study group was determined to have positive specific igE, it is obtained that 70 percent of this positive specific igE group has sensitiveness to house dust mite antigen.Asthma bronchiale cases have specific igE positiviness level and total igE level and total eosinophil counts were significantly higher than the control group.Patients with asthma and allergic rhinitis revealed significant correlation with specific igE Df, total igE and eosinophil counts, furthermore significant correlation was behold between specific igE Dp, total igE and eosinophil counts.In our study compared to other allergens, sensitivity against house dust was much more higher and it is coherent with other studies in literature.The usefullness of evaluating total serum igE or allergen specific igE for reasons of diagnosis and management is mutable however serum igE level is an important indicator of the intensity of inflammatory processes in allergic diseases and serum igE, total eosinophil counts and specific igE are helpful for diagnosis and developing therapeutic strategy in allergic diseases."} +{"text": "Bovine Leukemia Virus (BLV) is distributed worldwide and causes important economic losses on dairy farms. Currently, there are no effective vaccines or antivirals against BLV. Egg yolk antibodies (IgY) has many advantages over mammalian IgG. Despite the higher yields, they are non-invasively extracted from egg yolk, do not cross react against mammalian antigens or activate the mammalian complement system. In this work we evaluate the reactivity of Igy antibodies against Bovine Leukemia Virus p24 core protein and against the whole virus particle. Hens were immunized by intramuscular inoculation with purified p24 or the virus particle until the development of high antibody-titers. Total IgY was purified from egg yolks by ammonium sulfate precipitation. The purified egg yolk antibodies strongly reacted with BLV particles from a persistently infected cell line, with supernatants from ex vivo cultures of PBMCs from natural infected animals and also with purified p24 by both ELISA and Western blot. These data suggest that chicken IgY may be a suitable platform to produce large amounts of anti-BLV antibodies for diagnostic systems. Furthermore, the use of IgY for passive immunization against BLV infection should also be explored in order to develop new strategies to control the disease in cattle."} +{"text": "ICR-CTSU introduced EDC in 2012, requiring revision of systems developed for paper case report form (CRF) management. The challenges encountered and benefits realised with EDC are described.EDC does not allow central data cleaning at point of entry or traditional manual CRF tracking upon receipt so alternative approaches are required. EDC specific site training is required and site staff need to be encouraged to submit data immediately following participant visits, instead of batching data entry. Database user access and internet browser compatibility monitoring systems are required to ensure only current site staff have access and are using a supported browser. Conventions for CRF design and submission require revision to facilitate EDC use at sites.EDC allows logic checks to highlight discrepancies to site staff during data entry. Data can be reported in real time, ensuring safety data are readily available for central review, advantageous for high risk trials. Queries can be sent instantly with reduced turnaround times for data clarification. EDC provides an audit trail with data entry only by authorised individuals; challenging with CRFs if signatures are illegible. EDC also results in fewer transcription errors and illegible data issues, a frequent source of data queries on CRFs. Using EDC reduces paper management time and requires less physical storage space.The benefits of EDC outweigh the challenges however it requires continual reassessment and re-evaluation of novel processes as they are developed and implemented."} +{"text": "Choosing to undertake a CT scan relies on balancing risk versus benefit, however risks associated with CT scanning have generally been limited to broad anatomical locations, which do not provided adequate information to evaluate risk against benefit. Our study aimed to determine differences in radiation dose and risk estimates associated with modern CT scanning examinations when computed for clinical protocols compared with those using anatomical area.Technical data were extracted from a tertiary hospital Picture Archiving Communication System for random samples of 20\u201340 CT examinations per adult clinical CT protocol. Organ and whole body radiation dose were calculated using ImPACT Monte Carlo simulation software and cancer incidence and mortality estimated using BEIR VII age and gender specific lifetime attributable risk weights.Thirty four unique CT protocols were identified by our study. When grouped according to anatomic area the radiation dose varied substantially, particularly for abdominal protocols. The total estimated number of incident cancers and cancer related deaths using the mean dose of anatomical area were 86 and 69 respectively. Using more specific protocol doses the estimates rose to 214 and 138 incident cancers and cancer related deaths, at least doubling the burden estimated.Modern CT scanning produces a greater diversity of effective doses than much of the literature describes; where a lack of focus on actual scanning protocols has produced estimates that do not reflect the range and complexity of modern CT practice. To allow clinicians, patients and policy makers to make informed risk versus benefit decisions the individual and population level risks associated with modern CT practices are essential. Over the four decades since the introduction of Computed Tomography (CT) scanning, diagnostic imaging technological advancements have been a potent factor driving innovations in medicine Large scale data capture of CT scanner radiation dose in databases have been proposed to enable institutional benchmarking, optimization of CT protocols, and quality control Estimates of radiation dose and cancer risk from CT scanning were classically undertaken using \u2018typical\u2019 anatomically based CT protocol/machine settingsThe aim of our study was to examine the radiation dose associated with modern CT scanning examinations by anatomical location versus actual clinical protocol to demonstrate the degree of variation in radiation dose and its impact on population burden and risk estimates.A cross-sectional, observational, retrospective study design with technical CT data collected from a large metropolitan tertiary/teaching hospital in Western Australia (using two 64-slice CT machines of the same make and model), via the Picture Archiving Communication System (PACS), on adult diagnostic CT scanning protocols (excluding extremities) identified by way of discrete protocol code/naming conventions used by the institution. Prior to collection of the data the study was approved by the Western Australian Department of Health Human Research Ethics Committee, with a waiver of informed consent for the retrospective review of electronic medical records.st January and 30th April 2011. Rarely fewer than twenty cases were identified within the collection period; in this event all cases were included in the study. If the technical parameters appeared particularly variable up to forty cases were collected. A sample of 20 cases is at least double studies using self-report data have used A random sample of 20 to 40 cases from each protocol identified was collected on scans performed between 1Values of CTDIvol (inclusive of automated tube current modulation) and DLP were used to calculate the organ specific dose (mGy) and effective dose (mSv) for each sequence using the ImPACT dosimetry calculation software The age and gender specific lifetime attributable risk (LAR) inferable from a single exposure was estimated for the mean, lowest and highest dose across protocols and each anatomical area. This was achieved using the protocol specific organ dose and the age/sex-specific risk coefficients from tables 12D-1 and 12D-2 of the BEIR VII report As expected there was a large variation in the estimated risk of incident cancers and cancer related mortality across anatomical areas driven bWhen anatomical area mean dose was considered . Our study has provided comprehensive information about the radiation dose and risk burden of modern CT scanning in order to facilitate incorporation into clinical decision making.This study has demonstrated modern CT practice comprises multiple protocols, across most anatomical areas, resulting in a wide range of radiation doses. This variation in radiation dose interacted substantially with the lifetime attributable risk ascribed to ionising radiation, greatly influencing the number of incident cancers and cancer related mortality estimated to be associated with CT scanning. The effective radiation dose, and subsequent risk, resulting from a single CT examination is highly dependent on machine parameters (ie the protocol used) and the radiosensitivity of the anatomical area scanned. Both of these factors need to be considered when dosimetric and risk assessments are undertaken. Using anatomical area for dose and risk estimates does not accurately account for the significant differences between protocols. The end result is an inaccurate perception of risk to individual patients but also the impact of CT radiation burden at a population level.The majority of authors when reporting CT radiation dose either state a broad range (eg 10 to 100 mGy A limitation of our study is that our method of estimating effective and organ dose did not include size specific dose estimation (SSDE) methods since information regarding the body habitus of the patients included in the study were not available. However, for this study the use of SSDE would not change the deviation between the two methods because it concerns the same patient groups. In addition, while it is very important to account for patient size when estimating individual patient radiation dose In our study we aimed to estimate the average effective radiation dose for each adult CT protocol using a random sample of adult patients. The use of random sampling methodology was used to capture any underlying variation in doses produced for each scanning protocol, comparable to other published research Detailed information regarding patient numbers according to CT protocols from routinely captured administrative data has allowed for cancer and cancer related mortality attributable to CT scanning to be estimated, for individual protocols and by anatomical area. The risk estimates reported in this study are not based on epidemiological data of actual malignancies in populations of patients receiving CT scans (such information is unavailable). The estimates are extrapolations of the attributable cancer risk models developed in the BEIR VII report While the BEIR VII report provides a framework for estimating age, sex and organ specific cancer risks from a radiation exposure it does not fully account for underlying pathology and life expectancy. The BEIR VII risks should be considered representative of the independent effect of radiation dose and can only be said to account for competing risks included in the original BEIR VII models. The estimated number of incident cancers and cancer related mortality are presented here as a demonstration of the magnitude of the effect on risk estimates of using protocol rather than anatomic area dose. There is substantial difficulty in estimating population cancer risk as noted by the International Organization for Medical Physics (IOMP) Radiation dose and risks associated with CT scanning have commonly been presented using broad anatomical locations without consideration for the diversity of modern CT examinations performed within each region. This leaves referring clinicians and patients with limited or simplistic information to evaluate risk against benefit. Our study demonstrated the insufficiency of presenting radiation dose according to anatomical areas, rather than specific CT protocols, using rigorous CT technical data sources and established risk estimation methods. Lack of focus on actual clinical scanning protocols has produced dose estimates that do not reflect current clinical practice, therefore to improve risk versus benefit decision making differentiation of the associated radiation dose resulting from the variety of services present in modern CT is essential."} +{"text": "Inguinal hernias in girls are often irreducible when they contain ovaries. Rarely the hernial sacs may have unusual contents like vermiform appendix, uterus and urinary bladder. We report a case of a female infant who presented with bilateral irreducible inguinal hernias presumed to be due to ovaries. However at exploration, the hernial sacs contained bilaterally an omental mass with calcifications. Presence of mucin with meconium- laden macrophages in the mass on histology suggested an antenatal intestinal perforation. To the best of our knowledge no such case has been reported in a female neonate. We present this rare case and discuss the unusual findings and the outcome. Most inguinal hernias in female infants are reducible and indirect; however, the hernia can become incarcerated. In the absence of bowel obstruction, ovaries are the most likely content seen in female infants [1]. We report a case of a female infant who presented with bilateral irreducible inguinal hernias presumed to be due to ovaries. However at prompt exploration, the hernial sacs contained bilaterally an omental mass with calcifications. Histopathological examination confirmed the omental mass to be consistent with meconium peritonitis. We report this unusual case and herein discuss the unusual findings and the outcome. A preterm female baby born at 30 week gestational age was delivered by emergency caesarean section in view of maternal pre-eclampsia. Antenatal foetal scans did not reveal any abnormality. She was initially intubated for respiratory distress syndrome but was self ventilating by day 9 of life. She passed meconium on day 2 of life and was opening her bowels regularly. Abdominal and genitalia examination were unremarkable. Feeds were commenced on day 9 and were established slowly. She had an AXR to check the position of the umbilical catheter on day 2 of life and this did not reveal any abnormality.At 6 weeks of age she was referred to us with bilateral incarcerated inguinal hernias. She had no evidence of bowel obstruction and clinically there were firm masses on both groins suggesting ovary as the most likely content. Bilateral inguinal herniotomy was carried out promptly. However, Intra-operatively on either side the inguinal hernial sacs contained omentum attached to an irregular, grey coloured mass with nodules distally. This mass was excised, healthy omentum returned and a standard herniotomy carried out on both sides. Post-operatively she recovered well and was discharged the following day. Histopathological examination confirmed the excised mass to be made up of omentum with areas of dystrophic calcification reminiscent of the saponification with mucin and meconium-laden macrophages; consistent with antenatal perforation of intestine . At 3 months of follow-up, she remains well and opening her bowels regularly. Incarcerated inguinal hernias in female infants are commonly due to herniated ovaries in the absence of bowel obstruction. Ovaries are reported in almost 90 \u2013 100% of girls less than 1 year of age with incarcerated inguinal hernias [1]. Although, the ovary is not at great risk of strangulation, there is an increased risk of torsion [27%] and therefore prompt surgery is recommended [1]. Given such a high incidence of irreducible ovaries in female infants with incarcerated inguinal hernias, it is extremely rare to find hernial contents other than ovaries. Meconium laden omental mass in inguinal hernias have been reported in many male infants [2-4], however not so in female infants. In our patient, meconium laden grey omental masses were noted to herniate through the inguinal canal bilaterally. The mass was excised off a normal looking omentum proximally on both sides and herniotomy carried out. The only case report in literature on a female infant similar to such a presentation was a term infant born with a right sided labial swelling consistent with meconium hydrocele within the canal of Nuck [5]. In the absence of signs of an acute bowel perforation, asymptomatic antenatal bowel perforation with meconium peritonitis should be considered in such cases.Meconium peritonitis is a sterile process resulting from in utero intestinal perforation. Any pathology causing bowel obstruction in utero can lead to meconium peritonitis; however the incidence is low and is reported almost as 1 in 30000 [6]. Antenatal bowel obstruction is commonly seen with intestinal atresias, but can be transitory as seen in cystic fibrosis with meconium ileus. In utero ischemia of bowel has also been reported to cause bowel perforation and subsequent meconium peritonitis [7]. Antenatal ultrasound can reveal findings suggestive of meconium peritonitis. Kamata et al described three types of meconium peritonitis based on ultrasound findings: Type 1: Meconium ascites, Type 2: Giant pseudocyst, Type 3: Peritoneal calcification and small pseudocyst. In their same series of 20 patients, intra-abdominal calcifications were seen in only 25% of the patients [8] suggesting that not all cases of meconium peritonitis have findings on antenatal ultrasound scans and therefore antenatal diagnosis of meconium peritonitis can be missed. In our patient routine antenatal ultrasound scans failed to reveal any evidence of meconium peritonitis. Clinical manifestation of meconium peritonitis after birth depends on the extent of peritonitis and the underlying abnormality of bowel. It can present as giant meconium pseudocysts [9] or meconium stained inflammatory masses. A patient with an unusual sequela of meconium peritonitis involving massive extension of a processes vaginalis to the contralateral anterior abdominal wall has been reported [4]. However, meconium peritonitis does not always produce significant inflammation and may resolve spontaneously without any sequelae. Estroff et al reported 2 cases of meconium peritonitis detected antenatally that resolved spontaneously without any sequelae [10]. Ekinzi et al reported a male infant who was incidentally found to have meconium mass at bilateral inguinal hernia surgery [2]. The abdomen was explored and loose small bowel adhesions with viable small bowel were noted. They recommended that with such findings in the absence of bowel obstruction, further abdominal exploration is unnecessary. In our patient, we did not explore the abdomen and performed a routine herniotomy excising the herniated omental mass. Our patient has remained well since the surgery and to date has not had any complications. In conclusion, meconium peritonitis can be asymptomatic and can present as omental mass herniating through the inguinal canal. Abdominal x-rays might not reveal intra-abdominal calcifications in all patients, as in our case. Clinically asymptomatic neonates with omental mass found incidentally at inguinal herniotomy can be managed with inguinal herniotomy alone and further exploration of bowel we believe is unnecessary. Source of Support: NilConflict of Interest: None"} +{"text": "Cardiac MRI is increasingly utilized in patients with congenital heart disease; however, the expertise to perform and interpret these studies is not universally available, despite an increasing population of congenital heart survivors. This retrospective analysis describes our experience providing CMRI services on-site versus over a distance of 250 miles.Our technique utilized the syngo Expert-i (Siemens) remote control software. Our configuration included a T3, high speed dedicated line with secure communication to achieve remote control of the scanner console without lag, immediate transfer of DICOM images, and to support secure voice and video over internet (VOIP). The remote site utilized a standard hospital-issued PC with a graphics card upgrade to install the Expert-i and VOIP software. The local site installed VOIP software on a laptop with built-in webcam. Figure We performed a retrospective descriptive analysis of our experience providing congenital cardiac MRI services both locally and from a remote location using the same physician providers.Patient demographics and scan details are listed in Table This experience suggests that remote delivery of cardiac MRI services for the congenital heart population is feasible and can be done with comparable success and safety to a traditional \"local\" model. We also suggest the necessary configuration to provide such remote CMRI services with readily available hardware and software."} +{"text": "Patients with systemic sclerosis may develop borderline pulmonary arterial pressure. The clinical relevance of this condition is not always clear. Reported data support the evidence that this subgroup may represent an intermediate stage between normal pulmonary arterial pressure and manifest pulmonary arterial hypertension, a serious complication in scleroderma. Recognizing the clinical relevance of borderline pulmonary arterial pressure increase in scleroderma patients, future studies should aim for clear evidence for diagnostic and therapeutic algorithms for this population. In their recent study, Visovatti and colleagues present In fact, every physician who has observed the dramatic deterioration of patients with pulmonary arterial hypertension (PAH) and successive right ventricular failure would urge for the earlier recognition and therapy of this devastating condition. About 10% of all scleroderma patients may develop PAH , which -If we assume that the increase of PAP is a process lasting for a longer period of time, there must be a phase of transition from normal (mean PAP \u226420 mmHg) pulmonary hemodynamic conditions to PAH (mean PAP \u226525 mmHg). Patients in this so-called 'borderline' range may represent the early stage of PAH. Earlier studies found that such patients were more likely to develop pulmonary hypertension than patients with mean PAP \u226420 mmHg, with a hazard ratio of 3.7 . The ratThe analysis of Visovatti and colleagues includesAmong the DETECT population, 15% of all patients presented with borderline PAP hemodynamics. Although this number may be different in the general scleroderma population, due to the strict inclusion and exclusion criteria of the DETECT study , the borIn addition to the borderline elevation of resting PAP, another specific hemodynamic situation in scleroderma patients needs careful interpretation: exercise-induced PAP increase. Earlier studies showed that this may be a frequent condition among scleroderma patients and clinical deterioration and the development of PAH are frequent in this population . In a reThe most important question remains open: should targeted PAH therapy be offered to scleroderma patients with borderline PAP or exercise-induced PAP increase? Unfortunately there has been no clinical study investigating patients with borderline PAP so far and only two small studies have selected patients with exercise-induced PAP increase ,9. The rBased on a series of studies indicating borderline hemodynamics has an important role in scleroderma patients with regard to the development of PAH and potentially for early treatment, future studies should aim for clear evidence for diagnostic and therapeutic algorithms for this patient population. This may contribute to a substantial prognostic improvement for patients with scleroderma who develop pulmonary vasculopathy"} +{"text": "In the central nervous system, NMDA receptors play a pivotal role, however, their role in pancreatic islets has been largely unexplored or is controversial. We hypothesized that NMDA receptors are involved in glucose stimulated insulin secretion from beta cells and might serve as novel drug targets for diabetes treatment.in vitro and in vivo. Subsequently, insulin secretion from isolated mouse and human islets as well as blood glucose and plasma insulin concentrations were analyzed in mice and via a clinical trial in type 2 diabetic patients.We generated mice with a pancreas-specific deletion of all NMDA receptors. The phenotype of these mice and their pancreatic islets were characterized in terms of insulin secretion, glucose tolerance and calcium oscillations. We also pharmacologically inhibited the NMDA receptors in vitro and improves glucose tolerance in vivo. The pharmacological inhibition of NMDA receptors selectively increases glucose stimulated insulin secretion in vitro and improves glucose tolerance in C57Bl/6 and diabetic mice. In addition, we performed a clinical trial in patients with type 2 diabetes and provided evidence that the NMDA receptor antagonist Dextromethorphan (DXM) lowers blood glucose levels and increases insulin secretion without the risk of hypoglycemia.Functional NMDA receptors are expressed in insulin producing cells. A pancreas specific NMDA receptor knockout selectively increases glucose stimulated insulin secretion We show for the first time that NMDA receptors can be targeted genetically and pharmacologically in mice and men to selectively increase glucose stimulated insulin secretion with improvement of glucose tolerance. We uncovered a new role for NMDA receptors in the pancreas and showed that inhibiting these receptors might serve as a useful treatment of human diabetes in pediatric and adult patients."} +{"text": "Fragility fractures are a major problem resulting in high morbidity and mortality in the older population. Over 80% of such injuries are caused by low energy trauma in patients with underlying osteoporosis. The first-year mortality rate of hip fracture ranges from 12% to 36%; only one-third of patients return to their prefracture functional status eventually and one-third require further nursing home care.Within this special issue we have a variety of articles focussed on fragility fracture care. One article is about a new approach in treating atypical fractures. Other two articles deal with hip fractures, whereas one is showing that the routine use of cemented stems for femoral neck fracture treatment on the elderly does not lead to higher complication rates and the other article describes that the collapse following the use of a DHS for treating such fractures does impact mobility but not survival of these patients. A very interesting operative strategy is shown for proximal tibial fractures allowing the patients immediate full weight bearing after operation. One more article is focussed on the outcome of elderly hip fracture patients following prolonged ICU treatment.In summary we have an interesting collection of valuable articles in this upcoming field. The editors want to thank the authors for their contributions.Christian KammerlanderHitendra K. DoshiWolfgang B\u00f6ckerMarkus Gosch"} +{"text": "Apart from ICRC, Jinnah Postgraduate Medical Center, Ziauddin University and Jinnah Sindh Medical University had participated in this study and the report was formally released at a meeting in Karachi on November 13Principal investigator Prof. Lubna Baig Dean, APPNA institute of Public Health who has now also been appointed Pro Vice Chancellor of JSMU and his team has done a commendable job in pointing out the reasons for violence against healthcare professionals (HCPs) and Healthcare facilities (HCFs). The most important reasons highlighted in this study include unreasonable expectations 56.1%), unexpected outcome (42.6%), communication failure (55%), and human error (53.7%) besides substandard care (35%). Poor quality of service provided by the healthcare facilities besides low capacity of healthcare professionals are also reported to have contributed to these violent incidents. The report has also pinpointed that a vast majority of the perpetrators of this violence were the patient\u2019s attendants.%, unexpe1In fact increasing commercialization and corruption along with wide spread unethical practices by the medical profession has also lead to increased violent incidents.6Violent incidents against healthcare professionals have also been reported in other countries in this region i.e. India, Bangladesh and Pakistan by Ali Khawaja and Hira IrfanViolence against healthcare professionals has also been reported from Saudi Arabia, Kuwait and Australia.Media should also follow some code of ethics while reporting such events because continuous negative reporting will eventually go against the interests of the patients whose interest they wish to safeguard. Already healthcare professionals are now reluctant to handle serious cases with the result that many precious lives which they could have saved are being lost. Serious cases are often referred to tertiary care facilities. Transporting these patients takes some time which could make a difference in the eventual outcome of such cases. Government authorities also need to wake up and initiate steps to implement some of the above and other recommendations by the research team of ICRC which in addition to Prof. Lubna Baig included Prof. Kamran Hameed, Dr. Kausar S. Khan, Dr. Seemin Jamali and Dr. Syeda Kausar Ali. In many cases no additional funding is required but just ensuring judicious use of existing facilities and resources and administrative measures will go a long way in improving the situation to a great extent immediately while long term plans can be prepared and implemented later on."} +{"text": "Distinct preference of species for habitats is most often driven by long term differences in demographic rates between habitats. Estimating variation in those rates is key for developing successful conservation strategies. Stochastic events can interact with underlying variation in habitat quality in regulating demography but the opportunities to explore such interactions are rare. Whimbrels in Iceland show a strong preference for sparsely vegetated riverplains. Such habitats in Iceland face various threats, e.g., climate change, river regulation and spread of alien plant species. In this study we compared demographic parameters of breeding Whimbrels between riverplains and other habitats before, during and after volcanic eruption events to estimate the importance of the habitats for the species and the effect of ash deposit on breeding success. We found that an estimated minimum of 23% of the Icelandic population of Whimbrels and c. 10% of the world population of the species breed in riverplain habitats in Iceland. Whimbrels bred consistently at much higher densities in riverplain habitats than in other habitats and riverplains also had higher densities of pairs with fledglings although the proportion of successful breeders was similar between habitats. Predation by livestock may have had a considerable negative effect on breeding success on our study sites. Breeding was negatively affected by the volcanic activity, probably through the effects of ash on the invertebrate food supply, with breeding success being gradually worse closer to the eruption. Breeding success was equally affected by volcanism across habitats which differed in underlying habitat quality. This study gives an example of how populations can be regulated by factors which operate at different spatial scales, such as local variation in habitat quality and stochastic events which impact larger areas. Habitat specialisation is one of the major factors contributing to species vulnerability to habitat loss . SpeciesPluvialis apricaria) (52%), Purple Sandpiper (46%), Whimbrel (Numenius phaeopus) (40%) and Ringed Plover (Charadrius hiaticula) (32%) )lifornia . It is aVanellus vanellus) and Curlews (Numenius arquata) were present and defended against aerial predators [But high density could also be beneficial when it comes to defending against aerial predators. Whimbrels are very apt flyers and aggressively mob much larger birds and highredators .High breeding density in riverplains might also be a response to resource abundance being higher there than in other habitats e.g. \u201371) alth alth71])For many birds, like most waders, which show strong adult philopatry and nataDue to these limitations it seems probable that higher density in riverplains is achieved through breeding success and/or survival being higher there on average. Whimbrels are long lived birds and the evolution of habitat selection probably only requires slight differences in productivity or survival over long time periods. It can therefore not be excluded that Whimbrels on riverplains have a higher individual fitness on average that went undetected in this study.2. The Icelandic population is thought to consist of 250 thousand pairs [Almost half of the estimated world population of Whimbrels breeds in Iceland and evidence suggests that riverplains are under threat so estimating the relative importance of this key habitat for population demographics is important for successful conservation. Riverplains in Iceland constitute around 8% of Iceland\u2018s lowlands . In thisnd pairs and fromThe two volcanic eruptions which occurred half-way through the study provided an opportunity to assess the short-term effect of volcanism on Whimbrel demography. A clear positive relationship between distance to volcano and breeding success, suggests a link with the amount of ash deposition. A likely driver of this relationship is the negative effect which volcanic ash has on the invertebrate food stock of birds although it seems that these effects are generally short-lived ,60 and tS1 FileDensity during nesting and chick rearing for Whimbrels on each of the study sites.(PDF)Click here for additional data file.S2 FileSuccessful and unsuccessful pairs on each of the study sites and the distance between sites and Eyjafjallajokull(PDF)Click here for additional data file.S3 FileInformation about nests found on the main study sites.(PDF)Click here for additional data file."} +{"text": "Morone chrysops \u00d7 M. saxatilis) to navigate downstream past an artificial barrier. Fish were released either individually or in groups into the flume using flow conditions that approached the limit of their expected swimming stamina. We compared fish swimming behaviors under solitary and schooling conditions and measured risk as the time individuals spent exposed to the barrier. Solitary fish generally turned with the current and moved quickly downstream past the barrier, while fish in groups swam against the current and displayed a 23-fold increase in exposure time. Solitary individuals also showed greater signs of skittish behavior than those released in groups, which was reflected by larger changes in their accelerations and turning profiles. While groups displayed fission-fusion dynamics, inter-individual positions were highly structured and remained steady over time. These spatial patterns align with theoretical positions necessary to reduce swimming exertion through either wake capturing or velocity sheltering, but diverge from any potential gains from channeling effects between adjacent neighbors. We conclude that isolated performance trials and projections based on individual behaviors can lead to erroneous predictions of risk exposure along engineered structures. Our results also suggest that risk perception and behavior may be more important than a fish's swimming stamina in artificially modified systems.Artificial barriers have become ubiquitous features in freshwater ecosystems and they can significantly impact a region's biodiversity. Assessing the risk faced by fish forced to navigate their way around artificial barriers is largely based on assays of individual swimming behavior. However, social interactions can significantly influence fish movement patterns and alter their risk exposure. Using an experimental flume, we assessed the effects of social interactions on the amount of time required for juvenile palmetto bass ( Nearly 65% of surveyed rivers and their aquatic habitats are threatened by human activity and climate change, with extinction rates among fresh water fish species rivaling those of past geological events Human barriers, such as dams, water diversion facilities and pumping stations can expose fish to a variety of dangers, including physical harm from collisions, exhaustion from swimming exertion Engineering and operational efforts predominantly rely upon estimates of average individual swimming stamina and behavior to expedite the safe passage of a region's fish. The design objective is to enable a fish's capacity to avoid flows or structural designs that would otherwise either impede its progress or increase its mortality risk Most juvenile fish form into groups of varying social and physical organization Morone chrysops \u00d7 M. saxatilis hybrid, were selected for their availability and propensity to display polarized schooling behavior when young. We began by determining individual swimming performance using ramped velocity tests to establish the water velocity necessary to challenge the average individual's swimming stamina (experiment I). Subjects were then released upstream of a behavioral barrier under solitary and social conditions to evaluate how neighbors altered individual swimming behaviors and, subsequently, impacted risk exposure (experiment II). We found that risk exposure varied dramatically between fish swimming by themselves or within schools, where risk was measured as the time taken to navigate successfully downstream past the barrier. A fine-scale kinematic analysis further revealed how individual behaviors varied under each treatment. We conclude by discussing how the observed patterns relate to existing theory and empirical evidence concerning fish swimming behavior.In this study we tested the hypothesis that social interactions alter the swimming behaviors of fish when they are forced to navigate past an artificial barrier. Palmetto bass, a All work was conducted within the U.S. Bureau of Reclamation's water lab in Denver, CO, and was included within BHL's dissertation work at USU. The BOR facility did not have an Institutional Animal Care and Use Committee (IACUC) in place and so all handling procedures followed the ethical standards outlined by the National Research Council Juvenile palmetto bass were obtained from Keo Fish Farms, AR, and maintained in flow-through cylindrical tanks , fed daily to satiation and experienced light: dark cycles typical of summer months in the northern hemisphere. Experiments were conducted in a 16 m flume . Water tWe determined the expected swimming stamina of our subjects using the critical swimming speed paradigm. Critical swimming speed, The speed observed during the final successfully completed interval .Our primary response variables were the critical swimming speeds (https://knb.ecoinformatics.org/).The secondary variables used to characterize individual swimming behavior included: rheotaxis , while grouped fish predominantly faced upstream and Pink (O. nerka) salmon generally increases with the size of the schools they form Palmetto bass share diet and habitat preferences with their maternal species in the wild and integrate themselves into striped bass spawning migrations Salmo salar) and American Shad (Alosa sapidissima) to navigate through bypass weirs decreases with increases in the size of the schools they form, with the majority of fish breaking off from larger groups and passing their respective barriers in pairs or alone Data also suggests that the size of the group may play a role. For example, the ability of Atlantic salmon , with spatial positioning from a school's front to rear being negatively correlated with the constituents' MS Consider that \u2018leaders\u2019 within groups can either possess information, or merely display bold tendencies In conclusion our findings demonstrate that schooling can enhance individual risk exposure for fish swimming through artificial environments. Future efforts would benefit from exploring how group size, hydraulic gradients, and structural complexity influence schooling behavior and individual exposure times along manufactured structures and obstructions in aquatic systems. While a thorough understanding of the fluid dynamics within schools remains out of reach, including basic social interactions when modeling animal movements may improve our ability to provide reliable risk assessments.File S1This file contains supporting information for the article and also contains Figure S1\u2013Figure S5. Figure S1, The influence of interaction range, r, on the size (a) and number (b) of groups observed. Figure S2, Time series of rheotactic patterns observed in the solitary and social travel conditions. Figure S3, Time series of observed swimming velocity and acceleration in both the solitary and social conditions. Figure S4, Time series of nearest neighbor values, d1. Figure S5.(PDF)Click here for additional data file."} +{"text": "Strategies aiming at light sedation are associated with decreased times on mechanical ventilation. However, awake or easily aroused patients may be prone to have greater prevalence of posttraumatic stress disorder. This systematic review and meta-analysis aimed to evaluate the safety of light sedation strategies in the prevalence of posttraumatic stress disorder.We searched MEDLINE, Scopus and Web of Science from inception to November 2014 for randomized controlled trials which compared a light sedation strategy with a deeper sedation strategy and addressed posttraumatic stress disorder prevalence as a specific outcome.I2 = 40 %).Five studies fulfilled our inclusion criteria and were included in the meta-analysis. Two studies compared daily sedation interruption with usual care (92 patients), two studies compared a light sedation protocol with daily sedation interruption (47 patients) and one study compared light and deep sedation (102 patients). Compared with usual sedation care/deep sedation, neither daily interruption of sedation nor a light sedation protocol were associated with increased risks on long-term PTSD prevalence. Heterogeneity was moderate (Light sedation strategies seem to be safe in terms of posttraumatic stress disorder prevalence. However, the small number of included trials and patients may not be sufficient to drive strong statements. Ongoing large trials will be able to answer this question."} +{"text": "This study explored and describes the factors that may influence how patients react to source isolation from others during hospitalization.The study also sought to determine how background variables such as gender, age and previous hospitalization affect source isolation.This qualitative study used content analysis to review data collected from interviews with five patients.- The conceptual framework describes antibiotic resistance and infection control from a public health perspective and explored its prevention in Denmark.- The theoretical framework describes how patients experience an infection acquired by exposure to drug-resistant bacteria, as well as subsequent isolation.The limited space, lack of contact with people resulted in patient monotony and anxiety.- Women showed greater concern about precautions against infection, and about risk of transmitting disease to visitors.- Men outwardly resigned themselves to the situation and did not speculate about infection precautions. Men had a more rational approach, and tended to cope better when isolated.- Younger patients seemed to have better coping strategy during isolation.- Elderly patients felt sad and lonely.- Patients developed their own strategies for coping with source isolation.Hospitals need more alternatives to prevent negative psychological affects due to isolation without compromising infection prevention. Hospitals should update their personnel at all organization levels, and focus on room facilities in the ward, contact time and improved information. Risk assessment should be individualized for each patient.None declared."} +{"text": "Publication of clinical research findings in prominent journals influences health beliefs and medical practice, in part by engendering news coverage. Randomized controlled trials (RCTs) should be most influential in guiding clinical practice. We determined whether study design of clinical research published in high-impact journals influences media coverage.We compared the incidence and amount of media coverage of RCTs with that of observational studies published in the top 7 medical journals between 1 January 2013 and 31 March 2013. We specifically assessed media coverage of the most rigorous RCTs, those with >1000 participants that reported \u2018hard\u2019 outcomes. There was no difference between RCTs and observational studies in coverage by major newspapers or news agencies, or in total number of news stories generated . Large RCTs reporting \u2018hard\u2019 outcomes did not generate more news coverage than small RCTs that reported surrogate outcomes and observational studies . RCTs were more likely than observational studies to attract a journal editorial , but less likely to be the subject of a journal press release . Large RCTs that reported \u2018hard\u2019 outcomes did not attract an editorial more frequently than other studies , nor were they more likely to be the subject of a journal press release .The design of clinical studies whose results are published in high-impact medical journals is not associated with the likelihood or amount of ensuing news coverage. Randomized controlled trials (RCTs), particularly those that are large and assess \u2018hard\u2019 outcomes, provide the most rigorous evidence to guide clinical practice . In contPublication of clinical research findings in prominent medical journals is often accompanied by media coverage, including that by outlets with large circulations. Such publications can also be accompanied by an editorial or a journal press release, each of which potentially increases the visibility and impact of the source article. Press releases from journals and academic institutions strongly influence the content of news stories in the lay media about the source research \u20135. Mediahttp://www.healthnewsreview.org/), but few studies have examined the relationships between research publications and news stories. A recent report suggested that newspapers in the United States with high readerships preferentially cover observational studies rather than randomized trials, and favour reporting of lower quality observational studies intended to substitute for a clinical endpoint [and] expected to predict clinical benefit (or harm\u2026) based on epidemiologic, therapeutic, pathophysiologic, or other scientific evidence\u201d . \u201cHard\u201d We assessed 171 source articles that reported 86 observational studies and 85 RCTs, 100 accompanying editorials and 584 news stories . NEJM doObservational studies were less likely than RCTs to attract an accompanying editorial but more likely than RCTs to generate a journal press release (18 source articles reported large (>1000 participants) RCTs with \u201chard\u201d outcomes. Journal and media coverage of these trials did not differ from that generated in response to the 153 observational studies and RCTs with smaller samples size and/or surrogate endpoints Click here for additional data file."} +{"text": "To the Editor: Adenoviruses commonly infect vertebrates, including humans and nonhuman primates clustered with SAdV-A species, showing 81%\u201386% nt and 90%\u201393% aa identity . One strFor more accurate characterization and for possible species determination of adenovirus strains, it is beneficial to sequence the entire hexon gene. In our previous study (Our phylogenetic analysis also identified another possible new simian species: 5 strains, which were previously reported as \u201cunassigned adenoviruses,\u201d were isolated from Old World monkeys living in captivity and in the wild . To defiOur results demonstrated sequence conservation in the hexon genes of some adenovirus strains but also high diversity among other strains in a colony of captive monkeys. Our findings of close sequence relatedness between simian and human adenovirus strains suggest the interspecies transmission of these viruses and highlight the continuing risk for new and emerging infections in humans."} +{"text": "Food allergy is apparently increasing and it is estimated that 3\u20134% of adults have food allergy. Shrimp is one of the most common causatives in seafood allergy. Patients with shrimp allergy may exhibit life threatening anaphylactic reactions. Tropomyosin is a known important allergen in shrimp. The IgE-binding epitope in shrimp tropomyosin, cross-reacts with other allergenic invertebrate tropomyosins in house dust mites and cockroaches (Per a 7).this study was undertaken to evaluate the results of immunotherapy to Mites upon allergic reactions to shrimp allergen in a 40 years old female suffering from combined allergy to mites and shrimp. The patient had a 10 years history of severe allergic reactions after eating shrimp; for which she was hospitalized several times to receive intravenous steroids and antihistamines. Despite avoiding consumption of shrimp for several years she continued to have allergic rhinitis symptoms. The last episode of allergic reactions to shrimp occurred while cooking shrimp not eating it.Allergy skin prick testing against a panel of 30 common allergens including Mites and shrimp revealed a strong positive wheal and erythema reaction to both shrimp and D. farinae . Allergen specific serum IgE testing also revealed elevated serum specific IgE to both allergens. The patient started subcutaneous allergen specific immunotherapy (ASIT) against D. farinae using Omega labs allergy shots. Six months after ASIT there was a significant reduction in the size of the wheal and erythema and also significant decrease of serum specific IgE values for both allergens.Subcutaneous immunotherapy against mites may desensitize patients against shrimp allergy."} +{"text": "During this interactive hands-on workshop the participants will view complete imaging examinations on workstations in a clinical routine setting and discuss findings with two tutors.Cases will include those with typical findings as well as pitfalls and problems.Particular attention will be paid to the correct assessment of findings that alter the therapeutic concepts -3."} +{"text": "The rapid progress in high throughput sequencing technology has significantly enriched our capability to study mitochondria genomes. Other than performing mitochondria targeted sequencing, an increasingly popular alternative approach is to utilize the off-target reads from exome sequencing to infer mitochondria genomic variants including SNP and heteroplasmy-9. HowevWe designed a specific study using targeted mitochondria sequencing data as a gold standard to evaluate the practicality of SNP and heteroplasmy detection using exome sequencing and RNAseq data. Five breast cancer cell lines were sequenced for mitochondria targeted sequencing, exome sequencing, and RNAseq. Furthermore, we examined three mitochondria alignment strategies: 1) align all reads directly to the mitochondria genome; 2) align all reads to the nuclear genome and mitochondria genome simultaneously; 3) align all reads to the nuclear genome first, then used the unaligned reads to align to the mitochondria genome.Our analyses found that exome sequencing can accurately detect mitochondria SNPs and can detect a portion of the true heteroplasmies with a reasonable false discovery rate. RNAseq data on the other hand had a lower detection rate of SNP but higher detection rate for heteroplasmy. However, the higher false discovery rate makes RNAseq a less ideal source for studying mitochondria compared to exome sequencing data. Furthermore, we found that aligning all reads directly to the mitochondria genome reference or aligning all reads to the nuclear genome and mitochondria genome references simultaneously produced the best results.Exome sequencing and RNAseq data can be potentially mined for mitochondria variants. Overall, exome sequencing provides less false discovery than RNAseq for mitochondria variant detection, making it a more desirable choice. In conclusion, our study provides important guidelines for future studies that intend to use exome sequencing or RNAseq data to infer mitochondria SNP and heteroplasmy."} +{"text": "In a recent issue of this journal Dr. Oymak and her colleagues presented a clinically and genetically well-studied 5-year-old boy who was seen with severe microangiopathic hemolytic anemia without laboratory findings of renal involvement despite complement factor H gene mutations .Because of Yeneral\u2019s extensive review on atypiThough in our patient with thrombotic thrombocytopenic purpura renal involvement was documented at the beginning but not in the last two recurrences, neither serum nor urinary findings indicated kidney involvement .Although the discussions of Dr. Oymak et al. are well taken, the term \u2018microangiopathic hemolytic anemia\u2019 is covering the syndrome to a large extent as suggested by George and Nester ."} +{"text": "Progressive weakness and degeneration of skeletal muscles caused by genetic alterations fall into the category of muscular dystrophy. Muscular dystrophy occurs worldwide and affects all races. The overall incidence of muscular dystrophy varies among forms, as some forms are more common than others. Muscle loss and weakness are not necessarily caused by genetic alteration. Skeletal muscle inactivity, denervation, cancer-associated cachexia, and physiological responses to fasting or malnutrition cause skeletal muscle mass loss through imbalance in synthesis and breakdown of proteins. Several genes have been identified that are directly or indirectly involved in various muscle wasting. Studies performed in human and animal models have substantially contributed to our knowledge of molecular mechanism of muscle degeneration but still these findings are inadequate for developing effective therapy. Therefore, precise dissection of molecular mechanisms provides the way for the development of therapeutic interventions for muscular dystrophies as well as for skeletal muscle loss.\u03b21) in skeletal muscles. TGF\u03b21 is recently shown to be a key player in skeletal muscle atrophy and endomysial fibrosis. E. Guadagnin et al. demonstrated that TGF\u03b21 alone can induce Tyr705 phosphorylation of STAT3 in skeletal muscle cells, and higher pSTAT3 (Tyr705) leads to severe phenotype in transgenic TGF\u03b21 mice.In this special issue, we intended to publish research and review articles on exploring molecular mechanisms and target identification for treatment of muscle diseases. This issue will give insight into cellular and molecular mechanisms, activation of signaling pathways, how activation of these pathways causes muscle dysfunction, and subsequent disease symptoms. The review article published in this special issue discusses the animal model for muscular dystrophy associated with dystroglycan , followed by an article focusing on inflammation status and nutrition in Duchenne muscular dystrophy (DMD) patients . The other three articles are related to cellular and molecular modeling of skeletal muscle loss. These articles describe recent advancement in the skeletal muscle research field as well as possibility for developing tools in therapeutic intervention. A study conducted by E. Guadagnin et al. provides new insight into the role of transforming growth factor beta 1 is highly expressed in skeletal muscle and served as extracellular matrix receptor. Mutations in components of the DG complex are cause of several muscular dystrophies such as mutations in one of the DG complex genes,Hampered calcium signaling has been often reported in muscular dystrophies, which led to proteolysis of muscle protein induced by calcium ions. The assessment of intracellular calcium event is important for understanding the molecular mechanisms underlying muscular dystrophies. To understand the precise mechanism of calcium signaling in muscle cells, there is a need of robust cellular model. N. Smolina et al. have examined myotubes as a model of adult skeletal muscle for studying evoked calcium release. In addition, the authors also assessed the possibility of this cellular model for studying functional mutation effects through lentiviral transduction. Therefore, primary murine myotubes may serve as a useful cellular model for investigating calcium signaling.\u03b2 signaling on muscle regeneration with treadmill training in SCI through Smad3 downregulation, providing early indicators of efficient reloading in SCI model.C. Baligand et al. identified the key molecular pathways activated during muscle remodeling after spinal cord injury (SCI) and locomotor training in a rat model since molecular events associated with changes in muscle mass after SCI are not known completely. In this study the authors have performed genome wide expression profiling of soleus muscles at multiple time points after SCI in the well-characterize rat model. Their expression data suggest the involvement of TGF-beta/smad3 signaling in association with decrease in muscle mass observed with SCI, while the BMP pathway was activated during treadmill training. This study may provide insight into effects of BMP signaling activation and TGFArticles published in this special issue will provide new insights into understanding the pathophysiology and novel therapeutic target identification of skeletal muscle diseases. As the understanding of skeletal muscle diseases improves with time, new findings will further enrich the current knowledge of these diseases, ultimately helping to develop effective therapy."} +{"text": "Most current single nucleotide polymorphism (SNP) genotyping methods are still too slow and expensive for routine use in large association studies that requires hundreds or more SNPs in a large number of DNA samples and for diagnostic purpose. Clinical implementation and ultimate use of nucleic acid biomarkers necessitate development of pragmatic detection assays that deliver adequate sensitivities, mutation selectivity and high throughput capabilities in a rapid, robust and cost effective manner. In the present study we have developed a sensitive and cost effective genotyping technique with computer aided tool for allele discrimination and implemented it to genetic epidemiology study.Modified allele specific oligonucleotide hybridization assay with elimination of DNA isolation step for PCR and novel allele discrimination method was used to screen folate metabolism gene polymorphisms in acute lymphoblastic leukemia patients and normal individuals. The plasma homocysteine and global DNA methylation was performed by HPLC method.The developed novel allele discrimination method along with the newly developed image analysis software increases the sensitivity and elimination of DNA isolation step for PCR reduces the cost of the genotyping technique. The average kappa value of all designed probes was > 0.81. The technique was successfully designed for both diagnosis and research purpose. The evaluation of effect of gene polymorphism on plasma folate, homocysteine level and global DNA methylation explored that the only MTHFR C677T gene polymorphism is associated with elevated homocysteine level in healthy young adults. The genotyping of 22 folate metabolism genes explored that only RFC1 80GA and RFC1 80AA genotypes were significantly associated with increased risk of Acute Lymphoblastic Leukemia (n=203) when compare to healthy individuals (n=245) in south Indian population.The developed visual based allele specific oligonucleotide hybridization assay will be useful in clinical diagnostic system where large scale screening is often necessary without any sophisticated detection systems. RFC1 80GA and RFC1 80AA genotypes were significantly associated with increased risk of acute lymphoblastic leukemia. In young adults, only MTHFR C677T polymorphism is associated with elevated homocysteine level."} +{"text": "Mitochondrial dysfunction (bioenergetic failure) is knownt to contribute to the development of multiorgan failure in acute sepsis. The defect is mainly at the level of respiratory complex I , 2.To assess whether mitochondrial dysfunction in skeletal muscle perists until protracted critical illness and whether it contributes to the development of ICU-acquired weakness.Muscle biopsies were obtained from critically ill patients with severe ICUAW (defined as MRC score < 24 on two separate tests) and from otherwise healthy controls undergoing elective hip replacement. 50-100mg of the native sample was homogenized by a technique which perserves mitochondria in their cytosolic context . Oxygen In skeletal muscle of patients with protracted critical illness and severe ICUAW we have found the activity of complex II and III to be >200% of activity of healthy controls. Function of complexes I and IV was identical as were global mitochondrial function/integrity indices.Upregulation of complex II may represent an adaptive phenomenon as skeletal muscle during critical illness is more reliant on fatty oxidation (which feeds electrons to respiratory chain via complex II), because of insulin resistance and impaired oxidative glucose disposal.In patients with severe ICUAW we have demonstrated increased activity of respiratory chain complexes downstream to complex I and intact global mitochondrial function. This may represent an adaptation to insulin resistance."} +{"text": "Review HRMRI techniques for pelvic evaluation of cervical uterine carcinoma using phased-array coil.Discuss the accuracy of high resolution MR for diagnosis and staging of cervical uterine carcinomaDiscuss the correlation between MR findings, FIGO staging and treatment strategyDiscuss the correlation between MR features and histopathology findings in resected tumours\u2022 Uterine cervical carcinoma\u2022 FIGO staging\u2022 High resolution MRI\u2218 Dedicated protocol\u2218 Primary tumour detection\u2218 Myometrial invasion\u2218 Lymph node involvement\u2218 Parametrial invasion\u2218 Bladder and rectal invasion\u2218 Vaginal involvement.\u2022 Treatment strategies\u2022 Histopathathology correlationCervical cancer remains a major threat to women's health worldwide. MR is the imaging modality of choice to depict the primary tumour and assess local extent. Comparing the radiological findings with the postoperative histological reports, high resolution MR with a dedicated protocol demonstrated to be useful for primary tumour detection and for the assessment of myometrium invasion, lymph node commitment, parametrium invasion, bladder and rectal infiltration and vaginal involvement. This ability of MRI to demonstrate accurately the local extension of the tumour in patients with cervical cancer has become a useful tool to identify prognostic risk factors such as the depth of the infiltration, the tumour volume and the commitment of the adjacent structures. A correct evaluation of these factors is crucial for choosing and planning the most appropriate treatment."} +{"text": "We embrace with enthusiasm the recent study by Roderburg et al. identifyWe recently argued that currently used markers of systemic inflammation such as C-reactive protein and procalcitonin are substantially cleared during continuous renal replacement therapy (CRRT) and thus may lose sensitivity to evaluate the degree and evolution of inflammation . OPN is More information regarding an eventual significant \u201closs\u201d of OPN by filtration and/or membrane adsorption during CRRT is imperative before this protein can be accepted as a valid prognostic biomarker. We are grateful for the letter from Honore et al. raising the important question of an eventual \u201closs\u201d of OPN by filtration during (C)RRT in critically ill patients. We cannot fully exclude this bias but we have performed additional analyses on our cohort of critically ill medical patients , suggestTaken together, our results do not support the concern that OPN serum levels might not be a valid prognostic parameter in patients with acute kidney injury undergoing RRT. Nevertheless, we cordially thank Honore et al. for their valuable comment on our article, as further prospective trials considering renal failure and replacement should be conducted to properly address this question."} +{"text": "Best solution to solve ideal revascularisation remains a dilemma for complex Coronary artery disease. However, emerging minimally invasive off pump CABG technique can provided optimum solution for this condition.To evaluate significance of Minimally invasive CABG using \" LIMA to LAD first \" in staged hybrid approach in patients with complex Coronary artery disease with compromised LV function and associated co morbid conditions.Between December 2012 and 2014 total 28 patients were taken up for Surgery using off pump MIDCAB and MICS CABG technique. LIMA was harvested in all cases using harmonic scalpel and target LAD grafting was done using polypropylene 7-0 or 8 - 0 depending on luminal size and quality of LIMA. All patients were evaluated with preoperative risk stratification and standard work up protocol including consent to convert in sternotomy, on pump or any other life support system in demand.Out of 28 patients, 27 (97%) had successful LIMA to LAD grafting achieved. All patients were operated off pump method.Relief in angina was observed in 60 per sent cases. Nine patients (30 %) underwent staged PCI after six months of the CABG. They also had smooth course and improved outcome during and after the intervention.Using current Minimally invasive off pump CABG technique with staged hybrid PCI option for non - LAD lesions, complex Coronary artery lesion can be efficiently managed by the heart team with better outcomes in short term. In addition,this procedure may provide safe and cohesive environment for the Surgeons and intervention cardiologists to work together for best interest of the patients."} +{"text": "This is a comment on \u201cNew insights into the Steen solution properties: breakthrough in antioxidant effects via NOX2 downregulation\u201d .The addressed paper investigated reactive oxygen species (ROS) production and NADPH oxidase (NOX2) activity in platelets, polymorphonuclear leukocytes, and lymphocytes-monocytes, while being incubated in buffer solution and in Steen solution. The results showed significant role of Steen solution to reduce ROS production and to downregulate NADPH oxidase activity . HoweverDuring ex vivo lung perfusion (EVLP), antagonizing ROS production would be important for the attenuation of cytokine production, which may constitute the stimulus for subsequent graft dysfunction and rejection after transplantation .ATP channels [During graft ischemia, the absence of flow would be translated into membrane depolarization and increased ROS production, with associated increased NOX2 activity and inhibition of KTo antagonize ROS during EVLP, antioxidants or NOX2 inhibitors might be used. However, caution should be taken with that because EVLP might be considered as an ischemic preconditioning model. This hypothesis is supported by the observation that lung grafts are tolerant to both cold and normothermic post-EVLP ischemia . In suchAccordingly, the use of Steen solution with its antioxidant potential and its inhibitory effect on NOX2 during the graft cold static preservation phase would be expected to result in a better outcome than that of the currently applied protocols.+ channel agonists would have a dual benefit through decreasing ROS production during initial ischemia and graft conditioning and protection during the subsequent ischemic-reperfusion injury [In addition, the presence of albumin and dextran in Steen solution would provide further protection for the graft during cold preservation. Furthermore, supplementation of Steen solution with Kn injury , 5.+ channel agonists) during cold static graft preservation as well as EVLP [Formerly this year, a new EVLP protocol (Shehata protocol) was introduced by the author which recommended the use of Steen solution (supplemented with antioxidants and K as EVLP . The res"} +{"text": "An internal pilot study is the first part of a substantive RCT where trial parameters are examined and data contribute to the final analyses. This two-phase design provides an opportunity to review pre-agreed \u2018progression criteria\u2019 that determine whether the internal pilot should continue into the main trial. Selecting appropriate criteria is critical but little is known about this area. Key issues in selecting progression criteria when designing RCTs with an internal pilot phase are considered.Opinions of stakeholders were elicited via a workshop to facilitate discussion regarding optimal use of internal pilots to inform efficient RCTs. This was informed by a literature review of pilot work.Three common progression criteria to be considered in pragmatic RCTs with an internal pilot were considered: (i) recruitment; (ii) protocol adherence; (iii) completeness and quality of outcome data. Pre-agreed progression criteria provide an opportunity to review the viability of completing the main trial within the planned timetable and budget, and should be considered alongside other indicators of feasibility rather than simply whether absolute targets were met. Open dialogue between the funder, trial team and steering committee is desirable to address emerging issues and optimally inform decisions about the main trial. This talk will be illustrated with examples, including from surgical trials.Careful selection of progression criteria may inform optimal design of RCTs with an internal pilot phase. Flexibility when reviewing progression criteria is necessary when considering the viability of proceeding to the main trial."} +{"text": "Sakr and colleagues report seemingly disappointing data on the effects of selenium in severe sepsis . MortaliHowever, there are good reasons for selenium supplementation in sepsis; the selenium status declines with the disease especially in nonsurvivors , reducinYet some major questions prevail. Is it sufficient to balance selenium decline (substitution)? Are larger dosages required for overcoming compromised selenoprotein biosynthesis (supplementation)? Are bolus dosages of selenite key to health benefits (pro-oxidative)? For me, the third option has been specifically disproven by the data .So why does this study seemingly contradict the positive findings of Angstwurm and colleagues ? An answYasser SakrCritical Care[We read with interest the letter by Dr Schomburg concerning our article published recently in ical Care. He inteDr Schomburg also suggests that selenium supplementation may be beneficial in patients with selenium deficiency , and refThe authors declare that they have no competing interests."} +{"text": "Furthermore, hypotension (systolic BP < 80 mm Hg) was present in 44% of children with severe acidosis (base deficit >15). These findings also indicate important baseline differences in two cohorts of children studied. We agree that reconsideration of guidelines for acute fluid management is warranted, particularly when current recommendations await an adequate evidence base. Nevertheless, conflicting opinions on the question of volume status in children with severe malaria can be satisfactorily resolved only through prospective randomized trials that include both fluid resuscitation and control groups. While the design and conduct of such trials will involve considerable challenges, optimal fluid management will never be resolved on the basis of theoretical consideration alone.The study by Planche et al."} +{"text": "Health information exchange and multiplatform health record viewers support more informed medical decisions, improve quality of care, and reduce the risk of adverse outcomes due to fragmentation and discontinuity in care during transition of care. An example of a multiplatform health record viewer is the VA/DoD Joint Longitudinal Viewer (JLV), which supports the Department of Veterans Affairs (VA) and Department of Defense (DoD) health care providers with read-only access to patient medical records integrated from multiple sources. JLV is intended to support more informed medical decisions such as reducing duplicate medical imaging when previous image study results may meet current clinical needs.We estimated the impact of provider usage of JLV on duplicate imaging for service members transitioning from the DoD to the VA health care system.We conducted a retrospective cross-sectional study in fiscal year 2018 to examine the relationship between providers\u2019 use of JLV and the likelihood of ordering duplicate images. Our sample included recently separated service members who had a VA primary care visit in fiscal year 2018 within 90 days of a DoD imaging study. Patients who received at least one imaging study at VA within 90 days of a DoD imaging study of the same imaging mode and on the same body part are considered to have received potentially duplicate imaging studies. We use a logistic regression model with \u201cJLV provider\u201d (providers with 1 or more JLV audits in the prior 6 months) as the independent variable to estimate the relationship between JLV use and ordering of duplicate images. Control variables included provider image ordering rates in the prior 6 months, provider type, patient demographics , and clinical characteristics (Elixhauser comorbidity score).P=.005). This effect is robust across multiple specifications of linear and logistic regression models. The provider\u2019s practice pattern of ordering image studies and the patient\u2019s health status are powerful confounders.Providers known to utilize JLV in the prior 6 months order fewer duplicate images relative to providers not utilizing JLV for similar visits over time (odds ratio 0.44, 95% CI 0.24-0.78; This study provides evidence that adoption of a longitudinal viewer of health records from multiple electronic health record systems is associated with a reduced likelihood of ordering duplicate images. Investments in health information exchange systems may be effective ways to improve the quality of care and reduce adverse outcomes for patients experiencing fragmentation and discontinuity of care. Health information exchange (HIE) allows health care providers and patients to access and share patient-level electronic health information between different health care settings -3. When Minimizing orders for unnecessary duplicate medical image studies is important for improving health care efficiency, reducing unnecessary time burdens on patients, and attenuating adverse health outcomes caused by excessive medical radiation, such as increased risk of cancer -13. HoweAn example of integrated health record viewers is the Joint Longitudinal Viewer (JLV), formerly known as the Joint Legacy Viewer (version 2.2). As a web-based graphical user interface, JLV supports the Department of Veterans Affairs (VA) and Department of Defense (DoD) health care providers with an integrated, read-only view of health data from the VA and DoD systems as well as VA community partners . ReleaseWe conducted a retrospective cross-sectional study in fiscal year 2018 to examine the relationship between provider use of JLV and the ordering of potentially duplicate image studies. The analysis compared duplicate imaging ordered by JLV-using and non\u2013JLV-using providers of VA outpatient primary care visits in fiscal year 2018 for recently separated service members. We conducted the study for VA quality improvement and program evaluation purposes, and therefore, the study was exempt from Institutional Review Board review.Recently separated service members who had at least one VA primary care visit in fiscal year 2018 within 90 days of an imaging study conducted at DoD were eligible to be included in the sample. We excluded VA primary care visits that were compensation and pension exams or not provided by physicians, physician assistants, or nurse practitioners. We also excluded DoD imaging studies if the primary diagnosis was cancer because duplicate diagnostic images were likely to be clinically appropriate and recommended by providers for patients with cancer. Patients who received at least one imaging study at VA within 90 days of a DoD imaging study using the same imaging mode and on the same body part were considered to have received potentially duplicate imaging studies .VA clinic stop codes were used to identify outpatient primary care visits. Compensation and Pension exams were identified using the secondary stop code (450) and the appointment type (Compensation and Pension) and were excluded from the VA primary care visits. Audit logs acquired from the JLV system were assessed to determine a provider\u2019s JLV utilization during a specific VA primary care visit and the provider's JLV utilization history over the 6 months prior to the visit.The independent variable \u201cJLV encounter\u201d indicated whether a JLV audit was linked to the patient on the primary care visit date. The independent variable \u201cJLV provider\u201d indicated whether the provider had 1 or more JLV audits in the 6 months prior to the visit date. Endogeneity is likely to be a problem in the estimation of the association between \u201cJLV encounter\u201d and duplicate imaging because unobserved confounders such as patient complexity are related to both JLV use and ordering duplicate image studies during the primary care visit. We estimated the direct (proxy) relationship between \u201cJLV provider\u201d and duplicate imaging to deal with the potential endogeneity problem. We also used a 2-stage statistical model by using JLV providers as an instrumental variable to estimate the causal relationship between JLV encounter and duplicate imaging. The categorization of JLV providers was based on the provider\u2019s prior interactions with other patients and indicated the provider\u2019s propensity to view health records through JLV. Thus, this variable was independent of the observed and unobserved characteristics of the patient under study. This is especially true in the VA setting where patients are arbitrarily assigned to primary care providers. Current Procedural Terminology codes indicating imaging procedures were categorized by mode and body part to compare VA and DoD imaging records and identify potential duplicate images. Following Vest et al , the depDescriptive statistics were calculated to explore the distributions of duplicate image ordering, the provider\u2019s rate of ordering images in the prior 6 months, the patient\u2019s Elixhauser comorbidity score, and other covariates. In our primary statistical model, we used a logistic regression to estimate the relationship between the provider\u2019s JLV use in the prior 6 months (yes/no) and duplicate imaging (yes/no). In an alternative specification, we used instrumental variables to focus on JLV use in the actual primary care visit. To deal with the potential endogeneity problem that unobserved patient characteristics might confound that relationship, we used a 2-stage residual inclusion (2SRI) logistic regression model to estimate the relationship between the provider\u2019s JLV use during the primary care visit (yes/no) and the ordering of duplicate imaging studies (yes/no) with JLV provider as the instrumental variable.\u00b5e) calculated from the first stage were included in the second stage model because 2SRI models with Anscombe residuals generate less biased estimates for rare outcomes compared to 2SRI models with other forms of residuals [More formally, we wished to estimate the relationship between duplicate imaging and use of JLV during the primary care visit, controlling for potential confounders, including provider imaging rate in the prior 6 months, provider type (physician or physician assistant/nurse practitioner), patient age, gender, Elixhauser comorbidity risk score, time (month), and facility (equation 1). This model could produce biased estimates because the decision to use JLV during the visit could be simultaneously determined with the decision to order a duplicate imaging study due to unobserved confounding factors such as patient complexity. To address this problem, we estimated the first stage model, which related JLV use during the visit to the provider\u2019s JLV use history (the JLV provider variable). Then, we estimated the second stage model, which related duplicate imaging to JLV use during the visit. Anscombe residuals , patient age, gender, Elixhauser comorbidity risk score, time (month), and facility X\u00b5: Unobserved confounding factor such as patient complexityX\u03b5: ResidualW: The instrument of JLV provider and observed exogenous variables\u00b5e: Anscombe residual calculated from the first stage model estimatesXWe tested the robustness of the result by using different model specifications, including ordinary least squares models on the relationship between JLV provider and duplicate imaging and linear 2-stage least squares models on the relationship between JLV encounter and duplicate imaging using JLV provider as an instrumental variable. We used Stata 15.1 (StataCorp LLC) to conduct the statistical analysis.Overall, JLV use has increased since fiscal year 2015. Rapid growth of monthly JLV audits was observed in fiscal year 2018 . Table 1P=.005; average incremental effect=\u20130.05) of ordering duplicate image studies. Provider history of ordering images and patient Elixhauser comorbidity scores were strong confounders of the relationship between JLV use and duplicate imaging. Providers with high rates of ordering images in the prior 6 months were more likely to order duplicate images . Patient Elixhauser comorbidity scores of 3 or more were significantly associated with a reduced likelihood of receiving duplicate imaging services . In a loP<.001). In a test of the coefficient on the instrument, a Cragg-Donald Wald F statistic greater than 10 indicates that the instrument is strong enough [P<.001). The Cragg-Donald Wald F statistic of 43.68 is greater than 10 and suggests that provider past use of JLV was a strong instrument. This is a necessary condition for JLV provider to serve as an instrumental variable for JLV encounter in our 2-stage specifications.In the 2SRI analysis, the results of our first stage model indicateg enough . In a liP=.03; average incremental effect=\u20130.16) in the likelihood of ordering duplicate images, controlling for patient and provider characteristics, time effects, and facility random effects (P=.009) compared to providers with low rates of ordering medical image studies. Patient Elixhauser comorbidity scores of 3 or more were significantly associated with a reduced likelihood of receiving duplicate imaging procedures .Our main finding that JLV use had a significant effect on reducing duplicate imaging was robust using different model specifications, including 2-stage least squares models estimating the association between JLV encounter and duplicate imaging (see Table S2 in This study using national data from VA and DoD found that providers who viewed integrated patient health records from multiple settings were less likely to order potentially duplicate imaging studies for patients who had prior imaging studies conducted within 90 days. Based on results from our primary analysis, providers with a history of using JLV were 5 percentage points less likely to order duplicate images during a VA primary care visit for recently separated service members compared to providers who did not have a history of using JLV. Using the JLV provider as an independent variable, we were able to reduce potential endogeneity due to unobserved confounders that were associated with both JLV use during the primary care visit and ordering of duplicate images.Our results were consistent with previous findings that use of HIE systems was associated with a reduction in repeat imaging studies and thatOur study has several limitations. First, we focused on VA primary care visits and images within the 90-day follow-up period of a DoD image and therefore were unable to capture duplicate images in other settings such as community-based clinics. Further research could examine duplicate image studies ordered during different types of outpatient and inpatient encounters to improve the generalizability of the results. Second, limited by the administrative data source, we could not determine whether the identified duplicate imaging procedures were unnecessary. In some cases, providers may need to examine repeat image studies for serial changes in disease status or order follow-up imaging studies based on recommendations in the patient\u2019s previous imaging reports. Thus, some of the duplicate image studies we identified might have been clinically appropriate. We mitigated this limitation by excluding patients with cancer diagnoses and ensuring the consistency of the definition of duplicate imaging studies among the providers who used and did not use JLV. Third, restricted by data access, we could not adjust for HIE through another widely used health information viewer, VistaWeb, which was recently decommissioned. We tried to overcome this limitation by focusing on VA primary care visits in fiscal year 2018\u2014the year when we observed rapid growth in JLV utilization after the VA\u2019s transition from VistaWeb to JLV. Fourth, our result was not robust when we changed the definition of JLV provider to providers with 10 or more JLV audits in the 6 months prior to the VA primary care visit, suggesting the heterogeneity of JLV benefits by frequency of use.Organizational fragmentation and discontinuity of care have been linked to increased costs and adverse outcomes in VA and other health care settings . Our finIn conclusion, this study provides evidence that adoption of a longitudinal viewer of health records from multiple electronic health record systems is associated with a reduced likelihood of ordering duplicate image studies. In future studies, the association between health information viewers and other types of duplicate medical tests and care coordination metrics such as follow-up of suspicious lung nodules could be investigated to more fully illustrate the impact of HIE on quality and efficiency of care."} +{"text": "The SCHEMA consortium has identified ten genes in which severely damaging variants substantially increase schizophrenia risk.To characterise the clinical features of carriers of variants causing complete loss of function (LOF) of these genes.This research was conducted using the UK Biobank Resource and 200,000 exome-sequenced volunteers were screened to identify carriers of LOF variants in these genes. For these subjects, data fields were extracted which reflected educational and occupational functioning as well as clinical features including diagnoses and medication.CACNA1G were commoner than in SCHEMA cases, suggesting this was not a real schizophrenia susceptibility gene. 159 subjects carried LOF variants in one of the other nine genes and overall they did not have poorer educational or occupational functioning or increased mental or physical health problems. Detailed examination revealed that one had schizophrenia, one had psychotic depression and two had a developmental disorder. Otherwise, a number of subjects had features of minor mental illness such as depression or anxiety and these rates were somewhat increased in subjects carrying LOF variants in HERC1, of whom more than half reported having consulted their GP for such problems. However the majority appeared to be entirely normal from a neuropsychiatric point of view.LOF variants in Although particular genetic variants can substantially increase the risk of schizophrenia, most people carrying them are entirely normal. This further supports the concept of schizophrenia as a distinct illness rather than representing the extreme of a trait which is present in the population.No significant relationships."} +{"text": "Classic bioecological models of human development highlight the influence that five layers of context have on individuals\u2019 function and growth. Modern versions specifically highlight five specific aspects of context that influence both how older adults negotiate their daily lives and how they age. Using a collection of empirical findings obtained from our analyses of longitudinal panel data from the German SOEP, experience sampling data from iSAHIB, and screenome data from the Stanford HSP, we illustrate how differences in individuals\u2019 engagement with green spaces (earth), ambient air pollution (wind), and smartphones (fire) may contribute to differences in daily emotional well-being and developmental changes in life satisfaction. We then use these examples to elucidate shortcomings of the hierarchical models used to frame investigations of context and development for the past 40 years \u2013 and suggest the classic models be renewed rather than continually recycled."} +{"text": "The past two decades have brought significant medical advances in antiretroviral therapies (ART) for people living with HIV/AIDS (PLWHA). This has enabled PLWHA to live longer and healthier lives than ever before. In fact, nearly half of all PLWHA in the United States are age 50+ and HIV is treated as a chronic disease, instead of a terminal illness. Despite these advancements older adults living with HIV/AIDS (OALWHA) are still a highly stigmatized population who faces challenges with their health and overall well-being. This symposium will highlight recent research on factors that play a role in successful aging among OALWHA. Our first presentation leverages a quantitative dataset of OALWHA and examines the relationship between childhood sexual abuse and ART adherence, with a particular focus on substance abuse as a potential mediator. Our second presentation features a qualitative study with OALWHA that explores their conceptions of successful aging. Our third presentation includes a sample of medical case managers who serve OALWHA and examines the feasibility of using a cognitive screening tool with this population. Cognitive screening with OALWHA can help provide early indicators of cognitive decline and initiate early intervention. Finally, our fourth presentation includes a sample of older women living with HIV/AIDS and examines their support needs and resources, particularly to help understand their complex caregiving needs. Findings from these four studies advance our understanding of OALWHA and their needs to ensure continued successful aging. This symposium informs further research and direct practice with this population."} +{"text": "The present cocktail of a bleak economic outlook combined with high inflation and rising interest rates has raised questions as to whether a new euro debt crisis might emerge. Euro area sovereign spreads have widened, but not to \u201cunwarranted\u201d levels, unlike the situation that prevailed during the European debt crisis. A replay of the funding fears, which drove the euro area debt crisis a decade ago, seems unlikely today with the extensive toolkit now in place."} +{"text": "There has been increasing policy interest in changing dietary patterns to reduce diet-related diseases and improve population health. Meanwhile, the food choices people make every day have a determining impact on the climate change, with food systems responsible for a third of global greenhouse gas emissions. Current policies focused on dietary health are designed, implemented and evaluated in relative isolation, and there is a critical open question concerning the extent of possible synergy with an additional focus on carbon removal.We analysed the changes in UK households\u2019 food purchases from an online, randomized control experiment (n = 3933) designed to contrast respondents\u2019 current food purchase behaviour with that under a range of potential tax and labelling policies targeting improvement in dietary health, alone or combined with those designed to reduce carbon emissions. We assessed changes in the healthiness of food baskets between interventions through indicators of: i) purchase of calories; ii) % of calories purchased from 23 food groups; and iii) relative changes in nutrient composition of food purchased.Food labelling and fiscal measures for both health and decarbonisation have a positive impact on dietary health, by reducing the calorie content of food purchases (p < 0.001). Adding carbon reduction considerations into health policies achieves nutritional improvement by further reducing fat and increasing fibre, resulting in a reduction of up to 193 kcal/person/day (95%CI: 172-214).With an additional focus on planetary health, the combined tax and food labelling policies could achieve a reduction in calorie content at a magnitude close to the Public Health England's estimate of average excess calories consumed by adults .\u2022\u2002Policies focused on achieving both nutrition and carbon reduction goals could achieve greater improvements in food choices and produce win-win scenarios.\u2022\u2002There is a need for greater dialogue and policy development between public health and environmental researchers, practitioners and policy makers."} +{"text": "Lower extremity deep venous thrombosis (DVT) is prevalent amongst hospitalized patients and is associated with significant morbidity and mortality POCUS for DVT has been utilized by non-radiologist physicians and non-vascular technicians and validated in various settings including the emergency department, intensive care unit and hospital floor Our curriculum consisted of a half-hour didactic session followed by a half-hour session of facilitated scanning on inpatients who were identified prior to the session. The didactic session included a review of ultrasound basics as well as a detailed overview of the POCUS for DVT protocol including video clips of negative and positive scans (Supplementary File S1). Beyond describing the protocol, specific attention was focused on proper identification of vascular anatomy, potential DVT mimics, as well as limitations of POCUS for DVT. The second half of the session focused on facilitated scanning of inpatients who were identified and amenable to practice scans prior to the session. These patients had lower extremity duplex ultrasound exams ordered or already completed prior to the educational session to allow for post scanning correlation of POCUS findings. We found that that a facilitator could be paired with approximately five or six interns at the bedside during a half-hour scanning session, which allowed all learners a chance to practice compression ultrasonography. Appropriate facilitators included credentialed faculty or senior residents with prior experience with POCUS for DVT. Facilitators were provided with a validated checklist of procedural steps to observe and evaluate interns\u2019 ability to correctly identify vascular anatomy (Supplementary File S2). While we did not utilize the checklist beyond serving as a tool to ensure immediate training efficacy regarding skill acquisition, it may serve as a useful instrument to validate this or similar curriculum and longitudinally determine if learners are retaining material appropriately. Time constraints within the resident didactic schedule are a remaining barrier to incorporating POCUS for DVT. This one-hour introductory curriculum provides a time and resource effective approach to teach the three-point compression protocol to residents. We believe that this model can be easily incorporated into didactic schedules of most residency programs. Further study is needed to determine whether this curriculum is effective for achieving competency in the POCUS exam for DVT detection and longitudinal skill retention. None.Supplementary File S1Supplementary File S2"} +{"text": "The identification and manipulation of spatially identified neuronal ensembles with optical methods have been recently used to prove the causal link between neuronal ensemble activity and learned behaviors. However, the standardization of a conceptual framework to identify and manipulate neuronal ensembles from calcium imaging recordings is still lacking.We propose a conceptual framework for the identification and manipulation of neuronal ensembles using simultaneous calcium imaging and two-photon optogenetics in behaving mice.We review the computational approaches that have been used to identify and manipulate neuronal ensembles with single cell resolution during behavior in different brain regions using all-optical methods.We proposed three steps as a conceptual framework that could be applied to calcium imaging recordings to identify and manipulate neuronal ensembles in behaving mice: (1)\u00a0transformation of calcium transients into binary arrays; (2)\u00a0identification of neuronal ensembles as similar population vectors; and (3)\u00a0targeting of neuronal ensemble members that significantly impact behavioral performance.The use of simultaneous two-photon calcium imaging and two-photon optogenetics allowed for the experimental demonstration of the causal relation of population activity and learned behaviors. The standardization of analytical tools to identify and manipulate neuronal ensembles could accelerate interventional experiments aiming to reprogram the brain in normal and pathological conditions. The main advantage for interventional experiments is the high spatial resolution,,Previous experiments have demonstrated that the activation of a single neuron rather than a group of neurons could evoke some behavioral readout,In this review, we propose three steps as a conceptual framework for interventional experiments during behavior: (1)\u00a0transformation of calcium transients into binary arrays; (2)\u00a0identification of neuronal ensembles as similar population vectors; and (3)\u00a0targeting of neuronal ensemble members that significantly impact behavioral performance .The goal of this review is to propose a conceptual framework for the identification and manipulation of neuronal ensembles related to learned behaviors using simultaneous two-photon calcium imaging and two-photon optogenetics. In the next sections, we describe the main steps of this conceptual framework and the restrictions and considerations to identify and manipulate neuronal ensembles from calcium imaging recordings during behavior.2,,,\u2013in vivo experiments stills represents an unavoidable limitation to design interventional experiments in behaving mice with single spike synchrony. Recently, it has been demonstrated that spike inference from calcium transients compared with electrical recordings produced different interpretations of population analyses and individual neuron properties, highlighting that spike inference should be cautiously considered at least for interventional experiments in behaving mice.,,,,in vivo in the same experimental conditions in which interventional experiments are performed. A high threshold could originate sparse population activity requiring more trials for the identification of neuronal ensembles, whereas a low threshold could make ensemble identification challenging due to spurious correlations. The main advantages of the binary representation of population activity proposed here are the reduction of processing times,,Even though last generation genetically encoded calcium indicators can report a single action potential3,,,,,,The bursting activity of recorded neurons could be visualized as a binary matrix of size ,,,A simple but robust visualization of the similarity between population vectors is the implementation of similarity maps.,,The goal of neuronal ensemble identification for interventional experiments is to find groups of neurons with coordinated activity that repeat at different times and that have a causal relation to learned behaviors.\u2013,,,,\u2013Recently, different algorithms from calcium imaging recordings have been used to study population activity in mice4Interventional experiments have used two different approaches to recall neuronal ensembles related to learned behaviors.On the one hand, a probabilistic graphical model was used to identify neurons with pattern completion capability that can recall neuronal ensembles associated with the correct performance of the learned behavior.\u2013On the other hand, a different approach was the selection of targeted neurons based on the averaged activity of all of the recorded neurons in the trials related to the learned task.Both approaches relied on the identification of specific groups of neurons from calcium imaging recordings and the activation of selected neurons using two-photon optogenetics. These studies suggest that the optogenetic activation of a handful group of neurons is sufficient for triggering widespread neuronal ensembles that can modulate behavioral performance.5\u2013The causal relation between neuronal ensembles and learned behaviors has been demonstrated recently in different brain areas.\u2013The conceptual framework to identify and manipulate neuronal ensembles proposed in this review was implemented for the analysis of calcium transients that represent bursting activity disregarding spike rates. It has been extensively shown that spike rates from electrophysiological recordings provide detailed information of brain computations. However, the technology to control single action potentials in many neurons simultaneously is still under development, so interventional experiments until now have not used single spike synchronization to drive learned behaviors.,,Analyses on raw calcium transients to identify neuronal ensembles related to behavior lack biological interpretability because correlations on raw calcium transients introduce artifacts due to the slow decaying phase of calcium fluorescence.,,It is important to highlight that neuronal ensemble analyses related to behavior should be validated by controlled experimental conditions; in other words, for \u201cx\u201d controlled experimental conditions, there should be at least \u201cx\u201d neuronal ensembles that match accordingly each experimental condition.,On the other hand, performing independent analyses on the same datasets could be useful for validating the identification of neuronal ensembles. For example, after the identification of neuronal ensembles using any method on population vectors, the correlation between the neurons that belong to a defined neuronal ensemble could be used to corroborate that the neurons identified as an ensemble indeed have coordinated activity,,,,,,It is worth mentioning that two-photon optogenetics can produce spurious activation of nontargeted neurons within a radius of Despite that several laboratories can record and manipulate neuronal populations simultaneously, the approaches to identifying which neurons should be targeted are heterogeneous among research groups. The conceptual framework proposed here for the identification and manipulation of neuronal ensembles in behaving mice could provide a first step to standardizing metrics across laboratories and experimental conditions.,,The next generation of interventional experiments should consider not only the recalling of ensembles at behavioral time scales,,,Finally, it has been proposed that the alteration of neuronal ensembles could be related to movement deficits,"} +{"text": "Technology is transforming how older adults find health-related support and get connected with others who cope with similar healthproblems. This study examined the relationship between virtual peer health support and alternative appraisals of illness experience in old age among people who use the Internet for cancer related support . Regression models with interaction terms examined whether and to what extent virtual peer health support was associated with differential illness appraisal as well as and post-diagnosis self-reappraisal. Results demonstrated that a) perceived benefits of virtual peer health support is a significant predictor of self-reported self-reappraisal b) appraisals of illness experience as a traumatic event and/or as a life challenge are both significantly associated with virtual peer health support , and c) appraisal of illness experience as a personal growth growth opportunity is not significantly associated with virtual peer health support . These results suggest the statistical interactions of the virtual peer health support with illness appraisals are significant predictors of self-reappraisal post-diagnosis. Specifically, those who appraised cancer to have been a traumatic experience and/or life challenge for them perceived virtual peer support to be positively influential on their self-perception after cancer diagnosis, which is in contrast to those who perceived cancer experience as an opportunity for personal growth and thus possibly not deriving further significant additional benefits from virtual peer support."} +{"text": "Given comparable fracture torque and the similarity in fracture patterns for fracture trials using composite samples versus cadaveric femurs, the use of composite femur models may be a reasonable choice for postoperative periprosthetic femoral fracture studies within certain limitations.Postoperative periprosthetic femoral fracture following hip replacement has been the subject of many varied experimental approaches. Cadaveric samples offer realistic fit and fracture patterns but are subject to large between-sample variation. Composite femurs have not yet been validated for this purpose. We compared the results of composite femurs to cadaveric femurs using an established methodology. In vitro postoperative periprosthetic fracture results using axial-rotational loading were compared between 12 composite femurs and nine fresh frozen femurs, which were implanted with an otherwise identical collarless (6 composite vs 4 cadaveric) or collared (6 composite vs 5 cadaveric) cementless femoral stem using identical methodology. Fracture torque and rotational displacement were measured and torsional stiffness and rotational work prior to fracture were estimated. Fractures patterns were graded according to the Unified Classification System. Fracture torque, displacement, torsional stiffness and fracture patterns for cadaveric and composite femurs were similar between groups. There was a trend for a greater rotational displacement in the cadaveric groups, which lead to a decrease in torsional stiffness and a significantly greater rotational work prior to fracture for all cadaveric specimens (collarless stems: 10.51 [9.71 to 12.57] vs 5.21 [4.25 to 6.04], Studies use a range of methodology with the choice of specimen largely between composite femur1013 and cadaveric specimens.15 The majority of studies, regardless of specimen choice use fracture loads at the moment of fracture as the primary outcome.The risk of PFF is strongly associated with a range of cementless stem implant characteristics6 but this limits the experiment to the testing of one variable per pair of femurs. Composite femurs are a valid and highly uniform specimen choice when comparing whole bone composite femurs to whole bone cadaveric specimens17and do not require ethical approval or specialised handling and storage techniques to use effectively. However, validation has largely focused on the mechanical properties of the complete femur and validation to date relies on a single study comparing composite femur models to a single cadaveric femur trial.Cadaveric samples can offer realistic implant fit, loading and fracture patterns but these benefits are offset by the large between-sample variation which occurs when testing groups of cadaveric specimens. Variability can be overcome using pairwise comparison of results in bilateral femur pairs,In this study we aimed to compare in-vitro results of PFF simulated methods using composite femur specimens to results using cadaveric specimens using identical loading protocols.To assess the validity of a composite femur model, results from tests using fresh frozen femur specimens were compared to results from tests using an osteoporotic femur composite model. Specimens from the fresh frozen cadaver trials have been published previously as part of a separate study.This study was performed in accordance with local ethical guidelines and regulations of Hamburg University School of Medicine and the University of Leeds. Fresh frozen human female femora were dissected within 48-hours post-mortem, frozen at \u221220\u00b0C (2 freeze-thaw cycles per specimen), defrosted overnight before biomechanical testing and kept wet with saline solution and impermeable covers. and specimens were stripped of soft tissue and underwent computer tomography scanning to screen for pre-existing bony pathology which would bias results and to estimate bone mineral density.Preparation was been previously described\u2018Osteoporotic femur\u2019 models are intended to mimic the specific biomechanical properties of a real osteoporotic femur and were selected since they were likely to more closely match those in the cadaveric testing group. Pre-operative implant size selection and neck cut to recreate preoperative offset and leg length was planned using proprietary software following plain anteroposterior radiographs with a 25\u2009mm diameter scaling ball. Preparation and implantation was performed according to manufacturer\u2019s guidance by a single experienced surgeon (JL) to minimize variability.Composite femurs contained 10 PCF low-density cancellous, thin walled low-density cortical shell, overall length 45.5\u2009cm, and 16\u2009mm hollow canal.After preparation, distal femoral resection was performed so that 40\u2009mm of specimen remained between the stem tip and the distal fixative. Specimens were fixed into steel pots using a rapid setting resin fixative in an identical alignment to those in the cadaveric group . Femurs were implanted with an appropriately sized fully coated cementless femoral stem with and without a medial calcar collar in accordance with manufacturer guidelines and underwent visual inspection (composite femurs) or CT (cadaveric specimens) to screen for intraoperative fractures. and details of this adaptation have been previously published. In the adapted method the femoral stem had a 36\u2009mm femoral head attached to the trunnion which was then held in a custom clamp . In composite femur trials rotation was applied directly to the femoral head using a custom clamp that additionally ensured that the rotation axes was aligned to the femoral axes. and each trial was recorded to establish fracture mechanism using video camera equipment .Fracture torque and rotational displacement were measured and torsional stiffness (rotary displacement divided by torque) and rotational work prior to fracture were estimated (area under rotatory displacement torque curve). Fractures were classified according to the unified classification system (UCS)p\u2009<\u20090.05. Comparisons were stratified by implant collar, since this has been shown to affect the mechanical properties prior to fracture.6Data were tested for normality using a Shapiro-Wilks test and comparisons between cadaveric and composite femur groups were conducted using a Mann-Whitney U test, with significance set at p range 1.0 to 0.1) and a significantly greater rotational work prior to fracture in cadaveric versus composite femurs .The baseline demographics for cadaveric femur donors are shown in Fracture resulted in UCS B2 fractures in all trials. Subjective assessment of fracture pattern demonstrated similar patterns of fracture in cadaveric and composite femur testing and 3, aThis study has demonstrated comparable fracture torque and fracture patterns between composite femur and cadaveric femur trials. Rotational work in cadaveric femurs was greater than that recorded in composite femurs with the same loading regimen. This was largely because cadaveric femur trials fractured at a greater median rotational displacement but this difference did not reach statistical significance. with some important differences. Whilst the fracture torque was comparable between composite and human femurs the composite femurs appear to be stiffer than cadaveric counterparts and fracture occurred at smaller angular displacements in comparison to cadaveric specimens. Whole human femur stiffness follows a rate dependent relationship, with strength and stiffness increasing with loading rate. The stiffness of whole composite femurs has been found to be constant over a range of loading rates and comparable to human specimens in torsional and axial loading. When the stem is placed under axial load, the stem can move independently of the femur under high loading rates6 and the implant-femur construct stiffness is dependent on the mechanical properties of the stem, the stem-bone interface and the bone. The internal foam of the composite femur is homogenous and does not represent the variation seen in mechanical strength and mineral density within human femurs. This may make the stiffness of foam adjacent to the stem greater than that which is seen in human specimens and reduce relative stem-femur displacement under high load conditions. In addition the coefficient of friction between a stem and artificial bone is dissimilar to human trabecular bone and may lead to differing results when loads are transferred across the stem-bone interface. During preparation of the cadaveric femurs it was noted that the foam did not behave in a similar way to normal trabecular bone. The Corail hip system uses an impaction broaching technique to prepare the femur for implantation. The broaching technique was not easy to replicate in the composite femur and the foam did not appear to compress against the broach in a similar way. In addition, foam particles which are broached tended to fall into the void in the central portion of the composite femur specimen. Absence of a compressed trabecular layer in the composite femur is likely to change the stem-bone interface mechanics and may account for some differences in rotational stiffness seen in this study.This study confirms that the results obtained with composite femur specimens are largely comparable to those results from testing using cadaveric femur trials in this study and elsewhere using similar methods,26 unrealistic patterns and also unrealistic stability when the mechanical properties of \u2018fixed\u2019 composite femur fractures are tested. In this study, the pattern of fracture between composite and human femurs in an axial loading model was very similar and in agreement with a small study including just one cadaveric trial. This would suggest that the failure mechanism is similar between composite and human femurs during axial loaded PFF simulations.Previous studies assessing neck of femur fracture patterns in composite femur models have found fracture patterns which are both consistent with cadaveric and embalmed femursFor researchers hoping to use similar composite femur specimens mentioned in this study it is worth commenting on the practical constraints experienced by the authors. The Osteoporotic composite femur specimens had a very thin cortical shell which in comparison to the cancellous foam were incredibly fragile and in future work, care should be taken during manipulation, preparation and implantation of the femur since inadvertent fracture is much more likely than standard composite femurs which represent adult anatomy. The small sample sizes reduce the power of our comparisons and further work should seek to validate composite femur models with larger sample sizes. This study utilised two different fixatives, which may have contributed to the variation between experiments. Although the loading methodology has been used widely in previous work, further work should seek to improve loading conditions to maximise repeatability and validity to typical periprosthetic fractures. We were not able to quantify relative motion between stem and femur, which would have enabled interesting comparison of implant behaviour during rotational loading. Future work should seek to integrate methods which allow accurate quantification of implant displacement and foam deformation. This study only compares the results with a torsional loading model. Even though this model has been used previously and produces clinically valid fracture patterns around cementless stems, testing should also look to validate the use of composite femurs with a range of loading methods.The main limitation in this study is small sample sizes in both cadaveric and composite femur groups. Given the precious resource which cadaveric samples represent, this is a common drawback of biomechanical testing. Reassuringly the results of the composite femur tests are also similar to other results, which used a similar, although non-identical methodology.Given the reduced variability of results and comparable fracture torque and the similarity in fracture patterns from the fracture trials using composite samples versus cadaveric femurs, the use of composite femur models is a reasonable choice where maximum fracture torque is the outcome of choice."} +{"text": "Previous studies have disproportionately focused on caregivers\u2019 negative experiences while overlooking the positive aspects of caregiving especially for caregivers of older adults with cognitive impairment. Therefore, we aim to identify how positive aspects of caregiving varied by care recipients\u2019 cognitive status and caregivers\u2019 relation to care recipients . We applied multilevel mixed-effects models on pooled three-wave data from the National Study of Caregiving and the National Health and Aging Trends Study . The findings suggested that dementia and spouse caregivers had worse relationship with their counterparts. Overall, future research needs to study caregiver\u2019s experience integratively and focuses on caregiver\u2019s individual need. Policy makers need to fulfill caregiver\u2019s demands by establishing socially supportive programs."} +{"text": "In the article on computed tomography (CT) volumetrics in liver cirrhosis Romero\u2010Crist\u00f3bal et\u00a0al. investigated the relationship between changes in liver\u2010spleen volume and features of cirrhosis in patients with compensated or decompensated liver cirrhosis who either underwent liver transplantation or partial hepatectomy for hepatocellular carcinoma (HCC).The authors showed in this Spanish cross\u2010sectional single center study that changes in liver volume, liver segmental volume ratio and liver\u2010spleen volume ratio evaluated by CT reflected the course of disease progression through the different stages of liver cirrhosis. Specific changes in volumetrics appeared to be related to compensated cirrhosis, compensated cirrhosis with development of portal hypertension and decompensated liver cirrhosis. Interestingly, these changes were independent of the predictive value of histological level of liver fibrosis. The authors concluded that the observed changes in liver and spleen volumes correlate with the different clinical stages in the course of liver disease progression. In turn, this would suggest that CT volumetrics of liver and spleen could be a readily available non\u2010invasive tool to provide prognostic information in cirrhotic patients.These findings are of utmost importance, as one of the most essential challenges clinicians face in guiding treatment of patients with liver cirrhosis is stratifying the risk of disease progression, the development of decompensating events or even outcomes in hepatocellular carcinoma.Central, and current gold standard, in the risk stratification of patients with liver cirrhosis is estimating hepatic venous pressure gradient (HVPG) with HVPG measurement.The strategy of volumetrics would provide an alternative to transient elastography in risk stratification in cirrhotic patients, without the risk of hemodynamic alternations being responsible for negatively influencing its predictive capacity. These techniques could however be implemented complementary and the overall diagnostic accuracy might improve using these techniques simultaneously.Interestingly, volumetrics appear to be able to classify the stage of liver disease independent of the level of liver fibrosis as there was no association observed between volumetrics and intensity of fibrosis in this cohort of cirrhotic patients that underwent liver transplant or partial hepatectomy. This would suggest that fibrosis and parenchymal extinction are two independent factors affecting disease course, which in turn could explain that some patients with less severe fibrosis (but still F4) with intense parenchymal extinction may present a decrease of liver volume. As a result of the effect of volumetric changes independent of the predictive value of fibrosis stage, patients at risk of decompensation could potentially be more effectively identified additively using volumetrics.Future study prospectively comparing different non\u2010invasive techniques should determine which tools should be used when in order to provide a patient\u2010tailored risk assessment."} +{"text": "Though aging in place is a stated goal for many older adults, complications of advanced disease or disability can derail these plans. Little is known about the current preparedness of older adults to age in place. The January 2022 National Poll on Healthy Aging surveyed adults age 50-80 about their home and available supports. Findings indicate that while 80% of adults believe they have the necessary features in their home to support aging in place, less than one in five had homes that were wheelchair accessible and fewer than half had other common safety and accessibility features. Older adults were most confident they could obtain help with household chores (51%) and were least confident about getting assistance with personal care (33%). Persons with disabilities (n=683) and those living alone (n=639) reported the most concerns. Several opportunities exist to better prepare older adults to successfully age in place."} +{"text": "Psychological work with cognitive beliefs were shown to be beneficial for patients with somatoform disorders and unexplained somatic complaints . There is still a question of whether these results are specific or common for different kind of interventions including psychoanalytic psychotherapy .The aim was to reveal dynamics of illness perception after group analysis psychotherapy comparing to psychoeducation in patients with somatoform disorders.100 patients with somatoform disorders were randomized to psychoeducation intervention and to the group analysis psychotherapy . Before and after treatment they filled Screening for somatoforms symptoms and Illness Perception Questionnaire - Revised .2 (Groups) \u00d7 2 (Time: Before / After) ANOVA with repeated measures revealed major effect of time with both groups demonstrated equal decrease in somatoform symptoms during treatment . Patients from both groups after treatment appraised their illnesses as having shorter duration without cycles, less severe consequences on their lives, reported increase in treatment control, understanding of their illness and decrease in emotional reactions . In group analysis condition only patients demonstrated increased beliefs that psychological and risk factors could impact their illness .Patients with somatoform disorders almost equally benefitted from both psychoeducation and group analysis but group analysis psychotherapy led to better awareness of psychological and risk factors of their illness."} +{"text": "Botulism is a rare illness caused by Clostridium botulinum toxin with a na\u00efve case fatality ratio of 40-50%. There is no coordinated collective worldwide reporting on cases and comparatively few recommendations on case management. This study examined 14 European botulism treatment guidelines.A ten-language search was conducted to examine European botulism guidelines. The guidelines were classified by differential diagnosis advice; expert advice access; mention of causalities; contract tracing; biological sampling method; and treatment access rapidity. The guidelines were linearly represented on a probability pathway. Quantified probabilities were entered into the algorithm. Probabilities for algorithmic delay or deviance were estimated or mathematically modeled against Hamiltonian, Ford- Fulkerson and Kruskal pathways. Case recognition was deemed proportional to the availability of information at point of care and produced a hazard function related to a Bayes\u2019 probability model.Two guidelines did not display all diagnostic information in one place, and six European nations had incomplete descriptions of the chain of causality linking botulism cases: factorially reducing the Borel algorithmic likelihood of diagnosis through contact tracing and decreasing the affectable survival chance. Another limitation was specialist advice and treatment availability in a 48-hour window. Survival probability models to the quoted na\u00efve minimum constraint of a 60% survival factor were depicted, with pharmacokinetic tendential to an exponential decay model. This highlighted the importance of well-constructed case management and logistical stockpiling methods.In botulism poisoning the 48-hour window is cited as crucial to patient survival chances, to this extent, the availability of clear diagnostic criteria including causation considerations, expert advice access and logistically considered therapeutic stockpiles could improve survival probability.\u2022\u2002An international standard for botulism guidance may further improve botulism case identification and survival rates.\u2022\u2002National botulism guidelines with direct contact method to an expert and with strategic positioning of therapeutic stockpiles may reduce time to treatment and improve survival chances."} +{"text": "Plant domestication can be viewed as a form of co\u2010evolved interspecific mutualism between humans and crops for the benefit of the two partners. Here, we ask how this plant\u2013human mutualism has, in turn, impacted beneficial interactions within crop species, between crop species, and between crops and their associated microbial partners. We focus on beneficial interactions resulting from three main mechanisms that can be promoted by manipulating genetic diversity in agrosystems: niche partitioning, facilitation, and kin selection. We show that a combination of factors has impacted either directly or indirectly plant\u2013plant interactions during domestication and breeding, with a trend toward reduced benefits arising from niche partitioning and facilitation. Such factors include marked decrease of molecular and functional diversity of crops and other organisms present in the agroecosystem, mass selection, and increased use of chemical inputs. For example, the latter has likely contributed to the relaxation of selection pressures on nutrient\u2010mobilizing traits such as those associated to root exudation and plant nutrient exchanges via microbial partners. In contrast, we show that beneficial interactions arising from kin selection have likely been promoted since the advent of modern breeding. We highlight several issues that need further investigation such as whether crop phenotypic plasticity has evolved and could trigger beneficial interactions in crops, and whether human\u2010mediated selection has impacted cooperation via kin recognition. Finally, we discuss how plant breeding and agricultural practices can help promoting beneficial interactions within and between species in the context of agroecology where the mobilization of diversity and complexity of crop interactions is viewed as a keystone of agroecosystem sustainability. Spatial complementarity has been described in trees where canopy stratification\u2014significant height difference\u2014in mixtures of Eucalyptus globulus and Acacia mearnsii reduces competition for light in comparison with monocultures, as shown by an increase of diameter growth and above\u2010ground biomass in mixtures compared to sole\u2010stands , from differences in plant development such as phenology and therefore timing in resource use , and from differences in plant resource needs uptake. Such specialization reduces the opportunities for complementarity between varieties using different forms of nitrogen (Cantarel et al., , Violle, et al., The strong loss of genetic and phenotypic diversity that crops experienced during domestication and breeding (Glemin & Bataillon, TB1 gene introgressed in a modern maize background confers greater phenotypic plasticity and responsiveness to light than the maize allele (Lukens & Doebley, Complementarity can arise from phenotypic plasticity when co\u2010occurrence of species and genotypes promote trait differentiation that leads to stable coexistence (Turcotte & Levine, 3Facilitative interactions describe the effect of a species on its local environment in such a way that it improves the growth and development of other species (Callaway, , George, Dupuy, et al., 3+ in the rhizosphere when there is Fe deficiency, enhancing Fe availability for other intercropped species (Dai et al., Beneficial interactions through direct facilitation can occur in crops when one component of the mixture provides a physical support to the others, reducing the risk of lodging. This has been demonstrated in variety of mixtures in barley (Creissen et al., Aegilops accessions than in Triticum aestivum cultivars (Neelam et al., Indirect evidence suggests that human selection might have shaped belowground facilitative interactions among plants Figure . For ins42 fixation (Li et al., Many crops benefit from positive interactions with soil microbes that can indirectly trigger synergies within and among plant species. For instance, legumes benefit from the capacity of fixing atmospheric N through symbiosis with a group of soil bacteria collectively called rhizobia. The association of non\u2010N\u2010fixing crops with legumes triggers facilitation processes resulting in increased N availability for the former via rhizobial N fixation in the latter. For instance, root exudates from maize promotes faba bean nodulation and N2\u2010fixing bacteria (Kiers et al., Responsiveness of crops to their micro\u2010organism partners may have been affected by agronomic practices and/or selection on domesticated traits Figure . Indeed,Studies documenting plant interactions with fungi indicate that domestication has impacted more strongly the composition of fungal communities than bacterial ones (Leff et al., 5Increase in frequency over generations of competitive phenotypes is a frequent outcome of natural selection within populations. Yet, groups of competitive phenotypes of the same species may have a low productivity due to stronger competitive interactions among plants and higher investment in resource harvesting at the expense of seed production. Such a negative correlation between individual competitiveness and seed production of the group is a classic prediction of evolutionary game theory (see Anten and Vermeulen for a reThe very few studies investigating phenotypic variation on plant height along a domestication gradient suggest that tall competitive phenotypes have first increased in frequency in emerging crop species Figure . Such tePromoting high relatedness requires dedicated selection schemes that are unlikely to have been mobilized before the onset of pedigree selection at the end of the 19th century Allard, . The pedCooperation arising from kin selection can be facilitated by the existence of kin recognition that allows cooperative behaviors to be directed preferentially toward kin (Hamilton, 6Modern agriculture faces the need to maintain high quantity and quality production in a context of increasing food demand, while reducing the environmental costs due to massive use of chemical inputs Altieri, . PromotiPromoting beneficial interactions in agrosystems will be facilitated by the identification of the traits that underlie them. This has been a central focus in ecology (Navas & Violle, First, we still know very little about both above and belowground traits involved in beneficial interactions within and among species. In particular, root traits that have been largely neglected in breeding programs might offer new opportunities to develop more beneficial crops.2 fixation in the former and soil N acquisition in the latter, a complementarity effect that disappears with the application of N fertilizers (Rodriguez et al., Second, mechanisms underlying beneficial interactions are likely to be dependent on environmental conditions. Indeed, we might expect niche partitioning and facilitation to play a stronger role in more limiting conditions, as described in the framework of the Stress Gradient Hypothesis\u2014SGH (Maestre et al., Third, traits involved in plant\u2010resource acquisition, and therefore potentially important for complementarity and facilitation, can display trade\u2010offs between them. For example, root diameter correlates positively with the amounts of carboxylates and phosphatase activity in the rhizosheath as well as with AMF colonization across 16 crop species in limiting soil P (Wen et al., Fourth, our current understanding on plant\u2013plant interactions mediated by microorganisms is still very limited. A recent study suggests that maize plants trigger AM fungal colonization in mutant neighboring plants displaying deficiency in the mycorrhizal Pi uptake pathway, through nutrient delivery to the CMN (Fabianska et al., Finally, it is likely that favorable trait combinations will differ depending on the objectives targeted by farmers. For instance, the traits affecting durum wheat mixture performance were different when considering yield or grain quality (Montazeaud, Violle, et al., Niche partitioning, facilitation, and kin selection all depend on genetic similarity among interacting plants and can thus be promoted by manipulating genetic diversity. Yet, whereas niche complementarity and facilitation are promoted by genetic dissimilarity among plants, kin selection relies on high genetic similarity among interacting plants and often leads to monomorphic stands with only one cooperative phenotype maximizing group productivity (Montazeaud, Rousset, et al., Such idea fits with the many ecological and agronomic studies showing that phenotypic diversity can have both positive and negative effects depending on the trait. For instance, the relative performance of durum wheat genotype mixtures was positively impacted by between\u2010genotypes differences in seminal root branching intensity, but negatively impacted by differences in tiller number (Montazeaud, Violle, et al., Promoting beneficial interactions arising from niche partitioning and facilitation within and between species calls for extending the genetic and functional diversity currently used in agrosystems. The evolutionary history of crops has been characterized by recurrent bottlenecks and directional selection on genes that determine desirable phenotypes, leading to massive loss of genetic diversity (Gaut et al., Arabidopsis thaliana has been shown to promote stand\u2010level productivity, through an effect on plant\u2010soil interactions (Wuest & Niklaus, An alternative approach to promote beneficial interactions is the use of trait\u2010blind approach. This has been the rationale of the \u201cmixing ability\u201d approach, where mixing ability of the mixture components are estimated based on their observed performances, and used to assemble relevant components (see Barot et al. for addiThe authors declare no conflict of interest."} +{"text": "Subjective memory impairment, defined as self-reported difficulties in recall and learning, doubles the risk of Alzheimer\u2019s Disease and related dementia, despite being weakly related to objective memory decline. Because of its strong stability over time, it may be possible that subjective memory impairment reflects earlier life risk factors for dementia such as adverse childhood experiences. It is reported that over a fifth of older adults worldwide experienced physical abuse during childhood. Previous cross-sectional studies suggest physical abuse is associated with later cognitive impairment. Still unclear, are the longitudinal associations between childhood abuse and subjective memory impairment in later life. Using a sample of adults drawn from the Health and Retirement Study we assessed associations between reported physical abuse by a parent before the age of 18 and subjective memory impairment (current memory problems and perceived memory decline) over periods of up to 18 years. Generalized linear mixed models examined longitudinal associations between childhood physical abuse and subjective memory impairment while controlling for depressive symptoms and other empirically relevant covariates. Experiencing childhood physical abuse was associated with increased likelihood of reporting more current memory problems and perceived memory decline in later life . Findings suggest childhood physical abuse is associated with subjective memory impairment, a strong predictor of dementia. Understanding early life conditions, including adverse childhood experiences may help explain associations between subjective memory impairment and dementia risk."} +{"text": "Chronic pain has been dubbed a place where mental and physical health meet. The emotional and social needs of young people experiencing Chronic Pain can be vast, yet families often fall through cracks in services. Other families receive a plethora of support options from psychological practitioners, allied health professionals and social care, but with laser focus on the medical answer and fix decline all potential referrals.Historically psychology received numerous referrals for young people who declined to meet with us, leaving our colleagues and families feeling unheard, frustrated stuck and alone. How to support families/colleagues without seeing children individually?Ed experiencing paralysis and pain in dominant arm and hand:\u2022 extensive investigations and specialist consultations sought, including scans and neurological opinion - nothing medically concerning identified.\u2022 Previous referral to another tertiary psychology service made and group therapy offered.\u2022 Over the course of the investigations reports of pain and A&E attendance increased, whilst school attendance decreased -> referred to chronic pain service.Input offered at Evelina London:\u2022 2 x Multidisciplinary clinic (chronic pain diagnosis given at first appointment).\u2022 1 x Pain education workshop.\u2022 Multiple physiotherapy and occupational therapy appointments.\u2022 3 x referrals to psychology over preceding 9 months with concerns about mood and engagement.Progress:Initial gains in first months\u2022 Pain and paralysis reduced to dominant hand only.\u2022 Strength and range of motion restored to shoulder and arm.\u2022 School attendance increased.However, in recent months A&E attendances and reports of pain were increasing, with service engagement decreasing.Requests for individual psychology for Ed had been made by various team members, but Ed had not taken up offered psychology conversations or appointments. This contributed to expressions of frustration, worry and hopelessness in the team who wanted individual psychology for Ed \u201cto move things forward\u201d.3rd Multidisciplinary Clinic appointment:With all therapists and doctors in attendance the psychologist took time to explore Ed and his mother's daily lives outside pain, their interests, joys and skills. They explored the family's explanations for pain, experience of services so far, roles they felt investigations, doctors, occupational therapy, physiotherapy and psychology had, could and should play.Amongst other things particular emphasis placed on:\u2022 Privileging family's views.\u2022 Using patient's language.\u2022 Regular summarising.\u2022 Placing professionals then family members in listening positions and exploring what each had heard the other say.The psychologist endorsed Ed's view that psychology had no role to play in his care.Was Ed in desperate need of 1-1 psychology to move forward?For this family the psychological intervention was with both family and healthcare system. The psychologist used a narrative approach, positioned as a curious \"outsider\", with the patient as an expert in their own care. Beginning with a foundation of identity outside pain, followed by facilitated witnessing of ideas and experiences for family and healthcare professionals. Thus, new insights, a shared understanding and plan could develop.With this approach it emerged referrals to psychology coupled with no simple medical explanation and treatment fed fears professionals thought \"pain was all in Ed's head\". This shook confidence in the healthcare team and reinforced messages the family experienced family, school and friends as giving. Around this time Ed's progress stalled.Whilst family described previous negative experiences of psychologists, current physiotherapy was described as developing \"strength and mobility\", occupational therapy as \"really understanding\" and giving \"useful exercises/advice\", cheerleading throughout a difficult journey. Doctors offered (another) detailed review of investigations and physical examination reiterating pain, but not damage were present. The psychologist thoroughly explored and all things psychology might add, none of which the family felt were useful. This choice was thoroughly validated and respected.During the appointment \"thinking break\" (family not present) the team were surprised by the family's positive interpretations of care offered thus far. It was hypothesised not feeling believed/validated was a very difficult place to \"recover\" from and ideas generated of what validation/being believed would look like for this family. Given the family's vocalised block to progress was \"the hand\", perhaps is was a hand specialist rather than a psychologist?The family were pleased to see joint occupational and hand therapy resulting in \"full recovery\". Throughout the professionals were offered regular psychological consultation and team discussion time.There are many ways to peel the chronic pain orange.\u2003When faced with often overwhelming needs of young people living with chronic pain and sometimes overwhelming needs of referring health and social care systems it can be hard to hold onto this idea. Initially referrers thought individual psychology was indicated, what was offered and created change was a psychologically informed multidisciplinary appointment in which the family's worries were given space, validated and answered, whilst their right to decline psychological support was validated and bolstered. Alongside ongoing psychological consultation to professionals.Pain is all in my head, no-one believes me, no-one understands.\u2003Are powerful ideas that can be detrimental to family-healthcare relationships, whether stated directly, implied or internally voiced. They can represent a significant barrier to families accessing psychology directly.We may wholeheartedly believe people experience pain that cannot reliably be seen or measured externally, but that does not mean we are experienced that way. For me this case highlighted the importance of\u2022 checking out beliefs about symptoms.\u2022\u2009gauging whether ongoing/regular validation and reassurance is needed and in what form.\u2022 even celebrating someone's successes in the face of pain even suggesting someone can move on with their life can be experienced as invalidating/not getting it. Families need a voice in their won paths forward.Is what you thought you said what they thought you meant?\u2003Whether with families or professional you can never underestimate the value of checking whether the same thing was heard differently.This is written from the position of a psychologist embedded within a predominantly non-pharmacological multidisciplinary chronic pain service, which informed the intervention. Other cases have invited/necessitated different approaches. It would be interesting and helpful to invite discussion around the variety of ways similar/different road blocks have been bypassed, removed or otherwise."} +{"text": "Patient portals are made available and widely promoted in healthcare systems in the USA and Europe. These technologies can help patients access healthcare, receive timely treatment, and manage their health through services such as appointment booking and repeat prescription ordering. However, it is not clear if all patients who need the services are using them. This study explored patient portal use (online appointment booking and repeat prescription ordering features) and patient characteristics among NHS England GP practice patients.The study used cross-sectional participant-level data from the GP Patient Survey (GPPS) of 2018, 2019, and 2020. Performing multilevel regression analysis, we explored the association between patient portal feature use and ethnicity and deprivation and controlled for eight other patient characteristics and one GP practice level characteristic, and modelled GP practice as a random effect in the model.In the fully adjusted model controlled for all patient characteristics and GP characteristics, participants of the Black and Other ethnic groups were less likely to have used online appointment booking and online repeat prescription ordering compared to the White ethnic group. Association with patient portal use increased proportionally with reduced deprivation ranking.In NHS England GP practices, certain ethnic minority groups and high deprivation ranking is associated with a reduced likelihood of using patient portals. If patient portals are the only route to access services, it is likely to lead to inequalities in use by some patient groups introducing unfair access to the services. Patients could continue to be provided with alternatives to patient portals to prevent potential inequities in access to services.\u2022\u2002Patient portals are widely used in the healthcare system and can benefit all patients given that disparities are prevented by understanding patient groups who cannot access portals.\u2022\u2002Understanding patient groups less likely to use patient portals could help adapt healthcare system services and meet the needs of all patient groups."} +{"text": "Sexual dysfunction is a frequent adverse drug reaction (ADR) of antidepressants that considerably affects quality of life and adherence to therapy. We previously investigated the potential underlying neurofunctional mechanisms by neuroimaging methods and revealed a dampening of the dopaminergic mesolimbic-mesocortical reward system along with decreased sexual functioning under serotonergic antidepressants compared to placebo.Within a combined pharmacoepidemiologic and pharmacodynamic approach, we examined the association between serotonin transporter (SERT) affinity of various antidepressants and corresponding alterations in sexual desire as ADR.Using disproportionality analyses, reporting odds ratios (RORs) were calculated for reports indicating decreased sexual desire as ADR under the antidepressants. The data were extracted from the WHO global database of individual case safety reports VigiBase and several MedDRA terms were grouped for \u201cSexual Desire Disorders\u201d. For the pharmacodynamic assessment, we calculated Pearson correlation coefficients between SERT affinity and corresponding RORs16 signals were detected for \u201cSexual Desire Disorders\u201d. We observed a statistically significant (r (20) =. 65, p = 0.001) association between SERT affinity and decreased sexual desire. Higher SERT affinity was associated with higher risk of sexual desire.While sexual dysfunctions under serotonergic medication were previously described, we now elaborated that in particular attenuated sexual desire as ADR is associated with SERT affinity of the antidepressant. These results strengthen our previously described neurofunctional model regarding sexual dysfunction under antidepressant medication and indicate that the specific SERT affinity of the antidepressant drug should be considered in clinical practice to minimize the risk of this ADR.No significant relationships."} +{"text": "Caregiver burden is well understood as an important contributor to caregiver health. However, little is known about how positive aspects of caregiving and the quality of caregivers\u2019 relationships with care recipients might play a role in caregiver health. The study aimed to examine whether positive caregiving and caregivers\u2019 relationship with care recipients were associated with caregiver mental health (depression and anxiety) and perceived general health. The sample consisted of 2,652 family caregivers in the National Study of Caregiving (NSOC) III (2017) providing care to older adults. A series of multiple regression models with covariate adjustments were performed to examine the associations. Results indicated that positive aspects of caregiving predicted caregiver mental health but did not predict perceived general health. Caregivers\u2019 relationship with care recipients and caregiver burden significantly predicted caregiver mental health and perceived general health . After controlling for caregiver burden, only caregivers\u2019 relationship with care recipients remained a significant predictor of caregiver mental health and perceived general health . Our results suggest that positive caregiving perceptions and quality of relationships between caregivers and care recipients are linked to better caregiver mental health. Interventions to reduce caregiver burden, including strategies to help caregivers maintain positive attitudes and positive relationships with care recipients, might be beneficial to improving caregiver health."} +{"text": "Gastroduodenal artery (GDA) aneurysm is a rare vascular condition that is potentially life-threatening. It is associated with significant morbidity and mortality and most often encountered in patients with chronic pancreatitis. Other etiologies including atherosclerosis, peptic ulcer disease, alcohol abuse, cholecystectomy and absence of celiac axis, have been reported. False and true GDA aneurysms account for about 1.5% of all visceral artery aneurysms however are associated with a risk of rupturing and hemorrhage up to 75%. Mainstay of GDA aneurysm management involves aneurysm repair with open surgery with vessel ligation or endovascular repair with coil embolization or stent placement, depending on the patient\u2019s comorbidities and overall hemodynamic stability. The use of percutaneous thrombin injection via computed tomography (CT) and ultrasound has also been described in the literature and is being increasingly used to promote clot formation and pseudoaneurysm occlusion as a less invasive option.To present a case of endoscopic ultrasound guided thrombin injection as an effective and safe management strategy for GDA pseudoaneurysms.At this time, there are limited data in the literature on endoscopic ultrasound guided thrombin injection for pseudoaneurysms. To our knowledge, this is the first case report describing a patient with recurrent gastrointestinal bleeding from a duodenal ulcer, who had undergone multiple endoscopic and angiographic procedures, with subsequent development of a gastroduodenal pseudoaneurysm unrelated to pancreatitis which was successfully managed by occluding the pseudoaneurysm with thrombin injection under endoscopic ultrasound guidance. EUS guided thrombin injection is a viable alternative to traditional interventional radiology guided thrombin injection for management of pseudoaneurysms.In clinic follow-up three weeks after thrombin injection, our patient continued to do clinically well, without abdominal pain or symptoms. Repeat CT angiogram demonstrated the GDA pseudoaneurysm had remained thrombosed, indicating successful endoscopic obliteration. He had no further signs of gastrointestinal bleeding.EUS guided thrombin injection is a novel approach that has been presented as an option in the management of bleeding gastroduodenal pseudoaneurysms, especially when radiology guided or surgical options are higher risk or have previously failed. The paucity of available data highlights the need for ongoing clinical studies comparing the different therapeutic strategies that can effectively and safely manage these rare but life-threatening vascular entities.NoneNone Declared"} +{"text": "Emotional distress increasingly represents a major burden in children and adolescents (C&A), especially in conflict zones where its prevalence is estimated to reach 70%. Resilience training programmes (RTPs) are interventions that seek to enhance resilience in individuals or groups pursuing mental distress prevention. Literature suggests RTPs be particularly effective in C&A; however, their effectiveness and value for public health are still unclear.A scoping review was performed in order to summarize evidence regarding the implementation and effectiveness of RTPs in children and adolescents. A search string has been built according to the PICO model and launched on PubMed, PsycInfo, Academia databases. Additional references were identified by a hand-search in Google Scholar. Studies included were narratively summarized according to topics that emerged.18 articles were finally included in the review. Main issues were 1) RTPs seem to be more effective in adolescents rather than in children; 2) COVID-19 pandemic has raised the attention towards RTPs in C&A; 3) beyond conflict zones their implementation is increasingly recognized in supporting C&A management of daily stressors and traumas also in C&A with disabilities; 4) school is identified as the key setting for RTPs; 5) the high heterogeneity in RTPs approaches, contexts and study samples limits a conclusive effectiveness assessment.Our findings highlighted how RTPs are increasingly recognized as a tool to improve C&A cognitive and behavioral functioning and resilience to external stressors, getting greater interest in the COVID-19 pandemic. Despite relevant theoretical support and promising study results, RTPs still lack strong evidence supporting their embracement by policymakers and effective implementation in public health policy. In order to not miss this chance, more efforts are needed in strengthening RTPs conceptualization and cost-effectiveness studies.\u2022\u2002RTPs are a promising tool to enhance the resilience of children and adolescents gaining increasing interest due to the COVID-19 pandemic.\u2022\u2002More studies are needed to provide a strong evidence base that supports their acknowledgment by policymakers and their implementation in public health policies."} +{"text": "Because cannabinoids are usually amorphous solids, the thought that some of them may also exist in distinctly different crystal polymorphic forms might at first seem unusual. However, this commentary provides compelling evidence and precedent for the likely existence of cannabinoid crystal polymorphism. Cannabis have been characterized by numerous spectroscopic tools over many decades seemingly \u201camorphous\u201d solids. Consequently, the proposal that any cannabinoid might exist in multiple crystal forms known as \u201cpolymorphs\u201d would seem very unlikely. However, the purpose of this commentary is to challenge that perception and explore the possibility of cannabinoid crystal polymorphism.The structurally diverse substances comprising the ever-growing collection of cannabinoids found in As mentioned, crystal polymorphism is defined as the existence of one or more distinctly different crystal forms of the same substance and it can manifest itself with both complex and uncomplicated molecules. Whatever the structural details, crystal polymorph creation is controlled by the intricate choreography of both nucleation kinetics and thermodynamic stability. Historically, it is commonly accepted that crystal polymorphism was first discovered for the very simple compound benzamide by the collaboration of noteworthy nineteenth century chemists Friedrich Wohler and Justus von Liebig in 1832. One can only imagine their amazement as they surprisingly observed the fine needles of one benzamide crystal polymorph convert to the rhombic crystals of another polymorph. Almost two centuries later, benzamide crystal polymorphism continues to fascinate current researchers . Two crystal polymorphs (designated \u03b1 and \u03b2) of it were discovered and characterized by X-ray crystallography (Kamlekar and Swamy Interestingly, the cannabinoid receptors have been an important docking location for several structurally diverse compounds which have exhibited crystal polymorphism. The lipid Importantly, the discovery of cannabinoid crystal polymorphs would clearly be of more than mere academic interest. The very real possibility exists that such cannabinoid crystal polymorphs might possess greater stability or possibly improved bioavailability properties. It has been stated (Haleblian and McCrone Cannabis cannabinoids will also exhibit crystal polymorphism.Based on the foregoing evidence, it is reasonable to conclude that at least some"} +{"text": "Roughly a million cases of herpes zoster (HZ) occur annually in US adults, andnearly one third of these patients will experience postherpetic neuralgia (PHN) lastingfor \u22651 month [The initial report on the clinical trial for zoster vaccine indicated that it waspartially protective in adults \u226560 years of age . The plaThe Journal of InfectiousDiseases [The article by Levin et al in this issue of Diseases providesTwo studies of participants in the Shingles Prevention Trial indicated that CMIbut not IgG levels correlated with reduced HZ morbidity \u201311. Alth"} +{"text": "The retraction has been agreed following an investigation based on allegations raised by a third party, which revealed that some images in this article are inappropriate duplications of images from previously published articles [The above article published online on 01 July 2017 in Wiley Online Library (articles , 4. Thus"} +{"text": "Ethnic health disparities exist in the context of pregnancy and childbirth, suggesting that women of Turkish origin in Germany have higher risks for some adverse maternal health and child developmental outcomes. Stress is believed to be a relevant pathway by which migration may be associated with these risks. In this study, we tested associations of Turkish origin with stress biology and psychological stress experiences during pregnancy.140 pregnant women (33 of Turkish/26 of other origin) participated in a prospective cohort study that was carried out in Bielefeld and Berlin . Inflammatory markers CRP and IL-6 from venous blood samples and diurnal cortisol profiles from salivary cortisol samples were derived and participants completed the Perceived Stress Scale (PSS) and Center for Epidemiologic Studies Depression Scale (CESD) at two study visits during pregnancy . Multilevel models were conducted to account for the nested data structure due to repeated measurements.Compared to non-migrant women, women of Turkish origin had significantly higher inflammatory levels , a blunted cortisol awakening response , a flatter diurnal cortisol slope , and higher PSS and CESD scores during pregnancy after adjusting for socioeconomic factors.The results of our study suggest higher stress at the biological and psychological level in pregnant women of Turkish origin. Stress is a risk factor for pregnancy complications and poor birth and child developmental outcomes. To reduce such unequally distributed risks, interventions for stress reduction are needed that are tailored to women of Turkish origin.\u2022\u2002Women of Turkish origin in Germany have elevated psychological and biological stress levels during pregnancy compared to non-migrant women.\u2022\u2002This finding underscores the need for targeted interventions to reduce stress in this high-risk group."} +{"text": "Remote cerebellar hemorrhage as a rare complication of supratentorial surgery was already first described in the 1970s by Yasargil. Its incidence ranges from 0.2% to 0.4% after supratentorial craniotomies. Although its incidence is low, the volume of reports with remote cerebellar hemorrhage in the literature has been growing in recent times. The authors report here a new case of a controlateral remote cerebellar hemorrhage after 24 hours of supratentorial craniotomy for a solitary brain metastasis of a pulmonary adenocarcinoma in a 59 year-old male patient with unbalanced high blood pressure.Supratentorial craniotomy, Remote cerebellar hemorrhage, CT scan Remote cerebellar hemorrhage (RCH) as a rare complication of supratentorial surgery was already first described in the 1970s by Yasargil The authors report here a new case of a controlateral remote cerebellar hemorrhage after 24 hours of supratentorial craniotomy for a solitary brain metastasis of a pulmonary adenocarcinoma in a 59 year-old male patient with unbalanced high blood pressure.A 59-year-old male patient known to have unbalanced high blood pressure with no other particular medical or surgical history was admitted to our department of neurosurgery for progressive onset of raised intracranial pressure made of constrictive headache and bilateral blurred vision associated to recent lower left limb heaviness. The patient as his family denied any prior traumatic or epileptic event. Upon examination, our patient was fully awake and apyretic. His neurocognitive function assessment revealed a slurred speech made of poor pronunciation of words, mumbling, and change in speed during a conversation. The rest of the neurological exam showed a predominantly crural left hemiparesis not exceeding 4/5. An immediate brain magnetic resonance imaging (MRI) showed aThe patient underwent an urgent gross total tumor resection through a frontotemporal craniotomy. Intraoperatively he presented a significant intraoperative systolic arterial hypertension managed by vasodilator drugs, which made it possible to continue excision in complete safety.The postoperative course was fortunately uneventful. The enhanced postoperative control computed tomography (CT) scan performeThe histopathological examination of the specimen concluded to a cerebral infiltration by an adenocarcinoma whose immunohistochemical profile was in favor of pulmonary origin.The patient continued to improve his neurological deficit and was subsequently discharged from our department 5 days later and transferred to the radiotherapy department for further investigation and further management.As noted above, RCH was initially described in surgeries at the supratentorial level Although the physiopathology of RCH is not elucidated, most authors agree on its venous origin, since per- or postoperative CSF loss would play an important role Some risk factors are debated according to authors: peroperative position The treatment could be expectant as in the case of our patient; however, this may require surgical measures such as placement of external CSF shunts and/or posterior fossa decompression Although cerebellar hemorrhage is associated with 8.4% morbidity From a functional point of view, RCH remains a rare complication of supratentorial neurosurgery which must be suspected in case of postoperative complications even when it is a minimally invasive surgery. Diagnosis is confirmed by cerebral imaging and its prevention requires controlled depletion of CSF during surgery and maintenance of normal perioperative blood pressure. Its treatment is often symptomatic, allowing most often favorable outcomes.Written informed consent was obtained from the patient for publication of this case report and any accompanying images."} +{"text": "Developing effective visual analytics systems demands care in characterization of domain problems and integration of visualization techniques and computational models. Urban visual analytics has already achieved remarkable success in tackling urban problems and providing fundamental services for smart cities. To promote further academic research and assist the development of industrial urban analytics systems, we comprehensively review urban visual analytics studies from four perspectives. In particular, we identify 8 urban domains and 22 types of popular visualization, analyze 7 types of computational method, and categorize existing systems into 4 types based on their integration of visualization techniques and computational models. We conclude with potential research directions and opportunities."} +{"text": "GSA\u2019s revised KAER Toolkit for Primary Care Teams is an important resource, yet the complexities of depressive and cognitive symptoms in aging individuals create particular challenges for generalist behavioral health providers. Mental health practitioners and their patients can benefit from evidence-based clinical intervention materials that address the intersection of depression and brain health concerns. This presentation highlights treatment strategies and clinical tools from the new Brain Health module of the revised 2021 client workbook Treating Later-Life Depression: A Cognitive Behavioral Therapy Approach from Oxford University Press. Examples will be presented of large print within-session \u201cLearn pages\u201d that inform both providers and patients about normative cognitive aging and ways in which cognitive functioning can be affected by depression. Between-session \u201cPractice forms\u201d will also be demonstrated that address lifestyle factors to promote brain health, consideration of whether to complete a cognitive evaluation, and strategies to manage brain health changes."} +{"text": "Depression during pregnancy leads to deterioration of the mothers\u2019 and the fetus\u2019 health.To explore women\u2019s perception and attitude towards using antidepressants during pregnancy and identify the factors that influence decision making regarding antidepressants use.A cross-sectional survey of 991 subjects using convenient sampling. All study subjects were invited to fill out an electronic questionnaire, KAAUH staff and PNU female associates who were less than 18 years old were excluded. Answers were reported using 5-point Likert scale. The responses were summed up to give a total score for each respondent. The cutoff point is 75%. Respondents who scored above or equal 75% of the total score was considered as positive perception or favorable attitude.The majority of women had negative perception and favorable attitude towards using antidepressants during pregnancy reaching 64%. While, women with positive perception and favorable attitude represented about 20% of the study subjects. The main factors influencing decision making were, education specialty and subject history of diagnosis with any psychological disorder. Social stigma, religious believes and fear of addiction were reported by surveyors to be the reason influencing their perception and attitude about antidepressants use .This study reveals that although Saudi women reflect a negative perception towards using antidepressants during pregnancy, yet they have a favorable attitude once depression during pregnancy becomes an issue.No significant relationships."} +{"text": "Gene transfer using adeno-associated viral (AAV) vectors has made tremendous progress in the last decade and has achieved cures of debilitating diseases such as hemophilia A and B. Nevertheless, progress is still being hampered by immune responses against the AAV capsid antigens or the transgene products. Immunosuppression designed to blunt T cell responses has shown success in some patients but failed in others especially if they received very high AAV vectors doses. Although it was initially thought that AAV vectors induce only marginal innate responses below the threshold of systemic symptoms recent trials have shown that complement activation can results in serious adverse events. Dorsal root ganglia toxicity has also been identified as a complication of high vector doses as has severe hepatotoxicity. Most of the critical complications occur in patients who are treated with very high vector doses indicating that the use of more efficient AAV vectors to allow for dose sparing or giving smaller doses repeatedly, the latter in conjunction with antibody or B cell depleting measures, should be explored. Gene transfer using vectors based on adeno-associated viruses (AAV) has achieved lessening of symptoms or even cures of diseases caused by single gene defects such as Leber\u2019s congenital amaurosis, a congenital form of blindness , or hemoImmune mediated rejection of AAV vector transduced cells can in part be prevented by immunosuppressants given during or shortly after gene transfer . ImmunosAlthough we have gained over the last two decades a better understanding of innate and adaptive immune responses to natural infections with AAV or AAV vector-mediated gene transfer using different serotypes applied at various doses to different organs some basic questions remain unanswered.This manuscript reviews the different types of immune responses that are elicited by AAVs and how they translate into lack of efficacy or even worse toxicity Figure\u00a01AAVs are dependoviruses that naturally infect primates and other species where they can replicate with the assistance of a helper virus such as an adenovirus. As a genus they have a wide host cell range for both resting and dividing cells although different serotypes of AAVs or AAV vectors have a more restricted tropism; for example, in humans AAV1 vectors have high tropism for muscle while AAV2 vectors readily infects lung and brain cells . TropismAAVs carry a single stranded DNA genome with two open reading frames (ORFs) encoding regulatory proteins as well as the capsid antigens. The genome is flanked by invert terminal repeat (ITR) sequences. In AAV vectors the two ORFs are deleted and replaced with an expression cassette for a therapeutic protein or RNA; during viral production the deleted sequences as well as essential sequences from a helper virus are provided in trans. Upon injection antigens from the capsids of AAV vectors are present in the inoculum but they, unlike the transgene product, are not actively produced by the transduced cells. This is turn limits the duration of presentation of capsid antigens to the immune system till the proteins are degraded, which according to clinical trial results may take months .nd exposure to the same pathogen elicits very similar responses.Viruses or viral vectors carry either within their antigens or their genome pathogen-associated molecular patters (PAMP), which are absent in mammals and therefore recognized as threats by so-called pathogen recognition receptors (PRR), which are located on the cell surface, within endosomes or the cytosol . ExpressComplement, which is part of the innate immune system but relies in some respects on effector molecules of the adaptive immune system provides a crucial defense mechanism against viruses. It can kill or neutralize viruses directly, it can aggregate viruses, promote their phagocytosis by leukocytes, and in concert with virus-specific antibodies kill infected cells thereby stopping production of new viral progeny . Three pAntigen-specific adaptive immune responses come up more slowly than innate responses and depending on the antigenic load, site of antigen exposure and strength of the inflammatory reaction can take one to several weeks before they become detectable in blood. B cells recognize linear or conformational epitopes on soluble or surface-bound antigens. Na\u00efve B cells initially differentiate into short-lived plasma cells, which mainly produce IgM antibodies that have not undergone affinity maturation. With the help of follicular T helper cells antigen-exposed B cells then form germinal centers where they undergo class-switching and affinity maturation followed by differentiation into long-lived plasma cells and memory B cells. Long-lived plasma cells home to lymphatic tissues and bone marrow, the latter provides them with a niche where they can survive for decades. Memory B cells mainly stay within lymph nodes or spleens. Upon reencounter of their antigen, they either proliferate and undergo additional rounds of affinity maturation or they assume effector function and start secreting antibodies. Memory antibody responses typically come up faster than primary responses, peak responses are higher and more sustained, and antibodies show increased avidity to their cognate antigen. They are also less dependent on T cell help, which is essentially for induction of primary affinity matured antibody responses \u201331.+ T cells while peptides bound to MHC class II molecules trigger a CD4+ T cell response. MHC class I molecules are present on most cells while MHC class II molecules are only expressed by cells of the immune system such as macrophages, dendritic cells, and B cells. Upon activation depending on the type of the inflammatory reaction and the strength of T cell receptor (TcR) signaling CD4+ T cells differentiate into different subsets, i.e., Th1 cells, which promote CD8+ T cell responses, Th2 cells,which drive B cell activation, Th17 cells, which induce strong inflammatory responses and activate neutrophiles, Th22 cells, which play a role in protecting skin against infections and regulatory T cells (Tregs), which dampen effector T cell responses and are crucial to maintain tolerance against self-antigens -g or lyse antigen-expressing cells through the release of perforin and granzyme B. Once the antigen-expressing cells have been removed, most activated CD8+ T cells undergo apoptosis, the remaining cells differentiate into different subsets of memory cells: effector memory cells circulate, they are still partially activated and can commence effector functions immediately without further proliferation; central memory cells home to lymph nodes, they are \u2018resting cells\u2019 and upon encounter of their antigen proliferate before they become effector cells. Tissue resident memory cells can be found in nearly all tissues; they provide rapid local protection by immediately assuming effector functions once they encounter their cognate antigen \u00a0with hemolytic anemia,\u00a0low platelet counts, and\u00a0hemolytic uremic syndrome (HUS) leading to kidney damage and mutations in the gene encoding superoxide dismutase 1 (SOD1) were treated with a single intrathecal infusion of an AAV vector encoding a microRNA targeting SOD1. One patient, who was treated with prednisolone as of the day of gene transfer, showed increases in circulating AAV capsid-specific T cells by about 4 weeks after treatment and concomitantly developed neurological symptoms which according to MRI scans were consistent with DRG toxicity. A second patient was given a more aggressive immunosuppressive regimen; his T cell responses were low and came up later, he developed no neurological symptoms, and his MRI was unremarkable \u201382.+ T cells contribute to DRG.In another trial two patients under immunosuppression were given an rhAAV10 vector expressing survival motor neuron 1 intravenously for treatment of SMA type 2 and again one of the patients developed DRG toxicity . It is fhttps://pharmaphorum.com/news/myocarditis-case-mars-sarepta-dmd-gene-therapy-readout/). In a second trial by Sarepta Therapeutic using an AAVrh74 vector for mini dystrophin 1 out of 20 patients also developed myocarditis , which resolved upon steroid treatment. Although the etiology of AAV vector-induced myocarditis is not yet fully understood is seems feasible that the inflammatory milieu of the damaged muscle tissues in DMD patients favors induction of an immune response to the AAV vectors\u2019 transgene product or alternatively that the added inflammatory reaction induced by the AAV vectors promotes auto-reactive T cell responses to muscle cells.As described above, an inflammatory milieu is essential to trigger adaptive immune responses and upon AAV gene transfer this is triggered by PAMPs present on or in the AAV vectors. Some disease such as DMD are characterized by increased secretion of pro-inflammatory cytokines by muscle infiltrating leukocyte responding to muscle fiber degeneration which maImmune system-mediated toxicity continues to challenge successful gene transfer by AAV vectors especially when high doses are required to correct the targeted genetic disease. Immunosuppression, which could be further optimized, has been used successfully to blunt some of the AAV vector-induced immune responses but in other cases has failed. Even more worrisome are some of the more recently described serious or even fatal adverse events such as TMA, fulminant hepatoxicity or myocarditis. TMA is caused by excessive complement activation, but it remains unknown how this pathway is activated upon AAV gene transfer and if genetic variants that affect the complement system played a role. T cell-mediated severe hepatoxicity upon systemic transfer of very high doses of AAV vectors is to be expected by the fatalities observed in patients with pre-existing liver damage may have had a different etiology as remains to be investigated in more detail.There is no easy pathway to avoid immune-mediated toxicity in response to transfer of very high doses of AAV vectors; our immune system has evolved over the eons to view viruses as threats and responds accordingly by getting rid of them and the cells they have infected. Every war causes collateral damage and the battles the immune system wages against viruses or AAV vectors are no exception. At this stage our focus may have to shift away from the use of excessively high AAV vector doses. Our efforts should concentrate on developing vectors that can more efficiently transduce cells, achieve higher transgene product expression or deliver transgene with higher activity ; either The author confirms being the sole contributor of this work and has approved it for publication.The author consults for several Gene Therapy companies and holds equity in Virion Therapeutics.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."} +{"text": "We draw from theories of identity and stress to examine the impact that siblings have on caregivers\u2019 psychological well-being. Using data collected from 404 caregivers nested in 231 families as part of the Within-Family Differences Study, we conduct mediation analyses to examine whether perceived sibling criticisms are associated with caregivers\u2019 depressive symptoms (a) directly and/or (b) indirectly through sibling tension. Qualitative data from the same caregivers give insight into the processes underlying statistical associations. We found an indirect relationship whereby perceived sibling criticisms were associated with greater sibling tension, which in turn was associated with higher depressive symptoms. Qualitative interviews show that efforts to mitigate the negative impact of sibling criticisms can lead to caregiver strategies that fuel sibling tension. These findings demonstrate how identity processes, as well as the family networks in which caregiving takes place, shape the experiences and consequences of parent care."} +{"text": "Exposure to stressors in childhood is theorized to sensitize individuals to stressors experienced later in life. One way that stress sensitization might manifest is through greater affective reactivity to daily stressors. In turn, greater affective reactivity to daily stressors has been associated with poorer health and increased mortality risk. The present pre-registered investigation tested greater affective reactivity to daily stressors in later life as a potential mediator of the association between early life stressors and mortality risk in a sample of 144 men from the VA Normative Aging Study. Partially consistent with our hypotheses, greater early life psychosocial stressors were associated with greater positive (but not negative) affective reactivity to daily stressors in later life. However, neither early life psychosocial stressors nor affective reactivity to daily stressors were significant predictors of mortality risk. We will discuss implications of these findings for theories of stress experience across the lifespan."} +{"text": "While none of the included studies provided support that the interventions improved quality of life overall, the authors did find evidence that nursing led education in stoma care improved the self-efficacy significantly.Jensen et al's ERAS nursing perspective in radical cystectomy, which describes the increasingly important role of shared decision making in oncology care, where ERAS nurses help patients faced with difficult treatment decisions that require them to weigh efficacy, safety, and quality of life.Majumdar et\u00a0al. from Memorial Sloan Kettering Cancer Center implemented an updated ERAS protocol in total mastectomy patients which included a 35% increase in total intravenous anesthesia usage and near elimination of ketorolac. These changes resulted in a statistically significant reduction in reoperation due to hematoma formation.Lu et\u00a0al. aimed to develop an ERAS protocol for lung cancer surgery tailored to the Chinese national context and extended their study search to Chinese databases including Chinese Biomedical Literature Database (Sinomed) and China Academic Journals (CNKI) among others.,Balfour et al. who discuss that twenty-five years on, we are still seeing barriers to expansion of the ERAS nurse role in today's hospitals.Finally, it is now established that nursing workload actually decreases with the increasing ERAS compliance,Nursing is central to the successful implementation of ERAS. The nursing role will continue to evolve within the ERAS program just as the ERAS guidelines themselves change and as ERAS teams innovate toward improvements in clinical outcomes for patients and healthcare systems globally.None declared."} +{"text": "Calcium channel blockers (CCBs) have been used in the treatment of pulmonary hypertension since 1980s . In 1992versus vasodilator-nonresponsive idiopathic PAH . The recent research of this Topic showed that TIMP-1 elevation could serve as a circulating biomarker to identify PH among COPD patients.A positive acute response is defined as a reduction of the mean PAP \u226710\u00a0mmHg to reach an absolute value of mean PAP \u226640\u00a0mmHg with an increased or unchanged cardiac output . Studiesthic PAH . More biJiang et al.). This recent research showed that exercise-based rehabilitation (aerobic exercise training) may benefit the change from baseline at Week 26 in right ventricular stoke volume (RVSV), determined by pulmonary artery from CMR.The recent work highlighted multiple ways to predict the long-term outcome of pulmonary hypertension patients. This Research Topic includes a study which found that besides hemodynamics, exercise capacity was valuable in the evaluation of the long-term outcome of pulmonary hypertension patients . This recent research showed that Levosimendan treatment could effectively improve acute decompensated right heart failure and systemic hemodynamics in connective tissue disease-associated pulmonary arterial hypertension patients, with positive effects on survival in hospital.Besides the CCBs that have been predominantly used like diltizem, nifedipine and amlodipine, other medicine related to calcium signaling have also been used in pulmonary hypertension . LevosimDai et al.). This finding expanded the mutational spectra of CCDC40 gene related Kartagener\u2019s syndrome which is vital for screening and genetic diagnosis of the disease.Many gene mutations are involved in the etiology of pulmonary hypertension such as BMPR2. In this topic, we reported a rare case of pulmonary hypertension caused by Kartagener\u2019s syndrome with a novel homozygous nonsense mutation in CCDC40 gene (Calcium is involved in the etiology, mechanism and therapy target in pulmonary hypertension. CCBs can be used in first diagnosed pulmonary arterial hypertension and calcium sensitizing agent can be used in the late-stage pulmonary hypertension with right heart failure. More researches should be investigated in the future, such as: whether CCBs can be used in other types of pulmonary hypertension? are there calcium signaling related gene mutation or biomarkers are related to pulmonary hypertension? whether new drugs can be developed targeting calcium signaling such as calcium channels or calcium related receptors?"} +{"text": "CpG dinucleotides. The length of the reads makes it comparatively easy to look at patterns consisting of multiple loci; here, we exploit this property to search for regions where one can define subpopulations of molecules based on methylation patterns. As an example, we run our clustering algorithm on known imprinted genes; we also scan chromosome 15 looking for windows corresponding to heterogeneous methylation. Our software can also compute the covariance of methylation across these regions while keeping into account the mixture of different types of reads.Nanopore reads encode information on the methylation status of cytosines in https://github.com/EmanueleRaineri/cvlr.simon.heath@cnag.crg.euBioinformatics Advances online. NA12878 (Lymphoblastoid cell line) sequenced by the Nanopore consortium the reads mapping on each window; the median absolute difference in methylation between the clusters at each locus is in WhatsHap, we haplotagged all the reads mapping onto chr15 and computed the median absolute methylation difference between haplotypes in windows of 5\u2009kb. There were 17 windows where cvlr could not give results and WhatsHap could, and 905 windows where WhatsHap did not give any result and cvlr did. The threshold of 0.53 gives 909 peaks when using cvlr and WhatsHap X, with n rows representing reads and d columns corresponding to genomic positions. Each position ijX can be 0 (unmethylated) 1 (methylated) or \u22121 (unknown). Reads are clustered (into k clusters) via a mixture of multivariate Bernoulli distributions. To this purpose, we use an Expectation\u2013Maximization (EM) algorithm using the method described in The E step computes the posterior probability that a given read belongs to a certain cluster; the M step updates the To verify that the software works as expected, we simulate 100 reads covering 1000 positions; methylation values are generated first by assuming that the reads form two clusters corresponding to an uniformly low and uniformly high methylation levels. We then check that the simulated clusters are recovered correctly by cvlr-cluster, even in the presence of missing values. We repeat the same procedure with clusters formed by reads which are methylated or not methylated at alternating (non-overlapping) positions. The error rate is Megalodon meg. Enhancers were taken from cvlr to measure methylation heterogeneity along the human genome; although we used imprinted regions as a benchmark, cvlr can be run to detect subpopulation of reads regardless of whether they are due to an allelic effect and does not need a preliminary phasing step. As mentioned above, this is important in practice because when phasing many reads cannot be assigned to an haplotype simply because they contain no informative SNPs; on the other hand, methylation measurements are dense along the genome and each read bears a methylation pattern.We downloaded all the test data we used from wgs and processed them with our internal pipeline which is based on vbac101_Supplementary_DataClick here for additional data file."} +{"text": "In both scale and impact, population ageing has far reaching implications for our planet, not least as a major driver of population growth and the ever-increasing human demands on natural resources and ecosystems. This fundamentally impacts sustainable development efforts to eradicate poverty, achieve food security, build inclusive and resilient communities, and ensure sustainable consumption. The overarching connections between global ageing and sustainability are clear: a focus on sustainable healthy ageing is fundamental to a healthy planet. Our responses to date have however largely been disconnected. To progress this dual agenda, our work aims to i) assess whether current national/international strategies addressing healthy ageing include a strategic focus on sustainability; ii) present the evidence for such alignments; and iii) develop a framework of sustainable actions and aligned policy.A mixed-methods approach using content and applied thematic analysis was utilised to examine strategy documents, and develop an analytical framework derived from relevant theory to guide quantitative and qualitative analysis of the resultant data. Evidence themes were developed iteratively during analytical phases. Findings informed the development of the framework.We identified and analysed 36 strategies published from 2000 to 2021 containing over 600 wide-ranging policies. No strategies and only a minority of policies included a strategic sustainability focus. A larger subset made reference to links between ageing and sustainability or environmental elements yet these were largely theoretical and not carried through in the key strategic approaches or resulting polices.This work provides valuable insights into strategic approaches to foster sustainable healthy ageing and identifies levers for greater alignment and sustainable action. The recently declared 2021-30 UN Decade of Healthy Ageing provides an ideal platform for action.\u2022\u2002While the evidence for strong alignment is unequivocal, global healthy ageing and sustainability agendas are largely disconnected.\u2022\u2002By strengthening the links between healthy ageing and sustainability agendas, stakeholders across sectors can reinforce and design approaches that meet human needs while protecting our planet."} +{"text": "Experiencing challenging events without a partner who can be supportive can be hard and change perceptions of the singlehood experience. We conducted growth curve analyses predicting intercepts and slopes of singlehood satisfaction from ten major life events (MLEs). Single people were followed longitudinally over 10 years (2008-2017) and provided information on MLEs and singlehood satisfaction. People were fairly satisfied with singlehood (Mintercept=6.52/10), and satisfaction increased over time (Mquadratic slope=.01). Some difficult events were associated with declines in singlehood satisfaction. However, responses to MLEs were overall complex\u2014unemployment was related to lower levels of satisfaction but unrelated to changes in satisfaction. Seemingly positive events were also related to lower levels of satisfaction. Results for each MLE will be discussed in line with additional qualitative data on single people\u2019s perceptions of change."} +{"text": "Brain circuits undergo substantial structural changes during development, driven by the formation, stabilization, and elimination of synapses. Synaptic connections continue to undergo experience\u2010dependent structural rearrangements throughout life, which are postulated to underlie learning and memory. Astrocytes, a major glial cell type in the brain, are physically in contact with synaptic circuits through their structural ensheathment of synapses. Astrocytes strongly contribute to the remodeling of synaptic structures in healthy and diseased central nervous systems by regulating synaptic connectivity and behaviors. However, whether structural plasticity of astrocytes is involved in their critical functions at the synapse is unknown. This review will discuss the emerging evidence linking astrocytic structural plasticity to synaptic circuit remodeling and regulation of behaviors. Moreover, we will survey possible molecular and cellular mechanisms regulating the structural plasticity of astrocytes and their non\u2010cell\u2010autonomous effects on neuronal plasticity. Finally, we will discuss how astrocyte morphological changes in different physiological states and disease conditions contribute to neuronal circuit function and dysfunction. Astrocytes are highly complex cells which display structural plasticity. Here we review recent findings linking astrocyte structural plasticity to brain function and behavior. On the contrary, the induction of Ca2+ fluctuations by activation of exogenous Gq\u2010coupled receptors caused an increase in PAP motility \u2010cofilin\u20101 pathway. NKCC1 induces the phosphorylation of cofilin\u20101, which regulates actin polymerization. When phosphorylation of cofilin\u20101 was inhibited, so was LTP\u2010induced PAP shrinkage which could cause cytoskeletal rearrangements. Some Ca2+ transients in PAPs are IP3R2\u2010independent , a secondary messenger mediating the activation of protein kinase A (PKA), modulates a subgroup of Ca2+ oscillations during astrocyte hypertrophy , which increases the entire cell volume and the number of branches. However, such structural changes are not observed when mice simply repeat what they have already learned , it was shown that Gq or Gs activation have opposing effects on recent memory formation and supraoptic (SON) nuclei of the hypothalamus. Substantial structural and functional plasticity happens in the PVN and SON of female mice which are lactating. These changes are driven by both neuron\u2013neuron and neuron\u2010astrocytes contacts , a morphologically distinct sub\u2010population of OT receptor\u2010expressing astrocytes can communicate through gap junctions upon OT release to promote positive emotional states in a 24\u2010h period , stratum radiatum (s.r.), and the SCN in the dark (D) phase of the circadian rhythm compared to the light (L) phase sleep features, which are important for memory consolidation , an oscillatory pattern of cortical neural activity during NREM sleep. This Gi\u2010driven increase in SWA regulates sleep depth but not duration , hypertrophy of astrocytic processes in response to injury was remarkably reduced compared to WT. This effect was accompanied by a more dramatic loss of synaptic proteins and axon degeneration in GFAP\u2212/\u2212Vim\u2212/\u2212 mice around the injury site around a traumatic injury site in the CNS to protect surrounding healthy tissues levels. The reconstitution of Kir4.1 expression was sufficient to decrease the hyperexcitability of striatal medium spiny neurons and improve HD\u2010associated motor deficit patients lesions are hypertrophic , a heterozygous mutation in GFAP causes hypertrophy and accumulation of cytoplasmic protein inclusions called Rosenthal fibers within astrocytes is not known. Are all astrocytes responding in the same way to neurotransmitters, or is there regional heterogeneity in their structural plasticity? These questions remain to be answered.We also lack information about the specific pathways utilized to reorganize the cytoskeleton of PAPs, to regulate the release of gliotransmitters and synaptogenic factors, and to promote the uptake of ions and neurotransmitters from the extracellular space. Neurotransmitter mediated GPCR signaling in astrocytes could induce the observed structural changes. So far GPCR signaling within astrocytes were mostly performed by using overexpression of exogenous designer GPCRs to induce dramatic intracellular calcium elevations. The endogenous GPCR\u2010signaling within PAPs is poorly understood.Future experiments to understand the molecular pathways regulating astrocytes' structural and functional responses are needed.In the disease context, the cellular mechanisms observed in reactive astrocytes largely overlap with the experience\u2010dependent or physiological state\u2010driven changes in astrocytes. However, reactive astrocytes differ in the magnitude of the observed structural changes. In brain injury or neurodegeneration, astrocyte hypertrophy goes beyond a threshold that causes massive changes in gene expression profile, overexpression of GFAP and vimentin, and even disruption of astrocyte territories. Because of their relevance to disease, these cellular mechanisms have received much attention, but we are still far from understanding their function and utility. What is the threshold between physiological astrocyte plasticity and astrocyte reactivity? How can astrocytes regulate the magnitude of their structural responses? Addressing these unanswered questions may reveal\u00a0new mechanisms regulating astrocyte structural plasticity. These insights have the potential to pinpoint the causes of and discover new treatments for brain diseases.The authors declare no potential conflict of interest."} +{"text": "Transplant International is characterized by anticipation of the developments lying ahead and attempt to grow through generated opportunities. In an increasingly digital world and amid growing concerns for the environment, we adopted a paperless format years before our competitors. We have taken advantage of the potential offered by digitalization and designed a journal with a completely novel appearance, with the animated covers as a hallmark TOP 10 2022 MOST DOWNLOADED ARTICLESTOP 10 2022 MOST VIEWED ARTICLESEDITORS\u2019 PICKS: 2022 COVER ARTICLES"} +{"text": "Children and adolescents with learning disability need multi-disciplinary input when they present with challenging behaviours or mental health disorders. This patient group needs specialist skills from the clinicians and professionals to support and meet their needs. There is no commissioned Child and Adolescent Learning Disability Mental Health Service (CAMHS) in Waltham Forest and support comes from Specialist Tier 3 generic CAMHS which comprises of Emotional Difficulties Pathway, Neurodevelopmental Disorders Pathway and recently developed Behaviour Pathway which mostly comprises of specialist parenting training/interventions. To identify gaps in service provision for children and young people with learning disability presenting with challenging behaviours and/or mental health needs in Waltham Forest as there is no formally funded CAMHS learning disability service in the locality.All children and young people under 18 with learning disability under Waltham Forest CAMHS with ASD/ADHD and other neurodevelopmental disorders who meet the project criteria are included. Project criteria include 1) Main diagnosis of Learning Disability , likely to have low IQ with cognitive impairment.), with or without associated other Neurodevelopmental disorders or other mental health disorders and 2) Engages in challenging behaviours. Challenging behaviours defined as behaviours significantly limiting engagement in daily & family life, education and/or social activities, and have persisted for at least a period of 3 months. Data were collected from electronic recoding system of individual patients; using a data collection sheet on level of learning disability; comorbid neurodevelopmental or emotional and mental health disorders; profession of allocated clinician; joint working with discipline; involvement with social care; allied health professionals input; presenting difficulties and severity; CETR or hospital admissions; referral to National services; What interventions offered ; if medications offered, was it used as first line and how long for; parents\u2019 view on medication management.As we have expected, medication management were used as first line and there were limited offers of behaviour support. Joint working with social care, speech therapy and occupational therapy but with limited input especially occupational therapy in cases with high sensory needs. It was unclear with the cognitive assessment and diagnosing the learning disability in the population under 16s.There is a service gap for CAMHS learning disability population and more joint working needed among relevant health professionals."} +{"text": "In summer 2020, 68 clinicians in the New England area caring for Veterans with dementia as part of a specialty team were surveyed regarding caregiver support services, with a 46% response rate (n=31). When faced with the need to abruptly discontinue in-person dementia support services, the majority of respondents offered caregivers support via telephone rather than video telehealth. Only 4 of 31 (13%) mentioned offering video visits for the first time to replace face-to-face visits. Clinician choice of modality largely reflected shifts among preestablished communication modalities/patterns and were influenced by clinician perception that older patients and their caregivers would prefer the telephone. Clinicians without experience using video telehealth with older adults were unlikely to offer video visits despite evidence that many older adults are willing and able to participate. Assessing caregiver wish/ability to participate in video visits may inform and shape clinician choice of telehealth modality."} +{"text": "Trichosurus vulpecula)\u2014a marsupial for which social learning has not previously been investigated. In large outdoor pens, we presented wild-caught \u2018demonstrator\u2019 possums with puzzle devices containing an attractive food reward; 2 of 8 demonstrators accessed the reward the first night the puzzle was presented and another three succeeded on later nights. Meanwhile, \u2018observer\u2019 possums in adjacent pens watched the demonstrators for five nights and then were given the opportunity to solve the puzzle themselves; 15 of 15 succeeded on their first night (a highly significant improvement). This experiment thus provides strong evidence of social learning by common brushtail possums. Future research should investigate whether information about aversive stimuli (such as traps and toxic baits) can similarly be transmitted between possums by social learning; if so, this could have important implications for possum pest control.Social learning can reduce the costs associated with trial-and-error learning. There is speculation that social learning could contribute to trap and bait avoidance in invasive species like the common brushtail possum ( Our expThe observer possums may have learned to associate the demonstrators\u2019 food rewards with the colour of the target dome; however, the importance of colour as a cue is unclear as little is known about colour vision in common brushtail possums. Vlahos proposedOur research provides, to our knowledge, the first evidence of social learning capabilities in common brushtail possums. These social learning abilities may help explain why some recalcitrant individuals in NZ populations are difficult to eradicate. We urge caution on this point, however, until there is evidence possums' social learning of a positive stimulus can indeed be generalized to social learning of negative encounters, for example, sub-lethal interactions with traps and toxins. This question should be a priority for future research because of its relevance to NZ's current Predator Free initiative."} +{"text": "Greetings to our avid readers. September brings us seasonal changes and a sense of contemplation as we count the slim pages left on the calendar.Our issue article offers inspiration and insight as a thoughtful reflection on 2020, the International Year of the Nurse and Midwife. The invitation to uncover our nursing stories and share our collective voice has already gained more than 1,500 views on KJWHN\u2019s online website. The Korean Society for Women Health Nursing also responded to the author\u2019s nudge, by putting out a call for nurses and nursing students to submit written and/or artistic expressions of their stories. Please join us for the December issue for a glimpse of selected works.On another note, September also signaled the start of a new academic semester and accreditation season for nursing schools in Korea. In addition to coronavirus disease 2019 (COVID-19) dragging on, I suspect these factors may have affected submissions and a slower review process. Although the number of articles has decreased, we trust you will find the variety of topic clusters informative.Midwifery role development: An English article on professionalism and job satisfaction among Korean midwives according to their practice site, highlights midwives\u2019 current limitations and discusses solutions.\u2022 Reproductive health: Nursing students are often exposed to stress and fatigue and a study examined factors affecting premenstrual symptoms in this particular group.\u2022 Maternal health: A study explored the narratives of preterm birth experiences through text network analysis. Readers may find it interesting to see how MAXQDA software visualized and aided identification of clinical symptom expressions.\u2022 Nursing education: A study tested a labor care high-fidelity simulation program for nursing students who had not yet experienced clinical practicum. With on-site direct learning an ongoing challenge due to COVID-19, the authors\u2019 report on its effects on knowledge, critical thinking, and clinical performance, may be helpful for nurse educators.\u2022 Women\u2019s mental health: A study investigated suicide ideation among female late adolescents in rural areas, an underserved group. Affective factors and personal experiences were found as key.\u2022 We hope this issue will inspire you to share your nursing stories (and manuscripts) with others."} +{"text": "Williams syndrome is a autosomal dominant disorder associated with deletion of multiple genes on the long arm of chromosome 7. It affects one in every 25000 live births and is associated with elfin facies, cardiac abnormalities like supra valvular aortic stenosis and mental impairment. Williams syndrome is diagnosed in about sixty percent patients of supravalvular aortic stenosis. This patient presented to us with the characteristic \u2018elfin facies\u00b4 which is shown in the image as having a large mouth, widely spaced eyes, maloccluded teeth, patulous lips broad forehead with a short and upturned nose. Supravalvular aortic stenosis (SVAS) involves a narrowing of the ascending aorta above the level of coronary arteries. Narrowing of the peripheral pulmonary arteries is also present in SVAS with Williams syndrome but it does not progress over time. Clinical findings on examination showed an ejection systolic murmur with no associated ejection click. There was a disparity in the blood pressure between both upper limbs, the right arm systolic blood pressure exceeded that of the left arm by 24 mmHg. The chromosomal defect results in deficiency in elastin. This also results in lax skin and stiff joints with a hoarse voice. Supravalvular AS eventually requires valve replacement when the patient becomes symptomatic."} +{"text": "Peripheral artery disease (PAD) is caused due to buildup of atherosclerotic plaques, typically in the leg arteries, preventing adequate blood circulation and ultimately claudication. A previous study showed that the vertical ground reaction force (VGRF) curve is significantly flatter in claudicating patients, resulting in a lower and less fluctuant center of mass when ambulating. Patients with PAD also demonstrate significantly decreased propulsion forces in the anterior\u2013 posterior direction. Assistive tennis shoes can potentially assist push-off by substituting for muscle forces using energy stored in a carbon fiber plate or metal spring within the shoe. This study aims to examine how the CF and SL shoes impact walking performance in patients with PAD. A total of ten patients with PAD performed a progressive treadmill test using a pressure-instrumented treadmill for each shoe type: i) standard shoes, ii) CF shoes, and iii) SL shoes. We calculated the peak VGRF for three subjects to date as an average of ten stance phases for the beginning of the walking condition (pain free condition). Preliminary results from three subjects showed that patients with PAD generated a greater peak VGRF wearing CF and SL shoes compared to normal shoes during the heel contact and push-off . In future, we will calculate the VGRF for the remaining patients in pain free and pain conditions and conduct statistical analysis to identify significant differences among shoe types."} +{"text": "Interdisciplinarity is a key element for modern cancer treatment. This is not only true for the interaction between various medical disciplines, but also a necessity due to the everlasting subspecialization within the field of surgery itself. Increasing surgical capabilities and technical advances within all specialized surgical disciplines have dramatically changed the face of modern surgical approaches to cure patients with malignant diseases .While basically until the middle of the last century the sole removal of malignant tumors in any part of the human body was a challenge that had to be mastered by general surgeons on their own, it is now common sense and practice that specialized surgeons join the effort and come together to remove even advanced tumors and allow for safe simultaneous reconstructions \u20134. The vPeng et al.). Also, even when a tumor cannot be completely cured, interdisciplinary surgery can offer improve the quality of life in palliative situations . By their very nature, these growing surgical specialties and subspecialties tend to be consolidated in academic centers and larger urban regions where most teaching and training occurs .Not only has the practice of surgery changed and undergone a significant evolution over the past 4 decades , butCianci et al.). By integrating various surgical disciplines into tumor surgery more radical tumor resections can therefore be more safely performed and interdisciplinary reconstructions optimize the outcome of the individual patient`s treatment along with increased quality of life despite radical and oncologically sufficent cancer surgery.While on the one hand technical advances such as the evolution of minimally invasive surgery has been an important milestone in the field of surgical oncology or which has almost totally globally replaced open gastrectomy in treating gastric cancer, the individual knowledge of technically advanced instruments and tools, including high defintion imaging techniques is continuousy contributing to push the limits of possible resections and reconstructions forward over the course of the 20th and 21st century, based largely on the focus on specific organ systems or anatomic regions or specific surgical techniques /peripheral primitive neuroectodermal tumors (pPNETs). This entities are extremely rare, and the current understanding of these tumors is poor. The authors aim to illustrate the clinical characteristics of primary spinal ES/pPNETs and to discuss prognostic factors by survival analysis. They show that otal en bloc resection can significantly improve PFS for primary spinal ES/pPNETs and adjuvant radiotherapy was a favorable factor for PFS in their patients. Total en bloc resection and adjuvant radiotherapy considerably improve overall survival (OS) for patients with primary spinal ES/pPNETs.The special issue comprises relevant hot topics and variants of interdisciplinary surgical oncology. Thiele et\u00a0al. compare the pros and cons of various perineal reconsturctive techniques following the resection of anorectal malignancies, which may result in extensive perineal/pelvic defects that require an interdisciplinary surgical approach involving reconstructive surgery. Their experience with either a myocutaneous gracilis flap (MGF) or a gluteal fold flap (GFF) compares the outcome regarding clinical key parameters. They conclude that MG-flaps and GF-flaps prove to be reliable and robust techniques for perineal/pelvic reconstruction. They suggest a decision-making based on distribution of adipose tissue for dead space obliteration, intraoperative patient positioning, and perforator vessel quality/distribution.Horch et\u00a0al. This interdisciplinary approach can minimize the downside of abdomino-perineal resection or exenteration especially in women when parts of the vagina need to be resected. Derived from their experince with over 300 patients receiving pelvic and perineal reconstruction with a transpelvic vertical rectus abdominis myocutaneous (tpVRAM) flap they found that the tpVRAM flap is reliably perfused and helps to reduce long term wound healing desasters in the irradiated perineal/vaginal/gluteal region for relapsing or far advanced rectal and anal cancers in female patients with previous irradation prior to the surgical resection has been described in detail by l region Figure\u00a01Steiner et\u00a0al. analyzed the interdisciplinary treatment of breast cancer which is based on the histological tumor type, the TNM classification, and the patient\u2019s wishes. They demonstrate that following tumor resection and (neo-) adjuvant therapy strategies, breast reconstruction represents the final step in the individual interdisciplinary treatment plan. Their analysis comprises data from autologous microsurgical breast reconstruction with the deep inferior epigastric artery perforator (DIEP) or the muscle-sparing transverse rectus abdominis myocutaneous (ms-TRAM) flap. I a retrospective study focusing on the safety of autologous breast reconstruction upon mastectomy using abdominal free flaps in an academic university hospital they show a high success rate with comparatively few complications. Using preoperative computer tomography angiography, intraoperative fluorescence angiography, titanized hernia meshes for rectus sheath reconstruction, and venous coupler systems, autologous breast reconstruction with DIEP or ms-TRAM free flaps is a safe and standardized procedure in high-volume microsurgery centers.Tan et\u00a0al. studied the effectiveness and safety of the enhanced recovery after surgery (ERAS) protocol vs. traditional perioperative care programs for breast reconstruction. Ten studies were included in their meta-analysis. Their results suggest that ERAS protocols can decrease LOS and morphine equivalent dosing; therefore, they discuss that further larger, and better-quality studies that report on bleeding amount and patient satisfaction are needed to validate their findings.Weitz et\u00a0al. studied reconstructions of complex scalp after ablative resection or by post-traumatic tissue loss, that can make a simultaneous interdisciplinary two-team approach complicated, which is considered a major disadvantage regarding safety and operation time. Finally their data leed to the assumption that parascapular flap seem to be a good alternative for reconstruction of complex tumor defects of the scalp besides the latissimus dorsi flap. Stable long-term results and little donor site morbidity are enabled with good aesthetic outcomes and shorter operation time in an interdisciplinary two-team approach.Cao et\u00a0al. assessed the impact of enhanced recovery after surgery (ERAS) protocols in pancreaticoduodenectomy. They found no significant increase in mortality, readmission, reoperation, or delayed gastric emptying. Therefore they come to the conclusion that their analysis revealed that using ERAS protocols in pancreatic resections may help decrease the incidence of pancreatic fistula and infections. Furthermore, ERAS also reduces length of stay and cost of care. This study provides evidence for the benefit of ERAS protocols. Weber et\u00a0al. describe that craniofacial osteosarcomas (COS) and extracranial osteosarcomas (EOS) show distinct clinical differences. They conclude that the reduced Gli1 expression in COS could be interpreted as reduced activation of the Hedgehog (Hh) signaling pathway. The increased M1 polarization and reduced Hh activation in COS could explain the low incidence of metastases in these osteosarcomas.Zheng et\u00a0al. aimed to compare survival outcome after receiving radiofrequency ablation (RFA) and surgical resection (SR) for solitary hepatocellular carcinoma (HCC) with size large as 5 cm. They found that by applying several effective sensitivity analyses, OS and CSS were similar between the patients with tumors smaller than 3 cm receiving RFA and SR. But SR may be a superior treatment option with better long-term outcome than RFA in patients with tumor measuring 3.1-5 cm.Liang et\u00a0al. performed a retrospective study to identify the prognostic significance of time to local recurrence (TLR) with regard to overall survival (OS) and survival after local recurrence (SAR) in patients with soft tissue sarcoma (STS) of the extremity and abdominothoracic wall. From their results they conclude that in patients with STS of the extremity and abdominothoracic wall, ELR after R0 resection indicated a worse prognosis than those with LLR, and TLR can be considered an independent prognostic factor for OS and SAR. Furthermore, local recurrence was significantly influenced by the depth and the histopathological grading of the primary tumor, and reoperation after local recurrence could improve survival, which means salvage surgery may still be the preferred treatment when there are surgical indications after recurrence.The contributions to this special issue highlight recent advances and approaches to the art of interdisciplinary oncological surgical and show how the challenges go along with functional organ or tissue preservation or restoration/reconstruction to maintain the highest possible QOL without reducing the aim of oncologic radicality.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication."} +{"text": "Quantification of ancient human intelligence has become possible with recent advances in polygenic prediction. Intelligence is a complex trait that has both environmental and genetic components and high heritability. Large-scale genome-wide association studies based on ~270,000 individuals have demonstrated highly significant single-nucleotide polymorphisms (SNPs) associated with intelligence in present-day humans. We utilized those previously reported 12,037 SNPs to estimate a genetic component of intelligence in ancient Funadomari Jomon individual from 3700 years BP as well as four individuals of Afanasievo nuclear family from about 4100 years BP and who are considered anatomically modern humans. We have demonstrated that ancient individuals could have been not inferior in intelligence compared to present-day humans through assessment of the genetic component of intelligence. We have also confirmed that alleles associated with intelligence tend to spread equally between ancestral and derived origin suggesting that intelligence may be a neutral trait in human evolution. Z-score obtained through METAL software ) based on Fisher exact test .We have demonstrated first ever insight into genetic component of intelligence through PGS in ancient individuals from around 3700\u20134100 BP. The performed calculations indicate a possibility that people living on the territory of modern Hokkaido and Russia in that period being not less intelligent than modern humans. Absolute IQ values inferred from PGS in Afanasievo individuals and Funadomari Jomon individual tends to be within the 95% range of mean general human population suggesting similarity of intelligence of humans living 3700 BP and modern humans. Although intelligence PGS of Afanasievo family tend to fluctuate on the lower tail of normal distribution of the scores of 1000 Genome project these scores translate to absolute IQ values within mean of general population given the low variance explained by intelligence PGS forming essential cognitive functions. IQ measures have been implicated with survival, adaptation to environment, and mental functioning . DigitalDNA.Land platform has previously demonstrated evaluation of intelligence PGS using GWAS findings based on 72 SNPs . Likely A common approach in studying quantitative traits like intelligence in humans has been based on monozygotic and dizygotic twins . PreviouIntelligence as a phenotypic trait with underlying effects of DNA polymorphism has been likely shaped by evolutionary processes. Majority of mutations in genes affecting underlying cognition used in this study tend to interact in extremely complex networks with higher activity in hippocampal as well as somatosensory neurons . Since nWe demonstrated that genomic data from ancient individuals can be used to evaluate a genetic component of intelligence. Funadomari Jomon as well as Afanasievo family individuals demonstrated intelligence PGS as well as IQ scores in line with modern humans. DNA evidence may indicate a possibility of intelligence being a neutral trait in human evolution suggesting that ancient individuals living 3700\u20134100 years BP could have been as intelligent as modern humans."} +{"text": "Care givers of patients with severe mental disorders have been shown to be under heavy stress burden that reflect itself through various heterogenous psychiatric symptoms that may mimic PTSD with associated negative impact on interpersonal relations and work performanceto assess the prevalence of PTSD symptoms among care givers of patients with severe mental illness70 patients care givers of sevely mentally ill patients compred to control 70 care giver of patients with chronic debilitating medical illness were recruited from outpatient of the university hospital outpatient facilities, random selection. Severe mental illness was defined by Global assessment of function GAF score above 50 and duration exceeding 2 years. Both groups were subject to Zarit burden interview to assess stress burden and post traumatic stress diagnostic scale PDS to assess PTSD symptomats43% of care givers of severly mentally patients showed moderate to severe burden on the Zarit scale compared to only 10% among care givers of medically ill patients, this difference was statistically significant . Among care givers of severly mental patients showed moderate to severe score on post traumatic stress diagnostic scale compared to 0% among those taking care of medically ill patients. this difference was statistically significant Stress burden among care givers of patients with severe mental illness is high and may manifest symptoms of post traumatic disorder. This highlight the importance of particular psychological support and assessment among care givers of patients with sever mental illnessNo significant relationships."} +{"text": "The retraction has been agreed following an investigation based on allegations raised by a third party, which revealed that some images in this article are inappropriate duplications of images from previously published articles [The above article published online on 19 January 2016 in Wiley Online Library (articles , 4. Thus"} +{"text": "Breast and axillary surgery after neoadjuvant systemic treatment for women with breast cancer has undergone multiple paradigm changes within the past years. In this review, we provide a state-of-the-art overview of breast and axillary surgery after neoadjuvant systemic treatment from both, a clinical routine perspective and a clinical research perspective. For axillary disease, axillary lymph node dissection, sentinel lymph node biopsy, or targeted axillary dissection are nowadays recommended depending on the lymph node status before and after neoadjuvant systemic treatment. For the primary tumor in the breast, breast conserving surgery remains the standard of care. The clinical management of exceptional responders to neoadjuvant systemic treatment is a pressing knowledge gap due to the increasing number of patients who achieve a pathologic complete response to neoadjuvant systemic treatment and for whom surgery may have no therapeutic benefit. Current clinical research evaluates whether less invasive procedures can exclude residual cancer after neoadjuvant systemic treatment as reliably as surgery to possibly omit surgery for those patients in the future. \u2022Breast and axillary surgery after neoadjuvant systemic treatment has evolved.\u2022Choice of axillary surgery depends on lymph node status before and after treatment.\u2022Optimal management of exceptional responders to neoadjuvant treatment is unclear.\u2022Clinical research aims to reliably exclude residual cancer without surgery.\u2022For exceptional responders, breast cancer surgery may be omitted in the future. On the one hand, NST often shrinks the tumor before surgery which allows for surgical downstaging and less invasive breast-conserving surgery to spare our patients relevant treatment-associated morbidity . On the In this review, we provide a state-of-the-art overview of breast and axillary surgery after NST from both, a clinical routine perspective and a clinical research perspective. We will focus on the surgical management of four scenarios : patient2The axillary surgical management of breast cancer patients has undergone multiple changes since the introduction of NST. In the adjuvant setting, we learned that even leaving some tumor behind (non-sentinel lymph node metastasis for patients who undergo SLNB instead of ALND) does not impair oncologic safety but spares our patients relevant treatment-associated morbidity . In the For patients with initial cN0 status, SLNB after NST is recommended .For patients with cN\u00a0+\u00a0status, SLNB initially showed a high risk of leaving tumor behind . HoweverPatients who present with histopathologic residual axillary disease (ypN+) as determined by axillary surgical staging (SLNB or TAD) are recommended completion ALND, as retrospective registry studies suggest that the omission of ALND in case of ypN\u00a0+\u00a0status results in inferior survival . HoweverNotably, guideline recommendations for routine axillary surgical management have changed based on the results of diagnostic clinical trials \u2013 to date, there is no high-quality evidence on actual (long-term) oncologic outcomes like local control or survival for de-escalated axillary staging following NST. The general assumption is that a false-negative rate of <10% will not translate into impaired oncologic outcomes which, in fact, could be observed for de-escalated axillary staging in the adjuvant setting ,28,36,37Current clinical research in breast cancer axillary staging also focuses on patients with (y)cN0 disease and the question of whether there is a specific group of patients who may not benefit at all from axillary staging and whom we could spare this procedure. A clinical trial (NCT04101851) has just commenced evaluating whether it is oncologically safe to omit SLNB in triple-negative and HER2-positive breast cancer patients with radiologic and pathological complete response in the breast after NST . We know3Not only the axillary but also the breast surgical management of breast cancer patients has been influenced by the introduction of NST. Although breast conserving surgery (followed by radiotherapy) showed equivalent survival compared to mastectomy in the neoadjuvant and adjuvant setting, some evidence suggests that \u201cless surgery\u201d after NST may be associated with higher local recurrence rates . One shoThe increasing applications and efficacy of NST led to another pressing question in breast surgical care: How should we deal with the growing number of patients who achieve a pathologic complete response to NST ? Modern therapy regimens showed pathologic complete response rates of 60\u201370% for HER2+ and triple-negative breast cancer in large phase III trials and there is growing evidence that also patients with high-proliferative Luminal B breast cancer may benefit from NST ,10. As aSeveral diagnostic tools have been evaluated to exclude residual cancer after NST. We learned that imaging after NST is not accurate enough to replace surgery: ultrasound and mammography show high rates of missed residual cancer (about 20%) and although MRI and PET-CT miss less cancer they show high rates of false-positive findings which restricts the clinical applicability ,41. Rece4While the surgical management of the primary tumor in the breast and in the axilla has been mainly considered independent during the past decades, more recent evidence suggests, that axillary lymph node metastasis are mainly driven by the biology of the primary tumor . As the 5Breast and axillary surgery after NST for women with breast cancer has undergone multiple paradigm changes within the past years. For axillary disease, ALND, SLNB, or TAD are nowadays recommended depending on the lymph node status before and after NST. For the primary tumor in the breast, breast conserving surgery remains the standard of care. The clinical management of exceptional responders to NST is a pressing knowledge gap due to the increasing number of patients who achieve a pathologic complete response to NST and for whom surgery may have no therapeutic benefit. Current clinical research evaluates whether less invasive procedures can exclude residual cancer as reliably as surgery to possibly omit surgery for those patients in the future.There was no funding for this article.Not required.None."} +{"text": "To the Editor,On November 1 2022, on the Food and Drug Administration (FDA) virtual public meeting of the Center for Devices and Radiological Health (CDRH), the Anesthesiology and Respiratory Therapy Devices Panel of the Medical Devices Advisory Committee discussed several recent studies reporting pulse oximeters to provide less accurate readings in the case of people with darker skin pigmentations [Therefore, any medical equipment that leverages near-infrared light interacting with the skin should not show discordance in its performance across people with different skin tones.2) of the hemoglobin present primarily in small vessels (<\u20091\u00a0mm in diameter) such as the capillary, arteriolar and venular bed of the cerebral cortex. Already in 2005, Wassenaar et al. [2 should be interpreted with caution, as melanin clearly interferes with the quality of the reflected NIRS signal. In modern cerebral oximeters, wavelengths have been added to enable a more precise determination of hemoglobin and to be less sensitive to skin pigmentation. Very recently, the robustness of two commercial NIRS-based cerebral oximeters (using four or five wavelengths) to variations in skin pigmentation was evaluated using a tissue-simulating phantom; unexpectedly increasing melanin content decreased saturation values displayed by both devices [We think the problem with pulse oximeters is also relevant for other type of noninvasive optical oximeters. The noninvasive estimation of cerebrovascular blood oxygen saturation (mostly by a sensor attached to the forehead) by near-infrared spectroscopy (NIRS)-based cerebral oximetry equipment has been increasingly used in many clinical areas such as (1) cardiac, vascular and thoracic surgery for detecting cerebral hypoxia\u2013ischemia as well as developing interventions to avoid and improve hypoxic brain injury and 2) intensive care for monitoring and modifying brain oxygenation in patients at risk of hypoxic ischemic brain injury after cardiac arrest intensiv. NIRS-bar et al. highlighConsidering the extensive clinical use of cerebral oximetry, we strongly suggest that the FDA and the other regulatory bodies should deliberate also how to improve NIRS-based cerebral oximeters to best ensure accuracy for all skin pigmentations and eventually recommend labeling them with warnings about their limitations. Furthermore, we urge manufacturers producing optical cerebral oximeters to validate their devices and sensors regarding their robustness against skin pigmentations of different strength."} +{"text": "Revisiting the Teaching Nursing Home is a two-year pilot project to address the long-term care workforce shortage by introducing nursing students to geriatric nursing while also improving quality of care within nursing homes. The initiative has multiple components: enhanced clinical rotations for nursing students with partner schools of nursing, implementation of the Institute for Healthcare Improvement Age-Friendly Health System \u201c4M\u201d quality improvement model, and an online learning network. Undergraduate and graduate nursing students at three Schools of Nursing participated in clinical experiences at regional nursing homes. Students completed an \u201cactivity feedback\u201d form after each clinical rotation at the nursing home or related activity, such as a session about the 4Ms or quality improvement/assessment. The activity feedback form asked students to share their most important takeaway and suggestions for improvement. Data from 340 feedback forms was coded qualitatively using conventional and directed content analysis using the American Association of Colleges of Nursing (AACN) Essentials for Professional Nursing Education. Multiple coders and audit trials were used to establish rigor. Students\u2019 takeaways encompassed 7 of 8 key concepts in the AACN Essentials; Knowledge for Nursing Practice, Person-Centered Care, and Interprofessional Partnerships were most frequently mentioned. Students provided numerous suggestions for improving their clinical experiences including facilitated learning from instructors and supported engagement with nursing home staff. In conclusion, the program addressed many of the core competencies designated by AACN. One recommendation that flows from these findings is to enhance the role of clinical preceptors in the nursing home setting to facilitate mentored training."} +{"text": "The use of firefighting foam containing per- and polyfluoroalkyl substances (PFAS) has resulted in environmental contamination in three Australian communities. We examined whether people who had lived in these communities had higher rates of selected cancers and causes of deaths than those who had lived in comparison areas without known contamination.The three exposure areas of interest were in Katherine (NT), Oakey (Qld) and Williamtown (NSW). We identified those who ever lived in exposure areas by linking street addresses in these areas to addresses collected in Medicare (1983-2019)\u2014a consumer directory for Australia\u2019s universal healthcare system. We also identified a sample of those who had lived in selected comparison areas. Exposed and comparison populations were then linked to Australia\u2019s national cancer and death registries. We estimated standardised incidence ratios (SIRs) for 23 cancers, four causes of death and three control outcomes, adjusting for sex, age and calendar time of diagnosis.We observed higher rates of prostate cancer 1\u00b736\u20132\u00b724) in Katherine; laryngeal cancer , kidney cancer and coronary heart disease (CHD) mortality in Oakey; and lung cancer and CHD mortality in Williamtown. We also saw elevated SIRs for control outcomes\u2014outcomes not known or thought to be associated with PFAS exposure. SIRs for all other outcomes and overall cancer were similar across exposure and comparison areas.There was limited evidence to support an association between PFAS exposure and risk of cancer. There was modest evidence of an association with CHD mortality, which merits further study given the links between PFAS and elevated blood lipids."} +{"text": "Antipsychotic medications such as risperidone, olanzapine and aripiprazole are used to treat psychological and behavioural symptoms among dementia patients. Current evidence indicate prescription rates for antipsychotics vary and wider consensus to evaluate clinical epidemiological outcomes is limited. This study aims to investigate the potential impact of atypical antipsychotics on the mortality of patients with dementia.A retrospective clinical cohort study was developed to review United Kingdom Clinical Record Interactive Search system based data between January 1, 2013 to December 31, 2017. A descriptive statistical method was used to analyse the data. Mini Mental State Examination (MMSE) scores were used to assess the severity and stage of disease progression. A study specific cox proportional hazards model was developed to evaluate the relationship between survival following diagnosis and other variables.A total sample size of 1692 patients were identified using natural language processing of which, 587 were prescribed olanzapine, quetiapine, or risperidone (common group) whilst 893 (control group) were not prescribed any antipsychotics. Patients prescribed olanzapine and Risperidone showed similar risk of death , . Patients prescribed Quetiapine showed no significant association . Factors associated with a lower risk of death were elevated MMSE score at diagnosis along with other demographic factors such as women and being of a Caucasian British group .A significant mortality risk was identified among those prescribed olanzapine and risperidone which contradicts previous findings although the study designs used were different. Comprehensive research should be conducted to better assess clinical epidemiological outcomes associated with diagnosis and therapies to improve clinical management of these patients."} +{"text": "Bilateral mydriasis is usually associated with severe brain stem damage or drug-induced sympathomimetic stimulation.Herein we report it as a unique neurologic complication of Hodgkin\u2019s lymphoma.A 23-year-old woman presented at our emergency department with dilated pupils unresponsive to light stimuli. MRI and CT scans showed bilaterally enlarged lymph nodes in the mediastinum and supraclavicular compressing the carotid artery on both sides. The histologic examination of lymph node biopsy specimens confirmed the diagnosis of Hodgkin\u2019s lymphoma.Pathologies around the carotid artery causing oculosympathetic spasm should be considered among the possible causes of a mydriasis, especially when other common causes like brain stem impairment are excluded. Dilated and unreactive pupils are caused by parasympathetic paralysis or sympathetic stimulation. Reasons for a parasympathetic paralysis are often anticholinergic drugs like scopolamine or atropine or oculoSympathetic stimulation can be a result of sympathomimetic drugs like cocaine or amphetamine .Mydriasis as a result of impairment of the cervical sympathetic nerve chain is described in only very rare cases , 5 and hHere, we present the case of a young woman with bilateral mydriasis as the first clinical manifestation of Hodgkin\u2019s lymphoma and enlarged pericarotid lymph nodes causing oculosympathetic spasm.A previously healthy 23-year-old woman presented with bilateral mydriasis Fig.\u00a0. The mydMRI scans of the brain 4\u2009weeks prior to admission and on day of admission conducted with gadolinium contrast were unremarkable.MRI scans of the cervical spine and chest 15\u2009days prior to admission showed enlarged lymph nodes in the mediastinum and supraclavicular on both sides.The result of histopathologic examination of a lymph node biopsy specimen showed classical Hodgkin\u2019s lymphoma.18F-FDG PET/CT performed for staging revealed further pathological lymph nodes on the left axillary, the right pelvic area and retroperitoneal. Mediastinal and cervical lymph nodes were found significantly enlarged compressing the carotid artery on both sides examination demonstrated a lymphocytic pleocytosis (10 cells/\u03bcl) with normal protein and glucose levels. Polymerase chain reaction (PCR) for herpes simplex virus (HSV) -1 and\u2009\u2212\u20092 was positive with low genome concentration (<\u2009500 genome copies/ml). Flow cytometric analysis of CSF leukocytes showed a normal cell distribution and no signs of meningeosis lymphomatosa.18F-FDG PET/CT for follow-up assessment and restaging showed a complete remission. There was a slight improvement of the bilateral mydriasis.Therefore, the patient was treated with 18\u2009day acyclovir therapy and a chemotherapy with escalated BEACOPP . After approximately four months and four cycles of escalated BEACOPP a new We describe the case of a patient presenting with bilateral mydriasis and first diagnosis of Hodgkin\u2019s lymphoma.Pathophysiologically, we consider the enlarged lymph nodes as irritating the pericarotid sympathetic nerves, causing oculosympathetic spasm and ultimately resulting in autonomic dysfunction clinically manifested by pupillary dilation.The pupillary dilator muscle is innervated by the sympathetic nervous system . The firPostganglionic sympathetic fibres ascend from the superior cervical ganglion, along the walls of the internal carotid artery and innervate the ciliary ganglion activating the pupillary dilator muscle.Pathologies of the brain stem as a potential differential diagnosis were clinically unlikely since all other brain stem reflexes, like the corneal reflex, remained intact and were eventually excluded by two unremarkable MRI scans of the brain.We assume the detection of HSV-DNA in the CSF with low genome concentration to be a reactivation, possibly due to altered immune system in Hodgkin\u2019s lymphoma disease, and in no causal link to the mydriasis.The mild improvement of the mydriasis after the start of chemotherapy supports our hypothesis. However, as in most observational case studies a causality cannot be proved with absolute certainty..Neurologic complications from Hodgkin\u2019s lymphoma are overall rare. Direct neurologic dysfunction can be caused by metastases of the central nervous system and depending on the site of metastasis result in cranial nerve palsies, headaches and gait disturbance. Indirect affection of the central or peripheral nervous system by paraneoplastic syndromes can cause cerebellar degeneration and clinically present with dysarthria, nystagmus and ataxia .The case presented herein shows a bilateral mydriasis as the primary clinical manifestation of Hodgkin\u2019s lymphoma.To the best of our knowledge, this is the first reported case of mydriasis in association with Hodgkin\u2019s lymphoma. Other reports described Horner syndrome in a patient with Hodgkin\u2019s lymphoma with enlarged mediastinal lymph nodes and oculIn conclusion, this case emphasizes the importance of detailed diagnostic, especially imaging of structures around the sympathetic pathway, in cases of autonomic dysfunction such as mydriasis."} +{"text": "Knowing which elements in the environment are associated with various opportunities and dangers is advantageous. A major role of mammalian sensory systems is to provide information about the identity of such elements which can then be used for adaptive action planning by the animal. Identity-tuned sensory representations are categorical, invariant to nuances in the sensory stream and depend on associative learning. Although categorical representations are well documented across several sensory modalities, these tend to situate synaptically far from the sensory organs which reduces experimenter control over input-output transformations. The formation of such representations is a fundamental neural computation that remains poorly understood. Odor representations in the primary olfactory cortex have several characteristics that qualify them as categorical and identity-tuned, situated only two synapses away from the sensory epithelium. The formation of categorical representations is likely critically dependent on\u2014and dynamically controlled by\u2014recurrent circuitry within the primary olfactory cortex itself. Experiments suggest that the concerted activity of several neuromodulatory systems plays a decisive role in shaping categorical learning through complex interactions with recurrent activity and plasticity in primary olfactory cortex circuits. In this perspective we discuss missing pieces of the categorical learning puzzle, and why several features of olfaction make it an attractive model system for this challenge. Because physical information arriving at sensory organs does not completely specify the structure of the environment, however, the brain is tasked with inferring what that structure is based on previous experience, a process that requires learning and memory. Knowledge about identity is useful not only for scene interpretation, but for assigning and predicting specific action-outcome contingencies that reduce uncertainty about environment dynamics. Identity-tuned representations are categorical in the sense that the neural response remains unchanged under variation along certain axes of the sensory input space. Categorical sensory representations that provide inference about stimulus identity in the presence of variable, incomplete or ambiguous sensory data are well established across a range of sensory and non-sensory cortical areas. Most of these responses are found in higher-order cortices, several steps away from the sensory input organs. This is in part because the sensory feature axes that these representations become invariant toward tend to be specifically encoded in lower-order cortices. It is hard to understand the transformations that take place during the formation of categorical representations when there are multiple intervening synaptic steps between sensory input and the neural response. The rodent olfactory system offers an attractive model system for studying categorical identity-tuned representations and their formation as this functionality is implemented already in the first cortical processing step, two synapses from the sensory organ.In a complex and changing world, sensory signals can be extremely rich in information and therefore challenging for an animal to process rapidly and adaptively. One way for the brain to mitigate this is through abstraction: sensory structure can be categorized by grouping relevant signals into simpler representations, thereby facilitating interpretation of the sensory scene. Sensory signals emanating from unitary phenomena such as objects, conspecifics or contexts tend to covary in unique ways, meaning correlation structures within the sensory scene can be exploited to encode Odor representations in the primary olfactory cortex are less like low-level feature detectors in other primary cortical counterparts, and more like representations found in higher-order cortices where representational structure principally reflects intrinsically relevant encoding axes such as perceptual categories.The rodent olfactory system has evolved to detect and perceptually classify a staggering range of volatile chemical configurations present in the environment. Olfactory sensory neurons (OSNs) in the nasal epithelium each express one of an estimated 1500 distinct genes devoted to the mouse olfactory receptor (OR) complement . There iOne synapse downstream from the olfactory epithelium, activity in the main olfactory bulb (OB) is organized into anatomically segregated glomeruli that are clusters of axons from OSNs that terminate on dendrites of mitral and tufted cells that then project directly to olfactory cortical areas. Each glomerulus receives inputs from a single type of OR-OSNs. This labeled line organization is not preserved one synapse further downstream in the primary olfactory cortex. Multiple cortical areas can be included in the term primary olfactory cortex (O1) on the criterion that they receive direct input from the OB. Here we focus on the anterior portion of O1 aO1, see . This rade novo odor representations depending on the situation. At the posterior end of O1 lies LEC, one of the main cortical inputs to the hippocampus. Information from the olfactory system therefore has an unusually direct route into the hippocampus. Although the O1 includes many distinct structures, in this perspective our focus is on the aO1 where characteristics of categorical learning have been best documented.Multiple cortical structures receive direct bulbar inputs and may thereby be considered part of the primary olfactory cortex. These include anterior olfactory cortex , pirifoIn the rat OB, exposure to a mixture of two monomolecular odor components causes neural cross-habituation to the isolated mixture components in subsequent exposures . OB respResponses to familiarized odor mixtures remain stable even if single mixture components are omitted, a process termed pattern completion . Patternper se. Instead, most natural odors are mixtures of molecules that become useful for adaptive behaviors through association. While individual odor molecules may belong to several distinct phenomena, it is the joint probability distribution of molecules across exposures that qualifies unique odor representations. But how do these representations form? Because of the high dimensionality and discrete nature of the olfactory input space, and the synthetic format of aO1 representations, it is experimentally straightforward to design unique odor stimuli for aO1. Using novel odor stimuli offers a way to carefully study the formation and characteristics of categorical representations within aO1.The rather unusual primary cortical attributes of aO1 make sense considering that natural odors are not monomolecular, and behavioral interpretation of natural odors is not usually defined by their molecular structure Categorical learning is a statistical and dynamic process that depends on experience and perceptual demands. It is thought that categorical representations emerge with experience through associative learning that extracts and binds together invariant feature axes from distinct stimulus configurations. To become separable in neural activity space, these feature axes should minimize overlap between distinct representations. Categorical sensory configurations can this way be thought of as distributions living in a high-dimensional feature space, and determining the combination of feature axes that best define and separate various distributions represents a statistical learning problem . Becausede novo odor representation? To incorporate a novel stimulus into an existing representation, the statistics of this representation must be changed to include the new mixture distribution. A functional requirement for generating de novo stimulus representations is to separate the novel stimulus representation from existing representations. Pattern completion in aO1, which reflects stimulus generalization, depends on recurrent circuits that implement attractor dynamics, and experimentally inhibiting recurrent connectivity in aO1 changes several features of the attractor landscape affects both behavioral and neural measures of odor discrimination and generalization. ACh selectively inhibits intrinsic activity in recurrently connected cortices without affecting afferent input responses , proposeNoradrenaline (NA) also affects odor learning and is involved in novelty processing. Like ACh, NA activation selectively suppressed aO1 recurrent activity while leaving afferent inputs unaltered , and wasde novo categorical representations. Abrupt destructive interference between new learning and previously learned representations is termed catastrophic forgetting [as opposed to advantageous forgetting receives a particularly strong direct projection from the hippocampus that might take part in novelty detection. However, the speed with which rats can detect and respond to odor novelty is faster than a single sniff (50\u2013100 ms) . InactivRodent olfaction provides an excellent model system for understanding how the brain flexibly generates novel categorical representations and how these are integrated with existing representations. The aO1 is a unimodal sensory area positioned only two synapses from the sensory organ, minimizing synaptic non-linearities that otherwise obscure transformations from sensory inputs to cortical responses. Unlike in the hippocampus, where novelty detection unfolds with exploration and is therefore more difficult to determine with temporal precision, novel odor stimuli and their detection by the experimental subject can be precisely timed. Novel odor stimuli with predefined feature correlations can readily be designed using odor mixtures, making it trivial to repeatedly provide novel stimuli to the same subject. It may therefore be possible to disentangle activity motifs on the population level that are specific to novelty detection and continual categorical learning versus the stimuli themselves.Several outstanding questions about categorical learning in aO1 remain.(1)de novo odor representations, and if so, does that reformatting reflect statistical learning rules that dynamically optimizes separability between representations?Do familiar odor representations in aO1 undergo reformatting in response to (2)How does neuromodulatory activity change population dynamics on short (seconds) and intermediate (minutes-hours) timescales within aO1?(3)Can catastrophic forgetting of established odor representations be induced by manipulating modulatory activity in aO1 during continual learning?(4)How does aO1 activity develop across sniffs during first exposure to a novel odor?(5)What distinguishes subsections of aO1 activity during categorical learning; is acquisition and storage of representations implemented by separate neural populations?3 neurons (Making progress will require recording large ensembles of aO1 neurons that are trackable across recording sessions. Furthermore, understanding the dynamics of population activity on the timescale of individual sniffs requires that the recording technique offers high temporal resolution, and that odor delivery is controlled with high temporal precision. Recent advances in electrophysiological recording techniques, such as Neuropixels silicon probes that all neurons , offer a neurons and a goIn conclusion, categorical learning represents a fundamental neural computation that is attractive on several levels across several academic disciplines, and the olfactory system offers unique experimental advantages toward understanding this neural computation.The original contributions presented in this study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.TS and HS conceptualized and wrote the manuscript. Both authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."} +{"text": "Zhou and colleagues are commended for their innovative research on the tolerability of \u201clow-viscosity\u201d fibre supplements in symptomatic diabetic gastroparesis patients . Using aFurthermore, recent reports have found the treatment efficacy of gastric peroral endoscopic myotomy for gastroparesis to substantially diminish over time , and theAnother clue in solving the gastroparesis riddle are the landmark observations by Burkitt and others who correlated low stool weights with impaired intestinal transit times (ITTs) . For insBased upon these reports and our experience , we beli"} +{"text": "The influenza-specific antibody repertoire is continuously reshaped by infection and vaccination. The host immune response to contemporary viruses can be redirected to preferentially boost antibodies specific for viruses encountered early in life, a phenomenon called original antigenic sin (OAS) that is suggested to be responsible for diminished vaccine effectiveness after repeated seasonal vaccination. Using a new computational tool called Neutralization Landscapes, we tracked the progression of hemagglutination inhibition antibodies within ferret antisera elicited by repeated influenza A/H3 infections and deciphered the influence of prior exposures on the de novo antibody response to evolved viruses. The results indicate that a broadly neutralizing antibody signature can nevertheless be induced by repeated exposures despite OAS induction. Our study offers a new way to visualize how immune history shapes individual antibodies within a repertoire, which may help to inform future universal influenza vaccine design. Thenfection C, demonsIn summary, by tracking the changes in the inhibition profile of ferret antisera induced by repeated influenza A/H3 infections, we demonstrated that a broadly neutralizing antibody could be guided along the map after a series of infections. We further show that prior immune history can heavily influence the ferret antibody repertoire. In ferrets exposed to two or more viruses, a broadly neutralizing antibody signature that potently inhibited all infection strains was, nevertheless, produced at the expense of de novo HAI antibodies H. While"} +{"text": "Experiencing a greater number of stressful days is associated with a heightened risk of cardiovascular disease. This study examined whether positive affect reactivity, the trait-like change in positive affect in response to daily stressors, moderates the association between the number of stressful days and blood pressure. Participants were 664 adults from the National Study of Daily Experiences II (Midlife in the United States sub-study). Participants reported stressors and positive affect across 8 days and provided resting blood pressure measures during a separate clinic visit. A greater number of stressful days was associated with increased systolic blood pressure, but only among individuals with higher positive affect reactivity . Results suggests that individuals who maintain positive affect when experiencing stressors may have lower risk of heightened systolic blood pressure, contributing to the growing evidence that positive affective reactivity may be protective against daily stress."} +{"text": "Pteropus medius bat roosts identified near the locations of human Nipah cases in Bangladesh during 2012\u20132019. Pooled bat urine was collected from 23 roosts; 7 roosts (30%) had >1 sample in which Nipah RNA was detected from the first visit. In subsequent visits to these 7 roosts, RNA was detected in bat urine up to 52 days after the presumed exposure of the human case-patient, although the probability of detection declined rapidly with time. These results suggest that rapidly deployed investigations of Nipah virus shedding from bat roosts near human cases could increase the success of viral sequencing compared with background surveillance and could enhance understanding of Nipah virus ecology and evolution.Knowledge of the dynamics and genetic diversity of Nipah virus circulating in bats and at the human-animal interface is limited by current sampling efforts, which produce few detections of viral RNA. We report a series of investigations at Henipavirus) that has caused outbreaks of neurologic and respiratory disease in humans and livestock in Bangladesh, India, Malaysia, Singapore, and the Philippines is the major reservoir of Nipah virus in Bangladesh and India at any given time values from qRT-PCR were not reported for all tests, we used the proportion of positive aliquots as a proxy for the intensity of virus shedding in bats, assuming that roosts with higher virus concentrations in urine would produce more positive aliquots. We then analyzed changes in the proportion of positive aliquots across roosts along 2 time axes. We aligned dates to the number of days since the presumed exposure date of the first human spillover Nipah case associated with each roost site. We then aligned roost-sampling dates to the number of days since the start of the calendar year for comparison. We fit binomial linear models to estimate the probability of detecting a Nipah virus\u2013positive aliquot at each roost along each time axis.P. medius bat roost in Faridpur District during 2007\u20132012 as part of a longitudinal study; from visits to different roosts throughout Bangladesh during 2006\u20132011 as part of a cross-sectional spatial analysis; or as part of Nipah virus outbreak investigations in 2009, 2010, and 2012. Urine samples were either collected from individual bats or from underneath roosts. For these comparisons, we considered each roost visit as a discrete sampling event, including repeat visits to the same roost. Ignoring the initial visits to 7 roosts near 5 suspected human cases that were Nipah virus\u2013negative, the 23 roosts in our study were sampled across 47 visits. We made comparisons between studies for the number of sampling visits with positive Nipah detections and the number of positive urine samples across all sampling visits or during the first visit to each roost. We evaluated comparisons by using a \u03c72 test of proportions or Fisher exact test. We considered statistical tests significant if p values were <0.05.To evaluate the utility of sampling bat roosts near human Nipah virus cases as a surveillance approach, we compared the rate of successful Nipah virus detections from this study to data reported by Epstein et al. was 0.66 (95% CI 0.42\u20130.84) . This prRoost urine samples from our study and individual urine samples from longitudinally sampled roosts in Epstein et al. ; the detNipah virus spillover from bats occurs sporadically in Bangladesh, so surveillance that optimizes viral detection in bats is a challenge. In contrast with cross-sectional or longitudinal bat roost surveillance used previously . RepeateThis study also provides critical information about the timing of Nipah virus shedding in bats in Bangladesh. Longitudinal surveys have shown that Nipah virus shedding from bats is sporadic throughout the year Table 2.P. medius across Bangladesh, but increasing the number of detections is still crucial, especially given the few Nipah virus isolates currently available (n = 11). Reactive roost investigations could be complemented with additional roost surveys outside of surveillance areas to learn more about Nipah virus transmission and genetic diversity in bat populations across Bangladesh.Our case investigations were also limited to the catchment area of 3 surveillance hospitals and the winter seasonality of Nipah virus spillover surveillance. This design systematically misses virus shedding events at bat roosts outside the surveillance area or during seasons when humans are not drinking fresh date palm sap (This study provides proof of concept that reactive investigations of bat roosts near human Nipah virus cases can complement ongoing surveillance efforts and could increase the likelihood of viral detection and sequencing. Improvements in virus detection would aid in characterizing the genetic diversity of Nipah viruses circulating in bats and identify novel genotypes that might pose pandemic threats. Furthermore, these data provide evidence that viral shedding can continue for weeks after an initial spillover event, posing a hazard for additional contamination. Precise knowledge of when bats are shedding Nipah virus could be used to deploy public health campaigns more efficiently, such as by using barriers to prevent bat access to date palm sap (Additional information about Nipah virus detection at bat roosts after spillover events, Bangladesh, 2012\u20132019Additional data used in study of Nipah virus detection at bat roosts after spillover events, Bangladesh, 2012\u20132019"} +{"text": "Addressing physical inactivity for older Hispanics/Latinos with mild cognitive impairment (MCI) is a public health priority, since MCI increases the risk of developing Alzheimer\u2019s Disease and related dementias (ADRD). Compared to non-Latino Whites, Hispanics/Latinos are 2X more likely to develop ADRD. One promising approach to promoting physical activity includes use of mobile health strategies that may deliver Spanish-language information and culturally relevant motivational messages to enhance intrapersonal/interpersonal factors for health behavior change. The purpose of this study is to test mHealth strategies as a mechanism to deliver booster sessions for reinforcing physical activity goals/progress among older Latinos with MCI who complete the Tiempos Juntos intervention treatment. Our hypothesis is that culturally adapting mHealth strategies may improve efficacy and maintenance of physical activity effect; as well as cognitive health outcomes (6 months post-intervention). Results among the first wave of participants will be discussed, including challenges and opportunities for future research. While the use of mHealth is not new, these approaches commonly exclude individuals with limited-English proficiency and thus, designing interventions that center the needs of older Hispanics/Latinos with MCI/ADRD and other historically excluded communities, is an important first step in promoting physical activity and advancing cognitive health equity."} +{"text": "The authors contacted the Editorial Office requesting a retraction due to an oversight in the clinical trial registration process and the recruitment of subjects before it was approved, which is not in accordance with formal clinical trial procedures. All authors agreed to retract this article.The article, \u201cThe 95% effective dose of nalbuphine in patient-controlled intravenous analgesia for patients undergoing laparoscopic total hysterectomy compared to equivalent sufentanil\u201d,"} +{"text": "Very old adults (80+) are the fastest growing population worldwide. Children of the very old adults may see their prolonged relationship with parents as a benefit but also as burden . Using a sample of older children (N = 219) from the Boston Aging Together Study and Korean Aging Together Study, we investigated the factors associated with older children\u2019s (aged 62\u201376) reports of burden in their relationships with very old parents (aged 81\u2013101), focusing on how family relations were imbedded in different cultural contexts. Overall, American older children showed lower levels of burden, compared to Korean older children. The factors associated with burden differed by country; support given to parents and relationship quality were associated with American older children\u2019s burden, whereas support received from parents, familism, and negative relationship quality were associated with Korean older children\u2019s burden."} +{"text": "Dendrocopos major were found exploiting galls of the elm balloon\u2010gall aphid Eriosoma lanuginosum by eating both the insects and their honeydew. Literature offers sparse and indirect information to explain this behavior. Taking both aphids and honeydew from within galls is reported here for the first time for a bird species. Only one other vertebrate, the Eurasian red squirrel Sciurus vulgaris, would show similar behavior. In contrast with the seeming rarity of such a feeding way, likely advantages in the double food source suggest it may have been overlooked.An adult female and a young great spotted woodpecker Great spotted woodpeckers were found exploiting galls of the elm balloon\u2010gall aphid by eating both the insects and their honeydew, a behavior reported here for the first time for a bird species. In contrast with the seeming rarity of such a feeding way, likely advantages in the double food source suggest it may have been overlooked. She flew directly to an exposed cluster of galls in a growth of field elms 3Sciurus vulgaris would be the only other vertebrate behaving similarly, when the number of aphids and the amount of honeydew in galls of the elm\u2010currant aphid Eriosoma ulmi are largest ; Investigation (lead); Validation (lead); Visualization (lead); Writing \u2013 original draft (lead); Writing \u2013 review & editing (lead).None declared."} +{"text": "Gusher or perilymphatic fistula is a complication seen during stapedectomy or stapedotomy. It is characterized by sudden perilymphorrhage immediately after platinotomyA female 38 year old patient complained of mostly left hypoacusis for the past 4 years; mild bilateral tympanic membrane retraction was present. Audiometry disclosed moderate left conduction loss and slight right conduction loss; immitance testing revealed an A curve and absence of stapedian reflexes bilaterally. The diagnosis was otosclerosis and left stapedectomy was recommended. Perilymph gushed out during microperforation of the platina of the stapes (gusher). The procedure was interrupted and fat from the left auricle was used for orifice tamponing, which stopped lymphorrhage.This complication of stapedotomy is an unexpected event and there are no warning signs that could alert the surgeonGusher is a rare complication of otological surgery. When present, it becomes difficult to complete the surgical procedure; no true benefits have been observed to justify continuing the operation."} +{"text": "Given that assessment tools vary widely across researchers and clinicians, it has been daunting to identify distinct patterns in outcomes across diverse cancer types and to implement systematic neurocognitive screening tools. This review aims to operationalize A comprehensive literature review was conducted to examine the existing research on cognitive late effects and biopsychosocial risk factors in order to conceptualize processing efficiency skill trends in childhood cancer survivors.While a frequently reported pattern of neurobiological (white matter) and cognitive (working memory and processing speed) disruption is consistent with processing efficiency skill impairment, these weaknesses have not yet been fully operationalized in this population. We offer a theoretical model that highlights the impacts of a host of biological and environmental factors on the underlying neurobiological substrates of cancer survivors that precede and may even predict long\u2010term cognitive outcomes and functional abilities following treatment.The unified construct of processing efficiency may be useful in assessing and communicating neurocognitive skills in both outcomes research and clinical practice. Deficits in processing efficiency may serve as a possible indicator of cognitive late effects and functional outcomes due to the unique relationship between processing efficiency skills and neurobiological disruption following cancer treatment. Continued research along these lines is crucial for advancing childhood cancer outcomes research and improving quality of life for survivors. Skillful processing efficiency enables one to think and learn effectively, as cognitive resources in the mental workspace can be \u201cfreed up\u201d to address more challenging cognitive tasks as they arise. Processing efficiency skills can be considered a distinct construct separate from the umbrella classification of executive functioning skills posits that anxiety and worry significantly disturb task performance; processing efficiency skills decline in anxious states due to the investment of additional cognitive resources to maintain accuracy, with both working memory and processing speed uniquely contributing to this effect . In fact, the effects of aggressive cancer treatments can be conceptualized as an acquired brain injury due the significant impacts on cerebral white matter and vasculature , treatment approach , and time off treatment. Over time, disruptions to developing neurobiological substrates coupled with external factors and general increase in cognitive and environmental demands as survivors age ultimately lead to the presentation of cognitive late effects. These long\u2010term cognitive deficits are observed in a significant portion of childhood cancer survivors (estimates up to 60%) and lead to functional impairment across settings, including academic, vocational, social, and general adaptive skills , as intact processing efficiency skills are believed to \u201cfree up\u201d cognitive reserve for higher\u2010order tasks. It is unlikely that deficits in processing efficiency directly cause these other cognitive deficits; rather, processing efficiency may act as a tool for detecting early disruption in neurocognitive functioning that may later fully manifest as these downstream cognitive late effects.Another clinically meaningful application of this work will be to explore the translational impacts of individual differences in processing efficiency deficits in pediatric cancer survivors. It is anticipated that as deficits in processing efficiency increase, there will be greater likelihood of impairments across activities of daily living and quality of life, ultimately driving need for additional services, clinical care, and follow\u2010up. Individual differences, such as demographic and environmental factors, are expected to drive some of the variability in processing efficiency skills following cancer treatment. Neuroimaging research specifically investigating the relationships between processing efficiency skills and white matter integrity and volume in survivors would be especially compelling. Longitudinal investigations of processing efficiency across time will yield crucial evidence for understanding the trajectories of disruptions to cognitive skills and possibly provide clues for the changes to underlying neurobiological substrates following cancer treatment.Interventions may specifically target processing efficiency skills in this population. For example, a cognitive remediation program focused on targeted skill development in the domain of working memory yielded positive benefit that translated to improvements in parent\u2010reports of learning problems among a group of school\u2010aged ALL and brain tumor survivors (Hardy et\u00a0al., 8Research on cognitive late effects among survivors of pediatric cancer has gained recent and warranted momentum. Processing efficiency, an integrated and nuanced cognitive skill that has the potential to be an emerging hallmark sign of cognitive disruption in this population, remains largely understudied. Within this review, we have presented compelling theoretical evidence for the utility of processing efficiency assessment in pediatric cancer survivors. A novel conceptual framework for considering and describing processing efficiency skills among childhood cancer survivors has been proposed. It is well\u2010documented that biological influences, treatment approach, age of onset, and time off treatment are each uniquely associated with cognitive late effects, and that these risk factors are suspected to act through disruption to neurobiological substrates\u2014primarily, white matter disruption. The framework offered here outlines the associations between neurobiological disruptions and the increase in environmental and cognitive demands that typically accompany cognitive late effects. Utilizing a standard approach for assessing and communicating processing efficiency skills across outcomes research and within routine clinical practice may allow for distinct patterns in neurocognitive outcomes to surface, with findings that may hold significant translational impacts for cognitive and adaptive outcomes. More ambitiously, outcomes of a processing efficiency screening measure may even serve as unique and sensitive tool for monitoring the emergence of cognitive late effects. Future directions of this line of research should take aim at providing empirical support for this characterization of processing efficiency impairment in childhood cancer populations while also considering associations with complex biopsychosocial factors known to impact survivorship outcomes.The authors of this paper have no conflicts of interest to declare, financial or otherwise. All contributors have read and approved this submission.https://publons.com/publon/10.1002/brb3.2809The peer review history for this article is available at"} +{"text": "Global shocks from the COVID 19 pandemic have disproportionately impacted children and young people (CYP) and their families. Underlying health disparities have widened as a result of disruptions to healthcare, education and economic impacts. There are compelling arguments to engage children and young people in rebuilding society and systems from emerging public health threats including the covid 19 pandemic, the climate crisis and conflicts. Children and young people like others in society have a right to be involved in decisions that impact their lives and health but are too often unheard. Engaging those affected most by health policy ensures relevance and can improve adoption of interventions. Involving children and young people in strategic decision making can also improve their citizenship skills and improve health literacy. Although many children and young people have contributed to debates and publicly demonstrated their collective views on a variety of issues such as climate change, movements including #timesup, #Blacklivesmatter and #Metoo, their voices are seldom heard in policy and decision making arenas. In 2021, EUPHA CAPH and EUPHANxt sections collaborated to host the inaugural Engaging the Unheard Stakeholder workshop, supporting 2 young people aged <25 to attend the EPH and lead the workshop. (links). CAPH is now committed to make this a regular feature as part of our action plan to support young voices in public health.\u2022\u2002Develop insight into innovative ways to support and engage young people in public health policy making.\u2022\u2002Practise advocacy skills to facilitate young people's voices in research & policyParticipants will hear from young people who are championing activism or have been involved in shaping public policy.Interactive skills building breakout session involving role play and scenarios to challenge their unconscious bias and learn skills as \u2018active bystanders\u2019 to empower young people\u2022\u2002Despite major movements where children and young people have demonstrated their views on a variety of issues such as climate change, their voices are seldom heard in policy and decision making arenas.\u2022\u2002Public health professionals can advocate, support and engage children and young people in public health policy making but lack awareness and skills in advocacy to do this effectively."} +{"text": "Societies nowadays, including Greece, are usually multicultural. Health professionals should therefore be properly trained to consider patients\u2019 beliefs, attitudes and particular needs depending on their different cultural background.To identify the features that the culturally competent professional should have in order to understand better the nature of cultural competence and its importance to mental health professionals in early intervention of immigrants\u2019 mental health problems.A literature review has been made through PubMed database.The development of cultural competence is a continuous process. Culturally competent professionals should have the following features: a) Understand the concept of culture and the way individuals\u2019 cultural background affect their feelings and their intercultural interactions. b) Choose appropriate collaboration strategies with people from different cultural backgrounds. c) Accept diversity and respect patients\u2019 differences, demands and choices without criticism while providing them the proper care. d) Be fair and take care of all patients without any distinction regardless of the language they speak. e) Familiarize themselves with issues related to mental health and illness and encourage patients to explain how their illness affects their lives. Culturally competent mental healthcare professionals should seek more than the provision of caring without prejudice. They should respect the positive contribution of cultural origin and identity to people\u2019s well-being, learn their life stories and develop a relationship of trust with each patient separately.Cultural competence might help mental health professionals to understand and provide adequate services with respect to patients who come from a different cultural background.No significant relationships."} +{"text": "Natural and manmade emergencies have become more frequent over the past 20 years and will continue to pose serious risks to public health and safety. Older adults are more adversely affected by\u2014and less prepared for\u2014emergencies than younger adults. However, little is known about the risk factors impeding emergency preparedness among older adults. This study uses the ecological systems approach to explore factors associated with emergency preparedness and how those factors influence adults 60 and older. The study analyzed cross-sectional data taken from 690 community-dwelling older adults who participated in Wave 5 of the National Poll on Healthy Aging. The sample was broken down into the following two groups for comparison: individuals aged 60\u201369 (n = 383) and individuals 70 or above (n = 307). The self-reported measures of sociodemographic characteristics, physical health, mental health, and previous experience of a disaster were utilized via regression analysis to predict emergency preparedness. Emergency preparedness was assessed using X dichotomous questions . The results revealed that living alone and having a Hispanic background were negatively associated with emergency preparedness among those aged 60\u201369, while mental health status was positively associated with emergency preparedness among those aged 70+. Previous experience of a disaster positively impacted emergency preparedness among the sample. Implications for policy and practice focus on shifting the perspective of the disproportional risks for older adults around emergencies to one that values and supports older adults\u2019 strengths and insights."} +{"text": "The changes preceding and initiating uncomplicated separation of the posterior hyaloid membrane (PHM) from the retina and,The pathological variations of this process that influence so many of the vitreoretinal disorders dealt with today and thereby their management.The relationship between the vitreous body, posterior vitreous detachment (PVD) and retina has been the focus of much research for over 100 years [Historically, much has been theorised on the combined biological changes of syneresis and synchisis leading in some ill-defined manner to weakening adhesion at the vitreoretinal interface, inducing the outer vitreous cortex to separate from the inner limiting membrane of the retina. In contrast, more recent research \u201314 has dThe patient was referred aged 32 with a serous macular detachment linked to an optic dis pit and vision reduced to counting fingers (CF). The vitreous was attached and the patient underwent pars planar vitrectomy, endolaser and gas tamponade which was not successful in resolving the serous detachment and her vision remained at CF. After further consultation the patient opted for a further attempt at pars planar vitrectomy, endolaser and gas tamponade with a different surgeon, but with the same result and failure to close the link with persistent serous macula detachment.A year elapsed without change and the patient requested a third attempt at vitrectomy, gas tamponade and endolaser. On this occasion, the surgery was successful in resolving the macular detachment and surprisingly her vision improved from CF to 6/9.Her crystalline lens remained clear for a further 10 years until she required cataract surgery which was uneventful and restored her vision to 6/9 which she retained for a further 10 years.Twenty years after her three previous vitrectomy procedures she re-presented with temporal photopsia, a sudden onset of new floaters and a shadow in her vision. Examination revealed a detached PHM, including a Weiss ring and an inferior rhegmatogenous retinal detachment with a single horseshoe tear. The detachment did not involve any of the area previously associated with the optic disc pit and was successfully repaired with a combined vitrectomy and buckle procedure and has remained stable for 2 years with a visual acuity of 6/12.in absentia.This case highlights the curious but well recognised phenomenon that \u201ctrue\u201d PVD (separation of the PHM) can occur independently from, and in the absence of, the vitreous body\u2014PVD When it occurs, PVD is usually benign causing few or minor symptoms in most individuals but it can also be the precursor to more serious secondary pathological conditions including but not exclusively, retinal tears, retinal detachment, cellophane maculopathy, vitreomacular traction syndrome and macular hole.Research over the last 20 years \u201313 has iThe cellular factors differentiating benign from pathological PVD remain poorly understood. Our current research is investigating the baseline functional transcriptome characteristics of laminocytes within the PHM associated with uncomplicated, physiological PVD when compared with PVD in association with pathological vitreoretinopathies such as cellophane maculopathy, macular hole and retinal detachment using RNA sequencing (RNA-Seq). PHM samples from patients with either uncomplicated or pathological PVD collected during surgery have been processed for RNA extraction, library preparation and next generation sequencing. Provisional differential gene expression analysis shows significantly marked differential of expressed genes (DEG) in all pairwise analyses with the overwhelming majority of the DEGs upregulated in each of the pathological PVD groups Fig.\u00a0.Fig. 2VoGene ontology enrichment analysis is now underway to identify the biological processes and molecular functions that may be over (or under) represented in derived DEG sets in conjunction with a secondary batch of RNA-Seq on fresh and historical tissue samples being analysed using spatial transcriptomics. This innovative technology permits the measurement of gene transcripts \u201cin situ\u201d at distinct spatial locations in the PHM at near-cellular resolution, with the potential to provide a more comprehensive and unique transcriptomic profile of the laminocyte than can be obtained from bulk RNA-Seq alone. Analyses of these singular and combined approaches should provide new insights on a near single cell basis differentiating physiological from pathological PVD."} +{"text": "Nearly 30% of adults aged 60 or older suffer from knee osteoarthritis (KOA) that causes significant pain and disability. Walking is considered a \u201cgold standard\u201d treatment option for reducing KOA pain and maintaining joint mobility. However, pragmatic trials have shown that walking increases pain for some and relieves pain for others. The mechanism by which walking is helpful for KOA pain is unclear. The purpose of this study was to gain a better understanding of the mechanisms underlying walking for knee pain. We conducted a pre-test/post-test study using quantitative sensory testing to measure pressure pain sensitivity at the knee and examined protein signatures and measured energy metabolism in platelets in six adults with KOA before and after six weeks of walking three days/week at 100 steps/minute. All participants identified as Black/African American, five were female, average age 57\u00b15.8. Pressure pain sensitivity increased for three participants and decreased for three participants. Protein signatures among KOA participants indicated differences in immune and energy metabolism pathways. Proteins in the energy metabolism pathways were significantly downregulated after walking in participants whose pain increased compared to participants whose pain decreased. Platelet energy metabolism was also lower among participants whose pain increased as compared to participants whose pain decreased. One goal of developing individualized interventions for KOA pain is to elucidate the mechanisms by which self-management interventions impact pain. The addition of therapies that target cellular energy metabolism may lower pain with walking among Black adults with KOA."} +{"text": "The use of agrochemicals is increasingly recognized as interfering with pollination services due to its detrimental effects on pollinators. Compared to the relatively well-studied chemical toxicity of agrochemicals, little is known on how they influence various biophysical floral cues that are used by pollinating insects to identify floral rewards. Here, we show that widely used horticultural and agricultural synthetic fertilizers affect bumblebee foraging behavior by altering a complex set of interlinked biophysical properties of the flower. We provide empirical and model-based evidence that synthetic fertilizers recurrently alter the magnitude and dynamics of floral electrical cues, and that similar responses can be observed with the neonicotinoid pesticide imidacloprid. We show that biophysical responses interact in modifying floral electric fields and that such changes reduce bumblebee foraging, reflecting a perturbation in the sensory events experienced by bees during flower visitation. This unveils a previously unappreciated anthropogenic interference elicited by agrochemicals within the electric landscape that is likely relevant for a wide range of chemicals and organisms that rely on naturally occurring electric fields. Significance StatementFlowers exhibit morphological and biophysical adaptations that attract pollinators. A recently discovered ability of several insect species to detect and use electric fields surrounding flowers uncovered the biological significance of natural electric fields. Here, we show that commonly applied synthetic fertilizers and the neonicotinoid pesticide imidacloprid alter the biophysical properties of flowers, affecting both the magnitude and the dynamics of floral electrical cues. These changes result in reduced bumblebee foraging, reflecting a perturbation in the sensory events experienced by bees during flower visitation. Linking fertilizer application to an alteration in pollinator behavior reveals the direct interference by human-made chemicals on how an organism can perceive its physical environment and offers insights into potentially wide-spread perturbations of the biologically relevant electrical environment.Flowers produce a diverse range of cues and attractants to pollinators that collectively promote localization and pollination . These cThe widespread use of chemicals in agriculture and horticulture constitutes a pervasive and substantial source of pollution , 15. AgrSpray applications with insecticides and fungicides have indeed been linked to an immediate temporary decline in bee foraging , 29, as Spray applications conceivably affect a wide range of biological and physical properties relevant to flower-pollinator interactions that can involve both the plant surface as well as its surrounding air . The mag2O) containing realistic fertilizer concentrations and a control dH2O-only application were measured on a background of white paper with self-adhesive covering film. This spectrophotometric test revealed no difference between treatments in purple molds in laboratory flight arenas where the surrounding feeding platforms were sprayed F-dH 2O (5\u2009mL/L\u22121). Sucrose consumption was quantified relative to control feeding platforms sprayed with dH2O-only (see \u201cGeranium pratense) with F-dH2O and subsequently coated the flowers with positively charged, colored particles released as an aerosol close to the corolla. The electrostatic deposition pattern of colored particles consistently differed between untreated flowers and flowers treated with F-dH2O in response to a fertilizer spray application. Although a measurement closer to the flower is required to capture the true magnitude of floral E-field change, this measurement revealed that spray applications of fertilizers can instantly increase floral E-fields followed by a slow decay over the course of several minutes of flowers and thereby misguide bees. To test this, the reflectance spectra of spray applications with demi-water . Although it remains speculative how electric alterations operate at larger spatial and temporal scales, the potential of human-made chemicals to alter a complex set of interlinked biophysical processes likely carries implications beyond pollination since natural E-fields are pervasive and intrinsically linked to a wide array of organisms and biological processes , 44\u201349.pgac230_Supplemental_FilesClick here for additional data file."} +{"text": "Policy choices, aims and priorities are defined by the dominant belief system among stakeholders in a policy area . Traditional assessments of health systems performance have focused on the institutional configurations, processes and resources in place . We argue that the more latent dimension of \u2018stakeholder perceptions\u2019 is crucial to understanding why seemingly effective health system policy designs underperform during implementation. This paper brings in new evidence (2020) from an elite population study of 261 stakeholders in Public Health policy in Greece . We questioned stakeholders on: their conceptual understanding of public health, public health determinants, the field\u2019s policy responsibilities and jurisdiction, threats and enabling factors to policy advancement, and public health systemic performance in Greece. Our findings highlight that: 1) stakeholder beliefs converge regarding drivers for effective policymaking - most prominently resources, expertise, coordination and management - but respondents also identify their scarcity in the Greek public health system (GPHS); 2) stakeholders converge regarding systemic pathogenies - most prominently managing inequalities and monitoring - and rank all legally stated functions of the GPHS poorly on average; 3) stakeholders agree that the existing paradigm in Greek public health policy does not promote the holistic perspective to health but most maintain a medicine-centric view of public health. We conclude that despite Greece enjoying a progressive public health system policy design, the mismatch between the system\u2019s stated aims and the prevailing anachronistic stakeholder perceptions has hampered effective policy development. Future research should zero in on the importance of installing a co-oriented culture among stakeholders to achieve success in the performance of health systems.\u2022\u2002Stakeholder perceptions are crucial to understanding why seemingly effective health system policy designs under-perform during implementation.\u2022\u2002Despite Greece enjoying a progressive public health system policy design, the mismatch between the stated aims and the prevailing anachronistic stakeholder perceptions has hampered policy development."} +{"text": "They acknowledge that most surgeons rely on clinical judgment when performing revisionary cases of this type and that complications may result from an assumption that the dominant circulation emanates from medial intercostal vessels arising from the internal mammary artery. They site 8 consecutive cases, the majority of which were reconstructive to demonstrate their point that preoperative MRI prior to revision augmentation/mastopexy prevents nipple-areolar compromise and wound healing complications (Video). Four cases were presented in detail differing in clinical detail. History, MRI data, operative plan, and 2-week results were shown. Patients 1 and 3 had prior augmentation and periareolar mastopexies, Patient 3 had multiple revision augmentations and a prior circumvertical mastopexy, and Patient 4 had nipple-sparing mastectomies and device reconstruction without any prior mastopexy. None of the cases required significant elevation of the nipple or extensive undermining of skin flaps. In most of the cases, there appeared to be significant blood supply from medial perforators and in Case 3 from medial and lateral perforators. I could see no reason why either perforating system would require disruption in any of these cases, so I remain unconvinced that the preoperative MRI was of clinical relevance here. I appreciate that some surgeons may take comfort from knowledge of the existing blood supply in revisional breast cases like the ones demonstrated but see no reason for the expense unless the surgeon intends to incise a nipple pedicle for rotation or transfer to the upper pole of the breast. In my experience, this is generally not warranted and unwise. There may be cases where a device was placed via an upper hemispheric periareolar incision dividing the superior blood supply with a periareolar mastopexy and the patient requires an exchange with nipple transposition and a more formal vertical or inverted T mastopexy. Preoperative MRI would clearly be beneficial in cases such as these.This paper attempts to illustrate the value of preoperative magnetic resonance imaging (MRI) in helping surgeons plan secondary augmentation/mastopexy.ojac075_Supplementary_DataClick here for additional data file."} +{"text": "Sander lucioperca is an important fish species in the development of European aquaculture. The aquaculture of a fish species can be facilitated by knowing the genetic variability within and among populations. Here, we assessed the genetic background of 8 wild populations along with 13 broodstocks of pikeperch through a combination of genetic markers. We underlined that current broodstocks have a genetic diversity similar to wild populations. When focusing on genetic differentiation, we highlight that European pikeperch populations are divided into two groups: one predominantly present in Northern Europe and around the Baltic Sea and another mainly in Central Europe. Broodstocks appear to contain fish of a single origin with only a few exceptions. Ultimately, we have proposed baseline information about genetic diversity of pikeperch along with a genetic tool that can help pikeperch producers manage and improve their fish stock.The pikeperch b gene sequences were also used to infer phylogeographic relationships. Results show that on average, the domesticated populations do not exhibit significantly lower levels of genetic diversity compared to the wild ones and do not suffer from inbreeding. Nuclear data provide evidence that pikeperch populations in Europe belong to at least two genetically differentiated groups: the first one is predominantly present in Northern Europe and around the Baltic Sea, while the second one comprises populations from Central Europe. In this second group, Hungarian origin populations constitute a differentiated stock that needs special consideration. Aquaculture broodstocks analyzed appear to contain fish of a single origin with only a few exceptions.The pikeperch is a freshwater/brackish water fish species with growing interest for European aquaculture. Wild populations show signs of decline in many areas of the species natural range due to human activities. The comparative evaluation of genetic status in wild and domesticated populations is extremely useful for the future establishment of genetic breeding programs. The main objective of the present study was to assess and compare the genetic variability of 13 domesticated populations from commercial farms and 8 wild populations, developing an efficient microsatellite multiplex tool for genotyping. Partial cytochrome Sander lucioperca) is a temperate predatory freshwater fish species that tolerates brackish waters )For the studied domesticated pikeperch populations, our results show that current fish stocks do not seem to suffer from inbreeding nor strong genetic diversity loss. This means that (i) current broodstock management methods seem to allow maintaining genetic diversity within stocks, and (ii) current domesticated fish stocks could provide a solid basis for future selective breeding programs.The present overview of genetic background in wild and domesticated pikeperch populations could be further supported by complementary analyses, including higher sample sizes and stocks originating from a broader distribution area as well as a higher number of genetic markers (such as SNPs) coming from whole genome or reduced representation sequencing methodologies.Current results already provide valuable insight for fish farmers. First, knowing genetic divergences between domesticated fish stocks paves the way (i) to develop accurate pedigree assignment and . Nevertheless, this can result in outbreeding depression . IndeedThird, considering genetic divergences between wild and domesticated populations can potentially improve the prevention of environmental risk near farming locations. Special attention should be paid to the risk of escapees in areas where farmed stocks and wild neighboring populations are genetically distinct to avoid nuisances observed in other fish species e.g., ,60). Wit. Wit60])"} +{"text": "Australia has a universal health insurance scheme covering part costs for private mental health care and which supports the public system. The Medical Benefits Schedule (MBS) schedule provides a recommended fee for each service, the amount the Australian Government thinks the service should cost. Many patients still pay a gap fee for the service. Similarly a system for medications, the Pharmaceutical Benefits Scheme (PBS) subsidises the cost of medicines for most medical conditions. As new evidence emerges in the treatment of psychiatric conditions, it is important that the MBS and PBS are updated so patients receive subsidised best practice treatment.To provide an overview of RANZCP efforts to expand treatment availability through evidence and advocacy to government.The RANZCP made submissions to the independent Medical Services Advisory Committee (MSAC) requesting an MBS listing for repetitive transcranial magnetic stimulation (rTMS) for treatment of antidepressant medication-resistant major depressive disorder. Submissions were made to the independent Pharmaceutical Benefits Advisory Committee (PBAC) to request ability to prescribe quetiapine in 25mg ranges for maintenance therapy.Following RANZCP submissions, the MSAC supported public funding for initial treatment with rTMS for adults with major depression who have tried antidepressant medicine or psychological therapy and remain unwell. The PBAC has recommended changes allowing prescription of 25mg quetiapine tablets for maintenance therapy for acute mania, bipolar 1 disorder and in the treatment of schizophrenia following RANZCP submission.The RANZCP has achieved access to treatments to provide optimal symptom relief for people living with mental illness."} +{"text": "The social media platform Reddit is a contemporary context where we have an opportunity to identify problems experienced by people regarding different aspects of life. The platform is virtually anonymous which might make users discuss their problems more freely. Reddit is divided in subreddits where different subjects are discussed and the discussions are controlled by creators and moderators. I have identified a quite active subreddit targeted towards recovering addicts of benzodiazepines; r/benzorecovery.* To analyze strategies of recovery in user narrative * To identify techniques commonly used and the how they are described * To construct metadata in order to assess how frequent the discussion of a different techniques areTechnically, what is done in this study, is adding mark-up metadata to different discussion. A rudimentary form of analysis suitable with a larger digital corpus where content metadata is added (Gilliland Swetland 2000). The metadata is constructed through a hermeneutical method in which the researcher analyses the subreddit.Answering question like: Example: DIY-tapering; different ways to limit drug use by using less. 1) how common are discussion of taperings in relation to other subjects? 2) Is tapering commonly discussed together with other subjects and techniques?Using a method of categorization and metadata mark-up we could gain a good understanding of the problems among recovering benzodiazepine addicts. We will also have the possibility to identify concepts that addicts themselves discuss and relate these to professional concepts thus creating better possibilities of communication between professionals and clients.No significant relationships."} +{"text": "Scenedesmus sp. CHK0059 on Strawberry Microbiota Community, the funding acknowledgment was partially incorrect as published.In the article titled Effect of AcknowledgmentsThis work was supported by the Rural Development Administration (PJ014934).AcknowledgmentsThis work was supported by the Rural Development Administration (PJ015641)."} +{"text": "Dementia caregivers can experience negative affect when interacting with their care recipients. However, few studies have examined the specific factors that predict caregiver negative affect in this dyadic context. We hypothesized that deficits in care recipients\u2019 emotional functioning would be associated with increased intensity of caregivers\u2019 negative affect during interactions with their care recipient. Caregivers (Nf100) reported on two aspects of their care recipients\u2019 emotional functioning: (1) emotion recognition (the ability to recognize other people\u2019s emotions), and (2) negative emotional reactivity . Dyads then visited the laboratory and engaged in a 10-minute conversation about an area of conflict in their relationship. Caregivers then watched recordings of their conversation while rating the valence and intensity of their experienced affect using a rating dial. We used these ratings to quantify changes in caregivers\u2019 emotional valence across the course of the conversation. Caregivers of care recipients with greater deficits in emotion recognition demonstrated greater increases in negative affect across the conversation. In contrast, care recipients\u2019 negative emotional reactivity was not related to changes in the valence of caregivers\u2019 affect across the conversation. Findings remained significant even after accounting for caregiver baseline valence ratings, biological sex, and age, as well as the care recipients\u2019 diagnosis and level of cognitive impairment. Results reveal the important role that care recipients\u2019 deficits in emotion recognition play in caregivers\u2019 emotional lives. Caregivers\u2019 negative affect may be more likely to increase when their care recipient has deficits in emotion recognition."} +{"text": "The rapid growth of the aging demographic requires students entering the workforce to be adept at engaging with older adults in a professional capacity. The Age Friendly Community (AFC) and Age Friendly University (AFU) initiatives present an opportunity for researchers and students to work alongside older residents to improve Age Friendly practices, policies, and infrastructure in their local communities. Age Friendly Lowell developed an Action Group (AG) of 25 Lowell residents aged 50y+, who provide feedback and guidance for 12 graduate students, as they develop measurement tools for a comprehensive community assessment. Students work with AG members to identify, pilot test, and adapt evaluation tools and learn innovative strategies for recruitment. This experiential learning structure is mutually beneficial, as residents lend their expertise, while students learn to echo those concerns throughout their work. This structure serves as a unique and effective model for other communities pursuing AFC and AFU designations."} +{"text": "Arterioportal malformations, a rare type of vascular malformation, have significant associated morbidity and mortality. Management requires a carefully thought out approach by a multidisciplinary team. Low resource settings have an added challenge of limited treatment options and consumables.We report a case of a 14-month-old male with failure to thrive due to a congenital hepatic arterioportal fistula. He was successfully treated via an endovascular approach with metallic coil embolization.Hepatoportal fistula, a rare hepatic vascular malformation, has limited treatment options which can further be restricted by overall patient wellness. Minimally invasive endovascular treatment options can offer a high rate of success and reverse the morbidity associated with the disease as was seen with our case. Hepatic arterioportal malformations is a communication seen directly between the portal vein and hepatic artery and can be divided into congenital and acquired. Congenital ones are a rare type of vascular malformation with one study reporting prevalence of less than 10% was placed retrogradely into the right common femoral artery. Intraarterial hourly heparin bolus was administered to prevent thrombosis at the access site and lower limb. Access to the celiac axis and then common hepatic artery was obtained using hydrophilic standard angled guide wire and 4 Fr Cobra 2 catheter . The left intrahepatic arterioportal malformation was demonstrated receiving blood supply from the left hepatic artery and draining into left portal venous vein Fig.\u00a0.Fig. 3DiA decision was made to perform coil embolization of the main feeder as distally possible using a 2.4Fr microcatheter (Progreat by Terumo) to deploy a 4\u2009mm detachable micro-coils . There was some flow still noted and hence a second 5\u2009mm detachable micro-coil was put in place which stopped the flow or microsphere (Tasar et al. The choice of embolization agent depends on the type of arterioportal malformation, size and number of the feeding arteries, location of the fistula which further determines the accessibility (Oguslu et al. One of the complications to consider include migration of the embolization agent past the site of the arterioportal malformation and into a distal tributary of the portal vein which may cause portal venous thrombosis and subsequent infarction. The risk for migration of embolization increases with increasing size of the arterioportal malformation and high flow rates (Oguslu et al. The prognosis if left untreated results in arterialization of the portal vein with early onset of portal hypertension, hepatoportal sclerosis and fibrosis of the portal radicles further contributing to portal hypertension and fatal hepatic necrosis.Hepatoportal fistula, a rare hepatic vascular malformation, has limited treatment options which can further be restricted by overall patient wellness. Minimally invasive endovascular treatment options can offer a high rate of success and reverse the morbidity associated with the disease as was seen with our case."} +{"text": "Failing to graduate high school is linked to many risk factors, including family history academic achievement. This research examines how important an older sibling's academic achievement is in predicting whether a younger sibling will graduate high school.This study used linkable administrative databases housed at the Manitoba Centre for Health Policy (MCHP). The cohort consists of 33,843 individuals born in Manitoba between April 1, 1983 and March 31, 1994, who stayed in the province until at least their 20th birthday, had at least one older sibling, and had no missing values on key variables. Logistic regression, controlling for a variety of confounders, is used to determine how much having an older sibling who didn't graduate high school impacts the odds of a younger sibling not graduating high school. The adjusted odds of not graduating high school within 6 years of entering grade nine for individuals who had at least one older sibling who did not graduate high school was 4.81 times higher than for individuals whose older sibling(s) graduated high school. Individuals living in low income neighborhoods at birth or age 18, individuals living in rural northern Manitoba at birth or age 18, and individuals who moved before age 18 were significantly less likely to finish high school. High school graduation rates for those living in the lowest income quintile at age 18 whose older siblings graduated high school were higher than those living in the highest income quintile at age 18 and had at least one older sibling who did not graduate high school.The influence of an older sibling's educational achievement has significant implications for younger siblings' odds of high school graduation. This is likely due to social learning (younger sibling modeling actions of older sibling), and the shared parental influence and social risk experienced by both siblings."} +{"text": "In the United States, over 3.5 million home health aides provide care and assistance to older adults and people with disabilities living in the community . These providers are often tasked with emotionally and physically demanding work and are poorly compensated for their labor. In this poster I discuss the connection between the emotional demands of home health care jobs and their compensation through a quantitative content analysis of online job advertisements for home health aide positions (n=312). This research addresses two primary research questions: 1) what are the prevailing pay rates for home health aides and how do they compare to living wages? 2) what is the relationship between emotion work requirements and pay rates in home health aide job advertisements? In about 57% of the job advertisements hourly wages were not sufficient to meet the living wage level according to the MIT Living Wage Calculator. Using linear probability models, I found that there is no statistically significant difference between pay rates listed by home health agencies requiring emotional labor and home health agencies not requiring emotional labor while holding education, experience, and licensure requirements constant. Stratifying the data by home health agency/company size yielded no statistically significant results. These findings support and expand upon existing literature that home health aides are undercompensated for their work regardless of agency size, and provide evidence for the need for fair compensation and support."} +{"text": "Previous research confirmed high rates (20-89%) of non-adherence to medication among psychotic and bipolar patients. Results suggests that positive attitude to treatment has the highest influence on patients\u2019 adherence and significant differences between treatment related attitudes and treatment adherence of psychotic and bipolar patients were found.The aims were to compare treatment related attitudes and treatment adherence between psychotic (schizophrenia spectrum) and bipolar patients; to evaluate the relationship between treatment related attitudes, illness perceptions and health locus of control in psychotic and bipolar populations.Treatment attitude was evaluated with the Drug Attitude Scale (DAI). Treatment adherence was rated by doctors on Clinical Global Impression (CGI) Scale. Illness perceptions were evaluated with the Illness Perception Questionnaire for Schizophrenia (IPQS) and health locus of control with the Multidimensional of Health Locus of Control Scale \u2013Form C (MHLC) at the end of inpatient care.Number of participants was 51. Data indicated more positive treatment attitude in bipolar patients than in psychotic patients. MHLC scores indicated significant role in symptoms control for chance and \u201epowerful\u201d persons in psychotic patients. IPQS scores indicated that bipolar patients rather have perceptions about treatment influencing symptoms than psychotic patients. Treatment related attitudes were strongly influenced by perceptions about controllability of symptoms by treatment.Bipolar patients had more positive treatment attitude and perceptions about effectiveness of treatment on symptoms. This illness perception about controllability of symptoms by treatment was the strongest determinant of positive treatment attitude in this study.No significant relationships."} +{"text": "Poor access to quality health services, especially in urban slums, is a global challenge. Given similar challenges in Nairobi's Kibra informal settlement area, we collaborated with the Langata/Kibra sub-county health management team to conduct a pilot program for improving the quality of child health services delivered by health care providers (HCPs). The pilot introduced a digital mHealth platform to HCPs working in Kibra informal settlement area in Nairobi. This mHealth platform was compliant to WHO's recommended guideline for integrated management of newborn and child illnesses (IMNCI) and was designed to help sick child assessment, diagnosis and management by HCPs. We aimed to determine if using this digital platform, coupled with supportive supervision and community outreach, would lead to improve compliance to the IMNCI guideline for assessment, diagnosis and treatment of sick children. We conducted baseline (February 2019) assessment, trained selected HCPs on the mHealth platform on handheld android tablets, conducted end line (March 2020) and measured any change in HCP's compliance to IMNCI guidelines. Total 89 HCPs were the mHealth platform users during end line assessment. When asked about the choice of antibiotic for treating childhood pneumonia, we found proportion of HCPs who preferred Amoxycillin dispersible tablet, the recommended treatment for childhood pneumonia, increased from 3% at baseline to 38% at end line. Proportion of HCPs who were aware that antibiotics should NOT be used for the management of simple diarrhea increased from 14% (at baseline) to 50% (at end line). At end line, more than 90% HCPs were found compliant in their practice to IMNCI guidelines for sick child assessment, diagnosis and management. These results demonstrate the use of the IMNCI compliant mHealth platforms as a potential important effective way to improve capacity and compliance among HCPs who are serving communities like Kibra informal settlement in Nairobi, Kenya.\u2022\u2002WHO recommended IMNCI compliant mHealth platform enables health care providers to offer quality child health care.\u2022\u2002Using mHealth platform to ensure WHO\u2019s IMNCI guideline implementation by health care providers might have potential impact on saving sick children\u2019s lives from preventable deaths."} +{"text": "Regional anaesthesia in patients on antithrombotic drugs \u2013 a joint ESAIC/ESRA guideline. This clinical practice guideline serves as a useful decision aid for Nordic anaesthesiologists providing regional anaesthesia to adult patients on antithrombotic drugs.The Clinical Practice Committee of the Scandinavian Society of Anaesthesiology and Intensive Care Medicine endorses the clinical practice guideline The individual domain totals were: Scope and Purpose 95%; Stakeholder Involvement 56%; Rigour of Development 67%; Clarity of Presentation 79%; Applicability 43%; Editorial Independence 79%; Overall Assessment 75% Figure\u00a0.The breakdown of the individual appraisers (de\u2010identified) is available in Supporting Information Material 4Agreement between the SSAI CPC appraisers was acceptable and the overall assessment of the guideline was good. There were issues related to stakeholder involvement and applicability, which were only covered briefly. Furthermore, the timing of post\u2010procedural dosing of antithrombotic agents was not covered in detail, and readers need to refer elsewhere for guidance on this.The guideline can be used in daily clinical practice in the Nordic countries without major adaptation or modification, noting the limitations in applicability above.Regional anaesthesia in patients on antithrombotic drugs \u2013 a joint ESAIC/ESRA guidelineThe clinical practice guideline 5Regional anaesthesia in patients on antithrombotic drugs \u2013 a joint ESAIC/ESRA guideline.The SSAI CPC endorses the clinical practice guideline All authors drafted, revised and approved the manuscript.Appendix S1 Supporting InformationClick here for additional data file."} +{"text": "Sex\u2010specific dominance reversals (SSDRs) in fitness\u2010related traits, where heterozygotes' phenotypes resemble those of alternative homozygotes in females versus males, can simultaneously maintain genetic variation in fitness and resolve sexual conflict and thereby shape key evolutionary outcomes. However, the full implications of SSDRs will depend on how they arise and the resulting potential for evolutionary, ecological and environmental modulation. Recent field and laboratory studies have demonstrated SSDRs in threshold(\u2010like) traits with dichotomous or competitive phenotypic outcomes, implying that such traits could promote the emergence of SSDRs. However, such possibilities have not been explicitly examined. I show how phenotypic SSDRs can readily emerge in threshold traits given genetic architectures involving large\u2010effect loci alongside sexual dimorphism in the mean and variance in polygenic liability. I also show how multilocus SSDRs can arise in line\u2010cross experiments, especially given competitive reproductive systems that generate nonlinear fitness outcomes. SSDRs can consequently emerge in threshold(\u2010like) traits as functions of sexual antagonism, sexual dimorphism and reproductive systems, even with purely additive underlying genetic effects. Accordingly, I identify theoretical and empirical advances that are now required to discern the basis and occurrence of SSDRs in nature, probe forms of (co\u2010)evolutionary, ecological and environmental modulation, and evaluate net impacts on sexual conflict. Core ambitions in evolutionary biology are to identify key processes that maintain genetic variation in fitness and that shape the outcome of evolutionary sexual conflict are of direct interest because they constitute one key mechanism that could both maintain genetic variation and ameliorate sexual conflict Fry . SSDRs aFundamental questions of whether dominance of beneficial versus detrimental alleles can directly evolve and/or simply arises as an intrinsic property of nonlinear genotype\u2010phenotype (or genotype\u2010fitness) maps have been widely considered and historically generated considerable controversy. One key contention was that, since dominance manifests in heterozygotes, a relatively high frequency of heterozygosity is required to generate appreciable selection on dominance and hence any possible dominance evolution, yet sufficient heterozygosity may not generally arise traits. I then demonstrate how these properties can readily generate phenotypic SSDRs that arise as intrinsic consequences of sexual dimorphism and/or competition without necessarily requiring either direct SSDRs in underlying allelic effects or directly concave genotype\u2010fitness maps. I achieve these objectives using illustrative caricatures of traits, quantitative genetic architectures and study designs reported in the four recent empirical studies but also on the variance in liability. This is because the mean and variance jointly define the proportion of individuals whose liabilities exceed the threshold and hence express the alternative phenotype Figure\u00a0. Hence, Given these well\u2010established properties, the potential for threshold(\u2010like) traits to generate SSDRs on observed phenotypic scales, with or without explicit genetic SSDRs acting on underlying liability scales, can be outlined with broad reference to the four recent empirical studies.Theory on SSDRs, involving either evolution of direct dominance modifiers or intrinsic effects of nonlinear fitness landscapes, primarily envisages large\u2010effect loci that detectably affect fitness Fry . CorrespIn salmon, genome\u2010wide association studies using relatively high\u2010density SNP data revealed a large\u2010effect locus, VGLL3, where alternative alleles substantially affect the occurrence of maturation and hence result in maturation age in both sexes could then substantially exceed the threshold in both sexes, meaning that most males and females will mature now, potentially still with some phenotypic sexual dimorphism at the large\u2010effect locus relative to the threshold and the resulting sex\u2010specific frequencies of the alternative phenotypes. Here, Figure\u00a0Given this scenario, Figure\u00a0Such SSDRs can then be further modulated by the variance in liability and by the degree of sexual dimorphism in the variance . For exaThese scenarios imply that ongoing evolution of the degree of sexual dimorphism in the mean and/or variance in liability, or simply environmental effects on the mean and/or variance and resulting phenotypic sexual dimorphism, could alter the emerging degree of phenotypic SSDR. For example, if there were less sexual dimorphism in mean baseline liability than illustrated in Figure\u00a0ST, Czorlich et\u00a0al. Indeed, temporal and/or spatial variation in sexual dimorphism in liability could readily arise in nature if the form of (sex\u2010specific) selection varies among environments, potentially driving evolution of (sex\u2010specific) plasticity and resulting phenotypic outcomes. For example, mean salmonid maturation ages and degrees of anadromy and sexual dimorphism commonly vary among populations and even among cohorts dominance modifiers can invade in Drosophila melanogaster. Such survival is clearly a key fitness component, implying selection for increased immunity. However, increased immunity may trade\u2010off against reduced mating success, particularly in males phenotypic sexual dimorphism. Then, 65+ generations of experimental evolution, where parents in each generation comprised individuals who survived bacterial challenge, successfully generated lines that were more resistant, with much higher survival rates and still little phenotypic sexual dimorphism. This evolutionary response indicates substantial additive genetic variation underlying survival, with no evidence of sex linkage , while male hybrids showed relatively low survival rates (closer to the stock than the resistant lines). These patterns imply polygenic SSDR, at least assuming the hybrids are relatively heterozygous at numerous loci compared to the stock and resistant lines . Simple scenarios suggest that they potentially can, due to the key property that mean phenotypic values of threshold traits depend on both the mean and variance in liability Fig.\u00a0.Drosophila stock population has some sexual dimorphism in both mean and variance in liability, such that males have lower mean and higher variance than females lines will have disproportionately high and low success, respectively, while females and males from male\u2010beneficial lines will have disproportionately low and high success, respectively. Simple simulations show how opposite nonadditive effects on fitness can then emerge in females versus males, readily generating a negative cross\u2010sex correlation in line cross covariance and hence apparent phenotypic SSDR Figure\u00a0. Indeed,Such outcomes depend on the shapes of the relationships between genetic value and competitive reproductive success in each sex and on the relative mean value of the reference population against which competitive reproductive success is assayed traits can readily generate phenotypic SSDRs that broadly caricature those observed in recent empirical studies, even given purely additive genetic effects on underlying scales. Given polygenic quantitative genetic architectures that include large\u2010effect loci, simply the presence of sexual dimorphism in mean baseline liability relative to the threshold can generate phenotypic SSDRs traits. While the occurrence of sexual dimorphism in trait means is widespread and very well known, the possibility that there can be sexual dimorphism in genetic and/or environmental trait variances is also embedded in core aspects of evolutionary quantitative genetic theory and increasingly evidenced in diverse empirical systems, resulting from some degree of sex\u2010specific autosomal as well as sex\u2010linked genetic effects traits should not necessarily be viewed as fixed properties that could act as alternatives to evolved sexual dimorphism in resolving sexual conflict. Rather, they can be viewed as evolutionarily, ecologically, and environmentally labile outcomes that could emerge from, and potentially coevolve with, degrees of sexual dimorphism and reproductive systems. While it has long been established that dominance relationships emerge as intrinsic properties of nonlinear biological systems, such systems are often considered to be relatively fixed or stable traits. Obvious examples include the occurrence of maturation, seasonal migration, diapause, resistance to disease, survival, alternative reproductive tactics and the development of alternative morphologies Roff . Forms oAccordingly, the potential for threshold\u2010like traits to generate strong phenotypic SSDRs, including through coevolutionary feedbacks involving genetic architectures, forms of sexual dimorphism and reproductive systems, should now be more explicitly examined, both theoretically and empirically. Such work can aim to more clearly distinguish key points: the degrees to which SSDRs can in principle arise through combinations of intrinsically nonlinear genotype\u2013phenotype maps and/or explicit genetic dominance modification and the degrees to which such SSDRs are actually likely to be expressed, to be dynamic and to act as predominant forces that could widely maintain genetic variation and resolve sexual conflict given forms and impacts of heterozygosity arising in nature.Multiple opportunities for theoretical advances are evident. First, we can examine whether, by facilitating emergence of phenotypic SSDRs, threshold traits with sexual dimorphisms in liability could actually facilitate invasion and maintenance of stable polymorphisms for large\u2010effect mutations or complexes of linked genes with sexually antagonistic phenotypic effects. We can then examine whether such invasions can feed back to shape the form and plasticity of underlying sexual dimorphism. To date, the dynamics of genetic architectures involving large\u2010effect loci or gene clusters have been examined in the context of local adaptation and migration\u2010selection\u2010drift balance traits to be formally considered through new models that jointly track the (co)evolution of multiple routes to generate and resolve sexual conflict, including sexual dimorphisms and complex reproductive systems.There is also considerable scope for future empirical studies to explicitly examine the basis and modulation of phenotypic SSDRs in threshold(\u2010like) traits. First and most obviously, we should more explicitly distinguish whether observed phenotypic SSDRs (or lack of SSDRs) result from direct SSDRs acting on underlying liability scales, from the properties of defined nonlinear genotype\u2010phenotype or genotype\u2010fitness maps (given purely additive underlying genetic effects), or both. This distinction requires estimating appropriate liability\u2010scale fixed effects and variance components and back\u2010transforming onto observed phenotypic scales, which has not yet been a primary focus of empirical studies of SSDRs in threshold traits . Such anSecond, we should more explicitly quantify the degree to which phenotypic SSDRs are modulated by ecological and environmental conditions, thereby treating SSDRs as dynamic rather than fixed entities. This could be achieved, for example, by manipulating environmental conditions that affect the degree of sexually antagonistic selection or the degree or form of competition for reproductive success. Any experimental design to reveal SSDRs requires major efforts, even without any ambition to replicate across different conditions traits in wild populations. This objective will require attention to how locus\u2010specific or genome\u2010wide heterozygosity arises and to the overall phenotypic consequences of such heterozygosity. It has been widely emphasized that substantial heterozygosity is required for the evolution of dominance modifiers (Otto and Bourguet J.M.R. conceived the ideas and wrote the manuscript.There are no primary data associated with this conceptual manuscript. The R code underlying the illustrative figures is provided as Supporting Information.Associate Editor: T. ConnallonHandling Editor: T. ChapmanS1. Derivations of SSDRs given a threshold trait with a large\u2010effect locusS2. Parameter valuesS3. Illustration of the emergence of SSDRs given uniform distributions of liabilitiesS4. Emergence of genome\u2010wide SSDRs in a full diallel line\u2010crossS5. Empirical estimation of SSDRsClick here for additional data file.Supplementary informationClick here for additional data file."} +{"text": "Intraoperative indocyanine green (ICG) near\u2010infrared fluorescence guidance is a type of optical imaging technology currently available to facilitate a better understanding of surgical landmarks. Therefore, ICG has been utilized intraoperatively during laparoscopic or robotic urologic procedures to help identify relevant anatomy and assess perfusion.In this article, Imai et\u00a0al described the useful application of ICG for the horseshoe kidney during laparoscopic partial nephrectomy.In addition, the authors reported that retroperitoneal laparoscopic partial nephrectomy was successfully performed in the modified supine position and modified port placement, which provided an excellent operative field. The authors might choose a retroperitoneal approach with the modified supine position because of the large experience with radical nephroureterectomy in this position for upper tract urothelial tumors,The author does not have any conflicts of interest to declare."} +{"text": "Ixodes holocyclus) antiserum are commonly observed by veterinarians and can lead to significant morbidity and potentially fatal. A purified antiserum canine IgG concentrate was chromatographically prepared and aseptically formulated in single doses containing the equivalent of 5\u2009mL of unrefined tick antiserum (TAS). The IgG was used for slow intravenous infusion into clinically affected cats at multiple veterinary clinics on the eastern seaboard of Australia. Overall, 72/76 (95%) of cats survived hospital discharge, an efficacy comparable to published data. A subset of 22 cats previously treated with unrefined TAS and considered high risk were included in the dataset. The safety profile was excellent with 0/76 acute adverse reactions although 2/76 (2.6%) developed mild facial swelling within 2\u2009h of infusion that responded to the antihistamine. In conclusion, cats intravenously infused with purified IgG from canine TAS did not exhibit the expected frequency of acute adverse reactions during infusion and it was both safe and effective for the treatment of tick paralysis in cats.Acute adverse reactions in cats administered unrefined canine paralysis tick ( IgGimmunoglobulin GPTASpurified tick antiserumTAStick antiserumIxodes holocyclus).Cats frequently succumb to lower motor neurone paralysis from salivary gland neurotoxins found within engorged Australian paralysis ticks research permit PER7250.Veterinarians were invited to participate in the study of purified tick antiserum (PTAS) in cats, to directly substitute the PTAS\u2010like\u2010for\u2010like with TAS and to follow their usual clinical treatment protocol. A dose recommendation of one vial was made and administration of additional vials was made at the discretion of the attending veterinarian. Clinical severity scores of gait and respiration were performed 1\u20134 by the clinician.The PTAS purification process was reproducible. The final IgG formulation migrated on an agarose gel as a single peak in the gamma globulin zone with the complete absence of canine serum albumin Figure\u00a0. The visSurvival rates at discharge varied as expected with severity Table\u00a0 but wereThe safety profile was excellent with no acute adverse reactions observed in 76 cats treated with 1 to 2 vials. Veterinary practice protocols varied in the duration of infusion and ranged between 30 to 240\u2009min. Facial swelling, presumably caused by angioedema, is a recognised issue with homologous and heterologous plasma transfusion in cats and dogs.Cats previously treated with unrefined TAS should based on theory, be at higher risk of acute anaphylactic reactions to subsequent TAS infusions. Although the dataset presented here is small, cats previously treated with TAS experienced no acute adverse reactions to PTAS. This observation is encouraging and supports the use of PTAS in this potentially higher\u2010risk group has to merit in reducing infusion risk. However, there may be benefits other than explicitly in cats that have had prior TAS treatment because prior exposure does not explain those adverse reactions reported in na\u00efve cats. Further studies to examine specific IgE responses and their role in acute reactions are required.A number of APVMA licensed highly effective tick paralysis preventatives have become available since 2018 to cat owners and veterinarians, focusing the clinical need on refinement of ever more effective and safer treatments for those feline cases that do present to veterinarians.This study has provided preliminary evidence that the use of PTAS in cats can prevent acute adverse reactions from occurring compared to rates in published data, without compromising efficacy.PTAS was manufactured by Padula Serums, a company owned by the author Andrew Padula. Boehringer Australia generously provided funding to undertake this research with the endorsement of the Australian Paralysis Tick Advisory Panel."} +{"text": "A growing body of evidence has identified potential modifiable risk factors for Alzheimer\u2019s disease and related dementias (ADRD). In 2021, the National Plan to Address Alzheimer's Disease included a new goal to promote healthy aging and address risk factors to help delay onset or slow progression of ADRD. Applying a robust public health approach to ADRD risk reduction can help achieve meaningful progress at the population level. The activities outlined in the Building Our Largest Dementia (BOLD) Infrastructure for Alzheimer\u2019s Act (P.L. 115-406) are designed to create a uniform national public health infrastructure with a focus on various issues including risk reduction. The purpose of this session is to illustrate a public health approach to ADRD risk reduction, including its current status along with future directions and priorities. An overview of the National Plan\u2019s new goal regarding ADRD risk reduction (McGuire) and data highlighting the current burden of key modifiable risk factors in the United States along with important disparities (Omura) will be presented. Holt will describe how ADRD risk reduction is integrated into the work of BOLD funding recipients, and Head will present experiences implementing public health activities that support ADRD risk reduction in the field along with successes and lessons learned. Finally, priorities and future directions for a public health approach to ADRD risk reduction will be presented from the perspective of the CDC\u2019s Building Our Largest Dementia Infrastructure Public Health Center of Excellence on Dementia Risk Reduction (Baumgart)."} +{"text": "This case series reveals a number of young adults, whom after chronic use of recreational drugs, suffer the life-long consequence of severe chronic mental illness.\u2022 Review the illicit drugs that are commonly associated with psychotic symptoms. \u2022 Highlight exposures theorized to impact genetics associated with DSM 5 diseases. \u2022 Compare trends in illicit drug use during the worldwide COVID pandemic.A literature review is used to examine the impact of COVID pandemic on illicit drug use in metropolitan cities in European countries and compare the trends with what is seen by the consult liaison psychiatry service at a metropolitan community hospital in the USA.In European Countries with data available, there were measurable differences in which illicit drugs were used most during the COVID 19 pandemic. In the US this data is not readily available at the time of submission for proper comparisson.Although definitive comparrison is pending, the results of extensive illicit drug use demostrate a high comorbidity with psychotic spectrum disorders in the DSM 5.No significant relationships."} +{"text": "Telehealth services tends to be used relatively infrequently by minority populations, thereby exacerbating health inequalities. This study examines the individual, circumstantial and environmental factors that facilitate or hinder usage of telehealth among Israeli Arabs, who constitute 21% of the Israeli population.Data was collected through a telephone survey among the adult Arab population in October 2020 with 501 respondents (42% response rate). Analysis included logistic regression.Most of the Arab population use the internet several times a week (93%) and have a smartphone (96%). The most popular telehealth service was telephone appointments with a doctor (66%). Two thirds have never used the health plan\u2019s mobile application, though most have no objection to using chat (75%) or video conversation (51%) with a medical professional. The most significant barrier to using telehealth is lack of awareness of services such as ordering medicines (23%). Conversely, factors that facilitate the use of telehealth include previous acquaintance with the doctor (91%); Arabic services (82%); and recommendation by health professionals (79%). Multivariate analyses indicate a strong positive correlation between education and the use of telehealth for written correspondence with a known health professional (p = 0.001).Telehealth services which are already used widely by the Israeli-Arab population, should be retained and developed further. In parallel, digital health literacy and linguistically and culturally adaptation of digital services should be promoted. Awareness of those services should be enhanced through culturally adapted marketing and via recommendations from the family doctor.\u2022\u2002Identification of the barriers to the use of telehealth services among minority populations can help service providers reduce usage gaps between minority and majority population.\u2022\u2002The use of telehealth services should be simplified to suit people with a low digital health literacy."} +{"text": "What is the topic of this review?Studies using cardiovascular magnetic resonance imaging and echocardiography to investigate cardiac alterations at rest and during exercise\u2010induced physiological stress in adults born preterm.What advances does it highlight?People born preterm have a greater long\u2010term cardiovascular risk, which may be explained in part by their cardiac structural and functional alterations. They have potentially adverse alterations in left and right ventricular structure and function that worsens with blood pressure elevation; an impaired myocardial functional reserve; and an increase in diffuse myocardial fibrosis that may drive their lower diastolic function.Preterm birth accounts for more than 10% of births worldwide and associates with a long\u2010term increase in cardiovascular disease risk. The period around preterm birth is a rapid and critical phase of cardiovascular development, which might explain why changes in multiple components of the cardiovascular system have been observed in individuals born preterm. These alterations include reduced microvascular density, increased macrovascular stiffness, and higher systolic and diastolic blood pressure. Cardiac alterations have been observed in people born preterm as early as neonatal life and infancy, with potentially adverse changes in both left and right ventricular structure and function extending into adulthood. Indeed, studies using cardiovascular magnetic resonance imaging and echocardiography have demonstrated that preterm\u2010born individuals have structural cardiac changes and functional impairments. Furthermore, myocardial tissue characterization by cardiovascular magnetic resonance imaging has demonstrated an increase in left ventricular diffuse myocardial fibrosis in young adults born preterm, and under acute physiological stress, their myocardial functional reserve assessed by echocardiography is reduced. The preterm heart is also more susceptible to chronic systolic blood pressure elevation, with a significantly greater increase in left ventricular mass as systolic blood pressure rises observed in preterm\u2010born compared to term\u2010born young adults. Given these known, potentially adverse acute and chronic cardiac adaptations in the preterm\u2010born population, primary prevention strategies are needed to reduce long\u2010term cardiovascular disease risk in this subgroup of the population. The postnatal window may offer an opportunity for intervention given its importance for cardiac remodelling. CMR imaging in preterm\u2010born young adults who had been randomized at birth to different milk feeding diets showed that those who were randomized to an exclusive human milk diet postnatally (9In conclusion, preterm birth associates with a unique cardiac phenotype that may put these individuals at increased risk of cardiovascular disease. The number of people born preterm each year continues to rise, as does the percentage of individuals surviving. While much is known about the immediate costs and pressures preterm birth places on healthcare systems, little is known about this in the longer term. Primary prevention strategies to reduce risk in this group are needed and, in order to be implemented at scale, will require input from multi\u2010disciplinary teams including the affected patients.None."} +{"text": "Interrogation of the human proteome in a highly multiplexed and efficient manner remains a coveted and challenging goal in biology. We present a new aptamer-based proteomic technology for biomarker discovery capable of simultaneously measuring thousands of proteins from small sample volumes (15 [mu]L of serum or plasma). Our current assay allows us to measure ~800 proteins with very low limits of detection (1 pM average), 7 logs of overall dynamic range, and 5% average coefficient of variation. This technology is enabled by a new generation of aptamers that contain chemically modified nucleotides, which greatly expand the physicochemical diversity of the large randomized nucleic acid libraries from which the aptamers are selected. Proteins in complex matrices such as plasma are measured with a process that transforms a signature of protein concentrations into a corresponding DNA aptamer concentration signature, which is then quantified with a DNA microarray. In essence, our assay takes advantage of the dual nature of aptamers as both folded binding entities with defined shapes and unique sequences recognizable by specific hybridization probes. To demonstrate the utility of our proteomics biomarker discovery technology, we applied it to a clinical study of chronic kidney disease (CKD). We identified two well known CKD biomarkers as well as an additional 58 potential CKD biomarkers. These results demonstrate the potential utility of our technology to discover unique protein signatures characteristic of various disease states. More generally, we describe a versatile and powerful tool that allows large-scale comparison of proteome profiles among discrete populations. This unbiased and highly multiplexed search engine will enable the discovery of novel biomarkers in a manner that is unencumbered by our incomplete knowledge of biology, thereby helping to advance the next generation of evidence-based medicine."} +{"text": "Loneliness in the aging population is a concern, as increased loneliness is associated with decreased cognitive function and increased neuropathology. Less is understood about the relationship between loneliness and cognitive resilience. Cognitive resilience is defined as the discordance between a person\u2019s actual and expected cognition given their neuropathology and can be estimated by extracting residuals from a model regressing cognition on neuropathology. Using data from two longitudinal aging cohorts (MAP/MARS), we estimated cognitive resilience proximate to death and cognitive resilience over time to use as the key outcomes. We then regressed these two cognitive resilience indicators onto loneliness level and slope. Higher baseline loneliness and increasing loneliness over time were both associated with lower cognitive resilience. Our results suggest that loneliness should be included into resilience-based prevention models, and interventions aimed at optimizing cognitive function across older adulthood should include loneliness reduction as a potential area of focus."} +{"text": "We identified analytic limitations in a recent meta\u2010analysis and re\u2010examined the efficacy and safety associated with immune checkpoint inhibitors (ICIs) in unresectable hepatocellular carcinoma (HCC) compared with standard therapies. Our findings mostly contradict conclusions from the previous study, suggesting the need for continuing the investigation of ICIs in HCC with additional clinical evidence. The predictive interval, which also takes into account the between\u2010study variation in overall uncertainty quantification, can be exploited in sample size determination and power calculation for the planning of future HCC ICI studies."} +{"text": "Recent research shows that not only do life course socioeconomic status and engaging in leisure activities have independent effects on cognitive performance of older adults, but also there are significant interaction effects between them. What is less clear is whether these effects, both separate and interactions between them, vary by age among older adults. We use data from the Chinese Longitudinal Healthy Longevity Survey 2005-2014 and a Generalized Linear Mixed Model to examine these relationships. Results show that engaging in leisure activities is significantly associated with cognitive impairment even into very old ages (85+), although such associations are partially absorbed by life course SES. Furthermore, interaction effects of leisure activities and life course SES are also detected across all age groups, although they vary by specific activities. Virtually all interaction effects point to one direction: individuals of higher life course SES enjoy extra benefits from engaging in leisure activities."} +{"text": "Though safety is a critical component of all surgical training, the uniquely subjective nature of aesthetic surgery makes it an insufficient metric for evaluating the competency of future aesthetic surgeons. To date, there is a paucity of studies examining subjective clinical outcomes arising from RACs.5 Such outcome studies should be encouraged as they are invaluable in shaping aesthetic education (Video).This original article6 As this demand grows, so too will the number of nonplastic surgeons who offer cosmetic services. As aesthetic surgeons, we must embrace all procedures our specialty has to offer, whether surgical or nonsurgical. A dedicated aesthetic fellowship should be encouraged by program directors and strongly considered by those residents hoping to incorporate aesthetic surgery into their future practices. Additional focused training will increase confidence in performing the full spectrum of aesthetic surgery procedures and assure our continued ownership of this discipline.Aesthetic surgery is an exceedingly diverse discipline, the demand for which is only increasing.ojac080_Supplementary_DataClick here for additional data file."} +{"text": "The mechanism of thermal transport can be enhanced by mixing the nanoparticles in the base liquid. This research discusses the utilization of nanoparticles (tri-hybrid) mixture into Carreau\u2013Yasuda material. The flow is assumed to be produced due to the stretching of vertical heated surface. The phenomena of thermal transport are modeled by considering Joule heating and heat generation or absorption involvement. Additionally, activation energy is engaged to enhance heat transfer rate. The mathematical model composing transport of momentum, heat and mass species is developed in Cartesian coordinate system under boundary layer investigation in the form of coupled nonlinear partial differential equations. The complex partial differential equations are converted into coupled nonlinear ordinary differential equations by using the appropriate similarity transformation. The conversion of PDEs into ODEs make the problem easy to handle and it overcome the difficulties to solve the PDEs. The transformed ordinary differential equations are solved with the help of help of finite element scheme. The obtained solution is plotted against numerous involved parameters and comparative study is established for the reliability of method and accuracy of obtained results. An enhancement in fluid temperature is recorded against magnetic parameter and Eckert number. Also, decline in velocity is recorded for Weissenberg number and concentration is controlled against higher values of Schmidt number. Furthermore, it is recommended that the finite element scheme can be implemented to handle complex coupled nonlinear differential equation arising in modeling of several phenomena occurs in mathematical physics. Bhatti and Abdelsalam2 studied behavior of Ree\u2010Eyring liquid under action of magnetic parameter considering by irreversible process. Bilal et al.3 discussed features of mathematical model containing microorganisms inserting approach of hybrid nanoparticles whereas flow has been induced by wavy fluctuating heated disk. Elmaboud and Abdelsalam4 captured model based on generalized Burger's liquid using effects of magnetic parameter in an annulus. Abumandour et al.5 studied peristaltic thrusting in the presence of nanoparticles trough vertical pipe including thermal-viscosity. Haq et al.6 captured consequences of energy transfer inserting SWCNTs in trapezoidal cavity solved by finite element method. The involvement of nanoparticles for the enhancement of heat transport in thermal exchanges under several important effects computational study was presented by Bondareva et al.7 to analyze the thermal transport by mixing different nanoparticles indifferent phase changing materials. Mallawi and ullah8 examined the continuation of slip in Darcy\u2013Forchheimer inclined plane medium containing the mixture of hybrid nanoparticles. They derived the flow governing equation in the form of partial differential equations by engaging boundary layer survey and solved the resulting transformed ordinary differential equations by incorporating similarity transformation with the help of homotopic procedure. They monitored the decline in velocity field by augmenting the values of slip parameter. Algehyne et al.9 studied the inclusion of ternary hybrid nanoparticles mixture in pseudo-plastic material past over a heated porous surface in the presence of heat generation and modified heat flux (Cattaneo\u2013Christov model). They analyzed the shear thinning and thickening behavior of considered model for different values of power law index. They engaged a powerful numerical scheme namely finite element procedure to handle the model equations. They found the description in fluid velocity against porosity parameter.Transport of heat in fluid flows has much importance due to their usage in many industrial applications. Researchers have great interest on these medium and mechanisms which are favorable for thermal transport. Carreau Yasuda liquid is known as non-Newtonian martial which is applicable in colloidal suspension, manufacturing, engineering problems and fermentation industry. Bhatti et al.10 implemented magnetic field in energy transfer phenomena based on natural convention inserting approach of hybrid nanoparticles and nanofluid. They investigated comparative analysis among nanofluid and hybrid nanoparticles. Khan and Pop11 studied the stretched viscous nanofluid boundary value problem past over a linear stretching sheet. They have shown the increase in temperature against Brownian motion parameter and depreciation in concentration field. Moreover, validity of result is expressed by comparing the obtained solution as a limiting case of previously published findings. Rajagopal et al.12 presented the numerical study on second grade liquid past over a stretching sheet. Chabani et al.13 used hybrid nanoparticles to obtain maximum amount of energy transfer in triangular enclosure. Shafiq et al.14 developed model hyperbolic tangent liquid inserting role of bioconvective flow with nanoparticles over porous surface. Saeed et al.15 investigated thermal enhancement in couple stress liquid using hybrid nanoparticles approach in the presence of Darcy\u2013Forchheimer theory. Saeed et al.16 studied physical significance of nanoparticles over a stretching surface via convective energy transfer. Ullah et al.17 used the Lie group similarity analysis to handle the problem of non-Newtonian model with thermal transport. They mentioned that higher values of magnetic retard the flow. Some important contributions are covered in21. Elkoumy et al.22 discussed influences of Maxwell liquid in heat energy in the presence of peristaltic flow under action of Hall current and magnetic field. Abdelsalam23 studied electro-magnetically containing swimming sperms in the presence self-propulsion in heated channel. Eldesoky et al.24 discussed thermal aspects in peristaltically induced incorporating slip conditions in catheterized heated pipe. Bhatti and Abdelsalam25 studied impacts of hybrid nanoparticles in peristaltic propulsion under action of magnetic effects. Marzougui et al.26 captured features of magnetic field in entropy generation and energy transfer in the presence of nanofluid. Pushpa et al.27 determined phenomena of convective flow inserting approach of nanofluid in thin baffle. Rasool et al.28 discussed model of Second grade liquid considering features of thermal radiation and viscous dissipation using Darcy\u2013Forchheimer model. Shafiq et al.29 investigated mechanism magnetic field in the presence of Darcy\u2013Forchheimer theory in nano-Casson material. Kumar et al.30 studied phenomena regarding enhancement of heat transfer in ferromagnetic flow in the occurrence of hybrid nanoparticles considering with solar radiation. Ganesh Kumar et al.31 performed modeling of heat transfer implementing nanoparticles considering various shapes effects over a moving heated frame numerically simulated by least square approach. Kumar et al.32 analyzed study of thermal energy enhancement due to carbon nanotubes in heated channel (convergent/divergent) under the occurrence of Darcy\u2013Forchheimer medium. Souayeh et al.33 investigated characterizations of thermal radiation in energy transfer involving rheology of ferromagnetic in the presence of nanomaterial based on dusty fluid using slip conditions. Kumar et al.34 discussed features of Maxwell liquid in Double-diffusive free approach related to conservative heat transfer including thermal radiation over a heated surface. Kumar et al.35 studied double diffusion phenomena in convective flow including Casson liquid involving the concept of thermal radiation using slip conditions and thermal radiation. Kumar et al.36 discussed thermal features in viscoelastic fluid inserting nanofluid under action of thermal radiation implementing convective boundary conditions past a stretching heated surface. Ganesh Kumar37 developed energy transfer model in term of three dimensional flows in the presence of thermal radiation and nanoparticles over heated surface. Kumar et al.38 investigated impacts of non-Newtonian liquid considering nanofluid and slip factor across a heated surface. Ali et al.39 analyzed modeling of blood behavior in stenosis simulated by finite difference approach. Khan et al.40 modeled problem regarding energy transfer including anomalous diffusion amorphous semiconductors implementing fractional calculus. Hussain et al.41 studied mixed convective heat transfer in presence of CNTs nanofluid considering reactions and variable viscosity. Irfan et al.42 evaluated thermal aspects in term of convective heat transport using new mass flux approach in Carreau liquid. Ali et al.43 studied dynamics behavior regarding Josephson junction using fractional calculus procedure. Hussain et al.44 discussed features of thermal flux in Jeffery flow using concept of heat source past stretching surface. Hussain et al.45 studied thermal and solute effects in hybrid nanoparticles using chemical reaction over curved surface involving various shapes effects. Irfan et al.46 investigated theoretical analysis of Carreau liquid in heat and mass fluxes using new concept of activation energy under magnetic influence. Rafiq et al.47 studied thermal features of Maxwell liquid in the presence of thermal radiation including suspension of nanoparticles.Modeling of fluid flows over a stretching surface got remarkable consideration due to their industrial wider applications. For instance, Dadheech et al.Literature survey is reported in \u201cModeling under several important physical aspects with similarity analysis is included in \u201cUtilized scheme is explained in \u201cResults are analyzed in \u201cAvailable study shows that no one attempted the ternary hybrid nanoparticles mixed Carreau\u2013Yasuda model numerically via finite element scheme along with activation energy. The utilization of viscous dissipation, Joule heating in the mixture of ternary hybrid nanoparticles is an important novel contribution to the existing body of knowledge. This report is organized as follows.48 are considered below:The rheology of Carreau Yasuda liquid is observed;Triple species along with activation energy and chemical reaction is addressed;Heat generation phenomena is taken out;Effects related viscous dissipation and Joule heating are considered;Composite of Figure\u00a0An enhancement in motion of Tri-hybrid nanoparticles in Carreau Yasuda liquid over a vertical heated surface is observed which is shown in Fig.\u00a049 are formulated as:Physical actions are visulaized in form PDEs and non-linear PDEsDesired transformations in the presence of hybrid nanoparticles areIt is noticed that Eq.\u00a0 is used Dimensionless BCs areIt is noticed that present developed problem is known as non-Newtonian model in the presence of Carreau Yasuda material. The present model can be reduced in Newtonian model by implanting mentclass2pt{minimEquations\u00a0\u201316) are are16) a in Eqs. \u2013(16). ThDrag force (skin friction coefficient) in view of Carreau Yasuda liquid under the action of hybrid nanoparticles areRate of heat transfer in the presence of hybrid nanoparticles isConcentration gradient at wall of surface isNumerous of applications of finite element method are investigated in computational fluid mechanics problems;Complex types of geometries are tackled by FEM;Physical problems based on applied science are developed by FEM;It has ability to discretize the derivatives with very ease;An important role of FEM is that to solve various types of boundary conditions;FEM requires low investment and time rather than others numerical techniques.Step-I: Equations (quations \u20137) with withStepquations \u2013(7) on oStep-II: Weak form is developed using linear shape functions by implementing Galerkin finite element method. The residuals of present analysis are formulated asThe shape functions are developed asStep-III: Assembly approach is utilized for the development of stiffness element whereas assembly approach is performed via assembly procedure of FEA. Stiffness elements are derived asStep-IV: Picard linearization approach provides transformed algebraic system (linear equations).Step-V: Finally, system of linear algebraic equations is numerically solved within computational tolerance which simulate problem in iteration manner. It is mentioned that exact solution of developed model is not available. The stopping criterion is defined in Eq.\u00a0. Therefo49 neglecting impacts of tri-hybrid nanoparticle, viscous dissipation, Weissenberg number and heat generation number. Good agreements are found among present work and published work49 whereas comparative study is carried out by Table 48.It is noticed that comparative numerical values of Nusselt number is validated with published study50. A detail and comprehensive discussion for concentration, flow behavior and heat energy is discussed below.Aspects related to triple-diffusion species and energy transfer are studied towards a stretching heated sheet. Rheology of Carreau Yasuda material is inserted into fluid particles along with base fluid called engine oil inserted hybrid nanoparticles under an influence of magnetic field. Thermal aspects are based on heat generation, viscous dissipation and Joule heating phenomena whereas concentration aspects are considered as activation energy and chemical species. Such complex developed model is numerical simulated using finite element method. It noticed that ranges of parameters Flow of tri-hybrid nanoparticles is measured against variation in magnetic field number, Weissenberg number and fluid number whereas these influences are noticed by Figs.\u00a0Figures\u00a0Figure\u00a0The estimation of mass diffusion is investigated versus an influence of Schmidt number, activation number, chemical reaction number and Lewis parameter considered by Figs.\u00a0Table Convergence of problem is investigated carried by 300 elements;Higher viscosity is generated versus higher numerical values of magnetic number, fluid parameter and Weissenberg number;Maximum production in view of heat energy is obtained using argument impacts of magnetic parameter, Eckert number and heat generation number;Diffusion into fluid particles is slow down versus higher values of Schmidt number, Lewis number and chemical reaction number are increased;Ternary hybrid nanoparticles approach is visualized most significant to boost heat energy into fluid particles rather than hybrid nanoparticles approach;Coolant in automobiles and dynamics of fuel are applicable involvement of ternary hybrid nanoparticles;Remarkable achievement in heat transfer process which is applicable in industrial technologies, hybrid powered engines, fuel cells, microelectronics and industrial technologies;Developing model is applicable in printing process, electronic devices, temperature measurements, engineering process and food making, coolant in automobiles, fluidic dynamic process and solar systems.Developed model including double diffusion effect over a moveable vertical surface is considered. A rheology related to Carreau Yasuda martial is analyzed along with Darcy\u2019s Forchheimer model is addressed. A finite element scheme is utilized to simulate required results. The desired consequences are captured below."} +{"text": "Materials and Methods section. We omitted the source (Africa Rice Center) of the original material from which the QTL mapping population used in this study was developed and misstated the purpose for developing that original material. It was not targeting Madagascar specifically but all P deficient environments in Sub-Saharan Africa.In the published article, there was a mistake in the Materials and Methods, Plant Material, 1st paragraph. This sentence previously stated:A correction has been made to \u201cAn initial breeding population targeting P deficient upland environments in Madagascar had been developed from a cross of P efficient genebank accession DJ123 with P inefficient upland variety Nerica4.\u201dThe corrected sentence appears below:\u201cAn initial breeding population targeting P deficient upland environments in SSA had been developed from a cross of P efficient genebank accession DJ123 with P inefficient upland variety Nerica4 by the Africa Rice Center and shared with FOFIFA for further advancement and evaluation in Madagascar.\u201dA correction has also been made to Acknowledgments. The sentence below has now been added:\u201cWe acknowledge Dr. Nani Drame (Former Africa Rice Center staff) for sharing seeds of the Nerica4 x DJ123 mapping population with FOFIFA.\u201dThe authors apologize for these errors and state that this does not change the scientific conclusions of the article in any way. The original article has been updated."} +{"text": "Since 2019, the RAISE Family Caregiver Advisory Council (FCAC) has met regularly to carry out its work of developing a national family caregiving strategy. This strategy incorporates five goals to support family caregivers, and key actions that a range of stakeholders can carry out centered around these goals. This overview will describe the RAISE FCAC\u2019s work in developing the national family caregiving strategy, and highlight the development of recommendations and key actions to support the fourth goal, \u201cFamily caregivers\u2019 lifetime financial and employment security is protected and enhanced.\u201d This goal\u2019s recommendations include supporting caregivers through flexible workplace policies; supporting affordable long-term services and supports; supporting financial education and planning; and reducing overall negative financial impacts of caregiving short and long-term."} +{"text": "We explore the use of Quantum Machine Learning (QML) for anomaly detection at the Large Hadron Collider (LHC). In particular, we explore a semi-supervised approach in the four-lepton final state where simulations are reliable enough for a direct background prediction. This is a representative task where classification needs to be performed using small training datasets \u2014 a regime that has been suggested for a quantum advantage. We find that Classical Machine Learning (CML) benchmarks outperform standard QML algorithms and are able to automatically identify the presence of anomalous events injected into otherwise background-only datasets."} +{"text": "Remote activity monitoring (RAM) technology has the potential to allow at-home caregivers to track the behaviors and activities of persons with dementia in real-time, thus facilitating more proactive symptom management. The aim of the present study was to assess whether RAM technology was associated with reductions in negative health transitions and service utilization for persons with Alzheimer\u2019s disease or related dementias (ADRD). An embedded experimental mixed methods design was used that included 179 caregivers who were followed over a 1.5 year study period. Participants were randomly assigned to receive the RAM technology system or usual care. Follow-up surveys were administered on a bi-annual basis over an 18-month period that collected information on dementia caregiver and care recipient characteristics and outcomes. We developed multilevel mixed effects models to estimate odds ratios for binary outcomes and categorical outcomes . In adjusted models, RAM technology use was statistically and significantly associated with lower odds of emergency department visits (p<.05) and less frequent falls (p=.05) for people living with dementia over the 1.5 year study period. Technologies that prevent or delay the onset of negative health events via improved care management and monitoring may enhance the care of persons with dementia. As dementia continues to pose an array of challenges for someone living with ADRD, RAM or similar technologies may offer a solution to the conundrum of dementia care."} +{"text": "To assess the job and training satisfaction of junior doctors working in Mental Health placements in Derbyshire; to highlight areas of good practice and identify areas that need improvement to enhance their working experience.This is an ongoing Cycle of Quality Improvement to address Juniors Doctors enjoyment of work and job satisfaction. On a 25 point questionnaire we sought feedback as open response, graded response and free text. Questions were formulated using suggestions from Royal College of Psychiatrists Supported and Valued Review and BMA Fatigue and Facilities Charter. Advised areas of improvement from the previous 2017 Quality Improvement project were also reviewed and incorporated into the questionnaire design.All junior trainees working between December 2020 to April 2021 in Derbyshire Healthcare NHS Foundation Trust were sent the questionnaire.Official end of placement feedback from January-December 2020 was also compared to our findings.15 doctors completed the questionnaire.Areas of trainee-reported satisfaction included training on management of common psychiatric conditions (73%), weekly teaching sessions (100%), ability to organise leave (100%).Areas of dissatisfaction included training on management of psychiatric emergencies (40%), poor regularity of supervision (53%), inadequate access to phlebotomy services (66%), ability to take adequate breaks (66%) and ability to fulfil training requirements (40%).Discrepancies were noted in responses to similar questions in our questionnaire compared to the official end of placement feedback, with greater trainees answering with negative responses in this project.This project highlighted areas of high satisfaction for trainees and showed specific areas for improvement. Trainees responses have been reviewed with Educators and Trust Management for collaborative solutions, pilot schemes and future QI projects identified.Observer bias was noted, with greater numbers of doctors answering similar questions negatively when feedback was anonymous, suggesting that they may be giving more honest answers when their identity is concealed."} +{"text": "Women from ethnic minorities who experience mental health problems during the perinatal period are disproportionately represented in involuntary care. They have poorer access to community care but have higher engagement with services once accessed. Their pathways to accessing perinatal mental health care remain underexplored.To investigate the pathways to perinatal mental health services for women across different ethnic groups, including number of caregivers encountered and time elapsed between referrals.Analysis of patient records and routine service data from community and inpatient perinatal mental health services in the United Kingdom. Use of an adaptation of the WHO\u2019s pathway encounter form.Women from ethnic minority groups experience increased levels of complexity on their journey to accessing perinatal mental health care. We will present a detailed analysis of patient and service characteristics.Referral pathways to perinatal mental health services need to be optimised for women from underrepresented groups."} +{"text": "Callous-unemotional (CU) trait is a characteristic of conduct disorder. As CU-like behaviours emerge from early childhood, this could potentially be predicted early on in life. There is debate in whether general or specifically fear expression processing was impaired in those with CU traits. No studies investigated subliminal emotion processing in those with CU traits. Hence, this study addressed two questions. Firstly, we investigated whether attention to general facial expression or fearful expression is related to future CU behaviours. Secondly, we examined whether subliminal emotion processing can predict CU behaviours alongside supraliminal emotion processing by comparing EEG data to CU behaviours.We performed EEG on 7 months old infants using fearful and happy faces as stimuli to investigate whether attention bias to general facial expression or fearful expression is related to future CU behaviours through the Nc component (300\u2013600ms). We also used both subliminal and supraliminal eliciting techniques to determine whether there are any differences in terms of prediction of CU behaviours. The ERP data were then compared with behavioural data, including aggression and empathy scores, collected when the participants reach 14 to 18 months old through the infant-toddler version of the Multidimensional Assessment of Preschool Disruptive Behavior (MAP-DB) and the infant empathy and prosocial behaviour (IEPB) questionnaires.A total of 18 infant participants were included in our analyses. There is a significant interaction between emotion and empathy for the Nc component, but not aggression. Infants with low empathy paid less attention to fearful facial expressions compared to happy facial expressions while those with high empathy paid more attention to fearful facial expressions compared to happy facial expressions. Moreover, subliminal and supraliminal emotion processing had similar ERP eliciting ability.Our study showed those with less empathy have a different pattern of attention bias to emotional expression and are less sensitive to fear emotion. Attention bias to emotional expression during infancy could be used to predict CU behaviours during toddlerhood. Being able to predict CU behaviours before their occurrence could help identify those in need of early intervention and help identify potential participants for longitudinal studies that could aid the development of interventions and understanding of CU behaviours. Furthermore, subliminal and supraliminal emotion processing has a similar predicting ability for CU behaviours. This is the first study that investigated subliminal emotion processing in infants with CU behaviours. Future studies would need to include a larger sample size to verify our findings."} +{"text": "The standard of care treatment in advanced ovarian/fallopian tube cancer involves a combination of surgery to achieve complete cytoreduction and platinum\u2010based chemotherapy. The British and European guidelines for the surgical management of advanced ovarian/fallopian tube cancer advise maximal surgical effort cytoreductive surgery. This may require four\u2010quadrant surgery including multivisceral resection techniques such as peritoneal stripping, diaphragmatic resection, removal of bulky pelvic/para\u2010aortic lymph nodes, splenectomy, liver and/or liver capsule resection and bowel resection. It is recognized that the delivery of adequate surgery for ovarian cancer frequently requires gynaecological oncologists to work together with colorectal surgeons and with surgeons from other specialities, including upper gastrointestinal (UGI) surgeons.This statement sets out a framework for joint working for gynaecological oncologists and colorectal and UGI surgeons. The Royal College of Obstetricians and Gynaecologists\u2019 curriculum for subspeciality training in gynaecological oncology is outside the remit of this document. However, we understand that this is under a cycle of review. As key stakeholders the ACPGBI, ASGBI, AUGIS and BGCS are committed to inputting into this process.Colorectal\u00a0\u00b1\u00a0UGI surgical input into gynaecological oncology cases must be formally recognized in job plans and appropriately resourced. It is no longer acceptable for colorectal input into gynaecological oncology cases by colorectal surgeons to be performed pro bono.Gynaecological cancer centres will identify at least one, but ideally two colorectal surgeons with a specific interest in this area of joint working. For RCOG\u2010approved gynaecological oncology training centres one colorectal surgeon will be responsible for the colorectal training of RCOG gynaecological oncology subspeciality trainees. There will be reciprocal arrangements for gastrointestinal surgery trainees to be supported by gynaecological oncologists to achieve the relevant knowledge and competences in gynaecological disease incorporated in the \u2018Intermediate & Final Stage Syllabus\u2019 of the Intercollegiate Surgical Curriculum for General Surgery .Published evidence confirms the challenge in accurately predicting the need for colorectal surgical input at ovarian debulking surgery, given the poor predictive value of cross\u2010sectional imaging. Nevertheless, every effort should be made to involve colorectal surgeons ahead of surgery if this is anticipated. This may take the form of a formal multidisciplinary team discussion or may simply involve joint discussion between consultant colleagues. All associations agree that colorectal and UGI surgeons should only be called to theatre for planned procedures without advance notice in exceptional circumstances.The extent of support required by colorectal and other surgeons for the gynaecological oncology surgery team for the provision of maximal effort cytoreductive surgery for advanced ovarian cancer will vary depending upon the skills and experience of the gynaecological oncologists. These arrangements should be agreed with the colorectal and UGI surgery teams and ratified through appropriate clinical governance processes. In some centres, gynaecological oncologists will perform the majority of bowel surgery independently, whereas in others colorectal surgeons will be involved more frequently. In both models, arrangements for postoperative care and management of complications must be explicitly detailed and agreed with both gynaecological oncology and colorectal/UGI surgical departments.Postoperative management of joint gynaecological oncology/colorectal cases will follow documented enhanced recovery after surgery protocols as agreed locally. Both teams will ensure that communication exists with the duty surgical teams. It is accepted that specific individuals will be unlikely to be able to provide 24/7 cover for this group of patients.Postoperative complications arising in elective patients after major gynaecological oncology resections should be managed in a timely fashion and may require liaison with the general surgery on\u2010call team. Patients with intra\u2010abdominal complications should be managed in line with the Trust\u2019s emergency laparotomy pathway with regard to resuscitation and cross\u2010sectional imaging. Preoperative, operative and postoperative management of these cases requiring emergency laparotomy should be performed in line with the recommendations of the National Emergency Laparotomy Audit (NELA). It is planned that these cases will now form part of the NELA database.Patients undergoing surgery by both gynaecological oncology and colorectal/UGI surgeons should be subject to joint regular audit and morbidity and mortality meetings. Patients will be submitted to national registries where appropriate. It is hoped that such registries will expand in the fullness of time to cover large\u2010bowel\u2010related issues and patient reported outcome measure data.No ethics committee approval was required for this work."} +{"text": "African Americans are more likely to have Alzheimer\u2019s Disease (AD) yet less likely to be included in AD research. Additionally, memory complaints may signal the clinical genesis of AD. Interventions such as physical activity that can help individuals maintain their cognitive functioning as they age have merit in research. The objective of this study is to examine the influence of physical activity of various intensities on episodic memory in African American older adults at the interindividual and intraindividual levels over time. We used data from the Health and Retirement Study over 12 years. Our analyses included an indicator of episodic memory and we examined physical activity of three intensities as predictors while controlling for relevant demographic and health variables. Preliminary data indicates that African American older adults who engaged in more frequent physical activity (regardless of intensity) had better episodic memory. Individuals who engaged more frequently in physical activity had a slightly greater rate of decline than those who engaged less frequently in physical activity. Physical activity may act as a buffer against cognitive decline for older adults. Intensity need not be vigorous to observe changes."} +{"text": "Emergency medical services (EMS) systems have developed alternative disposition processes for patients vs taking patients directly to an emergency department (ED). Studies show that patients favorably support these alternative options but have not included the perspectives of caregivers of children. Our objective was to describe caregivers\u2019 views about these alternative disposition processes and analyze whether caregiver support is associated with sociodemographic factors.We surveyed a convenience sample of caregivers in a pediatric ED. We asked caregivers 15 questions based on a previously validated survey. We then conducted logistic regressions to determine whether sociodemographic factors were associated with levels of support.We enrolled 241 caregivers. The median age of their children was five years. The majority of respondents were non-Hispanic Black (57%) and had public insurance (65%). We found that a majority of respondents supported all alternative EMS disposition options. The overall level of agreement for survey questions ranged from 51\u201393%. We grouped questions by theme: non-transport; alternative destinations; communication with EMS physician; communication with primary care physician and sharing records; restricted EMS role; and shared decision-making. Regression analyses for each theme found that race/ethnicity, public insurance, and patient age were not significantly associated with the level of support.Most caregivers were supportive of alternative EMS disposition options for children with low-acuity complaints. Support did not vary significantly by respondent race/ethnicity, public insurance status, or patient age. Emergency medical services (EMS) call volumes have increased to more than 20 million annual EMS responses in the United States9EMS Agenda 2050 envisages that in the future, \u201cEMS and its partner agencies will coordinate to provide the most appropriate care to the patient, with transport to a healthcare facility being just one option.\u201d12 In 2019, the Centers for Medicare & Medicaid Services (CMS) launched the Emergency Triage, Treatment & Transport (ET3) model. The ET3 provides incentives for EMS agencies to develop and assess protocols for Medicare patients so that they may be assessed at the scene (including with the use of telemedicine) and not transported or transported to a primary care office.15Industry experts and federal funding agencies have recommended pilot studies of alternative EMS disposition processes.Successful implementation of alternative EMS disposition processes will require understanding the perspectives of patients and caregivers.We conducted a cross-sectional survey of caregivers presenting to an urban, academic pediatric ED between August 2018\u2013January 2019. This study took place at a freestanding children\u2019s hospital with a Level I pediatric trauma center with an annual volume of approximately 90,000 emergency patient encounters. The hospital receives almost all EMS pediatric transports from the District of Columbia, and the majority of pediatric EMS transports from two neighboring counties in Maryland. Our institutional review board approved this study.We used a previously validated survey developed by Munjal et al.Population Health Research CapsuleWhat do we already know about this issue?Adult patients are supportive of alternative EMS dispositions for non-emergent calls.What was the research question?Are caregivers supportive of including children in alternative EMS disposition programs?What was the major finding of the study?Most caregivers are supportive of including children in alternative EMS disposition programs.How does this improve population health?Including appropriate children in alternative EMS disposition programs could provide more efficient and patient-centered care.We decided a priori to collect an initial sample of approximately 250 patients to enable us to perform multivariable modeling with 12 predictor variables for the outcome of caregiver agreement (assuming at least 50% respondent agreement). The primary objective of our study was to describe the overall level of support for specific components of an alternative EMS disposition process. We decided a priori to group \u201cagree\u201d and \u201cstrongly agree\u201d responses together. The secondary objective of our study was to determine whether support for components of an alternative EMS disposition process was associated with race/ethnicity or insurance status. We used bivariable regression analyses for each survey question to determine the association with race/ethnicity and insurance status.all questions grouped within a theme. We decided a priori to adjust our final multivariable regression models for patient demographic factors, including age, race/ethnicity, gender, insurance status, state of residence, and other patient encounter variables. Other encounter variables included in the regression analysis were as follows: arrival by ambulance on day of survey completion; use of an ambulance in the prior three years; day of week; hour of arrival; and Emergency Severity Index (ESI) triage level on the date of visit. All statistical analysis was conducted using SAS software version 9.3 .We then grouped questions into six themes . We repeated the bivariable logistic regression analyses based on respondents who agreed with We enrolled 241 caregivers. The median patient age was five years (interquartile range 18 months\u201310 years), and 56% were male. The most common racial/ethnicity responses were non-Hispanic Black (57%) and Hispanic (26%). Most patients were enrolled in public insurance programs (65%). These sample characteristics are similar to overall ED patient demographics at our institution. Almost one-quarter of caregivers stated they had called 911 in the prior three years, while only 14% of respondents had arrived in the ED by ambulance on the day of survey enrollment .The overall level of agreement for survey questions ranged from 51\u201393%. For ease of interpretation, we grouped questions into themes that addressed specific components of alternative EMS disposition processes: non-transport; alternative destinations; communication with EMS clinicians; communication with primary care physicians and sharing of medical records; a restricted role for EMS; and shared decision-making. These themes align with those used in previously published literature using this survey.Participants were told that a 911-nurse triage line involves a nurse speaking with parents after they have called 911, to determine whether an ambulance is needed. After hearing this brief description, 61% of caregivers agreed with the statement \u201cI would feel comfortable speaking to the nurse triage line operator by telephone and following their advice\u201d (Q15). We found that 63% of caregivers agreed with the statement \u201cI would prefer my child received an urgent appointment at a clinic or primary care doctor\u2019s office rather than being transported to the emergency room if the nurse triage line operator determines that they do not need to go to the hospital\u201d (Q14) .We used White, Non-Hispanic, and private health insurance as our reference group in separate bivariable analyses and did not identify any significant association between those variables and caregiver level of support for any survey question. We ran additional bivariable regression analyses for all other covariates and did not find any variables with a significant association with the level of caregiver support. In our adjusted models, we similarly did not identify any patient or encounter variables associated with support for any specific survey question or compoA majority of caregivers in this study were supportive of including children in alternative EMS disposition processes. Our results do not support our hypothesis that child age, race/ethnicity, and insurance status would be associated with the level of caregiver support for any aspect of an alternative EMS disposition process. There is currently very little literature regarding caregiver preferences for alternative EMS dispositions for children and no data regarding caregiver attitudes toward a 911-linked nurse triage line. The levels of support for alternative EMS disposition processes in our study are similar to the findings in previous studies with adults.Previous data from our institution shows significant rates of low-acuity pediatric EMS utilization.Even though a higher proportion of pediatric EMS calls are for low-acuity complaints than adult EMS calls, children have been excluded from the vast majority of community paramedicine programsThere are several limitations to our study. First, this was a single-center study undertaken in an urban area with most respondents identifying as Black or non-Black Hispanic. These findings should not be applied to other populations. Second, this data was collected before the coronavirus 2019 pandemic. Families and EMS agencies have been eager to reduce unnecessary EMS transports and ED visits during the pandemic,Additionally, there are specific limitations related to our survey methodology. We may have selection bias, as this was a sample of caregivers in the ED when RAs were available to enroll participants. While our patient sample had similar demographics to overall ED patient data, social factors affecting the use of EMS may be different for children arriving overnight. Low-acuity pediatric EMS calls are more common overnight than during usual office hours.Caregiver support for alternative EMS disposition processes for children is similar to published rates for adult patients. We found high levels of support for most components of an alternative EMS disposition process, although almost half of caregivers were opposed to being left at the scene if EMS determined transport was not necessary. Levels of support did not vary significantly with caregiver insurance status or race/ethnicity. Our study directly refutes the assertion that caregiver expectations should automatically preclude children from being included in alternative EMS disposition programs. Further qualitative studies should explore why caregivers have variable levels of support for the component parts of an alternative EMS disposition process. Caregiver perspectives could also be used to develop specific alternative EMS disposition protocols that are patient centered. These protocols would then need to be prospectively evaluated."} +{"text": "We report human monkeypox in a man who returned to the United States from the United Kingdom and reported no sexual contact. He had vesicular and pustular skin lesions but no anogenital involvement. The potential modes of transmission may have implications for the risk of spread and for epidemic control. The 2022 multicountry monkeypox outbreak has been linked primarily to intimate contact among men who have sex with men . The palmar vesicle was unroofed; a swab of the expressed clear fluid tested positive for nonvariola orthopoxvirus DNA by quantitative PCR (qPCR) and was confirmed as monkeypox virus DNA by qPCR specific for clade 2/3 (West Africa) monkeypox to clarify viral shedding. We detected virus DNA in a saliva sample, as well as from patient-collected conjunctival and rectal swabs using both the non-variola orthopoxvirus and clade 2/3 monkeypox virus qPCRs. Lesions resolved by day 26 after symptom onset.This patient tested positive for monkeypox virus DNA from several nonlesion samples. The nasopharyngeal and saliva findings are noteworthy because the patient did not report respiratory symptoms. In addition, the detectable viral DNA in the rectal swab specimen in the absence of visible anal lesions or pain indicates a potential for sustained sexual transmission, although the viral DNA levels were low; contamination during self-collection cannot be ruled out. We were unable to assess whether internal rectal lesions were present. This case highlighted the distinctiveness of clinical manifestations as they indicated potential routes of transmission during the 2022 multicountry outbreak of monkeypox. This patient did not report recent sexual contact, did not have evidence of genital lesions or inguinal lymphadenopathy (This case also demonstrates the importance of local monkeypox virus testing, rather than centralized testing in public health or commercial reference laboratories. Local testing enabled diagnosis in <12 hours and immediate notification to local and state public health authorities for isolation and contact tracing. Additional information about monkeypox in a patient without viral prodrome or sexual exposure, California, USA."} +{"text": "This article reviews microbial carbon dioxide fixation for the purpose of making useful chemicals from a chemical engineering perspective.Natural organisms have evolved to fix carbon dioxide for their survival purposes, and producing artificial autotrophic organisms might provide for more beneficial microbes as cell factories for biotechnology.2 fixation in biotechnology published in FEMS Microbiology Letters. The issue includes studies of naturally occurring bacteria and engineered enzymes.This link is to a thematic issue compendium dealing with CO2 fixation.This review article deals with recent advances in studies of microbial organelles for CO2 fixation.This article discusses largely unknown sources of carbon dioxide fixation, sometimes denoted as microbial COHeterotrophs catalyse many different carboxylation reactions. This has often been ignored as a contribution to large\u2010scale carbon dioxide fixation, but the report suggests it is significant.Here, it is discussed that electrons from solid\u2010phase conductive surfaces can stimulate photosynthetic carbon dioxide fixation.2 fixing enzymes described to date.This report investigates enoyl\u2010CoA carboxylases/reductases as some of the most efficient COThis link takes viewers to an interesting video on synthetic carbon dioxide fixation.There are many efforts to sequester carbon dioxide from the atmosphere. This report seeks to be comprehensive and approaches the topic from a range of synthetic chemical and biological perspectives.2 fixation serves to provide information of adaption for an autotrophic lifestyle.This engineering of COThis report is a metagenomic study focussed on carbon dioxide fixation and rhodopsin proteins in microbes found in mats in hypersaline lakes.This is a popular science article describing high\u2010flux carbon dioxide fixation by a modified microbial enzyme."} +{"text": "Squamous Cell Carcinoma of the temporal bone is an uncommon entity accounting for fewer than 0.2% of all tumors of the head and neck and is associated with a poor outcomeA 57 year old patient presented at the ENT OPD of Hamidia Hospital, Bhopal with complaints of discharge from right ear since 6 months, right preauricular swelling since 15 days and right sided facial nerve palsy since 15 days. On otologic examination there was a diffuse bony hard swelling of about 5 \u00d7 5cm involving the right preauricular and zygomatic region. External auditory canal was completely obliterated by a fleshy polypoidal mass covered with discharge. Tuning fork tests revealed a moderate conductive hearing loss on right side.High resolution CT scan of temporal bone and head revealed a large soft tissue mass showed post contrast enhancement involving middle ear cavity and external auditory canal . ExtensiBiopsy was taken from external auditory canal and revealed the mass as Squamous cell carcinoma (adenoid pattern). Owing to the poor cardiovascular and pulmonary status as well as unresectability surgery was deferred. Patient underwent high dose induction chemotherapy with Methotrexate(400mg/kg) and Mitomycin(10mg/sq m) followed by radiotherapy, dose of which was 7000 rads with brain exposure lessened by 1000 rads. Tumor shrinked and patient's facial palsy recovered however complete remission could not be achieved and patient succumbed to his disease within 3 months of presentation.Chronic otitis media and cholesteatoma are common in patients with temporal bone cancers and have been implicated as etiologic factorsSpecific radiographic information is crucial for accurate preoperative staging. A fine-cut (1 mm) high-resolution CT scan of the temporal bone should be obtained. MRI with gadolinium enhancement can be helpful as it delineates soft tissue interfaces. An audiogram is obtained prior to performing any major procedure on the ear or temporal bone. To date, no staging system for temporal bone malignancies is universally acceptedPrimary radiation is ineffective as curative treatment. Postoperative radiation treatment may be indicated in advanced diseaseThe optimal management of temporal bone cancer remains unclear because of continued debate regarding staging, the utility of preoperative radiographic evaluation, the extent and nomenclature of surgical procedures and the use of adjuvant radiation. The limited number of cases of temporal bone malignancies at each individual institution precludes definitive conclusions regarding the optimum protocol for management."} +{"text": "The retraction has been agreed upon following an investigation based on allegations raised by a third party, which revealed inappropriate and unexplained duplications between this and a previously published article [The above article published online on 03 November 2020 in Wiley Online Library ( article . Thus, tThe first author of this article admitted to adding the corresponding author to this paper without their consent."} +{"text": "Evaluate the effectiveness of the virtual JRS interventionMeasure discharge rates after JRS intervention to examine if attendance at JRS at the point of entry into CAMHS can lead to more timely discharge due to targeted early interventionTo capture parent and clinician feedback focusing on the challenges and improvements that have occurred due to this adapted delivery of servicesCOVID-19 resulted in dramatic shifts in how interventions are provided within mental health services, creating the opportunity to virtually deliver JRS groups to parents of young people attending Belfast CAMHS. This is a sensory attachment intervention that facilitates the process of self-regulation and co-regulation through the use of food, sensory activities and an enriched environment provision. It is currently facilitated by CAMHS clinicians via video calls over 4 consecutive weeks.A systematic database search was conducted examining number of parents who have attended overall; weekly attendance; Did Not Attend rate; length of time between CAMHS initial assessment and JRS intervention; number of families discharged after JRS and number of families allocated to partnership/medic after JRS.CAMHS clinicians (not directly involved in facilitating JRS intervention) gathered qualitative feedback from families .132 parents were invited between March-December 2020. 41 families have been discharged, 60 families have been allocated to partnership or medic and 31 are awaiting future JRS groups due to non-engagement, or a further review by JRS facilitators or a CAMHS clinician that they are already allocated to.Five parents provided positive qualitative feedback.As JRS has engaged a high number of parents in a relatively short time -period, it would be helpful to further explore its effectiveness as a first line intervention in CAMHS, thereby informing service delivery moving forward."} +{"text": "Clinical practice management guidelines for early-stage endometrial cancer suggest surgical staging, including histological assessment of lymph nodes.Surgical staging is a surgical credo that\u2014despite lack of randomized evidence\u2014has remained standard practice for more than three decades. Since its adoption in 1988 by the International Federation of Gynecology and Obstetrics (FIGO), surgical practice has transitioned into sentinel node biopsy using indocyanine green and near-infrared imaging.The international ENDO-3 trial aims to deal with this knowledge gap. It randomizes patients to hysterectomy with or without sentinel node biopsy.\u201cI want peace of mind\u201d, \u201cI want all my cancer removed\u201d) and fear that not removing lymph nodes puts them at risk. Others voice equally strong concerns against it .Some patients voice strong opinions for or against surgical staging through sentinel node biopsy even before specific information is provided. Some have a strong preference for lymph node removal (High-level evidence informing patients of the benefits and disadvantages of sentinel node biopsy are not yet available. ENDO-3 will provide patients with the necessary high-level information relating to the advantages and potential detriments of sentinel node biopsy compared with no lymphadenectomy, thus quantifying key risks, including the requirement for full lymphadenectomy; the need for post-operative treatment; operative blood loss and adverse events including lymphedema; and the impact on survival probability.ENDO-3 will address the knowledge gap that has been present for over 30 years and will be critical to inform both clinicians and patients. It will ensure that the choice of surgical management of endometrial cancer by clinicians and patients is supported by robust evidence leading to optimal health outcomes."} +{"text": "Previous studies have examined associations of cardiometabolic factors with depression and cognition separately.To determine if depressive symptoms mediate the association between cardiometabolic factors and cognitive decline in two community studies.Data for the analyses were drawn from the Rotterdam Study, the Netherlands (n = 2940), the Whitehall II study, UK (n = 4469) and the Canadian Longitudinal Study on Aging, Canada .Mediation analyses suggested a direct association between cardiometabolic factors and cognitive decline and an indirect association through depression: poorer cardiometabolic status at time 1 was associated with a higher level of depressive symptoms at time 2 , which, in turn, was associated with greater cognitive decline between time 2 and time 3 .Evidence from three independent cohort studies suggest an association between cardiometabolic dysregulation and cognitive decline and that depressive symptoms tend to precede this decline.\u2022\u2002Cardiometabolic dysregulation and depression might increase cognitive decline.\u2022\u2002The association between cardiometabolic dysregulation and cognitive decline might be mediated by depression."} +{"text": "A previously healthy 45-year-old man presented to the emergency department with bilateral knee pain and inability to extend his knees after a slip and fall on ice. The clinical diagnosis of bilateral quadriceps tendon rupture was confirmed by computed tomography (CT) of bilateral knees. The patient underwent successful operative repair the following day.Bilateral quadriceps tendon rupture is rare and can be difficult to diagnose due to the impossibility of comparing the affected to the unaffected limb. Plain radiographs are usually not helpful, but ultrasound, CT, and magnetic resonance imaging may be used to confirm the clinical diagnosis. A 45-year-old man with no significant past medical or surgical history presented to the emergency department after a mechanical fall. He took no daily medications and had a body mass index (BMI) of 44.2 kilograms per squared meter. The patient slipped on ice while walking and felt \u201cpops\u201d over both knees while falling. Exam demonstrated bilateral knees with no obvious swelling or bruising. He had good passive range of motion and mild tenderness to palpation superior to the patella on both knees. The patient was unable to perform straight leg raise with either leg.Plain radiographs of bilateral knees demonstrated mild soft tissue swelling over bilateral superior patellae but no other evidence of injury . ComputeBilateral extensor mechanism rupture is rare and can be difficult to diagnose, with an initial missed rate reported up to 30\u201350%.3Tendon rupture often occurs due to indirect trauma. While attempting to regain balance the quadriceps rapidly contracts with the knee flexed. Rupture is likely from the maximum quadriceps contraction rather than the fall itself.2What do we already know about this clinical entity?Simultaneous bilateral quadriceps tendon rupture is a rare event, especially in patients without significant comorbidities.What is the major impact of the image(s)?Plain radiographs are rarely diagnostic but ultrasound, computed tomography, or magnetic resonance imaging may be helpful to make the diagnosis in the emergency department.How might this improve emergency medicine practice?Include bilateral quadriceps tendon rupture in the differential diagnosis when evaluating a patient whose clinical presentation is consistent with this rare entity."} +{"text": "There are more complications in transbronchial lung cryobiopsy than in a conventional transbronchial lung biopsy. Respiratory endoscopists should be aware of the potential complications, including rare complications such as hemothorax. There are more complications in transbronchial lung cryobiopsy than in a conventional transbronchial lung biopsy. Respiratory endoscopists should be aware of the potential complications, including rare complications such as hemothorax. A man in his 80s visited our hospital with fever and dyspnea. Chest computed tomography (CT) revealed diffuse ground\u2010glass opacities in both lungs Figure\u00a0. IntravaComplication rates are higher in TBLC than in conventional biopsy in patients with interstitial lung diseases, with pneumothorax and moderate/severe bleeding rates of 12% and 39%, respectively.Toshiyuki Sumi is the guarantor of the clinical content of this submissionNone declaredThe authors declare that appropriate written informed consent was obtained from the patient for the publication of this report and any accompanying images"} +{"text": "Good medical practice encompasses teaching students which is a core competency for trainee doctors. The aim of this project was to assess and improve junior doctor participation in undergraduate psychiatry teaching.2 surveys were conducted: 1) Psychiatry-related trainee doctors working in Severn Deanery were emailed a questionnaire to assess their involvement in undergraduate teaching, including barriers and motivators for teaching; 2) doctors with a formal role in teaching were sent a questionnaire to explore their views on recruiting trainee doctors to teach. Questionnaires consisted of multiple answer questions, matrix questions and qualitative free text answer questions. Trainees were then delivered a presentation advertising teaching opportunities. The impact of this on recruitment into psychiatry undergraduate teaching was reassessed by questionnaire.44 responses were received to the first survey; 13 to the second. The most common answer trainees gave for factors that prevented involvement with teaching students was \u201cunaware of teaching opportunities,\u201d and \u201clack of overall availability due to clinical commitments.\u201d The most common factor chosen as a motivator for involvement was \u201cnotification of session date/timing early in placement\u201d and \u201cprotected teaching time in job-plan.\u201d The results highlighted difficulties recruiting trainee doctors to teach, resulting in tutors reducing, cancelling or adapting sessions due to lack of support.This project identifies barriers and motivators of trainee doctor involvement in undergraduate medical education. To ensure lasting participation of trainees in medical education, support is needed for protected time to teach in clinical roles."} +{"text": "COVID-19 as a public mental health emergency has exacerbated existing mental health inequalities. The UK government invited local authorities with areas of high deprivation to apply for a year of funding, in order to address the mental health impacts of COVID-19 and incentivize investment in prevention and promotion interventions for better mental health. South Tyneside Council in North East England made a successful bid to the Better Mental Health Fund (BMHF), and distributed grants to 7 organisations delivering 13 programs. A qualitative evaluation of these programs aimed to answer the following questions:1. How were the funded programs implemented?2. What difference did they make to local beneficiaries?3. How might these programs and their impacts be sustained into the future?4. Has the BMHF led to any wider impacts on organisations and local communities?In-depth interviews with individuals, pairs and groups were conducted online or in person with service providers and beneficiaries. Non-verbatim transcripts were made from recordings, checked with verbatim transcripts from Teams and Zoom, and analyzed thematically to generate a coding frame. Throughout the analysis, comparisons were made between organizations and programs.Fifteen interviews involving 22 participants lasting up to an hour each were conducted. The main themes identified as impactful were 1) community approaches based on supportive and good relationships between the local authority public health lead and participating organizations (mainly voluntary agencies), enabling 2) capacity-building for mental health resilience and 3) community empowerment. This was despite the short turnaround of the grant application process, limited time to deliver on targets, and anxieties about future sustainability.Short-term funding can build capacity in mental health resilience in deprived areas if administered by public health leaders who relate well with provider organizations.\u2022\u2002Public health leaders who relate well with provider organizations are key drivers of community health promotion strategies that include mental health capacity building.\u2022\u2002Qualitative methods used in evaluations can inform public health commissioning by capturing the benefits and challenges of short-term funding for interventions promoting community mental health."} +{"text": "To determine whether there are any gaps in cardiometabolic monitoring within primary or secondary care for people prescribed antipsychotic medication. A well-established system of cardiometabolic monitoring and checks has been implemented for patients with psychosis and bipolar in secondary care. It was unclear whether patients without these diagnoses were receiving the same level of monitoring.Data were collected retrospectively from case notes of service users under CMHRS Reigate. We included all patients from three GP practices (100 patients) and identified all who were prescribed antipsychotics and their diagnoses. The GP practices were contacted to determine whether a system was in place to flag physical health monitoring requirements for service users on antipsychotics regardless of diagnosis. The results were used to calculate the potential number of patients across the entire trust who were at risk of not receiving cardiometabolic monitoring.24/100 patients were prescribed antipsychotics without a diagnosis of psychosis or bipolar. 11/24 had a diagnosis of Emotionally Unstable Personality Disorder. Quetiapine was the commonest antipsychotic. None received routine cardiometabolic monitoring.The total caseload for all 11 adult community teams in the Trust is 2434. If prescribing and monitoring practices are similar 584 individuals may be affected.2/3 GP practices responded. Both confirmed that they would only conduct cardiometabolic monitoring when taking over prescribing/on discharge from secondary care if specifically requested to do so.This service improvement project has identified a significant group of patients who aren't automatically offered cardiometabolic monitoring in secondary care.Private correspondence from Professor David Taylor confirms that these patients would also benefit from monitoring when prescribed doses that are more likely to cause adverse effects (Quetiapine > 150mg/Olanzapine >5 mg Risperidone >2mg)Secondary services need to identify these patients and include them in routine cardiometabolic monitoring.Secondary services need to work closely with primary care to ensure that responsibility for checks is agreed and handed over when necessary."} +{"text": "Eating disorders (EDs) constitute serious mental illnesses with high morbidity, lifetime mortality and associated stigma due to the label of mental illness. The sparse research assessing adolescents\u2019 knowledge of and attitudes towards EDs highlights their low understanding of these conditions.The proposed study aims to bridge this gap by investigating adolescents\u2019 knowledge of and attitudes towards EDs as this will inform young people\u2019s engagement with ED services.Participants aged 12-18 will be randomly assigned a vignette depicting either a male or female 15-year-old displaying symptoms of anorexia nervosa (AN) or binge eating disorder (BED). They will be asked to select what they believe the condition described in the vignette is from a pre-determined list. They will then be informed of the correct diagnosis before completing a series of scales designed to assess their attitudes towards EDs. Participants\u2019 own potentially disordered eating behaviours will be assessed using the ED risk composite (EDRC) subscale from the EDI-3.It is expected that BED will be less likely to be correctly identified compared to AN, eliciting more stigma and male vignette subjects will be seen more negatively than female vignette subjects. Also, it is expected that participants with higher EDRC scores will have more knowledge of and less negative attitudes towards EDs than those with lower EDRC scores.This study will highlight the need for education around EDs targeted at adolescents to increase their knowledge and awareness, providing them with factual information ought to reduce stigma and negative attitudes and beliefs about EDs.No significant relationships."} +{"text": "This was the third time replicating the Preferences for Activity and Leisure (PAL) Card quality improvement project, but the first conducted entirely during the height of the pandemic. Nursing home providers attempted n=174 PAL Cards and completed n=166 (96%). Feedback from surveys with n=68 staff who came in daily contact with residents were collected by project champions. Staff had worked on average 6 years, indicated that they remember being been told about PAL Cards (81%), noticed them (94%), reported that the information on the PAL Card helped them start a conversation with a resident (79%), and helped them provide care (59%). PAL Cards are a feasible method to communicate important resident preferences to staff who come in daily contact with residents and may not have access to electronic health records. Recommendations for practice will be discussed including staff recommendations for improvements."} +{"text": "Latent variable models have recently been used to extract latent dementia index scores using cognitive and functional ability data. Yet, no cross-cultural comparisons have been conducted. This analysis tests metric measurement invariance of a latent dementia indicator (LDI) among older adults in Mexico and the US using different statistical criteria. We then test whether demographic risk factors relate with the LDI similarly depending on criteria used to test measurement invariance. Data included the MexCog and Harmonized Cognitive Assessment Protocol . The LDI is a latent variable measured using 13 cognitive and 10 functional common items. Measurement invariance was tested in multiple group confirmatory factor analyses using both nested chi-square difference tests and comparisons of comparative fit indices (CFI). Covariances between demographic risk factors (age and education) and the LDI were compared across models achieved using different measurement invariance criteria. Full metric invariance of the LDI was achieved using CFI comparisons but not with chi-square comparisons, which required nine loadings to be freely estimated across studies to achieve partial metric invariance. Regardless of criteria used to test measurement invariance, age and education correlated with the LDI in both studies in expected directions with similar parameter estimates regardless of approach used to test measurement invariance. The LDI may be a valid tool to compare associations between risk factors and dementia in the HCAP and MexCog. The LDI related with demographic factors in expected ways and findings were robust to statistical approach used for measurement invariance testing."} +{"text": "This pilot qualitative study aimed to investigate exercise habits and assess defecatory dysfunction among adult survivors of rectal cancer with and without stomas. Patients were eligible for the study if they had stage I\u2013IV rectal cancer, and less than 5 years had elapsed since surgery. We conducted semi-structured interviews with outpatients visiting two general hospitals in Japan and inquired about their diets, defecation, and exercise habits. The interview data were transcribed verbatim, interpreted, and abstracted to generate coding units; we divided the responses into categories and subcategories. Eleven patients had stomas inserted after surgery while six did not. Content analysis identified four categories common to patients with and without stomas: [diet control], [coping with defecation dysfunction], [compromising with defecation dysfunctions], and [maintenance of exercise habits]. Our results suggest the need for intervening among rectal cancer survivors to address eating habits to alleviate defecation dysfunction and exercise habits to maintain physical function. In clinical practice, symptom relief and exercise instruction may improve the well-being of cancer survivors with bowel dysfunction. Although it may sound strange, I walk about 40 min a day as a hobby. I walk about 5,000 steps a day,\u201d indicating that were maintained even after surgery. [Perceived difficulty of exercise after colostomy] consisted of , , , and (\u201cAfter all, I am handicapped because the stoma is compressed when crouching or picking stuff up,\u201d reflecting . Additionally, another participant stated \u201cI hate that I can only walk slowly because of exercise dysfunction caused by pain or neuropathy due to comorbidity and chemotherapy. I hate that I became a bit weak. I like sports but not extreme ones. I had so many things that I wanted to accomplish, but now it is impossible for me,\u201d reflecting .In the stoma group, five categories of exercise habits were identified; these were combined into two broad categories: [maintenance of exercise habits] and [perceived difficulty of exercise after colostomy] . , [coping with defecation dysfunctions], [compromising with defecation dysfunctions], and [maintenance of exercise habits]. In contrast, two categories were identified for survivors without pre- and postoperative exercise habits: [difficulty coping with defecation dysfunctions] and [perceived difficulty of exercise after colostomy]. In a systematic review of 25 cases, Neuberger et al. concludeThe participants in this study used [diet control] to [cope with defecation dysfunctions]. Previous studies found that dietary fiber ameliorates diarrhea, constipation, and fecal incontinence , suggestDietary management is the most common approach for ameliorating defecatory dysfunction. However, this approach requires clinical guidelines because patients\u2019 decisions so far have been based on their own experiences and assumptions; as such, the relationship between food types and defecation patterns remains uncertain . These iAdditionally, [compromising with defecation dysfunctions] led to [maintenance of exercise habits]. In contrast, those in the non-stoma group who did not exercise either before or after surgery had a seven-fold higher frequency of bowel movements and [difficulty coping with defecation dysfunctions], suggesting that defecation dysfunctions due to low anterior resection syndrome negativeOur current study revealed that, in the non-stoma group, and were issues of concern, indicating a need for more specific guidance regarding exercise. Moreover, was identified as a problem in the non-stoma group. In contrast, was a concern in the stoma group. These findings suggests that exercise intervention is necessary to prevent a decline in physical function. Furthermore, Neuberger et al. reportedThis study demonstrated that the [perceived difficulty of exercise after colostomy] caused by was common for those who did not exercise before or after surgery and those who discontinued exercise postoperatively. Asnong et al. investigCollectively, our findings illustrated the importance of a safer and more feasible combined intervention program incorporating eating, bowel movement, and exercise habits by identifying predictors of exercise based on physical activities and guidThe lower QoL in the stoma group may stem from emotional distress likely owing to changes in excretion habits because of the presence of a colostomy bag and defecation dysfunction. Additionally, patients in the stoma group experienced higher levels of fatigue and pain than did those in the non-stoma group on the symptom scales. These results conform to [perceived difficulty of exercise after colostomy] consisting of as identified during our interviews. A systematic review and meta-analysis of 19 trials on the effect of exercise among individuals with colorectal cancer by Singh et al. found thWe first evaluated defecation dysfunction and exercise habits in survivors of colorectal cancer with and without stomas. The study strength lies in its novelty, and our findings may provide fundamental clinical evidence to promote nutrition education and exercise habits in survivors with postoperative defecation dysfunction. Interventions for exercise habit formation may also improve the QoL . HoweverOur survey of lifestyle habits among rectal cancer survivors with and without stomas found that the participants engaged in [diet control] to [cope with defecation dysfunctions] and [maintained exercise habits] by [compromising with defecation dysfunctions]. Those without [maintenance of exercise habits] had [difficulty coping with defecation dysfunctions] and experienced [perceived difficulty in stoma management].Our findings suggest the need for providing interventions to improve eating and exercise habits to maintain physical function and alleviate defecatory dysfunction in survivors of rectal cancer, including those with and without stomas."} +{"text": "Cognitive functioning fluctuates daily throughout adulthood. Lapses in mindfulness can have cognitive consequences, which may be impacted by how old a person feels each day. Subjective age was examined as a mediator in the within-person relationship between mindfulness and cognition. 107 younger adults and 116 older adults completed reports of mindfulness and subjective age and tests of inductive reasoning and episodic memory for 8 consecutive days. Within-person multilevel mediation models indicated that daily subjective age mediated the relationship between daily mindfulness lapses and both indicators of daily cognition across ages. However, the mediation effect was stronger for younger adults on inductive reasoning but was stronger for older adults on episodic memory. These results show that daily changes in subjective aging are an important mechanism for daily cognition, with differential impact based on age and cognitive component."} +{"text": "INFORM (Improving Nursing home care through Feedback on perfoRMance data) is a tested research intervention targeting care managers that demonstrated positive two year follow up results and has subsequently been shaped into an operationally acceptable \u201cimplementation package\u201d. This package or innovation is being scaled up in one Canadian province\u2019s total Long-Term-Care (LTC) home population with in depth process evaluation during the first cohort of LTC homes. This evaluation will, among other things, assess sector needs for adaptation (vs fidelity). At its core INFORM is designed to address managers\u2019 learning needs with respect to using data to make positive change in a continuous learning loop. We will discuss the transformation of a research intervention to a sector innovation and report on interim process evaluation results."} +{"text": "Anal intercourse (AI) has been reported to be the riskiest among other sexual intercourses in spreading human immunodeficiency virus (HIV) and the risk could be minimized by the use of condoms. Whilst AI is believed to be practised mainly by men who have sex with men, AI has also been reported to occur in heterosexual relationships. However, data on condom use during heterosexual AI are inadequate in sub-Saharan Africa.A scoping review of English language published articles on condom use during heterosexual anal sex, whose studies were conducted in Sub-Saharan Africa from January 2010 to May 2020 was conducted. Articles were searched systematically on PubMed and Google Scholar electronic databases. Heterosexual AI was defined as penile penetrative anal sex between a man and a woman regardless of the sexual orientation of the 2 parties involved in the act of heterosexual AI.A total of 21 studies were eligible for analysis. Most of the studies (17 out of 21) reported females to be involved in heterosexual AI whilst 9 out of 21 studies reported males to be involved in heterosexual AI. The lifetime prevalence estimate of condom use during heterosexual AI ranged from 29%\u201397.5%. Other prevalence estimates of condom use during heterosexual anal intercourse were reported over various recall periods which were: 12 months' recall period with prevalence estimates ranging from 2.9%\u201359%; prevalence estimates for the past 3 months which ranged from 50%\u201394.4%; 1 month's recall period with prevalence estimates ranging from 5%\u201396% and prevalence estimates for the last intercourse experienced ranging from 1%\u201355%. Condom use during heterosexual AI was generally low and/or inconsistent among female sex workers (FSWs), men who have sex with men and women (MSMW) and some women in the general population. There were no risk factors identified in the study for the inconsistent or low use of condoms during heterosexual AI.Evidence from this study suggests condom use during heterosexual AI could be fairly low especially among groups such as FSWs, MSMW and some women in the general population. Risk factors for using condoms inconsistently or using condoms less during heterosexual AI are not clear. Heterosexual anal intercourse and condom use during the AI practice is generally an under-studied subject in Sub-Saharan Africa. Future studies need to explore on heterosexual AI and condom use practices during AI comprehensively so that there can be concrete evidence on the subject which will inform targeted interventions aimed at reducing HIV among heterosexual populations in SSA. The Acquired immunodeficiency syndrome (AIDS) pandemic is still a burden especially in sub-Saharan Africa (SSA). Existing evidence has shown that globally, in 2018 about 67.5% people living with the Human Immunodeficiency Virus, HIV (virus which causes AIDS) were living in SSA In many parts of the world especially the developing world including SSA, anal intercourse (AI) is believed to be mainly practiced by men who have sex with men. However, anal sex has been reported to be practised too among heterosexual couples which has been corroborated by various studies particularly from the United States of AmericaThis scoping review's main aim was to synthesize current evidence on condom use during heterosexual anal intercourse in SSA. Scoping reviews map present literature to a certain research topic, examining the research activity in a topic area and identifying gaps in the existing literature The scoping review was undertaken following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Articles were searched on PubMed and on Google Scholar databases. English language articles published from January 2010 to May 2020 were searched on PubMed using the search terms: heterosexual OR women OR female AND anal AND AND behaviour AND condom use AND Africa as the Medical Subject Headings (MeSH) terms. English language articles published from January 2010 to May 2020 were further searched on Google Scholar using the key words: heterosexual, women, anal sex behaviour, condom use, Africa with the exact phrase \u2018sub-Saharan African countries\u2019. A list of all the identified references was made in Zotero (a reference managing software) and duplicates were removed. Titles were first screened and those which were clearly irrelevant were discarded and abstracts were then screened. Full text articles were opened if the abstract had reported AI and if study was done in SSA or if study area was not specified. Full text articles were also opened from the abstracts whose study areas were not specified in case the studies were done in SSA. Full text articles were screened for condom use during heterosexual AI in sub-Saharan Africa. Eligible articles' bibliographies were screened for any additional relevant articles.Studies were included if: they were published in English language from January 2010 to May 2020, they were original studies, they were conducted in sub-Saharan Africa, they reported condom use during heterosexual AI and if the studies were based on quantitative or both quantitative and qualitative data. The studies had to contain at least a quantitative section so that the research questions on estimating prevalence of condom use during heterosexual AI and identifying risk factors for inconsistent condom use during heterosexual AI could be answered.Studies were excluded if: they were not original studies, they reported heterosexual AI but did not report condom use during heterosexual AI, if they reported both homosexual and heterosexual AI in a way that the 2 were indistinguishable, if they were conducted solely or partly outside SSA and if they did not report quantitative data. Heterosexual anal intercourse was defined as the penile-anal penetrative intercourse between a man and a woman regardless of the sexual orientation of the 2 parties involved in the heterosexual AI.A total of 21 articles were included in the final analysis. Most of the studies were from South Africa (9 studies), followed by Kenya (4 studies), Nigeria (2 studies) and single studies each from Tanzania, Cote d'ivoire, Senegal, Eswatini and Mozambique. One additional study was conducted in 3 countries; South Africa, Zimbabwe and Uganda. The common study design was the cross sectional study (12 articles), followed by cohort studies (4 articles), clinical trials (2 articles) and single studies each based on observation, in-depth interviews and mixed methods. Over 50% of the studies were based on convenience sampling. All the studies' responses on sexual behaviour were self-reported by participants. The common data collecting technique was the questionnaire; 9 interviewer administered, 6 self-administered and 2 which were not specified how they were administered. The studies' sample sizes ranged from 83 to 4965 though the sample sizes of those who practiced heterosexual AI differed by study. Only 1 study of the 21 focused exclusively on heterosexual AI. The prevalence estimates for heterosexual AI ranged from 1.1% among 501 men who have sex with men and women (MSMW)Prevalence estimates of condom use during heterosexual anal sex were reported disaggregated by recall periods. Apart from the lifetime recall period, a total of 4 recall periods were identified from the various studies analysed. The four recall periods include the 12 months' recall period, 3 months' recall period, 1 month's recall period and the last intercourse experienced. Study groups and their area were also specified on the prevalence estimates since the studies were focusing on various groups and from various places.The lifetime prevalence estimate for condom use during heterosexual AI ranged from 29%, among female sex workers (FSWs) in Kenya18 to 97.5%, among men and women in South AfricaCondom use during heterosexual AI was generally low among FSWs and among some men who have sex with men and women (MSMW) populations with prevalence estimates ranging from about 3% to about 70%All the 21 studies did not report any risk factors of inconsistent condom use during heterosexual AI. However, one study from Tanzania identified the following reasons for not using condoms consistently during AI from focus group discussions and these include; to avoid reducing pleasure during the intercourse, to prove loyalty to one's partner, unavailability of condoms nearby, inconvenience of condoms during AI (that is anus too dry to use condoms) and some participants perceived AI to be less risky in transmitting HIVOur findings identified the prevalence estimates for condom use during heterosexual AI in SSA to vary but mostly low. Over 60% of the reviewed studies' various prevalence estimates for condom use during AI ranged from about 3% to about 70% with only 2 articles in that range reporting estimates of about 70% and the rest below 60%. Populations such as FSWs, MSMW and women in general are known to be HIV high risk groupsIt is not clear why condom use during heterosexual AI was identified to be lower among men and women in general populations in studies conducted outside South Africa than in studies conducted in South Africa. However, condom use during heterosexual AI might be low in some countries other than South Africa possibly due to cultural variations which either encourage or discourage condom use as also identified in another study that social norms might either encourage or discourage condom useNone of the reviewed studies identified risk factors for inconsistent or low condom use during heterosexual AI. However, some risk factors identified for heterosexual AI could be indirect risk factors of inconsistent or low condom use during heterosexual AI for example experiencing sexual violence/coercionEvidence from our study shows that anal sex is being practiced during heterosexual intercourse in SSA though its prevalence is not clear. However, extensive studies on heterosexual AI in SSA are deficient including studies on condom use during heterosexual AI in SSA. Only 1 article of the 21 reviewed studies conducted a study exclusively on heterosexual AI. It is therefore imperative for future studies to explore on heterosexual anal intercourse and condom use during the anal intercourse comprehensively, especially given that AI is the riskiest in transmitting HIV. Concrete evidence on heterosexual AI and condom use during the AI practice can provide crucial information which will inform appropriate and targeted interventions aimed at averting HIV/AIDS in SSA.Our review only included articles based on published studies. Studies from earlier than 2010 were excluded because we wanted more recent data on the practice. Also, various recall periods and heterogeneity of study populations made it harder to compare prevalence estimates. Most studies used interviewer administered questionnaires to collect data which are less private and this might have led to social-desirability bias such as underreporting AI practises. Most of the studies employed convenience sampling and some recall periods were long which could have led to recall bias. As a result of the above limitations, our study's findings can only be used as preliminary evidence on heterosexual AI practice. Our study however, managed to identify some gaps for instance we identified that data on the risk factors of inconsistent or low condom use during heterosexual AI are deficient and that there is a general lack of studies on condom use during heterosexual AI in SSA. Some of the limitations mentioned above such as use of interviewer administered questionnaires and using convenient samples can also be treated as gaps in the heterosexual AI research which should be addressed by future researchers.Future studies on heterosexual AI to use self-administered tools if possible to collect data so as to minimize desirability bias given the sensitive nature of the AI subject.Future studies on heterosexual AI to try and sample participants randomly so that the samples can be representative of the target population and hence the findings can be generalized to the target population.To minimize recall bias, researchers in future could avoid asking participants to recall their sexual behaviours which would have happened a long time ago. Generally, more studies need to focus on exploring heterosexual AI and condom use during the practice in SSA so that there can be concrete evidence on the practice which will inform targeted interventions aimed at reducing HIV among heterosexual populations in SSA.Evidence from this study suggests condom use during heterosexual AI could be fairly low and the most vulnerable groups for inconsistent or low condom use during heterosexual AI being FSWs, MSMW and some women from the general population. Risk factors for inconsistent or low condom use during heterosexual AI are not explicit. Since most studies employed convenience samples, used interviewer administered questionnaires to collect data which are less private and some recall periods in the studies were long, we therefore recommend;"} +{"text": "The aim of the project was to improve the routine incorporation of driving advice based on Driver and Vehicle Licensing Agency (DVLA) guidance into discharge planning by responsible inpatient teams. This would optimize patient safety, demonstrate good clinical practice and minimize/prevent the emergence of accidents/unfair loss of licenses/unfair attribution of driving accidents caused by people who have been under recent or ongoing inpatient care.The following questions: \u201cDo you have a valid license\u201d, \u201cDo you own/have access to a vehicle\u201d, \u201cDo you currently drive\u201d were developed as a standard template for gathering patients\u2019 driving information.Barriers to ward discharge discussionsTrust-wide communications via screensaver and circularThese questions were embedded within:Answers to these questions were to be clearly documented on patient's records to serve as prompts for the responsible discharging team to take up providing the appropriate advice.After a specified period, the electronic discharge notification (EDN) database was searched for patients with relevant diagnosis who were discharged from all the general adult/older adult acute inpatient wards within a specified period. The patients\u2019 records were then checked for documentation of relevant driving information evidenced by documentation of answers to the screening questions as well as recorded evidence of DVLA discussion/advice held since date of diagnosis or admission.should have their driving licence status recorded during their admissionshould have their access to a vehicle recorded during their admissionwith a relevant mental health diagnosis should have a record of advice regarding driving given in bespoke and DVLA informed manner during ward discharge planning by the responsible discharging teamshould have documentation of the outcome of the driving advice given by the responsible team in their recordsThe standards audited against were all patients:28 patients with relevant DVLA notifiable mental health conditions were audited. 11% (n = 3) had driving licence status recorded. 14% (n = 4) had access to a vehicle recorded. 7% (n = 2) had driving advice given. Only one patient had outcome of driving advice recorded. No best practice was identified.Documentation of driving information, DVLA signposting advice and outcome for patients with relevant mental health diagnosis is a crucial part of patient risk assessment and management as these patients are not free from posing a driving risk on discharge. The trust is implementing actions to improve the routine incorporation of driving advice based on DVLA guidance into discharge planning."} +{"text": "As healthcare increasingly shifts to home and community-based settings, informal caregiver responsibilities are increasing beyond assistance with activities of daily living to include complex care procedures previously performed by licensed caregivers in clinical settings. With an aging population, increasing numbers of older adults are assuming a caregiving role and these older adult caregivers are performing complex care procedures such as wound care. The negative physical and mental health consequences of caregiving for older adult caregivers are well documented in the literature. However, access to and use of resources are associated with better physical and mental health. Past research on caregiving resources has utilized pre-determined resource variables. Little is known about older adult caregivers\u2019 salient thoughts on resources important to caregiving and performing complex care procedures. This study utilized thematic analysis of qualitative interview data to identify themes and patterns related to resources as described by older adult caregivers. The following seven themes related to resources needed or utilized were identified: 1) expert guidance from healthcare professionals; 2) written instructions; 3) relationships with healthcare professionals for obtaining wound care supplies; 4) additional durable medical equipment; 5) financial resources; 6) coverage for caregiver personal time; and 7) select persons for social and emotional support. Older adult caregivers need and use a variety of resources when providing wound care. As increasing numbers of older adults choose to \u2018age in place\u2019, the importance of adequate resources to sustain care recipients and their caregivers in the home setting is critical."} +{"text": "Narrative analysis has been used extensively in qualitative research, but photo analysis is relatively new. In the present study, older adults took photographs and wrote narratives for 15 days about something that brought meaning and purpose to their lives. This presentation explains how both methods were paired together and the challenges of seeking Institutional Review Board (IRB) when new and \u201cunproven\u201d approaches are used. In our case, IRB reviewers were particularly concerned with identifying information in photographs and recommended that personal information should be blurred when sharing photographs. Discussion of how using diverse qualitative methods can create opportunities for new dissemination methods and partnerships with other disciplines will also be noted. In our study, several pop-up art exhibits of participant photos and narratives were hung in public community spaces to promote greater discussion and reflection on purpose and meaning in later life."} +{"text": "Background: The rise of antimicrobial resistance has made it critical for clinicians to understand antimicrobial stewardship principles. We sought to determine whether the opportunity to participate in an American Board of Pediatrics Maintenance of Certification Part 4 (MOC4) quality improvement (QI) project would engage pediatricians and improve their knowledge about antimicrobial stewardship. Methods: In August 2019, a new clinical algorithm for acute appendicitis, spearheaded by the antimicrobial stewardship program (ASP), was implemented at UCSF Benioff Children\u2019s Hospital Oakland to standardize care and optimize antimicrobial use. Medical staff were invited to participate in a QI project evaluating the impact of this algorithm. Data were collected for the 2 quarters preceding implementation (baseline), for the quarter of implementation (transition period), and for the quarter after implementation. Participants were offered MOC4 credit for reviewing these 3 cycles of data and associated materials highlighting information about antimicrobial stewardship. An initial survey was given to participants to assess their baseline knowledge via 4 questions about antimicrobial use in surgical patients completed the QI project and the second survey. Between surveys, there was significant improvement in knowledge about the appropriate timing and duration of surgical antibiotic prophylaxis (Table Conclusions: Participation in this MOC4 QI project resulted in significant improvement in knowledge about antimicrobial use in surgical patients, and the activity was perceived as a highly effective way to learn about antimicrobial stewardship. QI projects that leverage MOC4 credit can be a powerful tool for engaging pediatricians and disseminating education about antimicrobial stewardship.Funding: NoDisclosures: None"} +{"text": "Inappropriate prescription of antibiotics remains a major contributor to the global antimicrobial resistance crisis despite clear linkages between antibiotic utilization and resistance spread. This study aims to better understand the simultaneous and independent effect of previous prescription behavior, patient expectation, and clinical uncertainty on antibiotic prescribing. This discrete choice experiment was embedded within a routine organizational climate survey administered to all physicians working in the Tuscany healthcare system administered between Nov 11 and Nov 20, 2019 . Participants were provided with a patient encounter vignette and subsequently asked to in which of two alternatives they were more likely to prescribe antibiotics. The two alternatives varied in levels of clinical uncertainty, patient expectations, and the physician\u2019s past behavior. We fitted a conditional logistic regression model. Respondents included 1,436 hospital-based physicians, of which 52% were female, 78% practiced in a general hospital setting, and 33% were between the ages of 50 and 59. Results show that the odds of prescribing antibiotics decrease when a patient requests it and increase when the physician has prescribed antibiotics to a patient under similar circumstances previously . We found no significant effect of clinical uncertainty on the odds of prescribing antibiotics . We show that patient expectation has a significant negative association with antibiotic prescribing among hospital-based physicians. Our findings inform the design of antibiotic stewardship programs in Tuscany and highlight the importance of cultural context in shaping the physician\u2019s disposition when confronted with patient expectations. We suggest shared decision-making to improve prudent prescribing without compromising on patient satisfaction.\u2022\u2002Health administrators should address patient expectations when designing hospital antibiotic stewardship programs.\u2022\u2002Physicians\u2019 past prescribing behaviour influences antibiotic prescribing decisions and should be considered during intervention design."} +{"text": "Dear Editor,WAS gene that impair or abolish expression of the WAS protein (WASp).Wiskott\u2013Aldrich syndrome (WAS) is a severe X\u2010linked primary immunodeficiency disorder with an incidence of between 1 in 50 000 and 1 in 250 000 live births.WASp is a key regulator of the actin cytoskeleton. It coordinates the assembly of actin filaments in response to cell signalling events. The fact that WASp is only expressed in all haematopoietic cells (other than red blood cells) explains the broad range of associated clinical manifestations. Impairments in WASp function have been shown to impair multiple cellular processes in both myeloid and lymphoid lineages, including cell adhesion, cell migration, phagocytosis, immune synapse assembly,It is generally accepted that children lacking WASp are candidates for curative allogeneic haematopoietic stem cell transplantation (HSCT) or (if available) gene therapy via genetically modified autologous stem cell transplantation. In fact, patients who fully lack WASp do not survive beyond their second or third decade of life.In the two largest studies recently performed by the Primary Immune Deficiency Treatment ConsortiumHow does HSCT compare with gene therapy? Several trials of gene therapy for classical WAS are ongoing. Although early studies were hampered by late\u2010onset haematological malignancies due to insertional mutagenesis,3. Two parameters govern the level of myeloid chimerism observed after gene therapy: the intensity of the conditioning regimen and the level of correction of the gene\u2010modified autologous CD34+ cells. It may therefore be beneficial to intensify conditioning if toxicity can be abrogated. Monoclonal antibodies against B cells and plasmacytoid cells can further deplete the autoimmune compartment without increasing toxicity, and monoclonal antibodies against stem cells (whether conjugated to various drugs or not) might also improve these results. Nevertheless, patients with the same level of gene correction in the drug product can differ in long\u2010term engraftment of corrected cells, possibly as a result of damage to stem cells through chronic inflammation and autoimmunity. This question should be investigated further to improve the overall outcome and platelet correction. Last, impressive results in the absence of adverse events have been obtained in two adults treated by gene therapy. Moreover, thymic function was maintained in these two patients, showing that the immune compartment can be successfully rebuilt. Gene therapy might therefore be a particularly attractive alternative to allogeneic transplantation in older patients with severe disease.Of course, this enthusiasm should be tempered by the small number of patients who have undergone gene therapy and the variable correction of the platelet count reported to date. In our small cohort, only the patients with high levels of engraftment with transduced cells achieved a normal platelet count. All the others had a count below 50 000/mm"} +{"text": "Background: Penicillin allergies are frequently reported and are associated with adverse clinical and antimicrobial stewardship outcomes. Allergy delabeling, either by patient history or skin testing and oral challenge can facilitate removal of penicillin allergy label. However, penicillinallergies are often reinstated in the medical record and data is limited about how and why this occurs. In our center, the departments of allergy and infectious diseases utilize an allergist nurse practitioner for penicillin allergy delabeling. We investigated the prevalence of penicillin allergy reinstatement following removal and associated factors thereof. Methods: We performed a retrospective observational study of patients who previously had penicillin allergy removed by the allergist nurse practitioner between August 2020 and May 2021 (250 days). Patients were followed for a minimum of 8 months and up to 16 months after penicillin allergy removal. We then assessed whether the allergy was reinstated. Clinical characteristics were compared between patients with penicillin allergy reinstated and not reinstated using the \u03c72 and Mann-Whitney U test. The primary end point was prevalence of penicillin allergy reinstatement following removal. Results: During the study period, 81 patients had penicillin allergy removed, but it was later reinstated in 19 patients (23%) (Fig Conclusions: Penicillin allergies were redocumented in almost one-quarter of patients, most frequently by a nonphysician team member and without acknowledgement of prior removal. Patients who undergo skin testing may be less likely to continue to report a penicillin allergy to medical staff compared to those whose allergy is removed based on history. Increased interactions with the healthcare system may have contributed to having the allergy reinstated.Funding: NoneDisclosures: None"} +{"text": "The correct sentence is: We found that participants who knew a person affected by leprosy had lower mean EMIC-CSS scores and therefore lower levels of perceived stigma, compared to participants who did not know a person affected by leprosy (14.2 vs 17.3, The seventh paragraph in the Discussion section misreports the study findings. The paragraph should read: In our study participants who knew a person affected by leprosy and those who were a close contact of someone affected perceived lower levels of stigma. Knowing a person affected also appeared to reduce the desire for social distance towards leprosy patients, but this effect did not quite reach statistical significance in the multivariate analysis. Nevertheless, it seems that knowing someone affected could potentially improve personal attitudes towards and reduce fear of person affected.There is an error in the first column heading in In Tables"} +{"text": "Tighter glycaemic targets may be of benefit for women with GDM and their infants. Barrier and enabler identification prior to implementation of tighter glycaemic targets for women with GDM may support a successful transition.A cross-sectional questionnaire survey was conducted among Key Informant Health Professionals in ten hospitals in New Zealand. The survey assessed what was currently working using less tight glycaemic targets; what barriers and enablers were considered likely when introducing tighter glycaemic targets and whether these perceptions differed by health professional groups.Sixty Key Health Informant Health Professionals completed the survey. When using the lower glycaemic targets, participants considered that women with GDM found the targets easy to use and that collaborative collegial support was effective. No significant barriers were identified. Perceived enablers identified prior to implementation of tighter targets included receiving collegial support , attending education sessions , use of pocket prompt cards , availability of wall charts and glycaemic target reminder stickers . For health professionals referring into the Diabetes in Pregnancy Service effective communication was considered important. Perceived barriers were confusion over glycaemic targets use (27 (45%), not being informed of the glycaemic target change , non-involvement with multidisciplinary decisions and increased difficulty of blood glucose control for women . Overall, barriers and enablers between Health Professional groups did not differ.Key Informant Health Professionals reported effective communication as a key perceived enabler and that woman would find it more difficult to control their blood glucose concentrations. Education sessions, multidisciplinary engagement, wall charts and stickers were considered effective to overcome the perceived barriers. Further research is needed to assess if the barriers perceived were realised and if the perceived enablers supported the implementation of the tighter glycaemic targets effectively. Gestational diabetes mellitus (GDM) is increasing globally . GDM sigThere has been a growing interest in the literature of identification of perceived barriers and enablers that are key to guide effective implementation of practice and behaviour change , 16. TraIn 2014 the New Zealand Ministry of Health published clinical practice guideline recommendations for health professionals on the screening, diagnosis and treatment of gestational diabetes in New Zealand . The guiWhat has worked well and not so well using less tight glycaemic treatment targets amongst Key Informant Health Professionals caring for women with GDM?What are Key Informant Health Professionals\u2019 perception for barriers and enablers prior to the implementation of tighter glycaemic targets?Are there differences of enabler and barrier perception prior to implementing tighter glycaemic targets between Key Informant Health Professionals\u2019 groups?A cross-sectional questionnaire survey was conducted among Key Informant Health Professionals recruited from the ten hospitals collaborating in the Target Trial from nine DHB\u2019s providing different levels of maternity care and Diabetes in Pregnancy services in New Zealand between February and November 2016 (N = 60). Six DHB\u2019s were geographically spread over the North Island and three DHB\u2019s were in the South Island ). The birth rates for the DHB\u2019s varied with a range from 1386 (Lakes DHB) to 8287 (Counties Manukau DHB) births per annum .The study was approved by the New Zealand Health and Disability Ethics committee (HDEC) Ref. 14/NTA/163, research registration number 1965. Locality agreements were obtained from all DHB\u2019s.At each of the 10 hospitals one Key Informant from six health professional groups providing care for women with GDM were identified by the Target Trial site investigator. The six health professional groups included: endocrinologist or diabetes physician, obstetrician, clinical nurse specialist: diabetes or diabetes midwife, diabetes dietitian, hospital midwife and community midwife (lead maternity carer (LMC) midwife). The Key Informant Health Professional had to have been associated with the provision of care for women with GDM for at least four months. The identified Key Informants were contacted by the researcher (RM) and informed about the study and their consent sought for participation.Participants completed a questionnaire survey, designed for this study, before the implementation of tighter glycaemic treatment targets for women with GDM at their hospital. The questionnaire survey comprised 16 questions and included demographic characteristics of the Key Informant Health Professionals, their thoughts relating to what worked well and what was challenging using the less tight glycaemic treatment targets for women with GDM at their hospital. The questionnaire survey asked the participant\u2019s perception of how the implementation to tighter glycaemic treatment targets may work well or be challenging. The tighter glycaemic targets were fasting plasma glucose \u22645.0mmol/L; 1 hour postprandial \u22647.4mmol/L; 2 hours postprandial \u22646.7mmol/L) . After fThe administration of the questionnaire survey was linked to the timing of the stepped wedge randomised trial design of the TARGET Trial . Site viThe data were entered into an Excel spreadsheet and analysed using Pivot Tables in Microsoft Office Excel 2016. Descriptive statistics and frequencies were used to analyse the Key Informant Health Professionals\u2019 demographics and questionnaire survey responses.All 60 Key Health Informant Health Professionals approached gave consent to participate in this questionnaire survey. All participants answered all the questions. Demographic characteristics of the participants showed that the majority were female (83%), the mean time of the Key Informant Health Professionals practising in their profession was 20.4 years (SD 10.2) and the mean years providing care for women with GDM was 13.2 (SD 7.8) . While aOver half of the Key Informant Health Professionals identified that women with GDM in their care found the lower glycaemic targets easy to adhere to , successfully controlled the woman\u2019s capillary blood glucose (CBG) concentration , and there was collaborative collegial support in using the same glycaemic treatment targets . Study folders and education materials that were available to support and guide the use of the less tight glycaemic treatment targets were considered to be working well by 16 (27%) Key Informant Health Professionals .The questionnaire survey invited participants to add other comments in free text boxes and 11 (18%) opted to do so . A rangeKey Informant Health Professionals identified only minor challenges with the use of the less tight glycaemic treatment targets . Poor glThe Key Informant Health Professionals reported perceived enablers for staff to implement use of tighter glycaemic treatment targets as attending education sessions for staff , use of small pocket prompt cards , receiving collegial support , regular reminders of the targets in use and wall posters detailing the targets in use .For women with GDM who would be using tighter glycaemic targets, Key Informant Health Professionals perceived those women would be more likely to adhere to the tighter treatment glycaemic targets because they would believe that this was better for their baby and that they would have a better birth outcome . No incrHospital services might possibly be impacted by implementing tighter glycaemic treatment targets. Key Informant Health Professionals saw enabling the implementation through effective communication , an increase in multidisciplinary engagement and through effective information dissemination .In New Zealand, community midwives, also known as Lead Maternity care (LMC) providers, provide autonomous care for pregnant women in their care, unless a referral or hand-over to specialists\u2019 care is required. Community midwives often continue the care for women with GDM in a shared care arranged with their diabetes and obstetrician colleagues. A change of glycaemic treatment targets may have an impact on community midwives\u2019 provision of care. The two main perceived enablers by Key Informant Health Professionals were effective communication and involvement with multidisciplinary decisions .Further enablers were listed by 41 (68%) participants in the free text box who perceived clinic room wall charts (25) and stickers (24) that detailed the treatment targets in use to be the most effective means for successful implementation of tighter glycaemic targets by the sFor the women, other perceived enablers centred around information to be provided in their own language (4) or visual (3) to enhance the women\u2019s understanding of their glycaemic targets and GDM in general. Having a free pick-up service for clinic appointments was perceived as enabling successful use of tighter glycaemic targets for women with GDM (3).Most participants perceived no other enablers were needed for the wider hospital services, with \u2018business as usual\u2019, as changing to tighter glycaemic treatment targets would not impact on hospital resources .Effective communication, perceived as enablers for community midwives, was further supported with additional comments in the free text box by Key Informant Health Professionals, that included evidenced based information sharing (9), electronic communication (2) and multidisciplinary study days (5) . Three pParticipants reported perceived barriers for the implementation of tighter glycaemic targets for staff, women, wider hospital and community midwives .The top two perceived barriers for staff were identified as confusion over which glycaemic targets to use and different treatment thresholds being used by different health professionals . For the women with GDM, perceived barriers reported were difficulty to control capillary blood glucose (CBG) concentration and inability to attend more frequent clinic appointments , which would be necessary if the perception of increased difficulty of blood glucose control was realised. Ineffective communication was perceived as a barrier for other hospital services and community midwives . Lack of resources for the hospital and non-involvement with multidisciplinary decisions for community midwives were repOther perceived barriers identified was the belief that the information on change to use of tighter glycaemic targets may not be disseminated to all relevant staff (5) . For theThe biggest perceived barrier for LMC community midwives centred around not being informed of the treatment target change (7) by the health professionals providing the diabetes care for the pregnant women and the inability to attend specialists\u2019 appointments with the women (4) .Endocrinologists , obstetricians (7 (70%), hospital midwives and community midwives , most frequently perceived education sessions and collegial support would enable staff to accept, support and implement tighter glycaemic targets . While dAll the endocrinologists perceived women would respond well to use of tighter glycaemic targets, as women would believe that it will be better for their infants, and similarly perceived by 80% (8) of clinical diabetes nurse specialist or diabetes midwives, hospital midwives and diabetes dietitians . ObstetrHospital wide perceived enablers were reported as effective communication and an increase in multidisciplinary engagement by all Key Informant Health Professionals. There was a small variation between Key Informant Health Professionals with 80% (8) obstetricians and clinical diabetes nurse specialist, 70% (7) endocrinologists or diabetes physician, 60% (6) diabetes dietitians, hospital midwives and LMC community midwives supporting these enablers. A health cost reduction was only identified as a substantial enabler by the obstetricians that may affect the wider hospital services . There wMany pregnant women with GDM in New Zealand are cared for by community midwives, who refer the women once diagnosed with GDM to the Diabetes in Pregnancy Service, usually at the nearest tertiary or secondary hospital. Introducing tighter glycaemic targets and any prescribed treatments need to be communicated to the LMC community midwife, who often provides ongoing pregnancy care. In this context it is seems reasonable that effective communication with community midwives was perceived as the highest important enabler among all Key Informant Health Professionals . AdditioThere were considerably fewer perceived barriers identified by all Key Informant Health Professionals compared to enablers. The most important perceived barrier identified for staff was the possible confusion over which glycaemic targets to use, by 60% (6) endocrinologists/diabetes physicians and obstetricians, by 50% (5) community midwives, by 40% (4) clinical nurse specialists: diabetes or diabetes midwives and by 30% (3) both by diabetes dietitian and hospital midwives . AdditioFor women with GDM, the major perceived barrier by the Key Informant Health Professional was the potential increase in difficulty of controlling their blood glucose concentrations. Ninety percent (9) of the clinical nurse specialists: diabetes or diabetes midwives, the diabetes dietitians and community midwives reported this perceived barrier and 70% (7) each of the endocrinologists/diabetes physicians, obstetricians and hospital midwives Key Informant Health Professionals . Only thGreat variations with low percentage results were identified between Key Informant Health Professionals reporting perceived barriers for the wider hospital services . ResultsIn-effective communication was reported as a perceived barrier not only for hospital services but for community midwives . Over haThis study was seeking to answer three research questions by identifying current enablers and barriers and the perception to implementing tighter glycaemic targets for women with GDM from Key Informant Health Professionals involved in care for women diagnosed with GDM. The results aimed to inform the implementation of tighter glycaemic treatment targets prior to the commencement of the TARGET Trial .What has worked well and not so well using less glycaemic treatment targets amongst Key Informant Health Professionals caring for women with GDM?When using the lower glycaemic treatment targets participants considered that women with GDM found the targets easy to use and that collaborative collegial support was effective. No significant barriers were identified. In the free text option, stickers and wallcharts were identified as working well. These enablers were identified in multiple areas in the survey and would benefit consideration to use when implementing tighter glycaemic treatment targets for women with GDM.What are Key Informant Health Professionals\u2019 perception for barriers and enablers prior to the implementation of tighter glycaemic targets?The second research question was analysed in subsections for easier perceived enabler and barrier identification from different GDM care providers and services.For staff involved in using/recommending glycaemic targetsSimilar to the findings on the use of less tight glycaemic targets, participants identified collegial support as the main perceived enabler, closely followed by education sessions and pocket prompt card for health professionals involved with the care of women with GDM. Additionally, over half of the participants who opted to answer in the free text option section commented that clinic wall charts and stickers detailing the tighter glycaemic targets would be seen as enabling. Nearly half of the participants identified confusion over which glycaemic targets to use as a perceived barrier, with an additional third of participants perceiving that different treatment thresholds would be used by different health professionals. The identified perceived enablers would potentially address these barriers and can be used to guide the implementation of tighter glycaemic treatment targets for women with GDM.For women using the tighter glycaemic targetsWhile the less tight glycaemic targets were identified as working well, it was perceived by most participants that tighter glycaemic targets would be more difficult to achieve for women with GDM. Despite this concern, the majority of Key Informant Health Professionals perceived that the tighter glycaemic targets would be acceptable to women with GDM, as women would believe these targets would be better for the baby and their own health, as well as women would believe that they would have better birth outcomes. These findings indicate that women with GDM benefit from receiving clear information, but a study to explore women\u2019s views on enablers and barriers to tighter glycaemic targets is warranted.For wider hospital servicesOver two thirds of Key Informant Health Professionals perceived effective communication and increased multidisciplinary engagement would enable the implementation of tighter glycaemic successfully. There was a small perceived concern about needing more clinic rooms if women required more antenatal appointments and more hospital beds for admissions as there may be a higher induction of labour with tighter glycaemic target recommendations. Although \u2018business as usual\u2019 was perceived by nearly half of the participants who opted to enter further perceived enablers in the other free text box, a third of participants perceived an overall health cost reduction, whilst a tenth of participants were concerned about a perceived overall health cost increase. Cost-benefit analysis and resource demand when changing to tighter glycaemic treatment targets may be an area to consider when planning to address potential enablers and barriers.For LMC, community midwivesEffective communication was the most significant perceived enabler for LMC community midwives, followed by involvement with multidisciplinary decisions. This was conversely reflected in the perceived barrier identification by participants for tighter glycaemic targets implementation identified as in-effective communication and non-involvement with multidisciplinary decisions. Communication and multidisciplinary engagement have been shown to be effective tools for behaviour change and implementation of clinical recommendations and skills .Are there differences of enabler and barrier perception prior to implementing tighter glycaemic targets between Key Informant Health Professionals\u2019 groups?The third research question attempted to elicit if the health professional groups providing care and services for women with GDM identify different significant enablers and barriers as these results may be useful for targeting different professional groups with different implementation strategies for a change over to tighter glycaemic targets for women with GDM.For staff involved using glycaemic targetsDifferent Key Informant Health Professionals groups identified different perceived enablers for staff involved with glycaemic treatment targets. Education sessions were perceived as important enablers to implement tighter glycaemic targets for women with GDM by all community midwives, over half of the hospital midwives, obstetricians and endocrinologists or diabetes physicians. For clinical nurse specialists, diabetes or diabetes midwives and hospital dieticians this was not a significant enabler. Collegial support was identified by all Key Informant Health Professionals as an enabler for the implementation of tighter glycaemic targets, with the highest indicator from diabetes dieticians and clinical nurse specialists: diabetes or diabetes midwives and endocrinologists or diabetic physicians. What collegial support means for each of the health professional groups was not clear and warrants further research.Regular reminders and pocket prompt cards were identified by the majority of the obstetricians and community midwives and by over half of the endocrinologists or diabetic physicians. Except for the community midwives, all other Key Informant Health professionals, commented additionally in the free text section for perceived enablers the provision of clinical wall charts and stickers listing the tighter glycaemic targets.PowerPoint presentations were not seen as enabling by any of the Key Informant Health Professionals. While the barrier identification did not identify any significant areas, half of the endocrinologists, obstetricians and community midwives anticipated confusion amongst health professionals over which glycaemic targets to use. These results indicate the importance of supporting different health professional groups with different resources to enable them to implement the tighter glycaemic treatment targets for women with GDM.For women using the tighter glycaemic targetsThe majority of Key Informant Health Professionals perceived that woman diagnosed with GDM would not find it easy to accept the tighter glycaemic targets, as it would be more difficult to control capillary blood glucose concentrations. This did not differ significantly across all Key Informant Health Professionals groups. It was perceived that women would manage this challenge, as once women with GDM were provided with in depth information to understand the reason for tighter glycaemic targets, they would believe it was good for their health, better for their infant and that they would have better birth outcomes. While these beliefs were enablers identified highly across all Key Informant Health Professionals, all endocrinologists or diabetes physicians had this perception. Nearly half of the clinical nurse specialist, diabetes or diabetes midwives perceived that tighter glycaemic target would require more frequent clinic visits and perceived this as a barrier, as women with GDM may not be able to attend increased clinic appointments. This was of low concern for all other Key Informant Health Professional groups. Health literacy and access to health services was always a concern and is an area that would need careful attention when implementing tighter glycaemic targets.For wider hospital serviceOver half of the obstetricians and diabetes dieticians perceived that implementing tighter glycaemic targets would enable an increase of evidence information dissemination for the wider hospital service. All Key Informant Health Professionals perceived an increase in multidisciplinary engagement across the hospital and significantly identified effective communication as an enabler and ineffective communication by half of the obstetricians. Lack of resources for the wider hospital was perceived by over half of the LMC community midwives but was not defined what this may be for the wider hospital services.For LMC* community midwivesEffective communication for LMC community midwives were identified as significant perceived enablers by all Key Informant Health Professionals. Half of the obstetricians perceived that LMC community midwives need effective access to expert advice and involvement with multidisciplinary decisions. Clinical nurse specialist, diabetes or diabetes midwife perceived significantly that LMC community midwives need involvement with multidisciplinary decisions. These results corresponded with the perceived barriers response from all Key Informant Health Professionals of ineffective communication and non-involvement with multi-disciplinary decisions. Half of the LMC community midwives opted to comment in the free text identifying a perceived barrier of community midwives not being informed of treatment change. These results highlight the importance of clear communication and multidisciplinary involvement especially for LMC community midwives who usually continue to be involved with antenatal care.The survey questionnaire results have contributed to the understanding of barriers and enablers perception by Key Informant Health Professionals providing care for women diagnosed with GDM. The overarching perceived barriers and enablers by participating Key Informant Health Professionals identified the importance of regular and clear communication and multidisciplinary health professional involvement when implementing change to use of tighter glycaemic targets and the perception that women diagnosed with GDM may find it more difficult to control their blood glucose concentrations.There is currently a lack of international studies and in New Zealand addressing enabler and barrier identification from a Health professional perspective for implementing any glycaemic treatment targets for women with GDM. Previous studies within the diabetes field from Australia and the USA, not specifically in women with GDM, have reported sub-optimal communication and lack of professional relationships between health professionals as barriers to effective implementation when change is required , 29. A qKey Informant Health Professionals in this study reported simple, inexpensive enablers that may support the perceived communication concerns and the desired multidisciplinary approach. Education sessions, multidisciplinary engagement, wall charts and glycaemic target reminder stickers were perceived to be effective to overcome the perceived barriers. Further investigation is needed after the implementation of the tighter glycaemic targets to assess if the perceived enablers supported the implementation of tighter glycaemic targets effectively.No perceived enablers were identified by Key Informant Health Professionals of the identified barrier perception that women diagnosed with GDM will find it more difficult to control their blood glucose concentrations. As this was reported by the majority of participants the question arises if this message is conveyed to women diagnosed with GDM at their first clinic appointment. This could have a potential negative impact on women\u2019s attitude towards understanding and accepting their glycaemic targets. Clinician\u2019s attitude, beliefs and knowledge about diabetes can influence diabetes management and patient\u2019s perception . A recenThe strength of the current study is the survey of Key Informant Health Professionals across six professional groups involved in gestational diabetes care at 10 hospitals across New Zealand with a 100% response rate. Given the lack of research in this area in New Zealand and internationally, this study is exploratory and encourages further follow-up research. Often studies single out one health care provider group. Women diagnosed with GDM have care provided by several health professionals groups. Therefore, it is important to identify collectively and individually from involved health professional groups what barriers or enablers are perceived before implementing tighter glycaemic targets. This can inform effective strategies to enable the implementation of change collectively or with a health professional specific approach. This will enhance effective care for women with GDM.A limitation of the current study is the small sample size, therefore the study findings cannot be generalised and applied to all New Zealand Diabetes in Pregnancy Services and their involved health professionals. Accessing all health professionals providing care for women with GDM at all New Zealand hospitals was too difficult, hence access to Key Informants was considered the best option at the ten hospitals who consented to participate in the Target Trial. It is recognised that women who have been diagnosed with GDM need to be included into research about enablers and barriers to implementing tighter glycaemic targets. As part of the TARGET study this has been conducted and is published elsewhere , 35.Key Informant Health Professionals reported effective communication and multidisciplinary health professional involvement as key perceived enablers. A perceived barrier for women with GDM was identified as women would find it more difficult to control their capillary blood glucose concentrations, but that women will believe it is better for their health and the health of their infant. Education sessions for staff and women, multidisciplinary engagement, wall charts and stickers were perceived to be effective to support the identified enablers and to overcome the perceived barriers. This study identifies that an effective strategy for implementing tighter glycaemic treatment targets for women with GDM needs to be carefully planned. Further research is needed after the implementation of the tighter glycaemic treatment targets for women diagnosed with GDM to assess if the barrier perceptions were realised and if the perceived enablers supported the implementation effectively.S1 File(PDF)Click here for additional data file."} +{"text": "Although sometimes caregiving is associated with closeness in marriage, at other times the stresses may affect marriage relationships negatively. In the current study we explore how caring for a partner with dementia, and subjective burden are associated with marital quality. We further explore how dementia care and subjective burden might interact (suggesting a pileup of stress) to affect marital quality. Using data from 1,066 spousal caregivers that participated in the NSOC study and their corresponding care recipients from the NHATS study, the current analysis explored cross-sectional associations between spousal caregiving and partner care recipient characteristics predicting positive and negative marital quality. Results suggested that dementia classification and subjective burden were associated with lower positive marital quality and higher negative marital quality. The relationship between subjective burden and positive marital quality was moderated by whether or not the care recipient had dementia. Specifically, when dementia was present, the negative association between subjective burden and positive marital quality was stronger. Spousal caregivers often carry substantial burden to help their partners with activities of daily living. Care provision can alter the marriage relationship in negative ways when perceived burden is high. The current study suggests that the negative association between burden and positive marital quality is even stronger when caring for a spouse with dementia. Gender differences need further exploration, as well as how these patterns play out in early vs. later stages of dementia and across time."} +{"text": "Elliott and Reynolds). Although there are many treatment options for esophageal cancer this issue mainly focused on chemotherapy. In this special issue we received many research and review articles on chemo and associated therapies to treat this cancer and two of these review articles by Luo et al and Huang et al sheds light on the role of PI3K/Akt/mTOR signalling pathway in development and pathophysiology of esophageal squamous cell carcinoma (ESCC). They also suggested that either PI3K/Akt/mTOR or their downstream eukaryotic translation initiation factors (eIFs) may act as potential therapeutic targets to treat this disease. A study by Xiao et al discussed the possible brain metastasis of ESCC and concluded that development of characteristics of brain metastases is rare in these patients and suggested that local or specific territorial treatment is associated with improved overall survival. Another in silico study by Zhao et al predicted the overall survival and benefits of chemotherapy using Deep Learning (DL)-based protein features in gastric cancer and they also demonstrated the advantages of DL-based workflow in gastric cancer molecular subtyping along with its possible therapeutic application. Study by Yang et al suggested that the esophageal cancer patients who are intolerable to surgery or who are under the impact of old age or geriatric patients (aged \u226580 years), should prefer chemoradiotherapy (CRT) as a preferable treatment option compared to other therapies.Esophageal cancer is the cancer of esophagus and is one of the most deleterious cancers and sixth most common cause of carcinogenesis related mortality worldwide . AlthougFeng et al discussed the clinical efficacy of neoadjuvant chemotherapy (NACT) combined with Laparoscopic gastrectomy (LG) for locally advanced adenocarcinoma of the esophagogastric junction and concluded that these combined therapies does not increase the risk of postoperative morbidity and mortality when compared with LG alone (https://pubmed.ncbi.nlm.nih.gov/34660265/). A population study associated SEER analysis by Yang et al revealed that ESCC subjects with organ specific metastasis other than liver or bone have more benefits from local ablative treatment (LAT) with systemic chemotherapy.\u00a0A study by Kermani et al concluded that in ESCC patients\u2019 predictive or anticipating value of endoscopic results, observations, impressions and biopsy after neoadjuvant CRT are insufficient for assessing overall complete pathological response after neoadjuvant treatment and they also suggested that additional methods are required for overall assessment of the treatment and its impact.\u00a0A phase II randomized study by Wang et al compared the preoperative concurrent CRT versus NACT or neoadjuvant chemotherapy for locally well-developed later stage gastric cancer (LAGC) patients in a single center-based data. In contrast to this report another study by Zheng et al compared the side effects and effectiveness of chemical drugs Lobaplatin-based versus Cisplatin-based adjuvant chemotherapy data from multicenter study and based on their analysis, Lobaplatin plus docetaxel might be a better choice of drug for adjuvant chemotherapy particularly for ESCC. Finally, A systematic review and meta-analysis by Xia et\u00a0al. concluded that consolidation chemotherapy (CCT) after Concurrent chemoradiotherapy (CCRT) significantly increases over all long-term survival and disease progression-free survival of patients with nonsurgical esophageal cancer and could provide them astonishing overall survival benefits.A retrospective, propensity score-matched short-term study by Finally, these elegant research and review articles increased the current knowledge and added additional information about benefits and drawbacks of different therapeutic options for patients with advanced esophageal cancer.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication."} +{"text": "To the Editor,We would like to present several ideas and comments regarding \u201cAccuracy of echocardiographic estimations of right heart pressures in adult heart transplant recipients\u201d recently published in a clinical cardiology journal. The authors have examined the accuracy of\u00a0noninvasive parameters of right side pressures including estimated right atrial pressure (eRAP) and pulmonary artery systolic pressure (PASP) in adults who underwent heart transplants.However, these measures might serve as potential predictors of clinical prognosis as well as correlates of invasive RAP. Furthermore, the authors have not clearly determined the severity of right ventricle dysfunction, which might affect both RAP and PASP in patients. We also suggest adjusting the correlations using Natriuretic peptides with atrial and ventricular origin as well as RA volume."} +{"text": "Adult children often report intergenerational ambivalence that may be exacerbated when parents need increasing support. Evaluations or appraisal of providing support may mediate the links between stress and outcomes. Using structural equation modeling, we assessed the relationship between change in parental disability and intergenerational ambivalence through adults\u2019 perceptions of the stress and reward of providing help to parents. Participants included 369 adults who provided information on 478 parents from Waves I and II of the Family Exchanges Study. The association between change in parental disability and intergenerational ambivalence was explained through stress appraisal; greater parental disability led to higher stress appraisal which led, in turn, to greater intergenerational ambivalence. The model did not significantly differ by race. Results show that stress, rather than reward, appraisal is an essential factor in determining relationship quality as parental care needs emerge."} +{"text": "Prior use of antimicrobials, in particular broad-spectrum antimicrobials, is associated with the development of Clostridium difficile infection (CDI). Our previous work has demonstrated increased risk with cumulative exposure but there is limited evidence on specific patterns of cumulative antimicrobial prescribing prior to infection. Understanding this pattern will help inform antimicrobial stewardship. This study aims to investigate the prescribing patterns for CDI cases with more than 4 weeks cumulative antimicrobial exposure during the 6 months prior to their CDI date.We linked three Scottish patient-level data sets: laboratory confirmed CDI (ECOSS), prescriptions for antimicrobials in primary care (PIS) and all hospital admissions (SMR01). From ECOSS all cases of CDI reported in the period August 2010 to July 2013 were identified. Each CDI case was linked to SMR01 to allocate case type and to PIS for previous antimicrobial prescriptions. Visual representation of temporal exposure indicating both duration and drug type of each antimicrobial dispensed during the 6 month period before the infection was produced to clearly understand the pattern of prescribing.In the study period, there were a total of 1557 community acquired CDI cases without recent (prior 3 months) hospitalisation. Among them, 287 (18%) cases had more than 4 weeks prior cumulative antimicrobial exposure accumulating a total of 1311 dispensed prescriptions. Cases had an average 4 prescriptions and 2 different types of antimicrobials dispensed. The timeline plot shows that repeated short duration prescriptions contributed more to cumulative exposure than long duration prophylaxis. The most common antimicrobials prescribed were amoxicillin (38% of cases), trimethoprim (30%) and flucloxacillin (27%). The median duration per prescription for each of these was less than 2 weeks. The antimicrobials that had a median duration of around a month per prescription were oxytetracycline (3%) and clindamycin (6%). This study of a national linked patient level data set contained sufficient numbers of cases to enable investigation of the prescribing pattern in individuals with highest risk cumulative exposure. This study uses NHS Scotland\u2019s developing Infection Intelligence Platform which will place Scotland as a world leader in the use of health informatics to support infection control and antimicrobial stewardship."} +{"text": "Patient was started on cefepime and vancomycin while awaiting results of blood culture that grew methicillin\u2010susceptible Staphylococcus aureus. She continued treatment with cefepime and had her central line removed. On T+50 parents reported the appearance of a new blistering rash on her hands. Skin examination demonstrated multiple 1\u20132\u00a0mm deep seated noninflammatory vesicles located on the distal digits and metacarpophalangeal joints of both hands and pad of the right thumb on admission showed a neutrophil count of 8940\u00a0\u00d7\u00a010norovirus\u2010related urticaria presenting with generalized erythematous wheals associated with pruritus. Dermatologic manifestations of EBV are reported more commonly in patients with infectious mononucleosis (IM). IM patients more commonly develop a faint erythematous macular rash early in the course or a maculopapular rash associated with the inappropriate use of amoxicillin. There are reports of IM presenting with erythematous papulovesicules, but it is uncommon. Vesicular lesions associated with Herpes simplex 1 or 2 infection are characterized by grouped vesicles on an erythematous base ring. In severely immunocompromised patients with deficient cellular immunity, the risk of extensive mucocutaneous disease and disseminated infection is higher. Transplant patients are at risk of developing herpes simplex virus (HSV) infection due to reactivation. Lastly, sucking blisters are noninflammatory vesicles with thick walls that contain sterile fluid. Lesions are usually located on dorsal areas of fingers, hands or wrists and can be bilateral. The observation of the infant sucking the involve areas helped guiding with the diagnosis. In the absence of lesions in other areas and with lack of systemic symptoms sucking blisters became the most likely diagnosis. No biopsy was performed due to well apearing baby. Fever subsided and antibiotics were stopped after 10 days with negative blood culture. Rash is persistent with a healthy looking infant without other manifestations.In an early posttransplant (<100 days) patient, our major concern was acute graft versus host disease (GVHD). Due to the limited distribution of rash and the lack of systemic symptoms, it became less likely. Although GVHD can present as bullous skin lesions, it is associated with severe disease presentation. Second in our list were infectious causes. There are reports of The authors declare they have no conflicts of interest.The authors received no specific funding for this work.A written informed consent was obtained from the parents of the patient to publish the case and images in a medical journal."} +{"text": "The findings from a recent systematic review which investigated medical nutrition education throughout the world found that it was insufficiently incorporated into medical education, regardless of country, setting or year of medical education.2A pilot intervention to improve Year\u20104 medical students' knowledge and communication skills about nutrition and diet was developed and implemented by a multidisciplinary team from the University of Auckland, New Zealand. The pilot was included in the 1\u2010week General Practice Observed Patient Simulation (GPOPS) teaching block primarily focused on communication skills development in the context of simulated general practice consultations. This was considered an ideal place to introduce and practice nutrition\u2010related curriculum content and skills.Online nutrition and Motivational Interviewing (MI) resources An introduction to the 5As of Obesity ManagementHealthy Conversation Skills (a patient\u2010centred approach to support life behaviour change) including a video to enhance healthy conversations skillsAn MI tutorial to practice skills\u2010related to nutrition using role play with a peerA simulation weight management/Pre\u2010Diabetes case with a standardised patient (actor) to practise MI interviewing and nutrition counselling skills followed by a debrief with a tutorThe education intervention included:3On completion of each GPOPS teaching block, students were invited to complete a questionnaire to evaluate their perceptions of the resources provided and to canvas their opinions on the types of nutrition skills development that they would find useful. Additionally, students completed NUTCOMP, a reliable and validated questionnaire that assessed self\u2010perceived competence of primary health professionals in providing nutrition care to patients with lifestyle\u2010related chronic disease.The MI resources provided to students in GPOPS were perceived most useful for increasing knowledge and confidence to counsel in nutrition. Similarly, observation of the patient simulation case increased students' perceived confidence to counsel patients. Opportunities for naturally incorporating and reinforcing nutrition skills suggested by the students included small group activities during pre\u2010clinical years involving problem\u2010based learning and role\u2010playing to strengthen communication skills. Students also suggested that consideration be given to the importance of interdisciplinary teaching and faculty collaboration to appropriately link, sequence and frame curricular content to create an integrated nutrition curriculum. Students' self\u2010perceived nutrition competence scores demonstrated positive attitudes towards nutrition, lack of confidence in nutrition knowledge and skills and moderate confidence to counsel in nutrition.The pilot intervention aimed to integrate a modest amount of nutrition content focused on communication skills relating to diet and nutrition within the time constraints of the GPOPS teaching block. It complimented without compromising teaching of other important related aspects of lifestyle intervention."} +{"text": "We are grateful for these comments on this important aspect. Helbing et al. convinciIn our opinion, neglecting the impact of biotic and abiotic stressors represents a main cause for conflicting results in sepsis research. Hence, the study mentioned by Helbing et al. revealing increased mortality of LPS-treated mice at housing temperature below thermoneutrality sharply How to interpret these contradictory results? One obvious explanation might be the different LPS doses applied in various studies. Whereas Ndongson-Dongmo et al. used 10\u00a0Sepsis patient studies incidentally also uncover contrasting results about the effects of ambient temperature on the morbidity and mortality of critical illness. In this context, the controversy regarding targeted temperature management in patients after cardiac arrest\u2014an ultimately severe stress event\u2014might also shed light on the problem: whereas initial reports \u20136 revealIn sum, we share the opinion of Helbing et al. to inclu"} +{"text": "In clinical practice, urologists encounter adrenal gland metastasis from other malignancies occasionally. Most cases are presented with unilateral metastatic lesions in the adrenal gland, although limited cases are demonstrated with bilateral adrenal metastasis. Usually, unilateral resection of the adrenal gland with metastatic lesion could be indicated in case the patient only has unilateral adrenal gland metastasis and clinically permitted general condition and acceptable prognosis due to adrenal function preservation even after unilateral adrenal surgical removal. Based on experience, patients with lung or gastric cancer are frequently consulted by the urologic department for adrenal metastasis treatment. Recently, both cancers have been attempted to be treated with immunotherapy and combined immunochemotherapy using a molecular target drug.In this case study, the patient underwent combined systemic lung cancer treatment and laparoscopic right adrenalectomy. Subsequent laparoscopic left adrenalectomy and systemic treatment led to the 4\u2010year progression\u2010free survival.The case study reminds urologists to have courage to conduct resection of both sides of the adrenal glands to achieve a cancer\u2010free condition with permanent adrenal hormone replacement after screening the patient's background, general conditions, and drug compliance.The authors declare no conflict of interest."} +{"text": "Structural stigma toward sexual minority individuals varies widely across countries and is associated with psychosocial health outcomes. Yet, the association of changes in country-level structural stigma over time, as has recently characterized many European countries, with such outcomes is largely unknown. The current study examined the association between change in structural stigma from 2012 to 2019 across European Union countries and change in life satisfaction among sexual minority individuals during the same period. Secondary analyses examined whether changes in structural stigma differentially benefitted some subgroups of sexual minority individuals more than others.The current study analyzed data from sexual minority respondents living in 28 European countries.Adjusted multilevel models showed that life satisfaction had improved among sexual minority individuals in all countries between 2012 and 2019 , but the improvement was stronger among those living in higher stigma countries compared to those living in lower stigma countries. Changes also varied by relationship status; the strongest improvement in life satisfaction as a function of improvement in structural stigma was found among sexual minority individuals in a relationship.Although life satisfaction has increased during the past decade among sexual minority individuals living in Europe, significant variation in this change exists across countries as a function of country-level structural stigma and individual sociodemographic characteristics. The findings support the relevance of structural stigma for sexual minority individuals\u2019 life satisfaction and call for further research to understand the differential impact of structural stigma across sexual minority subgroups."} +{"text": "A 2003 SJPHC opinion paper on Frontline Ideas titled \u2018Joining the mobile revolution\u2019 promotedThe SJPHC paper of the year 2019 \u2013 \u2018Having to learn this so late in our lives\u2026\u2019 Swedish elderly patients\u2019 beliefs, experiences, attitudes, and expectations of e-health in primary health care\u2019 shows thp\u2009=\u2009.61). The proportion of participants who reached SBP targets in the intervention and control groups were 28% and 31%, respectively (p\u2009=\u2009.52). Medication changes, number of antihypertensives and health care utilisation were also similar in both groups. Yet, results of blood pressure measurements presented in a box-plot-diagram in the paper showed a possible positive effect of the intervention regarding home SBP. Also, checklist and text message support were considered useful and important by most participants.To improve antihypertensive drug treatment, SMS was tested in the Check and Support study, a cluster randomised control trial (RCT) of 111 patients 30\u201375\u2009years old with newly diagnosed hypertension in Finland . The intA Swedish six\u2009month RCT pilot study of SMS-messages for treatment of hypertension included 60 primary care patients aged 40\u201380\u2009years with hypertension . Four liA main difference between the interventions were the number of messages delivered to the patients which were higher in the Swedish study. The Finnish patients were younger and newly diagnosed with hypertension whilst the Swedish patients had hypertension since before and thus a much lower baseline blood pressure value than the Finnish patients. These two recent Nordic RCTs of SMS to improve outcome in hypertension treatment in primary care did not give clear evidence for SMS being a useful tool even if especially the Swedish study showed promising results. Why? We believe that this is probably explained by underpowered design preventing the detection of small benefits of the interventions and the Swedish study indeed being a pilot intervention.Another explanation may be a low motivation to lifestyle modifications in patients without manifest cardiovascular disease which makes primary prevention a difficult challenge. The Australian RCT TEXTME studied lifestyle-focused SMS in patients with previous coronary heart disease and found significant improvement in several cardiovascular risk factors including reduced low density lipoprotein cholesterol and systolic blood pressure . HoweverAs most conclusions we also conclude that more evidence is needed in this field of support for hypertension treatment by using the SMS text function of the ever-present personal cell phone as a convenient digital telemedicine tool."} +{"text": "Psychiatric disorders continue to be on the rise around the globe. Meanwhile, efforts and investments directed to early diagnosis and appropriate interventions for mental health problems are lagging resulting in \u2018substantial loss of human capabilities and avoidable suffering\u2019 . A majorPsychiatric conditions are often particularly reflected through, and exacerbated by, difficulties in social functioning in everyday life . Yet, cuWe argue that a thorough understanding of the real-world manifestations and implications of psychiatric disorders is the key for the field to move towards the development of more effective personalized assessments and interventions. To address this, we outline a general multilevel framework for deep behavioural phenotyping to guide the scientific inquiry into the behavioural and neural mechanisms of psychopathology and to delineate specific therapeutic interventions Fig.\u00a0. This apThe most debilitating symptoms of psychiatric disorders may only manifest in everyday life, where patients must cope with multiple aspects of life simultaneously (illustrated by the red scale at the bottom of Fig.\u00a0Importantly, these passive measures of daily behaviour can be complemented by subjective ecological momentary assessments providing real-time self-reported measures of patients\u2019 well-being. Such subjective momentary evaluations may prove crucial to understanding changes in passively monitored behavioural patterns. For example, while single passive measures may not be sufficiently discriminative, negative mood evaluations in combination with reductions in locomotive activity, social application usage and speed of typing may represent early signs of a depressive episode. Detecting such patterns offers a chance for early intervention to avoid hospitalization (arrow 1. In Fig.\u00a0While such digital phenotyping can address macroscopic aspects of social and motor functioning, it lacks specificity for assessing how these problems relate to difficulties the patients face during real-life social interactions. To address this, recent studies have started to objectively measure behaviour during social interactions using motion tracking techniques to detect e.g. individual differences related to interaction success and behaTo date, most studies focusing on the neural bases of psychiatric disorders performed categorical comparisons of psychiatric disorders while overlooking the extensive variability in how the disorders manifest in individual patients. Whilst replicable brain-based group differences appear to exist , group-lSeveral approaches could be used to measure the neurobiological and physiological correlates of psychiatric disorders from stable anatomical properties to short-term functional changes during naturalistic experimental conditions (we illustrate some options in Fig.\u00a0Such insights from combining behavioural and neuroimaging markers of psychopathology may be key for detecting clinically applicable biomarkers for psychiatric disorders that can feed back into clinical practice (grey arrow in Fig."} +{"text": "Emotional interactions may change as a result of dementia and negatively impact caregivers\u2019 mental health. We examined 62 dementia caregivers\u2019 emotional experiences during conflict with their partners, including 22 individuals with behavioral variant frontotemporal dementia (bvFTD), 20 with Alzheimer\u2019s disease (AD), and 20 with a language variant of frontotemporal dementia (language variant). Dyads engaged in a 10-minute conversation involving conflict. Caregivers watched recordings of their conversation while rating their emotional valence using a rating dial. Caregivers of individuals with bvFTD reported greater decreases in positive emotion across the conversation relative to caregivers of individuals with AD and language variants. Caregivers who reported greater decreases in positive emotion across the conversation had higher levels of depressive symptoms, even after accounting for their partner\u2019s diagnosis and level of cognitive impairment. Findings suggest bvFTD caregivers experience the greatest declines in positive emotion during conflict, with potential implications for depression."} +{"text": "Couple therapy continues to gain in stature as a vital component of mental health services. The linkage of relationship distress to disruption of individual emotional and physical well-being emphasizes the importance of improving and extending empirically based strategies for treating couple distressTo evaluate the efficacy of dialectical behavior therapy \u201cDBT\u201d in outpatients couples with emotional dysregulationTwenty couples presented with marital distress and at least one of them suffers from emotional dysregulation assigned at their convenience or according to immediate availability of treatment slot to a couple DBT group. Arabic version of DERS was used for assessment of emotional dysregulation before and after intervention. Dyadic Adjustment Scale was used for assessment of marital adjustmentBoth male and female partners showed significant improvement in marital adjustment and emotional regulation. Female partner showed significant higher change amplitude in both scales. Female partners showed significant improvement in all DERS subscales except for (GOALS) subscale (significant decrease), while male partners showed significant improvement in (IMPULSE), (AWARNESS), (STRATEGIES) and (CLARITY) subscalesDialectical behavioral therapy for couples is an effective approach to couples with emotional dysregulation in one or both partnersNo significant relationships."} +{"text": "Home care (HC) aides provide health and personal care services that enable older adults to live at home as they age. Aides are increasingly in demand as our population ages rapidly. Yet there is an aide shortage due in part to instabilities in HC work organization and to occupational safety and health (OSH) hazards, resulting in aides losing work time or leaving their jobs. Aide injuries and illness interrupt the continuity of care delivery and HC hazards can put clients and family caregivers at risk. This research synthesizes recent findings of aide OSH studies in order to identify risks and preventive interventions to improve both HC aide and client safety. Mixed methods were used to characterize aide OSH hazards including needlesticks, musculoskeletal strain, experiences of violence, COVID-19 and other infections and harmful disinfection practices. Improving aide OSH can contribute to improved care services enabling older adults to live at home."} +{"text": "While intergenerational models of adult health contend that children\u2019s educational attainments influence the health of their parents, background characteristics that predict both can confound the results. Data from the National Longitudinal Study of Adolescent to Adult Health Parent Study are used to examine how having no children who completed college influences parents\u2019 self-rated health and depressive symptoms. We use propensity scores to assess this relationship net of potential confounders and test for heterogeneity in the consequences associated with having no children who completed college. Having no children who completed college is negatively associated with parents\u2019 self-rated health and positively associated with depressive symptoms. Among parents with the highest propensity for having no children who complete college, the consequences on depressive symptoms are greatest. These findings are important given the call for investments in children\u2019s educational opportunities as a vehicle for promoting health among adults and their older parents."} +{"text": "To reduce monthly bed days for children and young people (CYP) aged under 18 years admitted to adult psychiatric beds by 50%Early senior psychiatric CAMHS review for all CYP admitted to adult psychiatric beds (same or next working day)Increased access to CAMHS medical records for out of hours staffAdmission of all appropriate under 16's to paediatric beds instead of adult mental health bedsShort test of change of staffing CAMHS specialist nurses over a weekendDevelop alternative non-health crisis support/bed for CYPDevelop Personality Disorder (PD) pathwayQI tools used included driver diagram, stakeholder analyses, process mapping, ishikawa diagram, pareto chart and interviews with CYP and carers to gather qualitative data. Monthly data were collected on all admissions of CYP to adult mental health beds. Change ideas/ process changes included:Early senior CAMHS psychiatric review was associated with a reduction in CYP admitted to adult mental health beds from a median of 20 days a month to 2 days a month without an associated increase in CAMHS inpatient admissionsPareto chart showed that Personality Disorder (PD) was the commonest diagnosisAccess to CAMHS medical records for all out of hours psychiatric medical staff was increased from 13% to 100%Routine admission to paediatrics for all under 16's was agreed with paediatric medical and nursing managers but not sustainably implementedThere were no acute referrals to the CAMHS specialist nurses over the single weekend short test of changeDevelopment of an alternative non-health crisis support/bed and development of a Personality Disorder (PD) pathway is still in processThe primary outcome measure was successfully met with the median bed days of CYP admitted to adult mental health beds sustainably reduced from a median of 20 days to 2 days. This was associated with the implementation of routine early senior psychiatric CAMHS review and increased access to CAMHS health records for all medical staff providing psychiatric out of hours assessments. The change ideas including development of different admission pathways , weekend CAMHS specialist nurses service and development of a personality disorder pathway were not implemented sustainably. The pathways of care around CYP presenting in crisis are complex. Making sustainable improvements in complex adaptive systems is complex and challenging but not impossible."} +{"text": "A 50\u2010year\u2010old man with a history of multiple episodes of tachycardia and complete left bundle branch block (LBBB) morphology underwent electrophysiological studies and catheter ablation. The earliest retrograde activation in response to premature ventricular stimulation was observed simultaneously in the His bundle (HB) region and proximal coronary sinus (CS), without decremental conduction Figure\u00a0. RetrogrGenerally, shortening of the HA interval during tachycardia coinciding with the resolution of complete LBBB morphology (Coumel's law; Figure\u00a0Our observations are explained as follows: during the ventricle capture alone, retrograde conduction was predominantly through the accessory pathway. In contrast, during the HB and ventricle capture, the activation wavefront propagated through the fast pathway and reached the atrium along the CS recording earlier than while propagating through the working myocardium and bundle branch and reaching the ventricular myocardium along the CS recording. Therefore, para\u2010Hisian pacing with direct HB capture was associated with a shorter stimulus to atrial activation interval than in the absence of direct HB capture.Retrograde conduction over the fast pathway during para\u2010Hisian pacing in presence of the posteroseptal accessory pathway is often observed.The accessory pathway was successfully ablated at the left posterior mitral annulus.The authors have no conflicts of interest to disclose.Approval was obtained from the local ethics committee.The patient has provided signed consent for publication.None."} +{"text": "The COVID-19 pandemic was the first pandemic with an enormously dramatic global impact after the world went digital. Never before we got so much data about a disease in such a timely fashion . HumanitInfectious Disease Forecasting including Effects of Confinements and Vaccinationfor Increasing Epidemic Preparedness in Public Health,for the Detection of Diseases andin Genome SequencingAIRole of AI in Contact TracingAI-Assisted TestingGenerating Recommendations for Individuals\u2019 HealthSituation AwarenessSentiment Analysis and Trustworthiness of Information During EpidemicsArea Editor Sven Groppe University of L\u00fcbeck, GermanyAfter organizing two special issues , 5 targe"} +{"text": "Maternal health remains a major issue of concern in Sierra Leone. In the main referral maternity institution, Princess Christian Maternity Hospital (PCMH), up to 25% of maternal deaths occur during or shortly after transit from another health facility. There is an urgent need to improve referral systems between peripheral health units (PHUs) and PCMH. Our aim was to pilot and evaluate an eHealth tool facilitating referral of obstetric emergency cases through effective teleconsultation between PHUs and PCMH.A web application was designed to capture unclear or complicated delivery cases at PHUs and request respective telemedical counselling from the referral institution PCMH. The eHealth tool was piloted at 10 PHUs in Western area urban and rural in August 2021. Necessary devices were provided and delivery staff was trained to use the app. In December 2021, we conducted focus group discussion with 3-6 delivery staff at five PHUs and at PCMH to evaluate utilization and outcomes of the tool.All participants perceived the eHealth tool as an improvement of referral procedures. Response time from PCMH after a request for counselling from a PHU was mostly <30 minutes. The main perceived advantage of the tool was the systematic documentation of obstetric complications and procedures. This relieved staff from fear of wrong treatment accusations, and recorded communication with PCMH made processes and responsibilities transparent. Another important benefit was PCMH staff being already prepared to receive a specific emergency case after use of the app, thus reducing the \u2018third delay\u2019 within the referral facility. As a major obstacle to smooth referral despite the eHealth tool, a lacking ambulance system was mentioned as a critical gap.Exceedingly positive user experiences with this simple tool seem to make an expansion to more PHUs worthwhile. Benefits of using the app in more remote districts in Sierra Leone should be further investigated.\u2022\u2002Delivery staff in Sierra Leone was capable of using a web app for telemedical counselling in a useful and effective manner.\u2022\u2002The eHealth tool was perceived as very helpful in systematically and transparently documenting emergency delivery cases and treatment procedures."} +{"text": "This review aims to provide an overview of evidence on feasibility and effectiveness in diverse populations of eHealth physical activity (PA) community engagement (CE) interventions. Increasing global PA levels would have a substantial positive impact on population health. Given their diffusion, eHealth technologies may address certain barriers to PA and reach wide audiences. The most recent Italian guidelines on PA highlight inequalities in health, which can be addressed using CE models. The potential scalability of successful eHealth CE interventions and the scarcity of previous reviews on the topic are reasons which convinced us to work on this paper.This mixed-methods systematic review utilized the Joanna Briggs Institute methodologies. Primary quantitative outcome measures were minutes of PA per week. Qualitative outcome measures included self-efficacy and user engagement. Data were processed using a segregated convergent design. A narrative summary and a meta-aggregation were performed for synthesizing quantitative and qualitative data respectively. Only the interventions where CE principles were fulfilled were analyzed.Quantitative evidence supported effectiveness and feasibility of interventions to improve PA outcomes and related proxy indicators across studied populations. Qualitative findings suggest the utility of peer-support and that from other health care providers.Implementing CE in future PA interventions will be critical for producing an effective digital application with the potential for considerable impact in the real world. If supported by central governments and the European Union, entities such as primary care hubs and local health units with their professionals and CE capabilities may play the key role in implementing evidence.\u2022\u2002eHealth PA CE interventions work better when peer support takes place.\u2022\u2002Health systems could pursue these prevention strategies for population health gains."} +{"text": "We thank Gomby for the Gomby's assertioWe leveraged HOLC maps because they are valuable data for capturing lending discrimination. Faber , a spatiThe studies Gomby cites usWe conclude that \u201chistorical redlining has an enduring impact on cardiovascular risk among Black adults in the United States,\u201d a statement that was supported by the associations we documented in our study. Regardless of whether HOLC caused redlining or is just a reflection of redlining practices, the fact remains that this discriminatory practice may have an intergenerational impact on the current-day cardiovascular health of Black people. We employed several epidemiological methods to assess the robustness of our results, including rigorous control for confounding, conducting sensitivity analyses with various outcome measures, and utilizing three different exposure assessment methods. We anticipate that future research will enhance our findings."} +{"text": "These angioscopic findings might reveal the risk of vascular injury from excessive laser irradiation to strong adhesion tissue in the same region.Cardiovascular implantable electronic device (CIED) removal is indispensable for treatment of CIED infection.A 57-year-old man with dilated cardiomyopathy was admitted to our hospital owing to need for TLE for pocket infection of his cardiac resynchronization therapy defibrillator. He underwent cardiac resynchronization therapy defibrillator implantation 4 years prior and had catheter ablation therapies performed 4 times for atrial fibrillation and atrial tachycardia.We performed TLE under general anesthesia. We peeled off adhesions with all leads from axillary vein to right atrium using 12F and 14F excimer laser sheaths and successfully extracted all leads, which were covered with adhesive tissue. Before and after using the excimer laser sheaths, we examined the condition of the venous endothelium in between the innominate vein and above the superior vena cava by nonobstructive angioscopy through a femoral vein approach. Before use of the excimer laser sheaths, normal venous endothelium was smooth and white ; howeverAfter appropriate antibiotic administration and wound care, his wound completely healed and he was discharged from our hospital.From these angioscopic findings, we directly recognized the risk of vascular injury after TLE with excimer laser sheaths. Vascular injury is one of the most serious complications during TLE procedures."} +{"text": "Dear Editor,At present, there is no noninvasive prenatal testing (NIPT) method available for pregnant women that one partner of couples carry balanced chromosomal rearrangements (BCRs). Here we present a novel NIPT method for detecting foetal chromosomal rearrangements based on haplotype analysis of maternal cell\u2010free foetal DNA (cffDNA) and demonstrate its effectiveness and potential for clinical application in a cohort of 46 patients.BCRs are estimated to occur in approximately 0.5% of newborn infantsCore family\u2010based haplotype linkage analysis has long been used to indirectly diagnose monogenic diseases, such as in preimplantation genetic testing (PGT) and NIPT.To determine the parental allele inherited by the foetus, we first established the parental haplotypes through target\u2010region sequencing genotyping data of the parents and one relative based on Mendel's law Figure\u00a0. This stTo further estimate the accuracy of the inferred foetal haplotype using this NIPT method, SNP genotypes those inferred from maternal plasma DNA sequencing were validated using direct sequencing results of foetal genomic DNA obtained from amniocentesis or blood. The average accuracy of inferred maternal and paternal alleles was 98.8% Table , which wTo validate the effectiveness of this proposed NIPT method, the results were confirmed by karyotyping analysis of foetal cells. Among all patients, 29 pregnant women underwent invasive prenatal amniocentesis in the second trimester and 7 pregnant women had their newborns' umbilical cord blood testing at birth, our results revealed that all foetal karyotypes were completely consistent with those detected using the NIPT method Figure\u00a0. FurtherThe study strengths include its high accuracy and large sample size in the cohort, the rearranged chromosomes involve almost all 22 pairs of autosomes, proving that the noninvasive method is universal for different aspects of chromosomal rearrangements. To the best of our knowledge, this is the first report about a noninvasive approach to detect foetal balanced chromosomal rearrangements using maternal circulating cell\u2010free foetal DNA.In conclusion, we develop a novel haplotype\u2010based NIPT method for couples with BCRs and demonstrated this approach is applicable and universal for detecting a wide spectrum of chromosomal rearrangements in foetus. The proposed new NIPT\u2010SR technology provides an option for these pregnant and will be an important supplement to the noninvasive prenatal diagnosis system for genetic diseases, reducing the possible miscarriage risk of IPD and facilitating the prevention of birth defects.The authors declare that they do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted.Supporting InformationClick here for additional data file."} +{"text": "Introduction: Adaptive Treatment Strategies warns therapists of patients at risk of treatment failure to prompt an adaption of the intervention. Internet-delivered Cognitive Behavioural Therapy (ICBT) collects a wide range of data before and during treatment and can quickly be adapted by adjusting the level of therapist support. Objectives: To evaluate how accurate machine learning algorithms can predict a single patient\u2019s final outcome and evaluate the opportunities for using them within an Adaptive Treatment Strategy. Methods: Over 6000 patients at the Internet Psychiatry Clinic in Stockholm receiving ICBT for major depression, panic disorder or social anxiety disorder composed a training data set for eight different machine learning methods . Symptom measures, messages between therapist and patient, homework reports, and other data from baseline to treatment week four was used to predict treatment success for each primary symptom outcome. Results: The Balanced Accuracy for predicting failure/success always were significantly better than chance, varied between 56% and 77% and outperformed the predictive precision in a previous Adaptive Treatment Strategy trial. Predictive power increased when data from treatment weeks was cumulatively added to baseline data. Conclusions: The machine learning algorithms outperformed a predictive algorithm previously used in a successful Adaptive Treatment Strategy, even though the latter also received input from a therapist. The next steps are to visualize what factors contributes most to a specific patient\u2019s prediction and to enhance predictive power even further by so called Ensemble Learning.No significant relationships."} +{"text": "There is evidence health utilization increases after incident dementia, particularly toward the end of life. However, less is known about utilization in the years before dementia. Our study objectives were to compare outpatient emergency department (ED) and inpatient hospital utilization in the six years preceding incident dementia compared to a reference group without dementia. We obtained data on n=5,547 Beneficiaries from the Health and Retirement Study-Medicare linked sample, and defined dementia using a validated algorithm. Those with and without dementia were balanced on confounders using inverse probability weighting applied to longitudinal Generalized Estimating Equation models. We found persons with dementia had greater odds of ED and inpatient hospital usage in the years preceding dementia compared to those without dementia across a comparable timespan. This study provides evidence to suggest greater healthcare burden may exist before manifestation of dementia."} +{"text": "The proportion of the world\u2019s population over 60 years of age is projected to nearly double in the next two decades, but the advances in the life span bring us new challenges, the functional deficit and impaired life quality in the elderly. Sarcopenia is a progressive decline in muscle mass and strength. One of the risk factors in this process is mitochondrial reactive oxygen species and oxidative stress. In a recent study, we tested the causal effect of targeting hydrogen peroxide within mitochondria to illuminate the role of mitochondrial oxidative stress in sarcopenia . To specThe findings in the study demonstrate that scavenging mitochondrial hydrogen peroxide improves mitochondrial functions that are critical for cellular respiration and calcium homeostasis. The decreased OXPHOS capacity in mice lacking superoxide dismutase 1 (Sod1KO) was fullDisruption at neuromuscular junction is widely considered as one of the key drivers of sarcopenia. An important question in this process is the role of oxidative stress from mitochondria. Animals with nerve transection surgery that results in denervation exhibit 30\u201340 folds increase in reactive oxygen species from skeletal muscle mitochondria . This daTargeting mitochondrial hydrogen peroxide attenuates muscle atrophy and contractile dysfunction in a mouse model of early onset of sarcopenia , 5. AtroIn conclusion, recent evidence using various transgenic mouse models clearly illustrates the potential of targeting mitochondrial oxidative stress in prevention and attenuation of sarcopenia. These animal models have increased antioxidant enzymes within skeletal muscle but targeting neuronal mitochondrial hydrogen peroxide might also protect against neuromuscular junction disruption and neurogenic atrophy as an independent mechanism. Future research should narrow our targets for interventions and pharmacological therapies that can help increase active and healthy lifespan in the elderly."} +{"text": "A primary disruption of the bodily self is considered a core feature of schizophrenia patients (SCZ). The \u201cdisembodied\u201d self would be underpinned by an inefficient body-related multisensory integration mechanism occurring in the Peripersonal Space (PPS). PPS is a plastic sector of space surrounding our body, whose extent is altered in SCZ. Although PPS represents a malleable interface marking the perceptual border between self and others, no study has investigated the potential alteration of its plasticity in SCZ.We investigated the PPS extension and its plasticity in SCZ and their potential correlations with the clinical scales.Thirty SCZ and thirty healthy controls (HC) underwent a multisensory task to estimate PPS boundary before and after a motor training. Patients were also administered the Positive And Negative Syndrome Scale (PANSS) and the Examination of Anomalous Self-Experience (EASE).Data confirm a narrower PPS extent in SCZ than in HC, whereas no differences in PPS expansion was found in the two groups after the motor training . Positive symptoms were associated directly with PPS extent and inversely with PPS plasticity. No associations were found between PPS and EASE domains. Figure1: Graphical representation of PPS expansion in SCZ and HC. Both panels show individual normalized sigmoid fitsThe present study suggests a narrower PPS extent and a preserved PPS plasticity in SCZ with respect to HC. Both PPS extent and plasticity are related to the severity of positive symptoms. These results highlight the potential role of rehabilitation interventions in order to improve patients\u2019 weakened body boundary.No significant relationships."} +{"text": "Space occupying lesions compromising frontal lobes usually may produce in the first place psychiatric symptoms such as progressive change of personality and/or symptoms suggestive of depressive episodes. Thus they can be misdiagnosed and mistreated.A case report is presented as well as an updated review of frontal lobe tumor diagnosis and treatment literature.We present the case of a 45 years-old male patient with no relevant medical history who arrives at the mental health center due to behavioral disorders, depressive mood, workplace absenteeism and personal hygiene neglect in the last 3 months.Since the clinical picture was compatible with depressive disorder the patient was treated with psychotherapy and antidepressant drugs with no remission. Due to the treatment absence of response he attends emergency services where he is performed a craneal tomograpy (CT) where a right frontal lobe tumor (FLT) is observed.In early stages FLT are sometimes presented as psychological mood or anxiety disorders without accompanying neurologic deficits. Thus, mental health professionals should be aware that psychological symptoms might be a presentation of organic disease of the brain and in some cases organic screening and hence brain imaging should be considered.No significant relationships."} +{"text": "Mucoceles are benign lesions, covered by pseudostratified epithelium, that affect paranasal sinuses. Most of them occur in the frontal sinus (60%). Bilateral involvement is extremely rareA male patient aged 37 years with a history of cranioencephalic trauma 21 years ago, reports a left frontal tumor beginning 4 months ago. Magnetic resonance imaging revealed a lesion suggesting mucocele . The patMucoceles develop following obstruction of the drainage ostia, growing slowly within the sinuses, eventually eroding adjacent bone structuresMucoceles are mostly located in the frontal sinus, although bilateral involvement is extremely rareThe diagnosis is made based on the clinical history, physical examination and radiologic imaging. Symptoms vary in frontal ethmoid mucocele from absence of symptoms to incapacitating pain, headache and visual disturbancesComputed tomography shows an isodense homogeneous image, with loss of the normal sinus contour that is not enhanced with contrast if not infectedMagnetic resonance imaging is indicated if there is any doubt in diagnosis. The typical finding is a T1 hyposignal area T1 and a T2 hypersignal area, however any combination of signal intensities may be found depending on the presence of blood particles or the degree of hydration of the contentCurrently ample marsupialization of the mucocele and simple drainage of the sinus by endoscope has been done with excellent surgical results. The advantages include low morbidity, low risk of complication and a rare recurrence rateEndoscopic treatment seems to be the best treatment option. However, for technical reasons anatomical variants may preclude ample marsupialization done only endoscopically."} +{"text": "The increasing global burden of mental disorders has led to rising demand for mental healthcare services. Effective resource management is essential to ensure safe and timely access to care. Electronic health records (EHRs) provide a real-time source of data on clinical presentation and prognostic factors that could be harnessed to provide clinicians with actionable insights to prioritise mental healthcare delivery. We describe the development and evaluation of MaST, an EHR data visualisation tool that provides information to clinicians on risk of mental health crisis defined as an admission to a psychiatric hospital or acceptance into a community crisis service.(i) To develop an EHR-data driven risk prediction tool for risk of crisis. (ii) To evaluate predictive performance in a real-world clinical setting.The risk of crisis algorithm was developed and evaluated with EHR data from six UK NHS mental health providers using Ordered Predictor List propensity scores grouped into 5 quintiles. The predictor variables were clinical and sociodemographic factors including previous mental health service contacts.Data from 2,620 patients contributed to algorithm development which was subsequently tested on data from 107,879 patients. The risk of crisis algorithm performed well with an overall accuracy for predicting the greatest risk of crisis (top quintile) ranging from 64% to 80%.The MaST algorithm accurately predicted risk of mental health crisis in UK community mental health services. EHR data visualisation tools can provide actionable insights to clinicians to prioritise mental healthcare delivery in real-world clinical practice.This study was funded in full by Holmusk."} +{"text": "Elimination of dead or live cells take place in both a healthy and diseased central nervous system (CNS). Dying or dead cells are quickly cleared by phagocytosis for the maintenance of a healthy CNS or for recovery after injury. Live cells or parts thereof, such as the synapses and myelin, are appropriately eliminated by phagocytosis to maintain or refine neural networks during development and adulthood. Microglia, the specific population of resident macrophages in the CNS, are classically considered as primary phagocytes; however, astrocytes have also been highlighted as phagocytes in the last decade. Phagocytic targets and receptors are reported to be mostly common between astrocytes and microglia, which raises the question of how astrocytic phagocytosis differs from microglial phagocytosis, and how these two phagocytic systems cooperate. In this review, we address the consequences of astrocytic phagocytosis, particularly focusing on these elusive points. Phagocytic targets are mostly common between astrocytes and microglia.Various factors determine whether either of the glial cell types phagocytose the targets.Interaction of astrocytes and microglia may provide microglia with a phagocytic advantage. The inhibitory effects of microglia on astrocytic phagocytic activity have also been supposed when astrocytes engulf dead cells. As described in a previous section, intravital live imaging showed territories of astrocytes and microglia when cell death was induced in a single neuron. Both glial cells respect each other's phagocytic territory; however, astrocytes invade the primary microglial territory to engulf dead neurons upon microglial depletion (Damisah et al.,\u00a0Conversely, several reports have suggested that microglia inhibit astrocytic phagocytosis Figure\u00a0. A speci5Microglia have been regarded as phagocytes in the CNS from the period when Rio\u2010Hortega introduced the microglial concept (del Rio\u2010Hortega,\u00a0Elucidation of glial phagocytosis may provide new therapeutic strategies. Regarding synapses, abnormal synaptic pruning by microglia during development causes an increased or decreased number of synapses, which is related to autism spectrum disorder and schizophrenia, respectively (Filipello et al.,\u00a0To promote the clearance of dead cells, enhancement of lysosomal biogenesis as well as upregulation of phagocytic receptors in astrocytes is expected to be a therapeutic target (Chandra et al.,\u00a0Metabolic reprogramming of astrocytes could be another therapeutic target for accelerated clearance of dead cells. Regarding microglia, they have metabolic flexibility (Bernier et al.,\u00a0Further studies may provide clues for the therapeutic application of astrocytic phagocytosis to the efficient clearance of extracellular protein aggregates as well as dead cells in the CNS.Hiroyuki Konishi: Writing \u2010 original draft preparation. Schuichi Koizumi: Writing \u2010 review & editing. Hiroshi Kiyama: Writing \u2010 review & editing.All authors declare no conflicts of interest."} +{"text": "Distinguishing between support and care, this study investigated how different types of past support exchanges with children were associated with older parents\u2019 care receipt and expectations. Older parents reported on tangible, non-tangible, and childcare support exchanges with each of their adult children in two waves of the Family Exchanges Study (2008 and 2013). Multilevel, within-family, logistic regression models were estimated. Parents with functional limitations more likely received care from children whom they received more tangible support from at the prior wave. Parents without current limitations more likely named children whom they previously provided childcare support to and received more tangible support from as their expected future caregiver. These findings emphasize continuity in the transition from receiving tangible support to receiving and expecting care from adult children. The importance of older parents\u2019 childcare support given to adult children also highlights reciprocity in intergenerational care exchanges."} +{"text": "Metronomic dosing for pancreatic cancer combination therapy treatment association with reduced neutropenia.This research aims to evaluate the use of combination therapy for shrinking tumor size in patients with metastatic pancreatic cancer using gemzar/paclitaxel combination therapy for treatment. Patients with pancreatic cancer were treated and observed in a cancer outpatient clinic. Gemzar/Paclitaxel combination therapy was used in lower than recommended dose. Patients were educated on the risk of neutropenia associated with combination chemotherapy. Labs were repeated every month to assess the tumor size, metastasis of tumor growth to other organs and neutropenia associated with combination chemotherapy.Patient in the clinic undergoing chemotherapy showed that using gemzar/paclitaxel combination therapy has reduced chance of neutropenia when used with low dose intervals in metastatic pancreatic cancer.We observed that using metronomic dosing in patients using combination therapy for pancreatic cancer shows decreased neutropenia compared to others who did not receive metronomic dosing on combination treatment with gemzar/paclitaxel. While neutropenia is decreased, patients also have decline in the metastasis of the tumor to other organs and shrinkage in the size of tumor."} +{"text": "Sleep problems and loneliness both increase with age and undercut older adults\u2019 health and well-being. Loneliness is a predictor of sleep problems, however, research in this area often fails to also consider the role of relationship quality. The current study therefore focuses on spousal relationship quality as it has been shown to have a proximal influence on sleep. We examined relative effects of physical and emotional facets of relationship quality\u2013namely affectionate touch and emotional support (in addition to loneliness). We further examined the potential for relationship quality to buffer effects of loneliness on sleep. We utilized data from a nationally representative sample of older adults from the National Social Life Health and Aging Project (NSHAP). Participants were partnered (N = 559) and completed a novel sleep module that included subjective and objective markers of sleep. Upon controlling for demographics and mental and physical health, a different pattern of findings emerged for each facet of sleep. For subjective sleep, older adults who were more lonely reported more insomnia symptoms, but only when spousal emotional support was low. For objective sleep, older adults who reported more affectionate touch experienced less WASO. These findings suggest that loneliness that occurs in the context of low emotional support from spouses is particularly damaging for older adults\u2019 subjective sleep quality. Further, affectionate touch from spouses may represent an important intervention target for promoting felt security that helps older adults stay asleep throughout the night."} +{"text": "Tertiary metabolic health services are in high demand as people with severe obesity increase. Once predetermined health goals have been achieved patients must transition to community-based care to urgently free up capacity in tertiary services. Maintenance of successful outcomes achieved via tertiary services is therefore important to limit rates of relapse back to these services.This qualitative project explored community-based care needs to help individuals living with obesity maintain health gains. An interview schedule guided one-on-one interviews with patients and staff from metabolic clinics in Sydney, Australia.We interviewed 22 patients and 13 clinicians. A lack of appropriate and consistent clinical support in the community was identified by patients and clinicians. Most clinicians agreed primary care was key to successful maintenance care. Lack of primary care understanding of appropriate management and support for patients with obesity, lack of bariatric equipment and limited funding for allied health were all seen barriers to appropriate support beyond their clinics. Patients were highly reluctant to transition from tertiary clinics and reluctant to engage with community-based care due to experience of limited clinical/social support and bariatric equipment, demeaning clinical interactions, lack of care coordination and being stigmatised. Support groups outside of the clinic were also identified important in mitigating social isolation and stigma. Both patients and clinicians felt support groups have potential to provide important supplementary help to individuals with obesity outside tertiary settings.Currently, individuals aiming to maintain their weight are likely to struggle in the context of existing community care provisions. Integrated, community-based and affordable models of care are needed now to allow tertiary metabolic services discharge their patients safely.\u2022\u2002Tertiary obesity services are at capacity.\u2022\u2002Subsequent community care for people wth obesity needs to be mote appropriate tp promote weight maintenance."} +{"text": "Suicide is an international public health problem and a leading cause of death for youth and adults, worldwide. Prevention efforts in health care systems create opportunities for identifying medical patients with occult suicidality. Detecting suicide risk among patients in medical settings can be a challenge, but successful suicide risk screening programs have been demonstrated in hospital settings.This presentation will discuss how a suicide risk screening tool that was developed for the pediatric emergency department was tested and then implemented in other medical settings in order to leverage healthcare providers as partners in combating the public health crisis of youth suicide..Implementation and quality improvement projects in various medical settings that have adapted the ASQ will be described. Effective management of pediatric patients that screen positive for suicide risk and how mental health clinicians can best be utilized in efficient ways will also be discussed.Average time to administer the ASQ was 20 seconds. Positive screen rates across ED, inpatient and outpatient settings ranging from 2-14% equating to one additional psychiatric consultation per week. The ASQ Toolkit was developed to help medical providers implement screening including scripts for nurses, flyers for parents and a brief suicide safety assessment (ASQ BSSA) to operationalize next steps for patients at risk.The medical setting is a key venue for youth suicide risk detection and linking patients with effective interventions. Mental health clinicians have a role in guiding non-mental health providers in the identification and management of patients found to be at risk.No significant relationships."} +{"text": "Decidualized ovarian endometrioma is a rare phenomenon that occurs during pregnancy. A 43-year-old pregnant woman with an endometriotic cyst increased owing to desmoplasia presented to us urgently with abdominal pain and was performed a cesarean section at 36 weeks and 1 day of pregnancy. The left ovarian cyst was noted to be partially ruptured, and the pathological diagnosis was an endometriotic cyst with desmoplasia. Endometriotic cysts may enlarge during pregnancy owing to desmoplasia and rupture in the last trimester of pregnancy, causing acute abdomen. Decidualized ovarian endometrioma is a rare phenomenon that occurs during pregnancy and must be differentiated from ovarian cancer A 43-year-old woman with 2 previous pregnancies and one lactation presented a 40-mm ovarian endometriotic cyst, noted before the following pregnancy . At the A decidualized ovarian endometrioma should be carefully distinguished from a malignant transformation, as it may show increased size and papillary projections on ultrasound imaging. MRI is useful to determine whether a malignant transformation occurred in endometriosis. The presence of a nodule with contrast effect is a finding suspicious for malignant transformation A previous review reported that a decidualized ovarian endometrioma diagnosed with MRI rarely causes complications that require surgery during pregnancy In conclusion, this case shows that endometriotic cysts may become desmoplastic and enlarged during pregnancy, requiring differentiation from malignancy. Endometriotic cysts increasing in size may also rupture in the last trimester of pregnancy and cause acute abdomen.Informed consent has been obtained from all individuals included in this study."} +{"text": "Subjective memory impairment, defined as self-reported difficulties in recall and learning, doubles the risk of Alzheimer\u2019s Disease and related dementia, despite being weakly related to objective memory decline. Because of its strong stability over time, it may be possible that subjective memory impairment reflects earlier life risk factors for dementia such as adverse childhood experiences. It is reported that over a fifth of older adults worldwide experienced physical abuse during childhood. Previous cross-sectional studies suggest physical abuse is associated with later cognitive impairment. Still unclear, are the longitudinal associations between childhood abuse and subjective memory impairment in later life. Using a sample of adults drawn from the Health and Retirement Study we assessed associations between reported physical abuse by a parent before the age of 18 and subjective memory impairment (current memory problems and perceived memory decline) over periods of up to 18 years. Generalized linear mixed models examined longitudinal associations between childhood physical abuse and subjective memory impairment while controlling for depressive symptoms and other empirically relevant covariates. Experiencing childhood physical abuse was associated with increased likelihood of reporting more current memory problems and perceived memory decline in later life . Findings suggest childhood physical abuse is associated with subjective memory impairment, a strong predictor of dementia. Understanding early life conditions, including adverse childhood experiences may help explain associations between subjective memory impairment and dementia risk."} +{"text": "Social connectedness has been linked to decreased rates of cognitive decline in later life. However, recent work suggests that particular social network characteristics may buffer against age-related degeneration. The present study analyzes social network and structural MRI data of 176 older adults from the Social Networks and Alzheimer\u2019s Disease (SNAD) study. Results indicate that increased social bridging is associated with greater grey matter (GM) volume in several limbic structures. Increased social bonding is associated with greater GM volumes in several cerebral cortex structures as well as greater volumes in some components of the limbic system. Most notably, the effects of bridging are primarily lateralized in the left hemisphere while the effects of bonding are observed mostly in the right hemisphere. These results suggest that the neurocognitive benefits of social connectedness depend on the preponderance of bridging and/or bonding ties in older adults\u2019 social networks."} +{"text": "The Cannabis and Older Persons Study has examined the increasing use of cannabis among Americans over 60 years old since 2016. This year\u2019s symposium presents varied methodological approaches researchers have used to better understand the harms and benefits associated with cannabis use among older Americans. Divya Bhagianadh examines the association between cannabis legalization policies across the Untied States and corresponding use of end of life programs and services. Julie Bobitt analyzes interviews provided by older adults in Illinois and defines how attitudes held by medical doctors and other clinical care providers are critical to shaping cannabis use among older adults. Brian Kaskie considers the role of cannabis policy clusters. Jacobo Mintzer reviews findings from a controlled trial providing two cannabinoids to treat agitation among persons with Alzheimers in hospice. Thorsten Rudroff examines cannabis use among a sample of older persons at risk for falls. These studies reflect how cannabis use among older persons continues to grow and diversify, and how researchers have use different approaches to advance scientific understanding of determinants and outcomes, both desirable and undesirable, associated with cannabis use among older adults. This symposium offers policy makers and health care leaders a balanced perspective. On one side, we discuss how prevention and treatment efforts for substance use disorder, including cannabis use disorder, must increase proportionally to the increasing number of older cannabis users. On the other side, we consider how more than 5 million older Americans, especially those with pain, find some benefit in using cannabis."} +{"text": "Gestational diabetes mellitus (GDM) is a significant, global public health problem. Subsequent strain on healthcare systems is widespread and multidisciplinary care may be inadequate. We assessed current nutrition management of GDM in a large, metropolitan maternity hospital in Melbourne, Australia and associations between the model of dietetic care and maternal and neonatal health outcomes.Hospital medical record data from The Women\u2019s Hospital, Melbourne for women with GDM (July 2105-May 2017) was retrospectively analysed. Adjusted linear and logistic regression were used to assess associations between the number of dietitian consultations and maternal and neonatal health outcomes.Half of all women received two consultations with a dietitian. Nineteen percent of women received three or more consultations and of these women, almost twice as many were managed by medical nutrition therapy (MNT) and pharmacotherapy (66%) compared with MNT alone (34%). Odds of maternal complications increased with number of consultations (p = 0.008). Lower odds of infant admission to the Neonatal Intensive Care Unit were observed among women receiving one , two , or three+ dietitian consultations , compared to no consultations.The optimal schedule of dietitian consultations for women with GDM in Australia is unclear. Alternative delivery of nutrition education for women with GDM such as telehealth and utilisation of technology may assist in relieving public health and healthcare system pressures and ensure optimal pregnancy outcomes.\u2022\u2002Delivering medical nutrition therapy through individual consultations does not deliver a linear benefit to women with GDM and their offspring.\u2022\u2002Alternative delivery modes are needed to optimise outcomes for healthcare services and their patients."} +{"text": "The functional luminal imaging probe (FLIP) utilizes high-resolution planimetry to provide information regarding esophagogastric junction (EGJ) diameter, EGJ distensibility, and reactive contractile patterns of the esophageal body. This is an FDA-approved measurement tool utilized to both diagnose and measure various upper gastrointestinal disorders. While patients are sedated during FLIP panometry, significant respiratory variations can affect the quality of FLIP panometry results.\u202fNasal continuous positive airway pressure (CPAP)\u202fcan be utilized to prevent intraoperative or postoperative hypoxia in obese patients as well as those with obstructive sleep apnea (OSA).\u202fIn this retrospective chart review, we compared obese patients with a diagnosis of OSA who underwent FLIP panometry utilizing nasal CPAP as airway management against a group who underwent the same procedure with a nasal cannula to evaluate the incidence of hypoxia, hypercapnia, variation in cardiovascular dynamics, and the quality of FLIP panometry readings. Functional luminal imaging probe (FLIP) panometry is a novel tool to further evaluate the esophageal function as well as guide management and treatment in patients with dysphagia or other esophageal symptoms ,2.\u00a0PlaceIn this retrospective chart review, 50 obese patients with an apnea-hypopnea index (AHI) > 15, who were candidates for gastric bypass surgery at Baylor University Medical Center (BUMC) with a chart diagnosis of OSA, who underwent upper endoscopy with FLIP panometry from April to July 2021, were included.\u00a0This retrospective chart review analysis was IRB Quality Improvement approved by our institution. Based on provider comfort and experience, some patients received nasal CPAP as preferred airway management and others received nasal cannula instead. Group 1 (25 patients) had the nasal CPAP utilized for airway management, while group 2 (25 patients) had the nasal cannula utilized for airway management during FLIP panometry.\u00a0In this study, group 2 was selected as the control group due to the use of a nasal cannula as the standard method of airway management and sedation.\u00a0The nasal CPAP settings were standard set for these patients at pressure support of 10\u00a0cmH2O\u00a0and oxygen flow of 10 L/min. The nasal cannula settings were also standard set with an oxygen flow of 4 L/min. All patients had a Patient State Index (PSI) value of 25-50 from SEDline with optimal hypnotic sedation using propofol. The baseline past medical history for each group is displayed in Table Table Figure This retrospective chart review highlights the potential benefit of utilizing nasal CPAP for airway management in obese patients with OSA undergoing FLIP panometry under IV sedation. Aside from their use intraoperatively for airway management, CPAP machines have been a well-established treatment for patients outside of surgery with OSA as they have been shown to decrease overall blood pressure, daytime sleepiness, risk of heart failure, and stroke . BesidesIn summary, in this retrospective chart review, we compared obese patients with a diagnosis of OSA who underwent FLIP panometry utilizing nasal CPAP as preferred airway management against a group who underwent the same procedure utilizing the standard nasal cannula as airway management to evaluate the incidence of hypoxia, hypercapnia, variation in cardiovascular dynamics, and the quality of FLIP panometry readings. This study has potential limitations. With a larger sample size, a more accurate statistical analysis could be attempted to compare the intraoperative vitals of the study groups. In addition, the nature of the study type produced many limitations owing to the design of a retrospective chart review as there could have been potential biases in our data and results. Being a retrospective chart review, the possibility of a selection bias is present due to convenience sampling and thus each group may not accurately represent the general population. We understand that to further evaluate the superiority of nasal CPAP as preferred airway management during FLIP procedures in patients with OSA, we must further undergo a randomized prospective study analysis. The nasal CPAP\u2019s ability to provide a steady flow of oxygen or steady maintenance of pressure keeps the patient's airways open continuously throughout the FLIP panometry procedure, thus preventing intraoperative hypoxia and hypercapnia as well as minimizing respiratory variations, which may affect FLIP panometry findings.\u00a0CPAP is able to increase intrapleural pressure thereby preventing constriction of the lower esophageal sphincter to decrease RRCs.\u00a0Moreover, as the incidence of RRCs is significantly decreased in the nasal CPAP group, the risk of aspiration is hypothesized to follow and decrease as well. Therefore, by minimizing episodes of intraoperative hypoxia and hypercapnia, we can blunt any intraoperative movements caused by hypercapnic-induced sympathetic activation and thereby improve the quality of the FLIP panometry readings."} +{"text": "We defined enrichment seeking as the capacity for adults to engage in the novel and intellectually challenging activities for exploring diverse experience. Although enrichment seeking is associated with cognitive resilience in older adulthood, the tendency for older adults to adopt explorative behavior decreases with age in concert with decline in executive control. The goal of our study was to examine to what extent older adults can adjust their cognitive foraging performance under stress by inducing relaxation through the task environment. We used cognitive foraging games (including word search puzzle games) to understand how older adults optimize their search performance by the tradeoff between exploration (seeking new information) and exploitation (staying at old information). A modified Trier paradigm, where game performance was observed by a neurologist via video conferencing, induced stress; relaxation was manipulated by turning on an electric fireplace in the study room. The study followed a 2 (relaxation) X 2 (stress) within-subjects experimental design. Sixty-one adults played games on a tablet under stress / no stress conditions with and without the fireplace in a simulated smart home environment. The interaction effect of relaxation and stress on cognitive foraging performance was significant, indicating that with the relaxation elements in the environment , older adults are likely to have better cognitive foraging performance under stress. The association can be in part explained by individual differences in cognitive abilities and strategies. Implications on facilitating enrichment seeking through environmental factors are discussed."} +{"text": "There are known risk factors that are associated with the onset and exacerbation of musculoskeletal (MSK) conditions and pain. Physiotherapists are uniquely placed to deliver brief interventions with their patients. Healthy Conversation Skills is the main training component of the Wessex approach to Making Every Contact Count. Despite its potential for promoting MSK health and wellbeing, there is no evidence to support its acceptability within MSK services. This is the first known study to explore the use and perceptions of the Wessex model of MECC HCS within MSK services. A mixed method design was used. Phase one employed an online questionnaire, open to all professionals trained in MECC HCS, consisting of items relating to implementation outcomes. Barriers and facilitators to delivery were explored and mapped to the Theoretical Domains Framework. Phase two invited physiotherapists for a follow-up interview and qualitatively explored their acceptability of delivering MECC HCS to patients with MSK conditions. MECC HCS was found to be highly acceptable, appropriate, and feasible. Physiotherapists reported using their skills at least daily but missed opportunities for delivering MECC HCS were evident. Barriers mapped mostly to \u2018Environmental Context and Resources\u2019 on the Theoretical Domains Framework. Qualitative themes developed during phase two were: \u2018Recognising the patient as the expert supports change', \u2018MECC HCS improves physiotherapy practice', \u2018MECC HCS shared problem solving reduces workload', \u2018time as a perceived barrier to MECC HCS\u2019 and \u2018system-level support needed to sustain MECC HCS'. MECC HCS is a promising brief intervention for supporting people with MSK conditions. Further rollout of this intervention may be beneficial for meeting the goals of the NHS and Public Health England in prevention of MSK conditions and promotion of MSK health. Barriers associated with sustainability must, however, be addressed.\u2022\u2002Making Every Contact Count Healthy Conversation Skills is considered a highly acceptable brief intervention for supporting behaviour change in people with musculoskeletal conditions.\u2022\u2002Organisational, system-level barriers to implementation must be addressed in order to increase sustainability and enhance future roll out of the brief intervention."} +{"text": "Leaving the home to access medical care may result in undue burden for patients with dementia and other serious illnesses and their caregivers. While home-based medical care (HBMC) may be beneficial for many older adults, it is not clear how to best identify individuals who could benefit from such services. Using the 2015 NHATS linked to Medicare claims we estimated prevalence across multiple overlapping subtypes: Individuals who have moderate/severe dementia; are homebound; have serious illness; are frail; rely on assistive devices; have high caregiving needs; those with minimal primary care and high ED use; and those who met previously established criteria for Independence at home. Using these criteria, more than half of community-dwelling older adults could benefit from HBMC and more than 25% meet multiple criteria. Medicare and other payers can benefit from targeted identification of patients who could benefit from HBMC."} +{"text": "Its morphogenesis relies on the rearrangement of a primordial structure that progresses through successive steps, ranging from neurogenesis to synaptogenesis. With form comes function and disruption of some developmental steps can lead to cortical malformations associated with a wide spectrum of clinical presentations. The cellular and molecular mechanisms underlying cerebral cortex morphogenesis are intricate and the exquisite cellular layout of the cortical wall results from the orchestrated migration of neural precursors born in distinct germinative forebrain regions.Cell migration not only brings precursors to their final position in the cortex but also promotes transient interactions with other cells, thereby conferring additional roles to those played once integrated into the cortical network.st wave vOPCs are born in overlapping germinative compartments and migrate together in the forebrain to reach the developing cortical wall. We thus assessed whether these cell populations may crosstalk during embryogenesis. While cINs navigate within the cortex in organized streams, 1st wave vOPCs undergo random walking and later disperse via dynamic gliding along the blood vessels (BVs) that are growing in the forebrain.Along this line, our laboratory discovered that some oligodendrocyte precursor cells (OPCs) steer the migration of cINs in the forebrain via direct contact.Here, we unravelled a transient and non\u2010canonical role for 1st wave vOPCs \u2013 whose number is greatly reduced after birth by apoptosis \u2013 in guiding cIN migration during embryogenesis. Class A plexins and their ligands are implicated in various aspects of cortical development and our study demonstrates how some axon guidance cues are reused in a different biological context. The idea that some migrating precursors rely on each other for reaching their individual target areas is likely to apply more generally. Collectively, these data show that the establishment of cellular crosstalk between migrating precursors is required for proper brain morphogenesis. Importantly, accumulating evidence shows that disruption of some of these crosstalks or the emergence of pathological ones contribute to the onset and progression of neurological disorders."} +{"text": "The intentional use of drugs before or during sexual intercourse (chemsex) is a phenomenon of special importance in the MSM (men who have sex with men) population due to its impact on mental, physical and sexual health. Sexual health issues related to chemsex practice have been described such as difficulties in achieving sober sex, erectile dysfunction or problems with sexual desire.To describe the sexual health interventions for patients with chemsex practices in the NGO Apoyo Positivo in Madrid. We describe the main sexual problems.Descriptive analysis.The main sexual problems were dissatisfaction in sexual intercourse without substance and difficulties with sexual desire activation (70%); compulsive sexual behaviour (70%), difficulties with sexual orientation and non normative gender expression, difficulties in erection (34%), premature ejaculation (7%) and delayed ejaculation (10%).Chemsex is a phenomenon that needs a multidisciplinary approach and mental and sexual health must be taken into account. \u201cSexo, Drogas y Tu\u201d is a model of collaborative approach which is a pioneering intervention developed by an NGO in Spain."} +{"text": "Previous caregiving research has focused on a single care recipient; however, no research has explored the potential simultaneous care needs of fathers and mothers in the same family. Drawing from gender role theory, we use qualitative data from 76 spousal dyads from the Within-Family Differences Study to compare fathers\u2019 and mothers\u2019 explanations of which adult child they prefer as their future caregiver. In 72% of families, fathers and mothers did not share the same care preferences suggesting that care preferences are dispersed among multiple adult children within the same family. In families in which fathers and mothers shared care preferences, 75% chose the same daughter. Additional findings showed that fathers\u2019 explanations for a preferred caregiver aligned with mothers\u2019 explanations; however, differences were identified based on children\u2019s gender. These findings shed light on the similarities and differences in parental care preferences and underscore the influence of gender in family care networks."} +{"text": "Geriatrics care in medical intensive care units (MICU) establishes a unique opportunity in early screening of High Risk Elderly (HRE) patients admitted for critical care. Many MICUs do not have a standard protocol to screen for HRE patients as part of their daily huddle. Our program is a quality improvement initiative to improve early identification of HRE patients in the MICU. HRE patients were identified based on nursing specific screening triggers at one of the Regional Hospitals of a large teaching hospital in Northeast Ohio.The program was designed as a part of geriatrics care expansion at regional hospital sites. Identified patients were discussed in daily huddles to determine unique geriatric needs in caring for these patients. A geriatrics co-management team was engaged in comprehensive geriatric assessments and care transition when it was needed. Geriatrics care in MICU demonstrates a unique opportunity in early identification of HRE patients. This helps to support a patient -centered approach in caring for critically ill elderly patients. The program would lay foundations in early screening for risk factors and optimizing elderly care in MICU."} +{"text": "Our cases highlighted the importance of an immediate diagnosis and bailout strategy.\u2022Once hemoptysis occurs during HBA, the electrophysiologists should be aware of any PV injury, and the immediate use of noninvasive positive pressure ventilation should be considered as a bailout strategy.\u2022When we encounter massive air on the fluoroscopic image and ST elevation during HBA, we should perform an emergent coronary angiography as soon as possible. A thrombus aspiration device may be an alternative option when mechanical aspiration via a coronary guiding catheter has failed.The hot balloon is one of the widely used balloon ablation technologies for the treatment of atrial fibrillation (AF) in Japan, and the balloon size can be adjusted to occlude the pulmonary vein (PV) ostia. With the increased use of this balloon ablation technology, complications have rarely occurred during HB ablation (HBA). We experienced 2 cases that had severe complications that were successfully resolved by a specific bailout strategy. This report aimed to share these experiences and bailout treatments.2 12 L/min via a reservoir face mask). Those vital signs did not recover despite a large-volume infusion and oxygen administration with a self-inflating bag. According to the fluoroscopic image with the guidewire peripherally positioned in the PV, we suspected the occurrence of PV injury . During the occlusion of the left superior PV, ST elevation was detected on the inferior leads and she ,During the balloon-based ablation, it has been reported that guidewire-related complications are rarely caused by a guidewire being positioning deep in the distal PV or left atrial appendage, as manifested by a PV hemorrhage or LA perforation.The HRS/EHRA/ECAS/APHRS/SOLAECE expert consensus statement reported the incidence of air embolisms is less than 1% and cerebral infarctions/transient ischemic attacks 0\u20132%.Both PV injury and air embolisms as complications during HBA are rare but potentially fatal. Our cases highlighted the importance of an immediate diagnosis and a bailout strategy that can potentially resolve those complications."} +{"text": "Premature infants with fetal growth restriction are at a significantly higher risk for developing bronchopulmonary dysplasia.Increased activity of renin\u2013angiotensin\u2013aldosterone system and oxidative stress damage are common mediators.Angiotensin converting enzyme inhibitors and melatonin (antioxidant) are promising therapies, which are physiologically suited to address the disease pathophysiology.Fetal growth restriction (FGR) affects a significant proportion of pregnancies delivered prematurely. Generally, FGR refers to birthweight <10th centile for gestational age and sex, accompanied by absent/reversed Doppler in systemic arteries. Placental insufficiency (failure to supply adequate oxygen and nutrients to the developing fetus) is the dominant underlying pathology. Premature infants are at increased risk for developing bronchopulmonary dysplasia (BPD), with approximately 20\u2010fold greater risk of mortality and/or perinatal morbidity in FGR preterm infants. A linear trajectory between higher incidence of BPD and severity of FGR has been demonstrated. FGR increases the risk for BPD; adverse respiratory outcomes attributable to the FGR state being independent of the degree of prematurity. Arrested development of capillaries and alveoli, leading to reduced gas\u2010exchange surface and dysmorphic pulmonary arteries are hallmarks of \u2018new BPD\u2019. Pulmonary hypertension (PH) complicates the management of infants with severe BPD, and is characterized by persistent vasoconstriction and structural remodelling of the pulmonary blood vessels.Involvement of vasculature has been proposed as an important instigating hypothesis for BPD in FGR infants.Prominent amongst the mediators are heightened RAAS and ROS, and their status as therapeutic targets is further discussed (RAAS [angiotensin converting enzyme\u2014ACE] and ROS [melatonin]). Figure\u00a0Hypoxia/ischemia/inflammation instigated oxidative stress damages the alveolo\u2010capillary membrane and endothelium. Oxidative stress is involved in the development of BPD in preterm infants (\u2191 in oxidative damage to lipids and proteins in lung tissue and \u2193 levels of antioxidants). Pre\u2010eclamptic pregnant women have reduced melatonin levels (possibly explaining suppressed antioxidant capacity found in preeclampsia). Melatonin is a broad\u2010spectrum antioxidant and a potent free radical scavenger, with pulmonary level vasodilator properties. In chronically hypoxic rats, daily administration of melatonin alleviated PH and reversed vascular remodelling, and attenuated right ventricular pressures, and oxidative and inflammatory parameters.In conclusion, evidence from experimental models supports the vascular constructs between FGR and BPD. Further refinement in the selection of physiologically appropriate drug and dose, and timing of administration , needs prospective analysis.None declared."} +{"text": "The COVID-19 pandemic has exacerbated racism against racial minorities and widened racial/ethnic disparities in health outcomes and access to health care services. This study analyzed cross-sectional data (N=219) collected from custodial grandparents via Qualtrics Panels in February 2022 to understand the role of race and perceived racial discrimination in contributing to custodial grandparents\u2019 depressive symptoms and access to health care services. Results indicated that a higher level of perceived racial discrimination was positively associated with grandparents\u2019 more depressive symptoms, but it was also associated with lower odds of custodial grandparents\u2019 access to health care services. Furthermore, racial/ethnic disparities in depressive symptoms and access to telemental health services among custodial grandparents were identified. Results imply the importance of addressing racial/ethnic disparities in depressive symptoms and access to health care services among custodial grandparents."} +{"text": "To the Editor,1The clinical implication of ambulatory intermittent inotropes therapy is receiving great concern thus far given that an incremental number of patients refractory to guideline\u2010directed medical therapy (GDMT) are not candidates for cardiac replacement therapy. Laufer\u2010Perl et al. demonstrated that repetitive, intermittent, and short\u2010term milrinone therapy was safe and potentially efficacious in ambulatory patients with advanced heart failure who were intolerant to maximum GDMT.In their study, participants received IV furosemide and IV iron supplementation if necessary during routine clinic visits concomitantly to milrinone therapy.In general, those with advanced heart failure complaining of New York Heart Association functional Class IIIb\u2013IV indicate in\u2010hospital treatment. Were there any critical reasons why they were not hospitalized and remained as outpatients?In their study, many patients had intolerance to maximum GDMT at baseline.According to the lists of medications,The prescription of SGLT2 inhibitor was significantly increased following the initiation of milrinone therapy. However, given that SGLT2 inhibitors have very recently been recognized as one of the GDMT, their incremental prescription rate might have been due to incremental awareness and willingness of the physicians rather than the stabilized hemodynamics by milrinone."} +{"text": "Epidemic transition, sustained costs and health workforce shortage challenges have led numerous countries to strengthen primary care (PC) and implement new models of care. Faced with declining numbers of general practitioners (GPs), France has introduced medical assistants (MAs) in 2019 to guarantee access to care and maintain workforces in deprived areas. Trained to perform administrative and clinical tasks delegated by a physician, MAs are expected to optimize medical time and improve working conditions in practices. How does French model of MAs impact quality and productivity in GPs\u2019 practices and articulate with other policies? We conducted a qualitative case study in 6 pilot practices to explore the effects of MAs\u2019 work , complemented with views from public policy makers and health professional unions (9 interviews). MA was defined as a function centered on physicians\u2019 needs, accessible both to administrative staff and nursing professions. MAs with a clinical profile performed a wider range of tasks, were more prone to perform clinical tasks and build developed interactions with patients, and seemed better fitted for chronic disease care management. Recruitment of MAs by physicians is supported with grants that decrease yearly while practice productivity is expected to rise. In general, a gain of efficiency in daily workload enabled GPs to slightly increase their productivity. However, for most GPs, it primarily helped them to maintain high workload without burning out. Although MAs with clinical background seem better suited for patients\u2019 needs, recent figures have shown that more than half of MAs employed are former secretaries. If in-person secretaries could be endorsed with further administrative duties, MAs could hold a more clinical role in PC teams including physicians and allied health professionals. Other aspects than productivity must be taken into account in a support policy.\u2022\u2002Regardless of productivity objectives to attain, hiring MAs can relieve physicians\u2019 workload and stress, preventing them from burning out and guarantee access to care in deprived areas.\u2022\u2002MA\u2019s clinical profile could have a stronger impact on public health issues such as chronic disease care management."} +{"text": "Evidence-based interventions offering meaningful benefits to informal caregivers of people living with dementia (PLWD) would be attractive to office-based practitioners if pragmatic linkages could be made between these interventions and outpatient care settings. This presentation will explain experiences and lessons learned in an ongoing pilot study in which we are: pragmatically identifying and inviting caregivers of PLWD to join online dementia education and support programs; collecting and storing caregiver outcomes data into electronic health records where these data are accessible to clinicians. Participating outpatient health care settings are a geriatrics practice at UConn Health and a memory care clinic at Emory Healthcare. Caregivers recruited at both sites participate in either Tele-Savvy or Caregiving During Crisis programs. Outcomes data will inform effects of program participation on caregivers\u2019 competence and stress, and help clinicians gain insights into caregivers\u2019 capacity to manage PLWD. Implementation evaluation strategies and results also will be discussed."} +{"text": "The perinatal period is an optimal time to intervene for achieving smoking cessation in expectant parents and offers multiple health benefits for women and the newborn. While Behavior Change Technique (BCT) interventions are a promising approach to support pregnant smokers to quit smoking, effectiveness of these interventions among expectant and new fathers is not equally well documented. Better understanding of the potential utility of these BCT interventions for this group is important for the development of effective gender-sensitive programmes.This systematic review examines the existing evidence on effectiveness of BCTs on smoking cessation outcomes when offered to expectant and new fathers (child < 1 year) both through individual and/or couple-based interventions. Eight databases were searched for peer-reviewed articles. Studies were subjected to systematic retrieval and quality-assessment by two independent reviewers.We identified 9 randomised control trial studies that fulfilled the inclusion criteria. In terms of quit outcome data, 8 studies reported biochemically verified quit rates for men. While 5 BCT interventions targeted expectant/new fathers, 3 were directed to couples and 1 primarily focused on women with a component directed at men. Though most of the interventions were found to be effective, they showed small significant positive effects on cessation outcomes. Findings are suggestive of gender specific interventions being more likely to have positive outcomes. High heterogeneity across the studies made it difficult to determine the most effective BCT approach.This review suggests that use of BCT interventions for smoking cessation among expectant and new fathers is effective in achieving positive quit rates; however, these studies are limited. Further research is needed to determine the most effective BCT approach associated with smoking cessation among this group.\u2022\u2002BCT interventions for smoking cessation among expectant and new fathers are a promising approach to increase quit rates.\u2022\u2002Future research needs to develop evidence based BCT interventions for smoking cessation specifically targeting expectant and new fathers to inform policy and practice."} +{"text": "Smoking is a major contributor to socioeconomic inequalities for a wide range of health conditions. A link between smoking and dementia has been uncertain but prior evidence indicates socioeconomic inequalities in both smoking and dementia, so Raggi et al.,Inequalities in smoking can arise from a combination of inequalities in smoking uptake during youth and young adulthood and inequalities in smoking cessation among adults.,When implementing policies or interventions aimed at reducing smoking rates, a more consistent focus on socioeconomic inequalities would also be beneficial. For example, a recent adult-focused review of population-level measures found almost half of the 68 included studies had not been designed to assess intervention impact by socioeconomic position, and many had sub-optimal methodological characteristics for assessing this.Other open questions also remain. While this paper presents strong evidence that inequalities in smoking by occupational grade contribute to occupational grade inequalities in dementia,MJG wrote the manuscript.MJG received funding from the UK Medical Research Council (MC_UU_00022/2) and Scotland's Chief Scientist Office (SPHSU17). Funders had no role in preparation or submission of the manuscript."} +{"text": "The death of a family member may trigger exacerbations among individuals with serious mental illness (SMI). We hypothesized that bereavement would be associated with SMI exacerbations among bereaved partners and adult children diagnosed with SMI. Using linked population-based registries in Denmark, we identified partners and adult children diagnosed with schizophrenia, schizoaffective disorder, bipolar disorder, and major depression in the five years preceding the family member\u2019s death. Generalized estimating equations were used to estimate the odds of SMI exacerbation two years after decedent death. Partners had increased odds of SMI exacerbation at 3 months into bereavement compared to 9-12 months prior to partners\u2019 death . Children with a history of SMI had lower odds of SMI exacerbation in the second year of bereavement. Sociodemographic characteristics and co-occurring alcohol and substance abuse disorders were associated with higher odds of SMI exacerbations. These findings have implications for targeted bereavement support."} +{"text": "Little is known about the conditions that foster greater positive affect in the daily lives of spousal dementia care dyads in the early stages of dementia. This study aimed to examine the extent to which multiple indicators of health, including activities of daily living needs, quality of life, and the person with dementia\u2019s behavioral symptoms were associated with each partner\u2019s positive affect in daily life. Using secondary baseline data from a randomized controlled trial testing a stress reduction intervention in 63 couples (N=126), we examined whether individuals\u2019 multiple health indicators were associated with their own positive affect (actor effects) and their partner\u2019s positive affect (partner effects). Actor partner interdependence model results showed that for both persons with dementia and spouses, actor quality of life was the greatest predictor of positive affect, controlling for all other actor and partner health indicators =3.36, p=.001)."} +{"text": "Translation of messenger RNAs into proteins by the ribosome is a fundamental step in gene expression. In bacteria, it is possible to accurately predict the rate of translation initiation from the sequence surrounding a gene\u2019s start codon using thermodynamic models of RNA folding and ribosome binding. These predictions have applications in a range of fields, from systems biology studies that aim to understand and model bacterial physiology to synthetic biology studies that seek to reprogram bacterial cells. For example, metabolic engineers can design ribosome binding site (RBS) sequences to tune the expression of different enzymes in a pathway and thereby optimize the production of a desired chemical compound by cells. OSTIR (Open Source Translation Initiation Rates) is a Python package and command line tool for predicting translation initiation rates in bacteria. Several software programs exist for predicting translation initiation rates in bacteria , but nonEscherichia coli; (4) OSTIR supports multi-FASTA and CSV input files for batch processing.OSTIR is open source software (GPLv3) derived from the RBS Calculator v1.0 codebase . OSTIR wUpdating OSTIR to be compatible with ViennaRNA and newer RNA folding energy parameters required refitting coefficients in the underlying thermodynamic model . After m"} +{"text": "Cultural reproduction theory posits that cultural resources transmit to the next generation, suggesting a lingering effect of parental influences on cultural experiences in adulthood. Further, middle-aged adults\u2019 cultural engagement may not only be influenced by their own childhood experiences but also their spouses\u2019 experiences. This study extends our understanding of childhood and midlife cultural engagement of married couples, using a sample of 1,271 couples (age 49\u201366) from the 2012 Korean Baby Boomer Panel Study and Korean Forgotten Generation Study. Results from Actor Partner Interdependence Models showed that beyond one\u2019s own childhood cultural engagement, spouse\u2019s childhood cultural engagement was associated with levels of perceived cultural engagement at midlife (for both husbands and wives) and number of arts and cultural activities at midlife (only for husbands). Given the cross-spousal associations in cultural engagement among Korean middle-aged couples, both spouses\u2019 cultural resources need to be considered for policy implications."} +{"text": "The authors have suggested that giant cell arteritis could be diagnosed on clinical grounds rather than relying on temporal artery biopsy (TAB). While we agree with the authors' conclusions, it also needs a pointer that around 25% of patients of giant cell arteritis (GCA) can present with visual loss alone due to arteritic anterior ischemic optic neuropathy (AAION). Patients who do not comply for proper visual acuity assessment due to old age or an underlying neurocognitive disorder may lead to a delay in diagnosis of AAION. Classical presenting features [Figure 1A\u20131F] like chalky white optic disc edema, jaw claudication, and scalp tenderness may not always exist. AAION is a rare disease in the Asian population hence a strong suspicion of its presence is required for averting the potentially blinding sequel. In this era of resorting to noninvasive modalities of establishing diagnosis, there is a need to explore such markers in elderly population where GCA-causing AAION is common. Standard fluorescein angiography available in most clinical settings shows choroidal ischemia with delayed filling of the optic disc [Figure 1B]. These changes in optic nerve head may possibly be visualized using a noninvasive technique like optical coherence tomography (OCT) that may show thinning of the retinal nerve fiber layer. OCT-angiography may reveal reductions in vessel density and vessel tortuosity in AAION with worse values than non-arteritic anterior ischemic optic neuropathy (NAION). OCT angiography availability everywhere is an issue yet it currently stands as a noninvasive marker in nearly all retinal, optic nerve head-related disorders in the current times. Further, every patient on presentation undergoes hematological and biochemical testing for systemic evaluation. In a study by Inanc et al, neutrophil lymphocyte ratio (NLR) seemed as a reliable index to differentiate between NAION and AAION. NLR value was found significantly higher in AAION patients in their study. This was also observed in an 80-year-old female [Figure 1A\u20131E] who presented with classical features of AAION due to GCA and had NLR ratio of 3.84 , especially those in aorta, correlated with poor treatment outcomes in GCA and early treatment intensity could be determined by their presence or absence. Careful analysis of magnetic resonance angiography revealed attenuated caliber of internal, external, and common carotid artery apart from both temporal arteries [Figure 1F]. These LVLs seem a reliable index for diagnosing GCA. Although TAB remains a part of diagnostic work-up in most settings, the procedure is inconvenient to elderly patients and has potentially high false negative rate in its ability to diagnose GCA. In addition, ability to detect temporal artery on ultrasound and to secure a TAB is not possible in all centers, especially in rural areas.It seems logical to investigate for noninvasive markers for diagnoses of GCA in the elderly based on anecdotal reports in literature.This is with reference to the recently published article by Aghdam et al.Nil.There are no conflicts of interest."} +{"text": "For students in their final year, it is an opportunity to prepare for clinical practice by exploring different clinical environments and expanding their understanding of medicine in a global context. With the COVID\u201019 pandemic causing strict border closures, students were prevented from travelling internationally and interstate, thus reducing medical elective opportunities and making it difficult to fulfil the traditional curriculum requirements.2Three Australian final year medical students pioneered a 2\u2010month medical elective in the field of virtual rural medicine. Students joined the teams at vCare and the Virtual Rural Generalist Services (VRGS) which support communities in western New South Wales, Australia, the largest health district in the state.3This novel placement was an opportunity for students to experience critical care and emergency medicine whilst obtaining a new skill set in virtual care. Direct supervision from experienced health professionals allowed students to learn critical thinking and clinical reasoning by observing senior clinicians evaluate complex scenarios and make real\u2010time decisions. Overcoming challenges due to virtual consulting as well as treating deteriorating patients in the setting of resource limitations (i.e. no on\u2010site imaging) and coordinating retrieval logistics, was a unique educational experience. Many skills learnt will be transferable throughout a medical career such as increased familiarity with virtual health care technology and processes and would have been difficult to learn under certain constraints of traditional medical training. Students acknowledge reduced opportunity to practice procedural skills and the need to adopt virtual examination skills. However, as virtual medicine becomes fundamental for clinical practice, preparing medical students for virtual practice holds increasing importance. Medical students around the world can consider virtual electives as rich learning environments which have the potential to help them develop global perspectives in preparation for medical practice."} +{"text": "Healthcare providers underestimate the willingness of adults to engage in a healthier lifestyle to potentially slow the progress of the disease and the willingness of patients to participate in research. Few adults recognize the impact lifestyle modifications have on the risk for cognitive decline and dementia, but some significant differences exists among perceptions amongst diverse communities. While most adults are willing to modify selected brain-healthy behaviors, relatively few currently engage in brain-healthy behaviors all or most of the time. Numerous discrepancies exist between the realities of dementia and overall feelings about a diagnosis. Among the more startling findings is 48% of adults believe they will likely have dementia \u2014 far more than will actually develop it. Health care providers substantially overestimate the worry that adults age 40 and older would feel if they had dementia. While one in five adults (19%) said they would feel ashamed or embarrassed if they had dementia, a staggering seven in 10 providers (69%) said their patients would feel ashamed or embarrassed. These negative perceptions by healthcare providers carry over into the interactions they have with patients when dealing with cognitive function. Nine in 10 adults age 40 and older (91%) want to be told of a dementia diagnosis, but only 78% of providers said they always tell patients the truth. There is a recognition by everyone that early diagnosis is beneficial, but most adults over 40 are not aware there are treatments available for dementia. More than half of adults do not know that dementia cannot be cured."} +{"text": "Disadvantageous socioeconomic circumstances and minor mental health problems have both been associated with mental disorders, such as depression, but their joint contribution remains unknown.The Helsinki Health Study baseline survey (2000\u201302) of 40- to 60-year-old employees was linked with antidepressant medication data from registers of the Social Insurance Institution of Finland. The analyses were made using logistic regression with first prescribed antidepressant medication purchase during a 10-year follow-up as the outcome. Minor mental health problems were measured by the emotional well-being scale of the RAND-36. Odds ratios were calculated for joint association of the lowest quartile of the emotional well-being scale of the RAND-36 and socioeconomic circumstances. Childhood , conventional and material (housing tenure and current economic difficulties) socioeconomic circumstances were examined. This study included 5450 participants.Minor mental health problems dominated the joint associations. Minor mental health problems were associated with antidepressant medication irrespective of socioeconomic circumstances whereas only low income, current economic difficulties and living in rented housing showed an association without minor mental health problems at baseline. Marital status, working conditions and BMI and health behaviours had only minimal contributions to the associations.Minor mental health problems were consistently and strongly associated with antidepressant medication and dominated the joint associations with socioeconomic circumstances. Paying attention to minor mental health problems might help prevent mental disorders such as depression. A meta-analysis concluded that disadvantageous socioeconomic circumstances were associated with the onset of depression and increased the risk of persistent depression.Both disadvantageous socioeconomic circumstances,,,,Conventional socioeconomic measures namely education, occupational class and income have been associated with antidepressant medicationWe aimed to examine the joint associations of past and present socioeconomic circumstances and minor mental health problems with antidepressant medication among midlife and ageing employees. We used a multiple framework of socioeconomic circumstances by including childhood socioeconomic circumstances namely parental education and childhood economic difficulties, conventional measures of socioeconomic circumstances namely education, occupational class and income and material circumstances namely housing tenure and current economic difficulties.This study is part of the Helsinki Health Study (HHS) among the employees of the City of Helsinki,n\u2009=\u20096603, 74% of the participants) who consented to the linkage. Purchases of prescribed antidepressant medication were followed from the day of returning the baseline questionnaire until 10 years, until the date of antidepressant medication purchase or until death (n\u2009=\u200984). Participants with antidepressant medication purchases during the 3 years preceding the baseline survey were excluded (n\u2009=\u2009751). After exclusions due to missing data on minor mental health problems or covariates (n\u2009=\u2009373) the study included 5450 employees of whom 4211 were women and 1239 men. There was item-non-response on measures of socioeconomic circumstances and the final numbers in individual analyses and \u2018high\u2019 . Childhood economic difficulties were measured by asking whether there were economic difficulties in the family before the respondent turned 16 (yes/no). Respondent\u2019s own education was divided into \u2018low\u2019 and \u2018high\u2019 . Occupational class was based on the job title and divided into \u2018low\u2019 and \u2018high\u2019 . Monthly household income was dichotomized by the median into \u2018low\u2019 and \u2018high\u2019. Housing tenure was divided into owner-occupiers and renters. Current economic difficulties were measured by asking (i) how often the respondent had enough money to buy clothing and food needed by the family, and (ii) how much the respondent had difficulties in paying bills. A combined variable was formed: \u2018No difficulties\u2019 and \u2018difficulties\u2019 the latter including both occasional and frequent difficulties.Minor mental health problems were measured by the emotional well-being scale of the RAND-36.The outcome measure of the study was the first purchase of prescribed, reimbursed antidepressant medication. Medication purchases were classified according to the Anatomical Therapeutic Chemical system by WHO.2. Problem drinking was measured by the CAGE-scale .Age and gender were included as covariates. Marital status was divided into \u2018single\u2019, \u2018married or cohabiting\u2019 and \u2018divorced or widowed\u2019. There was a single-item question inquiring how mentally strenuous the respondent considered the work with response alternatives ranging from \u2018very light\u2019 to \u2018very heavy\u2019. A similar question inquired physical strenuousness of the work. The four response alternatives were reduced to three groups. Smoking was divided into smokers and non-smokers. Body mass index (BMI) was calculated from self-reported weight and height and divided into under 25, between 25 and 30 and above 30\u2009kg/mDifferences in the distributions of antidepressant medication purchases by socioeconomic variables and mental health problems were tested by the chi-squared test. The associations between socioeconomic circumstances, minor mental health problems and antidepressant medication were further analyzed by logistic regression analysis yielding odds ratios (OR) and their 95% confidence intervals (95% CIs). The first reimbursed antidepressant medication purchase during a 10-year follow-up served as outcome variable. The SAS statistical program version 9.4 was used in performing the analyses .P\u2009=\u20090.0251) was found for the association between education and antidepressant medication. This association was presented separately for men and women but otherwise the analyses were made on pooled data.First, we fitted models separately for the associations between socioeconomic circumstances and antidepressant medication and between minor mental health problems and antidepressant medication. Interactions for gender were tested and statistically significant interaction were calculated using the following equation: S\u00a0=\u00a0OR /[ + ]. A synergy index above 1 suggests that the joint association is synergistic, a synergy index =1 suggests an additive association and a synergy index below 1 an antagonistic association.To examine the synergistic interactions between socioeconomic circumstances and minor mental health problems the synergy indices whereas the majority (83%) had not experienced any childhood economic difficulties . High owThe age- and gender-adjusted associations of socioeconomic circumstances and minor mental health problems with antidepressant medication in S\u2009=\u20091.22).The joint associations of socioeconomic circumstances and minor mental health problems with antidepressant medication are presented in The joint associations of socioeconomic circumstances and minor mental health problems with antidepressant medication after adjusting for all socioeconomic circumstances simultaneously are in This study sought to examine the joint associations of socioeconomic circumstances and minor mental health problems with antidepressant medication. The significance of socioeconomic circumstances was small and minor mental health problems dominated the joint associations. Thus, employees with minor mental health problems showed strong associations with antidepressant medication largely irrespective of socioeconomic circumstances. Of the socioeconomic circumstances, material resources and current economic difficulties had the greatest contribution as they were associated with antidepressant medication even without minor mental health problems at baseline.Our study underlines the importance of minor mental health problems to antidepressant medication irrespective of socioeconomic circumstances. Our results expand those of previous research as the outcome, antidepressant medication, portrays a medical diagnosis and seeking treatment. The results confirm previous findings showing that individuals with a risk of clinical depression might be recognizable by screening instrumentsThe synergy indices concerning the joint associations of minor mental health problems and household income and housing tenure were slightly synergistic suggesting that advantageous material socioeconomic circumstances might protect from minor mental health problems developing into more severe depression whereas the double burden of minor mental health problems and disadvantageous socioeconomic circumstances increases the risk. The finding should, however, be interpreted with caution as the confidence intervals partly overlapped. A previous study suggested that people in advantageous socioeconomic circumstances might be treated more often than people in lower socioeconomic circumstances despite similar needs for treatmentAmong the socioeconomic circumstances our results emphasize the importance of material resources as household income and living in rented housing were associated with antidepressant medication even without minor mental health problems at baseline. Being better off financially might enable different alternatives both at work and in leisure time and provide freedom from financial restrictions. Also current economic difficulties were associated with antidepressant medication independent of minor mental health problems at baseline. These results confirm those of a previous study using the HHS data that examined the associations between multiple socioeconomic circumstances and antidepressant medication with a 5-year follow-up time.,To shed light on the mechanisms behind the associations, the contribution of various covariates was examined. Interpersonal relationships have been found to prevent depressive symptoms.The strengths of the study include a relatively large dataset, a prospective study design and an opportunity to use a broad multi-domain approach to socioeconomic circumstances. The measure of depression used in the study, namely antidepressant medication was not optimal since it also measures treatment and not just incidence. On the one hand, it is a reliable register-based measure based on medical assessment without self-report bias and non-response. On the other hand, it is unlikely to detect all employees with diagnosed depression as part of the episodes of depression is treated by psychologies and psychotherapists. However, severe cases of depression are likely to be included as the current guidelines recommend medical therapy in such cases. Antidepressant medication is also used for other purposes, such as against chronic pain and anxiety disorders, but the most common indication is depression. We were unable to include a measure of depression not based on registers but a previous study found associations between socioeconomic circumstances and mental health using both self-reported measures of depression and antidepressant medication.Of the original 8960 study participants 5450 were included in the study. The main exclusion was due to not consent to register linkage. According to the non-response analysis there were only small differences in consenting to the data linkages although men were more eager to consent. Another major reason for exclusion was due to omitting employees having purchased antidepressant medication 3 years preceding the baseline. Due to the exclusions employees in poorer physical and mental health might have selected out and the associations between minor mental health problems and antidepressant medication might be underestimates. The target population included only individuals employed at baseline and the most disadvantaged people suffering from mental health problems could not be covered. The associations might thus be stronger within the general population. The baseline data were collected in 2000\u201302 and studies with more recent data are needed to confirm whether the findings hold still in the present day.Our findings stress the importance of minor mental health problems to antidepressant medication irrespective of socioeconomic circumstances. We also noted the importance of material resources and current economic difficulties as they were associated with antidepressant medication even without minor mental health problems at baseline. In conclusion, preventing minor mental health problems and less so, also improving socioeconomic circumstances might have protective influences against more severe depression.EURPUB online.A.S. was supported by the Emil Aaltonen Foundation. E.M. was supported by The Finnish Work Environment Fund (grant number 190256). O.R. was supported by the Juho Vainio Foundation.Conflicts of interest: None declared.Key pointsMinor mental health problems dominated the joint association of past and present socioeconomic circumstances and minor mental health problems with antidepressant medication among midlife municipal employees.Of the socioeconomic circumstances, the study stressed the role of material resources and current economic difficulties as they were associated with antidepressant medication even without minor mental health problems at baseline.Preventing minor mental health problems and less so, also improving socioeconomic circumstances might have protective influences against more severe depression.ckac048_Supplementary_DataClick here for additional data file."} +{"text": "Lay and professional people may use terms for mental health and mental health problems differently, causing difficulties in adequately addressing associated needs. Despite the public health issue of increased mental health problems among adolescents, there is limited research on perceptions of mental health concepts among young people. This study aimed to explore conceptual views of mental health and mental health problems among adolescents.During October and November 2020, a total of 32 adolescents (15-18 years old) living on Sweden's largest island Gotland were interviewed in focus groups or individual interviews. The interviews were semi-structured and audio recorded. Data were analysed thematically according to Systematic Text Condensation.Three themes emerged from the analysis: Mental health is about how we feel; One's mental health depends on one's situation, thoughts and ways of coping; and Mental health problems should be taken seriously and can get severe. The adolescents described mental health as an overarching concept encompassing both positive mental health and mental health problems. Mental health problems were perceived as something other than normal challenges in life, however ranging from minor problems to severe illness. Good mental health was understood as a condition with absence of mental health problems and presence of symptoms of positive mental health.The adolescents\u2019 had a complex and holistic understanding of mental health concepts, consistent with definitions used by the World Health Organization and Swedish authorities. They suggested both positive mental health and mental health problems to be considered when assessing and discussing their mental health. Further, the results highlight the need of support for young people on how to cope with difficulties in life and support for those suffering from minor mental health problems.\u2022\u2002The adolescents\u2019 understanding of mental health and mental health problems were highly consistent with current accepted definitions of the concepts.\u2022\u2002According to the participants, both positive mental health and mental health problems should be considered simultaneously to understand and address adolescents\u2019 mental health."} +{"text": "Myelin is essential to nervous system function, playing roles in saltatory conduction and trophic support. Oligodendrocytes (OLs) and Schwann cells (SCs) form myelin in the central and peripheral nervous systems respectively and follow different developmental paths. OLs are neural stem\u2010cell derived and follow an intrinsic developmental program resulting in a largely irreversible differentiation state. During embryonic development, OL precursor cells (OPCs) are produced in distinct waves originating from different locations in the central nervous system, with a subset developing into myelinating OLs. OPCs remain evenly distributed throughout life, providing a population of responsive, multifunctional cells with the capacity to remyelinate after injury. SCs derive from the neural crest, are highly dependent on extrinsic signals, and have plastic differentiation states. SC precursors (SCPs) are produced in early embryonic nerve structures and differentiate into multipotent immature SCs (iSCs), which initiate radial sorting and differentiate into myelinating and non\u2010myelinating SCs. Differentiated SCs retain the capacity to radically change phenotypes in response to external signals, including becoming repair SCs, which drive peripheral regeneration. While several transcription factors and myelin components are common between OLs and SCs, their differentiation mechanisms are highly distinct, owing to their unique lineages and their respective environments. In addition, both OLs and SCs respond to neuronal activity and regulate nervous system output in reciprocal manners, possibly through different pathways. Here, we outline their basic developmental programs, mechanisms regulating their differentiation, and recent advances in the field. Oligodendrocytes (OLs) and Schwann cells (SCs) share some characteristics including transcription factors, myelin components, and responsiveness to neuronal activityOLs and SCs use unique mechanisms to form myelin and possess differential capacities to respond to external factorsThe lineage and environment of OLs and SCs may account for differences in their developmental plasticity and regenerative capacity. Myelin is an essential component of the nervous system. Myelin has been canonically known to facilitate saltatory conduction in axonal fibers and speed up electrical conduction When and where do OLs and SCs originate from? (2) What are the key pathways that regulate CNS and PNS myelination? and (3) What are recent advances in our understanding of CNS and PNS myelinating glia? Attempting to answer these questions is crucial to uncovering differences in their regenerative capacity and finding novel therapeutic strategies that can stimulate myelination in each or both systems.1Most OLs are originated through the following basic differentiation program: 1) after neurogenesis, neural stem cell\u2010derived radial glia become specified as OL precursor cells (OPCs); (2) OPCs proliferate and migrate across the CNS at specific time points known as \u2018waves\u2019; (3) a subpopulation of OPCs undergo partial differentiation into pre\u2010myelinating OLs while many remain OPCs until adulthood; and (4) pre\u2010myelinating OLs form compact myelin sheaths and become mature myelinating OLs. Each of these stages is defined by a growing set of cellular markers, as shown in Figure\u00a0 after neOPC specification and production occurs at different time points and locations across the CNS. In the mouse spinal cord, the first wave of OPCs originate from the ventricular germinal zones at E12.5. These OPCs are derived from the ventral pMN progenitor domain that also generates motor neurons originate from neural crest cells (NCCs) and have unique and flexible developmental pathways that allow for distinct, reversible differentiation outcomes. The basic developmental program for axon associated SCs is as follows: (1) NCCs specify into Schwann cell precursors (SCPs); 2) SCPs develop into immature SCs (iSCs), which can then differentiate into at least four potential cellular fates; (3) a subset of iSCs differentiate into myelinating SCs which wrap the axons, and another subset differentiates into non\u2010myelinating SCs, including but not limited to Remak SCs and perisynaptic SCs myelination in response to neuronal activity; (2) experience\u2010dependent myelin plasticity; and (3) myelinating glia modulating neuronal activity (see Figure\u00a05.1Activity\u2010dependent mechanisms showing communication between axons and myelinating cells were first demonstrated through co\u2010culture experiments using mouse DRG neurons and OL\u2010 and SC\u2010lineage cells. Action potentials in mouse DRG neurons induced adenosine receptor activation in OPCs, subsequently inhibiting proliferation and promoting myelination by activating synthesis of myelin proteins (Stevens et al.,\u00a0Studies using neuronal stimulation in mice clearly showed that adaptive myelination also occurs the mammalian brain. Optogenetic activation of neurons in the premotor cortex led to thicker myelin in associated white matter tracts (Gibson et al.,\u00a02+ waves in SCs in vitro (Stevens et al.,\u00a02+ to promote myelination in early postnatal rat nerves (Ino et al.,\u00a0+, K+, and Cl\u2212 signaling (Heredia et al.,\u00a0In the PNS, the differentiation state of SCs is known to be highly plastic and responsive to external cues, as evidenced by the robust injury response in peripheral nerves. As such, it remains difficult to disentangle SC differentiation from the variety of external cues including immune cells and the basal lamina. Early work using cultured mouse DRGs and SCs showed that electrical stimulation and ATP release from neurons initiated intracellular Ca5.2Experiential paradigms better recapitulate the complex, multicellular response to external inputs to the nervous system. Studies by Makinodan et al. and Liu Mechanistically, NRG1 and its receptor ErbB3 have been implicated in the OL response to social isolation in adolescent mice (Makinodan et al.,\u00a0While most research on experience\u2010dependent myelination in the PNS has been confined to resistance and aerobic exercise paradigms, recent evidence has shown parallel results as studies in the CNS, where increasing external stimuli tend to promote myelination. In rats, load\u2010carrying tasks and ladder\u2010based resistance training increased myelin sheath thickness in the tibial nerve (Krause Neto et al.,\u00a05.3Recent progress in the field has clearly demonstrated a reciprocal relationship between neurons and OLs. Seminal studies clearly showed that oligodendrogenesis and differentiation are necessary for higher behavioral functions in mice, including social interaction, learning and memory, as well as motor learning tasks (Makinodan et al.,\u00a0A receptor in mice led to a loss of post\u2010synaptic currents in OPCs and changes in myelin structure and OL morphology; however, the downstream mechanisms mediating these effects remain unknown. Studies in zebrafish have identified the role of OPCs in the formation and tuning of visual circuits in development (Xiao et al.,\u00a0The interplay between neurons and OLs and mechanisms by which OLs modulate neuronal function are only beginning to be explored. Myelination may regulate neuronal activity through modulation of conduction velocity (Kato et al.,\u00a0SCs are known to play active roles in modulating neuronal function during PNS development due to their multipotency. Myelinating SCs in mice have been found to regulate conduction speed and through restricting axonal diameter (Eichel et al.,\u00a06On the surface, OLs and SCs seem to play similar roles as the sole producers of myelin in the nervous system. However, they comprise two highly distinct cell types that undergo unique developmental programs and utilize different strategies to maintain plasticity in their respective systems. The CNS maintains OL lineage cells at multiple stages of differentiation to serve different purposes: mature OLs to provide myelin, and OPCs to serve as a highly responsive pool of stem\u2010like cells that play a variety of roles. On the other hand, the PNS utilizes the plasticity of the neural crest\u2010derived SCs to produce both myelinating and non\u2010myelinating cell fates. Fully differentiated SCs depend on environmental cues to maintain their phenotypes and can radically change their phenotypes in response to injury.Owing to their cell lineage and unique environments, the molecular machinery used over the course of OL and SC development diverge greatly. However, key aspects of their differentiation programs are similar, including the production of many common myelin components as well as mechanisms that allow them to respond to neuronal activity. Understanding both the shared and distinct characteristics of each cell lineage will be essential to identifying appropriate therapeutic strategies for both the CNS and PNS."} +{"text": "Smoking, physical inactivity, low fruit and vegetable consumption, and obesity are common in Kosovo. The Accessible Quality Healthcare project is implementing a primary healthcare intervention that entails nurse-guided motivational counselling to facilitate lifestyle behaviour change. This study assesses the uptake of counselling and the impact on health behaviours and participants\u2019 stages of health behaviour change as well as describes experiences and perceived benefits of motivational counselling.Study participants (n = 907) were recruited from 12 municipalities participating in the Kosovo Non-Communicable Disease Cohort study. For the quantitative study, data on lifestyle behaviours, use of counselling, and stages for behavioural change were used. For the qualitative study, in-depth interviews were conducted with 26 cohort participants who had undergone motivational counselling.Motivational counselling was received by only 22% of the eligible participants in the intervention municipalities. Unhealthy behaviours remain high even in persons who underwent counselling . According to the qualitative study results, the participants that received counselling were very satisfied with the services but requested additional services such as group physical activity sessions and specialized services for smoking cessation.More tailored and additional primary healthcare approaches in accordance with patients\u2019 views need to be considered for the motivational counselling intervention to reach patients and efficiently facilitate lifestyle behaviour change.\u2022\u2002The following tailored approaches are suggested: a) strengthened referral mechanism b) specialized services for smoking cessation; c) delivery of group physical activity sessions.\u2022\u2002More tailored and additional primary health care approaches in accordance with patients\u2019 views need to be considered for the motivational counselling intervention."} +{"text": "This qualitative study describes results from a semistructured interview investigating technical capabilities and barriers related to pediatric data sharing and confidentiality that was administered to health care organizations participating in the California Pediatric Informatics Collaborative. This research letter describes the formation and activities of this work group and provides results from a semistructured interview investigating final decisions of participating health care organizations, their technical capabilities, and barriers to implementation.SRQR) reporting guideline for qualitative studies.This qualitative study conducted semistructured interviews via video teleconference between April 7 and September 30, 2021. The study was approved by Stanford University with a waiver of informed consent because it was considered non\u2013human participant research. This study followed the Standards for Reporting Qualitative Research conducted the interviews and performed the data analysis. Questions were divided into general topics regarding portal configuration (including configurations for children aged <12 years and adolescents aged 12-17 years), ability to technically parse data elements to block access, and implementation challenges (eMethods in the Our study had an 81% response rate (13 of 16 organizations). Final participants included 5 stand-alone children\u2019s hospitals, 3 large integrated delivery networks, 3 safety-net health care systems, 1 community health care system, and 1 federally qualified community health center. All 13 participants agreed about which information should be electronically released to pediatric patients younger than 12 years and their proxies in an ideal state of data sharing. The implemented portal release patterns were compared for adolescents and their proxies . Within The creation of the California Pediatric Informatics Collaborative promoted early collaboration among important stakeholders across the state with regard to deciphering the 21st Century Cures Act final rule in the context of California state laws on adolescent consent. Participating organizations praised the sharing of both the general approach to data sharing and specifics about the technical capabilities of the data sharing portal. Our interviews suggested that participating health care institutions in California agreed about which information should be released to patients younger than 12 years, adolescent patients, and patients\u2019 proxies. However, given difficulties with data segmentation and technical infeasibility, no institution, regardless of organizational type, has yet to successfully implement its entire ideal state of data sharing.Study limitations include a predominance of Epic Systems electronic health records and difficulties in ascertaining granular details of confidential data sharing. Continued collaboration is necessary to identify a unified approach to health information sharing that will empower patients while protecting adolescent confidentiality. The creation of novel technical capabilities by electronic health record vendors to allow granular segmentation of data release will be important in this process. Future research is warranted to address the technical limitations of pediatric information sharing."} +{"text": "MRI and molecular biomarkers have been proposed to identify predictors of neurodegeneration and risk factors for disease progression. Early signs of axonal dysfunction have come to light including impaired mitochondrial trafficking, structural axonal changes, and synaptic alterations. With sustained inflammation as well as impaired remyelination, axons succumb to degeneration contributing to CNS atrophy and worsening of disease. These studies highlight the role of chronic demyelination in the CNS in perpetuating axonal loss, and the difficulty in promoting remyelination and repair amidst persistent inflammatory insult. Regenerative and neuroprotective strategies are essential to overcome this barrier, with early intervention being critical to rescue axonal integrity and function. The clinical and basic research studies discussed in this review have set the stage for identifying key propagators of neurodegeneration in MS, leading the way for neuroprotective therapeutic development.Axonal loss in multiple sclerosis (MS) is a key component of disease progression and permanent neurologic disability. MS is a heterogeneous demyelinating and neurodegenerative disease of the central nervous system (CNS) with varying presentation, disease courses, and prognosis. Immunomodulatory therapies reduce the frequency and severity of inflammatory demyelinating events that are a hallmark of MS, but there is minimal therapy to treat progressive disease and there is no cure. Data from patients with MS, Immune System Diseases > Molecular and Cellular PhysiologyNeurological Diseases > Molecular and Cellular PhysiologyThis article is categorized under: BioRender.com).Chronic demyelination results in impaired axon metabolism and function, ultimately leading to progressive neurological decline. Neuroprotective strategies aim to protect axons from inflammatory\u2010mediated destruction, thereby improving axonal integrity and clinical disability (Image created in MS is a leading cause of disability in young adults and is estimated to affect 2.8 million people worldwide perivascular demyelination, (2) subependymal/pial demyelination, and (3) neuroaxonal degeneration , primary progressive (PPMS), and secondary progressive (SPMS) MS. Within each clinical course, patients may experience variable disease activity, which is evidenced by new or increasing neurologic dysfunction or the appearance of new/enlarging T2\u2010weighted gadolinium enhancing lesions on magnetic resonance imaging (MRI), indicative of disease activity has been proposed to discriminate MS lesions from MRI mimics such as microvascular disease and migraine by identifying a blood vessel in the center of a T2 hyperintense lesion in the cerebrospinal fluid (CSF) are included in the most recent revision of the McDonald criteria to aid in diagnosis of MS by detecting B cell activation , and T1\u2010weighted with or without gadolinium enhancement have been used both to diagnose MS and to monitor evidence of disease activity imaging of the retina measures the structural integrity and thicknesses of its distinct cellular layers are elevated in progressive MS patients and associated with disease duration and clinical disability secondary neuroaxonal loss as a sequela to prior disease activity; (2) disease activity affecting the regions infrequently imaged ; and (3) limited imaging resolution or obscuration by artifact. Neurodegenerative changes can be visualized by cortical thinning, whole and regional brain atrophy, and spinal cord atrophy . As mentioned, thalamic atrophy measured by MRI and neuronal loss in Despite these advances in predicting disease progression, the underlying mechanisms driving neurodegeneration must be elucidated in order to develop neuroprotective strategies. Animal models of demyelinating disease have provided useful insight into the relationship between demyelination, axonal injury, and neurodegeneration and how to promote remyelination and axonal repair.3Myelinated axons require proper support from oligodendrocytes in order to cluster sodium channels for saltatory conduction which increases signal transmission speed , which is an adaptive immune\u2010mediated model whereby CNS\u2013infiltrating myelin\u2013reactive T cells initiate autoimmune demyelination and axon loss proliferate in response to inflammation and are found in MS lesions ; writing \u2013 original draft (supporting). Kedar Mahajan: Conceptualization (supporting); writing \u2013 original draft (supporting); writing \u2013 review and editing (supporting). Tara DeSilva: Conceptualization (lead); funding acquisition (lead); resources (lead); supervision (lead); validation (lead); writing \u2013 review and editing (lead). The authors declare no competing interests."} +{"text": "Pandemic are known to generate traumatic events, such as job losses or violence [1]. Several studies have shown that epidemics and related health measures lead to an increase of acute stress disorders (ASD), post-traumatic stress disorders (PTSD), anxiety and depression in the adult population [2]. In the pediatric population, few studies have been carried out on the psychiatric outcomes during and after epidemics and associated measures [3].The aim of this study was to explore ASD symptoms during stay-at-home and Covid 19 pandemic and its impact on children and adolescent mental health.Sixty participants were included in this longitudinal study [4]. The measures consist in an emergency semi-directed interview designed to assess symptoms of ASD according to the age of children.Patients\u2019 age modulated psychiatric outcomes. Children under the age of six shown more developmental regressions and more restlessness than older ones. Children from 6 to 12 years were characterized by more oppositional behaviors than adolescents. Finally, adolescents were characterized by more social isolation than younger ones. Other symptoms appear to be more stable across ages: sleep disturbance, fear behavior and somatization.Young children experienced more externalized symptoms (opposition and agitation) and developmental regressions than older children [5]. Thus, it appears necessary during pandemic to take into account the psychiatric consequences of confinement to reduce psychosocial long-term outcomes in particular in younger patients who appeared to develop specific and age-related psychiatric disorders."} +{"text": "Autistic Spectrum Condition is often characterized by the presence of deficits in social interaction. An abnormal attentional processing may explain these difficulties, as it has been suggested that individuals with autism spectrum conditions may have problems with orienting attention to socially relevant stimuli and/or inhibiting their attentional responses to irrelevant ones.The aim of the current study is to shed light on this issue by the assessment of the attentional orienting and inhibitory control to emotional stimuli .An antisaccade task (with both prosaccade and antisaccade blocks) was applied to a final sample of 29 children with autism spectrum conditions and 27 children with typical development.The main findings were: i) children with autism spectrum condition committed more antisaccade error when seeing angry faces than happy or neutral faces, while children with typical development committed more antisaccade errors when seeing happy faces than neutral faces, and ii) latencies in the prosaccade and antisaccade blocks were associated with the severity of autism symptoms.These results suggest that children with autism spectrum conditions show an impaired inhibitory control when angry faces are presented. This bias to negative high-arousal information is congruent with affective information-processing theories suggesting that threatening stimuli induce an overwhelming response in autism. From a clinical perspective, therapeutic strategies that focus on shifting attention to emotional stimuli may improve autism symptomatology and their socials functioning."} +{"text": "Antibody diversification and selection of B cells occur in dynamic structures called germinal centers (GCs). Passively administered soluble antibodies regulate the GC response by masking the antigen displayed on follicular dendritic cells (FDCs). This suggests that GCs might intercommunicate via naturally produced soluble antibodies, but the role of such GC\u2013GC interactions is unknown. In this study, we performed in silico simulations of interacting GCs and predicted that intense interactions by soluble antibodies limit the magnitude and lifetime of GC responses. With asynchronous GC onset, we observed a higher inhibition of late formed GCs compared to early ones. We also predicted that GC\u2013GC interactions can lead to a bias in the epitope recognition even in the presence of equally dominant epitopes due to differences in founder cell composition or initiation timing of GCs. We show that there exists an optimal range for GC\u2013GC interaction strength that facilitates the affinity maturation towards an incoming antigenic variant during an ongoing GC reaction. These findings suggest that GC\u2013GC interactions might be a contributing factor to the unexplained variability seen among individual GCs and a critical factor in the modulation of GC response to antigenic variants during viral infections. Antibody responses result in the generation of memory B cells and long-lived plasma cells from structures called germinal centers (GCs) in secondary lymphoid organs . GCs areSeveral mechanisms act to regulate the magnitude and efficiency of GC responses. Antigen amount has been suggested to impact antibody production , affinitRegulation of GCs by soluble antibodies is also extensively studied in the context of developing broadly neutralizing antibodies. Administering antibodies targeting immunodominant epitopes can suppress the GC response to these epitopes and shift the focus of GCs towards rare epitopes, thus opening up possibilities for therapeutic interventions . HoweverA simulation framework was developed was also considered, where the soluble antibodies were not allowed to mask the antigen on FDCs. In control simulations, both GC\u2013GC interactions via soluble antibodies and the feedback on individual GCs due to self-produced antibodies were ignored.The antibody concentration was multiplied with \u00d7 10\u221218 , 10\u221217 a\u22125 M bound to the soluble antibodies. The immune power (IP) ,43 was chttps://doi.org/10.5281/zenodo.6568368 (uploaded on 21 May 2022).Simulations were performed using C++ and resuFirstly, we investigated whether GC\u2013GC interactions inhibited only individual GC reactions or also the overall GC response. We simulated GCs that were initialized asynchronously with a sIn the network of GCs with asynchronous onset, individual GCs showed differences in sensitivity to antibody mediated inhibition and the sensitivity increased with the delay of GC initiation B,F,G. WhIn the absence of GC\u2013GC interactions, variability observed in the size of individual GCs at any given time point is primarily due to asynchronous GC onset B, but thAs the exact mechanisms contributing to asynchronous onset and founder cell composition of GCs is unknown, we tested whether the sensitivity of late GCs to antibody mediated inhibition was reduced if the late GCs were seeded by high affinity founder cells. Such high affinity founder cells could be memory cells produced from early GCs. We chose high affinity founder cells for GCs 7 and 8 that were initialized at late time points. The founder cell affinity of other GCs was randomly chosen resulting in relatively lower average affinity as in the previous simulations. Higher affinity founder cells partly counteracted the reduction in maximum size A, plasmaThis result was derived for a single epitope in a primary GC response. Although it is not known whether memory B cells produced from early GC reactions might participate in late GCs of a primary immune response, our results suggest that late GCs can overcome a strong antibody feedback when founded by high-affinity memory cells when compared to lower affinity na\u00efve B cells.Given the potential role of GC\u2013GC interactions in generating variability in the lifetime, kinetics and affinity maturation of individual GCs, we investigated whether GC\u2013GC interactions also lead to differences in affinity maturation to individual epitopes in the presence of multiple epitopes. We tested the affinity of plasma cells at the end of the GC response from individual GCs in the presence of two epitopes (shape space positions 3333 and 5555) in equal proportion with founder cell positions randomly chosen anywhere in the shape space. With no interaction between GCs, plasma cells from individual GCs had similar affinities to both the epitopes, suggesting that individual GCs support affinity maturation to diverse epitopes. GC\u2013GC interactions increased differences in plasma cell affinity between the two epitopes in individual GCs . Late foInjecting soluble antibodies against the immunodominant epitope has been shown to shift the focus of in silico GC reactions towards the sub-dominant or rare epitope . To inveWe tested whether the small increase in affinity maturation towards rare epitope enhanced the GC efficiency (IP), which is a combined measure of quality and quantity of plasma cells produced (see methods). The IP of the overall GC response towards the rare epitope was not enhanced by GC\u2013GC interactions (not shown). This result was robust against variation of the total amount of available antigen. Due to the higher sensitivity of late GCs to soluble antibodies, these GCs are short-lived, and their production of plasma cells is greatly diminished. Thus, a small increase in affinity of plasma cells was insufficient to enhance the efficiency of late GCs towards neutralizing the rare epitope.Previous sections showed that GC\u2013GC interactions induce differences in kinetics and epitope specificity of individual GCs. We have previously predicted that GCs induced in a primary immune response upon immunization with sheep red blood cells have highly heterogeneous lifetimes that could be explained by differences in founder cell composition and antigen availability among individual GCs . To testTaken together, our results suggest that depending on the antigen complexity, antibody-mediated GC\u2013GC interactions lead to differences in individual GC kinetics, quality and quantity of plasma cells in an initiation time dependent manner or a characteristic shift in affinity maturation towards specific epitopes.Antibody-mediated decrease in the longevity of GC responses and output production might act as a self-regulatory mechanism that is limiting the magnitude of GC responses. As this mechanism acts by limiting antigen accessibility on FDCs, we tested whether a persistent deposition of antigen on FDCs is able to overcome the effects of GC\u2013GC interactions on the overall GC response. Starting from the simulation with low GC\u2013GC interaction and synchronous GC onset as in , we simuWe hypothesized that GC\u2013GC interactions modulate the GC responses to an incoming antigenic variant during viral infections. We initialized GCs synchronously with a single epitope at shape space position 3333 and persistently added an antigenic variant (epitope 2) from day 7 of the GC reaction. Relatedness of epitope 2 to epitope 1 was varied by choosing shape space positions at a distance of 2, 4 or 8 mutations from epitope 1.Irrespective of the GC\u2013GC interaction strength and relatedness of epitope 2 to epitope 1, average affinity of plasma cells towards epitope 1 was not enhanced compared to simulations with no GC\u2013GC interactions at day 25 of the GC reaction . This suIncrease in the affinity of plasma cells towards epitope 2 was only transient, and over a long-term remained lower than the plasma cell affinity in simulations without GC\u2013GC interaction due to accelerated shutdown of GCs . These fModulation of GCs by soluble antibodies is an important strategy for developing broadly neutralizing antibodies ,30. HoweAt the level of individual GCs, GC\u2013GC interaction led to variability in lifetime, kinetics and affinity maturation of GC reactions due to differences in GC onset times. GC initiation dynamics impacted the variability between single GCs by modulating GC\u2013GC interactions. Given that the GC initiation dynamics might depend on experimental settings , we expeBehavior of late GCs was highly sensitive to the output of early GCs due to communication by soluble antibodies. It is important to note that the sensitivity of late GCs might vary depending on the plasma cell production dynamics of early GCs, which contributes to the early pool of soluble antibodies influencing other GCs. A temporal shift in the memory and plasma cell production during the GC response has been described , but theIn silico late GCs attained a larger size in the presence of memory B cells as founders when compared to na\u00efve B cells. It is unknown whether the memory B cells from early GCs participate in late GCs formed within the response to a primary immunization and needs future investigation. In the case of secondary GC responses with multiple epitopes, memory B cells recognizing the immunodominant epitope experience higher inhibition by antibody feedback compared to na\u00efve B cells specific to a different epitope . IgM+ meIn addition to the exchange of soluble antibodies and potential exchange of memory B cells, GCs might also communicate with each other by the exchange of Tfh cells . The rolPreviously, we have suggested that GC lifetimes and output could vary among individual GCs within the same lymphoid organ and that these differences cannot be solely explained by GC\u2013GC interactions . Our preGC\u2013GC interactions might be a major factor regulating antigen availability and limiting GC lifetime. Antibody feedback has been shown to limit booster responses to vaccination against malaria at sub-protective titers but promote the diversification of B cell responses to subdominant epitopes . Our simTesting the effects of GC\u2013GC interactions identified in this study by experimental modulation of the antibody secretion or stability would help verify the predictions. For example, persistent addition of antigenic variant with varying degree of similarity to the existing antigen would enable detection of changes in B cell affinity maturation due to GC\u2013GC interactions. As the effects predicted vary depending on conditions such as whether there is a persistent deposition of antigen, extent of synchronicity between GC initiation times and antigen complexity, experiments will have to be performed under different immunization conditions. The characteristic differences in the behavior of individual GCs predicted in this study might also facilitate the experimental analysis and enable testing the relevance of GC\u2013GC interactions by analyzing single GCs in a lymphoid organ. As variability among single GCs could arise due to other mechanisms, identifying other potential mechanisms that lead to such heterogeneity and investigating the contribution of each mechanism would help characterize the causes of GC\u2013GC heterogeneity. Systematically injecting antibodies at diffeIn our previous study , we showIn this study, we have assumed that antibodies produced are well-mixed throughout the organism. It remains to be explored in experiments whether antibodies secreted by plasma cells in the GC area result in a locally higher concentration of antibodies inside GCs. Direct diffusion of antibodies from a GC to neighboring GCs was also neglected in this study. While the mechanisms considered in the GC model are common to GCs in spleen and lymph nodes, initiation times and number of GCs in the asynchronous setting were estimated using experimental data from the spleen. We expect a similar qualitative effect of GC\u2013GC interactions in lymph nodes, however whether the permeability and local concentration of antibodies differ in the GCs of spleen and lymph nodes needs further investigation. Relative contribution of different antibody isotypes towards antibody feedback due to differences in properties such as tissue permeability and half-lives is unclear and needs further investigation with a detailed model of isotype switching ,56 that In the present study, we focused on the antibody mediated regulation of GCs by epitope masking. Other mechanisms of regulation of immune responses by antibodies have been identified such as Fc\u03b3-mediated inhibition . Moreove"} +{"text": "From a public health political science perspective there are alternative routes to HiAP. Besides the \u2018classic\u2019 HiAP approach seeking government internal departmental alignments, facilitating and rewarding bottom up social initiatives by communities, or marketed health innovations by commercial health-related consultancies and enterprises, and regional (socio-) economic innovation networks that after a developmental stage can start to pressure multi-level governments for action. A final route lies along the litigation path (Think of climate agreements and governments being held accountable by the judiciary to comply with agreed emissions reductions etc.)"} +{"text": "Individuals often experience improvements in emotional well-being into old age. Understanding mechanisms contributing to these emotional outcomes in daily contexts can inform ways to support healthy aging. Development is embedded within various contexts that shape individuals\u2019 experiences. Novel perspectives are emerging on how to conceptualize context and the way it can contribute to emotional development during the aging process. This symposium illustrates four innovative ways to consider contextual contributions to emotional well-being across adulthood. The first talk will use experience sampling to illustrate age differences in how daily situations contribute to emotion regulation related processes, showing that older adults can more easily distinguish between emotions when in familiar situations. The second talk will take a fresh perspective on psychosocial contexts by distinguishing between types of social interactions in couples, highlighting the important role of affection for well-being in adulthood. The third talk will introduce the idea that the body itself provides context for emotional processes, showcasing that the way this context affects emotional experience changes as individuals age. The fourth talk will center on how renewing our classical developmental models of context in modern ways can help to overcome shortcomings of previous research and provide insight into how engagement with environmental features contributes to well-being across the lifespan. In sum, this symposium features innovative perspectives on how context can be leveraged to gain a deeper understanding of psychosocial development into old adulthood and illustrates specific ways individuals can navigate their social world to preserve or improve mental health across adulthood."} +{"text": "Owing to perceived poor outcomes and limited benefit from reperfusion therapies, acute stroke patients presenting with large infarct core on baseline imaging were consistently excluded from thrombolytic and endovascular therapy trials for the last two decades \u201310. Priovia thrombolytic or endovascular reperfusion justified by their risk-benefit balance? And third, how can secondary injury and complications of acute large infarcts be minimized?Elucidating the best management of a recently established or evolving large infarct comes down to three key questions. First, what are the optimal imaging modalities and criteria to identify a \u201clarge core\u201d? Second, are attempts at reducing the infarct core size Koopman et al. present evidence that perfusion imaging is a reliable early biomarker to predict clinical outcome in this patient population. Yang et al. continue the discussion with evidence that patient age and perfusion profile may potentially define a ceiling to treatment benefits (Chen et al. propose a model based on demographic and imaging biomarkers (Hu et al. suggest a promising role for low-dose tirofiban (Hassan et al. present data from the COMPLETE registry to reaffirm the importance of time in limiting infarct growth and improving clinical outcomes (DeHoff and Lau then round off our discussion with a review of medical management of cerebral edema resulting from large hemispheric infarcts (This review on the topic of acute strokes with large infarct core examines all three aspects. benefits . To addromarkers . As the irofiban . Meanwhioutcomes . DeHoff infarcts .The world of cerebrovascular medicine currently eagerly awaits the outcomes of randomized controlled trials on endovascular therapy for acute strokes with large infarct cores \u201324. WhilAll authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication."} +{"text": "Loneliness is prevalent among older adults and is associated with increased risks for morbidity and mortality. This study examined what types of social interactions could reduce loneliness for older adults and who would benefit the most from social interactions. We used data from 312 community-dwelling older adults (aged 70 to 90 years) who completed ecological momentary assessments (EMA) five times a day for 16 consecutive days using smartphones , as part of the ongoing Einstein Aging Study (EAS). At each EMA, participants reported their social interactions in the past 3 to 4 hours and their current feelings of loneliness. Results from multilevel models revealed that older adults reported lower levels of loneliness on occasions when they had pleasant social interactions (p<.000) or interactions with family (p=.001) in the past few hours, compared with occasions when they had no social interaction. In contrast, they reported higher levels of loneliness if they had unpleasant social interactions in the past few hours (p=.004). These within-person (WP) effects of social interactions on momentary loneliness were significantly moderated by participants\u2019 trait levels of loneliness and neuroticism; and were significantly stronger among those with higher (vs. lower) trait loneliness (ps <.001) or neuroticism (ps <.042). Other personality traits did not moderate any WP association. These results highlight the importance of having pleasant social interactions and frequent interactions with family for reducing older adults\u2019 loneliness in daily life, especially for those higher in trait loneliness and neuroticism."} +{"text": "Cervus canadensis; 29%), and were dominated by gray wolves (40%) or humans (39%) as predators of interest. Emerging literature suggests that we can utilize predation risk for conservation with top\u2010down and bottom\u2010up approaches. It is less clear whether fear\u2010related changes in physiology have population\u2010level fitness consequences or cascading effects, which could be fruitful avenues for future research. Conflicting evidence of trait\u2010mediated trophic cascades might be improved with better replication across systems and accounting for confounding effects of ungulate density. Improving our understanding of mechanisms modulating the nature of trophic cascades likely is most important to ensure desirable conservation outcomes. We recommend future work embrace the complexity of natural systems by attempting to link together the focal areas of study identified herein.Because ungulates are important contributors to ecosystem function, understanding the \u201cecology of fear\u201d could be important to the conservation of ecosystems. Although studying ungulate ecology of fear is common, knowledge from ungulate systems is highly contested among ecologists. Here, we review the available literature on the ecology of fear in ungulates to generalize our current knowledge and how we can leverage it for conservation. Four general focus areas emerged from the 275 papers included in our literature search (and some papers were included in multiple categories): behavioral responses to predation risk (79%), physiological responses to predation risk (15%), trophic cascades resulting from ungulate responses to predation risk (20%), and manipulation of predation risk (1%). Of papers focused on behavior, 75% were about movement and habitat selection. Studies were biased toward North America (53%), tended to be focused on elk ( In this review, we highlight the need to diversify research efforts on the ecology of fear in ungulates. We show that most of the literature found with our search disproportionately focused on a few prey and predator species, especially in North America, while several species and systems were highly underrepresented. Moreover, ungulates are widespread globally, important economically and ecologically, and are often sympatric with large, apex predators that are of conservation concern . Thus, broad review and understanding of the state of research into the ecology of fear is warranted, particularly given the interest in using the ecology of fear for conservation or broad\u2010scale . We defined physiological responses as any change in the physiology of ungulates as a result of predation risks, including changes in body chemistry or disease risk. We defined a trophic cascade as occurring when predators altered ungulate behavior, resulting in the release of plants from herbivory , while fewer focused on physiological effects of fear and only three (1%) on manipulation of predation risk for wildlife management . Fewer studies were conducted in Europe , Sub\u2010Saharan Africa , and other world regions .The literature search yielded 275\u00a0studies relevant to the ecology of fear in ungulates Appendix . While mt Figure . More th3.1n = 216) into two subtopics: movement and habitat selection and vigilance and herding .In the presence of predators, prey generally alter their behavior to become more difficult to capture, detect, or encounter. Antipredator behaviors are a complex suite of innate and learned behavioral responses, which can be individual or species\u2010specific , and Grant's gazelle (Nanger granti) prefer areas with high grass biomass to areas of high visibility during droughts shows that in the absence of clouded leopards, bearded pigs (Sus barbatus) were more nocturnal than when leopards were present, perhaps indicating the bearded pigs alter their activity pattern to decrease predation risk spatial and temporal behavior reporting that roe deer avoid areas of high chronic predation by Eurasian lynx (Lynx lynx) at night but not during the day in summer because lynx activity is low due to human disturbance during the day by changing their foraging decisions at the scales of vegetation types and specific features of the vegetation type such as edges. At finer scales, many studies have documented behavioral responses to predation risk related to forage selection and quality. For example, bison (Bison bison) reduced selection of high\u2010quality foraging sites as wolf risk increased in winter traded off forage abundance for safety cover. Similarly, some studies have linked behavioral effects of predation risk to fine\u2010scale landscape features and vegetative cover. For example, Nubian ibex (Capra nubiana) perceived greater risk of predation as their distance from cliff and slope edges increased, and their perception of risk decreased with vegetative cover avoided the space use of all predators to reduce probability of encounter. Concomitantly, larger species only avoided areas of intense space use by lions (Panthera leo) and leopards (Panthera pardus). The authors concluded that ungulates used a simple behavior rule: avoid areas used by sit\u2010and\u2010pursue predators (lion and leopard) but increase wariness in areas used by cursorial predators . Similarly, other studies using predator excrement at foraging areas monitored with camera traps demonstrated red deer (Cervus elaphus) were not only apparently able to discern hunting mode from the type of excrement present, but also used different antipredator behaviors to mitigate risk of each threat habitat use was dependent on predation risk from wolves, though they acknowledged several underlying explanations that could have been confounding . Similarly, Samelius et al. (Lynx lynx) had limited effects on habitat selection of roe deer (Capreolus capreolus) in Sweden. The authors suggested their results provided evidence for the complexity of prey responses to risk and that such responses likely were variable between ecosystems and predator\u2013prey constellations and non\u2010parous females did not alter space use, while those giving birth did so nearer to paved roads avoided by brown bears increase vigilance when close to predators in places where predator encounter probability is high. Vigilance also depends on herd size because herding ungulates generally rely on group vigilance so that individuals can spend less time scanning for predators as group size increases into two subtopics: diet quality and nutrition and fitness and physiology .Ungulates must balance forage acquisition and risk avoidance, which necessitates interplay between physiology and behavior switched to an abundant low\u2010quality food in response to stress from coyotes suppressed dik dik (Madoqua guentheri) populations but did not result in trophic cascades to the plant community, likely because of herbivore diversity in the system. Furthermore, surrogate predators, either introduced or invading, may or may not cause trait\u2010mediated trophic cascades similar to that of native predators, even if they have the same general hunting mode. As an example, coyotes have recently expanded their range across eastern North America , recent literature has reported coyote selection against behavioral traits of white\u2010tailed deer that had presumably evolved as an adaptive response to red wolves did not result in a trophic cascade on aspen in Arizona, perhaps because the density of wolves was too low relative to elk densities .Predicting the strength of trophic cascades is complicated because a multitude of factors affects this phenomenon predator removal or exclusion, (2) predator reintroduction, and (3) ungulate exclusion resulted in population increases in invasive herbivores and decreases in biodiversity. Finally, in a review, Estes et al. . Predators may limit disease spread by reducing host densities or selecting infected individuals ; Investigation ; Methodology ; Writing \u2013 original draft (lead). Carolina Baruzzi: Data curation (lead); Investigation ; Methodology ; Visualization (lead); Writing \u2013 original draft (supporting). Marcus A. Lashley: Conceptualization ; Investigation ; Methodology ; Writing \u2013 original draft (supporting).Appendix S1Click here for additional data file."} +{"text": "Lifestyle behaviors are important determinants of healthy brain aging. Research has not fully explored how sleep quality and physical activity may differentially influence specific domains of cognitive function. The present study aimed to estimate the relative influence of sleep quality and physical activity on cognitive performance in three domains in a sample of older adults. Older adults without cognitive impairment (N= 160) wore an accelerometer for 7 days in a free-living environment. We used average vector magnitude counts per minute to measure total physical activity (TPA), and average wake after sleep onset (WASO) to measure sleep quality. We created cognitive composite scores from neuropsychological data using confirmatory factor analysis. We regressed cognitive scores onto TPA and WASO with age and education entered as covariates. Higher amounts of physical activity and better sleep quality were associated with better executive function = 11.12, p < .001). Neither physical activity nor sleep quality was associated with verbal memory or attention. Results suggest that more physical activity and improved ability to stay asleep may benefit executive function, but not other cognitive domains. Future studies should clarify the interaction and mechanisms of action between health behaviors and cognitive performance in older adults."} +{"text": "Family caregivers of persons with dementia (PWDs) use social media to obtain and share health information. Yet relatively little is known about the types of information that these caregivers share. We address this gap by analyzing caregivers\u2019 posts (N = 401) in two subreddits, r/Alzheimers and r/dementia, which represent two subgroups on the social media platform Reddit. Our research questions were as follows: (1) What are these caregivers\u2019 main purposes in posting? (2) What types of health information do the caregivers exchange online? (3) What are the characteristics of online posts that receive the caregivers\u2019 greatest attention? Content and correlational analyses revealed that over half of users (57%) posted regarding specific types of health information that they desired to seek and share, whereas the remaining users posted information for sharing only. The health information most commonly posted fell into the following three categories: psychosocial health information, information about patients\u2019 daily care, and characteristics of health conditions. The more health information types that a post contained, the greater the number of people who participated in the subreddit discussions. Further research should examine how social media meet PWD caregivers\u2019 needs for information support."} +{"text": "To audit VTE risk assessment compliance across psychiatric inpatient wards at three different sites within Surrey and Borders Partnership NHS Foundation Trust (SABP), and to highlight the importance of completing VTE risk assessments for psychiatric inpatient safety and care as set out by NICE guidelines (2019).Numbers of VTE risk assessments completed and VTE risk assessments not completed were collected via SABP electronic mental health records. Percentage compliance for each ward and hospital involved in the study were calculated. Chi square statistical t tests were conducted using Excel to check for associations between type of ward (older adult and working age) and VTE risk assessment completion.A total of 3004 patients were included in the study. Ages ranged from 18\u201382 years of age, and both males and females included in the study. A total of 2060 were working age (WA) patients (aged 18\u201364 years) and 944 were older adults (OA) (aged > 65 years).Across all three sites, more than 90% of all inpatients admitted between May 2018 and October 2020 did not have a formal VTE risk assessment completed. Across all sites, less than 1% of all inpatients had a completed VTE risk assessment done within 24 hours, as recommended by the NICE guidelines. Older Adult wards showed better compliance with VTE risk assessment completion with 38% of patients on one OA ward having had a completed VTE risk assessment, and 28% on another completed OA ward. Being admitted to an OA ward was strongly associated with VTE risk assessment completion (p < 0.05).OA wards have hosted QI programmes with regards to VTE risk assessment which may be why VTE risk assessment was more likely to have been completed on OA wards. VTE risk assessment compliance overall is inadequate across all sites included in the study. Recommendations include further education for all ward staff on how, why and when VTE risk assessment should be completed, greater accessibility of an improved VTE risk assessment form and for QI initiatives on OA wards to be rolled out on WA wards. These findings have been presented and discussed at regional Trust teaching days, and this audit will be repeated in one year."} +{"text": "He was diagnosed with nonepisodic angioedema with eosinophilia (NEAE), which spontaneously resolved after 2 months . NEAE in men or involving body parts other than the lower extremities is unusual (5). Although its exact pathophysiology remains unknown, the female predominance and occurrence of this disease following infection or drug exposure in some patients are suggestive of NEAE being a consequence of an aberrant immune response to various exogenous stimuli in genetic susceptible individuals under the influence of sex hormones (4). Corticosteroid therapy is generally reserved for patients with severe symptoms or marked eosinophilia. However, regardless of treatment, even the most affected patients completely recover within few months after presentation.Nonepisodic angioedema with eosinophilia is characterized by nonrecurrent peripheral angioedema with eosinophilia and normal IgM levels along with lack of fever, weight gain, and internal organ involvement. It mainly affects young women from Japan, Korea and Thailand NoneThatchai Kampitak contributed to manuscript preparation, patient care, and discussionIRB approval was not required for this studyInformed consent has been obtained from the patient"} +{"text": "National guidelines recommend adults >75 engage in shared decision making (SDM) around colorectal cancer (CRC) screening because of the uncertain benefit to risk ratio. There are no decision tools to support CRC decision making for adults >75 years with low health literacy (LHL). The purpose of this mixed-methods study was to better understand the perspectives of adults >75 with LHL on SDM around CRC screening and to obtain their feedback on an existing higher literacy CRC decision aid. Utilizing the Brief Health Literacy Screening Tool to identify participants with LHL, semi-structured interviews were conducted with 30 adults. Findings indicate that 80% of participants were non-Hispanic Black and 42% had < high school degree. 76% felt they would benefit from CRC screening despite their age. Themes related to CRC screening included lack of knowledge of options and harms, but a desire to understand more to better take care of their health."} +{"text": "While previous studies evince a strong link between family bereavement and worse cardiovascular functioning, factors that may influence the association remain unexplored. This study examined the relation between experiencing the death of an immediate family member and heart rate variability (HRV) and whether the associations differed by sleep quality. The sample included respondents from the Midlife in the United States (MIDUS) Biomarker Project who reported losing an immediate family member \u2013 parents, spouse, siblings, or children \u2013 within a year before project (n = 94) and those who did not experience any deaths (n = 872). Results showed that the death of a family member was associated with worse HRV only among those who reported having a poor sleep quality and not for those with good sleep quality. These findings suggest that poor sleep quality may indicate psychophysiological vulnerability for those who experienced the death of an immediate family member."} +{"text": "The Primavera is considered amongst the greatest and controversial artistic masterpieces worldwide painted by renaissance artist Sandro Botticelli. The aim was to identify any underlying medical foundations for the painting.Observational study.The painting reveals, a \u2018butterfly\u2019 malar rash, bilateral ptosis and a clear neck swelling consistent with a goitre in the figure of Flora. This could be explained by concomitant Graves\u2019 disease and systemic lupus erythematosus, or other presentations of multiple autoimmune syndrome.These findings highlight the likely presentation of the earliest pictorial depictions of thyroid disease with systemic lupus erythematosus and emphasize the exactitude of depiction demonstrated by Botticelli in renaissance era. The Primavera or \u2018Spring\u2019 painted during the Early Renaissance (circa late 1470\u2013early 1480) by Sandro Botticelli demonstrates a clear neck swelling consistent with a goiter Fig.\u00a0b with biDifferential diagnosis of the goiter with ptosis and the malar rash could represent the individual depicted may have suffered from concomitant thyroid and malar-causing autoimmunity. This could be explained by concomitant Graves\u2019 disease (GD) and malar-associated systemic lupus erythematosus (SLE) or Hashimoto\u2019s thyroiditis (HT) and SLE . The preFurther support of Botticelli\u2019s allegory of spring in this piece could include the finding that if the SLE was pathology in the character of Flora, there is seasonal evidence that this condition is more prevalent in Spring, and most conspicuously the malar rash is most identifiable in this season. These findings highlight the depth of genius in this great artist and strengthen the understanding of scientific principles captured in the magnificent renaissance artistic portrayals."} +{"text": "Very thick left ventricular papillary muscles (PAMs) may kiss each other and premature ventricular contractions (PVCs) can originate from the sides of the PAMs facing each other. In such a setting, mapping of those PVCs is confusing and rendering catheter ablation challenging. Very thick left ventricular papillary muscles (PAMs) may kiss each other and premature ventricular contractions (PVCs) can originate from the sides of the PAMs facing each other. In such a setting, mapping of those PVCs is confusing and rendering catheter ablation challenging. A 73\u2010year\u2010old woman with premature ventricular contractions (PVCs) had very thick left ventricular papillary muscles (PAMs) kissing each other. The PVC origin at the septal side of the anterolateral PAM that faced the posteromedial PAM, rendered mapping confusing. This case illustrated an unusual challenge in catheter ablation of PAM PVCs.A 73\u2010year\u2010old woman with symptomatic idiopathic premature ventricular contractions (PVCs) exhibiting a right bundle branch block and right inferior axis QRS morphology Figure\u00a0 underwenCatheter ablation of ventricular arrhythmias originating from the papillary muscles (PAMs) is often challenging because their origins are located deep inside thick PAMs.The authors declare no conflict of interest.Both authors made a substantial contribution to the preparation of this manuscript and approved the final version for submission. TY: drafted the manuscript. KA: acquired the images and performed the literature search.Written informed consent was obtained from the patient. This case study was completed at the University of Alabama at Birmingham. The Institutional Review Board approved all types of the studies regarding ventricular arrhythmias originating from the papillary muscles.The authors have confirmed during the submission that the patient consent has been signed and collected in accordance with the journal's patient consent policy."} +{"text": "Nicrophorus vespilloides. Parental care, produced from many individual behaviors, should be influenced by changes of expression of multiple genes, and one suggestion is that the genes can be predicted based on knowledge of behavior expected to be precursors to parental care, such as aggression, resource defense, and mating on a resource. Thus, testing gene expression during parental care allows us to test expectations of this \u201cprecursor hypothesis\u201d for multiple genes and traits. We tested for changes of the expression of serotonin, octopamine/tyramine, and dopamine receptors, as well as one glutamate receptor, predicting that these gene families would be differentially expressed during social interactions with offspring and associated resource defense. We found that serotonin receptors were strongly associated with social and aggression behavioral transitions. Octopamine receptors produced a complex picture of gene expression over a reproductive cycle. Dopamine was not associated with the behavioral transitions sampled here, while the glutamate receptor was most consistent with a behavioral change of resource defense/aggression. Our results generate new hypotheses, refine candidate lists for further studies, and inform the genetic mechanisms that are co\u2010opted during the evolution of parent\u2013offspring interactions, a likely evolutionary path for many lineages that become fully social. The precursor hypothesis, while not perfect, does provide a starting point for identifying candidate genes.Understanding the genetic influences of traits of nonmodel organisms is crucial to understanding how novel traits arise. Do new traits require new genes or are old genes repurposed? How predictable is this process? Here, we examine this question for gene expression influencing parenting behavior in a beetle, Predicting gene function of nonmodel organisms is necessary to understanding how novel phenotypes arise and how conserved genetic mechanisms are. We tested whether neurotransmitter receptor expression can be predicted from known associations from previously studied organisms during parental care. Some neurotransmitters showed high predictability , while other did not . The octopaminergic system is generally associated with increased aggression and again expected these would be differentially expressed when aggression is highest, during resource defense and parenting. The dopaminergic system is also strongly associated with direct parental care with the same expectation as the octopamine/tyramine receptors. Finally, we profiled the glutamate receptor, nmdar1, which is associated with the transition into direct parental care, as seen before with N. vespilloides with an expectation of changed expression during active parenting.Our aim was to select pathways that might be differentially expressed over a reproductive cycle based on the heuristic provided by the precursor hypothesis. This would provide evidence of their possible involvement, but will not provide direct evidence of specific functions. It does, however, test the precursor hypothesis and provide a starting point for further studies of specific gene functions. We surveyed the gene expression of serotonin (5HT), octopamine (OCT), dopamine (Dop), and glutamate (NMDA) receptors during the behavioral transitions across a single reproductive cycle. These neurotransmitters were chosen for their identified functions with other species. For example, the serotoninergic system is associated with periods of increased sociality as serotonin increases ad libitum once a week after adult eclosion.We used beetles from an outbred colony of 2.2We assayed gene expression from female whole head samples collected at specific points across a reproductive cycle of age\u2010matched individuals search as the endogenous reference gene. We have previously shown that tbp was stable across these behavioral transitions by standardized cDNA input amount into individual reactions . No treatment was individually statistically significantly different from Virgins.The expression of serotonin receptor 2 was statistically significantly different across the behavioral states . The specific contrast showed that there was a statistically significant decrease between the Resource Preparation and Parental Care treatments compared with the other treatments without aggression or parental care . Expression of the Direct and Indirect Parental Care state was statistically significantly lower than in Virgins .The expression of serotonin receptor 7 was statistically significantly different across the behavioral states . The specific contrast showed that there was a statistically significant increase between the Resource Preparation and Parental Care treatments compared with the other treatments without aggression or parental care . No treatment was individually statistically different from Virgins.The expression of 3.2octopamine alpha receptor was statistically significantly different across the behavioral states . Expression of the Resource Preparation treatment was statistically significantly decreased compared with Virgins .The expression of octopamine beta receptor 1 was statistically significantly different across the behavioral states . The specific contrast showed no statistically significant difference between the Resource Preparation and Parental Care treatments compared with the other treatments without aggression or parental care . No treatment was individually statistically significantly different from Virgins.The expression of octopamine beta receptor 2 was not statistically significantly different across the behavioral states . The specific contrast showed no statistically significant difference between the Resource Preparation and Parental Care treatments compared with the other treatments without aggression or parental care .The expression of octopamine beta receptor 3 was statistically significantly different across the behavioral states . The specific contrast showed no statistically significant difference between the Resource Preparation and Parental Care treatments compared with the other treatments without aggression or parental care . Expression of the Resource Preparation treatment was statistically significantly decreased compared with Virgins .The expression of octopamine/tyramine receptor 1 was statistically significantly different across the behavioral states . The specific contrast showed a statistically significant decrease between the Resource Preparation and Parental Care treatments compared with the other treatments without aggression or parental care . The Mated treatment was statistically significantly increased compared with Virgins . The Resource Preparation treatment was statistically significantly decreased compared with Virgins . The Direct & Indirect Parental Care treatment was statistically significantly decreased compared with Virgins .The expression of octopamine/tyramine receptor 2 was statistically significantly different across the behavioral states . The specific contrast showed no statistically significant difference between the Resource Preparation and Parental Care treatment compared with the other treatments without aggression or parental care . The Resource Preparation treatment was statistically significantly decreased compared with Virgins . The Direct and Indirect Parental Care treatment was statistically significantly decreased compared with Virgins . The Post\u2010Care treatment was statistically significantly decreased compared with Virgins .The expression of 3.3dopamine receptor 1 was statistically significantly different across the behavioral states . No treatment was individually statistically significantly different from Virgins.The expression of dopamine receptor 2 was not statistically significantly different across the behavioral states . The specific contrast showed no statistically significant difference between the Resource Preparation and Parental Care treatments compared with the other treatments without aggression or parental care .The expression of dopamine DDR2 receptor 2 was not statistically significantly different across the behavioral states . The specific contrast showed no statistically significant difference between the Resource Preparation and Parental Care treatments compared with the other treatments without aggression or parental care .The expression of 3.4glutamate receptor subunit 1 was statistically significantly different across the behavioral states . The Resource Preparation treatment was statistically significantly decreased compared with Virgins .The expression of 4N. vespilloides. All of these neurotransmitter systems are expected to influence social interactions, affiliative and aversive behavior. We expected the strongest pattern changes in expression during Resource Preparation, where resource defense and indirect parental care are highly increased, and Direct & Indirect Parental care, where social interactions are highly increased along with resource defense based on studies from other organisms. Gene expression changes for serotonin, octopamine, and the nmdar1 receptor(s) had differences across the behavioral transitions into and out of active parenting. In contrast and against expectation, dopamine receptors had no strong differences across the behavioral transition sampled here. All of the serotonin receptors also had differences between the Resource Preparation and Direct and Indirect Parental Care treatments compared with the others. More broadly, the evolution of parent\u2013offspring interactions is hypothesized as a likely evolutionary pathway for animals from solitary to social life history ; Data curation ; Formal analysis ; Investigation ; Methodology ; Project administration ; Visualization ; Writing\u2010original draft ; Writing\u2010review & editing . Daven Khana: Investigation ; Methodology ; Writing\u2010review & editing . Annika Carter: Investigation ; Methodology ; Writing\u2010review & editing . Elizabeth C. McKinney: Data curation ; Investigation ; Methodology ; Writing\u2010review & editing . Allen J. Moore: Conceptualization ; Formal analysis ; Funding acquisition ; Project administration ; Visualization ; Writing\u2010review & editing .Appendix S1Click here for additional data file."} +{"text": "Olive oil is a traditional product of the Mediterranean diet. The nutritional properties and remarkable health benefits associated with olive oil consumption explain the growing worldwide use of this product in people\u2019s diets . In fact1HNMR profiling database using monocultivar reference olive oil for the analysis of olive oil blends [Despite the aim of this Special Issue encompassing three key topics\u2014specificity, authenticity, and adulteration\u2014no contributions were received regarding the theme of specificity. Thus, this editorial has collected papers giving an interesting outlook on several promising methodologies, including NMR-based approaches ,3, chroml blends .All these papers actively contribute to creating a more complete picture of the situation and could open new avenues in the field of food authenticity and traceability."} +{"text": "An audit was conducted to assess if thorough risk assessments had been documented in electronic clinical record notes (ECR) clerking for new patients in two acute mental health wards. Risk assessment is a vital part of admission clerking and when done well it can prevent early incidents and aid the ward nursing team greatly. During induction, junior doctors are advised to document assessed risks when clerking a new patient. A screening of the risks on admission could help determine the levels of observations required to minimise the identified risks whilst the patient awaits their first ward review.The NHS numbers for the 30 current inpatients across male and female acute psychiatric wards were gathered at the time of the audit (February \u2013 March 2020). Admission clerking was analysed for a clear statement of patient risk to self, others or property. Within these categories quantitative results were obtained on how often the risk of self-harm, self-neglect, absconding, vulnerability or aggression was documented. The term \u2018risk\u2019 was used for each patient on their ECR notes to search for risk assessments in all entries other than admission clerking.12 out of the 30 patients had a junior doctor risk assessment documented in their clerking (40%). 14 patients had no mention of risk assessment on admission (47%) and their first formal risk assessment was documented only in their senior ward review. Of the 12 assessments completed in clerking; all assessed self harm/suicide risk and violent risk to others, 1 mentioned risk of absconding, 8 mentioned risk of illicit substance use and 8 mentioned vulnerability. It was unclear if the risks documented were based on current or historic presentation. Junior doctors were anonymously surveyed following this audit and reported they did not feel confident in how to document a risk assessment or whether to document negative findings.Clear documentation of risk assessment being performed was lacking in over half of junior doctor admission clerkings. When risks were assessed it was mainly violence/self harm risk documented not vulnerability and physical health risks. Based on these findings we have designed more comprehensive teaching on risk assessments and a template for how to complete a risk assessment. We feel the use of a template will ensure all elements of risk are clearly considered even if they are not present currently. This is being reaudited to assess if the changes have impacted the quality of risk assessment conducted."} +{"text": "Since 2016, the Cannabis and Older Persons Study has examined the increasing use of cannabis among Americans over 60 years old. Our current work dives into particular groups of cannabis users and explores outcomes related to medical conditions and symptoms. This symposium also features a range of methodological approaches from an analysis of the BRFSS caregiving and cannabis modules, a convenience sample of more than 4,000 older cannabis users enrolled in the Illinois Medical Cannabis Program and qualitative interviews conducted with aging veterans. Kanika Arora examines the association between informal caregiving and marijuana use and whether this association varies by age. Julie Bobitt shares findings from 32 interviews with older Veteran cannabis users. Alton Croker examines cannabis use as a complement or alternative to palliative care. HyoJung Kang clusters negative outcomes experienced by older persons who use cannabis. Brian Kaskie compares cannabis use among persons with Multiple Sclerosis (N=135) and persons diagnosed with arthritis (N=582) or cancer (N=622). While we certainly find reason to remain concerned that cannabis use alone and co-occurring use with prescription opioids may contribute to increased rates of substance misuse and other undesirable outcomes among older adults, we find it increasingly difficult to overlook the benefits many persons derive when taking cannabis as a method to manage pain or address other medical conditions. At this point, public policy officials and program administrator should strive to strike a balance between addressing cannabis harms relative to promoting benefits such as opioid reduction and diversion."} +{"text": "Purpose\u2003Ischemic stroke is a relatively rare complication of giant cell arteritis often accompanied by vessel stenosis. Our purpose was to compare the location of internal carotid artery stenosis in GCA patients by performing a literature review suggesting a specific and characteristic pattern.Methods\u2003We performed a PubMed research including all articles and cited articles reporting cases and case series about giant cell arteritis patients with internal carotid artery stenosis and ischemic strokes.Results\u2003In this case series 39 cases were included. We found a clear tendency of giant cell arteritis-related stenosis to be in the intracranial segments (35/39 (89.7%)). Only in 8/39 (20.5%) patients there was further involvement of extracranial segments. Many cases (27/39 [69.2%]) showed a bilateral involvement.Discussion\u2003This literature review reveals a specific pattern of internal carotid artery involvement in patients with giant cell arteritis and ischemic strokes. To our knowledge this pattern has not been reported as a sign strongly pointing toward giant cell arteritis before. We have not found case reports mentioning other common types of vasculitis reporting this involvement pattern.Conclusion\u2003Internal carotid artery stenosis and ischemic stroke is a rare complication in patients with giant cell arteritis. Considering the characteristic features of bilateral distal internal carotid artery stenosis giant cell arteritis should be suspected which potentially leads to an early diagnosis and immunotherapy. Ischemic stroke in giant cell arteritis (GCA) is a well-known complication. The incidence rate ranges between 2.8 and 7%.In this literature review we want to discuss the characteristic localization of GCA-related bilateral intracranial internal carotid artery (ICA) stenosis comparing radiological and histopathological findings of previous studies. The possible challenges of diagnosing this disease will be demonstrated.We want to present our own case of a 58-year-old female patient with suspected temporal-artery-biopsy-negative GCA who suffered recurrent bihemispheric strokes and hemodynamic impairment of both hemispheres while the only manifestation site was both intracranial carotid arteries. Despite immunosuppressive treatment the patient could not be prevented from experiencing new strokes.Our aim was to perform a literature review of cases with internal carotid artery involvement in GCA and ischemic stroke. A PubMed research was performed including articles and cited articles about selected cases of patients describing stenosis of both or one ICA in patients with GCA and ischemic strokes at the time of diagnosis while searching for the terms \u201cgiant cell arteritis,\u201d \u201cGCA,\u201d and \u201ccarotid artery,\u201d \u201cischemic stroke\u201d or \u201cintracranial involvement.\u201d The patients had to be diagnosed according to plausible clinical features and current guidelines, by a positive temporal artery biopsy (TAB) or histological proof of giant cells in other large vessels combined with clinical features attributable to GCA. Another criterion was that the segment or segments of stenosis were mentioned or could be figured out by analyzing the provided vascular imaging or autopsy.Although other signs of inflammation can be drawn to attentionThis literature review revealed a clear tendency of GCA to cause bilateral intracranial stenoses (mainly cavernous and [para]clinoid segment) in the case of ICA involvement. The patients' features are shown inA 58-year-old female patient was admitted to the emergency room complaining of sudden onset palsy of the left arm and leg. No headache was present. She had a history of hypertension. We diagnosed a diabetes mellitus type II and dyslipidemia. CT angiography and MRI/MR angiography revealed bilateral stenosis of both intracranial ICA pronounced on the right side, a perfusion deficit of the right hemisphere and bilateral new infarcts also pronounced on the right side. A diagnostic cerebral angiography, and MRI showed bilateral infarcts and smooth and mostly concentric bilateral distal ICA stenosis, a 70% stenosis of the right internal carotid artery in the C3 to C6 segments, a 60% stenosis of the left ICA in the same segments, and a partial supply of the right middle and anterior cerebral artery territories by crossflow at the anterior communicating artery . LaboraWe found that internal carotid artery involvement in GCA with ischemic strokes follows a characteristic pattern with bilateral mostly symmetrical distal ICA stenosis or occlusion .To our knowledge this is the first systematic review examining case series and case reports about GCA patients with ischemic stroke and ICA stenosis/occlusion. This bilateral distal internal carotid artery involvement pattern was mentioned before to be a possible manifestation in GCA patientsIt has been reported before that patients with large-vessel-giant-cell-arteritis have less headache, jaw claudication or visual symptoms, are younger than GCA patients with temporal arteritis and their TAB specimens are less likely to yield positive resultsWe scarcely found comparable report cases in other categories of vasculitis . In one case of Beh\u00e7et's disease a patient had bilateral proximal ICA occlusion.To a lesser degree ICA stenosis can occur in the short proximal intradural course but rarely involves purely intradural vessels.The incidence rate of ischemic stroke in patients with GCA has been repeatedly reported ranging between 2.8 and 7%.It has been repeatedly reported that at the time of diagnosis patients are more likely to have an ischemic stroke in the vertebrobasilar region rather than carotid perfused region with an estimated ratio of around 5:1Stenosis and VWE might pose a higher risk for ischemic strokes in GCA patients but has not been studied systematically yet. Caselli and Hunder 1988 investigated the occurrence of ischemic strokes in GCA patients during a 3-year study period and found a higher incidence rate of ischemic strokes in patients with carotid disease; however, the latter was defined only by bruits and/or diminished pulses. To our knowledge there are no studies comparing the degree of stenosis (including VWE) with the incidence rate of ischemia. Early treatment seems to be important: a retrospective database study showed a strong focus of GCA-related strokes with a fivefold-increased risk during the active phase of the disease.As mentioned by the Chapel Hill Consensus Conference 2012 authors, \u201cif the features of a vasculitis that is confined to one organ indicate that it is a limited expression of one of the systemic vasculitides, this vasculitis should be considered a limited expression of that category of vasculitis rather than an independent SOV (single organ vasculitis).\u201d"} +{"text": "Baystate Health\u2019s Geriatrics Workforce Enhancement Program (GWEP) postponed implementation of Group Medical Visits focused on falls reduction for older adults in Springfield, Massachusetts due to COVID-19 and quickly shifted efforts to participate in Dartmouth\u2019s Falls Prevention Training Program. Long standing GWEP Community Based Organizations (CBOs) were consulted, and all believed that the virtual Tai Ji Quan Moving for Better Balance\u00ae (TJQMBB) program would combat social isolation and improve older adults\u2019 comfort with technology in addition to reducing falls during the COVID-19 pandemic. Baystate\u2019s GWEP was able to reallocate grant dollars to support the purchase of equipment for CBOs to deliver TJQMBB virtually. While many challenges continue to arise, the innovative and collaborative approach between the two GWEPs and Baystate\u2019s CBOs leveraging Administration for Community Living falls prevention funding has led to high level engagement and rapid implementation. Dartmouth\u2019s model capitalizes on and strengthens existing GWEP partnerships with its CBOs."} +{"text": "To provide awareness of safety concerns around use of alcohol hand sanitiser on a mental health ward, and to consider ways of improving how learning for a serious adverse incident in one trust can better be communicated to other trustsDD a male patient with history of paranoid schizophrenia alongside historic illicit drug use and current alcohol dependency admitted detained to Bluestone hospital following bizarre behaviour at a wake. Had been non-compliant with medication. Transferred to PICU due to going AWOL and returning under influence of alcohol.2nd April overnight staff noted him to become over-sedated, presenting with slurred speech and appeared under influence of alcohol \u2013 transferred to A + E due to deteriorating GCS \u2013 was intubated, and transferred to ICU. Blood alcohol level was 373. Several empty bottles of hand sanitiser from dispensers on ward found in his room and he later disclosed he had accessed further alcohol hand sanitiser in sluice while washing clothesSAI learning outcomes from one healthcare trust in Northern Ireland not currently routinely shared with other trustsLiterature review carried out to search for reports of similar incidents \u2013 1 previous review article suggesting one death and 11 other major complications from consumption of alcohol hand sanitiser over 5 year period 2005-2009.Quality improvement steps implemented to address this riskWard policy was reviewed to ensure patients no longer had unsupervised access to wash clothesLiaised with Infection Control to assess the need for alcohol hand sanitiser to be available to patients given the ward is effectively a community settingIntoxication policy reviewed and education sessions on this provided to all medical and nursing staffRegional regular PICU staff update seminar launched for purpose of bringing PICU staff from across Northern Ireland together to share learning from SAIs and casesInfection control agreed alcohol hand sanitiser dispensers could be removed from wards and kept only in locked nursing office with use of visitors.Learning from this case shared with other trusts locally at newly launched regional PICU update seminarNo further incidents to datePatient access to alcohol hand sanitisers found to be a significant safety risk in PICU settingFollowing implementation of quality improvement steps no further incidents of patients swallowing alcohol hand sanitiserImproved awareness of risk of alcohol intoxication on ward with nursing staff escalating concerns to on-call doctor more frequently"} +{"text": "OBJECTIVES/GOALS: Atrial fibrillation (AF) not only is the most common sustained cardiac arrhythmia in clinical practice placing patients at increased risk for thromboembolic events. Hispanics despite having a higher risk factor burden for developing AF have a lower overall incidence and prevalence of AF when compared to Non-HispanicWhites (NHW). European ancestry in the African American population was found to be an independent predictor for developing AF. Consequently, we have decided to evaluate if European ancestry is an independent risk factor for Puerto Rican Hispanics(PRH) METHODS/STUDY POPULATION: This project is a secondary analysis of a Puerto Rican population sample (n = 250) fromThePharmacogenetics of Warfarin in Puerto Ricans StudyandAGenomic Approach for Clopidogrel in CaribbeanHispanics; and1000GenomeProject to establish a control group of healthy PRH population. We will evaluate the presence of 111 known single nucleotide polymorphisms(SNP)associated with AF Europeans and determine the frequency in PRH population sample.Using admixture informatic markers (AIM) analysis will determine the percentage of admixture. Statistical analysis will include the use of Pearson Product-MomentCoefficient correlation analysis and multivariate regression. For admixture will use Maximum LikelihoodEstimation Markov Chain Monte Carlo models RESULTS/ANTICIPATED RESULTS: We anticipate that higher-frequency of AF associated European SNPs and overall higher percentage of European admixture will be associated with atrial fibrillation in PRH patients. DISCUSSION/SIGNIFICANCE OF IMPACT: The expected outcomes for this study are to identify the frequency of known genetic loci associated with AF Europeans and validate their use PRH population for machine learning risk factor models."} +{"text": "Der neunte Arm des Oktopus by German drugstore magnate Dirk Rossmann. Certain elements of brand-name authorship are applicable here, but the market power of Rossmann goes far beyond name recognition. Beyond the typical marketing for a \u2018big book\u2019 in the sense of contemporary trade publishing, Rossmann flooded the campaign with his own funds. This contribution approaches an unlikely case study through a trilateral interdisciplinary perspective , underlining the unequal footing on which books enter the market.This paper considers the success of the thriller Der neunte Arm des Oktopus (The Ninth Arm of the Octopus)\u00a0))and publDer neunte Arm des Oktopus at the time of its publication, thus explaining large parts of the high demand. As a downstream effect of high sales figures, Rossmann\u2019s book quickly reached a top position on the Spiegel bestseller list shortly after its release date. This will have reinforced the market breakthrough that had already occurred, since bestseller lists and popularity rankings allow consumers to learn (indirectly) about product quality by observing other consumers\u2019 purchase decisions ( diapers\u201d .\u00a0Ar"} +{"text": "Alcohol use across the life course provides some physical and psychological benefits when used in moderation. As a social model of care, assisted living (AL) communities emphasize autonomy; yet, we do not know how this philosophy extends to drinking. Using ethnographic and interview data from a larger 5-year NIA-funded study in four diverse AL communities designed to identify best practices for the meaningful engagement of AL residents with dementia, we examine how residents, families, and staff interpret residents\u2019 rights about alcohol use and how staff and families facilitate or limit alcohol use of residents with dementia. Findings indicate staff and families frequently rely on a narrative of \u201cwatchful oversight\u201d to limit or restrict alcohol consumption while simultaneously affirming the social connection of drinking . We discuss the implications of our findings for research and practice aimed at promoting meaningful engagement and quality of life among persons with dementia."} +{"text": "The aim was to improve the experience of transgender patients in a general adult inpatient setting, through delivering practical 'bitesize' ward-based staff training. This training was to improve awareness of issues faced by transgender patients, knowledge around gender dysphoria, and increase confidence in discussing these issues appropiately with patients.Staff from a range of disciplines attended sessions held on the ward in small groups; these bite size sessions were delivered in under 20 minutes making them easy to fit around clinical commitments.All attendants rated increased confidence in their skills and ability to support transgender patients.Improved staff training specifically focussing on transgender patients can contribute towards improved care for this patient group; this should form part of a wider strategy including clear operational policy and supportive environments."} +{"text": "Journal of Cachexia, Sarcopenia, and Muscle.All authors certify that they comply with the ethical guidelines for authorship and publishing in the We have read with great interest the recent excellent article by Freire and colleagues, which comprehensive analysed the gene expression profiling of cachexia\u2010inducing factors (CIFs) in 12 human cancers.Firstly, the authors limited their study to The Cancer Genome Atlas (TCGA) and Genotype\u2010Tissue Expression databases (GTEx) and did not investigate other international public databases with mass expression profiling, including Gene Expression Omnibus (GEO), International Cancer Genome Consortium (ICGC), and ArrayExpress. Selection of a restricted subset of databases for characterizing the expression profile of secretome genes can lead to biased results, even wrong conclusions. Meanwhile, authors just concentrated on the mRNA profiling rather than protein expression levels of cachectic soluble factors. It will be better to analyse the protein expression levels of these genes based on reverse\u2010phase protein arrays (RPPAs) from The Cancer Proteome Atlas (TCPA)Secondly, the authors explored the expression profile of these 25 CIFs based on tumour purity caculated by ABSOLUTEP\u00a0<\u00a00.1) using a stepwise selection/backward elimination process for multivariate Cox analysis.Finally, the authors found that the altered expression of these 25 CIFs can predict the poor overall survival of 12 cancer types using the univariate Cox analysis. We do not think the evidence is clear enough, because this analysis was performed without considering any other clinical characteristics. It is better to perform univariate Cox analysis for clinicopathological factors and then incorporate the prognostic factors identified by univariate Cox analysis (In conclusion, the authors analysed a valuable question regarding the CIFs in human cancers, but the results of this study should be explained with caution due to the limitations mentioned above."} +{"text": "This study addressed three questions: 1) Is living in Chinatown associated with better cognition among Chinese older immigrants? 2) Is the association moderated by education, acculturation level, and social engagement? 3) Does the association vary by preferred language , an important indicator of heterogeneity among Chinese immigrants? Data were derived from the Population Study of Chinese Elderly in Chicago . Results showed that Chinese older immigrants who lived in Chinatown had significantly poorer cognition than those who didn\u2019t, and such a difference was largely due to educational differences between the two groups. Higher education or acculturation buffered the influence of Chinatown residence on cognitive health, but only among those who speak Mandarin. The findings indicate that living in an ethnic enclave may have a negative impact on cognitive function of Chinese older immigrants. The findings also reveal the sources of heterogeneity within the population."} +{"text": "This case series examines how individuals who died by suicide responded to a survey question regarding access to firearms before death. We stratified findings by firearm and other suicide methods, because opportunities for suicide prevention may differ between these groups. The Kaiser Permanente institutional review board approved this study and waived the need for patient informed consent, because use of this protected health information involved no more than a minimal risk to the privacy of individuals. This study followed the reporting guideline for case series.This population-based case series utilized Washington State death records and electronic health record data to identify Kaiser Permanente Washington patients who received outpatient care within the year prior to suicide death. Cause-of-death indicators for firearm vs other suicide means were defined using During the observation period, 236 ambulatory care patients died by suicide, including 114 (48%) who died by firearm , of whom 104 (91%) had utilized care in 1 or more clinics using the MH questionnaire with the firearm question . Sixty-sOur findings have important implications for health care systems that are considering firearm access screening to support suicide prevention. First, this study underscored the potential reach of standardized firearm access questions in primary care clinics, which implemented routine use of this question during the study and where the highest proportion of firearm suicide decedents were seen prior to death. Second, the decision to only ask primary care patients with a MH or SUD diagnosis about firearm access likely resulted in missed opportunities, as many firearm suicide decedents did not have these diagnoses.5 However, more than half of those who answered the question and who subsequently died by firearm suicide reported no firearm access. A study limitation is that we cannot determine whether these individuals acquired access after answering the question or answered no despite having access. Nevertheless, prior qualitative findings suggest that transparency about how firearm access information will be used and building clinician competency and clinician\u2013patient trust may encourage honest reporting, open dialogue, and improved patient-centeredness of this practice.6 Assessing patients\u2019 plans to acquire firearms may also be useful. Future work is needed to test language and strategies designed to encourage patient-reported firearm access.This study also highlighted the need to improve how health care systems ask standardized questions. Most patients who died by firearm suicide answered the firearm access question when they received it, confirming our prior finding that patients will answer this standardized firearm access question."} +{"text": "Human lifespan is a multifactorial trait resulted from complicated interplay among many genetic and environmental factors. Despite substantial progress in clarifying many aspects of lifespan\u2019 variability the mechanism of its multifactorial regulation remains unclear. In this paper we investigate the role of genes from integrated stress response (ISR) pathway in such regulation. Experimental studies showed that persistent cellular stress may result in cellular senescence (for proliferating cells), or in apoptosis (for post-mitotic cells) which may affect health and lifespan in laboratory animals. These studies also showed which ISR genes are likely to interplay to produce joint effects on these traits. Note that in humans, the interplay between these genes does not necessarily influence these traits. This is because biological mechanisms regulating these traits in laboratory animals and humans may differ. This means that, when possible, the experimentally detected connections promising for human applications, should be verified using available human data before their testing in expensive clinical trials. In this paper we used HRS data to test connection between SNPs from the EIF2AK4 gene that senses cellular stress signals and the DDIT3 gene from the apoptosis regulation part of the ISR. We found genome wide significant associations between interacting SNPs from these genes and longevity. This result shows that available human data may be successfully used for making important steps in translation of experimental research findings towards their application in humans. Following this strategy may increase efficiency of clinical trials aiming to find appropriate medications to promote human health and longevity."} +{"text": "The Sage Resource Project aimed to broaden the pool of researchers who include the voice of older adults using long-term services and supports (LTSS) in research processes. We developed training to build researcher capacity to engage older adults through the development of Sage Model research advisory boards. Methods included training strategies for learning mode, design, duration, and emphasis of content that were informed by results of a researcher needs assessment and input from 2 older adult research advisory boards. Over 100 researchers registered for a 4-webinar series. All respondents to webinar evaluations (22) reported learning about topics that aligned with webinar objectives and had interest in engaging older adult stakeholders and/or developing an older adult research advisory board in the future. Representatives from five universities expressed interest attending online interactive workshops to build advisory boards. Lessons learned identify directions for research on best practices for developing older adult advisory groups."} +{"text": "In the pharmaceutical industry, competing for few validated drug targets there is a drive to identify new ways of therapeutic intervention. Here, we attempted to define guidelines to evaluate a target\u2019s \u2018fitness\u2019 based on its node characteristics within annotated protein functional networks to complement contingent therapeutic hypotheses.We observed that targets of approved, selective small molecule drugs exhibit high node centrality within protein networks relative to a broader set of investigational targets spanning various development stages. Targets of approved drugs also exhibit higher centrality than other proteins within their respective functional class. These findings expand on previous reports of drug targets\u2019 network centrality by suggesting some centrality metrics such as low topological coefficient as inherent characteristics of a \u2018good\u2019 target, relative to other exploratory targets and regardless of its functional class. These centrality metrics could thus be indicators of an individual protein\u2019s \u2018fitness\u2019 as potential drug target. Correlations between protein nodes\u2019 network centrality and number of associated publications underscored the possibility of knowledge bias as an inherent limitation to such predictions.Despite some entanglement with knowledge bias, like structure-oriented \u2018druggability\u2019 assessments of new protein targets, centrality metrics could assist early pharmaceutical discovery teams in evaluating potential targets with limited experimental proof of concept and help allocate resources for an effective drug discovery pipeline.The online version contains supplementary material available at 10.1186/s12859-021-04342-x. Pharmaceutical companies strive to select suitable targets and minimize attrition. This has driven more than two decades long efforts towards the identification and annotation of \u2018druggable\u2019 fractions of the genome \u20133. A semMany naturally existing networks, including biological signaling networks, exhibit an approximate scale-frThe application of graph theory to the analysis of biological networks has been largely focused on the \u2018architecture\u2019 of biological signaling , 21. SomThese studies highlighted a few general trends and some contradictions. Depending on the analysis and networks \u201331 it waAs annotated biological signaling or protein interaction networks are influenced by their underlying data sources, annotation method, and completeness, so may be the outcome of analyses applied to these networks. Recognition of this possible source of bias through cross comparison of different networks or generation of consensus networks was limited in previous studies , 12.In the current study, we attempted to address discrepancies apparent from the comparison of previous studies and implement a broad evaluation of node centrality metrics along with parallel comparison of multiple source networks/databases. We reasoned that such comparative inspection would minimize any bias derived from their different annotation sources and assembly strategies. In our analysis we evaluated whether any network positioning and centrality features would discriminate \u2018ideal\u2019 target proteins, associated to selective marketed drugs not only from the entire proteome, but also from other proteins of potential pharmaceutical interest. Additionally, we dissected comparisons between network characteristics of drug targets versus other proteins over their respective target classes to identify differences that would not merely arise from the biased target class composition of drug targets. Last, we evaluated the entanglement between protein nodes characteristics within annotated functional networks and their literature enrichment as a measure of the knowledge bias that may influence the outcome of these analyses.Previous studies demonstrated that drug target proteins in general exhibit higher centrality within signaling networks than other proteins \u201312. To iWe first analyzed these sets of proteins by calculating their node parameters within the String database network , 34 across the entire dataset and class-specific comparisons we repeated the above described evaluation of node descriptors from additional networks generated through diverse annotation strategies; (2) we compared differences in centrality between Phase4 and all targets (subdivided in their respective target classes) against their relative number of literature references (within each network and across networks); (3) we compared dataset-wide correlations between centrality metrics of individual nodes and number of associated references to the probabilities of increased Phase4 targets centrality within each network.We sought to address the critical prospect that this result could be a First, we analyzed node centrality parameters for additional publicly available protein signaling networks Table : String This assessment indicated that differences in centrality between Phase4 and all targets identified in the String0.7 network were not completely robust to switch of protein functional network sources, partially reconciling the conflicting conclusions of earlier studies \u20137, 12, tlog , Table log of Phase4 targets was higher than that of comparable targets for other target classes , indicating a higher degree as the dominant centrality metric for these target classes. This combined parameter exhibited generally minor differences between Phase4 and other GPCRs suggesting that the increased centrality of Phase4 GPCRs reflects with minimal deviation the reverse correlation between nodes degree and topological coefficient.We measured differences between Phase4 and all targets after degree \u2018normalization\u2019 of the nodes\u2019 topological coefficient .Projections of the original network degree distributions of the \u2018nuclear receptors\u2014Phase4\u2019 and \u2018nuclear receptors\u2014all targets\u2019 protein sets onto the normal degree distribution of random networks were obtained by selecting in each random network two pairs of random sets of nodes of equal size as the nuclear receptors sets . Each random set met the following criteria \u2009<\u2009log(Kmean). Normality of the selected random sets was verified using the Shapiro\u2013Wilk test.This procedure approximates a log scaling of the original String0.7 degree distribution but circumvents the variation in relative distributions after scaling caused by mean(logKWe designed Knime workflowP\u2009=\u20090.05 were noted as meaningful and highlighted in Table The full list of GO annotations for the human proteome (goa file) was uploaded in a Knime workflow where GO terms were ranked by frequency and filtered to include 95% of all associations, resulting in 25 high frequency GO terms. Gene identifiers were matched to the String07 network and GO terms associations were enumerated by target status and protein class. Relative enrichment or depletion of terms between protein groups was assessed by evaluating the normalized ratios and differences between individual GO terms associated to different groups of proteins . A statistical analysis of these differences was performed by assessing the statistical power of the observed effect sizes relative to the sample size of each group of proteins. Differences exhibiting\u2009>\u20090.8 statistical power with target Additional file 1. Supplementary Figures 1 to 15, Supplementary tables 1 to 7.Additional file 2. Excel spreadsheet containing network analysis data, GO term definitions and output tables for predictive models of drug target proteins.Additional file 3. Knime workflows utilized to generate predictive models of drug target proteins from network centrality metrics, GO terms and annontated disease associations."} +{"text": "Direct care workers are paid health care professionals who provide hands on assistance with daily activities to persons with disabilities in home, community, and institutional settings. Many workers are employed by direct care agencies, but little is known or understood about the organizational attributes of these agencies. We describe results from a mixed mode survey of n=1112 residential care agency administrators in Maryland to assess organizational and direct care worker characteristics. Preliminary findings indicate that half of direct care agencies\u2019 revenue comes from Medicaid and roughly 40% of clients are living with dementia. Administrators report challenges managing dementia-related behaviors (70%), communicating with persons living with dementia (63%) and interacting with family caregivers (63%). Findings from this work will inform the development of an organizational level intervention that targets training and support of direct care workers."} +{"text": "Recruitment efforts just after the recent COVID crises brought in several new graduate nurses. They had limited clinical exposure during COVID-19 resulting in difficulty transitioning into practice providing safe patient care. As a result, these nurses lacked the fundamental knowledge needed to care for acutely ill burn and wound patients resulting in the new graduate registered nurses (NGRNs) feeling overwhelmed at the bedside. These findings coincide with assessments noted in Kavanagh and Sharpnack\u2019s (2021), article identifying only 9% of NGRNs were practice ready, with 7% failing to recognize urgency or a change in a patient\u2019s condition.In order to achieve the designated American Burn Association (ABA) competencies, our center designed a program based on Patricia Benner\u2019s Novice to Expert nursing theory. Additionally, we divided the competencies into achievable goals and domains using the Donna Wright's nursing competency model.StaRN program: didactics/simulation/skills/unit orientation one on one with a preceptorCompetency based staged orientation program for new staffBurn Specific Education includes:1) Burn and complex wound care didactic2) Burn specific High-fidelity simulation scenario utilizing critical care equipment promoting critical thinking and critical reasoning skills3) Task trainers4) On-going preceptor education5) Nurse Extern programNGRNS arriving at our unit in early 2020 were found to be incapable of performing clinical tasks in the burn ICU (BICU) setting at the level of competency recommended by the ABA. We immediately placed this cohort into the revised training program incorporating Benners Novice to Expert Theory and Wright\u2019s Competency Model. Of the 25, 17 were able to be placed in the BICU (68%), and 8 were able to transfer to a lower level of care (progressive care/med-surg). All 25 were given extended orientation . We will follow this group to determine retention rates.Current levels of competencies by the ABA creates gaps in care for graduate nurses entering the workforce with deficits. Applying Benner's Novice to Expert Theory and Wright\u2019s Competency Model to modify approaches to training helps identify gaps in care, addresses areas that are weak for the nurse, and help guide the graduate nurse through stages of expertise to arrive more confidently at the level of competency expected by the ABA."} +{"text": "The literature is lacking in theoretically grounded techniques to teach interprofessional skills specific to caring for older adults. This presentation details how Wagner\u2019s Chronic Care Model and the Constructivist/Active Learning theoretical frameworks were used in the design of an interprofessional education. The content of the education was modeled after Wagner\u2019s chronic illness care model that advocates changes in processes and organizational structures to promote interprofessional team practice. The educational intervention follows a Constructivist/Active learning framework delivered in a simulation format. Constructivist approaches encompass active learning and guided experiential learning procedures, methods well-suited to our scaffolded simulation educational experience."} +{"text": "Ipsilateral recurrent laryngeal nerve paralysis is one of the rare complications during the superior mediastinal node dissection for lung cancer. However, very few reports of contralateral recurrent laryngeal nerve paralysis during the procedure are available.Two women aged 74 and 80\u00a0years developed hoarseness after undergoing right upper lobectomy and right superior mediastinal node dissection for primary lung cancer. Postoperative laryngoscopy in the two patients confirmed left vocal cord paralysis.Node dissection is performed in the standard procedure for right upper lobe lung cancer. At this time, care must be taken not to cause damage not only to the recurrent laryngeal nerve on the ipsilateral side but also to the recurrent laryngeal nerve on the contralateral side. Vocal cord paralysis may occur as a complication during lung cancer surgery due to recurrent laryngeal nerve injury during the superior mediastinal node dissection. Lymphadenectomy during surgery for right upper lobe lung cancer involves superior mediastinal node dissection, but very few reports of the left recurrent laryngeal nerve paralysis during surgery for right-lung cancer are available. This paper reports two cases of left recurrent laryngeal nerve paralysis after surgery for the right upper lobe lung cancer.A 74-year-old woman was admitted to our hospital with suspected primary lung cancer in the right upper lobe clinical T1bN0M0, stage IA2. Right upper lobectomy and superior mediastinal node dissection were performed under general anesthesia with selective lung ventilation using a double-lumen tube. Nodes in the right upper paratracheal nodal station (# 2R) and right lower paratracheal nodal station (# 4R) were dissected. Nodes in # 2R and # 4R showed no swelling, but were removed contralaterally along the anterior surface of the trachea , neuropathy may be caused by exclusion by operation in the vicinity or energization using an energy device in the vicinity.Stimulation by intubation may be the cause of hoarseness rather than the operation of the surgical field. Arytenoid cartilage subluxation and recurrent laryngeal nerve injury are considered the causes of hoarseness due to intubation . HoweverCareful manipulation is required to prevent left recurrent laryngeal nerve injury during right superior mediastinal node dissection with contralateral recurrent laryngeal nerves in the vicinity if lymphadenopathy is significant.The right lower paratracheal node dissection over the right lateral wall of the ascending aorta should be avoided when the lymphoadenopathy in the vicinity is not suspected. The right lateral wall of the ascending aorta is defined as the left side boundary plane for the # 4R station. Clearance beyond the right wall of the ascending aorta can cause contralateral recurrent laryngeal nerve damage and should be avoided.Node dissection is performed in the standard procedure for right upper lobe lung cancer. At this time, care must be taken not to cause damage not only to the recurrent laryngeal nerve on the ipsilateral side but also to the recurrent laryngeal nerve on the contralateral side."} +{"text": "This study examined perceived quality of life in Chinese older adults living with cognitive impairment in a group of urban Chinese older adults and explore its associations with caregivers\u2019 characteristics. Questionnaires were administered in person to 300 caregiver-care recipient dyads from three urban communities in mainland China in 2019. The 40-item Alzheimer\u2019s Disease-related Quality of Life tool asked caregiver respondents to indicate care recipients\u2019 life conditions. Higher levels of caregiving burden and more depressive symptoms amongst caregivers were significantly associated with lower quality of life of care recipients. The results suggested that reducing caregivers\u2019 burden and depressive symptoms are essential to promote quality of life of care recipients. Formal support from health professionals, service organizations, and communities are urgently called for to promote the wellbeing of Chinese families affected by cognitive impairment."} +{"text": "There is limited evidence on effect of high and low dose oxytocin used for labor induction on perinatal outcomes. We compared perinatal outcomes among pregnant mothers who received the two different oxytocin regimens and identified risk factors associated with adverse perinatal outcomes.Facility based comparative cross-sectional study was conducted in four hospitals of Ethiopia over eight month\u2019s period during 2017/2018 year with 216 pregnant women who received high and low dose oxytocin for labor induction. Socio-demographics, reproductive characteristics of mothers and perinatal outcomes data were collected and entered into Epi-data version 3.1 and then exported to SPSS version 20 for cleaning and analysis. Chi-square test and logistic regression were done to see the effect of different oxytocin regimens on perinatal outcome. The result was presented using 95\u2009% confidence interval of crude and adjusted odds ratios. P-value\u2009<\u20090.05 was used to declare statistical significance.Higher adverse perinatal outcomes and higher non-reassuring fetal heart rate pattern was observed among mothers who received high dose oxytocin compared to mothers who received low dose oxytocin. Using high oxytocin dose , caesarean birth , instrumental birth , and antepartum hemorrhage were risk factors of adverse perinatal outcomes.There was significance difference in the occurrence of adverse perinatal outcomes among pregnant mothers who received high and low dose of oxytocin. Using high dose oxytocin, antepartum hemorrhage, caesarean birth and instrumental birth were associated with increased risk of adverse perinatal outcomes. We recommend using low dose oxytocin for better perinatal outcomes. Labor induction is aimed at achieving vaginal birth when the benefits of giving birth out-weigh the risk of continuing the pregnancy to the mother and perinatal outcome , 2. OxytOxytocin regimen can be either high dose or low dose depending on the starting dose, the amount and rate of sequential increases in the dose of oxytocin . IntervaTo date, there is no strong recommendation of a particular dosage of oxytocin regimen for labor induction \u201312. AlthFacility based comparative cross-sectional study was conducted in four hospitals of Ethiopia namely Jimma University Medical Centre (JUMC), Shanan Gibe general hospital, Arbaminch General Hospital and Kuyu General Hospital over eight months period during 2017/2018 year. JUMC is a tertiary teaching hospital with high birth rate capacity about 4300 births per year and uses high dose oxytocin regimen while the other three were with lower birth rate capacity utilizing low dose oxytocin regimen. Arbaminch general hospital has 1500 births per year, Kuyu general hospital about 1350 births per year and Shenen gibe general hospital has nearly 1400 births per year. The methodology used in the current study is similar to that of another study related to the same project which has been published in BMC Pregnancy and Childbirth has showed statistically significant association with occurrence of adverse perinatal outcome at P-Value\u2009<\u20090.05 ] were found to be associated with adverse perinatal outcome at Using high dose oxytocin was associated with increased risk of developing adverse perinatal outcomes by 2.5 times compared to low dose oxytocin regimen. Neonates born from laboring mothers who received high dose of oxytocin were more at risk of developing adverse neonatal outcomes. This finding was inconsistent with other studies that showed no significant difference on perinatal outcomes with regard to oxytocin regimen , 11, 12 Cesarean birth and instrumental birth were also associated with increased risk of developing adverse perinatal outcomes by 9.4 times and 8 times compared to vaginal mode of birth in this study. This is consistent with other studies that showed increased risk of perinatal morbidities , 16. OneSimilarly, antepartum hemorrhage (APH) as indication of IOL was associated with increased risk of developing poor perinatal outcomes by 18 times compared to post-term indication. The fact that APH chiefly abruption-placenta, causes severe perinatal morbidities, severe neonatal acidaemia & cerebral palsy might have led to poor perinatal outcomes [The limitation to this study was, it was conducted in a teaching and public health facilities where complicated pregnancies come to tertiary center JUMC in this case. The parity of the study participants were not matched among the two groups proportion of primi-gravida being higher at high dose center. It is known that primi-gravidity is associated with higher adverse perinatal outcome compared with multiparas. These two factors might have affected the finding.It was found that oxytocin regimen didn\u2019t show significant association with induction success and adverse maternal outcome in another article of the same project unlike adverse perinatal outcome that increase with high dose oxytocin , 17.High dose oxytocin regimen, antepartum hemorrhage, caesarean delivery and instrumental delivery were associated with increased risk of developing adverse perinatal outcomes. The study favored use of low dose oxytocin as it\u2019s associated with decreased risk of adverse perinatal outcomes without affecting induction success and maternal outcome."} +{"text": "Pure periorbital electrical injuries are uncommonly reported and may cause both immediate and delayed complications. These injuries are rare and pose a difficult challenge for both ophthalmologist and plastic surgeon. Here we report an unusual case of pure periorbital electrical injury in a 12-yr old boy while drinking water from water dispenser. Future prognosis of periorbital burn injury depends on duration of exposure, mechanism of injury, tissue damage, quality of treatment and risk of infection2.In patients with severe burn usually the face will be affected and facial burn lesions will lead to psychological and physical morbidity. Ocular and periorbital injuries are reported in 20% facial thermal burnsA 12-year old boy referred to our clinic in Shiraz University of Medical Sciences, Shiraz, Iran with deep electrical burn at medial side of left periorbital region and left medial canthus. He was accidentally electrocuted while drinking water directly from water dispenser in the school yard . On examETHICAL APPROVAL Informed consent was obtained from this patient. All procedures performed in this patient were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.3. Patients with extensive burn require fluid resuscitation upon admission and in the emergency phase patients with facial burn should be fully examined to rule out periorbital injuries4. Patients with severe facial burn often have large total body surface areas burned and therefore require a prolonged intensive care unit course with periods of medical instability that may prevent timely return to the operating room. In addition, these patients may require prolonged periods of sedation that may inhibit their ability to protect their own corneas.Electricity is a serious environmental and health hazard especially in developing countries5,6. Current eyelid management protocols include aggressive lubrication in the days following injury and timely excision and skin grafting with thick split-thickness or full-thickness skin grafts7. In addition, aggressive treatment of ectropion with repeated grafting is also part of modern eyelid burn protocols8.Management of burned eyelids can provide challenges in both acute and reconstructive burn periods. Suboptimal eyelid burn management can result in potentially devastating complications including severe eyelid contracture, ectropion, corneal ulceration, and even vision lossThe reported case had a rapid and complete healing thanks to the timely and appropriate treatment.Pure electrical periorbital injury is an uncommon event. It is important to note that by taking simple measures electrical eye injuries are preventable. Therefore holding a good public awareness program not only can save eyes but also lives."} +{"text": "Caregivers of persons living with Alzheimer\u2019s disease and Alzheimer\u2019s disease-related dementias benefit from unique interventions to address their different needs. While information on which interventions best meet specific needs exists, less is known about how to match caregivers with those interventions. To address this research gap, we tested Care to Plan (CtP) within a large healthcare system. After care navigators guided caregivers through twenty CtP tailoring questions to identify caregivers\u2019 greatest needs, the online tool provided the intervention type best suited to meet their needs, along with region-specific information on available programs. This mixed methods analysis evaluated the utility of the CtP tool with 20 family caregivers of PLWD after a 1 month follow-up. Most caregivers agreed that the CtP tool was helpful (85%) and would recommend the tool to other caregivers (90%). Caregivers also said they valued being able to discuss the CtP recommendations with the care navigator (95%). However, only 65% said they found services that met their needs or planned on using services recommended by CtP. Interview data indicate time constraints and restricted availability of resources due to COVID-19 precautions reduced caregivers\u2019 abilities to pursue some recommendations. In addition, the stage of dementia experienced by their care recipient may explain why others found CtP less useful. However, these caregivers noted the potential utility of the resources for their future care planning needs. A larger evaluation of the CtP tool within the healthcare system is ongoing."} +{"text": "African Health Sciences which is rich in innovative research and practice papers from all over the world. We have purposely kept away from Covid-19 this season because it was distorting our perception of ill health in Africa and beyond.Welcome to this June 2021 issue of Escherichia coli and Klebsiella pneumoniae isolates from type 2 diabetic patients with urinary tract infections keeping the Klebsiella theme aliveKlebsiella again! It is to a paper on the prevalence and antimicrobial susceptibility of extended-spectrum beta lactamases-producing Escherichia coli and Klebsiella pneumoniae isolated in selected hospitals of Anyigba, NigeriaHence we have focused largely on infectious diseases. Our first paper comes from Egypt. El- Domany and colleaguesBut our next paper is on \u2018bacterial profile and their antimicrobial susceptibility patterns in patients admitted at Madda Walabu University Goba Referral Hospital, Ethiopia.\u20196Asefa and others8This is followed by papers on urinary tract infections in TurkeyThen follows the prevalence and density of malaria parasitaemia amongst HIV individuals in Nigeria;16Muzanyi and colleagues19Now to cancer. We have several oncology papers. First, Uganda authorsWe continue this NCDs work with papers on diabetes mellitus. These include risk factors for type 2 diabetes mellitus in NigeriaNCDs continue. We have papers on \u2018circulatory microRNA expression profile for coronary artery calcification in chronic kidney disease patients;\u201931Now to the clinical. Nigerian clinicians have a paper on the relationships between \u2018cardiovascular signs and neurological signs in asphyxiated neonates.\u201937We have several papers on injury: traumatic spinal cord injury and clinical complications;42We bring you several papers on sexuality: a new grading system for female sexual dysfunction in Egyptian women49The remaining papers are on diverse issues such as availability of low vision services in Ghana;African Health Sciences: the home of truly free online publishing in Africa!We thank all our readers, authors, reviewers, our team of highly dedicated volunteers, collaborators, partners and colleagues for their immeasurable contribution to"} +{"text": "Mild cognitive impairment (MCI) research faces challenges to successful enrollment, especially with clinical trial studies. This study explores researchers\u2019 experiences recruiting from a U.S. Alzheimer\u2019s Disease Center for a pilot, platform trial of biopsychosocial interventions for MCI dyads. Individuals with MCI that met the inclusion criteria for the study were invited to participate (n=39). Thematic analysis of recruitment case notes was utilized to track participants\u2019 and study partners\u2019 interest in participation. In most cases, participants with MCI were interested and willing to enroll and study partners were not. Recruiting persons with MCI and their study partners for clinical trials research may require specialized communication messaging such as education about how interventions address the needs of MCI, along with training on the relationship of MCI to cognitive decline. This presentation highlights effective strategies to engage study partners into recruitment for MCI research such as creating more flexible participation roles and offerings."} +{"text": "Coping strategies are important factors that influence caregivers\u2019 mental health outcomes. The purpose of this study is to examine the association between coping strategies and caregiver burden and depression among Chinese caregivers of older adults with cognitive impairment. Data came from structured interviews with 300 primary family caregiver-care recipient dyads in Wuhan, China. We used OLS to examine the association between coping strategies and caregiver burden and depression. More positive reframing and acceptance were associated with lower caregiver burden, whereas more self-distraction was associated with higher caregiver burden. More positive reframing was associated with lower caregiver depression, whereas higher self-distraction and religion were associated with higher caregiver depression. Findings of this study suggest that a psychosocial intervention package that emphasizes on enhancing positive reframing skills and affirming acceptance may be effective in reducing caregiver burden and depression among Chinese caregivers of older adults with cognitive impairment."} +{"text": "Stenella longirostris longirostris) rest by day in protected bays that are increasingly popular for recreation. Because more frequent interactions of people with these dolphins is likely to reduce rest for dolphins and to explain recent decline in dolphin abundance, the National Oceanic and Atmospheric Administration (NOAA) proposed stricter rules regarding interactions with spinner dolphins near the main Hawaiian Islands and plans to increase enforcement. Simultaneous investment in public education about both interaction rules and their biological rationale has been and is likely to be relatively low. To test the hypothesis that more educational signage will reduce human-generated disturbance of dolphins, a paper questionnaire was distributed to 351 land-based, mostly unguided visitors at three dolphin resting bays on Hawai\u2018i Island\u2019s west coast. Responses indicated that visitors wanted to see dolphins, were ignorant of interaction rules, were likely to read signs explaining rules and their biological rationales, and were likely to follow known rules. Therefore, investment in effective educational signage at dolphin resting bays is recommended as one way to support conservation of spinner dolphins on Hawai\u2018i Island\u2019s west coast and similar sites in the Hawaiian archipelago.Spinner dolphins on Hawai\u2018i Island\u2019s west coast ( Gorilla beringei beringei) mortality increased with transmission of respiratory pathogens from tourists and/or guides, marine iguana (Amblyrhynchus cristatus) immunological capabilities decreased with an increase in tourism intensity, and bottlenose dolphin (Tursiops sp.) abundance decreased with an increase in tour boat visitation . Such studies would help us understand when and to what extent educational signs are effective conservation tools.S1 File(DOCX)Click here for additional data file.S2 File(XLSX)Click here for additional data file."} +{"text": "To ensure future sustainability, cities need to consider concepts of livability and resident wellbeing alongside environmental, economic and infrastructure development equity. The current rapid urbanization experienced in many regions is leading to sustainability challenges, but also offers the opportunity to deliver infrastructure supporting the social aspects of cities and the services that underpin them alongside economic growth. Unfortunately, evidence of what is needed to deliver urban wellbeing is largely absent from the global south. This paper contributes to filling this knowledge gap through a novel interdisciplinary mixed methods study undertaken in two rapidly changing cities (one Thai and one Kenyan) using qualitative surveys, subjective wellbeing and stress measurements, and spatial analysis of urban infrastructure distribution. We find the absence of basic infrastructure unsurprisingly causes significant stress for city residents. However, once these services are in place, smaller variations in social and environmental conditions begin to play a greater role in determining differences in subjective wellbeing across a city. Our results indicate that spending time in urban greenspaces can mitigate the stressful impacts of city living even for residents of informal neighborhoods. Our data also highlights the importance of places that enable social interactions supporting wellbeing\u2013whether green or built. These results demonstrate the need for diversity and equity in the provision of public realm spaces to ensure social and spatial justice. These findings strengthen the need to promote long term livability in LMIC urban planning alongside economic growth, environmental sustainability, and resilience. With the global transition to urban living, cities need to become sustainable in the broadest sense, which increasingly includes concepts of wellbeing and quality of life alongside environmental and economic considerations . The HabRapidly developing cities in low-middle- income countries (LMIC) represent unique challenges and opportunities for the delivery of such sustainable development. The current rapid urbanization of sub-Saharan Africa is putting pressure on natural resources and the environment, increasing environmental- and climate change-related vulnerabilities, urban poverty and the proliferation of informal settlements . These cUnfortunately, such unplanned growth often outpaces infrastructure provision and occurs at the expense of a city\u2019s ecological foundations, undermining resident\u2019s wellbeing and the city\u2019s sustainability . AddressWellbeing supporting environments that promote mental health allow individuals realize their own abilities, cope with the normal stresses of life, work productively and fruitfully, and contribute to their community . ResearcThis paper explores these multiple dimensions of city developments impacts on resident\u2019s wellbeing in LMIC contexts. We present results from two complementary LMIC cities exploring the interaction of urban form on wellbeing. Our findings address knowledge gaps that call for greater granularity of data to explore interactions with income, gender and environment . Our ana1. How are objective aspects of wellbeing (distributed according to socioeconomic and sociodemographic characteristics) related to subjective assessments of wellbeing (life satisfaction)?2. How is the relationship between subjective wellbeing mediated by the quality of urban environments?3. What are the implications for urban development to achieve equitable wellbeing improvements?We addressed these topics in relation to three interlinked questions:To investigate these questions in real world settings two comparable but contrasting secondary cities of the Global South were selected as representative examples in which to explore these concepts and the county capital. Nakuru lies at an altitude of 1,850\u00a0m and has a Mediterranean climate (K\u00f6ppen-Geiger climate classification is Csb) remaining temperate throughout the year with no annual dry season. According to the County Integrated Development Plan 2018-2022, Nakuru town had an estimated population of 405,000 in 2018 which is expected to reach 458,000 by 2022 (a 13% increase). It has a mixture of built environments, including informal and unplanned settlements and both green and blue spaces. Rapid growth in Nakuru is putting development pressure on the public realm including greenspace.Udon Thani in northeast Thailand is a small city of 130,000 residents facing rapid development due to its strategic location near the Laotian border. The city has a tropical savanna climate (K\u00f6ppen-Geiger classification Aw) with warm dry winters followed by a 6-month monsoon season. Through the Udon Charter for 2029, a multi-stakeholder vision for the city, the city is committed to achieving six policy points, driven by the objective of becoming a green city focused on MICE . It seeks to have a walkable urban core, invest in green transport and green infrastructure including parks and public realm spaces.Wellbeing can be considered a key component for a person\u2019s quality of life and encapsulates both objective and subjective elements. The objective dimensions define wellbeing in terms of quality-of-life indicators including access to basic needs resources and social attributes . The subjective dimension emphasizes people\u2019s own life evaluations including satisfaction and happiness . SubjectThis paper reports on the findings from two surveys detailed below: a bespoke neighbourhood survey investigating dimensions of socio-economic, environmental and wellbeing conditions (see 2.2.4.1 and 2.2.4.2 below); and a validated scale questionnaire exploring individual mood effects in different urban settings (see 2.2.4.3). All the survey tools received individual ethical approval via the relevant University of York, UK committee and participants gave informed consent. To facilitate accurate completion surveys were translated into local languages appropriate for each city.The wellbeing survey was carried out across diverse neighbourhoods (six in Nakuru (during November 2018 dry season) and seven in Udon Thani (during December 2018 warm season)), identified in collaboration with city officials and local project partners, which represented a cross-section of local environmental, social and economic conditions ranging from central to suburban locations, including fully to partially serviced areas in terms of public utilities neighbourhood . Adults (over the age of 18) were recruited through on-street intercepts in each neighbourhood aiming for a gender balanced sample.To assess the impact of different types of urban spaces upon mood, a young (18\u201330\u00a0years) gender balanced, self-reported healthy, cohort of residents were recruited. This cohort was purposively selected to control for impacts of ageing on mobility and wellbeing as these participants also undertook recordings of heart rate variability (reported on in an upcoming paper) in different urban locations. Participants undertook transect walks between a busy built public realm space (market) and a quieter greenspace (public park) via other important infrastructure (e.g. bus interchange). These start and end points were selected to maximise the contrast in terms of type of public realm space\u2013green vs grey; busy vs quiet. To control for the effects of direction the cohort was randomly sub-divided to undertake the walk in opposing directions . Transect walks and mood surveys were undertaken in april 2019 during Udon Thani\u2019s hot season and Nakuru\u2019s wet season. Walks were only undertaken on dry days and in early morning to avoid high temperatures.The neighbourhood survey included questions on the impact on respondent\u2019s wellbeing of eleven different environmental and social factors. The impacts ranged from large (scored 1) to no impact (4) on a forced four-point Likert scale. By summing the participant\u2019s response scores across the eleven variables, a composite indicator of objective wellbeing was created. The raw data was scaled for graphing to range between greater than zero and the range maximum by subtracting the integer value of the minimum objective wellbeing score for each city. This improves visualization but means the graphed values are city specific and should not be directly compared utilized the Short Warwick Edinburgh Mental Wellbeing Scale (SWEMWBS) that assesses subjective wellbeing through seven questions rated on a five-point Likert scale which have been validated for construct validity . The scaStress is inevitable and healthy factor of life. However, the duration and frequency of stress as well as someone\u2019s belief and ability to return to a non-stressed state has significant implications for overall health and wellbeing. The Perceived Stress Scale (PSS), is a measure of sub-chronic stress which evThe urban settings survey used the Acute Subjective Mood measured by the University of Wales Institute of Science and Technology (UWIST) Mood Adjective Checklist (MACL) to determine acute subjective mood changes between our two key locations (market and park). MACL is a 24-item checklist that gives an acute psychometric measure of hedonic tone , stress and arousal, shown as three scores. Respondents are required to complete the questionnaire before and soon after completion of activity to ensure measurement of momentary shifts in mood. The arousal scale measures feelings of subjective energy. The stress scale measures feelings of subjective tension and the hedonic tone scale measures overall pleasantness of mood and is associated with feelings of somatic comfort and wellbeing. Scores are obtained from summation of individual item scores pertaining to each of the three mood components.The age and gender distribution of survey participants in each city can be seen below in Natural urban spaces, often referred to as urban greenspace (UGS), have been defined as vegetated urban spaces . Whilst climateengine.org) to determine the mean NDVI values for the 12\u00a0months prior to the survey period to assess the most recent variations in greenness that could affect wellbeing. These images were clipped to official neighbourhood boundaries for both cities and the distributions of 29\u00a0m pixel values determined for input to statistical tests.To assess the quantity of greenspace satellite imagery processed to depict vegetation ) was accessed. Landsat imagery was processed by Climate Engine related to subjective assessments of wellbeing (life satisfaction)?\u2019 All the significant statistical analysis presented in this results sections are in data.worldbank.org) varied from $7808 for Thailand to $1816 in Kenya indicating significant overall differences in average living standards and economic prosperity between the two countries. Our indicators of socio-economic conditions (relative affluence) confirmed these key differences for our two case study cities. Our Kenyan city has a statistically significant higher number of self-employed and tenants compared to Thailand where more residents were employees and homeowners. Access to basic services were reported as unproblematic across Udon whereas there were significant impacts from lack of access to infrastructure including water in Nakuru. The objective wellbeing scores represent a continuum from the most affluent neighbourhood in Nakuru having similar scores to the least affluent in Udon (see GDP per capita in 2019 (Udon see .Employment status (employed versus self-employed) determined which neighbourhood residents can afford to live in. There was no significant difference in objective wellbeing except between the extremes of the best serviced district (Shabab) and the least affluent . Whilst this indicates similar infrastructure conditions across the majority of Nakuru neighborhoods analyzing by gender reveals significant differences in women\u2019s objective wellbeing scores (whilst men\u2019s do not vary significantly). Access to water, water quality and solid waste pollution were the most important differences in basic services and environmental conditions identified between the semi-informal neighborhoods and planned, more affluent locations.Tenancy status varied by neighbourhood indicating differences in home ownership levels across the city. Objective wellbeing varied significantly by neighbourhood. These results confirmed our sample neighborhoods had varying levels of affluence. Of the socio-environmental factors assessed, only traffic congestion was perceived to be having a \u2018somewhat negative\u2019 impact on wellbeing.Our subjective wellbeing metrics varied within our case study cities by neighbourhood and with gender.Perceived stress tracks with affluence and objective wellbeing metrics and varied significantly between the most (Section 58) and least affluent (Kaptembwo) neighborhoods. This indicates that the absence of basic infrastructure and employment uncertainty has a significant psychological impact on daily life.Our objective wellbeing data indicates that differences in the impacts from social conditions including the incidence of crime and anti-social behaviour between neighborhoods could be underlying factors affecting stress level variations. These take on a gendered dimension with significant differences in women\u2019s PSS between Kaptembwo (large to somewhat negative crime impacts (1.7); somewhat negative anti-social behaviour impacts 2.4) and Shabab (somewhat negative crime (2.26) and anti-social behaviour (2.46) impacts) see . Within and ShabWellbeing scores and stress were not significantly correlated with age.The SWEMWBS are lowest for the extreme\u2019s of high and low objective wellbeing neighborhoods indicating lower overall life satisfaction in these locations see with theBoth cities perceived stress results can be characterized as \u2018moderate\u2019. Perceived stress in Nakuru (mean score 18.25) was significantly higher than in Udon Thani (mean 14.24), however, subjective wellbeing scores were only marginally different. This highlights that even when urban conditions are a source of persistent stress, longer term personal life satisfaction can remain high.The following sections results present findings relevant to our initial research question of \u201chow is the relationship between subjective wellbeing mediated by the quality of urban environments?\u201dIn our survey, two aspects of the quality of physical environments were considered; availability of greenspace and public realm spaces both of which can be used for recreation promoting both physical and mental health.Our spatial analysis showed statistically significant differences in NDVI values between neighborhoods see . These wFor those participants who did utilize greenspace, spending greater than 2\u00a0h per week in natural environments led to significant improvements in subjective wellbeing (SWEMWBS) (from scores of 26.9 (\u00b14.5) to 28.3 (\u00b15.2)). This 2\u00a0h threshold links to recommended \u201cdoses\u201d of greenspace use found inThe survey findings identified a weak but significant association between neighbourhood and walking distance access to public realm spaces. Shabab, the best planned neighbourhood, reported the greatest accessibility . The survey also indicated that increased availability of public realm space led to greater use by residents. These results highlight the unequal distribution of public realm assets leading to different opportunities for residents to access sociable community spaces.The only significant effect of undertaking the transect walk was upon men\u2019s hedonic tone who ended their route in the park. Hedonic tone indicates feelings of happiness or sadness and this result suggests there maybe gendered effects to the benefits from public greenspaces.Our spatial analysis indicated differences between neighborhoods in terms of their extreme greenness or extreme greyness see . These dUse of greenspace did not lead to any significant differences in subjective wellbeing measures (SWEMWBS and PSS). The majority (67.5%) of respondents were making some recreational use of greenspace indicating this behaviour was ubiquitous, however, 65.7% (n = 375) of respondents were spending less than the two 2-h per week threshold. For those who did spend time in greenspace there was no significant relationship or time related benefit on subjective wellbeing or stress scores.There was a significant difference in the perceived accessibility of public realm spaces by neighbourhood. In general, those neighborhoods on the periphery had less access to public spaces than inner city locations. Approximately 70% of respondents are making use of public realm spaces for recreation, however, greater equality of provision could increase this usage. There was no significant impact on the use or length of time spent in public spaces for recreation on wellbeing or stress.Looking at the influence of route on the participants in the transect walk, those who began their walk in the park and ended in the market did not see a significant change in hedonic tone (happiness). However, participants who began their transect walk in the market and ended in the park saw a significant decrease in hedonic tone (indicating increased sadness). These decreases effected both men and women significantly. These results contradict the findings from Nakuru where there were hedonic benefits attributed to greenspace for men.Our results address how urban quality affects both objective and subjective wellbeing outcomes. The variation in our measured scores between neighborhoods across both cities confirms that our sample sites have a diversity of economic affluence allowing us to usefully compare how objective aspects of local environmental conditions interact with the subjective wellbeing of residents in LMIC settings.In Kenya, our data indicates how informal and poorly implemented infrastructure has resulted in unequal access to the provision of basic services. Further exploration of these results highlight that poor water access and quality, and solid waste pollution contribute to measurable differences in objective wellbeing impacts between planned and less affluent districts. However, across all neighborhood\u2019s people perceived the limited water access and crime incidence were undermining their wellbeing demonstrating that some challenges are ubiquitous.In comparison, in Thailand, overall infrastructure and socio-economic conditions were largely un-problematic. However, despite the effects on objective wellbeing being marginal there were a greater number of differences between neighborhoods related to variations in air quality, noise pollution and traffic congestion. This indicates that as relative affluence increases, the significance of marginal inequalities between neighborhoods can become more pronounced. Our Udon Thani data also mirrors findings from other middle income countries (Colombia) that mid-In Nakuru, subjective wellbeing predominantly lay in the \u2018good\u2019 range (scores of 26\u201328) whilst pSignificantly, our Kenya case results demonstrates that residents who make regular use of greenspace (greater than 2-h per week) show benefits to their subjective wellbeing independent of their neighborhoods conditions. This indicates the psychologically restorative benefits of greenspace can offset stress even for those living in informal settlements. In Thailand we did not find associations between wellbeing improvements and greenspace use. This could indicate that other factors influencing ability to spend time in greenspace that also affect stress or wellbeing, are masking any benefits of time spent in natural surroundings. The Thai satellite data revealed, whilst there were variations in greenness between the city-centre and peri-urban fringe, most neighbourhoods had significant levels of vegetation. We hypothesize an alternative explanation for these findings that as urban vegetation is more equitably distributed across a city, routinely exposing people to nature, spending time specifically in greenspace has less discernable mental health benefits. The young cohort of Udon Thani transect walk participants indicated a subjective preference for a sociable retail space over a city park again indicating that perhaps greenspace may be less appreciated when it is widely available.Our mixed-method approach highlights the complexity of these inter-relationship; however, they do identify a prioritization for urban planners when considering delivery of life satisfaction improvements. Our cross-city comparison highlights that delivering basic needs infrastructure or services universally must always be the primary city development priority. However, once these services are widely available urban form and management require greater attention. Wellbeing effects associated with variations in these factors begin to take on a gendered and age dimension independent of neighbourhood affluence (employment and housing status). This conclusion is supported by other studies undertaken in higher income locations .Our findings demonstrate that accessible public realm greenspace and neighbourhood greenery can offset some of the negative impacts on wellbeing of urban living even in challenging environments (socio-economic conditions) including informal settlements counteracting some income related health inequalities . This suOur results also highlight that neighbourhood greening needs to be culturally appropriate and relevant for local communities including the urban poor . This suOur results highlight that distributing greenery throughout cities enables a wider cross-section of residents to enjoy benefits to their underlying wellbeing without needing to spend dedicated time in specific parkland destinations . This imSuch distributed greenspace would also ensure equity in other ecosystem service benefits such as urban cooling; shading; biodiversity increases; and surface water flood mitigation . UnfortuCross-cutting development issues by their complex nature benefit from an integrated, multi-sector, consultative approach to problem solving if identified solutions are to be resilient in the cThe cross-sectional survey data used in this analysis represents a snapshot of conditions at a particular moment. Cities and communities are dynamic - investigating wellbeing\u2019s relationship to changing urban environments would therefore benefit from a long-term longitudinal approach, similar to cohort studies from health sciences. Including a wider range of quantitative data with which to compare subjective wellbeing results and environmental perceptions would also provide a more robust picture of the relationship between people and cities. This could include measuring environmental factors known to affect wellbeing such as air and noise pollution, temperature, and humidity, but also quantitative recording of locally relevant socio-economic conditions such as crime or fluctuations in employment levels. Also improving our understanding in a more nuanced way of the interactions of people and places beyond home neighborhoods would explore temporal and seasonal dimensions. Incorporating more qualitative data from participants would add significant richness and additional context to the findings. Results from a complementary survey undertaken by the paper authors in both case study cities on the cultural ecosystem services that different urban spaces provide addresses this shortfall to a certain extent . We alsoThis study contributes to filling data gaps from LMIC secondary cities on the impacts of urban living on resident\u2019s wellbeing. Our data highlights that delivering basic services to all neighborhoods should be the initial priority. Once these amenities are provided inequalities in the availability of other infrastructure and socio-cultural conditions begin to impact life satisfaction and stress. Our findings indicate that enabling residents to spend 2\u00a0hours per week in greenspace may generate similar wellbeing benefits to those identified in European studies. Improving equitable access across cities by dispersing green infrastructure should therefore be a key target for urban planners. Our Thai findings indicate that accessible greened spaces that support social interactions should be the preferred model for implementing these recommendations to support wellbeing for the widest cross-section of city residents.Rapidly changing cities need to take greater account of the impacts urban form have upon human health and wellbeing. Ensuring equitable access to greenspace entails city authorities prioritize maintaining existing green infrastructure whilst protecting locations that will enable the inclusion of public realm spaces as the urban area expand. Adding improved neighbourhood scale data on human health and wellbeing benefits to the understanding of other ecosystem services provided by urban nature could justify such protection. Our findings indicate that such evidence could counterbalance significant densification pressures driven by cities ambitions to improve efficiency through the conversion of natural spaces into conventional economic assets. Expanding nature provision as cities evolve rather than expensively retrofitting greenspace into built infrastructure is a more cost-effective strategy for LMICs. Further evidence is needed of the financial costs of poor mental health or the economic gains resulting from access to green infrastructure from a wider cross-section of LMIC cities to strengthen these recommendations ensuring they become a key development issue and priority for urban authorities."} +{"text": "Use of this data in conjunction with metabolomic and genomic results to predict response to lisinopril or ondansetron has been completed. METHODS/STUDY POPULATION: Study population consists of >2000 subjects recruited from the Emergency Medicine Specimen Bank at University of Colorado Denver (UCD). All patients presenting to the emergency department are approached to participate which significantly increases demographic diversity of our study populations. Clinical data is pulled from Health Data Compass data to be able to deliver de-identified). Effectiveness of lisinopril and ondansetron were investigated using metabolomic data collected via ultra-high performance liquid chromatography mass spectrometry and genomic data from Illumina chip technology to find relevant correlations. RESULTS/ANTICIPATED RESULTS: Obtaining retrospective clinical data from data warehouses comes with significant challenges to be addressed. Verifying all clinical variables from patient EHRs is a crucial step that requires extensive quality control steps. As well, ensuring data validity, appropriateness of data points pulled as relate to the study criteria and identifying alternate EHR data points is needed. Chart review is a critical step necessary to surmount these challenges. Additionally, use of retrospective EHR data often necessitates the development of novel definitions of clinical effectiveness that can be abstracted from the EHR\u2013 such as how to determine decrease in nausea without a visual analogue scale. DISCUSSION/SIGNIFICANCE OF IMPACT: Utilizing data warehouses to deliver EHR data provides a valuable tool for completing retrospective precision medicine projects. The validation of definitions for clinical outcomes identifiable retrospectively are necessary and provide novel guidance for future studies.OBJECTIVES/GOALS: Utilizing clinical electronic health record (eHR) data pulled"} +{"text": "COVID-19-associated pulmonary aspergillosis (CAPA) is a recognized but incompletely characterized secondary fungal infection reported to be associated with increased mortality in patients in the ICU. Case definitions have been published but lack clarity: clinical and radiological features are non-specific and rely heavily on mycological tests for which both a timely availability and accuracy pose an issue contributing to the wide range of incidence reported of 4%\u201334%. In April 2021, we introduced a CAPA guideline for ICU patients at St George's University Hospital including when to suspect and how to investigate for CAPA . Only one patient had an underlying host risk factor (B cell lymphoma). Of those defined with probable/possible CAPA, average time from intubation to treatment was 18.4 days (range 8\u201331) and from onset of COVID symptoms to treatment was 28.5 days (range 18\u201349). Three of 21 patients were treated empirically for CAPA and all 3 died prior to further investigation and 15 patients following an MDT were felt to have unlikely CAPA. Serum BDG and GM were negative in all patients with probable/possible CAPA.Twenty-one patients were prescribed the specified antifungals. All had CT imaging and varying microbiological investigations. Real-time clinical decisions on empirical antifungal prescribing for CAPA (start or stop) were made by a multidisciplinary team (MDT) comprising Microbiology and ICU physicians in light of clinical, microbiology and radiology findings. Nine of 21 patients prescribed empirical antifungals had radiological evidence of CAPA. The St George's CAPA guideline would have picked up two out of seven cases; two cases had antifungals started in another hospital so the utility of this guideline could not be assessed. Based on analysis of these cases the following changes were made to the guideline: (i) to assess patients \u22658 days post ICU admission (rather than \u226510 days) and (ii) assess patients following \u22653 days post appropriate antibiotic therapy (rather than following \u22655 days). Serum BDG and GM were negative in all patients with probable/possible CAPA prior to starting treatment therefore negative serum markers do not exclude diagnosis; this is consistent with experience reported elsewhere. Radiological presentations of CAPA were variable and wide ranging, and BAL sampling was not always possible, therefore we found the MDT approach including radiology, infection, respiratory and ICU invaluable in making decisions regarding treatment. This helped to reduce the use of fungal diagnostics, antifungal agents and appropriately manage patients with potential CAPA. The rationalization of antifungal use by the introduction of this guidelines was particularly important and also expedited the introduction of in-house fungal diagnostics."} +{"text": "Quality of clozapine clinic appointmentEffectiveness of clozapine clinic serviveCompliance with BCUHB guidelines for physical health monitoring in clozapine clinicsWe retrospectively audited 40 case notes 10 each from 4 differtent CMHT clozapine clinicsMy role in the Project & How does this represent my practice?I was the audit and overall lead for this projectI formulated the audit tool and registered my project with Audit Registration TeamI lead data collection and compilation of resultsThis audit followed up from a Coroner's investigation for a clozapine clinic patientClozapine is used for Treatment Resistant Schizophrenia but needs close monitoring due to potentially fatal side effectsNICE recommends annual monitoring of weight, blood pressure, waist measurement, blood glucose and plasma lipid levelsHas the patient been seen in the past year by clinician to monitor response to clozapine treatment?Has the clozapine plasma level been measured during the last year of treatment?Is brief MSE & Risk assessment documented during review?Has Life style modification advice been provided?Has annual physical health been completed?Has Annual CTP/CPA been completed and documented?Has the patient been allocated a named care coordinator?Has clozapine side effects monitoring been documented?Clozapine is a superior medication for the treatment of refractory schizophrenia and is also be effective for other conditionsClozapine is underused due to a variety of barriers related to the drug and its properties, the health care system & regulatory requirementsThis service evaluation/quality improvement project provides the framework for clozapine clinics evaluation and recommends strategies for improvement"} +{"text": "Species of carabid (ground) beetles are among the most important postdispersal weed seed predators in temperate arable lands. Field studies have shown that carabid beetles can remove upwards of 65%\u201390% of specific weed seeds shed in arable fields each year. Such data do not explain how and why carabid predators go after weed seeds, however. It remains to be proven that weed seed predation by carabids is a genuine ecological interaction driven by certain ecological factors or functional traits that determine interaction strength and power predation dynamics, bringing about therefore a natural regulation of weed populations. Along these lines, this review ties together the lines of evidence around weed seed predation by carabid predators. Chemoperception rather than vision seems to be the primary sensory mechanism guiding seed detection and seed selection decisions in carabid weed seed predators. Selection of weed seeds by carabid seed predators appears directed rather than random. Yet, the nature of the chemical cues mediating detection of different seed species and identification of the suitable seed type among them remains unknown. Selection of certain types of weed seeds cannot be predicted based on seed chemistry per se in all cases, however. Rather, seed selection decisions are ruled by sophisticated behavioral mechanisms comprising the assessment of both chemical and physical characteristics of the seed. The ultimate selection of certain weed seed types is determined by how the chemical and physical properties of the seed match with the functional traits of the predator in terms of seed handling ability. Seed density, in addition to chemical and physical seed traits, is also an important factor that is likely to shape seed selection decisions in carabid weed seed predators. Carabid responses to seed density are rather complex as they are influenced not only by seed numbers but also by trait\u2010based suitability ranks of the different seed types available in the environment. The study elucidates the possible mechanisms that bring weed seeds and carabid predators together, and then, it highlights the key ecological factors that determine strength and dynamics of seed predation interactions. This suggests C.\u00a0cilicrus larvae can potentially depress populations of C.\u00a0cyanus in the field and carabids is more complex. Some of the early carabid literature had proposed that carabid weed seed predators locate weed seeds upon random encounter carabid species rely upon most to guide their food location and selection responses. Despite this uncertainty however, the evidence strongly hints that chemoperception is likely the top candidate for roles related to food searching and food choice in carabid predators , as well as volatile chemicals specific to prey itself or to its pheromones , indicating that they were able to discriminate among the seed species offered in the olfactometer based on odor alone. Moreover, the species of carabid weed seed predators were able to excavate weed seeds buried down to 1\u00a0cm below soil surface, without considerable loss in seed\u2010finding efficiency seed foraging should not be mutually exclusive in weed seed predation interactions as frequency\u2010dependent models presume ; Methodology ; Resources ; Writing\u2010original draft . Christian J. Willenborg: Funding acquisition ; Project administration ; Supervision ; Writing\u2010review & editing ."} +{"text": "Xylotrupes beetles. By using a previously published phylogeny with 80 Xylotrupes taxa, we estimated the transition rates between the two phenotypic states . Based on the estimated transition rates, we then simulated possible phenotypic outcomes between sympatric species. We found that sympatric species were equally likely to evolve the same versus distinct phenotypic states based on the estimated transition rates given the phylogeny. The empirically observed number of sympatric species showing different phenotypic states can be explained by evolutionary contingency alone. We discussed the importance of applying phylogenetic comparative methods when studying phenotypic evolution and more generally to investigate the effect of stochastic processes before making deterministic inferences.Character displacement that leads to divergent phenotypes between sympatric species has been hypothesized to facilitate coexistence and promote the accumulation of biodiversity. However, there are alternative evolutionary mechanisms that may also lead to the evolution of phenotypic divergence between sympatric species; one of the mechanisms is evolutionary contingency. We studied the evolution of the presence and absence of a major male horn phenotype, which may have ecological implications for promoting coexistence between sympatric beetles, across geographic populations from different Sympatric closely\u2010related species may exhibit different adaptive phenotypes, where character displacement is often invoked to explain such pattern. However, the evolution of distinct male horn phenotypes in sympatry can be explained by evolutionary contingency in Xylotrupes beetles. It is important to account for stochastic effect when study character evolution. A major male horn phenotype refers to a longer thoracic than cephalic horn , and X. sp from Luzon (XSP), the empirical accounts of sympatric species that evolve distinct phenotypic state can range from 4 to 6 . The garbage model resulted in a terrible fit to our data (lnL\u00a0=\u00a0\u221247.64538), which is much worse than those of ER and ARD models. Therefore, we used results from the ER model for ancestral state estimation . Furthermore, the simulated distribution of the number of sympatric taxa that evolved different phenotypes peaked around 4 and 5 out of 9 possible instances can frequently result in sympatric species pairs that exhibit different phenotypic states. Note that we are not downplaying the importance of character displacement and competition nor species interaction in promoting phenotypic divergence and coexistence; instead, our results aim to bring an often neglected evolutionary force, that is, stochasticity and contingency, into the discussion when studying character evolution using phylogenetic comparative methods.The evolution of distinct phenotypic states in traits that may have evolutionary or adaptive significance between closely related sympatric species has long been an example for niche partitioning that facilitates coexistence Losos,\u00a0. HoweverLerista lizards did not fit the empirical data statistically better than another one assuming equal transition rate (ER), implying that the transition rate could be treated as the same between the two directions. Additionally, the ancestral states at deeper nodes could not be effectively estimated without uncertainty Figure\u00a0. For exay Figure\u00a0. As menty Figure\u00a0. While cXylotrupes beetles, even though our results demonstrated that evolutionary contingency alone can explain the evolution of difference in the presence and absence of a major male phenotype in sympatry. Previous studies have shown that the pattern of character displacement was more apparent in genitalic structure, but less so in male horn structures in the Giant Rhinoceros beetle, genus Chalcosoma and X. florensis (XFS) can be found on Sangeang Island, where a major male phenotype is absent in both taxa ; Data curation (lead); Formal analysis (lead); Funding acquisition (lead); Investigation (lead); Methodology (lead); Project administration ; Resources ; Software (lead); Supervision (lead); Validation (lead); Visualization (lead); Writing\u2010original draft (lead); Writing\u2010review & editing (lead). Brett Morgan: Data curation ; Formal analysis (supporting); Methodology (supporting); Resources (supporting); Visualization (supporting); Writing\u2010review & editing (supporting)."} +{"text": "After revolutionizing neuroscience, optogenetic therapy has entered successfully in clinical trials for restoring vision to blind people with degenerative eye diseases, such as retinitis pigmentosa. These clinical trials still have to evaluate the visual acuity achieved by patients and to determine if it reaches its theoretical limit extrapolated from ex vivo experiments. Different strategies are developed in parallel to reduce required light levels and improve information processing by targeting various cell types. For patients with vision loss due to optic atrophy, as in the case of glaucoma, optogenetic cortical stimulation is hampered by light absorption and scattering by the brain tissue. By contrast, ultrasound waves can diffuse widely through the dura mater and the brain tissue as indicated by ultrasound imaging. Based on our recent results in rodents, we propose the sonogenetic therapy relying on activation of the mechanosensitive channel as a very promising vision restoration strategy with a suitable spatiotemporal resolution. Genomic approaches may thus provide efficient brain machine interfaces for sight restoration. The retinal prosthesis PRIMA provides today the best restored visual acuity in blind patients. This retinal prosthesis has been tested in patients affected by dry age-related macular degeneration allowing a prosthetic visual acuity between 20/460 and 20/565 in 4 patients with the ability to fuse the central infrared artificial vision and the natural peripheral vision.,Following these initial studies, we have evaluated the possibility to express the microbial opsin with improved light sensitivity CatCh\u2013Others have similarly created an image converter,Retinal ganglion cells are characterized by a large anatomic and functional diversity resulting in different light responses, which cannot be reproduced simultaneously and individually in each cell type by optogenetics. All retinal ganglion cells become ON cell types indifferently from their original cell types. To offer more variability, several strategies were proposed to target cells at a higher level in the retinal information processing sequence. For instance, bipolar cells were targeted to express ChR2 using a specific promoter,As microbial opsins require very high light intensities and they could potentially induce a negative immune response, various laboratories have expressed mammalian opsin ectopically to restore vision. Melanopsin was the first opsin to be targeted in retinal ganglion cells because this opsin is already present in a small population of intrinsically photosensitive retinal ganglion cells with non-visual functions.,Finally, optogenetic gene therapy was also combined with cell therapy because photoreceptors generated from induced pluripotent stem cells are not yet able to generate active photosensitive outer segments. This strategy is appealing as it enabled us to transplant active photoreceptors in advanced states of retinal degeneration. Transplantation of engineered photoreceptors expressing inhibitory microbial opsins restored visual responses and behavior in blind animals.,For diseases leading to blindness following the loss of the optic nerve, cortical visual prostheses were developed in the 1960s by Brindley and Lewin with success in eliciting phosphenes in the visual field\u2013,,,Optogenetic therapy appeared as an obvious non-contact alternative for the distant activation of cortical neurons from the brain surface. Expression of a microbial opsin in the visual cortex was found to activate cortical neurons even in the cortex depth following surface illumination of the brain.\u2013\u2013For future clinical applications, it should be considered that deep brain optogenetic stimulation is difficult due to light absorption and scattering in the brain tissue,\u2013,\u2013\u2013,In a recent study, we have proposed to use ultrasound waves instead of light to activate cortical neurons following targeted expression of a mechanosensitive ionic channel. This strategy of ultrasound activation of neurons was named sonogenetics, as it relies on the genetic expression of a mechanosensitive protein coupled to the ultrasound stimulation. Ultrasound waves are well known to propagate into the tissue depth and even deep into the non-human primate visual cortex, as it has been demonstrated in our study by brain cortical functional ultrasound imaging.Genomic strategies based on gene therapy expressing ectopically photosensitive and mechanosensitive ionic channels may provide high resolution brain-machine interfaces. Optogenetics is already in clinical trials with results demonstrating restored partial vision in patients affected by retinitis pigmentosa. Further analyses are needed to assess if patients can reach the theoretical visual acuity (20/72) estimated in ex vivo studies on the non-human primate retina. The results of ongoing clinical trials will thus decide if optogenetics can provide a convincing alternative to retinal prostheses for restoring vision. Similarly, sonogenetic therapy offers an alternative to cortical prostheses for a deep non-contact cortical stimulation from above the dura mater. Further studies are needed to evaluate the safety and efficacy of this strategy prior to launching clinical trials. Despite these current limitations, the results of our study suggest that sonogenetics holds great hope for a novel generation of brain machine interface for cortical visual restoration and other neurological applications."} +{"text": "To the Editor:Congenitally corrected transposition of great arteries (CCTGA) is a rare but important condition, especially in the field of adult congenital heart disease. Although many cases of CCTGA have various associated conditions such as ventricular septal defect (VSD), pulmonary stenosis (PS), and tricuspid valve abnormalities,,We read with interest the report published by Osakada et\u00a0al., who presented a case report of CCTGA in a patient who was diagnosed at 88 years of age."} +{"text": "Audit carried out to assess whether or not patients had been asked about their smoking status during admission onto an acute adult mental health ward, as well as if they had received any smoking cessation advice or offered nicotine replacement therapy.Physical health outcomes in patients with serious mental illness (SMI) are consisitently worse than the general public This is due to multiple factors; adverse effects of medication (including metabolic syndromes with psychotropics) as well as poor lifestyle factors such as smoking statusPatients with an SMI are 3\u20136 times more likely to die due to coronary artery disease. 70% of patients in inpatient psychiatric units are smokers, a strong independent risk factor for cardiovascular disease.Smoking cessation is a potent modifiable risk factor that can prevent mortality and reduce morbidity.A cross-sectional review of all 34 inpatients across four general adult acute psychiatric wards.Patient records were explored using the Aneuran Bevan Health Board admission proformas to identify evidence of smoking status and whether advice was offered.Smoker but not given cessation advice n = 13 (38%)Not asked about smoking n = 11 (32%)Smoker and given cessation advice n = 4 (12%)Non-smoker n = 6 (18%)Patients were asked about their smoking status the majority of the time (68%) but provision of advice or nicotine replacement therapy was only done in 14% of potential smokers (identified smokers and patients not asked about smoking status).A consideration to be taken into account is that on admission, a patient's physical health status may be unknown, with the additional difficulty of a patient's acute distress complicating the physical examination, smoking status and modification of patient's smoking status may not be the highest priory in that context.Data regarding asking about smoking were different amongst wards, potentially signifying differences between assessors willingness to ask about smoking status.There is a lack of smoking cessation literature available on the wards and patients are often unaware of what options are available to quit smoking.The audit simply determined whether or not assessors were documenting smoking status, it does not measure the quantity or quality of smoking cessation advice provided.Further quality improvement projects should be launched, with focus groups as the intial step at further investigating inpatient smoking rates, as well as attempting to reduce them in a more systemic way."} +{"text": "Reduced energy is a hallmark feature of aging. Maintaining higher energy late in life may be a key adaptive strategy to the challenges that accompany older age and ultimately promote resilience. Perceived lack of energy is often construed as synonymous with fatigue, and energy and fatigue are frequently considered opposite aspects of the same phenomenon. However, evidence suggests that energy and fatigue have distinct underlying neurobiology. Further exploration of the energy/fatigue dichotomy is needed in community-dwelling older adults free of neuropathologies and clinically overt conditions. This symposium will first present clinical and epidemiologic justifications for operationalizing energy as a separate construct from fatigue and then will provide evidence on the underlying neurobiological correlates. Taken together, our results suggest perceived energy: a) overlaps with but is distinct from lower fatigability (Katz); b) may signal resilience against age-related declining mood and gait speed despite self-reported tiredness (Ehrenkranz); c) appear negatively influenced by Alzheimer\u2019s neuropathology (Dougherty); and d) may reflect a distinct spatial distribution of brain functional connectivity (Hengenius). Thus, this symposium will explore energy as a mechanism related to yet distinct from fatigue and its implications for both healthy aging and neuropathological processes."} +{"text": "The coronavirus disease (COVID-19) pandemic has magnified inevitable physical, mental and social health consequences, especially in Hispanic older adults who experience health disparities and ageism. Even though physical distancing has been adopted as a key strategy to help reduce further spread of COVID-19, prolonged periods of physical distancing may worsen existing health problems. This study aims to explore how the COVID crisis affected diverse older adults. An explanatory sequential mixed methods design was utilized. Quantitative data were collected by questionnaires via Qualtrics survey and qualitative data were collected by individual phone interviews with four open-end questions. One in 4 older adults lives alone and one in 20 has no friend to call on. More than half of the participants were afraid of COVID and a fourth of them were afraid of losing their life to COVID. Participants identified keeping themselves busy as key to staying healthy during the pandemic."} +{"text": "This study examines older couples\u2019 dyadic patterns of informal and formal advance care planning (ACP) and determines the associations of these patterns with their own and spousal characteristics. Using data from the 2014 and 2016 Health and Retirement Study, we performed a) latent class analysis to identify distinctive ACP engagement patterns and b) multinomial regression models to describe related characteristics of older couples . We identified four dyadic patterns of ACP engagement: a) high ACP engaging couple (45%); b) high engaging husband \u2013 low engaging wife (13%); c) high engaging wife \u2013 low engaging husband (11%); and d) low engaging couple (31%). Engagement in informal and formal ACP was associated with both individual and spousal factors: Older couples with advanced age or higher levels of education and wealth were more likely to engage in both informal and formal ACP, whereas only wife\u2019s high level of constrain or husband\u2019s greater number of depressive symptoms was associated with discordant ACP engagements. Couple-based approach to promote ACP merits older couples with limited resources or poorer psychological health in both or either spouse."} +{"text": "To quantify the potential impact of engaging religious leaders in promoting safe burial practices during the 2014\u20132016 Ebola virus disease outbreak in Sierra Leone.We analysed population-based household survey data from 3540 respondents collected around the peak of the outbreak in Sierra Leone, December 2014. Respondents were asked if in the past month they had heard an imam or pastor say that people should not touch or wash a dead body. We used multilevel logistic regression modelling to examine if exposure to religious leaders\u2019 messages was associated with protective burial intentions if a family member died at home and other Ebola protective behaviours.Of the respondents, 3148 (89%) had been exposed to faith-based messages from religious leaders on safe Ebola burials and 369 (10%) were unexposed. Exposure to religious leaders\u2019 messages was associated with a nearly twofold increase in the intention to accept safe alternatives to traditional burials and the intention to wait \u2265\u20092\u00a0days for burial teams . Exposure to messages from religious leaders was also associated with avoidance of traditional burials and of contact with suspected Ebola patients .Public health messages promoted by religious leaders may have influenced safe burial behaviours during the Ebola outbreak in Sierra Leone. Engagement of religious leaders in risk communication should be prioritized during health emergencies in similar settings. Within each cluster, households were randomly selected, followed by the selection of two participants from each household. The head of the household was always selected in addition to a second random household member who was either an adult woman (aged 25 years or older) or a young person (aged 15\u201324 years). Consent was obtained from a parent or guardian before any child\u2019s involvement in addition to obtaining assent directly from the child. \u201cIn the past month, have you heard an imam/pastor say that during this period people should NOT touch or wash a dead body?\u201d Other independent variables we selected for analysis were the demographic characteristics of respondents: region of residence, age, sex, educational level and religion.Our main independent variable was respondents\u2019 exposure to religious leaders\u2019 messages promoting safe Ebola burials. This information was obtained in the survey using the question: We selected five outcome variables for analysis. Three outcome variables reflected behavioural intentions if a family member died at home: (i)\u00a0intending to accept safe alternatives to traditional burials; (ii)\u00a0intending to avoid touching or washing the corpse; and (iii)\u00a0intending to wait 2 or more days for a burial team to arrive. Two outcome variables reflected protective behaviours against Ebola transmission: (iv)\u00a0avoiding unsafe traditional burials and (v)\u00a0avoiding physical contact with suspected Ebola patients. We selected these five outcome variables because they closely reflected key aspects of attitudes and behaviour that were promoted in the faith messages delivered by religious leaders. Further descriptions of the independent and outcome variables we selected for inclusion in our analyses are provided in the data repository.We generated unweighted frequencies and proportions for all independent and outcome variables. We conducted multilevel logistic regression modelling to account for the intraclass correlation among respondents from the same geographical cluster. We used multilevel logistic regression models to examine the association between exposure to religious leaders\u2019 messages and the three binary outcomes on safe burial intentions and the two binary outcomes on protective behaviours against Ebola transmission. In addition, we fitted two multilevel logistic regression models to further investigate the association between intention to wait for a burial team for 2 or more days and respondents\u2019 protective behaviours. All multilevel logistic regression models were adjusted for respondents\u2019 region of residence , age , sex , religion and educational level . We performed statistical analysis using Stata statistical software, version 16 .We obtained ethical approval for our study from Sierra Leone Research and Scientific Review Committee. The Center for Global Health at the United States Centers for Disease Control and Prevention determined that the assessment was part of the public health response to the Ebola outbreak in Sierra Leone, and was determined to be non-research. The secondary data analysis protocol was further approved by the ethical review board at Karolinska Institutet, Stockholm, Sweden (DNR 2018/1276\u201331).A total of 3540 respondents participated in the survey (98% of the 3612 people who were asked to participate). One third of the respondents were younger than 25 years, with an approximately equal distribution of males and females reported that they had heard faith-based messages from religious leaders on safe Ebola burials and 369 (10%) had not heard these messages (data were missing for 23 survey respondents).Overall, 3049 respondents (86%) intended to accept safe alternatives to traditional burials if a family member died during the Ebola outbreak. Exposure to religious leaders\u2019 messages was positively associated with accepting a safe alternative to traditional burials intended to avoid touching or washing the corpse of a family member, regardless of exposure to religious leaders\u2019 messages . None of the demographic variables were significantly associated with this outcome.Half of the respondents intended to wait up to 2 or more days for the burial team\u2019s arrival if they had a family member who was suspected of having Ebola. The odds of expressing this intention were almost twofold greater among respondents who were exposed to faith-based messages . Demographically, compared with respondents who self-identified as Muslims, those who self-identified as Christians had nearly a 20% decrease in their odds of intending to wait at least 2 days for the burial teams to arrive .When asked unprompted about their protective behaviours against Ebola, 1673 (47%) of the respondents cited that they were avoiding unsafe traditional burials. Exposure to religious leaders\u2019 messages was associated with avoiding unsafe traditional burials among respondents exposed to faith-based messages compared with those who did not receive messages respondents were avoiding physical contact with suspected Ebola patients to prevent Ebola transmission. Exposure to religious leaders\u2019 messages was associated with avoiding Ebola patients . Residing in the eastern region was also associated with this outcome compared with residing in the western region. Respondents with primary and secondary education had greater odds of avoiding Ebola patients when compared with those without any education .Of those who intended to wait 2 or more days for burial teams\u2019 arrival, 1670 were more likely to avoid unsafe burials compared with those 1542 who did not intend to wait that long . Compared with the western region, residing in any of the three provincial regions was associated with avoiding Ebola patients: southern ; eastern ; northern . Having secondary education or higher was also associated with this outcome .The results from this nationwide household survey conducted around the peak of the Ebola outbreak in Sierra Leone suggest that religious leaders may have been effective risk communicators during the outbreak response. Exposure to messages about safe burials from religious leaders was significantly associated with intending to adhere to safe burial measures, including accepting safe alternatives to traditional burials and intending to wait 2 or more days for a burial team to bury the corpse. Behaviourally, exposure to religious leaders\u2019 messages was significantly associated with engaging in protective behaviours such as avoiding unsafe traditional burials and avoiding physical contact with suspected Ebola patients. Those who intended to wait for a burial team were also more likely to avoid unsafe traditional burials and physical contact with suspected Ebola patients.,Unsafe traditional burials played a key role in the transmission of Ebola during the outbreak.,,Intention to wait for 2 or more days for a burial team\u2019s arrival may have been essential in minimizing the risk of unsafe burials. While government policy stated that all burial teams must respond to deaths within 24 hours, in practice they were sometimes unable to respond within this time frame.In our study, 10% of respondents reported they were not exposed to religious leaders\u2019 messages at the time of the survey. This relatively small comparison group likely reflects the overall success of the religious leader\u2019s communication campaigns during the outbreak. Apart from communicating Ebola messages through their preaching in mosques and churches, religious leaders were also involved in various media campaigns on radio and television to communicate the Ebola message during the outbreak.,,,Trust is an essential component of effective risk communication during an outbreak and is likely one of the reasons why religious leaders were effective risk communicators during the Ebola outbreak.Limitations to this study include the potential for participants to provide socially desirable responses regarding their intentions and behaviours, especially given that the survey was conducted after intensified social mobilization efforts around the peak of the outbreak in the country. Due to the cross-sectional nature of the data, we cannot establish causality with the available data. Exposure to multiple information sources during the outbreak may also have made it difficult for respondents to discern the source of messages. Therefore, exposure to faith-based messages could have been conflated with exposure to other sources of information on Ebola burial prevention. To account for this, our primary independent variable was a question from the knowledge, attitudes and practices survey which was specific to religious leaders. A strength of this study is the large, national random sample obtained around the peak of the outbreak with a very high response rate. In the absence of quantifiable empirical data on the potential impact of religious leader engagements during any Ebola outbreak to date, our findings have important global public health significance by establishing a strong association between religious leaders\u2019 messages and protective Ebola behavioural outcomes.In conclusion, effective risk communication is a vital pillar in building a foundation of trust during health emergencies. Our results highlight the potential impact of religious leaders, who are trusted and respected figures in Sierra Leone, as effective risk communicators during the Ebola outbreak. Engaging religious leaders in risk communication in similar contexts may have a measurable impact in increasing protective behaviours that slow disease transmission during health emergencies. During the coronavirus disease-2019 (COVID-19) pandemic, effective risk communication and a cohesive interdisciplinary approach to health communication are essential. Protective behaviours such as physical distancing are again important components of the outbreak response."} +{"text": "The purpose of this pilot project was to explore the experience of an intergenerational learning environment focused on healthy aging for nursing students and older adults. Intergenerational learning experiences provide opportunities for individuals from different age groups to communicate and participate in learning activities together. The growing population of older adults calls for increased geriatric nursing expertise. Nursing students\u2019 attitudes toward older adults are often negative though, and result in decreased interest in geriatric nursing. The opportunity to transform nursing students\u2019 perspectives on older adults has the potential to improve nursing care for older adults, and the number of nurses focused on geriatric nursing care. This qualitative inquiry used a convenience sample of 10 participants from a cross-listed university course on healthy aging for baccalaureate nursing students and older adult members of a lifelong learning institute. Semi- structured focus group interviews were conducted. Narrative transcripts were analyzed using an inductive approach. Analysis illustrated improved nursing students\u2019 perspectives of older adults and aging. A similar theme was noted for older adults\u2019 perspectives of younger adults. The importance of social interaction within an intergenerational learning environment and the need for opportunities to challenge ageist perspectives was illustrated. Increased exposure to healthy older adults, personally and professionally, may increase nursing students\u2019 interest in geriatric nursing and improve nursing care for older adults. Future research should examine more specifically how intergenerational learning experiences can decrease ageism, improve nursing students\u2019 and nurses\u2019 perspectives on older adults, and improve nursing practice for older adults."} +{"text": "World report on disability.This year marks the tenth anniversary of the ,,World report on disability revealed that half of the people with disabilities in 51 surveyed countries could not afford needed health care and that, overall, people with disabilities were more likely to incur catastrophic health expenditures.People with disabilities typically need more health care than the rest of the population because they require disability-related services and assistive devices on top of general health services.Social health protection is critical to achieving sustainable development goal (SDG) 3 \u2013 to ensure a healthy life for all \u2013 and its target 3.8 \u2013 to achieve UHC.,,,Social health protection programmes are increasingly being implemented in middle-income and even low-income countries.,Second, programmes must assess whether covered health-care services and products have the range and quality to adequately meet the health needs of people with disabilities. Many existing programmes provide insufficient coverage for disability-related services and assistive devices or only cover services of lesser quality.,Third, programmes must assess whether people with disabilities seeking health care are receiving sufficient financial protection. Even if fully covered for all their needed services and products, people with disabilities may still face impoverishment from frequent or high user fees and are more likely to incur indirect costs.We now need further research to understand how social health protection affects people with disabilities and to learn how to design disability-inclusive programmes that will lead to success in attaining SDG\u00a03 and UHC."} +{"text": "To the Editor\u2014Thank you for the opportunity to respond to the letter by Jia andWang regarding our earlier publication [lication .We appreciate the comments made by Jia and Wang, especially those recognizing our novelstrategy of integrating the in vitro activity and lung concentration ofhydroxychloroquine (HCQ) using a physiologically based pharmacokinetic (PBPK) model tooptimize dose regimens. The time between the determination of anti\u2013severe acuterespiratory syndrome coronavirus 2 (SARS-CoV-2) activity of HCQ in vitro and therecommendation of dose regimens of HCQ and chloroquine (CQ) using PBPK simulations wereless than 1 week, and our clinicians almost immediately used these recommended humandoses to evaluate drug efficacy and safety in coronavirus disease 2019 (COVID-19)patients in China (ChiCTR2000029899). This would be extremely difficult without PBPKmodels.We agree with Jia and Wang that the \u201capplication of PBPK \u2026 must rely on rigorouspharmacokinetic mechanism and reasonable assumption.\u201d We declared assumptions andlimitations of the model, and indicated that future studies are underway to update themodels .50)\u201d suggests that Jia and Wang may nothave carefully read or understood our approach and the assumptions presented in thepaper. We described HCQ dose regimen optimization in the Methods section as follows: \u201cina recent clinical trial, 500 mg of chloroquine phosphate given twice daily was shown tobe effective on study day 5 . This dosing regimen forchloroquine was used as the target for dose optimization for hydroxychloroquine.\u201dAlthough we calculated the RLTEC for each compound (CQ and HCQ), weultimately used relative potency between the 2 compounds to facilitate HCQ\u2019s dosingrecommendations, rather than judging whether HCQ is effective or not. As compared toconventional methods that predict clinical efficacy based on in vitro and in vivo dataof the same compound, our approach heavily relied on the emerging clinical antiviraleffect by CQ [The comment \u201cthe target tissue (lung) concentration of HCQ was overestimated andmismatched the in vitro activity (ECd later) . Even fod later) demonstr50) of HCQ against SARS-CoV-2 [We agree with Jia and Wang that \u201cin vitro activity was significantly affected byexperimental factors.\u201d Unfortunately, our group was 1 of the first reporting halfmaximal effective concentration (ECFinally, we would like to reiterate our response to an earlier letter to the editor:\u201calthough one can employ modeling and pharmacology concepts to predict the likelihood ofclinical efficacy from in vitro data, given the inherent limitations of any modelingapproach and assumptions being made, in vitro efficacy can only be ultimately confirmedthrough clinical trials. To this end, any modeling analysis has to fit for purpose\u201d."} +{"text": "Castalia ambigua Lamarck, 1819 (Hyriidae) and Anodontites elongatus (Mycetopodidae), are presented on the basis of hydraulic variables linked with the riverbed in six 500\u2010m reaches in an eastern Amazonian river basin. Within the reaches, there was strong habitat heterogeneity in hydrodynamics and substrate composition. In addition, we investigated stressors based on landscape modification that are associated with declines in mussel density. We measured hydraulic variables for each 500\u2010m reach, and landscape stressors at two spatial scales (subcatchment and riparian buffer forest). We used the Random Forest algorithm, a tree\u2010based model, to predict the hydraulic variables linked with habitat suitability for mussels, and to predict which landscape stressors were most associated with mussel density declines. Both mussel species were linked with low substrate heterogeneity and greater riverbed stability (low Froude and Reynolds numbers), especially at high flow (low stream power). Different sediment grain size preferences were observed between mussel species: Castalia ambigua was associated with medium sand and Anodontites elongatus with medium and fine sand. Declines in mussel density were associated with modifications linked to urbanization at small scales (riparian buffer forest), especially with percent of and distance from rural settlements, distance to the nearest street, and road density. In summary, the high variance explained in both hydraulic and landscape models indicated high predictive power, suggesting that our findings may be extrapolated and used as a baseline to test hypotheses of habitat suitability in other Amazonian rivers for Castalia ambigua and Anodontites elongatus and also for other freshwater mussel species. Our results highlight the urgent need for aquatic habitat conservation to maintain sheltered habitats during high flow as well as mitigate the effects of landscape modifications at the riparian buffer scale, both of which are important for maintaining dense mussel populations and habitat quality.Novel insights into habitat suitability for two Unionida freshwater mussels, Random Forest algorithms with complex hydraulic variables and landscape stressors predicted that Amazonian mussels are associated with riverbed stability at high and low flows and mussel density is higher with low substrate heterogeneity and hydrodynamic energy. Local riparian buffer modification by urbanization leads to mussel density declines, and there is an urgent need for landscape and riverscape management and conservation for maintenance of mussel populations and habitat quality. We extracted a set of thirteen landscape variables using layers from OpenStreetMap (https://www.openstreetmap.org) available via the OpenLayers plugin in QGIS 3.12 to take into account possible nonindependence of the randomly selected replicates (plots) within each site and potential pseudoreplication regression in the es Table\u00a0 to predies Table\u00a0.x\u00a0+\u00a01) transformation on the raw mussel density data to focus over the lower range of individuals which is more ecologically meaningful than over the higher range samples and were used to create an oob estimation of the generalized error in the model for the oob samples. Random Forests compute an importance value for each predictor, which gives information about the relationship between this predictor and the response variable to evaluate this relationship 70.5% of the total variance for Castalia ambigua and 49.7% for Anodontites elongatus. The high variance explained and low oob generalized error indicated high predictive power of our model. Both species had the same subset of important predictors 69.3% of the total variance for Castalia ambigua and 86.8% for Anodontites elongatus. The high variance explained and low oob generalized error indicated high predictive power of our model. Both species had the same subset of important predictors and Anodontites elongatus . We suggest that Castalia ambigua, for having a more robust shell, supports habitats with relatively higher hydrodynamics, similar to those of sculptured mussel species in temperate rivers appear not to be important in predictive models to describe suitable habitat for mussels for Anodontites elongatus and especially Castalia ambigua, which underwent greatest declines in density and Margaritifera margaritifera (restricted distribution) in rivers of Bavaria, Germany and Anodontites elongatus . Therefore, changes in Amazonian land\u2010 and riverscapes at different spatial scales may affect mussel density. For example, altered riparian buffer cover may decrease mussel density and the number of suitable habitats , shelter during high flow (low stream power), and low substrate heterogeneity. Mussel species showed distinct preferences for sediment type: The authors declare no conflicts of interests.Diego Simeone: Conceptualization ; Formal analysis ; Writing\u2010original draft (lead); Writing\u2010review & editing . Claudia Helena Tagliaro: Conceptualization ; Writing\u2010review & editing . Colin Robert Beasley: Conceptualization ; Formal analysis ; Writing\u2010review & editing ."} +{"text": "The COVID-19 pandemic has posed challenges to safely engaging older adults in volunteer activities. This research explored a unique partnership between a Retired Senior and Volunteer Program (RSVP) and a school of nursing to administer a telehealth virtual simulation training for nurse practitioner students. Semi-structured interviews were carried out with nursing simulation coordinators and volunteers after the telehealth simulation exercise. The purpose of this research was to identify principles of successful virtual volunteer engagement for telehealth simulations. This initial pilot study encompassed debriefing interviews with volunteers (N = 3) and interviews with simulation coordinators (N = 2). Three major themes emerged within the response coding: 1) the benefits of virtual simulation volunteering, 2) technology as a facilitating factor and challenge, and 3) unique volunteer management considerations. Both volunteers and coordinators noted that volunteers derived positive emotional benefits and new insights from their participation. Coordinators discussed the \u201cauthenticity\u201d factor that older adults brought to the simulation experience as a benefit to engaging older adult volunteers. Technology sub-themes included accessibility considerations, experience with the online format, and other logistical considerations in conducting telehealth simulation. Volunteer management sub-themes encompassed volunteer skills and motivations, the perceived successful aspects of training, and improvements for future simulations. Volunteers discussed an interest and connection to healthcare and education as a motivating factor for their participation in the telehealth simulation. This small scale pilot research will be expanded through future simulation activities to continue to identify principles of practice for engaging older adults in virtual volunteerism."} +{"text": "Replicative errors, inefficient repair, and proximity to reactive oxygen species production sites make the mitochondrial DNA (mtDNA) susceptible to damage with time. mtDNA mutations accumulate with age and accompany a progressive decline in organelle function. We lack molecular biology tools to manipulate mtDNA, thus we explore the possibility in vivo of utilizing allotopic expression, or the re-engineering mitochondrial genes and expressing them from the nucleus, as an approach to rescue defects arising from mtDNA mutations. This study uses a mouse model with a mutation in the mitochondrial ATP8 gene that encodes a protein subunit of the ATP synthase. We generated a transgenic mouse with an epitope-tagged recoded and mitochondrial-targeted ATP8 gene expressed from the nucleus. Our results show that the allotopically expressed ATP8 protein in the transgenic mice is robustly expressed across all tested tissues, successfully transported into the mitochondria, and incorporated into ATP synthase. We are currently evaluating if allotopic expression of ATP8 will functionally rescue the behavioral and bioenergetic defects in ATP8 mutant mice. Translating allotopic expression technology into a mammal and demonstrating systemic functional rescue will lend credence to utilizing allotopic expression as a gene therapy in humans to repair physiological consequences of mtDNA defects that may accumulate with age."} +{"text": "Although long distance caregivers (LDCs) are starting to be recognized as a subgroup of care partners experiencing unique challenges and stresses, it is unknown 1) what types of supportive services LDCs use for themselves and 2) what factors are associated with supportive service use in this understudied caregiving population. In our sample of 304 LDCs (Mage=56.9), the most frequently utilized service was video phone/webcam systems to monitor the care recipient (CR). Guided by Andersen\u2019s Model of Health Care Utilization and using multiple hierarchical regression analysis, younger age of the LDC (a predisposing factor) and need-related characteristics were associated with greater use of supportive services. Enabling factors were not associated with service use. These study findings can help inform how to engage LDCs in supportive service utilization."} +{"text": "An ongoing activity that cuts across several courses in the Gerontology Certificate Program at our College is the completion of implicit association exercises focused on age. Most college students show a distinct preference for those who are younger adults. It is difficult to get across to these students that the construct of being an adult is appropriate for all people beyond adolescence without relevance to age. College students enrolled in healthcare programs often have distorted views of aging and may not fully appreciate that all adults may share common aspects of their current lives. We describe qualitative analyses of reflections taken from an undergraduate psychology course that included a service learning component involving older adult learners. The service learning lessons focused on victimization associated with fraud and scamming. The classroom structure involved round table discussions with direct contact between college students, older adults and local law enforcement personnel. Reflective practices were used to integrate course content (development in adulthood) into this service learning activity. We report on qualitative data taken from student reflections. Content analyses of reflective essays identified five themes which operated to produce stronger identification between age groups: frequency of being scammed across all 21 participants; insight that learning continues across the lifespan; understanding that broad learning challenges impact people (for different reasons) at both ends of the adult age spectrum; respect for adoption of strategies that facilitate learning/compensate for cognitive changes that occur with aging; acknowledgement that familiarity breaks down barriers between people."} +{"text": "To examine historical changes in views on aging, we compared matched cohorts of older adults within two independent studies that assessed differences across a two-decade interval, the Berlin Aging Studies and the Midlife in the United States Study . Consistent across four different dimensions of individuals\u2019 subjective views on aging in the Berlin Aging Studies and corroborated with subjective age felt in the MIDUS, there was no evidence whatsoever that older adults of today have more favorable views on how they age than older adults did two decades ago. We discuss reasons for our findings, including the possibility that individual age views may have become increasingly decoupled from societal age views."} +{"text": "Human Immunodeficiency Virus (HIV) and Simian Immunodeficiency Virus (SIV) are associated with severe perturbations in the gut mucosal environment characterized by massive viral replication and depletion of CD4 T cells leading to dysbiosis, breakdown of the epithelial barrier, microbial translocation, immune activation and disease progression. Multiple mechanisms play a role in maintaining homeostasis in the gut mucosa and protecting the integrity of the epithelial barrier. Among these are the secretory IgA (sIgA) that are produced daily in vast quantities throughout the mucosa and play a pivotal role in preventing commensal microbes from breaching the epithelial barrier. These microbe specific, high affinity IgA are produced by IgA+ plasma cells that are present within the Peyer\u2019s Patches, mesenteric lymph nodes and the isolated lymphoid follicles that are prevalent in the lamina propria of the gastrointestinal tract (GIT). Differentiation, maturation and class switching to IgA producing plasma cells requires help from T follicular helper (Tfh) cells that are present within these lymphoid tissues. HIV replication and CD4 T cell depletion is accompanied by severe dysregulation of Tfh cell responses that compromises the generation of mucosal IgA that in turn alters barrier integrity leading to commensal bacteria readily breaching the epithelial barrier and causing mucosal pathology. Here we review the effect of HIV infection on Tfh cells and mucosal IgA responses in the GIT and the consequences these have for gut dysbiosis and mucosal immunopathogenesis. Human and Simian Immunodeficiency Virus infections are associated with dramatic changes in mucosal tissues such as the gastrointestinal tract (GIT) with massive replication and depletion of CD4 T cells during the acute stages of infection \u201317. Vira11 \u2013 1013 bacteria found in the lumen of the GIT with the gut associated lymphoid tissue (GALT) hosting ~ 80% of IgA producing plasma cells in the body , 60. MicsIgA is abundant in mucosa associated lymphoid tissues (MALT) found in other mucosal sites such as the female genital tract , the LN Unlike the isolated lymphoid follicles in the lamina propria that can generate IgA responses in a T cell independent manner, most of the T cell dependent induction of IgA responses occurs within the Peyer\u2019s patches and mesenteric lymph nodes \u201373. T ceTfh cells are a subset of CD4 T cells that mainly reside in the germinal centers of lymph nodes and provide help to differentiating B cells during the germinal center reaction . Tfh celHIV and SIV infections are characterized by loss of CD4 T cells that compromises the immune system with viral reservoirs persisting throughout the life of an infected subject , 92\u2013104.Other studies have reported that HIV specific Tfh significantly expand during chronic HIV infection . SimilarIn contrast to the accumulation of Tfh in a subset of infected subjects, numerous studies have reported that HIV and SIV infection were characterized by a depletion of Tfh cells that was associated with altered B cell responses. Mesenteric lymph node Tfh cells were lost very early during SIV infection that was accompanied by a loss of GC and memory B cells , 123. OtDespite early antiretroviral therapy, Tfh cells remain major reservoirs in lymphoid tissues and peripheral blood , 131. StMucosal IgA plays a critical role in protecting the mucosa from invasive bacteria that colonize the GIT . IgA areEnterobacteriaceae (The decrease in intestinal IgA is consequential given its central importance in preventing gut microbes from accessing the intestinal epithelium. Gut commensal bacteria and their metabolites play a role in inducing effector T cells and maintaining immune homeostasis in the GIT \u2013167. Hegeriaceae . Others eriaceae have shoeriaceae , 175. Imeriaceae , 176.\u00a0Mieriaceae reportedBacteroides and Firmicutes and an increase in the prevalence Proteobacteria (Prevotella during chronic HIV infection (Enterobacteriaceae and Moraxellaceae similar to that of HIV infected patients who have low CD4 T cell counts. Lower microbial richness and diversity was associated with poor CD4 T cell reconstitution in HIV infected subjects undergoing treatment with HAART (HIV infection is associated with changes in the composition of the gut microbiota , 178 chaacteria \u2013181. Othacteria and Prevnfection \u2013186. Gutnfection . SIV infnfection , and macth HAART , 190. NuIn conclusion, the dysregulation of Tfh cells that compromises B cell responses especially the secretion of microbe specific IgA is one of the likely drivers of gut microbial dysbiosis during HIV infection. This dysregulation that accompanies high levels of HIV replication, loss of mucosal CD4 T cells, breakdown of the integrity of epithelial barrier significantly alters mucosal immune homeostasis leading to microbial translocation, immune activation and progression of disease. Strategies that can preserve and maintain Tfh responses in the mucosa could potentially restore high affinity mucosal IgA that could aid in protecting the mucosal epithelial barrier from invasive dysbiotic bacteria.OO and JM wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Urbanization is rapidly altering landscapes worldwide, changing environmental conditions, and creating novel selection pressures for many organisms. Local environmental conditions affect the expression and evolution of sexual signals and mating behaviors; changes in such traits have important evolutionary consequences because of their effect on reproduction. In this review, we synthesize research investigating how sexual communication is affected by the environmental changes associated with urbanization\u2014including pollution from noise, light, and heavy metals, habitat fragmentation, impervious surfaces, urban heat islands, and changes in resources and predation. Urbanization often has negative effects on sexual communication through signal masking, altering condition\u2010dependent signal expression, and weakening female preferences. Though there are documented instances of seemingly adaptive shifts in trait expression, the ultimate impact on fitness is rarely tested. The field of urban evolution is still relatively young, and most work has tested whether differences occur in response to various aspects of urbanization. There is limited information available about whether these responses represent phenotypic plasticity or genetic changes, and the extent to which observed shifts in sexual communication affect reproductive fitness. Our understanding of how sexual selection operates in novel, urbanized environments would be bolstered by more studies that perform common garden studies and reciprocal transplants, and that simultaneously evaluate multiple environmental factors to tease out causal drivers of observed phenotypic shifts. Urbanization provides a unique testing ground for evolutionary biologists to study the interplay between ecology and sexual selection, and we suggest that more researchers take advantage of these natural experiments. Furthermore, understanding how sexual communication and mating systems differ between cities and rural areas can offer insights on how to mitigate negative, and accentuate positive, consequences of urban expansion on the biota, and provide new opportunities to underscore the relevance of evolutionary biology in the Anthropocene. Urbanization is ubiquitous and alters many environmental features, but we are just beginning to understand the implications for sexual communication in city\u2010dwelling animals. Our review discusses existing literature on how aspects of urbanization affect sexual selection and communication. One crucial driver of this change is urbanization, with over 2.5 billion additional residents predicted to inhabit cities by 2050 that are critical for successful reproduction. While there is a rich body of research investigating how sexual communication is affected by environmental conditions , and endocrine\u2010disrupting chemicals from pharmaceuticals that did not directly address urbanization. We were left with 58 relevant papers from this search that are summarized in Table We conducted a search in ISI Web of Science that included the following search terms, TOPIC: \"sexual selection\" or \"sexual signal*\" or \"mating behavio*\" or \"courtship\" or \"sexual communication\" or \"mate choice\" or \"male\u2010male competition\" AND TOPIC: \"urban*\" or \"city\" This search was last updated March 3, 2021, and yielded 327 papers in total. We determined the relevance of each paper and excluded reviews, meta\u2010analyses, empirical studies that did not explicitly examine the city as a selective agent on sexual traits, and studies that broadly investigated consequences of anthropogenic impacts but not urbanization Below, we highlight key themes in these studies and others that focused on environmental factors associated with urbanization to synthesize our current understanding of how urbanization affects sexual communication and to identify important areas for future research.4Urbanization encompasses a complex suite of environmental changes, including pollution from noise, light, and metals; new physical structures that fragment habitats and change ambient conditions; and shifts in community structure and resource quality Figure . Below, 4.1Cities experience much more background noise than rural areas, largely due to air and vehicle traffic showed a weaker preference for pair\u2010bonded males when exposed to high\u2010amplitude white noise, suggesting that normally monogamous songbird populations may show more extra\u2010pair behavior in areas with more anthropogenic noise , females do not express typical preferences for low\u2010frequency male songs in noisy conditions, thereby weakening sexual selection , which in turn reduced the intensity of coloration involved in sexual signaling. This is important because it shows that noisy urban conditions can disrupt non\u2010acoustic animal communication. Birds reared under noisy conditions have also been shown to reduce investment in brain regions associated with song learning do not move away from artificial light even though it significantly reduces mate attraction, suggesting a maladaptive response are less selective about male signal quality under illuminated conditions but show no response to longer wavelength (\u2265597\u00a0nm) ambient light, suggesting that replacing broad\u2010spectrum lighting with longer wavelength lighting could help this species (and maybe other fireflies) thrive in urban settings express reduced carotenoid\u2010based coloration when living near sources of heavy metal pollution relative to conspecifics in less\u2010polluted areas at the cost of survival in urban parks in Oregon (USA), females closest to the edges were in the best condition and had the longest tail length because these areas were closer to anthropogenic food sources like bird feeders , higher temperatures during development are associated with greater pheromone production typical of higher quality males engage in greater web\u2010building behaviors at higher temperatures songs attenuated faster and reverberated more in urban areas use colorful plastic items like straws, bottle rings, wires, and other human\u2010associated items in their displays provides a compelling case study of how urban\u2010associated resource shifts can create an evolutionary trap present in rural areas produces carotenoid\u2010rich fruit that is highly abundant during the molting season. Though this plant allows males to express high levels of coloration, cardinals that nest in this shrub experience much higher levels of predation . In Arizona, these birds feed on small seeds in the desert, but have access to large, hard sunflower seeds from bird feeders in urban areas under natural conditions because carotenoids cannot be synthesized de novo and instead must be obtained through the diet Hill, . House f4.8To our knowledge, there appear to be no studies that have explicitly investigated how changes in predation regimes impact sexual communication in urban areas. However, these impacts are likely common. Urbanization alters predator\u2013prey interactions for many species by reducing the prevalence of specialist species and those at higher trophic levels in Panama represents one of the few documented examples of the rapid evolution of a sexual signal . These birds have historically lived in rural mountain habitats, but recently found a population in the suburban region of San Diego captured from roadsides as nymphs and reared under common conditions produced higher frequency songs compared to those captured from quieter habitats . However, much of the novelty of urban environments likely comes from complex interactions among multiple anthropogenic influences. A recent study ; writing\u2013review and editing (lead). Adam D. Kay: Conceptualization (supporting); writing\u2013review and editing (supporting). Marlene Zuk: Conceptualization (supporting); writing\u2013review and editing (supporting)."} +{"text": "Our interesting electrocardiogram has two qRS morphology without features of preexcitation suggesting two atrio ventricular node conduction system. All cardiologists should be aware of this feature in heterotaxy syndrome as reentrant supraventricular tachycardia may develop in these patients. Twelve\u2010year\u2010old male diagnosed as heterotaxy syndrome with complete atrioventricular canal defect planned for intracardiac repair presented with the following electrocardiogram . Both types of qRS are narrow and hence non preexcited. There are no features of any hemiblock. So most probably ventricle is activated in two different conduction system which are oriented in different direction. There are reports of presence of dual atrio ventricular (AV) node in some complex congenital heart disease most commonly with heterotaxy syndrome with right atrial isomerism and some cases of discordant atrioventricular connection.One study using 3D electroanatomic mapping system in patients of heterotaxy syndrome with atrioventricular septal defect has showed the presence of two AV nodes one at the superior and other at inferior aspects of the common AV junction connected by a sling of conduction tissue along the ventricular border of the AV septal defect. They have shown the presence of two separate discrete His potential decremental and adenosine sensitive conduction and inducible AV reciprocating tachycardia involving both AV nodes.The authors declare no conflict of interests for this article."} +{"text": "The University of Southern Indiana (USI) GWEP uniquely embeds Area Agencies on Aging (AAA) care coordinators within primary care settings to invite the participation of aging patients in advance care planning (ACP), among other health interventions. Two subsequently developed features of the USI GWEP\u2019s ACP initiative emerged to address the What Matters metric of the 4Ms: 1) Patients are invited to engage in What Matters Most conversations through multiple touchpoints that frame Medicare Wellness Visits with a Deaconess provider and introduce a free, online ACP platform, Prepare for Your Care. 2) Provider, patients and families are supported in having ACP conversations with the dedication of a new Advance Care Planning facilitator position. Certified in Respecting Choices and jointly funded by the GWEP and Deaconess, the ACP facilitator supports individuals in navigating these essential healthcare conversations about balancing quality care with quality of life."} +{"text": "Little is known about whether care recipients\u2019 and their spousal caregivers\u2019 own pain influence the marital quality perceived by caregivers. Considering that experiencing and witnessing pain may be related to marital distress, we hypothesized that care recipient and caregiver pain would be associated with caregivers\u2019 greater increases in marital conflict over time. We focused on 264 spousal caregivers of older adults with chronic illnesses or disability from the 2015 and 2017 National Study of Caregiving. Sixty-nine percent of care recipients and 54% of caregivers in this study were bothered by pain at baseline. Findings revealed that caregiver and care recipient pain at baseline were both associated with caregivers\u2019 higher marital conflict at follow-up. These findings suggest the importance of accounting for not only care recipients\u2019 pain but also spousal caregivers\u2019 own pain when examining caregivers\u2019 marital quality."} +{"text": "Science provided a novel method to produce metallic GNRs by inserting a symmetric superlattice into other semiconductive GNRs. This finding will broader the applications of GNRs both in nanoelectronics and fundamental science.Isolated graphene nanoribbons (GNRs) usually have energy gaps, which scale with their widths, owing to the lateral quantum confinement effect of GNRs. The absence of metallic GNRs limits their applications in device interconnects or being one-dimensional physics platform to research amazing properties based on metallicity. A recent study published in Science reported an ingenious method to produce metallic GNRs [Unlike the gapless semimetal of graphene , the gralic GNRs based onI/dV point spectrum of a sGNR is shown in Fig.\u00a0In this work, Rizzo et al. in University of California at Berkeley used the precursor molecule 1 (Fig.\u00a0This work provides a smart strategy for realizing the metallicity in GNRs to act as a candidate used in logic devices. In future, as a direct measurement of metallicity, variable temperature conductivity experiments can be further considered. Additionally, the performance comparison between these metallic GNRs and traditional metals like copper used in interconnect technology is also meaningful for the applications in nanoelectronics. Finally, considering the possible formation of junctions between these metallic GNRs and ordinary semiconductive GNRs, whether Ohmic contact or Schottky contact can be formed should also deserve extra efforts."} +{"text": "Salmonella Typhimurium infections. The isolates were closely related genetically to each other by whole genome sequencing (WGS) analysis and related to isolates from two previous outbreaks of S. Typhimurium infections linked to pet hedgehogs . The Public Health Agency of Canada identified 31 cases highly related by WGS to U.S. cases, also linked to hedgehog contact in children aged <5 years. Eleven (26%) of 42 patients with available information were hospitalized, and no deaths were reported. Among 36 interviewed patients (or their parents or guardians), 30 (83%) reported hedgehog contact before becoming ill. Seven of 13 patients reported awareness of the risk for Salmonella.Hedgehog purchase locations were available for 20 of the 36 patients interviewed and included U.S. Department of Agriculture\u2013licensed breeders,Salmonella strain has continued to cause disease despite targeted outreach to hedgehog breeders and industry groups during two previous outbreaks with the strain linked to hedgehogs (Salmonella among hedgehogs and to limit transmission from hedgehogs to humans. CDC recommendations to pet owners during this outbreak focused on handling hedgehogs safely, including proper hand hygiene (Salmonella prevalence in animal populations to evaluate sanitation and husbandry practices and monitoring hedgehogs for Salmonella through diagnostic testing.This particular Salmonella in hedgehogs is complicated because of asymptomatic carriage and persistent or intermittent fecal shedding; however, Salmonella mitigation is possible through prevention and control measures focused on good sanitation and husbandry practices (Salmonella transmission from hedgehogs and advise that hedgehogs might be inappropriate pets for children aged <5 years. The pet industry, veterinarians, and public and animal health officials could collaborate to help prevent disease transmission to humans by establishing and disseminating information on ways to reduce the prevalence of Salmonella in hedgehog breeding colonies intended for use in the pet industry.Prevention and control of"} +{"text": "Gata binding protein 2 (GATA2) and 80% of patients with GATA2 mutations develop myeloid malignancy before the age of forty. Although GATA2 is established as one of the key regulators of embryonic and adult hematopoiesis, the mechanisms behind the leukemia predisposition in GATA2 haploinsufficiencies is ambiguous. The only curative treatment option currently available is allogeneic hematopoietic stem cell transplantation . However, allo-SCT can only be applied at a relatively late stage of the disease as its applicability is compromised by treatment related morbidity and mortality (TRM). Alternatively, autologous hematopoietic stem cell transplantation (auto-SCT), which is associated with significantly less TRM, might become a treatment option if repaired hematopoietic stem cells would be available. Here we discuss the recent literature on leukemia predisposition syndromes caused by GATA2 mutations, current knowledge on the function of GATA2 in the hematopoietic system and advantages and pitfalls of potential treatment options provided by genome editing.Inherited bone marrow failure syndromes (IBMFS) are monogenetic disorders that result in a reduction of mature blood cell formation and predisposition to leukemia. In children with myeloid leukemia the gene most often mutated is GATA2 are the most common genetic defects in pediatric MDS genetic factors in the complex network of blood regulation is essential to design non-invasive and preventive treatment options for IBMFS patients.IBMFS are a heterogeneous cluster of disorders manifested by an ineffective blood production and concurrent cytopenias that eventually result in a hypoplastic bone marrow (BM). These syndromes constitute an increased propensity to develop hematological malignancies such as myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) nuclease platforms, improve rapidly and progress toward efficient therapies for several genetic diseases GATA(A/G) DNA sequence and other protein partners through two multifunctional zinc finger (ZF) domains; ZF1 and ZF2 that are encoded by exon 4 and exon 5, respectively syndrome . Currently, despite the improving definition of the phenotypic characteristics of GATA2 deficiency syndromes and high penetrance of myeloid malignancy, the mutational background and phenotypic outcome observed in these patients do not correlate, suggesting that additional events are important for disease progression 10.5 and none survive beyond E11.5, due to severe anemia. Chimeras of WT and Gata2\u2212/\u2212 embryonic stem (ES) cells show that Gata2-null cells cannot contribute to hematopoiesis in adult blood, fetal liver, BM and thymus revealing a requirement for Gata2 in embryonic hematopoiesis and in the first transplantable hematopoietic stem cells (HSCs) that differentiate from HE promotes proliferation in hESCs, but quiescence in hESC-derived HSCs , these mutations would need to be restored at the endogenous locus, requiring HDR as repair mechanism. Therefore, optimizing an editing strategy by using large HDR donor templates that cover various GATA2 mutation regions found in patients or the whole gene, containing homologous regions covering several exons, could provide treatment for a substantial group of GATA2 patients. An efficient method for gene correction in HSCs with CRISPR/Cas9 and large HDR donor delivered by rAAV6 was used to correct a HBB gene mutation causing sickle cell disease and has potential to correct GATA2 mutations in HSCs using the same strategy and DISCOVER-seq (Discovery of in situ Cas Off-targets and VERification by Sequencing), are shown to overcome these obstacles and could be used to efficiently identify OTEs that might result from GATA2-editing strategy before its clinical translation that might occur in undesired parts of the DNA. Detection of OTEs with whole genome sequencing are often challenging due to high background of random reads in combination with low sequence depth (<10-fold) . Currently, clinical trials are performed where CRISPR/Cas9 is used to remove erythroid expression of the fetal hemoglobin repressor BCL11A in the treatment of hemaglobinopathies, implicating a highly promising potential for genome editing to treat various hematological disorders of leukemogenic driver mutations to help us choose an appropriate time frame and strategy to treat these patients using genome editing. If leukemic driver mutations arise early during hematopoietic development, targeting leukemic clones will be challenging.Although in vivo Gata2+/\u2212 models have not developed an MDS/AML phenotype (Ling et al., Gata2+/\u2212 models could provide more insight, since this would challenge the HSC compartment and increase the chances of additional events that would promote leukemogenesis to occur.GATA2 mutations or any other gene mutations that predispose to hematological malignancies when potentials and risks of these tools are tested sufficiently prior to the actual patient treatments. In addition to their potential for gene therapy discussed in this review, CRISPR base and prime editing technologies are also fantastic tools for basic research to introduce additional predicted leukemia driver mutations to HSCs in GATA2 haploinsufficiency models in order to identify their potential role in malignant transformation.Base editing and prime editing are the recent promising and rigorous refinements of genome editing technologies which could provide and improve a patient specific mutation correction for CK and EP wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Prenatal adversity is hypothesized to increase risk of Autism Spectrum Disorder (ASD) via epigenetic changes. Maternal stress in late pregnancy may alter offspring neurodevelopmental outcomes by disrupting a unique period of rapid neurogenesis. Observational studies reporting an environmentally mediated programming pathway face challenges in drawing causal inferences including passive gene-environment correlation. This project aims to use a quasi-experimental genetically informed design to assess if reported correlations between maternal prenatal stress and offspring ASD traits were due to maternally inherited factors or consistent with a potentially causal prenatal exposure effect. No previous cross-fostering studies have assessed the effects of prenatal stress on childhood ASD.This study used an in-vitro fertilization cross-fostering sample with pregnant mothers related (n = 365) or unrelated (n = 111) to their offspring (mean age = 9.84 years). Prenatal stress was assessed using a subjective Likert scale during pregnancy. Questionnaires examined maternally rated offspring ASD traits using the Social and Communication Disorders Checklist. Birth weight and gestational age from medical records were used as comparison outcomes to validate the measure of stress as evidence suggests they are influenced by environmental factors. Correlations from multiple regression models were examined in relation to magnitude of effect size as well as significance. This is partly due to small sample size and that cross-fostering designs rely on comparing magnitudes of associations between related and unrelated groups. An interaction term was used to test the difference in the strength of association between related and unrelated mother-child groups.Subjective assessment of prenatal maternal stress showed construct validity as it was associated with low birth weight and reduced gestational age . Subjective late pregnancy stress was associated with increased offspring ASD traits in the whole sample and in the related and unrelated mother-child subgroups. Non-significant interaction terms demonstrated that the mechanisms underlying the association between maternal stress and ASD and birth outcomes are likely to be similar and environmentally driven in the different conception groups.Findings demonstrate the utility of genetically informed designs in disentangling inherited factors from environmental influences in the study of prenatal risk factors. Correlations between maternal prenatal stress and offspring ASD being present in both related and unrelated mother-child groups indicate an environmental link that is consistent with a potential causal effect. Associations detected are of imperative use for clinicians and policymakers, as they can guide the implementation of early psychosocial care for families at high liability."} +{"text": "We present the case of a patient with lung adenocarcinoma stage IVB diagnosed as orbital apex syndrome (OAS) associated with intraorbital metastasis of lung cancer. When patients with lung cancer have diplopia, ptosis or ocular motility disorder, identifying OAS is important. A 53\u2010year\u2010old male patient with lung adenocarcinoma stage IVB complained of decreased right vision and diplopia when he was hospitalized to begin chemotherapy. His right eyeball was protruding and right eyelid was drooping Figure\u00a0. NeuroloNone declared.Takashi Ookuma and Ryota Kikuchi designed the research; Shinji Abe, Ryota Kikuchi, Hiroyuki Takoi and Takashi Ookuma analysed the data; and Takashi Ookuma and Ryota Kikuchi wrote the paper.The authors declare that appropriate written informed consent was obtained for the publication of this manuscript and accompanying images."} +{"text": "A 90\u2010year\u2010old Japanese man presented with back pain after falling. Imaging tests revealed compression fracture of the lumber vertebrae with diffuse idiopathic skeletal hyperostosis (DISH), and surgical intervention was performed. Back pain is common in primary care setting, and primary care physicians should recognize this condition well. A 90\u2010year\u2010old Japanese man presented to the emergency department complaining of backache and difficulty in walking after falling on his buttocks. On physical examination, severe tenderness over the lumbar vertebrae was found. There were no neurological deficits or pain in the pelvis or femurs. Compression fracture of the lumbar vertebrae was suspected. A computed tomography (CT) scan was performed to evaluate structural abnormalities, and injury of ligament with ossification with diffuse idiopathic skeletal hyperostosis (DISH) was detected Figure . MagnetiDISH is a noninflammatory disease where spinal ligaments and entheses become calcified.Back pain is one of the most common symptoms in a primary care setting.In summary, vertebral compression fracture in patients with DISH should be treated surgically because of the potential for neurological complications. Primary care physicians should remember which types of fracture may be candidates for surgery and refer patients with suspected vertebral compression fractures with DISH to specialists for further evaluations.The authors have stated explicitly that there are no conflicts of interest in connection with this article.Informed written consent was obtained from the patient for publication of this report and any accompanying images."} +{"text": "The Choosing Wisely Canada campaign raises awareness amongst physicians and patients regarding unnecessary or inappropriate tests and treatments. Using an online survey, members of the Pediatric Otolaryngology Subspecialty Group within the Canadian Society of Otolaryngology \u2013 Head & Neck Surgery developed a list of nine evidence based recommendations to help physicians and patients make treatment decisions regarding common pediatric otolaryngology presentations: (1) Don\u2019t routinely order a plain film x-ray in the evaluation of nasal fractures; (2) Don\u2019t order imaging to distinguish acute bacterial sinusitis from an upper respiratory infection; (3) Don\u2019t place tympanostomy tubes in most children for a single episode of otitis media with effusion of less than 3\u2009months duration; (4) Don\u2019t routinely prescribe intranasal/systemic steroids, antihistamines or decongestants for children with uncomplicated otitis media with effusion; (5) Don\u2019t prescribe oral antibiotics for children with uncomplicated tympanostomy tube otorrhea or uncomplicated acute otitis externa; (6) Don\u2019t prescribe codeine for post-tonsillectomy/adenoidectomy pain relief in children; (7) Don\u2019t administer perioperative antibiotics for elective tonsillectomy in children; (8) Don\u2019t perform tonsillectomy for children with uncomplicated recurrent throat infections if there have been fewer than 7 episodes in the past year, 5 episodes in each of the past 2\u2009years, or 3 episodes in each of the last 3\u2009years; and (9) Don\u2019t perform endoscopic sinus surgery for uncomplicated pediatric chronic rhinosinusitis prior to failure of maximal medical therapy and adenoidectomy. Delivery of efficient healthcare has become increasingly important. The Choosing Wisely Canada campaign raises awareness amongst physicians and patients regarding unnecessary or inappropriate tests and treatments. The Canadian Society of Otolaryngology \u2013 Head & Neck Surgery (CSOHNS) is a proud partner of Choosing Wisely Canada and has previously released recommendations for Head and Neck Surgical Oncology , OtologyThe CSOHNS is dedicated to improving patient care through the support of education, promotion of research, dissemination of information, scientific advancement of the society, and the maintenance of high professional and ethical standards. The Pediatric Otolaryngology Subspecialty Group (POSG) of the CSOHNS maintains these same core values and strives to promote evidence-based care amongst physicians and patients through the development of a Choosing Wisely Canada recommendation list.Pediatric otolaryngology complaints are commonly seen in many different settings. These recommendations aim to reduce unnecessary or inappropriate tests or treatments and may be relevant to various providers including general practitioners and otolaryngologists encountering pediatric otolaryngology patients.The current list was created by members of the POSG of the CSOHNS. Members were chosen based on their ongoing affiliation with an academic teaching hospital and their subspecialty training in pediatric otolaryngology. An initial list of 25 recommendations regarding unnecessary tests and interventions were compiled, along with sourced material to support the recommendations. Members of the group, considered to be national leaders within the subspecialty, were then asked to complete an electronic survey providing feedback on each recommendation and ranking their merit. The survey was constructed using Opinio .Each recommendation was rated by individual PSOG members based on five commonly evaluated factors: potential to affect clinical practice, cost-benefit ratio, potential to cause harm, evidence supporting recommendation, and pervasiveness of test/intervention . A scaleNasal fractures are one of the most common facial fractures in the pediatric population . The decUncomplicated acute bacterial sinusitis (ABS) is a diagnosis that is made based on clinical criteria and has a low prevalence amongst children presenting with respiratory symptoms \u201310. AlthThe American Academy of Pediatrics recommends diagnosing pediatric ABS when a child with an acute upper respiratory tract infection presents with (1) persistent illness , (2) a worsening course , or (3) severe onset .Although tympanostomy tube insertion can be associated with short-term quality of life improvements, the natural history of otitis media with effusion (OME) is sufficiently favorable and the majority of uncomplicated OME cases in children will spontaneously resolve within 3\u2009months \u201315. CaseIn most cases, medical treatment using antihistamines, decongestants, systemic antibiotics and steroids have shown little to no effect on the long-term outcomes of uncomplicated OME in children \u201319. In aThe use of unnecessary oral antibiotics can promote antibiotic resistance and increase the risk of opportunistic infections . TopicalCodeine has been associated with a high rate of adverse drug reactions in children , 27. ThiAdministration of perioperative antibiotics for children undergoing elective uncomplicated tonsillectomy shows no significant benefits in regard to common post-tonsillectomy morbidities , 32. OveFor children who have a lower number of recurrent throat infections, tonsillectomy has significantly less benefit when compared to those with more frequent infections, and many children with recurrent throat infections naturally improve without intervention , 34. TheWhile endoscopic sinus surgery (ESS) has been found to be an effective therapy in children with uncomplicated chronic rhinosinusitis, comparable outcomes can be achieved with medical therapy and adenoidectomy , 37. A sThese recommendations are not hard \u201crules\u201d but are rather intended to promote discussion amongst physicians and patients regarding evidence-based approaches to reduce inappropriate or unnecessary treatment for pediatric otolaryngology patients. Relevance for patients revolves primarily around providing a concise description as to why practitioners may opt for or against certain treatments. In addition to these recommendations being open access on their website, Choosing Wisely Canada actively promotes their campaigns through social media and posters for use in clinicians\u2019 offices, both of which patients may be exposed to. Clinicians should carefully tailor treatments and discussions with patients based on each individual patient\u2019s unique presentation and circumstances. These recommendations are not meant to establish payment or coverage by insurers."} +{"text": "Amnestic mild cognitive impairment (aMCI) is marked by episodic memory deficits, which is used to classify individuals into early MCI (EMCI) and late MCI (LMCI). Growing evidence suggests that individuals with EMCI and LMCI differ in other cognitive functions including cognitive control, but these are less frequently studied. Using a semantic Go/NoGo task, we examined differences in cognitive control between EMCI and LMCI on behavioral (accuracy and reaction time) and neural measures. Although no behavioral differences were observed between the groups, EMCI and LMCI groups differed in patterns of neural oscillations for Go compared to NoGo trials. The EMCI group showed differences in theta power at central electrodes and alpha power at central and centro-parietal electrodes between Go and NoGo trials, while the LMCI group did not exhibit such differences. Furthermore, the LMCI group had higher theta synchronization on Go trials at central electrodes compared to the EMCI group. These findings suggest that while behavioral differences may not be observable, neural changes underlying cognitive control processes may differentiate EMCI and LMCI stages and may be useful to understand the trajectory of aMCI."} +{"text": "Sleep is an important behavior in the prevention and management of chronic conditions in later life. Marital status may account for variability in sleep quality, but little is known about this association in the later part of life or how transitions into and out of marriage are related to changes in sleep quality. This study used the resource model and crisis model as frameworks to understand how marital status and marital transitions were related to sleep quality in mid to late life and whether these findings differed by gender. Interview data from 2,872 participants 50-74 years old from the ORANJ BOWL, a longitudinal panel study in New Jersey, were used. Marital status and sleep quality were examined in two waves approximately 10 years apart. All analyses controlled for health and sociodemographic characteristics. Weighted regressions revealed that individuals in committed romantic relationships and women had worse sleep quality than those in other marital status groups and men (p<.005). Weighted fixed effects regressions revealed that compared to individuals who remained married, individuals who remained divorced or widowed or who became widowed had better sleep quality, whereas those who became divorced had worse sleep quality (ps<.05); individuals who transitioned into marriage had better sleep quality than those who remained divorced or widowed (ps<.03). Findings differed depending on the index of sleep quality examined. Efforts to understand which middle-aged and older adults are most vulnerable to sleep disturbances can inform the design of interventions to promote better sleep quality."} +{"text": "Odocoileus hemionus) are currently being colonized by ERVs, which provides an opportunity to study ERV dynamics at a time when few are fixed. We previously established the locus-specific distribution of cervid ERV (CrERV) in populations of mule deer. In this study, we determine the molecular evolutionary processes acting on CrERV at each locus in the context of phylogenetic origin, genome location, and population prevalence. A mule deer genome was de novo assembled from short- and long-insert mate pair reads and CrERV sequence generated at each locus. We report that CrERV composition and diversity have recently measurably increased by horizontal acquisition of a new retrovirus lineage. This new lineage has further expanded CrERV burden and CrERV genomic diversity by activating and recombining with existing CrERV. Resulting interlineage recombinants then endogenize and subsequently expand. CrERV loci are significantly closer to genes than expected if integration were random and gene proximity might explain the recent expansion of one recombinant CrERV lineage. Thus, in mule deer, retroviral colonization is a dynamic period in the molecular evolution of CrERV that also provides a burst of genomic diversity to the host population.All vertebrate genomes have been colonized by retroviruses along their evolutionary trajectory. Although endogenous retroviruses (ERVs) can contribute important physiological functions to contemporary hosts, such benefits are attributed to long-term coevolution of ERV and host because germline infections are rare and expansion is slow, and because the host effectively silences them. The genomes of several outbred species including mule deer ( Retroviruses are unique among viruses in adopting life history strategies that enable them to exist independently as an infectious RNA virus or as anThe life history strategy adopted by retroviruses indicates why this virus family has been so successful in colonizing host germline. Retroviral replication requires that the viral RNA genome be converted to DNA and then integrated into the genome of an infected cell . As withThe retroviral life cycle also demonstrates how ERVs can affect host biology . ERVs reThe coding portion of a new ERV can be eliminated from the genome through nonallelic homologous recombination (NAHR) between the LTRs, which are identical regions that flank the viral coding portion. A single LTR is left at the site of integration as a consequence of the recombination event and serves as a marker of the original retrovirus integration site . Most ERAlthough in humans most ERV colonization events occurred in ancestral species, acquisition of new retroviral elements is an ongoing or conteOdocoileus hemionus) ERV because we have extensive data for prevalence of CrERV loci in northwestern US mule deer populations (Odocoileus virginianus) , all endinianus) . In thisc value of 3.41\u2009pg) experimentally determined genome size of reindeer (Rangifer tarandus) .n\u2009=\u200970 was manually blocked to accommodate the variable regions in the retrovirus env. The right panel of env insertions and deletions characteristic of each lineage (see env variable structure of each reconstructed CrERV). The resultant tree shows four well-supported CrERV lineages, each diverged from a common ancestor since the split of mule deer and white-tailed deer; these results are consistent with the phylogenetic structure of CrERV based on a partial genome alignment reported previously relative to Lineage A env while retaining the transmembrane region (TM) . The phylogenetic history of Lineage B CrERV recorded in the mule deer genome indicates that all members that share this env structure arose approximately 150 ka relative to the env of Lineage A CrERV. CrERVs with this env configuration represent 9% of coding CrERV in MT273. Because the prevalence of Lineage Bi is high in mule deer, this group could represent the ancestral state for Lineage B CrERVs. The second group appears to have a unique env not found in any other CrERV lineages CrERV in the data set that have the signature 59\u2009bp insertion \u201c\u201d in env online ct 500 ka online. t 500 ka online, env insertion \u201cc\u201d with Lineage C but lacks deletion \u201ce,\u201d . In this region, several CrERV, which we provisionally classified as Lineage B because they carried the prototypical env deletion \u201cd\u201d of SU form a monophyletic group that is affiliated with Lineage A CrERV .There is good support for recombination between Lineages A and B in a region spanning a portion of A CrERV . Therefore, to determine if any CrERV had an integration site preference near genes, we determined if the number of CrERV loci observed to be within 20 kb of a gene differed from that expected if the distribution was random. There are significantly more observed insertions that fall within 20 kb of the translation start site of a gene than would occur randomly (P\u2009=\u20090.002891). Additionally, several recombinant CrERVs are close to a gene . The recombinant breakpoint within this monophyletic group is identical, suggesting that an interlineage recombinant most likely expanded by retrotransposition. Notably, two CrERV in this recombinant cluster were only found in M273, indicating expansion was a recent event. There are other clusters of CrERV with Lineage B env affiliated with Lineage A CrERV that have different breakpoints in this partial phylogeny, suggesting that interlineage recombination is not a rare event. Recombination between an XRV and ERV is also a well-documented property of retroviruses , and an outgroup genome (hg19). Genome alignments were performed with lastz under thJN592050) using bwa mem. Mates of reads that mapped to the reference CrERV were extracted and then mapped to the WGS assembly using bwa mem. Mates mapped to the WGS assembly were then clustered using the \u201ccluster\u201d function of bedtools. Anchoring mate pair clusters on both sides of the insertion site were complemented by junction fragments to localize CrERVs. Based on the RACA data, CrERVs that sit between scaffolds were also retrieved in this manner. CrERV reads were then assigned to their corresponding cluster and were assembled using SeqMan (DNASTAR). Sanger sequencing was performed to complement key regions used in CrERV evolutionary analyses. All reconstructed CrERV sequences used in the phylogenetic analyses are included in CrERV locations and sequences were retrieved based on junction fragment and long-insert mate pair WGS data. The long-insert mate pair WGS reads were mapped to the reference CrERV (GenBank accession number CrERV sequences of interest were initially aligned using the default setting of muscle , manuallWe simulated 274 insertions per genome to approximate the average number of CrERVs in a mule deer . The simMolecular Biology and Evolution online.msab252_Supplementary_DataClick here for additional data file."} +{"text": "Friendship plays a crucial role in maintaining social connectedness in late life. Volunteering helps older adults to stay socially engaged and often times provides the opportunity to meet and make new friends. A small literature suggests that volunteering may be associated with friendship, but many studies are limited by reliance on small, non-probability samples and simplistic analytic approaches. The literature is also unclear on how volunteering behaviors relate to specific characteristics of friendships and whether there are gender differences that condition these relationships. Using the 2014 and 2018 waves of the Health and Retirement Study , we investigate whether volunteer status and hours volunteered in 2014 are associated with friendship characteristics in 2018 among community-dwelling adults aged 50 years and above . We also examine whether gender moderated these relationships. Volunteer status and hours in 2014 were positively associated with the number of close friends and contact frequency in 2018. Only those who volunteered 200 hours or more in 2014 were positively associated with friendship quality in 2018. Regarding gender differences, men who volunteered 200 hours or more in 2014 had higher friendship quality in 2018 than women, while women who volunteered 100-199 hours in 2014 had greater contact frequency in 2018 than men. Hence, our results suggest volunteering is integral in shaping late-life friendships and volunteering might be more critical for understanding friendship characteristics among older men and women."} +{"text": "The findings reveal clear evidence that knowledge-oriented interventions and supportive devices to improve menstrual health need to be developed and tested. This includes materials that dispel harmful social beliefs and taboos that discriminate against all who menstruate, but even more profoundly affect those with disabilities. An attempt to quantify a \u2018menstruation interference score\u2019 found those with no disability reported greater daily interference from menstruation, although those with disabilities suffered greater restrictions. Alternative indicators to measure intervention effects and quality of life evaluation are thus needed. Studies around costs of interventions, benefits, and return on investment are required to inform implementation strategies and policy frameworks. Additionally, Wilber and colleagues prior review identified evidence that forced cessation of menstruation through hysterectomies occurs among disabled, and warrants urgent attention.\u2022Persons with disabilities suffer greater disadvantages in care and support of their menstruation.\u2022Caregivers lack knowledge-based guidance and supportive devices which deleteriously impacts adequate menstrual health and hygiene practices.\u2022Interventions are urgently needed to provide dignity and care and ameliorate social stigma and taboos which increase isolation and restrictions among those with disabilities.None."} +{"text": "Sedentary behavior may adversely affect physical and cognitive health of older adults in assisted living (AL). Replacing sedentary behavior with light physical activity (PA) could help them maintain functional abilities and independence. We interviewed AL residents to obtain their guidance regarding the implementation of an intervention to reduce sedentary behavior. Here we report the results of a thematic analysis exploring contextual factors that may influence intervention implementation. We interviewed 20 residents and identified 7 themes. The first was attitudes and beliefs about PA. Most residents believed PA was important, but some lacked motivation or confidence to perform PA. Another theme was attitudes and beliefs about aging, as some residents felt discouraged about aging and uncertainty about how much PA they could safely perform. Abilities of AL residents was seen as an important consideration. It was noted that residents have a wide range of abilities and this could present challenges in planning a PA program appropriate for all residents. Social influences for PA should be considered, as residents may find encouragement from family or other residents. Space for being active is another factor because it is typically limited within AL. We found that some residents wanted more challenging exercise classes than currently provided by their facility. Finally, residents described limited opportunities for PA due to the nature of the AL environment. This thematic analysis brings attention to important factors that could influence the implementation of PA interventions with the AL population."} +{"text": "Some significant problems related to undergraduate education in biochemistry and molecular biology magnified by COVID-19 had been recognized previously AAAS\u00a0. Taking Erin Dolan highlighted the power of integrating Course-based Undergraduate Research Experiences (CUREs) into undergraduate course experiences to influence student retention and improve students\u2019 likelihood of graduating from university and majoring in a STEM discipline Fig.\u00a0. These CLuciane Vieira de Mello demonstrated the benefits of requiring students to engage in reflective practice to enhance their life and employability skills discussed post-pandemic scenarios for learning and teaching. Considering the need to enhance face-to-face education with digital approaches, synchronously and asynchronously, the presentation considered the advantages and disadvantages of each format and established the imperative that new designs are framed under active learning paradigms presented the \u201cBiokimi App\u201d to support student learning of hepatic glycolysis and gluconeogenesis regulations (Oliveira et al."} +{"text": "We apply the adaptive leadership framework for chronic Illness in a care partner\u2013assisted intervention to improve oral hygiene of older adults with cognitive impairment. Care partners receive four coaching sessions which we recorded and transcribed verbatim. We will describe how our team of seven investigators codes the data using a priori codes from the framework. The data from 17 care-partners contributes 68 individual sessions for coding. We have two subgroups of 7 individuals with mild dementia (MD) and 10 with mild cognitive impairment (MCI). We will discuss the plan for multiple comparisons such as (a) longitudinal across 3 months of intervention, b) within MD and within MCI and c) between MD and MCI. To illustrate, we will discuss our approaches to reaching coding consensus and rigor and will present results of the within group analyses. Finally, we will discuss next steps and the end products we aim to achieve."} +{"text": "Given the current health service constraints, a review of dietetic services within maternity care is warranted.Globally, there has been a renewed focus on addressing gestational weight gain (GWG). In Australia, the Department of Health pregnancy care guidelines recommend women be offered routine weighing and receive brief nutritional and physical activity support during antenatal care visits. Women gaining weight outside the Institute of Medicine (IOM)\u2019s weight gain reference values are further recommended to be referred to a dietitian. However, professional and organizational barriers, including an absence of weight gain referral pathways and limited workforce resources, exist with the translation and scaling of these recommendations into practice. This study aimed to explore patterns of GWG among a cohort of Australian pregnant women and to determine if pregnancy weight gains of above or below 2 kg or 5 kg in the second and third trimester can be used to predict total GWG outside recommendations. Sensitivity, specificity, negative, and positive likelihood ratios were calculated. The most predictive time point was 24 weeks\u2019 gestation using the minimum weight change parameter of +/\u22122 kg, demonstrating reasonable sensitivity and specificity , resulting in 55% ( In 2009, the Institute of Medicine (IOM) published revised guidelines for gestational weight gain (GWG) in response to the increasing prevalence of overweight and obesity within the United States (US) population . These gn = 1034) multiethnic Australian cohort of pregnant women with a diagnosis of gestational diabetes mellitus (GDM), conducted by Barnes et al., identified that 61% of the women in this cohort evidenced high rates of early EGWG with only 44% achieving their GWG targets within the IOM recommendations by the time they gave birth . . n = 103Although further research is required to test the referral process, our model suggests that there is potential for weight gains of 2 kg or greater identified at two consecutive second trimester time points occurring at approximately 19 and 24 weeks\u2019 gestation to be used to identify women at risk for high total GWG and risk of GDM. This supports the Academy of Nutrition Dietetics GDM evidenced practice-based guidelines by increasing the scope of recommendations to encompass screening and detection of at risk women prior to GDM diagnosis for early glycemic control and GWG management . The detn = 29) had returned to or weighed less than their pre-pregnancy BMI [n = 37) returned to or weighed less than their pre-pregnancy BMI at 12 months post-partum, with GWG significantly predicting weight retention at 12 months [Although there is limited time during the third trimester (after 30 weeks\u2019 gestation) for providing more than brief dietetic assessment and follow up, there is potential scope for the identification of late pregnancy weight gains above the IOM recommendations to be used for identifying women at risk of adverse birth outcomes as well as post-partum weight retention. A previous analysis of the WATCH cohort conducted by Martin et al. identified that only 22% of women followed up at 6 months post-partum and objectively measured weights obtained during WATCH study pregnancy care visits.2 was derived from a sample of n = 7 women and should be interpreted with caution. Further research is now required within larger multiethnic cohorts of pregnant women.Total GWG was measured at 36 weeks\u2019 gestation and as such, may not accurately reflect total pregnancy weight gain immediately prior to giving birth. We acknowledge that the use of self-reported pre-pregnancy weight to inform pre-pregnancy BMI and weight gain targets as limitations; however, a systematic review by Headen et al. concludeIncongruencies exist with the translation of GWG guideline recommendations into maternity care, particularly at the service provider level. A deficit of screening and referral pathways to identify women most in need of dietetic support to achieve appropriate GWG is evident. The current analysis suggests that weight changes of +/\u22122 kg detected at one second trimester time point or across two second trimester time points may be useful to inform dietetic referral practices and promote GWG guideline adherence. Given the professional and organizational barriers that exist with dietetic resourcing within antenatal care, a stepped approach for dietetic referral is warranted, allowing for service providers to prioritize resources to meet demand. The implementation of referral pathways early in the second trimester may additionally allow \u201cat-risk\u201d women to receive early dietary behavioral support to achieve appropriate weight gain targets and glycemic control, potentially reducing their risk of having a large or small for gestational age infant and optimizing overall maternal and infant health. Further research is now required to test the predictive value of the weight change referral parameters within larger multi-ethnic cohorts of pregnant women."} +{"text": "ABSTRACT IMPACT: This work has the potential to help clinicians decide which infants exposed to in utero opioids, will need to be treated early or can be discharged home early based on their risk, thus reducing prolonged hospitalization OBJECTIVES/GOALS: To develop and validate a prediction model with inclusion of clinical and demographic risk factors to identify infants with NAS likely to need pharmacotherapy. METHODS/STUDY POPULATION: A pooled cohort of 761 infants from 5 different studies including 2 trials and 3 observational cohorts will be used to develop the model.All infants >than or equal to 37 weeks gestational age born to mothers with history of OUD will be included. Infants with congenital disorders and severe medical and surgical illnesses will be excluded. Multivariable mixed effects logistic regression modeling will be performed to predict the need for pharmacologic treatment for NAS. Candidate variables will be included based on clinical knowledge and previously published data. Model performance will be evaluated by measuring discrimination using Area Under the Curve (AUC) statistics and calibration. Model will be internally validated using boot strap validation. RESULTS/ANTICIPATED RESULTS: Pending data analysis DISCUSSION/SIGNIFICANCE OF FINDINGS: Opioid Use Disorder in pregnancy has resulted in concurrent rise in NAS incidence. NAS affects opioid exposed infants variably and accurate prediction of its severity and need for treatment remains elusive. Known clinical and demographic factors can predict the need for NAS therapy in opioid exposed infants, aiding clinical decision making."} +{"text": "To suggest a link between sertraline and urinary side effects in a Sheffield Child and Adolescent Mental Health Service population.Evidence suggests that Serotonin has an important role in bladder control through central and peripheral neurological pathways. Increased serotonergic activity leads to parasympathetic inhibition, which results in urine retention. It is through this mechanism of action and their effect on pre-synaptic serotonin 1A and peripheral 5-HT3 receptors that SSRIs were observed to have anti-enuretic effect. At low 5-HT concentrations, micturition is inhibited whereas at high levels, an excitatory effect is achieved. This may suggest a dose-dependent relationship between Sertraline and urinary side effects.Inclusion criteria:Under 18 years of ageOn SertralineReported urinary side effectsExclusion criteria:Above 18 yearsNot on SertralineAssociated urinary problemsDid not report urinary side effectsClinical records of eligible patients were accessed to gauge temporal relationship between initiation of sertraline and reported urinary side effects.Three cases were identified in the authors\u2019 clinical practice at Sheffield CAMHS that were suggestive of a link between sertraline and urinary side effects.It's important for clinicians to bear in mind the genitourinary side effects of SSRIs, which may be debilitating for patients in the CAMHS population. It's equally important for us as clinicians to educate young people and their parents about these potential side effects and how they can be managed. It has also been observed that higher doses of Sertraline have shown a possible link between onset of urinary side effects."} +{"text": "Late-life depression is a serious public health concern in the U.S., especially as the population ages. To improve care coordination and increase the number of providers working to improve depression outcomes, primary care clinics and community-based organizations (CBOs) can partner and improve care. Addressing social determinants of health is one area CBOs can help respond to but there are other ways CBOs can bring value to these partnerships with primary care clinics. As part of a qualitative evaluation of the Care Partners Project, 84 key informant interviews and 20 focus groups were conducted over five years with selected primary care physicians, care managers, administrators and psychiatric consultants. These data were coded and organized using an inductive and deductive thematic analysis approach. CBOs contributed to care through 1) adding new services that focus on clients\u2019 social needs that were foundational to effective depression care; 2) strengthening core aspects of existing care; 3) incorporating a lay health workforce to enhance care; and/or 4) adding home visits that supported deeper understanding of patient\u2019s life context, enhanced trust and improved access to care. CBOs can enhance depression care through increasing access and quality of care. Findings can inform conversations about the value CBOs offer when partnering with health care systems and improve partnership efforts. Such conversations are worth revisiting as organizations deepen their connections and work together over time."} +{"text": "Recruitment and engagement of racial/ethnic minority older adults in clinical trials is crucial to expand implementation of evidence-based interventions for disability prevention. Public Health measures to counteract COVID-19 pandemic have increased the challenges on reaching this population. This study seeks to comprehensively evaluate a set of recruitment strategies to enroll Latino, Asian and African American older adults with symptoms of depression and anxiety during the first year of a randomized clinical trial. A partnership of three academic sites across the U.S. involving several collaborations with community agencies recruited racial/ethnic minority older adults using different strategies involving bilingual interviewers calling from hospital research dataset and community agencies\u2019 list of clients, referrals from primary care providers or psychotherapy waitlist. In this presentation we will report various recruitment and retention data including individual and organizational predictors of successful recruitment as well as challenges across all three sites."} +{"text": "This paper explores how gradual retirement impacts inequality later in life, with a focus on transitions from career to bridge employment. We use 26 years of longitudinal data from the Health and Retirement Study to document the various pathways that older Americans take when exiting the labor force, and examine how bridge employment impacts non-housing wealth and total wealth, including the present discounted value of Social Security benefits. We find that gradual retirement in the form of bridge employment neither exacerbates nor mitigates wealth inequalities among Americans who held career jobs later in life. We do find evidence that wealth inequalities grow among the subset of older career workers who transition from career employment to bridge employer at older ages. These findings provide quantitative evidence that bridge employment at older ages is taken by those who need to continue working financially and those who continue working for nonpecuniary reasons."} +{"text": "ABSTRACT IMPACT: This work sheds diagnostic insight on patients with idiopathic rare disease and has the potential to further their care and treatment as a result. OBJECTIVES/GOALS: Correct diagnosis is imperative to treating patients with idiopathic, suspected genetic conditions, yet sequencing approaches leave up to 70% of these patients undiagnosed. We sought to improve diagnosis rates for a cohort patients referred for sequencing by characterizing deleterious variants within the 5\u2019UTR. METHODS/STUDY POPULATION: We retrospectively analyzed whole exome sequencing (WES) data from 472 unsolved rare disease patients within the Mayo Clinic Center for Individualized Medicine to identify variants within the 5\u2019UTR that affect the presence of upstream open reading frames (uORFs). uORFs are short regions that typically influence downstream gene translation by sequestering ribosomes. We specifically searched for variants with the potential to disrupt existing uORFs or introduce new uORFs within the 5\u2019UTR, and developed a pipeline to annotate these variants with information including GnomAD allele frequency and gene loss of function intolerance (pLI) score. To aid in variant interpretation, we applied two deep learning tools to predict variant impacts on transcript ribosome load (TITER and FramePool). RESULTS/ANTICIPATED RESULTS: Our pipeline identified a median of 21 variants per patient that were predicted to have a deleterious impact on the translational efficiency of protein coding transcripts, primarily by introducing new start codons within the 5\u2019UTR or by altering the Kozak consensus of existing start codons. A median of 10 of these variants occur upstream of haploinsufficient genes with an existing disease association. We also identified a subset of variants that are predicted to introduce translationally active N-terminal extensions to protein coding transcripts, with the potential to disrupt protein localization and processing. DISCUSSION/SIGNIFICANCE OF FINDINGS: This work demonstrates that analysis of 5\u2019UTR variants can be incorporated into existing WES pipelines, and identifies a group of variants with potential significance to patient disease. Further experimental evidence is necessary to ascertain the pathogenicity of these variants."} +{"text": "To survey the prevalence of monitoring of vitamin D on an inpatient ward.To audit the treatment if there is identified vitamin D deficiency or insufficiencyTo compare differences between findings in auditsAll inpatients admitted to Milford centre between August 2019 and August 2020 were selected as part of the sample size.Data were collected by FY1 and FY2Patients\u2019 laboratory results were accessed to determine vitamin D levels.E-notes were used to conclude who were vitamin D sufficient or deficient for treatmentThe standard for the audit were as per:Management of vitamin D deficiency or insufficiency in adults \u2013 CKS (2018)Vitamin D and bone health: a practical clinical guideline for patient management and Scientific Advisory Committee on Nutrition (SACN) guidelineThe above was based on National Osteoporosis Society (NOS) guideline 201748/188 patients had vitamin D levels measured36/48 patients had sufficient vitamin D levels12/48 patients were either deficient or insufficient12/12 patients were treated where found deficient or insufficient202090/115 patients had vitamin D levels measured47/90 patients had sufficient vitamin D Levels43/90 patients had either insufficient or deficient vitamin D levels22/43 patients had treatment documented in noted where found deficient or insufficientDifficult to make comparisons with previous audit due to difference in number of patients testedVitamin D is routinely tested on Milford ward on admission hence the large number compared to the last audit52% had noted to have sufficient levels of vitamin DConcerning were results that only 51% of those deemed to have insufficient or deficient were treated based on notesPotential reasons could be:Prescribed in medication card and not documented in notes.Vitamin D results checked in another ward, no supplementation given, and then transferred to Milford house.Patients refused treatment but not documented adequately.Patient discharged before results were received due to quick aroundResults were deemed insufficient in terms of the range but very close to normal hence decision made not to start supplementationResults to be disseminated with medical and nursing colleaguesRe-audit in September 2021"} +{"text": "This study examines older couples\u2019 dyadic patterns of informal and formal advance care planning (ACP) and determines the associations of these patterns with their own and spousal characteristics. Using data from the 2014 and 2016 Health and Retirement Study, we performed a) latent class analysis to identify distinctive ACP engagement patterns and b) multinomial regression models to describe related characteristics of older couples . We identified four dyadic patterns of ACP engagement: a) high ACP engaging couple (45%); b) high engaging husband \u2013 low engaging wife (13%); c) high engaging wife \u2013 low engaging husband (11%); and d) low engaging couple (31%). Engagement in informal and formal ACP was associated with both individual and spousal factors: Older couples with advanced age or higher levels of education and wealth were more likely to engage in both informal and formal ACP, whereas only wife\u2019s high level of constrain or husband\u2019s greater number of depressive symptoms was associated with discordant ACP engagements. Couple-based approach to promote ACP merits older couples with limited resources or poorer psychological health in both or either spouse."} +{"text": "Introduction: New clinical guidelines recommend comprehensive and timely postpartum services across 3 months after birth. Research is needed to characterize correlates of receiving guideline-concordant, quality postpartum care in federally qualified health centers serving marginalized populations.Methods: We abstracted electronic health record data from patients who received prenatal health care at three health centers in North Carolina to characterize quality postpartum care practices and to identify correlates of receiving quality care. We used multivariable log-binomial regression to estimate associations between patient, provider, and health center characteristics and two quality postpartum care outcomes: (1) timely care, defined as an initial assessment within the first 3 weeks and at least one additional visit within the first 3 months postpartum; and (2) comprehensive care, defined as receipt of services addressing family planning, infant feeding, chronic health, mood, and physical recovery across the first 3 months.Results: In a cohort of 253 patients, 60.5% received comprehensive postpartum care and 30.8% received timely care. Several prenatal factors and postpartum factors were associated with timely postpartum care. The most important correlate of comprehensive services was having more than one postpartum visit during the first 3 months postpartum.Discussion: Identifying best practices for quality postpartum care in the health center setting can inform strategies to reduce health inequities. Future research should engage community stakeholders to define patient-centered measures of quality postpartum care and to identify community-centered ways of delivering this care. Patients cared for in community health centers have been found to have fewer racial and ethnic disparities in birth outcomes compared with the general U.S. population.To contribute to the nascent literature on quality postpartum care since the change in clinical guidance, we conducted an exploratory analysis to understand current postpartum care practices in the community health center setting. We used electronic health record (EHR) data from patients who received prenatal health care at three community health centers in North Carolina to characterize guideline-concordant, quality postpartum care and to identify correlates of receiving this quality postpartum care. We examined associations between health center, provider, and patient characteristics and quality postpartum care informed by the recent ACOG guidelines.32 Approximately 86% of patients at these centers had a household income at or below 100% of the poverty line and nearly half were uninsured.We used EHR data from perinatal patient encounters of people who received prenatal care at one of three outpatient community health centers who gave birth or experienced a miscarriage or stillbirth in 2018. The three community health centers belong to a network of community health centers serving 14 rural and urban counties across central North Carolina. They served nearly 50,000 patients in 2018, and 75% of these patients were of non-white race or of Hispanic/Latino ethnicity, compared with 29.4% of North Carolina residents.This exploratory study was conducted in collaboration with three facilities in this community health center network whose diverse site characteristics and provider teams facilitated data abstraction from a single EHR system to generate findings that will be relevant across different contexts. These facilities provide outpatient prenatal and postpartum care and coordinate intrapartum care with local hospitals where their patients deliver.33 of patient-level, provider-level, and health center-level predictors of postpartum care use in marginalized populations to enumerate potential variables associated with receiving guideline-concordant quality postpartum care.We drew from our prior systematic review34 which is secure and compliant with the Health Insurance Portability and Accountability Act (HIPAA). The lead researcher (K.W.) created separate REDCap data collection forms for storing demographic data as well as encounter data from prenatal visit notes, intrapartum hospital notes scanned into the EHR, and postpartum visit notes.Targeting these key variables, we abstracted discrete data and free text on patient encounters from the GE Centricity EHR software into a Research Electronic Data Capture (REDCap) structured database. REDCap is a web application for building and managing online surveys and databases,For each patient who received prenatal care at one of the three health centers and gave birth or experienced a miscarriage or stillbirth in 2018, we abstracted data from all visits during their pregnancy through 12 weeks postpartum. Data abstraction involved mostly free text review owing to the lack of discrete fields for many of the variables of interest and the lack of a standardized template in the EHR for documentation of the postpartum visit. Data collection forms were edited to incorporate input from health care providers and information technology administrators at the community health network, and appropriate variable construction was confirmed with technical support from the North Carolina Translational and Clinical Sciences Institute.The lead researcher (K.W.) trained a team of medical scribes employed at the community health centers to abstract data into the structured REDCap database. Training involved K.W. double-coding 20 charts entered by each abstractor to review and discuss discrepancies to improve agreement before proceeding. Record audits were also carried out throughout the data abstraction process of approximately four records per data abstractor each month to verify data quality over time. This study was approved by the Institutional Review Board at the University of North Carolina at Chapel Hill (IRB #19-0423) and by the board of the community health center network.18 we classified patients as receiving quality postpartum care based on separate dichotomous variables for timeliness and comprehensiveness of care. First, we classified each patient as receiving timely postpartum care if they had both an initial assessment within the first 3 weeks postpartum and at least one additional visit within the first 3 months postpartum. Second, we classified each patient as receiving comprehensive postpartum care based on ACOG's suggested components of a postpartum care plan,35 as long as each of the five components were documented at any visit within the first 3 months postpartum:Based on the 2018 ACOG recommendations,(1) sexuality, contraception, and birth spacing ; (2) discussion of infant feeding status and/or offer of infant feeding support, (3) chronic disease management for patients with a documented chronic health issue at the first prenatal visit (including a tobacco use screen among patients who reported current use or a history of tobacco use); (4) mood ; and (5) physical recovery from birth and/or pregnancy-related health issues, including a blood pressure check in the first 10 days among patients with hypertensive disorders .Patient characteristics extracted from the EHR included parity, maternal age, insurance status, chronic diseases , medical complications beginning during pregnancy , delivery type, and a dichotomous variable for exclusive breastfeeding status at the first postpartum visit (exclusive vs. any/none).36 and the consistency of the health care provider (defined as the number of unique providers seen across pregnancy). Based on patient data and a Qualtrics survey sent to all the health care providers identified in our data set , we classified whether the patient and provider had concordant race/ethnicity and whether or not the provider was able to communicate in the patient's preferred language.Health center characteristics included an identifier of the facility where a patient's first prenatal visit in the community health center network occurred to control for site-level differences: two sites are located in urban counties and one in a rural county; the number of staff ranges from 45 to 85 across the sites; the number and type of provider team differs by site, and only one site has resident family physicians who have hospital privileges and attend some deliveries. Additional health center characteristics included the time since delivery at which the postpartum visit was scheduled and any enabling services used by the patient .37 We chose this modeling framework instead of logistic regression because odds ratios would overestimate risk/prevalence ratios of primary interest, and difficulties translating odds ratios from logistic regression into risk or prevalence ratio estimates.37We present the associations between prespecified variables and the two quality postpartum care outcomes of interest. We used multivariable log-binomial regression to estimate risk ratios (RRs) for prenatal variables and prevalence ratios for postpartum variables, because postpartum characteristics were ascertained cross-sectionally with outcomes at the postpartum visit.i.e., timely and comprehensive postpartum care). We present crude and adjusted estimates of association along with 95% confidence intervals (CIs) to assess precision. To avoid the 38 we constructed one directed acyclic graph40 , 4 with miscarriage, and 1 with stillbirth. Descriptive characteristics of the cohort up to the birth event are given in Nearly 74% of the cohort was Hispanic/Latina, 11% non-Hispanic Black, almost 10% Asian (mostly Burmese immigrants), and \u223c5% non-Hispanic white. Most of the sample were married or partnered (72%) and multiparous (80%). Most patients received adequate prenatal care or better (69%). Baseline prevalence of chronic health issues was 25%, with mood or anxiety disorder being most common (19%). Among the 36% of patients who experienced a medical complication during pregnancy, anemia was most common (17%). Vaginal delivery occurred for 76% of patients in the cohort.Most patients saw a family medicine physician (53%) or a family nurse practitioner (39%) at their first prenatal visit, and 43% of patients only saw this one provider for all their prenatal care. Only 12% of the sample were racially and ethnically concordant with their first prenatal health care provider; however, this health care provider almost always (92%) spoke the patient's preferred language.At the health center level, \u223c60% of patients received enabling services at some point across their pregnancy, and almost one-third received these services at their first postpartum visit. Data on the time since delivery at which the postpartum visit was scheduled were only available for 91 patients, and \u223c64% of this sample had a postpartum visit scheduled in the first week.Of the 253 patients in our sample, 89% returned for at least one postpartum visit in the first 3 months and 42% had an initial contact with the health care provider in the first 3 weeks. Comprehensive postpartum care was more prevalent than timely care, and most patients were asked about their infant feeding status and/or offered support; counseled on family planning and/or offered a contraceptive; screened for a mood or anxiety disorder; and asked about birth or pregnancy-related health issues.Fewer patients received support for infant feeding (whether breast or formula feeding) from the provider or a lactation consultant; a blood pressure check within 10 days; or a tobacco use screen . Almost Approximately one-third of the sample (31%) received quality postpartum care as defined by timeliness. Patients who had received adequate or \u201cadequate-plus\u201d levels of prenatal care engagement or had a documented mood or anxiety disorder during pregnancy had increased likelihood of receiving timely postpartum care . PatientN\u2009=\u200991). Exclusive breastfeeding and receiving enabling services at the time of the first postpartum visit were both associated with a higher prevalence of timely postpartum care.The racial/ethnic concordance of patient and provider were associated with this outcome. Timely care was more common for patients who saw more than one health care provider across pregnancy . Having a postpartum visit scheduled in the first week postpartum versus after the first week was associated with more timely postpartum care, although data on scheduling was only available for a small proportion of the sample (Three-fifths (61%) of the sample received quality postpartum care defined as receiving all recommended services across any number of visits in the 3 months postpartum. Whereas attending an early postpartum visit in the first 3 weeks was not associated with receiving comprehensive postpartum services, attending a higher number of postpartum visits was associated with a higher prevalence of receiving comprehensive services that addressed all recommended health domains . ReceiviUsing novel EHR data from the community health center setting, we characterized quality postpartum care defined through clinical guidelines on the broad structure of the timing and component domains. We found that less than one-third of patients receive quality postpartum care defined by the timeliness of services, whereas nearly two-thirds of patients receive quality postpartum care as defined by comprehensive service measures.42 the majority of patients in our community health center setting received all recommended components of postpartum care. The most prevalent postpartum services included family planning, follow-up regarding recovery from pregnancy and birth, and documentation of infant feeding status.In contrast to the low prevalence of services provided during comprehensive postpartum visits in a national sample,Fewer patients received more time intensive support for infant feeding from the provider or a lactation consultant, and fewer patients with chronic health issues received targeted follow-up, especially in the case of tobacco use or hypertensive disorders requiring an early postpartum blood pressure check. Among patients who received comprehensive services, most also engaged in timely care; however, the low proportion of patients engaging in timely care overall may be a result of the recent shift in clinical guidance recommending early and ongoing postpartum care beyond a single visit at 6 weeks.Timely postpartum care and comprehensive postpartum services are two proxy definitions of quality postpartum care for which different factors appear to be predictive. In this exploratory analysis, timely postpartum care was associated with several patient characteristics, including adequate prenatal care use, documentation of a mood or anxiety disorder during pregnancy, and exclusive breastfeeding at the first postpartum visit; one provider characteristic, receiving prenatal care from a family nurse practitioner; and two health center characteristics, early appointment scheduling and receiving enabling services at the first postpartum visit .43\u201349 Having a mental health disorder has been previously found to correlate with higher postpartum visit attendance where enhanced mental health services are available51; having a prenatal mood or anxiety disorder may similarly increase timely postpartum care engagement in the community health center setting where enabling services are typically co-located with primary care. Alternately, documentation of a mental health disorder may reflect provider-level engagement, serving as a proxy for the provider's familiarity with the patient and their health history, which is a known predictor of increased health care use following birth.52Adequate prenatal care use has been found to predict postpartum visit attendance across numerous studies.53 Future qualitative research should explore patient and provider perspectives on potential mechanisms underlying the association between provider type and timely postpartum care.We found that patients cared for by family nurse practitioners more frequently engaged in timely postpartum care. Nurse providers may be more likely to encourage timely postpartum follow-up or may indirectly increase timely follow-up by spending more time with their patients and establishing strong rapport. A recent survey of a national sample of postpartum care providers found that nurse-midwives and family medicine physicians spend significantly more time with postpartum patients than obstetrician/gynecologist physicians, with nurse-midwives reporting the most time spent with patients.54 and trouble understanding the health care provider55 are associated with lower postpartum visit attendance. Future research in community health center communities can complement EHR analyses with patient perspectives of providers' cultural and linguistic competency to further investigate patient\u2013provider relational predictors of quality postpartum care.We did not observe an association between the racial/ethnic or language concordance of the patient and provider and timely postpartum care. This concordance may be less salient where the majority of providers speak the patient's preferred language. However, previous research has found that perceived discrimination during intrapartum care57In addition, given the significant proportion of Latina and Burmese mothers in our patient population, future qualitative research can explore issues related to acculturation and the effects of the sociopolitical environment that may impact access to quality care for immigrant families served by community health centers.13 Receipt of enabling services was measured in the early postpartum and may therefore reflect characteristics of patients who would be more likely to return for timely postpartum care, such as parity, insurance status, or maternal age. However, our sample was restricted to a population of mostly low-income and uninsured families for whom socioeconomic characteristics were relatively homogenous and not associated with either quality postpartum care outcome; therefore, these characteristics are not likely to explain the association between receiving enabling services and timely postpartum care.Whereas receipt of enabling services in the prenatal period was not associated with timely postpartum care, postpartum access to these nutrition, infant feeding, and behavioral health services appears to be associated with timely postpartum care, possibly owing to the intersecting maternal and infant health issues that can arise in the early postpartum.59 and access to patient navigators who schedule postpartum visits during the intrapartum hospitalization51 are associated with increased postpartum visit attendance.Whereas data on the timing of scheduling for the postpartum visit were not available for the entire sample, having a postpartum visit scheduled in the first week after delivery was associated with a higher likelihood of timely postpartum care. We do not know whether these missing data result from a lack of early scheduling or a lack of documentation alone; however, other studies have found that postpartum visit appointment reminders60\u201362 Future research can experiment with the timing of visit scheduling to test the effect of early timing on measures of quality postpartum care.In settings where perinatal health services are co-located with pediatric and adult primary care, offering dyadic services such as interconception care at regular well child visits may improve maternal risk assessment and offer more frequent opportunities for health care engagement across the postpartum period.18 More frequent postpartum visits facilitate multiple opportunities for providers to address intersecting health issues as they arise over time.The only correlate of quality postpartum care defined as receipt of comprehensive services was the number of postpartum visits attended across the first 3 months after delivery. This finding reinforces the importance of providing ongoing postpartum care across numerous patient/provider contacts instead of a single postpartum visit at 6 weeks to meet the complex health needs of new parents.63 and contraception initiation64 in other settings, attending an early visit was not associated with receipt of comprehensive postpartum services in this study. The high prevalence of receiving comprehensive postpartum services in our cohort may have contributed to the lack of significant associations with other variables.Although attending an early 2-week postpartum visit is associated with improved breastfeeding support65 Our data abstractors were medical scribes working in the community health center setting who were familiar with the EHR, provider notes, visit flow, and where to identify relevant data. By abstracting data across all visits across pregnancy through 3 months postpartum, we were also able to identify detailed descriptions of the services provided across multiple postpartum visits, which are not available when relying on codes for billing or diagnosis.Documentation of comprehensive services may have been high in our sample because we conducted comprehensive medical record reviews for data abstraction rather than utilizing administrative or claims data.Our data abstraction involved mostly free text review to identify and abstract exposure and outcome data, and the availability of variables of interest may have been limited by inconsistent provider documentation. EHR data are not collected for research purposes and often lacks consistent but important patient demographic details such as immigration status, length of time living in the United States, education level, or employment status. We surveyed health care providers for their race/ethnicity and spoken language ability to complement some missing data with potentially informative provider characteristics.The diverse site characteristics and provider teams of the three sites in our sample facilitated the use of a single EHR to generate findings that may be relevant across different community health center contexts. However, more integrated systems are needed to improve care coordination, document services received outside the community health center, and track referrals for appropriate follow-up care. We were able to confirm receipt of enabling services within the community health center network, such as behavioral health, dental, or nutrition services, but we were not able to link the EHR to external software and relied on provider notes to document receipt of additional services, such as WIC or lactation support.We were also unable to link the EHR data from the postpartum parent with their infant and are therefore missing any infant feeding or mental health information that may have been documented at well child visits. In the early postpartum period, maternal and infant health behaviors and outcomes are especially interconnected and characterizing the quality of postpartum care requires adapting postpartum visits and EHR documentation to support dyadic health care.66 We used a causal modeling approach to test associations between exploratory patient, provider, and health center characteristics and outcomes of interest, controlling for all measured confounders that could distort these estimates (Data abstraction from the EHR of a network of community health centers allowed us to capture patient encounters from postpartum people often missing from research using hospital system or claims data owing to high levels of postpartum insurance loss.stimates . Our est18Future research can build upon our findings to test these suggested associations between patient, provider, and health center characteristics and quality postpartum care outcomes, especially where factors such as early visit scheduling or the availability of enabling services are amenable to intervention. Finally, we defined quality postpartum care informed by guidelines developed through clinician consensus on the broad structure of postpartum care timing and component domains.Our findings suggest that different exposures may correlate with quality postpartum care differently depending on how quality care is defined. Future research should engage patients, health care providers, and other stakeholders from marginalized communities to define quality postpartum care measures and identify community-centered practices and policies to improve access to this quality care.In our community health center setting, we found a high prevalence of quality postpartum care as defined by comprehensive service measures, but a lower prevalence of timely postpartum care. Our findings suggest that prenatal factors such as adequate care use and an engaged patient\u2013provider relationship, and postpartum factors such as early appointment scheduling, exclusive breastfeeding status, and use of enabling services may increase timely postpartum care. Future research should engage community health center stakeholders to further explore these preliminary findings to translate clinical guidelines for settings serving the most marginalized populations. Identifying best practices from these settings can inform postpartum care strategies to improve maternal health equity."} +{"text": "Physical performance is associated with cognitive function in later life, but few studies have examined the prospective association of physical performance with incident dementia. We studied 4539 community-dwelling National Health and Aging Trends Study (NHATS) participants aged \u226565 years with data on demographics and the Short Physical Performance Battery (SPPB) in 2011, who were followed through 2014. Our outcome was dementia diagnosis from a validated NHATS algorithm. We applied survey weights to make results nationally representative and performed Cox regression analyses. After adjustment for potential confounders, lower baseline SPPB scores were associated with incident dementia . Slower gait speed was the SPPB component most strongly associated with incident dementia . We found that poorer physical performance was linked to incident dementia in a cohort of older adults. More research is needed to examine the effect of improving physical performance on the prevention of dementia."} +{"text": "The COVID-19 pandemic unleashed a relentless stressor on the human species with numerous deadly risks. These risks have been disproportionately threatening to the health and wellbeing of older adults. Since April 2020, we have been studying how the pandemic has affected the emotional experiences of older and younger adults broadly in several studies. For instance, in one study, we found that older adults (N=176) experienced fewer negative emotions and coped with greater levels of agency than younger adults (N=181). In additional work, we have been examining how these age differences differ for older workers versus retirees as well as in minority populations. This work broadly supports and illuminates our recent theoretical framework that focuses on how evaluative appraisal processes underlie and contribute to age differences in emotional experience generally, but especially in the context of the stress experienced during a global pandemic."} +{"text": "Podocyte damage is a hallmark of glomerular diseases, such as focal segmental glomerulosclerosis, typically associated with marked albuminuria and progression of renal pathology. Podocyte structural abnormalities and loss are also linked to minimal change disease and more common diabetic kidney disease. Here we applied the first\u2010time scanning ion conductance microscopy (SICM) technique to assess the freshly isolated human glomerulus's topology. SICM provides a unique opportunity to evaluate glomerulus podocytes as well as other nephron structural segments with electron microscopy resolution but in live samples. Shown here is the application of the SICM method in the live human glomerulus, which provides proof of principle for future dynamic analysis of membrane morphology and various functional parameters in living cells. Primary and secondary processes of adjacent podocytes intertwine, creating a slit diaphragm that tightly covers glomeruli capillaries. This diaphragm has a complex morphology and forms the final barrier for blood filtration contributing to size selectivity and permitting permeability to molecules smaller than albumin. A reduction in podocyte number and foot process effacement was reported in focal segmental glomerulosclerosis, minimal change disease and diabetic nephropathy.2For the experiment, we used a part of the cortical area from the discarded transplant human kidney dissected and stored in Wisconsin preservation solution followed by the incubation in an oxygenated physiological saline solution (PSS).3SICM image quality is comparable to SEM and allows precise examination of glomerulus surface structure, including blood vessels and podocytes, and the architecture of the secondary foot processes.The application of the SICM technique for investigating the three\u2010dimensional surface structure of the soft tissue samples, including glomerulus and podocyte cells, was initially introduced by Nakajima et alIn summary, the method described here has excellent potential for the studies of glomeruli and other freshly isolated nephron segments. Furthermore, it shows that it is possible to use human kidney samples without elaborate preparation to evaluate morphological changes in the study of pathologies.Dr Andrew Shevchuk is a shareholder and receives the consulting fees from the ICAPPIC Ltd. All other authors declared no competing interests.Ruslan Bohovyk: Conceptualization ; Data curation (lead); Formal analysis (lead); Writing\u2010original draft ; Writing\u2010review & editing . Mykhailo Fedoriuk: Data curation (supporting); Formal analysis (supporting); Writing\u2010review & editing . Elena Isaeva: Conceptualization (supporting); Data curation (supporting); Formal analysis (supporting); Writing\u2010original draft (supporting); Writing\u2010review & editing . Andrew Shevchuk: Conceptualization (supporting); Software (supporting); Writing\u2010original draft (supporting); Writing\u2010review & editing . Oleg Palygin: Conceptualization ; Formal analysis (supporting); Methodology (supporting); Project administration (supporting); Writing\u2010original draft (supporting); Writing\u2010review & editing . Alexander Staruschenko: Conceptualization (lead); Project administration (lead); Resources (lead); Supervision (lead); Writing\u2010original draft ; Writing\u2010review & editing .The data that support the findings of this study are available from the corresponding author upon reasonable request."} +{"text": "We measured the pupil response to a light stimulus subject to a size illusion and found that stimuli perceived as larger evoke a stronger pupillary response. The size illusion\u00a0depends on combining retinal signals\u00a0with contextual 3D information; contextual processing is thought to vary across individuals, being weaker in individuals with stronger autistic traits. Consistent with this theory, autistic traits correlated negatively with the magnitude of pupil modulations in our sample of neurotypical adults; however, psychophysical measurements of the illusion did not correlate with autistic traits, or with the pupil modulations. This shows that pupillometry provides an accurate objective index of complex perceptual processes, particularly useful for quantifying interindividual differences, and potentially more informative than standard psychophysical measures. Although atypical perception is not a diagnostic criterion for Autism Spectrum Disorders (ASD), growing evidence shows that autistic individuals have different perceptual styles than neurotypicals. Perhaps the best known aspect of this consists of a preference for local details in children and adults with ASD, who often outperform controls in tasks requiring the discrimination of fine-grained visual features abstracted from their global context, like the embedded figure task or visual search tasks . We expect that the contextual effect should vary across individuals, being reduced in individuals with stronger autistic traits (higher AQ scores).Comitato Etico Pediatrico Regionale\u2014Azienda Ospedaliero-Universitaria Meyer\u2014Firenze (FI) \u201cunder the protocol \u201cFusione di Informazioni Multisensoriali\" v4/04.10.2018\u201d] and were in accordance with the Declaration of Helsinki.None of the authors have conflicts of interest to declare. The research reported here involved human participants, who gave their written informed consent to the participation in this study. Experimental procedures were approved by the regional ethics committee or pupil response .Emmert\u2019s law states that objects with the same retinal image will look larger if they appear to be located further away Boring . A clearNeuroimaging work in humans questionnaire, consistent with Chouinard et al. who alsoThere is growing interest in using objective indices, such as pupillometry, to quantify the peculiarities of autistic perception. Several studies have attempted to use the dynamics of the simple pupillary light response (evoked by an isolated light flash) to dissociate individuals with and without ASD (Daluwatte et al. Our findings are coherent with two other recent reports (Pome et al. This follows other examples of lack of correlation between pupillary light responses and perceptual responses. For example, Benedetto and Binda showed tIn conclusion, our findings show that pupil responses provide an accurate objective index of complex perceptual processes. They are more effective than perceptual estimates and particularly useful for quantifying interindividual differences that could be also extended to clinical population in order to measure individual perceptual processes with an objective and non-invasive technique."} +{"text": "This study evaluated the life course processes through which early life racial discrimination (ELRD) and racial centrality interact to shape allostatic load (AL) among African American (AA) adults aged 50+ in the Nashville Stress and Health Study (N=260). Adolescent ELRD was associated with greater racial centrality in adulthood and conferred 35% greater risk of high adult AL; greater centrality was also linked to high adult AL. Centrality accounts for 24% of the association between ELRD and AL. ELRD and centrality interact to shape adult AL, such that racial centrality is protective against high AL for adults who experienced racial discrimination as children or adolescents. Findings highlight the multiple pathways through which race-related stressors and psychosocial resources interact to shape physiological dysregulation in later life and underscore the health significance of racial identity for older AAs."} +{"text": "Sentinel bleeds in head and neck cancer patients present as an ominous symptom often necessitating urgent endovascular embolization. However, this approach can be complicated in patients who have previously undergone head and neck cancer ablation and reconstruction, thus altering the standard arterial vascular supply. Herein we describe an innovative method of internal maxillary artery (IMA) access in a patient with a sentinel bleed who previously underwent proximal external carotid artery (ECA) rerouting for free flap reconstruction. The open retrograde superficial temporal artery approach for IMA embolization is minimally invasive and effective and should be considered for head and neck cancer patients at risk of hemorrhage from distal ECA branches without a proximal ECA embolization option. Sentinel bleeds in head and neck cancer patients present as an ominous and urgent symptom that can occur due to vascular complications such as pseudoaneurysm, thrombosis or arteriovenous fistula following prior surgical manipulation, tumor progression or chemoradiation , 2. The A 43-year-old male underwent tooth extraction for jaw pain and experienced heavy bleeding; prompting imaging that revealed a large mass of the left maxilla, invading the hard palate and orbital floor. He underwent prophylactic endovascular coil embolization of his left IMA prior to biopsy of the mass to prevent further hemorrhage. He was diagnosed with a solid type adenoid cystic carcinoma of the left maxilla, T4N0M0. He was referred to Otolaryngology-Head and Neck Surgery and underwent hemimaxillectomy with a three-segment stacked fibula free flap reconstruction guided by preoperative virtual surgical planning. The vascular pedicle did not have sufficient length to anastomose to branches of the external carotid artery, so a vein graft was used to create a Corlett loop reaching from the neck to the reconstructive site. Initial attempts to anastomose to the facial artery were complicated by thrombosis; thus, the distal external carotid artery was used and the flap successfully revascularized. The patient recovered uneventfully and underwent adjuvant proton irradiation with concurrent chemotherapy. Following the completion of radiation, he developed dehiscence of the posterior extent of the reconstructed palate that was successfully managed with a palatal obturator.Eight months after treatment completion, the patient removed a small wire from his maxillectomy cavity that was consistent with a vascular coil from his previous IMA embolization. CT angiography was obtained notable for partial coil extrusion from a left IMA branch, with a separate vascular blush concerning for pseudoaneurysm of another IMA branch . SeveralDue to inability to access patient\u2019s IMA via the ECA as it had previously been utilized for free flap reconstruction, options considered included endoscopic exploration of the maxillectomy cavity for direct vessel ligation; neck exploration or percutaneous approach to access the distal stump of the ECA; or retrograde access for IMA endovascular embolization via the undissected STA. The decision was made to attempt retrograde access via the STA.The patient underwent a left preauricular incision to isolate the STA from surrounding parotid parenchyma by the Head & Neck Surgery team. Under microscopic guidance, the STA was cannulated with an 18-gauge angiocatheter by the NThe patient tolerated the procedure well and was discharged on postoperative day 1 in stable condition. In follow-up on postoperative day 10, he had no further evidence of epistaxis or other symptoms.Endovascular embolization techniques offer an efficient and successful option for managing urgent bleeding difficult to access otherwise. However, in head and neck cancer patients, vascular anatomy is often altered in the context of prior ablative and reconstructive surgery, posing significant challenges to standard approaches for arterial embolization. We describe an innovative method of IMA access in a patient who previously underwent proximal ECA rerouting for free flap reconstruction. As free flap reconstruction is increasingly standard of care for the complex defects created by head and neck cancer ablation, this technique is a useful alternative to access branches of the distal ECA when it has been previously ligated or rerouted proximally.There is one previous similar report, to our knowledge, of open retrograde STA access to the IMA, for successful embolization using polyvinyl alcohol particles of an arteriovenous malformation with persistent hemorrhage after previous ECA embolization . AnotherThe open retrograde STA approach for IMA embolization is minimally invasive and effective and should be considered for head and neck cancer patients at risk of hemorrhage from distal ECA branches without a proximal ECA embolization option."} +{"text": "Education about the heterogeneity of the older adult population is an important step for reducing ageist attitudes. As many undergraduate students view gerontology as an unrelatable discipline, educators are tasked with identifying innovative strategies to make course content engaging. The purpose of this presentation is to share an emerging educator\u2019s experience with creating a novel essay assignment. Based off the International Movie Database (IMDb), the Gerontological Movie Database (GMDb) Review encourages students to use their knowledge to evaluate how older adults are portrayed in films. Explicitly, students must 1) choose a film that focuses on older adult characters and 2) apply key gerontological concepts to critique the film\u2019s representation of aging. Though movie reviews are not a typical genre of writing, this assignment increases students\u2019 understanding of how their perception of aging, coupled with master narratives embedded within today\u2019s culture, influences the construction of age."} +{"text": "Purpose. This article examines the role of family context in shaping the influence of childhood maltreatment on later life psychological well-being in the cultural context of Chinese society. Method. Data were drawn from the China Health and Retirement Longitudinal Study (CHARLS) baseline. Maltreatment was measured by corporal punishment by either mother or father in childhood. We used family violence, parents\u2019 family socioeconomic status (SES) and mental health to represent family context. Result. Our ordinary least square regression analysis shows that corporal punishment administered by a mother was associated with higher depressive symptoms in later life while being hit by father did not result in higher depressive symptoms. Family contexts had residual (\u201clong arm\u201d) influence on respondents\u2019 mental health: violence in the family, including being hit by siblings and witnessing violence between parents contributed significantly to higher depressive symptoms. Conclusion. Corporal punishment by parents had long term effects on mental health of their children in later life. Cultural values, such as filial piety did not eliminate the negative impacts of being hit in childhood on mental health in later life. Family contexts including violence between parents also played important roles in shaping the relationship between child maltreatment and mental health in later life. Implication. Our study offers important insights about the complex matrix of cultural traditions, social circumstances and diversity in dealing with child rearing stress and their consequences for later life mental health."} +{"text": "Frailty is a common characteristic of patients undergoing transcatheter mitral valve repair (TMVR). It is unclear whether the physical vulnerability of frail patients translates into increased procedural health care utilization.n\u2009=\u2009107, 47%) compared to non-frail patients showed significantly higher total costs , with a difference in means of 2,317 \u20ac. Frailty was the only clinical baseline characteristic with significant association with total costs. Higher total costs in frail patients were attributable primarily to longer stay on intermediate/intensive care unit , but also to costs of clinical chemistry and physiotherapy. The prolonged stay on intermediate/intensive care unit in frail patients was attributable to postprocedural complications such as bleeding, kidney injury, infections and cardiovascular instability.Frailty was assessed using the Fried criteria in 229 patients undergoing TMVR using the MitraClip system at our institution and associations with total costs and costs by cost centers within the hospital incurred during periprocedural hospitalization were examined. Frail patients (Frailty is associated with a mean 32% increase of hospital costs in patients undergoing TMVR, which is primarily the result of a prolonged recovery and increased vulnerability to complications. These findings are valuable for a hospital\u2019s total cost calculation and resource allocation planning. Since frailty is regarded a potentially reversible health state, preventive interventions may help reduce costs in frail patients.The online version contains supplementary material available at 10.1007/s00392-020-01789-5. The implications of frailty for the health care sector gain increasing attention in the context of ageing populations. Frailty is a complex clinical syndrome, which describes a decrease of physiological reserves that come along with an increased vulnerability to stressors . AlthougPotential medical stressors where an increased vulnerability of frail people might become particularly relevant are surgery or interventional procedures. In patients undergoing percutaneous coronary intervention or cardiac surgery, frailty was associated with higher mortality, vascular and bleeding complications and renal failure \u20137. TheseSo far it is unclear whether \u201clow stress\u201d procedures like TMVR have an impact on the complication rate, recovery from complications and resulting treatment costs in highly vulnerable patients. We have recently demonstrated that the risk of major cardiac or vascular complications was not increased in frail compared to non-frail patients undergoing TMVR . HoweverConsecutive adult patients undergoing TMVR using the MitraClip system at our institution between May 2014 and October 2016 were eligible for this retrospective analysis of prospectively assessed data on frailty in patients undergoing TMVR. Treatment decision for TMVR with MitraClip was taken by a multidisciplinary heart team after preoperative risk assessment based on objective risk scores, clinical parameters and morphological suitability for MitraClip according to current guidelines compared to non-frail patients and significantly higher total costs excluding implant costs .When defining frailty by disability in IADL, results were virtually the same. Patients with disability had significantly higher total costs than in the US [median 2 (1\u20135) days] [\u201315 days]In order to estimate the resource use, data on costs was based on a case-based lump sum which did not accurately reflect the real net effect of frailty on incurred costs. However, calculations based on individual patient data would have required a different study design applying a detailed process of cost calculation which was beyond the scope of this analysis.Frailty is associated with a mean 32% increase of hospital costs in patients undergoing TMVR, which is primarily the result of a prolonged recovery and increased vulnerability to complications. These findings are valuable for a hospital\u2019s total cost calculation and resource allocation planning. Since frailty is regarded a potentially reversible health state, preventive interventions may help reduce costs in frail patients.Supplementary file1 (DOCX 19 KB)Below is the link to the electronic supplementary material."} +{"text": "Population-based health disparities studies requires improved research design and appropriate research questions for investigation that will inform evidenced-based interventions and prevention strategies. The NIA Division of Neuroscience is committed to supporting new studies that 1) invests in health priorities as reflected by needs of minoritized populations ; 2) examines Alzheimer\u2019s Disease and cognitive changes across the individual lifespan; and 3) understands intersectionality of cohorts and minimize potential biases in participant selection. This brief session will outline updated steps to permit critical thought about the use of the NIA\u2019s Health Disparities Framework to examine relevant biological, sociocultural, behavioral and environmental across multiple levels of influence."} +{"text": "Because of the increasing incidence of elder abuse and financial exploitation, Adult Protective Services (APS) cases open for these individuals often relay on capacity evaluations conducted by a clinician to facilitate legal assignment of a surrogate decision maker. Despite this growing need, the number of physicians willing and capable of performing them is limited. Barriers reported by physicians reportedly impair their ability to conduct these evaluations include absence of relevant case information and lack of knowledge about the process itself. Geriatricians and related clinicians often perform these assessments. Sharing best practices with internists and family physicians may help overcome these barriers. A survey of geriatric medicine providers was conducted to identify essential components and questions necessary in the assessment of general decision making capacity. Twenty-nine providers at 6 academic institutions in Ohio responded to the survey and its follow-up inquiries. Though variability existed in evaluation styles and content between providers, a uniform set of recommendations was able to be generated. A total of 13 different summary recommendations were generated from this survey. Necessary components to these evaluations include (1) performance of cognitive testing (2) obtaining collateral information regarding functional status from another trusted individual (3) assessing the individual\u2019s insight into any reported functional impairments or safety concerns by explaining discrepancies between that individual\u2019s own observations and reported concerns from the trusted individual, and (4) using hypothetical situations to assess a person\u2019s judgment and reasoning in addressing any gaps in care or safety concerns raised during the interview."} +{"text": "Mobile genetic elements (MGEs) sequester and mobilize antibiotic resistance genes across bacterial genomes. Efficient and reliable identification of such elements is necessary to follow resistance spreading. However, automated tools for MGE identification are missing. Tyrosine recombinase (YR) proteins drive MGE mobilization and could provide markers for MGE detection, but they constitute a diverse family also involved in housekeeping functions. Here, we conducted a comprehensive survey of YRs from bacterial, archaeal, and phage genomes and developed a sequence\u2010based classification system that dissects the characteristics of MGE\u2010borne YRs. We revealed that MGE\u2010related YRs evolved from non\u2010mobile YRs by acquisition of a regulatory arm\u2010binding domain that is essential for their mobility function. Based on these results, we further identified numerous unknown MGEs. This work provides a resource for comparative analysis and functional annotation of YRs and aids the development of computational tools for MGE annotation. Additionally, we reveal how YRs adapted to drive gene transfer across species and provide a tool to better characterize antibiotic resistance dissemination. A systematic resource for tyrosine recombinase annotation is presented. Comparative sequence analysis of the protein family enables the functional classification of these enzymes and the identification of mobile genetic elements in bacterial genomes. Some MGEs hijack host\u2010encoded Xer proteins domain binds the recombination DNA site, and the catalytic (CAT) domain catalyzes all DNA cleavage and joining reactions required for recombination was built. Profile HMM is a probabilistic model used to describe characteristic sequence features of the alignment. This profile HMM was then used as a new query in the next search cycle. This iterative procedure allows identification of distantly related homologues of the original query of the identified ICEs and look for such undisrupted sites in closely related genomes. As functional ICEs can move to new genomic sites, successful identification of naive sites can provide ultimate confirmation of their identity and mobile nature. However, identification of such naive sites requires recent mobility of the ICE and may also be challenged by a limited availability of complete genome sequence data for related species in public databases. Nevertheless, we found naive sites for 18 out of the 49 ICEs, which further validates these elements and indicates their recent activity Dataset .Tn916 (23), IntP2 (17) and IntSXT (14) subgroups. To further analyze the detected ICEs, we next reconstructed the phylogeny of their YRs and plotted the genetic structure of their respective conjugation machineries genes within newly identified and previously reported ICEs Fig\u00a0. Xis regTaken together, successful identification of new ICEs confirms the predictive value of our classification system for automated annotation of YR function and demonstrates its utility to improve characterization of the bacterial mobilome.dif DNA sequences as a query in a jackhmmer iterative search against the rp15 (v.2019_08) database with different e\u2010values (from 1e\u201005 to 1e\u201040). Our aim was to include as many remote Xer homologues as possible, which is possible through the jackhmmer procedure . This script uses protein accession numbers as an input, retrieves their genomic position, expands this position 100\u00a0Kb usptreams and downstream, downloads all proteins from this region, and runs hmmsearch against these proteins using user provided collection of profile HMMs (such as conjugation machinery protein profile HMMs). The results of this run can be directly visualized using iTol web server insertion sites for confirmed ICEs in closely related genomes (Appendix Fig All ICEs identified in the study are presented in Dataset GS performed the study. GS, AB, and OB designed the study and wrote the manuscript. All authors read and approved the final manuscript.The authors declare that they have no conflict of interest.AppendixClick here for additional data file.Dataset EV1Click here for additional data file.Dataset EV2Click here for additional data file.Dataset EV3Click here for additional data file.Dataset EV4Click here for additional data file.Source Data for AppendixClick here for additional data file.Review Process FileClick here for additional data file."} +{"text": "As time spent at home has significantly increased during the pandemic, reports of household conflict has also risen among people living with others . One solution to alleviate the potential stress of increased time with others could be carving out time to oneself. The present study investigated how living conditions are associated with everyday desire for solitude and whether daily solitude experience comes with improved daily emotional well-being in people living with others. Furthermore, it also explored whether relationship quality is associated with solitude experience in a similar manner as living conditions. To do so, we used repeated daily life assessments from a lifespan sample collected during the early pandemic (April to August 2020). Findings indicate that neither living conditions nor relationship quality were directly associated with daily desire for solitude, but higher relationship well-being was related to low preference for solitude when measured as an individual trait. In addition, relationship quality significantly moderated everyday solitude\u2013affect links: higher relationship quality was related to reduced negative affect during solitude, and conflict was related to increased positive and decreased negative affect on solitude as compared to non-solitude days. The results imply that it is the subjective experience of relationships rather than objective living conditions that shape daily affective quality during solitude."} +{"text": "To the EditorHuang and colleagues demonstrated that coronary artery calcium score (CAC) improved coronary artery disease (CAD) risk prediction, compared to conventional risk scoring, even in the absence of cardiovascular risk factor screening.The authors discussed the cost\u2010effectiveness. The cost of CAC is cheaper than that of computed tomography coronary angiogram. Given that approximately 65% of their study population had risk scores <10% after application of CAC, many patients can avoid unnecessary computed tomography coronary angiogram and save costs without detailed cardiovascular risk factor screening.The authors stated that the CAC\u2010incorporated risk models had approximately comparable sensitivity and specificity to functional stress tests in predicting CAD.Lastly, some patients might have any previously implanted devices including pacemaker and cardiac resynchronization therapy with/without a defibrillator. Accurate calculation of CAC might be challenging due to artifact and the prognostic impact of CAC might decrease in such a cohort."} +{"text": "To introduce and assess the impact of balint groups on core medical trainee (CMT) doctors working within an acute medical trust.A high rate (80%) of dissatisfaction and burnout has been reported amongst trainee doctors. This has had a significant impact on recruitment with a large proportion of foundation doctors delaying their application into core specialist training. Of those already in training, up to 50% have reported taking time, out citing burnout as a cause. Balint groups are a form of reflective practice groups looking at the doctor-patient interaction. For core psychiatric trainees these groups are a mandatory part of their training.We piloted a total of three balint groups over a period of three months amongst CMT doctors based at an acute medical trust in London. A specialty registrar (ST6) in psychiatry facilitated the balint groups. Balint facilitators received supervision from a consultant psychiatrist in psychotherapy. CMT doctors were given questionnaires at the beginning of session one and emerging themes later explored. The questionnaires used were taken from the \u2018Bristol Trainee-led Balint Group Scheme\u2019.The pre-questionnaires showed that all CMT doctors surveyed believed psychological factors play an important role on patient presentation and recovery. 14/19 (74%) agreed or strongly agreed that a doctor's reaction to a patient directly influenced care. All doctors agreed or strongly agreed that it was important to reflect on a patient's emotional experience, as it was crucial to their development as a doctor.CMT doctors found balint groups useful as it provided them a space, which was not routinely offered to discuss challenging cases. Themes that emerged included a lack of support and difficulties maintaining boundaries when treating complex patients. Litigation was a recurring theme with many trainees reporting anxieties and a lack of support. Trainees reported guilt and worries that they were not doing enough for their patients. These themes appeared to have a direct impact on training experience and burnout.With increasing burnout and dissatisfaction amongst junior doctors, balint groups provide a unique approach to supporting junior doctors within medical specialties. The current pilot has demonstrated that CMT doctors can make use of balint groups in an effective way. We recommend that balint groups should become an integral part of specialist medical training. Psychiatrists can play a central role in supporting the health and well being of medical trainees through balint group facilitation."} +{"text": "Primary aimTo explore whether naltrexone/acamprosate had been considered for each patient completing alcohol detoxification.Secondary aimswhat proportion of those offered agreed to be prescribed acamprosate/naltrexonewhether these patients were being adequately followed up in terms of prescriptionNICE guidelines recommend that all patients who undergo a successful alcohol detoxification programme should be considered for treatment with acamprosate or oral naltrexone. This audit studied the proportion of patients considered for acamprosate or naltrexone treatment in a North-East Addictions Service.There is a significant evidence base for both naltrexone and acamprosate in the maintenance of abstinence in patients with alcohol addiction. NICE recommends the consideration of both medications for patients following successful alcohol detoxification from alcohol. The addictions service at Plummer Court in Newcastle upon Tyne has a comprehensive pathway for alcohol detoxification patients, which involves multiple reviews by keyworkers and medics. The attendance at these appointments is often poor, and it is often unclear whether these patients have been offered anti-craving medication.A list of patients referred for inpatient or outpatient alcohol detoxification between June to August 2018 (n = 23) was curated. The progress notes were reviewed for any evidence that there had been clinical consideration of acamprosate/naltrexone. If evidence was found that the discussion had taken place, the notes were further scrutinised to assess if the client had accepted a prescription. The clinical documentation was further reviewed to see if follow-up for anti-craving medication was in place.There was evidence that anti-craving medication had been considered in 47% of patients during the treatment processIn all but one case, acamprosate was offered rather than naltrexoneIn cases where medication was offered, it was accepted in all but one caseAnti-craving medication was universally well toleratedThere was considerable difficulty with assessing who was following up the prescription. On scrutiny of the notes, several GPs had contacted addictions services stating that they would not prescribe acamprosate because of local policy prohibiting its prescription from Primary Care (this policy is in fact no longer current)Practice changed to offer patients monthly follow-up with addictions services for six monthsTemplate letter sent out to GPs with discharge from addictions requesting acamprosate prescription, outlining current policy and offering support if GPs not comfortableAudit presented to medical team. Treatment pathway amended to specify medical team's role in offering anti-craving medication at initial appointmentRe-audit in six months"} +{"text": "A best evidence topic has been constructed using a described protocol. The three-part question addressed was: In [patients undergoing bariatric surgery], is [intraperitoneal local bupivacaine during the operation ] associated with [ lower pain score and decrease in post operative pain medications]?The search has been done and six randomized trial studies are considered to be appropriate to answer this question. The outcome assessed is the value of intraperitoneal bupivacaine in bariatric surgery in terms of effect on the pain score and post operative analgesia. We concluded that intraperitoneal bupivacaine causes improvement in both the pain score and post operative analgesia. \u2022Use of intraperitoneal bupivacaine, during bariatric procedures, improves post operative pain score.\u2022There is decrease in the post operative analgesia when intraperitoneal bupivacaine is used in the bariatric operations.\u2022Different ways can be used to apply intraperitoneal bupivacaine in the bariatric surgery field. A BET p2You are going to perform a laparoscopic bariatric surgery in an obese patient. You are anticipating that the patient will have post operative pain after such a procedure, and you understand that pain control is imperative for patient's recovery and length of stay . This is3In [patients undergoing bariatric surgery], is [intraperitoneal local bupivacaine during the operation ] associated with [lower pain score and decrease in post operative pain medications]?4The search was conducted as following: Embase 1974 to 06/December/2021 and MEDLINE\u00ae 1946 to 06/December/2021 using the OVID interface: [intraperitoneal] AND [local anaesthesia OR local anaesthetic OR bupivacaine] AND [bariatric surgery OR bariatric procedure OR weight loss procedure]. The search was limited to the English language and human studies. Studies which are non-randomized and conference abstracts were excluded from this review.5A total of 8020 papers were found using OVID. Out of those 8020, 8010 were excluded because they were irrelevant based on titles and abstracts or duplicates. Ten full-text articles were screened and examined for eligibility. Of those ten, six randomized trials papers were identified to provide the best evidence to answer the question.6: Not applicableNot applicable.None."} +{"text": "The purpose of this audit was to identify all women being prescribed Sodium Valproate under the Bassetlaw Local Mental Health Team (LMHT) caseload to see how well latest prescribing guidelines are being met, and help set up a system allowing efficient monitoring of Sodium Valproate prescribing in the future.Despite early concerns regarding potential teratogenicity, Sodium Valproate became a widely used anticonvulsant and mood stabiliser and is licensed for use in Epilepsy, Migraine prophylaxis and Bipolar affective disorder. Research evidence now shows its use in pregnancy increases risk of neurodevelopmental disorders to 40%, and serious birth defects to 10%. Despite research finding these risks prescribing practice did not significantly change. To better reflect these findings in clinical practice in 2018 the Pharmacovigilance Risk Assessment Committee recommended Sodium Valproate should not be used in pregnancy unless they have a form of epilepsy unresponsive to other anti-epileptic drugs, and all with childbearing potential should be enrolled in a pregnancy prevention programme (PPP). This was endorsed by UK Medicines and Healthcare Devices Regulatory Agency in April 2018 with launch of the PPP.Standards:Must be offered counselling about risks of valproate to unborn child and importance of effective contraception.Annual specialist Review by a specialist now mandatoryRisk acknowledgement form must be updated at least annually.The electronic RiO records for all female patients on the Bassetlaw LMHT caseload in the year 2019 were checked to identify those prescribed Valproate. For those prescribed Valproate, evidence of annual risk acknowledgement form, date of last appointment, underlying diagnosis and contraceptive method was checked. This data was stored together on an excel file and used to create a patient list to help allow future monitoring.From 594 female patients identified, 27 (4.5%) were prescribed Sodium Valproate. Of these, 14 (52%) had PPP documentation uploaded, 24 (89%) had been reviewed within the last 12 months, and 13 (48%) had no documentation of contraceptive method.This audit helped highlight there is likely a large population of patients not yet on the Pregnancy Prevention Programme. Creating a monitoring system in excel for female patients being prescribed Valproate can help improve adherence to latest guidelines, with a colour coding system to highlight those needing risk acknowledgement forms/appointments within the next three or six months. Educating patients and other healthcare professionals about risks will also help improve prescribing practice and avoid use in pregnancy."} +{"text": "Replicative errors, inefficient repair, and proximity to reactive oxygen species production sites make the mitochondrial DNA (mtDNA) susceptible to damage with time. mtDNA mutations accumulate with age and accompany a progressive decline in organelle function. We lack molecular biology tools to manipulate mtDNA, thus we explore the possibility in vivo of utilizing allotopic expression, or the re-engineering mitochondrial genes and expressing them from the nucleus, as an approach to rescue defects arising from mtDNA mutations. This study uses a mouse model with a mutation in the mitochondrial ATP8 gene that encodes a protein subunit of the ATP synthase. We generated a transgenic mouse with an epitope-tagged recoded and mitochondrial-targeted ATP8 gene expressed from the nucleus. Our results show that the allotopically expressed ATP8 protein in the transgenic mice is robustly expressed across all tested tissues, successfully transported into the mitochondria, and incorporated into ATP synthase. We are currently evaluating if allotopic expression of ATP8 will functionally rescue the behavioral and bioenergetic defects in ATP8 mutant mice. Translating allotopic expression technology into a mammal and demonstrating systemic functional rescue will lend credence to utilizing allotopic expression as a gene therapy in humans to repair physiological consequences of mtDNA defects that may accumulate with age."} +{"text": "Telomerase ribonucleoprotein was discovered over three decades ago as a specialized reverse transcriptase that adds telomeric repeats to the ends of linear eukaryotic chromosomes. Telomerase plays key roles in maintaining genome stability; and its dysfunction and misregulation have been linked to different types of cancers and a spectrum of human genetic disorders. Over the years, a wealth of genetic and biochemical studies of human telomerase have illuminated its numerous fascinating features. Yet, structural studies of human telomerase have lagged behind due to various challenges. Recent technical developments in cryo-electron microscopy have allowed for the first detailed visualization of the human telomerase holoenzyme, revealing unprecedented insights into its active site and assembly. This review summarizes the cumulative work leading to the recent structural advances, as well as highlights how the future structural work will further advance our understanding of this enzyme. Tetrahymena thermophila consisted of repetitive TTGGGG sequences . Th. Th56]. Tribolium castaneum TERT . Th. Th83]. On its own, each H/ACA heterotetramer forms a similar assembly as seen in previous structures Figure . They arThe above observation has several important implications. Each of the two hairpins of the H/ACA RNA contributes differently to the H/ACA RNP assembly. Changes made to the 3\u2032 hairpin, which reduce its protein binding affinity, were detrimental to the accumulation of both hTR and the canonical H/ACA snoRNAs . On the The inter\u2013tetramer interactions also explain why dyskeratosis congenita mutations found in the H/ACA RNP specifically result in telomere maintenance defects rather than ribosome and spliceosome biogenesis defects . These dTCAB1 provides further protein\u2013RNA affinity enhancement on the 3\u2032 H/ACA hairpin of telomerase . Here weAlthough we gained unprecedented mechanistic insights into human telomerase, when compared with the understanding of other RNPs gained through structural studies such as the ribosome or splicTetrahymena telomerase at different stages of the catalytic cycle have been recently captured [Many questions regarding how repeat addition processivity is achieved still remain to be answered: How does telomerase initiate on a telomeric DNA substrate? How many base-pairs are maintained in the active site at each stage of the catalytic cycle? What signals termination upon the addition of a full GGTTAG repeat? How is the product DNA substrate repositioned in the active site for another round of repeat synthesis? How do G-quadruplexes affect telomerase activity ? Althougcaptured , the humin vitro [Telomerase recruitment to telomeres is mediated by the shelterin complex, which binds mammalian telomeric DNA specifically ,77,95\u201397in vitro . Future The association of histone H2A\u2013H2B dimer with the catalytically essential CR4/5 RNA domain in human telomerase is unexpected . Future The biogenesis of cellular telomerase holoenzyme requires a complex pathway that is not fully understood , 99. ProThe low natural abundance, complexity and flexibility of human telomerase had made its structure determination an intractable problem for many years. The \u2018Resolution Revolution\u2019 in cryo-EM has allowed these hurdles to be overcome, and the telomerase field has entered the structural era. This first atomic view of human telomerase holds promise for drug design studies and provides a structural framework for future studies on human telomerase catalytic cycle, telomere recruitment and regulation.Telomerase ribonucleoprotein resolves the end-replication problem to maintain genome stability. Telomerase dysfunction and misregulation are implicated in cancers and ageing. Therefore, it is crucial to understand how telomerase functions at a molecular level.The first detailed visualization of the human telomerase holoenzyme using cryo-EM accounts for numerous genetic, biochemical and cell biology data and provides mechanistic insight into telomerase assembly and function.Future structural studies combined with biochemical and biophysical methods and cell biology will be required to address further questions regarding the molecular mechanism and regulation of human telomerase."} +{"text": "We congratulate Margus and colleagues on their interesting study documenting emergency medicine physicians\u2019 use of Twitter preceding surges in COVID-19 cases [On Twitter, there is no specific criteria listed for verification of medical professionals. Instead, verification is based on notability criteria associated with representing a notable individual or brand . These cIn 2010, the American Medical Association issued guidelines regarding the ethical use policy to aid physicians in navigating social media . HoweverAs time moves forward, the use of social media in medicine will continue to expand beyond prediction as will its potential pitfalls. Margus and colleagues\u2019 article brings u"} +{"text": "Very little data exists on salvage surgery in previously unresectable or metastatic disease treated with initial immunotherapy. Only a handful of case reports/series regarding surgery for advanced lung cancer after immunotherapy mention the technical challenges involved. We report the case of a 67\u2010year\u2010old female with a left lung squamous cell lung cancer revealed by computed tomography\u2010guided biopsy. Treatment started with chemotherapy followed by immunotherapy in which a partial response was recorded. Subsequent salvage lingulectomy with the thoracoscopic approach was performed. The patient fully recovered and shows no sign of recurrence at follow\u2010up 16\u2009months on. Our case discusses the surgical tactics involved in the procedure, highlights similar findings encountered in the literature, and contributes to the few reports therein. Thoracoscopic intrapericardial lingulectomy following immunotherapy can be safely performed even in the presence of pulmonary hilar fibrosis. The use of immune checkpoint inhibitors (ICI) has been gaining ground in the treatment of non\u2010small cell lung cancer (NSCLC) worldwide. Early studies of these drugs in patients with advanced NSCLC suggest that long\u2010lasting sound treatment responses in such cases can be expected.A 67\u2010year\u2010old woman presented to our hospital with cough and progressive dyspnea. A computed tomography (CT) of the chest revealed an ill\u2010defined left lung mass with obstructive atelectasis and massive pleural effusion Figure . Her resConsidering the residual tumor after treatment, the patient underwent salvage surgery 4 months after the last cycle of immunotherapy. A double\u2010lumen endotracheal intubation was performed with right decubitus positioning. The patient underwent left three\u2010port thoracoscopic approach. Diffuse intrapleural adhesions and dense hilar fibroses were encountered, which were considered remnants of the previous malignant pleural effusion and probably a postimmunotherapy phenomenon Figure and enabThe use of immune checkpoint inhibitors (ICI) is gaining traction in the treatment of NSCLC. Hence, a full understanding is imperative due to its potential of becoming a part of mainstay treatment. Several prospective pilot studies revealed the viability of resection after ICI therapy.Under similar circumstances, we performed intrapericardial division of the lingular vein followed by stapling the remaining hilum due to severe adhesions between the lingular bronchus and artery. In this manner, the danger of pulmonary air leakage and vascular injury can be circumvented. In cases reported by Bott et al. and Fujishita et al., the possibility of a fusion between the pulmonary artery and bronchus following the administration of an ICI has been documented. The former authors cut both artery and bronchus together with a single stapler,Despite the way immunotherapy has reshaped the treatment approach to NSCLC, dense fibrosis and adhesions of pulmonary hilum may be anticipated during operations based on previous surgical experiences. However, more evidence is still required to validate the data through ongoing clinical trials. From our viewpoint, the merits of intrapericardial division of pulmonary vessels to avoid directly managing/dissecting hilar structure can be considered. Furthermore, thoracoscopic segmentectomy or even lobectomy can be performed safely with careful manipulation using proper surgical techniques.The authors report no conflict of interest.None declared."} +{"text": "Individuals living at home with dementia often rely on a team of caregivers and health care providers. Yet little is known about how the role of paid caregivers within this team is determined. We identified patients with moderate to severe dementia (n=9) and conducted individual interviews with their care teams (n=27) to explore perspectives on paid caregiver roles. Participants disagreed on who determined the paid caregiver\u2019s role. Agencies were perceived to set limitations on the scope of care (particularly by physicians) but agency care plans were often seen as inadequate and failing to capture important nuances of care. Most family caregivers believed they should guide what paid caregivers did in the home, while most paid caregivers reported relying on their own experience and knowledge. Understanding and addressing these differing perceptions is critical to improving the quality of paid care in the home."} +{"text": "Among Latinx older adults, our current understanding of barriers to eye exam often fails to consider the impact of patient and provider burnout which can decrease treatment adherence and recommendation receptivity in this group. The purpose of this study was to examine correlates of eye exam frequency among Latinx older adults in South Los Angeles and explore associations reflecting patient and/or provider burnout. Data analysis was informed by the Secret Self-Management Loop and the Burnout Dyad conceptual models. This secondary analysis used data collected from a convenience sample of non-institutionalized Latinx adults 55+ in South LA (n=165) and used multinomial regression analysis. Outcome variable is recency of eye exam, independent variables are self-reported health, including diabetes mellitus diagnosis, and either patient or provider burnout . Variables associated with Provider Burnout, appear to represent a larger influence on eye examination frequency then variables associated with Patient Burnout, with the most influential factor being provider recommendation. A surprising finding was the number of participants who had never received this recommendation from a provider (21%). One-third (32%) of participants with diabetes mellitus had not had an eye examination within 12 months and almost one-fifth (13%) of participants with diabetes who had received this recommendation had not received the exam. Further exploration is needed to support a better understanding of how both patient and provider burnout impacts adherence to eye examination and other preventive care recommendations for diabetes mellitus among Latinx older adults."} +{"text": "Recruiting persons living with dementia (PLWD) and their caregivers (dyads) into research is challenging and costly. The purpose of this study was to better understand factors that influence dyads decisions to enroll in a clinical trial. We used Ajzen\u2019s Theory of Planned Behavior (TPB) to develop a qualitative interview guide and analyze the data with a directed content analysis. We conducted semi-structured telephone interviews with 12 PLWD and 9 caregivers who all enrolled in one clinical trial. Aligning with the TPB we found the following positively influenced enrollment: 1) wanting to learn, in-person meetings with knowledgeable staff, and the money always helps (attitudes toward joining); 2) to support another person (perceived norm); and 3) easy to participate . Flexible scheduling and the study taking place in the home was comfortable and convenient for participants. Findings can inform future recruitment efforts and research studies."} +{"text": "NICE guidelines and Maudsley prescribing guidelines both stipulate that patients over the age of 65 prescribed lithium or antipsychotic medication should have their bloods and physical parameters monitored regularly. There is currently no provision from the community mental health teams in Edinburgh to provide this monitoring, which falls to the patients GP. Following an initial data collection, it was found that there was no monitoring advice being provided on immediate discharge letters (IDLs) for patients discharged from two functional old age psychiatry inpatient wards at the Royal Edinburgh Hospital. This patient group often have comorbid medical conditions and therefore monitoring of their psychotropic medication is especially important. The aim of the QI project was for 100% of patients discharged from thesewards on lithium or antipsychotic medication to have appropriate advice documented on their immediate discharge letter (IDL) with regards to medication monitoring.Data were collected monthly by reviewing the notes of all discharged patients to determine the frequency at which medication monitoring advice was documented on IDLs from the two wards. A proposed new template for discharge letters which included advice on medication monitoring was discussed and agreed with the old age psychiatry team in Edinburgh. This was disseminated to the appropriate medical staff members and was included in induction packs for junior doctors. Following this a new \u201ccanned text\u201d template was implemented to automatically populate the discharge letter with advice depending on whether they were antipsychotics/lithium/neither.IDLs for 91 patients discharged between May 2020 and February 2021 were reviewed. Baseline data showed that 0% of patients (n = 15) had appropriate monitoring advice documented on their IDL. Following initial introduction of monitoring advice to the induction pack for junior doctors, the mean frequency of completed advice on IDLs was 50.9% across 6 months. Following implementation of the canned text, the frequency of completed advice on discharge letters for February 2021 was 100% (n = 7).This QI project has been successful in improving the rate of appropriate advice for antipsychotic and lithium monitoring being provided on immediate discharge letters. It is hoped that this will help reduce adverse effects associated with antipsychotics and lithium in older psychiatric patients. Further work could be done on determining the frequency that the advised monitoring is being carried out."} +{"text": "To assess the level of understanding and difficulties encountered when obtaining sexual health details of their patients among mental health clinicians.People with mental health problems, especially those treated with psychiatric medication experience greater rates of sexual difficulties than those in the general population. Mental health practitioners need to examine personal beliefs and attitudes about sexuality among people with mental health problems. Providing information about sexuality and sexual practice benefits and enhances the quality of life of people with mental health problems. Therefore taking a sexual history should be an integral part of psychiatric assessment.An online survey consisted of 17 questions to cover 3 areas of objectives mentioned above was created using Survey Monkey. A link to the survey was emailed to all the clinicians who perform psychiatric assessments. Response collection and data analysis was performed by the trust IT team.Total of 54 clinicians participated in the survey representing nurses, junior, middle grade doctors and consultants. Almost all stated that mental health patients have capacity to make appropriate decisions about their sexual behaviour patterns. 43% thought people with mental health problems don't have similar patterns of sexual behaviour compared to people without mental health problems. 11% stated that people with mental health problems do not experience greater rates of sexual difficulties than those in the general population. Nearly a third did not believe that telling patients about potential sexual side effects may lead to poor compliance. Nearly 70% stated taking a sexual history should be an integral part of psychiatric assessment. 44% reported lack of knowledge and skills when talking about sexual health and 33% avoided asking about sexual health due to lack of knowledge. Half of the clinicians avoided asking about sexual health due to the fear of embarrassing or causing distress to patients while 16% avoided asking about sexual health due to self-embarrassment. 65% talk about sexual health issues only if patients brought them up.During last 3 clinical encounters majority never asked about sexual difficulties, high risk behaviour and drug side-effects related to sexual difficulties. A significant proportion of clinicians never asked about contraception from their female clients.Survey revealed majority of mental health clinicians lack understanding and skills about sexual health issues highlighting the importance of raising awareness among clinicians about sexual health issues."} +{"text": "Memecylon, in Sri Lanka, an island, using species occurrences and climate data. We aim to determine which climate variables explain current distribution, model how climate change impacts the availability of suitable habitat for Memecylon, and determine conservation priority areas for Sri Lankan Memecylon. We used georeferenced occurrence data of Sri Lankan Memecylon to develop ecological niche models and assess both current and future potential distributions under six climate change scenarios in 2041\u20132060 and 2061\u20132080. We also overlaid land cover and protected area maps and performed a gap analysis to understand the impacts of land\u2010cover changes on Memecylon distributions and propose new areas for conservation. Differences among suitable habitats of Memecylon were found to be related to patterns of endemism. Under varying future climate scenarios, endemic groups were predicted to experience habitat shifts, gains, or losses. The narrow endemic Memecylon restricted to the montane zone were predicted to be the most impacted by climate change. Projections also indicated that changes in species\u2019 habitats can be expected as early as 2041\u20132060. Gap analysis showed that while narrow endemic categories are considerably protected as demonstrated by their overlap with protected areas, more conservation efforts in Sri Lankan forests containing wide endemic and nonendemic Memecylon are needed. This research helped clarify general patterns of responses of Sri Lankan Memecylon to global climate change. Data from this study are useful for designing measures aimed at filling the gaps in forest conservation on this island.Recent climate projections have shown that the distribution of organisms in island biotas is highly affected by climate change. Here, we present the result of the analysis of niche dynamics of a plant group, Memecylon, to develop ecological niche models and assess future potential distributions under six climate change scenarios. Our results provided insights into patterns of responses of Sri Lankan Memecylon to global climate change. Additionally, data from this study are useful for designing measures aimed at filling the gaps in forest conservation on this island.With the aim of understanding how climate change affects the distribution of island\u2010dwelling plants, this research used occurrence data of a Sri Lankan woody plant group, The remaining Memecylon are confined to wet or dry forests. Memecylon also shows a wide range of ecological diversity in different locations, including the highest mountain of the island, Pidurutalagala , the dry lowland forests , and coastal areas . Due to its diversity across the island and high regional endemism, this plant group represents an ideal model to investigate niche differentiation and the possible impact of climate change on vegetation in different climate zones. Currently, Sri Lanka has set aside a considerable proportion of land for conservation What are the patterns of distribution of Sri Lankan Memecylon under current climate conditions? (2) How will climate change impact the availability of suitable habitats of Memecylon, and what will be the main driving factors of these changes? And (3) Which areas should be targeted for conservation as indicated by Memecylon distribution in the face of climate change in Sri Lanka? Endemic species are reported as highly vulnerable to climate change in the Indian Ocean and has a land extent of 65,525\u00a0kmAdditional GPS points from plant locations were collected during fieldwork from June to August 2017 from randomly selected forests representing several climate and elevation zones of Sri Lanka were removed using R packages spocc, scrubr, and spatstat may introduce bias because those occurrence points may not adequately capture the range of environmental conditions in which a species might occur . We also removed two species (Memecylon gracilimum Alston and Memecylon macrocarpum Thwaites) in which occurrence data were found only from a single location. Using these filtering criteria, 21\u00a0Memecylon taxa were used for analyses for all bioclimatic variables within the species\u2010specific buffer zones were estimated to avoid collinearity between them using R packages raster and rgdal from General Circulation Models (GCMs): Beijing Climate Center Climate System Model (BCC\u2010CSM1\u20101) and Model for Interdisciplinary Research on Climate (MIROC5) and for each, we used 2.6, 4.5, and 8.5 Representative Concentration Pathways (RCPs) (30 arc\u2010second resolution); these data were obtained from WorldClim v.1.4 applied to two GCMs (BCC\u2010CSM1\u20101 and MIROC5) over two time periods (2050 and 2070) (selection of these models were explained under climate data selection). Projections were carried out on MaxEnt extrapolating into parts of environmental space from which there are no training samples and lowland narrow endemic . Nonendemic Memecylon which contain suitable habitats within the wet zone of the island and showed a strong contribution of precipitation\u2010related bioclimatic variables were subclassified as wet zone nonendemics. Nonendemic Memecylon which contain suitable habitats within the dry zone and showed a strong contribution of temperature\u2010related bioclimatic variables were subclassified as dry zone nonendemic. By the above\u2010defined standards, we found four species to be wide endemics, eight as lowland narrow endemics, three as montane narrow endemics, and an additional three species each were categorized as wet\u2010zone and dry\u2010zone nonendemics, respectively.Sri Lankan Bremer, , area of2) for each species between current and future distributions were evaluated.In each endemic category, the threshold models of species were overlapped on each other and the overlapping area under the current condition was calculated using customized R scripts. These overlaps showed regions where niche conditions are suitable for two or more taxa. Threshold models of each taxon in 12 future models were also overlapped using the above procedure, and changes in the distribution of suitable habitat , we overlapped current individual suitable areas of Memecylon species by endemic categories. The resulting raster files of species richness were reclassified to indicate the number of species in each grid cell using the R package raster conservation forest category and (2) all conserved category, were extracted and separately merged using QGIS. The resulting conservation maps were cropped using QGIS to fit the extent of the richness map. Percent areas of conservation forests and all conserved areas were then estimated using the raster calculator in QGIS. By comparing the forest cover and protected area results, the area of unprotected forest falling within the richness area in each Memecylon category was estimated. This comparison was repeated for the predicted future (2050 and 2070) richness areas based on the most optimistic climate scenarios using current protected area maps; this analysis assumes that the current protected areas remain the same for 2050 and 2070. Finally, the unprotected forests which require conservation were determined by superimposing the current richness maps of all endemism categories of Memecylon, forests cover, and protected area maps on QGIS.A protected areas map of Sri Lanka from the World Database on Protected Areas (WDPA) to 2524\u00a0m and permutation importance ), and one wide endemic wet zone species (Memecylon urceolatum Cogniaux) were predicted to undergo a total decrease under future climate scenarios model shown in Figure 3.5Memecylon with the protected area and forest maps resulted in information relevant for conservation. Land area extracted from protected areas and land\u2010cover maps are provided in Dryad. The area estimations after superimposing these extracted areas with Memecylon richness areas are provided in Figures 2 . When the species richness areas , the percentage overlap with all types of protected lands decreased are susceptible to habitat loss ; Data curation (lead); Formal analysis ; Funding acquisition ; Investigation (lead); Methodology (lead); Resources ; Writing \u2013 original draft (lead); Writing \u2013 review & editing . Narayani Barve: Conceptualization ; Formal analysis ; Methodology (supporting); Writing \u2013 review & editing . Hashendra Kathriarachchi: Resources ; Writing \u2013 review & editing . Bette Loiselle: Conceptualization ; Methodology (supporting); Supervision ; Writing \u2013 review & editing . Nico Cellinese: Conceptualization ; Funding acquisition ; Project administration (lead); Resources ; Supervision ; Writing \u2013 review & editing ."} +{"text": "Engaging with Aging is an emerging framework proposed by Carnevali which provides a new lens to understand an active, conscious daily living process of coping with age-related changes (ARCs) taken on by older adults. Study aims were to 1) describe the ARCs experienced by community-dwelling older adults; 2) identify the strategies and resources used by older adults to accommodate the daily living challenges caused by the associated ARCs; and 3) evaluate the framework of EWA based on findings from aims 1 and 2. We conducted semi-structured interviews with 29 participants aged 64 to 98 online due to COVID-19 restrictions. We used a virtual card sort to assist data gathering. Fifteen ARCs were mentioned by participants and their corresponding adaptations were discussed. We found that older adults linked their adaptations to their ARCs based on their changing capacities and needs. Commonly used adaptations included conserving energy, utilizing tools or technology, and being more conscious before and while taking actions. The challenges caused by COVID-19 in implementing the adaptations were also discussed . Our study substantiates the EWA framework by showing the commonality among older adults in linking ARCs with adaptations. Implications for clinicians and researchers include using EWA to help older adults identify personalized solutions that fit their capacities. Our study is late-breaking as we recently finished data analysis and the information included was not yet available by the previous submission deadline."} +{"text": "The implementation of pharmacogenetic tests including multiple gene variants has shown promising potential as a decision-making tool for optimizing psychopharmacological treatment regimens and reducing treatment costs. However, the varying clinical validity of gene variants included in pharmacogenetic test batteries, and inconsistencies in their translation into medical recommendations between commercially available pharmacogenetic tests, complicates their rational implementation. Thus, there is a need for well-designed, reproducible studies documenting the clinical significance of the various genetic variants. Callegari and colleagues show in CES1) variant identified to date that would be expected to alter the therapeutic results of CES1 substrate drugs, such as methylphenidate [CES1 gene variation only partially explains the pharmacokinetic variability [CES1 expression and activity would be desirable before this gene variant was included in a pharmacogenetic test battery. Another example is the CYP2C19 haplotype *1F, which is associated with increased metabolism, but apparently only becomes phenotypically significant in the presence of additional inducers [In recent years, there has been a trend towards the combination of several gene variants rather than using them individually. This makes good sense on a theoretical level as several potentially competing genetic variants relevant to the current pharmacological treatment can be detected simultaneously. However, it also involves combining gene variants with different clinical validity, some of them without broader consensus on how the result should be translated into a medical decision. For example, SNP G143E rs71647871) is the only Carboxyesterase 1 and DPWG (The Dutch Pharmacogenetics Working Group). Batteries of pharmacogenetic tests should not be implemented in clinical practice as a kind of black box test where it is unknown whether the effect obtained is actually due to the pharmacological treatment adapting the pharmacogenetic test result, or is confounded by the doctor, for example, giving the patient\u2019s pharmacological treatment greater attention."} +{"text": "To review available standards for physical health monitoring in people taking clozapine To audit current practice against standards To identify changes in practice and facilitate a re-audit to assess impact of any changesStandard: CG178 Psychosis and Schizophrenia in Adults: Prevention and Management \u2013 NICE, February 2014Target:100%Exceptions: NoneSample: The original audit included all 58 patients from the Worcester clozapine clinic, as per October 2018. The re-audit reviewed a random sample of all patients attending the clozapine clinics in Worcester, Kidderminster and Redditch, as part of Worcestershire Health and Care NHS Trust, as per October 2019. A total of 66 patients were selected.Data Source: Carenotes and ICEAreas of good practice:Monitoring of HbA1c and FBC remains goodThere has been an improvement in monitoring alcohol use, substance misuse and side effectsAreas requiring improvement:There continues to be limited recording of respiratory rateThere has been a decline in recording temperature, BMI and concomitant therapiesPotential reasoning for missing data includes:Staff not knowing the monitoring requirements, which is more likely to be an issue when staff members running the clinics change frequentlyMonitoring being completed but not documentedPatients\u2019 refusal of monitoringData being recorded in alternative locations including general practice, without communication between servicesPatients moving between teams or having inpatient stays may disrupt monitoring regimeLIMITATIONSThis audit assumes all patients involved to be on a stable dose of clozapine with routine monitoringSome patients may have been transferred between teams or inpatients during the period of data collectionThere is no scope to record when patients refuse monitoringWe may not have access to all notes such as those from general practice for data collectionRECOMMENDATIONSInduction programme for junior doctors to include education on clozapine monitoringTraining for staff involved in clozapine clinics to ensure better understanding of monitoring requirementsProcurement of ECG machines for each site and relevant training for nursing and medical staffCollaboration with GPs for shared dataRe-audit in 1 year"} +{"text": "PLOS Biology uses threespine stickleback to detect a genomic signature of ecological incompatibilities.As we uncover the ubiquity of hybridization in nature, determining how natural selection acts on hybrids has newfound importance for speciation. A study in As we uncover the ubiquity of hybridization in nature, determining how natural selection acts on hybrids has newfound importance for speciation. This Primer explores a study in PLOS Biology that uses threespine stickleback to detect a genomic signature of ecological incompatibilities. A long-standing goal in evolutionary biology is to understand how natural selection acts on hybrids and assess how often selection against hybrids can contribute to reproductive isolation among species. Selection against hybrids is often classified as intrinsic or extrinsic based on whether hybrids exhibit low fitness across many environments or only in certain conditions, respectively . These 21 hybrids with mismatched traits. These mismatches may occur because which parent an F1 resembles can differ between ecologically important traits . Dominasmatches ; examplesmatches ,8). Howe2 hybrids through manipulative crosses, rear replicate hybrid populations in multiple environments, then track traits and fitness for each individual in each environment to generate fitness landscapes that are environment specific.While an intriguing possibility, the standard of evidence required to demonstrate such complex extrinsic incompatibilities is substantial; researchers must generate hundreds of F2 generation is also predicted to leave distinct genomic signatures. If particular combinations of homozygous genotypes lead to lowered fitness by creatively leveraging previously published studies and unpublished data to look for genomic signatures of ecological incompatibilities [2 hybrids exhibit unusual jaw morphologies that combine distinct traits from either parental species . These exhibit; .To identify genome-wide signatures of ecological incompatibilities, Thompson and colleagues calculated how much heterozygosity pond-raised and lab-raised hybrids exhibited, and how much these levels of heterozygosity deviated from expected . Under With new tools in hand, what are the next steps for understanding ecological incompatibilities? While Thompson and colleagues present a straightforward method for detecting ecological incompatibilities, amassing more examples will require substantial effort, and in many systems will be unachievable. Nonetheless, as more examples of ecological incompatibilities accumulate, 2 key areas of research will emerge: What are the mechanisms underlying ecological incompatibilities and how important are they for speciation?Determining how selection acts on specific traits/trait combinations will be essential to understand how selection is acting against hybrids. This will require the nitty-gritty of building environment-specific fitness landscapes and tracking both the targets and agents of natural selection in hybrid populations. Moreover, a comprehension of the genetic architecture of the trait(s) involved in ecological incompatibilities will be a key step in elucidating the importance of this reproductive barrier. Understanding if the traits involved in ecological incompatibilities have largely additive genetic architectures with epistatic effects on fitness, the number and effect size of the loci involved, and the strength of epistatic selection can give insight into how much of the genome is expected to be involved in reproductive isolation, as well as how many hybrid individuals\u2014and in what generation\u2014low fitness is expected to manifest. The answers to these questions have crucial implications for the efficacy of ecological incompatibilities to limiting gene flow in nature. Lastly, assessing how common these types of incompatibilities are and when in the speciation process they tend to evolve are central for determining their importance in speciation. The latter 2 goals will require amassing many examples of ecological incompatibilities, both across taxonomic groups as well as lineage pairs of differing divergence times within a group. While much work is needed, the creative reuse of previously published datasets presented by Thompson and colleagues is a reminder that applying genomic technologies to previous experiments may serve as a fruitful avenue forward."} +{"text": "In our manuscript entitled \u2018Neoadjuvant Chemotherapy Switch in Borderline Resectable/Locally Advanced Pancreatic Cancer\u2019 we report on our cumulative high-volume institutional experience with CS, including frequency, indications, and outcomes of patients who require CS after initial FL NAC prior to curative-intent surgery.5Neoadjuvant chemotherapy (NAC) is considered for patients with borderline resectable/locally advanced (BR/LA) pancreatic adenocarcinoma (PDAC). A chemotherapeutic regimen that is both tolerable and demonstrably effective is of utmost importance as oncologic outcomes in these higher risk patients highly correlate with subsequent pathologic treatment responses.5We were able to demonstrate that a substantial proportion (30%) of patients required a chemotherapeutic switch after initial FL NAC due to the lack of objective treatment responses and/or chemotherapeutic toxicities. Of those undergoing CS, a majority were able to proceed to curative-intent surgery after subsequent CS. We found no major preoperative or perioperative differences between those undergoing resection after FL NAC compared with those who required CS. Importantly, there were no differences in pathologic or survival outcomes between cohorts, suggesting that such a CS strategy does not incur an oncologic detriment and can potentially be used to salvage those patients in whom FL chemotherapy is not tolerable or is ineffective.The presented results contribute to our overall understanding of appropriate neoadjuvant treatment sequencing and highlight the continued evolution of what constitutes an optimal treatment strategy for patients with anatomically borderline resectable or locally advanced PDAC. Based on these results, we suggest that CS be considered for all patients with initial suboptimal responses or in those who develop treatment-limiting toxicities to FL as a significantly important neoadjuvant strategy prior to consideration of surgical resection in order to improve outcomes for these patients. We anticipate the use of such a strategy combined with evolving NAC response metrics will improve the optimal selection of patients who could potentially benefit from curative-intent surgical resection."} +{"text": "Researchers hypothesize that how people react to daily stressful events partly explains the personality-health relationship, yet no study has examined longitudinal associations between these factors. The current study examined the role of negative affect reactivity to daily stressors as a mediating pathway between personality and physical health outcomes using three waves of data spanning 20-years from a nationwide probability sample of 1,176 adults. Results indicate that Wave 1 neuroticism was associated with greater negative affect reactivity at Wave 2, which then predicted the development of chronic conditions and functional limitations at Wave 3. Higher conscientiousness was associated with less negative affect reactivity, which in turn predicted better physical health at Wave 3. Negative affect reactivity partially mediated both personality traits and physical. These findings highlight the usefulness of using a daily stress framework for understanding how personality impacts health over time, which has important implications for disease prevention."} +{"text": "Circadian rhythm disturbances (CRD) are commonly seen in people living with dementia. A clear understanding of the role of CRD in dementia etiology will be beneficial by exploring the exogenous factors and endogenous factors . This symposium will apply a chronobiological approach to study exogenous and endogenous factors that influence circadian rhythm and their effects on sleep and neuropsychiatric symptoms in persons living with dementia (PLWD). Four paper presentations will use secondary data analysis of data from the Healthy Patterns Clinical Trial (NCT03682185), a randomized controlled trial of a home-based activity intervention designed to improve circadian rhythm disorders in PLWD. We will first describe the circadian rhythm pattern reflected by endogenous factors , then examine salivary cortisol (endogenous) and white light intensity (exogenous) and on subjective sleep and neuropsychiatric symptoms (including depression) in PLWD, respectively. In session 1, we will present cortisol diurnal rhythm pattern in PLWD using a cross-sectional design. In session 2, we will discuss the relationship between salivary cortisol indicators and depressive symptoms. In session 3, we focus on the association between diurnal cortisol slope and neuropsychiatric symptoms using the baseline data. In session 4, we describe the association between evening white light exposure and subjective sleep. The discussant will describe how these findings build on our understanding the nature of circadian rhythm disturbance in dementia and inform future research and treatment approaches."} +{"text": "Progressive decline in adult stem cell function is a feature of aged tissues and contributes to multiple age-related diseases. Of particular interest are bone marrow mesenchymal stem cells, also known as bone marrow stromal cells (BMSCs), which are multipotent, and have significant therapeutic potential for age-related disease. BMSCs isolated from older individuals show decreased differentiation and characteristics of cell senescence. We and others find that inhibition of the mTOR pathway prevents senescence and extends BMSC proliferation. We have examined miRNA profiles in rapamycin-treated BMSCs to identify miRNAs that may be needed to maintain \u201cstemness\u201d and facilitate differentiation in this population in undifferentiated cell populations. The analysis reveals a distinct miRNA profile induced by rapamycin associated with enhanced differentiation capacity."} +{"text": "Previous research suggests the prevalence of mental health conditions among medical inpatients may be as high as 38%. Anecdotally, junior doctors report lacking the confidence, knowledge and skills to assess and treat patients with psychiatric conditions. Identifying this unmet need offers potential to improve standards of care and achieve parity of esteem between psychiatric and medical conditions within the general hospital. Aims:To assess self-reported preparedness of newly-qualified Foundation Doctors to care for patients with acute or chronic psychiatric symptoms in comparison to physical health conditions.In September of each year , a survey was cascaded to all incoming Foundation Year 1 Doctors. For each respective year there were 1673, 961 & 1301 respondents. Respondents were asked to rate their agreement with statements on a Likert scale. Statements pertaining to mental health included \u201ca) I am competent in acute mental health care provision, b) I am competent in chronic mental health care provision\u201d and \u201cI feel confident in prescribing the following drugs; c) drugs for mental health problems\u201d. Comparison statements assessed confidence caring for medically unwell patients, performing practical procedures and prescribing drugs for physical health conditions.Preparedness for acute and chronic mental health were lower than both physical health comparison items; preparedness to care for patients with critical illness and preparedness to perform practical procedures .Confidence prescribing mental health drugs was lower than all other comparison items These results identify a disparity in foundation doctors\u2019 self-reported preparedness to treat acute and chronic mental health conditions and prescribe psychotropic medications, compared to a variety of physical health domains. To our knowledge this is the first large-scale study to empirically test a potential discrepancy between newly-qualified doctors\u2019 preparedness to treat patients\u2019 mental and physical health needs. Medical school education and foundation training may therefore present a fruitful opportunity to improve care for patients with psychiatric conditions within general hospital settings."} +{"text": "ABSTRACT IMPACT: Optimizing the use of endotracheal aspirate cultures (EACs) has the potential to improve the care of complex mechanically ventilated children by improving testing practices and avoiding unnecessary antibiotic treatment for false-positive results. OBJECTIVES/GOALS: An electronic survey has previously been employed to characterize the practices and attitudes around blood cultures among critically ill children. The objective of this work was to develop and pilot a new survey as a tool to understand practices and attitudes that could inform quality improvement initiatives to optimize EAC practices. METHODS/STUDY POPULATION: Informed by prior experience of diagnostic stewardship of EAC in other settings and using a similar structure to the blood culture practice survey, we developed an electronic self-administered survey sent to respiratory therapists, advanced practice providers, and physicians at the Johns Hopkins All Children\u2019s pediatric intensive care unit. RESULTS/ANTICIPATED RESULTS: A total of 27 of 87 clinicians (37%) responded to the survey . Responses indicated samples are typically collected by respiratory therapists via in-line or open suctioning (tracheostomy). Most respondents did not feel EACs could lead to unintended negative consequences (71%), agreed practices vary between people (89%), and felt an algorithm would help align the clinical team (79%). Most respondents agreed some clinicians may be reluctant to change practice (82%) and may not change practice due to concern for missing diagnosis of ventilator-associated pneumonia or tracheitis (78%). Surveillance cultures were not used in this unit and there were no prior EAC diagnostic stewardship efforts. DISCUSSION/SIGNIFICANCE OF FINDINGS: This survey captured practices, perceptions and barriers to changes that will inform the implementation of quality improvement initiatives to optimize EAC use in this unit. Future studies can consider utilizing an electronic survey to describe practice variation, clinician believes and attitudes about EAC testing in ventilated patients."} +{"text": "Wear and surface microgeometry aspects of fiber-reinforced hybrid composite dry friction clutch facings are revealed in a novel way: after different, real life automotive tests during their lifetime. This study examines and reveals the tribological response of friction material surfaces to real life application conditions with two different facing diameters and in two directions. Along the increasing activation energy scale, wear values increased according to two different trends, sorting tests into two main groups, namely \u2018clutch killer\u2019 and \u2018moderate\u2019. Wear results also highlighted the influence of mileage and test conditions, with clutch killer tests also creating considerable wear-more than 0.1 mm-at inner diameters: 1% higher wear was generated by 90% higher mileage; another 1% increment could be caused by insufficient cooling time or test bench-specific conditions. Surface roughness values trends varied accordingly with exceptions revealing effects of facing size, friction diameter and directions and test conditions: small (S) facings produced significantly decreased Rmax roughness, while large (L) and medium (M) size facings had increased roughness values; Rmax results showed the highest deviations among roughness values in radial direction; tests run with trailer and among city conditions resulted in more than 2% thickness loss and a 40\u201350% roughness decrease. In automotive applications, the purpose of utilizing a clutch is to transfer kinetic energy in the form of modulated torque of a rotating crankshaft coupled to a power source towards a transmission system and further to the wheels of the vehicle. The commModern hybrid composites consist of a great variety of components, which makes their identification and characterization rather complex.These components of a hybrid composite material are usually sorted into groups based on their mechanical role and function. However, one single component usually affects many different characteristics besides the targeted aspect: even reinforcing fibers play a critical role regarding tribological\u2013friction and wear\u2013properties . FurtherOn the other hand, filler materials with various volume percentages were found by Basavarajappa et al. to make Even agriculture fiber wastes such as corn, sugar bars and palms fibers were found to be capable of increasing the friction coefficient while decreasing wear in an investigation by Bakry et al. , with thpv (surface pressure multiplied by velocity) values towards severe conditions affected running-in COF fluctuation. Besides these approaches, Abdullah et al. ) and rotational velocity difference ) between coupling surfaces, as stated in Equation (1) [m) and the clamp load (Fm):Determining surface activation energy for these tests provides an opportunity to compare and highlight their intensity and tribological effects. During the time (t) the clutch pedal is released, the dissipated energy for a single engagement indicated by temperature rise is a function of transmitted clutch torque (Ttion (1) :(1)E=\u222b0tdyn is the dynamic rolling radius, i1stgear is the first gear ratio, idiff is the differential ratio, neng is the engine speed, Tengmax is the maximum engine torque, \u03c7 is an engine type-dependent torque factor and the drag torque Tdrag is function of the vehicle mass.Thermal load of the clutch can be calculated according to Equation (3) :(3)W=12\u00b7pv value describing the different clutch engagement profiles [For a certain diesel engine passenger car with a total weight of ~1300 kg and manual transmission with a 1st gear transmission ratio of 4.23 and differential ratio of 3.46, operating a clutch with facing outer/inner diameters of \u00d8228/\u00d8160 mm, the energy values for actuation in different circumstances and as building blocks of prescribed automotive tests are as However, during the tests described above many clutch engagements occurred and many tests were run in repeated cycles with different blocks from those in 2 of the friction surface) of each investigation could be calculated and a hypothetical comparative scale could be created, as Recreating the preliminary tests from these actuation profiles shown in TH\u2013VRS\u2013VRTS\u2013VC\u2013VTC\u2013VR.Based on this, the intensity scale of the preliminary tests is as follows: Taking mileage into consideration, the total energy can be calculated. Different sized dry friction fiber-reinforced clutch facings\u2013from which our laboratory pin-on-disc test samples were cut\u2013had been preliminarily loaded according to the matrix illustrated in Mileage is a common parameter to mark the lifetime of automotive components. Multiplying it with test intensity and activation surface values, the total surface activation energy could be calculated for all of our samples.To examine or prove the test intensity order hypothesis, with which a tribological load consequence and prediction map was to be set up for dry friction single disc clutches, first the wear values of the clutch discs after the tests were examined. Along the non-linear Joule scale two trends can be observed to be governing wear results after different test types, so measured data were separated accordingly: clutch killer and moderate usage. Comparing results, it can be concluded that the more total energy was applied to the system, the more wear was generated, with some exceptions, highlighting effects of other parameters from the system and conditions. In this paper all results are illustrated along activation energy values, though not in proportional scale.Clutch killer tests created significantly higher wear values at inner diameters;TH wear result shows that mileage has a strong influence on wear besides applied energy, and a test condition such as the test bench causes higher heat stress than in real life;On the other hand, TH test bench conditions produced less difference between inner and working diameter wear values;Analyzing wear values VTC showed wear conditions were severe even after lower mileage test runs and remained more intense even at high mileage runs: VTC tests run with trailer created engagements in non-optimal contact positions regarding the clutch friction surface, as dw wear was about three times higher than di wear regardless energy or mileage;The same phenomenon appeared in the VTRS test, causing a higher wear rate at lower mileage but with smaller diameter facings;Tests with actuation profiles happening among city traffic conditions (\u2018C\u2019) meant an extra energy load on the system, similar to test bench conditions, as shorter cool down opportunities were present.The following conclusions can be drawn examining the data:Moderate tests created significantly lower wear values at inner diameters;VR tests run in cars driven by professional drivers operating with presumably shorter shifting periods (slip) generated lower wear values;By more or less the same mileage smaller diameter, VR-tested facing (size S) showed higher wear values than greater diameter facings.VC results suggest that if the system was applied with loads from non-professional drivers, but for a long time (mileage), wear vales would be similarly moderate as those caused by professional drivers.Examining Wear values at di and dw diameter changed more or less according to the same trend, though working diameter material loss was higher considering relative values;Comparing inner and working radius values, the conical position of the facing at the beginning of the engagement was clear from lower wear values at the inner radius: di wear was always less than dw wear;Within the same test type category, the more energy applied, the higher the wear values;Though the more energy applied, the higher the wear losses, the exact amount was highly influenced by driving styles and the rapidity of energy load build-up at vehicle starts.Overall, it can be stated, that:t = 4.8 mm and Ra, Rz and Rmax was determined. Examining surface roughness values of friction facings after the different tests and comparing the results with characteristics describing the surfaces of the unused friction material gives us another opportunity to judge the hypothetical test intensity scale. Therefore, measurements were carried out utilizing the MarSurf surface roughness measurement device equipped with a PHT 350 header. During these measurements measuring length was LRa surface roughness values at inner (di) and outer (dw) diameter in radial direction are illustrated in Tangential direction Ra surface roughness distribution is illustrated in Measured then separated Rz surface roughness values are illustrated in Rmax results are illustrated in Examining the surfaces by visual inspection, the differences at inner diameter were clear. Radial and tangential direction surface roughness values decreased on the working diameter along the increasing applied energy scale, with some exceptions due to different parameters governing their application. Trends observed from wear results were present and Rmax values highlight them more significantly than Ra or Rz results: clutch killer low energy and mileage test effects were even more significant with increased Rmax values on both inner and working diameters in radial and axial direction, proving the theory of torn-up surfaces.There was a difference between radial and tangential Ra results regarding the amount of surface energy value from which roughness started to decrease along the Joule scale. This could be the result of the fact that radial measurement crosses through diameters with slightly different load and wear conditions.Inner diameter results highlight the presumably present non-parallel position of mating surfaces due to tapering: test results with trailer produced higher roughness values on inner diameter than on outer diameter in low energy and mileage tests (VTRS), while the opposite was true after the initial running-in phase ;The inner diameter trend showed more deviations that could be caused by the tapering occurring during multiple repeated shifts during which deformation influenced contact areas, so that it resulted in drastically different wear at different diameters;VR test results produced the lowest roughness values;Within VR results, small size facing roughness values were the smoothest;After VR tests, where professional drivers were operating the clutch, di and dw results were not that different, highlighting the possibility of less slip and deformation (remaining conicity) caused by heat stress;However, working diameter Ra roughness values did not decrease linearly along the Joule scale: in low mileage and energy tests the trend was decreasing, so similar mileage tests resulted in similar Ra values, and smaller size facings were characterized by lower roughness values after energy and mileage tests similar to those on larger size facings;Radial direction Rz surface roughness values slightly decreased on the working diameter along the increasing applied energy scale;Inner diameter Rz values followed the same trend that describes inner diameter Ra characteristics;Tangential direction Rz surface roughness values slightly decreased as well on the working diameter along the increasing applied energy scale;Working diameter radial direction Rmax values more or less followed the other dw surface roughness characteristics, while inner diameter values did not show as much deviance from dw values as other features;The same conclusions can be drawn from tangential direction Rmax values.The following can also be seen from the measurements:The fact that tapering and deformation induced higher deviation at inner diameter results in drastically different wear at different diameters is clearly highlighted;Comparing Ra difference values in radial direction highlights more intensive surface roughness value changes by inner diameter than working diameter;In the tangential direction, low energy or mileage can cause deviations from the trend along the Joule-scale;Rz deviation values produced similar results; both clutch killer and moderately operated test results had a good correlation with Ra trends at inner and outer diameter, respectively, considering both radial and tangential directions, even with percentage values taken into account, though tangential direction deviations on the inner diameter were relatively smaller.Low energy and mileage results can lead to increased Rmax values on the inner diameter both in radial and tangential direction, as VTRS results suggest;Only test bench TH investigation increased the working diameter tangential Rmax value significantly, highlighting the fact that test rig results do not necessarily recreate real conditions-induced results regarding all aspects.Detailing surface roughness deviation as a percentage, the following can be seen from the Ra, Rz and Rmax results:As clutch killer tests increased wear along the Joule scale on the working diameter, so the surfaces became smoother in radial and tangential direction as well regarding Ra. However, as TH results highlight, high mileage or lack of proper cooling (test bench) can lead to increasing tangential Ra surface roughness at the working diameter, probably due to rapid thermal overloads at certain spots tearing up the surface and causing remaining macroscopic deformations. Additionally, a steeper wear trend is paired with less decreasing Ra values in the radial direction.Moderate tests led to less wear on the working diameter that was paired with decreasing Ra surface roughness in both directions along the activation energy scale, though deviations could be observed when there was significant inner diameter wear that also led to increased inner diameter Ra surface roughness both in radial and tangential directions. Among the test condition results, smaller size facings suffered higher wear that was paired with lower tangential and radial Ra surface roughness.Inner diameter results after clutch killer tests highlight the presumably present non-parallel position of mating surfaces due to tapering: test results with a trailer produced higher radial Ra roughness values on the inner diameter than on the outer diameter in low energy and mileage tests (VTRS), while the opposite can be stated after the initial running-in phase .Moderate tests caused less wear at the inner diameter, hence the hardly changing Ra values in both directions. However, when there was wear, Ra could increase above the reference level, as mentioned.Tribological aspects of fiber-reinforced hybrid composite dry friction clutch facings were examined in a novel way: after different, mainly non-laboratory automotive tests, that were characterized by different surface activation energy values. Tests were carried out according to automotive industry best practice, then transmission systems were disassembled so friction facings were able to be examined regarding their thickness loss and surface roughness to characterize tribologically induced changes of the friction surface. Thickness loss\u2013wear and changes of microgeometry characteristics\u2013and surface roughness values Ra, Rz and Rmax from two different diameters (inner and working) were compared with the aim of highlighting the main governing parameters influencing these values during clutch facing lifetime.Wear values showed double increasing trends based on test types: two groups in the analyzed tests could be sorted into: \u2018clutch killer\u2019 and \u2018moderate\u2019 applications. The former consisted of tests during which a trailer was attached to the vehicle or there were hill starts on purpose , or a similar load case was simulated on a test bench (TH). Starting the vehicle upwards on a hill and/or with a trailer led to high energy loads due to longer slippage times, being the root cause of so-called tapering that creates considerable wear\u2013above 0.1 mm\u2013also at inner diameters. The other group was formed by tests in which professional drivers were operating the clutch, mainly on test tracks, or a customer drove the car for more than a year with high mileage among mainly constant conditions.\u2018Clutch killer\u2019 inner diameter wear results followed an increasing trend along the Joule scale. The relative material loss was influenced by the mileage and test conditions: 1% higher wear was generated by 90% higher mileage; another 1% increment could be caused by insufficient cooling time or test bench-specific conditions.Increased inner diameter Ra results paired with low or zero thickness loss suggested torn-up surfaces due to rapid, low mileage applications of the clutch. The highest relative roughness decrease was caused by the tests resulting in one of the highest relative thickness losses. The same trend describes \u2018clutch killer\u2019 inner diameter Rz values in the radial direction. Rmax values in the radial direction at the inner diameter differed from only those of lower energy or lowest mileage tests: roughness increased by 20\u201360%, highlighting surface tear. Overall, tests run with a trailer and in city conditions resulted in more than 2% thickness loss and a 40\u201350% roughness decrease.Tangential direction Ra values at inner diameter followed more closely the trends seen from wear results with more intensive roughness decrease paired with higher thickness loss, with only the lowest energy and mileage tests resulting in increasing roughness. Changes in Rz values followed the same trend. Rmax results highlight the low energy roughness increase as well, but differed from other values of the 15,000 km VTC results.\u2018Moderate\u2019 inner diameter wear results followed a less steep increasing trend compared to \u2018clutch killer\u2019 values along the Joule scale. The highest energy and mileage application led to the highest thickness loss: more than 0.3 mm after 140,000 km and more than 3.7 GJ.Inner diameter radial direction Ra and Rz values decreased less and less along the non-proportional Joule scale with Rmax values resulting in a roughness increase in the highest energy test in this group.In the tangential direction, changes of the three roughness values followed the same trend, but lacking wear, no correlation can be drawn. The Ra trend showed less moderate changes.Comparing \u2018clutch killer\u2019 and \u2018moderate\u2019 roughness result trends at the inner diameter, by the former, tangential results followed more closely wear result trends, while for the latter, radial value trends behaved following an inverse trend regarding percentage change.\u2018Clutch killer\u2019 working diameter wear results followed an increasing trend along the Joule scale. Mileage or test-specific conditions did not have such influence that it was present among inner diameter results.Working diameter \u2018clutch killer\u2019 Ra results in the radial direction showed an increase in lower energy tests. Rz and Rmax results showed sensitivity to facing size: small (S) facings produced significantly decreased roughness, while large (L) and medium (M) size facings had increased roughness values. Rmax results showed the highest deviations among roughness values in the radial direction.In the tangential direction, none of the roughness value change trends followed wear behavior, but Ra and Rz showed sensitivity to test conditions, resulting in roughness increase after the TH test.\u2018Moderate\u2019 working diameter wear results followed a less steep increasing trend compared to \u2018clutch killer\u2019 values along the Joule scale, just like those of the inner diameter.Radial direction working diameter roughness value trends were very similar to inner diameter trends with Rmax correlating better with the other two roughness results along the energy scale.The three tangential direction working diameter roughness trends were the same, with the small size facings producing the highest decrease in Ra and Rz roughness values as they had higher thickness loss compared to medium size facings with similar surface activation energy.Comparing the two different trends from the roughness point of view, working diameter value trends did not show the difference between tangential and radial behavior that was present among inner diameter results.Different activation energy-induced changes in surface microgeometry of automotive dry clutch facings are heavily influenced by operating parameters such as mileage and driving style and also affected by facing size. The hypothesis that more activation energy results in an increased amount of wear and smoother surfaces is only right if those conditions and deviation in behavior between different diameters is considered.Future investigations will further examine tribological behavior and its effects during the component\u2019s lifetime via pin-on-disc tests. The final aim is to extend the prediction capability of currently utilized contact models coupled with the thermomechanical and tribomechanical analyses considered."} +{"text": "Adolescent and young adult (AYA) enrollment in cancer clinical trials (CCT) is suboptimal. Few studies have explored site level barriers and facilitators to AYA enrollment on CCTs and the efficacy of interventions to enhance enrollment. A cross sectional survey was developed by the COG AYA Oncology Discipline Committee Responsible Investigator (RI) Network to identify perceived barriers and facilitators to enrollment, as well as opportunities to improve enrollment. Associations of barriers and facilitators to enrollment with program demographics were assessed. The survey was sent to all AYA RI Network members (n\u2009=\u2009143) and quantitative and thematic analyses were conducted. The overall response rate was 42% (n\u2009=\u200960/143). Participants represented diverse institutions based on size, presence or absence of dedicated AYA programs, and proximity and relationship between pediatric and medical oncology practices within the institution. The most frequently cited barriers to enrolling AYAs in CCTs were administrative logistical issues (45%), disparate enrollment practices (42%) and communication issues (27%) between pediatric and medical oncology and perceived limited trial availability (27%). The most frequently reported facilitators to enrollment included having strong communication between pediatric and medical oncology (48%), having a supportive research infrastructure (35%) and the presence of AYA champions (33%). Many barriers and facilitators were similar across institutions and AYA program types. Shared barriers and facilitators to AYA CCT enrollment exist across the landscape of cancer care settings. Interventions aimed at increasing coordination between pediatric and medical oncology clinical trials offices and providers have high potential to improve site-level AYA enrollment. Despite a growing focus on addressing disparities in AYA cancer care and outcomes, few studies have assessed factors contributing to the low enrollment of AYAs into CCTs. Even fewer studies have assessed the efficacy of interventions to improve enrollment. AYA cancer biology, tolerance to intensive treatment and survival outcomes for specific malignancies differ in comparison with older adults and younger children, strongly supporting the need to identify optimal treatment and supportive care approaches in the AYA population4.Cancer clinical trials (CCTs) are vital for studying disease biology and improving survival and health-related quality-of-life outcomes; however, only 2\u20135% of all AYAs with cancer enroll in a CCT7. These include global issues such as the perception of limited availability of relevant CCTs, regional issues such as lack of referral of AYA patients to centers with National Cancer Institute (NCI)-CCTs and institutional-level issues such as not activating CCTs due to the regulatory burden and cost of study activation and conduct. Additional suggested barriers at the site level include lack of eligibility screening procedures, limited communication between medical and pediatric oncologists, limited knowledge and comfort with other NCI Clinical Trials Network (NCTN)-CCTs, as well as time and economic constraints to open and enroll AYAs on CCTs. In addition psychosocial barriers such as stress/distress, developmental and emotional maturity, feeling ill, experimentation play a role as well12. While many publications have proposed potential barriers to enrollment, data reporting actual barriers are sparse12.The reasons for limited AYA CCT enrollment, even amongst the ones eligible for trials while not well understood, have been hypothesized in recent reviewsIn 2018, the Children\u2019s Oncology Group (COG) AYA Oncology Discipline Committee developed an international network of AYA Responsible Investigators (RIs) consisting of\u2009>\u2009140 individuals from demographically and geographically diverse sites that serve a variety of distinct roles at their respective institutions such as physicians, nurse practitioners, nurse navigators and research staff. Institutions included free-standing children's hospitals, sites with pediatric and medical oncology on shared or separate campuses, and sites located in community and urban settings.13. The primary mechanism through which the AYA RI Network supports enrollment is providing education and peer support to institutional AYA RIs. To achieve its goal, the AYA RI Network hosted a series of informal webinars allowing RIs to share barriers and facilitators to AYA enrollment, as well as unique challenges to AYA enrollment based on site-specific factors. The purpose of this survey was to systematically identify shared barriers and facilitators to AYA accrual in CCTs as reported by members of the COG AYA RI Network and assess associations with program demographics to inform institutional and national interventions aimed at increasing AYA CCT enrollment.The primary purpose of the AYA RIs is to optimize AYA enrollment onto COG-led trials, and other NCTN trials in which COG is participating, at their sites. AYA RI responsibilities are focused on implementing steps to facilitate clinical trial enrollment of AYAs treated within their institution as previously describedA cross sectional survey was developed by the COG AYA Oncology Discipline Committee RI Network leadership to identify perceived barriers and facilitators to enrollment, as well as opportunities to improve enrollment. The survey was piloted with five AYA oncology stakeholders and revised as needed for content, readability and clarity. The survey consisted of two parts: the first comprised twelve questions on demographic information, including program characteristics, institutional structure and the relationship between pediatric and medical oncologists; the second comprised four free text questions on participants\u2019 perspective on: 1) institutional barriers to enrollment; (2) facilitators to enrollment; and (3) possible solutions to improve AYA accrual to CCTs at the institutional level; and (4) cooperative group level. Of note, participants could have their responses placed into multiple categories and multiple answers were permitted with a brief description of the study and an embedded, clickable link to the survey. One RI from each institution in the RI Network was sent the survey. In case of non-response, the survey was redistributed two additional times within 4\u00a0weeks from initial survey distribution (December 2019\u2013January 2020). All methods were carried out in accordance with relevant guidelines and regulations. The study was approved as exempt by the Prisma Health Upstate IRB. Participation in the survey was voluntary and signed informed consent was not obtained for individual participants as it would be the only link between the participant responses and their identity.Demographic information was summarized for all respondents. Response frequencies and proportion of total responses were calculated for all categorical variables. Means and medians were calculated for all continuous variables. Responses to the free text questions were reviewed and themes for each of the four free text questions were identified by two study investigators . The themes were reviewed and consensus was achieved among three study investigators . For each question, responses were subsequently independently categorized into the previously agreed upon themes . When there was disagreement in the categorization of responses, agreement was sought between the two raters ; if consensus was not reached, a third, independent investigator (MR) categorized the response. Fisher exact test and Chi Square analysis were used to assess the association between demographic variables and perceived barriers and facilitators to enrollment. Two-sided p value\u2009<\u20090.05 was considered statistically significant.The overall survey response rate was 42% n\u2009=\u200960/143) and 97% of these respondents (n\u2009=\u200958) completed the entire survey. The participants represented a diverse group of institutions based on size, presence of an AYA Program and geographic proximity between pediatric and medical oncology or one in development (n\u2009=\u200914) reported that pediatric and medical oncology providers work in the same building, 48% (n\u2009=\u200928) were located on the same campus and 19% (n\u2009=\u200911) were located on separate campuses. Three quarters of respondents (n\u2009=\u200943) stated they had a single IRB at their institution to approve both pediatric and medical oncology trials. Forty-three percent (n\u2009=\u200925) of respondents stated they had formal joint tumor boards between pediatric and medical oncology for all or some oncologic diagnoses. Almost all participants reported having ad hoc discussions with their medical oncology colleagues about AYA patients. When asked how often their medical oncology colleagues enrolled their patients onto COG trials, 45% (n\u2009=\u200926) responded that this occurred \u2018frequently/occasionally,\u2019 while 41% (n\u2009=\u200924) responded that this \u2018rarely/never\u2019 occurred.Participants identified several perceived barriers to enrolling AYAs at their local sites with 102 total responses provided by 60 participants were associated with specific perceived barriers and facilitators to enrollment , enhancing medical and pediatric oncology engagement 20%), disseminating information on trials (17%), fostering the development of AYA CCTs (15%), fostering process changes at the NCTN/NCI (10%) and clarifying site enrollment procedures (7%) may be underlying reasons for poor cross-enrollment by medical oncologists, as documented in previous studies. Importantly, lack of available AYA CCTs is only partially responsible for lower cross-enrollment as some medical oncology programs with similar availability of AYA CCTs also had similar suboptimal accrual rates9. Large national efforts, including the reorganization of NCTN which, by consolidating the network groups and supporting closer collaboration, fostered cross network enrollment, have been undertaken and are in process to address this barrier. In our survey, availability of AYA CCTs was often noted to be dependent on the primary oncology service, pediatric or medical oncology. The referral patterns, insurance contracts, administrative policies implementing existing age cut offs for obtaining cancer care largely determine where AYAs get their care and impact their treatment. AYA trials may be more likely to be opened by pediatric oncology sites compared with medical oncology sites20 and adolescents treated by adult medical oncologists are less likely to be enrolled in clinical trials16. The hospital treatment setting, community vs academic, also plays a major role. Over 90% of cancer patients under the age of 15 are treated at a tertiary care center versus less than 20% of 15\u201340-year-old cancer patients22. Community oncology practices, where the majority of AYAs are treated, often do not have access to AYA CCTs. Furthermore, there is often limited communication between the pediatric and medical oncologists in the community cancer care setting, and thus, there is often limited knowledge of locally available CCTs21. In addition, the 18-year-old lower age limit of eligibility continues to limit younger AYA participation in CCTs evaluating novel targeted therapies, including immunotherapy trials, and this needs to be further addressed at the national level22.Perceived lack of availability of AYA CCTs is a shared barrier and has been suggested by multiple prior studies23. Tumor boards represent an opportunity to review open trials, identify eligible patients and identify optimal treatment approaches for patients with rare cancers and/or complicated presentations. Regular attendance and visibility of AYA team members at shared multidisciplinary tumor boards (MTBs) is crucial to establishing referral networks, enabling ongoing screening of eligible patients and facilitating enrollment24. One example is the EORTC-SPECTA, a virtually conducted molecular profiling MTB specifically focused on recruiting AYA patients with newly diagnosed relapsed high-grade gliomas and high-grade bone and soft tissue sarcomas. A virtual central pathology review with a clinically-validated molecular profiling report is provided to referring clinicians to improve access to novel drugs for AYAs25. Similarly, virtual MTBs may help knowledge sharing across disciplines and improve collaboration between providers and geographically-limited centers as evidenced during the ongoing pandemic26.The current survey further identified potential areas for high-yield interventions to enhance enrollment. Based on this information, key targets for local intervention could include: (1) improving communication between pediatric and medical oncology; (2) employing AYA-specific personnel, such as a patient navigator; and (3) implementing an AYA CCT screening process. Robust communication between pediatric and medical oncology services was perceived as a strong facilitator to CCT enrollment. Increased interaction via tumor boards have been effective in fostering communication between different services and disciplines29. However, the financial implications of developing an AYA program and allocating additional resources to clinical research are barriers. Philanthropic and institutional financial and non-financial support is often needed to launch such initiatives.AYA programs also foster communication between pediatric and medical oncology. While mostly limited to single institution reports, AYA programs appear to be associated with improved CCT participation due to dedicated staff connecting AYA patients with CCTs30. In this role, navigators can help bridge the knowledge gap of available AYA CCTs across pediatric and medical oncology departments, as is being studied by the AYA Program in Utah31.Patient navigators and supportive care professionals are well-poised to identify and address needs, values and communication styles of AYA cancer patients and survivors can serve as a conduit for identifying eligible patients for CCTs and relieve some responsibility from the primary medical team. One paramount role of the patient navigator is to serve as a first point of contact for the patient\u2019s care team, with the ability to collaborate with other departmentsThe development of screening procedures to capture eligible AYAs can also enhance AYA CCT enrollment. Implementation of a standard operating procedure for screening at a cancer treatment site improved access and referral to NCTN AYA CCTs presenting to different oncology providers at an academic site. Additional studies are needed to identify optimal screening procedures.A framework for interventions based on the information obtained from the survey has been presented in Table 10. Through a series of webinars, these groups have sought to increase interaction amongst AYA oncology providers to disseminate information on available trials and provide guidance on overcoming local barriers to AYA CCT enrollment. Expanding pediatric and medical oncology provider participation in these webinars might be an opportunity to further increase the reach of these educational endeavors.While increasing the availability of studies is an important step to increasing enrollment, provider variability in practice and awareness may be more challenging to address at a national level given the diversity of clinical practices and patient populations. However, addressing provider hesitancy and lack of collaboration is critically important. Our survey suggests that education on AYA CCT enrollment processes and trial availability may help to overcome some of the provider hesitancy in offering CCTs to AYAs. The AYA RI Network and NCORP have developed efforts to directly address this gap in knowledgeThis study has a few limitations. The RI Network consists of individuals who have an interest in addressing disparities in AYA enrollment and are likely more aware about AYA trials and enrollment processes than their colleagues. Thus participant responses may not be fully representative of other stakeholders, particularly those at sites that are not participating in the AYA RI Network. Further, the RI Network consists mostly of pediatric oncologists, and while the current survey did not assess participants\u2019 role, it is likely that the survey captured a limited number of perspectives from medical oncology stakeholders. It will be extremely important in future studies to obtain a broader perspective from medical oncologists working in varied practice settings on the barriers, enablers, and the feasibility of the proposed strategies to improve accrual. However, stakeholders representing the supportive disciplines and regulatory office were surveyed to capture different perspectives. In addition, the survey focused on medical oncologists cross-enrolling on COG trials and did not include perspectives on pediatric oncologists or medical oncologists enrolling AYA patients on other network group trials. Further studies evaluating medical oncology stakeholder perspectives are needed.To our knowledge, the extent to which barriers and facilitators to AYA CCT enrollment are shared among institutions has not been previously reported. The shared barriers and facilitators identified in this study provide prime targets for potential intervention to improve enrollment. Studies addressing, and not just describing, the dismal enrollment of AYA CCT enrollment are urgently needed and our survey highlights starting points to begin to optimize the process.Supplementary Information 1.Supplementary Information 2."} +{"text": "For older workers, having a retirement plan is important for a successful transition. Social awareness of the problems encountered by older women during retirement remains low. Women have limited retirement resources due to their unequal work experience, and older women with access to fewer retirement resources often postpone their retirement. This research examined how the timing of older women\u2019s retirement was influenced by their retirement resources as well as their marital status. The study used 2014 HRS and RAND data, and collected sample of women aged 50-62 years old who worked either full or part time . Respondents were female (56%), white (63%), married (70%), and working full time (82%). Guided by the theory of planned behavior (TPB), multiple regression analysis examined gender differences in predicting older adults' retirement timing. TPB included three sub factors: attitudes toward retirement, subjective norm, and perceived behavioral control. Logistic regression analyzed the effects of respondents\u2019 expectations of retirement . The findings indicated that the TPB model works similarly for men and women but there is a difference according to marital status. Unmarried women are less likely to have accumulated financial resources and more likely to anticipate a later retirement (1.4 years) than married women and are also less likely to set an expected timing for retirement (p<05). Such a robust research agenda would provide key information for government agencies and policymakers and contribute to the development of retirement planning models or retirement education programs for older women."} +{"text": "Alcohol use is typically associated with impaired cognitive functioning on tasks related to attention and concentration. However, it remains unclear whether these impairments persist across days in ways that are noticeable to the individual. We examined this using the daily diary project of the Midlife in the United States Refresher cohort. Participants completed 8 nights of telephone-based diaries (Mdiaries=6.87) that included questions about daily alcohol use (\u201chow many drinks did you have today?\u201d) and five items assessing concentration rated on a scale . Using autoregressive multilevel models, we examined how same and previous day alcohol use related to perceived difficulties with concentration. Greater total alcohol use over the diary period was related to reports of concentration problems though current day and previous day alcohol use were not. The association between previous day use and concentration problems was qualified by an interaction with total alcohol use . Individuals who drank less alcohol in general, experienced greater perceived concentration problems following the days on which they did drink relative to those who drank more alcohol across the diary period . This relationship did not vary based on age, sex, or education. These results suggest that daily alcohol use could impair concentration across days, particularly for those adults who tend to consume less alcohol."} +{"text": "Aggressive myectomy through transapical approach not only relieves basal and midventricular obstructions but also may augment diastolic dysfunction in hypertrophic cardiomyopathy.See Article page 71.The video clip by Sun and colleagues,First, how do we determine whether symptoms are secondary to LVOT obstruction or diastolic dysfunction? In reality, many patients suffer from a variable combination of both pathologies, and failure to recognize the latter may result in residual symptoms, even though transaortic septal myectomy is an excellent septal reduction therapy for relief of LVOT obstruction,Second, does an aggressive myectomy relieve diastolic dysfunction? There is a clear relationship between diastolic dysfunction and heart failure in nonobstructive HCM, and this should be evaluated with echocardiography and invasive hemodynamic catheterization to identify this larger group of patients with HCM. The Mayo group has championed a transapical approach. Their combined transaortic and transapical approach has been shown to abolish any cavitary obstruction and augment left ventricular diastolic function with good short-term outcomes.This 24-year-old male patient might have received a heart transplantation had he been treated elsewhere. While our own institutional data of 41 patients with end-stage HCM managed with heart transplantation suggest it is a good option,"} +{"text": "Needle phobia is a common issue amongst children, particularly with long-term illnesses requiring repeated interventions. Psychological interventions such as coping strategies and distraction techniques can be effective in many of these children, but is not universally successful. Recent research has reportedthat the use of virtual reality (VR) headsets as a distraction technique can be effective at alleviating both pain and fear in children receiving intravenous (IV) injections, cannulation and venepuncture. Although use of VR distraction is becoming more widespread in some disciplines (such as paediatric oncology/A&E), it is yet to be widely adopted in paediatric rheumatology.We report the case of a 9-year-old boy (ME) who in early 2019 developed psoriatic juvenile idiopathic arthritis (JIA) and an associated severe, widespread plaque psoriasis causing him substantial discomfort and distress. He has been treated with subcutaneous adalimumab biosimilar 40\u2009mg fortnightly and oral methotrexate 20\u2009mg weekly, alongside Keppra 1000\u2009mg twice daily for epilepsy. Although he is just tolerating the subcutaneous adalimumab, he is requiring intermittent steroid joint injections under general anaesthetic for recurrent flares of his polyarticular arthritis and regular monitoring phlebotomy. He has been developing progressive needle phobia over the past 8 months, with superimposed anxiety about general anaesthetics with particular fear of the anaesthetic provoking an epileptic seizure.Despite psychological intervention to teach coping strategies and standard play therapy distraction techniques, both phlebotomy and cannulation for general anaesthetics have become more distressing and challenging for him. During two recent day case admissions for joint injections, despite premedication with sedatives, distraction techniques and psychology interventions, he became so distressed that attempts to anaesthetise him had to be abandoned. This has resulted in prolonged periods of continuing joint inflammation and pain. Discussion with local paediatric oncology colleagues resulted in a suggestion of using recently acquired VR headsets as an effective distraction technique. He has had a previous positive experience of using VR headsets in a play environment. He is receiving VR training intervention from the oncology team and is now fully engaged with the idea of using VR to distract him from negatively focussing on his next planned general anaesthetic intervention.Unmanaged needle-related procedural pain in children is associated with increasing pain and stress in subsequent procedures and fear/avoidance of medical care which can affect compliance in chronic disease management. The psychological distress for children with needle phobia can add to existing psychological impact from JIA and increase family stress of caring for an ill child. The cost of poor management of needle-phobic children can be considerable, with cancelled or increased time for procedural interventions and restricted therapeutic interventions. Needle phobias in childhood may persist into adulthood.Employing distraction techniques when children undergo medical procedures can help them reduce anxiety. VR is a method of captivating an individual\u2019s interest in a virtual environment and can help divert attention from potentially distressing situations in the real world. Recent studies have reported VR as an effective distraction technique in children. Chan (2019) reports that VR significantly decreased the pain and post procedural anxiety experienced by children aged 4\u201411 years old during venepuncture/IV cannulation compared to controls. The majority of children in the VR group expressed interest in utilising VR again in the future, to improve their experience of IV injections. Chen (2020) subsequently reported a randomised controlled trial which significantly reduced pain and fear experienced from IV injections by the children in the VR group.There are cost implications to both buying VR headsets/software and training play specialists/specialist nurses to use the equipment effectively. Furthermore, the acceptability of using VR with regards to the child\u2019s age, engagement with age-appropriate software packages and whether a passive VR or a more interactive \u2018gaming\u2019 experience is preferred should be considered. The use of VR is not a panacea, but is potentially effective as an additional new tool in clinicians\u2019 efforts to reduce the impact of needle phobia.Addressing needle phobia in children can require novel strategies.VR has shown success as a distraction technique to alleviate pain and fear in children undergoing needle related procedures.The implementation of VR in paediatric rheumatology has cost and staff training implications.The use of age-appropriate software and an individual child\u2019s preference for passive/active interaction with VR should be considered."} +{"text": "Caregiving activities often lead to positive and negative appraisal for caregivers. Caregivers may limit social participation due to caregiving activities. Changes in level of activity participation could have profound consequences for caregiver\u2019s valence. However, little is known about how activity participation could moderate the association between these caregiving appraisals and emotional valence. Data came from the National Study of Caregiving (Round 1 and 2), a nationally representative study of caregivers. Referencing Lawton\u2019s two-factor model (1990), we examined both the level and changes in activity restriction interacting with positive and negative caregiving appraisals to predict both valence across two waves. Consistent with two factor models, findings revealed level and changes in activity restriction moderated the relationship between caregiving appraisal and outcomes for both valences. These findings highlight the role of activity restriction as a target to reduce negative valence and improve positive valence for caregivers."} +{"text": "While antimicrobial resistance (AMR) continues to be a major public health problem in Pakistan, data regarding trends of resistance among pathogenic bacteria remain scarce, with few studies presenting long-term trends in AMR. This study was therefore designed to analyse long-term AMR trends at a national level in Pakistan.We report here results of a comprehensive analysis of resistance among pathogens isolated from blood and CSF, between 2011 and 2015. Susceptibility data were obtained from a local laboratory with collection points all across Pakistan (Chughtai Laboratory). Resistance proportions to most commonly used antimicrobials were calculated for each pathogen over a period of 5 years.Acinetobacter species demonstrated highest resistance rates to all tested antimicrobials, a sharp increase in carbapenem resistance was the most noticeable (50%\u201395%) between 2011 and 2015. Our results also highlight the presence of third and fourth generation cephalosporins resistance in Salmonella enterica serovar Typhi in Pakistan. Interestingly, where a rise in AMR was being observed in some major invasive pathogens, decreasing resistance trends were observed in Staphylococcus aureus to commonly used antimicrobials.While Overall pathogens isolated from blood and CSF between 2011 and 2015 showed an increase in resistance towards commonly used antimicrobials."} +{"text": "PatienUsing an ultrasound-guided vascular access technique, both left and right brachial veins were accessed and conventional 8F short sheath was positioned .The diagnostic catheter was positioned under fluoroscopy guidance from the left brachial access in both cases; a decapolar steerable catheter in the proximal coronary sinus in patient 1 and a nonsteerable octapolar catheter in patient 2 . In bothAccess to the right atrium and subsequent mapping of the arrhythmia substrate was successful in both patients and depicted a reentrant tachycardia around an atretic tricuspid annulus in patient 1. A linear lesion was deployed from the absent tricuspid annulus to the inferior caval vein, which terminated the atrial tachycardia and rendered the patient noninducible.In patient 2, a left lateral bidirectionally conducting accessory pathway was located in left lateral position. The magnetic catheter was subsequently advanced via the peripheral arterial access and retrogradely advanced into the left ventricle and also the left atrium has potential cost-saving implications.Our technique offers an alternative option to enable electrophysiologic procedures to be performed from exclusively peripheral access sites. This could potentially transform electrophysiological procedures in a similar way as interventional procedures, which moved from femoral to radial access in recent years.\u2022Catheter ablation can be safely and effectively be carried out via vascular access from the arms.\u2022This novel peripheral access route is an alternative in patients with blocked or absent inferior access.\u2022This technique requires remote magnetic navigation to allow versatile navigation of the ablation catheter.\u2022To avoid injury at the access sites vascular access should be performed using ultrasound guidance.Two patients with blocked femoral vascular access underwent successful catheter ablation using remote magnetic navigation via vascular access from both arms. This technique excludes the risk for pneumothorax, allows immediate mobilization, and was successfully used for both right- and left-sided arrhythmias."} +{"text": "This Yale Aortic Institute lecture provides \u201ctips and pitfalls\u201d regarding echocardiographic assessment of the aorta. This lecture guides By convention, the ascending aorta is measured by echo from leading edge to leading edge. \u201cLeading edge\u201d connotes the edge of the aortic wall that is the closest to the probe (at the top of the inverted \u201cV\u201d of the ultrasound image).By transthoracic echo (TTE), the leading edges are the outer anterior wall and inner posterior wall. By transesophageal echo (TEE), the leading edges are the outer posterior wall and inner anterior wall.Aortic measurements should be taken (by convention) in diastole (when the aorta is moving least).Simple TTE is 70 to 85% sensitive in diagnosing ascending aortic dissection. TEE sensitivity approaches 100%, though the tracheal carina imposes a blind spot on TEE, impeding visualization of distal ascending aorta and proximal aortic arch.While computed tomography angiography may be superior for defining full anatomic extent of aortic dissection, echocardiography is superior in assessing functional consequences such as mechanism and severity of aortic regurgitation, evidence of myocardial ischemia when complicated by coronary dissection, or evidence of tamponade physiology when pericardial effusion is present.Reverberation artifact can mimic a dissection flap. A true flap moves independently of the outer aortic wall which can be confirmed by one-dimensional motion mode. Color flow respects a true flap but does not respect a reverberation artifact.Assessment can be done for bicuspid aortic valve morphology in systole, not diastole. In diastole, when the valve is closed, the raph\u00e9 can make a bicuspid valve appear as trileaflet. Doming in the parasternal long axis (PLAX) view and an eccentric closure line on PLAX M-mode should also raise suspicion for bicuspid aortic valve.Video 1The Yale Aortic Institute lecture by Dr. Sarah Hull on the \u201ctips and pitfalls\u201d of echocardiographic assessment of the aorta."} +{"text": "The woody vegetation, in particular trees, is facing difficult times in many regions across the globe. Unprecedented rapid increases in average temperatures along with increasingly frequent periods of extreme drought and heat outrun the acclimatory capacities of many tree species and other woody plants. Observations of increased tree mortality and severe forest decline are accumulating in all forested biomes and quesMore than a decade ago, the seminal paper by PL) relatively constant during changing environmental conditions (soil and atmospheric drought), more anisohydric species let PL covary congruently with environmental fluctuations (Pst) is reached (Ppd) thus corresponds to the soil water potential in the soil horizon where water is taken up from storage, linking leaf-level responses to whole-plant regulation. The main finding is that there is a trade-off between stem capacitance and stem NSC storage, where species that make greater use of stem water storage have a smaller NSC storage pool. Less isohydric water potential regulation is associated with greater NSC storage depletion during the dry season, most likely as a consequence of osmotic adjustment. More isohydric species, by contrast, are characterized by greater stem water storage use. Surprisingly, and contrary to what the hydraulic framework would predict, more isohydric species showed an accumulation of NSC during the dry season, apparently because the use of stem water allowed earlier leaf flushing and higher photosynthetic rates. As such, the contribution by Jiang et\u00a0al. provides an additional dimension to research on water regulation and drought responses and suggests a mechanistic explanation for why NSC storage pools often\u2014but not always\u2014decline during drought . These cPst is reached (Ptlp). The maintenance of water loss via declining PL is facilitated by this biochemical mechanism that increases the concentration of osmotically active substances to lower the osmotic potential for preventing turgor loss in living plant cells versus pre-dawn (Ppd) leaf water potential (blue triangular area in Pmd is below their Ppd over a wider range of water potentials, which results in a larger HSA. A low slope \u03b2 in this relationship (Pmd has to approach Ppd after complete stomatal closure (Pst driven by osmotic adjustment may result in certain degree of plasticity in this trait (Pst indicates that it may be useful as a complement to hydraulic safety margins based on stomatal response (cf., Despite ongoing controversies about the usefulness of the iso/anisohydry concept , it appetionship , for exationship . Notably closure , which tis trait . Furthernse cf., , as theyPtlp in response to seasonally declining PL. Future research should assess the role of starch conversion in response to abscisic acid signals (The study by Jiang et\u00a0al. highlights the importance of a holistic whole-tree approach that acknowledges the interdependency of the water and carbon balance of trees. As identified by signals as a regAnswering such questions would also feed back into the hydraulic framework of"} +{"text": "Comorbid conditions can complicate healthcare access, yet little is known about the impact of cognition on accessing those services. This study aims to examine the effect of gender on health service utilization among the aging population. Data from the National Health Interview (2019) Survey were utilized to assess the impact of gender on the use of physician and dental services among older adults managing cognitive impairments. We estimated logistic regression models controlling for demographic, health/physical functioning, and psychological predictors. Findings indicated older male respondents managing cognitive impairments were less inclined to utilize both physician (p<.05) and dental (p<.01) health services on a yearly basis as compared to their female counterparts. Household income and health insurance coverage respectively were the strongest structural factors limiting access to both physician and dental services, with lower levels of educational attainment being an additional barrier for dental service utilization. Respondents with low self-rated health were inclined to utilize physician more than dental services . It is imperative geriatric practitioners adopt a more integrative approach to health, especially among the aging population managing chronic disabilities, where additional community supports are warranted to promote and maintain physical and oral health."} +{"text": "Although physical activity throughout life is one of the most reliable predictors of healthy aging, can less consistent or favorable trajectories also improve cognition trajectories among older adults? Drawing from accumulation theories, we use longitudinal data from the Health and Retirement Study and Life History Mail Survey to examine the early antecedents of cognitive decline and the extent to which different life course physical activity profiles can slow such a decline. Results from latent class analysis reveal seven distinct profiles: consistently low, consistently high, consistently average (reference), improvers, decliners, midlife motivators, and previously athletic \u201ccouch potatoes.\u201d Growth curve modeling analyses show that membership in the consistently high class and midlife motivators were associated with better cognition initially and over time, with no difference between the two classes. Additionally, though poor health and learning problems in childhood were associated with worse initial cognition, physical activity does not mediate the relationship."} +{"text": "Engaging with Aging is an emerging framework proposed by Carnevali which provides a new lens to understand an active, conscious daily living process of coping with age-related changes (ARCs) taken on by older adults. Study aims were to 1) describe the ARCs experienced by community-dwelling older adults; 2) identify the strategies and resources used by older adults to accommodate the daily living challenges caused by the associated ARCs; and 3) evaluate the framework of EWA based on findings from aims 1 and 2. We conducted semi-structured interviews with 29 participants aged 64 to 98 online due to COVID-19 restrictions. We used a virtual card sort to assist data gathering. Fifteen ARCs were mentioned by participants and their corresponding adaptations were discussed. We found that older adults linked their adaptations to their ARCs based on their changing capacities and needs. Commonly used adaptations included conserving energy, utilizing tools or technology, and being more conscious before and while taking actions. The challenges caused by COVID-19 in implementing the adaptations were also discussed . Our study substantiates the EWA framework by showing the commonality among older adults in linking ARCs with adaptations. Implications for clinicians and researchers include using EWA to help older adults identify personalized solutions that fit their capacities. Our study is late-breaking as we recently finished data analysis and the information included was not yet available by the previous submission deadline."} +{"text": "Thunnus orientalis) during seasonal foraging migrations in the California Current. We tracked 242 tuna across 15 years (2002\u20132016) with high-resolution archival tags, estimating their daily energy intake via abdominal warming associated with digestion (the \u2018heat increment of feeding\u2019). The poleward extent of foraging migrations was flexible in response to climate variability, allowing tuna to track poleward displacements of thermal habitat where their standard metabolic rates were minimized. During a marine heatwave that saw temperature anomalies of up to +2.5\u00b0C in the California Current, spatially explicit energy intake by tuna was approximately 15% lower than average. However, by shifting their mean seasonal migration approximately 900 km poleward, tuna remained in waters within their optimal temperature range and increased their energy intake. Our findings illustrate how tradeoffs between physiology and prey availability structure migration in a highly mobile vertebrate, and suggest that flexible migration strategies can buffer animals against energetic costs associated with climate variability and change.Animal migrations track predictable seasonal patterns of resource availability and suitable thermal habitat. As climate change alters this \u2018energy landscape\u2019, some migratory species may struggle to adapt. We examined how climate variability influences movements, thermal habitat selection and energy intake by juvenile Pacific bluefin tuna ( In respWhile there are benefits to optimizing foraging migrations to exploit known productive areas, there can be costs to strategies that rely on the predictability of resources . Ocean sMobile marine vertebrates must strike a balance between the need to find prey, and the need to minimize energetic costs associated with locomotion, foraging and thermoregulation. Migrations can therefore reflect a need to remain within an ambient temperature range that minimizes energy expenditure and maximizes physiological performance. This could constrain the ability of animals to migrate to areas of high prey availability that fall outside a narrow thermal range ,21. EnviThunnus orientalis) are large pelagic fish that can reach sizes of up to 3 m and 450 kg at maturity. This species is highly mobile and can undertake multiple trans-oceanic migrations during their lifetimes between breeding grounds off the coast of Japan, and productive feeding grounds in the California Current system )We found that Pacific bluefin tuna foraging migrations were highly responsive to anomalous climate conditions. Deviations from the average migration path of approximately \u221260 and +100 km occurred when conditions across the California Current were anomalously warm or cold respectively, and one tuna foraged up to 1500 km further north than average in response to warm water anomalies during the 2015 marine heatwave. This outpaces even the extreme displacement of thermal habitat observed during this time . This mic), indicating an active behavioural tradeoff between energetic costs and benefits conferred by foraging in cooler, more productive waters. This parallels observations of fine-scale behavioural strategies by albacore tuna, where individuals stay on the warm side of a front to maintain an elevated body temperature, but make regular forays into the cold side, to maximize prey capture and thus energy intake [b). Studies have suggested that compressed pockets of cool, high-quality foraging habitat remained available in parts of the California Current during the heatwave, aggregating prey and providing increased foraging opportunities for mobile species [b and b).Although thermal habitat selection appears to be a principal driver of migration in bluefin tuna, we showed that tuna did not migrate flexibly with the sole purpose of remaining within their metabolic minimum zone. Tuna spent almost twice as many periods in \u2018cool\u2019 waters below this zone, than in \u2018warm\u2019 waters above this zone c, indicay intake . Unexpec species . Our fina), in line with observations of reduced primary productivity, and low forage fish and krill abundance during this period [2c, d and 4b). Declines in local prey availability or quality, coupled with a lack of migratory flexibility, appear to explain why some top predators including sea lions, grey whales and some seabirds experienced starvation-induced mass mortalities in the California Current in the years immediately following this event [The negative spatial energy intake anomalies that Pacific bluefin tuna experienced in 2015 suggest that the quality of the foraging landscape was generally reduced in the California Current during the heatwave a, in lins period . Tuna cois event \u201354. FutuPleuroncodes planipes [Comprehensive diet analyses on Pacific bluefin tuna throughout the California Current would also help to resolve some of the uncertainty inherent in using estimates of energy intake based on the HIF, which can be sensitive to factors including the type of prey being digested ,35. For planipes ), with tplanipes . While tOur findings that juvenile Pacific bluefin tuna migrate flexibly in response to climate variability and maintain a high energy intake under anomalously warm ocean conditions have important implications for managing this population. Bluefin tuna fisheries operating in the California Current have seen a recent rise in catches, including during the 2014\u20132016 marine heatwave . These iClick here for additional data file."} +{"text": "Age-related disability and diseases are known to be delayed in people living to 100 years or more. Changes in the immune system with age are known, including in cell type composition and gene expression differences. To further explore changes in extreme longevity subjects, we investigated peripheral blood immune system cell subpopulations across age and extreme longevity at a single cell resolution. We performed an integrative analysis of public scRNA-seq datasets to define consensus cell types of longevity and age, and classified cell types in our novel New England Centenarian Study dataset. We integrated these datasets together to investigate cell type specific differences at a composition and gene expression level. Our findings identified higher cell type diversity in extreme longevity subjects compared to younger age groups, but no significant difference among younger age groups demonstrating that overall composition differences are unique to longevity. We identified novel differences in myeloid and lymphocyte populations; Extreme longevity subjects have higher composition of CD14+ Monocytes, Natural Killer cells, and T gamma delta populations and lower composition of CD16+ Monocytes and dendritic populations. We characterized gene expression differences between extreme longevity and younger age groups and differences in aging across younger age groups. We found that extreme longevity cell type specific signatures overlapped with the aging signatures by at least 50%. We identified unique genes to extreme longevity that are enriched for pathways specific to immune activation and inflammation, suggesting a protective mechanism for centenarians through efficient activation and regulation of immune subpopulations in peripheral blood."} +{"text": "Our studies of US national-level samples across adult lifespan as well as older adults in California and in Italy\u2019s Cilento region have found a consistently strong inverse correlation between loneliness and wisdom, especially its compassion component. Loneliness and social isolation are associated with worse physical and mental health while the reverse is true for wisdom and compassion. Follow-up of older adults in San Diego during the Covid-19 pandemic showed no change in this pattern. While the effects of the pandemic and the necessary social distancing were heterogeneous, older adults generally handled these stresses better than younger adults, with less loneliness and greater compassion. Our recent studies assessing EEG responses to emotional stimuli as well as alpha and beta diversity in gut microbiome showed opposing biological patterns characterizing loneliness and wisdom. I will also present preliminary data from a compassion training intervention to reduce loneliness among older adults."} +{"text": "Information and communication technology (ICT) use has been associated with well-being among older adults. This link is often attributed to the fact that technology use facilitates connecting with social relations generally. What is less known, however, is the extent to which distinct dimensions of social relations uniquely influence how ICT use affects health. Thus, the importance of weak ties is receiving increased attention. Using data from the Detroit-based Social Relations Study collected in 2015, we examine the extent to which separate dimensions of weak ties (contact frequency and network size) mediate and moderate the link between technology use and depressive symptoms among adults age 65+ (n=213). A greater number of less close relations mediated the link as it was associated with technology use and fewer depressive symptoms. A moderating effect was also found as technology use was associated with fewer depressive symptoms only among those with lower contact frequency."} +{"text": "Insects tend to feed on related hosts. Coevolution tends to be dominated by interactions resulting from plant chemistry in defense strategies, and evolution of secondary metabolisms being in response to insect herbivory remains a classic explanation of coevolution. The present study examines whether evolutionary constraints existing in host associations of economically important fruit flies in the species\u2010rich tribe Dacini (Diptera: Tephritidae) and to what extent these species have evolved specialized dietary patterns. We found a strong effect of host phylogeny on associations on the 37 fruit flies tested, although the fruit fly species feeding on ripe commercially grown fruits that lost the toxic compounds after long\u2010term domestication are mostly polyphagous. We assessed the phylogenetic signal of host breadth across the fruit fly species, showing that the results were substantially different depending on partition levels. Further, we mapped main host family associations onto the fruit fly phylogeny and Cucurbitaceae has been inferred as the most likely ancestral host family for Dacini based on ancestral state reconstruction. The present study examines whether evolutionary constraints existing in host associations of economically important fruit flies in the species\u2010rich tribe Dacini (Diptera: Tephritidae) and to what extent these species have evolved specialized dietary patterns. We found a strong effect of host phylogeny on associations on the 37 fruit flies tested, although the fruit fly species feeding on ripe commercially grown fruits that lost the toxic compounds after long domestication are mostly polyphagous. Further, we discussed the role of secondary metabolisms in feeding preference, and other suites of traits in the specialization of fruit fly dietary patterns. To obtain the interacted hosts of the tephritid species, we extracted records from the pest\u2010oriented database of the Centre for Agricultural and Biosciences which reports fruit flies occurring and feeding on plants. Host associations were cross\u2010checked for completeness with the COFFHI database . Synonyms on the host list were eliminated and only accepted names adopted for each plant species. In general, our database of interactions between fruit flies and host plants contains 1,841 associations, including 37 Dacini fruit flies and 706 host species belonging to 87 families.The tribe Dacini, primarily comprises species of three genera model of evolution, values of K\u00a0>\u00a01 imply strong phylogenetic signals, and K\u00a0=\u00a00 implies the absence of phylogenetic conservatism. While there is no clearly defined K value cutoff in which to apply phylogenetic comparative methods, nonsignificant value of <1, or more conservative <0.5, are typical for characters that are phylogenetic independent. Pagel's \u03bb examines the effect of ancestral relatedness on character evolution, with a value close to 1 indicates phylogenetic signal and a value approaching 0 indicates phylogenetic independence.To test whether host breadth and namely states of polyphagy would be phylogenetically distributed across the tephritid species tree, we used Blomberg's \u03bb Pagel,\u00a0 to asses10 (phylodistance\u00a0+\u00a01) according to empirical experiments from Gilbert et al. (\u03b20) and slope coefficient (\u03b21) and the 95% confidence interval were obtained from the mean coefficients across all 1,000 sets from these regressions for all fruit flies.We took a logistic regression approach to calculate the probability of a host plant being infected by, or found in association with, a particular tephritid species , and its relationship with phylogenetic distances. We compiled associations at the genus level, and the associations were modeled as a binary trait . In the regression analysis, we only considered fruit fly species with three or more plant associations to make it more reasonable, and host phylogenetic distances were calculated in million years. We integrated the associations between fruit flies and host plants with the matrix of pairwise phylogenetic distances among host genera to investigate the role of host phylogeny. To calculate the possibility of fruit fly host affiliation, we applied the phylogenetic distance as predictor variable , 2.4We applied the DEC model \u201ctwo families\u201d and \u201cfour families\u201d were not apparent using the K estimate, but signals were detected under the \u03bb estimate . The character (feeding on) \u201cthree families\u201d showed strong phylogenetic signals using both the te Table\u00a0. SignifiThe probability that two host genera sharing one tephritid species declined significantly with phylogenetic distance, supporting that Dacini fruit flies prefer to feed on closely related plants Figure\u00a0. The logZeugodacus and Dacus.There were 87 families being used as hosts; however, only a handful of families are \u201cimportant\u201d host families which might play a decisive role in the evolutionary history of Dacini fruit flies. It contained nine main host families exclusively in the ancestral host state reconstruction analysis for studying purpose. Families of Cucurbitaceae and Myrtaceae were the most frequently used two families in terms of number of associated host plants belong to these families; families of Cucurbitaceae and Rutaceae were the most commonly used two families in terms of number of fruit fly species feeding upon these plant species. In addition, host use family Cucurbitaceae was distributed widely in the diet on genera of Bactrocera, and two Zeugodacus species Zeugodacus cucurbitae and Zeugodacus tau were not included in the reconstruction, because that these fruit flies feeding on more than 20 host families seemed to be out of limitations due to shifts in geographic distribution and could do little to estimate ancestral hosts. There were 25 tephritid species that had been expected to be contributed to estimate the ancestral host state in the DEC model. We mapped main host families onto simplified phylogeny of tephritid species, most likely ancestral host states were shown on each node by host shifts, that fruit odors as key traits help distinguish among respective host plants that can evolve as adaptive syndromes may be relevant to plant\u2013insect interactions. Although ripe, fleshy fruits function primarily to attract seed dispersers, they must also defend against diverse communities of frugivores. In particular, fruits can employ a variety of strategies such as ripening in seasons in which herbivores population densities are low or, reducing nutrient content and evolving a thick protective exocarp . Alternatively, inducible volatile organic compounds involved in host recognition may function as indirect defenses is an evolutionarily dynamic phenomenon and host breadth should be treated as a continuum (expand or shrink over evolutionary time), its evolution is thus particularly difficult to test due to the inherent uncertainties. It requires systems approaches that could be capable of handling the practical situation of preference and performance on a case\u2010by\u2010case basis especially for highly polyphagous species which exhibit a great degree of variability in their host use patterns. However, combining host utilization with information of bioinformatics and phylogenetic analyses is still a proven approach to investigate insect species dietary pattern and feeding strategy. Furthermore, although the association data included were cross\u2010checked, the identification of these fruit flies on hosts might actually be incorrect or based upon accidentally incidence on putative hosts . The existence of complexes could also confuse fruit fly host affiliations. We assigned the host records at the level of host genus or family that could be more reliable and reasonable for the analyses, and dubious host records are omitted. Nonetheless, an extremely polyphagous fruit fly might consist of a complex of cryptic species, leading to overestimation of host breadth in the fruit flies. However, it means that the conservatism should be more evolutionarily constrained than we investigated.5We found that Dacini fruit flies feeding on economically important host plants which normally lost secondary metabolisms after long\u2010term domestication still show specialized host associations, implying a decrease in the possibility of host sharing with increasing phylogenetic distance of the host plant taxa. Earlier study proposed that polyphagous fruit flies should be able to infest a broad range of phylogenetically distant host plants without absolute limitations and overcome plant defenses with little effort. Our study disaccords with this view and suggests that host specialization cannot be explained solely by secondary metabolites. Cucurbitaceae was recognized as the most probably ancestral host family in ancestral host reconstruction analysis. Diverse associations outside ancestral hosts for extremely polyphagous fruit flies are potentially attributable to capabilities of long\u2010distance dispersal and being generalist opportunists.The authors declared no conflicts of interest.Jiayao He: Conceptualization ; Data curation (supporting); Funding acquisition ; Methodology (supporting); Visualization (supporting); Writing\u2010original draft (supporting); Writing\u2010review & editing (lead). Ke Chen: Conceptualization ; Funding acquisition (supporting); Project administration ; Supervision ; Validation ; Visualization . Fan Jiang: Conceptualization ; Funding acquisition (supporting); Investigation ; Validation (lead); Writing\u2010review & editing . Xubin Pan: Conceptualization ; Investigation ; Project administration ; Supervision (lead); Writing\u2010review & editing .Data S1Click here for additional data file."} +{"text": "There is an imperative for conservation practitioners to help biodiversity adapt to accelerating environmental change. Evolutionary biologists are well\u2010positioned to inform the development of evidence\u2010based management strategies that support the adaptive capacity of species and ecosystems. Conservation practitioners increasingly accept that management practices must accommodate rapid environmental change, but harbour concerns about how to apply recommended changes to their management contexts. Given the interest from both conservation practitioners and evolutionary biologists in adjusting management practices, we believe there is an opportunity to accelerate the required changes by promoting closer collaboration between these two groups. We highlight how evolutionary biologists can harness lessons from other disciplines about how to foster effective knowledge exchange to make a substantive contribution to the development of effective conservation practices. These lessons include the following: (1) recognizing why practitioners do and do not use scientific evidence; (2) building an evidence base that will influence management decisions; (3) translating theory into a format that conservation practitioners can use to inform management practices; and (4) developing strategies for effective knowledge exchange. Although efforts will be required on both sides, we believe there are rewards for both practitioners and evolutionary biologists, not least of which is fostering practices to help support the long\u2010term persistence of species. Although practitioners may not always have the time or capacity to access the primary literature themselves, better access to management\u2010relevant primary research with clear recommendations for conservation practice can assist those in boundary\u2010spanning roles and the knowledge practitioners need for decision\u2010making (e.g. identifying effective solutions), even for highly applied disciplines like restoration ecology and invasion biology of >1000 to maintain evolutionary potential , whereas longer\u2010term outcomes can require more proactive strategies that promote functional and resilient ecosystems. Supporting the adaptive capacity of species requires transitioning away from such reactive management strategies promote applied evolutionary biology through research focused on questions relevant to biodiversity science and policy, and encouraging researchers to actively transmit their findings to policy\u2010makers (Hendry et al., Recent efforts to understand the uncertainties practitioners have about building adaptive capacity have revealed a range of questions for which the current evidence base cannot provide clear guidance (e.g. how transferable is knowledge about adaptive capacity across populations and related taxa?; Aitken & Whitlock, Another strategy that can be highly effective at influencing management practices is to embed scientists within management agencies and conservation organizations Figure , where t6Practitioners are open to changing their management practices to support the adaptive capacity of species, but they need help to achieve the required shift in accepted methods, policies and practices. Despite arguably having less of a history of engaging with conservation practitioners (Smith & Bernatchez, None declared."} +{"text": "ABSTRACT IMPACT: Our goal is to identify bacterial biomarkers of adverse Clostridioides difficile infection outcomes OBJECTIVES/GOALS: We characterized microbiota features of Clostridioides difficile infections (CDIs) and will investigate the association between bacterial taxa and adverse outcomes, which includes severe and recurrent CDIs. METHODS/STUDY POPULATION: 1,517 stool samples were collected from patients diagnosed with a CDI at the University of Michigan along with 1,516 unformed and 910 formed stool control samples. We characterized the microbiota of the 3,943 stool samples by sequencing the V4 region of the 16S rRNA gene and used the Dirichlet Multinomial Mixtures method to cluster samples into community types. Severe CDI cases were defined using the Infectious Diseases Society of America criteria and recurrent CDIs were defined as CDIs that occurred within 2-12 weeks of the primary CDI. We will use machine learning to examine whether specific bacterial taxa can predict severe or recurrent CDIs. We will test 5 machine learning models with 80% training and 20% testing data split. RESULTS/ANTICIPATED RESULTS: Similar to findings from a previous study with 338 samples, we found there was no difference in diversity between CDI cases and unformed controls and samples from the 3 groups clustered into 12 community types. To investigate the bacterial taxa that are important for predicting adverse CDI outcomes, we will select the best machine learning model based on performance and training time and examine how much each feature contributes to performance. We anticipate the large number of CDI cases in our cohort and robust machine learning approaches will enable us to identify more bacteria associated with adverse outcomes compared to other studies that have attempted to predict CDI recurrence with fewer CDI cases. DISCUSSION/SIGNIFICANCE OF FINDINGS: Adverse CDI outcomes are a significant source of the morbidities, mortalities, and healthcare costs associated with CDIs. Identifying bacterial biomarkers of severe and recurrent CDIs could enhance our ability to stratify patients into risk groups and may lead to the development of more targeted therapeutics."} +{"text": "Siphateles bicolor snyderi), which has been rendered endangered by introgession from the introduced Lahontan Tui Chub (Siphateles bicolor obesa). Using geometric morphometric analysis of 457 individual Tui Chub from thirteen populations, we found that both native and introgressing parent taxa exhibited divergent body and caudal fin shapes in lake versus stream habitats, but their trajectories of divergence were distinct. In contrast, introgressed populations exhibited intermediate body and caudal fin shapes that were not differentiated by habitat type, indicating that introgression has eroded phenotypic divergence along the lentic\u2013lotic gradient throughout the historic range of the Owens Tui Chub. Individuals within hybrid populations were less morphologically variable than those within parent populations, suggesting hybrid adaptation to selective agents other than water flow or loss of variance by drift.Introgressive hybridization may erode phenotypic divergence along environmental gradients, collapsing locally adapted populations into a hybrid swarm. Alternatively, introgression may promote phenotypic divergence by providing variation on which natural selection can act. In freshwater fishes, water flow often selects for divergent morphological traits in lake versus stream habitats. We tested the effects of introgression on lake\u2013stream morphological divergence in the minnow Owens Tui Chub ( Introgressive hybridization can either erode or promote adaptive phenotypic divergence along environmental gradients. We tested the effects of widespread introgressive hybridization on morphological divergence between lake and stream habitats in the minnow Tui Chub. We found that hybridization has eroded morphological divergence, but comparisons of morphological variance between parent and hybrid populations suggest this outcome may not be maladaptive. The extent to which these populations diverge phenotypically to meet the demands of their local environment affects population fitness and can substantially alter ecological processes was historically abundant in a variety of lentic and lotic habitats following geographic isolation which has been maintained since at least the late Pleistocene , the minnow Owens Tui Chub . We used geometric morphometrics of body shape from three perspectives and measured caudal fin aspect ratio to test whether Owens, Lahontan, and hybrid Tui Chub exhibit morphological divergence between lake and stream habitats and compared the trajectory and magnitude of phenotypic divergence in the two parental taxa and their hybrids. We then evaluated the effects of introgression on intrapopulation morphological variation by comparing hybrid and parental populations.22.1We collected fish in July and August in 2019 and 2020, except for one site which we sampled in February 2021, using beach seines and backpack electrofishing. We sampled one lake and one stream population from each of the parent subspecies (Owens and Lahontan), and four lake and five stream populations of putatively introgressed populations separately to avoid potential scaling issues associated with allometric models of combined landmark sets as a fixed covariate to account for allometry, habitat type (lake or stream) as a fixed effect, and site as a nested random effect using the \u201cprocD.lm\u201d function in the R package \u201cgeomorph\u201d , though the magnitude of divergence was greater for Lahontan Tui Chub exhibited body shape divergence between lake and stream populations for all three perspectives population means were bounded within the convex hull connecting the four parental population means. Linear models provided no evidence for lake\u2013stream divergence in body shape for the introgressed populations . In the introgressed populations, only fish length affected caudal fin aspect ratio (p\u00a0<<\u00a0.001) Table .3.3Morphological disparity within populations was highest in the lake populations of the parent subspecies Figure\u00a0. Hybrid 4Introgressive hybridization may erode phenotypic divergence along environmental gradients, collapsing locally adapted populations into a hybrid swarm. Alternatively, introgression may promote divergence by providing variation on which natural selection can act. Our results indicate that hybrid Tui Chub are intermediate to the parental subspecies in morphology and exhibit less divergence between lake and stream habitats. This result indicates that hybridization may impede adaptation to divergent selection imposed by lotic versus lentic environments, although the hybrids may differ from the parent species in terms of other ecological functions or characteristics.The implications of this finding for local adaptation and population fitness rest in part on which point we sampled along the time course of hybridization\u2010induced evolutionary change. Gene flow from distinct taxa via introgression is at first a variance\u2010generating process that produces a suite of phenotypes with variable fitness due to recombination between parental genomes ; Data curation (lead); Formal analysis (lead); Funding acquisition (lead); Investigation (lead); Methodology (lead); Project administration (lead); Resources (supporting); Supervision (supporting); Visualization (lead); Writing\u2010original draft (lead); Writing\u2010review & editing . Danielle C. Hankins: Data curation (supporting); Investigation (supporting); Writing\u2010review & editing (supporting). Jonathan B. Shurin: Funding acquisition (supporting); Resources (lead); Supervision (lead); Writing\u2010review & editing .Appendix S1Click here for additional data file.Appendix S2Click here for additional data file."} +{"text": "Following publication of the original article , it was The figures and the related content have now been corrected in the published article, and the corrected figures and corrected content can be found detailed below:AbstractResults: The qRT-PCR result showed that GPC3 expression was much lower in the LUSC tissues than that in the control group. IHC results further showed that GPC3 was more negatively expressed than positively expressed in LUSC tissues.\u201d\u201cMethods\u201cThe paraffin sections of cancer tissues were collected from 10 patients undergoing pulmonary malignant tumour surgery at the Liaoning Cancer Hospital and Institute between June 2018 and June 2020.\u201dResultsGPC3 expression was much lower in the LUSC tissues than that in the control group paraffin-embedded LUSC tissues, while positive staining was observed in the remaining cases Fig. . This suThe authors thank you for reading this correction and apologize for any inconvenience caused."} +{"text": "Chronotype has been linked to poor cognitive outcomes and mortality among older adults. Although previous studies indicate an association between personality and sleep, little is known about associations between personality and chronotype in older adults. We examined the association between personality and objective sleep midpoint (a measure of chronotype) in 463 older adults aged 73.5 \u00b17.7 from the National Social Life, Health, and Aging Project who completed the Midlife Developmental Inventory Personality scale and three nights of wrist actigraphy, from which we derived participants\u2019 average sleep midpoints. After adjusting for demographics, higher conscientiousness was associated with earlier sleep midpoint . Associations for other traits were not significant. Findings link conscientiousness to chronotype and raise the possibility that earlier sleep timing may partially account for associations of conscientiousness with health outcomes. Further studies are needed investigating the role of personality in links of sleep and circadian factors with health."} +{"text": "Base on effective medium theory we introduce a multi sites method for calculation of realistic energy bands of strongly correlated systems. We found due to approximated self energy, the density of states that obtained directly by calculated local Green function does not reflects system energy bands truly. By using this method we investigated how electrons repulsion renormalizes graphene bands. Graphene realistic bands calculated in both the dynamical mean field theory (DMFT) and four sites beyond super cell approximation for different repulsions. Our calculated interacting graphene bands illustrate a semi metal to a Mott insulator anti ferromagnetic phase transition at repulsions A and 6. Transition metal oxides are a class of materials that electron\u2013electron Coulomb repulsion in their valance d-wave orbitals is strong. Conventional single electron band theories break down for these systems. Although theoretical calculations of these systems properties are hard but their applications is wide7. How electron\u2013electron Coulomb repulsion modifies electronic band structure of these systems is a big change in strongly correlated systems. Usually in the calculations of physical quantities of such systems two different methods finite size approximation15 and effective medium theory are using25. For a honeycomb graphene lattice by using Hubbard model with on site electrons repulsion and a finite size quantum Monte Carlo calculations for 28.Strongly Correlated electrons are responsible for notable properties such as unconventional high 29. Dynamical mean field theory (DMFT) is a single site approximation widely used for any interaction strength u17. In the DMFT inter sites correlation is neglected. Multi sites, Nc, dynamical cluster approximation (DCA) for including multi sites correlation is introduced21. Although they claimed that DCA recovers exact self energy 22. The DCA coarse grained self energies 25 for disorder systems introduced. In the EMSCA relation between real space and k-space grained self energies are mth maximum with mth band energy 28, our results show that graphene bands are very sensitive to electrons Coulomb repulsion, hence a semi metal to a Mott insulator phase transition occurs at very low repulsions in comparison to graphene band width.In the effective medium theories the electron\u2013electron interaction effects replaces by an effective medium that identifies by a self energy i and i and j lattice sites. We start our investigation by a Hubbard model for a strongly correlated system which is given by,In the imaginary time mentclass2pt{minimThe single particle equation of motion corresponding to Eq. in the eIn the real and spin spaces the following relation between interacting single particle Green function entclass1pt{minimarom Eqs. 6\\documenFor solving Eq. we intromentclass2pt{minimmentclass2pt{minim25By applying EMSCA to the interacting system, Eq. reduces Equation could beS, in the following partition function Z,S is action defined byEquation could bementclasspt{minimaRelation between non interacting Green function By imaginary time Fourier transform of Dyson Eq. we have1Equation denotes By substitution Eq. in the aNow we apply EMSCA that keeping interaction in the central super cell By inverse imaginary Fourier transform of Eq. we have2By imaginary time Fourier transform of first and third terms of right hand side of Eq. we have2To complete loop of super cell single particle average Green function by Eqs. and 11)\\document21. By dividing imaginary time M small portions 21 hence impurity super cell two particle Green function 21 and To solve Eq. we shoul17In the actual calculation of imaginary time discretized of Eq. the follInverse imaginary Fourier transform of Eq. given byi and j sites are inside same super cell and second term including all self energies that i and j sites are inside different super cellsl are integer numbers. Deriving process of k-space and real space self energy and average Green function EMSCA is as follows. We divide the k-space self energy mentclass2pt{minimBorn\u2013von Karman periodicity condition along lattice lengths implies thatFor other lattice lengths we haveSince The wave vectors that satisfying Eq. simultanBy real space Fourier transformation of sing Eq. it is eaence Eq. convertsSo in the FBZ we have nth region self energy values are equal. Figure Now by dividing FBZ to entclass1pt{minimanth regionFor graphene reciprocal primitive vectors areEquation implies By applying Eq. to the GA initial guess for self energy usually 0.calculating coarse Green function Calculate cavity Green function from Real space and imaginary time Fourier transformation Calculate real space super cell from Eq. .Calculate real space super cell average Green function by Calculate Calculate new self energy from Go to step 2 and repeat whole process until convergence.Analytical continuation of self energy to obtain Calculate Algorithm for implementation of method is as follows: Process of extracting band structure from calculated 16For an exact effective medium system with whole lattice sites, imaginary part of self energy goes to zero, tion Eq. 1659\\docum grain in the first Brillouin zone is continuous but at grain boundaries are discontinuous. Although density of states could be calculated from calculated local Green function mth band energy at wave vector In general, effective medium self energy Figure Now we apply our method to a graphene interacting system with two different cluster sizes ) in Eq. and analt in which the fake states are eliminated. To high light advantage of our method we compared the direct DMFT calculated density of states N(E) with density of states obtained from calculated realistic valance and conduction bands. Our results show that by increasing electrons energy repulsion u valance and conduction bands separated but steel valance band is completely full by both spin up and down electrons while conduction band is empty. Our results show that in the DMFT the critical value of repulsion to have a semi metal to an anti ferromagnetic Mott phase transition is t first we applied four sites 25. Then we approximate First we calculate realistic band structure of this system in the DMFT. Figure ) in Eq. all bandFor strongly correlated systems we introduced a method for calculation of continues self energy in the whole first Brillouin zone that allows us to calculate renormalized band structure. By using this method the realistic renormalized band structure of Hubbard model of an interacting electrons graphene lattice obtained. Our results show that graphene bands are sensitive to electrons Coulomb repulsion even at low repulsions. Until now people taught that all states obtained by approximated self energies even by DMFT are acceptable but we proved that this method should be corrected to complete band structure calculation process. Our method and results open new perspective on physics of strongly correlated system."} +{"text": "The risks to older adults in nursing homes (NHs) and assisted living communities (ALCs) exposed to disasters are evident in prior research. However, little research has been conducted to understand the factors related to facilities\u2019 vulnerability. This research examined NH and ALC experiences during Hurricane Irma in 2017. Qualitative interviews were conducted with representatives of facilities (N=100), transcripts were analyzed using Atlas.ti version 8. Team members met to reach consensus on codes and major themes and subthemes, which they analyzed using a conceptual model designed to identify factors related to the disaster vulnerability in long-term care (LTC). We found physical factors are important, but physical strength is not enough. Multiple social/organizational factors are critical. Results indicate managing a major disaster and protecting LTC residents involve social and organizational connections across a range of groups from staff and family members to emergency mangers and neighborhood associations."} +{"text": "Assisted living serves as a substitute for nursing home residents with low care needs, especially in markets with a high proportion of dually eligible Medicare beneficiaries. This study examines trends in the acuity of residents in assisted living communities over time in comparison to nursing homes to characterize how substitution has affected the resident compositions of both settings. We also examine how trends in acuity are shaped by dual eligibility. Using Medicare claims data, we identify cross-sectional samples of beneficiaries in each setting from 2007-2017. The proportion of residents in assisted living with high care needs has increased 18% in assisted living communities compared to 8.7% in nursing homes. Acuity levels are higher among dually eligible assisted living residents compared to assisted living residents who are not dually eligible. Policy makers and administrators should examine whether assisted living is prepared to provide care for an increasingly acute population."} +{"text": "Proceedings of the National Academy of Sciences of the United States of America, 119, e2116136119), this review attempts to balance this bias by emphasizing on non\u2010visual perception of asymmetry. In conclusion, we discuss the methodological challenges associated with testing the role of multimodal cues in detecting mate asymmetry, and highlight the importance of considering ecological, behavioral, and evolutionary aspects of animals while interpreting empirical data that test such hypothesis.The postulates of developmental instability\u2013sexual selection hypothesis is intensely debated among evolutionary biologists, wherein despite a large amount of empirical data, evidence for or against it has been largely inconclusive. A key assumption of this hypothesis is that animals assess symmetry in potential mates as an indicator of genetic quality , and consequently use this information to discriminate against those with higher asymmetries while choosing mates. However, the perceptional basis that must underlie such discriminatory behavior is not clearly defined. It is also argued that since asymmetry levels in natural populations are very low, the low signal\u2010to\u2010noise ratio would make accurate assessment of symmetry both difficult and costly. Rather than attempting to validate this hypothesis or even as to whether animals assess mate symmetry, this review simply aims to examine the plausibility that animals perceive symmetry (directly or indirectly) and consequently discriminate against asymmetric mates in response to perceived irregularities during courtship. For this, we review mate choice and courtship literature to identify potential sensory cues that might advertise asymmetry or lead to discrimination of asymmetric individuals. Although signaling associated with mate choice is commonly multimodal, previous studies on asymmetry have mainly focused on visual perception. In the light of a recent study (Vijendravarma et al., 2022, We review how multimodal non\u2010visual cues can signal morphological asymmetry of potential mates during courtship in diverse species across taxa. We also discuss how extended phenotypes and mate choice copying aid females to indirectly assess and discriminate against asymmetric males. With the current emphasis on multimodal communication during courtship, this review is well timed to rekindle interest in the role of symmetry during mate choice. Caecil2.4Thamnophis sirtalis) compete to align themselves alongside a female's body and cloaca use tips of their long antennae to determine the abdominal tip of females while mating, and both sexes preferentially choose mates with symmetric antennae with altered non\u2010intromittent genital spines fail to achieve copulation and have reduced pre\u2010 and post\u2010mating success regarding the internal environment and potential noxious stimuli to elicit appropriate behaviors display leg rubbing behavior during copulation that is speculated to provide tactile cues to females for post\u2010mating mate discrimination apply pheromones from inguinal glands directly to the female during amplexus, suggesting again that tactile and chemosensory cues can be communicated simultaneously . In some species, these EPs are built by individuals of one sex solely to attract mates or as a part of their courtship ritual, functioning as a sexual trait, wherein the male's reproductive success is correlated with the quality of structures they build. These EPs have several benefits and drawbacks that are reviewed in Schaedelin and Taborsky . Most imTorquigener sp.), wherein the male (12\u00a0cm long) constructs a complex arena that is 2\u00a0m in diameter, and takes over 7 to 9\u00a0days to complete build sand pyramids and sand dunes to attract females to their mating burrows . Males s Figure ). Respons Figure .Even prior to the formulation of the DI\u2010SS hypothesis, female satin bower birds were shown to prefer bowers that were symmetrical Figure , and thi3.2Poecilia reticulata) show preference to copy mate choice of older females is yet another indirect way through which asymmetric individuals may be discriminated during courtship. Individuals indulging in mate choice copying tactically overcome their inexperience in mate choice or mitigate the costs of such assessments, but can indirectly bias their choice for symmetric mates if the model had preferred a symmetric mate. The ability to assess potential mate quality increases with age as a consequence of learning, so mate choice copying by naive individuals might set a benchmark on quality of preferable mates for future mating's. Importantly, its known that females copy not only other females\u2019 preferences for certain males but can generalize these preferences to other males with similar traits (reviewed in Vakirtzis ). FurtheRattus norvegicus) and female mice (Mus musculus) prefer mating with recently copulated males that they detect using chemosensory cues Polak, , and finDrosophila were shown to discriminate against males with low levels of wing asymmetry through the consequent asymmetry in male courtship song features female preference was assayed under different mate choice paradigms; (c) males that were competed in the mate choice assays were randomly chosen from the same rearing condition; (d) FA level in males that were successful or unsuccessful in securing a mating was determined post\u2010female choice; (e) manipulation of different female sensory modalities was used to determine the sensory basis of female preference for male symmetry; and (f) genetic and surgical manipulation of male symmetry was used as additional FA\u2010inducing methods to corroborate the findings ; Funding acquisition ; Methodology ; Project administration (lead); Resources ; Validation (lead); Visualization (lead); Writing \u2013 original draft (lead); Writing \u2013 review & editing (lead). Pierre Leopold: Conceptualization (supporting); Funding acquisition ; Project administration (supporting); Resources ; Writing \u2013 original draft (supporting); Writing \u2013 review & editing (supporting)."} +{"text": "Livestock grazing is often perceived as being detrimental to the quality and functioning of dryland ecosystems. For example, a study in a semiarid Kenyan savanna proposed that cattle form bare spaces throughout the landscape, which indicate ecosystem degradation. Other studies, conducted in north\u2010eastern Spain, where climatic conditions range between semiarid and Mediterranean subhumid, reported that sheep and goat trails have increased the emergence of rill erosion processes. Sometimes, this negative perception is extended to include wild, large ungulate herbivores as well. Here, we challenge this perception by highlighting the generally nonadverse and even ameliorative impacts of moderate animal rate on geoecosystem functioning of hilly drylands. Specifically, trampling routes formed across hillslopes by grazing animals\u2014being either domesticated livestock or native large herbivores\u2014transform the original two\u2010phase vegetation mosaic of shrubby patches and interpatch spaces into a three\u2010phase mosaic. The animal routes increase the complexity of ecosystem, by strengthening the spatial redistribution of water and soil resources at the patch scale and decreasing hydrological connectivity at the hillslope scale. As a consequence, the animal routes improve functioning of hilly drylands and increase their resilience to long\u2010term droughts and climatic change. Therefore, instead of viewing the animal routes as degraded spots, they should be perceived at a wider perspective that allows to properly understand their overall role in sustaining dryland geoecosystems. Livestock animals and wild ungulates form trampling routes across the landscape, transforming the original two\u2010phase vegetation mosaic into a three\u2010phase mosaic. The routes increase the complexity of ecosystems and decrease hydrological connectivity at the hillslope scale, thus improving ecological functioning and sustaining dryland durability under long\u2010term droughts and climatic change. Therefore, two\u2010phase mosaics, also known as source\u2013sink ecosystems, are prevalent and form a patchy vegetation cover Spach, regulate its cover vegetation. Despite the expected increase in the ecosystem's NPP, this chain of effects decreases ecosystem complexity, reduces plant species diversity, and lowers the economic value of rangelands How do lithology, topography, and soil type affect route formation and pattern?; (2) How are the morphology and patterns of routes regulated by regional climatic conditions (in the long term), and by local climatic fluctuations (in the short term)?; (3) What is the optimal cover of animal routes to maximize forage production while sustaining geoecosystem functioning?; (4) What is the best animal rate to form the optimal cover of routes?; (5) How does the routes' optimal cover change for hillslopes with different inclines or of different shapes (concave vs. convex morphology)?; (6) How does the routes' optimal cover change for hillslopes with different aspects ?; (7) How does the routes' optimal cover change across climatic gradients?; and (8) How is the impact of animal routes on the spatial distribution of soil\u2010water at the patch and hillslope scales regulated by the abovementioned issues?These and other questions necessitate thorough research of this topic, which has direct implications for elemental cycling, ecosystem health, and environmental sustainability. Global climatic change, with the forecasted aggravation of aridity in the world's deserts and the expansion of dryland areas ; project administration (lead); resources (lead); supervision (lead); writing\u2010original draft (lead). Hezi Yizhaq: Conceptualization (supporting); software ; writing\u2010original draft ; writing\u2010review & editing . Yagil Osem: Conceptualization ; investigation ; validation ; writing\u2010review & editing . Eli Argaman: Conceptualization ; investigation ; writing\u2010original draft ; writing\u2010review & editing .Appendix S1Click here for additional data file."} +{"text": "Disproportionately affected by numerous relational stressors , older African American couples frequently find solace in religion and each other. Research notes that both married and cohabiting couples effectively respond to difficult situations by sharing the ownership of a stressor and organizing a collaborative, collective response. However, little is known about the influence of religion on shared coping experiences, particularly among older African American couples. This study examined dyadic data from the Strong African American Couples Project to capture the influence of relational sanctification on the communal coping practices of married and cohabiting older African American couples. The sample included 194 African American couples (146 married and 48 cohabiting) between the ages of 50 and 86 years. With the use of Actor Partner Independence Models, this study found that men\u2019s sanctification predicted both their own communal coping and their partner\u2019s communal coping. However, there were no significant effects when women\u2019s sanctification was used as a predictor of communal coping among older African American couples. These findings are both important and novel, because these relationships had never before been examined within the United States, much less among older African American couples. Similar to existing research among majority White couples, this research finds that men\u2019s religiosity may be a more influential predictor of relational outcomes than women\u2019s religiosity. Such findings offer a valuable foundation for future studies seeking to consider how relational sanctification and communal coping may impact other outcomes associated with the romantic relationships of older African Americans."} +{"text": "Long-term care is a challenging environment for quality improvement due to the high resident acuity, wide variation in resident needs, and wide variation in types and backgrounds of the large staff across three daily shifts. We report results from a learning collaborative undertaken to improve care quality and staff quality improvement skills in the VA CLCs through development of high functioning relationally coordinated teams operating in accord with person-centered care principles. The collaborative included 27 CLCs. Over 9 months leadership teams completed action assignments supported by 5 workshops and regular group coaching calls. Evaluation included fidelity monitoring , satisfaction questionnaires, and review of the VA quality measures (CLC Compare). Pre-post participant evaluations revealed a significant increase in positive responses to the question \u201cto what extent do you think applying these new skills/knowledge will improve quality in your CLC?\u201d and positive responses trending toward significance in ratings of abilities to apply new skills. Open-ended survey comments were positive and indicated change in understanding and practice: \u201cutilizing the daily huddle to facilitate real time communication afforded the team a proactive approach to providing care and reducing acute exacerbations. We are able to avert, evaluate as a real time team and make it happen in the now not as a look back.\u201d; \u201cdefinitely unified front-line staff and CLC leadership.\u201d Some changes were achieved in CLC Compare quality scores )."} +{"text": "While recent Phase 3 glioblastoma (GBM) trials have failed to establish novel therapies, they potentially provide a high-quality source of external control patients treated with temozolomide. We consider hybrid two-stage adaptive designs that leverage these external controls to safely accelerate Phase 3 GBM trials. The basic strategy is that first patients are randomized 1:1 between the control and experimental arms, then an interim check measures similarity between the trial's control patients and potential external controls, and finally if this interim similarity is high the randomization ratio is changed accordingly and the external controls are used in the final analysis. An extensive simulation study is conducted to assess operating characteristics and we discuss when these hybrid designs can accelerate GBM therapy development while maintaining strict error control."} +{"text": "Past research has shown that childhood adversity (CA) affects the health of older adults; however, the biological mechanisms underlying this association are unclear. Though past research has implicated DNA methylation (DNAm), studies utilizing representative data from older adults and reliable DNAm measures are needed to answer key questions about how stable DNAm changes associated with CA are in later life. Methylation risk scores (MRSs) are an emerging tool that can be used as biomarkers of exposure and as a dimension reduction approach for mediation analyses. This study clarifies the association between CA and later life health by generating MRSs for childhood adversity based on an epigenome wide association study conducted in an independent sample and validating that measure in a nationally representative sample of older adults living in the US from the Health and Retirement Study (HRS), including 2016 methylation data from the HRS Innovative Subsample of the Venous Blood Study. For these 4,018 respondents, DNAm was assessed in whole blood using the Illumina Infinium Human Methylation EPIC BeadChip microarray. Results indicate that retrospective report of childhood SES is significantly associated with an MRS for CA after controlling for demographic factors , suggesting that DNAm signal from CA persists across the life course into older adulthood. This study helps clarify the biological processes underlying the association between CA and adult health."} +{"text": "This project develops a tailored and adaptive virtual reality platform to innovatively promote older adults\u2019 disaster preparation in a socially engaging environment. The platform serves the following purposes: 1) assist older adults to develop tailored household emergency preparedness plans, 2) simulate extreme weather conditions and warnings for older adults to practice disaster response and develop relevant knowledge and skills as well as test and revise their emergency preparedness plans, 3) use the process as a social engagement tool to reduce social isolation and promote a sense of community. The virtual environments are designed in Unity to simulate extreme weather conditions/natural disasters and older adults are guided to use the HTC VR headset and experience the selected disaster scenario. The pilot VR platform will be tested among community-dwelling older adults in the Dallas-Fort Worth Metropolitan Area."} +{"text": "Although informal caregiving for older adults (OAs) can increase knowledge and awareness about one\u2019s own aging , it can also negatively impact caregivers\u2019 physical health and emotional wellbeing and have spillover effects on school, work, and marriage . Despite the recent trend of family caregiving for OAs by young adults (YAs), research about these young caregivers is scarce. The present study focused on YAs\u2019 perceptions on aging. We hypothesized that YAs who provided at least three months of caregiving tasks for OAs would hold more awareness and negative perceptions on their own aging, as measured by a modified version of the Brief Aging Perceptions Questionnaire , compared to those who did not. We recruited 234 YAs between the ages of 18 - 40 and had them complete a survey via Amazon Mechanical Turks. About one third (32.1%) had caregiving experience. Results of independent t-tests revealed that caregivers scored higher on awareness of aging and negative consequences/control . Scores of positive consequences/control did not differ between the two groups. Our findings indicate the need for psychological interventions designed to help young caregivers integrate their caregiving experiences with less negative aging perceptions. Future research should examine the direct effects of caregiving experience on perceptions of aging between young and middle-aged adults."} +{"text": "Since the treatment of postmenopausal breast cancer patients with aminoglutethimide caused hypothyroidism with an unexpectedly high frequency previous treatment was suspected to contribute to hypofunction of the thyroid. Serum thyrotropin, triiodothyronine and free thyroxine index were compared between breast cancer patients who had undergone irradiation of regional lymph nodes and non-irradiated breast cancer patients, as well as patients having endometrial or colorectal carcinoma. Subclinical and clinical primary hypothyroidism was significantly more frequent in breast cancer patients who had previously received irradiation on supraclavicular lymph nodes comprising a minor part of the thyroid. Testing for the presence of autoantibodies against thyroid tissue components gave no evidence for radiation-induced autoimmune thyroiditis. Drugs suppressing thyroid hormone synthesis like aminoglutethimide may frequently cause myxedema in such irradiated women, especially at postmenopausal age."} +{"text": "We report our results of a selective approach to primary direct appositional vaginal repair versus transverse rectus abdominis flap repair (TRAM) in patients with extensive rectal/anal cancer or in cases with primary cancer of cervix, vagina or vulva involving the anal canal and anal sphincters.with total mesorectal excision between May 2002 and September 2005, were studied.Eighteen female patients with a median follow-up of 14 months (range: 2\u201336 months) undergoing extended abdominoperineal reconstruction following pelvic exenteration. Exenterative procedures were performed in 2 cases of primary vaginal cancer, following Wertheim hysterectomy for carcinoma of the cervix with recurrence after radiation and in 2 further cases of anal cancer with extensive pelvic recurrence after primary chemoradiation. Fifteen cases are alive on follow-up with no evidence of disease; 2 patients who had recurrent carcinoma of the cervix and who underwent TRAM flap reconstruction, have recurrent disease after 5 and 6 months of follow-up, respectively.Twelve patients underwent an extended abdominoperineal resection with hysterectomy and vaginectomy, with 6 patients undergoing primary TRAM flap reconstruction one case of wound breakdown after primary repair underwent external beam and intracavitary irradiation primarily with wound breakdown of a primary repair followed by a delayed pedicled graciloplasty. TRAM flap reconstruction has been reserved in our unit for patients undergoing total pelvic extenteration. In general, we would recommend the use of TRAM flap reconstruction in younger sexually active patients where there has been external irradiation combined with brachytherapy.Our experience shows that careful primary closure of an extended abdominoperineal resection wound is effective and safe. Our Most This approach may also be used successfully in patients who have undergone combined external radiation and brachytherapy or where repeat irradiation for recurrent carcinoma has been utilized [Perineal wounds in such patients are at considerable risk for dehiscence and delayed healing when primarily repaired, sometimeutilized . The comutilized which prutilized . The simutilized where thSimple extension of the abdominoperineal resection with high posterior vaginectomy has beenprimarily . All patients with rectal and anal carcinoma received preoperative chemoradiation with a median radiation dose of 50 cGy with anal cancer patients routinely receiving elective bilateral inguinal irradiation. Patients with cervical carcinoma also received intracavitary radiation (21 cGy in 6\u201313 fractions delivered to point A). The chemotherapy used in each case of rectal cancer was 5-Fluorouracil (425 mg/m2) plus Leukovorin rescue (20 mg/m2) with the anal cancers undergoing a modified Nigro r\u00e9gime utilizing 5-Fluorouracil alone [who have recurrent disease after 5 and 6 months of follow-up respectively and for TRAM flap was 26 days (range 19\u201363 days). Mesh insertion was not used for any case undergoing a TRAM flap with one patient having an incisional hernia of the donor site at one year of follow-up. The same patient had apical cutaneous flap necrosis requiring minor d\u00e9bridement and dressings which extended her hospital stay. One patient undergoing simple extended abdominoperineal resection for an advanced cervical carcinoma, , experienced breakdown of the vaginal repair necessitating a delayed graciloplasty 4 months after the initial procedure which proved successful. No patient was sexually active through the period of follow-up.The outcome is reported of a select group of patients presenting to a specialist colorectal unit with extensive rectal carcinoma following neoadjuvant therapy, recurrent anal squamous cell carcinoma after primary chemoradiation and primary or recurrent cervical and vaginal carcinoma involving the anus where the surgical decision was made either for extended abdominoperineal resection with high posterior vaginectomy or for total pelvic exenteration with TRAM flap perineal reconstruction. No patients underwent neovaginal reconstruction using the TRAM flap technique. All patients received preoperative radiation with 2 cases receiving both external and intracavitary radiotherapy. Eleven out of 12 patients with extended vaginectomy showed primary healing and 5 of the 6 cases undergoing TRAM flap reconstruction healed primarily.The only case of dehiscence resulting in a patient undergoing combined external and intracavitary irradiation being successfully treated with a pedicled graciloplasty. The TRAM flap has been reserved in our patients only for those undergoing total pelvic exenteration and although its unselected use has been shown to be safe as a primary treatment, [In selected cases it may be combined with a myoperitoneal flap to reduce flap-related morbidity [In this complicated setting using high dose perineopelvic radiation, delayed perineal healing may be expected with chronic perineal sinus formation at 3 months after abdominoperineal excision being reported in between 15\u201365% of patients -18. Our eatment, the simpeatment, . In seleorbidity . Alternaorbidity or the uorbidity which aporbidity .None of our patients were sexually active during the period of follow-up. It is well known that the more extensive the pelvic surgery for cancer, the greater the effect on body image and self-perception of attractiveness. The greatest restriction on postoperative quality of life appears to be imposed by such disturbances in sexual function, particularly where the surgery is non-reconstructive in nature [In this respect, myocutaneous flap reconstruction appears in such patients to provide the greatest satisfaction with the lowest morbidity [No one in our group underwent neovaginal reconstruction, where it has been suggested that sexual activity is able to be resumed in about half of the patients of whom over 80% will do so within the first postoperative year [It is accepted that there is considerable psychosexual morbidity in some patients undergoing total exenteration and our n nature . In thisorbidity . No one ive year . In geneAndrew P Zbar: Assisted in the format and design of the paper and provided patients and surgical expertiseRadhakanth K Shenoy: Evaluation of the patientsAntonio Chiappa: Assisted in patient management and performed the literature searchAll authors read and approved the final manuscript."} +{"text": "Pancreatic metastases from previously treated renal cell carcinoma are uncommon. Surgical resection of pancreatic metastasis remains the only worthwhile modality of treatment.A case where pancreatic metastasis from previously resected right sided renal cell carcinoma was resected with a subtotal left pancreatectomy is described. An unusual feature was the presence of a large splenic vein tumor thrombus extending into the portal vein with associated portal hypertension. The patient underwent an uneventful portal vein resection with primary anastomosis.This is possibly the first documented case of portal vein renal tumor thrombosis in a case of isolated pancreatic metastasis from previously operated renal cell carcinoma in published world surgical literature. Pancreatic metastases from previously treated renal cell carcinoma are known but uncommon. Surgical resection of pancreatic metastasis remains the only worthwhile modality of treatment. We describe our experience where a pancreatic metastasis from previously resected right sided renal cell carcinoma was resected with a subtotal left pancreatectomy with portal vein resection and primary anastomosis for a renal tumor thrombus in the splenic and portal vein.th postoperative day. Final histopathology confirmed a metastatic renal cell carcinoma to the pancreas with tumor thrombus in the portal vein scan, was diagnosed to have a pancreatic mass with evidence of portal hypertension and a portal vein thrombosis Figure . In viewOur case raised three questions. First, is a radial resection justified in view of a locally advanced (metastatic) pathology? Second, is a major pancreatic resection safe enough in these patients and last but not the least, the rarity of our intraoperative findings that prompted us to write this report.et al., reported a mean survival of 61 months after resection for metastasis to the pancreas from renal cell carcinoma [Despite the unsatisfactory survival in our case, isolated pancreatic metastasis from renal call carcinoma are best treated by surgical resection. While the 5 \u2013 year survival for metastatic renal cell carcinoma is poor and hovers around 9% , aggressarcinoma . This coPancreatectomy with portal vein resection is well described. A number of large studies have evaluated the safety of pancreatectomy with resection of tumor infiltrated retropancreatic vessels ,8. The c\"renal cell carcinoma, pancreatic metastasis, portal vein thrombosis\" did not reveal a single documented case of splenic and portal vein thrombosis in pancreatic metastasis from a previously treated renal cell carcinoma.Portal hypertension in primary renal cell carcinoma is extremely rare and a medline search of recent literature revealed a solitary report of a hypernephroma infiltrating the pancreas, causing splenic vein thrombosis with resultant gastric varices as a manifestation of portal hypertension . HoweverWhile our group has recently published a single center experience with metastases to the pancreas from renal tumors , this ra"} +{"text": "Objective: Our laboratory previously demonstrated that asymptomatic vaginal colonization during pregnancy is a factor predisposing patients to subsequent symptomatic vulvovaginal candidiasis. It is unknown whether symptoms result from strain replacement or a change in host relationship to the original colonizing strain. This study wasundertaken to determine whether Candida albicans isolates from asymptomatic women could be responsible for subsequent symptomatic vaginitis.Methods: We retained isolates of C. albicans from women followed longitudinally through pregnancy, and identifiedsix pairs of cultures from women who were colonized without symptoms and who later became symptomatic(average time 14 weeks). We used a random amplification of polymorphic DNA (RAPD) analysis to determinewhether isolates from our study patients were genetically similar or dissimilar.Results: Analysis of these pairs of yeast strains by RAPD revealed that five of the six women had symptomsapparently due to the same yeast strain that was found initially as a commensal strain. To increase the power ofthese observations, we also performed RAPD analysis on six randomly selected yeast strains from other women inthis study who had not become symptomatic to determine whether any of these unrelated strains matched strainsfrom those women who became symptomatic.Conclusion: Symptomatic yeast vaginitis is usually due to strains of C. albicans already carried in the lower genitaltract, underscoring the need to understand regulation of growth and virulence of the organism in vivo."} +{"text": "Archival paraffin embedded material was used to examine whether additional quantitative criteria would be helpful to discriminate between histologically benign and malignant rat mammary tumours. To this end nuclear DNA content expressed as DNA ploidy index (DI) was measured using flow cytometry (FCM). A total of 63 benign and malignant mammary tumours were investigated. Thirteen out of 38 (34%) mammary carcinomas were DNA aneuploid against 0 out of 25 benign mammary tumours. Aneuploidy was not significantly increased in tumours showing histological signs of greater malignancy such as cribriform-comedo type or invasive growth. In addition to DI other quantitative criteria indicative for malignancy, such as mitotic count and nuclear morphometric characteristics, were estimated in 24 benign and malignant tubulopapillary tumours, a category where the histological classification may be difficult. It appeared that five out of nine noninvasive tubulopapillary carcinomas and six out of seven invasive carcinomas had abnormal values for either DI, mitotic count or nuclear area or for a combination of these parameters. Each single parameter however was abnormal only in a minority of the malignant tumours. In this respect our data are in accordance with the fact that rat mammary carcinomas are clinically and histologically less malignant than their human counterparts."} +{"text": "The messenger RNAs for the angiogenic acidic and basic fibroblast growth factors are expressed at a significantly higher level in samples of human benign neoplastic and hyperplastic tissue than in samples from breast cancers. However, approximately one in four malignant breast cancer samples contain basic fibroblast growth factor mRNA at the same level as in the benign lesions when basic fibroblast growth factor mRNA levels are corrected with respect to levels of expression of glyceraldehyde-3-phosphate dehydrogenase mRNA. A similar proportion of human malignant breast cancer cell lines express a high level of basic fibroblast growth factor mRNA. The results suggest that some malignant breast cancers and their constitutive carcinoma cells express abundant levels of basic fibroblast growth factor mRNA. The resultant production of basic fibroblast growth factor by breast cancer cells within some tumours may contribute to their development."} +{"text": "There is no information on the effect of food or concurrent drug administration on the bioavailability of oral etoposide, despite the fact that treatment is frequently administered over several days and most often in combination with other cytotoxic agents. The influence of these factors has been studied in 11 patients, receiving combination cytotoxic therapy for extensive small cell lung carcinoma. Neither food nor concurrent oral or intravenous chemotherapy had a significant effect on the mean plasma concentrations of etoposide, achieved following oral administration. Wide variation in peak plasma concentrations and in area under the concentration time curve (AUC) occurred both between and within patients. It appears unnecessary for patients receiving etoposide (at 100 mg) to fast prior to drug administration. Furthermore, oral etoposide (at 100 mg and at 400 mg) may be given in combination with other cytotoxic agents without compromising its bioavailability."} +{"text": "Fluorescence in situ hybridisation (FISH) has been used increasingly for gene mapping and ordering probes on interphase and metaphase preparations. The association of consistent chromosomal aberrations with certain malignancies allows the possibility of using interphase cytogenetics as a diagnostic tool. In small round cell tumours of children accurate diagnosis may be difficult using existing methods. We have therefore evaluated the diagnostic potential of this technique when applied to the characteristic t found in Ewing's sarcoma and peripheral neuroectodermal tumour (ES and PNET). Interphase nuclei were prepared from normal human foreskin fibroblasts (HFF), two Ewing's sarcoma cell lines and several fresh tumour biopsies. DNA probes each side of the breakpoint at 22q12 were labelled with biotin and digoxygenin, hybridised to chromosomes in interphase and detected in different colours. Measurements between pairs of signals arising from each copy of chromosome 22 were taken and statistical analysis performed. There was a highly significant difference (P < 0.0001) between the two populations of measurements obtained ). Studying four tumours and one further ES line (blind) it was found that median values from 30 nuclei could correctly identify which samples contained the t. This application of interphase cytogenetics contributes a reliable, accurate and conceptually simple diagnostic test for ES and PNET. It may now be applied to other tumours with characteristic translocations, amplifications or deletions when suitable probes are available. This approach is likely to become a routine in clinical diagnosis."} +{"text": "Collagen breakdown, and thus bone resorption, can now be assessed by measuring the urinary excretion of the collagen crosslinks, pyridinoline (Pyd) and deoxypyridinoline (Dpd). In a pilot study we measured Pyd and Dpd in 20 patients with breast cancer, ten with known bone metastases and ten with no recognised metastases in bone or elsewhere after 1 year's subsequent follow up. Eight out of the ten patients with metastases had crosslink excretion values higher than the reference interval, but so did some patients without known metastatic disease. For both crosslinks there was a clear correlation with serum alkaline phosphatase activity measured at about the same time. We consider that measurement of urinary collagen crosslink assays may have a place in the early detection of metastatic spread to bone."} +{"text": "The in vitro cytotoxicity and oncogenic potential of both native and acid leached asbestos fibres were studied using the C3H 10T1/2 cell model. Both native and leached fibres induced a dose-dependent toxicity. At high fibre concentrations, acid leached fibres were significantly less toxic than their untreated counterparts. While asbestos fibres alone do not induce oncogenic transformation at the concentration examined, it was found that both leached and native fibres substantially enhanced the oncogenicity of gamma-irradiation in a more than additive fashion. Although no significant chromosomal aberrations or sister chromatid exchanges (SCE) were found in asbestos treated cultures, a significantly higher number of SCEs was observed in cells treated with both asbestos and radiation compared to cells receiving radiation alone. The results suggest that the enhancement in radiation induced oncogenicity by asbestos fibres may be attributed to the mere physical presence of the fibres rather than any chemical contaminants the fibres may contain. Furthermore, the carcinogenicity of asbestos may be unrelated to genotoxicity."} +{"text": "Cell division occurs during normal human development and aging. Despite the likely importance of cell division to human pathology, it has been difficult to infer somatic cell mitotic ages because direct counting of lifetime numbers of divisions is currently impractical. Here we attempt to infer relative mitotic ages with a molecular clock hypothesis. Somatic genomes may record their mitotic ages because greater numbers of replication errors should accumulate after greater numbers of divisions. Mitotic ages will vary between cell types if they divide at different times and rates.Age-related increases in DNA methylation at specific CpG sites (termed \"epigenetic molecular clocks\") have been previously observed in mitotic human epithelium like the intestines and endometrium. These CpG rich sequences or \"tags\" start unmethylated and potentially changes in methylation during development and aging represent replication errors. To help distinguish between mitotic versus time-associated changes, DNA methylation tag patterns at 8\u201320 CpGs within three different genes, two on autosomes and one on the X-chromosome were measured by bisulfite sequencing from heart, brain, kidney and liver of autopsies from 21 individuals of different ages.Levels of DNA methylation were significantly greater in adult compared to fetal or newborn tissues for two of the three examined tags. Consistent with the relative absence of cell division in these adult tissues, there were no significant increases in tag methylation after infancy.Many somatic methylation changes at certain CpG rich regions or tags appear to represent replication errors because this methylation increases with chronological age in mitotic epithelium but not in non-mitotic organs. Tag methylation accumulates differently in different tissues, consistent with their expected genealogies and mitotic ages. Although further studies are necessary, these results suggest numbers of divisions and ancestry are at least partially recorded by epigenetic replication errors within somatic cell genomes. Human \"age\" can be characterized by chronological age (time since the zygote) and mitotic age . Somatic cells within an individual have similar chronological ages but their mitotic ages may differ because different cell types divide at different rates and times. Cell division occurs during normal human development and aging, and many diseases are associated with excessive numbers of divisions. Despite the likely importance of cell division to human pathology, it has been difficult to measure mitotic ages because human lifetimes are long and direct observations or experimental manipulations are impractical.Here we attempt to infer relative somatic cell mitotic ages with a molecular clock approach . DivisioIt may be possible to replace the 5' to 3' order of bases with the 5' to 3' order of CpG DNA methylation because genome replication also involves the duplication of epigenetic patterns. Methylation also exhibits somatic inheritance , but is DNA methylation patterns may be directly compared between different aged individuals because methylation is removed early in development (before implantation) at most CpG islands ,9. This Replication errors may represent the major source of age-related methylation because genomes are regularly duplicated in mitotic tissues. In addition, cell division appears to be required for in vitro de novo methylation . PotentiSpecimens were obtained from 21 autopsies performed at the Los Angeles County-University of Southern California Medical Center . Approximately 500 ng of DNA was bisulfite treated for 12 hours at 50 C using an agarose bead method . Bead siApproximately 10% of the agarose bead containing bisulfite treated DNA was amplified for each PCR 42 cycles) of three different CpG rich loci or tags . The other cirrhotic liver (Pt Q) had high CSX tag methylation but not remarkably high SOX10 methylation. The liver with the highest CSX tag methylation was from a patient with AIDS (Pt L), with no obvious liver pathology. The two kidneys with high CSX tag methylation had benign nephrosclerosis, which was not observed in kidneys from individuals of similar age with lower CSX methylation. Average CSX and SOX10 tag methylation was higher in kidneys with benign nephrosclerosis, but the average age of individuals with benign nephrosclerosis was also higher, consistent with the observation that nephrosclerosis is more common in the elderly .Trend lines for average CSX or SOX10 tag methylation with age for heart, brain, liver and kidney are illustrated in Figure Although much is known about human development and aging, the exact fates of somatic cells are somewhat mysterious because it is difficult to observe cells throughout a lifetime. Potentially somatic cell mitotic ages may be inferred by counting numbers of replication errors using a molecular clock approach . Here wePrior studies of mitotic adult tissues (intestines and endometrium) demonstrated age-related tag methylation consistent with methylation representing replication errors -13. CellCell division occurs rarely in normal adult liver, perhaps once a year . ConsistA molecular clock approach can compare genomes in cells of widely different phenotype. For example, sequences potentially infer ancestry across all three kingdoms of life . SimilarDifferent tags may have different replication error rates and attain different levels of methylation. Tags with higher error rates better document early development because the mitotic ages of neonatal and infant tissues are relatively low. For example, SOX10 tags become nearly fully methylated by infancy and therefore only minimal changes are possible even with further increases in mitotic age. By contrast, tags such as CSX with lower error rates exhibit fewer changes early in life but more readily document further mitotic age changes during aging.Mitotic age contains additional information about somatic cell genealogy because only certain cell types divide during the differentiation from a zygote to present day cells. Somatic cell genealogy can be divided into three successive phenotypic phases \u2013 development from the zygote, a stem cell phase, and differentiation Figure . GenealoThe first pattern, observed in non-mitotic adult tissues such as the brain and heart, is a static genealogy. Mitotic age remains constant during chronological aging, and replication errors found in adult cells primarily represent divisions during earlier organ developmental and differentiation. Liver and kidney also have this general pattern, but further stem or differentiated cell divisions may occur during adult life from normal cell turnover or in response to cell loss.A second pattern, observed in mitotic epithelium such as the intestines and endometrium, is a continuous genealogy that expands as replication errors accumulate throughout life. Replication errors likely accumulate primarily in adult stem cells because stem cell lineages are long lived and numbers of divisions during development and differentiation are usually similar regardless of chronological age. For example, intestinal development is limited to the neonatal period and differentiated cells in the intestines divide only a few times and survive about a week . TherefoA third pattern, observed in hair follicles, is a punctuated genealogy. There is a lack of a measurable age-related increase in average replication errors despite high hair follicle mitotic activity . This paSelection can alter error frequencies by favoring or eliminating some types of errors. Methylation can potentially confer selection because promoter methylation is associated with loss of expression, and promoter methylation is another mechanism to silence tumor suppressor genes in cancer . The locReplication errors are stochastic and the methylation of a single allele is relatively uninformative. Our analysis sampled a relatively small number of tags from only a few different loci, but illustrates the potential for \"somatic cell clocks\" to record mitotic ages. More information can be obtained with more tags from the same \"clock\" locus or from multiple \"clock\" loci. Methylation may not be independent between CpG sites and error rates may vary between CpG sites within a tag or between cell types. However, with most replication error mechanisms, average methylation should generally correlate with mitotic age. Another biological source for tag variation is the sampling of heterogeneous cell types. In this study, organ fragments containing uncertain mixtures of different cell types were analyzed. Ideally tags from small homogeneous clones of cells woulThe present study provides tentative evidence that somatic cell mitotic ages may be recorded within their genomes by methylation changes at certain CpG rich loci. The exact mechanisms responsible for methylation changes at our \"epigenetic molecular clocks\" are uncertain, but many changes appear to represent replication errors. Mitotic age in actively dividing adult tissues may largely depend on the behavior of their stem cells because numbers of divisions during development and differentiation are limited. There are a number of challenges to inferring genealogies from replication errors, but such an approach potentially allows for systematic studies of human development and aging without prior experimental interventions.The author(s) declare that they have no competing interests.MWC, CLE, and JYK performed most of the experiments and helped write the manuscript. ASY and GCK supplied specimens. KDS and ST helped with the statistical and quantitative analysis. DS analysed the data and wrote the manuscript.The pre-publication history for this paper can be accessed here:"} +{"text": "I agree with Stevens that free radicals and changes in oxidative state in cells could play an important mediating role in some biological effects of nonionizing electromagnetic fields (EMFs), such as DNA damage. Certainly, cellular iron metabolism affects these processes. Genetic damage in cells can lead to malignancy and cancer. However, excessive cumulative genetic damages could also result in cell death. One possibility is that EMFs may be useful in treating cancer."} +{"text": "A single left coronary artery with right coronary artery arising from either left main stem (LMS) or left anterior descending artery (LAD) or circumflex artery (Cx) is an extremely rare coronary anomaly. This is the first report of separate origins of proximal and distal RCA from LAD and circumflex arteries respectively in a patient with a single left coronary artery. This 57 year old patient presented with unstable angina and severe stenotic disease of LAD and Cx arteries and underwent urgent successful quadruple coronary artery bypass grafting. The anomalies of right coronary artery in terms of their origin, number and distribution are reviewed. A 57 year old male presented with unstable angina. Risk factors included hypertension and hypercholesterolaemia. Significant past history included multiple episodes of deep vein thrombosis and pulmonary embolism. Coronary angiography demonstrated a single coronary artery arising from left coronary sinus fig which diPatient was taken to theatre for urgent CABG. Operative findings confirmed the following: Proximal RCA was arising as a branch from the proximal LAD after the first diagonal, crossed the RVOT and gained the anterior aspect of the right AV groove to anastomose with the distal RCA which arose as a continuity of circumflex artery. LAD gave off two further branches to supply the right ventricle Fig . The disIn a study of 126,595 patients who underwent coronary angiography over a period of 28 years, from 1960 to 1988, at Cleveland Clinic, Yamanaka et al reported coronary artery anomalies in 1686 1.3%) patients. 1461 (87%) had anomalies of origin and distribution, and 225 (13%) had coronary artery fistulae [% patientRight coronary anomalies can involve origin or distribution of the artery. When right coronary artery arises anomalously from the left coronary sinus -6, it ofDouble right coronary arteries have been anecdotally reported. They can be different to differentiate from a high take off of a large right ventricular branch , both thA single left coronary artery arising from the left coronary sinus is a rare anomaly with right coronary artery arising from either the left main stem or the branches downstream from the main stem. If arising from left main stem, as also when arising from a separate ostium in the left coronary sinus, the anomalously arising right coronary artery can course anterior, posterior or in between the great arteries, the last course constituting a malignant type of anomaly due to extrinsic compression. Arteaga described the third case of the Shirani-Roberts subtype IB4 anomaly with the right coronary artery arising from the left main stem and coursing behind the aorta to reach the right heart . Ajith eRight coronary artery arising from left anterior descending artery in the absence of a normally situated right coronary ostium is considered a variant of single left coronary artery and ten such cases have been described in world literature -33. In mShammas et al reported two cases of a single left coronary artery with continuation of circumflex as the distal right coronary artery without stenotic disease . In a seThus, in a circulation where there is a single left coronary artery and absent right coronary ostium, the right coronary artery tends to originate as a single vessel either from the left main stem or from the LAD or circumflex systems. Yamanaka et al reported 31 cases of single left coronary artery and categorised them into two groups ["} +{"text": "In Japan, the emergency medical system is categorized into three levels: primary, secondary, and tertiary, depending on the severity of the condition of the patient. Tertiary care centres accept patients who require 24-h monitoring. In this research, the average travel times (minutes) from the centroids of all municipalities in Japan to the nearest tertiary care centre were estimated, using the geographic information system. The systems affecting travel time to tertiary care centres were also examined. Regression analysis was performed to determine the factors affecting the travel time to tertiary care centres, using selected variables representing road conditions and the emergency transfer system. Linear regression analysis was performed to identify specific benchmarks that would be effective in reducing the average travel time to tertiary care centres in prefectures with travel times longer than the average 57 min.The mean travel time was 57 min, the range was 83 min, and the standard deviation was 20.4. As a result of multiple regression analysis, average coverage area per tertiary care centre, kilometres of highway road per square kilometre, and population were selected as variables with impact on the average travel time. Based on results from linear regression analysis, benchmarks for the emergency transfer system that would effectively reduce travel time to the mean value of 57 min were identified: 26% pavement ratio of roads , and three tertiary care centres and 108 ambulances.Regional gaps in the travel time to tertiary care centres were identified in Japan. The systems we should focus on to reducing travel time were identified. Further reduction of travel time to tertiary care centres can be effectively achieved by improving these specific systems. Linear regression analysis showed that a 26% pavement ratio and three tertiary care centres are beneficial to prefectures with an average time longer than the mean score, to achieve a reduction of travel time. Measures for reducing travel time need to be considered in policy-making to re-evaluate the current locations of tertiary care centres to provide equality of access to emergency medicine. In Japan, the emergency medical system has been provided systematically as a result of the Medical Care Law enacted in 1985, to ensure that \"anyone can receive appropriate emergency medical care anytime, anywhere\". The actual framework and infrastructure of emergency medical care have been developed through Medical Care Planning, which is ruled by Medical Care Law to establish the provision of the health care system in Japan. Medical Care Planning specifically states the requirement of, \"securing and maintaining the emergency medical care system\" . \"TertiaAs tertiary medical care targets patients with serious conditions, travel time to the tertiary care centre has a significant impact on the survival rate of patients, especially when the condition involves cerebral haemorrhage, subarachnoid haemorrhage, acute myocardial infarction, acute heart failure, pneumonia, or cardio pulmonary arrest (CPA) . HoweverIt is estimated that the travel time to tertiary care centres is affected by the current road conditions ,8 as welHistorically, research on travel time in medical accessibility has utilized GIS, and a number of studies have been published on various types of health service. In studies on accessibility to general practitioners, Lovett et al. examinedN = 47), and Figure A histogram of average travel time (minutes) is shown in Figure Population values are shown in a histogram Figure and the 2) of each prefecture was also analysed . The variable with Pearson's or Spearman's correlation of less than 0.8 and with meaningful interpretation, i.e., the \"number of primary hospitals per million capita\" was used to adjust for population. To adjust for size the same criteria as in the case of adjusting for population were used; the following variables were adjusted: \"population density (population/size)\", \"kilometres of general road per square kilometre\", \"kilometres of highway road per square kilometre\", \"total kilometres of all roads per square kilometre\", \"kilometres of impassable road per square kilometre\", \"average coverage area per tertiary care centre\", \"average coverage area per fire station\", \"average coverage area per ambulance\", \"average coverage area per emergency team\", \"average coverage area per emergency team with EMT\", \"average coverage area per EMT\", \"average coverage area per emergency crew\", \"average coverage area per emergency medical specialist\", \"average coverage area per tertiary hospitals\", \"average coverage area per secondary hospitals\", and \"average coverage area per primary hospitals\" Population: \"total kilometres of all roads per square kilometre\" and \"population density\"2): \"mountain area (km2)\"2) Size (km3) Kilometres of general road per square kilometre: \"total kilometres of all roads per square kilometre\", \"kilometres of impassable road per square kilometre\", \"average coverage area per emergency team\", and \"average coverage area per ambulance\"4) Total kilometres of all roads per square kilometre: \"average coverage area per ambulance\", \"average coverage are per emergency team\", \"average coverage area per emergency team with EMT\", \"average coverage area per secondary hospitals\" and \"population density\"5) Average coverage area per tertiary care centre: \"average coverage area per emergency team with EMT\", \"average coverage area per EMT\", \"average coverage area per emergency crew\", \"average coverage area per tertiary hospitals\" and \"population density\"6) Average coverage area per fire station: \"average coverage area per ambulance\", \"average coverage area per emergency team\", \"average coverage area per emergency crew\" and \"population density\"7) Average coverage area per ambulance: \"average coverage area per emergency team\", \"average coverage area per emergency team with EMT\", \"average coverage area per EMT\", \"average coverage area per emergency crew\", \"average coverage area per tertiary hospitals\", \"average coverage area per secondary hospitals\" and \"population density\"8) Average coverage area per emergency team: \"average coverage area per emergency team with EMT\", \"average coverage area per EMT\", \"average coverage area per emergency crew\", \"average coverage area per tertiary hospitals\", \"average coverage area per secondary hospitals\" and \"population density\"9) Average coverage area per emergency team with EMT: \"average coverage area per EMT\", \"average coverage area per emergency crew\", \"average coverage area per secondary hospitals\", \"average coverage area per tertiary hospitals\" and \"population density\"10) Average coverage area per EMT: \"average coverage area per emergency crew\", \"average coverage area per emergency medical specialist\", \"average coverage area per tertiary hospitals\", \"average coverage area per secondary hospitals\" and \"population density\"11) Average coverage area per emergency crew: \"average coverage area per tertiary hospitals\" and \"population density\".Therefore, variables with correlation coefficient of more than 0.8 which were excluded from the regression analysis were: \"population density\", \"total kilometres of all roads per square kilometre\", \"mountain area\", \"kilometres of impassable road per square kilometre\", \"average coverage area per emergency team\", \"average coverage area per ambulance\", \"average coverage area per emergency team with EMT\", \"average coverage area per emergency crew\", \"average coverage area per EMT\", and \"average coverage area per tertiary hospitals\" Table and 7.p = 0.150), and would not be at a statistically significant level (p = 0.150) when other variables were added into the model. Consequently, \"average coverage area per tertiary care centre\", \"kilometres of highway road per square kilometre\", and \"population\" were selected. As a result of multiple regression analysis with these variables (Table R2 = 0.7001 (Table Stepwise (forward-backward) selection was performed in order to refine variables contributing to the model Table . Variables Table , adjuste01 Table . The fin01 Table .y = a + bx), where x is the emergency transfer system and y is the average travel time. The x value of each regression equation was calculated in all emergency teams, 69%; ambulances, 108; emergency teams, 90; EMTs, 262; general road per kmThe demand for emergency medicine in Japan has been increasing; in 1996 the number of emergency patients transferred to hospitals was 324,000, whereas in 2003 that number increased to 457,000 . In NagaIn Japan, focus on the need for emergency medicine coincided with the formulation of the Government's Medical Care Planning around 1985. At that time, establishment and development of the health care system was designed with focus on quantity. Thus, one of the drawbacks of such efforts is that resources regarding emergency medicine are not efficiently allocated. This may result in some situations where tertiary care is not accessible. As such, while the emergency medical system in Japan has been developed, regional gaps by prefectures were found in the number of emergency medical specialists as well as facilities . The lacThis study analysed equitable access to emergency care; thus the issue of quality has not been examined. As adequate quality as well as quantity of health care provision is necessary for equitable access to emergency care, further studies on quality are required.As the population of Japan is rapidly aging, the characteristics of diseases are changing correspondingly. Thus, the emergency medical system requires further research to ensure equitable care going forward. We believe the results of our study provide beneficial information to health policy administrators in formulating Medical Care Planning and, in general, in the decision-making process for health policy. By enhancing the emergency transfer system as we have set out here, travel time to emergency care would be reduced, ultimately leading to an improvement in the survival rate of emergency patients.Official data on the outcome of emergency medical patients is not available. Specifically, the survival rate and the number of patients transferred to tertiary care centres have not been disclosed. Thus we performed this research using officially available data, such as the development of roads and the status of the emergency transfer system, as factors that impact travel time. In the near future, the collection and disclosure of such outcome data will be desirable. One reason for the unavailability of outcome data could be because the emergency transfer system is dual-administered by the FDMA and the MHLW. If this dual system is generating inefficiencies, further review of this issue may be necessary.In this research, \"travel time\" represents only Process 4 of the emergency transfer system , and to ultimately perform multiple regression analysis, the selection of variables was conducted as follows:2) or population, or be kept unadjusted, correlation analysis (Spearman and Pearson) between population and size (km2) with each variable was performed. Based on the correlation analysis, the variables used for regression analysis of average travel time were selected. Hokkaido was excluded from the analysis as an outlier.(1) To determine if the variables should be adjusted for size (km(2) Single regression analysis of the average travel time was performed using variables selected from the correlation analysis.(3) Correlation analysis between variables was performed using the variables selected for the single regression analysis, and candidate variables for multiple regression analysis were refined.(4) Stepwise selection was performed in order to refine variables contributing to the model. Consequently, multiple regression analysis was performed using selected variables and factors impacting average travel time.t values, and statistical significance. This was performed in order to estimate the benchmarks in the emergency transfer system that would be effective in reducing the average travel time to tertiary care centres in prefectures with travel times longer than the average 57 min.(5) As a result of the single regression analysis in (2), linear regression was performed to model the relationship between the average travel time and variables with negative SAS9.1.3 and the SPSS 12.0 were used for the statistical analyses.This paper examined and modelled average travel time (minutes) to tertiary care centres in Japan. Using GIS software , we constructed geographical plots representing the centroids of 2594 municipalities and tertiary care centres. Based on travel distance (not straight line distance) using road network information and average travel speed , travel times (minutes) to tertiary care centres were calculated. Consequently, travel distance (km) and travel time (minutes) from the centroid of all municipalities to the nearest tertiary care centre were estimated. Travel times to tertiary care centres in each municipality were then aggregated by the 47 prefectures, and the average travel time (minutes) for each prefecture was calculated.Travel time (minutes) used in this research does not include the whole time required in the full procedure of the emergency transfer system, which is measured as the time required from the first response to the emergency call to the start of treatment after the patient is transferred to the tertiary care centre. The emergency transfer system in Japan is coordinated by both the fire department system and the emergency medical system. Specifically, once computer-assisted emergency dispatch operators respond to emergency calls, the nearest available ambulance is dispatched from the fire station to the scene of the incident. When the ambulance arrives at the emergency scene, the emergency crew determine the nearest available hospital and the ambulance will go to that hospital. In this study, travel time is defined as the time taken from the determination and confirmation of hospital to arrival at the tertiary care centre. As shown in Figure We estimated the travel time assuming the use of private car or ambulance. Public transport, such as railway or bus, and other means of transport such as helicopters were not included in this study. Generally speaking, in most cases of emergency transfer, the percentage transported by ambulance was 99.9%, and helicopter transport accounted for 0.04% . In remoUsing the above-mentioned travel time, our study was undertaken to identify:(1) the average travel time (minutes) to a tertiary care centre for all prefectures in Japan and the regional gaps;(2) the effects of development of the emergency transfer system and the effect that improvements in road conditions would have on the travel time (minutes) to tertiary care centres;(3) benchmarks for the emergency transfer system that would be effective in reducing the average travel time (minutes) to tertiary care centres in prefectures with travel times longer than the average 57 min.The author(s) declare that they have no competing interests.MM collected data and carried out the analysis. KH organized the collection of information on tertiary care centres. AH collaborated in the development of the paper. KK organized the management of the study."} +{"text": "This prospective study of 63,090 Norwegian women with 124 cases of thyroid cancer diagnosed during 1961-1989 revealed no strong associations with reproductive factors. Late last birth was related to increased risk, whereas no association was noted with parity. A long reproductive period was related to increased risk of papillary carcinomas, whereas a decreased risk of follicular carcinomas and other adenocarcinomas was observed in women with early menarche and late menopause. The risk of thyroid cancer was significantly increased among women in the occupational category 'fishing, ships officers and crew'. Our results are consistent with a modest effect of certain reproductive factors upon thyroid cancer development."} +{"text": "The crucial role of non-plasminogen dependent serine proteinases is tissue invasive and cytolytic functions of Walker 256 cancer cells has been documented using a rat urinary bladder invasion and a 125I-labelled fibroblast cytolysis assay. The invasive capacity of these cancer cells was abrogated by non toxic concentrations of the serine proteinase inhibitors, diisopropylfluorophosphate and phenylmethylsulfonylfluoride, but not by metallo or cysteine proteinase inhibitors. Although tumour cell collagenase activity and plasminogen activator were demonstrated, these proteolytic enzymes were not essential in these in vitro assays. These results suggest that different categories of proteinases play specific roles in the complicated process of cancer invasion."} +{"text": "In order to investigate allele loss in colorectal tumours we have developed a rapid technique which overcomes most of the problems associated with radioactive Restriction Fragment Length Polymorphism (RFLP) analysis of allele loss. We utilise microsatellite length polymorphisms which are highly informative and are closely linked to loci of interest. Sequences containing microsatellites can be amplified from normal and tumour DNA pairs by a polymerase chain reaction (PCR) in which one of the primers is fluorescently labelled. This enables us to detect the products on polyacrylamide gels run on an automated DNA sequencer using dedicated software, by which results are automatically quantitated in terms of peak size, height, and area. Using this technique we have analysed 26 normal tissue: cancer pairs for allele loss at two loci linked to the adenomatous polyposis coli (APC) gene on chromosome 5q. Repeated assays yielded identical results for each pair. Allele loss was found in 10 out of 25 informative samples (40%)."} +{"text": "Flow cytometry and histopathology were utilised in evaluating 50 primary and 16 metastatic colorectal carcinomas to determine the influence of heterogeneity and proportion of dying cells on pathological assessments. A new procedure was developed for staining unfixed whole cells with acridine orange and ethidium bromide to quantify DNA and RNA content and number of dead and dying cells. Attempts were made to reduce interobserver variation in histological assessment and to determine whether flow cytometry could refine current grading and staging procedures. Interobserver variation in grading was not improved by estimating proportions of differing grades in multiple samples from individual tumours. Considerable heterogeneity was observed within tumours although this was less apparent when defining ploidy status than histological grade. No consistent differences were observed between superficial and deep parts of tumours or between primary and secondary tumours by either method of analysis. The proportion of dead and dying cells varied widely between tumours but there was no correlation with tumour grade or stage. Non-diploid tumours were not of more advanced stage or poorer histological grade than diploid tumours. Since ploidy status may be an important prognostic factor, analysis of colorectal carcinomas by flow cytometry could be of greater value than conventional grading and staging procedures."} +{"text": "Sympatric speciation\u2014the divergence of populations into new species in absence of geographic barriers to hybridization\u2014is the most debated mode of diversification of life forms. Parasitic organisms are prominent models for sympatric speciation, because they may colonise new hosts within the same geographic area and diverge through host specialization. However, it has been argued that this mode of parasite divergence is not strict sympatric speciation, because host shifts likely cause the sudden effective isolation of parasites, particularly if these are transmitted by vectors and therefore cannot select their hosts. Strict sympatric speciation would involve parasite lineages diverging within a single host species, without any population subdivision.Here we report a case of extraordinary divergence of sympatric, ecologically distinct, and reproductively isolated malaria parasites within a single avian host species, which apparently occurred without historical or extant subdivision of parasite or host populations.This discovery of within-host speciation changes our current view on the diversification potential of malaria parasites, because neither geographic isolation of host populations nor colonization of new host species are any longer necessary conditions to the formation of new parasite species. Plasmodium and HaemoproteusMalaria parasites comprise a diverse group of protozoans that infect reptiles, mammals and birds, and that are transmitted through the bite of different families of blood-feeding insect vectors Haemoproteus parasite lineages that has occurred within a single bird host species, the blackcap Sylvia atricapilla. To illustrate such occurrence, we (i) determine the distribution of parasite lineages among 47 passerine species that are sympatric to the blackcap, showing that most blackcap parasites are exclusive to this species, (ii) determine the evolutionary relationships of parasites found in blackcaps and its closest relatives, demonstrating that many blackcap parasites are included in a monophyletic group that apparently diverged within the blackcap, (iii) analyse whether such group of parasites are reproductively isolated entities, and (iv) analyse the geographic structuring of genetic variation of blackcap parasites, showing that the probability that this group of parasites evolved in allopatry is very slim.Here we describe a case of great diversification of b gene of malaria parasites found in European passerine birds to broadly investigate the phylogenetic diversity of this group of organisms. Our survey included 4470 individual birds of 47 European species, and 1911 parasite infections, each one involving one to four parasite lineages distinguished through their DNA sequences Plasmodium (45 lineages) or the Haemoproteus (92 lineages) genera by comparing their DNA sequences with those of parasites identified by microscopy.We sequenced part of the mitochondrial cytochrome 25). This index of parasite richness was higher in blackcaps (R25\u200a=\u200a9.6\u00b10.06) than in any other widely sampled bird species , despite of the fact that both species are sympatric over most of their ranges and were often trapped at the same sites , which is the closest extant relative to the species pair formed by blackcaps and garden warblers Given that blackcaps harboured many more of these monophyletic parasites than garden warblers, we suspected that parasites might have diversified in blackcaps after both bird species diverged from each other, and then some parasite lineages switched host from blackcaps to garden warblers . To furtHaemoproteus parasite flock. Both cytochrome b and DHFR-TS genes produced identical phylogenies, although two mitochondrial lineages shared identical nuclear sequences (P\u200a=\u200a0.0095). Random effects are also unlikely to explain linkage between nuclear and mitochondrial haplotypes determined in single infections of the same parasite lineages . This result supports the hypothesis that the diversification of blackcap malaria parasites occurred without long-term population subdivision.Blackcaps were sampled in seven breeding populations distributed between southern Spain and Sweden, but there was no geographic structure in the genetic variation contained in the parasite flock infecting this species methods We detected malaria infections by amplification of 479 bases of the parasite cytochrome b sequences was tested by 5000 permutations of parasite haplotypes among populations We analysed the genetic structure of the blackcap parasite flock using an analysis of molecular variance (AMOVA) n>40 birds and >25 scored infections), which were used to estimate intraspecific parasite richness (25). Average curves and R25 values (\u00b1S.E.) were derived from 1000 richness curves constructed by randomly changing the order in which individual hosts were screened. While the number of parasite lineages found in one species depended on the number of infections scored , R25 was independent of sample size .Aside from blackcaps, we extensively sampled 14 other species (richness , Table 1"} +{"text": "The article \u201cAgricultural Task and Exposure to Organophosphate Pesticides among Farmworkers\u201d seems toIn the abstract, Little is known about pesticide exposure among farmworkers, and even less is known about the exposure associated with performing specific tasks.The investigators open weakly by ignoring the substantial exposure (amount per person) data available related to work tasks of handlers [Pesticide Handlers Exposure Database; We commend n = 211; 2\u20136 years of age). These data do not permit estimation of dose, and they prohibit full evaluation of the relationship of exposure from parents\u2019 work tasks or other sources to the dimethyl metabolites from residential exposures, particularly diet (rly diet . It seemrly diet than to rly diet proposedEHP or otherwise made available to investigators.The data presented by"} +{"text": "The modern generation of transthoracic defibrillators now employ impedance compensated biphasic waveforms. These new devices are superior to those with monophasic waveforms and practice is currently switching to biphasic defibrillators for the treatment of both ventricular and atrial fibrillation. However, there is no universal guideline for the use of biphasic defibrillators in direct current cardioversion of atrial fibrillation. This article reviews the use of biphasic defibrillation waveforms for transthoracic cardioversion of atrial fibrillation. Electrical cardioversion is a commonly performed procedure for the treatment of atrial fibrillation (AF) that has been successfully employed since the 1960\u2019s . InductoIt was recognised subsequently that biphasic waveforms offered distinct advantages over monophasic waveforms in ventricular defibrillation . These results stimulated further research into biphasic waveforms for defibrillation of AF, where similar results were reported for internal and external cardioversion.Some physicians have questioned whether biphasic waveforms offer significant advantages over monophasic defibrillators. It has been noted that monophasic waveforms result in similar rates of cardioversion by the maximum energy of a treatment protocol. It is already known that DCC does not result in cardiac damage at currently applied energies . In addiMany studies have shown that lower energy impedance compensated biphasic (ICB) waveforms are equivalent or superior to the higher energy monophasic waveforms 5-13]. -13. 5-13Manufacturers are no longer producing monophasic defibrillators. These older devices will be superseded and replaced by those with biphasic waveforms. This will result in an inevitable shift in practice toward biphasic DCC of atrial arrhythmias.Antero-posterior (AP) and antero-apical (AA) electrode positions are commonly employed during DCC of AF. There has been considerable debate as to whether any one position is optimal. Historically, AP configurations have been considered to provide a better \u201cshock vector\u201d through the atria compared with the AA configuration ,19]. Se. Se19]. TTI is known to be an important determinant of both atrial and ventKirchhof and colleagues have recently examined the midline AP configuration with non-impedance compensated monophasic and impedance compensated biphasic waveforms using hand-held paddles (a factor that is known to reduce TTI) . This poThere are 2 distinct approaches to energy selection for DCC. Some physicians feel that it is best to select the highest possible energy in order to maximize the chance of initial success, minimize the number of shocks and lessen the exposure to sedative agents. Others prefer to follow an escalating energy protocol. Lown\u2019s rationale for this approach was to minimise post-shock arrhythmia . This meAt present each manufacturer recommends different energy selection protocols for their devices. Whilst lower energy shocks may be efficacious in patients with AF of short duration (1 week or less), published studies show that shocks of \u2265150 J will result in a success rate of ~80% . Two manRecent clinical trials have demonstrated that a rate control and anticoagulate strategy is acceptable for many patients with AF. The AFFIRM and RACEDirect current cardioversion of AF using biphasic waveforms is highly efficacious and is superior to monophasic waveforms. All manufacturers\u2019 devices have been shown to be effective for this procedure. The choice of electrode position appears to be less important when impedance compensated biphasic waveforms are employed for DCC. An initial energy of at least 150 J should be selected for DCC of AF, although only a small minority of patients will benefit from shocks of greater than 200 J."} +{"text": "Forty patients with metastatic adenocarcinoma of the prostate were evaluated for response to treatment with aminoglutethimide plus cortisone acetate. All had relapsed from or failed to respond to primary endocrine treatment with orchidectomy or stilboestrol. Nineteen patients (48%) showed subjective response, in most cases relief of bone pain. Side effects limited treatment in only 3 patients. We conclude that aminoglutethimide plus cortisone acetate is a useful addition to the treatment available for this difficult group of patients. The mechanism by which this treatment has a beneficial effect remains unclear."} +{"text": "The small intestine is constructed of many crypts and villi, and mouse studies suggest that each crypt contains multiple stem cells. Very little is known about human small intestines because mouse fate mapping strategies are impractical in humans. However, it is theoretically possible that stem cell histories are inherently written within their genomes. Genomes appear to record histories (as exemplified by use of molecular clocks), and therefore it may be possible to reconstruct somatic cell dynamics from somatic cell errors. Recent human colon studies suggest that random somatic epigenetic errors record stem cell histories . Potentially age-related methylation also occurs in human small intestines, which would allow characterization of their stem cells and comparisons with the colon.Methylation patterns in individual crypts from 13 small intestines (17 to 78 years old) were measured by bisulfite sequencing. The methylation patterns were analyzed by a quantitative model to distinguish between immortal or niche stem cell lineages.Age-related methylation was observed in the human small intestines. Crypt methylation patterns were more consistent with stem cell niches than immortal stem cell lineages. Human large and small intestine crypt niches appeared to have similar stem cell dynamics, but relatively less methylation accumulated with age in the small intestines. There were no apparent stem cell differences between the duodenum and ileum, and stem cell survival did not appear to decline with aging.Crypt niches containing multiple stem cells appear to maintain human small intestines. Crypt niches appear similar in the colon and small intestine, and the small intestinal stem cell mitotic rate is the same as or perhaps slower than that of the colon. Although further studies are needed, age-related methylation appears to record somatic cell histories, and a somatic epigenetic molecular clock strategy may potentially be applied to other human tissues to reconstruct otherwise occult stem cell histories. The human small intestine is the longest segment of the gastrointestinal tract, approximately five to six meters long in adults. Although it is three to four times longer than the colon, small intestinal tumors are rare, forming about 1% of all gastrointestinal malignancies -4. Stem Although there are likely to be many stem cells per crypt -4, intesStem cell turnover by a niche mechanism ,6 can exHuman stem cell studies are difficult because the powerful visible fate-marking strategies of animal models are impractical. Most human crypts are visually homogeneous and therefore consistent with either immortal or niche stem cell mechanisms. A study of human colon crypts after therapeutic radiation was consistent with stem cell niches because the frequencies of mutant heterogeneous crypts progressively declined after radiation . SimilarOne method that can reconstruct the past without direct serial observations infers ancestry from present day sequences. The past is encoded in sequences because errors may accumulate in a clock-like fashion (the molecular clock hypothesis ) and recMutations are rare in normal cells, but recent studies illustrate that stem cell ancestries may be automatically recorded by random somatic epigenetic errors that frequently accumulate with aging . Methyla2 patches of fresh small intestines obtained from 13 patients undergoing surgery for non-small intestinal diseases . After a given number of stem cell divisions, eight alleles are randomly sampled from each simulated crypt, as in the experimental approach. Niche and immortal scenarios were identical except niche stem cells also exhibit symmetric divisions (P1 < 1.0). Total niche stem cell numbers remain constant because divisions that produce two differentiated daughters are balanced by divisions that produce two stem cell daughters (P0 = P2). Symmetric divisions tend to reduce crypt diversity because methylation patterns can be lost through lineage extinction.The Paneth cell compartment was not specifically modeled because its dynamics are uncertain and these cells would be rarely sampled because their relative numbers are small (~2\u20134 Paneth cells per crypt section ). PanethMethylation of CSX or MYOD \"tags\" can be converted into a 5' to 3' binary code, with \"0\" representing an unmethylated site and \"1\" representing a methylated site. For example, an unmethylated CSX tag (eight CpG sites or 256 different possible tags) is \"00000000\" and a fully methylated MYOD tag (5 CpG sites or 64 different possible tags) is \"11111\". Tags can be summarized with three statistics that reflect numbers and types of stem cell divisions:1) Percent Methylation. Numbers of methylated tag sites can be summarized by percent methylation. For example the CSX tag \"01010101\" is 50% methylated. In general, percent methylation reflects numbers of divisions since birth.2) Unique Tags per Crypt. Diversity, or numbers of unique tags among the eight tags sampled from a crypt, reflects numbers of stem cells and stem cell lineage survival. Greater numbers of stem cells or longer-lived stem cell lineages would lead to greater crypt tag diversity.3) Intracrypt Distance. This is the average number of site differences between crypt tags (Hamming distance). For example, the distance between CSX tags 00000011 and 11000000 is four. Intracrypt distance is another measure of crypt diversity.Crypts of Lieberk\u00fchn and villus fragments were isolated from fresh human small intestine Figure and indiDifferent tags within the same intestine are consistent with stochastic errors that accumulate independently in different cells. To extract ancestral information encoded by seemingly random tags within a single intestine, crypts were modeled assuming stochastic methylation errors and either immortal or niche stem cells (see Methods). The primary difference between the models is that the probability of symmetric division (yielding two stem cell P2) or two differentiated daughters (P0)) is zero with immortal stem cells, whereas the probability of symmetric division is greater than zero with niche stem cells better fit the small intestine data and less intracrypt diversity than immortal stem cells Figure . Small iNiche stem cells defined by ancestry are physically intangible because common ancestors are defined by past events and no longer exist. Stem cells with immortal lineages are more readily identified because their pasts and futures are predictable. However, niche stem cell fates are unpredictable because all may potentially become common ancestors but only one will. The inability to predict niche survival may help explain why some adult stem cells have been so difficult to isolate or characterize. A niche somatic cell tree has few branches because random stem cell turnover eventually \"prunes\" all niche lineages except one Figure . Such boOther mechanisms may also be consistent with crypt methylation patterns and the assumptions of our model remain unverified . For exaOne strategy to test empirically a stochastic model that inherently yields scattered results is to examine similar but biologically distinct entities. Both small intestine and colon crypts are thought to be maintained by stem cell niches ,6 and thMouse studies suggest that stem cell division rates are higher in the small intestines than the colon ,3,20. HoThere is no definitive evidence supporting a somatic cell molecular clock hypothesis, but a priori a slower small intestinal stem cell division rate is more consistent with human intestinal cancer epidemiology , becauseMethylation patterns suggest that niches containing multiple stem cells maintain crypts throughout the lower gastrointestinal tract. Small intestine stem cells appear to divide at equivalent or slower rates relative to the colon, and niche dynamics remain stable during aging. Although methylation tags only indirectly track stem cell dynamics and their exact interpretations are uncertain, they potentially allow for the systematic investigation of any intestine without prior experimental intervention. A somatic cell tree must underlie all human tissues and studies based on the hypothesis that certain somatic methylation errors record somatic cell histories may better define how cells divide and die during normal and abnormal aging.The author(s) declare that they have no competing interests.JYK performed most of the experiments and helped write the manuscript. KDS and ST helped with the statistical and quantitative analysis. DS analyzed the data and wrote the manuscript.The pre-publication history for this paper can be accessed here:"} +{"text": "EHP, Barr et al. used data from the Third National Health and Nutrition Examination Survey (NHANES III) to establish reference ranges for urinary creatinine for specific age and demographic categories (Urinary creatinine is almost universally employed to adjust concentrations of urinary analytes for variations in hydration status. In the February 2005 issue of tegories . They reOur study in Bangladesh on 1,650 adults revealed that urinary creatinine concentrations are significantly correlated with plasma folate concentrations\u2014particularly among males, who had a higher prevalence of folate deficiency than females in Bangladesh . AlthougIn summary, we concur with"} +{"text": "You might say that a mother\u2019s influence begins even before her offspring were just a twinkle in dad\u2019s eye. During egg cell development , the mother deposits gene products\u2014consisting largely of messenger RNAs (mRNAs) and some proteins\u2014into the developing egg. These mRNAs take over after fertilization to orchestrate the earliest stages of embryogenesis. As the embryo develops, the fertilized egg (or zygote) cuts the apron strings as it activates its own genome\u2014which contains both maternal and paternal genes\u2014and relies less on the pre-existing maternal mRNAs.To navigate this maternal-to-zygotic transition (MZT) successfully, the zygote must integrate signals that control pre-existing maternal mRNA transcripts with those that activate the zygotic genome. How the embryo manages this phased transition from one class of regulatory mechanisms to another has long remained obscure.Drosophila melanogaster. By matching gene transcripts with their corresponding DNA template across the genome, the researchers identified classes of zygotically and maternally expressed genes and show that the embryo combines (zygotic) transcription with message degradation to produce the localized patterns of gene expression required for organizing the early embryo.In a new study, Stefano De Renzis, Eric Wieschaus, and colleagues describe an innovative experimental approach to determine the maternal-versus-zygotic provenance of mRNA transcripts during MZT in the fruit flyThe early zygote consists of a single membrane filled with nuclei that go through repeated rounds of division before a membrane encases each nucleus to form discrete cells. Maternal gene products direct embryonic development until nuclear division 13, when the zygotic genome assumes control.The details of zygotic genome activation have remained elusive partly because maternal transcript degradation proceeds alongside zygotic gene transcription. Investigators have traditionally classified increased (or up-regulated) transcripts as zygotic , stable transcripts as maternal, and down-regulated transcripts as degraded maternal transcripts. Because this interpretation doesn\u2019t account for concomitant degradation and transcription\u2014mRNA levels can drop or stay the same even with a zygotically active genome if maternal transcripts disappear at the same time\u2014De Renzis et al. developed an approach to distinguish the two mechanisms clearly.They first measured gene expression levels at 1-hour intervals over the first 3 hours of embryogenesis, starting with the unfertilized egg (0\u20131 hour) through nuclear cycle 14 (2\u20133 hours), just after the zygotic genome takes over . By the 14th nuclear cycle, many of the transcripts had either increased or decreased in abundance\u2014but are these changes the result of transcription, degradation, or both?Fruit fly embryos can survive the loss of entire chromosomes and display distinct morphologies when parts of their chromosomes are removed, allowing the researchers to see the onset of zygotic activation. They started by using fly stocks with deletions in the left arm of the second chromosome (2L)\u2014which accounts for roughly 20% of the genome\u2014then compared mRNAs from these embryos with those with intact 2L. Of the transcripts with reduced levels in the mutant embryos, roughly 90% correspond to genes located on 2L: deleting the arm removed the DNA templates required to make the transcripts, ergo the mRNAs are derived from zygotic transcription. Those transcripts corresponding to 2L genes that were detected in cycle 14 embryos, the researchers concluded, did not depend on 2L and thus must be the products of maternal transcription.Next, the researchers analyzed mRNA profiles in embryos missing either the entire second chromosome, the third chromosome, or the X chromosome. Since flies have only three major chromosomes, they could distinguish between zygotic and maternal contributions across the entire genome. They identified mRNAs whose levels were directly related to the presence of each chromosome and classified these transcripts as zygotically derived.To chart the fate of maternal transcripts, the researchers compared mRNA levels from early untreated embryos with those from embryos missing one of the three chromosomes. The presence of about 30% of maternal transcripts was significantly reduced by cycle 14. For about a third of these cases, zygotic transcription compensated for the degraded transcripts, keeping expression levels at cycle 14 fairly constant, and demonstrating the pitfalls of using transcription level changes alone as an indication of zygotic activity.About a third of the genes identified as zygotic have no maternal counterparts (most of these genes are transcription factors), but the rest, which coexist with maternal transcripts, have no particular functional bias. The newly transcribed genes may fulfill roles the maternal genes can\u2019t, either by virtue of their cellular location or the timing of their expression (or simply because they were never intended to meet the later demands of development). These possibilities are supported by the finding that many zygotic genes show distinctive expression patterns at cycle 14, coinciding with the MZT. Since the expression levels for these genes had either stabilized or decreased during the MZT, the researchers concluded that maternal degradation and zygotic transcription occurred in tandem to influence their patterned expression.The researchers go on to show that maternal transcripts and zygotic genes have distinct regulatory elements: one controls maternal transcript degradation, the other zygotic gene transcription. Activation of the zygotic genome\u2014which accounts for roughly 20% of transcripts at cycle 14\u2014begins when a protein deposited in the egg by the mother during oogenesis binds with the regulatory motif shared by the early zygotic genes.This study raises a number of avenues of investigation, such as identifying the key regulatory genes responsible for calling the silent zygotic genome into action\u2014a watershed moment for the developing embryo. Future studies can also explore whether the developmental patterns identified here hold true for other animals."} +{"text": "Case histories from 20 patients undergoing postchemotherapy \"second look\" laparotomy for metastatic epithelial cell carcinoma of the ovary were reviewed in an attempt to evaluate the usefulness of this procedure and its likely impact on patient survival. The patient population comprised 18 patients treated with a combination of cisplatin, adriamycin and cyclophosphamide (PACe) and 2 patients treated with chlorambucil. The findings at second look were often predictable, and related to the adequacy of initial surgery. Complete tumour regression identified a group of patients with a relatively good prognosis. However in most patients residual tumour was found which rarely proved resectable. Second line chemotherapy was poorly tolerated, and appeared to have little impact on the disease particularly after combination chemotherapy had been used initially. There was little evidence that second look surgery itself positively contributed to survival. This procedure and its timing should be regarded as experimental and a suitable subject for randomised clinical trials."} +{"text": "We have investigated the oestrogen receptor (ER) status of 20 cervical adenocarcinomas by immunocytochemistry for ER protein and non-isotopic in situ hybridisation for ER mRNA. Both methods, which are applicable to paraffin sections, were developed and validated in breast carcinomas with known ER content. Six cervical adenocarcinomas contained immunocytochemically demonstrable ER protein; all contained ER mRNA, but staining was less intense in poorly differentiated areas of four tumours. This disparity between protein and mRNA detection needs further investigation as does the possibility that oestrogens may play a role in the pathogenesis of cervical adenocarcinoma."} +{"text": "EHP 112:1768\u20131771][.Bronchiolar epithelium cells known as Clara cells are thought to help defend airways against damage; Clara cell protein (CC16) is a lung-specific protein thought to protect the respiratory tract from inflammation. Studies have shown a positive association between ozone exposure and increased concentrations of CC16 in blood serum, leading researchers to suggest that ozone exposure damages the lung epithelium, causing it to \u201cleak\u201d proteins such as CC16. Now Birgitta Json Lagerkvist of Ume\u00e5 University in Sweden and colleagues present findings that complicate that idea Lagerkvist and colleagues report that children who regularly visited indoor swimming pools actually showed decreased concentrations of CC16 both before and after sessions of exercise outdoors in ambient levels of ozone. The researchers theorize that repeated exposure to disinfection by-products around indoor swimming pools may damage Clara cells, decreasing production of CC16 and possibly masking any effect that ozone exerts on CC16 levels.This report is part of a set of studies examining possible changes in CC16 serum levels in relation to ambient ozone exposure as well as exposure to other environmental agents such as the chlorine around swimming pools and its by-products. In addition to the current study, which examined children in the summer, three other studies have examined children in winter, adults in summer, and adults in winter.Subjects were screened using blood samples, lung function tests, and questionnaires. The final 57 subjects were 33 boys and 24 girls aged 10\u201311 years who had no asthma, pollen allergy, or initial decreased lung function. One subset of the 34 children had visited an indoor pool for at least one hour per month for six months or longer; the remaining 23 children had not.The children exercised lightly outdoors for two hours on the Ume\u00e5 University campus, where the ambient ozone was a moderate 77\u2013116 micrograms per cubic meter. Lung function testing and blood sampling were conducted both before and after exposure.The researchers saw no impairment of lung function in any of the children, and in looking at the entire study group, they did not find any statistically significant relationships between ozone exposure and CC16 concentrations. Among the children who did not visit swimming pools, there was a marginally significant tendency toward such a correlation. However, the children who regularly visited chlorinated indoor swimming pools showed significantly lower CC16 serum concentrations both before and after ozone exposure, compared to those who didn\u2019t visit pools.The authors say this may indicate that repeated exposure to disinfection by-products around indoor swimming pools damages Clara cell function. This theory is supported by previous studies, including one by report coauthor Alfred Bernard that showed an association between regular pool attendance among people taking swimming lessons and reduced CC16 concentrations. The researchers speculate that if regular indoor pool attendance does decrease CC16 production, then this effect may mask any increased leakage of CC16 caused by ozone. They further suggest that such masking may have happened in this study, indicated by the slight tendency to show a correlation between ozone exposure and increased CC16 concentrations only in the children who did not visit swimming pools.They conclude that Clara cell damage associated with indoor pool use may diminish the anti-inflammatory effect of CC16 in the lung. Another previous study by Bernard showed increased incidence of asthma among children who regularly visited indoor pools, and the authors of the current report call for further investigation of the possibility that exposure to disinfection byproducts around indoor pools can play a role in inducing asthma through impaired Clara cell function."} +{"text": "Carcinoembryonic antigen (CEA) is an oncofetal antigen whose function in the progression of colorectal carcinoma remains unclear although recent studies suggest it participates in homotypic cellular adhesion. We have previously shown that 40 micrograms of CEA injected intravenously into athymic nude mice enhances experimental metastasis in liver and lung by two human colorectal carcinoma cell lines that are injected intrasplenically 30 min later. The metastatic potential of another three moderately to highly metastatic colorectal carcinoma cell lines and of one weakly metastatic line has now been analysed in this model. CEA pretreatment only enhanced colony formation by cell lines that were weakly metastatic in untreated nude mice; it did not affect experimental metastasis by highly metastatic lines. CEA pretreatment enhanced the retention of 125I Idudr-labelled weakly metastatic tumour cells within the liver and lungs 4 h after intrasplenic injection but not the retention of highly metastatic tumour cells or inert latex beads. A significant correlation existed between the formation of experimental metastases and the early retention of tumour cells within the liver after intrasplenic injection. Aggregation did not appear to be important for retention in liver because CEA did not aggregate colorectal carcinoma cells in vitro. Also CEA did not alter natural host effector cell function in a cytolysis assay in vitro. We suggest that CEA facilitates liver colonisation by three of eight human colorectal carcinomas in athymic nude mice by increasing the hepatic retention of tumour cells. The potential mechanisms by which CEA may increase the retention of tumour cells in the liver are discussed."} +{"text": "The new study by Boyle and colleagues provides important data on basic science mechanisms involved in pain and inflammation . Their dBecause the present study suggests that spinal delivery may be more effective than systemic delivery when attempting to intervene in spinally-mediated inflammatory mechanisms, the authors note the potential importance of developing compounds that may bypass the blood-brain barrier. The present author speculates that the rapid and significant clinical effects noted following perispinal administration of etanercept in small pilot studies suggest that perispinal administration of p38 inhibitors may also allow these compounds to reach the spinal cord and dorsal root ganglia in therapeutically effective amounts ,8. It is"} +{"text": "Regional chemotherapy for colorectal liver metastases has not demonstrated a convincing survival benefit over systemic chemotherapy. This may be due to poor delivery of chemotherapeutic drugs to hypovascular liver tumour. Since vasoactive agents may influence hepatic blood flow this study investigated the effects of systemic and regional vasoconstrictors on the delivery of a regionally delivered marker in an experimental model of liver tumour. Systemic administration of angiotensin II caused a significant retention of marker in normal liver, but not in tumour compared to controls. Regional delivery of angiotensin II and phenylephrine caused significantly greater retention of marker in tumour than liver with an overall 4-fold increased retention of marker one minute after its injection. Ninety minutes after injection there was still significant retention of marker compared to control animals. Regional delivery of hepatic artery vasoconstrictors increase delivery of marker and may increase delivery of chemotherapeutic drug to liver tumour."} +{"text": "In a population-based study serum sialic acid level was examined in relation to subsequent development of central nervous system (CNS) tumours (229 cases). Significantly increased sialic acid concentration was found in men with a malignant CNS tumour diagnosed within 8 years of analysis, compared with corresponding matched controls. These findings suggest that the tumour existed at the time of examination which is supported by a negative linear association between sialic acid level and the time from screening to tumour diagnosis."} +{"text": "A number of amiloride analogs can sensitise wild type Chinese Hamster ovary (CHO) cells to the cytotoxic action of vinblastine, daunomycin, puromycin or colchicine. Some of these analogs also have weak sensitising effects on the multidrug resistant CHO cell line, CHRC5. The unusual feature of most of the active amiloride analogs is that they are more potent in reversing the intrinsic multidrug resistance (MDR) phenotype of CHO cells than their acquired MDR characteristic. Human HeLa cells that do not exhibit intrinsic MDR are not affected by these agents. Several of the amiloride analogs have a greater effect in increasing adriamycin uptake in wild type CHO cells than they do with CHRC5 cells. The differential effect of amiloride analogs on intrinsic versus acquired MDR characteristics of Chinese hamster cells suggests some differences in the underlying resistance mechanisms."} +{"text": "Six different immunisation regimes have been used to generate spleen cells with reactivity against human pancreatic exocrine cancer. Immunised spleen cells were fused with an NSO/1 myeloma line and supernatants from these hybridomas selectively screened for monoclonal antibodies which bound predominantly to a pancreatic cancer cell line (GER). The spleen cells from hairy litter mates immunised with pancreatic cancer xenograft homogenates and viable GER cells generated 13% of hybridoma supernatants which showed some selectivity for GER pancreatic cancer cells in a fixed cell ELISA assay. The other methods produced only 4% of hybrids with selectivity for GER cells. The antigen distribution on gluteraldehyde fixed cells was similar to that found for viable cell monolayers but many antigens were unstable on formalin fixation. Immunohistochemical staining of GER cells grown on glass slides showed a heterogeneity of antigen distribution with up to 70% of the cells exhibiting a vesicular pattern of staining. Fifty percent of the antibodies which bound to GER cells were also reactive against antigens in formalin-fixed paraffin-embedded tissue sections of the original GER tumour. Monoclonal antibody DD9E7 identified an antigen expressed on 12/14 pancreatic adenocarcinomas. The antibody showed strong staining of malignant luminal membranes and cytoplasm. The antigen was also present in normal salivary and sweat glands, and colon and breast carcinomas, but its tissue distribution was unlike that of CEA or EMA. The expression of this antigen in 12/14 of pancreatic carcinomas suggests that DD9E7 may be a useful reagent for pancreatic tumour detection."} +{"text": "The long term prognostic significance of oestrogen receptors was assessed in a prospective study of 767 patients presenting between the years 1975 and 1981 with stage 1 and 2 breast cancer treated by mastectomy with either full axillary dissection or nodal sampling. Oestrogen receptor binding was determined by a dextran coated charcoal method and median follow up was 11 years. Oestrogen receptors were present in 396 (54%) of tumours. Absence of oestrogen receptors was associated with tumours of high histological grade, but there was no relationship between nodal status or tumour size. Oestrogen receptor status did not predict survival for the group as a whole or when stratified by nodal status. In multivariate analysis both nodal status and tumour size were powerful independent prognostic factors, but oestrogen receptors failed to achieve statistical significance."} +{"text": "This study was aimed at determining whether tumour DNA content measured by cell image analysis could provide additional prognostic information when compared to that provided by flow cytometry. Sections cut from paraffin blocks of tumours from 101 patients with node negative breast cancer were analysed by both methods and the results related to other prognostic variables and to patient relapse and overall survival. DNA ploidy measured by flow cytometry classified 46 tumours as diploid and 55 as aneuploid, whereas by cell image analysis 30 were diploid and 71 aneuploid (P less than 0.002). There were 20 tumours with discrepancies between the two methods; 18 of these were tumours with only one peak in flow analysis, but determined to be aneuploid with image analysis. DNA content as measured by both methods was significant for predicting relapse and survival by log-rank test, as were tumour histological grade, c-erbB-2 expression and tumour size. Multivariate analysis showed DNA ploidy measured by flow cytometry to be the only variable of independent significance (P less than 0.02) for both relapse and overall survival. Compared with cell image analysis, flow cytometry demonstrated a significantly higher proportion of diploid tumours, which may be related to differences in the internal standards applied to each method. We suggest that cell image analysis techniques can provide more sensitive information on the DNA content of tumour cells by direct measurement of nuclear DNA density of both normal lymphocytes and tumour cells in the same section. However, although image analysis appears to be more sensitive than flow cytometry in detecting DNA aneuploidy, the image technique appears to lack the specificity of flow cytometry in correlation with clinical outcome."} +{"text": "Tumour radioiodine concentration has been compared with serum thyroglobulin (Tg) and, in a few cases, with tumour complement of thyrotrophin receptors in patients with differentiated thyroid carcinoma. All tumours examined possessed TSH receptors. In most the complement was similar to that of normal thyroid tissue although all but one of the tumours had no detectable 131I concentration in vivo even with excess TSH stimulation. Elevated serum Tg (patient taking T4 in suppressive dose) was generally associated with tumours which had 131I concentrating function when stimulated by excess TSH. Some patients, however, had high serum Tg concentration but only low or indetectable tumour 131I uptake. We conclude that (a) measurement of tumour TSH receptor complement is unlikely to be useful in clinical management as tumours which do not significantly concentrate 131I in vivo may have a normal TSH receptor complement and (b) the capacity to secrete Tg is usually associated with 131I concentration but quantitatively the relationship varies considerably between tumours."} +{"text": "The evolutionary role of postcopulatory sexual selection in shaping male reproductive traits, including sperm morphology, is well documented in several taxa. However, previous studies have focused almost exclusively on the influence of sperm competition on variation among species. In this study we tested the hypothesis that intraspecific variation in sperm morphology is driven by the level of postcopulatory sexual selection in passerine birds.Using two proxy measures of sperm competition level, (i) relative testes size and (ii) extrapair paternity level, we found strong evidence that intermale variation in sperm morphology is negatively associated with the degree of postcopulatory sexual selection, independently of phylogeny.Our results show that the role of postcopulatory sexual selection in the evolution of sperm morphology extends to an intraspecific level, reducing the variation towards what might be a species-specific \u2018optimum\u2019 sperm phenotype. This finding suggests that while postcopulatory selection is generally directional across avian species, it also acts as a stabilising evolutionary force within species under intense selection, resulting in reduced variation in sperm morphology traits. We discuss some potential evolutionary mechanisms for this pattern. The evolutionary role of postcopulatory sexual selection in shaping male reproductive morphology, physiology and behaviour is well documented in several taxa Sperm are amongst the most variable cells across animal taxa Notomyx alexis) for example, intermale variation in sperm head morphology is greater than that of the closely related species, Pseudomys australisTaeniopygia guttata) and Eurasian bullfinch (Pyrrhula pyrrhula) may be the result of relaxed postcopulatory sexual selection.Theoretical models of sperm size evolution intraspecific variation using a phylogenetic framework. Using data for 18 species of passerine bird, two indices of intraspecific variation in sperm morphology and forMost sexually selected traits are under directional selection In contrast to precopulatory sexually selected traits traits, there is almost no evidence that sperm morphology traits are influenced by environmental factors or are condition dependent In the following section we consider three possible evolutionary mechanisms that might favour an \u2018optimum\u2019 sperm morphology under strong postcopulatory sexual selection, through sperm competition Under intense sperm competition, a more competitive ejaculate will always be favoured and an optimum sperm design might be linked to maximising sperm function . The size of two particular sperm components, (i) the flagellum (the sperm's \u2018motor\u2019), and (ii) the midpiece (the sperm's \u2018powerhouse\u2019) have been theoretically linked with sperm function From a male's perspective, success in postcopulatory competition depends on achieving a balance in the theoretical trade-off between sperm size and numbers Two types of genetic factors may influence variation in sperm morphology and account for the pattern observed. First, negative genetic correlations between different sperm components may constrain sperm design and reduce the likelihood of achieving an \u2018optimum\u2019 design, especially when postcopulatory sexual selection is relaxed Drosophila, variation in sperm design would decrease, whereas under reduced polyandry the reverse would be true. Although such selection studies have been conducted While our results are consistent with the idea that sperm competition and/or cryptic female choice account for the degree of intermale variation in sperm morphology, the relative importance of these two processes remains to be established. If cryptic female choice is important, we might predict the variation in the dimensions of female sperm storage tubules will be less in species with high levels of female polyandry. Another prediction is that in artificial selection experiments in which the degree of female polyandry is increased, as in some studies of intraspecific variation in sperm morphology in a comparative framework. The fact that both intermale sperm size and design variability decrease with the level of postcopulatory sexual selection suggests that the latter is a strong stabilizing force in the evolution of avian sperm morphology. This is consistent with theoretical predictions for the effect of selection on variability of condition-independent traits In conclusion, this is the first study to explicitly test the role of post-copulation sexual selection as an evolutionary force acting on rI range 0.90 to 0.99 We investigated intraspecific variation in sperm morphology in 18 species of passerine bird. Two methods were used to obtain sperm samples for morphometric analysis: (i) from the faeces of males in reproductive condition To date, most studies of sperm morphology have focused on total sperm length Malurus cyaneus), for low and high levels respectively n , the potential confound of allometry in testes mass and body mass across the n\u200a=\u200a18 species sampled was controlled for by incorporating both CTM and BM as predictors in the model. The term BM never showed a significant effect but was retained (see Relative testes size (testes size controlled for body size) and level of extrapair paternity (EPP) are the two most widely used indices of the intensity of sperm competition ined see .All the statistical and simulation analyses were conducted using R v.2.3.1 Methods S1Adequate Sample Size Simulations and Sources of Data(0.03 MB DOC)Click here for additional data file.Figure S1n at which the solid lines level off is different in the two cases.Bootstrapped estimate of the intraspecific coefficient of variation (CV) in sperm tota length (solid lines) against sample size, in species under (i) low or (ii) high sperm competition. The dashed lines correspond to the CV estimate using the complete sample for each species. Note that the (0.04 MB TIF)Click here for additional data file."} +{"text": "A consecutive series of 644 women who presented with breast nodularity between 1976 and 1982 have been followed up to determine their rate of subsequent breast cancer. Fifteen women have developed breast cancer, 14 of these were among 352 women with an aspirated cyst (relative risk 4.4). Women with multiple cysts had the highest risk and women with breast nodularity had no excess risk. Review of histology specimens from those women who had undergone biopsy showed an excess of florid epithelial hyperplasia in women who subsequently developed breast cancer and women with multiple aspirated cysts were more likely to have florid epithelial hyperplasia. Multiple cysts are clinical markers of histological breast proliferation and women who have had multiple breast cysts aspirated have an increased risk of breast cancer and should be advised to practice regular self examination."} +{"text": "This study aims to select the radiopharmaceutical vehicle for targeted radiotherapy of neuroblastoma which is most likely to penetrate readily the centre of micrometastases in vivo. The human neuroblastoma cell line NB1-G, grown as multicellular spheroids, provided an in vitro model for micrometastases. The radiopharmaceuticals studied were the catecholamine analogue metaiodobenzyl guanidine (mIBG), a specific neuroectodermal monoclonal antibody (UJ13A) and beta nerve growth factor (beta NGF). Following incubation of each drug with neuroblastoma spheroids, autoradiographs of frozen sections were prepared to demonstrate their relative distributions. mIBG and beta NGF were found to penetrate the centre of spheroids readily although the concentration of mIBG greatly exceeded that of beta NGF. In contrast, UJ13A was only bound peripherally. We conclude that mIBG is the best available vehicle for targeted radiotherapy of neuroblastoma cells with active uptake mechanisms for catecholamines. It is suggested that radionuclides with a shorter range of emissions than 131I may be conjugated to benzyl guanidine to constitute more effective targeting agents with potentially less toxicity to adjacent normal tissues."} +{"text": "A detailed study was performed in 14 patients with epithelial ovarian tumours using the satellite probes 33.15, 228S and 216S to investigate the nature of somatic changes and frequency with which clonal changes could be demonstrated during metastasis and progression. Somatic changes were evident in approximately 70% of ovarian tumours, the most common being a deletion or reduction in intensity of a band suggesting loss of heterozygosity. Additional changes that were observed included increased intensification of single bands and the appearance of novel DNA fragments. Somatic alterations were seen following digestion of DNA with methylation resistant restriction endonucleases indicating that methylation differences alone could not account for all of the somatic changes. Using DNA fingerprint analysis ovarian tumours were shown to be heterogeneous with different DNA patterns observed in different sites in five of eight patients. Generally, within an individual patient the primary and metastases appeared to share a DNA fingerprint pattern with minor variations occurring in different sites suggesting that different populations have derived from a common stem line. This study clearly demonstrates that DNA fingerprint analysis is a sensitive method to detect somatic changes in tumour DNA and for investigating the development of clonal heterogeneity in ovarian tumours."} +{"text": "Cyclic AMP binding proteins were measured in the primary tumour from 100 patients with non-disseminated breast cancer selected on the basis that sufficient tumour material was available for analysis. These measurements have been related to factors of established prognostic value and to the patients' disease-free interval and survival. There was a wide variation in amounts of binding proteins in different tumours. Values were significantly higher (P less than 0.05) in oestrogen receptor-negative tumours but no statistically significant correlations were apparent between levels and tumour grade or whether the patients had lymph node metastasis or adjuvant treatment. However, levels were significantly higher in patients whose disease recurred within 3 years of primary treatment as compared with those who remained disease-free. Using a retrospectively determined cut off point of 8 pmol mg-1 cytosol protein, it was shown that patients with tumour cyclic AMP binding in excess of this value had a significantly greater chance of developing recurrent disease and poorer survival rates than those with lower levels. This remained true when other prognostic factors were taken into account in a multivariate analysis. It is suggested that the level of tumour cyclic AMP binding may be an independent prognostic factor for patients with early breast cancer."} +{"text": "A recent trial by the MRC Lung Cancer Working Party used physician assessments to compare two palliative schedules of radiotherapy in lung cancer. A prospective study has been undertaken on a subset of these trial patients to see how physician assessments of symptomatic relief and general condition correlate with patient perception of therapeutic response. In 40 patients followed up monthly from presentation until close to death, good agreement was found between doctors and patients on change in specific physical symptoms and overall physical condition. Doctors were poor judges of life quality at presentation but appeared able to identify relative improvement or deterioration in overall quality of life. In conclusion, physician assessments may constitute valid end-points for radiotherapy trials comparing palliative schedules in lung cancer."} +{"text": "To determine the influence of inflammation on salivary secretion. Secretion by salivary glands involves interactions between nerves, blood vessels and salivary cells. The present study investigated the effects of inflammation on rat submandibular gland function following acute ductal obstruction.Under recovery anaesthesia a metal clip was placed on the main duct of the submandibular gland. After 24 h salivary secretion was evoked by nerve and methacholine stimulation. For recovery experiments the clip was removed after 24 h and the animal left to recover for 3 days when salivary function was again assessed.In vitro analysis of cells from 24-h ligated glands revealed normal changes in intracellular calcium (the main secondary messenger involved in fluid secretion) in response to methacholine stimulation. Protein secretion from isolated cells indicated some changes in particular to methacholine-induced protein secretion although a significant protein secretion was still seen in response to isoprenaline \u2013 the main stimulus for protein secretion.By 24 h of obstruction an inflammatory infiltrate had developed within the obstructed gland and stimulated salivary flows were just 20% of the normal secretion, whilst protein secretion and ion reabsorption were also severely impaired. If ductal obstruction was removed after 24 h the salivary function returned to normal after 3 days of recovery. This report demonstrates reversible salivary inhibition associated with an inflammatory infiltrate within the salivary gland. Saliva forms a film over teeth and mucosal surfaces in the mouth and perfSj\u00f6gren's syndrome and HIV The rat submandibular gland has been used as a model to investigate the effects of obstructive diseases and a metal clip was placed on the submandibular duct through a small incision in the floor of the mouth. Following suturing animals were left to recover for 24 h before being placed under terminal anaesthesia for the procedures listed below. In three rats the parotid duct was ligated in the same way as for the submandibular duct. In a separate series of experiments, following 24 h of submandibular duct obstruction six rats were again placed under recovery anaesthesia to remove the metal clip obstructing the duct. Three days following the removal of the clip rats were placed under terminal anaesthesia to collect saliva as described below. In total 29 rats were used for the experiments described in this paper.Twenty-three adult male Wistar rats weighing 275\u2013325 g were placed under recovery anaesthesia for 5 min each. Salivas were collected on ice into pre-weighed tubes to calculate salivary flow rates.\u03bcg min\u22121 kg\u22121 body weight. Saliva samples were collected as described above from both left and right submandibular ducts simultaneously.An intravenous cannula was inserted into femoral vein during induction of anaesthesia and this was connected to a calibrated syringe pump, as previously described or isoprenaline (10\u22126m) for 30 min under continuous oxygen at 37 \u00b0C as described previously (Following ligation glands were removed from anaesthetized rats and digested by collagenase (5 mg/20 mL buffer) as previously described and stimulated with increasing doses of methacholine (10\u22128\u201310\u22126m), then ionomycin (13 \u03bcm) and finally manganese chloride (2 mm) to calibrate levels of dye within each cell. Fluorescence levels were assessed in cells with an acinar morphology using a confocal microscope \u2013 relative fluorescence levels are expressed relative to the maximum and minimum values obtained as (fluorescence \u2212 minimum)/.Another aliquot of cells from the collagenase-digested glands were loaded with the calcium-sensitive dye Fluo 4 AM . Analysis of the methacholine-induced submandibular salivas revealed that in addition to reduced saliva production there was also a similarly reduced output (concentration \u00d7 flow rate) of total protein and IgA, although the reduced IgA output was not as great as for total protein it was still significantly reduced or methacholine (10\u22125m) than in the absence of the autonomimetics. However, cells from the previously ligated gland produced mixed results. Isoprenaline still caused a significant increased secretion of IgA and peroxidase compared with the unstimulated cells from the ligated gland although the protein secretion response to methacholine was no longer significant.Cells from the unoperated submandibular gland secreted significantly more IgA and peroxidase with iso\u22128m methacholine stimulation.Despite altered protein secretion in response to the cholinergic agonist methacholine in acinar cells from the ligated gland intracellular calcium concentrations in response to methacholine were similar for cells from the control and previously ligated glands . Figure Three days following removal of the ductal ligature methacholine-stimulated salivary flows from the previously ligated glands were improved compared with 24 h ligated glands but was still less than the contralateral side when expressed as total flow per gland. The previously ligated glands were now approximately 30% lighter than the unoperated control gland . When exet al. 1999, The 24-h time point for ductal ligation was chosen as a previous study further down the secretory tree. Ion reabsorption is generally regarded as occurring at one rate so that the slower the flow of saliva the greater the reabsorption of ions. Despite a much lower salivary flow rate salivas from the ligated gland had greater concentrations of sodium and chloride, in agreement with in vivo . Isoprenaline caused a significant secretion of IgA and peroxidase by cells from unoperated control glands, as shown previously (in vivo secretion results) of peroxidase and IgA secretion was affected \u2013 levels were no longer different to secretion by unstimulated cells. The difference between IgA and peroxidase secretion from the same cells may well relate to the different storage and transport mechanisms of these proteins, for a review see in vitro is in contrast to the changes in intracellular calcium in acinar cells caused by methacholine stimulation. Cells from control and ligated glands both produced a typical biphasic response \u2013 the first peak relating to calcium release from internal stores, the plateau reflecting influx of calcium from outside the cell we have avoided any obvious atrophy, although this cannot be completely eliminated. The use of whole-body methacholine stimulation has bypassed any effect of oedema on the nerve\u2013salivary cell neuro-effector junction. In contrast to the extreme salivary hypofunction demonstrated The authors have no conflicts of interest."} +{"text": "David Healy raises intriguing questions regarding the rapid increase in bipolar diagnoses and the use of \u201cmood stabilizing\u201d medications . AlthougA widely disseminated advertising campaign for aripiprazole (Abilify) claimed that it worked in the brain \u201clike a thermostat to restore balance\u201d . InteresSince the product information insert approved by the United States Food and Drug Administration (FDA) lists the mechanism of action as \u201cunknown\u201d , this adRegarding unipolar depression, we recently argued that antThe presentation of metaphorical explanations as scientific consensus in consumer advertising has not been publicly addressed by the relevant professional associations. In fact, we observe that a cooperative relationship exists between industry and medical facilities, even highly esteemed ones: the Mayo Clinic Web site on depression, sponsored by Wyeth Pharmaceuticals (makers of venlafaxine) explains the treatment of depression via the serotonin metaphor .Such bioreductionistic and highly arguable advertisements for psychiatric treatments imply much about the disorder they are licensed for. As Dr. Healy suggests, consumers who view such advertisements are likely to characterize their problems in a manner congruent with industry promotion and to request well-advertised pharmaceuticals as treatment. At a bare minimum, increased medicalization will result; in some cases, disease mongering may indeed be an appropriate characterization.Such consumer advertising is only possible in the absence of vigorous government regulation or outcr"} +{"text": "Recombinant human erythropoietin (rHuEpo) has recently become available for the treatment of chronic anaemia, including that associated with cancer. Carcinoma NT in CBA mice causes a progressive anaemia which can be overcome by daily injections of recombinant human erythropoietin (rHuEpo). This model was used to study the effect of haematocrit on tumour blood flow, growth rate and radiosensitivity, in mice with haematocrits ranging from approximately 38% (control) to 65% (20 U/day rHuEpo). Tumours showed a small but significant slowing in growth rate with higher haematocrit. In vitro studies showed rHuEpo had no direct effect on the growth of NT cells. Tumour blood flow was measured by two methods in each mouse (133Xe clearance and 86Rubidium uptake). Blood flow showed a tendency to decrease with increasing blood viscosity although this effect was not significant despite the large differences in haematocrit. Although tumour doubling time was prolonged despite the large differences in haematocrit. Although tumour doubling time was prolonged with increasing radiation dose, from 0 (sham irradiated) to 35 Gy, haematocrit was not found to influence the growth delay. This was attributed to adaptation of the tumour during the relatively slow change in the haematocrit. rHuEpo is being considered for clinical use in anaemic cancer patients. Our data suggest that this treatment will correct haematocrit with no effect on tumour radiosensitivity."} +{"text": "PLoS Clinical Trials [We wrote to l Trials followinl Trials in orderl Trials to the eThe authors' follow-up letter reiteratPhase III studies must be based on specific and relevant phase II studies. These preferably assess intervention versus standard treatment and optimise dosing strategy while rigorously and proactively noting adverse events . HoweveFailure to address any of the specific points in our letter impairs the ability of readers to review primary data for themselves. Repeating arguments for phase III studies on albumin in severe malaria does not make them more compelling."} +{"text": "Many colorectal liver metastases are hypovascular, and their low level of perfusion is associated with limited drug uptake and poor response rates with regional chemotherapy. We have previously shown that hepatic arterial vasoconstrictors may increase drug delivery to liver tumours, but the underlying haemodynamic changes have not been defined. Using intraoperative laser Doppler flowmetry (LDF) we have assessed the effect of intraarterial angiotensin II (AI) on tumour blood flow in ten patients with colorectal liver metastases. Measurements were performed during placement of infusion catheters for regional chemotherapy. Blood flow was recorded continuously with a Periflux PF3 perfusion monitor via a probe held on the tumour surface, following hepatic arterial infusion of 15 micrograms AII over 90 s. Six patients with isolated small metastases (< 5 cm in diameter) showed increases in flow, which reached a peak at 170-240 s from the start of AII infusion, and which were closely correlated with the corresponding increase in arterial pressure . Of the four patients with large confluent tumour deposits, two showed smaller transient increases in flow over the first 60 s of AII infusion and two had no measurable flow response. Increased blood flow following AII infusion may increase the exposure of tumour to therapeutic agents. This study suggests that both tumour size and the effect upon systemic arterial pressure may be important determinants of the blood flow response to AII. LDF may provide useful information about the potential of AII and other vasoconstrictors to enhance targeting precision."} +{"text": "The value of ethics education have been questioned. Therefore we did a student survey on attitudes about the teaching of ethics in Swedish medical schools.Questionnaire survey on attitudes to ethics education with 409 Swedish medical students participating. We analyzed > 8000 words of open-ended responses and multiple-choice questions using classic grounded theory procedures.In this paper we suggest that medical students take a proximity morality stance towards their ethics education meaning that they want to form physician morality \"on the job\". This involves comprehensive ethics courses in which quality lectures provide \"ethics grammar\" and together with attitude exercises and vignette reflections nurture tutored group discussions. Goals of forming physician morality are to develop a professional identity, handling diversity of religious and existential worldviews, training students described as ethically naive, processing difficult clinical experiences, and desisting negative role modeling from physicians in clinical or teaching situations, some engaging in \"ethics suppression\" by controlling sensitive topic discussions and serving students politically correct attitudes.We found that medical students have a proximity morality attitude towards ethics education. Rather than being taught ethics they want to form their own physician morality through tutored group discussions in comprehensive ethics courses. Medical ethics education differs from other subjects and the importance of formal courses in ethics has been questioned . Some meWe designed a questionnaire survey in order to elucidate how medical students view the ethics education in medical schools ,5. Many st, 5th and 11th (last) term participated. The survey consisted of 14 items of which 10 had a total of 59 multiple-choice response options and generous space for open-ended comments, and 4 items were open-ended only, see Table We did a survey on attitudes towards the medical ethics education during 2005 as a request from the delegation of medical ethics of the Swedish Society of Medicine. Swedish medical students from the 1The overall response rate to the questionnaire survey was 36%, and varied between different centers from 13% to 83%, with a total of 409 respondents, 308 women (75%) and 101 men (25%). More than half (220/409) of the respondents gave one or more written open comments amounting to > 8000 words. These comments were transcribed into Word from handwritten text.At some centers a whole term would drop out since the responsible teacher failed to hand out the survey. Yet, the response patterns of the different questionnaire items did not differ significantly between schools with low and high response rates when different logistic regression models were applied to the data ,5. The mWe analyzed open-ended comments and multiple-choice responses by classic grounded theory (GT) procedures -12. SincDiscovery of Grounded Theory from 1967 . Fit hasIn this study we analyzed student attitudes and \"what was going on\" in the medical ethics education based on survey data. The medical students argued for a proximity morality stance, or forming morality \"on the job\". This morality forming is ideally done in comprehensive ethics courses with tutored small groups. Forming physician morality requires \"ethics grammar\" provided by selected high quality lectures, and impulses from attitude exercises and vignette reflections in \"ethics labs\". Patient cases and clinical issues are thus discussed in interactive groups that help to deal with emotionally difficult clinical situations. To desist negative role modeling is another function of the ethics courses where reflected professionalism is developed for diverse medical students in a heterogeneous world.On the job morality forming in medical school is typically done in interactive discussion groups. These groups have a support network function where medical students are allowed professional role growth within a permissive context where ethical and value-laden issues are discussed and tried. The structure ideally consists of tutored groups that repeatedly work with case study approaches, discuss ethical principles, and continue during internship. Within a frame resembling the clinical setting students grow their own ethical attitudes and shape their individual physician morality. Group discussions provide good training for handling ethical difficulties since real world medical ethics consist of unique complex situations often involving several people. One goal of interactive ethics group discussions is to understand what appropriate physician behavior is.\"ethics discussion forums should be based on tutored small groups to prevent people with strong views from dominating\" last term student.\"(we need) group discussions with teachers making sure that everyone develops decent ethical values as physicians\" fifth term student\"every section could end with ethical discussions related to the specific subject, psychiatry/internal medicine/surgery\" last term studentForming physician morality also includes quality lectures on ethics, preferably by professional ethicists. These lectures provide students with a basic \"ethics grammar\" about ethical principles and concepts. This feeds the interactive group discussions and improves their quality concerning ethical issues.\"Professional lecturers from the faculty of arts (are wanted)\" first term studentIn the \"ethics lab\" students work with practical, sometimes challenging attitude exercises and vignette reflections. These stimulate critical thinking about current ethical problems in clinical training. It requires that participants position themselves ideologically, and for some attitude exercises also physically. Attitude exercises are often done in case studies.\"A case is presented and different opinions (re the case) represented by four different corners. One can go to any corner and argue against the other corners and eventually change corners\" last term student.Why would medical students want to form physician morality on the job? The deliberate forming of a physician morality is necessary for several reasons. A number of student responses dealt with arguments for ethics education in general and forming physician morality on the job in particular:The professional identity of future physicians requires moral reflection.\"An open discussion forum on difficult issues and professional identity conflicts would make us better physicians.\" last term studentSmall groups during clinical training \u2013 discussing the professional physician role and work issues \" (on suggested ethics education during later internship) last term student\"Medical students are different. Some are ethically naive, or not interested in ethics, and others even described as socially \"autistic\". The importance of ethics education is obvious for these groups.Only autistic people need ethics education\" last term student\"Diversity. We live in a society with increasing diversity and multiple religious views.\"What is it really like in our secularized country? How can we say something is right when we don't share the same values\" fifth term studentProcessing difficulties. A group discussion format of ethics education helps in processing tough experiences from the clinical setting.\"We underestimate the power of what we can do for each other during the education.\" last term student\"Small groups discussing everyday problems and ethical issues in the workplace\" (on suggested ethics education during later internship) first term studentDesisting negative role modeling. By defying ethics suppression and politically corrected ethics the influences of physicians/teachers as poor role models may be addressed and negative role modeling dealt with in the interactive groups. Some teachers and physicians were described as being \"masters of opinion control\" trying to neutralize discussions about ethically sensitive topics by putting the lid on discussions, and defending politically correct opinions.\"I prefer a good clinician instead of zealous, ideologically motivated people\" fifth term student.\"Teachers gave too little space for own views \u2013 there was a correct key for the discussion\" last term studentIn a statistical analysis of the survey presented elsewhere we saw a\"Personally I'm always ready to learn, although I do not always like being taught.\" W Churchill (1874 \u2013 1965)ethics grammar. These lectures provide default ethical principles nurturing group discussions together with attitude exercises and vignette reflections in ethics labs . These interactive discussion groups also have a support network function. Here students process ethical problems in an environment where physician morality is allowed to form and grow on the job. Hence, rather than being served ideologically stained opinions students prefer to reflect and discuss different ethical attitudes. As an example of their proximity morality one could say they want to bake their own moral cakes instead of being served ethical cookies [The quote illustrates the students' attitudes towards medical ethics education in this study. We propose that medical students by a proximity morality stance want ethics education on the job to help them in the learning process of becoming physicians. In this process they form their own physician morality rather than being taught ethics. This ideally takes place in comprehensive ethics courses where tutored groups openly discuss and reflect on difficult ethical topics and moral dilemmas. High quality lectures are interwoven to give an cookies .ethics grammar and input from ethics labs . In a review of ethics teaching the authors were nihilistic about its effects and suggested that critical determinants of physician identity operate not within the formal curriculum but in a subtler, less officially recognized \"hidden curriculum\" [In a British study of university students' expectations of teaching the students hoped for more interaction between students and teachers . They alriculum\" . Also, mriculum\" . The goariculum\" . As a rericulum\" . SimilarThe method for this study was classic grounded theory (GT) aiming at generating conceptual hypotheses of the resolution of a main concern and not describing a studied area. Yet, most studies citing GT fail to fulfill its aims -11. In ath term students gave the largest quantitative input of qualitative data and thus had a comparatively larger impact on theory generation. Whether this was a limitation is questionable. In our view it gave us more valuable longitudinal data. To use interview data by theoretically sampling outside of the survey might improve the model. We tried to compensate for this by also sampling dichotomized multiple-choice survey data in accordance with the GT maxim \"all is data\". The survey data consisted of structured responses, which is not recommended in classic GT. This is another argument for expanding the theoretical model with interview data. For possible future application in medical schools we thus intend to refine and modify the model and develop it through interaction with medical students and teachers.This paper suggests a proximity morality model showing how medical students want their ethics education in medical school, but does not take into account their teachers' views. Also, our study is limited by the qualitative data being only written comments in an otIn this study we present a tentative conceptual model of proximity morality guiding medical students to shape their own medical ethics education \"on the job\". We suggest that medical students rather than being taught ethics want to form their own physician morality. This is typically done in comprehensive ethics courses with tutored group discussions. Here high quality lectures supply \"ethics grammar\" and together with attitude exercises and vignette reflections in \"ethics labs\" nurture discussions on different ethical issues. This helps students to develop a professional physician identity, handling diversity of religious and existential worldviews, training students described as ethically naive, processing difficult clinical experiences, and desisting negative role modeling.The author(s) declare that they have no competing interests.RL and NL conceived the study. All authors helped in designing the survey questionnaire. RL and NL were responsible for coordinating the survey. HT did the grounded theory analysis and drafted the manuscript. NL, RL and KS gave input to the data analysis and manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:"} +{"text": "The monoclonal antibodies HMFG1 and HMFG2 identify antigens of the milk fat globule membrane which are also found on breast epithelial cells. Immunohistochemical staining was performed using both antibodies on formalin fixed, paraffin embedded sections of 93 breast carcinoma, 36 histologically benign lesions and 29 histologically normal breast tissue blocks. In both normal and benign breast disease the staining was largely extracellular whilst in malignant tissue the staining was variable and often intracellular. Nine carcinomas did not stain with either antibody. The staining patterns of malignant tissues were graded and no correlation was found between the grades and survival or indices of prognosis, This study indicates that with the present methods available for grading staining patterns, although of diagnostic value, these monoclonal antibodies are unlikely to assist in determining either the degree of tumour differentiation or prognosis in breast carcinoma."} +{"text": "Fifteen patients with locally advanced refractory breast cancer have been treated with recombinant leucocyte interferon for up to 12 weeks. Toxicity was considerable with the initial dosage schedule employed but became acceptable after reducing the starting dose by 50%. Minor side effects occurred in all patients and major CNS toxicity in six. Nine patients showed some evidence of tumour regression at 4 weeks. Only two of these were still responding at 12 weeks. Response was unrelated to the length of previous history, oestrogen receptor status or previous responsiveness to cytotoxic or hormone therapy."} +{"text": "We have examined by immunohistochemistry the ability of breast carcinomas to produce pepsinogen C, an aspartyl proteinase usually involved in the digestion of proteins in the stomach. A total of 113 out of 245 breast tumours (46%) were positive for pepsinogen C immunostaining. There was a significant association between pepsinogen C and oestrogen receptors with proteinase levels higher (HSCORE) in oestrogen receptor positive tumours than in oestrogen receptor negative. There was also a significant association between pepsinogen C and histological grade, pepsinogen C levels being higher in well and moderately differentiated breast carcinomas than in poorly differentiated tumours. On the basis of these results, we suggest that pepsinogen C may be useful as a marker of good prognosis in breast cancer."} +{"text": "Previous studies showed that murine polymorphonuclear leukocytes (PMNs) lyze tumour cells in the presence of wheat germ agglutinin or actinomycin D. This paper reports studies on whether a soluble factor participates in PMN-mediated cytolysis dependent on lectin or a chemotherapeutic drug. Tumour lysis was observed with supernatants from PMN cocultured with wheat germ agglutinin or actinomycin D. The supernatant from cultures of PMNs alone was not cytotoxic, but addition of these agents to the supernatant induced tumour lysis. PMNs released a soluble factor spontaneously into the medium and cytolysis was induced by a combination of this factor and wheat germ agglutinin or actinomycin D. This factor was not an oxygen metabolite, but a protein with a molecular weight of approximately 100 K daltons. These results suggest that a soluble factor(s) from PMNs participates in tumour killing in cooperation with appropriate reagents."} +{"text": "Dr. Gallagher et al wrote 22Baseline electrocardiogram of patients with atriofascicular or atrioventricular pathways (pseudo-Mahaim) are charElectrocardiogram of fasciculoventricular pathway is characterized by normal frontal plane axis like an anteroseptal accessory pathway (0\u00b0 to +75\u00b0) with a sThe major findings are the slow conducting and decremental properties that can be assessed during right atrial pacing: AH interval lengthens, HV interval shortens and QRS widens until a steady value is achieved. Faster atrial stimulation does not increase preexcitation, but can prolong AV conduction time until block occurs . DecremeVentricular stimulation usually discloses ventriculoatrial conduction from the A-V node. The vast majority of atriofascicular and atrioventricular pathways have only anterograde conduction ,31. AdenThere are plenty of electrophysiologic and anatomic data supporting the concept that at least atriofascicular pathways are accessory AV nodes. We have seen a patient with an electrophysiologic profile suggestive of the presence of an atriofascicular pathway without conduction through the AV node. This patient had unexplained syncope with a baseline ECG showing LBBB-like pattern with normal PR interval . Atrial Associated conditions: Mahaim fibers (AF/ AV) very often occurs in the setting of Ebstein's disease 10 to 40%) and associated accessory pathways are a commom finding (up to 30%). Fasciculoventricular pathways also occurs very often in association with accessory pathways. I have reviewed the cases reported since 1981 0 to 40% ,23,33,34\u00ae SR2 or SR3, which improves stability. The potential may be as large as the His bundle potential or small, narrow with low amplitude. Catheter ablation at a site with \"M\" potential is likely to be successful (Searching for the \"M\" (Mahaim) potential along thccessful . We 20]\u00ae SR2 or ccessful , but ocaccessful . We haveActivation mapping of the earliest local ventricular potential is feasible in short atrioventricular pathways like in fast conducting AV accessory pathways. Atrioventricular decremental pathways with a long course often shows extensive arborization over a wide area of ventricular muscle . TargetiShortest stimulus-QRS interval as assessed by atrial stimulation at a constant pacing rate along the atrial aspect of the annulus was the gold standard mapping method before mapping of \"M\" potential had been reported. Stimulation sites remote from the atrial insertion of the accessory pathway result in long stimulus-QRS interval due to the amount of interposed atrial tissue. We do not use this method because it is time consuming and very inaccurate because it is difficult to stimulate from many sites at the same distance from the annulus and stimulating atrial tissue requires good contact with the tip, which is not always possible.Extrastimulus mapping during antidromic tachycardia. Finding an atrial site where the longest coupled premature extrastimulus causes resetting, or assessing the amount of advancement of the QRS following application of a fixed atrial extraestimulus coupling interval. Similar to the previous technique it looks for a site in the atrial annulus with the least interposing tissue separating it from the accessory pathway proximal insertion. Likewise shortest stimulus-QRS technique is an inaccurate and tedious method.Some authors ,37 reporElectroanatomic mapping (noncontact mapping) ,38 can bSome particular features are unique to Mahaim fibers: Mapping of the atrial insertion by ventricular stimulation is usually not possible because those decremental pathways do not conduct retrogradely. Atrioventricular connections can be located by mapping the site of earliest ventricular activation on the annulus, as with other anterogradely conducting accessory AV pathways. On the contrary, atriofascicular pathways or even the long atrioventricular pathways with distal (nonannular) insertion cannot be mapped in this way. To worse matters these decremental pathways are unusually sensitive to mechanical trauma. Inadvertent knocking of the ablation catheter against the annulus can result in transient abolition of conduction through the pathway from minutes to hours ,35. The Radiofrequency current should be applied during atrial pacing to enhance preexcitation and making it easier to assess conduction block at the AP. Stability is improved during atrial pacing as compared with ablation during antidromic tachycardia when catheter is likely to move with tachycardia termination. MAT is a common and expected event and can also cause catheter displacement. Before \"M\" potential mapping technique became the gold standard mapping technique some authors favored targeting the ventricular insertion to avoid MAT and maintain a better catheter stability .Catheter ablation have been very successful particularly when ablating at a site with \"M\" potential or assessing earliest delta-V interval in atrioventricular decremental pathways ."} +{"text": "The chemotherapeutic agents 2'-deoxy-5-azacytidine (DAC) and cisplatin (cDDP) have been shown in vitro to be synergystic in their cytotoxicity toward human tumour cells. We have investigated possible molecular mechanisms underlying this synergy using the plasmid pSVE3 in vitro and after transfection into CMT3 cells. Increased binding of cDDP to DAC-substituted DNA generated in vivo was confirmed by flameless atomic absorption spectrophotometry (FAAS). The plasmid used in these experiments was unmethylated suggesting that DAC was effective in enhancing cDDP binding to DNA without acting as a hypomethylating agent, but by directly changing the topology of DNA. The role of DNA methylation in cDDP binding was studied using methylated and unmethylated plasmid incubated in vitro with cDDP. Restriction analyses and FAAS measurement of bound platinum indicated that methylated DNA bound more cDDP than unmethylated DNA. In addition, in vivo studies confirmed the in vitro observations since replication of methylated plasmid was inhibited to a greater extent than unmethylated plasmid."} +{"text": "In vitro drug sensitivity testing, both by optical colony counting and by a [3H]-TdR incorporation assay, was performed on human tumour cells proliferating in soft agar cultures. Cells from two different human tumour cell lines, 5 different human tumour xenografts, and 94 different primary human tumour specimens of various histologic types were studied. Regression analysis comparing the results of the colony counting assay and the [3H]-TdR assay revealed good to excellent correlations between the two assay endpoints for quantitating the effect of in vitro anticancer drug exposure for a large number of different agents. The presence of pre-existing tumour cell aggregates complicates the performance of the optical colony counting assay. The [3H]-TdR incorporation assay is more sensitive and reproducible than the colony counting assay when performed on samples containing a large number of initially seeded tumour cell aggregates."} +{"text": "Pregnant women are famously exhorted to faithfully take their daily prenatal vitamins, which often contain iron and other minerals. But new research suggests that a weekly iron supplement coinciding with the renewal of the small intestine\u2019s mucosal lining cells (where nutrient absorption occurs) works better than a daily supplement and prevents problems resulting from too much iron at the wrong times.Maternal iron deficiency and anemia early in gestation can result in premature birth and low birth weight. These, in turn, can trigger further problems ranging from slow physical growth and motor development to impaired emotional control. In severe cases, both maternal and fetal survival can be threatened at or near birth. Thus, there exists a near-global public health policy of maternal iron supplementation during pregnancy.Archives of Medical Research. A team including nutritionist and epidemiologist Esther Casanueva of the National Institute of Perinatology Isidro Espinosa de los Reyes (INPerIER) in Mexico City and colleagues elsewhere in Mexico City and California studied 116 women receiving prenatal care at INPerIER. All had come to INPerIER for prenatal care by gestational week 20.The new study appears in the July 2006 issue of 12 once a day; the others took double this dose once a week. The researchers checked the women\u2019s levels of hemoglobin (which transports oxygen) and ferritin every four weeks through the end of pregnancy.None of the women were anemic at that point, but 66% had low levels of ferritin , suggesting low iron nutritional status. Half took 60 mg of iron as ferrous sulfate with 200 \u03bcg of folic acid and 1 \u03bcg of vitamin BMore of the women taking the weekly dose were mildly anemic (with hemoglobin levels shown not to carry any risk for mothers and infants) compared with the women taking the daily dose. However, by weeks 28 to 36, women taking the daily supplement had a significantly higher prevalence of hemoconcentration, a condition defined as hemoglobin levels above 145 g/L. Ironically, both early gestational iron-deficiency anemia and hemoconcentration later in pregnancy increase the risk of premature birth and low birth weight. Thus, the researchers suggest that excess iron supplementation can cause the same problems it is supposed to correct.Animal studies suggest that excess iron can also trigger formation of free radicals in the intestinal mucosa and other tissues, and that both iron deficiency and iron overload can damage nuclear DNA and mitochondrial DNA. This kind of damage has been implicated in cancer induction.The intestinal mucosa is renewed every 5 to 6 days and will absorb as much iron as necessary to maintain iron balance; however, mature cells will stop absorbing iron entirely if they are flooded with it, even if there is an iron deficit. \u201cMaintaining a high iron environment in the intestine by ingesting significantly more iron than needed every day overwhelms this safety system,\u201d says coauthor Fernando E. Viteri. A more subtly calibrated iron supplementation during pregnancy may be as effective as current public health recommendations, and perhaps safer."} +{"text": "EHP 115:226\u2013230; K\u00e4rrman et al.][.Studies have found assorted perfluorinated compounds (PFCs)\u2014the persistent chemicals in such products as nonstick coatings\u2014in samples of human blood and milk, but what isn\u2019t clear is how efficiently the chemicals transfer between these two media. To address this gap, researchers in Sweden compared PFC levels in blood serum and milk samples to better understand the lactational transfer of these compounds Previous animal and human studies have shown that mothers can pass certain PFCs to fetuses and infants. That these compounds can find their ways into humans at the earliest stages is cause for concern because the PFCs perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA), which have infiltrated ecosystems from Asia to Antarctica, have been linked in laboratory animals to effects that include liver and testicular cancer, developmental defects, immune disruption, neuroendocrine effects, and birth defects.The team collected milk and blood samples from 12 women at three weeks postpartum. The team also compared PFC levels from this relatively small sample to levels in human milk samples collected from 1996 through 2004 from groups of 25 to 90 women per year.The team found eight PFCs in the current serum samples and five in the current milk samples. All of these milk samples contained PFOS and perfluorohexanesulfonate. Some also contained PFOA, perfluorooctanesulfonamide, or perfluorononanoic acid. These patterns and levels were similar to those detected in the earlier milk samples.The scientists calculated that the breast milk PFC concentration averaged about 1% of the corresponding maternal serum concentration. They write that the estimated levels of PFCs that infants received from mothers (about 200 ng per day) could represent a substantial exposure, and call for further studies of the potential hazards of PFCs in breast milk.They also found that the relationship between serum and milk PFC levels depends on the specific compound. These differences, the scientists caution, may not necessarily indicate the efficiency at which the different compounds travel from whole blood to milk. Variables such as how readily each compound concentrates in blood plasma rather than whole milk may affect the ratios."} +{"text": "Human-carnivore conflict is challenging to quantify because it is shaped by both the realities and people\u2019s perceptions of carnivore threats. Whether perceptions align with realities can have implications for conflict mitigation: misalignments can lead to heightened and indiscriminant persecution of carnivores whereas alignments can offer deeper insights into human-carnivore interactions. We applied a landscape-scale spatial analysis of livestock killed by tigers and leopards in India to model and map observed attack risk, and surveyed owners of livestock killed by tigers and leopards for their rankings of threats across habitats to map perceived attack risk. Observed tiger risk to livestock was greatest near dense forests and at moderate distances from human activity while leopard risk was greatest near open vegetation. People accurately perceived spatial differences between tiger and leopard hunting patterns, expected greater threat in areas with high values of observed risk for both carnivores. Owners\u2019 perception of threats largely did not depend on environmental conditions surrounding their village . Surveys revealed that owners who previously lost livestock to carnivores used more livestock protection methods than those who had no prior losses, and that owners who had recently lost livestock for the first time expressed greater interest in changing their protection methods than those who experienced prior losses. Our findings suggest that in systems where realities and perceptions of carnivore risk align, conservation programs and policies can optimize conservation outcomes by (1) improving the effectiveness of livestock protection methods and (2) working with owners who have recently lost livestock and are most willing to invest effort in adapting protection strategies to mitigate human-carnivore conflict. An important challenge in conserving large carnivores in human-dominated landscapes is overcoming human-wildlife conflict arising from people\u2019s real or perceived threats to their livelihoods and personal safety ,2. MethoStakeholders that can distinguish between threats from carnivores with different hunting behaviors could potentially implement carnivore-specific management strategies to more effectively reduce conflicts. If such stakeholders have suffered livelihood losses from carnivores in the past, they may also be more receptive to investing in mitigation efforts to prevent future conflicts and thus represent a high-priority demographic for human-carnivore coexistence initiatives. Yet it is currently unclear whether people can accurately discriminate predation risks imposed by co-occurring carnivores with different hunting characteristics, and how previous livelihood losses to carnivores affect their efforts to mitigate future human-carnivore conflict.Panthera tigris) and the leopard (Panthera pardus), in Kanha Tiger Reserve in central India. Tigers hunt primarily in dense forests with low levels of human presence whereas leopards pursue prey in more open, human-dominated landscapes [We investigated these questions by assessing landscape-scale predation threats and protection efforts for livestock from two large carnivores with different hunting characteristics, the tiger using the MASS, MuMIN and R DAAG packages.We developed prediction maps of observed attack risk by using sites of carnivore attacks on livestock in the study site to build spatial prediction models of attack risk for tigers and leopards (separate model for each species). Sampling and modeling methods for calculating observed attack risk are described in detail in ,18. ModeTo assess how livestock owners perceive and respond to attack risk, we interviewed people who reported that their livestock were killed by tigers or leopards during the study period for financial compensation by the local Forest Department. In an effort to prevent livestock owners from retaliating against carnivores, the Forest Department financially compensates owners for livestock killed by wild carnivores. To receive compensation, a livestock owner must locate and report the livestock carcass to the Forest Department within 48 h, and an officer then visits the site to record evidence of the death. Though not all livestock owners choose to report lost livestock . We also asked owners whether their household had previously lost livestock to wild carnivore). We also asked owners to describe what methods they used to protect livestock and what methods they would use in the future.We asked owners to rank their perceptions of tiger and leopard attack risk in four major land-use categories: village, agricultural fields, field-forest edge vegetation and forest. Each land-use category was assigned a ranking of 1 (low), 2 (moderately high), 3 (high) or 4 (very high). Not all owners felt capable of assessing risk levels in every land-use category, resulting in unequal sample sizes among land-use categories and mapped the perceived risk in village to this land-use category. We quantified the variation in owners\u2019 responses by calculating the proportion of all responses that differed from the median perceived risk in each land-use category.We expected that previously losing livestock to carnivores would strengthen the accuracy of a person\u2019s understanding of risk (i.e. that perceived attack risk would more closely resemble model predictions of observed attack risk) than owners who had not previously experienced a carnivore attack. To test whether owners\u2019 risk perceptions changed if their household\u2019s livestock had been previously depredated, we ran one-way ANOVAs on the perceived risk for each land-use category. We also expected that owners who had previously lost livestock to carnivores would use more protection methods and that owners who lost livestock for the first time might be more open to trying new methods for protecting livestock. We ran one-way ANOVAs to test the effect of a previous attack on owners\u2019 past and future use of livestock protection methods.We explored spatial associations between model predictions of observed carnivore attack risk on livestock and owners\u2019 perceptions of attack risk. We calculated the mean observed risk within 500 m of surveyed village centers and tested its role in predicting the perceived risk of owners from each village using ordinal logistic regression with chi-square tests to indicate model fit. To compare the spatial distributions of observed and perceived risk, we sampled the observed risk and the median perceived risk in each pixel across the landscape and used boxplots to examine differences across the study area. We ran Kruskal Wallis tests with Bonferroni correction followed by Dunn\u2019s multiple comparisons post-hoc tests to test for differences in observed risk between perceived risk levels.P = 0.001 for tiger; P < 0.001 for leopard; Models of attack risk revealed that tiger and leopard kills were associated with unique sets of landscape features , forming2(3) = 142,342, P < 0.001; Dunn\u2019s test P < 0.001) and leopard (\u03c72(3) = 64,029, P < 0.001; Dunn\u2019s test P < 0.001; Owners\u2019 perceptions of tiger and leopard risk closely mirrored spatial distributions of observed risk from both carnivores across the landscape. Owners perceived the lowest risks from tigers in village and gradually increasing risks across land-use categories with denser forest and less human infrastructure , a gradi2 < 0.05; 2 < 0.05; 2 < 0.05; 2 < 0.05; 2 < 0.05; 2 < 0.05; 2 = 1.00 for all tests).Owners\u2019 perceptions of leopard risk were unaffected by the spatial location , while all other protection methods (livestock enclosures and hired herders) were used equally . When asked what protection efforts they would change in the future, significantly more owners who lost livestock for the first time (21%) showed interest in changing grazing areas compared to owners with previous losses ; all other future changes did not differ by previous experience had previously lost livestock to tigers or leopards. Previous losses did not affect owners\u2019 perceptions of tiger or leopard threats in any land-use category predictably modify their escape tactics in response to a predator\u2019s hunting mode and habitat domain, adapting movement, vigilance and foraging behaviors to optimize survival across the landscape \u201322. PreyIn central India and many landscapes worldwide, livestock owners and park managers simultaneously apply multiple strategies to protect livestock from a community of predator species ,19. Thisfor the first time are most willing to change. Programs and policies aimed at reducing livestock losses to carnivores should therefore prioritize owners who have recently lost livestock for education and subsidies since these people are likely to be more receptive to devoting time and resources to improving their livestock protection strategies. This information should be combined with other known factors associated with more positive attitudes towards carnivores, such as higher level of education, younger age, larger village size, greater agricultural production, smaller holdings of large-bodied livestock stock, greater diversity of income sources and greater benefits associated with carnivore presence [The association between owners\u2019 previous experience of losing livestock to carnivores and their efforts to protect livestock offers insight into an important factor driving behavioral change that could improve the effectiveness of conservation programs and policies aimed at reducing human-carnivore conflict. Owners who had previously lost livestock invested extra effort into protecting their animals by sending family members to herd grazing livestock (in addition to hiring herders and using enclosures like owners who had no previous experience with depredation). Sending a family member to accompany livestock\u2013which could jeopardize the person\u2019s safety \u2013represen health) ,26,27.Our results also showed that owners\u2019 perception of threats largely did not depend on the dominant land-use or frequency of observed kills near their villages. However, people living in southern and forested areas perceived greater tiger risk in forest and owners living in areas with a higher observed attack risk perceived lower risk in forest. Because dense forest is a strong indicator of kills, owners living near forests likely observe more tiger attacks in forest than people living in more open land types and thus consistently associate forest with high tiger risk. In contrast, owners living in areas with a high frequency of observed kills, regardless of the surrounding land type, may observe tigers killing livestock over a greater variety of land types and therefore less frequently associate forest explicitly with high risk. This suggests that conservation practitioners may need to adapt strategies for reducing livestock depredation in different areas based on local perceptions of risk. In addition, owners showed greater uncertainty in their perceptions of leopard (than tiger) risk across all land-use categories, possibly due to the extensive distribution of high leopard observed attack risk across the landscape. Because leopards utilize a broader variety of habitats than tigers, hunting in both forested and human-dominated areas , their ssame carnivore species vary across landscapes and which factors shape people\u2019s spatial understanding of\u2013and responses to\u2013carnivore threats. Integrating spatial, ecological and social perspectives will be key to formulating a comprehensive framework for resolving human-wildlife conflict.Communities in southern Asia are renowned for being some of the most tolerant towards carnivore conservation globally ,29. ThisOur sample for measuring perceived risk was limited to owners of livestock killed by tigers and leopard and we recognize that results do not account for the perceptions of all livestock owners or all people living in the study area. Because the villages in Kanha are small, ranging from 100\u20132,000 people per village (Kanha Tiger Reserve Forest Department 2012), and people openly share stories and opinions about tigers and leopards , we do not expect that the perceptions of owners with depredated livestock differed substantially from other people in the community. However, if the process of locating livestock carcasses in the landscape refined owners\u2019 spatial understanding of carnivore risk, we would expect the people we interviewed to reflect the most accurate perceptions of risk in the study area. Furthermore, we recognize that people\u2019s perceptions of carnivore risk may not be homogeneous across a single land-cover type yet also acknowledge that a coarse-level of understanding can be valuable for informing further investigation. We encourage future studies to survey the broader community in order to incorporate more diverse perspectives into conservation decision-making.Our findings provide spatial insight into the relationships between humans and carnivores by showing that people who have lost livestock to carnivores can accurately estimate threats from carnivores at a landscape scale. This has significant implications for conservation because programs and policies aimed at reducing human-carnivore conflict can channel efforts towards improving the effectiveness of livestock protection methods rather than educating stakeholders on where to implement them. Our study also reveals that livestock owners are particularly motivated to invest in livestock protection methods if they or their families previously lost livestock to carnivores, especially in the recent past. These people should be prioritized for receiving training or financial subsidies to improve livestock protection methods in order to optimize conservation outcomes.Because understanding informs action, the spatial alignment between perceived and observed carnivore risk suggests that people take precautionary measures to protect livestock relative to the level of risk at a given site and their first-hand experience with losing livestock to carnivores. The fact that livestock depredation continues to occur underscores the complexities that link people\u2019s perceptions of risk with their motivation to reduce threats, as well as the inherent challenges of preventing carnivore attacks. Comparing the realities and perceptions of carnivore threats represents one important step towards strategically enhancing efforts for mitigating human-carnivore conflict in ways that facilitate coexistence with large carnivores.S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file."} +{"text": "Right hemisphere damaged patients with and without left visual neglect, and age-matched controls had objects of various sizes presented within left or right body hemispace. Subjects were asked to estimate the objects\u2019 sizes or to reach out and grasp them, in order to assess visual size processing in perceptual-experiential and action-based contexts respectively. No impairments of size processing were detected in the prehension performance of the neglect patients but a generalised slowing of movement was observed, associated with an extended deceleration phase. Additionally both patient groups reached maximum grip aperture relatively later in the movement than did controls. For the estimation task it was predicted that the left visual neglect group would systematically underestimate the sizes of objects presented within left hemispace but no such abnormalities were observed. Possible reasons for this unexpected null finding are discussed."} +{"text": "Recently, Cordula Lukas and colleagues from TU Dortmund have revisited the relationship between the occurrence of urinary bladder cancer and polymorphisms of xenobiotic metabolizing enzymes . CurrenThe majority of these occupational bladder carcinomas are associated with exposure to aromatic amines and azo dyes. Lukas et al. (2017) analyzeAssociations with polymorphisms have been studied in more than 1800 diseases and thousands of SNP associations have been found but typically SNPs only explain a minor share of the variance (Liaqat et al., 2015; Hashemi"} +{"text": "In Ghana, priority-setting for reproductive health service interventions is known to be rudimentary with little wider stakeholder involvement. In recognizing the need for broad stakeholder engagement to advance reproductive care provision and utilization, it is necessary to jointly study the varied stakeholder views on reproductive care services.We applied an ethnographic study approach where field data was collected between March-May 2015 in three rural districts of northern Ghana. Data was collected among women with recent births experiences (n = 90), health care providers (n = 16) and policy actors (n = 6). In-depth interviews and focus group discussions was applied to collect all data. Each stakeholder participant\u2019s audio file was transcribed, and repeatedly read through to identify similar and divergent views in data. A coding scheme guided coding processes. All transcripts were then imported into QSR NVivo 11 for further analysis.Four themes emerged. Women participants accentuated that sex and sexuality values of men have changed over time, and drives gender roles, parity levels and decision making on reproductive care needs at community levels. Sexual stigma on reproductive care reduces the willingness of women to voice poor experiences related to their previous reproductive experiences. All stakeholders\u2019 highlighted clinical treatments for post-abortion care are minimally covered under the fee exemption policy for antenatal and postnatal care. Policy processes on service delivery protocols still is top-down in Ghana.Health teams working to improve sexual and reproductive health care must find suitable context strategies that effectively work to improve women reproductive care needs at their operational levels. Private sector participation and informal community support clutches are encouraged to advance the delivery of reproductive care services. In sub-Saharan Africa (SSA), sexual reproductive health behavior of women is influenced by multiple individual and societal needs . The douIn Ghana, priority-setting for reproductive health service interventions is known to be rudimentary with little wider stakeholder involvement , 7. ReceIn recognizing the need for broad stakeholder engagements to advance reproductive care provision and utilization, it is necessary to jointly study the varied stakeholder views on reproductive care services. Reproductive care provision and use in this study includes user\u2019s right to information, access to safe effective, affordable and effective strategies of birth control. Sexual reproductive health education, advocacy and reproductive health strategies for fertility control are included in this broad concept as well. Specifically, there has been growing need to understand the shifting expectations of user preferences on reproductive care services . PrioritTo identify these gaps, views of users of reproductive health services (women), health staff and policymakers were analyzed concurrently in three rural districts of northern Ghana. At the health policy level, this study provides stakeholder opinions of how women reproductive care shape reproductive behavior, choices, decision making, and priority-setting for policy decision making in rural Ghana. Our results add to the current knowledge on how various stakeholders\u2019 roles, decision making and priorities impact reproductive health services provision and use in Ghana.This study employed an ethnographic design. In ethnographic study designs, the voice of participants is an important source of data gathered throughout the investigation process , 19. We Three districts in addition to the capital of the Upper East region of Ghana form the study setting. The Upper East region is one of the 10 administrative regions in Ghana. The Upper East region currently has 13 administrative divisions under Ghana\u2019s decentralization structure. Major ethnic groups in the region include Bimoba, Bissa, Buli, Frafra, Kantosi, Kasem, and Kusasi. The health system in the Upper East region is organized into a 4-tier system typical of most regions in Ghana; regional, district, sub-district and community levels often called CHPS centres. Three districts; Bongo, Talensi, and Nabdam were purposively selected for this study. The 3 districts were selected due to poor indices of maternal and reproductive health outcomes among the 13 administrative divisions in the region. Proximity and resource availability for data collection also influenced choice of districts. In each of these districts, three remote facilities providing reproductive health services in each district were selected . We also included Bolgatanga, the capital of the Upper East region as one of the settings because some policy interviewees resided in this area.Data were collected among three groups of stakeholders: women with records of recent births (2 years prior to study), health staff, and policymakers. Focus group discussions (FGDs) were conducted with women and health staff while in-depth interviews (IDIs) were held with policymakers (public and private). To select women participants, midwives in the included facilities provided information on 15 women per facility with recent birth experiences using data from antenatal and postnatal facility registers. In total 135 women were selected as potential participants in all 3 districts. At each district level community, meetings were held across all 9 facility settings to select final women participants for the study. A final inclusion checklist with the following criteria was used to rank final participants at each facility: woman should be physically present at meetings, consent to participate, interested in the study and able to share prior knowledge related to the study aim. At each facility meeting, 10 participants with high rating towards study were selected. A convenience sample of 30 eligible women per district took part in the FGDs (3 FGDs per district with 10 women in each FGD). Thus, in total 90 women took part in the study. Each woman participant was provided a participant consent form and was asked to complete this form through signing or thumbprint before all interviews started. Across the three facilities per district, health staff in one of the facilities was selected. In these district facilities, one midwife, a senior staff in-charge of facility, and facility nurses (on average 3\u20137 persons per district) responsible for providing reproductive health services at these facility centers were recruited as district health staff participants. Policymakers were later recruited and included 2 private health policy program managers and 4 public sector policymakers. The two private health policy managers worked in health facilities under the umbrella Christian Health Association of Ghana (CHAG) across our study settings. All four public sector policymakers worked in public health facilities across study districts. All four policy actors read and signed a consent information sheet before the start of each interview. FGDs with women were conducted in one main district local language; Talensi , Nabdam (Nabit) and Bongo (Grune). All IDIs were conducted in English. FGDs and IDIs were conducted using sample structured guides see .In conducting the interviews, the ethnographic approach allowed for continuous inductions and verification of all stakeholder views. FGD and IDI audio files were transcribed verbatim into English by two research team members (the principle researcher and an assistant). We used two research team members to minimize single level biases in our analysis. Transcripts from all stakeholder groups were repeatedly read by the principle researcher and the assistant to identify similar and divergent views in data. All individual stakeholder transcripts were finally checked for accuracy and consistency with the original audio files. The research team then developed a coding scheme to guide the coding for each stakeholder group response based on the aim of the study.To ensure consistency among the two coders, we developed coding rules to facilitate the process. An initial coding for transcripts was undertaken by the principle researcher and the assistant using the principles for open coding . All traAll participants were provided with detailed information about the study and how data are collected, used and stored in the study. During the data collection, we ensured that accurate and adequate data on the study aim was captured. We also ensured strict compliance by all research team members to prevent unauthorized access to participant\u2019s data, loss, destruction or damage to any data collected. Study approval was granted by the Institutional Review Board of the Navrongo Health research Centre, Ghana with ID number: NHRCIRB202.Parity levels of the 90 women in the FGD ranged between 1 and 7 births. Only 13 women ever had a home birth. Four women reported pregnancy miscarriages. Six women reported they ever sought post abortion care. Two women indicated they had to pay for post-abortion care services. Ever use and discontinuation of contraceptive use was reported by 18 women. Few women however knew where to obtain related services such as sexual transmitted infection (STIs) information and treatment. No woman reported to have been examined for any related reproductive cancers. The health staff was represented in the study by 3 midwives, 3 senior health nurses and 10 junior community/enrolled nurses with varied levels of working experience within the Ghana Health Service. Policymaker\u2019s participants were responsible for policy dissemination at facility, community, district and regional level.Four main themes emerged from the analysis in NVivo: 1) gender roles on reproductive care needs, 2) the experiences on meeting reproductive care needs, 3) the expectations about reproductive care needs, and 4) the policy setting and decision making processes on reproductive care needs. Relevant abstracted findings based on the 4 themes are presented below. The results highlight comparative synthesis of participants\u2019 views showing consensus and aberrations across each stakeholder group.5). However, some women\u2019s views show that their ability to economically support male spouses to meet family care needs, gives them leverage to covertly meet certain reproductive care needs (3).This theme emerged only among women participants. re needs w3.10. When male health staff power relations were quizzed, women indicated that the impact of male nurse staff on their reproductive behavior was limited. The majority of women however indicated that male community volunteers assisted and provided them feedback on addressing reproductive needs (11).Gender roles in this study also showed women expression of health staff cluttered controls over women at health facility centres. Some women indicated that they had been skipped or ignored by the staff, or received insufficient attention by nurses during post-partum family planning care. This often happened if health staff perceived that women challenged the status-quo when receiving reproductive services as asserted by women. Some women also expressed that were skipped during antenatal visits by female nurses as expressed in ve needs , w11.6).Furthermore, women views on care and support beyond reproductive services provision at the community level were varied with that of health staff. Women were of the opinion that services delivery on reproductive health did little to address psychological aspects of care. Most women accentuated that services, which address their emotional and physical self-worth concerns, were absent in most facilities they often seek care. In the absence of psychological support, strong family opposition to women making independent reproductive choices has an impact on women\u2019s preferences. Women indicated that negotiating in case of familial opposition is often difficult see , w6.1, 4).The experiences-related theme was observed among all three stakeholder groups see . User ex1, 6). Women reported a lack of facility space for services related to infertility counseling, and STI diagnosis for women. Views among health staff and policy makers largely show that most public health facilities are unable to diagnose conditions such as reproductive cancers and offer post abortion services in this facility or the district (1). Women (n = 10) who experienced pregnancy terminations (induced abortion or miscarriage) paid high fees to obtain post-abortion care needs, which was never a rewarding experience. A woman recounted paying USD 25.00 to obtain post-abortion care services (2).Policymakers in responding to post-abortion fee cost indicated that clinical treatments for post-abortion care were minimally covered under the fee exemption policy for antenatal and postnatal care (2). Policy makers and health staff support women suggestions that abortion and post-abortion care services should be a part of the care continuum for reproductive health services.Women asserted that the health staff did not differentiate between adolescents and women with higher parity desires , w1, 6. district , -hs1. Wservices , w2.Polital care , p2. Pol3). Few women reported that the health staff\u2019s unaccommodating attitudes towards young women and adolescents seeking abortion care, lowered the trust among new users.Additionally, poor women-health staff communication was observed to contribute to the unwillingness of women to use contraception as highlighted by women , w3. Few5). Although health staff did not respond directly to women perceived views on women-health staff relations, they expressed their views that they undertook their duties in conformity with facility procedures for engaging clients who visit for services (5). In instances where health staff cannot meet women expected reproductive service preferences, users and local actors are often unable to explain what services are better met at community facility centres (5).Specifically, women mentioned they have been traumatized by bad experiences when they accompanied their young adolescent girls for antenatal care , w5. Altservices ,\u201d-hs5. I centres , hs5.On how women-health staff relations are better managed to improve quality of reproductive services delivery at the policy level, policymakers and health staff both agreed that health facility level evaluations of staff performance are one way to monitor reproductive and other general services delivery. Women agreeing to this however indicated that these feedbacks of evaluations are sometimes not immediate, often exacerbating trust and relational issues between health staff and users at facility levels. Never the less, policymakers reported periodic community engagements help to understand user\u2019s preparedness and demands for quality and satisfactory service needs.3). Social stigma on the use of some reproductive services often contributed to negative experiences with reproductive demands at facility and community levels as agreed by both women and health staff. Stigmatization may sometimes reduce the willingness of women to voice poor experiences related to their previous reproductive experiences.Policymakers recognized that new health infrastructures prioritize user needs, such as adolescent reproductive friendly centres spaces to cater for individualized needs in new public facilities. The need for privacy to reduce the negative stigma among intended users, particularly counseling for HIV/AIDS services has improved , p3. Soc2 & 3). Health staff indicated that comprehensive reproductive care can be met when several skilled para health staff is available to provide services (6). Health staff attributed health system inability to meet this demand as partly accounting for these concerns raised by women. Policymaker\u2019s views substantiated the health staff perspectives, emphasizing services such as shortcomings in abortion care, infertility treatment, and physical infrastructure deficits, which have to change dramatically to meet user preferences (1 & 2). Even when reproductive health services provided are desired by a user, the inability to ask questions and be provided feedback on appropriate method use limit meeting women desired contraceptive needs. A women recount a health staff rhetorically saying \u201cI couldn\u2019t do anything\u201d. This was agreed by many women as a common response when they push further on meeting certain expected services at the facility level (4).The expectations-related theme was catalogued based on all three stakeholder groups. Women\u2019s views here reflect assertions that the health system has overly emphasized and relied on products and services to meet their reproductive health needs. Examples of how facility level structures meet or fail to meet women\u2019s needs are reported by health staff and policy makers. At the policy level, what needs to be done more to meet user needs was highlighted by women discussants. Women views expressed reflect their expectations for more information sharing by health staff in meeting health effect concerns, particularly among contraceptive service users. The majority of women indicated they are hardly provided with adequate and appropriate information on how to overcome negative side effects. Services were more focused on family planning with little attention paid to sexual disease prevention, opportunistic infections such as reproductive cancers and infertility treatments , w2 & 3.services , hs6. Heferences , p1 & 2.ty level , w4.4). Health staff views showed that some women rely on the few available private medical centres to meet such basic service needs. Some health staff agreed partly to these women expressions of unmet reproductive expectations. This was driven by health staff assertion that they lacked appropriate counseling skills beyond family planning (1).Additionally, the demand for more organized integrated services rather than visiting the facility severally for different reproductive services is reported by women. Health staff responses on integrated services show the lack of knowledge about women\u2019s basic infertility needs by health staff and the lack of facility level infrastructure add to the inability to meet integrated reproductive services for women. Policy makers cited individualized counseling as a great challenge to health staff, despite improvements in skills training on the job for health staff , hs4. Heplanning , hs1.5 & 6). Both health staff and policy makers affirmed that the contextualization of most reproductive health targets would improve expectations of psychological support for reproductive care (7 and p6). In addition, women again alluded to expectations of experiencing services delivered to them at closer proximities and at their home. Health staff expressed the view that local level arrangement at the facility level tailored towards increasing patient-health staff contact hours, so as to address more user complaints and unfriendly relations often reported by women is one of the ways to improve patient-centered reproductive care needs (5). A broad consensus on the existence of informal groups and their role in influencing the use of reproductive health care services at community level was asserted by each stakeholder participant. Policy makers recognized the growing concerns about informal social support at some community levels, and their active engagements with some health staff to provide them community based reproductive counseling services. More importantly, policy maker\u2019s views indicate the need for health system to be more accountable and socially supportive to meet reproductive needs (8).Women views on informal group structures supported policy makers expression for the formal recognition and adoption of informal social support groups such as mother-to-mother support groups to provide them formal health advocacy information and psychological support. Health staff views supported women and policy maker assertions, indicating that informal group structures such as the mother-to-mother group\u2019s help to address myths and misinformation on reproductive services offered at facility level (8)Other expectations\u2019 concerning the delivery of reproductive care services is related to emotional and psychosocial care for users and how the health system is supporting meet this need. Women recounted timely information sharing by health staff to influence their reproductive care choices should be a top priority among service providers. In addition, health staff and policy makers agreed to women views of the need to improve social support and care on reproductive services provided at facility levels. Women views indicate health staff\u2019s inabilities to address their psychological needs create low self-esteem for intended users. This impacts women\u2019s abilities to realize their desired fertility choices. Health staff avowed that unmet women reproductive psychological needs are not prioritized in meeting reproductive care needs at facility levels , hs5 & 6re needs , p5. A bve needs , p8.Womety level , hs81). Policy implementers and program managers only have the opportunity to prioritize a few activities from reimbursements received from the National Health Insurance Authority as reported by public policymakers (4). Most activities prioritized at this level are for reproductive advocacy (5). Health staff views on policy setting show that they sometimes do little to influence reproductive decision making at policy level. Health staff asserted that most policies just come from the top, sometimes neglecting women pressing reproductive needs at the facility or community level. Health staff recalled how refresher trainings are the only periods they are introduced to reproductive service or products (2). The policy processes only involve health staff when they have to disseminate skills for service improvements as policy makers make an argument (6). Women on the other hand in two of our study districts acknowledged that they have been asked to participate in health program manager\u2019s advocacy meetings in the past, but few policy inclusion changes have occurred. Women posited that although they have provided suggestions on how health staff and policy makers can better address their reproductive needs, they have become tired of these policy-provider engagements since their inputs are minimally incorporated into the services delivery . Health staffs opinions blamed policy implementers at the district level for doing little to improve women reproductive care needs (5).This theme emerged from varied expression among the 3 stakeholder groups in the study. Policy maker\u2019s consensus on policy setting and reproductive services provision supports that most policies are driven by donor funding. Hence most decision making priorities align to who provides funding and to which areas funders allocate their funds , p1. Polcymakers , p4. Mosadvocacy , p5. Heaproducts , hs2. Thargument , p6. Womdelivery , w1,2, 4re needs , hs5.6). Some policy makers recalled how the Sector Wide Approaches (SWAp) and Millennium Development Goal (MDGs) targets have made contraceptive services available in some districts. SWAp in health were developed in the early 1990s with an aim to respond to widespread dissatisfaction with fragmented donor-sponsored projects in Africa. Ghana health sector SWAP formed the basis for investments and actions by the Ministry of Health and donor partners. Despite shortcomings with SWAps, the approach has been shown to have supported alignment, harmonization and improved accountability between donors and country governments who adopted the approach. The MDG period in Ghana also witnessed reproductive interventions in the MDG Accelerated Framework (MAF), reduction of maternal mortality, comprehensive antenatal clinic programs, safe motherhood, and prevention and management of safe abortion programs. A challenge admitted by policymakers is the poor application of economic evaluations to inform future prioritization of reproductive care services (10). This offers a challenge in estimating effectively on-going targets for reproductive health interventions at the public-private health sector levels. Private health policy makers view support the need for improved guidelines on reproductive policy setting standards for effective delivery of services by health staff (11).Another key highlight of policy setting processes and how this impacts on reproductive care among policy makers and health staff was the role of political leadership. Policy makers expressed the views that commitments at the socio-political level have driven major reproductive care needs over the last decade , p6. Somservices , p10. Thth staff , p11.5). Women indicated their decisions and choices based on certain service demands should drive providers and policy managers to take steps to make these services friendly and available even to the few women who need them (8). Health staff Health staff stated that the decision making for protocol dissemination of reproductive services has little involvement with facility level staff. Women views also indicate they had no ideas on what standards and protocols dissemination plans are used in providing services for them (8). Both women and health staff views reflect the need for better information flow and provider centered training needs to better address user concerns and needs. Overall, women recognized that their involvement in reproductive policy decision making was limited. Women views show their high positive ratings on friendly and prompt reproductive services compared with their direct involvement in policy setting for reproductive care at the facility level. Health staffs observed that their influence on women reproductive care choices is based on conditions that influence services use at the facility level. Most health staff admitted that some services are largely not provided because of logistical needs. All policymakers (public and private) strongly agreed that reproductive health targeting must be broad with clear guidelines to ensure reproductive care needs are properly contextualized.Women expressions reflect little involvement of community level actors and leaders in policy setting and decision making , w5. Womeed them , w8. Heafor them , hs8. BoThis study has examined stakeholder\u2019s views related to experiences, expectations, and policy decision making processes that influence reproductive care in three rural district settings in Ghana. In addition, women\u2019s view of how gender roles influence reproductive care was unearthed. While women\u2019s access to and use of reproductive care services may be influenced by broad social and economic determinants, the experiences, expectations, and policy processes enumerated by all stakeholders\u2019 in our findings impact rural women reproductive care choices in rural Ghana. Overall, stakeholder consensus based on expressed experiences highlight the need for a more facility based resource allocation to meet facility based reproductive plans. Health staff views sharply differed from women assertions that services provided were non-differentiated. The reasons cited for this were that facility spaces and health staff were limited. Stakeholder\u2019s opinions differed on what levels of counseling were adequate to meet user reproductive care expectations. Women viewed most counseling services as inadequate, and not tailored towards individual needs. Health staff and policymakers however were of the opinion that current counseling services were adequately designed, stressing that these counseling services were only limited due to low numbers of health staff to patient ratios at health facilities. To ensure current health sector policies on counseling meet user expectations, there is a need for consensus among key stakeholders on counseling components for reproductive care services. We discuss further under each results theme stakeholders views and how the policy processes meet or limit meeting user preferences for reproductive care.Our findings on gender roles reflect women views on how gender influences reproductive health care in many societies in sub-Saharan Africa. Women\u2019s views show that strong external friendship and ties outside the traditional family environment as a positive enhancer for their first acceptance and adoption of family planning services. Even in communities where health advocacy and education is provided by health staff, interfamilial associations influence women\u2019s reproductive decision making and choice of care. Few narrations of households or communities where women lack economic power, fertility decisions are the preserve of the male spouse in consultations with other male external family heads. Even when it comes to birthing care, consultations in a traditional context require women acquiesce to final decisions prescribed by these traditional heads. Although many women admitted that male sexuality values have changed over time, this is limited to the family level where women\u2019s contribution to economic livelihood is strong. Our findings corroborate several gender studies in developing countries that show that the women\u2019s ability to exercise control at the household level improves their chances to use contraceptives and facility based birthing services \u201329. Our Our findings show that reproductive services non-differentiation at the point of service provision influences individual reproductive choices and use. There was consensus by all stakeholders on the need for service differentiation support the policy to provide reproductive services by taking into consideration the socio-demographic background of users and the group or social milieu of the intended user . ServiceAnother important finding was stakeholders shared views on fee payments for post abortion care and management at the facility level by users. Women indicated that full fee payments were demanded before a patient is attended for pregnancy loss/miscarriage. Although some policymakers and health staff acknowledged this phenomenon, they sought to provide reasons why these demands exist. Policymakers and health staff views expressed here however did not indicate a justification of these experiences by women. Policy makers acceded to a need to fully include post-abortion care and management into the National Health Insurance policy benefit package.Furthermore, health staff expressions on some facility level incapacity to provide a broad range of reproductive services is not helpful in addressing stigma and myths of family planning issues at the community levels. Health staff arguments points to the fact that in situations where reproductive services for STIs, abortion/post-abortion care, and infertility treatments are limited in scope. Myths on what define sexuality and womanhood in the society further foil social stigma on care seeking for such services as reiterated by health staff. Policymakers and health care staff attestation that some reproductive care services remain unmet and not currently provided remain more of human capital and finance need in Ghana. Health sector arrangements that diversify current and future reproductive human resource and financial needs will assist in many more services available for users.One highlighted expectation by health staff and women was the need for health services to address reproductive opportunistic infection needs such as reproductive cancers and infertility treatment. Both stakeholder groups agreed that these services are mostly non-existent as part of service components offered. Private health providers play active roles in delivering a broad range of reproductive services absent at public sector level as agreed by policymakers and health care staff. However, since private facilities tend to be more urbanized, rural women seeking abortion and infertility treatments face distance barriers aside existing social stigma and norms for these services. Individualized reproductive care was also admitted by policymakers and health staff as difficult to attain partly to limited staff numbers and diagnostic facilities. This is documented as a major barrier in delivering maternity care services in Ghana \u201343.Another repeated expectation among all stakeholder groups was the need to improve systems that are more integrative and supportive for reproductive service users. This study provides evidence that the non-availability of psychological support creates low self-esteem among intended users. While women expressed optimism that information and counseling enabled them to address clinical reproductive concerns, these counseling services were limited in supporting them to address individual psychological needs. The inability of the formal health system to address these specific psychological stress needs has a propensity to lower, or cause user withdrawal for several reproductive care services. Our finding here corroborates existing findings that a psychological burden is the most common reason for patients to withdraw from a facility treatment center for infertility . In thisWomen and health staff views were divided on how effective counseling services enabled users to attain their desired fertility goals. While women expressed that counseling services were limited in supporting them to address individual psychological needs, health staff were of the view that this challenge existed due to limited para health professionals to provide such services. The inability of the formal health system to address these specific psychological stress needs has a propensity to lower reproductive care services use. Our finding corroborates existing findings that a psychological burden is the most common reason for patients to withdraw from a facility treatment center for infertility .Lastly, adopting participatory approaches for reproductive services delivery is acknowledged by all stakeholder groups as vital for improving reproductive services. The mother-to-mother support (MTMS) group evidence from our study draws more evidence of the important role played by informal community structures to promote health and well-being. Our study typifies studies across developing countries that support bottom-up approaches to delivery of community health services\u201350. It aThe policy processes for informed decision making on reproductive care services is argued largely by policy makers to be driven by previous and current donor funding needs. Health staff views align to top to bottom approaches on policy processes making it difficult for them to prioritize context reproductive care needs for intended users. Despite the health sector policies to ensure bottom-up planning processes, views from policy makers and health staff on reproductive care services agree to bottom-up planning gaps. Health staff at facility levels arguments was based on limited and delayed budgetary allocations for effective monitoring and reproductive health outreach activities in communities. Policy makers in agreeing to health staff indicates irregular insurance reimbursement further leaves most facilities ineffective in meeting basic to comprehensive reproductive service needs. An existing study on priority setting on sexual health in Ghana evidenced that reproductive policy setting is rudimentary, with little involvement from donors and advocates . This suWomen indicated little involvement in policies on their expected needs, despite their involvement in health committee meetings with facility and district health providers. Agreeably, health staff also indicated most reproductive policy decisions come from the ministry, with little space for other stakeholder inputs. Health staffs influence on women reproductive care choices based on child birthing conditions at facility level, and not through involvement in policy processes on reproductive care. Health staff views impugned policy makers at district levels fail to recognize user concerns on policy processes on reproductive care services. Policy maker\u2019s views differed with health staff assertions, indicating deficits in health sector funding and logistical needs account for perceived inefficiency at the district levels. Furthermore, policy makers agreed with health staff concerns that reproductive health targeting still remain limited in scope, since most reproductive care guidelines and protocols have not been contextualized and fully operational in all facilities.Some major policy processes and decisions as argued by policy makers and supported by health staff and women have been driven by political commitments over the last decades. Specifically, many safe motherhood interventions implemented during the MGD period were influenced by political reform processes. One such policy is the free fee exemption policy for delivery care and the National Health Insurance Scheme in Ghana. These policies provided impetus for many health sector goals for improving sexual reproductive health needs in Ghana, 54. PolOverall, stakeholder views on the policy processes and its effects on user preferences for comprehensive and friendlier reproductive services show agreements centered on a top-down linear policy process and decision making. Largely, improvements in most maternal interventions have been made possible because of political commitment and other national and international agreements on improving sexual and reproductive health in Ghana. Despite this, gaps in meeting user individualized and psychological needs exist due to weak enforcement of the policy processes related to health staffing and health facility infrastructure needs. Reproductive health policy strategies that gives greater emphasizes on improving health staff capacity to contextualize reproductive working protocols at facility levels can help to meet user preferences on reproductive care services.Our study has some limitations. First, notwithstanding the saturation observed in our interviews, we may have still missed out important categories that could have emerged among stakeholders in our study. We also acknowledge the possibility of health staff and policy implementer\u2019s biases in trying to withhold information on the study aim. Our triangulation of views have minimizes potential errors on stakeholder narratives. We acknowledge the inability of our study to investigate views from persons at the health ministry level and other public-private partners involved in implementing reproductive health strategies. Nevertheless, the three group of stakeholders examined in this study represent a good fit when examining district level stakeholders on sexual and reproductive care needs in any rural context in Ghana.This study explored stakeholder\u2019s views on experiences, expectations, and decision making on reproductive care among rural Ghanaian women. Gender mainstreaming approaches in Ghana should develop structures and systems within the health delivery system that will create conditions of mutual respect among provider-user, and empower women in their healthcare seeking behaviors. In addition, health teams working to improve sexual and reproductive health care must find suitable context strategies that will effectively work to improve women reproductive care needs at their operational levels. From our results, accounts of services non-differentiation of services among adolescents and older women group\u2019s calls for the need for health providers at facility levels to adjust health services to target and meet specific needs for different user groups. Whiles we support the inclusion of post-abortion care and post-pregnancy termination management issues into the National health insurance benefit package, a broad program scheme that ensure sexual and reproductive health needs are truly subsidized or provided essentially at lower fee cost will benefit rural population groups.To support accountability and equity in reproductive services provision at facility levels, the local health systems need to be resourced to evaluate health staff working attitudes in relation with user needs and satisfied reproductive care demands. To upscale services using the private sector contributor approach, policies that target increased private sector participation may result in more multi-faceted reproductive services care provisions, something not entirely available in most public sector health facilities.Health sector leads such as the Ministry of Health and Ghana Health Service should drive the need to close the inclusiveness gap on sexual and reproductive care sector policy planning processes and needs. Stakeholder consensus from this study also advocates strongly for clear guidelines on delivery reproductive care to be contextualized to meet user care needs. Health counseling and the need for individualized care have increasingly been evidenced as an entry approach to reach new users of health services. To fully reach individualized care at community level, health systems must have community support clutches such as the mother-to-mother support groups evidenced from our study. This is important to provide community level psychological support and delimit cluttered relationships that may exist between provider-user during formal facility access hours."} +{"text": "Stage I and II testicular germ cell tumors (GCTs) are almost always cured with appropriate treatment and most ongoing research regarding these tumors focuses on minimizing treatment toxicity. The management of clinical stage I testicular GCTs has grown more complicated due to the emergence of a brief course of chemotherapy as an additional treatment option for stage I seminomas and stage I nonseminomas. In addition, growing concern about radiation-induced cancers and other late toxicity has dulled enthusiasm for radiotherapy as a treatment for stage I seminomas. However, recent randomized trials have shown that radiotherapy doses and field sizes can be lowered without compromising cure rates and it is possible that this reduction in radiation exposure will reduce the rate of secondary cancers. At this point in history, stage I patients have three treatment options following radical orchiectomy: adjuvant chemotherapy , surveillance, and either retroperitoneal lymph node dissection (for nonseminomas) or radiotherapy (for pure seminomas). Clinical studies have made it possible to identify subgroups of patients at high and low risk for relapse and this has made it possible to tailor treatment decisions to the individual patient's postorchiectomy relapse risk."} +{"text": "Biomedical research is contributing significant role in the field of biomedical engineering and applied science. It brings research and innovations to a different level. This study investigated artificial human blood \u2013synthetic plasma liquid as conductive medium. Keeping in mind the conductivity of synthetic plasma, astable multivibrator as well as differential amplifier circuit were demonstrated. The circuits were given normal input voltages at regular temperature and ideal conditions. The result shows desired response which supports the novel concept. For both the circuits, phase shift of 180\u00b0 achieved by analysing biological electronic circuits. Majority of human body consists of water and blood particles like white cell, red cell, platelets. Liquid blood is responsible for supply of nutrients such as glucose, amino acids, and fatty acids ). An artificial human blood named synthetic plasma was made in the laboratories of applied science to investigate and analyse its behaviour in order to realise liquid based electronic circuits.Human blood based liquid memristor is made possible and feasible for research and analysis . ElectroPhysical model of human tissue skin based memoristor and their Network is possible for biomedical analysis . This liThe human body can be analyzed as a transmission medium for electrical current by means of numerical simulations and measurements. Properties of predefined tissue layer and variations related to human body geometry were analysed. Significant transmission achieved between two thorax with typical signal to noise ratio . Human bSynthetic plasma has electrically charged particle ions. Under the effect of external emf, they acquire dynamism, face collisions and thus manifest resistance R. Similar manner, electrically charged particle ions are distantly placed have dynamic emf field between them and thus manifest capacitance C. Silicon rubber tubes of various distances with different diameter were filled with synthetic plasma. Tube capacitance C and resistance R were realized by capacitor meter and multimeter.1 and C2 ) are charging and discharging but not at the same time so according their polarities, transistors Q1 and Q2 are on and off but again not at the same time so as a result, circuit gives two stable states with regular interval.Figure Designing of human tissue (skin) based transistor is feasible now a days where different point of skin can be treated as three terminals of the transistor . ActuallFigure As observed from the figure, all tubes are filled with equal plasma level. For realising registers/capacitors, 15 ml to 20 ml liquid in 12 mm diameter transparent glass test tubes were analysed. Similar manner, realising transistors, 40 ml to 50 ml liquid in 25 mm diameter transparent glass test tubes were analysed. Wooden stand is used for mechanical support.Figure This time, similar approach has been applied by the authors to achieve electronic circuit. Five tubes were taken to realise three resistors and two transistors as they were filled with synthetic plasma liquid . Here also synthetic plasma plays a major role for designing the components and finally whole biological electronic circuit.Figure The proposed circuits of astable multivibrator and differential amplifier shows desired response which supports feasibility of liquid based electronic circuits. The presented idea can be extended to develop pure biological human implantable circuits which can regulate our body function and heart bits. Of course, research related to human implanting needs critical care to develop similar circuit.Synthetic plasma liquid has been used as conducting medium to investigate biological electronic circuits . The result supports presented biomedical approach to design biological electronic circuit. Presented circuits are environmentally friendly and support a very novel idea to design pure biological circuit from human blood. Using this novel concept, an extra ordinary research can be made possible to design first human implantable biological electronic circuit. Presented research has very high applications in the field of biomedical science and medical society."} +{"text": "Schizophrenia is associated with a higher prevalence of cannabis use and cigarette use. However, it is unknown to what extent these associations are due to a shared genetic aetiology. We therefore aim to examine how schizophrenia genetic risk associates with patterns of cigarette and cannabis use in adolescence.We analysed repeated measures of cigarette and cannabis use during adolescence in a sample of 5,300 individuals in the Avon Longitudinal Study of Parents and Children (ALSPAC) birth cohort who had at least 3 measures of cigarette and cannabis use between ages 14\u201319 years. Cigarette and cannabis use data were summarised using longitudinal latent class analysis to identify longitudinal classes of substance use, and associations between polygenic scores for schizophrenia and resulting classes were assessed.The schizophrenia polygenic score based on single nucleotide polymorphisms (SNPs) meeting a discovery sample threshold of p \u2264 0.05 was associated with late onset cannabis use as compared to non-use but not with early onset or cigarette only use latent classes. This association persisted after excluding the CHRNA5-CHRNA3-CHRNB4 nicotinic receptor gene cluster , a locus which has previously been found to strongly associate with schizophrenia.This study found that genetic risk of schizophrenia (as captured by polygenic scores) is associated with late-onset cannabis use but not with other smoking phenotypes in adolescence in ALSPAC. Possible explanations for these results are that schizophrenia and cannabis use have a shared genetic aetiology or that biological risk of schizophrenia leads to cannabis use through secondary mechanisms. These secondary mechanisms may include stress of childhood behavioural problems occurring as a result of biological processes underling schizophrenia. Future analyses involving mediation models may shed some light on factors influencing patterns of substance use in individuals with a high genetic liability for schizophrenia."} +{"text": "Urinalysis is an integral part of a thorough patient evaluation. Change in urine characteristics can give clues to help solve some of the diagnostic challenges faced by physicians. We discuss a case of a benign cause of green discoloration of urine caused by propofol infusion, which reversed following its discontinuation."} +{"text": "We report on 12 near-term babies from three families in which an unexplained transient respiratory distress was observed. No known risk factor was present in any family and no sequelae were recorded at follow-up. The most common causes of respiratory distress at birth are Neonatal Respiratory Distress Syndrome (NRD) and Transient Tachypnea of the Newborn (TTN), and their cumulative incidence is estimated to be about 2%. Genetic factors have been identified in NRD (surfactant genes) or suggested for TTN (genes affecting lung liquid clearance). Survivors from NRD may develop clinically relevant sequelae, while TTN does not cause any problem later in life. Our cases do not immediately fit NRD or TTN, while familial recurrence suggests the existence of a previously unreported subgroup on patients with respiratory distress for which autosomal-recessive inheritance is likely."} +{"text": "The article titled \u201cAdvanced Hepatic Fibrosis in Fatty Liver Disease Linked to Hyperplastic Colonic Polyp\u201d uses theThe two articles are similar regarding the study population, but there is a significant difference between them regarding the main findings; the first article published at IMAJ showed the association between the two diseases. The second published at CJGH showed novel and interesting association between the degree of fibrosis in steatohepatitis (not the disease itself) and hyperplastic polyp."} +{"text": "All participants were administered faux pas recognition written stories. The dysexecutive PD patients performed less accurately than both HC and executively unimpaired PD individuals on all faux pas story questions (p < 0.05); the executively unimpaired PD group performed as accurately as the HC group on the ToM tasks. Results of the study clearly demonstrate that PD is not tout court associated with ToM impairments and that these may occur in PD patients as a function of the degree of their executive impairment. Our findings also indirectly confirm previous data on the role of the prefrontal regions in mediating ToM capacities.Understanding the mental states of others entails a number of cognitive processes known as Theory of Mind (ToM). A relationship between ToM deficits and executive disorders has been hypothesized in individuals with Parkinson's disease (PD). The present study was aimed at investigating the effect of dysexecutive deficits on ToM abilities in PD patients without dementia. Participants included 30 PD patients and 30 healthy subjects (HC). PD patients were divided into two groups according to their executive test performance: patients with poor (dysexecutive group;"} +{"text": "Despite a large volume of evidence supporting the use of dual antiplatelet therapy in patients with acute coronary syndrome, there remains major uncertainty regarding the optimal duration of therapy. Clinical trials have varied markedly in the duration of therapy, both across and within trials. Recent systematic reviews and meta-analyses suggest that shorter durations of dual antiplatelet therapy are superior because the avoidance of atherothrombotic events is counterbalanced by the greater risks of excess major bleeding with apparent increases in all-cause mortality with longer durations. These findings did not show significant heterogeneity according to whether patients had stable or unstable coronary heart disease. Moreover, the potential hazards and benefits may differ when applied to the general broad population of patients encountered in everyday clinical practice who have markedly higher bleeding and atherothrombotic event rates. Clinicians lack definitive information regarding the duration of therapy in patients with acute coronary syndrome and risk scores do not appear to be sufficiently robust to address these concerns. We believe that there is a pressing need to undertake a broad inclusive safety trial of shorter durations of therapy in real world populations of patients with acute coronary syndrome. The clinical evidence would further inform future research into strategies for personalised medicine. A ruptured or eroded coronary atherosclerotic plaque is the principal underlying cause of an acute coronary syndrome. The greatest \u2018at risk\u2019 period is during this early phase of plaque instability and healing, with recurrent event rates peaking in the first month. By 3 months, the plaque has usually stabilised, healed and subsequent event rates return to the background rates seen in patients with stable coronary heart disease.In an acute coronary syndrome, thrombus formation occurs under conditions of high shear stress and is principally driven by platelet aggregation . This doGiven aspirin\u2019s remarkable success, it is perhaps unsurprising that adjunctive antiplatelet therapies have been investigated to build on these benefits, especially as there are multiple mechanisms of platelet activation beyond the cyclo-oxygenase pathway . HoweverThe benefit of dual antiplatelet therapy following an acute coronary syndrome was established by the CURE,The evidence for dual antiplatelet therapy in patients with stable coronary heart disease is less distinct. In the CHARISMA trial,Current EuropeanIn recent trials of patients treated with newer generation drug-eluting stents, shorter durations of dual antiplatelet therapy (3 months to 6 months) were non-inferior to 12Beyond 12 months, there remains a residual risk of local and systemic atherothrombotic complicationsMeta-analyses of trials using dual antiplatelet therapy in patients receiving intracoronary stents have compared short (3\u20136 months), 12-month and prolonged (>12\u2009months) durations of therapy.The optimal duration of dual antiplatelet therapy is dependent on the balance between preventing future atherothrombotic events and the increased risk of bleeding from continued treatment. Following an acute coronary syndrome, predictors of atherothrombotic risk include ST deviation, diabetes mellitus, smoking status, left ventricular ejection fraction, stent type, number of stents and complexity of coronary artery disease. Subgroup analyses have attempted to identify if any of these factors influence outcomes with regard to duration of dual antiplatelet treatment.In the EXCELLENT trial,Audit of the British Cardiovascular Society Intervention database indicates that in 2013/2014, a half of all percutaneous coronary intervention procedures were associated with residual disease (\u22651 stenosis of >50% severity), fulfilling the criteria for incomplete revascularisation. Patients with acute coronary syndrome and incomplete revascularisation have a residual burden of coronary disease that is a substrate for recurrent plaque rupture, coronary thrombosis and future cardiac events.Large registries and trials have shown that major bleeding is associated with an increase in mortality that could potentially negate the benefits of dual antiplatelet therapy in acute coronary syndrome.Currently there are variations in local and regional dual antiplatelet therapy practices that are confusing for patients, primary care physicians and cardiologists. Indeed, while EuropeanFor clinicians and healthcare providers, there remains much uncertainty regarding the default duration of dual antiplatelet therapy for most patients with acute coronary syndrome. Current guidelines are largely based on evidence that predates potentially important technological advances, including second-generation drug-eluting stents, while in recent trials, only a minority of patients presented with an acute coronary syndrome and many of these studies were underpowered to detect differences due to low event rates. Selected populations included in randomised controlled trials have lower rates of bleeding and non-cardiovascular death than the general population , since pIncreased thrombin generationNewer generation P2Y12 inhibitors provide more effective antithrombotic protection than clopidogrel but at the cost of increased bleeding. Given the greater expense of these agents and the marked temporal decline in thrombotic risk that is evident over the first few months, an early \u2018switch\u2019 from ticagrelor or prasugrel to clopidogrel after 1 month to 6 months has been advocated. While evidence from small cohortGeneric recommendations for length of dual antiplatelet therapy derived from a protocolised intervention in clinical trials will inevitably expose some patients to an excessive duration of treatment and disadvantage other patients by withdrawing therapy that protects them from myocardial infarction. The critical clinical question is therefore: can individuals who are more or less likely to benefit from shorter or longer durations of treatment be identified? While several risk tools have been developed to help determine the future incidence of coronary thrombotic events and major bleeding episodes for an individual, the DAPT ScoreUsing data from the DAPT trial, the DAPT Score was developed to determine the net clinical benefit of extending dual antiplatelet therapy from 12 months to 30 months . Thirty-Variables included in risk tools tend to predict both bleeding and myocardial infarction. High platelet reactivity was originally reported to identify patients at an increased risk of future atherothrombotic events.It has been 15 years since the CURE trial demonstrated the benefit of dual antiplatelet therapy following an acute coronary syndrome and yet the optimal duration remains uncertain. With regard to thrombotic complications, recent clinical trials and meta-analyses suggest that with newer generation drug-eluting stents, 3 months to 6 months of dual antiplatelet therapy is non-inferior to 12 months of treatment. Prolonged treatment (>12\u2009months) reduces the risk of stent thrombosis, myocardial infarction and possibly cardiovascular death but at the cost of increased major bleeding and with no net mortality benefit. However, these potential hazards and benefits of intervention may differ when applied to the general broad population of patients encountered in everyday clinical practice who have higher bleeding and atherothrombotic event rates.While ongoing randomised clinical trials may address some of the residual uncertainties in select subgroups, we believe there is a pressing need to undertake a broad inclusive trial of shorter durations of therapy in broad populations of patients with acute coronary syndrome. Such a trial will need to be able to explore specific subgroups, such as those who are medically managed, undergo percutaneous coronary intervention or have coronary artery bypass graft surgery, as well as enable better identification of atherothrombotic and bleeding risks from real world data to inform a more personalised approach to decisions regarding treatment duration."} +{"text": "Gastric antral vascular ectasia (GAVE) and its nodular antral gastropathy (NAG) variant is a unique lesion associated with hypergastrinemic hormonal alterations that may be compounded by concurrent proton pump inhibitor (PPI) therapy. The use of octreotide as a somatostatin analogue and its role in the down regulation of variousenteric hormones has been well documented however its use in the management of NAG has not been widely reported. We herein present a case where octreotide induced gastrin down-regulation as well as PPI cessation facilitated NAG resolution. Proton pump inhibitor (PPI) therapy can be very effective in the symptomatic and endoscopic remission of acid-peptic disorders. However, despite its increasingly widespread use; their side effect profile is just beginning to be unveiled. Here we present a case where hormonal disarray in conjunction with PPI therapy produced deleterious effects on nodular gastric antral vascular ectasia (GAVE). This case also highlights the importance of understanding the pathophysiologic basis of different disease states so as to provide comprehensive optimal therapeutic patient care.A 49-year-old Caucasian male was referred from an outside facility for further evaluation of refractory iron deficiency anemia secondary to chronic intermittent melenic gastrointestinal bleeding. Despite multiple blood transfusions and iron supplementation, his anemia continued to worsen. Upper endoscopy performed at an outside facility described features of chronic gastritis hence proton pump inhibitor therapy was instituted. Despite this the frequency of melenic stools increased. Repeat upper endoscopy reported multiple friable antral polyps with histopathologic analysis reporting changes consistent with adenomatous tissue.Our patient\u2019s past medical history was notable for chronic active hepatitis C infection, coronary artery disease and chronic kidney disease. The patient was post pancreatic and renal transplantation. A liver biopsy in 2001 demonstrated mild fibrosis consistent with chronic viral hepatitis C. Medications on referral to our institution included Nexium 40 mg daily, Iron sulfate 325 mg twice daily, ASA 81 mg, Carafate 1 g four times daily and cyclosporine 100 mg daily.Helicobacter pylori were all negative and the patient\u2019s PPI was adjusted to twice daily. Recurrent melenic gastrointestinal bleeding continued to occur despite multiple sessions of cautery with APC and snare polypectomies of the antral lesions. During the patient\u2019s 12-month clinical course, four APC procedures were performed. Furthermore, serial EGD evaluations observed a temporal increase in the size and associated friability of the nodular, polypoid lesions after each cautery treatment. The size of the polypoid lesions increased from an average size of 6 mm at initial presentation to 20 mm after 8-months into his clinical course paralleled the growth of these polypoid antral lesions. With an increasing size of polypoid growth and continued recurrent melenic bleeding requiring periodic transfusions; additional strategies to combat these antral lesions were needed. We then decided to address the hormonal stimulation of these lesions and first discontinued the proton pump inhibitor agent and started an H2-blocker, Ranitidine 150 mg twice daily. An interval EGD noted stable antral lesions, but not significantly changed; thus additional hormonal treatment with Octreotide therapy was subsequently commenced with a dramatic effect on the antral lesions noted. Immediately after this intervention, a significant decrease in size and number of the antral lesions was observed, with only several 2 to 3 millimeter faintly erythematous nodules observed in the antrum without friability or oozing. In tandem, the levels of serum gastrin decreased exponentially as well with the initiation of Octreotide and the discontinuation of the PPI. Subsequent EGD studies confirmed marked improvements in the antral vascular ecstatic pattern observed since discontinuation of the PPI agent and initiation of the Octreotide therapy . The patGastric Antral Vascular Ectasia (GAVE) was first described in 1953 by Ryder et al .The pathogenesis of the condition still remains an enigma; however two schools of thought are currently in vogue with the latter gaining more popularity. The first involves a predominantly mechanical component with the view that repeated prolapse of the antral mucosa by forceful peristalsis results in repeated obstruction of mucosal and submucosal blood flow leading to vascular ectasia; a view supported by the nearly equidistant and unique linear pattern of the blood vessels seen .The second theory favors a biochemical origin borne out of the observation that the condition was more prevalent in patients with chronic liver disease. Recent studies have made a clear distinction between chronic liver disease as opposed to portal hypertension as the implicating factor. It was noted that GAVE lesions disappeared post liver transplant; however, portal pressure reduction strategies had no effect -5. It isTreatment options for GAVE are diverse, however first line management is generally cautery given endoscopically such as with Argon Plasma Coagulation (APC) as it commonly provides resolution of bleeding, anemia and ablation of friable lesions , 7. NoduGastrin is a linear peptide hormone produced by G-cells of the antrum and duodenum. Its effects include the stimulation of gastric blood flow, parietal cell maturation, increased antral muscle mobility with fundal growth and trophic effects on the GI tract. Reports of enterochromaffin cell hyperplasia and carcinoid tumors in hypergastrinemia settings have raised a safety concern in humans , 11. ItsGastric antral vascular ectasia and its NAG variant is a unique lesion associated with hormonal alterations that largely remain to be characterized. It has been demonstrated to be associated with a hypergastrinemia and when compounded by chronic proton pump inhibitor therapy which itself can lead to elevated gastrin, the ensuing \u2018gastrinemic state\u2019 may lead to untoward effects as evidenced in this case. Our case illustrates an instance where proton pump inhibitor therapy may have actually had deleterious effects. It also demonstrates the expanding use of Octreotide and the importance of understanding disease pathophysiology at a pathophysiologic level in order to provide optimal comprehensive therapeutic care."} +{"text": "Data that defines IGHV (immunoglobulin heavy chain variable) germline gene inference using sequences of IgM-encoding transcriptomes obtained by Illumina MiSeq sequencing technology are described. Such inference is used to establish personalized germline gene sets for in-depth antibody repertoire studies and to detect new antibody germline genes from widely available immunoglobulin-encoding transcriptome data sets. Specifically, the data has been used to validate [1]) the inference process. This was accomplished based on analysis of the inferred germline genes\u2019 association to the donors\u2019 different haplotypes as defined by their different, expressed IGHJ alleles and/or IGHD genes/alleles. The data is important for development of validated germline gene databases containing entries inferred from immunoglobulin-encoding transcriptome sequencing data sets, and for generation of valid, personalized antibody germline gene repertoires. Specifications TableValue of the data\u2022The data is valuable for development of computational inference approaches that feature improved confidence in the outcomes of the inference process.\u2022The data is valuable for development of validated immunoglobulin germline gene databases.\u2022The data is valuable for validation of computational inference of personalized antibody germline gene repertoires.\u2022The data is valuable for the analytical process preceding studies of evolution of immune responses.1The data of this article summarize the identity and accession numbers of sequencing data files , the siz2https://github.com/ukirik/giggle. Immunoglobulin gene names and sequence numbering complies with the nomenclature defined by the International ImMunoGeneTics information system\u00ae (IMGT) (http://www.imgt.org) IgM heavy chain variable domain-encoding gene repertoires were isolated by RT-PCR from transcriptomes of PB and BM collected out of season of most seasonal allergens from six allergic subjects"} +{"text": "Glucocorticoid (GC) hormones play significant roles within homeostasis and the chrono-dynamics of their regulatory role has become increasingly recognised within dysregulated GC pathology, particularly with metabolic phenotypes. Within this article, we will discuss the relevance of the ultradian homeostatic rhythm, how its dysregulation effects glucocorticoid receptor and RNA polymeraseII recruitment and may play a significant role within aberrant metabolic action. In Cushing disease cohorts, excessive constant GC production drives significant over-representations of obesity, diabetes mellitus and dyslipidaemia phenotypes 3GCs act via the ligand activated intracellular transcription factor, the glucocorticoid receptor (GR). GR dynamic regulation closely tracks the rising and falling levels in each GC pulse, and may therefore be highly sensitive to altered GC patterns associated with dysregulated GC phenotypes. In vivo studies observed pulsatile GR recruitment to the period circadian clock 1 (PER1) gene's GC response element (GRE) and similar pulsatile nascent RNA production 4In the brain, the ultradian GC rhythm has been shown to play a critical role in maintaining the physiological, behavioural and molecular response to an acute stress. Replacement of a pulsatile GC rhythm with a matched constant infusion significantly impaired the adaptive response to the stressor 5Ultradian GC action is highly sensitive to disruption and therefore could be equally integral to development of metabolic phenotypes. In support of this, GR and Pol2 recruitment in vitro and in vivo can be differentially modulated depending upon the pattern of cort exposure. New evidence currently emerging suggests that the pattern of delivery, not just the dose, should be considered when treating patients with sGC and that benefits could be garnered from \u2018ultradian\u2019 patterned therapies.The authors declare that they have no competing interest."} +{"text": "Breast cancer patients are presenting at advanced stages for oncological treatment in Nigeria and World Health Organization predicted developing countries\u2019 breast cancer incidence and mortality to increase by year 2020.Prospective observational hospital based study that enrolled breast cancer patients from catchment area of an oncology service hospital in Nigeria between 2007 and 2013. Patients\u2019 demographics, breast cancer burden and health care giver presentation variables were analysed for causal factors of seeking medical help and what determines commencement of effective oncological treatment.patients\u2019 matrimonial setting, breast cancer awareness and mode of discovery of breast symptoms are patient related factors that determines their choice of health care providers and, determinant of effective oncological treatment is patient first contact health care provider.Forty-six patients were enrolled, 19.6% of them presented primarily to oncologist while 80.4% presented secondarily for oncological treatment. There is a significant difference in presentation time for oncological treatment between primary (M =11.56 \u00b1 5.21 weeks) and secondary presentation (M= 52.56 \u00b1 10.27weeks). Tumor burden of those that presented secondarily were significantly more advanced and, univariate analysis reveals that: Patients\u2019 bio-characteristics that determine their choice of health care provider should be incorporated into community breast cancer sensitization drives. Additionally, there is a need for a government agency assign the task of accrediting and defining scope of enterprise of health care institutions and their health care providers in our pluralist health system. Breast cancer is the commonest cancer among women worldwide and the most common cause of cancer related morbidity and mortality \u20133. ThereThis is a cross-sectional hospital based study carried out at Wesley Guild Hospital (WGH) in Ilesa, between January 2007 and December 2013. Ilesa is a cosmopolitan town situated in Osun States in south western Nigeria. The town has good roads network connection and transport system, it health system is pluralist made up of orthodox and modern health facilities and the modern health facilities consist of private and government owned hospitals. WGH is one of the governments owned hospital and is a satellite hospital of Obafemi Awolowo University Teaching Hospitals Complex (OAUTHC), Ile-Ife, Nigeria. Eligibility for the study is being a breast cancer patient presenting at the surgical oncology unit of WGH consenting to recruitment and data collected to be used for research purpose. Inclusion criteria were: being domicile in Ilesha and malignant breast lesion. Exclusion criteria were: being domicile outside Ilesha, benign breast lesion and inability to provide adequate information because of karnofsky performance status. Data obtained were patients\u2019 demographics, clinical and laboratory details of breast cancer burden using American Joint Committee on Cancer (AJCC) staging system and evolution of patient's presentation to health care provider(s). Patients were subdivided into two groups whether they presenting primarily or secondarily for oncological treatment Data obtained were entered into the proforma designed for the study and analysed using Statistical Package for Social Sciences version 22. Descriptive statistics were employed for univariate analysis and, independent samples t test, nonparametric tests and regression analysis for multivariate analysis.their matrimonial setting, breast cancer awareness and mode of discovery of breast symptoms and, the significant determinant whether a breast cancer patient will have effective oncological treatment is their first healthcare provider (Forty-six patients met the inclusion criteria. They were all females; nine of these patients presented primarily in WGH while the remaining 37 presented secondarily for oncological treatment. Details of their demographic bio- characteristics are as shown in provider .Presentation of breast cancer patients at advanced stage is a major contributor to increasing morbidity and mortality in the developing countries , 17. RevThe result of this study shows the dominant role of a health care provider at first patients\u2019 contact as determinant of effective oncological treatment, though being tag qualified health care personnel is at the prerogative of the government who is the licensing body; unfortunately, 80.4% of the patients presented outside the setting where they can access oncological management. Health care providers\u2019 documentation of patients\u2019 disease in Matrimonial setting, breast cancer awareness and mode of discovery of breast symptoms are significant patients\u2019 bio-characteristics that determine their choice of health care provider. These factors should be considered in community breast cancer sensitization drives. Additionally, non oncologist health care providers in our pluralist health system will need re-training in oncology and their scope of enterprise should be properly defined by appropriate government institution.Presentation of breast cancer patients outside the setting where they can access early and effective oncological treatment seems to forms the basis of advanced stage presentation. Seventy to eighty percent of breast cancer patients in Nigeria present primarily at oncological centre for treatment with locally advanced or metastatic disease;Breast cancer awareness is the determinant of early presentation to a health care provider;Determinant of effective oncological treatment is patient presentation for treatment.Only about twenty percent of breast cancer patients in this study present primarily at oncological centre for treatment and about sixty percent of them presented with early breast cancer;Breast cancer awareness, patient's matrimonial setting and mode of discovery of breast symptom are the determinants of early presentation to a health care provider;Determinant of effective oncological treatment is the health care provider at first patient/health care provider contact."} +{"text": "Flexible gastro-intestinal (GI) endoscopy is an integral diagnostic and therapeutic tool in clinical gastroenterology. High quality standards for safety, patients' comfort, and efficiency have already been achieved. Clinical challenges and technical approaches are discussed in this short review.Image enhanced endoscopy for further characterization of mucosal and vascular patterns includes dye-spray or virtual chromoendoscopy. For confocal laser endoscopy, endocytoscopy, and autofluorescence clinical value has not yet been finally evaluated. An extended viewing field provided by additional cameras in new endoscopes can augment detection of polyps behind folds. Attachable caps, flaps, or balloons can be used to flatten colonic folds for better visualization and stable position.Variable stiffness endoscopes, radiation-free visualization of endoscope position, and different overtube devices help reducing painful loop formation in clinical routine. Computer assisted and super flexible self-propelled colonoscopes for painless sedation-free endoscopy need further research. Single-use devices might minimize the risk of infection transmission in the future.Various exchangeable accessories are available for resection, dissection, tunneling, hemostasis, treatment of stenosis and closure of defects, including dedicated suturing devices. Multiple arm flexible devices controlled via robotic platforms for complex intraluminal and transmural endoscopic procedures require further improvement. Standard procedure for GI endoscopy has not changed much during the last decades. The tip of the flexible insertion tube can be bent vertically and horizontally by steering wheels via Bowden cables. Manual Insertion and retraction can be combined with rotation of the entire endoscope. Light is transmitted from the connected processor to the endoscope tip from where a chip sends back image signals from the lens to a monitor. Channels allow insufflation of the GI lumen, aspiration of fluid content, and rinsing of the lens. A larger instrumentation channel accommodates various diagnostic and therapeutic accessories. Design and cover of the endoscope allow efficient disinfection before re-use. Flexible endoscopy combines accurate mucosal visualization and therapy for the price of higher invasiveness than imaging techniques and wireless capsule endoscopy. Recommendations for technical and clinical quality standards in GI endoscopy have been established Advanced optical systems for precise visual diagnosis and multiple diagnostic and therapeutic accessories which can be applied and exchanged on demand are further important features. Robotic platforms for steering of multidimensional endoscopes have already been developed.Screening for colorectal cancer (CRC), one of the three most common types of cancer worldwide, is one of the major indications for flexible GI endoscopy Other indications for GI endoscopy are gastro-esophageal reflux disease including potentially premalignant Barrett's esophagus, gastro-duodenal ulcers, gastric cancer, treatment of small bowel bleeding, and diagnosis of inflammatory bowel disease.Clinical tasks in flexible endoscopy are optimal characterization of lesions for targeted management, improving adenoma detection, avoiding incomplete endoscopy, reducing pain and need for sedation, infection prevention, and improvements in therapeutic endoscopy. This short review discusses clinical needs and technical solutions already achieved or under development , address22.1high definition (HD) endoscopy in narrow band imaging . Other modalities like i-scan , Flexible Spectral Imaging Color Enhancement , and Storz Professional Image Enhancement System involve real-time electronic post-processing algorithms for spectral color selection. By blue light imaging c and in I; Fuji) e narrow I; Fuji) d combineConfocal laser endoscopy (CLE) uses blue laser light brought to direct contact with the mucosa after intravenous injection of fluorescein allows visualization of details down the level of nuclei by contact light microscopy after staining the mucosa with methylene blue and crystal violet. The system was proposed as a miniprobe device and has also been integrated into flexible endoscopes (Olympus). A novel computer aided diagnosis system provided automatic classification of colonic polyps based on identification and characterization of nuclei during processing of EC images HD endoscopy is recommended for routine CRC screening, and real or virtual chromoendoscopy of the entire colon in high risk situations as surveillance of long standing ulcerative colitis or polyposis syndromes 3Up to 20\u201340% of adenomas are missed during standard colonoscopy Approaches to detect adenomas hidden behind colonic folds by expanding the standard forward-viewing angle of 140\u2013170\u00b0 or by mechanic manipulation of folds during endoscopy are described below and summarized in 3.1Full Spectrum Endoscopy(FUSE) colonoscopy platform uses a standard colonoscope with two additional cameras and light sources build into the left and right side of the distal end . A single image is combined from a standard forward viewing lens and the additional convex shaped lens. A feasibility study suggested potential for a higher ADR In a similar matter, Olympus developed a prototype colonoscope with an Omniview modus of the single use, self-propelled pneumatic Aer-O-Scope combines a 57\u00b0 forward viewing lens and a 44\u00b0 lateral view circular around the entire central axis. However, in a first clinical trial there was no diagnostic benefit over standard colonoscopy Third Eye Retroscope is an auxiliary device with camera and light source. After insertion through the working channel of a standard colonoscope it is angulated 180\u00b0 allowing an additional retrograde view of the colon with significant increase in adenoma detection of 11\u201323% Third Eye Panoramic (Avantis) with two side viewing cameras and light sources is attached to the tip of a colonoscope is a single use silicone rubber device attached to the distal end of the colonoscope. Flexible flaps stretch and straighten out folds during withdrawal is a similar single use device using rubber arms instead of flaps to straighten out the mucosa has a balloon permanently integrated in the distal end of a standard colonoscope. If inflated during withdrawal it straightens out folds similar to EndoRings or Endocuff. A multicenter randomized study reported an 16% increased ADR compared to a standard colonoscope The cap attached to the endoscope tip had no benefit in ADR in a large randomized trial study Colonoscopy assisted by a transparent 3.3Auto fluorescence imaging has been described as a tool to highlight GI neoplasia. A metaanalysis found no increase in ADR with AFI compared to WLE Endoscopy with 3.4To increase complete visualization of colonic mucosa, optic feedback on missed areas could be useful. For this purpose, an algorithm creating 3D images with color-coding of improperly visualized areas in a synthetic colon model based on brightness intensity analysis of endoscopic images has been proposed Technical aids discussed in 4Loop formation and luminal distension are main reasons for incomplete or painful colonoscopy. Technical approaches to tackle this issue are discussed below and summarized in 4.1Sedation, manual external compression, positioning of the patient and straightening and rotation of the endoscope may be useful in preventing loops and obtaining complete investigations.Variable stiffness colonoscopes allow manual control of stiffness by applying tension to integrated cables. After passing a flexure with a more flexible endoscope, increasing rigidity can prevent loop formation during further advancement. The rate of complete colonoscopies was significantly higher compared to a standard colonoscope Additionally, 4.2ScopeGuide (Olympus) is a positioning system emitting electromagnetic signals from special colonoscopes or inserted miniprobe to a detector beside the patient. From the signals a 3D real-time localization image of the colonoscope is displayed on a monitor resembling a virtual X-ray visualization The commercially available NeoGuide Endoscopy System Inc. developed a computer assisted prototype colonoscope consisting of multiple fully articulated segments in the insertion tube. A computer algorithm calculates their position in relation to each other to generate a real-life 3D image of the insertion tube to reduce looping 4.3Device assisted endoscopy (DAE) methods have been developed to reduce loops by pleating the long and hardly assessable small bowel over an overtube Single balloon endoscopy, Olympus) or on the tip of both overtube and endoscope used to keep position by gripping the small bowel. A spiral endoscopy overtube translates continuous rotation into forward movement while pleating the small bowel. A new prototype endoscope with a motor driven, force controlled rotating distal spiral segment (Olympus) demonstrated feasibility in patients, allowing complete enteroscopy with a single oral approach in some of them balloon assisted enteroscopy with a standard colonoscope assisted by a through the scope (TTS) balloon has been reported Several DAE methods are the gold standard for therapeutic endoscopy of the small bowel. They may also be applied in case of incomplete colonoscopy to facilitate complete inspection of the entire colon. Variable stiffness colonoscopes are widely used in routine, Scope guide is available commercially.4.4Insufflation of CO2 instead of air through the scope diminishes patient's discomfort and pain during colonoscopy as it is absorbed up to 160 times faster Extensive insufflation of air causes pain and hampers endoscope progression due to a larger lumen. 4.5Another approach to reduce pain and discomfort during colonoscopy is the development of super flexible self-propelling devices. They potentially avoid necessity of sedation by smoothly following the loops without painful stretching of the bowel.Invendo SC20 , a single-use colonoscope including a working channel and controlled by motor rollers is a super-flexible self-propelling and self-steering endoscope tip advancing through the colon loops by controlled air pressure between inflated balloon in the rectum and at the tip of the endoscopy. Mucosa is inspected during manual withdrawal of the scope. Biopsy and polypectomy are not possible. Complete colonoscopy was possible in a clinical study in 55/56 patients without sedation Endotics System operates in a similar manner. A remotely controlled, single-use colonoscopy probe crawls through the colon by repeatedly adjusting its length mimicking an inch-worm are sinColonoSight a hybrid solution had been developed. The reusable colonoscope does not need disinfection as a single-use cover including the working channel prevents the endoscope from contact with potentially infectious agent. No bacterial contamination of endoscope was found With the However, none of these single use flexible endoscopes has entered clinical routine yet.6Flexible endoscopy is indispensable for treatment of GI lesions. The therapeutic spectrum has increasingly been extended, during routine procedures as well as by referrals to complex interventions potentially avoiding surgery. Through the scope (TTS), over the scope (OTS) and over the wire (OTW) accessories are available for various purposes.6.1Polypectomy of adenomatous polyps detected at colonoscopy is integral part of screening procedures. Standard TTS instruments are biopsy forceps for histology including complete removal of lesions up to 5\u00a0mm, and polypectomy snares with electrocautery for larger polyps. Flat and laterally spreading adenomas can be lifted by submucosal injection of saline before endoscopic mucosal resection (EMR) with a snare. Stain added to the saline may be useful for better delineation of the margins. Larger lesions have to be resected in parts . Although IEE can correctly classify most lesions accurate, histology is still important for correct classification . After injection of fluid into the submucosal space and circular incision of the mucosa around the lesion, the submucosa is dissected underneath the lesion using various types of TTS knifes for incision and dissection, needles for repeated injection, and graspers for coagulation of vessels Superficial malignant lesions of the mucosa can be resected en-bloc by 6.2Small perforations of the GI wall including those after endoscopic resection or after surgery can be treated endoscopically in selected patients. Multiple TTS metallic clips are useful to adapt wound margins. Larger defects can be closed with a TTS loop used to adapt several clips at both sides of the defect. Alternatively, a larger clip mounted over the scope clip (OTSC) may close margins sucked into the cap of the device c, d. A f6.3GI strictures can often be dilated with hydrostatic balloons advanced through the scope. Bougies are advanced over a wire placed through the endoscope after withdrawal of the scope, as well as larger pneumatic dilation balloons or self-expanding metal stents POEM) has been introduced as alternative to surgery of hypertrophic lower esophageal sphincter in achalasia causing dysphagia. The esophageal submucosa is dissected after incision of the superficial mucosa to create a tunnel allowing advancing the endoscope distally for incision of the deep muscular layer of the hypertrophic sphincter. Finally, the mucosal defect is closed by clips Recently, PerOral Endoscopic Myotomy system Transoral Gastroplasty OverstitchEndomina, Endoscopy is used for a long time to guide percutaneous placement of feeding tubes in patients with jeopardized nutrition. However, nowadays, obesity and diabetes are increasing challenges. Hence, similar tubes have even been used to empty the stomach in obese patients. Endoscopically placed gastric balloons reduce the gastric lumen temporarily. Permanent gastroplasty is typically performed surgically but has also been performed endoscopically by stapling devices as experimental articulating circular endoscopic improved obesity and diabetes, but proximal fixation hooks in the duodenum may cause adverse events The endoscopically implanted duodeno-jejunal bypass sleeve 6.6Complex endoscopic resection of large or unfavorably positioned lesions is augmented by a stable endoscope position. A robotic platform additionally uses an intuitive interface to control motorized endoscope movements and deflection of the tip via joystick or touch pad A double channel endoscope allows limited triangulation with an additional grasper. Procedure times for ESD could be shortened in a Japanese series Natural Orifice Transluminal Endoscopic Surgery (NOTES) aims to reduce invasiveness of traditional transabdominal surgery by accessing the peritoneal cavity through natural lumina as stomach, colon or vagina 2 requires higher flow rates than provided by standard endoscopes for distension of the GI lumen. Adopting high flow devices with pressure control from laparoscopy could be helpful Anubiscope\u2122 (Storz) EndoSamurai (Olympus) robotic platform translates bimanual actions into movements of the two flexible small caliber instruments in a flexible endoscope. This system outperformed a conventional dual channel flexible endoscope The Cobra and TransPort flexible endoscopy systems provide a platform for transluminal endoscopy. The TransPort device with 4 working channels allows simultaneous application of flexible endoscopes and surgical instruments Master And Slave Transluminal Endoscopic Robot (MASTER) platform translates bimanual steering to a flexible endoscope with two instrumentation arms with nine degrees of freedom Direct drive endoscopic system (DDES) allows to bimanually directing two instruments in an endoscopic sleeve via a robotic platform by one operator Endoeye Flex 3D, Olympus) The principle of a semi flexible laparoscope creating 3D images from two separate cameras (Most cases of NOTES have been performed as hybrid transvaginal cholecystectomy still with limited transabdominal augmentation. Transgastric and transcolonic access have also been used but are not yet ready for use in clinical routine 7Wireless technique of capsule endoscopy, the first line diagnostic tool for the small bowel is already available for diagnostic endoscopy of the upper and lower GI tract. However, detailed characterization of lesions including histology and therapy will require flexible endoscopy as gold standard for the next years. An endoscope combining possible advantages would be a single-use, super flexible, self-propelled device for a pain free procedure without sedation. Optional increase in rigidity could provide a stable position for therapy. Ideally, a small caliber endoscope provides ample working channels with sufficient size to easily apply and exchange all appropriate accessories. Additionally, real-time image processing programs could assist in lesion detection and characterization. Dedicated robotic platforms might further augment intraluminal and transmural complex therapies augmented by 3D imaging."} +{"text": "This review aims to provide clinicians and researchers with a condensed update on the most important studies in the field during 2017. We present the academic output measured by active clinical trials and peer-reviewed published manuscripts. The most important and contributory manuscripts were summarized for each diagnostic entity, with a particular focus on manuscripts that describe translational research that have the potential to improve clinical care. Finally, we highlight the importance of collaborations in adrenal tumor research, which allowed for these recent advances and provide structures for future success in this scientific field. Clinically unapparent adrenal tumors are found in 2\u201310% of the population worldwide. Cases that do require treatment are enriched in risk populations particularly those with hypertension or genetic risk factors and paragangliomas , 177 on adrenocortical tumors and 18 that fell into a general adrenal tumor category. In this review, we have referenced 110 of these manuscripts and selected three prominent topics that we felt deserved special attention . We alsoet al. studied adrenogonadal development during weeks 6\u201310 and characterized the processes of testis determination, onset of steroidogenesis and primordial germ cell development. Their genomic atlas of human adrenal and gonad development proposed new candidate genes for adrenal and reproductive disorders aims to map all the human proteins in cells, tissues and organs using integration of various omics technologies. It has employed more than 26,000 antibodies on multiple tissues, cells and pathological states. The consortium now presented their data on the adrenal gland with RNA sequencing of tissue homogenates that identified 253 genes with an elevated expression pattern compared to other tissues PET/CT could improve diagnosis PGLs . Both cl et al. performed a prospective study on the effects of surgical resection on carotid intima-media thickness and left ventricular mass in 50 PPGL patients , those with PCCs had a higher rate of comorbidities including congestive heart failure, chronic lung disease and malignant hypertension with poor survival associated with male sex and synchronous metastases . In addi131I-meta-iodobenzylguanidine (MIBG) (SDHB patients had a higher response rate to CVD compared to those without SDHB mutations (Efficacy and safety of peptide receptor radionuclide therapy (PRRT) (SDHx) were thought to have almost complete penetrance for PPGL. This figure has now been adjusted, a finding of major importance when designing surveillance protocols for healthy carriers: the overall penetrance of SDHB mutations was estimated to be 21% at the age of 50 years and 42% at the age of 70 years (SDHA-related manifestation in non-index patients was 13% at age 40 years (FH mutations) was assessed in 182 cases from 114 families and found only two cases of PPGL patients with SDHB mutation had an open approach. The underlying argument was to preserve cortical function in patients with a high risk of bilateral PCC having a low risk of metastatic disease (RET and VHL carriers), whereas those with a low risk of bilateral PPC but with risk factors for metastases (including SDHB and large tumor size) could benefit from an open approach to maximize probability of radical resection. A second study show that patients with NF type 1 had more volatile intraoperative course and more severe complications, probably related to larger tumors and abundant catecholamine secretion that resulted in a high proportion of open resections and proposed a modified GAPP with addition of SDHB staining to be useful for the prediction of the metastatic potential and prognosis in PPGL (Koh https://gdc.cancer.gov) and serves to accelerate research in this rare entity. Disease-causing mutations or gene fusions occurred in 73% of cases. At least two novel disease-causing genes were identified: MAML3 and CSDE1. Integrated analysis classified PPGL into three main subtypes: kinase signaling, pseudohypoxia and Wnt altered. A forth subtype, cortical admixture, was also detected but is thought to reflect a signal from non-tumoral cells.The Cancer Genome Atlas described results from 173 PPGLs that were analyzed with six different molecular profiling technologies, the most comprehensive molecular characterization of PPGL ever performed have already allowed PPGL to be included into several pan-cancer analyses; the first underscore that PPGL genomes exhibit relatively low number of somatic mutations as well as copy number segmentations compared to other tumors . The secTERT structural rearrangements expression and mechanisms of alternative lengthening of telomeres in the highest proportion of cases as a driver of APA, macrolides were shown to selectively inhibit mutant KCNJ5 opening, which might provide the option for improved diagnosis and treatment and was associated with reduced DNA methylation at the CYP11B1 promoter that may result in CYP11B1 transcription and hypercortisolemia has been proposed as a surrogate for overall survival in ACC. This study found that most patients with metastatic ACC and long survival times had PR within the first 6 months of systemic therapy, and almost all within the first year. The absence of response after that period could be considered as a treatment failure achieved rapid control of Cushing syndrome induced by cortisol-secreting ACC in three patients (ERCC1, involved in DNA excision repair, was investigated as a predictive biomarker of platinum-based chemotherapy in 146 ACC but demonstrated negative results (One hundred forty-five ACC received gemcitabine based chemotherapy, PR or stable disease was achieved in 4.9 and 25.0%, respectively (et al. investigated DNA methylation and Sbiera et al. investigated VAV2 gene expression (Comprehensive characterization of ACC biology was previously achieved by TCGA and ENSAT consortiums that together proposed a robust molecular classification (TP53 mutations and showed similar prognosis and outcome regardless of mutation status. Ki67 index was a promising prognostic biomarker also in pediatric ACC (A study of 60 pediatric ACCs investigated the impact of germline Independent studies show that circulating tumor DNA can be found in a subset of ACC with high tumor burden (MUTYH-associated polyposis had adrenal lesions; two were hyperfunctioning. Among four patients that underwent adrenalectomy, three had benign tumors and one was oncocytic of uncertain malignant potential (et al. were able to detect this MUTYH deficiency signature in ACC (SDHx deficiency.Five out of 21 patients with The TCGA dataset has now allowed researchers not involved in the adrenal field to characterize ACC and compare it to other cancers. Three different studies compared the mutational landscape , patternCDK4 and CDK6 inhibitors were suggested to be candidate drugs for treatments of ACC (The aurora kinase inhibitor AMG 900 increased apoptosis and chemosensitivity to anticancer drugs in the NCI-ACC cell line (VAV2 was mentioned earlier as a prognostic factor, this study revealed molecular mechanisms involved and suggest that blocking VAV2 may be a new therapeutic approach to inhibit metastatic progression (Rottlerin was introduced as a novel chemotherapy agent (Analyses of the mTOR and SSTR2 pathways in ACC cell lines H295R and SW13 (ATR-101 was found to inhibit cholesterol efflux and cortisol secretion by ATP-binding cassette transporters, causing cytotoxic cholesterol accumulation in ACC . This coA mouse xenograft model of metastatic ACC (Clinical and basic research on adrenal tumors is an active field that generated very promising advances during 2017. Prominent examples include an improved understanding of adrenal tumor molecular pathogenesis as well as the introduction of new classifications, molecular markers and tracers for functional imaging. We also highlight international collaboration as a key factor that is likely to accelerate improvements in treatment and outcome of patients with these tumors.J C received lecture honoraria from Novartis.J Cs research position is funded by Akademiska Sjukhuset, Uppsala, Sweden."} +{"text": "Transcatheter aortic valve implantation (TAVI) has expanded rapidly as a novel therapeutic strategy for patients with severe symptomatic aortic stenosis. This procedure is already well established in patients at intermediate or high\u00a0risk for conventional surgical valve replacement and, with multiple studies currently evaluating TAVI in low-risk patients, use of this treatment is likely to continue to expand worldwide. While long-term outcome data are lacking, recent results from the Placement of Aortic Transcatheter Valves (PARTNER) trial indicated promising results at 5\u2009years post-implantation, with TAVI valves demonstrating similar haemodynamics to surgical bioprosthetic valves and no evidence of structural valve deterioration on echocardiography.Heart manuscript, Del Trigo et alIn their\u00a0This study does have some limitations. The cohort largely comprised patients with well-functioning valves, indeed even those patients diagnosed with valve haemodynamic deterioration had only mild bioprosthetic stenosis, with transvalvular gradients <40\u2009mm Hg. As a consequence, there were too few adverse clinical events in patients with valve haemodynamic deterioration in the matched population (n=7) for meaningful analysis. Another important limitation is that bleeding rates in this cohort were not available, and\u00a0it is therefore not possible to balance the apparent benefits of anticoagulation on valve durability against the risk of major bleeding. Finally, this is not a randomised controlled trial and there will therefore be both known and unknown confounders that might account for the observed differences.et al is therefore important because of the key mechanistic questions raised. In the absence of anticoagulation, do bioprostheses with evidence of leaflet thrombosis calcify and degenerate more quickly? Can histological or imaging studies confirm the association between leaflet thrombosis and early calcification activity? Are surgical bioprostheses also susceptible to similar mechanisms of degeneration? Research must now focus on gaining a greater understanding of the link between valve thrombosis and degeneration, with the hope that ultimately this will help inform optimal management to prolong bioprosthetic valve longevity and improve patient outcomes.Nevertheless, the authors are to be congratulated on investigating this important area and highlighting the intriguing hypothesis that anticoagulation therapy post-TAVI may protect against subsequent valve deterioration. This requires a clear mechanistic explanation. Bioprosthetic valve degeneration is predominantly driven by calcification: how might this be linked to antithrombotic therapy? One potential answer is valve leaflet thrombosis. While leaflet thrombosis causing abrupt valve failure occurs in just 1% of TAVI valves, subclinical valve thrombosis has been observed in 7%\u201310% of implanted TAVI valves."} +{"text": "Impaired analysis of signal conflict and congruence may contribute to diverse socio-emotional symptoms in frontotemporal dementias, however the underlying mechanisms have not been defined. Here we addressed this issue in patients with behavioural variant frontotemporal dementia and semantic dementia relative to healthy older individuals (n = 20). We created auditory scenes in which semantic and emotional congruity of constituent sounds were independently probed; associated tasks controlled for auditory perceptual similarity, scene parsing and semantic competence. Neuroanatomical correlates of auditory congruity processing were assessed using voxel-based morphometry. Relative to healthy controls, both the bvFTD and SD groups had impaired semantic and emotional congruity processing (after taking auditory control task performance into account) and reduced affective integration of sounds into scenes. Grey matter correlates of auditory semantic congruity processing were identified in distributed regions encompassing prefrontal, parieto-temporal and insular areas and correlates of auditory emotional congruity in partly overlapping temporal, insular and striatal regions. Our findings suggest that decoding of auditory signal relatedness may probe a generic cognitive mechanism and neural architecture underpinning frontotemporal dementia syndromes. \u2022Socioemotional changes in frontotemporal dementia may reflect altered signal decoding.\u2022To address this, we varied semantic and emotional signal relatedness in sound scenes.\u2022Frontotemporal dementia patients had impaired semantic and emotional scene decoding.\u2022These deficits correlated with atrophy of distributed cortico-subcortical regions.\u2022Signal processing approaches may help deconstruct complex dementia phenotypes. Successful decoding of such scenes depends on resolution of the sensory mixture to enable a coherent behavioural and emotional response. Competing or conflicting signals present an important challenge to this enterprise. Signal conflict are often ambiguous and require active contextual decoding to prepare an appropriate behavioural response . The reqHere we addressed the processing of signal conflict and congruence in auditory environments in two canonical syndromes of frontotemporal dementia, bvFTD and SD relative to healthy older individuals. We designed a novel behavioural paradigm requiring decisions about auditory \u2018scenes\u2019, each comprising two competing sound sources in which the congruity or incongruity of the sources was varied along semantic (identity relatedness) and affective dimensions independently. We constructed \u2018model\u2019 scenes that would simulate naturalistic processing of the kind entailed by real world listening while still allowing explicit manipulation of the stimulus parameters of interest. The stimulus dimensions of semantic and emotional congruity were anticipated to be particularly vulnerable to the target syndromes, based on an extensive clinical and neuropsychological literature in auditory and other cognitive domains . StructuWe hypothesised firstly that both bvFTD and SD would be associated with impaired detection and affective valuation of auditory signal relatedness, given that these syndromes show qualitatively similar semantic and affective deficits when required to integrate information from social and other complex auditory signals . We furt22.1MAPT, three C9orf72); no other pathogenic mutations were identified. CSF examination was performed in six patients with sporadic bvFTD and in five patients with SD: profiles of CSF neurodegeneration markers in these cases provided no evidence for underlying AD pathology based on local laboratory reference ranges . In total 14 patients in the bvFTD group had either a pathogenic mutation, consistent CSF neurodegenerative markers or both. Clinical brain imaging (MRI or CT) revealed variably asymmetric but compatible profiles of atrophy across the patient cohort in which the congruity of the two sounds was varied independently along two dimensions; semantic (whether the sounds would be likely or unlikely to occur together) and emotional . The procedure we followed in preparing the auditory scene congruity tests is diagramed in Individual sounds were obtained from on-line digital databases to sample semantic categories of human nonverbal sounds, animal sounds, natural environmental noises and artificial noises (machinery and tools).Pairs of sounds were superimposed using Goldwave\u00ae software, further details of stimulus synthesis are in Supplementary Material on-line. The resulting auditory \u2018scenes\u2019 comprised four conditions in a factorial matrix; semantically congruous \u2013 emotionally congruous, ScEc ; semantically incongruous \u2013 emotionally congruous, SiEc ; semantically congruous \u2013 emotionally incongruous ; semantically incongruous \u2013 emotionally incongruous, SiEi . Auditory scene stimuli were edited to fixed duration (8\u00a0s) and mean intensity level. Based on an initial pilot experiment in healthy older individuals , a final set of 60 auditory scene stimuli was selected from a larger set of 193 candidate auditory scenes, using criteria of > 80% correct identification of both constituent sounds in each scene and rated likelihood and pleasantness of the scene (the sound combination) by the healthy pilot group.The final auditory scene stimuli were arranged to create two tests, each incorporating the four sound conditions , but requiring a decision on either the semantic congruity or the emotional congruity of the sound scenes. A forced-choice response procedure was used in both tests. Stimuli for each test are listed in 2.2.2In order to interpret participants\u2019 performance on the auditory scene tests, we created control tests to probe auditory perceptual similarity processing, auditory scene analysis and semantic knowledge of individual sounds.In the perceptual similarity control test, we assessed each participant's ability to perceive acoustic similarity and variation between two sounds. Concatenated sounds were presented such that the sequence of sounds either comprised a single sound source or two sound sources of a single kind . The individual acoustic tokens comprising the sequence were always varied . Thirty trials sampling different semantic categories were presented; the task on each trial was to decide if the thing making the sound changed or remained the same. This task served as a control both for the perceptual analysis of constituent sounds and the decision-making procedure used in the tests of semantic and emotional congruity judgment.In the auditory scene control test, we assessed each participant's ability to parse superimposed sounds. We adapted an existing test requirinIn the auditory semantic (sound identification) control test, we assessed each participant's ability to identify and affectively evaluate individual sounds. All 43 constituent sounds composing the auditory scene stimulus set were presented individually; the task on each trial was to match the sound to one of three pictures representing the sound source , a closely semantically related foil and a distantly semantically related foil . In addition, the participant was asked to rate the pleasantness of each sound on a Likert scale .2.3All stimuli were delivered from a notebook computer running MATLAB\u00ae via headphones (Audio-Technica\u00ae) at a comfortable listening level for each participant in a quiet room. Within each test, trials representing each condition were presented in randomised order. Participants were first familiarised with each test using practice examples (not administered in the subsequent test) to ensure they understood the task instructions and were able to comply reliably. Participant responses were recorded for offline analysis. During the tests no feedback was given about performance and no time limits were imposed.2.4t-tests or Wilcoxon rank sum tests, where assumptions for the t-test were materially violated .All behavioural data were analysed using Stata12\u00ae. Demographic characteristics and general neuropsychological data were compared between participant groups using Fisher's exact test or (for continuous variables) either two sample On the perceptual similarity, auditory scene control and auditory semantic control tests, the proportion of correct responses was analysed using a logistic regression model owing to a binary outcome (correct / incorrect), with robust standard errors to account for clustering by participant. Mean overall pleasantness ratings of individual sounds on the auditory semantic control test were compared between participant groups using linear regression with bias corrected, accelerated confidence intervals from 2000 bootstrap replications due to the skewed distribution of the data. In each model, participant group was included as a categorical predictor and age, gender and reverse digit span (an index of executive and auditory working memory function) were included as nuisance covariates.In order to interpret the processing of auditory scene congruity in the main experiment, we wished to take into account whether the constituent sounds in a scene were identified correctly. Data for the semantic and emotional congruity decision tasks on auditory scene stimuli were pre-processed using data from the auditory semantic (sound identification) control test. For each participant, congruity decisions were scored only for those scene stimuli containing sounds that were both identified correctly when presented in isolation in the auditory semantic control test. Analyses were therefore based on different subsets of the scene stimuli in each participant group pleasantness ratings of both sounds individually and the interaction of the sounds in an auditory scene. This model allowed us to go beyond any abnormal rating of individual sound pleasantness in the disease groups, to assess group differences in the rating of sound combinations. To account for violated normality assumptions, the analysis used bias corrected, accelerated confidence intervals based on 2000 bootstrap replications.In separate post hoc analyses, for each patient group separately we assessed for correlations between key cognitive measures of interest using Spearman's correlation coefficient. Specifically, we assessed the extent of any correlation between semantic and emotional scene congruity performance; between semantic scene congruity and individual sound recognition performance; and between congruity decisions and performance on a standard test of nonverbal executive function (WASI Matrices), a standard index of semantic competence score) and a surrogate measure of disease severity score)A threshold p < 0.05 was accepted as the criterion for statistical significance in all analyses.2.5Volumetric brain MRI data were acquired for 27 patients on a Siemens Trio 3Tesla MRI scanner using a 32-channel phased array head-coil and a sagittal 3-D magnetization prepared rapid gradient echo T1-weighted volumetric sequence . Volumetric brain images were assessed visually in all planes to ensure adequate coverage and to exclude artefacts or significant motion. Pre-processing of patient brain MR images was performed using the Segment routine and the DARTEL toolbox of SPM12 . Normali2.6Using the framework of the general linear model, multiple regression was used to examine associations between voxel intensity (grey matter volume) and behavioural variables of interest over the combined patient cohort. In separate design matrices, voxel intensity was modelled as a function of participant scores on the semantic and emotional congruity tasks and the perceptual similarity, auditory scene and auditory semantic control tasks. In all models, age, gender, TIV, syndromic group and reverse digit span were included as nuisance covariates. For each model, we assessed both positive and negative (inverse) grey matter associations of the behavioural variable of interest. Statistical parametric maps were thresholded at two levels of significance: p < 0.05 after family-wise error (FWE) correction for multiple voxel-wise comparisons over the whole brain; and p < 0.05 after FWE correction for multiple voxel-wise comparisons within defined regions of interest based on our prior anatomical hypotheses.The anatomical regions used for small volume correction in order to more fully delineate the profile of atrophy in each patient group.As a reference for interpreting the correlative analysis, we conducted an additional, separate analysis to assess disease-related grey matter atrophy profiles in each of the patient groups, comparing patients\u2019 brain MR images with brain images acquired in the healthy control group using the same scanning protocol. Groups were compared using voxel-wise two-sample 33.1The participant groups did not differ for age (p = 0.07) or educational background (p = 0.25) and the patient groups did not differ in mean symptom duration (p = 0.32). Gender distribution differed significantly between groups, males being significantly over-represented in the bvFTD group relative to the healthy control group (p = 0.019); gender was incorporated as a nuisance covariate in all subsequent analyses. The patient groups showed the anticipated profiles of general neuropsychological impairment .3.23.2.1Performance profiles of participant groups on the perceptual similarity, auditory scene and auditory semantic control tests are summarised in 3.2.2Performance profiles of participant groups on the congruity decision tests are summarised in In the semantic scene congruity task there was an overall significant performance difference between participant groups (p < 0.0001). Both the bvFTD and SD groups performed significantly worse than healthy controls (p = < 0.001); there was no significant performance difference between patient groups nor evidence of an overall significant interaction between group and condition (p = 0.62).In the emotional scene congruity task , there was again an overall significant performance difference between participant groups (p = 0.0001), both the bvFTD and SD groups performing significantly worse than healthy controls in the congruous and incongruous conditions with no significant performance difference between patient groups. There was no evidence of an overall significant interaction between group and condition (p = 0.14). However, the SD group trended toward a greater performance discrepancy between conditions than was shown by the healthy control group (p = 0.053). This effect was driven by relatively more accurate performance for scenes containing emotionally congruous sounds.3.2.3Individual ratings of auditory scene pleasantness in the emotional congruity test are plotted in The SD group rated auditory scenes overall as significantly more pleasant than did the healthy control group while ratings of overall scene pleasantness by the bvFTD group did not differ significantly from healthy controls\u2019 ; the two patient groups rated sound scenes similarly for overall pleasantness .The healthy control group exhibited an additive emotional effect of combining sounds into scenes (a significant positive interaction of sound pleasantness ratings) relative to individual sound pleasantness rated separately. Emotionally congruous auditory scenes were significantly more likely to be rated as pleasant than predicted from the individual sound ratings alone . This interaction effect was significantly stronger in healthy controls than in either patient group . Indeed, neither patient group showed evidence of the effect .The healthy control group rated semantically congruous auditory scenes as significantly more pleasant than semantically incongruous scenes . This effect was replicated in the bvFTD group . The effect was significantly stronger in healthy controls than in either patient group but did not differ significantly between patient groups .3.2.4Accuracy of semantic and emotional auditory scene congruity decisions were significantly positively correlated in the bvFTD group , but not the SD group . Accuracy of semantic scene congruity judgment and constituent sound identification (on the auditory semantic control task) were significantly positively correlated in the bvFTD group but not the SD group . Semantic scene congruity judgment was significantly positively correlated with general executive capacity (WASI Matrices score) in the SD group , though not the bvFTD group ; with general semantic competence (BPVS score) in the bvFTD group but not the SD group ; and with a global measure of cognitive function (MMSE score) in both patient groups . Emotional scene congruity judgment was significantly positively correlated with WASI Matrices score in the bvFTD group but not the SD group ; with BPVS score in both patient groups ; and with MMSE score in both patient groups .3.3The patient groups showed the anticipated group-level, disease-related grey matter atrophy profiles: these encompassed bi-hemispheric prefrontal, anterior cingulate, insular and anterior temporal cortices and subcortical structures in the bvFTD group and leftward-asymmetric, predominantly antero-mesial temporal areas in the SD group see .Significant grey matter associations of behavioural measures for the combined patient cohort are summarised in FWE corrected for multiple comparisons over the whole brain), posterior cingulate, posterior and anterior superior temporal, insular, medial prefrontal and inferior frontal cortices and caudate nucleus . Impaired accuracy of judging the emotional congruity of auditory scenes was associated with grey matter loss in bi-hemispheric, anterior cortico-striatal areas including anterior superior temporal sulcus, insula, putamen and caudate nucleus .Impaired accuracy of judging the semantic congruity of auditory scenes was associated with grey matter loss in distributed, bi-hemispheric cerebral regions including precuneus, left supramarginal and premotor cortices was associated with grey matter loss in prefronto-temporo-parietal regions including supplementary motor, anterior and posterior cingulate and posterior superior temporal cortices. Impaired sound identification was associated with grey matter loss in left inferior frontal cortex.4Here we have shown that patients with bvFTD and SD have impaired processing of semantic and emotional congruence in auditory scenes relative to healthy older individuals. Both patient groups exhibited a similar profile of impaired congruence decisions about sound scenes. These deficits were evident after controlling for general executive, auditory semantic and auditory perceptual competence and not attributable to impaired identification or disordered affective valuation of individual constituent sounds. Taken together, our findings support the hypothesis that processing of auditory semantic and emotional relatedness is comparably impaired in both bvFTD and SD. Although there was no strong evidence overall for a specific condition effect, the SD group showed a tendency to more accurate determination of emotional congruity than incongruity in auditory scenes, suggesting a partial awareness of affective relatedness that was lost in the bvFTD group; in addition, performance in decoding the semantic and emotional congruity of auditory scenes was correlated in the bvFTD group but not the SD group, suggesting that the underlying processes are at least potentially dissociable. Previous work in SD and bvFTD has largely addressed the impaired semantic and affective coding of individual sensory objects, for which these syndromes show distinctive profiles of impairment. In contrast, the processing of semantic and affective relatedness might plausibly engage higher-order, associative and regulatory mechanisms, instantiated in extensive brain circuitry and jointly vulnerable in both syndromes. We therefore argue that the convergent deficits shown by our bvFTD and SD groups on these high-order semantic and affective tasks are consistent with previous studies of sensory object processing in these syndromes. The present findings corroborate a growing body of evidence for impaired processing of conflict and congruence in the auditory and other domains in bvFTD and SD, including striking impairments of socio-emotional signal decoding .The present paradigm demonstrates a generic mechanism relevant to decoding of sensory signals in natural environments that might underpin a range of difficulties that patients both with bvFTD and SD experience in the more complex scenarios of daily life . WhereaIn addition to impaired cognitive decoding, as anticipated both the bvFTD and SD groups here showed altered affective valuation of auditory scenes. The SD group (though not the bvFTD group) tended to rate auditory scenes overall as more pleasant than did healthy controls. While this appears somewhat at odds with the high reported frequency of daily life sound aversion in this syndrome , it is cThe overlapping but partly separable neuroanatomical correlates of semantic and emotional congruity processing identified here suggest a framework for understanding the brain mechanisms that process different dimensions of auditory signal relatedness. These neuroanatomical substrates are in line with our experimental hypotheses and with previous neuroanatomical work in auditory and other modalities. Processing of both semantic and emotional auditory congruence had substrates in anterior temporal and insula cortices that are likely to constitute \u2018hubs\u2019 for processing signal patterns and salient deviations based on prior expectations or stored templates . These rThe processing of auditory emotional congruence had an additional correlate in striatal structures broadly implicated in the evaluation of emotional congruence and reward . AlthougA further potentially relevant issue is the lateralisation of cerebral regional atrophy profiles, which showed considerable variation across our patient cohort . Based oThe neural correlates of auditory semantic and emotional congruence decisions here overlapped with cortical associations of performance on the auditory control tasks, suggesting that these regions may be engaged as a functional network and that particular network components may play a more generic role in the analysis of stimulus relatedness. Performance on the auditory scene analysis control task had a substrate in temporo-parietal junctional and supplementary motor areas known to be fundamentally involved in parsing and monitoring of the auditory environment in healthy and clinical populations . The temWe regard this study as establishing proof of principle for the utility of the auditory congruence paradigm: the study has several limitations and suggests a number of directions for future work. Group sizes here were relatively small; studying larger cohorts would increase power to detect effects, particularly differences between syndromic groups (such as the bvFTD and SD groups here). The present findings have not established any strong specificity of auditory congruence deficits for particular neurodegenerative syndromes. There would be considerable interest in comparing these frontotemporal dementia syndromes with other syndromes and diseases, in order to assess the specificity of behavioural and neuroanatomical profiles of auditory signal relatedness processing for particular neurodegenerative pathologies. Alzheimer's disease, for example, might be expected to show a quite different profile of auditory conflict signalling based on available neuropsychological and neuroanatomical evidence . EquallyAcknowledging the above caveats, this study suggests that auditory scene decoding may be a useful model paradigm for characterising the effects of dementias on signal processing in the more complex scenarios of daily life. From a clinical perspective, effective treatment of the dementias will likely depend on an accurate picture of the disability these diseases produce, in domains such as social and emotional cognition that are most sensitive to patients\u2019 everyday functioning ; this inNil conflicts of interest declared."} +{"text": "Prevent symptom relapse and promote functional recovery are the two main goals of early intervention services in first episode of psychosis. Identify patterns of recovery will be important in developing and implementing targeted recovery focused interventions. The goal of this study was to explore trajectories of recovery following a first episode of psychosis.A sample of 373 FEP patients was followed over 3 years. Recovery profiles in terms of symptomatic and functional remission were explored. Relapses during follow-up were considered.Four recovery trajectories were identified: good stable (26%), good unstable (21%), poor unstable (10%), poor stable (43%). Those who met criteria for good stable recovery more likely have less severe baseline negative symptoms and not be diagnosed with schizophrenia ; short DUP and low premorbid IQ increased the likelihood of good unstable recovery; less severe baseline negative symptoms and single status increased the likelihood of a poor unstable recovery when these three trajectories were compared with a poor stable recovery. Poor unstable trajectory was significantly associated with a high relapse rate (73%).Our results shed light on identifying different recovery profiles in FEP. Despite evidence for early intervention effectiveness, we should explore ways to prevent relapse and improve long-term recovery, particularly attending the role of timing in the design of interventions."} +{"text": "Cardiogenic shock can occur due to compression of the four pulmonary veins and the left atrium by a mediastinal tumor. Steroid infusion can be a temporary alternative therapy before obtaining a definite diagnosis and performing an intervention with stents to dilate the pulmonary veins. An 81\u2010year\u2010old man who had undergone left upper lobectomy due to squamous cell carcinoma 7 years ago was referred to our emergency department because of severe dyspnea. Physical examination showed signs of cardiogenic shock and a chest X\u2010ray revealed lung congestion. An echocardiographic evaluation revealed decreased left atrial cavity size due to a compressive tumor, and ECG\u2010gated cardiac computed tomography (CT) was subsequently performed that revealed a large tumor (40 mm minor axis diameter) in the posterior mediastinum and compression of the left atrium and pulmonary veins (PVs) (Fig. Single PV stenosis due to a compressive or invasive tumor was previously reported in a few case reports KY and FF: equally contributed to the conception of the work. KY: wrote the first draft of the manuscript. All authors: revised it critically and approved the final version to be published.None declared."} +{"text": "Arterial complications following traumatic knee injury are relatively rare but mandate timely recognition and treatment to avoid significant comorbidity and medicolegal ramifications. In this report we describe a case of acute thrombotic occlusion of the popliteal artery occurring after knee dislocation, successfully repaired by intimal fixation and a limited venous patch reconstruction. We present a review of local practice in screening vascular injuries following knee dislocation, aligned with a review of the literature and considerations for practice. Popliteal artery injury following blunt trauma to the lower extremity has been shown to range from 28% to 46% \u20133. WithoThis paper describes a case of knee dislocation with popliteal artery vascular injury and its timely recognition, investigation, and focused open surgical management.A 58-year-old male mechanic was admitted via the emergency department (ED) one hour after bilateral knee injury with presumed spontaneous relocation of bilateral knee dislocations. Whilst standing at the front of one car working under the bonnet, a coworker started the vehicle (in gear) causing it to jolt forward pinning him against another car, causing a distraction crush injury to both knees (at the level of the vehicle bumpers). The patient was placed in bilateral vacuum splints and brought to hospital by ambulance. Plain radiography of the right knee identifiDespite significant distracting injuries, the ED clinicians quickly identified the absence of peripheral pulses on the right foot and alerted the vascular on call team. Clinical assessment identified normal pulses throughout the left lower limb in the presence of left ligamentous knee disruption and only a femoral pulse on the right with an asymmetric ABPI , with an incomplete sensorimotor deficit below an unstable knee joint. Following administration of an intravenous heparin bolus , a CT angiogram [CTA] as shown in Following multidisciplinary case review and with patient informed consent, we proceeded to the emergency operating theatre for revascularisation of the right lower limb (@18:30).Following our standard preprocedural case plan outline and World Health Organisation (WHO) safety surgery checklist, the great saphenous vein (GSV) position and patency were confirmed and marked under ultrasound for use as a vascular conduit. General anaesthesia was induced alongside prophylactic intravenous Co-amoxiclav (1.2 grams) followed by a urinary catheter. The patient was positioned supine, on our standard X-ray compatible surgical table, and the left leg was immobilised in a cricket pad splint. The right leg underwent hair removal over the incision site (over the GSV) and was prepared with 2% alcoholic chlorhexidine gluconate from the groin to ankle; the foot was placed in a Bogota bag for monitoring. . The popliteal artery was identified and controlled using silastic vessel slings at the below knee popliteal inflow component where a pulse was palpable and distally, at the anterior tibial artery origin, between which the popliteal vessel wall was thrombosed, creating a dark blue hue.Using a standard medial approach to the popliteal vessels, an incision was placed 1-2\u2009cm posterior to the medial border of the tibia, starting at the tibial tuberosity and extending distally. Subcutaneous fat and fascia were sharply divided, preserving GSV in the wound limits. To reach the popliteal fossa, gastrocnemius muscle was retracted dorsally and deep fascia dividedA longitudinal arteriotomy was performed over the area and extended into healthy vessel in both directions (2\u2009cm each). The intimal disruption was resected back to healthy vessel in both directions and tacked using multiple Prolene 7.0 sutures. The GSV was harvested in the wound edge over a 6\u2009cm length and splayed for use as a tension-free patch on the popliteal artery using a Prolene 6.0 suture. Backflow and inflow were tested and flushed once again before completion of the patch anastomosis, with limb reperfusion following preparation and discussion with the anaesthetic team. Immediate arterial perfusion was noted with a corresponding blood pressure drop, end tidal carbon dioxide rise, and short run of tachyarrhythmia.Severe ischaemia lasting longer than 4\u20136 hoursPreoperative shockFirm compartments when palpatedDecreased sensibility and/or motor functionCompartment pressure > 30\u2009mmHgDisproportionate pain localised distal to the vascular injuryAll four lower leg compartments were explored, through an extension of the medial tibial incision and the placement of a lateral incision anterior to the fibular. As all compartments remained soft after perfusion in the setting of a short ischaemic time, we elected to close incisions with interrupted nonabsorbable sutures (Ethilon 3.0) to expedite healing, whilst retaining the ability to release the sutures should compartment syndrome develop postoperatively. As a guide we advocate the following as indicators where fasciotomy is likely to be necessary: compatibility angst in MR but also generates artefact which may negatively impact imaging interpretation for orthopaedic reconstruction.Preoperatively, the decision was taken to avoid use of ligaclips and skin staples as the patient was under consideration for early ligamentous intervention and therefore required preoperative MRI. Use of such devices often causesPostoperatively, after revascularisation, the patient had a cricket pad splint applied with confirmation of joint space restoration with intraoperative fluoroscopy by our trauma and orthopaedic team. He was managed on continuous intravenous heparin (target APTR 2.0\u20134.0) for 3 days before switching to prophylactic low molecular weight heparin (Enoxaparin 40\u2009mg once daily) and commenced on aspirin (75\u2009mg once daily). He was medically fit for discharge by day 5 after operation with palpable pedal pulses but did not leave hospital until day 11 after operation due to equipment limitations for discharge.Subsequent MRI of the knees identifiBlunt trauma to the lower extremity can cause popliteal artery injury in up to 46% of cases \u20133. In thRecommendations for the management of suspected vascular injuries in the lower limb have evolved from mandatory exploration of all suspected injuries to routine imaging . In our The use of cross-sectional imaging angiography is more often indicated after blunt trauma than penetrating because clinical examination is much more challenging in the setting of extensive soft tissue and nerve damage . Thus CTIn a review of our contemporaneous practice (2015-current), we analysed a prospectively collated orthopaedic database for trauma admissions, cross referencing data with electronic imaging picture archiving and communication system (Sectra-PACS) and patient records. We identified seven knee dislocations (not including this case report); five were imaged for vascular pathology and only one was pathological, demonstrating popliteal occlusion requiring an interposition graft and external fixation in a patient with an incomplete sensorimotor deficit.Hard signs , includiSoft signs , such asClearly, injury of the popliteal artery is detrimental to distal limb perfusion and associated with poor outcomes, particularly in those with prolonged ischaemic time. The timely recognition of extremity arterial injury should be considered in the context of the following:A recent systematic review identified a lack of consensus among practitioners regarding the diagnostic and treatment algorithm for vascular injury in the context of knee dislocation . We recoIn patients with popliteal injury, the goal should be to permanently restore continuity of the artery without stenosis or tension. A previous study reported that simple thrombectomy alone was insufficient and 71% of patients required a revascularisation (bypass) procedure . TherefoEarly diagnosis and accurate initial therapy are essential for limb salvage in the context of popliteal injury following knee trauma which mandates an understanding of the underlying biomechanics of vessel injury in the popliteal, good working relationships across the multidisciplinary team and clear communication."} +{"text": "Analysis of spatial and temporal genetic heterogeneity in human cancers has revealed that somatic cancer evolution in most cancers is not a simple linear process composed of a few sequential steps of mutation acquisitions and clonal expansions. Parallel evolution has been observed in many early human cancers resulting in genetic heterogeneity as well as multilineage progression. Moreover, aneuploidy as well as structural chromosomal aberrations seems to be acquired in a non-linear, punctuated mode where most aberrations occur at early stages of somatic cancer evolution. At later stages, the cancer genomes seem to get stabilized and acquire only few additional rearrangements. While parallel evolution suggests positive selection of driver mutations at early stages of somatic cancer evolution, stabilization of structural aberrations at later stages suggests that negative selection takes effect when cancer cells progressively lose their tolerance towards additional mutation acquisition. Mixing of genetically heterogeneous subclones in cancer samples reduces sensitivity of mutation detection. Moreover, driver mutations present only in a fraction of cancer cells are more likely to be mistaken for passenger mutations. Therefore, genetic heterogeneity may be considered a limitation negatively affecting detection sensitivity of driver mutations. On the other hand, identification of subclones and subclone lineages in human cancers may lead to a more profound understanding of the selective forces which shape somatic cancer evolution in human cancers. Identification of parallel evolution by analyzing spatial heterogeneity may hint to driver mutations which might represent additional therapeutic targets besides driver mutations present in a monoclonal state. Likewise, stabilization of cancer genomes which can be identified by analyzing temporal genetic heterogeneity might hint to genes and pathways which have become essential for survival of cancer cell lineages at later stages of cancer evolution. These genes and pathways might also constitute patient specific therapeutic targets. Malignant tumors can display a high degree of spatial and temporal genetic heterogeneity , 2. GeneThe identification of only a few and specific mutations in colon cancer has suggested that malignant progression proceeds with the continuous accumulation of a limited number of oncogenic mutations followed by clonal expansion of the mutated subclones resulting in a linear multistep process of cancer evolution . SubsequChromosome aberrations such as aneuploidy, more complex chromosomal rearrangement as well as abundant gene copy number variations can be found in early stages of malignant progression and these structural mutations seem to get stabilized at later stages of somatic cancer evolution and clinical progression. This has been demonstrated in many tumors such as malignant melanoma, breast cancer, pancreatic cancer and prostate cancer , 2, 4\u201311Point mutations seem to be acquired more steadily but at least in melanoma, more advanced tumors do not always display higher mutation loads , 13.Why do cancer cells acquire most structural mutations and probably most driving point mutations during early stages of carcinogenesis where molecular and histologic signs of genome destabilization such as atypical mitoses are less apparent compared to metastasized cancer cells?What are the mechanisms responsible for the apparent stabilization of the cancer genomes at later stages of malignant progression?This insight into somatic cancer evolution raises two major questions:Aneuploidy has fascinated generations of pathologists as atypical mitoses, a key process leading to unequal distribution of chromosomes are visible under the microscope in cancer samples . In addiThe analysis of subclone fate during somatic cancer evolution in different cancers could demonstrate that subclones may evolve through independent mutations but targetting the same genes, chromosome regions or the same molecular pathways , 20\u201325. A continuous accumulation of gene mutations and structural mutations within a cancer subclone lineage should be expected under the assumption that carcinogenesis as well as cancer progression represents an evolutionary process. In the absence of strong selection, mutations loads in progenitor cells should increase proportionally depending on the mutation rate. As fidelity of DNA replication as well as fidelity of equal chromosome distribution during mitosis should decrease in cancer cells with increasing damage of genes implicated in DNA repair, DNA replication and mitosis, the mutation rates of both structural mutations and gene mutations should increase during cancer progression . TherefoIn most cancers a specific mutation may be detected which is present in all cancer cells and thereby defines the monoclonal origin of the cancer. Often, this mutation is cell type specific, targets an oncogene or inactivates both copies of a tumor suppressor gene, induces proliferation and thus leads to an initial clonal expansion. This initial and positive selection is mediated by the microenvironment and the differentiation status of the cell which together allow that a specific mutation results in cell proliferation. In most cases this expanded cell clone must accumulate further mutations leading to sequential or parallel subclone formation for full malignant conversion. Parallel evolution in this second stage indicates that selection indeed plays a significant role in cancer evolution.In Darwinian evolution, mutation acquisition in subclones creates subclone heterogeneity which is then reduced by positive selection of the fittest subclones and negative selection of clones with lower fitness or lethal mutations. Detectable genetic heterogeneity as well as mutation load is therefore modified by the selective pressure. The high degree of genetic heterogeneity, the already significant mutation load in early stage cancers as well as the selection of mutated genes suggest that the initial clonal expansions are positively selected by the requirements of the microenvironment and by the advantages of genetic instability. In the majority of all human cancers, genetic instability is present in the form of chromosomal instability; a minority of cancers evolves through genetic instability at the nucleotide level. Genetic instability can be detected already in early cancers and even in cancer precursors. At later stages, both forms of genetic instability may be present simultaneously in cancer cells or subclones . The earEarly selection for chromosomal instability will lead to early acquisition of structural aberrations which confer a fitness advantage. Additional structural aberrations may occur when proliferation approaches the Hayflick limit and shortening of telomeres impedes regular chromosome distribution during mitosis. Overcoming the Hayflick limit can lead to telomere-driven chromosomal instability and most probably represents an additional non-linear event leading to profoundly rearranged cancer genomes.As it is very unlikely that genetically unstable cancer cells stabilize their genomes at later stages through uprated mechanisms of gene and chromosome repair, one must hypothesize that with accumulating mutation load in genes and increasing chromosomal aberrations, cancer cells progressively lose their tolerance towards additional mutation acquisition. Negative selection of additional mutations is sufficient to explain the observed stabilization of cancer genomes at later stages of cancer progression within an evolutionary context. Rasnick has postulated in 2002 that there exists a point of maximum disorder of the genome that still sustains life . A similNot all chromosomal rearrangements will result in negative constraints. Doubling of genes and especially whole genome doubling leading to tetraploidy seems to relieve negative constraints of chromosomal instability . Genome Figure\u00a0During early somatic cancer evolution, oncogenic pathways may be targeted independently in different subclone lineages through individual activating mutations. In the monoclonal state, hemizygous activating mutations may suffice for clonal expansion. If two different mutations target the same pathway in two subclones of similar size, each mutation might represent less than 25% of the detected DNA sequences given the admixture with normal cells in all cancer samples. With additional subclone formation, driving mutations might be further diluted and thereby escape detection. Detection of hemizygeously or homozygeously deleted tumor suppressor genes might become even more demanding when present in only a fraction of subclones [Genetic analysis of multiple areas of a primary tumor will reduce admixture of subclones, if present, and thereby enhance the sensitivity of detection of driving mutations as well as detection of gene deletions Fig.\u00a0. It willIf one assumes the premise that the cancer genome is stabilized at later stages of cancer progression by negative selection, then the genetic analysis of multiple areas of one metastasis will probably not provide additional information. In contrast, analysis of different metastases in one patient will allow distinguishing individual subclonal lineages as a metastasis should represent a monoclonal proliferation originating from one single cell of one lineage Fig.\u00a0.Analysis of the temporal heterogeneity during somatic cancer progression by sampling the primary tumor and its metastases or by analyzing metastases before and after therapy will also allow distinguishing individual subclone lineages Fig.\u00a0. FurtherThe amount of biological information which can be deduced from analyzing genetic heterogeneity in cancer samples depends on the applied molecular techniques. Used methods for molecular profiling vary significantly with regard to their spatial resolution, the amount of parallel information they are able to deliver and their requirement for input tissue. Accordingly, the gain in biological information obtained by analyzing multiple cancer specimens or multiple single cells also depends on the used technique Table\u00a0.Table\u00a01AComparative genomic hybridization (CGH) will detect imbalances of genetic content which resulted from aneuploidy or from complex structural rearrangements. As CGH covers the whole genome, it is able to provide information on the presence, amplification or loss of genetic material at multiple chromosomal loci . This paSingle nucleotide polymorphism (SNP)-arrays and loss-of-heterozygosity (LOH)-microsatellite analysis represent techniques which enable parallel quantification of gene copy number alterations at multiple genetic loci but also permit assigning alleles to autologous chromosomes. Both techniques should therefore allow a more sensitive detection of parallel evolution and a better resolution of the underlying structural rearrangements compared to CGH. SNP-arrays and LOH analysis still require significant amounts of input DNA when multiple genetic regions are analyzed.Single cell analysis of structural rearrangements can be achieved by fluorescence in situ hybridization (FISH), but this technique provides information on only a limited number of genetic loci which reduces its ability to differentiate between multiple subclones.Detection of gene mutations by Sanger sequencing has a high sensitivity for identifying driver mutations in oncogenes or inactivating mutations in tumor suppressor genes. Sanger sequencing may be performed on single cells but the amount of input DNA rises with the number of analyzed genes. Subclones can be identified by individual mutations only when multiple areas of a primary tumor or the primary tumor and its metastases are analyzed. Subclones with the same set of driving mutations but with different chromosomal rearrangements may not be distinguished by Sanger sequencing alone. As cancers contain only few driving mutations despite a high overall mutation load, Sanger sequencing of oncogenes or tumor suppressor genes will only provide a limited amount of information on genetic heterogeneity compared to CGH or SNP-arrays and it does not confer information on structural rearrangements which is necessary to identify conserved structural mutations or conserved essential genes during cancer progression.As each technique has its specific limitations, combination of different techniques has been advocated for reconstruction of somatic cancer cell evolution and detection of genetic heterogeneity but this approach further enhances the amount of input DNA which in turn reduces its spatial resolution .A breakthrough in analyzing genetic heterogeneity as well as structural rearrangements has been provided by next generation sequencing (NGS) , 2, 5, 6Mutations in cancers may also be detected in the serum of the patients (=liquid biopsy) as tumors release their DNA by necrosis or apoptosis to the blood stream in the form of cell free DNA. Although DNA detected by liquid biopsy should represent a mixture of subclones, sampling of cell free DNA at different time points and before and after treatment should enable the identification of subclones as well as of adaptive mutations conferring resistance to treatment .Genetic heterogeneity, has been made responsible for primary and secondary resistance to classic as well as targeted therapies . WhetherToday, the conventional approach is to screen one cancer specimen for druggable mutations by Sanger sequencing or by NGS prior to initiation of targeted therapy and to repeat genetic analysis on a recurrent tumor sample in case of resistance. In this case, chances are elevated that de-novo mutations detected in the recurrence are responsible for resistance and might represent additional therapeutic targets. An approach which might gain importance in the future is the monitoring of tumor protein release to the serum during targeted or immune therapy and performing genetic analysis of cell free DNA as a liquid biopsy whenever a rise in serum tumor markers indicates enhanced tumor proliferation due to resistance to therapy Fig.\u00a0. This apNon-linearity of mutation acquisition during somatic cancer evolution as well as parallel evolution of cancer subclones have been described in many human cancers and they have been designated with different adjectives. Some authors focused on the primary role of aneuploidy in carcinogenesis; others have emphasized the complexity of chromosomal structural rearrangements while some have identified restrictive or permissive effects of the cancer architecture on somatic evolution. The common denominator is that cancer genomes are shaped by selective pressures and tend to become very complex already at early stages of somatic cancer evolution. Most structural and numeric chromosomal aberrations found in early cancers tend to persist at later stages despite ongoing genetic instability. Apparent stabilization of unstable cancer genomes can be explained by negative selection.Positive selection can be easily identified at early stages of carcinogenesis and it is reflected by identification of a limited number of driving mutations in oncogenes and tumor suppressor genes. The identification of driving mutations in cancers has led to the development of targeted therapies which have significantly enlarged the armamentarium against this deadly disease. Unfortunately, many patients still succumb to cancer despite the use of targeted therapies. Genetic heterogeneity, in part due to parallel evolution, has been identified as one culprit. It has been proposed that negative selection might play an important role at later stages of cancer progression. The overwhelming numbers of mutations found in cancers do not target classic oncogenes or tumor suppressor genes. It is tempting to assume that some, if not most of these mutations are not innocuous but mediate the putative negative selection effects at later stages of cancer progression by reducing functional genome redundancy. These mutations or the remaining non-mutated pathways could represent additional therapeutic targets. The challenge will be to identify them against a background of bystander mutations.The leading paradigm in cancer evolution is that genetic instability results in genetic heterogeneity leading to a phenotypically diverse pool of cancer subclones upon which selection can act. Identification of selection during cancer evolution has and will continue to lead to identification of therapeutic targets. Positive or negative selection cannot be assessed directly in clinical specimen but may be deduced by the analysis of genetic heterogeneity during cancer evolution. Incorporating genetic heterogeneity in the analysis of malignant tumors should therefore greatly enhance the information content of molecular analysis of cancer genomes, and help identify patient-specific therapeutic targets."} +{"text": "The vesicle SNARE VAMP8 on mucin granules within goblet cells is specifically activated following infection with the protozoan parasite Entamoeba histolytica that is known to induce potent hyper-secretion and coordinates mucin exocytosis. This secretion event is critical in fending off a pathogen, as cells lacking VAMP8 are prone to increased E. histolytica colonization and cytolysis through apoptosis. Failing coordinated mucus exocytosis and subsequent epithelial barrier destruction, the host mounts an immune response as a last line of defence.The intestinal mucosa encounters a barrage of ingested insults within the host yet under homeostasis elegantly facilitates nutrient absorption and sustenance of the commensal microbiota. An essential defence mechanism employed by the host is limiting the spatial niche various microbes may occupy as executed by the fluid mucus layer. Pathogens that violate their restricted niche within the intestinal mucosa are first expelled by robust mucus secretion from goblet cells thus by-passing the need for an immune response. Surprisingly, while many pathogens are known to exert hyper-secretion of mucus from goblet cells, the mechanisms governing this event remain elusive. In a recent report by Cornick Similar to neuronal exocytosis of neurotransmitters, SNARE mediated release of MUC2 utilizes the vesicle SNARE VAMP8 on mucin granules to likely bind cognitive SNARE receptors on the plasma membrane to facilitate membrane fusion. While constitutive release of mucin was dependent on VAMP8, pathogen-induced mucus hyper secretion was drastically reduced in VAMP8 deficiency.The colonic mucus barrier forms a bimodal layer consisting of an inner firmly adherent layer immediately above the epithelial cells and an outer less dense layer that is heavily colonized by commensal microbiota. Goblet cells continually replenish the mucus layer that is constantly eroded by luminal bacteria by producing and releasing the primary component of mucus, MUC2. Secretion of MUC2 from goblet cells has long been postulated to follow classical exocytosis despite no empirical evidence to suggest so. Indeed, the molecular machinery responsible for MUC2 release from goblet cells was not discovered until recently when Cornick E. histolytica responsible for amebiasis is a colonic pathogen that presents asymptomatic in most individuals. We hypothesize that E. histolytica remains restricted to the luminal niche in these individuals and once the parasite breaches the mucus layer to interact with epithelial cells it exerts a pathogenesis profile to invoke disease in a small subset of infected hosts. The integrity of the mucus layer therefore serves as a critical first line of innate host defence against invasion by E. histolytica and indeed mice lacking Muc2 mucin display a more severe disease. Similarly, VAMP8-mediated mucin exocytosis is equally important during infection with E. histolytica, as mice lacking Vamp8 have increased apoptosis and cytolysis of epithelial cells due to aberrant mucin release. This followed classical apoptosis pathways as characterized by Caspase 3, 9 and PARP cleavage. Clearly this demonstrates the importance of both constitutive mucin release to maintain the barrier as well as induced mucin secretion in response to a pathogen to fend off imminent threats. E. histolytica therefore serves as an important model to investigate the role of coordinated VAMP8-dependent SNARE exocytosis in goblet cells that has broad implications to many intestinal pathogens that induce mucus secretion. It is likely to conceive that genetic susceptibility loci that alter the ability of goblet cells to execute proper mucin exocytosis would therefore render the host susceptible to infection from a variety of intestinal pathogens.The protozoan parasite E. histolytica, invading parasites are well poised to subvert inflammatory macrophages and translocate to extra-intestinal sites such as the liver and brain. A synopsis of how VAMP8 deficiency leads to abrogated mucin release from goblet cells and the downstream consequences is summarized in Figure 1.Perturbation of the mucin secretion SNARE machinery has detrimental effects on the ability of the host to innately protect against luminal pathogens; a burden that is ultimately passed to members of the myeloid compartment to control an invading threat. Indeed, in mice lacking Vamp8 we noted a more aggressive pro-inflammatory cytokine release characterized by Il-1\u03b1, Il-1\u03b2 and Tnf-\u03b1 compared to mice with intact mucin exocytosis. In the context of E. histolytica infection we do not know how these kinases activate the SNARE complexes. Analogous to other models of non-neuronal exocytosis, this event likely stems from phosphorylation of SNARE proteins and/or chaperones.The study reviewed here highlights the first report of regulated SNARE exocytosis in goblet cells to afford mucin secretion. While the study focuses on disease pathogenesis, the consequences of VAMP8 deficiency on abrogating mucin exocytosis constitutively to maintain homeostasis remains elusive. Further studies should evaluate the plasma membrane SNARE receptor complex implicating an involvement for SNAP and Syntaxin core machinery proteins. Additionally, while the role of kinases in driving mucin secretion in goblet cells is established following"} +{"text": "Efferocytosis (the phagocytosis of apoptotic self cells) is a key mechanism in the resolution of inflammatory processes such as community-acquired pneumonia (CAP). Efferocytosis therefore represents a modifiable target for therapy aimed at enhancing intrinsic recovery mechanisms. It is currently not known which patients recovering from CAP would mostly benefit from a strategy aimed at enhancing efferocytosis.We recruited a cohort of patients with CAP admitted to a hospital in Liverpool. One month into recovery, subjects were invited for research bronchoscopy and bronchoalveolar lavage. An ex vivo efferocytosis assay was performed by challenging alveolar macrophages with autologous, apoptotic neutrophils. The percentage of alveolar macrophages that had undergone efferocytosis was determined by flow cytometry. We conducted a multivariable regression using a linear mixed effects model to determine which clinical parameters were most closely associated with efferocytosis.We observed high rates of comorbidity among this CAP cohort. Efferocytosis was measured in 22 subjects. We assessed multiple combinations of clinical parameters for association with efferocytosis and found the best-fitting model included an interaction between smoking status and prior statin use\u2014smoking being associated with decreased efferocytosis and statin use with increased efferocytosis. These effects were modified by an association between efferocytosis and body mass index (BMI), such that as BMI increased so did efferocytosis.This is the first study to measure efferocytosis in patients recovering from CAP. The results suggest that smokers with low BMI have impaired efferocytosis and may benefit from a statin to boost recovery. Among the 80% of patients who survive an admission with community-acquired pneumonia (CAP), a proportion suffers prolonged symptoms.Subjects recruited from two UK Hospitals between February 2011 and March 2013 had CAP (British Thoracic Society definition), were aged >16\u2005years and were recruited within 24\u2005hours of their first dose of in-hospital antibiotic. We excluded patients requiring invasive ventilation, requiring renal replacement therapy, with cystic fibrosis (CF), non-CF bronchiectasis, lung cancer, lung metastases, advanced cancer of any type, immunocompromise (including systemic corticosteroids), those treated palliatively or admitted within 14\u2005days. At 1\u2005month, subjects were invited for bronchoscopy with bronchoalveolar lavage (BAL) and were excluded if they were at increased risk of complications. BAL was performed as previously published with 200\u2005ml saline instilled into the right middle lobe (RML) bronchus.Ex vivo autologous apoptotic neutrophils were derived by published protocol.10.1136/thoraxjnl-2016-208505.supp1supplementary dataThe flow cytometric gating strategy (see online Of 169 subjects recruited, efferocytosis was analysed in 22 S3 reveaThis is the first study of efferocytosis during recovery from CAP. After adjustment for BMI, the strongest associations with efferocytosis were smoking status and statin use prior to CAP.Strengths of this study include the use of autologous neutrophils as a pathophysiologically appropriate apoptotic target for the efferocytosis assay, the use of linear mixed effects modelling to quantify the contributions of experimental and between-patient variations and the flow cytometric method used. Limitations include the small study size, lower median age and lower range of severity than that in clinical practice. It is not known if CAP in humans has an effect on efferocytosis or whether any such effect would be local or generalised, but we consistently lavaged the RML, and three of the patients were recovering from RML CAP. It is possible that recent local inflammation by the RML CAP may have affected rates of local efferocytosis; however, in our univariate analysis, there was no statistically significant difference in efferocytosis associated with RML involvement.Previous studies have found that cigarette smoke affects molecular pathways that lead to the activation and membrane localisation of the enzyme Rac which facilitates the cytoskeletal rearrangements needed for efferocytosis.We also showed a novel association between BMI and efferocytosis. Previous studies have shown that reduced CAP mortality is associated with high BMIOur study suggests that smokers with CAP and low BMI may benefit most from augmented efferocytosis; a statin would be an appropriate candidate for such a trial."} +{"text": "Their stabilisation is crucial to prevent shearing forces causing flap failure. This can be challenging using traditional dressings so different stabilisation methods have been described including plaster of Paris and topical negative pressure dressings. These are disadvantaged by limiting flap observations and wound care. External fixation was first described for this indication nearly half a century agoIn order to stabilise an ipsilateral pedicled groin flap to resurface a palmar hand defect, a Hoffmann style external fixator was used with pins inserted into the ipsilateral distal radius and anterior superior iliac spine . The fraExternal fixation to immobilise pedicled flaps provides robust stabilisation of the flap with excellent access for observation and wound care. Ease of application and removal facilitates flap management as well as definitive patient rehabilitation and hand therapy. Potential risks of bone fracture, muscle cramps and pin site infection can be minimised by appropriate placement/adjustment, wound care and patient counselling.Consent was secured from the patient and the UK Ministry of Defence for publishing this report and the clinical photographs."} +{"text": "The lifetime risk of suicide and suicide attempt in patients with schizophrenia are 5% and 25%\u201350%, respectively. Understanding the suicide risk factors is of great significance in research and clinical practice. The current systematic review is the first attempt to examine and demonstrate the associations between the core negative symptom of blunted affect and suicide in people with schizophrenia. We believe this review may have important implications for suicide epidemiology and helps us improve prevention tools.A comprehensive search strategy using PRISAMA guidelines was used to identify potential studies and data that met inclusion criteria. We searched original studies published since 2016 via MEDLINE (R) from 1946 to February 2016, EMBASE from 1947 to February 2016, and PsychINFO from 1806 to February 2016. Inclusion criteria were met if an article reported any kind of correlation between negative symptoms and suicide ideation, attempted suicide or completed suicide in patients with schizophrenia. The used search terms were: schizophreni* AND suicid* AND negative symptom* OR affective symptom* OR expressed emotion* OR emotional internal*. Studies with original data related to the blunted affect and suicide in schizophrenia were examined by manual reviewing.The initial search found 878 papers about negative symptoms and suicidal behaviour. From those only 12 papers fulfilled the inclusion criteria. Eight of twelve eligible papers found a positive association between blunted affect and suicide in schizophrenia indicating the link between social isolation and blunted affect with suicide (p<0.018), the impact blunted affect has on completed suicides on female population with schizophrenia (p<0.034) and the link between blunted affect and suicide in the stage of hospital admission (p<0.001). Two of twelve papers report no significance between blunted affect and suicide. One paper shows blunted affect did not have direct relation with suicide but can lead to the development of a suicidal behaviour. The last paper demonstrates blunted affect is important as suicide risk factor in schizoaffective disorder only.Based on the best available data, our results demonstrate a challenging link between blunted affect or related emotional disturbances and suicide in schizophrenia. Despite major suicide risk factors such as hopelessness, positive symptoms and depression, blunted affect is another factor we need to consider as it relates to social engagement and emotion regulation which are essential elements for eliminating suicides and improve interventions in psychiatry. Our outcomes may help with future development of preventive strategies and tools to combat suicide but also with gaining better understanding around what determines suicidal behaviour in schizophrenia."} +{"text": "Disseminated Intravascular Coagulopathy (DIC) is the most common coagulopathy in patients with prostate cancer. Though rare, it could be fatal without treatment. Literature suggests that there is significant activation of fibrinolytic pathway. Pathophysiology of DIC in patients with prostate cancer is not completely understood. We present here a case of chronic DIC in a patient with metastatic androgen independent prostate cancer. His DIC was managed successfully with a combination of aminocaproic acid and low weight molecular heparin. The use of low molecular weight heparin may make management of chronic DIC in prostate cancer more feasible in an out patient setting."} +{"text": "The data reported here support the manuscript Nuske et al. (2017) Specifications TableValue of the data\u2022This data shows the differences in dietary fungal species of different mammals and hence their relative contribution to the dispersal of these species. Future studies can confirm these trends with targeted sampling of both mammalian fungal specialists and generalists.\u2022Bettongia tropica and Potorous longipes diets; further studies can target these species to confirm whether the absence of fungal specialists results in lower dispersal rates.This data lists fungal species which only occur in endangered \u2022Further studies can also target the listed fungal species in the data for the development of genetic markers or reference libraries to study gene flow and population genetic diversity in relation to different dispersal modes.12Data were gathered from literature (references in Endoptychum sp.) that could not be equated to modern genera.For each data point in each study, the location of the study was used as the lowest grouping variable. Data across studies were compared by pooling data together if they occurred within 100\u00a0km from a random central point. In comparisons, fungal names included both formally published and as yet unpublished names, identified at least to genus (value=1 in \u2018Cf\u2019 column of"} +{"text": "While tin vitro experiments and pre-clinical animal studies. APR- 246 is currently being tested in phase II clinical trials for platinum resistant high-grade serous ovarian cancer with mutated p53 [Other innovative approaches being pursued currently to target the GOF p53 mutants in cancer cells includes identifying small molecules and peptides that can bind to the mutant p53 proteins and induce conformational changes in the mutant protein that converts it to a more WT-like conformation . This prated p53 .Despite the promise of the current batch of mutant p53 targeting therapeutics, it is important for us to continue our efforts to identify new ways to selectively target mutant p53 in cancer cells as the extant molecules could face challenges during their clinical translation or have issues with development of drug resistance upon prolonged use. Pharmacological targeting of mutant p53 by small molecules is a rapidly evolving field that holds tremendous promise and potential to pave the way towards the development of novel anti-cancer agents that would allow personalized treatment based on the p53 mutation status of the patient's tumor. Understanding how the various GOF mutant forms of p53 differ, their specific roles in cancer progression, and developing novel therapeutics and strategies to target them selectively should certainly be priorities worth investing going forward."} +{"text": "It is now well established that resistance exercise stimulates muscle protein synthesis and promotes gains in muscle mass and strength. However, considerable variability exists following standardized resistance training programs in the magnitude of muscle cross-sectional area and strength responses from one individual to another. Several studies have recently posited that alterations in satellite cell population, myogenic gene expression and microRNAs may contribute to individual variability in anabolic adaptation. One emerging factor that may also explain the variability in responses to resistance exercise is circadian rhythms and underlying molecular clock signals. The molecular clock is found in most cells within the body, including skeletal muscle, and principally functions to optimize the timing of specific cellular events around a 24 h cycle. Accumulating evidence investigating the skeletal muscle molecular clock indicates that exercise-induced contraction and its timing may regulate gene expression and protein synthesis responses which, over time, can influence and modulate key physiological responses such as muscle hypertrophy and increased strength. Therefore, the circadian clock may play a key role in the heterogeneous anabolic responses with resistance exercise. The central aim of this Hypothesis and Theory is to discuss and propose the potential interplay between the circadian molecular clock and established molecular mechanisms mediating muscle anabolic responses with resistance training. This article begins with a current review of the mechanisms associated with the heterogeneity in muscle anabolism with resistance training before introducing the molecular pathways regulating circadian function in skeletal muscle. Recent work showing members of the core molecular clock system can regulate myogenic and translational signaling pathways is also discussed, forming the basis for a possible role of the circadian clock in the variable anabolic responses with resistance exercise. Skeletal muscle encompasses ~40% of total bodily mass and is a highly malleable tissue with the capacity to alter its structure and metabolism in response to internal and external stress signals such as muscle contraction and nutrition has led to the development of terms such as \u201cnon,\u201d \u201clow,\u201d \u201cmoderate,\u201d \u201chigh,\u201d and \u201cextreme\u201d responders to describe the divergent magnitude in response of a particular individual to a given exercise stimulus cluster analysis as a method to group participants into \u201cextreme,\u201d \u201cmodest,\u201d and \u201cnon-responder\u201d clusters based on changes in The same research group recently reported that altered ribosome biogenesis responses may be implicated in regulating the extent of myofiber hypertrophy with resistance training as well as intracellular processes are temporally coordinated into rhythms coinciding with the 24-h solar cycle and BMAL1 (brain muscle arnt-like 1) targets both positive and negative loops. In particular, CK1\u03b5 phosphorylates PER1 to induce a conformational change that negates nuclear entry of PER1 that leads to its cytoplasmic accumulation and subsequent degradation targets BMAL1 for phosphorylation followed by subsequent ubiquitination and degradation via the proteasomal pathway , vascular endothelial growth factor (VEGF), and monocyte chemoattractant protein-1 (MCP-1) conditions, allowing it to sense fluctuations in energy expenditure associated with contractile activity. Following increased calcium-induced contractile activity, CLOCK translocates to the nucleus to regulate gene expression, suggesting myocardium contraction may directly modulate the molecular clock that in turn regulates cardiac muscle contractile activity can be regulated independently of the SCN as evidenced by differences in gene expression between the exercised and non-exercised legs. However, there are a number of limitations associated with these findings that must be considered. Firstly, there were only four male participants in this study from a non-homogenous age bracket (31\u201351 years) that were untrained. Whether the effects of the circadian clock exert similar effects in younger (20\u201330 years), older (>65 years), highly trained resistance individuals or females is unknown. Another considerable limitation was that there were only two muscle biopsy time points which reduces the capacity to detect peak and trough times in the expression of the circadian-regulated core clock genes. Indeed, the authors failed to detect Cry1 and BMAL1 expression from the two samples obtained. Regardless, available evidence collectively indicates that the regulation of circadian rhythms in skeletal muscle by resistance exercise modulates the molecular profile of adaptation responses that may, over time, influence key physiological responses such as muscle hypertrophy and increased strength.Considering the current evidence demonstrating the interplay of the molecular clock machinery with growth and hypertrophy responses in skeletal muscle, it stands to reason the circadian clock may also play a critical role in regulating the established molecular mechanisms mediating muscle growth responses with resistance exercise. The premise of a circadian influence on muscle growth is largely based on accumulating evidence showing BMAL1, CLOCK, Rev-erb, and ROR\u03b1 to transcriptionally regulate other genes not directly implicated in circadian function. The downstream targets of these circadian clock genes, termed clock-controlled genes (CCGs) as a skeletal muscle-specific clock controlled gene through its activation by CLOCK: BMAL1 or post-intervention increases in MyoD and myogenin transcripts, respectively . Lipton and colleagues recently showed in mouse embryonic fibroblasts that the phosphorylation of BMAL1 by p70S6K at the Serine 42 site induces BMAL1 to interact with the translational machinery in the cytosol and subsequently stimulate cellular translation independent of BMAL1's established role as a transcription factor are firstly required. Additionally, most studies investigating the capacity for exercise to alter circadian rhythms have centered on endurance exercise and much less is known about the potential for resistance exercise to modulate molecular clock function in skeletal muscle. The optimal time of day to perform resistance exercise to induce the greatest synergistic anabolic effect of resistance exercise on myogenic regulatory factor expression, translational signaling transduction and optimal hormonal milieu responses is unknown. This information could provide novel insight to the mechanisms that may contribute to heterogeneity in anabolic-related adaptation responses with resistance training as well as progress the future development of individualized exercise training programs to maximize adaptation responses. Additionally, the potential for an \u201coptimal\u201d time to perform resistance exercise in conjunction with circadian clock pathways and hormonal regulation raises the prospect of an \u201canabolic periodicity\u201d such that certain times of the day may provide a greater overall cellular and systemic environment to maximize resistance exercise adaptation responses. This proposal is of practical relevance to strength and powerlifting athletes who are needed to maximize anabolic training outputs for optimal performance outcomes.vastus lateralis. Thus, additional analyses in other activated muscle groups that involves the simultaneous investigation of the circadian molecular clock may yield new insight to additional mechanisms underlying heterogeneity in muscle growth responses with resistance training. Another relevant factor in the circadian regulation of anabolic responses with resistance exercise is the interaction with nutrients. Feeding of nutrients, like exercise, is a known entrainment factor of skeletal muscle and slow (Type I) fibers which is likely a result of established differences in activity levels, substrate metabolism and functional properties between muscle fiber types (Dyar et al., The author confirms being the sole contributor of this work and approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Preexposure Prophylaxis for the Prevention of HIV Infection \u2014 2014: a PHS Clinical Practice Guideline* and a Clinical Providers\u2019 Supplement\u2020 are now available online.The documents The guideline and supplement are intended for use by clinicians in the United States providing medical care for persons without human immunodeficiency virus (HIV) infection who are at substantial risk for acquiring it by their sexual or injection drug use behaviors. The guideline is the first federal resource that provides comprehensive, evidence-based information about the provision of daily oral antiretroviral preexposure prophylaxis (PrEP), including how to identify patients with indications for PrEP, guidance for safe prescribing practices, monitoring clinical safety for patients taking PrEP medications, and supporting medication adherence and the reduction of HIV risk behaviors. The supplement provides additional tools and information that might be useful to clinicians prescribing PrEP."} +{"text": "HAPs) often has consequences for behavior, physiology, and fecundity. These fitness effects suggest there is potential for an evolutionary response by vertebrates to HAPs. Here, we explore the toxicological HAP\u2013vertebrate relationship in an evolutionary framework and discuss the potential for vertebrates to adapt to or even co\u2010opt the effects of plant\u2010derived chemicals that influence fitness. We lay out several hypotheses about HAPs and provide a path forward to test whether plant\u2010derived chemicals influence vertebrate reproduction and evolution. Studies of phytochemicals with direct impacts on vertebrate reproduction provide an obvious and compelling system for studying evolutionary toxicology. Furthermore, an understanding of whether animal populations evolve in response to HAPs could provide insightful context for the study of rapid evolution and how animals cope with chemical agents in the environment.Living plants produce a diversity of chemicals that share structural and functional properties with vertebrate hormones. Wildlife species interact with these chemicals either through consumption of plant materials or aquatic exposure. Accumulating evidence shows that exposure to these hormonally active phytochemicals ( However, we will consider HAPs to more broadly include plant chemicals and mixtures with agonistic or antagonistic effects on a range of endocrine outcomes including lipid metabolism, steroid or thyroid hormones, prolactin, or luteinizing hormone decreased egg\u2010laying rate and egg mass, whereas in older ducks, the daidzein diet increased egg\u2010laying rate, although those eggs had decreased yolk volume and lower hatchability androgenic and (anti)estrogenic properties in yeast assays, with oak in particular showing strong estrogenic and antiandrogenic in vitro properties populations are locally adapted to polychlorinated biphenyls have become locally adapted to toxic road salt contamination populations are more adapted to pesticides if they live near agriculture at the embryonic stage if their intestinal microflora includes bacteria that produce favorable isoflavone metabolites populations impacted by road salt contamination , endogenous endocrinology , and developmental stage would be the especially useful.This work necessitates also understanding the agonistic and antagonistic properties of HAPs and HAP mixtures as they interact with different physiological pathways . Fully understanding the physiology of HAPs in vertebrate systems requires a third area of research, investigating the evolutionary history of hormonally active molecules (HAPs and vertebrate hormones) and their receptors. The timing of when different ligands and receptors evolved would clarify whether HAPs are adaptations or exaptations in plants.Fourth, to assess evolutionary consequences for vertebrates, research should evaluate whether HAP exposure promotes or reduces lifetime fecundity and offspring survival. Assessing whether different HAP exposure results in fitness differences between populations is a key step for inferring fitness effects of HAPs.The fifth area of research involves testing whether individuals within populations vary in their susceptibility to HAPs and whether different susceptibilities explain variation in fitness. Related questions would ask whether individuals from populations exposed to HAPs have higher HAP tolerances than individuals from other environments and whether populations have the capacity to adapt to HAPs or are already locally adapted. For species with long life spans, this latter step may be particularly challenging due to the logistical constraints of rearing animals from different populations to maturity or for multiple generations for common garden or reciprocal transplant experiments. However, modern molecular techniques may allow us to infer patterns of adaptation through genomic or transcriptional variations among populations , protein production and structures (proteomics), and cell or tissue metabolites (metabolomics). Prior work has called for increasing genomics work in the study of hormonally active chemicals may be understood more fully in the light of evolution. There may be fitness advantages in being able to respond to diverse environmental signals, such as HAPs, which convey contextual environmental information. If HAPs increase in plant foods due to drought\u2010induced stress, for example, that stress might be signaled to animals through their diet and enable endocrine\u2010regulated acclimation to environmental change, including altered reproduction and metabolism. As global climate change progresses, HAP\u2010related mechanisms may play an important role in how animals respond. Because HAPs represent relatively natural interactions among plants and animals, they can provide useful evolutionary insight into broader toxicological mechanisms and responses.There are no data associated with this manuscript to archive."} +{"text": "An 80-year-old woman with a medical history of diabetes and duodenal cancer presented to our emergency department (ED) complaining of sudden severe dyspnea after vomiting. She was alert and oriented on arrival, but showed tachypnea and poor oxygenation. Inspiratory stridor was evident. A computed tomography (CT) revealed a dilated esophagus with food bolus and intraluminal air compressing the trachea at the level of the sternoclavicular joint . Her symThis case presented reversible, severe tracheal compression by dilated esophagus with no functional abnormality. Cases of tracheal compression by dilated esophagus with structural diseases such as hiatal herniationWhat do we already know about this clinical entity?Cases of tracheal compression by dilated esophagus with hiatal herniation or esophageal achalasia have been reported.What is the major impact of the image(s)?Reversible tracheal compression by dilated functionally normal esophagus was evident on the first CT.How might this improve emergency medicine practice?Spontaneous esophageal dilation is rare, but worth considering when we see the ED patients complaining of sudden severe dyspnea.Documented patient informed consent and/or Institutional Review Board approval has been obtained and filed for publication of this case report."} +{"text": "This study aims to investigate autonoetic consciousness associated with episodic autobiographical memory in patients who had undergone unilateral medial temporal lobe resection for intractable epilepsy. Autonoetic consciousness, defined as the conscious feeling of mentally travelling back in time to relive a specific event, was assessed using the Remember/Know (R/K) paradigm across different time periods as proposed in the autobiographical memory task developed by Piolino et al. (TEMPau task). Results revealed that the two patient groups gave reduced sense of reliving (R) responses and more familiarity (K) responses than healthy controls. This poor autonoetic consciousness was highlighted when patients were asked to justify their Remember responses by recalling sensory-perceptive, affective or spatiotemporal specific details across all life periods. These results support the bilateral MTL contribution to episodic autobiographical memory covering the entire lifespan, which is consistent with the multiple trace theory of MTL function . This study also demonstrates the bilateral involvement of MTL structures in recalling specific details of personal events characterized by autonoetic consciousness."} +{"text": "This data article presents files supporting calculation for urban heat island (UHI) inclusion in building performance simulation (BPS). Methodology is used in the research article \u201cFrom urban climate to energy consumption. Enhancing building performance simulation by including the urban heat island effect\u201d Specifications TableValue of the data\u2022Presented urban weather data enable researchers to improve building simulations for the cities of Lima, Guayaquil, Valparaiso and Antofagasta by considering the UHI effect.\u2022Presented PCA data enable researchers to downscale urban climate to building level for building performance simulation purposes in other locations of the world.\u2022Presented UWG data permit to generate similar urban scenarios in other locations of the world.\u2022Building Performance Simulation models are useful to conduct similar building simulations in other locations of the world.1PCA: 4 excel files (one for each city), containing urban parameters for each sample and the following Principal Component analysis done to group the samples.UWG: 17 Extensible Markup Language (xml) files containing the urban description of each group .TRNSYS: 3 models for building performance simulation in.bld format and 3 simulation studio files for Trnsys.EPW: 21 weather files containing the information needed to run building simulation . 4 files are rural and 17 are urban (one for each group described above). Presented data are files needed for the inclusion of urban heat island (UHI) in building performance simulation (BPS). BPS needs weather files to obtain the thermal demand of buildings; normally weather files are standard files, obtained from monitoring stations close to the location. However, urban climate is different from this standard climate, which is often a rural climate . To include UHI effect, urban climate has to be downscaled to building level by a four steps methodology 2To generate urban weather files to be used in building performance simulation, a four step methodology was developed: 1) cities are divided in zones of influence of the Sea and then a random distribution of 24 samples is done; 2) each sample was analyzed by using Archgis and Google Street View to obtain some important urban parameter ; 3) a PCA TRNSYS is a validated tool to conduct BPS; UWG is a relatively new tool useful to obtain urban weather files. However, the parameters needed by UWG have to be obtained from direct observation of urban morphology. The use of Urban Tissue Categories enables to represent a city with a relatively small number of morphologies. Each of these morphologies can be translated into a specific urban climate and a related weather file can be generated. These files allow to more accurate performance simulations of buildings placed in the urban environment. The importance of the PCA analysis in avoiding correlations between different urban variables makes these data very useful for further studies in other locations."} +{"text": "A patient who had a small bowel mesentery perforation following insertion of a suprapubic catheter (SPC) is described. He had no bowel complaints immediately following the procedure, but presented 10 weeks later with insidious onset bowel obstruction due to the kink caused by the catheter. This complication occurred despite cystoscopy control and adequate bladder distension prior to the procedure. This isolated case illustrates the fact that regardless of the ease and frequency of SPC insertion, complications do occur."} +{"text": "Diabetic nephropathy is a major risk of end-stage kidney failure and is associated with greater morbidity and mortality, predominantly with augmented cardiovascular disease. Many complex factors relate to the progression of nephropathy of diabetic patients .Current investigations have focused on the optimization of renin-angiotensin system blockade in patients with diabetic nephropathy using combinations of drugs that target this pathway, however further studies have focused on the potential of new therapies that either target various pathways up-regulated by hyperglycemia or other targets believed to promote progression of diabetic nephropathy.et al, entitled \u201cwhether vitamin D3 is effective in reducing proteinuria in type 2 diabetic patients? D3 concentrations were meaningfully lower in females than in males have been completely observed by the authors.None declared."} +{"text": "AMI) is to return blood flow into the occluded coronary artery of the heart, a process defined as reperfusion. However, reperfusion itself can increase mortality rates in AMI patients because of cardiac tissue damage and dysfunction, which is termed \u2018ischaemia/reperfusion (I/R) injury\u2019. Mitochondria play an important role in myocardial I/R injury as disturbance of mitochondrial dynamics, especially excessive mitochondrial fission, is a predominant cause of cardiac dysfunction. Therefore, pharmacological intervention and therapeutic strategies which modulate the mitochondrial dynamics balance during I/R injury could exert great beneficial effects to the I/R heart. This review comprehensively summarizes and discusses the effects of mitochondrial fission inhibitors as well as mitochondrial fusion promoters on cardiac and mitochondrial function during myocardial I/R injury. The comparison of the effects of both compounds given at different time\u2010points during the course of I/R injury are also summarized and discussed. Finally, this review also details important information which may contribute to clinical practices using these drugs to improve the quality of life in AMI patients.The current therapeutic strategy for the management of acute myocardial infarction ( Numerous studies have shown that during the reperfusion period mitochondria undergo fission and that there is an absence or reduction in mitochondrial fusion In this review, we have comprehensively summarized and discussed the effects of mitochondrial fission inhibitors and mitochondrial fusion promoters on cardiac and mitochondrial function during myocardial I/R injury and have compared the effects of both compounds given at different time\u2010points to generate important information, which will contribute to the use of mitochondrial dynamics modulators in clinical practices for improving the quality of life in AMI patients.Mitochondria are known to play an important role in myocardial I/R injury It has been shown that ischaemia induced excessive mitochondrial fission and fragmentation via altered proteases, SUMOylating or phosphorylation via Drp1 activation. Phosphorylation of Drp1 at serine 616 by oxidative stress, Cyclin\u2010dependent kinase 1 (CDK1) and Protein kinase C\u2010\u03b4 (PKC\u03b4) activated mitochondrial fission, whereas phosphorylation of Drp1 at serine 637 inhibits mitochondrial fission Alteration in Drp1 expression initiates fission by post\u2010translational modification In addition to increased mitochondrial fission during I/R injury, decreased mitochondrial fusion has been shown to promote mitochondrial fragmentation under this condition In summary, the disturbance of mitochondrial dynamics is a crucial phenomenon in myocardial I/R injury that leads to larger infarct volumes, cardiac cell death and dysfunction. The best way to improve and reduce the impact of these injuries is to modulate the structural changes of the mitochondria by inhibition of mitochondrial fission or promotion of mitochondrial fusion or both.in vitro, ex vivo and in vivo studies have shown that attenuation of mitochondrial morphological changes in myocardial I/R injury models can potentially be achieved using the inhibitor of Drp1 in the form of pharmacological and non\u2010pharmacological intervention in vitro, ex vivo and in vivo studies.Reports from Mitochondrial fission is caused by increased phosphorylation of Drp1 at serine 616 and decreased phosphorylation of Drp1 at serine 637 Previous studies demonstrated that anoxia\u2010reoxygenation induced cardiomyocyte death by increasing Drp1 phosphorylation at serine 616, dephosphorylation at serine 637, ROS production and the release of lactate dehydrogenase (LDH) Non\u2010pharmacological interventions have been demonstrated to effectively inhibit mitochondrial fission. For example, inhibiting Drp1 function decreased mitochondrial metabolism and improved mitochondrial membrane potential without altering ATP levels ex vivo studies are summarized and shown in Table The beneficial effects of mitochondrial fission inhibition following I/R injury in in vivo cardiac I/R injury studies are summarized and shown in Table The beneficial effects of inhibiting mitochondrial fission prior to ischaemia in in vivo report supports the role of inhibiting mitochondrial fission in attenuating cardiac I/R injury.In addition, there is only one study demonstrating the beneficial effects of mitochondrial fission inhibition by a non\u2010pharmacological intervention given prior to ischaemia in cardiac I/R injury Table . AdenoviThe timing of the administration of treatment is an essential determinant of its therapeutic efficacy against I/R injury. Several studies using pharmacological and non\u2010pharmacological interventions to inhibit mitochondrial fission found that the greatest efficacy was ensured if treatment was given prior to ischaemia in vitro study by Dong et al., they reported that mitochondrial fission inhibitor Mdivi\u20101 given at the onset of reperfusion attenuated apoptosis, but increased total cell death in HL\u20101 cells in vivo and ex vivo studies suggest that interventions at the onset of reperfusion may also exert cardioprotective effects.Currently, there is only one study that reports the benefit of mitochondrial fission inhibition during the ischaemic period of cardiac I/R in rats via transfection with Mfn1 or Mfn2 or with Drp1K38A into HL\u20101 cells At this time, there are two reports available on the effect of mitochondrial fusion promoters given in cardiac I/R injury in vitro, ex vivo and in vivo models by reducing infarct size, preventing cardiomyocyte apoptosis and improving cardiac function. However, the effects of cardiac mitochondrial fusion proteins modulation in the heart are still limited and unclear. In addition, there are many gaps in the knowledge surrounding mitochondrial dynamics modulation, including the temporal influences of mitochondrial dynamics modulation during I/R injury. These dynamics will require future intensive investigations before clinical application can occur.Mitochondrial dynamics play crucial roles in both the normal heart and those with I/R injury. Inhibition of mitochondrial fission in myocardial I/R injury has been shown consistently to provide cardioprotective effects in The authors declare that they have no conflict of interest."} +{"text": "This data article provides genome statistics, phylogenetic networks and trees for a phylogenetic study of Southern Hemisphere Buccinulidae marine snails Specifications TableValue of the data\u2022Summary statistics for whole mitochondrial DNA sequences and 45S nuclear ribosomal genes are presented because such information for gastropods is currently rare, and base bias is known to influence phylogenetic inferences.\u2022Phylogenetic reconstructions (from short-length DNA sequence data) presented here include multiple buccinid and buccinulid taxa not included in the main-text trees, and may be useful to future evolutionary studies of Neogastropoda.\u2022DNA sequence variation and phylogenetic trees are provided because Southern Hemisphere taxa are currently under-sampled.\u2022The proportion of variable DNA sites for a selection of mitochondrial and nuclear genes from buccinulid whelks are compared. This information can improve genetic marker selection for future molluscan studies.1The data presented here originates from a phylogenetic study of Southern Hemisphere whelks cox1 genes and nuclear ribosomal 28S RNA gene. This short-length sequence data was acquired via PCR amplification and Sanger sequencing using universal primers. Sequence alignments used for analyses presented in the main text are attached to this paper.32 putative buccinulid and buccinid marine snails, as well as three fasciolariid snails used as a phylogenetic outgroup, were high-throughput sequenced on the Illumina 2500 platform. Sequence data was assembled to provide mitochondrial (mtDNA) genomic and 45S nuclear ribosomal DNA (rDNA) sequence data for most taxa, although some individuals failed to successfully sequence for the entire mtDNA or rDNA. This data was complemented with short-length sequence data from the mitochondrial 16S RNA and Using these sequence alignments, we present maximum-likelihood and Bayesian phylogenetic reconstructions for the sampled buccinulid whelks. These phylogenetic trees are alternative reconstructions that can be compared to trees presented in the main text. Splits networks are also estimated using the mtDNA genomic and nuclear ribosomal RNA sequence data. The proportion of variable sites per sequence length for a set of mitochondrial and nuclear ribosomal genes is investigated as well, which provides insight towards marker information for recent and distant evolutionary change in neogastropods , Fig. 9.2The DNA extraction, purification, sequencing method and routine for sequence assembly is provided in the main text"} +{"text": "Late failure of systemic right and single ventricles is difficult to predict. Approximately 1/3 of subjects with congenitally corrected transposition of the great arteries (ccTGA) have congestive heart failure by the fifth decade and 2/3 of subjects with ccTGA and significant associated defects have congestive heart failure by the age of 45 years. Perfusion defects have been identified at rest in 55 % of subjects with systemic right ventricles by dipyridamole sestamibi and with exercise in 45 %, which are not due to endovascular obstructive coronary disease. Subjects with perfusion defects typically have worse right ventricular function, but not always. It may be tied to coronary flow reserve and coronary mismatch in these hypertrophied ventricles. The objective of this study was to assess the technical feasibility of stress perfusion imaging by CMR and calculate coronary flow reserve in adults with systemic right or single ventricles.With IRB approval and informed consent, 14 adult patients with either a systemic right ventricle or a single ventricle underwent CMR with vasodilator stress perfusion imaging using adenosine (140 mcg/kg/min). Data collection included stress perfusion imaging, measurement of cardiac volume metrics and rest perfusion imaging at least 20 minutes after adenosine. Global myocardial blood flow (MBF) in ml/min/g and coronary flow reserve (CFR) were quantified using a fully quantitative model constrained deconvolution. Subjects underwent additional data collection, including lab work, 6-minute walk test and ECG.Table Myocardial blood flow and coronary flow reserve are quantifiable with current techniques regardless of ventricular morphology. This technique shows promise as a potential method of risk stratification of this population."} +{"text": "Teen depression was associated positively with teen smoking when controlling for sensation seeking behaviors. Maternal smoking was also directly linked to adolescent smoking . These findings underscore a potentially important role of sensation seeking in the origins of adolescent smoking, and clarify pathways of influence with regard to maternal attitudes and behaviors in subsequent teenage nicotine use.The purpose of this study was to examine maternal and adolescent depression, maternal and teen sensation seeking, and maternal smoking, and their associations with adolescent smoking. Data were collected from a sample of 47 male and 66 female adolescents (ages 11\u201418 years) and their mothers from three different health clinics. The findings indicated that maternal sensation seeking was linked indirectly with adolescent smoking through teen sensation seeking, both of which were significantly associated with teen smoking (\u03b2 = 0.29,"} +{"text": "Superabsorbent hydrogel polymer having 35% water retention ability was analyzed with three replicates. Hydrogel increased the water restoration capability of agricultural soil.Synthetic polymer was exploited as water-superabsorbent hydrogel and helped to conserve water in the agricultural soil. The hydrogel polymers were synthesized the carboxymethyl cellulose (CMC) and starch in addition to SiO The The dataset of this article described the consequence of SiO22 nanoparticles and analyzed for water retention capacity. Carboxymethyl cellulose sodium salt (CMC) and starch soluble in addition to SiO2 (20\u00a0nm) nanoparticles (enhance the surface area) were used to synthesize superabsorbent polymer films and aluminum sulfate octadecahydrate was used to establish crosslink in between polymer composite Vigna mungoSynthesis of hydrogel polymer was conducted with SiO"} +{"text": "The cancer statistics in Iran showed that breast cancer has increased in last two decades; therefore, it becomes most common cancer that affects women. Breast cancer has first rank and fifth rank among cancer incidence and cancer mortality in Iranian women respectively , 2. In oAn inequality cancer burden is borne by low socioeconomic status (SES) provinces and these provinces have higher incidence rates . For canWe motivated to find high rate clusters of breast cancer in Iranian women using by cancer provinces data from 2008 national registry of cancer (NCR) in Iran. Women population of each province was obtained by the 2006 national census data from Statistical Centre of Iran.We addressed this objective by Kulldorff spatial scan statistic in SaTScan software. As shown in Tehran and Kohgiluyeh and Boyer-Ahmad provinces have highest and lowest ratio of observed/expected (obs/exp) breast cancer obs/exp=1.92 and obs/exp=0.25 respectively. Breast cancer in women differently distributed across provinces and province based characteristics such as SES can impede this geography disparity. The results are relevant for answering which provinces can be targeted for screening and health care allocation and policy decisions."} +{"text": "Incomplete bladder emptying or dysfunctional voiding is a common lower urinary tract dysfunction encountered in children. Alpha blocker therapy is used to facilitate bladder emptying in the adult population and has likewise been applied to the pediatric population. Alpha blocker therapy seems well tolerated in children and appears efficacious towards improving bladder emptying in a variety of pediatric voiding disorders. Long-term follow-up and further investigation are warranted in order to validate the role of alpha blocker therapy in pediatric dysfunctional voiding."} +{"text": "Working memory and cognitive control deficits are hallmarks of schizophrenia. It is not known specifically how gating mechanisms that regulate memory may be disrupted in schizophrenia. Gating mechanisms determine what task-relevant information is selected into working memory while distractors are left out (input gating) and which items stored in working memory are selected for the rule or goal at hand (output gating). The current study investigated whether patients are able to perform the same cognitive control task that is able to dissociate input and output gating processes in a general population, and explored whether schizophrenia patients inappropriately use suboptimal cognitive control strategies (e.g. output gating when input gating can be used).Patients (n=5) with schizophrenia or schizoaffective disorder were recruited from the Providence VAMC. Participants completed a computer-based cognitive control task. In this task, participants remembered a target item from a sequence of two items in order to select a response. A context (rule) cued which item was relevant to remember, and was presented first in the stimulus sequence (context first) or last (context last). On selective trials, one item in the trial was relevant. On global trials, both items in the trial were relevant.Patients were able to complete the task with minor modifications to adjust for ability to understand the task rules. Preliminary results of reaction time data suggest that patients were challenged at increased cognitive load. Patients performed poorly on trials where participants could use only an input gating strategy (selective first). Preliminary data also suggest that performance in patients tended to be slightly worse for selective first trials where the distractor was presented before the relevant item .The current study supports the feasibility of using the cognitive control task selected to investigate gating mechanisms in the schizophrenia patient population. Preliminary data suggest disruption in the ability for patients to optimally use gating strategies and handle cognitive load. Future research will seek to reproduce these preliminary results in a larger sample, as well as compare patient performance to an age-matched control population directly. By understanding how gating mechanisms are disrupted in the patient population, we may be able to better develop therapeutic interventions such as cognitive training strategies to treat cognitive dysfunction in schizophrenia patients."} +{"text": "GHG) emissions and contribute to climate change mitigation by substituting for fossil fuels; yet delivering significant GHG savings will require substantial land\u2010use change, globally. Over the last decade, research has delivered improved understanding of the environmental benefits and risks of this transition to perennial bioenergy crops, addressing concerns that the impacts of land conversion to perennial bioenergy crops could result in increased rather than decreased GHG emissions. For policymakers to assess the most cost\u2010effective and sustainable options for deployment and climate change mitigation, synthesis of these studies is needed to support evidence\u2010based decision making. In 2015, a workshop was convened with researchers, policymakers and industry/business representatives from the UK, EU and internationally. Outcomes from global research on bioenergy land\u2010use change were compared to identify areas of consensus, key uncertainties, and research priorities. Here, we discuss the strength of evidence for and against six consensus statements summarising the effects of land\u2010use change to perennial bioenergy crops on the cycling of carbon, nitrogen and water, in the context of the whole life\u2010cycle of bioenergy production. Our analysis suggests that the direct impacts of dedicated perennial bioenergy crops on soil carbon and nitrous oxide are increasingly well understood and are often consistent with significant life cycle GHG mitigation from bioenergy relative to conventional energy sources. We conclude that the GHG balance of perennial bioenergy crop cultivation will often be favourable, with maximum GHG savings achieved where crops are grown on soils with low carbon stocks and conservative nutrient application, accruing additional environmental benefits such as improved water quality. The analysis reported here demonstrates there is a mature and increasingly comprehensive evidence base on the environmental benefits and risks of bioenergy cultivation which can support the development of a sustainable bioenergy industry.Perennial bioenergy crops have significant potential to reduce greenhouse gas ( Studies have reported life\u2010cycle GHG savings ranging from an 86% reduction to a 93% increase in GHG emissions compared with fossil fuels willow and poplar have low nitrogen input requirements , can sequester soil carbon due to reduced tillage and increased belowground biomass allocation, and can be economically viable on marginal and degraded land, thus minimizing competition with other agricultural activities and avoiding iLUC effects and woody crops (SRC poplar and willow) varied significantly, dependent on historic and current fertilizer rates, prior land use and time since planting calculated for four feedstocks individually.Appendix\u00a0S1. Method for calculation of net greenhouse gas (GHG) intensity for four biofuel production scenarios (illustrated in Fig.\u00a0Click here for additional data file."} +{"text": "Prenatal exposure to infection is increasingly recognized to play an important etiological role in neuropsychiatric and neurological disorders with neurodevelopmental components, including schizophrenia, autism, bipolar disorder, and mental retardation. The adverse effects induced by prenatal infection may reflect an early entry into a deviant neurodevelopmental route, but the specificity of subsequent disease or symptoms is likely to be influenced by the genetic and environmental context in which the prenatal infectious process occurs. The epidemiological link between prenatal infection and increased risk of neurodevelopmental disorders also receives strong support from experimental work in animal models. These models are based on maternal gestational exposure to specific infectious agents such as influenza virus or immune activating agents such as the bacterial endotoxin lipopolysaccharide (LPS) or the viral mimic poly(I:C).Converging evidence form these models suggests that prenatal immune activation can negatively affect early foetal brain development and change the offspring\u2019s neurodevelopmental trajectories, which in turn can lead to the emergence of behavioral and cognitive disturbances in later life. Modelling the human epidemiological association between prenatal infection and increased risk of neurodevelopmental disorders in animals has also greatly advanced our understanding of the underlying mechanisms. According to the prevailing view, cytokine-associated inflammatory events, together with downstream pathophysiological effects such as oxidative stress and (temporary) macronutrient and micronutrient deficiency, seem critical in mediating the post-acute effects of maternal infection on the foetal system.Recent findings have further implicated epigenetic processes as possible molecular mechanisms translating the negative effects of prenatal immune activation on the offspring. Not only does prenatal immune activation cause long-lasting epigenetic modifications such as altered DNA methylation and miRNA expression, but it also causes a transgenerational transmission of behavioral and neuronal abnormalities without additional immune exposures.Prenatal infection and associated developmental neuroinflammation may have a pathological role in shaping neurodevelopmental disease risk across generations."} +{"text": "Based on their reanalysis of our whole exome sequencing data, the ZNF23 rs531705739 variant segregated with affected members in the kindred and was not present in unrelated spouses. They also report a noncoding region that segregates with affected members but do not specify the sequence.We read the article by Gerhard and colleagues with interest.ZNF23 rs531705739 variant (T40R) segregates with six affected family members, two additional family members who developed thyroid cancer during surveillance do not have the variant variant in affected members with familial non-medullary thyroid cancer, the HABP2 rs7080536 variant (G434E) completely segregates in all the affected members in the kindred including the two newly diagnosed members during surveillance (Fig.\u00a0We performed Sanger sequencing of peripheral blood DNA from the kindred to experimentally validate the findings of Gerhard et al. Although we could validate the"} +{"text": "To explore feedback processes of Western-based health professional studenttraining curricula conducted in an Arab clinical teaching setting.Thisqualitative study employed document analysis of in-training evaluation reports(ITERs) used by Canadian nursing, pharmacy, respiratory therapy, paramedic,dental hygiene, and pharmacy technician programs established in Qatar. Sixexperiential training program coordinators were interviewed between Februaryand May 2016 to explore how national cultural differences are perceived toaffect feedback processes between students and clinical supervisors. Interviews were recorded, transcribed, andcoded according to a priori cultural themes.Document analysis found all programs\u2019 ITERs outlinedcompetency items for students to achieve.Clinical supervisors choose a response option corresponding to theirjudgment of student performance and may provide additional written feedback inspaces provided. Only one program required formal face-to-face feedback exchangebetween students and clinical supervisors. Experiential training programcoordinators identified that no ITER was expressly culturally adapted, althoughin some instances, modifications were made for differences in scopes ofpractice between Canada and Qatar.\u00a0 Powerdistance was recognized by all coordinators who also identified both studentand supervisor reluctance to document potentially negative feedback inITERs. Instances of collectivism weredescribed as more lenient student assessment by clinical supervisors of thesame cultural background. Uncertainty avoidance did not appear to impactfeedback processes.Our findings suggest that differences in specificcultural dimensions between Qatar and Canada have implications on the feedbackprocess in experiential training which may be addressed through simple measuresto accommodate communication preferences. Document analysis is systematic procedure for identifying, reviewing, and deriving useful information and understanding from existing documents.The study participants included the experiential coordinators of the six health professional programs delivering Canadian curricula in Qatar:\u00a0 UC-Q nursing, QU CPH pharmacy, and CNA-Q respiratory therapy, emergency medical services, dental hygiene, medical radiography and pharmacy technician programs.\u00a0 Individuals were first contacted by email whereby the study and its objectives were summarized.\u00a0 Experiential coordinators expressing interest in participation were then contacted by telephone to confirm their participation and schedule an interview at their place of work.\u00a0 The consent form was then sent by email for their review and signed when the interviews took place.,Semi-structured interviews were conducted in person following a question topic guide informed by Hofstede and Hall\u2019s cultural theoretical frameworks see .16,20 ThAll instruments used as ITERs by clinical supervisors to assess students in the health professional programs, as well as any form students submit in turn as evaluation of their clinical supervisor were described and categorized. The semi-structured interview discussions were audio-recorded and transcribed verbatim by one researcher and then verified by a second researcher attending the interview.\u00a0 Finalized interview transcripts were evaluated independently by two researchers using directed content analysis whereby various portions of the transcribed data were divided into units at the level of the phrase, sentence, or paragraph and examined for ideas and messages.Seven documents were obtained from these six programs and are summarized in Several examples of cultural impact on feedback processes were given in the interviews with the 5 Canadian and 1 American experiential program coordinators.All coordinators described issues arising that were consistent with of power distance effects whereby students were unwilling to give constructive criticism to clinical supervisors.\u201cSome students are shy and are afraid to give feedback because it might get them in trouble.\u201d (Participant 3)\u201cThe other thing is the authority, especially if the student is Qatari and the preceptor is non-Qatari. Sometimes they think if this is a Qatari student and if I do not say good things, I will be in trouble\u201d (Participant 5)\u201cStudents might be hesitant to report some issues and when they come later with preceptor concerns [they do so] without naming names because they will have to work with them after graduating\u201d (Participant 2)Once anonymous means were made available in some programs, such as blinded (to the preceptor) submission in an electronic system, the veracity of such feedback was felt by coordinators to have increased. Paradoxically, availability of such blinded feedback mechanism was often countered with student reluctance to record supervisor or site recommendations on these ITERs (low context).\u00a0 Coordinators of all programs perceived student and clinical supervisor preference to interact in-person with the faculty clinical instructors or experiential program coordinators to share potentially negative information or experiences.\u00a0\u201c[Students] are happy to speak to [coordinators] but less comfortable documenting it\u2026because of the perception that documenting makes it more permanent\u201d (Participant 1)\u201cI suspect [supervisors] will wait over repeated attempts before rating achievement; they are not going to document each time the student fails the competency before the end\u201d\u00a0 (Participant 5)A few instances could be attributed to greater collectivism in Qatar than in Canada. Discrimination in performance when paired students were sharing an internship supervisor was sometimes not discernable when submitted ITERs are compared.\u201cFor the most part, the comments are similar when there are more than one student at a site. Unless the student is very poor, then I do see differing grades and comments\u201d (Participant 6)One coordinator also described occasions when unsatisfactory student behavior (e.g. absenteeism) was recorded by clinical supervisors, which would be contradicted in reports by other preceptors who shared the student\u2019s ethic background.\u201cWe had one [Arab] student only showing up in the morning. Feedback from Arab preceptors was that he was there, but his other [Asian] preceptors told us that he was not\u201d (Participant 5)No participant could offer any example of how differences in uncertainty avoidance might tangibly affect feedback in this setting.,-If we consider experiential training fundamental to health professional education, then feedback is central to the enterprise. Clinical supervisors often have dual roles to facilitate learning opportunities and role model care in the internship setting, as well as advise students how they might improve their performance.Our findings illustrate various approaches to experiential feedback among the different disciplines with established Canadian curricula in Qatar. Different measures of achievement for student competencies were outlined (as levels or dichotomous ratings), as well as expectations and formal mechanism for students to evaluate their clinical supervisors.\u00a0 Review of ITER documents and discussion with experiential program coordinators underscore how the quantity and veracity of feedback may be affected by the medium of exchange.\u00a0 In the program continuing to use paper-based ITER completion (pharmacy), especially when in-person \u201c360 degree\u201d feedback between students and clinical supervisors was expected, experiential program coordinators described a disconnect between what they read in the submitted evaluations and what they learned separately from students when given an opportunity. When there is anonymous or indirect evaluation, the candour of both student and supervisor commentary increases and prior evidence of power distance is essentially eliminated. Programs at CNA-Q use an electronic system akin to ONE45 used for medical resident evaluation throughout Canada, whereby ITERs are completed by clinical supervisors online. Additionally, many CNA-Q program ITERs have a relatively higher proportion of technical competencies as part of the experiential training or removing endotracheal tube (respiratory therapy)) when compared to nursing or pharmacy and as such, student assessment was considered less subject to cultural influences by the experiential program coordinators interviewed.,That the student\u2019s or supervisor\u2019s true perspective is not documented in writing would be consistent with preferences of a low-context culture, such as Qatar. However, obtaining quality narrative is not exclusive to this Arab setting. Study of the written record to support assigned scoring on Canadian medical resident ITERs has shown gaps in the quality of feedback, which may be improved with directed faculty development program.We could not delineate many instances of how differences in cultural dimensions of individualism and uncertainty avoidance reported between Canada and Qatar might impact feedback. Lack of expected group-oriented evaluations in this collectivist environment could in part be attributed to the discrete nature of technical competencies evaluated coupled with the low number of paired student or supervisor compositions among most of the programs . Literature seems lacking on this phenomenon within health disciplines, particularly study of how physician supervisors might differentiate assessment among medical students or residents who often train concurrently in the medical model. Inquiry has been into how in the opportunistic environment of patient care in experiential learning, clinical supervisors assign tasks to different levels of learners, but not subsequent evaluation.Members of cultures high on uncertainty avoidance are risk averse and prefer predictable environments. While we hypothesized that students and clinical supervisors in Qatar would therefore eschew processes and circumstances whereby negative feedback might be exchanged, it is possible that the structured mechanisms delineated by each health professional program were instead accepted as rules for the group norm and so members complied with its expectations.,A number of limitations to this exploratory study merit consideration. We relied heavily on Hofstede\u2019s cultural theoretical framework mapping dimensions of a nationality\u2019s preferences, but must acknowledge, as he does, that individual variations exist. While we have described Canadian and Arab cultural features and associated behaviours as monolithic, not all members of a population strictly ascribe to its outlined model. Our research question presupposed that the Arab students would be supervised by local supervisors of Arab-origin and we did not previously take into account the number of Canadian clinical faculty members that we discovered were embedded within care settings as instructors. It is unknown if their feedback behaviours would differ from local supervisors and how this would subsequently affect experiential program coordinators overall impressions. Similarly, all coordinators interviewed were of North American-origin and may perceive cultural aspects of feedback through a different lens. Full appreciation of feedback processes is limited by document analysis and experiential program coordinators interview alone and we intend to conduct further study with the students and clinical supervisors of these programs.\u00a0 Finally, our research question did not incorporate inquiry into student reflection or self-assessment, which forms an important component of many health professional experiential training curricula in Canada. Although Canadian physician training has not yet taken foothold in the Middle East, overseas partnerships are in place elsewhere and understanding of how medical student and resident feedback is considered, even in seemingly like-minded cultures, is essential for quality assurance.Our results have implications within this Arab context whereby existing health professional programs would benefit from purposeful revision of ITER format and documentation, but also for Western-oriented curricula entering the regional market to educate these and students of other health disciplines in the future.\u00a0 For example, preferences specific feedback process in experiential training may be accommodated when anonymous and electronic systems are in place.\u00a0 Health professional programs engaging in cross-border partnerships within Asian and African contexts may also inform cultural adaptation of experiential training feedback processes from our experiences. Finally, this research may also contribute to understanding of feedback preferences of foreign health professional students or graduates entering Western-oriented education and care settings.We have explored how power distance, collectivism, uncertainty avoidance and cultural contexts might influence communication between clinical supervisors and health professional students in Qatar.\u00a0 Our findings suggest that differences between Canadian and Middle East cultural dimensions have implications on the feedback process in experiential training which may be addressed through simple measures to accommodate communication preferences.This study was funded by a Qatar University internal study grant.The authors declare that they have no conflict of interest."} +{"text": "Electrolyte concentration in sweat depends on environmental context and physical condition but also on the pathophysiological status. Sweat analyzers may be therefore the future way for biological survey although how sweat electrolyte composition can reflect plasma composition remains unclear. We recruited 10 healthy subjects and 6 patients to have a broad range of plasma electrolyte concentrations and pH. These variables were compared to those found in sweat produced following cycling exercise or pilocarpine iontophoresis, a condition compatible with operating a wearable device. We found no correlation between plasma and sweat parameters when exercise-induced sweat was analyzed, and we could identify a correlation only between plasma and sweat potassium concentration when sweat was induced using pilocarpine iontophoresis. We tested measurement repeatability in sweat at 24hr-interval for 3 days in 4 subjects and found a great intra-individual variability regarding all parameters in exercise-induced sweat whereas similar electrolyte levels were measured in pilocarpine-induced sweat. Thus, electrolyte concentration in sweat sampled following physical activity does not reflect concentration in plasma while pilocarpine iontophoresis appears to be promising to reproducibly address sweat electrolytes, and to make an indirect evaluation of plasma potassium concentration in chronic kidney disease and arrhythmia. Sweat analysis is considered as the future way for biological survey5: sweat can be collected in vivo via non-invasive means using easily wearable devices, and sweat is assumed to reflect the body\u2019s biochemical environment. This would be of special interest when continuous monitoring is necessary, in ambulatory conditions or for out-door uses. Sweat sample processing is indeed generally simple and does not require any treatment prior to analysis. Yet, a main limitation of the use of sweat in a context of in vivo monitoring is production of a sufficient volume of sweat for analysis7.Sweat composition varies depending on environmental context, physical condition and body regulatory systems but also on the pathophysiological status10 and testing drug or ethanol consumption11. Sweat analysis is also currently developed to monitor diabetic, schizophrenic and tumorigenic prognostic markers2. Many efforts are therefore ongoing to design fast, reliable and user-friendly methods to access the body\u2019s biochemical status via sweat, and hence to directly address pathophysiological conditions. One such attempt is development of alternative approaches of non-invasive collection and manipulation of metabolites released in sweat by skin via the use of osmotic properties of hydrogels contained in micropatches14. These approaches are based on nanosized hydrogel particles functionalized with affinity reactive baits and are designed to investigate complex body fluids such as sweat. Those formats can concentrate up to 104-fold and isolate in one single step low abundance biomarkers prior to analysis whereas unwanted high abundance analytes are excluded concomitantly14.Sweat analysis is already routinely used for the diagnosis of cystic fibrosis5. In this work, the authors obtained comparable results using the sweat-based colorimetric procedure they described and standard biochemical techniques used to analyse sweat. Their report, the last of several others in the field (reviewed in ref.3), is quite significant and promising as it describes a device providing embedded chemical analyses. The article raises however at least two important issues.A major progress in the development of a monitoring equipment was recently made. A group described an innovative colorimetric approach to measure various biological parameters in sweat samples collected in a context of physical exercise performed to increase sweat sample volume7 but the influence of this step on sweat composition is rarely considered. Sweat sample volume is generally increased using physical exercise. Exercise modifies however sweat composition16 and may interfere with the use of sweat to address steady-state physiological parameters and their pathological variations. Sweat production is also sometimes induced using application on skin of pharmacological compounds such as pilocarpine17. This alkaloid agent stimulates sweat gland secretion and is routinely used via iontophoresis in cystic fibrosis diagnosis for sweat chloride titration17. There are obviously many reasons why pilocarpine iontophoresis may influence sweat characteristics1. Additionally, each of these two common procedures probably modifies sweat composition in a different manner.First, inducing sweat production is an essential step in the development of sweat-based monitoring devices5 often takes for granted that sweat composition reflects plasma composition but such relationship remains unclear. Yet, following storage in the sweat glands, secretion into the sweat and transport to the skin surface, sodium and chloride are for example partially and selectively reabsorbed during transportation. These mechanisms can be differently affected by various disorders19.Second, the literature in the field including its latest reportsIn this study, we addressed two key-points in the development of a reliable sweat-based monitoring of the body\u2019s biochemical composition: i) how physical exercise and pilocarpine iontophoresis influence electrolyte concentration and pH of sweat? ii) can sweat analysis address plasma electrolyte levels?Ten healthy subjects and 6 patients were selected according to their plasma electrolyte levels to constitute a panel encompassing a broad range of values when pilocarpine iontophoresis was used to stimulate sweat production according to an established procedure followed for cystic fibrosis diagnosis, a correlation was found between plasma potassium level and sweat potassium level, no relationship being found regarding sweat and plasma levels for the other electrolytes and pH values; (ii) when cycling exercise was used to stimulate sweat production, no correlation was found between sweat and plasma parameters; (iii) similar chloride, potassium and sodium concentrations were measured in sweat at 24hr-interval for 3 days using pilocarpine iontophoresis, which indicates measurement repeatability, whereas a great intra-individual variability was found following cycling exercise.1. Accordingly, the advantage gained by pilocarpine iontophoresis over physical exercise may be due to the fact that the alkaloid tends to unify sweating conditions among the subjects whereas native sweating during physical exercise is highly dependent on various individual parameters, including physical condition, body temperature, food, emotional state and environment16. These latter factors may be also responsible for the poor measurement repeatability found in our work using exercise-induced sweat whereas pilocarpine iontophoresis significantly improved repeatability, an important property for in vivo assays used in clinical settings.Sweat induced by pilocarpine iontophoresis results from local peripheral cholinergic stimulation whereas central, peripheral and thermal mechanisms influence sweat rate and composition in a context of physical exercise18, even following pilocarpine iontophoresis. Additional substantial reasons can be probably found in the presence within the skin of extrarenal mechanisms that regulate electrolyte homeostasis and act via sodium and chloride sequestration in the skin interstitium21. Finally, it is of note that the low inter-individual variability we found here further supports a previous study that reported low variability within subjects regarding sweat sodium level contrary to potassium level7.The discrepancy observed here between plasma and sweat concentrations of most electrolytes but potassium is probably largely due to selective filtering of electrolytes inherent to the function of the sweat gland22, which can be evaluated probably only by plasma osmolality or at least natremia19. In this case, simply considering increased sodium level in sweat as a marker for dehydration during a long run may lead to misinterpretation. Yet, a smartphone application was recently proposed to indirectly monitor hydration via pH changes in sweat and indicate during physical exercise the proper time for hydration23. Our results do not however support this use as we found here that sweat pH-measurements were not reproducible in the same subject in spite of similar exercise conditions.Although the results we present here further highlight pilocarpine as a key component to produce sweat as part of ambulatory monitoring, it remains that the discrepancies found here between plasma and sweat levels of several electrolytes challenge the rapid marketing of sweat analyzers. Additional concerns include for example the fact that dehydration reflects a loss of water leading to hypertonicity25. Potassium concentration in sweat is often found, or considered, to be similar to plasma and poorly vary with sweat rate1. For some experts in the field, potassium level in final sweat\u2009>\u20098\u2009mM may be therefore indicative of sample evaporation whereas for others usual potassium level in final sweat is in the 2\u20138/10\u2009mM range (review1). We found here a rather high mean of sweat potassium levels, in agreement with others7 (\u224813\u2009mM) who additionally reported high inter-individual variability.Of particular interest is our finding that sweat potassium level was correlated with plasma potassium level. This point is quite interesting because reliable, non invasive monitoring of potassium concentration in the body is of paramount importance in a context of ambulatory monitoring as small changes in potassium affect heart rhythm level, for example in end-stage kidney disease patients26; (iii) we did not normalize sweat volume because we considered that the Macroduct system strongly limits per se evaporation; (iv) we did not take into account genders although it was found in particular that sodium level is lower in male sweat7.There are at least four limitations to our work: (i) we addressed a low number of subjects. Yet, if a routine sweat testing is expected to monitor plasma electrolyte level, then the data obtained should be consistent and relevant in the vast majority of subjects. Major discrepancies were manifest here although our panel was small. Thus we consider that our findings did not validate the proof of concept tested here except for plasma potassium level; (ii) sweat collected using occlusive devices may contain non native sweat electrolyte concentrations because of electrolyte leaching from the stratum corneum of the skin in these conditions25.Overall, the merit of this study lies in the fact that it addressed a generally neglected question by evaluating the relationship between plasma and sweat composition, this link being generally taken for granted. The results we present here indicate that electrolyte concentration in sweat sampled following physical activity does not properly reflect plasma electrolyte levels, and hence cannot be used without careful consideration for biological survey, in particular for monitoring hydration state. We show however that pilocarpine iontophoresis is a promising way to reproducibly address sweat electrolytes, and to make an indirect evaluation of plasma potassium level, a marker that is particularly relevant in arrhythmia and chronic kidney disease25. The protocol was approved by the Ethics Committee of our institution (CPP Sud Mediterran\u00e9e) and the study was carried out in accordance with the Code of Ethics of the World Medical Association (Declaration of Helsinki). Procedures were carried out with the adequate understanding and written consent of the subjects.We recruited 10 healthy subjects and 6 patients who were attending the hemodialysis center of the hospital to treat end-stage kidney disease , a disorder that is generally associated with abnormal plasma levels of electrolytes7, using a FinnPipetteTM because we considered that this approach largely respects native sweating conditions. Alternatively, pilocarpine iontophoresis was performed in both healthy subjects and patients in the same time slot according to a standard procedure followed for cystic fibrosis diagnosis17. We made use of the Macroduct system 3700 (ELITechGroup) that delivers during 10\u2009min a safe and optimal quantity of pilocarpine for gland stimulation . The skin was carefully cleaned prior to the experiment using soap and then tap water and a cotton pad prior to extensive rinsing with distilled water and drying with a sterile towel. Both electrodes were placed on the anterior size of the forearm as recommended by the manufacturer. Sweat collection was achieved using the Macroduct\u00ae sweat collection system, a device that consists of a concave disk and a spiral plastic tube that collects sweat. Whole blood samples (3\u2009mL) were collected in parallel from the cubital vein at the end of the cycling period when cycling exercise was used to induce sweat production, or at the beginning of sweat production when pilocarpine stimulation was used.Sweating was induced following physical exercise consisting in cycling exercise . Prior to exercise, a medical interview was carried out to verify that all subjects had breakfast 3\u20134\u2009hours before the cycling exercise and no special diets, pathological disorders or medication. After a resting period of 1\u2009hour in an air conditioned room around 25\u2009\u00b0C, cycling exercise was performed at this temperature between 10 and 11\u2009h AM in conditions that enable sufficient sweating for sampling as determined as part of our practice of exercise stress test in cardiology settings. We collected sweat in the lower back region as often doneTo test reproducibility, 4 healthy subjects performed the cycling exercise (see above) at 24\u2009hours interval for 3 days and electrolytes were measured as described above. The same protocol was also followed by replacing the cycling exercise with pilocarpine stimulation.Plasma and sweat electrolytes were measured using a Cobas 8000 analyzer (Roche Diagnostics\u00ae) and dedicated programs . Specimen values higher than upper limit of the range were assessed following dilution.Quantitative variables were expressed as mean\u2009\u00b1\u2009standard deviation (SD) and range as indicated. Correlations between biological parameters were quantified and tested using Pearson rank correlation coefficient. Linear regression was assessed and r2 value determined. A p value\u2009<\u20090.05 was considered significant. Analysis was performed using Prism, a GraphPad Software.all data presented in this study are available upon request.we thank the AMIDEX foundation."} +{"text": "Dear Editor, Luteolin is a naturally found flavone, which is obtained from numerous plant species . Luteolin vitro and in vivo studies (Table 1In this letter, conclusive evidences have been presented for the potent antioxidant activity of luteolin reported in various The authors declare no conflict of interest."} +{"text": "Mouse mammary tumour viruses (MMTVs) may have a role in a subset of human breast cancers. MMTV positive human breast cancers have similar histological characteristics to neuroendocrine breast cancers and to MMTV positive mouse mammary tumours. The purpose of this study was to investigate the expression of neuroendocrine biomarkers \u2013 synaptophysin and chromogranin, to determine if these histological characteristics and biomarker expression were due to the influences of MMTV.Immunohistochemistry analyses to identify synaptophysin and chromogranin were conducted on a series of human breast cancers in which (i) MMTV had been previously identified and had similar histological characteristics to MMTV positive mouse mammary tumours and (ii) MMTV positive mouse mammary tumours.The expression of synaptophysin and chromogranin in MMTV positive mouse mammary tumors were all positive (7 of 7 specimens \u2013 100% positive). The expression of synaptophysin and chromogranin in MMTV positive human breast cancers was much less prevalent (3 of 22 \u2013 14%). There was no expression of synaptophysin and chromogranin in the normal breast tissue control specimens.It is not possible to draw any firm conclusions from these observations. However, despite the small numbers of MMTV positive mouse mammary tumours in this study, the universal expression in these specimens of synaptophysin and chromogranin proteins is striking. This pattern of synaptophysin and chromogranin expression is very different from their expression in MMTV positive human breast cancers. The reason for these differences is not known.The high prevalence of positive expression of synaptophysin and chromogranin in MMTV positive mouse mammary tumours and low expression of synaptophysin and chromogranin in MMTV positive human breast cancers indicates that MMTV is not usually associated with neuroendocrine human breast cancers.The online version of this article (doi:10.1186/s13027-017-0135-8) contains supplementary material, which is available to authorized users. This evidence is as follows: (i) identification of MMTV\u2013like gene sequences in breast cancer tissues is associated with a 15 fold increase in breast cancer [controls , 3, controls , 7, (v) controls \u201311, controls , 14, .Twenty breast cancer archival formalin fixed breast cancer specimens were selected because in previous studies MMTV envelope gene sequences had been identified in these specimens . The MMTThirty eight normal breast specimens from breast reduction surgery were used as controls. The donors of these specimens were on average younger than the breast cancer patients and therefore should be considered as a comparison group and not age matched controls.Seven mouse mammary tumours in which MMTV envelope gene sequences had previously been identified were used for comparative purposes .The automated Tissue-Tek Prisma system were used for haematoxylin and eosin staining and Ventana BenchMark Ultra were used for the identification of synaptophysin and Chromogranin A proteins. Synaptophysin and chromogranin A antibodies were used on formalin fixed paraffin embedded specimens. Both synaptophysin and chromogranin A proteins stain the cell membranes and cytoplasm of the target cells. Pancreatic tissues were used as positive controls for each specimen. Omission of the antibodies was used as negative controls.The non-parametric Kolmogorov-Smirnov test was used to test the significance between the human breast cancer and mouse mammary tumour synaptophysin and chromogranin positivity.The outcomes are presented in Table\u00a0The expression of synaptophysin and chromogranin in MMTV positive mouse mammary tumours and MMTV positive human breast cancers are shown in Fig.\u00a0negative human breast cancers are also similar to MMTV positive mouse mammary tumours. The synaptophysin and chromogranin positive MMTV positive human breast cancers are characterised by intensely stained nuclei which occupy most of the cell, the cells are mostly round and regular in size and are clumped together without glandular acini or lumen. MMTV negative human breast cancers are not similar to MMTV positive mouse mammary tumours.The similar histological characteristics of MMTV positive and neuroendocrine marker positive human breast cancers and MMTV neuroendocrine positive mouse mammary tumours are shown in Fig.\u00a0One human breast cancer specimen was positive for both Synaptophysin and Chromogranin. As shown in Fig.\u00a0We have demonstrated that (i) MMTV positive mouse mammary tumours are all synaptophysin and chromogranin positive (7 of 7 specimens \u2013 100% positive), (ii) in MMTV positive human breast cancers only 3 of 22 (14%) are synaptophysin or chromogranin positive, (iii) there was no expression of synaptophysin and chromogranin in the comparative normal breast specimens and (iv) MMTV positive (and either synaptophysin and chromogranin positive or negative) human breast cancers have similar histological characteristics to neuroendocrine human breast cancers and to MMTV positive mouse mammary tumours.The expression of synaptophysin and chromogranin proteins in MMTV positive human breast cancers was much less prevalent than in MMTV positive mouse mammary tumours.These are confusing observations and it is not possible to draw any firm conclusions. However, despite the small numbers of mouse mammary tumours in this study, the universal expression in these specimens of synaptophysin and chromogranin proteins is striking. This pattern of synaptophysin and chromogranin expression is very different from the MMTV human breast cancers. The reason is not known.The expression or secretion of synaptophysin and chromogranin proteins by endocrine (hormone) producing cells is presumably in response to a neurological or other external stimulus. However, the substantial differences in the expression of synaptophysin and chromogranin in MMTV positive human breast cancer and MMTV positive mouse mammary tumours indicate that MMTV is not usually associated with neuroendocrine human breast cancers.It has been suggested by Wiedenmann et al. and lateThe high prevalence of positive expression of synaptophysin and chromogranin in MMTV positive mouse mammary tumours and low expression of synaptophysin and chromogranin in MMTV positive human breast cancers indicates that MMTV is not usually associated with neuroendocrine human breast cancers.Additional file 1: Table S1.Selected MMTV positive human breast cancers. Synaptophysin and chromogranin expression. (XLSX 11 kb)Additional file 2: Table S2.Mouse mammary tumours. Synaptophysin and chromogranin expression (XLSX 10 kb)Additional file 3: Table S3.Normal human breast specimens. MMTV negative. Synaptophysin and chromogranin (XLSX 11 kb)"} +{"text": "The article by recovered 11 differentially expressed probesets.While we agree with (i) Utilize the technical replicates as a random-effect variable in LIMMA.(ii) Utilize the technical replicate as a random variable in a repeated-measures ANOVA.(iii) Utilize a linear mixed-effects model to account for the technical replicate as a random effect while accounting for treatment as a fixed effect.Each of the aforementioned methodologies is readily accessible through R\u2019s base or Bioconductor packages.Biostatistics online), the dataset was initially analysed with ComBat including the treatments as covariates as required by the SVA package .Upon further investigation, a variety of different preprocessing and batch correction methods all yield far more differentially expressed genes than the method proposed by In summary, we present three different methods for analyzing microarray data that utilize technical replicates while correcting for batch effects. These methods yield consistent results, and therefore represent robust alternatives to traditional batch correction methods in datasets subject to the concerns articulated by Supplementary DataClick here for additional data file."} +{"text": "Patients with frontal lobe damage and cognitively normal elderly individuals demonstrate increased susceptibility to false facial recognition. In this paper we review neuropsychological evidence consistent with the notion that the common functional impairment underlying face memory distortions in both subject populations is a context recollection/source monitoring deficit, coupled with excessive reliance on relatively preserved facial familiarity signals in recognition decisions. In particular, we suggest that due to the breakdown of strategic memory retrieval, monitoring, and decision operations, individuals with frontal lobe impairment caused by focal damage or age-related functional decline do not have a reliable mechanism for attributing the experience of familiarity to the correct context or source. Memory illusions are mostly apparent under conditions of uncertainty when the face cue does not directly elicit relevant identity-specific contextual information, leaving the source of familiarity unspecified or ambiguous. Based on these findings, we propose that remembering faces is a constructive process that requires dynamic interactions between temporal lobe memory systems that operate in an automatic or bottom-up fashion and frontal executive systems that provide strategic top-down control of recollection. Executive memory control functions implemented by prefrontal cortex play a critical role in suppressing false facial recognition and related source memory misattributions."} +{"text": "Traumatic axonal injury (TAI) is an important pathoanatomical subgroup of traumatic brain injury (TBI) and a major driver of mortality and functional impairment. Experimental models have provided insights into the effects of mechanical deformation on the neuronal cytoskeleton and the subsequent processes that drive axonal injury. There is also increasing recognition that axonal or white matter loss may progress for years post-injury and represent one mechanistic framework for progressive neurodegeneration after TBI. Previous trials of novel therapies have failed to make an impact on clinical outcome, in both TBI in general and TAI in particular. Recent advances in understanding the cellular and molecular mechanisms of injury have the potential to translate into novel therapeutic targets. Multiple therapeutic targets are emerging that offer the potential to reduce secondary brain injury at a cellular level. These include cytoskeletal and membrane stabilisation, control of calcium flux and calpain activation, optimisation of cellular energetics, and modulation of the inflammatory response.Wallerian degeneration, as occurs following an axonal injury, is an active, cell-autonomous death pathway that involves failure of axonal transport to deliver key enzymes involved in NAD biosynthesis.Chronic microglial activation occurs following traumatic brain injury (TBI) and may persist for decades afterwards. This ongoing response has been linked to long-term neurodegeneration, particularly of white matter tracts.Phagoptosis is the process whereby physiologically stressed but otherwise viable neurons are phagocytosed by microglia in response to a range of eat-me signals induced by tissue injury. TBI (see http://www.cdc.gov/traumaticbraininjury/data). TBI is an emerging research priority, with large North American and European comparative effectiveness research studies enrolling several thousands of patients and looking at a broad range of research questions secondary brain injury and associated pathophysiological responses , due to impaired Immunohistochemical analysis for APP accumulation is currently the gold-standard clinical and experimental technique for assessment of TAI Neurofilaments are the dominant intermediate filament of axons; they are produced in the neuronal soma and transported throughout the neurite. Structurally they are obligate heteropolymers assembled from a central rod domain surrounded by the neurofilament triplet proteins in vitro stretch reduced secondary axotomy The calcineurin inhibitor FK506 (tacrolimus) is used in humans as an immunosuppressive agent to reduce the risk of organ rejection. It inhibits phosphatases and hence attenuates the effects of dephosphorylation-dependent proteases on the neuronal cytoskeleton, including, neurofilament compaction and spectrin/ankyrin degradation Microtubule stabilisers including paclitaxel (Taxol) have also been suggested as potential neuroprotective agents and there is evidence that they may affect the rate of axonal degeneration Spectrin is a key cytoskeletal element whose breakdown leads to the formation of specific, quantifiable, stable \u03b1II spectrin fragments of 145 kDa and 150 kDa . The SBP145 breakdown product is brain specific and is found in contusions with brain necrosis, but an isolated TAI is also sufficient to stimulate its generation. The rise in breakdown products may occur within 15\u00a0min and is reliably demonstrated within 3\u201324\u00a0h Ankyrins are a family of adaptor proteins that link the spectrin\u2013actin complex to integral membrane proteins, a function vital to the maintenance of ion channels within the plasma membrane. Proteolytic degradation of ankyrin-G following axonal injury may result in mislocalisation of sodium channels in nodal regions. It might also encourage instability of the axolemma through altered binding of neurofascin, a member of the L1 immunoglobulin superfamily of cell adhesion molecules Injury to axons in the central nervous system can lead to death of the whole neuron, although this may vary by neuronal subtype and with the distance of the injury from the cell body Wallerian degeneration (WD) . This is related to ion (WD) . WLD shos mice demonstrate reduced physical evidence of TAI, less evidence of axonal transport disruption , and delayed motor and cognitive impairment. These findings are consistent with WldS-sensitive degeneration following TAI but this has not been definitively confirmed in vivo protection through inhibition of the WLD pathway, with reduction of behavioural deficits and axonal APP aggregates in the corpus callosum When subjected to a TBI, WLDin vivo screening approach for neurodegenerative disease modifiers seems to be proneurogenic and antiapoptotic in vivo work in models of blast injury and TBI have shown that P7C3 can provide neuroprotection and/or preservation of function There are no currently available modulators of SARM1. However, as the WD pathway is increasingly understood other therapeutic targets may emerge within the pathway and offer new treatment opportunities. One example is P7C3 and related compounds; this aminopropyl carbaxole discovered using an unbiased When a central nervous system axon is stretched there is an acute increase in intracellular calcium primarily derived from intracellular stores. This is followed by a more gradual and long-lasting dysregulation of intracellular calcium metabolism in vivo results with this agent in rodent models of TBI have been encouraging in vitro after a stretch injury and exhibits neuroprotective effects following TBI in animal models but has not been tested in humans. The exact mechanisms of action of this agent are debated but may partially involve inhibition of p38 mitogen-activated protein kinase (p38-MAPK) activation or cathepsin B- and tBid-mediated mitochondrial cell death triggering Attempts to use agents such as Kollidon VA64 to reseal microdefects in the plasma membrane following injury aim to guard against mechanoporation-related calcium damage. Initial in vivo proteolysis of neurofilaments, although the exact steps in such a mechanism have not been fully described ex vivo \u2013 a failing that may limit human translation Excitotoxicity has a long history of being implicated as a secondary brain injury mechanism contributing to neuronal death following trauma. Excitotoxic cell death is intimately linked to downstream calcium ion influx modulation and dysregulation, which is mediated in part by calpains Axonal stress is associated with reduced mitochondrial movement, disruption of cristae, and swelling, coalescence, or fragmentation of mitochondria N-acetyl cysteine Attempts to improve mitochondrial energetics have included the use of in vitro stretch modelling is cardiolipin peroxidation. The compound XJB 5131 has been shown to reduce lipid oxidation and caspase activation in a rat cortical contusion model, with subsequent improvement in both lesion size and functional measures Another mitochondrial target identified with Creatine is an endogenous amino acid, administration of which can increase stores of the high-energy metabolite phosphocreatine. Creatine may directly act on central nervous system axons and modify response to brain injury by inhibiting calcium-induced activation of the mPTP, maintaining ATP levels, preserving normal mitochondrial membrane potentials, and reducing intramitochondrial calcium levels TAI triggers a complex cascade of inflammatory response with release of cytokines, chemokines and growth factors by microglia, astrocytes, and neurons 7 receptors localised to astrocytic end feet and colocated with aquaporin 4 expression. P2X7 activation leads to IL-1\u03b2-mediated exacerbation of local neuroinflammation, reactive gliosis, and cytotoxic oedema. It also results in membrane poration and increased tissue damage as a result of enhanced calcium influx 7 and might downregulate this damaging response up to 4\u00a0h post-injury Purinergic signalling through cognate transmembrane receptors represents a core part of the neuroinflammatory response. Adenosine signals via P1 receptors while ADP and ATP activate P2 receptors. Stretched cells, including astrocytes, can release their intracellular stores of ATP, which activates ionotropic P2X11C](R)PK11195 Alzheimer's disease (AD) . Similarphagoptosis has emerged. Molecules that are expressed by stressed neurons and known to directly trigger the phagoptotic response include phosphatidylserine and desialylated glycoproteins. Alternatively, opsonins and receptors including MertK, MFG-E8, galectin-3, protein S, and GAS6 can act as intermediaries Phagocytosis and debris clearance is required for maintenance of tissue homeostasis, regeneration, and plasticity but may be detrimental when occurring in excess in vitro and in vivo models that exists to examine various aspects of TAI has contributed to substantial advances in mechanistic knowledge of axonal injury and death pathways cerebral microdialysis and imaging technologies, to be certain that results obtained in TBI models are applicable in clinical settings. Crucial questions remain but the hope is that increased knowledge gained from an improved mechanistic understanding of injuries will translate into effective therapies and improved clinical outcomes.Outstanding QuestionsIs increasing total neuron survival the optimal target to improve outcomes in TAI or are poor outcomes more a failure of function and connectivity at the level of the individual cell or large network?Why is there progressive white matter volume loss following TAI and does microglial phagoptosis contribute?To what degree is primary or secondary axotomy responsible for dysfunction following TAI?Do degenerating axons trigger death in adjacent axons? If so, how is this mediated?s or SARM1 knockout offer protection in model systems of mechanistic or prognostic importance?How does TBI contribute to the development of neurodegenerative diseases ?Why do proteins including amyloid \u03b2 and TDP43 increase following TAI and are these detrimental?Can aspects of the inflammatory (cytokine/chemokine) response be modulated to alter neuronal survival and patient outcomes?What is the role of autophagy in TAI?What is the optimal biomarker for TAI ?Despite increasing interest in and research into TAI, there has so far been a notable lack of translation into efficacious patient therapies"} +{"text": "How much do patients know about everyday matters that can affect their health? A study of 200 patients attending a rural G.P. surgery was undertaken to find out.It revealed some ignorance about the link between smoking and heart disease especially among female smokers (22% for this group). It showed a great deal of ignorance about alcohol in relation to health. Patients had a poor understanding of the relative alcohol content of different beverages, diseases related to excessive drinking and safe limits of alcohol consumption."} +{"text": "Female sex workers have been disproportionately affected with HIV and anal sexual experience elevate their vulnerability. Anal intercourse has more risk of HIV transmission than vaginal intercourse for receptors that coupled with low condom and proper lubricant use behavior during anal sex. Besides majority of them did not understand HIV transmission risk of anal intercourse. In Ethiopia, studies on anal sexual experience is almost none existent, so the purpose of this study is to explored anal sexual experience and HIV transmission risk awareness among female sex worker in Dire Dawa, Eastern Ethiopia.Qualitative study with thematic analysis approach was conducted among 18 female sex workers and recruitment of study participants performed until saturation of information. The principal investigator conducted in-depth interviews using local language (Amharic) and it was recorded on audio recorder. Tape recorded data was transcribed and translated to English and entered into open code version 3.4 for coding and theme identification. Data collection conducted simultaneously with data analysis.Female sex workers practiced anal sex for different themes like financial influence, coercion, intentionally, peer pressure and as a sign of intimacy and love. Coercion, negative attitudes, poor awareness about HIV transmission risks of anal sex and protection capacity of condom and proper lubricants are the identified themes for not using condom and proper lubricants during anal sex by female sex workers. Inaccessibility and unavailability of health services for issues related to anal sex was the core reason for female sex workers\u2019 misperception and risk anal sexual experience.Female sex workers practiced anal sex without risk reduction approaches and they did not understand exacerbated risk of anal sex to HIV transmission. Stakeholders including ministry of health need to incorporate potential awareness raising tasks and programs about risk of anal sex and methods of risk reduction for female sex workers.The online version of this article (doi:10.1186/s41256-017-0047-6) contains supplementary material, which is available to authorized users. Since late twentieth century, HIV has become a major global public health problem particularly in Sub Saharan Africa including Ethiopia . HIV hurHIV transmission risk on per act unprotected receptive anal intercourse is 20 times higher than receptive vaginal intercourse . BesidesStudies conducted in previous years among FSWs in Ethiopia, focused on exploring and determining risk sexual behaviors giving emphasis to vaginal sex , 14\u201316. HIV intervention programs in Ethiopia did not give due emphasis to anal sexual behavior of female sex workers and related risk reduction approaches . The finQualitative study was conducted in Dire Dawa town where HIV prevalence was higher than national level, 4% Vs 1.5% . The priThe first three study participants or seeds were recruited through agents. Then each study participant recruited one or two other FSWs who have anal sexual experience. During the recruitment process, first the recruiter individual would tell new study participants about the study and as the principal investigator would meet them for further information. Then if they became voluntary to participate, the recruiter person would give recruited women\u2019s phone number to the principal investigator. Then the principal investigator met each recruited participant in place, they would be accessed easily and can ensure privacy to talk. Study participants were informed about the purpose of the study and confidentiality of the information they provided. We took informed written consent from each study participant prior to each interview. Each study participant was paid about 13$ USD for compensating the business they loss while they participate in the study which is estimated based on the minimum amount of money they were paid for per act vaginal intercourse, which was at least two times lower than that clients paid for per act anal sex. Besides, we provided extra 4$ USD to recruiter individuals for successfully recruiting one eligible FSW to the study.Then the principal investigator collected data with in depth interviews which, were conducted with local language (Amharic) using semi structured interview guide. Each interview took 1 hour to one and half hour. To increase trustworthiness the principal investigator approached them friendly; moreover we provided brief description about the study for each study participant to enable them speak freely and show interest on the research before conducting the interview. Interviews were performed in place and time that agreed by both parties to ensure safety and privacy for study participants. During interviews, we forwarded crosschecking questions to them to be sure with their responses and consistency of their ideas.The following are some of the operational definitions used in this study.Anal Intercourse/sex: penetrative anal sex by a male sexual partners\u2019 penis or putting his penis in female sex workers anus .Female sex workers (FSWs): is female who had sex for exchange of money in cash .Risk awareness: understand true risk of HIV transmission through anal intercourse or be aware as anal intercourse carry exacerbated risk for HIV transmission than vaginal intercourse .Data analysis was performed with thematic analysis approach and it was executed concurrently with data collection. At the end of each interview we listened the tape recorded interviews repeatedly and then it was transcribed. Then after, we removed jargon data and translated the transcribed data to English. The principal investigator read the translated data repeatedly and transferred in to Open Code software version 3.4., for easy of coding and theme identification. Open coding and themes identification was performed inductively. Then major themes were identified from the existing sub-themes and we used major themes for the final analysis. In subsequent interviews, codes and themes were modified based on the codes and themes from prior interviews or new codes and themes were emerged from the data.Study participants had mean age of 24.8\u00a0years, ranging from 18 to 39\u00a0years. Except one, all study participants had formal education. About ten of 18 FSWs had at least one child. Four of them were peer educators and two women identified themselves, as they were diagnosed positive for HIV. Moreover about ten and five FSWs were venue based and phone based commercial sex workers respectively. Duration of their stay in commercial sex work varies from one to 10 year. Coerced sex and poor decision-making power: clients engaged in anal sex with FSWs forcefully without the will of FSWs. Different situations increased FSWs vulnerability for experiencing coerced anal sex like being drank of FSWs and unsafe hotel or gust houses.\u2026I would not engage in anal intercourse whatever money he paid me. You know why, because the money he gave me might not even cover medical bills for problems happened on me due to my anal sexual experience\u2026 I just engaged in anal intercourse once while I was very drunk. Did you hear me? After that moment, I did not want to try it again. That day, I only remembered the sex we did once and as I slept after that, since I was very drunk. Then I shouted and disturbed the surrounding due to the pain I felt when he inserted his penis in my anus when I was in deep sleep\u2026 There are small hotels here in Dire Dawa where nobody came for help when we (FSWs) shout, I hate spending a minute in these hotels. I feel excited when clients took me to standard hotels, because standard hotels always registered full identification information of their gusts. So these men will go nowhere hurting me\u2026 R (venue based FSW)Intentionally: they practiced anal intercourse to enjoy sexual pleasure particularly among FSWs who developed extreme desire for anal sex due to their repeated anal sexual experience. Besides these women did so to get relief from unpleasant sensation they experienced when they avoided anal sex. Female sex workers also practiced anal sex once they adapt it considering it as part of commercial sex work, though it was not pleasurable for them.\u2026nothing influenced me to practice sexual intercourse from behind; I did it on my will. Even sometimes, I feel ashamed to ask men to have sex with me from behind, when I want to satisfy my sexual desire. I need men to practice sex with me from behind, though I do not request them to do so since I feel ashamed for developing desire for anal intercourse. I never asked men to do so but if any client requested me for anal intercourse, I will accept his offer with pleasure\u2026 H (phone based FSW)\u2026Variety of men, like their skin color, came to us sex workers to engage in sexual intercourse, so there would be some men who wanted to practice anal sex. When they request me for anal sex, I will accept their offer since it is my job to satisfy my clients\u2026 Q (venue based FSW)As sign of intimacy and love: the fourth major theme that FSWs practiced anal sex was to express their intimacy and love to men. They experienced anal sex with specific regular clients and partners for these reasons....When my spouse came home looking tired, he always wanted to have anal intercourse and I did not deny him from doing so since he was my real husband. I should practice sexual intercourse with my husband in a way he want to do with me... F (venue based FSW)\u2026for me, I have about seven regular clients to practice anal sex alone. They have the power to pay whatever I ask them. Mostly I do not engage in anal intercourse with other customers. Actually, I do not like practicing anal intercourse with other men other than those seven men whom I like very much \u2026 N (HIV Positive Street based FSW)Peer influence: female sex workers who never experienced anal intercourse mostly influenced by their peers who have extensive anal sexual experience particularly when they try and practice anal intercourse for the first time.....It was not new for my friends to practice anal intercourse. One of my friends told me as one of the man would pay me well if I will go with him and practice anal intercourse. She influenced me to try it with him and I did it because of her... E (venue based FSW)Nine of the 18 FSWs use condom inconsistently during anal intercourse and three themes emerged to explain their inconsistent condom use behavior. The first theme is poor decision-making power of women and they practiced unprotected anal sex due to objection of their sexual partners for not using condom. These objections from FSWs\u2019 sexual partners vary from coercion and rape to influencing FSWs to accept their offer for unprotected anal intercourse by threatening them, as they would not pay for the vaginal sexual intercourse they already practiced..... He just inserted his penis through my anus when I was in state of deep sleep... Nooooo he did not use, he did not use any condom ... it is mandatory to use condom ... Some standard hotels did not put condom at bedrooms. Actually, I always have condom with me... R (venue based FSW)...mostly men did not want to use condom during anal intercourse. They thought anus would be relaxed and would loss its tightness if they used condom, so they prefer unprotected anal sex since they do not like anus to lose its tightness. Moreover, mostly they do not want lubricates applied on condom... When I told them, as we should protect ourselves from different problems by using condom, you know what they would say, \u2018a real sex is when you do it freely and without condom\u2019.... A (venue based FSW)Pleasure principle: Female sex workers practiced unprotected anal intercourse to enjoy maximum pleasure from their sexual engagement. This because they develop negative attitude towards condom and perceive as condom will decrease their pleasure during sexual intercourse.\u2026Whenever I agree with a client for anal intercourse, primarily I ask them how they will practice it and whether they will ejaculate out of my anus or not. Some clients want to ejaculate inside my anus. If the client agree not to ejaculate inside me, I will offer him what he want. You know why I suggest them to ejaculate outside my anus it is because I dislike using condom since condom causes dryness during intercourse. If you ever tried condom, the lubricant it has would fad up as the intercourse prolonged. Therefore, condom do not give me comfort during intercourse. If they do not ejaculate inside me, I do not care whether they use condom or not or whoever the client is. You know why I dislike condom, the lubricant condom has, has very disgusting smell and I hate it. I did not use condom mostly\u2026 E (venue based FSW)Misperceptions about condom: female sex workers believe using condom during anal sexual intercourse is not necessary and they did not use it. They thought so in two scenarios in which either they do not aware the risk of HIV transmission through anal intercourse or since they believe as they are no more at risk of HIV though they practiced unprotected anal sexual intercourse.\u2026 My clients tell to me, as anal intercourse do not transmit any disease including HIV. Mostly I do not use condom during anal intercourse since I know, as there is no risk of HIV from practicing unprotected anal intercourse \u2026 C (venue based FSW)\u2026 I only fear not to be pregnant whenever I went with a man to practice sexual intercourse. I do not fear other issues; even I do not care about HIV since I am already infected with HIV. I am taking anti retro viral drugs currently\u2026 N None of the study participants used proper lubricants though about 11 of them used petroleum-based lubricants like Vaseline consistently. About three themes are identified to explain why FSWs did not use compatible lubricants during anal intercourse.Lack of awareness about lubricants: it is the first theme that enable FSWs to practice anal sex without proper lubricant. They did not use proper lubricants since they did not aware its advantage for minimizing HIV transmission risk during anal intercourse and the proper type of lubricants. Petroleum-based lubricants are the commonly used lubricants for anal intercourse by FSWs and this is because they misunderstand petroleum based lubricants as proper type of lubricant and they do not aware their adverse effects. Moreover, they also perceive as condom has adequate lubricant for practicing anal sex and misperceived as additional lubricant is not necessary if they practice anal sex with condom. Lack of awareness about proper lubricant use during anal sex among FSWs attributed to poor health information dissemination tasks from health professionals on issues related to anal intercourse. Besides FSWs themselves did not use opportunities to discuss with health service providers about proper lubricant use when they attended health facilities for other services.\u2026condom itself has lubricant and I think using condom is enough. Since I use condom always, I never thought to use additional lubricant \u2026 D (venue based FSW)\u2026 We use Vaseline adding it on the condom, because condom will lose its lubricating capacity gradually during intercourse. The anal mucosa is sensitive and easily traumatized, the Vaseline that will be added on the condom will lubricate anus. Mostly, I use Vaseline as lubricate\u2026I never thought about side effects from using Vaseline\u2026 B (phone based FSW)Negative attitude about lubricants: Some of FSWs are not interested to use any type of lubricant though they aware the advantage of using additional lubricants with condom. The negative attitude of FSWs for lubricant raise from their previous experiences, fear of side effects and misperceptions....I heard from my friends, as there is lubricate that can be used during anal intercourse... Nevertheless, I did not use it... Even the lubricant of condom is difficult for my vagina, give alone using lubricants that I did not know well... G (venue based FSW)Coerced anal sex: female sex workers practiced anal intercourse without proper lubricants in times they experienced anal intercourse due to rape. They were not able to use lubricants in such scenarios since they were not able to deal with their sexual partner and convince their sexual partners to use proper lubricants.... I never use any thing as lubricant. Sometimes men might lubricate their penis with saliva and before inserting it on me. Men know as, anal sex is painful since they can observe my suffering on my face while we practice it. If the man understand my suffering and believe as I have no power to oppose him from practicing anal intercourse, at least he will lubricate his penis with saliva... A (venue based FSW)Female sex workers understand risk of HIV transmission through anal intercourse in three schemes. The first group of FSWs perceived anal sex as HIV risk free sexual behavior. They believe as, there should have contact between sperm and vaginal cavity to transmit HIV during sexual intercourse....how HIV could be transmitted through anus? How anus will have contact with vagina and uterus and transmit HIV; because anus is located on behind in opposite to vagina and uterus, which are located on the front. I think HIV does not transmit through eating together and practicing anal intercourse with HIV positive person, the two have no difference... F (venue based FSW)The second group of FSWs perceived as there is risk of HIV transmission through anal intercourse but they do not aware its exacerbated risk for HIV transmission. They perceive risk of HIV transmission during anal intercourse is either minimal or comparable with HIV transmission risk through vaginal intercourse.... Many individuals thought as HIV transmit only through normal sex, the route that God allow for us to practice sexual intercourse. However, to my level of understanding, anus is also part of my body so it can transmit HIV like the one it will be transmitted through vaginal intercourse... I think both vagina and anus had equal chance for transmitting HIV... I (phone based FSW)The third group of FSWs aware about the elevated risk of HIV transmission during anal sex. They thought the exacerbated risk of HIV transmission during anal intercourse is attributed to high vulnerability of anus for friction and trauma due to thin layer and tight nature of anus. Beside this fragility of anal mucosa during intercourse precipitated with lack of anal secretion.... HIV can be transmitted through anal intercourse more than it can be transmitted through vaginal intercourse. There is more friction and more pain during anal intercourse than during vaginal sex since anus is very fragile and has a very thin wall. I think anal mucosa is the one that can be traumatized and ulcerated easily than vaginal mucosa. When we see vagina it produce its own secretion during intercourse. There is no secretion from anus during anal intercourse... A (venue based FSW)Financial influences were one of the major themes that FSWs engaged in anal sex in this study. These financial influences raise from either financial problem or high payment for anal intercourse. Provision of extra money was a common reason for numerous FSWs to practice anal intercourse in other studies too , 20. FinCoercion and limited decision-making power of female sex workers to forbid clients from practicing anal intercourse was the other theme that FSWs practiced anal intercourse. Clients forcefully practiced anal sex with FSWs in other studies too , 17, 23.When clients forced FSWs to practice anal intercourse, they did not use risk minimization methods like condom and proper lubricants. Other studies discussed that FSWs were less likely to use condom if they practiced anal intercourse forcefully . Even inHalf 9/18) of study participants did not use condom consistently during anal intercourse. This finding is consistent with study conducted in North West Ethiopia and in India, which discussed as only around half of FSWs were using condom during anal intercourse of study. NeverthNegative attitudes towards condom and misperceptions on importance of condom during anal intercourse were discussed by FSWs for practicing unprotected anal intercourse. They misperceived significance of using condom during anal intercourse due to lack of awareness about risk of anal intercourse for HIV transmission. This finding is supported by study conducted in Kenya where female sex workers practiced unprotected anal sex due to their negative attitude towards condom . Poor heSimilarly, none of the study participants used proper lubricants during anal sex and Vaseline was the commonly used lubricant. Lack of awareness about lubricants, negative attitude for lubricants and misperception as condom is sufficient protection measure during anal intercourse are the major themes for not using proper lubricants by female sex workers during anal sex. They lack awareness on the proper types of lubricant to be used during anal sexual practice and adverse effects of using petroleum based lubricants.These themes are routed from not understanding the role of proper lubricant in minimizing the elevated HIV transmission risk during anal intercourse. Poor knowledge of FSWs about advantage of proper lubricants for anal sex precipitated with their lack of health services including information dissemination about proper lubricants. Give alone health services provided at the grass root level of the health system, distribution of proper lubricants is not intended by government in its 5 year national HIV/AIDS prevention strategic plan from 2015 to 2020 , 13, 31.Female sex workers inadequately understand HIV transmission risk of anal intercourse. Majority (13/18) study participants do not understand its exacerbated risk for HIV transmission and most of them (9/13) did not aware as HIV can be transmitted through anal intercourse. The worst scenario was when peer educators and HIV positive FSWs who are under HIV care and treatment did not understand risk of HIV transmission through anal intercourse. They do not attend health services for issues related to anal intercourse. Beside, health professionals do not address these issues during their discussion with FSWs about HIV. Therefore, FSWs perception on anal sex related issues including on risk minimization approaches are mannered on their experience and the information they got from their clients and peers.Misunderstanding risk of HIV transmission through anal intercourse among FSWs is commonly discussed in other studies as well. Female sex workers discussed about non-HIV related risks of anal intercourse rather than risk of HIV transmission when they were asked about possible risks of anal intercourse in studies conducted in Uganda and India , 32. MorThe study has certain limitations. Though we tried to increase its trustworthiness through different approaches, we did not used participant checking to get confirmation from participants on the analysis of the data collected from them. Since anal sex is not a socially accepted behavior, there might be social desirability bias on the information gained from study participants. Therefore readers need to consider these things while they use this article.Female sex workers practice risk anal sex for different reasons without protective measures and they do not understand its exacerbated risk for HIV transmission. This will hinder the countries and global plan on fighting against HIV. So, we recommended Dire Dawa city administration health bureau, Ethiopian federal ministry of health and other stakeholders to consider anal intercourse as one route of HIV transmission and incorporate appropriate intervention programs including behavioral change communication activities for these group of population. Moreover, we recommend further research on estimating contribution of anal intercourse for new HIV infection at national level and innovative approaches of HIV prevention on female sex workers who practice anal intercourse that might inform policy and implementation."} +{"text": "Recently, Peter Jansen from the Department of Gastroenterology and Hepatology and colleagues published a comprehensive review about the mechanisms leading to cholestatic liver disease . A key For toxicologists the perhaps most important lesson learned from this review is that cholestatic liver disease may have an ascending and a descending pathophysiology. For example primary sclerosing cholangitis and primary biliary cholangitis begins with early lesions 'downstream' in bile ducts which leads to bile salt-mediated injury 'upstream' in liver parenchyma. In contrast, most forms of drug induced cholestasis have a descending pathophysiology, where damage of hepatocytes represents the initial key event.Considering this classification it may be justified that in vitro systems to identify hepatotoxic compounds focus on hepatocytes primarily causes damage to bile ducts, while parenchymal damage occurs as a secondary event (Fickert et al., 2007[Therefore, insufficient sensitivity in currently performed in vitro screens for hepatotoxicity may be a consequence of neglecting compounds acting by an ascending pathophysiology, where cholangiocytes and not hepatocytes represent primary targets."} +{"text": "Chest pain is a leading reason patients seek medical evaluation. While assays to detect myocyte death are used to diagnose a heart attack , there is no biomarker to indicate an impending cardiac event. Transcriptional patterns present in circulating endothelial cells (CEC) may provide a window into the plaque rupture process and identify a proximal biomarker for AMI. Thus, we aimed to identify a transcriptomic signature of AMI present in whole blood, but derived from CECs. Candidate genes indicative of AMI were nominated from microarray of enriched CEC samples, and then verified for detectability and predictive potential via qPCR in whole blood. This signature was validated in an independent cohort. Our findings suggest that a whole blood CEC-derived molecular signature identifies patients with AMI and sets the framework to potentially identify the earlier stages of an impending cardiac event when used in concert with clinical history and other diagnostics where conventional biomarkers indicative of myonecrosis remain undetected. While efforts to identify and reduce risk factors for atherosclerotic heart disease remain the focus of primary prevention, the inability to accurately and temporally predict acute myocardial infarction (AMI) impairs our ability to further improve patient outcomes3. The current diagnostic evaluation for the presence of coronary artery disease relies on functional testing, which detects flow-limiting coronary stenosis, but it has been known for decades that most lesions underlying AMI are only of mild to moderate luminal narrowings prior to acute plaque rupture and not obstructing coronary blood flow6. Accordingly, there is an urgent need for improved diagnostics of the underlying arterial plaque dynamics, fissure and rupture8. Increased numbers of circulating endothelial cells (CEC) are known to be present not only in patients with AMI but also with unstable angina \u2013 marked by the absence of traditional biomarkers of myonecrosis - and may provide a window into the pathophysiologic state preceding an acute atherothrombotic event and the development of myonecrosis10.Despite the significant reduction in the overall burden of cardiovascular disease (CVD) over the past decade, CVD still accounts for a third of all deaths in the United States and worldwide each year4. Prior to plaque rupture most atherosclerotic plaques responsible for acute coronary syndromes are not physiologically significant and there is no current diagnostic modality for accurate identification of unstable plaques11. Differential gene expression patterns of leukocytes have previously been used successfully in the assessment of stable coronary disease14. Additionally, microarray-derived gene expression patterns in whole blood and PMBCs of patients presenting with AMI have been studied, and CEC-specific gene expression has been examined in patients with metastatic carcinoma and systemic sclerosis21. However, the prior studies in AMI were limited by their size and predictive ability. Here, we focus on CECs as a potential source of gene markers for AMI given their temporal elevation in the peri-plaque rupture process. Elevated numbers of CECs have been implicated by our group and others in the pathophysiology leading to acute myocardial infarction25. In fact, while absent in stable angina, increased CECs have been noted not only in AMI, but also in unstable angina, a condition of plaque instability without elevated biomarkers of myonecrosis 9. Additionally, CEC elevations during AMI are known to be completely independent of the traditional measurements of troponin and CK-MB10. Thus, our primary motivation for initiating our study of gene expression of CECs is that they may be regarded as a biomarker temporally preceding myonecrosis and a transcriptomic signature derived from these cells, and detectable in whole blood, may provide the key to earlier identification of AMI.The transition from stable atherosclerotic disease to a ruptured plaque with acute thrombo-occlusive disease is multifactorial and has been the subject of great study. It is thought to involve a combination of physical and biochemical factors10. The median CEC count was elevated in AMI patients with 82.5 cells/mL whereas the median for healthy volunteers was 9.5 cells/mL (p\u2009<\u20090.0001 by Mann-Whitney) and healthy control volunteers (n\u2009=\u200928). CECs were enriched from whole blood using CD146+ immunomagnetic separation and enumerated using the CellSearch system as previously described26. CMPs have previously been shown to be associated with an increased risk of CVD and adverse cardiovascular clinical outcomes in patients with known CAD possibly by promoting procoagulant and inflammatory pathways29. In this group of AMI patients (n\u2009=\u200914) and healthy volunteers (n\u2009=\u200914), CMPs were elevated in AMI , using novel AC electrokinetic methodology previously utilized in the oncology space, as an additional and independent marker for AMI in a separate subset of patients10 and gene expression determined via microarray. Markers were initially filtered based on biological function (see Methods) in order to account for expression differences correlated with co-morbidity differences in our cases vs controls not necessarily indicative of the presence of AMI. Initial marker discovery was performed with elastic net regression in a discovery set of enriched CECs from healthy control volunteers (n\u2009=\u200922) and AMI patients (n\u2009=\u200921 (Table\u00a0\u22126), but was less influential on the overall discriminative model (coefficient 0.0283). A model built around the expression levels of these 11 genes effectively discriminated myocardial infarction from healthy control as illustrated in ROC-curve analysis . As a broad assessment of the general gene pathways altered during AMI we also conducted a gene set enrichment analysis (GSEA) on the microarray data , platelet aggregation and GPCR1 ligand signaling , are highly upregulated in AMI.We next replicated this 11-gene model in a separate cohort of control volunteer n\u2009=\u200925) and AMI patient n\u2009=\u200923) samples acquired, processed and sent for microarray analysis independently of our discovery cohorts (Table\u00a05 and AMI13) Fig.\u00a0. It shou samples Following the designation of 11 candidate genes on microarray gene expression analysis of enriched CECs as markers for AMI, we asked if the top performing genes in this molecular signature could be assessed directly from whole blood. By examining the whole blood gene expression patterns we would obviate the specialized cell sorting done prior to microarray. To this end, RNA was isolated from whole blood of the same patients (control and AMI) utilized in the microarray replication study (above) with the addition of 14 new AMI patients following RBC lysis from which cDNA was prepared for qPCR analysis (n\u2009=\u200944 AMI and 29 control) Table\u00a0. An impoThe expression levels for many of the original genes determined in enriched CEC microarray remained significantly elevated in whole blood samples of patients with AMI compared to healthy control volunteers Fig.\u00a0. HeparinFinally, given the differences in age, sex and co-morbid diseases apparent in this first cohort of healthy controls compared to AMI patients we validated this gene expression model in a completely independent cohort of patients presenting with AMI (n\u2009=\u200945) as compared to a new cohort of age and sex-matched control patients (n\u2009=\u200936) Table\u00a0. The maj31. Our ultimate goal is to identify a simple, whole blood molecular signature that would not rely upon the endpoint of AMI and myocardial cell death but rather reflect the underlying acute biologic process leading to atherosclerotic plaque rupture and AMI. Here we present the initial steps towards that goal in the designation of a robust gene-based molecular signature for the identification of AMI. We began our search in a specific population of cells, circulating endothelial cells (CEC), that have been identified in increased numbers not only in patients with AMI but also in patients with unstable angina who have not yet manifested biomarker evidence of myonecrosis32. As such, CECs can be considered a potential signal of the active peri-plaque rupture process that eventually leads to acute atherothrombotic occlusion of the entire vessel and AMI. While our prior work had validated the findings from Mutin et al. and introduced a novel method for identifying and enumerating CECs, we sought to move beyond enumeration and fully characterize the transcriptome of CECs from patients with AMI so as to generate a specific molecular gene signature that would effectively differentiate AMI from control9. These findings may prove useful for future advances in the discovery of diagnostics for an impending acute coronary syndrome, which will require prospective assessment in at-risk patients who present to an acute care setting with chest pain, suspect AMI, but do not exhibit biomarker signs of myonecrosis.In the acute setting, the diagnosis of AMI relies upon detecting necrotic cardiomyocytes, as reflected by troponin or creatine kinase MB-fraction assays in addition to pathognomonic electrocardiographic changes. Yet each year a number of patients who present to an emergency room with chest pain do not manifest these signs and are discharged, only for some of them to manifest an MI or sudden cardiac death in subsequent daysThe initial phase of this study identified 11 genes upregulated in AMI in samples enriched for CECs as determined by gene expression microarray with excellent discrimination. This 11-gene signature was subsequently replicated in an independent cohort of patients with AMI and control volunteers without a loss of power. However, the performance in this initial phase must be tempered by the fact that these comparisons were carried out in patients on separate extremes of the health spectrum: young volunteers without chronic disease and patients presenting with heart attack \u2013 a design that may increase statistical power if co-morbidity stratification across the cohorts is appropriately addressed. Additionally, the requirement for specialized cell sorting is a barrier to translating this finding to a point-of-care diagnostic setting.Accordingly, we then asked if the expression profiles of these genes could be detected from whole blood using qPCR. In whole blood, seven of these genes showed continued expression differences that when analyzed using the elastic net remained significant to the combined molecular signature for discriminating AMI. We observed model coefficient variability depending upon the comparison being made; AMI vs healthy controls or AMI vs age-matched disease controls. However, while the coefficients vary in effect size, their predictive power is conserved and was validated across the different comparisons, as demonstrated by the ROC curves where training and testing were performed in disparate cohorts. Further, supporting the non-reliance of this signature on myonecrosis was that the performance of the seven genes of the signature remained unchanged if not marginally superior in a subset of patients presenting to a single center that had no elevation of their cardiac specific biomarkers at the time of presentation.The determination of candidate genes from microarray analysis was completed by comparing the gene expression dynamics of two very separate populations, healthy controls and patients having AMI. The age and sex differences in addition to the dissimilarities of underlying co-morbid disease or medications of these populations could partly have magnified the discriminative ability of the original 11-gene model in initial testing. The initial AUC values we report in the discovery and validation cohorts in microarray analysis may reflect this magnified discriminative power. However, we would argue that any biases that are not reflective of AMI status would dampen the predictive power observed in our final age and sex matched validation cohort. We addressed this possibility in our final qPCR analysis of the 7-gene model in whole blood using an age and sex matched control cohort of patients with cardiovascular disease for which the model was attenuated though remained significantly robust. Also, given the limited sample size for this study, ethnic differences were not explored.33. Additionally, even by using advanced non-invasive imaging tools to identify and then potentially intervening on high risk plaques, those with the greatest potential of rupture or fissure leading to AMI, would not eliminate the majority of future cardiac events34. While gene expression analysis has previously been combined with traditional clinical risk factors to improve determining the likelihood of stable obstructive coronary disease in non-diabetic patients, that classifier does not indicate or predict impending clinical events12. Likewise, several other groups have completed gene expression analysis of whole blood and PBMCs from patients in the setting of AMI to identify the genes with greatest expression differences, but none have reported a similar discriminatory performance as the molecular signature reported here in whether from enriched CEC microarray or whole blood qPCR18.Currently, there exists no biomarker, diagnostic study or advanced clinical decision making algorithms that foretell a plaque rupture event leading to AMI. Physicians have imperfect tools to calculate ten-year and lifetime risk of potential cardiovascular events based on various epidemiologically derived, population-based risk factors including hypertension, dyslipidemia, diabetes mellitus, age and baseline inflammatory markers, but nothing that places this probability on a more temporal scaleWhile the inability to accurately identify patients in an acute care setting destined for heart attacks before they fully manifest is a limitation to our study, it is also the driving force behind this study. The logical next step will be the prospective clinical validation of this CEC-derived, whole blood molecular signature for AMI in a large cohort of patients presenting to acute care settings with symptoms and high clinical suspicion for AMI, but without accompanying ECG or biomarker signs of myonecrosis. However, the seven-gene molecular signature presented herein may indeed provide a window into the biologic underpinnings of AMI that may precede current biomarkers and potentially lead to changes in the way we approach patients with chest pain symptoms in the future.The study population consisted of patients aged 18\u201380 years old of both sexes who presented to one of five San Diego County medical centers with the diagnosis of acute myocardial infarction (AMI). Healthy control patients between the ages of 18 and 35 without a history of chronic disease and diseased control patients (with known but stable cardiovascular disease) of between the ages of 18\u201380 years old were recruited to outpatient clinical centers affiliated with The Scripps Translational Science Institute (STSI) through which Institutional Review Board (IRB) approval for all aspects of this study was obtained. All experiments were performed in accordance with relevant guidelines and regulations. Recruitment of all patients occurred from February 2008 through July 2014, and experiments were conducted with patient samples in phases as separate cohorts. Informed consent was obtained from all subjects in this study. All AMI cases met strict diagnostic criteria including chest pain symptoms with electrocardiographic (ECG) evidence of ST-segment elevation of at least 0.2\u2009mV in two contiguous precordial leads or 0.1\u2009mV in limb leads in addition to angiographic evidence of obstructive CAD in the setting of positive cardiac biomarkers. Our sample sizes were above the calculated threshold of 12-samples at an alpha 0.01, estimated using an established microarray calculator to detect at least two-fold difference with a power of 0.8 and standard deviation of 0.7. This study is registered with ClinicalTrials.gov (NCT01005485).CMPs were isolated from patient plasma using electric current and quantified using a fluorescent microscope with a charge-coupled device camera. Additional details are provided in the Online Appendix.32. The samples were maintained at room temperature and processed within 36\u2009hours of collection. The CellTracks\u00aeAutoPrep\u00ae system was used in conjunction with the CellSearch\u00aeCEC kit and the CellSearch\u00aeprofile kit (Veridex) to immunomagnetically enrich and enumerate CD146+ CECs as previously described35. The enriched CEC samples were analyzed with the CellTracks\u00aeAnalyzer II and the number of CECs in the sample determined. For CEC microarray profiling, the AutoPrep tube with the sample from the CellTracks\u00aeAutoPrep\u00ae system was removed and placed into the MagCellect Magnet for ten minute incubation. With the tube still in the MagCellect Magnet, the supernatant liquid was aspirated without disrupting the ferrofluid bound cells from which RNA was subsequently isolated. For whole blood samples in EDTA tubes leukocytes and cellular debris was obtained for RNA isolation following RBC lysis with Erythrocyte Lysis Buffer .Early after arrival to an acute care setting, arterial blood was collected from AMI patients into both EDTA containing and CellSave tubes in the cardiac catheterization laboratory following the placement of an arterial sheath prior to the introduction of any guide wires or coronary catheters. Prior work has shown no effect of access site differences on CEC acquisitionMicroarray analysis was performed in three separate experiments each with even numbers of cases and controls to minimize potential batch effects. Enriched CEC\u2011derived RNA was isolated using Trizol Reagent . Labeled target antisense RNA (cRNA) and double stranded cDNA using the Ovation\u2122 RNA Amplification System V2 was prepared from enriched CEC RNA samples. Purified cDNA underwent a two-step fragmentation and labeling process using the Encore Biotin Module (NuGEN). The amplified cDNA targets were hybridized to Affymetrix human U133 Plus 2.0 array to assess expression levels of over 47,000 independent transcripts . Following hybridization, arrays were washed and stained before scanning on the Affymetrix GeneChip Scanner from which data was extracted using the Affymetrix Expression Console. Signal intensities from each array were normalized using the robust multichip average expression measure technique.36. Quality controls were conducted with the affy and affyQCReport R packages. A Gaussian mixture clustering of the principal components of the expression data detected eight outliers (five AMI and three control), which were discarded under accession code GSE66360.All microarray data are available from the Gene Expression Omnibus database . The cDNA was pre-amplified using ABI TaqMan PreAmp (Applied Biosystems) and the selected candidate genes were assessed using the qRT\u2011PCR. PCR data of Ct values were exported for further analysis. \u0394Cts normalized by GAPDH were applied entered into an elastic net model to predict acute myocardial infarctionSUPPLEMENTARY MATERIAL"} +{"text": "Clinical trials offer seriously ill patients a chance at receiving investigative therapies containing new or innovative treatments for cancer. These trials can yield extremely valuable information regarding optimal therapies for specific cancers. Yet it\u2019s baffling to consider the fact that less than 5% of adults with cancer participate in clinical trials. And even more baffling is the fact that despite enrollment in clinical trials, almost one in five publicly funded clinical trials in oncology fails to recruit enough participants to provide reliable data.As oncology advanced practitioners, we sometimes care for patients on clinical trials. Those of us who work in academic or community centers are familiar with the rigors and characteristics of clinical trial participation. Still others of us will help patients obtain referrals to clinical trials offered by other institutions. Caring for patients on clinical trials requires adherence to strict guidelines and reportage.Unfortunately, securing adequate patient accrual in phase III trials is difficult. A recent study determined that one-third of Clinical Trials Cooperative Group phase III trials closed with insufficient accrual . BennettThe researchers examined data from 787 phase II/III adult NCTN-sponsored trials that had started between the years 2000 and 2011. The study defined low accrual trials as those that closed with or accrued less than 50% of their target enrollees. The potential predictors were determined from the literature review and interviews with experts; the final predictors were identified using stepwise regression. All statistical tests used for the study were two-sided. Eighteen percent of the NCTN-sponsored trials closed with low accrual (less than 50% of their targets) 3 or more years after initiation of the study. The results demonstrated that a model of 12 trial-level risk factors had calibration for prediction of trials with low accrual and predictor selection strategies . Some ofTrials requiring patients to give a tissue sample or undergo a biopsyTrials in which patients know they are unlikely to receive a potentially new drug or therapy Phase III trials The researchers note that the algorithm developed from the study can help to predict how an NCTN trial might have better success in enrolling participants and how those factors could be incorporated into trial design.Difficulties in achieving optimal patient accrual numbers for adult oncology clinical trials have been an ongoing problem in cancer care. Much of the research in this area previously focused on the deterrents noted by physicians and patients affecting accrual once the trial has opened . The stuThose advanced practitioners working in centers or community practices should be aware of difficulties in patient accrual for selective trials and the need for higher participation to gain adequate information to affect cancer therapy. The algorithm developed by Bennette and colleagues represents an effort to identify predictive factors affecting potential accrual, and incorporation of these factors may aid in optimal trial design. Patients with cancer may have improved outcomes when enrolled in clinical trials. Better design can and should increase those numbers, offering patients improved cancer therapy."} +{"text": "A tendon is a tough band of fibrous connective tissue that connects muscle to bone, designed to transmit forces and withstand tension during muscle contraction. Tendon may be surrounded by different structures: 1) fibrous sheaths or retinaculae; 2) reflection pulleys; 3) synovial sheaths; 4) peritendon sheaths; 5) tendon bursae. Tendons contain a) few cells, mostly represented by tenoblasts along with endothelial cells and some chondrocytes; b) proteoglycans (PGs), mainly decorin and hyaluronan, and c) collagen, mostly type I. Tendon is a good example of a high ordered extracellular matrix in which collagen molecules assemble into filamentous collagen fibrils (formed by microfibrils) which aggregate to form collagen fibers, the main structural components. It represents a multihierarchical structure as it contains collagen molecules arranged in fibrils then grouped in fibril bundles, fascicles and fiber bundles that are almost parallel to the long axis of the tendon, named as primary, secondary and tertiary bundles. Collagen fibrils in tendons show prevalently large diameter, a D-period of about 67 nm and appear built of collagen molecules lying at a slight angle (< 5). Under polarized light microscopy the collagen fiber bundles appear crimped with alternative dark and light transverse bands. In recent studies tendon crimps observed via SEM and TEM show that the single collagen fibrils suddenly changing their direction contain knots. These knots of collagen fibrils inside each tendon crimp have been termed \u201cfibrillar crimps\u201d, and even if they show different aspects they all may fulfil the same functional role. As integral component of musculoskeletal system, the tendon acts to transmit muscle forces to the skeletal system. There is no complete understanding of the mechanisms in transmitting/absorbing tensional forces within the tendon; however it seems likely that a flattening of tendon crimps may occur at a first stage of tendon stretching. Increasing stretching, other transmission mechanisms such as an interfibrillar coupling via PGs linkages and a molecular gliding within the fibrils structure may be involved."} +{"text": "Spontaneous coronary artery vasospasm is one of the important causes of acute chest pain syndromes. The diagnosis of diffuse multifocal spasm can be quite challenging and it could be easily mistaken for diffuse coronary artery disease. The use of intracoronary nitroglycerin can relieve spasm and reveal the real extent of coronary artery disease. Herein we present a case presenting with acute myocardial infarction due to severe coronary artery spasm that had even received fibrinolytic therapy. Multiple narrowing was shown during coronary angiography and the patient was scheduled for percutaneous coronary intervention (PCI). But after intracoronary (IC) injection of nitroglycerin, all of lesions disappeared completely and the diagnosis of coronary spasm was confirmed. He had On admission, vital signs were stable and physical examination was unremarkable except for the presence of S4 and a mild diffuse end-expiratory wheezing.Laboratory data showed mild leukocytosis and a high troponin I level. In echocardiographic evaluation there was mild left ventricular systolic dysfunction with ejection fraction of 45% associated with anterior and anterolateral wall hypokinesia and a mild mitral regurgitation. The patient was scheduled for cardiac catheterization which was 5 hours after receiving thrombolytic therapy.Coronary angiography was performed via right radial access and the findings were: normal left main trunk and left anterior descending artery (LAD), first diagonal (D1) was large with significant midpart narrowing at 2 sites , Arrows,After discussing the situation, we planned for multivessel PCI on D1, OM and PDA. D1 lesion was targeted first but after intracoronary (IC) injection of nitroglycerin, the lesion disappeared completely. The whole study was repeated after IC nitroglycerin administration and the result was patent coronary arteries without any narrowing . So, dia3 Endothelial dysfunction seems to be the most important contributing factor.4 Although patients with coronary spasm generally have a good prognosis even during acute coronary syndromes but the presence of multivessel coronary spasm could be a predictor of adverse prognosis.5Spontaneous coronary artery vasospasm is described as a transient narrowing or occlusion of an artery. It has the incidence of 0.3-3% during coronary angiography and is an important cause of morbidity.6 It could be mistaken for diffuse obstructive atherosclerotic disease7 and result in unnecessary revascularization therapies such as coronary artery bypass grafting (CABG) or PCI.7Multivessel coronary spasm is very uncommon and normal coronary vasculature is present in one third.6The most common manifestations of coronary spasm are related to episodic periods of myocardial ischemia which can lead to myocardial infarction, malignant arrhythmias and even sudden cardiac death in severe forms. Usually the only atherosclerotic risk factor is cigarette smoking.8One of the important issues is to distinguish between spontaneous coronary spasm and catheter induced spasm. Spontaneous coronary spasm can occur in any coronary artery and far from the catheter tip in different lengths. It has irregular border and is eccentric. Finally it is associated with angina, hypotension, arrhythmia, and ST segment elevation in electrocardiogram. In contrast catheter induced spasm is often asymptomatic and occurs at the catheter tip and usually in right coronary artery. Its shape is almost invariably concentric with smooth borders and less than 2 mm in length.7 There are some case reports in the literature that invasive procedures are performed for these patients. Ahooja and Thatai presented a case of diffuse multi vessel vasospasm resulting in CABG because it was misdiagnosed as atherosclerotic obstructive coronary artery disease. The only risk factor they could find was a positive urine screening test for opiates and benzodiazepines, but not for cocaine or its metabolites.2 Another report by Guardado et al was a case of two vessel disease that presented with cardiogenic shock immediately after adhoc PCI due to diffuse coronary spasm. The patient was a 50-year-old man, smoker and hypertensive, with chronic renal failure on routine dialysis, without history of cocaine or other illicit drug abuse.7 It seems that smoking plays a role in multifocal coronary spasm in these cases as well as our case.Diffuse multifocal spasm diagnosis can be quite challenging and could be easily mistaken for diffuse coronary artery disease.Our patient presented with acute myocardial infarction due to severe coronary artery spasm and even received fibrinolytic therapy. Multiple narrowing was shown during coronary angiography and the patient was scheduled for PCI. If IC nitroglycerin was not administered before angioplasty the serial of unnecessary managements for this patient could be completed (e.g. thrombolytic therapy and PCI).It is suggested to administer intracoronary nitrates in every patient with the suspicion of coronary spasm before terminating the diagnostic assessment of coronary arteries. Maybe routine injection of IC nitroglycerin before angioplasty procedure would be helpful in diagnosing some cases of coronary artery spasms. It could help to evaluate the extent of coronary disease better and aid the physician for optimal management and prevent unnecessary high risk interventions. This case highlights the value of intracoronary nitroglycerin injection before angioplasty.Occasionally, vasospasm can occur extensively either in the presence or absence of obstructive lesions and pose a serious therapeutic dilemma. The use of intracoronary nitroglycerin can relieve spasm and reveal the real extent of coronary artery disease. Therefore appropriate use of nitroglycerin and heightened clinical suspicion could impede unnecessary interventions and help in the optimal therapy for these cases.This study was approved by the ethics committee, Shiraz University of Medical Sciences, Shiraz, Iran. Informed consent form was signed by the patient.All authors declare no competing financial interests exist."} +{"text": "Trust is an essential condition for exchange. Large societies must substitute the trust traditionally provided through kinship and sanctions in small groups to make exchange possible. The rise of internet-supported reputation systems has been celebrated for providing trust at a global scale, enabling the massive volumes of transactions between distant strangers that are characteristic of modern human societies. Here we problematize an overlooked side-effect of reputation systems: Equally trustworthy individuals may realize highly unequal exchange volumes. We report the results of a laboratory experiment that shows emergent differentiation between ex ante equivalent individuals when information on performance in past exchanges is shared. This arbitrary inequality results from cumulative advantage in the reputation-building process: Random initial distinctions grow as parties of good repute are chosen over those lacking a reputation. We conjecture that reputation systems produce artificial concentration in a wide range of markets and leave superior but untried exchange alternatives unexploited. Trust problems hamper mutually beneficial exchange across a broad swath of contexts. Trust is an issue whenever exchange requires that one party \u2013 the \u201ctrustor\u201d \u2013 first expose herself to the risk of abuse by the other party \u2013 the \u201ctrustee\u201d. Abuse may involve failed delivery, compromised quality, shirking, theft, physical violence, or disclosure of sensitive information1256Reputation systems can enable exchange when other mechanisms fall short51112131415161018510Here we study an overlooked side-effect of reputation systems: Reputation building exhibits a form of cumulative advantage212223242526272829We tested the emergence of reputation cascades in a laboratory experiment. The experimental protocol was checked and approved by the IRB of Stony Brook University (CORIHS #2014-2787 F). The experiment was subsequently carried out in accordance with the approved protocol. 336 subjects played games in groups of four trustors and four trustees . A game T points for the abusing trustee. Games were played in two trust conditions. In the condition \u201cTrust Problem\u201d, T was 80 or 100 .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations."} +{"text": "The 36Cl data reveal that individual faults typically accumulate meters of displacement relatively rapidly over several thousand years, separated by similar length time intervals when slip-rates are much lower, and activity shifts between faults across strike. Our rates agree with continuum deformation rates when averaged over long spatial or temporal scales but over shorter timescales most of the deformation may be accommodated by <30% of the across-strike fault array. We attribute the shifts in activity to temporal variations in the mechanical work of faulting.Many areas of the Earth\u2019s crust deform by distributed extensional faulting and complex fault interactions are often observed. Geodetic data generally indicate a simpler picture of continuum deformation over decades but relating this behaviour to earthquake occurrence over centuries, given numerous potentially active faults, remains a global problem in hazard assessment. We address this challenge for an array of seismogenic faults in the central Italian Apennines, where crustal extension and devastating earthquakes occur in response to regional surface uplift. We constrain fault slip-rates since ~18 ka using variations in cosmogenic Many areas of the Earth\u2019s crust deform by distributed extensional faulting, not only in low-lying rift settings but also in areas of high topography1234567125Across the central Italian Apennines active e47810w6.2 and October 30th Mw6.6 events that ruined towns and villages around Amatrice and Norcia (in the Provinces of Rieti and Perugia). Some workersA key observation that helps us address this question is that there is a clear asymmetry in the distribution of historical seismicity since 1349\u2009A. D. . The 13436Cl concentration along exhumed faults planes have been used to infer the timing of earthquakes on extensional faults modelling approach to obtain the best fit model for the full post-LGM slip history as well as to estimate confidence intervals on these fits. The novelty of using this Bayesian approach is that it does not require initial identification of slip events from subtle 36Cl variationsAlong several large extensional faults we sampl36Cl concentration with height up a bedrock scarp should vary systematically with the average fault slip-rate the rate of decrease in concentration with depth in trench and (iii) the rate of increase in 36Cl concentration with height on the scarp itself, should all be larger. Thus if faults slipping at different rates are plotted together, we expect an overall \u2018fanning\u2019 pattern of 36Cl profiles to be observed compare well with rates predicted by assuming that the total extension rate (3\u2009mm/yr36Cl concentrations in the top samples at 7 of the 8 sites are consistent (given that weathering precludes sampling the full height) with the maximum 36Cl concentration and hence SRV based on our own sensitivity study that we infer from the 36Cl data further imply that at any given time only a small fraction of the total fault population , faults in the southwestern part of the central Apennines fault array have, over periods of several thousand years, slipped at rates significantly greater than the Holocene average rate while over other, similar length time intervals, these faults have been moving much more slowly or were temporarily quiescent. The overall summed across-strike strain-rate is maintained because when one fault slows another across strike becomes more active, e.g., sites PESC and FRAT and quie411The periods of fault activity documented here are characterised by cumulative slip consistently larger in amplitude (many meters) than that generated by individual earthquakes and our field observations and 3 exWe apply dissipation analysis24\u22121), currently confined to a zone only ~50\u2009km wide on the northeast flank of the mountains, are the explanation for the skew in historical earthquake shaking and may even be interpreted as deformation associated with a \u2018single\u2019 fault system, as previous authors have suggestedFinally, the dissipation analysis can reco36Cl data reveal evidence for distributed deformation across both flanks of the central Italian Apennines but with significant temporal variability in fault slip-rates, and thus earthquake activity, that can be explained by the principal of minimum work. The implication is that the recent concentration of seismic activity on the northeast flank of the Apennines may persist for several thousand years but ultimately represents just one \u2018snap-shot\u2019 of a naturally complex deformational response to regional surface uplift that has, in the past, led to both flanks rupturing in major earthquakes. Slip-rate variability over multiple earthquake cycles can now be quantified and is essential to understand seismic hazard in areas of distributed extensional faulting because short term slip-rates, over the last few thousand years, can be significantly higher than both decadal (geodetic) and longer term geologic estimates may suggest.In summary, the 36Cl profile strongly constrains the slip history and elapsed time (See Detailed site characterisation and 3 watime See . Where atime See yielded in situ-produced cosmogenic 36Cl AMS (Accelerator Mass Spectrometry) targets from carbonate bedrock samples broadly followed the method in ref. 36Cl data were then used to model fault slip histories by embedding the Matlab\u00ae code developed in ref. ave and SRV for each site. The LiDAR and GPR datasets were used to constrain the site geometry parameters scale and measurements caused by earthquakes with magnitudes less than 5.8 were removed (due to incomplete data for these events). The records were projected onto a transect orientated southwest-northeast (225\u00b0) perpendicular to the mean strike of faults in the central Apennines and plotted in 5\u2009km bins along this transect .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations."} +{"text": "Graft Versus Host Diseases (GVHD) is the leading cause of morbidity and mortality after allogenic hematopoietic stem cell transplantation, occurring in up to 75% of patients . AccordiA 63 years old female patient with history of acute myelogenous leukemia underwent unrelated donor graft rejection followed by matched unrelated donor peripheral blood stem cell transplant presented with diarrhea. The patient underwent colonoscopy that showed mildly congested and erythematous colonic mucosa. Biopsy from the colon showed focal crypt abscess, apoptotic bodies, cryptal destruction and laminar propria fibrosis consistent with graft versus host disease grade 2. CT of the abdomen and pelvis was done and showed a bowel wall thickening due to edema involving multiple loops of jejunum, however the mucosa remained enhancing. The patient was diagnosed based on the clinical presentation with severe gut GVHD.Because the patient continued to have diarrhea and work up for infectious diarrhea was negative, a repeated colonoscopy was done and showed a granular mucosa with ulcerations involving the entire the colon. A tubular structure was seen floating in the colon and looked like a worm . It was We concluded that the tubular structure represented a sloughed gut tissue induced by severe gut GVHD."} +{"text": "Deficits in auditory-verbal memory have been reported by the vast majority of published research in schizophrenia and also detected in first episode psychosis (FEP), confirming they are already present at the early stages of the illness. However, the specific neurocognitive constructs underlying defective verbal memory and their neuroanatomical correlates remains poorly understood in schizophrenia spectrum disorders patients.Data on the Rey Auditory Verbal Learning Test (RAVLT), a widely used verbal memory measure that provides a range of performance indexes to evaluate distinct memory processes including: a) acquisition/learning b) sensitivity to interference c) retrieval; d) retention or rate of forgetting; e) and retrieval efficiency was available for 388 FEP patients and 184 healthy controls (HC). In 218 FEP patients and 146 HC, structural magnetic resonance imaging data were analysised using Voxel based morphometry (VBM) toolbox.The FEP group showed significantly lower results on acquisition/learning, delayed recall as well as higher rates of forgetting. They also exhibited a significant sensitivity to retroactive but not proactive interference. We also found significant correlations between bilateral frontal lobe morphometry and proactive interference as well as between right frontal lobe morphometry and retroactive interference. Rate of forgetting was significantly correlated with right occipital cortex morphometry. Those with higher rates of forgetting and proactive and retroactive interference demonstrated further gray matter reductions in frontal and occipital cortical areas.The application of a process oriented approach to the neuropsychological evaluation of verbal memory allows a finer-grained analysis of the neurocognitive constructs underlying defective verbal memory in FEP. The results suggest specific relationships between different neuroanatomical structures and discrete memory processes with these structures playing an important role in verbal memory deficits found in FEP."} +{"text": "A 35 year old male presented to the emergency department with a four day history of a right sided facial droop and expressive dysphasia. His admission MRI brain showed an acute cortical infarct with vasogenic and cytotoxic edema affecting the precentral gyrus of the left frontal lobe, on Fluid Attenuation Inversion Recovery T2-weighted imaging (Figure Admission bloods showed an elevated ESR and anaemia, while blood cultures unexpectedly grew Granulicatella adiacens. A transoesophageal echocardiogram showed vegetations involving the mitral and aortic valves. Both valves were repaired and postoperatively he complained of dyspnoea. A CT pulmonary angiogram was negative for pulmonary emboli but revealed multiple large splenic artery aneurysms, a clinically unexpected but significant complication of infective endocarditis (Figure Granulicatella adiacens is a nutritionally variant streptococcus. To date 29 cases of infective endocarditis due to Granulicatella species have been reported in the literature. Although this species has frequently been associated with complications of infective endocarditis such as emboli, perivalvular abscess and heart failure, there currently are no reported cases of splenic artery mycotic aneurysms due to Granulicatella. The true incidence of splenic artery aneurysms is not known and quoted range is anything from up to 10.4%. It is the third most common intraabdominal aneurysm after abdominal aorta and iliac arteries, and the commonest visceral aneurysm. The exact aetiology is unknown but unlike aortic aneurysms is not thought to be due to atherosclerosis. Splenic artery aneurysms are associated with conditions affecting the liver or portal venous system including portal hypertension, cirrhosis, liver transplantation, conditions affecting the vessel wall such as arteritis, collagen vascular disease, arterial fibroplasia and inflammatory or infectious disorders.Most cases like this one are clinically asymptomatic and discovered incidentally on imaging performed for another purpose. Typically when symptoms are present it is due to rupture and presents with abdominal pain and haemodynamic instability. Although an infrequent complication of infective endocarditis if a splenic artery aneurysm ruptured it is a serious and potentially fatal complication. Splenic artery aneurysms are four times more common in females, half of those that rupture are in pregnant females and have a mortality of up to 90%. Treatment is indicated for aneurysms that cause symptoms, more than 30 mm and in pregnant or young females .The author declares that they have no competing interests."} +{"text": "We describe a patient who underwent nephrectomy for an enhancing right renal mass that was subsequently pathologically confirmed as right renal splenosis. Since renal splenosis is quite rare and has previously been reported only in the left kidney, we did not consider splenosis in our differential diagnosis during the evaluation of the renal mass. Magnetic resonance imaging, as well as radionucleotide scan using 99mTc-labelled red blood cells, has been utilized for identifying ectopic splenic tissue. An elevated index of suspicion must be present in patients with a history of splenectomy or traumatic splenic rupture to avoid undue nephrectomy."} +{"text": "The ability to generalize over naturally occurring variation in cues indicating food or predation risk is highly useful for efficient decision-making in many animals. Honeybees have remarkable visual cognitive abilities, allowing them to classify visual patterns by common features despite having a relatively miniature brain. Here we ask the question whether generalization requires complex visual recognition or whether it can also be achieved with relatively simple neuronal mechanisms. We produced several simple models inspired by the known anatomical structures and neuronal responses within the bee brain and subsequently compared their ability to generalize achromatic patterns to the observed behavioural performance of honeybees on these cues. Neural networks with just eight large-field orientation-sensitive input neurons from the optic ganglia and a single layer of simple neuronal connectivity within the mushroom bodies (learning centres) show performances remarkably similar to a large proportion of the empirical results without requiring any form of learning, or fine-tuning of neuronal parameters to replicate these results. Indeed, a model simply combining sensory input from both eyes onto single mushroom body neurons returned correct discriminations even with partial occlusion of the patterns and an impressive invariance to the location of the test patterns on the eyes. This model also replicated surprising failures of bees to discriminate certain seemingly highly different patterns, providing novel and useful insights into the inner workings facilitating and limiting the utilisation of visual cues in honeybees. Our results reveal that reliable generalization of visual information can be achieved through simple neuronal circuitry that is biologically plausible and can easily be accommodated in a tiny insect brain. We present two very simple neural network models based directly on the neural circuitry of honeybees. These models, using just four large-field visual input neurons from each eye that sparsely connect to a single layer of interneurons within the bee brain learning centres, are able to discriminate complex achromatic patterns without the need for an internal image representation. One model combining the visual input from both eyes showed an impressive invariance to the location of the test patterns on the retina and even succeeded with the partial occlusion of these cues, which would obviously be advantageous for free-flying bees. We show that during generalization experiments, where the models have to distinguish between two novel stimuli, one more similar to a training set of patterns, that both simple models have performances very similar to the empirical honeybee results. Our models only failed to generalize to the correct test pattern when the distractor pattern contained only a few small differences; we discuss how the protocols employed during training enable honeybees to still distinguish these stimuli. This research provides new insights into the surprisingly limited neurobiological complexity that is required for specific cognitive abilities, and how these mechanisms may be employed within the tiny brain of the bee. Apis mellifera) display an impressive visual behavioural repertoire as well as astounding learning capabilities. Foragers rely on visual and olfactory cues identifying rewarding flowers. Being able to recognise informative cues displayed by flowers can be assumed to facilitate fast and efficient decision-making. Indeed, honeybees can be trained to discriminate by an impressive range of visual cues; symmetry . Despite our models\u2019 extreme simplicity, they largely predicted the honeybees\u2019 generalization performances accurately for a majority of the tested pattern pairs. Our simulated bees did fail to generalize when the two test patterns were very similar . HoweverApparently sophisticated cognitive abilities are often seen as a result of an equally complex neuronal architecture. However, here, this view is fundamentally challenged. Despite honeybees having a tiny brain consisting of less than one million neurons (as compared to eighty-six billion neurons in the human brain ), they sst, 2nd optic ganglia), similar to those found in other insect medullas , 2019, 2Due to the difficulties attaining electrophysiological recordings from honeybees during visual learning tasks , 48\u201350 wConsequently, to allow us to compare our model simulation results directly against the empirical honeybee experimental results we make the following assertion: our models\u2019 simulated bee performances for any given experiment are directly correlated to the average Kenyon cell similarity ratio of all simulation trials for that experiment. In this way if a model\u2019s average Kenyon cell similarity ratio for a given experiment were 0.64 then its simulated bee\u2019s overall experimental performance for selecting the correct test pattern would be 64%. It would have been possible to implement a probabilistic \u2018Monte Carlo\u2019 style binary response for the simulated bees to choose either the corrector incorrect test pattern per trial and subsequently calculate the proportion of correct choices (as with honeybees). However, this would have added probabilistic variability, whereas the Kenyon cell similarity ratio values are variant on just the small amount of synaptic noise applied to the lobula orientation-sensitive neuron to Kenyon cell connections , therefore this additional probabilistic step was judged an unnecessary and potentially detrimental complication. The above assertion does have some limitations when assuming a direct comparable mechanism within the honeybee brain see , but nonIt would have been desirable to assess how our models correlated with the honeybees\u2019 relative performances over all of the tested experiments. Each set of the original honeybee generalisation experiments , 20 only"} +{"text": "In vitro, however, especially cells cultured under neurosphere conditions reveal a larger potential and long\u2010term self\u2010renewal under the influence of growth factors. This is rather well comparable to reactive astrocytes in the traumatic or ischemic brain some of which acquire neurosphere\u2010forming capacity including multipotency and long\u2010term self\u2010renewal in vitro, while they remain within their astrocyte lineage in vivo. Both reactive astrocytes and endogenous NSCs exhibit stem cell hallmarks largely in vitro, but their lineage differs in vivo. Both populations generate largely a single cell type in vivo, but endogenous NSCs generate neurons and reactive astrocytes remain in the astrocyte lineage. However, at some early postnatal stages or in some brain regions reactive astrocytes can be released from this fate restriction, demonstrating that they can also enact neurogenesis. Thus, reactive astrocytes and NSCs share many characteristic hallmarks, but also exhibit key differences. This conclusion is further substantiated by genome\u2010wide expression analysis comparing NSCs at different stages with astrocytes from the intact and injured brain parenchyma. GLIA 2015;63:1452\u20131468Here, we review the stem cell hallmarks of endogenous neural stem cells (NSCs) during development and in some niches of the adult mammalian brain to then compare these with reactive astrocytes acquiring stem cell hallmarks after traumatic and ischemic brain injury. Notably, even endogenous NSCs including the earliest NSCs, the neuroepithelial cells, generate in most cases only a single type of progeny and self\u2010renew only for a rather short time As we know, the term \u201cstem cell\u201d is among the most disputed definitions and yet everybody knows exactly what it is Ledford, \u2014reminiscin vitro is an artifact because their cellular in vivo counterparts self\u2010renew only for a little while as they continuously adopt lineage biases. Thus, ESCs are a prime example of stabilizing a fate in vitro, which is rather transient in vivo , but are also able to form specialized cells (differentiation). The prototype examples of this definition are embryonic stem cells (ESCs) that show a virtual unlimited self\u2010renewal, but can also give rise to all the cells of the embryo proper and are therefore pluripotent the first ancestors to appear are the neuroepithelial cells (NECs). They are mostly amplifying the pool of stem and progenitor cells of the CNS initially and adult NSCs Table . They shIn vivo and in primary cultures in vitro most RGCs generate only a single type of progeny, most of them neurons, some glia only, and similar to the NECs around 16.7% generate both neurons and glia , while gliogenesis now prevails position as subventricular zone (SVZ). Only very few regions located mostly in the telencephalon have already a visible SVZ at embryonic stages that comprises however neuronal progenitor cells and is most visible in regions with a large neuronal output that forms predominantly at postnatal stages and along the lateral wall of the lateral ventricle. During embryonic development, the latter region is called ganglionic eminence (GE) at the lateral wall of the lateral ventricles and the secondary neurogenic zone, the subgranular zone (SGZ) of the DG in the hippocampal formation or ischemic brain injury. The first report incorporation or Ki67 immunostaining within the first week after stab wound injury labeled astrocytes and isolation of labeled reactive astrocytes by FACS showed that up to 1 in 18 viable GFP+ cells could form a neurosphere , but each neurosphere generates neurons. Conversely, only one\u2010third of all neurospheres derived from reactive astrocytes generate neurons may resemble endogenous NSCs from the same or different regions.To answer these questions more globally and in an unbiased manner, genome\u2010wide expression analysis should be well suited. While the above review shows key similarities between NSCs and reactive astrocytes in terms of some marker genes Table and funcThe limitation of the few \u201cmarker genes\u201d examined so far becomes immediately obvious when alleged \u201cNSC markers,\u201d such as nestin, Sox2, DSD1, and BLBP, are examined in reactive astrocytes Table . Thus, tSuch genome\u2010wide expression analysis could not only shed some light onto global similarities between these cell types but also help to understand which of these cells are more similar to each other. For example, one may expect that adult NSCs and embryonic NSCs may be most similar given their neurogenic progeny, localization at the ventricle, epithelial hallmarks was used to isolate RGCs from the developing cerebral cortex both at mid\u2010neurogenesis and at the end of neurogenesis (E18) of astrocytes will allow us determining their subtype identity and possibly predicting why some subtypes do not react even to a strong injury while others react by polarization and yet others by proliferation (Bardehle et al.,"} +{"text": "Synonymous rare codons are considered to be sub-optimal for gene expression because they are translated more slowly than common codons. Yet surprisingly, many protein coding sequences include large clusters of synonymous rare codons. Rare codons at the 5\u2019 terminus of coding sequences have been shown to increase translational efficiency. Although a general functional role for synonymous rare codons farther within coding sequences has not yet been established, several recent reports have identified rare-to-common synonymous codon substitutions that impair folding of the encoded protein. Here we test the hypothesis that although the usage frequencies of synonymous codons change from organism to organism, codon rarity will be conserved at specific positions in a set of homologous coding sequences, for example to tune translation rate without altering a protein sequence. Such conservation of rarity\u2013rather than specific codon identity\u2013could coordinate co-translational folding of the encoded protein. We demonstrate that many rare codon cluster positions are indeed conserved within homologous coding sequences across diverse eukaryotic, bacterial, and archaeal species, suggesting they result from positive selection and have a functional role. Most conserved rare codon clusters occur within rather than between conserved protein domains, challenging the view that their primary function is to facilitate co-translational folding after synthesis of an autonomous structural unit. Instead, many conserved rare codon clusters separate smaller protein structural motifs within structural domains. These smaller motifs typically fold faster than an entire domain, on a time scale more consistent with translation rate modulation by synonymous codon usage. While proteins with conserved rare codon clusters are structurally and functionally diverse, they are enriched in functions associated with organism growth and development, suggesting an important role for synonymous codon usage in organism physiology. The identification of conserved rare codon clusters advances our understanding of distinct, functional roles for otherwise synonymous codons and enables experimental testing of the impact of synonymous codon usage on the production of functional proteins. Proteins are long linear polymers that must fold into complex three-dimensional shapes in order to carry out their cellular functions. Every protein is synthesized by the ribosome, which decodes each trinucleotide codon in an mRNA coding sequence in order to select the amino acid residue that will occupy each position in the protein sequence. Most amino acids can be encoded by more than one codon, but these synonymous codons are not used with equal frequency. Rare codons are associated with generally slower rates for protein synthesis, and for this reason have traditionally been considered mildly deleterious for efficient protein production. However, because synonymous codon substitutions do not change the sequence of the encoded protein, the majority view is that they merely reflect genomic \u2018background noise\u2019. To the contrary, here we show that the positions of many synonymous rare codons are conserved in mRNA sequences that encode structurally similar proteins from a diverse range of organisms. These results suggest that rare codons have a functional role related to the production of functional proteins, potentially to regulate the rate of protein synthesis and the earliest steps of protein folding, while synthesis is still underway. As a result, rare codons are generally associated with slower translation rates and are typically considered deleterious, due to their negative impact on high level gene expression [3] and sometimes lower translational accuracy [4]. The conventional view holds that selection favors common codons, which are considered translationally optimal, but a low level of rare codons is incorporated due to random mutational drift and weak selection [5]. However, the distribution of rare codons is non-random: clusters of synonymous rare codons are widespread in the coding sequences of most prokaryotic and eukaryotic species [7]. Clustering would be expected to exacerbate negative effects of rare codons. This suggests that the distribution of rare and common codons may be shaped by selection and plays a functional role in protein production.Most amino acids are encoded by multiple codons, but these synonymous codons are not used with equal frequency. Rare codons generally correlate with lower levels of cognate tRNA, or weaker codon:anticodon interactions [9], solubility [10] and co-translational modifications [11] of encoded proteins, and is hypothesized to regulate targeting of exported proteins [13]. Codon usage can also affect translational efficiency indirectly via mRNA structure effects at the 5\u2019 end of coding sequences [18]. Within coding sequences, an intriguing hypothesis suggests that rare codons may slow translation rate to coordinate proper co-translational folding of the nascent polypeptide chain [23], potentially to simplify the folding energy landscape for multi-domain proteins [25]. Such effects have been observed for in vitro translation reactions of some proteins [26].Supporting a functional role for synonymous rare codons, altering synonymous codon usage has been shown to adversely affect the expression level [27], codon pair bias [28], low sequence divergence between recently duplicated genes , and potentially other unknown sources of synonymous codon usage bias. Moreover, altering synonymous codon usage can affect gene expression in diverse ways [29]. In addition to the effects described above, synonymous mutations can also affect translational accuracy [31], splicing efficiency [33], and introduce undesirable nucleotide motifs such as internal Shine Dalgarno sites [34]. Some large clusters of synonymous rare codons have no measurable effect on protein folding [6]. In addition, even the rarest codons still encode \u22651% occurrences of an amino acid, challenging the identification of statistically significant usage patterns for functionally important rare codons against the background of neutral drift.While previous studies have suggested that synonymous codon usage is functionally important for some proteins, it is not yet clear in which cases codon usage results from selection versus random drift. Efforts in this direction have been stymied in part because many past analyses of synonymous codon usage neglected to account for specific known biases in synonymous codon selection, including the percent GC content at the third nucleotide position of a codon [i) occur in clusters [6], in order to produce larger translation rate changes than a single codon, and (ii) occur at similar positions amongst homologous proteins across the tree of life, as homologous proteins often have similar three dimensional structures [35]. Under this hypothesis, evolution would select for codon rarity at a particular position in an alignment of mRNA sequences without necessarily conserving a specific DNA or protein sequence. To test whether synonymous rare codon clusters are conserved during evolution, we developed a rigorous set of criteria, including an appropriate null model and statistical tests, to analyze codon usage in all open reading frames of 76 diverse, fully sequenced genomes. This analysis revealed a widespread conservation of synonymous rare codon clusters, particularly amongst water-soluble proteins, across diverse species. Most conserved rare codon clusters were found within conserved protein structural domains, rather than between domains. These results indicate that synonymous rare codons are frequently subject to positive selection, and have functional importance across the tree of life.We hypothesized that synonymous rare codons that are important for co-translational protein folding might . Species were selected to span as much of the tree of life as possible in order to keep DNA identity low, as species with high DNA identity may have diverged too recently for synonymous codon conservation to be reliably detected. Protein sequences from these 76 ORFeomes were assigned to homolog families, and the sequences within each family were aligned . To reduce potential false-positive results arising from recent gene duplications , homolog families were trimmed to include only one sequence from each organism was collected for each of 76 diverse eukaryotic, archaeal, and bacterial species with fully sequenced genomes [16] , but does not require fitting to an adjustable parameter.The conserved codon usage patterns we sought to identify in these homolog families are those that do not alter the encoded amino acid sequence, as amino acid sequence changes can alter protein function, binding and/or stability. For this reason, we used the %MinMax algorithm or other, unknown factors. Hence in the second step of the statistical analysis the homolog families with significant rare codon co-occurrence were filtered to remove families where co-occurrence did not differ significantly from co-occurrence found in random reverse translations (RRTs), a Monte Carlo simulation method we developed to control for rare codons that co-occur for reasons other than rarity co-occurrence of rare codon clusters after adjusting for these effects , indicating that these homolog families contain regions of conserved codon rarity.The broad analysis described above determined that rare codon clusters in general show a non-random distribution, with 26% of homolog families showing significant rare codon co-occurrence , indicating widespread conservation of rare codon clusters within the interior of coding sequences.In the third step of the statistical analysis, we filtered the dataset to determine what fraction of rare codon conservation arises due to previously observed conservation of rare codons at coding sequence termini in many species ), perhaps because the analyses used small sets of proteins with solved structures. To broadly test whether CRCCs are enriched at or near the boundaries of protein structural domains, the locations of CRCCs were compared to the locations of annotated SCOP [45] and CATH [46] domains in proteins with PDB structures and domains predicted from gene sequences [47]. Surprisingly, this analysis revealed that CRCCs are significantly under-enriched near domain boundaries . Hence the major function of CRCCs does not appear to be to separate the co-translational folding of entire domains. Instead, CRCCs often occurred at positions where a translational pause would expose a smaller structural sub-domain outside of the ribosome exit tunnel . Crucially, small structural motifs such as these often fold much faster than an entire domain [52], and hence might be more sensitive to the small differences in the rate of appearance of the nascent chain achievable via synonymous codon selection.If CRCCs function to modulate co-translational folding of the encoded protein, we hypothesized that their positions might correlate with the locations of conserved structural features, particularly domain boundaries. Previous investigations of correlations between rare codons and domain boundaries have arrived at conflicting conclusions . The majority occurred inside conserved domains, suggesting that these rare codons are in fact conserved and do not result from neutral drift in regions where amino acid content is non-critical. However, although most CRCCs occurred inside conserved domains, a subset of CRCCs did show a small but statistically significant enrichment outside known conserved domains. This result highlights the complexity and challenges of a truly comprehensive analysis of synonymous codon usage. Going forward, novel computational methods will be required to distinguish between CRCCs with different roles.It has been hypothesized that rare codons are enriched in unstructured regions of proteins due to reduced selection for translational accuracy in these regions , and it has been argued that the fitness effects of synonymous codon changes are too small to result in selection in species with a small population size [59]. However, more recent studies have shown that synonymous codon changes can have phenotypic effects [61] and that synonymous codon usage in eukaryotes is at least partly the result of selection [62].Synonymous mutations were once thought to be neutral. This assumption is the basis of the frequently used K6], however the functional significance has remained controversial, as have the evolutionary reasons . In recent years there has been a growing consensus that rare codons at the 5\u2019 termini of coding sequences are conserved and increase translation efficiency [43]. In contrast, conservation of non-terminal rare codons is still under active debate, in part because of the many origins of codon usage bias, which can make it challenging to distinguish conservation of codon rarity versus other aspects of codon bias. For example, although a previous study identified low average codon usage frequencies within Pfam domain alignments [36], it was not determined whether these codons occurred more frequently than expected by random chance. Further, this study considered only absolute codon usage frequencies, which means that conservation of rare amino acids , which are by definition encoded by codons that are rare in an absolute sense, can lead false-positive results. Likewise, Pechmann et al. analyzed rare codon conservation in several closely-related yeast species using a codon usage metric that compares tRNA supply with demand (as determined by mRNA levels) and found some evidence for conservation [63], although the relatively recent divergence of these species may make it more challenging to detect significant conservation.Beyond synonymous codon selection in general, whether there are detectable patterns of codon usage within ORFs is still an open question. Previous studies have shown that the distribution of rare codons in ORFs is non-random in most organisms . While rare codons in unstructured regions can also function to promote co-translational folding [64], their presence in such locations is also consistent with the hypothesis that rare codons exist due to mutational drift in genome regions under less selection for translational speed or accuracy [53]. The CRCCs identified by our study are not enriched at domain boundaries in human or E. coli coding sequences. If rare codons do function to separate the folding of protein structural units, these foldable units do not necessarily correspond to a defined domain. Our data set serves as a starting point for more detailed structural and functional analyses, including the effects of mRNA secondary structure on translation rate and co-translational folding of the encoded protein.The conservation of rare codon clusters suggests they serve a functional role. Given the diverse effects of codon usage, it is likely that CRCCs have multiple functions. The hypothesis that codon usage modulates co-translational protein folding led to the expectation that the locations of rare codon clusters might be correlated with protein structural features. Studies examining correlations between codon usage and protein secondary structure have identified an enrichment of rare codons in unstructured regions and common codons in conserved, structured regions [65]. These results are consistent with results showing that substituting common codons for rare codons in the sequences encoding bacterial and fungal circadian regulatory proteins adversely affects the circadian clock and cell growth rate [21]. Of note, codon sensitivity in the fungal clock protein FRQ is localized to portions of the coding sequence encoding a predicted intrinsically disordered region (IDR) [64]. The functions of many IDRs include binding to other proteins and nucleic acids, often to regulate proliferation and cell cycle events [66]. Intriguingly, we found that CRCCs are enriched in proteins with binding functions , suggest that synonymous codon usage may provide a mechanism to regulate cell growth and development across diverse species.The association between CRCCs and the processes of growth and development is particularly intriguing, given that the coding sequences of cell cycle-regulated proteins are enriched in rare codons in general [21], and both tRNA levels and the codon usage of highly expressed genes vary with cell growth rate and cell cycle stage [67]. The results reported here should be broadly useful toward the development of a mechanistic understanding of how synonymous codon usage can affect various aspects of protein biogenesis.In conclusion, conservation of rare codons is a widespread phenomenon, and occurs in structurally and functionally diverse protein families. Homolog families with CRCCs were enriched in specific structural and functional categories. CRCCs were more likely to be found in water-soluble nuclear and cytosolic proteins rather than membrane proteins, suggesting a possible connection with domain organization and folding in the cytosol. Proteins with CRCCs are enriched in functions associated with development and cell growth. This association is particularly intriguing, given that codon usage has been shown to affect circadian growth rhythms of an organism [68] constructed for species with fully sequenced genomes. A separate tree was constructed for species from each domain of life using 16S or 18S rRNA sequences aligned using MUSCLE [42]. 18S or 16S rRNA sequences were obtained from the Green Genes [69] or Silva rRNA [70] databases. Certain eukaryotic species were not present in the Silva database and their 18S rRNA sequences were obtained from NCBI. Based on these initial trees, closely related species were removed and the analysis repeated in order to maximize species diversity. Species for the final data set were chosen based primarily on diversity (to include representative species from the main branches of each tree) and secondarily on the significance of the organism . Trees were drawn using Plottree [68] for an unrooted tree. The final 76 species used for rare codon conservation analysis are listed in , and include 24 bacteria, 26 archaea, and 26 eukaryotes.To minimize DNA identity within our dataset, we evaluated phylogenetic trees .For each of the 76 selected species, the set of all annotated protein coding sequences in the fully sequenced genome (the ORFeome) was collected. Most ORFeomes were obtained by downloading the coding sequences corresponding to all protein coding genes in the species genome from the NCBI database. To avoid fragments not corresponding to full reading frames, only those gene sequences with length equal to an integer multiple of 3 were included in the final ORFeomes. If the same gene identifier was associated with >1 sequence , only the longest sequence was used. 41]. Families were edited to remove potential false positives arising from paralogs by including a maximum of one protein sequence from each species. The representative sequence was chosen at random and other sequences from the same species were discarded. For each resulting homolog family, protein sequences were aligned using MUSCLE [42].Families of homologous genes from the 76 ORFeomes were assembled using OrthoMCL [6], which was designed to identify clusters of synonymous rare codons. %MinMax compares actual codon usage to hypothetical sequences encoding the same amino acids using either the most common (%MinMax = +100) or most rare (%MinMax = -100) synonymous codons for the species of origin. To identify clusters of rare codons, %MinMax scores were averaged over a sliding window of 17 codons, and one or more consecutive windows with %MinMax < 0 were considered a rare codon cluster. For each cluster, the \u201cpeak\u201d was defined as the window with the minimum (most negative) %MinMax score.Overall codon usage for each species was determined by counting occurrences of each codon in the corresponding ORFeome. To calculate the relative codon usage along each gene, we used the %MinMax algorithm predictably altered translation rate. Rare synonymous mutations led to an increase the [YK]/[KB] molar ratio, indicating slower translation rates. (B) The geometric mean of tAI values for the same mutations in (A) similarly predicted slower translation rates.((PNG)Click here for additional data file.S3 FigA. Average length of human and E. coli proteins in homolog families with or without CRCCs. B. Length differences do not explain lower percentage of membrane proteins or higher frequency of rare codons (larger average %%Min) in sequences from homolog families with CRCCs. Graphs compare the full set of analyzed human sequences, human sequences from homolog families with CRCCs , and a length-matched control set (similar lengths to CRCCs set but no CRCCs). %TMH = percentage of proteins with \u2265 1 transmembrane helix predicted by TMHMM. Average %%Min = average percent of sequence windows containing RCCs (%MinMax < 0).(TIF)Click here for additional data file.S4 FigE. coli homolog is shown (PDBID 2B0C), color-coded as for Fig 4, to indicate portions of the protein outside the ribosome exit tunnel and able to fold at two rare codon-induced translational pauses. Locations of these CRCCs are indicated on the alignment by arrows. The contact maps indicate amino acids pairs that are in contact (distance \u2264 6 \u00c5) in the three-dimensional structure.Green bars in the heatmap indicates the location of rare codons and p-values for co-occurrence of rare codons in the sequence alignment . The structure of the (TIF)Click here for additional data file."} +{"text": "Exosomes are small vesicles that were initially thought to be a mechanism for discarding unneeded membrane proteins from reticulocytes. Their mediation of intercellular communication appears to be associated with several biological functions. Current studies have shown that most mammalian cells undergo the process of exosome formation and utilize exosome\u2010mediated cell communication. Exosomes contain various microRNAs, mRNAs and proteins. They have been reported to mediate multiple functions, such as antigen presentation, immune escape and tumour progression. This concise review highlights the findings regarding the roles of exosomes in liver diseases, particularly hepatitis B, hepatitis C, liver cirrhosis and hepatocellular carcinoma. However, further elucidation of the contributions of exosomes to intercellular information transmission is needed. The potential medical applications of exosomes in liver diseases seem practical and will depend on the ingenuity of future investigators and their insights into exosome\u2010mediated biological processes. Hepatitis B virus (HBV) and hepatitis C virus (HCV) are two types of viruses that infect the liver and replicate in hepatocytes Liver cirrhosis is the 14th most common cause of death worldwide and is an increasing cause of morbidity and mortality in developed countries Hepatocellular carcinoma is the most common primary liver cancer. Approximately, 80% of HCC cases are associated with chronic HBV or HCV infection and liver cirrhosis Exosomes were first identified in the intracellular production of small vesicles containing specific plasma membrane proteins in maturing mammalian reticulocytes via exosomes and thus restore the antiviral state in hepatocytes. The antiviral response induced by IFN\u2010\u03b1 could be transmitted from LNPCs to HBV\u2010infected hepatocytes via exosomes containing antiviral molecules. Exosomes mediate and enhance the anti\u2010HBV treatment effects of IFN\u2010\u03b1 HBV is a member of the Hepadnaviridae family that exclusively infects hepatocytes Recent studies highlighted the importance of exosomes in cell\u2010to\u2010cell communication via the transfer of proteins, mRNAs and microRNAs Exosomes mediate cell\u2013cell communication In addition to enhancing HCV transmission to hepatocytes, HCV\u2010related exosomes are also involved in the innate immune response and immune escape HCV\u2010related exosomes are also involved in immune escape to treat HCC in rats demonstrated significant suppression of HCC development. The anti\u2010tumour response was mainly mediated by natural killer (NK) cells and could be enhanced by ADMSC\u2010derived exosomes Heat shock proteins (HSPs) are a family of highly conserved proteins More and more studies have revealed the roles of exosomes in liver diseases. The functions of exosomes mainly depend on the cells that receive exosome signals and contents In summary, one category of exosomes may be related to the development of liver diseases and may be valuable in diagnosis. Another category of exosomes plays a role in promoting hepatitis, cirrhosis and HCC. Targeting different types of exosomes may help clinicians to better control liver diseases. However, further studies will be needed to gain full knowledge of exosome formation and function to better utilize their capabilities.All authors confirmed that there is no conflicts of interest."} +{"text": "In addition, patients with WMA showed increased perfusion in the putamen compared with patients without WMA and with healthy controls . The authors suggest that these morphological and functional brain abnormalities could be caused by the systemic mastocytosis and might be related to neuropsychiatric symptoms of systemic mastocytosis patients.In their article entitled \u201cNeuroimaging evidence of brain abnomalities in mastocytosis\u201d Boddaert et al.2. Suspicion of rather prevalent disease (up to 17%)3 of primary aberrant mast cell activation and only limited proliferation, now termed mast cell activation syndrome (MCAS), arose a decade ago. Similar mutational menageries have been detected in systemic mastocytosis and MCAS4 engendering the term of mast cell activation disease (MCAD). Thus, MCAD comprises a heterogeneous group of multifactorial, polygenic (genetic and epigenetic) disorders5 characterized by aberrant release of variable subsets of up to 200 different mast cell mediators together with accumulation of either morphologically altered and immunohistochemically identifiable mutated mast cells due to mast cell proliferation (systemic mastocytosis and mast cell leukemia), or morphologically ordinary mast cells due to decreased apoptosis (MCAS and well-differentiated systemic mastocytosis).Primary mast cell disease, i.e., a disease due to pathologically altered mast cells, has long been thought to be just the one rare proliferative disease of systemic mastocytosis with its several subtypes2. Although we did not yet analyze systematically the occurrence of WMA in our more than 500 MCAS patients we have seen similar alterations in the brain (Fig. The investigation of the prevalence of WMA in patients with the common variant of MCAD, i.e., MCAS, would be the logical completion of the study by Boddaert et al. which, however, has not yet been performed. Our Interdisciplinary Multicenter Research Group on Mast Cell Diseases Bonn is specialized in diagnosing MCAS according to current criteriaain Fig. as repor"} +{"text": "Women in the managerial-engineering group showed fewer musculoskeletal disorders of the upper extremity compared with the other groups and also had significantly stronger handgrip. Our findings encourage us to recommend hand dynamometer testing as a useful diagnostic tool to determine loss of handgrip strength.The aim of this study was to determine if handgrip strength might be used as a diagnostic tool in musculoskeletal disorders of the upper extremities in women working in an industrial environment. The setting was an electronic factory with four groups of women (n = 101) in a factory assembling electronic components. Handgrip strength was measured using a Jamar\u00ae hydraulic hand dynamometer. The study investigated grip strength in managers-engineers, cable wiring, circuit board assembly, integrated circuits women at 90? elbow flexion and 180? elbow extension. Women seeking or receiving medical care for musculoskeletal disorders of the upper extremities or neck showed significant declines ("} +{"text": "ATP) channels are metabolic sensors with channel activity promoted by decreases in ATP and\\or increases in MgADP. The most studied are channels present in cardiac myocytes and pancreatic \u03b2 cells but KATP currents also exist more widely in a range of tissues and organs [ATP channel populations. Kir6.1 is less studied and thought to underlie the current in vascular smooth muscle cells, however it appears to be ubiquitously expressed [ATP currents are also present in endothelial cells but defining their physiological role is difficult; pharmacological tools and global knockout mice will also lead to effects on vascular smooth muscle and nerve endings [ATP channel subunit Kir6.1 in endothelium (eKO) and studied the phenotype of the mice [ATP currents in endothelial cells when studied using patch-clamp [ATP sensitive potassium and the Orai\\STIM1 complex [ATP channels containing Kir6.1 are activated by protein kinase A through direct channel phosphorylation and this is important for the action of vasodilators in smooth muscle cells [ATP currents could be activated in endothelial cells by an adenosine receptor agonist and indeed found this to be the case. In contrast, in eKO mice this did not occur. KATP channels are also directly sensitive to metabolism and this could contribute to the response. Although metabolic sensitivity can be demonstrated for Kir6.1 containing channels, we have viewed them as more prominently regulated by hormonal cellular signalling pathways [A critical question is how is the metabolic signal sensed and transduced by the endothelial cells. We explored one possibility, namely that release of adenosine from ischaemic tissues may bind to the adenosine family of G-protein coupled receptor. It has been established that Kle cells . The A2 ATP channel? KATP channels can clearly influence endothelial cell membrane potential sufficiently to lead to membrane hyperpolarisation and calcium entry. Thus there is the potential for them to be involved in modulating endothelial cell biology more broadly. For example, intracellular calcium in endothelial cells is important for barrier function, immune surveillance for pathogens and angiogenesis [ATP channels as this mechanism could contribute to a range of physiological processes. A second issue is what influence the endothelial channel more widely has in the vasculature. For example, does it shape resting blood pressure in some way or act to prevent endothelial dysfunction? These are all questions that can be pursued using murine models with conditional endothelial deletion of KATP channel subunits.Is there more to be learnt about the role of the endothelial Kogenesis . There aogenesis . In addi"} +{"text": "Pinus sylvestris L.) could be related to changes in ambient ozone concentration when the impact of tree dendrometric parameters and crown defoliation are accounted for. More than 200 dominant and codominant trees from 12 pine stands, for which crown defoliation had been assessed since 1994, were chosen for increment boring and basal area increment computing. Stands are located in Lithuanian national parks, where since 1994\u201395 Integrated Monitoring Stations have been operating. Findings of the study provide statistical evidence that peak concentrations of ambient ozone (O3) can have a negative impact on pine tree stem growth under field conditions where O3 exposure is below phytotoxic levels.This study aimed to explore if changes in stem increment of Scots pines ("} +{"text": "Considerable success was achieved in 2016 to obtain fertile offspring starting with mouse ES/iPS cells, however the specification of human ES/iPS cells into PGCLCs in vitro is still not achieved. Human ES cells will not yield patient-specific gametes unless and until hES cells are derived by somatic cell nuclear transfer (therapeutic cloning) whereas iPS cells retain the residual epigenetic memory of the somatic cells from which they are derived and also harbor genomic and mitochondrial DNA mutations. Thus, they may not be ideal starting material to produce autologus gametes, especially for aged couples. Pluripotent, very small embryonic-like stem cells (VSELs) have been reported in adult tissues including gonads, are relatively quiescent in nature, survive oncotherapy and can be detected in aged, non-functional gonads. Being developmentally\u00a0equivalent to PGCs , VSELs spontaneously differentiate into gametes in vitro. It is also being understood that gonadal stem cells niche is compromised by oncotherapy and with age. Improving the gonadal somatic niche could regenerate non-functional gonads from endogenous VSELs to restore fertility. Niche cells can be directly transplanted and restore gonadal function by providing paracrine support to endogenous VSELs. This strategy has been successful in several mice studies already and resulted in live birth in a woman with pre-mature ovarian failure.Infertile couples including cancer survivors stand to benefit from gametes differentiated from embryonic or induced pluripotent stem (ES/iPS) cells. It remains challenging to convert human ES/iPS cells into primordial germ-like cells (PGCLCs) Making gametes in a Petri dish by directed differentiation of human pluripotent embryonic and induced pluripotent stem cells (hES/iPS) is considered one of the most important goals of stem cells research to help infertile couples attain biological parenthood. Research efforts by several groups across the globe have been focused to use ES cells grown in a Petri dish to differentiate into gametes for almost 3\u20134 decades based on when mouse , 2 and hThere exists an additional novel population of pluripotent stem cells termed very small embryonic-like stem cells (VSELs) in all adult organs including testis and ovary, which can also be differentiated into gametes in vitro. Pluripotent VSELs in reproductive tissues were recently reviewed and reasBeing developmentally\u00a0equivalent to PGCs, which are natural precursors to the gametes, VSELs are attractive alternative,\u00a0pluripotent stem cells in adult gonads to obtain gametes. However, VSELs are not yet widely accepted by the reproductive biologists because of their small size and scarce nature. A recent update on fertility preservation published in September 2017 does notGeijsen et al. isolatedOur group has reported VSELs in adult human and mousVSELs that survive chemotherapy were reported to spontaneously differentiate into sperm since thTo address the above mentioned limitation of possible contamination with mature germ cells, Shaikh et al. documentw) in vitro [Galdon et al. publishein vitro . The train vitro . Elhija in vitro could prin vitro used juvSemen preservation prior to oncotherapy in male cancer patients is advocated for fertility preservation and testicular tissue biopsies are cryopreserved for young pre-pubertal boys . The fieIt is evident from above review that it will be a long time before we could obtain human sperm starting with ES/iPS cells whereas human VSELs need to be differentiated in vitro into sperm. Another major concern is the highly inefficient nature of the process and pregnancy outcome in mice using gametes differentiated in vitro. Our group has further proposed that rather than isolating VSELs from azoospermic testicular biopsies and differentiating into sperm in vitro, a better approach will be to manipulate the VSELs that survive oncotherapy to restore spermatogenesis in vivo . Anand ehttps://clinicaltrials.gov/ct2/show/NCT02041910) but the outcome is still awaited.These results of restoring spermatogenesis by transplanting niche cells\u00a0are ready to initiate clinical trials and will bring about a paradigm shift in our current approach to oncofertility. There may be no need to cryopreserve testicular tissue from pre-pubertal individuals prior to oncotherapy nor to be concerned about infertility as a side effect. Providing healthy niche cells to non-functional testis could restore testicular function. But well-planned clinical studies need to be undertaken to confirm the beneficial effects observed in mice for possible translation to humans. This will involve transplanting autologus mesenchymal cells (from any source including bone marrow) into the azoospermic testis via inter-tubular route to study the effect. Few trials are registered using this approach and have been extensively used to generate primordial follicles using innovative strategies. In fact OSCs were detected and published initially in a landmark paper by Tilly\u2019s group in 2004 . PluripoZou et al. establis\u2018Eggbert\u2019 was born by culturing primordial follicles isolated from newborn mice in vitro using a two-step culture method. The pup however, developed health problems including obesity and neurological abnormalities . Later tAnother alternative is to develop artificial ovary that implies 3D culture of preantral, immature follicles in a scaffold which could be a source of oocytes upon transplantation . This apIt is evident from the above description that research is progressing on several fronts to produce gametes and provide fertility options to women with premature ovarian failure including cancer survivors. Human ES/iPS cells will require more research to reach the clinics whereas a baby has already been born by transplanting autologus mesenchymal cells in the non-functional ovary presumabwhether the transplanted cortical tissue slices are a source of oocytes or do they induce regeneration of the intact non-functional ovary or both.Besides banking oocytes and embryos in adult females prior to oncotherapy, ovarian cortical tissue slices are cryopreserved in young and unmarried girls and also if the cancer is hormone sensitive and oncologists cannot wait to initiate treatment , 100. GeWe believe that the transplanted cryopreserved\u00a0ovarian\u00a0tissue could also regenerate the non-functional ovary. There are several lines of evidence to support this. Oktay et al. reportedThese results in females\u00a0Table are veryIt is intriguing to note that several somatic organs , 122\u2013131Research needs to progress in various directions to make gametes and help infertile couples including cancer survivors to attain biological parenthood. Changing life style has resulted in delayed childbearing and also individuals who undergo gender reassignment require fertility options. Considerable progress has been made and still lot more time is required to obtain gametes from ES/iPS cells for clinical use. Cryopreserved ovarian cortical tissue transplantation has been largely successful however use of cryopreserved pre-pubertal testicular tissue has not yet reached the clinics. At this juncture, present review offers a novel alternative to restore gonadal function from endogenous stem cells by providing them a healthy niche. This is achieved by transplanting autologus mesenchymal cells in the non-functional gonads and gametes will be developed in vivo from endogenous VSELs. Pros and cons of various approaches\u00a0to address infertility are mentioned in Fig. Table\u00a04 VSELs were reported for the first time in 2006 by Ratajczak\u2019s group and are pluripotent stem cells in adult tissues. They have been extensively studied in hematopoietic system and survive total body irradiation in mice similar"} +{"text": "Urinary tract infection (UTI) is an exceedingly common problem prompting seven million office visits and one million hospitalizations in the United States each year (1). Advances in the understanding of both host and bacterial factors involved in UTI have led to many improvements in therapy. While there have also been advances in the realm of antimicrobials, there have been numerous problems with multiple drug resistant organisms. Providing economical care while minimizing drug resistance requires appropriate diagnosis, evaluation, and treatment of urinary tract infections."} +{"text": "Aedes mosquitoes. The data presented in this article propose environmental layers suitable for mapping RVF vector habitat zones and livestock migratory routes. Using species distribution modelling, we used RVF vector occurrence data sampled along livestock migratory routes to identify suitable vector habitats within the study region which is located in the central and the north-eastern part of Kenya. Eleven herds monitored with GPS collars were used to estimate cattle utilization distribution patterns. We used kernel density estimator to produce utilization contours where the 0.5 percentile represents core grazing areas and the 0.99 percentile represents the entire home range. The home ranges were overlaid on the vector suitability map to identify risks zones for possible RVF exposure. Assimilating high spatial and temporal livestock movement and vector distribution datasets generates new knowledge in understanding RVF epidemiology and generates spatially explicit risk maps. The results can be used to guide vector control and vaccination strategies for better disease control.Rift Valley fever (RVF) is a zoonotic disease affecting humans and animals. It is caused by RVF virus transmitted primarily by Specifications TableValue of the data\u2022Vegetation seasonality, topography, soil types and climatic data can be used to understand ecological characteristics of mosquito habitats as a factor for RVF propagation.\u2022Livestock movement patterns can be used to explore the role of animal movement in RVF propagation.\u2022The datasets can be integrated and used to identify risk zones for RVF hence, improve the effectiveness of intervention strategies against the disease.1This article presents datasets used to map exposure of pastoralist to RVF vectors along their migratory routes. 22.12.2Aedes, Anopheles, Mansonia, Culex, Aedeomyia and Coquillettidia. Sampling was done during long and short rains and each sampling site was considered an occurrence point for species distribution modelling as shown in 3We downloaded pre-processed 16-day NDVI and monthly MOD16 Evapotranspiration (ET) time series data for 2001\u20132015 from University of Natural Resources and Life Science, Vienna portal 4The data variables and methods are summarized in We used species distribution modelling technique to map vector habitat suitability. This was achieved by associating the occurrence data with environmental layers resultin"} +{"text": "A 43-year-old female with history of systemic lupus erythematosus, prior cytomegalovirus esophagitis treated with ganciclovir, and long segment Barrett's esophagus (Prague class C8 M9) with high grade dysplasia treated with radiofrequency ablation presented to the hospital with hematemesis. An upper gastrointestinal endoscopy showed multiple esophageal ulcers with active arterial spurting which could not be controlled with endoscopic interventions including placement of hemostatic clips. An emergent angiogram demonstrated actively bleeding saccular dilations (pseudoaneurysms) in the esophageal branches of the lower thoracic aorta as well as left gastric artery for which gelfoam and coil embolization was initially successful. Due to recurrence of massive bleeding, she subsequently underwent emergent esophagectomy and bipolar exclusion. Pathology demonstrated submucosal hemorrhage, esophagitis with dysplastic Barrett's mucosa, and an ulcer containing cytomegaloviral inclusions. We report the first case of arterial bleeding from periesophageal pseudoaneurysms as well as use of angiographic embolization for arterial bleeding in the esophagus. A 43-year-old female presented to the hospital with hematemesis and hemorrhagic shock. She had a medical history of systemic lupus erythematosus (SLE), prior cytomegalovirus esophagitis treated with ganciclovir (DNA negative after therapy), and long segment Barrett's esophagus (Prague class C8 M9) with high grade dysplasia treated with radiofrequency ablation in the past.The patient was resuscitated with fluids and blood products. An upper gastrointestinal endoscopy showed multiple esophageal ulcers with active arterial spurting which could not be controlled with endoscopic interventions including placement of hemostatic clips . An emerShe eventually underwent surgical resection of a necrotic esophagogastric junction and thoracic esophagectomy with bipolar exclusion for recurrent bleeding about 2 weeks later. A cervical esophagostomy was created at the level of the left clavicle. Pathological examination demonstrated submucosal hemorrhage, esophagitis with dysplastic Barrett's mucosa, and an ulcer containing cytomegaloviral inclusions .We report the first case of arterial bleeding from periesophageal pseudoaneurysms as well as use of embolization for arterial bleeding in the esophagus.Upper gastrointestinal bleeding from the esophagus is a common emergent condition in gastroenterology and often presents with hematemesis. Variceal bleeding and esophagitis are common causes of upper GI bleeding while vascular lesions are a relatively uncommon cause , 2. BleeThe exact etiology of pseudoaneurysms in our patient is not clear but we speculate that vasculitis from SLE and recurrent cytomegalovirus may have contributed in its formation. We found only 2 case reports of SLE related pseudoaneurysms of the abdomen in which pseudoaneurysms developed as a sequel to pancreatitis , 4. ThorWhile endoscopy is the mainstay for the diagnosis and management of patients with upper gastrointestinal bleeding, a small cohort may fail endoscopic therapy. Transcatheter embolotherapy is an increasingly common intervention being performed for such patients .Transarterial embolization in the esophagus for nonvariceal bleeding is a very rare intervention with only 5 cases of ulcer related bleeding reported in the literature . There aWe present a rare case of bleeding from periesophageal pseudoaneurysms which were treated with transcatheter arterial embolization. To our knowledge, this entity as well as embolization as a management option has not been described before."} +{"text": "Ethanol is a widely used psychoactive drug whose chronic abuse is associated with organ dysfunction and disease. Although the prevalent metabolic fate of ethanol in the human body is oxidation a smaller fraction undergoes nonoxidative metabolism yielding ethyl glucuronide, ethyl sulfate, phosphatidylethanol and fatty acid ethyl esters. Nonoxidative ethanol metabolites persist in tissues and body fluids for much longer than ethanol itself and represent biomarkers for the assessment of ethanol intake in clinical and forensic settings. Of note, the nonoxidative reaction of ethanol with phospholipids and fatty acids yields bioactive compounds that affect cellular signaling pathways and organelle function and may contribute to ethanol toxicity. Thus, despite low quantitative contributions of nonoxidative pathways to overall ethanol metabolism the resultant ethanol metabolites have important biological implications. In this review we summarize the current knowledge about the enzymatic formation of nonoxidative ethanol metabolites in humans and discuss the implications of nonoxidative ethanol metabolites as biomarkers of ethanol intake and mediators of ethanol toxicity. \u00a9 2016 IUBMB Life, 68(12):916\u2013923, 2016 The consumption of ethanol has a widespread social tradition among many populations worldwide. Whereas moderate ethanol intake has been regarded beneficial to cardiovascular health, chronic alcohol abuse is associated with an increased risk of pancreatitis, cardiomyopathy, liver disease and cancer EtG is formed by transfer of a glucuronyl moiety from uridine 5\u2032\u2010diphospho (UDP)\u2010glucuronic acid to ethanol Fig. . This rein vitro. Among them, UGT2B7 and UGT1A9 exhibited highest ethanol glucuronidation activities Km values of ethanol glucuronidation exceed physiologically attainable ethanol concentrations in vitro studies using recombinant SULTs, members of the 1A, 1B, 1C, 1E and 2A subfamilies are able to catalyze sulfonation of ethanol. Based on its high expression in liver SULT1A1 has been suggested to be a major contributor to hepatic EtS formation in vivo is currently unknown.The human genome encodes for 22 UGTs, which are divided into three subfamilies, termed UGT1A, UGT2A and UGT2B. UGTs are localized at the endoplasmic reticulum with active sites facing the lumen. Each UGT isoform shows a tissue\u2010specific expression pattern. Liver, gastrointestinal tract and kidney express the highest levels of UGT isoforms EtG and EtS exhibit extended half\u2010lives in body fluids as compared to nonmetabolized ethanol and have been used as biomarkers for recent ethanol intake and abstinence monitoring. After a single event of ethanol intake the time frame of detectable serum EtG and EtS exceeds that of blood ethanol by 4\u20138 h in vitro and to cause allodynia in rats after intrathecal administration in vivo remains to be established.Phase II modifications increase water solubility and facilitate excretion of metabolites PEth is formed by transphosphatidylation of phospholipids with ethanol, which was first observed by Alling et al. in vitroin vitroin vivo has not been addressed. Cell culture studies indicate that PEth turnover occurs at slower rates compared to PA indicating that PEth is more resistant to further metabolic conversions 2, PC phospholipase C and PA phosphohydrolase The transphosphatidylation of phospholipids and ethanol is catalyzed by phospholipase D (PLD) Due to slow PEth elimination rates, detection of blood PEth permits verification of ethanol intake even after several days of abstinence. The majority of blood PEth is associated with erythrocytes whereas only a minor fraction can be found in leukocytes and plasma 2+in vitro including Na+/K+\u2010ATPase, protein kinase C, phospholipase C and cytosolic phospholipase A2Because PEth formation occurs at the expense of PA upon activation of PLD it has been suggested that this reaction interferes with PLD\u2010mediated cellular processes in vitro and to accumulate FAEE in vivo after ethanol intake. Among them, highest FAEE levels have been consistently reported in liver and pancreas. Detectable amounts of FAEE are formed within minutes in cultured cells and perfused organs and dose\u2013response relationships have been observed for FAEE synthesis rates and extracellular ethanol concentrations FAEEs are formed through the enzymatic esterification of ethanol with FAs. These ethanol metabolites have been described first by Goodman and Deykin in total body lipid extracts of rats acutely intoxicated with ethanol and were later found in multiple tissues of rodents subjected to acute or chronic ethanol exposure O\u2010acyltransferase (AEAT) transfers acyl moieties from acyl\u2010CoA to ethanol esterifies ethanol with free FAs whereas acyl\u2010CoA\u2010ethanol\u2010nol Fig. 69, 78, FAEE depositions in hair have been proposed as suitable markers for the retrospective detection of alcohol abuse and the monitoring of abstinence 2+ levels ultimately associated with cellular dysfunction and cell death in vitro and in vivoSince the identification of FAEE in organs commonly damaged by ethanol such as heart, brain, pancreas and liver, an increasing number of studies linked FAEE formation to ethanol toxicity. Toxic cellular effects ascribed to FAEE include inhibition of cell proliferation, destabilization of lysosomes, mitochondrial depolarization and induction of apoptosis in vitro studies. The characterization of enzymes and signaling pathways mediating effects of nonoxidative ethanol metabolites in vivo is thus inevitable to improve our understanding of nonoxidative ethanol metabolites as biomarkers and bioactive molecules.Although the quantitative contribution of nonoxidative pathways to human ethanol metabolism is low, the resultant metabolites have important analytical and biological implications. The detection of nonoxidative ethanol metabolites in body fluids, hair and neonatal matrices provides a valuable tool for the monitoring and retrospective assessment of ethanol intake. Different elimination rates of nonoxidative ethanol metabolites permit a wide range of analytical time frames for the verification of ethanol intake ranging from hours to months after termination of ethanol consumption. The enzymatic reaction of ethanol with cellular lipids generates bioactive metabolites such as FAEE and PEth, which have been shown to interfere with cellular signaling pathways and organelle function and may therefore contribute to specific manifestations of ethanol toxicity. Although considerable research has been performed regarding the enzymology of nonoxidative ethanol metabolism our current knowledge is limited mainly to"} +{"text": "We are usually able to recognise novel instances of familiar faces with little difficulty; yet unfamiliar face recognition can be dramatically impaired by natural within-person variability. Unless otherwise prompted, naturally varying instances of an unfamiliar face are often perceived as belonging to multiple different people . In Experiment 1 participants sorted naturally varying images of unfamiliar faces into their separate identities; half were told that only two target identities were present (constrained), while half were given no indication of the number of targets (unconstrained). Results indicate that unconstrained participants sorted images into 7.5 identities (mean 5 images per pile). On a subsequent matching test, participants who had performed a constrained sort were more accurate than unconstrained sorters, although matching task accuracy was greater for identities seen in the sorting task than for completely novel faces. To investigate this form of learning, Experiment 2 replicated the design using equally complex non-face stimuli (photographic negative faces from Experiment 1). Results indicate that while sorting and matching accuracy was generally poorer, matching task accuracy was greater for learnt than novel negatives. The implications of these findings are discussed with regards to the importance of within-person variability in developing stable face representations."} +{"text": "Also, the magnitude of improvement from Baseline to Post-Drainage on few specific tests of learning and recall significantly predicted the magnitude of improvement after shunt surgery on the same tests . Results indicate that testing before and after temporary drainage may be useful in predicting which patients are less likely to improve in memory with shunting.Studies of the cognitive outcome after shunt insertion for treatment of Normal Pressure Hydrocephalus have reported widely mixed results. We prospectively studied performance of 60 patients with Normal Pressure Hydrocephalus on a comprehensive battery of neuropsychological tests before and after shunt surgery to determine which cognitive functions improve with shunt insertion. We also administered a subset of cognitive tests before and after temporary controlled drainage of cerebrospinal fluid to determine if change on this brief subset of tests after drainage could predict which patients would show cognitive improvement three to six months after shunt insertion. There was a significant improvement in learning, retention, and delayed recall of verbal memory three to six months after surgery (using paired t-tests). The majority (74%) of patients showed significant improvement (by at least one standard deviation) on at least one of the memory tests. Absence of improvement on verbal memory after temporary drainage of cerebrospinal fluid had a high negative predictive value for improvement on memory tests at 3\u20136 months after surgery (96%;"} +{"text": "He introduced to medicine the terms Szpital Starozakonnych Czyste in Warsaw, called the Jewish Hospital, where until 1911 he was an assistant at the Department of Neurology established by Edward Flatau (1868\u20131932)\u2014creator of the First Polish Neurological School was significant in differentiating neurodegenerative conditions such as AD and senile dementia [In subsequent work he demonstrated similarities and differences between senile dementia and Alzheimer\u2019s disease (AD) . He alsodementia .Teofil Simchowicz\u2019s scientific achievements include as well work on neuroendocrinology . Additio"} +{"text": "We describe a 69-year-old patient with superior altitudinal hemianopia who contentiously denied having any visual impairment after stroke in the lower banks of both calcarine fissures. Although the patient did not produce intentional responses to visual stimuli in the blind fields, he showed reduced reaction times to stimuli presented in the inferior visual fields when they were primed by identical stimuli in the superior blind fields. Furthermore he showed left extinction to the double stimulation and delayed reaction times for left unprimed stimuli in the inferior fields. Based on these findings we discuss the possibility that blindsight and right hemisphere damage might be both necessary conditions for denying bilateral blindness."} +{"text": "Reactive oxygen species (ROS) production and the resultant shift in the cellular redox state can exert a biphasic effect on cellular functions such as survival, migration or proliferation. At high levels, ROS can induce broad DNA and protein damage which can result in cell death, but at lower levels ROS act as signaling molecules that sustain proliferation or activate specific stress-response pathways. The molecular mechanisms underlying the activation of specific signaling pathways and transcriptional programs are not fully understood but one key mechanism is the oxidation of critical cysteine residues in transcription factors which in turn affect DNA binding and transcriptional activity.Redox regulated transcription factors have been implicated in carcinogenesis and tumor progression. The NRF2 transcription factor regulates antioxidant defenses and is sequestered in the cytoplasm by KEAP1, but oxidation of cysteines in KEAP1 leads to the release of NRF2 and the induction of antioxidant and cytoprotective enzymes which enhances chemoresistance of cancer cells .Our recent work demonstrates that the pluripotency transcription factor OCT4 is also directly regulated by the cellular redox state [Glutamine metabolism is upregulated in both ESCs and certain types of cancers , 6. In aOur findings demonstrate that the glutamine availability affects the cellular redox state and in turn regulates transcription factors such as OCT4. Suppression of glutamine metabolism could reduce pluripotency of stem cells and possibly cancer stem cells -although the role of pluripotency transcription factors in malignancies remains controversial- and other redox-sensitive transcription factors that are critical for tumor formation and progression such as NRF2 and p53 could be affected as well.A major unanswered question is how oxidation/reduction of individual transcription factor cysteine residues is regulated. Cysteine oxidation depends on the local electrostatic environment, with positively charged amino acids like arginine or lysine enhancing reactivity. Therefore, even within the same protein, not all cysteines have the same reactivity. Cysteine oxidation is also readily reversible, for example by thioredoxins. A recent proteomic paper has shown that Thioredoxin-1 specifically interacts with a number of oxidized proteins. In lungs exposed to hyperoxia, a method to induce ROS, a total of 17 Thioredoxin-1 interacting proteins were identified [Transcription factors that regulate stem cell function and tumor growth can act as sensors for shifts in cellular metabolism and the redox state. This highlights the complex interplay of metabolic and redox pathways but also provides novel mechanistic insights and therapeutic targets in stem cells or tumor cells."} +{"text": "Type-I CRISPR-Cas systems are abundant antiviral defense systems of bacteria and archaea. The hallmark sequences of these systems are short CRISPR RNAs (crRNAs) that contain spacer sequences which guide an interference complex termed Cascade toward their viral DNA target are located in intergenic regions and 391 (92.7%) are present in coding regions is acknowledged.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Age related macular degeneration (AMD) is a visual disease that affects elderly population. It entails a progressive loss of central vision whose consequences are dramatic for the patient\u2019s quality of life. Current rehabilitation programs are restricted to technical aids based on visual devices. They only temporarily improve specific visual functions such as reading skills. Considering the rapid increase of the aging population worldwide, it is crucial to intensify clinical research on AMD in order to develop simple and efficient methods that improve the patient\u2019s visual performances in many different contexts. One very promising approach to face this challenge is based on perceptual learning (PL). Through intensive practice, PL can induce neural plasticity in sensory cortices and result in long-lasting enhancements for various perceptual tasks in both normal and visually impaired populations. A growing number of studies showed how appropriate PL protocols improve visual functions in visual disorders, namely amblyopia, presbyopia or myopia. In order to successfully apply these approaches to more severe conditions such as AMD, numerous challenges have to be overcome. Indeed, the overall elderly age of patients and the reduced cortical surface that is devoted to peripheral vision potentially limit neural plasticity in this population. In addition, ocular fixation becomes much less stable because patients have to rely on peripheral fixation spots outside the scotoma whose size keeps on evolving. The aim of this review article is to discuss the recent literature on this topic and to offer a unified approach for developing new rehabilitation programs of AMD using PL. We argue that with an appropriate experimental and training protocol that is adapted to each patient needs, PL can offer fascinating opportunities for the development of simple, non-expensive rehabilitation approaches a large spectrum of visual functions in AMD patients. Age-related macular degeneration (AMD) is the leading cause of visual impairments in elderly population in western countries and affects several million of people worldwide. Starting progressively over 50 years, the end-stage AMD results in loss of central vision, usually in both eyes, with retinal scotomas extending beyond 20\u00b0 of diameter appeared. These approaches were quite successful at improving peripheral reading in patients focused on exercises aimed at improving muscle control, eye movements and fixation . Older participants might indeed show smaller training-related improvements because of reduced neural plasticity the retinal lateral connections might be affected by the macular degeneration; and (2) the spontaneous cortical reorganization taking place after the lesion might produce differences in perceptual and training effects in AMD patients with respect to healthy participants. From the electrophysiological and neuroimaging perspectives, spontaneous cortical reorganization in AMD is a controversial topic: earlier electrophysiology studies agreed upon consistent evidence for cortical reorganization after retinal lesion , affordable (it does not require expensive equipment) and comfortable (participants are trained with demanding but not uncomfortable session). Moreover PL does not just produce a temporary increase of performance, but it also promotes neural plasticity and cortical reorganization. Even more, combined approaches coupling PL with brain stimulation (electric or magnetic) promise to reduce the time needed for a significant improvement, delineating scenarios in which few weeks of training can produce long lasting changes in visual functions. They should contribute to develop efficient and appropriate rehabilitation programs to increase visual abilities and therefore quality of life of AMD patients. Figure All authors partipated in the writing of this review.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Enhanced recovery may be viewed as a comprehensive approach to improving meaningful outcomes in patients undergoing major surgery. Evidence to support enhanced recovery pathways (ERPs) is strong in patients undergoing colorectal surgery. There is some controversy about the adoption of specific elements in enhanced recovery \u201cbundles\u201d because the relative importance of different components of ERPs is hard to discern . There is evidence that specific approaches to fluid management are better than alternatives in patients undergoing colorectal surgery; however, several specific questions remain.In the \u201cPerioperative Quality Initiative (POQI) Fluids\u201d workgroup, we developed a framework broadly applicable to the perioperative management of intravenous fluid therapy in patients undergoing elective colorectal surgery within an ERP.We discussed aspects of ERPs that impact fluid management and made recommendations or suggestions on topics such as bowel preparation; preoperative oral hydration; intraoperative fluid therapy with and without devices for goal-directed fluid therapy; and type of fluid. We recommend unrestricted access to clear fluids for oral intake up to 2\u00a0h before the induction of anesthesia to maintain hydration while minimizing the risk of aspiration.We recommend that the clear fluid used to maintain oral hydration contain at least 45\u00a0g of carbohydrate to improve insulin sensitivity (except in type I diabetics due to their insulin deficiency state). We suggest that complex carbohydrate be used when available.We recommend that clinicians avoid administration of intravenous fluids to replace preoperative \u201cfluid losses\u201d in patients who received iso-osmotic bowel preparation provided there was unrestricted intake of clear fluids for up to 2\u00a0h before the induction of anesthesia. There is no evidence that iso-osmotic mechanical bowel preparation leads to adverse effects on preoperative volume status.We recommend against the use of hyper-osmotic or hypo-osmotic bowel preparations prior to surgery since there is no benefit relative to iso-osmotic bowel preparation and there may be adverse effects on preoperative volume status.5.We recommend the application of a hemodynamic framework to guide clinical decision-making during surgery. We have developed such a framework and suggest that the use of intraoperative goal-directed fluid therapy (GDFT) is likely to be safe in the majority of patients undergoing major colorectal surgery. GDFT has little risk, and the use of advanced hemodynamic monitoring equipment may enhance clinical decision-making when compared with the use of conventional monitors.6.We suggest that the advanced hemodynamic monitoring equipment used to guide clinical decision-making intraoperatively be selected based on a combination of surgical patient and institutional factors since such monitoring can minimize both hypovolemia (by promoting therapy in volume responders) and hypervolemia (by restricting therapy in non-responders).7.We recommend that in isolation, intraoperative oliguria should not trigger fluid therapy, as low urine output is a normal physiologic response during surgery and anesthesia. We also recommend that intraoperative oliguria be investigated and that absolute (as opposed to relative) hypovolemia be ruled out.8.We recommend that intraoperative and postoperative anuria warrant immediate attention since anuria is pathological.9.We recommend that fluid management strategies focus on the following: first, identifying if there is a clinical problem that can be solved by fluid therapy and then identifying what fluid and how much is appropriate. Rather than treating every instance of abnormal hemodynamic values , clinicians must establish causation based on available information about the patient and clinical context.10.We recommend that therapy attempt to reverse the most likely cause of a hemodynamic derangement. Absolute hypovolemia may or may not be responsible for observed hemodynamic abnormalities. For instance, stroke volume variation above 13\u00a0% soon after the induction of anesthesia and with the institution of mechanical ventilation should prompt consideration of vasodilation (relative hypovolemia) rather than as the cause of fluid responsiveness. The patient may hence require vasoconstrictors rather than bolus fluid therapy provided clear fluids have been consumed preoperatively and iso-osmotic bowel preparation has been used.11.We recommend the use of buffered isotonic crystalloids for the treatment of hypovolemia in patients undergoing colorectal surgical procedures. We acknowledge that the restrictions on the use of starch solutions are based on extrapolations from the critical care literature.12.We suggest that patients tolerating fluids orally after surgery be given unrestricted access to such fluids as this increases patient satisfaction and as it is likely that intravenous fluid administration offer no added benefit.13.We suggest that the hemodynamic framework utilized intraoperatively be extended into the postoperative period to the extent possible, in situations where patients might benefit from such postoperative monitoring (high-risk patients or those with significant blood loss or complications during surgery).Outcomes such as complication rates, readmissions, and length of stay may be highly variable across different centers conducting colorectal surgery , was invited to participate. In total, 32 experts from around North America and Europe met in Durham, NC, on March 4\u20135, 2016, to iteratively discuss the evidence supporting enhanced recovery paradigms and develop consensus statements with practical recommendations for clinicians.all the experts present at the first POQI meeting.A list of relevant questions was collectively developed and circulated electronically prior to the meeting. Based on literature searches performed by members, questions were formulated. In the first plenary session, the POQI perioperative fluid management subgroup presented these questions to the entire POQI workgroup, to receive feedback and assistance in refining the questions. The subgroup then worked together to formulate answers to these questions, supported by evidence when available and by expert opinion when no clear evidence was available. These were presented in the second plenary session. After receiving feedback, the subgroup refined a series of consensus statements, which was then reviewed with and modified by the entire POQI group in the final plenary session. This manuscript is based on these multiple rounds of feedback from Based on both discussions (held prior to the conference) and the literature (identified by the participants), the following questions were considered most relevant to perioperative fluid management before, during, and after colorectal surgery within an ERP:(i)What are the effects of preoperative oral intake of clear solutions (containing complex versus simple carbohydrates) up to 2\u00a0h prior to the induction of anesthesia?(ii)Does mechanical bowel preparation contribute to preoperative hypovolemia?(iii)Is urine output a valid indicator of perioperative fluid needs?(iv)Is there a rational approach to intraoperative fluid management based on the current evidence?(v)Which types of fluids should be used intraoperatively?(vi)How do variations in surgical and anesthesia technique affect intraoperative fluid management?(vii)How should fluid therapy be managed postoperatively?It has known that both simple and complex carbohydrate-containing solutions prevent protein catabolism following exercise , the oral intake of clear liquids should occur more than 2\u00a0h prior to the induction of anesthesia. ASA guidelines recommend modification of preoperative fasting on an individual basis in the presence of \u201cgastroesophageal reflux disease, dysphagia symptoms, or other gastrointestinal motility disorders.\u201d may lead to hypovolemia due to gastrointestinal losses prior to colorectal surgery, was supported by a study comparing ten subjects randomized to Picolax (magnesium citrate (a hyper-osmotic laxative) and sodium picosulfate (a stimulant)) versus not. There was significantly more orthostasis and tachycardia in the group that received the hyper-osmotic bowel preparation based on transpulmonary thermodilution, after induction of anesthesia. The authors concluded that hypovolemia was likely to be present , the mean cardiac index was 2.66\u00a0L/mModern MBP techniques typically utilize iso-osmotic agents, which in theory do not produce dehydration (no osmotic shift in fluids toward the bowel lumen). When combined with the emphasis on intake of clear fluids up to 2\u00a0h before surgery (see question i) in compliance with the ASA Fasting Guidelines, concerns related to the impact of MBP on volume status are minimal. There is no need for fluid therapy to treat presumed fluid losses from iso-osmotic MBP and starvation. The shift in practice away from empiric administration of fluid therapy toward therapy based on the detection of \u201cfluid responsiveness\u201d has further diminished arbitrary preoperative intravenous hydration. If clinicians are able to rapidly identify hypovolemia intraoperatively, excessive preoperative fluid losses can be detected and managed objectively. Since it has not been established that iso-osmotic MBP predisposes patients to hypovolemia, and since clinicians can identify patients in whom MBP might have produced excessive fluid losses promptly group, is more challenging to interpret as abnormal, especially when other indicators of overall tissue perfusion are normal is a natural response to anesthesia and surgery. The resorptive actions of vasopressin on the collecting duct in nephrons lead to the retention of water with accompanying oliguria\u2014this may not indicate organ dysfunction . Minimally invasive cardiac output monitoring-guided fluid management has been studied in multiple randomized controlled trials in patients undergoing diverse procedures . Several investigators have examined device-guided GDFT in the modern era of ERPs. Three such groups independently tested a \u201czero balance\u201d or \u201crestrictive\u201d strategy against conventional minimally invasive cardiac output monitoring -guided GDFT within the context of colorectal ERPs, and all found no difference in the length of stay or incident complications to crystalloid in critically ill patients\u2014found no difference in the primary outcome of 28-day mortality . Several surgical maneuvers as well as by anesthetic interventions , utilization of thoracic epidural analgesia with local anesthetics) may impact measures of \u201cfluid responsiveness.\u201d Understanding the physiologic implications of these maneuvers can help clinicians contextualize changes in device-based measures and avoid the use of fluid therapy based only on the presence of \u201cfluid responsiveness\u201d without attendant absolute hypovolemia.While Trendelenberg (\u201chead down\u201d) positioning has been used for over a century in an effort to improve hemodynamics by augmenting venous return, its intraoperative use is primarily for better visualization of the operative site. Immediately after placing a patient in the head down position, there is a transient increase in right ventricular preload and stroke volume from increased venous blood flow (from the lower extremities and unstressed compartments). This subsequently leads to increased left ventricular output and cardiac output measurements taken 3\u20135\u00a0min after initiating the head down position show an increase in cardiac index respiratory variation as guides to \u201cfluid responsiveness.\u201dIntraoperative mechanical ventilation may now incorporate PEEP most commonly used for open abdominal surgical procedures. TEA with local anesthetics can leads to hypotension from reduction in venous return (sympathectomy with venodilation) and decreases in cardiac index and calories (through fluids or solid food)). In such fasting patients, replacement fluids must be provided intravenously. With ERPs a change in philosophy has occurred with surgeons often allowing oral intake immediately after surgery\u2014provided there is no active nausea/vomiting. Given unrestricted access to oral fluids, patients can regulate their intake to preserve intravascular volume (as long as their thirst mechanisms are intact). Isolating the impact of this paradigm shift toward early postoperative oral intake is a challenge primarily because of the heterogeneity among various published colorectal ERPs. For instance, in a recent meta-analysis examining the characteristics of 13 colorectal ERPs, 9 of 13 centers allowed MBP, and only 8 of 13 centers protocolized perioperative fluid administration after abdominal surgery is directly related to both length of stay and the incidence of complications ?Is there a clinical outcome difference between simple versus complex carbohydrate loading?https://clinicaltrials.gov/ct2/show/NCT01424150), the focus of which is liberal versus restrictive fluid administration but which plans to examine the effect of GDFT using a statistical test of interaction. However, this study does not specifically focus on GDFT in the context of ERPs.Is a protocolized \u201crestrictive\u201d or \u201czero balance\u201d technique equivalent to GDFT? This may be answered to some extent by the ongoing RELIEF trial (What risk stratification tool(s) best predict outcomes in patients undergoing colorectal surgery and what are the clinical and financial implications of using risk stratification to influence monitoring decisions and hemodynamic management in patients undergoing colorectal surgery?Do colloids offer any benefits over crystalloid for intraoperative GDFT in non-septic patients undergoing colorectal surgery?Are potential benefits of chloride-restrictive electrolyte solutions demonstrable in prospective studies and are there differences within available choices of such solutions ?Iso-osmolar bowel preparation is unlikely to lead to preoperative hypovolemia requiring intravenous fluid therapy provided patients are given unrestricted access to clear fluids orally. In patients that present to the operating room in a hypovolemic state, rapid detection is feasible by dynamic indicators of fluid responsiveness such as arterial (or plethysmographic) respiratory variation. Inclusion of carbohydrates in preoperative oral fluids is likely to improve insulin sensitivity (particular when complex carbohydrates are used) and may reduce protein catabolism. Anuria is abnormal and requires immediate attention. In general, oliguria is common during and after anesthesia and surgery and should trigger diagnostic efforts but not fluid therapy until hypovolemia is established as the cause. Intraoperative fluid therapy should be based on a framework where all available information is integrated to determine if there is a physiologic problem requiring reversal. Low tidal volumes and PEEP alter the sensitivity and specificity of dynamic indicators of fluid responsiveness but have a minimal impact on cardiac index at levels commonly utilized in the operating room. To the extent possible, the approach to intraoperative fluid management should continue postoperatively."} +{"text": "Obtaining adequate venous access is a recurrent problem in patients where long term access is required for the intravenous administration of prolonged antibiotics or parenteral nutrition. Assessment of vein adequacy and patency is clinically important in these patients.Time Resolved Angiography with interleaved stochastic trajectories (TWIST) is a technique that creates a sequential series of multiplanar images during passage of intravenous contrast Figure . TWIST hWe retrospectively reviewed patients who successfully underwent TWIST MR angiography at our centre for the evaluation of central venous patency with a view to obtaining access. 70% of patients were on long term total parenteral nutrition (TPN) for intestinal failure, the remaining 30% were patients with cystic fibrosis requiring long term lines for antibiotic administration).TWIST MRA in parallel with the GeneRalized Autocalibrating Partially Parallel Acquisitions (GRAPPA) algorithm was performed using a 1.5T Siemens Magnetom Avanto MRI scanner. Gadolinium contrast (dose of 0.1 mls per kg), was administered at a rate of 2 mls per second for each examination.All studies were diagnostic and in all cases vascular occlusive disease was confirmed. In 84% of all cases an appropriate site for access was recommended, with successful implementation in 84%. Access was not attempted in the remaining patients. In one patient, the technique was performed to identify a suspected superior vena caval obstruction Figure . One patWe believe that TWIST with GRAPPA parallel acquisition could be used successfully to non-invasively and efficiently image patients with more complex vascular access issues, including those requiring long term total parental nutrition (TPN)."} +{"text": "BioEssays, Hamid and Makeyev explore an interesting new idea regarding the evolutionary origins of regulated mRNA turnover pathways In this issue of The hypothesis is supported by recent results that NMD not only regulates cellular transcripts but also viral RNAs NMD and other regulated mRNA decay pathways need to correctly identify target mRNAs. Interestingly, a similar molecular recognition problem of selecting RNA molecules is faced by the innate immune system. Defence against RNA viruses often involves cellular recognition of viral RNA molecules. This triggers direct effector functions such as degradation or sequestration of viral RNAs as well as the activation of signalling pathways that induce an antiviral state"} +{"text": "Plant\u2010associated microbiomes have tremendous potential to improve plant resilience and yields in farming systems. There is increasing evidence that biological technologies that use microbes or their metabolites can enhance nutrient uptake and yield, control pests and mitigate plant stress responses. However, to fully realize the potential of microbial technology, their efficacy and consistency under the broad range of real\u2010world conditions need to be improved. While the optimization of microbial biofertilizers and biopesticides is advancing rapidly to enable use in various soils, crop varieties and environments, crop breeding programmes have yet to incorporate the selection of beneficial plant\u2013microbe interactions to breed \u2018microbe\u2010optimized plants\u2019. Emerging efforts exploring microbiome engineering could lead to microbial consortia that are better suited to support plants. The combination of all three approaches could be integrated to achieve maximum benefits and significantly improved crop yields to address food security. Increased productivity can also significantly contribute to various other SDGs including SDG 6 (clean water and sanitation), SDG 9 , SDG 13 (climate Action) and SDG 15 (life on land). To meet the food requirement for a global population exceeding 9 billion by 2050, crop productivity needs to increase by 70\u2013100%. Conventional intensive agricultural practices that depend on inorganic fertilizers, pesticides and other chemical inputs have increased yield but also contributed to soil degradation, loss of biodiversity, increased susceptibility of crops to pests/pathogens and negative environmental impacts which, together, have significant consequences for human health and food security modulates the colonization of specific microbes in guts and can restore healthy status has enormous potential to manipulate host microbiomes in order to enhance the effectiveness of disease management. Engineering plant/soil\u2010optimized microbes and plant/soil\u2010optimized microbiomes that can be used as inoculum for different crops in different soils can also be achieved by artificial ecosystem selection. While not yet applied in industrial settings, there is evidence that soil microbiomes adapt to their crops over time leading to improved plant\u2013microbe interactions , emerging microbial\u2010based solutions can potentially transform sustainable agriculture. Given that agriculture has been central to the success of Matthew Wallenstein is a cofounder of Growcentia, Inc."} +{"text": "Management of ambiguous genitalia is highly controversial. This condition was known previously as intersex and presently as disorders of sex development (DSD). There is no consensus regarding the choice, timing and method of sex assignment in neonates with DSD. Consensus conferences could not unify the views of various stakeholders and third parties. This article philosophically examines the nature and origin of such controversies. Misconception, bias and conflicting priorities are identified as the three cardinal sources of controversies. Conceptual duality of sexes, confused notion of sex and gender, bias towards penetrative intercourse, conflict between utopian ideals and reality, unwillingness to compromise are identified as perpetuators of controversies. Suggestions are made regarding sex assignment in various types of DSD based on the understanding of published literature and the author\u2019s personal experience. INTRODUCTIONSex of a newborn is typically assigned at birth on the basis of genital appearance. Therefore, children with ambiguous genitalia frequently require reassignment of sex either because of incorrect original labeling or because of subjective dissatisfaction with the sex of rearing (gender dysphoria). Since sex is a fundamental attribute of human life its reversal after original assignment is fraught with emotional, social and existential turmoil. As many as 65% of parents required psychological support at diagnosis.[5] Consequently considerable disagreement exists regarding the choice, timing and method of sex assignment. Biological complexity of the issue is further complicated by the involvement of advocacy groups and sensationalizing media.[8] These third parties vociferously accuse physicians guilty of genital mutilation and human rights violations. Doctors have even been sued for alleged impropriety of sex reassignments. This volatile atmosphere was feared to adversely affect the wellbeing of affected individuals. Therefore, in 2005, a consensus conference was organized in Chicago to unify the views of various stakeholders.[11] Paradigm shift of the conference was emphasis on sex assignment based on genetic and molecular criteria rather than gonadal function.[12] Old terms such as hermaphroditism, intersex and ambiguous sex were discarded in favour of the newly proposed nomenclature \u201cdisorders of sex development (DSD)\u201d. Unfortunately, even after a decade of consensus statement, controversies refuse to die down. Focusing only on the controversies rather than their origin could be responsible for this vexatious situation. Misconception, bias and conflicting priorities are the three cardinal pillars of any controversy. Sex, being a taboo subject, has no dearth of this evil combination. Complexity of sex reassignment can be better understood if approached in the light of these 3 perpetuating factors. This article is intended to philosophically examine the origin, factuality and possible solutions of controversies pertinent to the management of ambiguous genitalia in newborns.Misconceived Duality of sexesThe fundamental flaw of sex assignment is the conceptual duality of sexes. In fact sex of an individual is determined by a conglomeration of factors such as chromosomal pattern (XX vs. XY), nature of gonads (ovary vs. testis), predominance of circulating sex hormones (estrogen vs. androgen), topographic anatomy of genitalia and secondary sexual characters.[15] Usually genital appearance and phenotype are influenced by sex hormones secreted from gonads which in turn are genetically programmed by chromosomal arrangement.[16] Therefore harmony between various determinants of sex is presumed and individuals are neatly categorized into male or female. Problem arises when there is discordance between the various factors. For example, in complete androgen insensitivity syndrome (CAIS) the individual is chromosomally a male with 46XY and has bilateral testes which secrete androgen; but due to receptor deficiency circulating testosterone fails to effect male phenotype. Consequently the individual will externally look like a female with fully developed breasts and labial folds.[17] Contrastingly, in congenital adrenal hyperplasia (CAH) the individual is genetically a female with 46XX and gonads are typically ovaries; but due to deficiency of steroidogenic enzymes excess testosterone is produced thereby leading to virilization. Therefore, girls with CAH will have fused labia mimicking scrotum and hypertrophied clitoris mimicking penis.[18]. Permutation of sex determining factors (Table 1) suggests that sex is a spectrum rather than two neatly packed compartments.[19] Conventional male and female are at the extremes of the spectrum with innumerable shades of sexes lying between them. Surgical reduction of the enlarged clitoris in CAH and excision of testes in CAIS are basically attempts of trimming the individual to suit one of the two artificial categories.Conflicts of recognizing sex as a spectrumGrowing voices emphasize recognition of individuals as they are. Although this viewpoint is logically and scientifically ideal, it presents enormous conflicts with established social and ethical principles. Recognizing sex as a spectrum will result in chaos. For example, women upliftment programs will face serious setback because of the overlapping definition of females among various shades of sexes. Disrupting the smooth social order of the majority is as equally unethical as neglecting the needs of DSD individuals. A possible compromise of the conflict is to group all intermediary sexes under \u201cthird gender\u201d.[20] Even the Supreme court of India has recently promulgated the constitutional rights of \u2018third gender\u2019.[21] However this concept of third gender may not be rational. Homogeneity of components is a prerequisite of defining a group. It may not be logically tenable to include diagonally opposite conditions such as CAH and CAIS under the same umbrella of \u2018third gender\u2019. Inclusion of transgenders under this third group adds to the confusion as their problems are very different from that of DSD patients. Therefore, until a radical shift occurs in the societal thinking, rigid compartmentalization of sexes as male and female is indispensable. The conflict between societal outlook and individual preference is best resolved by personalizing the decision of sex reassignment irrespective of the underlying DSD. For example, CAH patients may be assigned to either male or female sex depending upon their individual psychosexual inclination and social circumstances. However genital appearance is no guide to decide the sex of rearing. Misconception: Sex versus genderPhilosophically an individual is made-up of body (soma) and mind (psyche). As Harry Benjamin succinctly put it, \u2018Sex is what you see and gender is what you feel\u2019.[22] Both sex and gender are usually concordant in majority of individuals. For example, men behave manly and are attracted towards women while its converse is true of women. This implies that male and female brain must be functioning differently.[23] The greatest blow to the understanding of DSD came when feminists, in their enthusiasm to establish equality of sexes, denied this difference of brain functioning. John Money\u2019s theory of gender neutrality at birth[26] indirectly endorsed the feminist view of equality. Interestingly his theory became popular in 1960\u2019s coinciding with the second wave of feminism. According to him both boys and girls are born without any predilection towards social or sexual role play and their subsequent gender-specific behavior is purely determined by social nurturing. Overwhelming importance given to nurture over nature led to bizarre sex reassignments. Boys with aphallia, micropenis and exstrophy were castrated and feminized [27-30] citing Money\u2019s theory as excuse. Evidence for the fallaciousness of Money\u2019s theory came from his own patient. One David Reimer was born male and he lost his penis in infancy due to a complication of circumcision. Money, confident of his nurture theory, advised him to be brought up as female. Reimer who underwent feminizing genitoplasty was followed up by Milton Diamond.[31] During adolescence Reimer increasingly felt uncomfortable to identify himself as female and he opted for sex reversal operation. Thus nature is proved to prevail over nurture.The exact mechanism as to how the nature determines gender is poorly understood.[32] Preliminary evidences suggest that fetal brain is masculinized by prenatal exposure to androgen.[33] Peak testosterone levels between 8 to 21 week of gestation appears to facilitate androgen imprinting of male fetal brain.[34] Although androgenization of body and brain often concur, discordance is not unknown. For example, failed or defective androgen imprinting of brain could probably explain homosexuality in an otherwise healthy male.[35] Drawing analogy from this hypothesis, high levels of circulating testosterone is believed to cause varying degree of androgen imprinting in CAH.[36] Thus CAH women with fully virilized brain will have male sexual orientation while those with poor androgen imprinting retain their feminine inclination .[36] Using a single yardstick for sex reassignment in these subgroups will not only be inappropriate but also disastrous. Sex assignment is relatively easy when sex and gender are congruent than when they are discordant with each other. For example, CAH females with fully developed \u201cpenis-like\u201d clitoris and male sexual orientation can be assigned to male sex. But those with slightly prominent clitoris but strongly androgenized brain or vice versa will pose severe dilemma.[37] Hindu philosophy appears to have the solution for this puzzle. In Hinduism \u2018atman\u2019 (soul or psyche) is considered superior to \u2018sarira\u2019 (body). Psychosexual orientation rather than bodily anatomy should prevail in sex reassignments. Contended mind may adjust with defective body while a healthy body is unlikely to cope up with resentful mind. In essence, sex should be tailored to suit gender.Bias towards penetrative sexual intercourseSurgical alteration of external genitalia to suit the assigned gender is frequently biased towards feminization. Reconstructing a penis with erectile capacity is technically more challenging than creating a receptive vagina.[38] Popularity of neo-vaginoplasty over neo-phalloplasty indirectly influences sex reassignment. For example, male neonates with inadequate penis such as congenital aphallia, exstrophy, traumatic penile loss and micropenis are often (erroneously) assigned to female sex irrespective of their genetic makeup and gonadal function. On the other hand, enlarged clitoris encroaching upon the vaginal inlet is either resected or reduced in CAH patients. These approaches probably reflect our unconscious bias towards penetrative intercourse.Evolutionarily sex is intended to be penetrative for sperm transfer and reproduction. However, mankind has moved far from evolutionary purposes. As Masters and Johnson [39] remarked, sex is now intended not only for reproduction but also for recreation and relationship. For the latter two functions vaginal penetration is not essential. In fact, alternate sexual behaviors such as masturbation and oral sex are as equally enjoyable as penetrative sex.[39] Therefore, an intact albeit inadequate genitalia is probably better than an insensate sex organ. Orgasmic difficulty of DSD patients is often attributed to neonatal operative injury of genital nerves. As much as 39% of CAH patients reported orgasmic difficulty despite clitoris preserving genitoplasty. On the other hand 100% those who did not undergo any genital operation reported satisfactory orgasm.[40] These findings suggests that neonatal genitoplasty should be aimed to provide sensually enjoyable organs rather than cosmetically acceptable genitals.Conflict between utopian ideals and realityTiming of sex reassignment is a highly contentious issue.[41-43] It is caught between the utopian ideals of allowing affected individuals to decide for themselves at puberty and the practical problems of raising these children with gender uncertainty.[44] Long periods of indecisiveness is feared to leave them with confused gender identity and social ridicule. Initial gender of rearing is found to be a better predictor of adulthood gender identity and contentedness. However, the greatest hurdle is the inability of neonates and infants to express their psychosexual orientations.Ability to predict future sexuality of infants is an interesting proposition to solve the problem of early sex assignment in DSD individuals. It is suggested that the degree of androgen imprinting of brain and hence a future male inclination can be predicted to some extent from genital appearance and toy preference. For example, CAIS patients with fully feminized genitalia are more often satisfied with female sex assignment.[49-50] Insensitivity of cerebral androgen receptors could be a logical explanation of this. In CAH, most of those with Prader 4 and 5 type (fully virilized) genitalia are more dissatisfied with female sex assignment than those with lesser Prader-score although both the group develop unambiguous female identity if the gender is assigned before 24 months of age. Significant difference in the toy preference of boys and girls is thought to correlate with androgen imprinting and future psychosexual orientation.[53] A similar observation of gender-specific toy selection in primates implies that the phenomenon is probably a biological characteristics rather than a mere effect of parental rearing.[54] Therefore it is suggested that children who prefer male-type toys may be assigned to male sex and vice versa. However our current understanding of sexuality prediction is far from completeness. Genital appearance and androgen levels were found to correlate well with gender specific social role play but not sexual orientation.[47] Similarly, gender-specific toy preference is well correlated with gender identity but not with sex role-play.[55] It is well known that gender identity is different from sexuality.[56] CAH girls who may behave boyish may still be feminine in their sexual outlook. From these observations it appears that the degree of androgen imprinting of brain may not be uniform; probably it differs not only between individuals but also between different areas of the brain in the same individual. Further research is needed to test this hypothesis. Until then who should be responsible for decision making on behalf of DSD neonates is the looming question. In many other spheres of life such as choice of school education, food and vaccination parents take surrogate decision in the best interest of their offspring. Therefore it may not be inappropriate for parents to decide upon the sex of their children when it is ambiguous. However, problems arise when parents take decision under social pressure, misconception, bias or ignorance. For example, in developing countries like India, social stigma is more for an inadequate female than for a deficient male. Sexually handicapped male may still earn a livelihood, can effectively evade sexual abuse and can openly experiment with his sexuality; but the same is not true of DSD neonates raised as females. Therefore, many parents request male sex assignment in CAH despite knowing the possibility of fertility if the child is raised as female.[57] More intriguingly male sex assignment is requested even in CAIS despite acknowledging the futility of such decision. Parental health education, psychosocial support and governmental welfare schemes may mitigate such inappropriate decisions by parent.Conflict between procreativity, sexuality and sociabilityGonads are meant for procreation while external genitalia offers sexual pleasure and concordant secondary sexual characters enhance social interaction. Achieving harmony between all the three components is a utopian ideal desired in every DSD neonates; [58] but the harsh reality necessitates sacrificing one or two of them to achieve successful outcome. For example, retained testes of partial androgen insensitivity syndrome (PAIS) males may facilitate future procreation by assisted reproductive techniques. However mismatched external appearance at adolescence consequent to testosterone secreted from the testes will stigmatize the individual and adversely affect sociability. Prioritizing is easier if the gonads carry high risk of malignancy such as that of streak gonads. [59] In such cases \u201csafety of life\u201d principle negotiates all other ethical dilemma. But gonadectomy is fraught with serious ethical problem when the risk of cancer is low as it is in non-hormonal DSD. When faced with conflicting priority between sexuality and procreativity the former should be given preference over the latter as exemplified by the meaningful life of infertile couple who are otherwise healthy. Research data indicate that DSD patients with social acceptability and career success are satisfied with their gender of rearing irrespective of sexual satisfaction or procreative ability.[60] Success achieved by sacrificing a few is more endurable than failure resulting from attempted preservation of all.ConclusionAcknowledging that utopian ideals are different from reality is an essential prerequisite of resolving the controversies of sex reassignment. Willingness to compromise in case of competing priorities, elimination of misconceptions and overcoming bias are necessary adjuncts. Sex reassignment should be personalized for each patient irrespective of the underlying disease. Physical appearance should be tailored to suit psychosexual orientation. When it is impossible to know the mental inclination of neonates and infants, informed decision of parents should prevail. Prolonged uncertainty of gender is better avoided and early sex assignment is recommended although it may be far from ideals. Sex assignment should be aimed to preserve sociability, sexual satisfaction and procreative ability in that order of importance. Genitoplasty should be aimed to provide sensually enjoyable organs rather than cosmetically acceptable genitalia. Further research on the nature of androgen imprinting of brain and ability to predict it in infancy may add more clarity to the understanding of gender development and sex assignment. Nevertheless, interference of social activism with medical science will be detrimental for elucidation of truth.Source of Support: NoneConflict of Interest: NoneDisclaimer:Views expressed in this article reflect the author\u2019s understanding of neonatal sex assignment rather than any official recommendation."} +{"text": "Female College students in Korea are having changes in their bodies, transforming into more westernized shape from improvement of diet and improvements in their physique. Their average height and weight have gradually increased compared to 20 years ago. In 2016, the average height was 160.9 cm and the average weight was 57.1 kg for Korean female college students . Other countries female college students recorded higher significance level (P<.001) than Korean female college students in health level, indicating that other countries female college students have higher interest in health related standards. Other countries female college students also had higher significance level (P<.001) in exercise preferences and exercise status than Korean students, meaning that other countries students possess higher willingness to exercise.In recent years, Korean female college students are having more interest in seeking for easier methods to decrease body fat and outer beauty rather than being involved in physical activities. It is speculated that there will be differences between Korean and other countries female college students in the same age group. P<.001) than Korean female college students. Furthermore, among total of 149 students who exercised more than once a month, the comparison on these two groups showed that other countries female college students had three times significantly longer exercise duration level (P<.000) than Korean female students, as the other countries female college students persisted for 3 years and 1 month while Korean female college students lasted 1 year and 1 month. However, no significant differences were seen in exercise frequency, single exercise time and sedentary time between the two groups.The result of studying whether sufficient physical activities achieved shows that other countries female college students are more actively involved in physical activities by recording higher significant level (In conclusion, other countries female college students are more active and willing to maintain good health from exercise compared to Korean female college students. Current Korean female college students value outer beauty rather than physical health. Therefore, it is important to change Korean female college students\u2019 perspective on beauty. It is necessary to provide Korean female college students with effective programs to improve their health level and more information guide within campuses for better accessibility in the future. Moreover, it would be desirable to keep students in the habit of sharing and planning health improvement plans and actively participating in physical exercises."} +{"text": "Plasmodium falciparum cause an accumulation of specific responses against various antigens that correlate with a decreased risk of clinical malaria episodes. However, small effect sizes and the often polymorphic nature of immunogenic parasite proteins make the robust identification of the true targets of protective immunity ambiguous. Furthermore, the degree of individual-level protection conferred by elevated responses to these antigens has not yet been explored. Here we applied a machine learning approach to identify immune signatures predictive of individual-level protection against clinical disease. We find that commonly assumed immune correlates are poor predictors of clinical protection in children. On the other hand, antibody profiles predictive of an individual\u2019s malaria protective status can be found in data comprising responses to a large set of diverse parasite proteins. We show that this pattern emerges only after years of continuous exposure to the malaria parasite, whereas susceptibility to clinical episodes in young hosts (< 10 years) cannot be ascertained by measured antibody responses alone.Antibodies are thought to play an essential role in naturally acquired immunity to malaria. Prospective cohort studies have frequently shown how continuous exposure to the malaria parasite P. falciparum malaria is of fundamental importance for malaria control and elimination efforts. The identification of parasite antigens that could potentially be considered as vaccine targets often relies on prospective cohort studies where observed infection rates are related to measured immune responses. However, what is unknown, is how these population-level associations between antibody titres and protection from severe malaria can predict the risk of an infection for an individual. We therefore analysed three sets of cohort-based immune profiles using a machine learning approach in order to identify distinct immune signatures that are predictive of protection at the individual-level. Our results show that even statistically significantly associated responses fail to provide robust information about an individual\u2019s risk of malaria and that machine learning approaches should be considered more prominently alongside traditional methods for analysing these complex and high dimensional datasets.Understanding naturally acquired immunity against P. falciparum endemic areas develop protection against clinical and symptomatic infections over years of repeated exposure. Since the first experimental evidence demonstrating how passively transferred immunoglobulins from immune adults can dramatically reduce parasitaemia in infected recipients P. falcip(PDF)Click here for additional data file.S1 Script(R)Click here for additional data file."} +{"text": "This study was to investigate the Heart Rate Variability(HRV) and emotional response to positive and negative audiovisual stimulation in patients with chronic schizophrenia and healthy control groupAmong 253 chronic schizophrenic patients admitted in 00 Hospital in 00 city by psychiatrist, 104 patients were informed about this research and consented. Those who met this study criteria were randomly selected. 35 healthy control consisted of peoples that did not have past and present history of mental and physical illness. Positive and negative affect and HRV were compared between chronic schizophrenia and healthy control groups, and positive and negative affect and HRV to positive and negative audiovisual stimulation were measured according to planed research process. Positive and negative audiovisual stimulation was defined by an art therapy professionalist and a psychiatrist as 10 positive and negative pictures. 3 positive and negative musics were shown to two groups for 4 minutes simultaneously. Positive and negative audiovisual stimulation were shown to two groups during 1-week intermission. HRV was measured with Ubpulse H3, an equipment for autonomic nervous system test made by Laxtha company and also analyzed by frequency domain analysis. Emotional Empathy Scale(EES) and Positive Affect and Negative Affect Schedule (PANAS) of two groups were measured at baseline and after positive and negative audiovisual stimulation. Global Assessment of Functioning Scale(GAF) and Positive and Negative Syndrome Scale(PANSS) of chronic schizophrenia group were measured by a psychiatrist.1) Positive affect of patients group were significantly lower than control group, negative affect of patients group were significantly higher than control group. Low Frequency(LF), High Frequency(HF), and Total Power(TP) of HRV in patients group were significantly lowered than control group at baseline.2) 7 subscales of emotional empathy scale were lowered in patients group compared to control group.3) Positive affect of patients group was significantly less increased compared to the control group after positive audiovisual stimulation, negative affect of patients group was significantly less decreased to the control group after positive audiovisual stimulation.4) Positive affect of patients group was increased after negative audiovisual stimulation, but positive affect of control group was significantly decreased compared to the patients group after negative audiovisual stimulation. There was no significant difference in negative affect between two groups after audiovisual stimulation.5) LF of patients group was significantly higher than control group after positive audiovisual stimulation, HF and TP of patients group were significantly lowered than control group after positive audiovisual stimulation.6) LF of patients group was significantly higher than control group after negative audiovisual stimulation, HF and PT of patients were significantly lowered than control group after negative audiovisaul stimulation.Patients with schizophrenia showed lower positive affect, higher negative affect, and lowered HRV parameters compared to the control group. They also showed lower empathy ability and inappropriate and non-contexual response. Schizophrenic patients represented hypersensitive sympathetic nervous system activity and lowered parasympathetic nervous system activity to the audiovisual stimulation. These results suggested that schizophrenic patients would show higher negative affect, less adaptive autonomic nervous system and hypersensitive or sharp to audiovisual stimulation, and decreased relaxation ability after stimulation. Audiovisual stimulation in integrative arts therapy program for schizophrenia might have avoid overactive sympathetic stimulation and recommend activate parasympathetic stimulation. Integrative art therapy for schizophrenia must be sufficiently relaxed, empathetic, and promote positive affect during therapeutic process."} +{"text": "Cardiac perfusion MRI enables quantification of myocardial blood flow in ml/min/g. However, signal intensity (SI) saturation of the arterial input function (AIF) leads to underestimation of the AIF and errors in perfusion quantification. Dual bolus experiments avoid this by using an unsaturated dilute bolus to measure the AIF and have been shown to be accurate against gold standard microsphere measurements. Despite the advantages associated with absolute quantification, dual bolus has seen limited adoption outside of a few large academic centers in part because it is difficult to implement. Set up requires either two injectors or a complex preloading scheme, both of which add time and complexity to a procedure that can be sensitive to experimental error. To calculate perfusion, the AIF from the dilute bolus is scaled to match the myocardial SI data from the full bolus using the ratio of contrast concentrations between the full and dilute boluses at the left ventricle. This is typically assumed to be the dilution ratio used to mix the dilute bolus, and any imprecision during mixing can translate into large errors in AIF height and thus perfusion calculations. Modifying the dual bolus technique so that it is more tolerant of experimental error might facilitate more widespread adoption outside of large research centers. Here we present a method for empirically determing contrast ratios that can be applied retropspectivley to dual bolus perfusion data to reduce experimental error.11 dual bolus (0.005 and 0.05 mmol/kg) firstpass perfusion studies were conducted on 5 dogs during rest and adenosine vasodilated stress on a 1.5 T clinical scanner. Myocardial blood flow was quantified by injected microspheres. Dual bolus correction was performed two separate ways: 1) using an assumed ratio of 10, 2) using least squares fitting to find the concentration ratio that minimized the differences between the dilute and full AIFs in the unsaturated tail region Fig of the SIncorporating the tail correction to empirically determine contrast concentration ratios increased correlation against microsphere perfusion values from 84 to .90 Fig . The 95%Using empirically determined contrast ratios improved MR perfusion correlation against gold standard reference values. Becaue this method does not assume a fixed dilution ratio between the two boluses, it is much more tolerant to any imprecision in experimental setup. This reduces the logistical burden on the staff carrying out a dual bolus experiment, which may facilitate more widespread adoption of this proven quantitative technique into busier clinical environments."} +{"text": "TCD) is a clinical tool for stratifying ischemic stroke risk by identifying abnormal elevations in blood flow velocity (BFV) in the middle cerebral artery (MCA). However, TCD is not effective at screening for subtle neurologic injury such as silent cerebral infarcts. To better understand this disparity, we compared TCD measures of BFV with tissue\u2010level cerebral blood flow (CBF) using arterial spin\u2010labeling MRI in children with and without sickle cell disease, and correlated these measurements against clinical hematologic measures of disease severity.Transcranial Doppler ultrasonography or controls . After conversion from BFV into units of CBF, a strong association was observed between TCD and MRI measures . While BFV in the MCA showed a lack of correlation with arterial oxygen content, an inverse association was observed for CBF measurements.There was no significant association between BFV in the MCA cannot be used as a surrogate marker for tissue\u2010level CBF in children with sickle cell disease. Therefore, TCD alone may not be sufficient for understanding and predicting subtle pathophysiology in this population, highlighting the potential clinical value of tissue\u2010level CBF.This study demonstrates that"} +{"text": "During 2016 in Guangzhou, China, we detected infectious avian influenza viruses (AIVs) in 39.8% of samples from chicken carcasses slaughtered at live poultry markets but none from carcasses supplied to supermarkets by facilities bypassing live poultry markets. Promoting supply chains with high biosecurity may reduce the risk for zoonotic AIV transmission. A total of 1,230 swabs were collected from the oropharynx, cloacal cavity, and visceral cavity of chicken carcasses supplied from the LPM system .During July\u2013December 2016, we also sampled chilled chicken carcasses supplied from the private poultry slaughtering industries that bypass the LPM system. Chicken carcasses were sampled from 3 different supermarket chains once each month; 147 swabs were collected in virus transport media . The quaThe AIV-positive rates detected from fresh chicken carcasses varied by market type . Rates oWe detected significantly higher viral loads in oropharyngeal swabs than in cloacal or visceral cavity swabs of chicken carcasses sold at the dressed poultry stalls or the retail markets . Most AIOur results agree with results from a previous study that reported detection of AIV RNA from chicken carcasses sold at retail and dressed poultry stalls in Guangzhou (p = 0.172 by Fisher exact test) from live poultry sold at the same wholesale market as that detected from the chicken carcasses at the dressed poultry stalls (67 [55.4%] of 121) . The resIn conclusion, our data suggest that chicken carcasses may pose a substantial zoonotic risk for AIV infection even in the absence of direct contact with live poultry. Central slaughtering might not by itself eliminate zoonotic risk if the source poultry have high rates of virus carriage. The LPM system in China continues to provide venues and opportunities of poultry mixing from different sources that facilitate AIV persistence and amplification despite interventions, such as market rest-days and banning the holding of live poultry overnight, that aim to reduce such a risk ("} +{"text": "We highlight the implications this has for Blair Thymallus arcticus) compared with rainbow trout (Oncorhynchus mykiss). While rainbow trout exposed to 17 ppt salinity were able to adjust osmoregulation successfully and establish homeostasis, Arctic grayling showed increasing serum sodium and chloride over time, which eventually led to mortality. They conclude that the probable impacts of highly saline water spills or discharge into freshwater habitats should be based on species-specific salinity tolerances, rather than the response of the euryhaline rainbow trout (ow trout . This isWe agree with the main conclusions and conservation implications of this article but are writing here to address an inaccuracy. Arctic grayling are described as having a \u2018strict(ly) freshwater existence\u2019 , and the authors suggest that \u2018Arctic grayling have lost the ability to execute the necessary osmoregulatory mechanisms to cope with higher salinity environments.\u2019 Although Arctic grayling is indeed a freshwater species, there is evidence that some populations can tolerate saline waters. A \u2018strictly freshwater existence\u2019 would preclude the use of any brackish (0.5\u201329 ppt) habitats; however, in the Arctic this species is known to move through brackish waters during migration experienced very little mortality, whereas 19% of fish from a southern population died has evolved an anadromous form in several locations in the Baltic Sea \u2014previously considered the least tolerant salmonid to saltwater\u2014have recently been discovered (Local adaptation to salinity tolerance is common in salmonids and other genera of fishes (ion died . FurtherAlthough the specimens used by In conclusion, we would like to acknowledge again the well-designed and -conducted study of"} +{"text": "Preventing healthcare-associated infections and reducing their avoidable impact on health systems is critical today to make facilities safer for patients worldwide . In addith May 2017 Global Annual Hand Hygiene Day, WHO urges policy makers, top-level managers, IPC specialists and other health professionals to focus on the fight against AMR spread, by building ever stronger hand hygiene and IPC programmes (Table\u00a0Hand hygiene is at the center of effective infection prevention and control (IPC) to combat AMR spread . The Worth May 2017 campaign [We encourage health facilities worldwide to endorse the WHO's 5Let\u2019s fight antibiotic resistance together; it's in our hands."} +{"text": "The concept of delivering nucleic material encoding a therapeutic gene to the heart has arduously moved from hypothesis to a variety of high potential clinical applications. Despite the promise however, the results achieved have yet to be realized due to several problems that persist in the clinic. One of these identified problems is the need for an efficient delivery method which facilitates complete cardiotropism and minimizes collateral effects. Additional parameters impacting gene delivery that most need to be improved have been identified as follows: (1) Increasing the contact time of vector in coronary circulation permitting transfer, (2) Sustained intravascular flow rate and perfusion pressure to facilitate proper kinetics, (3) Modulation of cellular permeability to increase uptake efficiency, and once in the cells (4) Enhancing transcription and translation within the transfected cardiac cells, and (5) Obtaining the global gene distribution for maximum efficacy. Recently it was hypothesized that use of cardiopulmonary bypass may facilitate cardiac-selective gene transfer and permit vector delivery in the arrested heart in isolated \u201cclosed loop\u201d recirculating model. This system was named molecular cardiac surgery with recirculating delivery (MCARD). The key components of this approach include: isolation of the heart from systemic organs, multiple pass recirculation of vector through the coronary vasculature, and removing the residual vector from the coronary circulation to minimize collateral expression. These attributes unique to a surgical approach such as MCARD can effectively increase vector transduction efficiency in coronary vasculature. In the previous 25 years, there has been a substantial increase in the understanding of the aims of gene therapy, development of transgenic models of various diseases and synthetically packaging nucleic material in a variety of biologic therapeutics. Recently in parallel with clinical development, there have been numerous organized efforts to improve gene delivery techniques specifically tailored for cardiovascular gene therapies. Three major conclusions from previously published data are as follows: (1) Even the best engineered vectors such as those containing cardiac-specific promoter cannot limit the delivery of viral capsids to collateral organs or non-target tissue in the heart, (2) The route of administration of gene transfer is equally or more important than the vector or promoter system in larger species, and (3) Optimal gene transfer can be defined in terms of transfer ratio to the target organ versus inadvertent collateral transfer to evaluate efficiency. Thus, as additional cardiac gene therapies have moved into clinical trials, the drug delivery aspects specific to gene therapies have thrived opening and entirely new device field.Despite significant interest and investment to optimize delivery however, an ideal cardiac gene delivery route of administration with complete cardiotropism and no collateral effects does not exist. Intravascular and intramyocardial delivery has been shown to limit transduction to the heart with varied results since systemic leakage is unavoidable given single pass gene transfer. Moreover, very rapid dilution featuring dramatically reduced vector concentration via larger circulating blood volume is typically observed. In addition, decreases in bioavailability are a problem as subsequent gene therapy products not reaching the heart are either absorbed by collateral organs or inactivated by the immune system. Thus, delivery systems addressing these concerns would offer substantial improvement for overall therapy with a key focus on several parameters.The gene therapy parameters impacting delivery that most need to be improved have been identified as follows: (1) Increasing the contact time of vector in coronary circulation permitting transfer, (2) Sustained intravascular flow rate and perfusion pressure to facilitate proper kinetics, (3) Modulation of cellular permeability to increase uptake efficiency, and once in the cells (4) Enhance transcription and translation within the transfected cardiac cells, and (5) Obtaining the global gene distribution for maximum efficacy.Selective coronary catheterization with antegrade delivery was first tested in a rabbit model . Today, The next advancement was selective intra-coronary retrograde delivery through the coronary sinus . The useAs a response to address the shortcomings of catheter based systems, researchers hypothesized that utilizing advanced device concepts leveraging consistent flow perfusion would provide a better means to increase bioavailability and reduce first pass effects. The ultimate device concept of this proposal is \u201cclosed-loop\u201d recirculatory systems, which allowed separation of the coronary vascular bed from the systemic . RecentlUltimately, one leading group in the field of cardiac gene delivery developed an isolated \u201cclosed loop\u201d recirculating model. This system was named molecular cardiac surgery with recirculating delivery (MCARD) and demonstrated effectiveness in the large animal permitting vector recirculation for 20 min . The keyin situ, using CPB with high-pressure retrograde coronary sinus infusion with multiple-pass recirculation of vector through the heart results in an increase of several orders of magnitude in cardiac marker gene activities compared with controls, receiving retrograde infusion of adenovirus without CPB and without cardiac isolation [Summarizing, it has been demonstrated that complete surgical isolation of the heart solation . The prisolation . \u201cClosedMinimally invasive cardiac surgical and or image guided interventional techniques and tools would serve to broaden the base of potential patients that would benefit from cardiac gene therapy. Currently, there is a revolution in cardiac surgery featuring advanced robotic applications that access the heart with minimal incision and access points. Additionally, minimally invasive grafts, valve replacements and other supporting devices would offer a means to integrate \u201cgene eluting\u201d or other inspired combination devices applied in patients."} +{"text": "August 21\u201325, 2017, marks the fourth annual Contact Lens Health Week. In collaboration with partners from the clinical, public health, industry, and regulatory sectors, CDC is promoting healthy wear and care practices to reduce the risk for eye infections among the approximately 45 million persons in the United States who wear contact lenses. Research after outbreaks of rare but serious eye infections in the United States has indicated that these infections occur most often in contact lens wearers who do not take proper care of their contact lenses, indicating a need to promote safer wear and care (MMWR describes CDC\u2019s first-ever population-based estimates of contact lens\u2013related risk behaviors in persons aged 12\u201317 years (referred to here as adolescents) in the United States. Approximately six in seven adolescents reported at least one behavior putting them at risk for a serious contact lens\u2013related eye infection. Encouraging adolescents to adopt healthy contact lens wear and care habits might help them maintain healthy habits into adulthood.A report in this issue of https://www.cdc.gov/contactlenses.Although most contact lens wearers receive the benefits of vision correction, contact lenses can pose an infection risk, especially if they are not worn and cared for properly. Practicing proper contact lens hygiene and regularly visiting an eye care provider are important behaviors for keeping contact lens wearers\u2019 eyes healthy. Additional information on Contact Lens Health Week and the proper wear and care of contact lenses is available at"} +{"text": "Saccharomyces cerevisiae) towards homeopathically potentized Arsenicum album, a duckweed nosode, and gibberellic acid. The three test substances were applied in five potency levels and compared to controls (unsuccussed and succussed water) with respect to influencing specific growth parameters. Five independent experiments were evaluated for each test substance. Additionally, five water control experiments were analyzed to investigate the stability of the experimental setup (systematic negative control experiments). All experiments were randomized and blinded. Yeast grew in microplates over a period of 38\u2009h in either potentized substances or water controls with 250\u2009mg/l arsenic(V) added over the entire cultivation period. Yeast's growth kinetics were measured photometrically. The test system exhibited a low coefficient of variation . Succussed water did not induce any significant differences compared to unsuccussed water. Data from the control and treatment groups were both pooled to increase statistical power. In this study with yeast, no significant effects were found for any outcome parameter or any homeopathic treatment. Since in parallel experiments arsenic-stressed duckweed showed highly significant effects after application of potentized Arsenicum album and duckweed nosode preparations from the same batch as used in the present study, some specific properties of this experimental setup with yeast must be responsible for the lacking response.This study investigated the response of arsenic-stressed yeast ("} +{"text": "Adherence to prescribed medications is associated with improved clinical outcomes for chronic disease management and reduced mortality from chronic conditions were significantly more adherent with their medication regimen 12 months after hospital discharge (89%) compared with patients not receiving team-based care (74%). Patients reported that team-based care improved their comfort in asking clarifying questions, raising concerns about their medication regimen, and collaborating in developing their treatment plan , compared with beneficiaries insured by another health plan with the same third-party prescription drugs administrator that did not reduce or eliminate copays for the same medications. These improvements, while modest, could result in significant cost savings in the prevention of acute events and progression of major chronic conditions if scaled to larger populations , because treatment lowers the amount of virus circulating in the blood, which improves the patient\u2019s health and reduces the risk of transmitting HIV to others by >90% increased first-fill medication adherence by 10% compared with those using paper prescriptions portal. Use graphs to demonstrate challenges and successes with treatment regimensChoose lower cost medicationsUse step-therapy protocols that are developed by a multidisciplinary team and are standardized across the organizationControl access to pharmaceutical marketingMake the patient\u2019s payer-specific formulary available in the EHR to inform medication selectionUse generic medication substitutionProvide assistance in paying for medications Consult social workers to assist with adherence barriersMinimize medication complexityChoose once-a-day and combination medicationsEngage in dialogue about costs versus convenience Monitor side effectsBe creative in addressing concernse.g., if concerned about swollen feet, use a diuretic, if appropriatee.g., if concerned about medication causing abnormal potassium level, use a combined angiotensin-converting enzyme (ACE) inhibitor and a diuretic to normalize potassiumWhen to monitor side effectsAt visitsAt prescription renewals, using a standard documentation templateAfter hospital discharge: automated alerts for new medicationsConsult pharmacistsFor complex medication regimens or side effectsAfter hospital discharge regarding patients who are on high-risk medicationsShow effectiveness of the medications in lowering blood pressureEmpower patient to record blood pressure readings at homeProvide booklets to record readingsFor patients with financial hardships, provide free home blood pressure monitorsOffer free blood pressure clinicsAutomatically upload blood pressure readings into the EHRMonitor medication adherenceEncourage patients to document their medication-taking behaviorUse EHR systems that can show medication fill historyAutomate adherence monitoring using payer medication claimsReview adherence information during visits. Patients\u2019 knowing that a clinician is monitoring adherence is at least as important as a patient seeing the resultsAlthough a range of interventions have demonstrated improved medication adherence and health outcomes during the study period, few studies have shown that these benefits were maintained over time ensuring access to providers across the continuum of care and implementing team-based care; 2) educating and empowering patients to understand the treatment regimen and its benefits; 3) reducing barriers to obtaining medication, including cost reduction and efforts to retain or re-engage patients in care; and 4) use of health information technology tools to improve decision-making and communication during and after office visits. Understanding root causes of medication nonadherence and cost-effective approaches that are applicable in diverse patient populations is essential to increasing adherence and improving long-term health impact."} +{"text": "Complex ventricular-arterial (VA) relationships in patients with double outlet right ventricle (DORV) make preoperative assessment of potential repair pathways challenging. The relationship of the ventricular septal defect (VSD) to one or both great arteries must be understood and this influences the choice of surgical procedure [1] In neonates and infants with DORV, Computed Tomography (CT) is often performed due to the ability to get high spatial resolution and ECG gated images [2], however it is possible to get the necessary information from Magnetic Resonance (MR) imaging with an added advantage of avoiding exposure to ionizing radiation. Both CT and MR allow image acquisition in three dimensions (3D) but traditional viewing of the anatomy using the multiplanar reformatting is actually done in two dimensions (2D). Volume rendering from either modality may also be performed, but typically only the external vascular anatomy is depicted. We hypothesized that it is possible to accurately define the intracardiac anatomy in infants with DORV using virtual and physical 3D printed (rapid prototyped) models created from either MR or CT and this can both aid in better defining potential VA pathways and may assist in surgical decision making.Virtual and physical 3D models were generated for three patients with DORV. Non-ECG-gated 3D spoiled fast gradient echo sequence MR angiography was used for two patients. Retrospective ECG gated CT angiography images acquired in diastole were used in the third patient (to better define the coronary arteries given the suspicion of a single coronary artery by echocardiography). Blood pool segmentation Figure was perfThe VSD and VA relationships were well visualized in all three patients using both the virtual and physical models Figure . The modConstruction of 3D models in patients with DORV is feasible and allows for extensive examination and surgical planning. This may facilitate a focused and informed surgical procedure and improve the potential for successful outcome. For purposes of DORV, non-gated MRA is sufficient to delineate the VA relationships adequately for 3D printing and enhanced clinical decision-making. CT imaging should be reserved for only those patients where additional information like coronary artery anatomy is desired."} +{"text": "Stroke is a known cause of cognitive impairment but the relationship betweenasymptomatic carotid artery stenosis and cognitive function is not clear. Themain risk factors for vascular disease are also related to carotid stenosis andcognitive impairment. The association of high-grade stenosis of the internalcarotid artery with cognitive impairment is related to silent embolization andhypoperfusion, but it may also be present without evidence of infarction onmagnetic resonance imaging. Carotid stenosis treatment may lead to a decline incognitive function due to complications related to the procedures(endarterectomy or stenting). On the other hand, reperfusion may improvecognitive impairment. The best treatment choice is unclear, considering possibledeterioration of cognitive function related to carotid artery stenosis. There isinsufficient evidence to consider cognitive impairment an important factor indetermining the therapy for carotid stenosis. With aging populations, the most important unmodifiable risk factor forcognitive disorders, cerebrovascular risk factors and disease, have becomesignificant modifiable factors, particularly in the development of stroke-dementiaassociation.2 Possiblemechanisms involved include silent embolization and hypoperfusion.3The main risk factors for vascular disease are also related to cognitive impairment.Hypertension, diabetes mellitus, cigarette smoking, and dyslipidemia are associatedwith an increased risk of carotid artery disease. Some studies have suggested thatstenosis of the internal carotid artery may be an independent risk factor forcognitive impairment. High-grade stenosis of the internal carotid artery may beassociated with cognitive impairment even without evidence of infarction on magneticresonance imaging and is therefore suspected as an independent risk factor fordementia.2 A possibleassociation considering improved cognitive function and better cerebral perfusionhas been hypothesized, whereas subclinical microembolic cerebral patterns occurringduring revascularization may worsen cognitive function.2Carotid endarterectomy (CEA) or stenting in patients with severe carotid stenosisreduces stroke risk.3According to population studies, some degree of carotid disease can be found in morethan 75% of men and around 60% of women older than 65 years of age. Overallprevalence of over 50% carotid stenosis in the same age group is 7% in men and 5% inwomen. The prevalence of cognitive impairment also increases with aging.3The diagnosis of advanced carotid stenosis is mostly reached in symptomatic patientsbased on the presence of stroke or transient ischemic attack (TIA). However, thecognitive status in these patients is often overlooked, although if cognitiveimpairment is present, such patients should probably be consideredsymptomatic.In this paper, we review some studies that have evaluated the association betweencognitive decline and advanced carotid disease, possible mechanisms involved in thisrelationship, and how to evaluate it in clinical practice.The presence of cognitive deficit is associated to loss of one or more cognitivefunction. The cognitive functions are linked to different neural synapses andanatomic brain regions. In clinical practice, the Mini-Mental State Examination(MMSE) is the most commonly applied screening test and most frequently used tostratify the severity of cognitive impairment and dementia. The main problem is thatthe MMSE does not differentiate among cognitive functions.4Patients with severe carotid disease without previous stroke or TIA can have subtlecognitive abnormalities that are undetectable by the MMSE and may only be identifiedby applying specific neuropsychological tests.2The use of well-defined neuropsychological tests allows the evaluation of specificcognitive functions linked to specific neural systems.5 As an example, the Montreal Cognitive Assessment (MoCA), a morecomprehensive test than the MMSE, is capable of identifying reduced cognitive statusin patients with asymptomatic ICA stenosis.6These neuropsychological tests can reveal cognitive decline even in patients withcarotid atherosclerosis characterized by number of plaques and total plaques, andnot severe stenosis, suggesting that subclinical carotid atherosclerosis mayincreases the risk of cognitive decline.7Transcranial Doppler may be used as a functional parameter for intracranialsubclinical atherosclerotic changes, measured by the breath holding index (BHI).Performance on the BHI test is decreased and related to impaired cerebrovascularreactivity in patients with early cognitive decline.2The vascular risk factors are alsorisk conditions for stroke, carotid stenosis and dementia. Theoretically, a carotidstenosis may be a direct cause of a reduced level of cognitive functioning, or itmay act only as a marker of intracerebral or generalized atherosclerosis. Therelationship of asymptomatic stenosis with cognitive impairment isunclear.8An evaluation of the relationship between cognitive decline and atherosclerosis wascarried out in the 1975 Framingham Offspring Study participants. The resultssuggested that internal carotid artery intima-media thickness may be a marker forcognitive impairment and be associated with higher prevalence of silent cerebralinfarcts and of extensive white matter hyperintensity.9The Troms\u00f8 study evaluated subjects without history of stroke and with Carotidstenosis measured by ultrasonography that showed right-sided or bilateral narrowingof \u226535%. This group was compared to subjects with and without carotidstenosis. The study demonstrated that subjects with carotid stenosis hadsignificantly lower levels of performance on several subsets of cognitivetests.10The Cardiovascular Health Study evaluated individuals with no history of stroke,transient ischemic attack (TIA) or carotid endarterectomy. Internal carotid arterystenosis was measured by duplex ultrasonography. The study found that high-grade(\u226575%) stenosis of the left internal carotid artery was associated withcognitive impairment and cognitive decline during follow-up, even in participantswithout cerebral infarction on MRI. These observations support the notion that evenasymptomatic carotid stenosis may be an independent risk factor for cognitiveimpairment and decline.4The effect of carotid stenosis on cognitive functioning remains largely unexplainedalthough it is believed that asymptomatic advanced carotid stenosis should beconsidered an independent risk factor for decline in cognitive functioning. Ifasymptomatic advanced carotid stenosis indeed causes cognitive impairment, thedecision on surgical treatment might consider cognitive evaluation using aneuropsychological test as a clinical definition of symptomatic carotid disease andmay be considered as important as the effective treatment of vascular risk factorsin stroke/TIA free patients with carotid stenosis.The main mechanisms related to cognitive impairment in carotid stenosis areembolization and hypoperfusion.2The presence of silent brain infarction is related to an increase risk of dementia.Silent brain infarcts may be related to carotid stenosis, embolization orhypoperfusion. They may also be present in asymptomatic carotid stenosis and arerelated to lacunar infarcts secondary to microangiopathy and cardiovascular riskfactors.11 On the other hand, theTroms\u00f8 Study and the Cardiovascular Health Study observed that cognitiveimpairment in patients with carotid stenosis was independent of vascular lesionsobserved on MRI.10Embolization may be detected by middle cerebral artery monitoring on transcranialDoppler. A study comparing Alzheimer's disease, vascular dementia and controls,observed that spontaneous cerebral emboli were significantly more frequent inpatients with both Alzheimer's disease and vascular dementia.9Brain hypoperfusion may contribute to the onset of clinical dementia and is observedin patients with severe heart failure and also with carotid stenosis. TheTroms\u00f8 Study results showed a significant relationship between cognitiveimpairment and degree of carotid stenosis.In most studies, cognitive function after carotid intervention has shown significantimprovement. By contrast, some studies showed no change in cognitive function whileother reports have noted cognitive decline due to surgical procedures.12The effect of carotid procedures on cognitive function is not fully understood butchange in cognition is being increasingly recognized as an important outcomemeasure. A systematical literature review of 32 papers reporting on neurocognitionafter carotid procedures showed that assessment of cognition after carotidrevascularization is probably influenced by many confounding factors such aslearning effect, type of test, type of patients, control group, and lack ofconsensus in defining improvement or impairment after either carotid artery stenting(CAS) or carotid endarterectomy (CEA). Based on recent evidence, it is likely thatcarotid endarterectomy as well as carotid artery stenting do not affectneuropsychological function.13 The general anesthesia carotid procedures have also beenlinked to early cognitive decline that is temporary in nature.14 Recent studies on the mechanism ofcognitive decline associated with hyperperfusion have shown that cerebralhyperperfusion after CEA results in postoperative cortical neural loss thatcorrelates with postoperative cognitive impairment even in the absence of MRIlesions.15 Otherpredictors of neurocognitive decline after CEA include advanced age, diabetes,obesity, preoperative monocyte count and presence of the APOE-\u03b54allele.18Some studies have shown cognitive decline after carotid endarterectomy withmechanisms related to cerebral hyperperfusion after carotid endarterectomy, while inasymptomatic cases MRI does not always disclose structural brain damage associatedwith postoperative cognitive impairment.19A recent study evaluated cerebral blood flow using phase contrast magnetic resonanceangiography and cognitive testing preoperatively, and at 1, 6, and 12 monthspostoperatively. Patients with baseline impairment of MCA blood flow were morelikely to experience improvement in flow after revascularization. Improvement in MCAblood flow was associated with greater cognitive improvement in attention andexecutive functioning.21The benefit of the carotid procedure for prevention of brain infarct, expected fromreduced embolism and improved hemodynamics, with regard to cognitive functionremains unclear. Improvement in cognitive function following carotid reconstructionmay be greater in patients with low-flow-endangered brains than in those withhemodynamically insignificant stenosis that are frequently clinicallytreated.Carotid artery stenosis and atherosclerosis are risk factors for cognitiveimpairment. When there is symptomatic or severe artery stenosis, carotidinterventions benefit stroke risk but may leave cognitive performance unchanged, orlead to a decline or improvement. There is no evidence to support the performance ofprophylactic carotid endarterectomy or carotid stenting with the aim of preventingcognitive decline in otherwise asymptomatic patients.The literature on cognitive outcome after carotid revascularization is complex andfurther studies investigating specific populations of patients with carotid stenosiswill help elucidate whether carotid endarterectomy or carotid stenting is moreappropriate for a given patient considering the cognitive function and risks afterthe procedure.Future studies should consider standardizing neuropsychological outcomes using auniform battery of tests across multiple centers. Studying relatively homogeneousgroups of patients may help to reduce the variability inherent to cognitivestudies."} +{"text": "Astrocytes support neuronal function by providing essential structural and nutritional support, neurotransmitter trafficking and recycling and may also contribute to brain information processing. In this article we review published results and report new data suggesting that astrocytes function as versatile metabolic sensors of central nervous system (CNS) milieu and play an important role in the maintenance of brain metabolic homeostasis. We discuss anatomical and functional features of astrocytes that allow them to detect and respond to changes in the brain parenchymal levels of metabolic substrates (oxygen and glucose), and metabolic waste products (carbon dioxide). We report data suggesting that astrocytes are also sensitive to circulating endocrine signals\u2014hormones like ghrelin, glucagon\u2010like peptide\u20101 and leptin, that have a major impact on the CNS mechanisms controlling food intake and energy balance. We discuss signaling mechanisms that mediate communication between astrocytes and neurons and consider how these mechanisms are recruited by astrocytes activated in response to various metabolic challenges. We review experimental data suggesting that astrocytes modulate the activities of the respiratory and autonomic neuronal networks that ensure adaptive changes in breathing and sympathetic drive in order to support the physiological and behavioral demands of the organism in ever\u2010changing environmental conditions. Finally, we discuss evidence suggesting that altered astroglial function may contribute to the pathogenesis of disparate neurological, respiratory and cardiovascular disorders such as Rett syndrome and systemic arterial hypertension. Astrocytes are versatile CNS sensors capable of detecting various metabolic and endocrine signals.Astrocytes modulate the activities of the brainstem respiratory and autonomic neuronal circuits that ensure metabolic homeostasis. A complex hierarchy of behavioral, physiological and biochemical mechanisms ensure adequate delivery of metabolic substrates and effective elimination of metabolic waste products from all tissues of the body Fell, .The central nervous system (CNS) plays a key role in the maintenance of energy homeostasis. This function requires specific sensors that can rapidly respond to perturbations in the metabolic milieu. A series of seminal studies identified groups of neuronal metabolic sensors located in discrete brain areas, particularly in the hypothalamus and the brainstem and certain neuronal cues with increases in intracellular [Ca2+] in the arterial blood is sensed by the peripheral oxygen chemoreceptors located in the carotid bifurcation and in the aortic arch (in some species). The chemosensitive glomus cells of the carotid body are traditionally considered to be the primary (and only) respiratory oxygen sensors in mammals. When activated by hypoxia, carotid bodies initiate a chemoreflex that results in activation of the respiratory and sympathetic circuits located in the brainstem. This leads to rapid respiratory and cardiovascular responses directed towards restoring the arterial PO2 were found to trigger robust increases in astroglial [Ca2+]i and [Ca2+]i in cultured astrocytes showed that a decrease in PO2 causes a significant decrease in \u0394\u03c8m and that this response precedes increases in [Ca2+]i , mitochondrial antioxidant (MitoQ) or ROS scavenger (\u03b1\u2010tocopherol). Subsequent pharmacological analysis of Ca2+ responses suggested a feasible hypoxia\u2010sensitive signaling pathway: in astrocytes hypoxia leads to inhibition of mitochondrial respiration, increased production of free radicals, lipid peroxidation, activation of phospholipase C and recruitment of Ca2+ from IP3\u2010sensitive intracellular stores . Although we do not know what was the brain tissue PO2 in these conditions, these fascinating data suggest that the brain can operate in a very low oxygen environment (the participants were able to perform complex tasks), with parenchymal PO2 sufficiently low to trigger astroglial activation and downstream sympathetic, respiratory and regional cerebrovascular responses (see below).It was reported that the PO2 facilitate exocytosis of ATP\u2010containing vesicles by the activities of the sympathetic and parasympathetic branches of the autonomic nervous system. Groups of sympathoexcitatory (pre\u2010sympathetic) brainstem neurons, including bilateral populations of catecholaminergic C1 neurons, are essential for the generation of cardiovascular sympathetic tone PO2 is the key metabolic factor that determines the direction of cerebral arteriole response that follow astroglial [Ca2+]i elevations were not able to respond to systemic hypoglycemia with increased glucagon secretion reported data suggesting that astroglial insulin signaling modulates hypothalamic glucose sensing and systemic glucose metabolism. The authors demonstrated that ablation of insulin receptors in hypothalamic astrocytes reduced glucose\u2010induced activation of pro\u2010opio\u2010melanocortin neurons and impaired physiological responses to changes in glucose availability. Following systemic glucose administration, cerebrospinal fluid accumulation of glucose and insulin were found to be reduced in mice lacking astroglial insulin receptors. The authors concluded that brain glucose sensing and, therefore, systemic glucose metabolism are controlled, at least in part, by insulin acting at hypothalamic astrocytes. Moreover, the data reported by Garcia\u2010Caceres et al. in the arterial blood is directly proportional to the rate of CO2 production and inversely proportional to the rate of CO2 elimination by the respiratory system. Increased CO2 production and/or impaired CO2 elimination facilitate generation of hydrogen ions (respiratory acidosis), a condition that needs to be rapidly corrected by adaptive changes in the ventilatory and cardiovascular activities in order to ensure effective CO2 removal.Metabolic homeostasis also requires effective elimination of waste products. Carbon dioxide is generated in proportion to the metabolic rate and the amount of metabolic substrates utilized. At rest, our body produces \u223c12\u00a0mmol\u00a0kgPCO2/pH are monitored by specialized pH\u2010sensitive neurons residing in the brainstem are focused on a group of pH\u2010sensitive neurons of the retrotrapezoid nucleus (RTN) located near the ventral surface of the brainstem . This view is supported by the results of the experimental studies which demonstrated that the permanent loss or acute silencing of RTN neurons abolishes or significantly reduces ventilatory CO2 sensitivity were found to be preceded by Na+ entry, reduced by inhibition of the Na+/HCO3\u2212 cotransport (NBC) or Na+/Ca2+ exchange (NCX) and abolished in the absence of extracellular Na+. Acidification\u2010induced Ca2+ responses were also dramatically reduced in brainstem astrocytes of mice deficient in the electrogenic Na+/HCO3\u2212 cotransporter NBCe1 , since pharmacological agents which block functional connexin hemichannels have little effect on acidification\u2010induced Ca2+ responses in brainstem astrocytes in anesthetized and mechanically ventilated rats was reported to trigger release of ATP from the ventral brainstem surface gene lead to a neurodevelopmental disorder called Rett syndrome, which is characterized by irregular breathing pattern and blood gas instability observed in some other pathological conditions.Impaired astroglial mechanisms may contribute to the development of abnormal breathing patterns observed in some prototypical neurological disorders. In humans, mutations of the transcriptional regulator methyl\u2010CpG\u2010binding protein 2 (2. Howarth and colleagues (2017) reported that in anesthetized mice, increases in the level of inspired CO2 trigger [Ca2+]i responses in cortical astrocytes, which in turn may evoke cerebral vessel dilations via stimulation of COX\u20101 activity followed by PgE2 release and its action on cerebrovascular smooth muscle cells is the only circulating peptide known to stimulate appetite and increase food intake. Ghrelin is mainly produced and released by oxyntic glands of the gastric fundus and its CNS actions increase food intake, produce weight gain, and promote adiposity via increased production of orexigenic neuropeptides such as neuropeptide Y and Agouti\u2010related peptide (AGRP) by the neurons of the arcuate nucleus of the hypothalamus antagonist [(D\u2010Lys3)\u2010GMPR\u20106] express functional ghrelin receptors and respond to physiological concentrations of ghrelin with elevations in intracellular [Ca4.22+]i . This hypothesis can be tested in the future by recording the sympathetic and cardiovascular effects of GLP\u20101R activation within the sympathoexcitatory brainstem regions in conditions when astroglial signaling pathways are blocked using molecular approaches.There is evidence that the central effects of GLP\u20101 analogs are associated with excitation of pre\u2010sympathetic C1 neurons, increases in central sympathetic drive, systemic arterial blood pressure and heart rate bulk\u2010loaded with the Ca2+ indicator Oregon Green\u2010488 BAPTA\u20101 AM (OGB\u2010488). Astrocytes were identified by labeling with sulforhodamine 101 (SR101) as described previously were abolished by either MRS2179 or apyrase are sensitive to key hormonal factors whose central actions provide important information about the nutritional state and energy demands of the organism. It remains to be determined whether these sensitivities are exclusive features of astrocytes residing in brain areas involved in metabolic control. It also remains to be determined whether chronic exposure to the elevated levels of these hormones may alter astroglial function and signaling mechanisms. This may have a significant impact on the control of feeding behavior and cardiorespiratory homeostasis and ultimately contribute to the pathogenesis of metabolic and/or cardiovascular disease.52 and glucose), metabolic by products (CO2), and hormonal metabolic factors involved in the CNS mechanisms controlling food intake and energy balance. In the brainstem, astrocytes modulate the activities of the neuronal circuits responsible for the generation of the respiratory and autonomic rhythms and the development of the adaptive changes in breathing and sympathetic nerve activity in conditions of increased metabolic demand. The key signaling molecule which mediates communication between astrocytes and the brainstem cardiorespiratory networks appears to be ATP, although other gliotransmitters may also play a role. Furthermore, there is evidence that altered astroglial function may contribute to the pathogenesis of disparate respiratory and cardiovascular disorders such as Rett syndrome, heart failure and systemic arterial hypertension.There is growing evidence to suggest that astrocytes actively monitor CNS metabolic milieu and contribute to the development of adaptive physiological respiratory, cardiovascular and behavioral responses which maintain metabolic homeostasis. Anatomical and functional features of astrocytes allow them to detect and respond to changes in the brain parenchymal levels of metabolic substrates (O"} +{"text": "Over the last 20 years a number of studies have been published using progressive eccentric exercise protocols on motorized ergometers or similar devices that allow for controlled application of eccentric loads. Exercise protocols ramp eccentric loads over an initial 3 weeks period in order to prevent muscle damage and delayed onset muscle soreness. Final training loads reach 400\u2013500 W in rehabilitative settings and over 1200 W in elite athletes. Training is typically carried out three times per week for durations of 20\u201330 min. This type of training has been characterizes as moderate load eccentric exercise. It has also been denoted RENEW (Resistance Exercise via Negative Eccentric Work by LaStayo et al., There are generally acknowledged modalities to train either aerobic capacity (endurance training) or the capacity of muscles to develop force (strength training). Endurance training aims at improving the overall capacity of the organism to perform aerobic work by activities such as running, bicycling, or cross-country skiing involving a significant muscle mass. Effective exercise intensities are characterized by individual target heart rates to be achieved in training sessions lasting typically 30 min or more for 5 days per week. Strength training targets individual muscles or muscle groups whereby effective protocols use 1\u20133 sets of 8\u201312 near maximal contractions per individual muscle of muscle groups in 2\u20133 training sessions per week , has become popular as a time-efficient mean for improving cardiorespiratory and metabolic capacities milliseconds such as drop jumps and similar plyometric exercises are most prone to massive and acute muscle damage. It can be calculated that a drop jump from 1 m in altitude loads the Achilles tendon and associated muscles with several thousands of watt of negative power to decelerate the body upon landing on the ground. The current evidence suggests that drop jumps and plyometric exercises are effective in increasing kicking distance, speed and jumping ability in soccer players by which this is achieved and the relative contribution of muscles and tendons remains open. A change in titin isoform pattern observed with eccentric exercise in the rat experiment has been taken to suggest a role for this protein in increasing muscle stiffness we decided to perform a \u201cproof of principle\u201d study for the use of moderate load eccentric exercise programs in coronary patients. The main initial concern of cardiologists was that eccentric exercise could lead to high pressure in the central circulation.n = 10), and percutaneous transluminal coronary angioplasty (n = 9) or coronary bypass surgery studied by Meyer et al. with heart failure that performed either stair ascending or descending for 6 weeks, three times per week. Gremeaux et al. , could hold considerable promise both for women and men to moderate load eccentric exercise on an eccentric ergometer , in a study including 62 elderly men and women of average age 81 years skis. Using accelerometers fixed to the sternum of athletes during world cup competitions we measured peak accelerations in excess of 3 g during turns. The centrifugal forces in a carved turn must be taken-up essentially only by the outer leg. This means that in 80 kg athletes, leg extensors must be capable of eccentrically resisting forces up to 3000 N were subjected to a progressive eccentric exercise protocol involving the elbow flexors (Reid et al., Parkinson's disease were randomized to a 12-weeks progressive moderate load eccentric training program or to an active control group (Dibble et al., multiple sclerosis were randomized to either a standard exercise group or to a standard exercise and progressive moderate eccentric exercise group for 12-weeks (Hayes et al., Progressive eccentric exercise training programs with small numbers of participants have been used for various neurological disorders. A group of 14 children with Moderate load eccentric exercise or RENEW has been used effectively as a rehabilitative tool for medical conditions in which central energy supply is of concern. Its effectiveness in achieving muscle strength and volume gains at low metabolic loads has amply been demonstrated. Moderate load eccentric exercise can lead to similar improvements of muscle performance than conventional strength training protocols but at lower joint loads. This makes it a valuable tool in orthopedic rehabilitation and injury prevention. There is a distinct lack of large-scale multi-center studies prospectively exploring eccentric exercise modalities making the currently available evidence essentially based on exploratory-type studies. We also need to acknowledge that the basic physiological mechanisms by which muscle produce force when lengthened are still debated (Hoppeler, The author confirms being the sole contributor of this work and approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Phase contrast magnetic resonance (PCMR) has been shown to provide accurate measurement of differential branch pulmonary artery (BPA) flow in patients with congenital heart disease when compared to the gold standard, lung perfusion scintigraphy (LPS). Although most studies have compared the ratio of net BPA flow volume (NFV) to LPS, one study found that forward flow volume (FFV) correlated more accurately. PCMR quantification of pulmonary venous (PV) flow has also been shown to be feasible and highly reproducible. We sought to calculate differential pulmonary blood flow using PV flow and compare the results with both NFV and FFV calculations.Retrospective review of 56 patients with congenital heart disease who had phased contrast flow quantification of both branch pulmonary arteries and pulmonary veins during the same study. FFV and NFV of RPA and LPA, as well as their ratios were calculated and were compared to RPV and LPV flow volume.There were 22 males, and the median age was 12.2 years of age. 37 patients had tetralogy of Fallot and the remainder had other conotruncal abnormalities requiring intervention to the right ventricular outflow tract. Agreement between NFV and PV was superior than FFV and PV . The BPA flow ratio calculated by both NFV and FFV had good agreement with PV flow ratio .Differential lung perfusion calculated using the PV flow method has excellent agreement with that calculated using PA flow measurements. This technique may be useful in patients where accurate measurement of BPA flow may not be feasible ."} +{"text": "Deep sternal wound complications are uncommon after cardiac surgery. They comprise sternal dehiscence, deep sternal wound infections and mediastinitis, which will be treated as varying expressions of a singular pathology for reasons explained in the text.This article reviews the definition, prevalence, risk factors, prevention, diagnosis, microbiology and management of deep sternal wound infections and mediastinitis after cardiac surgery. The role of negative pressure wound therapy and initial and delayed surgical management is discussed with special emphasis on plastic techniques with muscle and omental flaps. Recent advances in reconstructive surgery are presented.Deep sternal wound complications no longer spell debilitating morbidity and high mortality. Better understanding of risk factors that predispose to deep sternal wound complications and general improvement in theatre protocols for asepsis have dramatically reduced the incidence of deep sternal wound complications. Negative pressure wound therapy and appropriately timed and staged muscle or omental flap reconstruction have transformed the outcomes once these complications occur. Deep sternal wound complications are uncommon after cardiac surgery. Deep sternal wound infections (DSWIs) are invariably accompanied by varying degrees of sternal dehiscence and mediastinitis. Alternately, even a purely mechanical sternal dehiscence will quickly get secondarily infected unless rewiring is undertaken expeditiously. In this article, therefore, deep sternal wound complications will be presumed to include deep sternal wound infections (DSWIs), sternal dehiscence (SD) and mediastinitis, and will be treated as a singular entity which might have varied expression rather than qualitatively different pathologies. The author recognises that mediastinits and deep sternal wound infections after cardiac surgery can occur without frank sternal dehiscence, but what invariably perpetuates a deep sternal wound infection is a communication of presternal tissues with mediastinum.Deep sternal wound infections, sternal dehiscence and mediastinitis have moved from being fearsome complications of cardiac surgery to manageable problems. Negative pressure wound therapy has revolutionised the ward-based management of deep sternal wound infections. Pectoralis, rectus and latissimus flaps and omental grafts are commonest forms of sternal reconstruction although free flaps, intercostal perforator flaps, breast flaps and allogeneic bone grafts have been used.A literature search was done using Healthcare Databases Advance Search comprising in particular PubMed and Medline databases up to June 2017 using MeSH headings: Sternal Wound Infections, Sternal Dehiscence, Sternal Reconstruction, Latissimus Dorsi Flaps, Omental Flaps, Rectus Abdominis Flaps, Pectoral Flaps, Negative Pressure Wound Therapy.We present a review of prevalence, risk factors, prevention, diagnosis, microbiology and pathogenesis of sternal wound infections and mediastinitis. We discuss management of early and later, and therefore mostly infected, dehiscences and the central contribution of negative pressure wound therapy. The crucial role of various muscle flaps including pectoralis, rectus and latissimus flaps and omental grafts is specifically elaborated upon. Also presented is a review of some newer approaches that have been recently introduced to manage this complication that can prove particularly devastating if not treated early and well.Deep sternal wound complications occur in 0.8% to 1.5% patients and as high as 8% when bilateral IMAs are used . Cheung Risk factors for deep sternal wound complications include breaches in theatre asepsis, long operations , postopePrevention of sternal wound complications should be the cornerstone of all cardiac surgery. A precise aseptic technique, reducing personnel traffic in theatres, shorter and less complex procedures and heightened awareness of asepsis in theatre and ICU all reduce the prevalence of infection . ProphylDiagnosis of deep sternal wound infection, dehiscence or mediastinitis is generally made clinically. Disproportionate pain on coughing, persistent wound discharge, appreciation of movement of two sternal halves relative to each other unmasked by deep inspiration or coughing are the usual warning signs. Indeterminate malaise, fever, rising WBC count and CRP, deteriorating renal function, widened mediastinum and displaced sternal wires are occasionally seen. CT scan showing sternal disruption and retrosternal fluid with air pockets is diagnostic although some edema and clot is present in mediastinum routinely after cardiac operations .Gardlund et al. identified three different types of postoperative mediastinitis in cardiac surgery with respect to microbiology and pathogenesis: 1. mediastinitis associated with obesity, chronic obstructive airway disease and sternal dehiscence, typically caused by coagulase negative staphylococci 2. mediastinitis following peroperative contamination of mediastinal space, often caused by staph aureus 3. Mediastinitis mainly caused by spread from concomitant infection in other sites during postoperative period, often caused by gram negative rods . A largeThe principles of the treatment of wound infections are standard and involve drainage of infected spaces, debridement of necrotic tissue, antibiotic therapy and strategies to achieve closure of sternal space. Quite often the treatment has to be individualised based on the infecting organism, patient\u2019s status and, most crucially, the depth of infection . ImmediaP\u00a0=\u00a00.021). In DSWI infected with MRSA, in particular, NPWT significantly reduced in-hospital mortality caused by DSWI . Equally importantly, NPWT as a bridge therapy to tissue flaps may play a major role in treating DSWI and improve prognosis for patients with MRSA-infected sternal wounds [Negative pressure wound therapy (NPWT) applies subatmospheric pressure to the surface of wound, removes excessive fluid, decreases wound edema, accelerates wound healing and granulation tissue formation, stabilises chest wall, increases sternal blood flow and has been shown to improve early and long term survival in patients with deep sternal wound infection. It has emerged as one of the most significant developments in the last two decades in the management of sternal wound infections , 32. Alll wounds . DebreceAll sternal dehiscences, whether sterile or infected, causing frank sternal instability result in considerable pain, respiratory distress and occasionally haemodynamic compromise and need to return to theatre for immediate reoperation. This involves removal of all sternal wires, suture and necrotic material. If sternum is generally healthy and well vascularised, rewiring after limited sternal debridement suffices. There is no substitute to native healthy sternum in front of heart. The author is not a great believer in continuous warm saline, povidone-iodine or antibiotic irrigation postoperatively. Their benefit is doubtful but therA real challenge in sternotomy wound complications comes from patients who develop early infected sternal dehiscences with substantial loss of sternal bone with considerable mediastinal instability and relative movement of the residual sternal plates. This is obviously not amenable to rewiring and despite filling the residual space with muscle flap there might still be residual relative movement of sternal plates with mediastinal instability. Sternal debridement followed by NPWT till mediastinal stability is achieved by tissue adhesions followed by reconstructive flaps is probably preferable in these patients but careful and close observation is required.Most patients with infected sternotomy wounds with loss of sternal bone or soft tissue will require reconstructive surgery requiring mostly muscle flaps individually or in combination, preferably after a few weeks following initial surgery, allowing for mediastinal adhesions and stability. The most commonly used flaps are pedicled pectoralis major, rectus abdominis and omental flaps. Less commonly pedicled latissimus dorsi flaps may be used. Rarely free flaps with AV loops may be used. Anecdotally, internal mammary artery perforator (IMAP), superior epigastric artery perforator (SEAP), intercostal artery perforator (ICAP) flaps, breast flaps, falciform ligament flaps or free gracilis flaps have been employed. Bilateral pectoralis major flaps based on thoracoacromial artery will cover most of the sternal wound except occasionally the lower 1/4th. The sternal and costal origins of the muscle are dissected off the bone and clavicular origin left intact. If the insertion of the pectoralis major tendon to the anterior lip of the humeral intertubercular sulcus is divided in the deltopectoral groove, the muscle can be mobilised inferomedially to reach the lowest part of the sternal wound. Pectoralis major flaps are the workhorse flaps for sternal reconstruction and can reach almost all sternal wounds. Apart from occasional seromas and rare incidences of shoulder weakness when the insertion is divided, they are relatively free of side effects. Pedicled rectus abdominis flaps are especially suited to the lower 1/4th of the sternal wound and are often used in combination with the pectoral flaps. They obviously need a long abdominal incision unless harvested laparoscopically. They are based on the superior epigastric artery and therefore a contralateral flap is used when LIMA has been used although there is evidence of reasonable collateral supply even when a flap ipsilateral to IMA harvest is used. Rectus abdominis flap is easy to harvest and is a turn over flap not requiring considerable tissue dissection but may lack muscle mass to support extensive sternal loss. Omental flaps based on right gastroepiploic artery and unusually on left gastroepiploic artery provide more vascular tissue potentially with greater infection neutralising properties and have been used extensively. There have been anecdotal reports of their use when there is obvious infection of the heart and great vessels. Their obvious disadvantages include the need to enter peritoneal cavity in the presence of mediastinitis and rare herniations. They however can be harvested laparoscopically. Latissimus dorsi flaps provide extensive bulk and can be employed as both muscle and myocutaneous flaps and are uniquely suited when there is massive sternal loss due to necrosis. They clearly need large harvesting incisions but do not disrupt blood supply to sternum and parasternal tissues.Wu et al. reported 19 patients who underwent bilateral pectoralis major flaps for deep sternal wound infections (DSWI) out of which 4 needed additional rectus abdominis (RA) flap because the pectorals would not reach the inferior part of the wound. No patient showed infection recurrence . PremingKuntscher et al. reported on the versatility of 7 superiorly based vertical musculocutaneous rectus abdominis flaps for reconstruction of osteocutaneous defects following sternal osteomyelitis and tumour resection, with occasional requirement of arterial and/or venous recharging, with no flap loss . Netschen\u00a0=\u00a011), a single pectoralis major flap and omentoplasty (n\u00a0=\u00a020) and bilateral pectoral flaps only (n\u00a0=\u00a01) with no early or late flap failures [n\u00a0=\u00a08) and anterolateral thigh perforator free flaps (n\u00a0=\u00a04) anastomosed to the right gastroepiploic vessels harvested in 42% patients laparoscopically with uneventful healing [Lovich et al. described their experience of 20 patients with postcardiotomy sternal wound infections 17 of whom underwent omental pedicle grafts with excellent results. A left subcostal incision began to be used for omental harvesting to avoid incisional hernia towards the later part of their practice . Brabendfailures . Milano failures . Parissifailures . Pieri efailures . Dorseif healing . Kaul et healing . Omental healing .Meland et al. described intrathoracic transposition of extrathoracic skeletal muscle in intrathoracic infections associated with infection, leakage or bleeding of airway, lung parenchyma, esophagus, heart or great vessels . Tizian Taeger et al. described 8 patients with extensive deep sternal osteomyelitis requiring total sternectomy where the large defect was reconstructed by free flap transplantation using the vastus lateralis myocutaneous flap, rectus abdominis and bipedicled latissimus dorsi/parascapular flap. As the local recipient vessels were depleted in all patients, the pedicle of the flap was anastomosed to a high-flow and short-length subclavian arteriovenous loop as recipient vessel in all cases . Wang etCooper et al. described the use of inferomedial fasciocutaneous breast flaps to reconstruct sternal dehiscence with concomitant soft tissue loss . De Fontn\u00a0=\u00a010), calva (n\u00a0=\u00a01) and only crushed spongy bone (n\u00a0=\u00a02) along with transverse titanium plates and bipectoral flaps as a promising and easily applied method in serious sternal bony loss [Pancholy et al. used sternal reconstruction with sternal plating and local use of bone morphogenetic protein (BMP) and demineralised bone matrix, preceded by NPWT and followed by pectoral flaps to achieve good sternal stability as seen at 3 and 6\u00a0months follow up . Kalab eony loss .There are a number of studies which either describe use of a combination of flaps or which offer comparative analyses of the various types of flaps for sternal reconstruction. Lindsey described his experience with 47 patients with DSWE undergoing reconstruction with pectoral and rectus flaps and observed striking differences between outcomes of flap reconstruction within or beyond 5\u00a0days of wound drainage and debridement, with 23% major wound complications in the first group when flap reconstruction was done within 5\u00a0days of dehiscence . DescribAll cardiac surgeons should be able to construct pectoralis major muscle flaps or myocutaneous advancement flaps which are the work-horse sternal reconstruction flaps. This is in the anatomical area of their usual site of activity and will address around 80% of all sternal reconstructions. The pectoralis major needs to be dissected off from the chest wall origin and that is all that needs to be done for the advancement flaps. For the pectoralis major muscle flap, the muscle in addition needs to be dissected from the overlying subcutaneous tissue and then stitched to the contralateral muscle in front of the rewired or debrided sternum. Only a small number of patients with extensive sternal defects will need a further rotation of the muscle inferomedially and this can be done easily by a separate incision in the dectopectoral groove and dividing the insertion of the pectoral head to the lateral lip of the bicipital groove of the humerus and anterior lip of deltoid tuberosity. It is important to preserve the pectoral branch of the thoracoacromial artery which is the main blood supply to the pectoralis major. When there is substantial sternal loss inferiorly, rectus abdominis flap might need to be constructed in addition to the pectoralis flaps. This is done by a long midline abdominal incision and dissecting off the muscle within the sheath based on the superior epigastric artery after dividing it inferiorly. Usually the side contralateral to the LIMA harvest is taken. This can also be done by a cardiac surgeon easily. Cardiothoracic surgeons who have reasonable experience of thoracic surgery should be well within their comfort zone to harvest latissimus dorsi myocutaneous flaps for anterior turn-over when encountering substantial sternal and soft tissue loss. The pedicle is based on thoracodorsal artery. All cardiothoracic surgeons who have had general surgical training will be able to harvest greater omentum based on right gastroepiploic artery and using it for reconstructions in inferior or total sternal problems. It is particularly important to pay attention to the greater curvature of the stomach and the middle colic artery in the transverse mesocolon.In general, many cardiac surgeons will perform most of the above procedures with a plastic surgeon. However, I do feel that as cardiac surgeons we should endeavour to train ourselves to be able to treat all the site-specific wound complications that we encounter.Better understanding of risk factors that predispose to deep sternal wound complications and general improvement in theatre protocols for asepsis have dramatically reduced the incidence of deep sternal wound infections, sternal dehiscence and mediastinitis following cardiac surgery. Negative pressure wound therapy (NPWT) and appropriately timed and staged muscle or omental flap reconstruction have revolutionised the outcomes once these complications occur."} +{"text": "We read with interest the article by Jacob and colleagues on the rRBC may use different contradictory pathways to modulate microvascular flow by modifying nitric oxide (NO) bioavailability, which is responsible for precapillary dilatation and capillary perfusion.On the one hand, erythrocytes may reduce NO bioavailability through hemoglobin NO scavenging; on the other hand, enhancing hematocrit may increase viscosity and wall shear stress (WSS), a crucial agonist enabling endothelium NO release.Thus, in hypoxic conditions, RBC may sense tissue oxygen tension and release vasodilatory agents such as NO or ATP.In moderate hemodilution, blood viscosity is reduced, cardiac output increases, wall shear rate increases and WSS is unchanged (WSS\u2009=\u2009WSR\u2009\u00d7\u2009viscosity). NO bioavailability increases because of reduced NO scavenging by erythrocytes, leading to systemic vasoplegia. In extreme hemodilution (hematocWe have known since 1999 from the results of a trial using modified human hemoglobin in traumatic hemorrhagic shock that modWe do not share the authors\u2019 opinion that it is \u201cbetter to choose hemorrhagic rather than septic shock for yourself\u201d. Guidelines for traumatic hemorrhagic shock management do not c Nicolas Morel and Marie Moisan have drawn attention to the special but confounding role played by RBC in modulating regional microcirculatory flow distribution. As they correctly point out, contributory effects include changes in blood viscosity and scavenging or provision of vasoactive metabolites. Such influences must be expected, especially in conjunction with transfusions of RBC in situations of treatment or prevention of shock . AlthougThese few examples of the problems still associated with understanding the mechanisms by which RBC may influence regional flow distribution illustrate the continued necessity of conducting well-planned clinical studies for the improvement of patient outcome in situations of shock. Although at first sight the approach to management of traumatic hemorrhagic shock seems more clear than that of septic states, Morel and Moisan have provided good reasons why this is not resolved."} +{"text": "Genome editing technologies enable scientists to modify DNA sequence at specific genomic loci in various cells and species. There are several editing tools, including homing endonucleases fused with cytidine deaminases was directed to specific genomic locus and replace a C with a T (or replace G with A) within a defined window (Komor et al., Protein and Cell, two groups demonstrated that base editing can be applied to generate mouse models with precise modification very efficiently (Kim et al., Including an exciting piece of work published in this volume of These studies highlighted that base editor can serve as a powerful tool to generate precise point mutations in animal models. However, in addition to the desired precision base substitution, other types of mutations such as indel and proximal-site deamination are often generated. The next step would be to perfect the system to enable precise base editing at a single base resolution with high efficiency, without introducing extra mutations."} +{"text": "Aeromonas hydrophila and two strains of Serratia proteamaculans after searches in the GenBank\u00ae database were analyzed. The analysis which included 104 other bacteria strains, was carried out using molecular evolutionary genetic analysis (MEGA 7.0) software.Here the data on evolutionary relationships of persistent bacteria from water tanks and their close relatives are shown. Curated sequences of the hypervariable region of ribosomal ribonucleic acid (rRNA) obtained from a strain of Specifications TableValue of the Data\u2022Data shows phylogeny of water tank bacteria and other species from several sources.\u2022Selection of strains for comparative whole-genome analysis can be facilitated by the data.\u2022Data is useful for further investigations of weak or strong biofilm producers during fouling of water tanks.1A. hydrophila , HG328350 and HG328352 (S. proteamaculans) from previous work Sequences deposited in the Genbank\u00ae under accession numbers Listeria monocytogenesPseudomonas aeruginosaPseudomonas fluorescensPseudomonas species Updated searches were carried out after which the top hits showing sequences from closely related culturable strains were selected for each genus and then subjected to phylogenetic analysis with MEGA software, version 7"} +{"text": "Streptococcus involving her lower extremities. She was hospitalized, and blood cultures grew out group B Streptococcus. She received treatment with intravenous levofloxacin and vancomycin and demonstrated clinical improvement. However, careful inspection of the area of cellulitis on her lower extremities revealed papillary lesions consistent with a condition known as papillomatosis cutis lymphostatica ?This is the first case of papillomatosis cutis lymphostatica reported in the emergency medicine literature.How might this improve emergency medicine practice?This image will help clinicians recognize and treat papillomatosis cutis lymphostatica. In the ED, recognition of this rare complication would allow clinicians to have an appropriately high level of suspicion for cellulitis."} +{"text": "Cell surface adhesion receptors of the syndecan family initiate intracellular events through clustering of receptors. This crucial clustering occurs through receptor dimerization or oligomerization, which is mediated by receptor transmembrane domains. However, the exact role of the transmembrane domain during receptor activation is not fully understood. Researchers have not yet determined whether the transmembrane domain functions solely in the physical aspects of receptor clustering, or whether the domain has additional functional roles. Here we review recent advances in understanding the functionality of transmembrane domain\u2014dependent oligomerization of syndecan cell adhesion receptor."} +{"text": "As a chronic immune complication, celiac disease has a broad spectrum of clinical manifestations and gluten ingestion as an external trigger will induce the onset of this disease in genetically predisposed individuals. Because of the complex nature of celiac disease and various cascades of immunological pathways, therapies which are tend to target a single pathway or factor, often have unsatisfactory results. Thus, it should be considered that the new emerging area of cellular therapy by targeting multiple pathways may hold the key for treating celiac affected patients with complicated forms of this disease. The aim of this review is to discuss different pathways which are affected by celiac disease and to compare how various strategies, mainly cellular therapies, can regulate these pathways. Celiac disease is a prevalent and immune mediated intestinal disorder with complicated genetic backgrounds . The onsIntestinal RegenerationThe emerging area of cellular therapy for CD is mainly base on the stem cell therapy which has the advantage of targeting multiple pathway and has yielded the promising results. It is crucial to bear in mind that the intestinal tract has a highly regulated process for the regeneration mainly due to the harsh environment which it is exposed. All the differentiated epithelial cells of the intestine derived from a single intestinal stem cell (ISC) (CD133+/Lgr5+ crypt cell) compartmStem cell transplantation is an effective treatment for patients with severe refractory autoimmune diseases compare to conventional treatments like ineffective GFD regimen for patients with refractory CD and enteropathy- associated T cell lymphoma . ConsideHematopoietic Stem CellHSCs are a heterogeneous population of cell that are derived from mesoderm and can produce both the myeloid and lymphoid lineages of blood cells. HSCs are shown to play a prominent role in mucosal healing due to tMesenchymal Stem CellMesenchymal stem cells (MSCs) are multi-potent stromal cells that can differentiate into a variety of cell types like myocyte, adipocyte, osteoblast and chondrocyte . They caThe Epithelial BarrierMaintaining the selective permeability is achieved by the complex interplay among epithelial cells in intestine. Epithelial barrier is consisting of both tight and adherent junctions that connecting the adjacent enterocytes. Pro- inflammatory cytokines can disrupt both complexes via phosphorylation and in turn increase the permeability of intestine due the opened tight junctions. Studies have shown that in mouse model of colitis, MSCs can exert their protective effect by reassembling claudins which have the most prominent role in tight junctions and thus maintaining the epithelial barrier . MoreoveNatural Killers and Intraepithelial LymphocytesThe intraepithelial lymphocytes are normally found sparsely across small intestinal mucosa and have a crucial role in innate immune response. Their increasingly activation in CD leads to epithelial damage. Through production of IFN-y, perforin and granzymes, CD8+ TCRaB+ cells induce apoptosis in enterocytes . Also thAntigen-Presenting CellsHLA genes are the most powerful susceptibility determinants for CD predisposition . HLA-DQ2B cell-lymphocyteIntestinal plasma cells produce the IgA specific for tissue transglutaminase and gluten derived peptides which is the hallmark for active CD. MSCs derived chemokine involving CCL2 and CCL7 inhibit STAT 3 and thus can suppress B cells proliferation and differentiation into plasma cells . MoreoveT Reg (Regulatory T cells)In vitro studies have shown that MSCs can express various extension of FoxP3 depending on culture condition that in turn increases T regulatory population. FurtherT Cell ResponseMSCs induce its beneficial effect through immunomodulation of T cell response by shifting the Th1/ Th2 ratio toward Th2 profile . IFN-y eAs it has been demonstrated in this review, by possessing a wide range of immunomodulation properties, MSCs can target almost all the mechanisms involved in CD pathogenesis. Although it needs to be considered that MSCs apply their actions in a specific mucosal microenvironment. Due to their cell to cell interaction and by releasing a wide range of immunoregulatory substances, it\u2019s not essential for MSCs to be persistence in the damaged mucosa. It is worth mentioning that the most common rout of MSCs delivery in their therapy is via intravenous injection and in many occasions it has been shown that MSCs were trapped in the lungs due to their big size. Furthermore, studies have shown no biological differences regarding the sources which MSCs are obtained, but MSCs transplantation is an invasive procedure and there are many potential limitations when they are obtained from bone marrow and adipose tissue. These limitations have given rise to the need of obtaining MSCs from other sources with unlimited donors, including umbilical cord blood, amniotic fluid, amnion and placenta which has the best proliferative properties. Finally, further methodological variables such as the rout, doses and intervals of administration need to be tuned for the best approach before therapeutic prospect of using MSCs as the clinical therapy for celiac disease."} +{"text": "Tumors comprise a variety of specialized cell phenotypes adapted to different ecological niches that massively influence the tumor growth and its response to treatment.glioblastoma multiforme, a highly malignant brain tumor, we consider a rapid proliferating phenotype that appears susceptible to treatment, and a dormant phenotype which lacks this pronounced proliferative ability and is not affected by standard therapeutic strategies. To gain insight in the dynamically changing proportions of different tumor cell phenotypes under different treatment conditions, we develop a mathematical model and underline our assumptions with experimental data.In the background of We show that both cell phenotypes contribute to the distinct composition of the tumor, especially in cycling low and high dose treatment, and therefore may influence the tumor growth in a phenotype specific way.Our model of the dynamic proportions of dormant and rapidly growing glioblastoma cells in different therapy settings suggests that phenotypically different cells should be considered to plan dose and duration of treatment schedules. Whether such conversions occur spontaneously or can be induced specifically or randomly by extrinsic or intrinsic mechanisms is poorly understood. We thus assumed a spontaneous event which can be modeled by a constant rate.An important aspect of our model is the conversion between the different cell phenotypes. Recent studies suggest that dormant cells may originate from \u201cnormal\u201d tumor cells by currently intensively investigated mechanisms e.g. \u201353). As . As 53])Using our theoretical approach we showed that dependent on the applied treatment strength an equilibrium balance between rapidly proliferating cells and dormant cells is eventually reached. At this fixed point and with low dosage or no treatment, mostly rapidly proliferating cells dominate the population, similar to the findings of Basanta and colleagues . At stroSeveral previous models discuss the effect different treatment schedules on various aspects of the cancer growth like angiogenesis , 54 or eIn this study, we have developed a theoretical model to predict the tumor growth kinetics under different treatment strengths including a dormant cell phenotype and underlined our theoretical approach with experimental data. Using our model which allows for phenotypic conversion, we could simulate how different tumor cell phenotypes proportionally contribute to the growing tumor mass in cycling treatment schedules. Additionally, we could observe that switching between high and low dosage treatment remarkably affects tumor growth in a frequency dependent way.Thus, the dynamic proportions between cell phenotypes should be taken into account in the optimization of treatment schedules in order to control tumor growth."} +{"text": "Objective: To clarify whether agraphia or alexia occurs in lesions of the left posterior middle temporal gyrus.Methods: We assessed the reading and writing abilities of two patients with this lesion using kanji (Japanese morphograms) andkana (Japanese syllabograms).Results: Patient 1 first presented with pure alexia more impaired for kana after an infarction in the left middle and inferior occipitalgyri and right basal occipital cortex, and after a second infarction in the left posterior middle temporal gyrus adjoining the firstlesion he showed alexia with agraphia for kanji and worsened alexia for kana; kanji alexia recovered over the following six to 10months. Patient 2 presented with alexia with agraphia for kanji following a hemorrhage in the left posterior middle and inferiortemporal gyri, which resolved to agraphia for kanji at two months after onset. Kana nonword reading was also slightly impaired,but became normal by six months post-onset. In both patients, kanji agraphia was mostly due to impaired character recall.Conclusion: The present patients demonstrate that damage to the left posterior middle temporal gyrus alone can cause agraphiafor kanji. If the adjacent mid fusiform/inferior temporal gyri (Area 37) are spared, the kanji alexia is transient."} +{"text": "Acute hepatitis E virus infection during pregnancy has a high fatality rate in developing countries. Little data are available on chronic infection in pregnant women. We report a case of chronic hepatitis E during treatment with infliximab and azathioprine, without adverse event during pregnancy and with spontaneous resolution after delivery. Hepatitis E virus (HEV) genotype 1 causes a high number of deaths of pregnant women in developing countries since May 2014 led her physician to suspect viral hepatitis; HEV infection was later diagnosed in September 2014 by detection of HEV IgM and RNA in plasma , panel APersistence of HEV has not been reported among patients receiving infliximab or azathioprine. However, HEV persistence was reported in a patient receiving azathioprine combined with oral steroids , panel B10 copies/mL, and ALT returned to reference range and a >3-log decrease of HEV RNA , panel BBecause of the high rate of severe acute hepatitis E reported in pregnant women in developing countries, we monitored the patient for negative outcomes during gestation but found none. This finding is consistent with the small number of reported HEV infections during pregnancy in industrialized countries (Innate immunity has been suggested as essential for severe outcomes of acute HEV infection during pregnancy (The risk for HEV vertical transmission seems dependent on viral load ("} +{"text": "A high\u2010throughput chemical genetic screen of chemical compounds from several commercial libraries has revealed that lithocholic bile acid (LCA), and some other bile acids, can slow yeast chronological aging . The robLCA and other bile acids are mildly toxic molecules that cause a so-called \u2033hormetic\u2033 stress response in animals; because bile acids elicit chemical hormesis, they act as endobiotic geroprotective regulators that can delay the onset and slow the progression of animal aging . Yeast cWe then used the selected long-lived yeast mutants for a laboratory test of evolutionary theories of programmed or non-programmed aging . ProgramIn sum, a laboratory test of evolutionary aging theories provided evidence that yeast cells have evolved some active mechanisms for limiting their lifespan upon reaching a certain chronological age. Furthermore, it seems that these mechanisms can drive the evolution of yeast longevity towards maintaining a finite yeast lifespan within ecosystems. One could hypothesize that these mechanisms may involve the ability of the parental wild-type strain to secrete into growth medium certain compounds capable of inhibiting growth or even killing long-lived yeast mutants. Because these compounds may be responsible for the maintenance of a finite yeast lifespan within ecosystems, it would be important to identify them."} +{"text": "We aimed to study whether previously described impairment in decision making under risky conditions in patients with Parkinson's disease (PD) is affected by deficits in using information about potential incentives or by processing feedback (in terms of fictitious gains and losses following each decision). Additionally, we studied whether the neural correlates of using explicit information in decision making under risk differ between PD patients and healthy subjects. We investigated ten cognitively intact PD patients and twelve healthy subjects with the Game of Dice Task (GDT) to assess risky decision making, and with an fMRI paradigm to analyse the neural correlates of information integration in the deliberative decision phase. Behaviourally, PD patients showed selective impairment in the GDT but not on the fMRI task that did not include a feedback component. Healthy subjects exhibited lateral prefrontal, anterior cingulate and parietal activations when integrating decision-relevant information. Despite similar behavioural patterns on the fMRI task, patients exhibited reduced parietal activation. Behavioural results suggest that PD patients\u2019 deficits in risky decision making are dominated by impaired feedback utilization not compensable by intact cognitive functions. Our fMRI results suggest similarities but also differences in neural correlates when using explicit information for the decision process, potentially indicating different strategy application even if the interfering feedback component is excluded."} +{"text": "Background. Brain computer interface (BCI) is a combination of software and hardware communication protocols that allow brain to control external devices. Main purpose of BCI controlled external devices is to provide communication medium for disabled persons. Now these devices are considered as a new way to rehabilitate patients with impunities. There are certain potentials present in electroencephalogram (EEG) that correspond to specific event. Main issue is to detect such event related potentials online in such a low signal to noise ratio (SNR). In this paper we propose a method that will facilitate the concept of online processing by providing an efficient filtering implementation in a hardware friendly environment by switching to finite impulse response (FIR). Main focus of this research is to minimize latency and computational delay of preprocessing related to any BCI application. Four different finite impulse response (FIR) implementations along with large Laplacian filter are implemented in Xilinx System Generator. Efficiency of 25% is achieved in terms of reduced number of coefficients and multiplications which in turn reduce computational delays accordingly. A brain computer interface (BCI) is a communication system that allows humans to interact with their surroundings, without any involvement of nerves and muscles, by using certain control signals generated by brain that are stored in the form of electroencephalogram (EEG) . BCI creThere are different brain imaging and brain signal acquisition techniques are available that can be invasive or noninvasive. Noninvasive BCI systems do not require any type of surgery, and these are often preferred over invasive methods . These tThe EEG signals reflect a noninvasive method to record electrical activity of brain which can be termed as neurophysiology of associated task. EEG contains huge amount of data but there are certain potentials present in EEG that are specifically related to an event. Such potentials are known as event related potentials. One of the main concern of BCI system is to detect these potentials with minimum delay. But to detect these potentials from EEG is a challenging task because of very low signal to noise ratio. As EEG is prone to movement artifacts and noise of different frequencies. There are different movement artifacts removal techniques that can be used to detect an activity from such noise . Few popMost BCIs consist of three major portions as shown in We propose FIR implementation for band pass filtering along with large Laplacian spatial filtering in hardware friendly environment. Zero phase filtering is used to avoid any delays associated with FIR filter.Flipping coefficient methodFFT methodSystolic Multiply Accumulator or manual filter implementation 1Manual filter implementation 2.Four alternate methods are proposed to simulate zero phase filtering results with minimized latency in a hardware friendly environment using Xilinx System Generator: Xilinx System Generator is an efficient way for providing cosimulation environment. It is compatible with MATLAB Simulink. There is large amount of Digital Signal Processing (DSP) blocks available which includes FIR compilers, multipliers, adders, delays, and many more . Black bThe rest of the paper is organized in the following way. Second section is for proposed methodology. The methodology section is further divided into two subsections, first subsection explains spatial filtering and second subsection includes band pass filtering using four proposed methods. Third section explains cosimulation and selected hardware. Last section is for results and conclusion.xi are the weights given to different channels of EEG signal to form a single surrogate channel. Nch depicts the number of channels. According to equation, Channel 1 was given maximum priority and maximum weight will be assigned to it.Multichannel data that is acquired from acquisition unit has very low SNR, so to enhance signal to noise ratio a spatial filter known as large Laplacian filter is used. This will create a single surrogate channel with enhanced SNR via localization. Different weights are assigned on the basis of channels .(1)xi=1,N channel implementation is shown in In XSG this task is performed using Black Box in which a Verilog code is translated into MATLAB module as shown in Most important phase of a BCI is band pass filtering. As EEG contains the brain activity over passage of time and contains ERPs, it contains different frequency waves and abnormalities as well. So in order to detect targeted signals we limit our focus to specific frequency band via band pass filtering , 19.Signals such as ERPs can be of very low potentials and lie in very narrow band. These studies require filter with very sharp cutoff due to which IIR filter is preferred but IIR cannot be implemented in hardware and FIR filter requires very large number of coefficients to achieve such cutoff. In current work focus is on very narrow band FIR filter that suffices for the requirement of narrowest range of 0.005 to 0.4 discussed in previous works , 16. AftFirst MAC performs forward filtering then before using second MAC filter the output of first MAC filter is reversed and supplied to second MAC filter; this results into zero phase filtering. In XSG FIR compiler 5.0 is used for filtering; it takes FIR coefficients as parameter and filters the input signal. If we try to implement zero phase filtering using FIR compiler, then we will require two FIR compilers: first compiler will filter input signal and then before using second compiler it will be required to flip the output of first compiler. For flipping, buffering of signal will be required which will require extensive memory. An alternative to this method can be achieved using simple mathematics.\u2217 show the convolution operation. In frequency domain the above equation can be represented as The following equations show zero phase filtering: y = MAC filter with original coefficients (x)\u2009y2 = MAC filter with reverse coefficients (y).\u2009We can achieve zero phase filtering in the following way:x; second MAC filter uses reverse FIR coefficients and filter sequence y (output of first filter).In this method first MAC filter uses original FIR coefficients and filter sequence Benefit of this method is reduced delay caused by flipping and buffering complete data. In Xilinx System Generator, we have to generate a dedicated block for this buffering to store complete data. But using the proposed method 1, flipping FIR coefficients offline and supplying to FIR compiler reduce delay and remove buffer. Main focus is to implement proposed methods on hardware using Xilinx System Generator (XSG). In XSG the above task is achieved by using two FIR compilers 5.0 in cascade. One compiler uses original FIR coefficients and the other FIR compiler uses Flipped FIR coefficients as shown in FIR compiler provided in XSG can implement a filter up to 1024 coefficients on hardware using cosimulation file. So this technique can be verified in simulations but cannot be implemented on actual hardware.Method one uses two FIR compilers which is a waste of resources; we can perform the above task by using only one FIR compiler. If we see into the mathematics of zero phase filtering the same task can be performed by taking FFT of FIR filter coefficients, then taking square of FFT, and then performing IFFT.Then applying MAC filter to input sequence with step 3 produced coefficients. Same drawback as defined in previous section highlighting limitation of FIR compiler 5.0 in XSG.FIR compiler 5.0 only works for 1024 taps. Implementing bandpass filter of 5000 taps using FIR compiler 5.0 is not possible. It works only in simulation but when we move towards hardware cosimulation it results in an error \u201c5000 taps exceeds the limitation of FIR compiler 5.0.\u201d Above compiler uses \u201cDistributed Arithmetic\u201d or \u201cSystolic Multiply Accumulator.\u201d Distributed Arithmetic method is very difficult to implement using basic blocks given in Xilinx System Generator library. But Systolic Multiply Accumulator can be easily implemented using basic blocks. Xilinx implementation of this method is shown in This manual filter implementation overcomes the limitations of predefined FIR compiler of XSG and if above architecture is implemented it will require 5000 taps to design filter of required range (0.05\u20130.4). In this technique 4999 adders and 5000 multipliers are used. By discarding coefficients close to zero, for this method adders are reduced to 2499 and multipliers to 2500. Utilizing symmetry of filter coefficients we move towards 2nd efficient manual implementation.N multiplications and (N \u2212 1) additions. In contrast, the architecture in N/2] multiplications and approximately N additions. This significant reduction in the computation workload can be exploited to generate efficient filter hardware. Further reduction in coefficients is achieved by discarding coefficients that are very close to zero. Filter coefficients are symmetric so we can use the following methodology. This technique uses 2499 adders and 1250 multipliers and reduced number of coefficients from 5000 to 1250. Instead of implementing this filter using the architecture shown in For creating a cosimulation file, Xilinx System Generator block is used. It has many options; first option is compilation; programmer is required to select the target device for which system generator is supposed to create simulation file. In this case as there is extensive MAC engine so we have selected extreme DSP kit (vertex 4 FPGA Family). Vertex 4 FPGA has dedicated DSP slices for efficient implementation of multipliers and accumulators while Spartan family FPGAs lack this feature. In language selection option we have selected VHDL. After that by XSG it generates cosimulation files. This process takes some time depending on complexity of Simulink model. During the process it checks the model status and simulation time and then performs compilation and generation. All steps required in chip making are done, for example,\u2009\u2009netlist generation, mapping, HDL compilation, design hierarchy analysis, and low level synthesis.Comparison between previously implemented techniques (using IIR) and four proposed FIR methods is shown in p is number of coefficients of proposed method. NCo is number of coefficients of original method.Efficiency of proposed method is calculated in comparison to previous available techniques. Response of IIR filters was replicated using FIR filter and it contains 5000 coefficients which is then considered as benchmark to validate results. Our proposed method reduces the number of coefficients without any significant loss in signal and MRCP can easily be captured from this method. p = 1250 and NCo = 5000 so using equation we get efficiency of 25%.From Similar implication can be made for other event related potentials as well. We are using MRCP as an example but this can easily be extended for other ERPs. Proposed method with these results shows that we can move from offline filtering to real-time on device filtering.In this work, an effort is made to implement preprocessing steps of any BCI system that requires sharp cutoff band pass filtering in XSG to provide fast reliable hardware based filtering. Replicating response of a sharp cutoff filter in FIR domain requires very large number of coefficients. Filter used in , 16 is BResults show that all proposed methods produce satisfactory results in simulation environment. Method 4 is the best of all with least latency to replicate the response of IIR filters that were used in previous studies, after performing preprocessing steps in hardware environment and after validating results. In future work we will be looking into the artifacts removal techniques of EEG. Another direction is to focus on processing aspect of ERPs such as matched filtering and classification techniques."} +{"text": "CSR triangle of plant strategies. The weak degree of trait coordination displayed by the heath vegetation species contradicted our expectation of high trait coordination in stressful environmental habitats. The distinct biogeographic origins of the species occurring in the study region and the prevalence of a regional environmental filter coupled with local homogeneous conditions could account for prevalence of trait independence we observed.A core question involving both plant physiology and community ecology is whether traits from different organs are coordinated across species, beyond pairwise trait correlations. The strength of within\u2010community trait coordination has been hypothesized to increase along gradients of environmental harshness, due to the cost of adopting ecological strategies out of the viable niche space supported by the abiotic conditions. We evaluated the strength of trait relationship and coordination in a stressful environment using 21 leaf and stem traits of 21 deciduous and evergreen woody species from a heath vegetation growing on coastal sandy plain in northeastern South America. The study region faces marked dry season, high soil salinity and acidity, and poor nutritional conditions. Results from multiple factor analyses supported two weak and independent axes of trait coordination, which accounted for 25%\u201329% of the trait variance using phylogenetically independent contrasts. Trait correlations on the multiple factor analyses main axis fit well with the global plant economic spectrum, with species investing in small leaves and dense stems as opposed to species with softer stems and large leaves. The species\u2019 positions on the main functional axis corresponded to the competitor\u2010stress\u2010tolerant side of Grime's Variation in plant form and function creates the basis for species coexistence, plasticity, and evolvability from the images of scanned leaves. Three leaves per individual per species were fixed in 70% (v/v) ethanol until anatomical analysis was performed. Freehand transections of each leaf blade were obtained and stained with alcian blue and safranine. Leaf thickness, mesophyll layer, palisade and spongy parenchyma, and cuticle were measured (in \u03bcm) using a Nikon Eclipse E200 microscope. We also measured the content of starch and nonreducing soluble sugars (predominantly sucrose) in 200\u00a0mg of fresh leaves for each individual per species using the Antrona method with the number of litterfall traps in which species were found. Most abundant species had similar mean and coefficient of variation values as rare species , as suggested by the low coefficients of correlation using multiple imputation with chained equations (MICE) through predictive mean matching with the \u201cmice\u201d function from the mice package and the megatree R20120829. Branch lengths were assigned to the initial megatree using the \u201cbladj\u201d function in Phylocom 4.2, with angiosperm nodes aged according to Wikstrom, Savolainen, and Chase to two orders of magnitude Tables . RemovinStems presented higher within\u2010organ correlations than leaves Table\u00a0. Pairwisn\u00a0=\u00a033) using 14 traits produced only one significant axis using 21 species. The axes accounted for 41% of trait variance using the raw dataset and 53% using phylogenetically independent contrasts coupled with high radiation exposure and long dry seasons , but present more drought\u2010prone semi\u2010arid species from the Caatinga and Cerrado savannas.Despite weak trait coordination, the pattern of trait correlations on the MFA's main axis fits well with the global plant economic spectrum, which supports small\u2010leaved species with dense stems facilitating slower strategies and opposes large\u2010leaved species with soft stems facilitating faster ecological strategies large leaves were not richer in acquisitive anatomical traits, as suggested by the MFA analysis. Further investigations in systems under distinct degrees of stress will contribute to understand the generality of whole\u2010plant trait coordination within local communities in stressful environments.According to the leaf economic spectrum, higher leaf area is related to cheap resource\u2010acquisitive leaves that are more efficient in carbon uptake and have lower water economy (Wright et\u00a0al., All mean trait data used in this manuscript are present in Tables None declared.JS and AS conceived the ideas, the methodology, and wrote the manuscript; JS collected and analyzed leaf and stem trait data; AC collected litterfall data; EV contributed with the leaf biochemistry analysis; JL contributed with the leaf anatomy analysis. All authors contributed critically to the drafts and gave final approval for publication.\u00a0Click here for additional data file."} +{"text": "Liver transplantation is the best treatment option for early-stage hepatocellular carcinoma, liver cirrhosis, fulminant liver failure, and end-stage liver diseases. Even though advances in surgical techniques and perioperative care have improved postoperative outcomes, perioperative cardiovascular complications are a leading cause of postoperative morbidity and mortality following liver transplantation. Ischemic coronary artery disease (CAD) and cardiomyopathy are the most common cardiovascular diseases and could be negative predictors of postoperative outcomes in liver transplant recipients. Therefore, comprehensive cardiovascular evaluations are required to assess perioperative risks and prevent concomitant cardiovascular complications that would preclude good outcomes in liver transplant recipients. The two major types of cardiac computed tomography are the coronary artery calcium score (CACS) and coronary computed tomography angiography (CCTA). CCTA in combination with the CACS is a validated noninvasive alternative to coronary angiography for diagnosing and grading the severity of CAD. A CACS > 400 is associated with significant CAD and a known important predictor of posttransplant cardiovascular complications in liver transplant recipients. In this review article, we discuss the usefulness, advantages, and disadvantages of CCTA combined with CACS as a noninvasive diagnostic tool for preoperative cardiac evaluation and for maximizing the perioperative outcomes of liver transplant recipients. Since the first successful liver transplantation was reported in 1963 . As the The American College of Cardiology/American Heart Association guidelines recommend cardiovascular evaluation for individuals undergoing noncardiac surgery . As withA scientific statement from the American Heart Association and the American College of Cardiology Foundation gives a class I recommendation to screen all potential liver transplant candidates for cardiovascular disease initially with a history and physical examination . NoninvaCCTA has a negative predictive value of 97\u201399% for predicting the absence of obstructive CAD , 19. In Preoperative testing for cardiovascular evaluation is highly recommended in liver transplant candidates with a history of cardiovascular disease, alcoholism, or diabetes mellitus, especially in patients > 60 years of age . HoweverPreoperative resting electrocardiography is a noninvasive test used to obtain diagnostic and prognostic information on liver transplant recipients . StandarPreoperative cardiopulmonary exercise testing is a safe, noninvasive method to determine the cardiopulmonary reserve in liver transplant recipients. The preoperative cardiopulmonary reserve assessed by submaximal cardiopulmonary exercise testing represents a sensitive and specific predictor of early survival after liver transplantation . In patiDobutamine stress echocardiography has been introduced as an initial screening test for coronary heart disease. The American Association for the Study of Liver Disease recommends dobutamine stress echocardiography as an effective screening tool for evaluating CAD in patients undergoing liver transplantation . Current\u03b2-blocking agents for the prevention of esophageal variceal bleeding in end-stage liver disease has been found to be a common cause of failure to achieve the target heart rate in dobutamine stress echocardiography. The previously reported results of dobutamine stress echocardiography were inconclusive in 19\u201321% of patients on \u03b2-blocking agents . A . A 73]. CCTA combined with CACS is well tolerated in comparison with the stress test and is a useful noninvasive technique for assessing CAD in patients with end-stage liver disease. The prognostic value of CCTA is comparable to that of dobutamine stress echocardiography and it has a negative predictive value of 95% for major adverse cardiac events in the 1-year posttransplant follow-up period in orthotopic liver transplant recipients . Kong etHowever, there is still limited information on the predictive ability of the CACS with respect to perioperative outcomes in patients undergoing liver transplantation . More stNoninvasive CCTA reduces the need for invasive coronary angiography and can be safely used in the perioperative cardiovascular risk assessment of liver transplant candidates during the posttransplant follow-up period . CACS isThe necessary radiation dose for CCTA has been found previously to be in the range of 8\u201321\u2009mSv, which is higher than that associated with conventional coronary angiography (2\u20135\u2009mSv) . HoweverNotwithstanding diverse clinical experiences and various advances in knowledge, no gold standard has yet been developed for cardiac evaluation in liver transplant candidates. Due to an equal or high incidence of CAD associated with significant morbidity in these patients, the development of a screening protocol with a reliable predictive value is still required. Given that the clinical applications that can be used in liver transplant candidates remain limited, CCTA combined with CACS seems to be a reliable screening option for preoperative noninvasive evaluation of CAD in liver transplant recipients with diabetes mellitus or \u2265 2 traditional risk factors for CAD. Coronary angiography can be performed in liver transplant recipients with coronary artery stenosis \u2265 50% on CCTA or CACS > 400. In addition, CCTA combined with CACS provides useful information for predicting posttransplant cardiovascular complications in patients undergoing liver transplantation."} +{"text": "These limitations should be considered when counseling pregnant women about the risks and benefits of testing for Zika virus infection during pregnancy. This updated guidance emphasizes a shared decision-making model for testing and screening pregnant women, one in which patients and providers work together to make decisions about testing and care plans based on patient preferences and values, clinical judgment, and a balanced assessment of risks and expected outcomes.CDC has updated the interim guidance for U.S. health care providers caring for pregnant women with possible Zika virus exposure in response to 1) declining prevalence of Zika virus disease in the World Health Organization\u2019s Region of the Americas (Americas) and 2) emerging evidence indicating prolonged detection of Zika virus immunoglobulin M (IgM) antibodies. Zika virus cases were first reported in the Americas during 2015\u20132016; however, the incidence of Zika virus disease has since declined. As the prevalence of Zika virus disease declines, the likelihood of false-positive test results increases. In addition, emerging epidemiologic and laboratory data indicate that, as is the case with other flaviviruses, Zika virus IgM antibodies can persist beyond 12 weeks after infection. Therefore, IgM test results cannot always reliably distinguish between an infection that occurred the definition of possible Zika virus exposure has not changed and includes travel to, or residence in an area with risk for mosquito-borne Zika virus transmission or sex with a partner who has traveled to or resides in an area with risk for mosquito-borne Zika virus transmission. These areas can be found on the CDC \u201cZika Travel Information\u201d webpage.*For these recommendations, Key recommendations include the following:before and during the current pregnancy, at every prenatal care visit.1) All pregnant women in the United States and U.S. territories should be asked about possible Zika virus exposure CDC recommends that pregnant women not travel to any area with risk for Zika virus transmission. It is also recommended that pregnant women with a sex partner who has traveled to or lives in an area with risk for Zika virus transmission use condoms or abstain from sex for the duration of the pregnancy.2) Pregnant women with recent possible Zika virus exposure and symptomsof Zika virus disease should be tested to diagnose the cause of their symptoms. The updated recommendations include concurrent Zika virus nucleic acid test (NAT) and serologic testing as soon as possible through 12 weeks after symptom onset.with ongoing possible Zika virus exposureAsymptomatic pregnant women should be offered Zika virus NAT testing three times during pregnancy. IgM testing is no longer routinely recommended because IgM can persist for months after infection; therefore, IgM results cannot reliably determine whether an infection occurred during the current pregnancy. The optimal timing and frequency of testing of asymptomatic pregnant women with NAT alone is unknown. For pregnant women who have received a diagnosis of laboratory-confirmed Zika virus infection \u226510 and dengue virus PRNT <10 results]) any time before or during the current pregnancy, additional Zika virus testing is not recommended. For pregnant women without a prior laboratory-confirmed diagnosis of Zika virus, NAT testing should be offered at the initiation of prenatal care, and if Zika virus RNA is not detected on clinical specimens, two additional tests should be offered during the course of the pregnancy coinciding with prenatal visits.3) 4) Asymptomatic pregnant women who have recentwithout ongoing possible exposure are not routinely recommended to have Zika virus testing.possible Zika virus exposure but Testing should be considered using a shared patient-provider decision-making model, one in which patients and providers work together to make decisions about testing and care plans based on patient preferences and values, clinical judgment, a balanced assessment of risks and expected outcomes, and the jurisdiction\u2019s recommendations. Based on the epidemiology of Zika virus transmission and other epidemiologic considerations , jurisdictions might recommend testing of asymptomatic pregnant women, either for clinical care or as part of Zika virus surveillance. With the decline in the prevalence of Zika virus disease, the updated recommendations for the evaluation and testing of pregnant women with recent possible Zika virus exposure but without ongoing possible exposure are now the same for all areas with any risk for Zika virus transmission.5) Pregnant women who have recent possible Zika virus exposure and who have a fetus with prenatal ultrasound findings consistent with congenital Zika virus syndrome should receive Zika virus testing to assist in establishing the etiology of the birth defects. Testing should include both NAT and IgM tests.6) The comprehensive approach to testing placental and fetal tissues has been updated. Testing placental and fetal tissue specimens can be performed for diagnostic purposes in certain scenarios . However, testing of placental tissues for Zika virus infection is not routinely recommended for asymptomatic pregnant women who have recent possible Zika virus exposure but without ongoing possible exposure and who have a live born infant without evidence of possible Zika virus\u2013associated birth defects.with ongoing possible Zika virus exposure is not warranted7) Zika virus IgM testing as part of preconception counseling to establish baseline IgM results for nonpregnant women because Zika virus IgM testing is no longer routinely recommended for asymptomatic pregnant women with ongoing possible Zika virus exposure.CDC continues to evaluate all available evidence and will update recommendations as new information becomes available.Zika virus is a mosquito-borne flavivirus that is closely related to dengue, West Nile, Japanese encephalitis, and yellow fever viruses have been reclassified to indicate that Zika virus transmission has been interrupted . However, in light of the limitations of serologic testing , for pregnant women without a previous diagnosis of laboratory-confirmed Zika virus infection, including those with laboratory evidence of flavivirus infection or laboratory evidence of presumptive Zika virus or flavivirus infection from the Food and Drug Administration (FDA) for use on nontissue clinical specimens.Several assays can be used to detect Zika virus IgM antibodies in serum or cerebrospinal fluid. Zika virus IgM tests can be difficult to interpret because of false-positives and cross-reactivity with other flaviviruses, especially in persons who were previously infected with or vaccinated against a related flavivirus mosquito season after introduction of Zika virus, testing becomes more complex. Given the evolving situation and the many uncertainties, the updated testing algorithms for symptomatic and asymptomatic pregnant women Figure emphasizitations . To addritations have alsBOXbefore and during the current pregnancy. Health care providers should ask about the presence of symptoms of Zika virus disease , and place, duration, and type of travel to assess a woman\u2019s potential for exposure to Zika virus and other flaviviruses .Pregnant women with possible Zika virus exposure should be asked about their risk for exposure both before the current pregnancy and one that occurred during the current pregnancy.It is important to ascertain whether a woman had exposure to Zika virus before the current pregnancy because Zika virus immunoglobulin M (IgM) antibodies can be detected for months after an infection. A positive Zika virus IgM result could indicate antibodies from infection before the current pregnancy, thus limiting the ability to distinguish between an infection that occurred It is important to ascertain whether a woman had exposure to flaviviruses other than Zika virus before the current pregnancy because a positive IgM result might have been caused by cross-reactivity from a previous flavivirus exposure.Health care providers and counselors should provide appropriate pretest counseling to inform decisions on whether to test; Zika virus test results should be interpreted within the context of known limitations.A negative Zika virus IgM test result, if performed during the recommended time frame, in the setting of a negative Zika virus nucleic acid test (NAT) result, provides some reassurance of absence of Zika virus infection during the current pregnancy. However, a negative Zika virus IgM test result should be interpreted within the context of the limitations of the assay.When plaque reduction neutralization testing (PRNT) is indicated and performed during the recommended time frame, a negative PRNT result in the setting of a negative NAT result indicates that there is no laboratory evidence of Zika virus infection.before and during the current pregnancy. Health care providers should ask about presence of symptoms of Zika virus disease and place, duration, and type of travel to assess a woman\u2019s potential for Zika virus exposure. Data from other mosquito-borne illnesses indicate that intensity of transmission, duration of travel, and type of travel influence the likelihood of infection , specifically results . Zika viPregnant women with recent possible Zika virus exposure and symptoms of Zika virus disease. Testing for Zika virus infection is still recommended for pregnant women with symptoms of Zika virus disease and possible Zika virus exposure, with the main goal of establishing a diagnosis that accounts for their symptoms, or ruling out Zika virus infection so that an alternative diagnosis can be considered. Negative test results should prompt evaluation for other causes, which might include dengue virus or chikungunya virus infection, depending on the symptoms and epidemiology of circulating viruses.Concurrent NAT (serum and urine) and serologic testing (serum) is recommended for pregnant women as soon as possible, through 12 weeks after symptom onset . ReportsFor women who seek care >12 weeks after symptom onset, Zika virus IgM testing might be considered; however, a negative result does not rule out an infection during pregnancy because IgM levels decline over time. A positive result should be interpreted within the context of the known limitations of serologic testing.with ongoing possible Zika virus exposure.Asymptomatic pregnant women For asymptomatic pregnant women with ongoing exposure to Zika virus, testing for Zika virus infection should be offered as part of routine obstetric care because it might identify acute infection during pregnancy IgM testing has diagnostic limitations; 2) Zika virus NAT testing of serum does not reflect persistence in other body fluids . The current understanding of Zika virus shedding in genital secretions is limited but . For asymptomatic pregnant women with recent possible Zika virus exposure , but without ongoing possible exposure, testing for Zika virus infection is not routinely recommended. However, testing should be considered using a shared decision-making model, one in which patients and providers work together to make decisions about testing and care plans based on patient preferences and values, clinical judgment, a balanced assessment of risks and expected outcomes, and the jurisdiction\u2019s recommendations. Health care providers should consider potential exposure risk factors when deciding whether to advise testing. These include symptoms, type and length of possible exposure, Zika virus transmission trends at location of possible exposure and the use of prevention measures . Jurisdictional recommendations may take into account the epidemiology of Zika virus transmission and other epidemiologic considerations in areas with risk for Zika virus transmission and, therefore, might include a routine recommendation to test asymptomatic pregnant women either for clinical care or as part of Zika virus infection surveillance.without ongoing possible exposure to address the increased probability of false positive results in the setting of the declining prevalence of Zika virus disease should be considered to assess fetal anatomy, particularly fetal neuroanatomy, and to monitor growth. A study of 17 pregnancies in symptomatic women with laboratory-confirmed Zika virus infection and adverse fetal outcomes in Colombia and a summary of eight published studies of 37 pregnancies reported a median of 18 weeks from symptom onset to prenatal diagnosis of microcephaly and 119\u2013238 days (mean\u00a0=\u00a0163 days), respectively, from maternal symptom onset to provide insight into the potential etiology of the fetal loss or infant death , which cwithout ongoing possible exposure, it is critical that pediatric health care providers inquire about possible maternal and congenital Zika virus exposure for every newborn. Infants born to mothers with possible Zika virus exposure during pregnancy but who did not receive testing, including asymptomatic pregnant women with recent possible Zika virus exposure but without ongoing possible exposure, should receive a comprehensive physical examination, including standardized measurement of head circumference and newborn hearing screen, as part of routine pediatric care. In addition, based on the level of possible Zika virus exposure , the provider should consider whether further evaluation of the newborn for possible congenital Zika virus infection is warranted, in which case, a head ultrasound, and ophthalmologic assessment should be considered. Based on results of the evaluation, testing of the infant for Zika virus infection could be considered.Interim guidance for the evaluation of infants with congenital Zika virus exposure has been previously published; infants who meet one or more of the published criteria for testing for congenital Zika virus infection should be tested and evaluated in accordance with the updated CDC interim guidance for the evaluation and management of infants with possible Zika virus infection . Recommendations for outpatient management during the first 12 months of life include monitoring of head circumference and development and are provided in the updated CDC interim guidance for the evaluation and management of infants with possible Zika virus infection (This guidance also applies to infants born to mothers with negative maternal testing in the setting of ongoing possible Zika virus exposure or a possible Zika virus exposure that occurred more than 12 weeks before maternal testing (CDC recommends that pregnant women avoid travel to any area with risk for Zika virus transmission. To prevent Zika virus infection during pregnancy, all pregnant women and their partners should receive counseling on prevention measures including strategies to prevent mosquito bites and sexual transmission of Zika virus ("} +{"text": "Prenatal calcification of the inferior vena cava (IVC) and renal veins is a rare condition with unclear etiology and prognosis. It occurs with renal vein thrombosis in utero and is associated with congenital anomalies and abnormal prenatal hemodynamic status. We report a rare case of prenatal IVC and renal vein calcification in a normal neonate without any history of compromised prenatal or perinatal condition, or significant deterioration of kidney function."} +{"text": "In developing countries like South Africa processed geographic information systems (GIS) data on land suitability, is often not available for land use management. Data in this article is based on a published article \u201cThe strategically located land index support system for humans settlements land reform in South Africa\u201d Specifications TableValue of the data\u2022This data is useful because, it maps out well-located land in South Africa to establish smart human settlements.\u2022The data can be used to facilitate decision making for human settlements land reform, and other land use management needs.\u2022The maps and the data are useful for other researchers, urban planners and policy makers.\u2022The maps provide a visual picture of strategically located land in South Africa.\u2022The data is useful in the on-going debate on how successful has the new democratic dispensation in promoting spatial integration.1The SLLI ranges from 0 to 100 where a value close to zero implies unstrategically located land to establish human settlements whereas a value close to 100 implies highly strategic land to establish human settlements. Similarly, 2The strategically located land index was developed using geographic information system and multi-criteria decision analysis (GIS-MCDA) techniques. Fourteen spatial criteria and datasets relating to human settlements underwent a GIS-MCDA process as described in"} +{"text": "Objective: To clarify the characteristics of neuropsychiatric symptoms in patients with idiopathic normal pressure hydrocephalus (iNPH).Methods: Neuropsychiatric symptoms of 64 iNPH patients with mild triad symptoms from three kinds of hospitals were evaluatedwith the Neuropsychiatric Inventory (NPI) and compared with 126 patients with Alzheimer\u2019s disease (AD).Results: The most frequently observed neuropsychiatric symptom in the iNPH patients was apathy followed by anxiety and aggression. No symptom was more prevalent or more severe in iNPH than in AD. The severity of cognitive impairment was correlated with both aberrant motor activity and apathy.Conclusions: Neuropsychiatric symptoms were mild in patients with iNPH and apathy was the most prevalent symptom. The correlation between neuropsychiatric symptoms and cognitive impairment in iNPH appears to arise from a common pathologyin the frontal lobe."} +{"text": "Rapid progress has been made in obtaining an inventory of Parkinson\u2019s Disease (PD) associated genes. The challenge now is to understand their interactions and on which physiological pathways they converge. Two major themes have emerged; influence upon mitochondrial dynamics and on trafficking within the endocytic pathway . Parkin Broader links between Parkin and mitochondrial health have been made in both cardiac maturation and following myocardial infarction . Parkin in vivo. Ubiquitylated proteins from mice, flies or human cells can then be isolated by Neutravidin beads under stringent washing conditions. This provides greater peptide coverage of substrates although the actual site of ubiquitylation is not always identified [Discovery-based proteomic approaches offer the opportunity to identify E3-ligase substrates in an unbiased manner. Quantitation of protein levels may reflect differences in turnover rate but can also report indirect effects on transcription/translation. Alternatively ubiquitylated peptides can be directly detected by a signature diGly modification following trypsinisation. We have recently used an alternative method (BioUb) whereby a tagged form of ubiquitin is stably expressed and biotinylated entified .Acute mitochondrial depolarisation provides the only known trigger for Parkin activation and therefore offers a means to facilitate the identification of substrates. Using this procedure in tissue cell culture models has revealed a broad swathe of mitochondrial substrates, with some bias towards modification with ubiquitin chains linked through Lys6 . Our resInterest in Parkin function is clearly broadening and unbiased proteomics screens offer opportunities to open up new biology. Methods have been developed to detect direct substrates and advances in label-free proteomics offer opportunities for tissue wide analyses. Looking forward, these might have less specific emphasis on the brain as the relevance to other types of pathophysiology is now clear."} +{"text": "We tested the notion that patients at high risk for progression to Alzheimer's disease (AD) display relatively isolated memory deficits by assessing the relationship between memory and fluency performances in a sample of 92 geriatric subjects with cognitive complaints and normal to mild clinical presentations. Patient groups were formed on the basis of memory test scores. Patients with normal memory scores also performed normally on fluency tests, and their fluency scores were significantly higher than those of patients with low memory performances. Patients falling between these two groups in memory abilities also displayed intermediate level fluency performances. Whereas the normal memory group performed at equivalent levels on semantic and phonemic fluency tasks, both the impaired memory group and the intermediate group displayed relatively greater weaknesses in semantic fluency. This pattern is similar to that seen in AD. Since the impaired memory patients meet criteria for Amnestic Mild Cognitive Impairment, these findings suggest that memory deficits in \u201cpre-clinical\u201d AD are likely to be accompanied by fluency weaknesses, with semantic fluency weaknesses predominating."} +{"text": "Investigations of developing enamel crystals using Atomic and Chemical Force Microscopy have revealed a subunit structure. Subunits were seen in height images as collinear swellings about 30 nM in diameter on crystal surfaces. In friction mode they were visible as positive regions. These were similar in size (30\u201350 nM) to collinear spherical structures, presumably mineral matrix complexes, seen in developing enamel using a freeze fracturing/freeze etching procedure. More detailed AFM studies on mature enamel suggested that the 30\u201350 nM structures were composed of smaller units, ~10\u201315 nM in diameter. These were clustered in hexagonal or perhaps a spiral arrangement. It was suggested that these could be the imprints of initiation sites for mineral precipitation. The investigation aimed at examining original freeze etched images at high resolution to see if the smaller subunits observed using AFM in mature enamel were also present in developing enamel i.e., before loss of the organic matrix. The method used was freeze etching. Briefly samples of developing rat enamel were rapidly frozen, fractured under vacuum, and ice sublimed from the fractured surface. The fractured surface was shadowed with platinum or gold and the metal replica subjected to high resolution TEM. For AFM studies high-resolution tapping mode imaging of human mature enamel sections was performed in air under ambient conditions at a point midway between the cusp and the cervical margin. Both AFM and freeze etch studies showed structures 30\u201350 nM in diameter. AFM indicated that these may be clusters of somewhat smaller structures ~10\u201315 nM maybe hexagonally or spirally arranged. High resolution freeze etching images of very early enamel showed ~30\u201350 nM spherical structures in a disordered arrangement. No smaller units at 10\u201315 nM were clearly seen. However, when linear arrangements of 30\u201350 nM units were visible the picture was more complex but also smaller units including ~10\u201315 nM units could be observed.Conclusions: Structures ~10\u201315 nM in diameter were detected in developing enamel. While the appearance was complex, these were most evident when the 30\u20135 nM structures were in linear arrays. Formation of linear arrays of subunits may be associated with the development of mineral initiation sites and attendant processing of matrix proteins. Enamel comprises highly ordered crystals of substituted hydroxyapatite. These are of regular size and shape, densely packed with their long c-axes parallel and arranged in bundles, the enamel prisms. The precise mechanism of initiation and growth of these crystals is unclear.Early transmission electron microscope (TEM) data suggested that crystals formed immediately outside of the ameloblast membrane, immediately after matrix secretion, appearing as thin needles or plates of maturation stage enamel crystals. AFM revealed contiguous regular 30\u201350 nM globular swellings along maturation stage enamel crystals, redolent of the globules shown by freeze etching but which had subsequently fused and crystallized (Kirkham et al., These appeared more obviously as the globules formed linear arrays, possibly reflecting matrix processing associated with transition from amorphous mineral to crystals.In addition, however, later high resolution AFM indicated that the 30\u201350 nM globular structures comprised smaller ~15 nM subunits arranged in roughly hexagonal or possibly spiral patterns (Robinson et al., Freeze etching of early enamel was reported by Robinson et al. . BrieflyAFM was carried out as described previously (Kirkham et al., As previously reported, the data shown illustrates the presence of 30\u201350 nM diameter globules in secretory enamel, arranged randomly or in linear arrays (Robinson et al., However, high resolution AFM images of mature enamel also revealed previously unreported 15 nM substructures within the ~30 nM globules arranged in roughly hexagonal or perhaps spiral patterns (Robinson et al., Approximately 15 nM units of enamel structure have also been reported using other techniques. Diekwisch reportedper-se but are more likely to be mineral matrix complexes. That they appeared more clearly when 30 nM globules lined up suggests that matrix processing may be involved in alignment and mineral precipitation see below (Fang et al., These 15 nM structures may be amorphous mineral Figure It is not yet known precisely how the amorphous material is initiated and temporarily stabilized. Initiation may occur within the 15 nM subunits if ionic peptide side chains, for example, the C terminal peptide of amelogenin and/or its phosphate group are turned inward and the subunits held together by hydrophobic interaction Figure .In vitro investigations using amelogenin (Fang et al., The rapid loss of the hydrophilic C terminal of amelogenin and loss of the phosphate group have been implicated in the transformation from stabilized amorphous mineral to crystalline phase (see Kwak et al., The significance of clustering of 15 nM initiation sites is significant from a number of points of view. From the viewpoint of enamel structure, clustering into 30\u201350 nM units delineates the ultimate crystal width and thickness thus outlining tissue volume to be occupied by crystals. This is important since the matrix is ultimately removed.This also has implications for enamel caries since the fused interface between these units would lead to increased acid solubility due to crystalline discontinuity (see Robinson et al., Wistar rats were maintained and killed according to local and national animal regulations. University of Leeds Dental School Animal Committee. Human extracted teeth were obtained from the tissue bank at Leeds dental School. Teeth were obtained according to national and local guidelines permission obtained at source.CR designed and set up both investigations and was largely responsible for freeze etch studies. SC carried out and advised upon AFM and CFM investigations. Both authors interpreted data and discussed implications, both were involved in writing.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Healthcare interventions, and particularly those in public health may affect multiple diseases and significantly prolong life. No consensus currently exists for how to estimate comparable healthcare costs across multiple diseases for use in health and public health cost-effectiveness models. We aim to describe a method for estimating comparable disease specific English healthcare costs as well as future healthcare costs from diseases unrelated to those modelled.We use routine national datasets including programme budgeting data and cost curves from NHS England to estimate annual per person costs for diseases included in the PRIMEtime model as well as age and sex specific costs due to unrelated diseases.The 2013/14 annual cost to NHS England per prevalent case varied between \u00a33,074 for pancreatic cancer and \u00a3314 for liver disease. Costs due to unrelated diseases increase with age except for a secondary peak at 30\u201334 years for women reflecting maternity resource use.The methodology described allows health and public health economic modellers to estimate comparable English healthcare costs for multiple diseases. This facilitates the direct comparison of different health and public health interventions enabling better decision making. Disease specific healthcare costs for use in health and public health economic models can be calculated using one of two different approaches: adding together the costs of all components of a patient\u2019s care, such as staff and equipment (a micro-level or bottom-up approach), or allocating an overall healthcare budget to specific diseases (a macro-level or top-down approach).Guidelines exist for how to estimate costs for health economic cost-effectiveness models, however these do not provide specific advice on how to identify costs when evaluating the impact on multiple diseases simultaneously where the comparability of data sources for different diseases is important ,2. WithoA second challenge is how to quantify the age and sex specific future economic consequences of individuals becoming unwell from diseases unrelated to those explicitly modelled following interventions that may prolong life ,11. InclThe aim of this paper is to describe a method for estimating comparable disease specific and unrelated future healthcare costs using routinely available data in England for use in health and public health economic modelling. We present both the method and the results of applying this method to 11 diseases included in an existing multistate life table model.A case study is used to illustrate the methodology: disease specific costs are estimated for the 11 diseases simulated by the multistate life table model, PRIMEtime , and foDisease specific costs are derived from 2013/14 NHS England programme budgeting data which reports expenditure by clinical commissioning groups , and accounts for around two thirds of the total NHS England budget , an, an9], aFinally, in the absence of any UK studies estimating unrelated healthcare costs by age and sex, results are compared with Blakely et al. who estimated healthcare expenditure for diseases unrelated to tobacco consumption for the New Zealand population by age and sex using bottom-up unit patient care costs . The magIn summary, we describe a novel approach to estimating NHS England costs for multiple diseases and unrelated future healthcare costs using routinely available data. The key strength is that we use a consistent approach to estimating costs across multiple diseases meaning that the cost implications of different interventions affecting these diseases can be directly compared. This approach can be applied to other health and public health economic models that estimate the economic consequences of an intervention affecting multiple diseases in England.S1 File(DOCX)Click here for additional data file."} +{"text": "Psychotic disorders are associated with serious deterioration in functioning even before the first psychotic episode. Also on clinical high risk (CHR) states of developing a first psychotic episode, several studies reported a decreased global functioning. In a considerable proportion of CHR individuals, functional deterioration remains even after (transient) remission of symptomatic risk indicators. Furthermore, deficits in functioning cause immense costs for the health care system and are often more debilitating for individuals than other symptoms. However in the past, CHR research has mostly focused on clinical outcomes like transition and therefore, functioning in CHR patients is under-investigated. The current study aims at predicting functioning at a single subject level applying multi pattern recognition to clinical data for the first time.PRONIA is a prospective collaboration project funded by the European Union under the 7th Framework Programme (grant agreement n\u00b0 602152). Considering a broad set of variables as well as advanced statistical methods, PRONIA aims at developing an innovative multivariate prognostic tool enabling an individualized prediction of illness trajectories and outcome. Seven university centers in five European countries and in Australia participate in the evaluation of three clinical groups as well as healthy controls.In the current study, we analysed data of 114 CHR patients. Functioning was measured by the \u2018Global Functioning: Social and Role\u2019 Scales (GF S/R). Features were derived from the large pool of clinical data that were assessed in PRONIA including questionnaires measuring CHR criteria as well as psychopathology, family history of psychotic disorders or treatment and various self-rating scales. Feature Elimination method of a strict Wrapper was used to identify most predictive variables from the multitude of clinical data included into the analysis.Balanced Accuracy of predicting social functioning in CHR patients was acceptable . In contrast, applying the strict wrapper model revealed worse prediction performance for role functioning. Which might indicate that predicting level of role functioning requires more information than social functioning. As expected, prior functioning levels were identified as main predictive factor but also distinct protective and risk factors were selected into the prediction models.Identifying single predictive variables is in purpose of a much more efficient prognostic process. Moreover, understanding the mechanisms underlying functional decline and its illness related pattern might enable an improved definition of targets for intervention. Future research should aim at further maximisation of prediction accuracy and cross-centre generalisation capacity. In addition, other functioning outcomes as well as clinical outcomes need to be focused on."} +{"text": "We argue that FBOs are poised to meet health promotion and health communication needs among African American women who face social barriers in health.Low birth weight (LBW) rates remain the highest among African Americans despite public health efforts to address these disparities; with some of the highest racial disparities in the Midwest (Kansas). The Developmental Origins of Health and Disease (DOHaD) perspective offers an explanation for how LBW contributes to racial health disparities among African Americans and informs a community directed health communication framework for creating sustainable programs to address these disparities. Trusted community organizations such as faith-based organizations are well situated to explain health communication gaps that may occur over the life course. These entities are underutilized in core health promotion programming targeting underserved populations and can prove essential for addressing developmental origins of LBW among African Americans. Extrapolating from focus group data collected from African American church populations as part of a social marketing health promotion project on cancer prevention, we theoretically consider how a similar communication framework and approach may apply to address LBW disparities. Stratified focus groups ( Racial disparities in low birth weight (LBW) are pervasive among minority populations, especially among African Americans (AA) . For exaFBOs are trusted, affinity organizations that serve as health promotors and health communicators within a community. Inequities in health outcomes such as LBW are exacerbated by inequitable health information among minority populations . ThroughEarly childhood LBW may have long lasting health implications and lead to adult racial health disparities . Risk faAlthough the behavioral factors that increase LBW have become more well-known , the challenges to limiting the impact of social determinants endure. Social disadvantage has cumulative impacts throughout the life course with the long-term effects of LBW potentially becoming worse over time, resulting in increased health disparities. For example, while medical advances continue to increase survival rates from LBW, the same social factors that increased risk to LBW remain , which may increase and compound risk whereby children born LBW may receive fewer and less effective investments in post-natal care and remain exposed to health-compromising, disadvantaged environments ,13. ExpoTraditional public health promotion has helped to mitigate barriers among AA populations through disease prevention promotion and health communication programs but gaps remain because messages and delivery of current public health efforts are often not transferrable to the target culture, are devoid of existing policies designed to protect and or promote health equity and are focused on downstream factors . While tNew and emerging research shows the promise of employing dynamic communication similar to SM that informs and translates DOHaD \u201cfindings from the bench to population-level health promotion and disease prevention (p. 170)DOHaD communication combined with SM principles that integrate community and societal partners move beyond single-level communication. Impacting public health efforts through a DOHaD communication and SM framework addresses the public health issue of LBW on multiple levels and has the potential to incorporate multifaceted views of not only the traditional communicators involved in the communication process but less-dominant individuals who are from within segments of the population. Community organizations, such as FBOs, that are trusted and influential among AA have the capacity to impact public health outcomes through organizationally provided social support. These organizations have traditionally been champions for vulnerable populations; they are also demonstrated health promotors and health communicators for numerous health issues and have been instrumental in helping to impact health behavior and improve public health ,19. ExtrWe aim to determine how FBOs are poised to meet public health communication needs among AA women. Building on previous results from a social marketing health promotion project involving AA church populations in the Kansas and Missouri bi-state area , we drawn = 78) that were stratified by state (Kansas or Missouri); area of the city (urban or suburban) and by gender . The sample was purposive and drawn from a group of 124 AA women from 14 churches who participated in the parent study. Women who were included in the sample were AA, between the ages of 35 and 70 who either attended church or lived in the bi-state areas of Kansas and Missouri . Women who participated were part of a larger Community-Based Participatory Research (CBPR) study that explored attitudes, beliefs and behavior intention toward colon cancer prevention and perceptions of FBOs as marketers of cancer prevention health promotion among AA church populations. The present study sample included 9 focus group interviews as a social marketer of health promotion of disease prevention were included. Authors applied emergent themes from the focus groups to explore how DOHaD communication within a social marketing framework has potential applications to issues of LBW. The authors took a multi-method approach to interpreting, analyzing and coding data collected through focus group discussions. Three individuals followed an open coding process where each coder read transcripts multiple times to look for emerging themes among five topic areas; these were pre-determined through work with a community advisory board and were slightly modified based on feedback after the first set of focus groups. The topic areas stayed the same throughout data collection and included: Experiences with Cancer; Experiences with Colon Cancer; Colon Cancer Prevention, Diagnosis and Treatment; as well as the two primary areas of focus for this study: Barriers to Screening; and Faith-Based Prevention Messages. Some questions included: How does cost figure into whether or not to get screened? How comfortable would most people be with bringing these things (screening) up with their doctor? What do you think is most effective in getting cancer prevention messages across to church members? How can the church be used to transmit health prevention messages? What is your opinion of the church serving as a sponsor of cancer prevention promotion materials?Coders were from diverse backgrounds and included two AA females and an Indian female . The diversity of views and perspectives addressed validity and reliability of the coding and the coding process. Coders met two times over the course of four months to compare and contrast categories discovered during their individual open coding process. The open coding process and subsequent contrast and comparison enabled contextualization and categorization of data. The primary and secondary author subsequently took these results to apply a grounded theory approach to explore how women interpreted FBOs as social marketers of disease and prevention among church populations. While grounded theory traditionally occurs when the researcher collects data and analyzes the data , here itThe authors drew parallels between cancer health disparities and LBW and also well-documented barriers and facilitators to proactive health behavior among AA women. FBOs Help\u00a0Reduce Medical Cost WorryThe following three salient themes emerged about health care access, cost and FBOs as SM within a DOHaD context:(Participant from Church 7)\u2014\u201cI have to weigh the cost of screening with costs of providing for others, medicine, etc. Screening becomes low priority and takes a back seat.\u201dThe first salient theme among the sample was the worry of health care costs. In some cases women indicated that they had to forgo paying for preventive care to purchase grocery and or clothing for their family to avoid the burden of medical bills. It was important that they also had utilities paid and rent before taking care of other preventive medical needs.FBOs are Trusted\u00a0Marketers of Preventive InformationThe undue burden of cost worry among this population has been a perpetual barrier and contributes to inadequate healthcare over the life course. Among AAs, the cost of healthcare and related barriers have presented persistent challenges and increased vulnerability; the inequitable status begins in infanthood and is perpetuated throughout life. Addressing cost through FBOs within a DOHaD communication framework may mitigate some of these barriers to existing views of the \u201chigh\u201d cost of care and living well. Moreover, rather than considering health care and general situational challenges as separate costs, clearer, directed and segmented communication could help to clarify the importance of how both current health conditions and stressors both increase risk of LBW.(Participant at Church 4) \u201cI think that anything\u00a0that your church sponsors or are participating in that you would want to participate in it.\u201d(Participant at Church 1) A testimony is a greater sales pitch than\u00a0a regular commercial.\u201dFBOs Are Trusted Networks of Health InformationParticipants were provided with a definition of social marketing and an explanation of what it means to be a sponsor. They were subsequently asked pointed questions about whether they saw FBOs as a social marketer of health and who they thought was the most trusted individual within the organization to deliver this type of health information .and delivery of the health information. The opportunity to take pertinent health information and to strategically place pro-health, pro-social ideas and suggested health behavior within trusted, affinity networks has potential to bolster appeal, increase positive perceptions and reduce suspicion and non-trustworthy communication and endorsement.(Participant from Church 2) \u201cOur pastor\u2019s endorsement is important because he\u2019s our leader, the protector of our souls and we believe him.\u201d(Participant at Church 5) \u201cThe more you hear health messages,\u00a0the more you are likely to listen,\u201d(Participant from Church 7) \u201cYou know I asked (Unnamed Cancer Organization) to come over here for the health fair, okay and I asked them to come over and do screenings in a black neighborhood for a black church, black woman on the telephone asking, ok? I was turned down cold.\u00a0Turned straight down\u2026 I was over in Mission, Kansas and they were doing it over there. You see what I\u2019m saying?\u201dSM principles applied within a DOHaD perspective involves the delivery and deliberate strategic messaging by trusted organizations and individuals to enhance the promotion of DOHaD. Currently, DOHaD is visible in some health promotion/communication programs but only a few programs incorporate trusted entities into the creation type of health information may be building blocks for integration of FBO marketed and tailored information about LBW risk factors. Incorporation of these factors into health communication programs increase the probability of combating developmental health disparities among an underserved population.The strategic incorporation of social marketing principles into health communication research, practice and programming has far reaching implications for AA concerning LBW. Salient themes among the sample show FBOs are trusted marketers of disease prevention and are also seen as gatekeepers of pertinent public health information; FBOs were seen as trusted entities to broker positive and/or life-threatening information. The sample also overwhelmingly looked to pastors within these FBOs as credible voices for health promotion. Applied to a DOHaD perspective, FBOs are positioned for promulgating health information about LBW throughout several phases of life for the mother and infant. Secondly, key conceptual components were identified for strategic media advocacy through SM segmentation among AA women. Pinpointing control and/or behavioral beliefs about worry of cost of care, trust in the health communicator and also trust in the The primary limitation of this research was the sample focus. The sample ranged in ages outside of primary childbearing years (between 35 and 70 years of age) with primary queries focused on cancer prevention and faith-based messages from FBOs. Nonetheless, we found the drawing on this sample allowed us to theoretically apply findings to broader issues of cumulative disadvantage and identified similar challenges applicable to LBW. Future research would benefit from interviews specifically pertaining to LBW. Through additional analyses in the context of LBW, we identified overarching themes of fears, held beliefs and barriers. While clearly not the same, the issues of cancer and LBW disparities are both prevalent among this population and may occur and impact AA women over the lifespan. In fact, several younger women in this sample referred to reproductive screening when asked about health care access and preventive screening. Women expressed that routine screening that began as a youth, was a common and expected practice but not always welcomed. Public health campaigns designed to reach vulnerable populations with DOHaD elements are either scarce or are not strategically woven into messaging strategies and delivery of health information. These deficiencies often derive from poor or little planning that excludes culturally relevant factors for the target audience and multiple avenues to effectively communicate positive health behavior change. Public health campaigns that harness communication science to optimize reach and impact will be the most successful ,24. UsinFBOs have historically and traditionally been shown to influence opinions and behaviors among AA. As demonstrated, SM serves as a useful tool utilized by community organizations to appeal to AA as trusted health communicators of prevention and health behavior. By addressing key components of SM (such as segmentation), FBOs have the ability to strategically communicate effective coping strategies to reduce both LBW disparities, as well as long-term outcomes among this and other vulnerable segments of the population. The infrastructure of FBOs affords a strong social network that reinforces positive, trusted and reciprocated relationships that are ecological in nature. Health communication programs/interventions that incorporate community input, involvement and feedback throughout the development, implementation and evaluative phases address key issues of cost and trust; factors that we suggest are highly applicable to multiple health outcomes. Our findings in a previous cancer study highlight how communicating trust and intervention strategies through strategic marketing represents one critical way that FBOs can potentially assist in reducing LBW. The DHOaD perspective suggests long-term implications of LBW and highlight LBW as a key objective for long-term public health improvements. Health communication must recognize the barriers imposed by social factors that increase LBW risk. Disadvantaged and segregated communities increase exposures to pollution, infectious disease , racism, gender-based violence, and stress that increase the risk of LBW infants. For example, research has suggested that increased exposure to influenza may be especially detrimental to pregnant mothers . StressoFBOs have the potential to address these health risks in a myriad of ways. Previous work has shown that they encourage positive health behaviors such as encouraging prayer as a beneficial stress reduction strategy they offLow socioeconomic status continues to influence LBW even when physiological pathways, behavioral antecedents, and medical solutions are identified. While FBOs may be able to increase access and utilization of health care or promote adaptive coping, more efforts will still be needed to reduce increased exposure to disadvantage and offset challenging environments. Drawing on focus group discussions with AA women, we draw parallels with health screening and suggest that community partnering in innovative health promotion approaches such as SM approaches remain necessary to overcome the barriers presented through discriminatory experiences and community-level distrust of the medical establishment among AA women. While enthusiastic attempts to establish DOHaD remain critical, efforts to understand the cultural and social mediation of maternal and infant health remains an issue of social inequality . Additio"} +{"text": "Chitosan is a cheap resource, which is widely used in biomedical applications due to its biocompatible and antibacterial properties. In this study, composite nanofibrous membranes of chitosan (CS) and poly (PVA) loaded with antibiotics at different ratios were successfully fabricated by electrospinning. The composite nanofibers were subjected to further analysis by scanning electron microscopy (SEM). SEM images revealed that the volumetric ratio of CS/PVA at 50/50 achieved an optimal nanofibrous structure compared with other volumetric ratios, which indicated that this CS/PVA electrospun scaffold has great potential to be used for infection related wound dressing for skin tissue regeneration. Chronic dermal wounds, such as infected diabetic foot ulcers, represent a major health problem that affects millions of people worldwide and induces billions of dollars in social and economic costs; the poor treatment outcomes result in high healthcare costs. Such data explain the large research efforts now focused on developing new therapeutic approaches to improve wound healing (PVA) solution were prepared as described before (Zhou et al., Chitosan and PVA had been widely investigated for a very long time due to their biocompatible and antibacterial properties. SEM images of nanofibers resulting from different ratio of CS/PVA are shown in Figure In this study, CS/PVA nanofibers were successfully prepared by electrospinning different ratios of CS/PVA solutions. SEM images showed that nanofibers had larger and more nanobeads formed with increasing concentrations of CS, while narrower and breaking nanofibers could be observed if the mixed solution was more than 75% PVA. The results from SEM images showed that the ratio of CS and PVA at 50/50 achieved a nanofibrous structure the most similar to natural tissues. These novel electrospun scaffolds have the potential to be used for infection related wound dressing for skin tissue regeneration.MW Performed experiments. AR, Designed and written the paper. TW, Designed and edited.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Targeted therapies have substantially improved the survival of patients with metastatic clear cell renal cell cancer. No prognostic or predictive biomarkers are available. Comprehensive genetic profiling offers the opportunity to define prognostic and predictive signatures aiming at a more personalized approach to treatment.In this prospectively conducted cohort study, tumor tissue and liquid biopsies are sampled at baseline and upon first and second progression under systemic treatment. Samples will be analyzed by whole-exome sequencing to generate prognostic and predictive patterns for systemic therapies.This study is aiming at exploring genetic profiles with prognostic and predictive value in metastatic renal cell cancer patients. Clonal evolution facilitating resistance to systemic treatment will be investigated by repeat biopsies. Kidney cancer is genetically diverse between different patients. Further, within a single affected individual, multiple genetic clones develop in the tumor and different metastases. Current treatment standard is molecularly targeted, albeit currently no genetic signatures can predict if an individual will benefit from treatment. This study aims at defining genetic profiles of kidney tumors of individual patients at the start of systemic therapy and at the timepoints of treatment failure. The aim will be to define markers that could help to choose the best therapy at baseline and at tumor progression. Renal cell carcinoma (RCC) accounts for about 2\u20133% of all cancers and shows its highest incidence in Western countries . About 7Over the last decade, the availability of targeted therapies has led to a substantial improvement in outcome for patients with metastatic RCC . InhibitThe MORE trial is a prospective biomarker study designed to investigate molecular alterations in patients with metastatic RCC. Whole-exome analysis of tumor samples and circulation tumor DNA will be performed at baseline and upon progression under systemic treatment with approved agents according to the European guidelines of urology by using whole-genome sequencing of primary and biopsy tissue samples as well as liquid biopsies from therapy-naive patients at baseline and after first and second drug treatment.To investigate the molecular alterations occurring under targeted drug treatment and to learn about drug resistances, baseline liquid biopsies and tissue biopsies will be analyzed and compared with mutational patterns occurring under first- and second-line targeted therapy. Potential molecular targets for personalized therapy of progressive disease should be analyzed to improve patient care and outcome.clinicaltrials.gov (NCT02208128) and on the National Study Register (DRKS0006193).The study is approved by the ethics committee at the Heidelberg University Medical Faculty (S-539/2013). The study is listed on A feasibility analysis was performed with four patients before implementation of the full study. Tissue and liquid biopsies generated sufficient DNA for analyzing mutational patterns .Patient selection and study design: this NGS-based trial is a monocenter prospective cohort study with explorative character to investigate molecular alterations under drug treatment in patients with metastatic RCC. Treatment-naive patients with metastatic clear cell RCC are eligible.The study will include a maximum of 100\u00a0patients with metastatic RCC on the assumption of a maximum of 20\u00a0patients to be recruited annually within a period of 5 years. The study is open for recruitment and is expected to recruit until 2022. The eligibility criteria are shown in An average response period of 11\u00a0months per drug is assumed according to the literature available . AccordiDNA from tumor tissue and a liquid biopsy at baseline, after first and second progression under standard of care drug treatment according to physician's choice will be obtained and subjected to whole-exome sequencing. Progress evaluation is performed according to RECIST and a tumor biopsy will be performed on one accessible progressing lesion in order to obtain vital tumor for DNA preparation.Since there is no clear evidence for a clinical benefit with any approved drug in the third-line situation, the treatment of choice in third line will be based upon the obtained mutational patterns, in other words, the approved targeted drug will be chosen that best covers the pathways altered by detected mutations. This includes immunotherapy which could be related with tumor mutational burden as a potential predictive signature. The MORE study diagram and workflow is shown in g for 10\u2009 min at 10\u2009\u00b0C, and plasma supernatant will be stored at -80\u2009\u00b0C until use. DNA extraction will be from 500 \u2009\u03bcl aliquots of frozen plasma using the QIAamp Blood Mini Kit according to manufacturer's protocol. Analysis and detection of mutation in ct-DNA will be performed according to previously published algorithms [Sample collection: standard treatment of patients with metastatic RCC includes a cytoreductive nephrectomy. Small samples of tumor tissue as well as samples of healthy kidney tissue will be obtained from tumor preparation. In addition to a standard blood sample, 10 ml of blood will be obtained for circulating tumor-DNA (ct-DNA) diagnostics. Processing of blood for ct-DNA collection will be performed as described previously [gorithms ,12. 2 weIsolation, quantification and quality control of genomic DNA, fresh frozen surgical tumor tissue and biopsy samples (10\u201330\u00a0mg) will be mechanically disrupted and homogenized using the TissueRuptor . Genomic DNA will be extracted using the QIAamp DNA Mini Kit (Qiagen) according to the manufacturer's protocol. As germline control, genomic DNA from matched blood cells will be isolated by the QIAamp DNA Blood Mini Kit (Qiagen). DNA concentrations will be determined using the Qubit , and DNA integrity will be assessed using the TapeStation .Sequencing libraries will be prepared from 200\u00a0ng genomic DNA. Prior to library preparation, all DNA samples will be sheared to an average fragment length of 150\u00a0bp using the Covaris S220 ultrasonicator. Exome-enriched sequencing libraries will be prepared using the Agilent SureSelectXT Human All Exon V5+UTR kit (low input protocol). Library sizes and qualities will be evaluated before and after capture by Bioanalyzer 2100 analysis using the High Sensitivity DNA Kit (Agilent) and quantified using the Qubit dsDNA HS Assay kit (Thermo Fisher Scientific). All libraries will be subjected to 100 bp paired-end sequencing on the Illumina HiSeq 2000 v3 at the DKFZ Genomics and Proteomics Core Facility. Selected variants will further be validated by bidirectional Sanger sequencing on an ABI 3130 Genetic Analyzer (Thermo Fisher Scientific) using the BigDye Terminator v1.1 Cycle Sequencing Kit (Thermo Fisher Scientific) as described previously [A small feasibility cohort of four patients was subjected to sequencing of primary and metastasis upon first progression according to the requirements of the funding body (HIPO-POP Heidelberg). The feasibility to obtain sufficient tumor DNA by biopsy for NGS could be demonstrated . We coulVHL gene. However, apart from VHL, a very diverse intra- and inter-individual genetic heterogeneity has been observed when different regions of single tumors and different metastatic sites have been analyzed [BRCA and PI3K mutations. The current standard of care in first line at our center is sunitinib or pazopanib for patients with a reduced performance status. In the near future, immune-checkpoint inhibitors as well as newer tyrosine kinase inhibitors (TKIs) like cabozantinib will likely be approved. In second line, nivolumab is currently the standard of care with rare cases receiving cabozantinib or lenvatinib and everolimus if the clinical situation requires a fast objective response. Hence, to prevent the cohorts with a specified therapy from getting too small we aim at treating larger groups of patients uniformly. Further, analysis of molecular signatures with regard to predictive factors will have to consider comparing groups of different modes of action-targeted agents versus immune-checkpoint inhibitors as well as analyzing TKIs followed by checkpoint-inhibitor and checkpoint-inhibitor followed by TKI sequences, respectively. In later lines of treatment, approved targeted substances will be chosen according to mutations found in the individual patient. The recommendations for a personalized therapy based on genetic findings within this study will be made by our molecular tumor board, taking into consideration the functional relevance as well as the allelic frequencies of detected genomic alterations (at least 20%) and the availability of an approved drug with target specific activity. Clinical follow-up within our study is aiming at the validation of the molecular tumor board recommendation.Clear cell renal carcinoma is a genetically heterogeneous disease. Founding genetic alterations that can be detected in most patients are genetic or epigenetic changes leading to a biallelic loss of function of the analyzed . Moreoveanalyzed ,5. The MSince the analysis of primary tumor or a single metastatic site at baseline or progression may not detect the relevant clone driving progression in other sites, we will obtain liquid biopsies in parallel for detection and mutational analysis of ct-DNA. This relatively new technique may help to avoid the necessity for invasive biopsies in the future . HoweverThe MORE study will comprehensively gather whole-exome data of tumors and circulating DNA at baseline and under first and second progression together with all clinically relevant data of RCC patients under systemic treatment. It aims at a better biomarker understanding, better genomic subgrouping and the development of a more tailored therapy approach at choosing specific drugs for individual patients.Several target substances and immune checkpoint inhibitors are active and approved in renal cell carcinoma.Renal cell carcinoma is clonally heterogeneous.No molecular biomarkers predictive for any type of treatment are defined.Clonal evolution develops under therapy leading to progression.The described study aims at whole-exome sequencing of tumor and circulating tumor-DNA in blood to detect molecular biomarkers."} +{"text": "We read with interest the article by Zanaty et al. \u201cFailure of the Pipeline Embolization Device in Posterior Communicating Artery Aneurysms Associated with a Fetal Posterior Cerebral Artery\u201d .The authors presented a case series of 4 patients who had been treated with the pipeline embolization device for posterior communicating artery (PCoA) aneurysms across several institutions. However, their review of the previously published literature is incomplete and indeed this phenomenon has been described by our own series of 4 patients several months earlier . Soon, t Journal of Neurosurgery [All 12 cases (4 in each series) involved posterior communicating artery aneurysms that incorporated a fetal origin of the posterior cerebral artery (PCA). The large flow demand of the fetal PCA causes flow across the aneurysm and into the distal PCA territory, preventing successful flow diversion. Without diversion of flow, the aneurysm remains patent and treatment fails. The same concept applies to other aneurysms incorporating \u201cend vessels\u201d; we recently presented this concept at the AANS 2016 meeting [osurgery ."} +{"text": "Advances in targeted immune therapeutics have profoundly improved clinical outcomes for patients with inflammatory arthropathies particularly rheumatoid arthritis. The landscape of disease that is observed and the treatment outcomes desired for the future have also progressed. As such there is an increasing move away from traditional models of end\u2010stage, chronic disease with recognition of the need to consider the earliest phases of pathogenesis as a target for treatment leading to resolution and/or cure. In order to continue the discovery process and enhance our understanding of disease and treatment, we therefore need to continuously revisit the animal models we employ and assess their relevance and utility in the light of contemporary therapeutic goals. In this review, we highlight the areas where we consider new developments in animal models and their application are most required. Thus, we have contextualised the relevant mouse models and their use within the current concepts of human inflammatory arthritis pathogenesis and highlight areas of need. Rheumatoid arthritis (RA) is a prototypic inflammatory arthritis \u2013 it is a chronic, painful and disabling inflammatory disease principally resulting in damage to joints and surrounding connective tissues. The processes underlying initiation and perpetuation of disease involve a complex interplay between genetic and environmental factors. Consequently, current research focuses on how these factors lead to perturbations in innate and adaptive immune responses, changes in tissue stroma activity, and ultimately to joint disease. Our current knowledge can be summarised in a step\u2010wise pathogenesis map that depicts distinct phases or processes in RA The continuing elucidation of the pathogenesis map relies on data obtained from clinical and animal model studies. Debate over the validity of animal models can arise when contrasting results arise from human studies, questioning the validity of animal models. The publication of data stating mouse models poorly reflect human genomic inflammatory responses It is clearly unrealistic to expect a single model of arthritis to fully recapitulate human disease. However, individually they allow molecular and spatiotemporal dissection of various pathological processes of RA development that would otherwise be impractical, or unethical in a clinical setting. Employing combinations of models offers the potential to better mirror the complexities of human disease, reflecting different environmental, genetic and temporal contributions to clinical heterogeneity. Which models to combine will ultimately be dependent on the scientific question being asked. By mapping existing models onto different stages of human disease, appropriate systems can be selected to investigate major steps in disease initiation and progression. Additional benefits of this type of mapping are to highlight knowledge gaps in pathogenesis and hence where new models may be required. The generation of a comprehensive RA pathogenesis map will require in\u2010depth analysis of existing models and will rely heavily on generation of detailed standardised protocols. The importance of developing pathogenesis maps, the refinement and standardisation of current models and ethical considerations have recently been discussed in detail elsewhere In addition to the clear definition of models relevant to specific disease stages, there must be greater appreciation of processes regulating transition from stage to stage. While key cells and molecules contributing to pathology have been highlighted, we lack knowledge pertaining to the underlying dynamics of the immune response. Candidate triggers can be highlighted by correlative patient studies but the development of current and new models will be critical in defining mechanisms by which they drive pathogenesis.With successes in the treatment of established RA, one critical therapeutic objective is now restoration of antigen\u2010specific tolerance in RA ptpn22, cd40, ctla4 and cd28 in disease pathogenesis. Yet a comprehensive picture integrating these susceptibility loci with mechanisms leading to breach of self\u2010tolerance in preclinical RA is lacking.Critical roles for the adaptive immune system in the development of RA are supported in part by predisposing genetic traits. In addition to the long\u2010standing association with human leukocyte antigen (HLA)\u2010DR4 alleles, genome wide scanning studies implicate q haplotype HLA class II mouse models are relevant to initial breach of tolerance mechanisms including shaping of the TCR repertoire and profiling T\u00a0cell epitopes arising from autoantigens PTPN22). PTPN22 encodes lymphocyte tyrosine phosphatase (Lyp), with the 1858 C to T polymorphism representing one of the highest risk associations after HLA class II ptpn22 polymorphisms to RA pathogenesis. Yet these risk allele models do not spontaneously develop overt arthritis unless in the context of an additional autoreactive TcR transgene or injection of arthritogenic antigen in adjuvant. Additional factors are required to work in tandem with genetic predispositions in the development of autoimmune T and B\u00a0cell responses.Models of additional risk associated alleles include those relating to polymorphisms in protein tyrosine phosphatase non\u2010receptor type 22 (\u2212/\u2212) Porphyromonas gingivalis was seen to accelerate joint inflammation and systemic markers of inflammation in CIA P. gingivalis exacerbation of RA are unclear. However, it is postulated that P. gingivalis infection contributes to autoreactivity through post\u2010translational modification of proteins and the development of antibodies against citrullinated peptides (ACPA) SKG mice, which carry a point mutation of ZAP70 gene, spontaneously develop chronic autoimmune arthritis stemming from altered TcR signaling and consequent perturbations in thymic selection events. Notably, these arthritis prone mice require environmental triggers for disease onset. Rearing of SKG mice in specific pathogen\u2010free or germ\u2010free conditions requires fungal or intestinal microbiota respectively to establish the development of arthritis The lung has been implicated as an important site mediating environmental contributions to pathogenesis. Notably, smokers are at a higher risk of developing RA and once afflicted will progress more aggressively Clearly genetic susceptibility alone is insufficient for the development of autoreactivity. Environmental contributions such as mucosal damage, microbiome perturbations and/or chemical/microbial mediated post\u2010translational protein modifications contribute to RA risk. Animal models represent an important opportunity to combine these aspects in a fuller exploration of preclinical pathogenesis concepts.It is clear that patients can exhibit the preclinical autoimmune phenotype for many years in an essentially asymptomatic state. Yet the cues that drive the transition to overt articular inflammation and the mechanisms of immune cell accumulation in the joint remain enigmatic. Biomechanical stress, hypoxia and trauma have been proposed as important factors in transition to articular localization. Physical trauma has been reported as being significantly associated with RA onset Direct joint damage and consequent microbleeding facilitate arthritis development in mice expressing a variant of the IL\u20106 signaling transducer gp130 Deposition of immune complexes in articular surfaces has a clear role in promoting local inflammation and recruitment of immune cells. Recent studies employing CIA implicate key roles for Th17 helper activity in suppressing sialtransferase activity in B\u00a0cells Chronic joint inflammation is one of the hallmarks of clinical disease, with the majority of patients presenting at this stage. A picture of key cellular and soluble mediators is beginning to emerge based on the success of several biologics such as anti\u2010CD20, CTLA4\u2010Ig and TNF\u03b1. Despite this, there is a paucity of models displaying chronic disease hampering mechanistic study.\u0394ARE mice back\u2010crossed to a RAG1\u2212/\u2212 background still develop erosive polyarthritis, indicating a key role for this cytokine downstream of T and B\u00a0cell responses TNF\u2010overexpressing mice are consistently reported as developing chronic pathology with several lines contributing to our understanding of TNF\u03b1 activity in disease \u2212/\u2212 and SKG mice have been shown to be T\u00a0cell dependent and model chronic disease, data pertaining to adaptive mechanisms beyond disease onset are scarce.Although the CIA model, IL\u2010RAActivation of the adaptive immune response clearly occurs early in disease onset, yet how T and B\u00a0cells might contribute to ongoing chronic pathology is unclear. The acute self\u2010resolving nature of antibody\u2010induced models suggests that T and B\u00a0cell activity is required for ongoing production of autoantibodies. Yet even intact models such as CIA do not readily develop chronic disease. Induction of chronic relapsing CIA in DBA/1 mice can be achieved by immunisation with autologous type II collagen. Antibody responses to particular antigenic epitopes of type II collagen have also been implicated in development of chronic CIA in vitro work using primary human cells, with the recent genomic profiling of mouse synovial fibroblasts from TNF\u2010Tg mice demonstrating comparison with human data demonstrates significant commonalities A final aspect of chronic disease warranting deeper investigation is the contribution of the articular stromal compartment. Synovial fibroblasts , mast cells, lymphatic and blood vessels, make up the joint lining and contribute to its normal function Clinical observations including elevated lymphangiogenic factors in RA synovium, increased numbers of lymphatic vessels, yet reduced lymphatic flow implicate a role for the lymphatic system in RA pathogenesis Disease models have been extremely useful in identifying key cell populations and molecules contributing to pathogenesis (for extensive review see Advances in bioimaging equipment and techniques are already adding spatial and temporal information to mouse models of arthritis. Notable examples include multiplexed longitudinal non\u2010invasive imaging of disease onset and progression Criticism of animal models lies often in their inability to fully recapitulate all of the complex processes leading to disease. Cases of conflicting data between mouse and human studies continue to fuel debate . NeverthThe authors declare no financial or commercial conflicts of interest.ACPAanti\u2010citrullinated peptide antibodiesAIAantigen induced arthritisCIAcollagen induced arthritisCIAIcollagen antibody induced arthritisHLAhuman leukocyte antigenLEClymphatic endothelial cellLyplymphocyte tyrosine phosphatasePTPN22protein tyrosine phosphatase non\u2010receptor type 22RArheumatoid arthritisSMCsmooth muscle cell"} +{"text": "The spread of breast cancer cells to bone and survival in this new metastatic environment is influenced not only by the genetic signature of the cells, but also multiple host cells and soluble factors produced locally (paracrine) or from distant sites (endocrine). Disrupting this metastatic process has been evaluated in clinical trials of the bone targeted agents bisphosphonates and denosumab and have shown that these agents reduce the recurrence of breast cancer in postmenopausal women only, suggesting the efficacy of the drugs are influenced by levels of reproductive endocrine hormones. The molecular mechanism driving this differential effect has not been definitively identified, however, there is evidence that both reproductive hormones and bisphosphonates can affect similar paracrine factors and cellular components of the bone metastatic niche. This review focuses on how the ovarian endocrine hormone, inhibin, interacts with the paracrine factors activin and follistatin, abundant in the primary tumour and bone microenvironment, with subsequent effects on tumour cell survival. Inhibin also affects the cellular components of the bone microenvironment primarily the osteoblastic niche. Recent evidence has shown that bisphosphonates also alter this niche, which may represent a common mechanism by which inhibin and bisphosphonates interact to influence disease outcomes in early breast cancer. Further research is needed to fully elucidate these molecular mechanisms to enable understanding and future development of alternative bone targeted treatments with anti-tumour efficacy in premenopausal women. The survival of tumour cells during this process is influenced by their genetic signature and a plethora of host cells and soluble factors 1.1in vivo accumulates rapidly in the bone marrow indicating that it can distribute to bone In vitro, FSH increases osteoclast differentiation in vivo treatment of ovariectomised 14-week old mice with an antibody to \u03b2-subumit of FSH, blocking its biological activity, prevents OVX-induced bone loss after 4 weeks of treatment. Dynamic histomorphometry showed inhibiting FSH increases all bone formation parameters and inhibits bone resorption parameters Menopause is characterised by a decrease in ovarian oestradiol and inhibins with an increase in pituitary follicle stimulating hormone (FSH). The decline in inhibins drives the increase in bone turnover that occurs in early menopause and although inhibins are not abundantly expressed in bone, radiolabelled inhibin A administered intravenously 1.2in vivo studies Inhibins do not have an identified intracellular downstream signaling pathway but bring about their effector functions by inhibiting ligand: receptor interaction of the soluble paracrine factors activin and TGF\u03b2, abundant in both the primary tumour and bone microenvironment 1.3in vitro and inhibits proliferation Activin is secreted by breast cancer cell lines Hormone interaction with bisphosphonates; the bisphosphonate zoledronic acid (ZA) has been shown to increase activin's biological activity in breast cancer cells in vitro and in vivo, enhancing its tumour suppressive effects vs CT alone 1.4vs 29.5%) in vivo data showing that lowering activin levels with a soluble activin receptor type IIA fusion protein prevents the formation of bone metastases from MDA-MB-231 cells Disseminated tumour cells are detectable in the bone marrow of a third of patients with early breast cancer without any clinical manifestations of bone metastasis Hormone interaction with bisphosphonates; Clinical trials have shown that bisphosphonates decrease the number of bone marrow DTCs in marrow aspirates from breast cancer patients 1.550% of patients with detectable DTCs will relapse during 10 years post diagnosis Hormone interaction with bisphosphonates; Disrupting the interactions between DTCs and the bone stromal cells was evaluated in phase III randomised clinical trials of adjuvant bisphosphonates, and a large meta-analysis of these adjuvant bisphosphonate trials involving >18,000 patients showed bisphosphonates prevented breast cancer recurrence in bone , at other distant sites and improved breast cancer mortality in women who were postmenopausal when treatment started 2in vivo models that expanding the Ob niche with the use of parathyroid hormone, increased the number of DTCs in bone from sub-cutaneous prostate tumours in vivo study evaluating the effects of a single dose of zoledronic acid (100\u00a0\u03bcg/kg) showed that the drug significantly reduced Ob number which influenced where intracardiac injected MDA-MB-231 breast cancer cells home to, with a preference demonstrated for Ob rich areas Reproductive endocrine hormones such as inhibin affect breast cancer cell survival in the primary tumour and affect tumour homing and survival in the bone microenvironment. The molecular mechanism driving this effect of hormones in bone is likely to be multifactorial by modification of both paracrine factors and the cellular components of the bone metastatic niche. If the Ob niche is key to the maintenance of dormancy and survival of tumour cells then factors that affect the size of this niche can potentially determine outcomes for breast cancer cells in this environment. There is evidence from 2.1\u2022Which female hormone(s) affect the direct anti-tumour efficacy of bisphosphonates in primary tumours and the indirect anti-tumour efficacy of bisphosphonates in bone, in particular what are the roles of activin and inhibin?\u2022What are the cellular/molecular drivers of tumour growth in the pre- and postmenopausal bone microenvironments?\u2022Is the differential effect of menopause on the anti-tumour efficacy of osteoclast inhibitors specific to breast cancer patients only or does this apply to other tumours such as lung cancer?"} +{"text": "Unfortunately, upon publication of this article it was n\u201cIn our data, we expect a relatively weak phylogenetic signal of CV body mass and thus low values of \u03bb as the amount of body fat is phenotypically plastic and can undergo quick and extensive adaptive modifications in response to food availability and local environment. Therefore, closely related species might have very different CV body masses depending on their habitats .\u201dThis should read:\u201cIn our data, we found a surprisingly weak phylogenetic signal of CV body mass and thus low values of \u03bb for the model residuals, indicating that the phylogenetic disposition for fat disposition is partially masked by habitat-caused variation . The fact that we still found significant relationships between CV body mass and allomaternal care would then make our case even stronger, because it implies that the underlying effect must be very strong.\u201dThe original article has now been updated and the publisher apologizes for any inconvenience caused."} +{"text": "The purpose of the modified repositioning appliance was to overcome the shortcoming of existing design for repositioning protruded premaxilla in a child with bilateral cleft lip and palate.The basic principles of design were similar to Latham\u2019s appliance but the surgical pinning of premaxillary segment was avoided and instead acrylic splint was prepared.This technique avoids any invasive procedure, is useful to reposition protruded premaxillary segment in bilateral cleft lip and palate cases specifically in child who reports late with deciduous dentition. Presurgical orthopedics is routinely required in the management of complete bilateral cleft lip and palate (BCLP) cases, which have markedly protruded premaxillary segment. The initial step in the management of BCLP is to reposition the protruded premaxilla prior to surgical correction. Repositioning protruded premaxilla serves dual advantages; first it prevents excessive tension at suture line following surgical correction of lips and secondly provides psychological benefit to child because of early esthetic improvement.1 where he utilized an intraoral prosthesis with a head bonnet and extaoral strap for repositioning of protruded premaxillary segment. Since then, arrays of appliances/methods6 have been introduced to reposition the premaxilla in BCLP cases. Georgiade and Latham (1975)3 developed an intraoral premaxillary repositioning appliance, which was later modified by Millard and Latham7 and came to be known as intraoral elastic chain premaxillary repositioning appliance (ECPRA) or more frequently Latham\u2019s appliance. This appliance consists of acrylic pads over the maxillary segments connected posteriorly by an expansion mechanism. The premaxil-lary segment is retracted with elastic bands attached to a pin inserted in the premaxillary bone, just anterior to the premaxillovomeral suture. In BCLP patients where commonly posterior alveolar segments are collapsed, Latham\u2019s appliance had an added advantage of achieving posterior expansion along with repositioning of protruded premaxillary segment. Latham\u2019s appliance, inserted on an average at 2-month-of-age, relocates the segments over 3 to 4 weeks.8 Removal of the appliance is immediately followed by functional surgery.Any procedure undertaken at neonatal age to remold or reposition the skeletal or soft tissue segments so as to simplify the surgical procedures in a cleft lip and palate case are commonly referred to as \u2018neonatal maxillary orthopedics or presurgical orthopedics\u2019. The concept of presurgical neonatal maxillary orthopedics was first introduced by McNeil (1950),The case presented in this article is of a child aged 4 years and 2 months with complete bilateral cleft lip and palate. Neonatal maxillary orthopedics treatment is customarily initiated within first 6 months after birth, but many times we may come across situations where a child reports at an older age with deciduous dentition, therefore compounding the existing problem. Since appliance which uses oral pinning and traction could have caused interference with growth or damage to developing permanent tooth buds, therefore in this case it was decided not to use any pinning of premaxillary segment. Hence, a modified noninvasive repositioning appliance was fabricated for repositioning of protruded premaxillary segment, although the basic principles of design were similar to Latham\u2019s appliance.The patient Juber aged 4 years and 2 months reported with complete bilateral cleft lip and palate with markedly protruded premaxillary segment shifted to the left side and 2. I9Measurements were preformed by utilizing following reference points .Right and left cleft widths were measured.Right cleft width (RCW): the distance between R and R\u201dLeft cleft width (LCW): the distance between L and L\u201d.Following were the recorded measurements on pretreat-ment study models :Dental impressions of the maxillary and mandibular arch were taken using customized impression tray and rubber base impression (polysiloxane) material. Impressions were poured in white dental stone An acrylic maxillary occlusion split was fabricated on right and left segments, and hooks with 0.9 mm stainless steel wire were placed in the deciduous second molar region on both side, these hooks were directed distally for attachment of right and left elastic bands. A transpalatal arch was incorporated to stabilize the maxillary segments . On the premaxillary segment, model acrylic cap was fabricated with bilateral stainless steel hooks in the most lateral aspect which were directed mesially . A cut was made in this splint for the erupted left deciduous incisor. The tissue\u2019s side of the premaxillary splint was relined by perma soft denture reliner for cushioning of sensitive soft tissue. The maxillary occlusion splint was cemented on both maxillary segments with GIC cement. Orthodontic elastic bands were secured from hooks on maxillary splint to the premaxillary splint applying a force of 200 gm on each side .The patient was checked weekly and elastic bands were adjusted to reposition and align the premaxillary segment. Within 8 weeks sufficient amount of distal repositioning of premaxillary segment was achieved to 8.After treatment right cleft width was 15.29 mm and left cleft width was 12.91 mm.Since appliance which uses oral pinning and traction can cause interference with growth and may damage developing tooth buds, therefore, in this case it was decided not to use any pinning of premaxillary segment. As the maxillary width was normal, there was sufficient space to retract the premaxillary segment, therefore self expansion of lateral palate was not included in the treatment plan. The premaxillary cap splint was prevented from dislodgement by applying a blob of composite on the labial surface of incisor after placing the splint. Appliance was worn successfully by the patient, following were the problems encountered: Frequent breakage of elastic due to masticatory forces for this parent was trained to change the elastics on daily basis. Patient was recalled every week, and premaxillary segment was removed, cleaned and replaced, at the end of treatment minor bruises were seen.10 Bitter (1992),4 Millard and Latham8 believed that these repositioning appliance for alignment of alveolus segment are beneficial not only for lip and nose reconstruction but also for the occlusion as well. Other authors, Bertcovitz (1996),11 Henkel and Grundlach (1998)12 considered that this results in more malocclusion then when there is no orthopedic treatment.Controversy exists with regard to treatment and the dental occlusion,All considered, the facilitation of lip and nose reconstruction in the difficult case makes presurgical orthopedics with improved technique worthwhile, not only because of reduced tension at the suture line and less need for soft tissue undermining but also because it does eliminate the necessity for additional lip adhesion surgery."} +{"text": "Formal thought disorder is one of the fundamental features of schizophrenia. Early onset schizophrenia (EOS) is strongly related to poor prognosis and illness outcomes. The aim of this study is to investigate the relation of EOS and formal thought disorder in schizophrenia.This research was a retrospective study. Data regarding the patients with schizophrenia were obtained from two separate studies conducted at Dokuz Eylul University.Thought disorder scores were compared between 32 patients with early onset schizophrenia and 120 patients with adult onset schizophrenia . Also, we looked at the effect of Duration of Untreated Psychosis (DUP). We further categorized these two sets as short DUP and long DUP groups.Schizophrenia patients with early onset showed significantly higher scores compared to adult onset schizophrenia patients with regards to poverty of speech and peculiar sentences .Early onset schizophrenia patients had significant formal thought disorder abnormalities. Formal thought disorder may have some developmental characteristics which indicate an important dimension related to prognosis and outcome of schizophrenia. Also, DUP may have potential effect over formal thought disorder."} +{"text": "The symbols for the new IUPAC elements named in November 2016 can introduce subtle ambiguities within cheminformatics software. The ambiguities are described and demonstrated by highlighting inconsistencies between software when handling existing element symbols. Nh), 115 Moscovium (Mc), 117 Tennessine (Ts), 118 Oganesson (Og). Cheminformatics libraries typically use a centralised dictionary of elements to store and look up symbols in the periodic table. Na\u00efvely adding the new element symbols to this table can introduce unexpected behaviour.On the 28th November 2016 the International Union of Pure and Applied Chemistry (IUPAC) approved the names and symbols of four new elements: 113 Nihonium but NH (secondary amine) may now unexpectedly be picked up as Nihonium from the internal dictionary.A more subtle problem may arise with the symbol Support for the SMARTS query language is available in many closed and open-source cheminformatics toolkits. A potential area for ambiguity is again found with Nihonium and the interpretation of other transfermium symbols. Transfermium symbols were officially named after the initial release of the Daylight SMARTS toolkit and in sThe problem occurs due to the implicit conjunction between adjacent primitive expressions. The new symbol [Nh] could be interpreted as #113] (element 113) or [N&h] (Aliphatic nitrogen and at least one implicit hydrogen). Table 13 (elemeA pragmatic approach to handling the new elements or perhaps all high atomic number elements with a very short half-life could be to simply ignore them. Whilst these elements are unlikely to have a practical application it is unsatisfactory to simply ignore them and we hope this commentary highlights that care should be taken when supporting the new symbols in cheminformatics software."} +{"text": "Hb S) gene. This erroneous belief may lead to the delivery of a homozygous Hb SS infant if the partner has a high risk of being a Hb S gene carrier when marriages of close relatives occur, as in some populations like Eti-Turks.Hematopoietic stem cell transplantation (HSCT) with non-myeloablative regimens has become more feasible and it is currently performed more frequently in adult patients with sickle cell disease (SCD) . Hsieh reportedSCD is the most common hereditary disease worldwide. The impaired microcirculation caused by rigid erythrocytes leads to considerable mortality and severe morbidity if not managed appropriately . The onlWhen young SCD patients want to marry after full recovery with transplantation, some of them tend to hide their disease intentionally or due to a lack of awareness. A complete blood count and hemoglobin electrophoresis test as part of the screening done before marriage may give normal results. Consequently, affected germ cells may be overlooked. This may result in giving birth to affected children. The transplant team is responsible for providing sufficient information about these issues."} +{"text": "Physical examination revealed a loud ejection systolic murmur at the left sternal edge. A transthoracic echocardiogram showed turbulent flow and increased velocity at the right ventricular outflow tract (RVOT). The right atrium and ventricle were dilated and there was severe tricuspid regurgitation Figure . A cardiThere is a large mass causing near occlusion of the main PA. This mass extends distally into proximal left and right PA. This mass also extends proximally into pulmonary valve and proximal posterior aspect of the RV out flow tract.On T1W images, the tumor shows heterogeneous low-signal while on T2W sequences, it exhibits a mixed signal with hyperintensity as well as areas of intermediate to low intensity or tumor embolism from pulmonary or other malignancies. Pulmonary artery sarcoma mimicking as PE, pulmonary artery aneurysm or Takayasu's disease has been reported in the literature. In our case, the absence of thromboembolism risk factors, together with heterogeneous tumor enhancement and invasion into the wall of main pulmonary artery as well as its branches are suggestive of a malignant process instead of PE."} +{"text": "PNBI). The report considers the uses and merit of magnetic resonance imaging (MRI) in the primary assessment of PNBI. The traditional technique of cranial ultrasound as the first modality of choice can have several limitations, which includes a lower temporal resolution in its ability to differentiate grey\u2010white matter distribution patterns, lower spatial resolution in its ability to accurately map white matter fibre tracts and distribution patterns which are critical in white matter injury pathological events. In this specific case report, MRI was useful for the assessment of haemorrhagic brain injury post partum.Therefore, should MRI be considered, the primary imaging modality in these cases when the concerns about PNBI is presented? This case study explores the current trends in MRI neonatal brain imaging and advancements being made in this field.This case report aims to extend analytical thinking and clinical reasoning of clinicians and radiographers when presented with diagnosing premature neonatal brain injuries ( Magnetic resonance imaging (MRI) for premature neonatal brain injuries (PNBI) is a recurrent request that we face at many clinical centres in Australia and New Zealand in my experience. Our team of neonatologists, paediatricians and paediatric radiologists utilise radiology and its modalities for correct and timely diagnosis of PNBI. Commonly a routine cranial ultrasound is performed, however, ultrasound has limited temporal and spatial resolution when attempting to image white matter injuries. Cranial MRI of preterm neonates is often a more beneficial imaging modality.The patient's caregiver has provided consent for his case to be reported and published.A patient presented from the Neonatal Intensive Care Unit for MRI following concerns that they had experienced PNBI as a result of traumatic brain haemorrhage, following delivery. A previous cranial ultrasound study was inconclusive other than some enlarged hydrocephalus. MRI was utilised to investigate this neonate more intensively.A routine paediatric neonate head MRI protocol was performed on a General Electric (GE) Signa HDxt 1.5 Tesla (GE MRI) System using an 8 channel GE head coil. A vacuum beanbag was used to support and minimise head motion. The child was fed and a sucrose solution given prior to the scan to calm them down for sleep, which is described as a feed and sleep type scan. A routine neonatal paediatric head protocol was used, which includes the following sequences;Localiser 3\u2010plane scoutTR3400, TE104)T2 transverse DWI b0\u2010b1000 transverse T1 sagittal T2 coronal Post\u2010partum ultrasound report: \u201cno obvious appearance of germinal matrix periventricular haemorrhage\u201d. The lateral, third and fourth ventricles are larger than normal ranges which suggest, there is something pathological occurring in this specific case. There was therefore a need for a cranial MRI to evaluate the presence of PNBI.MRI report: \u201cMarked ventricular dilation of the lateral, third, fourth ventricles, extending into a prominent infra cerebrospinal fluid space. This is likely to be secondary to extensive intra\u2010ventricular haemorrhage which arises from germinal matrix/choroid plexus regions. Cerebellar atrophy, but no definite structural abnormality of the cerebellum is seen.\u201d This MRI report demonstrates that haemorrhage due to PNBI is visualised in the lateral, third and fourth ventricles, structurally the brain ventricles are enlarged. Pathologically it demonstrates PNBI with specific periventricular leukomalacia (PVL) injury.MRI demonstrated PNBI accurately in this case report, haemorrhage is clearly visualised on the examination images, in comparison to the previous ultrasound study performed. It was also able to diagnoses PVL injury more accurately. PNBI consists of multiple mechanisms these are mainly, periventricular haemorrhagic infarction and PVL.The T2 coronal (Fig. The diffusion\u2010weighted transverse MRI image with a bipolar gradient value of b1000 Fig. B was difIn this case, germinal matrix intra\u2010ventricular haemorrhage has occurred, and secondary to that there is post\u2010haemorrhagic hydrocephalus which may likely additionally be associated with PVL white matter injury. This imaging highlights the importance of the use of MRI in diagnosing PNBI. Recent literature in peri/prenatal risk factor patterns of PNBI concluded that measured prenatal risk factors did not predict the pattern of brain injury. Additionally, they could not predict the clinical and 30 months neuro\u2010developmental outcomes or patterns of brain injuries in neonatal infants.Ultrasound is useful in some abnormal structural injury cases as it has the benefits of being widely available, accessible and cost\u2010effective in comparison to MRI. Being able to perform multiple cerebral ultrasounds over the duration of the patients stay by definition would increase its sensitivity and specificity on first look. However, literature suggests increased frequency of performing cranial ultrasound was not found to increase the detection of white matter injuries.With the advancements in critical neonatal care medicine, survival of preterm infants is associated with increased rate of neurodevelopmental impairment, which is currently 3\u20134 times that of the general population.The mortality rate for childhood stroke is about 1 per 4000 and rates are highest in infants under the 1\u2010year\u2010old age group.A recent MRI study, using DTI focused specifically on white matter tract migratory development and found that perinatal cerebral white matter injury seems to have major deleterious effects on the subsequent development of fibre tracts both in the cerebral white matter and more cortically. The ultimate impact of PNBI in the new\u2010born should be considered as a function not only of tissue destruction but also of impaired subsequent brain development.Rose et al.PNBI mortality rates are relatively low and tolerance of such injury events is remarkable. The prenatal and perinatal risk factors often do not correlate to injury types and little is known about the developmental effects on PVL white matter injuries at such a young age. In this specific case report, haemorrhagic hydrocephalus from the germinal matrix and choroid plexus region secondarily may have led to white matter PVL injury. A neurosurgical intervention was needed. Ultrasound plays little part in white matter injury detection, however, may be effective in determining long\u2010term survival outcomes of PNBI patients. This case report emphasises the uses of MRI in PNBI as a very useful tool for primary diagnosis and management of this complex neonatal age group and pathological process of white matter\u2010type injuries. Little is known about the longer\u2010term effects of such an injury on the development of the brain and lifelong disabilities associated with them, however studies suggest that these injuries are playing a larger role in society as advancements in modern neonatal care increase survival rates. This case report aims to increase thinking and clinical reasoning of clinicians and radiographers when presented with similar cases. I hope that this case report sparks interest and awareness of the thought processes involved in investigating PNBI.The authors declare no conflict of interest."} +{"text": "Chrysanthemoides monilifera ssp. rotundata DC. Norl.), a South African shrub invader. We used the same bitou propagule pressure across all treatments and monitored invasion success and resource availability for 13 months. Contrary to our predictions, we found that functional richness did not mediate the number of bitou individuals or bitou cover and functional identity had little effect on invasion success: there was a trend for the grass single functional group treatment to supress bitou individuals, but this trend was obscured when grasses were in multi functional group treatments. We found that all constructed communities facilitated bitou establishment and suppressed bitou cover relative to unplanted mesocosms. Abiotic resource use was either similar among planted communities, or differences did not relate to invasion success (with the exception of light availability). We attribute invasion resistance to bulk plant biomass across planted treatments rather than their functional group arrangement.Biotic effects are often used to explain community structure and invasion resistance. We evaluated the contribution of functional richness and identity to invasion resistance and abiotic resource availability using a mesocosm experiment. We predicted that higher functional richness would confer greater invasion resistance through greater resource sequestration. We also predicted that niche pre-emption and invasion resistance would be higher in communities which included functional groups similar to the invader than communities where all functional groups were distinct from the invader. We constructed communities of different functional richness and identity but maintained constant species richness and numbers of individuals in the resident community. The constructed communities represented potential fore dune conditions following invader control activities along the Australian east coast. We then simulated an invasion event by bitou ( Plant invasion may be predicted in part by stochastic events such as dispersal (e.g. ) or by hIn order to understand the role of biological interactions in invasion, researchers focused initially on invader attributes which contribute to invasion success such as high growth rates, efficient resource use or high resource allocation to reproduction e.g. , 8). Mor. Mor8]).Plant composition may have greater importance than richness in determining invasion outcomes. This idea has been established at the species level , and theMuch of the theoretical work examining biotic resistance\u2013where resident communities reduce the success of invaders \u2014has consFunctional group effects on invasion are often considered similar to species effects . HoweverChrysanthemoides monilifera ssp. rotundata) as our system invader. Bitou is a South African shrub species that has invaded large areas of coastal eastern Australia. It has been the subject of intensive management by practitioners but activities have often focused on reducing biomass rather than establishing functionally diverse native communities after control ).We predicted that:Communities with higher functional richness would be less invaded than those with lower functional richness;Abiotic resource availability would be lowest in the most functionally rich planted treatment, highest in the unplanted treatment and intermediate for the low and moderate functional richness treatments;Communities represented by functional groups similar to the invader would be less invaded than communities where all functional groups were distinct from the invader.3 of sand) in each tank. The fertiliser addition was repeated in April 2008. We measured total nitrogen ) in October 2007 to allow soil chemistry to re-equilibrate after the setup disturbance and to allow release of the fertiliser. Resultant average soil nitrogen concentrations were low (0.002\u20130.024 mg TKN/g) and comparable to fore dune conditions ).All planted treatments supported significantly more bitou individuals than the unplanted treatment. We attribute this pattern to a nurse effect among extant native species (e.g. ). Bare gFacilitation of invasives by native species is increasingly recognised as an important interaction in plant communities and it mHowever, we did not see facilitative effects of native functional groups when considering bitou cover: all native communities similarly suppressed bitou cover. Our findings agree with a meta-analysis of competition studies which reported that native species interactions regulate invader abundance rather than completely repel invaders .P = 0.06). There was a complementary pattern of reduced bare ground cover among communities with grass representation. So grassed communities may make up for reduced vertical biomass with increased horizontal coverage. Below ground biomass effects at the end of the experiment were modest: the only significant difference at the 0\u201320 cm level (where we expect strong resource competition) was greater biomass for the GS treatment compared with the unplanted treatment. These results combine to suggest that bulk above-ground plant cover may affect invasion levels more than functional group arrangement. This interpretation is supported by the negative relationship between native and bitou biomass at the final time period, and the finding that functional richness treatment does not affect the relationship between bitou biomass and native biomass (ANCOVA results with the Unplanted treatment removed). In a related mesocosm arrival order study, we found that functional identity and historical dominance were unimportant in determining invasion resistance ),vs. unplanted treatments. This result may indicate that abiotic resources were not limiting in any of our communities: natives, the invader and different growth forms used abiotic resources in a similar, non-limiting way. Alternatively, bitou may be acting as an equaliser across treatments\u2013allowing all functional richness treatments to optimise resource use. This argument is supported by final total above ground biomass (native and bitou biomass) results: the unplanted treatment had similar final total biomass to most planted treatments. The only significance we found was that unplanted and G communities had lower total biomass than the HS community.Contrary to our second prediction, we found that abiotic resource availability did not vary consistently among the functional richness treatments. And resource availability in the unplanted treatment was often indistinguishable from planted treatments. For example, soil moisture levels and nutrient availability could not be explained by functional richness or the coarser metric of planted It is noteworthy that there were discrepancies in the results for bare ground cover and PAR at ground level at the end of the experiment. Bare ground cover was significantly greater in the unplanted treatment than all planted treatments. However averaged PAR values for each mesocosm yielded similar values across all treatments, and when PAR was categorised into sub-optimal, optimal and supra-optimal values, the unplanted treatment had significantly more sub-optimal PAR counts (i.e. lower light penetration) than expected. So bare ground cover is not a direct proxy for PAR at ground level. These findings raise an unexpected methodological consideration for diversity studies which measure resource availability. Light interception is dependent on both total above ground biomass and the geometric configuration of the canopy , so usinContrary to our third prediction, inclusion of the shrub functional group in a community did not improve invasion resistance relative to communities that did not have shrub representation. Our results therefore do not support the concept that greater niche overlap improves invader resistance (e.g. ). Our reWe observed greater intra- than inter-specific competition for the shrub invader as demonstrated by the hump-shaped response of bitou individuals over time in the unplanted treatment compared with the pattern of individuals increasing and then transitioning to a relatively stable level for most planted treatments. While these results demonstrate that competition may contribute to community structure, the biotic effects are modest with no significant effect on invasion resistance through niche pre-emption. It is interesting that intra-species competition was more important than inter-species competition for bitou individuals and may indicate that bitou is a strong invader .Our results indicated that functional richness does not confer resistance. Our design controlled the invasion event, so we can exclude propagule pressure as an explanatory variable. And we have not found evidence for niche overlap as a significant factor in invasion resistance. We offer two possible explanations for the result that all our planted communities had similar resistance in terms of bitou cover and, to a lesser extent, number of bitou individuals. Firstly, invader identity may be more important than resident diversity in determining community resistance to invasion. Bitou may be a \u201cstrong\u201d invader and its We found that invasion resistance was unrelated to native functional group richness or identity in our constructed communities. Native constructed communities influenced the invader more strongly through suppression of adult biomass than through inhibition of seedling establishment. While the grass single FG treatment showed a trend of reducing bitou individuals relative to the HS multi FG treatment, in general all planted treatments had significantly more bitou individuals than the unplanted treatment, and this pattern remained consistent over time. Conversely, all planted treatments had significantly lower bitou cover than the unplanted treatment. These findings indicate that 1) extant native biomass significantly reduces invader biomass compared with bare ground; (2) increasing functional richness does not increase resistance to invasion; and (3) regardless of native functional richness, invader individuals are able to establish and potentially founder future populations following disturbance. Grass, herb and shrub growth forms dominate coastal fore dune communities. Our findings suggest that these growth forms have similar resource acquisition and may form a large single guild\u2014at least in terms of invasion resistance. As a result, invasion outcomes in native communities may be dictated more by chance extant n than bioS1 File(XLSX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file."} +{"text": "With continued economic growth and expanding mortgage markets, until recently the pattern across advanced economies was of growing homeownership sectors. The Great Financial Crisis (GFC) has however, undercut this growth resulting in the contraction of homeownership access in many countries and the revival of private renting. This paper argues that these tenure changes are not solely a consequence of the GFC, and therefore, reversible once long-term growth returns. Rather, they are the consequences of more fundamental changes especially in labour markets. The very financialisation that fuelled the growth of homeownership has also led to a hollowing out of well-paid, secure jobs\u2014exactly those that fit best with the taking of housing loans. We examine longer-term declines in labour market security across Europe from before the GFC, identifying an underlying correlation between deteriorated labour market conditions and homeownership access for young adults. While variations exist across European countries, there is evidence of common trends. We argue that the GFC both accelerated pre-existing labour insecurity dynamics and brought an end to offsetting such dynamics through the expansion of credit access with the likelihood of a return to an era of widespread homeownership growth starkly decreased. Union of Homeowners and its consequences improvements in economic prosperity, government policies and financial transformations played an important intervening role in homeownership sector growth. Government policies have directly influenced the possibilities and attractiveness of different tenure choices wealth, labour market changes have had the clearest adverse impacts on young adults. Existing evidence from the US and the UK show a decrease in relative incomes of younger people since before the crisis provides a useful sample with variation in labour market conditions for younger adults, young homeownership entry rates and levels of mortgage credit access. While the analysis remains exploratory and the data does not allow a long-term longitudinal analysis, variation that does remains across countries (see descriptive statistics in Table\u00a0p\u00a0<\u00a00.01). Secondly, model (b) provides some support\u2014albeit only moderately significant (p = 0.08)\u2014for the association between higher total income inequality levels and lower homeownership rates seeming to corroborate notions of a hollowing out of the middle class undermining widespread ownership potential (i.e. Atterhog p\u00a0<\u00a00.05) with lower homeownership levels (model c). This supports the importance of employment term stability, which would affect accessing mortgage loans beyond absolute income as well as motivations for housing commitments when labour instability might necessitate future moves.The results (see Table\u00a0While remaining an indirect proxy, the mortgage-debt-to-GDP ratio reflects the expectation that countries with more developed and accessible mortgage markets display more opportunity for access to homeownership. While countries that retain more marketised mortgage markets are associated with overall higher youth homeownership rates, at the individual level, this is still expected to be largely constrained to the most economically advantaged sectors of society. In fact, notwithstanding higher overall levels, related research suggests such contexts may have more unequal homeownership access. Lersch and Dewilde find thaThe empirical analysis presents initial evidence across Europe of a significant correlation at the macro-level in terms of degrees of labour market inequality and insecurity in entry to homeownership for young adults. Taken in conjunction with the evidence on continuing trends towards constrained labour market opportunities and an unlikely\u2014or undesirable\u2014return to deregulated mortgage credit expansion, this fundamentally questions the likelihood of any resumption of homeownership growth or notions of widespread owner-occupation as a housing solution for the masses. These exploratory results encourage more detailed future empirical research into the link between underlying transformations of the labour and housing markets and how this results in diverging homeownership opportunities for current and future generations of households.While not negating variations that remain across countries, the analyses bring attention to common labour and housing market trends across the sample of European countries. The evidence presented points to a future for most European countries in which continued trajectories at levels that may be referred to as \u2018mass homeownership\u2019 are unlikely. In the years since the financial crisis of 2007, there has been a realisation that homeownership has become increasingly unattainable for large sectors of the population. Indeed, in many advanced economies\u2014both within and beyond Europe\u2014the secular expansion of the preceding decades alongside the ideological optimism in a socio-political project of widespread homeownership, has given way to stagnation or decreases. While older generations have sometimes been shielded by these transformations, stark reductions in homeownership entry have been evident among younger cohorts in many countries. Nonetheless, there has often been an implicit or explicit assumption that such trends are largely a product of the recent economic downturn so that an eventual post-GFC recovery will see rebounding opportunities where homeownership once again becomes widely accessible. Even if homeownership may persist as a majority tenure\u2014considering that many who previously entered homeownership will remain owner-occupiers for years to come\u2014where large proportions of younger generations are increasingly excluded from property ownership, we contend such a divided housing sector can no longer describe notions of mass homeownership. The argument presented here is that expectations of a return to growth have neglected fundamental longer-term labour market transformations, only partly masked by a regime of unstable credit expansion to riskier and riskier households in the pre-crisis \u2018boom\u2019 years. Across advanced economies, evidence on labour market transformations reveal long-term shifts towards growing inequality and increases in precarious employment contracts\u2014underscored by the reduction in wage shares accruing to labour. Younger cohorts appear especially affected; their position being characterized by growing income poverty and decreasing employment participation and thus an on-going reduction in the well-paid and stable jobs required for taking on mortgage credit and entering homeownership. These fundamental changes to the labour market clearly challenge any notion of a future \u2018return to normal\u2019 characterised by widespread entry to homeownership.Notwithstanding longer-term labour market deterioration, homeownership rates in many advanced economies actually expanded in the mid-noughties (Andrews and Caldera Sanchez The consequences are wide reaching. Housing careers are central to life-course trajectories with, in many contexts, homeownership epitomizing a realisation of full adulthood independence. Stunted or unstable housing careers may have significant impacts on current quality of life, potential future economic wellbeing, and other spheres of adulthood transition (see Arundel and Lennartz primes rather than the subprimes.\u201d Labour market polarisation has thus become amplified in the property market. One crucial dimension has been that a limited sector of the population has increasingly not only been successful in owning their own property but have further purchased other people\u2019s homes\u2014what may be termed a rising \u2018generation landlord\u2019 (Ronald et al. There is a further possibility that the developing housing systems not only reflect disparity across consumers but will themselves act as a stimulus to inequality. Increasing divergence in labour market opportunities and homeownership access has seen housing wealth become a key dimension of rising inequality (Allegr\u00e9 and Timbeau These interrelated labour and housing market transformations have been spurred on by common shifts towards growing financialisation and neo-liberal marketization. This points to a contradiction at the heart of homeownership sectors in many developed countries. Increasingly financialised housing markets, the growth of mortgage credit and a relaxation of down-payment constraints both motivated home purchase and effectively moved the tenure down the income distribution (Andrews and Caldera Sanchez Beyond growing disparities on the housing market\u2014epitomized by notions of a \u2018generation rent\u2019 versus a \u2018generational landlord\u2019\u2014labour market and housing system transformations may very well undermine the viability of the economic system upon which it is predicated. What is happening to homeownership can be seen as part of a wider crisis of capitalism. While lower wages may help individual firms to compete by driving down production costs and provoke similar measures among competitors, wages are not only a component of production, but also the source of demand. More generally, wider inequality will have a negative impact on aggregate demand and economic growth (see, for example, Mason"} +{"text": "The current unprecedented expansion of infrastructure promises to enhance human wellbeing but risks causing substantial harm to natural ecosystems and the benefits they provide for people. A framework for systematically and proactively identifying the likely benefits and costs of such developments is badly needed. Here, we develop and test at the subregional scale a recently proposed global scheme for comparing the potential gains from new roads for food production with their likely impact on biodiversity and ecosystem services. Working in the Greater Mekong\u2014an exceptionally biodiverse subregion undergoing rapid development\u2014we combined maps of isolation from urban centres, yield gaps, and the current area under 17 crops to estimate where and how far road development could in principle help to increase food production without the need for cropland expansion. We overlaid this information with maps summarising the importance of remaining habitats to terrestrial vertebrates and (as examples of major ecosystem services) to global and local climate regulation. This intersection revealed several largely converted yet relatively low-yielding areas , where narrowing yield gaps by improving transport links has the potential to substantially increase food production at relatively limited environmental cost. Concentrating new roads and road improvements here while taking strong measures to prevent their spread into areas which are still extensively forested could thus enhance rural livelihoods and regional food production while helping safeguard vital ecosystem services and globally significant biological diversity. Proposals for new infrastructure in developing countries are typically muted on its environmental impacts, while environmentalists typically say little about its potential benefits for people. This study explores a more conciliatory approach by trying to identify where beneficial infrastructure might be expanded at least environmental cost. Focusing on the Greater Mekong Subregion of Southeast Asia, we intersected agricultural, social, and environmental layers to map variation in the potential for new roads to enhance food production and in their likely impacts on biodiversity and ecosystem services. In several areas well-planned road expansion has the potential\u2014if linked to strong habitat protection elsewhere\u2014to help boost agricultural production at limited environmental cost. In others , new roads would risk marked environmental harm, often for little agricultural gain. By mapping specific proposals onto our data layers, we were also able to identify planned roads that might deliver low-impact improvements in food production and others that risk disproportionate environmental costs. We hope these analyses can help guide future road planning in this exceptionally diverse, rapidly developing area. The world is undergoing an unprecedented expansion of human infrastructure. For example, more than 450 new hydropower dams are planned for the Amazon, Congo, and Mekong basins alone . LikewisNew infrastructure can bring substantial benefits to people \u201310 but iInfrastructure development\u2014especially road construction\u2014is often aimed at enhancing food production and reducing poverty. New and upgraded roads can reduce the transport costs of agricultural inputs and of getting products to market, lower postharvesting food waste, and increase access to technological improvements ,19\u201322. TThis recognition of the potential gains from promoting new roads in places where they can most enhance farming while avoiding highly sensitive areas has been formalised in a new \u201cglobal road-mapping strategy\u201d . In prinTo address this gap, our paper adopts, refines, and tests the approach of the global road-planning strategy to examine its potential to contribute to finer-scale decision making on road investment, with three overarching aims:To examine the generality and practicality of the approach by applying it to a highly biodiverse but rapidly developing subregion\u2014the Greater Mekong \u2014rather tTo explore the sensitivity of the results to the data layers used to create these surfaces, to how they are combined, and to the type of benefits from road development which they consider.To use the framework to characterize specific roads which have been proposed as development priorities in the Greater Mekong Subregion (GMS) and to identify a subset that seem well situated to address poverty and food production concerns at potentially limited environmental cost and others that appear less beneficial.Our analysis suggested substantial potential for increasing food production in the GMS through closing existing yield gaps, without any change in the area under production. Across the 17 crops we considered, increasing yields to those attained in climatically similar areas could raAlmost the entire GMS falls within the Indo-Burma Hotspot , and as Overlaying our aggregate surfaces suggested that the balance of likely environmental costs and food production benefits from road growth varies widely across the GMS . Areas wThe data layers we used inevitably have errors, were compiled for different purposes, and could defensibly be integrated in other ways than we have considered here. However, three sorts of sensitivity tests indicateDespite the relatively coarse resolution of our analysis, overlaying the locations of 43 road development schemes onto our intersection of the potential benefits and costs of new roads can shedOur analyses indicate that even within a global biodiversity hotspot there iNevertheless, considerable care is required to realise this potential. In many areas . Our fi2 Greater Mekong Subregion . The GMS is densely settled (with over 320 million inhabitants), culturally diverse, and biologically exceptional 60]. This(EPS)Click here for additional data file.S7 FigData from . Note th(EPS)Click here for additional data file.S8 FigMean values for the potential food production benefit of grid cells in a 10km buffer around each of 43 proposed new roads or road improvements, plotted against their mean potential environmental cost (a); and values for each grid cell adjacent to roads TH1 (b) and CMB2 (c). See (TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 TableRoad locations are from and mapp(DOCX)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.S2 Data(ZIP)Click here for additional data file."} +{"text": "For some time, estrogen has been suspected to play a negative role in anterior cruciate ligament (ACL) fibroblast biosynthesis; however, reports on this issue have been controversial. In a recent study, our group demonstrated a negative combined effect of estrogen and mechanical loading on the gene expression of major extracellular matrix component molecules in ACL fibroblasts."} +{"text": "Distinct characteristics of cancer cells govern tumour initiation and progression. The fact that a tumour represents a new entity that is pathologically interposed between normally differentiated tissues generates aberrant mechanical interactions that affect each of the well\u2010known hallmarks, but also tends to form an additional feature of their own during tumour progression via integrin/focal adhesion kinase (FAK)/Src signalling, which promote cytoskeletal remodelling, development of increased intracellular tension and ultimately create positive feedback loops and a vicious circle between ECM and the cytoskeleton via Src, FAK and calpain\u20102 activation, but also promotes cell migration by up\u2010regulating the physically\u2010associated ECM protein tenascin\u2010C, which in turn induces the mammalian target of rapamycin (mTOR) signalling pathway Genetic mutations and other factors such as tissue injury, chronic inflammation and accumulated mechanical stress reprogram and activate differentiating regulatory circuits that were normally silent in adults v\u03b23 integrin expression through assembled fibronectin, underlining the effect of mechano\u2010induced integrins in the role of CAFs during tumour invasion However, new evidence highlights the role of the tumour's microenvironment in increased stiffness of the ECM as a critical modulator of mechanosignalling that promotes invasion and metastasis. Cancer\u2010associated fibroblasts (CAFs) are the most abundant type of stromal cells and favour cancer cell invasion. CAFs require the activation of the mechano\u2010induced YAP transcriptional coactivator to maintain a cancerous state, which further enhances ECM stiffening and augments YAP activity to promote invasion and angiogenesis via activation of \u03b21 integrin/c\u2010Jun N\u2010terminal kinase (JNK) signalling axis Nowadays, the imperative need of elucidating such mechanisms is to find ways to overcome the acquired resistance of cancer cells to chemotherapeutic agents. The impact of biomechanical cues between cancer cells, ECM and the stroma emerges as a vital factor of insensitivity to such agents. Increased ECM stiffness produces resistance to several anticancer drugs, among them Raf inhibitors, whose efficacy is diminished in stiffer, collagen\u2010rich tumour ECM In the light of these findings and poor results from integrin\u2010targeting clinical efficacy, novel molecules emerge that regulate mechanotransduction as potential therapeutic targets. Polycystin\u20101 and polycystin\u20102 form complexes and are involved in acquisition of aggressive phenotypes in colorectal cancer, but they are also involved in osteosarcoma pathobiology In summary, an ever\u2010increasing volume of studies support the impact of tumorigenic mechanical stresses and corresponding aberrant mechanical perception by cells on cancer initiation and progression, unveiling mechanisms that need further documentation. Nonetheless, future projects should focus on elucidating the mechano\u2010induced signalling networks that integrate the interactions between ECM, cancer cells and their surrounding stroma, unravel mechanisms of drug resistance attributed to these interactions and also reveal novel mechanosensitive molecules as candidates for therapeutic targeting and bypassing networks of treatment resistance.The authors confirm that there is no conflict of interest."} +{"text": "Discoveries from the Human Genome Project have invigorated discussions of epigenetic effects\u2014modifiable chemical processes that influence DNA\u2019s ability to give instructions to turn gene expression on or off\u2014on health outcomes. We suggest three domains in which new understandings of epigenetics could inform innovations in health promotion research: (1) increase the motivational potency of health communications ; (2) illuminate new approaches to targeted and tailored health promotion interventions ; and (3) inform more sensitive measures of intervention impact, . We suggest a three-step process for using epigenetics in health promotion research that emphasizes integrating epigenetic mechanisms into conceptual model development that then informs selection of intervention approaches and outcomes. Lastly, we pose examples of relevant scientific questions worth exploring. Over the last two decades, discoveries from the Human Genome Project (HGP) and visions for applying genomics in everyday medical care (aka precision medicine) have invigorated discussions of epigenetics updates in health risk information that could improve the motivational potency of health communications ; (2) illuminate new approaches to targeted and tailored interventions ; and (3) inform more sensitive measures of intervention impact . Additionally, we discuss feasible ways to use epigenetics in health promotion interventions and related research methods, and provide practical \u201chow to\u201d steps for getting there. But, first, we go under the hood with a technical overview of epigenetic concepts that could inform these innovations.Epigenetic mechanisms are the chemical processes that influence the ability of deoxyribonucleic acid (DNA) to give instructions and influence whether phenotypes associated with gene variants become manifest physically or clinically gets added to the histone tail , which in turn contributed to cancer and other disease etiology can be interrogated to indicate global methylation, rapidly and relatively economically updates in health risk information that could improve the motivational potency of health communications, (2) the development of new approaches to targeted and tailored interventions, and (3) novel measures of intervention impact. In this section, we expand on these points, offering several examples of how epigenetics might inform the future of health promotion research to validate self-reported behavior change where possible and to minimize related threats to validity when evaluating intervention effectiveness. Similarly epigenetic methylation processes could be assessed to indicate whether self-reported intervention adherence is concordant with physiological processes that might improve intervention adherence or benefits of sustained behavior change . These new approaches could give evidence of whether improvements in self-reported initiation and maintenance of behavior change deemed statistically Bryan and colleagues are among the few research teams that evaluated the effect of participating in health promotion interventions and its association with epigenetic processes that may be biologically beneficial for the health outcome of interest or influenced by intervention participation. Additionally, these approaches could enable evaluation of whether the intervention group or some subgroup of individuals based on intervention adherence level or convoy characteristics show patterns of methylation consistent with a conceptual model or hypothesis. Thus, methylation patterns offer a measure of epigenetic modifications that may be more sensitive to intervention effectiveness.Incorporating epigenetic-related biomarkers into health promotion interventions has been done relatively infrequently. However, those who have succeeded used a systematic approach that we have summarized in a three-step process. The process begins with development of a conceptual model that emphasizes the defined \u201cexposome\u201d most germane to the health outcome and target population , household factors and mood state . Thus, a focused social ecological model might be constructed using epigenetic mechanisms to link levels of influence to intervention adherence. For example, the conceptual model would consider epigenetic mechanisms that may be prompted by or encourage intervention adherence within a specific context. In turn, inclusion of epigenetic assessment could illuminate whether, the intervention group or some subgroup of participants show patterns of methylation that vary in accordance with a multi-level conceptual model or hypothesis. Additionally, the model could posit potential effect moderators such as exposure convoys or adherence level.Imagining potential methylation patterns requires some understanding of the families of genes that could plausibly be influenced by the intervention. This is important because the aim is not to evaluate all pathways but to parsimoniously consider those most plausible and specific to the influence levels under consideration. Lastly, a conceptual model can guide decisions about the appropriate comparison groups against which the influence of the intervention on epigenetic processes would be evaluated.For example, Bryan et al. proposedOne limitation of Bryan et al. model isOnce the conceptual framework has been determined, the next step is to specify the intervention elements where influence could be identified via epigenetic mechanisms. Building on Bryan et al. example For example, Crujeiras et al. hypothesIn this step, the researcher decides on the optimal epigenetic assessment and timing of measures. These considerations would logically build upon the prior steps in suggesting where and when epigenetic changes might occur if prescribed adherence levels were attained.A prospective study conducted by Bryan et al. offers aThe researchers selected a priori the CpG islands linked to breast cancer acquisition and progression, and then developed a \u201ccomposite measure\u201d of these sites. Several sources were used to identify the epigenetic markers that comprised the composite measures. For example, genes and variants gleaned from a literature review included those studied among breast cancer patients taken from tumor cells and those suggested as possible preclinical markers for breast cancer. Other potential CpG sites were identified through an online annotation file available from Illumina; additional genes were identified that had been suggested to play a functional role in breast cancer development. In all, the researchers settled on 21 genes and 45 markers to analyze for their association with improvements in physical activity and hypothesized that DNA methylation across these sites would be negatively associated with self-reported physical activity levels (based on the PAR) and cardiovascular fitness (based on VO2 max). Saliva samples were collected prospectively at three-month intervals up to a year after intervention participation. DNA extracted from the saliva was analyzed via a commercially available Illumina platform that enabled methylation patterns to be evaluated (Bryan et al., Whether epigenetics can be used into improve health promotion interventions and research in the ways we have suggested raises numerous scientific questions worth exploring. A compelling advantage of pursuing this translational research is that emerging discovery in epigenetics may illuminate modifiable mechanisms that link different levels of influence on health and intervention benefits overlooked by current measures. Tractable research questions and related development of testable multi-level conceptual frameworks could move the field beyond the predominant focus of intervention research targeting a single level of influence. Accordingly, integrating epigenetics into health promotion research will call for intervention and methodological accommodations. Health promotion researchers can take the lead in keeping such research in the forefront of precision medicine discussions that will be increasing in the decade ahead. Indeed, the potential for launching a new generation of conceptual models, interventions and related methods informed by genomic discoveries should embolden us to gain the skills needed to engage in and advocate for this arena of translational research."} +{"text": "Understanding genetic factors contributing to the pathophysiology of BMFS has enabled the identification of causative genes and development of diagnostic tests. To date more than 40 mutations in genes involved in maintenance of genomic stability, DNA repair, ribosome and telomere biology have been identified. In addition, pathophysiological studies have provided insights into several biological pathways leading to the characterization of genotype/phenotype correlations as well as the development of diagnostic approaches and management strategies. Recent developments in bone marrow transplant techniques and the choice of conditioning regimens have helped improve transplant outcomes. However, current morbidity and mortality remain unacceptable underlining the need for further research in this area. Studies in mice have largely been unable to mimic disease phenotype in humans due to difficulties in fully replicating the human mutations and the differences between mouse and human cells with regard to telomere length regulation, processing of reactive oxygen species and lifespan. Recent advances in induced pluripotency have provided novel insights into disease pathogenesis and have generated excellent platforms for identifying signaling pathways and functional mapping of haplo\u2010insufficient genes involved in large\u2010scale chromosomal deletions\u2013associated disorders. In this review, we have summarized the current state of knowledge in the field of BMFS with specific focus on modeling the inherited forms and how to best utilize these models for the development of targeted therapies. S Bone marrow failure syndromes are characterized by a common phenotype of peripheral cytopenia and/or hypoplastic bone marrow. Great strides have been made in the last 20 years both scientifically and clinically resulting in identification of more than 40 causative genes and improved transplant outcomes. In this review, we summarize the most recent findings achieved through application of animal models and stem cells which have generated important insight for disease physiopathology and improved patient care.Bone marrow failure syndromes (BMFS) are rare diseases characterized by peripheral cytopenias and/or hypoplastic bone marrow and can either be inherited or acquired BMFS have a broad clinical spectrum, sharing the failure of hematopoietic stem cells (HSCs) to produce functional blood cells and can affect patients of all ages Although rare, the clinical impact of BMFS is undoubtedly significant. Experimental approaches to increase our understanding of these disorders are thus essential. Moreover, since many of the causative genes play important roles in the development and maintenance of the hematopoietic system, studying their dysfunctions may provide further insights into the production mechanisms of blood and immune cells. Thus, investigations using animal and cellular models of the group of diseases reviewed herein are of great value.Several strategies may be applied to the generation of animal models of BMFS with murine models being most typically applied. Genetically modified mice can be generated by either direct pronuclear injection of exogenous DNA into fertilized zygotes or injection of genetically\u2010modified murine embryonic stem cells (ESC) into a blastocyst. Direct pronuclear injection is technically demanding and often results in multiple, random integrations of the injected DNA into the genome, and the resulting disease phenotypes can vary depending on the expression level of the injected transgene. Mouse ESCs have the advantage that they can be genetically modified by means of homologous recombination, a process by which a fragment of genomic DNA introduced into a mammalian cell can recombine with the endogenous homologous sequence. This process is known as \u201cgene targeting.\u201d When such genetically modified ES cells are introduced into a preimplantation embryo, they can contribute to all cell lineages of the resulting chimeric animal. If this contribution also comprises germ cells and the chimeras are capable of breeding, it is possible to establish lines of animals that are both heterozygous and homozygous for the genetic alteration introduced into the ESCs. This process can be used to add DNA sequences to specific genomic loci (knock\u2010ins), a protocol most often used to generate cell lines with gene\u2010specific reporter systems Gene editing of murine ESCs has been used to create a number of genetically modified animals carrying mutations associated with some of the more common forms of BMFS. Despite the effectiveness of gene editing, these models do not always demonstrate all the mechanistic or symptomatic problems shown by humans. However, they have generated useful data on the mechanisms of BMFS. An overview of animal models created to date is provided in Table FANCA), B (FANCB), C (FANCC), D1 (FANCD1/BRCA2), D2 (FANCD2), E (FANCE), F(FANCF), G (FANCG), I (FANCI/KIAA1794), J (FANCJ/BRIP1), L (FANCL), M (FANCM), N (FANCN/PALB2), P (FANCP/SLX4/BTBD12), O (FANCO/RAD51C), S (FANCS/BRCA1), and T (FANCT/UBE2T) have been cloned The hallmark of FA is hypersensitivity to DNA cross\u2010linking agents, intolerance to oxidative stress and frequent chromosomal aberrations pointing to a DNA damage response defect, all of which are used as diagnostic tests. To date 17 complementation groups have been identified and the genes encoding these groups A which mimics many features of FA including peripheral cytopenia, reduced fertility, dysmorphic features, ocular abnormalities, hydrocephalus, chromosomal instability, accumulation of damaged chromosomes, hypersensitivity to DNA crosslinking agents and abnormal lymphopoeisis FANC genes remains a significant problem. The potential greater susceptibility of mice to sustain and retain DNA damage and/or the presence of alternate regulatory mechanisms for FANC proteins in humans, indicate that murine FA models may not be optimal tools to understand the pathophysiology of FA and develop novel treatments. Furthermore, the nature of mutations in various types of FA is extremely heterogeneous, including point mutations, small insertions/deletions, splicing mutations, and large intragenic deletions, which makes it difficult to replicate exactly all human mutations through targeted gene knock\u2010ins/outs in the mouse system.Targeted single deletions in mouse of various genes such as DKC1, TERT, TERC, TINF2, WRAP 53, NOP10, NHP2, CTC1, and RTEL1DKC1), TERC and TERT, and mutations that impair telomerase recruitment in genes such as TIN2DKC is the first disorder to be etiologically linked to mutations in the telomere pathway hTERT) cause defects in stem cell proliferation, organ regeneration and incidence of cancer in humans. Patients with telomerase dysfunction including DKC, frequently develop aplastic anemia whereas telomerase\u2010null murine models display only modest hematopoietic defects.Gene editing of murine ESC has been used to model loss of function for most of these genes; however concerns about differences in telomere maintenance between mouse and human can limit the utility of these models. Although the telomeric DNA sequence is identical in both species, abnormalities in telomere maintenance and in telomerase function do not coincide in phenotype in humans and mice. Most strains of laboratory bred mice have telomeres 5\u201010 times longer than humans, whereas absence of telomerase activity is only phenotypically present over several generations in mice and even heterozygous mutations affecting the telomerase reverse transcriptase subunit in mice that already have short telomeres is a more effective means of replicating the DKC phenotype. Similarly, double knockout of Pot1b (an ortholog of the \u201cprotection of telomeres protein\u201d encoding gene) and Terc results in enhanced telomere degradation (rather than progressive telomere shortening) and results in premature death, BMFS, significant anemia, leukopenia and thrombocytopenia. That such double knockout is necessary to create a DKC\u2010like phenotype calls into question once more the validity of murine models of human disease. Further, the question of how stem cell failure occurs at all in these models also arises. Loss or functional failure of HSC clearly occurs after significant erosion of telomeric DNA but whether this is due to induction of senescence by critically short telomeres is not yet clear. Other mechanisms to account for the dysfunctional HSC of DKC patients have been proposed such as defects in ribosome biogenesis leading to defects in the processing of 18s rRNA Nop10 and this model does show enhanced HSC apoptosis but to our knowledge; however this has not been attempted in mice.Early models of DKC (hypomorphic DKC1) display a DKC\u2010like phenotype reflected in the increased evidence of tumors in the mammary glands and lungs (not in gut and skin as in human DKC patients), splenomegaly, dyskeratosis of the skin, anemia, yet in the early generations there is no obvious telomerase dysfunction DKC1 in de novo DKC) and in some cases dominant negative mutations have been reported in addition to loss of function mutations. Whilst, loss of function mutations can be modeled with targeted gene approaches in mice, partial loss of function and haploinsufficiency are difficult to mimic unless the human mutation is introduced into the mouse germ line or ESC using the most recent gene editing technique (for example Crispr/Cas9 method). This however can be easily superseded by the iPSC disease modeling approach which enables the assessment of human mutations in a dish by reprogramming of patient specific somatic cells.A further complication with disease modeling of DKC using targeted gene knockdown in mice is the nature of human mutations. In most documented cases to date, partial loss of function as well as haploinsufficiency require regular platelet and red blood cell transfusions for bone marrow failure Tpp1 were embryonic lethal The severity of the mutations is also an important aspect which cannot always be matched between mouse models and human patients. This is best exemplified by a rare and severe form of DKC, Hoyeraal\u2010Hreidarsson syndrome, which is caused by mutations in a subset of genes, including RPS19 gene mutations are found in 25% of DBA patients RPS19 can affect the synthesis of the ribosomal protein by altering transcription, splicing, or translation RPS19 genes in Saccharomyces cerevisiae yeast leads to complete arrest of small ribosomal subunit synthesis RPS19 mutations. This defect in pre\u2010RNA maturation was also observed in CD34\u2212 hematopoietic precursors, skin fibroblasts, and lymphoblastoid cell lines. As shown in Table Rsp19\u2010/\u2010 homozygous mice are embryonic lethal, whilst heterozygous mice either are normal or show a mild macrocytic anemia (depending on the nature of targeted gene event), indicating that these models are unable to truly recapitulate the RPS19 haploinsufficiency described in the human DBA patients. Mice heterozygous for missense mutations of Rsp19 and for a similar ribosomal component Rsp20 show mild macrocytic anemia indicating possible HSC dysfunction. Other models including antisense oligonucleotide knockdown of Rpl11 in Zebrafish show significant effects on early hematopoietic development and defects in adult hematopoiesis such as reduced formation and maturation of erythroid cells which is similar to the phenotype of DBA. Another informative model is the conditional Rsp6 mouse where homozygous deletions of Rsp6 were achieved through CD4\u2010driven Cre recombinase resulting in abrogation of T cell development. In contrast to homozygous deletions, Rps6 haploinsufficiency although it did not impact T cell maturation, it affected their proliferation. Together these data suggest that blood cells with Rsp6 haploinsufficiency may cope with ribosome synthesis under low cell proliferation; however this is severely compromised in tissues characterized by rapid proliferation (such as the hematopoietic system).Mutations in ribosomal protein genes that encode structural components of the ribosome responsible for the correct assembly of the ribosomal subunits are associated with the abnormal pre\u2010rRNA maturation patterns in DBA patients Sbds) and ribosomal protein 18 (Rps18) which are central to ribosome function are unsurprisingly embryonic lethal SBDS gene which shares 97% homology with an adjacent pseudogene, SBDSP which contains deletions and nucleotide changes preventing generation of a functional protein. 75% of patients with SDS have SBDS mutations which are acquired as result of gene conversion events from the pseudogene SBDSP. Patients homozygous for SBDS mutations have not been identified, suggesting that complete loss of SBDS is likely to be lethal in humans. This is corroborated by animal studies which have shown that loss of Sbds gene in mice results in early embryonic lethality and more latterly, induced pluripotent stem cells (iPSCs), are capable of indefinite in vitro expansion while retaining the ability to differentiate into cell types characteristics of most tissues found in the developing embryo. Reprogramming somatic cells to pluripotency by ectopic expression of four transcription factors expressed by ESC was first reported in 2006 Differentiation of pluripotent stem cells toward patient specific hematopoietic cell types is the basis of modeling inherited BMFS. However, the major challenge is the generation of fully functional HSCs. Several studies have described the generation of hematopoietic progenitors, but on further differentiation in both in vitro and in vivo experimental models, such progenitors show a preference toward myeloid differentiation so high long term engraftment efficiency and multi\u2010lineage differentiation of functional HSC in the bone marrow of immune compromised mice remains challenging FANCD2 and FANCAThe processes of reprogramming needed to generate hiPSCs is dependent on enhanced cellular proliferation during the initial stages of reprogramming which requires the activation of DNA repair pathways to ensure maintenance of genomic stability. Thus, it is quite difficult to derive hiPSCs from somatic cells of patients suffering from DNA repair disorders, which renders the development of a hiPSCs based model of FA difficult to achieve. Initial studies suggested that reprogramming of FA patient specific hiPSCs was only possible after genetic complementation of the donor fibroblasts prior to reprogramming or when reprogramming was performed under hypoxic conditions DKC1, TERC, TERT, and TCAB1 which display normal karyotypes and hallmarks of pluripotency as described earlier TERC mutations. However, attempted upregulation of an hTERT gene carrying heterozygous mutations merely produces a dysfunctional hTERT protein, which cannot function effectively within the telomerase holoenzyme complex. For this reason, hiPSCs lines derived from patients with such hTERT mutations display shortened telomeres following extended culture which ultimately prevents their self\u2010renewal. This phenotype is more pronounced in hiPSCs lines derived from patients with X\u2010linked DKC where the mutation of DKC1 blocks telomerase assembly and disrupts telomere elongation DKC1 mutation; hiPSCs lines carrying Q31E and \u0394L37 mutations maintained the same telomere length as the parental fibroblasts; however the A353V mutant DKC\u2010 hiPSCs presented shorter telomeres compared to the parental fibroblasts suggesting that this A353V mutation has a more severe effect on the telomere maintenance process.hiPSCs have been derived from DKC patients with mutations in RPS19 and RPL5 genes DBA\u2010hiPSCs have been generated from patients with mutations in SBDS expression which display dysfunctional ribosome assembly SBDS expression in some patient specific lines led to absence of the pancreatic phenotype and presence of hematopoietic defects only, similar to previous clinical observations in SDS patients, thus further corroborating the genotype\u2010phenotype correlation. Despite this observed variability, both the pancreatic and/or hematopoietic phenotype could be rescued either by the forced expression of the SBDS transgene or by the use of protease inhibitors in the culture media, thus providing a clear example of drug\u2010reversible phenotype using the hiPSC model for these patients and providing novel therapeutic insights.hiPSCs have been generated from patients with reduced ELA2 and HAX1HAX1 and ELA2 in the respective isogenic hiPSCs lines. In a recent study, Nayak et al. identified the mislocalization of the ELA2 gene encoding protein neutrophil elastase (NE) as the inductor of the UPR/ER stress, dysfunctional differentiation and apoptosis and demonstrated that the SCN phenotype could be corrected by addition of the NE inhibitor called sivelestat to the culture media of the hiPSC\u2010derived myeloid progenitors. Together these studies indicate that SCN\u2010hiPSCs provide an excellent platform for high\u2010throughput screening of drugs to reverse various congenital neutrophil disorders as well as better understanding of disease pathogenesis which can be clinically exploited to achieve therapeutic responses using lower doses of G\u2010CSF combined with targeting to correct NE mislocalization.Several groups have reported the generation of patient specific hiPSCs carrying mutations in genes involved in SCN such as Recent progress in the field of genome editing is giving iPSC technology a solid framework for disease modeling and for future clinical therapies. Previous gene therapies carried out via transient or constitutive expression of transgenes were undermined by the possibility of transgene integration into loci that conferred uncontrolled cellular proliferation resulting in tumorigenesis. To date the development of robust and efficient human genome engineering tools including zinc\u2010finger nucleases (ZFNs), transcription activator\u2010like effector nuclease (TALEN) and Crispr\u2010Cas9 systems which permit in situ gene editing with reduced risk of off\u2010site mutagenesis has propelled forward the genome engineering approaches FANCA gene in wildtype hESC to create a FA disease model and restoration of specific mutations in the FANCC gene in wild type fibroblasts via the Crispr/Cas9 system have been reported REV7 using the Crispr/Cas9 approach and showed that this resulted in increased cellular hypersensitivity to DNA interstrand cross\u2010link drugs (ICL) as well as an impaired ability of the mouse hematopoietic progenitor cells to form hematopoietic colonies in Colony\u2010Forming Unit (CFU) assay, resulting in characterization of a new gene in the FA pathway FANCA defective fibroblasts and later generation of disease\u2010free iPSC which upon hematopoetic differentiation could generate similar number of hematopoietic colonies compared to healthy cord blood progenitor cells To date, a handful of studies have reported the successful use of genome editing tools for the study of BMFS. For example, ZFN mediated disruption of the DKC1 in patient specific IPSC as well as introduction of DKC1 mutation in wild type iPSC. As expected, the corrected DKC1\u2010iPSC showed high telomerase activity, whereas DKC1 mutant\u2010 iPSC showed lower telomerase activity than wildtype controls. Interestingly, this study also provided data that supported the link between DKC1 and Wnt signaling pathway by showing restoration of telomere length, telomere capping and reduction of p53BP1 telomere foci upon application of the Wnt agonist CHIR99021 in DKC\u2010iPSC, indicating that both gene editing and manipulation of signaling pathway provide potential avenues for treatment of BMFS.Application of iPSC technology as a platform for gene correction and drug screening was also reported for DKC To date, allogeneic HSC transplantation from HLA\u2010identical healthy donors remains the only curative therapy for BMFS patients. The possibility of performing gene correction approaches in patients HSCs together with an improved yield of HSC expansion so that sufficient numbers of cells for autologous transplantation can be achieved, offers a revolutionary prospect for the field of BMFS. This will require a reduction in off\u2010target mutagenesis; however with the pace the field is progressing, one would hope that this is within our reach very soon.MPL gene, resulting in loss of megakaryocytes (MK) in the bone marrow, severe thrombocytopenia and development of fatal BMF later in life. Hirata et al. investigated the mechanisms involved in the MPL signaling and the development of MK/Erythrocyte progenitor (MEP) by generating hiPSCs from CAMT patients carrying MPL mutations CAMT is characterized by the loss of function or deletion of the thrombopoietin (TPO) receptor encoded by the MDS is the most common form of primary BMF. The somatic loss of the long arm of chromosome 7 (del (7q) is one of the most characteristic chromosomal abnormalities in MDS. Recently, Kotani et al. derived hiPSCs clones that displayed normal karyotype as well as clones with del(7q) from MDS patients In the last 20 years, great progress has been achieved in identifying new genetic causes/susceptibilities of inherited BMFS, which has resulted in better genotype\u2010phenotype correlations, improved diagnosis and clinical treatments. A large number of mouse models have been created enabling generation of insights into disease mechanisms and pathology. Nevertheless, the inability of gene knock\u2010ins/out to mimic the precise nature of human mutations and especially happloinsufficiency together with differences in DNA damage tolerance, telomere length and short life span have undermined their utility in mimicking the full spectrum of BMFS Fig. . The advS.H. and D.M.S.: Conception and design, manuscript writing, final approval of manuscript; G.E.K. and S.A.: manuscript writing, final approval of manuscript; S.S., L.A., and M.L.: Conception and design, manuscript writing, final approval of manuscript, fund raising.The authors no potential conflicts of interest."} +{"text": "Pericardial effusion after midline cardiac surgery may be transudative or exudative. The exudative infective or haemorrhagic variety requires early surgical intervention. However there are rare cases of collections like chylomediastinum which should be ruled out. Their low incidence prompts to establish protocol for evaluating postoperative pericardial collections, which includes echocardiography and biochemical analysis of aspirate. The same is important from the perspective of management as chylopericardium may be successfully managed without surgical intervention by aspiration, pig tail insertion, dietary and medical management, which we demonstrate through our rare case which occurred after midline double valve replacement. Symptomatic pericardial effusion after month of midline cardiac surgery is an emergency, mostly fearing infective or hemorrhagic collection, more so in undereducated patients on anticoagulation with underprivileged unhygienic living conditions, not so rare in developing countries. Although transudative pericardial effusion either reactionary or because of cardiac failure should always be investigated, there are other causes, which must be ruled out before embarking the invasive way back into the mediastinum. Chylo-pericardium is a rare cause of pericardial collection after a midline valve surgery, even in absence of any excessive dissection in mediastinum, neck or around left superior vena cava or breach in pleura. There are few case reports highlighting their anecdotal incidence and their management strategy.1 We report chylo-mediastinum in a young patient 2 months after double valve replacement, and highlight the importance of systematic investigation and successful non-surgical management.A 24-year-old shopkeeper from rural India, was symptomatic for severe rheumatic mitral stenosis since the age of 16 years and underwent balloon mitral valvotomy in 2009. He was on medical management and regular follow up when he became symptomatic in January 2017 with NYHA class II Dyspnoe on exertion progressing to class III. He was euthyroid and had no other co morbid medical or surgical illness. Echocardiography was suggestive of severe aortic stenosis and severe mitral stenosis and underwent double valve replacement with Sorin Bicarbon 27 mm bileaflet mitral prosthetic valve and St Jude Medical Regent 21 mm bileaflet aortic prosthetic valve in April 2017 through midline sternotomy. Pleura were not breached during the procedure. Patient required mediastinal lavage on post-operative day three in view of mediastinitis. He further required bilateral pleural aspiration on postoperative day five for pleural collection, which was serous in nature with no growth on culture. The patient recovered uneventfully and discharged with echochardiography suggestive of adequate valve function, no pericardial collection and no evidence of pleural collection. The patient became symptomatic again after two months with progressive dyspnoe on exertion class III. There was no history of fever but a documented weight loss of 5 kg. On investigations he was diagnosed to have large pericardial collection with preserved prosthetic valve and myocardial function, Right Atrium and Right Ventricle showed diastolic collapse. There was no pleural collection. On pericardial aspiration, 900 ml of chylous fluid was drained and pigtail catheter was secured in pericardial cavity . Fluid aChylopericardium or the collection of chyle in the pericardium, post valve replacement through midline sternotomy is sporadically reported.1-3 Chylous fluid leak significant to cause collection is indicative of thoracic duct injury or one of its major channels. Thoracic duct, classically is not encountered in operative field during valve replacement. Mechanisms causing this rare complication are not confirmed and proposed to be due to traction on the duct from manipulation of the heart and great vessels. Rarely thrombosis at the junction of the left jugular and subclavian veins may obstructs thoracic duct drainage.1 Very rarely connections develop between the pericardial sac and a lymphatic leak.1 Delayed presentation is suggestive of slow accumulation of chyle supporting the hypothesis of opening up of new lymphatic connections.4 The diagnosis of chylopericardium is confirmed by chemical analysis of drained chylous milky fluid cholesterol level greater than 110 mg/dL, a cholesterol-to-triglycerides ratio of less than 1 and a triglyceride level greater than 500 mg/dL. It is hard to extrapolate that intervention in form of mediastinal lavage or pleural aspiration could have resulted in the chylopericardium in our patient. Can repeated sternal wire closure in a setting of mediastinitis inadvertently causes lymphatic injury to cause the collection is a farfetched assumption. Although it should be investigated whether infection or mediastinitis is an etiology as many of the cases reported have had either intervention for infection, carditis or developed early post operative infection or reintervention1,5 which incidentally was in our case as well.The management can be medical or surgical. The site of culprit leak can be investigated with the help of computer tomography based lymphangiography, invasive angiography or nuclear lymphoscintigraphy. This also quantifies the leak and guides in appropriate management once the leak does not decrease with conservative medical management. We did not consider the diagnostic imaging as our patient responded to medical management within 48 hours. Medical management includes fluid and electrolyte management, parentral nutrition or Medium Chain triglyceride diet, somatostatin infusions.6 Surgically creation of a pericardial window tides over the crisis but definite surgical management is the ligation of thoracic duct above the diaphragm preventing repeated leaks, while the chyle gets diverted through other channels. A combination of medical management and pericardial drainage either multiple aspirations or pigtail may be done if there are facilities of repeated evaluation and round the clock operation facilities for surgical intervention if required. This \u2018watchful masterly inactivity\u2019 proved successful in our case and avoided an invasive surgical intervention.A pericardial collection after a midline surgery large enough to cause symptoms is generally a warning sign. It must be investigated with a protocol of radiological investigation and cytological analysis after stabilizing the patient clinically. Transudative pericardial effusion either reactionary or because of cardiac failure should always be ruled out and medical management initiated, an infective or hemorrhagic collection might require an urgent surgical intervention, rare collections of chyle should be kept in mind which can be successfully managed without reentering the mediastinum surgically, not only putting the prognosis of patient in better perspective but also conserving the much needed resources in a developing countryAn informed consent was obtained for publication this case.All authors declare no competing financial interests exist."} +{"text": "Dear editoret al. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4975131/), and I would like to address some related comments. Frequently clinicians report adult cases of patients with opportunistic infections as disseminated tuberculosis and/or fungal infections in patients consider as immunocompetent based mainly in the absence of human immunodeficiency virus infection (HIV negative). Immunocompetence is more complex than absence of HIV infection and involves a normal capacity to develop an immune response following the exposure to an antigen or broadly a normal immune response, but usually immunocompetent is define as the opposite of immunodeficiency. In the report authors said \"Our aim is to report the case of an immunocompetent patient diagnosed with Mycobacterium tuberculosis and Candida albicans co-infections\" but my deliberation is Do we make in the clinical practice all the efforts to consider a patient as immunocompetent?I read a case report about Tuberculosis and fungal co-infection in a previously healthy patient published in Colomb Med by Fontalvo Mycobacterial, fungal and other opportunistic infections force the clinician to rule out a large list of conditions associated with secondary immunodeficiency as infectious agents , drugs , metabolic diseases , malignancies and environmental conditions 3). This feature is found frequently in patients with anti-cytokines autoantibodies and is related with self-antibodies to gastric parietal cell and to intrinsic factor producing pernicious anemia Interestingly the patient presented had mild macrocytic anemia (hemoglobin 10.7 g/dL and mean corpuscular volume 103 \u03bcm"} +{"text": "The treatment landscape of metastatic renal cell carcinoma (mRCC) dramatically changed with the introduction of vascular endothelial growth factor receptor (VEGFR) tyrosine kinase inhibitors (TKI) . DespiteA primary challenge with TKIs is the balance of clinical efficacy and associated toxicities of long-term therapy. Treatment breaks were incorporated into the development of VEGFR TKIs to limit toxicities, suggesting that continuous treatment is not always necessary and that extended periods off treatment might be feasible without compromising clinical outcomes. Retrospective and prospective data support the feasibility of intermittent TKI dosing with extended breaks.A retrospective analysis investigated whether patients with mRCC treated with TKI (sunitinib or sorafenib) who achieve complete response (CR) on treatment, can take a treatment break until relapse . Of 53 pSimilarly, the impact of treatment breaks of 3 months or longer for reasons other than progressive disease (PD) in mRCC patients receiving VEGFR TKI was evaluated in a retrospective study of 112 patients [The concept of prolonged treatment breaks was prospectively investigated in a randomized discontinuation trial of sorafenib, in which mRCC patients with stable disease after 12 weeks of sorafenib were randomly assigned to receive placebo or to continue sorafenib. The progression free survival (PFS) in patients who were assigned to placebo but then crossed over at progression to restart sorafenib, was similar to patients who were continued on sorafenib. Patients who were in the placebo arm had more tumor growth but the antitumor effect of sorafenib was maintained upon reinitiating treatment further highlighting that extended breaks from therapy do not necessarily compromise clinical outcomes .Additional prospective data supporting the feasibility of intermittent TKI dosing with extended breaks in patients with mRCC was recently published . PatientA phase II/III clinical trial in UK is ongoing with a goal of randomizing 1000 mRCC patients to standard dosing or intermittent dosing of sunitinib . The oveIn summary, TKIs have changed the treatment landscape of RCC and will continue to play an important role in the treatment of RCC in the future. Challenges in TKI delivery include mitigating long-term toxicities while maintaining clinical efficacy, and strategies like prolonged treatment breaks are critical to optimize the delivery of TKIs."} +{"text": "Kuru is an acquired human prion disease that primarily affected the Fore linguistic group of the Eastern Highlands of Papua New Guinea. The central clinical feature of kuru is progressive cerebellar ataxia and, in sharp contrast to most cases of sporadic Creutzfeldt\u2013Jakob disease (CJD), dementia is a less prominent and usually late clinical feature. In this regard, kuru is more similar to variant CJD, which also has similar prodromal symptoms of sensory disturbance and joint pains in the legs and psychiatric and behavioural changes. Since a significant part of the clinicopathological diversity seen in human prion disease is likely to relate to the propagation of distinct human prion strains, we have compared the transmission properties of kuru prions with those isolated from patients with sporadic, iatrogenic and variant CJD in both transgenic and wild-type mice. These data have established that kuru prions have prion strain properties equivalent to those of classical (sporadic and iatrogenic) CJD prions but distinct from variant CJD prions. Here, we review these findings and discuss how peripheral routes of infection and other factors may be critical modifiers of the kuru phenotype. C) to an abnormal isoform, designated PrPSc (PRNP), infection with prion-infected tissue or by rare sporadic events that generate PrPSc , Gerstmann\u2013Str\u00e4ussler\u2013Scheinker disease, fatal familial insomnia, kuru and variant CJD and homozygosity confers genetic susceptibility to both sporadic and acquired forms of CJD . Codon 1PRNP genetic factors have also been revealed suggest that there are several different human PrPSc conformations, referred to as molecular strain types and these can be further classified by the ratio of the three PrP fragments seen after protease digestion. PrPSc types 1\u20133 are seen in classical (sporadic or iatrogenic) CJD brain, while type 4 PrPSc is uniquely seen in vCJD brain or methionine (M) at residue 129 and in wild-type mice , lack a transmission barrier to classical (sporadic and iatrogenic) CJD prions, regardless of the codon 129 genotype of the inoculum has been strongly supported by molecular and neuropathological analysis of human prion transmissions to conventional and transgenic mice. Transgenic mice expressing only human PrP have shown that residue 129 polymorphism constrains both the propagation of distinct human PrPent size . By content size and stroent size .Sc type and transmission rates of kuru prions and classical CJD prions, type 3 PrPSc from kuru or sporadic CJD brain propagated faithfully in 129VV Tg152 mice (Sc was also observed in 129VV Tg152 mice inoculated with the type 2 PrPSc kuru isolate (Sc and codon 129 methionine when crossing a PRNP codon 129 transmission barrier; however, further transmissions of CJD isolates with this molecular strain type will be required to investigate this directly (In agreement with the close similarities of both PrP152 mice . In thes isolate . This swSc in 129VV Tg152 mice contrasts sharply with that seen in vCJD-inoculated 129VV Tg152 mice propagating type 5 PrPSc. In both clinically affected and sub-clinically infected 129VV Tg152 mice, the propagation of type 5 PrPSc is only accompanied by the occurrence of large, non-florid, PrP plaques in the corpus callosum with an absence of PrP plaques or diffuse PrP deposition in other brain areas (Wadsworth et al. The pattern of neuropathology associated with the propagation of type 3 PrPet al. Molecular and biological strain typing studies have established that kuru prions have molecular strain types (Parchi et al. et al. et al. et al. PRNP mutation (Despite the close molecular and biological similarity of kuru prions and sporadic CJD prions, both the clinical presentation and the neuropathology of kuru are distinct from the majority of patients with sporadic CJD. While rapidly progressive dementia is the defining clinical feature of approximately 70 per cent of cases of sporadic CJD (mutation .PRNP genetic loci that exert a major influence on prion disease incubation time have been mapped in mice (In addition to the route of exposure, other factors may also influence the neuropathology and clinical features of kuru. Age is an important determinant of survival in sporadic and inherited prion diseases ("} +{"text": "Sir,According to the recent guidelines set by Medical Council of India (MCI), it is now compulsory to have psychiatry posting for 14 days as part of rotatory internship, besides completing three hours per day for 14 days of psychiatry posting during the fifth or sixth semester for MBBS undergraduates (UGs). In many medical colleges, where there are postgraduate (PG) degrees in psychiatry, UG teaching is not given importance. The previous symposium on UG teaching in psychiatry has highlighted the importance of this issue very clearly.1 There arThe medical colleges which do not have psychiatric departments can have their UGs posted to the nearest government psychiatric treatment centers. Research shows greater exposure to and working with mentally ill persons during medical training decreases fear and creates a positive attitude towards caring for the mentally ill.5 The MCIThis effort helps in creating awareness about various mental disorders and in decreasing stigma attached towards psychiatry. We, as psychiatry professionals, need to devote time and , keep ourselves well versed with current knowledge about our subject and conduct ourselves in dignified way."} +{"text": "Gold(III) complexes are emerging as a new class of metal complexes with outstanding cytotoxicproperties and are presently being evaluated as potential antitumor agents. This renewed interest is the resultof recent studies in which various gold(III) complexes have been shown to be stable under physiologicalconditions and to manifest relevant antiproliferative properties against selected human tumor cell lines. Thepharmacological investigation of some representative gold(III) complexes has been extended to consider theireffects on the cell cycle and to reveal induction of apoptosis. Remarkably, preliminary studies suggest thatthe interactions in vitro of gold(Ill) complexes with calf thymus DNA are weak whereas significant bindingto model proteins takes place. Our findings imply that the mechanism of action of cytotoxic gold(Ill)complexes might be substantially different from that of clinically established platinum compounds."} +{"text": "The formation of 4-hydroxynonenal and similar aldehydic products of lipid peroxidation, which play a role as mediators of inflammatory processes, was clearly demonstrated in rat hepatocytes treated with CBrCl3. It may be assumed that haloalkane toxicity is connected with the biological effects of those inflammation mediatory aldehydic compounds.Bromotrichloromethane (CBrCl"} +{"text": "To clarify the relationship between percutaneous transhepatic cholangioscopic findings such as papillogranularsurface and vascular dilation, which are reportedly characteristic of carcinoma, and thepattern of spread for bile duct carcinomas, we compared endoscopic photographs with histologicalfeatures of biopsy specimens in 57 regions of specimens from 35 patients with malignant stenosis ofthe bile duct. Regions with a papillogranular surface were associated with noninvasive mucosal carcinomasand papillary proliferation of superficial epithelia significantly more often than regions withoutsuch a surface (P<0.0001). The sensitivity and specificity of the papillogranular surface tononinvasive mucosal carcinoma was 79 and 95%, respectively, that of papillary proliferation of superficial epithelia was 100 and 98%, respectively. Regions with vascular dilation were associatedwith invasive carcinoma significantly more often than regions without vascular dilation (P<0.0001).The sensitivity and specificity of vascular dilation to invasive carcinoma were 90 and 86%, respectively.Results indicate that a papillogranular surface is related to noninvasive mucosal carcinomaswhile vascular dilation is related to invasive carcinomas. However, a papillogranular surface waseven more closely related to papillary proliferation of superficial epithelia."} +{"text": "A correlative histocytological study was made of 6 patients with palatal carcinomata and 342 patients with palatal lesions (primarily leukoplakias) associated with reverse smoking from the Srikakulam district of Andhra Pradesh. Among 6 histologically diagnosed carcinomata only 2 showed cytological findings typical of carcinoma. Of the 46 atypias diagnosed histologically among the other palatal lesions, only 6 (13%) were diagnosed cytologically. Our findings show that cytological examination of precancerous and cancerous lesions located on the hard palate, which is a highly keratinized area of the oral cavity, may not be reliable enough for revealing premalignant or malignant changes."} +{"text": "Endoscopic sclerotherapy has been used to control acute variceal haemorrhage which persists despiteconservative therapy, prevent recurrent variceal haemorrhage in patients with a history of oesophagealhaemorrhage, and to prevent a haemorrhage in patients with oesophageal varices who never bled.\t\tIn this short paper I will cover our personal experience with more than 2000 patients receivingparticularly paravariceal endoscopic sclerotherapy of bleeding esophageal varices, and especiallypresent the results of our prospective and controlled randomized trials (Table 1) and underlinethe thesis that endoscopic sclerotherapy and surgical procedures for patients with portalhypertension are complementary supporting measures or options."} +{"text": "The macrophage migration inhibition test has been used to study the immune responses of guinea-pigs immunized with injections of whole cells of both an allogeneic and a syngeneic hepatoma grown as established cell lines in tissue culture.A clear dose-response relationship between tumour cell concentration and migration inhibition was seen in immunized animals and no significant migration inhibition was seen in control animals. There was no cross reaction between the two tumours used. There was no cross reaction between whole isolated normal liver cells and tumour cells, or between foetal liver cells and tumour cells. Whole isolated liver cells from carbon tetrachloride damaged livers caused some degree of migration inhibition in both normal and immunized guinea-pigs but, taking this into account, they did not appear to cross react with hepatoma cells."} +{"text": "The high-throughput screening and drug discovery paradigm has necessitated a change in preparation of natural product samples for screening programs. In an attempt to improve the quality of marine natural products samples for screening, several fractionation strategies were investigated. The final method used HP20SS as a solid support to effectively desalt extracts and fractionate the organic components. Additionally, methods to integrate an automated LCMS fractionation approach to shorten discovery time lines have been implemented."} +{"text": "Tumour necrosis factor (TNF) has been found to be an important immunomodulator. Among other functions TNF activates natural killer (NK) cells and stimulates monocytes/macrophages in an autocrine fashion. TNF production and NK activity in peripheral blood mononuclear cells were determined in a clinical phase I study in which recombinant human (rh) TNF was administered as a continuous infusion weekly for a period of 8 weeks. Even though TNF production and NK activity were significantly reduced directly after rhTNF infusion the effect proved to be transient and most pronounced at the first rhTNF administration. One day after completion of the rhTNF infusion the peripheral cells released more TNF into the supernatant compared to TNF activity immediately before the rhTNF infusion. This effect was conspicuous in non-stimulated cultures. After repeated rhTNF infusions both stimulated and non-stimulated TNF production of the peripheral blood mononuclear cells was increased. NK cell activity was also enhanced after repeated cycles of rhTNF administration as compared to early rhTNF treatment. Thus, repeated rhTNF infusions lead to a stimulatory effect on TNF production and NK activity of peripheral blood cells."} +{"text": "Twenty-two asymptomatic women with rising CA 125 levels after chemotherapy for ovarian cancer were entered into a trial of isotretinoin combined with calcitriol. Tumours were evaluated according to precise criteria based on serial CA 125 levels and by comparing regression slopes of CA 125 before and during therapy. There was no evidence based on CA 125 of any responses or significant change in tumour growth rate."} +{"text": "Extensive experience with isotransplants of 27 different tumours , all of strictly spontaneous origin in laboratory bred mice of low cancer strains CBA/Ht and WHT/Ht, has revealed no evidence of tumour immunogenicity. Of approximately 20,000 maintenance transplants, none failed and none regressed; of almost 10,000 carefully observed tumours arising from small or minimal inocula of tumour cells, none spontaneously regressed. The number of injected viable tumour cells required to give a 50% probability of successful transplantation (the TD50) ranged from approximately 1 cell to greater than 10,000 cells among the 27 tumours; high TD50 values, which were dramatically reduced by various procedures having no immunological significance, did not signify active \"resistance\" of the hosts. In the case of all of 7 randomly selected tumours, prior \"immunization\" of recipients with homologous lethally irradiated cells increased their tumour receptivity. Several experiments using various tumours failed to give evidence that immunity could be non-specifically induced or that a massive preponderance of lymphocytes from specifically sensitized mice could inhibit tumour transplantation or growth in vivo; no trace of \"resistance\" to tumour was adopted by isogeneic recipients of lymphocytes from regional nodes of tumour bearers. A limited review of the recent literature on tumour immunity shows that practically all the animal data presented in support of a general theory of tumour immunogenicity or to provide a basis for active clinical immunotherapy have been obtained from transplanted tumour systems which entail artefactual immunity associated with viral or chemical induction of the tumours or their allogeneic transplantation. It is suggested that isotransplants of spontaneously arising tumours are the only appropriate models of human cancer and that any genuine rapport between the animal laboratory and the clinic requires their exclusive use."} +{"text": "The boronated aromatic amino acids were shown to be potent hypolipidemic agents in mice lowering bothserum cholesterol and triglycerides after 16 days. Selective compounds were as effective as the clinicalstandards. Furthermore, the compounds were effective anti-inflammatory agents reducing local and central painas well as suppressing LPS induced endotoxic shock in mice. These agents inhibited lysosomal andproteolytic enzymes of the liver and macrophages as a part of their mechanism of action."} +{"text": "Monoclonal antibodies to carcinoembryonic antigen (CEA) promise improved specificity for the measurement of this widely expressed human cancer antigen. A mouse monoclonal antibody binds weakly to CEA in perchloric acid extracts of tumour but strongly to CEA similarly isolated from serum, and its spectrum of cancer detection differs from conventional antisera."} +{"text": "New disinfection methods include a persistent antimicrobial coating that can be applied to inanimate and animate objects (Surfacine), a high-level disinfectant with reduced exposure time , and an antimicrobial agent that can be applied to animate and inanimate objects (superoxidized water). New sterilization methods include a chemical sterilization process for endoscopes that integrates cleaning (Endoclens), a rapid (4-hour) readout biological indicator for ethylene oxide sterilization (Attest), and a hydrogen peroxide plasma sterilizer that has a shorter cycle time and improved efficacy (Sterrad 50)."} +{"text": "Mus musculus domesticus), the major urinary protein (MUP) gene cluster provides another highly polymorphic scent signal of genetic identity Animals might be able to use highly polymorphic genetic markers to recognize very close relatives and avoid inbreeding Mus musculus domesticus) rather than genetically homogeneous laboratory mice that are hybrids of three Mus subspecies derived from an extremely small pool of founders Our experimental design met a set of stringent requirements to establish whether mice use major histocompatibility complex (MHC) and/or major urinary protein (MUP) to avoid inbreeding. First, genetic recognition\u00a0and mate preferences need to be demonstrated against normally variable genetic and environmental backgrounds typical of natural populations. We therefore used wild house mice (2 with abundant cover and food). Tissue samples for genotyping were obtained from all F1 founders prior to release and from their parents, allowing us to establish the separate MHC and MUP haplotypes of each heterozygous founder. The combinations of males and females available as potential mating partners covered the full range of possible MHC and MUP haplotype sharing . The strong deficit in successful matings between mice of the same MUP genotype also accounts for the weak deficit in matings between full sibs imprinting on maternal haplotypes would allow animals to avoid inbreeding with a greater proportion of kin than the use of self-haplotype sharing alone There were fewer offspring per successful mating with full sibs than random expectation was nonsWhy should mice avoid mates that share both MUP haplotypes but not those that share one haplotype? In our experiment, the sharing of both MUP haplotypes was a good predictor of whether a potential mate was a full sib or not: 31% of full-sib dyads shared both MUP haplotypes versus only 5% of half-sib dyads . HoweverThe use of MUP alone was sufficient to explain inbreeding avoidance in this study. Although the deficit in mating between mice of the same MUP type was very strong, there was no evidence that animals used genetic\u00a0markers other than MUP to improve their level of inbreeding avoidance. Further, there was no evidence to support the previously untested hypothesis that animals could increase the range of relatives avoided by imprinting on the separate haplotypes carried by their mother The use of MUP as a genetic marker for inbreeding avoidance (whether a pre- or postcopulatory mechanism) will promote genome-wide heterozygosity that includes MHC. Despite widely held assumptions in the literature that MHC-based scents are used by females to avoid inbreeding and promote MHC heterozygosity Two broad outcomes derive from this study. First, it challenges the widely held assumption that MHC scents provide a general mechanism across vertebrates to avoid inbreeding and promote MHC heterozygosity. This idea has arisen largely from studies of laboratory mice under extremely abnormal genetic, social, and environmental conditions. Instead, normal wild mice use\u00a0a set of species-specific urinary proteins to avoid inbreeding that has evolved to provide optimized characteristics for effective signaling through their urine scent-marking system. Given the importance of reliable identity information for mate selection and reproductive success, we should expect animals to evolve signals that are most appropriate for reliable communication in that species. The only known function of MUPs is in scent communication, and they are produced at very high abundance in urine by mice of both sexes but with particularly strong investment by adult males Mus species Mus macedonicusThe second general implication is that the ability to recognize kin as a mechanism to avoid inbreeding might be more variable across species than previously considered. Mate-selection mechanisms to avoid the deleterious consequences of inbreeding are only likely to be important where kin encounter each other as adults"} +{"text": "We hypothesized that patients with recurrent mood disorders who are sensitized to tree pollen , in comparison to those who are not sensitized, would report larger negative changes in mood during exposure to tree pollen in spring. We also hypothesized that differences between high and low tree pollen periods in self reported allergy symptoms would correlate positively with differences in self reported depression scores. We present 1-year preliminary data on the first 51 patients with unipolar or bipolar disorder . Ratings of mood and allergic disease status were performed once during the peak airborne pollen counts and once during the period of low airborne pollen counts, as reported by two local pollen counting stations. Linear regression models were developed to examine associations of changes in depression scores (dependent variable) with tree pollen sensitization, changes in the allergy symptom severity score, adjusted for gender and order of testing. We did not confirm the hypothesized relationship between a specific tree pollen sensitization and changes in mood during tree pollen exposure. We did confirm the hypothesized positive relationship between the changes in allergy symptoms and changes in subjects' depression scores (adjusted p<0.05). This result is consistent with previous epidemiological evidence connecting allergy with depression, as well as our recent reports of increased expression of cytokines in the prefrontal cortex in victims of suicide and in experimental animals sensitized and exposed to tree pollen. A relationship between"} +{"text": "A total of 107 patients with bronchial carcinoma have been studied for the presence of potential circulating tumour markers which might be used as indicators of recurrence after primary treatment. Plasma carcinoembryonic antigen (CEA) levels were estimated in every patient and, after a preliminary hormone screening study, plasma calcitonin (CT) and parathyroid hormone (PTH) levels were assayed in 66 patients. Oat-cell tumours proved to be of particular interest in that CEA levels greater than 40 microgram/l were measured in 40.6 percent and CT levels were elevated in 75 percent. Longitudinal studies point towards the possible use of elevated marker levels as guides to therapy when all other features of recurrent disease are lacking. It is clear that no ideal tumour marker exists for bronchial carcinoma but in an individual case an abnormal level of one or more marker substances may provide a valuable aid to treatment."} +{"text": "Previous studies while demonstrating the presence of blood group isoantigens on normal prostatic epithelium have failed to identify such antigens on malignant prostatic tissue. Using a series of blood group specific monoclonal antibodies directed towards the A, B, H and Y antigens we have reinvestigated blood group isoantigen expression in both benign prostatic hypertrophy and prostatic adenocarcinoma. Results obtained from areas of benign prostatic hypertrophy are in broad agreement with those published however though we were unable to detect either A or B blood group isoantigens Type 2H and Y isoantigens were identified in 10 of the 12 tumours. These findings, while differing from previously reported results, lend support to the suggested connection between ontogenesis, oncogenesis and blood group isoantigen expression and also support the proposed link between Type 2 structures and malignant transformation."} +{"text": "Numerous studies have indicated that the plasma membrane plays an important role in the development of resistance to anthracycline and vinca alkaloid drugs (pleiotropic resistance). We have previously shown that pleiotropically resistant Ehrlich ascites tumour cells, which are of epithelial origin, have a significantly increased plasma membrane traffic (endo/exocytosis) to the endosomal compartment compared to sensitive cells. The present study, using the same ultrastructural morphometric technique, has demonstrated a similar significant difference in plasma membrane traffic between daunorubicin resistant and sensitive P388 cell lines (which are of lymphoid origin). Furthermore, we have shown that this difference between the P388 sublines is accompanied by an approximately 4 fold increase in the plasma membrane area participating in recycling together with an increased endosomal volume, number and membrane area in resistant cells. Plasma membrane traffic in resistant cells was significantly inhibited by the calcium channel blocker verapamil, a well known modulator of anthracycline resistance, but unaffected by daunorubicin itself. The confirmation of this phenotype in an additional pleiotropically resistant cell type with a different histogenesis further supports a hypothesis of endosomal drug trapping and vesicular extrusion as a possible resistance mechanism."} +{"text": "Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosaandKlebsiella pneumonae.Acylhydrazine derived furanyl and thienyl Schiff bases and their Cu(II) complexes have been prepared andcharacterized on the basis of their physical, spectral and analytical data. The preferred enolic form of theSchiff base function as a tetradentate ligand during coordination to the metal ion yielding a square planarcomplex. The Schiff bases and their complexes with different anions were tested for their antibacterialactivity against bacterial species such as"} +{"text": "Stroke and cerebrovascular disorders are important causes of morbidity and mortality in children; they are already amongst the top 10 causes of childhood death and are probably increasing in prevalence. Acute treatment of stroke syndromes in adults is now evidence based. However, paediatric stroke syndromes are far less common and the differential diagnosis is very wide, but the individual health resource implications are much greater because of the life-long treatment costs in survivors. Recognition and consultation with a paediatric neurologist should be rapid so that children can benefit from regional services with emergency neurological, neuroradiological and neurosurgical intervention and paediatric intensive care. This review focuses on the epidemiology, presentation, differential diagnosis, generic/specific emergency management and prognosis of acute stroke in children. Its aim is to educate and guide management by general paediatricians and to emphasise the importance of local guidelines for the initial investigation and treatment and appropriate transfer of these children. Recent epidemiological data suggest incidence rates of 2\u20135/100\u2009000 children/year for childhood stroke (at least 300 a year in the United Kingdom),5Haemorrhagic stroke includes12\u201322\u201324Ischaemic stroke is commonly arterial (AIS)15There are similarities and differences between the predisposing conditions and intermediate risk factors for haemorrhagic stroke , AIS fi, VST fi and stro23\u2013At least a third of cases of childhood stroke occur in the context of infection,28The very different nature of the vascular, coagulation and nervous systems in neonates, infants and children means that clinical and radiological presentations are different from adults.AVMs are defi\u201334Arterial dissection occurs a32Transient cerebral arteriopathy (TCA) 3, 1C4 re30Moyamoya is the Japanese for \u201cpuff of smoke\u201d and describes a cerebral arteriopathy with bilateral severe stenosis/occlusion of the terminal internal carotid arteries (ICAs) associated with the development of basal collateral vessels.\u20136\u201320The cerebral veins drain into \u201csuperficial\u201d or \u201cdeep\u201d cerebral venous sinuses. Sinus blood flow rates are dictated by mean arterial pressure (MAP) and thrombosis therefore occurs more commonly in hypotension. The pathogenesis of VST in neonates remains uncertain but may relate to anatomical distortion during childbirth as well as dehydration and infection. In older children trauma, malignancy or sepsis play a larger role . Dehydra15Vein of Galen malformation (VGAM) is an embryonic arterio-venous fistula occasionally presenting with catastrophic neonatal heart failure which within centres with an experienced multidisciplinary team may be endovascularly palliatedSturge Weber syndrome (SWS) is characterised by a facial capillary haemangioma and venous angiomata of the leptomeninges/choroids associated with intractable epilepsy (which may require hemispherectomy)These are diagnoses of exclusion which should only be made after discussion with a paediatric neurologist or intensivist.There may be a family history. EEG usually shows unilateral slow background activity.The demyelination is usually obvious on MRI. Intravenous methyl prednisolone probably reduces the duration of the illness and perhaps improves long term outcome.This is characterised by seizures, disorders of consciousness, visual abnormalities and headaches associated with posterior white matter abnormalities on CT/MRI and has been described after acute chest syndrome in SCD,There are often clinical clues to the aetiology of metabolic stroke, for example persistent vomiting, hypoglycaemia or diabetes. Organic acidaemias, urea cycle disorders and mitochondrial disorders can cause stroke-like episodes with imaging abnormalities in an atypical vascular distribution. Homocystinuria and Fabry disease are usually associated with cerebrovascular disease.Although stroke in childhood is relatively rare, its clinical presentation is usually obvious to the paediatrician, who can investigate both obvious and subtle presentations and initiate emergency medical management. Hemiplegia, headache, seizure or altered levels of consciousness may all herald a potentially reversible or lethal medical or surgical stroke emergency. If a stroke syndrome is suspected, initial resuscitation in conjunction with the local anaesthetic/intensive care team should be followed by consultation with regional paediatric neurological services before further imaging, investigation or treatment are planned and instituted. Although stroke is traditionally defined as a neurological deficit lasting for \u2a7e24 h, many children with a TIA lasting \u2a7d24 h have had a recent cerebral infarction/haemorrhage on imaging. In addition to the underlying diagnosis , the timIf immediate transfer is deemed necessary after discussion with the on-call neurosurgeon or paediatric neurologist, liaison with the regional paediatric intensive care unit (PICU) is advisable. Children may present in coma, status epilepticus, or with signs of intracranial hypertension or imminent herniation mandating liaison with local anaesthetic teams/regional PICUs, and appropriate retrieval/transport of children to regional centres for further imaging/management.No studies have specifically examined the effect of the loss of cardio-respiratory integrity on stroke outcome in children. However, based on principles which would be applied to the care of any acutely ill child including the maintenance of adequate oxygenation (non-invasively estimated by pulse oximetry), cardiac output, systemic and cerebral perfusion pressure, and tight control of blood glucose and body temperature should be the aim. Hypertension should not be treated unless intracranial pressure is monitored if there is a space-occupying lesion, and should only ever be lowered slowly.Children whose level of consciousness deteriorates should be ventilated and transferred to the nearest neurosurgical/PICU in case they require drainage of a haematoma, ventriculostomy for hydrocephalus or craniectomy for intractable intracranial hypertension. Management may be guided by intracranial pressure monitoring, which should be considered in children who remain sedated and in whom there is radiological/clinical suspicion of a space-occupying lesion. Seizures in the acute phase should be managed aggressively in accordance with conventional algorithms and local guidelines as they significantly increase the cerebral metabolic rate for oxygen and can thus unfavourably affect the substrate supply\u2013demand balance. Consideration should be given to continuous cerebral function monitoring in paralysed children.Haemorrhagic stroke or AIS with mass effect should be excluded by emergency CT, which might also show some evidence of focal ischaemic damage but often only from 24 h after presentation. If CT is not available immediately at the local site, discussion with a tertiary centre is mandatory and urgent consideration should be given to transfer of the child, even if this can only be achieved safely by intubation and ventilation. The regional PICU should be involved in this decision, as this is an emergency. If immediately available, MR with diffusion weighting has advantages over CT as haemorrhage can be diagnosed or excluded 2, RPLS 2, hemiplThe management strategy should ensure optimal intravascular volume, normothermia and normoglycaemia. A neurosurgical opinion is mandatory for the discussion of haematoma drainage and the management of complications, including hydrocephalus, vasospasm, perihaematomal oedema and brain shift. Intracranial pressure monitoring and osmotherapy targeted at maintaining an adequate cerebral perfusion pressure may be required. Fluid restriction is not advisable initially but may be initiated if there is evidence of inappropriate ADH release. Vasospasm may complicate subarachnoid haemorrhage, is detectable by transcranial Doppler (TCD) and treatable with calcium channel antagonists. Blood pressure control is a controversial topic as perfusion pressure must be maintained while the risk of recurrent haemorrhage may mandate avoidance of hypertension before definitive vascular treatment. If there is an underlying AVM or aneurysm, the recurrence riskAll stroke syndromes are potential neurosurgical emergencies and should be discussed with a consultant paediatric neurologist on presentation. Further management and any transfer may involve liaison with the nearest available PICU.Acute ischaemic arterial stroke\u00b1haemorrhage\u00b1mass effectAcute venous stroke\u00b1haemorrhage\u00b1venous infarction\u00b1mass effectPrimary haemorrhagic stroke\u00b1mass effectNon-accidental injurysubdural haematomastrangulation with compression of internal carotid arteryPosterior leukoencephalopathy (hyper/hypotension or immunosuppression)Unilateral hemispheric cerebral oedema, for example secondary to diabetes, hyperammonaemia (ornithine carbamoyl transferase deficiency)Hemiplegic migraine (but diagnosis of exclusion \u2013 migrainous symptoms seen in cerebrovascular disease)Post-ictal (Todd\u2019s paresis)short duration so neuroimaging essential if persistentchildren with prolonged seizures may develop permanent hemiparesis with seizures (hemiseizure-hemiplegia-epilepsy)Acute disseminated encephalomyelitisBrain tumourEncephalitis, for example secondary to Herpes simplex Rasmussen\u2019s encephalitisMitochondrial encephalopathy with stroke-like episodesAlternating hemiplegia\u20138Some children presenting with AIS/VST are candidates for acute interventions after ne6The population with sickle cell disease provides an ideal model for proactive stroke prevention as the majority of strokes are predicted by TCD. Blood transfusion is a mainstay of stroke prevention\u2013Despite a little published experience,6\u20136\u20138The use of anti-coagulation remains controversial. Children are probably at less risk of haemorrhage than adults and there is a case for acute anticoagulation in AIS.6Magnetic resonance imaging (MRI) (including diffusion and perfusion), arteriography (MRA), venography (MRV)to exclude haemorrhageto define extent and territory of infarctMRA to define vascular anatomy of circle of Willis and neck vesselsT1-weighted spin echo of the neck with fat saturation sequence to exclude dissectionMRV to exclude venous sinus thrombosisdiffusion imaging to differentiate acute from chronic infarctionperfusion imaging to demonstrate areas of abnormal cerebral blood flow, blood volume and mean transit timeCT scan to exclude haemorrhage if MRI not available acutely; consider CT venographyConventional angiography if:haemorrhage without coagulopathy and cause not obvious on MRA or MRVischaemic stroke, MRA normal and fat-saturated T1-MRI of the neck does not demonstrate dissectionIf intracranial hypertension persists or there is evidence of impending herniation despite maximal medical therapy, decompressive craniectomy should be considered for AIS, VST and stroke mimics. Patients with hydrocephalus secondary to large cerebellar infarcts may need ventriculostomy or cerebellectomy.Physio-, occupational and speech therapists should be available for children soon after stroke as part of the multidisciplinary team. Long term rehabilitation should include cognitive,As there is a wide range of differential diagnoses, which may be difficult to recognise,"} +{"text": "Penile reconstructive surgery has always been a notablechallenge in the field of genitourinary reconstruction because of the fact thatit attaches huge importance to the functional and aesthetic aspects of thisorgan and the genital region as a whole. During the last decades, many operative procedures andtheir modifications for the treatment of both congenital and acquired anomaliesare published. Based on the establishedprinciples of reconstruction, as westrive forward in this difficult art, we also feel the need to adapt to growing demands of rewriting the stiffchallenges of techniques, their modifications,the long-term results, and futuristic issues which encompass thissubspeciality.hypospadias.Repair of this anomaly has become easier with Snodgrass principle for distaland buccal mucosa graft combined with genital flaps for proximal forms.However, complication rate is still not minimized to be satisfied.Heidelberg group in the article entitled \u201cHypospadias\u201d withtheir vast experience had discussed all aspects of hypospadias in details as itstands today commencing from its understanding as a developmental anomaly toits presentation, preparation, operative techniques as they stand today based on well defined historic and currentconcepts. Their overview emphasizes the fate of urethral plate in variousdistal and proximal surgical methods algorithmically to bring aboutsatisfactory planning and execution of each treatment arm. The authors have ingreat details described their preferred approach of longitudinal preputial orpenile flap in both distal and proximal hypospadias and presented its visualand schematic understanding.One of the most common congenitalanomalies is Reinberg et al. in \u201cTunneled tunica vaginalisflap for recurrent urethrocutaneous fistulae\u201d in a retrospective clinical analysispresent their 5-year data on a set of 12 boys who underwent tunneled tunicalvaginalis flap repair for recurrent fistulae in all locations. Another groupfrom Detroit have presented a unique group of patients in their 5-year studywhere they discuss a subset of \u201ciatrogenic strictures\u201d presenting in adulthood resulting fromchildhood hypospadias surgeries. The complexity of these cases is increasedmanifold due to reasons of delayed presentation of many decades, varyinglengths, as well as poor healing capacity due to scarred territory from previous surgical attempts and associated complicating factors of renal failure and fistulae.intersex, now namedas disorders of sex development, presents results of updated etiological andoutcome data as well as refined surgical procedures, as discussed in \u201cAdult urethral stricturedisease after childhood hypospadias repair.\u201d Ambiguousgenitalia play a role in gender differentiation, andsurgical treatment should give answers for psychosexual questionnaire of this population. Gupta et al. in \u201cMale genitoplasty for intersex disorders\u201d have come up witha large series review to discuss dilemmas in gender reassignment in intersexdisorders in the childhood age group. In their review which includes majority ofcases of proximal hypospadias, authors advocate early and complete chordeecorrection in childhoood for satisfactory phallic growth. The issue of genderreassignment is a delicate one in paediatric age group and needs to take intoconsideration the community, parental wishes, the state of child, and itsphenotypic gender of being reared, as well as the age of presentation. Inmasculanising genitoplasty satisfactory hormonal treatment, removal orpreservation of mullerain structures and parental counselling are key features.It involves a dedicated teamwork and close yet longterm followup forcorrections with changing age and hormonal management.The current management of patientswith Peyronie's disease. The majority of procedures areusually followed with some loss of length. Using radical geometrical principlesin creating and fashioning the graft with appropriate size leads to precisecorrection with penile lengthening. Thefirst review from Aboseif et al. \u201cReview of the surgical approaches for Peyronie's disease:corporeal plication and plaque incision with grafting\u201d judgesour understanding of surgical approaches to date in managing Peyronie's plaque\u2014from plication tografting. Their review article focuses on surgical evolution and compares thetwo methods from all recent publications thus judging the advantages of bothtechniques. A rationale approach today would be proper counseling of each well-informedpatient and a surgeon capable of the armamentarium of techniques to choose fromas required in an individual case with intraoperative decisions which hold thekey. More complex and multiple deformities would benefit with a combination ofthe techniques described to ensure complete correction.The degree of curvature, the type ofdeformity, erectile dysfunction, and penile length are all characteristics thatare assessed in choosing the best surgical intervention in Inanother paper, \u201cPeyronie'sreconstruction for maximum length and girth gain\u2014geometrical principles,\u201dEgydio et al. redefine their concept of straightening by geometrical principlesin yet another landmark study of 521 treated patients over 8 years who achievedsatisfactory sexual health with the performed straightening and enlargementtechnique divided into two arms of use of bovine pericardial grafts and thecorresponding arm with additional penile prosthesis. A small subgroup ofpatients with grafting alone who developed deterioration of erectile function in postoperative followup weretreated with penile prosthesis to satisfactory outcomes. The penile disassemblydescribed earlier is a huge assistance in exposing even the most distaldeformities for complete correction. As we strive toward the search of an idealgraft for penile reconstructive surgeries, the use of bovine pericardial graftswith its advantages as demonstrated in such large series safely givesconfidence of yet another biocompatible graft with long-term results. Prospectivecomparative studies with other contemporary surgical techniques would go a longway in furthering our knowledge toward ultimate patient satisfaction inPeyronie's disease.Incontrast, Piacentini et al. in \u201cPreservation of cavernosal erectile function after soft penileprosthesis implant in Peyronie's disease: long term follow up\u201d present an excellent retrospective review ofintermediate results of 6 years in 12 patients who have been treated withsemirigid prosthesis alone for Peyronie's disease without any plaque treatment.Their study is based on the hypothesis which heavily relies on the residualcavernosal tissue that becomes a peripheral component to the prosthesis andplays a healing role in further straightening as well as being available forfuture pharmacologic therapy.erectile dysfunction. Technologicalimprovement of penile prosthesis lead to their rising popularity, but strictindications and rigorous surgical principles for implantation should always berespected (followed) in order to avoid unnecessary surgery and possiblecomplications with severe psychological consequences. Thenext exciting section of this issue is devoted to the difficult art andcontemporary world of penile prosthesis. Aboseif et al. in \u201cA preliminary report oncombined penoscrotal and perineal approach for placement of penile prosthesiswith corporal fibrosis,\u201d in acomprehensive retrospective study of 15 patients, review their state-of-the-arttechniques for penile reconstruction in difficult penile prosthesis placement. Theirgroup is divided into two treatment arms of severe Peyronie's tunical fibrosisand the other of severe tunical fibrosis of previous prosthesis-relatedcomplications. Thedata are very interesting and larger prospective studies for longer duration could throw more light to ourcontinued search for ideal techniques. In another review, \u201cPenile corporealreconstruction during difficult placement of a penile prosthesis,\u201d theydiscuss their preliminary results in combined penoscrotal and perineal approachfor complex corporal fibrosis spanning 3 patients. Finally, Bettocchi et al. in\u201cPenileprosthesis: what should we do about complications?\u201d madea review of penile prosthesis implantations, indications, and, especially, a discussionabout possibilities in the treatment of complications.A wide variety of medications,devices, and surgical interventions are available for patients with penile carcinoma is an aggressivedisease with significant treatment-associated psychosexual morbidity. Despitehigh control rates with radical surgical approaches, organ-sparing surgeryshould be considered to achieve better psychosexual life. Lisbongroup in \u201cOrgan preserving surgery forpenile carcinoma\u201d broughtforth the idea of balance between the functional and anatomical aspects of thisorgan weighing against any compromise with local oncolgical radicality in thismalignancy. They explore all possibilities of penile preserving approaches inpenile cancers by various operative and nonoperative interventions in properlyselected patient with a word of caution that the approach to organ preservationcan be applied to low-grade and low-stage tumours until more evidence emergesfrom prospective studies. To further attempts at proper organ preservationtechniques, the recent description of penile disassembly and excision\u2014reconstructionwould go a long way in achieving satisfactory results.Last but not least, Finally,Hoebeke et al. in \u201cReconstructive surgery for severe penile inadequacy: phalloplastywith a free radial forearm flap or a pedicled anterolateral thigh flap?\u201d have summarized their experience on thedevastating condition of severe penile inadequacy in young males. Phalloplastyremains a challenge in reconstruction and is the hallmark achievement intranssexual surgery and severe penile deficiency that results from trauma,micropenis, and penile amputations. This series describes the procedure andconsequences of 11 young males who underwent radial forearm flap oranterolateral thigh flap neophallus reconstruction.Miroslav L. Djordjevic"} +{"text": "Correlated with this overall pattern of immune response, membrane immunization did not elicit tumour rejection reactions. These findings are relevant to current views that humoral factors operate antagonistically to limit cell mediated immunity to tumours. A further relevant feature was the observation that membrane immunization, eliciting a prominent humoral immune reaction, conditioned the recipients so that they subsequently failed to elicit a tumour rejection immunity on treatment with irradiated tumour cells.The principal expression of immunity elicited in syngeneic rats immunized with rat hepatoma membrane fractions was the development of a tumour specific antibody response. This antibody was demonstrable by membrane immunofluorescence staining of viable hepatoma cells in suspension and the sera exhibited complement dependent cytotoxicity for cultured hepatoma cells. In the absence of complement, however, membrane immune sera were highly \u201cblocking\u201d, protecting plated hepatoma cells from attack by sensitized lymph node cells. The cell mediated immune response elicited by hepatoma membrane immunization was weak, as evaluated by the colony inhibitory activity of lymph node cells for hepatoma cells"} +{"text": "A survey of rabies postexposure prophylaxis administered by local health departments for a 1-year period showed that very few patients received treatment as a result of exposure to a confirmed rabid animal. Most prophylaxis was administered for contact with domestic animals in situations where existing recommendations for quarantine or laboratory testing of the animal were not followed. Because rabies in domestic animals in Kentucky is uncommon, these findings suggest that had the existing recommendations been followed, the prophylaxis would have been unnecessary in most cases."} +{"text": "Using reduced-impact timber-harvesting practices in legally logged tropical forests would reduce global carbon emissions by 0.16 Gt/year at a modest cost and with little risk of \"leakage\" (increased carbon emissions elsewhere). Most ongoing discussions of REDD focus on tropical deforestation, while the potential carbon saving from reduced forest degradation is mostly disregarded [2 emissions can be achieved by improving forest management in the tropics, and argue that this cost-effective approach to mitigation should be included in the new climate change agreement.Negotiations leading up to an international climate change agreement to replace the Kyoto Protocol in 2012 have included consideration of reduced emissions due to deforestation and degradation (REDD). This option has figured prominently in the \u201croad map\u201d toward such an agreement that was agreed upon during the 13th Conference of Parties to the United Nations Framework Convention on Climate Change held in Bali in December 2007 . In cont forests , and are forests , the benThe potential global contribution of improved tropical forest management to carbon retention is substantial. Using information on intensities and intervals of logging, area of production forest , and the\u22121) . Most ofhttp://www.unfccc.int/). Given that improved forest management is currently practiced in less than 5% of tropical forests [Programs promoting global reductions in carbon emissions through improved forest management should easily satisfy the United Nations Framework Convention on Climate Change criteria for biomass projects .Click here for additional data file."} +{"text": "Chlamydia trachomatis infection were revealed as significant risk factors for CIN after adjustment for HPV antibodies.To identify the risk factors for cervical intraepithelial neoplasia (CIN), we reanalysed the data from our previous case\u2013control study by adjusting for human papillomavirus (HPV) antibodies. Unlike our previous study based only on HPV DNA, smoking and Infections by oncogenic human papillomaviruses (HPVs) are established as a major risk factor for cervical intraepithelial neoplasia (CIN) and invasive cervical cancer (ICC). However, only a fraction of infected women develop cervical cancer, suggesting the involvement of additional cofactors in cervical carcinogenesis. In numerous case\u2013control studies various epidemiological factors have been suggested as relevant to CIN and ICC, although the results from these studies have not been entirely consistent . In epidTo address this issue, we reanalysed the data from our previous case\u2013control study of CIN by adjusting for HPV DNA and antibodies.Chlamydia trachomatis was determined by using an enzyme immunoassay (EIA) kit that does not detect antibody to Chlamydia pneumoniae. The adjusted odds ratios (ORs) and 95% confidence intervals (CIs) were estimated by a logistic regression analysis. The analysis was carried out using JMP 4.0J statistics package . The P-values obtained in all tests were considered significant at <0.05.The present study was conducted on women who previously participated in a Japanese case\u2013control study of CIN. The details about this case\u2013control study have been provided elsewhere , although the relative risk for CIN was considerably different between HPV DNA positive and seropositive women and CIN development because of the low prevalence of OC users among study subjects. The effect of smoking on CIN development did not appear to vary between HPV-positive and HPV-negative subjects, while the effect of C. trachomatis infection was stronger among the former.Human papillomavirus status among cases and controls was determined by using HPV DNA testing and HPV capsid serology . Since HPV DNA testing at a single time point cannot identify past infections, the analysis based on HPV DNA may miss cofactors determining whether HPV infection is cleared or becomes persistent. In fact, a very recent study reported that smoking is associated with a reduced probability of clearing oncogenic HPV infections (C. trachomatis infection with persistent HPV infections may be supported by several studies suggesting the modulation of host immunity by smoking (With regard to smoking and Although the DNA adjustment enhanced the CIN risk among current smokers as well as the antibody adjustment, the association of smoking with an increased risk of CIN was not statistically significant in the DNA-based analysis. This may be due to the small numbers in the statistical analysis.C. trachomatis infection are significant cofactors for CIN development after adjustment for HPV antibodies. Simi-larly, associations of smoking and C. trachomatis infection with cervical neoplasia have been consistent in case\u2013control studies employing HPV capsid serology (Unlike our previous study based on HPV DNA ("} +{"text": "The subcutaneous growth of the Lewis lung tumour in C57BL mice chronically exposed to fresh cigarette smoke was increased above that in age-matched control mice. When murine sarcoma virus (Harvey) induced tumour cells were introduced to the lungs of groups of BALB/c mice, only mice chronically exposed to fresh cigarette smoke died with tumour cells in the lungs. Tumour cell growth in mice during short term cigarette smoke exposure was indistinguishable from that in controls."} +{"text": "Periodontal disease is a chronic adult condition. Bacteria implicated in the etiology of this disease causes destruction of connective tissue and bone. As a result of stimulation by bacterial antigen PMN produces free radicals via respiratory burst as a part of host response to infection. Patients with periodontal disease display increased PMN number and activity. This proliferation results in high degree of free radical release culminating in heightened oxidative damage to gingival tissues, periodontal ligament and alveolar bone. Damage mediated by free radicals can be mitigated by \u201cANTIOXIDANT DEFENSE SYSTEM \u201c. Physiological alteration and pathological states produced by free radicals depend on disequilibrium between free radical production and antioxidant levels leading to oxidative stress.Hence this study has been designed to estimate the TOTAL ANTIOXIDANT CAPACITY in patients with PERIODONTITIS and healthy control subjects Oxygen is required for all living organisms for their survival. But at the same time it is potentially toxic. Salvemini has described oxygen as a double edged sword. It is vital to life but at the same time because of its highly reactive nature it is capable of becoming part of potentially damaging molecules called free radicals. Oxygen is the ultimate electron acceptor in mitochondrial electron transport chain where flow of electrons ultimately produces energy in the form of ATP. The leaked out electrons are exposed to oxygen leading to formation of free radicals.A free radical may be defined as \u201cany species capable of independent existence that contains one or more unpaired electrons\u201d. This makExogenous sources - air pollutants, ozone, radiation, chemicals, toxins, pathogenic microorganisms.due to leaks in electron transport chain in mitochondria during oxidation of food stuffsinflammatory cells produce free radicals by a process of respiratory burst during phagocytosisenzymes which indirectly produce free radicals.Free radicals cause tissue damage by a variety of different mechanisms which includeDNA damagelipid peroxidationprotein damageoxidation of important enzymes [e g anti proteases]stimulation of pro inflammatory cytokines releaseReactive oxygen species [ROS] encompasses other reactive species which are not true radicals but are nevertheless capable of radical formation in the intra and extra cellular environment. E. g: Hydrogen peroxide, Hypochlorous acid, singlet oxygen, ozone.The living organism has adapted itself to an existence under a continuous efflux of free radicals. Among the different adaptive mechanism the antioxidant defense mechanism is of major importance.Antioxidants are \u201cthose substances which when present in lower concentrations compared to those of an oxidisable substrate, will significantly delay or inhibit oxidation of that substrate\u201d.The different possible mechanisms by which antioxidants may offer protection against free radical damage includeprevention of formation of free radicalsinterception of free radicals by scavenging the reactive metabolites and converting them to less reactive moleculesfacilitating the repair of damage caused by free radicalsproviding a favourable environment for effective functioning of other antioxidants.Antioxidant defense system is very dynamic and responsive to any disturbance taking place in redox balance of body. Antioxidants can be up regulated and neutralize free radicals formation that could take place due to oxidative stress. Transcription factors such as nuclear factor -kb and activating protein 1 are redox sensitive. Redox poSmaller changes in redox state - trigger gene transcription events which lead to tissue damage secondary to induction of pro inflammatory state.Larger upward shift in the pro oxidant / antioxidant ratio bring direct damage to vital biomolecules and structures .2]2]The body has a sophisticated antioxidant defense system to cope with free radical formation under normal conditions and thereby maintaining redox balance. However, when there is not an excess of antioxidant defense and an overproduction of free radicals or a drop in level of antioxidants it will lead to an imbalance and cause deleterious effects a situation known as oxidative stress .4]4]Periodontitis is a term used to describe an inflammatory process initiated by plaque biofilm that leads to loss of periodontal attachment to root surfaces and adjacent alveolar bone which ultimately results in loss of tooth.The primary etiological agent is specific, predominantly gram negative anaerobic facultative bacteria within subgingival biofilm. These baAmong the host responses leukocytes serve as the initial host defense against periodontal pathogens. After stimulation by bacterial pathogens neutrophils produce free radicals. Periodontal tissue destruction is caused by an inappropriate host response to these microorganisms and their products. More specifically due to oxidative stress.Hence this study has been designed to estimate the serum total antioxidant capacity in periodontitis and health.The objectives of the study is to investigateTotal antioxidant capacity in serum of patients with periodontal disease.Total antioxidant capacity in serum of patients without periodontal disease.To compare the total antioxidant capacity in serum of patients with and without periodontal disease.Subjects reporting to Department of Periodontics , A.B. Shetty Memorial Institute Of Dental Sciences , MangaloreSample size of 60 subjects are taken and divided into 2 groups of 30 eachGroup 1: Control group: 30 subjects with healthy periodontal conditionsGroup 2: Study group: 30 subjects with clinically diagnosed periodontitisClinical attachment loss \u22655mm measured using Williams periodontal probeBleeding on probingControls who are periodontally healthyPatients who had not undergone any periodontal treatment for atleast 6 months prior to samplingAll measurements and samples are taken before starting any periodontal therapySubjects who require antibiotic or anti inflammatory drug therapyHistory of any systemic diseaseSubjects who are pregnant and pre eclampticSubjects with a history of smoking and tobacco consumptionSubjects with vitamin supplementsSubjects who regularly use mouth washes.The total antioxidant capacity of clinical samples is measured using spectrophotometric quantitation through formation of phosphomolybdenum complex.Venous blood samples collected are centrifuged at 3000 rpm for 15 minutes and the supernatant serum is collected.An aliquot of 0.1.ml of sample solution containing a reducing species was combined in an eppendrof tube with 1ml of reagent solution .The tubes are capped and incubated in a thermal block at 95 degree centigrade for 90 minutes. After the samples are cooled to room temperature, the absorbance of aqueous solution of each was measured at 695nm against a blank.A study was conducted in Department of Periodontics, A. B. Shetty Memorial Institute of Dental Sciences, Mangalore to evaluate and compare Total Antioxidant Capacity in serum in chronic periodontitis patients and healthy subjects with a sample size of 60 each subdivided into case and control groups of 30 each.The group statistics showed a mean of 28.5052 for cases and 50.5955 for controls in serum . The stuThe present study was conducted in the department of Periodontics, A.B. Shetty Memorial Institute Of Dental Sciences, Mangalore to evaluate the total antioxidant capacity in serum in chronic periodontitis patients and healthy subjects with a sample size of 60 subdivided into case and control of 30 each showed statistically significant difference between case and control groups.Reactive oxygen species are associated with pathogenesis of variety of inflammatory diseases and have a role (direct or indirect) in tissue damage.Periodontal disease occurs in predisposed individuals with an aberrant inflammatory and immune response to microbial plaque. NeutrophChronic inflammatory conditions are associated with increased oxidative stress with phagocytes [particularly neutrophils] being implicated in disease pathogenesis because of generation of oxidative burst during phagocytosis and killing.Plaque bacteria and their products are source of factors that could stimulate neutrophils infiltrating the periodontal tissues. EnhancedDiseased sited will be associated with increased levels of a variety of cytokines and chemokines produced by inflammatory cells and normal resident cell population with in periodontal tissues.A variety of pro inflammatory cytokines , growth factors and lipopolysaccharides have a priming effect on human neutrophil oxidative burst.Although all cells produce ROS during normal physiological functions it is phMajority of tissue destruction in periodontitis is considered to be the result of an aberrant inflammatory / immune response to microbial plaque and involve prolonged release of ROS and neutrophil enzymes.ROS generation in periodontal disease causes bone resorption, degrade connective tissue, increases matrix metallo proteinases activity causing an imbalance.Traditionally ROS production by phagocytes has been associated with the defense of body to infection as they are essential for efficient killing of microbes.By contrast, ROS generation at high levels can cause oxidative stress with in tissues and result in direct damage to cells and extracellular matrix. ProductsNuclear factor kb and activator protein, the two redox sensitive transcription factors are of potential importance in the pathogenesis of periodontal disease.They can be activated by a variety of stimuli including bacterial products, viral proteins, cytokines, growth factors, oxidative stress. After acIn our study the results showed that the total antioxidant capacity in serum in periodontitis patients was significantly lower when compared to healthy subjects . The reset al. [2004] studied the total antioxidant capacity of serum in periodontitis and control subjects and found higher serum total antioxidant capacity for healthy controls than periodontitis cases.[Broke is cases.et al. [2004] investigated total antioxidant capacity of serum and concluded that the total antioxidant capacity in periodontitis was lower than in health and suggested a negative correlation between total antioxidant capacity and periodontal parameters.[Pavlica rameters.et al. [1997] studied serum samples in periodontitis and control groups and concluded that prevalence of periodontitis was positively associated with decreased serum antioxidant capacity.[Chapple capacity.Oxidative stress lies at the heart of periodontal tissue damage that results from host microbial interactions, eitheras a direct result of excessive ROS activityantioxidant deficiencyactivation of transcription factors and the creation of pro inflammatory statewhile a myriad of possible mechanisms leading to periodontal destruction exist, the influence of free radicals and antioxidants cannot be overlooked undoubtedly.Several avenues of enquiry now exist for the development of antioxidant based approaches to periodontal therapy which includes traditional routes of increasing the antioxidant capacity of periodontal tissues and newer routes based on modulation of transcription factorsThis array of pathways provides opportunities to develop novel antioxidant therapies that target the free radicals and which function not only as antioxidants in the traditional sense but also as powerful anti inflammatory agents."} +{"text": "Lancet346: 796\u2013798). In mice infected with the mouse mammary tumour virus (MMTV), immunosuppression also reduces the incidence of mammary tumours. DNA with 95% identity to MMTV is detected in 40% of human breast tumours Cancer Res55: 5173\u20135179). These findings led us to ask whether the incidence of HBC could be correlated with the natural ranges of different species of wild mice. We found that the highest incidence of HBC worldwide occurs in lands where Mus domesticus is thse resident native or introduced species of house mouse. Given the similar responses of humans and mice to immunosuppression, the near identity between human and mouse MTV DNA sequences, and the close association between HBC incidence and mouse ranges, we propose that humans acquire MMTV from mice. This zoonotic theory for a mouse-viral cause of HBC allows testable predictions and has potential importance in prevention. \u00a9 2000 Cancer Research CampaignIncidence of human breast cancer (HBC) varies geographically, but to date no environmental factor has explained this variation. Previously, we reported a 44% reduction in the incidence of breast cancer in women fully immunosuppressed following organ transplantation"} +{"text": "A significant positive correlation was found between AI and the change in tumour oxygenation following radiotherapy . The lack of correlation between apoptosis and hypoxia may occur because the Eppendorf measures both acute and chronic hypoxia, and the relative ability of acute hypoxia to induce apoptosis is unknown. These results indicate that cell death via apoptosis may be a mechanism of tumour reoxygenation during radiotherapy. \u00a9 2000 Cancer Research CampaignA relationship between hypoxia and apoptosis has been identified in vitro and in experimental tumours. The aim of this study was to investigate the relationship between apoptosis, hypoxia and the change in oxygenation during radiotherapy in human squamous cell carcinoma of the cervix. Forty-two patients with locally advanced disease underwent pretreatment evaluation of tumour oxygenation using an Eppendorf computerized microneedle electrode. Twenty-two of these patients also had a second evaluation of tumour oxygenation after receiving 40\u201345 Gy external beam radiotherapy. Paraffin-embedded histological sections were obtained from random pretreatment biopsies for all 42 patients. Apoptotic index (AI) was quantified by morphology on TUNEL stained sections. No correlation was found between pretreatment measures of AI and either the median pO"} +{"text": "Fifteen patients undergoing surgery for Stage IIb malignant melanoma were randomly allocated either to a group who received a vaccine of BCG mixed with irradiated autologous melanoma cells, or a control group who received no further treatment. All patients were monitored sequentially for immunological competence and tumour-directed immunity, using a wide range of techniques, and the results were compared retrospectively with their clinical course. Three months after surgery, there was a trend towards inhibition of PHA-induced lymphocyte transformation by autologous serum in patients who developed recurrent tumour within 12 months after treatment. Serum from patients who remained tumour-free for 12 months did not inhibit stimulation of autologous lymphocytes by PHA. Apart from this test, no other immunological parameters correlated either with clinical course or with the type of treatment received."} +{"text": "In vitro studies of the cell cultures, during and after histone treatment, suggested that cellular \u201ctransformation\u201d rather than selection was effected by the crude histone preparation. Cells from the primary tumours of both control and test groups appeared morphologically identical but after sub-culture in vitro they retained their respective growth characteristics on reinoculation.Neonatal hamster kidney cells (BHK21/C13), challenged in monolayer culture for three days with crude rat liver histone, have been shown to exhibit increased malignant characteristics when injected subcutaneously into hamsters. In contrast to the controls, the challenged cells produced tumours which invaded either the epidermis or the body wall of their hosts and frequently caused extensive visceral metastases."} +{"text": "Functional medical imaging promises powerful tools for thevisualization and elucidation of important disease-causingbiological processes in living tissue. Recent research aims todissect the distribution or expression of multiple biomarkersassociated with disease progression or response, where the signalsoften represent a composite of more than one distinct sourceindependent of spatial resolution. Formulating the task as a blindsource separation or composite signal factorization problem, wereport here a statistically principled method for modeling andreconstruction of mixed functional or molecular patterns. Thecomputational algorithm is based on a latent variable model whoseparameters are estimated using clustered component analysis. Wedemonstrate the principle and performance of the approaches on thebreast cancer data sets acquired by dynamic contrast-enhancedmagnetic resonance imaging."} +{"text": "August and Wistar rat lymph node cells were found to respond well to PHA stimulation and in mixed lymphocyte culture, as determined by an increased incorporation of 3H-thymidine. August rat lymph node cells were also stimulated by incubation with irradiated syngeneic tumour cells. Allogeneic Wistar rat lymph node cells produced a larger response to the August tumour cells. The response of syngeneic and allogeneic lymph node cells was reduced by pretreating the tumour cells with a Wistar anti-tumour serum. Pretreating the tumour cells with sera from normal or tumour bearing rats also reduced the response of syngeneic lymph node cells but did not reduce the response of allogeneic lymph node cells."} +{"text": "Adaptive divergence between populations in the face of strong selection on key traits can lead to morphological divergence between populations without concomitant divergence in neutral DNA. Thus, the practice of identifying genetically distinct populations based on divergence in neutral DNA may lead to a taxonomy that ignores evolutionarily important, rapidly evolving, locally-adapted populations. Providing evidence for a genetic basis of morphological divergence between rapidly evolving populations that lack divergence in selectively neutral DNA will not only inform conservation efforts but also provide insight into the mechanisms of the early processes of speciation. The coastal plain swamp sparrow, a recent colonist of tidal marsh habitat, differs from conspecific populations in a variety of phenotypic traits yet remains undifferentiated in neutral DNA.Here we use an experimental approach to demonstrate that phenotypic divergence between ecologically separated populations of swamp sparrows is the result of local adaptation despite the lack of divergence in neutral DNA. We find that morphological (bill size and plumage coloration) and life history (reproductive effort) differences observed between wild populations were maintained in laboratory raised individuals suggesting genetic divergence of fitness related traits.Our results support the hypothesis that phenotypic divergence in swamps sparrows is the result of genetic differentiation, and demonstrate that adaptive traits have evolved more rapidly than neutral DNA in these ecologically divergent populations that may be in the early stages of speciation. Thus, identifying evolutionarily important populations based on divergence in selectively neutral DNA could miss an important level of biodiversity and mislead conservation efforts. Understanding how populations adapt to local ecological conditions is not only a central theme of evolutionary biology Melospiza georgiana) is an adaptive or plastic response to divergent selection. By raising individuals from different environments in a common laboratory environment (common garden) we can differentiate between adaptive and plastic responses to divergent environments. If phenotypic divergence is maintained in experimental populations, then we can conclude that differences are due to underlying genetic divergence.An increasing number of studies, however, report an absence of differentiation between populations when using molecular genetic markers in taxa that exhibit significant morphological divergence sufficient to be classified as subspecies or even species M. g. nigrescens, coastal) are recent colonists of tidal marshes of the mid-Atlantic estuaries and have larger bills and have both grayer and blacker plumage than populations found in inland fresh water marshes Swamp sparrows provide an excellent system in which to test the hypothesis that geographic variation results from genetically-based adaptation to a recent ecological shift from inland freshwater marshes to tidal salt marshes. North American tidal marshes are primarily post-glacial geologic features characterized by tidal flooding, high salinity, and a biotic community specialized for these conditions Despite differences in morphology and life history, there is no divergence between inland and coastal populations in a variety of molecular genetic markers that are presumably selectively neutral; allozymes Melospiza georgiana) is an adaptive or plastic response to divergent selection. By raising individuals from different environments in a common laboratory environment (common garden) we can differentiate between adaptive and plastic responses to divergent environments. If phenotypic divergence is maintained in experimental populations, then we can conclude that differences are due to underlying genetic divergence.Although some success has been reported in searching for candidate genes whose expression effects adaptive traits in birds such as bill morphology As observed in wild populations, we found that experimental adults from coastal populations had significantly larger bills than experimental adults from inland populations in plumage coloration. Coastal adults had significantly more black plumage (non-breeding) on the head, back and eye line , and flaPrevious studies have found that 100% of specimens collected in the wild could be correctly assigned to subspecies using a Discriminant Function Analysis (DFA) based on bill size and plumage characteristics The timing of molt was also significantly different between experimental populations , fig. 1.This study provides evidence for a genetic basis to explain morphological and behavioral differences between inland and coastal populations of swamp sparrows consistent with a local adaptation hypothesis despite the lack of divergence in neutral genetic markers. While maternal effects cannot be completely ruled out, genetic explanations for differences between populations are nonetheless supported and a plastic response to the environment is ruled out. We do not have evidence that any of these traits confer a direct fitness advantage to swamp sparrows. However, strong correlation between environment and suites of traits in closely related species is evidence of similar responses to selection Delayed molt, which also has a genetic basis, appears to be an adaptation to allow coastal plain swamp sparrows to undertake more nesting attempts in a longer breeding season. The extended breeding in tidal marsh sparrows is hypothesized to mitigate the observed high nest failure and decrease in clutch size in tidal marsh sparrows, which may also be a response to higher levels of predation Lack of detectable genetic divergence in light of evolutionary divergence in swamp sparrows suggests that selection is likely to be strong in tidal marsh populations with adaptation occurring relatively quickly and possibly in the presence of gene flow The results of this study call into question the expectation of reciprocal monophyly of selectively neutral DNA to identify locally adapted subspecies because the stochastic processes responsible for these patterns do not account for the more rapid diversification in genes under selection. There are a growing number of examples of morphological divergence between subspecies in which researchers have been unable to detect divergence in a variety of selectively neutral loci Irrespective of the specific adaptive significance of morphological and behavioral divergence between populations of swamp sparrows, differences in bill size and plumage as well as the timing of reproduction and molt in experimental groups provide evidence of divergence that is the result of local adaptation to different environments rather than to phenotypic plasticity. Because adaptive differences occur in the absence of detectable divergence in neutral markers, we hypothesize that selection on these traits in swamp sparrows is sufficiently strong to override recent divergence and/or ongoing gene flow from inland populations. These results are strongly suggestive of rapid divergence of coastal plain populations with strong selection that is consistent with incipient ecological speciation We located the nests of breeding swamp sparrows in Woodland Beach Wildlife Area, Delaware and The Glades, Garrett County Maryland (inland population). Nests were monitored until hatching and we collected nestlings when they reached 4 days of age. We collected 17 nestlings (5 nests) from MD and 17 nestlings (6 nests) from DE. We transported nestlings to indoor animal care facilities at the Smithsonian National Zoological Park, Washington, DC where we hand reared them under identical conditions on 12D:12L photoperiod. Nestlings fledged at approximately 10 days and were transferred into group cages. Once nestlings reached independence at approximately 18 days, they were transferred into individual cages (18\u2033L \u00d79\u2033D \u00d710 \u00bd\u2033H) where they remained into adulthood on natural photoperiod cycles. All birds were sexed by genetic assignment ad libitum and hand feeding diet was reduced to once every 3 hours. At, day 24 hand feeding diet ceased. Fresh food diet was provided ad libitum along with adult diet (see below) for one week and then fresh food was reduced gradually over the course of the three weeks until birds were on adult diet by approximately day 60. Fresh food diet was a combination of soaked seed, fresh peas, tofu, and egg food. This diet provided birds with a variety of items to choose from while they were becoming independent. Adult diet was ad libitum dry seed mixture, 6\u20138 mealworms every other day and egg food with shell and multi-vitamin once per week. They were provided with grit that contains a calcium supplement.Nestling diet was a mixture of raw lean ground beef, whole grain baby cereal, raw wheat germ, hard boiled egg, carrot, calcium supplement, iron supplement, multi-vitamin supplement, and powdered milk. Nestlings were hand-fed once every half hour until day 10, then once every hour. At day 18, fresh food (see below) was introduced 575 nm\u2013700 nm). Digital photographs were taken under identical lighting conditions in the laboratory with a Panasonic 35 mm camera. Digital photographs were analyzed using the masking tool of Corel Paint photo editing software to estimate the amount of black coloration in the back and head. Sections of digital photographs were sampled from the back and head and the masking tool was used to estimate the number of pixels that were black for each section. The amount of black was estimated as the number of black pixels/total number of pixels of the section. Two independent observers performed photo analyses with highly repeatable results . We estimated the percentage of the eye line that contained chestnut feathers.For spectrometeric analyses, we analyzed a patch of 8\u201310 feathers pulled from the flank and affixed to a black background in such a way as to mimic natural arrangement of feathers. We recorded spectral data with an Ocean Optics S2000 spectrometer using a micron fiber-optic probe at a 90\u00b0 angle to the feather surface. Ambient light was excluded with a cylindrical metal sheath attached to the probe tip with the probe held at fixed distance of 6 mm from the feather surface. The reading area was 2-mm diameter of light illuminated with a tungsten-halogen bulb (visible light source). We generated reflectance data relative to a white standard . Using spectra acquisition software, OOIBase, we recorded 5 spectra sequentially and averaged the spectra to reduce noise. This process was repeated five times by lifting the probe and replacing it at random locations on the feather sample. An average was taken from the five scans. We quantify flank coloration as the slope of the line of the reflectance spectra in the orange-red portion of the each spectrum or an arcsine transformation (percent chestnut in eye-line), or compared with Mann-Whitney U test (crown class). We used a general linear mixed model in SAS"} +{"text": "Ventilator-associated pneumonia (VAP) is pneumonia in patients who have been on mechanical ventilation for > or =48 hours. VAP is most accurately diagnosed by quantitative culture and microscopy examination of lower respiratory tract secretions, which are best obtained by bronchoscopically directed techniques such as the protected specimen brush and bronchoalveolar lavage. These techniques have acceptable repeatability, and interpretation of results is unaffected by antibiotics administered concurrently for infection at extrapulmonary sites as long as antimicrobial therapy has not been changed for <72 hours before bronchoscopy."} +{"text": "The initial management of posterior urethral injuries is controversial. Options of management include immediate surgical realignment, early realignment using minimally invasive techniques or simple suprapubic catheter (SPC) placement followed by delayed urethroplasty. The latter method has been preferred by most urologists but the last couple of decades have seen increasing reports of early urethral realignment which have provided better if not similar results as SPC placement. In this article a detailed analysis of studies involving primary realignment has been presented to reinforce the argument in favor of this approach. Mouraviev et al.,[Immediate urethral realignment has been proposed to result in a much lower rate of subsequent stricture formation. Elliot and Barret reportedet al., comparedv et al., comparedet al.,[et al.,[Since the early 1990s, several investigators\u201314 have ,[et al., comparedet al.,[et al.,[Porter et al., used mag,[et al., used mulThese studies strengthen the argument for immediate or early urethral realignment.Initial placement of a SPC is associated with a prolonged period of catheter drainage . Almost all patients managed by this technique will develop a pelvic fracture urethral distraction defect which requires extensive urethroplasty, the facility for which may be limited only to centers of excellence. Also there is a 10-12% failure rate associated with urethroplasty even in these centers.In posterior urethral injuries due to pelvic fracture, impotence and incontinence are a result of the primary injury and not due to the type of initial management. Initial SPC placement is a safe technique but has an almost 100% incidence of stricture formation requiring extensive urethroplasty subsequently. With this approach patients have to wait three to six months for delayed repair and have to bear with the complications of long-term SPC placement. This approach may be used by surgeons inexperienced in primary alignment or where such facilities are unavailable.In the stable patient with posterior urethral injury, primary urethral realignment, either surgical or endoscopic, should be the treatment of choice since it results in a less than 50% incidence of stricture and an acceptable rate of impotence and incontinence Tables and 2. T"} +{"text": "The fluorescent redox probe hydroethidine was accumulated and metabolised about five times faster in aerobic than in hypoxic mammalian cells. Patterns of fluorescence in Chinese hamster V79 spheroids also indicated that internal hypoxic cells were less able to metabolise the drug; toxicity was observed in cells only when cell fluorescence exceeded about 500 times background. In medium equilibrated with air or nitrogen, cell accumulation of the stain was rapid, and began to plateau after 30 min; loss of ethidium was initially rapid, with a slower component after 30 min, and transfer of the metabolite ethidium between stained and unstained cells was observed after 2 h co-incubation. Sorting cells from irradiated spheroids on the basis of ethidium fluorescence provided good separation of aerobic radiosensitive and hypoxic radioresistant cells, although separation using the perfusion probe, Hoechst 33342, was superior. Similar experiments with the murine SCCVII squamous cell carcinoma suggested that hydroethidine might be a useful indirect stain for locating hypoxic cells in experimental tumours when used in combination with a perfusion probe such as Hoechst 33342."} +{"text": "When the clonogenic survival of mouse haemopoietic stem cells (CFU-S) and leukaemia L1210 cells growth as ascites tumours are compared after being heated in vitro and assayed in vivo by spleen-colony assay, there is no significant difference in the terminal slopes of the survival curves. The shoulders of the survival curves differ, but this may be explained by differences in cell kinetics. By contrast, L1210 leukaemic marrow cells are considerably more susceptible to the lethal effects of hyperthermia (43 degrees C) than either normal marrow stem cells or L1210 leukaemic cells grown as ascites tumours. Moreover, the killing of L1210 ascites cells by hyperthermia can be enhanced by heating L1210 ascites cells with an equal number of normal marrow cells, or as upernatant removed from heated marrow cells. Most cells in lukaemic marrow are normal, and it is postulated that the increased thermal sensitivity of L1210 cells in leukaemic marrow is caused by diffusible factors released by heating normal marrow cells."} +{"text": "Well differentiated liposarcoma is a low grade tumour, with no metastatic potential unlessdedifferentiation supervenes. When superficial, it recurs locally only occasionally after marginal excision. We present apatient in whom bilateral childhood retinoblastoma was followed by later development of massive confluent areas of lowgrade liposarcoma and lipomatous tissue affecting the upper extremities and trunk. We discuss the role of mutations in theretinoblastoma gene (RB1) in linking these conditions and demonstrate the surgical management of an extremely unusualand challenging case."} +{"text": "The failure to observe a significant effect of ploidy on radiation sensitivity is due to the complex and multifactorial basis of radiation sensitivity. When we determined the relationship between survival and radiation-induced chromosome aberration frequency, a measure independent of most other modifiers of sensitivity, we observed a direct relationship between ploidy and mean lethal aberration frequency. The mean lethal frequency of aberrations increased from about 1 for diploid cells to about 2 for tetraploid cells. The mean lethal frequency of aberrations was independent of DNA repair variations. These observations demonstrate that changes in DNA ploidy are an important contributor to radiation sensitivity variations in human tumour cell lines. Therefore, any battery of predictive assays should include DNA ploidy measurements. \u00a9 1999 Cancer Research CampaignThe contribution of DNA ploidy to radiation sensitivity was investigated in a group of eight human tumour cell lines. As previous studies suggest, while more aneuploid tumours tend to be more radioresistant, there is no significant relationship between ploidy and radiation sensitivity (SF"} +{"text": "Any link between pancreatic carcinoma and chronic pancreatitis could reflect the malignant potential of a chronic inflammatory process. Four patients with ductal adenocarcinomas had a long history of pancreatic pain (median duration 5 years) and showed clearcut evidence of chronic pancreatitis \u201cdownstream\u201d of the tumour. Four were alcoholics and two heavy smokers. These four cases arose within a surgical series of approximately 250 patients with chronic pancreatitis, giving an incidence of 1.6 per cent. The incidence and anatomical distribution of carcinoma and chronic pancreatitis could possibly be consistent with a casual relationship."} +{"text": "Pulmonary growths produced by intravenous injection of methycholanthrene-induced sarcoma cells were controlled or suppressed by specific immunostimulation with BCG-sarcoma cell inocula at subcutaneous sites or by intravenous treatment with BCG alone. The response was dependent upon the immunogenicity of the treated tumour so that in comparison, intravenous injection of BCG enhanced rather than suppressed pulmonary growths of transferred cells of several weakly immunogenic tumours including a spontaneously arising sarcoma and an epithelioma as well as a carcinogen-induced mammary carcinoma."} +{"text": "In this prospective study, a quantitative determination of histamine and tryptase in nasal secretions after nasal phosphate buffered saline (PBS) and allergen challenge was performed in 18 atopic patients who were compared with ten non-allergic healthy volunteers. The aim of the study was to determine the normal and pathological concentrations of these important mediators in nasal secretions. The second objective was to test the relevance of these two mast cell secreted mediators after nasal challenge. Results showed that the concentrations of tryptase in almost all samples were under the minimal detection limit (< 0.5 \u03bcU/g) and only a sigrtificant increase of tryptase occurred immediately after nasal allergen challenge in the patient group. Histamine concentration significantly increased after every nasal PBS challenge in the control group, as well as in the patient group after both PBS and allergen challenge. On the other hand, a rapid onset of sneezing and increase in nasal airway resistance was experienced only in the patient group after nasal allergen challenge, but did not occur after PBS challenge even though the histamine concentrations significantly increased in both groups. This study suggests that tryptase is a more preferable marker than histamine in quantitative monitoring of mast cell activation especially during the early phase nasal allergic reaction."} +{"text": "The role of operation, particularly pancreaticojejunostomy, in the treatment of abdominal pain fromchronic pancreatitis is controversial, but relief of pancreatic duct obstruction may decrease the rate ofpancreatic organ failure. Our results over 6 years in 13 carefully selected patients suggest that pancreaticdrainage does relieve pain but is less effective in preventing pancreatic exocrine failure. Pain was theindication for operation in all patients."} +{"text": "Our results suggest that gutderived smooth muscle cells may represent an important source of proinflammatory prostanoids but not leukotrienes during inflammatory states of the intestine. The inhibition of prostanoid activity by thiourea may be mediated by suppression of cyclooxygenase activity in this cell line.The contribution of smooth muscle cells as a potential source of eicosanoid production during inflammatory states remains to be elucidated. We investigated the effect of trinitrobenzene sulfonic acid (TNB), a known pro-inflammatory agent, on jejunal smooth muscle cell eicosanoid production. Human gut-derived smooth muscle cells (HISM) were incubated with TNB for 1 hour. Additionally, some cells were preincubated with either dimethylthiourea, or indomethacin for 1 hour before exposure to identical concentrations of TNB. Incubation with TNB led to significant increases in PGE"} +{"text": "Cardiac computed tomography (CT) has been a hot topic for yearsbecause of the clinical importance of cardiacdiseases and the rapid evolution of CT systems. In this paper, wepropose a novel strategy for controlled cardiac CT that mayeffectively reduce image artifacts due to cardiac and respiratorymotions. Our approach is radically different from existing onesand is based on controlling the X-ray source rotation velocity andpowering status in reference to the cardiac motion. Wetheoretically show that by such a control-based intervention thedata acquisition process can be optimized for cardiac CT in thecases of periodic and quasiperiodic cardiac motions.Specifically, we formulate the corresponding coordination/controlschemes for either exact or approximate matches between the idealand actual source positions, and report representative simulationresults that support our analytic findings."} +{"text": "Seventeen patients with Hodgkin's disease who had a staging laparotomy (SL) within 2 months of the completion of initial chemotherapy are presented. Only 1 patient had a positive laparotomy. Postchemotherapy SL allows any residual active disease to be assessed, but the incidence of positive finding may be small, and such findings are unlikely to alter subsequent management. SL following chemotherapy is therefore not recommended either for patients in clinical remission or for patients with evidence of relapsed disease."} +{"text": "Levamisole has been examined for its ability to control local growth and pulmonary metastases of transplanted rat tumours. The compound did not suppress subcutaneous growth of 3-methylcholanthrene induced sarcomata when administered systemically in a variety of regimens, or when injected in admixture with tumour cells. In addition, levamisole treatment failed to suppress pulmonary growth of intravenously transferred sarcoma cells or spontaneous pulmonary metastases appearing after surgical removal of a transplanted epithelioma."} +{"text": "Monolayer cultures have been established from a poorly differentiated carcinoma of the lung. Homogeneous cell growth and morphology have been maintained for over 18 months through more than 80 subculture passages, and the cells have been found to produce both immunoreactive calcitonin and an immunoreactive carcinoembryonic antigen-like material."} +{"text": "In vitro studies of both techniques havedemonstrated an effective, broad spectrum antibiotic effect including most silver-resistant strains.Over 100 cases of recalcitrant osteomyelitis have been treated with an overall success rate ofapproximately 65% and no evidence of argyria.A study of the use of free silver ions as an antibacterial and antifungal agent administered toinfected local wounds has been conducted over the past two decades. A variety of iontophoretictechniques has been employed utilizing either pure silver wires or several types of silvered nylonfabrics as anodes in a direct current electrical circuit. A new type of silver nylon has recently beenevaluated for the same use without iontophoretic current."} +{"text": "The quality of immediate repair of common bile duct injuries with or without tissue loss occurring duringelective cholecystectomy is crucial and maybe the sole factor behind future stricture formation with itsconsiderable morbidity and mortality. Successful repair of iatrogenic common bile duct injuries has beenachieved by immediate saphenous vein grafts in two patients with cystic duct avulsion, in one patientwhose duct was split by a balloon catheter, and in one patient where a segment of the duct was resected.Follow-up for 5 years demonstrated that the grafting remained sound and produced no complications.Consequently, the immediate repair of iatrogenic bile duct injuries using vein grafts deserves consideration."} +{"text": "Eleven patients with disseminated midgut carcinoid tumour disease were subjected to hepatic artery embolisation. In six patients, lymphocytosis with a predominance of NK cells occurred and the cytotoxic activity of isolated lymphocytes increased. A relation between NK cell accumulation and subsequent radiological and biochemical response was observed, and it is suggested that anti-tumour mechanisms other than ischaemia may contribute to the therapeutic response in these patients."} +{"text": "Intracardiac echocardiography (ICE) is a useful imaging modality that is now being used more widely to assist in the percutaneous closure of atrial septal defects (ASD) and patent foramen ovales (PFO).A 42 year old lady with a history of transient ischaemic attacks and migraine underwent percutaneous closure of an ASD. Intraprocedural ICE demonstrated a mammoth billowing multiperforated interatrial septal aneurysm in association with a secondum ASD.ICE provides excellent adjuvant imaging during percutaneous closure of intracardiac shunts, in this case demonstrating a 'mammoth' interatrial septal aneurysm. Intracardiac echocardiography (ICE) is a useful imaging modality that is now being used more widely to assist in the percutaneous closure of atrial septal defects (ASD) and patent foramen ovales (PFO).A 42 year old lady with a history of previous transient ischemic attacks and migraines was found to have a secundum ASD and an aneurysmal interatrial septum on contrast transthoracic echocardiography. She then went on to undergo percutaneous closure of the ASD, during which ICE demonstrated a dramatic giant multiperforated aneurysmal interatrial septum with a secundum ASD on Doppler flow imaging Figures , 2, 3.An aneurysmal interatrial septum is thought to be the harbinger of potential embolic thrombus and its presence increases the risk of a stroke significantly particularly in association with a PFO and is thus of clinical importance . AdjuvanThis case demonstrates a \"mammoth\" billowing multiperforated interatrial septal aneurysm in association with a secondum ASD and illustrated elegantly by ICE.The author(s) declare that they have no competing interests.All authors contributed to the preparation of this manuscript."} +{"text": "Dear Editor,et al.[We read with interest the article \u2018Optical coherence tomography (OCT) in a patient with chloroquine- induced maculopathy\u2019 by Korah et al.We must congratulate the authors for the excellent article but we would like to make a few points.Why was the patient started on chloroquine rather that hydroxychloroquine? It is well-documented that hydroxychloroquine has a lower incidence of eye complications compared to chloroquine.Was the patient regular about her follow-up schedules? What were the findings in the examination done just before the complications were noted? Did the patient have any corneal deposits? Corneal deposits though innocuous are the most commonly described ophthalmological findings following chloroquine therapy. Was a viExamination and perimetry reliably showed chloroquine toxicity and yet the patient was not told to stop chloroquine but the drug was changed to hydroxychloroquine. Hydroxychloroquine was stopped and methrotrexate started after four months when the patient complained of subjective symptoms. In our opinion chloroquine/hydroxychloroquine should have been stopped earlier.Any proposed screening test needs to detect retinal lesions at a stage where intervention can reverse the condition or prevent deterioration. If the cases of retinopathy detected by monitoring fail to respond to cessation of therapy, the monitoring does not have a useful role.In this case the optical coherence tomography (OCT) findings of significant thinning were seen in a patient who had well-documented changes on fundus examination and perimetry examination. It would be interesting to know if the thinning occurs before any other change can be noted on regular examination. If not, OCT has no role whatsoever.Multifocal ERG (mfERG) is a very sensitive test for detection of early retinal abnormalities under chloroquine/hydroxychloroquine therapy. Multifocal ERG can reliably detect retinal functional loss associated with chloroquine/hydroxychloroquine retinopathy. In some patients the mfERG showed reduced response amplitudes when other functional tests or morphologic examinations were conducted. In addition, follow-up studies demonstrated a decline of retinal function when using hydroxychloroquine and improvement of retinal function after discontinuation of treatment. In our o"} +{"text": "Breastfeeding-associated inflammatory breast diseases appear especially during the first twelve weeks postpartum and are the most common reason for early cessation of breastfeeding. It also becomes increasingly evident that these inflammatory mammary diseases are triggered or perpetuated in a large part by psychosocial stress. Immunological processes taking place during this cascade in the mammary gland and consequences for the breastfeed newborn are mostly yet unknown. This review summarizes insights from studies on modulation of cytokine levels in breast milk during inflammatory processes like milk stasis and mastitis systematically. It also gives an overview on possible pathological effects, which these cytokine changes in the breast milk might have on the newborn. The WHO suggests a six-monthperiod of breastfeeding to all breastfeeding mothers . EvidencThese breastfeeding-associatedinflammatory breast diseases appear especially during the first twelve weekspostpartum and are the most common reason for an early cessation ofbreastfeeding . ChangesImmune mediators such ascell subsets or cytokines involved in this perpetuating inflammation in themammary gland in humans are mostly unknown.In veterinary medicine, a number of scientists work on the subjectfocussing on bovine mastitis in experimental studies, since bovine mastitiscauses enormous economic damage in the dairy industry , and henIt is generally believedthat inflammatory breastfeeding-associated mammary diseases may be triggered oraggravated by psychosocial stress, as observed for example in veterinary medicine. Here, exposure to experimental stressors such as regrouping and relocation resulted into mastitis in animals, asdescribed for lactating ewes . In humaWhatare the possible pathophysiological mechanisms of stress-dependent breastdiseases during lactation? The increased secretion of catecholamines instressed mothers impairs the release and access of oxytocin to the mammarygland and the action of oxytocin on the secretory epithelium . The relStressfullevents may also cause immune supression in the mammary tissue. T-lymphocytes haveregulatory functions or act directly on foreign antigens because of producingcytokines. T-lymphocytes are strongly involved in the defense against bacterialinvasion during mastitis . These cAt birth the immunesystem of the neonate is primed towards a Th2 dominance. Within the first 2years of life, the immune system is activated, probably via childhoodinfections, leading to a naturally occurring shift from Th2 to Th1 immunity.Intestinal mucosa is also premature in the first two years. Thus higherconcentrations of proinflammatory Th-1-cytokines in the breastmilk maylead to local and systemic immunological effects of the newborn .Maternal stressperception is likely intimately linked to stress reactions of the newborn.Published data indicate that the offspring may develop somatic diseases , well deIf a major part of breast diseases, as proven in our own surveys, is caused by stress and atthe same time the prevalence of these diseases is relatively high ,a changThe aim of this systematic review was to show detectable changes ofcytokines in breast milk during inflammatory processes of mammary tissue likemastitis and possible pathological effects of these mediators on the newborncaused by breastfeeding.Search strategy 1: (\u201cmastitis\u201d [MeSH Terms] ORmastitis [Text Word]) AND AND(\u201ccytokines\u201d [MeSH Terms] OR cytokines [Text Word]); searchstrategy 2: (\u201ccytokines\u201d [MeSHTerms] OR cytokines [Text Word]) AND (\u201clactation\u201d [MeSH Terms]) OR(\u201cbreast feeding\u201d [TIAB] NOT Medline [SB]) OR \u201cbreastfeeding\u201d [MeSH Terms] OR LACTATION [Text Word]) AND . All articles published in German or English between 2002until 2007 were included. This periodwas established to get the most current results of this field of research.Veterinary and human studies, experimental and clinical surveys were analysed. An overview on the process of research andselection is shown in In order to identifypublished evidence addressing the topics (1) cytokines detectable ininflammatory processes like a mastitis in breast milk and (2) pathologicaleffects of cytokines in the breast milk on the newborn, literature databases were searched. The following search strategies were developedto identify the publications most sensitively. Searching according tothe above-described key word strategy yielded a total of 191 publications(titles or abstracts): after selection of the literature, 16 publicationsremained see , 10 of wResults from the firstsearch strategy are presented in Results from the second search strategy arepresented in An increase of cytokines in breast milk has been reported from differentstages of maternal lactation: on the one hand, maternal diseases duringpregnancy\u2014likepre-eclampsia or allergies\u2014can lead to arise in cytokines of breast milk . In addiWhat are possible pathophysiological mechanisms for the shown increaseof interleukines in breast milk during inflammatory processes? The proportionof T cells in mammary tissue normally declines during lactation and the numberof T cell subsets varies significantlyduring this period. The proportions of several cell populations are lower in milk than in blood following parturition, while theproportion of WC1+ cells is higher in milk . But an What kind of biological mechanism could be made responsible for that? Soon the one hand, a rise in cytokines of breast milk is useful to activate amechanism of maternal self-defence against infectious processes in theglandular tissue . On theThe review shows evidence of increased cytokinesin breast milk during inflammatory processes like mastitis and possiblepathological effects of these higher cytokine-concentrations on the newborn. Acorrelation between these consequences on state of health and special interleukinsin breast milk could not be detected in the current literature and should beinvestigated in further studies."} +{"text": "WR 1065, 2-[(minopropyl) amino] ethanethiol is an effective scavenger of free radicals. When present during irradiation it reduces cellular DNA damage as analysed by alkaline elution from filters. The same technique indicates that without irradiation, WR 1065 has no effect of DNA integrity. Using nucleoid analysis, where DNA damage is detected at the level of replicon clusters, WR 1065 distorts replicon supercoiling without breaking the DNA molecule. This confirmational change in nucleoid structure occurs with no detectable change in nucleoid protein content. It is proposed that perturbation of replicon supercoiling affects the process of normal DNA synthesis and strand break rejoining, allowing a longer time for the accurate repair of DNA damage."} +{"text": "Catheter ablation is increasingly used to treat patients with atrial fibrillation (AF). Ablation of ganglionic plexi is often performed to reduce vagal innervation and has been shown to confer a better long-term outcome in terms of AF recurrence. We report a case of a patient having AF ablation with a profound vagal response, suggesting ganglionic plexus ablation, who subsequently developed ventricular fibrillation after programmed ventricular stimulation. Reduced vagal modulation is known to predispose to ventricular arrhythmias and vagal denervation following AF ablation may predispose to ventricular arrhythmias and requires further study. Catheter ablation is increasingly used to treat patients with atrial fibrillation (AF) . During A 55-year old man with no past medical history was found to be in fast AF following a leg injury. He underwent successful external cardioversion following a normal trans-esophageal echocardiogram; the latter revealed no evidence of intra-cardiac thrombus, preserved biventricular systolic function and no significant valvular lesions. His CHADS2 score was 0 and he was commenced onto warfarin therapy following electrical cardioversion. The AF recurred 36 hours later and he was commenced on oral flecainide which reverted him back to sinus rhythm but his palpitation continued. Subsequent Holter monitoring revealed episodes of paroxysmal AF triggered by atrial premature beats (APBs), a narrow complex tachycardia and a regular broad complex tachycardia at a similar rate thought to represent a supraventricular tachycardia with aberrant conduction . As he rUnfortunately, he remained symptomatic with PAF and was unwilling to commence amiodarone given its long-term side effects and opted for percutaneous catheter ablation therapy. Baseline electrophysiology study revealed concentric decremental retrograde and antegrade conduction; dual AV node physiology was not demonstrated and tachycardia was not induced despite use of intravenous isoprenaline infusion, repeat curves and burst pacing. Given his documented PAF, pulmonary vein isolation (PVI) was performed using the Ensite NavX\u2122 electro-anatomic mapping system . Ablation was carried out at the ostia of all four pulmonary veins. During the ablation procedure the patient went into AF; ablation was carried out near areas of complex fractionated atrial electrograms around the left superior and inferior pulmonary veins which produced profound bradycardia . All fouDuring catheter ablation for AF, bradycardic responses are often seen, especially at and around the pulmonary veins. These have been regarded as 'vagal reflexes' and abolition of these reflexes during ablation are believed to represent vagal denervation . These pCardiac autonomic modulation is known to have a significant influence on initiation of ventricular arrhythmias and sudden cardiac death. We recently reported that vagal stimulation decreases susceptibility of the heart to VF, while sympathetic stimulation increases it . DisturbThe strategy of vagal denervation using ganglionic plexus ablation during AF ablation to improve long term success requires further study. Medium and long term follow up to exclude a predilection to ventricular arrhythmias is required to ensure no deleterious long term effects."} +{"text": "Here, we report the success of our method and demonstrate the feasibility of deciphering the putative metabolic products of uncharacterized PKS clusters found in newly sequenced genomes. Profile Hidden Markov Model analysis has revealed distinct sequence features that can distinguish modular PKS proteins from their iterative counterparts. For iterative PKS proteins, structural models of iterative ketosynthase (KS) domains have revealed novel correlations between the size of the polyketide products and volume of the active site pocket. Furthermore, we have identified key residues in the substrate binding pocket that control the number of chain extensions in iterative PKSs. For modular PKS proteins, we describe for the first time an automated method based on crucial intermolecular contacts that can distinguish the correct biosynthetic order of substrate channeling from a large number of non-cognate combinatorial possibilities. Taken together, our in silico analysis provides valuable clues for formulating rules for predicting polyketide products of iterative as well as modular PKS clusters. These results have promising potential for discovery of novel natural products by genome mining and rational design of novel natural products.Sequence data arising from an increasing number of partial and complete genome projects is revealing the presence of the polyketide synthase (PKS) family of genes not only in microbes and fungi but also in plants and other eukaryotes. PKSs are huge multifunctional megasynthases that use a variety of biosynthetic paradigms to generate enormously diverse arrays of polyketide products that posses several pharmaceutically important properties. The remarkable conservation of these gene clusters across organisms offers abundant scope for obtaining novel insights into PKS biosynthetic code by computational analysis. We have carried out a comprehensive in silico genome mining. These results also have interesting implications for rational design of novel natural products using a biosynthetic engineering approach.Polyketide synthases (PKSs) form a large family of multifunctional proteins involved in the biosynthesis of diverse classes of therapeutically important natural products. These enzymes biosynthesize natural products with enormous diversity in chemical structures by combinatorial use of a limited number of catalytic domains. Therefore, deciphering the rules for relating the amino acid sequence of these domains to the chemical structure of the polyketide product remains a major challenge. We have carried out bioinformatics analysis of a large number of PKS clusters with known metabolic products to correlate the chemical structures of these metabolites to the sequence and structural features of the PKS proteins. The remarkable conservation observed in the PKS sequences across organisms, combined with unique structural features in their active sites and contact surfaces, allowed us to formulate a comprehensive set of predictive rules for deciphering metabolic products of uncharacterized PKS clusters. Our work thus represents a major milestone in natural product research, demonstrating the feasibility of discovering novel metabolites by A number of recent theoretical studies have demonstrated the utility of in silico analysis in providing novel insights into the mechanistic details of polyketide biosynthesis as well as in identifying novel natural products by genome mining. Computational analysis of polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) proteins have provided valuable clues for development of knowledge-based methods for identification of catalytic domains in PKS Mycobacterium tuberculosisIt is well known that polyketide synthase (PKS) gene clusters can generate enormously diverse array of polyketide products by making use of various biosynthetic paradigms like, modular organization of sets of catalytic domains or iterative catalysis of condensation steps using single set of catalytic domains in silico identification of polyketide products of uncharacterized PKS clusters, the computational method should also take into consideration various different paradigms employed by PKS biosynthetic machinery in silico methods should be capable of predicting from sequence information, whether a given PKS cluster is iterative, the number of iterative chain condensation steps catalyzed by it and crucial amino acids which control the number of iterations.Thus, these studies have established that knowledge based computational approaches can play a powerful role in elucidation of novel secondary metabolite biosynthetic pathways. However, for cat) of chain transfer of otherwise disfavored substrates by as much as 100-fold et alIn contrast to type I iterative PKSs where a single multifunctional enzyme is involved in biosynthesis of the polyketide product, biosynthesis in type I modular PKS clusters often involve multiple ORFs, each containing several modules. Therefore, predicting the correct order of substrate channeling between various ORFs is crucial for deciphering the final metabolic product of a modular PKS cluster. Several lines of experimental evidence reveal that inter subunit interactions between C-terminal docking domain region of the upstream ORF and N-terminal docking domain region of the downstream ORF, play a crucial role in channeling of substrates from upstream domains to downstream domains in silico prediction of polyketide products. Is it possible to distinguish between modular and iterative PKS from their sequence alone? Can we predict the number of iterations a given iterative PKS protein would catalyze and identify crucial amino acid residues that control the number of iterations? Is it possible to predict the correct order of substrate channeling between various ORFs in a modular PKS cluster? We have carried out profile Hidden Markov Model (HMM) analysis of KS domains to identify signature profiles which can decipher whether a PKS protein is modular or iterative. Structural modeling of KS domains of iterative PKS proteins and analysis of their active site pockets have given novel insight into the structural features that dictate the number of iterations catalyzed by a PKS protein and crucial amino acids which control them. Similarly, comparative analysis of crucial inter polypeptide contacts between cognate and non-cognate pairs of ORFs based on the three dimensional structure of the docking domains have given novel clues for prediction of the correct order of substrate channeling.In this work, we have carried out a detailed comparative analysis of the experimentally characterized modular and iterative PKS clusters with known polyketide products to address following major questions relating to KS domains are the most conserved among various catalytic PKS domains and are responsible of catalysis of the chain condensation step. We have analyzed them in detail to identify class specific conserved patterns which distinguish modular and iterative PKS systems. For KS domains, the total dataset comprised of 217 pure modular KS domains, 82 pure iterative domains, 19 enediyne, 43 trans-type and 34 KS domains from hybrid NRPS-PKS clusters. Apart from the sequences of 20 experimentally characterized bacterial type I modular clusters included in our earlier analysis E. coli KAS-II protein were used as the templates for modeling these iterative KS domains. Since 1B3N was a ligand bound structure belong to the MSAS type PKSs that perform three iterations. Intermediate sized cavities (\u223c800\u00c53) belong to the napthopyrone (NAP) like PKSs that iterate from five to eight times. The largest cavities, 1780\u00c53, were observed for the T-Toxin models that perform 20 iterations. 3) and surround the ligand analogue cerulenin. A comparison of the modeled structures with the template FAS KS structure revealed that in case of MSAS and NAP, the backbones of the models had not altered significantly during modeling Click here for additional data file.Figure S2Superposition of backbones of iterative KS domain models on structural templates(0.24 MB DOC)Click here for additional data file.Figure S3Genomic order vs biosynthetic order(0.09 MB DOC)Click here for additional data file.Figure S4The four helix bundle structure of DEBS docking domain(0.21 MB DOC)Click here for additional data file.Table S1Scoring scheme for docking domain interactions(0.07 MB DOC)Click here for additional data file."} +{"text": "Perinephric lymphangioma is rare disorder that may be confused with various forms of renal cystic diseases and urinomas. In this disorder a developmental malformation results in failure of developing lymphatic tissue to establish normal communication with the rest of lymphatic system. Once there is restricted drainage of lymphatic fluid the lymphatic channels dilate to form cystic masses that may be unilocular or multilocular and may be seen unilaterally or bilaterally .This condition presents with various signs and symptoms or can be just an incidental finding which in presence of misleading clinical history may be confused with other diseases. CT scan with delayed cuts and USG guided aspiration with biochemical analysis of fluid will help us in arriving to final diagnosis. Perinephric lymphangioma is rare disorder that may be confused with various forms of renal cystic diseases and urinomas. In this disorder a developmental malformation results in failure of developing lymphatic tissue to establish normal communication with the rest of lymphatic system. Once there is restricted drainage of lymphatic fluid the lymphatic channels dilate to form cystic masses that may be unilocular or multilocular and may be seen unilaterally or bilaterally .This condition presents with various signs and symptoms or can be just an incidental finding which in presence of misleading clinical history may be confused with other diseases. CT scan with delayed cuts and USG guided aspiration with biochemical analysis of fluid will help us in arriving to final diagnosis.A 50-year-old postmenopausal female with multiple episodes of urine retention and uterine prolapse consulted the department of gynecology. On physical examination, she was well built, apyrexic with normal blood pressure and pulse of 78 beats per minute. Her systemic examination was unremarkable. Local genital examination revealed grade-4 uterine prolapse with decubitus cervical ulcer, grade-2 rectocele, and grade-3 cystocele. Before the patient was planned for vaginal hysterectomy with colpoperineorrhaphy, a preoperative ultrasonographic evaluation was performed at the department of radiodiagnosis. The ultrasonography (done on Siemens Adara Sonoline ultrasound machine) revealed bilateral septated perinephric fluid collections with otherwise normal study. With ultrasonographic features and clinical history of urine retention, a provisional diagnosis of perinephric urinoma was made; however for confirmation, intraveous urography and a contrast enhanced CT scan of the abdomen were performed. The result of intravenous urography was unremarkable. A subsequent CT scan (Siemen's sensation 64 slice CT) showed bilateral perinephric fluid collection of attenuation 5\u201310 HU . No otheRenal lymphangiomatosis is an extremely rare developmental disorder of lymphatic system surrounding kidneys. They can occur in any location where lymphatics are normally present. The frequency of lymphangiomas are 75% in head and neck, 20% in axillary region, and 5% at other less common sites. Retroper3511318Perinephric lymphangiomas can present with a number of nonspecific symptoms, and high blood pressure however can present as an incidental finding for which nothing needs to be done till patient remains asymptomatic. The close differential diagnosis is perinephric urinoma which can be ruled out with a contrast enhanced CT scan with delayed cuts, however, biochemical examination of aspirated fluid is also vital in reaching a final diagnosis."} +{"text": "A stathmokinetic method has been used to determine the cell cycle parameters, particularly the potential tumour doubling time, of a murine fat pad sarcoma. Additional information has been obtained by determining the percentage of labelled mitoses (PLM). A technique which simultaneously demonstrates autoradiographically labelled S phase nuclei and histochemically localized acid phosphatase activity has also been used at light microscope level to compare these parameters: acid phosphatase activity was demonstrated in tumour cells and macrophages. Single cell deletion by apoptosis has been investigated as distinct from necrosis. Condensed, dying apoptotic cells, have been found in proliferative areas of tumour that are not under physiological stress. The analysis of apoptosis indicated a previously unsuspected variation in apoptotic activity with tumour weight. Cell death by apoptosis initially rose as the tumour grew, but after the tumour reached a threshold weight it declined dramatically, and finally remained stable. This may reflect an initial attempt at autoregulation of tumour size which ultimately fails. Apoptosis was estimated to account for an average of 7% of the total cell loss rate in this tumour."} +{"text": "Autocrine growth factor secretion has classically been considered as a mechanism by which tumour cells achieve autonomous growth. However, there is now considerable evidence that autocrine circuits operate in the growth regulation of normal adult tissues. Here we consider the possible advantages to the normal epithelial cell of utilising such an external growth factor circuit and suggest that autocrine growth factor secretion, when viewed in a multicellular context, could paradoxically form part of a mechanism for preventing tumour development."} +{"text": "Nasopharyngeal carcinomas (NPC) are common in Hong Kong and southern China but rare in Western countries. Telomerase activation is common in human cancers but has not been reported previously in NPC. Telomerase activation in NPC was determined using the sensitive TRAP (telomerase rapid amplification protocol) assay in 45 nasopharyngeal biopsies in four xenografted NPC tumours established in nude mice and in five in vitro NPC cell lines. Telomerase activation is common in NPC and can be detected at high frequencies (85% in primary tumours and 100% in recurrent tumours). The frequency of telomerase activation was lowest in NPC biopsies without lymph node involvement (60%) compared with those with positive lymph node involvement (100%), and the difference is statistically significant . All the xenografted NPC tumours and in vitro NPC cell lines were strongly positive for telomerase activity. Our results suggest that telomerase activation is common in NPC and it may be useful as a diagnostic marker in the detection of tumour cells in nasopharyngeal biopsies. The high frequency of telomerase activation in stage I NPC (80% positive) suggests that it is an early event in tumour progression."} +{"text": "The vascular architecture of four different tumour cell lines transplanted subcutaneously in mice was examined by means of microvascular corrosion casting in order to determine whether there is a characteristic vascular pattern for different tumour types and whether it differs significantly from two normal tissues, muscle and gut. Three-dimensional reconstructed scanning electron microscope images were used for quantitative measurements. Vessel diameters, intervessel and interbranch distances showed large differences between tumour types, whereas the branching angles were similar. In all tumours, the variability of the vessel diameters was significantly higher than in normal tissue. The quantitative data provide strong evidence for a characteristic vascular network determined by the tumour cells themselves. \u00a9 1999 Cancer Research Campaign"} +{"text": "The section on Capacity Building describes the journal\u2019s work to assist scholarly journals in developing countries in overcoming obstacles to publication. Closely related to this section is a third area, Journal Partnerships, which describes EHP\u2019s program of fostering scientific exchange through wider dissemination of scientific information to broader audiences.The website contains four main sections. Global Access describes EHP content is provided in languages other than English. Since 2001, a Chinese-language edition of EHP has been published quarterly in partnership with the Shanghai Municipal Center for Disease Control and Prevention. Selected journal content is also available in Portuguese in the Brazilian public health journal Ci\u00eancia & Sa\u00fade Coletiva and in Spanish in the Chilean occupational and environmental health publication Ciencia y Trabajo. A Palestinian medical journal, Annals of Alquds Medicine, also has recently begun translating selected EHP news articles into Arabic.Translated Content offers easy access to the various forums in which"} +{"text": "The effects of aspirin, vitamin B2 and warfarin as potential blockers of the ruthenium binding sites inHSA were investigated through UV/visible, circular dichroism (CD), fluorescence spectroscopy and theinductively coupled plasma-atomic emission spectroscopy ICP(AES). The studies on the interactions ofseveral biologically relevant molecules with HSA have shown that drugs like aspirin or warfarin maystrongly influence the interaction of serum protein with anticancer drugs. It can derive from the influence ofthe drug on protein conformation or binding close to binding site of anticancer drug. Aspirin, vitB2 andwarfarin bind to IIA subdomain leading to partial blocking of the ruthenium binding site in HSA."} +{"text": "Meiotic chromosomes in an oocyte are not only a maternal genome carrier but also provide a positional signal to induce cortical polarization and define asymmetric meiotic division of the oocyte, resulting in polar body extrusion and haploidization of the maternal genome. The meiotic chromosomes play dual function in determination of meiosis: 1) organizing a bipolar spindle formation and 2) inducing cortical polarization and assembly of a distinct cortical cytoskeleton structure in the overlying cortex for polar body extrusion. At fertilization, a sperm brings exogenous paternal chromatin into the egg, which induces ectopic cortical polarization at the sperm entry site and leads to a cone formation, known as fertilization cone. Here we show that the sperm chromatin-induced fertilization cone formation is an abortive polar body extrusion due to lack of spindle induction by the sperm chromatin during fertilization. If experimentally manipulating the fertilization process to allow sperm chromatin to induce both cortical polarization and spindle formation, the fertilization cone can be converted into polar body extrusion. This suggests that sperm chromatin is also able to induce polar body extrusion, like its maternal counterpart. The usually observed cone formation instead of ectopic polar body extrusion induced by sperm chromatin during fertilization is due to special sperm chromatin compaction which restrains it from rapid spindle induction and therefore provides a protective mechanism to prevent a possible paternal genome loss during ectopic polar body extrusion. It is known that male and female germ cells undergo different processes of meiotic divisions and post-meiotic remodeling to produce gametes with striking differences in size, shape, chromatin packaging state, cell cycle, and etc. An oocyte undergoes two rounds of extreme asymmetric meiotic division following one round of DNA replication, producing a large sized haploid egg and discarding the other half of the chromosomes into two small polar bodies designated for degeneration. Female meiosis in many animal specieses is not complete at ovulation but is arrested at metaphase of the second meiosis (MII), waiting for fertilization to reinitiate the meiosis II. At fertilization, a sperm not only delivers a haploid paternal genome to the egg but also triggers resumption and completion of meiosis II by inducing Ca2+ oscillations required for egg activation) Because of the special DNA packaging and testis-specific chromatin remodeling during spermatogenesis, sperm chromatin behaves differently from its female counterpart during fertilization and early embryo development We and others showed previously that the cortex of mouse eggs is inductive to a general chromatin signal by inducing assembly of a distinct cortical structure, characterized by formation of a cortical actin cap Chromosomes play important roles in determining asymmetric meiotic divisions in the oocytes by inducing spindle formation and establishing a special cortical domain, characterized by formation of an actin cap surrounded by a myosin ring and exclusion of cortical granules and microvilli from the region and disr2 which mimics the fertilization-induced Ca2+ oscillations to induce egg activation To determine if the observed fertilization cone formation was due to sperm-egg membrane fusion during fertilization, we injected demembraned sperm chromatin into egg cortex distant from the pipette penetration site , enabling a robust extrusion of unwanted maternal DNA during meiosis. However, induction of ectopic polar body by a fertilizing sperm chromatin poses a risk of paternal genome loss, as we have shown that sperm chromatin has a high frequency of being extruded with the ectopic polar body. This would generate a situation reminiscent of gynogenesis, where the paternal genome is eliminated from the egg after fertilization It is well known that sperm chromatin undergoes extensive chromatin remodeling and reorganization after completion of meiosis during spermatogenesis. The paternal genome is repacked with a male-specific nuclear protein, protamine, which makes sperm chromatin highly compacted compared to its maternal counterpart. It has been thought that repackaging of sperm chromatin with protamines is important for preserving DNA integrity ICSI has been used in treating human infertility for almost twenty years. It should be noted that in most cases, only mature spermatozoa are used for ICSI. Injection of immature round spermatids or earlier stages of spermatocytes into eggs results in very low rates of fertilization and development to term It is interesting to note that while the sperm chromatin-induced spindle formation is discriminatively repressed in the fertilized eggs, induction of cortical polarization by sperm chromatin is not affected. This suggests that the signals that emanate from the chromosomes to induce spindle formation and cortical reorganization are qualitatively and quantitatively different. Regardless, it remains unclear what biological function of the sperm chromatin-induced cortical cone is during fertilization. It appears that sperm incorporation into an egg does not require pre-formation of a cortical cone, and disruption of cone formation does not affect fertilization In summary, post-meiotic remodeling and repackaging of sperm chromatin may play a previously unappreciated role in orchestrating cytoskeletal assembly but limiting its ability to induce spindle formation during fertilization, thus constituting a protective mechanism to prevent ectopic polar body extrusion and a potential of paternal genome loss during fertilization.The experimental animals were handled in accordance with good animal practice as defined by the National Institute of Health of the United States and guidelines of Institutional Animal Care and Use Committee (IACUC) and all the animal work was approved by the IACUC committee at the Stowers Institute for Medical Research, protocol #2007-0013.Female mice of CD1 at ages of 4\u20136 week-old received superovulation treatment by injection of pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) as described For in vitro fertilization, eggs were removed of zona pellucidia by brief treatment in acidic Tyrode solution 2+ oscillations and egg activation Mature spermatozoa were collected from cauda epididymes as described above for the in vitro fertilization. The collected sperm were sonicated in Triton X-100 to remove tail and plasma membranes, plus ionic detergent treatment to produce demembraned sperm heads for microinjection Microinjection was performed by using micromanipulators and a Piezol system . A single demembraned sperm head, round spermatid nucleus, or a cluster of 3\u20135 DNA beads were separately injected into the cortex distant from the MII chromosome/spindle, aiming to induce ectopic formation of a cortical cap and a bipolar spindle as described previously 2 in air for different periods of time before egg activation to induce anaphase onset and polar body extrusion.The injected eggs were cultured in M16 (Chemicon) at 37\u00b0C in an atmosphere of 5% CO2 in Ca2+ free CZB Eggs were either parthenogenetically activated by using 10 mM SrClEggs were fixed, immunostained and mounted on the slides as described previously Figure S1Complete cytoplasmic abscission of ectopic polar body induced bt sperm chromatin/spindle. Confocal images showing complete cytoplasmic abscission of the ectopic polar body induced by sperm chromatin-spindle. (A\u2013C) Different confocal sections of an egg showing ectopic polar body at 7 o'clock position (arrows) and PbII at the 11 o'clock position. Note the complete separation of cytoplasm membrane between ectopic polar body (C arrow) and the fertilized egg. The sperm chromatin-injected egg was activated by IVF and the extra DAPI staining spots are surface bound sperm.(1.10 MB DOC)Click here for additional data file."} +{"text": "The DNA-binding bisbenzimide fluorochrome Hoechst 33342 is being used routinely in radiobiological studies to assess cell kinetic parameters and tumour blood flow. However, there are reports in the literature which indicate that exposure to this compound can affect the radiation sensitivity of tumour cell populations. In this investigation, it was found that staining murine tumour cells in vitro with H33342 at concentrations greater than 0.1 microM before irradiation resulted in radioprotection. The protection factor calculated for fibrosarcoma cells stained with 10 microM H33342 was 1.7. Varying the time between radiation treatment and exposure to the fluorochrome demonstrated that the effect rapidly changed to radiosensitization when staining was performed subsequent to irradiation. Cells in transplanted KHT tumours were stained in vivo by intravenous administration of H33342 to determine whether the radiation sensitivity of these populations might also be modified. Flow cytometric analysis of suspensions prepared from tumours stained in this manner revealed that recovered cells exhibited a greater than 100-fold range in fluorescence intensities. These suspensions were irradiated in vitro and the cells were then fractionated according to fluorochrome content using cell sorting. Little evidence for a radioprotective effect was observed when these subpopulations were assessed for survival, even when tumour-bearing mice were given doses of H33342 which approached the LD50. Further analysis demonstrated that insufficient amounts of the fluorochrome were taken up by cells during in vivo staining to attain levels required for radioprotection. However, our results indicate that the amount of H33342 accumulated by cells may affect the radiation sensitivity of populations exposed to high concentrations of this fluorochrome, such as those required to achieve stoichiometric binding to DNA."} +{"text": "Rising demand for aesthetic adhesive restorations has led to wide use of composites. A multilayer technique is recommended for success of these restorations. The surface layer of composite coming in contact with air forms a superficial sticky layer called oxygen inhibited layer, upon polymerization, allowing resins from both sides to cross the interface and form an interdiffusion zone. The present study was sought to test whether oxygen inhibited layer increases or decreases the shear bond strength at the interface of composites.A microhybrid composite Esthetic \u2013X was used in this study. A cylindrical mold of composite, five mm thick and eight mm long, was prepared and embedded in acrylic resin molds after curing. This was placed in distilled water for two hours and sheared in universal testing machine at a cross head speed of one mm/sec.Data analyzed statistically to determine the significant difference between the groups. Mean and standard deviation values were estimated for the study groups and compared by one way ANOVA.No significant difference in shear bond strength of composites cured with and with out oxygen inhibited layer.The presence or absence of oxygen inhibited layer made no significant difference in shear bond strength of composite resins. Typically, dental composites are random copolymers of Bis \u2013 GMA propane (Bis-GMA), triethyleneglycol dimethacrylate (TEDGMA) and filled with various inorganic filler particles. Bis-GMA and TEGMA are bifunctional monomers that harden by free radicle induced polymerization. This reaction is strongly inhibited by free radicle scavengers such as oxygen.2 The inhOxygen inhibited layer is always present when bonding agent or composite is polymerized in air. The oxygen inhibited layer is primarily composed of unreacted monomers and oligomers and possesses a liquid-like consistency. This layer not only readily adopts the overlying material to increase contacting area but also allows materials on both sides to cross the interface and blend together to form an interdiffused zone where copolymerization can take place to produce a chemical bond. All these actions will tend to strengthen layer-layer interaction.For years it was a common perception that an oxygen- inhibited layer is required before adding more layers of bonded composite. Based on the principle of molecular interaction, one might easily reason that an oxygen-inhibited layer should improve the interfacial bonding between two contacting polymers.\u20137Reports on how the oxygen-inhibited layer affects bond strength have been inconsistent. A few studies reported that the presence of an oxygen-inhibited layer made no significant differences to bond strength.\u20139This study aimed to evaluate whether the presence of oxygen inhibited layer increases or decreases the shear bond strength.Resin composites used in this study were Esthetic\u2013X which is micro hybrid and samples were cured in Q-Lux (Dentsply) curing unit for 20 seconds per layer. Sixteen composite samples with standard dimensions of five mm (diameter), and eight mm (height) were prepared.Samples were embedded in acrylic resin blocks so that the interface of two increments is at resin substrate junction to facilitate testing in universal testing machine. Each sample consisted of two layers of four mm each cured for 20 seconds.Samples were divided into two groups of eight samples each.Group 1 \u2013 Presence of oxygen inhibited layer.Samples were prepared by placing first increment of composite in a gelatine capsule without cellophane strip and cured, subsequent layer of composite added by pushing the first increment in gelatine capsule so that oxygen inhibited layer is in between the subsequent increments of four mm.Group 2 (n = 8) \u2013 Absence of oxygen inhibited layer.Samples were prepared as above and cured in gelatine capsule with cellophane strip between the subsequent increments of 4 mm.After photo polymerization the samples were conditioned in distilled water for two hours and specimens were sheared to failure in universal testing machine at cross head speed of 1 mm/min.Bond strength calculated = F (force)/A (area).2,; A= cross sectional area of interface, r = Radius of sample.A = \u03a0rThe average bond strength for each group was calculated.Data was analyzed between both the groups to determine the significant difference between them. Mean and standard deviation values were estimated for the study groups and compared by one way ANOVA.P > 0.05) at the interface between the increments.Group 1 consisting of composite samples cured with oxygen inhibited layer showed no significant difference in shear bond strength compared to group 2 cured without oxygen inhibited layer Studies have shown a positive correlation indicating oxygen inhibited layer increases bond strength by formation of covalent bond within interpenetrating network.7 However8In the present study shear bond strength of composite resin (Esthetic\u2013X) was measured with or without oxygen inhibited layer. It is found that presence of oxygen inhibited layer made no significant difference in shear bond strength. The resin in the oxygen inhibited layer has the same composition as the uncured resin, except that the photoinitiator system, commonly camphorquinone (CQ) and amine, has been consumed or decomposed. The rateX hybrid composite used in present study composed of Phenyl propandion (PPD) as initiator and filler particle size is smaller compared to traditional macrofilled composites. It has been found that interfacial bond strength decreases as filler loading changes from highly filled to microfilled. IncreaseThis study used micro hybrid composite with improved photoinitiator and reduced filler particle size as compared to conventional composites, reducing the thickness of oxygen inhibited layer by complete interdiffusion of oxygen inhibited layer with fresh composite overlay.Taking these observations into consideration it can be concluded that the presence or absence of oxygen inhibited layer does not have influence on bond strength.Under the limitations of the present study and contrary to common perception, it can be concluded that the presence of oxygen inhibited layer made no significant difference in shear bond strength of composite resins."} +{"text": "Bacillus subtilis is a sporulating Gram-positive bacterium that lives primarily in the soiland associated water sources. The publication of the B. subtilis genome sequence andsubsequent systematic functional analysis and gene regulation programmes, together withan extensive understanding of its biochemistry and physiology, makes this micro-organisma prime candidate in which to model regulatory networks in silico. In this paper we discusscombined molecular biological and bioinformatical approaches that are being developed tomodel this organism\u2019s responses to changes in its environment."} +{"text": "To the Editor: Anthrax was introduced into Australia in 1847 near Sydney, New South Wales, and spread along stock routes throughout New South Wales and southern Queensland horses. Annually, 6\u201312 properties are affected in unrelated incidents; where cattle are involved, generally only 1\u20133 animals per property are affected (Bos taurus) with peracute anthrax and 1 horse died on 11 properties in the Rouchel area, 20 km east of Aberdeen in the Hunter Valley and 350 km from the anthrax belt flies were seen on any carcass, and the small number of carcasses and relatively large distances between some properties made mechanical transmission with ocular inoculation by nonbiting flies unlikely , a mechanism that has been implicated in wildlife epidemics of anthrax (We are currently unable to resolve this epidemiologic conundrum. However, our experience is a timely reminder that veterinary public health authorities should be on high alert for possible anthrax when unexpected livestock deaths follow flooding in areas where anthrax has historically occurred."} +{"text": "Sir,We have previously reported that CA IX (a marker of hypoxia) is an independent prognostic marker in early-stage breast cancer . Taking this into account, CA IX remained an independent prognostic factor.Data regarding adjuvant chemotherapy were available for 126 of the 144 patients. Of these, 39 received adjuvant chemotherapy (mostly CMF). Of 31 patients with CA IX positivity and available data, only nine received adjuvant chemotherapy. Thus, based on these numbers, we are unable to draw conclusions regarding a predictive role for CA IX and benefit from adjuvant chemotherapy.We certainly agree that the potential predictive value of CA IX in determining the most appropriate adjuvant therapy is interesting and reiterate our concluding remarks:\u2018\u2026CA IX expression may serve as a predictive factor to guide the selection of the most appropriate adjuvant treatment modality\u2019 and: \u2018Randomised studies with translational endpoints are required to further elucidate the prognostic and predictive value of CA IX\u2019.et al suggesting that CA IX-positive patients derive little or no benefit from standard treatments raise the important question whether these patients should therefore receive no treatment, or are actually candidates for targeting with potentially more effective treatment. We recognise that the utility of CA IX expression is currently experimental and the issue of both prognostic and predictive status will only be clearly resolved when investigated in a large series of patients treated within randomised trials of different therapies.The comments by Span"} +{"text": "To the Editor: The authors presented a therapy for patients with acute cardiopulmonary failure of gaining interest,The clinical value of mechanical support as the optimal treatment for patients with acute circulatory failure currently is under intensive debate. As a result, current guidelines for the use of aortic counter pulsation in acute circulatory failure are questioned.45With novel techniques of mobile, easy to use heart lung support systems like LIFEBRIDGE B2T it is possible to provide full cardiopulmonary support within several minutes after the patients shows first signs of circulatory crisis.The cardiac surgeon no longer needs to get to the patient in circulatory crisis, since the rapid extracorporeal cardiopulmonary support can safely be established by a team consisting of an experienced interventional cardiologist or intensive care specialist who implants the femoral cannulas using Seldinger technique and a trained nurse that sets up the system in a semi-automated guided process of priming the system. This procedure serves as a powerful method of avoiding irreversible consequences of cardiogenic shock such as multi-organ failure or disease deterioration. After rapid normalization of arterial blood pressures and oxygenation a reasonable time window for the patient is opened. Extensive diagnostics including CT imaging can be performed under stable hemodynamic and metabolic conditions that lead to optimal organ protection. Once the diagnosis is made and no spontaneous recovery is achieved, the underlying disease can be treated , if required under extracorporeal support.In case of other treatment options patients can safely be transferred to remote hospitals with specialized treatments such as transplant programs or assist programs. Currently, the ECLS (extracorporeal life support or ECMO transports usually are very complex and guidelines for staff and patient safety during transport are not met. However, the Conformit\u00e9 Europ\u00e9enne-marked emergency cardiopulmonary support systems LIFEBRIDGE B2T system is the first and only HLM approved to date for mobile use according European norm EN 1789. With the availability of these novel systems, the clinical use of extracorporeal circulation outside the operating room to treat circulatory or pulmonary failure is expected to increase rapidly in the future, and unaddressed clinical needs in acute intensive care will be met."} +{"text": "Choriocarcinoma can be imaged by external scintigraphy after intravenous administration of radiolabelled antibody directed against human chorionic gonadotrophin (HCG). The purpose of this study was to investigate whether antibody imaging was sufficiently sensitive and specific to improve the selection of patients for surgical resection of localised deposits of drug resistant or recurrent choriocarcinoma. Eighteen patients with raised serum HCG concentrations in whom the site of tumour was not known were investigated by antibody imaging and conventional imaging methods. When the tumour appeared localised, resection was attempted. Tumour was found at all sites in which both antibody imaging and conventional imaging methods were positive. Antibody imaging gave false positive results in 2 of 18 patients and false negatives in 5. Computerised tomography was false positive in one case and false negative in 2. In these patients, antibody imaging gave true negative and true positive results respectively. Of 8 patients with positive antibody imaging in whom resection was attempted, 5 achieved sustained complete response with up to five years follow up. It is concluded that antibody imaging is useful in selection of patients for surgery in drug resistant or recurrent choriocarcinoma."} +{"text": "A detailed casenote review was performed on 55 patients registered with testicular non-seminomatous germ cell tumours (NSGCT) between 1983 and 1988 under the Scottish Cancer Registration Scheme and who had died by 1992. Details of all aspects of clinical management relating to their NSGCT and death details were extracted and summarised. An assessment was made on whether the patients' management had been optimal. An analysis of 5 year survival rates by the five Scottish oncology centres demonstrated significant differences between centres . Some patients in all centres were assessed as having received suboptimal treatment, but two centres performed less well than the other three. There is a suggestion that the number of patients treated suboptimally decreases with increasing number of patients seen, but this does not reach statistical significance."} +{"text": "To obtain functional evidence for DCC as a tumour suppressor associated with endometrial cancer, the human DCC cDNA encoding a complete open reading frame (ORF) was transfected into highly tumorigenic human endometrial carcinoma cells, HHUA and Ishikawa in which DCC expression was completely deleted. Reconstituted expression of DCC in HHUA had little effect on in vitro growth, but suppressed tumour formation in mice completely. The clones from Ishikawa had abundant DCC expression similar to that in normal endometrium. Their growth in vitro was suppressed and showed apoptotic phenotype. Lower levels of DCC expression in the prolonged passaged clones did not induce apoptosis, but still had the potential to suppress tumorigenicity. These observations imply a role of DCC in regulation of normal endometrial cell growth, and categorize DCC as the tumour suppressor gene for endometrial cancer. \u00a9 2000 Cancer Research Campaign"} +{"text": "The detection of bone marrow involvement might be of prognostic value and may influence therapeutic decisions in small cell lung cancer. By unilateral bone marrow aspiration and biopsy, evidence of bone marrow metastases is seen in 15-30% of patients with this disease. Since magnetic resonance imaging of the lower body and immunostaining with monoclonal antibodies have recently been shown to be very sensitive detection methods, we investigated the value of these two techniques in detecting bone marrow involvement in 35 consecutive patients with small cell lung cancer. The results were compared to those obtained with conventional cytohistological analysis. In all cases when cytology and/or bone marrow biopsy were positive, monoclonal antibodies immunostaining and magnetic resonance imaging also detected malignant cells. Furthermore, evidence of bone marrow involvement was shown with magnetic resonance imaging and/or immunostaining in 10 of 26 cases (38%) where routine procedures were unable to detect malignant cells. In one of these 26 patients, magnetic resonance imaging and immunostaining provided the only evidence of metastatic disease. These data suggest that the rate of bone marrow metastases is underestimated by routine procedures. Further investigation is needed to determine whether or not these new non-invasive methods have prognostic value or affect therapeutic choices in small cell lung carcinoma."} +{"text": "Mean trait anxiety scores were higher than general population data but not significantly different from published data from other screening samples. Overestimators and underestimators reported significantly different risk estimates (i.e. increased accuracy) after counselling, but significant inaccuracies persisted. Over- (n = 12) and underestimators (n = 60) were still inaccurate in their risk estimates by a factor of 2 after counselling. Thirty per cent of the sample scored above the cut-off (5/6) for case identification on a screening measure for psychological distress, the General Health Questionnaire (GHQ). GHQ scores were significantly lower after counselling with no evidence of increasing risk estimate causing increased distress. The risk of distress after counselling was greater for younger women and those who were more distressed at first presentation. The counselling offered was effective in increasing the accuracy of risk perceptions without causing distress to those who initially underestimated their risk. It is worrying that inaccuracies persisted, particularly as the demand for service has since reduced the consultation time offered in this clinic. Further work is needed to evaluate alternative models of service delivery using more sophisticated methods of assessing understanding of risk. \u00a9 1999 Cancer Research CampaignWomen referred to a familial breast cancer clinic completed questionnaires before and after counselling and at annual follow-up to assess their risk estimate and psychological characteristics. The aims were to determine whether those who attended the clinic overestimated their risk or were highly anxious and whether counselling influenced risk estimates and levels of distress. Women ("} +{"text": "Public-health policy is inconsistent in its approach to the sexually transmitted disease human immunodeficiencyvirus (HIV). Nearly every health agency has politicized the reporting, finding, and contacting of HIV cases. There is also no consistency among the various state health departments and the various federal health agencies. Until we have a uniform health policy that treats HIV infection as every other reportable sexually transmitted disease, we will make little progress toward controlling its inevitable increase in both cases and costs."} +{"text": "Intraoperatively blue light (405 nm) was applied to the operation site. Sections of the excised tumour and some lymph nodes were prepared and analysed with a fluorescent microscope. All primary mammary tumour tissues showed significantly higher fluorescence intensity than surrounding normal mammary tissue. Fluorescence of the mammary tumours could also be discriminated macroscopically and intraoperatively. Fluorescence intensity in nonmetastatic lymph node tissue was higher in 2 out of 3 patients than in primary tumour tissue. By photodynamic diagnosis using aminolevulinic acid we were able to reliably distinguish primary mammary tumours from normal mammary tissue microscopically and macroscopically in all our patients. We suggest that photodynamic diagnosis with aminolevulinic acid for breast tumours should be further investigated and developed for intraoperative use and may well be a simple tool for better intraoperative diagnosis and recognition of tumour margins. We hypothesize that lymph node metastasis of breast tumours will not be detectable by this method. \u00a9 2001 Cancer Research Campaign http://www.bjcancer.comPhotodynamic diagnosis is of increasing interest for diagnosis in oncology. It is based on a more intense incorporation of a fluorescent dye in tumours compared to normal tissue. As a feasibility study we investigated the effectiveness of oral application of 5-aminolevulinic acid for photodynamic diagnosis of human primary mammary tumours. The study included 16 patients with palpable breast tumours. Aminolevulinic acid was administered at a concentration of 40 mg kg"} +{"text": "Anaesthesiologists are increasingly confronted with patients who had a recent coronary artery stent implantation and are on dual anti-platelet medication. Non cardiac surgery and most invasive procedures increase the risk of stent thrombosis especially when procedure is performed early after stent implantation. Anaesthesiologist faces the dilemma of stopping the antiplatelet therapy before surgery to avoid bleeding versus perioperative stent thrombosis. Individualized approach should be adopted with following precautions. i) In a surgical patient with a history of percutaneous coronary intervention (PCI) and coronary stent, determine the date of the procedure, the kind of the stent inserted and the possibility of complications during the procedure. ii) Consider all patents with a recent stent implantation Any decision to postpone surgery, continue, modify or discontinue antiplatelet regimes must involve the cardiologist, anaesthesiologist, surgeon, haematologist and the intensivist to balance the risk and benefit of each decision. The incidence of coronary artery disease (CAD) has dramatically increased in India during the recent years to the extent that during the past 30 years the CAD rates have tremendously increased.Currently, over 90% of all PCI involve placement of stentsRevascularization with coronary balloon angioplasty may cause vessel spasm and abrupt closure due to vessel recoil. The deployment of stents after angioplasty reduces the risk of abrupt vessel closure by sealing coronary artery dissection. Stent deployment also reduces long term risk of restenosis by preventing elastic recoil and negative vessel remodeling.Bare Metal Stents (BMS)Drug Eluting Stents (DES)Two major types of coronary artery stents are commonly deployed:There is high incidence of late stent restenosis with BMS. In fact, restenosis is a side-effect of the normal healing process with the growth of the scar tissue around the stent mesh in a process called neointimal hyperplasia, which in some cases, can lead to occlusion of the coronary lumen. In patients receiving a BMS, in-stent restenosis requiring repeat intervention occurs in 12 \u2013 20% of cases, especially it peaks at three months7To prevent restenosis, drug eluting stents (DES) were designed, by coating a standard coronary stent with a thin polymer containing an antiproliferative substance that inhibits smooth muscle proliferation and neointimal hyperplasia within the stented segment.9two major types of DES being inserted:Sirolimus Eluting Stents (\u2018Cypher\u2019 stent) orPaclitaxel Eluting Stents (\u2018Taxus' stent).Currently there are (SES) Sirolimus Eluting Stents: Sirolimus (rapamycin) is a macrolide antibiotic with potent immunosuppressive and antimitotic properties, which binds to its cytosolic receptor FKBP 12, and inhibits down-regulation of cyclin dependent kinase inhibitor p27 kipl. This blocks transition from G1 to S phase in the cell cycle and inhibits vascular smooth muscle cell proliferation and migration.(PES) Paclitaxel Eluting Stent: Paclitaxel is a potent anti-tumor drug which inhibits microtubule formation during cell division.DES has been shown to have as good safety profiles as BMS in the short to medium term (6 \u2013 12 months). The mechanism of obstruction of DES is different from that of BMS. In DES the stent struts remain uncovered, hence prone to thrombosis. However in BMS, the pathophysiological mechanism of obstruction is restenosis with neointimal hyperplasia and DES inhibits this process.14Antiplatelet therapy is mandatory for patients after coronary artery stenting as platelets play a major role in thrombus formation after coronary stenting. Coronary artery stents have been shown to be associated with a very high risk of thrombosis.1416Mechanism of action Clopidogrel and ticlopidine are metabolized to an active compound in the liver which inhibits P2Y 12 adenosine diphosphate (ADP) platelet receptor, thus inhibiting binding of fibrinogen to platelet glycoprotein IIb/IIIa receptor complex. This prevents platelet aggregation by ADP stimulation.Clopidogrel therapy is initiated prior to or immediately following stenting procedure. A loading dose of clopidogrel 300 mg should preferably be given at least six hours prior to stenting procedure.For Bare metal stents. Loading dose of 300-600 mg of clopidogrel is given before implantation of a BMS. After the procedure aspirin, 75-100mg and clopidogrel 75mg are continued for 4-6 weeks. Low dose aspirin therapy is continued for life.For Drug eluting stents : After the loading dose of 300-600mg of clopidogrel, 75 mg should be continued for a minimum of 3 months after implantation of sirolimuseluting stent and 6 months after a paclitaxel eluting stent. Aspirin dose and regime remain the same. Recent guidelines suggest dual antiplatelet therapy to be continued till one year.21Contraindications to clopidogrel: Clopidogrel is contraindicated in patients with active pathological bleeding. Intracranial haemorrhage is an infrequent complication. Arare complication reported after clopidogrel was thrombotic thrombocytopenic purpura (TTP).The potential risk of stent thrombosis in patients with coronary stents who experience resistance to antiplatelet medication must be considered in the perioperative setting.Clopidogrel resistance: The term clopidogrel resistance encompasses patients for whom drug does not achieve its pharmacological effect. Failure of therapy reflects patients who have recurrent thrombotic events despite receiving therapy. Causes of clopidogrel resistance are listed in No standard validated method is available to measure clopidogrel efficacy.23There is inter individual variability of platelet inhibition by antiplatelet agents which may lead to clopidogrel resistance, as clopidogrel is a pro-drug, which requires activation by cytochrome 450 isoenzyme CYP3A4 24In a study by Aggarwal et al on platelet function using optical light aggregometry has shown that only 50% had a definitive response to clopidogrel.2 by irreversible acetylation of cyclooxygenase-1 (COX -1) and thus fail to prevent platelet activation and aggregation.2 a marker of aspirin resistance, has been shown to be associated with increased incidence of vascular events.31Resistance to aspirin has also been described. Aspirin fails to reduce platelet production of thromboxane AKaluza et al first time in the year 2000 documented high risk of surgery in patients having recent insertion of coronary artery stents33Analyzing the outcome of the study undertaken by Sharma et al, on 27 patients who underwent non-cardiac surgery within 3 weeks after BMS implantation 6 out of 7 patients, in whom the thenopyridine was stopped for more than 5 days died compared to only 1 of 20 patients who continued thenopyridine therapy (p<0.001).37However in a single center series of 38 patients who underwent 41 major and 18 minor non cardiac surgeries at a median of 9 months after successful DES implantation no major adverse events were seen by Compton et al.th day. Platelets are not activated in the post operative period, but they are more prone to being activated as demonstrated by aggregation studies and the platelet count is significantly increased on 7th day.42Thromboelastogrpahy has shown evidence of hypercoagulability during surgery which may lasts for 7 days in post operative period, evident in the form of decrease reaction time (r-time), increase in clot strength, with continuous post operative increase in maximum amplitude (MA).Thus three factors play important role in acute stent thrombosis as shown in Patients taking dual antiplatelet therapy may show increased risk of major bleeding complication. Increased bleeding time is seen when combined therapy of clopidogrel and aspirin is used, through synergistic antiplatelet actions.34What is the overall risk of coronary adverse events in patients with stents if antiplatelet therapy is discontinued? There are no such reports which quantify this statement. There are few reports documenting adverse events when antiplatelet therapy was withdrawn. Kaluza et al showed that non-cardiac surgery soon after BMS placement was linked to a very high rate of adverse events.46The American College of Cardiology (ACC), American Heart Association (AHA), European Society of Cardiology (ESC), Society for cardiovascular Angiography and Interventions (SCAI) and several other societies engage in production of guidelines in the area of cardiovascular diseases from time to time. These guidelines attempt to define the practices that meet the need of most patients in most circumstances. The aim of the guidelines is to improve the patient care. The ultimate judgment regarding the care of the particular patient is to be made by the clinician keeping in mind all the circumstances.American College of Cardiology and American Heart Association (ACC/AHA) has laid down guidelines-A delay of 4-6 weeks between the BMS and elective non cardiac surgery is recommended to allow at least partial endothelization of the stent but not more than 12 weeks when restenosis may begin to occur.-In patients treated with DES, elective surgical procedures with significant risk of bleeding should be deferred up to one year. In emergent non-cardiac surgery that requires stopping clopidogrel, the guidelines recommend continuing aspirin therapy if possible, and restarting clopidogrel as soon as possible.-Risk of stopping the antiplatelet therapy should be weighed against the benefit of reduction of bleeding complication. If there is need to stop antiplatelet therapy, clopidogrel should be stopped for as minimum time as possible and restarted early. Aspirin should be continued perioperatively. Luckie et alApproach to a patient for major surgery following recent coronary artery stenting Regarding perioperative approach in patients with coronary stents there is no accepted standard or optimal approach for management.When was the PCI done?What is the type of stent?How many stents were placed?Was the revascularization complete?Drug regime and any irregularities of the treatment?History of any adverse cardiac event/stent thrombosis?Urgency of surgery? / Can the surgery be delayed?Bleeding risk during surgery?History of conditions prone to stent thrombosis Whether antiplatelet medication is to be maintained in perioperative period or stopped before operation?The key questions in such patients during pre anaesthesia evaluation areAn important aspect of pre-operative visit is patient education. Patient should be explained the importance of antiplatelet therapy, the need to discontinue and to restart the therapy after surgery.Investigations for platelet count and platelet function should be undertaken. Whole blood and platelet concentrates should be arranged prior to surgery.Elective surgical procedures: It is the consensus that all elective procedures should be delayed for at least 4-6 weeks in patients who have received BMS. Preanaesthetic evaluation for elective surgery in patients with DES plays a significant role in decision making and risk assessment. Earlier reports suggested that if there is no major risk of bleeding, all elective surgeries within 6 months after DES implantation should be managed similar to urgent surgery. However the current consensus is that elective surgeries should be delayed for 12 months after DES placement or PCI prior to non-cardiac surgery has previously been widely practiced, to reduce peri operative cardiac complications/adverse reactions in patients with known coronary artery disease. However, currently this practice is reduced as the data suggest that pre operative revascularization has little impact on peri operative adverse cardiac events when stenting is performed solely for this purpose.If a patient with coronary artery disease is known to require non cardiac surgery, the first question to ask is whether the patient really needs revascularization. The CARP (Coronary Artery Revascularization Prophylaxis) study suggests that revascularization may not be necessary for a large number of patients without an unstable coronary syndrome or other high risk features. Pre operative prophylactic coronary revascularization should be considered as per ACC/AHA guidelines The risks and benefits of each strategy should be assessed, keeping in account patients symptoms, comorbidities, and coronary anatomy, degree of associated ischaemia and urgency and type of surgery to be performed.Ongoing development in stent technology may render concerns regarding the long duration of anti-platelet therapy necessary following DES implantation obsolete. A number of different pro-healing surfaces are becoming available which may allow much more rapid and complete endothelialization of the stented segment. The Genous-R stent consists of a standard stainless steel stent, which is coated in a matrix containing monoclonal antibodies targeted specifically at the CD34 receptor. This receptor is exclusive to the surface of endothelial progenitor cells (EPC), which are preferentially captured onto the stent surface. Once attached to the stent surface, the EPCs mature into endothelial cells, rapidly creating a smooth endothelial surface within the stented segment without the risk of restenosis.51One should learn from this review that patients with recent coronary artery stents are on antiplatelet therapy and are on high risk of peri-operative complication. The anaesthesiologist faces the dilemma of stopping the antiplatelet treatment before operation to avoid bleeding versus risk of post operative stent thrombosis. When faced with a patient who requires major non-cardiac surgery and has a coronary stent, the risk of stent thrombosis needs to be assessed against both the potential risk of bleeding and adverse consequences.Till now there is no definite evidence that bleeding in non-cardiac surgery is not common and not a troublesome complication when compared with incidence of cardiac events. With high level of scientific evidence anaesthologist, surgeon and cardiologist should establish local treatment algorithms for the management of patients who have had previous PCI and coronary stent and are now to undergo surgery and follow the recommendations of current guidelines. Major noncardiac surgery should be avoided for 4-6 weeks for bare metal stents unless immediately life saving. After DES implantation, surgery within 3 \u2013 6 months also substantially increases the risk of cardiac complications.Non cardiac surgery should be delayed for 12 months. Even beyond 12 months, report of the late stent thrombosis suggest, that at least one antiplatelet agent should be continued perioperatively. Surgery which cannot be delayed should be performed on dual antiplatelet medication whenever possibleThe multifaceted approach inclusive of cardiologist, surgeon, anaesthesiologist and haematologist should be used with each patient in order to provide maximum individual benefits. Each patient is different and treat the patient and not the stent."} +{"text": "There are several stigmas on the resting surface electrocardiogram that are indicators of past myocardial injury. Broad QRS pattern with bundle branch block, Q waves, persistent ST elevation are some of those facsimiles which may at times even be considered as definitive signs of left ventricular impairment.We would like to focus here on a lesser known entity of the surface electrocardiogram - the fragmented QRS complex. This marker of myocardial injury may often be the only electrocardiographic marker in patients with non-Q myocardial infarction and in patients with resolved Q wave. It can also be a reliable pointer to left ventricular functional compromise.Fragmented QRS electrocardiograms were for the first time recorded from canine hearts with experimentally induced acute ischemia and healing. It was found that fragmented electrocardiograms were more frequently observed in healed myocardial infarctions more than 2 weeks old, than in preparations from 5 day old infarcts .The asynchronous excitation of muscle fibers causing fragmentation of electrocardiogram may be due to the poorly inter-connected muscle bundles; separated by high resistance intercellular connective tissue caused by the healing process. This effect is most pronounced at the bordering areas of necrosis where the connective tissue invades the surviving 'islands' of muscle, hence causing separation and distorted orientation of muscle fibers. Any form of gross structural abnormality, like large chamber dilatation, may also cause a similar picture. Flowers et al have eveIt has been observed in various studies ,4 that fDas MK et al using myBroad premature ventricular complexes (\u2265160 ms) with notched QRS (notch separation >40 ms) was found to be a reliable marker of a global form of ventricular dysfunction involving ventricular mass, chamber size or function . It may Flowers et al found a Fragmented QRS electrocardiogram is an independent predictor of left ventricular function. It is a marker of higher stress myocardial perfusion abnormalities and functional deterioration . This waQRS fragmentation with or without Q waves was found to predict a higher mortality and recurrent cardiac events than either Q wave alone or resolved Q wave without QRS fragmentation ,11. HencFragmented QRS on the resting surface electrocardiogram is a simple, fast and inexpensive modality of non invasive investigation that can be of great value in predicting the cardiac status and prognosis of an individual being evaluated for coronary artery disease."} +{"text": "Splenocytes from inbred Wistar rats bearing a syngeneic squamous cell carcinoma (Spl) were fractionated by several techniques to characterize the lymphoid cells cytotoxic to the tumour in vitro. The anti-tumour cytotoxicity is presumably mediated primarily by T lymphocytes because it was greatly reduced by removal of T lymphocytes with heterologous anti-T serum plus complement but not by removal of other cell types. Cytotoxicity could be blocked at the tumour cell but not at the effector cell by sera taken late in tumour growth. Sera taken earlier in tumour growth could induce cytolysis of tumour cells by normal splenocytes but only if the tumour cells were treated with serum and washed before addition of the effector cells. Although splenocytes from normal and tumour-bearing rats were equally effective at lysing antibody-coated target cells it is unlikely that this mechanism is important in vivo as sera from early in tumour growth onwards contained factors (immune complexes?) which inhibited antibody-induced lymphocytolysis."} +{"text": "The clinicopathological features and surgical treatment of biliary carcinoma around the major hepatic duct confluence arising after pancreatoduodenectomy (PD) due to initial bile duct carcinoma are described in three patients. Occurrence of biliary carcinoma more than 12 years after initial surgery and a histological finding of cholangiocellular carcinoma mixed with hepatocellular carcinoma suggested metachronous incidence of biliary carcinoma after PD. Extended right hemihepatectomy with complete removal of the residual extrahepatic bile duct and segmental, resection of the jejunal loop were carried out safely without operative death or severe postoperative complications. Two patients died of tumor recurrence 6 months after surgery, and the remaining patient is currently living a normal life without evidence of recurrence 17 months after surgery. These surgical procedures are a therapeutic option in patients with biliary carcinoma around the major hepatic duct confluence arising after PD."} +{"text": "To the Editor: I read with interest the article by Halton and Graves on the economics of catheter-related blood stream infections with its attendant economic implications.>90%, which results in essentially the same serum/tissue levels as if when administered by IV. Because all parenteral antimicrobial agents do not have an oral formulation, clinicians should select an equivalent oral agent with the same spectrum as its parenteral counterpart to treat most serious systemic infections infections are important from a clinical and economic perspective. Clinicians should consider oral antimicrobial agents more frequently instead of having CVC lines placed for IV drug administration. Currently, oral agents are available to treat nearly all pathogens, even those formerly only treatable with intravenous antimicrobial drugs. In his letter Cunha suggests that oral antimicrobial drug therapy is safer and less expensive than intravenous therapy via central venous catheters (CVCs) . Financial costs or cost-savings are important, but not the sole consideration for a decision maker ("} +{"text": "Bioluminescent imaging has proven to be a valuable tool formonitoring physiological and pathological activities at cellularand molecular levels in living small animals. Using biologicaltechniques, target cells can be tagged with reporters encodingseveral kinds of luciferase enzymes, which generate characteristicphotons in a wide spectrum covering the infrared range. Part ofthe diffused light can reach the body surface of the small animal,be separated into several spectral bands using appropriatefilters, and collected by a sensitive CCD camera. Here we presenta bioluminescence tomography (BLT) method for a bioluminescentsource reconstruction from multispectral data measured on theexternal surface, and demonstrate the advantages of multispectralBLT in a numerical study using a heterogeneous mouse chestphantom. The results show that the multispectral approachsignificantly improves the accuracy and stability of the BLTreconstruction even if the data are highly noisy."} +{"text": "Bombesins are potent growth factors for murine Swiss 3T3 cells. Using these cells in chemically defined conditions we have been able to characterise the bombesin receptor and the early signals preceding DNA synthesis. We describe two substance P analogues substance P and substance P which competitively block the binding of bombesins to their receptor and all the events leading to mitogenesis. Bombesins are secreted by human small cell lung cancers (SCLC) and may act as autocrine growth factors for these tumours, so the development of peptide bombesin antagonists could have therapeutic implications. We demonstrate that the antagonists can reversibly inhibit the growth of SCLC in vitro, with relatively little effect on other lung tumours."} +{"text": "A micro-assay designed to assess the capacity of peripheral blood mononuclear cells to differentiate in vitro into mature macrophages is described. In patients with \"final common pathway\" malignant melanoma, there was a highly significant deficiency in macrophage precursors (MPs). By conventional morphological criteria such patients did not show a significant monocytopenia. Serum factors do not seem to contribute to the MP defect in the patients. We conclude that these patients have an intrinsic functional defect in their peripheral blood monocytes, but the mechanisms responsible for this defect are as yet unknown."} +{"text": "Photodynamic therapy (PDT) utilizing Photofrin is proving to be effective for the treatment of early stage lung cancer. However, wider clinical applications of Photofrin as a photosensitizer for various cancers are hampered by potentially serious and prolonged skin photosensitivity. To prevent these side effects and reduce the hospitalization period, we recently gave reduced doses of Photofrin by bronchial arterial infusion. Five patients with endoscopically evaluated minimally invasive carcinoma of the lung were given 0.7 mg/kg of Photofrin by bronchial arterial infusion 48 hr before PDT. Complete remission was obtained in all 5 cases and no case showed skin photosensitivity when exposed to sunlight under careful surveillance at one week after PDT."} +{"text": "Based on an independent forward model in fluorescent tomography, aparallel reconstructed scheme for inhomogeneous mediums with unknown absorptionproperty is proposed in this paper. The method considers the two diffusion equationsas separately describing the propagation of excited light in tissues with and withoutfluorescent probes inside. Then the concentration of fluorophores is obtained directlythrough the difference between two estimations of absorption coefficient which can beparallel inversed. In this way, the multiparameter estimation problem in fluorescenttomography is transformed into two independent single-coefficient determinedschemes of diffusion optical tomography (DOT). Any algorithms proved to beefficient and effective in DOT can be directly applied here. In this study theabsorption property is estimated from the independent diffusion equations by agradient-based optimization method with finite element method (FEM) solving theforward model. Simulation results of three representative occasions show that thereconstructed method can well estimate fluorescent property and tissue absorptiondistribution."} +{"text": "Recurrent fetal losses indicate screening for antiphospholipid antibodies, especially after the third consecutive fetal loss, or when they occur after 12 weeks gestation or when the mother presents with thrombosis or other ailments of antiphospholipid syndrome. Fetal loss may be caused by thromboses of placental vasculature. There is no agreement concerning the mechanism of thromboses: protein C pathway and/or annexin V are the best candidates. When fetal loss occurs early during gestation, murine models suggest that antiphospholipid antibodies can also act on trophoblasts by inhibiting syncytia formation. Among the high risk patients with more than two fetallosses, an association of aspirin and heparin given early during gestation is successful in 70\u201380% of cases."} +{"text": "Nontuberculous mycobacteria (NTM) have been recognized as an important cause of disease in immunocompromised hosts. Pulmonary disease caused by NTM is increasingly recognized in previously healthy persons. Investigation of pulmonary disease affecting a family of five identified an indoor hot tub as the source of NTM-related disease."} +{"text": "Chromosome bands did not result with urethane, a compound which does not produce cytotoxicity or cause transformation by direct exposure of cells. Non-carcinogenic chemicals which do not inhibit cell multiplication also failed to produce bands. Cytogenetic analysis following removal of the carcinogens from the cultures indicated non-banded uniformly stained chromosomes. It is concluded that metaphases with chromosome bands are more likely to be the result of nonspecific toxicity rather than being related to the carcinogenic properties of the chemicals.After 24 hours of treatment of human peripheral leucocyte cultures or Syrian hamster embryonic secondary cultures with some known chemical carcinogens at concentrations that produce transformation"} +{"text": "A comparison of the cytotoxic effectiveness of adriamycin incorporated into ion exchange microspheres with conventional chemotherapeutic use of adriamycin was carried out in a rat tumour model. Drug microspheres were targeted to the tumours by embolisation into the arterial supply of the hind limb bearing the tumour. Microspheres were found to embolise in tumour tissue at concentrations of up to 39 times that of the surrounding normal tissue. As a result, adriamycin microsphere therapy was found to retard significantly (P less than 0.01) tumour growth rates compared to growth rates associated with similar doses of adriamycin delivered as free drug rather than bound to controlled release microspheres. Equivalent sham microsphere treatments showed no significant difference in tumour growth rates compared with the control group. Adriamycin loaded on to ion exchange microspheres holds strong potential for treatment of human malignancy."} +{"text": "We discuss the case of a 63 years old female who required repeated intubation due to recurrent pulmonary edema. She was found to have hypertrophic cardiomyopathy with a gradient of 82 mmHg across the left ventricular outflow tract. Initially adequate rate control and treatment with negative inotropes did not help her condition. Finally a dual chamber pacemaker implantation and atrioventricular node modification lead to successful extubation. Hypertrophic cardiomyopathy has been reported as a cause of failure to wean from mechanical ventilator .We repor63-year-old Caucasian female, with past medical history of failure of transplanted kidney, persistent atrial fibrillation, hypertension and hypothyroidism was admitted to the intensive care unit with acute respiratory failure secondary to pulmonary edema. She was intubated and treated with intravenous diuretics. An echocardiogram revealed severe hypertrophic cardiomyopathy with a left ventricular outflow gradient of 82 mm Hg . She wenLung congestion and low cardiac output syndromes are commonly encountered in critically ill and mechanically ventilated patients. These disturbances may impede weaning from mechanical ventilator and require repeated intubations if left unaddressed. Proper management of congestive heart failure in mechanically ventilated patients has been associated with favorable outcomes, and by optimizing the treatment patients have successfully been weaned from mechanical ventilators. Hypertrophic Cardiomyopathy (HOCM) may be one of the conditions responsible for such a scenario and the routine treatment for systolic heart failure may be detrimental in such patients.Adamopoulos et al reports In patients who are refractory to medical management permanent pacing in the form of dual chamber pacing (DDD) has been introduced as an alternative treatment modality. By altering the pattern of ventricular depolarization pacing may result in decreased left ventricular outflow tract gradient and improve functional status in such patients .The imprOur patient in addition to hypertrophic cardiomyopathy had atrial fibrillation which might have independently contributed to worsening of the outflow gradient. Inspite of good rate control during atrial fibrillation in addition to negative inotropic medication, patient continued to have repeated intubations. Although alcohol septal ablation is a recommended procedure in a selected group of patients, it was not contemplated in this patient who was critically sick. The long-term results of dual chamber pacing in hypertrophic cardiomyopathy are controversial ,4. ThereIn patients with difficult weaning and extubation from a mechanical ventilator due to congestive heart failure in an intensive care unit setting, an underlying cause including hypertrophic cardiomyopathy should be ruled out."} +{"text": "The former were tested in two leukemia celllines: chronic myelogenic leukemia (K562) and human promyelocytic cell line (U937). They showed to haverelatively high toxicity in K562 cells and a relatively low cytotoxicity in U937 cells, as assessed by bothMTT and Trypan Blue assays. The five molybdenum complexes were tested in human promyelotic U937 cellline and they showed to have high toxicity.Cytotoxicity and cell growth inhibition studies were performed for five distinct cobalt(ll)[Co"} +{"text": "Avian influenza, caused by influenza virus A (H5N1), continues to be a source of outbreaks among avian species and of sporadic human cases that result in a high case-fatality rate. These historically unprecedented outbreaks have raised serious global concerns for both animal health and human health. Significant progress in the research of avian influenza has occurred in the past decade, but unanswered questions remain. How does avian influenza cross species barriers and acquire transmissibility among humans? How can we minimize the risk of emergence of a pandemic virus? Will subtype H5N1 maintain its virulence in humans when it becomes a pandemic virus? This book helps readers understand what is known and what remains to be known about avian influenza.The book contains 19 articles written by leaders in avian influenza research. The authors provide a comprehensive and updated review of current knowledge on avian influenza, with particular emphasis on H5N1. The articles cover various aspects of avian influenza, including its epidemiology and ecology as well as control strategies for potential outbreaks of avian influenza in Asia and Europe. Some articles describe the molecular mechanisms of interspecies transmission and virulence in birds and humans. Both interspecies transmission and virulence are determined by many molecular changes in different genes, but the mechanisms for interspecies transmission and virulence are not completely understood. Other articles address timely and important issues such as vaccine development and antiviral resistance.All pandemic influenza viruses in humans originated from avian influenza viruses. Understanding how an avian virus can become a pandemic virus that causes devastating effects on human health is critical. This book is a valuable reference for scientists and public health specialists who work in either animal health or human health."} +{"text": "Urodynamic evaluation in the assessment of women complaining of urinary incontinence remains controversial with recent UK National Institute of Health and Clinical Excellence guidance maintaining that it is unnecessary prior to surgery for women with a primarily stress leakage. Other experts contend it should be part of routine preoperative assessment since it establishes a diagnosis, allows more careful patient counseling and predicts surgical outcome.To summarize current literature to define the evidence level on which these conflicting opinions are based.A systematic literature search was performed and retrieved publications summarized in a narrative evidence review using both original papers and previous reviews.Five hundred and one primary research papers and 65 previous reviews were retrieved. The findings were summarized in a narrative comprising overview, description of methods of bladder and urethral pressure measurement, and a summary of the literature concerning four key questions.The level of evidence was low regarding answering each of the questions posed, preventing firm conclusions. Urodynamic findings do correlate with relevant symptoms and, to some extent, with symptom severity, giving reasonable diagnostic accuracy. There is no reliable evidence that preoperative urodynamic diagnosis improves outcome from surgery for stress incontinence although it is likely to facilitate preoperative discussion. Tests to differentiate sphincter deficiency and urethral hypermobility are not currently recommended due to poor validity and reproducibility. This along with the current use of mid-urethral tapes as the universal primary surgical procedure means differentiation is not a necessity. Preoperative diagnosis of detrusor overactivity does not appear to worsen surgical outcome in women with a primary symptom of stress leakage. Large, well-designed prospective studies are now underway to provide a definitive answer to these questions. Urinary incontinence has a significant impact on the quality of life of the affected individual and carries a substantial financial burden on any healthcare system. A shift to less invasive techniques has meant surgery is more accessible to patients and is being performed with increasing frequency. A key clinical research question in this area is where urodynamic diagnostics tests should be placed in the assessment pathway. This review highlights relevant findings from current literature to help guide clinicians regarding the use of cystometry in the investigation of urinary incontinence.The lower urinary tract which includes the bladder, urethra and sphincter mechanisms has two key functions - storage and voiding. Satisfactory storage requires intra-vesical pressure to remain lower than the pressure required to open the urethra. This is achieved by the elastic properties of the connective tissues of the bladder wall and the ability of the detrusor to increase its muscle fiber length which together ensure high compliance. Reflex sphincter activity guards against sudden intra-vesical pressure increases, such as those caused by standing and coughing, by maintaining urethral closure pressure higher than bladder pressure. When micturition is appropriate external urethral sphincter and pelvic floor relaxation initiates urine flow which is then augmented by detrusor contraction. Normal cystometry shows a low and stable bladder pressure during storage with no phasic activity and an appropriate increase in pressure during voluntary voiding.In an effort to standardize terms and improve assessment, the International Continence Society (ICS) has published a number of standardization reports which define incontinence according to symptoms and findings on cystometry Boxes and 2.11\u20134Blaivas categorized urodynamic stress urinary incontinence into four types and subtypes according to findings on video cystometry .5]5]Any situation where intra-vesical pressure is higher than urethral closure pressure will result in urinary leakage and this can be due to either decreases in urethra closure pressure caused by urethral dysfunction or increases in bladder pressure caused by detrusor dysfunction or a combination of both. Given this simple unifying urodynamic explanation it is not surprising that clinical tests measuring bladder or urethral pressure have been developed to delineate urodynamic diagnosis for women with urinary incontinence.To critically review current literature concerning the usefulness of urodynamic testing for people with urinary incontinence in terms of diagnosis, communication and prognosis.We carried out a systematic search of Medline using the MESH terms \u2018urodynamics\u2019 and \u2018urinary\u2019 and \u2018incontinence\u2019 between the years 1990 and 2009. We also searched references listed in existing reviews. The title and abstracts were checked by one author and full papers retrieved if relevant information regarding the use of urodynamics and incontinence assessment were found.The high number of papers retrieved highlights how broad this field is . An overFilling and voiding cystometry is the most commonly performed procedure. It has a number of accepted and debated indications for use .This test involved the placement of fluid-filled or solid-state pressure transducers into the bladder and rectum together with a fine catheter to fill the bladder artificially; allowing simultaneous measurement of intra-abdominal and intra-vesical pressure. Subtraction of these recorded pressures calculates the subtracted bladder pressure (detrusor pressure). While the bladder is filled artificially observations and measurements regarding detrusor overactivity, bladder capacity, bladder sensation, compliance and provoked or unprovoked leakage can be made. At the maximum cystometric capacity a voiding study is performed measuring the pressure and flow characteristics of micturition. Filling of the bladder with radio-opaque contrast and the use of fluoroscopic screening during filling, provocation and voiding is termed video urodynamics; this allows urinary leakage to be observed together with anatomical detail of the outlet during provocation and voiding. Provocation testing is ideally performed at a bladder volume > 150 ml using standardized increases in intra-abdominal pressure produced by coughing or valsalva maneuver to measure the pressure required to overcome urethral opening pressure; abdominal leak point pressure (ALPP) or valsalva leak point pressure (VLPP). Alternatively, provocation can be used to induce involuntary detrusor contractions such as turning on a water tap.Ambulatory bladder monitoring is performed using the same principles as conventional cystometry but without any artificial filling; the patient\u2019s bladder fills normally by their own urine production. This allows miniaturization of monitoring equipment leaving the patient free to move around. It is thought to be more representative of day-to-day life and may give more accurate information regarding lower urinary tract function. Usually, at least two filling and voiding cycles are captured over a four-hour period during which the patient drinks normally and keeps a diary of symptoms and events while a portable recording device continually measures intra-abdominal and intra-vesical pressure. The traces should be interpreted with the patient present to allow more diagnostic information to be collected. The catheter positions should be checked periodically.Urethral pressure profilometry is a technique designed to measure urethral closure pressure and is defined as the difference between the intra-luminal pressure in the urethra and the intra-vesical pressure at rest or during stress. For continence to be achieved, urethral pressure must exceed intra-vesical pressure at all times, except for micturition. In the static test a catheter with two separate transducers is placed with the top one in the bladder and the more distal one in the mid-urethra connected to a gradual infusion. In the dynamic test the catheter is withdrawn until the maximum urethral pressure is reached. Stress tests are used to illicit leakage.Urethral retro-resistance profile is a noninvasive technique involving the retrograde infusion of fluid against the closed sphincter allowing measurement of the pressure required to open the sphincter. Only a few studies have evaluated this technique but initial results were thought to be promising; differentiating women with incontinence from continent controls. There is also evidence it may be able to categorize patients into symptom severity but further studies are needed to replicate the findings of the originators of the technique.The ICS have detailed precise reports on urodynamic techniques in an effort to create standardized methodology; these should be consulted for more in-depth information.10\u201312The goal of urodynamics is to reproduce the patient\u2019s symptoms and provide a physical explanation of their cause; in this case to demonstrate incontinence and to differentiate between sphincter weakness \u2013 urodynamic stress incontinence, and involuntary bladder activity \u2013 detrusor overactivity incontinence (diagnosis). Various parameters are then used to try and distinguish between symptom severity, urethral hypermobility and intrinsic sphincter deficiency, and identify patients who have detrusor overactivity to facilitate discussion of treatment options (communication). The final aim is to be able to predict treatment outcome enabling the patient and clinician to choose the most appropriate intervention (prognosis).2O, or detrusor overactivity without urodynamic stress urinary incontinence in case of suspected stress urinary incontinence.[Lemack and Zimmern studied the predictive value of the Urogenital Distress Inventory (UDI-6) questionnaire for urodynamic outcomes in a retrospective study of 174 women. No single question was able to predict patients who had urodynamic stress incontinence or detrusor overactivity incontinence on cystometry. Combining a high score to the question related to leakage with physical activity and a history of previous incontinence surgery identifies 91% of \u2018critical\u2019 diagnoses in women with a predominant symptom of stress incontinence, urodynamic stress urinary incontinence with detrusor overactivity, abdominal leak point pressure < 60 cmHntinence.et al, performed a literature review including 5192 women with incontinence from 23 studies to identify how well incontinence symptoms related to urodynamic findings. The sensitivity was 0.82, 0.69 and 0.51 for stress, urgency and mixed urinary incontinence respectively. They therefore suggested that cystometry was more accurate for the diagnosis of women with stress incontinence. The specificity of urodynamics was similar in all the three groups.[Colli e groups.et al, including 3428 women found that only 9% could be classified with stress urinary incontinence through the King\u2019s Health Questionnaire.[Another large retrospective study by Digesu ionnaire. Of this A small observational study by Fitzgerald and Brubaker correlatet al, using a small cohort of 30 patients showed that detrusor overactivity is not easily reproducible on repeat cystometry with 10% of patients having stable detrusor pressure on their second investigation; in addition 70-80% of patients had significant changes in other measured urodynamic parameters.[Hashim and Abrams explored whether urgency was related to a urodynamic diagnosis of detrusor overactivity in a study of 1809 patients with overactive bladder syndrome. They fouAll the evidence is conflicting; symptom scores do not appear to reliably predict urodynamic findings and are therefore not a replacement for cystometry in the assessment of patients with incontinence.Cystometry can characterize detrusor overactivity incontinence and urodynamic stress incontinence with an accuracy ranging from 60\u201390%, and the results help facilitate discussion of treatment options with the patient. It remains uncertain, however, how well a preoperative urodynamic diagnosis predicts outcome of treatment and consequently whether testing is cost-effective.There are no large randomized trials comparing patients undergoing surgery for stress urinary incontinence with or without preoperative urodynamics, only small retrospective studies. One study of 212 women undergoing retropubic surgery for stress urinary incontinence compared three groups; Group One had basic assessment and video cystometry, Group Two basic assessment only, and Group Three had basic assessment and cystourethroscopy. Follow-up was over a 14-year period by annual questionnaire. No difference was found in postoperative continence rates between the three groups.Laurikainen and Kiiholma performed mid-urethral tape procedures (TVT) on 191 patients based on a clinical assessment alone and compared the outcome to historical cohorts of women who underwent preoperative cystometry. They found no difference in cure rates for incontinence (88% in both groups).It is clear that guidelines are based on small retrospective studies which did not show that urodynamics predicted outcome. To improve the level of evidence for this contention, large prospective randomized studies comparing standardized urodynamics with clinical assessment are currently being planned and conducted in the UK and United States.Currently, the ICS does not recommend sub-categorization of urodynamic stress incontinence according to pressure or anatomical criteria such as Blaivas score, ALPP or VLPP.A reliable and reproducible marker of severity of stress urinary incontinence has long been searched for with clinical measures such as questionnaires, pad tests and incontinent episodes, and urodynamic measures such as abdominal leak point pressurenone and urethral pressure profilometry all having been investigated.2O correlated with poor surgical outcome.[It is well documented that women with stress urinary incontinence have much lower maximum urethral closure pressures than those without, with the magnitude of difference being dependent on symptom severity.22 One st outcome. The wide outcome.et al, demonstrated an inverse relationship between abdominal leak point pressure and severity of stress urinary incontinence.[2O and Blaivas Type Three urodynamic stress incontinence, a group with abdominal leak point pressure 60-89 cmH2O and Blaivas Type Two stress urinary incontinence and finally a group with abdominal leak point pressure > 90 cmH2O and Blaivas Type One incontinence. They suggested that the measurement could guide surgeons on which intervention to use; autologous bladder neck slings for women with values < 60 cmH2O (Type Three), for example. A later study examined 79 women who underwent both abdominal leak point pressure measurement and urethral pressure profilometry; all had urodynamic stress urinary incontinence without previous surgery and were planned to undergo insertion of mid-urethral tape (TVT).[2O for abdominal leak point pressure and 30 cmH2O for maximum urethral closure pressure were used to categorize the women. No statistical association between abdominal leak point pressure and maximum urethral closure pressure was found; suggesting they are measuring different pathophysiological events. Lemack has subsequently published many papers concerning the diagnostic accuracy and clinical usefulness of both abdominal leak point pressure and urethral profile profilometry[et al, reported a study of 362 women who underwent mid-urethral tape insertion (TVT) which found no difference in outcome when stratified into intrinsic sphincter deficiency and urethral hypermobility groups according to abdominal leak point pressure and maximum urethral closure pressure measurements.[Abdominal leak point pressurenone measurements are reproducible but the lack of standardized methodology makes it difficult to compare studies. An early study by McGuire ntinence. Using a pe (TVT). Cutoff vfilometry28 in whiurements. In a furet al, have reviewed the literature on treatment of mixed urinary incontinence to gauge the impact that a prior diagnosis of detrusor overactivity had on treatment outcome.[et al. conclude that patients with mixed urinary incontinence can be treated with colposuspension and tape procedures. If urgency symptoms persist they can be treated with conventional treatment modalities. A further insight was made by an additional study of 35 women with mixed urinary incontinence which found that those with persistent detrusor overactivity after surgery also had a low maximum flow rate preoperatively.[Patients with stress urinary incontinence often have concurrent symptoms of urgency and frequency defining overactive bladder syndrome, and in a proportion this will be reflected by a finding of detrusor overactivity on cystometry. It is of some concern that the preoperative presence of detrusor overactivity will prejudice the outcome of surgery for presumed outlet weakness. Lai outcome. They quo outcome.\u201335 Lai eratively. These smet al, commissioned by the UK National Health Service found few well-powered primary studies, making valid conclusions difficult.[Although significant research regarding clinical and urodynamic assessment of urinary incontinence exists, an extensive and authoritative review by Martin ifficult. A thorouThe degree to which cystometry affects surgical decision-making and prediction of outcomes is unclear. The evidence was of low level; consisting mainly of small case series. This, together with a lack of standardization in urodynamic terms and techniques in the literature makes meaningful interpretation and comparison of studies difficult. The limited evidence available does point to a degree of correlation between symptom severity, urodynamic diagnosis and surgical outcome. The validity and clinical usefulness of urethral closure pressure assessment remains subject to much discussion due to poor reproducibility, uncertain ability to differentiate between intrinsic sphincter deficiency and urethral hypermobility, and lack of influence of deciding on the primary surgical procedure. There is therefore a widely acknowledged need for large, well-designed prospective studies to address these issues and these are now in progress."} +{"text": "Immunocytochemical staining with monoclonal antibody NCRC 11 of formalin fixed paraffin embedded tumour tissue has been studied in 444 cases of primary breast cancer with a minimum follow period of 6 years. The relationship between extent of staining, assessed on a four point scale, and patient survival has been confirmed. There are significant relationships between staining and both histological grade and oestrogen receptor status. No association has been shown between staining and lymph node stage or tumour size. Simplification of staining assessment by modification to two staining groups still allows significant separation of patients into prognostic groups and incorporation into an existing prognostic index."} +{"text": "One of the major problems met for the conception of antiviral or antiparasitic drugs is toreach a high level of selectivity towards the pathogenic agent versus the host. We shall describetwo synthetic approaches where main group organometallics have been used towards this goal. Aseries of nucleoside sila-analogues was synthesized as potential therapeutic agents designed toinhibit HIV Reverse Transcriptase. In a second approach novel organosilicon derivatives havebeen synthesized as mimics of antisense oligonucleotides.Infectious agents, namely viruses or parasites, more or less use cellular machinery. Thereforetherapeutic agents must interfere with biochemical mechanisms or possess high affinity towardsspecific molecular cellular components, to reach selectivity.We thought that main group organometallics could show many advantages for designingbiologically active molecules in this field. They allow a high synthetic flexibility for the modulationsof physico-chemical properties and they show a mechanistic behaviour which may be close to theone of several heteroelements present in living organisms such as sulfur or phosphorus.We tried to use this approach towards two directions involving the synthesis of organosiliconderivatives i.e:-the synthesis of organosilicon derivatives as inhibitors of HIV Reverse Transcriptase,-the synthesis of organosilicon precursors of modified antisense oligonucleotides."} +{"text": "Subcellular fractions from an aminoazo dye induced rat hepatoma (D23) were examined for their ability to evoke rejection responses in syngeneic hosts to transplanted tumour cells and to induce the production of humoral antibody. Membrane fractions isolated by zonal centrifugation and displaying an increased activity of tumour specific antigen , as well as crude membrane fractions and purified tumour cell ghosts, all elicited tumour specific antibody demonstrable by membrane immunofluorescence staining of viable hepatoma D23 cells. Tumour cell nuclei or soluble cytoplasmic protein were, however, lacking in this capacity. Resistance to tumour cell challenge was not observed in rats treated with any of the hepatoma D23 subcellular fractions administered by various routes either alone or in admixture with bacterial adjuvants. These findings are relevant to current views that tumour immunity may be more optimally achieved by inoculation of intact (viable or attenuated) tumour cells."} +{"text": "These findings suggest that testing cloacal or oral swabs might be a low-resource approach to detect WNV in dead birds.We evaluated if postmortem cloacal and oral swabs could replace brain tissue as a specimen for West Nile virus (WNV) detection. WNV was detected in all three specimen types from 20 dead crows and jays with an average of >10 West Nile virus [WNV]) transmission throughout the eastern United States Since 1999, surveillance of bird deaths has become a standard epidemiologic method for detecting the spread and continued presence of West Nile virus on cloacal and oral swabs of corvidGiven that birds with acute WNV infection frequently shed the virus in cloacal or oral cavities \u20136 and thCorvus brachyrhynchos), 4 Fish Crows (Corvus ossifragus), and 4 Blue Jays (Cyanocitta cristata), that had died (or in one case had been euthanized after being moribund for 24 hours) after experimental infection with the New York 1999 strain of WNV. , especially corvids, for detection of infectious virus particles or RNA in brain or other viscera. We have shown that postmortem oral and cloacal swabs, in addition to brain, are effective samples to collect for WNV detection in experimentally infected corvids. A potential implication of these findings, pending field trials using corvids and other species routinely collected as part of avian mortality surveillance, is that WNV may be detected by simply collecting swabs from carcasses and forwarding the swabs (frozen) to a virology laboratory for testing. Eliminating multiple steps currently necessary for WNV testing of bird carcasses may conserve valuable public health resources and reduce the risk of exposure for laboratory personnel."} +{"text": "An explicit formal-ontological representation of entities existing at multiple levelsof granularity is an urgent requirement for biomedical information processing. Wediscuss some fundamental principles which can form a basis for such a representation.We also comment on some of the implicit treatments of granularity in currentlyavailable ontologies and terminologies ."} +{"text": "During echocardiographic examination, respiration induces cyclic physiological changes of intracardiac haemodynamics, causing normal variations of the right and left ventricle Doppler inflows and outflows and physiological variation of extracardiac flows. The respiration related hemodynamic variation in intra and extracardiac flows may be utilized in the echocardiography laboratory to aid diagnosis in different pathological states. Nevertheless, physiologic respiratory phases can cause excessive translational motion of cardiac structures, lowering 2D image quality and interfering with optimal Doppler interrogation of flows or tissue motion.This review focuses on the impact of normal respiratory cycle and provocative respiratory maneuvers in echocardiographic examination, both in physiological and pathological states, emphasizing their applications in specific clinical situations. Respiration induces cyclic physiological modification of intracardiac haemodynamics. Changes are related to variations in intrathoracic and intraabdominal pressure, systemic and pulmonary venous return, intrapericardial pressure, pericardial constraint and interdependence between the four cardiac chambers. With inspiration intrathoracic and intrapericardial pressures decrease. This results in augmented right ventricular filling and stroke volume and, as the total pericardial space is limited, a compensatory decrease in left ventricular stroke volume occurs in early inspiration. With expiration, intrathoracic and intrapericardial pressures increase, resulting in mild decrease in right ventricular diastolic filling and a subsequent increase in left ventricular filling.Because of the constraining effect of the pericardium on the combined volume of the four cardiac chambers, respiratory variation in intrapericardial pressure results in reciprocal variation in the filling of both ventricles .With inspiration the anteroposterior diameter of the chest increases, and the lungs inflate and expand particularly anteriorly, partly filling the space between the heart and the thoracic cage. Inspiratory movements do not only decrease the amount of cardiac tissues that lies close to the sternum through anterior pulmonary expansion, but also produce a posterior displacement and rotation of the heart relative to a fixed echo beam .Expiration will give a better parasternal and often apical access to the heart by lungs deflation and diastolic (D) flow velocities as well as retrograde A velocity compared to apnea. Figure .The normal forward biphasic flow velocity pattern in the superior vena cava (SVC) with systolic flow velocity greater than diastolic flow velocity, shows inspiratory changes similar to those of hepatic vein flow.Respiratory variations are minimal for pulmonary vein flow Figure Diaphragmatic traction associated with respiration changes the position of intracardiac structures in relation to a fixed Doppler sample volume causing errors in velocities measurement.The Doppler sound beam should be oriented as parallel as possible to the flow, guided both by the 2D image (sometimes assisted by color flow imaging). Small (<20 degrees) deviations in angle produce mild (<10%) errors in velocity measurements. Although these errors may be acceptable for low-velocity flows, when Doppler is used to derive pressure gradients even a small error in velocity measurement can lead to significant underestimation of the gradient because of the quadratic relation between velocity and pressure gradient. The effects of respiration can be minimized by taking measurements during short periods of apnea or by averaging multiple consecutive beats ,8.The phase of respiration affects Doppler Tissue Imaging (DTI) recordings in the same manner because the shift in heart position during respiration over a fixed Doppler sample volume leads to inaccurate recording of the annular velocities Figure .Whenever possible, the echocardiographer should obtain annular DTI recordings during end-expiratory apnea Figure . Once thEvaluation of the inferior vena cava (IVC) from the subcostal view is part of the routine TTE examination. The diameter of the IVC decreases in response to inspiration with minimal size observed at end inspiration when the negative intrathoracic pressure leads to an increase in RV filling from the systemic veins. IVC size is significantly influenced by patient position, being largest in the right lateral position, intermediate in the supine position, and smallest in the left lateral position . The dia\u2022 The normal IVC diameter is less than 1.7 cm and there is a 50% decrease in the diameter when the RA pressure is normal (0\u20135 mm Hg).\u2022 A dilated IVC (>1.7 cm) with normal inspiratory collapse (>50%) is suggestive of a mildly elevated RA pressure (6\u201310 mm Hg).\u2022 When the inspiratory collapse is less than 50%, the RA pressure is usually between 10 and 15 mm Hg.\u2022 Finally, a dilated IVC without any collapse suggests a markedly increased RA pressure of greater than 15 mm Hg. Figure .A small IVC with spontaneous collapse is often seen in the presence of intravascular volume depletion .In a recent paper, Brennan et al evaluated the IVC diameter in patients undergoing right heart catheterization . The mea(1) High collapsibility with a small or normal-sized IVC; RAP is very likely low (<5 mm Hg).(2) High collapsibility with a large IVC or normal collapsibility with a small/normal-sized IVC; RAP is probably between 0 and 10 mm Hg(3) Normal collapsibility with large IVC; RAP is 10 to 15 mm Hg(4) Low collapsibility with a large IVC; RAP is clearly high (10\u201320 mm Hg)(5) RAP in patients with low collapsibility and a normal-sized or small IVC should be interpreted as indeterminate.There are several additional conditions to be considered when evaluating the IVC:\u2022 Athletes have been shown to have dilated IVCs with normal collapsibility index\u2022 In mechanically ventilated patients, the relationship between IVC and RAP is controversial; it was shown that a dilated IVC did not always indicate a high RA pressure Nonetheless a small IVC (< 1.2 cm) had a 100% specificity (with a low sensitivity) for a RA pressure of less than 10 mm Hg. It was also suggested that the IVC diameter measured at end expiration and end diastole using M-mode echocardiography correlates better with RA pressure than IVC diameter at end-expiration without ECG synchronization .In patients with chronic obstructive pulmonary disease (COPD) pulmonary hypertension (PH) is sometimes difficult to assess by direct evaluation using the tricuspid regurgitant Doppler velocity because of emphysema or mediastinal deviation. An alternative method of respiration-aided evaluation of PH was proposed by Kunichika et al . AuthorsTransmitral Doppler flow parameters are widely used in the assessment of LV diastolic function. It is well known that with spontaneous respiration small changes <15%) in transmitral peak flow velocities occur in healthy subjects. While some authors found no alteration in the ratio of early-to-late peak flow velocity (E/A) % in tran.The potential effects of spontaneous respiration on mitral inflow Doppler patterns in patients with abnormal LV diastolic function are poorly understood. Tsai et al. observedIn diseased ventricles, progressive shortening of the transmitral DT and increasing E/A ratio can be seen with decreasing ventricular compliance and increasing left atrial pressure. Impaired relaxation is frequently masked by elevated filling pressures resulting in a pseudonormal flow pattern (E/A ratio >1). The Valsalva maneuver has been found to effectively unload the heart and unmask an impaired relaxation pattern and high filling pressures in patients with a baseline pseudonormal flow pattern. This maneuver is accomplished first by cessation of breathing at any point in the respiratory cycle when assessment of mitral inflow is optimized. Next, the participant exerts a firm contraction of the abdominal muscles to force air against the closed glottis for approximately 10 seconds thereby increasing the intrathoracic pressure. In order to be properly performed, the Valsalva maneuver should be standardized. This can be done by attaching a mouthpiece (e.g. the tube of a syringe from which the plunger has been detached) to a sphygmomanometer and asking the patient to blow into it in order to raise the column of Hg to 40 mm and keep it stable at this value for 10 seconds . This reChanges in transmitral E/A ratio during the Valsalva maneuver across different stages of diastolic dysfunction were recently described by Reagan et al .Because the E/A ratio increases as filling pressures rise it is generally assumed that an E/A ratio <1 (impaired relaxation pattern) indicates lower or even normal filling pressures when compared with patients who have a pseudonormal or restrictive filling pattern. However, Schwammenthal et al demonstrated that patients with an E/A ratio of <1 can nonetheless have severely elevated filling pressures. In these patients LV relaxation is so severely impaired that E/A ratio remains <1 despite increased filling pressures. The standardized Valsalva maneuver can unmask the presence of elevated filling pressures in patients with a baseline impaired relaxation pattern. The A wave will increase during the maneuver in patients with elevated filling pressures in proportion to the level of LV end-diastolic pressure, and independent of the baseline transmitral flow pattern .Figure The increased pericardial pressure in cardiac tamponade produces reciprocal respiration-related changes in right and left ventricular volumes, diastolic filling, and systolic emptying that can be documented by echocardiography. The normal effects of respiration are accentuated such that venous return and right-sided filling occur during inspiration as intrathoracic pressures fall, providing a pressure gradient from the systemic veins to the RA. Because the total intrapericardial volume is fixed by the pressurized effusion, this increased inspiratory RV filling causes the interventricular septum to shift to the left, exaggerating the normal decrease in LV filling volume and, hence, stroke volume (ventricular interdependence). Thus, in tamponade, left heart filling occurs preferentially during expiration when there is less filling of the right heart. The small normal respiratory variation in left ventricular (LV) stroke volume and systolic arterial pressure <10 mm Hg) is markedly accentuated in cardiac tamponade, resulting in the clinical finding of \"paradoxical pulse\" 0 mm Hg i-24.Echocardiography is particularly useful in demonstrating the exaggerated phasic variation in cardiac volumes and flows caused by tamponade . The diaRespiratory variation in tricuspid and pulmonary flow is more dramatic than mitral and aortic flow, but there is progressive impairment in all intracardiac flows as the degree of tamponade worsens: with inspiration, the RV early diastolic filling is augmented (>25%), while LV diastolic filling diminishes >15%). The flow velocity integral in the pulmonary artery increases with inspiration, while the aortic flow velocity integral decreases (>10%) [5%. The fThe hepatic vein flow pattern may also reflect the exaggerated respiratory phase dependency of right ventricular filling. The loss of forward flow in the hepatic veins during the expiration phase of the respiratory cycle with flow out of the hepatic veins confined to the early inspiratory phase can be observed in patients with hemodynamically significant pericardial effusion . SuperioIn constrictive pericarditis, the rigid pericardium impedes the transmission of intrathoracic pressures to the cardiac chambers. During inspiration there is a lower driving force from the lungs into the left side of the heart and the LV becomes underfilled. The constricting pericardium also results in an increase interventricular interaction, so that with LV volume decrease, there is a corresponding increase in right ventricular volume see AddIn Doppler echocardiographic studies the dissociation of intrathoracic and intracardiac pressures is manifested by an inspiratory increase in peak tricuspid flow velocity and a simultaneous decrease in mitral flow velocity, with opposite changes occurring in expiration .\u2022 PW Doppler mitral inflow: high E velocity, E/A ratio > 2, short E wave deceleration time (EdT < 160 ms); inspiration: decrease E velocity >25%, prolonged IVRT >25%; expiration: opposite changes Figure \u2022 PW Doppler tricuspid inflow: E>A; inspiration: increased tricuspid E velocity >35%, characteristic phenomenon increased TR velocity Figure \u2022 PW Doppler recordings of hepatic vein flow: inspiration-minimally increased S and D; expiration: decreased diastolic flow/exaggerated atrial reversal waves Figure \u2022 PW Doppler recordings of pulmonary vein flow: S/D ratio = 1, inspiration: decreased PV S and D waves, expiration: opposite changes\u2022 SVC Doppler usually shows a diastolic dominant pattern, minimal respiratory variation as right atrial pressure is constantly elevated throughout the respiratory cycle by the thickened, constraining pericardium\u2022 Inspiration: aortic velocity decreases (-14 \u00b1 5%), pulmonary artery velocity increases (16 \u00b1 4%)\u2022 Dilated IVC with reduced inspiratory change in diameter\u2022 Respiratory changes in the proximal aortic waveform in constrictive pericarditis, similar to those described in the LV outflow tract and transmitral pattern, are reported by some authors : an inspThere are some clinical and hemodynamic situations that cause difficulties in revealing the significant respiratory Doppler findings for constrictive pericarditis as follows:notably elevated left atrial pressure, respiratory variation of the Doppler inflows may not be present unless preload is reduced by head-up tilt or diuretics [\u2022 In patients with iuretics Not all patients with surgically proven constriction actually have increased respiratory variation of Doppler inflows [\u2022 inflows .atrial fibrillation some authors [\u2022 In patients with authors reportedafter pericardiectomy\u2022 The same pattern of constriction, with respiratory variations, can persist for variables time periods chronic obstructive pulmonary disease (COPD) [\u2022 Patients with e (COPD) without Respiratory maneuvers are very useful in echocardiographical differential diagnosis constrictive pericarditis (CP) \u2013 RCM (restrictive cardiomyopathy) as follows because ventricular filling is limited mainly by a noncompliant restrictive myocardium rather than a constrictive pericardium. In addition, these patients usually have markedly increased left atrial and pulmonary venous pressure. Thus, the normal inspiratory intrathoracic pressure decline may cause minimal change in pulmonary venous and left atrial pressure.cardiac tamponade, constrictive pericarditis, chronic obstructive pulmonary disease, but also by acute right ventricular dilatation due to right ventricular infarction or pulmonary embolism. Most of these conditions can be distinguished by clinical and morphological echocardiographic features can be reliably detected with contrast echocardiography, using agitated saline; the transthoracic and transesophageal echocardiography evaluation for detecting a patent foramen ovale is performed during normal respiration and during Valsalva maneuver During Valsalva, atrial shunting from right to left will begin during the release phase (phase III) . The ValThe right-to-left shunt of a large atrial septal defect may be nearly continuous, whereas for smaller atrial septal defects the appearance of contrast in the left atrium may be phasic, coordinated with the respiratory cycle; during inspiration, right heart filling increases, thus increasing the flow of contrast into the right atrium.In patients with obstructive hypertrophic cardiomyopathy the obstruction of the LV outflow tract is dynamic and may be mild or nonexistent at rest. Echocardiography during Valsalva maneuver can unmask latent gradients in patients without resting obstruction and reveal the basis for drug treatment choices. During the strain phase of Valsalva maneuver, due to the decrease in preload, end-diastolic left ventricular volume and afterload, SAM occurs earlier in systole, mitral-septal contact lasts longer and left ventricular outflow tract gradient increases Figure .Due to left ventricular volume decrease during Valsalva maneuver in patients with mitral valve prolapse mitral regurgitation starts earlier in systole and severity of mitral regurgitation increases.The M\u00fbller maneuver, the opposite of Valsalva, less often used in echo examinations, is performed by forcibly inspiring while the nose is held closed and the mouth is sealed for about 10 seconds. Because the maneuver exaggerates inspiratory effort, right-sided filling is augmented and can be use for the augmentation of tricuspid regurgitation.\u2022 In the absence of normal sinus rhythm, respiratory variation of Doppler flow cannot be assessed. An irregular heart rhythm does not allow a meaningful interpretation of the influence of respiration on transvalvular Doppler flow.\u2022 The Valsalva maneuver should only be performed in a standardized manner. Lack of standardization of the Valsalva maneuver can limit its feasibility and, more importantly, can lead to wrong conclusions.\u2022 The use of Valsalva maneuver is limited in certain conditions: lack of patient' cooperation; patient's inability to adequately increase intrathoracic pressure because of medical illness, recent operation, sedation (eg during TEE).\u2022 The decrease in image quality sometimes encountered during Valsalva maneuver or with normal respiration may interfere with the interpretation of the findings. However, appropriate training and regular use of respiratory maneuvers help in this regard.Using normal respiratory phases and provocative respiratory maneuvers can be very useful in characterizing normal cardiac function parameters as well as various cardiac disorders Table . The extThe authors declare that they have no competing interests.CG conceived of the review, participated in its coordination and revised the final draft of the manuscript. CCB revised the literature, prepared the final draft of the manuscript and provided echocardiographic illustrations. MI revised the literature and prepared the first draft of the manuscript. AC revised the manuscript critically for important intellectual content. BAP participated in the coordination of this review and revised the manuscript critically for important intellectual content. All authors read and approved the final manuscript.Changes in the quality of the echocardiographic image with inspiration. Transthoracic parasternal long axis view of the left ventricle recorded with normal respiration revealing a decrease in the quality of the echocardiographic image with inspiration.Click here for fileImprovement in the quality of the echocardiographic image recorded during held end expiration. Transthoracic parasternal long axis view of the left ventricle recorded during held end expiration showing a significant improvement in the quality of the echocardiographic image.Click here for fileInfluence of pulmonary interference on echocardiographic visualization of cardiac structures during normal respiration. Transthoracic apical 4 chamber view recorded during normal respiration. During inspiration pulmonary interference does not allow the visualization of cardiac structures.Click here for fileInfluence of expiration on echocardiographic visualization of cardiac structures. Transthoracic apical 4 chamber view. Expiration allows apical access to the heart by lung deflation.Click here for fileInfluence of inspiration on echocardiographic image in subcostal view. Subcostal 4 chamber view. Inspiration brings the diaphragm down, improving imaging of the heart.Click here for fileExcessive translational motion of the heart with normal respiration. Transthoracic short axis view recorded during normal respiration. The heart moves medially with inspiration.Click here for fileImage acquisition during suspended respiration (held end-expiration) in a patient with excessive translational motion of the heart with normal respiration. Transthoracic short axis view. Excessive translational motion can be avoided by acquiring images during held end-expiration.Click here for fileLack of variation of the inferior vena cava diameter during deep inspiration in a patient with dilated cardiomyopathy and severe pulmonary hypertension. The subcostal view adjusted to demonstrate the long axis of the inferior vena cava (IVC) in a patient with dilated cardiomyopathy and severe pulmonary hypertension. There is no inspiratory variation of the IVC diameter during deep inspiration.Click here for fileLack of variation of the inferior vena cava diameter during brief sniff in a patient with dilated cardiomyopathy and severe pulmonary hypertension. Evaluation of the inferior vena cava (IVC) from the subcostal view during brief sniff in a patient with dilated cardiomyopathy and severe pulmonary hypertension demonstrates no variation of the IVC diameter.Click here for filePhasic variation in cardiac volumes visualized in transthoracic basal short axis view caused by cardiac tamponade. Transthoracic basal short axis view in a patient with cardiac tamponade demonstrates diastolic collapse of the free walls of the right atrium and right ventricle.Click here for filePhasic variation in cardiac volumes caused by cardiac tamponade, visualized in transthoracic apical 4 chamber view. The intermittent collapse of the free walls of the right atrium and right ventricle visualized in the transthoracic apical 4 chamber view in a patient with cardiac tamponade.Click here for fileIncreased interventricular interaction in a patient with constrictive pericarditis. Transthoracic apical 4 chamber view in a patient with constrictive pericarditis reveals the increased interventricular interaction (with left ventricular volume decrease there is a corresponding increase in right ventricular volume).Click here for file"} +{"text": "Beside general requirements for modern automated systems, immunoassay automation involves specific requirements as a separation step for heterogeneous immunoassays. Systems are designed according to the solid phase selected: dedicated or open robots for coated tubes and wells, systems nearly similar tochemistry analysers in the case of magnetic particles, and a completely original design for those using porous and film materials."} +{"text": "Pneumococcal pneumonia is a life-threatening disease with high mortality and morbidity among children under 5 years of age, the elderly and immunocompromised individuals worldwide. Protection against pneumococcal pneumonia relies on successful regulation of colonisation in the nasopharynx and a brisk alveolar macrophage-mediated immune response in the lung. Therefore, enhancing pulmonary mucosal immunity through mucosal vaccination might be the key to prevention of pneumococcal infection. Current challenges include a lack of information in humans on mucosal immunity against pneumococci and a lack of suitable adjuvants for new vaccines. Data from mouse models, however, suggest that mucosally active vaccines will enhance mucosal and systemic immunity for protection against pneumococcal infection. Streptococcus pneumoniae (the pneumococcus) is a Gram-positive aerobic commensal bacterium which forms part of the normal flora in the nasopharynx Pneumonia accounts for 19% of all under 5 year old deaths worldwide, which makes it the most deadly infectious illness for this age group treating pneumococcal disease rather than preventing it, but with the current increase in antibiotic resistance and the HIV pandemic, it is widely accepted that prevention is the key to minimising the disease burden Pneumococcal pneumonia is treatable using antibiotic therapy. However, where treatment is delayed or unavailable mortality is high et al. unpublished). The currently licensed 7-valent conjugate vaccine (containing 7 capsular polysaccharides conjugated to a diphtheria CRM197 protein) is being used as part of childhood immunisation programmes in several countries but others are waiting for the licensing of 10-valent and 13-valent vaccines. The disadvantages of PCVs are that they are expensive, have limited serotype coverage, can be associated with an increase in disease caused by serotypes not included in the vaccine and are less effective against radiological pneumonia (20\u201337% efficacy) Vaccination offers the most efficient and cost-effective method of preventing this disease. However, there are more than 90 pneumococcal serotypes which make development of a vaccine to provide universal protection a big challenge. There are two formulations of pneumococcal vaccines that have been licensed thus far: polysaccharide vaccines (PPVs) and protein conjugate vaccines (PCVs). The 23-valent pneumococcal polysaccharide vaccine, which contains purified capsular polysaccharide antigens from 23 serotypes, offers some protection against invasive pneumococcal disease in adults but is not effective in either children less than 2 years of age or immunocompromised adults There are several key developments that would result in a breakthrough in the global control of pneumococcal disease. Use of the PCV is an important landmark This review will focus on recent advances in our understanding of mucosal immunity relevant to pneumococcal infection and, in particular, the critical immune responses that must be augmented by new vaccines.S. pneumoniae involving phagocytes (neutrophils and macrophages), B cells and T cells rapidly eliminates colonisation, whereas a poor mucosal immune response results in protracted colonisation S. pneumoniae.Pneumococcal colonisation of the upper respiratory tract precedes infection of the lower respiratory tract, but is normally asymptomatic and not usually followed by disease S. pneumoniae. CRP is an acute-phase protein which is mainly found in serum and elevated during inflammation S. pneumoniae and Haemophilus influenzae), which includes activation of complement by the classical pathway, enhancing opsonisation and inhibition of the attachment of bacteria to epithelial cells Innate factors play a crucial role in the host defence against colonisation with S. pneumoniae and H. influenzae) suggest that successful clearance of pneumococci in the nasopharynx resulted from opsonisation by complement, followed by phagocytosis by neutrophils which were recruited to the mucosal surface S. pneumoniae and subsequently played an important role systemically It is known that complement plays a role in protection against pneumococcal infection through the promotion of opsonophagocytosis The innate response includes cellular responses from neutrophils and macrophages. It has been demonstrated in murine models that pneumococcal colonisation of the upper respiratory tract triggers an acute inflammatory response characterised by a robust influx of neutrophils into the lumen of the paranasal spaces S. pneumoniae secrete a zinc metalloprotease which inactivates IgA1 (a subclass of IgA). Furthermore, the cleaved IgA1 fragment might assist translocation of the opsonised bacteria across the host respiratory epithelium Nasopharyngeal colonisation stimulates the production of secretory IgA antibodies and serum IgG S. pneumoniae (+ T cells (MHC-II knockout mice) show prolonged carriage, suggesting an important role for CD4+ T cells rather than antibody-mediated immunity Several recent studies suggest that other mechanisms of protection against pneumococcal carriage are required, in addition to antibody-mediated immunity. Firstly, the course of an experimental colonisation is not affected in mice that are unable to produce pneumococcal-specific antibody eumoniae . The int+ T cells (Th17) which produce IL-17A et al. showed that blocking IL-17A in mice models reduced immunity to pneumococcal colonisation following intranasal immunisation with cell wall polysaccharide in vivo against pneumococcal carriage Studies have shown that immunity to pneumococcal colonisation is mediated by a specific subset of CD4et al. showed that primary challenge with pneumococci in mice generates CD4+ T cell memory, resulting in enhanced Th17-mediated recruitment of neutrophils after secondary pneumococcal challenge IL-17A-mediated protection against pneumococcal colonisation results in recruitment of neutrophils into the upper airway lumen to clear bacterium If pneumococci colonising the human nasopharynx are aspirated into the distal airways and alveolar air spaces, they will interact with pulmonary defence mechanisms. The bacteria will either be cleared or cause disease. Excessive replication of the bacteria in the alveoli triggers infiltration of immune cells which \u2013 if not properly regulated \u2013 impairs gas exchange resulting in the clinical syndrome of pneumonia.The process of bacterial clearance in the lung is a highly regulated process \u2013 an excessive response might potentially lead to tissue damage, whereas a weak response leads to exponential growth of the pathogens. The primary host immune defence against small numbers of pneumococci during early infection is phagocytosis Alveolar macrophages are the first cells that combat pneumococci during early infection in vitro to suggest that alveolar macrophages are able to present antigens to T cells, although less effectively than other antigen-presenting cells (APCs) + T cells as a result of antigen recognition in the absence of co-stimulation It is still unclear whether antigen presentation occurs in the lung, in the draining lymph nodes, or both, and whether alveolar macrophages are part of this antigen presentation process. There is data When the alveolar bacterial load rises above a critical threshold, alveolar macrophages cease to perform effective opsonophagocytosis and produce an increased proinflammatory cytokine response dominated by TNF-\u03b1 and IL-8 When a proinflammatory signal (TNF-\u03b1 and/or IL-8) is produced in the alveolus, there is upregulation of adherence molecules on endothelial cells which bind to their receptors on neutrophils. This results in rolling of neutrophils along the endothelial wall and transmigration into the alveolar space in a process called chemotaxis in vivo coincided with the phase when bacterial growth ceased T cells are also recruited in high numbers to the lung in late infection \u2013 the peak of T cell infiltration in the lung during intranasal pneumococcal infection in mice Following clearance of pneumococci from the lungs, neutrophils, some macrophages and T cells undergo rapid apoptosis. Macrophage apoptosis leads to reduced TNF-\u03b1 expression, which in turn results in reduced neutrophil recruitment and enhanced neutrophil apoptosis In summary, mucosal responses are critical in regulating pneumococcal carriage and defence against infection. Therefore, enhancing pulmonary mucosal immunity might be an effective strategy in the prevention of pneumococcal disease.S. pneumoniae induces both mucosal and systemic humoral and cellular immune responses Salmonella enterica serovar Typhimurium enhances protection against S. pneumoniaeMucosal exposure to + T cells and independent of antibody and bacterial serotype S. pneumoniaeMucosal immunisation of experimental animals has been shown to elicit protection against carriage S. pneumoniae pathogenesis, and several are particularly relevant to protection against carriage et al. demonstrated that a fusion conjugate, including cell wall polysaccharide coupled to pneumolysin and PsaA, delivered intranasally with cholera toxin, protected mice against experimental pneumococcal colonisation An ideal mucosal vaccine would include several pneumococcal proteins such as pneumolysin, PspA, PsaA or PspC Pneumococcal conjugate vaccine is less effective against pneumonia than against invasive pneumococcal disease Lactococcus lactis increased the clearance rate of S. pneumoniae from the lung and prevented invasion of pneumococci into blood L. lactis increased phagocyte activation in lung, blood and bone marrow of the vaccinated mice Mucosal vaccination has shown promising results in protection against pneumococcal lung infection There are also some data to suggest that mucosally administered vaccines might actually provide better protection against both mucosal and systemic disease than conventional parenteral (systemic) vaccines. Immunisation of mice with lactococcal PspA vaccine elicited better protection against respiratory pneumococcal challenge than conventional parenteral PspA vaccine in intraperitoneal sepsis and intranasal respiratory infection models In summary, there are encouraging data to support the role of mucosal vaccination in protection against both mucosal and systemic pneumococcal disease. However, lack of a suitable adjuvant is a major obstacle to success, but cytokine adjuvants might be useful We have discussed the role of mucosal immunity and reviewed available data on mucosal immunisation against pneumococcal disease. There is evidence from murine studies to suggest that mucosal immunisation against pneumococci induces mucosal and systemic immunity more effectively than parenteral vaccination. The data from humans, however, are insufficient to draw firm conclusions. Further studies using lung, nasal and other mucosal samples from humans are needed.S. pneumoniae. These correlates of protection might then help in predicting efficacy to future vaccines. Research questions for future work in the field are outlined in Immediate priorities include the need to address the role of T cell-mediated immunity against pneumococcal colonisation and infection in humans. Such data might clarify the human correlates of protection or immunity to Strategic decisions regarding future pneumococcal vaccines need to determine whether to focus on improving the current conjugate vaccines or developing new pneumococcal protein based vaccines. Pneumococcal conjugate vaccines are currently being used in several developed countries because they provide good protection against systemic disease. Their main disadvantage is that they have limited serotype coverage and are less effective against mucosal disease. By contrast, pneumococcal protein based vaccines have the potential to offer universal coverage as well as offer protection against both mucosal and systemic disease if delivered through the mucosal route. Vaccination still remains the key to minimising the high burden of pneumococcal disease worldwide. We believe that alternative routes of immunisation might help in improving the efficacy of pneumococcal vaccines to both mucosal and systemic disease."} +{"text": "This case report describes a laparoscopic sacral colpopexy using Mersilene mesh in a patient with complete vaginal vault prolapse. Mersilene mesh was placed as a hammock between the vaginal apex and the anterior surface of the sacrum, using intracorporeal needles and an extracorporeal knot tying technique. Minor modifications are made from the traditional abdominal approach, because the patient had previously undergone a pelvic lymphadenectomy and vaginal cuff radiation for a stage IB grade 1 adenocarcinoma of the endometrium."} +{"text": "Heat shock proteins are highly conserved proteins present in organisms ranging from bacteria to man. They are both dominant microbial immunogens and among the first proteins produced during mammalian embryo development. Since bacterial and human heat shock proteins share a high degree of amino acid sequence homology, it has been suggested that sensitization to bacterial heat shock proteins during an infection may result in autoimmunity to human heat shock proteins. Infertile couples seeking in vitro fertilization (IVF) may have been previously sensitized to bacterial heat shock proteins as a consequence of an asymptomatic upper genital tract infection. Due to daily clinical monitoring and precisely timed fertilization these patients are an ideal study group to investigate the effect of prior sensitization to heat shock proteins on preimplantation embryo development and implantation failure. Immune sensitization at the level of the cervix to the 60 kD heat shock protein (hsp60) has been associated with implantation failure in some IVF patients. Similarly, the highest prevalence of circulating hsp60 antibodies among IVF patients was found in the sera of women whose embryos failed to develop in vitro. To more directly assess whether humoral immunity to hsp60 influenced in vitro embryo development, a mouse embryo culture model was established. Monoclonal antibody to mammalian hsp60 markedly impaired mouse embryo development in vitro. These data suggest that immune sensitization to human hsp60, possibly developed as a consequence of infection, may adversely affect pregnancy outcome in some patients."} +{"text": "Human leukaemia cells isolated from peripheral blood were employed as targets for natural killer (NK) cells obtained from healthy donors and the effect of pretreatment of leukaemia cells with Actinomycin D on lysability was analysed in a chromium release assay. In 8/14 leukaemia cell samples a substantial enhancement of specific release could be repeatedly obtained by exposure of leukaemia targets to Actinomycin D for 4 h. The phenomenon was seen both with interferon-treated and untreated NK cells and could be demonstrated with fresh, as well as, liquid nitrogen stored leukaemia cells. In contrast, lysis of two leukaemia cell lines could not be further enhanced and no release was seen from normal lymphocyte targets or mitogen-induced blasts. Cold target inhibition studies indicate that enhanced killing is mediated by the same kind of natural killer cell, which is active against the Molt4 and K562 leukaemia cell lines."} +{"text": "When visual input has conflicting interpretations, conscious perception can alternate spontaneously between competing interpretations . There i \u25ba Structure of superior parietal lobe (SPL) predicts switch rate in perceptual rivalry \u25ba White-matter integrity in SPL correlates with individuals' switch rate \u25ba Deactivation of SPL with transcranial magnetic stimulation slows perceptual rivalry We recorded subjective reports of spontaneous alternations for an ambiguous rotating structure-from-motion (SFM) stimulus that evokes bistable perception fluctuating between two rotation directions A 10]. A. A10]. AWe found a significant negative correlation between cortical thickness and percept duration across individuals in the bilateral superior parietal lobule and bilateral postcentral gyrus . The negTo cross-validate our findings, we conducted a voxel-based morphometry (VBM) analysis of GM density on the sThe differences between cortical thickness and GM density analyses could be due to a number of factors such as measurement of different aspects of GM structure . On the Having established a significant correlation between an individual's bistable percept duration and GM thickness/density in the bilateral parietal cortex, we next examined the contribution of WM integrity to individual differences in the bistable percept duration. This analysis used an independent data set of diffusion-weighted images (DWIs). The FA values derived from DWIs provide an estimate of the integrity of WM tracts . To inveThe results of this analysis are shown in Our findings so far only establish that brain structure is correlated with fluctuations in conscious perception and cannot on their own determine whether these structural variations also play a causal role in generating such perceptual switches. In addition, our finding of variability in homologous bilateral cortical structures could conceivably reflect unrelated covariation between hemispheric structures. For example, GM density in parietal regions correlates with the homotopic region in the contralateral hemisphere . TherefoTherefore, we now investigated whether the anatomical loci that we identified also played a causal role in mediating switches by using transcranial magnetic stimulation (TMS). In\u00a0separate sessions, we temporarily disrupted the function of right SPL or left SPL by delivering continuous theta-burst stimulation (cTBS) . If\u00a0actiRepeated-measures analysis of variance on the changes in percept duration with respect to cTBS revealed that there was a significant difference among the stimulation conditions = 15.2, p < 0.01). Post hoc least significant difference tests revealed that cTBS over the right SPL and the left SPL significantly increased percept durations compared to the control condition . These rTaken together, our findings reveal that differences in brain\u00a0structure in focal regions of bilateral parietal cortex can\u00a0account for individual differences in conscious visual perception, and that the same regions showing such structural variability also play a causal role in such perception. Between-participant variability in bistable perception has been documented, but the neural basis underlying the variability has been neglected for many years . The resThe SPL loci identified here are anatomically very close to those parietal regions that show transient activations when participants shift their attention between locations or\u00a0betweOur findings offer a possible neural account for hitherto unexplained relationships that have been found between switch rate and the effects of psychiatric mood disorders, normal aging, or brain damage. For example, patients with bipolar disorder show cortical thinning compared with matched controls in multiple cortical regions, including a region close toA recent study that compared monozygotic and dizygotic twins found that about half (52%) of the variability in spontaneous switches in perceptual rivalry can be accounted for by genetic factors . Our preHow differences in the structure of SPL result in differences in perceptual switch rate remains unclear. One possible mechanism is the differences in the strength of feedback signals from SPL to early sensory areas that reset the neuronal activities supporting the current percept. If the SPL is large and strongly connected to early visual areas, the impact of the feedback signals would also be stronger and therefore trigger perceptual switches at a higher rate.In the present study, we used SFM as a representative bistable stimulus. Thus, whether our findings generalize to\u00a0a broader range of bistable stimuli remains an open question. However, previous studies suggest the existence of neural substrates common to a range of bistable stimuli. For example, temporal pattern of alternations reported during binocular rivalry is highly correlated with that of motion-induced blindness . MoreovePrevious functional neuroimaging and stroFinally, our work shows that intervention with TMS may be a particularly powerful approach for validating findings from independent structural analyses of the brain. Our approach of linked anatomical and functional studies may be particularly relevant and useful for addressing a situation that may arise in large-scale correlational studies such as VBM. Highly specific interventional studies with TMS can be combined with highly sensitive correlational methods such as VBM and FA to confirm the causal link between the behavior of interest and brain regions identified by structural analyses.Taken together, our findings reveal that in humans, the cortical thickness and GM density in remarkably focal regions of parietal cortex can account for differences in how conscious visual perception fluctuates over time, and that the same regions showing this structural variability also play a causal role in bistable perception. We speculate that other aspects of conscious visual perception may similarly have an unexpected motif in human brain structure.Full details of the methods used are provided in the A total of 52 healthy volunteers were recruited for the individual differences experiment; 12 healthy volunteers were recruited for the TMS experiment. We obtained written informed consent from all participants, and the experiments were approved by the local ethics committee (University College London).Reconstruction of the pial surface and GM/WM boundary was performed for T1-weighted magnetic resonance (MR) images via the fully automated procedure implemented in FreeSurfer software . The thihttp://www.fil.ion.ucl.ac.uk/spm). Subsequently, we performed diffeomorphic anatomical registration through exponentiated lie algebra (DARTEL) [The same set of T1-weighted MR images were used for the VBM analysis. The MR images were first segmented for gray matter and white matter by using the segmentation tools in SPM8 ((DARTEL) for inteFA was calculated by using Functional Magnetic Resonance Imaging of the Brain (FMRIB)'s Diffusion Toolbox (FDT v2.0) applied to the diffusion-weighted MR images. FA images were coregistered with a standard template (FMRIB58). The standardized FA images were smoothed with an isotropic Gaussian kernel (FWHM = 8 mm) for multiple regression analysis. We used SVC based on the coordinates of parietal sites revealed in the VBM analysis. We used p < 0.05 corrected for the small volume as the criterion to detect voxels with a significant correlation with an individual's percept duration.The stimuli and procedure for the TMS experiment were identical to the experiment used for estimating individuals' percept duration for the correlation studies above. Mean percept duration was compared before and after cTBS. Participants completed three blocks reporting their ambiguous motion percepts before the TMS session and another three blocks immediately after. The three stimulation sites were tested on separate days, and the order of the sites was randomized for each subject."} +{"text": "Thirty-five patients with pure seminoma, and 34 patients with teratoma but without any postoperative evidence of residual or recurrent tumour, consistently had normal serum AFP levels (less than 25 ng/ml). Of 84 patients with active teratomas, 56 (67%) had serological evidence of AFP production. Ten patients with histological evidence of pure yolk sac tumours all had raised levels. Teratomas containing yolk sac (elements may or may not be associated with raised serum levels. Trophoblastic (choriocarcinomatous) elements in a teratoma were not normally associated with high values. Fourteen patients with teratomas had elevated levels in the absence of histologically detectable yolk sac elements. Serum AFP levels often became elevated before clinical evidence of recurrence, so that AFP can act as an effective marker of the course of the disease and its response to therapy in many patients, but recurrent or progressive disease may be present in the absence of raised levels."} +{"text": "Perpetrators of Factitious Disorder by proxy are usually driven by motives such as garnering attention, mobilizing sympathy, acting out anger or controlling others. Widespread media coverage provides an opportunity for fulfilling all these needs. We describe a case of Factitious Disorder by proxy with a rather unusual ocular complaint. Circumstantial evidence indicates that the presentation may have been influenced by a similar case from the same locality in the preceding month, which received extensive media attention. The role of media on shaping psychopathology is discussed. Comparisons are drawn with other media influenced cases reported in the recently."} +{"text": "This review surveys the methods available for assessing liver blood flow,examines the different parameters being measured and outlines problems of applicability and interpretationfor each technique.The study of hepatic haemodynamics is of importance in understanding both hepatic physiology anddisease processes as well as assessing the effects of portosystemic shunting and liver transplantation. Theliver has the most complicated circulation of any organ and many physiological and pathologicalprocesses can affect it\t\tThe classification of these techniques is to some extent arbitrary and several so called \u201cdifferent\u201dmethods may share certain common principles. The methods reviewed have been classified into twogroups (Table 1): those primarily reflecting flow through discrete vessels or to the whole organ and thoseused to assess local microcirculatory blood flow. All techniques have their advantages and disadvantagesand in some situations a combination may provide the most information. In addition, because of themany factors affecting liver blood flow and sinusoidal perfusion, readings in a single subject may varydepending on positioning, recent food intake, anxiety, anaesthesia and drug therapy. This must beborne in mind if different studies are to be meaningfully compared."} +{"text": "Cell structure abnormalties are found in acute leukaemia and preleukaemic states. Studies on bone marrow cells and peripheral leucocytes of 4 patients with idiopathic acquired sideroblastic anaemia showed patterns in cell culture similar to those reported in acute leukaemia: 2 of these patients later developed leukaemia. Other patients with idiopathic, secondary or congenital sideroblastosis showed no such cell culture abnormalities, and none developed leukaemia. Studies such as this suggest that cell culture methods detect altered cellular function preceding overt leukaemia and that these abnormal findings may be helpful in the evaluation of patient groups with an increased incidence of leukaemia."} +{"text": "Gallbladder cancer is a common malignancy of thebiliary tract. It is the fifth common malignancy of thegastrointestinal tract in United States [1] and third inNorthern India [2]. Despite such high prevalence,there is scanty published literature about this diseasein indexed journals. Therefore, this article is intendedto provide a brief overview of gallbladder cancer riskfactors, based mainly on published evidence fromanalytical epidemiology and recent research findingsof biologists and practising oncologists. Furthermore,an attempt has been made to establish an associationbetween different causative factors and the occurrenceof the disease."} +{"text": "The aim of this study was to examine pulmonary function after acute lymphoblastic leukaemia in childhood and identify risk factors for reduced pulmonary function. We studied a population-based cohort of 94 survivors of acute lymphoblastic leukaemia in childhood who were in first remission after treatment without spinal irradiation or bone marrow transplantation. Pulmonary function test results were compared with reference values for our laboratory, based on 348 healthy subjects who had never smoked from a local population study. A median of 8 years after cessation of therapy (range 1-18 years) the participants had a slight, subclinical, restrictive ventilatory insufficiency and reduced transfer factor and transfer coefficient. The changes in lung function were related to younger age at treatment and to more dose-intensive treatment protocols that specified more use of cranial irradiation and higher cumulative doses of anthracyclines, cytosine arabinoside and intravenous cyclophosphamide than previous protocols. We conclude that, 8 years after treatment without bone marrow transplantation or spinal irradiation, survivors of childhood acute lymphoblastic leukaemia in first remission were without pulmonary symptoms but had signs of slight restrictive pulmonary disease including reduced transfer factor. The increased dose intensity of many recent protocols for childhood acute lymphoblastic leukaemia may lead to increased late pulmonary toxicity."} +{"text": "A biopsy gun which can be operated by one hand has been evaluated at post-mortem to determine itsaccuracy in biopsying impalpable lesions within the liver under intraoperative ultrasound control. Of 20impalpable metastases identified positive histology was obtained in 90% demonstrating that thistechnique is of value in identifying and localising metastases in the liver."} +{"text": "We tested 46 fully vaccinated children in two day-care centers in Israel who were exposed to a fatal case of pertussis infection. Only two of five children who tested positive for Bordetella pertussis met the World Health Organization's case definition for pertussis. Vaccinated children may be asymptomatic reservoirs for infection."} +{"text": "In addition core biopsies were obtained from the same tumour on two separate occasions (n = 10). A highly significant correlation was found in counts performed by the same observers at different times and between two different observers. No significant correlation was found in counts of core biopsies and tumour sections taken either simultaneously or subsequently. No correlation was found between counts of sequential core biopsies. Study findings suggest that, although microvessel counts may be assessed reproducibly by the same and different observers, counts performed in core biopsies do not accurately reflect those of overall tumour, limiting their potential as predictive or prognostic markers. \u00a9 1999 Cancer Research CampaignAssessment of tumour vascularity in core biopsy specimens may be a useful predictor of response to primary therapy. This study addresses practical methodological issues regarding accuracy of tumour vascularity assessments in different breast cancer specimens. Issues addressed in the study are variation caused by (i) inherent observer variation in the method, (ii) tumour heterogeneity and (iii) previous surgical manipulation of tumours. Microvessel counts were performed by two observers on separate occasions and by two different observers. Counts were performed on core biopsies and tumour sections taken simultaneously ("} +{"text": "Adherent, predominantly phagocytic, mononuclear cells expressing spontaneous cytotoxic activity against diverse target cells in vitro were present in various tissues of different strains of rats and mice. Cells with such natural killer capacity were thus everywhere readily available for mobilization and activation. The inherent spontaneous killer capacity of adherent mononuclear phagocytes can be abrogated by silica particles in vitro, and can be considerably enhanced by appropriate stimuli in vivo. Spontaneous cytolysis mediated by unstimulated mononuclear phagocytes was consistently manifested only after a lag phase of 12-20 h and was quite nonspecific; there was no cogent correlation between susceptibility to lysis and transformation."} +{"text": "Release of TNF in mice infected by an aerosol of influenza virus was significant after administration of bacterial lipopolysaccharide (LPS) at 72 h, whereas administration of homologous influenza virus produced only modest amounts of TNF at 96 h. Significant production of TNF was observed 48 h after intravenous administration of infectious influenza in response to LPS but not with the homologous virus. TNF induced after influenza virus infection could be blocked by a specific murine anti-TNF monoclonal antibody. Higher TNF production following aerosol influenza infection correlated with peak titres of influenza virus in the lungs of infected mice and with enhanced generation of luminoldependent chemiluminscence.Increased morbidity and mortality occur regularly during influenza epidemics. The exact mechanisms involved are not well defined but bacterial superinfection of influenza virus infected patients is considered to play an important role. In the present study, the effect of influenza virus infection on"} +{"text": "Dear Sir,I read with interest the article \u2018Tuberculous Sarcoidosis\u2019 by Shah JRAuthor has stressed on considering \u2018tuberculous sarcoidosis\u2019 as a definite clinical entity where two diseases present simultaneously or another disease develop in a course of existent disease. I thanks the author, for awaring the clinicians about this distinct entity for personal reason, that now I can suspect retrospective diagnosis of \u2018tuberculous sarcoidosis\u2019 in three cases in my previous clinical experience. Two AFB smear positive and tuberculin reactive pulmonary cases with noncaseating granulomas on tissue biopsy, required oral corticosteroid in view of poor response with antituberculosis drugs and another case, a 10 year old male child with lymphocyte predominant exudative pleural effusion and tuberculin reactivity developed cervical lymphadenopathy, nine months after completion of six months antituberculosis therapy with adequate response. The scalene node biopsy showed noncaseating granulomas and a second course with antituberculosis drugs resulted in poor response and patient lost to follow up. Although none of these cases could be investigated for sarcoidosis as diagnosis of tuberculosis was confirmed and the term \u2018tuberculous sarcoidosis\u2019 was neither familiar to me nor is mentioned in the standard textbooks of respiratory medicine till date. Since tuberculosis is rampant in our country and may present with various unusual clinical and radiological features, the diagnosis of other coexistent diseases is often overlooked. Lack of infrastructure, financial constraints, and lack of newer diagnostic techniques at most centre of our country, despite adequate clinical material and trained medical persons are possible causes for these situations. The recent studies on possible association of tuberculosis and sarcoidosis are interesting and encouraging further research in this field.+ CD4+ T cells at the sites of granulomatous inflammation, consistent with a MHC-restricted antigen driven processSarcoidosis has pathologicOne of the strongest arguments against a potential role of mycobacteria in sarcoidosis pathogenesis is inability to detect microorganism on histological staining or by culture from pathologic tissues. The possible explanations for negative microbial workup noted in sarcoid lesions could be very low concentration of the bacteria's, ultra-slow growth pattern, and lack of certain nutritional requirements etc. or the sarcoidosis pathogenesis may reflect an immune response to infectious antigens that might not be dependent upon actively replicating organisms. Recently newer diagnostic techniques have been emerged for assessing presence of microorganisms in pathologic tissues. Polymerase chain reaction (PCR) analysis of pathologic tissue for 16S r RNA serve as an alternative means of identifying putative infectious agents that are difficult to isolate. One study of PCR analysis for conserved Mycobacterium 16S r RNA and rpo B sequences revealed the presence of slow-growing mycobacteria such as M tuberculosis and M avium, as well as unique 16S r RNA and rpo B sequences that suggest a novel mycobacteriumAnother mechanism to identify the causative microorganism is use of antigen specific immune responses to microbial antigens and this has been. recently utilized to identify novel infectious agent in SARSThese recent studies of successful molecular analysis and humoral immunity to mycobacterial antigens from sarcoidosis patients have renewed interest in a potential role of mycobacteria in sarcoidosis and support the hypothesis that mycobacterias may have a causal role in \u2018some\u2019 sarcoidosis patients. Further molecular studies with positive and negative controls and investigation of genetic risk factors will be important, in order to explain why some patients are found to have an association with microbial antigens and others are not. In conclusion, we must be flexible on the diagnosis of two co existent diseases and possibility of \u2018tuberculous sarcoidosis\u2019 may be considered whenever such clinical situation arises and supported by complete investigation backup."} +{"text": "Traumatic injury to the extrahepatic biliary system is rare and usually diagnosed at laparotomy when it isassociated with other visceral injuries. Isolated gallbladder rupture due to blunt abdominal trauma is evenrarer. The clinical presentation of gallbladder injury is variable, resulting in a delay in diagnosis andtreatment. Awareness to the possibilty of trauma to the extrahepatic biliary system enables early surgicalintervention and eliminates the high morbidity associated with delated diagnosis. A 5 year old child with isolated gallbladder rupture caused by blunt abdominal trauma is presented."} +{"text": "It was long believed that Mahaim pathways represented nodo-fascicular or nodo-ventricular connections. However, this misconception was challenged when patients underwent surgical or catheter ablation of the AV node but remained pre-excited. Electrophysiology (EP) studies showed these pathways to be right sided decrementally conducting atrio-fascicular accessory pathways with the atrium forming a part of the antidromic tachycardia circuit. Mahaim pathways are usually reported to occur on the right side. We report a patient who presented with a broad complex tachycardia thought to be ventricular tachycardia; however during EP study this was shown to be an antidromic atrioventricular tachycardia utilising a left free wall pathway that demonstrated 'Mahaim-like' properties and was successfully ablated. The pathway was shown to have long conduction times with no retrograde conduction, had an effective refractory period longer than the AV node and its conduction was only evident during antidromic AVRT. It also had a decremental antegrade property and was responsive to intravenous adenosine. These 'Mahaim-like' features are very unusual and rarely reported on the left side. Mahaim fibres are accessory pathways that usually cause an antidromic atrio-ventricular re-entrant tachycardia (AVRT) with a left bundle branch block pattern. We report a case of a patient presenting with a broad complex tachycardia and right bundle branch block pattern that was thought to be ventricular tachycardia on resting 12-lead electrocardiogram (ECG) initially but was found to be due to an antidromic AVRT utilising a left sided accessory pathway with Mahaim-like properties that was successful ablated. A 44-year old woman presented with a two hour history of sudden onset palpitation while walking her dog but no syncope. She had no significant past medical history of note but a paternal uncle had died suddenly at the age of 50 years and her father had been diagnosed as having cardiomyopathy and had an implantable cardioverter defibrillator implanted several years previously. She was taking no regular medication. On arrival into hospital she had a blood pressure of 110/60 mmHg and pulse rate of 215 beats per minute; there were no signs of cardiac failure. A resting 12-lead electrocardiogram (ECG) revealed a regular broad complex tachycardia, which was diagnosed as ventricular tachycardia ; this di The patient was referred for electrophysiological assessment and treatment. Following counselling and informed consent, she was admitted for electrophysiology study which was carried out under local anaesthesia and intravenous sedation. Catheters were inserted via femoral venous access with a decapolar catheter placed in the coronary sinus (CS) and quadripolar catheters placed at the right ventricular apex and His bundle position. Programmed stimulation demonstrated concentric, decremental retrograde and antegrade conduction. A spontaneous tachycardia was observed during the study with the same morphology as the clinical tachycardia . The tac In 1938, Mahaim described the existence of islands of conducting tissue extending from the AV node into the ventricular myocardium . Since tElectrophysiology studies confirmed these pathways to be right sided decrementally conducting atrio-fascicular accessory pathways with the atrium forming a part of the antidromic tachycardia circuit . Several Mahaim pathways are usually reported to occur on the right side. Only sporadic cases of left-sided antegrade decremental pathways have been reported including one left posterior , one lefWe report a left free wall pathway with slow decremental antegrade conduction which was only evident during antidromic AVRT which was initially thought to be ventricular tachycardia. There was no retrograde conduction of the pathway and it was responsive to adenosine; these features have rarely been reported for a left free wall pathway and may represent a variant demonstrating 'Mahaim-like' properties. The utility of a late atrial premature stimulus which reset the tachycardia is highlighted in making the correct diagnosis."} +{"text": "The antigen specific cell mediated cytotoxicity of MSV immune spleen lymphocytes to 51Cr labelled murine lymphoma cells was wholly abolished by pretreatment of the spleen cells with anti-theta antibody and complement. Early during the immune response to MSV the cytotoxic acitivity was inhibited by incubation of immune lymphocytes with \"late progressor\" or \"early regressor\" serum. Immune lymphocytes at later times were more refractory to such inhibition by serum blocking factors. Although unfractionated cytotoxic lymphocytes, irrespective of the time after MSV infection at which they were tested, were inhibited by soluble tumour associated antigen (TAA), a subpopulation of cytotoxic T cells was identified which was inhibited neither by antigen nor serum."} +{"text": "It is estimated that 75% of all women will experience at least 1 episode of vulvovaginal candidiasis (VVC) during their lifetimes. Most patients with acute VVC can be treated with short-term regimens that optimize compliance. Since current topical and oral antifungals have shown comparably high efficacy rates, other issues should be considered in determining the most appropriate therapy. It is possible that the use of short-duration narrow-spectrum agents may increase selection of more resistant organisms which will result in an increase of recurrent VVC (RVVC). Women who are known or suspected to be pregnant and women of childbearing age who are not using a reliable means of contraception should receive topical therapy, as should those who are breast-feeding or receiving drugs that can interact with an oral azole and those who have previously experienced adverse effects during azole therapy. Because of the potential risks associated with systemic treatment, topical therapy with a broad-spectrum agent should be the method of choice for VVC, whereas systemic therapy should be reserved for either RVVC or cases where the benefits outweigh any possible adverse reactions."} +{"text": "A method for assigning functions to unknown sequences based on finding correlations between short signals and functional annotations in a protein database is presented.This approach is based on keyword (KW) and feature (FT) information stored inthe SWISS-PROT database. The former refers to particular protein characteristicsand the latter locates these characteristics at a specific sequence position. In this way,a certain keyword is only assigned to a sequence if sequence similarity is found inthe position described by the FT field. Exhaustive tests performed over sequenceswith homologues (cluster set) and without homologues (singleton set) in the databaseshow that assigning functions is much \u2019cleaner\u2019 when information about domains (FTfield) is used, than when only the keywords are used."} +{"text": "Using a highly sensitive chemiluminescent enzyme immunoassay, we have evaluated the measurement of serum prostate-specific antigen (PSA) as a potential diagnostic test for differentiation between women with breast cancer and those with benign breast disease. In a controlled study consisting of 284 women with well-documented patient files and matched for age and long-term place of residence, serum samples collected from 90 women with histologically confirmed breast cancer, 94 women with benign breast disease and 100 controls were analysed. Serum total PSA levels in benign breast disease and cancer patients are not statistically different from those of healthy controls. Total PSA levels decrease with age in normal controls and breast cancer patients but not in those with benign breast disease. The total PSA concentration decreases after menopause in healthy women, though not in patients with breast cancer or benign breast disease. Total PSA bore no relation to the histological type or grade of the tumour or the disease stage of the breast cancer patients. In benign breast disease, all mastopathy patients had normal total PSA, whereas elevation of the values was observed in 7% of fibroadenoma patients. Our results show that serum total PSA cannot be used to distinguish between healthy women and/or women with breast cancer or benign breast disease. \u00a9 1999 Cancer Research Campaign"} +{"text": "Regionally administered vasopressors might increase tumour chemotherapy uptake by differentially constricting normal and tumour blood vessels, leading to a selective increase in blood flow to the tumour. In this study, we compared the effects of the vasopressors angiotensin II, vasopressin and endothelin I and the vasodilator calcitonin gene-related peptide (CGRP) by continuously measuring liver parenchymal and tumour blood flow during a 30-min regional vasoactive infusion in a rat HSN liver metastasis model. Vasopressin and angiotensin II produced a vasoconstriction that decreased despite continued infusion, while endothelin I infusion led to prolonged vasoconstriction with a more gradual onset. CGRP infusion resulted in increased vessel conductance but a reduction in blood flow due to systemic hypotension. The tumour to normal flow ratio (TNR) was transiently increased during infusion of all pressors, but only endothelin I produced sufficient change to result in a rise in average TNR throughout pressor infusion. Continuous liver and tumour blood flow measurement throughout vasoactive infusion demonstrated that the extent and the duration of blood flow change varied with the agents assessed. No vasoactive agent increased tumour blood flow, but endothelin I had the most suitable vasoactive properties for enhancing tumour uptake of continuously infused chemotherapy."} +{"text": "Portopulmoanry hypertension (POPH) is a form of pulmonary arterial hypertension (PAH) associated with portal hypertension with or without underlying chronic liver disease. POPH is increasingly recognized and recent evidence suggests that it is one of the leading causes of PAH. The pathophysiology of POPH is poorly understood although the pathological changes in pulmonary vasculature in advanced POPH are similar to those seen in idiopathic pulmonary hypertension. The prognosis in patients with liver disease who also suffer from significant POPH is considered to be poor. Higher degree of pulmonary artery pressure (PAP) may preclude a patient from liver transplant as mortality in these patients is high. The treatment with vasodilator therapy has shown to improve both hemodynamics and clinical outcome in POPH in retrospective studies and in some case series. The aim of medical management is to bring PAP <35 mmHg that may make a patient with POPH and advanced liver disease eligible for liver transplant, which otherwise would have been denied because of high PAP. Portopulmonary hypertension (POPH) is defined as pulmonary arterial hypertension (PAH) complicated by portal hypertension, with or without advanced hepatic disease. It is classified as group 1 in current classification of pulmonary hypertension. Although it affects only 2\u20135% of the population suffering from portal hypertension, its clinical implications are enormous. The prevThe outcome of LT in the presence of POPH is poor with a 35% reported mortality rate in LT recipients having a mean pulmonary artery pressure (mPAP) >35 mmHg. A patient with significant pulmonary artery pressure (PAP) may be denied the opportunity for transplant unless the mPAP is brought below 35 mmHg with medical treatment. In patients who do undergo successful LT, there can be resolution of pulmonary hypertension with time.\u22125 and a normal or decreased pulmonary artery wedge pressure (PAWP) < 15 mmHg are considered to have POPH according to the European Respiratory Society Pulmonary Hepatic Vascular Disorder Task Force 2004 Consensus Report [Patients who present with portal hypertension and have an increased mPAP >25 mmHg at rest, elevated pulmonary vascular resistance (PVR) >240 dyne/sec/cms Report .\u22125 can be used in order to define POPH in the presence of hyperdynamic circulatory state.[Approximately 30\u201350% of the patients with cirrhotic liver disease have low systemic vascular resistance and high cardiac output. In these patients, pulmonary arterial pressures may be elevated due to increased cardiac output. These patients have lower values of PVR. Some investigators propose that a cutoff of PVR >120 dynes/s/cmry state. ElevatedCertain risk factors are found to be associated with development of POPH. Female sex and autoimmune hepatitis were associated with an increased risk of POPH, while patients with hepatitis C infection had a lower risk. In additP = 0.03). Twelve patients underwent LT and five-year survival for the nine patients receiving therapy for POPH was 67% compared to 25% in three patients who were not pretreated with prostacyclin therapy.[The survival of untreated patients with POPH is very poor. In a recent retrospective study from the Mayo Clinic Liver Transplantation Group, they identified 74 POPH patients from 1994 through 2007, and categorized them into three groups: 1) no therapy for POPH or LT; 2) therapy for POPH alone; and 3) therapy for POPH followed by LT. Five-year survival in 19 patients who received no therapy for POPH and no LT representing the natural history of POPH was 14%, with 54% dying within one year of diagnosis. The median survival in 43 of the 74 patients (58%) who received medical therapy for POPH and did not undergo LT was 46 months and five-year survival was 45%, being significantly better than the patients who were not selected to receive medical therapy for POPH , all patients being screened for LT should be evaluated for POPH by transthoracic echocardiography (TTE). In a larAfter initial screening with TTE, definitive diagnosis should be made by right heart catheterization that includes measurements of mPAP, PAOP, cardiac output (CO), and calculated PVR. Acute vaThe diagnosis of POPH by right heart catheterization before LT is crucial as data suggest an increased risk of death following LT in patients with POPH if the mPAP exceeds 35 mmHg. It is suggested to consider specific vasomodulating treatment for POPH if pre-liver transplant mPAP is greater than 35 mmHg. Patients with mPAP of less than 35 mmHg can be transplanted without undergoing vasodilator treatment for POPH.\u201327Right heart catheterization is also important to help classify severity of POPH and therIn addition to the general psychosocial issues, keeping optimum oxygen saturation is crucial. Hypoxemia may worsen POPH through pulmonary vasoconstriction, and therefore supplemental oxygen should be considered for all patients with hypoxemia to maintain oxygen saturation higher than 90% at all times.Diuretics have to be used with caution in patients with POPH. Although they are useful in reducing the increased intravascular volume commonly present in chronic liver disease, they can reduce the cardiac output by decreasing the right ventricular preload.Digoxin has been shown to improve cardiac output acutely in idiopathic pulmonary hypertension. Its effiThere is a favorable response with anticoagulation in other forms of PAH especially idiopathic pulmonary hypertension and chronic thromboembolic pulmonary hypertension for its ability to slow disease progression. Anticoagulation is traditionally not recommended in patients with POPH. This is Transjugular intrahepatic portosystemic shunt (TIPS) may worsen POPH because of acute increase in preload causing increased cardiac output and mPAP. This leads to worsening right ventricular strain and dysfunction. It is not recommended in the patients with POPH.Very limited data exist in term of medical therapy in POPH. Most of the studies are case series or case reports.Calcium channel blockers are not recommend in POPH, as they could potentially increase the hepatic venous pressure gradient and worsen portal hypertneion.33Prostanoids have been shown to be effective in the treatment of POPH. Epoprostenol is the best studied prostanoid in POPH. Pulmonary hemodynamics associated with POPH can be improved by infusion of Epoprostenol. In moderate to severe POPH, intravenous Epoprostenol results in significant improvement (both acute and long-term) in PVR, mPAP, and cardiac output. Pulmonary hemodynamics may be improved and brain natriuretic peptide and human atrial natriuretic peptide decrease to normal levels during epoprostenol therapy. Epoprostenol may significantly improve pulmonary hemodynamics and facilitate acceptance of patients who may otherwise be denied LT as a result of POPH.\u201337 As usCombination therapy with intravenous Iloprost and oral Bosentan, a dual endothelin-1 receptor antagonist, might extend the survival of selected patients suffering from POPH and recurrent right heart failure. BosentanSildenafil (Revatio) is reported to be effective in decreasing pulmonary vascular resistance.\u201349 SildeCombination vasodilator therapy has been shown to be effective in patients with idiopathic PAH. Its role\u22125 before proceeding to liver transplant. Perioperative mortality in patients with mean PAP >35 mmHg is significantly higher compared to those with mPAP < 35 mmHg.[The goal of therapy in patients with POPH, who are candidates for liver transplants, is to reduce mPAP <35 mmHg and the PVR <400 dynes/s/cm 35 mmHg.POPH is a type of PAH associated with portal hypertension. The proposed pathophysiology includes hyperdynamic pulmonary circulation leading to shear stress of pulmonary vasculature causing obstructive vasculopathy and increased pulmonary resistance. The diagnosis of POPH is suggested by TTE and is confirmed by right heart catheterization. Mortality in advance liver disease in the presence of pulmonary hypertension is high. Every effort should be made to treat these patients with vasodilator therapy to reduce PAP and PVR, and ultimately improve the right heart function. Treatment with a combined approach of pulmonary arterial vasomodulator therapy and liver transplant may improve long term survival in patient with POPH. Randomized controlled trials are needed to determine the future direction in management of POPH."} +{"text": "It has been suggested that patients with bleeding varices and hypersplenism will show significant improvementsin leucocyte and platelet counts following distal splenorenal (Warren) shunt surgery. Whilst this maybe true in the short term, this report shows that in the long term hypersplenism is not relieved, whereas thelienorenal shunt is associated with a return of normal haematological values."} +{"text": "Aspergillus fumigatus (Af) cause an allergic lung disease in humans. This study was carried out to determine the effect of environmental tobacco smoke (ETS) on a murine model of allergic bronchopulmonary aspergillosis (ABPA). BALB/c mice were exposed to aged and diluted sidestream cigarette smoke to simulate 'second-hand smoke'. The concentration was consistent with that achieved in enclosed public areas or households where multiple people smoke. During exposure, mice were sensitized to Af antigen intranasally. Mice that were sensitized to Af antigen and exposed to ETS developed significantly greater airway hyperreactivity than did mice similarly sensitized to Af but housed in ambient air. The effective concentration of aerosolized acetylcholine needed to double pulmonary flow resistance was significantly lower in Af + ETS mice compared to the Af + AIR mice. Immunological data that supports this exacerbation of airway hyperresponsiveness being mediated by an enhanced type 1 hypersensitivity response include: eosinophilia in peripheral blood and lung sections. All Af sensitized mice produced elevated levels of IL4, IL5 and IL10 but no IFN-\u03b3 indicating a polarized Th2 response. Thus, ETS can cause exacerbation of asthma in ABPA as demonstrated by functional airway hyperresponsiveness and elevated levels of blood eosinophilia.Involuntary inhalation of tobacco smoke has been shown to aggravate the allergic response. Antibodies to fungal antigens such as"} +{"text": "Prognosis of 16 patients with hepatic tumors and angiographically proven arterioportal fistulas wasanalysed in relation to treatment. Six patients received only conservative therapy; they all died ofvariceal bleeding in the course of two months after angiography. Hepatic resection was performed infour patients; three of them are still alive 13\u201352 months later including two free of both the tumor andportal hypertension. Hepatic artery embolization was carried out in six patients. All of them died in 2\u201336 months after the procedure, but only two from gastroesophageal hemorrhage. It is concluded that prognosis of arterioportal fistulae in liver neoplasms is poor due to hyperkineticportal hypertension and following variceal bleeding. Hepatic resection of both the tumor and the fistulais the treatment of choice. In unresectable cases hepatic artery embolization will decrease the risk ofvariceal hemorrhage."} +{"text": "These observations suggest thatinjection sclerotherapy for oesophageal varicesresults in disturbances of gastric emptying thatmay contribute to the pathogenesis of portal hypertensivegastropathy.Bleeding from portal hypertensive gastropathy(PHG) has been estimated to account for upto 30%of all upper gastrointestinal haemorrhage in patientswith cirrhosis and portal hypertension. Althoughportal hypertension seems to be an essential prerequisite,the precise mechanisms responsible forthe development of PHG are unknown. The aim ofthis study was to examine the role of injection sclerotherapyof oesophageal varices in the developmentof PHG. Gastric emptying was studied using aradionuclide test meal with the emptying characteristicsof a slow liquid in 57 patients with cirrhosisand/or portal hypertension (median age 53 yrs), ofwhom 34 had received injection sclerotherapy fortheir oesophageal varices and 20 normal healthyvolunteers (median age 42 yrs). As vagal damage isassociated with more rapid emptying of liquids,despite hold up of solids, this technique might beexpected to demonstrate such damage if gastricemptying was accelerated. The results indicated thatthere was no difference in the rate of gastricemptying between normal healthy volunteers andportal hypertensive patients. However, patients whohad received injection sclerotherapy emptied theirstomachs faster than those who had not ("} +{"text": "Outpatient Laparoscopic Cholecystectomy was attempted in 98 patients selected from 266 patientspresenting for elective cholecystectomy (37%). Two patients required admission following conversion to\u201copen\u201d Cholecystectomy, one patient was admitted for observation because of a technically difficultLaparoscopic Cholecystectomy and 16 patients were admitted because of refractory nausea andvomiting in the early post-operative period. Seventy-nine patients (81%) were able to be dischargedhome within 4 to 6 hours of surgery, with only one patient requiring readmission to hospital because ofthe onset of nausea and vomiting. There were no post-operative complications attributable to theoutpatient experience. We believe this approach to elective gallbladder pathology can be safelyaccomplished in selected patients and will be increasingly utilized in the future."} +{"text": "Protein structure determination of soluble globular protein domains has developed into an efficient routine technology which can now be applied to generate and analyze structures of entire human protein families. In the kinase area, several kinase families still lack comprehensive structural analysis. Nevertheless, Structural Genomics (SG) efforts contributed more than 40 kinase catalytic domain structures during the past 4\u00a0years providing a rich resource of information for large scale comparisons of kinase active sites. Moreover, many of the released structures are inhibitor complexes that offer chemical starting points for development of selective and potent inhibitors. Here we discuss the currently available structural data and strategies that can be utilized for the development of highly selective inhibitors. The interplay of 518 human kinases and the phosphatase family creates a signalling network that controls most signalling processes through reversible phosphorylation 2+ binding motifs \u201cVIAK\u201d and \u201cDFG,\u201d the catalytic HRD motif and the activation segment. A large number of kinases are activated by phosphorylation of the activation segment which is typically disordered in its inactive state and assumes a stable structure suitable for substrate binding in its phosphorylated active state Eukaryotic protein kinase domains (ePKs) possess highly conserved architecture comprising an N-terminal lobe with the conserved regulatory helix \u03b1C, and a larger mainly \u03b1-helical C-terminal lobe A. The ac2http://www.sgc.ox.ac.uk/research/kinases/) and other resources Currently there are 136 unique human catalytic domain structures in the protein databank (PDB) . However3Structural comparison is a powerful method for the identification of new mechanisms underlying enzymatic regulation. However, structural features may be influenced by the crystalline state of the protein. High throughput technologies offer the possibility to generate several structures of the same protein or closely related isozymes that differ in crystal contact regions adding confidence to the interpretation of structural data. For instance, regulation of kinase autophosphorylation by activation segment exchange between two adjacent catalytic domains was initially observed in the crystal structure of the kinase CHK2 4per se does not guaranty more favourable selectivity profiles and many unexpectedly cross reacting targets have been identified for type II inhibitors as well. Other strategies aim to target unique active site features, for instance small residues located in the hinge gatekeeper position that open an additional binding pocket. In addition, targeting allosteric binding sites that are located outside the conserved ATP binding pocket is an emerging strategy that has generated very selective inhibitors One of the most successful strategies for the development of selective inhibitors is targeting of diverse inactive conformations of kinases. These inactive states comprise the DFG-out state, in which the conserved phosphate binding motif \u201cDFG\u201d changes conformation opening a large allosteric binding pocket. This conformation is crucial for the tight binding of the first approved kinase inhibitor gleevec to inactive ABL but the binding mode was only recognized long after this drug had been developed Highly potent and selective ligands can also be developed using organometallic inhibitors. Metal centres offer a large chemical diversity by their ability to coordinate a wide variety of ligand spheres. This property made it possible to design ligands that showed outstanding shape complementarity to kinase active sites and that bind with sub-nanomolar potency to the protein kinases PIM1 and GSK3beta 2+ binding motif Asp-Phe-Gly (DFG), which is replaced by Asp-Tyr-Thr (DYT), and the Ala-Pro-Glu (APE) motif usually found at the C-terminus of the activation segment. In addition, haspin shares only weak sequence homology with ePKs and contains a highly divergent kinase domain with several unique inserts Finally, certain human ePKs have very diverse active sites and share less than 25% homology with other kinase family members. The diversity of the ATP site and the lack of certain motifs that are otherwise highly conserved in ePKs makes the design of selective inhibitors less challenging. A number of these diverse kinases have interesting links to disease. One examples for such an atypical kinase is haspin A number of large cross screening panels have been developed recently that allow extensive profiling of kinase inhibitors 5In recent years high throughput structure determination efforts provided a large resource of structural and chemogenomic information for the design of kinase inhibitors. Multiple structures determined in complex with different inhibitors locked kinase catalytic domain structures in a variety of different conformations. Comparison of these diverse conformational states provided insight into the dynamic features of kinases and unravelled new mechanisms of regulation."} +{"text": "In all 6 different murine tumours of spontaneous origin, a high proportion (22-95%) of the regional lympgh nodes draining small intradermal tumours gave rise to tumours after their isogeneic transplantation as whole nodes. In separate experiments with 4 of these tumours, equivalent tumour-bearing mice had their tumours surgically excised and were observed for the development of regional nodal corresponding frequency of tumour formation by transplanted nodes. After high-dose radiotherapy of intradermal carcinomas, there was a progressive fall in the incidence of positive regional node transplants from 48 to 96 h after irradiation. It is concluded that continual lymphatic dissemination of viable cancer cells is characteristic of malignant tumours, but that there is a relatively small chance of such cells giving rise to nodal metastatic growth. Related studies showed that the ability of a small number of cancer cells to give rise to tumours was very much greater if they were incorporated in a lymph node at transplantation than if they were transplanted directly as a suspension."} +{"text": "With more experience and better management, the incidence of complications like stone formation after continent urinary diversion is uncommon today. We report a case of large stone bulk in a patient who underwent this surgery 10 years back and who suggested the formation of stones in the pouch herself by sounding them. Proper counseling, regular pouch irrigation and follow-up are essential in any kind of diversion. Occasionally, urinary diversion is needed in cases of complex vesicovaginal fistula who have failed repeated repairs. We report one such case that underwent continent urinary diversion and reported 10 years later when she noticed a gritty sensation on passing catheter that was later found to be due to a large stone bulk in the pouch.A 20-year-old woman presented with vesicovaginal (VVF) and rectovaginal fistula (RVF) due to obstructed labor. She underwent a temporary transverse colostomy followed by two failed attempts of VVF repair. One year later, the RVF was repaired successfully by the vaginal route and a simultaneous continent cutaneous urinary diversion procedure (right colon pouch with Mitrofanoff continence mechanism) was performed. She was then lost to follow-up and could not come even for colostomy closure due to poor socioeconomic condition. She presented after 10 years when she noticed a gritty sensation while passing catheter for pouch emptying. On evaluation, she had normal renal functions, with serum creatinine of 1.0 mg%, while an intravenous urogram revealed a large pouch full of stones . In viewStone formation is a known complication of all reservoirs.\u20133 This pThis case demonstrates that if continent urinary diversion is planned, the patient should be counseled well about the need of regular self-catheterization, pouch irrigation and regular follow-up even if asymptomatic and may be advised to report any abnormal gritty sensation while draining the pouch by catheter. This grittiness may be experienced while passing the catheter or may even be experienced while withdrawing the catheter-sometimes better in the latter situation as the pouch becomes free of liquid urine and stones come close to each other and the catheter within the collapsing pouch. Although the stones were smooth surfaced, the passage or withdrawal of catheter through a significant stone burden can still produce gritty sensation to an intelligent patient."} +{"text": "Wolbachia are ubiquitous inherited endosymbionts of invertebrates that invade host populations by modifying host reproductive systems. However, some strains lack the ability to impose reproductive modification and yet are still capable of successfully invading host populations. To explain this paradox, theory predicts that such strains should provide a fitness benefit, but to date none has been detected. Recently completed genome sequences of different Wolbachia strains show that these bacteria may have the genetic machinery to influence iron utilization of hosts. Here we show that Wolbachia infection can confer a positive fecundity benefit for Drosophila melanogaster reared on iron-restricted or -overloaded diets. Furthermore, iron levels measured from field-collected flies indicated that nutritional conditions in the field were overall comparable to those of flies reared in the laboratory on restricted diets. These data suggest that Wolbachia may play a previously unrecognized role as nutritional mutualists in insects. Wolbachia are bacteria that infect millions of insect species worldwide. Wolbachia aren't infectious, but are maternally inherited symbionts passed from mother to offspring. To infect a host population, Wolbachia behave as reproductive parasites and alter the host reproductive system in a manner that increases infected female reproductive success. Some strains of Wolbachia, however, cannot manipulate their host's reproductive systems\u2014yet they can successfully infect insect populations. How is this possible? Here we show that a Wolbachia strain that naturally infects Drosophila melanogaster, and induces very low levels of reproductive parasitism, can also act as a nutritional mutualist. When D. melanogaster flies were reared on normal diets, we observed no cost or benefit associated with the Wolbachia infection. But, if we reared flies on diets containing either very low or high amounts of iron, Wolbachia-infected flies produced more eggs than uninfected flies. As wild-caught flies contain low amounts of iron, our results suggest that flies in the wild should benefit from their Wolbachia symbiont. Wolbachia pipientis is arguably the most abundant endosymbiont in the insect world Wolbachia infected females have mated to males carrying an unrelated Wolbachia strain Wolbachia infected females possessing a reproductive advantage over uninfected females and as a result Wolbachia is able to invade host populations Wolbachia invasion. This paradigm, however, is based primarily on experimental work with a limited number of Wolbachia strains that induce strong CI in Drosophila simulansWolbachia strains, including all of those recovered from D. melanogaster, induce very weak and variable CI especially under field conditions Wolbachia invasion in the field. Moreover, strains of Wolbachia have been indentified in D. simulans that induce no CI at all, yet these strains have managed to invade host populations Wolbachia infection that in time will be lost Wolbachia infections in Drosophila melanogaster have been observed to act positively upon non-reproductive fitness traits, such as the extension of adult lifespan or protection against viral and fungal pathogens Wolbachia infected Drosophila melanogaster, none has been identified CI is considered to be the major driving force behind Wolbachia also infect filarial nematodes, where the bacterium is an obligate mutualist required for successful reproduction and development of the worm Brugia malayi revealed that it lacked a complete biosynthetic pathway for both heme and riboflavin Wolbachia strain, wBm contained a complete suite of genes for both pathways wBm, providing further support for the hypothesis that this pathway may be a key point of interaction in the association and offering a potential explanation for the basis for the obligate mutualism Wolbachia strain, wMel that infects D. melanogasterWolbachia for heme biosynthesis, the microbe could supplement host stores or play a role in iron homeostasis. Iron is an essential micronutrient required for a diverse range of metabolic processes Wolbachia infection alters fitness of the model insect host, Drosophila melanogster when reared under varying levels of dietary iron to test the hypothesis that Wolbachia may function as a nutritional mutualist as well as a reproductive parasite in insects.Although insect hosts are not dependent upon D. melanogaster was responsive to our dietary manipulations as measured by mass-spectrophotometry. Flies reared on high iron diets contained approximately twice as much total iron as those reared on cornmeal diet . The total content of eight other biologically relevant metals was used to specifically chelate iron(II) D. melanogaster flies.To investigate if periment , Expt 3 D. melanogaster were reared on diets that contained high levels of iron, due to the addition of FeCl3 we observed a significant reduction in fecundity for both infected and uninfected fly lines relative to flies reared on cornmeal diets. However, the presence of Wolbachia in flies on high iron diets resulted in significant gains in fecundity in two independent experiments . Therefore we conclude that the fitness benefits conferred by Wolbachia were in response to the high iron content.When Wolbachia effects on male fertility in response to changes in dietary iron were performed using Wolbachia infected and uninfected BNE lines reared on Tea, BPS and FeCl3 diets. In no experiment did we observe a cost or benefit to male fertility associated with Wolbachia infections (data not shown), and conclude that Wolbachia only benefits female fecundity and not male fertility during periods of iron deficiency or overload.Assessments of Wolbachia strains that infect filarial nematodes are one such example, and are thought to provide their host with essential vitamins, nucleotides and cofactors, including heme Wolbachia, and evolutionary analyses have previously identified signatures of positive selection on pathway genes Wolbachia and their hosts, we experimentally determined if Wolbachia could influence iron homeostasis and female fecundity in an insect host.Metabolic provisioning of hosts by endosymbionts is commonly observed in obligate associations Wolbachia had no effect on Drosophila melanogaster fecundity when reared on cornmeal diets, consistent with previous observations Wolbachia did provide a significant fecundity benefit to female Drosophila when subjected to low or high iron environments in the majority of experiments conducted. This is the first report of a Wolbachia conferred compensatory effect during periods of nutritional stress or deficiency to an insect host. The observed variability in fecundity measures is consistent with previous experiments in D. melanogaster, which have shown that laboratory measurements of fecundity are highly sensitive to local assay conditions, and are notoriously difficult to replicate even under controlled laboratory conditions Wolbachia fecundity advantage in perturbed iron environments and the observed low iron content of flies from the wild, it is likely that the results of the laboratory experiments reported here may have ecological relevance, providing a variable but positive fitness benefit to Wolbachia infected flies across a range of environments. Previous studies have shown that if Wolbachia can simultaneously induce cytoplasmic incompatibility and increase female fecundity, the rate at which Wolbachia invades na\u00efve host populations is increased Given the observed Wolbachia might provide protection against oxidative stress.Benefits observed under high iron conditions, while not ecologically relevant based on the estimates of total iron in field caught flies, are interesting mechanistically. Increases in dietary iron result in an increase in oxidative stress for most insects Drosophila melanogaster strain BNE was derived from field caught female flies from Brisbane, Australia, and is described in detail elsewhere wMel infection introgressed by crossing to yw67c23 females. Subsequent offspring were backcrossed with males derived from the original field collection for a minimum of five generations to re-establish the original BNE genetic background. All flies were maintained at \u223c25\u00b0C on a 12/12hr light/dark schedule throughout the study. Tetracycline treatments were performed as described previously Wolbachia infection. To reconstitute gut flora, stock bottles containing cornmeal fly diet were seeded with non-tetracycline treated males for a period of three days. These males were then excluded from the diet and newly emerged tetracycline treated adult flies allowed to mate and lay eggs on the diet. Assessments of fecundity were performed at least three generations post tetracycline treatment and reconstitution of gut flora to minimise maternal or grandmaternal mitochondrial effects Camellia sinensis; 3 Tetley tea bags infused in 1litre of water for 5 minutes Drosophila larvae was reduced by iron chelating agents 3 solution to the cornmeal fly diet to a final concentration of 10mM 3 could influence fecundity.The D. melanogaster larvae were introduced to vials containing modified or cornmeal fly diets at low densities (50\u201380 larvae) and reared to adulthood at 25\u00b0C. Virgin males and females were collected and maintained separately on the same diet for a period of three days. Individual crosses among males and females of identical infection status were allowed to mate once within a 60-minute window. Once mating was complete, males were discarded and mated females allowed to oviposit onto molasses plates seeded with yeast for a period of three days. A new plate was introduced every 24 hours and the total number of eggs laid was scored. To determine the impact of Wolbachia infection on female fecundity under iron limitation or overload, females reared on modified diets were mated with males reared on cornmeal diet. The reciprocal cross permitted assessment of male fertility. Once the total number of eggs laid over the three-day period had been scored, comparisons of fecundity between Wolbachia infected or uninfected Drosophila were made using parametric (ANOVA) or non-parametric (Mann-U Whitney) tests where appropriate.Females were allowed to lay eggs onto molasses/agar plates in the absence of yeast. First instar The total content of biologically relevant metals present in flies reared on each of the food types or collected from the field, were determined using inductive coupled plasma mass-spectrometry (ICP-MS) at the Advanced Centre for Isotope Research Excellence at the University of Queensland. The only metal responsive to diet was iron. Pools of 10 female flies were used for each analysis and replicated ten times for lab reared flies or four times for field caught flies. Flies were caught using modified banana traps, such that flies were attracted to the bait but excluded from feeding upon it to ensure that total iron levels were not affected."} +{"text": "A prospective trial with concurrent controls was designed to assess the effects of specific active immunotherapy in patients receiving intermittent cytotoxic chemotherapy (DTIC + Vincristine) as an adjuvant to surgery in Stage IIB malignant melanoma. The treated group received monthly irradiated allogeneic melanoma cells and BCG, and the controls BCG only. Sixteen patients in the treatment arm had a median relapse-free interval of 5 months, compared to 8 months in 12 controls given chemotherapy and BCG, and because of this we felt that continuation of the study was unjustified on ethical grounds. Although all the controls who relapsed did so at distant sites, 7/11 patients given specific active immunotherapy relapsed initially within the lymphatic drainage area of the primary tumour. The median intervals from starting treatment to relapse at distant sites, and the median survival were identical in the 2 groups. We conclude that immunotherapy comprising irradiated allogenic melanoma cells as employed in this study does not prolong survival in surgically treated Stage IIB malignant melanoma and may even promote early, local relapse."} +{"text": "Identifying a preclinical phase of Alzheimer\u2019s Disease (PCAD) that is distinct from cognitive changes in healthy aging continues to be a major research focus. Combining neuropsychological and neuroimaging methodologies should improve our ability to differentiate healthy from pathological aging, although studies that utilize both methods often result in equivocal findings, possibly due to variability in cognitive test performance that may be capturing distinct phenotypes. One method of capturing this cognitive variability is to utilize contrasting neuropsychological tests to identify subgroups representative of distinct cognitive phenotypes, and determine whether differences in brain morphometry support these classifications. We review several approaches to defining cognitive subgroups, and we consider the possibility that cognitive asymmetry might provide one means of identifying both functional and structural changes associated with aging and dementia."} +{"text": "Cellular responses to hypoxia include modulation of respiration rate and up-regulation of genes which encode for angiogenesis factors. We tested whether human malignant glioma cells vary in their response to hypoxic stress over the range of oxygen concentrations which exist in tumours. In five cell lines tested, decreased oxygen availability resulted in decreased rates of oxygen utilization, however substantial differences in the magnitude of the response were observed. Northern blot analysis was used to study induction of vascular endothelial growth factor mRNA in response to hypoxia. In two cell lines, modest hypoxia increased vascular endothelial growth factor mRNA levels compared with those of aerobic controls. In two additional cell lines, vascular endothelial growth factor mRNA was constituitively expressed under aerobic conditions and was not further increased by hypoxia. These findings demonstrate that differences in the response to hypoxia exist among human malignant glioma cell lines and suggest that therapies designed to exploit tumour hypoxia may have varying effects in tumours with different hypoxic stress responses. \u00a9 1999 Cancer Research Campaign"} +{"text": "Significant opportunities and challenges are presented when transitioning from managing laboratory automation development of pharmaceutical products at a single site to collaborative management with multiple domestic and international sites. Prior to integrating Glaxo and Burroughs Wellcome about two yearsago, each company had expertise in laboratory automation, but neither had a strategy for consistent business-justified laboratory automation. The approach for international harmonization of automation development of pharmaceutical test methods that the integrated company has adopted is presented. Some items to consider before undertaking a company-wide automation development harmonization programme are offered for consideration. Experiences encountered and future planned benefits are discussed."} +{"text": "Antibody was detected by membrane immunofluorescence tests in sera of rats bearing an ascitic variant of a transplanted hepatoma, and in concentrated cell-free ascitic fluid. Ascites hepatoma cells were also shown to have immunoglobulin, possibly tumour specific antibody, bound to their surface. The kinetics of antibody responses to ascites hepatoma and hepatoma cells from solid tumours were compared: both tumour types gave positive reactions by the third day after implantation; antibody was present throughout subsequent tumour growth with the ascites whereas antibody was not detected after tumour became palpable in rats injected with hepatoma cells from solid tumour. Antibody responses to ascites tumour were investigated in rats bearing solid hepatoma tumour. Subcutaneous hepatoma did not influence the antibody response to ascites, but rats bearing intraperitoneal tumours showed a diminished serum antibody response to ascitic hepatoma."} +{"text": "Endoscopic resectional techniques for colon cancer are undermined by their inability to determine lymph node status. This limits their application to only those lesions at the most minimal risk of lymphatic dissemination whereas their technical capacity could allow intraluminal or even transluminal address of larger lesions. Sentinel node biopsy may theoretically address this breach although the variability of its reported results for this disease is worrisome.Medline, EMBASE and Cochrane databases were interrogated back to 1999 to identify all publications concerning lymphatic mapping for colon cancer with reference cross-checking for completeness. All reports were examined from the perspective of in vivo technique accuracy selectively in early stage disease .Fifty-two studies detailing the experiences of 3390 patients were identified. Considerable variation in patient characteristics as well as in surgical and histological quality assurances were however evident among the studies identified. In addition, considerable contamination of the studies by inclusion of rectal cancer without subgroup separation was frequent. Indeed such is the heterogeneity of the publications to date, formal meta-analysis to pool patient cohorts in order to definitively ascertain technique accuracy in those with T1 and/or T2 cancer is not possible. Although lymphatic mapping in early stage neoplasia alone has rarely been specifically studied, those studies that included examination of false negative rates identified high T3/4 patient proportions and larger tumor size as being important confounders. Under selected circumstances however the technique seems to perform sufficiently reliably to allow it prompt consideration of its use to tailor operative extent.The specific question of whether sentinel node biopsy can augment the oncological propriety for endoscopic resective techniques cannot be definitively answered at present. Study heterogeneity may account for the variability evident in the results from different centers. Enhanced capacity (perhaps to the level necessary to consider selective avoidance of en bloc mesenteric resection) by its confinement to only early stage disease is plausible although not proven. Specific study of the technique in early stage tumors is clearly essential before proffering this approach. Advances in technological capability have made feasible the local resection of small colonic tumors by intraluminal and even transluminal endoscopy -4. AlthoSentinel node biopsy would seem on first principles well suited to address this breach as it fulfills a similar role in tumors of the breast and skin. This technique has also been recently proposed to accompany endoscopic dissection of early gastric cancers in order to enhance functional outcome by minimizing the extent of surgical resection -13. AdjoTo date however no comprehensive study or review has been performed from the perspective of using lymphatic mapping to facilitate minimally resective techniques for early stage colon tumors. Analyses to date have instead focused primarily on the capacity of the technique to predict recurrence risk through the upstaging of conventionally node negative disease after standard operation has been performed -24. The Note: Rectal cancers lie outside the premise of this review as the anatomical arrangement of the mesorectum precludes against intraoperative nodal biopsy for rectal cancer. Furthermore, violation of the mesorectum may also compromise any subsequent attempt at formal oncological resection (and hence negatively impact upon patient outcome) should this be indicated by the pathology of the resected specimen.st January 1999 (the year of the earliest series published on the technique in colon cancer) and the 30th July 2008. The Cochrane library and EMBASE databases were also directly searched in a similar fashion. The following expanded Medical Subject Headings were used-'sentinel node', 'lymphatic mapping', 'colon cancer', 'colon tumo(u)rs', 'colorectal cancer/tumo(u)rs', 'large intestine' and 'gastrointestinal' . The reference list of all full publications so identified along with that of consensus papers, review articles, editorials and relevant book chapters were cross-checked for additional relevant publications. Data contained in meeting abstracts were not studied as these were judged unlikely to present sufficient detail for extraction required by our study protocol. Only English language publications were included . Finally only those studies that used vivo methodology alone for both the injection and the identification of salient sentinel nodes were analyzed . Intraoperative marking of the sentinel node rather than actual excisional biopsy allowed inclusion however as clearly the intent and purpose is the same.The following strategy was used to identify relevant publications regarding experience with lymphatic mapping in human patients with colon cancer . Otherwise the paper was excluded. In cases of mixed populations, where possible, only data relating to the in vivo mapping and biopsy of sentinel nodes in colon cancer were extracted. If not possible, the data was included with note made of the circumstances. Finally any further formal results analyses or additional hard data from the Methods, Results or Discussions sections of the studies was also recorded to allow for subsequent analysis and consideration.All data extraction was performed by two authors (RAC and JL) with cross-checking to ensure validation. When any disparity or disagreements arose the investigators met with the third author JM as final adjudicator to resolve the debate. The fields for data capture were pre-specified before analysis of the studies identified. Data pertaining to patient demographics, technique methodology and sentinel node efficacy by binary classification for patients with colon cancer undergoing lymphatic mapping were prepared on Microsoft Excel datasheets. Data from papers that explicitly declared themselves further sub-analyses of a previous study were included with the prior publication. Studies from the same authors but which did not declare themselves to contain overlapping cohorts were analyzed separately although are flagged in the subsequent tables to indicate that this is possibility. When quantitative results were not presented and were not extractable only that data that was useful to this analysis was extracted , true negative (both sentinel and non-sentinel nodes clear), false negative (sentinel node clear but non-sentinel nodes involved). The term 'false positive' is not appropriate in studies regarding sentinel node in cancer because the presence of isolated macrometastases in the sentinel node confers node positivity on the patient. Nor, given the experimental nature of sentinel node biopsy and the biological uncertainty of the significance of micrometastases, is this term appropriate for use when micrometastases alone are present in the sentinel node. Instead the term upstaging is used to better reflect the standing of sentinel nodes that are immunohistochemically positive when other non-sentinel nodes are clear of disease.The following definitions were therefore used to ascribe the performance rates of sentinel node biopsyDetection rate \u2013 refers to the number of times a sentinel node was actually identifiable = (Number of successful attempts to retrieve a sentinel node/Number of attempts to retrieve a sentinel node)*100%.Accuracy rate refers to the ability of the sentinel node to reflect the overall status of the lymph basin (whether positive or negative) = [*100%].Sensitivity refers to the number of times the sentinel node reflects the fact that disease is present in the non-sentinel nodes = (Number of patients with tumor-involved sentinel nodes/Number of patients with any lymph node containing tumor)*100%.false negative rate reflects the proportion of patients in whom no cancer was identified in the sentinel node but who had nodal deposits found in their non-sentinel nodes compared to the total number of those who had tumour containing metastases in non-sentinel nodes = *100%.The Upstaging rate refers to the number of cases in which sophisticated analysis of the sentinel node reveals tumor deposits that otherwise would not have been detected = *100%.There were no randomized controlled trails identified by our search methodology. Sixty-three clinical studies regarding lymphatic mapping and sentinel node biopsy for colon cancer in human patients were published in the English language during our review period of interest. Nine of these studies however actually utilized a primarily ex vivo lymph node identification technique (only the dye injection was performed in vivo and the surgeon made no attempt intraoperatively to identify any mapped nodes). These studies -37along. A furthThis left fifty-two studies warranting consideration for inclusion in this study ,47-97se. AlthougIn total fifty two diagnostic studies patient age while eleven gave no breakdown of the patient population by gender. The age breakdown of the other studies show no especially striking data (mean age 69 years) although three studies have an unexplained clear preponderance (>70%) of males as have two of females among their cohorts. This suggests that their populations may be somewhat atypical. Only four studies present data regarding the BMIs of their patient population \u2013 a potential important discrepancy that may induce error in both detection and false negative rates as intra-abdominal obesity may obscure discolored nodes in the mesentery. Finally, only 25 (50%) studies examined colon cancer in isolation. The vast majority (26) of the other studies also included rectal cancer. These studies, despite usually declaring the proportions of each tumor studied, most often did not present result sub-analysis. While the mean number of colon cancers studied in each publication is 68, 30 studies included less than 50 of such patients while 14 comprised less than 30 patients with colon cancer.Despite the stated aim of most studies being the evaluation of the utility of lymphatic mapping for staging node negative tumors, 19 studies made no attempt to exclude patients with distant metastases or indeed evident mesenteric deposits let alone grossly involved lymph nodes. Indeed some actually specifically included such patients. Of the remainder, seven excluded only patients with distant metastases while nine required the patient had only 'clinically localized' or 'resectable' disease to allow their inclusion. Of the twelve papers that exclude gross nodal and distant metastases, five include T4 disease while five do not profile their tumors by T-stage. Overall, at least 21 studies include T4 tumors within their cohorts. Furthermore, 25 of the studies that present such data possess high T3 and T4 to T1 and T2 ratios. Only five studies specifically consider tumor length or diameter as a factor that may affect performance parameters. Interestingly, four of these studies conclude that false negative rates are considerably more likely in patients with larger tumors while the fifth only examined this by taking 4 cm as a cut-off point for analysis. Only one study considered how tumor size may relate to the quantity of dye needed to adequately map it and found a significant positive correlation. Although the mean number of resected nodes in each study often is adequate , 21 studies include patients who have had considerably less nodes than this resected in their 'definitive' operation while 25 do not state either the mean or range of the non-sentinel nodal harvest. This raises concern over the quality control mechanisms in place to ensure the standard of the oncological operation performed in these studies.Only five studies specifically sought to ensure surgeon experience in the technique prior to commencing their study . This is likely particularly pertinent in the ten multicentre studies, only four of whom specifically sought to ensure minimum practical experience among their participants . Injection methodology overall was relatively similar. 45 protocols utilized an intraoperative subserosal injection of colorimetric mapping agent while three employed a submucosal injection. Twenty eight studies utilized isosulfan blue 1% in isolation while ten used Patent Blue alone and one used Vital Blue. Eight studies used a radioisotope as a mapping agent and the majority injected this agent submucosally preoperatively by additional endoscopy. Two studies also incorporated fluorescein while one used indocyanine green. Six studies specifically included laparoscopic operations with three employing this approach exclusively. All commenting authors agreed however that the technique is easily performed regardless of operative approach and adds minimally to overall operative time. The meaDespite the fact that the variation in accuracy and sensitivity rates is frequently decried, only fifteen publications specifically included analysis of their false negative rates with low detection rates also had false negative rates greater than 20% . Of the 43 studies with detection rates > 90%, only eleven (c. 25%) also had false negative rates greater than 20%.2 . None of the other studies presented any data in this latter regard.Of those with detection rates < 90%, two were multicentric trials. Neither these nor six of the seven single centre studies stated they validated surgeon expertise prior to commencing patient enrollment. Furthermore each of these studies was composed of less than 60 patients. Five studies included a proportion of rectal cancers approximating 15% of the population within their study cohorts. Four studies had marked T3/4 to T1/2 preponderance . Only three studies of those with detection rates > 90% included such high proportions of locally advanced disease but one of these specifically excluded clinically apparent lymphadenopathy while the other did not contain any T4 cases. Furthermore one other study included patients with liver metastases and even obvious mesenteric deposits and direct nodal invasion by the primary while every patient in another study was conventionally node positive. Finally the patients of one report had a mean BMI above 25 kg/mOf the sixteen studies with false negative rates above 20%, twelve presented no critical analysis of their false negative rates. Nor did any of these studies place any emphasis on surgeon experience in their stated inclusion criteria. Four studies were performed on a multicentre basis but none explicitly ensured surgeon expertise prior to commencement (in one such study the mean number of operations per surgeon was less than three) and nine studies included non-colonic tumors in between 10 and 26% of the cohort size. Furthermore one study specifically commented that tumors adherent to the retroperitoneum were included while five had T3/4 tumors accounting for approximately 80% of their patient cohorts. Three had a mean tumor size of greater than 4.2 cm with one concluding that its false negatives case were associated with significantly bigger tumor sizes . One additional study also found a strong trend in favor of an association between false negative rate and larger tumor sizes . Two studies had a mean number of resected lymph nodes of eight and nine respectively while at least twelve studies included some patients with less than ten nodes in their resected specimens (eight including some with five or less nodes examined). Two others presented no mean data on this subject and three presented no range. Four did not serially section the sentinel node while six did not employ immunohistochemistry or RT-PCR.With respect to the fifteen publications with false negative rates between 10 and 20%, only three studies included specific analysis of their rates. Of the fifteen, two were multicentric trials and both sought to ensure surgical expertise and had false negative rates each of 11 and 12%. Nine studies included rectal cancers in their cohorts (in between 9% and 74% of their cohorts). Four studies had no explicit exclusion criteria while six sought only to exclude distant disease deposits. No study provided any data on patient BMI. Only one study specifically excluded T4 disease. Eight had a significant (>60%) T3/4 preponderance (being >70%). The mean number of resected lymph nodes was greater than ten in fourteen. Four studies included patients with less than this number while the one presented no data regarding the range. Two studies did not employ serial sectioning of the identified sentinel nodes and two did not utilize immunohistochemical or RT-PCR methods of examination.Sufficient lymph basin resection is an oncologic sine non qua of operation with curative intent for solid organ malignancies as nodal status remains the primary portent of prognosis and adjuvant therapy prescription. For colon cancer, staging propriety by convention demands that this equate to en bloc resection of the entire mesenteric lymphatic delta. While the manner of performance of standard resectional operation for colon cancer by laparoscopy or laparotomy means that the extent of access is already determined (and so supplementing bowel resection with full mesenteric resection is readily facilitated) this is not for case for endoscopic resectional techniques. Therefore these techniques are currently limited to the address of benign lesions or neoplastic disease with minimum likelihood of lymphatic dissemination. A minimally invasive means of reliably confirming the lymph node status could greatly enhance the oncological propriety of these approaches and extend their indications towards their actual technical capacity. While it is clear that sentinel node biopsy is not indicated to minimize the extent of therapeutic lymphadenectomy in colon cancer (i.e. the resection of nodes containing tumor deposits), it could theoretically have a role in helping select those with truly early stage disease for endoscopic resection.Furthermore, although it is often considered that adjoining mesenteric lymph node dissection to the intestinal resection impacts little on the patient undergoing conventional oncological operation for colon cancer -103, thiafter conventional radical operation[The focus of sentinel node biopsy in colon cancer has therefore been on upstaging conventionally node negative patients operation. This meoperation. AlthougThe first evident confounder to obtaining clarity regarding the accuracy of the technique is the number of publications emanating from single centers. Although it seems likely that these seven centers have overlapped their patient cohorts at least to some extent in successive publications, the exact proportions is rarely explicitly declared. Equally however it cannot be assumed that these studies entirely overlapped their experiences and so to ensure fairness and transparency every study meeting the inclusion exclusion is included in this study with those coming from the same center being flagged in the Tables. The next main obstacle within each publication complicating deliberation of the technique's applicability for early stage colon cancer is the marked contamination of rectal cancers throughout the evidence base. The consequences of doing so without presentation of complete subgroup analyses gives an artificial impression of reduced feasibility and accuracy rates overall because lymphatic mapping is clearly more arduous and less reliable in this site. FurtherSurgeon expertise and experience has already been determined a major feature for lymphatic mapping in breast cancer and seemCertain groups have however clearly managed to overcome confounding factors of the technique and consistently obtain negative predictive values of similar quality to those that currently justify conservative resection fields in other specialties. It is perhaps no surprise that these investigators tend to fastidiously analysis their false negative results as did the pioneers of the technique in breast cancer and melanoma. On the other hand, other authorities have not hesitated to declare the technique in colon cancer either invalid or of dubious clinical value,122 on tThe general tendency of a relationship between advancing stage an increased likelihood of metastases being present in non-sentinel nodes also supports the basic contention that lymphatic mapping in colon cancer may be particularly efficacious in germinal cancers \u2013 a perspective made particularly compelling by the inherent suitability of early stage disease for truly minimally invasive resective techniques. The lesions that could be resected by endoscopic means are by definition smaller and therefore likely confined to a single lymphatic delta. Furthermore T3 or T4 disease identifiable by staging is not feasibly resected endoscopically and so the tumor stages with the highest frequency of being node-positive are excluded. The fact that 20\u201340% of patients with T3/4 lesions but without demonstrable lymph node metastases actually die of their disease also supports exclusion of these patients from non-radical operation. De facto confinement of the patient cohort to T1 and T2 stage disease (perhaps by including adjunctive staging measures such as endoscopic ultrasound) may theIf sentinel node biopsy is ever to be used as means to alleviate mesenteric resection in cases that are truly node negative, consideration must be given to the cases where the sentinel node is positive or indeed falsely negative. In the former case, subsequent radical lymphadenectomy should still be performed. Ideally therefore the sentinel node analysis should be performed intraoperatively to allow direct progress to the definitive excisional surgery . Considerable precedent exists for such analysis in breast cancer -131 and Sentinel node mapping could never substitute for a properly performed oncologic colorectal resection when this is indicated. The concept however that lymphatic mapping may have sufficient capability to provide the oncological proprietary for the curative surgery for early stage cancers without en bloc mesenteric resection seems biologically plausible but cannot yet be definitively judged given the lack of clarity and consistency in the literature to date. Specific study of the technique in those early stage tumors likely to be selected for endoscopic resection is clearly therefore essential before this approach can be considered in clinical practice.The authors declare that they have no competing interests.RAC conceived of the study, participated in the study design and performance and composed the manuscript. JL contributed to the study design and performance and guided the manuscript composition. JM conceived of the study and participated in its design and coordination. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:"} +{"text": "Redo open-heart surgery and sternal reentry in patients with previous deep sternal wound infection (DSWI) and absence of sternal integrity can be a delicate and morbid task due the lack of a dissection plane between the heart and the surrounding soft tissues. Delayed sternal reconstruction and osteosynthesis with horizontal titanium plating fixation (Synthes) following vacuum assisted therapy (KCI) has recently been proposed and adopted for the treatment of DSWI. We present such a case of a patient who was successfully reoperated for valve replacement three years after coronary artery bypass grafting complicated by DSWI and initially treated with titanium plate fixation. Deep sternal wound infection remains a feared complication of cardiac surgery still associated with significant morbidity and mortality. Furthermore, there is a lack of consensus for its definitive management ,2. We haS.Aureus DSWI which was referred to our center and managed with aggressive debridement, VAC therapy and then delayed sternal wound reconstruction with horizontal titanium plate fixation in 2005 at an outside hospital. This initially uneventful procedure was followed by a n Figure and pectThree years later the patient was investigated following complaints of recurrent chest pain and shortness of breath. A repeat coronary angiogram showed that the two vein grafts were occluded while the left internal thoracic artery directed to the left anterior descending coronary artery remained patent and functional. Severe aortic stenosis was documented by echocardiography with an aortic valvular area of 0.6 cm2 and respective maximal and mean gradients of 59 and 38 mmHg. A transapical aortic valve replacement and percutaneous coronary dilatation were initially considered but a formal midline sternotomy finally chosen, as it was felt he was a good candidate for conventional surgery.After removing the locking pin and cutting the titanium plates along the sternal midline, sternal re-entry could be performed uneventfully with the oscillating saw while the plates were pulled upward Figure . The plaDeep sternal wound infection will remain a costly and worrisome complication of cardiac surgery despite better care of septic patients and management with muscle flaps. Sternal preservation with osteosynthesis has previously been suggested to make cardiac reoperation a safer procedure. HoweverSince 2002, we have adopted the routine use of negative pressure wound therapy after debridement in patients presenting with DSWI in our center, and have been able to better preserve sternal integrity with this approach. It usually takes 2 weeks following initial and repeat debridements to obtain negative wound cultures in these cases and then a full sternal reconstruction can then be performed with titanium plates covered with pectoralis myocutaneous flaps even after partial bone losses from the manubrium or each hemi-sternum.With increasing life expectancy in industrialized countries, reoperation is a likely possibility in open heart surgery patients often presenting with degenerative valvular pathologies such as aortic stenosis. The volume of transapical aortic valvular replacement has been rapidly increasing worldwide and will be an option in these patients but this procedure is also carrying its own risk and at this point in time is still offered to patients who are not good surgical candidates. If the CABG: Coronary artery bypass grafting; VAC: Vacuum Assisted Therapy/Negative pressure wound therapyThe authors declare that they have no competing interests.All authors read and approved the final manuscript.Data from all patients operated at the IUCPQ are entered in the surgical data base without patient's name and according to current ethical rules of the research center."} +{"text": "Objective: The authors performed a prospective evaluation of staging laparoscopy with laparoscopic ultrasonography in predicting surgical resectability in patients withcarcinomas of the pancreatic head and periampullary region.Summary Background Data: Pancreatic resection with curative intent is possible in a select minority of patients who have carcinomas of the pancreatic head and periampullary region. Patient selection is important to plan appropriate therapy and avoid unnecessary laparotomy in patients with unresectable disease. Laparoscopic ultrasonography is a novel technique that combines the proven benefits of staging laparoscopy with high resolution intraoperative ultrasound of the liver and pancreas, but which has yet to be evaluated critically in the staging of pancreatic malignancy.Methods: A cohort of 40 consecutive patients referred to a tertiary referral center and with a diagnosis of potentially resectable pancreatic or periampullary cancer underwent staging laparoscopy with laparoscopic ultrasonography. The diagnostic accuracy of staging laparoscopy alone and in conjunction with laparoscopic ultrasonography was evaluated in predicting tumor resectability .Results: \u201cOccult\u201d metastatic lesions were demonstrated by staging laparoscopy in 14 patients (35%). Laparoscopic ultrasonography demonstrated factors confirming unresectable tumor in 23 patients (59%), provided staging information in addition to that of laparoscopy alone in 20 patients (53%), and changed the decision regarding tumor resectability in 10 patients (25%). Staging laparoscopy with laparoscopic ultrasonography was more specific and accurate in predicting tumor resectability than laparoscopy alone .Conclusions: Staging laparoscopy is indispensable in the detection of \u201coccult\u201d intraabdominal metastases. Laparoscopic ultrasonography improves the accuracy of laparoscopic staging in patients with potentially resectable pancreatic and periampullary carcinomas."} +{"text": "Bronchoalveolar lavage (BAL) is a useful diagnostic tool in interstitial lunge diseases (ILD). However, differential cell counts are often non specific and immunocytochemistry is time consuming. Staining of glyoproteins by periodic acid Schiff (PAS) reaction may help in discriminating different forms of ILD. In addition, PAS staining is easy to perform. BAL cells from patients with idiopathic pulmonary fibrosis (IPF) (n = 8), sarcoidosis (n = 9), and extrinsic allergic alveolitis (EAA) (n = 2) were investigated. Cytospins from BAL cells were made and cells were stained using Hemacolor quick stain and PAS staining. Lymphocytic alveolitis was found in sarcoidosis and EAA whereas in IPF both lymphocytes and neutrophils were increased. PAS positive cells were significantly decreased in EAA compared to IPF and sarcoidosis (P < 0.05). No significant correlation between PAS positive cells and inflammatory cells was observed. These results suggest that PAS staining of BAL cells may provide additional information in the differential diagnosis of ILD. Further studies ware warranted to evaluate PAS staining in larger numbers of BAL from patients with ILD. Bronchoalveolar lavage (BAL) provides an important diagnostic tool that can facilitate the diagnosis of various diffuse lung diseases. It can be used to determine inflammatory cell profiles and detect pathogens . BAL cytPAS (periodic-acid-Schiff) staining is used for detection of structures that contain high concentrations of carbohydrate macromolecules typically found in connective tissue, mucus, and basal laminae. In BAL PAS staining is mostly used to diagnose alveolar proteinosis . In ILD BAL samples of patients with proven idiopathic pulmonary fibrosis (IPF) (n = 8), with sarcoidosis (n = 9), and with extrinsic allergic alveolitis (EAA) (n = 2) were obtained with the help of flexible bronchoscopy. Table In short, a total of 200 ml of sterile saline solution was instilled in 20 ml-aliquots into the middle lobe and aspirated thereafter. Following standard techniques cytospinFigure The demographics of the groups show that patients with IPF and EAA were markedly older than patients with sarcoidosis. This is not unusual in clinical practice since sarcoidosis is more often observed in younger people and IPF is typically found in older patients ,7. ThereThe findings in the present study demonstrated a marked decrease in PAS positive BAL cells in patients with EAA compared to other interstitial lung diseases such as IPF and sarcoidosis. However, we only investigated a very small number of EAA patients but both patients had a low percentage of PAS positive BAL cells (25% and 26%). This analysis of individual data suggests that in PAS positivity of BAL cells may be in fact reduced in EAA but this has to be further investigated with larger numbers of samples.We did not find a significant correlation between PAS staining and a single cell type and I did not do double staining to differentiate PAS positive cells. Therefore it is not clear which inflammatory cells mostly stained positive. Future studies will have to identify whether macrophages, lymphocytes or neutrophils are the main source of PAS positive material. Another option is that PAS positive proteins undergo phagocytosis by alveolar macrophages. In fact, several proteins (eg. surfactant proteins) are secreted by pneumocytes . These pIn the present study IPF, sarcoidosis and EAA were not further subdivided into different stages of disease due to the small numbers of patients. It may be interesting to look whether percentage of PAS positive cells is associated with different stages of disease . However, this was beyond the scope of the present investigation.So far no experiments on PAS staining in other interstitial lung disease such as pneumoconiosis or Langerhans cell histiocytosis were performed but these studies would clearly be very helpful to further evaluate the role of PAS staining in discrimination of ILD. Own experiments on patients with allergic bronchopulmonary aspergillosis (ABPA) and pneumonia revealed a mean of PAS positive cells of 70.5% and 47.5%, respectively. It is interesting that in pneumonia that is commonly associated with mucus hypersecretion the mean rate of PAS positive cells was lower than in BAL cells from patients with sarcoidosis or IPF. This further underlines the fact that PAS staining identifies glycoproteins and not only mucin proteins.In conclusion the present findings suggest that PAS stain of BAL cells may be helpful in differential diagnosis between EAA and other ILD. Although the numbers of patients investigated in this study was very small we think that further studies are warranted to evaluate the use of PAS stain in different forms of ILD. Should future studies prove that PAS can discriminate different ILD then this would be a very easy and less expensive tool for BAL-based differential diagnosis.BAL: bronchoalveolar lavage; EAA: extrinsic allergic alveolitis; IPF: idiopathic pulmonary fibrosis; PAS: periodic acid SchiffThe authors declare that they have no competing interests.HPH planned and performed the experiments. HPH and PZ did the data analysis and wrote the manuscript."} +{"text": "Adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) levels of peripheral blood mononuclear cells were measured in 34 patients with various types of solid tumours. The mean ADA activity was found to be significantly lower than in controls (P less than 0.005). Patients with nonresectable tumour or with recurrence after radical surgery showed low ADA levels, while patients operated upon and without recurrence had enzymatic activity not different from that of normal controls. The mean value of PNP activity was similar to that of normal controls; no differences were observed between operated patients without recurrence and cases with nonresectable tumour or with recurrence after surgical treatment. No effects on ADA and PNP levels appeared to be induced by chemotherapy."} +{"text": "A photoexcited titanium dioxide surface has a strong ability to decompose water into hydrogen and oxygen. We have studied this effect in order to use it to kill cancer cells in vitro and in vivo. A distinct cell killing effect was observed on cultured T-24 human bladder cancer cells treated with titanium dioxide particles and 300-400 nm UV light irradiation. Titanium dioxide plus UV light also dramatically suppressed the tumour growth of T-24 cells that were implanted in nude mice. Cells cultured on the titanium dioxide electrode were also killed under UV irradiation when the electrode was anodically polarised, suggesting that photogenerated holes are involved in the cell killing. The cell killing effect caused by titanium dioxide particles plus UV light irradiation was significantly hampered in the presence of L-cysteine and catalase, scavengers of hydroxyl radicals and hydrogen peroxide respectively. Transmission electron microscopic observations showed the titanium dioxide particles to be distributed on the cell surface and inside the cells. These results suggest that titanium dioxide particles under UV light irradiation produced photogenerated holes on the surface yielding hydroxyl radicals and hydrogen peroxide inside or outside the cells and the cells were then killed by the action of these highly oxidising molecules. The possible application of photoexcited titanium dioxide particles to cancer treatment as a new anti-cancer modality is discussed."} +{"text": "PAF is a potent inflammatory compound known to stimulate the release of various cytokines involved in rheumatic diseases. Elevated blood PAF levels are reported in these patients. We report that serum PAF acetylhydrolase activity (AHA) levels are decreased in patients with rheumatoid arthritis or osteoarthritis as compared to healthy controls. Serum and synovial fluid AHA levels were correlated in these patients. The present study suggests the potential role of AHA in controling systemic and/or local PAF levels in patients with rheumatic diseases."} +{"text": "The transgenic animals had lower IGF-I levels, were smaller in terms of body size and weight, and exhibited decreased tumour incidence relative to controls. The demonstration that both body size early in life and breast cancer incidence are influenced by experimental perturbation of the GH\u2013IGF-I axis in a transgenic model provides evidence that variability between individuals with respect to these hormones underlies the relationship between body size early in life and breast cancer risk observed in epidemiological studies. \u00a9 2001 Cancer Research Campaign http://www.bjcancer.comSeveral reports have provided evidence that body size early in life is positively correlated with risk of subsequent breast cancer, but the biological basis for this relationship is unclear. We examined tumour incidence in transgenic mice expressing a growth hormone (GH) antagonist and in non-transgenic littermates following exposure to dimethylbenz["} +{"text": "This study was designed to investigate cerebellar lobular contributions to specific cognitive deficits observed after cerebellar tumor resection. Verbal working memory (VWM) tasks were administered to children following surgical resection of cerebellar pilocytic astrocytomas and age-matched controls. Anatomical MRI scans were used to quantify the extent of cerebellar lobular damage from each patient's resection. Patients exhibited significantly reduced digit span for auditory but not visual stimuli, relative to controls, and damage to left hemispheral lobule VIII was significantly correlated with this deficit. Patients also showed reduced effects of articulatory suppression and this was correlated with damage to the vermis and hemispheral lobule IV/V bilaterally. Phonological similarity and recency effects did not differ overall between patients and controls, but outlier patients with abnormal phonological similarity effects to either auditory or visual stimuli were found to have damage to hemispheral lobule VIII/VIIB on the left and right, respectively. We postulate that damage to left hemispheral lobule VIII may interfere with encoding of auditory stimuli into the phonological store. These data corroborate neuroimaging studies showing focal cerebellar activation during VWM paradigms, and thereby allow us to predict with greater accuracy which specific neurocognitive processes will be affected by a cerebellar tumor resection."} +{"text": "In vitro fertilization is an upcoming speciality. Anaesthesia during assisted reproductive technique is generally required during oocyte retrieval, which forms one of the fundamental steps during the entire procedure. Till date variety of techniques like conscious sedation, general anaesthesia and regional anaesthesia has been tried with none being superior to the other. However irrespective of the technique the key point of anaesthesia for in vitro fertilization is to provide the anaesthetic exposure for least duration so as to avoid its detrimental effects on the embryo cleavage and fertilization. Finally, on 25th July 1978 LOUISE JOY BROWN, the 1st successful test tube baby was born. Since then there has been continuous refinement in the fertility drug protocols and the techniques to retrieve eggs. As a result, IVF success rates began to climb slowly reaching 25-30%In-vitro fertilization (IVF) started 30 years back when Lesley and John Brown, a young couple from Bristol were unable to conceive for 9 years. Lesley had blocked Fallopian tubes. On 10In-Vitro Fertilization is a broad term for the technique of ultrasound directed Oocyte retrieval (UDOR) or Trans Vaginal Follicle Aspiration (TVFA) and fertilization in the laboratory with transfer of embryos back into the uterus.Broadly speaking IVF involves the following stepsOvarian stimulationEgg collectionSperm processing &Fertilization & embryo transfer1980's witnessed a drastic change from the use of laparoscope to vaginal ultrasound probe for egg retrieval. Although this technique of using Vaginal ultra-sound probe is less invasive and associated with higher pregnancy rates, it forms one of the most stressful and painful components of the entire assisted reproductive treatment23Pain during oocyte retrieval is caused by the puncture of the vaginal skin and ovarian capsule by the aspirating needle as well as manipulation within the ovary during the entire procedureCoexisting illness-Patients presenting in the IVF clinic needs to be investigated for any co morbid illnesses. In India tuberculosis is the most important cause of infertility, so we need to know the drug interactions of anti tubercular drugs with the anaesthetic agents. These patients are generally kept on aspirin or heparin so as to prevent the hypercoaguable state occurring as a result of gonadotrophic injections. Aspirin should ideally be stopped 3 days prior to egg retrieval procedure. In case our patient is on heparin, we need to know the Activated prothrombin time.Thyroid can also be a cause of infertility so it becomes mandatory to assess the thyroid function tests and take appropriate anaesthetic precautions.Some of the patients might be receiving treatment forpsychomotor disorders like depression and are on anti depression drugs like Selective serotonin reuptake inhibitors(SSRI), tricyclins or drugs like tragadone, bupropion. It is therefore important to adjust the dosages of anaesthetic agents especially narcotics accordingly.Anxiety \u2013 Another major challenge for the anaesthetistis to allay the anxiety. The patients presenting in the IVF clinic are under high degree of social and psychological stress. Majority of them are in late thirties, and the immense family pressure makes them more susceptible to psychomotorillness like depression and psychosis. Moreover this problem is further aggravated by the hormonal manipulation occuring during in vitro fertilization.Thus it becomes important to provide them with a comfortable environment so as to extract complete medical and pharmacological history. Nowadays many upcoming IVF centres have a provision for isolated rooms for the pre-anaesthetic checkups. One must remember that proper preoperative counseling is very important in allaying anxiety in such patients.Presently anaesthesia for assisted reproductive tecnique is emerging as a speciality in itself. Patients presenting for IVF can have varied causes for infertility like pelvic inflammatory disease due to tuberculosis, chlamydial infection, history of previous pelvic surgery, tubal blockage or endometriosis. Therefore the patients undergoing this treatment are thoroughly evaluated for the cause of infertility and appropriate treatment instituted. A thorough pre anaesthetic evaluation is required to identify any comorbid illness.Monitored sedation with/without local anaesthesiaGeneral AnaesthesiaRegional AnaesthesiaA survey conducted by Bokhari et al in U.K showed the use of sedation in 46% of the centres, general anaesthesia in 28%, regional anaesthesia with sedation in 12% while a cocktail regime was followed by the rest 14%Monitored anaesthesia is relatively easy to deliver, drugs are well tolerated and best suited in day care settings. However, it has its own risks of cardiac, respiratory and anaphylactic complications.In USA, 95% of the programs use conscious sedation as a part of monitored anaesthesia care10In UK, 84% of the centres now use sedation11Monitored anaesthesia technique with remifentanil resulted in a higher pregnancy rate than GA with alfentanil+ propofol or isoflurane + propofol for maintenanceHadimioglo etal had studied various combination of sedation regimens for oocyte retrieval. and found no significant difference between propofol +fentanyl, midazolam+fentanyl and propofol+fentanyl in the recovery characteristics142O and opioids can be an option for anaesthesiologists. Hammadeh etal in 1999, showed a higher retrieval of oocytes with remifentanil + propofol or isoflurane based general anaesthesia than with sedation with midazolam, diazepam or propofolInvariably allanaesthetic agents being used in general anaesthesia have been detected in follicular fluid, raising concerns regarding their use. However, with recent studies documenting the safe use of the agents, balanced anaesthesia with NWhile selecting a desired agent our main concerns are:-Whether the substance enters the follicular fluid?What are its toxic effects on the fertilization and clevage and pregnancy rates?\u2022 PropofolWidely being used in assisted reproduction and its effects on the fertilization, embryo clevage and pregnancy rates has been extensively studied. Propofol has added advantages of antiemetic property along with faster recovery.Though earlier studies had documented adverse effects of increased exposure to propofol on clevage of oocytes17In addition, there was no significant difference found in fertilization rate, clevage and embryo cell number, implantation rate as compared to thiop entone. Except a trend towards low fertilization rate with longer exposure to anaesthetic drug19\u2022 Role of Nitrous \u2013 OxideIts role still remains controversial. Gonen etal found out that nitrous oxide has deleterious effect on IVF outcome2O inactivates methionine synthetase thereby decreasing the amount of thymidine available for DNA synthesis in dividing cells. However, as the inactivation of methionine proceeds slowly in the human liver, the effect of N2O is minimal. Further more, the low solubility of N2O exposes the oocytes to this gas for a brief duration. Rosen etal in 1987 found no significant difference between the fertilization or pregnancy rates when comparing isoflurane with O2 which was further confirmed by Matt et al2223While, Hadimioglu N et al in 2002 showed nitrous oxide actually increase the rate of IVF by reducing the concentration of other potentially toxic and less diffusible anaesthetic drugs\u2022 BenzodiazepineMidazolam is the most commonly used benzodiazepine. Although minimal amount of this benzodiazipine are found in follicular fluid, no detrimental effects have been proven so far5\u2022 NarcoticsIn recent years, various opioids have been used as a part of regime in conscious sedation and monitored care for anesthesia in assisted reproductive technique.Fentanyl or alfentanil were found to be favourable agents when used in combination with propofol by Hadimioglu etal in 2002. Fentanyl has minimal penetration into follicular fluid26\u2022 KetamineA randomized prospective study, found the combination of midazolam and ketamine a good alternative to general anaesthesia2O resulting in decreased clevage rates and increased abortionsMajority of studies have shown detrimental effect of halogenated fluorocarbons with N2O and isoflurane anaesthesia on human IVF pregnancy rateMatt etal in 1991 found no significant effect of NIt constitutes either central neuraxial blockade or the peripheral nueral block.Paracervical block with different doses of lidocaine with sedation has been used by anaesthetist for egg retrievalSpinal anaesthesia is also an effective method. Martin et al in 1998 had used low dose hyperbaric 1.5% lidocaine (45mg) spinal with low dose fentanyl 10mcg for egg retrieval38Epidural anaesthesia also forms a viable option but does not demonstrate any advantage over intravenous sedationBupivacaine compared favorably to lidocaine in all aspect except taking approximately 30 min longer to micturition and to dischargeHormonal response to follicular puncture is fully attenuated by regional anaesthesia and partially by technique requiring sedationIt is a traditional Chinese medicine, nontoxic, relatively affordable, therapy with possible indications as an adjunct in assited reproduction with the following beneficial effects:SympathoinhibitoryIncreased beta-endorphin levelsAntidepressant, anxiolyticNeuroendocrine effect on hypothalamic \u2013 pituatry-ovarian axisIncreased uterine blood flowElectroacupuneture has been used with along with paracervical block for analgesia during oocyte retrievalThe technique employed in aspiration of the oocyte and laborotry manipulations have all been modified and updated. Which is better, sedation or general anaesthesia is more of a personal preference. But the anaesthetic which is important to the comfort level both for the patient and the gynaecologist to maximize the harvesting of oocytes plays an important role in the successful outcome.How Safe are Anaesthetic Agents? With the coming up of large prospective trials documenting safe use of drugs like propofol, opioid, the newer anaesthetics have lost their inhibitions regarding the use of these agents, thereby widening the scope of more rationale anaesthesia in IVF and extending our services to this developing sub-speciality.The key to anaesthesia in IVF is to aim for pharmacological exposure of shortest duration with minimal penetration to follicular fluid."} +{"text": "A recently introduced assay of cell mediated immunity and humoral inhibitory factors has been evaluated in colorectal cancer patients. Using a perchloric acid extract of adenocarcinoma of the large bowel as antigen, 16/27 patients with colorectal cancer had significant cellular reactivity when their separated peripheral leucocytes were tested in homologous AB serum. In autologous serum only 7/27 had significant reactivity; 6/20 patients with a variety of other malignancies showed sensitization to the colorectal antigen preparation. It is concluded that the leucocyte adherence inhibiton test may offer a simple method of assaying for serum blocking factors in sequential studies but will be of little value in the diagnosis of large bowel cancer."} +{"text": "An awareness and understanding of the presence of an additional root and unusual root canal morphology is essential as it determines the successful outcome of endodontic treatment. Aberrations in root canal anatomy are commonly occurring phenomena. A thorough knowledge of basic root canal anatomy and its variation is necessary for successful completion of endodontic treatment. This report points to the importance of looking for additional roots and canals because knowledge of their existence would enable clinician to treat a case successfully that otherwise might end in failure. An awareness and understanding of the presence of additional roots and unusual root canal morphology is essential as it determines the successful outcome of endodontic treatment. In spiteet al. Martinez-Berna, Ruiz-Badanelli studied 338 maxillary first molar's and reported three cases of six canals with three canals in the mesio buccal root two in disto buccal and one in palatal root.[3et al. analyzed endodontic treatment in 16 maxillary molars and of six extracted teeth with two palatal roots and classified these 22 molars into three types, according to root separation level and their divergences.[Anatomic characteristic of permanent maxillary molar is generally described as a group of teeth with three roots, one palatal and two buccal. The occurrence of second mesiobuccal canal is a common variation. Beatty reported a first maxillary molar with five root canals, three of them located in mesio buccal root. Bond et al root.3 Christieergences.A 24-year-old male patient with pain in 26 was referred to the Department of Endodontics. Medical history was non contributory. Clinical, radiographic and pulp testing examination revealed that tooth was symptomatic and patient requires endodontic treatment. The preoperative radiograph revealed the presence of an additional palatal root . The patA 21-year-old male patient was referred to the Department of Endodontics, with pain in 16. Medical history was non contributory. Clinical, radiographic and pulp testing examination revealed that tooth was symptomatic and patient requires endodontic treatment. Anesthetizing the tooth, a conventional endodontic access opening was made. After removing the coronal pulp and probing with a DG 16 three principal root canal orifice mesiobuccal, distobuccal, palatal and in addition a small hemorrhagic point was noted adjacent to the palatal orifice. The conventional triangular access was modified to a trapezoidal shape to improve access to additional canal and by evaluating radiograph from different angulations, it was concluded that this tooth was having an additional palatal root. The working length of each canal was estimated by means of an apex locator , and confirmed with intra oral periapical radiograph. The canals were initially instrumented with #15 nickel titanium files (Dentsply Maillefer) under irrigation with 3% sodium hypochlorite and 17% EDTA. Coronal flaring was carried out by using Gates Glidden drills (number 3 and 2 Dentsply Maillefer). Cleaning and shaping were done using hand nickel titanium file system (Dentsply Maillefer). The canals were obturated with AH plus resin sealer and gutta-percha points using lateral condensation technique and tooth was restored with a posterior composite filling .et al.[Anatomical variations can occur in maxillary permanent molars. Christie et al. reportedType 1 \u2013 Buccal roots are often cow-horn shaped and less divergent. It has two widely divergent palatal roots, often long and tortuous.Type 2 \u2013 Roots are shorter run parallel and have blunt root apices.Type 3 \u2013 Root morphology is constricted with mesiobuccal, mesiopalatal, distopalatal canal engaged in one web of root dentin. The distobuccal root seems to stand alone and may diverge to distobuccal.et al. classification.The present two cases come under Type 1 Christie Slowey also reported maxillary molar with two palatal roots. LibfieldThe etiology behind formation is unclear. In supernumerary roots its formation could be related to external factors during odontogenesis or penetrance of atavastic gene. Curzon suggests that additional rooted molar trait has high degree of genetic penetrance.In mandibular molars also there could be variation in root morphology. Carlsen and Alexandersen suggest that if additional root is present distolingually it be termed Radix Entomolaris If additTo determine for presence of additional root there should be slight different approach besides normal procedural protocol and the clinician should look for following signs which might indicate towards the presence of additional root \u2013Cervical prominence \u2013 it could be detected through periodontal probingExtra cusp \u2013 which is present in combination with cervical prominenceRadiographic examination \u2013 radiograph should be taken at different angulationsCT scanThe variation in root or root canal morphology, especially in multirooted teeth, is challenging for diagnosis and successful endodontic therapy. The knowledge of common anatomic characteristics and their possible variation is fundamental. Knowledge of unknown variation like the case discussed is essential as non treatment of one additional root or root canal can lead to failure of endodontic treatment. The presence of an additional palatal root is rare but still an unforeseen eventuality and whenever an endodontist is confronted with unusual difficulties and patient complains of persistent post medicament pain, the possibility of an extra canal or root has to be borne in mind. The present report emphasizes the need for the endodontist to be ever vigilant and knowledgeable about aberrant anatomical situations."} +{"text": "Dear Editor,et al., on manual small incision cataract surgery (MSICS) under topical anesthesia with intracameral lignocaine.[We read with great interest the article by Gupta gnocaine. The authThe authors have mentioned that sensitivity to lignocaine and inability to understand verbal commands were the only contraindications for topical anesthesia. However, certain patients who although can follow verbal commands, are either apprehensive or uncooperative and so may not be suitable candidates for topical anesthesia. Besides, patients with nystagmus would also be a contraindication to topical anesthesia.The authors have not mentioned the dose of intracameral lignocaine, whether it was given as a single injection or in divided doses and whether supplementary anesthetic eye drops were used during surgery. It is also mentioned in the study that 0.5% lignocaine with or without preservative is \u2018safe\u2019 for intracameral use. Regular 2% lignocaine and 2% lignocaine viscous contain methylparaben and propylparaben as preservatives. These preservatives have been shown to cause corneal endothelial toxicity in animal models although the effects are temporary. There is also no mention of pre- and postoperative endothelial cell count. Thus it would be premature to say that lignocaine with preservative is a \u2018safe\u2019 drug for intracameral injection without investigating this aspect.[In the present study, 2% lignocaine viscous was used after draping and was allowed to remain in contact with the ocular surface for about one minute after which surgery was commenced. It is not mentioned whether 2% lignocaine was washed before starting the surgical procedure or not and also whether one tube was used for single or multiple patients. Lignocaine 2% viscous can easily get contaminated if used on a shared basis thus posing a risk of postoperative endophthalmitis. Application of 5% povidone-iodine solution three times, five minutes apart after putting 2% lignocaine viscous and then thoroughly washing the conjunctival sac with sterile water before starting surgery, can reduce the risk of postoperative endophthalmitis and toxic anterior segment syndrome secondary to seepage of preservatives into the anterior chamber.4The authors have stated in their study that peribulbar anesthesia causes instability of anterior chamber secondary to raised intraocular pressure due to increased intraorbital volume. We feel that raised intraocular pressure is not responsible for anterior chamber instability since in the standard Blumenthal technique of MSICS, the intraocular pressure remains more than 35 mm of Hg throughout the procedure without compromising anterior chamber stability. On the contrary, constant lid squeezing, residual sensations and blepharospasm secondary to bright microscope light may be the causes for intraoperative anterior chamber instability rather then peribulbar anesthesia.["} +{"text": "Approximately 28% of patients with cancer pain die with severe untreated pain despite effective multidisciplinary techniques that should treat these patients effectively. Despite Interventional pain management procedures are most often used in concert with standard analgesic regimens to reduce opioid side effects or gain better analgesic efficacy. The interventional analgesic technique may be used at any time during the course of cancer treatment but is often employed during the more aggressive phases of disease. IntervenBefore jumping into interventional technique, patients should receive appropriate trials of opioid and non-opioid analgesics. A complete pain evaluation with pain history, appropriate physical examination (including a neurologic examination), and a tentative etiology for the pain is necessary prior to any invasive procedures. A neurologic examination identifies any preexisting neurologic deficits as well as areas of minor motor or sensory reduction, Informed consent is a prerequisite, especially with the neurolytic blocks that may result in permanent motor/sensory deficits. A local anesthetic block is usually performed prior to any \u201cpermanent\u201d neurolytic procedure to evaluate the likelihood of success and identify neurologic deficits that may be intolerable.Virtually all studies, to date, recommend the proper selection of patients so that less invasive analgesic techniques may be utilized as first-line therapy.The wide variety of interventional pain management techniques available in the present situation should encourage physicians to consider \u201csomething further\u201d in almost all cases of cancer pain. The appropriate use of anesthesiologic interventions to help manage cancer pain will improve the quality of life for all patients.There is a need for research to determine clear indication and benefit from interventional pain management techniques in cancer pain patients."} +{"text": "In order to determine the significance of local oestrogen biosynthesis within the breast, aromatase activity has been measured in adipose tissue from the breasts of women with either benign (n = 36) or malignant breast disease (n = 51). Particulate fractions from all samples possessed aromatase activity, but levels in adipose tissue adjacent to malignant tumours were significantly higher than those in tissue close to benign breast lesions (P less than 0.0001). Elevated aromatase activity in adipose tissue from breast cancer patients may be of importance in view of the central role played by oestrogen in the natural history of breast cancer."} +{"text": "Vegetarian diets have decreased incidence rates of chronic non-communicable diseases that have been associated with burden on medical systems worldwide. Previous studies propose a relationship between meat consumption and high medical expenditure; however, there is a dearth in the literature with strong evidence supporting the hypothesis between two factors. Using the data from Taiwan\u2019s National Health Insurance Database this study is the first one comparing medical expenditure between vegetarian and non-vegetarian groups among older adults in Taiwan. In the National Health Insurance Database there were 2127 vegetarians matched with 4254 non-vegetarians based on age and gender from 2005 to 2014. Vegetarian diet is associated with approximately 10 \u2013 15% lower total and outpatient medical expenditure. Vegetarians had lower medical expenditure incurred by diabetes and metabolic diseases, mental diseases, and genitourinary diseases. Implication for future research will be discussed at the presentation."} +{"text": "Primary cilia are sensory organelles which co-ordinate several developmental/repair pathways including hedgehog signalling. Studies of human renal allografts suffering acute tubular necrosis have shown that length of primary cilia borne by epithelial cells doubles throughout the nephron and collecting duct, and then normalises as renal function returns. Conversely the loss of primary cilia has been reported in chronic allograft rejection and linked to defective hedgehog signalling. We investigated the fate of primary cilia in renal allografts suffering acute rejection.Here we observed that in renal allografts undergoing acute rejection, primary cilia were retained, with their length increasing 1\u00a0week after transplantation and remaining elevated. We used a mouse model of acute renal injury to demonstrate that elongated renal primary cilia in the injured renal tubule show evidence of smoothened accumulation, a biomarker for activation of hedgehog signalling. We conclude that primary cilium-mediated activation of hedgehog signalling is still possible during the acute phase of renal allograft rejection. Renal primary cilia are sensory organelles that co-ordinate signalling pathways involved in proliferation and differentiation, including hedgehog (Hh) and wingless (Wnt) . CanonicRenal allograft rejection occurs when the recipient\u2019s immune system mounts an immune response against a non-self renal tissue and can destroy a graft. Here we examined renal primary cilia and graft function (assessed by serum creatinine and urine output) in serial biopsies from human renal allografts suffering acute rejection controlled by immunosuppressive drugs. We also explored primary-cilium mediated hedgehog signalling in the context of acute renal injury using a mouse ischemia/reperfusion model.Tissue was obtained from needle biopsies taken from human renal allografts suffering acute rejection. The use of biopsy material was approved by the St Vincent hospital Human Ethics committee. Allograft recipients were on standard triple immunosuppression therapy of cyclosporin, mycophenolate mofetil and prednisolone. Paraffin embedded biopsies were obtained between 0 and 40\u00a0days post transplantation. Rejection was assessed from two needle biopsy series by an experienced pathologist (PAH) and one was categorized as acute cellular rejection and one as antibody-mediated rejection. Acute rejection was diagnosed by histology and C4d immunostaining. The type and severity of rejection was rated using the Banff scale . Graft function data , and pathology reports) were obtained for each allograft biopsy series.Primary cilia were visualized and measured as previously described . For eacp\u2009<\u20090.05 Values are expressed as mean\u2009\u00b1\u2009SEM.Cilium length data were analyzed using a one-way ANOVA with an accompanying Tukey\u2019s post hoc test performing intergroup comparisons. Statistically significant differences within segments examined were defined as Immunostaining for the Hh signaling pathway component Smo was conducted in mouse kidneys (n\u2009=\u20093 for sham and IR) due to the need to use fixed and frozen material that was not available for human biopsy series.Mouse studies were approved in advance by a Monash University Animal Ethics Committee and were performed in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. The induction of renal ischemia/reperfusion injury and kidney collection was as described previously . KidneysLocalization of Smo to primary cilia in sections was as described previously . PrimaryPrimary cilium length was assessed in the aquaporin-1 positive proximal and aquaporin-1 negative remaining segments of the nephron in two biopsy series from human renal allografts undergoing rejection Figs.\u00a0. Cilium Primary cilia borne by epithelial cells in ischemia/reperfusion injured mouse kidney were elongated and showed punctate accumulations of Smo Fig.\u00a0a. In conWe investigated the fate of primary cilia during rejection in biopsy series from human renal allografts suffering acute rejection. Primary cilia were maintained on epithelial cells of the renal tubule and collecting duct and became longer with the onset of rejection injury.These findings are in keeping with our previous observations that renal injury causes the elongation of primary cilia on epithelial cells throughout the kidney . We and A study by von Toerne et al. investigAn important distinction may be that von Toerne et al. studied Two biopsy series (one acute cellular rejection and one antibody-mediated rejection) were examined and ciliary smoothed was the only marker of hedgehog activation studied."} +{"text": "Nearly 20% of adults age 50 and older report experiencing age discrimination by a healthcare professional. Numerous harmful health consequences flow from ageism in healthcare encounters, including failure to accurately diagnose and respond to treatable conditions such as pain and depression. Despite this, little is known about ageism among healthcare professionals, including whether job role and work setting influences attitudes to older patients. This study aims to use relational ageism theory to explore the relationship between personal aging anxiety among healthcare professionals and their attitudes to older patients, including potential moderating factors of job role and work setting. A convenience sample of healthcare professionals working for a small regional health system in the US (N = 145) completed an online survey using the Aging Anxiety Scale to measure personal aging anxiety and the Geriatric Attitudes Scale to measure negative attitudes to older patients. Regression analyses demonstrated that personal aging anxiety significantly predicted attitudes to older patients, with greater anxiety associated with more negative attitudes. Job role significantly predicted attitudes to older patients with physicians\u2019 attitudes being more negative compared to other disciplines, while work setting was not predictive of attitudes toward older patients. This research confirms the need to provide ageism awareness training for healthcare professionals and to include in this training an exploration of internalized attitudes to aging. Further research with a larger, more representative sample is indicated to test replication and to better understand how to overcome job role risks of ageism among healthcare professionals."} +{"text": "Hospice delivers care to a substantial and growing number of individuals with primary and comorbid dementia diagnoses. Dementia diagnosis and racial/ethnic minority status are risk factors for hospice disenrollment. However, little research examines racial/ethnic disparities and other risk factors for hospice disenrollment among hospice patients with dementia. This paper uses multinomial logistic regression to explore sociodemographic and functional status risk factors for hospice disenrollment among 3,949 home hospice recipients with primary or comorbid dementia. Results indicate that patients with a primary dementia diagnosis, racial/ethnic minority groups, and those higher functional status have elevated risk of disenrollment due to hospitalization, disqualification, and electively leaving hospice care. Additional research is needed to understand why primary dementia diagnosis and underrepresented racial/ethnic status are associated with multiple kinds of hospice disenrollment so that hospice practice can be tailored to respond to the needs of these individuals."} +{"text": "While many prior studies have evaluated the antecedents and consequences of changes in disability, few have considered the social context. As nearly 60% of older adults currently live with a spouse or intimate partner, it is important to examine spousal influences on disability. This study examined spousal associations in self-reported disability using data from the Precipitating Events Project, an ongoing longitudinal study of 754 initially nondisabled community living adults age 70 and over who have had monthly assessments of functional status since 1999. We hypothesized that one spouse\u2019s level of disability would be associated with increases in the other spouse\u2019s subsequent disability. We used the Actor Partner Interdependence Model (APIM), a statistical modeling framework that accounts for the interdependence in two-person data and tests the associations of both self (actor) and partner influences on outcomes. We used multilevel, longitudinal APIMs to examine lagged associations in spouses\u2019 monthly reports of disability in 13 activities of daily living in the 37 married couples. As hypothesized, one partner\u2019s prior disability level was significantly associated with the other partner\u2019s (the actor\u2019s) subsequent disability level after controlling for the actor\u2019s prior disability level. Also, when both couple members had higher levels of prior disability, they were particularly at risk of subsequent increases in disability . Incorporating partner disability level in modeling individuals\u2019 outcomes provides greater precision in predicting future disability levels."} +{"text": "CDC will continue to update recommendations as new information becomes available.Zika virus infection can occur as a result of mosquitoborne or sexual transmission of the virus. Infection during pregnancy is a cause of fetal brain abnormalities and other serious birth defects ( Aedes aegypti mosquito, Zika virus can also be transmitted through unprotected sex with an infected partner. As of July 3, 2018, 52 cases of confirmed sexual transmission of Zika virus infection have been reported in the United States since 2015 (https://www.cdc.gov/zika/reporting/case-counts.html). Most documented reports of sexual transmission have involved transmission from a man to a woman of Zika virus appeared to enhance viral dissemination to the female reproductive tract, compared with subcutaneous inoculation during the periconceptional period have been associated with infection in the fetus and adverse pregnancy outcomes; although in some cases, the timing of infection relative to conception was uncertain or their last possible Zika virus exposure (if asymptomatic) before trying to conceive with their partner .CDC continues to recommend shared patient-provider decision making, in which couples and health care providers work together to make decisions about timeframes to wait before trying to conceive after possible Zika virus exposure. Some couples might choose to wait shorter or longer periods after possible Zika virus exposure, based on individual circumstances , clinical judgment, and a balanced assessment of risks and expected outcomes. Other guidance for preconception counseling and prevention of sexual transmission of Zika virus after possible Zika virus exposure remains unchanged (Zika virus infection during pregnancy is a cause of serious birth defects. CDC previously released interim guidance on preconception counseling and prevention of sexual transmission of Zika virus in October 2016.CDC now recommends that men with possible Zika virus exposure who are planning to conceive with their partner wait at least 3 months after symptom onset or their last possible Zika virus exposure before engaging in unprotected sex. This updated timeframe also applies to prevent sexual transmission of Zika virus.These recommendations provide couples planning pregnancy with updated timeframes expected to reduce the risk for fetal Zika virus infection."} +{"text": "We read \u201cEarly Direct Antiglobulin Test Negativity After Bendamustine and Rituximab Treatment in Chronic Lymphocytic Leukemia: Two Cases\u201d . Eren an In their comments, Won Sriwijitalai and Viroj Wiwanitkit pointed out that the possibility of false negative direct antiglobulin test (DAT) was not denoted in our paper in which we shared our experience about the achievement of early DAT negativity in patients receiving bendamustine and rituximab treatment for chronic lymphocytic leukemia ,2. AlongBest Regards,"} +{"text": "Evidence suggests that depressive symptoms among older adults were associated with cognitive impairment and affect cognitive decline over time, while physical activity was associated with lower risk of cognitive decline or have positive effect on cognitive function. The purpose of this study is to examine whether physical activity could mediate the effects of depressive symptoms on the cognitive function of older adults. Data from the 2014 Health and Retirement Survey (HRS) of older adults \u2265 60 years were used. Hierarchical regression was conducted to examine the relationship between depressive symptoms, physical activity, and cognitive function. Mediation analysis was used to examine whether physical activity could mediate the effects of depressive symptoms on cognitive function. Regression results indicated that increased depressive symptoms was associated with poorer cognitive function, while increased moderate and mild physical activity were associated with better cognitive function. Mediation analysis indicated that the direct effect of depressive symptoms on cognitive function was significant. The indirect effect of depressive symptoms on cognitive function mediated by moderate and mild physical activity were also significant. Findings suggest that physical activity could potentially improve the cognitive function of older adults who have depressive symptoms. Moderate and mild physical activity could benefit older adults with depressive symptoms and reduce the risk of cognitive decline. Frail, disabled or chronically ill older adults are less likely to participate in vigorous physical activity, but they could benefit from moderate or mild physical activity and have better cognitive health."} +{"text": "Advance care planning requires older adults to contemplate what kind of medical care they would prefer if faced with serious illness. Those decisions involve weighing risks of medical treatments with quality of life. The purpose of this project was to explore older adults\u2019 tolerance for risk in medical treatment scenarios. Thirty-three adults over age 60 were presented with four medical scenarios reflecting increasingly severe diseases . For each, they were asked whether they would accept a treatment with increasing risk of death . Participants also recorded demographic characteristics and completed a physical and mental health measure. Older adults were more willing to accept hypothetical treatments with lower risk of death , and they were more willing to accept treatments when faced with more severe diseases . There were no significant associations between risk tolerance and demographic and health characteristics. These results suggest that older adults are diverse in their willingness to accept medical treatments risks, at least when presented with hypothetical medical scenarios. Medical professionals and family members who collaborate with older adults should be aware of this diversity of preferences and check assumptions based in demographic and health characteristics. Given these nuances, interventions to promote communication about medical preferences are essential to patient-centered advance care planning and medical decision making."} +{"text": "Severe acute malnutrition (SAM) has been considered as the complex nutritional problem within developing countries. Alleviating its occurrence also exists in an anxiety. A series of studies were conducted to disclose evidences and documented by here author. Moreover key messages were abstracted with this review easing access of texts.Escherichia coli; while usage of raw milk has been common do. Besides the mean severe household food insecurity was 6.5% and practice of family planning was 30%; whilst family size subsists as predictor for household food insecurity. The habits of exclusive breastfeeding, timely initiation of complementary feeding and apt complementary feeding were 78%, 34% and 11%, respectively with awareness as predictor. On the other hand SAM has been recognized as problem in children and treated mainly in outpatient therapeutic program by curative foods. Yet the provided foods were shared due to severe household food insecurity causing SAM recovery rate intolerable. So children get severely underfed by multidimensional determinants and need multifaceted strategies starting from awareness creation and alleviating household food insecurity.Due to pitiable sanitary practices 30% of cow milk had massive bacterial isolates like Overall eleven investigations were carried out in and around Wolaita Zone within Southern Ethiopia in the last 5\u00a0years. The key findings of the studies were appropriately documented in reliable scientific journals. Unfortunately the evidences in papers had links by indicating valid determinants of SAM burden among under five children. Furthermore the documented findings strengthen the concepts of \u201cone health\u201d and \u201cmultifaceted\u201d nature of nutritional problems \u201311.Among the subjects examined by the investigator; bacterial loads of dairy cow milk, household food insecurity and the state of family planning practices were a few to cite , 9, 11. However as shown elsewhere findings had trends of key messages towards indicating versatile nature of severe nutritional problem in under five children. We recognize that nutritional problems are multifaceted, but devoid of adaptable intervention measures in developing countries including Ethiopia. So the impacts of interventions were hardly visible. Besides the findings from different research questions specify balanced messages querying multi-dimensional projects to alleviate nutritional problems among under five children.Recognizing the merged documentation eases accessibility of texts and fastens the opportunities of appropriate interventions, the investigator decided to abstract key messages and point out costly intervention directions based on own findings. Therefore this short text was prepared from eleven research articles documented by the present author from 2013 to 2018. This document eases access of costly evidences for stakeholders and policy makers in favor of apt interventions of versatile nutritional problem in the setting.Overall 11 studies were carried out in and around Wolaita Zone, Southern Ethiopia by the investigator. Based on respective study questions; community based cross sectional, retrospective cross sectional and retrospective cohort study designs were implemented. Different sample sizes were calculated using the standard statistical formula and/or professional statistical packages still based on specific research questions. The sample sizes were ranging from 349 to 794 as per precise study queries and it was considered as representative for the source population.Standard probability sampling technique was chosen for each study and/or design effect was used to compensate parallel effects. Data were collected by close supervision by apt field staffs. Then it was decisively managed in Excel, Epinfo, EpiData, SPSS and/or Stata statistical package software\u2019s. Data were analyzed by univariate, bivariate and multivariate regressions as per need. The effect measures used were adjusted odds ratio and adjusted hazard ratio to fix the predictor variables after controlling likely confounders. Manuscripts were set and presented in varied workshops. Besides the well prepared manuscripts were disseminated in peer reviewed scientific journals. Still to ease access of evidences, this text was set by abstracting key findings of the records.Overall 3% of under five children were severely malnourished based on the findings documented in the studies setting. Over 85% of those SAM children were treated in OTP which is part of community based management of acute malnutrition (CMAM). Once admitted, children receive ready-used therapeutic food weekly in view of their weight and other helpful therapies until discharge as per SAM management protocol [Staphylococcus aureus, Streptococcus agalactiae and Escherichia coli. Inadequate sanitation was the key predictor for bacterial loads in cow milk [Just about 30% of dairy cow milk had massive public health important bacterial isolates such as cow milk . Besidescow milk . Then agcow milk .The usage of long acting reversible contraceptives as family planning way was also identified as 30% and maternal education had effect on its usage . BesidesOn the other hand the level of SAM in children was well-known as public health problem in the studies setting and managed in CMAM mainly by outpatient form . Yet theThe nutritious food option for children next to breast milk is cow milk, but using the foul raw cow milk has been common practice in the studies setting. So the pathogenic bacteria in cow milk were likely to cause diarrheal diseases which are instant cause of SAM in children. Besides household food insecurity has been known source of SAM incidence and also for its poor recovery rate due to sharing and selling of curative foods. Likewise apt IYCF practices were considered as vital for alleviating the incidence of SAM, but evidences show vast gaps in implementation of IYCF practices and the key obstacle for poor IYCF do was identified as still poor awareness. On the other hand CMAM is the easiest inclusive program for SAM management, but the validations shown as routine indicators were far out of international sphere standard yardsticks Fig.\u00a0.Fig.\u00a01ThThe author bears in mind that other casuals also exist as nutritional problems are multifaceted; however children get severely malnourished perhaps owing to unhealthy consumption practices and/or due to severe household food insecurity and/or as result of poor IYCF practices based on the findings from here investigator. Besides SAM management by CMAM has been challenged by sharing and selling therapeutic foods perhaps because of household food insecurity and the habit of eating together. Yet family size was key predictor of household food insecurity, but the practice of family planning was silly boosting household food insecurity and the rate of SAM in children again Fig.\u00a0.The state of household food insecurity in urban setting argues calming food markets and creating job prospects. The sizeable severe household food insecurity in rural setting as well desires actions to pick up food production and productivity in the area. Besides awareness must be created on family planning do as family size enhances household food insecurity and be likely casual for SAM incidence among children. Moreover to reduce public health impacts, appropriate works needed in sanitary do above all on the usage of cow milk for children. The practice of exclusive breastfeeding was worthy but extra efforts darling in women education and awareness creation, as mothers think insufficiency of breast milk for their children. On the other hand timely initiation of complementary feeding and apt complementary feeding were also negligible. So nutrition specific care services and women education must be monitored. OTP admits large extent of SAM cases per year; however the routine performance indicators were intolerable. Thus focused outreach activities and monitoring of service provisions as per executive protocol be elective. Last but not least it\u2019s wise that evidences by the present investigator update the multifaceted nature of nutritional problems inquiring versatile intervention measures in the field.Letting unnamed folds of SAM burden identified by other researchers secure and lack of follow-up studies incorporated by the investigator."} +{"text": "Wound botulism is a potentially lethal condition that can cause paralysis. Its association with black tar heroin is a well\u2010established fact. It is essential to alert clinicians in recognizing the patients with history of injection drug abuse presenting with clinical features of botulism early on admission for prompt diagnosis and treatment. We present 2 cases (48\u2010year\u2010old Hispanic Man & 34\u2010year\u2010old Hispanic Woman) both with past medical history of heroin abuse was brought to the emergency department with acute respiratory failure, proximal muscle weakness of upper and lower extremity, neck flexor muscle weakness, and diplopia. Urine drug was consistent with opioids. Multiple areas of visible skin popping, a technique of injecting black tar heroin into extravenous subcutaneous sites are seen on the right and left areas of thigh associated with the development of botulismNone declared.IAQ: involved in manuscript writing; MAQ: involved in critical revision of the manuscript; ARV and DK: involved in patient care."} +{"text": "Each minute increases the chance of survival by 10%.A novel smartphone application (app) has been developed that can direct first responders to cardiac arrest victims more than three minutes before the emergency services arrive. European Society of Cardiology (ESC). It was released during EHRA EUROPACE \u2013 CARDIOSTIM 2017.The EHRA First Responder app was created by the European Heart Rhythm Association (EHRA), a registered branch of the \u2018Sudden cardiac arrest is lethal within minutes if left untreated,\u2019 said EHRA spokesperson Dr Christian Elsner. \u2018In Europe, the emergency services arrive around nine minutes after a cardiac arrest. Every minute earlier raises the probability of survival by 10% and reduces the risk of brain injury, which starts four minutes after cardiac arrest.\u2019If cardiopulmonary resuscitation (CPR) is initiated by a member of the public, this will in essence shorten the time between cardiac arrest and the urgently needed resuscitation measures. However, bystander resuscitation occurs in just of 30\u201360% of patients who have a cardiac arrest outside hospital.The EHRA First Responder app was developed to increase the rate of bystander resuscitation and reduce the time between cardiac arrest and resuscitation. Based on GPS tracking technology, the app is used by existing emergency services to locate trained \u2018app rescuers\u2019 and then automatically direct them to the scene of cardiac arrest. The target is for an app rescuer to arrive three to four minutes after the cardiac arrest.In a typical scenario, after the cardiac arrest, a bystander calls the emergency services. The operator dispatches an emergency crew and simultaneously locates nearby app rescuers. The nearest app rescuers are notified on their smartphones and the quickest responder is given directions, via the app, to the patient and then performs CPR. Other app rescuers can then additionally bring a nearby automated external defibrillator (AED).The app was tested in Lubeck, Germany, where around 600 app rescuers were recruited. In 36% of cardiac arrests, an app rescuer arrived more than three minutes before the emergency services. App rescuers were recruited through a local media campaign and 70% were already medically trained. The 30% without medical training took a basic lifesupport course and committed to retaking it every two years.\u2018Recruitment of the app rescuers was no problem at all because people want to help,\u2019 said Dr Elsner. Project organisers are now asking emergency dispatch units across Germany to connect to the app so that they have free access to the fleet of app rescuers.\u2018The software has a standard interface and can be easily connected to most emergency alert systems in Europe in just a few steps,\u2019 said Dr Elsner. \u2018We provide insurance for app users and we have a guarantee of data security from the German Department for Data Security in Schleswig\u2013 Holstein.Dr Elsner concluded: \u2018Ultimately we will roll the app out across Europe. We hope to raise bystander resuscitation rates to 70\u201390% and for cardiac arrest patients to be resuscitated in three to four minutes on average.\u2019www.firstresponderapp.comFor more information, visit:"} +{"text": "The data in brief provides a descriptive summary of the field data collected using Eco-health approach in order to support local effort aimed at creating information base for taking evidence-based decisions, especially in regard to wildlife conservation outside protected area and range resource management. The data were collected between June 2012 and July 2014 on a range of issues including wild animals, livestock, household income and cost of diseases control in cattle. In a nutshell the data article shows spatial pattern of a declining brucellosis prevalence in cattle linked to animal population density with increasing distance away from the Lake Mburo National Park (LMNP) boundary in southwestern Uganda. It is the trend of animal distribution in private land that the pastoralist communities perceived as influencing economic losses associated with diseases affecting cattle production. The pastoralists strongly believe that wild ungulates grazing with cattle outside the park on a daily basis present a potential risk of disease transmission which adversely affects their cherished source of livelihood. This article refers to \u201cBrucellosis in cattle and micro-scale spatial variability of pastoral household income from dairy production in south western Uganda. Acta tropica\u201d, Acta Tropica, 2018. Specifications TableValue of the data\u2022The data variables indicate unique circumstances of brucellosis transmission in cattle and household income that might inform a monitoring plan for local disease control.\u2022The data provides information evidencing strong concerns the local communities have regarding the presence of wild species of animals on their private farms and ranches around Lake Mburo National Park in southwestern Uganda.\u2022Therefore, the data in this article allows other interested researchers access and use of raw facts in different ways that might extend statistical analysis and subsequently lead to a more comprehensive understanding of pastoralists\u2019 development trajectory at the wildlife-livestock nexus.11.1The dataset in this article contains variables such as spatial pattern of wild animals outside the park, livestock species reared in Lake Mburo conservation area and economic losses pastoralist communities incur due to limitations imposed on cattle production by diseases. The 1.2A population survey of wild ungulates was carried out along 3 transect lines in order to determine any spatial association between location of animals and homesteads from Lake Mburo National Park (LMNP) boundary.Wild ungulates sighted on livestock grazing farms/ranches along a distance gradient from the LMNP were counted and recorded from June 2012 to July 2014), using a standard method described by Buckland et al."} +{"text": "Enhalus acoroides collected from Singapore and Peninsular Malaysia to test the hypothesis that fungal communities are homogeneous throughout the study area. Seagrass samples were separated into different structures , and a sediment sample was collected next to each plant. Amplicon sequencing of the fungal internal transcribed spacer 1 and subsequent analysis revealed significant differences in fungal communities collected from different locations and different structures. We show a significant pattern of distance decay, with samples collected close to each other having more similar fungal communities in comparison with those that are more distant, indicating dispersal limitations and/or differences in habitat type are contributing to the observed biogeographic patterns. These results add to our understanding of the seagrass ecosystem in an understudied region of the world that is also the global epicenter of seagrass diversity. This work has implications for seagrass management and conservation initiatives, and we recommend that fungal community composition be a consideration for any seagrass transplant or restoration programme.Marine fungal biodiversity remains vastly understudied, and\u00a0even less is known of their biogeography and the processes responsible for driving these distributions in marine environments. We investigated the fungal communities associated with the seagrass Enhalus acoroides.Examining marine fungi associated with the seagrass, Quality\u2010filtered reads were then used to estimate and correct sequencing errors, and remove de novo\u2010detected chimeras within the DADA2 package. Contaminant sequences found in negative controls were removed using the prevalence method in the decontam R package . Any sequences matching nonfungal taxa were removed. The remaining ESVs that were taxonomically assigned as fungi were used in all downstream analyses within the phyloseq R package structured fungal community, a permutational multivariate analysis of variance (PermANOVA) test was performed using the adonis function in the ge Chen, . Networkhttps://github.com/gzahn/Enhalus_Fungi, and all raw sequences associated with this work have been deposited at the National Centre for Biotechnology Information under the BioProject Accession PRJNA517736.All analysis code and outputs for this project, including our taxonomic reference database can be found at 3E.\u00a0acoroides in Singapore and Peninsular Malaysia show that hosts sampled from different localities harbor significantly different fungal communities ,\u00a0and several major oil refineries and petrochemical facilities are located on offshore islands. It is extremely likely the unique environment of Singapore influences the fungal communities associated with the samples collected here.The idea that habitat differences in the marine environment can drive population differentiation has been invoked to explain genetic structuring and the high diversity of coral reefs in Southeast Asia Benzie, . CorrespAll sampling locations and plant structures are primarily dominated by fungi from the classes Dothideomycetes, Eurotiommycetes, Agaricomycetes, and Saccharomycetes. Fungi from these classes are frequently observed in marine environments and are often associated with seagrasses and from Singapore under permit numbers NP/RP 18\u2010035 & NP/RP 18\u2010035a.\u00a0Click here for additional data file.\u00a0Click here for additional data file.\u00a0Click here for additional data file."} +{"text": "This report includes CDC recommendations for mitigating a reduction in tuberculosis (TB) testing capability resulting from the anticipated Aplisol shortage tuberculin antigens licensed by the Food and Drug Administration (FDA) for use in performing tuberculin skin tests. This time frame is the manufacturer\u2019s current estimate and is subject to change. The manufacturer notified CDC that they anticipate an interruption of supply of Aplisol 5 mL beginning in June 2019, followed by an interruption of the supply of Aplisol 1 mL in November 2019. The expected shortage of Aplisol 1 mL could occur before November 2019 if demand increases before then. Information on the status of this supply interruption will be updated at FDA\u2019s Center for Biologics Evaluation and Research\u2013Regulated Products: Current Shortages website are used for detecting Two FDA-approved PPD tuberculin antigen products are available in the United States for use in performing TSTs: Tubersol (Sanofi-Pasteur) and Aplisol. In controlled studies, the concordance between the two products is high (CDC recommends the following three general approaches to mitigate a reduction in TB testing capability resulting from the expected shortage of Aplisol:\u2022 Substitute IGRA blood tests for TSTs. Clinicians who use the IGRA blood tests should be aware that the criteria for test interpretation are different from the criteria for interpreting TSTs (\u2022 Substitute Tubersol for Aplisol for skin testing. In studies, the two skin test products give similar results for most patients (\u2022 Prioritize allocation of TSTs, in consultation with state and local public health authorities. Prioritization might require the deferment of testing some persons. CDC recommends testing only for persons who are at risk for TB (M. tuberculosis infection status (Although overall test concordance is high, switching between PPD skin test products or TSTs and blood tests in serial testing might result in apparent conversions from negative to positive or reversions from positive to negative that might be attributable to inherent interproduct or intermethod discordance rather than change in In settings with a low likelihood of TB exposure, the deferment of routine serial testing should be considered in consultation with public health and occupational health authorities. Annual TB testing of health care personnel is not recommended unless there is a known exposure or ongoing transmission ("} +{"text": "A 35 years old woman presented at 8-week gestation for her first prenatal visit. She had no complaints at the time. While her past obstetrical history showed two prior cesarean sections. Transvaginal ultrasound was performed showing fortuitously a cesarean scar ectopic pregnancy with a gestational sac implanted in the myometrium between the bladder and the anterior wall of the uterus (A) and associated with a periovulatory hyper-vascularization in color doppler (B). This aspect was confirmed in 3D Ultrasound which showed a gestational sac bulging through the anterior wall of the uterus into the bladder with diminished myometrial layer between the bladder and the sac (C). The outcome was favorable after conservative surgical treatment by laparotomy associated with a triple preventive ligation because of the high risk of bleeding. Cesarean scar pregnancy is so a rare iatrogenic complication of cesarean section; however, her incidence is actually growing due to the increase in cesarean section rates. The diagnosis is usually made via ultrasound showing an empty uterine cavity and cervical canal, a gestational sac located anteriorly at the isthmus, and evidence of a functional trophoblastic/placental circulation on color Doppler. Early diagnosis is crucial to avoid severe vital and functional complications such as hemorrhage, early uterine rupture and hysterectomy, so its recognition is important to keep fertility of young woman."} +{"text": "Although prior research has linked perceived neighborhood characteristics to cognition, scant research has investigated underlying mechanisms regarding how neighborhood characteristics impact cognition. One pathway, in particular, may be through mental health outcomes. Poorer neighborhood characteristics have been independently linked to greater depressive and anxiety symptoms, which may, in turn, be risk factors for cognitive decline in later life. The current study examined direct and indirect effects of perceived neighborhood characteristics on cognitive functioning through anxiety and depressive symptoms using longitudinal data from the Health and Retirement Study (2010\u20132014). Results revealed that higher social cohesion was associated with better memory and executive functioning through lower anxiety and depressive symptoms. Physical disorder was associated with worse episodic memory and executive functioning through greater anxiety symptoms. These findings highlight the importance of neighborhood context for promoting both mental and cognitive health outcomes in older adulthood."} +{"text": "We note the author(s) of the letter entitled \u201cDoes \u2018overall catastrophic health care expenditure\u2019 make sense?\u201d Meta-analyses often include studies that are different from each other in important ways; hence heterogeneity may also be due to differences in study design or patient characteristics across studies . HeterogHeterogeneity is one of limitations that researchers deal with when combining the results of individual studies, while it could be our greatest ally when we explore its sources . HeterogSignificant statistical heterogeneity arising from methodological diversity or differences in outcome assessments suggests that the studies are not all estimating the same quantity. However, the heterogeneity does not necessarily suggest that the true intervention effect or the analyzed Index varies. In particular, heterogeneity associated solely with methodological diversity would indicate the studies suffer from different degrees of bias .In our study , heterogNevertheless, meta-analysis of sensitive issues such as catastrophic health expenditure should cite with caution due to the possible bias in primary research results and presence of possible heterogeneity sources."} +{"text": "Our study evaluated and contrasted responses to 25 content areas essential to the primary care of older adults by medical students and residents, and identified attitudes toward aging amongst students and residents. One hundred and thirty-six medical students and 61 Internal Medicine residents completed a survey including the 25-item Geriatrics Clinician-Educator Survey and 18-item Images of Aging Scale. Students and residents rated importance and knowledge for content areas from 1 (low) to 10 (high). Gap scores reflecting the difference in ratings between importance and knowledge were calculated. The Images of Aging scale ranges between 0 (furthest from what you think) and 6 (closest to what you think). Results indicated that students and residents reflected similar beliefs about the importance of content areas, but students provided lower ratings in knowledge. Students revealed larger gap scores in areas that reflected general primary care , whereas residents revealed larger gap scores in areas that reflected specialists\u2019 expertise . Attitudes toward older adults did not differ appreciably between students and residents. In sum, primary care topics applicable for any age demographic were rated as most important by first-year medical students and Internal Medicine residents. Topics relevant to older populations \u2013 particularly those requiring specialists\u2019 knowledge of or requiring sensitive discussion with older adults \u2013 were rated as less important and were less well mastered."} +{"text": "Fluid volume and hemodynamic management in hemodialysis patients is an essential component of dialysis adequacy. Restoring salt and water homeostasis in hemodialysis patients has been a permanent quest by nephrologists summarized by the \u2018dry weight\u2019 probing approach. Although this clinical approach has been associated with benefits on cardiovascular outcome, it is now challenged by recent studies showing that intensity or aggressiveness to remove fluid during intermittent dialysis is associated with cardiovascular stress and potential organ damage. A more precise approach is required to improve cardiovascular outcome in this high-risk population. Fluid status assessment and monitoring rely on four components: clinical assessment, non-invasive instrumental tools , cardiac biomarkers (e.g. natriuretic peptides), and algorithm and sodium modeling to estimate mass transfer. Optimal management of fluid and sodium imbalance in dialysis patients consist in adjusting salt and fluid removal by dialysis and by restricting salt intake and fluid gain between dialysis sessions. Modern technology using biosensors and feedback control tools embarked on dialysis machine, with sophisticated analytics will provide direct handling of sodium and water in a more precise and personalized way. It is envisaged in the near future that these tools will support physician decision making with high potential of improving cardiovascular outcome. By natu,,,,,,,,,,,,,Sodium and fluid accumulation that may occur in dialysis patients over time due to repetitive positive fluid imbalance is responsible for chronic extracellular fluid overload with its,Assessing fluid status of dialysis patients is not an easy task from a clinical perspective. In that context, it is interesting to note that over time several tools have been proposed to asses,1. Clinical assessment focusing on fluid status, hemodynamic stability, and patient perception was the first attempt to address this issue in developing the concept of \u2018dry weight\u2019,,,Subsequently, several tools have been proposed to help physicians in refining clinical acumen and defining more objectively \u2018dry weight\u2019 of dialysis patients2. Instrumental or technology-based tools use various non-invasive ways to assess volemia, fluid status, or hemodynamic surrogate indicators.,,Inferior vena cava diameter (IVCD) and collapsibility has been proposed to monitor intravascular volume and right atrial pressure or central venous pressure in dialysis patients with interesting findings,,Relative blood volume change (RBV) and refilling rate capacity during dialysis assessed by online blood volume sensor has been also proposed for fluid management. In expert hands, this tool provides useful information on individual patient volume status to facilitate hemodynamic guidance,,,,,,,Bioimpedance approach has been proposed over the last few years as a more objective way to assess fluid status in dialysis patients,More recently, it has also been proposed to extend the use of lung ultrasound in chronic hemodialysis patients for tracking silent fluid accumulation in the lung interstitium (extravascular edema). Interlobular septa thickening due to water accumulation reflects US beam and generates visible B line bundles (comet-like tail). A simple counting of these B lines provides an estimate of lung water excess and predictive value for patient outcomes,,,Sodium MRI has been introduced quite recently in the field of sodium and fluid assessment in chronic kidney disease patients in dialysis to assess tissue sodium accumulation,,,,,,,,3. Cardiac and vascular biomarkers have been used extensively in an attempt to disentangle fluid status and cardiac dysfunction in dialysis patients. Atrial natriuretic peptides are the most popular ones for assessing fluid overload,,,,,4. In recent past years, several researchers have develop algorithms to quantify sodium and water mass transfer during hemodialysis sessions using either mass balance equations based on the law of conservation of mass within the dialysis/patient systemOptimal management of fluid and sodium imbalance in dialysis patients is achieved by adjusting salt and fluid removal through dialysis and salt intake restriction, and fluid gain between dialysis sessions. This is the conventional approach obtained by adjusting \u2018dry weight\u2019 according to clinical judgment and complementary tools including dialysate sodium prescription adaptation described earlier. However, this approach may be hampered by the discontinuous nature of the HD treatment and/or patient intolerance to fluid and sodium removal. An obvious solution would be to increase time and/or frequency of dialysis sessions in patients with high inter-dialytic weight gains and/or intolerance for fluid removal, as this has been shown to reduce intradialytic hemodynamic stress. However, this approach will not always be possible for financial or logistic reasons, or because of the wish of the patient.,Modern technology using biosensors and sophisticated analytics provide tools for handling directly sodium and water during hemodialysis session in a more precise and personalized way that have potential for improving patient outcomeComplementary clinical studies on a large scale should help to better characterize dialysis patients in term of diet sodium intake over prolonged time period and explore effects of this precise sodium and fluid management approach on patients intermediary and clinical endpoint outcomes.,,,Dialysis adequacy concept has evolved over time and based on patient outcomes. Due to more efficient hemodialyzers, more technically advanced hemodialysis machines, and wider use of ultrapure dialysis fluid, efficiency and biocompatibility of renal replacement therapy have improved tremendously"} +{"text": "Older adults with visual impairments experience barriers to participation that could impact their own health and wellbeing. Participation limitations that are associated with poor vision and unsupportive environments have been associated with objective health outcomes, including chronic disease and secondary outcomes such as fall injuries. In this study, we assessed the association between functional vision impairment and self-rated health (SRH). We also tested the mediating role of participation in that relationship. We conducted analysis with covariates representing six domains included in the International Classification of Functioning, Disability, and Health (ICF). We computed ordinal logistic regression models using two waves (2011 and 2016) of the National Health and Aging Trends Study (NHATS). Vision status was assessed via self-reported measures of visual functioning . \u201cParticipation\u201d was operationalized using 6 items that assessed participation in social and community-based activities . SRH (measured in 2016) was assessed using a single item asking participants to rate their health on a scale ranging from excellent to poor. We assessed key relationships while holding ICF\u2019s other health dimensions constant. Functional vision status was statistically associated with SRH in models containing all covariates. Participation variables reduced but did not eliminate the effects of vision, suggesting a partial mediating effect\u2014that is, part of the association between vision and SRH was explained by participation factors. These results point to the importance of developing community support and reducing barriers to participation by older adults with functional vision impairments."} +{"text": "Data in this article are related to the chemical characterization of various oak wood samples. Data have been obtained by the application of Fourier Transform Infrared (FTIR) spectroscopy and pyrolysis-gas chromatography-mass spectrometry (Py\u2013GC\u2013MS) to living tree species and shipwreck wood fragments. Measurements were performed on individual rings in order to facilitate the understanding of the variability in wood chemical composition along the radial cores, i.e. the same kind of material traditionally used for dendrochronological analysis. The data in this article is labelled according to the anatomical sections of the wood where the samples were taken. The experimental background and the results can be found in the related research article, \u201cChemometric tools for identification of wood from different oak species and their potential for provenancing of Iberian shipwrecks (16th\u201318th centuries CE)\u201d . Specifications tableValue of the data\u2022The FTIR and Py\u2013GC\u2013MS data in this paper refer to wood chemical composition through the molecular characteristic.\u2022Data in this paper provide valuable wood chemical detail related to differences between sapwood, transition wood and heartwood; between various wood species; and also between sound wood and underwater archaeological wood.\u2022This data are useful for researchers in order to extend the statistical analyses.1Quercus spp.) species. Samples were collected in different forests in the Cantabrian and the Basque country regions in Spain. Unknown species of oak wood fragments were collected from various shipwrecks with Iberian shipbuilding features.Data presented here include FTIR and Py\u2013GC\u2013MS values of living tree wood and shipwreck wood samples, from four oak (\u22121) for sound and shipwreck woods. Band selections were carried out by considering only FTIR peaks related to the main wood chemical constituents (polysaccharide and lignin). The second excel file (relative abundances of pyrolysis products) contains the relative abundances of pyrolysis products for sound and shipwreck woods, expressed as relative proportions of total peak area; again, focused on polysaccharide and lignin compounds, in addition to several minor products. The third file contains the extended heartwood spectral dataset (see article Data are subdivided in three excel files. The first excel file (relative intensities in the fingerprint region) contains the relative FTIR-ATR intensities of the main absorption bands of the fingerprint region (1800-800\u202fcm2Fourier Transform Infrared spectroscopy (FTIR) and pyrolysis-gas chromatography-mass spectrometry (Py\u2013GC\u2013MS) are the two analytical techniques applied for wood chemical characterization in this article. More details about the analytical techniques can be found in"} +{"text": "In addition, these quiescent cells were tumorigenic upon serial transplantation and were resistant to a variety of clinically relevant chemotherapeutic drugs. Our study is the first to demonstrate a quiescent MM cell niche and the effects of functional interactions between quiescent MM cells and their microenvironment [Efforts to differentiate mechanisms underlying tumor quiescence and proliferation have occurred mostly in leukemia , and in ke cells . These cironment .+ MM cells isolated from the OS niche compared to proliferating PKH-CD138+ MM cells (CD138 is a plasma cell marker) or PKH+ MM cells from other niches. A search of the integrated cancer microarray database revealed that TRIM44 mRNA expression is significantly upregulated in MM compared to normal or MGUS (a precursor stage of MM) [Molecular profiling of these cells revealed a relatively unknown protein, tripartite motif (TRIM) containing 44, TRIM44, which is highly expressed in quiescent PKHe of MM) , 7. ThisWhat are the implications of this finding? Dysregulation of protein ubiquitination and deubiquitination pathways is one of the pathological features of MM . MM cellThe TRIM family represents a large family of pattern recognition receptor, and select TRIM proteins have roles as regulators of autophagy as well as cargo receptors . Out of Furthermore, our work provides a framework to further dissect mechanisms of quiescence and proliferation within the tumor microenvironment. Are particular cells within the microenvironment predisposed to tumor dormancy? What cues trigger tumor cells to exit their quiescent state and become more proliferative? What factors in the microenvironment are involved in maintaining tumor quiescence? These questions are still unanswered but further understanding how tumors remain in quiescence, which allows them to escape effects of chemotherapies, will likely provide a valuable tool to prevent cancer relapse in patients."} +{"text": "As a means of enhancing experiential educational opportunities for adult-gerontology nurse practitioner students who are prepared to manage the complex care of older adults, interactive simulation videos were developed using the eLearning authoring tool H5P to create learning experiences for students that can be used either in face to face classroom experiences or embedded in learning management systems. H5P is a web-based authoring tool that helps faculty build interactive course content. H5P activities provide instant feedback to students, allowing them to self-assess their understanding of the dynamic video simulation case. With funding through the Health Resources and Service Administration Advanced Nursing Education Workforce grant, four video simulation cases were developed that address emerging chronic care conditions in an older women who aged 15 years presenting initially with signs of hypothyroidism, progressed to early frailty, through moderate dementia and eventually along with her daughter face end of life health care issues. Partnering with the university instructional design experts, nurse practitioner faculty created questions that were inserted throughout the video as a means of keeping students engaged in problem-solving and decision making. A faculty handbook that described the case scenario with the interactive questions with suggested discussion questions was developed for each video simulation. The adult-gerontology primary care nurse practitioner competencies addressed in each case are identified in the handbook. Recommendations for the interactive question format will be presented and QR codes with access to direct viewing of the videos will be presented on the poster."} +{"text": "Background: Puerto Ricans have the highest likelihood of psychiatric disorders among Latinos. This study developed and evaluated a prototype depression literacy curriculum; culturally grounded with perspectives and narratives of Puerto Rican older adults. The way a person determines need for services and decides to seek help has been found to be influenced by their perceptions of services and providers. McGuire (1989) presents the Communication Persuasion Model (CPM) that takes into account how persuasive communication changes attitudes and behaviors of consumers. Using the CPM as a theoretical foundation, this study presented a culturally grounded story through a Virtual Reality (VR) platform. Methods: A script was developed based on narratives of Puerto Rican older adults about depression. Filmed in 360\u00b0 format and enhanced with supporting imagery, participants were presented two versions of the video, one with a VR headset and the other with a smartphone. Two focus group interviews were conducted with community-dwelling Puerto Rican older adults (n=14) in Orlando, FL. Results: Participants preferred the VR headset and found it was beneficial to educate about depression because it felt more immersive and encouraged an environment conducive to identifying their own experiences about depression. They noted that presenting the material with a case narrative was more culturally sensitive for the population. All participants needed minor assistance with operating technology. Conclusions and Implications: A narrative approach to depression literacy may be effective in personalizing messages. Assisted VR technology with supporting imagery may be efficacious and standardize positive messages to underrepresented and low resource populations."} +{"text": "Chronic inflammatory demyelinating polyneuropathy (CIDP) is characterised by significant clinical heterogeneity and as such reliable biomarkers are required to measure disease activity and assess treatment response. Recent advances in our understanding of disease pathogenesis and the discovery of novel serum-based, electrophysiologic and imaging biomarkers allow clinicians to make more informed decisions regarding individualised treatment regimes.As a chronic immune-mediated process typified by relapse following withdrawal of immunomodulatory therapy, a substantial proportion of patients with CIDP require long term treatment with intravenous immunoglobulin (IVIg), a scarce and expensive donor-derived resource. The required duration and intensity of immunoglobulin treatment vary widely between individuals, highlighting both the heterogeneous nature of the underlying disease process as well as the variable pharmacologic properties of IVIg.This review outlines the use of multimodal biomarkers in the longitudinal evaluation of nerve injury and how recent developments have impacted our ability to predict both response to immunoglobulin administration and its withdrawal. Chronic inflammatory demyelinating polyneuropathy (CIDP) is the most common chronic immune-mediated neuropathy worldwide, with an estimated prevalence of 2\u20137 per 100,000 people .The current approach to diagnosis and treatment of CIDP has several significant limitations. While foremost amongst these is the lack of a safe, efficacious and readily accessible therapy, the lack of effective disease biomarkers has resulted in an inability to stratify patients to individualised treatment regimes. While clinical response to treatment can provide an indication of disease activity, objective measures of assessing therapeutic effect and predicting outcome are limited, and this has many implications on assessing the required duration and intensity of immunoglobulin therapy.The initial diagnosis of CIDP is clinical, with patients presenting with a characteristic pattern of weakness and areflexia that evolves over a period of more than 2 months. Confirmation of diagnosis is made by demonstrating evidence of peripheral nerve demyelination, most commonly by electrophysiological testing with supportive findings on cerebrospinal fluid analysis and rarely nerve biopsy.In lieu of well-defined objective measures of disease activity, a variety of scoring systems based on functional status have been developed to quantify disease severity. Commonly used scales include the Inflammatory Neuropathy Cause and Treatment (INCAT) overall disability sum score which relies on self-reported impairment in undertaking activities of daily living and the Medical Research Council (MRC) Muscle Sum Score which relies on examiner-based evaluation of strength in a variety of muscles and anti-contactin 1 (anti-CNTN1) antibodies have been identified in approximately 3\u201310% of patients with chronic infammatory polyneuropathies \u201318. PatiTesting for different immunoglobulin classes of paranodal antibodies may be useful in evaluating patients with a phenotype of aggressive, younger-onset inflammatory neuropathy (even if this resembles a Guillain-Barr\u00e9 Syndrome) particularly in the setting of either treatment resistance or clinical relapse following an initial response to IVIg therapy. While transient IgM responses to neurofascin can be seen in patients with GBS, the presence of IgG4 antibodies appears to be extremely specific for an eventual diagnosis of CIDP , 18. It Despite the promise shown by these discoveries, the identification of IgG4 paranodal antibodies in patients with CIDP remains rare, and while early indications of a specificity approaching 100% make them an invaluable tool for assessing patients with suggestive clinical presentations, more ubiquitous biomarkers are clearly necessary for routine clinical use .Although the scarcity of detectable antibodies in CIDP mean they are an impractical method of measuring disease activity, the quantifiable serologic response to immunoglobulin treatment has been proposed as an alternative surrogate biomarker.The mechanisms by which IVIg exerts a regulatory effect on the dysimmune response in CIDP has not been completely established, though in-vivo studies have suggested it may neutralize pathogenic autoantibodies, inhibit complement binding and possibly act directly on the myelin sheath to enhance remyelination . These pThere is mounting evidence to support an immunoglobulin dose-response relationship in inflammatory neuropathies, with higher serum levels after IVIg administration associated with a superior treatment response , 24. CerFollowing the results of the Intravenous Immunoglobulin CIDP Efficacy (ICE) trial which established IVIg as an effective therapy for CIDP, the standard approach to initiating treatment has been to commence with an induction dose of 2\u2009g/kg IVIg over 2\u20135\u2009days. This recommendation however does not take into account patient-specific pharmacokinetic responses, and adjusting treatment dose based on immunoglobulin levels may help guide more effective individualised treatment regimes. A study of change in immunoglobulin G level (\u2206IgG) following treatment actually demonstated wide variability between patients unrelated to weight or body mass index . As suchComprising pooled IgG derived from between 1000 and 15,000 different donors, IVIG preparations consist of predominantly monomeric IgG with small percentages (5\u201315%) of dimeric complexes. This is relevant as increased dimeric fractions have been reported to be associated with increased incidence of adverse effects such as hypotension . IntriguAlthough their clinical utility in routine practice is yet to be established, other emerging immunoglobulin-specific biomarkers include level of sialylated IgG and the ratio of sialylated/agalactosylated IgG-Fc levels. These relate to the percentage of carbohydrate binding to the immunoglobulin Fc region, the primary site involved in both complement activation and antibody-dependent cell-mediated cytotoxicity . PatientMany individuals with CIDP require long term maintenance treatment with IVIg to effect disease stability and studies of serum IgG levels in treatment responders have revealed that patients actually achieve a steady state wherein both pre- and post-treatment IgG levels are almost identical . The impWhile electrophysiology has an established role in the diagnosis of CIDP, its ability to monitor response has not been as clearly defined. Furthermore, the degree of nerve demyelination and axonal injury at diagnosis does not appear to predict either response to treatment or long-term disability . From a Studies involving patients with biopsy proven CIDP demonstrated that while electrophysiologic changes were present in all participants at diagnosis, serial nerve conduction studies at 4, 8 and 12\u2009months did not correlate well with improvement as measured by functional scores . DespiteThe discordance between clinical status and neurophysiology was most apparent in motor and sensory studies of median and ulnar nerves in the upper limb . While mThis discrepancy can in part be attributed to the poor relationship between sensory deficits and functional impairment. While distal sensory deficits in patients with CIDP often persist and occasionally worsen despite treatment , this rarely contributes to functional disability and as such it comes as no great surprise that the correlation between sensory studies and functional scores is poor .Despite being a chronic disease, long term electrophysiologic monitoring data in CIDP has not been widely published. Observational studies up to 24\u2009months after diagnosis suggest that certain electrophysiologic parameters and in particular resolution of conduction block may be useful in monitoring disease activity but the clinical utility of using such selective electrophysiologic biomarkers is difficult to assess and unlikely to reflect an efficient use of resources .post-hoc analyses of the ICE study [Use of a composite electrophysiologic score assessing averaged compound motor action potential (CMAP) amplitudes in a number of motor nerves appeared to correlate with treatment response in CE study . FurtherThe approach to patients who remain clinically stable but who develop interval deterioration in nerve conduction studies is controversial. While observational data has suggested that extensive axonal degeneration is a marker of poor outcome, the role for escalating immunotherapy in the setting of clinical stability has not been adequately studied . AlthougMultiple explanations for the disparity between electrophysiologic demyelination and disease activity have been postulated to explain why evidence of new demyelinating lesions does not always parallel clinical decline. CIDP remains a dynamic process that involves constant segmental demyelination and remyelination, and it is likely this constant spectrum of nerve injury cannot be easily assessed through electrophysiology at any single time point. Biopsy studies revealing so-called \u2018onion bulb\u2019 formations which represent repetitive Schwann cell induced proliferation provides strong evidence for a continuous regeneration routine at the microscopic level which cannot be easily quantified with the use of nerve conduction studies.While \u2018onion bulb\u2019 formation is a pathologic biopsy finding, there is evidence that quantifiable nerve root hypertrophy as measured by magnetic resonance imaging may be a possible radiologic correlate and the advent of advanced imaging techniques such as nerve ultrasound and magnetic resonance neurography have opened the way for multimodal assessments of nerve pathology .Ultrasonography provides precise measures of nerve anatomy in addition to information regarding fascicular related muscles and blood vessels. Use in compressive neuropathies like carpal tunnel syndrome has highlighted its high sensitivity in detecting subclinical nerve damage even in patients without symptoms . FurtherSpecificity in CIDP diagnosis can be increased through the use of ultrasound in addition to electrophysiology. One study comparing patients with diabetic polyneuropathy fulfilling electrophysiologic criteria for CIDP to those with clinically diagnosed CIDP demonstrated that cross-sectional area as measured on ultrasound reliably distinguished these two groups apart .In addition to being a useful adjunct in diagnosis, longitudinal changes in nerve ultrasound characteristics appear to correlate well with clinical status and amelioration of nerve enlargement during treatment with IVIg concordant with clinical improvement may thus be a promising surrogate biomarker of disease stability .There are however several technical factors that can limit ultrasound use. In particular, meaningful sonographic evaluation of the nervous system is only possible at accessible sites in the distal extremities and its ability to accurately visualise proximal structures like the lumbosacral plexus and spinal nerves is extremely limited. This is particularly pertinent in conditions like CIDP, as electrophysiologic markers at disease onset (delayed F-waves and absent H-responses) strongly suggest the process begins as a proximal polyradiculopathy that only subsequently extends distally.Using magnetic resonance imaging, it is possible to assess these proximal areas and thus identify structural evidence of peripheral nerve pathology. Early MRI studies involving gadolinium enhanced T1-weighted sequences described enhancement of the cauda equina (in up to 70% of cases) and less commonly nerve root enlargement however concluded that these features could not correlate with either clinical signs or disease severity . Modern As assessed by MR neurography, patients with CIDP had significantly enlarged cross-sectional areas and signal intensity in nerves of the lumbosacral plexus, the sciatic nerve at the level of the thigh and major nerves of the upper limb when compared with normal controls and this suggests it can be used as a highly specific diagnostic aid . While cIn addition to examining nerve pathology, adjunctive myopathic imaging in patients with CIDP may help assess disease severity, with structural alterations in muscle tissue composition as a consequence of denervation also correlating closely with clinical weakness . SeveralWhile both nerve signal change and structural nerve or muscle measurements provide valuable diagnostic data and indirect estimations of disease activity the emerging use of advanced functional MRI techniques involving diffusion tensor imaging (DTI) promises to yield additional quantitative information which can be used to monitor disease activity in CIDP. By using diffusion sensitizing-gradients to measure proton diffusion within individual voxels DTI allows visualization of nerve fibre tracks and recent studies have shown promise in its ability to provide quantitative data on myelin sheath integrity \u201347.Clinical genomics is starting to play an increasingly important role in our understanding of disease pathogenesis and has offered the potential for an increased application of high-precision medicine in a variety of autoimmune conditions.Differential gene expression in CIDP as measured through micro-array based analysis of human sural nerve biopsies has provided information on a number of up-regulated genes in these patients . As suraOne study suggested that five genes were reliably upregulated in skin biopsy samples in CIDP patients, with the Allograft Inflammatory Factor-1 gene most significantly associated. While it remains unclear what triggers this upregulation, all these genes have been implicated in the immune cascade . UnfortuThere are early indications that genetic profiling may open novel monitoring strategies during treatment with IVIg. Pilot data involving patients treated with immunoglobulin demonstrated that IVIg drove the down-regulation of a number of genes involved in the systemic inflammatory response however these findings need to be validated in larger populations until more definitive conclusions can be drawn .Current criteria for immunoglobulin use mandate considering cessation of IVIg in all patients after 12\u2009months of treatment. Discontinuing immunoglobulin therapy can however be associated with recrudescence of a significant symptom burden, and relapse rates of up to 45% at 6\u2009months have been reported . Of partObservational data of clinical characteristics in patients with CIDP revealed no significant difference in topography of weakness , type of neuropathic disturbance (sensory vs motor) or pre-treatment severity between individuals who could be weaned from therapy and those who were treatment dependent . This stWhile serum-based and imaging biomarkers have shown promise in being able to quantify disease activity, their role in predicting response to a discontinuation of therapy has not been determined performed a post-hoc analysis of demyelinating changes in patients who subsequently relapsed with electrophysiologic data obtained at the initial commencement of IVIg and just prior to discontinuing treatment .Although limited by small sample size the results obtained in each group at these two time-points were highly suggestive. All patients who relapsed demonstrated evidence of new demyelinating features between the two studies thus indicating that the accumulation of an electrophysiologic burden of disease may predict treatment dependency. Interestingly, 60% of patients in the No-Relapse group also accumulated demyelinating features, which suggests that number of new lesions in itself is not a particularly specific finding. Comparing the actual type of interval demyelinating change between the two groups however suggested that F-wave latency and distal compound motor action potential duration, which had odds ratios of 14.40 and 21.00 respectively, may be more potent biomarkers for predicting relapse following treatment withdrawal .The search for reliable biomarkers of disease activity in CIDP continues. While the discovery of specific paranodal antibodies which conform to phenotypic presentations of disease and predict response to immunomodulation represents a major advance in our approach to individualised treatment strategies, more ubiquitous biomarkers that provide robust longitudinal measures of neuropathic injury are clearly required.Immunoglobulin treatment dependence has significant physical and psychological consequences on patients in addition to profound financial implications at a population level, due to both inherent costs of treatment administration but also downstream effects driven by long-term patient disability.Promising genomic studies, serum-based assays, electrophysiological techniques and imaging modalities are emerging at a rate commensurate with our greater understanding of CIDP pathogenesis. How effectively the use of these various complementary investigations translates into a practical multimodal approach that has the ability to predict an individual\u2019s response to immunoglobulin treatment however still remains to be seen."} +{"text": "Cardiac embolism is presumed to cause a significant portion of cryptogenic strokes. Transesophageal echocardiography may detect intracardiac thrombi, but this remains a rare finding, possibly because remnant clots dissolve spontaneously or following thrombolysis. Cardiac imaging within cerebral CT angiography might offer an alternative method for thrombus detection within hyperacute stroke assessment. In a proof-of-concept study we analyzed records of patients aged \u2265 60 years that presented with suspected stroke and underwent extended cerebral CT angiography as part of their emergency assessment. CT imaging of patients with ischemic stroke or transient ischemic attack (TIA) and atrial fibrillation and of those with embolic strokes of undetermined source (ESUS) was reviewed for intracardiac clots and other cardiac or aortic pathology. Over a period of 3 months 59 patients underwent extended CT angiography for suspected stroke, 44 of whom received a final diagnosis of ischemic stroke or TIA. Of those, 17 had atrial fibrillation, and four fulfilled ESUS criteria. Thrombi were detected within atrial structures on CT angiography in three cases. In two ESUS patients complex atheromatosis of the proximal ascending aorta with irregular and ulcerating plaques was detected. Cardiac imaging within emergency cerebral CT angiography is feasible and can provide valuable diagnostic information in a patient group that might not routinely undergo transesophageal echocardiography. A small change to emergency assessment could potentially uncover cardioembolic pathology in cases that would have remained cryptogenic otherwise. A significant proportion of embolic strokes of undetermined source (ESUS) are probably cardiogenic, but cardioembolic pathologies can elude standard assessments . TransesAn alternative method for detecting left atrial thrombi (and other cardiac pathology) is computed tomography (CT) , but ECGScan range of emergency CCTA for acute stroke in elderly patients included the heart for a period of 3 months at our center. The potential for detecting intracardiac clots in patients with large artery occlusion or transient ischemic attack (TIA) and atrial fibrillation as well as ESUS was investigated.\u22121 ; corresponding radiation doses were thus 5.01 mSv .Our results demonstrate that co-imaging of cardiac structures within emergency CCTA in elderly patients with ischemic stroke or TIA and atrial fibrillation or ESUS can indeed visualize intracardiac clots before thrombolytic treatment, as well as other emboligenic pathologies such as ulcerating plaques of the ascending aorta.The main advantage of cardiac imaging within routine CCTA compared to dedicated ECG-gated cardiac CT is the minimal delay of only a few extra seconds, and the sparing of additional contrast agent. A potential disadvantage is lower image quality due to heart movement artifacts and variable contrast filling since bolus timing is optimized for cerebral arterial visualization . HoweverThe additional radiation exposure during cardiac imaging added to CCTA has to be considered. In our study the median DLP and estimated radiation dose for the entire extended CCTA were less than average for cardiac CT angiography . The lifOur study design does not allow estimations of the diagnostic yield of extended CCTA, pre-treatment prevalence of remnant left atrial clots, or direct comparisons with other methods. However, our findings serve as a proof of principle: intracardiac clots can be detected on extended emergency CCTA before they are potentially dissolved by thrombolysis or flushed into the circulation. We looked at patients with known atrial fibrillation to increase pretest probability for cardiac thrombi. However, the biggest clinical benefit of this method would be expected in patients with cryptogenic stroke, where detecting a cardiac thrombus has profound implications for acute treatment and secondary prevention (long-term anticoagulation). Prospective multi-center studies should explore this idea in the future. In the long run, a \u201cone-stop shop\u201d multimodal CT program could be developed to optimize emergency stroke diagnostics.The datasets generated for this study are available on request to the corresponding author.The study protocol was approved by the ethics committee of the Medical Faculty of Ruhr University Bochum. This was a retrospective study based on routine clinical data, so written informed consent was not acquired.SP had the idea for the study and served as principal investigator, performed data analysis and wrote the first draft of the manuscript. US and WW contributed to the analysis. IK and JA contributed to clinical and radiological data acquisition and analysis. All authors revised and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Epigenetic changes are one of the Hallmarks of Aging. DNA methylation is a key epigenetic mark that has been shown to change during aging. Several \"clocks\" have been developed whereby changes in DNA methylation can be used to predict chronological, and perhaps, biological age. This symposium will focus on recent advances in understanding how and why changes in DNA methylation occur during aging and whether these changes play a causal role in age-related functional declines and disease."} +{"text": "It has been demonstrated that order-entry systems inquiry of the necessity of repeat laboratory testing can significantly decrease variability of test ordering.3 This study shows that providing Clinical Teaching Units (CTUs) with cost-analysis weekly, laboratory orders would decrease without compromising patient care through increasing residents\u2019 knowledge of laboratory costs in Canada.Routine daily phlebotomy is often ordered for admitted patients after the established diagnostic algorithm and plan have been made and can often act as a replacement for direct patient care.Internal Medicine Residents were asked to estimate the costs incurred from phlebotomy related tests per patient per day (PPPD), which was done by completion of a survey distributed to key informants. Throughout the six-month study, four Internal Medicine CTUs were given weekly expenditure reports detailing their respective and average spending. Both in-hospital mortality rates and hospital readmission rates were tracked as surrogates of patient safety. The primary endpoint measured was laboratory test expenditure which reflected the number of total labs ordered. Time-series analysis, over the six-month period was performed to determine peak and trough spending.Residents surveyed were unable to accurately estimate laboratory costs . Spendin2This Canadian study shows that reductions in test ordering can be demonstrated by simply displaying this unique variable (PPPD) and comparing teams\u2019 expenditure. These results support implementation of a \u201cChoosing Wisely\u201d curriculum to Canadian trainees as there is currently no mechanism to regulate investigation ordering.This study was limited to a single academic tertiary care centre, which constrains the generalizability of the findings. Due to the lack of a control group, there may be confounders such as the progression of an in-training-physician\u2019s competency over time and seasonal disease variation. Increasing duration of data collection may reveal additional trends.Future studies can explore two programs of similar size in separate geographic locations (case versus control). This method might possess intrinsic biases, as residents may rotate through institutions monthly. By integrating financial stewardship and feedback into postgraduate medical education curricula, there is potential to facilitate the implementation of cost-conscious patient care as an essential component of the practice of medicine."} +{"text": "Radiofrequency energy has had widespread use for a variety of surgical procedures. Its application in orthopedic surgery initiated with shoulder instability. Over the last couple decades it has been applied as surgical tool for cartilage treatment as well. There have been significant gains in its technology and our understanding of its potential benefits. We address its history and advancements in becoming a surgical tool for cartilage lesions along with a review of recent long-term follow up studies. Articular cartilage within the knee provides a smooth gliding surface that protects subchondral bone by distributing pressures with a low frictional coefficient grade I lesions are superficial and rarely need surgical treatment or laser applications as described by Barber et al. . The useThe technology of RFE has been available for years in a variety of specialty procedures. Specifically the thermal energy is used to ablate abnormal tissue. Introduced in the 19th century to create neural tissue lesions , decreased subjective knee symptoms (knee and osteoarthritis outcome score (KOOS)) and decreased pain (VAS score) in the plasma layer group compared to mechanical shaver group at all time points. They concluded that RFE is superior to mechanical shaver.A randomized trial of 60 patients with grade 3 cartilage defects of the medial femoral condyle with concomitant meniscal injuries compared bipolar RFE to mechanical shaver for treatment of the cartilage defect (Spahn et al. A 4-year follow up study of this study continued to show positive results for plasma layer chondroplasty (Spahn et al. Furthermore, long-term follow up of this randomized trial continued to show differences between the mechanical and plasma layer treatment groups. The results of 10-year follow up demonstrated the coblation group had significantly delayed time to revision surgery of approximately 3\u2009years (Spahn et al. Gharaibeh et al., published the largest retrospective study to date evaluating the outcome of coblation therapy for cartilage lesions in 824 patients undergoing knee arthroscopy with safety profile and patient outcomes was the endpoints (Gharaibeh et al. It is critical to note, throughout the literature reviewed, the lesions studies have been Grade 2 and 3 lesions with the knee, if the fibrillations have penetrated passed the subchondral plate and/or demonstrate Grade 4 lesions, the efficacy of plasma layer remains unclear.Articular cartilage is essential within the knee to distribute pressure and decrease frictional coefficient during movement. Current treatment strategies of articular cartilage injury aim to remove free edges and stabilize the remaining cartilage. This debridement has traditionally been performed with mechanical shaving which risks removing of potentially healthy cartilage. Lesions treated with plasma layer demonstrated improved patient outcomes and reduced incidence of reoperations compared to mechanical shaving. Plasma layer used with appropriate settings and technique can be a safe surgical tool for the treatment of ICRS Grade 2 and Grade 3 lesions within the knee. With the primary of goal of decelerating the progression of cartilage lesions and improved patient outcomes, plasma layer may be a reasonable option to treat patient with Grade 2 and Grade 3 lesions."} +{"text": "Advances in open and endovascular techniques have resulted in novel approaches to repair of acute Type A aortic dissection. Hybrid arch procedures involve open arch resection and stent grafting of the descending aorta with stent graft insertion in one of two ways: Frozen or Staged. In this article, pros and cons of the two different paradigms of emerging hybrid arch techniques for acute Type A aortic dissections are discussed. Current debate regarding surgical repair of acute Type A aortic dissection (ATAAD) centers around whether an aggressive approach to the aortic arch should be undertaken, compared with an open distal anastomosis known as a hemi-arch. There are no randomized trials comparing these two approaches. While the results of surgery for ATAAD have improved over time, the most recent large registry datasets published in 2015 suggest a persistently high operative mortality of 15 to 20%.A variety of surgical techniques have been proposed under the broad classification of \u201chybrid arch\u201d repair, which involves surgical total or partial arch replacement and endovascular aortic stent graft deployment to treat a more extensive portion of the dissected aorta. These techniques raise the question of whether a more complex surgery can produce better outcomes.The goals of hybrid arch surgery in ATAAD are to resect the primary intimal tear and seal tears extending beyond the transverse aortic arch, as well as to cause false lumen obliteration of the descending thoracic aorta. Theoretical benefits include reductions in early malperfusion, in late distal aortic dilatation, in need for late aortic reintervention, and in mortality. These hybrid techniques may have particular value in patients with end-organ malperfusion, as studies have shown that this population has a fivefold increase in operative mortality compared with ATAAD without organ malperfusion.If hybrid arch procedures are going to be part of the future armamentarium of surgery for ATAAD, the optimal technique of stent graft deployment needs to be determined. During these hybrid arch procedures, after surgical arch replacement with a Dacron graft, the stent graft can be inserted in one of two ways: (1) through the open arch during circulatory arrest or (2) staged after separation from CPB using fluoroscopy and standard thoracic endovascular aortic repair (TEVAR) techniques.Industry has brought several new tools to market to facilitate hybrid arch procedures. Combination Dacron and stented grafts such as the Evita (JOTEC GmbH) and Thoraflex (Vascutek Ltd) prosthesis facilitate the insertion of a stent graft during circulatory arrest. Branched arch prostheses like the Bavaria and Lupiae grafts (Vascutek Ltd) facilitate creation of a proximal landing zone for the insertion of a stent graft post CPB. While both techniques aim to accomplish both exclusion of arch tears, as well as expansion of the distal true lumen and obliteration of the proximal thoracic false lumen, each has its own advantages and limitations .The \u201cfrozen stented elephant trunk\u201d technique has been widely published in the literature and has its own accepted acronym \u201cFET.\u201d The basis of this technique is antegrade deployment of the descending aortic stent graft through the open arch during hypothermic circulatory arrest . This cAdvantages of this technique over deployment post CPB include simplicity, in that a dedicated endovascular skill set is not required. In addition, this technique does not require the use of portable fluoroscopy equipment or a hybrid operating suite. As no pre- or postdeployment angiogram is done, potentially nephrotoxic intravenous contrast is not required. The large sewing collar that comes with these hybrid prostheses is advantageous, as it may facilitate an easier distal anastomosis. Finally, there is more data on this technique, as several large case series have shown \u201cacceptable\u201d morbidity and mortality, with potential long-term survival benefit.This technique involves endovascular stent graft deployment in a traditional TEVAR fashion with the use of fluoroscopy to identify landing zones.Advantages of deployment post CPB include radiographic confirmation of adequate proximal and distal sealing zones, detection of endoleaks, confirmation of no new tears (stent-induced new entry tear),As the surgical community recognizes ATAAD as a diffuse process that can affect every organ system, there is a move toward expanding past the most acutely life-threatening complication of proximal aortic repair and treating more segments of the aorta, and addressing clinically significant branch vessel involvement. In the discussion above, we have compared the advantages of graft deployment during circulatory arrest with stent graft deployment post CPB. At our centers, the ability to assess our repair with intraoperative fluoroscopy has led to therapeutic changes in the management of our patients with ATAAD. Our personal bias is to use intraoperative fluoroscopy at the time of hybrid arch repair for ATAAD and not to deploy the endograft blindly. Others have also advocated use of a hybrid room for ATAAD, whereby all diagnostic and therapeutic measures are available. There are concerns about endovascular stent grafts being used by clinicians who do not have the tool kit to diagnose and manage adverse outcomes at the distal landing zone. However, we recognize that the technique of FET can be more widely adapted by cardiac surgeons doing operations on an emergency basis while on call. Extended arch operations can be very challenging for the general cardiac surgeon who is usually performing these uncommon operations at inopportune timesOur opinion is that results of endovascular stent graft use in ATAAD may be optimal if performed by aortic surgeons who can identify the pathology, know the treatment options, and are capable of both open and closed techniques.Hybrid arch repair techniques have the potential to improve both perioperative and long-term outcomes in a challenging patient population. The goal of operative intervention for ATAAD is to minimize short-term mortality, while hopefully providing a close to normal life expectancy and quality of life in the long term. However, it remains to be seen which techniques will ultimately produce the best results for individual patients. A classification system of hybrid arch techniques will be required to compare outcomes. We propose that this classification system pays attention to method of stent graft deployment: during circulatory arrest or staged post-CPB. Future trials of surgery for ATAAD may address \u201chemi-arch\u201d versus \u201ctotal arch & descending stent graft\u201d or \u201cfrozen elephant trunk\u201d versus \u201cstaged endograft.\u201d"} +{"text": "The current study examined the association between intergenerational support exchange and marital satisfaction among older Korean couples. Prior work has not paid due attention to the fact that older parents and adult children often exchange various types of support in the context of marital relationships, and that provision or receipt of support could influence their marital relationships . Using the 2008 Actual Living Condition of the Elderly and Welfare Need Survey (ALCEWNS), a nationally representative survey of community-dwelling adults 60 years and older, we evaluated the links between marital satisfaction and each spouse\u2019s reports of emotional and instrumental support provided to or received from adult children. For analyses, a series of actor-partner interdependence models were estimated. Findings revealed that wives\u2019 marital satisfaction was associated with their husband\u2019s exchange of emotional support with adult children. By contrast, husbands\u2019 marital satisfaction was unaffected by their wife\u2019s emotional support exchange with adult children. More specifically, wives were more satisfied with their marriage when their husband reported providing greater emotional support to adult children than receiving it from adult children. In addition, wives indicated higher marital satisfaction when the couple provided similar levels of emotional support to their children. Provision or receipt of instrumental support had no bearings on marital satisfaction of either spouse. Taken together, our findings highlight how older couples may evaluate their relationship quality in the Korean cultural context."} +{"text": "A large body of research has found that individuals\u2019 attitudes toward aging may influence their future health. Previous research has found that age is associated with more negative expectations toward aging. However, it is possible that optimism, or generalized positive expectancies regarding future outcomes may play a role in expectations regarding aging. Optimism has been identified as a key component of successful aging. The purpose of this study was to compare expectations regarding aging among young adult and older adult age cohorts, controlling for optimism, and to investigate the differential relationships between optimism and expectations regarding aging by age cohort. Young adults (n = 130) and older adults (n = 335) completed a survey containing the Expectations Regarding Aging \u2013 12 and the Optimism-Pessimism Scale. Results found that, after controlling for optimism, older adults endorsed more negative expectations regarding aging. Comparison of the correlation coefficients between optimism and expectations regarding aging among age cohorts found that optimism was significantly associated with expectations regarding aging among older adults but not young adults and that this difference was significant. Taken together, the results suggest that older adults have more negative expectations for aging and that optimism may play a key role in older adults\u2019 expectations regarding aging."} +{"text": "Angina, the prototypic vasoocclusive pain, is a radiating chest pain that occurs when heart muscle gets insufficient blood because of coronary artery disease. Other examples of vasoocclusive pain include the acute pain of heart attack and the intermittent pains that accompany sickle cell anemia and peripheral artery disease. All these conditions cause ischemia \u2013 insufficient oxygen delivery for local metabolic demand \u2014 and this releases lactic acid as cells switch to anaerobic metabolism. Recent discoveries demonstrate that sensory neurons innervating the heart are richly endowed with an ion channel that is opened by, and perfectly tuned for, the lactic acid released by muscle ischemia."} +{"text": "Soil amendment with olive cake produced from olive mills waste (olive pomace/cake) is an ordinary practice in olive producing countries in the Middle East. It is used to improve soil physical and chemical properties as well as cheep waste management approach. But, the olive cake contains small percentage of residual oil which may affect water holding capacity of soil and penetration rate in agricultural lands. The data provided in this article shows the influence of adding olive pomace to clay and sand clay soils in terms of water holding capacity (WHC), penetration depth and accumulate intake. The penetration depths are shown in The penetration depths were read directly from the three transparent gradual scales cylinders (FEL5 Demonstration Infiltration Apparatus The water accumulate intake (mL) is shown in Normally, the clayey soil has less penetration and water intake than sandy clay soil as shown in 2Olive pomace addition on soil water holding capacity, penetration depth and accumulated intake were examined for clay and sandy clay Soils. The soils samples obtained from the top soils surfaces, crushed dried and passed through a 2 mm strainer to remove bulky fragments. The olive cake is shown in The olive pomace physicochemical properties are show in Soil-water-retention curves were obtained according to Ref.\u00a02.1D is the initial depth, IW and IS are the initial water and soil surface heights, respectively. HD is water depth as time elapsed and HW and HS are the heights of water and soil surface, receptively. AI is the accumulated intake calculated from Eq. FEL5 Demonstration Infiltration Apparatus \u2013 Issue 1"} +{"text": "This presentation identifies this chaos, and focuses attention on the issues to be addressed to facilitate descriptive and comparative scientific studies in the future. It is a call to action specifically to the medical arrhythmic community and its specialty societies to begin a quest to unravel the arrhythmic quagmire associated with \u201csilent atrial fibrillation.\u201dSilent or subclinical asymptomatic atrial fibrillation has currently gained wide interest in the epidemiologic, neurologic and cardiovascular communities. The association of brief episodes of paroxysmal atrial fibrillation or surrogate atrial arrhythmias which predict future clinical adverse events have been established. Nevertheless there exists a confounding array of definitions to indicate its presence without discrete indication of which populations should be examined. Moreover the term \u201catrial fibrillation burden\u201d (AFB) has emerged from such studies with a plethora of descriptions to prognosticate both arrhythmic and clinical adverse events. This presentation suggests clarification of diagnostic definitions associated with silent atrial fibrillation, and a more precise description of AFB. It examines the populations across the current disease and cardiovascular invasive therapeutic spectrum that lead to both silent atrial fibrillation and AFB. It describes the diagnostic methods of arrhythmia detection utilizing the surface ECG, subcutaneous ECG or intra\u2010cardiac devices and their relationship in seeking meaningful arrhythmic markers of silent atrial fibrillation. Whereas a wide range of The importance of clinical atrial fibrillation to all physicians has emerged during the last decade.Clinically in North America an appreciation of the precipitants of atrial fibrillation was recently reported to account for one\u2010third of all occurrences of atrial fibrillation, and resulted from surgery, infection, and myocardial infarction.2DS2\u2010VASc score to detect such susceptible persons.Although a variety of clinical risk markers have emerged to identify the patient at risk of atrial fibrillation during the past 3 decades,2DS2\u2010VASc. From such efforts have emerged the term \"silent atrial fibrillation\" (SAFib) which initially commutated the occurrence and detection of subclinical asymptomatic episodes of paroxysmal atrial fibrillation. To quantitate such episodes of asymptomatic SAFib there emerged the concept of \"atrial fibrillation burden\" (AFB). AFB has been represented by various arrhythmic markers, predominantly supraventricular arrhythmias, which prognosticate the development of atrial fibrillation and/or its outcomes. Whereas patients with symptomatic atrial fibrillation are usually discovered by medical attention resulting from symptoms associated with hemodynamic complaints, unfortunately SAFib may only present after the most serious of complications such as ischemic stroke or sudden death , intracardiac device patients, post\u2010RF ablation, and high CHA2DS2\u2010VASc score patients seemingly are clearly candidates to be identified in such a consensus document. What about the populations of obesity, sleep apnea, convulsive disorders, syncope, malignancy,post\u2010precipitant atrial fibrillation and inflammatory conditions? What diagnostic algorithm or criteria should exist for low risk populations?A diagnostic algorithm of examination in a specific population. Although some populations can employ the most simple of diagnostic methods , should there be a gradient guideline of cost\u2010effective diagnostic testing utilizing recommended surface ECG techniques before employing costly invasive devices in specific populations?When to examine in each population at risk.An authoritative consensus could guide a standardized time of examination and its duration for meaningful detection of SAFib\u2010A and SAFib\u2010O. This would better guide the scientific community in its requisition of tests and applications of therapies. It would render cost\u2010efficiencies to ongoing clinical studies and randomized trials.Establishment of Definitive Criteria for SAFib\u2010A and SAFib\u2010OBy establishing definitive criteria of SAFib\u2010A in specific populations a more robust scientific literature and knowledge base would be established. Perhaps a time\u2010threshold effect could be defined, whereby a greater burden of atrial fibrillation or longer episodes of atrial fibrillation should confer a greater risk of adverse outcomes. Prevention and therapies would be better guided and adverse outcomes hopefully avoided. The knowledge base of those SAFib\u2010O markers would be better appreciated by the medical community at large, and the overall medical community would be guided to lower the global burden of atrial fibrillation and is complications.Several issues come to the fore in addressing the clinical challenge of SAFib and include the following:This presentation is a call to action specifically to the medical arrhythmic community and its specific specialty societies to begin an effort to address the quagmire of SAFib. This dilemma currently exists, and should not be left to commercial market forces alone, but should receive thoughtful, incisive and cost\u2010effective attention from physicians for the benefit of their patients and society at large."} +{"text": "Parameters of genetic polymorphism show that the Cesky Fousek breed has a comparable rate of variation as other hunting breeds despite the low population size and severe historical bottlenecks. Clustering analyses reveal a unique genetic status as a distinct pointing dog breed and the relatedness of the breeds is in good concordance with historical data. The present study demonstrates that despite historical admixture among lineages, separate pointing breeds constitute genetically differentiated units, mirroring unique breeding stocks and pedigree isolation among specific breed clubs, reflecting differences in breeding programs under each association.Cesky Fousek is considered to be one of the oldest pointing dog breeds in Europe and has been appreciated for its versatile working skills. Because it faced extinction in the past, the Cesky Fousek was restored from German Wirehaired and Shorthaired Pointers. Additionally, the breed was recently used in the USA with the initial intent of improvement of the Wirehaired Pointing Griffon by the Bohemian Wirehaired Pointing Griffon Club of America. This study evaluates genetic diversity parameters of Cesky Fousek and compares them to the other continental pointing dogs that played a role in the formation of its gene pool. DNA from buccal swab and blood samples ( Canis familiaris) are the first domesticated animals that interacted with humans from the Paleolithic )The history of central European pointing dog breeds is characterized by recurrent founding events and admixture among lineages, for example the Cesky Fousek was used for creation of German wirehaired pointing dogs and later the German dogs were used to restore Cesky Fousek. In turn, Cesky Fouseks were used in the Bohemian Wirehaired Pointing Griffon Club of America to increase genetic variation of the original Wirehaired Pointing Griffon breed. Despite this reticulate demography, gene pools of particular breeds are recently well differentiated, as suggested by clustering analyses. These patterns could be ascribed to unique breeding stocks and pedigree isolation among particular clubs, related to differences in breeding programs under each association.S1 FigExample of pronounced differentiation in coat color in two breeds of the same historical origin\u2014(a) Deutsch Drahthaar; (b) German Wirehaired Pointer.(PDF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(PDF)Click here for additional data file."} +{"text": "MyelinJ can analyse single images or complex experiments with multiple conditions, where the ggpubr package in R is automatically used for statistical analysis and the production of publication quality graphs. The main outputs are percentage (%) neurite density and % myelination. % neurite density is calculated using the normalize local contrast algorithm, followed by thresholding, to adjust for differences in intensity. For % myelination the myelin sheaths are selected using the Frangi vesselness algorithm, in conjunction with a grey scale morphology filter and the removal of cell bodies using a high intensity mask. MyelinJ uses a simple graphical user interface and user name system for reproducibility and sharing that will be useful to the wider scientific community that study 2D-myelination https://github.com/BarnettLab/MyelinJ. For statistical analysis the freely available R and the ggpubr package are also required. MyelinJ has a user guide (MyelinJ is freely available at er guide and has Bioinformatics online. The MyelinJ ImageJ plugin we have developed is a freely available ImageJ and its degeneration is associated with spinal cord injury and several CNS diseases, most notably multiple sclerosis . Myelinae ImageJ for autoMyelinating cultures were made according to Background is first subtracted either using ImageJ\u2019s \u2018rolling ball\u2019 background subtraction, or using the neurite image as a mask to remove any bleed through of neurite fluorescence, followed by subtraction of pixels below a user provided threshold .MorphoLibJ library neurite density is calculated using the ImageJ filter \u2018normalize local contrast\u2019 (NLC). Alternatively, all standard ImageJ thresholding methods are also available to the user.% neurite density is calculated as:Total neurite pixels/Total pixels * 100.https://github.com/kassambara/ggpubr) for statistical analysis and the production of publication ready graphs. MyelinJ performs Welch\u2019s T test followed by correction for multiple testing using the false discovery rate (FDR). The user can choose between comparing all experimental conditions to each other or comparing all experimental conditions to control only.MyelinJ links ImageJ to R via the command line and uses the ggpubr package , being able to more accurately analyse both myelin sheaths and neurites. MyelinJ also offers automated calculation of % neurite density and % myelination in order to avoid human error, can analyse complex experiments where a summary of the results for each condition is provided and seamlessly links to R for the graphical representation of results and statistical analysis. In addition, MyelinJ analyses myelin sheaths significantly better than the Otsu ImageJ thresholding algorithm . This is the first time (to the best of our knowledge) that an ImageJ macro that can interact with the statistical package R has been made freely available. MyelinJ uses the ggpubr package to perform statistical analysis and produce publication quality graphs, providing a seamless analysis pipeline from raw images to graphical representation and statistical analysis for high throughput screens.This newly developed MyelinJ is a user friendly ImageJ macro for the analysis of fluorescent micrographs of 2D myelinating cultures providing quantification of the % of neurite density and the % of myelination. To the best of our knowledge, there are currently no other publicly available ImageJ macros for this analysis and MyelinJ marks a significant improvement upon the freely available CellProfiler pipeline and MS society of Great Britain (56).Conflict of Interest: none declared.btz403_Supplementary_DataClick here for additional data file."} +{"text": "Benevolent ageism has recently been recognized as a form of patronizing treatment that older adults experience because of the kind and incompetent age stereotype proposed by the Stereotype Content Model. However, there is limited research that examines older adults\u2019 experiences with patronizing treatment. The aim of this study was to conceptualize benevolent ageism based on older adults\u2019 experiences with items from an existing measure of ageism, the Ambivalent Ageism Scale, and additional items created by us that expand the measurement of benevolent ageist behaviors. In an internet-based sample of older adults who were 65 years old and older (N =135), the benevolent subscale of the Ambivalent Ageism Scale with our additional 10 items demonstrated excellent reliability (\u03b1 = .90). An exploratory factor analysis cleanly yielded a 4-factor solution that mirrored previous findings, (1) hostile ageism, (2) unwanted help, (3) cognitive assistance/protection, while introducing a new factor of (4) condescending endearment. The findings from this study have widened the scope with which ageism is viewed by examining older adults\u2019 experiences with ageism and conceptualizing characteristics of benevolence that older adults may face due to the widespread belief that they are kind and incompetent. The validation of a scale measuring individuals\u2019 experiences with ageism will provide insight as to whether older adults experience ageist behaviors that people report endorsing and if older adults receive unnecessary offers of help. A recipient\u2019s perspective of ageism will aid in the understanding of the insidious and benevolent characteristics of ageism within society."} +{"text": "Within diverse cohorts, African Americans (AA) demonstrate higher rates of Alzheimer\u2019s dementia (AD) and Alzheimer\u2019s dementia combined with multiple comorbid conditions. AA are also two times more likely to develop late-onset AD than whites and less likely to be diagnosed. Yet, our understanding of this disparity in cognition remains limited. Cognitive impairment (CI) and dementia are both underdiagnosed and underreported in primary care patients. The lack of early detection of cognitive and functional decline in high risk populations results in failure to provide care and interventions to members of vulnerable groups. This talk will focus on research that seeks to identify a more sensitive cognitive marker for early identification of cognitive impairment for AA and advances this objective by linking the cognitive marker to AD cerebral spinal fluid biomarkers and testing whether this association differs between AA and whites."} +{"text": "The data presented here are related to the research paper entitled \u201cEconomic Analysis of Natural Forest Disturbances: A Century of Research\u201d . Natural disturbances have always affected forest ecosystems, altering or disrupting the flows of goods and services provided by forests to human societies. Economic analysis can help private or public decision-makers take forest policy decisions by understanding the causes and consequences of forest disturbances, as well as evaluating tradeoffs in alternative policy scenarios. In consequence, the economic literature about natural disturbances is very rich and diversified.This paper describes a bibliographic database gathering some 340 scientific papers related to the economic analysis of forest natural disturbances. Papers have been inventoried primarily thanks to searches on databases using specific keywords and the dataset have been completed by searching through literature-cited sections of papers. Relevant papers have been manually encoded into a database (Excel file) taking into consideration each type of hazard and different economic approaches. Data cover papers published in English from 1916 to 2014. Specifications TableValue of the data\u2022Data cover 340 scientific articles in the field of economic analysis of forest disturbances.\u2022Data allow for literature reviews in the field of economic analysis of forest disturbances either by hazard or by economic approach.\u2022Data were used in 1The database is an original Excel file that includes some 340 scientific papers dealing with the economic analysis of forest natural disturbances. Data cover papers published in English from 1916 to 2014. Each line of the sheet represents an article; each column of the sheet represents a variable. Papers are encoded in the database according to 3 types of variables: variables concerning the references of the article; variables specifying the case study, if any; variables providing information on the method used by the author(s). 2\u2022Wildfire: wildfire OR fire OR prescribed burning OR fuel management OR fuel treatment OR fuel reduction OR fuel break\u2022Pest: pest OR epidemic OR infestation OR insect\u2022Pathogen: pathogen OR disease OR infection\u2022Storm: storm OR hurricane OR tornado OR cyclone OR typhoon OR wind OR windstorm OR windthrow OR gale OR straight line wind\u2022Wildlife damage: browsing OR debark\u2022Snow \u2013 Ice: snow OR ice\u2022Other abiotic disturbances: drought OR flood OR landslide OR volcano\u2022Other, multiple hazards: catastrophe OR damage OR mortality OR disturbance OR hazard OR risk OR stochastic OR uncertainty.We conducted a systematic literature research during the spring of 2014 using combinations of three keywords on four databases/search engines: JSTOR, ScienceDirect, Ingentaconnect and NRC Research Press. Each search combined the keywords for searching in papers\u2019 full-text as follows: Forest* AND Economic* AND specific keyword related to the analyzed disturbance. For each hazard, we tried the following specific keywords:Complementarily, we used the reference lists of the identified papers to add other relevant articles into the database. We only include articles that are published in English. We then collected 340 papers. The comprehensive list is presented in Note that we did not find any article using the defined keywords for the category \u201cOther abiotic disturbances\u201d. Thus, such disturbances do not appear in the database.\u201cOther, multiple hazards\u201d category gathers papers that we cannot classify into the previous ones because they deal with non-specified natural hazard or with several natural hazards or with another type of hazard . See"} +{"text": "In the article titled \u201cVertebral Arteriovenous Fistula: An Unwelcome Thrill\u201d , there wThe authors originally included the publication by Foreman et al. in their discussion on AV fistulas not associated with chiropractic manipulation, which was later removed during editing. Although there are many studies on patients suffering vertebrobasilar stroke and arterial dissection following chiropractic neck manipulation, temporal association does not necessarily equate causation. The authors praise Foreman et al. for their case report, which illustrates the danger of etiological assumption and thank them for drawing attention to the need for more rigorous investigation into causes of AV fistula formation."} +{"text": "Niviventer confucianus) via analysis of molecular variance and FST along three mountain slopes and tested the isolation\u2010by\u2010environment hypothesis using Mantel test and redundancy analysis. We found a consistent phenotypic divergence and marked genetic structure along elevational gradients; however, the species showed mixed patterns of size and skull shape trends across mountain zones. Individuals living at lower altitudes differed greatly in both phenotype and genotype from those living at high elevations, while middle\u2010elevation individuals showed more intermediate forms. The ecological parameters associated with phenotypic divergence along elevation gradients are partly related to species' ecological and evolutionary constraints. Fossorial and solitary animals are mainly affected by climatic factors, while terrestrial and more gregarious species are influenced by biotic and abiotic parameters. A novel finding of our study is that predator richness emerged as an important factor associated with the intraspecific diversification of the mammalian skull along elevational gradients, a previously overlooked parameter. Population genetic structure was mainly driven by environmental heterogeneity along mountain slopes, with no or a week spatial effect, fitting the isolation\u2010by\u2010environment scenario. Our study highlights the strong and multifaceted effects of heterogeneous steep habitats and ecologically based divergent selective forces in small mammal populations.Species distributed along mountain slopes, facing contrasting habitats in short geographic scale, are of particular interest to test how ecologically based divergent selection promotes phenotypic and genetic disparities as well as to assess isolation\u2010by\u2010environment mechanisms. Here, we conduct the first broad comparative study of phenotypic variation along elevational gradients, integrating a large array of ecological predictors and disentangling population genetic driver processes. The skull form of nine ecologically distinct species distributed over a large altitudinal range (100\u20134200\u00a0m) was compared to assess whether phenotypic divergence is a common phenomenon in small mammals and whether it shows parallel patterns. We also investigated the relative contribution of biotic (competition and predation) and abiotic parameters on phenotypic divergence via mixed models. Finally, we assessed the population genetic structure of a rodent species ( AnticipDivergent natural selection, briefly defined as selection arising from uneven ecological conditions that promote phenotypic and genetic shifts among populations, is a key source of biological diversity distributed over a wide altitudinal range and with distinct ecological attributes to assess whether phenotypic divergence is a common phenomenon in small mammals and whether it shows consistent parallel patterns. We chose skull morphology (size and shape) as a surrogate of overall morphological diversity as it is associated with the main sensory systems related to environmental perception and reflects changes in the dietary and ecological interactions across populations . Because of the large home range and generalist habits of our focal species, we expected weak or absent genetic structure along mountain slopes.22.1Soriculus) or along altitudinal gradients with similar climatic conditions. In the last case, to minimize putative effects of distinct evolutionary history, we selected individuals from closely genetic lineages distributed over a large altitudinal range and representing distinct ecological niches were selected to assess phenotypic variation (size and shape of the skull) across elevational gradients Table . All spegeomorph package version 3.0.5 . Based on the geographic coordinates of each individual, we extracted the altitude information via the raster R package at a 30\u2010s (~1\u00a0km2) spatial resolution , we used the database of sampling collections of the mammal group of the Institute of Zoology of the Chinese Academy of Science. This unique database includes specimens of small mammals systematically collected from several mountains in China. Detailed descriptions of the sampling protocols can be found in Wu et al. and Wen To evaluate the influence of predation risk on phenotypic variation, we compiled a list of predators of small mammals in China as a model due to its wide elevational range , broad climatic tolerance, and relatively high dispersal capability from 160 individuals comprising six populations covering an altitudinal range of 2,000\u00a0m . The delimitation of zones combined climatic and vegetation attributes and was based on Tang and Ohsawa , Zhong, geomorph R package with 10,000 permutations. In addition, we carried out pairwise comparisons between mean shapes and performed Tukey's honestly significant difference (HSD) test to assess the significance of morphological differences between pairs of elevation zones. We also explored size and shape trends across species to determine whether there was a consistent overall pattern. The relationship between size (centroid size) and altitude was visualized with scatterplots highlighting trends in each elevation zone. Skull shape deformation between zones was assessed via between\u2010group principal component analysis on skull phenotypes by creating a set of competing models ranked by the second\u2010order corrected Akaike information criterion (AICc) via the Barton, and testSummarizing shape information in univariate terms might lead to a loss of information or nonrandom (IBE) mechanisms, we performed two complementary tests . We found a mixed pattern of size trends across elevation zones. Rodents exhibited a marked decrease in size as altitude increased at low elevations, while shrews and moles were not affected or exhibited a weak positive correlation. Both groups presented contrasting patterns at middle elevations, and five of the seven species presented positive trends in size at high elevations differed significantly among populations. Similarly, AMOVA revealed significant population genetic structure . A large part of the genetic variation was attributed to variance within populations (59.9%\u201396.1%), whereas differences among elevation zones were low to moderate, albeit not significant between animals inhabiting low and high altitudes . Comparisons between populations from low and middle and middle and high elevations revealed generally moderate\u2010to\u2010low genetic differentiation or a weak effect on the landscape genetic structure of N. confucianus pattern across the nine species. This finding is consistent with that of previous studies on animals along elevational gradients at intraspecific shows a negative trend as elevation increases presents a positive trend were mainly associated with seasonal temperature and precipitation, while forest dwellers tended to be affected by multiple parameters.The ecological parameters associated with phenotypic divergence along elevational gradients are expected to be tied to species ecological and evolutionary traits drives phenotypic divergence at each elevation zone. Answering this question could provide us with a better understanding of the dynamic ecological interactions along altitudinal gradients , fitting the IBE scenario.None declared.AF conceived the idea; AF, ZW, and QY designed the research; ZW, JC, DG, LX, and QY conducted the field sampling; AF, ZW, JC, and DG curated the data; and AF analyzed the data and led the writing.\u00a0Click here for additional data file."} +{"text": "Building networks that are effective in linking older adults to supportive programs and services often involves challenges related to access, eligibility requirements, the elder's ability to understand enrollment processes, and lack of trust in service providers. For ethnic minority elders these challenges are often greater due to additional linguistic and cultural barriers. The four presentations on this panel address challenges to building effective service networks for ethnic minority elders using data derived from focus groups with members of these communities and those tasked with providing their care. The first presentation (Graham and Tseng) examines the Village model, a model designed to empower older adults, and asks why more Latino, African American and Asian elders do not participate. The second paper (\u00c5g\u00e5rd) looks at communication difficulties as a source for understanding the nature of cross-cultural discussions around end of life issues with ethnic minority patients. The third paper explores how caregiving programs for Alzheimer\u2019s Disease patients can be modified to better serve Arabic speaking caregivers. The final paper uses data collected among Spanish and Chinese (Mandarin) speaking elders to design a conceptual model which describes how ethnic minority and other elderly navigate the Long Term Care Services and Supports network. Our respondent will place these papers within the growing theoretical work on diversity and care support."} +{"text": "There is an increasing number of pediatric urgent care centers that are largely staffed by pediatric residency graduates. It is unclear if pediatric residency adequately prepares a physician to fully and successfully provide care in an urgent care setting. The goal of this study is to conduct an assessment of urgent care directors\u2019 perceptions of recent pediatric residency graduates\u2019 preparedness to successfully provide pediatric urgent care after graduation.This is a 2018 cross-sectional survey of all pediatric emergency medicine division chiefs in the United States and all pediatric urgent care directors who are members of the Society for Pediatric Urgent Care. An electronic survey was distributed consisting of eight multiple choice questions regarding perceived preparedness and knowledge gaps of recent pediatric residency graduates for independent practice in urgent care. Descriptive statistics were used to analyze results and qualitative data were analyzed via an inductive thematic approach.Forty-two percent (65/154) of surveys were completed. No respondents believed that a recent pediatric residency graduate would be adequately prepared to independently practice in a pediatric urgent care and 81% of respondents recommended some additional training. Most respondents described this training as important (46%) or very important (35%). Most respondents recommended between 6 months and 1 year as the appropriate amount of time to achieve competency.Despite the growing number pediatric residency graduates staffing pediatric urgent care centers, the majority of surveyed pediatric emergency medicine division chiefs and pediatric urgent care directors do not think that pediatric residency adequately prepares graduates to successfully provide urgent care to pediatric patients. We recommend further exploration of gaps in knowledge of recent pediatric residency graduates as a next step towards developing systems for further training for pediatric residency graduates to gain competency in urgent care management.The online version of this article (10.1186/s12913-019-4241-8) contains supplementary material, which is available to authorized users. In June 2017, there were 7639 urgent care centers (UCCs) in the United States, an increase in 5% from 2016 .The American Academy of Pediatrics Committee on Pediatric Emergency Medicine recommends that urgent care facilities have \u201cexperienced staff trained to provide critical support for ill and injured children until transferred for definitive care.\u201d AdditionThe survey was created with guidance from an educational researcher using survey development methodology . The finIn order to reach the maximum number of urgent care directors, we distributed the survey via email to members of the association of Pediatric Emergency Medicine North American Chiefs (PEMNAC), and to members of the Society for Pediatric Urgent Care (SPUC), an organization formed in 2014 for pediatric urgent care medical directors to develop best practices for ensuring clinical excellence and overall quality of care. A survey reminder was sent 2 weeks after the initial electronic mailing. Respondents were not incentivized for their involvement.We analyzed the quantitative data using descriptive statistics for central tendency (mean and SD), overall counts and percentages, and frequency distributions. Contingency tables (chi-squared) testing was used to evaluate differences in demographic characteristics between respondents as well as differences in perceived preparedness for independent practice. Analyses were conducted using Excel software . Qualitative data were analyzed via an inductive thematic approach. This quality improvement study was acknowledged and determined to be exempt from a standard review by the Children\u2019s National Health System Institutional Review Board per our institution\u2019s policy and with the need for consent waived.Of the 154 surveys sent, 80 responses were recorded. Fifteen respondents started but did not complete the survey, yielding a completed response rate of 42%. An additional two respondents stated that they were not affiliated with an urgent care center (UCC), leaving 63 total surveys in the final analysis.The majority of respondents directed urgent care sites that were affiliated with an academic institution (89%). Fifty-four percent surveyed identified their affiliated UCC as within a hospital setting and 35% were freestanding. The remaining 11% were described as freestanding UCC\u2019s, without an academic affiliation. The UCCs surveyed employed physicians with a variety of training, including pediatric residency graduates (89%), pediatric emergency medicine fellowship graduates (56%), emergency medicine residency graduates (30%), and family medicine residency graduates (12%). Forty-seven of our 63 respondents (75%) stated that their site either advertised for or hired a general pediatrician in the past year. Further demographic data are presented in Table\u00a0The majority of respondents described recent pediatric residency graduates as somewhat comfortable with the diagnosis and management of common urgent care clinical cases (68%), but very uncomfortable 16%) or somewhat uncomfortable (49%) with urgent care procedures. See Fig.\u00a06% or somNo respondents believed that a physician would be prepared to work independently in an UCC immediately upon graduating pediatric residency. The respondents recommended additional training ranging from 1 month to 3 years. Fifty-six percent of the survey participants felt that pediatric residency graduates needed more than 6 months of additional training in order to become competent in the field of pediatric urgent care. The average recommended time for additional training was 11\u2009months, and the median and mode of the responses were 12\u2009months.Consistent with their assessment that pediatric residency graduates needed additional time to attain competence in urgent care medicine, 81% of respondents considered additional training after residency either important (46%) or very important (35%) when hiring physicians to work in an UCC. Only 19% described additional training as unimportant (11%) or very unimportant (8%). See Fig.\u00a0Most respondents described clinical competency as the most important skill for urgent care providers with procedural competency as the next most important skill. Teaching, quality improvement and administrative skills were perceived as less important. See Fig.\u00a0In this needs assessment survey, we found that leaders in the field of pediatric urgent care believe that general pediatric residency graduates need additional training before they can achieve competence in urgent care. The estimated time range for this training varied widely; however, most survey respondents recommended that recent pediatric residency graduates have an additional 6 months to 1 year of training.The vast majority of survey respondents consider clinical competency the most important skill for a pediatric urgent care provider, followed closely by procedural competency. We believe these aspects of urgent care are best learned in a supervised environment. On the other hand, study participants considered the skills of administration, quality improvement, and teaching as less important. These can be safely acquired on-the-job and, while important, do not directly impact patient care.Our findings are timely because of the growing demand for urgent care services, due in part to the rising cost of emergency department visits and the shortage of primary care availability , 3. WithTo our knowledge, this is the first study evaluating the preparedness of pediatric residency graduates to provide care in a pediatric UCC. This adds to the developing set of literature around providing quality, patient-centered care in UCCs. Our results supplement the findings of another study, which examined the effects of establishing an urgent care curriculum and lecture series for residents in the primary care setting. Overall response to the content was positive, but more importantly, referral percentage to subspecialists decreased from 34% before the intervention to 31% after the intervention . InteresPediatric urgent care centers see a wide range of patient acuity and often provide care that encompasses some services normally administered in the primary care office and some traditionally provided in the emergency department. Pediatric urgent care providers are often expected to be competent in fracture management, wound care, abscess drainage and IV placement. In addition, between 1 and 5% of patients who initially present to an UCC will subsequently require transport to an emergency department ; consequThe majority of survey respondents recommend that recent pediatric residency graduates have an additional 6 months to 1 year of training to competently provide urgent care. The vast majority of survey respondents consider clinical competency the most important skill for a pediatric urgent care provider, followed closely by procedural competency. The majority of survey respondents consider administrative skills, quality improvement skills and teaching skills as less important. Recently, a new crop of pediatric urgent care fellowships has arisen to meet this demand. Further educational needs assessments will help refine these fellowships to more specifically meet the needs of recent pediatric residency graduates planning on working in pediatric UCCs.These new urgent care fellowships provide a supervised environment for pediatric residency graduates to gain the clinical and procedural competency needed to work in an urgent care setting. In addition, an academic setting can help fellows gain administrative, quality improvement and teaching skills.First and foremost, our survey completion rate was only 42%; however, this is comparable to the generally accepted 50% response rate in social research surveys and higher than the average 30% response rate to online surveys , 10. TheThe majority of academic urban and suburban urgent care directors do not perceive recent pediatric residency graduates as ready to provide competent care in an UCC without further training. This training can likely be accomplished with 6 months to 1 year of additional training in clinical and procedural skills. Future research should be directed at determining specific subsets of skills needed and most effective training methods.Additional file 1:Survey for urgent care directors. Includes the 10-question survey used for the needs-assessment study. (DOCX 17 kb)"} +{"text": "Develop Neurobiol, 2018After axonal injury, chromatolysis (fragmentation of Nissl substance) can occur in the soma. Electron microscopy shows that chromatolysis involves fission of the rough endoplasmic reticulum. In CNS neurons or in motor neurons or dorsal root ganglion neurons denied axon regeneration , chromatolysis is often accompanied by degranulation (loss of ribosomes from rough endoplasmic reticulum), disaggregation of polyribosomes and degradation of monoribosomes into dust\u2010like particles. Ribosomes and rough endoplasmic reticulum may also be degraded in autophagic vacuoles by ribophagy and reticulophagy, respectively. In other words, chromatolysis is disruption of parts of the protein synthesis infrastructure. Whereas some neurons may show transient or no chromatolysis, severely injured neurons can remain chromatolytic and never again synthesize normal levels of protein; some may atrophy or die. Ribonuclease(s) might cause the following features of chromatolysis: fragmentation and degranulation of rough endoplasmic reticulum, disaggregation of polyribosomes and degradation of monoribosomes. For example, ribonucleases in the EndoU/PP11 family can modify rough endoplasmic reticulum; many ribonucleases can degrade mRNA causing polyribosomes to unchain and disperse, and they can disassemble monoribosomes; Ribonuclease 5 can control rRNA synthesis and degrade tRNA; Ribonuclease T2 can degrade ribosomes, endoplasmic reticulum and RNA within autophagic vacuoles; and Ribonuclease IRE1\u03b1 acts as a stress sensor within the endoplasmic reticulum. Regeneration might be improved after axonal injury by protecting the protein synthesis machinery from catabolism; targeting ribonucleases using inhibitors can enhance neurite outgrowth and could be a profitable strategy Why does axonal injury result variably in axon regeneration or collateral sprouting, atrophy or cell death this can be accompanied by the disaggregation and/or disassembly of polyribosomes to leave a fine \u201cdust\u2010like\u201d powder Cragg, . Ribosomin situ hybridization show increases in rRNAs in motor neurons after peripheral nerve injury . IntereSteward, includinSteward, presumabSteward, . As willSteward, .A variety of ribonucleases might degrade RNAs during chromatolysis such as those in the secreted, vertebrate ribonuclease family is washed out or if an NMDA receptor antagonist is applied by which endoplasmic reticulum is fragmented is not known but there is evidence from other cell types that calcium\u2010dependent ribonucleases in the EndoU/PP11 family dynamically regulate endoplasmic reticulum only causes disassembly of polyribosomes into monoribosomes and does not cause degradation of monoribosomes into fragments with Ribonuclease 1 causes disassembly of polyribosomes (into monoribosomes) and degradation of monoribosomes Cellular stress leads to formation of stress granules in which mRNAs may be stored or degraded Wolozin, . Stress vice versa). For example, any floxed Ribonuclease gene could be deleted in sensory neurons expressing Advillin\u2010CreERT2 upon application of tamoxifen including Atg5 and Atg7 Targeting the ER stress response might modify chromatolysis; there are reports it can increase or decrease recovery after PNS or CNS injury including spinal cord injury and maintain low levels of key Regeneration\u2010Inhibiting Genes (RIGs) much of the protein synthesis machinery. Might this be feasible? Chromatolysis generally takes a few days to reach a maximum even when injury is within a few millimeters of the cell body (Matthews and Raisman, In conclusion, ribonucleases may contribute to chromatolysis, ER stress, ribophagy and reticulophagy after neuronal injury. Identification of which ribonucleases play deleterious role and which ribonucleases play pro\u2010regenerative roles could be an important step in developing new therapies for repair of nervous system injuries.This work was supported by a grant from the Wings for Life foundation and through a grant to the \u201cAxonRepair\u201d consortium from ERA\u2010NET NEURON that is co\u2010sponsored by the Medical Research Council. Thanks to Emeritus Professor Thomas Sears and Professors Mike Fainzilber and Simone Di Giovanni for providing feedback on a draft."} +{"text": "Pain is prevalent in late life and may cast negative impacts on older adults\u2019 sleep. We examined this link in older adults\u2019 everyday lives and asked whether receiving support on days when older adults had pain improved or worsened their sleep. We drew on the Daily Experiences and Well-being Study; over 300 adults aged 65+ reported on their pain, sleep and social support received throughout each day across 5 days. Multilevel models revealed that older adults in greater pain were more likely to nap throughout the day and to incur sleep disturbances at night. Older adults who slept better at night reported less pain the next day. The link between pain and sleep disturbances was stronger on days when older adults received support compared to days when they did not. This study adds to the literature regarding pain and sleep and explores what roles social factors play in this link."} +{"text": "Although it is well established that stress is negatively associated with cognitive functioning, less is known about age differences in the effects of stressors and anxiety on state anxiety and physiological reactivity . The current study examined state anxiety and cortisol reactivity during a series of cognitive tasks in a sample of younger (n=26) and older (n=29) adults. Participants completed the State-Trait Anxiety Inventory prior to cognitive testing and provided six salivary cortisol samples throughout one testing session: two cortisol samples prior to cognitive testing, three samples during testing, and one sample after testing. Six cognitive tasks were administered that measured attention span, declarative memory, and processing speed. Results indicated a significant interaction effect of age by time with younger adults\u2019 cortisol linearly decreasing during the testing session and older adults\u2019 cortisol showing a quadratic trend. A second interaction was found between age and state anxiety whereby older adults who reported more anxiety had higher cortisol levels during the cognitive testing session than both the older adults who reported low levels of anxiety and the younger adults. Only age (not cortisol or anxiety) was significantly related to cognitive performance. Results from this study suggest that standard cognitive testing could be anxiety producing for older adults, particularly for those who are already anxious. Future investigations should examine age-related differences in the processes linking anxiety and cortisol to specific types of performance, such as memory and attention."} +{"text": "Anabolic androgenic steroids may be associated with early coronary artery disease, according to research presented at the Brazilian Congress of Cardiology (SBC 2017). The annual congress of the Brazilian Society of Cardiology (SBC) was held in S\u00e3o Paulo from 3 to 5 November 2017. Experts from the European Society of Cardiology (ESC) presented a special programme.\u2018Anabolic androgenic steroid abuse among young people is a widespread problem worldwide, and adverse events such as sudden cardiac death and heart attack have been reported in athletes,\u2019 said lead author Francis Ribeiro de Souza, PhD student, Heart Institute , Medical School, University of Sao Paulo , Brazil. \u2018In Brazil, around one million people have used anabolic androgenic steroids at least once, and they are the seventh most commonly used drug in the country,\u2019 he added.This study examined whether anabolic androgenic steroids could be associated with early coronary artery disease. It also tested whether reduced high\u2013density lipoprotein (HDL) function could be a mechanism leading to coronary artery disease in anabolic androgenic steroid users.The study included 51 men with an average age of 29 years (range 23\u201343 years). Of those, 21 did weight lifting and had taken anabolic androgenic steroids for at least two years, 20 did weight lifting but did not take steroids, and 10 were healthy but sedentary.Participants underwent computed tomography coronary angiography to assess the presence of atherosclerosis in the coronary arteries.A urine test was performed in all participants to confirm steroid use. Blood samples were taken to measure lipid levels including HDL. The researchers used cell cultures to measure the ability of each participant\u2019s HDL to perform its normal function of removing cholesterol from the macrophages.The researchers found that 24% of steroid users had atherosclerosis in their coronary arteries, compared to none of the non\u2013users and sedentary participants. The steroid users with atherosclerosis also had significantly reduced HDL levels and HDL function.Mr Ribeiro de Souza said: \u2018Our study suggests that anabolic androgenic steroid use may be associated with the development of coronary artery disease in apparently healthy young people. Steroids may have an impact on the ability of HDL to remove cholesterol from macrophages, thereby promoting atherosclerosis.\u2019\u2018This was a small, observational study and we cannot conclude that steroid use causes atherosclerosis,\u2019 he continued. \u2018Larger studies with longer follow up are needed to confirm these results.\u2019Mr Ribeiro de Souza concluded: \u2018We observed coronary atherosclerosis in young anabolic androgenic steroid users, which in combination with lower HDL levels and reduced HDL function, could increase the risk of cardiovascular events. Greater awareness is needed of the potential risks of these drugs.\u2019Dr Raul Santos, scientific chair of SBC 2017, said: \u2018This study, despite its small sample size, is well done and calls attention to a possible important health problem in Brazil and elsewhere, since it shows not only the classical lipid disturbances induced by steroids but actually associates them with subclinical atherosclerosis presence, something that we are not supposed to find in young individuals.\u2019Professor Fausto Pinto, ESC immediate past president and course director of the ESC programme in Brazil, said: \u2018This is an important issue in cardiovascular prevention, which deserves further study. During SBC 2017, ESC experts highlighted hot topics in prevention and other fields of cardiology that were presented at ESC Congress 2017 in Barcelona."} +{"text": "There is a growing interest among aging services providers to better understand the pathways through which older adults and their caregivers navigate LTSS. Although there have been attempts at modeling this process they are often dependent on the quality of existing data, which can result in models which are incomplete and study samples that homogenize diverse older adult populations. These models face two challenges \u2013 1) the data may not include information about important elements of the LTSS navigation process, and 2) the actions of ethnic/cultural sub-groups may not be captured. This study uses a conceptual method called Social Interaction Modeling (SIM) to examine how older adults in two limited English-speaking communities (Spanish / Mandarin Chinese) navigate the use of LTSS and to evaluate disparities in service access. The findings will help to build a more comprehensive model which looks at service navigation among all older adults in Philadelphia."} +{"text": "Syphilis continues to affect a large number of pregnant women, causing substantial perinatal morbidity and mortality. It was estimated that approximately 1.36 million pregnant women globally were infected with syphilis, resulting in at least 520\u2009000 adverse pregnancy outcomes caused by mother-to-child transmission (MTCT) of the infection [Box 1Policy and resource\u2022 No policy on universal antenatal screening for syphilis;\u2022 Inadequate fund allocation;\u2022 Lack of coordination among stakeholders; and\u2022 Lack of integration with maternal and child health programmes.Socio environment\u2022 Stigma from family members and community;\u2022 Violence from sexual partners; and\u2022 Negative traditional/religious beliefs.Health system\u2022 Poor health care system;\u2022 Poor infrastructure;\u2022 Inadequate human resources/trained staff; and\u2022 Unavailability or stock-out of supplies .Programme management\u2022 Poor management system;\u2022 Lack of proper monitoring; and\u2022 Lack of support and supervision.OthersCurrent version of the global initiative for elimination of MTCT (EMTCT) of syphilis emphasizes the coverages of intervention services targeting pregnant women, ie, \u226595% antenatal care (at least one visit), \u226595% syphilis testing and \u226595% treatment coverage (at least one dose of intramuscular benzathine benzylpenicillin) for at least 2 years in order to achieve the goal of having a congenital syphilis rate \u226450 cases per 100\u2009000 live births. Increase of political commitment and financial investment from country governments and international donors, and early detection of infection by introduction of point-of-care (POC) tests in the first and second trimesters followed by treatment with benzathine penicillin have significantly facilitated the progress ensure the achievement of these intervention targets . SpecialSexually transmitted infections (STIs) other than HIV are usually neglected by many countries as a public health priority. Over the last two decades, the United States and China are the countries with national program specifically designed for control of syphilis although some of other countries initiated specific projects at national or subnational level, such as Attack of the Cursed Syphilis campaign to increase syphilis awareness in Toronto, and the National Gay Men's Syphilis Action Plan (NGMSAP) to increase syphilis testing among MSM at high risk in Australia . The UniBox 2Three screenings\u2022 Providing an active syphilis screening to patients at risk for sexually transmitted infections (STIs) at clinics where STI service is delivered.\u2022 Providing an active syphilis screening to clients who attend HIV voluntary counselling and testing (VCT) or methadone maintenance treatment (MMT) services followed by referral of syphilis-positives to STI clinics.\u2022 Providing an active syphilis screening as part of outreach services targeting high-risk groups and referral of syphilis-positives to STI clinics.One standardized care\u2022 Providing syphilis-infected patients with a standardized care in which treatment with at least one shot of benzathine benzyl penicillin (BBP) should be ensured, and behavioural interventions, and partner notification be delivered."} +{"text": "EDCs) can alter biological function in organisms at environmentally relevant concentrations and are a significant threat to aquatic biodiversity, but there is little understanding of exposure consequences for populations, communities and ecosystems. The pervasive nature of EDCs within aquatic environments and their multiple sub\u2010lethal effects make assessments of their impact especially important but also highly challenging. Herein, we review the data on EDC effects in aquatic systems focusing on studies assessing populations and ecosystems, and including how biotic and abiotic processes may affect, and be affected by, responses to EDCs. Recent research indicates a significant influence of behavioural responses (e.g. enhancing feeding rates), transgenerational effects and trophic cascades in the ecological consequences of EDC exposure. In addition, interactions between EDCs and other chemical, physical and biological factors generate uncertainty in our understanding of the ecological effects of EDCs within aquatic ecosystems. We illustrate how effect thresholds for EDCs generated from individual\u2010based experimental bioassays of the types commonly applied using chemical test guidelines [e.g. Organisation for Economic Co\u2010operation and Development (OECD)] may not necessarily reflect the hazards associated with endocrine disruption. We argue that improved risk assessment for EDCs in aquatic ecosystems urgently requires more ecologically oriented research as well as field\u2010based assessments at population\u2010, community\u2010 and food\u2010web levels.Endocrine\u2010disrupting chemicals ( I.et al., et al., et al., et al., Endocrine\u2010disrupting chemicals (EDCs) remain an active topic in contemporary ecotoxicology due to their proven environmental impacts and at higher levels of biological organisation (e.g. populations and food webs) overcomes several limitations associated with most current experimental ecotoxicology bioassays Danio rerio (Hamilton) populations that have been shown to alleviate negative individual reproductive effects of octylphenol exposure , polybrominated diphenyl ethers (PBDEs) and diclofenac have been shown to partition, accumulate and magnify within components of aquatic food webs in a behavioural response to oxazepam exposure resulted in enhanced consumption of its damselfly prey (Coenagrion hastulatum Charpentier), and in turn an increase in the transfer and bioaccumulation of oxazepam. These examples illustrate that ecological risks for some EDCs that affect ecosystem processes may be greater than commonly appreciated within aquatic ecosystems.Interactions between the direct effects of endocrine disruption and the subsequent transfer of EDCs through ecosystems may also occur, supporting that alterations in individual\u2010level effects may have consequences for wider biological systems et al., et al., et al. (+ channel) in Hyalella azteca (Saussure) populations meant that resistant individuals did not experience the neurotoxic effects observed in non\u2010resistant populations, instead exhibiting oxidative stress only at considerably higher pyrethroid concentrations. Varying levels of resistance were found across several populations. Adaptation has also been observed within fish assemblages and the Atlantic killifish (Fundulus heteroclitus L.) can adapt to polycyclic aromatic hydrocarbon (PAH) and PCB exposure in natural systems restricted inducible gene expression and was responsible for the observed resistance to EDC exposure than those populations which were not pre\u2010exposed under conditions within which non\u2010resistant individuals cannot survive. Within natural systems, adaptation of individuals or populations leads to an enhanced risk of bioaccumulation with increasing concentrations of EDCs. Adaptation to endocrine disruption indicates that organisms may be able to persist at environmentally relevant concentrations of EDCs, yet it also suggests potential for increased flux of EDCs through food webs. Changes in the bioaccumulation and transfer of EDCs potentially lead to increases in the body burden of higher trophic\u2010level organisms, increasing the likelihood of adverse effects across the aquatic food web.Resistance, and/or adaptation has significant implications for the potential broad\u2010scale effects of endocrine disruption in aquatic systems. A recent example in (3)et al., D. rerio individuals differed between exposure periods of 0\u201321 and 0\u201375 days post\u2010fertilisation eggs to an environmental oestrogen, bisphenol A (BPA), over a range of concentrations including 300 and 3000 ng l\u22121 resulted in lower energy levels in larvae to first feeding, reductions in specific growth and restricted food conversion ratios on gonad differentiation in D. rerio after a 5\u2010month depuration period post\u2010EE2 exposure exposure at 30 ng l\u22121. The length of both exposure and period for depuration thus appear to be important in weighing up the potential for biological impacts of EDCs in natural systems. The fact that EDCs can act through multiple pathways means that it is especially difficult to identify chronic and life\u2010stage\u2010specific effects . Whilst these taxa are convenient as study models, they may not necessarily allow the accurate prediction of effects within populations of longer\u2010lived organisms which may accumulate greater levels of EDCs over longer periods of time and have slower generational turnover, and thus a lower ability to adapt in response to toxicological impacts. Further efforts to understand long\u2010term exposure effects across a wider range of taxa are urgently required.Of note is the fact that contemporary assessments of EDCs in the laboratory are confined to a restricted range of short\u2010lived species suitable for experiments; for fish, notably (4)et al., et al., et al., et al., via simple additive\u2010effect modelling . TBT depressed the burst\u2010swimming response known to result from EE2 exposure, whilst EE2 influenced the alterations in the time spent in secluded areas generated by high concentrations of TBT. Consequently, when mixtures of EDCs combine with processes such as competition and predation, a range of complex and often unpredictable effects can result.Wastewater effluents and other pollutant sources are often composed of highly complex mixtures, and interactions between EDCs and of EDCs with other chemicals could alter their biological effects et al., et al., et al. (Rutilus rutilus L.) populations demonstrates the potential for interactive effects of multiple stressors. Here, the feminisation of individuals generated by endocrine disruption appeared to have negligible effects on population extinction risk, yet the combination of exposure and selective fishing practices resulted in significant increases in local population extinction rates. The feminising effect of oestrogenic EDCs in isolation does not always result in significant population effects , clotrimazole exposure (2000 and 10000 ng l\u22121) and inbreeding together had an additive effect, with a marked increase in the male\u2010skew of populations relative to the effects generated by individual stressors. The results of multiple\u2010stressor studies have indicated additive and synergistic interactions between stressors and endocrine disruption, but this depends on the level of biological organisation included et al., et al., D. rerio populations to EE2 exposure, differences in their breeding biology and response sensitivity were apparent. Inbreeding within laboratory fish stocks is a major issue for experimental assessments of EDCs, especially when intending to inform further research in systems involving outbred individuals and chemical contamination can have marked effects on the genetic diversity present within populations (Bickham (7)et al., et al., et al. (\u22121) increased microbial community biomass. A more commonly observed indirect mechanism is provided by the adverse effects of EDC exposure within predator assemblages and a subsequent top\u2010down cascade through the food web. Alterations in the structure of invertebrate communities have been recorded in response to failed recruitment of secondary\u2010consumer fish species when an entire Canadian lake was dosed with EE2 (5\u20136 ng l\u22121) over a period of three summers the limitations of using individual\u2010based bioassays to predict the effects of EDCs at population\u2010 and food\u2010web scales the need for research at a range of spatial and temporal scales to advance knowledge of broad\u2010scale ecological effects and risk assessment. The restricted scope of common experimental assessments has been highlighted previously et al., Contemporary research focuses on up\u2010scaling EDC exposure to populations and food webs within aquatic environments. The spatial coverage of these assessments, however, is restricted when using individual systems to exemplify the wider conditions present across the landscape. An example of this is the focus on WwTWs and their downstream impacts across aquatic systems. A focus on wild populations and the effects of regulated effluent discharges (containing EDCs) has made significant contributions to establishing the effects of effluent discharges on aquatic organisms across aquatic environments. However, a focus on WwTWs discharges has also led to limitations in our understanding of the spatial variation in EDC occurrence and their impacts within and between different types of aquatic systems. Up\u2010scaling research strategies to landscape scales to understand these spatial variations is much needed to extend our knowledge of the effects of EDCs within natural systems. This will enable improved impact and risk assessment, with practitioners able to assess more accurately the degree to which potential concerns vary across the aquatic environment. Water\u2010quality data regarding WwTWs discharges are available in many countries, consequently high\u2010risk WwTWs can be targeted for regulation and remediation. A range of techniques are available to achieve this objective, including spatial and statistical modelling. Modelling at extremely broad scales has identified variations in emission of steroidal oestrogens between catchments, highlighting spatial variation in effects (Zhang (2)et al. et al., et al., et al., Gammarus fossarum Fabricius) proved inconclusive as vitellogenin expression was shown to vary with unexplained environmental conditions to analytical quantification of environmental EDC concentrations (e.g. via gas chromatography mass spectrometry) is essential for advancing our understanding of endocrine disruption in natural systems. Such comparisons will allow evaluation of the robustness of biomarkers in assessing ecological risk from EDCs and stimulate the refinement of in vivo methods. The currently restricted focus on a few chemicals and organisms limits the ability of practitioners to utilise biomarkers for ecological risk assessment and environmental decision\u2010making et al., via genomic pathways . As well as allowing for broad\u2010scale analyses, these techniques enable a reduction in the previously large number of samples required for field\u2010based assessments to detect reproductive impacts and sex reversal at low EDC concentrations.A significant concern surrounding EDCs is the potential for impacts on the genetic structure of populations and thus on the integrity of wild populations The ecological effects of EDCs are currently investigated by effects assessments on individuals employing only a small number of different organisms under controlled experimental conditions. The environmental relevance of these findings is likely to be limited. Spatially and temporally up\u2010scaling these investigations within the aquatic environment is therefore vital in developing environmentally relevant knowledge and to provide supporting data for practitioners to make accurate risk assessments. The hormonal, sub\u2010lethal implications of EDC exposure could lead to a range of emergent effects resulting from ecological interactions.(2) We have highlighted the potential benefits of applying previously derived mechanistic knowledge at broader spatial and temporal scales to assess the ecological impacts of EDC exposure within natural systems. A range of abiotic and biotic characteristics and processes can alter the effects and transfer of EDCs within aquatic food webs and cause deviations of observed effects from those identified in experimental assessments. A range of indirect effects also occur within natural systems, thus accurate assessment of endocrine disruption risk within aquatic ecosystems requires an appreciation of ecological processes at a range of spatial and temporal scales.(3) Several limitations of experimental bioassay designs are highlighted by recent research assessing broad\u2010scale EDC exposure. Consequently, the results of experimental bioassays should be interpreted with caution as such investigations often poorly represent influential controls present in natural systems. It is suggested that chemical test guidelines and models developed using these bioassays may provide limited utility in assessing the impacts and risk associated with EDCs.(4) A complementary suite of assessments at a range of scales should be adopted within a multi\u2010tier integrated research strategy to promote the development of environmentally relevant knowledge suitable for use by practitioners. Understanding the various direct and indirect impacts of EDCs, across a range of different spatial and temporal scales, should allow us to determine more effectively the transfer and ecological effects of EDCs within natural systems. Increasing the effectiveness of empirical and experimental research through methods such as integrated frameworks is therefore an important development.(5) Future research should focus on expanding field\u2010based research across a range of different aquatic environments. To achieve this objective, however, methodological and theoretical advances are required to enhance their applicability to natural systems and to develop more comprehensive methods of risk assessment for EDCs.VIII.This work was supported by the Natural Environmental Research Council [NE/L002434/] (F. M. W.). The authors would like to thank two anonymous reviewers for their comments."} +{"text": "Saving for retirement and the ability to provide care for a loved one can be dramatically affected by student loan debt. Currently, approximately 44 million people of all ages in the United States carry the weight of over 1.4 trillion dollars of student loan debt. Student loan borrowers of all ages may experience lower financial preparedness for retirement as well as decreased ability to provide care for family members, including aging parents. While older adults hold a relatively small proportion of student loans, they are the fastest growing subset of student loan borrowers and have disproportionately high rates of student loan defaults. As a result of their defaults, the Social Security retirement benefits of Americans ages 65 and older experienced a 500% increase in offsets over the last decade. This presentation will spotlight an MIT AgeLab mixed methods study about how student loan borrowers between the ages of 51 and 75 experience student loans within family systems and perceive and prioritize longevity planning in light of their student loans. Data collected for this study include focus groups and a large national survey. Preliminary findings suggest that for older borrowers, student loans are generally one of several financial constraints that can inform spending and saving decisions. For most, student loan payments are regarded as stunting overall retirement savings while the minority regard the two separately. Older borrowers also tend to have increased financial and familial responsibilities, including caring for aging parents, that compete for borrowers\u2019 limited financial and temporal resources."} +{"text": "Digital image correlation was utilized with confocal laser scanning microscope images to better visualize crack initiation during tensile straining. This technique showed that cracks initiated earlier in the thicker areas of the film (crests) than in lower areas (troughs) because of a higher density of printing defects and the increased thickness.Printing of metallic films has been preferred over vacuum technologies for roll-to-roll processes because of faster processing times and lower processing costs. Films can be produced by depositing inks containing suspended metallic particles within a solvent and then heating the films to both remove the solvent and sinter the particles. The resulting printed structure and electrical and mechanical behavior of the printed films has been studied to better understand their electro-mechanical response to loading and eventual brittle fracture. This study evaluated the electro-mechanical behavior of 1.25- Electronic devices continue to be increasingly integrated into individuals\u2019 everyday life, with newly identified applications beginning to change design requirements. New requirements for devices have emerged including the need to reduce the relative electronic device weight and the ability of some devices to undergo bending and stretching while maintaining their electronic functionality. Manufacturing methods for flexible electronics, which are more bendable and/or stretchable than traditional electronic devices, are being developedTo optimize the R2R manufacturing processes further, researchers are exploring different film deposition methods including digital inkjet, gravure, and flexographic printing.The specific parameters of a heating process that removes the remaining solvent and sinters the metallic particles vary for each ink blend depending on the specific components. After heating, the resulting structure of the printed metallic films has been shown to contain pores and relatively high surface roughnesses compared with vacuum deposited films on the same substrates. Studies of their mechanical behavior without a post-deposition process suggest that these films behave as brittle materials.This study is focused on exploring a new approach to tracking mechanical damage in printed films and correlating the damage to the electrical behavior. The use of digital image correlation (DIC) with confocal laser scanning microscope (CLSM) laser intensity images will be demonstrated as well as how the local mechanical behavior correlates to the global electrical behavior. Printed films have a high surface roughness that creates a natural pattern necessary for DIC techniques. The DIC technique allows for local strain to be calculated by comparing CLSM laser intensity images taken during straining. The local strain measurements are then used to identify areas of high strain that then lead to fracture.\u03bcm-thick Ag lines that were 3\u00a0mm wide onto a DuPont Teijin Heat Stable substrate (OMET Varylex 530 system with PChem PFI-722 Ag nanoparticle ink). Further details of the printing process were described previously,A flexographic process was used to deposit 1.25-R/R0). The relationship between engineering strain and normalized resistance can be approximated by:13l is the gage length during straining followed by 2% straining steps up to a maximum engineering strain of 12%. At each predetermined step, a 4-min pause was necessary to image the same surface area at three different magnifications. Two types of images were captured with the CLSM technique that tracked either the laser intensity or sample height. For DIC, only the laser intensity images were utilized, and the three-dimensional DIC software GOM CorrelateA typical result of the relationship between normalized resistance and engineering strain for the printed Ag lines is shown in Fig.\u00a0What cannot be observed in Fig.\u00a0Closer inspection of the CLSM images taken at a higher magnification of a wave crest provides more details about crack initiation and propagation in flexographic printed films. At an engineering strain of 4%, it is difficult to observe cracks in the CLSM laser image Fig.\u00a0a even thInitially, only DIC can be used to evaluate the crack density (4\u20136% engineering strain) to directly observe cracks of the rough surface. However, the roughness does make DIC analysis possible to examine the early stages of crack growth in printed films Fig.\u00a0.Fig.\u00a04MeThe use of DIC as a characterization method of printed lines added insight into crack initiation and propagation within printed Ag lines. Surface images were collected using the CLSM technique, which provided clear, laser intensity images of printed surfaces with relatively large surface roughnesses. The combination of the CLSM laser images and DIC made it possible to more thoroughly examine crack initiation at the local scale in the printed lines. Using DIC, the crack densities were more easily evaluated and showed that cracks initiated earlier in the thicker areas of the film (crests) than in lower areas (troughs) because of a higher density of printing defects and the increased thickness. This insight will be used to alter processing routes to decrease the surface wave amplitudes and printing defects to reduce initiation sites for cracks and, therefore, indirectly reduce electrical degradation resulting from applied strain. These results lead the authors to suggest that this technique is valuable when studying the fracture of materials with relatively high surface roughnesses where it is difficult to observe fracture processes."} +{"text": "Stabilizing certified nursing assistant (CNA) employment is necessary for maintaining care networks and providing high quality of care for nursing home (NH) residents. This study\u2019s objective was to examine the relationship of high wages and empowerment practices on CNA retention. We used the 2015 Ohio Biennial Survey to construct a facility-level dataset of 547 NHs and estimated multivariable linear regressions. NHs that provided both high wages and high empowerment were associated with a 12.95 percentage-point improvement in the CNA retention rate . High wages and a high empowerment score did not have significant effects individually (p > .05). Retention rates were similar between NHs that lacked high wages and scored low on the empowerment scale, and NHs that provided one at a high level but not the other. Implications for better retaining CNAs require multiple empowerment practices combined with high hourly wages."} +{"text": "Social isolation is a critical public health issue that socially isolated individuals are at increased risk for mortality and deteriorated health. Those who acquire hearing loss in later life experience difficulties with communication, potentially leading to social isolation. However, less is known about the social consequences of age-related hearing loss, and few studies have assessed the influence of environmental factors on hearing loss and social isolation. The aims of this study are to examine: (1) the association between hearing loss and social isolation of older adults over time, and (2) the moderating effects of perceived neighborhood social cohesion and disorder on this relationship. We analyzed 2,080 community-dwelling Medicare beneficiaries aged 65 or above from Round 1 to 3 of National Health and Aging Trends Study. We conducted random coefficient models, entering hearing loss as a random coefficient. Older adults with hearing loss were less socially isolated than those without hearing loss. However, the effect of hearing loss on social isolation varied depending on perceived neighborhood social cohesion. Older adults with hearing loss who reported high neighborhood cohesion had significantly lower social isolation compared to those without hearing loss, while those with hearing loss who perceived low social cohesion had significantly higher social isolation. Our findings suggest neighborhood social cohesion can serve as a potential protective factor for older adults with hearing loss. This poster will propose neighborhood-level interventions that could supplement other services for those with hearing loss, such as assistive devices that are rarely covered by health insurance."} +{"text": "Neovascularization has been defined as the presence of reflux in a previously ligated sapheno-femoral or sapheno-poplital junction caused by the development of incompetent tortuous veins linked to the thigh or calf varicosities.Langenbeck already described in 1861 what we would now define as neovascularizationThe term neovacularization was first determined in 1987 by Glass as \u201crecurrence through growth of new vessels\u201d.Histopathology showed that after 6 weeks new venous vessels and after 18 weeks parallel new venous vessels were present and after 40 weeks there was a continuity of venous flow.With this he could demonstrate by histopathology that very thin-walled irregular new venous vessels did reconnect the previously transected ends of the great saphenous vein.,Neovascularization may represent a physiological healing process following venous surgery. Venous disconnection and altered venous hemodynamics may initiate neovascularization.Histological examination of the operative specimen helps to distinguish between different morphological subtypes of neovascularization and \u201cnormal\u201d remnants of venous vessels. Morphologically, various patterns exist such as typical neovascularization , venous Neovascularization is detected in duplex ultrasound as tortuous vessels leading to the stump or connecting end of the great or small saphenous vein. Neovascularization detected by duplex may or may not result in an intervention depending, amongst other factors, on the clinical presentation of the recurrent varicose veins of the patient. Other common causes for recurrent varicose veins apart from neovascularization are tactical error , technical error and of course disease progression .It is recommended to treat symptomatic recurrent varicose veins if indicated, by endovenous thermal ablation, ultrasound guided foam sclerotherapy or phlebectomies."} +{"text": "Understanding factors influencing centenarians\u2019 nutritional status can offer insight into effective nutrition interventions to improve quality of life among this population. This cross-sectional study was conducted to evaluate the moderating role of social support in the relationship between loneliness and nutritional status among Oklahoma centenarians (n=140). Nutritional status was assessed with the Mini Nutrition Assessment (MNA). Perceived social support was assessed with the 24-item Social Provisions Scale. Loneliness was examined with the 10-item UCLA loneliness scale. Ordinal logistic regression revealed that those who lacked social support were more likely to be at risk for malnutrition . Further, the interactive model revealed that centenarians who reported lack of support and loneliness were almost 2.8 times as likely to be at risk for malnutrition compared to their socially embedded counterparts (p<.01). Findings suggest that nutrition interventions offering centenarians opportunities to feel socially connected could improve their nutritional well-being."} +{"text": "More than half of Korean Americans living in the US are immigrants, these immigrants hold unique cultural perspectives, including collectivism and filial piety that originates from Korean culture. Every older adult has life experiences and background that build and shape their own wishes and values for their health care goals. Thus, a qualitative descriptive study was conducted using the Life Course Theory as a guiding framework to examine older Korean immigrants\u2019 health care goals and the influence of their life courses. Twenty six interviews from 13 participants were analyzed using content thematic analysis. Study rigor was ensured by audit trail, peer debriefing, and prolonged engagement. Data were organized under five overarching themes: health care priorities, time, location, linked lives, and turning point. Older Korean immigrants valued painlessness and being independent as health care goals . They experienced a dynamic historical period in Korea before immigrating to the US (Time). Once they reached the US, they were disconnected from their social support and traditional values (Location). Children and Korean churches constitute older Korean immigrants\u2019 primary support system once in the US (Linked lives). Their tumultuous life experiences contributed to their current perspectives on health care goals and priorities (Turning point). In studies of older immigrant populations, it is important to acknowledge individual differences while simultaneously understanding the general life history and cultural background behind individuals\u2019 values and perspectives. Life course approach provides both a contextual understanding of older adults\u2019 backgrounds and the trajectories of their individual life courses."} +{"text": "OBJECTIVES/SPECIFIC AIMS: Determine whether children with solid tumors maintain intact protective immunity to live vaccines during cancer therapy and after completing cancer therapy (postTx). METHODS/STUDY POPULATION: We will perform a prospective cohort study of children with solid tumors followed at the Puerto Rico\u2019s University Pediatric Hospital. Protective immunity will be measured with antibody titers against live vaccines at diagnosis, during cancer therapy, upon completion and 3 months postTx. RESULTS/ANTICIPATED RESULTS: We hypothesize that those patients with protective immunity to live vaccines prior to cancer therapy will lose it at the end of therapy. DISCUSSION/SIGNIFICANCE OF IMPACT: Loss of protective immunity to live vaccines has been reported in patients with hematologic malignancies after cancer therapy. This lack of protective immunity, which puts patients at higher risk of acquiring vaccine preventable diseases, has been limited studied in patients with solid tumors. The Center for Diseases Control has been established that it is safe to immunize cancer survivors with live vaccines 3 months post Tx. However, no clear guidelines for revaccination have been provided for this population. Understanding the protective immunity variation against live vaccines in children with solid tumors will allow us to identify the need for revaccination with live vaccines in this vulnerable population."} +{"text": "Lung agenesis is a rare congenital anomaly. The main etiology of the disease is unknown whereas genetic, iatrogenic and viral factors as well as vitamin A deficiency during early pregnancy may result in developmental failure of primitive lung bud causing unilateral pulmonary agenesis. Affected patients usually present with variable respiratory symptoms and recurrent chest infection at any age. Plain film demonstrates opaque unilateral lung while chest CT scan can definitely diagnosis the disease. The anomaly has three types. Type I is pulmonary agenesis, type II is called pulmonary aplasia and type III is pulmonary hypoplasia.Six patients with main complaint of dyspnea underwent contrast enhanced chest CT in radiology department of French Medical Institute for Mothers and children, Kabul and were diagnosed lung agenesis.Three patients were categorized as type II pulmonary agenesis (aplasia). Two patients, three months old boy and a seven year- old girl demonstrated right lung aplasia. Another patient boy of eighteen years old presented with left lung aplasia.Two boys of four and seven months of age were classified as type I pulmonary agenesis (agenesis).A boy of one year old was diagnosed pulmonary agenesis type III, right lung hypoplasia.Six patients were diagnosed with pulmonary agenesis by Chest CT scan. The clinicians should consider possibility of congenital pulmonary agenesis in dyspneic patients with opaque unilateral hemithorax in plain film. Pulmonary agenesis is an extremely rare congenital entity which can occur unilateral or bilaterally. Almost all cases are unilateral since bilateral agenesis in not compatible with life . UnilateSix patients whom underwent contrast enhanced chest CT scan in radiology department of French Medical Institute for Mothers and children in Kabul were diagnosed lung agenesis during 2015 to 2018.According to Boyden classification; two patients had type I pulmonary agenesis (agenesia) which was confirmed by total absent of unilateral lung, main bronchus and its pulmonary vessels. Two boys of four months and seven months of age with respiratory distress and history of recurrent chest infection showed evidence of right lung agenesis with mediastinal shift and right side position of the heart in both patients Fig. .Fig. 1a Three patients had type II pulmonary agenesis (aplasia) which is characterized by complete absence of unilateral lung and its pulmonary vessels with a small rudimentary blind ended main bronchus. A boy of three months of age with respiratory distress demonstrated right side aplasia Fig. . AnotherType III pulmonary agenesis (hypoplasia) was observed in a one year old boy with mild shortness of breath and opacity in the right upper lung zone. The chest CT images demonstrated partial right lung in the lower zone with displacement of heart in the right upper lung Fig. .Fig. 6CoFor the first time pulmonary agenesis was classified by Schneider which laAs this anomaly can occur at any age, the possibility of lung agenesis should be in differential diagnosis of patients having decrease to absent breath sounds with less or no movement of unilateral chest wall and opaque hemithorax in plain film. For confirmation, diagnostic imaging such as chest CT scan, MRI, bronchoscopy and chest angiography can be done. The early detection of the pulmonary agenesis is essential to reduce the development of fibrosis in patient\u2019s unilateral lung which can occur as result of recurrent chest infection. The surgical procedures should also be in consideration in presence of other congenital anomalies or complications."} +{"text": "Somphot Duangchatrasiri should be Somphot DuangchantrasiriAnak Pattanvibool should be Anak PattanaviboolIn \u201cImpact of prey occupancy and other ecological and anthropogenic factors on tiger distribution in Thailand's western forest complex,\u201d which was published in issue 5, March 2019, the surnames of the first and fourth authors were incorrect. The correct information is printed below."} +{"text": "The median (IQR) target attainment of high-dose protein was 75% (66\u201385) versus 94% (87\u201397) on days 1\u201315 for iNPN and scNPN regimens respectively (p < 0.01). The median (IQR) target attainment of standard dose protein was 77% (67\u201385) versus 94% (91\u201396) on days 1\u201315 for iNPN and scNPN regimens, respectively (p < 0.01). This was associated with improved weight gain and head growth . scNPN regimens have better target attainment for parenteral protein intakes than iNPN regimens.Neonatal parenteral nutrition (NPN) regimens that are individualised (iNPN) or standardised concentrated NPN (scNPN) are both currently used in preterm clinical practice. Two recent trials (one iNPN and one scNPN) each compared standard (control) and high (intervention) parenteral protein and energy dosage regimens and provided data about actual protein intake. We hypothesised that scNPN regimens would achieve a higher percentage of the target parenteral protein intake than their corresponding iNPN regimens. We calculated the daily individual target parenteral protein intake and used the daily parenteral protein intake to calculate the target attainment for protein intake in each infant for the two control (iNPN: Neonatal parenteral nutrition (NPN) is an essential element of preterm care. The potential for early nutritional deficits in very preterm infants has been long understood ,2 and prThe conventional NPN strategy has been based on individualised prescription and formulation to address the rapidly changing and variable fluid and electrolyte needs characteristic of the very preterm infant. This has the potential to subvert early nutritional strategy particularly with inexperienced neonatal PN prescribers ,17. CompStandardised versus individualised neonatal PN has been reviewed ,23, inclWhile unintended variation due to NPN prescribing practice has been investigated, little attention has been given to the process of PN administration, which may vary considerably from the original prescription given the time-lag between laboratory results being received and NPN manufactured, dispensed and connected to the patient. Even then, complex and rapidly changing fluid and electrolyte requirements may have altered the preterm infant\u2019s requirements. Drug infusions and fluid restriction can also limit the volume of fluid available for aqueous PN (aqNPN). This contains all protein (as amino acids) and other water-soluble nutrients and maintenance electrolytes. This effect can be reduced by concentrating the aqueous PN into a smaller volume and making up any additional fluid requirement with a supplementary infusion ,39,40,41Standardising and concentrating neonatal PN (scNPN) has the potential to address the problems of suboptimal nutrient administration but randomised controlled trial (RCT) comparisons of scNPN and iNPN are not feasible. Our recent single centre RCT comparing two standardised, concentrated neonatal PN (scNPN) regimens, showed that higher protein and energy intake can improve head growth . We haveThis analysis used data obtained during two previously ethically approved and published RCTs that shared the same single site, similar eligibility criteria and other methodology ,8. The kThe policies were used to calculate a daily target PN protein intake for each infant according to their original treatment allocation. The earlier and faster introduction of scNPN compared with iNPN resulted in much higher target protein intakes for the scNPN groups in the first 5 days of life. Daily parenteral and enteral protein intake data were used to calculate actual daily parenteral and total protein intake for the first 15 days of life. After day 15, the majority of infants in both RCTs were not PN dependent. Both studies shared the same intravenous fluid guidelines and aimeThe collection of growth data is described in the original RCT publications ,8. ChangInitiation and randomisation phase (day 1\u20135).Maximum PN phase (day 6\u201310).Transition to mainly enteral feeds phase (day 11\u201315).To calculate target attainment, the days where maximum aqNPN intake was required were identified for each infant. The actual daily parenteral protein intake for all maximum aqNPN days was divided by the target parenteral protein intake for all maximum aqNPN days to obtain percentage target attainment for the study period (day 1\u201315). This ensured each infant contributed a single data point to their group. The combination of incremental introduction of parenteral and enteral protein means that target attainment may be influenced by postnatal age. To explore this, the study period was divided into three phases:Target attainment (%) was calculated for the phases as above. Infants with no days of maximum aqNPN-dependence during a particular phase were excluded from analysis for that phase.The target attainment calculations were then repeated for all PN days where enteral feeds were less than 75 mL/kg/day rather than PN days where aqNPN was maximal. This provides information about the early transition phase from NPN to enteral feeding. The target protein intake was kept as the maximum parenteral protein intake, but the actual protein intake included both parenteral and enteral sources.t-test for normally distributed dated and Mann\u2013Whitney U tests for non-parametric data (percentage target attainment). Other comparisons used Fisher\u2019s exact test as appropriate.Control iNPN and scNPN groups and intervention iNPN and scNPN groups were compared using an unpaired p = 0.050; control groups only) and head growth .The demographic data for the two RCT have been reported in detail previously ,8. ThereAll 14-day survivors contributed to day 1\u201315 and day 1\u20135 data in the scNPN group but only data from 36 weeks CGA survivors was available in iNPN groups. The target attainment for parenteral protein intake in infants receiving full PN was statThe target attainment for parenteral protein intake in infants still dependent on NPN for the majority of their nutritional needs was statStandardised supplementary electrolyte infusion usage for scNPN groups is shown in This is the first paper to describe the effect of different methods of PN administration on the extent to which targets for parenteral protein (amino acid) administration are attained. It is clear that the scNPN regimen achieves parenteral protein intakes much closer to the intended parenteral protein target intake than the iNPN regimen, even though those target intakes are higher. A median target attainment of 75% (in the iNPN group) indicates that half the infants are \u201closing\u201d >25% prescribed parenteral protein intake when receiving maximum NPN. Not only does this contribute to suboptimal nutrition but it introduces unintended and unpredictable variation in nutritional intake between patients. The principle of concentrating the standardised NPN formulation to prevent this and optimise nutrient intake has been demonstrated. There is limited evidence that growth outcomes may be improved as a result. The reasons for lower target attainment with iNPN regimens are multifactorial but include limited aqNPN availability and the effect of additional drug infusions and fluid restriction. The iNPN regimen did not use computer-aided prescribing or otherThis paper illustrates the strengths of using the principle of target attainment focused on comparing expected versus achieved parenteral nutrient intake. It has the potential to raise national standards for NPN provision by including nutritional targets within neonatal service specifications to drive regular audits of NPN nutritional intakes. Setting regional and national standards are important initial steps to quality improvement . This haSuboptimal target attainment has important implications for patient safety as the efficiency of protein intake is a surrogate measure of all other aqNPN components, including trace elements and maintenance electrolytes and minerals. Thus, electrolyte derangement in the very preterm infant is more likely if half of infants are receiving >25% less maintenance electrolyte than intended as described in this paper for the iNPN regimen. The subsequent unpredictable variation in target electrolyte intake (and so risk of plasma electrolyte derangement) has potential implications for patient safety. The use of standardised supplementary electrolyte/mineral infusions as part of the scNPN regimens allows rapid response to electrolyte/mineral deficiencies without compromising nutrition. The standardised supplementary electrolyte/mineral infusion usage data for the scNPN provides important information about potential additional workload and costs for this approach to correcting electrolyte deficiency. Standardising allows infusions to be premanufactured reducing costs, workload and reducing risks. These data also provide the information to optimise the future scNPN electrolyte/mineral content to meet the needs of the maximum number of infants and so minimise the need for supplementary mineral/electrolyte infusions. Hypokalaemia and hypophosphataemia are associated with increased parenteral AA provision ,46,47,48One of the strengths of this study are that the data were collected as part of two randomised controlled trials in the same centre, using similar, clearly defined fluid, parenteral and enteral nutrition regimens. The two standard protein/energy groups and two high protein/energy groups had very similar prescribed intakes as part of the NPN guideline and the management of PN intolerance (insulin treatment for hyperglycaemia and triglyceride monitoring) was the same. Nevertheless, the compared groups were in different RCT from different epochs (5 years apart) raising the possibility of confounding factors arising from other aspects of clinical management. In addition, the scNPN protocol has a more aggressive approach to introducing and increasing AA than the iNPN regimen. While this has no effect on target attainment, it has clear potential to affect growth outcomes ,52, greaThe findings in this paper are consistent with several studies that have shown benefits for standardised PN formulation ,31,32,33ScNPN regimens improve parenteral protein intake and have the potential to improve patient safety when compared to iNPN regimens by increasing macronutrient target attainment and reducing variability during aqNPN administration."} +{"text": "Influenza vaccination is recommended annually for persons aged \u22656 months for the prevention and control of influenza . Ages weThe VAERS adverse event findings suggest that expired IIV does not pose additional risks for adverse events beyond those of seasonal IIV. Vaccine failure was not assessed. In most reports, factors that contributed to administration of expired vaccine were not specified; however, one cluster of reports from a pharmacy stated that four persons received expired vaccine doses that had been mistakenly shipped from another pharmacy. Seven reports detailed that patients were offered revaccination with the current season\u2019s influenza vaccine; of these, three confirmed revaccination.https://www.cdc.gov/vaccines/hcp/admin/storage/toolkit/index.html) (As a spontaneous reporting surveillance system, VAERS likely captures only a small fraction of expired IIV administered; therefore, this error might be more common than VAERS data indicate. CDC\u2019s Vaccine Storage and Handling Toolkit contains guidance pertaining to prevention of and mitigation of administration of expired vaccines and is available online (Vaccines should be inspected for expiration before they are administered or transported to other facilities. Facility vaccine coordinators need to be aware of the standard expiration date of June 30 for IIV and make plans for the safe disposal or return of any remaining doses of IIV after that date. Sometimes unused vaccine may be returned for credit, even if the doses must be discarded. State immunization programs or vaccine manufacturers should be contacted to determine whether such provisions apply. Any person who receives an expired influenza vaccine should be revaccinated with the current season\u2019s influenza vaccine."} +{"text": "Lesbian, gay, bisexual, and transgender people (LGBT) with advanced illness need culturally competent advanced care planning (ACP) services but often encounter structural and communication barriers. The aim of this study was to examine the ACP behaviors of LGBT people. An integrative rapid review method was used to search electronic databases for peer reviewed and non-peer reviewed publications between 2010 to 2017. Eight survey instruments comprising 30 prevalence estimates were analyzed. ACP discussions between LGBT people and their primary health care providers were rare, with an overall prevalence of 10%. Transgender people were 50\u201370% less likely than their LGB counterparts to have a living will or to have appointed a healthcare proxy. These results suggest there is a critical need for greater cultural competency among health care providers serving LGBT populations. Social workers can play a key role in advocacy and social justice for LGBT individuals with advanced illness."} +{"text": "Previous research has found a negative association between network size and age, suggesting that people experience greater isolation with advancing age. In this paper, we evaluate age differences in how individuals perceive their social worlds to be structured, rather than focusing solely on network size. A nationally represented sample of respondents reported on their own ties to their close personal network members as well as their perceptions of acquaintanceship between those members . We used social network analysis to assess how the structure of these relationships vary by respondent age. We find a positive association between respondent age and personal network size and a negative association between network members\u2019 ages and the number of ties respondents\u2019 perceive their members to have to each other. This effect significantly weakens as respondent age increases. Moreover, we find evidence of age-homophily, intergenerational contact spanning three generations in both ego-alter and alter-alter ties, and age differences in ego network composition. Our results suggest that the evolution of our social worlds across the life course shifts in terms of size and structure. While contemporary close personal networks may grow slightly with age, perceived social ties among one\u2019s network members become less cohesive and less diverse with age. We discuss these results in the context of recent findings that suggest aging uniformly insulates individuals from social contact from both structural and symbolic perspectives."} +{"text": "Anal squamous cell carcinoma (SSC) is a relatively rare malignancy with an increasing incidence over the last years. Chemoradiotherapy (CRT) has replaced surgery as treatment for primary anal SCC and is currently standard of care for primary anal SCC. Treatment with CRT leads to preservation of the anal sphincter and a 5-year survival rate up to 80%. Failure of CRT occurs in 20\u201330% of the patients, resulting in persistent or recurrent anal SCC. The only available treatment option to achieve durable local control and survival for persistent or recurrent anal SCC is salvage abdominoperineal resection (APR).This study confirmed that salvage APR for either persistent or recurrent anal SCC, after failed initial treatment with CRT, can achieve long-term survival and durable local control.The use of salvage APR is well established, but achievement of a higher rate of clear resection margins remains a challenge. Intraoperative radiation therapy could be of value to improve overall survival and local control, but further research is warranted.3The biggest challenge remains systemic treatment of unresectable or metastasized anal SCC. Current systemic chemotherapy schemes often are based on 5-FU and Cisplatin and show poor response and survival rates."} +{"text": "Previous studies have shown that there is a high frequency of antibiotic use in NH for advance dementia patients. However, research has shown limited clinical benefit from antimicrobial use for this population, and antimicrobial exposure increases colonization with drug-resistant bacteria in nursing homes. The aim of this study was to identify NH and resident level characteristics associated with antibiotic use for patients with advance dementia. Using data from an ongoing cluster RCT in 28 Boston NHs; Trial to Reduce Antimicrobial use in Nursing home residents with Alzheimer\u2019s disease and other Dementias (TRAIN-AD), testing a program intervention to improve management of infections in advanced dementia. These data are taken from baseline measurements 2 months prior to intervention, and individual nursing home residents with advance dementia are units of analysis (n = 425). We ran multivariable logistic regression model with antibiotic use as the outcome, adjusting for clustering at NH level, with NH and individual patient characteristics as independent variables. Analyses found residents were more likely to receive antibiotics if they resided in nursing homes that employed less intense infectious disease practices prior to baseline , and full-time nurse practitioners or physician assistants . Female patients also had higher odds of receiving antibiotics . These findings provide potential insight into the importance of education regarding stringent infectious disease practices for practitioners, particularly for patients with advanced dementia."} +{"text": "Older adults attend to more positive than negative content compared to younger adults; this \u201cage-related positivity\u201d effect is often thought of as a way older adults may be regulating their moods. However, attentional disengagement abilities decline with age, which may make positive looking more challenging for older adults in some cases. To evaluate links between early attentional processes and affect, 48 younger adult and 49 older adult participants reported levels of positive and negative affect on the Positive and Negative Affect Scale (PANAS) and completed a spatial cueing task evaluating attentional orienting and disengagement from emotional stimuli. Participants were tasked with responding to the location of a spatial target after seeing a cue that either appeared on the same (orienting) or opposite (disengagement) side of the screen. Multilevel modeling analyses were conducted using age and self-reported affect from the PANAS as predictors at level-2, and trial characteristics as predictors at level-1. Positive affect (PA) was unrelated to task performance for younger adults. Older adults reporting higher PA responded more slowly overall, and higher PA scores predicted similar response times to positive and negative stimuli on both trial types. Older adults reporting lower PA oriented attention more quickly to positive stimuli, but took longer to disengage from negative. These results suggest that there may be a relationship between the ability to flexibly disengage from negative content and PA for older, but not younger adults, and also highlight the importance of teasing apart specific attentional processes when evaluating positivity effects."} +{"text": "NGS) has several advantages over conventional Sanger sequencing for HIV drug resistance (HIVDR) genotyping, including detection and quantitation of low\u2010abundance variants bearing drug resistance mutations (DRMs). However, the high HIV genomic diversity, unprecedented large volume of data, complexity of analysis and potential for error pose significant challenges for data processing. Several NGS analysis pipelines have been developed and used in HIVDR research; however, the absence of uniformity in data processing strategies results in lack of consistency and comparability of outputs from different pipelines. To fill this gap, an international symposium on bioinformatic strategies for NGS\u2010based HIVDR testing was held in February 2018 in Winnipeg, Canada, convening laboratory scientists, bioinformaticians and clinicians involved in four recently developed, publicly available NGS HIVDR pipelines. The goal of this symposium was to establish a consensus on effective bioinformatic strategies for NGS data management and its use for HIVDR reporting.Next\u2010generation sequencing NGS read quality control (QC)/quality assurance (QA); (2) NGS read alignment and reference mapping; (3) HIV variant calling and variant QC; (4) NGS HIVDR reporting; and (5) extended data applications and additional considerations for data management. The consensuses reached among the participants on all major aspects of these blocks are summarized here. They encompass not only recommended data management and analysis strategies, but also detailed bioinformatic approaches that help ensure accuracy of the derived HIVDR analysis outputs for both research and potential clinical use.Essential functionalities of an NGS is being adopted more broadly in HIVDR testing laboratories, data processing is often a bottleneck hindering its generalized application. The proposed standardization of NGS read QC/QA, read alignment and reference mapping, variant calling and QC, HIVDR reporting and relevant data management strategies in this \u201cWinnipeg Consensus\u201d may serve as a starting guideline for NGS HIVDR data processing that informs the refinement of existing pipelines and those yet to be developed. Moreover, the bioinformatic strategies presented here may apply more broadly to NGS data analysis of microbes harbouring significant genomic diversity.While However, NGS also requires well\u2010defined bioinformatics strategies and tools that help to reliably convert raw NGS data into user\u2010interpretable HIVDR results. Notably, with the broad adoption of NGS, the sequencing itself has become relatively less challenging, while the data processing steps have become the primary bottleneck for its generalized application to HIVDR. Such challenges arise largely from: 1) high HIV sequence diversity high HIVDevelopment of such a consensus necessitates knowledge of NGS data characteristics, relevant bioinformatics skill sets, appreciation of the clinical relevance (or lack thereof) of minority variants and, importantly, extensive expertise and experience in performing NGS HIVDR data analysis. In this commentary, we report the outcome of an international symposium on bioinformatic strategies for NGS HIVDR testing, which was held in February 2018 in Winnipeg, Canada, convening bioinformaticians, scientists and clinicians from four NGS HIVDR pipeline teams, including: HyDRA from the National Microbiology Laboratory in Canada, PASeq.org from Institute for AIDS Research (IrsiCaixa) in Spain, MiCall from the British Columbia Centre for Excellence in HIV/AIDS in Canada and hivmmer from the Providence\u2010Boston Center for AIDS Research at Brown University in USA. Notably, HyDRA, PASeq.org and MiCall are freely available web interfaces and are used by many investigators worldwide, while hivmmer and several other pipelines are also freely available but still require advanced computational skills to execute automated data analysis with a short turnaround time; (2) accommodation of all relevant HIV genes and raw data from varied NGS platforms; (3) incorporation of essential quality assurance (QA)/quality control (QC) strategies to ensure data accuracy and reproducibility; (4) production of customizable and easy\u2010to\u2010interpret HIVDR reports that satisfy research, surveillance and clinical monitoring needs; (5) user\u2010friendliness requiring minimal or no bioinformatics experience; and (6) easy access with minimal additional cost to the end\u2010users. The Winnipeg Consensus covers the major bioinformatic strategies that help to satisfy these requirements.Although pipelines vary, some basic principles apply in NGS HIVDR data analysis. The analytic components of an NGS HIVDR pipeline were grouped into five sequential functional blocks: (1) NGS read QC/QA; 2) NGS read alignment and reference mapping; (3) HIV variant calling and variant QC; (4) HIVDR interpretation and reporting; and (5) analysis data management. Table\u00a0 NGS readNGS read QC/QA\u201d warrants that only high\u2010quality NGS reads are to be utilized in downstream HIVDR data analysis. Although all NGS platforms attach quality scores to individual basecalls, the additional NGS read QC/QA steps described in this consensus were deemed both necessary and effective in reducing false variant calling. Only basic read QC/QA strategies are described here and more stringent filtering may be required in certain cases.\u201cNGS read alignment and reference mapping\u201d addresses the needs for valid and accurate read alignment to designated reference sequence(s) that enables subsequent variant calling. Pipelines should at minimum support reference mapping of the whole HIV pol gene, which encodes the three main drug\u2010targeted HIV enzymes: protease (PR), reverse transcriptase (RT) and integrase (IN). Although not urgently required for HIVDR genotyping, it would be beneficial for pipelines to also accommodate full\u2010length HIV reference alignment, since many users are adopting NGS for partial or full\u2010length HIV sequencing beyond the pol gene. Notably, genetic variability in the HIV env gene poses more challenges for reference alignment strategies than the relatively conserved pol gene. Certain insertions and deletions (indels) in HIV\u20101 PR (near codon 35) and RT (near codon 69) genes are associated with drug resistance and such indels should be identified and reported for both HIVDR surveillance and clinical monitoring purposes \u201cHIV variant calling and variant QC\u201d imposes additional stringency on the calling of variants, which is especially important when minority variants are concerned. NGS errors may arise at multiple points during sample processing and NGS data processing \u201cNGS HIVDR interpretation and reporting\u201d is the only component designed specifically for HIVDR application, while all other blocks and associated strategies may find broader application, especially for genomic sequence analysis of microbes harbouring high genomic diversity, similar to HIV. This specific element of the pipeline streamlines the strategies to convert valid NGS\u2010derived amino acid variant data into end\u2010user\u2010interpretable HIVDR results. Two HIVDR report formats are recommended in this Consensus for addressing needs of either research\u2010oriented projects (a comprehensive report) or clinically oriented testing (a concise report). Ultimately, a customizable HIVDR reporting strategy is preferred for an optimal pipeline, allowing the users to construct a report of their preference. To facilitate comparisons and merging of data from different pipelines, a new standard amino acid variant file (aavf) format has been proposed (Appendix\u00a0https://github.com/winhiv/aavf-spec). Based on the variant call format (vcf) standard that has been universally adopted for recording nucleotide variants, the aavf report provides a compact summary of the amino acid variation obtained by conceptual translation of the NGS read pileup across the examined region of the HIV genome. It also contains information on the frequencies of matching codons (wild type or mutant), quality of the variant calling as well as the coverage of relevant loci. Although the specification is designed to fully accommodate the requirements for reporting of NGS\u2010based HIVDR testing, it is still suitably generic to serve as a general purpose file format for reporting amino acid variants for broader applications. A tool suite to parse aavf format is available at https://github.com/winhiv/PyAAVF.\u201cGeneral analysis data management\u201d deals with issues that concern both the data generator and the analysis provider, to protect the best interests of both parties, including formats and contents for data storage, software versioning, information traceability and data ownership policies.\u201cThis symposium was held at a time when NGS for HIVDR genotyping is increasingly being adopted by many laboratories for research, surveillance and clinical monitoring purposes. Although the functionalities and assembly of bioinformatics strategies applied in different pipelines vary, they share a common objective. The Winnipeg Consensus addresses the urgent needs for and starts the process of standardization of NGS HIVDR data analysis pipelines. It is noteworthy that most of the bioinformatics strategies described in the Winnipeg Consensus have already been incorporated in three of the assessed pipelines, which explains the high concordance among these pipelines when the same data sets were analysed An additional important outcome of this symposium was a consensus that a well\u2010characterized NGS HIVDR \u201cdry panel\u201d should be constructed in support of both pipeline development and validation applications. Such a dry panel would consist of a variety of simulated data files as well as empirical data sets derived from plasmids, artificial plasmid mixtures and patient specimens. It should also cover all major HIV\u20101 subtypes and signature DRMs at a wide range of frequencies, allowing the flexibility for end\u2010users to customize panels based on their needs. Such a comprehensive panel is currently under construction by the symposium participant teams and will become freely accessible to the public once established. In fact, a subset of the dry panel has already been used for a comparison of PASeq, HyDRA and MiCall Additional NGS HIVDR assay comparative assessment strategies, such as parallel testing of the same plasma specimens in different laboratories followed by analysis of the raw NGS data from each laboratory using all available pipelines, are also underway. This is in collaboration with the Virology Quality Assurance (VQA) programme supported by the Division of AIDS at the National Institutes of Health, USA, which provides quality assurance support for HIVDR laboratories worldwide It is acknowledged that some limitations exist in the Winnipeg Consensus, including: (1) it only addresses strategic issues concerning NGS data processing and subsequent report accuracy. Errors arising from pre\u2010analytical procedures remain to be minimized through comprehensive protocol validations 3In conclusion, we present here the Winnipeg Consensus on bioinformatic strategies for NGS HIVDR data processing. This consensus may serve as an initial baseline to standardize NGS data analysis with a specific focus on HIVDR genotyping, and inform the refinement of existing pipelines and those still in development. This initiative and its subsequent activities may help make such technologies routine for both research and clinical HIVDR monitoring purposes, and may serve as a useful starting point for further developing of NGS analysis pipelines with similar and alternative intended applications.The authors have no competing interests to declare.HJ, RP, PS, RH, GVD, RK and MNJ conceived and initiated the project. HJ, MNJ, NP, CJB and RK drafted this manuscript. All authors participated in the Winnipeg symposium and contributed to the manuscript revisions. MNJ, EE, CJB, MH, ERL, RC and EM led the discussions on varied bioinformatics issues at the symposium and summarized the consensus on the corresponding topics that were presented here. All authors contributed significantly to this study and have reviewed and approved the final version."} +{"text": "Xanthogranulomatous prostatitis as mimicker of prostate adenocarcinoma can cause a diagnostic dilemma, as presented in this case. Therefore, alongside histopathology analysis, multiparametric magnetic resonance imaging (mpMRI) would be useful in this situation by identifying and characterizing suspicious prostatic lesions before biopsy thereby supporting current recommendations on the use of mpMRI. Xanthogranulomatous prostatitis as mimicker of prostate adenocarcinoma can cause a diagnostic dilemma, as presented in this case. Therefore, alongside histopathology analysis, multiparametric magnetic resonance imaging (mpMRI) would be useful in this situation by identifying and characterizing suspicious prostatic lesions before biopsy thereby supporting current recommendations on the use of mpMRI. It is a very infrequent histopathological entity. The most common xanthogranulomatous inflammations are seen in the kidneys and gallbladder.23 to 9.5\u00a0\u00b5g/L(2018), a repeat prostate biopsy was planned. Digital rectal examination revealed an enlarged firm and nodular prostate (T2c). Transabdominal ultrasound outlined a lobular heterogeneous prostate measuring 90\u00a0cm3Xanthogranulomatous prostatitis is indeed a rare entity and rare mimicker of prostate adenocarcinoma.In this prostatic entity, a large amounts of histiocytes (foamy macrophages) are observed accompanied by multiple plasma cells and lymphocytes. The presence of histiocytes may cause confusion and may be diagnosed as hypernephroid pattern type adenocarcinoma of the prostate.The presence of extensive xanthogranulomatous prostatitis on the repeated biopsy specimens may indicate diffuse changes within the prostate which may involve a significant volume of the gland. Unfortunately, mpMRI facility is not yet available in our institution. The mpMRI often spots areas under\u2010sampled by the systematic transrectal ultrasound guided biopsy and is very helpful in characterizing lesions.Histopathology analysis including immunohistochemistry staining will definitely be helpful in distinguishing xanthoma cells from carcinoma when facing histopathological dilemma as xanthoma cells can appear like high\u2010grade prostatic carcinoma.In the literature, it may be described alone or with another concomitant condition. Xanthogranulomatous prostatitis has been reported associated with prostatic abscess.Management of xanthogranulomatous prostatitis is mainly conservative . Surgical management (TURP or open prostatectomy) is indicated if symptoms are significant and/or failed conservative management.In conclusion, xanthogranulomatous prostatitis as mimicker of prostate adenocarcinoma can cause diagnostic dilemma as presented in this case. However, alongside histopathology analysis, multiparametric magnetic resonance imaging (mpMRI) would be useful in this situation by identifying and characterizing suspicious prostatic lesions prior to biopsy hence supporting current growing recommendations on the use of mpMRI as diagnostic tool.In this particular case scenario, the dilemma may have been resolved if we had mpMRI available in our institution to characterize the lesions and probably aid in diagnosis of either combined pathologies (xanthogranulomatous prostatitis and prostate adenocarcinoma) or only xanthogranulomatous prostatitis with the possibility of the previous histopathology assessment being incorrect.None declared.AMM: involved in substantial contributions to conception and design of the case report; involved in acquisition of data; drafted the manuscript; involved in critical revision for important intellectual content; and approved the final version. SD: involved in critical revision for important intellectual content and approved the final version. RM: involved in substantial contributions to acquisition of data and drafting of part of the manuscript.Written informed consent was obtained from the patient for publication of this manuscript and accompanying pictures. A copie of the written consent is available for review by the Editor\u2010in\u2010Chief of this journal."} +{"text": "While natural communities are assembled by both ecological and evolutionary processes, ecological assembly processes have been studied much more and are rarely compared with evolutionary assembly processes. We address these disparities here by comparing community food webs assembled by simulating introductions of species from regional pools of species and from speciation events. Compared to introductions of trophically dissimilar species assumed to be more typical of invasions, introducing species trophically similar to native species assumed to be more typical of sympatric or parapatric speciation events caused fewer extinctions and assembled more empirically realistic networks by introducing more persistent\u00a0species with higher trophic generality, vulnerability, and enduring\u00a0similarity to native species. Such events also increased niche overlap and the persistence of both native and introduced species. Contrary to much competition theory, these findings suggest that evolutionary and other processes that more tightly pack ecological niches contribute more to ecosystem structure and function than previously thought. In light of recent empirical studies, it is now widely recognized that evolution and ecology (and their interplay) affect the response of communities to environmental changes, and that the two processes may happen on similar timescales4. This is especially true when environmental changes occur at large scale or have high amplitude , as selective pressures can then act efficiently to alter natural selection processes and/or species\u2019 coevolution.Historically, prediction and management in ecology have been thought to be constrained mostly by demographic and ecological processes, with evolution playing a much more limited role or happening on much longer time scales6 or on one interaction8. These studies strongly suggest evolution alters the fate of the studied population, which links the individual gene/organism level with population structure but leaves unanswered how such effects might propagate to higher levels of organization such as communities or ecosystems Are food webs assembled through invasions more or less persistent compared to food webs assembled by speciation events? (2) Is the structure of \u201cinvasion\u201d networks more or less similar than \u201cspeciation\u201d networks to empirically observed networks? Based on competitive exclusion principles that assert species are less likely to persist the more they share niches with resident species42, one expects that species introduced via speciation events cause more extinctions and are therefore less persistent than introductions of invaders who share less of their niche space with resident species. However, invasions may cause a larger disturbance than speciation events due to the greater functional difference between invaders and resident species. Therefore, one may expect that \u201cinvasion\u201d webs should be less persistent. Our results support this latter prediction and indicate that the mechanism can be more finely understood by accounting for distribution of vulnerability and generality of the newly introduced type. Our results also show that \u201cspeciation\u201d networks more closely resemble empirical food webs. These findings have important implications for invasion ecology and co-evolution within complex communities22.We use this framework of functional similarities and differences43 with 35 species with low, medium, and high levels of connectance along with three corresponding sets of species whose traits such as trophic niche width enabled them to be introduced and trophically linked to species in each web with minimal methodologically enforced changes to their connectance with the mean maximum similarity of the introduced species. Part of this pattern would seem to emerge from more general and vulnerable species having more interactions which would overlap with species already in the web and therefore correlate well with mean maximum similarity. Conversely, species with low generality and vulnerability would have less potential for overlap and therefore low mean maximum similarity. However, more trophic interactions can also increase differences between introduced and resident species which can counteract correlations between increased numbers of interactions and increased mean maximum similarity. Whichever is the cause of the strong correlations between the number of interactions of introduced species and their maximum similarity to resident species, our results suggest that sharing large numbers of interactions with resident species facilitates rather than inhibits their persistence in the networks and reduces the number of extinctions caused by their persistence. On the other hand, fewer and more unique sets of interactions appears to inhibit introduced species from persisting and increase the number of extinctions caused by their introduction. Additionally, possessing relatively unique sets of interactions appears highly unlikely due to chance alone and instead appears associated with the structure of the resident community rather than introduced species possessing unusual fundamental niche properties. This is illustrated by the many fewer introductions at the invaders\u2019 side of the similarity axis than at the speciated species\u2019 side gradient. In particular, webs dominated by speciation events had a higher proportion of successful introductions and fewer extinctions, which resulted in webs that tended to have more species, higher connectance and higher trophic levels. Compared to the low generality and vulnerability of invading species, species introduced by speciation had higher generality and vulnerability which suggests that speciation introduces more interactions than invasions. Despite the possibility for increased interaction density to increase differences among species, such density creates more trophic overlap and helps explain the high trophic similarity of speciated species of speciation dominated webs. The structure of speciation-dominated webs more closely resembled those of a set of empirical food webs. This suggests that evolutionary dynamics may play a larger role in the structure and function of food webs than previously thought but appear opposite to expectations based on competitive exclusion42, our findings may result from niches that enable species to persist being already occupied in our native webs which restricts successful introductions to be those that join residents of already occupied niches. Conversely, introduced species with different niches than residents are more likely to occupy niches that are dynamically unable to sustain species. Others have suggested that competition can cause convergent evolution to lead to sympatric speciation40 for which there is substantial field evidence58. Another possibility is that the relatively small non- or less overlapping parts of similar species\u2019 niches provide sufficient resources for their sustenance as in pollination networks60. High generality could help maintain this sustenance by enabling species to shift their feeding towards less shared resource species whose identity may vary during dramatic changes that may accompany repeated introductions. This key role of small parts of generalists\u2019 niches with a few strong links and many weak links is consistent with the stabilizing effect of low mean interaction strength of species with many interactions61. While future studies need to better explore these and other potential explanations, our findings add to other findings indicating that sharing much of one\u2019s niche with other species, as one may expect in cases of sympatric and parapatric speciation, does not appear to strongly prevent species from coexisting and dynamically persisting62.Absent potentially overstated difficulties of sharing niches64 and by greatly simplifying and reducing the number of trophic levels in the community\u2019s overall structure. A prominent example is the invasion by Burmese pythons of the Florida Everglades (USA) which substantially altered the abundance distribution of its prey65. Another is the invasion of Australia by cane toads whose toxicity reduced the abundances and reorganised the communities of the toads\u2019 predators66. These observations anecdotally suggest that invasions by species substantially different than native species can greatly alter the local network structure and diversity in nature qualitatively similar to those in the present study.Invasions in our simulations dramatically affect the food web in several ways similar to invasions in nature by causing secondary extinctions that severely reduce the diversity of the native community67 or need several attempts to become an effective invader68. Our results combined with the frequent implicit assumption that phylogenetic distance is strongly related to ecological similarity in general69 and to network similarity in particular71 suggests that invasion is easier for species that are phylogenetically close to one of the local species in agreement with recent data72 used in invasive species prevention schemes73.Beyond such effects on communities, invasions often fail by not persisting within the system as seen where invading species remain at low populations for extended periods75. However, our evolutionary rules differ from these former models by relying on interaction similarity of phenotypes that emerge from evolutionary and ecological processes such as speciation and long range dispersal rather than relying on more explicitly modeled dispersal16 or evolutionary dynamics of traits such as body size75, foraging traits34 or competition based on interaction distributions74. Still, as in our study, these evolutionary events introduce new species whose niches substantially overlap with established species but whose traits such as body size, predators, and prey may slightly differ. While the rules generating new species differ, the basic co-evolutionary structure of these studies share the fundamental components of introduced trait variation and selection based on interaction with all species in the community. The bioenergetic basis of these studies ensure that such variation is retained over multiple generations and, as such, is effectively inherited. Disparate approaches to studying these fundamental components of evolutionary dynamics all lead to at least somewhat empirically realistic structures. This consistency suggests that a key aspect this process, i.e., a large degree of niche similarity between new and native species or morphotypes, is important to assembling realistic networks.Our results showing that networks assembled through speciation events are more similar to empirical networks agrees with other community evolution models that showed that evolutionary dynamics allow realistic network structures to emerge76 which leads to more complex trophic structures. While this stabilizing effect of evolution may not be expected generally in diverse communities, it appears more likely in trophic networks77. Evolution in food webs leading to stable and complex structures has been observed in many different models, relying on very different sets of rules78. Introducing species into bipartite networks of plants and pollinators found more conventionally expected results where increased niche overlap among pollinators led to less persistence of native species79. However, this only occurred when there was an extraordinary difference in the invader\u2019s niche beyond niche overlap; the cost-free ability to feed twice the rate of native pollinators. This ability caused the invaders to extirpate all natives whose niches were a subset of the invader\u2019s niche and greatly reduce the abundance of other natives whose niches only partly overlapped the invader\u2019s niche who survived by shifting their feeding to plants not consumed by the invader. Higher trophic levels may reduce such dramatic effects by preventing competition within lower levels from extirpating species81 and weakening the strong interactions that destabilize complex food webs83. Our analysis advances this latter idea by suggesting that such balance more specifically concerns maintaining high levels of vulnerability and generality between new species and their progenitors.When the network is assembled by speciation events, community robustness increases as evidenced by fewer secondary extinctionsOur finding regarding \u201cspeciation\u201d and \u201cinvasion\u201d networks may be tested against natural systems greatly contrasting in their openness to migration. For very closed networks , species invasion may be quite rare, so that the network may mostly represent the effects of species local adaptation or coevolution. Our \u201cspeciation\u201d results may best match such closed networks in e.g., showing largely stable and complex structures. On the other hand, we expect very open networks may have much more frequent invasions causing extinction cascades as well as having a lower complexity with evolution playing a secondary role compared to invasions.84. New predators may alter defense strategies among native prey as has been seen where mussels evolved thicker shells in response to the invasion by the Asian crab85. Similarly, predators may evolve in response to an invasive prey as predators have to invasive cane toads86. Our use of average similarity of 30 different invaders and the spectrum from invasion- to speciation-dominated webs helps inform such interactions between invasion and speciation by suggesting how webs generated by more interactions between these two types of species introductions are intermediate between webs mostly generated by one type of introduction.We emphasize here our qualitive results largely because they are relatively insensitive to the uncertain boundary between similarity extremes that may distinguish invasion from speciation Fig.\u00a0. However87 and because drawing rates at random allow for infinite combinations. On the other hand, evolutionary dynamics may be trapped in local maxima of fitness which prevents introduced species or morphotypes from differing greatly from resident populations40. This greatly restricts the sampling of the possible parameter space by evolutionary processes. For these reasons, it has been suggested that allowing for a mix of invasions and evolution may be key to understanding community closure22. Our approach may be easily adapted to tackle this type of question by focusing how assembly changes over time rather than more simply comparing average outcomes between beginning and end states.This interplay of invasion and evolutionary processes may help predict future states of ecological networks e.g., whether communities become closed or otherwise resistant to future invasions. Simulated invasions often do not cause such community closure both due to cyclic behaviours58, in our case both in terms of resource and consumer species, the latter of which has received little previous attention. For example, Morlon et al.52 found that species in a region\u2019s 50 lake communities share prey much more often than expected if community composition resulted from randomly sampling all species from all 50 lakes within the region. Such findings taken together suggest that there are strong ecological and evolutionary mechanisms forcing species to fit within a relatively restricted architecture of trophic niches as described by theory such as that formalized by the trophic niche model in a food web a niche value and III (h\u2009=\u20092) responses. The amount that each resource j loses to consumer i is equal to the resource\u2019s density, 101. The last term of Eq.\u00a0i\u2019s biomass to consumer j which is equal to consumer\u2019s consumption rate divided by eji, the efficiency of that consumption.The second term in Eq.\u00a0mentclass2pt{minimS) and connectance values of 0.05, 0.10, or 0.20 for each set. Persistent webs where generated by initializing each species with randomly chosen biomasses between 10\u22122 and 10\u22123 and extirpating species whose abundance decreases below 10\u221230. The first 110 webs within the set parameterized within each connectance value that maintained a species richness of 30 after 2000 time steps were retained for the additional simulations steps. Within each connectance class, the first 100 webs were chosen to be subjected to introductions and the 10 remaining webs served as a source for introductions. We subjected each of the 100 webs to a sequence of 30 introductions by 30 different species randomly chosen from the 300 species within the 10 source webs. The position of each introduced species within the web emerged from following the niche model\u2019s rules relate to the traits of species residing in the web. Body masses of species and other parameters are chosen based on the scaling of body size with trophic level96 where each consumer-resource body size ratio is chosen from a normal distribution with mean of 102 (SD\u2009=\u2009101). Following ref. 93, we assigned B0\u2009=\u20090.5, yij\u2009=\u20098, eij\u2009=\u20090.45 for consumption of basal species, and eij\u2009=\u20090.85 for consumption of non-basal species. Following ref. 47, we assigned initial Sl\u2009=\u2009N1\u2009=\u2009N2\u2009=\u20091, Ki1\u2009=\u2009Ki2\u2009=\u20090.15, D\u2009=\u20090.25, ci1\u2009=\u20091, and ci2\u2009=\u20092.We created three sets of food webs by parameterizing the niche model with 35 species while maintaining the extinction threshold at 10\u221230. We repeated this procedure for each of the 300 persistent webs comprised of the 100 webs at the three levels of connectance.The first of the 30-species sequence was introduced after the initial 2000 time steps required to generate persistent webs at 57 using 10 measures of network structure. One property is simply the number of species within the food web (S). Two other properties are standard measures of food-web trophic interaction richness45: links per species (L/S) also referred to as link density; and directed connectance (C\u2009=\u2009L/S2) which equals the proportion of all possible trophic links that are actually realized. Five more properties indicate the fraction of the following types of species in a food web: top , intermediate , basal species ; cannibals 43; and omnivores 43. Trophic level is calculated as short weighted trophic level (SWTL), a measure of trophic level based on mere presence of links that accurately estimates trophic level based on quantitatively weighted links103. Two additional properties are the standard deviation of mean generality (GenSD) and vulnerability (VulSD) among species which quantify the variabilities of species\u2019 normalized predator and prey counts respectively104.We compared the structure of food webs among those primarily assembled by speciation, primarily assembled by invasion, and 19 well-known empirical food webs43. We classified each invasion sequence as invasion-dominated or speciation-dominated using the mean maximum similarity across all introduction events. We base this classification on the idea that new species generated by sympatric and parapatric speciation would be relatively similar to species already in the web while new species immigrating from the regional species pool would, on average, introduce species more trophically distinct and therefore less trophically similar15. Our use of mean maximum similarity recognizes that there are likely to be speciation and invasion events inconsistent with this pattern. Therefore, we are most interested in trends along the gradient from invasion-dominated to speciation-dominated webs labelled in terms of mean maximum similarity. We focus on several key questions related to differences in assembly dynamics across this gradient. First, is establishment success, which is defined as persistence to t\u2009=\u200910,000, different between speciation-dominated and invasion-dominated webs? Second, is turnover (proportion of extinctions relative to establishments) affected by mean maximum similarity? We were also interested in whether the final structure of the webs is different in webs dominated by invasion versus speciation. To answer this question, we focused on final species richness, final connectance, and final short-weighted trophic level. Differences between invasion and speciation dominated webs could be due to a number of differences in properties of the introduced species. In particular, we were interested in the relationship between average niche model parameters including ni, ci, and ri and mean maximum similarity within the web. We also determined the relationship between mean maximum similarity and average invader vulnerability and generality. Finally, we also examined these food\u00a0web properties to determine whether food web properties of webs dominated by invasion versus speciation differed from structural properties of 19 empirical food webs.We calculated 17 different measures of niche similarity between each introduced species and native species including similarity measured in terms of the number and fraction of shared consumer species and shared resource species between all native and introduced species and between all native and introduced species with which an introduced species shared one link. None of the 16 measures explained more variability of our results than \u201cmaximum similarity\u201d measured as the similarity between the introduced species and the species in the web with which it shared the highest fraction of both predators and preySupplementary Figure S1"} +{"text": "Health administrative databases can be used to quantify prevalence and incidence of neurodegenerative diseases and their impact on health service utilization outcomes at the population level. Algorithms based on diagnosis codes and health service patterns can be used to identify persons suspected to have a neurodegenerative disease. Previous studies have developed and validated algorithms to identify persons with Alzheimer\u2019s and related dementias using primary care medical records as the reference standard, however, little previous work has focused on developing algorithms for rare neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). This session will discuss challenges in developing algorithms to identify persons with neurodegenerative diseases accurately and opportunities to improve existing definitions using novel data sources including electronic medical record databases. Preliminary findings regarding the development of an ALS algorithm will be presented."} +{"text": "Optic perineuritis is a rare manifestation of herpes zoster ophthalmicus (HZO). Relative afferent pupillary defect (RAPD) is an important early clue to an impending nerve involvement, and robust clinical examination allows early detection of such rare metachronous manifestation of cutaneous HZ and institution of timely management for such sight\u2010threatening conditions. Optic perineuritis is a rare manifestation of herpes zoster ophthalmicus (HZO). Relative afferent pupillary defect (RAPD) is an important early clue to an impending nerve involvement, and robust clinical examination allows early detection of such rare metachronous manifestation of cutaneous HZ and institution of timely management for such sight\u2010threatening conditions. A 55\u2010year\u2010old woman presented acutely with monocular pain, blurring, and double vision in the right eye. Physical examination was remarkable for deviation of the right eye towards midline, and she was unable to abduct it laterally Figure . Other vThe neuro\u2010ophthalmological symptoms were preceded by vesicular eruption over her eye/forehead Figure and ipsiNone declared.MAA: wrote the manuscript; MM and PC: cowrote the manuscript; MBA: provided clinical care and supervised the study. All authors performed a critical revision of the manuscript and approved the final version of the manuscript.Informed consent was obtained from patient prior to publication."} +{"text": "Vital biological processes such as gene transcription and cell division may be severely impaired by inevitable entanglements ensuing from the extreme length and confinement of the genome. The family of topoisomerase proteins has independently evolved in different organisms to resolve these topological problems, yet no existing model can explain how topoisomerase alone can reduce the topological complexity of DNA in vivo. We propose that a synergistic mechanism between topoisomerase and a family of slip-link\u2013like proteins called structural maintenance of chromosomes (SMC) can provide a pathway to systematically resolve topological entanglements even under physiological crowding and confinement. Given the ubiquity of topoisomerase and SMC, we argue that the uncovered mechanism is at work throughout the cell cycle and across different organisms. Topological entanglements severely interfere with important biological processes. For this reason, genomes must be kept unknotted and unlinked during most of a cell cycle. Type II topoisomerase (TopoII) enzymes play an important role in this process but the precise mechanisms yielding systematic disentanglement of DNA in vivo are not clear. Here we report computational evidence that structural-maintenance-of-chromosomes (SMC) proteins\u2014such as cohesins and condensins\u2014can cooperate with TopoII to establish a synergistic mechanism to resolve topological entanglements. SMC-driven loop extrusion (or diffusion) induces the spatial localization of essential crossings, in turn catalyzing the simplification of knots and links by TopoII enzymes even in crowded and confined conditions. The mechanism we uncover is universal in that it does not qualitatively depend on the specific substrate, whether DNA or chromatin, or on SMC processivity; we thus argue that this synergy may be at work across organisms and throughout the cell cycle. Genomes are long polymers stored in extremely crowded and confined environments; the ensuing inevitable entanglements are thought to cause DNA damage, interfere with gene transcription and DNA replication, and interrupt anaphase, eventually leading to cell death \u20133. In vi5While it has been suggested that DNA supercoiling may provide a solution for this problem by promoting hooked DNA juxtapositions \u201316, thisHere we propose a mechanism for efficient topological simplification in DNA and chromatin in vivo that is based on the synergistic action of structural-maintenance-of-chromosomes (SMC)-driven loop extrusion \u201321 ? A possSI Appendix). A publicly available code (We use a well-established bead-spring polymer model to descrble code is used SI Appendix). The simulations are performed in LAMMPS (Each bead in our simulation is evolved through the Langevin equation n LAMMPS with m=\u03b3After the present paper was submitted for publication, we learned of a similar model simultaneously developed by Racko et al. .Supplementary FileSupplementary FileSupplementary FileSupplementary FileSupplementary FileSupplementary FileSupplementary FileSupplementary File"} +{"text": "The Late Palaeozoic insect superorder Palaeodictyopterida exhibits a remarkable disparity of larval ecomorphotypes, enabling these animals to occupy diverse ecological niches. The widely accepted hypothesis presumed that their immature stages only occupied terrestrial habitats, although authors more than a century ago hypothesized they had specializations for amphibious or even aquatic life histories. Here, we show that different species had a disparity of semiaquatic or aquatic specializations in larvae and even the supposed retention of abdominal tracheal gills by some adults. While a majority of mature larvae in Palaeodictyoptera lack unambiguous lateral tracheal gills, some recently discovered early instars had terminal appendages with prominent lateral lamellae like in living damselflies, allowing support in locomotion along with respiratory function. These results demonstrate that some species of Palaeodictyopterida had aquatic or semiaquatic larvae during at least a brief period of their post-embryonic development. The retention of functional gills or gill sockets by adults indicates their amphibious lifestyle and habitats tightly connected with a water environment as is analogously known for some modern Ephemeroptera or Plecoptera. Our study refutes an entirely terrestrial lifestyle for all representatives of the early diverging pterygote group of Palaeodictyopterida, a greatly varied and diverse lineage which probably encompassed many different biologies and life histories. The evaluation of available data from the morphology of some larval stages as well as adults from a few species among Palaeodictyoptera and Megasecoptera reveals direct and indirect evidence for amphibious or possibly aquatic lifestyles in certain taxa. This can be determined from different aspects of external morphology as mainly the presence of caudal tracheal gills in early larval instars and most importantly retention of rudimentary or functional tracheal abdominal gills by adults. We, therefore, presume that at least these genera were amphibious or aquatic in early larval stages, transitioning possibly into a semiaquatic mode in mature larvae (much like petalurid dragonfly larvae (e.g. nditions ,56, and"} +{"text": "Aging is associated with normative declines in cognitive resources that increase the costs associated with mobilizing resources in cognitively demanding activities. Selective Engagement Theory hypothesizes that changes in costs influence the motivation to engage in such activities in everyday life. We used an economic discounting task to examine the relationship between both objective estimates (systolic blood pressure responses) and subjective estimates (NASA Task Load Index) of cognitive costs in 78 older adults\u2019 (ages 64-85) decisions to engage in more or less demanding activities. Perceptions of costs were meaningfully tied to actual costs (SBP), but further influenced by personal or primed attitudes about aging. Interestingly, decisions to engage in demanding activities\u2014as reflected in discounting decisions\u2014were less influenced by effort expenditure in the activity than by perceptions of difficulty. These results underscore the role that negative stereotypes play in undermining motivation to engage in potentially beneficial activities."} +{"text": "Triple negative breast cancer (TNBC) is distinguished from other breast cancer subtypes by its lack of therapeutic targets, aggressive biology, and poorest survival rates. In the early breast cancer setting, cytotoxic chemotherapy remains the mainstay of systemic treatment options. Multiple clinical trials and meta-analyses have demonstrated that patients who achieve a pathological complete response (pCR) to neoadjuvant chemotherapy (NAC) have an excellent prognosis . TherefoWe rationalised that the most clinically relevant heterogeneity exists in patients with tumors that do not achieve a pCR, and that biomarkers that refine prognostic estimates and improve biological understanding in this population might help guide the development of hypothesis-driven clinical trials in the future. We recently reported results from our analysis that combined four data series of patients with TNBC who received NAC and did not achieve pCR, with a key focus on two highly prognostic biomarkers \u2013 residual disease TILs, and residual cancer burden (RCB) . This edOur study demonstrated that both RCB and residual disease TILs are powerful prognostic markers that can further refine prognostic estimates in this population. The best survival outcomes were observed for patients with minimal residual disease burden (RCB class I). Other studies have demonstrated that patients achieving RCB class I after NAC have a similar prognosis to those who achieve pCR , 7. Conc+ T cells [On the other hand, the worst disease outcomes were observed for patients with extensive residual disease burden (RCB class III), with a 5 years OS estimate of 31% (95% CI 24\u201340). The majority of these patients 82%) experienced disease recurrence, of which 72% had a recurrence which occurred early (within 12 months of definitive surgery). In our cohort, the median OS (from the time of recurrence) for patients who developed an early recurrence in our cohort (irrespective of RCB class) was a dismal 6.1 months (95% CI 5.2\u20138.8). This highlights an urgent need for further research into these poor prognosis patients who are essentially chemo-refractory. Notably, the efficacy of PD(L)-1 blockade in this population remains unclear as patients with an early recurrence were not eligible for the recently published IMpassion130 study [2% experi T cells . FurtherThe positive prognostic influence of residual disease TILs was greatest in patients with moderate residual disease burden (RCB class II), whereby prognosis could be stratified to outcomes similar to that of RCB class I and RCB class III depending on the quantity of residual disease TILs. The significant prognostic influence of residual disease TILs, and the heterogeneity in disease outcomes in this setting suggests that adjuvant immunotherapies, such as with PD(L)-1 blockade with chemotherapy could potentially result in survival gains for these patients. Further research into the tumor intrinsic mechanisms underpinning the presence or absence of residual disease TILs in RCB class II, as well as the immune checkpoint molecules that are frequently expressed on T cells, will further help refine therapeutic strategies for these patients.Beyond improved prognostic stratification, we believe that there is a need to find a more refined biomarker surrogate for patient survival than pCR alone. Therapeutic strategies that have led to significantly increased pCR rates to neoadjuvant treatments have not uniformly lead to significant survival benefits for patients . We ratiIn summary, we believe our data can be used to improve prognostic stratification, help refine target populations for research, and contribute to the design of future neoadjuvant clinical trials."} +{"text": "It has become apparent that the molecular and biochemical integrity of interactive families, genera, and species of human gut microflora is critically linked to maintaining complex metabolic and behavioral processes mediated by peripheral organ systems and central nervous system neuronal groupings. Relatively recent studies have established intrinsic ratios of enterotypes contained within the human microbiome across demographic subpopulations and have empirically linked significant alterations in the expression of bacterial enterotypes with the initiation and persistence of several major metabolic and psychiatric disorders. Accordingly, the goal of our review is to highlight potential thematic/functional linkages of pathophysiological alterations in gut microbiota and bidirectional gut\u2013brain signaling pathways with special emphasis on the potential roles of gut dysbiosis on the pathophysiology of psychiatric illnesses. We provide critical discussion of putative thematic linkages of Parkinson\u2019s disease (PD) data sets to similar pathophysiological events as potential causative factors in the development and persistence of diverse psychiatric illnesses. Finally, we include a concise review of preclinical paradigms that involve immunologically\u2013induced GI deficits and dysbiosis of maternal microflora that are functionally linked to impaired neurodevelopmental processes leading to affective behavioral syndromes in the offspring. A key goal of biomedical research is to identify a common set of causative factors that is directly linked to pathophysiological changes observed in major neurodegenerative diseases afflicting human populations. A hallmark example identifies the major etiological factor in Parkinson\u2019s disease (PD) as a relatively slow temporal loss of striatal dopaminergic (DA-ergic) transmission, resulting from a degeneration of neuronal somata located in the substantia nigra cells, that partially mediate homeostasis of innate immunity is a multifunctional biogenic amine with neuronal and endocrine signaling roles in a range of GI physiological pathways , Tph1 and Tph2, respectively . Intraluminal GABA production and release by commensal lactobacilli provides an essential protective mechanism against infective bacteria via GDAR inhibition. In effect, selective strains of commensal microbiota may differentially influence ENS activity via diverse GABA-ergic mechanisms (Hyland and Cryan Gamma-aminobutyric acid (GABA) expression within the gut mediates critical regulatory activities between the ENS and lymphoid tissues involved in immune competence via T cell responses , resulting in enhanced inflammatory Th1/Th17 responses that are reciprocally linked to severe dysbiosis of commensal microbiota situated at the apical surface of adjacent epithelial cells within the lumen of the gut . Prefrontal cortical and hippocampal regions are densely innervated by rostral noradrenergic projections from LC neurons and have been established to mediate complex cognitive behaviors that are effectively compromised in neurodegenerative and psychiatric diseases (Borodovitsyna et al. Several lines of preclinical and clinical evidence have made strong case for the involvement of dysregulated GI function with associated colonic inflammation and dysbiosis of gut microbiota in the etiology and progression of AfSD and related psychiatric disorders (Diaz Heijtz et al. C. bolteae, consistent with chronic dysbiosis of the microbiome (Shaw In light of these findings, analysis of fecal samples from children with autistic symptoms revealed augmented levels of three Clostridium clusters and one Clostridium species, ome Shaw . High coome Shaw . In thisome Shaw . Furtherome Shaw . From a ome Shaw . Dysbiosome Shaw . Relativome Shaw and fecaome Shaw on reducFrom a neurodevelopmental perspective, a recent preclinical rodent study has demonstrated that trans-placental exposure of the fetal brain to circulating bacterial peptidoglycan released from the maternal gut microbiome mediates cognitive and behavioral disorders in the offspring (Humann et al. Finally, dysbiosis of essential strains of gut microflora engendered by dysregulation of biogenic amine signaling pathways may negatively affect essential gut\u2013CNS communication processes at multiple physiological check points that modulate mitochondrial bioenergetics (Petra et al. Operationally, stereo-selective conformational matching between coupled physiological processes of bacterial microflora and intrinsic GI cells appear to support the conservation of a critically important set of chemical messengers required for existential regulation of homeostatic cellular processes within GI and CNS tissues (Stefano et al."} +{"text": "Critical Care Ankawi and coworkers nicely summarized the potpourri of extracorporeal techniques in septic patients ranging from (ultra) high-volume hemofiltration to modern adsorption devices and a combination of them [Septic patients do not usually die from their infection per se but rather from an overwhelming pathological host response to it. Given that treatment is limited to resuscitation strategies, anti-infectives, and source control , it is o of them . We enjo. A recent meta-analysis identified four randomized controlled trials and found an association with reduced mortality in adults [The theoretical rationale for TPE goes beyond the simple (surely important) elimination of circulating injurious molecules. The exchange of septic with healthy plasma might also replace consumed protective factors that are of importance to maintain microcirculatory flow and counterbalance vascular leak . About 2000 reports\u2014mostly case reports or series\u2014on TPE in sepsis have been published over the last 20\u2009yearsOur own group has just released prospective pilot data on feasibility, safety, and secondary efficacy endpoints in early and severe septic shock patients . Based oFigure"} +{"text": "Valve thrombosis \u2013 either biological or mechanical \u2013 is proved to increase patient\u2019s morbidity and mortality. No consensus exist on the best management in such cases.We report the case of a 69-year-old man presenting with a late thrombosis of a transcatheter aortic valve who was medically managed until he acutely worsened, developing myocardial ischemia and cardiogenic shock.This unlucky case raises a word of caution about the safety of a reactive management. Prosthetic valve thrombosis is known to carry relevant morbidity and mortality. Little is known about its occurrence after transcatheter aortic valve replacement (TAVR); all the information we have are derived from case reports and relatively small series of patients .The current postoperative management suggests dual antiplatelet therapy (DAPT) to reduce the risk of valve thrombosis, even if more and more evidences reveal the potential superiority of oral anticoagulation A more sA 69-year-old man was referred to our Emergency Department for resting dyspnea after 2 months progressive shortness of breath, 2 years after transcatheter aortic valve replacement (TAVR).St. Jude, MN, USA) for native valve endocarditis. Eleven years later (2015) he was re-operated for structural valve deterioration. In that occasion \u2013 due to the presence of extreme calcification of the aortic annulus and root \u2013 we had to replace the prosthesis with a 23\u2009mm Edwards Sapien 3 transcatheter valve under direct view, as previously described [In 1994 he underwent aortic valve replacement with a 23\u2009mm Biocor\u2122 valve . He took Aspirin 100\u2009mg/day with a good compliance. He had no history of hyper-coagulation state or previous documented thrombosis.2 of valvular area) and ipo-echogenic images evocative of intra-valvular thrombosis. A thoracic computed tomography (CT) confirmed the presence of valvular thrombosis accounts for any thrombus formation attached to or near a prosthetic valve that is not caused by infection and determining some degree of valve obstruction and dysfunction and that carries significant morbidity and mortality to the patient.Some degree of reduced leaflet motion is a detected in 10 to 15% of TAVR patients and generally resolves after anticoagulation therapy. AlthoughNo consensus exists regarding the best treatment option once the thrombus has developed.In this case the biggest challenge was balancing a potential life\u2013threatening condition caused by valve thrombosis and its initial dysfunction and the extremely elevated surgical risk due to the third cardiac operation after surgical implantation of a transcatheter valve due to porcelain aortic root and annulus. Thus, the first-line we choose was trying to manage the patient conservatively with systemic heparinization and close clinical and instrumental monitoring.In this case the localized aggressive tumor and the relevant postoperative bleeding may have impaired the hemostatic status of the patient, shifting it toward a pro-coagulant one.We speculate that the transcatheter valve forced against the stiff aortic ring of the previous transcatheter valve and the rigidity and narrowing of the degenerated homograft has caused a progressive thrombosis occurred within the stented portion of the valve towards the leaflets once the hemostasis perturbation occurred leading to the catastrophic consequence.From this unlucky case a word of caution arises about the medical management of such TAVR population; although a single case report does not provide wider conclusion we believe that a very close monitoring of the clinical ad morphologic valve features are mandatory while the surgical option still remains valid once an early evidence of no improvement after adequate anticoagulation therapy."} +{"text": "Providing plausible mechanisms to explain variation in the honesty of information communicated through offspring begging signals is fundamental to our understanding of parent\u2013offspring conflict and the evolution of family life. A recently published research article used comparative analyses to investigate two long\u2010standing hypotheses that may explain the evolution of begging behavior. The results suggested that direct competition between offspring for parental resources decreases begging honesty, whereas indirect, kin\u2010selected benefits gained through saving parental resources for the production of future siblings increase begging honesty. However, we feel that evidence for a role of kin selection in this context is still missing. We present a combination of arguments and empirical tests to outline alternative sources of interspecific variation in offspring begging levels and discuss avenues for further research that can bring us closer to a complete understanding of the evolution of offspring signaling. Across a diverse range of taxa, offspring direct behaviorally complex begging displays toward caregiving parents. The function and evolution of such behavior has intrigued biologists for decades, spawning a myriad of different explanatory hypotheses that make diverse assumptions about the balance of power between parents and their offspring may favor offspring who adopt strategies that facilitate the production of future siblings. Producing honest signals about current nutritional state to preserve parental resources for future broods Trivers is one pAlthough we agree that the death of one parent indeed reduces future indirect benefits, it is incorrect to assume the same for divorce, and we therefore question whether the conclusion that kin selection underlies begging honesty is correct. As demonstrated in Figure http://www.birdtree.org .Having established that parental divorce may not necessarily reduce the kin\u2010selected incentives of current offspring to beg honestly, we retested the hypothesis that high inclusive fitness benefits of future offspring select for honest signaling, using an identical set of species and the same sample size as Caro et\u00a0al. . To provAs we outline above and in Figure (1) Pair bond duration is associated with clutch size and offspring competitionAs outlined above, scramble competition for limited parental resources may be an important mechanism that decreases signal honesty ] produce broods of smaller size Parental divorce is linked to social mate competitionIf offspring begging honesty is related to competition for limited resources, species where sibling competition is more intense are expected to be less honest. Because individuals with relatively short lifespans and hence short pair bonds produce a large number of offspring in each reproductive attempt (Charnov and Krebs (3) Pair bond duration is associated with increased sexual conflict over careAmong bird species, parental divorce rates are linked to extra\u2010pair paternity (Cezilly and Nager A key principle of life\u2010history theory is that parents trade\u2010off investment in current offspring with investment in future offspring Stearns . This trInterspecific variation in honesty of begging signals is an important source of information to make inferences about how selection acts according to social and ecological circumstances. The frequently hypothesized role of kin selection in mediating parent\u2013offspring conflict Trivers , and thuAssociate Editor: A. GardnerHandling Editor: A. Gardner"} +{"text": "Dear Editor,presented a well conducted retrospective study including eighty-nine patients underwentpost-infarction left ventricular aneurysm repair and myocardial revascularizationperformed between 1996 and 2016. Ventricular reconstruction was performed usingendoventricular circular patch plasty (Dor procedure) or linear repairtechnique . In concordance with several published experiences, theyconcluded that the results of their study demonstrate that post-infarction leftventricular aneurysm repair can be performed with both techniques with acceptablesurgical risk and with satisfactory hemodynamic improvement. I guess that thisconclusion would cause a dangerous concept that both operative technique would be usedindependent of the aneurysm size.Kaya et al..Based on this concept we proposed a surgical variant technique to repair leftventricular aneurysms Jatene geometric reconstruction with semi-rigid bovine pericardial prosthesis, and 5)Attempts to compare different techniques without definitive proof of superiority amongthem. However, from safety and reduction of surgical time, the \"no patch\" surgicalvariants techniques would be useful for the decision whether to operate left ventricularaneurysm or akinesia,5.It is relevant to mention that there are, beside experiences around the world, convincingexperiences for ventricular reconstruction: 1) Direct suture; 2) Modification of theCooley technique with patch suture; 3) Dor patch plasty with septalexclusion pointed that \"the decision on which technique to use inthe repair was based on the size of the aneurysm during surgery and the extent of thescar tissue. In the case of smaller lesions without a marked aneurysmal sac, linearrepair was preferred, whereas endoaneurysmorrhaphy was performed in case of largerlesions with a marked neck and fibrotic sac\". This opportune observation perse is a clear introduction of considerable bias in comparative studies thatneed a great number of patients.Doctor Kaya et al."} +{"text": "OBJECTIVES/SPECIFIC AIMS: Exosomes are living nanoscale vesicles that can shuttle large amounts of bioactive cargo for intercellular communication. The potential of these nanovesicles to serve as both biomarkers for disease diagnosis and vehicles for delivery of therapeutics has only begun to be explored. To realize these potentials, molecular tools for effective exosome tracking and capturing must be invented in order to advance basic research and clinical translation. METHODS/STUDY POPULATION: We utilize a surface display strategy that enables exosome modification in living mammalian systems. By reconfiguring the surface protein CD63 or viral envelope glycoprotein VSV-G, we generate 3 topologically distinctive protein chimeras for exosome imaging and capture in mammalian systems. RESULTS/ANTICIPATED RESULTS: We have shown that these genetically encoded protein chimeras have the ability to correctly target and integrate into exosomes in cultured human cells. Furthermore, we have demonstrated that the secreted exosomes could be successfully captured by an affinity peptide intentionally displayed on the outer surface of exosomes. DISCUSSION/SIGNIFICANCE OF IMPACT: Our study highlights the potential of these fusion proteins for exosome tracking and provides novel genetic tools for exosome research and translation, one of which is loading protein therapeutics for targeted delivery."} +{"text": "Clinical trials following ethical guidelines that cater to the health needs of people living in LMICs are needed. Significant investment is required in research infrastructure and research-based higher education centers. Governments need to implement changes that reduce approval times and speed regulatory processes to attract more funding for clinical trials."} +{"text": "Providing training to assist employees with excelling in their job role may increase their job effectiveness, which then translates to improvements in an organizations\u2019 performance measures. In 2018, the Geriatric Scholars Psychology Program measured whether a multi-day course influenced the Psychologists\u2019 perceived job effectiveness using modified questions from Godat and Brigham\u2019s Reaction Measure. Ninety-two percent agreed the training assisted them with identifying and overcoming obstacles; helped them set goals to increase problem solving at their designated facilities; boosted their confidence in fulfilling their job duties; and the training should be made available to other employees who provide care to older Veterans on a regular basis. Findings from the 3-month follow-up assessment demonstrated that the positive effect on job effectiveness was sustained. Discussion will explore aspects of the course that may be key in improving perceived effectiveness and consider novel approaches to enhance this outcome."} +{"text": "The Clinical Practice Guidelines for Dementia in Australia provide evidence-based recommendations for the assessment, diagnosis, and care of people with dementia and their informal carers. The extent to which current Australian post-diagnosis care reflects these recommendations is not well understood. This brief report provides a snapshot of current practice related to three key recommendations from the Guidelines: occupational therapy, exercise, and informal carer support.n\u2009=\u20093) and allied health clinicians (n\u2009=\u200929) provided data about 1114 consultations with people with dementia and/or informal carers over a 9-month study period. Results showed that delivery of evidence-based dementia care remains a significant challenge in Australia. Clinicians found it difficult to tailor exercise interventions to overcome cognitive and organisational barriers to adherence during and between consultations. Occupational therapists primarily focussed on functional assessment rather than on delivering evidence-based interventions. Clinicians also found it difficult to identify and address the array of needs reported by informal carers, especially when the person with dementia is present during the consultation. Though these results are reported by a selected sample, they emphasise the need for innovative knowledge translation strategies to facilitate widespread quality improvement in post-diagnosis dementia care.Nursing Post-diagnosis care for people with dementia in Australia has been criticised for being insufficiently available, fractured, and focussed on managing impairments rather than promoting wellbeing , 2. RatePeople with dementia living in the community should be offered occupational therapy (reflecting evidence-based programs).People with dementia should be strongly encouraged to exercise.Carers and family of people with dementia should have access to programs that provide respite and support to optimise their ability to provide care for the person with dementia.Consistent with this, the Clinical Practice Guidelines for Dementia in Australia were deThe extent to which current Australian post-diagnosis care reflects these recommendations is not well understood. Some evidence suggests that Australian occupational therapists working with people with dementia prioritise home safety assessment at the expense of intervention . HoweverAs part of a larger implementation research project, described in detail elsewhere , we collThe aim of the translational research project from which these data are taken is to improve adherence to the three key recommendations from the Guidelines listed above. We have established three Quality Improvement Collaboratives (QICs) in which health professionals complete a structured online education package and develop a unique quality improvement plan for their work site. Clinicians then meet monthly with the other members of their collaborative for shared brainstorming to overcome any barriers to implementing their plan. Data presented here were conducted prior to clinicians participating in the online education package.Each month, participating clinicians complete a checklist about their first 10 consecutive consultations with people with dementia or informal carers (Carer Support collaborative). Ten consecutive consultations were considered an adequate sample of the clinician\u2019s practice each month. Clinicians in all collaboratives were asked to report the date of birth and gender of the client, the nature of the consultation , a brief summary of the consultation, whether a written treatment plan was provided, and any other resources or referrals provided. Clinicians in the Exercise collaborative additionally reported the activities they recommended, barriers to completion identified, and any strategies used or suggested to overcome these. Clinicians in the Occupational Therapy collaborative reported any assessments completed in the session and treatment strategies suggested. Clinicians in the Carer Support collaborative reported whether the carers\u2019 needs were specifically addressed in the consultation and if so, the nature of these and strategies suggested.Checklists were collated and analysed for themes by one author and checked by a second author. Both qualitative (e.g. the consultation summary) and quantitative data were examined for information about assessments, resources, and referrals provided. This information was grouped into categories descriptively (quantitative data) or using a content analysis approach of distilling words into content-related themes . A quanThirty-eight implementation clinicians joined the QICs and 32 provided data that is included here Table\u00a0. All butFeatures of the consultations are described in Table\u00a0Consultations by clinicians in the Exercise collaborative commonly included one or more recommendations of strength training (55%), balance training (37%), or cardiovascular training (53%). Mobility assessment (61%) was the most common reason for referral and massage was commonly provided (12%). Clinicians identified several barriers to the person with dementia completing physical exercise including insufficient availability of physical assistance (22%), low motivation or behaviour change (22%), cognitive impairment (9%), and inadequate therapist time to explain the activity (3%). Behavioural strategies to overcome these barriers were implemented in 54% of cases. These most often included using a family or paid carer to encourage the person (45%), written or verbal reminders (14%), and modifying the timing or type of activity (10%).Carers seen by clinicians in the Carer Support collaborative were mostly spouses (54%) or children (35%) of people with dementia; the remainder were other relatives or friends. Clinicians were not able to discuss carer needs in 22% of consultations because of sensitivities associated with having this discussion in front of the person with dementia or because there was not enough time. In cases where carer needs could be discussed, topics included carer stress (45%), inadequate support services (26%), difficulty managing challenging behaviours (21%), and the need for better understanding of dementia (8%). Only 22 consultation summaries (8.8%) noted that the carer had no concerns and was managing well. Most consultations by clinicians in the Carer Support collaborative included resource provision, usually information about available services (69%), referral (51%), counselling (35%), problem solving strategies (24%), strategies to manage changed behaviour (20%), and advanced care planning (18%). Referral to respite was made in nine per cent of cases.Consultations reported by clinicians in the Occupational Therapy collaborative were diverse and often included several components in a single consultation. Most common was one or more of assessment of needs (67%), equipment or assistive technology prescription (47%), falls or pressure injury prevention (31%), home modifications (32%), and advocacy . Less common was dementia-specific intervention such as skill building, memory strategies, cognitive stimulation, and identifying and modifying meaningful activities (24% of consultations).Improvements in post-diagnostic dementia care are a policy priority in Australia. The new Aged Care Quality Standards rolled out in July 2019 mandate that aged care providers demonstrate efforts to implement quality improvements into their services . HoweverThe results are consistent with international findings that clinicians find it difficult to overcome the barriers to physical activity for people with dementia . A recenResults also support previous findings that the needs of an informal carer can be difficult to identify where they cannot have a private consultation with a professional . AlthougA 2011 survey of Australian occupational therapists identified that therapists spent most of their time with people with dementia conducting assessment and advising about environmental modification . ResultsFew effective strategies have been identified to bridge the \u2018evidence-practice gap\u2019 in dementia care. The translational research project from which this data was taken aims to assess the utility of QICs in this context. Implementation clinicians are upskilled in quality improvement theory and methods via a structured online education package and expert feedback. They provide mutual support while designing and implementing a bespoke quality improvement plan in their setting . ChangesThere are limitations to this data. This is a selected sample of clinicians who are motivated to implement quality improvements in their practice. They are also self-reporting, so may overstate or overestimate the quality of their practice. However, that even highly performing clinicians experience barriers to best practice highlights the importance of quality improvement efforts in dementia care and the complexity of this type of work. Participating clinicians are also not wholly representative of the dementia care workforce in Australia. Broadly examining current practices with an unselected sample will be important to better understand the key gaps in practice. Finally, the data presented do not provide insight into the acceptability and uptake of support options provided to the service users. Service uptake is known to be low among people with dementia , 23 but"} +{"text": "Among radiation induced arterial complications, stenoses and occlusions are commonly reported. Radiation induced pseudoaneurysms\u00a0(PSA) and their management outcomes are rarely reported.A 48\u2009year old male underwent low anterior resection surgery for a\u00a0clinically staged\u00a0T2N0M0 rectal adenocarcinoma and adjuvant chemoradiation for the findings of lymphovascular invasion and focally positive distal margin 2 years prior to current admission. The patient now presented with syncope and anemia. The patient was hypotensive after an episode of hematochezia during the hospital stay. An urgent sigmoidoscopy revealed bleeding from friable necrotic rectal mucosa with focal pulsations along the left posterolateral aspect of the\u00a0rectal wall. An emergent pelvic angiogram revealed active extravasation from a 3\u2009mm PSA from the anterior division of left internal iliac artery. After coil embolization of the affected vascular branch on either side of the neck of PSA, there was no opacification of PSA or extravasation. The patient remained asymptomatic for 3 years.Radiation induced PSA must be considered in the absence of trauma. Endovascular coil-embolization of radiation induced PSAs from small caliber vessels can be an effective treatment. Among radiation induced vascular complications, arterial stenoses and occlusions are more commonly reported, however radiation induced pseudoaneurysms (PSA) are extremely rare and are scarcely reported showed persistent extravasation from a 3\u2009mm PSA from the branch of an anterior division of LIIA Fig. . In ordeAmong radiation induced vascular complications, arterial stenoses and occlusions are more commonly reported than perforations or PSAs (De Baere et al., There is paucity of data correlating radiation dose with vessel PSA formation. A prior case report (De Baere et al., Pre-operative radiotherapy is the preferred sequencing for patients with locally advanced rectal cancer compared to post-operative radiation. A prospective randomized trial has indicated that the adjuvant radiation therapy was associated with a higher incidence of radiation toxicity including anastomotic leakage and wound complications when compared to neoadjuvant radiation therapy. The study did not report or compare any radiation induced vascular complications (Sprenger et al., Endovascular approach has been preferred over the open surgical approach in the management of iatrogenic PSAs as it obviates the need for surgical incision and wide dissection in the vicinity of the injured vessel. Also in patients with pelvic cancers, access to iliac vessels is often difficult due to prior surgery or radiation. In the prior case reports, the management of bleeding radiation induced pelvic PSAs included 10\u2009mm covered stent placement in the common iliac artery PSA with only 9\u2009month follow up, temporary balloon occlusion of a PSA arising from the iliac bifurcation with subsequent surgical ligation and axillo-femoral bypass, bare tungsten coil packing of an external iliac artery PSA with subsequent recurrent hemorrhage which was later treated by external iliac occlusion and femoro-femoral bypass (De Baere et al., In our patient a 3\u2009mm PSA was arising from an approximately 3\u2009mm diameter branch of an anterior division of left internal iliac artery, most likely a middle rectal artery. Considering the small size and tortuosity of this affected end branch vessel, coil embolization to isolate the PSA sac was preferred. Coil embolization of PSA sac itself was avoided to prevent possible increase in rupture and bleeding from the PSA sac. For PSA arising from vessels with large caliber, covered stents can be used. Thus radiation induced PSA must be included in the differential diagnosis in the absence of trauma. Endovascular coil embolization of radiation induced PSAs arising from the small caliber pelvic vessels can serve as an effective treatment."} +{"text": "With interest we read the article by Pappa et al. about a 42yo male with late onset Pompe disease (LOPD), initially manifesting with progressive, proximal muscle weakness of the lower limbs and later with dilative arteriopathy of the renal artery . The folWe do not agree with the description of the abnormal MRA as fibromuscular dysplasia . Since sWe also do not agree with the notion that there is a typical gait of LOPD patients . WaddlinLOPD may also manifest in the myocardium as hypertrophic/dilative cardiomyopathy . Was dysThe patient received enzyme-replacement therapy (ERT) and improved within 2y of treatment with regard to the outcome parameter walking distance. Did ERT also exhibit a beneficial effect on dilative arteriopathy?The patient developed intracerebral bleeding under a double therapy with an antithrombotic agent and oral anticoagulation. Which compounds were actually given?Overall, the case could be more meaningful if arterial biopsy would have been taken, if the effect of ERT on dilative arteriopathy would have been assessed, if the guidelines for treating renal infarction would be revised, and if the patient would have been thoroughly investigated cardiologically."} +{"text": "To the editor,We read with interest the recent paper by da Silva et al. examining effects of antibiotic prophylaxis and risk of urinary tract infection for spinal cord injured patients undergoing urodynamic studies. The authors describe a multi institutional study involving 661 patients who underwent urodynamic evaluation over 2 years is not mentioned. It is stated that in the consideration of variables a numbers that several factors were included yet there is no baseline assessment of subjective symptoms based on patient questionnaires such as the neurogenic bladder symptom score . In theSpecific to our Spinal cord injury unit we routinely perform videourodynamic evaluation of spinal cord injured patients both as inpatients and outpatients and all undergo mandatory dipstick assessment prior to the procedure. If suggestive of infection the procedure is deferred but we do not prescribe antimicrobials pre investigation. Additionally we record bladder symptom scores at baseline with a validated questionnaire and repeat scores following definitive treatment to evaluate response .Yours Sincerely,Authors"} +{"text": "This data set contains local experimental data of heat flux, wall temperature and void data profiles for vertical subcooled flow boiling of refrigerant Novec 649 at a copper wall. This data article presents average boiling curves from single-phase convection to fully developed film boiling for six combinations of mass flux and subcooling. Void profiles are provided for void fraction, void detection frequency, void velocity and void ligament size for three characteristic states along each boiling curve. Thermocouples were used to measure heat flux and temperature. Optical single fiber and double fiber micro probes were used for obtaining void data profiles. A traversing mechanism was used to position the optical fiber micro probes relative to the heater surface. Specific enthalpy of evaporation Denotes a difference Thermal conductivity Density Surface tension Contact time Surface heat flux Specific heat capacity Frequency Mass flux Index variable Index variable Index variable Length Quantity Number of samples Pressure Sample rate Temperature Time Voltage Velocity Distance in wall normal direction critConditions at critical pointcuCoppergGas phaselLiquid phaseligVoid ligamentpIsobaricprobeRefers to a characteristic of a fiber optic probesatSaturation conditionssubRefers to inlet subcoolingthThresholdwRefers to the wallSpecifications tableValue of the data\u2022These data consist of average boiling curves and detailed quantitative local information about the void phase in the cross section of a flow channel for refrigerant Novec 649.\u2022The presented void profiles for void fraction, void detection frequency, void velocity and void ligament size can provide insights into the critical heat flux process in subcooled flow boiling.\u2022Average boiling curves and void profiles are valuable for the development and validation of new predictive numerical tools for the simulation of two-phase flow.\u2022With this data important CFD-validation data such as interfacial area density or sauter diameters can be derived.1In this data article, average boiling curves and void profiles are presented. The data were obtained from experiments using a copper heater mounted flush in the wall of a vertical flow channel. Measurements were done at three different positions along the direction of flow. Experiments were conducted at mass fluxes of 2Experiments were conducted with a coolant at a heated copper wall in a square flow channel at mass fluxes of 2.1Measurements were taken using a fluid loop with a vertically oriented test section, as shown schematically in A three-dimensional view of the test section is shown in 2.22.2.1To measure heat flux and wall temperature, there are three rows of thermocouples inside the copper bar. Each row consists of four thermocouples placed at different distances below the surface according to 2.2.2Three optical fiber probes were used simultaneously to measure void data at distances between The optical fiber probes were inserted into the flow channel at an angle of All void data are calculated based on a phase indicator function , at CHF and at fully developed film boiling (FDFB). The respective definitions as a function of wall superheat"} +{"text": "Foreign body ingestion is a scenario occasionally encountered in the emergency room. Pediatric and psychiatric patients are the two most common populations suffering from accidental or in some cases intentional ingestion of foreign bodies. Commonly, majority of cases require no specific treatment and the swallowed objects pass through the digestive tract spontaneously without causing any significant complications. Less than 1% of the cases complicates with gastrointestinal tract perforation, which are often caused by sharp objects, which warrants surgical intervention. The average time from foreign body ingestion to development of perforation was noted at 10.4\u2009days in previous reports. These cases often present in rapidly progressing peritonitis and are subsequently managed by emergent laparotomy. In this case report, we describe an accidental chopstick ingestion of a patient who initially was misdiagnosed and remained asymptomatic for nine months, then presented with acute abdomen.A 27-year-old man accidentally ingested a wooden chopstick and sought consult at a clinic. Negative abdominal plain film misled the physician to believe ingested chopstick was digested into fragments and passed out unnoticed. The patient presented acute abdomen caused by duodenal perforation nine months later and was subsequently treated with emergency laparotomy with primary duodenorrhaphy.Negative plain films are not sufficient to conclude a conservative treatment in foreign body ingestion. Computed tomography scan or endoscopic examinations should be done to rule out retained foreign body within gastrointestinal tract. Ingestion of foreign bodies may be encountered from time to time in clinical practice. Small objects like coins, buttons or toy compartments are frequently accidentally swallowed by children whereas particular objects are associated with intentional ingestion by psychiatric patients. In prior studies, around 1% of foreign body ingestion complicates with significant clinical sequela like gastrointestinal (GI) tract obstruction or hollow organ perforation . These cTo the best of our knowledge, few reports have described upper GI tract perforation associated with foreign body ingestion which developed more than half year from the time of ingestion to onset of symptoms . We inteA 27-year-old young man sought consult at the outpatient clinic of our hospital complaining of sudden onset of dull abdominal pain over the right upper quadrant (RUQ) right of 3-days duration. He denied nausea, vomiting, diarrhea, or any other gastrointestinal symptoms. Prior to his consult at our hospital, he initially sought consult at a local clinic on the second day of symptom onset and was prescribed with antispasmodics under the impression of acute gastroenteritis however there was persistence of the RUQ pain despite the intake of antispasmodics. Persistence as well as progression of the symptoms was noted which now began to radiate to back on the third day which prompted the patient to seek consult at our institution Table .Table 1The patient had stable vital signs at our outpatient clinic. He denied any chronic illness or surgical history. He denied psychological disorder as well, and drank only in social occasions. The patient however volunteered the history of the accidental ingestion of a wooden chopstick nine months prior to which he sought consult a few days after that incident. The attending physician requested for an abdominal plain film which turned out to be unremarkable and offered the explanation to the patient that the chopstick may have been digested into fragments and will pass out unnoticed. After that consult, patient remained asymptomatic for nine months.Upon examination, he presented with a mild abdominal tenderness over the RUQ with negative peritoneal sign. His chief complaint was the back pain with positive knocking pain was noted over the T12 to L2 level. Laboratory investigation revealed leukocytosis and elevated C-reactive protein (CRP) level (serum CRP: 164\u2009mg/dl). Abdominal computed tomography (CT) was done, which revealed a chopstick-shaped foreign body about 11\u2009cm in length penetrating the second portion of duodenum into retroperitoneal space. Focal fat stranding and tissue swelling were likewise noted but no free air or ascites before causing significant complication. Such history should alert clinical practitioners that lack of symptoms and negative plain films are not sufficient to conclude a conservative treatment in foreign body ingestions. Computed tomography scan or endoscopic examinations should be done to rule out retained foreign body within GI tract.Clinical presentation of our case after duodenal perforation was compatible with the statistic results in previous studies , 7, 8. H"} +{"text": "Previous research has found that older adults endorse higher levels of racist attitudes than younger adults. However, little extant research has explored how young adults may respond to an older adult expressing racist views. One factor that may drive young adults\u2019 responses is ageism, particularly stereotypes that older adults cannot handle disagreement or are incapable of changing their views. The purpose of this study was to investigate the relationships between ageism and young adults\u2019 likely responses to an older adult relative making a racist statement. College students completed an online survey in which they were given a scenario in which an older adult relative makes a racist statement and rated how likely they would be to respond in different ways. Factor analysis of the likely response items found four facets: confront, agree, avoid, and leave. Bivariate correlations found that ageism was associated with higher likelihood of agreeing or avoiding, and lower likelihood of confronting the older adult relative. There was no association between ageism and likelihood of leaving the situation. Young adults higher in ageism may be more likely to agree or avoid because of ageist stereotypes that older adults cannot handle disagreement or are incapable of change, and they may be more likely to agree with the racist statement because they may have higher levels of intolerance toward both older adults and other ethnic groups. Ageism may play a role in how young adults respond to older adults expressing intolerant views."} +{"text": "Bacteroidales 16S rRNA genetic markers. The microbial community composition revealed that Proteobacteria and Bacteroidetes (70\u201390% relative abundance) were the most dominant bacterial phyla, followed by Firmicutes, especially in waters exposed to anthropogenic faecal contamination. The core archaeal community consisted of Parvarchaeota (mainly in the tributaries of drinking water reservoirs) and Crenarchaeota . The aquatic microbial diversity was substantially reduced in water with severe faecal contamination. In addition, the community compositions diverge between waters with dominant anthropogenic or zoogenic pollution origins. These findings present novel interpretations of the effect of anthropo\u2010zoogenic faecal water contamination on microbial diversity in lotic ecosystems.Faecal contamination is one of the major factors affecting biological water quality. In this study, we investigated microbial taxonomic diversity of faecally polluted lotic ecosystems in Norway. These ecosystems comprise tributaries of drinking water reservoirs with moderate and high faecal contamination levels, an urban creek exposed to extremely high faecal pollution and a rural creek that was the least faecally polluted. The faecal water contamination had both anthropogenic and zoogenic origins identified through quantitative microbial source tracking applying host\u2010specific Faecal water contamination originates from anthropogenic and zoogenic sources associated with both point and nonpoint/diffuse pollution. In addition, the contamination occurs in groundwater and surface waterbodies through direct (faeces and droppings) and indirect pathways. These are the main transmission routes to water environments for various microbiota species derived from faecal pollution methods using quantitative polymerase chain reaction (qPCR) have been widely implemented into practice. The qPCR\u2010based MST techniques detect and quantify host\u2010specific genetic markers, which determine the origins of faecal pollution. The most tested, validated and applied markers in aquatic research are those derived from et al., et al., et al., et al., et al., et al., et al., et al., Surface water environments are more vulnerable to contamination than groundwater, and their microbial profiles represent the most important indicators of biological water quality. It is therefore highly important to assess faecal contamination impact (with particular emphasis on anthropogenic and zoogenic origins) on the profile of aquatic microbiota in the natural water ecosystems. In this regard, next\u2010generation sequencing (NGS), for example Illumina MiSeq, has been successfully employed for the characterization of microbial community structure concentrations. Therefore, our main focus was given to the most distinctive samples exposing extreme variation in E. coli counts (lowest vs. highest) and those with divergent origins of faecal pollution. The origin was defined through quantitative microbial source tracking (QMST) using host\u2010specific Bacteroidales 16S rRNA genetic markers and was further presented as the percentage contribution profile of the markers in faecal pollution. The methodology of contribution profiling and its scientific background have been described in greater detail elsewhere were investigated, and all of them revealed constant exposure to faecal pollution based on E. coli concentrations were found in the rural creek (RC), where the contribution profile of the tested Bacteroidales DNA markers showed a predominantly zoogenic origin , where the contribution profile of genetic markers in faecal water contamination exposed a dominant zoogenic origin . These OTUs were further classified into 74 microbial phyla, with a majority of bacteria (71) and three archaeal phyla.Proteobacteria and Bacteroidetes were the dominant populations (constituting 70\u201390% relative abundance) in the investigated lotic ecosystems. In addition, the typical human gastrointestinal microbiota represented by Firmicutes of Parvarchaeota was revealed in all three tributaries , while Crenarchaeota constituted the second largest phylum, representing up to 44% relative abundance in RC ecosystem. This phylum was largely dominated by the genus Candidatus Nitrososphaera, which responds positively to nitrogen fertilization and farmyard manure as revealed by permutational multivariate analysis of variance (PERMANOVA). A group of two closely related clusters composed of ecosystems T1\u2013T2 and RC\u2013T3 was identified. Distinguishably, the UC ecosystem exhibited noticeable divergent microbial structure in comparison with the aforementioned group and stands as a separate cluster. This analysis clearly unveils that microbiota in the most faecally contaminated UC ecosystem, influenced mostly by the anthropogenic pollution sources (Table Proteobacteria, Firmicutes, Bacteroidetes, Synergistetes, Tenericutes and Fusobacteria (David et al., et al., et al., A heat map of beta diversity Fig. illustraBoth analytical and statistical tests validate the results of this study, which as a whole presents novel interpretations of the effect of anthropo\u2010zoogenic faecal pollution on aquatic microbial diversity in lotic waters. The outcomes clearly answer the research question addressed in this work by demonstrating a substantial reduction of the microbial diversity in water with severe faecal contamination. In addition, the study shows that the community compositions are distinct between ecosystems exposed to anthropogenic or zoogenic pollution. To conclude, the primary finding obtained in the current study is that the higher faecal water contamination the lower aquatic microbial diversity.None declared."} +{"text": "Cerebral autosomal dominant arteriopathy with subcortical infarcts and leucoencephalopathy (CADASIL) syndrome is a genetically inherited condition most notably affecting the central nervous system in young adults. There is limited knowledge on its association with coronary arteries, and its association with spontaneous coronary artery dissection (SCAD) has not been previously reported.A 61-year-old woman who is known to have CADASIL syndrome presented with anterior ST-segment myocardial infarction and underwent emergency angiography. This showed appearance consistent with SCAD in the mid left anterior descending artery with tubular stenosis.The association between CADASIL syndrome and SCAD has not been previously reported. The similarity in the underlying pathophysiology of these two conditions makes this case intriguing. Spontaneous coronary artery dissection could be associated with cerebral autosomal dominant arteriopathy with subcortical infarcts and leucoencephalopathy (CADASIL) syndrome.Spontaneous coronary artery dissection is a result of arteriopathy and CADASIL syndrome is a result of angiopathy both similarly affecting the vascular system causing ischaemia and infarction.NOTCH3 gene.Cerebral autosomal dominant arteriopathy with subcortical infarcts and leucoencephalopathy (CADASIL) syndrome is an autosomal dominant inherited angiopathy thought to be caused by mutations in the We present a case of a patient with CADASIL syndrome who presented with an acute coronary syndrome; subsequent coronary angiography was highly suggestive of spontaneous coronary dissection (SCAD). Spontaneous coronary artery dissection is characterized by a false lumen forming within the layers of the coronary arterial wall restricting blood flow. The underlying mechanism for SCAD is unclear but it is regarded as a manifestation of a more widespread arteriopathy resulting in coronary arterial wall degradation with increased risk of development of compressive intramural haematoma due to vaso vasorum bleeding or frank intimal tear and subsequent ischaemic coronary events.Figure 1) with a raised initial high-sensitivity troponin level which was 1551\u2009ng/L (0\u201315\u2009ng/L). On auscultation, her chest was clear and heart sounds were essentially normal. Jugular venous pressure was normal and there was no peripheral oedema present. Her bedside focused transthoracic echocardiography showed anterolateral regional wall motion abnormalities with akinesia in the anterior segments with a visually estimated left ventricular ejection fraction of 35%. She underwent emergency radial coronary angiography which showed an abnormal appearance of the left anterior descending artery (LAD) suggestive of SCAD. She had a long segment of irregular filling of the mid anterior descending artery with a tubular stenosis. The proximal LAD, the circumflex, and right coronary arteries were angiographically normal. Given the appearance of thrombolysis in myocardial infarction Grade 3 flow in the LAD territory and recognizing the potential risks of percutaneous intervention in the setting of SCAD, she was, therefore, managed medically with dual antiplatelets (Aspirin and Clopidogrel) for 3\u2009months, Bisoprolol, Ramipril, and Atorvastatin . The duration of dual-antiplatelet therapy was limited as no stent was deployed and a reported natural history of vascular healing in SCAD. Intravascular imaging was not used to confirm the suspected angiographic diagnosis given the reported vascular complications with instrumentations in SCAD such as extending the coronary dissection with wire or imaging catheter and guide catheter iatrogenic dissection.,A 61-year-old woman who was normally fit and well with a background history of CADASIL syndrome presented to the emergency department with chest pain and feeling dizzy and unwell. She had no other risk factors for coronary artery disease. CADASIL diagnosis was made in a regional specialist neurological centre and she was under follow-up. Her cardiovascular examination revealed an initial blood pressure of 167/81\u2009mmHg, heart rate of 81\u2009b.p.m., and her electrocardiogram showed anterior ST-segment elevation and EAPCI and member of British Cardiovascular Intervention Society (BCIS).European Heart Journal - Case Reports online.Slide sets: A fully edited slide set detailing this case and suitable for local presentation is available online as Consent: The author/s confirm that written consent for submission and publication of this case report including image(s) and associated text has been obtained from the patient in line with COPE guidance.Conflict of interest: none declared.ytz136_Supplementary_Slide_SetClick here for additional data file."} +{"text": "Recent reports by Martincorena et al and Yokoyama et al reveal unanticipated dynamics of somatic evolution in the esophageal epithelium, with clonal expansions apparently driven by mutations in Notch1 dominating the epithelium even in middle\u2010aged individuals, far outpacing the prevalence of these mutations in esophageal cancers. We propose a model whereby the promotion of clonal expansions by mutations such as in Notch1 can limit more malignant somatic evolutionary trajectories until old ages. So how could loss of Notch1 function impede cancer development?The recent papers by Martincorena et al and Yokoyama et al provide important and surprising new insights into how somatic cells in our tissues evolve with age can impede access to more malignant evolutionary trajectories, even if such decoy peaks can still lead, albeit with reduced frequency or delayed kinetics, to malignancy Figure , reminisFor this model, we incorporate a previously substantiated concept\u2014that the fitness impact of mutations can be highly context\u2010dependent. In particular, previous studies have demonstrated that oncogenic mutations are differentially selected for in tissues of young and old mice, with adaptation promoted in the aged context , while being adaptive in progenitors without such drivers. Alternatively, Notch1 mutant clones could represent dead\u2010end cellular expansions, which lose further self\u2010renewal activity \u2014\u201cfitness sinks\u201d rather than fitness peaks (akin to nevi in the skin). Notch1 mutant clones could even promote carcinogenesis noncell autonomously, as Yokoyama et al propose . While the strength of natural selection to limit cancer and other manifestations of animal senescence wanes at older ages (Rozhok & DeGregori, In all, the reports by Martincorena, Yokoyama and colleagues reveal how natural selection may have sculpted somatic programs to minimize cancer risk and thus maximize individual fitness. Additional studies, both from the clinic and from experimental and computational models, will be required to delineate the potential role, if any, of decoy fitness peaks in tissue maintenance and tumor suppression, and how these mechanisms evolved.None declared."} +{"text": "Iatrogenic coronary artery dissection is a potentially life-threatening complication of cardiovascular interventions. The optimal management of iatrogenic coronary artery dissection is not clear; however, both conservative management and percutaneous or surgical revascularization have been performed depending on the patient's clinical status and the extent of dissection. We present the first reported case of right coronary artery dissection after Bentall procedure performed for ascending aortic aneurysm. Urgent percutaneous intervention using adjunctive coronary imaging was performed with excellent clinical recovery. In this article, we highlight coronary artery dissection after Bentall procedure as a possible complication, provide an insight into various options in its management, and review published data on iatrogenic coronary artery dissection. We also discuss the challenges in percutaneous treatment of coronary artery dissection with special focus on intracoronary imaging for accurate diagnosis and guidance in the management of this complex lesion. We present a 66-year-old Caucasian male with a history of hypertension and chronic type A aortic dissection who was found to have an enlarging aortic root measuring 5.2\u2009cm in diameter on an echocardiography done as part of surveillance of aortic dissection repair done 9 years ago using a tube graft with resuspension of the aortic valve . EchocarThe patient subsequently underwent modified Bentall procedure. This involved graft replacement of the aortic root, replacement of the aortic valve using a 27\u2009mm bioprosthesis , and reimplantation of coronary arteries into the graft using the button technique. Soon after sternotomy closure was done, he was found in cardiac arrest with ventricular fibrillation intractable to pharmacologic resuscitation and defibrillation. Mediastinal reexploration was immediately performed revealing a fibrillating heart with no evidence of obvious bleeding or injury. He was internally defibrillated, and normal sinus rhythm was achieved. The patient was systemically heparinized and stabilized with vasopressors and veno-arterial extra-corporeal membrane oxygenation (VA-ECMO). Urgent transesophageal echocardiography (TEE) was done that showed a new, severe dilatation of the right ventricle along with reduced ejection fraction but a normal left ventricle . The proA concern for iatrogenic injury to the coronary vessels prompted an emergent coronary angiography which revealed dissection of the right coronary artery (RCA) extending from the ostium down to its distal segment, sparing the bifurcation . A 3.5\u2009\u00d7His postoperative course was also complicated by a right-sided pneumothorax which required chest tube drainage and nonoliguric renal failure that were managed conservatively. The patient's clinical status showed marked improvement, and by the fourth day postsurgery, he was extubated and weaned off from hemodynamic support. A transthoracic echocardiography one week later showed improved right ventricular size and function . The patThe Bentall procedure for management of aortic root dilatation was first described in 1968 by Bentall and De Bono. This involves surgical replacement of the ascending aorta and aortic valve with composite tubular graft containing a prosthetic valve followed by reimplantation of coronary arteries into holes punched in the graft . The claReports regarding coronary artery dissection after the Bentall procedure are lacking in literature. We describe the first reported case of RCA dissection after the Bentall procedure. We think that coronary artery dissection in our case was probably related to either mechanical injury during excision and reimplantation of the coronary buttons or from instrumentation at the time of administration of cardioplegics into the coronary arteries .We performed literature review in PubMed using the search strategy \u201cIatrogenic[Title] AND Coronary[Title] AND Dissection[Title].\u201d This yielded 82 articles; out of which, 38 articles had detailed information on the type of management strategy for each patient with iatrogenic coronary artery dissection and thus were selected for review. Four of these articles were case series studies, and the rest were case reports. We identified a total of 48 patients who suffered iatrogenic coronary artery dissection (ICAD), 30 (62.5%) of which were females. The age group with the most number of patients reported was 61\u201370\u2009years. 47 patients developed ICAD as a result of coronary angiography whereas only one patient developed ICAD after a coronary artery bypass graft (CABG) procedure. We did not find any case of ICAD resulting from the Bentall procedure. 41 patients had some form of intervention either with stent placement (33 patients), CABG (6 patients), or both (2 patients) whereas only 6 patients had conservative treatment as part of the management of ICAD. One patient died before any management strategy could be instituted. Dissection occurred in the left coronary circulation in 32 (66.7%) and right coronary circulation in 16 (33.3%) patients. Intracoronary imaging with either intravascular ultrasound (IVUS) or optical coherence tomography (OCT) was employed in 15 patients for evaluation and guidance in management. These findings are summarized in The diagnosis of coronary artery dissection with angiography alone can be arduous since an angiogram is a two-dimensional luminogram and does not image the arterial wall that is affected in coronary dissection . Thus, aThe overall management of ICAD is often dependent on operator discretion and other clinical and patient variables including the severity of dissection and hemodynamic stability. A wide variety of management strategies that include conservative management, percutaneous intervention (PCI), emergent coronary artery bypass grafting, thrombolytic therapy, and cardiac transplantation have been done \u201319. HoweThere are multiple reasons for suboptimal results associated with PCI in coronary artery dissection. First, with dissection, it is difficult to advance a guidewire into the true lumen . Second,Some authors have recommended percutaneous intervention techniques to improve results in patients with coronary artery dissection. In case of a relatively focal lesion, selection of a longer stent that spans across both the proximal and distal edges of the lesion would be preferable . This wiIntracoronary imaging modalities can be of great value in guiding successful PCI. IVUS and OCT can be used to identify true lumen, intimal tears and extent of the dissection, and intramural hematoma. They can ensure proper placement of guidewire in the true lumen and help in selecting the appropriate size of coronary stent and its optimal deployment , 30. IntIatrogenic right coronary artery dissection should be considered in patients with ventricular fibrillation and acute right ventricular dilatation after Bentall procedure. Percutaneous intervention along with intracoronary imaging is a useful strategy to accurately diagnose and guide revascularization in these cases."} +{"text": "Making health care truly universal requires a shift from health systems designed around diseases and health institutions towards health systems designed around and for people.\u201d constitutes health service expertise through the formal inclusion of experiential knowledge from patients and/or communities, care providers and resource decision-makers, together on even footing with epidemiological studies and more on the support systems around women throughout the perinatal period. This illustrated how including stakeholder knowledge as a complement to published literature can broaden both the problem definition and the menu of interventions.Cognitive maps that account for interdependence between factors can act as a decision aid for complex processes like clinical care, where artificially isolating associations within a de facto network or results chain can diminish the contextual understanding and relevance of decisions (Napier et al. known about a relationship with observed data about that same relationship, by calculating a posterior distribution using Bayes\u2019 theorem (Goldstein To bring these different perspectives in conversation with one another, we drew on Bayesian analysis as a formal method to integrate stakeholder perspectives with published literature. Conventional Bayesian analysis elicits prior weights from experts by asking how likely they consider the occurrence of an event to be (Gelman et al. how social, economic and organizational contexts contribute to outcomes prioritized in the literature or in stakeholder maps (Pawson Step 4 requires that we understand cognitive maps as conceptual, not probabilistic models Mingers . Along ws Pawson . Stakehos Pawson . Bringins Pawson . Our demStep 5 focuses on the identification of care recommendations. Engaging stakeholders in the explanatory analysis in the previous steps creates space not only for different forms of knowledge about how a particular system works but also shifts the realm of possible improvement strategies Midgley .Weight of Evidence presents a rigorous and transparent approach to unpack differences, to identify how and when these differences arise and with what consequences.Moving toward more people-centered health services requires that we take better account of how people\u2019s understandings of determinants of poor health intersect with conventional biomedical evidence (Napier et al. We share this work as an invitation to include methodological innovations as part of our collective response to calls for more people-centered health systems (James et al."} +{"text": "Individuals with dementia may enlist help from paid caregivers to address increasingly complex care needs. Yet it is unclear what specific tasks paid caregivers help with and how this is related to the individual\u2019s experience of unmet care needs. We used data from the 2015 National Health and Aging Trends Study (NHATS) to examine the association between type and intensity of individual paid caregiver tasks and adverse consequences of unmet needs among community-dwelling adults with dementia. Nearly one half (46%) of those with any functional impairment reported an adverse consequence of an unmet need . Individuals who experienced adverse consequences were more likely to receive paid caregiving . Those who received paid care with an individual task (e.g. toileting) were more likely to report an adverse consequence related to that task . Paid caregivers provided a median of 15 hours of care per week. For those persons with dementia receiving less hours of care weekly, paid caregivers rarely helped with unscheduled or frequently recurring functional tasks like toileting or eating (<10% of the time). The help received by individuals with dementia was inadequate to meet their care needs. Paid caregiving will only be able to prevent adverse consequences of unmet care needs when the level of paid care provided is better matched to the care needs of the individual with dementia."} +{"text": "Older adults who experience food insecurity (4.6 million) often have worse health outcomes. Food insecure older adults consume less nutrients, which puts them at greater risk of developing chronic diseases. They are at increased risk of falls due the impact of poor nutrition on muscle mass, bone density, and balance. Low-income older adults are often forced to choose between buying groceries and paying other bills. The Supplemental Nutrition Assistance Program (SNAP) plays an important role in reducing food insecurity. SNAP enables older adults to buy the nutritious food they need, while freeing up resources to pay for everyday things to meet their health needs such as prescription drugs. Research shows that medication adherence increases when low-income older adults enroll in SNAP. Despite the beneficial impact of enrolling in SNAP, it\u2019s estimated that 55% of eligible adults age 60 and older are not participating in this critical program. To understand which older adults are missing out on SNAP, the National Council on Aging engaged researchers at Leading Age LTSS Center at UMass Boston to analyze data from the 2014 Health and Retirement Study. The results show that some of the most vulnerable older adult populations are less likely to participate in SNAP even though they are eligible . The findings suggest that more targeted outreach to these groups is needed to ensure that the most vulnerable populations of older adults access this critical benefit."} +{"text": "Many older adults rely on informal care networks to overcome challenges in life and maintain well-being. The composition and function of the informal care network may change as existing caregivers leave and new caregivers join the network over time. The majority of prior studies on caregiving to older adults are based on cross-sectional data and thus cannot examine changes in older adults\u2019 informal care networks. Although some have followed older adults\u2019 informal caregivers over time, they usually focus on primary caregivers, rather than the entire informal care network longitudinally. The newly available panel data on a nationally representative sample of caregivers from the National Study of Caregiving (NSOC) provide an excellent opportunity for researchers to understand how older adults\u2019 informal care networks change over time and what factors relate to discontinuation of care. Using the NSOC 2015 and 2017, we found that 70% of older adults experienced changed in informal care networks within two years. Only a small portion of spouses (6%) discontinued giving care to older adults, whereas 21% adult children, 56% other kin, and 77% nonkin stopped caregiving by 2017. We further examined how older adults\u2019 needs for support, caregivers\u2019 resources and constraints, and caregiving experiences were associated with discontinuation of care. This study is expected to advance gerontological research by broadening our understanding of informal caregiving in late life and providing practical implications on how to sustain informal care."} +{"text": "The employment of differential centrifugation to prepare crude fractions of subcellular particles from homogenates is often a necessary first step to a subsequent purification of one or more particles on a density gradient. Buoyant density gradient purification of peroxisomes or lysosomes for example is almost invariably carried out on a light mitochondrial fraction so as to eliminate smaller particles that may have similar densities. Unless they are first removed, large rapidly sedimenting particles in homogenates may also disturb shallow gradients designed to fractionate small low-density microsomes."} +{"text": "More recent studies demonstrate that these cells comprise a distinct CD4+ T cell subset, making it a therapeutic target for the treatment of lupus flares. Transcriptional analyses of this subset reveal proteins uniquely expressed by this subset, which may serve as therapeutic to deplete these cells, treating lupus flares.Lupus flares when genetically predisposed people encounter exogenous agents such as infections and sun exposure and drugs such as procainamide and hydralazine, but the mechanisms by which these agents trigger the flares has been unclear. Current evidence indicates that procainamide and hydralazine, as well as inflammation caused by the environmental agents, can cause overexpression of genes normally silenced by DNA methylation in CD4 Th. Th+ femThe replication of DNA methylation patterns during mitosis requires not only adequate levels of Dnmt1 during mitosis but also requires SAM, which donates the methyl group required to methylate the cytosines in the newly synthesized DNA strand . SAM is + T cell DNA methylation levels through mechanisms including decreasing Dnmt1 activity, either by inhibiting its enzymatic activity or decreasing Dnmt1 levels through effects on signaling pathways, or by decreasing the bioavailability of dietary methyl donors. This suggests a mechanism by which antioxidants such as N-acetylcysteine may be beneficial in lupus patients and that attention to proper dietary habits or supplements may also be beneficial in the management of lupus patients.Together, these studies indicate that exogenous agents can induce lupus flares by decreasing CD4"} +{"text": "Mitochondrial dysfunction is a hallmark of aging, but severe mitochondrial dysfunction leads to rare childhood disorders such as Leigh Syndrome. This session explores the similarities and differences between normative aging and mitochondrial disease and the potential for interventions to positively impact both conditions."} +{"text": "Then Coalition for Quality in Geriatric Surgery has completed both alpha and beta pilots which represent the initial efforts to launch this national quality initiative to improve the care of all older adults undergoing operations. The alpha phase included hospital stakeholders rating the feasibility of implementing standards related to evidence-based high quality surgical care for older adults on the topics of eliciting patient goals, completing a preoperative frailty risk assessment, educating healthcare professional about care specific to older adults, and implementing postoperative age-friendly care models. The beta pilot phase required 9 medical centers nationally to implement the evidenced based standards for high quality surgical care of older adults. Site verification visits were completed in the Summer 2018 which evaluated the effectiveness of each medical center\u2019s ability to implement each of the standards."} +{"text": "However, such techniques do not directly measure changes in metabolic activity during neuronal activation. Changes in the redox state of cerebral oxidised cytochrome c oxidase \u0394[oxCCO] measured by broadband spectroscopy may be a more specific marker of neuronal metabolic activity. This study aims to investigate the spatial distribution of \u0394[oxCCO] responses during the activation of the visual cortex in the healthy adult human brain, and reconstruct images of these changes.Functional hyperaemia, characterised as an increase in concentration of oxyhaemoglobin [HbO2], \u0394[HHb] and \u0394[oxCCO] were calculated by fitting the broadband spectra between 780 and 900 nm using the UCLn algorithm. Centre of gravity analysis was applied to the concentration data to determine the centres of activation for [HbO2], [HHb] and [oxCCO].Multi-channel broadband NIRS measurements were collected from the left visual cortex of four healthy volunteers using an in-house broadband spectrometer during an inverting checkerboard visual stimulation paradigm. \u0394 in the presence of a typical visual-evoked haemodynamic response in channels overlying the visual cortex. Image reconstruction of the optical data showed a clear and spatially localized activation for all three chromophores. Centre of gravity analysis showed different localisation of the changes in each of the three chromophores across the visual cortex with the x-y coordinates of the mean centres of gravity (across 4 subjects) of HbOThe spatial distribution of \u0394[oxCCO] response appears distinct from the haemodynamic response in the human visual cortex. Image reconstruction of \u0394[oxCCO] shows considerable promise as a technique to visualise regional variation in [oxCCO] in a range of scenarios. Several studies using Diffuse optical imaging have been able to produce detailed [HbO2] and/or [HHb] maps of various functional regions [2] and [HHb] provide information only on the haemodynamic responses to neuronal activity and do not inform directly on the changes in cellular metabolism associated with functional activation. responses resulting from functional brain activation, and (2) evaluate the possibility of reconstructing images of these responses. A visual stimulation paradigm was chosen given its capability to produce a highly repeatable and well-characterised functional activation response that has been corroborated across multiple imaging modalities, including broadband spectroscopy and functional magnetic resonance imaging assess t.A detailed description of the Near-infrared spectroscopy (NIRS) used in this study can be found elsewhere . In summVisual functional activation was achieved with a 4Hz inverting checkerboard delivering 20 s stimulation and 20 s black screen repeating over 10 epochs. The optode array was fixed horizontally with the fourth source location positioned over Oz (10/20 EEG position).2], \u0394[HHb] and \u0394[oxCCO] were derived using the UCLn algorithmNear-infrared spectroscopy (NIRS) over the wavelength range 780 nm to 900 nm. The wavelength dependence of the differential pathlength factor (DPF) was taken into account when resolving concentration changes, as described by Matcher et al. , \u0394[HHb] and \u0394[oxCCO]. Data at seventeen wavelengths were selected from the measured broadband spectrum (every 10 nm from 740 to 900 nm) to perform the reconstruction , [HHb] and [oxCCO] in all 32 channels over the left Oxidised cytochrome c oxidase (oxCCO)HHbHbO2Visual cortex of a single subject which is representative of the data acquired from all four subjects. A typical haemodynamic response to functional activation (increase in [HbO2] and decrease in [HHb]) was seen across different channels.Following ethics approval and volunteer consent, four healthy adults were studied. Figure x and y directions between centres of gravity of [oxCCO] and [HHb] and between [HHb] and [HbO2], suggesting that they have distinctly separate locations. In the case of [oxCCO] and [HbO2], there is no overlap in the y direction but some in the x direction, suggesting a less distinct spatial separation than that seen for [HHb]. The centres of gravity for all subjects are summarised in Table Figure t\u2009=\u200920 s shows the maximal changes during the stimulation period. These images show different localisations between [HbO2]/[oxCCO] and [HHb] changes, whereas a similar region is active for [HbO2] and [oxCCO], consistent with the centres of gravity analysis results.Figure 2], [HHb]) during activation of the Visual cortex. The regional separation of changes in each chromophore may reflect the effect of surrounding/overlying vasculature versus regions of direct metabolic activity. The distinct spatial localisation of HbO2 and HHb might be explained by the fact that the HbO2 signal is derived from arteries, capillaries and veins whereas the HHb signal is derived mostly from HHb within the region of interest and therefore affected by the difference in distribution of arteries and veins in the field of view. Such separation in the centres of gravity for HbO2 and HHb has previously been demonstrated by Koenraadt et al. [2] and [HHb] [We have demonstrated focal localisation of \u0394[oxCCO] that is discrete from the haemodynamic signal ([HbOt et al. . Furthernd [HHb] , but in"} +{"text": "To the Editor,One of the life-threatening situations during surgery is unintentional tracheal extubation of patient under general anaesthesia in position limiting face access and can cause serious complications if not followed by rapid reintubation . Sudden We would like to present the case of successful reintubation of patient in prone position using AirTraq Avant videolaryngoscope. In the 39\u00a0years female patient anaesthetized and intubated using standard Macintosh blade laryngoscope without any difficulties in supine position for elective lumbal spine surgery shortly after positioning in prone position for surgery a serious leak was detected in anaesthesia circuit due to malfunction of endotracheal tube cuff. We decided to attempt intubation in prone position using AirTraq Avant videolaryngoscope (Fig.\u00a0There is no other paper on using videolaryngoscopes for intubation of patients in prone position. Kaur et al. reported successful use of C-Mac videolaryngoscope for intubation of patient in lateral position . Wang etChannelled videolaryngoscopes may be more adequate in case of intubation patients in prone position, however further observation are needed."} +{"text": "The increasing popularity of social media and other online communities offers new possibilities for older adults to stay socially connected. This study examines the relationship of older adults\u2019 online social engagement and bonding as well as bridging social capital based on a survey of over 1,000 adults aged 60 and over. Social bonding refers to support obtained from existing strong social ties while social bridging is creating connections across varied social networks. We estimated three multi-stage regression models to examine these relationships when controlling for sociodemographic factors, as well as Internet experiences and skills. We then extended the regression models with Internet skills as a moderator. Findings show that older adults who engage more often in specific online social activities enjoy greater bridging social capital (both in offline and online contexts) than those who do so less often. Furthermore, Internet skills moderate the relationship between online social engagement and social capital. Specifically, older adults with greater Internet skills benefit relatively more from engaging in specific online social activities more often with respect to online social bridging. These results imply that digital inequalities may put older adults who are less skilled in using the Internet at a disadvantage when it comes to building social capital from online social engagement. Thus, while social media has potential positive implications for well-being among older adults, the current manifestation of this does not suggest equitable distribution of those benefits across different older users."} +{"text": "Isolation by distance (IBD) is a natural pattern not readily incorporated into theoretical models nor traditional metrics for differentiating populations, although clinal genetic differentiation can be characteristic of many wildlife species. Landscape features can also drive population structure additive to baseline IBD resulting in differentiation through isolation\u2010by\u2010resistance (IBR). We assessed the population genetic structure of boreal caribou across western Canada using nonspatial (STRUCTURE) and spatial (MEMGENE) clustering methods and investigated the relative contribution of IBD and IBR on genetic variation of 1,221 boreal caribou multilocus genotypes across western Canada. We further introduced a novel approach to compare the partitioning of individuals into management units (MU) and assessed levels of genetic connectivity under different MU scenarios. STRUCTURE delineated five genetic clusters while MEMGENE identified finer\u2010scale differentiation across the study area. IBD was significant and did not differ for males and females both across and among detected genetic clusters. MEMGENE landscape analysis further quantified the proportion of genetic variation contributed by IBD and IBR patterns, allowing for the relative importance of spatial drivers, including roads, water bodies, and wildfires, to be assessed and incorporated into the characterization of population structure for the delineation of MUs. Local population units, as currently delineated in the boreal caribou recovery strategy, do not capture the genetic variation and connectivity of the ecotype across the study area. Here, we provide the tools to assess fine\u2010scale spatial patterns of genetic variation, partition drivers of genetic variation, and evaluate the best management options for maintaining genetic connectivity. Our approach is highly relevant to vagile wildlife species that are of management and conservation concern and demonstrate varying degrees of IBD and IBR with clinal spatial genetic structure that challenges the delineation of discrete population boundaries. IBD was first described by Wright and indiRangifer tarandus caribou) is What model of population genetic structure best describes boreal caribou across a continuous distribution in western Canada? (2) Do IBR hypotheses associated with anthropogenic features , waterbodies, and wildfires contribute to the observed patterns of genetic variation? and (3) What boundaries best capture the patterns of spatial genetic variation and could be used to delineate MUs for conservation purposes? We apply both aspatial and spatial methods as part of a framework for delineating management units for vagile wildlife species that present a natural clinal pattern of population genetic structure.22.12. Local population boundaries outlined in the federal recovery strategy to identify tracks and foraging sites made by caribou in snow. A field crew subsequently visited each foraging site by helicopter to collect fecal pellets, and coordinates for each collection site were recorded. Our samples from the Boreal Shield ecozone in Saskatchewan were obtained from blood blots or vials collected from individual boreal caribou handled during radio\u2010collaring in the winters 2013\u20132014 across the study area was determined using the individual\u2010based Bayesian clustering program STRUCTURE version 2.3.4 ) and delta K (\u0394K) plots between genetic distance and the spatial distance among individuals. These variables are extracted using \u201cmgQuick\u201d function , observed heterozygosity (Ho), number of alleles across loci (A), allelic richness (Ar), and individual inbreeding coefficient (FIS) along with statistical significance were further calculated using SPAGeDi. Each cluster was further tested for allele frequency deviation from Hardy\u2013Weinberg equilibrium (HWE) and linkage equilibrium (LE) using the probability test in GENEPOP version 4.2 . Due to different levels of anthropogenic disturbance and general landscape heterogeneity across the study area, we tested landscape effects on genetic variation separately for each first\u2010order cluster identified by STRUCTURE and MEMGENE. Landscape variables hypothesized to cause landscape resistance to gene flow for boreal caribou across the study area included main roads, water bodies, and recent wildfires. It is known that various anthropogenic habitat disturbance features affect boreal caribou movements and distribution were a matrix of MEM spatial variables (mgQuick results) describing spatial components of genetic variation and a matrix of dummy explanatory variables coding the polygon membership of samples. The latter partitioned the sampling locations into hypothesized MUs across the study area. Four hypothesized population boundary scenarios were tested delineating the study area into five units. Only scenarios with the same number of units are directly comparable in this analysis, as there is no method available to penalize the addition of dummy variables representing unit membership that will inflate the proportion of variance explained. MU delineations were based on second\u2010order STRUCTURE (Scenario 1) and MEMGENE (Scenario 2) results, as well as local populations outlined in the federal recovery strategy while limiting partitioning of Manitoba into North and South ranges from western Manitoba and East (Cluster 2) Figure a. Furthe3.3K\u00a0=\u00a02 and ranged from 0.01 to 0.08 and Scenario 2 (reflecting second\u2010order MEMGENE results), followed closely by Scenario 4 Table , Scenari44.1FST and number of migrants per generation, which revealed higher genetic differentiation and lower levels of dispersal between clusters found further apart compared with neighboring central clusters. Additionally, the two central clusters Cluster 2A and Cluster 2B had low levels of genetic differentiation and were the only second\u2010order clusters found with loci deviating from HWE, providing further evidence of immigration and non\u2010random mating, and suggesting that individuals in these clusters do not form demographically independent groups. The high level of dispersal and low genetic differentiation identified between Cluster 2A in southeastern Saskatchewan and Cluster 2B in the Manitoba North range supported findings by Ball et al. (FST =0.03) between these areas. Additionally supported by our results was the genetic connectivity previously found in the Interlake and Bog regions in Manitoba and extending into southeastern Saskatchewan , but IBD was still moderate in the eastern part of the study area and within clusters with relatively high levels of landscape fragmentation . The strong level of IBD present within and among clusters suggests that dispersal is based heavily on geographic distance rather than social behavior that would support demographically and structurally independent groups. Because the boreal caribou ecotype is genetically different from neighboring barren\u2010ground and eastern migratory ecotypes (see Kl\u00fctsch et al., The significance of IBD across the study area and within each genetic cluster supported the hypothesis that boreal caribou maintain a natural clinal pattern of genetic structure. Spatial autocorrelation persisted across a large spatial scale supporting previous telemetry\u2010based studies that reported relatively large home range sizes for the ecotype (McLoughlin et al., l et al. that ideMEMGENE, a visualization approach based on the predicted values of a spatial regression rather than on cluster assignment, was able to confirm areas of genetic discontinuity in our study while further identifying finer\u2010scale genetic variation not detected by STRUCTURE. MEMGENE avoids the requirement that spatial genetic clusters are in HWE, which is central to the algorithm in the program STRUCTURE (Pritchard et al., 4.2Further evaluating IBD within a landscape genetics framework aided in explaining many of the spatial genetic discontinuities detected by STRUCTURE and MEMGENE while disentangling the confounding effects of IBR caused by landscape heterogeneity. For example, roads can help explain some of the genetic discontinuity detected between clusters, such as the highway running north between Clusters 1A and 1B in Saskatchewan and the high density of high\u2010use roads and highways in south\u2010central Saskatchewan surrounding Prince Albert National Park. Furthermore, the strong genetic differentiation in the Manitoba South range, known in this study as Cluster 2C, supports previous studies that found roads and waterbodies in this region to restrict boreal caribou dispersal (Ball et al., The contributions of both IBD and natural and anthropogenic landscape features in restricting gene flow were significant, suggesting that the relative importance of these processes in driving boreal caribou population structure can be difficult to partition. Disentangling the effects of IBD and IBR is difficult in regions presenting limiting resistance to gene flow (e.g., Ruiz\u2010Gonzalez, Cushman, Madeira, Randi, & G\u00f3mez\u2010Moliner, 4.3Analyzing multiple MU scenarios provided a comprehensive analysis of population\u2010level delineation based on patterns of genetic variation and revealed that current boreal caribou population units identified in the federal recovery strategy across our study area do not capture population genetic structure of the ecotype. Our analyses of genetic connectivity across the study area further confirmed that discrete population boundaries do not exist. Most evidently, the localized populations in the Manitoba North range have been found to be too genetically connected to be considered as independent herds. Also, range division of north and south Saskatchewan, based on ecozone boundaries, does not reflect the genetic connectivity results captured in our analyses. We therefore recommend that, together with other ecological factors affecting boreal caribou conservation and recovery (e.g., Environment Canada, 5We showed that IBD played a dominant role in shaping spatial patterns of genetic variation across the landscape. Landscape genetics was useful in explaining the genetic discontinuity detected and the contribution of natural and anthropogenic landscape variables in restricting gene flow. As a result, estimating the relative contribution of IBD and IBR on genetic differentiation helped characterize connectivity at and below the population level, revealing a baseline clinal pattern of genetic variation across the study area.Strong IBD results provided challenges for population\u2010level delineation under assumptions of spatial and demographic independence as defined for a local population in the federal recovery strategy. The combined resistance effect of the landscape variables evaluated here, and IBD, was not large enough to cause strong breaks in gene flow and higher level divergence that are needed to classify discrete populations or MUs (Palsb\u00f8ll et al., None declared.MM and PJW conceived and designed the project. Data collection was organized by DH, MM, TT, and PDM. PP analyzed the data. Extension of MEMGENE methods and support for data analysis were provided by PG. Reagents, materials, and other tools for analysis were contributed by PJW. All authors contributed critically to drafts and gave final approval for publication.https://doi.org/10.5061/dryad.7k2g187Data available from the Dryad Digital Repository: \u00a0Click here for additional data file."} +{"text": "Lifelong learning has been regarded as an important factor of promoting active engagement in later life for researchers and policy makers. Most of the studies tend to illustrate old learners as a homogeneous and self-resilient group of people to engage in lifelong learning. Few studies address older learners\u2019 social capital in affecting their decision of engagement and in sustaining their motivation. The study documented the existing social networks of older Singaporeans in lifelong learning programs and illustrated how social networks contributed their participation in learning. The mixed methods consist of in-depth interviews and two network instruments (Name Generator and Position Generator) based on 30 older Singaporeans (between 50 and 79 years old) who attended lifelong learning courses between 2016 and 2018. Interviews are transcribed and analyzed. The network instruments of are quantified and visualized. The findings show that older learners\u2019 networks included a mixture of social ties from family and friends. Learners\u2019 closeness with network members and their living arrangement with them influenced learners\u2019 involvement in learning and future planning. Single respondents who had more non-kin members in the networks reported to be more active due to their weak ties. Overlapping networks among couple learners increase the spousal support for learning. Learners who had wider ranges of social resources are associated with their interest in learning activities. The study suggests that advocating lifelong learning needs to take older adults\u2019 networks into considerations as networks represent the social forces that influence their decisions and motivations."} +{"text": "Simplified Particle Input ConnEction Specification (SPICES) is a particle-based molecular structure representation derived from straightforward simplifications of the atom-based SMILES line notation. It aims at supporting tedious and error-prone molecular structure definitions for particle-based mesoscopic simulation techniques like Dissipative Particle Dynamics by allowing for an interplay of different molecular encoding levels that range from topological line notations and corresponding particle-graph visualizations to 3D structures with support of their spatial mapping into a simulation box. An open Java library for SPICES structure handling and mesoscopic simulation support in combination with an open Java Graphical User Interface viewer application for visual topological inspection of SPICES definitions are provided. A molecular simulation task comprises three successive steps: The definition of a simulation job with all necessary input information (preparation step), the actual loop over discrete integration time steps to numerically solve the equations of motion and the analysis of the simulation record with all calculated results . The first (preparation) step of this triad has to provide data structures that can be leveraged by the algorithms of the second (simulation) step in an optimized manner to allow for a maximum performance of their interplay. This is commonly achieved by definition of adequate sets of arrays that encode all necessary molecular information like spatial positions or bonds of the interacting entities. The content of these arrays is usually provided by large tabular ASCII files that are often (at least partly) edited by hand. An example of these ASCII files may be found at for 1,2-Cheminformatics aims at supporting efficient and errorless human\u2013machine interfaces where adequate molecular structure representations are at heart of the discipline . The majIn order to contribute to the realization of a molecular fragment cheminformatics roadmap this worKey part of this work is a set of methods operating on an intuitive line notation for particle-decomposed molecular structures denoted SPICES (Simplified Particle Input ConnEction Specification). The SPICES design is derived from straightforward simplifications of the well-established SMILES representation for atom-based molecular connectivity \u201323. The The SPICES implementation extends the fragment structure representation proposal in . The synA SPICES representation may contain multiple independent parts , e.g. to represent aggregated molecular structures like the quaternary structure of proteins. Finally a [START] and an [END] tag may be attributed for spatial orientation in the simulation box, see Figs.\u00a0Spices.jar library supports all aspects of SPICES definition and handling. A Spices object may be created with at least an input structure string or in combination with additional information like a map of available particles. A syntax parser analyzes the provided line notation and returns detailed syntax error information if necessary by the methods isValid and getErrorMessage. SPICES properties like the frequency of particles or complete lists of particle neighbors are evaluated upon user request by the methods getParticleFrequencies or getNextNeighbors.The A function of specific importance is the spatial projection of topological SPICES into a simulation box to set up adequate start geometries. Since a mesoscopic simulation is driven by soft particle potentials (in contrast to atomic hard core repulsions for e.g. molecular dynamics), different particles may occupy the same exact spatial position as well as penetrate each other. Thus the possibly severe problems of particle entanglements or caging effects due to inadequate start geometries are considerably attenuated . NonethesetCoordinates and getParticlePositionsAndConnections: After creation of a Spices object from a SPICES line notation string arrays for the first (start) and the last (end) particle positions of all spatial linear 3D tubes as well as the bond length may be provided via the setCoordinates method. The first (start) particles of the linear chains always have the defined start positions whereas the last (end) particles may not necessarily reach the defined end positions if the length of the defined start/end straight line is longer than the accumulated bond lengths of the particles on the longest linear chain so that a 3D tube may be smaller than defined. On the other hand a 3D tube may be squeezed if the length of the defined start/end straight line is smaller than the accumulated bond lengths. Thus the calling code into the simulation box performs in less than a second using an ordinary scientific workstation or even a standard notebook computer.The sketched spatial projection see Fig.\u00a0 is accom file at ). The skWhereas line notations may be regarded as a reasonable compromise for a human\u2013machine interface their definitions are error-prone for complex branched or ring structures. A visual display of the topological particle graph with all its particle\u2013particle connections may considerably alleviate a correct SPICES definition, see Fig.\u00a0GraphStream library [SpicesViewer.jar is a GUI application (on top of Spices.jar and connection library SpicesToGraphStream.jar) for a topological SPICES display with the GraphStream library to analyze the influence of different graph settings and to demonstrate computational functions like zooming or graph image generation. Figure\u00a0SpicesViewer.jar GUI with a manually tailored SPICES graph visualization of the cyclic peptide Kalata B1 with 29 amino acids.A graphical visualization may be achieved by adequate application of open-source projects that provide chemical structure drawing capabilities. For instance the structure-diagram layout of the Chemistry Development Kit (CDK) \u201328 can b library is necesSpices.jar) in combination with a connection library (SpicesToGraphStream.jar) and a Java Graphical User Interface (GUI) viewer application (SpicesViewer.jar) for visual topological inspection and manipulation of SPICES molecule definitions. All libraries/applications are publicly available as open source published under the GNU General Public License version 3 [SpicesViewer GUI application, all Javadoc HTML documentations [This work provides a Java library for SPICES handling and mesoscopic simulation support (ersion 3 . The SPIntations and the ntations source cSpicesViewer GUI application demonstrates relevant use cases in detail with corresponding sample code. The new libraries may be utilized within scripting environments or become part of integrated mesoscopic simulation systems.The presented set of methods may alleviate molecular structure definitions for mesoscopic simulation tasks. The Future developments may address SPICES parsers that especially support the more difficult preparation of polymer systems, e.g. a PDB-to-SPICES parser for peptides and proteins provided in form of PDB files (actually, the SPICES string of the Kalata B1 peptide in Fig."} +{"text": "Controlling massive haemorrhage from morbidly adherent placenta (MAP) at caesarean section is a major surgical challenge to obstetricians. This study compares different intra-operative interventions to control haemorrhage from morbidly adherent placenta and its impact on maternal morbidity.n\u2009=\u200942) had only balloon tamponade, Group B (n\u2009=\u200940) had balloon tamponade and bilateral uterine artery ligation, in Group C (n\u2009=\u200943) all cases were managed by bilateral uterine artery ligation and inverting the cervix into the uterine cavity and suturing the anterior and/or the posterior cervical lips into the anterior and/or posterior walls of the lower uterine segment using the cervix as a natural tamponade.Retrospective analysis was done for baseline characteristics, intra-operative and postoperative complications of 125 patients with morbidly adherent placenta who had elective CS at 35\u201338\u2009weeks gestation in the period from 01/2012 to 01/2017. The included patients were categorized into three groups according to intra-operative interventions they had for controlling bleeding; Group A (P\u2009<\u2009\u00a00.001), less requirement of blood transfusion more than 4\u2009units , significant reduction in prolonged hospital stay over 10\u2009days and lower risk of coagulopathy , visceral injuries and need for hysterectomy .There were no differences of baseline characteristics of patients in all groups. Group C had significantly better outcomes as compared with groups A and B; less total blood loss contains supplementary material, which is available to authorized users. Morbidly adherent placenta (MAP) is one of the major causes of massive obstetric haemorrhage. It is a rare but potentially life-threatening complication of pregnancy. The steady rise of caesarean section (CS) delivery rates in recent years is associated with increasing incidence of both placenta previa and placenta accreta .The inciThe optimal management of placenta accreta spectrum remains controversial. Whilst hysterectomy or conservative management are recommended in case of confirmed MAP during caesarean section IntraopeThere is lack of consensus on the optimal uterine sparing surgical approach to reduce intraoperative bleeding if MAP is partially separated. Whilst electing for timely hysterectomy may be recommended and lifesaving this mayAs intraoperative bleeding from MAP is often massive and dramatically quick resulting in severe maternal morbidity and mortality it is of utmost importance to have a pre-planned approach to this surgical challenge that is effective and swift.In this study we report on our own centre experience of three procedures to try and reduce intraoperative bleeding and report on its effects on maternal morbidity and mortality postoperatively.This is a retrospective study reporting on patients with suspected MAP who had a repeat elective CS in Minia maternity university Hospital, Egypt in the period from 01/01/2012 to 01/01/2017.ClinicalTrials.gov NCT02590484. Registered 28 October 2015.The interventions in the study as a part of the trial that was registered on Patients included in this study were women with at least one previous caesarean section and placenta previa with suspect MAP who were keen to preserve their fertility if possible and were booked for elective repeat caesarean section. Cases were only included if partial separation occurred at CS resulting in major bleeding. The diagnosis of MAP was confirmed by histopathological study of the removed part of the placenta showing deep invasion of chorionic villi and presence of myometrial fibres Fig. .Fig. 1shPatients who had previous CS/ placenta praevia only with no features of MAP were excluded from this study. We also excluded patients with previous CS with placenta praevia/ accreta who required an emergency caesarean section due to major antepartum haemorrhage (APH) and patients who had preoperative diagnosis of placenta percreta who opted to have an elective hysterectomy or when placenta percreta is confirmed intraoperatively.The primary outcome for the study was the total volume of blood loss in the intra and postoperative period and the need for hysterectomy. The secondary outcomes were maternal morbidities composite including coagulopathy, need for massive blood transfusion (>\u20094\u2009units),length of hospital stay >\u200910\u2009days, and visceral injuries.Other secondary outcomes intended to be reported were maternal mortalities if any present and gynaecological complications as amenorrhoea,intrauterine adhesions.The ultrasound features used for suspicion of MAP were as described previously in literature including one or more of the following;Loss or thinning (\u20391\u2009mm) of the normal hypo-echoic retro-placental myometrial plane or thinning or disruption of the hyper-echoic uterine serosa bladder interface or presence of multiple placental lakes .Patients with equivocal ultrasound diagnosis placenta accreta or suspicion of placenta percreta had magnetic resonant imaging (MRI) scan to confirm or refute the diagnosis.The MRI features used to diagnose placenta accreta/percreta were as described previously; uterine bulging, heterogeneous signal intensity within placenta, dark intra-placental bands, focal interruption to myometrial wall,invasion of pelvic structures by placental tissue .All patients fulfilling the inclusion criteria had the following protocol of antenatal and intrapartum management.All patients with suspected MAP included in this study were admitted to inpatient department for close observations if they had a minor APH and administration of corticosteroids if <\u200934\u2009weeks gestation. Patients were given hematinic medications during their admission to keep their haemoglobin level above 12\u2009g/dl. At least 4 Cross matched blood units were always kept available. They were booked for elective repeat CS at 37-38\u2009weeks if they remain asymptomatic or their elective CS brought forward to 35\u201336\u2009weeks if they had any further minor APH.All patients were fully counselled regarding the risk of bleeding and surgical options at delivery if placenta accreta is confirmed including risk of hysterectomy.During the CS, the patients were either in supine or lithotomy position, then opening the skin with a vertical midline or transverse incision. This is followed by opening the anterior abdominal wall in layers. The urinary bladder was dissected downward. For most cases lower segment CS incision was used unless unexpected percreta was noted intraoperatively. After delivery of the foetus a short tentative attempt of delivery of the placenta was performed if it was a suspect MAP to confirm diagnosis. This was not done however if placenta percreta was confirmed intra-operatively.In the majority of patients encountered in our series we have not encountered a total placenta accreta where partial separation of the placenta is not possible.In case of partially separated placenta accreta with ensuing bleeding, further action depended on patient\u2019s fertility wishes and elective hysterectomy was done for patients who completed their family however; for patients who wished to preserve their fertility prompt blood transfusion, available uterotonic agents were injected. Patients had one of three intraoperative surgical interventions that evolved during the study period with group A being the earliest group and group C being the most recent cases. There was however some chronological overlap over these groups depending on the surgeon\u2019s preference and expertise.These procedures were used as primary surgical strategy to control bleeding however in case of continued bleeding from the placental bed; a timely emergency hysterectomy was done. Therefore, patients in this study were divided into three groups according to the intraoperative intervention to control bleeding;In which patients had only Bakri Balloon inserted transabdominally through the CS incision to tamponade the placenta bed in the lower uterine segment after inflation of the balloon with 250\u2009ml of saline and the balloon tubes were brought vaginally with vaginal packs inserted to keep the balloon in situ and avoid early expulsion. Uterus was closed over the balloon which was kept in for 24\u2009h and removed postoperatively in operating theatre. In which patients had bilateral uterine artery ligations as described before , 8. BrieIn this group, a combination of bilateral uterine artery ligations and cervical tamponade by elevating the cervix into the uterine cavity using Allis forceps then suturing the anterior and/or posterior cervical lip (s) into the anterior and /or posterior uterine segment (s) depending upon the site of bleeding by two or three simple interrupted stitches. Hegar dilator was inserted from the uterine cavity to confirm patency of the cervical canal. Closure of the uterine incision after control of bleeding .Additional file 1: Movie S1 Cervical tamponade in placenta accreta. This is a movie file showing detailed steps of cervical tamponade in which the cervical lips are used to control bleeding in placenta accreta. The video was recorded by the authors of this research article. (MP4 12121 kb)In group C, Follow up appointments at 3 and 6\u2009months was given after delivery where revision of history, clinical, speculum and ultrasound examinations was performed.In all patients preoperative, intraoperative and postoperative data were collected and used in this report.The collected data were statistically analysed using SPSS software version 20 .Descriptive statistics were done for continuous variables using mean, standard deviation, while they were done for categorical data by number and percentage.P value \u22640.05).Comparisons for continuous variables were done using one-way ANOVA test and post Hoc Tukey\u2019s Correction and Chi square test was used for categorical variables between groups when the cell contains more than 5, and Fisher exact test when the cell contains less than 5. The level of significance was taken at included 42 patients, Group B 40 patients and group C included 43 patients.There were no statistically significant differences in the baseline characteristics of the patients in the three intervention groups A, B and C as shown in Table P\u2009<\u20090.001).Table\u00a0Table In group C, at 3\u2009months appointment, there were no remarkable clinical, speculum and ultrasound findings in 37 cases. In two patients, the cervical lips were displaced upwards with no pathological coloposcopic and/or hysterocopic findings. Four cases lost follow-up.Thirty five patients (81.4%) were seen at 6\u2009months appointment, where menstruation was resumed in 33 (76.7%) patients while the other ten patients (23.3%) were amenorrheic that could be explained by lactation or another cause and currently they are under follow-up. Contact details of the hospital and research coordinator(s) were given to the patients in case of experiencing unusual symptoms.This study has shown that prompt use of a combination of bilateral uterine artery ligation and cervical tamponade are simple, cost effective and most effective ways of controlling bleeding due to MAP and has led to significant reduction of maternal morbidity and need for hysterectomy.Women included in this report were very keen to preserve their fertility and the options that were acceptable to them were either uterine sparing surgical interventions or conservative management. The latter approach (cutting the cord short and leaving placenta in situ) though has recently been recommended , 9, was A number of other studies have reported on the use of various interventions to stop bleeding following partial separation of MAP. These included preoperative insertion of uterine artery catheters to embolise blood vessels in the placental bed , internaIn 2007, Dawlatly and his colleagues published a case report in which they used the inverted cervical lip(s) to control bleeding from the placental bed and they succeeded to preserve the uterus and patient life . AnotherWe believe that these techniques should be used as a primary line surgical intervention for controlling bleeding due to MAP. The techniques described in this report are readily available in most settings, easy to learn and apply and do not require sophisticated equipment as in uterine artery embolization or special skills and expertise as in ligation of internal iliac artery. These surgical measures should be provided as a part of a comprehensive preoperative and intraoperative care bundle. We suggest using a stepwise approach of intraoperative control of bleeding utilizing combination of uterine artery ligation, balloon tamponade and use cervical inversion as first step. This can be escalated to other techniques as internal iliac artery ligation and/or uterine artery embolization if first line measures are inadequate with prompt timely recourse to hysterectomy as a life-saving measure if all other measures proved futile.The strenghths of our study is its comprehensive preoperative diagnostic work-up and comprehensive reporting on techniques with significant impact on maternal outcomes and relatively large sample size for this uncommon obstetric problem. Our study is limited by its retrospective nature which could have introduced an element of performance bias. This may have led to the apparent shortening of operating time in the group C as these patients were the most recent group with possible better performance as experience accumulating in dealing with similar emergencies in the other two groups. Although all groups were not significantly different in known baseline confounding variables it is possible that with accumulating radiological experience and more confident diagnosis of MAP, Ultrasound -confirmed cases had more directed counselling for hysterectomy leaving only suspected cases with partial accreta in the late stages of study period. This may have introduced an element of selection bias.A prospective controlled study to address these limitations however due to the uncommon nature of the problem and impact of other factors would be difficult to conduct.The combination of bilateral uterine artery ligation and using the cervix as a natural tamponade are very effective and simple methods in controlling bleeding resulting from partially separated MAP. Use of these surgical techniques in combination with other preoperative and intraoperative measures have led to less risk of maternal morbidities."} +{"text": "Acute decompensated heart failure (ADHF) is a difficult clinical problem with poor outcomes. High in-hospital mortality and readmissions remain a concern despite advancement of treatment options for heart failure , 2. The The previously dreaded ventricular arrhythmias in chronic heart failure have been managed much more effectively since the introduction of the implantable cardioverter\u2013defibrillators (ICDs) . HoweverRenal dysfunction in a heart failure patient is common and cardiorenal syndrome is now an established term. ADHF often brings with it worsening renal function and acute kidney injury that can raise concerns about the use of the much needed intravenous diuretic therapy as well as demand adjustment in ACE inhibitors, angiotensin receptor antagonists, or sacubutril valsartan . ProgresAlthough research involving hibernating myocardium is almost exclusively confined to the stable chronic heart failure population, the heart failure clinician frequently encounters it in ischaemic acute heart failures with a troponin rise. Ryan and Perera highlight the importance of considering coronary angiography if appropriate and where possible in ADHF for better management planning. We are also informed of the important REVIVED-BCIS2 trial, a UK multicentre study that randomises ischaemic heart failure into revascularisation by percutaneous intervention or optimal medical treatment and incorporates hibernating myocardium.The reviews in this series on complicated acute heart failure syndromes bring up exciting future directions in managing these difficult subsets which often overlap. Tackling these subsets effectively is likely to go a long way in reducing mortality and hospitalisation rates."} +{"text": "The decision to give glucocorticoids to patients who have septic shock is difficult because of conflicting randomized controlled trial (RCT) level I evidence. Nonetheless, there is some evidence of overuse of corticosteroids in septic shock.against corticosteroid use in patients who have responded adequately to norepinephrine [for corticosteroids in patients who do not respond to norepinephrine in septic shock.Early cohort studies found that there was an acquired corticosteroid deficiency in septic shock. A subsequent RCT found thnephrine . Similarnephrine recommenn\u00a0=\u2009164) of children who had systemic inflammatory response syndrome (SIRS), sepsis, or septic shock. The GCR expression levels were lower and the serum cortisol levels were higher in patients who had poorer outcomes. Where does this study leave the clinician who cares for patients with septic shock?One reason for conflicting evidence regarding responses to corticosteroids in septic shock is that different patients have different genomic, transcriptomic, and proteomic profiles that define different responses to corticosteroids in septic shock. Alder and colleagues recently made the hypothesis that peripheral leukocyte glucocorticoid receptor (GCR) expression and serum cortisol levels correlate with the response to glucocorticoids in pediatric septic shock (REF). They measured these biomarkers in a modest size prospective cohort \u2014and even establishing a gold standard for the diagnosis of septic shock . Alder aWhat are the next steps for validation of GCR expression as a predictive biomarker of corticosteroid administration in septic shock? Corticosteroids need to be evaluated in RCT(s) that are adequately powered to detect a significant interaction between (1) GCR expression and (2) use\u2014or not\u2014of corticosteroids. Some would argue, in part because of the storied controversy of steroids in septic shock, for a second confirmatory RCT to validate a GCR expression predictive biomarker. Recently completed RCTs such as those by Annane and colleagues and VenkAfter such confirmation and prior to widespread clinical use, many would recommend regulatory approval of a clinically validated GCR kit.We and others have similarly addressed predictive biomarkers such as genomics , cytokinpersonalize treatment with corticosteroids, vasopressin, and norepinephrine in septic shock , and protein levels could ock Fig.\u00a0 and even"} +{"text": "Breast cancer related lymphoedema (BCRL) is a common side effect of cancer treatment. Recently indocyanine green (ICG) fluorescent lymphography has become a popular method for imaging the lymphatics, however there are no standard protocols nor imaging criteria. We have developed a prospective protocol to aid in the diagnosis and therapeutic management of BCRL.Lymphatic imaging procedures were conducted in three phases. Following initial observation of spontaneous movement of ICG in phase one, manual lymphatic drainage (MLD) massage was applied to facilitate ICG transit via the lymphatics in phase two. All imaging data was collected in phase three. Continuous lymphatic imaging of the upper limb was conducted for approximately an hour and lymphatic drainage pathways were determined. Correlations between the drainage pathway and MD Anderson Cancer Centre (MDACC) ICG lymphoedema stage were investigated.One hundred and three upper limbs with BCRL were assessed with this new protocol. Despite most of the patients having undergone axillary node dissection, the ipsilateral axilla drainage pathway was the most common (67% of upper limbs). We found drainage to the ipsilateral axilla decreased as MDACC stage increased. Our results suggest that the axillary pathway remained patent for over two-thirds of patients, rather than completely obstructed as conventionally thought to be the case for BCRL.We developed a new ICG lymphography protocol for diagnosing BCRL focusing on identification of an individual patient\u2019s lymphatic drainage pathway after lymph node surgery. The new ICG lymphography protocol will allow a personalised approach to manual lymphatic drainage massage and potentially surgery. Breast-cancer related lymphoedema (BCRL) is a common side effect of cancer treatment causing physical, functional, psychological and financial challenges for individuals and impacting their quality of life \u20134. LymphRecently, Indocyanine Green (ICG) lymphography has become an alternate popular method for imaging the lymphatics. ICG lymphography was initially used for breast sentinel node biopsy . Its appThe above lymphoscintigraphy criteria for lymphoedema diagnosis cannot be applicable for ICG lymphography because penetration of the near infrared rays is limited to 2\u2009cm from the skin surface making it difficult and inconsistent to identify lymph nodes . HoweverDue to the requirement of new imaging criteria for the diagnosis of lymphoedema using ICG lymphography, we have developed a prospective protocol to aid in the diagnosis of BCRL, assist decision making for therapeutic management including ICG-directed manual lymphatic drainage (MLD) massage and define selection criteria for surgical options. The aim of this study to summarise initial findings obtained by the new ICG lymphography protocol in breast cancer related lymphoedema.A retrospective cohort audit was conducted, reviewing prospectively collected data from patients with BCRL who underwent ICG lymphography at the Australian Lymphoedema Education, Research and Treatment (ALERT) clinic at Macquarie University (MQ) between February 2017 and April 2018. Data were sourced from electronic medical records and this audit was approved by MQ Health Ethics Committee (Reference: MQCIA2018017). Written informed consent was obtained from all patients in this study.In three patients for the pilot study, we repeated the ICG imaging after 24\u2009h and compared with the images obtained with this protocol. If the patients had previous lymphoscintigraphy in the affected limb, both lymphoscintigraphy and ICG lymphography images were compared.The near infrared camera system was used for this study. Indocyanine Green was mixed with 5\u2009ml of saline. Four injection sites were used in the distal aspect of the upper limb on the affected side: first and fourth web spaces and ulnar and radial volar wrist regions Fig.\u00a0. These cIntradermal injections were performed with a 30-gauge needle and a 1\u2009ml syringe. At each injection site 0.05-0.1\u2009ml (0.25-0.5\u2009mg) of ICG solution was administered. A cryogenic numbing device was used immediately before each injection to reduce needle discomfort .Lymphatic scanning using the near infrared camera commenced immediately after the injections and imaging data was recorded using a digital video recorder . Lymphatic imaging of the upper limb was continuously conducted in each upper limb for approximately an hour.Lymphatic scanning was conducted in three phases.Observation of any spontaneous movement of ICG via the lymphatics for approximately 10\u2009min. Patients were encouraged to clench and unclench their hand ten times to facilitate lymphatic uptake of the ICG.Manual lymphatic drainage (MLD) massage was then performed by an accredited lymphoedema therapist to facilitate ICG transit via the lymphatics. This MLD is undertaken by the therapist and the patient\u2019s real time visualisation of the lymphatic vessels and areas of dermal backflow provides patient feedback of direction, speed and skin pressure required to move the ICG dye. Scanning focused on identifying lymphatic vessels and the competency of their valves, direction of dermal backflow extension, and identifying lymph nodes. We found that MLD facilitated dye movement more efficiently compared to post-injection exercise and delayed scanning although this was not formally evaluated. When lymphatic vessels were identified, their course was marked on the patient\u2019s skin with a coloured pen Fig. a. Phase Demarcation lines of dermal backflow were marked on the skin, and collection of imaging data through still photography with both near infrared and digital cameras were taken. to provide an image of the whole upper limb. The lymphatics were designated into two categories; lymphatic vessels in the subcutaneous tissue and dermal backflow which is reflux of lymph fluid into dermal lymphatics. Lymphoedema was diagnosed by the presence of dermal backflow. Although ICG lymphography was considered mainly for imaging the superficial lymphatics, the following observation helped us to interpret the images. For example, if the epitrochlear lymph node was identified in the medial elbow, the efferent lymphatic vessel of the node was known to run along the brachial artery in the upper arm . AlthougLymphatic drainage pathways were determined by the location of identified lymph nodes or extension of ICG to the skin regions via dermal backflow or lymphatic vessels where lymph nodes were located underneath. Lymphoedematous upper limbs were also classified by MDACC stage as 0: normal lymphatics, Stage 1: many patent lymphatic vessels with minimal patchy dermal backflow, Stage 2: moderate number of patent lymphatic vessels with segmental dermal backflow, Stage 3: few patent lymphatic vessels with extensive dermal backflow involving the entire arm, Stage 4: no patent lymphatic vessels seen with dermal backflow involving the entire arm with extension to the dorsum of the hand and Stage 5: ICG does not move from injection sites , 19.One hundred and seven upper limbs at-risk or affected by BCRL were examined in 103 patients . Three patients who were previously diagnosed with unilateral BCRL were found by our imaging criteria to demonstrate normal lymphatics (MDACC Stage 0) and an additional bilateral patient who had normal lymphatics on the side of the sentinel node biopsy were excluded. Our study cohort therefore consisted of 103 upper limbs examined in 100 patients. Patient characteristics are described in Table\u00a0We could frequently specify sites in the upper limb where the lymphatic vessel was obstructed. Dermal backflow was identified at these obstruction sites extending through the dermal lymphatics demonstrated more than one drainage pathway. Variations of drainage pathway patterns are summarised in Table\u00a0% demonstThe diagnosis of lymphoedema is often difficult by physical examination alone, especially in early stages. Patients with BCRL often complain, for example, of discomfort in specific areas of their upper limb instead of uniform changes or swelling in the whole limb. In this study, we introduced a new ICG lymphography protocol for the upper limb to help to identify areas with underlying anatomical changes that occur in lymphoedema. Our previous review study found that ICG lymphography had the potential benefit to elucidate the relationship between lymphatic drainage pathway and severity of lymphoedema . The draLymphoscintigraphy has been the standard imaging examination for lymphoedema , 22. HowAnother advantage of ICG lymphography is that some patients may indeed not suffer from lymphoedema. In our study four limbs in four patients were diagnosed as not having BCRL as they had normal lymphatic drainage without any dermal backflow. Future research should address the correlation of ICG lymphography with subclinical lymphedema detected by bioimpedance spectroscopy. Further, there is often a misconception that lymphatic drainage occurs away from the dissected axilla. In Abe\u2019s lymphangiography studies 13 of 19 patients (68%) showed patent lymph vessels passing through the axilla . This waConventionally, BCRL was thought to be caused by the complete obstruction of the lymphatic drainage to the ipsilateral axilla secondary to surgical and/or radiation intervention. Our results contradict this notion and suggest that the axillary pathway was restricted functionally instead of complete obstruction in over two-thirds of patients.We developed a new ICG lymphography protocol for diagnosing BCRL focusing on identification of an individual patient\u2019s lymphatic drainage pathway after lymph node surgery to guide MLD and to assist with selection criteria for lymphatic microsurgery. ICG imaging combined with MLD will allow a personalised approach to lymphoedema care.Additional file 1. Dermal backflow was identified with ICG lymphography.Additional file 2. Gentle MLD could move ICG via lymphatic vessels in mild lymphoedema.Additional file 3. Firmer MLD could move ICG via dermal backflow in severe lymphoedema."} +{"text": "Television viewing is a risk factor for obesity and poor physical health. By contrast, close ties to family and friends in late life are often beneficial. This study examined associations between social engagement and television viewing. Participants (N = 313) from the Daily Experiences and Well-being Study completed an initial interview about their social partners and participated in a 5 to 6 day intensive data collection including Ecological Momentary Assessments about their social contact and activities every 3 hours. Participants also wore Electronically Activated Recorders (EAR) which captured snippets of sound in the environment. Multilevel models using self report and EAR data revealed that participants were more likely to watch TV when they were with close family members than with friends or acquaintances. Findings from these multiple methods suggest that close family may encourage risks as well as benefits in late life."} +{"text": "Acridocarbus orientalis from Al Ain and Oman.\u201d The correct title should be \u201cAntioxidant, Lipoxygenase and Histone Deacetylase Inhibitory Activities of Acridocarpus orientalis from Al Ain and Oman.\u201dThe Molecules Editorial Office wishes to make the following erratum to this paper . There iWe apologize for any inconvenience caused to the readers by this mistake."} +{"text": "Clinicians rely on care partners to provide health care at home for people with dementia, who typically have multiple chronic conditions in addition to progressive cognitive decline. We examined the accuracy of care partners\u2019 knowledge of care recipients\u2019 medical conditions and medications, using a benchmark of \u2265 80% match. Of 100 care partners of people with dementia who were recently hospitalized for a major medical illness, nearly all rated their knowledge as high, but about half did not correctly identify care recipients\u2019 medical conditions or know medications, and one fourth did not understand the purpose for which medications were given. A key predictor of poor objective knowledge was care partners\u2019 cognitive status. These findings highlight the importance of objective assessment of care partner knowledge and skills by clinicians who provide health care and advance care planning for people with dementia."} +{"text": "The CAregiver Perceptions About CommunIcaTion with Clinical Team members (CAPACITY) instrument measures perceived quality of communication with the health care team and the extent to which caregivers believe that the health care team considers their capacity and preferences in decision-making. A higher score reflects higher perceived quality. This presentation highlights features of CAPACITY scores in a national survey of care partners of persons with unexplained cognitive impairment who sought an Amyloid PET scan. A positive scan (presence of amyloid) indicates probably Alzheimer\u2019s (n=1746). In addition to presenting its psychometric properties in this new sample, discussion focuses on the relationship between CAPACITY scores and a process measure of care that matters \u2013 accurate reporting of a new diagnosis. The CAPACITY score reflects an important domain of care quality that could be used more broadly to reflect the caregiver-centeredness of health care experiences and to predict patient outcomes."} +{"text": "Despite the beneficial effects of the SASP on certain physiological events such as wound healing and tissue repair, more studies have demonstrated that senescent cells can substantially contribute to pathological conditions and accelerate disease exacerbation, particularly cancer resistance, relapse and metastasis. To limit the detrimental properties while retaining the beneficial aspects of senescent cells, research advancements that support screening, design and optimization of anti\u2010aging therapeutic agents are in rapid progress in the setting of prospective development of clinical strategies, which together represent a new wave of efforts to control human malignancies or mitigate degenerative complications.Cellular senescence is a typical tumor\u2010suppressive mechanism that restricts the proliferation of premalignant cells. However, mounting evidence suggests that senescent cells, which also persist Senescent cells display several distinct features including a flattened and enlarged morphology, DNA segments with chromatin alterations reinforcing senescence (DNA SCARS), nuclear heterochromatin foci and senescence\u2010associated \u03b2\u2010galactosidase activity and therapy\u2010induced senescence (TIS) (Sieben, Sturmlechner, Sluis, & Deursen, In clinical medicine, anticancer agents not only triggers significant apoptosis of cancer cells but also causes substantial damage in the TME and induces typical TIS of the resident stromal cells, which cause therapeutic resistance via secretion of the SASP factors (Chen et al., in vitro and liver regeneration of a treatment\u2010inducible OIS mouse model in vivo, thus raising the possibility that transient therapeutic delivery of senescent cells could be harnessed to promote tissue regeneration (Ritschka et al., A new function of the SASP was recently discovered, which is linked with increased expression of stem cell markers and keratinocyte plasticity upon short term exposure of cells to the SASP ex vivo evaluation in ovarian tumor samples (Lheureux et al., Several recent studies provided a series of pilot evidence in specific clearing senescent cells, including single or dual treatment of senescent cells with quercetin/dasatinib, and pan\u2010BCL inhibition with ABT\u2010263/ABT\u2010737 (Chang et al., in vivo may serve as a \u201cmolecular\u201d marker for disease occurrence and guide patient stratification (Demaria et al., Cellular senescence occurs throughout lifespan, and senescent cells are beneficial to certain physiological and pathological processes including embryonic patterning, tissue repair, wound healing and immune surveillance. However, as address above, a steady accumulation of senescent cells in the tissue has adverse consequences, ultimately enhancing clinical morbidity. Thus, the abundance of senescent cells in vivo toxicity but enhancing overall efficacy. Such a therapeutic modality is desirable and holds the potential to enhance patient treatment efficacy while reducing adverse side effects that can be observed upon administration of each agent in a single dose. Finally, targeting senescent cells while simultaneously promoting tissue regeneration represents an optimal solution to remove senescent cells from individuals particularly those with advanced diseases or at later stage in life. In doing so, we are getting even closer to achieving the goal of a real \u201chealthy\u201d therapy against human cancer and aging.Despite all the recent findings from senescence and cancer research, there are several caveats before we move forward. Agents targeting senescent cells, especially SASP inhibitors, should be investigated meticulously to ensure continued maintenance of cell cycle arrest, as bypassing the crisis can inevitably promote carcinogenesis. As senescent cells also have certain health\u2010promoting functions, identification of the beneficial components of the SASP could lead to development of optimal strategies that preserve vital factors while depleting their detrimental counterparts derived from senescent cells. As a technical issue, achieving the balance between deleterious and beneficial impact of senolytics in cancer patients requires careful and rational design of administration regimens such as classic chemotherapy followed by senolytic treatment, each provided in metronomic cycles to minimize None Declared."} +{"text": "Lung cancer is the leading cause of cancer mortality in Australia. Guidelines suggest that patients with suspected lung cancer on thoracic imaging be referred for urgent specialist review. However, the term \u201csuspected\u201d is broad and includes the common finding of lung nodules, which often require periodic surveillance rather than urgent invasive investigation. The British Thoracic Society recommends that a lung nodule with a PanCan risk >\u200910% be considered for invasive investigation. This study aimed to assess which factors influence general\u00a0practitioners (GPs) to request urgent review for a lung nodule and if these factors concur with PanCan risk prediction model variables.A discrete choice experiment was developed that produced 32 individual case vignettes. Each vignette contained eight variables, four of which form the parsimonious PanCan risk prediction model. Two additional vignettes were created that addressed haemoptysis with a normal chest computed tomography (CT) scan and isolated mediastinal lymphadenopathy. The survey was distributed to 4160 randomly selected Australian GPs and they were asked if the patients in the vignettes required urgent (less than two weeks) specialist review. Multivariate logistic regression identified factors associated with request for urgent review.p\u2009<\u20090.0001), larger nodule size, presentation with haemoptysis or weight loss , recommendation for urgent review by the reporting radiologist and female GP gender . In low risk lung nodules (PanCan risk <\u200910%), there was significant variability in perceived sense of urgency. Most GPs (83%) felt that a patient with haemoptysis and a normal chest CT scan did not require urgent specialist review but that a patient with isolated mediastinal lymphadenopathy did (75%).Completed surveys were received from 3.7% of participants, providing 152 surveys (1216 case vignettes) for analysis. The factors associated with request for urgent review were nodule spiculation (adj-OR 5.57, 95% CI 3.88\u20137.99, Future lung cancer investigation pathways may benefit from the addition of a risk prediction model to reduce variations in referral behavior for low risk lung nodules. Lung cancer is the fifth most common cancer in Australia but is the leading cause of both cancer related mortality and morbidity , 2. SmokGiven the poor outcomes for many lung cancer patients and the ongoing costs to the healthcare system, best practice guidelines have been created to streamline and standardise the lung cancer diagnosis and treatment pathway. These pathways emphasise timely review and early involvement of a lung cancer specialist , 4. BothThe term \u201csuspected\u201d lung cancer is used in both Australian pathways but is not clearly defined and thus may incorporate lung nodules, where the risk of malignancy is low and the need for specialist review may not be urgent. A lung nodule is defined as a rounded or irregular opacity, measuring less than 30\u2009mm in diameter . With thBoth local and international research suggests that adhering to the suggested timeframes for urgent review of suspected lung cancer can be challenging. An audit of the respiratory service at our hospital several years ago demonstrated that 73% of suspected lung cancer referrals were reviewed within two weeks . HoweverThe PanCan (or Brock University) model is a probabilistic risk-based model that calculates the malignancy risk of a pulmonary nodule detected on first screening low dose CT scan, using both radiographic and patient characteristics. The model has an area under the curve of more than 0.90 and shows excellent predictive accuracy for lung cancer even in small nodules . The BriIn view of the high prevalence of lung nodules, the overall low cancer risk and the lack of clarity in published pathways surrounding the need for urgent specialist review, this study was designed to assess the referral behaviors of Australian general practitioners (GPs) using a discrete choice experiment (DCE) approach.To identify factors that influence GPs to request urgent specialist review for patients with a lung nodule on CT scan.To assess the proportion of GP responses that request urgent review for lung nodules with a PanCan risk >\u200910%.To assess the proportion of GPs requesting urgent review in the specific clinical scenarios of a) A patient with haemoptysis and a normal chest CT and b) A patient with mediastinal and hilar lymphadenopathy without a parenchymal lung lesion.Urgent review was defined as review by a lung cancer specialist within two weeks of referral for suspected lung cancer.The BTS recommendation that a lung nodule with a PanCan risk >\u200910% be considered for further investigation with positron emission tomography (PET) scan and / or biopsy was used as a surrogate for needing urgent (within two weeks) specialist review.An orthogonal main effects plan (OMEP) was used to create 32 individual lung nodule case vignettes. An OMEP is a set of combinations of dimensions and levels where, for dimension, the number of times each pair of levels appear is constant. Therefore, for scenarios where (for example) lung nodule spiculation is observed, the location of the nodule is split equally between upper lobe and not upper lobe. Each vignette included eight variables, four of which form the parsimonious PanCan risk prediction model . The stuEach GP was also presented with two additional case vignettes practitioner database. The email invite included some background information about the Cancer Council OCP and a link to the Qualtrics survey platform, where the GP would be presented with eight randomly selected case vignettes, as well as the additional vignettes regarding haemoptysis and lymphadenopathy. Consent was implied if the survey was completed. Reminder emails were sent at two and four weeks. Participants had the option of providing their contact details to win one of five $100 incentive vouchers. Ethics approval for the study was granted by the St John of God Human Research Ethics Committee (reference 1302).p values >\u20090.05. Ordinal vignette and demographic variables were considered as continuous independent variables for the model. Statistical analysis was performed using the SAS university edition with SAS Studio version 3.8 .A multivariate logistic regression model was used to identify factors significantly associated with request for urgent review that initially included all vignette and demographic variables and then applied stepwise exclusion of factors with A total of 4160 surveys were distributed and 157 GPs began the survey, giving a response rate of 3.8%. Five participants did not complete the survey and were excluded from further analysis, giving a final sample size of 152 GPs (3.7%). The participants\u2019 demographic information is described in Table\u00a0p\u2009<\u20090.0001). Increasing nodule size was also associated with request for urgent review , as was recommended urgent respiratory review from the reporting radiologist . Patient factors associated with request for urgent review included being a current smoker and presenting with haemoptysis or unintentional weight loss . Increasing patient age made request for urgent review less likely .The factors associated with request for urgent review are summarised in Table\u00a0Female GPs were almost twice as likely to request urgent review as male GPs and GPs who worked more hours per week were less likely to request urgent review .From the 1216 vignettes analysed, almost two thirds of responses (65%) were concordant with the BTS guidelines and correctly identified high or low risk lung nodules. The relationship between PanCan risk and the proportion of responses requesting urgent review is presented in Fig.\u00a0When asked if a patient with haemoptysis and a normal chest CT needed to be seen by a specialist within two weeks, most GPs answered \u201cNo\u201d (83%). When asked the same question of a patient with mediastinal and hilar lymphadenopathy but no parenchymal lung lesion, three quarters answered \u201cYes\u201d (75%).This study has examined for the first time the referral patterns for lung nodules amongst a cohort of Australian GPs and identified factors that influence a perceived need for urgency. There is evidence of wide variation in practice, presumably due to interpretation of nodule risk, as well as participant characteristics (such as gender and number of hours worked per week).Some of the factors associated with request for urgent review in this study were in keeping with recommended practice, whilst others were not. Table\u00a0This study has demonstrated that the radiology report and recommendation of the reporting radiologist has a significant impact on GP referral behavior and this finding has also been seen in other literature. Blagev et al. reviewed 1000 CT pulmonary angiogram reports and found that appropriate follow up of incidental pulmonary nodules increased from 0 to 29%, when the reporting radiologist provided an overt suggestion in the conclusion of the report, compared with the nodule only being mentioned in the body of the report . SimilarFemale GPs were almost twice as likely to request urgent review as their male counterparts and this finding has been seen to a lesser extent in Canadian literature. Three papers found that female primary care physicians made 8% more referrals and were 12 and 15% more likely to refer than males \u201319. The Two specific and clinically important scenarios that are not addressed in the Australian Cancer Council OCP or the GP investigation pathway are patients with haemoptysis and a normal chest CT and patients with mediastinal lymphadenopathy without a parenchymal lung lesion. Most GPs surveyed did not think a patient with small volume haemoptysis and a normal chest CT required urgent specialist review and this is in accordance with both local and international guidelines . The haeOverall, this study found more variation in responses in lung nodules with a PanCan risk <\u200910%, with up to 94% of responses still requesting urgent review of these low risk nodules. This may be because the investigation pathway recommended for Australian GPs suggests urgent review in any new or changing nodule, regardless of other clinical or imaging risk factors . Lung noThere are a number of limitations that should be considered in interpretation of this study. The low response rate (3.7%) may have led to selection bias, although participating GPs were representative of all the Australian States and Territories, except the Northern Territory. Response rates to physician surveys are often poor and have been reported to be declining, due to a number of factors, including lack of time, ineligibility and inaccuracy in registration details , 29. IntThis study demonstrates variability in the sense of urgency for referral for lung nodules and highlights that most concern is driven by nodule spiculation, patient presentation with haemoptysis or weight loss and the recommendation of the reporting radiologist. Standardised reporting of lung nodules by radiologists and the addition of estimated nodule risk to lung cancer investigation pathways may help to ensure an effective, timely patient journey from chest CT to specialist review and ensure that all patients are being managed in a more evidence-based fashion.Additional file 1. Lung nodule case vignettes."} +{"text": "Plasmodium vivax predominated (96%). Autochthonous malaria cases emerged in areas previously malaria-free. Heightened malaria control and a response to this humanitarian crisis are imperative.Mass migration from Venezuela has increased malaria resurgence risk across South America. During 2018, migrants from Venezuela constituted 96% of imported malaria cases along the Ecuador\u2013Peru border. Plasmodium spp. and transmitted by Anopheles mosquitoes, characterized by fever and hemolysis with chronic and fatal potential were detected in adults in El Oro Province and reported to the Ecuadorian Ministry of Health remain abundant in this area complicate treatment of dormant hypnozoites that cause relapse .We cannot definitively state whether the migrants from Venezuela were exposed to malaria in Venezuela or during transit. Regardless, this population represents a highly vulnerable group with complex treatment issues. Malaria should be considered in the differential diagnosis for febrile patients from Venezuela and for local populations in nearby parts of South America. The transience of the migrant population presents treatment follow-up issues. The incubation period for patients often exLocal ministries of health responded quickly to these cases and implemented case surveillance. However, reductions in resources after elimination of local malaria transmission in 2011\u20132012 severely limited malaria control efforts in Ecuador and Peru. Imported cases of malaria at the Ecuador\u2013Peru border region pose a serious threat of continued resurgence in local transmission. We urge international solutions for Venezuela\u2019s humanitarian crisis and augmentation of infectious disease surveillance and control along migration routes and in surrounding regions.Additional details on effects of political instability in Venezuela on malaria resurgence at the Ecuador\u2013Peru Border, 2018."} +{"text": "Surgical resection of the esophagus combined with neoadjuvant chemoradiotherapy is a generally recommended treatment strategy with curative intent for patients with non-metastasized esophageal cancer.max of the initial positron emission tomography/computed tomography as independent prognostic factors for early recurrence after trimodality therapy.The current study identified gender, poor tumor differentiation grade, signet ring cell adenocarcinoma, baseline clinical nodal status, and SUVAn important caveat of this prediction model based on clinicopathological characteristics is that it is not 100% accurate. As such, avoiding surgery based on its predictions may cause the very undesirably result of a missed opportunity for cure in patients with locoregional disease only. Further improvement in prognostication is desired to accurately stratify patients by potential benefit of surgery balanced with risk of occult distant metastases. In this context, quantification of circulating tumor DNA represents a novel approach for disease burden quantification that has the potential to complement prognostication in esophageal cancer. Furthermore, recent studies have shown that functional magnetic resonance imaging"} +{"text": "Currently, approximately 44 million people in the United States carry the weight of over 1.4 trillion dollars of student loan debt. As the cost of education continues to rise, the decision of taking on student loans is increasingly a family decision rather than an individual one. While the majority of research focuses on younger borrowers, little research has been done to understand the experiences of parents and grandparents taking on student loans for a loved one. In order to financially and emotionally manage this burden, borrowers may benefit from support from their social networks, including family and friends. For many, navigating these difficult conversations presents a challenge of its own. This presentation will spotlight an MIT AgeLab mixed methods study about how student loan borrowers between the ages of 51 and 75 experience and manage their student loans within family systems and how these loans may impact family dynamics. Data were collected through focus groups and a national survey. Preliminary findings suggest that older borrowers demonstrate several distinct communication typologies within their families in regards to finances, particularly regarding student loan accrual and repayment. Each of the four primary communication styles regarding loans impact borrowers\u2019 financial and emotional wellbeing throughout the life course, as well as perceived relationship dynamics. Moreover, older borrowers are more likely to report family conflict if student loans are less frequently discussed with family members. Findings also suggest strategies to help parents and grandparents facilitate conversations abound student loans based on their unique family communication styles."} +{"text": "Little is known about older adults\u2019 perspectives on measuring functional status . This study used a qualitative design to understand older Veterans\u2019 perspectives on measuring function in primary care settings. Thematic analysis of interviews conducted with 28 Veterans \u226565 years and 5 caregivers from one VA Medical Center identified several themes including: 1) importance and relevance of discussing function; 2) preferences for assessment method and 3) wording of questions . These findings suggest that effective approaches to measuring function must consider patient preferences on content and format and ensure that measurement is used to inform care. We applied these findings to develop an interprofessional intervention to improve functional status measurement for older Veterans in primary care."} +{"text": "Daily experience tells us that breast cancer can be controlled using standard protocols up to the advent of a relapse. Now new frontiers in precision medicine like liquid biopsy of cell free DNA (cfDNA) give us the possibility to understand cancer evolution and pick up the key mutation on specific cancer driver gene. However, tight schedule of standardized protocol may impair the use of personalized experimental drugs in a timely therapeutic window.Here, using a combination of deep next generation sequencing and cfDNA liquid biopsy, we demonstrated that it is possible to monitor cancer relapse\u00a0over time. We showed for the first time the exact correspondence from the increasing clonal expansion and clinical worsening of metastatic breast cancer.Thanks to liquid biopsy may be possible to introduce new experimental drugs in the correct therapeutic window which would lead in the near future to an effective treatment which otherwise remains challenging. PIK3CA mutations are found in 27% of cases of disease progression in breast cancer and CDKN2A . Five months later, a second cfDNA analysis highlighted an exponential increase of clones with the same pathogenic mutations driver mutations at late relapse . LikewisData reported here indicate that there is a tight therapeutic window useful for counteract final clonal expansion and that the minimally invasive cfDNA analysis allows a close and dynamic monitoring of clonal evolution. This is also supported by the mathematical model developed by Khan et al. . In our In conclusion, we demonstrated that multiple points cfDNA analysis reflects clonal evolution and allows track the evolving molecular landscapes of growing cancer cells by capturing broader molecular alterations that could hinder targeted treatments efficacy. The shorter turnaround time of cfDNA analysis and its high sensitivity and specificity are key factors to provide novel opportunities for adaptive personalised therapies, optimizing healthcare resources and enabling higher treatment efficacy and lower side-effects."} +{"text": "Our aim in this study was to explore whether and how siblings\u2019 marital and work status influence Japanese adult children\u2019s perceived responsibility for parental care. Within traditional familial institutions in Japan, married sons were expected to assume parental care responsibility. At the same time, such care arrangements built on gendered division of labor; sons served as family breadwinners, and their wives cared for their parents-in-law while out of the paid labor force. Yet, because of sociodemographic shifts such as a greater percentage of unmarried persons and a growing number of women who seek to maintain their job, it has been increasingly unclear which adult children can and should assume the role of parental caregiver. Using online survey data from 989 Japanese adult children who were all employees with no parental care experiences ever, we sought to clarify the influences of siblings\u2019 circumstances on whether these children anticipated assuming responsibility for conducting different care tasks for their parents. In doing so, we focused on how siblings\u2019 gender and work and marital status might combine to affect adult children\u2019s anticipation of parental care responsibility. A series of logistic regression analyses revealed that having a married brother made it less likely for adult daughters to anticipate assuming responsibility for conducting typical care tasks whereas for adult sons, having a single sister declined such anticipation. We discuss our findings in terms of how traditional familial institutions still impinge on Japanese adult children\u2019s views of parental care responsibility."} +{"text": "Long standing ostomy related complications such as parastomal hernia and stoma prolapse may be at a higher risk of developing spontaneous rupture and evisceration, especially in patients suffering from chronic cough. Such patients may need early refashioning of the stoma to prevent this serious complication. Parastomal evisceration is a very rare complication of stomas and to date, only few cases have been reported in the literature.A 51\u2009year old patient with chronic obstructive pulmonary disease (COPD) and extensive hidradenitis suppurativa of the perineum underwent a temporary defunctioning loop sigmoid colostomy and subsequent perineal skin excision and skin grafting. The ostomy was complicated by a parastomal hernia and stoma prolapse 6\u2009weeks post operatively. Five months later he developed spontaneous rupture of parastomal hernia and evisceration of small bowel. Urgent surgery was done and reduction of small bowel loops and re-siting of the sigmoid colostomy was done.Parastomal evisceration is an extremely rare life threatening stoma-related complication which requires urgent treatment. The creation of a permanent or temporary stoma is associated with varying complication rates ranging from 21 to 70% [Parastomal hernia occurs through an acquired defect of the abdominal wall due to a surgical incision which allows protrusion of abdominal viscera and the incidence differs with the type of intestinal stoma. The reported occurrence of parastomal hernia with loop colostomy is between 0\u201330.8% [Parastomal evisceration is an extremely rare complication with only few cases reported in the literature.A 51\u2009year old patient with chronic obstructive pulmonary disease (COPD) due to long term smoking and extensive hidradenitis suppurativa of the perineum underwent a temporary defunctioning loop sigmoid colostomy and subsequent extensive perineal skin excision and skin grafting.The ostomy was complicated by a parastomal hernia and stoma prolapse 6\u2009weeks post operatively. Conservative management was opted as the stoma was temporary and was functioning well. Five months later, while he was in hospital care for further excision of perianal skin and skin grafting, he developed acute onset pain at the stoma site with rupture of parastomal hernia and evisceration of small bowel loops Fig.\u00a0.There waten cases have been reported worldwide. Most of the previously published cases were associated with ileostomies [Parastomal evisceration is a very rare complication of stomas and to date, only ostomies \u20136 and foostomies \u201310.The mIn our patient, long term parastomal hernia and stoma prolapse may have caused ischaemia and weakening of the underlying abdominal wall and the overlying skin resulting in parastomal evisceration of small bowel. Furthermore, the increased abdominal pressure due to chronic cough(i.e. due to COPD) may have also contributed. Lolis et al. reportedParastomal evisceration is an extremely rare life threatening stoma-related complication which requires urgent treatment. Patients with COPD and long standing ostomy related complications such as parastomal hernia and stoma prolapse may be at a higher risk of developing this complication. Such patients may need early refashioning of the stoma to prevent this serious complication."} +{"text": "Researchers are challenged both when querying older adults about their lived experience and later when analyzing these rich interview data to characterize and preserve their truths. This symposium presents innovative strategies to meet these challenges in ways that deepen our understanding of older adults\u2019 lives within their networks of care and caring. Participants in two interpretive phenomenological studies were family caregivers. One examined the impact of the Virtual Dementia Tour\u00ae on participants\u2019 perceptions of their family member\u2019s lived experience of dementia, identifying changed realities and approaches to caregiving following their experience. The second analyzed in-depth interviews of aging caregivers about future care for their adult children diagnosed with autism spectrum disorders, finding that they had difficulty identifying caregiving support for the future while feeling the need to make plans and decisions now. Two mixed methods studies explored older adults\u2019 experience of their current environment. In the first, measures of social cognition and engagement informed the interpretation of combined phenomenological-hermeneutic and conventional content analyses of nursing home residents\u2019 responses to questions about their relationships, finding that they valued day-to-day social interactions as connections with longtime friends were maintained. The second study combined focus groups, in-depth interviews, and web-based surveys during the psychometric testing of the Person-Place Fit Measure for Older Adults to assess older adults\u2019 experience of aging in a particular place with people they find important. The strategies detailed in these four papers transformed more conventional approaches to deepen our understanding of older adults\u2019 experiences of their lives with others."} +{"text": "This case highlights the importance of recognizing any new soft tissue abnormalities in cancer patients with an indwelling pleural catheter (IPC) or who has had an IPC. This report also describes the first case of catheter tract metastasis (CTM) due to renal cell carcinoma (RCC) and the second case of CTM post\u2010IPC removal. The importance of recognizing new nodules or subcutaneous mass in patients who has had indwelling pleural catheter (IPC) or overlying the IPC site is highlighted. This report describes the first case of catheter tract metastasis (CTM) due to renal cell carcinoma (RCC) and the second case of CTM post\u2010IPC removal. A 54\u2010year\u2010old female had a left indwelling pleural catheter (IPC) placed in March 2017 for a recurrent pleural effusion from renal cell carcinoma (RCC). She achieved spontaneous pleurodesis within two months and her IPC was removed. Her pleural effusion recurred four months later and a second IPC was placed at a separate site, posterior to her initial IPC placement. In January 2018, 10 months after her first IPC placement (eight months after her first IPC was removed), despite targeted treatment with lenvatinib and everolimus, she noticed an area of skin discolouration associated with pain along the previously tunneled tract of her first IPC Fig. , confirmAppropriate written informed consent was obtained for publication of this case report and accompanying images."} +{"text": "There is little evidence to inform provision of enteral or parenteral nutrition to infants with hypoxic ischaemic encephalopathy (HIE) during and soon after therapeutic hypothermia; as a consequence, clinical practice is both variable and changing. A 2014 UK survey found that 79% 33 of 42) of responding neonatal units routinely withheld enteral nutrition during cooling; 3\u2009years later, a similar survey found that 41% (20 of 49) of responding units reported withholding enteral nutrition.3 of 42 oThe landmark trialsThe gut plays a crucial role in the pathophysiology of critical illnesses such as HIE and NEC. The epithelial lining of the gut acts as a barrier, and plays an important role in preventing translocation of bacteria and other damaging luminal contents into the systemic circulation; inflammatory mediators released by the enteric immune system have local intestinal and systemic effects. In this context, enteral nutrition, particularly with mother\u2019s own milk, may actually play a beneficial role by influencing structural and functional integrity of the gut, reducing systemic inflammatory responses and promoting proliferation of gut microbial diversity. The limited available clinical data support this: a retrospective matched case\u2013control study of 34 infants in the USA, which compared minimal enteral nutrition with withholding feeds during therapeutic hypothermia, found lower inflammatory cytokine concentrations and a reduction in days of parenteral nutrition and hospital stay in the enteral feeding group.et al found that 29% (14/49) of responding UK neonatal units report routine use of parenteral nutrition.In high-income settings, infants who receive therapeutic hypothermia receive some form of intravenous fluid support: commonly intravenous dextrose with electrolytes as required, or parenteral nutrition.that 29% 4/49 of rIn critically ill adults and children the provision of early parenteral nutrition is increasingly controversial, with accumulating evidence of harm caused by early parenteral nutrition. A systematic review of 18 adult randomised controlled trials, with a total of 3347 patients, found no effect on mortality but significantly higher rates of infection and longer average length of stay in patients in the parenteral compared with enteral nutrition groups.The evidence to guide nutritional practice for term infants undergoing therapeutic hypothermia is based on a few small studies, with inherent risk of bias. This limited available evidence suggests that careful introduction of enteral feeds during therapeutic hypothermia is safe, may beneficially modulate inflammatory responses and may be associated with earlier time to full enteral feeding and earlier discharge. Parenteral nutrition during critical illnesses in children and term neonates may do more harm by increasing the risk of serious infection and prolonging hospital stay\u2014but the degree to which this is applicable to infants with HIE undergoing therapeutic hypothermia is not known.https://doi.org/10.1186/ISRCTN47404296, registered pre-results) aims to address this uncertainty using UK population data held on the National Neonatal Research Database. Data from several\u00a0thousand infants who received therapeutic hypothermia during a 10-year period will be included. This study will form two groups that are matched on an extensive list of background variables using propensity scores. The intention of this approach is to reduce the risk of bias from confounders, facilitating the use of observational data to address the question \u2018what is the optimum enteral and parenteral nutrition strategy for newborns with HIE during therapeutic hypothermia?\u2019 While we remain unsure of how to deliver nutrition to infants with HIE, it is imperative to consider that enteral feeding could be a beneficial adjunct to therapeutic hypothermia while parenteral nutrition, although a reliable vehicle for nutrient delivery, may increase the risk of infection.Further research is needed to inform nutritional practice for this sizeable group of infants;\u00a0an ongoing National Institute for Health Research funded study ("} +{"text": "The causes of occupational accidents have been classified into unsafe conditions and unsafe behaviour. Interestingly, numerous authors have contributed to the issues of safety practices in managing building production process with different views on factors causing construction accident and insensitiveness to safety practices, but there have been a little efforts to bring together major causes and factors militating against safety practices in unified manners. Therefore, all identified forty nine factors from literature review Specification TableValue of the data\u2022The data pointed out different categories of factors militating against safety practices, the understanding of the data will enable government and policy makers in decision making and implementation in enhancing construction safety practices.\u2022This data will be helpful in any research that relates to construction safety practices in developing countries in order to establish measures for curbing factors militating against construction safety practices.\u2022The survey questionnaire will be useful in analyzing and averting anticipated project risks at planning stage and it will enable projects team to state the degree of confidence at which construction projects could be executed.\u2022The data will also serves as benchmark to compare findings of factors militating against construction safety practices from other developing countries.1Construction accidents remained an ongoing concern in the developing countries, despite the level of awareness in promoting Occupational safety practices over the decades 2, seven variables were identified from Fig. 8, highlighted seven variable from The data for this study covered medium and large scale construction firms operating in Lagos State. Lagos State remained one of fastest growing state in Africa, it is also a coastal zone with a tremendous increase in modern construction activities and development such as: Eko Atlantic city, Lekki free trade zone (Dangote petroleum refinery and Lekki deep sea port) and Lagos Island international airport Fig. 7Since there are no accurate records on number of construction activities in the study area, the study adopted random sampling techniques in selecting population for the study. 75 copies of structured questionnaire were circulated to construction professionals with vast knowledge and proven years of experience to survey their opinion. The study got 88% response rate which are 66 copies from the total copies of questionnaire administered and they were fully analyzed. The survey data were measured on five-point Likert scale Strongly Agree =5, Slightly Agreed =4 Agreed =3, Disagree =2 Strongly Disagreed =1. The identified forty nine variables were designed into closed ended questionnaire, ranked with Mean Scores and presented in figures and tables using Microsoft Excel to allow easy replication of this data., while the research method adopted is similar to that of The ranking of this factors have categorized the forty nine factors militating against safety practices as evidence in Lagos State, Nigeria. Details of the previous studies as related to this data article could be found in [33]"} +{"text": "Although urinary neutrophil gelatinase-associated lipocalin (uNGAL) is useful as a prognostic tool for initiating renal replacement therapy (RRT) , determiDespite successful recovery using CHDF for septic shock with AKI, postoperative CHDF was deferred until the morning of the following day due to reduced Cre and lactate levels. In current clinical practice, AKI is diagnosed by measuring Cre and/or blood urea nitrogen levels, but these markers are insensitive and late indicators of AKI . PreviouuNGAL may be a useful indicator for septic AKI because it is increased by not only AKI but also by inflammation , 6. AlthEarly evaluation of uNGAL levels during an operation may be useful information for prompt determination and preparation of postoperative initiation of RRT in patients with septic shock accompanied by AKI when lactate or Cre levels might be undetectable after perioperative management."} +{"text": "To the editor,We read with interest the recent paper by Mammadov et al. examining the concerns regarding histological changes in adult neurogenic patients who have undergone bladder augmentation with bowel interposition as an adolescent or in childhood .The authors report a small study involving 20 patients who underwent selective anatomic bladder biopsies following augmentation for either neurogenic bladder, exstrophy or bladder neck trauma.Two patients underwent open bladder biopsy as a simultaneous stone extraction procedure was planned. Neuropathic patients with a reconstructed or ablated urethra pose a challenge for the Urologist as they do not have dependant bladder drainage . SpecifiMammadov et al. reported no malignant histology in the study but did detect 2 cases of squamous metaplasia and 1 case of intestinal metaplasia .In 2011, Higuchi at al examined 250 surveillance cystoscopies and although 4 lesions were identified, none were malignant leading the authors to conclude that annual surveillance cystoscopy was not cost effective (The authors conclude by stating that surveillance cystoscopy in augmented patients less than 5 years post operatively is now limited to those with symptoms. This is a very pertinent clinical point. Hamid et al. detected no malignancy in a series of 92 augmented and substituted patients undergoing surveillance cystoscopy but detected higher rates of chronic inflammation in the augmented group . Further"} +{"text": "KCC2 dysfunction attenuates Cl\u2212 extrusion and impairs GABAergic inhibition, and can lead to neuronal hyperexcitability. Converging lines of evidence from human genetics have secured the link between KCC2 dysfunction and the development of epilepsy. Here, we review KCC2 mutations in human epilepsy and discuss potential therapeutic strategies based on the functional impact of these mutations. We suggest that a strategy of augmenting KCC2 activity by antagonizing its critical inhibitory phosphorylation sites may be a particularly efficacious method of facilitating Cl\u2212 extrusion and restoring GABA inhibition to treat medication-refractory epilepsy and other seizure disorders.Epilepsy is a common neurological disorder characterized by recurrent and unprovoked seizures thought to arise from impaired balance between neuronal excitation and inhibition. Our understanding of the neurophysiological mechanisms that render the brain epileptogenic remains incomplete, reflected by the lack of satisfactory treatments that can effectively prevent epileptic seizures without significant drug-related adverse effects. Type 2 K A seizure is a transient increase in the brain\u2019s electrical activity that may be triggered by a variety of factors, including medications are the mainstay therapy for epilepsy that aim to restore this balance in neuronal excitability by either suppressing excitatory neurotransmission or augmenting inhibition. Despite decades of medical research and development of novel third-generation AEDs, a third to a half of epilepsy patients on medications continue to have seizures type A receptors (GABAARs) that are highly permeable to Cl\u2212, and to a lesser extent, HCO3\u2212 is a key determinant of Cl\u2212 homeostasis in neurons of the central nervous system that were enriched among individuals of French Canadian origin with idiopathic generalized epilepsy-14 compared to controls. Both variants exhibited reduced Cl\u2212 extrusion capacity, although unlike the R952H variant, the R1049C variant exhibited normal surface expression with decreased intrinsic cotransporter activity. Both variants also showed decreased phosphorylation of the serine 940 (S940) residue and G551D variants were found as compound heterozygous mutations in two affected children from another family. All of the KCC2 mutations identified by St\u00f6dberg et al. (\u2212 extrusion and reduced cell surface expression. Follow-up studies identified eight additional recessive KCC2 mutations that cause EIMFS (Saitsu et al., \u2212 extrusion without altering cell surface expression and distribution (Saitsu et al., The strongest genetic evidence for KCC2 dysfunction in epilepsy is demonstrated by studies of patients in families with a severe infantile epilepsy syndrome termed epilepsy of infancy with migrating focal seizures (EIMFS; St\u00f6dberg et al., g et al. resulted\u2212 secondary to KCC2 dysfunction may be responsible for driving neuronal hyperexcitability underlying the development of epilepsy syndromes. The association between loss-of-function KCC2 mutations and epilepsy also suggests that augmenting KCC2 activity to enhance Cl\u2212 extrusion may confer the opposite effect of rendering neuronal cells more resistant to seizures, representing a potentially powerful therapeutic avenue for idiopathic epilepsy. Indeed, high levels of neural activity due to seizures may promote the intracellular accumulation of Cl\u2212 that exceeds the normal Cl\u2212 extrusion capacity of KCC2, leading to GABAergic depolarizing currents and loss of synaptic inhibition that underlie epileptogenesis (Ellender et al., \u2212 and restore neuronal inhibition in hyperexcitable states.The presence of KCC2 mutations in human epilepsy indicates that accumulation of intracellular Cl\u2212 extrusion from neurons (Friedel et al., in vivo, Moore et al. (\u2212 extrusion (Moore et al., via different pharmacological approaches will enable flexibility in the selection of the ideal KCC2 modulator that is tailored to the underlying epileptogenic process.Targeting critical phosphorylation sites of KCC2 regulation is one promising strategy to enhance KCC2 function for therapeutic benefit. KCC2 activity is bidirectionally regulated at key phosphorylation sites: S940 phosphorylation increases KCC2 function (Lee et al., e et al. generateAlthough efforts to identify KCC2 modulators reflect its promise as a druggable target in epilepsy, there remain several caveats. First, it is unclear which pharmacological approach or combination of approaches to enhance KCC2 activity would rescue the defects in expression and function due to KCC2 mutations observed in epilepsy syndromes. Second, it remains uncertain whether there may be unintended adverse effects that arise from increased KCC2 activity. Indeed, a major shortcoming of current AEDs is not only their inability to prevent seizures in a large population of patients but also their association with drug-related side effects such as cognitive disturbance (Park and Kwon, \u2212 extruder in mature neurons to establish an inwardly-directed electrochemical gradient of Cl\u2212 necessary for the maintenance of fast synaptic inhibition. In the settings of diminished KCC2 activity secondary to risk factor and causal mutations in human epilepsy patients, intracellular Cl\u2212 concentrations accumulate, leading to impaired hyperpolarizing responses that render neurons hyperexcitable. In contrast, increasing KCC2 function by overexpression or modulation of key phosphorylation sites confers an anticonvulsant effect. Altogether, the presence of KCC2 mutations in epilepsy coupled with preclinical proof-of-principle for KCC2 as a therapeutic target motivates a rich stream of future studies to further investigate the mechanistic roles of KCC2 in epileptogenesis and how manipulation of KCC2 activity can be leveraged pharmacologically for therapeutic benefit in epilepsy syndromes and conditions of hyperexcitation.Epilepsy is a common brain disorder characterized by recurrent and unprovoked seizures thought to be caused by neuronal hyperexcitability. A third to half of epilepsy patients continue to have seizures despite medications (Kwan and Brodie, PD, WD and KK reviewed the literature and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Researchers at the International Collaborative Infantile Spasms Study (ICISS) conducted a study to assess the developmental and epilepsy outcomes in infants treated with combination therapy for the diagnosis of infantile spasms. Researchers at the International Collaborative Infantile Spasms Study (ICISS) conducted a study to assess the developmental and epilepsy outcomes in infants treated with combination therapy for the diagnosis of infantile spasms. Hormonal therapy was either adrenocorticotropin (ACTH) or prednisolone. A total of 377 infants with infantile spasms and a hypsarhythmic (or similar) electroencephalogram (EEG) were randomly assigned to combination therapy (n = 186) and hormonal therapy (n = 191). Developmental and epilepsy outcomes were evaluated at 18 months blinded to treatment, with 181 infants in each treatment group. Analysis was completed by intention to treat.The Vineland Adaptive Behavior Scale (VABS) was used to measure the difference in developmental outcomes at 18 months. The mean VABS composite scores showed no significant difference in developmental outcomes between both treatment groups . There was also no significant difference in the presence of epilepsy reported by caregivers or as a result of antiepileptic treatment (including ketogenic diet) in the previous 28 days between both treatment groups ; there was also no significant difference in the presence of infantile spasms between both treatment groups .This study also reported that spasm cessation between days 14 and 42 of the treatment was associated with higher mean VABS scores and reduced probability of seizures at 18 months. In addition, delay to treatment time was associated with lower VABS scores and poorer epilepsy outcomes (p = 0.023). [COMMENTARY. This study concluded that there was no significant change in developmental or epilepsy outcomes at 18 months for infants treated with combination therapy versus hormonal therapy alone. However, the previous study by ICISS reported that combination therapy was more effective than hormonal therapy alone in treating spasms between days 14 and 42 of treatment and that early spasm cessation is a strong predictor of epilepsy outcomes .Infants treated with hormonal monotherapy who did not respond well were quickly given vigabatrin, which is an effective combination therapy that may enhance developmental and epilepsy outcomes at18 months. Evaluation of etiological causes for infants with infantile spasms in each cohort may also be informative and help identify subgroups that are more sensitive to combination therapy.In conclusion, this study supports both rapid and effective treatment of infantile spasms.The authors have declared that no competing interests exist."} +{"text": "The interaction of lipids (entry mechanism) with respect to both oxy- and deoxy-myoglobin was explored using unrestrained Molecular Dynamics simulations. The results indicated a spontaneous entry of both palmitic and palmitoylcarnitine molecules into the oxy-Mb structure at the main binding site, whereas in deoxy-Mb, both the lipid ligands move away from the protein surface. For the alternative binding locations, entry of the ligands was independent of the oxygenation state. Presented here are the tables with the myoglobin binding energies for palmitic acid and palmitoylcarnitine estimated using Alchemical Free Energy Perturbation approach for the key structures obtained in unrestrained Molecular Dynamics simulations. These data are referenced in the original article \u201cExploring the entry route of palmitic acid and palmitoylcarnitine into myoglobin\u201d, reference number YABBI7787. Specifications tableValue of the data\u2022Describes myoglobin\u05f3s new role in lipid transport along with oxygen carrier/storage.\u2022Addresses lipid release from myoglobin on deoxygenation.\u2022Suggests potential impact of the bound lipids on oxygen release from myoglobin.\u2022The results open new doors to study other globin members and their involvement with lipid metabolism.1The resented data illustrate the results of Molecular Dynamics simulations of the lipids interacting with mouse myoglobin in oxygenated and deoxygenated states. Here, we provide energy estimates for the binding using Free Energy Perturbation approach and visualization of multiple interaction events.2Unrestrained Molecular Dynamics simulations were performed using NAMD software 2.1FEP estimations were performed using FEP module of NAMD"} +{"text": "Neighborhood and residential characteristics can potentially influence mobility of older adults but associations may differ based on cognitive function. We tested whether neighborhood and residential characteristics derived from audits of Google Streetview images were related to 4-year incident mobility disability and whether these associations differed by cognitive trajectories (maintainer vs decliner over 14 years). In 260 participants from the Health ABC , Cox proportional hazard models tested associations stratified by cognitive trajectory, adjusted for demographics. Mixed compared to residential land use was associated with greater risk of mobility disability among cognitive decliners but not cognitive maintainers . Presence of slopes near the home and having a ramp at the home entrance were associated with lower mobility disability risk, again in decliners only. Lower cognitive function may increase vulnerability to poorer neighborhood and residential characteristics for mobility outcomes."} +{"text": "Prior research has suggested that exposure to objectively stressful events contributes to mental health disparities in older adulthood. Yet, in order to understand the extent to which some groups bear a disproportionate stress and mental health burden, we consider black-white differences in not only stress exposure, but also stress appraisal\u2014how upsetting the exposures are perceived to be across five domains . Data come from 6,019 adults ages 52+ from the 2006 Health and Retirement Study. Fully adjusted models show stress exposure and appraisal significantly and independently predicted anxiety and depressive symptoms. Race and stress exposure interactions show that exposure differently predicts anxiety and depressive symptoms while race and appraisal interactions show blacks and whites report similar increases in anxiety and depressive symptoms. Findings suggest stress exposure has varying consequences for mental health of whites and blacks, while stress appraisals have similar consequences across groups."} +{"text": "Nursing homes are increasingly becoming more racially/ethnically diverse yet racial disparities in resident\u2019s quality of life and quality of care continue to persist. One reason for these disparities is lack of culturally-sensitive care and racial/ethnic similarity between residents and staff. This study examines a case of a high proportion minority nursing home with racially/ethnically diverse staff to understand how shared culture among direct care staff and residents may influence care delivery. We used three months of participant observation, supplemented by in-depth qualitative interviews with 8 Hmong residents and 5 Hmong staff to explore the labor of culturally sensitive care in a large, urban NH. We discovered four themes: 1) Culturally sensitive care was often equated to fulfilling language needs for residents who didn\u2019t speak English. 2) Hmong staff members had to take the initiative to inform non-Hmong staff members how to care for Hmong residents. 3) Hmong staff members also had to communicate the culture of NH care and its limitations to Hmong residents and their families. 4) Hmong staff members have to advocate for the culturally relevant needs of Hmong residents. The findings of this case study illuminate that having staff members from diverse cultural backgrounds and meeting language needs of residents does not reflect the everyday practices of culturally sensitive care. This type of emotional labor can also result in higher levels of burn-out for staff of color. Additional research into what constitutes culturally sensitive care to NH residents and staff is needed."} +{"text": "The development of advanced technology for microarray-based chromosomal studies helped discover increased prevalence of genomic copy number variants (CNVs) in individuals with autism spectrum disorder (ASD). Chromosomal microarray analysis (CMA) is now an important tool for clinical investigations in patients with ASD. While this technology helps identify high proportion of CNV positive individuals among patients with autism, the clinical interpretation of such genomic rearrangements is often challenged by inconsistent genotype-phenotype correlations. Possible explanations of such inconsistencies may involve complex interactions of potentially pathogenic CNV with additional (secondary) CNVs or single nucleotide variants (SNVs). Other involved factors may include gender-specific effects or environmental contributions. Development of risk models for interpreting such complex interactions may be necessary in order to provide better informed genetic counseling to the affected families. The growing consensus that CMA is the most cost-efficient single genetic test for these patients led to the recommendation of CMA as a first-tier testing for ASD in expert guidelines , SHANK3 and NLGN4 are deletion 15q11-q13 associated with Prader-Willi and Angelman syndromes, deletion 22q11.2 associated with Velo-Cardio-Facial syndrome, deletion 22q13 associated with Phelan-McDermit syndrome. Typically, these previously known microdeletions are larger and are in the ASD category referred to as \u201csyndromic autism\u201d that is, the patients with such abnormalities tend to have congenital anomalies, facial dysmorphisms and other clinical manifestations in addition to autism. These ASD associated microdeletion syndromes almost always include intellectual disability, early developmental delays, muscle hypotonia, or other clinically recognizable manifestations. They are often recognized in the genetic clinic prior to performing molecular studies and may be confirmed with more targeted tests by the testing laboratory if it is not known to be associated with abnormal phenotype and if it does not include genes know to be associated with genetic conditions. The presence of such abnormality in unaffected parent increases the likelihood for the aberration to be benign. However parental inheritance does not definitely confirm the benign nature of such rearrangement. Further analysis of available databases may help identify other similarly affected individuals with rearrangements in the same chromosomal region. Such useful databases are DECIPHER, ISCA and Autism Chromosome Rearrangement Database or other sequence variants for autism riskAdditive contribution of sequence variants, outside the primary CNV in relevant genes for the overall ASD/cognitive disability risk load was demonstrated in previous reports. Suggested mechanisms for such effects are involvement of genes outside of the CNV or unmasking of recessive disorders affecting genes within the CNV may provide an efficient method to screen for CNVs and SNVs in both coding and non-coding regulatory genomic regions. WGS was already suggested as a first-tier testing for patients with developmental disabilities (Lionel et al., Analysis of environmental factors may also be necessary in order to better predict the risk for ASD. Evaluation of multiple variables may require more complex risk models. Such risk estimating models are already widely used in clinical oncology assays that aim to determine the risk for cancer progression based on multiple molecular markers (Alexander et al., MV wrote the manuscript.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Accumulating evidence suggests that TRM cells are found in diverse human solid cancers where they correlate with improved prognosis and can protect against tumour challenge in mice. However, the mechanisms through which these cells mediate cancer protection are poorly understood. In our recent study , 2019) we developed a melanoma model that allowed us to identify a critical role for TRM cells in the establishment and maintenance of tumour-immune equilibrium in skin. Our findings provide insight into the immune cell populations important for maintaining long-term tumour dormancy in peripheral tissues and imply that targeting TRM cells may serve as a novel cancer treatment strategy.The immune system can prevent tumour development by engaging in a process termed cancer immunosurveillance, during which immune cells such as T cells restrict tumour growth either by completely eradicating cancer cells in a process of \u2018elimination' or by suppressing cancer cell outgrowth by establishing a state of tumour-immune \u2018equilibrium'. Most cancers develop within epithelial layers of tissues but circulating T cells are largely excluded from these epithelial tissue compartments in the absence of infection or overt inflammation. In contrast, CD8 This method of tumour inoculation initially prompts melanoma growth within the epidermis and dermis before infiltration of the subcutaneous layer and eventual metastasis to skin-draining lymph nodes, thereby approximating primary melanoma development in human patients. In contrast to the complete tumour penetrance observed after transfer of B16 melanoma cells via subcutaneous or intradermal routes, we found that only approximately 60% of mice inoculated epicutaneously went on to develop macroscopically detectable melanomas. Whereas subcutaneously inoculated tumours became uniformly palpable within one week of melanoma cell injection, epicutaneous tumours displayed far more variable growth kinetics and were usually not visible until 2-3 weeks after melanoma cell transfer. In some cases, epicutaneous melanoma growth was delayed even further and mice did not develop tumours until several weeks after inoculation. Importantly, approximately 40% of mice remained completely free of apparent melanoma disease for up to several months following epicutaneous inoculation .In order to study immune responses to skin cancer, B16 melanoma cells have traditionally been grafted to mice via intradermal or subcutaneous routes, resulting in tumour growth beneath the epidermis or skin, respectively. While these models have proven highly valuable for studying immunity against rapidly progressing tumours, they nevertheless bypass the earliest developmental stages of cutaneous melanoma confined to the epidermis. In our recent study, we developed an epicutaneous melanoma model to study anti-cancer immune responses Figure 2). We found that the control and long-term maintenance of these dormant melanoma cells was strictly dependent upon immune cells including T cells, as mice deficient in factors required for T cell development and/or survival were highly susceptible to epicutaneous melanoma formation by comparison with wild-type mice, and ultimately failed to establish tumour dormancy. These findings suggest that B16 melanoma inoculation via the epicutaneous route might provide a unique opportunity to study the immune interactions involved in the induction and maintenance of cancer-immune equilibrium \u2013 a clinically relevant phenomenon that has typically proven difficult to study due to the paucity of murine transplantable tumour models in which tumour dormancy is observed.Closer inspection of the skin of these inoculated yet apparently \u2018tumour-free' mice using a variety of highly sensitive detection approaches, including bioluminescence imaging, low copy number PCR amplification or intravital 2-photon microscopy, revealed that many animals continued to harbour low numbers of viable melanoma cells in the epidermis for at least several weeks or months. These findings suggested that dormant melanoma cells could be maintained within the skin of epicutaneously inoculated mice for long periods of time without being completely eliminated . This mechanism of tumour control mirrors that employed by TRM cells poised to control reactivation of latent infections in barrier tissues where TRM cells curb local pathogen replication to prevent symptomatic lesions as opposed to driving complete eradication. TRM cells isolated from tumours can also highly express cytotoxic molecules and have been shown to kill autologous tumour cells in vitro but their ability to kill or eliminate tumour cells in vivo remains uncertain. Our findings raise the possibility that TRM cells might also contribute to the initiation or control of tumour dormancy often observed in human cancer patients burdened with minimal residual disease following surgery or chemo- or immunotherapy. As such, our work suggests that enhancing TRM cell responses using targeted immunotherapies might be a novel avenue through which to improve solid cancer treatments in patients.In summary, our work has revealed a novel mode of anti-tumour control employed by T"} +{"text": "Progressive systemic poisoning by gradually accumulated damaged cells has been proposed as a major contributor to mammalian aging. Our preclinical studies support the hypothesis that this process results from a failure of innate immunity-mediated eradication of cells with DNA damaged by intrinsic mechanisms caused by the epigenetic desilencing of endogenous retroelements. This model suggests two translational approaches to improve the counteract accumulation of damaged cells: (i) by pharmacological suppression of LINE1 reverse transcriptase activities \u2013 the main driver of expansion of \u201cretrobiome\u201d and the trigger of damaged cell-associated inflammation and (ii) by counteracting immunosenescence by innate immunity stimulation. Preclinical proofs of concept have been obtained for both using nucleoside reverse transcriptase inhibitors and immunostimulators acting via TLR5 activation. Preparations for clinical testing of these agents in the context of age-related pathologies is in underway."} +{"text": "Gastric cancer (GC) is the third leading cause of cancer-related death worldwide and is a major cause of cancer-related mortality in China . Since cEmerging evidence indicates that the mechanisms of circRNAs mainly include I) function as competing endogenous RNAs or miRNA sponges ; II) regThe biggest highlight of our research is that the circRNA expression signatures in gastric cancer plasma were explored by using circRNA microarray analysis using plasma samples from 10 GC patients, including 5 patients with no lymph node metastasis and the other 5 patients with lymph node metastasis, and 5 normal individuals. We demonstrate that circPSMC3 can be used as a circulating biomarker in GC, which has been shown to have resistance to RNase R digestion based on the high stability of circRNAS in plasma of GC patients.So can circPSMC3 be used as a therapeutic target for gastric cancer? Some factors should be taken into consideration for designing circRNAs as therapeutic targets: the general drug target expression abundance is high, and circPSMC3 expression level is relatively low in GC, so the target may be difficult to achieve at present. In addition, circRNAs may prove useful as predictive markers for chemotherapy sensitivity in tailored anticancer treatment . WhetherThe authors declare that they have no competing interests."} +{"text": "Tumor tissue mutational burden (TMB) has emerged as a promising predictive biomarker for immune checkpoint therapy. Measuring TMB from circulating tumor DNA (ctDNA) found in plasma is attractive in tissue-constrained indications. We compared the performance of two plasma-based commercial TMB assays including the effect of two different collection methods. Our findings suggest that the two plasma based TMB assays are highly correlated and they are also both correlated with a tissue-based TMB assay for relatively high TMB samples. The two collection methods are also found to be very comparable. Plasma-based TMB assays may be mature enough to be clinically useful in mCRPC and potentially other indications. Tumor tissue mutational burden (TMB) has emerged as a promising predictive biomarker for immune checkpoint therapy. Measuring TMB from circulating tumor DNA (ctDNA) found in plasma is attractive2 in indications such as metastatic castration resistance prostate cancer (mCRPC), where obtaining tissue can be challenging.Liquid biopsies are more convenient, less expensive and less risky to the patient than standard tumor biopsyet al.3 demonstrated that TMB can be accurately measured by sequencing targeted gene panels but that accuracy is compromised when the sequenced genome region (bait size) is less than 0.5\u2009MB. The Guardant Health (GH) Omni and Foundation Medicine (FMI) bTMB panels are plasma-based NGS assays containing sufficiently large bait sizes to measure TMB across a broad range of TMB values. Poor concordance on mutation detection between two commercial vendors was reported by Torga and Pienta previously4. We compared the performance of TMB determination of the two plasma assays in this study, including the effect of two different collection methods.Chalmers 5) were sent to GH and FMI for analysis. In addition, one set of samples from the same 20 subjects collected in EDTA tubes6 were also sent to GH to investigate the impact of different pre-analytical collection methods in determination of TMB. Matching formalin-fixed paraffin-embedded (FFPE) tissue from each subject (collected within twelve months of plasma collection) was analyzed by WES at Personal Genome Diagnostics. All methods were carried out in accordance with relevant guidelines and regulations. All experimental protocols were approved by Merck Ethics Review Committees. Informed consent was obtained from all subjects or, if subjects are under 18, from a parent and/or legal guardian.Replicate sets of plasma samples from 20 mCRPC patients between blood TMB assays (muts/MB) and tissue WES (muts/exome) was observed in general even for biopsy samples with low/medium TMB from the two plasma assays are different. This is likely due to difference in sequencing depth, TMB algorithm implemented and allele frequency cutoff used in the two assays. Fig.\u00a0et al.8 reported a large discordance in driver gene mutation detection between tissue and plasma ctDNA based assays. In this study, although mutation level concordance was not compared, good correlation between tissue and plasma ctDNA TMB is observed for both assays for high TMB samples. However, correlation is compromised between tissue and plasma ctDNA based assays for low/medium TMB samples. Given the biological difference between plasma and tissue and the magnitude of sequencing depth differences between these two assays, a good correlation between tissue WES and plasma assays may not necessarily be expected. This study suggests the clinical validity of plasma TMB and tissue TMB in immunotherapy be evaluated independently. Another important finding of this study is that EDTA plasma may be a suitable specimen for the determination of TMB. This is important information for investigators interested in performing retrospective analysis in studies in which plasma samples may not have been collected in Streck tubes. Although additional studies with larger sample size and clinical outcome data are warranted to further substantiate these preliminary findings and evaluate the clinical utility of plasma TMB, these preliminary results are promising and suggest plasma-based TMB assays may be mature enough to be clinically useful in mCRPC and potentially other indications.Kuderer"} +{"text": "Malignant gliomas are the most common primary brain tumors. They are highly aggressive tumors characterized by a recurrence rate of virtually 100%. Despite significant advances in neuroimaging and neurosurgical techniques, the median survival time of patients with glioblastoma multiforme remains 12 to 18 months. Malignant gliomas are characterized by rapidly dividing cells, which invade into the normal brain, and a high degree of vascularity. Recent experimental evidence indicates that tumor-related angiogenesis contributes significantly to the malignant phenotype."} +{"text": "We included 101 patients, whose mean times from injury baseline and follow-up examinations were 6.1 days (standard deviation [SD] 17) and 235.0 days (SD 71), respectively. Median UEMS recovery was 7 points (interquartile range 2\u201312). One of the predictor variables was not statistically significant in our sample; one group did not fit progressively improving UEMS scores, and three of five groups had medians that were not significantly different from adjacent groups. Overall accuracy was 75%, but varied from 82% among participants whose examinations occurred at <12\u2009h, to 64% at 12\u201324\u2009h, and 58% at >24\u2009h. A previous URP-CTREE model had limited ability to stratify an independent into homogeneous subgroups. Overall accuracy was promising, but may be sensitive to timing of baseline neurological examinations. Further evaluation of external validity in incomplete injuries, influence of timing of baseline examinations, and investigation of additional stratification strategies is warranted.Clinical trials of novel therapies for acute spinal cord injury (SCI) are challenging because variability in spontaneous neurologic recovery can make discerning actual treatment effects difficult. Unbiased Recursive Partitioning regression with Conditional Inference Trees (URP-CTREE) is a novel approach developed through analyses of a large European SCI database . URP-CTREE uses early neurologic impairment to predict achieved motor recovery, with potential to optimize clinical trial design by optimizing patient stratification and decreasing sample sizes. We performed external validation to determine how well a previously reported URP-CTREE model stratified patients into distinct homogeneous subgroups and predicted subsequent neurologic recovery in an independent cohort. We included patients with acute cervical SCI level C4\u2013C6 from a prospective registry at a quaternary care center from 2004\u20132018 ( Their results suggested that URP-CTREE might optimize future clinical trials by providing a data-driven approach to early patient stratification.Unbiased Recursive Partitioning regression with Conditional Inference Trees (URP-CTREE) is a novel approach to stratification that was developed through analyses of the European Multicenter study about Spinal Cord Injury (EMSCI) database.While the EMSCI URP-CTREE is a potentially promising tool for acute SCI clinical trials, the system has not been externally validated on an independent dataset. Such an external validation would be helpful for understanding how the URP-CTREE system might perform in a subsequent clinical trial with an independent cohort of patients. Therefore, we performed an external validation study to determine how well a previously reported URP-CTREE model stratified patients into distinct homogeneous subgroups and predicted subsequent neurologic recovery when applied to an independent cohort of patients' data from an ongoing prospective observational study of patients with acute traumatic cervical SCIs.13We performed a retrospective analysis of data that were prospectively collected at the Vancouver General Hospital as part of the Rick Hansen Spinal Cord Injury Registry (RHSCIR) in Canada. RHSCIR is an ongoing multi-center prospective observational study of patients with acute traumatic spinal cord injuries, and the Vancouver General Hospital is a major academic quaternary care referral center that is part of the RHSCIR network. We obtained local Research Ethics Board approval prior to enrolling patients, collecting data, and performing this study. Further descriptions of the RHSCIR data elements, procedures, governance structure, and privacy and confidentiality framework have been previously reported.8 We excluded patients with incomplete baseline or follow-up neurological examinations. We also excluded patients whose baseline neurological examinations were recorded more than two weeks after their injuries, or whose discharge neurological examinations occurred at less than 5 months post-injury.We included all patients enrolled from May 2004 to February 2018 that met the eligibility criteria reported by Tanadini and colleagues of presenting with complete American Spinal Injury Association (ASIA) Impairment Scale (AIS) A acute traumatic cervical spinal cord injuries with baseline motor levels at C4 to C6 on the right side of their bodies.8 We also extracted patients' age at injury, mechanism of injury, Glasgow Coma Scale (GCS) score, Injury Severity Score (ISS), and data on whether they were treated with surgery as descriptors of our cohort. Missing or ambiguous data were reconciled with local research coordinators, hospital health records, and medical chart abstraction whenever possible.All data were collected by trained research personnel and entered into the standardized local RHSCIR database before being exported to the RHSCIR national office for centralized quality checks. We extracted age, motor level (right body side), bilateral sensory and motor scores, and zones of partial preservation for analysis as baseline predictors.14 ISNCSCI total motor scores (TMS) can range from 0 (absent motor function) to 100 (intact motor function) and comprise UEMS (range 0\u201350) and lower extremity motor scores (range 0\u201350). Zones of partial preservation (ZPP) refer to segments caudal to the ISNCSCI neurological levels where there is partial preservation motor or sensory function. ISNCSCI records were processed through a validated computerized algorithm that maintained consistency and high quality.15 We considered baseline motor scores to be those obtained on admission to acute care and final motor scores to be those obtained at the time of discharge to the community from acute care or inpatient rehabilitation.Neurological examinations were performed according to the International Standards for Neurological Classification of Spinal Cord Injury (ISNCSCI) by trained physicians, nurse practitioners, or physiotherapists.12 It produces models with branch points that occur sequentially at predictors whose univariate associations to the outcome of interest are greatest, and whose splits at each predictor are most efficient in comparison to all other potential splits. Branching continues until the remaining predictors do not have statistically significant univariate associations. In this study, we applied the existing URP-CTREE model reported by Tanadini and colleagues and partitioned our sample according to the predictors and splits derived from EMSCI as if they were being used to stratify enrollment in a clinical trial.8URP-CTREE recursively partitions samples into binary tree configurations that maximize goodness of fit according to two-sample linear statistics.16 We also performed a sensitivity analysis with a threshold of 5 points because studies of complete SCI might implement a narrower change. Patients with missing data were excluded from each analysis and imputations were not performed. All tests of significance were two-tailed and p values of less than 0.05 were considered significant except when Bonferroni corrections were applied to adjust for multiple testing. Boxplots depict medians, the first and third quartiles , and outliers more than 1.5 IQRs beyond Q1 and Q3. Probability density plots present the probability of an enrolled patients being partitioned into each node. We performed our analyses with Excel 2011 , and IBM SPSS Version 21, 2012 .We report discrete variables as counts or proportions, normally distributed continuous variables as means with standard deviations (SDs), and skewed continuous variables as medians with interquartile ranges (IQRs). We used parametric tests for data with normal distributions and non-parametric tests for data without normal distributions. We tested univariate associations with the Pearson correlation coefficient and differences between medians with the Mann-Whitney U test. We considered recovery to be correctly predicted when final UEMS scores were within a pre-specified threshold of 9 points from the median in each group, because that threshold was recently used in the sample size calculation of a current definitive randomized controlled trial of a neuroprotective agent in patients with acute traumatic complete and incomplete cervical SCI.Of 1295 patients with acute traumatic SCIs who consented to enrollment and were subsequently discharged to the community from acute care or inpatient rehabilitation, we excluded 650 because their baseline motors levels were not at C4 to C6, 456 because their baseline injury severity was not AIS A, 18 because they had incomplete baseline data, 64 because they did not have a discharge neurological examination at \u22655 months post-injury, and six because they had incomplete outcome data . In totan\u2009=\u2009122) is shown in We applied the previously reported EMSCI URP-CTREE model, which partitioned our cohort into five stratified groups of predicted UEMS recovery . The firWe present probability density plots for the nodes from each dataset in n\u2009=\u200936; n\u2009=\u200963, mean time\u2009=\u200910\u2009h [SD 6], median UEMS recovery was 6 points [IQR 2 to 12]), one of the predictor nodes was not statistically significant, and four of five medians were not significantly different from their adjacent medians . Prediction accuracy among participants whose examinations occurred at less than 12\u2009h was 82%, 12 to 24\u2009h was 64%, and greater than 24\u2009h was 58%. In a sensitivity analysis with a threshold of 5 points rather than 9, overall accuracy was 49%, at less than 12\u2009h was 55%, at 12 to 24\u2009h was 64%, and greater than 24\u2009h was 32%.We performed an external validation study to determine how well a previously reported URP-CTREE model stratified patients into distinct homogeneous subgroups and predicted subsequent neurologic recovery when applied to an independent cohort. We found that the model had limited ability to stratify patients into distinct homogeneous subgroups and that overall accuracy for predicting final motor recovery was reasonably promising but may be sensitive to timing of baseline neurological examinations.17 our sample size was comparable to that described by Tanadini and colleagues in their index report on URP-CTREE,8 and the patients in our study had similar epidemiology and underwent similar management to patients at other specialized centers nationally and internationally.19 Further, the patients in our sample experienced a magnitude of motor recovery comparable to that reported elsewhere for cervical complete injuries.21Our study included data from only one site in the RHSCIR network, which raises the possibility that our findings may reflect a small sample size or have limited applicability to other centers and healthcare systems. However, our center is known to have the highest volume of acute traumatic spinal cord injury admissions in Canada,22 Reproduction of these findings in another cohort of patients with incomplete traumatic spinal cord injuries such as RHSCIR remains an important knowledge gap, and further research is warranted before those results are applied to the design of trials for novel interventions.We limited our analysis to patients with cervical AIS A injuries in order to specifically evaluate the external validity of the EMSCI URP-CTREE model in this population; therefore, our results do not directly inform about the external validity of URP-CTREE for patients with incomplete injuries. Nonetheless, our study has some indirect application to patients with injuries at other anatomical levels and with varying severity. Whereas challenges of predicting neurological outcomes and stratifying patients efficiently are likely to be even greater when baseline heterogeneity is increased, it was critically important that URP-CTREE be externally validated initially under ideal circumstances. Our sample of C4\u2013C6 AIS A patients closely resembled the sample from EMSCI and likely provided the most favorable cohort in which to evaluate the EMSCI model. In an analysis that compared data from 122 EMSCI patients with AIS B and C injuries to data from 83 patients who were enrolled in a randomized trial of GM-1 ganglioside (Sygen), Tanadini and colleagues reported similar distributions of 6-month UEMS scores using a URP-CTREE model with nodes for baseline UEMS and light touch sensory scores.23 However, our findings of similar UEMS among patients examined at less than 12\u2009h and 24\u2009h post\u2013injury in comparison to our total cohort UEMS recovery do not support this argument. Our specific finding of worse accuracy with delayed examinations seems counterintuitive because we expected early examinations to yield less accuracy in the context of an EMSCI model based on delayed examinations, but our study nonetheless highlights that the URP\u2013CTREE approach needs further refinement and assessment before being generally implemented in trials. The interpretation of results from studies with delayed baseline neurological exams may require caution, and data from studies with delayed neurological examinations may have limited applicability to the design of trials that require early enrolment.The EMSCI model was created using data from participants whose baseline examinations occurred at a mean of 8.1 days (SD 4.7) post-injury. Our cohort had earlier baseline examinations than the majority of patients in the EMSCI database, but they were still later than the requirements of typical acute clinical trials . Delayed neurological examinations may miss substantial early neurological recovery and introduce considerable bias, and our finding of worsening prediction accuracy with increasing intervals from injury to baseline examination suggests that this bias could occur according to a dose\u2013response relationship.7 In an analysis of 600 patients with prospectively collected ISNCSCI data from a single rehabilitation hospital in Italy, Scivoletto and colleagues applied several distribution\u2013based approaches to estimate clinical significance and found that 5\u2013 and 11\u2013 point motor score changes were associated with clinically significant improvements of 0.2 and 0.5 SD units, respectively.24 They suggested that the proportion of subjects who achieve clinically significant improvements should be a preferred outcome when comparing the effects of interventions. It is also plausible that MCIDs for SCI might vary with baseline level, severity, and even chronicity.25 Our sensitivity analysis showed decreased accuracy when we selected a smaller threshold, and we would have undoubtedly found increased accuracy if our threshold was greater. In general, it is difficult to say exactly how close to the original URP\u2013CTREE findings the results of this external validation study would need to be in order to confirm the robustness of the initial prediction. Ultimately, researchers looking to utilize this URP\u2013CTREE for clinical trial purposes will have to consider this in the context of their specific research question and study design.We implemented a threshold of 9 points to detect clinically important changes in UEMS prediction accuracy because it had been used elsewhere, but the minimum clinically important differences (MCIDs) in UEMS and TMS remain controversial in the literature. Some investigators have estimated MCIDs to be larger than 9 points, while others argue that smaller changes might be very meaningful if they occur at myotomes that impact quality of life.We excluded patients whose discharge neurological exams occurred at less than 5 months post\u2013injury in order to generate a cohort with at least approximately 6 months of follow\u2013up. We excluded patients whose discharge neurological exams occurred at less than 5 months post\u2013 injury in order to generate a cohort with at least approximately 6 months of follow\u2013up. This allowed us to replicate the follow\u2013up for the primary UEMS analyses of Tanadini and colleagues, which also was 6 months post\u2013injury. However, we did not replicate their secondary analysis that was based on 12\u2013month data because our RHSCIR cohort does not routinely include 12\u2013month motor score follow\u2013up. That secondary analysis partitioned patients into two groups on the basis of whether or not they were predicted to achieve neurological improvements by two or more motor levels. We were also unable to directly compare the distributions of UEMS for each node in our model to their original model because we did not have access to their original data. Nonetheless, the medians and distributions that we report in 23 further research is warranted to explore potential bias due to variations in baseline neurological examination timing, including in relation to timing of surgery.This study did not control for potential confounding due to timing of surgery or to timing of neurological examinations relative to surgery, and it is possible that these omissions could have influenced our results. For example, patients who underwent earlier surgery and patients whose baseline examinations occurred before surgery may have been more likely to experience greater apparent neurological recovery than those with delayed examinations. Although a recent observational study from RHSCIR found a beneficial effect of earlier surgery among patients with incomplete injuries but not complete injuries,8 did not include a detailed description of these factors and we did not have access to the EMSCI database in order to evaluate them directly. Further work is warranted to explore the feasibility of potentially merging the RHSCIR database, EMSCI database, and other sources in order to bridge this critical knowledge gap.We were also unable to explore or control for potential differences in factors that were not part of the EMSCI URP\u2013CTREE model but may have influenced neurological recovery, such as differences in baseline characteristics and treatments. The report by Tanadini and colleagues2 Our study suggests that the use of early (< 2 weeks post\u2013SCI) motor scores and the zone of partial preservation to predict 6\u2013month motor scores has definite merit, but the precision with which these early neurologic findings separated patients into distinct subgroups was rather modest.This external validation study supports a growing body of literature that has attempted to accurately and reliably predict outcomes after traumatic SCI. Recent reports have highlighted the importance of understanding baseline clinical heterogeneity, and have illustrated how failures to acknowledge differences among participants can undermine trials designed to evaluate promising therapies. For example, an analysis of 836 patients from the RHSCIR database found that clinically meaningful motor score recovery could be predictably related to a joint distribution of the neurological level of injury and injury severity, but it failed to identify a statistical difference in prognosis between high (C1\u2013C4) and low (C4\u2013C6) AIS A.11 applied URP\u2013CTREE to Graded and Redefined Assessment of Strength, Sensibility, and Prehension data from EMSCI in order to predict upper limb function and self\u2013care after acute SCI, but we are unaware of any other investigations of external validity in cervical complete patients. The practical utility of URP\u2013CTREE in comparison to established methods that are more familiar to researchers and clinicians remains unclear, although it would seemingly be appropriate to at least try implementing it in a prospective clinical trial in which early subject recruitment was undertaken (thus requiring early baseline neurologic assessment) to evaluate the utility of this approach for predicting outcome and potentially reducing sample size.URP\u2013CTREE is one of many statistical techniques that can be applied to datasets in order to predict outcomes. More common alternatives include conventional linear or logistic regression, generalized linear modeling, other types of machine learning techniques, and other decision tree\u2013based methods. To date, URP\u2013CTREE has seen only limited implementation in the medical literature and even less so in the field of spinal cord injury. Velstra and colleagues27 While the use of early motor scores to predict later recovery is conceptually appealing for clinicians, our application of this approach to an independent dataset of complete SCI subjects did not reveal the ability to clearly discern different motor score outcomes. Further study is therefore warranted to establish the parameters by which early motor scores may be used to substratify patients who are enrolled into clinical trials of novel therapeutics. The role of \u201ctiming of assessment\u201d is also something that requires further study, given that clinical trials of novel therapeutics in the acute setting often require intervention to be started within 12\u201324\u2009h . The variation in spontaneous recovery with such an early baseline examination may certainly alter how well such early assessments can be used to predict outcome.The need to predict neurologic outcome with better accuracy is a translational imperative for the SCI field to enable the evaluation of acute clinical interventions. As such, initiatives such as the URP\u2013CTREE are extremely valuable for the field. Improved prediction may be achieved by combining such clinical features with objective magnetic resonance imaging biomarkers and/or neurochemical biomarkers from cerebrospinal fluid or blood.While the recursive partitioning concept was developed to assist in the stratification of patients for acute clinical trials, it is acknowledged that the application could be much broader. The more accurate prediction of long\u2013term neurological outcomes is not only directly relevant to patients who suffer these injuries but also to their caregivers and the broad group of healthcare providers and healthcare administrators who are involved in their medical and rehabilitative treatment. A method for more accurately predicting motor score recovery at the outset would also help to establish realistic rehabilitation goals and guide subsequent physical and occupational therapy.A previously reported URP\u2013CTREE model had limited ability to stratify an independent cohort of patients with acute cervical AIS A injury into distinct homogeneous subgroups. Overall accuracy for predicting final motor recovery was reasonably promising, but may be sensitive to the timing of baseline neurological examinations. Further research is warranted to evaluate the external validity of URP\u2013CTREE among patients with incomplete injuries and to investigate additional strategies for accurately stratifying patients with acute SCI."} +{"text": "The causes and consequences of individual differences in animal behavior and stress physiology are increasingly studied in wild animals, yet the possibility that stress physiology underlies individual variation in social behavior has received less attention. In this review, we bring together these study areas and focus on understanding how the activity of the vertebrate neuroendocrine stress axis (HPA\u2010axis) may underlie individual differences in social behavior in wild animals. We first describe a continuum of vertebrate social behaviors spanning from initial social tendencies (proactive behavior) to social behavior occurring in reproductive contexts and lastly to social behavior occurring in nonreproductive contexts . We then perform a qualitative review of existing literature to address the correlative and causal association between measures of HPA\u2010axis activity (glucocorticoid levels or GCs) and each of these types of social behavior. As expected, elevated HPA\u2010axis activity can inhibit social behavior associated with initial social tendencies (approaching conspecifics) and reproduction. However, elevated HPA\u2010axis activity may also enhance more elaborate social behavior outside of reproductive contexts, such as alloparental care behavior. In addition, the effect of GCs on social behavior can depend upon the sociality of the stressor (cause of increase in GCs) and the severity of stress (extent of increase in GCs). Our review shows that the while the associations between stress responses and sociality are diverse, the role of HPA\u2010axis activity behind social behavior may shift toward more facilitating and less inhibiting in more social species, providing insight into how stress physiology and social systems may co\u2010evolve. Acute stress, such as what occurs during the process of acquiring blood samples, rapidly elevates GCs . Urinary, fecal, and excreta GCs to manipulate individual GCs axis. Elevated HPA\u2010axis activity can not only suppress the HPG axis and therefore inhibit reproductive behavior individuals tend to be more sociable than those that are more reactive . For example, exploration and boldness, both of which are measures of proactivity, are linked to group shoaling tendency in fish that were artificially selected for proactive personality, showed markedly lower integrated artificially selected for lower HPA\u2010axis reactivity exhibited a more proactive personality artificially selected for higher aggressiveness selected for more exploratory behavior, selected for HPA\u2010axis reactivity selected for elevated HPI axis reactivity selected for boldness of cases where proactivity and GCs were compared Table\u00a0 showed tSomateria mollissima: Seltmann et\u00a0al., Tamias striatus: Montiglio, Garant, Pelletier, & R\u00e9ale, We found little support for the prediction that the association between HPA\u2010axis activity and proactivity was dependent on the sociality of the species Figure\u00a0. In soci5.1.3These results from studies of wild animals match those from previous studies in captive animals, suggesting that this measure of social behavior (proactivity) is negatively associated with HPA\u2010axis activity and that species sociality did not modulate the relationship between HPA\u2010axis activity and social behavior. Baseline GCs rarely associated with proactivity whereas studies measuring stress\u2010induced GCs or fecal/feather GCs found a negative association with proactivity. Because these studies were correlational we cannot readily present hormones as a cause of these behaviors and we found no studies testing for the effect of experimental stress on proactive behavior.In contrast with studies in captive animals, a few (16%) comparisons in wild animals also found a positive correlation where proactive individuals had higher GCs. Most of the studies (4 of 6) were done with wild animals brought into captivity or were from semi\u2010natural populations is unequivocally sociable behavior and in highly social species, social behavior often has components of both proactivity and reactivity. While reactive individuals are always less sociable, proactive behavior may divide into two types: In less social species, proactive personalities can be sociable or aggressive are also known to be enhanced by GCs. We examined the evidence of whether parental care is positively or negatively associated with HPA\u2010axis activity and whether this relationship depends on the magnitude of the stress response.5.2.1Peromyscus californicus) showed more paternal behavior after mild separation stress whereas chronic variable stress impaired their care behavior exhibit the highest amount of parental care. Small increases in GCs should act as a motivational signal to increase parental care while extreme increases in GCs should lead to abandonment of young when potential future reproduction exists.5.2.2We located 37 studies of 23 vertebrate species that investigated the associations between GC levels and parental care behavior in wild animals Table\u00a0. Most ofZenaida macroura, Miller et\u00a0al., Tachycineta bicolor, Bonier, Moore, Martin, & Robertson, Parus major, Ouyang, Sharp, Dawson, Quetting, & Hau, The majority of studies supported our prediction that the relationship between HPA\u2010axis activity and parental care was nonlinear where slight increases in GCs elevated parental care but substantial increases in GCs decreased it Figure\u00a0b. Most cFicedula hypoleuca) with different dosages of corticosterone. Birds with slight experimental increases in GCs exhibited enhanced parental food provisioning to offspring but higher GC increases reduced feeding of young and even higher GC increases led to nest and territory abandonment. Studies in fish species that exhibit parental care also show that the probability of nest\u2010abandonment or egg cannibalism became less likely with low increases in GCs but increased under high increases in GCs of comparisons found no correlation between GCs and parental care and 6 comparisons (13%) found evidence contradictory to our prediction where baseline GCs were negatively correlated with parental care. Interestingly, all of these studies were done with birds and used the probability of nest\u2010abandonment as the proxy of parental care . In contrast with other measures of parental care , nest\u2010abandonment probability seems to increase linearly with increasing HPA\u2010axisactivity in birds. For example, even though higher baseline GCs increased chick\u2010feeding rate in Great Tits, higher baseline GCs also increased the probability that the nest was abandoned when coupled with decreased prolactin levels are contingent upon simultaneously low levels of prolactin are used to strengthen nonsexual social bonds. For example, in social species, affiliative behaviors occur between members of the same social group, independent of breeding season or reproductive partnership. We will refer to this type of affiliative behavior in a nonreproductive context as \u201cnonsexual pair\u2010bonding\u201d.Because social and sexual relationships mirror very different attributes of individual fitness, the association of GCs and affiliative pair\u2010bonding behavior might depend on sexual/nonsexual context. Furthermore, the type of a stressor may have differential effects on the association between GCs and bonding behavior. For example, nonsexual pair\u2010bonding behavior may be used to cope with social stressors through consolation and social support whereas environmental stressors might trigger more nonsocial responses and thus reduce affiliation. We therefore examined the evidence of whether sexual or nonsexual pair\u2010bonding behavior is enhanced or reduced by GCs and whether this association depends on the context or the social nature of the stressor .5.3.1Galea monasteriensis), increased GCs caused by partner separation triggered socio\u2010sexual affiliative behavior upon reunion and this was associated with subsequently reduced GC levels , a neuropeptide that increases production of GCs , individuals that had experienced social isolation before pairing (and consequently had higher baseline GCs), approached each other more frequently and spent more time in proximity with each other than those that did not experience social isolation engage in socio\u2010sexual affiliation behaviors under the stress of aggressive conflicts between group members can promote nonsexual pair\u2010bonding behavior. For example, bonobos had higher integrated GCs and less affiliative behaviors during weekdays compared to weekends when the zoo was closed can both increase HPA\u2010axis activity and promote nonsexual pair\u2010bonding behavior. However, nonsocial environmental stressors (e.g. exposure to predators or extreme weather) may not promote affiliative behavior. For instance, studies of zoo\u2010housed mammals report higher fecal/urine GCs and stress behavior coupled with a reduced rate of social behaviours with an increasing number of zoo visitors that had higher integrated GCs due to a death of close relative increased their rate of grooming others and the more they groomed the lower integrated GCs they had a month after the stressful incident. The only nonprimate study was also the only one that had an experimental approach. This study showed that Adele penguins treated with high dose GCs (within the stress\u2010induced range) subsequently increased their affiliative behavior of the comparisons in the studies we located were consistent with our prediction, derived from captive animal studies, that elevated HPA\u2010axis activity would increase nonsexual pair\u2010bonding behavior (Eulemur rubriventer) had higher integrated GCs stressors affected nonsexual pair\u2010bonding behaviors in wild animals Table\u00a0. When fos Tecot, , reduceds Tecot, within t5.3.4subsequent increased affiliative behavior increase preening of subordinate group members when entering areas of potential intergroup conflict, which may help \u201cto get the soldiers in line\u201d increased grooming in baboons but in a social context and often involves coordination among multiple individuals. For example, related or unrelated individuals may care for offspring they did not produce or group members in social carnivores may cooperate with one another to acquire food during group hunting.cooperative motivation. However, as the effects of GCs on cooperative behavior likely reflect not only motivation to cooperate but also the mechanistic capacity to do so found some evidence supporting our prediction that individuals with higher GCs would exhibit more alloparental care,\u00a0many of the comparisons of GCs and different types of alloparental behavior within these studies also found no significant association , males with higher pre\u2010breeding integrated GCs exhibited less alloparental care, but those with higher integrated GCs during the breeding period exhibited more alloparental care , individuals that had high pre\u2010breeding baseline GCs were more likely to become helpers affected alloparental care, some studies showed how the timing of stress might be important for its effect on alloparenting. For example, in wild banded mongooses event memory, (ii) synchrony with others, and (iii) social responsiveness to other group members is a coordinating force behind a functioning group. It is needed both in maintaining social bonds or group stability and performing a coordinated cooperative behavior like group hunting. Even though the existence of actual empathy in nonhuman species is controversial, social responsiveness plays a key role in social behavior in all species inhabiting complex social groups. Because GCs may enhance vigilance and responsiveness to external stimuli in general, it is not surprising that empathy correlates positively with hormonal stress responsiveness in humans switched to a more selfish and less cooperative behavior where they bite nutritious mucus from their interspecific client fish partner affects this relationship. Chronic elevations in GCs can reduce HPA\u2010axis responsiveness formed cooperative groups, whereas they usually are territorial in other areas. Similarly, an environmental stressor such as an increased threat of predation promotes cooperation (cooperative mobbing or brood care) in several bird species living in small groups way to ameliorate stressful environments. For example, Savini, Boesch, and Reichard found thAphelocoma coerulescens). Since GCs and prolactin may interact in motivating parental care under stress (see above), they may play an interactive role in alloparental care as well.Taken together, these studies suggest that elevated HPA\u2010axis activity may motivate the expression of cooperative behavior, though the relationship is complex. Specifically, the effects of GCs on alloparental behavior seem to be sex\u2010specific and, as we suggested above, GCs might have different effects on cooperative behavior on short versus longer timescales. Future studies should investigate the interactive effects of GCs and other hormones on group\u2010level cooperative behavior. For example, Schoech, Mumme, and Wingfield found th6inhibitory to more facilitating across a continuum of social behavior. Specifically, GCs seem to play an inhibitory role behind proactivity, be nonlinearly associated with parental care behavior, but show a facilitating role behind many forms of pair\u2010bonding affiliative behaviors and cooperation among group members , experience the highest benefits during adverse and stressful conditions, when they can allocate tasks among individuals and cooperation can elevate individual survival rates. On the other hand, less social species may experience the highest benefits during good conditions, when competition among group members is low.The We propose that the association between GCs and social behavior switches from inhibiting to facilitating during the evolution of highly social cooperative groups, mirroring the shifting balance of the costs and benefits of sociality. Following this, behavioral traits and endocrine systems might co\u2010evolve to achieve more synchronized stress responses and coordinated action among group members. Because group\u2010level cooperation is associated with out\u2010group competition, GCs might trigger increases in social behavior toward group members and aggression/avoidance toward other individuals at the same time affiliation to reduce it;Increased stress promotes (giving) affiliation to increase social support/bonding;Increased stress in other individuals promotes affiliation to increase social support/bonding and to protect self from aggression;Increased stress in others promotes concern for others and empathy and this promotes cooperative behavior;Increased stress within the group promotes more or less synchronized stress response and cooperation among individuals as a way to adaptively respond to the stressor.Overall, our review highlights the gap in our knowledge of how endocrine systems co\u2010vary with social behavioral profiles over evolutionary timescales where elevated HPA\u2010axis activity may reduce some types of social behavior but increase the expression of other types of social behavior found in group\u2010living species . These relationships are complex and may depend upon the environmental feature inducing the change in HPA\u2010axis activity or the magnitude of the increase in HPA\u2010axis activity. We highlight specific areas of future research and provide a predictive framework Figure\u00a0 for futuNone declared.A. Raulo did the literature analyses and plotted results. A. Raulo and B. Dantzer wrote and revised the manuscript.\u00a0Click here for additional data file.\u00a0Click here for additional data file.\u00a0Click here for additional data file.\u00a0Click here for additional data file."} +{"text": "This study aimed to explore the mediating effect of grip strength trajectory on the longitudinal association between regular exercise and weakness in grip strength using a dyadic approach. We used six waves of the Korean Longitudinal Study of Aging (KLoSA) collected every two years from 2006 to 2016. The sample was middle and old-aged Korean couples who participated in all six waves of the survey . The outcome variables were husbands\u2019 and wives\u2019 grip strength at Wave 6, coded as a binary variable . The mediating variables were husbands\u2019 and wives\u2019 trajectories of grip strength across Waves 1 and 5. Independent variables were three dummy variables indicating couple\u2019s participation in regular exercise . Reference group was both not doing regular exercise. Results showed several significant mediational pathways. For husbands, engagement in regular exercise of both spouses and only husband were associated with higher grip strength at Wave 1, and slower decline in grip across waves was related to lower likelihood of having a clinically weak grip strength at Wave 6. As for wives, engagement in exercise of both spouses was associated with higher grip strength at Wave 1 which in turn was related to lower likelihood of having a clinically weak grip strength at Wave 6. These results suggest longitudinal dyadic processes through which engagement in regular exercise affects weakness in grip strength among older Korean couples."} +{"text": "Alzheimer's disease (AD) is the most common cause of dementia and is characterized by a progressive decline of memory and other cognitive functions . Emerginprefrontal cortex was more significantly affected in male as compared to female AD mice. In contrast, hippocampal GABA shows dynamic changes during A\u03b2 pathology progression and its level was significantly elevated in old female as compared to old male AD mouse brain. To reveal the mechanism leading to high hippocampal GABA in female brain, we investigated the possible contribution of reactive astrocytes. Reactive astrocytosis is commonly observed in AD brain and much higher in female than male AD patients. While normal astrocytes do not contain much GABA, affected astrocytes around amyloid plaques become reactive and produce high amounts of GABA [lacunosum moleculare and in dentate gyrus of hippocampus [Our groups recently observed that progression of GABAergic dysfunction in an animal model of AD is clearly influenced by sex . Using s of GABA . Till no of GABA have recpocampus . The anaThe higher number of activated astrocytes and high amounts of hippocampal GABA in females may be associated with low estrogen levels in old females. Estrogen is known to act on hippocampal astrocytes of males and females via different mechanisms ,6. AdditThe differential influence of sex on hippocampal GABA levels during AD pathogenesis offers unique challenges from the perspective of treating memory decline. While the suppression of GABA production or release from reactive astrocytes would bring about desirable therapeutic effects on memory impairment in AD, different therapeutic strategies may be required for improving hippocampus-dependent memory functions in male and female AD patients."} +{"text": "Hepatic arterioportal fistulas are rare, abnormal, direct communications between hepatic artery and portal venous system. Treatment options shifted from surgery to endovascular interventions. Catheterization may be challenging. We report a case of a hepatic arterioportal fistula treated successfuly with Amplatzer Vascular Plug II via percutaneous transhepatic hepatic artery access after failed transfemoral approach.58\u2009year old woman presented with right heart failure, kidney insufficiency and massive ascites related to portal hypertension caused by hepatic arterioportal fistula. She had a history of previous abdominal surgery. Colour Doppler ultrasound and computed tomography revealed a giant portal vein aneurysm related to large hepatic areterioportal fistula. Endovascular treatment was planned. Catheterization of the hepatic artery could not be realized due to severe tortuosity and angulation of the celiac artery and its branches. Access to the hepatic artery was obtained directly via percutaneous transhepatic route and fistula site was embolized with Amplatzer Vascular Plug II and coils. Immediate thrombosis of the aneurysm sac and draining portal vein was observed. Patients clinical status improved dramatically.Transcatheter embolization is the first choice of the treatment of hepatic arterioportal fistulas but the type of the therapy should be tailored to the patient and interventional radiologist should decide the access site depending on his own experience if the routine endovascular access can not be obtained. Arterioportal fistulas (APFs) are rare vascular anomalies that consist of direct connection between mesenteric arterial structures to the portal veins. Majority of the APFs are asymptomatic. They may manifest with gastrointestinal bleeding, ascites, high \u2013 output heart failure, diarrhea. Portal hypertension and hepatic cirrhosis and Computed Tomography (CT) scan demonstrated a large APF between replaced left hepatic artery of the left gastric artery draining into the left portal vein causing a giant aneurysm formation.Phsyical and labarotory examination was as follows; pulse rate was 80 beats/min and arterial blood pressure was 160/90\u2009mmHg, no sign of jaundice. Liver function tests were within normal limits. Serum creatitine level was 2,5\u2009mg/dl . Hepatitis viral markers were negative for HBV, HCV and HIV.US examination demonstrated massive ascites, macrolobulation of the liver contour demonstrating chronic liver disease, splenomegaly 16\u2009cm) and large tubuler structures in the left lobe of the liver. Color Doppler Ultrasound (CDUS) showed direct arteriovenous fistulae between dilated left hepatic artery and left portal vein. There was a giant saccular aneurysm (130x90mm) originating from left portal vein. Right portal vein was dilated too and main portal vein showed hepatopedal flow direction . Angiography showed the direct fistulae between the left hepatic artery and the left portal vein through single large window Fig.\u00a0a. The seAt the following session an US guided percutaneous transhepatic puncture of the left hepatic artery was planned. We eliminate the option of transhepatic portal venous access because of the aneurysmal dilation of the portal vein, the risk of AVP migration was relatively high. At first step, a celiac artery catheterization was performed and baseline angiograms were obtained. Doppler examination demonstrated that portal vein showed aneurysmal dilation 33\u2009mm) and tortuous course immediately after the fistula localisation. Hepatic artery showed straight course and smaller diameter (17\u2009mm). Arterial access was achived under US guidance with AccuStick II introducer system . After arterial puncture with 21G needle 0.018\u2033 guidewire was advanced has been widely used in many clinical situations , direct puncture of the diseased segment can be safely performed under sonographic guidance. Direct visualization of the needle path will significantly decrease complication rates. AVP is a valuable device to get sufficient occlusssion in these dilated segments. Percutaneous transhepatic route provides a short and straight access that will facilitate advancing large diameter AVPs through the sheath easily.To our knowledge this is the first case in English literature that a hepatic APF is treated by endovascular approach through a percutaneous transhepatic arterial access. In our case transfemoral approach to the hepatic artery could\u2019t be realized because of the severe tortuosity and angulation of the celiac artery that is the main technical failure in the endovascular management of hepatic and mesenteric aneurysms and fistulae. Transcatheter embolization is the first choice of the treatment of APF but the type of the therapy should be tailored to the patient and interventional radiologist should decide the access site depending on his own experience if the routine endovascular access can not be obtained."} +{"text": "Effective and timely airway management is a priority for sick and injured patients. The benefit and conduct of pre-hospital emergency anaesthesia (PHEA) and advanced airway management remains controversial but there are a proportion of critically ill and injured patients who\u00a0require urgent advanced airway management prior to hospital arrival. This document provides current best practice advice for the provision of PHEA and advanced airway management.This best practice advice was developed from EHAC Medical Working Group enforced by pre-hospital critical care experts. The group used a nominal group technique to establish the current best practice for the provision of PHEA and advanced airway management. The group met on three separate occasions to discuss and develop the guideline. All members of the working party were able to access and edit the guideline online.This EHAC best practice advice covers all areas of PHEA and advanced airway management and provides up to date evidence of current best practice.PHEA and advanced airway management are complex interventions that should be delivered by appropriately trained personnel using a well-rehearsed approach and standardised equipment. Where advanced airway interventions cannot be delivered, careful attention should be given to applying basic airway interventions and ensuring their effectiveness at all times. Airway management can be classified as basic or advanced . Basic airway interventions should be performed for every patient with airway compromise; emphasis must be placed on performing the intervention well, with repeated assessment of its effectiveness. In some patients, basic interventions will be insufficient to provide adequate oxygenation and ventilation. If appropriately trained personnel are available, advanced airway interventions should be performed, prior to transfer to hospital , 2. The \u25aa Impending or actual hypoxia\u25aa Impending or actual acute hypercapnia\u25aa Threatened or actual loss of airway control\u25aa Severe agitation associated with head injury\u25aa Reduced level of consciousnessBased on published scientific reports and guidelines , 7, the The EHAC MWG suggests that the requirement for, and provision of PHEA should be assessed on an individual case basis. Where PHEA is indicated, it should be performed in a timely fashion and should not significantly delay transfer of the patient to hospital. These guidelines are designed for physicians; paramedics performing pre-hospital drug-assisted intubation should meet the requirements of their employers and professional governing bodies.Standards for best practice:\u25aa Physicians entering pre-hospital practice should have a minimum of 12 months experience of in-hospital anaesthetic practice and a minimum of 12 months experience in emergency medicine and acute medicine, before undertaking PHEA.\u25aa Pre-hospital emergency care services should have a written standard operating procedure (SOP) for the conduct of PHEA. All relevant personnel must be fully conversant with this document.\u25aa Pre-hospital emergency care services should provide appropriate training and competency assessment for all providers on an annual basis.\u25aa All providers of PHEA should be competent in paediatric advanced airway management.\u25aa The provider performing advanced airway management should be assisted by another member of the HEMS team with appropriate training for the safe delivery of PHEA, at all times.\u25aa PHEA should be withheld if the HEMS team do not have the correct skillmix required to ensure safe and effective delivery of the procedure.\u25aa Consultants in pre-hospital emergency medicine should be available for telephone advice at all times.\u25aa All practitioners delivering PHEA should maintain a logbook of individual cases.Further considerations:\u25aa A relevant PHEA course or thorough induction training should be undertaken prior to starting clinical practice.\u25e6 Only providers with competence and experience in the delivery of in-hospital drug assisted intubation should deliver PHEA.p\u2009<\u20090.05) [It is well recognised that poorly performed tracheal intubation is associated with an increased morbidity and mortality , 9. Repe\u2009<\u20090.05) . EHAC MW\u2009<\u20090.05) . All preStandards for best practice:\u25aa Environmental factors such as ambient light, noise and adverse weather conditions should be considered when deciding where and when to intubate the patient.\u25aa Factors that may influence intubation success should be optimised prior to the first intubation attempt. These include good access to the patient (360-degree access where possible), and optimal positioning of the patient on an ambulance trolley placed at the correct height for the operator.\u25aa Intubation should be performed prior to loading onto the aircraft unless adverse weather conditions prevent safe conduct. Intubation should only be performed in the aircraft provided there is no increased risk of an adverse event during the intubation procedure.\u25aa Intubation must not be planned for, or performed in, the flight phase of aeromedical transfers.\u25aa The triage decision and distance to destination hospital should be considered prior to intubation and discussed with the HEMS team.Further considerations:\u25aa Aviation regulations must be observed at all times.All factors influencing the success of an intubation attempt should be optimised prior to the first attempt including access to the patient, assembly of all required equipment, full monitoring and a verbalised management plan and triage decision. Whilst movement of sick or injured patients should be limited, the EHAC MWG strongly recommends that the patient should be moved to an area with adequate space to permit 360-degree access prior to intubation. Intubation should be performed outside the aircraft unless adverse events such as bad weather, low outside temperatures, or suboptimal light are considered likely to reduce the chances of a successful intubation. The patient should be placed on an ambulance trolley at an optimum height prior to any intubation attempts. The quality of laryngeal view, intubation and first pass success rates have been demonstrated to be optimal when the trolley is placed at chest height for the intubating clinician , 18. OncStandards for best practice:\u25aa Nasopharyngeal and oropharyngeal airways\u25aa Two working laryngoscope handles with two different sized Macintosh blades\u25aa Intubating bougie\u25aa Cuffed tracheal tubes in appropriate sizes\u25aa Spare tracheal tube \u25aa 10 or 20\u00a0ml syringe for cuff inflation (cuff checked prior to intubation)\u25aa Tube tie or tube holder\u25aa Bag-valve-mask with oxygen reservoir connected to oxygen\u25aa Carbon dioxide monitoring (colorimetric and / or quantitative)\u25aa Spare oxygen cylinder\u25aa Suction\u25aa Second generation supraglottic airway device (for failed intubation)\u25aa Surgical airway equipment \u25aa Paediatric laryngoscopes with appropriately sized laryngoscope blades \u25aa Uncuffed and cuffed tracheal tubes in appropriate size range for paediatric intubationFurther considerations:\u25aa VideolaryngoscopyThe following equipment is considered essential for all intubation attempts and should be carried by personnel who are qualified to perform PHEA:All pre-hospital systems must have all the required equipment available for each intubation attempt. The service should carry a range of laryngoscope blades and tracheal tubes in different sizes, appropriate for both adult and paediatric intubation. The tube size should be calculated prior to intubation. The use of a challenge-response equipment checklist is recommended.Videolaryngoscopy is used in an attempt to improve laryngoscopic view and increase the overall intubation success rate and first pass intubation rate. The purpose of videolaryngoscopy is to enable all medical personnel involved to observe visualisation of the glottis and participate in improving the view where possible with interventions such as external laryngeal manipulation and suction. Data on the performance of these devices in improving intubation success rates are limited in this role. Most studies suggest videolaryngoscopes offer a benefit in pre-hospital intubation , 20, thoStandards for best practice required:\u25aa PHEA should be performed using methods described in the standard operating procedure (SOP) for each individual service. Compliance with, or reasons for deviation from, the SOP should be formally documented.\u25aa A formal checklist for PHEA should be carried out and include confirmation of monitoring, equipment, drugs, and failed intubation management.\u25aa All required equipment should be assembled and checked prior to intubation.\u25aa Drug doses should be calculated prior to intubation and confirmed with the anaesthetic assistant.\u25aa Preoxygenation should be performed for at least 3\u00a0min before laryngoscopy.\u25aa Each service should have, and be familiar with, a robust failed intubation plan.Further considerations:\u25aa Apnoeic oxygenation.There is increasing evidence for the benefit of both checklists and SOPs for many complex interventions. The introduction of such documents into pre-hospital practice has been shown to be feasible , and resPHEA drugs should include an induction agent, an opioid and a rapid-onset muscle relaxant Careful consideration should be given to the type and dose of PHEA drugs, especially in unstable patients. Induction agents can also be omitted in imminent risk of cardiac arrest. Where possible the choice of agents used within individual services should be limited to improve familiarity with the PHEA process, promote the use of reproducible techniques, and reduce human error.2 above 95% during the apnoeic phase [Passive apnoeic oxygenation with high-flow (15\u00a0l/min) oxygen via nasal prongs is a low-risk procedure. It is currently practiced by a number of pre- and in-hospital services around the world with demonstrable benefit in sustaining SaOic phase and diffic phase . Nasal pic phase . Most stic phase and the The evidence for cricoid pressure is weak and relatively scarce. The authors recommend consideration of the use of cricoid pressure for PHEA under normal circumstances but practitioners should have a low threshold for removing it if the view at laryngoscopy is impaired.External laryngeal pressure and manipulation may be of benefit when attempting to improve the view.Standards for best practice:\u25aa Effective ventilation should be established and confirmed immediately following placement of the tracheal tube. Where available, a mechanical ventilator should be used in preference to hand ventilation, especially for longer transfers.\u25aa The presence of an end-tidal carbon dioxide trace should be confirmed immediately after tube placement.\u25aa The rate of ventilation should be titrated to end-tidal carbon dioxide.\u25aa The position of tracheal tube should be confirmed and documented.\u25aa The patient should be reassessed after each intervention or change of position, and before loading onto aircraft.\u25aa The HEMS crew member(s) should have access to the patient during flight\u25aa Intubation equipment and airway rescue equipment should be immediately available during flight.\u25aa Normoxia and normocapnia should be achieved for each patient. Low to normocapnia should be considered for patients with traumatic brain injury .\u25aa Ensure the patient is appropriately packaged with consideration given to clot stabilisation if bleeding, fracture immobilisation and maintenance of normothermia.\u25aa Anaesthesia should be adequately maintained.\u25aa The requirement for chest decompression should be considered.Further considerations:\u25aa Use of arterial blood gas monitoring.End-tidal carbon dioxide monitoring is mandatory for all intubated patients and lack of continuous capnography is considered to be associated with an increase in morbidity and mortality . InadequHypothermia in sick and injured patients is widely considered to be detrimental, contributing to systemic dysfunction. One study conducted in a pre-hospital setting observed higher rates of hypothermia (<\u200935\u00a0\u00b0C) in patients undergoing PHEA; the mortality rate was significantly higher in this patient group . The preStandards for best practice:\u25aa Pulse oximetry.\u25aa Noninvasive blood pressure.\u25aa Heart rate.\u25aa Continuous waveform and quantitative capnography.\u25aa Continuous temperature monitoring.The following should be considered mandatory for all patients:Further considerations:\u25aa LactateFull monitoring should be attached to the patient prior to induction of anaesthesia and a summary of the information obtained from the monitoring recorded in the patient\u2019s documentation . The tem\u25aa Night operations\u25aa Adverse weather or environmental conditions\u25aa Psychiatric patients\u25aa Pregnant patients\u25aa ChildrenPre-hospital management of sick and injured patients is associated with a wide variety of challenges and factors that may influence individual patient management. It would be impossible to produce guidelines that accounted for all variables that may be encountered. Certain circumstances may require deviation from best practice guidelines. All decisions about how to proceed should be case specific and following a dynamic risk-benefit assessment and with a senior clinician providing support for the decision-making process.The practice of PHEA is increasing and adequate data collection is essential to improve practice through local audit and clinical governance processes. The following variables have been suggested as part of the minimum dataset for collection and analysis .System variables:Highest level of EMS provider on sceneAirway equipment availableAnaesthetic agents availableMethod of transportationResponse timeProvision of adequate governance structurePatient variables:AgeGenderCo morbidityPatient categoryIndication for airway interventionVital signs pre induction of anaesthesia Post intervention variables:Post intervention ventilationVital signs post induction of anaesthesia Survival statusNumber of attempts at airway interventionComplications , cardiac arrest)Drugs used to facilitate procedureOverall intubation success rateDevices used in successful airway managementFurther considerations:Intubation success rate at first attemptManagement of failed intubationAll patient documentation should be completed and data collection should be tailored to the requirements and processes of individual systems.EHAC MWG recommends a standardised approach to pre-hospital emergency anaesthesia and advanced airway management described in a clear and simple Standard Operating Procedure that is followed by competent clinicians. Only personnel with sufficient experience and expertise should deliver pre-hospital advanced airway management. Standards for the pre-hospital procedure should not be inferior to those found in-hospital regarding equipment availability, patient monitoring and post-intubation care. Continuous audit of the Key Performance Indicators is essential to maintain these standards."} +{"text": "The activity of the GABAergic neurons of the thalamic reticular nucleus (TRN) has long been known to play important roles in modulating the flow of information through the thalamus and in generating changes in thalamic activity during transitions from wakefulness to sleep. Recently, technological advances have considerably expanded our understanding of the functional organization of TRN. These have identified an impressive array of functionally distinct subnetworks in TRN that participate in sensory, motor, and/or cognitive processes through their different functional connections with thalamic projection neurons. Accordingly, \u201cfirst order\u201d projection neurons receive \u201cdriver\u201d inputs from subcortical sources and are usually connected to a densely distributed TRN subnetwork composed of multiple elongated neural clusters that are topographically organized and incorporate spatially corresponding electrically connected neurons\u2014first order projection neurons are also connected to TRN subnetworks exhibiting different state-dependent activity profiles. \u201cHigher order\u201d projection neurons receive driver inputs from cortical layer 5 and are mainly connected to a densely distributed TRN subnetwork composed of multiple broad neural clusters that are non-topographically organized and incorporate spatially corresponding electrically connected neurons. And projection neurons receiving \u201cdriver-like\u201d inputs from the superior colliculus or basal ganglia are connected to TRN subnetworks composed of either elongated or broad neural clusters. Furthermore, TRN subnetworks that mediate interactions among neurons within groups of thalamic nuclei are connected to all three types of thalamic projection neurons. In addition, several TRN subnetworks mediate various bottom-up, top-down, and internuclear attentional processes: some bottom-up and top-down attentional mechanisms are specifically related to first order projection neurons whereas internuclear attentional mechanisms engage all three types of projection neurons. The TRN subnetworks formed by elongated and broad neural clusters may act as templates to guide the operations of the TRN subnetworks related to attentional processes. In this review article, the evidence revealing the functional TRN subnetworks will be evaluated and will be discussed in relation to the functions of the various sensory and motor thalamic nuclei with which these subnetworks are connected. The thalamus is a prominent diencephalic structure that contains a large number of nuclei Figure , each ofThe main body of the thalamus is made up of nuclei composed of glutamatergic projection neurons and GABAergic interneurons and excitatory depolarizing spikelets, mediated by gap junctions , were recorded in ssTRN neurons and were evoked from regions surrounding recorded cells\u2014these regions are spatially restricted and generally correspond to the extent of the dendritic arbor of a recorded cell . However, the activity of many neurons in the dorsorostral region of TRN increases upon arousal during wakefulness and is negatively correlated with sleep-related rhythms. Neurons in the dorsocaudal region of TRN send projections to a thalamic nucleus involved in sensory functions\u2014in rodents, cells in this region are connected to first order TC neurons in the dorsal lateral geniculate nucleus and internal globus pallidus (GPi) Figure , or glutin vitro slice preparations through the mouse thalamus, GABAA receptor-mediated IPSCs were recorded in VA and CL neurons and were evoked from AZ-related neural clusters in mtrTRN nucleus Figure , has beeIn summary, two functionally distinct subnetworks of TRN neurons have been identified according to their firing patterns during different behavioral states\u2014sleep and arousal Figure ; these sin vitro slice preparation taken in the horizontal plane through the rat thalamus . Because at any given moment the amount of sensory information available in the external environment far exceeds the processing capability of the brain, attentional mechanisms allow access to limited neural resources to select a small fraction of this information. Such attentional selection is controlled through exogenous \u201cbottom-up\u201d processes and endogenous \u201ctop-down\u201d processes TRN neurons that make up approximately four fifths of the cell population in the mouse TRN. In SOM+ TRN neurons, activation of ErbB4 normally reduces the strength of the glutamatergic drive that specifically arises from L6 CT neurons. However, in SOM-ErbB4 knockout (KO) mice, this reduction is lost resulting in an enhanced L6 CT\u2192TRN drive and thereby an enhanced TRN\u2192TC/TS inhibition. Complementing this genetic approach, mice were initially trained to make leftward or rightward movements to visual or auditory cues and were subsequently tested in a sensory discrimination task or in a rule-specific (\u201cattend to vision\u201d) discrimination task; correct responses were rewarded during training and testing. The sensory discrimination task contained \u201cauditory/auditory\u201d trials\u2014a relevant (reward-associated) auditory stimulus among distracting auditory stimuli cued a leftward or rightward movement\u2014and the rule-specific discrimination task contained \u201ccongruent\u201d or \u201cincongruent\u201d trials\u2014a relevant visual target and a previously relevant (now distracting) auditory stimulus cued the same (congruent) movement or opposite (incongruent) conflicting movements. Compared to wild-type (WT) mice, loss of ErbB4 in KO mice improves performance in auditory/auditory trials and impairs performance in visual/auditory incongruent trials\u2014these changes in performance do not occur in KO mice when the enhanced L6 CT\u2192TRN drive is reduced by blocking the postsynaptic delivery of the AMPA receptor subunit GluA4 in SOM+ TRN neurons. To account for the improved performance in auditory-auditory trials, enhanced TRN-mediated lateral inhibition of neurons responding to distracting stimuli in the ventral medial geniculate nucleus (vMGN)\u2014containing first order TC neurons that convey auditory information from the inferior colliculus to auditory cortex\u2014is proposed and previous reward history\u2014a steady improvement in performance over test trials in a task requiring intermodality attentional switching is consistent with the idea of a gradual extinction of an earlier action-selection bias mediated by BG circuits. Further work is required to test this and other possible compensatory mechanisms that allow substantial behavioral recovery following loss of NRG1/ErbB4 signaling in TRN neurons.ErbB4, an NRG1 receptor expressed in SOMFunctionally distinct neural subnetworks can occupy different TRN sectors as defined by different neuronal firing patterns during wakefulness and sleep (Halassa et al., Functionally distinct neuronal subnetworks in TRN are often widely distributed in sensory and motor sectors. Two such subnetworks are made up of either elongated or broad AZ-related neural clusters that provide convergent inhibitory inputs onto neurons in various thalamic nuclei (Lam and Sherman, JC wrote the manuscript and constructed the figures.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Immunoisolation of pancreatic islets is a technology in which islets are encapsulated in semipermeable but immunoprotective polymeric membranes. The technology allows for successful transplantation of insulin-producing cells in the absence of immunosuppression. Different approaches of immunoisolation are currently under development. These approaches involve intravascular devices that are connected to the bloodstream and extravascular devices that can be distinguished in micro- and macrocapsules and are usually implanted in the peritoneal cavity or under the skin. The technology has been subject of intense fundamental research in the past decade. It has co-evolved with novel replenishable cell sources for cure of diseases such as Type 1 Diabetes Mellitus that need to be protected for the host immune system. Although the devices have shown significant success in animal models and even in human safety studies most technologies still suffer from undesired tissue responses in the host. Here we review the past and current approaches to modulate and reduce tissue responses against extravascular cell-containing micro- and macrocapsules with a focus on rational choices for polymer (combinations). Choices for polymers but also choices for crosslinking agents that induce more stable and biocompatible capsules are discussed. Combining beneficial properties of molecules in diblock polymers or application of these molecules or other anti-biofouling molecules have been reviewed. Emerging are also the principles of polymer brushes that prevent protein and cell-adhesion. Recently also immunomodulating biomaterials that bind to specific immune receptors have entered the field. Several natural and synthetic polymers and even combinations of these polymers have demonstrated significant improvement in outcomes of encapsulated grafts. Adequate polymeric surface properties have been shown to be essential but how the surface should be composed to avoid host responses remains to be identified. Current insight is that optimal biocompatible devices can be created which raises optimism that immunoisolating devices can be created that allows for long term survival of encapsulated replenishable insulin-producing cell sources for treatment of Type 1 Diabetes Mellitus. Tregs have been successfully immobilized on islet surfaces through streptavidin-biotin interactions and dexamethasone-loaded hydrogels, the silk macrocapsules showed a strong macrophage polarization toward a M2 phenotype which might provide an immunopermissive environment for the implants. A more recent study demonstrate that 2-aminoethyl methacrylate hydrochloride coupled to alginate can reduce tissue responses (Somo et al., Morinda citrifolia Linn (Sousa et al., Lentinula edodes (Ren et al., Schizophyllum commune (Du et al., Also silk hydrogels have been shown to have immunomodulatory effects on macroencapsulated rat islets (Hamilton et al., Although encapsulation in permselective membranes is a field that is around for more than three decades, important new polymeric approaches have emerged during recent years that create optimism that a technology can be developed that provokes minimal tissue responses and allows long term survival of encapsulated cells. The technology has revisited together with new approaches for creating a replenishable cell sources for curing endocrine diseases such as T1D. Some of these sources involve the use of xenogeneic tissue which might be particularly challenging in an encapsulation setting as indirect antigen presentation might be involved (Shin et al., in vivo results are not yet available. Apart from impact of polymers and tissue responses, long-term maintenance of islet cell viability is an important issue that requires much more attention by the scientific community. This is essential for graft function but also for reducing tissue responses as dead cells release danger-associated molecular patterns that provoke local tissue responses (Paredes-Juarez et al., In addition to novel polymers to reduce tissue responses also other approaches have emerged. Some promising approaches are coating or co-encapsulation of nanoparticles for targeted and local drug delivery without systemical side-effects (Fang et al., Overall, current insight points to several potential successful strategies to reduce tissue responses against encapsulated islets grafts. This include novel or improved polymers but possibly also immune modulatory molecules or cells to allow long-term survival of encapsulated islet grafts. Although many long-term successes have been shown in several animal models there is consensus among insurance companies that 1-year survival is required with possibility to retrieve the graft before human application can be considered. To achieve this with in this review discussed approaches is to our opinion a realistic goal.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Rheological models such as Bingham Plastic or Power law models depict fluid behavior with points of the rheological relation which correspond to higher shear rates, but these models are fairly easy to solve for their specific descriptive parameters. Lower rpm (and hence shear rate), could be used to improve the performance and understanding of drilling mud at the lower shear rates prevailing in the wellbore. These data can be utilized in validating these rheological models and the essence of Equivalent Circulating Density (ECD) calculation in analyzing pressure drop in annular hole cleaning. Specifications TableValue of the data\u2022The data can be applied in developing and validating a formula that will optimize hole cleaning during drilling operations.\u2022The data can be used to develop annular pressure loss model.\u2022The data can be utilized in obtaining direct model for the calculation of Equivalent Circulation Density.\u2022The data can be used to compare and justify the advantages and disadvantages of existing rheological models.1Good hole cleaning practically depends on the type of weighting material and the model applied during drilling operation, Given the data in 2The laboratory work was carried out following the API standard and the materials used are tabulated in 1.Bingham Plastic Modela.Pressure Loss along Drill Pipe in CASINGb.Pressure Loss along Drill Pipe in OPEN HOLEc.Pressure Loss along Drill Collar2.Power Law Modela.Pressure Loss along Drill Pipe in CASINGb.Pressure Loss along Drill Pipe in OPEN HOLEc.Pressure Loss along Drill CollarGiven the experimental and field data, the pressure loss and equivalent circulating density was calculated using Bingham Plastic and Power Law models for the calcium carbonates weighting agent"} +{"text": "Human systems display sensitive dependence of initial condition. That is, even though two individuals may be similar in most regards, small differences between these individuals may have far reaching consequences later in life. In dynamical systems analysis, this sort of behavior is quantified with maximum Lyapunov exponents. These exponents quantify the degree to which small differences in initial condition between two systems affect trajectories of these systems later in time. Current methods for estimating maximum Lyapunov exponents are sensitive to noise and this sensitivity leads to estimation errors when researchers attempt to estimate these exponents on data obtained from human participants. Additionally, most current methods only allow for maximum Lyapunov exponent estimation using univariate time series. In this presentation, we present a method for using structural equation modeling for estimating latent maximum Lyapunov exponents from noisy multivariate time series and discuss applications of this method for analyzing human generated data."} +{"text": "Racial residential segregation may be a fundamental cause of health disparities in the U.S., and few studies employ objective measures of segregation to estimate its impacts on cognitive decline. Using data from 21,446 REGARDS participants in urban areas, we employed race-stratified growth curve models to examine how city racial segregation was associated with trajectories of cognitive decline over time. Controlling for demographics and health conditions/behaviors, higher segregation for blacks was marginally associated with lower cognitive function at baseline while higher segregation for whites was associated with better cognitive function . For both blacks and whites, there were no significant associations between segregation and rate of cognitive decline but neighborhood poverty was adversely related to cognitive function . Further research into mechanisms that contributes to heterogeneity in associations between racial segregation and cognitive function is needed to develop effective prevention interventions."} +{"text": "Relapsing polychondritis is a rare autoimmune condition characterized by episodic and progressive cartilaginous inflammation. Its clinical presentation is vastly divergent and can affect various organs. We report the uncommon case of large airway involvement in a patient presenting with shortness of breath on the background of diagnosed relapsing polychondritis. Computed tomography (CT) chest demonstrated thickening of the cartilaginous portions of the trachea and bronchi with sparing of the posterior membranes, consistent with tracheobronchomalacia and repeated cartilaginous destruction. High doses of systemic glucocorticoids, accompanied by continuous positive airway pressure, were required for treatment. We highlight the importance of identifying the extent of airways affected and definitive positive airway pressure support for relapsing polychondritis affecting major airways in addition to conventional therapy of immunosuppression. Relapsing polychondritis is a rare life\u2010threatening autoimmune condition. The exact incidence of this disease is unknown. Clinical presentation varies from auricular chondritis and polyarthritis to large airway involvement. The disease can be stabilized on immunosuppression; however, exacerbations may be life\u2010threatening depending on the organs involved. We present a previously undescribed occurrence of relapsing polychondritis with acute large airway involvement requiring short\u2010term positive airway pressure therapy in the context of a respiratory viral infection.A 57\u2010year\u2010old woman with a past history of relapsing polychondritis presented with acute shortness of breath and wheeze after a long\u2010distance flight.In her 30s, she was diagnosed with relapsing polychondritis based on McAdam's criteria On initial examination, her heart rate was 130 beats per minute (regular), blood pressure 160/90, respiratory rate 24 breaths per minute, and oxygen saturation 94% on room air. Clinical examination demonstrated a prominent saddle nose deformity but remained short of breath and subsequently desaturated to an oxygen saturation of 89%. Repeated venous blood gases indicated a normal pH and PaCOSix months following this episode, the patient has progressed well and is back on her initial immunosuppression regime with no further exacerbations.Relapsing polychondritis is a rare autoimmune condition characterized by episodic and progressive cartilaginous inflammation Poor prognostic factors have previously been recognized and include the presence of a saddle nose deformity, anaemia and systemic vasculitis Large airway involvement is common and is a major cause of mortality and morbidity, present in up to 50% of patients Systemic glucocorticoids are the mainstay of treatment to dampen the immune response in moderate to severe disease. Other forms of immunosuppression may be indicated depending on the response to prednisolone and other organs involved We highlight the clinical challenge of the management for this condition and raise awareness of tracheobronchial involvement in relapsing polychondritis. Positive airway pressure therapy may be used in the acute setting for exacerbations that are self\u2010limiting without the need for longer\u2010term therapy. This would provide immediate airway support, augmenting the slower\u2010acting immunosuppression.Appropriate written informed consent was obtained for publication of this case report and accompanying images."} +{"text": "With the aging of the Baby Boom generation, increasing numbers of older adults require assistance in their daily lives and most help comes from family members. Delays in childbearing mean many adult elder care providers are simultaneously raising children. Although past research has documented disparities in psychological distress and financial costs, less is known about the social costs resulting from elder caregiving and how this varies by parental status. We examine the social costs of elder caregiving by comparing elder and child care configurations to investigate three questions. First, do the daily time use patterns of elder caregivers differ by parental status? Second, do the daily time use patterns of elder caregivers differ by caregiving intensity? Third, does caregiving intensity moderate associations of elder caregiving and parental status on daily time use? We address these questions using nationally representative time diary data from the 2011-2017 American Time Use Survey (ATUS)."} +{"text": "Hoarding disorder in late life has been associated with increased risk for medical conditions and decreased ability to perform activities of daily living in the home; however, no studies have yet examined the relationship between geriatric hoarding and sleep. This study represents a secondary data analysis of older adults who received 26 sessions of group behavioral treatment for hoarding disorder . Baseline sleep disturbance was significantly associated with hoarding severity, even when controlling for inability to sleep in a bed due to household clutter level. However, no significant change in sleep disturbance was reported following completion of treatment and baseline sleep disturbance was not significantly predictive of change in hoarding symptom severity. Findings suggest that disturbed sleep quality is associated with greater hoarding symptom severity but does not preclude positive symptom change in treatment."} +{"text": "Because seniors represent a rising proportion of Emergency Medical Services (EMS) provider activity, there is a growing focus on determining how EMS providers can better serve the aging population. Multifamily senior housing properties benefit from reductions in resident turnover; EMS providers aim to improve community health and minimize unnecessary and low-priority callouts. Developing partnerships between the two service sectors can help both meet their individual goals while reducing health system costs. Drawing on experience with the R3 initiative, this study describes how a partnership between EMS providers and supported housing sites has led to reductions in ambulance transfers to hospitals and reinforced falls reduction programs within senior housing. The R3 program represented an effort to work closely with EMS providers and, by doing so, provides an example of how collaboration between the two sectors can work to the advantage of both parties by identifying residents who would benefit from intervention."} +{"text": "Education\u2019s ambiguous association with cognitive decline may be due to unmeasured effect heterogeneity, including variation across environmental contexts. In areas with more social and physical resources, education may play less of a role in shaping cognitive trajectories. In areas with fewer resources, educational capital may be more important for slowing cognitive decline. Using multilevel models, this paper examines whether education\u2019s impact on cognitive trajectories varies among neighborhoods defined by differential densities of social and physical resources. Findings suggest that education plays a consistent role in shaping cognition across contexts. Lower education is associated with lower cognitive function and marginal differences in rates of decline . However, these patterns are invariant across neighborhoods. Findings reiterate the importance of education for cognitive function in late life, and stimulate further research on other contextual factors that may affect rates of cognitive decline."} +{"text": "Clostridium difficile infections, multi-organ dysfunction, sepsis in neonates and\u2014most notably\u2014ventilator-associated pneumonia (VAP) . It. It11]. The safety of using probiotics in critically ill patients has not been fully established. This has been a concern ever since the publication of the PROPATRIA trial, which\u2014although criticised on multiple fronts\u2014showed an increased mortality in patients with predicted severe acute pancreatitis on probiotic prophylaxis . ResultaMany questions remain before confidence in rolling out probiotic or synbiotic treatment to critical care patients wide scale. Certainly, evidence suggest significant benefit for reducing VAP, but the question remains why? Perhaps answering this will open up avenues for better targeted therapy with reduced risk of side effects. Although prokaryotic lineages contribute the vast majority of the gut microbiome by abundance, important players are also potentially missed as the eukarya and viral microbiome remain incompletely charted. Furthermore, at what dose, and can a single probiotic be used in all populations and geographies? Our experience from past critical care trials proves this will be unlikely. Hence, we need more large-scale studies which can team up with specialists in microbiome research to analyse the mechanisms behind such outcomes.It has been over 100\u2009years since Metchnikoff first hypothesised that the heavy consumption of cultured yogurt by Belgian peasants may somehow account for their remarkable health and longevity . Today,"} +{"text": "The concept of cascading trophic interactions predicts that an increase in piscivore biomass in lakes will result in decreased planktivorous fish biomass, increased herbivorous zooplankton biomass, and decreased phytoplankton biomass. Though often accepted as a paradigm in the ecological literature and adopted by lake managers as a basis for lake management strategies, the trophic cascading interactions hypothesis has not received the unequivocal support that might be expected of a paradigm. Here we review field experiments and surveys, testing the hypothesis that effects of increasing piscivore biomass will cascade down through the food web yielding a decline in phytoplankton biomass. We found 39 studies in the scientific literature examining piscivore effects on phytoplankton biomass. Of the studies, 22 were confounded by supplemental manipulations and could not be used to assess piscivore effects. Of the 17 nonconfounded studies, most did not find piscivore effects on phytoplankton biomass and therefore did not support the trophic cascading interactions hypothesis. However, the trophic cascading interactions hypothesis also predicts that lake systems containing piscivores will have lower phytoplankton biomass for any given phosphorus concentration. Based on regression analyses of chlorophyll\ufffdtotal phosphorus relationships in the 17 nonconfounded piscivore studies, this aspect of the trophic cascading interactions hypothesis was supported. The slope of the chlorophyll vs. total phosphorus regression was lower in lakes with planktivores and piscivores compared with lakes containing only planktivores but no piscivores. We hypothesize that this slope can be used as an indicator of \u201cfunctional piscivory\u201d and that communities with extremes of functional piscivory (zero and very high) represent classical 3- and 4-trophic level food webs."} +{"text": "Little is known about how health information obtained from different types of social networks affect health behaviors. This study aimed to explore the effect of health information on osteoporosis management behaviors among White and Asian women from a social capital (SC) perspective using a variety of SC measures . Semi-structured interviews were conducted with 10 White and 10 Asian women aged 50 and over in 2016. Through content analysis, we found that SC possession was different between older White and Asian women, and SC utilization to obtain health information corresponded with their possession of SC. Comparing to other diseases, health information relevant to osteoporosis was less frequently communicated. Health information from different types of SC interactively shaped participants\u2019 behaviors. The findings suggest that culturally appropriate health interventions might improve older White and Asian women\u2019s self-management behaviors of osteoporosis."} +{"text": "Certain demographics, health conditions, and functional limitations are associated with increased older adult falls. Health conditions and functional limitations are potentially modifiable and may have underlying factors in common. This study\u2019s objective is to understand whether health conditions and functional limitations related to increased fall risk have common underlying factors that could be useful in designing interventions. Factor analysis and multivariate logistic regression were used to analyze 2016 Behavioral Risk Factor Surveillance Survey data for adults aged 65+ years. About half of those who reported difficulty dressing (58%), difficulty running errands alone (53%), difficulty remembering (51%) and depression (48%) reported falling compared to 30% of the general older adult population. Two common factors of cognitive and physical limitations were identified and scales created for each. When controlling for demographic characteristics, both cognitive and physical limitations scales were significantly related to a higher odds of falling ."} +{"text": "States vary in their overall rates of nursing home deficiency citations as well as deficiencies for actual harm or jeopardy . Civil Money Penalty (CMP) fines collected by the Centers for Medicare and Medicaid Services (CMS) are one enforcement action imposed to promote nursing home compliance with regulations. Collected CMP funds are redistributed to states for the sole purpose of improving nursing home resident care and quality of life through reinvestment in quality improvement projects. Using CASPER data available for US skilled nursing homes in 2015 and 2016 through the CMS QCOR database we examined the distribution of quality of care (QOC) and quality of life (QOL) deficiencies and CMP enforcement action across states. Guided by the systems framework for evaluating nursing home quality we further explored how contextual factors such as state spending for nursing home care, structural characteristics of facilities in states, and inadequate care processes indicated by deficiencies contribute to CMP enforcement actions and fines. Findings indicate that 27% of enforcement actions resulting in a CMP between 2015 and 2016 were imposed for a QOL deficiency while 61.7% represented QOC deficiencies. QOL deficiencies represented only 8% of the highest severity deficiency category but 81.7% of enforcement actions for QOC were for those causing immediate harm or jeopardy. QOC deficiencies are a focus of enforcement actions as they represent critical care processes influencing resident basic needs for hydration, ambulation, skin integrity and care for other special physical and behavioral needs."} +{"text": "Family caregiving is often characterized as a chronically stressful situation, and stress process models have been the dominant conceptual foundation underlying caregiving studies for decades. Recently, this perspective has been augmented with more positive views that emphasize potentially healthy and prosocial aspects of caregiving. Replicated findings from population-based studies show that caregivers have lower mortality rates than noncaregivers, consistent with the more balanced conceptual approach. The Caregiving Transitions Study is investigating 251 participants who transitioned into a caregiving role at some point between two blood samples taken 10 years apart in a national epidemiological study and 251 matched controls. Preliminary analyses confirm that caregiving leads to increased psychological distress. Ongoing analyses are examining changes in inflammatory biomarkers, health status, and positive aspects of caregiving. Findings will be examined alongside our recent meta-analysis of convenience samples that found caregiving to have small and inconsistent relationships with biomarkers of inflammation and immunity."} +{"text": "Here we present new computational and experimental methods to leverage the gene expression and neuropathology data collected from several large-scale studies of Alzheimer\u2019s disease . These data sets include diverse data types, including transcriptomics, neuropathology phenotypes such as quantification of amyloid beta plaques and tau tangles in different brain regions, as well as assessments of dementia prior to death. This meta-analysis is a complex undertaking because the available data are from different studies and/or brain regions involving study-specific confounders and/or region-specific biological processes. We have therefore taken neural network and probabilistic computational approaches that reduce the data dimensionality, allowing statistical comparison across all brain samples. These approaches identify gene expression changes that are significantly associated with clinical and neuropathological assessment of Alzheimer\u2019s disease. We then conduct in vivo validation of the genes through genetic screening of C. elegans models of Alzheimer's disease utilizing our automated robotic lifespan analysis platform. This approach allows for the greater leverage of existing Alzheimer\u2019s disease biobank data to identify deep genetic signatures that could help identify new clinical gene-expression markers and pharmacological targets for Alzheimer\u2019s disease."} +{"text": "This study draws upon social capital and intergenerational reciprocity concepts to better understand how grandparents\u2019 depressive symptoms are related to their provision of grandchild care, within the context of their expectations regarding adult children reciprocating caregiving needs in the future. Analyses used the 2014 Health and Retirement Study dataset. The sample consisted of 9,612 grandparents, 2,595 of whom were providing grandchild care. Linear regression models were used to analyze how depressive symptoms were influenced by grandchild care provision and expectations of future care from adult children. Future care is measured as expectations from (1) any adult child, and (2) from the same adult child for whom the older parent provides grandchild care. Provision of grandchild care was not significantly related to grandparents\u2019 number of depressive symptoms. Among grandparents who provided grandchild care, both expecting any adult child and expecting the same adult child were associated with reporting fewer depressive symptoms. Expecting any adult child to provide future care showed a stronger effect than expecting the same adult child to provide future care. The results suggest that expectations of general reciprocity within the family system, rather than specific dyadic reciprocity, may be more important for a caregiving grandparent\u2019s emotional well-being. Providing grandchild care while expecting future care from adult children can indicate a sense of social capital within an intergenerational family system. Expecting support reciprocity from adult children may be a protective factor that allows caregiving grandparents to feel more secure about their future care needs, and consequently, less depressed."} +{"text": "Since age related perturbations in gene expression profiles have been described and transcriptomic changes in specific biological pathways have been implicated in the aging process, we performed whole transcriptome sequencing on 4000 HRS participants using RNA obtained from Paxgene tubes collected during the 2016 interview. We will describe design and implementation of innovative quality control procedures to minimize technical variability in transcriptomic measurements and monitor analytical variation in large population studies such as HRS. We will also report the distribution of transcriptomic profiles according to various demographic characteristics and describe the prevalence of previously reported aging related transcriptomic signatures in HRS. We will describe the associations between transcriptomic profiles and other measures of biological aging in HRS and report how changes in cell composition can affect transcriptomic profiles observed in population studies such as HRS."} +{"text": "Older adults are often excluded from physicians\u2019 preventive health promotion recommendations. The influence of patient race and gender on the physician-patient relationship in maintaining good health has been extensively researched, but little is known about the influence of patient race and gender on physicians\u2019 health promotion advice to the elderly. This study explored whether patient race and gender influence primary care physicians\u2019 health promotion advice to older adults. The sample of 536 respondents was obtained from a NCI funded study of \u201cHealth Care Partnerships in Cancer Prevention and Care of Aged. Respondents were randomly selected community dwelling older adults who attend Senior Center programs, sponsored by Area Agencies on Aging (AAA) . Multivariate logistic regression analyses were used to control for sociodemographic and health factors while accounting for a complex sampling design. African American patients had greater odds than whites for receiving recommendations to eat a healthy diet (OR = 1.62) and exercise more (OR = 1.83). Elderly women reported fewer recommendations to eat a healthy diet (OR = .81) & exercise more (OR = .45) than did men. Notably, respondents of both races and genders believed that maintaining a healthy lifestyle is very effective. These findings demonstrate that African American and female older adults receive differential health promotional advice from physician and suggest the need for raising physician awareness about the value of prevention advice for all patients."} +{"text": "Sus scrofa). Hypothesizing that pig space use is primarily driven by forage availability, we predicted strong selection for the most nutritionally beneficial crops and resource types as agricultural seasons progressed. We deployed GPS collars on 13 adult feral pigs in the Mississippi Alluvial Valley to study resource selection in a fragmented agricultural landscape. We estimated resource selection using mixed-effect logistic regression to assess variation in selection across planting, growing, harvest, and fallow seasons.The spatiotemporal distribution of resources is a critical component of realized animal distributions. In agricultural landscapes, space use by generalist consumers is influenced by ephemeral resource availability that may produce behavioral differences across agricultural seasons, resulting in economic and production consequences and increased human-wildlife conflict. Our objective was to assess changes in habitat selection across seasons in an invasive generalist omnivore . We also detected seasonal dependencies in proportional coverage on the net probability of selection of a land unit , resulting in marked variation in predicted space use among agricultural seasons.We found that feral pigs varied resource selection across seasons, particularly for corn (These findings indicate behavioral changes in selection across agricultural seasons are driven by complex interactions between the availabilities of temporally dynamic resources and temporally static natural cover. Temporal variations in resource selection trends indicate seasonal responses to crop phenology which suggests a season-specific habitat functional response. The distribution of resources across a landscape is a primary factor governing animal space use patterns. Spatial variation in the distribution of resources is reflected through animal distributions and local densities ; howeverOdocoileus virginianus) primarily consume crops in the summer , and the amount of consumption depends not only on resource availability in spring and summer, but also quality and abundance of natural forage consumed throughout the winter in adjoining forested habitat in A and B. We also defined a hypothetical raster \u03b1 such that (1) \u03b1 consists only of values of 0 and 1 , and (2) values of 0 and 1 are arranged so as to maximize the difference between \u03b1 and the target raster B. The value \u0394i,j is the difference between cell and represents the maximum theoretical difference that could occur if A = \u03b1. The metric S is simply the l2-normalized difference between rasters from 0 (perfect dissimilarity) to 1 (perfect similarity) and may be interpreted as the percent similarity between rasters. This approach allowed us to mathematically quantify the degree of similarity between rasters without requiring statistical assumptions or hypothesis tests that are heavily influenced by sample size.Lastly, we used the resource selection models to generate maps of the relative probability of selection for any given location within the entire region for each agricultural season using the entire CDL and flowline data for the LMAV. We calculated proportional cover by all land-cover and crop types within a 100 m buffer for each cell of the CDL raster, and calculated distance to flowline from the center of each cell. We used the model predictions to create seasonal maps of the predicted relative probability of feral pig occurrence. Using mixed-effects models accommodates small sample sizes when extrapolating selection patterns to larger spatial extents by reducing individual bias . We alson = 8; n = 12; n = 11; n = 8; Out of 16 feral pigs originally fitted with GPS collars, 13 provided viable data once non-independent animals were removed . Only animals with relocation data that spanned an entire season were included in seasonal analyses which resulted in the following sample sizes: early growing < 0.5) when corn, soybean, and rice were in high abundance relative to wetlands . Some general trends emerged when modeling a quadratic effect for distance to flowline. The lowest selection probabilities for corn, rice, and soybeans consistently occurred when feral pigs were located 450 m from an established flowline except in the early growing season, when no significant quadratic effect was observed .To better understand the realized implications of our models for space use, we solved each seasonal model across the LMAV and produced heatmaps from the probabilities of selection per pixel . The heaWe found strong evidence of season-specific differences in resource selection driven by the seasonal availability of agricultural resources. Not only did overall selection strength for proportional cover across the landscape vary , but theThe habitat functional response can also be driven by thermoregulatory considerations, as has been demonstrated in other ungulates , and thiTriticum aestivum). Wetlands were consistently selected for in each season but less strongly in the early growing season which coincides with increased resource availability in double-cropped fields and seasonal flooding which increases waste grain availability [It is important to note that the \u201cother crop\u201d category has high selection throughout all but the harvest season . This calability . Spring lability , and lowOur results underscore the importance of the composition of the landscape for wildlife movements and space use. Selection for locations varying in proportional cover between crops and natural wetlands was highly dependent on the relative proportions of each, with a general trend that increasing proportionality by wetlands increases the likelihood of selection . This imOur findings suggest feral pigs change their space use trends by altering the relative probability of selection for the primary crops. In the early growing season corn is one of first resources planted, but rice and soybeans have higher probabilities of selection during the early growing season . This coWe detected a significant effect of distance to flowlines Figs and 3, wCapreolus capreolus) [Alces alces shirasi) [We expected decreased selection strength for corn at later stages of maturity when other crops become more nutritionally valuable relative to corn ,15; howepreolus) , and in shirasi) . In pigs\u00df-estimates that are nonsensical when viewed in isolation, as we observed here . Our results indicate the importance of studying animal responses to season-specific landscape composition as it can affect their movements and overall space use patterns, leading to season-specific mechanisms influencing a single ecological phenomenon. We also stress the importance of proper model interpretation and prediction from complex or strongly collinear data, particularly proportional coverage data. Acknowledging such collinearity can aid in identifying interesting ecological phenomena , and failure to do so can lead to erroneous or misleading conclusions regarding a species\u2019 ecology. Our work highlights the importance of carefully considering seasonality in animal preference and provides guidance on fitting models relevant to such studies, particularly in understudied regions for wildlife studies .S1 TablePercent cover by each CropScape land cover classification across the Lower Mississippi Alluvial Valley.(PDF)Click here for additional data file.S2 TableModel outputs from generalized linear mixed effect models of feral pig resource selection throughout agriculturally defined seasons during the day. A bolded value represents a significant coefficient.(PDF)Click here for additional data file.S1 FigSeasonal 100% minimum convex polygons used to sample availability for each individual in the late growing season to show the overall extent of the Lower Mississippi Alluvial Valley sampled.(PDF)Click here for additional data file."} +{"text": "According to the family systems theory, strains from parenting an adult with disabilities may spillover to parents\u2019 relationships with their other children and disrupt family dynamics and their well-being in later life. This study examined whether parental ambivalence toward their non-disabled children is greater in families of adults with disabilities [developmental disabilities (DD) or serious mental illnesses (SMI)] than families without an adult child with disabilities. The study also investigated whether ambivalence mediates the associations of having an adult child with DD or SMI on parents\u2019 health. Data were from the 2011 Wisconsin Longitudinal Study in which aging parents were asked about their relationship with each of their adult children. Multilevel regression models and multilevel structural equation models (MSEM) were estimated to analyze the data. Our findings showed that parents of an adult with SMI felt greater ambivalence toward their non-disabled adult children than comparison group parents of adults without disabilities, whereas no significant differences were found between parents of an adult with DD and comparison group parents. Parental ambivalence toward their non-disabled adult children played a significant indirect role in the negative association between having a child with SMI and parental physical and mental health, after adjusting for parent- and child-characteristics associated with parental health and/or ambivalence. The findings have implications for clinical practice with aging families of adults with disabilities and suggest the need for additional research to better understand intergenerational dynamics in these families."} +{"text": "Voluntarism has been portrayed as a productive and even transformative process whereby rural communities, households and older residents are able to meet the challenges of changing rural demographics. Yet, little attention has been paid to building a critical perspective on the complex and often-contested expectations placed on older rural volunteers. This paper focuses on the particular gap in understanding the contributions of older rural adults as a crucial resource in creating opportunities for aging in place and sustainable rural community development. Drawing on research into voluntarism in Canada\u2019s aging resource communities, this paper presents qualitative findings from innovative \u2018volunteer leadership biographies\u2019 with older residents who were involved in key voluntary sector initiatives to improve community development. The findings show how older volunteer leadership is embedded in both place (residency) and time (life course), revealing new dimensions to the problem of understanding volunteer leadership in an era of rural population change."} +{"text": "Sleep disruption is a key clinical issue in the dementias but the sleep phenotypes of these diseases remain poorly characterised. Here we addressed this issue in a proof-of-principle study of 67 patients representing major syndromes of frontotemporal dementia (FTD) and Alzheimer\u2019s disease (AD), in relation to 25 healthy older individuals. We collected reports on clinically-relevant sleep characteristics - time spent overnight in bed, sleep quality, excessive daytime somnolence and disruptive sleep events. Difficulty falling or staying asleep at night and excessive daytime somnolence were significantly more frequently reported for patients with both FTD and AD than healthy controls. On average, patients with FTD and AD retired earlier and patients with AD spent significantly longer in bed overnight than did healthy controls. Excessive daytime somnolence was significantly more frequent in the FTD group than the AD group; AD syndromic subgroups showed similar sleep symptom profiles while FTD subgroups showed more variable profiles. Sleep disturbance is a significant clinical issue in major FTD and AD variant syndromes and may be even more salient in FTD than AD. These preliminary findings warrant further systematic investigation with electrophysiological and neuroanatomical correlation in major proteinopathies. Symptom duration did not differ between the FTD and AD groups (p\u2009=\u20090.940). As anticipated, most (36/39) patients with AD but only two patients with FTD were taking an acetylcholinesterase inhibitor; frequency of antidepressant use did not differ significantly between the AD and FTD groups (\u03c7\u00b2(1)\u2009=\u20092.32, p\u2009=\u20090.165), while only one patient in the study was prescribed a benzodiazepine and none was prescribed a neuroleptic medication.3.2Usual daily rest periods for all individual participants are plotted in Time spent overnight in bed differed significantly among the FTD, AD and healthy control groups \u2009=\u20099.80, p\u2009<\u20090.001); Bonferroni post-hoc tests revealed that the combined AD group spent significantly longer on average overnight in bed than healthy controls ; whereas the combined FTD group did not differ significantly from healthy controls (p\u2009=\u20090.240). Comparing disease groups, the combined AD group spent on average significantly longer overnight in bed than the combined FTD group . When syndromic subgroups were compared separately to healthy controls, no significant differences in average time spent overnight in bed were identified.The usual time of retiring was on average significantly earlier in patients with FTD and AD than in healthy controls. These groups did not differ significantly in usual time of rising \u2009=\u20093.08, p\u2009=\u20090.054). No significant syndromic group differences in usual times of retiring or rising were identified.The lack of syndromic differences on sleep time measures may in part reflect the wide variation in bed periods in the patient groups. Inspection of the individual data suggests3.3Compared to healthy controls, both the combined FTD and combined AD disease groups showed significantly increased odds of experiencing difficulty sleeping . The combined FTD and AD groups did not differ significantly in their odds of experiencing difficulty sleeping .Compared to healthy controls, the FTD and AD syndromic subgroups showed variably increased odds of experiencing difficulty sleeping: this was most pronounced in the bvFTD subgroup while the SD and PNFA subgroups did not show a difference in odds compared to controls . Both AD syndromic groups showed a significant effect . Around 80% of patients with bvFTD and around 60% of patients in other syndromic groups experienced some difficulty sleeping .3.4Relative to healthy controls, both the combined FTD and AD disease groups and all syndromic subgroups showed significantly increased daytime somnolence . The combined FTD group was significantly more likely to have excessive daytime somnolence than the combined AD group . Around 80% of patients with FTD syndromes and 50% of patients with AD syndromes experienced excessive daytime somnolence .3.5The combined FTD and AD disease groups and syndromic subgroups did not differ significantly from healthy controls overall in their propensity to experience disruptive events associated with sleep. The combined FTD group showed higher odds of experiencing disruptive sleep events than the combined AD group ; there were no other significant correlations among sleep symptoms. Overall clinical disease (symptom) duration was not significantly correlated with the presence of sleep symptoms in either the FTD group or the AD group.4Here we have shown that sleep disturbance is a substantial clinical issue in diverse dementia syndromes representing the major phenotypes of FTD and AD. Difficulty falling or staying sleep at night and excessive daytime somnolence were significantly more frequently reported for patients with both FTD and AD than healthy age-matched individuals, occurring in over half of the AD group and around three-quarters of the FTD group. Patients with FTD were more likely to experience disruptive sleep events than patients with AD. Patients with FTD and AD habitually retired earlier and AD spent on average significantly longer in bed overnight than did healthy older individuals. Excessive daytime somnolence was significantly more frequent in the FTD group than the AD group overall. Our findings are comparable to estimates of the overall frequency of sleep disturbance in previous AD and FTD case series , corroboper se, circadian behaviour is likely to be influenced by the subjective distress attending a given \u2018objective\u2019 level of sleep disturbance, how this is modulated by social context and how it is communicated by patients to their caregivers. Both bvFTD and SD may lead to altered sensitivity and abnormal behavioural responses to homeostatic derangements [While we do not have direct neuroanatomical or histopathological correlation in this study, our findings are in line with the known pathological anatomy of these neurodegenerative disorders. Both FTD and AD are associated with pathological involvement of circadian and sleep regulatory networks traversing hypothalamus, basal forebrain and mesial temporal lobe and associated disruption of homeostatic drives and mechanisms . Disruptngements . All thrngements ,51: we pC9orf72 mutations (but not with other FTD mutations or AD syndromes) was a striking feature of this cohort. This observation is in line with previous reports of disordered REM behaviour in association with C9orf72 mutations [MAPT mutations, in human patients and in animal models [The present findings hint that sleep syndromes may have histopathological or genetic associations. The similarity of the sleep symptom profiles exhibited here by patients with tAD and its major syndromic variant PCA are in line with shared involvement of the temporo-parieto-subcortical \u2018default mode network\u2019 and withutations and accoutations ,58. It il models . With rel models . This apl models relate tl models : it is pl models ,66. Howeprima facie case for further systematic exploration of the 24\u2009-hour sleep-wake cycle as a source of candidate biomarkers in FTD. Moreover, sleep disruption is a major cause of caregiver burden and therefore a key management issue across dementia syndromes [Our findings have important clinical implications. Sleep is a viable target for development of novel biomarkers and incorporation into clinical trials; this is a theme that has emerged strongly in AD but not, so far, in FTD. The present data provide a yndromes ,42,67. Tyndromes . Along wOur study has several limitations that suggest our findings should be interpreted with some caution and which should motivate future work. The first priority is to validate our sleep symptom survey and the findings in this participant cohort prospectively in other cohorts. Administering the survey questions to a larger cohort of healthy individuals would be required to determine their sensitivity and specificity in discriminating disease effects from those of healthy ageing. Further, the present findings should be corroborated using standardised sleep assessment scales and grading of symptom frequency and severity, in larger patient cohorts and based on reports collected in parallel from patients and caregivers. Larger cohorts will be particularly important given the wide individual variation observed across and between syndromic groups within the spectrum of FTD and AD.More fine-grained analysis of sleep symptoms and a wider representation of symptoms will be leading priorities. For example, in this study we deliberately avoided including patients with a prior history of obstructive sleep apnoea in order to avoid the potentially confounding issue of dual diagnoses but sleep disordered breathing is itself likely to constitute an important risk factor in the pathogenesis of AD ,68. MoreOur sleep symptom survey was based on self-reporting of sleep symptoms by healthy controls versus second-person (caregiver) reporting for patients: use of a uniform symptom survey protocol across the participant cohort will be required to ensure that similar phenomena are indeed being captured in each participant group. While patient self-reports are potentially confounded by memory and behavioural impairments, these will be required to capture subjective correlates of sleep disruption (particularly in syndromes such as bvFTD and SD that are likely to disconnect subjective awareness from objective deficits) as well as to compare patients\u2019 experience more directly with that of healthy controls. Self-reporting by patients would facilitate ratings of symptom severity and frequency, which are problematic to achieve second-hand but ultimately necessary to provide a more detailed picture of sleep phenomenology in neurodegenerative diseases. It will also be important to relate sleep symptoms to other indices of daily life functioning and to clinical prognostic outcomes. Medication use is a further important and potentially highly relevant variable: in our cohort, both the AD and FTD groups were taking antidepressants while there was a clear disproportion in the use of acetylcholinesterase inhibitors by the AD group. This reflects widespread prescribing practice in people with dementia and thus \u2018real world\u2019 experience. However, both drug classes can potentially affect sleep quantity and quality and further work is required to differentiate pharmacological from endogenous disease effects.A further key requirement in order to evaluate and validate the biomarker potential of any sleep symptom scale will be to assess symptoms longitudinally and to ground symptom reports in objective pathophysiological measures of circadian motor activity (actigraphy) and most importantly, changes in electrophysiological sleep architecture using polysomnographic techniques, over the entire 24\u2009-h circadian cycle. This will also be essential for excluding subclinical and intercurrent (premorbid) sleep disorders. From a neurobiological perspective, there will be considerable value in correlating sleep phenotypes with neuroimaging modalities that can examine the structural and functional integrity of circadian networks in particular FTD and AD syndromes and with neurochemical assays that can address orexinergic and related candidate mechanisms which may drive sleep disruption in these diseases. Combining data from multiple specialist centres is a powerful tool for assessing phenotypic correlates of rare genetic disease subtypes, and potentially, for detecting sleep changes that may predate other clinical symptoms .5This first clinical comparison of patient cohorts representing canonical FTD and AD syndromes has underlined that sleep-related symptoms are a significant issue across these diseases, with some evidence for differentiation of pathologies. Our findings, though preliminary, provide proof-of-principle to justify undertaking future larger scale validation and correlative studies, in order to characterise in detail the role of sleep disturbance in promoting clinical symptoms and signalling pathophysiology in major dementias."} +{"text": "We found significant phylogenetic signal in structure and pattern at both spatial scales, along local elevational, and latitudinal gradients. Moreover, beta diversity was affected by different environmental variables in herbaceous and shrub species distributions across different spatial scales. Our results highlight the relative importance of local ecological mechanisms, including niche\u2010based deterministic processes as well as those of biogeographical processes, such as stochastic dispersal limitation and habitat specialization in plant assemblages of mountainous rangeland.The mechanisms determining community phylogenetic structure range from local ecological mechanisms to broad biogeographical processes. How these community assembly processes determine phylogenetic structure and patterns in rangeland communities across multiple spatial scales is still poorly understood. We sought to determine whether the structure of herbaceous and shrub assemblages along local environmental gradients (elevation) and broad geography (latitude) exhibited phylogenetic signal at different spatial scales, across 2,500\u00a0ha of a mountainous rangeland. We analyzed species distribution and phylogenetic data at two spatial scales: the community level (1\u00a0m At smalSeveral theories explain the mechanisms shaping local communities. Niche\u2010based theories posit that deterministic processes such as environmental filtering and biotic interactions affect plant communities, whereas neutral theories suggest stochastic processes, including historical processes and dispersal limitation Hubbell, . DispersThe relative importance of stochastic and deterministic processes in shaping rangeland plant communities remains particularly unclear across elevation and latitude (36\u00b040\u2032 and 36\u00b053\u2032N). We divided the study region into four elevation zones . Within each zone, we randomly placed 59 plots. To examine the relationship between elevation gradient and climate, we extracted mean annual precipitation and mean annual temperature for all of sample units from the WorldClim v1.4 database level, and the habitat level. Accordingly, we studied plant species composition in 1\u00a0m2 plot was assigned to one of the four habitat types in the study region via the proposed CART extension to handle our response variables De'ath, . MRT was De'ath, package To identify indicator species that have statistically significant associations with each habitat\u2010type, we used indicator species analysis and mean nearest taxon phylogenetic distance (MNTD) among species in each plot and each habitat to evaluate spatial changes in the phylogenetic structure of community and habitat herbaceous and shrub assemblages , and between habitats at the habitat scale . The first split based on elevation explained variation (50%) in community composition across all plots. High elevation plots were further segregated based on latitude showed were significantly associated with only one habitat. The number of species occurring in single habitats ranged from 9 (in habitat H2) to 14 species (habitat H4). In general, high elevation and latitude habitats had more species associated with them , while herbaceous and shrub assemblages in habitats H3 and H4 tended to be phylogenetically overdispersed than that of elevation on phylogenetic beta diversity after accounting for the effect of latitude . However, standard effect sizes of Dpw suggested a greater role for elevation in basal phylogenetic beta diversity is consistent with predictions that more phylogenetical overdispersion occurs in colder environments due to biotic interactions and environmental filtering (Qian et\u00a0al., Although competitive exclusion as a process has a strong effect on plant communities, facilitation also has an important role in shaping communities especially in environmentally harsher ecosystems such as rangelands (Soliveres et al. 2012; Cavieres et al. 2013; Valiente\u2010Banuet & Verdu, Niche\u2010based deterministic and neutrality\u2010based stochastic processes were important in our study, as indicated by the strong effects of latitude and elevation on the phylogenetic turnover of herbaceous and shrub species. However, geography was a better predictor of phylogenetic beta diversity at the local scale. Evaluations of terminal and basal phylogenetic turnover metrics with geographic distances indicate there are greater turnover within clades than among clades, even though both are significantly correlated with geographic distances which we infer to mean that the dispersal limitations are relatively conserved near the tips of the phylogeny. Therefore, stochastic assembly and dispersal limitation have more prominent roles in explaining variation at local scale (Gilbert & Lechowicz Although environment and geographic distance had strong associations with terminal and basal phylogenetic beta diversity metrics, we observed greater turnover among clades than within clades at the regional scale. Therefore, we infer environmental requirements are relatively conserved at shallow levels in the phylogeny. Moreover, high beta diversity between habitats indicated nonrandom patterns between habitats types, suggesting the dominance of particular species in each habitat type (Pitman et al., 5In conclusion, we found significant phylogenetic signal in the structure and turnover of herbaceous and shrub species distributions of a mountainous rangeland located in the Northeast of Iran. The structure of herbaceous and shrub assemblages at both the community and habitat scales indicated significant phylogenetic overdispersion across elevation and latitude due to niche\u2010based deterministic processes and species interactions on the local scale and environmental filtering and habitat specialization at the regional scale of herbaceous and shrub assemblages. We particularly noted a different importance to stochastic and deterministic processes on the distribution of species assemblages at different scales. The greater explanatory power of geographic distance and latitude than elevation suggests stochastic processes and dispersal limitation create greater phylogenetic turnover at the tips of branches at the local scale, but a stronger association of terminal PBD with elevation distance at the habitat scale suggests that environmental requirements are conserved shallower in the phylogeny.Data will be made available in the Dryad Digital Repository.None declared.MP performed the project, wrote the MS and analyzed all of data as the Ph.D. student. HE defined the project as the main supervisor and considered the whole analysis. JV collaborated as the co\u2010supervisor of the project. RS considered the phylogenetic data analysis and edited the MS as the advisor, also allocated his laboratory in Tokyo University to carry out molecular experiments.\u00a0Click here for additional data file."} +{"text": "Motor skill learning typically occurs in a period when the brain needs to navigate a body that is still growing and developing. How the changing body, neural circuit formation, and motor coding influence each other remains unknown. Songbirds provide excellent model systems to study motor skill learning. It has recently been shown that songbird vocal muscles double in speed during sensorimotor learning. Here we argue that these contractile as well as morphological changes stem predominantly from use and only secondarily from hormones or genetic programs. This implies that muscle training constrains skill-learning trajectories. As contractile muscle property changes must require altered motor codes for achieving the same acoustic targets, the final performance results from interactions between brain and body.Understanding how novel behaviors are learned remains a major challenge to modern neuroscience. Acquiring and mastering fine motor skills, from dexterity in piano playing to microsurgery or speech, can take weeks to months or even years and is strongly affected by injury, stroke, and developmental as well as neurodegenerative disorders. Most fine motor skill learning occurs postnatally from infancy to adolescence when the brain needs to navigate a body that is exhibiting large changes due to growth and development. Changes in neural coding and circuit development remain challenging to follow over meaningful timescales in single individuals and are thus typically studied during rather brief periods in adult subjects or durinThe brain does not function in isolation. All animal behaviors result from complex system-wide interactions between nervous system, body, and surrounding environment . Motor pHow the developing body influences circuit formation and neural coding in the brain and vice versa is still largely unknown . Recent In this opinion piece, we argue that contractile changes occurring in the vocal muscles of songbirds during song learning stem predominantly from interactions between brain and body. This implies that extensive training of syringeal muscles is essential to achieve their maximal performance and that the duration and trajectory of song learning are not solely set by neural circuit formation. Given that virtually all motor skills or at least their building blocks are acquired during times while the body is still changing, truly understanding motor coding and its pathologies requires rethinking and an embodied approach to understand motor learning.in vivo recordings show that over vocal development the premotor neurons gradually change their firing pattern from highly variable patterns into sparse high-frequency bursts (The sensorimotor phase of song learning in zebra finches takes \u223c2 months and starts when juveniles start producing subsong at 28 d post-hatching (DPH). Song development proceeds from subsong through plastic song to adult, so-called crystallized song, which is reached \u223c100 DPH . Over thy bursts . In aduly bursts and can y bursts .MYH) toward near-exclusive expression of MYH13 aka superfast myosin time course after birth. In songbirds, MYH13 expression changes over 2 months during sensorimotor learning available to the field, birdsong is an ideal system to embrace such an integrative approach."} +{"text": "Rapid detection of increases in HIV transmission enables targeted outbreak response efforts to reduce the number of new infections. We analyzed US HIV surveillance data and identified spatiotemporal clusters of diagnoses. This systematic method can help target timely investigations and preventive interventions for maximum public health benefit. Despite innovations in HIV prevention and treatment, HIV outbreaks do occur in the United States. Local public health staff identified >200 persons with HIV resulting from an injection drug use (IDU)\u2013associated outbreak in 2015 in Scott County, Indiana (The Centers for Disease Control and Prevention (CDC) recently began using HIV nucleotide sequence data from the National HIV Surveillance System (NHSS) to identify clusters of recent and rapid HIV transmission . For each state or county, we determined the total number of diagnoses during the most recent 12 months (January\u2013December 2016) on the basis of residence address at time of HIV diagnosis . We calcState-level alerts occurred for 4 (8%) of 50 states ; county-level alerts occurred for 143 (5%) of 3,142 counties nationwide . A mediaWe aimed to develop a spatiotemporal cluster detection method that could efficiently be used and adapted to identify potential increases in HIV transmission in different local contexts. We identified significant increases in HIV diagnoses across all regions, capturing alerts from counties with small, medium, and large baseline numbers of HIV diagnoses. Some counties had small increases in the number of diagnoses and large percentage increases; others had larger increases in numbers but smaller increases in percentages . IDU-attWe discussed our results with several state and local health departments that expressed interest in a robust, systematic method for routine identification of spatiotemporal clusters. They confirmed that this method identified alerts where they had recently begun responding and that new alerts provided actionable information regarding concerning HIV transmission increases.Small median and mean numbers of alerts suggest reasonable investigative loads for this method. Batching data into moving 12-month frames reduces alerts resulting from seasonal variability and data noise. The chronic nature of HIV infection means that related cases might not be diagnosed until months or years after infection, so the 12-month analysis frame might not capture all related diagnoses, but it does account for delays between diagnosis and reporting to surveillance systems. These delays need to be addressed differently across states . Reviewing testing history, partner services, contact tracing, and molecular data might help determine whether alerts represent clusters of recent infections that warrant investigation. Future evaluation will assess the extent to which this method identifies recent transmission and whether modifications might improve the method for different contexts.,\u2013The ideal cluster and outbreak detection system would use both case surveillance and molecular sequence-based approaches. Each method might help overcome the other\u2019s limitations. Although some alerts occurred in counties with large baseline HIV numbers, this method is less sensitive for these areas and might not capture all meaningful clusters. Analysis of sequence data is crucial for identifying transmission clusters in areas with larger numbers of cases and those distributed over broader geographic areas. However, this method is timelier than molecular methods and can provide state and local health officials with actionable data for early investigation. This factor might be particularly necessary for identifying increases in transmission associated with IDU, given increasing opioid use and the potential for rapid spread of HIV among vulnerable populations (In summary, we developed a systematic method to identify spatiotemporal clusters of HIV diagnoses. Routine use of this method in near real-time can automate detection of increases in HIV diagnoses meriting further investigation, helping state and local health departments prioritize and target HIV prevention and outbreak response efforts for maximum public health benefit."} +{"text": "Genomics and particularly mutational profiling have deepened our understanding of cancer pathology and laid the foundation for precision therapies as a major field in modern oncology. Comprehensive genetic profiling has even led to proposals to replace the current histology-based WHO tumor typing with molecular classes based on the rationale that similar molecular alterations have similar clinical relevance across cancers -3. HowevOne way to assess the functional impact of genetic alterations in cancer is to study their relationship with corresponding (phospho-)proteomic profiles. Using publicly available proteogenomic profiling data across major cancer types, we propose a computational approach that systematically evaluates if and to what extent genetic aberrations observed in tumors are associated with distinct proteomic profiles . Our anaTo achieve this, we further propose a combined experimental and computational systems proteogenomics approach that allows for a reduction of the mutational complexity and facilitates the identification of functionally relevant molecular aberrations that can be exploited to overcome resistance against targeted (mono-)therapy in individual patients . It combIn summary, the ability to interpret and assess the functional and clinical relevance of the increasingly comprehensive mutational profiling data accruing not only in research but also in clinical cancer diagnostics is reaching its limits when relying only on pre-clinical experimental data and knowledge on biological pathways. Moreover, the combinatorial complexity of potential druggable target combinations is incompatible with testing in classical clinical trials. As a solution, combining genomic with proteomic profiling and classical computational analysis as well as advanced machine learning techniques can contribute to identifying functionally and clinically relevant molecular alterations which in the future may complement molecular diagnostics for precision oncology."} +{"text": "Progradungula otwayensis produces two variations of cribellate silk in webs: ladder lines are stereotypically combed with the calamistrum while supporting rail lines contain silk that is naturally uncombed, spun without the intervention of the legs. Combed cribellate silk is highly extensible and adhesive suggesting that the reserve warp and cribellate fibrils brings them into tension only near or after the underlying axial fibers are broken. In contrast, these three fiber components are largely aligned in the uncombed threads and deform as a single composite unit that is 5\u201310x stronger, but significantly less adhesive, allowing them to act as structural elements in the web. Our study reveals that cribellate silk can occupy a surprisingly diverse performance space, accessible through simple changes in spider behavior, which may have facilitated the impressive diversification of web architectures utilizing this ancient silk.Web-building spiders are an extremely diverse predatory group due to their use of physiologically differentiated silk types in webs. Major shifts in silk functional properties are classically attributed to innovations in silk genes and protein expression. Here, we disentangle the effects of spinning behavior on silk performance of the earliest types of capture threads in spider webs for the first time. Moreover, spider silk is an outstanding biomaterial that has received significant attention3. Most prey capture webs consist of non-sticky ampullate silk structural elements and sticky capture threads that adhere to prey. Glue droplet coated viscid silk is most familiar and is the dominant capture silk in most orb- and cob-webs4. However, viscid silk evolved recently and many spiders instead use cribellate silk to make webs adhesive. Cribellate silk uses a mesh of very thin, dry nano-fibers around its axial threads to generate adhesion6. Cribellate silk is laboriously spun from the functionally co-dependent cribellum and calamistrum 9. Cribellate threads are composites of several interwoven fiber types. The most prominent fibers are the (1) axial fibers produced by pseudoflagelliform glands, (2) reserve warps and (3) the characteristic mass of nanofibers originating from the cribellum11. The reserve warps usually coil into a unique spiral morphology that is thought to enhance extensibility11. In a classic cribellate thread the nanofibers are combed into characteristic puffs using the calamistrum12. Removing the calamistrum does not hinder uloborid spiders from using their hind legs to manipulate and produce functional threads, but greatly reduces cribellar puffs, indicating that combing is a crucial stereotyped behavior in cribellate spiders13.Spiders are successful and diverse predators in terrestrial ecosystems14, and van der Waal\u2019s forces so that adhesion depends in part on the total number of cribellate fibrils per length of thread. The puffs produced by combing may increase the total surface area of cribellate fibrils thereby improving adhesion16. Their composite nature also makes the tensile behaviors of cribellate threads quite complex as the cribellate fibrils are typically very loosely laid down on the axial fibers\u00a0so that they stretch and deform somewhat independently of the axial\u00a0fibers themselves6, in contrast to viscid silk where the deformation of the glue droplets and the underlying axial fibers is tightly linked17. Both the puffs and how the cribellate fibrils are laid down are under behavioral control by spiders. However, the complex cribellate threads are not easily disentangled making it difficult to directly test the effects of each fiber component on thread performance.Cribellate threads adhere through physical interlock, capillary forcesProgradungula otwayensis. We show how the different spinning behaviors allow the same silk to be used both as a strong, stiff structural element and as a highly extensible and adhesive capture thread. Our data suggest that the uncombed thread represents a new functional type of silk thread due to the clear differences in mechanical and adhesive properties compared to combed cribellate threads.Here, we disentangle the effects of spinning behavior on silk performance of the earliest type of capture threads in spider webs. We describe naturally uncombed cribellate threads, produced without intervention of the hind legs, which are incorporated as a structural element within the web of the Otway odd-clawed spider P. otwayensis webs consist of a signal line, upper scaffolding and a prominent catching ladder18 23, although the magnitudes of these values may change on natural insect surfaces where cribellate nanofibers interact with epicuticular waxes to generate additional capillary forces14. The over ten-fold higher adhesion of combed silk in the ladder lines compared to uncombed silk in the rail lines is likely due to the increased surface area of cribellar puffs23. Combing therefore mediates a tradeoff in silk performance that allows the same fibers to act either as a relatively stiff structural element in webs (rail lines) or as very extensible adhesive elements (ladder lines).Adhesive force generated by 24, it is hard to imagine intermediate evolutionary morphologies that lead to this functionally correlated system. Our finding of cribellate silk produced without the intervention of the calamistrum relaxes the idea of an obligate functional correlation between both structures. At the same time, certain spiders are known to use special combs of setae in the hind tarsi to draw viscid silk from the anterior lateral spinnerets and wrap their prey26, thus it is conceivable that either precursor of the cribellum or the calamistrum may have appeared first in evolution.Because the cribellum and calamistrum are always found together in spidersP. otwayensis achieves a functional differentiation of silk through behavioural manipulation of the same silk type (combing vs. not combing), instead of using changes in silk protein expression typically seen in other species. The implications of replacing major ampullate structural threads with uncombed cribellate silk remain to be tested but the rail lines are much more extensible than ampullate silk, which likely has important implications for how these webs deform during prey capture and hence for the foraging ecology of these spiders. Prey capture behavior in P. otwayensis27 is similar to net-casting spiders (Deinopidae) where the web is highly elastic and actively used to ensnare prey28. This hunting strategy utilizes structural threads with high extensibility. Here we show that P. otwayensis has converged on highly extensible structural threads but by modifying the spinning behavior of capture silk rather than employing physiologically distinct silk types.In summary, Progradungula otwayensis silk was collected as described in29 at Great Otway National Park, Victoria, Australia. Silk images were obtained using the BK PLUS Lab system with a customized microscope lens (Ocellus) and 10x Mitutoyo objective mounted on a Canon 7D Mark II camera. Image stacks were processed using Zerene Stacker. Scanning electron microscope images were obtained with a field emission Zeiss Supra 40 in high vacuum, after sputter coating with AuPd.6. The low sample size is due to the rarity of this Australian endemic species, but still clearly demonstrates dramatic differences in silk properties. Samples were stretched at 1.5% extension s\u22121 at ambient temperature (~25\u2009C) and humidity (~30% RH). Extensibility was calculated as percent change in length and represents structural changes as well as material properties due to the multiple components of the silk composite coming under tension at different times.Tensile mechanics of seven combed and five uncombed threads from two individuals were determined using a Nano Bionix\u00ae tensile tester (MTS) to generate load-extension data as previously described30. Cribellate threads were mounted perpendicularly above a 2\u2009mm wide glass stage on the force plate. Fibers were pre-loaded to 50\u2009\u03bcN for 10\u2009seconds then pulled off at 0.1\u2009mm\u2009s\u22121.Adhesive properties of three combed and two uncombed threads from two individuals were determined using a Nano Bionix (MTS) similar to previous studies10 transformed data and analyzed with one random factors and a fixed effect. Tensile data (load and extension at breaking) was not transformed and analyzed with one random factor and fixed effect.We analyzed our data using two general linear mixed models in the R package \u2018lmerTest\u2019. Adhesion data (force and extension at detachment) was logSupplemental Tables"} +{"text": "Genomic aberrations, such as translocations, deletions or more complex \u201cchained\u201d rearrangements of DNA are frequently found in almost any cancer entity. These rearrangements can be clonal and stable during the course of disease \u2013 thus pointing to an initializing event \u2013 or subclonal, with specific rearrangements being present in specific fractions of cancer cells with fraction sizes varying during disease progression. In the latter scenario, genomic instability of the cancer genome results in longitudinal subclonal diversification during disease progression, contributing to high clonal dynamics and finally to the selection of treatment refractory clones and disease relapse. While genome rearrangements involving loss or amplification of cancer related genes are apparent drivers that give a growth advantage to the cell, also balanced rearrangements without loss of genetic material can contribute to clonal fitness by generating fusion proteins or by affecting the expression of genes adjacent to breakpoint junctions. Particularly in chronic lymphocytic leukemia, complex karyotypes often go along with bad prognosis and poor response to therapies. Resistance to novel Bruton\u2019s Tyrosine kinase (BTK) inhibitors (ibrutinib) or BCL2 inhibitors (venetoclax) is more frequent in cases with complex karyotypes , 2. Ibru"} +{"text": "Social ties are essential for survival but the mechanisms accounting for this link are unclear. This study examined links between daily interpersonal experiences and cardiovascular reactivity. A total of 34 participants (aged 40 to 80) completed ecological momentary assessment surveys every three hours for 4 days and wore a device that assessed heart rate (HR) and heart rate variability (HRV). Multilevel models revealed that a greater number of social interactions and negative social interactions predicted increased HR. Links between social interactions and cardiovascular reactivity varied by gender and race. A greater number of interactions and negative interactions predicted increased HRV among men and not women. A greater number of social interactions predicted increased HR among Black individuals and White women but not White men. Thus, social interactions appear to get under the skin via the cardiovascular system but in unique ways that vary by gender and race."} +{"text": "The HIV prevention cascade could be used in developing interventions to strengthen implementation of efficacious HIV prevention methods, but its practical utility needs to be demonstrated. We propose a standardized approach to using the cascade to guide identification and evaluation of interventions and demonstrate its feasibility for this purpose through a project to develop interventions to improve HIV prevention methods use by adolescent girls and young women (AGYW) and potential male partners in east Zimbabwe.We propose a six\u2010step approach to using a published generic HIV prevention cascade formulation to develop interventions to increase motivation to use, access to and effective use of an HIV prevention method. These steps are as follows: (1) measure the HIV prevention cascade for the chosen population and method; (2) identify gaps in the cascade; (3) identify explanatory factors (barriers) contributing to observed gaps; (4) review literature to identify relevant theoretical frameworks and interventions; (5) tailor interventions to the local context; and (6) implement and evaluate the interventions using the cascade steps and explanatory factors as outcome indicators in the evaluation design. In the Zimbabwe example, steps 1\u20105 aided development of four interventions to overcome barriers to effective use of pre\u2010exposure prophylaxis (PrEP) in AGYW (15\u201024\u00a0years) and voluntary medical male circumcision in male partners (15\u201029). For young men, prevention cascade analyses identified gaps in motivation and access as barriers to voluntary medical male circumcision uptake, so an intervention was designed including financial incentives and an education session. For AGYW, gaps in motivation (particularly lack of risk perception) and access were identified as barriers to PrEP uptake: an interactive counselling game was developed addressing these barriers. A text messaging intervention was developed to improve PrEP adherence among AGYW, addressing reasons underlying lack of effective PrEP use through improving the capacity (\u201cskills\u201d) to take PrEP effectively. A community\u2010led intervention (community conversations) was developed addressing community\u2010level factors underlying gaps in motivation, access and effective use. These interventions are being evaluated currently using outcomes from the HIV prevention cascade (step 6).The prevention cascade can guide development and evaluation of interventions to strengthen implementation of HIV prevention methods by following the proposed process. The study is being implemented in eight sites in Manicaland representing different socio\u2010economic strata; VMMC and PrEP were selected being relatively new methods of HIV prevention with high efficacy and potential to contribute more to the overall impact of combination prevention in Zimbabwe.https://clinicaltrials.gov (NCT03565575 and NCT03565588).Data collected between 1998 and 2013 in a general\u2010population cohort study The study includes three individual\u2010based interventions, in eight study sites, and a community\u2010based intervention implemented in two of these sites. The study addresses individual\u2010 and community\u2010level barriers to HIV prevention use \u2013 recognizing that HIV prevention behaviour is influenced by multiple factors acting at different levels Increasing effective use of VMMC helps to reduce HIV incidence in AGYW by reducing exposure to HIV infection from their male partners. VMMC is central in Zimbabwe's HIV prevention programme, but only 10% of men (15\u201049) are circumcised in Manicaland Previous HIV prevention cascade analysis using Manicaland Cohort data found that low VMMC uptake in the study population was largely due to low risk perception for HIV infection, suggesting gaps in motivation and poor local availability An intervention was developed where HIV\u2010negative young men (15\u201029) participate in an education session on HIV risks and reducing these risks through VMMC, run by a circumcised male \u201crole\u2010model.\u201d They are randomized to receive a fixed financial reward or the opportunity to participate in a lottery upon VMMC uptake. Participants receive a contribution towards transport costs for accessing VMMC and are referred to participating study clinics offering VMMC. The education session aims to increase motivation by improving HIV risk perception, knowledge and perceptions about consequences of VMMC. The financial incentive increases motivation by creating more positive consequences for uptake. Previous behavioural economics research showed lottery tickets may be more effective in increasing motivation than fixed financial rewards as individuals overweight small probability events Organizing the follow\u2010up data in the prevention cascade framework aids evaluation by providing data on possible reasons why the intervention may have failed to improve uptake. Changes in the HIV prevention cascade for VMMC will be compared between a baseline and follow\u2010up survey six months later, for men in the intervention and control groups using HIV risk perception and VMMC uptake as primary outcomes.No prior measurements of the HIV prevention cascade for PrEP were available for AGYW in the study population. Since PrEP is a new method of HIV prevention in Zimbabwe and is not widely available Earlier analyses of Manicaland Cohort data on risk among AGYW indicated unprotected sexual relationships with older men contribute to their high HIV incidence An intervention was designed whereby HIV\u2010negative AGYW 18\u201024) play an interactive counselling game 8\u201024 playEvaluation will compare the HIV prevention cascades for PrEP for AGYW in the intervention and control groups after six months using HIV risk perception and uptake of PrEP will be compared in the two groups after six months.Some factors contributing to gaps in the cascade lie outside of the individual's control, including influences by partners, peers, families, healthcare providers and social structures A community conversations (CCs) intervention Survey and qualitative data will be analysed to evaluate whether the CCs intervention impacted the specific barriers to motivation, access and effective use of PrEP and VMMC by AGYW and young men in the study populations. Prevention cascades will be constructed and compared for VMMC and PrEP in the intervention and control groups between the two CCs sites and the remaining six sites to assess the effectiveness of CCs.3We have outlined a generic approach to using the HIV prevention cascade to identify, develop and evaluate interventions and demonstrated feasibility of application using the example of a study testing interventions to strengthen implementation of VMMC and PrEP services in Manicaland.When developing interventions, a central benefit of the HIV prevention cascade framework is that it underscores the multitude of factors to be addressed potentially limiting effective use of prevention methods. As with PrEP in Manicaland, the prevention cascade framework is useful for organizing evidence from other methods and settings to guide thinking about barriers to be addressed in implementing a new method. While the cascade highlights bottlenecks and areas that require interventions to improve progress through the cascade, it does not determine the most suitable interventions \u2013 these must be based on theoretical frameworks, local circumstances and evidence from similar settings.When evaluating interventions, outcomes and process indicators can be defined corresponding to steps and reasons underlying gaps in the HIV prevention cascade, providing a standardized basis to compare intervention and control groups and over time. Cascade analysis can be useful for interpretation of trial results and identifying reasons for the success or failure of interventions. The HIV prevention cascade does not measure the impact of interventions. This is possible \u2013 as planned in the Manicaland Study \u2013 by mathematical modelling to generate estimates of population\u2010level impact. The cascade is being used in evaluating the implementation of individual and combination HIV prevention methods to estimate the overall impact of the interventions on HIV incidence.In the Manicaland Study, the HIV prevention cascade is being measured and interpreted using data from population surveys and qualitative investigations. Other data sources \u2013 for example, routinely collected health data \u2013 could also be used should be considered. As the HIV treatment cascade has aided a range of policy, programmes and research at multiple levels, we believe this cascade can provide a framework to identify gaps in prevention efforts and targets for interventions. We encourage this approach to inform the intervention development and believe this framework can support global efforts to reduce HIV incidence.S.G. declares shareholding in pharmaceutical companies (GSK and Astra Zeneca). R.T. declares personal fees received for consultancy for the International Decision Support Initiative. The authors declare no further potential competing interests.All authors have been involved in the design of the Manicaland Study, led by TBH and SG, LM and RS wrote the article, with input from all authors. All authors have read and approved the final manuscript."} +{"text": "Health workforce planning improves when activities in the education system align with labour market dynamics.The health workforce \u2018pipeline\u2019It suggests ways to measure gaps and progress, and highlights the essential role of education and continuing professional development to support service provision.Entry planning, Entry, Exist, Exit) highlight the four stages in the life cycle of the health workforce \u2013 each of which requires careful planning and adequate investment.The four E's includes establishing functional working conditions along with realistic financial and non-financial incentives.Universal health coverageUniversal health coverage also depends on the quality of their performance \u2013 initially achieved through mastery of appropriate competencies and maintained after graduation through ongoing continuing professional development. It is essential that health workers are fit for practice \u2013 and remain so."} +{"text": "Seniors aged 65 and older are at great risk of psychological distress given their functional decline, which is known to limit participation and engagement in community life. The purpose of this study is to examine whether higher indices of social capital have a positive impact on the mental health of older, ethnic Californians. We conducted a secondary analysis of data for 7,485 Californians 65 and older from the 2016 California Health Interview Survey (CHIS). A principal components analysis generated two social capital measures; one measuring safety and social cohesion, the other civic engagement. Hierarchical linear regression analyses were conducted to assess the independent effects of social capital subscales on the severity of psychological distress as measured by the Kessler-6 (K6).Respondents were on average moderately distressed, with small yet significantly higher K6 scores observed among African Americans, Asians, and Native Americans. The addition of our social capital variables in subsequent steps resulted in little yet significant change in explaining psychological distress with only neighborhood safety and social cohesion being inversely associated with K6 . The interaction between ethnicity and neighborhood safety and social cohesion resulted in non-significant associations with K6 scores for all ethnic minority subgroups; however, for African Americans the relationship with psychological distress actually increased significantly . Our findings suggest that specific types of social capital may be helpful in remediating psychological distress for certain ethnic minority groups."} +{"text": "Hair shaft abnormalities including woolly hair are traditionally diagnosed by clinical examination and light microscopy which involves plucking of multiple hairs for examination. This is usually an inconvenient procedure especially in children. Trichoscopy may be a useful tool allowing close visualization of multiple hairs without causing discomfort to the patients. Trichoscopic findings of woolly hair have been described only in few reports. We report all of the trichoscopic findings of this rare disorder in one case which have only been reported separately in previous reports. Woolly hair (WH) is a rare congenital hair structure abnormality characterized by strongly coiled hair localized to a site or involving the whole scalp in non-black people . Hair grA 6-year-old Asian girl presented with short, sparse hair over the scalp noticed since birth . The haiTrichoscopy was performed using Firefly DE300 Polarizing Handheld USB Digital Dermoscope and photographs were captured by MacBook Pro 2013. Trichoscopy revealed \u201ccrawling snake\u201d appearance, with short wave cycles and tricWoolly hair is a rare congenital abnormality of hair which was first described by Gossage in 1907 in a European family characterized by an extremely curly hair with average curl diameter of 0.5 cm [The presence of woolly hair in non-blacks is extremely rare . InheritHutchinson et al. classifiLight microscopy of the hair shafts in woolly hair revealed ovoid cross sections, 180-degree longitudinal twisting, trichorrhexis nodosa, and pili annulati .Trichoscopy is a newer method which allows visualization of hair shafts in vivo in high magnification without the need of pulling hair for microscopic examination . CurrentWe are reporting this case because of its rarity and highlight the use of trichoscopy as a convenient diagnostic tool. Trichoscopy allows a simple, quick, and noninvasive examination of a single hair or multiple hairs in vivo which is not possible with a light microscope."} +{"text": "Prior longitudinal research suggests that younger subjective age predicts better subsequent cognitive functioning and lower dementia incidence independent of chronological age. However, no research has investigated interactions between subjective age and chronological age. This study examined whether older adults with more youthful subjective age performed better on cognitive evaluations than those with older subjective age and whether associations differed as a function of chronological age. Data from 1,047 older adults aged 65 and older from the Health and Retirement Study\u2019s 2016 Harmonized Cognitive Aging Project were analyzed. Separate linear regressions estimated associations between subjective age and factor scores corresponding to five cognitive domains: executive function, episodic memory, language, visuospatial functioning, and processing speed. Covariates included sociodemographic characteristics and chronic disease burden. Interaction terms tested whether chronological age modified associations between subjective age and cognition. In the whole sample, younger subjective age was associated with better language. Significant interactions for all five domains revealed that associations were stronger and statistically significant for participants at the oldest chronological ages. The predictive value of subjective age may be highest among the oldest adults. These cross-sectional findings suggest that the oldest adults are most vulnerable to the detrimental effects of feeling older than one\u2019s chronological age, cognitive difficulties are more relevant for subjective age perceptions for the oldest adults, and/or subjective age better reflects consequential physiological deterioration in that group. 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Aoife De Br\u00fan was initially incorrectly named in the author list as Jack De Br\u00fan. This has now been corrected. Apologies for any confusion caused."} +{"text": "In the article titled \u201cCardiac Arrest following a Myocardial Infarction in a Child Treated with Methylphenidate\u201d , the aut"} +{"text": "Authors would like\u00a0to correct the errors in table\u00a02. Corrected version of Table The original article has been corrected."} +{"text": "The phrase given as \u201clow-education\u201d should have been \u201clow-employment.\u201d This article has been corrected.1In the Original Investigation titled \u201cAssociation Between a State Law Allowing Pharmacists to Dispense Naloxone Without a Prescription and Naloxone Dispensing Rates,\u201d"} +{"text": "The corrected figure is shown below and is listed as In the article titled \u201cAntroquinonol Exerts Immunosuppressive Effect on CD8by H2O2\u201d , there w"} +{"text": "The reproducibility of using the \u201chomemade\u201d monofilament (a nylon fishing line) proposed by Parisi et al. in the manuscript titled \u201cDiabetic foot screening: Study of a 3000 times cheaper instrument\u201d"} +{"text": "In the article titled \u201cClinical Characteristics of Visual Dysfunction in Carbon Monoxide Poisoning Patients\u201d , there w"} +{"text": "In the main text, there is also a typographical error where the SUMO site on BZR1 should read \u201cK280\u201d and \u201cK310\u201d instead of \u201cK320.\u201d The authors apologize for the errors.In Figure\u00a04D of this article, the label of y axis should be"} +{"text": "In the article titled \u201cControl of a Humanoid NAO Robot by an Adaptive Bioinspired Cerebellar Module in 3D Motion Tasks\u201d , there w"} +{"text": "In the original publication the author\u2019s name \u2018Dimitri Lavillete\u2019 is published incorrectly. The correct author name should be spelt as \u2018Dimitri Lavillette\u2019 is provided in this correction."} +{"text": "In the article titled \u201cFcgRIII Deficiency and FcgRIIb Defeciency Promote Renal Injury in Diabetic Mice\u201d , there w"} +{"text": "In the article titled \u201cSodium Tanshinone IIA Sulfonate Attenuates Erectile Dysfunction in Rats with Hyperlipidemia\u201d , there w"} +{"text": "Due to a production error, an incorrect reference citation \u201cVanlierde et al. 2016)\u201d was inserted in Table 5. It should be \u201cVisentin et al. (\u201d was insThe publisher apologizes for this mistake. The original article has been updated."} +{"text": "Authors would like to correct the typo found in the fourth sentence of the introduction from \u201corganisional\u201d to \u201corganismal\u201d. The original article has been corrected."} +{"text": "Authors would like to correct the 4th author name from \u201cJu-Yeon Lee\u201d to the correct version \u201cJoo-Yeon Lee\u201d.The original article has been corrected."} +{"text": "AB is AB, from the development of background and preliminary tools to the collection of miscellaneous formulas and facts on the reverse order laws in one place with cogent introduction and references for further study. We begin with the introduction of a linear mixed model \u03b2 in the model, and the description of connections between the two types of least-squares estimators and the reverse order laws for generalized inverses of AB. We then prepare various necessary matrix study tools, including a general theory on linear or nonlinear algebraic matrix identities, a group of expansion formulas for calculating ranks of block matrices, two groups of explicit formulas for calculating the maximum and minimum ranks of A, B, and AB by means of the definitions of generalized inverses, the block matrix methodology (BMM), the matrix equation methodology (MEM), and the matrix rank methodology (MRM).Reverse order laws for generalized inverses of matrix products are a classic object of study in the theory of generalized inverses. One of the well-known reverse order laws for a matrix product Mathematics; Matrix product; Orthogonal projector; Idempotent matrix; Generalized inverse; Reverse order law; Matrix-valued function; Linear statistical model; Ordinary least-squares estimator; Block matrix methodology; Matrix equation methodology; Matrix rank methodology A matrix X is called a A, denoted by ith, A is denoted by A by definition, but people are mainly interested in the following eight situations that involve the equation A in the literature , the matrix equation methodology (MEM), and the matrix rank methodology (MRM), and has solved many fundamental problems in the theory of generalized inverses of matrices by means of the BMM, MEM and MRM. The corresponding work includes simplifying and deriving various complicated and nuanced matrix expressions and equalities, characterizing various ROLs for generalized inverses of matrix products, and establishing thousands of closed-form formulas for calculating ranks of block matrices, sums and differences of matrices, etc cf. . On the A, B, and AB by means of the definitions of generalized inverses, the BMM, the MEM, and the MRM. This paper is organized as follows. In Section The main purpose of this study is to gather in a single document various known and novel formulas and facts on a family of 512 one-sided set inclusions associated with 2In this section, we exhibit some basic definitions and known results from least-squares estimation problems in linear statistical models that are related to ROLs. It is well known that parametric regression analysis is perhaps the most commonly employed tool in statistical data analysis and inference, while linear regression models belong to classic issues in statistical theory and are the common roots of many branches of current statistical theory. Although there has been a relatively systematical research about linear regression models and their applications in the past centuries, one can still propose many theoretical problems in this field and investigate these problems by way of various mathematical analysis tools. When using linear regression models to fit given data, unknown parameters in the models may not necessarily be assumed to be fixed, instead, to vary at more than one level, or to be given in nested forms. Multi-level hierarchical linear model is a feasible technique of fitting data that have a hierarchical structure. Because of the occurrence of parameters at more than one level, the inference of a multi-level hierarchical linear model involves various nested calculations of given matrices and vectors in the model. In fact, many problems in statistics and applications involve analyzing and manipulating this kind of data and models with nested structure cf. . In this\u03b2 in \u03b2 and the mean vector AB\u03b2 in (I)y satisfies \u03b2 and AB\u03b2 under U is an arbitrary matrix. Furthermore, the expectations and the covariance matrices of The standard method is to\u03b2)\u2a7e0 cf. . Hence itent cf. . Solving(II)\u03b1 as followsOn the other hand, we may first solve the least-squares problem Because of the two alternative model forms y in Note from Lemma 2.1Let the OLSEs of \u03b2 and AB\u03b2 inandbe as given in(2.5),(2.6),(2.7),(2.9),(2.15), and(2.17), respectively. Then the following 6 assertions holdandAB, and the three matrix equalities in AB, respectively. The equivalent statements in A and B. Thus, it is imperative to establish necessary and sufficient conditions for the four matrix equalities in The above derivations and discussions show some of the main applications of generalized inverses in statistical analysis, as well as a practical motivation of approaching the ROL problem in 3In this section, we introduce a series of known formulas, results, and facts from the general theory of generalized inverses of matrices that we are going to use as background materials throughout this paper. In order to describe the relationships between the matrix sets in Lemma 3.1Let. Then the following results hold.(a)The following equalities hold(b)The general expressions of the seven commonly-used types of generalized inverses,,,,,, andof A can be written in the following 7 matrix-valued functionswhereare arbitrary. In particular,If, the general expressions of the last seven generalized inverses of A incan be written aswhereis arbitrary. If, the general expressions of the last seven generalized inverses of A incan be written aswhereis arbitrary.(c)The following set inclusions holdand following matrix set equalities hold(d)The following rank equalities holdand the corresponding generalized inverse satisfying(3.32)\u2013can be constructed by means of the singular value decompositions of A.(e)The following matrix equalities holdwhereis arbitrary. In particular,Besides, the following matrix equalities hold(f)The following set inclusionshold for any matrices P and Q.(g)andhold for all.Lemma 3.2Letand. ThenIf, thenIf, thenIfand, thenIn particular,In view of the facts in Lemma 3.3Let, andbe a matrix expression composed of,, \u2026,. Then the following results hold.(a)if and only if(b)if and only if(c)if and only if(d)if and only if(e)if and only if(f)if and only if(g)and.(h)andf is invariant and.The assertions in V and W are arbitrary.Let A and B, respectively, except the unique case V, U, A and B except A and B are distinctively divided into the following three groups:U and V are variable matrices. It is helpful to display all the matrix products together, so that we can approach the right-hand sides of these matrix-valued functions uniformly.It is natural to see from the ROL in 4One of the most significant developments of matrix theory in past several decades has been the establishment of many analytical formulas for calculating ranks of matrix expressions that involve variable matrices and generalized inverses of matrices. The salient values of these rank formulas have been demonstrated in solving various complicated algebraic and computational problems in matrix analysis and applications. In this section, we present a collection of known and new formulas, results, and facts concerning ranks of matrices and their generalized inverses, which are solid basis of the MRM and BMM, and are indispensable to make the paper self-contained when establishing and simplifying various matrix expressions and equalities associated with ROL problems.Lemma 4.1Letand, and integer.(a)If, then(b)If, thenLemma 4.2Let,,, and. ThenLemma 4.3Let,, and. ThenIn particular, ifand, thenFurthermore, the following results hold.(a).(b).(c).(d).(e),, and.(f)andfor.Lemma 4.4Letand, and denoteand. Then the following range equalityholds. Consequently, the following rank equalitieshold. In particular, the following results hold.(a).(b).(c).Lemma 4.5Let,, and. ThenLemma 4.6Letand. Thenholds for alland. In particular, the following inequalitieshold, and the following two groups of equivalent facts holdThe two formulas in the following lemma are best known in elementary linear algebra.Lemma 4.7Let. ThenLemma 4.8Letand assume that. ThenLemma 4.9Letbe a pair of orthogonal projectors. ThenLemma 4.10Letbe given, and letandbe a pair of idempotent matrices.Lemma 4.11Let,,, andbe given. ThenHenceLemma 4.12Letand, and denoteThen the following inequalities holdProofThe first inequality in Lemma 4.13Let, and denoteThenProofFollows from Lemma 4.14Letandbe a pair of linear subspaces of, and letandbe the orthogonal projectors ontoand, respectively. Then the following two dimension formulashold. In particular, the following statements are equivalent:(a).(b).(c)and/or.(d)and/or.(e).5X if and only if X if and only if Matrix equations occupy a central place in the development of matrix theory. Because generalized inverses of a matrix are defined to be solutions of some/all of the four Penrose equations, B=0 cf. . As matrLemma 5.1Letbe a given linear matrix equation, whereandare known matrices, andis an unknown matrix. ThenIn this situation, the general solution ofcan be written in the following parametric formwhereis arbitrary. In particular,holds for allif and only ifand, or equivalently,.Lemma 5.2Letbe a given linear matrix equation, where,, andare known matrices. Then the following statements are equivalent:(a)Eq.is solvable for.(b)and.(c)and.(d)and.(e).In this situation, the general solution ofcan be written in the following parametric formwhereare arbitrary. In particular,holds for allif and only ifThe constructions of the two block matrices in Lemma 5.3Letbe a given linear matrix equation, where,,,, andare known matrices. Then the following results hold.(a)Eq.holds for allandif and only if the 5 given matrices satisfy one of the following 4 block matrix equalities:(b)Under the assumptions thatand,holds for all matricesandif and only if one of the following 3 block matrix equalities holdsSeveral multilinear versions of matrix identities were also established in Lemma 5.4Letbe a given multilinear matrix equation, where,,,,,, andare known matrices. Then the following results hold.(a)Eq.holds for allandif and only if one of the following 4 block matrix equalities holds:(b)Under the assumptionand,holds for allandif and only if one of the following 3 block matrix equalities holds(c)Under the assumptionand,holds for allandif and only if one of the following 3 block matrix equalities holds(d)Under the assumptionand,holds for allandif and only if one of the following 3 block matrix equalities holds(e)Under the assumptionand,holds for allandif and only if one of the following 4 block matrix equalities holdsLemma 5.5Letbe a given multilinear matrix equation, where A,,, andare known matrices of appropriate sizes,. Then the following results hold.(a)Eq.holds for all,, andif and only if one of the following 8 block matrix equalities holds(b)Under the assumptionand,holds for all,, andif and only if one of the following 5 block matrix equalities holds(c)Under the assumptionand,holds for all,, andif and only if one of the following 5 block matrix equalities holds(d)Under the assumptionand,holds for all,, andif and only if one of the following 4 block matrix equalities holds(e)Under the assumptionand,holds for all matrices,, andif and only if one of the following 8 block matrix equalities holdsLemma 5.6Letbe a given multilinear matrix equation, where A,,, andare known matrices of appropriate sizes,. Thenholds for all matrices,,, andif and only if one of the following 8 block matrix equalities holdsUnder the assumptionand,holds for all matrices,,, andif and only if one of the following 5 block matrix equalities holdsLemma 5.7Letbe a given multilinear matrix equation, where A,,,,, andare known matrices of appropriate sizes,. Then the following results hold.(a)Eq.holds for all matrices,,, andif and only if one of the following 16 block matrix equalities holds(b)The matrix equationholds for all matrices,,, andif and only if one of the following 3 block matrix equalities holds(c)The matrix equationholds for all matrices,, andif and only if one of the following 8 block matrix equalities holds:(d)The matrix equationholds for all matrices,, andif and only if one of the following 8 block matrix equalities holds:(e)The bilinear matrix equationholds for all matrices,,, andif and only if one of the following 6 block matrix equalities holds(f)The matrix equationholds for all matrices,, andif and only if one of the following 4 block matrix equalities holds:(g)The matrix equationholds for all matrices,, andif and only if one of the following 4 block matrix equalities holds:Lemma 5.8Letbe a given multilinear matrix equation, where A,,, andare known matrices of appropriate sizes,. Then the following results hold.(a)Eq.holds for all matrices,,, andif and only if one of the following 16 block matrix equalities holds(b)Under the assumptionsand,holds for all matrices,,, andif and only if one of the following 6 block matrix equalities holds(c)Under the assumption,,, and,holds for all matrices,,, andif and only if one of the following 6 block matrix equalities holds(d)The multilinear matrix equationholds for all variable matrices,,, andif and only if one of the following 8 block matrix equalities holdsWith a brief summary, we can say that 6The hundreds of matrix-valued functions in A and B, it can readily be seen from the definition of the Moore\u2013Penrose inverse that the product For a matrix expression ucts cf. for expoTheorem 6.1Letandbe given. Then the following results hold.\u23291\u232ais invariant \u21d4 or.\u23292\u232ais invariant \u21d4 or.\u23293\u232ais invariant \u21d4 or.\u23294\u232ais invariant \u21d4 or.\u23295\u232ais invariant \u21d4 .\u23296\u232ais invariant \u21d4 ororand.\u23297\u232ais invariant \u21d4 orand.\u23298\u232ais invariant \u21d4 or.\u23299\u232ais invariant \u21d4 orandorandorand.\u232910\u232ais invariant \u21d4 andorandorandorand.\u232911\u232ais invariant \u21d4 orandor.\u232912\u232ais invariant \u21d4 andorand.\u232913\u232ais invariant \u21d4 orand.\u232914\u232ais invariant \u21d4 oror.\u232915\u232ais invariant \u21d4 or,, and.\u232916\u232ais invariant \u21d4 or.\u232917\u232ais invariant \u21d4 orandor.\u232918\u232ais invariant \u21d4 ororand.\u232919\u232ais invariant \u21d4 oror.\u232920\u232ais invariant \u21d4 orand.\u232921\u232ais invariant \u21d4 or.\u232922\u232ais invariant \u21d4 oror.\u232923\u232ais invariant \u21d4 or.\u232924\u232ais invariant \u21d4 oror.\u232925\u232ais invariant \u21d4 andorandorandorand.\u232926\u232ais invariant \u21d4 orand.\u232927\u232ais invariant \u21d4 ororand.\u232928\u232ais invariant \u21d4 orandorand.\u232929\u232ais invariant \u21d4 orand.\u232930\u232ais invariant \u21d4 oror,, and.\u232931\u232ais invariant \u21d4 or,, and.\u232932\u232ais invariant \u21d4 .\u232933\u232ais invariant \u21d4 orand.\u232934\u232ais invariant \u21d4 orand.\u232935\u232ais invariant \u21d4 or.\u232936\u232ais invariant \u21d4 and.\u232937\u232ais invariant \u21d4 .\u232938\u232ais invariant \u21d4 or.\u232939\u232ais invariant \u21d4 .\u232940\u232ais invariant \u21d4 .\u232941\u232ais invariant \u21d4 andorand.\u232942\u232ais invariant \u21d4 orandorand.\u232943\u232ais invariant \u21d4 orand.\u232944\u232ais invariant \u21d4 and.\u232945\u232ais invariant \u21d4 and.\u232946\u232ais invariant \u21d4 or,, and.\u232947\u232ais invariant \u21d4 ,, and.\u232948\u232ais invariant \u21d4 ororand.\u232949\u232ais invariant \u21d4 oror.\u232950\u232ais invariant \u21d4 oror,, and.\u232951\u232ais invariant \u21d4 oror.\u232952\u232ais invariant \u21d4 or,, and.\u232953\u232ais invariant \u21d4 or.\u232954\u232ais invariant \u21d4 oror.\u232955\u232ais invariant \u21d4 or.\u232956\u232ais invariant \u21d4 and.\u232957\u232ais invariant \u21d4 or,, and.\u232958\u232ais invariant \u21d4 or,, and.\u232959\u232ais invariant \u21d4 .\u232960\u232ais invariant \u21d4 ,, and.\u232961\u232ais invariant \u21d4 .\u232962\u232ais invariant \u21d4 or.\u232963\u232ais invariant \u21d4 .ProofWe only give the proofs of Results \u23291\u232a, \u232954\u232a, and \u232963\u232a as representatives. The product It follows from By The following results can be shown by a similar way.Theorem 6.2Letandbe given, and denote. Then the following results hold.\u23291\u232aEq.is invariant \u21d4 or.\u23292\u232aEq.is invariant \u21d4 or.\u23293\u232aEq.is invariant \u21d4 .\u23294\u232aEq.is invariant \u21d4 or.\u23295\u232aEq.is invariant \u21d4 .\u23296\u232aEq.is invariant \u21d4 or.\u23297\u232aEq.is invariant \u21d4 or.\u23298\u232aEq.is invariant \u21d4 or.\u23299\u232aEq.is invariant \u21d4 ororand.\u232910\u232aEq.is invariant \u21d4 orand.\u232911\u232aEq.is invariant \u21d4 .\u232912\u232aEq.is invariant \u21d4 orand.\u232913\u232aEq.is invariant \u21d4 or.\u232914\u232aEq.is invariant \u21d4 or.\u232915\u232aEq.is invariant \u21d4 or.\u232916\u232aEq.is invariant \u21d4 or.\u232917\u232aEq.is invariant \u21d4 .\u232918\u232aEq.is invariant \u21d4 or.\u232919\u232aEq.is invariant \u21d4 .\u232920\u232aEq.is invariant \u21d4 or.\u232921\u232aEq.is invariant \u21d4 or.\u232922\u232aEq.is invariant \u21d4 or.\u232923\u232aEq.is invariant \u21d4 or.\u232924\u232aEq.is invariant \u21d4 ororand.\u232925\u232aEq.is invariant \u21d4 or.\u232926\u232aEq.is invariant \u21d4 orand.\u232927\u232aEq.is invariant \u21d4 or.\u232928\u232aEq.is invariant \u21d4 orand.\u232929\u232aEq.is invariant \u21d4 or.\u232930\u232aEq.is invariant \u21d4 or.\u232931\u232aEq.is invariant \u21d4 .\u232932\u232aEq.is invariant \u21d4 .\u232933\u232aEq.is invariant \u21d4 or.Besides, invariance properties of product of generalized inverses can also considered in other algebraic systems, see 7The simplest matrix equality for generalized inverses in Lemma 7.1Letbe given. Then the following 24 results hold.\u23291\u232a.\u23292\u232aand.\u23293\u232aand.\u23294\u232aand.\u23295\u232aand.\u23296\u232a.\u23297\u232a,,, and.\u23298\u232aand.\u23299\u232aand.\u232910\u232a,, and.\u232911\u232aand.\u232912\u232aand.\u232913\u232aand.\u232914\u232aand.\u232915\u232aand.\u232916\u232a,, and.\u232917\u232aand.\u232918\u232a.\u232919\u232a.\u232920\u232aand.\u232921\u232a.\u232922\u232aand.\u232923\u232aand.\u232924\u232a.Lemma 7.2Letbe given. Then the following results hold.and.and..andorand.or {,, and.and.andorand.or {,, and.orand.andorand.or {,, and.and.or.or {,, and.orand.\u23296\u232a.orand.orand.orand.and.and.and.orand.and.and.and.orand.orand..and.and.and.and..orand.orand.or,, and.and.or {and.and..orand.orand.\u232916\u232a..and.and....A and B. For example, applying The results in Corollary 7.3Letbe two orthogonal projectors. Then the following 23 results hold.\u23291\u232a.\u23292\u232a.\u23293\u232a.\u23294\u232aand.\u23295\u232a.\u23296\u232aholds.\u23297\u232a.\u23298\u232aholds.\u23299\u232a.\u232910\u232aholds.\u232911\u232a.\u232912\u232a.\u232913\u232a.\u232914\u232a.\u232915\u232aand.\u232916\u232a.\u232917\u232aholds.\u232918\u232aholds.\u232919\u232a.\u232920\u232aholds.\u232921\u232aand.\u232922\u232aand.\u232923\u232a.Corollary 7.4Letbe two orthogonal projectors. Then the following results hold.....or...or.or.or...or.or.\u23296\u232a.\u23297\u232aor...or...or......or.or.or.or...or.or.\u232916\u232a......Corollary 7.5Letbe given. Then the following results hold.or.or..or.or..\u23293\u232aor.\u23294\u232aor.or.or..or.or.or.or.or.\u23298\u232aor.or...\u232910\u232aor.\u232911\u232a.or...\u232913\u232aor.\u232914\u232aor.\u232915\u232a.or.or.or.or.or..\u232918\u232aor.or.or..\u232920\u232aor.\u232921\u232aor.The preceding results show that much work is involved in the establishments of the simplest matrix equalities for two generalized inverses. Hence the approaches on general matrix equalities for generalized inverses will become quite complicated for operations of more matrices and generalized inverses.8In order to characterize the set inclusions in It is obvious that Lemma 8.1Letandbe given. Then the following 64 matrix set identitieshold according to Lemmafor the eight commonly-used types of generalized inverses of A and B.Lemma 8.2Letandbe given. Then the productcan be written aswhere the block matrices P, J, and Q satisfy,, and.AB. Thus it is imperative to determine the maximum and minimum ranks of Note that the rank of the product Lemma 8.3Letandbe given, and denoteAlso useandto denote the maximum and minimum ranks of the productwith respect to the choice ofand, respectively. ThenNotice that the rank formulas in Lemma 8.4Letandbe given, and denote. Then the following results hold.(a)The following two rank equalities always hold for all the generalized inverses(b)The following 4 maximum rank formulas hold(c)The following 2 minimum rank formulas holdthe following minimum rank formula holds(d)The following 2 formulas hold for all the generalized inverses(e)The following 2 maximum rank formulas holdthe following maximum rank formula holds(f)The following 3 minimum rank formulas hold(g)The following rank equality holdsfor all the eight types of commonly-usedand.(h)The following rank equality holds(i)The following 2 maximum rank formulas holdthe following maximum rank formula holds(j)The following 3 minimum rank formulas hold(k)The following rank equality holdsfor all the eight commonly-used types of generalized inversesand.(l)The following 3 minimum rank formulas holdthe following 8 minimum rank formulas holdthe following 4 minimum rank formulas hold(m)The following 4 rank equalities hold for all the generalized inverses(n)The following 8 maximum rank formulas holdthe following 4 maximum rank formulas hold(o)The following 12 minimum rank formulas hold(p)The following rank equality holdsfor all the eight commonly-used types of generalized inversesand.(q)The following 4 minimum rank formulas holdthe following 8 minimum rank formulas holdthe following 4 minimum rank formulas hold(r)The following 4 rank equalities hold for all the generalized inverses(s)The following 8 maximum rank formulas holdthe following 4 maximum rank formulas hold(t)The following 12 minimum rank formulas hold(u)The following maximum rank equality holdsfor all the eight commonly-used types of generalized inversesand.(v)The following 2 minimum rank formulas holdthe following minimum rank formula holdsTheorem 8.5Letand, and denote,, and. The following rank formulas holdProofIt is easy to verify that Finally, we present two groups of simple or well-known equalities and facts concerning ranges and ranks of matrices, which are the fundamental tricks of the MRM and will be used in the derivations and simplifications of calculations of ranks of algebraic operations of matrices and their generalized inverses in the sequel.Lemma 8.6Letand,,. Then the following resultshold, and the following rank equalities hold9After sufficient preparations of the background materials and tools in the preceding sections, we are now at the point to address the 512 set inclusion problems that are formulated in ,4)} cf. . DespiteA and B. The first group of results in this section describe various equivalent statements for the 64 ROLs in For the first matrix set Theorem 9.1Letand. Then the following 165 statements are equivalent:\u232977\u232aand/or.\u232978\u232aand/or.\u232979\u232aand/or.\u232980\u232aand/or.\u232981\u232a.\u232982\u232aand/or.\u232983\u232aand/or.\u232984\u232aand/or.\u232985\u232aand/or.\u232986\u232aand/or.\u232987\u232aand/or.\u232988\u232aand/or.\u232989\u232aand/or.\u232990\u232aand/or.\u232991\u232aOne/all of the four facts:,,,.\u232992\u232aand/or.\u232993\u232aand/or.\u232994\u232aand/or.\u232995\u232aand/or.\u232996\u232aand/or.\u232997\u232aand/or.\u232998\u232aand/or.\u232999\u232aand/or.\u2329100\u232aand/or.\u2329101\u232aand/or.\u2329102\u232aand/or.\u2329103\u232a.\u2329104\u232aand/or.\u2329105\u232aand/or.\u2329106\u232aand/or.\u2329107\u232aand/or.\u2329108\u232aand/or.\u2329109\u232aand/or.\u2329110\u232aand/or.\u2329111\u232aand/or.\u2329112\u232aand/or.\u2329113\u232aand/or.\u2329114\u232ais invariant with respect to the choice of, i.e.,holds for all.\u2329115\u232ais invariant with respect to the choice of, i.e.,holds for all.\u2329116\u232a.\u2329117\u232a.\u2329118\u232a.\u2329119\u232a.\u2329120\u232aand/or.\u2329121\u232aand/or.\u2329122\u232aand/or.\u2329123\u232aand/or.\u2329124\u232aand/or.\u2329125\u232aand/or.\u2329126\u232aand/or.\u2329127\u232aand/or.\u2329128\u232aand/or.\u2329129\u232aand.\u2329130\u232aand/or.\u2329131\u232aand/or.\u2329132\u232aand/or.\u2329133\u232aand/or.\u2329134\u232aand/or.\u2329135\u232aand/or.\u2329136\u232aand/or.\u2329137\u232aand/or.\u2329138\u232aand/or.\u2329139\u232aand/or.\u2329140\u232aand/or.\u2329141\u232aand/or.\u2329142\u232aand/or.\u2329143\u232aand/or.\u2329144\u232aand/or.\u2329145\u232aand/or.\u2329146\u232aOne/all of the four facts:,,,.\u2329147\u232aand/or.\u2329148\u232aand/or.\u2329149\u232aand/or.\u2329150\u232aand/or.\u2329151\u232aand/or.\u2329152\u232aand/or.\u2329153\u232aand/or.\u2329154\u232aand/or.\u2329155\u232aOne/all of the four facts:,,,.\u2329156\u232aOne/all of the four facts:,,,.\u2329157\u232aOne/all of the four facts:,,,.\u2329158\u232a.\u2329159\u232a.\u2329160\u232a.\u2329161\u232aand/or.\u2329162\u232aThe matrix equationsand/orare solvable.\u2329163\u232aThe matrix equationsand/orare solvable.\u2329164\u232aThe matrix equationsand/orare solvable.\u2329165\u232aThe matrix equationsand/orare solvable.ProofSetting all sides of Setting all sides of By Applying Applying A and B in Replacing A and B in Replacing Applying Applying Applying Applying Applying It can be deduced from B with The following rank equalitiesIt follows from The equivalences of \u2329120\u232a\u2013\u2329125\u232a and \u2329130\u232a\u2013\u2329137\u232a respectively follow from By Applying It follows first from \u2329147\u232a thatBy By The equivalence of \u2329156\u232a and \u2329157\u232a follows from the definition of direct sum of linear subspaces.By The equivalences of \u232991\u232a and \u2329159\u232a\u2013\u2329161\u232a follow from Applying The commutativity of the product of two orthogonal projectors is a quite special topic that attracts much attention in matrix theory, and has important applications in many disciplines of mathematics. A rich variety of results regarding the commutativity problem were scattered in the literature cf. .Theorem 9.2Letand, and denote. Then the following 29 statements are equivalent:ProofThe equivalences of \u23291\u232a\u2013\u232916\u232a and \u232926\u232a\u2013\u232929\u232a follow from AB.The equivalences of Results \u232916\u232a and \u232926\u232a\u2013\u232929\u232a in Theorem 9.3Letandbe given, and denote,, and. Then the following results hold.(a)The following 6 statements are equivalent:(b)The following 9 statements are equivalent:(c)The following 9 statements are equivalent:(d)The following 9 statements are equivalent:(e)The following 5 statements are equivalent:(f)The following 5 statements are equivalent:(g)The following 9 statements are equivalent:(h)The following 5 statements are equivalent:(i)The following 5 statements are equivalent:(j)The following 5 statements are equivalent:(k)The following 5 statements are equivalent:(l)The following 5 statements are equivalent:ProofApplying Theorem 9.4Letandbe given, and denoteand. Then the following results hold.(a)The following 26 statements are equivalent:(b)The following 6 statements are equivalent:(c)The following 6 statements are equivalent:(d)The following 6 statements are equivalent:(e)The following 6 statements are equivalent:(f)The following 6 statements are equivalent:(g)The following 6 statements are equivalent:(h)The following 6 statements are equivalent:(i)The following 6 statements are equivalent:(j)The following 6 statements are equivalent:(k)The following 6 statements are equivalent:(l)The following 6 statements are equivalent:(m)The following 6 statements are equivalent:(n)The following 6 statements are equivalent:(o)The following 6 statements are equivalent:(p)The following 6 statements are equivalent:ProofThe equivalence of \u23291\u232a and \u232925\u232a in (a) follows from the definition of Applying By By It follows from Applying Replacing By By By It follows from \u232917\u232a thatReplacing Since Replacing Applying U if and only ifThe equivalences of \u23291\u232a\u2013\u23295\u232a in (b) follow from U if and only ifThe equivalences of \u23291\u232a\u2013\u23295\u232a in (c) follow from U if and only ifThe equivalences of \u23291\u232a\u2013\u23295\u232a in (d) follow from U if and only ifThe equivalences of \u23291\u232a\u2013\u23295\u232a in (e) follow from U if and only if The equivalences of \u23291\u232a\u2013\u23295\u232a in (f) follow from The equivalences of \u23291\u232a\u2013\u23295\u232a in (g) follow from The equivalences of \u23291\u232a\u2013\u23295\u232a in (h) follow from V if and only ifThe equivalences of \u23291\u232a\u2013\u23295\u232a in (i) follow from U and V if and only ifThe equivalences of \u23291\u232a\u2013\u23295\u232a in (j) follow from U and V if and only if The two groups of equivalent facts in \u23291\u232a\u2013\u23295\u232a of (k) and (m) follow from U and V if and only if The two groups of equivalent facts in \u23291\u232a\u2013\u23295\u232a of (l) and (n) follow from V if and only ifThe equivalences of \u23291\u232a\u2013\u23295\u232a in (o) follow from V if and only if The equivalences of \u23291\u232a\u2013\u23295\u232a in (p) follow from The following theorem can be established by a similar approach, and the details are therefore omitted.Theorem 9.5Letandbe given, and denoteand. Then the following results hold.(a)The following 26 statements are equivalent:(b)The following 6 statements are equivalent:(c)The following 6 statements are equivalent:(d)The following 6 statements are equivalent:(e)The following 6 statements are equivalent:(f)The following 6 statements are equivalent:(g)The following 6 statements are equivalent:(h)The following 6 statements are equivalent:(i)The following 6 statements are equivalent:(j)The following 6 statements are equivalent:(k)The following 6 statements are equivalent:(l)The following 6 statements are equivalent:(m)The following 6 statements are equivalent:(n)The following 6 statements are equivalent:(o)The following 6 statements are equivalent:(p)The following 6 statements are equivalent:10As continuation of the preceding work, we obtain the following results regarding the three groups of set inclusions for Theorem 10.1Letandbe given, and denoteand. Then the following results hold.(a)The following 3 statements are equivalent:\u23291\u232a.(b)The following 3 statements are equivalent:\u23291\u232a.\u23293\u232a.(c)The following 3 statements are equivalent:\u23291\u232a.\u23293\u232a.(d)The following 3 statements are equivalent:\u23291\u232a.\u23293\u232a.(e)The following 3 statements are equivalent:\u23291\u232a.\u23293\u232aor.(f)The following 3 statements are equivalent:\u23291\u232a.\u23293\u232aoror.(g)The following 3 statements are equivalent:\u23291\u232a.\u23293\u232a.(h)The following 3 statements are equivalent:\u23291\u232a.\u23293\u232a.(i)The following 3 statements are equivalent:\u23291\u232a.\u23293\u232a.(j)The following 3 statements are equivalent:\u23291\u232a.\u23293\u232aor.(k)The following 3 statements are equivalent:\u23291\u232a.\u23293\u232a.(l)The following 3 statements are equivalent:\u23291\u232a.\u23293\u232a.(m)The following 3 statements are equivalent:\u23291\u232a.\u23293\u232a.(n)The following 3 statements are equivalent:\u23291\u232a.\u23293\u232a.(o)The following 3 statements are equivalent:\u23291\u232a.\u23293\u232aor.(p)The following 3 statements are equivalent:\u23291\u232a.\u23293\u232a.(q)The following 3 statements are equivalent:\u23291\u232a.\u23293\u232a.(r)The following 3 statements are equivalent:\u23291\u232a.\u23293\u232a.(s)The following 3 statements are equivalent:\u23291\u232a.\u23293\u232aor.(t)The following 3 statements are equivalent:\u23291\u232a.\u23293\u232aor.(u)The following 3 statements are equivalent:\u23291\u232a.\u23293\u232a.(v)The following 3 statements are equivalent:\u23291\u232a.\u23293\u232a.(w)The following 3 statements are equivalent:\u23291\u232a.\u23293\u232a.(x)The following 3 statements are equivalent:\u23291\u232a.\u23293\u232aoror.(y)The following 3 statements are equivalent:\u23291\u232a.\u23293\u232a.(z)The following 3 statements are equivalent:\u23291\u232a.\u23293\u232aoror.(a1)The following 3 statements are equivalent:\u23291\u232a.\u23293\u232aor.(b1)The following 3 statements are equivalent:\u23291\u232a.\u23293\u232aor.(c1)The following 3 statements are equivalent:\u23291\u232a.\u23293\u232aororand.(d1)The following 3 statements are equivalent:\u23291\u232a.\u23293\u232aorand.(e1)The following 3 statements are equivalent:\u23291\u232a.\u23293\u232aorand.(f1)The following 3 statements are equivalent:\u23291\u232a.\u23293\u232aand.Theorem 10.2Letandbe given, and denoteand. Then the following results hold.(a)The following 3 statements are equivalent:\u23291\u232a.(b)The following 3 statements are equivalent:\u23291\u232a.\u23293\u232a.(c)The following 3 statements are equivalent:\u23291\u232a.\u23293\u232a.(d)The following 3 statements are equivalent:\u23291\u232a.\u23293\u232a.(e)The following 3 statements are equivalent:\u23291\u232a.\u23293\u232aor.(f)The following 3 statements are equivalent:\u23291\u232a.\u23293\u232aoror.(g)The following 3 statements are equivalent:\u23291\u232a.\u23293\u232a.(h)The following 3 statements are equivalent:\u23291\u232a.\u23293\u232a.(i)The following 3 statements are equivalent:\u23291\u232a.\u23293\u232a.(j)The following 3 statements are equivalent:\u23291\u232a.\u23293\u232aor.(k)The following 3 statements are equivalent:\u23291\u232a.\u23293\u232a.(l)The following 3 statements are equivalent:\u23291\u232a.\u23293\u232a.(m)The following 3 statements are equivalent:\u23291\u232a.\u23293\u232a.(n)The following 3 statements are equivalent:\u23291\u232a.\u23293\u232a.(o)The following 3 statements are equivalent:\u23291\u232a.\u23293\u232aor.(p)The following 3 statements are equivalent:\u23291\u232a.\u23293\u232a.(q)The following 3 statements are equivalent:\u23291\u232a.\u23293\u232a.(r)The following 3 statements are equivalent:\u23291\u232a.\u23293\u232a.(s)The following 3 statements are equivalent:\u23291\u232a.\u23293\u232aor.(t)The following 3 statements are equivalent:\u23291\u232a.\u23293\u232aor.(u)The following 3 statements are equivalent:\u23291\u232a.\u23293\u232a.(v)The following 3 statements are equivalent:\u23291\u232a.\u23293\u232a.(w)The following 3 statements are equivalent:\u23291\u232a.\u23293\u232a.(x)The following 3 statements are equivalent:\u23291\u232a.\u23293\u232aoror.(y)The following 3 statements are equivalent:\u23291\u232a.\u23293\u232a.(z)The following 3 statements are equivalent:\u23291\u232a.\u23293\u232aoror.(a1)The following 3 statements are equivalent:\u23291\u232a.\u23293\u232aor.(b1)The following 3 statements are equivalent:\u23291\u232a.\u23293\u232aor.(c1)The following 3 statements are equivalent:\u23291\u232a.\u23293\u232aororand.(d1)The following 3 statements are equivalent:\u23291\u232a.\u23293\u232aorand.(e1)The following 3 statements are equivalent:\u23291\u232a.\u23293\u232aorand.(f1)The following 3 statements are equivalent:\u23291\u232a.\u23293\u232aand.Theorem 10.3Letandbe given, and denote. Then the following results hold.(a)The following 5 statements are equivalent:\u23291\u232a.\u23293\u232a.\u23295\u232a.(b)The following 3 statements are equivalent:\u23291\u232a.\u23293\u232aor.(c)The following 3 statements are equivalent:\u23291\u232a.\u23293\u232aor.(d)The following 3 statements are equivalent:\u23291\u232a.\u23293\u232a.(e)The following 3 statements are equivalent:\u23291\u232a.\u23293\u232a.(f)The following 3 statements are equivalent:\u23291\u232a.\u23293\u232aoror.(g)The following 5 statements are equivalent:\u23291\u232a.\u23293\u232a.\u23295\u232a.(h)The following 5 statements are equivalent:\u23291\u232a.\u23293\u232a.\u23295\u232a.(i)The following 5 statements are equivalent:\u23291\u232a.\u23293\u232a.\u23295\u232a.(j)The following 3 statements are equivalent:\u23291\u232a.\u23293\u232aor.(k)The following 3 statements are equivalent:\u23291\u232a.\u23293\u232aoror.(l)The following 5 statements are equivalent:\u23291\u232a.\u23293\u232a.\u23295\u232a.(m)The following 5 statements are equivalent:\u23291\u232a.\u23293\u232a.\u23295\u232a.(n)The following 3 statements are equivalent:\u23291\u232a.\u23293\u232a.(o)The following 3 statements are equivalent:\u23291\u232a.\u23293\u232aor.(p)The following 3 statements are equivalent:\u23291\u232a.\u23293\u232a.(q)The following 5 statements are equivalent:\u23291\u232a.\u23293\u232a.\u23295\u232a.(r)The following 5 statements are equivalent:\u23291\u232a.\u23293\u232a.\u23295\u232a.(s)The following 5 statements are equivalent:\u23291\u232a.\u23293\u232a.\u23295\u232a.(t)The following 5 statements are equivalent:\u23291\u232a.\u23293\u232a.\u23295\u232a.(u)The following 5 statements are equivalent:\u23291\u232a.\u23293\u232a.\u23295\u232a.Since the product Theorem 10.4Letandbe given. Then the following results hold.\u23291\u232aand.\u23292\u232aholds for all,, andor.\u23293\u232aholds for allor,, and.\u23294\u232aholds for allor.\u23295\u232aholds for all,, andor.\u23296\u232aholds for all.\u23297\u232aholds for allororand.\u23298\u232aholds for allandor.\u23299\u232aholds for all,, andor.\u232910\u232aholds for allandor,,, andorandorand.\u232911\u232aholds for allandorandorand,, andorand.\u232912\u232aholds for allandororand.\u232913\u232aholds for alland,,, andorand.\u232914\u232aholds for allandorand.\u232915\u232aholds for allandoror.\u232916\u232aholds for allandor,, and.\u232917\u232aholds for allor.\u232918\u232aholds for allandororand.\u232919\u232aholds for allandororand.\u232920\u232aholds for allandoror.\u232921\u232aholds for allandorand.\u232922\u232aholds for allandor.\u232923\u232aholds for allandoror.\u232924\u232aholds for allandor.\u232925\u232aholds for alloror,, and.\u232926\u232aholds for allandorand,, andorand,, andorand.\u232927\u232aholds for allandor,,, and.\u232928\u232aholds for allandororand.\u232929\u232aholds for allandorandor,,, and.\u232930\u232aholds for allandorand.\u232931\u232aholds for allandoror,, and.\u232932\u232aholds for allandor,, and.\u232933\u232aholds for all.\u232934\u232aholds for allandorand.\u232935\u232aholds for allandand.\u232936\u232aholds for allandor.\u232937\u232aholds for allandand.\u232938\u232aholds for alland.\u232939\u232aholds for allandor.\u232940\u232aholds for alland.\u232941\u232aholds for all,, and.\u232942\u232aholds for alland,,, andorand.\u232943\u232aholds for allandorandor,,, and.\u232944\u232aholds for allandorand.\u232945\u232aholds for alland,,, and.\u232946\u232aholds for allandand.\u232947\u232aholds for allandor,, and.\u232948\u232aholds for alland,, and.\u232949\u232aholds for allororand.\u232950\u232aholds for allandoror.\u232951\u232aholds for allandoror,, and.\u232952\u232aholds for allandoror.\u232953\u232aholds for allandor,, and.\u232954\u232aholds for allandor.\u232955\u232aholds for allandoror.\u232956\u232aholds for allandor.\u232957\u232aholds for alland.\u232958\u232aholds for allandor,, and.\u232959\u232aholds for allandor,, and.\u232960\u232aholds for alland.\u232961\u232aholds for alland,, and.\u232962\u232aholds for alland.\u232963\u232aholds for allandor.\u232964\u232aholds for alland.11This section is completely devoted to the following famous ROL:In this section, we present a series of known and new formulas, results, and facts associated with Theorem 11.1Letand. Then the following 40 statements are equivalent:\u23291\u232a.\u23292\u232a.\u23293\u232a.\u23294\u232a.\u23295\u232aand.\u23296\u232aand.\u23297\u232aand.\u23298\u232aand.\u23299\u232a.\u232910\u232aand.\u232911\u232a.\u232912\u232a.\u232913\u232a.\u232914\u232a.\u232915\u232aand.\u232916\u232aand.\u232917\u232a.\u232918\u232aand.\u232919\u232aand.\u232920\u232a.\u232921\u232a.\u232922\u232a.\u232923\u232a.\u232924\u232aand.\u232925\u232aand.\u232926\u232a.\u232927\u232aand.\u232928\u232aand.\u232929\u232a.\u232930\u232a.\u232931\u232a.\u232932\u232a.\u232933\u232a.\u232934\u232a.\u232935\u232a.\u232936\u232aand.\u232937\u232aand.\u232938\u232aandfor any integer.\u232939\u232aandfor any integer.\u232940\u232a.ProofB and A lead to \u23292\u232a. Pre- and post-multiplying Pre- and post-multiplying AB yields Eq. If \u23291\u232a holds, then it is easy to verify the following four equalitiesIf \u23291\u232a holds, then it is easy to verify the following four equalitiesThe equivalence of \u23291\u232a and \u23298\u232a follows from Result \u23291\u232a obviously implies \u23299\u232a. Conversely, pre- and post-multiplying A with If \u23291\u232a holds, then it is easy to verify from \u23291\u232a\u2013\u23298\u232a and \u232910\u232a the following four groups of equalitiesThe equivalences of \u23291\u232a and \u232920\u232a\u2013\u232937\u232a can be established by a similar way.If \u23291\u232a holds, then it is easy to verify the following four equalitiesFinally, applying A, B, AB, and their Moore\u2013Penrose inverses, including the three pairs of nested equalities in \u23295\u232a, \u23296\u232a, and \u23297\u232a. These equivalent assertions link many equalities and facts together, so that people can touch the ROL from different aspects. Moreover, for each of these matrix equalities and matrix set inclusions, we can separately derive conditions under which the equality or matrix set inclusion holds, so that we are able to obtain various equivalent facts for The family of results in To further characterize the role of the first equality in Lemma 11.2Letand. Then the following 57 statements are equivalent:\u23291\u232a.\u23292\u232a.\u23293\u232a.\u23294\u232a.\u23295\u232a.\u23296\u232a.\u23297\u232a.\u23298\u232a.\u23299\u232a.\u232910\u232a.\u232911\u232a.\u232912\u232a.\u232913\u232a.\u232914\u232a.\u232915\u232a.\u232916\u232ais an orthogonal projector.\u232917\u232ais an orthogonal projector.\u232918\u232ais an orthogonal projector.\u232919\u232ais an orthogonal projector.\u232920\u232ais an orthogonal projector.\u232921\u232aandcommute.\u232922\u232aandcommute.\u232923\u232aandcommute.\u232924\u232aandcommute.\u232925\u232ais EP.\u232926\u232a.\u232927\u232a.\u232928\u232a.\u232929\u232a.\u232930\u232a.\u232931\u232a.\u232932\u232a.\u232933\u232a.\u232934\u232a.\u232935\u232a.\u232936\u232a.\u232937\u232a.\u232938\u232a.\u232939\u232a.\u232940\u232a.\u232941\u232a.\u232942\u232a.\u232943\u232ais an orthogonal projector.\u232944\u232ais an orthogonal projector.\u232945\u232ais an orthogonal projector.\u232946\u232ais an orthogonal projector.\u232947\u232ais an orthogonal projector.\u232948\u232aandcommute.\u232949\u232aandcommute.\u232950\u232aandcommute.\u232951\u232aandcommute.\u232952\u232a.\u232953\u232a.\u232954\u232a.\u232955\u232a.\u232956\u232a.\u232957\u232a.ProofIt is obvious by definition thatAB. Then it follows from It is easy to verify that By It is easy to verify by definition that The equivalence of \u232916\u232a and \u232919\u232a follows The equivalence of \u232917\u232a and \u232920\u232a follows By the idempotency of By By A with B with A with Replacing The following results can be shown by a similar approach.Lemma 11.3Letand. Then the following 57 statements are equivalent:\u23291\u232a.\u23292\u232a.\u23293\u232a.\u23294\u232a.\u23295\u232a.\u23296\u232a.\u23297\u232a.\u23298\u232a.\u23299\u232a.\u232910\u232a.\u232911\u232a.\u232912\u232a.\u232913\u232a.\u232914\u232a.\u232915\u232a.\u232916\u232ais an orthogonal projector.\u232917\u232ais an orthogonal projector.\u232918\u232ais an orthogonal projector.\u232919\u232ais an orthogonal projector.\u232920\u232ais an orthogonal projector.\u232921\u232aandcommute.\u232922\u232aandcommute.\u232923\u232aandcommute.\u232924\u232aandcommute.\u232925\u232ais EP.\u232926\u232a.\u232927\u232a.\u232928\u232a.\u232929\u232a.\u232930\u232a.\u232931\u232a.\u232932\u232a.\u232933\u232a.\u232934\u232a.\u232935\u232a.\u232936\u232a.\u232937\u232a.\u232938\u232a.\u232939\u232a.\u232940\u232a.\u232941\u232a.\u232942\u232a.\u232943\u232ais an orthogonal projector.\u232944\u232ais orthogonal projector.\u232945\u232ais orthogonal projector.\u232946\u232ais an orthogonal projector.\u232947\u232ais orthogonal projector.\u232948\u232aandcommute.\u232949\u232aandcommute.\u232950\u232aandcommute.\u232951\u232aandcommute.\u232952\u232a.\u232953\u232a.\u232954\u232a.\u232955\u232a.\u232956\u232a.\u232957\u232a.Combining the above two lemmas, we obtain the following principle results in this section.Theorem 11.4Letand. Then the following 132 statements are equivalent:\u23291\u232a.\u23292\u232a.\u23293\u232aand.\u23294\u232a, and.\u23295\u232aand.\u23296\u232aand.\u23297\u232aand.\u23298\u232aand.\u23299\u232aand.\u232910\u232aand.\u232911\u232aand.\u232912\u232aand.\u232913\u232aand.\u232914\u232a.\u232915\u232a.\u232916\u232aand.\u232917\u232aand.\u232918\u232aand.\u232919\u232a.\u232920\u232a.\u232921\u232aand.\u232922\u232aand.\u232923\u232a.\u232924\u232a.\u232925\u232aand.\u232926\u232aand.\u232927\u232aand.\u232928\u232aand.\u232929\u232aand.\u232930\u232aand.\u232931\u232aand.\u232932\u232aand.\u232933\u232aand.\u232934\u232aand.\u232935\u232a.\u232936\u232a.\u232937\u232a.\u232938\u232a.\u232939\u232aand.\u232940\u232aand.\u232941\u232aand.\u232942\u232aand.\u232943\u232aand.\u232944\u232aand.\u232945\u232a.\u232946\u232a.\u232947\u232aand.\u232948\u232aand.\u232949\u232a.\u232950\u232a.\u232951\u232aand.\u232952\u232aand.\u232953\u232a.\u232954\u232a.\u232955\u232aand.\u232956\u232aand.\u232957\u232aand.\u232958\u232aand.\u232959\u232aand.\u232960\u232aand.\u232961\u232aand.\u232962\u232aand.\u232963\u232aand.\u232964\u232aand.\u232965\u232aand.\u232966\u232aand.\u232967\u232aand.\u232968\u232aand.\u232969\u232a.\u232970\u232a.\u232971\u232a.\u232972\u232a.\u232973\u232a.\u232974\u232a.\u232975\u232a.\u232976\u232a.\u232977\u232a.\u232978\u232a.\u232979\u232a.\u232980\u232a.\u232981\u232a.\u232982\u232a.\u232983\u232a.\u232984\u232a.\u232985\u232aand.\u232986\u232aand.\u232987\u232a.\u232988\u232a.\u232989\u232a.\u232990\u232a.\u232991\u232aand.\u232992\u232aand.\u232993\u232aand.\u232994\u232aand.\u232995\u232aand.\u232996\u232a.\u232997\u232a.\u232998\u232a.\u232999\u232a.\u2329100\u232aand.\u2329101\u232aand.\u2329102\u232aand.\u2329103\u232aandare orthogonal projectors.\u2329104\u232aandare orthogonal projectors.\u2329105\u232aandare orthogonal projectors.\u2329106\u232aandare orthogonal projectors.\u2329107\u232aandare orthogonal projectors.\u2329108\u232aandare orthogonal projectors.\u2329109\u232aandare orthogonal projectors.\u2329110\u232aandare orthogonal projectors.\u2329111\u232aandare orthogonal projectors.\u2329112\u232aandare orthogonal projectors.\u2329113\u232aandcommute, andandcommute.\u2329114\u232aandcommute, andandcommute.\u2329115\u232aandcommute, andandcommute.\u2329116\u232aandcommute, andandcommute.\u2329117\u232aandcommute, andandcommute.\u2329118\u232aandcommute, andandcommute.\u2329119\u232aandcommute, andandcommute.\u2329120\u232aandcommute, andandcommute.\u2329121\u232aandare EP.\u2329122\u232aand.\u2329123\u232aand.\u2329124\u232aand.\u2329125\u232aand.\u2329126\u232aand.\u2329127\u232aand.\u2329128\u232aand.\u2329129\u232aand.\u2329130\u232aand.\u2329131\u232aand.\u2329132\u232a.ProofAB. Then by We only give proofs of some results in the list to illustrate the usefulness of the MRM and BMM in the establishment of the theorem. It is easy to verify that AB, we find by Since T by Let The equivalences of the remaining results follow from the combination of 12A, B, and AB by means of the three conventional yet distinguished methods in matrix theory\u2014the matrix rank method, the block matrix method, and the matrix equation method, which brings together many conclusions on the matrix set inclusions in As a summary, we conclude that this expository article studies primarily the relationships between j) and Bs,\u2026,t2A(s1(I)AB can be written as various mixed ROLs, such as,X and Y composed of A, B, and their generalized inverses. Hence there are enormous concrete equalities for generalized inverses of AB that can be formulated. Several special cases of these mixed-type ROLs for Moore\u2013Penrose inverses were proposed and approached X satisfying the following four equations (1) X is called a weighted A, denoted by ith, A is denoted by A according to the above definition. In this situation, it would be of interest to consider the extensions of the preceding results to various ROLs for weighted generalized inverses of a matrix product.As a direct extension of the Moore\u2013Penrose inverse, the weighted Moore\u2013Penrose inverse of a matrix (III)x is called an inner inverse of a. For an operator x, then it is unique, and is called the Moore\u2013Penrose inverse of a and denoted by a is regular \u21d4 Similar to generalized inverses of matrices, algebraists have defined Moore\u2013Penrose inverses of elements in other general algebraic frameworks, such as, semigroups, rings, operator algebras, Hilbert ules cf. . Withoutx=a\u2020 cf. . It is wists cf. . Thus itFurthermore, the present author remarks that although investigations on ROLs have evolved enough up to now, the formulas, results, and facts collected and proved, as well as the methods and techniques used and developed in this paper will have certain new impacts on the development of matrix equality theory of generalized inverses. The author hopes that the whole work in this paper can be utilized as a general guide to prompt many more consecutive approaches to matrix equalities composed of various complicated matrix operations and generalized inverses. Here we mention some groups of problems in this area for ongoing consideration.This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.The author declares no conflict of interest.No additional information is available for this paper."} +{"text": "While typesetting the entries of the Table\u00a01 were incorrectly aligned. The correct Table\u00a0The original article has been corrected."} +{"text": "Jeremy Snyder\u2019s email address is:"} +{"text": "The authors wish to make the following corrections to this paper :\u22122 day\u22121\u201d should be \u201capproximately 10.75 \u00b1 0.16 \u03bcg cm\u22122 day\u22121\u201d.The authors have found one inadvertent error in our abstract . In thisThe authors would like to apologize for any inconvenience caused to the readers by this change."} +{"text": "In the article titled \u201cThose Who Hear Music: Three Cases on Musical Hallucinations\u201d , there i"} +{"text": "The unit of JA content inshould be \u201cng\u00b7g\u22121\u201d instead of \u201cug\u00b7g\u22121\u201d. The corrected In the original article, there was a mistake in The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated."} +{"text": "The original version of this article unfortunately contained a mistake. Author's surname is written incorrectly \u201cvon Rueden\u201d in the original article. \u201cvon R\u00fcden\u201d is the correct spelling .The original article has been corrected."} +{"text": "In the original publication of the article, the header row of Table 3 was incorrectly published and \u201ctannin\u201d and \u201cQuercetin (Stem)\u201d were placed under \u201cCondensed\u201d. The right most headers should read \u201cCondensed tannin\u201d and \u201cQuercetin (Stem)\u201d, respectively.The original article has been updated."} +{"text": "BioMed Research International has retracted the article titled \u201cPotassium Channel Ether \u00e0 go-go1 Is Aberrantly Expressed in Human Liposarcoma and Promotes Tumorigenesis\u201d [It was raised to our attention that theThe authors could not be contacted."} +{"text": "In the original publication, Table 1 was processed incorrectly during the typesetting and publication process. The final column of Washing clothes should be \u201c\u221210\u201d and not \u201c\u22121\u201d in Table The original article has been corrected."} +{"text": "An author\u2019s name was incorrectly spelled as \u201cDaniel Verg\u00e9\u201d in the FEP-EMDR Research Group section. The correct spelling is \u201cDaniel Berg\u00e9.\u201d The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way.The original article has been updated."} +{"text": "The text \u201cwith a waiver of informed consent\u201d was added by mistake. The study did not require approval or consent because it was a quality improvement project. This article has been corrected.1In the Original Investigation titled \u201cAssociation Between Positive Results on the Primary Care\u2013Posttraumatic Stress Disorder Screen and Suicide Mortality Among US Veterans,\u201d"} +{"text": "In the article titled \u201cMaximum Phonation Time in People with Obesity Not Submitted or Submitted to Bariatric Surgery\u201d , there w"} +{"text": "A-vaue\u201d which should have read \u201cA-value\u201d. The corrected table is shown below and is listed as In the article titled \u201cThe Effect of Cochlear Size on Cochlear Implantation Outcomes\u201d , there w"} +{"text": "Due to a production error, an author name was incorrectly written as \u201cY. Maurice II Morillon.\u201d The correct name is \u201cY. Maurice Morillon II.\u201d The publisher apologizes for this mistake. The original article has been updated."} +{"text": "It should read \u201cBrewer thioglycolate medium \u201d.The authors regret this error."} +{"text": "In the article titled \u201cEfficient BFCN for Automatic Retinal Vessel Segmentation\u201d , the ord"} +{"text": "We show that the existential fragment of B\u00fcchi arithmetic is strictly less expressive than full B\u00fcchi arithmetic of any base, and moreover establish that its"} +{"text": "To improve clarity \u201cIt was not until 1917\u20131937, years after Edison\u2019s patent for the light bulb\u2026\u201d has been changed to \u201cIt was not until 1917, 37 years after Edison\u2019s patent for the light bulb\u2026\u201d. The HTML and PDF versions of the Article have been corrected."} +{"text": "Alternanthera sessilis\u201d [In the article titled \u201cIn Vitro Wound Healing Potential of Stem Extract of essilis\u201d , an area"} +{"text": "In the original publication of the article \u201cImplications of time\u2010series gene expression profiles of replicative senescence\u201d by Y.\u2010M. Kim et al., the name of the first author, You\u2010Mie Kim, was misspelled as \u201cYou\u2010Mi Kim.\u201dThe author would like to apologize for the inconvenience caused."} +{"text": "In Figure 3, the label under 1 and 5 should have appeared as \u201cFavorable to conventional system\u201d and \u201cFavorable to navigation system,\u201d respectively. This article has been corrected.In the Original Investigation titled \u201cAssociation of Image-Guided Navigation With Complete Resection Rate in Patients With Locally Advanced Primary and Recurrent Rectal Cancer: A Nonrandomized Controlled Trial,\u201d"} +{"text": "Following the publication of the original article , the aut#21 in ZBI-22 should be corrected from \u2018should do more\u2019 to \u2018could do a better job\u2019.#12 in ZBI-12 should be corrected from \u2018should do more\u2019 to \u2018could do a better job\u2019.The corrected Table"} +{"text": "After publication of our article we were The correct Tables\u00a0In Table\u00a0Additional file 1. Control population calculations and raw registration numbers."} +{"text": "This abstract contained reference to the drug \u2018azithromycin\u2019 where \u2018aztreonam\u2019 should have been stated.The authors apologise for any inconvenience."} +{"text": "In the article titled \u201cA Case of Severe Acute Kidney Injury Exacerbated by Canagliflozin in a Patient with Type 2 Diabetes\u201d , there w"} +{"text": "In the article titled \u201cDietary Lipids in Health and Disease\u201d , there w"} +{"text": "Epidemiology & Infection with the indication that A. Pich\u00f3n Riviere's surname should be indexed as \u2018Riviere, AP\u2019 instead of \u2018Pich\u00f3n Riviere, A.\u2019 and was originally published as such.The above paper was originally submitted to This has been rectified in the online PDF and HTML copies and re-indexed accordingly."} +{"text": "Oxidative Medicine and Cellular Longevity has retracted the article titled \u201cThe Healing of Oxidative Injuries with Trehalose in UVB-Irradiated Rabbit Corneas\u201d [Corneas\u201d due to cCorneas\u201d is dupliCorneas\u201d , despiteCorneas\u201d is also Corneas\u201d despite"} +{"text": "In the row for \u201cBlack, non-Hispanic,\u201d the numbers under \u201cPast 30 days binge drinking*\u201d should have read"} +{"text": "Hoyle AP Two randomised trials has established the use of Abiraterone (AA) as an alternative standard of care to Docetaxel treatment in men with metastatic Hormone Na\u00efve Prostate Cancer (mHNPC) with \u201chigh risk\u201d or high volume disease , 2. UnceThis trail uses the STAMPEDE platform design, There were a 34% survival benefit in the AA group also in the \u201clow risk\u201d mHNPC although the number needed to treat to prevent one death was 4 times higher in \u201clow risk\u201d group compared with the \u201chigh risk\u201d.This trial results support the use of abiraterone in mHNPC irrespective of \u201crisk\u201d or \u201cvolume."} +{"text": "Correction to \u201cCombination of chemically modified SDF\u20101\u03b1 mRNA and small skin improves wound healing in diabetic rats with full\u2010thickness skin defects\u201d.In Luo et al.,These are errors in Figure\u00a0The corrected Figures"} +{"text": "These should have appeared as \u201c\u2026e-cigarette use in patients with CVD decreased from 2014 to 2019\u2026\u201d and \u201c\u2026the use of e-cigarettes rebounded in 2020\u2026\u201d This article has been corrected.1In the Research Letter titled \u201cTrends in Electronic Cigarette Use Among US Adults With a History of Cardiovascular Disease,\u201d"} +{"text": "Author would like to correct below error to their publication.In Materials and methods section, first paragraph line 9 should read as \u201c\u2026\u2026..concentration 153\u2013199\u00a0mg/dl [17].\u201dThe original article has been corrected."} +{"text": "Author contributions and Acknowledgments statements appear below.Due to a production error, \u201cEliana Al Haddad\u201d and \u201cKyong Bok Choi\u201d were not included as authors in the published article. The corrected"} +{"text": "Please note the following corrections to this article:Two citations are now provided:Functional Use of Objects: \u201c\u2026pretend/imaginative (citation withheld for blind review).\u201d should say \u201c\u2026pretend/imaginative .\u201dUnder the heading Discussion: \u201c\u2026children on the autism spectrum (citation withheld for blind review).\u201d should say \u201c\u2026children on the autism spectrum .\u201dUnder the heading Typographical errors that are present but that do not alter interpretation of the findings are acknowledged."} +{"text": "The author of the article by Noreen F. Rossi\u00a0 noted anThe correct figure is as follows:"} +{"text": "Due to a production error, an author name was incorrectly presented as \u201cJiang Li.\u201d The correct name is \u201cLi Jiang.\u201dThe publisher apologizes for this mistake. The original article has been updated."} +{"text": "Incorrect Author NameDue to a production error, an author\u2019s name was incorrectly spelled as \u201cMelinique Wall\u201d. The correct spelling is \u201cMelinique Walls\u201d. The publisher apologizes for this mistake.The original version of this article has been updated."} +{"text": "In the article titled \u201cHighly Integrated Multiplexing and Buffering Electronics for Large Aperture Ultrasonic Arrays\u201d , there w"} +{"text": "The original version of this article unfortunately contained an error, and it has been corrected with this erratum.The co-author name of \u201cRobert Garofalo\u201d is misspelled as \u201c\u201cRobert Garafalo\u201d and there appears to be a formatting irregularity on page 4 under \u201ccase 2\u201d these corrections are updated.The original article has been corrected."} +{"text": "Abstract. This sentence previously stated:A correction has been made to Taricha granulosa were exposed to a 1 x 107 per 10 mL dose\u201d\u201cThe corrected sentence appears below:Taricha granulosa were exposed to a 1 x 107 per 10 mL dose\u201d\u201cThe authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated."} +{"text": "We at Biomarkers Journal had opportunity to report \u201ca clue of persoanlized immunotherapy\u201d . In the"} +{"text": "In Xiwen Li et\u2009al.,"} +{"text": "In the originally published version of this manuscript, there was an error in the first column of Table 4 whereby asterisks were placed in the wrong position: i.e., they were erroneously added next to the \u2018M\u0101ori\u2019 \u2018Pacific\u2019 and \u2018Asian\u2019 ethnicity and NZDep quintile \u20185\u2019 categories for TST and erroneously omitted from the \u2018M\u0101ori\u2019 and \u2018Pacific\u2019 ethnicity and \u20182 - <3hr\u2019 visual media categories for Night Wakings. These errors have been corrected."} +{"text": "We regret that the fourth author\u2019sname was misspelled in the final publication (both the main articleand the Supporting Information file). It should read \u201cPakornVaranusupakul\u201d rather than \u201cPakorn Varanasupakul\u201d."} +{"text": "In the published article, there was an error in the Funding statement. The grant number was stated as \u201cB202314017099\u201d but should be \u201cB202314010044\u201d. The correct Funding statement appears below."} +{"text": "An omission to the funding section of the original article was made in error. The following sentence has been added: \u201cOpen access funding was provided by the \u00c9cole Polytechnique F\u00e9d\u00e9rale de Lausanne.\u201dThe publisher apologizes for this mistake. The original article has been updated."} +{"text": "MMWR Editors were informed by authors of \u201cTiming of Introduction of Complementary Foods \u2014 United States, 2016\u20132018\u201d (On May 25,"} +{"text": "Jo\u017eef \u0160imenko was not included as an author in the original publication [lication . And thi"} +{"text": "Due to a production error, the word \u201cmicroglial\u201d was misspelled as \u201cmircroglial\u201d in the article title.The publisher apologizes for this mistake. The original article has been updated."} +{"text": "Crocodylus porosus)\u201d (pages 911\u2013935), the authors would like to update the Acknowledgments section as follows:In Evolutionary Applications 16:4, for the article by Lloyd\u2010Jones et al.\u00a0 entitled"} +{"text": "Due to a production error, two author names were incorrectly spelled as \u201cEugenia Stavropoulou\u201d and \u201cElisavet Bezirtzoglou\u201d. The correct spelling is \u201cEugenia Bezirtzoglou\u201d and \u201cElisavet Stavropoulou\u201d. The publisher apologizes for this mistake.The original version of this article has been updated."} +{"text": "In the article \u201cBME 2.0: Engineering the Future of Medicine\u201d , the com"} +{"text": "We thank the author for his interest in our case report \u201cMyofibrillar myopathy \u2013 a rare but important differential diagnosis of camptocormia in a patient with Parkinson\u2019s Disease\u201d . First o"} +{"text": "Due to a production error, the authors \u201cChangyan Ju\u201d and \u201cYanping Ma\u201d sharing first authorship was not indicated clearly with the \u201cdagger mark\u201d.The correct statement to follow is \u201cThese authors share first authorship\u201d.The publisher apologizes for this mistake. The original article has been updated."} +{"text": "After our paper was published online, we discovered a mistake ofstructure of \u201ctanzawaic acid D\u201d in"} +{"text": "This should read: \u201cYuchen Liu\u201d instead of \u201cYuchen Lin\u201d.This error has been corrected within the article."} +{"text": "This is a peer review submitted for the paper \u201cEvaluating Population Density as a Parameter for Optimizing COVID-19 Testing: Statistical Analysis.\u201dIn this paper , the autGenerally, the manuscript is properly structured and well understood.Change the subtitle \u201cPolicy Proposal\u201d to \u201cIntroduction\u201d or \u201cBackground.\u201d"} +{"text": "In the recent article by Lamichhane et al.\u00a0, the aut"} +{"text": "The authors would like to draw the reader\u2019s attention to a YouTube video2"} +{"text": "Evolutionary Applications 16:1, for the article by Konopi\u0144ski et al.\u00a0(In i et al.\u00a0 entitledWe apologize for the error."} +{"text": "Due to an error, the caption of Figure 1 contained two typos. It was originally published as:\u201cFor experimental strategies use to evaluate paced mating in female rats.\u201d\u201cFor\u201d has been corrected to \u201cFour\u201d and \u201cuse\u201d to \u201cused\u201d. The correct sentence appears below.\u201cFour experimental strategies used to evaluate paced mating in female rats.\u201dThe publisher apologizes for this mistake.The original article has been updated."} +{"text": "Sir, the recent publication on \u201cPublication Ethics\u201d is very interesting . The art"} +{"text": "We would like to thank Oguz Kutlu A for her close interest in our paper entitled \u201cAnogenital Distance in Turkish Newborns\u201d. In our study , we used"} +{"text": "We need to update the first and third addresses as shown above. Furthermore, \u201czero\u201d has been modified and replaced by \u201c\u2014\u201d in"} +{"text": "After publication of the article by G\u00fcc\u00fc et al. , it was"} +{"text": "In Figure six Figure\u00a0 here of"} +{"text": "In the article \u201cTable 4. Top 12 first authors\u201d was published with errors; the corrected Table is as follows:"} +{"text": "Sir,et al, with a great interest.[et al, concluded that \u201cCd exposure causes renal dysfunction, but oral administration of onion could prevent it.\u201d[et al.[I read the recent report by Ige interest. Ige et avent it.\u201d This anivent it.\u201d There is.\u201d[et al. Finally,"} +{"text": "There is a typo in the third sentence of the \u201cPatterns of viral migration\u201d subsection of the Results section. The sentence currently reads:\u201cThe ORF VI data supported spread from Greece to Turkey (BF \u200a=\u200a 230) and to Iran (BF \u200a=\u200a 128), and from Japan to USA (BF \u200a=\u200a 112).\u201dThe correct sentence should read:\u201cThere was also some support for spread from Turkey to Japan (BF \u200a=\u200a 14). The ORF VI data supported spread from Greece to Turkey (BF\u200a=\u200a 230) and Turkey to Iran (BF\u200a=\u200a 128), and from Japan to USA (BF \u200a=\u200a 112).\u201d"} +{"text": "Figures The correct order of the singing task labels is: \u201cHum\u201d (5\u201310 min segment), \u201cHymn\u201d (11\u201316 min segment) and finally \u201cMantra\u201d (17\u201322 min segment).Figures with correct labeling appear in this Erratum."} +{"text": "The word \u201cmitigated\u201d in the sentence \u201cWe showed that under aerobic conditions, compatible solutes accumulated by thermophilic bacteria confer IR resistance to enzymes"} +{"text": "The recent report by BPA is found in receipt paper and appeThere is a sense of d\u00e9j\u00e0 vu about this story: In the 1970s polychlorinated biphenyls (PCBs) were widely used in carbonless copy paper . PCBs weThe research of"} +{"text": "We welcome Sch\u00f6llnberger and Kaiser\u2019s comments on our review . The bioSch\u00f6llnberger et al. used multi-model inference to assesWe also question the validity of the threshold models In summary,"} +{"text": "In the article \u201cAssociations between Nighttime Traffic Noise and Sleep: The Finnish Public Sector Study,\u201d by Halonen et al.\u201d [Environ Health Perspect 120:1391\u20131396 (2012)], the last sentence of the \u201cConclusions\u201d was incorrect: The nighttime traffic noise level associated with insomnia symptoms in the total study population should have been \u201c> 55 dB\u201d instead of \u201c> 50 dB.\u201dEHP regrets the error."} +{"text": "Dear EditorI read with interest the recently published review article by Caremel et al. titled \u201c"} +{"text": "The Research Grant number in Acknowledgments section in page 9 stating that \u201cThis work was supported in part by Research Grant 20590847\u201d was wrong and the corrected one is \u201c23591070.\u201d"} +{"text": "The first name of author C\u00e9line Formosa is incorrect.The correct author name is C\u00e9cile Formosa."} +{"text": "Survey responses \u201cscammed\u201d (1) the following sentence: The notion of \u201cscamming\u201d took center-stage in the blogosphere's response to LOG12, although not all comments went so far as to suggest \u201c\u2026 there are no \u2018Human Subjects4.\u2019 \u201d should read \u201cThe notion of \u2018scamming\u2019 took center-stage in the blogosphere's response to LOG12.\u201d and footnote 4 should have been removed.A correction implemented in the PDF file of the article was missed out in the HTML. Hence the PDF and full text HTML versions do not correspond. Under the heading"} +{"text": "All instances of \u03a7\u039b\u0394\u039d19 should be read as CLDN19 throughout the \"Materials and Methods\" and \"Results\" sections."} +{"text": "This abstract has also been published as O42"} +{"text": "The value for \u201cTraffic load on major streets within 100\u2011m buffer\u201d for GINI/LISA South in Table 1 was incorrect in the manuscript originally published online. It has been corrected here."} +{"text": "There was an error in the first author's name. The correct spelling is Em\u0151ke-\u00c1gnes Horv\u00e1t."} +{"text": "The legends for The title of Figure 9 contains an error. \u201c11 trajectories\u201d should read \u201c19 trajectories.\u201d? Please see Figure 9 with the correct title and legend below."} +{"text": "There is an error in the presentation of author Vanesa Richarte Fern\u00e1ndez' name in the citation. The correct abbreviation is: \"Richarte Fern\u00e1ndez V\"."} +{"text": "Given that we have developed for emotional response -4 and a"} +{"text": "Dear Editor,We read \u201cEffects of raloxifene on bone metabolism in hemodialysis patients with type 2 diabetes\u201d by Saito et al. with a g"} +{"text": "The author\u2019s affiliation was wrong in . The \u201cNa"} +{"text": "There are errors in equations and other expressions in the Methods section. In each instance in which a lowercase Greek script theta (\u03d1) appears, it ought to be a lowercase Greek phi (\u03d5). Please view the complete, correct equations here:In the section titled \"Binary traits\":In the section titled \u201cCase/Control Studies\u201d:In the section titled \u201cLiability R2\u201d:"} +{"text": "In the paper \u201cQuality of Life and Sexual Health in the Aging of PCa Survivors\u201d there are 2 mistakes at page 9, in the paragraph \u201c5.4. Androgen Deprivation Therapy (ADT)\u201d: \u201c[162]\u201d has to be replaced by the words \u201cwith LHRH agonists,\u201d while \u201c[162]\u201d has to be shifted at the end of the second and third sentences ."} +{"text": "The article titled \u201cPrenatal Diagnosis of Concurrent Achondroplasia and Klinefelter Syndrome\u201d has been"} +{"text": "There are a number of errors in the \u201caOR (95%CI)\u201d columns for Tables"} +{"text": "Tritonia brain\u201d, some labels for species names are incorrectly omitted. Please see the In"} +{"text": "The phrases \u201clog hazard ratio\u201d and \u201clog HR\u201d appear incorrectly throughout the article and Supporting Information files, with the exception of the \u201cData analysis\u201d subsection, Fig 5, and Fig 6. The correct terminology in all other cases should be \u201chazard ratio\u201d and \u201cHR.\u201d"} +{"text": "Upon publication, it was noticed that in the original version of the article (L\u00f3pez S\u00e1nchez et al."} +{"text": "There are errors in the column labeled \u201cUnweighted number of respondents\u201d in both Tables"} +{"text": "The authors wish to add the following amendment and correction on their paper published in IJERPH : Page 6139, Line 3 in Paragraph 3: \u201c\u2026Israel, ,\u2026\u201d should read \u201c\u2026Israel and Palestine \u2026\u201d.The authors would like to apologize for any inconvenience caused to the readers by these changes."} +{"text": "The paper titled \u201cAdrenal Schwannomas: Rare Tumor of the Retroperitoneum\u201d has been"} +{"text": "After the \u201cCompeting interests\u201d section in the original version of this article , an \u201cAck"} +{"text": "Upon publication of the original article , the aut\u201cFigure 7\u201d should have read \u201cFigure 3\u201d (Overview of co-design process)\u201cFigure 3\u201d should have read \u201cFigure 4\u201d \u201cFigure 4\u201d should have read \u201cFigure 5\u201d (Agitation/restlessness heuristic)\u201cFigure 5\u201d should have read \u201cFigure 6\u201d (Ending life sustaining treatment heuristic)\u201cFigure 6\u201d should have read \u201cFigure 7\u201d (Providing routine care at the end of life heuristic)These figure numbers have now been updated in the original paper.We, the publishers, apologise for any inconvenience caused by these errors and would like to thank Dr Nathan Davies from University College London for bringing this to our attention."} +{"text": "The author name Martin Ridderstr\u00e5le was incorrectly spelt as Martin Riddestr\u00e5le during the production of the original article . This ha"} +{"text": "There is an error in the Abstract. The sentence that reads \u201cTransfection studies show that Xbp1Mist1 promoter.\u201d should instead read as follows: \u201cTransfection studies show that Xbp1 activates the Mist1 promoter.\u201d"} +{"text": "There are errors in Questions 1\u20133 in"} +{"text": "In the published paper titled \u2018\u2018N-Screen Aware Multicriteria Hybrid Recommender System Using Weight Based Subspace Clustering,\u201d in Figure\u2009\u20092, it was not \u201cDevice pynamic profile,\u201d it is \u201cDevice dynamic profile\u201d and it was not \u201cinforomation\u201d it is \u201cinformation.\u201d Also, in Algorithm\u20092, there was an issue with square root placement in Equation (ii). The right equation is as follows:"} +{"text": "The article titled \u201cBacterial Biodegradation of Crude Oil Using Local Isolates\u201d has been"} +{"text": "After the publication of this work it was nThe author names should appear as Aaron Taudt and Maria Colom\u00e9-Tatch\u00e9.This has been updated in the original article."} +{"text": "There is an error in the \u201clength of longitudinal periods\u201d section of Table 1 in the third column. The correct value is \u201c5yrs.\u201d Please see the corrected"} +{"text": "The ninth author\u2019s name is misspelled. The correct name is: Joan Rosell\u00f3-Catafau."} +{"text": "Table 3, under the arrow that connects \u201cVentral Striatum Activity\u201d with \u201cBody Mass Index\u201d it must read c\u20321 = 0.31 (without asterisks). Please find attached the amended Figure.We have found a small error in Figure The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "S. purpuratus.\u201d Please see the corrected There is an error in"} +{"text": "If his patterns are more permanent than theirs, it is because they are made with ideas.\u201d\u2014G. H. Hardy, \u201cA Mathematician\u2019s Apology\u201d The Art of Doing Science and Engineering: Learning to Learn life without a struggle\u2026is hardly a life worth living.\u201dThe true gain is in the struggle for excellence, and \u201ca life without such a goal is not really living but it is merely existing\u201d ."} +{"text": "After publication it was noted by the author that Figure\u00a05 in the article \u2018Contact-mediated intracellular delivery of hydrophobic drugs from polymeric nanoparticles\u2019 (Snipstad et al."} +{"text": "Following the publication of our original article we noticed that an error was introduced during the assembly of Fig.\u00a03a Fig.\u00a0a here. I"} +{"text": "The authors wish to make the following corrections to this paper : The author name \u201cKo\u0142odziej \u0141ukasz\u201d should be \u201c\u0141ukasz Ko\u0142odziej\u201d.The authors would like to apologize for any inconvenience caused to the readers by these changes."} +{"text": "We read the article titled \u201cRemote ischemic preconditioning delays the onset of acute mountain sickness in normobaric hypoxia\u201d with great interest phase becoming evident after 12\u201324\u00a0h and persisting for 3\u20134\u00a0days Bolli . In the"} +{"text": "The phrase \u201chazard ratios\u201d appears incorrectly throughout the article. The correct phrase should be \u201codds ratios.\u201dIn In"} +{"text": "The seventh author\u2019s name is spelled incorrectly. The correct name is Eric Chabri\u00e8re."} +{"text": "The following sentence should be included in the funding statement of this article:\"HG and RS thank the support of Funda\u00e7\u00e3o para a Ci\u00eancia e Tecnologia through the project with reference number UID/NEU/04539/2013.\""} +{"text": "In the Introduction under the subheading \u201cB. Adaptation of Dysarthric speech\u201d,"} +{"text": "The article titled \u201cHow to Perfuse: Concepts of Cerebral Protection during Arch Replacement\u201d has been"} +{"text": "Unfortunately, the original version of this article containeThe correct author list and the new \u201cAuthors\u2019 contributions\u201d section have been correctly included in this erratum."} +{"text": "The second author\u2019s name is spelled incorrectly. The correct name is: P\u00e1draig MacNeela."} +{"text": "Unfortunately, the original version of this article containeH\u00e5kon Hofstad was incorrectly listed as \u2018Hakon Hofstad\u2019. The original article has now been updated to reflect the correct author name."} +{"text": "In the paper titled \u201cAsymptomatic Liver Abscesses Mimicking Metastases in Patients after Whipple Surgery: Infectious Complications following Percutaneous Biopsy\u2014A Report of Two Cases\u201d the misspelled last name of the 2nd author was \u201cMayody\u201d and is corrected as above."} +{"text": "The author would like to update \u201cConflicts of Interest\u201d section of their previous publication as follo"} +{"text": "Researches have demonstrated that new graduates encounter a degree of \u2018\u2018emotional hardness\u2019\u2019 and cynicism during clinical placements, which can influence the quality of their provided care . Meleis"} +{"text": "We previously showed that canonical TGF\u03b2 signaling is regulated in part by the primary cilium, and that ciliary TGF\u03b2 signaling is upregulated in stem cells differentiating into cardiomyocytes . Ciliary"} +{"text": "The seventh author\u2019s name is spelled incorrectly. The correct name is Li Fell\u00e4nder-Tsai."} +{"text": "In the article titled \u201cCutaneous Plasmacytosis with Perineural Involvement\u201d , the aut"} +{"text": "In the published work , Figure Corrected Figure four Figure\u00a0 here:"} +{"text": "The following information is missing from the Funding section: Red de Investigacion en Actividades Prevent\u00edvas y Promoci\u00f3n de la Salud and the European Regional Development Fund."} +{"text": "The article titled \u201cAn Improved Ant Colony Optimization Approach for Optimization of Process Planning\u201d has been"} +{"text": "In Dillehay et\u00a0al. We apologize for this error."} +{"text": "In the article titled \u201cCircadian Control of Global Transcription\u201d , there w"} +{"text": "The references to \u201cwhite circles\u201d and \u201cred circles\u201d within the legends of"} +{"text": "The article titled \u201cIntramedullary Chondrosarcoma of Proximal Humerus\u201d has been"} +{"text": "The parameter k_f in Table 5 is incorrect. It should be given as 50000. Correspondingly the standard deviation for the parameter k_f should also be \u00b115440."} +{"text": "The article titled \u201cOral Carcinogenesis and Oral Cancer Chemoprevention: A Review\u201d has been"} +{"text": "The paper titled \u201cObesity as a Consequence of Gut Bacteria and Diet Interactions\u201d , publish"} +{"text": "The use of the term \u201crisk ratio\u201d is incorrect throughout the manuscript. The correct term should be \u201crate ratio.\u201d"} +{"text": "The sixth author\u2019s name is spelled incorrectly. The correct name is: Cl\u00e1udio P. Figueira."} +{"text": "In the PDF version of this article, the footer erroneously gives the published year and month as \u201cNovember 2007\u201d. This date in the PDF footer should read \u201cNovember 2014\u201d."} +{"text": "ErratumAfter publication of the original article the authIn Definition 1 (Petri net): \u201cE\u2009\u2286\u2009((P x T)\u222a(T x P))\u201d is the set of directed edges not \u201cE\u2009\u2286\u2009((P T) \u222a (T P))\u201d. A segment of the Legend in Figure two: \u201cA model of insulin receptor activation and recycling.\u201d was incorrect and has been removed. The Legend of Figure three was incorrect and the correct legend is: \u201cThe model of insulin receptor recycling according to Figure 2 is represented as a Petri net. Places are drawn as circles and transitions as black squares.\u201dThese mistakes have been updated in the original article as detailed in this erratum."} +{"text": "The author\u2019s full name is Isabel Rodr\u00edguez-Barraquer.The latter surname of the 6"} +{"text": "In the original publication of the article, the spelling of the first author name \u201cTanmay Paul\u201d was incorrectly published as \u201cTanamy Paul\u201d. The correct author name is \u201cTanmay Paul\u201d."} +{"text": "There is an error in the seventh author\u2019s name. Nicola de\u2019Liguori Carino\u2019s last name is de\u2019Liguori Carino, not Carino, as previously noted."} +{"text": "In \u201cMaterials and Methods\u201d under the heading \u201cTissue Fixing and Clearing\u201d we incorrectly described the composition of our Hoyer's solution. \u201c\u2026 Hoyer's solution \u201d should read \u201c\u2026 Hoyer's solution .\u201dThe author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "International Journal of Molecular Sciences [The authors wish to change the first author\u2019s name \u201cGuoling Li\u201d to \u201cGuoliang Li\u201d on the Page of their paper published in the Sciences . The aut"} +{"text": "Due to a publisher error in the original version of this article , \u20188A\u2019 wa"} +{"text": "The original version of this article was unfoFigure\u00a0Figure"} +{"text": "The primer MicroVP1-R1 should read as \u201c5\u2032-NCG YTC YTG RTA NCC RAA-3\u2032.\u201dThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "The bottom of In the Materials and Methods section beneath the header \u201cIdentification of functional gene classes,\u201d the subheader \u201cIron resistance proteins\u201d should instead be \u201cIron regulated proteins.\u201d"} +{"text": "The penultimate sentence of the Conclusions section is incorrect. The correct sentence is:Chloroflexi was negative at low temperatures (55\u201343\u00b0C), but positive at higher temperatures (75\u201355\u00b0C).\u201d\u201cThe relationship between Cyanobacteria and filamentous"} +{"text": "Please see the corrected In The value for \u201cdelta\u201d in the S1 Appendix(ZIP)Click here for additional data file."} +{"text": "In the publication of this article (Ar\u00e9touyap et al."} +{"text": "There are errors in the Funding section. The correct funding information is as follows: This research was supported by funding from New York University Abu Dhabi and Comisi\u00f3n Nacional de Investigaci\u00f3n Cient\u00edfica y Tecnol\u00f3gica (Republic of Chile), grants FONDECYT 1151077 and BASAL FB0008."} +{"text": "The word \u201cLeaving\u201d should have read \u201cLiving\u201d. The article has since been corrected online.In the article \u201cIndications to Hospital Admission and Isolation of Children With Possible or Defined Tuberculosis Systematic Review and Proposed Recommendations for Pediatric Patients Leaving in Developed Countries\u201d,"} +{"text": "The third author\u2019s name is incorrectly spelled. The correct name is Fr\u00e9d\u00e9ric Coin."} +{"text": "There are errors in the \u201cLocality\u201d column of"} +{"text": "The paper titled \u201cTherapeutic Management of the Hallux Rigidus\u201d , publish"} +{"text": "The text \u201c_ENREF_2_ENREF_3\u201d incorrectly appears at the beginning of the second sentence of the Introduction and should be deleted. The text \u201c_ENREF_81\u201d also incorrectly appears in the first sentence of the Results section and should be deleted."} +{"text": "The article titled \u201cTechnique of Intravesical Laparoscopy for Ureteric Reimplantation to Treat VUR\u201d has been"} +{"text": "Min Zhao should be listed as a corresponding author. Dr. Min Zhao\u2019s email address is: There is an error in"} +{"text": "The authors wish to make the following corrections to this paper :The authors\u2019 names:\u201cRipamonti Carla\u201d should be \u201cCarla Ripamonti\u201d;\u201cTrippa Fabio\u201d should be \u201cFabio Trippa\u201d;\u201cBarone Gloria\u201d should be \u201cGloria Barone\u201d;\u201cMaranzano Ernesto\u201d should be \u201cErnesto Maranzano\u201d;The authors would like to apologize for any inconvenience caused to the readers by these changes."} +{"text": "The World Health Organization defines \u201ccomplementary and alternative medicine\u201d (CAM) as a \u201cbroad set of health care practices that are not part of that country\u2019s own tradition and are not integrated into the dominant health care system\u201d . Complem"} +{"text": "There is a typo in Table\u00a01 the original version of this article (Guerchet et al."} +{"text": "In the Materials and Methods section, in the last sentence of the subsection titled \u201cMicroarrays\u201d, there is a missing series accession number. Instead of \u201cseries accession number pending,\u201d it should read \u201cseries accession number GSE52344.\u201d"} +{"text": "In this article the wrong figures appeared due to a database error\u00a0as figures 1 and 2; the updated Figs."} +{"text": "This article has been retracted: Aging has completed its investigation of this paper. The investigation found that the text and figures overlap with an article by different authors published in another journal [Figure 1C \u201csham\u201d image is identical with \u201cTAC\u201d image in Figure 2C from the Aging paper and the Figure 2E \u201cTAC\u201d image from the aforementioned other paper [Figure 2C also are identical to two panels of Figure 2E in the other paper [Figure 2B - H&E staining of the heart tissue - bottom panels \u201cTAC\u201d and \u201cTAC+Ad-AK045171\u201d are identical with the bottom \u201cTAC\u201d and \u201csham\u201d images of Figure 4F of the other paper [ journal , which hThe Administration of the Beijing Chaoyang Hospital, Capital Medical University was notified about the retraction by Aging Journal."} +{"text": "The original article was incorrectly published as a \u201cReview\u201d article. The correct article type is \u201cMeta-Analyses and Systematic Reviews.\u201dThe publisher apologizes for this mistake."} +{"text": "In the original article, Table\u00a02 incorrectly states that the study \u201cWepf (2021)\u201d was conducted in Sweden. The correct country classification of this study should read as Switzerland."} +{"text": "Modern Pathology 10.1038/s41379-022-01123-6, published online 04 August 2022Correction to: NWO) and (2) Sally Wyatt and Flora Lysen should be mentioned under \u2018\u2019contributions\u2019\u2019 with \u2018\u2019We thank Sally Wyatt and Flora Lysen for their valuable comments on this work.\u2019\u2019 The original article has been corrected.After the publication of the original article the authors came across two important errors in the manuscript: (1) probably an autocorrect has changed the funding agency\u2019s name from \u2018\u2019NWO\u2019\u2019 to \u2018\u2019NOW\u2019\u2019 (so the correct spelling is"} +{"text": "Originally, the article was published with error. The author \u201cLaiose Coady\u201d should be correctly spelled as \u201cLaoise Coady\u201d.The original article has been corrected."} +{"text": "PLOS ONE article [The Funding Statement for this article states t article does not article which waPLOS ONE Editors issue this Expression of Concern.Therefore, the \u200b\u200b\u200b\u200bWe regret that this concern was not identified and addressed prior to the article\u2019s publicat"} +{"text": "The original version of this In Table 2: Because of a formatting error, the mean values of three peptides in Set 2 were listed under Set 1 and the mean values for Set 3 were listed under Set 2.In Table 3: The values under \u201cPositive\u201d and \u201cNegative\u201d columns for the row describing \u201cSARS-CoV-2 negative nasopharyngeal swab samples (n\u2009=\u200930)\u201d were switched and the \u201cSpecificity\u201d was incorrectly represented as \u201c~\u2009100%\u201d instead of \u201c100%.\u201dThe authors regret these errors, which have now been corrected. The corrected Tables"} +{"text": "In the published article, there was an error in \u201c"} +{"text": "In Zhang et al.,The correct Figure\u00a0The Author's apologize for this error."} +{"text": "Alemayehu Kidane\u201d was not included as an author in the original publication [\u201clication . The cor"} +{"text": "For all plots, the summary point estimate represented a unit change of the outcome \u201cper 250\u00a0mL serving\u201d of the beverage.This error has been corrected."} +{"text": "In the recent article by Aagaard et al.\u00a0, the out"} +{"text": "Figure 2B should have been labeled \u201cInpatient days per beneficiary,\u201d not \u201cComposite spending per beneficiary.\u201d This article has been corrected.1In the Original Investigation titled \u201cGeographical Variation in Health Spending Across the US Among Privately Insured Individuals and Enrollees in Medicaid and Medicare,\u201d"} +{"text": "The relevant funding code should read \u201cCE140100008\u201d instead of \u201cCE14010008\u201d.These details have been corrected only in this correction notice to preserve the published version of record."} +{"text": "In Sasaki et al.,The corrected Tables\u00a0The authors apologize for this error."} +{"text": "In the published version of Sun et al.\u00a0, the wor5\u2010HT6R null mutation induces synaptic and cognitive defectsWe apologize for this error."} +{"text": "PLOS ONE article [The Funding Statement for this article states t article does not article which waPLOS ONE Editors issue this Expression of Concern.Therefore, the \u200b\u200b\u200b\u200bWe regret that this concern was not identified and addressed prior to the article\u2019s publicat"} +{"text": "In the recent article by Cai et al.\u00a0, the authttps://orcid.org/0000\u20100002\u20108348\u20106477"} +{"text": "Dear Sir,We read with interest the article by Leffler et\u00a0al.,"} +{"text": "The labels \u201cFigure 1\u201d and \u201cFigure 2\u201d should be switched to be consistent with the citations in the main text. This article has been corrected.1In the Original Investigation titled \u201cAssessment of Trends in Guideline-Based Oral Anticoagulant Prescription for Patients With Ischemic Stroke and Atrial Fibrillation in China,\u201d"} +{"text": "The authors would like to addthe section \u201cAcknowledgements\u201d."} +{"text": "In the article titled \u201cAmeliorating Effect of Klotho Protein on Rat Heart during I/R Injury\u201d , Acknowl"} +{"text": "Synthesis and photophysics of sulfide, sulfoxide and sulfone based D\u2013\u03c0\u2013A compounds\u2019 by Matias Mon\u00e7alves The authors regret that The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "In Yasuda et al,The description of Table\u00a0in the result section:\u201cTable\u00a0The text was incorrect and should have read:\u201cTable\u00a0The most commonly implemented infection control measures were \u201cwearing masks at all times during working hours\u201d (79%) and \u201crequesting employees not to come to work when they are not feeling well\u201d (75%). In contrast, relatively few companies had implemented \u201cprohibiting workers from eating at their own desk\u201d (17%).\u201dThe corrected Table\u00a0The authors apologize for this error."} +{"text": "The main title should have title case capitalisation with the first clause in speech marks. The correct title should read: \u201cThey Were Saying That I Was a Typical Chinese Mum\u201d: Chinese Parents\u2019 Experiences of Parent-Teacher Partnerships for Their Autistic Children.Reframing Autism should be an additional affiliation for co-author Melanie Heyworth.Co-author Chong Yeow should have a middle name (Chong Tze Yeow).The sentence \u201cEthical approval was gained from [BLINDED FOR REVIEW]\u201d should be deleted from the Procedure section (page 3).The word \u2018written\u2019 should be deleted from the following sentences: \u201cOnce parents had provided written informed consent\u201d and \u201cWritten informed consent was gained from all participants\u201d .The Theme 1 subheading should be in title case. The heading should read: \u201cTheme 1: \u201cChildren are Your Heart\u201d\u201cThey expressed the view that \u201cAustralia teachers don\u2019t want to talk about the bad things\u201d should read: \u201cThey expressed the view that \u201cAustralian teachers don\u2019t want to talk about the bad things\u201d.\u201cThey [teachers] just tell you that that the classroom is not for you\u201d should read: \u201cThey [teachers] just tell you that the classroom is not for you\u201dUnder Theme 2 , the following sentences should be amended:\u201cBecause we are from Chinese culture\u201d should read: \u201cBecause we are from a Chinese culture\u201d.Under Theme 4 , the following sentence should be amended:Please note the following corrections to this article:"} +{"text": "In the original publication in Table 1, the entry: \u201cNichirei Histofine RALK iAEP Kit\u201d should read as: \u201cNichirei Histofine ALK iAEP Kit\u201d. The correct version of Table The original publication has been corrected."} +{"text": "In the recent article by Gerschwitz\u2010Eidt et al.\u00a0, the autThe authors apologize for the error."} +{"text": "In the recent article by Ruiz\u2010Frau et al.\u00a0, the corThe authors apologize for the error."} +{"text": "In the recent article by Obrist et al.\u00a0, the lab"} +{"text": "In Vitro Study\u201d [1In the article titled \u201cAndrographolide Enhances Proliferation and Prevents Dedifferentiation of Rabbit Articular Chondrocytes: An o Study\u201d , the aut Study\u201d 1, but the"} +{"text": "Following publication of the original article , the autThe word \u201ctan\u201d should be corrected to \u201ctank\u201d in Fig.\u00a0The correct version of figure is given."} +{"text": "The ninth author\u2019s name is spelled incorrectly. The correct name is: Aleksandra Kr\u00f3likowska."} +{"text": "The word \u201cOncology\u201d should have appeared before \u201cClinical.\u201d The Supplement title has also been updated. This article has been corrected.1In the Original Investigation titled \u201cParticipation of Lower and Upper Middle\u2013Income Countries in Clinical Trials Led by High-Income Countries,\u201d"} +{"text": "We read with great interest the case report in by Bouaouina et\u00a0al,As Bouaouina et\u00a0al,Bouaouina et\u00a0alIn summary, we would like to caution about a proper interpretation of an FFR measurement and advise using meticulous technique to improve outcomes."} +{"text": "The original version of the article unfortunately contained a mistake in the sequence of the second and third authors.The incorrect authors name order is: Jennifer Moloney \u00b7 Margaret Walshe \u00b7 Julie ReganThe correct authors name order is: Jennifer Moloney \u00b7 Julie Regan \u00b7 Margaret WalsheThe author group has been updated above and the original article has been corrected."} +{"text": "Due to a production error, an author\u2019s name was incorrectly inserted as \u201cQiao Jie\u201d. The correct name is \u201cJie Qiao\u201d.The publisher apologizes for this mistake. The original version of this article has been updated."} +{"text": "The Guardian which publishes in English and appears in the UK and Addustour which publishes in Arabic and appears in Jordan. Following Paltridge\u2019s : tridge\u2019s taxonomy Media discourse has attracted the attention of researchers who are interested in analysing linguistic and rhetoric aspects of discourse and interjections .The first acknowledges the need to adequately meet readers\u2019 expectations of inclusion and disciplinary solidarity, addressing them as participants in an argument with reader pronouns and references to shared knowledge. The second purpose involves rhetorically positioning the audience, pulling readers into the discourse at critical points, predicting possible objections and guiding them to particular interpretations. These functions are mainly performed by questions, directives in the use of engagement strategies between English and Arabic newspaper editorials?Therefore, writers\u2014including editorialists\u2014write to express and impress by carefully attending to their readership so as to express their views and convince readers to buy into the position of the writer and/or the newspaper, in the case of editorialists. Lafuente-Mill\u00e1n contendsimagined receiver and the evolving text itself\u201d . It is essential to clarify the use of the word \u2018imagined\u2019 in the quote. Hyland produced by governments and hospitals during the COVID-19 pandemic. The results showed that both sets of data included engagement markers and that their use did not significantly vary based on the issuing agencies. In particular, the findings showcased that there were no differences in the use of engagement strategies by the two agencies and that \u201cthere \u2026 [was] little change in the use of engagement markers as the reader\u2019s role shifts from staff to citizen\u201d (p. 187).Yang investigBased on the foregoing, we notice that studies which contrastively explore the use and functions of engagement strategies/markers are scarce, and more so are studies that investigate engagement in Arabic and English editorialists. Thus, this study aims at comparing journalistic writing practices in two different languages (English and Arabic) by examining the use and functions of engagement strategies. Editorials are chosen in this research because of their subjectivity and explicitness in representing the newspaper\u2019s position which differentiates them from academic prose Hyland, .The Guardian which publishes in English and appears in the UK and Addustour which publishes in Arabic and appears in Jordan. A corpus of 80 editorials (40 from each newspaper) was constructed for analysis. The editorials were retrieved from the website of the two newspapers. The editorials which were published between 2020 and 2021 were selected for reasons of recency so that the conclusions describe the genre at hand more accurately. Each set of editorials was transferred into a Microsoft Word file for analysis. In the analysis phase, a mixed-method approach, utilising quantitative and qualitative measures, was adopted. The analysis is based on Paltridge\u2019s and directives (20.4%). There were also questions in the English editorials, with 8.8% of the strategies. However, no questions were found in the Arabic set of editorials. The least frequent engagement strategy in the English set was appeals to shared knowledge, with 2.1% which was exactly the same as that of the Arabic editorials (2.1%). In the Arabic set of editorials, 233 engagement markers were used with the absence of questions from this set of editorials, as just mentioned. The most frequent strategy in the Arabic editorials was reader pronouns, with 94% of the strategies. Table directives, with 2.6%; this was followed by appeals to shared knowledge, with 2.1% and personal asides, with 1.3%.As shown in Table pronouns 0.4% and U statistical analysis test was used to measure the differences and/or similarities between the two groups of data. The Mann\u2013Whitney U test compares two independent groups to determine if there are differences in medians between them. The results of the Mann\u2013Whitney U test, as shown in Table U test results also showed a significant difference between English and Arabic editorials in the use of reader pronouns. The results also revealed that the use of personal asides was similar in the two sets of editorials but showed significant differences between the two groups of editorials among the rest of the engagement strategies .As noted earlier, the Mann\u2013Whitney Now that we have presented the results quantitatively, we turn to the qualitative analysis of the data obtained from the two corpora. We present our analysis for each engagement strategy with illustrative examples to explicate their functional use in discourse.we and the second-person pronoun you in interacting and communicating with readers. Writers make use of reader pronouns as an engagement strategy to involve the reader and to make the text more compelling and persuasive . This might be attributed to the fact that \u201cinclusive pronouns can act as positive politeness devices by describing and/or critiquing common disciplinary practices and elaborating arguments on behalf of the community\u201d .And that is the best We now have fewer than 100 days before the United Nations\u2019s Cop26 climate change conference opens in Glasgow, when world leaders will be given one last, clear chance to limit climatic mayhem. .\u0646\u062d\u0646 \u0623\u0645\u0627\u0645 \u0645\u0631\u062d\u0644\u0629 \u062c\u062f\u064a\u062f\u0629 \u0648\u0627\u0639\u062f\u0629 \u0645\u0644\u064a\u0626 \u0628\u0627\u0644\u0645\u0634\u0627\u0631\u064a\u0639 \u0627\u0644\u062d\u064a\u0648\u064a\u0629\u060c \u0648\u0647\u064a \u0628\u0627\u0644\u0645\u062c\u0645\u0644 \u062d\u0635\u064a\u0644\u0629 \u0631\u0624\u0649 \u0645\u0644\u0643\u064a\u0629 \u0647\u062f\u0641\u062a \u0625\u0644\u0649 \u062c\u0639\u0644 \u0627\u0644\u0639\u0642\u0628\u0629 \u0645\u0642\u0635\u062f\u0627 \u0633\u064a\u0627\u062d\u064a\u0627 \u0648\u0627\u0633\u062a\u062b\u0645\u0627\u0631\u064a\u0627 \u0648\u062a\u062c\u0627\u0631\u064a\u0627\u060c \u0648\u0628\u0648\u0627\u0628\u0629 \u0628\u062d\u0631\u064a\u0629 \u0625\u0642\u0644\u064a\u0645\u064a\u0629\u060c \u062a\u0646\u0639\u0643\u0633 \u0622\u062b\u0627\u0631\u0647\u0627 \u0639\u0644\u0649 \u0645\u0639\u062f\u0644\u0627\u062a \u0627\u0644\u0646\u0645\u0648 \u0648\u062e\u0644\u0642 \u0641\u0631\u0635 \u0627\u0644\u0639\u0645\u0644 \u0648\u0627\u0644\u062a\u062e\u0641\u064a\u0641 \u0645\u0646 \u0623\u0631\u0642\u0627\u0645 \u0627\u0644\u0628\u0637\u0627\u0644\u0629. (\u201c\u0627\u0644\u0645\u0644\u0643 \u0641\u064a \u0627\u0644\u0639\u0642\u0628\u0629\u201c\u060c \u0627\u0644\u062f\u0633\u062a\u0648\u0631\u060c \u0662\u0660\u0662\u0661).\u0628\u0627\u0644\u0645\u062d\u0635\u0644\u0629 we are in front of a new promising stage which is full of vital projects. This stage is the outcome of the royal vision that aims at making Aqaba a tourist, economic and commercial destination as the regional coastal gateway. This new phase will impact the rates of growth and unemployment and it will create new job opportunities. .As a result, \u0646\u0627 \u0642\u0627\u062f\u0631\u0648\u0646 \u0639\u0644\u0649 \u0625\u062d\u062f\u0627\u062b \u0627\u0644\u062a\u063a\u064a\u064a\u0631 \u0627\u0644\u0625\u064a\u062c\u0627\u0628\u064a \u0639\u0628\u0631 \u0645\u0634\u0627\u0631\u064a\u0639\u0647\u0645. (\u201c\u0634\u0628\u0627\u0628\u0646\u0627 \u0642\u0627\u062f\u0631\u0648\u0646 \u0639\u0644\u0649\u201c\u060c \u0627\u0644\u062f\u0633\u062a\u0648\u0631\u060c \u0662\u0660\u0662\u0661).\u0634\u0628\u0627\u0628The use of reader pronouns refers to the way personal pronouns such as the inclusive Harwood, , p. 343.Harwood, argumentOur youth can create a positive change through their projects. .we in English and the plural independent personal pronoun in Arabic \u0646\u062d\u0646\u064f. In the three first examples, the editorialists used the pronoun we to express a view that is shared by the audience and to persuade them to accept it. The fourth example shows how the plural suffixal pronoun /naa/ \u0640\u0640\u0640\u0640\u0640\u0640\u064e\u0646\u0627 which means (our) can be used to address the reader as part of the dialogue created in the text.The above-mentioned examples illustrate how reader pronouns can be used as a strategy to engage the reader in the discussion, especially by the use of the plural pronoun U test showed no significant differences in the usage of personal asides between English and Arabic editorialists. The following example shows the use of personal asides in both sets of data:5.and that includes too-silent Britain\u2014must take the lead. .For the sake of the Lebanese people, and out of obvious self-interest, the international community\u20146.and when ministers say that they will do something\u2014many people understandably interpret that as meaning that it is safe to do something. .When the government tells people that they can do something\u20147.\u060c \u0648\u0647\u0648 \u0645\u0627 \u064a\u062f\u0641\u0639\u0646\u0627 \u062c\u0645\u064a\u0639\u0627\u064b \u0644\u062a\u0639\u0638\u064a\u0645 \u0647\u0630\u0647 \u0627\u0644\u0642\u064a\u0645 \u0627\u0644\u0646\u0628\u064a\u0644\u0629\u060c \u0648\u0647\u064a \u0631\u0633\u0627\u0644\u0629 \u062a\u062f\u0644\u0644 \u0623\u064a\u0636\u0627\u064b \u0639\u0644\u0649 \u0645\u062f\u0649 \u0627\u0647\u062a\u0645\u0627\u0645 \u062c\u0644\u0627\u0644\u0629 \u0627\u0644\u0645\u0644\u0643 \u0628\u0633\u064a\u0631 \u062a\u0637\u0648\u0631 \u0639\u0645\u0644 \u0627\u0644\u0645\u0631\u0643\u0632 \u0631\u063a\u0645 \u0639\u062f\u064a\u062f \u0627\u0644\u062a\u062d\u062f\u064a\u0627\u062a (\u201c\u0637\u0648\u0628\u0649 \u0644\u0643\u0627\u0641\u0644 \u0627\u0644\u064a\u062a\u064a\u0645\u201c\u060c \u0627\u0644\u062f\u0633\u062a\u0648\u0631\u060c \u0662\u0660\u0662\u0661).\u0644\u064a\u062c\u0633\u062f \u0647\u0630\u0627 \u0627\u0644\u062a\u0648\u0627\u0635\u0644 \u0645\u062f\u0649 \u0634\u0639\u0648\u0631 \u0627\u0644\u0642\u0627\u0626\u062f \u0628\u0623\u0628\u0646\u0627\u0621 \u0634\u0639\u0628\u0647, which motivates us all to maximise these noble values, a message that also demonstrates how much His Majesty the King is interested in the evolution of the Centre\u2019s work despite the many challenges.This communication reflects how much the leader feels about his people8.\u060c \u0641\u0644\u0627 \u0645\u0643\u0627\u0646 \u0644\u0644\u064a\u0623\u0633 \u0628\u064a\u0646\u0646\u0627\u060c \u0648\u0642\u064a\u0627\u062f\u062a\u0646\u0627 \u0648\u0634\u0639\u0628\u0646\u0627 \u0644\u0627 \u064a\u0639\u0631\u0641\u0648\u0646 \u0627\u0644\u0645\u0633\u062a\u062d\u064a\u0644 (\u201c\u0645\u0626\u0648\u064a\u0629 \u0627\u0644\u062f\u0648\u0644\u0629\u201c\u060c \u0627\u0644\u062f\u0633\u062a\u0648\u0631\u060c \u0662\u0660\u0662\u0661).\u0646\u062d\u062a\u0627\u062c \u0627\u0644\u064a\u0648\u0645 \u0643\u0645\u0627 \u064a\u0624\u0643\u062f \u062c\u0644\u0627\u0644\u0629 \u0627\u0644\u0645\u0644\u0643 \u0639\u0628\u062f \u0627\u0644\u0644\u0647 \u0627\u0644\u062b\u0627\u0646\u064a \u0625\u0644\u0649 \u0625\u062d\u064a\u0627\u0621 \u0627\u0644\u0631\u0648\u062d \u0627\u0644\u062a\u064a \u0628\u0646\u064a \u0628\u0647\u0627 \u0648\u0639\u0644\u064a\u0647\u0627 \u0627\u0644\u0623\u0631\u062f\u0646Personal asides are defined as comments made by the writer within an argument to direct the reader to another related idea; they form an abrupt stoppage to engage the reader into continuing the argument. Hyland and Jiang referred, there is no place for despair between us, our leadership and our people do not know the impossible.Today we need to revive the spirit upon which Jordan was built as His Majesty King Abdullah II assertsExamples (5)\u2013(8) illustrate the use of personal asides in English and Arabic editorials. The editorialists openly made a comment on the issues under discussion using parentheses and commas as in the abovementioned examples. In the case of editorials, personal asides are not quite personal. Since editorials represent the institution\u2019s viewpoint rather than the writer\u2019s personal viewpoint, personal asides are among the most explicit ways of offering this standpoint. It can be noticed from Examples (5) and (6) that the attention is drawn to the writer\u2019s comment through the use of personal asides. Khabbazi Oskouei , p. 128 9.Needless to say, it is children and women who suffer most from the missing provision, especially those from poorer backgrounds .10.,of course, accelerated\u2014such places continue to knit the social fabric together in vital ways. .Though often hit by the long-term shift to online retail\u2014which the pandemic11.\u0628\u0637\u0628\u064a\u0639\u0629 \u0627\u0644\u062d\u0627\u0644 \u0641\u064a \u0625\u0639\u0627\u062f\u0629 \u0627\u0644\u0628\u0648\u0635\u0644\u0629 \u0648\u062d\u0634\u062f \u0627\u0644\u062c\u0647\u0648\u062f \u0627\u0644\u062f\u0627\u0639\u0645\u0629 \u0644\u0639\u062f\u0627\u0644\u0629 \u0627\u0644\u0642\u0636\u064a\u0629 \u0644\u0641\u0644\u0633\u0637\u064a\u0646\u064a\u0629\u060c \u062d\u064a\u062b \u0636\u0631\u0648\u0631\u0629 \u0627\u0644\u062a\u0648\u0635\u0644 \u0625\u0644\u0649 \u062d\u0644 \u0639\u0627\u062f\u0644 \u0648\u0634\u0627\u0645\u0644 \u0644\u0647\u0627 \u0639\u0644\u0649 \u0623\u0633\u0627\u0633 \u062d\u0644 \u0627\u0644\u062f\u0648\u0644\u062a\u064a\u0646\u060c \u0648\u0648\u0641\u0642\u0627 \u0644\u0645\u0628\u0627\u062f\u0631\u0629 \u0627\u0644\u0633\u0644\u0627\u0645 \u0627\u0644\u0639\u0631\u0628\u064a\u0629\u060c \u0648\u0642\u0631\u0627\u0631\u0627\u062a \u0627\u0644\u0634\u0631\u0639\u064a\u0629 \u0627\u0644\u062f\u0648\u0644\u064a\u0629. (\u201c\u062d\u0631\u0635 \u0623\u0631\u062f\u0646\u064a \u0639\u0644\u0649 \u0627\u0644\u062a\u0636\u0627\u0645\u0646 \u0627\u0644\u0639\u0631\u0628\u064a\u201c\u060c \u0627\u0644\u062f\u0633\u062a\u0648\u0631\u060c \u0662\u0660\u0662\u0661)\u0647\u0630\u0647 \u0627\u0644\u0631\u0633\u0627\u0626\u0644 \u0627\u0644\u0645\u0644\u0643\u064a\u0629 \u0627\u0644\u0647\u0627\u062f\u0641\u0629 \u0625\u0644\u0649 \u062a\u0648\u062d\u064a\u062f \u0627\u0644\u0635\u0641 \u0627\u0644\u0639\u0631\u0628\u064a\u060c \u062a\u0635\u0628 naturally serve to restore the balance and mobilise efforts in support of the justice of the Palestinian cause. It is also necessary to achieve a just and comprehensive solution based on the two-state solution, in accordance with the Arab Peace Initiative and the resolutions of international legitimacy.These royal messages aimed at unifying the Arab class 12.\u062d\u062a\u0645\u0627\u064b. (\u201c \u0633\u0640\u0646\u0640\u0628\u0640\u0642\u0640\u0649 \u0645\u062a\u0633\u0644\u062d\u064a\u0646 \u0628\u062a\u0636\u062d\u064a\u0627\u062a\u0646\u0627\u201c\u060c \u0627\u0644\u062f\u0633\u062a\u0648\u0631\u060c \u0662\u0660\u0662\u0661)\u0644\u0645 \u064a\u0639\u062f \u0645\u0642\u0628\u0648\u0644\u0627 \u0627\u0644\u0633\u0643\u0648\u062a \u0639\u0646 \u062c\u0631\u0627\u0626\u0645 \u0627\u0644\u0645\u062d\u062a\u0644\u060c \u0648\u0635\u0627\u062d\u0628 \u0627\u0644\u0623\u0631\u0636 \u0648\u0627\u0644\u062d\u0642 \u0648\u0627\u0644\u0642\u0636\u064a\u0629 \u0633\u064a\u0646\u062a\u0635\u0631 Appeals to shared knowledge refer to a strategy that writers use to engage readers by stating a piece of information that is shared between the writer and reader. In simple words, the writer attempts to treat the reader as a fellow member by using appeals to shared knowledge Hyland, . A signiinevitably triumph and prevail.Silence upon the occupier\u2019s crimes is no longer acceptable. And the owner of the right and the land will The reader should not be infuriated by the flippancy or lack of seriousness in addressing him/her within the editorial, and the best method to overcome this point as an editorialist is to use appeals of shared knowledge. In Examples (9) and (10), the editorialist referred to the common ground with the reader by using some multi-word expressions such as \u2018needless to say\u2019 and \u2018of course\u2019. Examples in (11) and (12) show how single-word expressions and multi-word expressions can be used as appeals to shared knowledge in editorials. Hyland stated tshould and must). A significant statistical difference was found between the two languages in the use of directives. Directives were less in Arabic editorials (2.6%), compared to their English counterparts (20.4%). This finding is consistent with that of Al-Rickaby .We 14.should be widely shared. .Today, as in the past, responsibility 15.\u064a\u0633\u062a\u0648\u062c\u0628 \u0645\u0646\u0627 \u0645\u0632\u064a\u062f\u0627\u064b \u0645\u0646 \u0627\u0644\u0639\u0645\u0644 \u0648\u0627\u0644\u0625\u0646\u062c\u0627\u0632. (\u201c\u0627\u0644\u0625\u0635\u0644\u0627\u062d \u0627\u0644\u0625\u062f\u0627\u0631\u064a \u0645\u0641\u062a\u0627\u062d\u201c\u060c \u0627\u0644\u062f\u0633\u062a\u0648\u0631\u060c \u0662\u0660\u0662\u0661).\u0648\u0647\u0648 \u0645\u0646\u062c\u0632 \u0645\u062a\u0631\u0627\u0643\u0645 \u0648\u0625\u0631\u062b must be followed by more work and achievement.This is a cumulative achievement and legacy that 16.\u0648\u0639\u0644\u0649 \u0627\u0644\u062c\u0645\u064a\u0639 \u0627\u0644\u064a\u0648\u0645 \u0641\u064a \u0627\u0644\u0642\u0637\u0627\u0639\u064a\u0646 \u0627\u0644\u0639\u0627\u0645 \u0648\u0627\u0644\u062e\u0627\u0635 \u0645\u0633\u0624\u0648\u0644\u064a\u0629 \u0645\u0634\u062a\u0631\u0643\u0629. (\u201c\u0628\u0627\u0644\u0639\u0632\u064a\u0645\u0629 \u0648\u0627\u0644\u0625\u0635\u0631\u0627\u0631 \u062a\u0646\u0647\u0636\u201c\u060c \u0627\u0644\u062f\u0633\u062a\u0648\u0631\u060c \u0662\u0660\u0662\u0660).\u0625\u0646 \u0627\u0644\u062c\u0648\u0644\u0629 \u0627\u0644\u0645\u0644\u0643\u064a\u0629 \u0641\u064a \u0627\u0644\u062c\u0646\u0648\u0628\u060c \u062a\u062f\u0641\u0639 \u0643\u0644 \u0645\u0633\u0624\u0648\u0644 \u0625\u0644\u0649 \u0627\u0644\u0627\u0642\u062a\u062f\u0627\u0621 \u0628\u0646\u0647\u062c \u0627\u0644\u0642\u0627\u0626\u062f\u060c \u0641\u0627\u0644\u0623\u0631\u062f\u0646 \u0628\u0644\u062f \u062e\u064a\u0631 \u0648\u0639\u0637\u0627\u0621\u060c \u0648\u0633\u0648\u0627\u0639\u062f \u0623\u0628\u0646\u0627\u0626\u0647 \u0648\u0637\u0627\u0642\u0627\u062a\u0647\u0645 \u0644\u0627 \u062a\u062d\u062a\u0627\u062c \u0633\u0648\u0649 \u0625\u0644\u0649 \u0627\u0644\u062a\u0628\u0646\u064a \u0648\u0627\u0644\u0625\u0633\u0646\u0627\u062f\u060c Directives are used to guide the reader and advise on how to run an action in the real world. It should be mentioned that, generally, directives are used to express the need to take a move and perform a certain kind of action using some modal verbs and (16) from the Arabic set of editorials illustrate that modals of obligation can be used to reinforce the persuasive power of the editorial. The use of must shows the strong desire that the writer has to involve, engage, and guide the readers.In the first two examples from the English corpus, the modal verb 17.Why would its return solve anything? In 2011 a repressive, authoritarian government collapsed because it proved unable to meet people\u2019s demands. 18.Can we learn to appreciate our own old clothes as well as other people\u2019s? 19.Will the BBC successfully continue to prove that it is a valuable anchor to British society, providing trusted news, information and entertainment of the highest quality? 20.who do not? .And even if they take off, will options like these be like parental leave for fathers (too often scuppered by fear of censure), or \u201csleep hygiene\u201d\u2014which in practice can separate those who have a choice from those Questions or interrogative statements are classified as a type of engagement strategy that is used to give a sense of emotional involvement to the reader. Biber et al. , p. 207 In (17), the writer used a wh-question to highlight a point for further reflection in the future. The editorialist in the following two examples used a yes/no type of question. In yes/no questions, \u201cthe addressee is expected to supply a truth value by answering yes or no\u201d . In addition, editorialists in the Guardian intended to use questioning as a way of transmitting information or circulating knowledge. It was evident that editorialists in the two languages were concerned with the establishment of relationships with readers by means of engagement markers. Consistent with the literature, appeals to shared knowledge and directives are used to address the public and to have a broader audience. Editorialists attract the attention of their readers and get a larger readership thru direct communication or engagement with readers.This paper has investigated the engagement strategies employed in English and Arabic editorials collected from two newspapers in the UK and Jordan. Some statistically significant differences were found between the two languages in the use of some types of engagement strategies . The data analysed were then presented to understand the nature of engagement strategies in the editorial genre of the two languages (English & Arabic). Although Arabic editorialists in Indeed, journalistic texts among other forms of written language lend special significance to the audience. The journalistic genre focuses on the persuasion of the reader with the arguments put forward by the writer. The rhetorical devices and engagement strategies can be considered among these methods through which the writer can represent and convince the audience of his/her viewpoint in a journalistic text. In specific, editorials were designed to promote certain ideas and ideologies to the audience which makes these editorials revolve around the persuasion of the audience. Given that persuasion takes place in the editorial section of the newspaper, the analysis of newspaper editorials would help in identifying the linguistic features utilised in the persuasion of the audience from different cultures and language backgrounds. In fact, one of the linguistic features that can be utilised to establish a writer\u2013reader relationship is engagement markers. Hyland and Jiang , p. 29 dTeachers of writing for specific disciplines can benefit from the comparison of corpora of discipline-specific texts in different languages in order to identify potential pitfalls for their students. Such corpora comparisons thus help teachers to understand reasons for potential mismatches in the formulation of specific text types by students. (p. 19)Several factors are found to influence the use of engagement strategies and the ways of persuading the reader with a certain standpoint such as the cultural background and the native language of the writer and the reader. A wide range of studies discussed the idea that cultural background plays an important role in the process of writing and the perception of written texts, and this gave rise to what is known as \u201cintercultural rhetoric\u201d. The term \u201cintercultural rhetoric\u201d can be broadly defined as the influence of one\u2019s first language on the acquisition of second or foreign language writing. The usage of certain linguistic features like, engagement markers depends upon users\u2019 first languages or cultures. Another point to be mentioned here is that the contrastive analysis of editorials relates to the theory of linguistic relativity defined by Connor as \u201cthe notion that patterns of language and writing are culture-specific\u201d (p.1). Consequently, this study is a chance to reflect on culture-specific rhetorical strategies that influence the language of newspaper editorials. This study is significant because of its implications for language learning and teaching. Connor and Traversa write:The present study contributes to the understanding of how engagement strategies can be used in persuasive and argumentative texts. In fact, engagement strategies in opinion pieces can reflect how the writer is trying to be considerate of the reader\u2019s needs. What makes this research significant is that it compares the use of engagement strategies by two groups of writers from two different linguistic and cultural backgrounds. The contrastive analysis of the English and Arabic editorials adds to the knowledge about intercultural rhetoric. The findings of this study are of interest to teachers of professional and journalistic writing in both languages. Writing workshops for novice journalists might draw on these findings to raise awareness about engagement strategies in persuasive writing. Language training classes that equip journalists for the demands of a fast-changing multimedia industry should emphasize the role of engagement strategies in opinion articles. Instances and applications of engagement strategies in English and Arabic ought to be included in journalism diplomas and qualifications of the two languages.This paper contributes to the overall understanding of the concept of engagement in specialist kinds of texts . The comparison between the two languages adds more to the perception of the role of engagement strategies in argumentative writing. The findings reported here have an impact on the teaching of \u201cwriting for professionals\u201d courses, in which they could incorporate such engagement strategies. Engagement strategies as tools for addressing the reader\u2019s needs and expectations would help in achieving the desired outcome from editorials efficiently and effectively. These questions would help teachers of journalistic writing in both languages in training students and early career journalists to meet the expectations of their readers. The effective use of engagement markers in any language makes the editorial easier to understand and more attractive. If the editorialist is aware of the functions of engagement strategies and can use them efficiently, this would make the editorial section available to a broader readership. Carroll , p. 113 This study has pedagogical implications for the teaching of journalistic writing in both languages where early career journalists need to be trained on the usage of these engagement strategies. The findings of this study have a number of practical implications. In terms of material design, this study suggests the integration of engagement strategies in journalistic writing courses in both languages. The explication of engagement strategies to students of journalistic writing would help in raising awareness of their significance in persuasive and argumentative texts. Awareness of engagement markers\u2019 use would help learners of the two languages (English & Arabic) in enhancing their comprehension of the public discourse in such languages as well. Another implication of this study is that students of languages for specific purposes should be taught about engagement strategies as rhetorical devices used in building and supporting arguments. Richardson , p. 2 po"} +{"text": "PLOS ONE article [The Funding Statement for this article states t article does not article which waPLOS ONE Editors issue this Expression of Concern.Therefore, the \u200b\u200b\u200b\u200bWe regret that this concern was not identified and addressed prior to the article\u2019s publicat"} +{"text": "The affiliation list has been reorderedThe text \u201cA and data not shown\u201d in the caption of Fig.\u00a01 has been updated to \u201cFig.\u00a02A and data not shown\u201dThe original publication of this article has been"} +{"text": "PLOS ONE article [The Funding Statement for this article states t article does not article which waPLOS ONE Editors issue this Expression of Concern.Therefore, the \u200b\u200b\u200b\u200bWe regret that this concern was not identified and addressed prior to the article\u2019s publicat"} +{"text": "In the article by Yongxin Zhou et\u00a0al., (2021), the authors noted some minor errors in Figure\u00a0These corrections have no impact on the experimental outcome or conclusions."} +{"text": "In the article titled \u201cA Decision-Making Approach Based on Score Matrix for Pythagorean Fuzzy Soft Set\u201d , there w"} +{"text": "In the recent article by Hansen Wheat et al.\u00a0, the Ack"} +{"text": "PLOS ONE article [The Funding Statement for this article states t article does not article which waPLOS ONE Editors issue this Expression of Concern.Therefore, the \u200b\u200b\u200b\u200bWe regret that this concern was not identified and addressed prior to the article\u2019s publicat"} +{"text": "PLOS ONE article [The Funding Statement for this article states t article does not article which waPLOS ONE Editors issue this Expression of Concern.Therefore, the \u200b\u200b\u200b\u200bWe regret that this concern was not identified and addressed prior to the article\u2019s publicat"} +{"text": "In addition, \u201cwomen\u201d was changed to \u201cfemale physicians\u201d and \u201cmen\u201d was changed to \u201cmale physicians\u201d to clarify that participants reported sex rather than gender. This article has been corrected.1In the Research Letter titled \u201cFull-time Work Rates of Physicians With Physician Spouses vs Nonphysician Spouses in Japan,\u201d"} +{"text": "Changes to \u201cauthors who contributed equally\u201dIn the original publication , the aut"} +{"text": "In all of those table cells, \u201c1 [Reference]\u201d should have appeared as \u201c0 [Reference].\u201d In addition, the last portion of footnote \u201ca\u201d in Tables 2 and 3 was modified slightly after the second closing parenthesis to read: \u201c\u2026all models were estimated with clinician-level random intercepts.\u201d This article was corrected online.The Original Investigation titled \u201cAssociation of Project ECHO Training With Buprenorphine Prescribing by Primary Care Clinicians in Minnesota for Treating Opioid Use Disorder,\u201d"} +{"text": "The original version of this article unfortunately contained a mistake. The wrong name was given for a subgroup analysis.The corrected details are given below for your reading.The last sentence in the following section \u2018\u2019Myasthenia gravis specific scores and burden of disease\u2019\u2019 should read asThere were no substantial differences between patients younger or older than 45\u00a0years old at the time the study was performed.Instead of EOMG (Early onset Myasthena gravis) and LOMG (Late onset Myasthenia gravis), the subgroups in Table 4 are \u201cPatients\u2009\u2264\u200945\u00a0years old\u201d and \u201cPatients\u2009>\u200945\u00a0years old\u201d, respectively.The corrected Table"} +{"text": "Breeding Science 70: 462\u2013473 (2020)In the above article, there was a mistake in Table 3.The sequence of reverse primers was presented in the 3\u2032\u21925\u2032 direction (highlighted).Now the sequences are reverse complemented and corrected in the 5\u2032\u21923\u2032 direction (highlighted)."} +{"text": "The correct figure is:In the article, \u201cHepatic metastasis from a meningeal Hemangiopericytoma: A case report,\u201d"} +{"text": "In the original publication of the article the second author name should be \u201cCarlos Fern\u00e1ndez-del Castillo\u201d and not \u201cCarlos Fernandez-del Castillo\u201d."} +{"text": "In the article titled \u201cEarly Detection of Medical Image Analysis by Using Machine Learning Method\u201d , there w"} +{"text": "MMWR Recommendations and Reports \u201cMethodology of the YouthRisk Behavior Surveillance System \u2014 2013,\u201d the Republic of the MarshallIslands and the Republic of Palau were erroneously referred to as U.S. territories.Throughout the report, all references to \u201cterritories\u201d should have read\u201cterritories and freely associated states,\u201d and allreferences to \u201cterritorial\u201d should have read \u201cterritorial andfreely associated state.\u201dIn the"} +{"text": "PLOS ONE article [The Funding Statement for this article states t article does not article which waPLOS ONE Editors issue this Expression of Concern.Therefore, the \u200b\u200b\u200b\u200bWe regret that this concern was not identified and addressed prior to the article\u2019s publicat"} +{"text": "Lin et\u00a0al. would liThe corrected figures are reproduced below. Figure"} +{"text": "CT scan revealed a spontaneous intramural hematoma of the descending aorta (Figure A 72\u2010year old woman with a previous history of polycythemia vera presented with acute chest pain. The full blood count showed leukocytosis WBC 43\u00a0\u00d7\u00a01009/L withThe authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported."} +{"text": "Due to a production error, an author\u2019s name was incorrectly spelled as \u201cOmal El Ayoubi \u201c. The correct spelling is \u201cOmar El Ayoubi\u201d. The publisher apologizes for this mistake.The original version of this article has been updated."} +{"text": "In the recent article by Hagen et al.\u00a0, the autIt should have read as follows:"} +{"text": "In the recent article by Yang et al.\u00a0, the aut"} +{"text": "P. falciparum\u201d is incorrectly marked with a \u0482 symbol. This should instead be a superscript letter \u2018c\u2019, as on the adjacent rows.In Please see the corrected"} +{"text": "Section 2.2: Where it reads \u201cDmax\u2010Dbk,\u201d it should read \u201c(Dmax\u2010Dbk)/Dmax.\u201dSection 2.2: Where it reads \u201cTaking the sequence of normalized darkness values along a transect, we estimated its two enveloping lines,\u201d it should read \u201cFrom the sequence of averaged RGB pixel values corresponding to each transect, we calculated the Euclidean distance of each pixel's color coordinates to the color white and estimated the enveloping lines of the upper and lower extremes of these values.\u201dFigure 1\u2014legend: Where it reads \u201cBy calculating the distance between each of those pixels to the black,\u201d it should read \u201cBy calculating the distance between each of those pixels and white.\u201dFigure 1\u2014panel a: Where it reads \u201cEuclidean distance of each pixel to black\u201d in the y axes, it should read: \u201cEuclidean distance of each pixel to white.\u201dFigures 3\u20135, Figures S1\u2010S2, and appendix Tables A2\u2010A5: Where it reads \u201cCpa,\u201d it should read \u201cCbk,\u201d and vice\u2010versa.In the paper published by Lafuente et al.\u00a0, the belThe authors apologize for these errors."} +{"text": "The published article entitled \u201cTherapeutic effects of lentinan on inflammatory bowel disease and colitis\u2010associated cancer\u201d with original manuscript ID of JCMM\u201006\u20102018\u2010109.R1 contains an error in Figure\u00a0The authors apologized for the mistakes."} +{"text": "The phrase given as \u201cNearly 20% of individuals with OUD are not treated with a MOUD\u201d should have omitted the word \u201cnot\u201d to read \u201cNearly 20% of individuals with OUD are treated with a MOUD.\u201d This Viewpoint has been corrected.In the Viewpoint titled \u201cFederal and State Pharmacy Regulations and Dispensing Barriers to Buprenorphine Access at Retail Pharmacies in the US,\u201d"} +{"text": "PLOS ONE article [The Funding Statement for this article states t article does not article which waPLOS ONE Editors issue this Expression of Concern.Therefore, the \u200b\u200b\u200b\u200bWe regret that this concern was not identified and addressed prior to the article\u2019s publicat"} +{"text": "In the article by Yoshioka\u2010Maeda and Fujii , the autIn Table\u00a0We apologize for these errors."} +{"text": "In the recent article by Sloat et al.\u00a0, the fol\u201cKarin Kiontke was supported by NIH grant GM141395.\u201dThe authors apologize for the error."} +{"text": "Figure\u00a01 caption in the original publication contains a mistake.The original article has been corrected."} +{"text": "In the original publication of the article, the last author should be \u201cMitsumi Terada\u201d and not \u201cMistumi Terada\u201d.The original article has been corrected."} +{"text": "The original version of this article unfortunately contained a mistake in the figures C and D of the following section \u201c3. Conditioning\u201d of the Fig.\u00a0The corrected Fig."} +{"text": "In an articleIn Figure 5, the label of \u201cDFB\u201d in Figure 5C should be modified to \u201cCCR2[R]\u201d, the correct figure is presented below.The authors apologize for the error."} +{"text": "In this article, the bars of \u201cDenture\u201d in Fig."} +{"text": "Following the publication of the original article the authp\u2009<\u20090.0009)\u201d in the \u201cResults\u201d section of the \u201cAbstract\u201d\u201cDiagnostic yield was significantly higher among fetal samples than postnatal samples \u201d in the \u201cResults\u201d section of the main text.\u201cDiagnostic yield was significantly higher among fetal samples compared to postnatal samples \u201d.The correct information is the one reported in Figure\u00a02: \u201cDiagnostic yield was significantly higher among fetal samples than postnatal samples (38.7%, n\u2009=\u2009176/455; z\u2009=\u20093.55, The original article has now been updated with the \u201cResults\u201d section of the \u201cAbstract\u201d and \u201cResults\u201d section of the main text having been corrected as per Figure\u00a02."} +{"text": "In the article by Oki E et al, entitled \u201cTrifluridine/tipiracil plus bevacizumab as a first\u2010line treatment for elderly patients with metastatic colorectal cancer (KSCC1602): A multicenter phase II trial,\u201d"} +{"text": "In the article titled \u201cHealth-Related Quality of Life and Sleep Quality after 12 Months of Treatment in Nonsevere Obstructive Sleep Apnea: A Randomized Clinical Trial with Continuous Positive Airway Pressure and Mandibular Advancement Splints\u201d [\u201cHypopnea events were defined as \u226550% reduction in respiratory flow lasting \u226510\u2009s, with a simultaneous \u22653% reduction in peripheral blood oxygen saturation from baseline\u201d should be corrected to \u201cHypopnea events were defined as \u226530% reduction in respiratory flow lasting \u226510\u2009s, with a simultaneous \u22653% reduction in peripheral blood oxygen saturation from baseline.\u201d"} +{"text": "An author name was incorrectly spelled as \u201cAntonio Florentino Leite.\u201d The correct spelling is \u201cAnt\u00f4nio Leite Florentino.\u201dFurther, in the original article, we neglected to include the funder \u201cS\u00e3o Paulo Research Foundation (FAPESP),\u201d \u201c2021/00463-7\u201d to \u201cJos\u00e9 Lavres.\u201dThe authors apologize for these errors and state that these do not change the scientific conclusions of the article in any way. The original article has been updated."} +{"text": "In Schneiders et al.,\u00a0The online version has been corrected."} +{"text": "After Claude Becker\u2019s name, there should have been a superscript number \u201c2\u201d to indicate that Drs. H\u00fcther and Becker are the authors for correspondence. The placement of the two superscript numbers was inadvertantly switched during production, so that a \u201c1\u201d appeared after Claude Becker\u2019s name and a \u201c2\u201d appeared after Niklas Schandry\u2019s name. The article has been updated to accurately reflect the acknowledgment and authors for correspondence. The Publisher apologizes for the error.In the published version of this article, a superscript number \u201c"} +{"text": "The first name of the sixth author was misspelled. It should be \u201cAkifumi\u201d instead of \u201cAkihumi,\u201d so it reads \u201cAkifumi Onagi.\u201dThe graph was missing in Figure 6C. The correct figure is presented below.In an articleThe authors apologize for the errors."} +{"text": "An author name was incorrectly spelled as \u201cVer\u00f3nica Marti\u00ednez-Cerde\u00f1o.\u201d The correct spelling is \u201cVer\u00f3nica Mart\u00ednez-Cerde\u00f1o.\u201dThe authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated."} +{"text": "In the original publication of the article, the author name was published incorrectly as \u201cMiho Stephanie Kitazawa\u201d. The correct name is \u201cMiho\u00a0S.\u00a0Kitazawa\u201d.The original article was updated."} +{"text": "This article has been corrected.1In the Original Investigation titled \u201cValidation of a Clinical Decision Rule to Predict Abuse in Young Children Based on Bruising Characteristics\u201d"} +{"text": "The co-author \u201cNathan Ng\u201d should have been included."} +{"text": "SMN2 Inclusion\u201d has been corrected to say \u201cReal-time PCR assay to assess SMN2 copy number\u201d. The corrected In the original article , there w"} +{"text": "Case Reports in Anesthesiology has retracted the article titled \u201cIntrathecal Pump Implantation in the Cisterna Magna for Treating Intractable Cancer Pain\u201d [er Pain\u201d due to c"} +{"text": "On page 384 of the originally published version of this manuscript, the sentence\u201cThis creates an effect size metric ranging from \u22121 to 1 with 1 and \u22121 denoting perfect association with no noise (i.e. perfect positive and negative association) and 0 denoting no relationship considering the error present\u201dshould have read\u201cThis creates an unstandardized effect size metric ranging from \u2212\u221e to \u221e with values farther from 0 denoting larger effect sizes and 0 denoting no relationship considering the error present.\u201dThese details have been corrected only in this corrigendum to preserve the published version of record."} +{"text": "Dr. Hanslmayr\u2019s email address is:"} +{"text": "VOLUME 295 (2020), PAGES 16156\u201316165The name of the fourth coauthor of this article was misspelled. It should read \u201cDana Kocincova\u201d and not \u201cDana Kocinkova.\u201d"} +{"text": "Following the publication of the original article , the autThe \u2018Funding\u2019 section now states: \u201cSBW has the ERAPERMED2019-310 grant\u2014Personalized Mitochondrial Medicine: Optimizing diagnostics and treatment for patients with mitochondrial diseases\u201d.The \u2018Competing interests\u2019 section now states: \u201cThe authors declare that they have no competing interests\u201d.The \u2018Funding\u2019 and \u2018Competing interests\u2019 sections have already been updated in the original article."} +{"text": "When this paper first published, an author name was incorrectly spelled as \u201cAsish Pun\u201d. This should have been \u201cAshis Pun\u201d. This error has now been corrected online."} +{"text": "The word \u201cdecrease\u201d should have appeared as \u201cincrease.\u201d This article has been corrected.1In the Original Investigation titled \u201cAssessment of Pediatric Admissions for Kawasaki Disease or Infectious Disease During the COVID-19 State of Emergency in Japan,\u201d"} +{"text": "Following publication of the original article , an erroThe font of the first and fifth lines currently read: LThe font should read:\u00a0Furthermore an error was identified in formula (7).The formula currently read:\u00a0The formula should read\u00a0The original article has been"} +{"text": "M\u00f4noca Josiane Rodrigues-Jesus\u201d. The correct spelling is \u201cM\u00f4nica Josiane Rodrigues-Jesus\u201d.An author name was incorrectly spelled as \u201cThe authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated."} +{"text": "In the article titled \u201cRepurposing Napabucasin as an Antimicrobial Agent against Oral Streptococcal Biofilms\u201d , the aut"} +{"text": "The corrected Page 2224. The experimentalrate constant from ref 11 for the Diels\u2013Alderreaction of 1 was incorrectly described as \u201c440times faster\u201d; it should read \u201c24 times faster\u201d.Inaddition, on page 2224 the reactivity difference of TCO andBCN toward diene"} +{"text": "Following publication of the original article , Fig.\u00a05b"} +{"text": "The first author\u2019s name is spelled incorrectly. The correct name is: Olivier Harl\u00e9."} +{"text": "Due to a\u00a0production\u00a0error, there was an error in the Article Title. The final \u201cs\u201d of \u201cChallenges\u201d was missed from the end of the title.The publisher apologizes for this mistake.\u00a0The original version of this article has been updated."} +{"text": "TO THE EDITORWe would like to share with you our thoughts on \u201cFirst Admission Neutrophil\u2013Lymphocyte Ratio May Indicate Acute Prognosis of Ischemic Stroke.\u201d1In the conclusion of their abstract, Alpua et al. state that \u201cfirst admission NLR [neutrophil\u2013lymphocyte ratio] can be used for acute prognosis of ischemic stroke.\u201d3"} +{"text": "The authors have provided a corrected version here.\u03c1A for two configurations: melanin ghosts and film with hr = 0.\u201d should be corrected to \u201cWe repeated the simulations for the aforementioned values of \u03c1A for two configurations: melanin ghosts and film with hr = 1.\u201d.As a result, in the third paragraph of the subsection \u201cSpatial arrangement of melanin affects shielding effectiveness\u201d in the \u201cResults\u201d, the phrase reading \u201cWe repeated the simulations for the aforementioned values of"} +{"text": "In the Figure 3 legend, \u201cthe second from right\u201d should be revised as \u201cthe second from left\u201d."} +{"text": "In Al\u2010Qadi MM,n\u00a0=\u00a034).\u201dIn the bottom slab, \u201cIncluded,\u201d box \u201cStudies included in the qualitative synthesis (The text was incorrect and should have read:n\u00a0=\u00a034).Studies included in the analysis (The corrected Figure 1 is below:The author apologizes for this error."} +{"text": "The original version of this article was published with a mistake in one of the authors\u2019 names. \u201cAbdallah Musa Abdallah\u201d is the correct full name but this was initially published as just \u201cAbdallah Musa\u201d. This has now been corrected."} +{"text": "The same URL in the \u201cData and code availability\u201d section has been amended as well.Within the \u201cKey Resources Table\u201d the link for the \u201cSoftware, algorithm\u201d entry has been changed from"} +{"text": "The above article [1] published with an error in one of the headings of Table\u00a01.The second heading should not read \u201cEvaluation.\u201d The correct heading is \u201cWorkforce development.\u201dThe publisher apologizes for this error."} +{"text": "In the article titled \u201cRole of Oxidative Stress in Pathophysiology of Nonalcoholic Fatty Liver Disease\u201d , there w\u201cC oxidase (VI complex)\u201d should be corrected to \u201cC oxidase (IV complex).\u201dThe authors apologize for the error."} +{"text": "Coptis chinensis Franchin Polycystic Ovary Syndrome\u201d [In the article titled \u201cNetwork Pharmacology-Based Strategy for Predicting Active Ingredients and Potential Targets of yndrome\u201d , there aCoptis chinensis has a therapeutic effect on polycystic ovary syndrome, suggesting that it could be an alternative choice for polycystic ovary syndrome.\u201dIn the Abstract section, \u201cThese results indicated that WZYZP has a protective effect on spermatogenesis disorder, suggesting that it could be an alternative choice for male infertility therapy\u201d should be corrected to \u201cThese results indicated that Coptis chinensis.\u201dIn Section 2.9, \u201cWZYZP\u201d should be corrected to \u201cCoptis chinensis.\u201dIn the legend of Figure 7, \u201cWZYZP\u201d should be corrected to \u201cThe authors apologise for these errors and confirm that this does not affect the results or conclusions of the article."} +{"text": "In the article by Tozlu et\u00a0al. incorrecThe authors apologize for the error."} +{"text": "In Shi\u2010Bin Jiang et al."} +{"text": "We would like to share our ideas on the paper \u201cEvaluating Voice Assistants\u2019 Responses to COVID-19 Vaccination in Portuguese: Quality Assessment\u201d . In addiSer\u00f3dio Figueiredo et al assessed"} +{"text": "Donghong Ji\u2019s email address is:"} +{"text": "In the article titled, \u201cGli1+ Cells Residing in Bone Sutures Respond to Mechanical Force via IP3R to Mediate Osteogenesis\u201d ["} +{"text": "The article by Brevik et\u00a0al.\u00a0 was publThe author apologizes for the error."} +{"text": "In the original publication of the article, one of the author name was published incorrectly as \u201cJ\u00e9rome Lechien\u201d. The correct name is \u201cJ\u00e9rome R. Lechien\u201d.The original article was updated."} +{"text": "In the original publication of the article, corresponding author\u2019s last name was published incorrectly as \u201cZitttermann\u201d. The correct name is \u201cArmin Zittermann\u201d.The original article has been corrected."} +{"text": "In the article \u201cExposure to Environmental Tobacco Smoke and Cognitive Abilities Among U.S. Children and Adolescents,\u201d"} +{"text": "Although its title attracted me to read this paper , I was q"} +{"text": "Following the publication of this article it was bThe sentence: \u201c\u2026children were five times more likely to be stunted at non-NIP sites compared to non-NIP sites\u201d should instead read: \u201c\u2026 children were five times more likely to be stunted at non-NIP sites compared to NIP sites\u201d."} +{"text": "In the article titled \u201cInterrelationship between Sleep and Exercise: A Systematic Review\u201d , there w"} +{"text": "In Table 2, row 2, all kcat and kcat/Km units should be \u201cmin\u22121\u201d and \u201cmin\u22121\u03bcM\u22121,\u201d respectively, rather than be \u201cs\u22121\u201d and \u201cs\u22121\u03bcM\u22121.\u201d The scientific interpretations remain the same, only the units change. Our conclusions about the active site structure based on the kinetic analysis of the mutants do not change.The original article unfortunately has an author error in the units for"} +{"text": "The numbers \u201c17020013\u201d are superimposed over"} +{"text": "Jixiong Xu's name appeared incorrectly as Jix Iong Xu.In the article, \u201cInsulin autoimmune syndrome in a pregnant female: A rare case report\u201d"} +{"text": "In the article titled \u201cRecent Advances in Microwave Imaging for Breast Cancer Detection\u201d , Figure"} +{"text": "In the article titled \u201cGene Expression Profiles of Human Phosphotyrosine Phosphatases Consequent to Th1 Polarisation and Effector Function\u201d ["} +{"text": "Discussion section, in the following sentence:\u201c[\u2026] highly absorptive and moisture retentive dressings such as BETAplast, are a welcomed addition to wound management armamentarium.\u201dThe original article contains an error in the DThe mention of \u2018BETAplast\u2019 in this sentence should instead be \u2018Medifoam\u00ae Silver\u2019."} +{"text": "These same errors were found in Data S1 (tab \u201cFly_diet aa ratios\u201d). Finally, the molar ratio values for the in\u00a0silico-translated exome of the \u201canimal\u201d in Data S1 did not contain the values for"} +{"text": "In the above-mentioned article, the fifth author\u2019s given name should read as \u2018Nayia\u2019 instead of \u2018Naiya\u2019. The corrected spelling is shown above."} +{"text": "In the article titled \u201cAutophagy Inhibition Enhances Apoptosis Induced by Dioscin in Huh7 Cells\u201d , there w"} +{"text": "The \u03b1-tubulin displayed in the bottom panel of In"} +{"text": "Vernonia amygdalina: A Comparative Study\u201d [In the article titled \u201cAntidiabetic Effect of Young and Old Ethanolic Leaf Extracts of e Study\u201d , there w"} +{"text": "Salvia miltiorrhiza Alcohol Extract on Oral Squamous Carcinoma Cells\u201d [In the article titled \u201cAnticancer Effects ofa Cells\u201d , the nam"} +{"text": "In the article titled \u201cManagement Challenges in a Child with Chronic Hyponatremia: Use of V2 Receptor Antagonist\u201d , the nam"} +{"text": "In the article titled \u201cRheumatological Findings in Candidates for Valvular Heart Surgery\u201d , the nam"} +{"text": "The original article containsThe co-lead author, Kassaw Amssalu Tadesse\u2019s name is incorrectly displayed as \u2018Kasahun Amsalu\u2019; this should instead be displayed as \u2018Kassaw Amssalu Tadesse\u2019."} +{"text": "Following publication of the original article it came \u2013 In the \u201cConsent for publication\u201d section the sentence \u201cAll co-authors and participants have given their consent for publication of this article in Lipids in Health and Disease\u201d should be replaced with \u201cNot applicable\u201d\u2013 The section titled \u201cCompeting of interests\u201d should be corrected to \u201cCompeting interests\u201d\u2013 The title of the section labeled \u201cAuthor\u2019 contributions\u201d should be changed to \u201cAuthors\u2019 contributions\u201d\u2013 The sentence \u201cNone of the authors reported a conflict of interest related to this study\u201d should be removed from the end of the \u201cAuthors\u2019 contributions\u201d section.The original article has been corrected."} +{"text": "The authors would like to add the following sentence to the \u201cAcknowledgments\u201d section of their article :\u201cThe authors thank Programa de Apoio ao Desenvolvimento Cient\u00edfico da Faculdade de Ci\u00eancias Farmac\u00eauticas da UNESP-PADC for their financial support\u201d."} +{"text": "In the original publication Fig.\u00a03 w"} +{"text": "Unfortunately, the original version of this article containeThe correct author list and the new \u201cAuthors\u2019 contributions\u201d section have been correctly included in this erratum."} +{"text": "The article has since been corrected online.In the article \u201cA \u201cBone Marrow Score\u201d for Predicting Hematological Disease in Immunocompetent Patients With Fevers of Unknown Origin\u201d,"} +{"text": "Upon publication of the original article , it was \u2018Francesca\u2019 and \u2018Sangiuliano\u2019 were tagged in the article XML as \u2018given names\u2019 and \u2018Intra\u2019 as the \u2018surname\u2019, whereas \u2018Francesca\u2019 should be the only \u2018first name\u2019 and \u2018Sangiuliano Intra\u2019 the \u2018surname\u2019.This means that the PubMed citation should be \u2018Sangiuliano Intra F\u2019 instead of \u2018Intra FS\u2019.This has now been corrected in this erratum."} +{"text": "Upon publication of the original article it was h\u201cIt is anticipated that there will be a total of 442 participants across 16 sites in this research study over a four year period. Of the 312, it is estimated that 120 will be adults with a primary diagnosis of BPD attending Adult Mental Health Services across eight study sites.......\u201dThe correct text should have \u201c312\u201d replaced with \u201c442\u201d so that it reads:\u201cIt is anticipated that there will be a total of 442 participants across 16 sites in this research study over a four year period. Of the 442, it is estimated that 120 will be adults with a primary diagnosis of BPD attending Adult Mental Health Services across eight study sites.......\u201d This has since been formally noted in this Correction article."} +{"text": "Fungal Biology and Biotechnology in October 2014 we stated that \u2018This is a new golden age of discovery in the fungi, and an exciting time to take part of this adventure\u2019 with given clinical names such as \u2018journal mania\u2019, \u2018IF mania\u2019 and \u2018impactitis\u2019\u201d . The Ame"} +{"text": "In the article titled \u201cEpigenetic and Neural Circuitry Landscape of Psychotherapeutic Interventions\u201d , there w"} +{"text": "I think you mean \u2018indicating the point at which aerobic metabolism is inadequate\u2026\u2019 (The word \u2018anaerobic\u2019 should be corrected to \u2018aerobic\u2019.)I read your article with interest. However, a sentence under the subheading \u2018Cardiopulmonary exercise testing\u2019 in the methods section ("} +{"text": "Unfortunately, the original version of this article containeTables\u00a0Additional file"} +{"text": "Artemisia annua L. Is a Negative Regulator of ABA Signaling\u201d [In the article titled \u201cType 2C Phosphatase 1 of"} +{"text": "In the original version of this article , publishZolt\u00e1n VargaOriginally the author name has been published as:Zolt\u00e1n V. VargaThe correct author name is:The original publication of this article has been corrected."} +{"text": "The original article contained a major omission whereby Tables\u00a0As such, the original article has now been updated to include these tables."} +{"text": "In Table 1, the final column\u2019s p values for \u201cSocial Contact\u201d and \u201cLiving With Someone\u201d are incorrect. Please see the corrected"} +{"text": "The corrected table is as follows:In the article titled \u201cThe Controversial C5a Receptor C5aR2: Its Role in Health and Disease\u201d , there w"} +{"text": "In the article titled \u201cAutomatic Segmentation of Ultrasound Tomography Image\u201d , the aff"} +{"text": "In the article titled \u201cSubdural Empyema Complicating Bacterial Meningitis: A Challenging Diagnosis in a Patient with Polysubstance Abuse\u201d , there w"} +{"text": "Scientific Reports6: Article number: 2716110.1038/srep27161; published online: 06032016; updated: 06302017This Article contains typographical errors in the Acknowledgements section.\u201cWe would like to thank to Ad\u00e9la Jirk\u016f, Lubo\u0161 Ma\u0165a\u0161 and Jan Hlad\u00edk from Bioster Company for all their efforts and assistance with the gamma irradiation of our samples\u201d.should read:\u201cWe would like to thank to Ad\u00e9la Jirk\u016f, Lubo\u0161 Ma\u0165a\u0161 and Josef Hlad\u00edk from Bioster Company for all their efforts and assistance with the gamma irradiation of our samples\u201d."} +{"text": "In the original publication of this article Table\u00a01"} +{"text": "In the article titled \u201cNonfamilial Juvenile Polyposis Syndrome with Exon 5 Novel Mutation in SMAD 4 Gene\u201d , there w"} +{"text": "The author wishes to make the following correction to this paper :TM\u201d should be replaced with \u201cclonoSEQ\u00ae\u201d.In figure 3, the name \u201cLymphoSIGHT"} +{"text": "In the Methods section, the second equation under the heading titled \u201cEstimating the distribution of time from infection to diagnosis (TID)\u201d should say \u201cmin\u201d instead of \u201cmax.\u201d Please view the complete, corrected equation here:"} +{"text": "The last author\u2019s name is spelled incorrectly. The correct name is: Juarez Ant\u00f4nio Sim\u00f5es Quaresma."} +{"text": "In the article titled \u201cPlant MicroRNA Prediction by Supervised Machine Learning Using C5.0 Decision Trees\u201d the name"} +{"text": "Polymorphic ventricular tachycardia can be a\u00a0detrimental consequence of coronary vasospasm . The pat"} +{"text": "In the article titled \u201cA Survey of Eyespot Sexual Dimorphism across Nymphalid Butterflies\u201d , there w"} +{"text": "The fourth author\u2019s name is spelled incorrectly. The correct name is: Nikolaus Gr\u00e4ber."} +{"text": "PLOS has received notice from McObject LLC that \u201cExtremeDB\u201d is a registered trademark of McObject LLC. The correct phrase in each instance in the paper should instead be \u201cExtremophileDB\u201d, and the correct database link is"} +{"text": "The publication by Dorji et al. , \u201cMicros"} +{"text": "Also, the reference citations throughout the Supplemental Information PDF were incorrectly numbered as \u201c[S?]\u201d instead of \u201c[S1],\u201d \u201c[S2],\u201d etc. The PDF has now been updated online to include the Supplemental References list, and to correct the reference citations throughout. The authors apologize for the inconvenience."} +{"text": "Salmonella Anatum Infections Linked to Imported Hot Peppers \u2014 United States, May\u2013July 2016,\u201d on page 663, the footnote (\u00b6 ) at the bottom of the page should have read \u201cLouisiana (two)\u201d.In the report \u201cMultistate Outbreak of"} +{"text": "In the original publication the last\u201cSAEs that occur between trial entry and up to 28\u00a0days after completion of the study drug will be reported\u201d."} +{"text": "In addition, in the caption of Table 4, the word \u201cBVr\u201d should be replaced by \u201cBr\u201d and the word \u201cBVl\u201d should be replaced by \u201cBl\u201d. The correct versions of Algorithm 2 and Table After publication of the original article , the aut"} +{"text": "To the editor, Shanman\u2019s letter to the editor is absolutely correct to point out that phrases can be truncated when searching in PubMed. In our article, we wanted to depict, with a simplified search strategy, how it is not straightforward to translate a strategy from MEDLINE Ovid into PubMed syntax. It is important to note that the main focus of our article was not performance of truncation in PubMed; rather, we investigated whether supplementary searches of PubMed improved the currency of MEDLINE Ovid.).ti,ab.The actual MEDLINE Ovid search strategy used in practice had more synonyms and was designed to capture the terms \u201cprostatic\u201d as well as \u201cprostate\u201d:#1 \u201cprostate cancer\u201d[tiab] OR \u201cprostate cancers\u201d[tiab] OR \u201cprostate cancerous\u201d[tiab] OR \u201cprostate carcinoma\u201d[tiab] OR \u201cprostate carcinomas\u201d[tiab] OR \u201cprostate neoplasm\u201d[tiab] OR \u201cprostate neoplasms\u201d[tiab] OR \u201cprostate neoplasia\u201d[tiab] OR \u201cprostate tumor\u201d[tiab] OR \u201cprostate tumors\u201d[tiab] OR \u201cprostate tumour\u201d[tiab] OR \u201cprostate tumours\u201d[tiab] OR \u201cprostate malignant\u201d[tiab] OR \u201cprostate malignancy\u201d[tiab] OR \u201cprostate malignancies\u201d[tiab] OR \u201cprostate adenocarcinoma\u201d[tiab] OR \u201cprostate adenocarcinomas\u201d[tiab] OR \u201cprostate adenoma\u201d[tiab] OR \u201cprostate adenomas\u201d[tiab]#2 \u201cprostatic cancer\u201d[tiab] OR \u201cprostatic cancers\u201d[tiab] OR \u201cprostatic cancerous\u201d[tiab] OR \u201cprostatic carcinoma\u201d[tiab] OR \u201cprostatic carcinomas\u201d[tiab] OR \u201cprostatic neoplasm\u201d[tiab] OR \u201cprostatic neoplasms\u201d[tiab] OR \u201cprostatic neoplasia\u201d[tiab] OR \u201cprostatic tumor\u201d[tiab] OR \u201cprostatic tumors\u201d[tiab] OR \u201cprostatic tumour\u201d[tiab] OR \u201cprostatic tumours\u201d[tiab] OR \u201cprostatic malignant\u201d[tiab] OR \u201cprostatic malignancy\u201d[tiab] OR \u201cprostatic malignancies\u201d[tiab] OR \u201cprostatic adenocarcinoma\u201d[tiab] OR \u201cprostatic adenocarcinomas\u201d[tiab] OR \u201cprostatic adenoma\u201d[tiab] OR \u201cprostatic adenomas\u201d[tiab]#3 #1 or #2This was translated to run in PubMed as:With hindsight, we should have included prostat* in our concise example and described in detail how truncating more than one word in a phrase can have unexpected results in PubMed: neither prostat* cancer*[tiab] or \u201cprostat* cancer*\u201d[tiab] produce the expected results. Traditionally, we have used this method because of past experiences with unexpected results and issues with PubMed timing out. We have been overcautious with our use of quotation marks, as well as field tags, in order to disable PubMed\u2019s Automatic Term Mapping so as to have more control over how our searches are performed.prostate cancer*[tiab] OR prostate carcinoma*[tiab] OR prostate neoplas*[tiab] OR prostate tumor*[tiab] OR prostate tumour*[tiab] OR prostate malignan*[tiab] OR prostate adeno*[tiab] OR prostatic cancer*[tiab] OR prostatic carcinoma*[tiab] OR prostatic neoplas*[tiab] OR prostatic tumor*[tiab] OR prostatic tumour*[tiab] OR prostatic malignan*[tiab] OR prostatic adeno*[tiab]Taking on board the approach that Shanman suggested, a modified version of our search strategy might look like the following:Thank you for clarifying how phrase searching truncation is performed in PubMed. We will consider using this approach when designing search strategies for PubMed in the future.Since our investigations were completed, Ovid has introduced the \u201cEpub Ahead of Print\u201d segment to their MEDLINE suite. This raises the question of whether it is still necessary to conduct supplementary searches in PubMed to identify ahead-of-print articles, which would be worth investigating further."} +{"text": "BRD7 Forward 5\u2032\u2010GAGGCTGAGGTGTTCCAGAG\u20103\u2032BRD7 Reverse 5\u2032\u2010TCACCTGGAGTCACTTGCTG\u20103\u2032In Yoo Kim et\u00a0al. The authors confirm that there are no conflict of interests and wishes to apologize for any misunderstanding or inconvenience caused."} +{"text": "It read \u2018Unima'ki\u2019, but it should have read \u2018Unama'ki\u2019.The publisher apologises for this error."} +{"text": "Thank you for your interest in our manuscript. You are quite correct; it should be \u2018aerobic\u2019 rather than \u2018anaerobic\u2019. We apologise for any confusion caused."} +{"text": "In the article titled \u201cPrebiotics: A Novel Approach to Treat Hepatocellular Carcinoma\u201d , the sec"} +{"text": "The lines labeled \u201cvehicle\u201d and \u201ccAMP+VEGF\u201d are incorrectly switched in"} +{"text": "In Mi et\u00a0al. , the las"} +{"text": "The authors wish to make the following correction to their paper :The second author\u2019s name \u201cYufeng Zhou\u201d should be \u201cYunfeng Zhou\u201d.The authors would like to apologize for any inconvenience caused to the readers by this error."} +{"text": "In Orasanu et\u00a0al. , the las"} +{"text": "In the publication of this article , there aBMJ Case Reports has no value in the PubMed Indexed column\u2018 Should instead read: \u2018BMJ Case Reports in the PubMed Indexed column should have indicated \u201cYes\u201dbecause it is PubMed indexed\u20181. The error: \u2018The journal title BMJ Case Reports has value \u201cNo\u201d in the Open access column\u2018 Should instead read: \u2018BMJ Case Reports in the Open access column should have indicated \u201cOptional\u201d because it does have an option for open access for an extra fee\u20182. The error: \u2018The journal title This has now been included in this erratum."} +{"text": "The original version of this article unfortunIn Table\u00a0An updated version of Table"} +{"text": "After publication of the original article , the aut\u03c1\u201d when it should have been \u201cP\u201d. The correct version of Table\u00a0The title for the right-hand column of Table"} +{"text": "The term \u201cAkaike\u2019s Information Criterion\u201d is misspelled throughout the article. The correct term is \u201cAkaike\u2019s Information Criterion.\u201d"} +{"text": "We agree with Kennedy et al. that \u201cTC"} +{"text": "In the article titled \u201cOptimal Hemoglobin A1c Levels for Screening of Diabetes and Prediabetes in the Japanese Population\u201d ["} +{"text": "Page 1013: \u201c\u201d should read \u201c\u201dPage 1013: \u201c\u201d should read \u201c\u201dPage 1016: \u201cfml1 and pso2 showed a more accentuated sensitivity \u201d should read \u201cfml1 and pso2 showed a more accentuated sensitivity \u201dThe authors have mentioned the following corrections:The authors would also like to acknowledge Dr John Rouse for his contribution at the initial stage of the project: \u201cWe thank John Rouse for helpful advice on the project and for sharing unpublished results\u201d.The authors would like to apologise for any inconvenience caused."} +{"text": "Plasmodium and Helminth Coinfection and Possible Reasons for Heterogeneity,\u201d [In the article titled \u201cEpidemiology ofeneity,\u201d there we"} +{"text": "Dear Editor, The recent report on \u201chuman brucellosis\u201d is very interesting . Indeed,"} +{"text": "Thomas Bayes (1701\u20131761) was a Pr"} +{"text": "In the article titled \u201cPhysiologic Conditions Affect Toxicity of Ingested Industrial Fluoride\u201d , there w"} +{"text": "In the article titled \u201cPerillaldehyde Inhibits AHR Signaling and Activates NRF2 Antioxidant Pathway in Human Keratinocytes\u201d , there w"} +{"text": "Corrigendum:After publication the authors noticed that the corresponding author's surname was listed incorrectly. The author list and author contributions have been amended so that Pelayo Salinas de Le\u00f3n is now listed as Pelayo Salinas-de-Le\u00f3n."} +{"text": "I enjoyed Charles Schmidt\u2019s original and informative article on the \u201cYuck Factor\u201d . This ph"} +{"text": "There appears to be a resurgence of puerperal sepsis due to a historically important pathogen, group A \u03b2-hemolytic streptococcus."} +{"text": "The sixth author's name was incomplete. The correct name is: Valdil\u00e9a Gon\u00e7alves Veloso."} +{"text": "Sir,We read the article entitled\u201d Asymptomatic Meckel\u2019s diverticulum in adults: Is diverticulectomy indicated?\u201d with int"} +{"text": "Instead of \u201cMarc A. Weisskopf,\u201d it should be \u201cMarc G. Weisskopf.\u201dIn \u201cCumulative Exposure to Lead in Relation to Cognitive Function in Older Women\u201d The authors apologize for the error."} +{"text": "Throughout the text and figures all instances of the symbols \u201ct_ant_clear\u201d and \u201ct_syn_clear\u201d relating to Figure 4 and Figure S2 should instead read \u201cN_ant_double\u201d and \u201cN_syn_double\u201d respectively.The correct version of Figure 4 can be found here: The correct version of Figure S2 can be found here: Click here for additional data file."} +{"text": "To the Editor: I read the case report on \u2018Sheehan syndrome with reversible dilated cardiomyopaty\u20192"} +{"text": "Sir,I read the article \u201cTaare Zameen Par and dyslexic savants\u201d by Ambar"} +{"text": "Dear Editor,I was interested to read comments by Gopal about \u201cE"} +{"text": "The following information was missing from the Funding section: This study was funded by INSERM and Universit\u00e9 Fran\u00e7ois Rabelais de Tours."} +{"text": "There was an error in the author's name in the copyright statement of the article. His name should appear as Roberto Fern\u00e1ndez Gal\u00e1n."} +{"text": "We thank M\u00fcller et al. for their interest in our article and conc"} +{"text": "In the article titled \u201cLow Citrate Synthase Activity Is Associated with Glucose Intolerance and Lipotoxicity\u201d , there w"} +{"text": "The seventh author\u2019s name is spelled incorrectly. The correct name is: Dino Kr\u00f6ll."} +{"text": "In the article titled \u201cFrom Localized Scleroderma to Systemic Sclerosis: Coexistence or Possible Evolution\u201d , the fir"} +{"text": "Xenopus tropicalis Immature Sertoli Cells\u201d [In the article titled \u201cEpithelial-Mesenchymal Transition Promotes the Differentiation Potential of i Cells\u201d , due to"} +{"text": "In the original article mentioned above, the name of the sixth author was wrongly mentioned as \u201cVincenzo Briganti\u201d instead of \u201cVito Briganti\u201d.The original article has been corrected."} +{"text": "Porphyromonas gingivalis and Tannerella forsythia Stimulates an Immune Response but Not Arthritis in Experimental Murine Model\u201d [In the article titled \u201cSubchronic Infection of e Model\u201d , there w"} +{"text": "In the article titled \u201cProresolving Lipid Mediators: Endogenous Modulators of Oxidative Stress\u201d , there w"} +{"text": "The twenty-first author\u2019s name is spelled incorrectly. The correct name is: Gl\u00e1ucio Ricardo Werner de Castro."} +{"text": "Ginkgo biloba and Magnetized Water on Nephropathy in Induced Type 2 Diabetes in Rat\u201d [In the article titled \u201cProtective Effect of in Rat\u201d , there w"} +{"text": "Withania somnifera on Reproductive System: A Systematic Review of the Available Evidence [In the article titled \u201cEffects of Evidence ,\u201d there In the Results section, the sentence \u201cOf 459 recognized studies, 42 studies were included in the present study\u201d should be \u201cOf 190 recognized studies, 42 studies were included in the present study\u201d.n\u2009=\u2009114)\u201d should be \u201cRecords after duplicates removed (n\u2009=\u2009144).\u201d The corrected In"} +{"text": "In the published article, there was an error regarding the affiliation for \u201cJulio C. De Rose.\u201d Instead of affiliation \u201c3\u201d it should have been affiliation \u201c1.\u201dThe authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated."} +{"text": "The name of the third author \u201cPascal Kouyoumjian\u201d has been corrected to \u201cPascalKouyoumdjian\u201d. The publisher apologizes for any inconveniences."} +{"text": "Pseudomonas Pneumonia Linked to Use of a Home Humidifier\u201d [In the article titled \u201cCommunity-Acquired Cavitary idifier\u201d , the nam"} +{"text": "In the article titled \u201cMyoinositol: The Bridge (PONTI) to Reach a Healthy Pregnancy\u201d , the \u201cCo"} +{"text": "Dear Editor, we read the publication on \u201cresponsible conduct of research\u201d with great interest"} +{"text": "There are errors in the \u201cHemoglobin (mmol/L)\u201d values in"} +{"text": "Fraxinus rhynchophylla Hance Extract in a Mouse Model of Chronic Stress-Induced Depression\u201d [In the article titled \u201cAntidepressant Effect ofression\u201d , an inco"} +{"text": "An author name was incorrectly spelled as \u201cPeter Schoenheit.\u201d The correct spelling is \u201cPeter Sch\u00f6nheit.\u201dThe authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated."} +{"text": "Results from the CRATF study\u201d,"} +{"text": "In the article titled \u201cPregabalin Effect on Acute and Chronic Pain after Cardiac Surgery\u201d , the pre"} +{"text": "In the original publication of this article Fig.\u00a02A is incorrectly published. The correct version of Fig.\u00a0Under the section \u201cThe rationale for the investigation of CAR IS\u201d the line starting with \u201cthe surface of an tumour cell or infected target cell\u201d is incorrect, it should be read as \u201cthe surface of a tumour cell or infected target cell\u201d.Under the section of \u201cCOMPLIANCE WITH ETHICS GUIDELINES\u201d the line starting with \u201cThis article does not contain any studies\u201d is incorrect, it should be read as \u201cThis article does not contain any studies with human or animal subjects performed by any of the authors\u201d.Under the section \u201cDevelopment of \u201coff-the-shelf\u201d NK cell products\u201d the line starting with \u201cThe CAR-modified NK92 cell line may\u201d is incorrect, it should be read as \u201cThe CAR-modified NK92 cell line may serve as a future \u201coff-the-shelf\u201d.Under the section \u201cThe background of IS\u201d the line starting with \u201cbetween peripheral blood NK cells in the YTS cell line and various transfectants\u201d is incorrect, it should be read as \u201cbetween peripheral blood NK cells and various transfectants\u201d."} +{"text": "In the article titled \u201cSuccessful Pelvic Resection for Acetabular Hydatidosis\u201d , there w"} +{"text": "The ninth author name was incorrectly published in the original publication. The correct name should read as \u2018C\u00e9dric Poyet\u2019."} +{"text": "UCP1 Gene in a Severe Obese Population from Southern Italy\u201d [In the article titled \u201cSequence Analysis of the"} +{"text": "The original article [1] contained an error whereby the leftmost graph in Fig. 1a mistakenly had its x-axis labelled as \u2018CFST\u2019; this has now been corrected to \u2018CFSE\u2019."} +{"text": "The Author Affiliations and Author Contributions sections incorrectly listed the last name of Henrik Toft S\u00f8rensen, MD, DMSc, PhD, as \u201cToft S\u00f8rensen.\u201d The proper listing is \u201cS\u00f8rensen.\u201d This article has been corrected.1In the Original Investigation titled \u201cCardiovascular Disease Among Women Who Gave Birth to an Infant With a Major Congenital Anomaly,\u201d"} +{"text": "The authors would like to update the \u201cAcknowledgements\u201d section of their previous paper as follo"} +{"text": "Supplementary Material Data Sheet 1 as published. In the tab \u201cAustralia\u201d the value of cell M191 should be \u201c0\u201d instead of \u201c1\u201d and that of cell N191 should be \u201c1\u201d instead of \u201c0.\u201d The corrected Supplementary Material Data Sheet 1 has been replaced in the original article.In the original article, there was a mistake in In addition, there was a mistake in The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated."} +{"text": "In the article titled \u201cEpac1 Restores Normal Insulin Signaling through a Reduction in Inflammatory Cytokines\u201d ["} +{"text": "There is an error in the Funding statement. The correct number for Funda\u00e7\u00e3o de Amparo a Pesquisa do Estado de S\u00e3o Paulo (FAPESP) is \u201c2008/03969-4.\u201dThe authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated."} +{"text": "Pontogammarus maeoticus,\u201d which was published in volume 8 issue 19, October 2018, the scales for panels a and b are missing in Figure\u00a0In \u201cAre Ponto\u2010Caspian species able to cross salinity barriers? A case study of the gammarid"} +{"text": "The arrow between \u201cIrma-related media exposure\u201d and \u201cWave 2 adjustment,\u201d as well as the arrow between \u201cDegree of hurricane exposure\u201d and \u201cWave 2 adjustment,\u201d were previously pointing away from \u201cWave 2 adjustment\u201d and are now pointing toward. This article has been corrected online.In the article titled \u201cMedia Coverage, Forecasted Posttraumatic Stress Symptoms, and Psychological Responses Before and After an Approaching Hurricane,\u201d"} +{"text": "Due to a production error it was indicated that a safe therapeutic concentration of lithium was \u201c1 mol/L\u201d in the second sentence of the Introduction. The correct measurement is \u201c1 mmol/L.\u201dThe publisher apologizes for this mistake. The original version of this article has been updated."} +{"text": "In the article titled \u201cMitochondrial Serine Protease HTRA2 p.G399S in a Female with Di George Syndrome and Parkinson's Disease\u201d , the nam"} +{"text": "In this paperHere are Tables\u00a0We apologize for these errors."} +{"text": "In the article titled \u201cPrognostic Effect of Long Noncoding RNA NEAT1 Expression Depends on p53 Mutation Status in Cancer\u201d ["} +{"text": "Oncorhynchus mykiss) Eggs Subjected to the High Hydrostatic Pressure Treatment\u201d [In the article titled \u201cTranscriptome Analysis of Rainbow Trout (eatment\u201d , there w"} +{"text": "In the article titled \u201cThe Role of Parathyroid Hormone and Vitamin D Serum Concentrations in Patients with Cardiovascular Diseases\u201d , the \u201c\u2264\u201d"} +{"text": "In the article titled \u201cTyrosine Kinase Receptor Landscape in Lung Cancer: Therapeutical Implications\u201d , there w"} +{"text": "In the article titled \u201cImunocompetent Mice Model for Dengue Virus Infection\u201d , there w"} +{"text": "IL\u20101\u03b1 cleavage by inflammatory caspases of the noncanonical inflammasome controls the senescence\u2010associated secretory phenotype. Aging Cell. 2019; 18:e12946. In the article \u201cIL\u20101\u03b1 cleavage by inflammatory caspases of the noncanonical inflammasome controls the senescence\u2010associated secretory phenotype,\u201d a duplication of the image in Figure We apologize for the inconvenience caused."} +{"text": "In the article titled \u201cThe Role of Medications in Causing Dry Eye\u201d , there w"} +{"text": "In the article titled \u201cFormation of Silver Nanoclusters from a DNA Template Containing Ag(I)-Mediated Base Pairs\u201d , there w"} +{"text": "In both eTable 7a and 7b, the left column head should have read \u201cLumpectomy alone\u201d and the right, \u201cLumpectomy and radiation.\u201d The headers were inadvertently switched. This article has been corrected.1In the Original Investigation titled \u201cAssociation of Radiotherapy With Survival in Women Treated for Ductal Carcinoma In Situ With Lumpectomy or Mastectomy,\u201d"} +{"text": "Aster glehni Extract: In Vivo and In Vitro Effects\u201d [In the article titled \u201cAntiadipogenic Effects ofEffects\u201d , the nam"} +{"text": "The cyan line should have indicated \u201c0 adverse childhood experiences\u201d and the navy line should have indicated \u201c\u22653 adverse childhood experiences.\u201d This article has been corrected.1In the Original Investigation titled \u201cAssociation of Childhood Adversity With Differential Susceptibility of Transdiagnostic Psychopathology to Environmental Stress in Adulthood,\u201d"} +{"text": "In the article titled \u201cA Case of Bing\u2013Neel Syndrome Successfully Treated with Ibrutinib\u201d , the nam"} +{"text": "In the article titled \u201cEffect of Lead on Human Middle Ear Epithelial Cells\u201d , there w"} +{"text": "MMWR Editors were informed by the authors of \u201cSuicide Rates by Occupational Group \u2014 17 States, 2012\u201d (Recently,"} +{"text": "An author name was incorrectly spelled as \u201cRinc\u00f3n AFC\u201d. The correct spelling is \u201cCarrillo Rinc\u00f3n AF\u201d.The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated."} +{"text": "The second sentence under 5. Conclusions is missing the word \u201cnot\u201d and should read \u201c\u201cThose survival differences are predominantly observed among patients not undergoing LAD with left sided disease having worse CSS compared to patients with right sided tumor.\u201dIn the article, \u201cTumor laterality in renal cancer as a predictor of survival in large patient cohorts: A STROBE compliant study\u201d,"} +{"text": "In the article titled \u201cNonpharmacologic Interventions in Prevention and Treatment of Hypertension\u201d , there w"} +{"text": "In Wigner\u00a0et\u00a0al,The authors wished to apologize for any misunderstanding or inconvenience this may have cause."} +{"text": "In Tang et\u00a0al,The authors wished to apologize for any misunderstanding or inconvenience this may have caused."} +{"text": "In the article titled \u201cCFTR Expression Analysis for Subtyping of Human Pancreatic Cancer Organoids\u201d , there w"} +{"text": "In Peng et\u00a0al,The authors apologize for the inconvenience this may cause."} +{"text": "This article is published with the incorrect copyright holder name in the HTML article as \u201c\u00a9 Springer 2018\u201d. The correct copyright line should read \u201cThe Author(s) 2018\u201d (as it appears in the article PDF)."} +{"text": "The navy line should indicate \u201c1% \u2264 ER < 25%,\u201d and the tan line should indicate \u201c75% \u2264 ER \u2264 100%.\u201d This article has been corrected.1In the Original Investigation titled \u201cArtificial Intelligence Algorithms to Assess Hormonal Status From Tissue Microarrays in Patients With Breast Cancer,\u201d"} +{"text": "Following publication of the original article , the autNamely, in the \u2018America\u2019 section of the table the species \u2018Crested Porcupine\u2019 is matched with the country \u2018Brazil\u2019.However, it should be matched with \u2018Bosnia and Herzegovina\u2019 (and so be placed in the \u2018Europe\u2019 section).The corrected\u00a0Table"} +{"text": "Dr. Schroeder\u2019s email address is"} +{"text": "There is an error in the Methods section under the sub-heading \u201cStudy subjects.\u201d The first sentence of the second paragraph should read: This study was reviewed and approved by the Institutional Ethical Committee \u201cComitato di Etica dell\u2019Universit\u00e0 degli Studi di Parma.\u201d"} +{"text": "In the article titled \u201cInositol and In Vitro Fertilization with Embryo Transfer\u201d , the \u201cCo\u201cPublication was sponsored by Studio Medico Ass. Agunco.\u201d"} +{"text": "In the article titled \u201cSITbench 1.0: A Novel Switch-Based Interaction Technique Benchmark\u201d [\u201cOn the other hand, a benchmark application is also required to make performance evaluation of SITs by using a number of standard tests and empirical attributes.\u201d"} +{"text": "The original version of this article unfortunately contained a mistake in Fig.\u00a03 part labels, the label \u201cd\u201d was incorrectly labelled as \u201cc\u201d and the subsequent labels should be corrected as d, e, and f. The corrected Fig.\u00a0The original article has been corrected."} +{"text": "In Vivo Biocompatibility of PLGA-Polyhexylthiophene Nanofiber Scaffolds in a Rat Model\u201d [In the article titled \u201ct Model\u201d , an inco"} +{"text": "The author requests to correct errors in the original article . Area wh1. In conclusion section of Abstract:\u201cThis study confirms that MUAC can be used for both admitting and discharging criteria in CMAM programs with MUAC <\u2009115 mm for admission and MUAC >\u2009125 mm or at discharge (a higher discharge threshold could be used).\u201d Replaces\u201cThis study confirms that MUAC can be used for both admitting and discharging criteria in CMAM programs with MUAC <\u2009115 mm for admission and MUAC >\u2009=\u2009115 mm or at discharge (a higher discharge threshold could be used).\u201dAll \u201cMUAC >\u2009115 mm\u201d should be changed into \u201cMUAC > 125 mm\u201d under each of the discharge criteria column.Superscript \u201ca\u201d above the \u201cwell\u201d should be removed.The legend under the table should be removed.2. In Table\u00a01:"} +{"text": "In the article titled \u201cPulmonary Hypertension Secondary to Partial Anomalous Pulmonary Venous Return in an Elderly\u201d , there w"} +{"text": "An author name was incorrectly spelled as \u201cAlizae Abbas.\u201d The correct spelling is \u201cAlizeh Abbas.\u201d Additionally, the author name \u201cZain Y. Khan\u201d was incorrectly spelled. The correct spelling is \u201cZain Yar Khan.\u201dThe authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated."} +{"text": "The percentages for the \u201cNon-Saiga Users\u201d group are incorrect in"} +{"text": "In the Abstract, the words \u201cLogistic regression and\u201d in the last sentence of \u201cMethods\u201d section should have been removed so it reads \u201cKaplan\u2013Meier analysis results are reported.\u201dCOMT Val158Met genotype\u201d to \u201cTime to age of diagnosis of psychosis by gender.\u201dIn Figure In Lodhi et al. , the folThe correct Figure"} +{"text": "Burkholderia sp. VITRSB1 Isolated from Marine Sediments\u201d [In the article titled \u201cBiodegradation of PAHs by"} +{"text": "Chrysochromulina parva virus BQ2 labeled as \u201cPhaeocystis globosa virus (group II)\u201d should have been labeled \u201cPhaeocystis globosa virus (group I).\u201d Similarly, the two branches labeled as \u201cPhaeocystis globosa virus (group I)\u201d and \u201cPhaeocystis globosa virus\u201d adjacent to Chrysochromulina parva virus BQ1 should have both been labeled as \u201cPhaeocystis globosa virus (group II).\u201dIn the original article, there was a mistake in The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated."} +{"text": "Two author names were incorrectly spelled as \u201cPardis C. Sarbeti\u201d and \u201cEkene Muebonam\u201d. The correct spelling is \u201cPardis C. Sabeti\u201d and \u201cEkene Muoebonam\u201d.The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated."} +{"text": "In the article titled \u201cPolymorphisms in the Chicken Growth Differentiation Factor 9 Gene Associated with Reproductive Traits\u201d , there w"} +{"text": "In the article titled \u201cECG Markers of Hemodynamic Improvement in Patients with Pulmonary Hypertension\u201d , there w"} +{"text": "Footnote a for both tables should have read \u201cCigar use was categorized as 0 for never used and 1 for ever used\u2026\u201d instead of \u201cBlunt use was categorized as 0 for never and 1 for ever used...\u201d This article has been corrected.1In the Original Investigation titled \u201cAssociation Between Adolescent Blunt Use and the Uptake of Cigars,\u201d"} +{"text": "The article titled \u201cPlants as Useful Vectors to Reduce Environmental Toxic Arsenic Content\u201d was foun"} +{"text": "The text reading \u201c181 SUDC with seizure history\u201d should have read \u201c181 SUDC without seizure history.\u201d This article has been corrected.1In the Original Investigation titled \u201cPotential Role of Febrile Seizures and Other Risk Factors Associated With Sudden Deaths in Children,\u201d"} +{"text": "In the article titled \u201cSpontaneous Resolution of Symptomatic Hepatic Sarcoidosis\u201d , the nam"} +{"text": "The x-axis labels for panels g and h of"} diff --git a/PMC_clustering_472.jsonl b/PMC_clustering_472.jsonl new file mode 100644 index 0000000000000000000000000000000000000000..ce01c5d504c360edb04bd68a7461a7c8e8f68381 --- /dev/null +++ b/PMC_clustering_472.jsonl @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:4d0fc3f0f2e6309382b2222a88435603556f68403e90fe73385027269947eda6 +size 61221975 diff --git a/PMC_clustering_473.jsonl b/PMC_clustering_473.jsonl new file mode 100644 index 0000000000000000000000000000000000000000..bbb9edd22e67c0d71062b1873fbaa3176bcc1be3 --- /dev/null +++ b/PMC_clustering_473.jsonl @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:51af382f877ccf285884fc0ea7a5b8913fc4b2e2a629e97de3bd97872617a884 +size 44709270 diff --git a/PMC_clustering_474.jsonl b/PMC_clustering_474.jsonl new file mode 100644 index 0000000000000000000000000000000000000000..e08e2f124548dfee5c593d1702a5f8b88e950a6f --- /dev/null +++ b/PMC_clustering_474.jsonl @@ -0,0 +1,1457 @@ +{"text": "A 17-year-old adolescent with no medical history; documented to have a mild SARS-CoV-2 infection ; had chilblains-like lesions on the toes and asymOur patient had presented chilblains-like lesions and acral purpura concomitantly, followed few days later by maculopapular lesions with targetoid lesions reminiscent of erythema multiforme. Same presentations were reported: 02 cases with chilblains-like lesions evolving to erythemato-papular targetoid lesions ; maculop"} +{"text": "Scientific Reports, 10.1038/s41598-020-64125-x, published online 29 April 2020Correction to: This Article contains errors. The authors reports that samples were collected from allantoic fluid; they were collected from fecal samples. The authors also reported incorrect biosafety level for the facilities where the experiments were performed.In Materials and Methods, \u2018Sample sites and sample collection\u2019,\u201cAfter HA activity testing, viral RNA was extracted from the HA-positive allantoic fluid using QIAamp Viral RNA Mini Kit . Approval for this study was obtained from the Institutional Animal Care and Use Committee at Chungcheongbuk-do Veterinary Service Laboratory (#2019-2) and all experimental procedures from virus isolation to sequencing library preparation were conducted in approved biosafety level 3 (BSL3) facilities (KCDC-12-3-04) at Chungbuk Veterinary Service Laboratory.\u201dshould read:\u201cAfter HA activity testing, viral RNA was extracted from the HA-positive fecal samples using QIAamp Viral RNA Mini Kit . Approval for this study was obtained from the Institutional Animal Care and Use Committee at Chungcheongbuk-do Veterinary Service Laboratory (#2019-2) and all experimental procedures from virus isolation to sequencing library preparation were conducted in approved biosafety level 2 (BSL2) facilities (QIA-GA2-16-015) at Chungbuk Veterinary Service Laboratory joongbu.\u201d"} +{"text": "Dear Editor,Malaysian Journal of Medical Sciences (MJMS)\u2019 performance status in 2018 , followed by Turkey (five manuscripts), Saudi Arabia (five manuscripts) and Iraq (one manuscript) . As the As elegantly stated on the journal\u2019s introductory web page, MJMS accepts high-quality papers especially from low- and middle-income countries . Iran (u"} +{"text": "Genome-wide chromosome conformation capture based on high-throughput sequencing (Hi-C) has been widely adopted to study chromatin architecture by generating datasets of ever-increasing complexity and size. HiCBricks offers user-friendly and efficient solutions for handling large high-resolution Hi-C datasets. The package provides an R/Bioconductor framework with the bricks to build more complex data analysis pipelines and algorithms. HiCBricks already incorporates functions for calling domain boundaries and functions for high-quality data visualization.http://bioconductor.org/packages/devel/bioc/html/HiCBricks.html.Bioinformatics online. High-throughput sequencing (Hi-C) allows probing physical proximity between potentially any pair of genomic loci and has been widely adopted to characterize chromatin structure and function originally proposed by . These fHiCBricks provides a framework useful for bioinformaticians who aim to build a custom analysis procedure for Hi-C data, or need to integrate Hi-C data analysis into pre-existing R/Bioconductor pipelines. On the other hand, biologists with specific questions that can\u2019t be addressed by standard tools will find HiCBricks functionalities useful to perform targeted statistical analyses. HiCBricks enables easy assembling of custom pipelines, going from Hi-C matrices to rich graphical output plots.AIRC Start-up grant 2015 [16841 to F.F.] and AIRC fellowship [21012 to K.P.]Conflict of Interest: none declared.btz808_Supplementary_DataClick here for additional data file."} +{"text": "Adjustment of cerebral blood flow (CBF) to the increased oxygen and nutrient demands of active brain regions via neurovascular coupling (NVC) has an essential role in maintenance of healthy cognitive function. In advanced age, cerebromicrovascular oxidative stress and endothelial dysfunction impair neurovascular coupling, contributing to age-related cognitive decline. Recently we developed a resveratrol -containing fusogenic liposome (FL-RSV)-based molecular delivery system that can effectively target cultured cerebromicrovascular endothelial cells, attenuating age-related oxidative stress. To assess the cerebromicrovascular protective effects of FL-RSV in vivo, aged (24-monthold) C57BL/6 mice were treated with FL-RSV for four days. To demonstrate effective cellular uptake of FL-RSV, accumulation of the lipophilic tracer dyes in cells of the neurovascular unit was confirmed using two-photon imaging . NVC was assessed by measuring CBF responses (laser speckle contrast imaging) evoked by contralateral whisker stimulation. We found that NVC responses were significantly impaired in aged mice. Treatment with FL-RSV significantly improved NVC responses by increasing NO-mediated vasodilation. These findings are paralleled by the protective effects of FL-RSV on endothelium-dependent relaxation in the aorta. Thus, treatment with FL-RSV rescues endothelial function and NVC responses in aged mice. We propose that resveratrol containing fusogenic liposomes could also be used for combined delivery of various anti-geronic factors, including proteins, small molecules, DNA vectors and mRNAs targeting key pathways involved in microvascular aging and neurovascular dysfunction for the prevention/treatment of age-related cerebromicrovascular pathologies and development of vascular cognitive impairment (VCI) in aging."} +{"text": "A 49-year-old prior healthy male without any cardiovascular risk factors living in the western part of Switzerland, noted in mid of March 2020 anosmia and dysgeusia, similar to his wife and four people from his close family, with whom he had frequent contact and who were positive for SARS-CoV-2. Six weeks later, he presented to the hospital with new-onset of dyspnea NYHA 3, general weakness, intermittent epigastrical pain and nocturia without orthopnea nor fever. The retro-nasal SARS-CoV-2 PCR, 6 weeks after initial anosmia and dysgeusia was negative but the antibody IgG blood test for SARS-CoV-2 was positive. Computed tomography of the lungs showed no pulmonary embolism, no infiltrates but left heart congestion, suspected by previous thoracic X-ray (A) and pleural effusion (B). Echocardiography revealed diffuse hypokinesia with severely depressed left- and right-ventricular function. The patient showed elevated C-reactive protein, troponin and NT-proBNP. ECG showed dynamic T-wave changes (C) and after ruling out coronary artery disease, he was diagnosed with isolated peri-myocarditis using multiparametric cardiac magnetic resonance imaging (CMR). CMR showed diffuse thickening of the myocardium and pericardium due to edema confirmed with T2 weighted imaging and T2 mapping (D). Further, pericardial effusion could be seen and tissue characterization revealed diffuse LGE, elevated T1 mapping values and an elevated extracellular volume fraction of 38% , indicating diffuse fibrosis (E). Global myocardial strain of all heart chambers was diffusely impaired (F). No other cause was found as an underlying reason for the peri-myocarditis. In the clinical setting of suspected Covid-19 with respiratory symptoms and negative pulmonary imaging, elevated C-reactive protein and troponin should lead to the suspicion of isolated peri-myocarditis. CMR is the primary noninvasive imaging tool to assess peri-myocarditis, also in Covid-19 patients\u00a0["} +{"text": "Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe acute respiratory symptoms. Due to the lack of medical countermeasures, effective and safe vaccines against MERS-CoV infection are urgently required. Although different types of candidate vaccines have been developed, their immunogenicity is limited, and the dose and administration route need optimization to achieve optimal protection. We here investigated the potential use of human \u03b2-defensin 2 (HBD 2) as an adjuvant to enhance the protection provided by MERS-CoV vaccination. We found that immunization of human dipeptidyl peptidase 4 (hDPP4)-transgenic (hDPP4-Tg) mice with spike protein receptor-binding domain (S RBD) conjugated with HBD 2 (S RBD-HBD 2) induced potent antigen (Ag)-specific adaptive immune responses and protected against MERS-CoV infection. In addition, immunization with S RBD-HBD 2 alleviated progressive pulmonary fibrosis in the lungs of MERS-CoV-infected hDPP4-Tg mice and suppressed endoplasmic reticulum stress signaling activation upon viral infection. Compared to intramuscular administration, intranasal administration of S RBD-HBD 2 induced more potent mucosal IgA responses and was more effective for protecting against intranasal MERS-CoV infection. In conclusion, our findings suggest that HBD 2 potentiates Ag-specific immune responses against viral Ag and can be used as an adjuvant enhancing the immunogenicity of subunit vaccine candidates against MERS-CoV. Since 2000, numerous potentially lethal zoonotic human diseases have emerged due to novel coronaviruses, including severe acute respiratory syndrome coronavirus (SARS-CoV) in 2002, Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012, and SARS coronavirus 2 (SARS-CoV-2) in 2019. Owing to their pandemic potential, impact on global health, and lack of effective medical countermeasures, these coronaviruses have been included in the World Health Organization (WHO) Research and Development Blueprint list of priority diseases. The MERS-CoV outbreak in Korea in 2015 has caused significant morbidity and mortality, posing severe threats to public health and the economy [Most MERS-CoV vaccines under investigation involve inactivated virus, live attenuated virus, viral vector-based vaccines, recombinant virus subunits, and DNA vaccines . The mosHost defense peptides play important roles as primary gatekeepers protecting respiratory, oral, reproductive, and enteric tissues from various pathogens and maintaining tissue homeostasis ,11. AmonMERS-CoV infects the lower respiratory tract, leading to severe acute respiratory failure and progressive pulmonary fibrosis. Small experimental animals, such as mice, ferrets, and hamsters, are non-permissive for MERS-CoV infection due to structural differences in DPP4 at the interface with MERS-CoV S RBD. We previously generated a human DPP4-transgenic (hDPP4-Tg) mouse model for MERS-CoV infection . Pulmonaad libitum. All animal experiments were approved by the Institutional Animal Care and Use Committee of Jeonbuk National University and performed in accordance with the committee\u2019s guidelines. MERS-CoV (1\u2013001-MER-IS-2015001) was obtained from the Korean Center for Disease Control and Prevention (KCDC). All experiments using MERS-CoV were performed in accordance with the WHO\u2019s recommendations under biosafety level 3 conditions in a biosafety level 3 facility of the Korea Zoonosis Research Institute , Jeonbuk National University. Unless otherwise specified, the chemicals and laboratory wares used in this study were obtained from Sigma Chemical Co. and SPL Life Sciences , respectively.hDPP4-Tg mice were generated as previously reported , and allProduction of the recombinant MERS-CoV S RBD with or without HBD 2 at the C terminus of the S1 (residues 291\u2013725) domain based on the MERS-CoV S protein sequence was performed as described previously ,18.hDPP4-Tg mice were immunized intramuscularly in the hind leg with 5 \u00b5g/mouse of each recombinant protein dissolved in 50 \u00b5L phosphate-buffered saline (PBS) emulsified with an equal volume of Freund\u2019s complete adjuvant. Ten days after the first immunization, mice were boosted with the same immunogen emulsified with Freund\u2019s incomplete adjuvant. In addition, hDPP4-Tg mice were intranasally immunized once per week for five weeks with 1 \u00b5g each recombinant protein via the intranasal route under anesthesia. Control mice were immunized with the inoculum prepared identically but with PBS only. Sera were collected three days after the final immunization boost to measure MERS-CoV S RBD-specific Abs.p-nitrophenyl phosphate substrate was added. The absorbance at 405 nm was read using an ELISA plate reader . ELISA results were calculated using the standard curve.The levels of MERS-CoV S RBD-specific immunoglobulin G (IgG) in mouse sera were determined by ELISA. Briefly, 96-well ELISA plates were coated with S RBD protein (2 \u00b5g/mL) overnight at 4 \u00b0C and blocked with 5% nonfat dry milk at 37 \u00b0C for 2 h. For preparing the standard curve, the anti-mouse IgG coating antibody was coated onto an ELISA plate, unlike the serum sample. After adding serially diluted sera or standard mouse IgG to each well, plates were incubated at 37 \u00b0C for 1 h, followed by four washes with phosphate-buffered saline (PBS) containing Tween 20. Bound IgGs were incubated with alkaline phosphate-conjugated anti-mouse IgG at 37 \u00b0C for 1 h, and 2 incubator. MERS-CoV was passaged 12 times in Vero E6 cells and subsequently used to assess hDPP4-Tg mouse morbidity and mortality. Briefly, hDPP4-Tg mice and their transgene-negative littermates were anesthetized and inoculated intranasally with 104 or 105 plaque-forming unit (PFU) of MERS-CoV in a total volume of 20 \u00b5L. Infected mice were weighed and monitored every other day for weight loss and death. Although the scoring was not recorded, other clinical signs following viral infection, including physical appearance, abnormalities of behavior or movements, and decreased activity or enhanced responsiveness, were also observed. Subsequently, immunized hDPP4-Tg mice were challenged with 105 PFU of MERS-CoV intranasally and monitored for their survival, weight, and pathological changes for up to 10 or 14 days post-infection (dpi). Some infected mice were sacrificed at the indicated time points to obtain tissue specimens. These tissue specimens were used to assess the expression levels of target genes and mucosal IgA responses using real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and hematoxylin-eosin (H&E) staining.MERS-CoV was propagated in Vero E6 cells grown in Dulbecco\u2019s Modified Eagle\u2019s Medium supplemented with 10% fetal bovine serum (Thermo Fisher Scientific) at 37 \u00b0C in a humidified COLung tissues obtained from MERS-CoV- and sham-infected hDPP4-Tg mice at the indicated time points were immediately fixed in 10% neutral buffered formalin, transferred to 70% ethanol, and paraffin-embedded. Histopathological evaluation was performed on tissue sections deparaffinized and stained by H&E. We examined tissues for pathological signs, such as denatured and collapsed cell/tissue organization, hemorrhage in the interstitial space, infiltration of inflammatory monocytes, and change in alveolar septa after MERS-CoV infection.Lung tissue specimens from MERS-CoV-infected mice were weighed and transferred into individual vials containing TRIzol reagent (Thermo Fisher Scientific). The collected tissues were homogenized and subjected to total RNA isolation as previously described ; RNA wasp-Values < 0.05 were considered statistically significant.Statistical analyses were performed using Prism 7 . Data are expressed as means \u00b1 standard deviations (SDs). The statistical significance of numerical data was analyzed using two-way analyses of variance (ANOVA). 4 or 105 PFU of MERS-CoV. The mice were monitored every other day for survival and clinical symptoms, including weight loss (4 or 105 PFU of MERS-CoV showed rapid weight loss (data not shown); the mortality rates were 60% and 100% at 10 dpi and 12 dpi, respectively. All transgene-negative littermate mice survived without clinical illnesses after the inoculation of the same dose of MERS-CoV. In contrast to sham-infected mice, progressive lung damage was observed in MERS-CoV-infected hDPP4-Tg mice in a viral dose-dependent manner in the lungs of MERS-CoV-infected hDPP4-Tg mice and sham-infected hDPP4-Tg mice in the lungs of MERS-CoV-infected hDPP4-Tg mice. Furthermore, MERS-CoV infection in hDPP4-TG mice significantly upregulated Atf6 (p < 0.05) and Chop (p < 0.01) in lung tissues four dpi; however, their mRNA levels were reduced at six dpi. These results suggest that MERS-CoV infection in hDPP4-Tg mice leads to lung damage by triggering ER stress and fibrosis.Upon viral infection, large amounts of viral proteins are produced and accumulated in the ER, inducing ER stress . Pulmona-Tg mice C. Perk, p < 0.05) higher than in mice immunized with S RBD alone, suggesting the improved ability of HBD 2-conjugated Ag to induce humoral immune responses compared to that of S RBD alone by immunizing the mice intramuscularly . The levesponses A. Then w changes B,C. One-We also compared the ability of intranasal and intramuscular S RBD-HBD 2 immunization to induce S RBD-specific adaptive immune responses and protective immunity against MERS-CoV infection . For theNext, we assessed the pathological changes in the lungs of MERS-CoV-challenged mice after different immunization regimens A. After p < 0.05) higher in hDPP4-Tg mice intranasally immunized with S RBD-HBD 2 than in intramuscularly immunized mice. Although pIgR mRNA levels were higher in the lungs of hDPP4-Tg mice intranasally immunized with S RBD-HBD 2 than in mice intramuscularly immunized with S RBD alone, they were slightly lower than in mice intramuscularly immunized with S RBD-HBD 2.In the mucosa, the production of secretory IgA (SIgA) and polymeric Ig (pIg) receptor (pIgR)-mediated transport of SIgA plays an essential role in protecting against pathogen invasion and maintaining homeostasis in mucosal surfaces ,24,25. FPerk, Atf4, Chop, Atf6, and Xbp1 were significantly less upregulated in the lungs of MERS-CoV-infected hDPP4-Tg mice after immunization with S RBD protein with or without HBD 2 (Aft4 and Xbp1s were expressed in significantly (p < 0.01) lower levels in the lungs of intranasally immunized mice. These results suggest that HBD2-conjugation induces potent mucosal IgA responses, preventing ER-stress-mediated lung damage following MERS-CoV infection.Interestingly, the ER-stress-associated genes ut HBD 2 C. Althoup < 0.001 and p < 0.05; We also evaluated the ability of low-dose S RBD-HBD 2 intranasal administration to induce Ag-specific Ab responses and protect hDPP4-Tg mice against intranasal challenge with MERS-CoV . The serMERS-CoV has spread in 27 countries since the 2012 outbreak in Saudi Arabia; it infects the lower respiratory tract of humans, causing acute respiratory distress syndrome with approximately 34.5% mortality ,29. PreeIn this study, we investigated the potential use of HBD 2 as an adjuvant to strengthen Ag-specific adaptive immune responses in hDPP4-Tg mice . HBD 2 iThe role of mucosal immunity in the lungs and other primary infectious tissues has not been widely studied, although the respiratory mucosa is the main infectious site for numerous pathogens, including MERS-CoV. In this study, we intramuscularly or intranasally immunized hDPP4-Tg mice with S RBD protein with or without HBD 2 and investigated systemic and mucosal immune responses against MERS-CoV. Ag-specific Ab levels and protective immunity against intranasal viral infection were significantly potentiated in mice intranasally immunized with S RBD-HBD 2, although the Ag dose was lower than the dose used for intramuscular immunization . It is wER stress has been implicated in fibrotic remodeling by activating unfolded protein responses and pro-apoptotic pathways, as well as promoting inflammation in various tissues, including the lungs, liver, gastrointestinal tract, kidney, and heart. We previously demonstrated that, upon MERS-CoV infection, tissue damage elicits severe inflammatory responses and activate ER stress pathway components, including PERK, ATF4, CHOP, and ATF6. In this study, we showed that the administration of HBD 2-conjugated S RBD downregulated various ER stress-associated molecules and alleviated progressive pulmonary fibrosis in the lungs of MERS-CoV-infected hDPP4-Tg mice .Overall, these results suggest that the conjugation of HBD 2 with S RBD protein enhances systemic and mucosal immune responses that protect from MERS-CoV infection. Moreover, these findings indicate that immunization via the intranasal route might be superior in triggering protective local and systemic immunity against mucosal pathogens. Therefore, the use of HBD 2 as an adjuvant may represent a promising approach to enhance the immunogenicity and safety of subunit vaccine candidates against MERS-CoV and other mucosal viruses."} +{"text": "Caenorhabditis elegans in a cilia specific manner. Sequential targeting of lipidated Rab28 to periciliary and ciliary membranes is highly dependent on the BBSome and the prenyl-binding protein phosphodiesterase 6 subunit delta (PDE6D), respectively, and BBSome loss causes excessive and ectopic EV production. We also find that EV defective mutants display abnormalities in sensory compartment morphogenesis. Together, these findings reveal that Rab28 and the BBSome are key in vivo regulators of EV production at the periciliary membrane and suggest that EVs may mediate signaling between cilia and glia to shape sensory organ compartments. Our data also suggest that defects in the biogenesis of cilia-related EVs may contribute to human ciliopathies.Cilia both receive and send information, the latter in the form of extracellular vesicles (EVs). EVs are nano-communication devices that influence cell, tissue, and organism behavior. Mechanisms driving ciliary EV biogenesis are almost entirely unknown. Here, we show that the ciliary G-protein Rab28, associated with human autosomal recessive cone-rod dystrophy, negatively regulates EV levels in the sensory organs of Cilia are conserved microtubule (MT)-based organelles that extend from the surfaces of most eukaryotic cell types. Cilia serve a variety of functions that include motility and signal transduction, as well as the capacity to shed extracellular vesicles (EV) . DefectsCilia depend on several modes of intracellular transport to establish and control their molecular make-up . IntraflIn addition to its role as a signaling hub, the cilium is also an evolutionarily conserved site of extracellular vesicle (EV) biogenesis and shedding . Most EVEVs released from the tips of cilia are clearly ectosomal in origin . SeveralC. elegans nematode is an excellent in vivo system to study ciliary formation, specialization, and function including EV shedding\u00a0. Most of these organs are environmentally exposed via cuticular pores derived from surrounding glial cell processes , and an axonemal structure that displays tremendous diversity in terms of their MT arrangements between different cell-types and tomography reconstructions. EVs are also \u2018released\u2019 from cilia directly into the environment outside of the sensory organs, as observed by fluorescently-tagged EV cargos such as the TRP polycystin PKD-2::GFP and myristoylated peripheral membrane protein CIL-7 and shared inner labial (IL2) neurons are the only known ciliated EV-releasing neurons (EVNs) as defined by imaging of fluorescently-tagged EV cargos released outside the animal . These ein CIL-7 . Ciliaryin CIL-7 . In thesC. elegans amphid and phasmid cilia, RAB-28 is a BBS-8-dependent IFT cargo that associates with the periciliary membrane (PCM) when in a GTP-bound conformation and EVN cilia. We find that RAB-28\u2019s PCM association is dependent on the BBSome and, to a lesser extent, its regulator ARL-6, whilst its IFT transport requires the BBSome, PDL-1 (PDE6D ortholog) and ARL-3. We also identify cell-specific distinctions in how RAB-28 transport machinery is deployed, with PDL-1 dispensable for maintaining RAB-28\u2019s PCM levels in amphid/phasmid neurons but not EVNs. Functional analyses revealed that BBSome mutants phenocopy the amphid sensory compartment morphogenesis defects observed in RAB-28-disrupted worms. Importantly, loss of RAB-28 or BBSome genes, but not PDL-1, causes ectopic EV accumulation in the cephalic EVN sensory organ, suggesting that RAB-28 and the BBSome negatively regulate EV biogenesis and/or shedding at the PCM. This research reveals a BBSome-ARL-6-PDL-1 network for targeting RAB-28, a critical EV regulator, to specific ciliary membranes, thus allowing regulation of EVs at distinct sites. The members of this transport network as well as RAB-28 and the EV cargo PKD-2 are all well-established ciliary proteins associated with several human genetic diseases null mutant cilia. We also assessed GFP-RAB-28Q95L localization in a null mutant of arl-6, which regulates membrane-targeting of the BBSome in mammalian cells worms is indistinguishable from bbs-8(nx77) mutants; GFP signals are diffused throughout the neurons, with no detectable PCM enrichment or IFT movement (arl-6(ok3472) mutants display a weaker phenotype, with modestly reduced levels of RAB-28 at the PCM and a diffuse dendritic localization targeting of GFP-tagged RAB-28nt bbs-8 . To furtan cells . We founmovement . By contlization . Also, i twofold . These dC. elegans orthologs of PDE6d (PDL-1) and ARL3 (ARL-3). We found that whilst the PCM localization of GFP-RAB-28Q95L remains intact, the reporter\u2019s IFT motility is completely lost in pdl-1(gk157) and arl-3(1703) null mutants (Q95L in the phasmid cell bodies of pdl-1(gk157) mutants. Thus, like the BBSome, the lipidated protein shuttle PDL-1 is also required for RAB-28 association with IFT trains in amphid and phasmid neurons, although it is dispensable for RAB-28 targeting to the PCM of these cells.As RAB-28 is a prenylated protein, we investigated whether RAB-28 ciliary targeting requires the mutants . An addiarl-6 and pdl-1, we assessed RAB-28 localization in pdl-1(gk157);bbs-8(nx77) and pdl-1(gk157);arl-6(ok3472) double mutants. GFP::RAB-28Q95L localization and IFT behavior in pdl-1(gk157);bbs-8(nx77) mutants was found to be identical to that of bbs-8(nx77) single mutants, reproducing the complete loss of RAB-28\u2019s PCM association and IFT in that strain (bbs-8 is epistatic (masking) to pdl-1. Similarly, in pdl-1(gk157);arl-6(ok3472) worms, the reduced GFP::RAB-28Q95L PCM localization phenocopies that of the arl-6(ok3472) single mutant, although there are additional diffuse signals in the cilium and distal dendrite ;arl-6(ok3472) double mutant cilia (pdl-1(gk157) single mutant, the latter suggests that arl-6 and pdl-1 mutations are mutually suppressive, consistent with an opposing relationship in regulating the formation of RAB-28-positive IFT trains.To explore the genetic relationship between the BBSome, t strain , indicatdendrite . With reT trains . It is nnt cilia . Since dTaken together, these data reveal a ciliary trafficking pathway in amphid and phasmid neurons whereby activated RAB-28 is initially targeted to the PCM by the BBSome, and to a lesser extent ARL-6, and subsequently solubilized by PDL-1 for loading onto IFT trains, a step inhibited by ARL-6 (summarized in rab-28 in amphid neurons could be related to a ciliary extracellular vesicle (EV) pathway, and (ii) rab-28 expression is highly enriched in EVNs based on transcriptome analysis because: (i) we speculated previously that the cell non-autonomous roles of analysis . There a neuron) .rab-28 is expressed in EVNs, we examined transgenic male animals co-expressing the EVN reporter klp-6p::tdTomato (rab-28 5\u2019UTR (promoter) sequence (rab-28p::sfGFP). rab-28 is expressed in the IL2 neurons present in both males and hermaphrodites, as well as all 21 male-specific EVNs -containing mutants of the BBSome , arl-6 and pdl-1. In EVN cilia, GFP::RAB-28Q95L PCM localization was absent in bbs-5(gk507) and bbs-8(nx77) males, whilst reduced in arl-6(ok3472) and pdl-1(gk157) males , bbs-8(nx77) and pdl-1(gk157) mutant EVN cilia. Surprisingly, RAB-28Q95L IFT movement was also not detectable in the EVN cilia of arl-6(ok3472) males, which is in striking contrast to the increased frequency of RAB-28-positive IFT trains in amphid and phasmid neurons (pdl-1(gk157);arl-6(ok3472) double mutant EVNs revealed that GFP::RAB-28Q95L is diffusely localized in the cytoplasm, with no PCM enrichment males , similar7) males . Also, t7) males . Like in neurons . Analysirichment . Thus, pQ95L enrichment along with an additional RAB-28Q95L pool in the distal cilium. However, RAB-28 IFT events are infrequent in this cell type and positively regulated by ARL-6 mutant hermaphrodites. We found that bbs-5(gk507) and bbs-8(nx77) mutant hermaphrodites abnormally accumulate electron dense matrix filled vesicles in the sheath cell process similar to RAB-28T26N-overexpressing worms, although sometimes at higher levels (Q95L (GTP-preferring) (bbs-5(gk507) and bbs-8(nx77) mutant hermaphrodites consistently exhibit highly distended amphid compartments,2\u20133 times the size of WT,although with much darker staining of the extracellular matrix (ECM) (bbs-5(gk507) and bbs-8(nx77) mutant compartments each contain 11 ciliary axonemes, instead of the usual 10 (arl-6(ok3472) mutant; whilst there are electron dense deposits in the sheath cell at the ciliary middle segment, transition zone and PCMC levels of the amphid sensory organ, compartment size and ECM density are normal contains 10 rod-like cilia (from 8 neurons) located in an environmentally-exposed pore, formed by the dendrites and ciliary axonemes punching through surrounding amphid sheath and socket glial cell processes . Previour levels . Additioferring) , bbs-5(gix (ECM) . Also, ausual 10 . A weakee normal .rab-28-disrupted worms (overexpressing RAB-28T26N or RAB-28Q95L). As with rab-28, C. elegans BBS genes are expressed exclusively in ciliated neurons mutant males, total PKD-2::GFP levels at the CEM ciliary region from the PCMC to the tip are similar to control (rab-28(tm2636) males indicating a ciliary localization defective (Cil) phenotype. Specifically, unlike control animals where PKD-2::GFP is most intense at the PCMC region of the CEM cilium, rab-28(tm2636) mutants display abnormally high PKD-2::GFP levels at more distal parts of the CEM cilium region and the function . PKD-2 ifunction . In contfunction . Note thfunction . In rab- control . Howeverm region .rab-28(tm2636) cephalic sensory organ appears to extend beyond that predicted by sole localization to the relatively narrow CEM cilium, we suspected that this excess GFP signal derives from abnormally high levels of PKD-2::GFP-labeled EVs in the cephalic lumen. To determine if rab-28 regulates environmental EV release similar to IFT and tubulin code regulators (rab-28(tm2636) males. We found that control and rab-28(tm2636) males release similar numbers of PKD-2::GFP-labeled EVs, ruling out a function for RAB-28 in environmental EV release of PKD-2 and suggesting a possible defect whereby EV shedding into the sensory organ lumen is abnormally upregulated cephalic sensory organ abnormally accumulates large amounts of CIL-7::GFP in ciliary regions (rab-28(gk1040) worms (rab-28(tm2636) males release similar numbers of CIL-7::GFP-containing EVs in mounting media (To further explore a role for 27 EVNs . In cont regions . A simil0) worms . Wild-tyng media . These rrab-28 mutants, we found Cil defects in bbs-8(nx77) and, to a lesser extent, arl-6(ok3472) mutants, with the ciliary EV marker CIL-7::GFP accumulating at the PCMC and around the axonemal region of CEM cilia (bbs-7 mutant (pdl-1(gk157) mutants show no gross CIL-7 localization defects , arl-6(ok372) and pdl-1(gk157) mutant males to optimally preserve EVs. Each organ contains a single cilium from the CEM and CEP neurons and is environmentally exposed via a cuticular pore formed by the surrounding cephalic glial sheath and socket cells (The accumulation of EV cargo markers in the ciliary region of et cells . In wildet cells . These Eet cells .rab-28(tm2636) male worms, the distal part of the cephalic lumen is enlarged and accumulates an excess of EVs. When compared with control counterparts, these rab-28 mutant EVs are smaller and more uniform in size, and sometimes more electron dense, possibly indicating a change in composition (rab-28(tm2636) mutant retains normal exposure to the environment, indicating that pore blockage or a ciliogenesis defect is not the cause of EV accumulation in the distal lumen (rab-28(tm2636) worms retain the normal CEM ciliary axonemal AB tubule split that is important to EV release (rab-28(tm2636) mutant males produce and shed excessive amounts of abnormally stained and sized EVs into the cephalic sensory organ. These data suggest that RAB-28 regulates EV cargo sorting and production (biogenesis) and that RAB-28 acts as a negative regulator of EV shedding without affecting environmental EV release. Our findings also show that RAB-28 negatively regulates cephalic sensory compartment size, in agreement with what we previously reported for RAB-28 in the amphid organs male worms, the cephalic lumen is distended and filled throughout with abnormally large numbers of EVs (rab-28(tm2636) worms, the accumulated EVs in bbs-8(nx77) mutant lumens are more numerous, and more varied in size. We also observed electron dense matrix filled vesicles within seventy five percent of bbs-8(nx77) mutant cephalic sheath glia (rab-28(tm2636) mutants, the ultrastructure of bbs-8(nx77) mutant CEM cilia is grossly normal. Thus, like RAB-28, the BBSome also negatively regulates EV shedding in the cephalic organ and the size of the organ\u2019s lumen. The differences in the severity of the EV phenotype in bbs-8(nx77) and rab-28(tm2636) mutants, and in the appearance of EVs that accumulate in both these worms, indicate a greater role for BBS-8 in EV regulation, possibly because the BBSome regulates the trafficking of multiple EV regulators and not just RAB-28.In s of EVs . Compareath glia , matchinath glia . Similarrab-28 and bbs-8 mutants, we were surprised to observe ectopic EVs in the amphid sensory organ lumens of cryofixed male bbs-8 but not rab-28 mutants. We previously observed rare EVs in the region surrounding the distal-most segment of amphid channel cilia in males and have not observed release of FP-tagged EVs from amphid cilia of males or hermaphrodites (rab-28(gk1040) and bbs-8(nx77) mutant hermaphrodites male worms, although irregular shaped vesicular structures are occasionally observed (bbs-8(nx77) mutants to pdl-1 for RAB-28 PCM localization, suggesting that the BBSome functions upstream of PDL-1 in the ciliary RAB-28 trafficking pathway. Our transport data here are in agreement with previous observations that the BBSome does not depend on RAB-28 for localization to phasmid cilia RAB-28 and the BBSome are negative regulators of EV shedding at the ciliary base, and (ii) the PCM may be the site of RAB-28\u2019s EV-related function. Taken together, our data indicate the presence of a tuneable system of EV regulation in EVNs that consists of transport regulators that control the levels and localization of EV regulators in cells and cilia. Since the prevalence, pattern, and extent of the EV phenotype is more severe in BBSome vs RAB-28 deficient worms, our data also suggests that the BBSome targets additional EV regulators, beyond RAB-28, to EVN cilia.We previously proposed that ciliary RAB-28 may serve a role in EV biology . We now C. elegans, very little is known about EV ciliary base shedding versus environmental release. Mutants of previously identified cilia-related EV regulators such as KLP-6, CCPP-1, TTLL-11, TBA-6, and CIL-7 showed disruption of environmental EV release with an excessive EV accumulation within sensory organs in an intact animal.A tantalizing hypothesis is that RAB-28 and the BBSome fulfill this role via their EV regulatory function. Indeed, EV exchange between neurons and glia has been documented in vitro and in vivo . HoweverC. elegans rab-28 and bbs-8 gene mutants, together with reduced disc shedding and/or phagocytosis reported in Rab28 knockout mice EVs observed in elegans . Given oC. elegans Rab28 important to suppress EV shedding surrounding distal parts of cilia? What is the mechanism by which Rab28 and the BBSome regulate EV shedding? Why are Rab28 and the BBSome not packaged into EVs despite being closely associated with the biogenesis process? How are proteins sorted and packaged into ciliary EVs? What are the effectors and GAP/GEF regulators of Rab28? How might ciliary EVs interact with surrounding cells such as glia to regulate the shape and size of sensory organ compartments? Answers to these questions will lead to a better understanding of the fundamental biology of ciliary EVs, neuron-glia signaling, and ciliopathies with relatively unexplored EV phenotypes such as polycystic kidney disease, Bardet-Biedl Syndrome, and retinal dystrophy.Our studies raise several interesting questions: How and why is periciliary membrane-localized E. coli. Plates were incubated at either 20\u00b0C or 15\u00b0C to slow development. Reporter strains were crossed into mutant backgrounds and double mutants generated by standard crossing strategies. Mutations were followed by genotyping PCR.All strains were cultured according to standard protocols . BrieflyQ95L + unc-122p::gfp]oqEx304arl-6(ok3472); oqEx304[gfp::rab-28OEB972 Q95L + unc-122p::gfp]bbs-5(gk507); oqEx304[gfp::rab-28OEB970 Q95L + unc-122p::gfp]bbs-8(nx77); oqEx304[gfp::rab-28OEB805 Q95L + unc-122p::gfp]pdl-1(gk157); oqEx304[gfp::rab-28OEB806 Q95L + unc-122p::gfp]arl-3(tm1703); oqEx304[gfp::rab-28OEB807 Q95L + unc-122p::gfp]pdl-1(gk157); bbs-8(nx77); oqEx304[gfp::rab-28OEB971 Q95L + unc-122p::gfp]him-5(e1490); oqEx304[gfp::rab-28OEB973 Q95L + unc-122p::gfp]arl-6(ok3472); him-5(e1490); oqEx304[gfp::rab-28OEB974 Q95L + unc-122p::gfp]bbs-5(gk507); him-5(e1490); oqEx304[gfp::rab-28OEB975 Q95L + unc-122p::gfp]bbs-8(nx77) him-5(e1490); oqEx304[gfp::rab-28OEB976 Q95L + unc-122p::gfp]pdl-1(gk157); him-5(e1490); oqEx304[gfp::rab-28OEB977 Q95L + unc-122p::gfp]pdl-1(gk157); arl-6(ok3472); him-5(e1490); oqEx304PT621 him-5(e1490); myIs23[cil-7p::gCIL-7::GFP_3'UTR+ccRFP]PT2679 rab-28(tm2636); him-5(e1490)PT3189 arl-6(ok3472); him-5(e1490)OEB945 bbs-8(nx77); him-5(e1490)OEB947 rab-28(tm2636);him-5(e1490); myIs4 [PKD-2::GFP+Punc-122::GFP]PT2984 rab-28(tm2636);him-5(e1490); myIs23[cil-7p::gCIL-7::GFP_3'UTR+ccRFP]PT3265 pha-1(e2123); him-5(e1490); myEx905[rab-28p::sfGFP+PBX]PT3190 pha-1(e2123); him-5; myIs20 [klp-6p::tdtomato] myEx905[rab-28p::sfGFP+PBX]PT3356 rab-28(gk1040); him-5(e1490) myIs23OEB948 arl-6(ok3472);him-5(e1490) myIs23OEB949 pdl-1(gk157); him-5(e1490) myIs23OEB951 rab-28p::sfGFP and klp-6p::tdTomato (bbs-8 mutants), males were placed in levamisole for 7\u20138 min prior to imaging. Epifluorescence imaging was performed using an upright Zeiss Axio Imager D1m. Images were acquired using a digital sCMOS camera . The microscope was controlled by Metamorph 7.1 to acquire Z stacks. All images were analyzed using Fiji and the intensity levels at different points along the cilium were obtained for several animals. The intensity of each point along the cilium was then averaged across several cilia/animals. Only points with intensity values across all examined samples were plotted.myIs4[PKD-2::GFP] as described previously .Q95L localization, hermaphrodite or male worms were placed on 5% agarose pads on glass slides and immobilized with 40 mM levamisole. L4 males were isolated the previous day to provide virgin males for imaging. Epifluorescence imaging was performed on an upright Zeiss AxioImager M1 microscope with a Retiga R6 CCD detector (Teledyne QImaging). Confocal imaging was performed on an inverted Nikon Eclipse Ti microscope with a Yokogawa spinning-disc unit (Andor Revolution) and images were acquired using an iXon Life 888 EMCCD detector (Andor Technology). All image analysis was performed using Fiji. For kymography, time-lapse (multi tiff) movies of IFT along cilia were taken at 250 ms exposure and 4 fps. Kymographs were generated from multi tiff files using the KymographClear ImageJ plugin , PT3189 (rab-28(tm2636); him-5) and OEB947 (bbs-8(nx77); him-5) strains were collected as L4 males the day before freeze fixation to provide virgin day 1 adult males on the day of fixation. Males were subjected to high\u2010pressure freeze fixation using a HPM10 high\u2010pressure freezing machine . Males were slowly freeze substituted in 2% osmium tetroxide, 0.2% uranyl acetate and 2% water in acetone using RMC freeze substitution device (High pressure freeze fixation (HPF) and freeze substitution for TEM on CEM cilia: control CB1490 . SamplesTEM of the amphid sensory organs of chemically fixed BBS gene hermaphrodite mutants was perfTo quantify amphid sensory compartment size, the area of each amphid compartment was measured at the same section depth for each genotype. The depth chosen was at the level of the middle segments/transition zones of amphid cilia, as this is the point at which compartment size is most extensive in all cases. To assess whether a mutant accumulated an excess of matrix filled vesicles in the amphid sheath cell, the number and the size of the matrix filled vesicles in the amphid sheath were noted. Wild-type animals accumulate smaller matrix filled vesicles and therefore any mutant that accumulated noticeably large vesicles was scored as defective.rab-28(tm2636) animals. For measurement of EV numbers in the sub-distal region of the cephalic sensory organ lumen, EVs were counted between the regions where the CEM cilia have 18 singlet MTs up to the anterior-most section where CEM and CEP cilia share the lumen. For measurement of EV numbers at the TZ level (CEM cilium) of the cephalic sensory organ, EVs were counted between the region where all the TZ microtubules were doublets up to the region where all the TZ microtubules terminated. For measurement of EV numbers at the PCMC level (CEM cilium) of the cephalic sensory organ, EV numbers were counted in the region 140 nm posterior to where the TZ microtubules terminate. To further ensure that the same part of the sensory organ was being assessed in the above experiments, various features of the CEP and OLQ ciliary axonemes were used as landmarks.All quantifications of EV numbers were done from serial section TEM images of males of WT and C. elegans RNA preparation and RT-PCR mRNA was isolated from mixed-stage control (him-5) and rab-28(tm2636); him-5 mutants. cDNA amplified from both strains using NEB Protoscript II was used as template for PCR using gene-specific primers. The amplified PCR products from both genotypes were subsequently sequenced. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:The editor and reviewers found your delineation of the novel in vivo regulatory mechanisms for extracellular vesicle production exciting and compelling.Decision letter after peer review:eLife. Your article has been reviewed by Suzanne Pfeffer as the Senior Editor, a Reviewing Editor, and two reviewers. The following individuals involved in review of your submission have agreed to reveal their identity: Guangshuo Ou (Reviewer #1).Thank you for submitting your article \"A ciliary BBSome-ARL-6-PDE6D pathway trafficks RAB-28, a negative regulator of extracellular vesicle biogenesis\" for consideration by eLife. But they also provided concrete suggestions to improve the manuscript.The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission. Both reviewers think that the work is interesting and should potentially be published in Summary:In a 2016 PLOS Genetics paper, the Blacque and Leroux labs identified Rab-28 as a gene associated with cilia function and they made the unexpected discovery that overexpression of dominant negative Rab-28 in ciliated amphid pore neurons results in non-cell autonomous phenotypes. Namely, sheath cells accumulate dark matrix-filled vesicles in strains overexpressing dominant negative Rab-28 and the amphid pore is enlarged and filled with a light matrix in strains overpexpressing consitutively active Rab-28. As formation of the amphid pore is dependent on secretion of ECM by sheath cells, it was concluded that Rab-28 expression in ciliated neurons affects the function of sheath cells.bbs and rab-28 mutants accumulate extracellular vesicles (EVs) in a dilated pore filled with dark matrix. Accumulation of dark vesicles within the sheath cells is also observed in these mutants. The authors conclude that the overproduction of EVs by ciliated neurons affects matrix secretion by the sheath cells.The nature of the communication between ciliated neurons and sheath cells was left as an open question which the current paper aims to address. In the present manuscript, the authors analyze mutants of Rab-28, of the BBSome and of ARL6 and find that Rab-28 fails to accumulate in cilia and at the periciliary membrane (PCMC) in BBSome and ARL6 mutants. Characterization of the cephalic and amphid pore by fluorescence and electron microscopy indicates that Although the current data set is not fully demonstrative of a role of EVs in regulating the function of sheath cells, it is a valuable contribution that advances the thesis of EVs as means of intercellular communication in a physiological context. The quality of the work is generally high, the experiments are presented clearly, and the logic is well articulated.Essential revisions:bbs or rab-28 mutants; the author's hypothesis predicts that the sheath cell phenotypes will be suppressed. The Barr lab previously reported that EV production is drastically reduced in klp-6 and ift mutants.The major caveat of the current manuscript is the correlative nature of the argument that EVs mediate communication between ciliated neurons and sheath cells. The alternative hypothesis that secreted factors produced by ciliated cells affect the support cells is neither formulated nor tested. An important experiment would be to interfere with EV production in the rab-28 deletion mutant, which is likely a null allele'. The gk1040 allele was studied in this past publication. Yet, in the current manuscript, the same gk1040 allele and the additional allele tm2636 both exhibit pronounced defects at the level of the sheath cells and the amphid pore. Could the authors comment on this discrepancy in the Discussion section?1) Quoting from Jensen et al., 2016, 'sensory pore structure and function appears grossly normal in the ift27 is deleted. The model that Rab-28 functions downstream of the BBSome in the regulation of EV shedding is based on the observations that Rab-28 localization to cilia and the PCMC is reduced in BBSome mutants while localization of the BBSome remains normal in the rab-28(gk1040) mutant . In light of the newly found phenotypes of Rab-28 mutants in the current paper, it would be important to re-examine the localization and dynamics of the BBSome in Rab-28 mutants and in strains expressing Rab-28 variants.2) The authors propose that Rab-28 functions downstream of the BBSome. Have the authors tested the localization of the BBSome in Rab-28 mutants? This experiment is particularly relevant in light of the sequence similarity between Rab-28 and RabL4/IFT27 and prior findings in mammalian cells that the BBSome accumulates in cilia when bbs and rab-28 mutants originate from budding events at the periciliary membrane (microvesicle shedding) rather than from secretion of multivesicular bodies (exosome release). Have the authors looked at the distribution of CD63-GFP, a marker of ILVs and exosomes, in the rab-28 and bbs mutants? This is important. Is the release of EVs found in the amphid pore dependent upon ESCRT function?3) The authors propose that the EVs that fill the amphid pore lumen in Essential revisions:The major caveat of the current manuscript is the correlative nature of the argument that EVs mediate communication between ciliated neurons and sheath cells. The alternative hypothesis that secreted factors produced by ciliated cells affect the support cells is neither formulated nor tested. An important experiment would be to interfere with EV production in the bbs or rab-28 mutants; the author's hypothesis predicts that the sheath cell phenotypes will be suppressed. The Barr lab previously reported that EV production is drastically reduced in klp-6 and ift mutants.bbs-8 and rab-28 mutants depends on genes required for EV production. However, as of yet, we have not identified such genes. Please note that our previous work identified klp-6, ccpp-1, ttll-11, tba-6, and cil-7 as positive regulators of ciliary EV release into the environment, but with no loss of EV shedding abilities within the cephalic sensory organ in all these mutants . Thus, we have not identified mutants that completely block ciliary base EV shedding, and hence we cannot do the suggested epistasis experiment. To address this concern, we revised our discussion to address a possible role for secreted factors in regulating sensory organ morphogenesis. Future studies will be aimed at learning more about the origin and cellular targets of shed EVs, and the mechanisms that stimulate EV shedding to provide answers regarding the role of EVs in ciliated neuron-glia communication.Although we find that all mutants accumulating EVs also have pore expansion phenotypes, we agree that secreted factors, either alone or in combination with the EVs, could play a role in sensory pore morphogenesis. Indeed, as suggested by the reviewer, an excellent experiment to clarify EV-pore phenotype associations would be to examine if the expanded pore lumen in 1) Quoting from Jensen et al., 2016, 'sensory pore structure and function appears grossly normal in the rab-28 deletion mutant, which is likely a null allele'. The gk1040 allele was studied in this past publication. Yet, in the current manuscript, the same gk1040 allele and the additional allele tm2636 both exhibit pronounced defects at the level of the sheath cells and the amphid pore. Could the authors comment on this discrepancy in the Discussion section?rab-28(tm2636) worms. What we reported was an enlarged cephalic pore lumen and sheath cell defect in these worms. Perhaps the source of the confusion was a less-than-optimal presentation of our data. For this reason, we have made major changes in how the revised manuscript is organized, taking great care to ensure full clarity and distinction of cephalic and amphid organ phenotypes. The revised manuscript also now reports on tm2636 amphid sensory organ ultrastructure. Consistent with what we reported in 2016 for rab-28(gk1040), the tm2636 lumen size appears grossly normal when compared to the control . Thus, for amphid compartment size, we have no evidence of any difference or discrepancy between the gk1040 and tm2636 alleles. It is notable, however, that some abnormal matrix filled vesicles (MFV) occur in the amphid sheath of tm2636 worms , which is something we did not see in the gk1040 allele. There are two possible explanations for this distinction: (1) our analyses of tm2636 (current study) and gk1040 (2016 study) was done in males and hermaphrodites, respectively, and thus we cannot exclude sex distinctions in the matrix filled vesicle phenotype, and (2) unlike gk1040, the tm2636 alelle is likely not a null, based on RTPCR analysis of tm2636 transcript . This allele distinction could explain, at least in part, the difference in the sheath cell phenotype. All of the above is now clearly outlined in the revised manuscript. For clarity, we also now include Table 1 that summarizes the TEM and non-TEM phenotypes in all relevant genetic backgrounds.There appears to be some confusion. Our original submission did not report a disrupted amphid sensory organ phenotype in 2) The authors propose that Rab-28 functions downstream of the BBSome. Have the authors tested the localization of the BBSome in Rab-28 mutants? This experiment is particularly relevant in light of the sequence similarity between Rab-28 and RabL4/IFT27 and prior findings in mammalian cells that the BBSome accumulates in cilia when ift27 is deleted. The model that Rab-28 functions downstream of the BBSome in the regulation of EV shedding is based on the observations that Rab-28 localization to cilia and the PCMC is reduced in BBSome mutants while localization of the BBSome remains normal in the rab-28(gk1040) mutant . In light of the newly found phenotypes of Rab-28 mutants in the current paper, it would be important to re-examine the localization and dynamics of the BBSome in Rab-28 mutants and in strains expressing Rab-28 variants.rab-28 mutant alleles. In amphid and phasmid neurons, we found that BBS-8::GFP ciliary localisation is unaffected in the tm2636 and gk1040 alleles, the latter validating previous findings from Jensen et al., 2016. In EVNs, there is some evidence of a modest reduction in ciliary BBS-8::GFP levels in tm2636 but not in gk1040, possibly due to the allele differences described above. Nonetheless given technical challenges in measuring ciliary BBS-8::GFP levels in EVN cilia, and the fact that any defect is likely to be relatively small and restricted only to one allele, we do not have sufficient grounds to imply any role for RAB-28 in regulating BBSome localisation in a subset of cilia (i.e. those of EVNs). Thus, at this point, our conclusion that the BBSome regulates RAB-28 localization in non-EVNs, but not vice versa, stands.We thank the reviewers for raising this important point. As suggested, we re-examined BBS-8 localization in non-EVN (amphid and phasmid) and EVN neurons of both 3) The authors propose that the EVs that fill the amphid pore lumen in bbs and rab-28 mutants originate from budding events at the periciliary membrane (microvesicle shedding) rather than from secretion of multivesicular bodies (exosome release). Have the authors looked at the distribution of CD63-GFP, a marker of ILVs and exosomes, in the rab-28 and bbs mutants? This is important. Is the release of EVs found in the amphid pore dependent upon ESCRT function?C. elegans ciliary EVs are microvesicles . We now clearly state this in the revised Discussion section. In the final conclusion paragraph, we also note however, that we cannot exclude that some of the observed EVs could be of exosomal identity.Our genetic and electron microscopy data are consistent with these small EVs being ectosomes/microvesicles. We observed normal EV biogenesis, shedding and release phenotypes in ESCRT pathway mutants . With confocal microscopy and electron microscopy and tomography, we observe EVs in the lumen, suggesting that EVs are shed at the ciliary base into the lumen. Using EM, we do not see multivesicular bodies in the region of the ciliary base. Hence, these small ciliary EVs (average diameter 104.7 \u00b1 46.7 nm) are not likely exosomes. Instead, we observe omega-shaped vesicle budding from the ciliary base suggesting that these In the non-EVN amphid lumen, the ectopic EVs observed in the bbs-8 mutants are poorly characterized in terms of content, regulation, and function, and a determination of the mode of EV biogenesis in the amphid sensory organ cannot be made until we learn of the identity of the cargoes within. For these reasons we have not made any conclusions regarding the origin of the ectopic EVs in the amphid sensory compartment.C. elegans (tsp-7) as well as 21 other tetraspanin encoding gene paralogs. While CD63 is a common exosomal marker, a major challenge in the EV biology field is a dearth of markers that are specific for exosomes and for microvesicles/ectosomes. Most EV studies have used cultured cells or biofluids, and much effort has been invested in improving methodology for isolating and characterizing different EV subtypes using protein and lipid markers. Exosomes (30-150 nm in diameter) are generated through fusion of multivesicular bodies. Microvesicles or ectosomes (100-1000 nm) bud from the plasma membrane. EVs are heterogeneous and typically isolated based on size and density and are of unknown biogenic origin: available markers are not strictly specific for exosomes or microvesicles/ectosomes. Therefore, the terms small EVs (<200 nm) and large EV (>200 nm) are used ; Meldolesi, (2018). Hence the term \u201csmall EV\u201d encompasses both exosomes and small ectosomes/microvesicles . For all of these reasons, we are conservative in our diagnosis of exosome versus ectosome, and use the term \u201cEVs.\u201d We have added excerpts of this paragraph to the Introduction.Finally, in relation to the CD63 query, there is indeed an orthologue in"} +{"text": "Following publication of the original article , the autIncorrect author name upon publication:Yin-Luo GuYing-Luo GuThe correct author name:"} +{"text": "Scientific Reportshttps://www.doi.org/10.1038/s41598-020-70209-5, published online 06 August 2020Correction to: The original version of this Article contained a typographical error in the title of the paper,\u201cQuasi-probability information in an coupled two-qubit system interacting non-linearly with a coherent cavity under intrinsic decoherence\u201dNow reads:\u201cQuasi-probability information in a coupled two-qubit system interacting non-linearly with a coherent cavity under intrinsic decoherence\u201dThis error has now been corrected in the PDF and HTML versions of the Article."} +{"text": "State-of-the-art nanopore sequencing enables rapid and real-time identification of novel pathogens, which has wide application in various research areas and is an emerging diagnostic tool for infectious diseases including COVID-19. Nanopore translocation enables de novo sequencing with long reads (> 10 kb) of novel genomes, which has advantages over existing short-read sequencing technologies. Biological nanopore sequencing has already achieved success as a technology platform but it is sensitive to empirical factors such as pH and temperature. Alternatively, \u00e5ngstr\u00f6m- and nano-scale solid-state nanopores, especially those based on two-dimensional (2D) membranes, are promising next-generation technologies as they can surpass biological nanopores in the variety of membrane materials, ease of defining pore morphology, higher nucleotide detection sensitivity, and facilitation of novel and hybrid sequencing modalities. Since the discovery of graphene, atomically-thin 2D materials have shown immense potential for the fabrication of nanopores with well-defined geometry, rendering them viable candidates for nanopore sequencing membranes. Here, we review recent progress and future development trends of 2D materials and their \u00e5ngstr\u00f6m- and nano-scale pore-based nucleic acid (NA) sequencing including fabrication techniques and current and emerging sequencing modalities. In addition, we discuss the current challenges of translocation-based nanopore sequencing and provide an outlook on promising future research directions."} +{"text": "Androgen receptor (AR) signalling drives neoplastic growth and therapy resistance in prostate cancer. Recent clinical data show that docetaxel combined with androgen deprivation therapy improves outcome in hormone-sensitive disease. We studied whether testosterone and AR signalling interferes with docetaxel treatment efficacy in castration-resistant prostate cancer (CRPC). We found that testosterone supplementation significantly impaired docetaxel tumour accumulation in a CRPC model, resulting in decreased tubulin stabilisation and antitumour activity. Furthermore, testosterone competed with docetaxel for uptake by the drug transporter OATP1B3. Irrespective of docetaxel-induced tubulin stabilisation, AR signalling by testosterone counteracted docetaxel efficacy. AR-pathway activation could also reverse long-term tumour regression by docetaxel treatment in vivo. These results indicate that to optimise docetaxel efficacy, androgen levels and AR signalling need to be suppressed. This study lends evidence for continued maximum suppression of AR signalling by combining targeted therapeutics with docetaxel in CRPC. Recently, the combination of ADT with docetaxel has\u00a0also been introduced in the metastatic castrate-naive setting as this combination significantly increased overall survival.5 Conversely, docetaxel without ADT after radical prostatectomy did not delay disease recurrence.6 These clinical trials suggest that the antitumour efficacy of docetaxel in castrate-naive PCa\u00a0(CNPC) is improved by inhibition of AR-pathway signalling. In this study, we explore the impact of sustained androgen levels and/or AR-pathway signalling on docetaxel efficacy in CRPC. Moreover, we studied the underlying mechanisms of AR-pathway activation on docetaxel treatment efficacy by examining docetaxel tumour accumulation, target engagement and cell death induction.The treatment of advanced or metastatic prostate cancer (PCa) is focussed around androgen deprivation therapy (ADT), as testosterone and dihydrotestosterone promote neoplastic behaviour of PCa cells through androgen receptor (AR) signalling. While ADT almost invariably induces disease regression, the majority of prostate tumours will at some point become resistant due to AR-pathway aberrations.7 Both PC346C-DCC-K and the parental cell line PC346C do not express AR variants ,8 while VCaP-DCC-E shows increased expression of AR-V7 compared to VCaP. Furthermore, these cell lines do not express ABCB1 (P-glycoprotein), which has been shown to induce multidrug resistance.9 For in vivo experiments, NMRI nu/nu male mice were subcutaneously inoculated with PC346C-DCC-K cells and surgically castrated once tumours established (Supplementary Methods). After 1 week, mice received a testosterone pellet or treatment control. One injection of docetaxel (33\u2009mg/kg) or NaCl was given the following day. Tumour volume was monitored weekly by callipers and mice were euthanised before the humane endpoint was reached by cervical dislocation (details in Supplementary Methods).10 For in vivo accumulation studies, tumours were obtained 3 days after docetaxel treatment. Tumour samples were used to determine docetaxel accumulation by liquid chromatography with tandem mass spectrometry,12 \u03b1-tubulin acetylation by western blot and cell death by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining. Uptake of docetaxel by OATP1B3 was studied in Hek293T cells transiently expressing SLCO1B3, uptake of C14-docetaxel was measured in the presence or absence of testosterone. To assess the impact of AR-pathway stimulation on docetaxel sensitivity, cell viability assays were conducted. We exposed PC346C-DCC-K and VCaP-DCC-E cells in vitro to a dose range of docetaxel with or without androgens.The AR-positive CRPC patient-derived cell line models, PC346C-DCC-K and VCaP-DCC-E, were derived from the hormone-sensitive prostate cancer cell lines, PC346C and VCaP, respectively, through long-term propagation in castrate conditions.11 Indeed, testosterone-supplemented mice displayed a 40% reduction of docetaxel tumour levels: median of 2.8\u2009ng/mg tumour from testosterone-supplemented mice versus 4.4\u2009ng/mg in androgen-deprived animals . SLCO1B3 is frequently overexpressed in prostate cancer, and docetaxel and testosterone are both OATP1B3 substrates16 . Therefore, tubulin stabilisation by docetaxel treatment in testosterone-supplemented mice, albeit reduced, did not translate into cell death induction. We thus hypothesised that AR-pathway activation by testosterone further abrogates docetaxel efficacy. Indeed, a small but consistent survival advantage was achieved by R1881 under effective docetaxel concentrations in AR-positive CRPC cell lines . R1881 did not impact docetaxel response in AR-negative prostate cancer (PCa) cells AR-targeted therapies with taxanes in mCRPC.Supplementary files"} +{"text": "We report the complete genome sequences of five human coronavirus NL63 (HCoV-NL63) strains obtained using next-generation sequencing. The five HCoV-NL63 strains were obtained from hospitalized children with severe acute respiratory infection detected in Guangdong, China. This study provides several complete genomes of HCoV-NL63 and improves our understanding of HCoV-NL63 evolution in China. We report the complete genome sequences of five human coronavirus NL63 (HCoV-NL63) strains obtained using next-generation sequencing. The five HCoV-NL63 strains were obtained from hospitalized children with severe acute respiratory infection detected in Guangdong, China. This study provides several complete genomes of HCoV-NL63 and improves our understanding of HCoV-NL63 evolution in China. Coronaviridae, genus Alphacoronavirus, and was first discovered in 2004 (Human coronavirus NL63 (HCoV-NL63) is a member of the family in 2004 . HCoV-NL in 2004 , for entde novo assembly model, and the contig sequences were then extracted for subsequent analysis. Partial genome sequences of five HCoV-NL63 strains were obtained by NGS methods. Meanwhile, sets of specific primer pairs were designed and used to amplify the gap region of HCoV-NL63, which was used for genome assemblies using the SeqMan subprogram of the DNAStar software version 7.1.0 with default parameters (NC_005831.2) as estimated using MEGA version 5.10 software , 7 as forameters . Finallysoftware Fig.\u00a01)de novo aOnly two complete genome sequences of HCoV-NL63 associated with acute respiratory illness have been obtained and reported in China. The complete genome sequence data from our study will provide insight into the evolution and genetic diversity of HCoV-NL63 in China.MK334043, MK334044, MK334045, MK334046, and MK334047. The sequencing reads are available in the SRA database under BioProject accession number PRJNA601331.The complete genome sequences of the five newly identified HCoV-NL63 strains have been deposited in GenBank under the accession numbers"} +{"text": "Bacterial and archaeal CRISPR-Cas systems offer adaptive immune protection against foreign mobile genetic elements (MGEs). This function is regulated by sequence specific binding of CRISPR RNA (crRNA) to target DNA/RNA, with an additional requirement of a flanking DNA motif called the protospacer adjacent motif (PAM) in certain CRISPR systems. In this review, we discuss how the same fundamental mechanism of RNA-DNA and/or RNA-RNA complementarity is utilized by bacteria to regulate two distinct functions: to ward off intruding genetic materials and to modulate diverse physiological functions. The best documented examples of alternate functions are bacterial virulence, biofilm formation, adherence, programmed cell death, and quorum sensing. While extensive complementarity between the crRNA and the targeted DNA and/or RNA seems to constitute an efficient phage protection system, partial complementarity seems to be the key for several of the characterized alternate functions. Cas proteins are also involved in sequence-specific and non-specific RNA cleavage and control of transcriptional regulator expression, the mechanisms of which are still elusive. Over the past decade, the mechanisms of RNA-guided targeting and auxiliary functions of several Cas proteins have been transformed into powerful gene editing and biotechnological tools. We provide a synopsis of CRISPR technologies in this review. Even with the abundant mechanistic insights and biotechnology tools that are currently available, the discovery of new and diverse CRISPR types holds promise for future technological innovations, which will pave the way for precision genome medicine. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR- associated (Cas) proteins constitute an RNA-guided adaptive immune system found in several bacteria and most archaea. Over the past decade, detailed molecular mechanisms were discovered which established CRISPR-Cas not just as a phage defense system, but also as a regulator of bacterial physiology such as virulence and group behaviors. and a set of Cas proteins (Class 1) or a single multi-domain Cas protein (Class 2) .CRISPR-associated complex for antiviral defense), which comprises crRNA and multiple Cas proteins , along with crRNA for DNA cleavage (vs. RNA) and guide RNA requirements [crRNA or crRNA-tracrRNA or crRNA-scout (short-complementarity untranslated)] have also been observed in type V systems from RNA . Detailsfrom RNA .crRNA Processing: Typically, the CRISPR array is transcribed into a long pre-crRNA, which associates with Cas proteins for further processing into mature crRNAs. Class 1 systems require Cas6 to process pre-crRNA into individual crRNA molecules ((2) olecules ). Class olecules , type V-olecules . Certainolecules .Interference: This stage involves the sequence-specific targeting and cleavage of foreign DNA and/or RNA. The classification section above details relevant proteins and cleavage types. Structural and mechanistic details of CRISPR interference have been recently reviewed ((3) reviewed . Interfe(i) presence of PAM (protospacer adjacent motif), a DNA motif flanking the RNA-DNA complementary region in types I, II, and V; (ii) absence of RNA complementarity between the 5\u2032-tag of crRNA and 3\u2032 flank of the target RNA in type III; and (iii) presence of a protospacer flanking sequence (PFS), an RNA motif in the target RNA, in type VI on the cell envelope to evade host immune responses (blp (FTN_1104-1101) (blps are upregulated in cas9 deletion mutants (\u0394cas9) of Streptococcus agalactiae GD201008-001 (type II-A) and Riemerella anatipestifer (type II-C) (Streptococcus pyogenes GAS-M1T1-5448 (type II-A), \u0394cas9 produces less master regulator protein Mga, which in turn downregulates ScpA and SIC proteins, which are essential to inactivate the host complement immune defense which regulates capsule genes that confer antiphagocytic properties . The best studied example is the esponses . This mepe II-C) . In Stre defense . S. pyogoperties . Involveresponse ). The baNeisseria, Streptococcus, and Campylobacter \u25b3cas9 strains adhere poorly to mammalian host cells .Salmonella enterica serovar Enteritidis \u25b3cas3 (type I-E) transcriptome studies, Cas3 downregulates LsrF production, preventing degradation of quorum sensing (QS) signaling molecules by LsrF, ultimately increasing expression of the lsr QS operon (cas3 Streptococcus mutans UA159 (type I-C) showed downregulation of biofilm formation genes controlled by the VicR/K TCS, which is known to influence streptococcal tissue specificity to nick the bacterial genome, initiating a RecA-dependent SOS response that inhibits biofilm formation and swarming (C. jejuni (type II-C) (S. pyogenes (type II-A) (Neisseria meningitidis (type II-C) . Experipe II-C) , S. pyogpe II-A) , Neisserpe II-C) , and S. pe II-A) where \u25b3ccas1 E. coli to DNA damage (E. coli (type I-E) (cas9 S. pyogenes (type II-A) (Streptococcus (type II-A) (Riemerella anatipestifer (type II-C) (Myxococcus xanthus have shown that CRISPR systems are involved in cell-stress dependent sporulation (type I-C) and fruiting body development (type III-B) . These spe II-A) , Group Bpe II-A) , and Riepe II-C) have inde III-B) .Type III and type VI CRISPR systems promote indiscriminate RNA cleavage leading to PCD, a strategy employed when immune protection has failed . In typeCurrent studies indicate that CRISPR has functions beyond adaptive immunity, primarily in regulating genome content and gene expression. Horizontal Gene Transfer (HGT) can be negatively impacted in CRISPR-containing bacteria since the acquired DNA can be targeted by CRISPR. The effects of CRISPR on HGT are evolving. Some bacteria compensate limitations on HGT by maintaining a defective CRISPR locus or establishing mechanisms for CRISPR-tolerance . A diffevia genome remodeling and regulatory changes. CRISPR changes expression of S. pyogenes master regulators Mga and VicR and the TCS CovR/S, which then regulate immunomodulatory virulence factors that drive development of strains with different host tissue preferences and physiologies ranging from hypervirulent to carrier status with a single guide RNA (sgRNA) (The most widely used CRISPR-based gene editing system is (sgRNA) ). Cas9-b (sgRNA) .Cas12a and CASCADE gene editing systems complement those of Cas9 since their DNA cleavage mechanisms produce HDR-enhancing staggered ends and instill long-range deletions respectively .Staphylococcus aureus Cas9 for transcript level gene knock-down .\u00a0Nucleaectively ).Cas12a and Cas13 inherently cleave non-specific nucleic acids after RNA-mediated target binding ). Notabletc. . As newAs described here, CRISPR-Cas is a two-in-one mechanism for protection against intruding nucleic acids as well as for regulating bacterial physiology, including pathogenicity. Following the current discovery trends, genomic analyses will keep unearthing new CRISPR-Cas systems, sometimes even re-writing the existing rules . The meC. jejuni and by a grant from the Research Council of the University of Oklahoma Norman Campus to RR.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Both prostate-specific membrane antigen (PSMA) uptake and tumour blood flow (TBF) correlate with International Society of Urological Pathology (ISUP) Grade Group (GG) and hence prostate cancer (PCa) aggressiveness. The aim of the present study was to evaluate the potential synergistic benefit of combining the two physiologic parameters for separating significant PCa from insignificant findings.82Rb]Rb positron emission tomography (PET) TBF in PCa, the 43 patients that underwent clinical [68Ga]Ga-PSMA-11 PET were selected for this retrospective study. Tumours were delineated on [68Ga]Ga-PSMA-11 PET or magnetic resonance imaging. ISUP GG was recorded from 52 lesions.From previous studies of [68Ga]Ga-PSMA-11 maximum standardized uptake value (SUVmax) and [82Rb]Rb SUVmax correlated moderately with ISUP GG and with each other . A combined model of [68Ga]Ga-PSMA-11 and [82Rb]Rb SUVmax separated ISUP GG\u2009>\u20092 from ISUP GG 1\u20132 and benign with an area-under-the-curve of 0.85, 96% sensitivity, 74% specificity, and 95% negative predictive value. The combined model performed significantly better than either tracer alone did (p\u2009<\u20090.001), primarily by reducing false negatives from five or six to one (p\u2009\u2264\u20090.025).[PSMA uptake and TBF provide complementary information about tumour aggressiveness. We suggest that a combined analysis of PSMA uptake and TBF could significantly improve the negative predictive value and allow non-invasive separation of significant from insignificant PCa. Prostate cancer (PCa) is a heterogeneous disease, and consequently, an important challenge in PCa management is differentiation of clinically significant PCa from insignificant PCa that will not cause symptoms or affect the patient\u2019s lifetime . MultiplProstate-specific membrane antigen (PSMA) is a transmembrane protein, which is upregulated in most PCa. PSMA tracers are indisputably excellent for detection of PCa, both for detection of local recurrence and for staging of primary tumour, bone-, and lymph node metastases , 5. The The PSMA protein is an enzyme called glutamate carboxypeptidase, which splices polygammaglutamate-folate into folate and glutamate. In the small intestine, it is referred to as folate hydrolase, which enables freeing of folate that can be absorbed from the diet. In nervous tissue it is often referred to as N-acetyl-L-aspartyl-L-glutamate peptidase, which increases the concentration of the excitatory neurotransmitter glutamate in the extracellular space, and was studied as a potential new drug target in treating various neurological and psychiatric diseases . Two stuBlood flow is an essential physiologic parameter for cancer growth, and, hence, tumour blood flow (TBF) has been measured for characterization of tumours of various origins , amongst82Rb]Rb is an accessible clinical blood flow tracer for positron emission tomography (PET), which is used for myocardial blood flow measurement in many PET-Centres worldwide. As coincidental findings, increased [82Rb]Rb uptake was described in neuroendocrine tumours [82Rb]Rb PET for TBF measurement in PCa [82Rb]Rb TBF superior to apparent diffusion coefficient (ADC) from multiparametric (mp) magnetic resonance imaging (MRI) in differentiating between clinically significant and insignificant PCa [82Rb]Rb TBF Rb TBF .68Ga]Ga-PSMA-11- and [82Rb]Rb PET/computed tomography (CT). Hence, the aim of the present study was to evaluate whether the two physiologic parameters are associated and whether there is a synergistic benefit of combining them for separation of significant PCa from insignificant findings.Both PSMA-uptake and TBF in PCa correlate with PCa aggressiveness. However, the two physiologic parameters have not previously been studied together. From our previous studies regarding TBF in clinical PCa patients, we have access to a sub-cohort, who underwent both [82Rb]Rb PET/CT and clinical [68Ga]Ga-PSMA-11 PET/CT and thereby met the inclusion criteria for the present study. All patients were recruited at the time of primary staging and hence none had previous therapy. The designs of the three studies from which the patients originated varied, and hence, the available ISUP GG derived from either radical prostatectomy, MRI-guided biopsies, or trans-rectal ultrasound-guided biopsies. Twenty-four patients with in total 33 lesions had MR-guided in-bore biopsy. Nineteen patients had trans-rectal ultrasound-guided biopsy, and 23 patients underwent subsequent radical prostatectomy. Histopathological inflammation for each lesion was registered.Across our previous studies \u201333, 43 pThe study was approved by the institutional review board and all subjects signed an informed consent form.82Rb]Rb PET/CT scans but one were performed on GE Discovery MI Digital Ready PET/CT , and a single scan was performed on GE Discovery MI (5 ring) PET/CT. Details of the scan and reconstruction protocols have previously been described [82Rb]RbCl (1110\u00a0MBq) was injected at scan start by the Cardiogen-82 generator infusion system . The static image series of [82Rb]Rb PET (3 to7\u00a0min post-injection) were used for SUV analysis.All [escribed . A bolus68Ga]Ga-PSMA-11 PET/CT scans were performed one hour post-injection of 2.14\u00a0MBq [68Ga]Ga-PSMA-11 (68Ga-Glu-CO-Lys(Ahx)-HBED-CC) per kilogram body weight on a Siemens Biograph TruePoint PET/CT scanner . 3D PET acquisitions with 3\u00a0min per bed position from vertex cranii to mid-femur were performed. Low-dose CT for attenuation correction was performed with all common corrections applied using the TrueX reconstruction algorithm (4 iterations and 21 subsets) and a 3-mm Gaussian post-filter (XYZ).[The mpMRI scans were performed according to clinical guidelines and the PIRADS v 2.1 minimum protocol on 3\u00a0T p68Ga]Ga-PSMA-11 PET/CT scans and [82Rb]Rb PET/CT scans were co-registered using the CT scan as bridge. Co-registrations with the T2 weighted sequence of the mpMRI were also carried out in the 24 patients where mpMRI was available, again using the CT scan as bridge. Volume-of-interests (VOI) were defined in two different ways. First, the tumour VOIs were defined by the [68Ga]Ga-PSMA-11 activity. The threshold used for automatic VOI drawing on [68Ga]Ga-PSMA-11 PET varied between individual patients, as no universal threshold could be defined. In two patients with basal tumour location, the bladder activity was masked. An external tumour VOI defined by the mpMRI was applied in the 24 patients with mpMRI scan available. The tumour VOIs were transferred to the [82Rb]Rb PET/CT and/or [68Ga]Ga-PSMA-11 PET/CT scans for measurement of TBF and [68Ga]Ga-PSMA-11-uptake, respectively. The analyses were mainly performed on the data from [68Ga]Ga-PSMA-11-guided VOIs; forty-eight lesions could be automatically drawn from the [68Ga]Ga-PSMA-11 hotspot, whereas the last four lesions (benign and ISUP GG-1) displayed too low activity and was drawn manually, guided by MRI. The cohort with MRI-guided VOIs was analysed for using an external modality to test the models on less biased data.Ga-PSMA-11 SUVmax and [82Rb]Rb SUVmax for separation of ISUP GG was tested using ordinal and nominal logistic fit models.As ISUP GG is an ordinal scale, Spearman\u2019s rank correlation (rho) was applied for analysis of correlations involving ISUP GG. For continuous variables, Pearson\u2019s correlation (r) was applied. For correlation analysis, ISUP GG\u2009=\u20090 was used for benign lesions in some analyses. Receiver operating characteristic (ROC) analyses were used for calculating area under the curve (AUC), sensitivity and specificity. Negative predictive value (NPV) and positive predictive value (PPV) were calculated according to standard definitions. McNemar\u2019s test for difference between paired nominal data was used to compare the diagnostic accuracies. The interaction between Ga-PSMA-11 and [82Rb]Rb PET in many cases as shown in Fig.\u00a068Ga]Ga-PSMA-11-uptake and TBF could be inhomogeneous within the MRI-guided tumour VOI. In other cases, the [82Rb]Rb- and/or the [68Ga]Ga-PSMA-11-activity extended beyond the border of the MRI VOI.The distribution of patients in different ISUP GG\u2019s is displayed in Table 68Ga]Ga-PSMA-11 SUV-measures and mean values of [82Rb]Rb SUV-measures alongside correlations with ISUP GG are provided in Table Median values for [lues of 8RbRb SUV-lesions.68Ga]Ga-PSMA-11 SUVmax and [82Rb]Rb SUVmax are plotted against ISUP GG in Fig.\u00a068Ga]Ga-PSMA-11 SUVmax and [82Rb]Rb SUVmax, respectively. The results of the ROC analyses are found in Tables 68Ga]Ga-PSMA-11 SUVmax or [82Rb]Rb SUVmax is above their cut-off this results in a positive test. In contrast, if both are below their cut-off this results in a negative test. ISUP GG-0 and GG-1 consist of only six lesions in total, which makes the results in Table [max and 8RbRb SUVmmax and 8RbRb SUVm68Ga]Ga-PSMA-11 SUVmax (log-transformed) and [82Rb]Rb SUVmax were correlated . The correlations for SUVmean and SUVpeak were equivalent. As shown in Fig.\u00a0[68Ga]Ga-PSMA-11 SUVmax and [82Rb]Rb SUVmax for separation of ISUP GG . McNemar\u2019s tests for difference between the combined analysis and [68Ga]Ga-PSMA-11 SUVmax alone (p\u2009=\u20090.008) and [82Rb]Rb SUVmax alone (p\u2009=\u20090.025) to separate ISUP GG\u2009>\u20092 from ISUP GG\u2009\u2264\u20092 and benign lesions were significant.We tested a combined model of [ GG Fig.\u00a0. Both pa GG Fig.\u00a0. The int68Ga]Ga-PSMA-11 hotspots. Furthermore, analysis with weight for the number of lesions per patient and the removal of multiple lesions per patient were performed. Analysis with registration of the inflammatory lesions was performed. Neither of these control analyses affected the results.The model was subsequently tested on the sub-cohort with MRI-guided VOIs, considering the mpMRI as an external modality, to minimize the bias introduced by defining VOIs based on [68Ga]Ga-PSMA-11 uptake and increased blood flow were detected [68Ga]Ga-PSMA-I&T ligand.The correlation between 6GaGa-PSMAn Score) and by Cn Score) . Anothern Score) includedn Score) used the68Ga]Ga-PSMA-11 SUVmax in the present study (Table 68Ga]Ga-PSMA-11 uptake, are also seen in the present study (Fig.\u00a0The AUC, sensitivity and specificity for [dy Table were in udy Fig.\u00a0a.68Ga]Ga-PSMA-11 activity. As proposed by Zamboglou et al. [68Ga]Ga-PSMA-11 PET/MRI.Tumour delineation is not always identical between the three modalities used in the present study, as the MRI VOI is occasionally expanded, especially by the Rb complements other studies using quantitative pharmacokinetic analysis of the dynamic contrast-enhanced series of MRI, which found an improvement in the PPV of mpMRI for primary local staging of PCa -H2O [18F]-flourocholine [11C]-donepezil [11C]-acetate and app15O]-H2O , [18F]-focholine , 49, Ga-PSMA-11- and [82Rb]Rb uptake suggests that the tumour PSMA uptake may be flow-limited (Fig.\u00a015O-H2O PET, which is gold standard of perfusion, to characterize the composition of the PSMA signal. If the PSMA uptake rate is an appropriate reflection of TBF in PCa, an approach with early dynamic plus late static PSMA PET scanning may examine both PSMA uptake and TBF and provide valuable additional information of biological potential of the tumour in selected patient categories. This could be a further step towards PSMA PET as a one-stop shop in PCa imaging to assess both whole body tumour burden and biological potential of the primary tumour.We found it important that the two tracers performed with such similarity in the analyses. The correlation between tumour [ted Fig.\u00a0. We founPSMA uptake and TBF both correlate with PCa aggressiveness, but the number of false negatives in separating significant from insignificant PCa makes the methods insufficient for clinical use as a sole risk stratification parameter. The present study demonstrated an association between PSMA uptake and TBF, but that they also provided complementary insights into tumour biology. Thus, the present study suggests that a combined assessment of PSMA uptake and TBF could significantly reduce the number of false negatives and, hence, allow non-invasive separation of significant from insignificant PCa."} +{"text": "Multidetector computed tomography (MDCT) plays a key role in patient assessment prior to transcatheter aortic valve implantation (TAVI). However, to date no consensus has been established on what is the optimal pre-procedural imaging protocol. Variability in pre-TAVI acquisition protocols may lead to discrepancies in aortic annulus measurements and may potentially influence prosthesis size selection.The current study evaluates the magnitude of differences in aortic annulus measurements using max-systolic, end-diastolic, and non-ECG-synchronized imaging, as well as the impact of method on prosthesis size selection.Fifty consecutive TAVI-candidates, who underwent retrospectively-ECG-gated CT angiography (CTA) of the aortic root, directly followed by non-ECG-synchronized high-pitch CT of the entire aorta, were retrospectively included. Aortic root dimensions were assessed at each 10% increment of the R-R interval (0\u2013100%) and on the non-ECG-synchronized scan. Dimensional changes within the cardiac cycle were evaluated using a 1-way repeated ANOVA. Agreement in measurements between max-systole, end-diastole and non-ECG-synchronized scans was assessed with Bland-Altman analysis.Maximal dimensions of the aortic root structures and minimum annulus-coronary ostia distances were measured during systole. Max-systolic measurements were significantly and substantially larger than end-diastolic (p<0.001) and non-ECG-synchronized measurements (p<0.001). Due to these discrepancies, the three methods resulted in the same prosthesis size selection in only 48\u201362% of patients.The systematic differences between max-systolic, end-diastolic and non-ECG-synchronized measurements for relevant aortic annular dimensions are both statistically significant and clinically relevant. Imaging strategy impacts prosthesis size selection in nearly half the TAVI-candidates. End-diastolic and non-ECG-synchronized imaging does not provide optimal information for prosthesis size selection. Systolic image acquisition is necessary for assessment of maximal annular dimensions and minimum annulus-coronary ostia distances. Multi-detector row computed tomographic (MDCT) assessment of the aortic root plays an important role in pre-procedural planning of transcatheter aortic valve implantation (TAVI)\u20133. AortiThe 2012 Society of Cardiovascular Computed Tomography (SCCT) guidelines for TAVI planning have not been conducive in establishing a golden standard pre-procedural MDCT protocol . The guiAlthough it is generally accepted that the aortic root is a dynamic structure, which constantly changes shape and dimensions throughout the cardiac cycle , 5, 6, tThe ambiguity has not been solved in the 2019 version of the SCCT guidelines, where the recommended acquisition method is dependent on manufacturer and/or type of CT-scanner . MoreoveThe aim of the current study is to describe the systematic and random discrepancies in aortic root measurements which occur when using max-systolic, end-diastolic, and non-ECG-synchronized high-pitch imaging, and to evaluate the impact on prosthesis size selection.Pre-procedural imaging of fifty consecutive TAVI-candidates with severe and symptomatic aortic valve stenosis, who underwent pre-TAVI MDCT assessment of the aortic root between 01/2011 and 08/2012, were retrospectively included and assessed for aortic root measurements in 2018. Patients with a history of valve replacement were excluded from the data analysis.All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. The approval for this study was obtained from the Institutional Review Board and the local medical ethical research committee (METC). Due to the retrospective nature of this study a waiver of written informed consent was issued by the Institutional Review Board. The data were coded and analyzed anonymously. The local METC reference number is METC:2018\u20130351.nd generation dual-source MDCT : a low-pitch retrospective ECG-gated helical scan of the aortic root, followed by a high-pitch non-ECG-synchronized computed tomographic angiography (CTA) of the whole aorta. 120ml pre-warmed, monomeric, non-ionic, low osmolar iodinated CM was injected according to a previously published CM protocol using a 4-point Likert scale: grade-1: non-diagnostic: impaired IQ precluding appropriate evaluation of the aortic root due to severe motion artifacts; grade-2: diagnostic: reduced IQ due to motion artifacts, but sufficient for aortic root dimension assessment; grade-3: good: presence of motion artifacts, but ability to reliably assess annular dimensions fully preserved; grade-4: excellent: complete absence of motion artifacts.P) and annular area (DA), were calculated with commonly used formulas[Pre-TAVI measurements were assessed in a blinded manner on the retrospectively ECG-gated scans at all 10%-time intervals of the cardiac cycle (0\u2013100%) and on the non-ECG-synchronized scan. The anatomical structures of the aortic root were assessed in accordance with SCCT expert consensus guidelines. Effecti formulas.S1 Appendix) for the balloon-expandable and for the self-expandable trans-catheter aortic valve . The ESV-guideline uses the annular area and DA for prosthesis sizing whilst the MCV-guideline uses the annular diameter and perimeter. MCV-guidelines define the perimeter as: annulus diameter x \u03c0. Prosthesis sizing was done for both the measured perimeter and for the calculated perimeter .Theoretical prosthesis size selection was based on the annular measurements assessed in the 20% phase (max-systole) and 70% phase reconstruction (end-diastole) on the retrospectively ECG-gated scan, 10, 11,Statistical analysis was conducted using Statistical Package for Social Sciences version 23.0 . Categorical variables are expressed as frequencies and percentages. Continuous variables are expressed using mean\u2009values \u00b1SD. Dimensional changes within the cardiac cycle are evaluated with a 1-way repeated ANOVA and Fischer\u2019s Least Significant Difference post-hoc test. Agreement between imaging strategies is evaluated using Bland-Altman analysis. This analysis enables evaluation of the magnitude of discrepancies in size measurements between max-systolic versus end-diastolic /non-ECG-synchronized measurements. A paired t-test is used to calculate the mean difference between methods and the SD of the difference. The mean difference can be interpreted as systematic measurement error and the SD of the difference can be used for calculation of the 95% limits of agreement (LOA). The 95%LOA gives a direct indication of the degree of random measurement error and allows clinicians to assess whether the limits are small enough to be confident that one measurement method can replace another method. A two-sided p-value <0.05 was considered statistically significant. Agreement between suggested prosthesis sizes is expressed as the percentage of patients in which the same valve size would be selected based on two or all three different imaging protocols.Table 2. The study population consisted of 26 female and 24 male patients with an average age of 81\u00b15 years.Baseline characteristics are summarized in The mean attenuation value of the retrospectively ECG-gated scans was 506\u00b1106HU, with mean SNR 13\u00b14 and mean CRN 13\u00b14. Cardiac motion artifacts were completely absent (grade-4) in 30 scans (60%), and non-significant (grade-3) artifacts occurred in 20 scans (40%). The mean attenuation value of the non-ECG-synchronized scans was 314\u00b179HU, with mean SNR 13\u00b13 and mean CRN 9\u00b13. Cardiac motion artifacts were completely absent (grade-4) in 12 scans (24%), grade-3 artifacts occurred in 28 scans (56%), and grade-2 artifacts in 10 scans (20%).S2 Appendix. The distances between the aortic annulus and the left and right coronary ostia were the only dimensions that reached their maximum during diastole (50%/60% phase) and their minimum during systole (10% phase). All other aortic annulus and aortic root dimensions reached their maximum dimensions in systole (10\u201330% phase).A complete overview of mean annular dimensions as measured on retrospectively ECG-gated scans (0\u2013100%) and non-ECG-synchronized scans are listed in Fig 1 shows box-plots of dimensional changes in annular dimensions used for prosthesis size selection during the cardiac cycle. These demonstrate that the largest dimensions of the aortic annulus were measured during the max-systolic phases (10 and 20% phase), and that values decrease during diastolic phases. Max-systolic measurements were significantly larger than end-diastolic (p<0.001) and non-ECG-synchronized measurements (p<0.001).Fig 1). For annular area and DA the maximum measurement in the 20% phase was significantly larger compared to measurements in all other phases.Differences between two cardiac phases in max-systole (10 and 20%) for short diameter and measured perimeter were non-significant varied per annular dimension. There were no significant differences in measurements of annular area and DA between 50\u201390% phases (p = 0.068\u20130.494), diastolic short diameter between 40\u201390% phases (p = 0.065\u20130.518), and measured perimeter between 50\u201380% phases (p = 0.071\u20130.736).Diastolic measurements did not significantly differ . The interval of non-significant dynamic differences in diastole or 90% phase . Non-ECG-synchronized short annular diameter did not significantly differ from measurements assessed in 40\u201390% phases (p = 0.311\u20130.977). Similarly, annular perimeter measured on non-ECG-synchronized scan did not significantly differ from that measured at 40% phase (p = 0.986) or 60\u201390% phases (p = 0.062\u20130.528).Non-ECG-synchronized values corresponded best with diastolic measurements. Annular area and DTable 3 shows results of the Blant-Altman analyses. A systematic difference between max-systolic, end-diastolic, and non-ECG-synchronized measurements of relevant annular dimensions. No proportional bias was found (p\u22650.161). The 95% limits of agreement indicate the range wherein 95% of the discrepancies between two methods are situated. For example, for DA, max-systolic measurements may be 0.1mm below and up to 2.6mm above end-diastolic measurements, and for perimeter max-systolic measurements may be 1.7mm to 7.6mm above end-diastolic measurements.The 20% and 70% phases were selected to represent max-systolic and end-diastolic measurements, respectively. calculated-perimeter was 9.1mm (95%LOA: 2.2\u201316.0), 11.1mm (95%LOA: 2.8\u201319.5), and 11.5mm (95%LOA: 2.8\u201320.1) for max-systolic, end-diastolic and non-ECG-synchronized assessment respectively, with no proportional bias (p\u22650.352).The mean differences between measured perimeter and showed that, in case of discrepancies, end-diastolic and non-ECG-synchronized measurements always led to smaller prosthesis size selection. Pairwise comparison between end-diastolic and non-ECG-synchronized measurements and MCV (based on diameter and perimeter). Pairwise comparison of max-systolic versus end-diastolic and non-ECG-synchronized measurements compared to max-systolic measurements. When based on DA and using end-diastolic and non-ECG-synchronized measurements, one size smaller ESV would be selected in 16 (32%) and 13 (26%) patients respectively, compared to max-systolic measurements.When based on annular area, non-ECG-synchronized and end-diastolic measurements led to a selection of MCV one size smaller in 15 (30%) and 17 (34%) patients respectively, compared to max-systolic sizing. One patient would be considered unsuitable for a TAVI procedure based on the non-ECG-synchronized short annular diameter of 17.0mm, while max-systolic and end-diastolic measurements would lead to selection of 23mm MCV valve.When based on short diameter, end-diastolic and non-ECG-synchronized measurements led to a selection of MCV prosthesis size in 24 patients (48%) based on measured perimeter, and in 27 patients (54%) based on calculated-perimeter. Nine patients (18%) would be considered unsuitable for a TAVI using all three protocols, as the max-systolic, end-diastolic and non-ECG-synchronized perimeter measurements were all greater than 81.7mm. An additional 9 patients would be excluded from TAVI based on the max-systolic measured perimeter. Compared to the max-systolic assessment, one size smaller MCV would be selected for 6 patients (12%) based on the end-diastolic measurement, and for 5 (10%) patients based on the non-ECG-synchronized perimeter measurement.Max-systolic, end-diastolic and non-ECG-synchronized measurements would result in selection of the same Different cardiac imaging strategies result in discrepant aortic annulus measurements, affecting the theoretical selection of TAVI prosthesis size in almost half the patients, regardless of prosthesis type. Furthermore, the annular dimension chosen for the prosthesis size selection can additionally influence disagreement between acquisition methods or even limit suitability of the patient for a TAVI procedure. To the best of our knowledge, this is the first study to evaluate the differences between aortic annulus measurements derived from the retrospectively-ECG-gated and non-ECG-synchronized high-pitch MDCT scans in light of prosthesis size selection.Our results contradict the study by Bertaso et.al., who found that differences in annular dimensions measured during systole and diastole were unlikely to alter patient suitability for TAVI or clinical decision making regarding prosthesis size. In theiPrecise assessment of maximal aortic root dimensions and the minimum distance between the annulus and coronary ostia are desirable for TAVI planning in order to minimize the risk of post-procedural paravalvular leakage and/or coronary ostium occlusion . AlthougS3 Appendix). This shows that even small dynamic changes in annular dimensions during diastole influence prosthesis sizing, despite often being statistically non-significant. Systematic discrepancies between max-systolic and end-diastolic measurements in the current study are described separately for each prosthesis sizing dimension. Estimating the dynamism of the aortic annulus dimension is helpful in cases where the assessment of maximal annular dimensions in systole is not possible or technically feasible.Despite the fact that non-ECG-synchronized annular measurements relevant for prosthesis sizing were best correlated to the diastolic phases of the cardiac cycle, the prosthesis sizes derived from the non-ECG-synchronized measurements differed to those based on end-diastolic measurements in 18\u201332% of patients, depending on annular dimension or prosthesis type . The annular perimeter has often been proposed as the best-suited annular dimension for prosthesis size selection, due to negligible dimensional changes in patients with aortic stenosis[P)[Annular dimensions used for prosthesis sizing significantly affect the degree of prosthesis size agreement between max-systolic, end-diastolic and non-ECG-synchronized protocols. In this study, a higher agreement was observed in the theoretically selected prosthesis size when sizing was based on annular area or D stenosis. Howeverenosis[P). HoweverThe main advantage of prospective ECG-triggered (end-diastolic) and non-ECG-synchronized imaging is the shorter image acquisition time compared to retrospective ECG-gating, which potentially reduces both CM volume and radiation dose. The firThe TAVI-candidate population has a multitude of intrinsic characteristics such as multiple comorbidities and a variety in presence and degree of annular calcifications at the stenotic valve. These patient characteristics cannot be influenced. On the other hand, the current study shows that variability in pre-TAVI imaging and sizing dimensions can also significantly influence clinical decisions. These differences resulting from imaging protocols are a variable in TAVI planning which can and should be taken out of the equation through standardization.This is a single-center retrospective study of a relatively small number of TAVI-candidates, which may not have been a representative sample of the general TAVI population. A gold standard reference protocol for pre-TAVI MDCT assessment is non-existent. Since we only evaluated the current industry recommendations for self- and balloon-expandable trans-catheter devices, our results may not be extrapolated to valves of other manufacturers or different type of deployment. For the purpose of this study, we assumed that the prosthesis would expand to its nominal size. The retrospective character of this study does not allow the comparison of clinical outcome or severity of paravalvular leakage between ECG-gated and high-pitch acquisition. Clinical outcome and sizing related complications were not investigated. This should be addressed in future studies.The systematic differences between max-systolic, end-diastolic and non-ECG-synchronized measurements for relevant aortic annulus dimensions are both statistically significant and clinically relevant. End-diastolic and non-ECG-synchronized high-pitch imaging does not provide optimal information for TAVI planning and prosthesis size selection. Systolic image acquisition is necessary in order to obtain maximal annular dimensions and smallest annulus-coronary ostia distances.S1 Appendix2 = square millimeter) * perimeter = diameter x \u03c0.Click here for additional data file.S2 AppendixA = area derived diameter; DP = perimeter derived diameter; LCA = distance from annulus to left coronary artery ostium; RCA = distance from annulus to left coronary artery ostium; STJ = sinotubular junction).(D(PDF)Click here for additional data file.S3 AppendixGreen cells represent cases with agreement in prosthesis size between end-diastole and non-ECG-synchronized measurementYellow cells represent cases where smaller prosthesis size would be selected based on non-ECG-synchronized measurement compared to end-diastolic measurementBlue cells represent cases where larger prosthesis size would be selected based on non-ECG-synchronized measurement compared to end-diastolic measurementDiagonally crossed cells indicate cases where unsuitable annular dimensions would be assessed.(PDF)Click here for additional data file.S1 File(DOCX)Click here for additional data file."} +{"text": "Whether this population is induced in patients infected with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is unknown. Recent reports have suggested that SARS-CoV-2 convalescent patients experience a rapid decay in their antigen-specific serum antibodies, raising concerns that humoral immunity against this virus may be short-lived8. Here we show that in patients who experienced mild infections (n=73), serum anti-SARS-CoV-2 spike (S) antibodies indeed decline rapidly in the first 3 to 4 months after infection. However, this is followed by a more stable phase between 4- and 8-months after infection with a slower serum anti-S antibody decay rate. The level of serum antibodies correlated with the frequency of S-specific long-lived BMPCs obtained from 18 SARS-CoV-2 convalescent patients 7 to 8 months after infection. S-specific BMPCs were not detected in aspirates from 11 healthy subjects with no history of SARS-CoV-2 infection. Comparable frequencies of BMPCs specific to contemporary influenza virus antigens or tetanus and diphtheria vaccine antigens were present in aspirates in both groups. Circulating memory B cells (MBCs) directed against the S protein were detected in the SARS-CoV-2 convalescent patients but not in uninfected controls, whereas both groups had MBCs against influenza virus hemagglutinin. Overall, we show that robust antigen specific long-lived BMPCs and MBCs are induced after mild SARS-CoV-2 infection of humans.Infection or vaccination induces a population of long-lived bone marrow plasma cells (BMPCs) that are a persistent and essential source of protective antibodies Early reports documenting rapidly declining antibody titers in convalescent SARS-CoV-2 patients in the first several months after infection suggested that protective immunity against SARS-CoV-2 may be similarly transient8. It was recently suggested that SARS-CoV-2 infection may fail to elicit a functional germinal center response, interfering with generation of the long-lived plasma cells that maintain durable antibody titers11. However, more recent reports analyzing samples collected approximately 4 to 6 months after infection indicate that SARS-CoV-2 antibody titers decline more slowly15. Because durable serum antibody titers are maintained by long-lived bone marrow plasma cells (BMPCs), we sought to determine whether they were detectable in SARS-CoV-2 convalescent patients approximately 7 months after infection.Reinfections by seasonal coronaviruses occur 6\u201312 months after the previous infection, indicating that protective immunity against these viruses may be short-livedBlood samples were collected approximately 1 month after onset of symptoms from seventy-three SARS-CoV-2 convalescent volunteers , the majority of whom had experienced mild illness probes (Memory B cells (MBCs) form the other component of humoral immune memory. Upon antigen re-exposure, MBCs rapidly expand and differentiate into antibody-secreting plasmablasts. We examined the frequency of SARS-CoV-2 specific circulating MBC pool in the convalescent patients as well as in the healthy controls. We stained peripheral blood mononuclear cells with fluorescently labeled S probes and determined the frequency of S-binding MBCs among isotype-switched IgD) probes . S-bindi) probes . S-bindi) probes .5. This is consistent with recent data showing increased levels of somatic hypermutation in MBCs targeting the receptor binding domain of the S protein in SARS-CoV-2 convalescent patients at 6 months compared to 1 month after infection15.This study sought to determine whether SARS-CoV-2 infection induced antigen-specific long-lived BMPCs in humans. We detected SARS-CoV-2 S-specific BMPCs in aspirates from 14 of 18 convalescent patients, and in none from the 11 control participants. Frequencies of anti-S IgG BMPCs modestly correlated with serum IgG titers 7\u20138 months after infection. Finally, we showed that S-binding MBCs in blood of convalescent patients are present at similar frequencies as those directed against influenza virus HA. Altogether, our results are consistent with SARS-CoV-2 infection eliciting a canonical T-dependent B cell response, in which an early transient burst of extrafollicular plasmablasts generates an early wave of serum antibodies that decline relatively quickly once a peak titer is reached. This is followed by more stably maintained serum antibody levels that are supported by long-lived BMPCs. Our data suggest that SARS-CoV-2 infection induces a germinal center response in humans because long-lived BMPCs are thought to be predominantly germinal center-derived24, we do not know the fraction of the S-specific BMPCs detected 7 months after infection in our study encoding neutralizing antibodies. It is important to note, however, that correlation between serum anti-S IgG binding and neutralization titers has been documented25. Further studies will be required to determine the epitopes targeted by BMPCs and MBCs and how preexisting immunity to human coronaviruses could alter the immune response to SARS-CoV-2 infection and immunization. Finally, while our data document a robust induction of long-lived BMPCs after SARS-CoV-2 infection, it is critical to note that our convalescent patients mostly experienced mild infections. Our data are consistent with a recent report showing that individuals who recovered rapidly from symptomatic SARS-CoV-2 infection generated a robust humoral immune response26. Therefore, it is possible that more severe SARS-CoV-2 infections could lead to a different outcome with respect to long-lived BMPC frequencies due to dysregulated humoral immune responses. This, however, has not been the case in survivors of the 2014 West African Ebola virus outbreak in whom severe viral infection induced long-lasting antigen-specific serum IgG antibodies27.To our knowledge, the current study provides the first direct evidence for induction of antigen specific BMPCs after a viral infection in humans, but it does have some limitations. Although we detected anti-S IgG antibodies in serum 7 months after infection in all 18 of the convalescent donors from whom we obtained bone marrow aspirates, we failed to detect S-specific BMPCs in four donors. Serum anti-S antibody titers in those four donors were low, suggesting that S-specific BMPCs may potentially be present at very low frequencies that are below our limit of detection. Another limitation is while SARS-CoV-2 S protein is the main target of neutralizing antibodies28. Encouragingly, the frequency of S-binding circulating MBCs 7-months after infection was higher than those directed against contemporary influenza HA antigens. These data indicate that even at that late time point, more MBC and BMPC precursors may still be emerging from ongoing germinal center reactions. We have recently shown that germinal center reactions can persist for at least two months in the draining lymph nodes after non-adjuvanted seasonal influenza virus vaccination29. It is not unreasonable to assume that SARS-CoV-2 infection would foster a more persistent germinal center reaction, but future studies will be needed to precisely determine the dynamics of such responses. Overall, our data provide strong evidence that SARS-CoV-2 infection in humans robustly establishes the two arms of humoral immune memory: long-lived BMPC and MBCs. These findings provide an immunogenicity benchmark for SARS-CoV-2 vaccines and a foundation for assessing the durability of primary humoral immune responses induced after viral infections in humans.Long-lived BMPCs provide the host with a persistent source of preformed protective antibodies and are therefore needed to maintain durable immune protection. However, longevity of serum anti-S IgG antibodies is not the only determinant of how durable immune-mediated protection will be. Indeed, isotype-switched MBCs can rapidly differentiate into antibody secreting cells upon pathogen reexposure, offering a second line of defenseAll studies were approved by the Institutional Review Board of Washington University in St. Louis. Written consent was obtained from all participants. Seventy-four participants who had recovered from SARS-CoV-2 infection and eleven controls without SARS-CoV-2 infection history were enrolled . Blood s30. Briefly, mammalian cell codon-optimized nucleotide sequences coding for the soluble version of the spike protein of SARS-CoV-2 including a C-terminal thrombin cleavage site, T4 foldon trimerization domain, and hexahistidine tag cloned into mammalian expression vector pCAGGS. The spike protein sequence was modified to remove the polybasic cleavage site (RRAR to A) and two stabilizing mutations were introduced . Recombinant proteins were produced in Expi293F cells (ThermoFisher) by transfection with purified DNA using the ExpiFectamine 293 Transfection Kit (ThermoFisher). Supernatants from transfected cells were harvested 3 days post-transfection, and recombinant proteins were purified using Ni-NTA agarose (ThermoFisher), then buffer exchanged into phosphate buffered saline (PBS) and concentrated using Amicon Ultracel centrifugal filters (EMD Millipore).For flow cytometry staining, recombinant S was labeled with Alexa Fluor 647-NHS ester (Thermo Fisher). Recombinant HA from A/Brisbane/02/2018 (a.a.18\u2013529) and B/Colorado/06/2017 (a.a. 18\u2013546) expressed in 293 cells were purchased from Immune Technology and biotinylated using the EZ-Link Micro NHS-PEG4-Biotinylation Kit (Thermo Fisher); excess biotin and Alexa Fluor 647 were removed using 7-kDa Zeba desalting columns (Pierce).For ELISpot, plates were coated with Flucelvax Quadrivalent 2019/2020 seasonal influenza virus vaccine (Sequiris), tetanus/diphtheria vaccine (Grifols), or recombinant soluble Spike protein derived from SARS-CoV-2, expressed as previously describedex-vivo ELISpot was performed to determine the number of total, vaccine-binding, or recombinant Spike-binding IgG- and IgA-secreting cells present in BMPC and PBMC samples using IgG/IgA double-color ELISpot Kits according to the manufacturer\u2019s instructions. ELISpot plates were analyzed using an ELISpot counter (Cellular Technologies Ltd.).Direct Assays were performed in 96-well plates coated with 100 \u03bcL of Flucelvax 2019/2020 or recombinant Spike protein in PBS, and plates were incubated at 4 \u00b0C overnight. Plates were then blocked with 10% FBS and 0.05% Tween20 in PBS. Serum or plasma were serially diluted in blocking buffer and added to the plates. Plates were incubated for 90 min at room temperature and then washed 3 times with 0.05% Tween-20 in PBS. Goat anti-human IgG-HRP was diluted in blocking buffer before adding to wells and incubating for 60 min at room temperature. Plates were washed 3 times with 0.05% Tween20 in PBS, and then washed 3 times with PBS before the addition of peroxidase substrate . Reactions were stopped by the addition of 1 M HCl. Optical density measurements were taken at 490 nm. The half-maximal binding dilution for each serum or plasma sample was calculated using nonlinear regression (Graphpad Prism v8). The limit of detection was defined as 1:30.P-values < 0.05 were considered significant.Spearman\u2019s correlation coefficients were estimated to assess the relationship between 7-month anti-S and anti-influenza virus vaccine IgG titers and frequencies of BMPCs secreting IgG specific for S and influenza virus vaccine, respectively. Decay rates based on the log-transformed S and influenza virus vaccine titers were estimated using a longitudinal linear mixed model approach. Time since symptom onset (days) was included as a fixed effect in these models and subject-specific random intercepts and slopes were included to adjust for repeated measurements. Exponential decay rate was estimated as the slope for the fixed effect and the half-life was estimated as ln(0.5)/decay rate. In addition to the analysis of all time points in a single model, we assessed whether there was evidence of a change in decay rate over the course of observation for anti-S titer by separately analyzing only the first and second measurements, and only the second and third measurements. These models only included 2 time points per person, and we removed the random slope parameter from these models. This resulted in a single mixed model for the anti-influenza virus vaccine titer, and three separate models for the anti-S titer. All analyses were conducted using SAS 9.4 and Prism 8.4 (Graphpad), and Staining for flow cytometry analysis was performed using cryopreserved PBMCs. Cells were stained for 30 min on ice with biotinylated recombinant HAs diluted in in 2% FBS and 2 mM EDTA in PBS (P2), washed twice, then stained for 30 min on ice with Alexa 647-conjugated S, IgA-FITC , IgG-BV480 , IgD-SB702 , CD38-BB700 , CD20-Pacific Blue , CD4-BV570 , CD24-BV605 , streptavidin-BV650, CD19-BV750 , CD71-PE , CXCR5-PE-Dazzle 594 , CD27-PE-Cy7 , IgM-APC-Fire750 , CD3-APC-Fire810 , and Zombie NIR diluted in Brilliant Staining buffer (BD Horizon). Cells were washed twice with P2 and acquired on an Aurora using SpectroFlo v2.2 (Cytek). Flow cytometry data were analyzed using FlowJo v10 (Treestar). In each experiment, PBMC were included from convalescent and control participants."} +{"text": "In mice, antibodies to the SARS-CoV-2 RBD were elicited just as well by mosaic particles as by homotypic nanoparticles. The mosaic nanoparticles elicited antibodies that, beyond recognizing the strains displayed, also recognized mismatched strains.In the past 20 years, three betacoronaviruses thought to have originated in bats have caused devastating disease in humans. The global pandemic caused by the latest such virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), highlights the need to protect against other strains that could present a threat to humans. Cohen Science, this issue p. 735 Immunizing with nanoparticles that display diverse coronavirus receptor binding domains elicits cross-reactive and neutralizing antibody responses. Protection against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and SARS-related emergent zoonotic coronaviruses is urgently needed. We made homotypic nanoparticles displaying the receptor binding domain (RBD) of SARS-CoV-2 or co-displaying SARS-CoV-2 RBD along with RBDs from animal betacoronaviruses that represent threats to humans (mosaic nanoparticles with four to eight distinct RBDs). Mice immunized with RBD nanoparticles, but not soluble antigen, elicited cross-reactive binding and neutralization responses. Mosaic RBD nanoparticles elicited antibodies with superior cross-reactive recognition of heterologous RBDs relative to sera from immunizations with homotypic SARS-CoV-2\u2013RBD nanoparticles or COVID-19 convalescent human plasmas. Moreover, after priming, sera from mosaic RBD\u2013immunized mice neutralized heterologous pseudotyped coronaviruses as well as or better than sera from homotypic SARS-CoV-2\u2013RBD nanoparticle immunizations, demonstrating no loss of immunogenicity against particular RBDs resulting from co-display. A single immunization with mosaic RBD nanoparticles provides a potential strategy to simultaneously protect against SARS-CoV-2 and emerging zoonotic coronaviruses. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a newly emergent betacoronavirus, resulted in a global pandemic in 2020, infecting millions and causing the respiratory disease COVID-19 (Most current SARS-CoV-2 vaccine candidates include the spike trimer (S), the viral protein that mediates target cell entry after one or more of its receptor binding domains (RBDs) adopts an \u201cup\u201d position to bind a host receptor . The RBDMultivalent display of antigen enhances B cell responses and can provide longer-lasting immunity than monovalent antigens . ParticlACE2 target cells . Neutralization and ELISA titers were significantly correlated (fig. S4), which implies that ELISAs are predictive of neutralization results when viral entry receptor usage prevents accurate pseudotyped neutralization assays.We immunized mice with soluble SARS-CoV-2 spike trimer (SARS-2 S), nanoparticles displaying only SARS-2 RBD (homotypic SARS-2), nanoparticles co-displaying RBDs , or unconjugated nanoparticles (mi3). Immunoglobulin G (IgG) responses were evaluated after prime or boost immunizations by enzymMice immunized with soluble SARS-2 S trimer showed no binding or neutralization except for autologous responses against SARS-2 after boosting , consistWe next compared serum responses against matched RBDs (RBDs present on an injected nanoparticle) versus mismatched RBDs (RBDs not present on an injected nanoparticle) . In otheIn an experiment that demonstrated the advantages of mosaic versus homotypic SARS-2 nanoparticles, sera from mosaic-8\u2013immunized mice bound SHC014 and WIV1 RBDs significantly better after priming than sera from homotypic SARS-2\u2013immunized mice and retained better binding to SHC014 RBD after boosting . Thus, tTo further address whether RBD nanoparticles elicited antibodies that recognized totally mismatched strains and SARS-CoV-2 RBD mutants, we evaluated sera for binding to SARS, Yun11, BM-4831, and BtKY72 RBDs ; SARS-2 + splenic B cells from RBD nanoparticle\u2013boosted animals could simultaneously recognize RBDs from SARS-2 and Rs4081 (related by 70% sequence identity) (Using flow cytometry, we investigated the potential for cross-reactive recognition\u2014specifically, whether B cell receptors on IgGTo compare antibodies elicited by RBD nanoparticle immunization to antibodies elicited by SARS-CoV-2 infection, we repeated ELISAs against the RBD panel using IgGs from COVID-19 plasma donors . Most ofOur results confirm that multimerization of RBDs on nanoparticles enhances immunogenicity relative to soluble antigen ("} +{"text": "The recently emerged SARS-CoV-2 virus is responsible for the ongoing COVID-19 pandemic that has rapidly developed into a global public health threat. Patients severely affected with COVID-19 present distinct clinical features, including acute respiratory disorder, neutrophilia, cytokine storm, and sepsis. In addition, multiple pro-inflammatory cytokines are found in the plasma of such patients. Transcriptome sequencing of different specimens obtained from patients suffering from severe episodes of COVID-19 shows dynamics in terms of their immune responses. However, those host factors required for SARS-CoV-2 propagation and the underlying molecular mechanisms responsible for dysfunctional immune responses during COVID-19 infection remain elusive. In the present study, we analyzed the mRNA-long non-coding RNA (lncRNA) co-expression network derived from publicly available SARS-CoV-2-infected transcriptome data of human lung epithelial cell lines and bronchoalveolar lavage fluid (BALF) from COVID-19 patients. Through co-expression network analysis, we identified four differentially expressed lncRNAs strongly correlated with genes involved in various immune-related pathways crucial for cytokine signaling. Our findings suggest that the aberrant expression of these four lncRNAs can be associated with cytokine storms and anti-viral responses during severe SARS-CoV-2 infection of the lungs. Thus, the present study uncovers molecular interactions behind the cytokine storm activation potentially responsible for hyper-inflammatory responses in critical COVID-19 patients. COVID-19 is a severe acute respiratory disease caused by SARS-CoV-2, a recently identified member of the Coronaviridae family ,4,5. TheImmune cell development and activation depend on gene expression\u2019s dynamic regulation through complex transcriptional and post-transcriptional mechanisms . Long noGiven the role(s) of lncRNAs in host cell anti-viral inflammatory response regulation, we sought to identify lncRNAs that are co-expressed with human genes involved in immune-related processes during SARS-CoV-2 infection in the lungs. We thus identified common differentially expressed (DE) mRNAs and lncRNAs from various publicly available SARS-CoV-2 infected lung transcriptome datasets. We subsequently identified a key lncRNA-mRNA module enriched for elements of different immune-related pathways related to cytokine signaling based on weighted gene co-expression analysis (WGCNA). Network analysis also revealed four lncRNAs as potential hubs, thereby pointing to the possible association of these lncRNAs with cytokine signaling during SARS-CoV-2 infection and potential involvement in hyper-inflammatory responses during SARS-CoV-2 infection of the lungs. These findings could thus advance understanding of complex interactions behind the immune dysfunction experienced in severe cases of COVID-19. https://bigd.big.ac.cn/, accessed on 2 February 2021), Beijing Institute of Genomics (accession number CRA002390) [N = 3 per group). The raw reads were retrieved and converted to FASTQ using the SRA toolkit, version 2.10.7. Several sets of raw sequence reads were retrieved from Gene Expression Omnibus (GEO) datasets. GEO dataset GSE147507 contains data from cell lines infected with SARS-CoV-2, including Calu-3 adenocarcinoma cells and A549 cells supplemented with a vector expressing ACE2. GEO dataset GSE139516 contains data from the Calu3 cell line infected with Middle East respiratory syndrome coronavirus (MERS-CoV), while GEO dataset GSE148729 contains data from the Calu3 cell line infected with SARS-CoV-1 and SARS-CoV-2. Raw sequence data from BALF of COVID-19 patients were retrieved from the Genome Sequence Archive (A002390) . RNA-seqp-values. Genes with FDR < 0.05 and fold-change \u2265 2 were considered as being differentially expressed.Raw reads were aligned to the latest human genome build (GRCh38.p13), using reference annotations derived from GENCODE release 35 by STAR, v2.7.5 . To incrhttp://www.cytoscape.org/, accessed on 2 February 2021) [WGCNA approaches were usery 2021) was usedry 2021) plug-in ry 2021) . p-value cutoff <0.05. KEGG and REACTOME pathway databases were consulted during pathway enrichment analysis. Fisher\u2019s exact test followed by Benjamini\u2013Hochberg multiple testing correction was employed for selecting the GO category and the relevant pathway. After identifying the different modules in the co-expression network, gene set enrichment and pathway enrichment analysis were performed to explore functional categories associated with modules. Gene ontology (GO) and pathway enrichment analyses were performed using ShinyGO , MetascaTo understand host transcriptional dynamics in response to SARS-CoV-2 infection of the lungs, we employed the publicly available RNA-seq datasets from BALF samples from COVID-19 patients and SARShttp://coronascape.org, accessed on 2 February 2021)) [Gene set enrichment analysis was next conducted using Coronascape ) to reveaGenes involved in the same pathway or that participate in similar biological processes often present correlated gene expression patterns . To iden2 was maximized. Co-expression modules were then determined by the dynamic tree cut procedure using the dynamic branch-cutting algorithm with a robust measure of interconnectedness, using DynamicTreeCut and the WGCNA R library. A total of three modules were identified in the network, with each module being assigned a unique color label , as represented in To identify potential lncRNA-mRNA interaction networks among DE genes crucial for cytokine signaling during SARS-CoV-2 infection, co-expression analysis based on WGCNA was performed . Module To explore the biological functions of the three modules, functional enrichment analysis based on GO biological processes and the KEGG and Reactome pathways was performed using ShinyGO with an. The resBased on the above analysis and considering the importance of the blue co-expression module for cytokine signaling, a sub-network based on this co-expression module was constructed. Four lncRNAs in this module were significantly up-regulated in the SARS-CoV-2-infected samples. To understand the lncRNAs expression profiles and assess their relationships with protein-coding genes within this module, the co-expression network was constructed, and hub nodes in this module were analyzed. A sub-network of the four lncRNAs and 105 protein-coding genes which were strongly co-expressed in the blue module, was assembled a. Those \u221230) and MERS-infected Calu3 cells. WAKMAR2 was found to be differentially expressed only in MERS-infected Calu3 cells, while ENSG00000271646 was found to be differentially expressed only in SARS-infected Calu3 cells. Furthermore, we analyzed how many interactors of these four lncRNAs from the co-expression network derived from the blue module are also differentially expressed in SARS and MERS-infected cells. Among the 105 protein-coding interactors from the blue module, we observed 74 DE protein-coding genes in SARS-CoV-infected cells and 58 DE protein-coding genes in MERS-infected cells and their interactors in the SARS and MERS-infected Calu3 cell line to understand the degree of similarity of interactions mediated by these lncRNAs during other human coronavirus infections. We observed that among these four lncRNAs, EGOT was found to be significantly differentially expressed in both SARS correspond to the top nodes in this sub-network. This finding suggests that these four lncRNAs are strong interactors of cytokine-related genes in the network and their potential interactions are crucial for generating dysfunctional immune responses. Recent studies demonstrated that low expression of WAKMAR2 up-regulates NF-\u03baB signaling and enhances inflammatory cytokine production by keratinocytes in chronic wounds . In respOverall, our results suggest that these four DE lncRNAs are associated with cytokine signaling and hyper-inflammatory responses during severe SARS-CoV-2 infection of the lungs. Furthermore, our study reveals that the molecular mechanisms of dysregulated immune responses due to the SARS-CoV-2 infection are different from SARS and MERS, leading to the severity of the response to SARS-CoV-2. This study reveals potential associations of lncRNAs in cytokine signaling during the response to severe SARS-CoV-2 infection in the lungs, which indicates the translational potential of those lncRNAs. Our analysis provides novel insight in the search for potential mechanisms underlying immune dysfunction in response to cytokine storms during severe SARS-CoV-2 infection and could also shed light on how cytokine inhibitors could be useful for treating acute cases of COVID-19.The results reported here provide novel transcriptomic insight into host responses upon severe SARS-CoV-2 infection. Comprehensive lncRNA and mRNA transcriptomes in SARS-CoV-2-infected human lung epithelial cell lines and BALF from COVID-19 patients were profiled. Through co-expression network analysis, four DE lncRNAs were identified as hub nodes that strongly correlated to the protein-coding genes in this network and enriched for different immune-related processes related to cytokine and interferon signaling. These findings reveal the potential role of lncRNAs in regulating anti-viral responses during severe SARS-CoV-2 infection. This study could thus provide valuable transcriptomic insight into pro-inflammatory cytokine production and the hyper-inflammation caused by severe SARS-CoV-2-induced infection of the lungs. Still, further experimental studies are necessary to elucidate the specific functions of these lncRNAs in COVID-19 pathogenesis."} +{"text": "Antibodies are becoming a frontline therapy for SARS-CoV-2, but the risk of viral evolutionary escape remains unclear. Here we map how all mutations to SARS-CoV-2\u2019s receptor-binding domain (RBD) affect binding by the antibodies in Regeneron\u2019s REGN-COV2 cocktail and Eli Lilly\u2019s LY-CoV016. These complete maps uncover a single amino-acid mutation that fully escapes the REGN-COV2 cocktail, which consists of two antibodies targeting distinct structural epitopes. The maps also identify viral mutations that are selected in a persistently infected patient treated with REGN-COV2, as well as in lab viral escape selections. Finally, the maps reveal that mutations escaping each individual antibody are already present in circulating SARS-CoV-2 strains. Overall, these complete escape maps enable immediate interpretation of the consequences of mutations observed during viral surveillance. AntibodMost leading anti-SARS-CoV-2 antibodies target the viral receptor-binding domain (RBD), which mediates binding to ACE2 receptor , 6. We rhttps://jbloomlab.github.io/SARS-CoV-2-RBD_MAP_clinical_Abs/). REGN10933 and REGN10987 are escaped by largely non-overlapping sets of mutations in the RBD\u2019s receptor-binding motif . As might be expected, escape mutations generally occur in the antibody-RBD interface. However, structures alone are insufficient to predict which mutations mediate escape. For example, LY-CoV016 uses both its heavy and light chains to bind a wide epitope overlapping the ACE2-binding surface, but escape is dominated by mutations at RBD residues that contact the heavy chain CDRs (To determine if the escape maps could be rationalized from the structural interfaces of the antibodies and RBD, we projected the maps onto crystal or cryo-EM structures (ain CDRs , S6E\u2013G. ain CDRs , S6A\u2013D. ain CDRs . So overain CDRs , S6D,G.Overall, we have completely mapped mutations that escape some of the leading antibodies used to treat COVID-19. These maps demonstrate that prior characterization of escape mutations was incomplete: for instance, overlooking a single amino-acid mutation that escapes both antibodies in the REGN-COV2 cocktail, and failing to identify most mutations that arose in a persistently infected patient treated with the cocktail. Of course, our maps still do not answer the most pressing question: will SARS-CoV-2 evolve widespread resistance to these antibodies? While the presence of escape mutations in the patient treated with REGN-COV2 is ominous, other viruses that typically cause self-limiting acute infections undergo extensive within-patient evolution only in long infections of immunocompromised patients and not Supplement 11"} +{"text": "It can be the end result of persistent secondary hyperparathyroidism and is most commonly observed in patients with long-standing chronic kidney disease (CKD) and often after renal transplantation. Untreated HPT can lead to progressive bone disease, fibrocystic osteitis, and soft-tissue calcifications, along with other severe complications. In the 2009 Kidney Disease Improving Global Outcomes (KDIGO) guidelines, CKD-Mineral and Bone Disorder (CKD-MBD) is used to describe the broader clinical syndrome encompassing mineral, bone, and calcific cardiovascular abnormalities that develop as a complication of CKD. We report a 62-year-old female with a severe HPT evolved from advanced chronic kidney disease . Patient was evaluated with multimodality nuclear medicine functional imaging to assess hyperfunctioning parathyroid glands and bone lesions. Tc-99m-methoxyisobutylisonitrile (MIBI) dual-phase scintigraphy, Tc-99m-methylenediphosphonate (MDP) bone scan and Howev results ,10. Our"} +{"text": "A coding-complete genome sequence of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) isolate was revealed. The sample for the virus was isolated from a female patient from Dhaka, Bangladesh, suffering from coronavirus disease-2019 (COVID-19). A coding-complete genome sequence of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) isolate was revealed. The sample for the virus was isolated from a female patient from Dhaka, Bangladesh, suffering from coronavirus disease-2019 (COVID-19). Coronaviridae family and Betacoronavirus genus, is the causative agent of pandemic coronavirus disease-2019 (COVID-19). In Bangladesh, the rate of positive cases and the death toll from COVID-19 are increasing at an alarming rate (https://corona.gov.bd/). To understand the genomic characteristics of SARS-CoV-2 in Bangladesh, several isolates have been sequenced and deposited in GISAID (https://www.gisaid.org/). However, those isolates have been sequenced using a next-generation sequencing platform, except for the one we are reporting. In this study, we sequenced the viral genome by Sanger sequencing technology, which is a gold standard method and is necessary for thorough genomic analysis (Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a member of the The isolate (SARS-CoV-2/human/BGD/NIB_01/2020) was collected from an oropharyngeal specimen on 11 May 2020. The patient was a 28-year-old saleswoman who tested positive (via reverse transcriptase PCR [RT-PCR]) for COVID-19 with symptoms of cough, mild fever, and throat congestion. . The viral RNA was extracted directly from the patient\u2019s specimen using the PureLink viral RNA/DNA minikit (Invitrogen). The viral RNA was then converted into cDNA using a SuperScript VILO cDNA synthesis kit (Invitrogen).To cover the whole genome of the virus, 48 pairs of primers were designed by following two conditions: (i) their sequence is conserved among all the available SARS-CoV-2 isolates, and (ii) the terminal of the amplicons will overlap the adjacent amplicon . These pThe raw reads were assembled using DNA Sequence Assembler version 4 2013) (Heracle BioSoft) and verified with SeqMan Pro version 14.1 . After assembly, 48 contigs with 94 overlapping regions were obtained. These overlapping regions were visualized using CLC Genomics Workbench version 20.0.4 and merged with EMBOSS: merger (013 (HeraMT476385.1). From NCBI, the FASTA sequences of 7 mostly similar genomes from Bangladesh, India, Sri Lanka, and the United States were taken along with the reference genome. Another 16 genomes of SARS-CoV-2 that were isolated in Bangladesh were collected from GISAID (https://www.gisaid.org/). The genomes were aligned with MAFFT version 7 using default parameters RNA that is 29,724 nucleotides long. The NCBI BLASTN program showed tThe genome has 8 nucleotide differences from the closest isolate. Interestingly, except for isolate SARS-CoV-2/human/BGD/CHRF0001/2020, the other strains of SARS-CoV-2 from Bangladesh showed separate clades and distant genetic relations. The tree also demonstrated that our viral genome and three isolates from the United States share an ancestor .MT509958.The complete nucleotide sequence of this SARS-CoV-2 isolate (SARS-CoV-2/human/BGD/NIB_01/2020) has been deposited in GenBank under the accession number"} +{"text": "Biological membranes, in addition to being a cell boundary, can host a variety of proteins that are involved in different biological functions, including selective nutrient transport, signal transduction, inter- and intra-cellular communication, and cell-cell recognition. Due to their extreme complexity, there has been an increasing interest in developing model membrane systems of controlled properties based on combinations of polymers and different biomacromolecules, i.e., polymer-based hybrid films. In this review, we have highlighted recent advances in the development and applications of hybrid biomimetic planar systems based on different polymeric species. We have focused in particular on hybrid films based on (i) polyelectrolytes, (ii) polymer brushes, as well as (iii) tethers and cushions formed from synthetic polymers, and (iv) block copolymers and their combinations with biomacromolecules, such as lipids, proteins, enzymes, biopolymers, and chosen nanoparticles. In this respect, multiple approaches to the synthesis, characterization, and processing of such hybrid films have been presented. The review has further exemplified their bioengineering, biomedical, and environmental applications, in dependence on the composition and properties of the respective hybrids. We believed that this comprehensive review would be of interest to both the specialists in the field of biomimicry as well as persons entering the field. Biological membranes play crucial roles in cellular protection as well as in the control and the transport of nutrients . BiologiThe goal of this review was to highlight some of the recent significant advances in the development of hybrid biomimetic polymer-based films holding great potential for addressing the current niches and unanswered questions from the areas of biomedical, biosensing, or environment-related applications. We have presented these hybrid films in particular regarding their applications for targeted drug delivery, stimuli-responsive coatings, tissue engineering, and biosensing by utilizing their diverse physicochemical properties, such as catalytic and optical activity, as well as stimuli-responsive changes in their morphology, chemical composition, or electrical properties. We have focused in particular on hybrid films based on (i) polyelectrolytes, (ii) polymer brushes, as well as (iii) tethers and cushions formed from synthetic polymers, and (iv) block copolymers in combination with lipids, biomolecules, and chosen nanoparticles .First, we have described biomimetic hybrids based on polyelectrolytes and diverse biomacromolecules, natural polymers, and nanoparticles, followed by the presentation of solid-supported films based on polymer brushes produced either via bottom-up or top-down approach. Next, polymer-lipid tethered and cushioned composite films have been discussed with respect to their composition and properties. In the fourth part, hybrid membranes composed of block copolymers and lipids have been presented. For each system, the assembly approach has been presented, in particular layer-by-layer, bottom-up, and top-down , Langmuir\u2013Blodgett and Langmuir\u2013Schaefer methods, and some of the analytical methods commonly utilized for characterization of such films have been mentioned. The review has further exemplified their distinct properties and the properties-related applications for the development of biosensors ,28,29,30Polyelectrolytes (PE) are polymers, which, when dissolved in a polar solvent like water, bear a number of charged groups covalently linked to them, and they are classified as anionic, cationic, or ampholytic, according to whether the ionized polymer carries negative, positive, or both charges, respectively ,63,64,65The self-assembly of the films based on polyelectrolytes is typically achieved via layer-by-layer (LbL) deposition, which takes advantage of the unbalanced charge on the polyelectrolyte species while in an aqueous solution by alternating electrostatic layer-by-layer adsorption of cationic and anionic PE onto a substrate ,67,68,69Less commonly utilized methods for the fabrication of PE-based layers include surface-initiated polymerization , chemicaFor the characterization of the PE-hybrid films, standard spectroscopic techniques are typically used for identification and analysis of organic compounds, like Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR) , mass spHybrid PE-based composite films contain a variety of functional compounds, either natural, such as proteins , enzymes2 3\u2212/4\u2212 and [Ru(NH3)6]3+/2+ [63\u2212/4\u2212) at film assemblies anchored with a monolayer of lipid-like 11-mercaptoundecanoic acid (MUA) and augmented with PLL/PSS bilayers and citrate-stabilized AuNPs [Incorporation of metal NPs into the layers has been reported to facilitate and/or enhance the electron transport within the layer and ensure the electrical contact of biomolecules deposited onto an electrode surface. Incorporation of gold nanoparticles (AuNPs) via self-assembly of methanesulfonic acid (MSA)-stabilized AuNPs and PLL onto Au electrode has led to high sensitivity to the charge of the outermost layer for the permeability of the probe ions\u2014[Fe(CN))6]3+/2+ ,146. AuNed AuNPs has reveDue to the presence of an accumulated charge resulting from the dissociation of nucleic bases in an aqueous solution, nucleic acids can be considered as polyelectrolyte species. Many of their physical properties are strongly dependent on electrostatic interactions between the negatively charged DNA and the surrounding positively charged counterions . Complexb-PEG-b-PAEU), and loading them with DNA polyplexes on heparin has been applied for intradermal cutaneous delivery of DNA vaccines dibromide} bromide) (PFPBr)-AuNPs-ssDNA . M. M183]. The bottom-up approach (grafting \u201cfrom\u201d) refers to the in-situ polymerization from reactive species present on the substrate surface, such as immobilized initiators . DependiCurrently, ATRP is the most extensively used polymerization method for grafting polymers from solid supports to form polymer brushes since it can be tailored depending on the reversible activation-deactivation equilibrium between a metal complex and a polymer chain-end in the presence of the monomer. By using this method, poly-phenoxyethylmethacrylate (PHEMA), poly(hydroxypropylmethacrylate) (PHPMA), and poly(carboxybetaine acrylamide) (PCBAA) brushes of different lengths have been formed on glass substrates . PHEMA ab-poly(methyl methacrylate) (I-PS-b-PMMA) block copolymer brushes in situ using Cu (II)/ligand complex . It Phosphatidylserine and phosphatidylcholine (PC) membranes have been combined with PAH and PSS multilayers to allow the incorporation of colloidal nanoparticles with specific biological properties based on their content of viruses (Influenza A/PR8) or virus-like particles (Rubella) . These ab-PEO/DPPC hybrid films have been employed to accommodate the anticancer drug Paclitaxel for drug delivery applications PDMS poly(2-methyl-2-oxazoline)PDMS polydimethylsiloxanePDMSB polyPE polyelectrolytesPED polymeric enzyme detectionPEDOT-S polyPEI poly(ethylene imine)PEMA polyPEO polyethylene glycolPEOX poly(ethyl oxazoline)PET polyethylenterephthalatePFN poly{9,9-di[3-propyl]-2,7-fluorenyl-alt-1,4-phenylene dibromide}PFPBr poly bromide)PGMA poly(glycerol monomethacrylate)PHA polyhydroxylalkanoatesPHEMA poly-phenoxyethylmethacrylatePHPMA poly-hydroxypropylmethacrylatePIB polyisobutylenePIMP photoiniferter-mediated polymerizationPL photoluminescencePLA polylactidePLGA poly(lactic-co-glycolic acid)PLL poly(L-lysine)PMMA poly(methylmethacrylate)PMOXA poly(dimethylsiloxane)PMPC poly(2-methacryloyloxyethyl phosphorylcholine)PNA peptide nucleic acidPNMP poly(o-nitrobenzyl-methacrylate-co-methyl-methacrylate-co-poly(ethylene-glycol)-methacrylate)pNIPAAM poly(N-isopropylacrylamide)POEGMA poly(oligo (ethylene glycol) methacrylate)POMA polyPPMA polyPPO poly(propylene oxide)PS poly(styrene)PSBMA poly(sulfobetaine methacrylate)PSS poly (4-styrenesulfonicacid) sodium saltPtBA poly(tert-butyl acrylate)PTMPHFs trimethylphosphonium-substituted polyfluorenesPVC polyvinylchloridePVS polyQCM-D quartz crystal microbalance with dissipation monitoringRAFT reversible addition-fragmentation chain transferRGD arginylglycylaspartic acidS. epidermidis Staphylococcus epidermidisSALB solvent-assisted lipid bilayerSAMs self-assembled monolayersSC stratum corneumSEM electron microscopySOD superoxide dismutaseSPMA spiropyran-containing methacrylateSPR surface plasmon resonancessDNA single stranded DNASSTR3 somatostatin receptor type 3TDAB tetradodecylammonium bromideTEMPO 2,2,6,6-tetramethyl-piperidinyloxyTIPNO 2,2,5-trimethyl-4-phenyl-3-azahexane-3-oxylmT melting pointTMB 3,3\u2019,5,5\u2019-tetramethylbenzidineTNF-\u03b1 tumor necrosis factor \u03b1UV ultravioletVBTAC vinylbenzyl-trimethylammonium chlorideXPS X-Ray photoelectron spectroscopy"} +{"text": "To the Editor\u2014The rapid dissemination of severe respiratory coronavirus virus 2 (SARS-CoV-2) throughout the globe has been declared a pandemic. A lack of national and internationally agreed case definitions for healthcare-associated coronavirus disease 2019 (COVID-19) has led to inconsistencies in describing epidemiology, which limit comparisons.2 A median incubation period of 5 days has been accepted in COVID-19 guidance.3 Adapting established case definitions from other infectious diseases, such as Clostridium difficile infection (CDI), may help overcome variability.4 All cases with a positive nasopharyngeal real-time polymerase chain reaction (PCR) assay would therefore be described as either healthcare associated (HA) or community associated (CA).Hospital-onset healthcare-associated (HoHA) COVID-19 would define current hospitalized inpatients residing >14 days. Hospital-onset possible healthcare-associated (HoPHA) cases, in those residing between 3 and 14 days in the hospital, in the absence of suspected COVID-19 on admission. New cases diagnosed within 14 days of acute-care hospital discharge would be community-onset, healthcare-associated (CoHA) infection. Community-associated (CA) cases would refer to those diagnosed within 2 days or suspected on admission (diagnosed >2 days after admission) and no acute-care hospitalization within the previous 14 days. This group can be further subdivided into those who are independent and self-caring from their own home (CoCA) or those requiring social care, that is, social-onset community-associated (SoCA). Social care includes those requiring domiciliary care , admissions from care homes, community rehabilitation, and mental healthcare institutions.We retrospectively applied these definitions to 631 adult COVID-19\u2013positive patients (aged >16 years) at our acute-care institution in North London from March 1, 2020, to April 15, 2020 inclusive. The study was registered with our local clinical governance committee. Because all care was routine, in keeping with UK national guidance, ethical approval was not required. In total, HoHA, HoPHA, and CoHA cases accounted for 80 of 631 (12.68%) of all positive cases (Fig.\u00a05 Our data demonstrate that healthcare-associated COVID-19 has contributed an important number of cases patients during the height of a pandemic. Sequential screening of non\u2013COVID-19 hospitalized patients beyond this, possibly on a weekly basis up to 14 days after hospital discharge, may prove beneficial in further reducing the threat posed by SARS-CoV-2. Further validation of proposed definitions is required and according to the evolution of CDI definitions, amendments are likely.Recent NHS England guidance recommends screening all emergency hospital admissions on admission followed by a single repeat, for those testing negative, between 5 and 7 days after admission."} +{"text": "Simultaneous detection and isolation of up to three viable and highly-purified cytokine-secreting B-cell subpopulations is feasible, albeit with some signal loss, with fractions subsequently amenable to gene expression analysis and in vitro cell culture. This multiplexing CSA-Flow approach will be of interest in many human cellular immunology contexts aiming to functionally characterize cytokine-secreting immune cells, especially when sample volumes and cell numbers are limited.The ability to functionally characterize cytokine-secreting immune cells has broad implications in both health and a range of immune-mediated and auto-immune diseases. Low-frequency cytokine-defined immune-cell subsets can play key immune-regulatory roles, yet their detailed study is often hampered by limited clinical sample availability. Commonly used techniques including intracellular cytokine staining require cell fixation, precluding subsequent functional interrogation. The cytokine-secretion assay (CSA) can overcome this limitation, though has mostly been used for detection of relatively high-frequency, single-cytokine secreting cells. We examined how adaptation of the CSA in combination with multiparametric flow-cytometry (CSA-Flow) may enable simultaneous isolation of multiple, low-frequency, cytokine-secreting cells. Focusing on human B cells , we show that single-capture CSA-Flow allows for isolation of highly-purified populations of both low-frequency (IL-10 In the field of immunology, tools such as single-cell RNA sequencing (scRNA-Seq), ATAC-Seq and others continue to reveal previously unrecognized cellular functional heterogeneity, of relevance to both health and a variety of diseases9. Functional heterogeneity is also reflected in different cytokine expression profiles of immune cells, such as T cells, B cells, innate lymphoid cells and myeloid cells14. Indeed, particular cytokines have historically been used to define functionally distinct immune cell sub-populations12 and there is substantial interest in furthering the study of cytokine-expressing immune cells, including their mechanisms of differentiation, maintenance and plasticity16. Such knowledge would have important therapeutic implications across multiple immune-mediated conditions.Recent technological advances have enabled detailed examination of cells at single-cell resolution, which has served to extend as well as gain novel insights into both phenotypic and functional profiles of different cell types14, most meaningful study of cytokine-defined immune cells relies on isolation of viable cells that are amenable to activation. Indeed, the isolation of highly purified and viable cytokine-defined immune cells is essential for robust interrogation of individual cells such as by scRNA-Seq17, and is also required for other downstream applications such as the assessment of the impact of such cells on responses of other cells in vitro, or when introduced in vivo . However, the isolation of viable and purified cytokine-expressing immune cells has presented a technical challenge, as commonly used intracellular cytokine-staining (ICS) or flow-fluorescent in-situ hybridization approaches involve cell fixation and permeabilization25. The resultant loss of cell viability and integrity seriously limits the use of cells for comprehensive functional profiling and downstream applications25. While viable cells can be sorted based on certain cell surface-markers that have been reported to enrich for particular cytokine-expressing immune cells, such markers are inevitably neither fully sensitive nor specific33.Since cytokine expression is context- and activation-dependent36. CSA relies on bispecific antibodies (bsAbs) bound to the cell surface that capture secreted cytokines on viable cells, thereby preserving cell viability and integrity, in stark contrast to ICS. In essence, CSA typically involves four steps applied to the mixed population of cells: (1) activation ; (2) labelling with bsAbs that bind on one hand to a cell surface antigen (e.g. CD45 for immune cells) and on the other hand to the particular cytokine of interest; (3) secretion and capture of the cytokine and (4) detection of the cytokine-secreting cells with fluorochrome-conjugated anti-cytokine antibody for flow cytometry analysis was designed to overcome this limitation and to allow for the isolation of viable cytokine-defined immune cellsf cells: activati40).In this study, we demonstrate the utility of the CSA-Flow for\u00a0the detection and isolation of viable human cytokine-secreting cells including simultaneous sorting of both low- and high-frequency populations. To illustrate this approach, we focus our attention on human B cells as a growing body of work implicates cytokine-secretion by B cells as relevant in orchestrating immune responses in both health (e.g. host-protection from infection) and different autoimmune diseases stimulation with PMA and ionomycin, using either ICS or GM-CSF+ (CSA: 5.49 . ICS: 2.65 p\u2009=\u20090.0649) B cells B cells from peripheral blood mononuclear cells (PBMC) and then assessed, in parallel, the ability to detect individual, low-frequency cytokine-producing human B cells, using either intracellular cytokine staining (ICS) or the cytokine secretion assay (CSA). IL-10ICS Fig.\u00a0a or CSA ICS Fig.\u00a0b. The CS+ or GM-CSF+ B cells, fluorescence-activated cell sorting (FACS) was used to isolate the cytokine-producing B cells. Flow cytometry following sorting confirmed enrichment of\u2009>\u200995% B cell subpopulations. We found that the multiplexing successfully yielded highly purified cytokine-defined cell-subpopulations and GM-CSF+ (single-capture: 5.25 p\u2009=\u20090.0139) B cells subpopulation with either of the low frequency (IL-10+ or GM-CSF+) subpopulations, generally resulted in lesser decreases in yields of the low-frequency sub-populations than the decreased yields observed with combined detection of the two low-frequency cytokine secreting sub-populations by a broad range of cytokine-sorted B cells, including those expressing IL-10+or GM-CSF+ in isolating viable, low- and high-frequency cytokine secreting B cells, both individually or concurrently. Isolated cell fractions were highly purified, exhibited excellent viability, and could be integrated into downstream applications such as analysis of gene expression or further in vitro activation/cell culture. The assay relies on sequential antibody-based cell surface labelling, thereby preserving cell integrity and minimizing cell loss43. Our data suggests that cell purities and viabilities are excellent upon multiplexing but the downside can be variable loss of signal. We speculate that signal loss during assay multiplexing was due to saturation of CD45, the cell surface anchor for the bispecific capture reagents35, which may impact on assay sensitivity. Reagent titration is important in optimizing signal to noise for each of the different multiplexed cytokines , though this does not fully mitigate partial loss of signal. While this can impact yields of the isolated subpopulations, their purities (including of the concurrently isolated low-frequency populations) following the multiplexed CSA was nonetheless excellent, making them amenable to highly sensitive interrogation. We note relatively broad variation in the magnitude of cytokine expression by the isolated subpopulations, both at the RNA-expression and secreted-protein levels which, in addition to the expected inter-individual variability, may also reflect cellular-level heterogeneity46 which could be of interest to elucidate.Assay multiplexing is attractive for maximizing the output of potentially limited clinical sample volumes, and has been previously used with CSA to both isolate and distinguish distinct cytokine-secreting immune cells47, as the anti-cytokine antibodies can be pre-mixed with other cell surface antibodies and simultaneously applied to cells. Prior work with CSA has included pre-enrichment of cytokine-secreting immune cells using magnetic-based isolation49 which can facilitate the subsequent study of particularly rare populations, and additional applications have included studies of cytokine detection kinetics42, cellular plasticity of cytokine expression50 and interrogation of antigen-specific cytokine-expressing cells49. We envision that CSA-Flow can also be integrated in the workflow for next-generation and single-cell technologies. This would enable analysis of molecular pathways and transcriptional networks of cells secreting different cytokines, as well as analyses of cells secreting the same cytokine, in different contexts. As an example of the latter, distinct subsets of IL-17-expressing T cells have been shown to harbor homeostatic versus pathologic properties52. Further cellular state diversity could be gleaned from combining CSA-Flow, antigen receptor sequencing and single-cell RNA-seq, as it would allow to draw relationships between antigenic specificity and cell transcriptome among seemingly homogeneous functional cellular profiles4, extending prior antigen-specific CSA applications.CSA-Flow can be seamlessly integrated into multi-parametric FACS panels as part of complex cell-sorting strategiesIn summary, we present a method for the simultaneous isolation of viable low- and high-frequency cytokine-secreting human B cells using CSA-Flow, and highlight strengths and limitations of this approach. Application of this approach should provide users with an opportunity to simultaneously and more fully elucidate the biology of distinct cytokine-defined cell subpopulations, with in-depth analysis and further functional characterization predicated on isolation of highly purified and viable cells from limited clinical samples.53. B cells were selected from PBMC using CD19 microbeads (Miltenyi Biotec) according to manufacturer\u2019s recommendations. Typical purities routinely assessed by flow cytometry were\u2009>\u200998%. B cells were cultured in serum-free X-Vivo medium (Lonza) and plated in flat-bottom 24 well plate at 2\u2009\u00d7\u2009106/well in a total volume of 1500\u00a0\u03bcl of medium. Cells were activated with phorbol 12-myristate 13-acetate and Ionomycin for 4\u00a0h. In the case of intracellular cytokine staining (ICS), Golgi stop was added to cells at the start of stimulation.Fresh blood was obtained from healthy individuals recruited from the Montreal Neurological Institute and Hospital (MNI/H), McGill University and University of Pennsylvania. All subjects provided an informed consent as approved by the corresponding institutional ethics review boards. The study was approved by and carried out in accordance with the guidelines and regulations of the Institutional Review Board of the Montreal Neurological Institute (McGill University) and University of Pennsylvania and in accordance with the Declaration of Helsinki. Peripheral blood mononuclear cells (PBMC) were isolated from whole blood using Ficoll-Paque density gradient centrifugation and a strict standardized protocol41. Briefly, cells were stained with LIVE/DEAD fixable Aqua dead cell stain (Thermo Fisher Scientific) for 20\u00a0min on ice following which cell-surface marker staining was performed using mouse anti-human CD20 and mouse anti-human CD3 . Cells were then fixed and permeabilized using fixation/permeabilization buffer (BD Biosciences). Rat anti-human GM-CSF (clone: BVD2-21C11), rat anti-human IL-10 (clone: JES3-19F1) and mouse anti-human TNF (clone: MAb11) antibodies (BD Biosciences) or matching isotype controls were added and cells were incubated for 30\u00a0min on ice. Cells were washed and resuspended in FACS buffer (PBS/1%FCS) until analysis on a FACS LSR Fortessa (BD Biosciences).Intracellular cytokine staining (ICS) of human B cells was performed as previously described5 cells/ml and incubated while rotating (using a MACS rotor) at 37\u00b0 C/5% CO2 for 45\u00a0min. B cells were then placed on ice for 10\u00a0min to terminate the secretion phase. Cells were subsequently washed twice in ice-cold MACS buffer before labelling with detection antibodies for GM-CSF , IL-10 or TNF along with mouse anti-human CD20 (clone: 2H7) and mouse anti-human CD3 (clone: UCHT1) antibodies. We employed the anti-human TNF antibody as a detection antibody. B cells were washed once in ice-cold MACS buffer and analyzed on BD LSRFortessa (BD Biosciences). Alternatively, cytokine-secreting B cells were sorted on BD FACSAria (BD Biosciences). Data analysis was performed with FlowJo Software (TreeStar).For the cytokine secretion assay, cells were harvested and washed once in ice-cold MACS buffer (PBS/2\u00a0mM EDTA/5%BSA). B cells were then resuspended in serum-free X-Vivo medium and labelled with capture antibodies for GM-CSF , IL-10 or TNF-alpha (Miltenyi Biotec) for 10\u00a0min on ice. Cells were further diluted in pre-warmed medium at 1\u2009\u00d7\u200910Cytokine levels in cultured supernatants were measured by ELISA kits (BD Biosciences or Thermo Fisher Scientific) following manufacturer\u2019s protocols.(Hs00929873_m1), IL10 (Hs00961622_m1) and TNF (Hs01113624_g1). 18\u00a0s (Hs03003631_g1) was used as a housekeeping gene. Fold change calculations were performed using the \u2013\u0394\u0394CT method.Total RNA extraction was performed using RNeasy Plus Micro kit following manufacturer\u2019s protocols. The RNA was stored -80C and used to generate single-stranded cDNA in a standard reverse transcription (RT) reaction using high-capacity cDNA reverse transcription kit with RNase inhibitor (Thermo Fisher Scientific). Analysis of gene expression was performed using the following TaqMan probes: CSF2 All values are expressed as mean Supplementary Legends.Supplementary Information 1.Supplementary Information 2."} +{"text": "Human mesenchymal stem cells have been explored for their application in cell-based therapies targeting stroke. Identifying cell lines that stand as safe, accessible, and effective for transplantation, while optimizing dosage, timing, and method of delivery remain critical translational steps towards clinical trials. Preclinical studies using bone marrow-derived NCS-01 cells show the cells\u2019 ability to confer functional recovery in ischemic stroke. Coculturing primary rat cortical cells or human neural progenitor cells with NCS-01 cells protects against oxygen-glucose deprivation. In the rodent middle cerebral artery occlusion model, intracarotid artery administration of NCS-01 cells demonstrate greater efficacy than other mesenchymal stem cells (MSCs) at improving motor and neurological function, as well as reducing infarct volume and peri-infarct cell loss. NCS-01 cells secrete therapeutic factors, including basic fibroblast growth factor and interleukin-6, while also demonstrating a potentially novel mechanism of extending filopodia towards the site of injury. In this review, we discuss recent preclinical advancements using in vitro and in vivo ischemia models that support the transplantation of NCS-01 in human stroke trials. These results, coupled with the recommendations put forth by the consortium of Stem cell Therapeutics as an Emerging Paradigm for Stroke (STEPS), highlight a framework for conducting preclinical research with the ultimate goal of initiating clinical trials. Ischemic stroke poses as one of the leading causes of death and disability in the modern world . The curThe neuroinflammatory response that arises from an ischemic event plays a significant role in stroke pathology ,8,9. TheAlthough preclinical studies provide ample support for the use of MSCs in human clinical trials, two clinical trials using MSCs have failed to translate these findings in human stroke ,29. IntrTransplantation of NCS-01 cells in stroke models may help ameliorate some of these gaps in translation. In July 2019, NSC-01 cells received FDA approval for clinical application of intracarotid (ICA) transplantation in ischemic stroke patients . Here, wIn vitro studies demonstrate that NCS-01 cells dose-dependently protect cocultured primary rat cortical neurons and astrocytes subjected to oxygen-glucose deprivation (OGD), although an increase of NCS-01 cells over a threshold ratio of 1:1 did not significantly increase host cell survival . This tyAnother consequence of OGD that contributes to neuronal death and inflammation involves the dysfunction of the mitochondria ,44. MitoIn vitro studies also suggest that NCS-01 cells demonstrate a potentially novel mechanism through which their filopodia may exert therapeutic effects under stroke conditions. To investigate the mechanism of action behind NCS-01 filopodia, primary rat cortical cells exposed to OGD were cocultured with NCS-01 cells at various distances ranging from 0 mm to 2.04 mm . ImagingTo tease apart the effects of IL-6, bFGF, and NCS-01-derived filopodia in cell rescue, other studies were performed on various host cells, including primary cortical neurons, primary rat astrocytes, and primary rat endothelial progenitor cells (EPCs). Each cell type was subjected to OGD and treated with either (1) cell media only (control), (2) IL-6 + bFGF only, (3) NCS-01 cells only, or (4) a combination of IL-6 + bFGF + NCS-01 cells. All treatment groups exhibit improved mitochondrial activity compared to the control group, with the greatest activity seen in groups treated with NCS-01 cells only and IL-6 + bGFG only. Among the different host cell types, neurons display the greatest recovery, in that NCS-01 cell treatment renders significantly better therapeutic outcomes than IL-6 + bGFG. However, IL-6 + bFGF treatment and NCS-01 treatment afford comparable rescue for astrocytes and EPCs. Interestingly, the combination treatment groups (IL-6 + bFGF + NCS-01 cells) perform significantly worse than when each treatment was given alone for neurons and EPCs but not for astrocytes. Furthermore, in all three neuronal host cell lines, filopodia formation accompanies both the NCS-01 cell only treatment and the combination of IL-6 + bFGF + NCS-01 cells treatment, indicating that filopodia formation correlates with improved cell viability and mitochondrial activity. These results suggest that NCS-01 cell\u2019s therapeutic effects stem from the release of IL-6 and bFGF, and filopodia formation. Further manipulation of cytokine release, and facilitating or reducing filopodia formation, may elucidate more mechanisms of brain repair and its application to cellular therapy .Filopodia formation participates in neuroprotection by Rho-GTPase kinase inhibition on organotypic hippocampal slices subjected to ischemia . Similar5, 7.5 \u00d7 106, or 3.75 \u00d7 107 NCS-01 cells in a set concentration of 7.5 \u00d7 106 NCS-01 cells/mL via the ICA method of delivery ). As stated in the recommendations put forth by STEPS, preclinical efficacy studies specifying target patient profiles should be conducted parallel to phase I/IIa clinical trials in order to identify populations to be considered in subsequent phase IIb/III clinical trials [In July 2019, FDA approved NCS-01 cell transplantation in ischemic stroke patients and a multicenter clinical trial study evaluating its safety started in February 2020 ("} +{"text": "Interventricular dyssynchrony is typically assessed by pulsed-wave echocardiography (PW-Echo) as the delay between onset of aortic and pulmonary flow. Recent multicenter trials demonstrated the value of this interventricular dyssychrony to predict response to cardiac resynchronization therapy (CRT). In the present study, the ability of phase contrast magnetic resonance angiography (PC-MRA) was assessed to quantify interventricular dyssynchrony in comparison with PW-Echo.40 patients with stable heart failure NYHA Class 2 to 3, reduced ejection fraction (28 \u00b1 11%), with (n = 21) or without (n = 19) complete left bundle branch block were prospectively included. Transvalvular flow curves of the aortic and pulmonary valve were acquired by PC-MRA and PW-Echo. Interventricular delay was calculated for PC-MRA as the delay between onset of aortic and pulmonary flow in analogy to PW-Echo. Interventricular delays by PC-MRA were correlated with PW-Echo; agreement was assessed by Bland-Altman analysis.(A) and one patient without interventricular dyssynchrony (B) is illustrated by Figure patient A (PW-Echo = 146 ms) and 21 ms in patient B (PW-Echo = 6 ms).A strong correlation between interventricular delays by PW-Echo and PC-MRA was found . Bland-Altman analysis demonstrated a good agreement between both methods (Mean difference -6 \u00b1 15 ms). An example of the assessment of interventricular delays by PC-MRA in one patient with PC-MRA quantifies interventricular dyssynchrony comparable with PW-Echo. PC-MRA has the potential to identify responders to CRT."} +{"text": "Many serologic tests are now available for measuring severe acute respiratory syndrome coronavirus 2 antibodies to evaluate potential protective immunity and for seroprevalence studies. We describe an approach to standardizing positivity thresholds and quantitative values for different assays that uses z-scores to enable rapid and efficient comparison of serologic test performance. Measurement of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies has become increasingly important for assessing potential immunity as the coronavirus disease (COVID-19) pandemic evolves. Most immunoassays for SARS-CoV-2 antibodies yield quantitative converted to qualitative results, requiring a positivity threshold whose basis might be unclear when provided by the manufacturer. Using specimens from hospitalized patients with acute COVID-19 and archived pre\u2013COVID-19 serum samples, we established standardized positivity thresholds and quantitative values for multiple commercially available immunoassays, which enabled efficient screening comparison of serologic reagents.Remnant blood specimens were selected from a convenience sample of patients given diagnoses of COVID-19 by using a laboratory-developed reverse transcription PCR (https://www.euroimmun.com) with recombinant structural protein (spike [S] 1 domain) as target (http://www.epitopediagnostics.com) with nucleocapsid protein (NP) as target; ImmunoDiagnostics anti-SARS-CoV-2-NP IgG Kit with NP as target; and ImmunoDiagnostics anti-SARS-CoV-2-S1RBD IgG Kit (lot no. S0313) with receptor-binding domain (RBD) of the S1 protein as target. All testing was performed according to manufacturer\u2019s protocols.We used 4 commercial SARS-CoV-2 IgG ELISA kits: Euroimmun IgG Kit was considered positive to minimize false-positive results.To standardize results, optical density (OD) scores for each sample were converted to z-scores by using the equation z-score\u00a0=\u00a0(test OD \u2013 mean negative control OD)/mean negative control SD. For Euroimmun, the OD ratio was calculated by using a kit calibrator. Negative control serum samples had been collected during April 2015\u2013November 2019 from 25 healthy community blood donors. A conservative z-score A total of 23 samples were tested from a cohort of 11 patients with reverse transcription PCR\u2013confirmed COVID-19. The standardized results illustrate the differing sensitivities of the 4 assays . As expeWe provide serial results for 3 patients over the first 3 weeks after symptom onset . Using tAmong 25 negative control samples, 6 were positive by EPI-provided thresholds, but negative by the other tests, suggesting that the recommended EPI cutoff was inappropriately low. All 25 control results were included in EPI z-score calculations, and led to a positivity threshold higher than recommended by EPI. In contrast, our local population-based z-score cutoff was lower than the threshold recommended by EU. Despite these differences, qualitative results obtained by using manufacturer-supplied cutoffs and z-scores were identical for EU and EPI results for our limited sample set. The ID kits did not include a recommended positivity threshold, but use of a z-score of 3, and results generated by using the same local negative control samples as the other kits facilitated an unbiased comparison.,Three patients had discordant qualitative results for Euroimmun, EPI, and ImmunoDiagnostics NP assays. Patient 10 had nucleocapsid responses (EPI and ImmunoDiagnostics NP) but no S1 response (Euroimmun) detected, and patients 4 and 5 had nucleocapsid antibody responses just above positivity thresholds detected by 1 but not the other assays. Different studies have reported serologic results using in-house (>20 control samples tested once) should also find utility in that setting. Finally, careful evaluation of manufacturer-recommended positivity thresholds for SARS-CoV-2 qualitative antibody tests is warranted.Clinical assay validation is always required, but is particularly needed for COVID-19 antibody assays given the current emergency use climate with limited regulatory oversight. Use of pre\u2013COVID-19\u2013era reference specimens to calculate standardized z-score results for immunoassays with different or no manufacturer-recommended cutoffs, and a small sample of locally collected specimens from SARS-CoV-2\u2013infected persons enabled rapid comparison. As attention turns to calculated measurement of vaccine-induced responses, comparison of quantitative assays is likely to become important, and z-scores (with"} +{"text": "Partnering with families to develop function-focused plans for hospitalized persons with dementia (PWD) improves both the hospital experience and patient outcomes. This secondary analysis included patients enrolled in the intervention arm of the on-going Family-centered Function-focused Care (Fam-FFC).study. This study examined the goals co-established by family caregivers, PWD, and nurses to prevent hospital-acquired complications and promote functional and cognitive recovery. The influence of goal attainment upon delirium and physical function at discharge was also examined. The majority of patients (N=162) were female (65%), black (53%) with a mean age of 82.7 (SD= 8.2). Goal attainment ranged from -2 to 2; mean = -0.24 (SD= 0.75). The goals (N=432) represent three main areas: mobility, self-care, and cognitive stimulation. Controlling for age and admission function, goal attainment was associated with less discharge delirium but not discharge function. Results support the contribution of function-focused care to improving delirium outcomes."} +{"text": "The simultaneous performance of a secondary task while walking increases motor-cognitive interference and fall risk in older adults. Combining transcranial direct current stimulation (tDCS) with the concurrent performance of a task that putatively involves the same brain networks targeted by the tDCS may reduce the negative impact of dual-tasking on walking. We examined whether tDCS applied while walking reduces the dual-task costs to gait and whether this combination is better than tDCS alone or walking alone (with sham stimulation). In 25 healthy older adults (aged 75.7\u00b110.5yrs), a double-blind, within-subject, cross-over pilot study evaluated the acute after-effects of 20 minutes of tDCS targeting the primary motor cortex and the dorsal lateral pre frontal cortex during three separate sessions:1) tDCS while walking on a treadmill in a virtual-reality environment , 2) tDCS while seated (tDCS+seated), and 3) walking in the virtual-reality environment with sham tDCS . The complex walking condition taxed motor and cognitive abilities. During each session, single- and dual-task walking and cognitive function were assessed before and immediately after stimulation. Compared to pre-tDCS performance, tDCS+walking reduced the dual-task cost to gait speed (p=0.004) and other gait features , and improved (p<0.001) executive function (Stroop interference score). tDCS+seated and sham+walking did not affect the dual-task cost to gait speed (p>0.17). These initial findings demonstrate that tDCS delivered during challenging walking ameliorates dual-task gait and executive function in older adults, suggesting that the concurrent performance of related tasks enhances the efficacy of the neural stimulation and mobility."} +{"text": "This Correction follows an Expression of Concern relating to this article previously published by Portland Press.Biosci Rep (2019) 39(9), DOI: 10.1042/BSR20190159) would like to replace The authors of the original paper \u201cIsorhamnetin inhibited migration and invasion via suppression of Akt/ERK-mediated epithelial-to-mesenchymal transition (EMT) in A549 human non-small cell lung cancer cells\u201d ("} +{"text": "Myosin II and its regulator Rho-associated coiled-coil containing protein kinase (ROCK) are essential for cell invasion and metastatic dissemination. Our recent findings show that this molecular machinery is also involved in drug resistance in melanoma by playing a dual role: protection of tumor cells from reactive oxygen species (ROS) and DNA damage (intrinsic), and co-option of myeloid and lymphoid populations to establish immunosuppression (extrinsic). Myosin II-driven cell contractility relies on Rho GTPase signaling that through Rho-associated coiled-coil containing protein kinase (ROCK) increases phosphorylation of myosin light chain 2 (p-MLC2) and Myosin II activity.BRAFV600E being the most common) and therefore, benefit from targeted therapies against MAPK pathway (BRAF and MEK inhibitors). However, resistance inevitably arises in most patients within a year, in most cases due to ERK restoration.4Metastasis is responsible for most deaths of cancer patients. While there are some therapies that achieve impressive responses and extend patient survival, these are not long-lasting due to drug resistance, both intrinsic/primary and acquired. This is exemplified in cutaneous melanoma, a highly aggressive and metastatic skin cancer. Most melanoma patients harbor mutations in the mitogen-activated protein kinase (MAPK) pathway (BRAF-MEK-ERK) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), aimed to awake the immune system that will eliminate the tumor, has proven successful in a subset of melanoma patients. However, response rates are low (less than 40%) and a substantial proportion of responders relapse within 2\u00a0years.4 Interestingly, cross-resistance to both MAPK inhibitors (MAPKi) and immune checkpoint blockers (ICB) is driven by common alterations at the transcriptional level in regulators of metastatic features , extracellular matrix (ECM) remodeling).7 Therefore, in our latest study, we assessed if Myosin II and cytoskeletal remodeling could play a role in therapy-cross-resistance.8The last decade has seen a myriad of efforts in deciphering mechanisms of resistance to targeted and immunotherapies in melanoma.Rho GTPase signaling and cytoskeletal remodeling were top enriched processes in the phospho-proteome of melanoma cells after a 24\u00a0h treatment of melanoma cells with MAPKi. MAPK inhibition decreased Myosin II activity (p-MLC2 levels), regardless of genetic background . Surprisingly, 48\u00a0h after MAPK inhibition Myosin II levels were fully restored or even increased, while ERK activity was only partially recovered. This uncoupling of Myosin II from MAPK suggested that Myosin II could confer a survival advantage under therapy. In fact, overexpression of constitutively active MLC2 conferred resistance to BRAFi. Therefore, under the selective pressure of treatment, Myosin II uncouples itself from MAPK signaling to provide a survival advantage.MYH9) or light chains (MYL9/12B) with RNAi impaired survival of resistant cells. At this point, the prediction was that cross-resistant melanomas, having transcriptionally rewired their cytoskeleton, would be refractory to a second therapy but now much more sensitive to Myosin II blockade. In fact, anti-PD-1-resistant cell lines were more sensitive to Myosin II inhibition with ROCKi, suggesting that this vulnerability is an intrinsic feature of therapy-resistant cells regardless of the therapy. Survival of patient-derived cell lines resistant to sequential MAPKi+ICB was also impaired upon Myosin II inhibition, indicating some level of cross-resistance.We next wondered if restoration of Myosin II in resistant cells could become a new vulnerability. BRAFi-resistant cells were much more sensitive to Myosin II blockade using ROCK inhibitors (ROCKi) or ROCK1/2 RNAi. This increased sensitivity was observed in 2D and 3D environments in a panel of several models of drug resistance . Direct inhibition of Myosin II with a specific inhibitor (blebbistatin) and knockdown of either Myosin II heavy chain upregulated ROCK-Myosin II pathway genes. Melanomas resistant to anti-PD-1 had also higher Myosin II levels before therapy, suggesting its potential as a biomarker of lack of response. In fact, in The Cancer Genome Atlas (TCGA) melanoma database we found that higher expression of ROCK-Myosin II pathway correlates with poorer prognosis, indicating that higher Myosin II levels before therapy could identify more aggressive melanoma cells that would have a survival advantage later on under therapy.When we analyzed 12 paired samples from patients before and after therapy , using immunohistochemistry (IHC) we measured increased p-MLC2 levels in the resistant samples. This was accompanied by changes in the tumor microenvironment: increased matrix deposition (which could contribute to higher p-MLC2) and higher numbers of immunosuppressive populations, in particular CD206+ macrophages and forkhead box P3 (FOXP3+) regulatory T cells (Tregs). This tumor-supporting microenvironment could explain therapy failure . It will9 We found that MAPKi-resistant cells harbor deregulated ROS metabolism and defective DNA damage repair. We exploited these vulnerabilities and found that ROCKi induced high ROS, DNA damage, and a pronounced cell cycle arrest in BRAFi-resistant cells compared to the sensitive counterpart. ROCKi reduced pro-survival signals mediated by phosphorylated signal transducer and activator of transcription 3 (p-STAT3)-Mcl-1. All these combined effects led to cell death in migrating melanoma cells.ll death .We then tested ROCKi in pre-clinical therapy-resistant mouse models. Combination of ROCKi with BRAFi reduced growth of BRAFi-resistant melanoma xenografts in nude mice. This could be due to increased intrinsic cell death induced by ROCKi in resistant cells. Furthermore, ROCKi-treated tumors had reduced p-MLC2 and lower numbers of CD206+ macrophages , which could also contribute to reduced tumor growth in an extrinsic manner. Importantly, using an experimental metastasis assay, we found that pre-treatment with ROCKi impaired survival of BRAFi-resistant patient-derived melanoma cells in the lung. Altogether, these data suggest that ROCKi could be used to impair both primary tumor growth and metastatic dissemination of resistant melanoma cells.10 Interestingly, ROCKi reduced TGB-\u03b2 levels in immunotherapy-resistant cells in vitro, which could lead to dampening of immunosuppression in vivo.We next investigated if ROCKi could enhance ICB efficacy. Indeed, combination of ROCKi with anti-PD-1 induced more tumor regressions than single anti-PD-1. ROCKi relieved immunosuppression by decreasing CD206+ macrophages and FOXP3+ Tregs. We have previously described that transforming growth factor beta (TGB-\u03b2), a potent immunosuppressor, promotes Myosin II-driven contractility in melanoma.11 and, therefore, immunosuppression. In fact, anti-PD-1/PD-L1 therapies have been suggested to function also directly on macrophages.12Similar to the human setting, we observed variable responses to anti-PD-1 treatment in mice; therefore, we isolated a non-responder that was allografted into new recipient mice. In this model of anti-PD-1 resistance, cancer cells had increased Myosin II and had polarized all macrophages to CD206+ phenotype. Importantly, combination ROCKi+anti-PD-1 induced more tumor regressions and reduced numbers of FOXP3+ Tregs. Furthermore, ROCKi+anti-PD-1 decreased levels of PD-1 ligand in both tumor cells and CD206+ macrophages , which c1 further pre-clinical studies assessing dose-escalation, treatment schedule, and delivery modality will be needed in order to optimize ROCKi. Such studies will aim to assess superior responses of ROCKi as single therapy, or increase efficacy when combined with standard of care therapies. Our study raises the possibility that other MAPK-driven tumors and ICB-resistant cancers could be vulnerable to ROCK-Myosin II inhibition, which warrants further investigation.Our study shows that adaptation to therapy and development of resistance comes with a cost, since it involves profound cytoskeletal remodeling and Myosin II reactivation. Resistant cells, therefore, gain a new vulnerability, which can be exploited by targeting Myosin II with ROCKi . Given t"} +{"text": "Pten knockout in parvalbumin (PV)-expressing or somatostatin (Sst)-expressing neurons, two common subtypes of GABAergic neurons. We found that mice with deletion of Pten in either PV-neurons or Sst-neurons displayed social deficits, repetitive behaviors and impaired motor coordination/learning. In addition, mice with one copy of Pten deletion in PV-neurons exhibited hyperlocomotion in novel open fields and home cages. We also examined anxiety behaviors and found that mice with Pten deletion in Sst-neurons displayed anxiety-like behaviors, while mice with Pten deletion in PV-neurons exhibited anxiolytic-like behaviors. These behavioral assessments demonstrate that Pten knockout in the subtype of inhibitory neurons sufficiently gives rise to ASD-core behaviors, providing evidence that both PV- and Sst-neurons may play a critical role in ASD symptoms.Disrupted GABAergic neurons have been extensively described in brain tissues from individuals with autism spectrum disorder (ASD) and animal models for ASD. However, the contribution of these aberrant inhibitory neurons to autism-related behavioral phenotypes is not well understood. We examined ASD-related behaviors in mice with conditional Inhibitory neurons are highly impacted in autism spectrum disorder (ASD) \u20133, a neuPTEN), originally identified as a tumor suppressor gene, negatively regulates cell proliferation and growth by downregulating the phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway [PTEN germline mutations have been identified in individuals with ASD [Pten is widely expressed in both glutamatergic and GABAergic neurons during development and adulthood [Pten is embryonically lethal, mice with brain-region-specific Pten knockout or Pten haploinsufficiency have been investigated. These transgenic mice exhibited autism-related behaviors, including deficits in social interaction, repetitive behaviors, hyperlocomotion, and anxiety-like behaviors [Pten in subtypes of inhibitory neurons causes autism-behavioral phenotypes remains elusive.The autism-risk gene, phosphatase and tensin homolog on chromosome ten on social behaviors. We specifically deleted Pten in PV-neurons by crossing PV-Cre mice with Ptenflox mice to generate PV-Cre+/+/Ptenfl/+ mice (See Methods for details). Using these mice as breeders, we obtained offspring littermates with three genotypes, PV-Cre+/+/Pten+/+ (PV-Pten-WT), PV-Cre+/+/Ptenfl/+ (PV-Pten-Het), and PV-Cre+/+/Ptenfl/fl (PV-Pten-KO). These three types of mice all have homozygous Cre but different copies of Pten in PV-neurons. Using immunohistochemistry and Western blot, we confirmed that Pten was specifically reduced and deleted in PV-positive neurons of PV-Pten-Het and PV-Pten-KO mice, respectively on social behaviors. Using the same breeding strategy for PV-Pten mice, we obtained Sst-Pten littermates with three genotypes, Sst-Cre+/+/Pten+/+ (Sst-Pten-WT), Sst-Cre+/+/Ptenfl/+ (Sst-Pten-Het), and Sst-Cre+/+/Ptenfl/fl (Sst-Pten-KO). We also verified that Pten was specifically reduced and deleted in Sst-positive neurons of Sst-Pten-Het and Sst-Pten-KO mice, respectively . In addition, conditional Pten knockout in PV-neurons causes differences in behaviors that are characteristic symptoms often observed in autism, such as hyperactivity and impairment in motor coordination/learning. Similarly, conditional Pten knockout in Sst-neurons gives rise to disrupted motor coordination/learning. Interestingly, PV-Pten-KO mice displayed anxiolytic-like behaviors, whereas Sst-Pten-KO mice showed anxiety-like behaviors. These findings provide the behavioral evidence that Pten mutation in PV-neurons or Sst-neurons sufficiently induces ASD-related behaviors.Our study revealed that conditional deletion of Pten in PV-neurons or Sst-neurons. Reduced somatostatin expression in homozygous Sst-Cre mice has been previously reported [fl/fl mice, and found that PV-Pten-WT and Sst-Pten-WT mice did not exhibit any difference in most behavioral tests, except that Sst-Pten-WT mice displayed increased time spent in the center of the open field test in PV-neurons exhibit autism-related behaviors of varying severity [MeCP2 (Rett syndrome) or Scn1a (Dravet syndrome) in PV-neurons but not Sst-neurons [Pten in either PV- or Sst-neurons resulted in autism-like core behaviors, further suggesting that Pten plays an important role in the function of these two subtypes of neurons.Mice with deletion of several other genes . All these strains were maintained on C57BL6/J background. We crossed Ptenfl/fl mice with Sst-Cre+/+ mice to generate Sst-Cre+/-/Ptenfl/+ mice which were then backcrossed with Sst-Cre+/+ mice to generate Sst-Cre+/+/Ptenfl/+ breeders. By crossing the breeders we obtained three types of experimental mice, Sst-Cre+/+/Pten+/+ (Sst-Pten-WT), Sst-Cre+/+/Ptenfl/+ (Sst-Pten-Het) and Sst-Cre+/+/Ptenfl/fl (Sst-Pten-KO) mice. They were all homozygous for Cre but expressed two, one or no copy of Pten in Sst neurons. Using the same breeding strategy, we generated PV-Cre+/+/Ptenfl/+ breeders and their offspring, PV-Cre+/+/Pten+/+ (PV-Pten-WT), PV -Cre+/+/Ptenfl/+ (PV-Pten-Het) and PV-Cre+/+/Ptenfl/fl (PV-Pten-KO) mice. Both male and female littermates at the age of 4\u20138\u00a0weeks were used for behavioral assessments. There was no significant correlation between the behavioral performance and ages in PV-Pten or Sst-Pten WT mice or V\u2013VI (Sst+\u00a0cells) from matching sections (Bregma \u2212 1.7\u00a0mm) of three mice per genotype.Animals were anesthetized with isoflurane and perfused transcardially with 0.9% saline solution. Brains were fixed in 4% paraformaldehyde. Double immunohistochemistry with either anti-parvalbumin made in guinea pig or anti-somatostatin made in rat , and anti-PTEN made in rabbit were performed as described in coronPTEN expression levels were quantified by measuring the fluorescence intensity in parvalbumin or somatostatin positive cells with Fiji image analysis package . After bCerebellum (PV-Pten mice) or cortex (Sst-Pten mice) samples were homogenized in lysis buffer with protease inhibitor . 50\u00a0\u00b5g of total protein was separated in 4\u201320% mini-Protean TGX Stain-Free precast gels (BioRad) and transferred to nitrocellulose membranes. Antibodies used included rabbit anti-PTEN, mouse anti \u03b2-Actin , and rabbit and mouse (Novex) HRP-conjugated secondaries.Following procedures established in our previous work , a serieMice were placed in the center of an open field apparatus (40\u2009\u00d7\u200940\u00a0cm) and allowed to explore the field for 30\u00a0min. The movement was tracked using the Ethovision XT software. Distance moved and time spent in the center area and corner area were further analyzed. Self-grooming behaviors were also analyzed from the open field activity using mouse behavior recognition in the Ethovision XT.Home-cage activity was recorded and analyzed using Ethovision XT software. Mice were individually housed in a clean cage for at least 24\u00a0h for acclimation. Mouse behaviors were recorded in its home cage with the filter top removed for 30\u00a0min. The distance moved was determined by off-line analyses of the video recordings.The elevated plus maze was composed of two open arms (30\u2009\u00d7\u20095\u00a0cm) and two closed arms (30\u2009\u00d7\u20095\u00a0cm) with 15\u00a0cm wall height and 50\u00a0cm above the floor. Mice were individually placed in the center of the maze and facing one of the open arms. They were allowed to explore the open and closed arms of the maze for 10\u00a0min. The duration of time in arms was recorded. The percentage of time in open arms (or closed arms) was calculated as the duration in open arms (or closed arms)/total exploring duration.We used a modified apparatus to examine the social behaviors . The appTwelve glass marbles (3\u2009\u00d7\u20094 arrangement) were placed on the surface of the bedding (5\u00a0cm deep) in standard mouse cages. The cages were covered with filter top during the test. Each test mouse was placed in the cage and allowed to explore for 30\u00a0min. The number of buried marbles was counted manually. The marble was checked from side of the cage. If the marble was buried more than 2/3 deep, it was counted as buried.The two-session rotarod test was conducted on an accelerating rotarod over two days. Mice were allowed to become acclimated to the stationary rod for 60\u00a0s on the first day. Each test session consisted of three trials with 15-min inter-trial intervals (ITI). During each trial, mice were placed on the rotating rod (4\u00a0rpm) facing away from the direction of rotation. The rotation speed was accelerated from 4 to 40\u00a0rpm over 5\u00a0min. If the mouse fell off the rod within 10\u00a0s, the trial was repeated after 15\u00a0min. The latency to fall was recorded as the time delay between the start of the trial and the moment when mice fell off the rod or made a complete revolution on the rod.t-test was used for comparisons between two groups, and two-way ANOVA with repeated measures in one factor (rmANOVA) or one-way ANOVA was used for comparisons among three groups. Tukey\u2019s post hoc test was used for multiple comparisons after ANOVA test. For datasets with non-normal distribution, Wilcoxon matched-pairs signed rank test (Wilcoxon test) was used for comparisons between two groups (paired), and Kruskal\u2013Wallis test with Dunn\u2019s post hoc test was used for comparisons among three groups. One sample t-test or Wilcoxon signed-rank test was used for comparing the mean (or median) of a dataset with a hypothetical number. p-value\u2009<\u20090.05 was considered to be statistically significant.Data was analyzed with SPSS (v.20) and GraphPad Prism 9 Software. All datasets were tested for normality using the Shapiro\u2013Wilk test. For datasets with normal distribution, two-tailed Additional file 1: Figure S1. PTEN expression in parvalbumin positive neurons of PV-Pten mice. Figure S2. PTEN expression in somatostatin positive neurons of Sst-Pten mice. Figure S3. No pre-existing side preference during the habituation session in modified three-chamber social tests for both PV-Pten and Sst-Pten mice. Figure S4. Cre expression in PV- or Sst-neurons did not change most behavioral performance. Figure S5. No significant correlation between behavioral performance and ages in PV-Pten-WT mice. Figure S6. No significant correlation between behavioral performance and ages in Sst-Pten-WT mice. Figure S7. No sex effect in open-field test and marble burying test was observed in PV-Pten or Sst-Pten mice. Figure S8. No sex effect in social test, rotarod test and EPM test was observed in PV-Pten or Sst-Pten mice."} +{"text": "Unbiased in silico approaches applied to genome-wide data prioritized putative functional gene variants associating with treatment-resistant ophthalmoplegic myasthenia gravis (OP-MG). Although altered expression of genes harbouring these variants, or associated pathways, were shown in patient-derived transdifferentiated-myocyte models, gene expression in orbital-derived muscle was required to test the validity of the predictions.We sampled orbicularis oculi muscle (OOM) and one paralysed extraocular muscle (EOM) from six individuals with OP-MG during blepharoptosis and re-alignment surgeries, respectively. For controls, the OOMs were sampled from four individuals without myasthenia undergoing surgery for non-muscle causes of ptosis, and one non-paralysed EOM. Using a qPCR array, expression of 120 genes was compared between OP-MG and control OOMs, profiling putative \u201cOP-MG\u201d genes, genes in related biological pathways and genes reported to be dysregulated in MG cases or experimental MG models, and in EOMs of cases with strabismus. Normalization was performed with two stable reference genes. Differential gene expression was compared between OP-MG and control samples using the \u0394\u0394CT method. Co-expression was analysed by pairwise correlation of gene transcripts to infer expression networks.p\u2009=\u20090.72). In OOMs, significant downregulated expression of eight genes was observed in OP-MG cases compared with controls , including TFAM, a mitochondrial transcription factor, and genes related to the following pathways: atrophy signalling; muscle regeneration and contraction; glycogen synthesis; and extracellular matrix remodelling. Several microRNAs, known to be highly expressed in EOMs, are predicted to regulate some of these genes. Co-expression analyses of gene-pairs suggested high interconnectedness of gene expression networks in OP-MG muscle, but not controls . Significant inverse directions of gene-pair correlations were noted in OP-MG versus controls OOM networks involving most OP-MG genes overlapping prominently with muscle atrophy/contractility and oxidative metabolism genes.Overall, transcript levels were similar in OOMs and EOMs (The gene expression in orbital muscles derived from OP-MG individuals compared with normal controls, support the pathogenic hypothesis previously generated from whole genome sequence analyses. Repression of gene transcripts in OP-MG orbital muscle implicate tissue-specific regulatory mechanisms, which may inform future biomarker discovery approaches. Previously we identified individuals with otherwise characteristic myasthenia gravis (MG) and who responded to immune therapies as expected in their non-ocular muscles, but who remained with treatment-resistant ophthalmoplegia with/without ptosis , 2. ThesTo understand the underlying genetic pathogenesis of the OP-MG subphenotype we previously used next generation sequencing to dissect the molecular genetic landscape using an extreme phenotype approach i.e. OP-MG cases vs control MG. Genes associated with OP-MG were identified using single variant and gene-based cumulative variant statistical association analyses , 6. The FAM92A1 and PEF1, which were more frequent in OP-MG vs control MG genomes (p\u2009<\u20091\u2009\u00d7\u200910\u20135) . Increaress [in , and in ress [in . IL6 encgnalling . Therefo effects .PEF1 and FAM92A1, which featured prominently in the dysregulated gene correlation networks by phenotype, were genes with unknown function when prioritized and included in the custom panel. FAM92A1, has recently been found to be critical for mitochondrial ultrastructural integrity [PEF1 remains with poorly characterised function [Gene expression profiling in orbicularis oculi muscles showed the genes which were previously prioritized as OP-MG-associated variants, were differently regulated in the muscles of OP-MG cases compared with normal controls, and supported their involvement in OP-MG pathogenesis. The original gene discovery strategy used an \u2018extreme phenotype\u2019 in which all the cases had MG, but who differed only by their EOM resistance to treatment observed in the clinic. In the current study we aimed to validate expression differences in these putative OP-MG genes/pathways using orbital muscles from affected cases vs controls without MG. Differential gene repression and gene\u2013gene correlations differed significantly between the OOMs from OP-MG cases and control muscle. Importantly, genes such as ntegrity . Althougfunction , the strIn addition to assessing the putative OP-MG gene transcripts in orbital muscles, we were also interested in whether the transcriptional profile of genes known to represent MG/EAMG and strabismus-related pathways, may cross correlate with OP-MG gene transcripts. Here we included strabismus-related genes based on reports showing dysregulation in maligned EOMs (strabismus) in a non-MG context. However, recently RNA-sequencing of cultured human myoblasts revealed that AChR-antibodies significantly impacted genes related to extracellular matrix and actin/myosin cytoskeleton pathways , which oMYH2 which showed significant repression in the OP-MG OOMs, also featured prominently in the muscle genes regulated by AChR-antibodies [Genes related to \u2018muscle atrophy signalling\u2019 pathways at cellular level including those regulating muscle fibre size by protein synthesis , degradation (ubiquitin-proteosome and autophagy-lysosome pathways) and the balance maintained by the TGF\u03b2-BMP superfamily , were cotibodies . MYH2 istibodies . MuRF1, tibodies . The obstibodies . Steroidtibodies . Howevertibodies , 48, 49.DDX17 [DDX17, which was highly expressed in both EOMs and OOMs, is a master regulator in muscle splicing events and miRNA biogenesis [The differential gene downregulation of gene transcripts in OP-MG muscle compared to controls, implicate regulatory mechanisms mediated by miRs. Bioinformatic tools predict several miR-mRNA interactions by miRs which are highly expressed in EOMs and genes which showed differential repression in OP-MG muscle. Furthermore, OP-MG associated genes with putative functional 3\u2032 regulatory variants, such as DDX17 , 7 , the EOM allotype differs from OOMs in some respects notably fatigue resistance, multi-innervated fibres (compared to singly innervated fibres in OOMs) and EOMs are constantly regenerating , 15. NevThe profiling of patient-derived orbital muscle supports the previous unbiased, genome-driven hypotheses of candidate genes and pathways involved in the pathogenesis of OP-MG. The dysregulated gene expression in the orbital muscles of treatment-resistant ophthalmoplegic MG cases implicate pathways related to muscle contractility and mitochondrial homeostasis and strongly suggests altered extraocular muscle-specific regulatory events.Additional file 1. Supplementary Table S1 present the results of the quality of RNA extracted from the orbital muscles. Table S2 depicts the raw data informing the reference gene selection and Table S3 the genes which were highly expressed in extraocular muscle and orbicular oculi muscles. Supplementary Figure S1. Differentially co-expressed gene pairs between OP-MG and control orbicularis oculi muscle ranked by significance derived from Fisher\u2019s Z test. Figure S2. Scatter plot of gene expression levels in two extraocular muscles and nine orbicularis oculi muscles."} +{"text": "Osteoarthritis (OA) is a principal cause of disabling knee pain, and movement is a known exacerbator of pain in African Americans (AAs). Still, research has neglected to understand the relationship between pain with movement and its impact on function and mobility. Our previous study found significantly higher movement-evoked pain between AAs and White American (WAs). Therefore, this case-control observational study investigated inter-racial and intra-racial differences in movement-evoked pain in AAs and WAs (N= 28) who were 55-78 years-of-age . We measured pain intensity (0-10) pre/ante/post multiple performance-based functional activities; we report preliminary results for 7-meter GAITRite\u00ae walk and Stair climbs. Pain intensity was higher before and after the 7-meter walk and stair climbs in AAs, although not significantly different than WAs. We will conduct additional statistical tests for the remaining functional activities to identify potential differences and ethnic-specific factors that distinguish movement-evoked pain and function by race."} +{"text": "Many patients with coronavirus disease 2019 (COVID-19) have a hyperactive immune response (cytokine storm) which has been incriminated in multiorgan dysfunction (MOD). Interleukin-6 (IL-6) and granulocyte-macrophage colony-stimulating factor (GM-CSF) are the key cytokines involved in mediating systemic inflammation and triggering endothelial dysfunction. To limit these effects, IL-6 receptor inhibitors (IL6ri) have been used in COVID-19 patients. The best approach regarding the total number of doses in COVID-19 patients is still unclear. In this single-center retrospective study, we investigated if multiple doses of tocilizumab (TCZ) prevented deterioration of COVID-19 patients. Patients were divided into two cohorts based on the number of TCZ doses; cohort 1 (received one dose) and cohort 2 (received \u2265 two doses). In both cohorts, all-cause-mortality was the primary outcome. Of 270 hospitalized patients with COVID-19, 81 patients received TCZ. Fifty patients received one dose of TCZ and 31 received \u2265 two doses. All-cause-mortality in cohort 2 remained higher (41.9%) suggesting that there was no additional benefit of multiple doses of TCZ to prevent the primary outcome. In addition, multiple doses of TCZ did not change any other secondary outcome . Many patients with coronavirus disease 2019 (COVID-19) have a hyperactive immune response (cytokine storm) which has been incriminated in multiorgan dysfunction (MOD). These dysfunctions include acute respiratory distress syndrome (ARDS), acute renal failure (ARF), acute cardiac injury (ACI), and complications related to hypercoagulability and thrombosis. Interleukin-6 (IL-6) and granulocyte-macrophage colony-stimulating factor (GM-CSF) are the key cytokines involved in mediating systemic inflammation and triggering endothelial dysfunction which precipitates these events\u00a0-4. TocilAdult patients with confirmed SARS-CoV-2 infection by nasopharyngeal (NP) polymerase chain reaction (PCR) who were hospitalized from March 1 to August 20, 2020, were reviewed. Out of 270 patients, 81 received tocilizumab (TCZ) (IL6ri). Patients were divided into two cohorts based on the number of TCZ doses; cohort 1 (received one dose) and cohort 2 (received \u2265 two doses). Both TCZ doses were weight-based (400-800 mg). Data were extracted using the hospital electronic medical record retrospectively. Categorical variables were compared by conducting a chi-square test or Fisher's exact test while continuous ones were compared by conducting a median two-sample test. Statistical analysis was done using SAS software. In both cohorts, all-cause-mortality was the primary outcome. Secondary outcomes included rate of ICU admission, rate of intubation, AKI, ARDS, ACI, thrombotic events, septic shock, effect on inflammatory markers, and duration of total hospital stay. Missing values were adjusted using f variance.Of 270 hospitalized patients with COVID-19, 81 patients received TCZ. Fifty patients received one dose of TCZ and 31 received \u2265 two doses. Median age (years) for cohort 1 was 63.5 and cohort 2 was 65.0 . Cohort 1 was more likely to have male patients and the difference was statistically different . The patients in cohort 1 received TCZ on the third day (median) of hospitalization compared to cohort 2 who received the first dose on the second day of hospitalization. The median of lactate dehydrogenase (LDH), D-dimer, ferritin, and IL-6 at admission was higher in cohort 2 than cohort 1 among the hospitalized COVID-19 patients. Additional doses were given as a result of worsening respiratory status as measured by increasing O2 requirements. These additional doses were given at least 24 hours apart in cohort 2 compared to cohort 1."} +{"text": "Here, we report the complete gap-closed genome sequence for a model n-alkane-degrading anaerobe, Desulfoglaeba alkanexedens ALDC.Anaerobic alkane metabolism is critical in multiple environmental and industrial sectors, including environmental remediation, energy production, refined fuel stability, and biocorrosion. Here, we report the complete gap-closed genome sequence for a model Desulfoglaeba alkanexedens strain ALDC was obtained from a U.S. Navy oil wastewater storage facility . Genomic DNA was extracted using the genomic tip DNA extraction kit as described by the manufacturer. Libraries were prepared for Pacific Biosciences single-molecule real-time (SMRT) sequencing following the protocol for 20-kb template preparation using a BluePippin size selection system (https://www.pacb.com/wp-content/uploads/2015/09/Procedure-Checklist-20-kb-Template-Preparation-Using-BluePippin-Size-Selection.pdf) using a template preparation kit v1.0. The size-selected SMRTbell library was sequenced on the RS II platform using standard MagBead loading, P6-C4 chemistry, and 6-h data collection times. Postfiltering quality control analysis confirmed low adapter dimer levels and adequate read length, quality, and quantity to proceed with genome assembly using the HGAP2 algorithm, which was performed by the Washington State University Molecular Biology and Genomics Core Facility. The genome sequence of the single resulting contig was uploaded to the JGI Integrated Microbial Genomes (IMG) v4 pipeline for automated annotation and public access , 47 tRNA genes, and 9 miscellaneous categorized RNA genes. The 16S rRNA genes are 99.9% similar to each other and are both located on the minus strand at locus tags 111218 and 113015.The genome of assABCDE and masE, as well as ancillary genes for large and small subunits of methylmalonyl-coenzyme A (CoA) mutase, an ATP-dependent lysine-arginine-ornithine (LAO)/arginine-ornithine (AO) transport system, and an MmgE/PrpD family protein. Gene cluster 2 is on the plus strand and contains genes functionally homologous to those in cluster 1 but also contains assF and a gene coding for a putative carboxyl transferase. The assA1 and assA2 sequences were highly similar to each other, with a maximum amino acid sequence dissimilarity of 3.1%. AssA homologues were most closely associated with AssA1 of strain AK-01, with sequence identity of 77% and residue chemistry similarity of 88%. Alignment of each AssA protein sequence revealed the proposed catalytic glycine residue and single cysteine residue (distinct from the conserved tandem cysteine for pyruvate-formate lyases) conserved among known AssA homologues (Two distinct gene clusters encoding the requisite subunits for alkylsuccinate synthases were identified. Gene cluster 1 is on the minus strand and contains the requisite genes PRJNA541298 and GenBank accession number CP040098. Unassembled sequence reads are available under the accession number SRX7796451.This genome is available from the NCBI GenBank database under BioProject accession number"} +{"text": "Also, multiple sequence alignments of the spike (S) gene protein of selected candidate zoonotic coronaviruses alongside the S gene protein of the SARS-CoV-2 revealed closest evolutionary relationship (95.6%) with pangolin coronaviruses (S) gene. Clades formed between Wuhan SARS-CoV-2 phylogeny data and five others suggests viral entry trajectory while revealing genomic and protein SARS-CoV-2 data from Philippines as early ancestors.phylogeny of SARS-CoV-2 genomic data suggests profiling in diverse populations with and without the outbreak alongside migration history and racial background for mutation tracking and dating of viral subtype divergence which is essential for effective management of present and future zoonotic coronavirus outbreaks. Coronaviruses (CoVs) are enveloped viruses with a positive-sense, single-stranded RNA genome belonging to the coronaviridae family . CoVs arPhylogenetic analyses of 15 human CoV whole genomes revealed 2019 novel CoV (2019-nCoV) genome shares highest nucleotide sequence identity with SARS-CoV 79.7%) while its two evolutionarily conserved regions (envelope and nucleocapsid proteins) had sequence homology of 96% and 89.6% with same respectively . Hence, 9.7% whilInterestingly, conserved domains of CoVs have been indicated in literatures as vital entry targets in vaccine and drug development ,10. HoweComparison and analyses of conserved domain of 2019-nCoV/SARS-CoV-2 protein: reference number SARS-CoV-2 was retrieved from National Centre for Biotechnological Information (NCBI) database and query for its conserved domains (CDS) was launched using affiliated resources. Proteins with similar conserved domains were included in the subsequent multiple sequence alignment of spike gene of zoonotic coronaviruses investigated in this study.Building the spike (S) gene protein candidates from zoonotic coronavirus hosts: the identification of highly related contigs in a set of viral genomes from blast search of SARS-CoV like sequences in NCBI database (included in supplementary data) directed our subsequent search of spike proteins in the selected nine zoonotic coronaviruses. The CoV host spike proteins which were compared with SARS-CoV-2 (QHD43416.1) are: infectious bronchitis virus (IBV) (ADR51590.1), HCoV-229E (AII82124.1), transmissible gastroenteritis virus (TGEV) (AAQ02624.1), feline infectious peritonitis virus (FIPV) (AZH81408.1), porcine epidemic diarrhea virus (PEDV) (AKP16765.2), equine coronavirus (BAJ52885.1), murine hepatitis virus (MHV) (BAA11889.1), bovine coronavirus (CCE89341.1), pangolin coronavirus (QIQ54048.1), bat coronavirus (AWV67072.1). Their nucleotide and S gene protein sequences were pooled using NCBI resource tools while analysis was done using EMBOSS needle, clustal W2 and clustal omega respectively.Homology and phylogeny analysis of the S-protein genes in candidate zoonotic viruses: the identified spike gene protein sequences of animal coronaviruses were retrieved from submitted protein entries in NCBI database, homology analysis of the sequences was compared using clustal omega, EMBOSS needle while phylogenetic trees was constructed using the neighbor-joining method by CLUSTAL X software.SARS-CoV-2 sequence and phylogenetic analyses: in total, we culled the respective genomic and protein data of eight [of eight 2019-nCoof eight LC523807of eight LC523808of eight MT308702Conserved domains in SARS-CoV-2: four out of 29 domain hits generated from 2019-nCoV/SARS-CoV-2 CDD query were selected based on the E-value scores as seen in The region of 2019-nCoV domain which encodes nsp 11 spans from about 18046-19824bp. It was indicated in countering host innate antiviral response via inhibition of type I interferon (IFN) production using NendoU activity-dependent mechanisms in porcine reproductive syndrome viruses . The nspThey are SARS-CoV helicases that are chiefly concerned with RNA processing, DNA replication, recombination and repair, transcription and translation . A few pin-silico), in-vitro and in-vivo mechanisms [Identification of the origin, natural host (s) and evolutionary pathway of viruses which causes pandemics is essential to understand molecular mechanism of their cross-species interactivity and implementation of a proper control measure . Proteinchanisms ,37-39.in-vivo and in-vitro RNA recombination leading to vast genetic variability of positive strand RNA viruses has also been reported [Series of reported . Domestireported ,36 raisereported ,40 direcreported . HoweverPossible host-viral genetic recombination could alViral cellular mechanisms are vital factors necessary for replication during infection. Hence, identification of domains of viral entry and evasion of antiviral mechanisms in host cells is essential for development of effective therapeutic measures. Conserved domains that are vital targets sites for inhibition of SARS-CoV-2 viral entry and replication in host cells found in this study include nsp11, nsp 13, RdRp and corona super family while compounds such as RNA aptamers, ATP inhibitors, papain-like proteinase (plPRO) and 3C-like main protease-3CLpro etc. are viable indicated inhibitors of these domains; also, understanding the evolutionary pathway of the novel coronavirus transmission will not only help combat the current pandemic but assist in mutation tracking for identifying future zoonotic coronaviruses threats. The phylogenetic analyses of candidate zoonotic coronavirus (S) gene with SARS-CoV-2 revealed pangolin as the most recent ancestor which formed a sub-clade with bat S-gene suggesting interspecies recombination of CoV in bats and pangolins. Evolutionary pattern observed between SARS-CoV-2 genomic data from source of outbreak with recent entries analyzed in this study showed relative trajectory course of infection from source to other places except protein data from Philippines suggesting earlier existence of SARS-CoV-2 which should be further investigated. Also, genomic and protein data revealed racial viral subtype divergence and rapid rate of mutation despite the novelty of the outbreak. Precise dating of viral subtype divergence will enable researchers correlate divergence with epidemics and pandemics via viral sequence sampling for proper time-scale measurements of zoonotic threats in human populations. Therefore, there is an urgent need for large scale analysis and profiling of genetic data of SARS-CoV-2 in affected populations especially in Africa where there is paucity of genomic SARS-CoV data for effective therapeutic measures.Cell machinery for SARS-CoV-2 viral entry is through the binding of its surface proteins to host cell receptors-angiotensin converting enzyme 2 (ACE 2) in epithelial cells in hosts;Host factors accounts for a great deal of genome variability in viral recombinants which ranges from multi-resistance to evolutionary novelties;The level of interactions between the S protein and the virus receptor controls the host cell range.Conserved domains for inhibition of SARS-CoV-2 viral entry and replication in host cells found in this study include nsp11, nsp 13, RdRp and corona super family;Compounds such as RNA aptamers, ATP inhibitors, papain-like proteinase (plPRO) and 3C-like main protease-3CLpro etc. are viable indicated inhibitors of these domains;The phylogenetic analyses of candidate zoonotic coronavirus (S) gene with SARS-CoV-2 revealed pangolin as the most recent ancestor which formed a subclade with bat S-gene."} +{"text": "Ins2 gene (RIP-Cre25Mgn neurons) are required for central leptin signaling to reverse dyslipidemia, thereby hyperglycemia in insulin-deficient mice. Ablation of LEPRs in RIP-Cre25Mgn neurons completely blocks glucose-lowering effects of leptin in insulin-deficient mice. Further investigations reveal that insulin-deficient mice lacking LEPRs in RIP-Cre25Mgn neurons (RIP-Cre\u0394LEPR mice) exhibit greater lipid levels in blood and liver compared to wild-type controls, and that leptin injection into the brain does not suppress dyslipidemia in insulin-deficient RIP-Cre\u0394LEPR mice. Leptin administration into the brain combined with acipimox, which lowers blood lipids by suppressing triglyceride lipase activity, can restore normal glycemia in insulin-deficient RIP-Cre\u0394LEPR mice, suggesting that excess circulating lipids are a driving-force of hyperglycemia in these mice. Collectively, our data demonstrate that LEPRs in RIP-Cre25Mgn neurons significantly contribute to glucose-lowering effects of leptin in an insulin-independent manner by improving dyslipidemia.Leptin is a potent endocrine hormone produced by adipose tissue and regulates a broad range of whole-body metabolism such as glucose and lipid metabolism, even without insulin. Central leptin signaling can lower hyperglycemia in insulin-deficient rodents via multiple mechanisms, including improvements of dyslipidemia. However, the specific neurons that regulate anti-dyslipidemia effects of leptin remain unidentified. Here we report that leptin receptors (LEPRs) in neurons expressing Cre recombinase driven by a short fragment of a promoter region of Central leptin injections can maintain euglycemic ranges in insulin-deficient rodent models without exogenous insulin administration \u20136. Previ25Mgn neurons) are distinguished from AgRP neurons, and uniquely composed from several neuronal groups (25Mgn cells result from ablation of LEPRs in the CNS.A study using single cell RNA-sequence shows that GABAergic neurons in the ARC and median eminence (Arc-ME) complex are composed of distinct genetically-defined neuronal groups . AgRP nel groups . A RIP-C \u03b2-cells . However \u03b2-cells , 13 due \u03b2-cells , 15. Bec neurons , 16, the neurons \u201320. RIP-enditure , 20 and enditure , 19 in tide (TG) . Becauseide (TG) , metabol25Mgn neurons is very similar to that of leptin-responsive GABAergic neurons (25Mgn neurons only represent a portion of leptin-responsive GABAergic neurons because the phenotypic differences between mice lacking LEPRs in GABAergic neurons (GABA\u0394LEPR) and RIP-Cre25Mgn neurons (RIP-Cre\u0394LEPR) are enormous, for instance, body weight of GABA\u0394LEPR mice is near identical to that of db/db mice and examined whether i.c.v. leptin injection can lower hyperglycemia in these mice without insulin. We found that deletion of LEPRs in RIP-Cre25Mgn neurons blocks glucose- and lipid-lowering effects of leptin in insulin-deficient mice. Further, we found that administration of lipid-lowering compound acipimox can restore glucose-lowering effects of leptin in insulin-deficient RIP-Cre\u0394LEPR mice. Our results indicate that RIP-Cre25Mgn neurons are vital components for glucose-lowering effects of leptin through the regulation of lipid metabolism in an insulin-independent manner.The anatomical profiling of GABAergic RIP-Cre neurons , 9. GABAus (MTu) . Of notey weight . Based o25Mgn , RIP-Cre25Mgn::LeprloxTB/loxTB::RIPHerr-DTR, LeprloxTB/loxTB::RIPHerr-DTR (null control for RIP-Cre25Mgn::LeprloxTB/loxTB::RIPHerr-DTR), LeprWT/WT::RIPHerr-DTR and RIP-Cre25Mgn::LeprWT/WT::RIPHerr-DTR (wild-type control for RIP-Cre25Mgn::LeprloxTB/loxTB::RIPHerr-DTR), GcgloxTB/loxTB::RIPHerr-DTR, GcgWT/WT::RIPHerr-DTR (control for GcgloxTB/loxTB ::RIPHerr-DTR), and RIP-Cre25Mgn::RIPHerr-DTR::Ai9TB/\u2212. We used KAPA Mouse genotyping kits to determine genotypes. All genotyping primers and predicted band sizes are described in RIP-Cre25Mgn and Ai9 25Mgn were obtprflox/\u2212 and LeprDTR mice were obtescribed . To geneDTR mice were breo allele to ident25Mgn::LeprloxTB/loxTB::RIPHerr-DTR was measured by rodent fMRI as previously described . As previously described .To induce insulin deficiency, mice were treated with diphtheria toxin . DT was dissolved in sterile 0.9% NaCl solution at a concentration of 150 \u03bcg/mL and kept at \u221280\u00b0C until use. Each concentrated DT aliquot was diluted to 0.075 \u03bcg/mL in sterile saline and delivered intraperitoneally (i.p) at a dose of 0.5 \u03bcg/kg B.W. one time per day for 3 consecutive days to ablate pancreatic \u03b2-cells was dissolved in sterile phosphate-buffered saline and administered by intracerebroventricular (i.c.v.) infusion using osmotic pumps as previously described , 8. An o25Mgn neurons are required or sufficient for leptin's capacity to reduce lethality in insulin-deficient mice. Free fatty acids (FFAs), ketone bodies, TG, and glycerol in blood were measured by commercially available kits as described previously . Complementary DNA from 1 \u03bcg of input RNA was generated with the High Capacity cDNA Reverse Transcription Kits (Life Technologies). SYBR Green PCR master mix (Life Technologies) was used for the quantitative real time PCR analysis. Sequences of deoxy-oligonucleotides primers are outlined in 13C deisotoping, and quantitation were conducted using a custom programmed Microsoft Excel macro as previously described after considering the principles of lipidomics as described previously . Lipids pidomics , 38.Acipimox was i.p. administered at a dose of 100 mg/kg B.W. two times per day for five consecutive days. Control solution was sterile saline (0.9% NaCl).t-test, one-way ANOVA followed by Turkey's multiple comparison test, two-way ANOVA followed by one-way ANOVA (Tukey's multiple comparison test if the interaction was significant) or unpaired t-test in the same factor (if the interaction was not significant), or repeated measures ANOVA followed by unpaired t-test if the interaction was significant. For analysis of survival curves, Log-rank (Mantel-Cox) testing was used. Since the number of mice surviving declined over time, we were prohibited from utilizing repeated measures ANOVA . We, therefore, performed the statistical analysis and showed each day individually in Figures 2C\u2013H and Figure 4E. For all tests, statistical significance was set at a critical value of P < 0.05.Data are represented as the group mean \u00b1 S.E.M. as indicated in each figure legend. Statistical significance was determined using GraphPad PRISM software by unpaired \u0394LEPR mice did not show significant differences of blood glucose and FFAs, while they exhibited modest increases in body weight and higher circulating insulin and TG levels compared to WT group neurons, and mRNA levels of Pomc, Agrp, and Ins2 in the mediobasal hypothalamus a RIPHerr fragment transgene drives genes in lesser ectopic expression levels than other RIP fragments do into RIP-Cre25Mgn::RIPHerr-DTR::tdTomatoTB/TB mice, which allow us to visualize RIP-Cre25Mgn neurons by a red fluorescent reporter tdTomato within 2\u20133 weeks after the induction of insulin deficiency (\u0394LEPR mice administered leptin (RIP-Cre\u0394LEPR-LEP) was comparable to insulin-deficient WT mice administered leptin (WT-LEP) despite (WT-LEP) , 6, 8, anitiated . Food innitiated . Previounitiated . At Day mparable . These d. WT-LEP .25Mgn neurons is sufficient for leptin to exert its glucose-lowering effects. To do so, we generated mice re-expressing LEPRs in RIP-Cre25Mgn neurons (RIP-CreRA\u2212LEPR). RIP-CreRA\u2212LEPR mice had a similar body weight up to 14\u201315 weeks of ages compared to LEPRs null mice (Lepr\u0394) . We did not see any improvements of the survival rate, blood glucose, body weight of RIP-CreRA\u2212LEPR-LEP compared to Lepr\u0394-LEP . Startinpr\u0394 mice . Howeverrol mice . We chroepr\u0394-LEP , althoug\u0394LEPR-LEP, however, i.c.v. leptin injection lowered blood glucagon in insulin-deficient RIP-Cre\u0394LEPR mice (\u0394Null mice (GcgRA\u0394Null-LEP) and examined their blood glucose levels, survival rate, body weight, and food intake. I.c.v. PBS injection did not reverse hyperglycemia in insulin-deficient GcgRA\u0394Null mice (GcgRA\u0394Null-PBS) . We chroull-PBS) . Interesull mice .KO-PBS was ~60% at 25 days after induction of insulin deficiency of mice in the insulin-clamped condition dramatically reduced blood FFAs but not glucose levels in RIPHerr-DTR-induced insulin-deficient mice (\u0394LEPR-LEP (two times per day for 5 days) 10 days after leptin administration initiated (\u0394LEPR-LEP (RIP-Cre\u0394LEPR-LEP-Acip) compared to the control group , along wLEP-Sal) . Collect25Mgn neurons are required for lipid-lowering effects of leptin, thereby glucose-lowering effects in an insulin-independent manner. Our data suggest that glucagon signaling does not drive hyperglycemia in insulin-deficient mice lacking LEPRs in RIP-Cre25Mgn administered i.c.v. leptin leptin-responsive GABAergic neurons are located only in the ARC, DMH, and LHA, and (ii) GABAergic RIP-Cre25Mgn neurons are anatomically limited to the ARC, DMH, and MTu; therefore, ARC and DHM are only overlapped regions that match to the anatomical and chemical-classification profiling from previous studies. Although studies has shown that GABAergic ARC \u201359 and Dneurons) neurons 25Mgn neurons in the ARC (ARC RIP-Cre25Mgn neurons) are distinct from AgRP neurons , and none of them are AgRP neurons at the single cell level is still undetermined. In addition, the role of DMH RIP-Cre25Mgn neurons in the regulation of metabolism is completely unknown. Further studies will be warranted to pinpoint the specific neuronal group(s) within RIP-Cre25Mgn neurons that regulate fat metabolism in an insulin-independent manner.RIP-Cre neurons , which a neurons , 8. ARC behavior . Mice larol mice . Compare\u0394LEPR-LEP tended to be higher than WT-LEP (\u0394LEPR-LEP-Acip was significantly higher that RIP-Cre\u0394LEPR-LEP-Sal (\u0394LEPR-LEP (25Mgn neurons regulates lipid metabolism in an insulin-independent manner.Glucose-lowering effects of leptin in insulin-deficient rodents are abolished by lipids infusion . Perry an WT-LEP . Surpris-LEP-Sal , althougLEPR-LEP . This reThe CNS-peripheral pathway underlying hypothalamic regulation of lipid metabolism by leptin in the absence of insulin remains largely unclear. Decreased leptin levels by fasting trigger lipolysis in WAT via activation of HPA-axis, leading to glucose-counterregulatory actions in response to starvation-induced hypoglycemia . The sym25Mgn neurons significantly contribute to glucose-lowering effects of leptin in an insulin-independent manner by reversing aberrant lipid metabolism. It is still unclear whether LEPRs in RIP-Cre25Mgn neurons mediate effects of leptin on lipid metabolism such as increases of fatty acid oxidation and lipolysis in adipose tissues in the presence of insulin. Unraveling the mechanism by which LEPRs in RIP-Cre25Mgn neurons regulate lipid metabolism may pave a way to design new treatments for several forms of diabetes.In summary, our current study demonstrates that LEPRs in RIP-CreThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by The Institutional Animal Care and Use Committee of the University of Texas Southwestern Medical Center and the Institutional Animal Care and Use Committee of the University of Texas Health San Antonio. Written informed consent was obtained from the owners for the participation of their animals in this study.GcgloxTB/WT mice and edited manuscript. XH supervised experiments and edited manuscript. TF designed, performed, supervised, analyzed experiments, and wrote and finalized the manuscript. All authors contributed to the article and approved the submitted version.AS performed and analyzed experiments and edited the manuscript. JP designed, performed, and analyzed experiments, and edited the manuscript. MP performed and analyze experiments. SF performed experiments. DS generated The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "The development of autoimmunity results from a breakdown of immunoregulation and involves cellularly complex immune responses against broad repertoires of epitope specificities. As a result, selective targeting of specific effector autoreactive T- or B-cells is not a realistic therapeutic option for most autoimmune diseases. Induction of autoantigen-specific regulatory T-cells capable of effecting bystander (dominant), yet tissue-specific, immunoregulation has thus emerged as a preferred therapeutic alternative. We have shown that peptide-major histocompatibility complex (pMHC)-based nanomedicines can re-program cognate autoantigen-experienced T-cells into disease-suppressing regulatory T-cells, which in turn elicit the formation of complex regulatory cell networks capable of comprehensively suppressing organ-specific autoimmunity without impairing normal immunity. Here, we summarize the various pMHC-based nanomedicines and disease models tested to date, the engineering principles underpinning the pharmacodynamic and therapeutic potency of these compounds, and the underlying mechanisms of action. The development of autoimmune disease results from dysregulated immune responses to self that are triggered by ill-defined environmental cues in genetically predisposed individuals. Such immune responses lead to the activation and recruitment of effector autoreactive T-cells into specific tissues/organs, the recruitment of additional inflammatory cell types to the site, chronic inflammation and, eventually, tissue/organ dysfunction and/or destruction. Given the autoantigenic complexity of most autoimmune disorders, targeting of effector autoreactive T-cell specificities is not a realistic therapeutic option for the treatment of these diseases. An alternative includes promoting the formation and/or expansion of regulatory autoreactive T-cell clonotypes capable of effecting bystander immunoregulation (against the many non-cognate autoantigenic epitopes that are targeted in the course of a specific disease). Several approaches that are potentially capable of eliciting bystander immunoregulation have been described over the last decade, but the mechanisms of action of some of these remain unclear and their therapeutic efficacy has not been thoroughly tested in non-contrived models of spontaneous, polyclonal autoimmunity [reviewed in ].in vivo. Upon pMHC-NP-induced expansion, these cognate, mono-specific autoreactive T-cells elicit self-sustaining regulatory cell networks that efficiently suppress polyclonal autoreactive T-cell responses in several murine models of autoimmunity, without compromising normal immunity. In this mini-review, we discuss the key engineering principles behind the pharmacodynamic activity of these compounds, the mechanisms underlying their therapeutic activity, and the disease models in which we have documented efficacy (We have shown that profound and sustained ligation of antigen receptors on cognate effector autoreactive T-cells by nanoparticles (NPs) displaying multiple copies of disease-relevant peptide-MHC class I or class II complexes (pMHC-NP) can trigger their differentiation into regulatory T-cells efficacy Table 1.206\u2013214). This T-cell specificity plays a significant role in the progression of islet inflammation to beta cell destruction in NOD mice aimed at triggering the deletion of a highly prevalent and diabetogenic CD8+ T-cell population specific for residues 206\u2013214 of the islet-specific glucose-6-phosphatase catalytic subunit-related protein complexes, should be able to efficiently deplete cognate CD8+ clonotypes over a broad dose range. We further reasoned that, if this hypothesis were true, combinations of pMHCI-NPs targeting different CD8+ T-cell specificities should be able to substantially reduce the pool of beta cell killing effectors. Surprisingly, although treatment of NOD mice with the multi-specific pool of pMHCI-NPs had therapeutic effects, so did NPs exclusively displaying the IGRPKd pMHCI Table 1.The allelic complexity MHC class I loci in humans limits the translational significance of pMHCI-NPs for human immunotherapy, as numerous compounds would need to be developed to treat a significant fraction of the patient population for any given autoimmune disease.Our work with pMHCI-NPs suggested that treatment with these compounds harnesses a naturally-occurring negative feedback regulatory loop that might have arisen during natural evolution to oppose the progression of autoimmune inflammation. In turn, this idea suggested that such negative feedback regulatory loops might also exist in the autoreactive CD4+ T-cell compartment. This hypothesis predicted that treatment of autoimmune disease-affected mice with pMHCII-NPs would elicit the formation and/or expansion of autoantigen-specific regulatory CD4+ T-cells. Since there are strong associations between human autoimmune diseases and certain HLA class II types, and CD4+ T-cells play a central role in the initiation, progression and maintenance of most, if not all autoimmune diseases, we reasoned that these pMHCII-based compounds would have superior translational significance than their pMHCI-based counterparts.g7, IGRP128\u2013145/IAg7 or IGRP4\u201322/IAg7) could stably restore normoglycemia in spontaneously diabetic NOD mice (38\u221249/IAb or human proteolipid protein (hPLP)175\u2013192/DR4 complexes were able to reverse limb paralysis in these animals when administered at the peak of disease severity. Similar therapeutic effects were seen when using hMOG97\u2013108/DR4-IE-NPs to treat hPLP175\u2013192-induced EAE in HLA-DR4-transgenic C57BL/6 mice, demonstrating that the pMHCII displayed by these compounds need not have to target disease-initiating T-cells -like phenotype and transcriptional profile, as compared to murine Tr1-like cells described elsewhere . ExperimIn vitro studies using NPs of different sizes further indicated that the \u201coptimal\u201d pMHC valency values increased with NP size, indicating that pMHC density (number of pMHCs/surface area), rather than pMHC valency , is the most critical parameter demonstrated that the biological activity of pMHC-NPs produced with NPs of a given size is a function of pMHC valency (number of pMHC monomers per NP) . In vitrarameter . That isarameter . These oIn vitro, compounds displaying threshold and supra-threshold pMHC valencies/densities elicited very rapid (within 2h), vigorous and sustained (>24h) TCR signals, as compared to optimal concentrations of an agonistic CD3\u03f5 mAb or PMA/ionomycin, which triggered much slower responses that peaked at 14h and progressively decreased afterwards. Furthermore, imaging of pMHC-NP/T-cell interactions via transmission electron microscopy, super-resolution microscopy and scanning electron microscopy revealed that pMHC-NPs bind cognate T-cells as clusters of several NPs spanning ~100\u2013150 nm that progressively grew to ~400 nm, culminating in internalization of the NPs in intracellular vesicles, starting ~3 h after binding. Importantly, cluster formation was only observed when using NPs coated at threshold and supra-threshold pMHC valencies/densities. Thus, pMHC-NPs function as sustained TCR nanocluster-binding and microcluster-triggering devices cells and re-engineering of pMHC heterodimers as knob-into-hole-based Fc fusions addressed these limitations; KIH-based pMHC molecules are expressed at much higher yields than pMHCIIs heterodimerized using leucine zippers and can be purified to the desired levels of purity using protein A chromatography and additional polishing steps routinely used in the purification of biologics (Our first generation pMHC compounds involved the expression of recombinant pMHC molecules in iologics .i.e. in pancreas-draining lymph nodes) autoantigen-loaded dendritic cells (DCs) and myeloid APCs in an Interleukin-10 (IL-10)- and Tumor Growth Factor beta (TGF\u03b2)-dependent manner. Furthermore, recruitment of these antigen-specific Tr1 cells into the pancreas-draining lymph nodes of the treated mice promoted the formation/recruitment of interleukin-10 (IL-10)-producing CD1dhigh/CD5+ B-cells therapeutic activity. Thus, pMHCII-NP therapy elicits the formation of disease-specific regulatory cell networks capable of restoring immune homeostasis.Studies in T1D (and later confirmed in other disease models) showed that pMHCII-NP-induced/expanded Tr1 cells suppressed the pro-inflammatory and antigen presentation capacities of local and proximal (e.g. cholangitis) can spread to anatomic liver structures that are preferentially targeted in other liver autoimmune diseases (e.g. hepatitis). These observations begged the question of whether autoimmune liver disease-relevant pMHCII-NP compounds would be disease-specific (e.g. against PBC) or pan-liver autoimmune disease-specific (e.g. capable of blunting different liver autoimmune diseases).Autoimmunity in the liver manifests itself through various diseases, including primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC) and autoimmune hepatitis (AIH). In these diseases, unlike those discussed above, the autoimmune response recognizes ubiquitously expressed autoantigens, such as the mitochondrial pyruvate dehydrogenase complex-E2 component (PDC-E2) in PBC; or nuclear, cytoplasmic, or Golgi-enriched proteins, such as F-actin, formimidoyltransferase cyclodeaminase (FTCD), or cytochrome P450 (CYPD2D6) in AIH; or tropomyosin isoform 5 (hTM5) in PSC, among others. In addition, there is a significant subgroup of patients in which liver autoimmunity has features of both, cholestasis and autoimmune hepatitis, suggesting that autoimmune responses against certain autoantigenic targets in a given liver autoimmune disease (166\u2013181/IAg7- and PDC-E282\u201396/IAg7-NPs) blunted the progression of liver autoimmunity in NOD.c3c4 mice and (NODxB6.Ifng ARE-Del\u2212/\u2212) F1 mice, which spontaneously develop a form of liver autoimmunity that closely resembles human PBC blunted Ad-hFTCD-induced AIH in NOD mice as efficiently as mFTCD58-72/IAg7-NPs. CYPD398-412/IAg7-NPs could also blunt the progression of PSC in NOD.Abcb4\u2212/\u2212 mice results in the priming and recruitment of T-cell specificities targeting other autoantigens. Our work indicates that these secondary T-cell specificities can also be harnessed by pMHCII-NPs to blunt disease progression, as we had previously documented in EAE and CYPD398-412/IAg7-NPs (AIH-relevant). Neither of these two compounds triggered the expansion of cognate Tr1-like CD4+ T-cells, suggesting that pancreatic beta cells either did not shed the corresponding antigenic epitopes, or did so in amounts insufficient to generate epitope-experienced CD4+ T-cells and BDC2.5/IAg7-NPs (T1D-specific), but not MOG36-50/IAg7-NPs .We sought to first investigate these assumptions by treating NOD mice with PDC-E2 T-cells Table 1.166\u2013181/IAg7-NPs and CYPD398\u2013412/IAg7-NPs , or PDC-E294\u2013108/IAb-NPs and CYPD353\u2013367/IAb-NPs to blunt MOG36\u201355-induced EAE in NOD and C57BL/6 mice, respectively. These experiments indicated that, upon oligodendrocyte damage, both PDC-E2 and CYPD2D6 are delivered to proximal APCs for autoreactive CD4+ T-cell priming, enabling Tr1 cell generation by cognate pMHCII-NPs, their recruitment to the CLNs, and suppression of EAE in B6 mice having Ad-hFTCD-induced AIH and/or EAE revealed that Tr1 cell recruitment and therapeutic effects require local autoantigen expression had pharmacodynamic activity but lacked therapeutic activity; liver inflammation in these mice retained antigen-specific Tr1 cells non-specifically (Subsequent experiments using MOGpression Table 1.i.e. in co-morbid mice) may give rise to competitive autoimmunity where neither tissue will recruit a sufficient number of Tr1 cells beyond the suppression threshold (To better understand how pMHCII-NP-expanded Tr1 cells traffic to multiple sites of inflammation (in co-morbid mice), we developed a mathematical model composed of a system of nonlinear ordinary differential equations . We comptential) .): (3) liver inflammation has the potential to non-specifically draw T-regulatory cells away from sites of cognate autoantigen expression and autoimmune inflammation.Collectively, these data indicated that (1) autoreactive T-cells targeting ubiquitous antigens can be awakened by antigen shedding from different cells/tissues (scid/Il2rg\u2212/\u2212 (NSG) mice engrafted with peripheral blood mononuclear cells (PBMCs) from patients. Treatment of NSG mice humanized with PBMCs from DRB1*0301+ and/or DRB1*0401+ T1D patients with NPs displaying human IGRP13\u201325-DRB1*0301 or human pre-proinsulin (PPI)76-90/DRB1*0401 complexes resulted in the expansion of cognate IL-10-producing CD4+ T-cells co-expressing the Tr1 cell markers CD49b and LAG-3 . The nature of the autoantigen-experienced T-cell type that gives rise to cognate Tr1 cells in response to pMHCII-NP therapy remains unclear, and so do the mechanisms in vivo implies that de novo suppression of newly recruited (non-autoantigen-loaded) APCs is required for sustained immunoregulation. On the other hand, this allows new, non-immunosuppressed APCs to process and present pathogen-derived antigens to pathogen-specific T-cell specificities. In addition, during a local infection, antigens derived from local pathogens likely overwhelm the APCs\u2019 antigen presentation machinery, diluting expression of the Tr1\u2019s cognate pMHC below the threshold required for Tr1 cell-induced APC immunoregulation. Upon clearance of the infection, new incoming APCs would then regain the ability to present the Tr1\u2019s cognate pMHC, allowing these Tr1 cells to resume their anti-inflammatory activity.The ability of these compounds to suppress autoantigen-loading APCs may explain why they spare normal immune responses to pathogens. The short half-life of dendritic cells in vivo via pMHCII-NP therapy. Ideal clinical candidates are those displaying epitopes from prevalent tissue-specific autoantigens in the context of allelic MHCII types expressed by a significant fraction of the patient population. For autoimmune diseases with strong HLA class II associations, such as T1D or Celiac Disease, the choice of HLA type is straightforward. For diseases in which there is not a strong HLA class II allelic bias, the use of MHCII molecules encoded in oligomorphic HLA class II loci, such as DRB3, DRB4 and DRB5 loci is desirable. Up to three different pMHC-NP compounds would be sufficient to treat >80% of the patient population for any given autoimmune disorder. Notwithstanding the progress to date, candidate pMHCII selection remains a bottleneck that would benefit from the availability of improved, higher throughput methods capable of enumerating the frequency of defined pHLAII specificities (as opposed to peptide specificities regardless of HLA restriction) in patients\u2019 peripheral blood samples. Although we have provided compelling evidence supporting translational potential, we do not yet know whether these compounds will be effective in clinical trials.At the translational level, we have made significant progress in candidate pMHCII identification and selection for specific autoimmune diseases. Our experimental work in mice has suggested that most, if not all, CD4+ T-cell specificities recognizing autoantigenic epitopes expressed by the target tissue of a given autoimmune disease can be re-programmed into Tr1 cells Lastly, the work done to date begs the question of whether T-cell types other than CD8+ and CD4+ T-cells, such as invariant Natural Killer T-cells or Mucosal Associated Invariant T-cells, can also be re-programmed into autoimmune disease-suppressing cell types using MHC-based nanomedicines. The next few years should provide answers to these outstanding questions.The manuscript was written and edited by both co-authors. All authors contributed to the article and approved the submitted version.The authors\u2019 work summarized here was funded by the Canadian Institutes of Health Research (CIHR), Diabetes Canada, the Crohn\u2019s and Colitis Foundation of Canada, the Multiple Sclerosis Society of Canada (MSSC), ISCIII and FEDER , REEM (Red Espa\u00f1ola de Esclerosis M\u00faltiple), NEURON-ERANET , the Praespero Foundation, the Ministerio de Economia y Competitividad of Spain , and Generalitat de Catalunya (SGR and CERCA Programmes). The JMDRC is supported by Diabetes Canada.PS is the scientific founder of Parvus Therapeutics Inc. and has a financial interest in the company.The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "The enhanced performance was attributed to the improved morphology of PEDOT:PSS, which consequently increased the hole injection capability of the optimized ITO/(Cu-Li):NiCo2O4/PEDOT:PSS electrode.The performance of solution-processed organic light emitting diodes (OLEDs) is often limited by non-uniform contacts. In this work, we introduce Ni-containing solution-processed metal oxide (MO) interfacial layers inserted between indium tin oxide (ITO) and poly:poly(styrene sulfonate) (PEDOT:PSS) to improve the bottom electrode contact for OLEDs using the poly(p-phenylene vinylene) (PPV) derivative Super-Yellow (SY) as an emission layer. For ITO/Ni-containing MO/PEDOT:PSS bottom electrode structures we show enhanced wetting properties that result in an improved OLED device efficiency. Best performance is achieved using a Cu-Li co-doped spinel nickel cobaltite [(Cu-Li):NiCo Since the discovery of polymer light emitting diodes , importax, WOx, and MoOx. The improvement in device efficiency were interpreted in terms of energetic alignment with stair-like hole injection electrode for the super-yellow OLED devices. To provide a direct confirmation of the improved hole injection capability of the proposed ITO/(Cu-Li):NiCo2O4/PEDOT:PSS bottom electrode the corresponding OLED devices were measured and compared, using the photocurrent mapping technique (PCT) but not with SCS CuOx interface modification. The improved performance of Ni-based metal oxides indicated the influence of Ni/oxygen vacancies on the presented wetting properties. We show that a range of Ni-containing solution-processed MO interfacial layers between ITO and PEDOT:PSS can be used to improve the bottom electrode contact and OLED device efficiency. The OLED device performance based on ITO/Ni-containing MOs/PEDOT:PSS bottom electrode was strongly correlated not only with the common optoelectronic properties but also with the MOs wetting properties. The best performing co-doped (Cu-Li):NiCo2O4 interfacial layer exhibited adequate transparency, the highest conductivity of ~4 S cm\u22121 and the lowest static contact angle of ~6\u00b0. The wetting properties of co-doped (Cu-Li):NiCo2O4 resulted in a smooth and homogeneous PEDOT:PSS overlayer that provided intimate interfaces and improved the hole injection properties of the bottom electrode [ITO/(Cu-Li):NiCo2O4/PEDOT:PSS] for the super-yellow OLED devices, as experimentally verified by the presented photocurrent mapping and single carrier device studies. The incorporation of co-doped NiCo2O4 [(Cu-Li):NiCo2O4] as interfacial layer between ITO and PEDOT:PSS provided a high performance ITO/(Cu-Li):NiCo2O4/PEDOT:PSS bottom electrode that increased the super yellow OLEDs current efficiency and power efficacy by 12% and 11%, respectively. We believe that the proposed metal oxide-based interface modification can be used as a method to control wetting properties and to improve the bottom contact in a range of solution-processed light emitting diodes.The effect of appropriate solution-processed metal oxide interfacial layers between ITO and PEDOT:PSS on the performance of OLEDs was investigated. Improved wetting properties were achieved by using solution combustion synthesized Ni-based metal oxides [Cu:NiO"} +{"text": "The role of nuclear medicine in the management of oncological patients has expanded during last two decades. The number of radiopharmaceuticals contributing to the realization of theranostics/radiotheranostics in the context of personalized medicine is increasing. This review is focused on the examples of targeted (radio)pharmaceuticals for the imaging and therapy of neuroendocrine neoplasms (NENs), prostate cancer, and breast cancer. These examples strongly demonstrate the tendency of nuclear medicine development towards personalized medicine. The number of nuclear medicine examinations and therapeutic procedures is increasing with acceleration worldwide reflecting the growing importance of the field in the modern healthcare system. The growth and expansion of nuclear medicine relies on the development and availability of radiopharmaceuticals. High demand for radiopharmaceuticals with biological activity specific for a certain disease yielded personalized patient treatment approaches, in particular theranostics using molecular imaging for the disease staging and prediction of the efficacy of specific therapeutic interventions on individual basis as well as for the monitoring response to the treatment. Molecular imaging in nuclear medicine is presented by positron emission tomography (PET) and single photon emission computed tomography (SPECT), and in combination with endoradiotherapy it can be defined as radiotheranostics .123I/124I/131I Ga-SST/PET) is recommended for the diagnosis, staging, and patient selection for endoradiotherapy [68Ga]Ga-SST was found valuable not only for pre-operative assessment of resectable lesions and multiple unresectable lesions but also for intraoperative radio-guided resection [68Ga]Ga-SST/PET is also efficient tool for the scheduling of the treatment combining long-acting somatostatin analogue (SSA) therapy and endoradiotherapy Ga-SST/PET-CT has become the most promising non-invasive technique to study NENs and demonstrated superiority over such imaging agents as [123I]MIBG, [11C]-HTP, [18F]FDG, [18F]FDOPA, [111In]-pentetreotide, and [99mTc]-SST analogues [18F]FDG is used for measuring the tumor metabolic rate whereas [68Ga]Ga-SST provides information on SSTR expression guiding the biopsy [68Ga]Ga-SST/PET-CT and minor/no [18F]FDG uptake was associated with better prognosis Ga-SST/PET-CT were demonstrated over MRI and CT [68Ga]Ga-SST/PET-CT and [68Ga]Ga-SST/PET-MR showed similar PET image quality however uptake quantification was found more accurate on PET/CT and detection rate for bone metastases was higher Ga-SST analogues demonstrated necessity for accurate discrimination between cancerous and benign lesions, physiological and inflammatory uptake [68Ga]Ga-SST analogues in patients and healthy volunteers revealed low total effective dose allowing multiple examinations per year and no immediate or delayed toxicity Ga-SST PET images require high accuracy. The (semi)-quantitative assessment of the response, using [68Ga]Ga-SST PET/CT, to the endoradiotherapy with 177Lu- or 90Y-based somatostatin analogues and re-staging of the disease have entered clinical practice. Patient selection, prognosis, prediction of absorbed dose for radiotherapy, and treatment response based on [68Ga]Ga-SST PET/CT have been performed using various parameters such as maximum standardized uptake value (SUVmax), tumor-to-background SUVmax ratio (TBR), tumor-to-liver SUVmax ratio (TLR), tumor-to spleen SUVmax ratio (TSR), functional volume (FV), K-Patlak and Ki [max cut-off of 16.4 was proposed for patient stratification for endoradiotherapy [max and Ki-67 index indicates that [68Ga]Ga-SST PET reflects cell proliferation and helps guide disease management Ga-SST lesion uptake and treatment response monitoring [68Ga]Ga-SST PET and computed by summing the volumes of all pathological foci was suggested as prognostic biomarker with cut-off of 13.8 cm3 [68Ga]Ga-SST uptake than 50% of SUVmax within the volume of interest for each lesion, demonstrated prognostic value of survival [68Ga]Ga-SST PET/CT images was found valuable for prediction and prognosis with heterogeneity leading to lower survival, even though it is difficult to quantify [68Ga]Ga-SST/PET-CT. In the patient sub-group re-evaluated for recurrence, the treatment management was changed after [68Ga]Ga-SST/PET-CT in up to 25% of the patients [68Ga]Ga-SST/PET-CT [68Ga]Ga-SST/PET-CT led to the treatment change in staggering 90.9% of patients with suspected recurrence [68Ga]Ga-SST/PET-CT changed the tumor staging from non-malignant to metastatic disease . The tre16%\u201371%) and 50% 16%\u201371%) of the ppatients . OperatiT/PET-CT . [68Ga]Gcurrence . [68Ga]G disease 43]..68Ga]Ga-177Lu emits gamma particles that can be detected by SPECT for the dosimetry measurement and calculation, however not prior but during the therapy course. The [177Lu]Lu-DOTA-TATE dosimetry feasibility and impact on radiotherapy efficacy and outcome was demonstrated wherein the survival improved with increased treatment cycle number determined based on dosimetry vs 6.71 d (177Lu)) and thus different pharmacokinetic time window. Kinetic modeling could provide a solution wherein the early distribution time points could be acquired by [68Ga]Ga-SST/PET with high accuracy [99mTc-MDP in terms of sensitivity and specificity that are crucial parameters for staging accuracy and treatment planning. It is relevant for initial staging [max on [68Ga]Ga-PSMA PET/CT and PSMA expression in primary prostate cancer, determined histopathologically, was found and cut-off for SUVmax of 3.15 to discriminate tumor from normal prostate was recommended demonstrBED-CC)] . Since tBED-CC)] ,127,128. staging , early d staging ,131, and staging ,133 partommended . Early iommended . 68Ga]Ga-PSMA-11 and [177Lu]Lu-PSMA-617 for the patient selection and prediction of response is conducted Ga-ABY-025/PET-CT detected bone metastasis and primary tumor with high SUVmax. The HER2-overexpression in the metastasis was confirmed by IHC. Consequently, the treatment regimen was significantly changed. Moreover, the false positive finding in the axilla by [18F]FDG/PET-CT presented higher detection rate and image contrast, more favorable organ distribution and lower effective and absorbed doses Ga-ABY-025 PET-CT technology worldwide spreading is production and availability of the radiopharmaceutical [68Ga]Ga-ABY-025/PET-CT for non-invasive assessment of HER2-status in breast cancers in a multicenter setting .In order to facilitate standardized multicenter trials and to enable dissemination of this diagnostic methodology for routine clinical use, it is necessary to provide data evaluation methods that are independent on the variation of PET scanner characteristics. Intra-image normalization such as tumor-to-reference tissue ratio (T/R) was investigated and spleen was found the most accurate approach providing a simple and robust semi-quantification of HER2 expression . The splceutical . The aut68Ga]Ga-ABY-025 PET/CT has potential for therapy planning and treatment response monitoring, enabling adjustment of the treatment very early in the process.Reliable whole-body, quantitative assessment of HER2-receptor expression is crucial in order to identify patients with HER2-positive tumors that can benefit from HER2 targeted treatments . It is aThese examples of NENs, prostate cancer, and breast cancer management using targeted imaging and (radio)therapy have proven the concept of (radio)theranostics for clinical practice valid. Numerous publications report on the alteration of treatment regimen based on radionuclide imaging that provides non-invasive, whole body mapping of the specific target in a single examination that can be safely repeated multiple times for monitoring treatment response and disease progression. Pre-therapeutic determination of the absorbed doses to normal organs and lesions is essential for treatment planning. However, the use of short-lived radionuclides for the prediction of dosimetry for long-lived therapeutic radionuclides presents challenge and therapeutic dose planning based on the receptor expression quantification awaits prospective clinical studies to prove the concept.Nuclear medicine is becoming an important component in personalized patient treatment. Dissemination of the (radio)theranostic technology requires standardization and harmonization of the clinical protocols and data evaluation strategies, as well as accessibility and regulatory approval of radiopharmaceuticals."} +{"text": "The Greek Rheumatology Society and the Greek Association of Professional Rheumatologists (EREEPERE) has been issuing treatment Guidelines for rheumatoid arthritis (RA) since 2005. These Guidelines have been updated in 2009 and 2012.Special Committee of Diagnostic and Therapeutic Protocols in Rheumatic Diseases of ERE-EPERE and input from experts in the field. In the preparation of these Guidelines the most recent Guidelines from the American College of Rheumatology (ACR)1, the Recommendations and Treat To Target paradigm from the European League against Rheumatism (EULAR)5 were taken into account.Here we present the updated Guidelines for the treatment of RA prepared by the Rheumatoid arthritis is the most common, chronic, autoimmune inflammatory arthritis in the Greek population that, without timely and effective treatment, leads to permanent joint or extra-articular damage, disability, impaired quality of life and decreased survival.rheumatologist, and therapeutic choices are based on a shared decision process between the rheumatologist and the well-informed patient.RA is managed by the immediately after the diagnosis of the disease for better treatment outcomes and prevention of permanent joint damage.Treatment of RA should be initiated indices of disease activity such as the Disease Activity Score (DAS) 28 \u2013 ESR, (Supplementary Table 1).Assessment of disease activity should be made with established remission (DAS28-ESR < 2.6) or, if this is not possible, low disease activity (DAS28-ESR < 3.2) for all RA patients (Supplementary Table 2).Treatment targets include sustained 1\u20133 months (for those with moderate/high disease activity) or 3\u20136 months (for those with low disease activity or in remission).To achieve the above therapeutic goals, frequent monitoring of patients every 3\u20136 months after treatment initiation or modification.Treatment efficacy is assessed The criterion for changing or discontinuating treatment is the inability to achieve low disease activity (DAS28-ESR > 3.2).disease activity, patients\u2019 preferences, presence or absence of adverse prognostic factors, presence of comorbidities and the occurrence of side effects from the administered therapies.Treatment decisions are based on The following General Principles apply to the treatment of RA in daily clinical practice:Therapeutic stepsFigure 1. Most specifically:Step 1initial treatment step is the administration of conventional synthetic disease-modifying anti-rheumatic drugs (csDMARDs) as monotherapy:st option is methotrexate (MTX) at a dose of 15\u201325 mg/week pos or subcutaneously in combination with folic acid (5 mg/week pos).The 1leflunomide should be administered next.In patients with contraindications or intolerance/toxicity to MTX, sulfasalazine or hydroxychloroquine are the next therapeutic options.In patients with contraindications or intolerance/toxicity both to MTX and leflunomide, The glucocorticoids may be added for a short period of time with rapid dose tapering (up to 6 months).During treatment initiation or disease flares, monotherapy with a biologic (bDMARD), or its approved biosimilar) or a targeted synthetic (ts)DMARD) is given:Biologic DMARDs (bDMARDs)Anti-Tumour Necrosis Factor - anti-TNFs AdalimumabCertolizumab PegolEtanerceptGolimumabInfliximaborNon-anti-TNFsAbataceptSarilumab-EMA approved)IL-6 inhibitors (Tocilizumab or IL-1 inhibitors (Anakinra)orEMA-approved biosimilarsJanus Kinase (JAK) inhibitorTofacitinib(EMA approved)Baricitinib Upadacitinib (EMA approved)orRituximab: Only in patients with history of:- Lymphoproliferative diseases or- Demyelinating diseases or- Solid organ neoplasiasIn patients with contraindications or intolerance/toxicity in the above csDMARDs, Step 2csDMARD monotherapy and in the:Absence of adverse prognostic factors (RF and anti-CCP: - and DAS28: 3.2-5.1 and absence of joint erosions),orSwitching to nd csDMARD is recommendedAddition of a 2Presence of \u22651 adverse prognostic factors (Supplementary Table 3)approved biosimilar) or targeted synthetic(ts)DMARD is added:, a bDMARD (or its Biologic DMARDs (bDMARDs)Anti-Tumor Necrosis Factor - anti-TNFs AdalimumabCertolizumab PegolEtanerceptGolimumabInfliximaborNon-anti-TNFsAbataceptSarilumab-EMA approved)IL-6 inhibitors (Tocilizumab or IL-1 inhibitors (Anakinra)orEMA-approved biosimilarsJAK InhibitorTofacitinib(EMA approved)Baricitinib Upadacitinib (EMA approved)orRituximab: Only in patients with history of:- Lymphoproliferative diseases or- Demyelinating diseases or- Solid organ neoplasiasIn patients who fail Step 3who had failed \u22652 or combination of csDMARDs, a bDMARD (or its approved biosimilar) or tsDMARD is added:Biologic DMARDs (bDMARDs)Anti-Tumor Necrosis Factor - anti-TNFs AdalimumabCertolizumab PegolEtanerceptGolimumabInfliximaborNon-anti-TNFsAbataceptSarilumab-EMA approved)IL-6 inhibitors (Tocilizumab or IL-1 inhibitors (Anakinra)orTheir EMA-approved biosimilarsst JAK Inhibitor1Tofacitinib(EMA approved)Baricitinib Upadacitinib (EMA approved)orRituximab: Only in patients with history of:- Lymphoproliferative diseases or- Demyelinating diseases or- Solid organ neoplasiasIn patients st bDMARD1, a nd bDMARD2 (or its approved biosimilar) or a tsDMARD can be added:nd bDMARD2:Anti-Tumor Necrosis Factor - anti-TNFs AdalimumabCertolizumab PegolEtanerceptGolimumabInfliximaborNon-anti-TNFsAbataceptSarilumab-EMA approved)IL-6 inhibitors (Tocilizumab or IL-1 inhibitors (Anakinra)orEMA-approved biosimilarsst JAK inhibitor1Tofacitinib(EMA approved)Baricitinib Upadacitinib (EMA approved)In patients who had failed their stJAK Inhibitor, a 2ndbDMARD (or its approved biosimilar) or a nd tsDMARD,2 can be added:Anti-TNFs AdalimumabCertolizumab PegolEtanerceptGolimumabInfliximaborNon-anti-TNFAbataceptIL-6 Inhibitor (Tocilizumab or Sarilumab)AnakinraRituximab (after failure of anti-TNF)orEMA-approved biosimilarsnd JAK inhibitor2Tofacitinib(EMA approved)Baricitinib Upadacitinib (EMA approved)In patients who had failed the 1The recommended 3 steps in the treatment of RA are shown in The dose of MTX should be gradually increased up to 20\u201325 mg/week to achieve the therapeutic target. At doses greater than 15 mg/week, subcutaneous administration of the drug is preferred.Sarilumab) or their EMA-approved biosimilars and JAK inhibitors . More efficacy data regarding monotherapies are available for IL-6 and JAK inhibitors.In patients with contraindications, intolerance or toxicity to csDMARDs, administration as monotherapy of biological agents is indicated .In patients who fail the bio-original DMARDs, changing to their biosimilar is not recommended (or vice versa).sustained complete remission of the disease who are being treated with:csDMARD monotherapy:- a gradual csDMARD dose reduction,- and, only in exceptional cases, its discontinuationThe following may be attempted:Combination of a csDMARD and a bDMARD- a gradual dose reduction or an increase in the administration interval of the bDMARD, or- a gradual csDMARD dose reductionThe following may be attempted:Monotherapy with a bDMARD- a gradual dose reduction or increase in the administration interval of bDMARDThe following may be attempted:In patients with There are not adequate data so far to support the discontinuation of bDMARDs in patients with RA at remission.6.The recommended doses of the different DMARDs are shown in These Guidelines propose a 3-step approach to the treatment of RA targeting low disease activity (DAS28-ESR < 3.2) and always take into consideration the presence or absence of adverse prognostic factors, the presence of comorbidities, and the development of side effects during therapy. Therapeutic decisions should be the result of a shared decision process between the rheumatologist and the well-informed patient."} +{"text": "A 62-year-old Indian man with a strong family history of metabolic syndrome (MeTS) presented with asymptomatic, granular-surfaced, dark brown pigmentation on the cheeks . Nuchal/Although facial AN and MH may actually represent evolutionary standpoints on the morphological spectrum of cutaneous markers of MeTS (CMM) , labelin"} +{"text": "Effective strategy to mitigate the ongoing pandemic of 2019 novel coronavirus (COVID-19) require a comprehensive understanding of humoral responses against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the emerging virus causing COVID-19. The dynamic profile of viral replication and shedding along with viral antigen specific antibody responses among COVID-19 patients started to be reported but there is no consensus on their patterns. Here, we conducted a serial investigation on 21 individuals infected with SARS-CoV-2 in two medical centres from Jiangsu Province, including 11 non-severe COVID-19 patients, and 5 severe COVID-19 patients and 5 asymptomatic carriers based on nucleic acid test and clinical symptoms. The longitudinal swab samples and sera were collected from these people for viral RNA testing and antibody responses, respectively. Our data revealed different pattern of seroconversion among these groups. All 11 non-severe COVID-19 patients and 5 severe COVID-19 patients were seroconverted during hospitalization or follow-up period, suggesting that serological testing is a complementary assay to nucleic acid test for those symptomatic COVID-19 patients. Of note, immediate antibody responses were identified among severe cases, compared to non-severe cases. On the other hand, only one were seroconverted for asymptomatic carriers. The SARS-CoV-2 specific antibody responses were well-maintained during the observation period. Such information is of immediate relevance and would assist COVID-19 clinical diagnosis, prognosis and vaccine design. The ongoing outbreak of 2019 novel coronavirus (COVID-19), known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was first reported in Wuhan, China in Dec 2019 .In this respective study, we serially analysed the virus RNA test results in swab samples, along with anti-SARS-CoV-2 IgM and IgG responses among 21 COVID-19 patients at the Second Hospital of Nanjing and the Affiliated Hospital of Xuzhou Medical University in Jiangsu Province, China. Patients with suspected SARS-CoV-2 were confirmed after two sequential positive respiratory tract sample results. Throat swab samples were collected every 1\u20132 days. Anal swab samples were also obtained for RNA testing since 27 February 2020, as anal swab samples with prolonged viral shedding were observed during clinical practice . Viral R2 \u226493%); (c) abnormal blood gas analysis (PaO2/FiO2\u2009\u2264\u2009300\u2005mm Hg); or (d) severe disease complications including respiratory failure which requires mechanical ventilation, septic shock, or non-respiratory organ failure. The illness severity was defined according to the Chinese management guideline for COVID-19 (version 6.0) [The demographic information and disease severity of COVID-19 patients were obtained from their electronic medical records. Patients who had any of the following features during COVID-19 disease progression were classified as severe cases: (a) respiratory distress; (b) hypoxia non-severe COVID-19 patients, 5 (20.8%) severe patients, and 5 (20.8%) asymptomatic cases with SARS-CoV-2 infection. As of March 24, all patients have been clinical recovered and discharged. The characteristics of each group were summarized in The longitudinal antibody responses were also determined in our cohort . All of Only 1 (20%) out of 5 asymptomatic cases generated SARS-CoV-2 specific antibody responses , and thiOur current study has revealed important implications to understand the dynamic interplay between SARS-CoV-2 and humoral responses. First, seroconversion was observed in 100% (17/17) of symptomatic patients during the observation period, suggesting that the serological test could serve as a complementary testing assay to nucleic acid test for those symptomatic COVID-19 patients, especially given the potential false negative viral RNA results using throat swab samples. Nevertheless, we also noted only one person with humoral responses among asymptomatic carriers. Additional viral and immunological analyses are warranted to understand this observation. Better understanding on the biological significance of asymptomatic cases without seroconversion is urgently needed.Although it is generally considered a beneficial role of specific antibody response during viral infection, we did not identify a strong association of seroconversion and disease severity in our cohort. For both non-severe and severe COVID-19 cases, the viral specific antibody responses were detected. Meanwhile, our study revealed an early induction of antibody responses in severe cases. Consistently, a recent study also revealed that high level of antibody titer might be independently associated with a worse disease severity for COVID-19 patients . A similOur study also has several limitations. First, our study is limited by small sample size, thereby it is not known whether our finding could be generalized. Nevertheless, our cohort is representative of different COVID-19 disease spectrum, including non-severe cases, severe cases, and asymptomatic carriers. Furthermore, the quantitative viral load and titers of antibody response were not available, so the kinetics of viral shedding and the magnitude of antibody response during COVID-19 disease progression remained unknown in this study.Collectively, our study of serial nucleic acid and serological testing among various COVID-19 patients indicated distinct dynamic patterns among three groups of SARS-CoV-2 infected individuals. Our findings contribute to the evolving understanding of the sophisticated interaction between this emerging SARS-CoV-2 virus and host immune system."} +{"text": "In caregivers of Latina breast cancer survivors, contextual factors such as informational support, Anglo-orientation, and spiritual well-being may affect physical and mental health. The purpose of this study was to test if caregiver self-efficacy moderated relationships between contextual factors and health outcomes. A model, derived from Bandura\u2019s Social Cognitive Theory, included self-efficacy cancer knowledge (survivor) and self-efficacy symptom management (caregiver) as moderators of relationships between contextual factors and global health and depression. Secondary analysis of baseline caregiver data from an experimental study testing two psychoeducational interventions with Latina breast cancer survivors and their caregivers was conducted. Both self-efficacy measures were tested as moderators for relationships between contextual factors and health outcomes with fixed cutoffs . Caregiver participants (N=233) were 43 years on average (SD=13), primarily women (70%), low-income (78%), and of Mexican-American ethnicity (55%). Anglo-orientation was significantly associated with global health (r(233)=.27, p<.001) and depression (r(233)=-.13, p=.05). High levels of self-efficacy cancer knowledge strengthened the negative relationship between depression and Anglo-orientation, while a slightly positive relationship was noted at low self-efficacy levels. Informational support was significantly related to global health (r(233)=.39, p<.001) and depression (r(233)=-.43, p<.001). Self-efficacy symptom management strengthened the negative relationship between informational support and depression. Correlational and moderation relationships were not significant for spiritual well-being. Both caregiver- and survivor-focused self-efficacy affected relationships between contextual factors and depression in caregivers of Latina breast cancer survivors. Further research should address both types of self-efficacy in caregiver health."} +{"text": "To assess the ability of cine-MRI to identify potential responders to cardiac resynchronization therapy (CRT).40 patients with reduced ejection fraction (28 \u00b1 11%), with (n = 21) or without (n = 18) left bundle branch block underwent cine-MRI and tissue-doppler imaging echocardiography (TDI). Time to peak systolic velocity (TPV) was measured by TDI for the septal, anterior, lateral and inferior left ventricle (LV). Time to peak contraction (TPC) was assessed visually for corresponding regions by cine-MRI. Left ventricular (LV) dyssynchrony was defined as the maximum delay of TPV- and TPC between opposing regions for TDI and cine-MRI, respectively. Correlation and agreement between TDI and cine-MRI were calculated. The ability of cine-MRI to identify patients with relevant echocardiographic LV dyssynchrony (defined as >65 ms) was assessed by ROC analysis.A strong correlation between LV dyssynchrony by TDI and cine-MRI was found . Bland-Altman analysis showed a large difference of absolute values between both methods (mean difference 108 \u00b1 104 ms). ROC analysis revealed an optimal cut-off value for cine-MRI of > 145 ms to identify patients with relevant echocardiographic LV dyssynchrony . The ability of cine-MRI to discriminate between patients with and without relevant echocardiographic LV dyssynchrony was excellent .Visual assessment of LV dyssynchrony by cine-MRI can identify potential responders to CRT."} +{"text": "Multiple laboratory-developed tests (LDTs) and commercially available assays have emerged to meet diagnostic needs related to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. To date, there is limited comparison data for these different testing platforms. We compared the analytical performance of a LDT developed in our clinical laboratory based on CDC primer sets and four commercially available, FDA emergency use authorized assays for SARS-CoV-2 on a total of 169 nasopharyngeal swabs. Multiple laboratory-developed tests (LDTs) and commercially available assays have emerged to meet diagnostic needs related to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. To date, there is limited comparison data for these different testing platforms. We compared the analytical performance of a LDT developed in our clinical laboratory based on CDC primer sets and four commercially available, FDA emergency use authorized assays for SARS-CoV-2 on a total of 169 nasopharyngeal swabs. The LDT and Cepheid Xpert Xpress SARS-CoV-2 assays were the most sensitive assays for SARS-CoV-2 with 100% agreement across specimens. The Hologic Panther Fusion, DiaSorin Simplexa, and Roche Cobas 6800 failed to detect positive specimens only near the limit of detection of our CDC-based LDT assay. All assays were 100% specific, using our CDC-based LDT as the gold standard. Our results provide initial test performance characteristics for SARS-CoV-2 reverse transcription-PCR (RT-PCR) and highlight the importance of having multiple viral detection testing platforms available in a public health emergency. Since the first infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was detected in the United States in January 2020 , there hThe explosion of COVID-19 cases in the United States has highlighted the critical role diagnostic testing plays in medical and public health decision-making in containing and mitigating the SARS-CoV-2 pandemic. Reliable test results enable appropriate utilization of scarce hospital resources, including personal protective equipment (PPE) and negative-pressure isolation rooms, as well as public health resources for contact tracing or isolation decision-making . In rapiin vitro (Reverse transcription-PCR (RT-PCR) is the mainstay of SARS-CoV-2 detection in vitro . FDA EUAin vitro . FDA EUAHere, the performances of one LDT-EUA assay developed in our clinical laboratory and four FDA-EUA cleared assays were evaluated for detection of SARS-CoV-2. The FDA-EUA cleared assays included were Hologic Panther Fusion , DiaSorin Simplexa COVID-19 Direct (EUA), Cepheid Xpert Xpress SARS-CoV-2 (EUA), and Roche Cobas 6800 (EUA). The test performance characteristics of each RT-PCR were determined compared to those of our reference LDT assay.n\u2009=\u2009169) were collected from patient specimens submitted to the University of Washington Medical Center laboratories for clinical diagnostic testing. LDT performance was validated based on detection of 20 of 20 positive specimens sent by the Washington State Public Health Laboratory in early March. Residual clinical samples were used for validation/verification of each subsequent instrument, including a common panel of 26 specimens tested at the University of Washington (UW) by the UW CDC EUA-based LDT (CDC LDT), DiaSorin Simplexa (positive specimens only), Roche Cobas 6800, and tested at LabCorp Seattle on the Cepheid Xpert Xpress, and Panther Fusion (12 positive specemens only). Additional residual (n\u2009=\u2009115) specimens were tested at the UW on individual assays and compared to the reference method (LDT): Panther Fusion (RUO), n\u2009=\u200936; Panther Fusion (EUA)-UW, n\u2009=\u200920; DiaSorin Simplexa (EUA), n\u2009=\u200919; Cobas 6800, n\u2009=\u200940. Finally, 28 specimens were used to compare the SARS-CoV-2 assay on the UW Panther Fusion with the DiaSorin Simplexa assay. All same-sample comparisons were performed on specimens stored at 4\u00b0C for less than 72 h\u2009with no freeze-thaws. Inconclusive results (one of two targets detected) were considered positive due to the high specificity of all assays and limited cross-reactivity seen for SARS-CoV-2 primer sets. This work was approved under a consent waiver from the University of Washington Institutional Review Board.Nasopharyngeal (NP) swabs was extracted from 200 \u03bcl of viral transport medium (VTM) on the Roche MP96 and eluted in 50 \u03bcl of elution buffer. Real-time RT-PCR was set up on 5 \u03bcl of eluate using the CDC N1, N2, and RP primers and run on ABI 7500 real-time PCR instruments as reported previously (n\u2009=\u200936) and tested again following FDA authorization (n\u2009=\u200920). Both Panther Fusion RUO and EUA assays were slightly less sensitive than the CDC-based LDT, missing one positive/inconclusive sample in each sample set (TC) (>37) by the CDC LDT test. All 29 negative specimens generated \u201cNot detected\u201d results by the Hologic Panther Fusion SARS-CoV-2 assay.The Panther Fusion SARS-CoV-2 assay was tested first as research use only (RUO) reagents . When we compared SARS-CoV-2 detection on the DiaSorin Simplexa to the Hologic Panther Fusion, all 16 Hologic Panther Fusion positive specimens were detected by the DiaSorin Simplexa, while the DiaSorin Simplexa generated one additional positive result in the 12 specimens that were negative by the Hologic Panther Fusion (TCs of 36.8 (N1) and 35.8 (N2), confirming the DiaSorin Simplexa result.We next compared the DiaSorin Simplexa SARS-CoV-2 assay to our CDC-based LDT. All 19 specimens (11 positives and 8 negatives) demonstrated complete concordance between the two platforms with lowr Fusion . This diTCs of 38.0 (N1) and 37.4 (N2) in the LDT. Across the 20 positive specimens, TCs were only slightly higher on the Roche Cobas assay compared to the CDC-based LDT, with an average TC difference of 0.6 . One of 13 positive specimens was a presumptive positive on the Cepheid assay ; upon repeat per package insert, the N2 gene was detected at a TC of 42.7 but the E gene was not detected, yielding a positive result. The CDC LDT demonstrated 100% concordance with the Cepheid Xpert Xpress, also detecting the extremely low viral load specimen above as an inconclusive . No other assay detected SARS-CoV-2 RNA in this specimen. In addition, the DiaSorin Simplexa failed to detect a positive specimen that on repeat was detected only by the ORF1ab primer set.After performing the above pairwise comparisons, we next compared 26 specimens from another high-complexity hospital laboratory (LabCorp Seattle). All 26 specimens were also tested on the Cepheid Xpert Xpress SARS-CoV-2 assay . All spein vitro diagnostic real-time RT-PCR assays to detect SARS-CoV-2 in high-complexity clinical laboratories in one of the early U.S. epicenters of the COVID-19 pandemic. The results demonstrated excellent performance of a CDC-based LDT and the Cepheid Xpert Xpress, concurring with a previous evaluation that demonstrated high sensitivity of the E-gene and N2 primer sets used by the Cepheid assay (TC of >37), and one was the inconclusive specimen. Therefore, we conclude that all the tested assays show good sensitivity for the detection of SARS-CoV-2, with the UW CDC LDT and Cepheid Xpert Xpress SARS-CoV-2 assays having the best and similar sensitivity, followed by the Roche Cobas 6800, DiaSorin Simplexa, and Panther Fusion SARS-CoV-2 assays.This analysis compared the performance characteristics of several id assay . The PanTC positive specimens that resulted in a lower measured sensitivity for the Panther Fusion. In clinical practice, the minor differences in sensitivity are likely to have little effect on Hologic Panther Fusion SARS-CoV-2 assay performance on VTM specimens, given the TC ranges we have observed in our clinical populations.Our results are chiefly limited by the small sample sets used to compare these different assays as well as asynchronous comparisons that allowed only for pairwise comparisons early in the pandemic. For instance, these asynchronous panels most greatly affected our CDC LDT versus Hologic Panther Fusion comparison, which had a greater proportion of high-TCs that may be seen between platforms. For instance, recent reports have demonstrated a slightly higher analytical sensitivity of the Cepheid Xpert Xpress SARS-CoV-2 assay compared to the Roche Cobas SARS-CoV-2 test, and a slightly lower sensitivity of the DiaSorin Simplexa SARS-CoV-2 assay compared to a modified CDC assay, both of which are concordant with our data (Despite their limitations, these data provide a basis for differences in analytical sensitivity at different our data , 8. We aOur results provide an early assessment of performance characteristics of five separate assays for the detection of SARS-CoV-2. During March 2020, reagent availability for SARS-COV-2 RT-PCR assays was heavily constrained, necessitating more-limited assay comparisons. All platforms examined here had acceptable performance criteria for testing during the early part of this pandemic. As the supply chain for SARS-CoV-2 RT-PCR attempts to catch up with testing demand, we look forward to additional assay comparison data."} +{"text": "We identified abundant EGFR expression in 95% of Sq-BLCA without evidence for activating EGFR mutations. Both SCaBER and p-SCC cells were sensitive to EGFR tyrosine kinase inhibitors (TKIs: erlotinib and gefitinib). Combined treatment with anti-EGFR TKIs and varying chemotherapeutics led to a concentration-dependent synergism in SCC cells according to the Chou-Talalay method. In addition, the siRNA knockdown of EGFR impaired SCaBER viability suggesting a putative \u201cAchilles heel\u201d of Sq-BLCA. The observed effects seem Sq-BLCA-specific since non-basal urothelial cancer cells were characterized by poor TKI sensitivity associated with a short-term feedback response potentially attenuating anti-tumor activity. Hence, our findings give further insights into a crucial, Sq-BLCA-specific role of the ERBB signaling pathway proposing improved effectiveness of anti-EGFR based regimens in combination with chemotherapeutics in squamous bladder cancers with wild-type EGFR-overexpression.Recent findings suggested a benefit of anti-EGFR therapy for basal-like muscle-invasive bladder cancer (MIBC). However, the impact on bladder cancer with substantial squamous differentiation (Sq-BLCA) and especially pure squamous cell carcinoma (SCC) remains unknown. Therefore, we comprehensively characterized pure and mixed Sq-BLCA ( Bladder cancer is the 9th common cancer worldwide comprisiEGFR mutations ) [19], which may be due in part to heterogeneous cohorts and different histopathological and molecular subtypes . In the present study, we provide a rationale for combining EGFR inhibitors with standard chemotherapy as treatment strategy for pure and mixed squamous bladder cancers, characterized by a strong dependency on wild-type EGFR signaling. Functionally, we confirmed a central role of wild-type EGFR in squamous-differentiated bladder cancer cells, as they are vulnerable to perturbances of the ERBB signaling pathway in vitro. SCaBER cells, lacking activating mutations or amplifications of the EGFR gene, were highly sensitive to treatment with both anti-EGFR TKIs erlotinib and gefitinib. The corresponding IC50 value is very close to those reported for EGFR-mutated NSCLC cell lines (example for gefitinib sensitivity: PC-9 (del 746\u2013750) IC50\u2009=\u20090.0235\u2009\u00b5M) .Statistical analyses were performed using SPSS 25.0 and GraphPad Prism 5.0. Differences were considered statistically significant if the two-sided ion 1.0) , 37.Supplementary Information: Detailed description of methods.Supplementary Figure 1: ERBB receptor expression in urothelial, squamous bladder and head and neck cancer cell lines.Supplementary Figure 2: Densitometric evaluation of ERBB pathway activation and inhibition in SCaBER cancer cells.Supplementary Figure 3: Densitometric evaluation of ERBB pathway activation and inhibition in J82 cells.Supplementary Figure 4: ERBB signaling and receptor expression in HT1376 bladder cancer cells upon TKI treatment and EGF stimulation.Supplementary Figure 5: ERBB receptor expression after siRNA mediated knockdown of EGFR in J82 cells.Supplementary Figure 6: ERBB receptor expression upon combined treatment in SCaBER cellsSupplementary Figure 7: EGFR signaling in pSCC cells upon TKI treatment and EGF stimulation.Supplementary Table 1: Clinico-pathological data of urothelial, muscle-invasive bladder cancers without squamous characteristics used in this study.Supplementary Table 2: Detailed information on identified mutations in Sq-BLCA .Supplementary Table 3: Detailed information on amplification of EGFR and HER2/ERBB2 in Sq-BLCASupplementary Table 4: Molecular characteristics of utilized cell lines.Supplementary Table 5: p-SCC associated gene signature.Supplementary Table 6: Primer sequences for Sanger sequencing of FFPE Material.Supplementary Table 7: PCR primer sequences for ERBB receptor, ligand and target gene expression analysis (intron spanning)."} +{"text": "Teaching Point: A submucosal bladder wall lesion with high signal on T2-weighted MRI warrants blood and urine analysis to rule out a paraganglioma. A 57-year-old male presented with right flank colic pain. Non-contrast computed tomography (CT) Figure confirmeBlood and urine work-up for excess catecholamine production showed slightly elevated urine normetanephrine and noradrenaline, suggesting the diagnosis of a paraganglioma. A cystoscopic biopsy specimen was obtained and the pathology results showed a lesion morphologically resembling UCC but expressing synaptophysin, leading to the diagnosis of neuro-endocrine tumour (i.e. paraganglioma). A whole-body 18F-fluoro-deoxyglucose positon emission tomography coupled with CT (F-18 FDG PET-CT) Figure showed mBladder paraganglioma is an exceedingly rare tumour arising from the chromaffin cells of the bladder, which explains their intramural location. They account for 0.05% of bladder tumours and for less than 1% of paragangliomas ["} +{"text": "In the landmark analysis, haplo-SCT resulted in a lower 2-year cumulative incidence of relapse and superior 2-year leukemia-free survival and 2-year overall survival than chemotherapy. In the time-dependent multivariate analysis with propensity score adjustment, postremission treatment (haplo-SCT vs chemotherapy) was an independent risk factor for the CIR , LFS , and OS . In all subgroups, CIR was lower in haplo-SCT. Myeloablative haplo-SCT with ATG+G-CSF might be one of the preferred therapies for YA patients with standard-risk Ph-ALL.Human leukocyte antigen (HLA) haploidentical stem cell transplantation (haplo-SCT) as a postremission treatment for standard risk Philadelphia chromosome-negative acute lymphoblastic leukemia (SR Ph-ALL) in the first complete remission (CR1) has not been defined. In this multicenter, phase 3 study (NCT02042690), of the 131 consecutive Ph-ALL young adult patients without high-risk features who achieved CR1, 114 patients without HLA-matched donors received consolidation with an adult chemotherapy regimen (ClinicalTrials.gov. Registered on 23 January 2014, https://clinicaltrials.gov/ct2/show/NCT02042690 To the Editor:Philadelphia chromosome-negative acute lymphoblastic leukemia (Ph-ALL) is categorized as high risk (HR) with risk factors such as advanced age, elevated WBC count, and high-risk cytogenetic abnormalities. The remaining older adolescents and young adults without risk factors are AYA with standard-risk (SR) Ph-ALL and represent a group with lower cumulative incidence of relapse and better overall survival. Allogeneic hematopoietic stem cell transplantation , especially from human leukocyte antigen (HLA)-matched sibling donors (MSDs) or matched unrelated donors (MUDs), is one of the preferred options over chemotherapy in the consolidation treatment of Ph-ALL . Howeverhttps://clinicaltrials.gov as NCT02042690 using pretransplant ATG and granulocyte colony-stimulating factor (G-CSF)-stimulated grafts (ATG+G-CSF) or posttransplant cyclophosphamide (PT-CY) protocol was confirmed equivalent to HLA-matched SCT in ALL \u20136. Howev90 Suppl .In total, 131 consecutive Ph-ALL young adult patients without high-risk features who achieved CR1 were enrolled with a median follow-up of 32 months was excluded, those undergoing SCT after the landmark were included in the chemotherapy group, and the remaining patients (n = 99) were divided into the haplo-SCT group (n = 49) and chemotherapy group (n = 50) (Table SP = 0.0017), LFS , and OS continued to be better in the haplo-SCT group than in the chemotherapy group and improved LFS and OS compared with chemotherapy. Con1 FCM MRD (+ vs \u2212) was an independent risk factor for CIR and LFS . Diagnosis was an independent risk factor for OS. No independent risk factors identified for NRM. When stratified by Con-1 MRD and diagnosis, haplo-SCT decreased CIR in all subgroups while improved LFS and OS only in the Con-1 MRD+ and B-ALL subgroups but not in the Con-1 MRD\u2212 and T-ALL subgroups (Table SCox PH regression model was constructed considering the time of haplo-SCT as a time-dependent exposure based on PH test Table S. UnivariCurrently, haplo-SCT is only an optional rather than a preferred choice for postremission therapy compared with MSD or 10/10 MUD-SCT MSD-SCT is the preferred treatment for ALL, and MUD is also acceptable in most countries . This stThe present study might be one of the best available evidence to compare haplo-SCT and adult chemotherapy for YA SR Ph-ALL in CR1. Cautions must be taken in interpreting these results due to non-randomized design and a relatively small group of patients. haplo-SCT might become one of the preferred therapies for YA patients with SR Ph-ALL in the absence of MSD or MUD-SCT.Additional file 1:. Supplementary figures.Additional file 2:. Supplementary tables.Additional file 3:. Supplementary methods."} +{"text": "Although adefovir dipivoxil, stavudine, and cidofovir are virtually discontinued for clinical use, tenofovir disoproxil fumarate and tenofovir alafenamide remain the most important antivirals against HIV and HBV infections worldwide. Overall, the broad-spectrum antiviral potential of nucleos(t)ide analogues supports their development to treat or prevent current and emerging infectious diseases worldwide.Nucleoside and nucleotide analogues are essential antivirals in the treatment of infectious diseases such as human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), herpes simplex virus (HSV), varicella-zoster virus (VZV), and human cytomegalovirus (HCMV). To celebrate the 80th birthday of Prof. Dr. Erik De Clercq on 28 March 2021, this review provides an overview of his contributions to eight approved nucleos(t)ide drugs: (i) three adenosine nucleotide analogues, namely tenofovir disoproxil fumarate (Viread N-conjugation, halogenation) have been proposed to design novel nucleoside or nucleotide analogues, such as the acyclic fleximer analogues -9H-purin-9-yl)ethoxy)methyl] phosphonoyl) di-L-alaninate) [Despite its antiviral activity, PMEG was the most cytotoxic compound among phosphonylmethoxyalkyl derivatives, because its diphosphates could be incorporated into host DNA and block cellular DNA polymerase delta and epsilon . A subseor model . To incraninate) . In fiveaninate) . Despite\u00ae) for treating HCMV retinitis in AIDS patients, (ii) zalcitabine for HIV treatment, (iii) emtricitabine (Emtriva\u00ae) for HIV treatment, and (iv) lamivudine (Epivir\u00ae) for HIV and HBV treatment ide analogues have been approved for clinical use, including (i) cidofovir (Vistidereatment . This seCidofovir is an acyclic nucleoside phosphonate b licenseAmong the list of eight nucleos(t)ide analogues co-contr\u00ae)\u2014a monophosphoramidate prodrug of an adenosine analogue ide analogues. As a popular example of broad-spectrum antiviral activities, remdesivir inhibits many RNA viruses such as ebolavirus and respiratory pathogens including Middle East respiratory syndrome coronavirus, severe acute respiratory syndrome coronavirus (SARS-CoV), and SARS-CoV-2 . In regaanalogue e that acIn addition to the discovery of broad-spectrum antivirals with better potency, it is important to develop highly selective nucleos(t)ide analogues with minimal toxicity because antiviral nucleos(t)ides may exert toxicity through the disruption of natural nucleoside triphosphate pools and interfere with activities of host polymerases such as human mitochondrial DNA/RNA polymerases . Future"} +{"text": "However the low yield of ESC-derived cardiomyocytes (ESC-CMs) using the conventional differentiation method makes it difficult to perform a murine ESC line containing puromycin resistance gene under control of a cardiac specific promoter, sodium calcium exchanger (NCX) was used to generate ESC-CMs. ESC-CMs were labeled with Superparamagnetic iron-oxide nanoparticles (SPIO) for MRI detection. Reperfused myocardial infarction was induced in athymic rats. Infarction size was estimated by MRI post-op day1 to exclude animals with infarct size smaller than 10% or larger than 35%of the LV volume. At post-op day 7, labeled ESC-CMs (5\u201310 millions) were injected into infarction region. Control group was injected with vehicle. MRI scan was performed at post-op day 8 to confirm successful CM cell transplantation. Global cardiac function in ESC-CM and vehicle treated animals was assessed by MRI for 2 months. Immunohistology staining and electrophysiology were performed on postmortem hearts and ESC-CMs, respectively.a high yield of ESC-CMs was achieved with positive cardiac specific alpha-actinin in more than 90% of cells. The low proliferative capacity of ESC-CMs allows them to retain SPIO for serial MRI tracking. LV ejection fraction in ESC-CM treated rats at 1- and 2-month is significantly higher than that in the controls. IHC demonstrated formation of grafts in the host myocardium and gap junctions between grafted ESC-CMs and host CMs. See Figure Large numbers of highly pure ESC-CMs were obtained. Preliminary results suggest that ESC-CMs form grafts and improve LV function in the infarcted hearts."} +{"text": "Anticholinergic medications (A-chol) increase risk for falls; higher dopaminergic signaling may provide resilience to these effects. In 2489 older adults with 10 years of data on medication use, falls, and dopaminergic genotype (catechol-O-methyltransferase (COMT)), we assessed the association of A-chol use with recurrent falls (\u22652) over the subsequent 12 months using generalized estimating equations. Effect modification by COMT was tested; analyses were then stratified by COMT and adjusted for demographics and A-chol use indicators. During follow-up, 843 people reported recurrent falls. A-chol use doubled the odds of recurrent falls , with a suggested effect modification by COMT (p=0.1). The association was present in val carriers but not in met/met . Higher dopaminergic signaling may provide protection against the effects of A-chol use on fall risk."} +{"text": "Understanding how heart muscle cells function and developing new strategies for repairing the damaged heart are important challenges for tackling heart disease. Human cells from the heart are required in the laboratory for these investigations, but are difficult to obtain in large numbers from patients. As an alternative, stem cells can be used which can be cultured indefinitely in the laboratory and then turned into heart muscle cells. The current methods lead to a mixture of cell types, belonging to the different regions of the heart, creating difficulties when trying to understand the biology of a particular area of the heart, or for future applications when these stem cell-derived cell types are used to repair the heart. Genetic modification of stem cells provides a solution to this problem, as these techniques allow fluorescent markers, so-called reporters, to be inserted into key genes that are active in the different cell types. The different coloured reporters thus allow the identification and purification of specific cell types. In this review, we discuss the various methods that can be used to establish these reporter systems and highlight their applications in different aspects of cardiovascular medicine.Recent advances have made pluripotent stem cell (PSC)-derived cardiomyocytes an attractive option to model both normal and diseased cardiac function at the single-cell level. However, in vitro differentiation yields heterogeneous populations of cardiomyocytes and other cell types, potentially confounding phenotypic analyses. Fluorescent PSC-derived cardiomyocyte reporter systems allow specific cell lineages to be labelled, facilitating cell isolation for downstream applications including drug testing, disease modelling and cardiac regeneration. In this review, the different genetic strategies used to generate such reporter lines are presented with an emphasis on their relative technical advantages and disadvantages. Next, we explore how the fluorescent reporter lines have provided insights into cardiac development and cardiomyocyte physiology. Finally, we discuss how exciting new approaches using PSC-derived cardiomyocyte reporter lines are contributing to progress in cardiac cell therapy with respect to both graft adaptation and clinical safety. Pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), have revolutionised the study of developmental biology and diseases ,2,3,4,5.Although primary adult cardiomyocytes (CMs) have provided important insights ,17, theiThe ability to label individual cell types non-invasively using a genetically encoded fluorescent reporter offers the prospect of greater insight into cardiac differentiation and the isolation of pure populations of specific cell types. In this review, we will discuss how such fluorescent reporter lines are generated and how reporter cells can be used to study developmental cardiology, CM function and how they have advanced cardiovascular medicine. In order to produce a fluorescent reporter line, a transgenic construct is required comprising a fluorescent protein (FP) inserted downstream of a promoter or gene of interest (GOI). A reporter line can be generated as a transient line, where the transgene remains episomal, or as a stable line, where the transgene becomes integrated into the host genome, either site-specifically or randomly. A summary of the various methods is presented in A transient reporter line is produced by introducing the reporter construct as a non-integrating plasmid or vector, e.g., adenovirus into theThis method relies on the reporter construct being cloned, packaged and delivered via a genome-integrating viral system, e.g., a lentiviral vector Figure B or in tThis technique is relatively simple but possesses many biological and technical risks attributable to the random insertion into the genome . FirstlyIn order to avoid the undesirable effects of a random integration, fluorescent reporter constructs can be inserted into specific genomic sites. Homologous recombination (HR) C 26] is is26] isThe development of site-specific endonucleases including zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR associated protein 9 (Cas9) has transformed our ability to manipulate the genome . These cAAVS1) [Gt(ROSA)26Sor [LoxP or Frt sites, are inserted into the SHS, and equivalent sites are incorporated into the reporter construct, enabling the sequences to be efficiently exchanged through the activity of recombinases [With respect to site-specific integration of a fluorescent reporter, there are two common strategies. In the first strategy, the reporter construct can be introduced into a so-called safe harbour site (SHS) E, e.g., AAVS1) , chemokiSA)26Sor . These gSA)26Sor and the mbinases . This membinases ,34. A general disadvantage for all exogenous fluorescent reporter constructs is that a minimal promoter region is used. Subsequently, the fluorescence readout may not faithfully recapitulate the biological expression of the GOI. To avoid this issue, endogenous regulatory elements can be exploited for FP expression, allowing a higher fidelity between reporter and actual promoter activity. Using HR, FPs can be inserted downstream F of an eEach construct design has inherent advantages and disadvantages and the exact approach adopted should be tailored to the study. Random integration may be a sensible approach for studying gene function and pre-screening of biological effects. A master cell line is more suitable for lineage-specific labelling where multiple reporter lines are needed. Lastly, endogenous locus targeting is safer for projects that rely on the sensitivity of the reporter system. In vitro PSC-CM differentiation involves sequential stages of mesoderm induction, cardiac mesoderm determination, lineage specification and terminal differentiation . WNT/\u03b2-cBRY) which has been used to drive a GFP fluorescent reporter. In vitro differentiation of mouse ESCs (mESCs) revealed a multipotency of canonical WNT-activated Bry-GFP+ cells. Under cardiogenesis-promoting conditions, Bry-GFP+ cells with low expression of kinase insert domain receptor (Kdrlow) exhibit cardiogenic potential whilst, Bry-GFP+, Kdrhigh populations possess haematopoietic potential [In a developing mammalian embryo, the earliest cardiovascular progenitors are identified in gastrulation, where cells enter epithelial-mesenchymal transition and form mesendoderm. These progenitors express a mesodermal marker T-box transcription factor brachyury . Investigations which used a fluorescently-tagged MESP1-mCherry/ NK2 homeobox 5 (NKX2.5)-eGFP dual reporter hESCs [MESP1-mCherry expression followed by NKX2.5-eGFP expression. Enrichment of WNT5A, receptor tyrosine kinase-like orphan receptor 2 and frizzled class receptor 2 in MESP1-mCherry+ cells suggested roles of non-canonical WNT pathway in cardiac progenitor determination [The transition from common mesoderm towards cardiac mesoderm requires canonical WNT inhibition and non-canonical WNT activation. These intermediate cells are marked by an expression of mesoderm posterior basic helix-loop-helix transcription factor 1 (er hESCs demonstrmination .ISL1) is a potential SHF marker [ISL1-cre dsRed reporter hESC line [+ multipotent status with differentiation potential for CMs, smooth muscle, endothelial and epicardial lineages. This finding may highlight a common ancestor shared between pro-epicardial progenitor and SHF CPCs since time-course analysis showed that the ISL1 expression preceded the expression of SHF markers T-box transcription factor 1 and NKX2.5. NKX2.5/ISL1 or ISL1/epicardial-specific Wilm\u2019s tumour protein 1 dual reporter systems may be able to elucidate the diversification of ISL1+ progenitor and SHF CPCs further. More recently, CRISPR-Cas9-assisted generation of a NKX2.5-TagRFP/T-box transcription factor 5 (TBX5)-Clover dual reporter hiPSC line [NKX2.5-TagRFP+/TBX5-Clover+); presumptive SHF (the NKX2.5-TagRFP+/TBX5-Clover\u2212); pro-epicardial cells (NKX2.5-TagRFP\u2212/TBX5-Clover+), which diversified into nodal-like CMs and epicardial cells; and endothelial progenitors (NKX2.5-TagRFP\u2212/TBX5-Clover\u2212) (The nascent cardiac progenitors undergo another transitory stage towards more defined cardiac lineages termed the first heart field (FHF), which contributes to the left ventricle and a portion of the atria, and the second heart field (SHF), which gives rise to the right ventricle, atria, outflow tract and inflow tract . In the F marker . A studyESC line ascertaiPSC line has offeClover\u2212) .Taken together, the stage-specific fluorescent PSC-CM reporter cell lines offer an optical tracking strategy for cell identity and enable in vitro studies of cardiac development. Uniquely, hiPSC-CM reporter cells provide insights into human cardiogenesis without embryo donor requirements and without the concerns over species differences encountered with mouse models. Future multi-coloured reporter lines may enabDespite the refinement of in vitro cardiac induction protocols , PSC-CM Percoll gradient centrifugation allowed MYH6) [TNNT2) [NKX2.5 [+/Ca2+ antiporter (NCX1) [2+ transient (CT) reduction and action potential (AP) shortening [Genetically engineered reporter lines provide a robust alternative. Reporter lines carrying an antibiotic-resistant cassette under the control of cardiac-specific gene\u2019s regulatory elements, e.g., \u03b1-myosin heavy chain (MYH6) ,78,79,80 [TNNT2) , NKX2.5 [NKX2.5 and Na+/r (NCX1) allow efr (NCX1) ,76. Howeortening . MeanwhiNKX2.5 is an early transcription factor in the FHF and SHF CPCs and plays supportive roles in cardiac contraction [NKX2.5-GFP reporter hESC line [NKX2.5 expression is also detectable in non-cardiac tissues such as the spleen, stomach and thyroid [NKX2.5 may be advantageous.traction . An engiESC line allowed thyroid ; hence, MYH6-GFP+[MYH6-mCherry+ [MYH6 reporter systems are better suited for the isolation of early committed human CMs and vice versa for mouse CMs. The ability of sarcomere-specific reporters to aid in isolation of beating CMs was also recently reported for a number of key genes: \u03b1-cardiac actin (ACTC)-mCherry [TNNT2-firefly luciferase /puromycin resistance [TTN)-GFP hPSC lines [Fluorescent reporter transgenes under the control of a sarcomere gene/promoter offer a more cardiac-specific cell labelling. Isolated fluorescent MYH6-GFP+,49 or MYmCherry+ hPSC-CMsmCherry+ ,85,86. T-mCherry , TNNT2-fsistance and titiSC lines . In addiSC lines .NCX1 plays essential roles in intracellular Ca2+ flux and excitation-contraction coupling [NCX1-eGFP hPSC reporter line [NCX1-eGFP+ cells displayed both molecular and functional characteristics of typical hPSC-CMs.coupling . An NCX1ter line offered Irrespective of the pan-cardiac promoter used in these reporter systems, FACS purification yielded a mixture of atrial, ventricular and nodal CM subtypes. These reporter lines benefit cardiotoxic screening and developmental studies, but the heterogeneous nature of the cell population complicates chamber-specific drug discovery, disease modelling and therapeutic applications. Each subtype exhibits unique physiological characteristics , leadingMYL2), is the most attractive ventricular marker with an expression restricted to ventricular compartments throughout mammalian development [MYL2 PSC-CM reporter lines [MYL2 is considered a late cardiac gene, where its expression level increases consistently with the stage of CM maturation, with the upregulation seen being more pronounced than other maturation factors, such as \u03b2 myosin heavy chain [MYL2-FP+ cells is important to optimise the yield of ventricular-like cells.Myosin light chain 2 is ubiquitously expressed within the primitive heart tube and only becomes confined to atrial compartments at later developmental stages [MYL7-GFP hiPSC-CMs identified co-expression of MYL2 and MYL7 in 96% of the MYL7-GFP+ fraction [The selection of atrial-like cells is more challenging owing to the lack of a reliable genetic marker. Myosin light chain 7 expression is confined to atrial lineages [SLN-tdTomato+ hiPSC-CMs [SLN as an alternative atrial marker. Following this report, an SLN-driven voltage-sensitive fluorescent protein (SLN-VSFP) hiPSC reporter line was generated [SLN-based atrial cell purification has been emphasised due to the downregulation of the gene as the cells become more mature [During mammalian heart development, sarcolipin enhancer activity restricted to the atrioventricular conduction system [cGATA6-eGFP hESC reporter line [HCN4).PSC-derived nodal-like CM enrichment is demanding due to this subtype being a relatively minor population under WNT-modulated differentiation procedures . Investin system . An engiter line enabled SHOX2)-VSFP hiPSC line [HCN4 and nodal-type AP. SHOX2 promotes the transcription of HCN4 [SHOX2 was sufficient to induce endogenous SHOX2 expression and steered the differentiation of ESCs towards the nodal lineage [A transgenic short stature homeobox 2 for CT measurement is common, these chemicals are known to suppress cardiac functions [2+ reporters are being explored as we outline below.Electrophysiological measurements are not only a reliable strategy to identify individual CM subtypes, they also allow investigation of either genetically inherited or acquired functional cardiac defects. Patch-clamp electrophysiology is the gold standard AP recording technique , but it activity , which ttraction . Althougunctions and prevunctions . GeneticCiona intestinalis and a super ecliptic pH-sensitive GFP variant (pHluorin A227D) [CAG-Arclight transgene into hiPSC-CMs enabled the ubiquitous expression of the GEVI driven by a synthetic CAG control element [Arclight reporter showed improved signal-to-noise ratios and was less affected by photobleaching than conventional dyes. To facilitate simultaneous subtype identification and electrophysiological studies, a GEVI can be directed by a CM subtype-specific promoter. As an alternative, VSFP-based GEVIs [Arclight system, provides a more stable AP pattern, unaffected by movement artefacts and photobleaching.The development of genetically encoded fluorescent voltage-sensitive indicators (GEVIs) has opened a new window for electrophysiological studies. Arclight is a synthetic protein containing a voltage-sensing domain from n A227D) ,104. Ran element . The flu element ,106. Fured GEVIs rely on ed GEVIs to track2+ indicators (GECIs) have been engineered and tested in PSC-CMs [2+-modulating protein (CaM), a CaM-interacting peptide, e.g., myosin light chain kinase-derived M13 and a circularly permuted GFP [2+-induced Ca2+ release, cytosolic Ca2+ binds to CaM which promotes the formation of Ca2+-CaM-M13, resulting in increased fluorescence [2+ dyes is the stable expression of the transgene, facilitating observations of the chronic effects of pharmacological molecules, and non-cytotoxic properties [Several versions of genetically encoded fluorescent Ca PSC-CMs ,65,106. uted GFP . Followirescence . Similaroperties . The fidoperties ,106.Chlamydomonas reinhardtii-derived channelrhodopsin 2 protein (ChR2) mediates inward cation currents [CAG-humanised ChR2 (hChR2)-mCherry reporter hiPSC-CMs could be optically paced at different beat rates [The field of optogenetics has captured interest in neurophysiology because of the ability to track spatiotemporal activation of neurons . Upon blcurrents , resulticurrents . Differeat rates , offerinat rates . Furtherat rates allows aThe adult human heart has limited regenerative capacity to self-repair . hiPSC-CNkx2.5-GFP+ miPSCs confirmed the cardiac differentiation potential of the CPCs without teratoma formation [The proliferative capacity of stem cells is desirable for cardiac regeneration to compensate for the massive cell loss seen following transplantation . Howeverormation . Future MYL2-eGFP+ hPSC-CMs revealed signs of morphological maturation and connexin 43 (CX43) expression at the host\u2013graft interface [Host-graft adaptation is another concern for cardiac cell therapy. Importantly, fluorescent PSC-CM reporters provide not only a tool for tracking the cell fate after transplantation but also provide information about graft\u2013host structural and functional integration. Histological analysis of rat heart pre-intramyocardially injected with nterface . Since Cnterface , the preNotwithstanding, the immaturity of hPSC-CMs still represents a challenge for cardiac cell therapy. New methodologies focused on promoting more adult phenotypes are needed to ensure clinical safety and maximize therapeutic benefit.2+ fluxes and \u03b2-adrenergic response [2+ cycling and mitochondrial respiratory capacity while promoting sarcomere organisation [Several techniques have been developed to improve iPSC-CM maturity including biochemical, physical and environmental stimulation . Modulatresponse . Similarnisation .Unlike PSC-CMs, primary adult atrial and ventricular CMs do not beat spontaneously, but require an electrical signal from nodal CMs. Mimicking the in vivo situation, extrinsic electrical or optical (for optogenetics) impulses not only help to pace cultured CMs but also exert positive effects on the ultrastructural organisation and functional properties ,125.Extracellular matrix stiffness changes dramatically during cardiac development and has an impact on the mechanical force experienced by CMs . 3-dimenCX43 [Combinations of 3D hiPSC-CMs with biochemical or electCX43 may be bThe indefinite source of PSC-CMs has the potential to provide scalable CM production for cardiovascular research. However, immature and embryonic-like phenotypes as well as the heterogeneous nature of PCS-CMs present challenges for their use in drug discovery, disease modelling and transplantation. Stage-specific fluorescent PSC-CM lines generated by different transgenic methods have helped researchers monitor cardiac development and improved in vitro differentiation protocols. The discovery of additional CM maturation-promoting pathways will require multi-coloured reporter systems in combination with advances in 3D PSC-CM culture.PITX2 and KCNA5, in combination with atrial-promoting differentiation approaches including retinoic acid treatment and CRISPRa/CRISPRi-mediated transcriptional activation/inhibition of atrial-regulating pathways [CM surface markers are largely unknown and thus FACS-mediated PSC-CMs sorting by fluorescent markers provide a genetic-based solution for specific cell-type isolation. Purified fluorescent PSC-derived CPCs have been successfully differentiated into functional CMs in vivo, devoid of tumorigenic risk, promising clinical safety for cardiac regeneration. Novel fluorescent PSC reporter lines will help identify the various CPCs that give rise to unique cell-type compositions specifically required for treating different pathological conditions. Furthermore, the isolation of subtype-specific PSC-CMs will enable subtype-specific cardiogenic drug discovery and disease modelling. Unlike nodal and ventricular types, the atrial PSC-CM markers are not fully characterised. Further insights from novel fluorescent atrial PSC-CM reporter lines, such as pathways ,137, couLastly, optogenetics is an emerging biotechnology that has recently been applied in PSC-CMs for spatiotemporal CM activation and maturation. The combination of optogenetics with fluorescent physiological reporters allows an all-optical measurement of CM function that benefits high throughput drug screening. We look forward to seeing the implementation of combined optogenetics and physiological sensors with minimised optical crosstalk in cell transplantation, which will allow PSC-CM graft performance to be followed in vivo."} +{"text": "Older Veterans are at especially high risk of depression and social isolation due to COVID-19 stay-at-home orders and necessary safety precautions. We aimed to objectively measure differences in mood reports before and after COVID-19 stay-at-home orders in rural older Veterans. Participants age > 62 were enrolled in the Collaborative Aging Research using Technology (CART) initiative, a NIH and VA HSRD funded multi-site study examining the feasibility of unobtrusive remote sensing and monitoring of physical, cognitive, and health-related activities. The VA CART site consists of Pacific Northwest Veteran volunteers and their cohabitants. Weekly online health forms including questions about blue mood and loneliness were collected January \u2013 July 2020. A COVID stay-at-home order was instituted March 13 2020. Generalized estimating equations (GEE) with logit link was used to investigate differences in mood reports pre- and post- stay-at-home orders. 100 older volunteers completed 2441 health reports . Thirty-five percent were urban, 34% large rural, and 31% small rural using rural-urban commuting area (RUCA) scores urban: 1-3; large rural 4-6; small rural 7-10. After adjusting for covariates, incidence of blue mood and loneliness reports were significantly higher after stay-at-home orders . Results varied by rurality with large rural volunteers showing the largest increases. Real-world monitoring of weekly health reports may identify those at greatest risk for depression and social isolation in older Veterans and their cohabitants and is of particular relevance in rural settings, where access to specialty care is limited."} +{"text": "Streptococcus pneumoniae is important for monitoring of vaccine impact. Unfortunately, conventional and molecular serotyping is expensive and technically demanding. This study aimed to determine the ability of matrix-assisted laser desorption-ionisation time-of-flight (MALDI-TOF) mass spectrometry to discriminate between pneumococcal serotypes and genotypes . In this study, MALDI-TOF mass spectra were generated for a diverse panel of whole genome sequenced pneumococcal isolates using the bioMerieux VITEK MS in clinical diagnostic (IVD) mode. Discriminatory mass peaks were identified and hierarchical clustering was performed to visually assess discriminatory ability. Random forest and classification and regression tree (CART) algorithms were used to formally determine how well serotypes and genotypes were identified by MALDI-TOF mass spectrum.Serotyping of One hundred and ninety-nine pneumococci, comprising 16 serotypes and non-typeable isolates from 46 GPSC, were analysed. In the primary experiment, hierarchical clustering revealed poor congruence between MALDI-TOF mass spectrum and serotype. The correct serotype was identified from MALDI-TOF mass spectrum in just 14.6% (random forest) or 35.4% (CART) of 130 isolates. Restricting the dataset to the nine dominant GPSC (61 isolates / 13 serotypes), discriminatory ability improved slightly: the correct serotype was identified in 21.3% (random forest) and 41.0% (CART). Finally, analysis of 69 isolates of three dominant serotype-genotype pairs resulted in the correct serotype identification in 81.1% (random forest) and 94.2% (CART) of isolates.This work suggests that MALDI-TOF is not a useful technique for determination of pneumococcal serotype. MALDI-TOF mass spectra appear more associated with isolate genotype, which may still have utility for future pneumococcal surveillance activities.The online version contains supplementary material available at 10.1186/s12866-020-02052-7. Streptococcus pneumoniae, a globally important pathogenic bacterium ) and 19F (ST236 [GPSC23]). For serotype 23F, 29 isolates from the two dominant ST were included (ST9050 and ST10637 [both GPSC624]). Thirty-two discriminatory peaks were identified from a total of 562 peaks . Although multiple genotypes were included for each serotype, a dominant ST could be identified in several of them. The authors concluded that further work to determine the interaction between pneumococcal genotype and MALDI-TOF mass spectrum would be helpful. Analysis of 416 Brazilian isolates from six serotypes identified 10 major clusters by visualising a neighbour joining tree based on Pearson\u2019s coefficient isolates) and 46 global pneumococcal sequence clusters comprised of 13 serotypes.Additional file 5. Discriminant peak list / matrix derived from 69 pneumococcal isolates, comprising tree dominant serotype-genotype pairs.Additional file 6 Characteristics of Random Forest and CART algorithms for serotype- and genotype-associated MALDI-TOF mass spectra from 69 Streptococcus pneumoniae isolates from three serotype - global pneumococcal sequence cluster (GPSC) pairs.Additional file 7. A cluster dendrogram of serotype-organised MALDI-TOF mass spectrum data for the three dominant pneumococcal serotype-genotype pairs."} +{"text": "Drosophila, the transient receptor potential (TRP) superfamily of non-selective cation channels serve as polymodal cellular sensors that participate in diverse physiological processes across the animal kingdom including the perception of light, temperature, pressure, and pain. TRPM3 belongs to the melastatin sub-family of TRP channels and has been shown to function as a spontaneous calcium channel, with permeability to other cations influenced by alternative splicing and/or non-canonical channel activity. Activators of TRPM3 channels include the neurosteroid pregnenolone sulfate, calmodulin, phosphoinositides, and heat, whereas inhibitors include certain drugs, plant-derived metabolites, and G-protein subunits. Activation of TRPM3 channels at the cell membrane elicits a signal transduction cascade of mitogen-activated kinases and stimulus response transcription factors. The mammalian TRPM3 gene hosts a non-coding microRNA gene specifying miR-204 that serves as both a tumor suppressor and a negative regulator of post-transcriptional gene expression during eye development in vertebrates. Ocular co-expression of TRPM3 and miR-204 is upregulated by the paired box 6 transcription factor (PAX6) and mutations in all three corresponding genes underlie inherited forms of eye disease in humans including early-onset cataract, retinal dystrophy, and coloboma. This review outlines the genomic and functional complexity of the TRPM3_miR-204 locus in mammalian eye development and disease.First discovered in a light-sensitive retinal mutant of Drosophila melanogaster in 1969 . Similar, \u03b1-SMA . These oTXNIP), whereas similar numbers of anti-oxidative genes were upregulated or downregulated . Remarkably, in silico analysis predicted that miR-204 not only targeted the 3\u2032-UTR of pro-oxidative genes for transcriptional repression but also targeted the 5\u2032-TATA-box promoter-sequence of anti-oxidative genes for transcriptional activation [Besides EMT, miR-204 has been associated with differential regulation of oxidative stress-related genes in human age-related cataract . Microartivation . Thus, itivation .Meis2 transcript levels consistent with a role for miR-204 regulation in lens development and congenital cataract pathogenesis [In addition to age-related cataract and PCO, miR-204 was reported to be downregulated (>\u20094-fold) in central anterior LEC samples from young children (1\u20134\u00a0years) undergoing surgery for congenital (\u2018pulverulent\u2019) cataract that typically presents at birth or during infancy . Transfeogenesis .TRPM3 was discovered in a 5-generation Caucasian-American family segregating pediatric cataract with autosomal dominant transmission that mapped to chromosome 9q [TRPM3 (RefSeq variant-9) was not present in the Exome Aggregation Consortium (ExAC) database and was predicted in silico to exert damaging effects on the function of at least one channel isoform (RefSeq isoform-k). Transfection studies of a recombinant hTRPM3-GFP reporter construct harboring the human cataract mutation have revealed that the Ile>Met substitution introduced an alternative translation start-site located 89 codons upstream from the native methionine found in at least eight other TRPM3 transcript variants and channel isoforms . Thus, in addition to damaging effects on isoform-k function, the novel Ile>Met start-site may exert deleterious effects on multiple RefSeq channel isoforms by extending their N-termini with 89 novel amino-acids. Recently, a second missense mutation located in exon-29 of TRPM3 has been discovered in a Chinese nuclear family segregating pediatric cataract [The first unambiguous human disease association for osome 9q . Approxicataract . This hecataract .Efnb3) and an ephrin receptor (ola-Ephb2) that was reversed by miR-204 overexpression during retinal development in medaka embryos. Most miR-204 knockdown embryos (65%) displayed retinal ganglion cell (RGC) axon pathfinding defects that cause axons to invade other retinal layers rather than extend along the optic nerve fiber layer to vision centers in the brain. Conversely, miR-204 overexpression resulted in aberrant projection of axons to the contralateral optic nerve and ectopic rostral projection of axons to the telencephalon rather than the optic-tectum. Rescue of these RGC axon misguidance defects was achieved by co-injection of morpholinos for miR-204 and Ephb2 or Efnb3 suggesting that miR-204 participates in RGC axon growth and/or guidance, in part, by targeting the ephrin-B receptor signaling pathway [In medaka embryos, differential regulation of miR-204 has been directly implicated in retinal development , 169. BeTrpm3 transcript levels were also upregulated, particularly in the INL, upon light exposure. The physiological role of such light-induced regulation in the inner retina is unclear. However, in the case of miR-204, it has been speculated that high turnover facilitates assembly of new ribonucleoprotein complexes termed miRNPs to cope with transcriptional changes during neuronal activity [In mouse retinal neurons, miR-204 (and miR-211) has been found to be reversibly up/downregulated during light/dark adaptation independent of the circadian clock . During activity .TGFBR2 and SNAI2) that are known to promote EMT. Second, miR-204 inhibition decreased (~\u200980%) trans-epithelial electrical resistance associated with reduced claudin gene expression and tight-junction formation that is required to maintain the epithelial barrier function of the RPE. Third, miR-204 inhibition triggered apical membrane hyper-polarization resulting from ion-channel activity that is required for normal RPE-photoreceptor interactions during the visual cycle. Thus, as in the lens, miR-204 participates in preservation of the epithelial cell phenotype of the RPE [In human fetal (hf) RPE cells, the constitutively high miR-204 levels have been functionally linked with altered gene expression that maintains epithelial cell physiology . First, the RPE .MITF, TYR, TRPM1) and 11-cis retinal pigment regeneration in the visual cycle [Prolonged culture (4\u00a0months) of confluent ARPE-19 cells resulted in massive upregulation (>\u20092000-fold) of miR-204 (and miR-211) that was associated with differentiation of a native RPE phenotype including a cobblestone epithelial morphology and intense pigmentation . Subsequ, RDH10) . Strong , RDH10) . MiR-204, RDH10) .Meis2 [In developing mouse RPE, conditional loss of the RNase III nuclease Dicer1, which cleaves pre-miRNAs, resulted in significant depletion (~\u200911-fold) of miR-204 along with upregulation of several predicted target genes including Meis2 . Dicer1-Meis2 .Recently, mice lacking miR-204 were found to develop age-related (~\u20099\u00a0months) RPE/retinal defects including hyper-autofluorescent (white) deposits, abnormal light-induced electrophysiological responses, increased microglia migration to the RPE, and impaired phagocytosis of photoreceptor outer segments accompanied by rhodopsin build-up in the RPE . FurtherDownregulation of miR-204 (~\u20094-fold) has been reported in a rat model of advanced glaucomatous retinal damage experimentally induced by chronic elevation of intraocular pressure . In partIn a rat model of optic nerve crush injury, miR-204 upregulation in retinal blood vessels was accompanied by decreased expression of growth-associated protein-43 (GAP-43) . SimilarDownregulation of miR-204 (~\u20092.5-fold) has been observed in human retinoblastoma (RB) tissues and cell lines when compared to normal pediatric retinas . RestoraMIR204 has been found to underlie an inherited pediatric form of bilateral retinal (rod-cone) dystrophy and iris coloboma with or without congenital cataract segregating in a 5-generation British family [MIR204 that is essential for target-transcript recognition and downregulation or bright-light (photopic) ERG a-waves (photoreceptor-derived) and ERG b-waves (OPL-derived Muller and ON-bipolar cells) [Visual function testing has shown that loss of r cells) . Thus, ir cells) .Trpm1-null mice, which exhibited a profound deficit in pupillary light reflex (PLR), Trpm3-null mice displayed a more subtle attenuated PLR under both bright light (rod/cone/melanopsin-response) and dim light (rod/cone-response) conditions, consistent with a role in non-image photoreception [Trpm3-null mice exhibited rapid pupil constriction in response to bright light that was maintained during illumination they failed to achieve full pupil constriction . They also displayed an abnormal post-illumination pupil response with a more rapid pupil dilation compared to the sustained post-stimulus pupil constriction of wild-type. In response to dim light, pupil constriction in Trpm3-null mice was approximately 45% of that in wild-type. Muscarinic stimulation of the eye by topical administration of the cholinergic agonist, carbachol, resulted in complete pupil constriction suggesting that the ciliary and iris-sphincter muscles were not functionally impaired in Trpm3-null mice. Since TRPM3 was not detected in outer retina photoreceptors (rods and cones) or photosensitive (melanopsin-expressing) retinal ganglion cells (pRGCs) of the inner retina, it was proposed to play a more distal role in regulating pupillary responses to light that may involve retinal Muller glial cells and the ciliary body where TRPM3 was enriched [In contrast to eception . While Tenriched .2+ concentration changes in the sub-retinal space (or inter-photoreceptor matrix) between the RPE and photoreceptors during the visual cycle [MITF and TRPM1_MIR211 [In human RPE cells, the apical membrane co-localization of TRPM3 and tight-junctions (ZO1) may modulate junctional permeability and barrier function, whereas TRPM3 enrichment at the base of the primary cilium may contribute to sensing light-induced Caal cycle . Prolongal cycle . However1_MIR211 . Since i1_MIR211 .Ins2Akita/+) mice [Dramatic downregulation (>\u2009200-fold) of miR-204 has been detected during corneal wound healing following traumatic corneal epithelial injury (by cell scraping) in mice . Convers/+) mice . This mi/+) mice , 184. UnKleip-null corneas was correlated with strong upregulation of the proangiogenic factor angiopoietin-1 (ANGPT1) and its receptor tyrosine kinase (TIE2), but not with canonical vascular endothelial growth factors A-C (VEGFA-C). Bioinformatics analysis identified an miR-204 binding site in the ANGPT1 transcript and overexpression of miR-204 mimic in transfected vascular endothelial cells resulted in downregulation of ANGPT1 consistent with the latter acting as a miR-204 target-gene and supporting a role for the miR-204-Angpt1 pathway in Kleip-null corneal neovascular dystrophy [Loss of miR-204 expression (~\u200920-fold) has been reported in mice lacking Kelch-like Ect2-interacting protein (KLEIP)\u2014a genetic model of spontaneous corneal neovascular dystrophy . Such miystrophy . In addiystrophy . Conversystrophy . Similarystrophy . These uystrophy . CombineBCL2L2, BIRC2), activators of the endoplasmic reticulum (ER)-stress response , and mediators of the inflammatory response . When subject to oxidative-stress (H2O2) or ER-stress (tunicamycin), HTM cells overexpressing miR-204-mimic displayed increased apoptotic cell death, accumulation of oxidized protein (carbonylation) along with decreased expression of ER-stress markers, and inflammatory factors [FOXC1)\u2014a causative gene for anterior segment dysgenesis known as Axenfeld-Rieger syndrome with a ~\u200950% risk for high tension glaucoma\u2014along with several downstream FOXC1-target genes, notably MEIS2 [Microarray and qPCR analyses have associated downregulation of miR-204 (~\u20092.5-fold) with increased senescence in primary cultures of human trabecular meshwork (HTM) cells , 142. Si factors . Other mly MEIS2 . Furtherly MEIS2 . Overall2+ stores by store-operated calcium entry (SOCE) and in sustainability of ATP-mediated Ca2+ signaling in white matter glial cells (astrocytes and oligodendrocytes) derived from mouse optic nerve [TRPM3 channels have been shown to participate in replenishment of ER Caic nerve .2+ sensors in the photosensitive retina [TRPM3 underlies pediatric cataract with or without glaucoma and anterior segment defects, whereas mutation of MIR204 underlies retinal dystrophy and iris coloboma with or without cataract [PAX6 mutations in humans also underlie a variable pan-ocular phenotype(s) including aniridia (iris hypoplasia), foveal hypoplasia, anterior segment dysgenesis 5 (ASD5), late-onset corneal dystrophy, ocular coloboma, and congenital cataract [From \u2018light-blindness\u2019 in fruit flies (trp) to \u2018night-blindness\u2019 in humans (TRPM1), TRP channels have been identified as evolutionarily important Cae retina , 36. Rece retina . In humacataract , 83. Notcataract , 191. SuEfnb2-Ephb2 pathway [TGFBR2) and Wnt/\u03b2-catenin signaling [Mir-204 expression has been prominently associated with development and differentiation of multiple eye tissues. In the retina, differential regulation of miR-204 has been linked with development of the neural retina that mediates phototransduction, differentiation of the RPE that supports adjacent photoreceptor function and forms the outer blood-retinal barrier of the eye, and with retinal disease. First, miR-204 has been implicated in RGC axon guidance to the visual cortex, in part, by targeting the pathway , 169. SeTNNBIP1) , 162. ThTNNBIP1) . Fourth,TNNBIP1) . Fifth, TNNBIP1) . Sixth, TNNBIP1) , 181. SeTNNBIP1) .Meis2 and Ankrd13a [Pax6 drives lens epithelial cell fate determination by targeting genes involved in neurogenesis and cell motility [ALDH1A3), whereas miR-204 downregulation in congenital cataract implicated targeting of Meis2 [Differential regulation of miR-204 expression has been directly associated with development and differentiation of the crystalline lens that facilitates fine-focusing of images onto the retina and with clinically distinct types of cataract formation. First, miR-204 controls lens morphogenesis, in part, by targeting Ankrd13a , 170. Se, Myo10) . Third, of Meis2 \u2013176.Angpt1) [FOXC1) [Beyond retina and lens, differential regulation of miR-204 has been implicated in pathophysiology of the cornea that generates most of the eye\u2019s refractive/focusing power and the trabecular meshwork that drains aqueous humor outflow from the anterior eye. First, miR-204 downregulation promoted corneal epithelial wound healing, whereas miR-204 upregulation inhibited wound healing in diabetic keratopathy by targeting SIRT1 and CCND1 to inhibit the cell cycle , 184. SeAngpt1) \u2013187. Thi [FOXC1) \u2013143, 188 [FOXC1) .Clearly, miR-204 plays complex multifunctional roles in regulating ocular gene expression. While there is some overlap of miR-204 target genes/pathways in different eye tissues , so far most miR-204 targets appear to be cell and/or disease context dependent. However, despite its multiple target genes, functional loss of miR-204 does not appear to negatively impact eye development and differentiation in mice that eventually acquire (~\u20099\u00a0months) an AMD-like phenotype . Such sp2+ dynamics. In retinal neurons, TRPM3 has been proposed to act as a Ca2+ sensor in the OFF-pathway of bipolar cells and a subset of ganglion cells [2+ sensor for the sub-retinal space between the RPE and photoreceptors during the visual pigment cycle [2+ homeostasis (SOCE) in optic nerve glial cells [TRPM3 is widely expressed in eye tissues and has been tentatively implicated in both neuronal and epithelial cell Caon cells . Similarnt cycle . TRPM3 cal cells . Functioal cells .2+ homeostasis [2+\u2014believed to result from activation of a non-selective cation conductance\u2014has long been implicated in the pathophysiology of lens aging and cataract formation in humans and experimental animals [2+ dynamics in the lens and other anterior eye tissues including the ciliary body and iris.In the lens, loss of TRPM3 has not been associated with a cataract phenotype in mice, raising the possibility of functional redundancy or compensation by other lens TRP channels. For example, TRPV1 and TRPV4 have been shown to participate in maintaining an intracellular hydrostatic pressure gradient within the lens . By conteostasis . Elevate animals . Besides animals , mechano animals and osmo animals , it is c1325) was not enhanced by heat [In addition to cellular aspects of TRPM3 channel function and dysfunction in the eye, several molecular aspects warrant further investigation. First, since alternative splicing can alter the functional properties of TRPM3 channels , the exp by heat raising by heat \u201379.TRPM3 also developed high-tension glaucoma [Trpm3-null mice, but may cause unwanted side effects in humans including reduced insulin secretion and/or noxious heat insensitivity. However, none of the known TRPM3 channel agonists or antagonists are likely to be specific enough for pharmacologic studies in vivo [Second, the true physiological agonists and antagonists for TRPM3 channels in the eye remain elusive. The endogenous neurosteroid, PS, is the most widely used experimental agonist and it activates TRPM3 channels within physiological concentration and temperature ranges suggesting that integration of chemical and thermal stimuli to activate cation conductance may be physiologically and/or pharmacologically relevant. Since heat (37\u00a0\u00b0C) sensitizes TRPM3 channels to activation by PS at levels close to those in blood , such chemical-thermal synergy raises the possibility that circulating PS may act as an authentic agonist for TRPM3 channels at body core temperature . Howeverglaucoma . Severalglaucoma \u201374. TRPM in vivo .2+ influx from TRPM3 channels triggers a signaling cascade of MAPKs and stimulus response transcription factors that in turn alter expression of delayed response genes [Finally, aside from physiological or pharmacological activation and inhibition, the constitutive intracellular regulation and downstream signaling mechanisms of TRPM3 channels in ocular tissues remain to be elucidated. Using transfected (HEK293) cells, at least three intracellular mechanisms are believed to regulate TRPM3 channels. CaM binding and PIP hydrolysis serve as activators, whereas G\u03b2\u03b3-subunits act as inhibitors , 79, 101se genes . Althoug2+ dynamics during vertebrate eye development.Future multidisciplinary studies including genomics, transcriptomics, proteomics, and metabolomics of the TRPM3_miR-204 locus will likely provide new insights regarding ocular health and disease. Regardless of its precise ocular function(s) however, the TRPM3 gene along with that for miR-204 appear to have co-evolved as a target locus for PAX6 to coordinate regulation of gene expression with CaAdditional file 1.Table S1. Schematic summary of human TRPM3 transcript variants and protein isoforms. (A) RefSeq variants (1\u201323) and isoforms (a-w). (B) Predicted variants and isoforms . Gray boxes denote exons included in each variant and numbers denote amino-acid (AA) counts for each isoform. Asterisks indicate translation stop codons. NT, nucleotide. AA, amino acidAdditional file 2.Table S2. Schematic summary of mouse TRPM3 transcript variants and protein isoforms (A) RefSeq variants (1\u201330) and isoforms . (B) Predicted variants and isoforms . Gray boxes denote exons included in each variant and numbers denote amino acid counts for each isoform. Asterisks indicate translation stop codonsAdditional file 3. Table S3. Ocular expression profile of the TRPM3 and miR-204 genes in humans and mice"} +{"text": "Australian variant signatures were more diverse than USA samples, but still, clonal events were found in these samples. Mutations in the helicase, encoded by the ORF1ab gene in SARS-CoV-2 were predominant, among others, suggesting that these regions are actively evolving. Finally, we firmly urge that primer sets for diagnosis be carefully designed, since rapidly occurring variants would affect the performance of the reverse transcribed quantitative PCR (RT-qPCR) based viral testing.Here we aim to describe early mutational events across samples from publicly available SARS-CoV-2 sequences from the sequence read archive and GenBank repositories. Up until 27 March 2020, we downloaded 50 illumina datasets, mostly from China, USA (WA State) and Australia (VIC). A total of 30 datasets (60%) contain at least a single founder mutation and most of the variants are missense (over 63%). Five-point mutations with clonal (founder) effect were found in USA next-generation sequencing samples. Sequencing samples from North America in GenBank (22 April 2020) present this signature with up to 39% allele frequencies among samples ( As a proof of concept, in the early beginning of the outbreak in China, sequencing the virus from nine patients from Wuhan in China revealed 99.9% similarity among samples. That finding suggests 2019-nCoV originated from one source within a very short time, supporting clonality of spreading after 1 month of the initially identified case on 31 December 2019, in Wuhan city, China . As SARSpreading . In thisPRJNA601736 (Chinese datasets), SRA: PRJNA603194 (Chinese dataset) (PRJNA605907 (Chinese datasets) (PRJNA607948 (USA-WI State datasets), SRA: PRJNA608651 , SRA: PRJNA610428 (USA-WA State datasets), SRA: PRJNA612578 (USA-San-Diego dataset), SRA: PRJNA231221 (USA-WA State dataset) , SRA: PRJNA231221 (USA-MD State dataset), and SRA: PRJNA614995 (USA-UT datasets). All illumina SRA accessions until 27 March 2020 are depicted in https://github.com/cfarkas/SARS-CoV-2_illumina_analysis and were obtained from SARS-CoV-2 resource at GenBank: https://www.ncbi.nlm.nih.gov/genbank/sars-cov-2-seqs/.Raw illumina sequencing data were downloaded from the following NCBI SRA BioProjects: SRA: dataset) , SRA: PRatasets) , SRA: PRdataset) , SRA: PRNC_045512.2, using the following parameters: -D 20 -R 3 -N 0 -L 20 -i S,1,0.50. Illumina Raw reads from whole genome sequencing and amplicon sequencing were trimmed by using fastp tool in default mode against ult mode and aligult mode . GenBankult mode was usedult mode . Varianthttps://github.com/tseemann/snippy. Also, Variant effect annotation tool, employing the variant effect predictor algorithm (VEP) was employed to assess functional effects of variants on SARS-CoV-2 transcripts., Europe (n = 40) and North America were downloaded on 22 April 2020 from NCBI virus database (https://www.ncbi.nlm.nih.gov/labs/virus/vssi/#/) using as query \u201cSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2), taxid:2697049\u201d. All sequences are publicly available at https://github.com/cfarkas/SARS-CoV-2_illumina_analysis. Merged sequences were also aligned by using MAFFT multiple sequence alignment program version 7.271 were obtained from database on 11 Ja primers and ten primers designedSRR11059943) due to large gaps in genome coverage, as explained in displayed the same five-point variant signature of USA-WA samples, two samples contain the same variant signature presenting one deletion (SRR11397715 and SRR11397716) and one novel signature (SRR11397728) presents a SNP that creates a stop codon present the same mutational profiling with deletions in the stem loop of the virus and notably, one sample (SRR11397728) presents a SNP that creates a stop codon in the helicase protein rather than synonymous see , sheet 4tein see , sheet 5SRR11397719 and SRR11397721) presenting one founder synonymous SNP that occurs within the 2019_nCoV_N3_Forward_Primer hybridization region. In the first sample, we also found an SNP with low allele frequency that falls into the 2019-nCoV_N1_Reverse_Primer , each one with a fluorescent probe for reverse transcriptase quantitative PCR. We investigated if these primers hybridized at positions that fall within the variants reported herein from the 50 next-generation sequencing datasets. We aligned these primer sequences with SARS-CoV-2 reference genome and the 50 analyzed samples. We found two Australian clonal samples and we compared these results with variants observed in submitted GenBank sequences in NCBI viral portal up until 22 April 2020. As already reported with HIV and Chikungunya outbreaks, the founder effect of five-point variants was observed in almost all USA-WA samples obtained by next-generation sequencing. These mutations also have high allele frequencies (around 33\u201339%) in SARS-CoV-2 GenBank sequences from USA origin . These vn = 1,359). SARS-CoV-2 Australian variants were heterogeneous, but still, clonal events were found including one sample presenting the USA-WA signature, implying worldwide spreading of this signature. The efficiency of RT-qPCR testing can be potentially affected by founder variants, since several SNPs affecting one of three primers sets currently used in COVID-19 testing has been found. By the time of this publication, the available data could change the conclusions presented in this manuscript as a result of further viral variants arising.We describe here the early mutational events in SARS-CoV-2 virus by analyzing sequencing samples from China, USA, Australia and GenBank sequences submitted between 27 March and 22 April 2020. SARS-CoV-2 variants from the USA display five-point mutations with clonal (founder) patterns of spreading at a considerably high frequency among samples. The latter was verified by sequence analysis of SARS-CoV-2 sequences submitted to GenBank, since these five-point mutations displayed alleles frequencies of 33\u201339% among all USA GenBank SARS-CoV-2 sequences , collapsing all clades whose average branch length distance was below 0.0002. Asian sequences were highlighted in red, European sequences were highlighted in purple and North American sequences were not highlighted. The tree scale was shown upper.Click here for additional data file.10.7717/peerj.9255/supp-2Supplemental Information 2Sheet 1: 31 March 2020 next generation sequencing datasets available for SARS-CoV-2 in sequence read archive repository.Sheet 2: All Illumina datasets employed in this study including accession numbers and founder variants.Sheet 3: Merged Strelka2 variants from Next Generation Sequencing datasetsSheet 4: Variant Effect from USA NGS samplesSheet 5: Variant Effect from Australia NGS samplesSheet 6: GenBank variants (Worlwide)Sheet 7: Variant Effect Predictor output from GenBank variantsSheet 8: Primer IntersectionsClick here for additional data file."} +{"text": "We sequenced the metagenome of a biofilm collected near a leachate stream of the Marsberg copper mine (Germany) and reconstructed eight metagenome-assembled genomes. These genomes yield copper resistance through Cu(I) oxidation via multiple copper oxidases and extrusion through copper-exporting P-type ATPases. We sequenced the metagenome of a biofilm collected near a leachate stream of the Marsberg copper mine (Germany) and reconstructed eight metagenome-assembled genomes. These genomes yield copper resistance through Cu(I) oxidation via multiple copper oxidases and extrusion through copper-exporting P-type ATPases. Functional analysis revealed the presence of genes for copper-sensing transcriptional repressors CsoR and RicR, copper-exporting P-type ATPases such as ActP, CptA, and CopA, and oxidation enzymes, multicopper oxidases (MCOs), involved in copper homeostasis in all MAGs. The detoxification pathways for reactive oxygen species, toxins, and antibiotic compounds involve superoxide dismutase (SOD) and peroxidases, which degrade superoxide anion radicals, and mycothiol-mediated detoxification through the enzyme Mca with thiols .Raw sequencing data are available at the NCBI Sequence Read Archive (SRA) under accession number"} +{"text": "Thus, assignment of LR-5 provided 94% sensitivity and 81% specificity for HCC. LR-M provided 79% sensitivity and 97% specificity for non-HCC (ICCA and cHCC-CCA); and the sensitivity and accuracy were lower in differentiating HCC from non-HCC (tumor size <20 mm). LI-RADS v2018 category 5 and M reliably differentiated HBV-related HCC from ICCA. However, a substantial proportion of cHCC-CCAs were categorized LR-5 rather than LR-M. While management is controversial for these combined tumors, accurate prospective differentiation is desired for optimal treatment.The diagnostic performance of the Liver Imaging Reporting and Data System (LI-RADS) in differentiating hepatocellular carcinoma (HCC) from other hepatic malignancies has not been investigated in Chinese patients with chronic liver disease from hepatitis B virus (HBV) infection. The aim of this study was to evaluate the accuracy of the LI-RADS version 2018 in differentiating HCC, intrahepatic cholangiocarcinoma (ICCA), and combined HCC-cholangiocarcinoma (cHCC-CCA) in Chinese patients with HBV infection. Seventy consecutive HBV-infected patients with ICCA (n = 48) or cHCC-CCA (n = 22) who underwent contrast-enhanced magnetic resonance imaging (CE-MRI) between 2006 and 2017 were enrolled along with a comparison cohort of 70 patients with HCC and CE-MRI-matched for tumor size . Imaging feature frequencies for each tumor type were compared using Fisher\u2019s exact test. The classification accuracy of LR-5 and LR-M was estimated for HCC versus non-HCC (ICCA and cHCC-CCA). The interobserver agreement was good for LI-RADS categories of HCC and moderate for non-HCC. After consensus read, 66 of 70 (94%) HCCs were categorized LR-5 (including tumor in vein [TIV] with LR-5), while 42 of 48 (88%) ICCAs and 13 of 22 (59%) cHCC-CCAs were categorized LR-M (including TIV with LR-M) ( Hepatocellular carcinoma (HCC) is the most common primary liver cancer and the second leading cause of cancer-related death in the world . HepatitThe liver imaging reporting and data system (LI-RADS) is a comprehensive algorithm which provides tools for standardizing the imaging diagnosis of patients at risk for HCC, and it is now completely concordant with the American Association for the Study of Liver Diseases (AASLD) guidance for the definite diagnosis and management of HCC ,11. The This retrospective study was approved by the institutional review board of our institution with waiver of written informed consent requirement. From November 2006 to December 2017, patients with either cHCC-CCA or ICCA who met the following criteria were enrolled: a) multiphase contrast-enhanced (CE)-magnetic resonance imaging (MRI) on 1.5 T or 3 T MR scanner performed within 30 days before tissue sampling; b) image quality was acceptable as defined below; c) chronic HBV infection; and d) no history of any previous therapy for liver malignancy. For the control group, during the latter 1 year of the study period (between 2016 and 2017), patients meeting the above categories of the inclusion criteria were enrolled by matching them one-to-one with the patients with non-HCCs (ICCA and cHCC-CCA) according to tumor imaging size . Patient clinical information and laboratory tests were obtained from retrospective review of the medical record. 2). Afterwards, breath-hold 3D T1W gradient-recalled-echo imaging (liver acquisition with volume acceleration) was performed before and at multiple time points dynamically after injection of gadobenate dimeglumine (Bracco), gadopentetate dimeglumine (Bayer), or gadoxetate disodium (Bayer). A dual arterial phase (AP) was initiated 15\u201320 seconds after the contrast media arrived at the distal thoracic aorta using bolus triggering, a portal venous phase (PVP) was acquired at 1 minute after contrast injection, and a delayed phase (DP)/transitional phase (TP) was acquired at 3 minutes. Twelve MRIs included delayed hepatobiliary phase imaging.Patients were scanned supine on a 3T whole-body MR scanner and 1.5T whole-body MR scanner with an eight-channel phased-array coil centered over the liver. Precontrast sequences included breath-hold coronal fast imaging employing steady-state acquisition (FIESTA), breath-hold coronal single-shot fast spin echo (SSFSE), respiratory-triggered axial T2-weighted fast spin echo (FSE), breath-hold two-dimensional dual-echo T1-weighted gradient-recalled-echo images at nominal opposed/in phase echo times for 1.5 T and 3 T, and respiratory-triggered axial diffusion-weighted spin-echo echo-planar imaging with 2 b values . The radiologists were blinded to the radiology report and histopathologic diagnosis, but they were aware that each patient had a diagnosis of HCC, ICCA, or cHCC-CCA. In cases of patients with multiple pathologically confirmed lesions, the largest tumor was evaluated for each patient. Before reviewing the MR images, both radiologists were given 3 months of hands-on instruction regarding the details of LI-RADS v2018 . LI-RADSThe LI-RADS imaging features were subclassified as a) major features (MF) of HCC ; b) ancillary features (AFs) that may favor malignancy in general ; c) AFs that favor HCC in particular ; d) targetoid LR-M features ; and e) nontargetoid LR-M features (infiltrative appearance and necrosis or ischemia). In addition, three non-LI-RADS features were scored: liver surface retraction, biliary obstruction, targetoid appearance on T2WI of biphenotypic differentiation (p = 0.003). Necrosis or ischemia was significantly more frequent in ICCA than HCC and cHCC-CCA . In addition, ICCA and cHCC-CCA showed significantly higher frequencies of target appearance on T2WI, which is not currently one of ancillary features of LI-RADS v2018. ICCA showed significantly higher frequencies of liver surface retraction than HCC (p = 0.007). The interobserver agreement was good for LI-RADS categories for HCC; and moderate agreement was also achieved in the assignment of non-HCC malignancies could slightly improve diagnostic performance to differentiate HCC from non-HCC malignancies.Diagnosis performances of LR-5 and LR-M after consensus read for HCC or non-HCC are shown in The primary aim of our study was to evaluate the diagnostic accuracy of LR-5 and LR-M for differentiating HBV-related HCC from non-HCC malignancy. Using a large cohort of primary malignancies in an HBV population, we demonstrated that 94% HCCs were accurately categorized LR-5, while 88% ICCAs and 59% cHCC-CCAs were accurately categorized LR-M. Compared with the ideal of LI-RADS and results of other studies -21, we fLI-RADS v2018 was accurate for classifying most of HBV-related ICCA as non-HCC malignancy and proved high accuracy, specificity, and sensitivity, as has been previously reported . Most HBp = 0.018), this feature on T2WI has the potential supplementary value when some significant distortions appear in DWI images. Hence, we recommend it to be considered as a feature suggestive of non-HCC malignancy, though further research is needed to make it a distinct LR-M feature.In our study, we used a novel imaging feature, T2WI targetoid appearance. T2WI targetoid appearance is not defined in LI-RADS but is similar to targetoid appearance on DWI and could be used at the discretion of the radiologist as a feature suggestive of a non-HCC malignancy, which is one route for making the LR-M categorization. In our cohort, targetoid appearance on T2WI had a statistical difference between non-HCC and HCC. Although the frequencies of target appearance on T2WI (31/70) were lower than targetoid appearance on DWI (45/70) in non-HCC malignancy (Similar to prior reports (6 of 14 [43%] and 4 of 11 [36.0%] biphenotypic primary liver carcinomas misdiagnosed as HCC) ,29, in oWhile laboratory values are not currently incorporated into the diagnostic imaging algorithm, they may be helpful in challenging cases. As would be expected, AFP was significantly higher in cHCC-CCA than ICCA group, and CA19-9 was significantly higher in cHCC-CCA than HCC group. AFP and CA19-9 were simultaneously positive in the two miscategorized cHCC-CCA patients, which perhaps supports the possibility of cHCC-CCA. Ye et al. similarlOur retrospective study has several limitations. First, our study population was enriched to include only pathology-proven malignant lesions (either HCC or ICCA or cHCC-CCA), so they were not representative of a more general LI-RADS cohort which would be expected to have a spectrum of both benign and malignant observations. Second, our inclusion of 16 ICCAs from which tissue was obtained by percutaneous biopsy may have resulted in underrepresentation of cHCC-CCAs . Of course, biopsy has been regarded as a confirmative tool that can determine the treatment plan in LI-RADS . Third, In conclusion, LI-RADS v2018 performed well in discriminating HCC from non-HCC malignancy in an Asian cohort of chronic HBV-infected patients. HBV-related ICCAs were misclassified in a minority of instances , whereas cHCC-CCA proved more challenging, i.e., a substantial proportion (41%) of cHCC-CCAs was miscategorized LR 5. In addition, T2WI targetoid appearance could be considered as a feature suggestive of non-HCC malignancy. For these atypical non-HCC malignancies, biopsy is recommended for the prospective accurate differentiation before the optimal treatment decision."} +{"text": "In January 2020, the coronavirus disease 2019 (COVID-19) outbreak in Italynecessitated rigorous application of more restrictive safety procedures inthe management and treatment of patients with cancer to ensure patient andstaff protection. Identification of respiratory syndrome coronavirus 2(SARS-CoV-2) infection was a challenge during the pandemic owing to a largenumber of asymptomatic or mildly symptomatic patients.18F-FDG,18F-choline (FCH), and 68Ga-PSMA.We report 5 patients with unknown SARS-CoV-2 infection undergoing positronemission tomography (PET)/computed tomography (CT) with radiopharmaceuticalstargeting different tumor processes: 18F-FDG (mean SUVmax 5.4) than for the other tracers(mean SUVmax 3.5).In all patients, PET/CT showed increased tracer uptake in the lungscorresponding to CT findings of SARS-CoV-2 pneumonia. Quantitativeassessment of tracer uptake showed more elevated values for the glucoseanalogue Our findings suggest that PET/CT is a sensitive modality to hypothesizeSARS-CoV-2 pneumonia in patients with cancer, even when asymptomatic. Moredata are needed to verify the correlation among immune response toSARS-CoV-2 infection, clinical evolution, and PET results. Under the strictsafety measures implemented at the PET center, the number of potentiallySARS-CoV-2\u2013positive patients undergoing PET/CT was very low (1.6%), and nostaff member has been diagnosed with infection as of April 30, 2020. In this context, management of diagnosticsessions for patients with cancer required rigorous application of safety procedureswith more restrictions compared to routine activity in order to guarantee patientand staff protection.In January 2020, an outbreak of the novel coronavirus disease 2019 (COVID-19)occurred in Italy (2Identification of patients infected with severe acute respiratory syndromecoronavirus 2 (SARS-CoV-2) appeared early as a challenge of the pandemic, owing tothe lack of serologic tests and the absence of testing for infection in the homesetting. On the other hand, patients with positive chest computed tomography (CT)findings may have negative reverse transcription polymerase chain reaction (RT-PCR)testing for SARS-CoV-2.Detection of suspected cases is critical in patients with cancer before they visitthe positron emission tomography (PET) center, because they are particularlysusceptible to respiratory pathogens owing to potential immunosuppressive state andantitumor therapy.https://www.sirm.org/2020/03/30/covid-19-caso-69/), mostly nottested on RT-PCR, but variably presenting with chest CT findings compatible withpulmonary interstitial infiltrates, potentially associated with infection ordrug-related reactions.Correct management of a PET session presents many difficulties because of thecoexistence of asymptomatic SARS-CoV-2 infection and patients with mild symptomsbefore the PET scan . For PET scanning withWhole body PET/CT protocol included a topogram to define the field of view (FOV)established according to the tracer or the cancer type, followed by a low-dose CTscan for attenuation correction andanatomical correlation and a PET emission scan .Acquired data were reconstructed by Q Clear , a Bayesianpenalized-likelihood reconstruction algorithm (strength 350).Images were corrected for injected dose, tracer decay, body weight, and attenuationusing the low-dose CT scan. Informed consent was obtained from all patients.Review and analysis of attenuation-corrected PET and CT images were performed usingan Advantage Workstation 4.6 .Quantitative assessment of tracer uptake was performed by drawing volume of interest(VOI) over areas with abnormally high increases in uptake, focal and/orwell-circumscribed.Standardized uptake value (SUV) was calculated as the ratio of decay-correctedactivity in the VOI to the injected activity per unit body weight, as a simplifiedmeasure of tracer uptake.The SUVmax was calculated as the hottest voxel within the VOI.PET/CT characteristics of all patients are reported in Quantitative analysis of PET images is reported in 18F-FDG wasperformed to characterize a solid lung nodule (1 cm diameter) revealed by a previousCT scan 1 month previously in an 80-year-old man. The examination was performed 60minutes after intravenous tracer injection (263 MBq) from scull base to the pelvis.Increased tracer uptake was detected corresponding to subpleural ground-glassopacities (GGOs) on CT images was referred for PETfor restaging due to appearance of a suspicious nodule in the left breast.The patient previously (2017) underwent breast-conserving surgery for IDC located inthe upper-outer quadrant of the left breast. Follow-up ultrasonography revealed ahypoechoic area (1 cm diameter) near the surgical scar (June 2019); mammography wasnegative. Fine-needle aspiration of the lesion with ultrasound assistance classifiedit as benign (C2).18F-FDG (GLUSCAN\u00ae) wasperformed 60 minutes after tracer injection (257 MBq) from vertex to knees forrestaging breast cancer.In February 2020, the patient underwent tomosynthesis with evidence of benignalterations (American College of Radiology 2) and absence of microcalcifications orskin alteration suspected of cancer. Ultrasound confirmed the hypoechoic nodulehaving unchanged size. PET/CT scan with Supplementary Figure 1).Focal tracer uptake (SUVmax 6.5) was detected in the solid nodule of the left breast.Intense and diffuse uptake was revealed in both lungs corresponding to GGOs on CTimages .CT characteristics were typical of COVID-19 pneumonia as described by Xie et al.The highest SUV values were measured in the subpleural GGO areas; otherwise, lowuptake values were revealed in the isolated lesions in the parenchyma. Highintensity of tracer uptake present in mediastinal, hilar, and carinal lymph nodesrefer to infection (SUVmax 7).The patient was referred to the dedicated COVID-19 protocol at the UniversityHospital of Parma and subsequently home-quarantined because she did not show anysymptoms. Her general practitioner was alerted for peripheral management of thepatient and organization of pharyngeal swab for testing for SARS-CoV-2 infection.The swab was not performed, but the patient underwent a rapid diagnostic test forSARS-CoV-2 antibodies from a finger prick, which resulted positive forimmunoglobulin G (IgG).68Ga-PSMA PET/CT.A 65-year-old man had previously undergone radical prostatectomy for adenocarcinomaGleason Score 7 (4+3). During follow-up, his prostate-specific antigen (PSA) valueslowly increased, with an actual value of 0.47 ng/mL, leading to a need to restageprostate cancer with 3 according to current European Union Good Manufacturing Practices,4 current Good Radiopharmacy Practice,5 and European Pharmacopoeia. Dynamic images over the pelvis were acquired soonafter intravenous tracer injection (142 MBq). Whole-body scan was performed after anuptake time of 60 minutes from vertex to knee. PET/CT was negative for cancerlesions but revealed mild tracer uptake (SUVmax 3.2) in the subpleural region ofboth lungs, with greater extent in the right lung, corresponding to CT findings ofsubpleural GGOs in the dependent lung . Adenocarcinoma of the prostate was demonstrated in9/12 mapping samples with a Gleason Score 7 (3+4).18F-choline was performed for staging. Dynamic images of the pelvis were acquired soonafter intravenous tracer injection (278 MBq). The whole-body scan was performed 60minutes after intravenous tracer injection from scull base to mid-thighs.A PET/CT scan with Supplementary Figure 2).PET/CT showed double focal intense uptake in the prostate gland. Mild uptake of thetracer was revealed in the subpleural region of both lungs, with greater extent inthe right lung, corresponding to CT findings of GGOs and consolidation areas . Focal tThe patient\u2019s general practitioner was alerted for peripheral management andorganization of pharyngeal swab testing for SARS-CoV-2 infection. The patient washome-quarantined but swab was not performed by decision of the Health Department. Herecently underwent laboratory-based serologic testing, which was positive for IgGantibodies to SARS-CoV-2.18F-FDG (GLUSCAN\u00ae) tocharacterize three focal splenic lesions (diameter 8\u201310 mm) with inconclusive CT andcontrast-enhanced ultrasound. The examination was performed 60 minutes afterintravenous tracer injection from skull base to pelvis. PET/CT did not show anytracer uptake in the spleen but revealed intense/moderate uptake in the bilateralsubpleural regions corresponding to CT findings of GGOs in the posterior segments, with obvious extentin the right lung .A 57-year-old woman underwent PET/CT with The patient developed mild respiratory symptoms a few weeks later; she wasquarantined and recently underwent thoracic radiography, which was normal.To our knowledge, this is the first report of SARS-CoV-2 infection in patients withcancer detected by PET/CT using different tracers according to tumor type.In a 1-month period (March 2020), we performed 302 PET/CT examinations for patientswith cancer. Five patients (1.6 %) undergoing PET/CT revealed PET manifestationscorresponding to CT findings suspicious for SARS-CoV-2 infection. All of them wereasymptomatic on the day of PET/CT scan; one of them (case 4) questioned after theexamination reported a mild fever 3 weeks previously.In https://www.escardio.org/Education/COVID-19-and-Cardiology/diagnosing-the-first-covid-19-patient-in-italy-codogno).He presented symptoms immediately after the identification of theman known in Italy as Patient No. 1.6 After disappearance of symptoms, he underwent pharyngeal swab testing forSARS-CoV-2 infection, which resulted negative. A second swab was not performed. Someweeks later, the patient\u2019s wife presented symptoms of SARS-CoV-2 infection. She isnow awaiting serologic testing. Most patients home-quarantinedafter PET scan, awaiting swab testing, but it was performed only in one case(patient 3), due to the lack of symptoms. However, recent implementation ofserologic testing has allowed confirmation of previous SARS-CoV-2 infection in twocases (patients 2 and 4).Patient 3 was from Codogno, the first red zone area for SARS-CoV-2 infection in Italy at the site of infection,without excluding active disease. In inflamed tissue, glycolysis is enhanced7 with an upregulation of GLUT transporters, with amplified affinity for theirsubstrates as glucose analogue 18F-FDG.Persistence of tracer uptake many weeks after symptoms disappearance (patient 3) maybe explained by the presence of inflammatory cells accumulating 810 with a parallel rise in FDGaccumulation.810 However, inour patients, tracers targeting different metabolic process, such as membranebiosynthesis (18F-choline) or the overexpression of transmembraneglycoprotein (68Ga-PSMA), showed moderate to intense uptake in thetypical COVID-19 pneumonia.Specific infections, such as human immunodeficiency virus, cytomegalovirus, or denguevirus, may lead to increased expression of GLUT transporters in infectedcells,11 FCH accumulation in inflammatory tissue may be due to upregulation of cholinekinase in the activated macrophages.12Increased regional blood flow at the site of infection/inflammation may increaseavailability of PSMA ligand. Additionally, folate receptors overexpressed byactivated macrophages may interfere with the expression of folate hydrolase/PSMA.More data are needed to verify the correlation among immune response to SARS-CoV-2infection, clinical evolution, and PET results; patient 3 had negative swab aftersymptom disappearance but positive PET examination for bilateral subpleural GGOs,and his wife later developed symptoms of COVID-19.In our cases, different types of serologic testing were used, such as rapiddiagnostic test in patient 2 and laboratory-based test in patient 4, allowing us toestablish a previous SARS-CoV-2 infection. More homogeneous management of pharyngealswab and serologic testing could help infection tracing, especially in thevulnerable population of patients with cancer with PET/CT findings suspicious forSARS-CoV-2 pneumonia.18F-FDG uptake in lung lesions accompanied by nodal involvementdetectable on PET/CT images.13 Incidental findings of SARS-CoV-2 infection in asymptomatic patients withcancer undergoing nuclear medicine procedures was recently reported using hybridscanner single-photon emission CT/CT14 and PET/CT.15Recent case series about PET in patients with SARS-CoV-2 pneumonia showed high18F-FDG in all the reportedcases. We have shown that different radiopharmaceuticals, used according to tumortype, may be taken up by SARS-CoV-2 pneumonia. In our cases, quantitative assessmentof 18F-FDG uptake was higher in the parenchymal lesions than in theaccompanying nodal involvement. The SUVmax values of 18F-FDG were moreelevated compared to the other tracers assessed previouslyin preclinical experiments.12The PET tracer used was the glucose analogue These preliminary data show prolonged metabolic activity in the typical lung lesionsof SARS-CoV-2 infection, remaining many weeks after symptoms disappearance ornegative swab (patient 3), suggesting a potential role of PET/CT in monitoringtreatment efficacy and disease resolution. We hypothesize that quantitative resultsof tracer uptake measured by SUV may be referred to infection timing, with higherSUVs in the initial phase of pulmonary involvement (patient 2).Under the strict protective measures implemented at the PET Center, the number ofpotentially SARS-CoV-2\u2013positive patients undergoing PET/CT was very low, and nostaff member has been diagnosed with SARS-CoV-2 infection as of April 30, 2020, asconfirmed by negative results of serology testing (immunoglobulin M and IgG) forSARS-CoV-2, performed at the beginning of May, in all staff members.Click here for additional data file.Supplemental material, Supplemental_figures_1 for Unknown SARS-CoV-2 pneumoniadetected by PET/CT in patients with cancer by Maura Scarlattei, Giorgio Baldari,Mario Silva, Stefano Bola, Antonino Sammartano, Silvia Migliari, TizianoGraziani, Carla Cidda, Nicola Sverzellati and Livia Ruffini in TumoriJournalClick here for additional data file.Supplemental material, Supplemental_figures_2 for Unknown SARS-CoV-2 pneumoniadetected by PET/CT in patients with cancer by Maura Scarlattei, Giorgio Baldari,Mario Silva, Stefano Bola, Antonino Sammartano, Silvia Migliari, TizianoGraziani, Carla Cidda, Nicola Sverzellati and Livia Ruffini in TumoriJournalClick here for additional data file.Supplemental material, Supplemental_figures_3 for Unknown SARS-CoV-2 pneumoniadetected by PET/CT in patients with cancer by Maura Scarlattei, Giorgio Baldari,Mario Silva, Stefano Bola, Antonino Sammartano, Silvia Migliari, TizianoGraziani, Carla Cidda, Nicola Sverzellati and Livia Ruffini in TumoriJournal"} +{"text": "Mounting evidence suggests that maternal diet influences pregnancy and birth outcomes, but its contribution to the global epidemic of childhood obesity has not as yet been definitively characterized. We investigated whether maternal whole diet quality and inflammatory potential influence childhood adiposity.z-score\u2009>\u200985th percentile). Secondary outcomes were sum of skinfold thickness (SST), fat mass index (FMI) and fat-free mass index (FFMI). We used multivariable regression analyses to assess the associations of maternal DASH and E-DII scores with offspring adiposity outcomes in cohort-specific analyses, with subsequent random-effect meta-analyses.We harmonized and pooled individual participant data from 16,295 mother-child pairs in seven European birth cohorts. Maternal pre-, early-, late-, and whole-pregnancy (any time during pregnancy) dietary quality and inflammatory potential assessed with the Dietary Approaches to Stop Hypertension (DASH) score and the energy-adjusted Dietary Inflammatory Index (E-DII\u2122) score, respectively. Primary outcome was childhood overweight and obesity (OWOB) (age-and-sex-specific BMI 2. Higher early-pregnancy E-DII scores (more pro-inflammatory diet) tended to be associated with a higher odds of late-childhood [10.6 (1.2)\u00a0years] OWOB [OR (95% CI) 1.09 per 1-SD E-DII score increase], whereas an inverse association was observed for late-pregnancy E-DII score and early-childhood [2.8 (0.3)\u00a0years] OWOB [0.91\u00a0]. Higher maternal whole pregnancy DASH score was associated with a lower odds of late-childhood OWOB [OR (95% CI) 0.92\u00a0 per 1-SD DASH score increase]; associations were of similar magnitude for early and late-pregnancy . These associations were robust in several sensitivity analyses and further adjustment for birth weight and childhood diet did not meaningfully alter the associations and conclusions. In two cohorts with available data, a higher whole pregnancy E-DII and lower DASH scores were associated with a lower late-childhood FFMI in males and a higher mid-childhood FMI in females (P interactions <\u20090.10).The study mothers had a mean (SD) age of 30.2 (4.6)\u00a0years and a mean BMI of 23.4 (4.2)\u00a0kg/mA pro-inflammatory, low-quality maternal antenatal diet may adversely influence offspring body composition and OWOB risk, especially during late-childhood. Promoting an overall healthy and anti-inflammatory maternal dietary pattern may contribute to the prevention of childhood obesity, a complex health issue requiring multifaceted strategy.The online version contains supplementary material available at 10.1186/s12916-021-01908-7. Childhood obesity has reached epidemic proportions worldwide . ObesityThe Developmental Origins of Health and Diseases (DOHaD) concept posits that early life represents a window of opportunity to optimize health trajectory of the immediate offspring and subsequent generations. Indeed, exposure to severe (in response to famine) or mild in utero malnutrition has been associated with a higher risk of obesity/higher adiposity \u201310 and mDue to the complexity of human diet and becaMost studies to date examining dietary quality or inflammatory potential and childhood adiposity have been conducted in a single country \u201323, poteA UK study that followed women from pre-conception through pregnancy showed that maternal dietary intakes and patterns changed little from before pregnancy to early and late pregnancy . HoweverThis study involves seven mother-offspring cohort studies from five European countries within the ALPHABET consortium. These cohorts and longitudinal follow-up from a randomized controlled trial include the Lifeways Cross-Generation Cohort Study (Lifeways) ISRCTN16537904) and\u00a0the Randomised cOntrol trial of LOw glycaemic index diet during pregnancy study (ROLO) in Ireland (ISRCTN54392969); the study on the pre- and early postnatal determinants of child health and development (EDEN) in France; the Avon Longitudinal Study of Parents and Children (ALSPAC) and the Southampton Women\u2019s Survey (SWS) in the UK; the Polish Mother and Child Cohort (REPRO_PL) (NCT01861548) in Poland; and The Generation R Study (Generation R) (NTR6671) in the Netherlands \u201335. All 6537904 an\u2009=\u20095 cohorts) and late pregnancy . Since maternal diet was assessed during both early and late pregnancy in SWS, both were included and the average was taken to reflect whole pregnancy exposure. Whole pregnancy refers to dietary information assessed at any time point of pregnancy.Pre-pregnancy or antenatal dietary intakes of the study mothers were assessed using validated food frequency questionnaires (FFQ) (mean food items in ALPHABET: 137), which have been described in detail elsewhere . In ALPHz-scores for each dietary parameter were derived by subtracting the mean of the energy-adjusted regionally representative world composite database from the participants-reported amount and dividing this value by the parameter\u2019s representative standard deviation. The z-scores were then converted to proportions and then centred by doubling and subtracting 1. The resulting value was then multiplied by the corresponding food parameter-specific inflammatory effect score (derived from a comprehensive literature review of 1943 peer-reviewed articles) and summed to yield the overall E-DII score. A higher E-DII score indicates a more pro-inflammatory diet. The systematic review was conducted through a comprehensive search of the National Library of Medicine database from 1950 through 2010, and the literature search strategy along with inclusion criteria were described in detail in the DII development paper , maternal alcohol intake during pregnancy (yes/no), maternal parity (primiparous/multiparous) and child exact age at anthropometry measurement (in months) and sex . These data were originally abstracted from birth records or collected using questionnaires (interviewer- or self-administered).Potential confounders and relevant covariates were identified from literature and harmonized for subsequent analysis. These were maternal age at delivery (in years), maternal height (in cm), pre-pregnancy BMI (in kg/mn\u2009=\u2009396) with likely implausible energy intakes to avoid extreme misreporting . These associations were of similar magnitude for early- and late-pregnancy (instead of whole-pregnancy) dietary scores with slight variations in statistical significance (Table\u00a0The main associations (involving greatest numbers of mother-child pairs) between maternal whole pregnancy E-DII and DASH scores and late-childhood OWOB are summarized in Fig.\u00a0ce Table\u00a0.Fig. 1AIn general, no consistent associations were observed between maternal E-DII and DASH scores with regards to early- and mid-childhood OWOB. The only exception is that higher late-pregnancy E-DII score was associated with a lower odds of early-childhood OWOB [OR (95% CI) 0.91 ].\u03b2 \u00a0kg/m2 per 1-SD increase in E-DII score]. In contrast, a higher whole pregnancy DASH score was associated with a lower late-childhood FMI [\u03b2 \u00a0kg/m2]. Across different pregnancy periods, the point estimates were in the same direction and of comparable magnitude, though the sample sizes and statistical significance varied . No other apparent associations were observed for other periods or between maternal E-DII, DASH scores and childhood SST.Similar to the primary outcome, associations between maternal dietary scores and secondary adiposity measures were only observed during late-childhood Table\u00a0. A higheP\u2009=\u20090.73) and that on higher dietary quality vs. lower OWOB odds was 13.1% (P\u2009=\u20090.48). For the significant association between more pro-inflammatory diet and lower FFMI, the proportion mediated by dietary quality was 7.0% (P\u2009=\u20090.69). When a stricter childhood obesity outcome was used (>\u200995th sex-and-age-specific BMI z-score), most of the abovementioned point estimates were stronger, except for late-pregnancy E-DII and early-childhood obesity which was largely attenuated . In contrast, higher maternal pregnancy E-DII score was associated with lower [\u03b2 (95% CI) \u2212\u20090.10 \u00a0kg/m2], whereas higher DASH score was associated with higher [\u03b2 (95% CI) 0.06 \u00a0kg/m2], late-childhood FFMI in males .Some potential sex-interactions were noted, especially for mid- and late-childhood FMI and FFMI outcomes during pregnancy was associated with a lower odds of late-childhood OWOB and lower FMI. In contrast, a more pro-inflammatory (higher E-DII) diet during pregnancy was associated with lower late-childhood FFMI. Furthermore, sex interactions were observed such that the associations of higher maternal DASH, lower E-DII and higher late-childhood FFMI were stronger in male offspring, while associations of lower maternal DASH, higher E-DII and higher mid-childhood FMI were stronger in female offspring. Contrary to our hypothesis, dietary score associations at different pregnancy stages were quite similar. Thus, the discussion of results will be based on the whole pregnancy period with the highest number of participants.n\u2009=\u20091566), higher maternal Mediterranean Diet Score was associated with lower mid-childhood BMI z-score and SST [n\u2009=\u2009354), higher maternal diet quality from pregnancy through 3\u00a0months postpartum was associated with lower infant weight-for-length z-score from birth to 6\u00a0months and body fat % at 6\u00a0months [n\u2009=\u2009992), a more pro-inflammatory diet (higher DII scores) during pregnancy was associated with higher mid-childhood (median age: 7.7\u2009years) adiposity, e.g. FMI and SST, but attenuated after adjustment for covariates [n\u2009=\u20091078) reported that higher DII scores in obese mothers were associated with increased neonatal fat mass [Previous studies showed that in US and Greek children . However, there is variation of timing of adiposity rebound and children or adults who become obese have been reported as having an earlier adiposity rebound (around 3\u2009years old) in early childhood . Those wWhile a very useful and relevant measure, a higher BMI may not only arise from greater body fat, but also from higher fat-free\u00a0mass, making it an imperfect measure of adiposity , 70. TheThe large sample size and substantial efforts spent in harmonizing and curating data across multiple studies are the major strengths of our study. To our knowledge, the current study represents the largest multi-centre collaborative effort in investigating the influence of maternal dietary quality and inflammatory potential on childhood adiposity outcomes.However, some limitations are worth noting. Our study can mainly be generalized to European-born/White women in developed countries. Nonetheless, we did include studies from a range of geographical regions within Europe , in which some diversity in dietary intakes and sociodemographic characteristics was observed. Self-reported dietary data were used, which might have increased non-differential measurement errors that may bias results towards the null. Moreover, the FFQs were mainly validated for European-born/White women (e.g. in Gen R), potentially introducing heterogeneity and more measurement errors for non-European-born/non-White women. Indeed, in our analyses restricted to European-born/White population, the observed associations strengthened. We have applied a commonly used energy intake cut-off for pregnant women , 55 to eOur individual participant data meta-analysis within a large consortium suggests that pro-inflammatory, low-quality maternal pregnancy diets may adversely influence offspring adiposity and obesity risk, especially during late-childhood. Promoting overall healthy dietary pattern during pregnancy may have lifelong consequences for the offspring. Because most associations were observed at mid-childhood or later, future studies investigating in utero programming of childhood adiposity may benefit from a longer follow-up.Additional file 1: Table S1-S19 and Fig. S1-S2. Table S1 Characteristics of study participants according to included studies. Table S2 Characteristics of the cohorts in the ALPHABET consortium. Table S3 Food parameters included for E-DII generation. Table S4 Food items included for DASH score generation. Fig. S1 Boxplots of E-DII scores in included studies. Table S5 Availability of outcome measures. Fig. S2 Scatterplots of DASH score against E-DII score in each study. Table S6 Association between maternal E-DII and DASH scores (per 1-SD increase) and childhood OWOB- excluding non-European-born/non-White participants. Table S7 Association between maternal E-DII and DASH scores (per 1-SD increase) and secondary childhood adiposity measures- excluding non-European-born/non-White participants. Table S8 Association between maternal E-DII and DASH scores (per 1-SD increase) and childhood OWOB- excluding mothers with pregnancy complications. Table S9 Association between maternal E-DII and DASH scores (per 1-SD increase) and secondary childhood adiposity measures- excluding mothers with pregnancy complications. Table S10 Association between maternal E-DII and DASH scores (per 1-SD increase) and childhood OWOB- with mutual adjustment of E-DII and DASH. Table S11 Association between maternal E-DII and DASH scores (per 1-SD increase) and secondary childhood adiposity measures- with mutual adjustment of E-DII and DASH. Table S12 Association between maternal E-DII and DASH scores (per 1-SD increase) and childhood obesity (BMI z-score\u2009>\u200995th percentile). Table S13 Association between maternal E-DII and DASH scores (per 1-SD increase) and childhood OWOB- with further adjustment of birthweight. Table S14 Association between maternal E-DII and DASH scores (per 1-SD increase) and secondary childhood adiposity measures- with further adjustment of birthweight. Table S15 Association between maternal E-DII and DASH scores (per 1-SD increase) and childhood OWOB- with further adjustment of gestational age. Table S16 Association between maternal E-DII and DASH scores (per 1-SD increase) and secondary childhood adiposity measures- with further adjustment of gestational age. Table S17 Association between maternal E-DII (per 1-SD increase) and late-childhood OWOB and adiposity measures- with and without further adjustment for child E-DII score in cohorts with child E-DII data. Table S18 Pooled P-values for sex-interaction between maternal E-DII and DASH score and offspring adiposity outcomes. Table S19 Stratified estimates for other sex-interactions between maternal E-DII and DASH scores and offspring adiposity outcomes ."} +{"text": "Rhesus cytomegalovirus (RhCMV) strain 68-1-vectored simian immunodeficiency virus (RhCMV/SIV) vaccines are associated with complete clearance of pathogenic SIV challenge virus, non-canonical major histocompatibility complex restriction, and absent antibody responses in recipients previously infected with wild-type RhCMV. This report presents the first investigation of RhCMV/SIV vaccines in RhCMV-seronegative macaques lacking anti-vector immunity. Fifty percent of rhesus macaques (RM) vaccinated with a combined RhCMV-Gag, -Env, and -Retanef (RTN) vaccine controlled pathogenic SIV challenge despite high peak viremia. However, kinetics of viral load control by vaccinated RM were considerably delayed compared to previous reports. Impact of a TLR5 agonist on vaccine efficacy and immunogenicity was also examined. An altered vaccine regimen containing an SIV Gag-FliC fusion antigen instead of Gag was significantly less immunogenic and resulted in reduced protection. Notably, RhCMV-Gag and RhCMV-Env vaccines elicited anti-Gag and anti-Env antibodies in RhCMV-seronegative RM, an unexpected contrast to vaccination of RhCMV-seropositive RM. These findings confirm that RhCMV-vectored SIV vaccines significantly protect against SIV pathogenesis. However, pre-existing vector immunity and a pro-inflammatory vaccine adjuvant may influence RhCMV/SIV vaccine immunogenicity and efficacy. Future investigation of the impact of pre-existing anti-vector immune responses on protective immunity conferred by this vaccine platform is warranted. This vaccine was associated with 31.2% protection against HIV acquisition, with envelope (Env)-binding antibodies capable of antibody-dependent cytotoxicity implicated as an immune correlate of protection2. These encouraging results propelled further development of antibody-based approaches using the nonhuman primate (NHP) simian immunodeficiency virus (SIV) model with the goal of further optimizing protection through generation of antibodies with broadly neutralizing and/or Fc-receptor-mediated effector activity. These approaches, based on a wide assortment of viral and DNA vectors and recombinant protein immunogens, have shown modest protection against acquisition of challenge virus6. However, a news release by NIH/NIAD (2020) revealed that the HVTN 702 human clinical trial testing a vaccine approach based on the RV144 regimen, adapted to the subtype Clade C, and conducted in southern Africa, did not recapitulate the efficacy observed for the RV144 trial. Vaccine induction of Env-binding antibodies of sufficient potency and breadth to confer absolute protection against acquisition of divergent challenge viruses remains a significant challenge.Development of an efficacious human immunodeficiency virus (HIV-1) vaccine remains a high-priority goal. The only HIV-1 vaccine showing efficacy in human clinical trials remains the RV144 ALVAC-HIV (vCP1521) and AIDSVAX7. This approach has been tested with multiple viral vectors including adenovirus (Ad)5, Ad26, poxvirus, and rhabdovirus (rVSV), and all have demonstrated induction of strong antiviral T-cell responses in nonhuman primates. Notably, T-cell responses elicited by these viral vectors are biased towards a central memory phenotype8. A novel viral vectored- SIV vaccine encoding a near full-length SIV genome and based on the rhesus monkey rhadinovirus (RRV) was shown to induce both antibodies and T cell responses to SIV antigens9. One promising avenue for T-cell-based vaccines came from the unexpected results obtained using species-specific rhesus cytomegalovirus (RhCMV) as a vector for delivery of critical viral antigens in rhesus macaques (RM)10. Vaccines vectored by the RhCMV68-1 strain (RhCMV/SIV) demonstrated impressive vaccine efficacy in wild-type RhCMV-seropositive RM, with 50% of vaccinated animals showing profound early control and eventual clearance of virus from blood and tissues after challenge with pathogenic SIVmac23912. Of note, the parental RhCMV68-1 strain lacks the RhUL128 and RhUL130 ORFs, resulting in a loss of tropism for epithelial cells which is a change reported as necessary for vaccine immunogenicity16. Furthermore, analysis of RhCMV/SIV vaccine-induced cellular immunity revealed unconventional major histocompatibility complex (MHC)-II and MHC-E-restricted antiviral CD8 T-cell responses that break previous immunological paradigms18. More recent reports showed that further attenuation of the RhCMV68-1 vector to limit dissemination of RhCMV-vectored SIV vaccines maintained a high efficacy of 59% and similar T-cell immunogenicity20. Importantly, antibodies directed to SIV antigens delivered by this vaccine platform have been severely restricted or undetectable in vaccinated RhCMV-seropositive RM19; the mechanistic basis for complete dominance of T-cell over antibody responses remains unknown. These collective findings suggested an opportunity for improvement of efficacy for the RhCMV/SIV vaccine approach by addition of immunomodulators capable of promoting humoral as well as cellular responses.In contrast to antibody-based vaccines, cytotoxic T-cell-based vaccines focus on generation of HIV-specific T-cell responses of significant magnitude and breadth23. We previously described construction of RhCMV-Gag-FliC, which co-expresses codon-optimized SIVmac239 Gag fused to FliC derived from Salmonella enterica serovar Enteritidis and also deleted for the hypervariable domain24. These same studies confirmed stable replication of RhCMV-Gag-FliC and TLR5 agonist activity in vitro. Greater inflammation at the site of subcutaneous inoculation distinguished this vaccine when compared to parental RhCMV-Gag and supported adjuvant-associated modulation of innate responses24. Studies described herein will compare immunogenicity and efficacy of regimens including RhCMV-Gag versus RhCMV-Gag-FliC when administered to previously RhCMV-seronegative recipients.We hypothesized that activation of innate immunity during vaccination with RhCMV/SIV vaccines would augment vaccine-mediated immunogenicity and protective immunity. To this end, a TLR5 agonist was expressed in the RhCMV68-1 backbone as an in-frame fusion with SIV Gag and evaluated for its ability to increase protective efficacy against SIV challenge. The TLR5 agonist, bacterial flagellin (FliC), exhibits mucosal adjuvant activity for vaccines expressing multiple bacterial and viral antigens by enhancing antibody and, to a lesser degree, cellular responses to vaccine antigens, particularly when expressed as a fusion protein with the immunogen10 or RhCMV-Gag-FliC24 in RhCMV-seronegative RM. Three groups of Specific Pathogen Free level 2 (SPF-2), i.e., RhCMV-seronegative, adult female RM were vaccinated with either: empty vaccine vector , RhCMV68.1 SIV vaccine including RhCMV-Gag, RhCMV-Retanef (RTN), and RhCMV-Env (Group B), or RhCMV-Gag-FliC, RhCMV-Retanef, and RhCMV-Env (Group C) and oral routes with the goal of induction of systemic and mucosal immune responses.We compared immunogenicity and protective efficacy of RhCMV68-1-vectored SIV vaccine regimens including RhCMV-GagMamu B*17 in Group B exhibited particularly robust CD4 T-cell responses . Gag-specific CD8 T-cell responses were comparable between Groups B and C at this same time point. T-cell responses to Gag in group B were concentrated in the CD4 T-cell compartment at both time points. Despite mucosal delivery of the vaccine, Gag-specific T cell responses were not detected in mononuclear cell (MNC) preparations from colonic biopsies.SIV-Gag-specific T-cell responses were assessed in peripheral blood mononuclear cells (PBMC) using intracellular cytokine staining (ICS) and a pool of 15-mer overlapping peptides spanning SIVmac239 Gag. CD4 and CD8 T-cell responses based on tumor necrosis factor (TNF) and interferon (IFN)-\u03b3 expression as demonstrated by a gating strategy and representative scatter plots in Supp Fig. 26. In this study we assessed responses to a Mamu-E-restricted Gag \u201csupertope\u201d peptide (Gag69) to which all vaccinated RM responded in previous reports18. Our results for RhCMV-seronegative RM demonstrated both CD4 and CD8 T-cell responses to the Gag69 supertope peptide, with comparable frequencies in Groups B and C; responses increased after the second booster immunization and RhCMV-seropositive RM (n\u2009=\u20096) twice with 104 PFU RhCMV-Gag by the SC route. Results of this second study revealed an absence of anti-Gag antibody after two RhCMV-Gag immunizations in RhCMV-seropositive compared to detectable antibody in most RhCMV-seronegative RM . Superinfection of RhCMV-seropositive RM was confirmed by detection of de novo Gag-specific T cell responses and boosting of RhCMV-specific responses after a second immunization with RhCVM-Gag -binding antibodies as determined by a custom SIV Env-binding antibody multiplex assay (SIV-BAMA)31 was conducted 12\u00a0weeks after the final immunization . Analysis also revealed a positive correlation between peak and set point viral loads (P\u2009=\u20090.002) challenge using a previously described SIVmac251 stock and protocolion Fig.\u00a0a,b. Two ups Fig.\u00a0a. Among Hematologic parameters were not significantly different between experimental groups over time vaccinated with the RhCMV68-1-vectored SIV vaccine demonstrated superior control of virus loads after challenge, as defined by a significant decline in plasma viral loads . These noteworthy findings demonstrate an effect of anti-vector immunity and/or chronic RhCMV infection on humoral responses to vaccine antigens delivered by RhCMV vectors. We recently reported that chronic subclinical viral infections including RhCMV impacted blood immune profiles, gut microbiota and humoral immune responses to an influenza vaccine in RM32. Additionally, a recent report revealed that prior immunity to mouse CMV (MCMV) conferred a negative impact on vaccine-associated humoral immune responses and efficacy of a MCMV-vectored vaccine expressing a Friend virus (FV) Env immunogen (MCMV.env) in mice highly susceptible to FV33. Furthermore, these studies also reported that a longer time frame between vaccination and challenge was required for MCMV.env vaccine efficacy. It is important to note that although our studies also revealed an impact on vaccine-induced humoral responses conferred by vector immunity, anti-ENV-binding antibody responses post immunization were of low magnitude and anti-ENV antibody responses post challenge did not correlate with virus control. Because humoral immunity is considered a necessary component of a successful HIV vaccine, elucidation of mechanisms by which pre-existing immunity and chronic CMV infection impact such responses elicited by vaccines based on RhCMV or human CMV (HCMV) vectors is needed.An unexpected but significant finding for this vaccine trial involving RhCMV-seronegative RM was detection of anti-Gag antibodies in Group B animals, as well as anti-Env antibodies in Groups B and C Fig.\u00a0b,c. Obse23. However, this strategy failed to achieve enhanced immunogenicity in either systemic or mucosal tissues, or to induce protection against pathogenic SIVmac251. These outcomes were unexpected but yielded important insights regarding modification of CMV-based vectors. First, anti-Gag antibody responses were not detected in RM immunized with RhCMV-Gag-FliC but were detected in RM immunized with the RhCMV-Gag vaccine (23 Indian-origin and 1 Indian- and Chinese-origin mix) ranging from 3 to 7\u00a0years of age. MHC class I genotyping for Mamu was alleles was performed by the AIDS Vaccine Research Laboratory, University of Miami, Miller School of Medicine. SPF-2 RM were maintained as free of infection with RhCMV, simian foamy virus (SFV), herpes B virus (BV), type D simian retrovirus (SRV), simian T-lymphotropic virus (STLV), and SIV. A second immunogenicity trial for the RhCMV-Gag vaccine utilized one group of SPF-2 RM and a second age-matched group of six conventionally raised non-SPF RhCMV-seropositive RM .10. Construction and characterization of the RhCMV68-1 vector expressing a SIVmac239-Gag and Salmonella enterica serovar Enteritidis FliC (flagellin) fusion protein (RhCMV-Gag-FliC) were also previously reported24. All virus stocks were titered by our standard plaque reduction assay using telomerized-rhesus fibroblasts38, that differs from titering methods used by Hansen et al.10.The construction and characterization of the RhCMV/SIV vaccines expressing SIVmac239 Gag, RTN, and Env were previously described4 PFU per vaccine) and orally (105 PFU per vaccine) and boosted subcutaneously and orally (105 PFU per vector for both routes) at weeks 12 and 24. For immunogenicity studies in SPF-2 (n\u2009=\u20096) or non-SF (n\u2009=\u20096) RM, RhCMV-Gag was delivered subcutaneously at a dose of 104 PFU at weeks 0 and 16.For the vaccine trial, animals were primed with either RhCMV68-1 (control Group A) or RhCMV/ SIV vaccines subcutaneously isolation by Accu Paque gradient centrifugation . PBMC were cryopreserved by resuspension freezing media containing 10% dimethly sulfoxide (DMSO) in fetal calf serum for assay of T cell response assays. Mononuclear cells (MNC) from lymph nodes (LN) or intestinal mucosa were isolated after incubation in digesting solution containing 50 U/mL DNase I and 250 U/mL type I collagenase or 0.5\u00a0mg/mL type II collagenase (Sigma-Aldrich) for 30\u00a0min and mechanically disrupted in gentleMACS C tubes (Miltenyi Biotec) and 70\u00a0\u03bcm cell strainers (Thermo Fisher Scientific).28. Plasma IgG binding to SIVmac251 gp130 and gp70 V1V2, SIVmac239 gp120 and gp70 V1V2, SIVsmE660 gp140 and gp70 V1V2 were determined by a custom SIV binding antibody multiplex assay (SIV_BAMA) as previously described30.The binding IgG titers for RhCMV antigens and SIV Gag p27 were quantified by in-house ELISAs as previously described273-287(Gag69) peptide were used in the presence of co-stimulatory CD28 and CD49d monoclonal antibodies . Cells were incubated with peptides and co-stimulatory antibodies alone for 1\u00a0h, followed by adding Brefeldin A (Sigma-Aldrich) for an additional 8\u00a0h. Cells incubated with co-stimulatory antibodies in media with DMSO but without peptides served as a control for background subtraction. Whole blood or cells from T-cell assays were stained with fluorochrome-conjugated monoclonal antibodies against human cell markers including CD3 (SP34-2), CD4 (L200), CD8 (SK1), CD20 (L27), CD14 (M5E2), CD16 (3G8), TNF (Mab11), and IFN-\u03b3 (B27). Lysis and fixation of whole blood samples were performed by the TQ-Prep Workstation and IMMUNOPREP reagent system (Beckman Coulter). Data were analyzed and illustrated using FlowJo software (BD Bioscience). Cells were acquired using a LSRFortessa cell sorter operated by FACSDiva software (BD Bioscience) with a minimum acquisition of 105 live T cells. Data were analyzed using FlowJo software (BD Bioscience). Frequencies of responding cells found to be less than 0.01% were considered very low and equivocal.SIV-specific CD4 and CD8 T-cell responses were measured in PBMC and tissue MNC by flow cytometric intracellular cytokine analysis. Sequential 15-mer peptides (overlapping by 11 amino acids) comprising the SIVmac239 Gag and Rev, Tat, and Nef (provided by NIH AIDS Reagent Program) proteins or a 15-mer Gag3 TCID50 in rhesus 221 cells, was delivered IVAG to each RM every week until infected or up to 12 challenges. Infection was defined by detection of plasma virus load of 103 SIV RNA copies per mL. Plasma SIV viral loads were measured weekly, with challenge continued for one week after detection positive of SIV RNA. Plasma SIV RNA levels were measured by a gag-targeted quantitative real-time/digital RT-PCR, designed to prevent potential cross reactivity between RhCMV-Gag and SIV, as previously described41. A total of 6 replicate reactions were analyzed per extracted RNA sample for assay thresholds of 15 SIV RNA copies in 1\u00a0mL plasma.All RM in the vaccine trial were challenged IVAG with SIVmac251 at 12\u00a0weeks after the last boost using a repeated (weekly) low dose challenge protocol. In brief, 1\u00a0mL of 1:20 diluted pathogenic SIVmac251 stock , equivalent to a titer of 5\u2009\u00d7\u200910https://www.R-project.org/). Comparisons of matched samples were performed with Friedman nonparametric tests followed by Dunn\u2019s multiple comparison tests. Protection against acquisition of SIVmac251 infection was analyzed by logrank tests. Overall comparisons between the control and vaccine groups were performed with ANOVA parametric or Kruskal\u2013Wallis nonparametric tests. If the differences were significant, Dunnet\u2019s (for ANOVA) or Dunn\u2019s post tests were conducted to determine the significance of pairwise differences between groups. For all tests, P\u2009<\u20090.05 was considered significant.Statistical and graphic analysis and graphing were conducted with GraphPad Prism 8 software or in the R programming language and were approved in advance by the University of California Davis (UC Davis) Institutional Animal Care and Use Committee (IACUC). All experiments were performed in accordance with UC Davis relevant guidelines and regulations including approved Biological Use Authorizations required by the UC Davis Safety Services for handling of nonhuman primate tissues, SIV virus stocks, and RhCMV vaccine vectors.Supplementary file1"} +{"text": "The defining features of a neuron are its functional and anatomical connections with thousands of other neurons in the brain. Together, these neurons form functional networks that direct animal behavior. Current approaches that allow the interrogation of specific populations of neurons and neural circuits rely heavily on targeting their gene expression profiles or connectivity. However, these approaches are often unable to delineate specific neuronal populations. Here, we developed a novel intersectional split intein-mediated split-Cre recombinase system that can selectively label specific types of neurons based on their gene expression profiles and structural connectivity. We developed this system by splitting Cre recombinase into two fragments with evolved split inteins and subsequently expressed one fragment under the influence of a cell type-specific promoter in a transgenic animal, and delivered the other fragment via retrograde viral gene transfer. This approach results in the reconstitution of Cre recombinase in only specific population of neurons projecting from a specific brain region or in those of a specific neuronal type. Taken together, our split intein-based split-Cre system will be useful for sophisticated characterization of mammalian brain circuits. Drosophila ), and Dlx6-CCre-IRES-eGFP::Rosa26-LoxP-Stop-LoxP-TdTomato (Dlx6+ RTM). S3(A) Total distance travelled in open field chamber (cm), (B) Number of movements, (C) Number of stereotypy, which is the amount of repetitive movements, includeing grooming behaviour, (D) Time spent in center (s). We noted statistically non-significant differences (Mann-Whitney test) between Dlx6- RTM and Dlx6+ RTM in locomotor activity (p\u2009>\u20090.9999), movements (p\u2009=\u20090.9143), stereotypy (p\u2009=\u20090.2571) and time in center (p\u2009=\u20090.2571)."} +{"text": "Introduction: Cross-reactivity to SARS-CoV-2 antigenic peptides has been detected on T-cells from pre-pandemic donors due to recognition of conserved protein fragments within members of the coronavirus's family. Further, preexisting antibodies recognizing SARS-CoV-2 with conserved epitopes in the spike region have been now seen in uninfected individuals. High-dose Intravenous Immunoglobulin (IVIg), derived from thousands of healthy donors, contains natural IgG antibodies against various antigens which can be detected both within the IVIg preparations and in the serum of IVIg-receiving patients. Whether IVIg preparations from pre-pandemic donors also contain antibodies against pre-pandemic coronaviruses or autoreactive antibodies that cross-react with SARS-CoV-2 antigenic epitopes, is unknown.Methods: 13 samples from 5 commercial IVIg preparations from pre-pandemic donors ; Privigen (CSL Behring); Intratect (Biotest AG); IgVena (Kedrion S.p.A); and Flebogamma (Grifols S.A.) were blindly screened using a semi-quantitative FDA-approved and validated enzyme-linked immunosorbent assay (ELISA) .Results: Nine of thirteen preparations (69.2%), all from two different manufactures, were antibody-positive based on the defined cut-off positivity . From one manufacturer, 7/7 lots (100%) and from another 2/3 lots (67%), tested positive for cross-reacting antibodies. 7/9 of the positive preparations (77%) had titers as seen in asymptomatically infected individuals or recent COVID19-recovered patients, while 2/9 (23%) had higher titers, comparable to those seen in patients with active symptomatic COVID-19 infection (index > 2.2).Conclusion: Pre-pandemic IVIg donors have either natural autoantibodies or pre-pandemic cross-reactive antibodies against antigenic protein fragments conserved among the \u201ccommon cold\u201d - related coronaviruses. The findings are important in: (a) assessing true anti-SARS-CoV-2-IgG seroprevalence avoiding false positivity in IVIg-receiving patients; (b) exploring potential protective benefits in patients with immune-mediated conditions and immunodeficiencies receiving acute or chronic maintenance IVIg therapy, and (c) validating data from a recent controlled study that showed significantly lower in-hospital mortality in the IVIg- treated group. Efficient immune surveillance of infected or recovering populations from COVID-19 is important in the fight against the pandemic. Recent evidence suggests that anti-SARS-CoV-2 immunity not only occurs after an active infection, but may also precede an infection. Cross-reactivity to SARS-CoV-2 antigenic peptides has been detected on T-cells and B-cells from pre-pandemic donors owing to recognition of protein fragments conserved among common cold-related coronaviruses but, basIntravenous Immunoglobulin (IVIg), derived from thousands of healthy donors, contains natural and cross-reactive IgG autoantibodies against various antigens at various thresholds of detection owing to accumulation of low antibody concentrations from single individuals. These antibodies can be detected at clinically significant levels not only within the IVIg preparations but also in the serum of IVIg-receiving patients . WhetherWe examined if various commercial IVIg preparations contain anti-SARS-CoV-2-antibodies, due to cross-reactivity with pre-pandemic anti-coronavirus IgG antibodies or the existence of natural IgG antibodies, and accurately measured antibody titers to assess if they are clinically meaningful, compared to titers seen in COVID-19 infected patients.We tested 13 samples from 5 commercial IVIg preparations using a semi-quantitative FDA-approved, enzyme-linked immunosorbent assay (ELISA) that measures anti-SARS-CoV-2-antibodies directed against the S1 viral spike protein. The assay, according to the manufacturer, has a 98.5 % sensitivity and 99 % specificity. An independent serological survey has validated this method reporting 93% sensitivity and 100% specificity . We haveThe following IVIg preparations were screened: HyQvia ; Privigen (CSL Behring); Intratect (Biotest AG); IgVena (Kedrion S.p.A); and Flebogamma (Grifols S.A.). From one brand, 7 different lots were available and from another one 3 different lots. A total of 13 lots were screened. All preparations were coded by manufacturer and lot and used as liquid preparations in either 1:250 or 1:500 dilution to match normal human serum IgG concentration (0.5 mg/ml). No dissolution was needed ensuring the lack or aggregate formation. No colloids were added as all IVIg preparations tested contained protective colloids.Control sera were used from patients with various autoimmune neurological diseases, autoimmune neuropathies and non-COVID19 ICU-hospitalized patients from the serum Biobank of the Department of Pathophysiology, University of Athens Medical School. Ethical approval for Bio-banking had been granted from the University of Athens Ethics Committee.Nine of thirteen preparations 69.2%), all from 2 different manufactures, tested positive based on the manufacturer's cut-off . From on.2%, all To further validate our results, we tested as controls pre-pandemic archived sera from: (a) 49 neurological patients with autoimmune neuropathies; only one had a titer of 2.32, comparable to COVID-19 infected patients and the IVIg-positive preparations (whether this patient had been previously receiving immunotherapy with IVIg is however unknown); and (b) 30 ICU-hospitalized patients; all were anti-SARS-CoV-2 antibody negative.IVIg preparations, made from pre-pandemic healthy donors, exhibit cross reactivities with SARS-CoV-2 S1 antigens, with some preparations having clinically significant antibody titers comparable to those seen in COVID-19 infected patients. The findings suggest that the respective healthy IVIg donors had either natural autoantibodies or cross-reactive antibodies against antigenic protein fragments conserved among the other \u201ccommon cold\u201d -related coronaviruses, similar to the cross-reactivity observed for T-cells and B cells from unexposed donors using an FDA-approved assay, same as in other major serological surveys \u20139; (b) tIVIg has been effectively used in some COVID-19 patients including COVID-19- immune inflammatory syndrome resembling Kawasaki's disease but whetThe just published evidence that preexisting seasonal coronavirus antibodies in uninfected individuals cross-react with SARS-CoV-2 recognizing conserved epitopes in the spike region targeted by neutralizing antibodies, may have important ramification for natural infection and the Apart from a potential benefit, the observations are also important in assessing SARS-CoV-2 seroprevalence, emphasizing the need to ensure that sera from prior IVIg-treated patients may contain SARS-CoV-2-antibodies derived from IVIg rather than form acquired antiviral humoral immunity.It would be of interest in a future study to check for antibodies against the S2 subunit when kits with verified reliability become available. Most importantly, it will be relevant to examine if the noted anti-SARS-CoV-2 antibodies have a neutralizing activity.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.All authors contributed to conception and execution of the work, writing, and reviewing the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "The virus severe acute respiratory syndrome\u2013coronavirus 2 (SARS-CoV-2) can cause coronavirus disease 2019 (COVID-19), an influenza-like disease that is primarily thought to infect the lungs with transmission via the respiratory route. However, clinical evidence suggests that the intestine may present another viral target organ. Indeed, the SARS-CoV-2 receptor angiotensin converting enzyme 2 (ACE2) is highly expressed on differentiated enterocytes. In human small intestinal organoids (hSIOs), enterocytes were readily infected by SARS-CoV and SARS-CoV-2 as demonstrated by confocal- and electron-microscopy. Consequently, significant titers of infectious viral particles were detected. mRNA expression analysis revealed strong induction of a generic viral response program. Hence, intestinal epithelium supports SARS-CoV-2 replication, and hSIOs serve as an experimental model for coronavirus infection and biology Sarbecovirus subgenus (Severe acute respiratory syndrome (SARS), caused by coronavirus SARS-CoV, emerged in 2003 and recapitulate key aspects of the organ from which the ASCs derive. Since SARS-CoV and SARS-CoV-2 target the lung, we added virus to organoid-derived human airway epithelium cultured in 2D and observed that SARS-CoV and SARS-CoV2 readily infected differentiated airway cultures. . ImmunosHuman small intestinal organoids (hSIOs) are established from primary gut epithelial stem cells, can be expanded indefinitely in 3D culture and contain all proliferative and differentiated cell types of the in vivo epithelium , enterocACE2 mRNA expression differed greatly between the four conditions. EXP-hSIOs express 300-fold less ACE2 mRNA compared to DIF-hSIOs, when analyzed in bulk (fig. S2). BMP treatment induced 6.5-fold up-regulation of ACE2 mRNA compared to DIF treatment alone. Since this did not yield infection rate differences, the DIF-BMP condition was not analyzed further.+ EECs . We also did not notice infection of Goblet cells across culture conditions. At 60 hours, apoptosis became prominent in both SARS-CoV and SARS-CoV-2 infected enterocytes (fig. S5). ACE2 protein was readily revealed as a bright and ubiquitous brush border marker in hSIOs in DIF medium to visualize enterocyte brush borders, DNA (DAPI) and cleaved caspase 3 to visualize apoptotic cells. Generally, comparable rates of viral infections were observed in the organoids growing in all three conditions. We typically noted staining for viral components (white) in rare, single cells at 24 hours. At 60 hours, the number of infected cells had dramatically increased . InfecteF medium . In hSIOF medium . In immaUnsupervised transmission electron microscopy (TEM) , at the We then performed mRNA sequence analysis to determine gene expression changes induced by SARS-CoV and SARS-CoV-2-infection of hSIOs cultured continuously in EXP medium and hSIOs cultured in DIF medium. Infection with SARS-CoV-2 elicited a broad signature of cytokines and interferon stimulated genes (ISGs) attributed to type I and III interferon responses . An overFinally, the infection was repeated in a second experiment in the same ileal HIO line and analyzed after 72 hours. Analysis involved viral titration (fig. S12), confocal imaging (fig. S13), and RNA sequencing (fig. S14). This experiment essentially confirmed the observations presented above. A limited, qualitative experiment applying confocal analysis demonstrated infectability of two other lines available in the lab from independent donors (fig. S13). This study shows that SARS-CoV and SARS-CoV-2 infect enterocyte lineage cells in a human intestinal organoid model. We observed similar infection rates of enterocyte-precursors and enterocytes whereas ACE2 expression increases ~1000-fold upon differentiation at the mRNA level (fig. S2). This suggests that low levels of ACE2 may be sufficient for viral entry.SARS-CoV-2 is the third highly pathogenic coronavirus (after SARS-CoV and MERS-CoV) to jump to humans within less than 20 years suggesting that novel zoonotic coronavirus spillovers are likely to occur in the future. Despite this, limited information is available on coronavirus pathogenesis and transmission. This is in part due to the lack of in vitro cell models that accurately model host tissues. Very recently, it was shown that human iPS cells differentiated toward a kidney fate support replication of SARS-CoV-2 ("} +{"text": "SARS-CoV-2 (COVID-19) is associated with increased thrombosis. Here, wedemonstrate patterns of pulmonary vascular disease in COVID-19 includingclassical acute pulmonary embolism and subsegmental perfusion defects in theabsence of acute pulmonary embolism suggestive of microvascular thrombosis. The BritishThoracic Imaging Society Guidelines recommend unenhanced pulmonary angiography andCTPA. This protocol facilitates lung subtraction iodine mapping (CT-LSIM) for lungperfusion, a clinically sensitive tool in PE. We report the first CT-LSIM images inCOVID-19.Severe acute respiratory syndrome (SARS) caused by the coronavirus SARS-CoV-2(COVID-19) has a high mortality due primarily to respiratory failure. Recent studieshave highlighted increased thrombosis in COVID-19.At our institution, 10 patients (mean age (SD) 70 (16), 40% female) with COVID-19,confirmed on reverse transcription polymerase chain reaction (RT-PCR), underwentCTPA and CT-LSIM for suspected acute PE based on clinical assessment and elevatedd-dimer levels . AnalysiThree patients had confirmed PE on CTPA and CT-LSIM classical acute PE with central clot associated with lung infarctionand (ii) subsegmental perfusion defects in the absence of acute PE which is perhapssuggestive of microvascular thrombosis.Distinct from classical thromboembolic PE, a high proportion of in situ pulmonaryarterial thrombosis exists in COVID-19, and the pathophysiology is not fully understood.CT-LSIM is potentially widely available for the assessment of lung perfusion inCOVID-19. Further studies to understand the pathophysiology of pulmonary thromboticdisease in COVID-19 are required."} +{"text": "Scientific Reports 10.1038/s41598-020-63043-2, published online 14 May 2020Correction to: The original version of this Article contained errors.In the Abstract,\u201cGenome-wide transcription factor (TF) binding signal analyses reveal co-localization of TF binding sites based on inferred cis-regulatory modules (CRMs).\u201dnow reads:\u201cGenome-wide transcription factor (TF) binding signal analyses reveal co-localization of TF binding sites, based on which cis-regulatory modules (CRMs) can be inferred.\u201dIn addition, in the Methods section, under the subheading \u2018BICORN input\u2019,\u201cBinary TF-gene binding input is used because it is the signal format most commonly used by different resources.\u201dnow reads:\u201cBinary TF-gene binding input is used because it is the signal format most commonly provided by different resources.\u201dFinally, the Acknowledgements section in this Article was incomplete.\u201cThis work was supported by National Institutes of Health (NIH) grants CA149653 (to JX), CA164384 (to LHC) and CA149147 (RC), and by NIH-NIGMS grant R01GM125878 to AFN.\u201dnow reads:\u201cThis work was supported by National Institutes of Health (NIH) grants CA149653 (to JX), CA164384 (to LHC) and CA149147 (RC), and by NIH-NIGMS grant R01GM125878 to AFN. Note that open access publishing is supported by \"VT Open Access Subvention Fund\".\u201dThese errors have now been corrected in the HTML and PDF versions of the Article."} +{"text": "Skilled Nursing Facility (SNF) Value-Based Purchasing (VBP) ties post-acute payments to readmissions performance. Minority-serving SNFs tend to be poorly resourced and understanding the financial implications of SNF-VBP is important. Our study examined VBP payments and penalties among minority-serving SNFs. We conducted cross-sectional analysis using public data sources. We defined minority-serving as SNFs with >50% of residents who are African American (AA)/Black (n=764) or with >50% of residents who are Hispanic/Latino (n=164). Majority-White SNFs (>50% residents who are White) were the reference group . Outcomes examined were: being in top 20% of performance rankings; receiving a bonus; receiving a bonus that was above the median bonus dollar amount; being in bottom 20% of performance rankings; receiving a penalty; receiving a penalty that was below the median penalty dollar amount. Logistic models estimated the likelihood of experiencing each performance outcome for AA/Black-serving and Hispanic/Latino-serving SNFs in reference to majority-White SNFs. Results show that minority-serving SNFs not only perform worse but also experience greater negative financial impacts. Among those penalized, Hispanic/Latino-serving SNFs had the largest average penalty amounts: $32,575 compared to approximately $26,000 for both AA/Black- and White-serving SNFs. Hispanic/Latino-serving SNFs had 1.69 times the odds of receiving larger than median penalties and AA/Black-serving SNFs had 27% lower odds of receiving higher than median bonus payments. Average penalties approximate the average salary for a certified nursing assistant, the primary direct care worker in this setting. Results from this study raise concerns over long-term impacts. Alternative approaches to encouraging quality should be considered."} +{"text": "Chemotherapy modulates the anti-tumor immune response and outcomes depend on the balance of favorable and unfavorable effects of drugs on anti-tumor immunity. 5-Florouracil (5-FU) is widely used in adjuvant chemotherapy regimens to treat colorectal cancer (CRC) and provides a survival benefit. However, survival remains poor for CRC patients with advanced and metastatic disease and immune checkpoint blockade therapy benefits only a sub-set of CRC patients. Here we discuss the effects of 5-FU-based chemotherapy regimens to the anti-tumor immune response. We consider how different aspects of 5-FU\u2019s multi-factorial mechanism differentially affect malignant and immune cell populations. We summarize recent studies with polymeric fluoropyrimidines that enhance DNA-directed effects and discuss how such approaches may be used to enhance the anti-tumor immune response and improve outcomes. Immune surveillance is essential for limiting cancer incidence and an effective anti-tumor immune response is important for maintaining durable remissions in colorectal cancer (CRC) patients with locally advanced and metastatic disease. The most widely used drug for CRC treatment is 5-Fluorouracil (5-FU) ,2,3. BioIn this review, we discuss the survival benefit associated with adjuvant chemotherapy with 5-FU-based regimens for CRC in the cColorectal cancer (CRC) is a major cause of cancer-related mortality and causes 700,000 deaths annually worldwide thru a multi-step process 47], th, th47], DPYD [While rapidly proliferating malignant cells undergo thymineless death in response to 5-FU treatment , non-malDPYD . 5-FU-inDPYD , consistDPYD , which mDPYD . 5-FU inDPYD , consistDPYD and on mWhile patients deficient in 5-FU catabolism are at increased risk for 5-FU toxicity mainly from elevated levels of ribonucleotide metabolites, the products of 5-FU catabolism, and fluoMost, if not all, chemotherapy drugs affect the anti-tumor immune response to some extent . The extMyeloid-derived suppressor cells (MDSCs) are heterogeneous immature myeloid cells that fail to terminally differentiate and suppress the anti-tumor activities of T and NK cells . In respTregs are a sub-population of CD4+ T-cells that display immunosuppressive function. Specifically, TRegs suppress conventional T helper (Th) cells and contribute to maintenance of immunologic self-tolerance . TRegs iClinical studies show increased MDSC (CD33+CD11b+HLA\u2212DR\u2212) are present in tumor tissue relative to para-neoplastic tissue . FurtherAmong the chemotherapeutic drugs shown to selectively deplete MDSCs in pre-clinical studies are Gemcitabine (Gem) and 5-FUClinical studies demonstrated that 5-FU-based regimens modulate levels of immunosuppressive cells and that a favorable response is associated with chemotherapy-induced reduction in immunosuppressive cell populations ,88. ElevMDSCs and Tregs are important mediators of immunosuppression affected by chemotherapy but otheMalignant cells are induced by chemotherapy to undergo any one of several cell death processes . The mode of cell death is an important determinant in activating immunogenic cell death (ICD), an immune response capable of contributing to further tumor eradication. In general, the extent to which chemotherapy-induced malignant cell death is immunogenic depends on both the antigenicity of target malignant cells and adjuvanticity or propensity to enhance cross-presentation of cancer-specific antigens to CD8+ T-cells by dendritic cells via MHC-I. Antigenicity is determined, in part, by mutational burden, which depends on DNA mismatch repair among other factors. Microsatellite instable (MSI) CRC tumors, in general, having greater mutational burden than MSS disease. Recruitment of DCs to dying cancer cells is stimulated by the secretion of damage-associated molecular patterns (DAMPS) . A necesChemotherapeutic drugs may be classified as ICD-inducers, in part, based on vaccination assays in which mice injected with drug-treated tumor cells are protected against subsequent re-challenge with the same tumor. Using this and related criteria several chemotherapy drugs have been categorized as ICD-inducers including anthracyclines, cyclophosphamide, mitoxantrone and bortezomib . Recent 5-FU chemotherapy is immunosuppressive and a linear relationship between 5-FU plasma concentration and decreased leukocyte count was observed . A recenMultiple studies have investigated the potential of interferons (IFNs) to enhance the anti-tumor activity of 5-FU in colorectal cancer patients. Several clinical studies evaluated 5-FU in combination with the type I interferon IFN\u03b1. Clinical studies were initiated in response to pre-clinical studies that demonstrated synergy for the IFN\u03b1/5-FU combination towards CRC cells ,113. SynIndirect activation of interferon genes may also contribute to anti-tumor immunity thru activation of the STING (stimulator of interferon genes) pathway. STING activates interferon regulatory factor 3 (IRF3) and NF-\u03baB stimulating production of cytokines and type I interferons. STING is activated in response to sensing of foreign DNA by cyclic GMP-AMP synthase (cGAS). While the STING pathway primarily functions to sense foreign DNA and stimulate an immune response, STING also is activated in response to genomic DNA in the cytosol that could result from aberrant cell division or treatment-induced DNA damage. Radiation increases tumor production of IFN\u03b2 and antitumor efficacy of radiation depends on IFN signaling . FurtherThe mechanism by which STING activation enhances antitumor immunity involves DC activation and priming antitumor responses thru effector CD8+ T-cells. STING is predominantly expressed in DCs, macrophages, T-cells and epithelial cells . STING-dAn anti-tumor immune response including recognition of malignant cells by effector T-cells is essential for a durable response to any cancer treatment. Even in cases where activated T-cells that recognize tumor-specific antigens are present, the anti-tumor effect may be muted by upregulation of immune checkpoint molecules on the surface of malignant cells, effector T-cells and other cell populations. For some malignancies, including NSCLC and melanoma, the upregulation of PD-L1 by tumor cells and PD-1 and CTLA-4 by effector T-cells leads to inhibitory interactions that attenuate tumor-directed T-cell cytotoxicity and antibodies directed at inhibiting these checkpoint interactions have had a profound impact on outcomes. Immune checkpoint inhibitors (ICIs), such as Nivolumab and Pembrolizumab, directed against PD-1, Atezolizumab and Durvalumab directed against PD-L1 and Ipilimumab targeting CTLA-4 and PD-1 display strong efficacy in NSCLC . The actPre-clinical studies evaluating chemotherapy in combination with ICIs have shown that while favorable interactions can occur, effects depend on the tumor model, the type of chemotherapy and the ICI. Chemotherapy, including 5-FU , upregulTS inhibition is centrWe recently demonstrated the 2nd generation polymeric FP CF10 displayed significantly improved anti-tumor activity relative to 5-FU in an orthotopic colon cancer model consistent with the potential of polymeric FPs to provide a survival benefit for CRC patients. The realization of a clinical benefit for CF10 depends on multiple factors among which effects on anti-tumor immunity are of high importance. CF10 is expected to differ from 5-FU on its effects to anti-tumor immunity because mechanistically it is more DNA-directed while 5-FU exerts both RNA- and DNA-directed effects. The extent of the mechanistic difference between F10 and 5-FU was demonstrated by COMPARE analysis of data from the NCI60 cell line screen, which showed low correlation between these drugs consistent with dissimilar mechanisms . FurtherThe RNA-mediated effects of 5-FU cause GI-tract and hema5-FU and 5-FU-based regimens display a survival benefit in stage II, III and IV CRC. The effects of 5-FU to the anti-tumor immune response are an important consideration both in understanding efficacy achieved with 5-FU and in developing improved FP drugs and novel combinations that might improve survival, which remains dismal for CRC patients with late-stage disease. 5-FU exerts biological effects thru DNA-, RNA- and degradation metabolites . The ant"} +{"text": "CDT2 by CDT2 knockdown can be more potent in killing cSCC cells than targeting CRLs or CRL4s in general by RBX1 or DDB1 depletion. Suppression of the APC/C or forced APC/C activation by targeting its repressor EMI1 are both potential therapeutic approaches. We observed that cSCC cells can be selectively killed by small-molecule inhibitors of USP8 (DUBs-IN-3/compound 22c) and the NEDD8 E1 activating enzyme/CRLs (MLN4924/pevonedistat). A substantial proportion of cSCC cell lines are very highly MLN4924-sensitive. Pathways that respond to defects in proteostasis are involved in the anti-cSCC activity of p97 suppression. Targeting USP8 can reduce the expression of growth factor receptors that participate in cSCC development. EMI1 and CDT2 depletion can selectively cause DNA re-replication and DNA damage in cSCC cells.We performed a small interfering RNA screen to identify targets for cutaneous squamous cell carcinoma (cSCC) therapy in the ubiquitin/ubiquitin-like system. We provide evidence for selective anti-cSCC activity of knockdown of the E3 ubiquitin ligase MARCH4, the ATPase p97/VCP, the deubiquitinating enzyme USP8, the cullin-RING ligase (CRL) 4 substrate receptor CDT2/DTL, and components of the anaphase-promoting complex/cyclosome (APC/C). Specifically attenuating CRL4 There is a need for improved treatment for cutaneous squamous cell carcinoma (cSCC) in high-risk recessive dystrophic epidermolysis bullosa (RDEB) and immunocompromised patients, including transplant recipients and in the general population . This inThe ubiquitin/ubiquitin-like system plays a widespread role in regulating cellular pathways and processes. It contains multiple classes of proteins including E1 activating enzymes, E2 conjugating enzymes, E3 ligases, receptors for ubiquitin/ubiquitin-like proteins, ATPases, and proteases. Considerable work has been carried out to target this system for cancer therapy, and there are a growing number of small-molecule modulators.We have shown that proteasome and ubiquitin E1 inhibitors have therapeutic potential for cSCC . In thisA cell line derived from a primary RDEB cSCC (SCCRDEB4) was transfected with pools of four siRNAs targeting 1,186 ubiquitin/ubiquitin-like pathway-linked genes . Cell viTo further investigate the therapeutic potential of suppression of genes for which multiple individual siRNAs had a phenotype, we determined the effects of the siRNAs on viability and death in normal skin cells (fibroblasts and keratinocytes) and cell lines derived from metastasis in an RDEB (SCCRDEBMet) and a transplant patient (SCCTMet). A cytotoxic siRNA was used in each experiment as a control for transfection efficiency. In addition, we determined the extent of target protein knockdown in normal and cSCC cells. The effects of small-molecule inhibitors on viability and death in normal skin cells and cSCC cell lines were also evaluated.MARCH4 is a little-studied transmembrane E3 ubiquitin ligase that is localized to the Golgi apparatus . EctopicMARCH4 siRNAs had little effect on death in normal skin cells, whereas two MARCH4 siRNAs caused a reduction in viability and increased death in cSCC cell lines . Ubiquitination of receptors promotes ESCRT-mediated lysosomal trafficking. USP8/UBPY-mediated deubiquitination of some ESCRT-associated receptors can facilitate their recycling, and USP8 also regulates endocytic sorting by stabilizing ESCRT-0 proteins HGS, STAM, and STAM2 .USP8 siRNAs reduced viability and increased death in cSCC lines but had little effect in normal skin cells (USP8(A) depleted USP8 to the greatest extent, and this was associated with reduced expression of growth factor receptors MET, EGFR, and ERBB2, along with HGS and STAM2. siRNAs USP8(B) and (C) reduced expression of MET and STAM2. This is consistent with a role of USP8 in protecting these proteins from degradation.in cells a. The anin cells b. siRNA Small-molecule USP8 inhibitors have been identified including DUBs-IN-3/compound 22c . DUBs-INCDC20 drives progression through mitosis was the least effective in depleting CDC20 and had no effect on viability or death. Three CDH1 siRNAs depleted CDH1 without causing a high level of death in cSCC cells were used in this study to minimize off-target effects. Reverse transfection with synthetic siRNA duplexes (10 nM) was performed using Invitrogen Lipofectamine RNAiMAX (Thermo Fisher Scientific). The library used for the primary screen containing pools of four siRNAs per gene was detailed previously . AdditioInhibitors used in this work were DBeQ and NMS-873 , DUBs-IN-3/compound 22c , and MLN4924 .For the primary screen, viability was measured by ATPase assay 96 hours after siRNA transfection . Where iTo assess re-replication (>G2/M DNA content), fixed DAPI-stained samples were analyzed for DNA content using a NucleoCounter NC-3000 cell counter according to the manufacturer\u2019s instructions.Primary antibodies are listed in MARCH4 probe/primer set Hs00863129_m1 (Thermo Fisher Scientific). TBP was used for normalization.RNA was extracted using RNeasy columns and real-time PCR were performed as previously described using MAThe authors confirm that the data supporting the findings of this study are available within the article and its https://orcid.org/0000-0003-3020-4701Angela McHugh: https://orcid.org/0000-0002-9406-267XKenneth Fernandes: https://orcid.org/0000-0001-5891-7573Nerime Chinner: https://orcid.org/0000-0002-1066-7111Adel F.M. Ibrahim: https://orcid.org/0000-0001-7486-9284Amit K. Garg: https://orcid.org/0000-0002-4656-2653Garry Boag: https://orcid.org/0000-0003-4851-6658Lydia A. Hepburn: https://orcid.org/0000-0002-3292-4836Charlotte M. Proby: https://orcid.org/0000-0001-8536-6439Irene M. Leigh: https://orcid.org/0000-0001-8057-0641Mark K. Saville: The authors state no conflict of interest."} +{"text": "Severe acute respiratory syndrome coronavirus 2 readily transmits between domestic cats. We found that domestic cats that recover from an initial infection might be protected from reinfection. However, we found long-term persistence of inflammation and other lung lesions after infection, despite a lack of clinical symptoms and limited viral replication in the lungs. Previous studies have demonstrated the transmissibility of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) by direct or indirect contact between domestic cats Figure 5Because SARS-CoV-2 did not cause acute lethal respiratory disease in the cats in our study, cats are a compelling animal model for studying the long-term effects of nonfatal infections. Cats were infected with SARS-CoV-2 and euthanized at postinfection day 28 Figure 8t-test) panel B.In conclusion, SARS-CoV-2 replicated effectively in the upper respiratory tract in cats, and infectious virus was cleared from the lungs within 6 days of infection; however, histopathologic examination demonstrated chronic lung sequelae in cats even a month after viral clearance. After initial infection with SARS-CoV-2, cats were protected from reinfection, with no virus replication in respiratory organs and no additional lung damage.Additional information about protective immunity and persistent lung sequelae in domestic cats after SARS-CoV-2 infection."} +{"text": "Agrimonia pilosa-Ledeb leaves on non-lipid oxidative damage. The antioxidative activity of methanolic (MeOH) extract of the Agrimonia pilosa-Ledeb leaves on non-lipid oxidation, including liposome oxidation, deoxyribose oxidation, protein oxidation, chelating activity against metal ions, scavenging activity against hydrogen peroxide, scavenging activity against hydroxyl radical and 2\u2019-deoxyguanosine (2\u2019-dG) oxidation were investigated. The MeOH extract of the Agrimonia pilosa-Ledeb leaves exhibited high antioxidative activity in the liposome model system. Deoxyribose peroxidation was inhibited by the MeOH extract of the Agrimonia pilosa-Ledeb leaves and MeOH extract of the Agrimonia pilosa-Ledeb leaves provided remarkable protection against damage to deoxyribose. Protective effect of MeOH extracts of the Agrimonia pilosa-Ledeb leaves on protein damage was observed at 600 \u00b5g level (82.05%). The MeOH extracts of the Agrimonia pilosa-Ledeb leaves at 300 \u00b5g revealed metal binding ability (32.64%) for hydrogen peroxide. Furthermore, the oxidation of 2\u2019-deoxyguanosine (2\u2019-dG) to 8-hydroxy-2-deoxyguanosine (8-OH-2\u2019dG) was inhibited by MeOH extracts of the Agrimonia pilosa-Ledeb leaves and scavenging activity for hydroxyl radical exhibited a remarkable effect. From the results in the present study on biological model systems, we concluded that MeOH extract of the Agrimonia pilosa-Ledeb leaves was effective in the protection of non-lipids against various oxidative model systems.Present study was conducted to evaluate the antioxidative activity of the"} +{"text": "Potyvirus) infection. This motif is a putative interaction site for WD40 domain-containing proteins, including VARICOSE (VCS). We abolished the interaction site in HCPro by replacing glutamic acid (E) and arginine (R) with alanines (A) to generate HCProWD. These mutations partially eliminated HCPro-VCS co-localization in cells. We have earlier described potyvirus-induced RNA granules (PGs) in which HCPro and VCS co-localize and proposed that they have a role in RNA silencing suppression. We now demonstrate that the ability of HCProWD to induce PGs, introduce VCS into PGs, and suppress RNA silencing was impaired. Accordingly, PVA carrying HCProWD (PVAWD) infected Nicotiana benthamiana less efficiently than wild-type PVA (PVAWT) and HCProWD complemented the lack of HCPro in PVA gene expression only partially. HCPro was purified from PVA-infected leaves as part of high molecular weight (HMW) ribonucleoprotein (RNP) complexes. These complexes were more stable when associated with wild-type HCPro than with HCProWD. Moreover, VCS and two viral components of the HMW-complexes, viral protein genome-linked and cylindrical inclusion protein were specifically decreased in HCProWD-containing HMW-complexes. A VPg-mediated boost in translation of replication-deficient PVA (PVA\u0394GDD) was observed only if viral RNA expressed wild-type HCPro. The role of VCS-VPg-HCPro coordination in PVA translation was further supported by results from VCS silencing and overexpression experiments and by significantly elevated PVA-derived Renilla luciferase vs PVA RNA ratio upon VPg-VCS co-expression. Finally, we found that PVAWD was unable to form virus particles or to spread systemically in the infected plant. We highlight the role of HCPro-VCS containing multiprotein assemblies associated with PVA RNA in protecting it from degradation, ensuring efficient translation, formation of stable virions and establishment of systemic infection.In this study, we investigated the significance of a conserved five-amino acid motif \u2018AELPR\u2019 in the C-terminal region of helper component\u2013proteinase (HCPro) for potato virus A and the host protein VARICOSE (VCS) are linked in a manner that is important for suppression of RNA silencing, formation of potyvirus-induced RNA granules, translation of viral proteins, stability of virions, and development of systemic potato virus A (PVA) infection. The results suggest that HCPro and VCS belong to the core components of large RNP complexes regulating PVA infection. We suggest that these complexes protect viral RNAs in the cytoplasm after release from the replication complex and direct them to translation and intact to the viral particles. The host-virus relationship is a quintessential example of the evolutionary \u2018arms race\u2019. The most intriguing aspect of this molecular struggle is that while the hosts have large genomes with a huge range of defences, viral genomes in comparison are minuscule. Yet they successfully manage to hijack host cellular machineries to serve their propagation. In order to cope with the constant selection pressure and to include all the essential genetic information within a tiny genome, viruses encode proteins which are multifunctional in nature.For example potyviral helper component-proteinase (HCPro) is involved in interactions with multiple host proteins. It carries out many essential functions during the viral infection cycle. Albeit best known for its role as an RNA silencing suppressor, HCPro\u2019s primary functions include polyprotein processing ,2 and viin planta and negatively affected the assembly and stability of HCPro-VCS-associated multiprotein complexes. Furthermore, the virus carrying this mutation demonstrated major defects in multiple stages of infection. Cumulatively he results advocate the biological significance of the HCPro-VCS interaction in the PVA infection cycle. In addition, this study illuminates the formation and function of large RNA-protein assemblies in the regulation of infection.Here we show that the C-terminal region of HCPro contains a highly conserved five amino acid motif. This motif has the potential to interact with WD40 domain proteins, a large family of eukaryotic proteins ,20. The Potyvirus (Potyvirus) [http://elm.eu.org/), there are three motifs overlapping with \u2018AELPR\u2019 between amino acids 398\u2013406 \u2018TSAAEL\u2019, \u2018SAAELPRI\u2019 and \u2018ELPRI\u2019, which have the potential to act as a short linear motif capable of interacting with WD40 domain-containing proteins. In the stretch 5 in the alignment of https://cluspro.bu.edu/) between TuMV HCPro and VCS suggested the presence of the \u2018AELPR\u2019 motif at the site of interaction Fig 1B)Potyviruseraction . BecauseWD on PVA infection we measured viral expression levels from local and systemic leaves. Expression of the viral polyprotein was assessed from the activity levels of the RLUC reporter gene incorporated between the NIb and CP cistrons [WD accumulated with approximately five-fold lower efficiency than PVAWT (WD accumulation level was lower than that of wild-type HCPro (HCProWT) in the blot, which was in line with the difference in the polyprotein expression levels of PVAWD and PVAWT. The presence of monomeric HCProWD derived from PVAWD infection and eIF(iso)4E binding-deficient HCPro (HCPro4EBM) [\u0394HCPro and complemented the infection with GUS (OD600 = 1), HCProSDM (OD600 = 1), HCPro4EBM (OD600 = 1), HCProWD (OD600 = 0.3), or HCProWT (OD600 = 0.3). Neither HCProSDM nor HCPro4EBM could elevate RLUC expression from PVA\u0394HCPro showing no sign of complementation. HCProWD on the other hand promoted PVA\u0394HCPro gene expression significantly . For thificantly and HCPrnd PVAWT . Infiltrnd PVAWT . NotablyWD and PVAWT in systemically infected leaves. There was more than a 1000-fold reduction in the RLUC activity in PVAWD compared to PVAWT did not increase granule count either for HCProWD or HCProWT. Rather, a reduction in the granule count was noticed in the case of HCProWT, indicating that the potency of the PG-inducing property of HCPro does not necessarily correlate with the increased protein amount , and we suggested PGs have a role in suppression of RNA silencing . Since H for PGs , all at HCProWT , which sn amount . No gran 35S-GUS .WD mutation changed HCPro\u2019s ability to form these HMW complexes. For this, we adopted a similar strategy reported in Ivanov et al. [N. benthamiana plants were infected with PVAWD-Strep-RFP or PVAWT-Strep-RFP at OD600 = 0.1. Since PVAWD gene expression in systemic leaves was low (WD/WT-Strep-RFP was performed following the method described in [WT interactors including S\u2013adenosyl-L\u2013methionine synthase (SAMS), VPg and CI [WD samples compared to HCProWT document different variants of VCS. We used BLAST searches to identify three genes in the N. benthamiana genome with circa 50% identity to the Arabidopsis thaliana VARICOSE (Q9LTT8): VCS-A (Niben101Scf00654g02003.1), VCS-B (Niben101Scf39216g00007.1) and VCS-C (Niben101Scf21107g00015.1). We generated expression constructs of these three N. benthamiana genes along with their tagged versions to induce PGs, YFP-tagged VCS , and another PG marker P0CFP (OD600 = 0.1). Their localization was monitored via confocal microscopy at 3 dpi. The results reveal that HCProWT was able to sequester VCS into the PGs. Co-localization of the PG marker P0CFP [bona fide PGs. Unless the threshold effect prevented the detection of an overlap, HCProWD-Strep-RFP -induced cytoplasmic foci did not co-localize with either VCSYFP or P0CFP . In contrast to the HCProWT-Strep-RFP, top-most band containing VCS (marked with an asterisk) was missing in HCProWD-Strep-RFP samples. Interestingly, the top-most band was also absent in the silver stained gel and drastically reduced in the \u03b1-HCPro immunoblot in the HCProWD-Strep-RFP pull-down samples. A comparison between the input and the eluted samples indicated specific enrichment of HMW complexes upon purification complexes. To probe into this phenomenon further, we ran the RNase A degraded complexes longer in the gel and optimized the loading quantity to get a better look on the degraded complexes . PVA\u0394HCPro, PVAWD and PVA WT expression levels were then measured both in the control and VCS-silenced background. VCS silencing was validated via reverse transcription-polymerase chain reaction (RT-PCR) and did not change upon VCS silencing . Followiof PVAWT and apprgulation , showinggulation . In contilencing . Thus, t\u0394GDD-\u0394HCPro, PVA\u0394GDD-HCProWD and PVA\u0394GDD-HCProWT. The replication-deficient PVAs were used in this experiment to narrow down the effects of VCS, VPg and HCPro to aspects pertaining to PVA translation. More vigorous protein production from an unaltered RNA concentration, which affects the ratio of the protein and its mRNA, has been regarded as an indicator of release from translational repression [\u0394GDD-\u0394HCPro, PVA\u0394GDD-HCProWD and PVA\u0394GDD-HCProWT upon VPg and VCS overexpression. In this experiment, all the constructs were co-infiltrated and samples were collected at 3 dpi. The absolute values for RLUC activities and RNA levels were quantitated and are presented in We proposed in our previous paper that thepression . Therefo\u0394GDD-HCProWD and PVA\u0394GDD-HCProWT are equally enhanced by VPg (\u0394GDD-\u0394HCPro with VPg together with either GUS (OD600 = 1), HCProSDM (OD600 = 1), HCPro4EBM (OD600 = 1), HCProWD (OD600 = 0.3), or HCProWT (OD600 = 0.3). VPg did not enhance translation in the presence of HCProSDM and HCPro4EBM. However, in the presence of both HCProWD and HCProWT, VPg caused a similar extent of RLUC enhancement . For this, we sampled locally infected leaves at 3 dpi. In order to obtain equivalent infection PVAWD was infiltrated in a higher amount (OD600 = 1) compared to PVAWT (OD600 = 0.1). Immunocapture (IC)-qRT-PCR was performed to measure the abundance of vRNA assembled into VLPs or virions. Although RLUC activities were broadly similar, a drastic reduction in the amount of particles was noticed in PVAWD-infected plants as compared to PVAWT-infected plants -induced pinwheel structures . In processing bodies VCS acts as an enhancer of mRNA decapping and associates with RNP complexes containing the decapping proteins DCP-1 and DCP-2 [WD40-domain proteins are known for their regulatory roles. They are involved for example in cell division, cell fate determination, gene transcription and mRNA modification . Accordind DCP-2 ,40. In and DCP-2 . Similarnd DCP-2 ,41. Furtnd DCP-2 . Along tnd DCP-2 .WD\u2019s protease function was unaffected nor HCPSDM/4EBM producedshown in , . HCProWomplexes . Althougnfection . AlthougWD forms approximately five-fold less PGs compared to HCProWT. On similar lines PVAWD gene expression is approximately five-fold lower than PVAWT gene expression. Second, HCProWD is deficient in hairpin-mediated RNA silencing suppression P3-N-terminus fragment (P1-HCPro-P3) in the pGEM-T-Easy vector (Promega) using a two-step site directed mutagenesis described by Edelheit et al. [CGC TTC CAG CAA TTT TGG TTG-3\u2019; reverse primer- 5\u2019-CAA CCA AAA TTG CTG GAA GCG CGG CGG CAC TTG-3\u2019 (changed nucleotides are marked in bold). The mutated fragments (P1-HCProWD-P3 or P1-HCProWD-Strep-RFP-P3) were then used to replace the P1-HCPro-P3 fragment in PVA or PVAWT-Strep-RFP icDNAs in the pUC18 vector using SexAI and NruI restriction sites. A non-replicating variant PVA\u0394GDD-HCProWD was prepared by replacing the wild-type NIb gene with the mutated \u0394GDD version as described in Eskelin et al. [Agrobacterium mediated transformation the mutated PVA icDNAs were transferred into the binary vector pRD400 using SalI and KpnI restriction sites.Mutation in the WD40 domain-interacting motif of HCPro was introduced in the wild type P1-C-terminus-HCPro . The mutated 35S-HCProWD-nos fragment was cloned into pRD400 using HindIII restriction sites. Expression constructs for TwinStrep-tagged RFP fusions of HCProWT and HCProWD were generated from P1-HCProWT-Strep-RFP-P3 or P1-HCProWD-Strep-RFP-P3 fragments by PCR using the following primers: forward- 5\u2019-TCC GCT CGA GAT GAA ATG GTC TCA TCC ACA A-3\u2019, reverse- 5\u2019-CGC GGA TCC TCA CGA CTC TTT TTC GAA CTG-3\u2019. The primers were designed to add an Xho1 restriction site at the 5\u2019 end and both a stop codon and a BamH1 restriction site to the 3\u2019 end of the amplified fragment. XhoI-BamHI digested PCR products were ligated into similarly double-digested pHTT690H to generate 35S-TwinStrep-RFP-HCPro-nos and 35S-TwinStrep-RFP-HCProWD-nos. After sequence verification the expression cassettes were transferred into the pRD400 binary vector using HindIII restriction.For HCPro expression constructs, the mutation was introduced similarly in an interim vector containing 35S promoter-HCPro-Arabidopsis thaliana VARICOSE were identified with a BLAST search of the N. benthamiana genome v1.0.1 predicted cDNAs (https://solgenomics.net/). Three N. benthamiana VCS-like genes: Niben101Scf00654g02003.1 (VCS-A), Niben101Scf39216g00007.1 (VCS-B) and VCSC Niben101Scf21107g00015.1 (VCS-C) were Gateway-cloned into expression vectors. VCS-A and VCS-C were amplified with gateway-compatible primers from a N. benthamiana cDNA library generated from total RNA using Superscript III (Invitrogen). VCS-B was synthesized and cloned into the pUC57 vector by BaseClear, Netherlands. The forward primer for VCS-A and -C was 5\u2019-GGG GAC AAG TTT GTA CAA AAA AGC AGG CTT CAT GGC TTT CTT TGC AGA G-3\u00b4 and the reverse primers were- 5\u2019-GGG GAC CAC TTT GTA CAA GAA AGC TGG GTT TCA TTT ACT CAT CAA CAT CGA-3\u2019 and 5\u2019-GGG GAC CAC TTT GTA CAA GAA AGC TGG GTT TCA TTT ACT ACA GGT CAT CAG-3\u2019, respectively. VCS-B was amplified with the forward primer 5\u2019-GGG GAC AAG TTT GTA CAA AAA AGC AGG CTT CAT GGC TTC TTC TCC TGG C-3\u2019 and the reverse primer 5\u2019-GGG GAC CAC TTT GTA CAA GAA AGC TGG GTT TCA TTT ACT CAT CAA CAT CGA AT-3\u2019. The PCR products were cloned into pDONR-Zeo via a BP-reaction with a BP ClonaseII enzyme mix (Invitrogen). Positive entry clones were verified by sequencing and LR-cloned into the KpnI-linearized destination vector pMDC32 to generate native expression constructs. XhoI-linearised pGWB42 [Genes homologous to the d pGWB42 was usedPreparation of the infiltration mix as well as agroinfiltration was carried out following Ivanov et al. .oC for later analysis.Samples at designated dpi were collected using a cork borer of internal diameter 5-mm. Leaf discs were either directly used for luciferase assays using a Dual luciferase kit (Promega) as in Eskelin et al. or storeYFP fluorescence intensity was quantified directly from 5-mm leaf discs using a Tecan Infinite M200 monochromator-based pate reader following De et al. . Ex/Em wWD-Strep-RFP, HCProWT-Strep-RFP and VCSYFP in N. benthamiana epidermal cells was examined with confocal laser scanning microscopy using a Leica TCS SP5II instrument (Leica Microsystems). For imaging, leaf samples were mounted upside down on an objective slide with a drop of water and placed under a cover glass. All images were acquired with a 63x water immersion objective and in sequential scanning mode in order to avoid fluorophore bleed-through. YFP was excited with a 488 nm argon laser and RFP with a DPSS 561 nm laser. Emissions were detected at 525\u2013555 nm and at 570\u2013630 nm for YFP and RFP, respectively. CFP was excited with an argon laser at 458 nm and emission was recorded at 470\u2013500 nm. Line-averaging was adjusted to 4 to improve image quality. Co-localization analysis of VCSYFP with HCProWT-Strep-RFP and HCProWD-Strep-RFP was performed with the Fiji (ImageJ) image analysis software package using the co-localization threshold-function. HCPro-containing granules were selected as ROIs for the co-localization and results are given as % intensity of VCSYFP co-localizing with HCProWT-Strep-RFP or HCProWD-Strep-RFP.The expression and localization of HCProPG visualization and quantification was carried out using epifluorescence microscopy following the method described in Hafr\u00e9n et al. .WT-Strep-RFP or PVAWD-Strep-RFP infiltrated at OD600 = 0.1. GUS-infiltrated plants were used as negative controls. Leaves were harvested at 3 dpi and immediately frozen in liquid nitrogen. Subsequently, Strep-tag mediated purification of HCProWT-Strep-RFP or HCProWD-Strep-RFP was carried out following Ivanov et al. [oC to remove insoluble debris. Approximately 2 mL of cleared lysate was mixed with 50 \u03bcL resin suspension and incubated overnight with gentle rotation at 4 oC. The rest of the protocol is same as Ivanov et al. [HCPro-associated protein complexes were isolated from leaves infected with PVAv et al. with minv et al. . Final ev et al. .oC and thereafter subjected to standard SDS-PAGE followed by silver staining.Purified products obtained after elution from Strep-Tactin resin were subjected to DNase , Proteinase K and RNase A (Fermentas) treatment. Each reaction mix comprised 3 \u03bcL eluate and 1 \u03bcL DNase (1u/\u03bcL), Proteinase K (20 \u03bcg/\u03bcL) or RNase A (10 \u03bcg/\u03bcL). Final reaction volume was made up to 10 \u03bcL with recommended buffer / water. Reaction mixes were incubated for 1 h at 37 N. benthamiana VCS proteins used in this study was produced in rabbit (Biomatik) and used at 1 \u03bcg/mL dilution. The antibody recognizes peptide sequence \u2018TEGPDEEDKPQITGK\u2019 located near the N-terminal region of all three forms of VCS gene was used as the reference gene. PP2A was amplified with the following primers: forward- 5\u2019-GAC CCT GAT GTT GAT GTT CG-3\u2019, reverse- 5\u2019-GAG GGA TTT GAA GAG AGA TTT C-3\u2019.Omega E.Z.N.A. Plant RNA-purification kit (Product No. R6827-02) along with RNase-free DNase (Product No. E1091-02) was used to purify RNA from 100 mg of leaf tissues. Standard protocol for ic-qPVA particles were visualized with a Jeol JEM-1400 transmission electron microscope . 100 mg leaf tissue was ground in 100 \u03bcL 0.06 M phosphate buffer and incubated on ice for 1 h to let the cell debris settle. Carbon-coated EM-grids were incubated with the anti-CP antibody (1:100 dilution) for 1 h at room temperature. Excess antibody was washed with phosphate buffer. Subsequently, the antibody-coated grids were incubated for 5 min with supernatant collected from the leaf extracts after settling down of the debris. Grids were further washed with 20 drops of phosphate buffer and immediately stained with 2% Uranyl acetate for 15 sec. Excess stain was drained from the grids using filter paper and dried grids were used for visualization of the particles. EM images of the infected leaf tissues were visualized following L\u00f5hmus et al. .S1 Fighttp://www.wdspdb.com/wdsp/). (C) AT3G13300.1 and (D) AT3G13300.3. (E) Modelling the molecular docking of HCPro (from (B)) and VCS (from (C)) was carried out using ClusPro online server (https://cluspro.bu.edu/). Similarly, (F) presents the predicted interaction model between (B) and (D). The docking model in which the AELPR motif interacts with VCS was among the top predictions.Identification of motifs within the HCPro sequence with potential to interact with WD domain proteins (A) 119 potyviral HCPro amino acid sequences were aligned and searched for interaction motifs for WD domain proteins. Six highly conserved, putative WD domain -interacting motifs were found. These sequences and the percentage of their conservation among potyviruses are indicated in (A). \u2018C\u2019 and \u2018H\u2019 residues that are important for cysteine protease activity of HCPro are marked with arrow in the alignment shown in (A). Variation in the amino acids within each motif is presented in the table below. Motif no. 5, the AELPR sequence used in this study, is the most conserved one. (B) Localization of the motifs 3-5 within the crystal structure of the C-terminal domain of TuMV HCPro (PDB id-3RNV). Motifs 1 and 2 are outside the resolved structure and motif 6 is not conserved in TuMV HCPro. Hence, they could not be shown. Motifs 3 and 4 were merged and are highlighted with yellow whereas motif 5 is indicated with red. Ribbon diagrams of VCS proteins from Arabidopsis thaliana, obtained from WDSP database ((TIF)Click here for additional data file.S2 FigWD-Strep-RFP / HCProWT-Strep-RFP constructs were Agrobacterium infiltrated at different ODs . The RFP fluorescence level at Ex/Em = 555/584 nm was measured from the intact leaf discs with a microplate reader. Statistically significant differences between the samples are denoted by asterisks . In spite of the statistically significant differences in the HCProWD-Strep-RFP / HCProWT-Strep-RFP accumulation levels, they did not differ drastically.HCPro(TIF)Click here for additional data file.S3 FigYFP overexpression alone (ref. Agrobacterium cell count in the absence of HCPro.Control images showing the absence of PG formation by P0one ref. . GUS is (TIF)Click here for additional data file.S4 FigWT-Strep-RFP (A-C) / HCProWD-Strep-RFP (G-I) and VCSYFP and HCProWT-Strep-RFP (D-F) / HCProWD-Strep-RFP (J-L) and P0CFP to support the result presented in Presented are the pairwise overlays between HCPro(TIF)Click here for additional data file.S5 Fign = 6RFP and YFP fluorescence levels in the intact leaf discs measured at Ex/Em 555/584 nm and 500/530 nm, respectively, in a microplate reader. Statistically significant differences between the samples are denoted by an asterisk (**P < 0.01); (TIF)Click here for additional data file.S6 FigYFP co-localizing with HCProWD-Strep-RFP / HCProWT-Strep-RFP. Statistical significance was calculated from images taken from three independent sets of biological replicates .HCPro-containing foci (= PGs) were selected as region of interests (ROIs) for the co-localization and the results are given in terms of % (by intensity) of VCS(TIF)Click here for additional data file.S7 FigP < 0.01; N.S. stands for non-significant; n = 48).RFP fluorescence was measured at Ex/Em = 555/584 nm while YFP fluorescence was measured at Ex/Em = 500/530 nm from the intact leaf discs using a microplate reader. Statistically significant differences between the samples are denoted by asterisks (**(TIF)Click here for additional data file.S8 FigYFP. Significance of the differences between the samples is denoted by asterisk . (B-D) HCProWD-Strep-RFP aggregates, P0YFP and their overlay within a single cell under a 100X water immersion objective. (E-G) Reference PGs with HCProWT-Strep-RFP and P0YFP shows co-localization. Samples were visualized at 3 dpi. Experimental details are the same as in YFP were visualized at 3 dpi with an epifluorescence microscope using RFP and YFP filters respectively.(A) Percentage of HCPro aggregates co-localizing with P0(TIF)Click here for additional data file.S9 FigWD-Strep-RFP purified fraction (marked with arrow). Monomeric HCPro was also detected by the anti-HCPro antibody (marked with asterisk). Neither HCPro nor VCS could be detected from the input samples in the western blots. Therefore their presence in the inputs was studied by loading 40-times higher total protein amounts than in S9A Fig. (B) Both HCPro and VCS were detected faintly in the respective input blots.Both inputs and eluates containing equivalent amount of total protein , were loaded as indicated. Left panel shows the silver-stained gel of the inputs and eluates while the right panel shows the western blots of the same samples as investigated with anti-HCPro and anti-VCS antibodies (A). While the anti-HCPro western blot indicates strong enrichment of HCPro-containing HMW complexes, the anti-VCS western blot confirms the absence of the VCS from the uppermost HMW band from PVA(TIF)Click here for additional data file.S10 FigValidation of VCS silencing via semi-quantitative RT-PCR. Panel (A) corresponds to the sample sets shown in (TIF)Click here for additional data file.S11 Fig\u0394GDD-\u0394HCPro, PVA\u0394GDD-HCProWD and PVA\u0394GDD-HCProWT during VPg, VCS and VPg + VCS overexpression (B) Amount of RNA accumulation detected from non-replicating variants of PVA- PVA\u0394GDD-\u0394HCPro, PVA\u0394GDD-HCProWD and PVA\u0394GDD-HCProWT during VPg, VCS and VPg + VCS overexpression Validation of VCS overexpression during the experiments discussed in P < 0.05). Significance of the differences between the compared samples is denoted by asterisk .(A) Amount of RLUC activity detected from non-replicating variants of PVA, PVA- PVA(TIF)Click here for additional data file.S12 FigAgrobacterium carrying different HCPro variants were infiltrated as follows: silencing-deficient HCProSDM and eIF4E binding-deficient HCPro4EBM mutants at OD600 = 1, HCProWD and HCProWT at OD600 = 0.3. GUS was used as the control, as well as to balance Agrobacterium counts between the sets. PVA\u0394GDD-\u0394HCPro was infiltrated at OD600 = 0.05 and VPg infiltrated at OD600 = 0.3. Samples for RLUC quantitation were collected at 3 dpi. The number of plants per experiment was 6. Different letters above the bars indicate statistically significant differences (student's t-test P < 0.05).(TIF)Click here for additional data file.S13 FigP < 0.05, **P < 0.01, ***P < 0.001). For validation of VCS silencing in (TIF)Click here for additional data file.S14 Fig\u0394HCPro, PVAWD and PVAWT upon VCS-silencing (B) Amount of RNA accumulation detected from replicating variants of PVA- PVA\u0394HCPro, PVAWD and PVAWT upon VCS-silencing (C) Amount of RLUC activity detected from replicating variants of PVA- PVA\u0394HCPro, PVAWD and PVAWT upon VCS-silencing and VPg overexpression (D) Amount of RNA accumulation detected from replicating variants of PVA- PVA\u0394HCPro, PVAWD and PVAWT upon VCS-silencing and VPg overexpression (E) Amount of RLUC activity detected from replicating variants of PVA- PVA\u0394HCPro, PVAWD and PVAWT upon VCS overexpression (F) Amount of RNA accumulation detected from replicating variants of PVA- PVA\u0394HCPro, PVAWD and PVAWT upon VCS overexpression (G) Amount of RLUC activity detected from replicating variants of PVA- PVA\u0394HCPro, PVAWD and PVAWT upon VCS and VPg overexpression (H) Amount of RNA accumulation detected from replicating variants of PVA- PVA\u0394HCPro, PVAWD and PVAWT upon VCS and VPg overexpression. Significance of the differences between the compared samples is denoted by asterisk .(A) Amount of RLUC activity detected from replicating variants of PVA- PVA(TIF)Click here for additional data file.S15 FigWD-Strep-RFP (OD600 = 0.3) and PVAWT-Strep-RFP (OD600 = 0.1) infected N. benthamiana plants (sampled at 4 dpi), via a Assymetric Field Flow Fractionation (AF4) protocol previously described in Eskelin et al. (2019). (A) Upper panel presents AF4 elution profiles of representative PVAWD-Strep-RFP infected samples while the lower panel presents those of representative PVAWT-Strep-RFP infected samples. Polysome fraction from the eluate (between 15\u201370 min) were pooled together and concentrated using a 10kDa MW cutoff centrifugation filter (Amicon). (B) Concentrated eluates were subjected to SDS-PAGE and the gels were silver stained (upper panel) and subjected to western blotting with anti-HCPro antibody (lower panel). Silver stained gel images for concentrated eluates show clear enrichment of ribosomal proteins (marked in the image between 55-25 kDa). The western blot revealed signals corresponding to both HCProWD and HCProWT both in the monomeric (marked by \u2018*\u2019) and HMW region (marked by \u2018**\u2019). Interestingly, band for HCProWD-Strep-RFP at the HMW region was visibly lower than that of the HCProWT-Strep-RFP.Polysomes were purified from PVA(TIF)Click here for additional data file.S16 FigWD compared to the control virus TuMVWT. Student\u2019s t-test was used to calculate satistical significance . (A) Abundance of TuMV CP in systemically infected N. benthamiana at 14 dpi. Plants were infiltrated by TuMVWT and TuMVWD (OD600 = 0.5) and samples from systemic leaves were analysed by anti-TuMV CP ELISA (Agdia). (B) Relative TuMV particle abundance in systemically infected N. benthamiana leaves at 12 dpi (OD600 = 0.5). Particle abundance was measured by IC-RT-PCR.Systemic infection and particle abundance are reduced in TuMV(TIF)Click here for additional data file.S17 FigYFP, VCS-BYFP and VCS-CYFP were overexpressed independently in N. benthamiana (infiltrated at OD600 0.5). Samples were collected at 3 dpi followed by affinity purification by GFP trap (ChromoTek). The eluates were analyzed by SDS-PAGE and anti-VCS western blot (antibody dilution 1 \u03bcg/ml).\u03b1-VCS western blot showing that all three forms of VCS are recognized by the anti-VCS antibody. For this experiment VCS-A(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.S1 Reference(DOCX)Click here for additional data file."} +{"text": "This diagnostic study compares unsupervised home self-collected midnasal swabs vs clinician-collected nasopharyngeal swabs for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Increased diagnostics are urgently needed to contain the spread of coronavirus disease 2019 (COVID-19). Home self-collected swabs may increase testing access while minimizing exposure risk to health care workers and depletion of personal protective equipment, allowing for early community detection of COVID-19. A comparison of unsupervised home self-collected swabs with clinician-collected nasopharyngeal swabs for COVID-19 diagnosis has not been well described.STROBE) reporting guideline. Participants provided electronic informed consent. Study participants were recruited from symptomatic outpatients testing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)\u2013positive and symptomatic health care workers presenting to drive-through clinics enrolled at drive-through clinics, and 27 (15%) enrolled after a positive SARS-CoV-2 test. Among the 185 participants, 41 (22.2%) yielded SARS-CoV-2 positive test results via clinician-collected nasopharyngeal swab, home self-collected midnasal swab, or both. One hundred fifty-eight participants (85%) were health care workers, of whom 14 (9%) tested positive. Among participants with COVID-19, common symptoms included myalgia (33 participants [80.5%]), cough (28 participants [68.3%]), and fever (26 participants [63.4%]). Compared with clinician swabs, sensitivity and specificity of home swabs was 80.0% and 97.9% , respectively . Cohen \u03baP\u2009<\u2009.001) (P\u2009=\u2009.32). The median (interquartile range) Ct of the clinician swab was lower for true positives vs false negatives . Four of 5 false-negative swabs had Ct greater than or equal to 33. In a sensitivity analysis of all swabs with Ct less than or equal to 32, sensitivity of home swabs was 95%.Cycle thresholds of home swabs were positively correlated with clinician swabs . Time frUnsupervised home midnasal swab collection was comparable to clinician-collected nasopharyngeal swab collection for detection of SARS-CoV-2 in symptomatic patients, particularly those with higher viral loads. During this rapidly evolving pandemic, we enrolled 185 individuals presenting for SARS-CoV-2 testing, including 41 with positive test results. We used novel home-based swab self-collection and rapid delivery services, thus avoiding participant contact with the health care system.1 We observed false-negative results in samples with low initial viral loads.4 A home-based strategy should be targeted toward individuals early in illness, when risk of transmission is highest and care seeking less likely.Unsupervised home self-swab collection presents several advantages, including accessibility outside of the health care system and minimizing personal protective equipment use. This approach is safe and scalable in the pandemic setting, permitting widespread testing of symptomatic participants early in illness and the potential for prompt self-isolation and contract tracing. The sensitivity of home self-collection in this study was lower than previously described.5 Second, home self-collection often occurred 1 day after clinician collection, likely leading to samples with lower viral load. Third, many participants were health care workers, potentially limiting generalizability to the general population. Fourth, clinician-collected swabs are an imperfect criterion standard that may introduce bias.Limitations of the study include shipping at ambient temperature, which may have led to sample degradation. However, we have demonstrated stability of respiratory viruses at ambient temperatures up to 9 days.As societies reopen, expansion of testing is critical for preventing a global resurgence in COVID-19. Home swab collection has the potential to play a pivotal role in increasing testing access across the broader population."} +{"text": "Correction to: BMC Emerg Med 20, 56 (2020)https://doi.org/10.1186/s12873-020-00351-wThe Tokushukai Group Ethical Committee approved that our study had added patients with standard BCAA for a control group to patients with leucine enriched BCAA and additional opt-out consent of patients in the control group is obtained (TGE01445-024)."} +{"text": "A 79-year-old woman presented with a long history of peripheral eosinophilia. Previous right hemicolectomy for colonic polyposis was reported. Laboratory tests were notable for mild macrocitic anaemia and eosinophilia. \u03b22 microglobulin and serum tryptase levels were elevated. Serum immunofixation revealed IgA/kappa monoclonal protein. Bence-Jones protein was positive. Bone marrow (BM) biopsy revealed the coexistence of two neoplastic components. Cohesive clusters of bland-looking, spindle-shaped mast cells, representing 20% of marrow cellularity, were close to aggregates of mature plasma cells occupying 40% of marrow cellularity. Molecular analysis on marrow aspirate demonstrated KIT D816V mutation, TET2 mutation, monoallelic deletion of TP53/17p13 and trisomy of ATM/11q23. A bone density study revealed mild osteoporosis. Full skeletal X-rays and magnetic resonance imaging (MRI) of spine and hips showed multiple, small rarefaction areas and an old L1-L2 fracture, both ascribed to osteoporosis. The association of systemic mastocytosis (SM) and multiple myeloma (MM) is very uncommon. The coexistence of SM with MM placed our patient in the SM with associated clonal haematological non-mast-cell lineage disease (SM-AHN) subtype. Midostaurin therapy was started. Serum immunofixation revealed IgA/kappa monoclonal protein (21 g/L). Bence-Jones protein was positive. Bone marrow (BM) biopsy revealed two neoplastic components. Low- and high-power views of haematoxylin and eosin sections showed cohesive paratrabecular aggregates of bland-looking, spindle-shaped cells and three of the minor criteria combined with the presence of a C finding [The coexistence of SM with MM placed our patient in the SM with associated clonal haematological non-mast-cell lineage disease (SM-AHN) subtype. Midostaurin therapy (100 mg twice per day) was started.SM-AHN, a subtype of SM recognized in the current WHO classification, is defined as meeting the criteria for SM and for an associated haematological neoplasm as a distinct entity . SM-AHN"} +{"text": "The materials are thermally stable and exhibit good thin film forming abilities. Their optical and physicochemical properties were found to be strongly dependent on the structure of the side chains. Comparative studies with octahedral silsesquioxane (POSS) analogues (POSS-triazole-Py and POSS-amide-Py) emphasized the role of the specific double-strand architecture of the LPSQ backbone and distribution of side Py groups for their photo-luminescent properties. The new hybrid materials were tested as fluorescence energy donors to red-emitting dyes (Nile Red and Coumarine 6). All the silsesquioxanes studied were found to be able to transfer FL emission energy to Coumarin 6, irrespectively of their spatial structure. However, due to the differences in the wavelength range of FL emission, only LPSQ-triazole-Py were able to act as energy donors to Nile Red. The Py-grafted LPSQ may be also applied for development of soluble and highly emissive chemosensors. Their fluorescent nature was explored for the detection of Cu(II), Fe(III), Co(II), Ag(I), Hg(II), Mg(II), Ca(II), Pb(II) and Zn(II). The morphology of the side chains and hydrogen-bonding interactions influenced the sensing capacity of all the studied materials.Hybrid polymers containing pyrene (Py) units bound to linear poly(silsesquioxane) (LPSQ) chains through flexible linkers containing heteroatoms (LPSQ-triazole-Py and LPSQ-amide-Py) exhibit intense fluorescence emission, both in very diluted solutions (c = 10 With fast growing awareness of environmental issues, an increasing level of pollution in the natural environment and the search for new reproducible energy sources, the demand for technologies has been shifted to novel intelligent materials. Organic light-emitting diodes , organicGreen light emitting Py excimers are formed when geometrical overlap of aromatic rings is provided. Highly organized systems, such as crystals of Py-containing molecules emit almost exclusively excimer fluorescence in the solid state . Yet, prFluorescence spectroscopy is also widely used for environmental and biological studies due to the low detection limit, selectivity, rapid response and applicability for bioimaging ,12,13. PTherefore, of high interest are novel molecules or macromolecules that allow for efficient use of Py fluorophores in dilute solutions and suppress \u03c0-stacking-induced molecular aggregation of pyrene in solid state and concentrated solutions. One of the possible resolutions is preparation of Py labeled polymer materials. Various types of optically active polymers have been prepared to date\u2014e.g., Py functionalized polyacetylenes , poly between the polymer backbone and Py moieties. We thoroughly studied the influence of the side chains structure with regard to the optical properties of LPSQ (characteristic position of FL bands and intensity of emission) both in dilute solutions and in the solid state. In order to determine the role of characteristic architecture of LPSQ, we also studied properties of their octahedral core silsesquioxane analogues .The improved efficiency of interactions between side chromophores in LPSQ-R-Py allowed for efficient EnT to organic dyes [9-diethylamino-5-benzo[\u03b1]phenoxazinone (Nile Red) and 2H-chromen-2-one (Coumarin 6)]. The effect of different paramagnetic and diamagnetic metal cations on optical properties of the LPSQ-R-Py was also examined. Materials with triazole linkers in side groups exhibited high sensitivity toward Cu(II) and Ag(I) ions, and only slightly poorer sensitivity for Hg(II) and Fe(III). LPSQ-amide-Py detected Co(II), Fe(III) and Cu(II), yet the effect was not large. POSS-amide-Py was selective toward Co(II).3 and POSS-N3 were obtained via azidation of LPSQ-Cl and POSS-Cl, respectively. LPSQ-triazole-Py and POSS-triazole-Py were obtained via the copper(I)-catalyzed Huisgen cycloaddition of 1-ethynylpyrene to LPSQ-N3 or POSS-N3. (3-Azidopropyl)trimethoxysilane was prepared according to the modified literature procedure 0/[Cl]0 = 1/8. The obtained mixture of polyhedral azides was used for the further studies. The organization of side substituents in linear ladder structure differs significantly from those of any of the obtained polyhedral silsesquioxanes, and thus, we did not separate the POSS components of the mixture. LPSQ-Cl suffered similar redistribution of siloxane bonds induced by NaN3. The SEC diagram shows an increase in polydispersity with regard to LPSQ-Cl. Precipitation of the obtained material resulted in a product fraction (Y = 76%) of a narrow Mw/Mn (PDI = 1.3) and molecular weight Mn of 1900 g/mol (\u22121) and \u03bd(C=C) (1550 cm\u22121) appeared. The structures of LPSQ-triazole-Py and POSS-triazole-Py were confirmed with 1H, 13C and 29Si NMR (in solution and solid state) .n = 1000 g/mol, PDI = 1.2) was used as the precursor for macromolecules with Py chromophores grafted through linkers containing amide groups and formation of new bands characteristic of amides: \u03bd(C(O)N) (1700\u20131600 cm\u22121) and \u03bd(C-N) in combination with \u03b4(N-H) (1600\u20131500 cm\u22121). The amount of amide groups in side chains influenced the position of vibrational bands. Increasing the ratio of amide groups led to the shift of \u03bd(C(O)N) mode toward lower wavenumbers (from 1640 cm\u22121 for LPSQ-amide50-Py to 1618 cm\u22121 for LPSQ-amide100-Py). A similar position of the \u03bd(C(O)N) band was found for POSS-amide-Py (1624 cm\u22121). All the obtained materials were characterized with 1H, 13C and 29Si NMR spectroscopy (in solution and in solid state) (The reaction progress was monitored by FTIR and it was carried out until the planned conversion of carboxylic groups into amides occurred. The assignment of IR characteristic bands is presented in d state) .C+NO)/NN \u2265 3, where N\u2014number of atoms) octasiloxane and diamagnetic cations. Metal ions influence properties of molecules containing heteroatoms and play an important role in many biological processes ,73. InteThe fluorescence intensity of LPSQ-amide50-Py, LPSQ-triazole-Py and POSS-triazole-Py noticeably decreased with the addition of paramagnetic cations: Cu(II) and Fe(III) and Co(II). Changes in FL emission intensity were also observed for triazole-containing materials in the presence of diamagnetic Ag(I) and Hg(II) ions. Sensing ability of pyrene derivatives toward Cu(II) and Fe(III) stems from, respectively, reverse-PET phenomenon ,80 and eWhile the studied triazole-containing materials exhibited high sensing ability toward cations, the change in FL intensity for LPSQ-amide75-Py was influenced only slightly. This may be explained in terms of strong hydrogen bonds between the side substituents that make Py chromophores better packed and less accessible for metal cations. Indeed, LPSQ-amide50-Py detected the presence of Cu(II), Fe(III) and Co(II), although the effect was not large. Interestingly, POSS-amide-Py exhibited FL emission quenching only in the presence of Co(II) ions . The hign+-\u03c0 interactions and diamagnetic metal cations. Good photo-optical properties and processability of hybrid LPSQ-R-Py make them promising materials for possible applications in optoelectronics and metal cations sensing devices."} +{"text": "Recombination is proposed to be critical for coronavirus (CoV) diversity and emergence of SARS-CoV-2 and other zoonotic CoVs. While RNA recombination is required during normal CoV replication, the mechanisms and determinants of CoV recombination are not known. CoVs encode an RNA proofreading exoribonuclease (nsp14-ExoN) that is distinct from the CoV polymerase and is responsible for high-fidelity RNA synthesis, resistance to nucleoside analogues, immune evasion, and virulence. Here, we demonstrate that CoVs, including SARS-CoV-2, MERS-CoV, and the model CoV murine hepatitis virus (MHV), generate extensive and diverse recombination products during replication in culture. We show that the MHV nsp14-ExoN is required for native recombination, and that inactivation of ExoN results in decreased recombination frequency and altered recombination products. These results add yet another critical function to nsp14-ExoN, highlight the uniqueness of the evolved coronavirus replicase, and further emphasize nsp14-ExoN as a central, completely conserved, and vulnerable target for inhibitors and attenuation of SARS-CoV-2 and future emerging zoonotic CoVs. Recombination is an essential part of normal coronavirus replication, required for the generation of the sub-genomic mRNAs as well as defective viral genome (DVGs) and is also implicated in novel strain emergence. However, the molecular mechanisms and determinants of RNA recombination in CoVs are unknown. Here, we compare recombination in 3 divergent beta-coronaviruses; murine hepatitis virus (MHV), MERS-CoV, and SARS-CoV-2. We show that they have striking similarities in the populations of RNA produced and in the sequences surrounding recombination junctions. Further, we demonstrate that the coronavirus proofreading exoribonuclease (nsp14-ExoN) is required to maintain the rates and loci of recombination generated during infection. These data suggest that recombination and the coronavirus exoribonuclease are conserved and important determinants of replication that may be targeted for inhibition and attenuation to control the ongoing pandemic of SARS-CoV-2 and prevent future outbreaks of novel coronaviruses. The ongoing severe global pandemic of SARS-CoV-2, the etiological agent of coronavirus disease 2019 (COVID-19) underlines the importance of defining the determinants of coronavirus (CoV) evolution and emergence into human populations . FurtherCoronaviruses are a family of positive-sense, single-stranded RNA viruses with genomes ranging in size between 26 and 32 kb . During in vivo virulence, resistance to nucleoside analogues, and immune antagonism [In other RNA virus families including picornaviruses and alphaviruses, regulation of recombination has been mapped to replication fidelity determinants in the viral RNA-dependent RNA polymerase (RdRp) \u201332. In ctagonism ,42,43.in vitro with broadly similar patterns of recombination, and generate diverse yet similar populations of recombined molecules. We further demonstrate that genetic inactivation of MHV nsp14-ExoN results in a significant decrease in recombination frequency, altered recombination junction patterns across the genome, and altered junction site selection. These defects and alterations result in a marked change in MHV-ExoN(-) recombined RNA populations, including defective viral genomes (DVGs). These results support future studies aimed at illuminating the role of SARS-CoV-2 nsp14-ExoN activity in RNA recombination, the regulation of sgmRNA expression, and its contribution to novel CoV zoonotic emergence. Combined with the multiple critical integrated functions of nsp14-ExoN, the role in recombination further defines nsp14-ExoN as a conserved, vulnerable, and highly specific target for inhibition by antiviral treatments and viral attenuation.In this study, we sought to define the frequency and patterns of recombination of divergent \u03b2-CoVs SARS-CoV-2, MERS-CoV, and MHV, and to test the role of nsp14-ExoN in recombination. We used both short-read Illumina RNA-sequencing (RNA-seq) and long-read direct RNA Nanopore sequencing for all three viruses to show that they perform extensive recombination during replication We first sought to quantify recombination frequency and identify recombination patterns in zoonotic CoVs by sequencing both MERS-CoV and SARS-CoV-2 RNA. In three independent experiments for each virus, Vero cell cultures were infected with either MERS-CoV or SARS-CoV-2 until the monolayer displayed >70% virus-induced cytopathic effect (CPE). Total RNA from infected cells was isolated and poly(A)-selected to capture all viral RNA containing poly-A tails, including genomic, subgenomic, and defective viral genome (DVG) RNA molecules. Equal amounts of total cell RNA from each of the three independent experiments for each virus was sequenced by short-read Illumina RNA-sequencing (RNA-seq), and by long-read direct RNA Nanopore sequencing. The depth and low error rate of RNA-seq facilitated the detection and quantification of both high- and low-abundance unique junctions. Long-read direct RNA sequencing on the Oxford Nanopore Technologies MinION platform was used to sequence complete RNA molecules, to define the organization of junctions in the context of intact RNA molecules. By comparing short- and long-read RNA sequencing, we accomplished high-confidence detection and quantification of recombination junctions as well as description of the genetic architectures of molecules formed by the junctions.ViReMa (Virus Recombination Mapper) [ViReMa detected recombination events that generated deletions greater than 5 base-pairs and that were flanked by a 25 base-pair alignment both upstream and downstream of the junction site. ViReMa-detected junctions may be formed from either inter-molecular or intra-molecular recombination during replication. ViReMa aligned both recombined and non-recombined reads in the library and reported the total number of nucleotides aligned to the genome and all detected recombination junctions.For RNA-seq, reads were aligned to the respective viral genomes using a Mapper) . ViReMa ViReMa demonstrated nearly identical read coverages for MERS-CoV (1118) and SARS-CoV-2 (1122) (freq) was calculated for MERS-CoV and SARS-CoV-2 . Thus, Jfreq was not biased by the number of virus-mapping reads. Jfreq was multiplied by 104 to scale for library size and was reported as the number of junctions per 104 mapped nucleotides. MERS-CoV had a mean Jfreq of 37.80 junctions detected per 104 mapped nucleotides. SARS-CoV-2 had a mean Jfreq of 475.7 junctions per 104 mapped nucleotides (freq between the two viruses that were infected at similar multiplicity of infections (MOIs), were collected when the cells displayed similar levels of CPE, and had similar viral abundance in sequenced RNA. We considered the possibility that the observed >10-fold difference between Jfreq of each virus could be due to the replication capacity of the parental virus. We compared the number of unique junctions generated by each virus to remove any potential viral replication bias. SARS-CoV-2 generated an average of 56,082 unique junctions per experiment, while MERS-CoV generated an average of 19,367 unique junctions per experiment . Further2 (1122) . To quanRS-CoV-2 . Jfreq rleotides . This waperiment . Thus, bTo define the patterns of the detected recombination junctions, we mapped forward (5\u2019 \u2794 3\u2019) recombination junctions according to their genomic position Figs . Both MEWe next sought to define and quantify the populations of recombined RNA molecules produced in both MERS-CoV and SARS-CoV-2. SARS-CoV-2 sgmRNAs were identified by the location of recombination junctions within previously defined 65 base-pair regions containing the transcription regulatory sequence (TRSs) of each sgmRNA . SimilarFor each virus, the frequencies of DVGs, canonical sgmRNAs, and alternative sgmRNAs were normalized to total virus RNA. For both MERS-CoV and SARS-CoV-2, canonical and alternative junctions were detected for all sgmRNAs Figs . MERS-CoWe next calculated the mean recombination frequency at each genomic position by comparing the number of nucleotides in detected junctions (both start and stop sites) at that position, and normalized to nucleotide depth at that position. Further, we determined genomic positions with a mean recombination frequency greater than 50% . In MERSFor both SARS-CoV-2 and MERS-CoV, the nucleotide composition of the start and stop sequences resulting in junctions forming DVGs in MERS-CoV and SARS-CoV-2 was determined and compared to the expected nucleotide percentage based on the parental viral genomes . SequencWe next tested whether MERS-CoV and SARS-CoV-2 junction sites favored regions of sequence microhomology at recombination junctions, defined as 2\u201320 nt regions of identical overlap . The disWe performed direct RNA Nanopore sequencing on the same RNA used for short-read RNA-seq. We analyzed three independent experiments for each virus and sequenced 178,658 MERS-CoV RNA molecules and 1,725,862 SARS-CoV-2 RNA molecules that had 85.6% and 82.2% identity to the parental genome, respectively . To remoTo define the architectures of detected molecules, we filtered for junctions with at least 3 supporting Nanopore reads. For both viruses, junctions were categorized as either a DVG or sgmRNA junction using the same criteria as with the RNA-seq data. In MERS-CoV, we defined 5 distinct species, including 3 sgmRNAs and 2 DVGs . In SARSViReMa (AY910861.1). In both infected cell monolayers and viral supernatants, MHV-WT and MHV-ExoN(-) had similar mean coverages ranging between 1100 and 1700 reads (We previously have reported that the nsp14 exoribonuclease (nsp14-ExoN) activity is required for high-fidelity replication and proofreading for the \u03b2-CoVs murine hepatitis virus (MHV) and SARS-CoV \u201336. We s00 reads .freq was calculated as described for freq relative to MHV-WT in both infected cells and viral supernatant (Previous studies have shown that MHV-ExoN(-) has decreased genome replication compared to WT . We accoernatant . To addrernatant Figs. ThRecombination junctions were plotted according to their start (5\u2019) and stop (3\u2019) sites in infected cells and viral supernatant Figs . MHV-WT We next calculated and compared mean recombination frequency at each genomic position in MHV-WT and MHV-ExoN(-) . Both MHCompared with WT, MHV-ExoN(-) had significantly decreased frequencies of DVGs and both canonical and alternative sgmRNAs . MHV-ExoDESeq2 [We next identified junctions with altered abundances in MHV-ExoN(-) compared to MHV-WT using DESeq2 . MHV-ExoTo test whether MHV-ExoN(-) has altered sequence composition at its recombination junctions, we filtered DVG junctions and quantified nucleotide composition of adenosine (A), cytosine (C), guanine (G), and uracil (U) in the start and stop sequences flanking junction sites. Both MHV-WT and MHV-ExoN(-) demonstrated similar patterns of depletion and enrichment of nucleotides in infected cell monolayers and viral supernatant Figs and S6A.minimap2, MHV-WT datasets contained 102,367 viral molecules and MHV-ExoN(-) contained 19,445 viral supernatant RNA by direct RNA Nanopore sequencing. When reads were mapped to the MHV genome using d 19,445 . We valid 19,445 .MHV-ExoN(-) had a global decrease in the number of junctions across the genome Tables. While CoV recombination has long been proposed as a driver of novel strain emergence and is known to be a constitutive aspect of CoV replication, the diversity of recombination products and sequence and protein determinants had not previously been defined. In this study, we show the diversity of the CoV recombination landscape in the \u03b2-coronaviruses SARS-CoV-2, MERS-CoV, and murine hepatitis virus (MHV), and we demonstrate that loss of the nsp14 exoribonuclease activity in MHV results in decreased recombination and altered site selection of recombination junctions. Our results support a model in which nsp14-ExoN activity is required for normal recombination. Thus, nsp14-ExoN is a key component of CoV recombination, adding another essential function to the repertoire of those already reported for nsp14-ExoN, specifically CoV high-fidelity replication, RNA synthesis, resistance to antiviral nucleoside analogues, fitness, immune antagonism, and virulence.We show that MHV, MERS-CoV, and SARS-CoV-2 perform extensive recombination and generate diverse populations of RNA molecules, demonstrated by independent short-read Illumina RNA-seq and long-read, direct RNA Nanopore sequencing. These divergent group 2a (MHV), 2b (SARS-CoV-2), and 2c (MERS-CoV) \u03b2-CoVs demonstrated many strong similarities in their patterns of recombination junctions across the genomes and in the types of recombined RNAs produced. Specifically, the similarities across all three viruses in the nucleotide composition of sequences flanking DVG junctions and the common increased junction sequence microhomology support the conclusion that recombination mechanisms have been conserved across different evolutionary trajectories and host species specificity.There also were distinct recombination patterns for each virus that were confirmed across independent experiments and by agreement between RNA-seq and Nanopore datasets for all viruses. These differences most likely represent evolutionary divergence of recombination in distinct viruses or sub-genera represented by MHV, SARS-CoV-2 and MERS-CoV. However, it remains possible that observed differences could be impacted by the diversity of the original sample or replication in different cell types. SARS-CoV-2 stock virus was a low passage (P5) population from a clinical isolate that had been passaged in Vero cells, while MERS-CoV and MHV were low passage stocks generated from isogenic cDNA clones. It will be important for future studies to determine the role of the diversity of the viral population, cell environment, virus-specific RNA synthesis kinetics, and virus adaptation/evolution in viral recombination. The extent of the pandemic and availability of genetically diverse viruses will allow investigators to test whether patterns of SARS-CoV-2 recombination show alterations between early and later pandemic isolates, and if any identified differences correlate with or predict changes in other replication or pathogenesis.High-resolution analysis of DVG junctions produced during replication by MERS-CoV, SARS-CoV-2, and MHV reveals that a significant preference for a UUG motif, suggesting a possible conserved core sequence for DVG synthesis that differs from sgmRNA transcriptional regulatory sequences. These results support a model across multiple divergent \u03b2-CoVs in which DVGs result from recombination junction selection by the RTC based on both broadly similar sequence identity and specific sequence microhomology of 2\u201310 bp . This mopicornaviridae and alphaviridae that lack any exonuclease, low-fidelity mutant viruses have altered polymerase speed and processivity [MHV-ExoN(-) mutants showed decreased recombination junction frequency and altered populations of sgmRNAs and DVGs, demonstrating a previously unknown role for nsp14-ExoN in CoV RNA recombination. There is no precedent in RNA viruses for the regulation of recombination by a virus encoded exoribonuclease. In contrast, in DNA viruses such as poxviruses and herpesviruses, virus-encoded exonuclease activity stimulates recombination by single-strand annealing through both exonuclease degradation of nucleic acids and interactions with other proteins ,40. In tessivity and thesessivity ,55,56. O4-hydroxycytidine [The similarities between the patterns of recombination across divergent WT \u03b2-CoVs, along with the differences observed between recombination in MHV WT and ExoN(-) viruses, support the hypothesis that ExoN mutants will inform our understanding of the evolution of the unique CoV multi-protein polymerase complex. Specifically, the model of DVG synthesis defined in MHV, MERS-CoV, and SARS-CoV-2 will allow for the direct testing of the roles of DVGs in CoV replication. Further, the role of ExoN in CoV recombination, along with the previously defined roles of ExoN in RNA proofreading during replication, native resistance to nucleoside analogues, immune evasion, and virulence and pathogenesis, highlight nsp14-ExoN as conserved and vulnerable target for both antiviral inhibitors and virus attenuation. ExoN(-) viruses are profoundly more sensitive to a range of antiviral nucleoside analogues, including remdesivir, ribavirin, 5-fluorouracil, and \u03b2-d-N31/2801) ,38,57. N31/2801) ,12. The 31/2801) ,58,59. TCercopithecus aethiops Vero CCL-81 cells maintained in Dulbecco\u2019s modified Eagle medium (DMEM) (Gibco) supplemented to final concentrations of 10% fetal calf serum (Gibco), 100 IU/ml penicillin (Mediatech), 100 mg/ml streptomycin (Mediatech), and 0.25 mg/ml amphotericin B (Mediatech) were used for MERS-CoV-2 infection. Vero CCL-81 cells were obtained from ATCC. Vero E6 cells maintained in Dulbecco\u2019s modified Eagle medium (DMEM) (Gibco) supplemented to final concentrations of 10% fetal calf serum (Gibco), 100 IU/ml penicillin (Mediatech), 100 mg/ml streptomycin (Mediatech), and 0.25 mg/ml amphotericin B (Mediatech) were used for SARS-CoV-2 infections. Vero E6 cells were obtained from ATCC.DBT-9 cells were maintained at 37\u00b0C as described previously . DBT-9 cAll MHV work was performed using the recombinant WT strain MHV-A59 (GenBank accession number AY910861.1 Experime2 flasks were infected with either MHV-A59 or MHV-ExoN(-) at an MOI of 0.01 PFU/cell. Supernatant was harvested at either 16 hours post infection (MHV-A59) or 24 hours post infection (MHV-ExoN(-)) when the monolayer was >95% fused and remained intact. Infection supernatant was clarified by centrifugation at 1500 x g for 5 minutes at 4\u00b0C. Viral supernatant was purified on a 30% sucrose cushion by ultracentrifugation at 25,000 RPM at 4\u00b0C for 16 hours. The viral pellet was resuspended in MSE buffer . Viral RNA was extracted using the TRIzol-LS reagent according to manufacturer\u2019s protocols. RNA was quantified using the Qubit RNA HS assay. Supernatant data in this paper is the result of three experiments sequenced independently from the infected cell monolayer samples.Subconfluent 150-cm2 flask of DBT-9 cells was infected with either MHV-WT or MHV-ExoN(-) at an MOI or 0.01 PFU/cell. Monolayer was harvested at either 16 hpi (MHV-WT) or 24 hpi (MHV-ExoN(-)) when the monolayer was >95% fused and >75% intact. RNA was extracted with TRIzol according to manufacturer\u2019s protocols. Infected monolayer data in this paper is the result of three independent experiments sequenced independently.In three independent experiments, a subconfluent 150-cm2 flask of Vero CCL-81 cells was infected with MERS-CoV at an MOI of 0.3 pfu/cell. Total infected cell lysates were collected at 72 hpi with the monolayer >70% fused. RNA was extracted in TRIzol according to manufacturer\u2019s protocols.In three independent experiments, a nearly confluent 25-cm2 flasks of Vero E6 cells were infected at an MOI = 0.45 pfu/cell and cellular monolayers were harvested 60 hpi when the monolayer was >90% fused. RNA was extracted in TRIzol according to manufacturer\u2019s protocols.In three independent experiments, a total of 5 subconfluent 25-cmNext generation sequencing (NGS) libraries were generated using 2 \u03bcg of RNA of each sample. RNA was submitted to Genewiz for library preparation and sequencing. Briefly, after quality control, polyadenylated RNA was selected during library preparation. Isolated RNA was heat fragmented, RT-PCR amplified with equivalent number of cycles, size-selected, and libraries were prepared for 2 x 150 nucleotide paired-end sequencing performed (Illumina). Genewiz performed basecalling and read demultiplexing.RNA from ultracentrifuge-purified viral supernatant was prepared for direct RNA Nanopore sequencing on the Oxford Nanopore Technologies MinION platform according to the manufacturer\u2019s protocols. Libraries were sequenced on fresh MinION R9.4 flow-cells for 24 hours, or until the pore occupancy was under 20%. Viral supernatant RNA from three independent experiments was sequenced on three separate flow cells for both MHV-WT and MHV-ExoN(-). MERS-CoV RNA from three independent cultures was sequenced on three separate flow cells. SARS-CoV-2 RNA isolated from three independent infections was sequenced on three separate flow cells.Trimmomatic [parameters java -jar trimmomatic.jar PE sample_R1.fastq.gz sample_R2.fastq.gz output_paired_R1.fastq output_unpaired_R1.fastq output_paired_R2.fastq output_unpaired_R2_unpaired.fastq ILLUMINACLIP:TruSeq3-PE.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGIWINDOW:4:15 MINLEN:36). Reads shorter than 36 bp were removed and low-quality bases (Q score < 30) were trimmed from read ends. The raw FASTQ files were aligned to the MHV-A59 genome (AY910861.1), the MERS-CoV genome (JX869059.2), and the SARS-CoV-2 genome (MT020881.1) using the Python2 script ViReMa [python2 ViReMa.py reference_index input.fastq output.sam\u2014OuputDir sample_virema/\u2014OutputTag sample_virema -BED\u2014MicroIndelLength 5. The sequence alignment map (SAM) file was processed using the samtools [BAM) file (using command line parameters samtools depth -a -m 0 sample_virema.sorted.bam > sample_virema.coverage).Raw reads were processed by first removing the Illumina TruSeq adapter using mmomatic default on 0.15) using thsamtools suite tofreqJ) was calculated by comparing the number of nucleotides in detected recombination junctions to the total number of mapped nucleotides in a library. Nucleotides in detected recombination junctions were quantified as a sum of nucleotide depth reported at each junction in the BED file generated by ViReMa. Total nucleotides mapped to the MHV-A59 genome were quantified as a sum of nucleotide depth at each position across the genome in the tab-delineated text file generated by the samtools. Jfreq was reported as junctions per 104 nucleotides sequenced. Mean freqJ values were compared between MHV-WT and MHV-ExoN(-) and statistical significance determined by an unpaired student\u2019s t-test. Junctions were mapped across the genome according to their start (5\u2019) and stop (3\u2019) positions. These junctions were first filtered in the forward (5\u2019 \u2794 3\u2019) direction using the dpylr package (RStudio). The frequency of each junction was calculated by comparing the depth of the unique junction to the total number of nucleotides in all detected junctions in a library. Junctions were plotted according to the genomic position and colored according to log10 of the frequency using ggplot2 in RStudio.Recombination junction frequency for three independent sequencing experiments by a 2-way ANOVA statistical analysis with multiple comparisons corrected through statistical hypothesis testing using the Sidak test.freq) of each sgmRNA was calculated by dividing the number of nucleotides in a specific sgmRNA population by the total amount of viral RNA . This ratio is multiplied by 10,000 to scale for the number of nucleotides sequenced and is therefore expressed as the number of junctions per 104 mapped nucleotides. The filtered sgmRNA junctions were compiled and DVG junctions were filtered in RStudio by performing an exclusionary anti_join using dplyr on forward junctions identified in each sample. DVG Jfreq was calculated by dividing the number of nucleotides in DVG junctions by the total amount of viral RNA in a sample . The ratio is multiplied by 10,000 to scale for number of nucleotides sequenced and the frequency is expressed as the number of junctions per 104 mapped nucleotides. The percentage of canonical and alternative sgmRNA and DVG junctions was calculated by comparing the depth of all filtered sgmRNA or DVG junctions to the sum of all detected forward junctions. Mean percent canonical and alternative sgmRNAs and DVG was compared between three independent sequencing experiments in viral supernatant RNA. Statistical significance was determined by a 2-way ANOVA test with multiple comparisons and corrected by statistical hypothesis testing using the Sidak test.Forward recombination junctions were classified as either canonical sgmRNA junctions, alternative sgmRNA junctions or DVG junctions based on the position of their junction sites and filtered in Microsoft Excel. Briefly, junction start sites were filtered to those positioned within 30 nucleotides of the TRS-L for each virus. The stop sites were then filtered for those positioned within 30 nucleotides of each respective sgmRNA TRS. This window is supported by other reports defining the flexibility of the CoV transcriptome ,65. Canohttps://github.com/DenisonLabVU) and the R package DESeq2 [bioinfokit [DESeq2 package in RStudio and junctions with a significant alteration of abundance in MHV-ExoN(-) compared to MHV-WT were visualized as either red or green in the graph generated by bioinfokit.To compare the abundance of junctions in MHV-A59 and MHV-ExoN(-), the ViReMa output list of junctions was analyzed by in-house scripts [BED files listing deletions detected in each sequenced RNA molecule. Both BAM and BED files were filtered for full length molecules using samtools and Microsoft Excel, respectively. Full-length CoV molecules were defined as encoding coverage at in the 5\u2019 UTR and 3\u2019 UTR of the respective viruses. Nanopore junctions output in BED files were compared to junctions in ViReMa RNA-seq BED files to confirm its presence in both datasets. To account for noisiness in Nanopore datasets, a Nanopore junction was considered confirmed if at least 1 RNA-seq junction start and stop sites fell within 20 bp of the Nanopore start and stop sites, respectively. Filtering of Nanopore and RNA-seq datasets was performed in Microsoft Excel. BED files generated by the flair align module were parsed based on the number of junctions were identified. Nanopore reads containing only 1 junction were identified using Microsoft Excel and unique junctions were quantified in RStudio using base-R functions. Sequencing coverage maps were generated from samtools depth analysis of filtered BAM files. All junctions present in sequenced libraries were mapped in Sashimi plots generated by the Integrated Genome Viewer (IGV) [Live basecalling was performed by NanoPlot . Pass re of RNA) to generer (IGV) . JunctioS1 Figtrans (inter-molecular) recombination and cis (intra-molecular) recombination and produce 3 different types of molecules: subgenomic mRNAs (sgmRNAs) that are translated into structural and accessory proteins, defective viral genomes (DVGs) whose role in viral replication, innate immune antagonism, and viral evolution have not yet been defined, and infectious (complete) genome molecules. sgmRNAs are produced by recombination between transcription regulatory sequences (TRSs) across the genome. DVGs are produced by recombination between sites across the genome outside TRSs that result in sequence deletions. Complete genomes are generated by recombination at the same location between 2 co-infecting molecules. The CoV replication transcription complex (RTC) is shown in gray. (C) Internally deleted recombined RNAs (DVGs) are formed by a recombination junction . In this report, a start site refers to the position where the 5\u2019 segment ends and a stop site refers to the position where the 3\u2019 segment begins in the viral genome (blue line). Nucleotides sequences in the genome at both the start and stop sites are numbered according to their position relative to the break formed by the recombination junction (red line). (D) Results in this report support the model in which microhomology (yellow box) between the CoV DVG start and stop sites facilitates formation of the complete RNA molecule through translocation of the CoV RTC (gray).(A) Genome organization of MERS-CoV (gray), SARS-CoV-2 (violet), and MHV (white). Nonstructural (nsps 1\u201316) and structural and accessory open reading frames (ORFs) are labelled. The common 5\u2019 leader transcription leader sequence (TRS-L) is denoted with an unfilled red star. Body TRSs are labelled with filled red stars. (B) CoVs perform both (TIF)Click here for additional data file.S2 Figfreq) per 104 mapped nucleotides of MERS-CoV canonical and alternative sgmRNA species normalized to total viral RNA. N = 3, error bars represent SEM. (H) Junction frequency (Jfreq) per 104 mapped nucleotides of SARS-CoV-2 canonical and alternative sgmRNA species normalized to total viral RNA. N = 3, error bars represent SEM.RNA-seq libraries of (A) MERS-CoV and (B) SARS-CoV-2 were aligned to the viral genomes with ViReMa. Nucleotide depth was calculated at each position and represented as mean nucleotide depth (N = 3). (C) The number of unique junctions detected was compared between MERS-CoV and SARS-CoV-2. N = 3, error bars represent standard error of the mean. Unpaired student\u2019s t-test, *** p < 0.001. Individual recombination junction scatter plots of (D) MERS-CoV and (E) SARS-CoV-2. Recombination junctions were detected by ViReMa and forward (5\u2019 \u2794 3\u2019) junctions were identified by bioinformatic filtering. Junctions are plotted according to their 5\u2019 (start) and 3\u2019 (stop) positions and colored according to their frequency in the population of total junctions. Highly abundant junctions are magenta and opaque and low-frequency junctions are red and transparent. (F) Relative proportions of junctions forming DVGs, canonical sgmRNAs, and alternative sgmRNAs as a percentage of the total population of all recombined RNA in MERS-CoV (black) and SARS-CoV-2 (violet). N = 3, error bars represent SEM. 2-way ANOVA, *** p < 0.001, **** p < 0.0001. (G) Junction frequency (J(TIF)Click here for additional data file.S3 FigRNA-seq libraries of (A) MHV-WT and (B) MHV-ExoN(-) infected cell monolayer RNA were aligned to the viral genomes with ViReMa. Nucleotide depth was calculated at each position and represented as mean nucleotide depth (N = 3). (C) The number of unique junctions detected was compared between MHV-WT and MHV-ExoN(-). N = 3, error bars represent standard error of the mean. Unpaired student\u2019s t-test, ** p < 0.01. Individual recombination junction scatter plots of (D) MHV-WT and (E) MHV-ExoN(-). Recombination junctions were detected by ViReMa and forward (5\u2019 \u2794 3\u2019) junctions were identified by bioinformatic filtering. Junctions are plotted according to their 5\u2019 (start) and 3\u2019 (stop) positions and colored according to their frequency in the population of total junctions. Highly abundant junctions are magenta and opaque and low-frequency junctions are red and transparent. (F) Relative proportions of junctions forming DVGs, canonical sgmRNAs, and alternative sgmRNAs in MHV-WT (blue) and MHV-ExoN(-) (orange) infected monolayer RNA. N = 3, error bars represent SEM. 2-way ANOVA, ** p < 0.01, *** p < 0.001, **** p < 0.0001. (G) Ratios of canonical sgmRNA species in MHV-WT (blue) and MHV-ExoN(-) (orange) infected monolayer RNA. Each sgmRNA species is reported as a percentage of the total sgmRNA population. N = 3, error bars represent SEM. 2-way ANOVA, **** p < 0.0001. (H) Ratios of alternative sgmRNA species in MHV-WT (blue) and MHV-ExoN(-) (orange) infected monolayer RNA. Each sgmRNA population is quantified as a percentage of the total number of minor sgmRNA species detected. N = 3, error bars represent SEM. 2-way ANOVA, * p < 0.05, **** p < 0.0001.(TIF)Click here for additional data file.S4 FigRNA-seq libraries of (A) MHV-WT and (B) MHV-ExoN(-) viral supernatant RNA were aligned to the viral genomes with ViReMa. (C) The number of unique junctions detected was compared between MHV-WT and MHV-ExoN(-). N = 3, error bars represent standard error of the mean. Unpaired student\u2019s t-test, ** p < 0.05. Nucleotide depth was calculated at each position and represented as mean nucleotide depth (N = 3). Individual recombination junction scatter plots of (D) MHV-WT and (E) MHV-ExoN(-). Recombination junctions were detected by ViReMa and forward (5\u2019 \u2794 3\u2019) junctions were identified by bioinformatic filtering. Junctions are plotted according to their 5\u2019 (start) and 3\u2019 (stop) positions and colored according to their frequency in the population of total junctions. Highly abundant junctions are magenta and opaque and low-frequency junctions are red and transparent. (F) Relative proportions of junctions forming DVGs, canonical sgmRNAs, and alternative sgmRNAs in MHV-WT (blue) and MHV-ExoN(-) (orange) viral supernatant RNA. N = 3, error bars represent SEM. 2-way ANOVA, *** p < 0.001, **** p < 0.0001. (G) Ratios of canonical sgmRNA species in MHV-WT (blue) and MHV-ExoN(-) (orange) viral supernatant RNA. Each sgmRNA species is reported as a percentage of the total sgmRNA population. N = 3, error bars represent SEM. 2-way ANOVA, ** p < 0.01, **** p < 0.0001. (H) Ratios of canonical sgmRNA species in MHV-WT (blue) and MHV-ExoN(-) (orange) viral supernatant RNA. Each sgmRNA population is quantified as a percentage of the total number of minor sgmRNA species detected. N = 3, error bars represent SEM. 2-way ANOVA, *** p < 0.001.(TIF)Click here for additional data file.S5 Fig2(Fold Change) of abundance .Mean recombination frequency at each genomic position is shown for MHV-WT (blue) and MHV-ExoN(-) (orange). (A) 5\u2019 UTR, (B) the non-replicase nonstructural proteins (nsp1\u20136), (C) the replicase proteins (nsp7\u201316), (D) the structural and accessory proteins, (E) 3\u2019 UTR. Key RNA elements including the TRS-leader (TRS-L) and body TRSs (TRS1\u20137) are labelled. Positions with statistically significant differences in MHV-ExoN(-) recombination frequency were identified by a 2-way ANOVA with multiple comparisons. Recombination junction abundance was compared in MHV-ExoN(-) to MHV-WT by DESeq2 in infected cell monolayer RNA (A) and viral supernatant RNA (B). Volcano plots of junctions colored by statistical significance and by the log(TIF)Click here for additional data file.S6 Fig(A) Nucleotide composition was calculated and reported as the percent adenosine (A), cytosine (C), guanine (G), and uracil (U) at each position in a 30-base pair region flanking DVG junction start and stop sites in MHV-WT (blue) and MHV-ExoN(-) (orange) viral supernatant RNA. Each point represents a mean (N = 3) and error bars represent SEM. 2-way ANOVA with multiple comparisons corrected for false discovery rate (FDR) by the Benjamini-Hochberg method. * q < 0.05, ** q < 0.01, *** q < 0.001, **** q < 0.0001. (B) Distribution of microhomology overlaps in MHV-WT (blue) and MHV-ExoN(-) (orange) compared to an expected probability distribution (gray). The frequency of each overlap length is displayed as a mean (N = 3) and error bars represent SEM.(TIF)Click here for additional data file.S1 TableNumber of reads in RNA-seq libraries and mapped to viral genome reported for MHV, MERS-CoV, and SARS-CoV-2. The percent mapping to the viral genome is reported as a mean of 3 libraries, \u00b1 standard error of the mean (SEM).(PDF)Click here for additional data file.S2 TableFor direct RNA Nanopore sequencing of MHV, MERS-CoV, and SARS-CoV-2, the percent identity of aligned reads, the mean read length, mean read quality, the read length N50 (fiftieth percentile), number of total sequenced reads, number of mapped reads, and number of unique detected junctions are reported. The percentage of junctions detected in Nanopore reads also detected in RNA-seq datasets is also reported.(PDF)Click here for additional data file.S3 TableDirect RNA Nanopore reads spanning the entire SARS-CoV-2 genome are listed. The mapping start site (Read Start), mapping end site (Read End), and unique read identifier (Read Name) are all listed. Each read represents a single detection (Count), and contains most of the SARS-CoV-2 genome (Read Length).(PDF)Click here for additional data file.S4 TableDirect RNA Nanopore reads aligning to viral genome by minimap2. Individual reads are listed by read name. Genomic positions of read alignment are listed . Read segments aligning to the genome are noted (\u201c# Segments\u201d) and start positions and aligned segment lengths listed .(XLSB)Click here for additional data file.S5 TablePositions with significantly altered recombination frequency in MHV-ExoN(-) infected monolayer RNA compared to MHV-WT and in MHV-ExoN(-) viral supernatant RNA compared to MHV-WT as determined by a 2-way ANOVA with multiple comparisons are listed. Genomic regions are noted. (PDF)Click here for additional data file.S6 TableJunctions with altered abundance in MHV-ExoN(-) infected monolayer RNA compared to MHV-WT and in MHV-ExoN(-) viral supernatant RNA compared to MHV-WT are listed. P-values calculated by DESeq2. (XLSB)Click here for additional data file."} +{"text": "An 85-year-old Caucasian female with past medical history of hypertension, hyperlipidemia, polymyalgia rheumatica, coronary artery disease, Osler-Weber-Rendu syndrome (diagnosed 18 years ago), intermittent epistaxis and pulmonary arteriovenous malformation (AVM) was referred to the clinic for abnormal chest imaging. She underwent multiple bilateral AVM embolization 17 years ago. She had minimal shortness of breath without epistaxis, hemoptysis, hematemesis or hematochezia. One month ago, her chest X-ray revealed multiple embolization coils bilaterally (A). Computed tomography (CT) chest scan without contrast showed multiple embolization coils, multiple tortuous vascular malformations in both lungs with enlargement of left lower lobe AVM (B). Magnetic resonance imaging (MRI) brain 17 years ago did not show brain vascular malformation. The diagnosis of progressive Osler-Weber-Rendu or hereditary hemorrhagic telangiectasia (HHT) was made. She was referred to HHT center and underwent coiling. After that, she has been clinically stable and a repeat CT chest on the follow-up visit showed smaller left lower lobe AVM (B), so conservative management was planned."} +{"text": "Medicago truncatula (Medicago) is a model temperate legume used to study flowering time pathways. Like Arabidopsis thaliana (Arabidopsis), its flowering is promoted by extended periods of cold , followed by warm long day (LD) photoperiods. However, Arabidopsis flowering-time genes such as the FLOWERING LOCUS C (FLC)/ MADS AFFECTING FLOWERING (MAF) clade are missing and CONSTANS-LIKE (CO-LIKE) genes do not appear to have a role in Medicago or Pisum sativum (pea). Another photoperiodic regulator, the red/far red photoreceptor PHYTOCHROME A (PHYA), promotes Arabidopsis flowering by stabilizing the CO protein in LD. Interestingly, despite the absence of CO-LIKE function in pea, PsPHYA plays a key role in promoting LD photoperiodic flowering and plant architecture. Medicago has one homolog of PHYA, MtPHYA, but its function is not known.Flowering time is an important trait for productivity in legumes, which include many food and fodder plants. MtPHYA Tnt1 insertion mutant alleles indicates that MtPHYA has an important role in promoting Medicago flowering and primary stem elongation in VLD and LD and in perception of far-red wavelengths in seedlings. MtPHYA positively regulates the expression of MtE1-like (MtE1L), a homologue of an important legume-specific flowering time gene, E1 in soybean and other Medicago LD-regulated flowering-time gene homologues, including the three FLOWERING LOCUS T-LIKE (FT-LIKE) genes, MtFTa1, MtFTb1 and MtFTb2 and the two FRUITFULL-LIKE (FUL-LIKE) genes MtFULa and MtFULb. MtPHYA also modulates the expression of the circadian clock genes, GIGANTEA (GI) and TIMING OF CAB EXPRESSION 1a (TOC1a). Genetic analyses indicate that Mtphya-1 Mte1l double mutants flowered at the same time as the single mutants. However, Mtphya-1 Mtfta1 double mutants had a weak additive effect in delaying flowering and in reduction of primary axis lengths beyond what was conferred by either of the single mutants.Genetic analysis of two MtPHYA has an important role in LD photoperiodic control of flowering, plant architecture and seedling de-etiolation under far-red wavelengths in Medicago. It promotes the expression of LD-induced flowering time genes and modulates clock-related genes. In addition to MtFTa1, MtPHYA likely regulates other targets during LD floral induction in Medicago. Medicago truncatula (Medicago) is a model nitrogen-fixing legume and a diploid, self-fertile annual forage plant . The domains highlighted include the N-terminal extension (NTE), Per-Arnt-Sim (PAS), cGMP phosphodiesterase/ adenylate cyclase/FhlA (GAF), and phytochrome (PHY), which comprise the N-terminal photosensory core module. The C-terminal regulatory module consists of the PAS-related domain (PRD) containing two PAS repeats (PAS-A and PAS-B) and the histidine kinase-related domain (HKRD) [domains adopted from 27]. At: Arabidopsis thaliana, Ca: Cicer arietinum (chickpea), Lj: Lotus japonicas (Lotus), Mt: Medicago truncatula, Ps: Pisum sativum (pea), Tp: Trifolium pratense (red clover). Identical and similar residues are highlighted in black.Additional file 2. Table S1. List of primers.pdf."} +{"text": "Immune checkpoint inhibitors (ICIs) have led to major therapeutic advances in the management of malignancy. Despite promising outcomes for some cancers, ICIs are linked to unique side-effects known as immune-related adverse events (IrAEs). These may affect a wide array of organ systems. In particular, ICI-induced hepatitis is diagnostically challenging given its variable natural history and clinical manifestations. The onset of ICI-induced hepatitis often occurs between 6 and 14 weeks after treatment initiation and rarely exhibits delayed presentations or manifests after treatment cessation. We present a case of very delayed-onset ICI-induced hepatitis, stressing the importance of long-term surveillance for immune-indued hepatitis in patients initiated on ICIs even long after treatment cessation. Immune checkpoint inhibitors (ICIs) have markedly improved the prognosis of patients with some cancers. These novel agents augment the immune system by downregulating inhibitors of the anti-cancer immune response, including: cytotoxic T-lymphocyte-associated antigen 4 (CTLA4), program cell death receptor 1 (PD-1) and its ligand\u2014programmed cell death ligand 1 (PD-L1) [Written informed consent has been obtained from the patient to publish this paper.A 78-year-old female with stage IIIC breast cancer presented with subacute onset of jaundice. Her breast cancer had been treated with pembrolizumab for 5 months and ceased 7 months prior to presentation. She developed a prodrome of progressive fatigue and anorexia over 2 weeks. There were no recent medication changes other than oral mesalazine, which was commenced 3 months prior due to a new diagnosis of unspecified left-sided colitis confirmed during colonoscopy (possibly immune mediated). She was diagnosed with stage IIIB triple negative inflammatory breast cancer in 2015. This was treated with neoadjuvant chemotherapy and surgery with subsequent adjuvant radiotherapy. Approximately 12 months prior to her presentation, she developed a locally advanced right breast recurrence requiring neoadjuvant chemotherapy and PD-1 inhibitor (pembrolizumab) prior to double mastectomy. Initial biochemistry revealed a severe transaminitis. Her bilirubin was 199 \u03bcmol/L (<20 \u03bcmol/L) with an alanine transferase (ALT) of 1519 U/L (<40 U/L), gamma-glutamyltransferase (GGT) of 440 U/L (<60U/L) and alkaline phosphatase (ALP) of 208 (<130 U/L). She had preserved synthetic function with an international normalised ratio (INR) of 1.0 (<1.2) and an albumin of 31 g/L (34\u201354 g/L). Her viral hepatitis serology including hepatitis E virus, cytomegalovirus and Epstein\u2013Barr virus was negative. A liver autoantibody screen was non-contributory. Her immunoglobulin G (IgG) titre was 9.4g/L (7\u201316 g/L). A liver ultrasound and subsequent magnetic resonance cholangiopancreatography (MRCP) showed no structural abnormalities or metastatic disease. The patient was admitted for 2 weeks with a provisional diagnosis of drug-induced liver injury (DILI) secondary to mesalazine or immunotherapy. Mesalazine was ceased and she was managed expectantly. On Day 5, her liver function tests (LFTs) worsened, her bilirubin peaked at 205 \u03bcmol/L with an ALT of 1543 U/L. A liver biopsy revealed pan-lobular hepatitis with scattered apoptic hepatocytes, necro-inflammatory foci, lymphocytosis and foci of interface activity consistent with ICI-induced hepatitis . Given tConsidering her biopsy results and rapid resolution of liver function whilst on glucocorticoid therapy, a diagnosis of delayed immune-induced hepatitis secondary to pembrolizumab was made. The clinical manifestations and natural history of ICI-induced hepatitis are heterogenous. Acute hepatitis occurs in 2\u201310% of patients on ICI therapy, which typically presents as an asymptomatic transaminitis . OccasioICI-induced hepatitis can be pathologically difficult to differentiate from autoimmune hepatitis (AIH) or drug-induced liver injury (DILI). Contrasting with AIH, auto-antibodies including anti-nuclear and anti-smooth muscle antibodies may be negative in ICI-induced hepatitis. Similarly, serum IgG levels are normal or slightly elevated in ICI-induced hepatitis as exemplified by our case . Liver bManagement of ICI-induced hepatitis is based on the Common Terminology Criteria for Adverse Events (CTCAE), which grades severity of disease from 1\u20134 . Our patDelayed-onset IrAEs of any organ system are underrecognised and clinical vigilance is critical even after treatment cessation . The medICIs are becoming an integral part of cancer therapy, and monitoring for adverse events requires awareness from all physicians and not just oncologists. This case pertinently emphasises the importance of long-term surveillance for immune-induced hepatitis in patients initiated on ICI therapy. Attentive appraisal of symptoms, signs and investigations is required for prompt diagnosis of ICI-induced hepatitis allowing for the timely introduction of therapy. Systemic treatment is nuanced and further high-quality evidence is required to guide optimal therapy. We advocate for individualised surveillance strategies for delayed-onset IrAEs up to 12-months after treatment cessation. However, stringent evidence-based surveillance protocols remains an area of ongoing development. Delayed-onset ICI-induced hepatitis can occur up to 12 months post-treatment cessation;Autoimmune biomarkers are often negative in ICI-induced hepatitis and plasmacytosis or eosinophilia is uncommon in the biopsy cellular infiltrate;Low-dose glucocorticoid therapy may be warranted in select ICI-induced hepatitis patients, but further randomized control studies are required to assess optimal glucocorticoid dosing;Routine monitoring for delayed-onset IrAEs including liver function should be individualised and can extend up to 12 months post-treatment cessation."} +{"text": "Tolerance induction was unable to prevent increases in MCMV-specific CD8+ T cells or dissemination of viral IE-1 DNA. Our data suggest that latently-infected allografts are inherently more susceptible to inflammation that is associated with viral dissemination in pre-tolerized recipients. Thus, CMV latently-infected allografts require enhanced strategies to protect allograft integrity and viral spread.Transplantation tolerance is achieved when recipients are unresponsive to donor alloantigen yet mobilize against third-party antigens, including virus. After transplantation, cytomegalovirus (CMV) reactivation in latently-infected transplants reduces allograft viability. To determine if pre-tolerized recipients are resistant to viral dissemination in this setting, we transfused chemically-fixed donor splenocytes (1-ethyl-3- (3\u2032-dimethyl-aminopropyl)-carbo-diimide (ECDI)-treated splenocytes (ECDIsp)) to induce donor antigen tolerance without immunosuppression. In parallel, we implanted donor islet cells to validate operational tolerance. These pre-tolerized recipients were implanted with murine CMV (MCMV) latently-infected donor kidneys to monitor graft inflammation and viral dissemination. Our results indicate that tolerance to donor islets was sustained in recipients after implantation of donor kidneys. In addition, kidney allografts implanted after ECDIsp and islet implantation exhibited low levels of fibrosis and tubulitis. In contrast, kidney cellular and innate immune infiltrates trended higher in the CMV group and exhibited increased markers of CD8 The current standard of care following transplantation is the use of broad spectrum immunosuppressants . DespiteCMV is a member of the \u03b2-herpesvirus family that affects 30\u201397% of the population . Its reaIn this study, we investigated the efficacy of the donor ECDIsp tolerance strategy in a full MHC-mismatch challenge of transplant tolerance. This was tested by implanting a second same-donor solid-organ allograft into an already tolerance-induced animal. Clinically-relevant latently-infected CMV donor kidneys were utilized as solid-organ allografts. Our data suggest that latently-infected allografts are inherently more susceptible to graft inflammation and that tolerance strategies should be optimized in cases where donor organs are latently-infected with CMV.BALB/c and C57Bl/6 (B6) mice were obtained from Jackson Laboratory. All mice were housed under specific pathogen free conditions at Northwestern University\u2019s Center for Comparative Medicine. All procedures used were performed according to guidelines from the Association and Accreditation of the Laboratory of Animal Care International (AAALAC) of the National Institutes of Health (NIH). Northwestern has an Animal Welfare Assurance with the Office of Laboratory Animal Welfare (A3283-01). All protocols (protocol number IS00001426) were approved by the Northwestern University Institutional Animal Care and Use Committee. Eight- to ten-week-old BALB/c mice were infected intraperitoneally with 107 plaque-forming units of the repaired \u0394m157 MCMV recombinant virus. The \u0394m157 MCMV virus was used to avoid MCMV resistance in C57BL/6 mice through natural killer (NK) cell Ly49H receptor and the viral m157 glycoprotein interaction . C. CHI mon+ T cell frequency per mg of tissue and percent of total live cells relative to non-ECDIsp-treated recipient rejecting controls (Kcmv-Tx) grafts also trended lower. There were no significant differences in lymphocyte infiltrate between the ECDIsp-treated groups, but mean values were consistently lower for non-latently infected (K-Tx-ECDIsp) grafts A. ECDIspKcmv-Tx) A. Similahaustion . In this[+ cells B, consisC57BL/6 mice with \u0394m157 recombinant virus when compared to BALB/c mice infected with a well-established MCMV smith virus grafts compared to contralateral, non-latently infected (K-Tx-ECDIsp) grafts, and non-ECDIsp-treated latently-infected rejecting control (Kcmv-Tx), consistent with viral reactivation and replication (+ T cells. We observed an increase in both m38 and m45 MCMV-specific CD8+ T cells from the latently infected (Kcmv-Tx-ECDIsp) grafts (+ T cells have been implicated as memory inflation and a marker of prior infection. The significant increase of m38 specific CD8+ T cells in the latently infected (Kcmv-Tx-ECDIsp) grafts compared to the contralateral non-transplanted donor (Kcmv) grafts suggests prior viral reactivation within the latently infected grafts. Collectively, these data indicate that pre-tolerance induction under our conditions was permissive to viral reactivation and inflammation within the graft, followed by viral replication and dissemination.We next assessed markers of MCMV reactivation from latently-infected kidneys. As introduced above, we transplanted kidneys that were previously infected with \u0394m157 MCMV. Confirmation of latency in donors and prior to kidney transplant was assessed by measuring serum antibody production against MCMV grafts C,D. HighTaken together, our findings indicate that MCMV latently-infected allografts are more susceptible to viral dissemination and exhibit heightened inflammatory trends following transplantation into pre-tolerized recipients. Early ECDIsp treatment was effective at protecting primary allo-islet cells, even after challenge with a second latently-infected donor organ. Although protection from viral dissemination was not conferred, early ECDIsp infusion remarkably induced a state of hypo-responsiveness to subsequent organ implantation, even though this occurred months after the initial ECDIsp infusion. We speculate that reactivation of latent CMV within the kidney graft is likely attributed to reperfusion injury during allograft implantation . We propAt the level of the primary islet grafts, our data indicate stable intra-renal implantation, consistent with the lack of an allogeneic anti-islet response. ECDIsp-induced survival of two islet grafts in succession has previously been demonstrated . Our dat+ and CD8+ T cells. Despite this enhanced protection, both ECDIsp-treated cohorts exhibited heightened histologic scores of fibrosis, tubulitis, and inflammation, relative to the non-transplanted control. Elevations in inflammation could be explained by ischemia reperfusion injury following organ implantation [Within the second implantation group , ECDIsp treatment proved superior to the non-tolerance-induced group, where untreated transplants exhibited increased evidence of tubulitis, and elevated levels of graft monocytes, macrophages, and CD4antation , a potenantation ,33,34. N+ T cells [While ECDIsp was effective at inducing donor graft protection, recipients did not mount effective anti-CMV immunity, even though we measured increased levels of both m38 and acute phase m45 MCMV-specific T cells in the latently infected (Kcmv-Tx-ECDIsp) group, consistent with viral reactivation . One pos T cells ,37,38. O T cells . Further T cells . A likel+ T cells, which is consistent with reactive effector T-cells and transplant rejection [+ T cells that may facilitate viral reactivation. An alternative possibility to explain heightened graft inflammation is a decreased population of donor-specific tolerogenic cells. Within the myeloid compartment, we discovered a trend for increased macrophage expression of trained immunity marker Dectin-1. Nanobiologics that block inflammatory Dectin-1 have been shown to promote tolerance in a heart transplant model [+ dendritic cells were associated with acute transplant rejection [At the cell and molecular level, our study revealed a unique signature that distinguished latently and non-latently-infected transplants after pre-tolerance induction. Latently infected Kcmv-Tx-ECDIsp kidney grafts had reduced PD-1-expressing CD8ejection ,40. It wnt model . Separatejection . The DC-ejection ,42,43. + T cells in the graft suggest reactivation within the ECDIsp-treated latently infected grafts (Kcmv-Tx-ECDIsp). Another consideration relates to potential chronic endpoints after latent-CMV implantation. For example, our data suggest increased inflammation in the latently-infected group. Thus, it may be informative in future experiments to examine later stage pathophysiology and vasculopathy between non-latently infected (K-Tx-ECDIsp) and latently infected (Kcmv-Tx-ECDIsp) transplants. Future experiments may also examine the contribution of additional anti-donor myeloid and lymphoid cell subsets. For example, previous transplant models using ECDIsp have implicated expansion of Tregs and myeloid derived suppressor cells [Our study has important limitations to consider. This includes the potential for future experiments to provide enhanced statistical power to normalize data heterogeneity. This in and of itself is a significant challenge given the logistical complications of these complicated double transplant procedures, including due to recent and permanent disruptions from the COVID-19 coronavirus pandemic. An important limitation of our study\u2014due to the extended time frame of surgeries\u2014was that we did not directly isolate virus from the organs or measure viral RNA expression over multiple time points for a dynamic view of viral transcription and subsequent DNA replication. However, our high viral DNA copies in the ECDIsp-treated latently infected allografts (Kcmv-Tx-ECDIsp), while no virus was detectable in either the donor contralateral kidney (Kcmv) or latently infected rejecting control (Kcmv-Tx) affords reasonable confidence that the kidney allograft is the source of the viral genome. Additionally, the elevated MCMV specific CD8or cells . We alsoregs or the risk of graft versus host disease during mixed chimerism [In conclusion, we report MCMV dissemination after transplantation of latently infected kidneys into pre-tolerance-induced recipients. ECDIsp treatment remains an attractive approach to optimize donor specific tolerance, as for example it does not require ex vivo expansion of Thimerism ,45, alth"} +{"text": "Underlying AD-related neurodegeneration or shared risk factors may influence hearing loss; in an innovative approach we tested whether genetic risk for AD also influences functional hearing loss. We studied 401,084 UK Biobank participants aged 40-70, with Caucasian genetic ancestry, and enrolled 2007-2010. Participants self-reported hearing difficulty and were followed for AD diagnosis until 2018. A genetic risk score for AD (AD-GRS) was calculated as a weighted sum of 23 AD risk variants. In age-, sex-, and genetic ancestry- adjusted models higher AD-GRS was associated with problem hearing in ages 60+, but not ages <60 (p>0.05). Using the AD-GRS as an instrumental variable for AD diagnosis, we estimated that incipient AD increased probability of difficulty hearing at enrollment by 45% . Higher AD-GRS was associated with slightly higher odds of hearing difficulty in older adults. Genetics that predispose for AD also influence late-life hearing difficulty."} +{"text": "The current outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in China threatened humankind worldwide. The coronaviruses contains the largest RNA genome among all other known RNA viruses, therefore the disease etiology can be understood by analyzing the genome sequence of SARS-CoV-2. In this study, we used an ab-intio based computational tool VMir to scan the complete genome of SARS-CoV-2 to predict pre-miRNAs. The potential pre-miRNAs were identified by ViralMir and mature miRNAs were recognized by Mature Bayes. Additionally, predicted mature miRNAs were analysed against human genome by miRDB server to retrieve target genes. Besides that we also retrieved GO (Gene Ontology) terms for pathways, functions and cellular components. We predicted 26 mature miRNAs from genome of SARS-CoV-2 that targets human genes involved in pathways like EGF receptor signaling, apoptosis signaling, VEGF signaling, FGF receptor signaling. Gene enrichment tool analysis and substantial literature evidences suggests role of genes like BMPR2 and p53 in pulmonary vasculature and antiviral innate immunity respectively. Our findings may help research community to understand virus pathogenesis. Coronaviridae and genus Betacoronavirus. Furthermore, phylogenetic analysis suspected bat as the original host of the virus but possibility of intermediate host animal was also purposed [The current outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was confirmed in 12,10956 peoples and there has been 67,594 deaths reported worldwide till now [A brief study of previously identified coronoviruses indicated six human coronaviruses (HCoVs) types: 229E, OC43, NL63, HKU1, SARS-CoV and Middle East respiratory syndrome (MERS-CoV). SARS-CoV and MERS-CoV are of zoonotic origin, and they have been outbreaks earlier during 2003 (China) and 2012 (Saudi Arabia) , 6. The MicroRNAs (miRNAs) are small (~22 nt) non-coding RNAs that play role in post-transcriptional gene regulation by binding to complementary sites on mRNA. The binding may results either in inhibition of translation or complete cleavage of mRNA depending upon complementarity of hybridization , 11. TheThe role of miRNAs in inducing the lung pathology, a characteristic symptom of SARS- CoV was identified previously by analyzing deep sequencing data from the lungs of SARS-CoV-MA15-infected BALB/c mice. 18-22 nucleotide long small viral RNAs were identified from genomic region of SARS-CoV, interestingly it was found that these small RNAs target the host cellular mRNAs 3\u2019UTR specific target sequences and upon in vivo inhibition of these small viral RNAs by antisense inhibitor a significantly decrease in pulmonary inflammation was observed. . But theMainly two approaches have been used for computational miRNAs prediction: ab-intio based and homology based. Homology based approach depends on evolutionary conservation, and therefore limited in locating novel miRNAs in genome. Whereas ab-intio based approach scan for hair-pin loop fold in genome to detect novel pre-miRNAs therefore is more significant , 20. Till date, the RNA viruses-encoded miRNAs have been predicted in Hepatitis-A virus (HAV), Hepatitis-E virus (HEV), Dengue virus (DENV), ZIKA Virus ZIKV), Ebola virus, Japanese Encephalitis virus (JEV), Kyasanur forest disease virus (KFDV) and Nipah virus -28. TherGenome Data Retrieval & Analysis: The complete genome sequence of SARS-CoV-2 was retrieved from NCBI genome database (https://www.ncbi.nlm.nih.gov/genome/) using accession number: MN908947. Genome is positive sense single stranded RNA molecule with linear topology. It contains 29903 nucleotide (nt) base pair.An ab-intio based pre-miRNA prediction software package, Vmir (v2.3) was used for identification of SARS-CoV-2\u2019s pre-miRNAs. VMir package contains two individual modules: VMir analyzer and VMir viewer for prediction and viewing pre-miRNAs respectively . The anaIdentification of Potential pre-miRNAs & mature miRNAs: Potential pre-miRNAs were identified by using ViralMir , a SVM (support vector machine) based web-server. ViralMir was specially designed for viruses with SVM model for prediction has been trained on sequence and structural features of experimentally validated pre-miRNAs data set [ and minimum free energy (MFE) of pre-miRNAs [data set . The Mfoe-miRNAs . Mature e-miRNAs . Prediction of target genes & GO (Gene Ontology) analysis: Target prediction of mature miRNAs against human genome was done using in an online web based server, miRDB (http://mirdb.org/). The custom prediction module of server was used for predicting target genes in human. The server uses seeding approach and scans viral mature miRNAs against 3\u2019 UTR (untranslated regions) of human\u2019s genome for possible hybridization [dization . GO analdization , 35. NCBVMir analysis predicted a total of 1114 hair-pin like pre-miRNAs folds in SARS-CoV-2 genome that were filtered using filtering parameter as described in methodology above. After filtering done by VMir viewer only top 13 pre-miRNAs were selected for further study. Nine pre-miRNAs were found on direct strand whereas four pre-miRNAs on reverse strand. Additionally, all 13 pre-miRNAs were in length range 78-148 nt. The sequence, rank, score, length and orientation are listed in As ab-intio based tools have the limitation of false-positive pre-miRNAs prediction because of selection of the pseudo hair pin loops structures , 37 therAfter authentication of pre-miRNAs, Mature Bayes server was used for retrieving the mature miRNAs. A total of 26 mature miRNAs were obtained from 13 precursors on 5\u2019 and 3\u2019 stem location as shown in ) by custom prediction using mature miRNAs sequences which bind at 3\u2019 UTRs. We selected top scoring target genes with prediction score >80 because score above this threshold are most likely to be real and not required any other supporting evidence [Computational prediction of miRNA-mRNA binding depends on Watson-Crick base pairing which is mostly implemented using seed pairing approach . miRDB aevidence . Gene Ontology term for the target genes were identified by PANTHER database which cluster and group them into biological process , moleculTarget genes were further evaluated using Enrichr, a gene list enrichment analysis tool which retrieved total 82 pathways. On the basis of p value<0.1 few important pathways associated with targeted gene are listed in which arIn the genomic-age, there are now more ways to find and studying miRNA biology, the most trending one is the genome-wide identification of this small non-coding RNAs . The ab-The significance of miRNAs in viral induced respiratory infection and immune regulation has been established previously . Here inWe found significant pathways that may contribute to disease etiology for example apoptosis play an important role in physiological processes and pathogenesis of infectious diseases caused by viruses. nCoV-MD3 -3P target p53 which act as a main inducer of apoptosis pathway during viral infection . p53-depnCoV-MD241-3P target BMPR2 (bone morphogenetic protein receptor type 2) which involved in transforming growth factor (TGF)-\u03b2 signaling pathway. Upon viral infection, BMPR2 gets suppressed which result inhibition of pulmonary vascular homeostasis . The previous studies on different viral miRNAs and their target gene silencing explained interesting facts, particularly about disease etiology -51. Abov"} +{"text": "To the Editor:We would like to comment on Lebin and LeSaint\u2019s overview of chloroquine/hydroxychloroquine (CQ/HoCQ) toxicity and management.Lebin and LeSaint recommended the administration of high-dose diazepam (2 milligrams per kilogram intravenously over 30 minutes) in severely CQ/HoCQ-poisoned patients.Similarly, in a double-blind placebo-controlled study, diazepam did not reverse CQ-induced clinical and electrocardiographic effects in moderate intoxication.High-dose IV epinephrine infusion should also be used with caution. As stated,Because of their direct cardiotoxicity through voltage-dependent sodium- and potassium-channel blockade, CQ/HoCQ may be responsible for rapid-onset dysrhythmia.In conclusion, due to expected CQ/HoCQ overdoses following growing prescriptions in COVID-19 patients, physicians should keep in mind the importance of early intubation and mechanical ventilation when reading the remarkable Lebin and LeSaint\u2019s brief overview."} +{"text": "NANOG functions as the gateway for the generation of pluripotent stem cells (PSCs) in mice and humans. NANOG is a transcription factor highly expressed in pig pre-implantation embryos, indicating that it is a conserved pluripotency-associated factor. However, pig NANOG reporter PSCs have yet to be established, and the regulation of pluripotency by NANOG is not fully understood in this animal.NANOG tdTomato knock-in reporter positive PC-iPS cells were established using CRISPR/Cas9. The resulting cell line was treated with several cytokines and their corresponding inhibitors to identify pathways that regulate NANOG expression. The pathways examined were LIF (leukemia inhibitory factor)/IL6 (interleukin 6)-STAT3, FGF (fibroblast growth factor)/ERK, IGF1 (insulin-like growth factor 1)/PIP3 (phosphoinositide 3-kinase)-AKT, Activin A/SMAD, and BMP4 (bone morphogenetic proteins)/SMAD.In this study, pig NANOG expression in the pig, as is also the case in mice and humans. Activin A directly regulates the expression of pig NANOG via SMAD2/3; inhibition of this pathway by SB431542 resulted in inhibition of NANOG expression.Our experiments showed that the Activin A/SMAD pathway is directly associated with activation of Our results show that Activin A plays an important regulatory role in NANOG-mediated pluripotency in pig iPS cells. Activin A treatment may be therefore an effective method for de novo derivation of authentic embryonic stem cells (ESCs) from pig pre-implantation embryos.The online version of this article (10.1186/s13287-020-1588-z) contains supplementary material, which is available to authorized users. NANOG, OCT4, and SOX2 are key regulatory genes that encode the core pluripotency circuitry in mice, rats, and humans )|, calculated as NANOG tdTomato/WT PC-iPS, was 1 or more, with an adjusted p-value \u22640.05 (n\u2009=\u20093). Genes meeting these criteria are shown as red dots (if more abundant), blue dots (if less abundant), and gray (if relatively unchanged). Genes that are discussed in detail in the text have been labeled. B, Heat map of the clustering analysis of gene expression in NANOG tdTomato knock-in positive and WT PC-iPS cells. The color scale represents the fold-change in expression as |(log2[fold-change])|.: Figure S3. Transcriptome of NANOG tdTomato Knock-in reporter positive PC-iPS versus WT PC-iPS. A, Volcano plot showing distribution of fold-change values (x-axis) and logAdditional file 6NANOG tdTomato knock-in positive PC-iPS cells vs. WT PC-iPS cells. .: Table S3. Differentially expressed genes (DEGs) identified using RNA-Seq data obtained from Additional file 7NANOG tdTomato knock-in positive PC-iPS cells vs. WT PC-iPS cells.: Table S4. KEGG pathway enrichment analysis for differentially expressed genes identified using RNA-Seq data obtained from Additional file 8NANOG tdTomato knock-in positive PC-iPS cells vs. PC-iPS cells.: Figure S4. KEGG pathway analyses of differentially expressed genes identified by RNA-Seq in Additional file 9NANOG tdTomato knock-in positive PC-iPS cells treated with Activin A or SB431542. .: Table S5. Differentially expressed genes (DEGs) identified using RNA-Seq data obtained from Additional file 10NANOG tdTomato knock-in positive PC-iPS cells treated with Activin A or SB431542.: Table S6. KEGG pathway enrichment analysis for differentially expressed genes (DEGs) identified using RNA-Seq data obtained from Additional file 11NANOG tdTomato knock-in positive PC-iPS cells in the presence of Activin A or SB431542.: Figure S5. KEGG pathway enrichment analysis of differentially expressed genes identified by RNA-Seq in Additional file 12NANOG in mice, humans, and pigs.: Model of cytokine regulation of"} +{"text": "A 65-year-old woman with known diabetes and hypertension underwent a technetium methylene diphosphonate (Tc-99m MDP) bone scan with single photon emission computed tomography/computed tomography (SPECT/CT) for shoulder pain. She was initially treated for breast cancer and later for hepatocellular carcinoma. SPECT/CT showed MDP nonavid and scattered pulmonary ground-glass opacities bilaterally along with rounded nodular densities. Another 56-year-old patient who was newly diagnosed with right breast invasive ductal carcinoma underwent a bone scan with SPECT/CT, which revealed bilateral pulmonary infiltrates. Both patients later tested positive for Coronavirus Disease-2019 (COVID-19). Therefore, nuclear physicians should be watchful of findings related to COVID-19 on SPECT/CT thorax as this is becoming the new normal."} +{"text": "The point mutation in unc-108(csn2) is identical to that in the previously characterized loss-of-function allele unc-108(n3263), substituting a glutamine in place of a glycine that is conserved among Ras superfamily members . Similar to other unc-108(lf) mutants, csn2 animals move slowly (not shown). Here we show that unc-108(csn2) as well as previously characterized unc-108 alleles are SIS-defective . While the majority of wild-type N2 animals cease head movement, locomotion and pharyngeal pumping following exposure to damaging conditions, unc-108(lf) animals retain all of these activities. This coordinated impairment of sleep-associated behaviors argues against a role for UNC-108 downstream of the SIS-promoting ALA neuron, which acts via the collective action of neuropeptides with overlapping but distinct effects on the sub-behaviors of sleep .In a genetic screen for mutants defective in stress-induced sleep (SIS) we isolated unc-108(csn2) animals are resistant to EGF-induced sleep (Panel C), indicating that UNC-108 functions downstream of EGF signaling within the SIS pathway. Together these results suggest that UNC-108 functions within ALA.SIS is dependent on Epidermal Growth Factor Receptor (EGFR) activation within ALA, and sleep can be triggered not only by noxious conditions but also by forced overexpression of LIN-3/EGF . We found that C. elegans nervous system and is implicated in the recycling of receptors through the endocytic pathway as well as in dense core vesicle (DCV) maturation . We speculate that the UNC-108 SIS defect arises from deficits in EGFR trafficking or in the maturation of DCVs within the ALA neuron.UNC-108 is widely expressed within the unc-108(n777), ZH382 unc-108(n3263), PS5970 syIs197[hs::LIN-3c(cDNA) + myo-2p::DsRed + pha-1(+)];him-5. Strains available upon request: CVB30 unc-108(csn2), CVB31syIs197;unc-108(csn2).Strains available from the CGC: N2 Bristol, MT1656"} +{"text": "The results showed increases in the nitric oxide synthase activities. Western blotting of the tissue homogenate showed an increase in the nNOS level in the brain and tongue, and an increase in the endothelial NO synthase (eNOS) level in penis. However, there was little association with VEGF production in HUVEC endothelial cells and no relationship with TNF-\u03b1 which showed low levels.This study examined the mechanisms underlying the effects of the vasorelaxation active substance (VAS), dimethyladenosine-5\u2019-L-arabinose, and its partial purification fraction on nitric oxide synthase in improving erectile dysfunction with particular focus on the nitric oxide (NO)-cGMP pathways. Two rat models, 9-month-old SD rats and 11-month-old SD rats, were given VAS (40 mg/kg per day) for 4 days, The aqueous fraction of silworm male pupae extract; semi-purified VAS (100 mg/kg per day) for 10 days, respectively. The NOS activities of the following three enzymes were examined: neuronal NO synthase ("} +{"text": "The coronavirus infectious disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains a world health concern and can cause severe disease and high mortality in susceptible groups. While vaccines offer a chance to treat disease, prophylactic and anti-viral treatments are still of vital importance, especially in context of the mutative ability of this group of viruses. Therefore, it is essential to elucidate the molecular mechanisms of viral entry, innate sensing and immune evasion of SARS-CoV-2, which control the triggers of the subsequent excessive inflammatory response. Viral evasion strategies directly target anti-viral immunity, counteracting host restriction factors and hijacking signalling pathways to interfere with interferon production. In Part I of this review, we examine SARS-CoV-2 viral entry and the described immune evasion mechanisms to provide a perspective on how the failure in initial viral sensing by infected cells can lead to immune dysregulation causing fatal COVID-19, discussed in Part II. The Coronavirus disease 2019 (COVID-19) pandemic is caused by a novel virus termed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Viral entry into host cells is the first stage of infection and a promising drug target. After viral entry, viral sensing by the host cell is essential for the innate immune response to be triggered. However, SARS-CoV-2 can evade the host immune response by targeting cellular host factors. Therefore, it is pivotal to elucidate the mechanisms of viral entry, innate sensing and immune evasion of SARS-CoV-2, which can provide a basis for therapeutic development.To initiate infection, the spike (S) protein of SARS-CoV-2 engages the host cell receptor, angiotensin-converting enzyme II (ACE2), which is highly expressed in lung tissues. Viral materials released into the cytosol can be sensed by the host cell viral recognition machinery which subsequently activates the anti-viral interferon (IFN) pathway. However, SARS-CoV-2 encodes for proteins that counteract these viral recognition pathways or function as IFN antagonists, leading to reduced IFN signalling. In addition, subsets of patients present with genetic mutations in the TLR3 and IRF7 signalling pathways, which lead to defective IFN responses and a worse outcome. This impaired sensing of virus may allow viral replication, which leads to cell damage and systematic immune dysregulation.The coronavirus infectious disease 2019 (COVID-19) pandemic has had devastating global impacts on human health and the economy. Caused by a novel virus termed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), COVID-19 is heterogeneous in clinical presentation and outcome: it is estimated that 20% of patients are asymptomatic, most progress with mild infection and a minority experience acute respiratory distress syndrome . DiseaseViral entry into a host cell usually requires proteins in the viral envelope engaging with cell surface receptors. In COVID-19, SARS-CoV-2 spike (S) glycoprotein binds to the angiotensin-converting enzyme II (ACE2) receptorFollowing receptor engagement, fusion of SARS-CoV-2 with the host cell requires transmembrane serine protease 2 (TMPRSS2) to cleave the S protein at the S1/S2 cleavage site . S1 mediViral tropism determines the target cell of the virus. Therefore, it is important to note other putative receptors for SARS-CoV-2 entry including kidney injury molecule-1 (KIM-1\u2014highly expressed in kidney cells) , CD147 , CD4 4343 and CDFollowing virus entry, the viral genome is exposed, and viral proteins are synthesized by the host cell machinery for assembly of new virions. However, host cells can recognize this viral material (pathogen-associated molecular patterns\u2014PAMPs) via pattern recognition receptors (PRRs). PRRs initiate signalling cascades culminating in the production of pro-inflammatory cytokines and IFNs, which upregulate IFN-stimulated genes (ISGs) that further direct innate and adaptive immunity.in vitro infection with SARS-CoV-2 upregulated pathways for RIG-I signalling in Huh7 cells (liver cell line) [PRRs include cytosolic RNA sensors such as retinoic acid-inducible Gene I (RIG-I) and melanoma differentiation gene 5 (MDA-5), while cytosolic DNA triggers the cyclic GMP\u2013AMP synthase/stimulator of IFN genes (cGAS-STING) pathway. Indeed, ll line) and actill line) . SARS-Coll line) and polyll line) .et al. who found that females produced more IFN\u03b12 and that elevated innate cytokines correlated with disease progression, but only in females [Endosomal toll-like receptor 7 (TLR7) recognizes single-stranded viral RNA (ssRNA) and TLR3 binds double-stranded RNA (dsRNA) generated during viral replication. Therefore, inborn genetic errors affecting the TLR3 signalling pathway result in a defective type I IFN response and are associated with life-threatening COVID-19 . Sex dif females .in vitro infection of lung Calu-3 cells [Fine tuning of IFN responses appears to be key for COVID-19 outcome, as shown by dysregulated responses attributed to auto-antibodies against IFN found in 10% of severe patients . While I-3 cells , especia-3 cells . Similar-3 cells . These lOther innate activation pathways involved in SARS-CoV-2 sensing include NOD-like receptor (NLR) activation and tumor necrosis factor (TNF)-\u03b1 production . NLR famet al. found that NSP1, NSP3, NSP12, NSP13, NSP14, but also ORF3, ORF6 and structural M protein could inhibit the activation of the IFN-\u00df promoter after infection with Sendai virus [Viruses have evolved mechanisms to evade the activation of host innate immune responses . For exaai virus . SARS-Coai virus but the ai virus ; howeverai virus , 70\u201373.et al. extensively screened for ISGs acting as host restriction factors in the context of SARS-CoV-2 infection [Viral proteins also target cellular intrinsic mechanisms of defence, such as anti-viral host restriction factors: proteins that interfere with the viral life cycle. The C-terminus of SARS-CoV-2 NSP1 obstructs the mRNA entry tunnel of the 40S ribosomal subunit, resulting in translation shutoff of host mRNAs . Other vnfection . These Infection . SARS-Confection by targeThe mechanisms of SARS-CoV-2 immune evasion at the early stages of infection are shown in"} +{"text": "The apolipoprotein B/apolipoprotein A-I ratio was shown to be strongly related to the risk of myocardial infarction in several large-scale studies. The current study aimed at exploring the diagnostic and short-term prognostic values of apolipoprotein B/apolipoprotein A-I ratio in patients presenting with non-ST segment elevation acute coronary syndrome. One hundred patients with non-ST segment elevation acute coronary syndrome were prospectively enrolled, in addition to a matched group of 100 patients with chronic stable angina. Serum levels of total cholesterol, low-density lipoprotein, high-density lipoprotein, triglycerides, and apolipoproteins B and A-I were quantified in both groups. Patients with non-ST segment elevation acute coronary syndrome underwent coronary angiography.The mean age of the study population was 57 \u00b1 6\u2009years, 65% being males. The non-ST segment elevation acute coronary syndrome group showed significantly unfavorable lipid profile parameters, including apolipoprotein B/apolipoprotein A-I ratio. Higher apolipoprotein B/apolipoprotein A-I ratio was associated with more coronaries showing significant stenosis and more complex lesion morphology. Receiver operating characteristic curve analysis reached an optimal cut-off value of 0.93 for diagnosis of non-ST segment elevation acute coronary syndrome (sensitivity 70% and specificity 88%) and 0.82 for predicting the presence of multi-vessel disease (sensitivity 90% and specificity 97%).Apolipoprotein B/apolipoprotein A-I ratio is a useful tool of risk assessment in patients presenting with non-ST segment elevation acute coronary syndrome including prediction of coronary multivessel affection.Apolipoprotein B/apolipoprotein A-I ratio was shown to be strongly related to risk of myocardial infarction. Higher ratios of apolipoprotein B/apolipoprotein A-I were recorded in NSTE-ACS patients (versus stable angina patients). Higher apolipoprotein B/apolipoprotein A-I ratios were associated with more diseased coronaries and complex lesions. Apolipoprotein B/apolipoprotein A-I ratio is a useful tool for acute risk assessment in cardiac ischemic patients. Apolipoproteins are proteins linked with lipids within lipoprotein particles. They are known to play essential roles in lipoprotein metabolism. They transfer these hydrophobic molecules in aqueous media of plasma directing lipids to their target organs, activating or inhibiting enzymes implicated in lipid metabolism . ApolipoIn the current study, the authors sought to determine the role of apoB/apoA-I ratio in risk assessment of development of non-ST segment elevation acute coronary syndrome (NSTE-ACS) in patients presented with acute typical chest pain. We also explored the short-term prognostic value of apoB/apoA-I ratio in NSTE-ACS patients meant for coronary angiography and subsequent revascularization.A total of 100 patients presented with NSTE-ACS were enrolled on prospective basis. They were admitted to the coronary care unit (CCU) within the first 6\u2009h of onset of chest pain, in the period between December 2016 and February 2018. All the included patients met the clinical criteria of NSTE-ACS (acute coronary syndromes without ST segment elevation including unstable angina (UA) and non-ST segment elevation myocardial infarction (NSTEMI). NSTEMI is defined by the rElectrocardiographic assessment was performed on admission. A matched group of 100 patients with chronic stable angina was prospectively enrolled. The following patients were excluded: patients with prior history of acute coronary syndrome (ACS), with prior percutaneous coronary intervention, or coronary artery bypass graft surgery; patients with any myocardial disease other than ischemic; and those with history of lipid-lowering drug use (in NSTE-ACS group) or those on high intensity statins (for stable angina patients). Patients with contraindications for aspirin and/or clopidogrel use, limited life expectancy due to coexistent morbidities , and chronic liver or kidney disease were also excluded. Before enrollment, informed written consent was acquired from all patients and the study protocol was ratified by our local institutional human research committee, as it complies to the ethical guidelines of the Declaration of Helsinki quoted in 1975, as revised in 2008.Assessment of both segmental and global left ventricular systolic functions was performed by trans-thoracic echocardiography employing a GE Vivid 7 cardiac ultrasonic machine . Standard 2D, M-mode, and Doppler images were acquired using a 2.5-MHz phased array probe. Modified Simpson`s method was used to assess left ventricular (LV) ejection fraction. LV internal dimensions using M-mode and wall motion abnormalities were recorded. The standard 17-segment model was used to assess segmental wall motion abnormalities as applied by the American Society of Echocardiography . Each seVenous sampling for cardiac troponin I was taken upon CCU admission for patients belonging to the NSTE-ACS group. Patients with initial negative cardiac troponin I results were subjected to serial measurements (every 12\u2009h) for 48\u2009h. Plasma concentration of cardiac troponin I was measured using the Troponin I Ultra assay by Siemens ADVIA Centaur system . Detection value was 6\u2009pg/mL, with a 99th percentile of 40\u2009pg/mL, and a coefficient of variation of <\u200910% at 30\u2009pg/mL. The serum levels of total cholesterol, LDL-C, HDL-C, and triglycerides (TG) were measured using standard methods. Fasting samples were withdrawn for chronic stable angina patients, while samples from the NSTE-ACS patients were withdrawn upon CCU admission (within 6\u2009h of onset of chest pain). Serum apolipoprotein B and A-I levels were recorded using latex agglutination assays . The non-HDL-C concentration was calculated as the total cholesterol minus the HDL-C.Patients belonging to the NSTE-ACS group underwent coronary angiography within the first 48\u2009h after CCU admission. Vascular access was obtained through femoral artery puncture in all patients. Standard angiographic views were obtained. Subsequently, coronary revascularization strategies were tailored individually. Coronary angiographic data were interpreted by 2 independent operators, blinded to the study protocol. It included the number of vessels exhibiting significant luminal stenosis (\u2265\u200970% diameter reduction), maximum grading of luminal stenosis (percentage of diameter reduction), and worst morphologic lesion type defined according to the American College of Cardiology/American Heart Association (ACC/AHA) angiographic classification of coronary lesions .t test. For categorical data, Pearson\u2019s chi-square test was performed. Kappa (k) statistics with the confidence interval was used to assess inter-rater reliability (k was 0.95). ANOVA test was used to estimate the relationship between apoB/apoA-I ratio and each of the number of significantly diseased coronaries and worst atherosclerotic lesion type. P value was used to describe significance . ROC curve (receiver operating characteristic curve) was used to obtain the optimal cut-off value of ApoB/ApoA-I ratio in order to predict the presence of significant \u2265\u20092 vessel disease in NSTE-ACS patients. Statistical calculations were done employing the Statistical Package for Social Sciences (SPSS for Windows) software .The sample size was projected from the study of Krintus et al. , which eA total of 200 patients were subjected to the study protocol. The mean age of the whole study cohort was 57 \u00b1 6\u2009years, 130 (65%) being male patients. Both study groups were matched regarding age, gender, and risk factor of coronary artery disease. Forty-five (45%) patients among the NSTE-ACS group were finally diagnosed as non-ST segment elevation myocardial infarction. There was no recorded significant difference between both study groups regarding echocardiographic data, body mass index, and pre-enrollment medications , LDL-C , non-HDL-C , total cholesterol/HDL-C , and LDL-C/HDL-C . ApoB/apoA-I ratio among chronic stable angina patients showed weaker (less significant) positive correlations with the same parameters as follows: total cholesterol , LDL-C , non-HDL-C , total cholesterol/HDL-C , and LDL-C/HDL-C . ApoB/apoA-I ratio did not show any statistically significant correlation with age, echocardiographic parameters, HDL-C, TG, and TG/HDL-C, in both study groups.Among the NSTE-ACS patients, apoB/apoA-I ratio showed a significant positive correlation with total cholesterol (r = 0.95.Only 85 (85%) patients (NSTE-ACS group) showed angiographically significant coronary artery disease. Angiographic data were interpreted by 2 interventional cardiologists, who were not informed about the study protocol. Analysis of inter-observer variability revealed a close correlation between repeated interpretations, with a correlation coefficient of P = 0.02). Higher mean apoB/apoA-I ratio was significantly associated with more coronary arteries affection. Patients were re-classified according to frequency of presence of worst type of coronary lesions . Using ANOVA test, it was found that there was a significant difference between the three sub-populations regarding mean apoB/apoA-I ratio (P = 0.03). Higher mean apoB/apoA-I ratio was significantly associated with more complex lesion morphology (Table P = 0.07) showing significant stenosis. Using ANOVA test, it was found that there was a significant difference between the three sub-populations regarding mean apoB/apoA-I ratio (o P = 0.0. Higher 7) Table .Table 3Receiver operating characteristic curve analysis Fig. of apoB/The current study presents 2 different roles for apoB/apoA-I ratio in NSTE-ACS patients. The first one can be described as a tool of risk assessment of development of NSTE-ACS in patients presenting with acute typical chest pain. The other one presents a short-term prognostic tool in NSTE-ACS patients undergoing coronary angiography in terms of multivessel affection and lesion morphology which subsequently could affect the revascularization outcome. One of the well-known risk factors in ischemic patients is elevated level of LDL-C. Despite several studies indicating the necessity for LDL-C recording in patients at risk of ACS development, centering only on LDL-C is not suggested as an ideal diagnostic and/or therapeutic approach . The coeIf the total apoB reflects the possibly atherogenic lipoproteins and apoA-I transports the major anti-atherogenic HDL particles, the apoB/apoA-I ratio could reasonably provide a measure of the gross cholesterol balance . That waThe present study uniquely reported the relationship between apoB/apoA-I ratio and coronary angiographic features in NSTE-ACS patients. Higher apoB/apoA-I ratios were associated with more coronary arteries showing angiographically significant coronary artery disease and more lesion complexity. However, there was no significant correlation between apoB/apoA-I ratio and degree of luminal stenosis. We propose that NSTE-ACS is a setting of enhanced oxidative stress and elevated lipoprotein levels, conferring a pronounced pro-inflammatory thrombogenic milieu. On the other hand, a study by Tsimikas et al. reported a positive correlation between apolipoproteins and degree of coronary luminal stenosis . They shThe results of the current study have their assumed clinical implications. High apoB/apoA-I ratio (\u2265\u20090.82) recorded before coronary angiography in NSTE-ACS patients calls for proper readiness due to high probability of tackling a complex multi-vessel disease. This additionally implies the presence of an experienced interventionalist. Thus, apoB/apoA-I ratio could help risk stratification of NSTE-ACS patients upon CCU admission. These patients are expected to have unfavorable angiographic outcomes, in spite of initial and/or persistent negative troponin results. The current study reached a cut-off value of 0.93 for apoB/apoA-I ratio that predicted the final diagnosis of NSTE-ACS. This could be of special importance in patients with initial negative troponin results. This population of patients should be regarded as a high-risk category among ACS patients. Moreover, this could have an impact on pharmacological treatment in CCU.The concluded data presented in this study only apply for patients designated by inclusion and exclusion standards. This is a single-center study with a relatively small sample size. Serial sampling for apoB/apoA-I ratio, especially after coronary revascularization, was not included in the study protocol. Furthermore, follow-up of adverse cardiovascular events was also not included. Further multi-center studies with wider scope are needed for evaluation of long-term impact of high apoB/apoA-I ratio in revascularized NSTE-ACS patients.ApoB/apoA-I ratio is a useful tool for risk assessment in patients presenting with acute typical chest pain. High apoB/apoA-I ratio predicts multi-vessel coronary artery disease with complex lesion morphology."} +{"text": "The development of stimuli-responsive supramolecular micelles with high drug-loading contents that specifically induce significant levels of apoptosis in cancer cells remains challenging. Herein, we report photosensitive uracil-functionalized supramolecular micelles that spontaneously form via self-assembly in aqueous solution, exhibit sensitive photo-responsive behavior, and effectively encapsulate anticancer drugs at high drug-loading contents. Cellular uptake analysis and double-staining flow cytometric assays confirmed the presence of photo-dimerized uracil groups within the irradiated micelles remarkably enhanced endocytic uptake of the micelles by cancer cells and subsequently led to higher levels of apoptotic cell death, and thus improved the therapeutic effect in vitro. Thus, photo-dimerized uracil-functionalized supramolecular micelles may potentially represent an intelligent nanovehicle to improve the safety, efficacy, and applicability of cancer chemotherapy, and could also enable the development of nucleobase-based supramolecular micelles for multifunctional biomaterials and novel biomedical applications. Nano-sized drug delivery systems are widely used in pharmaceutical research and the clinic to enhance the therapeutic effects of anticancer drugs ,2,3,4. TThe creation of stimuli-responsive nanocarriers has opened the door to a new generation of anti-cancer drug delivery systems that are more intelligent and effective than conventional delivery systems ,30. ApprIn our previous studies, we synthesized an intelligent supramolecular polypropylene glycol (PPG) that possesses difunctional uracil-containing end groups (BU-PPG) by a simple one-step Michael addition reaction ,41. BU-PThe general materials, material preparations, and instrumentation used in this work are described in more detail in the 2), the intensity of the C=C double bond absorbance peak at 263 nm in the UV-Vis spectra of BU-PPG solution gradually decreased due to the [2\u03c0 + 2\u03c0] photocycloaddition reaction between uracil groups. The peak eventually leveled off after 60 min irradiation , tadiation . Moreoveadiation . These r50) value (4.12 \u00b1 0.22 \u00b5g/mL) than DOX-loaded non-irradiated micelles (4.42 \u00b1 0.30 \u00b5g/mL), suggesting the irradiated BU-PPG micelles can more rapidly cross the tumor cell membranes, where the acidic intracellular tumor environment subsequently triggers DOX release from the micelles, which induces programmed cell death. Another possible factor could be the higher thermoresponsive behavior of irradiated BU-PPG micelles, which confers a more rapid DOX release rate, compared to DOX-loaded non-irradiated micelles [50 values than free DOX (3.71 \u00b1 0.14 \u00b5g/mL); in the absence of the micelles as a drug delivery carrier, DOX can exert significant cytotoxic effects in normal surrounding cells [We hypothesized that DOX-loaded BU-PPG micelles would enable selective drug release in the acidic intracellular tumor microenvironment and improve the overall therapeutic efficacy of cancer chemotherapy. The cytotoxicity of non-irradiated and irradiated BU-PPG micelles and DOX-loaded micelles against non-cancerous NIH/3T3 fibroblast cells (a mouse embryonic fibroblast cell line) and HeLa cells was evaluated using the methyl blue thiazol tetrazolium (MTT) assay. No significant changes in cell viability were observed after 24 h incubation with a wide range of concentrations of non-irradiated or irradiated BU-PPG micelles at 37 \u00b0C and pH 7.4, even at high micelle concentrations of up to 100 \u00b5g/mL . These rmicelles . Howeverng cells . OverallTo obtain further insight into the cytotoxicity of DOX-loaded non-irradiated and irradiated BU-PPG micelles, we assessed cellular uptake using confocal laser scanning microscopy (CLSM) and flow cytometry. The distributions of DOX-loaded non-irradiated and irradiated micelles (4\u2009\u03bcg/mL) were examined in HeLa cells after 1, 12, and 24 h incubation using CLSM. As demonstrated in Next, we confirmed these observations using quantitative flow cytometry analysis. As shown in In order to clarify the apoptotic pathway and endocytic mechanisms underlying the chemotherapeutic effects of photosensitive supramolecular micelles, HeLa cells were treated with DOX-loaded non-irradiated or irradiated BU-PPG micelles for 1, 6, or 16 h. Annexin V-Alexa Fluor488 (Annexin V)/propidium iodide (PI) double-staining was used to identify and quantify apoptosis/necrosis by flow cytometry. As shown in In vitro cytotoxicity and fluorescence imaging studies indicated the DOX-loaded irradiated micelles were rapidly and efficiently taken up into cancer cells and led to a significantly greater reduction in cell viability and higher proportions of apoptotic cells than non-irradiated micelles. Annexin V/PI-staining and flow cytometric analysis further demonstrated that the presence of the photo-dimerized uracil moieties within the polymeric structures facilitated rapid cellular internalization of the irradiated micelles by HeLa cells. Moreover, the acidic intracellular environment significantly accelerated the release of encapsulated DOX and subsequently triggered high levels of cell death via apoptosis. Thus, the presence of the photo-dimerized uracil moieties within the irradiated micelles enhanced the cellular uptake of therapeutic drugs and led to high levels of apoptosis in tumor tissues under normal physiological conditions. Overall, these photosensitive uracil-based micelles represent a promising multifunctional drug delivery nanovector that may potentially improve the safety and efficacy of cancer chemotherapy.We reported the design of photosensitive BU-PPG polymers containing two uracil end-groups that overcome numerous issues related to existing nanocarriers. Owing to the presence of photo-dimerized uracil and hydrogen-bonded uracil dimers in the polymer structure, BU-PPG spontaneously self-organizes into spherical nanosized micelles in water and can encapsulate DOX at a high drug-loading content."} +{"text": "Eosinophils are key effector cells in allergic diseases. Here we investigated Mcl-1 (an anti-apoptotic protein) in experimental allergic airway inflammation using transgenic overexpressing human Mcl-1 mice (hMcl-1) and reducing Mcl-1 by a cyclin-dependent kinase inhibitor. Overexpression of Mcl-1 exacerbated allergic airway inflammation, with increased bronchoalveolar lavage fluid cellularity, eosinophil numbers and total protein, and an increase in airway mucus production. Eosinophil apoptosis was suppressed by Mcl-1 overexpression, with this resistance to apoptosis attenuated by cyclin-dependent kinase inhibition which also rescued Mcl-1-exacerbated allergic airway inflammation. We propose that targeting Mcl-1 may be beneficial in treatment of allergic airway disease. Eosinophils are key immune cells in the pathogenesis and propagation of allergic airway diseases, including eosinophilic asthma.We have shown that downregulation of Mcl-1 in human eosinophils occurs concurrent with induction of apoptosis following treatment with cyclin-dependent kinase inhibitor (CDKi) drugs, and that induction of eosinophil apoptosis attenuates allergic lung inflammation in mice in vivo.+ve/CD11c-ve/Ly6G-ve/Siglec-F+ve), BALF cells were incubated (5\u00d7106/mL) in serum-free Iscove\u2019s Modified Dulbecco\u2019s Medium (IMDM) for 48\u2009hours +/-AT7519, Q-VD-OPh as per figure legends.In vivo experiments were performed under the UK Home Office Animals (Scientific Procedures) Act 1986, following approval by local ethics committee. Female mice (Charles River or littermate controls)) and transgenic mice overexpressing human Mcl-1 (hMcl-1)Mouse bone marrow-derived eosinophils (bmEos) were generated from WT and hMcl-1 mice as described.Data expressed as mean\u00b1SEM, analysed using GraphPad Prism and FlowJo software. Significance accepted at p<0.05.-ve/PI-ve) was increased in hMcl-1 mice, with a concurrent reduction in apoptotic eosinophils 10.1136/thoraxjnl-2019-213204.supp1Supplementary dataWe next investigated whether enhanced eosinophil viability in the hMcl-1 mice in vivo was intrinsic to eosinophils or a secondary phenomenon due to potential alterations in the inflammatory milieu. Serum and IL-5-starvation of bmEos demonstrated that hMcl-1 bmEos had enhanced viability . Similar+ve eosinophils (The CDKi AT7519 increased apoptosis of airway eosinophils from hMcl-1 mice. This AT7519-induced apoptosis was caspase-dependent (inhibited by the broad-spectrum caspase inhibitor Q-VD) and was associated with reduced Mcl-1 expression . To asseinophils . H&E secinophils while noinophils . OverallUnderstanding mechanisms and regulatory processes controlling inflammation resolution is the focus of recent intense investigation.We report several novel findings that extend our understanding of eosinophil apoptosis in controlling inflammation and tissue injury in airway allergy. First, we show that overexpression of Mcl-1 leads to exacerbated allergic airway inflammation as determined by several inflammation parameters including BALF cellularity, eosinophil numbers, BALF protein and airway mucus production. Whether manipulation of Mcl-1 also leads to changes in airway hyperresponsiveness or remodelling remains to be determined, but is a logical future extension of our current work.Second, we show that Mcl-1 overexpression reduces eosinophil death both in vitro and in vivo in the context of airway allergy, extending our previous observations that Mcl-1 is a key regulator of neutrophil apoptosis,Finally, we show that the apoptosis protection conferred to eosinophils by Mcl-1 overexpression could be overcome by pharmacological lowering of Mcl-1 levels using indirect Mcl-1 inhibition with a CDKi. Furthermore, Mcl-1-exacerbated allergic inflammation in vivo could be rescued by the same approach suggesting that Mcl-1 targeting approaches are capable of overriding the complex pro-survival signals that exist in the inflamed lung. Of note, this is in contrast to glucocorticoid-induced eosinophil apoptosis which can be abrogated by the presence of cytokines such as IL-5.In summary, our results demonstrate Mcl-1 as a significant regulator of eosinophil longevity and the outcome of allergic airway inflammation in vivo. We propose that manipulation of Mcl-1 could be exploited for the treatment of allergic diseases such as eosinophilic asthma in humans."} +{"text": "In this paper, we present MachSMT, an algorithm selection tool for Satisfiability Modulo Theories (SMT) solvers. MachSMT supports the entirety of the SMT-LIB language. It employs machine learning (ML) methods to construct both empirical hardness models (EHMs) and pairwise ranking comparators (PWCs) over state-of-the-art SMT solvers. Given an SMT formula"} +{"text": "To the Editor\u2014Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), has spread worldwide [orldwide . TherefoDespite the hard work of the brave medical workers around the world, many patients continue to die of COVID-19 . Unfortu"} +{"text": "Serologic tests must be carefully evaluated to assess coronavirus disease spread and immunity in tropical regions.We used commercially available ELISAs to test 68 samples from coronavirus disease cases and prepandemic controls from Benin. We noted Diagnosis of the causative pathogen, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is based on reverse transcription-PCR (RT-PCR) to detect viral nucleic acid or serologic assays to detect SARS-CoV-2 antigens in early stages of disease had caused >41 million cases and >1.1 million deaths globally by October 2020, according to the World Health Organization days after RT-PCR confirmation of SARS-CoV-2 infection . We alsohttps://www.euroimmun.com) that rely on different antigens and antibody classes: SARS-CoV-2 nucleocapsid (N) antigen (IgG), spike 1 (S1) subunit (IgG and IgA), and Middle East respiratory syndrome coronavirus (MERS-CoV) S1 (IgG). We also used the SCoV-2 Detect IgG ELISA , an IgG-only S1 antigen-based test authorized for emergency use by the US Food and Drug Administration. Serum samples also were tested by using commercially available ELISA kits (Euroimmun) against the Zika virus (ZIKV) nonstructural protein 1 (NS1) antigen (IgG), the Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) (IgG), and the EBV viral capsid (CA) antigen (IgM and IgG), as well as real-time PCR tests for all human pathogenic Plasmodium species, EBV, and cytomegalovirus (CMV). Plaque-reduction neutralization tests (PRNTs) were performed by using similar methods for SARS-CoV-2 and ZIKV as described , depending on the ELISA used , panel AWhen summarizing all antibody classes, antigens, and kits among the 60 prepandemic controls, we observed 25.0% positive or borderline ELISA results , panel Bt-test revealed no statistically significant difference in the magnitude of antibody titers against common cold coronaviruses between SARS-CoV-2 ELISA-positive or ELISA-negative samples (p = 0.09 for HCoV-OC43 and p = 0.8 for HCoV-HKU1) Figure 2oV-HKU1) Figure 3We assessed SARS-CoV-2 antibody-based serologic diagnostics in Benin and noted unspecific reactivity in up to 25% of febrile patients, possibly due to acute malaria. Limitations of our study include the small sample size and limited patient metadata. Testing of serum samples for CMV and EBV by PCR might not have been sensitive due to lack of cell-associated viral nucleic acid; therefore, we cannot exclude potential herpesvirus reactivation affecting serologic testing. Nevertheless, our analyses point to acute malaria as the likely cause of the unspecific serologic reactivity, although we cannot exclude other coexisting conditions in the tropics, such as dengue virus, which also can affect testing (Unspecific reactivity in serologic tests might affect public health interventions in tropical regions, leading to overestimates of SARS-CoV-2 circulation in regions where malaria is endemic and to misidentification of SARS-CoV-2 hotspots. In addition, due to false-positive SARS-CoV-2 results, target populations for vaccine campaigns might be missed when vaccines become available, and coexistent diseases, such as malaria, might be overlooked, leading to higher mortality rates from endemic diseases (Additional information on the limited specificity of serologic tests for SARS-CoV-2 antibody detection, Benin."} +{"text": "PNBS can be converted into theranostic polymeric nanobiosensors (TPNBS) by incorporating therapeutic cargo, thereby enabling concomitant diagnoses and therapy of targeted diseases. The polymeric compartments in TPNBS play a significant role in the development and therapeutic efficacy of nanobiosensors. Polymers enhance the stability, biocompatibility, and selective or effective accumulation of nanobiosensors at desired pathological sites. The intrinsic pH sensitivity of either the polymers in TPNBS or the TPNBS themselves provides integrated potentialities such as cogent accumulation of TPNBS at the tumor, augmented tumor penetration, cellular uptake, and theranostic activation, including enhanced bioimaging signals and controlled release of therapeutics. In this review, we summarize recent developments in the design, preparation, and characterization of pH-responsive TPNBS and their ability to behave as efficient Cancer is a major threat to human health worldwide. According to recent statistics, in 2020, the United States is projected to have 1,806,590 new cancer cases and 606,520 cancer-related deaths . The canOxidative phosphorylation is a vital pathway of normal cell metabolism; it releases chemical energy from molecular oxygen bonds through the enzymatic oxidation of nutrients during adenosine triphosphate (ATP) synthesis . Oxygen Theranostic nanomedicine for cancer primarily involves the use of colloidal nanoparticles (NPs) ranging from 10 to 1000 nm in size . SynthetStimuli-sensitive nanotheranostics offer advantages over conventional passive or active tumor-targeting strategies. Therefore, different stimuli-responsive nanotheranostic agents have been developed . pH-respConventional cancer treatment methods such as chemotherapy, surgery, and radiotherapy fail to cure cancer completely as they are associated with the impairment of the host immune system, adverse drug reactions, poor patient adherence, and low therapeutic efficiency . Thus faIn recent years, pH-responsive PNBS offering magnetic resonance imaging (MRI), photoacoustic imaging (PAI) fluorescence imaging (FI), ultrasound imaging (USI), and capabilities of improved signal-to-noise ratios in tumors have been developed . Therapein vivo blood circulation, intratumoral penetration, and the subsequent tumor cell internalization of the NPs -encapsulated and photosensitizer (Ce6)-loaded hollow silica NPs (S) were prepared antibody significantly boosted the infiltration of cytotoxic T lymphocytes (CTLs) into remote tumors. It impeded their growth, demonstrating a robust abscopal effect encouraging metastasis inhibition. Similarly, pH-responsive charge reversing NPs featuring enhanced tumor targetability and mitochondrial accumulation for PD-immunotherapy were reported recently (4000-DMMA) using electrostatic interaction. Additionally, pH-responsive charge conversion was used to augment mitochondrial targeting, thereby improving PDT efficacy.The non-immunogenic polymer, such as polyethylene glycol (PEG), shielding on NPs creates an inert NP surface that enhances their blood circulation half-life and passive tumor accumulation . Althougprepared . The NP recently . Sequent2-HP), by using pH-sensitive benzoic-imine bonds for programmed tumor therapy. The prepared DOX@MSN-WS2-HP with the surface-decorated peptide tLyP-1 promotes the tumor-homing ability of 4T1 (2 and tLyP-1weraing WS2-HP. Charge-converted electropositive DOX@MSN-NH2 enabled efficient chemotherapy on surface tumor cells. The deep tumor-penetrating WS2-HP showed tumor suppression in the deep-seated tumor cells by near-infrared (NIR)-light-triggered PTT.y of 4T1 . The benb-poly(\u03b5-caprolactone) (PBA-PEG-PCL), and galactose-functionalized diblock polymer were mixed to prepare dual-ligand micelles to demonstrate this concept ratio during tumor-specific imaging . Thus fa and USI . In thisT1) and transversal (T2) or spin-spin relaxation times of protons are involved in MRI, where 2T \u2264 T1. The relaxation times (T1 or T2) of water protons in lesions can be sped up using an MRI contrast agent. The T1 and T2 contrast agents increase the S/N ratios or the contrast of the lesion for more accurate diagnoses of cancer-related pathological diseases. Therefore, the delivery of MR contrast agents to the tumor by using tumor acidity to improve the S/N of imaging has received significant research attention.Non-invasive imaging techniques, such as MRI, provide a high spatial resolution and detailed three-dimensional anatomical images . During T1 MR contrast agent, releasing PNBS composed of MnO2, has been extensively investigated for its ability to release Mn2+ ions in acidic tumors NPs for the pH-responsive MRI contrast on-off system for signal amplification in subcutaneous C26 tumor-bearing mice. CaP NPs were prepared via the mineralization of poly(ethylene glycol)-b-poly(glutamic acid) [PEG-b-P(Glu)] block copolymers with Mn2+, Ca2+, and HPO2+ ions. This Mn2+ readily interacted with surrounding proteins and slowly rotated the Mn-protein system, which enhanced the sharp contrast due to the increased T1 relaxivity of Mn2+. Additionally, the PEGMnCaP NPs rapidly and selectively brightened and detected hypoxic regions in solid tumors and detected invisible millimeter-sized metastatic tumors in the liver, owing to their pH-responsive signal amplification capabilities. According to another report, a T2 contrast agent, such as Fe3O4 NPs, was loaded in the hydrophobic core of a pH-sensitive polymer (PEG-poly(\u03b2-amino ester) (PAE) micelle and was released and exposed to water molecules at the acidic tumor. Furthermore, a shortened water proton relaxation time T2 responsible for contrast enhancement during MRI was reported has been reported (T2 contrast agent RIAs (120 nm), composed of the T1 contrast agent ultrasmall iron oxide nanoclusters (USIONCs), is disassembled back to USIONCs. The pH-dependent detachment of the i-Motif cross-linker is responsible for this intelligent contrast agent transformation, resulting in bright HCCs and a healthy dark liver owing to the inverse contrast-enhancing imaging mode transformation.For example, a c tumors . Mi et areported . Recentlreported appears as a straightforward solution. However, NBs have low stability and low echogenicity, and they are excessively small to efficiently scatter ultrasonic waves at the frequencies used in clinical applications. Gas-generating NPs are introduced as a contrast agent for US imaging to overcome these issues and generate sufficient USI contrast while maintaining the nanometer-scale size.Ultrasound imaging is portable, cost-effective, and enables real-time imaging. Despite these advantages, the low contrast in USI decreases its sensitivity, thereby limiting its diagnostic applications. Thus, contrast-enhanced US (CEUS), which utilizes 1\u201310-\u03bcm microbubbles (MBs) as the contrast agent, has been developed . However2) gas from mineralized calcium carbonate polymeric NPs , which significantly enhances the US signal of the FA-FRT-PFP through the acoustic droplet vaporization (ADV) effect, is produced by the PFP (2) generation for USI is the use of manganese dioxide (MnO2) NPs that can aptly regulate TME oxygenation owing to their hydrogen peroxidase properties and favorable behavior of breaking-up in mildly acidic and H2O2-rich TMEs as the inert PA matrix, doped with a pH indicator dye, boron-dipyrromethene (pH-BDP), and served as a PA enhancer and amphiphilic triblock copolymer (PEG-b-PPG-b-PEG) as a coprecipitant (0), the PA brightness of the 50 wt% pH-BDP-doped nanoprobe (SON50) was substantially amplified by a factor of approximately 3.1 at 680 nm. Simultaneously, its ratiometric PA signal (PA680/PA750) increased by approximately 3.1 times on varying the pH from 7.4 to 5.5 in vitro. Systemic administration of SONs permits non-invasive real-time ratiometric PAI of the pH with the amplification of brightness in tumors of living mice. Another interesting example of pH-responsive PAI amplification was reported using a polyaniline (PANI)-based theranostic agent composed of bovine serum albumin (BSA) and PANI to the emeraldine salt (ES) state in an acidic TME (pH < 7). Therefore, in healthy tissues (pH \u223c7.4), the BSA\u2013PANI assemblies exhibit low PAI signals and PTT effects during blood circulation, whereas they exhibited amplified PAI and enhanced PTT in acidic TMEs.cipitant . The pH-and PANI activity and extracellular in vivo pH has been reported , ANNA], its fluorescence is quenched while being attached to the surface of an MRI agent (Fe3O4 NPs) using the peptide substrate of MMP-9, representing the \u201coff\u201d state , which serves as an always \u201con\u201d internal reference dye of the resulting secondary ratiometric fluorescent system. The MMP-9 dependent fluorescence emission from ANNA was compared with that of continually emitting Cy5.5 for quantitatively mapping proteases activity across the entire tumor. Extensive imaging studies using these dual-ratiometric probes in a mouse model of human colon cancer revealed that MMP-9 overexpression and abnormal TME pH have spatio-temporal correlations. Additionally, the synergistic effect of these two characteristics largely controls the heterogeneous invasion of malignant tumors. Another example is the use of the pH-sensitive fluorescence on-off nanoprobe to achieve highly selective tumor imaging (2Br2BDP) and loaded in a nanomicelle composed of a cyclic RGD peptide-poly(ethylene glycol)-block-poly(lactic acid) (cRGD\u2013PEG\u2013PLA) unimers is used as a FI nanoprobe .Lower pH in the TME and overexpressed matrix metalloproteases (MMPs) are two indicators of tumor-associated abnormalities. Quantitative and real-time detection of multiple TME factors through non-invasive multimodality imaging is highly informative. Non-invasive visualizations of these abnormality indicators are an effective approach for studying and confirming abnormal tumor signatures reported . The nanf\u201d state . The flu imaging . A pH-acanoprobe . The flu2O2) ROS, singlet oxygen (1O2), peroxy radicals (\u2022O2H), and hydroxyl radicals (\u2022OH) are highly reactive or more cytotoxic to cancer cells. The overproduction of ROS damages lipid bilayers, proteins, and DNA associated with cancer cells and causes cell death and therapeutic efficacy in tumor tissues. Image-guided therapy using TPNBS enables disease detection, monitoring of disease progression, and evaluation of patient response to therapy. ROS-mediated cancer treatment methods, including light-induced PDT , US-indull death .2O2. Alternatively, a triplet excited photosensitizer can directly transfer its energy to molecular oxygen in its ground state (3O2) and convert it into powerful non-radical ROS, 1O2. Therefore, in addition to light and photosensitizers, oxygen is also a key constituent that determines the success of PDT. In poorly oxygenated tumor tissues (tumor hypoxia), the efficacy of PDT is limited ethyl methacrylate) (mPEG-b-PDPA- Cy7.5) micelles were encapsulated with a mitochondria-targeted photosensitizer, triphenylphosphonium-conjugated pyropheophorbide-a (TPPa). The resulting NPs were represented as M-TPPa. The fluorescent signal and photoactivity of M-TPPa were completely switched off through the hetero-FRET from TPPa to Cy7.5 molecules. Hetero-FRET facilitated an amplified photodynamic effect in the tumor tissues and suppressed systemic adverse effects to normal tissues. M-TPPa is exposed to the acidic intracellular environment of human HO8910 ovarian cancer cells. The protonation of the tertiary amino groups of its unimers leads to micelle degradation, followed by the efficient early endosomal escape of TPPa owing to its small molecular structure and high permeability across the lipid membrane. Finally, cationic TPPa relocates into the mitochondria to kill the cancer cells through in situ ROS generation upon laser irradiation, providing robust antitumor efficacy with reduced systemic adverse effects. M-TPPa exhibited a 111-fold increase in the fluorescent signal and a 151-fold enhancement in singlet oxygen generation in acidic cancer cells.In another study, the enzyme HAase was conjugated to a pH-sensitive traceless linker, 3-(bromomethyl)-4-methyl-2,5-furandione (MMfu), modified dextran (DEX), DEX-MMfu Wang et. The obtIn SDT, US-induced cavitation causes sonoluminescence or pyrolysis, which contributes to the production of ROS from sonosensitizers once it meets oxygen or water at the tumor tissues . Dependi2 generation. The degradation associated with the increase in osmotic pressure leads to the redistribution of TPNBS from the endosome to the cytoplasm. In addition to the endosomal escape, the bubbling and bursting of CO2 under the US stimulus results in cavitation-mediated irreversible cell necrosis as well as the destruction of blood vessels to further obstruct blood supply, enabling a \u201cbystander effect.\u201d The generation of ROS from HMME, triggered by US, causes cell apoptosis via SDT. Therefore, the same pH-responsive TPNBS are used for apoptosis and necrosis and to promote cell injury in a complementary manner. The generated CO2 having the echogenic property, is an effective US contrast agent for the identification of cancerous tissues.x) NPs can effectively regulate TME oxygenation owing to their hydrogen peroxidase properties and favorable breakup behavior in mildly acidic and H2O2-rich TMEs (x can breakup via an antioxidant (GSH) depletion reaction in mildly acidic pH through a transition metal-ion catalyzed Fenton-like reaction (2+ (2+ (3+ (+ (Chemodynamic therapy is a unique method for endogenously generating highly cytotoxic ROS (\u2022OH) from TME-rich neutral ROS and H2S gas. The anticancer effect of H2S gas, together with the ROS production due to CDT, enables image-guided cancer therapy in 4T1-luciferase mammary tumor xenograft bearing BALB/c mice with significant suppression of tumor growth. Similarly, a pH-activatable chemodynamic system [ultrathin Mn-oxides MnOx nanosheet (MnII)1(MnIII)3.4(MnIV)2.8O11.7] that can produce 1O2 under the trigger of acidity through a novel chemodynamic process has recently been developed NPs (SPNs); thus, the excited SPNs emit near-infrared (NIR) chemiluminescence through a process called chemical electron exchange luminescence (CIEEL). This process further amplifies the yield of 1O2 generation. In an acidic tumor environment, the MnOx-SPN system achieved satisfactory chemodynamic therapeutic outcomes in vitro and in vivo . The output of 1O2 generation was calibrated using ratiometric imaging of chemiluminescence/fluorescence, which provides more accurate in situ monitoring of the chemodynamic treatment process.Tumoral pH-responsive theranostic NPs are being developed for imaging guided CDT. For example, a size-controllable, biodegradable, and metastable \u03b3-phase MnS nanotheranostic (MnS@BSA) was prepared using BSA as a template for tumor pH-responsive therapy and therapeutic efficacy in acidic tumors with reduced systemic toxicity. Image-guided therapy using TPNBS enables disease detection, monitoring disease progression, and evaluating patients\u2019 responses to treatments. Numerous TPNBS with excellent theranostic functions have been reported recently in basic and preclinical research. These designs need to be relatively simple and biocompatible; most of them, however, are excessively complicated for their clinical translation. Until now, the tumor acidity has been mainly used for developing cancer theranostics. It should be emphasized that the tumor acidity plays a crucial role in tumor initiation, progression, and metastasis. Therefore, developing novel nanotheranostics that modulate tumor acidity would be a promising approach to overcome treatment resistance associated with the current TPNBS.EP, WU, and JP reviewed and evaluated the literature and wrote the manuscript. All the authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Magnetic Resonance Imaging (MRI) can be used to measure common carotid artery wall volume (CCA-CWV) and maximum wall thickness (CCA-CWT) that incorporates adventitial thickness in addition to intima-media thickness (CCA-IMT). Although, the predictors of CCA-IMT are well known, those of CCA-CWV and CCA-CWT have not been studied.We hypothesized that patients with higher CCA-CWV and CCA-CWT have lower high-density lipoprotein (HDL) levels and greater incidence of metabolic syndrome.One hundred and three subjects with IMT >0.65 mm underwent MRI of their carotid arteries using T2-weighted black-blood sequence transaxial slices to measure CCA-CWV and CCA-CWT. Blood pressure (BP), body mass index (BMI), fasting lipid profile and glucose were measured. Metabolic syndrome (MetS) was defined in patients as having \u2265 3 risk factors, BMI \u2265 30, TG \u2265 150 mg/dl, HDL \u2264 40 mg/dl in men and \u226450 mg/dl in women, BP \u2265 130/85 and fasting glucose \u2265 110 mg/dl.CCA-CWV correlated negatively with serum HDL levels , positively with BMI and diastolic BP independent of age, sex, the presence of diabetes, hypertension, hyperlipidemia and smoking. CCA-CWT correlated negatively with serum HDL levels and positively with presence of MetS independent of age, gender and history of smoking.Cardio-metabolic profile is a predictor of carotid artery disease as quantified by magnetic resonance imaging."} +{"text": "Asian/Pacific Island Nursing Journal would like to thank the following people who acted as peer-reviewers for the journal from January 2019 through December 2019.The Editor-in-Chief of Patricia AlpertAmnatsatsue KwanjaiBalakrishnan UmamaheswarBooranasuksakul UraipronNafanua BraginskyByon Ha DoChen Wei-TiChad CrossAlona Dalusung-AngostaFelicitas dela CruzRaymond FolenFaye GaryArtemio GonzalesMargaret Hattori-UchimaMargaret HeitkemperNaoko HikitaDianne IshidaIra Kantrowitz-GordonMerle Kataoka-YahiroKarina KatigbakMasashi KawanoJennifer KawiMay KealohaHeeyoung LeeDongmei LeeWen-Wen LeeRebecca LorenzNada LukkahataiMay-Ying LyJennifer NeversAngelina NguyenNa-Jin ParkAndrew ReyesKarol RichardsonReiko SakashitaTodd SetoSetyowatiLillian Tom-OrmeTsai Hsiu-MinNancy WoodsJoyce YangYonezawa Kaori"} +{"text": "Moreover, decitabine and guadecitabine induce the expression of immune checkpoint molecules in AML cells. In this review, the accumulating knowledge on the immunopotentiating properties of decitabine and guadecitabine in AML and MDS patients are presented and discussed. In summary, combination of decitabine or guadecitabine with NY-ESO-1 vaccine enhances vaccine immunogenicity in AML patients. T cells from AML patients stimulated with dendritic cell (DC)/AML fusion vaccine and guadecitabine display increased capacity to lyse AML cells. Moreover, decitabine enhances NK cell-mediated cytotoxicity or CD123-specific chimeric antigen receptor-engineered T cells antileukemic activities against AML. Furthermore, combination of either HMAs with immune checkpoint blockade (ICB) therapy may circumvent their resistance. Finally, clinical trials of either HMAs combined with cancer vaccines, NK cell infusion or ICB therapy in relapsed/refractory AML and high-risk MDS patients are currently underway, highlighting the promising efficacy of HMAs and immunotherapy synergy against these malignancies.Decitabine and guadecitabine are hypomethylating agents (HMAs) that exert inhibitory effects against cancer cells. This includes stimulation of anti-tumor immunity in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) patients. Treatment of AML and MDS patients with the HMAs confers upregulation of cancer/testis antigens (CTAs) expression including the highly immunogenic CTA NY-ESO-1. This leads to activation of CD4 Acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) are clonal stem cell disorders characterized by heterogeneous clinical outcomes due to underlying molecular and cytogenetic architectures , 2. ChemImmunotherapy has revolutionized cancer treatment where it harnesses and activates patient\u2019s own immune system to destroy cancer cells. Immunotherapy is termed as the \u201cfifth pillar\u201d of cancer therapy that complements or supersedes conventional first-line cancer therapy. However, progress in the application of immunotherapy in AML and MDS such as antibody-based therapy, cellular immunotherapy, immune checkpoint blockade (ICB) agents and cancer vaccines has been slower compared with solid tumors , 10. CurEpigenetics regulation is inherited DNA modifications and external alterations of the physical DNA structure that affect gene expression profiles without modifying nucleotide sequence. The epigenetics modifications include DNA methylation, hydroxymethylation, post-translational histone modifications and nucleosome remodeling \u201315. DNA The hypomethylating agents (HMAs) 5-azacytidine (azacitidine) and 5-aza-2\u2019-deoxycytidine (decitabine) are cytidine nucleoside analogs approved for the treatment of AML and MDS patients. These HMAs mimic cytosine to incorporate into DNA during cellular replication, forming a scaffold that traps DNMTs for proteasomal degradation. This leads to DNA hypomethylation that restores gene transcription particularly tumor suppressor genes in AML , 32. GuaA growing body of evidence has demonstrated the potential of HMAs to promote the efficacy of immunotherapy in AML and MDS patients. In this review, the immunopotentiating effects of decitabine and its dinucleotide guadecitabine to sensitize AML and MDS patients for immunotherapy are presented and discussed. The immunomodulatory properties of azacitidine in AML or MDS patients have been documented in experimental or clinical settings \u201338, and CTAs are a group of tumor-associated antigens (TAAs) whose expression profile is absent in normal adult tissues but highly expressed in normal testicular germ cells and placenta trophoblasts, as well as various types of cancers \u201346. CTAsin vivo against leukemia cells (NXF2). Bone marrow samples derived from primary acute leukemia patients (n=8) also showed upregulation of NXF2 mRNA expression following decitabine treatment, and NFX2 was also upregulated in all AML or MDS patients (n=9) treated with decitabine (NXF2 mRNA expression was associated with demethylation of its promoter region CpG islands in leukemia cells (K562 and U937). However, CTL responses against NXF2-positive AML cells following decitabine treatment was not demonstrated in the study due to lack of known epitope sequence of NXF2 when the study was conducted.Expression of CTAs is often very low or absent in myeloid leukemias due to hypermethylation of the gene promoters. In mouse leukemia cells (L1210), the expression of P1A, a mouse CTA, was upregulated by decitabine treatment. P1A-specific CTLs generated from decitabine-treated DBA/2 mice showed markedly increased cytotoxicity against leukemia cells (L1210) treated with decitabine, indicating that the compound could induce the production of autologous CTA-specific CTLs ia cells . Treatmecitabine . Consist+ AML cells (THP-1) with chidamide and/or decitabine induced sensitivity to CTLs that recognized PRAME peptides presented by HLA-A*0201 on AML cells, and susceptible to cytotoxicity by PRAME-specific CTLs whose expression is primarily upregulated by DNA demethylation and its expression has been associated with favorable outcomes in leukemias including AML . This sufic CTLs . However+TIGIT+CD226\u2212CD8+ T cells were associated with failure to achieve remission after induction chemotherapy and chronic myeloid leukemia (CML) K562) cell lines. In MDS patient samples, the compound increased CTA-specific CTL recognition of upregulated CTAs in bone marrow cells of MDS patients, along with enhanced CTL function and increased expression of major histocompatibility complex (MHC) class I and II proteins as well as ICAM-1 (a cell adhesion molecule that enhances binding with T cells for tumor lysis) . Nonethe2 cell liotherapy .in vitro , and in AML xenografts in vivo (U937 in SCID mice). The CTAs upregulation induced cytotoxicity by HLA-compatible CTLs specific for NY-ESO-1 with increased expression of pro-inflammatory cytokines (e.g. IFN-\u03b3 and TNF-\u03b1) by the CTLs. This might be achieved through upregulation of MHC class I and expression of co-stimulatory molecules required for CTAs presentation. Essentially, guadecitabine at near-equivalent molar doses as decitabine was as efficient as decitabine in promoting CTA and co-stimulatory molecules expression elicited a specific and time-dependent CTL responses in vitro, and that de novo expressed NY-ESO-1 protein was effectively processed and presented by day 6 post-treatment with decitabine. The authors also further showed that CTL responses were cytotoxic against decitabine-treated AML cells (U937), NY-ESO-1 positive AML cells (U937 pulsed with NY-ESO-1 p94-102 peptide), and HLA-B51/NY-ESO-1 positive melanoma cells (LB39) but without cytotoxicity against NY-ESO-1 negative cells (MZ-Mel-7) in vitro. These cell line studies suggest that NY-ESO-1-specific CTLs may be induced by decitabine treatment in AML patients.In human AML cells (Kasumi-1), treatment of decitabine induced the transcript expression of numerous CTA genes preferentially located on the X-chromosome including reatment . NY-ESO-2 daily for 10 days) showed increased NY-ESO-1 and MAGEA3/A6 expression regardless of clinical response, along with promoter-specific and global (LINE-1) hypomethylation. Critically, AML blasts isolated from HLA-A*0201+ AML patients treated with decitabine sufficiently stimulated HLA-A*0201-restricted NY-ESO-1-specific CTL responses as shown by increased levels of intracellular cytokines in HLA-A*0201/NY-ESO-1157-165 tetramer+ CD8+ T cells was combined with fludarabine and low-dose total body irradiation regimen (Dec/Flu/TBI). The Dec/Flu/TBI regimen demonstrated tolerable response with promising OS (53%) post-HSCT. Immunomonitoring showed that CTA-reactive (MAGE-A1/A2/A3 and PRAME) CTL responses post-HCT occurred more frequently in patients who received Dec/Flu/TBI than those with only Flu/TBI conditioning , indicating that decitabine increased CTA-specific T cell responses for improved responses in clinical settings patients . The standard decitabine regimen (20 mg/msettings . EssentiTargeting of surface receptors of dendritic cells (DCs) with antibodies can lead to increased immunogenicity. DCs are frequently adopted in cellular vaccine clinical trials due to their essential roles in presenting antigens to activate immune responses, and capable of conferring promising clinical responses in advanced cancers including AML shown in early phase clinical trials . A commo2/day) showed increased NY-ESO-1 expression in all seven patients investigated. NY-ESO-1-specific CD4+ and CD8+ T cell responses were achieved in 85.7% (n=6/7) and 57.1% (n=4/7) of the vaccinated patients, respectively. Moreover, NY-ESO-1+ myeloid cells from one of the patients on decitabine therapy activated cytotoxic responses from autologous NY-ESO-1-specific T cells express higher levels of DEC-205 and TLR3 than CD1c+ cDCs. In normal immune processes, CD1c+ cDCs promote Th2 and Th17 immune responses against extracellular pathogens and CD4+ T cell priming, while CD141+ cDCs induce Th1 immune responses with a role in CD8+ T cell priming against tumor cells , MDS patients receiving HLA-unrestricted NY-ESO-1 vaccine (anti-DEC-205-NY-ESO-1 fusion protein combined with poly-ICLC) every 4 weeks with decitabine at standard dose (20 mg/m T cells . CD141hior cells . Increascitabine . It was In vivo model (mouse AML cells TIB-49 in C57BL/6J mice) treated with guadecitabine displayed T cells with reduced PD-1 levels but increased IFN-\u03b3 expression, as well as decrease in myeloid-derived suppressor cell (MDSC) populations (i.e. DC/AML fusion vaccine) successfully stimulated anti-tumor responses via the expansion and infiltration of leukemia-specific T cells, leading to prolonged remissions in AML patients post-chemotherapy (PDCD1 gene (that encodes PD-1 protein) promoter demethylation.Treatment of AML cells (THP-1) with guadecitabine resulted in increased expression of HLA-A2.1 with increased antigen presentation. ulations . Personaotherapy . In theiotherapy . Interese.g. nivolumab, pembrolizumab), PD-L1 and CTLA-4 (e.g. ipilimumab) has revolutionized the therapeutic landscape of solid cancers. However, early phase I and II clinical trials of ICB therapy in AML patients have shown limited success and ICB has not been approved for treatment of AML patients. This is due to low immunogenicity of AML compared with solid tumors such as melanoma and NSCLC that are more immunogenic due to higher mutation rates (The co-inhibitory receptor PD-1 and programmed death ligand-1 (PD-L1) are key immune suppressive factors whereby activation of PD-1 by its ligand PD-L1 induces immune tolerance \u201371. CTLAon rates . Cancer on rates , resultion rates \u201382. Furton rates , 84.de novo methylation programs were reversed by decitabine-mediated DNA hypomethylation, leading to enhanced CTLs expansion during ICB therapy (In mice model infected with lymphocytic choriomeningitis virus (LCMV), decitabine treatment followed by blockade of PD-1 reinvigorated the function of exhausted gp33-specific (a dominant LCMV epitope) CTLs. In particular, sequential decitabine treatment and PD-1 blockade induced the proliferation of virus-specific CTLs, suggesting that exhaustion-associated therapy . In humas (KG-1) . An indes (KG-1) . These fHowever, the effect of decitabine on immune checkpoint receptors expression in actual clinical settings is unclear. Donor lymphocyte infusion (DLI) for relapsed AML patients following HSCT is not particularly effective where the overall remission rates are between 15%\u201342% and with low OS of approximately 15%\u201320% \u201390. More+ or CD8+ T cells, Tregs, NK cells, NKT cells, B cells, DCs, and MDSCs. PD-1 expression was not altered upon decitabine treatment, and no significant difference in its expression was observed between responders and non-responders to decitabine. Nonetheless, stimulated CTLs from responders produced significantly higher IFN-\u03b3 than non-responders, while non-responders showed higher expression of multiple immune checkpoints including TIGIT in CTLs and NK cells, and CD38 in CTLs and CD4+ cells (T-cell immunoreceptor with Ig and ITIM domains (TIGIT) is a novel coinhibitory receptor expressed by T and NK cells that binds to CD155, leading to impaired T or NK cell anti-tumorigenic functions . TIGIT i4+ cells . These se.g. through the FAS receptor) leads to apoptosis (KLRK1) is the best characterized NK cell activating receptor that recognizes tumor cells that express NKG2D ligands. A group of eight NKG2D ligands (NKG2DLs) have been identified comprising of MHC class I polypeptide-related sequence A (MICA), MICB, and the UL16-binding proteins (ULBP) 1\u20136 , death-inducing ligands such as FAS ligand, and TNF-related apoptosis-inducing ligand (TRAIL) present on the surface of NK cells. Binding of these ligands by cancer cells and viability were reduced by decitabine treatment in a dose-dependent manner (0.02\u201320 \u00b5M) . This wa+ hematopoietic stem and progenitor cells (HSPC-NK cells), and subsequently treated with low-dose decitabine preserved their proliferation and IFN-\u03b3 production capacity but azacitidine attenuated such effects. In NOD/SCID/IL2Rgnull mice, decitabine but not azacitidine was capable of potentiating infused HSPC-NK cells\u2019 antileukemia activity through upregulation of ligands in AML cells (THP-1) for NKG2D and DNAX accessory molecule-1 (DNAM-1) immunoactivating receptors on HSPC-NK cells. Moreover, decitabine increased the proliferation of HSPC-NK cells in their bone marrow, and combination therapy of adoptive HSPC-NK cells with decitabine was proposed for treatment of AML patients in AML cells with opsonized BI 836858. In serial marrow aspirates of elderly AML patients receiving decitabine, BI 836858-mediated ADCC was enhanced when compared with pre-decitabine treatment. This was mediated by increased mRNA expression of ligands to the activating receptor NKG2D, and blocking of NKG2DL receptor decreased BI 836858-mediated ADCC . Limitatin vivo. The authors subsequently generated membrane-bound IL-21 (mbIL-21) NK cells from normal donors expanded with mbIL-21+ K562 AML cells. The ex vivo expanded mbIL-21 NK cells showed effective lysis of primary AML blasts in vitro and in vivo (patient-derived xenograft mice) which was further enhanced by Fc-engineered anti-CD33 mAb combination reported this year, decitabine treatment followed by haploidentical NK cells infusion and IL-2 administration in R/R AML patients demonstrated that no donor-derived NK cells were detected post-NK cell infusion . The probination .e.g. low-dose decitabine) and cycles of decitabine required to achieve similar antileukemic effects by human NK cells. It is also unclear if low-dose decitabine also similarly activates T cell anti-tumor responses.In other types of blood cancer, decitabine also induced NK cell-mediated anti-tumor immunity through increased IFN-\u03b3 production against CML (K562) and Burkitt\u2019s lymphoma (Raji) cells . Azaciti+/CD11b+/HLA-DR- cells that form an immunosuppressive niche in cancers. MDSCs suppress anti-tumor responses through multiple mechanisms including depletion of amino acids required for T cell proliferation .+/CD11b+) in normal (BALB/c), mouse AML (WEHI-3 cells in BALB/c) and lymphoma (EL4 cells in C57/BL6) mice models with minimal changes to other immune effector cells . The compound induced apoptosis of MDSCs from normal BALB/c mice, and activated CD4+ and CD8+ T cell responses in the mice bone marrow through depletion of MDSC populations. More importantly, in the same study, an adoptive transfusion mouse model demonstrated that decitabine treatment was capable of inducing autologous T cell responses against leukemia cells in vivo (decitabine-treated WEHI-3 cells in BALB/c nude mice) by depleting MDSCs which remains an incurable malignancy that requires novel therapies. Transwell co-culture of mouse IL-6-secreting MM cell line MPC11 with MDSCs showed reduction in IL-6 production by mouse MM cell line (MPC11) and increased apoptosis occurred when the MDSCs were derived from decitabine-treated bone marrow cells compared with the control-treated (PBS) counterpart . These dMDSCs are classified into two major groups: M-MDSCs and granulocytic MDSCs (G-MDSCs) where each group morphologically and phenotypically resembles monocytes and neutrophils, respectively, and M-MDSCs confer stronger immunosupressive activities than G-MDSCs , 122. ItInterleukin-3 receptor (IL-3R) is a heterodimeric receptor comprising of the IL-3-specific alpha subunit and the beta subunit (CD131 or IL-3RB) that is shared by receptors for IL-5 and granulocyte-macrophage colony-stimulating factor . IL-3 in388IL-3) is an engineered fusion protein comprising of IL-3 to target CD123 fused with a truncated diphtheria toxin (DT) payload. Tagraxofusp triggers cellular cytotoxicity by delivering DT to CD123+ cells where DT escapes endosomes post-internalization and catalyzes ADP ribosylation of eukaryotic elongation factor 2 , leading to inhibition of protein synthesis that kills the cell where their combination prolonged survival of the mice compared with either agent alone is another therapeutic mAb against CD123 where it is a humanized, affinity-matured and Fc-engineered mAb for increased affinity to CD16 expressed by innate effector cells. Talacotuzumab potently induces AML patient\u2019s own NK cells to destroy AML blasts and LSC-enriched populations via ADCC , 135. Phvia ADCC . Howeverreatment . An indereatment . The autTP53 mutations, 47% (n=7/15) demonstrated CR to flotetuzumab and displayed increased tumor inflammation signature as well as CD8, inflammatory chemokine, and PD-1 expression compared with non-responders. These patients who achieved CR experienced prolonged survival (+ AML cells (In terms of bispecific antibodies (bsAbs), the following bsAbs targeting both CD123 and CD3 (CD123 x CD3 bsAb) with promising efficacy are currently being assessed in early phase clinical trials of R/R AML patients, and their combination with HMAs has yet to be investigated: i) Flotetuzumab (MGD006 or S80880) is a bispecific dual-affinity re-targeting antibody (DART) that recognizes CD123 and CD3\u03f5 where it redirects T cells to destroy CD123-expressing cells. It was granted orphan drug designation by FDA for the treatment of AML in January 2017 . In R/R survival . Phase IML cells . Currentde novo DNA methylation mediated by DNMT3A is one of the key mechanisms that restrains long-term T cell memory and induces cytotoxic T cells exhaustion (in vitro (THP1 cells) and in vivo (NSG mice bearing THP1 tumor xenografts). This was achieved by decitabine through inhibition of DNMT3A and DNMT1 expression, increased DNA hypomethylation and upregulated expression of genes that favored na\u00efve and memory T cells differentiation, resulting in enhanced CD123 CAR-T cells anti-tumor responses targeting the specific antigen, and this forms the basis of TCR-engineered T cell (TCR-T) therapy , 146. TCrelapsed . The GvLIn conclusion, decitabine and guadecitabine have shown promising immunopotentiating properties to improve the efficacy of immunotherapies Figure 2i.e. thalidomide, lenalidomide and pomalidomide) shown to confer antileukemic T cell immunity in AML and MDS , Universiti Sains Malaysia awarded to KKW.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Viral whole-genome sequencing (WGS) provides critical insight into the transmission and evolution of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Long-read sequencing devices from Oxford Nanopore Technologies (ONT) promise significant improvements in turnaround time, portability and cost, compared to established short-read sequencing platforms for viral WGS . However, adoption of ONT sequencing for SARS-CoV-2 surveillance has been limited due to common concerns around sequencing accuracy. To address this, here we perform viral WGS with ONT and Illumina platforms on 157 matched SARS-CoV-2-positive patient specimens and synthetic RNA controls, enabling rigorous evaluation of analytical performance. We report that, despite the elevated error rates observed in ONT sequencing reads, highly accurate consensus-level sequence determination was achieved, with single nucleotide variants (SNVs) detected at >99% sensitivity and >99% precision above a minimum ~60-fold coverage depth, thereby ensuring suitability for SARS-CoV-2 genome analysis. ONT sequencing also identified a surprising diversity of structural variation within SARS-CoV-2 specimens that were supported by evidence from short-read sequencing on matched samples. However, ONT sequencing failed to accurately detect short indels and variants at low read-count frequencies. This systematic evaluation of analytical performance for SARS-CoV-2 WGS will facilitate widespread adoption of ONT sequencing within local, national and international COVID-19 public health initiatives. Nanopore sequencing (ONT) has been used in SARS-CoV-2 studies, however adoption of ONT for SARS-CoV-2 surveillance has been limited due to common concerns around sequencing accuracy. Here, the authors perform a comprehensive evaluation of ONT analytical performance on 157 matched SARS-CoV-2-positive patient specimens and synthetic RNA controls. SARS-CoV-2 is a positive-sense single-stranded RNA virus with a ~30-kb poly-adenylated genome2. Complete genome sequences published in January 20203 enabled development of RT-PCR assays for SARS-CoV-2 detection that have served as the diagnostic standard during the ongoing COVID-19 pandemic4.Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative pathogen for COVID-19 disease5. Integration with epidemiological data identifies transmission networks and can infer the origin of unknown cases11. Largescale, longitudinal surveillance by viral WGS may also provide insights into virus evolution, with important implications for vaccine development15.Whole-genome sequencing (WGS) of SARS-CoV-2 provides additional data to complement routine diagnostic testing. Viral WGS informs public health responses by defining the phylogenetic structure of disease outbreaks16.WGS can be performed via PCR amplification or hybrid-capture of the reverse-transcribed SARS-CoV-2 genome sequence, followed by high-throughput sequencing. Short-read sequencing technologies enable accurate sequence determination and are the current standard for pathogen genomics. However, long-read sequencing devices from Oxford Nanopore Technologies (ONT) offer an alternative with several advantages. ONT devices are portable, cheap, require minimal supporting laboratory infrastructure or technical expertise for sample preparation, and can be used to perform rapid sequencing analysis with flexible scalability19. Although protocols for ONT sequencing of SARS-CoV-2 have been established and applied in both research and public health settings22, adoption of the technology has been limited due to concerns around accuracy. ONT devices exhibit lower read-level sequencing accuracy than short-read platforms25. This may have a disproportionate impact on SARS-CoV-2 analysis, due to the virus\u2019 low mutation rate (8\u2009\u00d7\u200910\u22124 substitutions per site per year26), which ensures erroneous or undetected genetic variants have a strong confounding effect.The use of ONT devices for viral surveillance has been demonstrated during Ebola, Zika and other disease outbreaksIn order to address concerns regarding ONT sequencing accuracy and evaluate its analytical validity for SARS-CoV-2 genomics, we have performed amplicon-based nanopore and short-read WGS on matched SARS-CoV-2-positive patient specimens and synthetic RNA controls, allowing rigorous evaluation of ONT performance characteristics.27. We first sequenced synthetic RNA controls that were generated by in vitro transcription of the SARS-CoV-2 genome sequence. The controls matched the Wuhan-Hu-1 reference strain at all positions, allowing analytical errors to be unambiguously identified. To mimic a real-world viral WGS experiment, synthetic RNA was reverse-transcribed then amplified using multiplexed PCR of 98\u2009\u00d7\u2009~400\u2009bp amplicons that enabled evaluation of ~95% of the SARS-CoV-2 genome. Eight independent replicates were sequenced on ONT PromethION and Illumina MiSeq instruments (see Methods section).Synthetic DNA or RNA reference standards can be used to assess the accuracy and reproducibility of next-generation sequencing assaysR2\u2009=\u20090.67; indel R2\u2009=\u20090.82; Supplementary Fig.\u00a0R2\u2009=\u20090.15; indel R2\u2009=\u20090.42), indicating that short-read sequencing errors were less systematic than for ONT libraries . We detected just two erroneous variant candidates in a single ONT library (Table\u00a0n\u2009=\u20098). All Illumina libraries exhibited perfect accuracy . Short-read sequencing was performed according to a pathogen genomics accredited diagnostic workflow in a reference NSW Health Pathology laboratory, enabling direct comparison of nanopore sequencing to the established standard for pathogen genomics.To further evaluate the suitability of ONT sequencing for SARS-CoV-2 genomics, we conducted rigorous proficiency testing using bona fide clinical specimens. We performed ONT and Illumina WGS on matched, de-identified SARS-CoV-2-positive cases collected at public hospital laboratories in Eastern & Southern New South Wales and Metropolitan Sydney from March to April 2020 (see Methods section). Selected specimens covered a range of SARS-CoV-2 lineages and viral titres genome coverage with both technologies for 157 matched positive cases . In general, ONT variant candidates identified by both pipelines were highly concordant with the Illumina comparison set. Illumina variants were detected with 99.17% sensitivity and 99.58% precision by Nanopolish, compared to 98.33% sensitivity and 99.24% precision by Medaka and 153/157 samples (97%), respectively of samples . Both sensitivity and precision of variant detection were strongly influenced by sequencing coverage, showing a sharp decline below ~50-fold coverage depth, with minimal improvement observed above ~60-fold , to generate triplicate (n\u2009=\u20093) data on both Illumina and ONT platforms. We measured reproducibility by performing pairwise comparisons of detected variant candidates between replicates for a given sample and perfect concordance for SNVs . Analysis of the SARS-CoV-2 synthetic RNA controls and replicate short-read sequencing libraries (see above) showed that sequencing artefacts in Illumina libraries could be misinterpreted as variants at read-count frequencies below ~20% , indicating that these were bona fide variants, rather than sequencing artefacts .34. Therefore, we next evaluated the detection of SVs in SARS-CoV-2 specimens with ONT sequencing. We used NGMLR-Sniffles to identify potential SVs in ONT libraries and validated these with supporting evidence from short-read sequencing (see Methods section).Large genomic deletions or rearrangements can have a major impact on virus function and evolution, however, there are currently just a few reported cases of SARS-CoV-2 specimens harbouring structural variants (SVs)S, M, N, ORF3, ORF6, ORF8 and orf1ab are relatively inexpensive, highly portable and require minimal associated laboratory infrastructure; (ii) enable rapid generation of sequencing data and even real-time data analysis; (iii) require comparatively simple procedures for library preparation and; (iv) offer flexibility in sample throughput, accommodating single , multiple or tens/hundreds of specimens per flow-cell26, accurate sequence determination is vital to correctly define the phylogenetic structure of disease outbreaks. With ONT sequencing known to exhibit higher read-level sequencing error rates than short-read technologies25, reasonable concerns exist about suitability of the technology for SARS-CoV-2 genomics. Moreover, public databases for SARS-CoV-2 data already contain consensus genome sequences generated via ONT sequencing, potentially confounding investigations that rely on these resources.Due to the relatively low mutation rate observed in SARS-CoV-2Nanopolish showing modest improvements over Medaka; (iii) a minimum ~60-fold sequencing depth was required to ensure accurate detection of SNVs, but little or no improvement was achieved above this level; (iv) false-positive and false-negative variants were typically observed at low-complexity sequences, with fidelity improved by excluding these problematic sites; (v) in contrast to consensus SNVs, ONT sequencing performed poorly in the detection of consensus indels or low-frequency variants (such variants should therefore be interpreted with caution); (vi) while the high indel error rate in ONT sequencing impedes accurate detection of small indels, long nanopore reads appear well-suited for the detection of large deletions and potentially other structural variants. Although SNVs alone are sufficient for routine phylogenetic analysis, small indels and large structural variants can profoundly impact gene function and are, therefore, of interest to studies of virus evolution and pathogenicity15.The present study resolves these concerns, demonstrating accurate consensus-level SARS-CoV-2 sequence determination with ONT data. We report that: (i) variants at consensus-level read-count frequencies (80\u2013100%) were detected with >99% sensitivity and >99% precision across 157 SARS-CoV-2-positive specimens, confirming the suitability of ONT sequencing for standard phylogenetic analyses; (ii) high accuracy and reproducibly was achieved by each of two alternative tools for ONT variant detection, with As the first systematic evaluation of nanopore sequencing for SARS-CoV-2 WGS, this study removes an important barrier to its widespread adoption in the ongoing COVID-19 pandemic. While short-read sequencing platforms remain the gold-standard for high-throughput viral sequencing, the advantages to portability, cost and turnaround-time afforded by nanopore sequencing imply that this emerging technology can serve an important complementary role in local, national and international COVID-19 response strategies.https://artic.network/ncov-2019). Briefly, reverse-transcription was performed on aliquots of synthetic RNA (at 106 copies per \u03bcL) using Superscript IV (Thermo Fisher Scientific) with both random hexamers and oligo-dT primers. Prepared cDNA was then amplified using multiplexed PCR with 98\u2009\u00d7\u2009~400\u2009bp amplicons tiling the SARS-CoV-2 genome . The controls comprise synthetic RNA generated by in vitro transcription (IVT) of the SARS-CoV-2 genome sequence, representing the complete genome in 6\u2009\u00d7\u2009~5\u2009kb continuous sequences. The controls used in this study are identical in sequence to the Wuhan-Hu-1 reference strain MN908947.3), allowing sequencing artefacts to be readily identified. Synthetic controls were prepared for sequencing via a protocol established by the ARTIC network for viral surveillance , were retrieved from storage and included in this study. Wherever relevant, ethical regulations for work with human participants with informed consent were observed, with oversight by HREC at South Eastern Sydney Local Health District . All specimens were nasopharyngeal swabs originating from patients in New South Wales during March\u2013April 2020. Specimens underwent total nucleic acid extraction using the Roche MagNA Pure DNA and total NA kit on an automated extraction instrument (MagNA pure 96). Reverse-transcription was performed on viral RNA extracts using Superscript IV VILO Master Mix (Thermo Fisher), which contains both random hexamers and oligo-dT primers. Prepared cDNA was then amplified separately with each of 14\u2009\u00d7\u2009~2.5-kb amplicons tiling the SARS-CoV-2 genome, as described elsewhere36. Primer sequences were trimmed from the termini of read alignments using iVar (1.0)37. Trimmed alignments were converted to pileup format using samtools mpileup (v1.9)38, with anomalous read pairs retained (--count-orphans), base alignment quality disabled (--no-BAQ) and all bases considered, regardless of PHRED quality (--min-BQ 0). Variants were identified using bcftools call (v1.9)38, assuming a ploidy of 1 (--ploidy 1), then filtered for a minimum read depth of 30 and minimum quality of 20. Variants were classified according to their read-count frequencies as consensus (>80% reads supporting the variant) or sub-consensus (20\u201380%) variants, with the latter further divided into high (60\u201380%), intermediate (40\u201360%) or low-frequency (20\u201340%). Variants at read-count frequencies below 20% were considered to be potentially spurious and excluded on this basis.Pooled amplicons were prepped for short-read sequencing using the Illumina DNA Prep Kit, according to the manufacturer\u2019s protocol. Samples were multiplexed using Nextera DNA CD Indexes and sequenced on an Illumina MiSeq. Within each sequencing lane, a blank sample was also prepared and sequenced, in order to monitor for contamination and/or index swapping between samples. The resulting reads were aligned to the Wuhan-Hu-1 reference genome (MN908947.3) using bwa mem (0.7.12-r1039)RAMPART (v1.0.6) software package39 was used to monitor sequencing performance in real-time, with runs proceeding until a minimum ~200-fold coverage was achieved across all amplicons. At this point, the run was terminated and the flow-cell washed using the ONT Flow Cell Wash kit (EXP-WSH003), allowing re-use in subsequent runs.ARTIC amplicons (~400\u2009bp) from the synthetic RNA controls were prepared for nanopore using the ONT Native Barcoding Expansion kit (EXP-NBD104). The longer amplicons (~2.5\u2009kb) used on SARS-CoV-2 patient specimens were prepared for nanopore sequencing using the ONT Rapid Barcoding Kit (SQK-RBK004). Both kits were used according to the manufacturer\u2019s protocol. Up to 12 samples were multiplexed on a FLO\u2010FLG001, FLO-MIN106D or FLO-PRO002 or flow-cell and sequenced on a GridION X5 or PromethION P24 device, respectively. In addition, a no-template negative control from the PCR amplification step was prepared in parallel and sequenced on each flow-cell (Supplementary Data\u00a0Guppy (4.0.14) and aligned to the Wuhan-Hu-1 reference genome (MN908947.3) using minimap2 (2.17-r941)40. The ARTIC tool align_trim was used to trim primer sequences from the termini of read alignments and cap sequencing depth at a maximum of 400-fold coverage. Consensus-level variant candidates were identified using each of two workflows developed by ARTIC (https://github.com/artic-network/artic-ncov2019), using Nanopolish41 or Medaka (0.11.5) to variants, respectively. Nanopolish variants candidates were filtered directly with the ARTIC artic_vcf_filter tool, while Medaka candidates were evaluated by LongShot (0.4.1)42 before filtering. Sub-consensus level variant candidates were identified using Varscan2 (v2.4.3)43.The resulting reads were basecalled using pysamstats, with any bases that differed from the Wuhan-Hu-1 reference sequence considered errors.For synthetic RNA controls, read-level quality metrics, such as sequencing error rates, were derived from read alignments using bcftools norm (1.9)38; (ii) multi-nucleotide variants were decomposed into their simplest set of individual components using rtg-tools vcfdecompose (3.10.1) and; (iii) indels at simple repeats were left-aligned using gatk LeftAlignAndTrimVariants (4.0.11.0). Variant candidates identified by Illumina/ONT could then be considered concordant based on matching genome position, reference base and alternative base/s. For a given case, variant candidates identified with ONT and Illumina were classified as true-positives (TPs), candidates identified by ONT but not Illumina as false-positives (FPs) and candidates identified by Illumina but not ONT as false-negatives (FNs). The following statistical definitions were used to evaluate results:The accuracy of variant detection by ONT sequencing was evaluated by comparison to the set of variants identified by Illumina sequencing in matched cases. To ensure consistent representation of variants across calls generated by different programs: (i) multi-allelic variant candidates were separate into individual SNVs/indels using NGMLR (v0.2.7)44. Sniffles (v1.0.11)44 was then used to detect candidate variants with a minimum length of 10\u2009bp and \u226520 supporting reads. To validate SVs detected with ONT alignments, split short-read alignments and discordant read-pairs were extracted from matched Illumina libraries using lumpy45. Variant candidates were then manually inspected to verify evidence from ONT and short-reads and assess breakpoint position reFurther information on research design is available in the Nature Research Reporting Summary linked to this article.solution.To identify structural variation, nanopore reads were re-aligned to the Wuhan-Hu-1 reference genome (MN908947.3) using the rearrangement-aware aligner Further information on research design is available in the Nature Research Reporting Summary linked to this article.Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Data 6Supplementary Data 7Supplementary Data 8Reporting Summary"} +{"text": "The papillary muscle (PM) is an integral component of the mitral valve apparatus. Acute or chronic myocardial infarction (MI) with PM ischemia is a primary factor leading to the occurrence of mitral regurgitation, with associated substantial morbidity and mortality , 2. PM-MWe hypothesize that multi-contrast delayed enhancement (MCDE) imaging will improve the identification of PM-MI in patients with acute and chronic MI, compared to conventional IR-FGRE imaging.Cardiac DE-MRI studies using both MCDE and IR-FGRE in patients with MI were reviewed. Twenty-three patients met the diagnostic criteria of PM-MI, as outlined below. All studies were performed on a 1.5 T GE Signa HDx system , which included a short-axis oblique (SAO) and two or four chamber SSFP studies. Both IR-FGRE and MCDE covering the whole LV in SAO were performed 10\u201320 minutes after double-dose bolus injection of Gd-DTPA. For IR-FGRE, the TI varied from 200 to 300 ms, depending on the null point of healthy myocardium. For MCDE, a segmented SSFP readout is used following an inversion pulse, providing 20 cardiac-phase-resolved images at varying effective TIs [PM-MI was considered if the following criteria were satisfied in the IR-FGRE or MCDE images: (1) the increased signal intensity of PM was similar to that of adjacent hyper-enhanced infarct segments; (2) the hyper-enhanced PM region was limited to the PM area defined by pre-contrast SSFP. The contrast between blood pool and hyper-enhanced LV infarct was rated as excellent (3), good (2) or fair (1) based on their differentiation.Based on the standard AHA 17-segment model , all patMCDE imaging provides better contrast between blood pool and infarcted myocardium, thus improving the determination of PM-MI that may help identify patients in whom the significant mitral regurgitation may affect morbidity and mortality."} +{"text": "Bruton\u2019s tyrosine kinase (BTK) inhibitors, drugs utilized in cancer, are being repurposed for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) (COVID-19). Recently, BTK inhibitors acalabrutinib and ibrutinib have been found to protect against pulmonary injury in a small group of patients infected with SARS-CoV-2. The high levels of pro-inflammatory cytokines found in the circulation of COVID-19 patients with severe lung disease suggest the involvement of the innate immune system in this process. Understanding the potential mechanism of action of BTK inhibition in SARS-CoV-2 is clearly of importance to determine how acalabrutinib, ibrutinib and possibly other BTK inhibitors may provide protection against lung injury. To the Editor:et\u00a0al. have also demonstrated that BTK is essential for NLRP3 inflammasome activation and contributes to ischemic brain injury [SARS-CoV-2 infects nasal and respiratory epithelial cells via attachment to angiotensin-converting enzyme 2 (ACE2) , 2 whichn injury . These fn injury \u201312. Inden injury . Previoun injury \u201316.A clinical study was recently conducted that reported on six Waldenstrom macroglobulinemia patients who were taking the BTK inhibitor ibrutinib and were subsequently diagnosed with COVID-\u00a019. These six patients exhibited only mild COVID-19-related symptoms . In this"} +{"text": "Cross-cultural validation of the FACE-Q Satisfaction with Facial Appearance Overall Scale (FACE-Q SFAOS) in Brazilian rhytidoplasty patientsIn the article Replace"} +{"text": "Respiratory motion in PET/CT leads to well-known image degrading effects commonly compensated using elastic motion correction approaches. Gate-to-gate motion correction techniques are promising tools for improving clinical PET data but suffer from relatively long reconstruction times. In this study, the performance of a fast elastic motion compensation approach based on motion deblurring (DEB-MC) was evaluated on patient and phantom data and compared to an EM-based fully 3D gate-to-gate motion correction method (G2G-MC) which was considered the gold standard.18F]FDG PET/CT examinations applying hardware-based respiratory gating. In addition, a dynamic anthropomorphic thorax phantom was studied with PET/CT simulating tumour motion under controlled but realistic conditions. PET signal recovery values were calculated from phantom scans by comparing lesion activities after motion correction to static ground truth data. Differences in standardized uptake values (SUV) and metabolic volume (MV) between both reconstruction methods as well as between motion-corrected (MC) and non motion-corrected (NOMC) results were statistically analyzed using a Wilcoxon signed-rank test.Twenty-eight patients were included in this study with suspected or confirmed malignancies in the thorax or abdomen. All patients underwent whole-body [max, SUV mean, MV, and contrast-to-noise ratio (CNR) for both reconstruction algorithms. Furthermore, both methods showed similar increases of 11\u201312% in SUV max and SUV mean after MC. The statistical analysis of the MC/NOMC ratio found no significant differences between the methods.Phantom data analysis showed high lesion recovery values of 91% (2 cm motion) and 98% (1 cm) for G2G-MC and 83% (2 cm) and 90% (1 cm) for DEB-MC. The statistical analysis of patient data found significant differences between NOMC and MC reconstructions for SUV max, SUV mean, and CNR after MC on clinical and phantom data. The fast elastic motion compensation technique DEB-MC may thereby be a valuable alternative to state-of-the art motion correction techniques.Both motion correction techniques deliver comparable improvements of SUV At the same time, PET has continuously been improved from a low-sensitive 2D imaging technology with poor spatial resolution into a high-resolution 3D technique with excellent signal-to-noise (SNR) characteristics using time-of-flight (TOF) capabilities , 28M durmax, SUV mean) and the metabolic 18F-FDG volume (MV) were reported. For tumour segmentation, SUV mean and the metabolic volume are based on a 50% threshold of SUV max of each lesion:To evaluate the effect of MC on quantitative data, volume-of-interest (VOI) defined on multiple lesions were analyzed, and changes (ratios of MC/NOMC) in standardized uptake values and maximum (MAX) activity concentrations were calculated together with the recovery coefficients (RC) between the reconstructed images and GT (at maximum expiration). To this end, MVs were defined on the GT images for both, e7 and EMrecon. These MVs were also used to analyze the corresponding NOMC and MC data. The RC was defined as the mean activity concentration divided by the mean activity concentration of the corresponding GT data.As image quality parameter, the contrast-to-noise ratio (CNR) of the lesions was analyzed using the following definition (SD = standard deviation). The background region was placed directly beside the respective lesion.max, SUV mean, MV, and CNR were analyzed with Wilcoxon signed-rank tests using Matlab . p values <0.05 were considered as statistically significant.For the statistical evaluation of differences in outcome between G2G-MC and DEB-MC, MC/NOMC ratios of SUV By visual inspection of the different motion and non-motion-corrected images Fig.\u00a0, both moMVs of the GT acquisitions result in 0.807 ml (e7) and 0.740 ml (EMrecon), respectively. Both motion-corrected reconstructions using DEB-MC and G2G-MC show similar improvements in quantitative lesion activity Table\u00a0. Both MCFigures\u00a0max and SUV mean in correspondence with a decrease in MVs after MC between NOMC and MC reconstructions for SUV max, SUV mean, MV, and CNR for both, EMrecon and e7 and elastic motion correction based on gate-to-gate motion estimation (G2G-MC), are equally applicable to clinical whole-body PET/CT data leading to quantitative improvements in SUV"} +{"text": "Cancer cells generate large amounts of lactate derived from glucose regardless of the available oxygen level. Cancer cells finely control ATP synthesis by modulating the uptake of substrates and the activity of enzymes involved in aerobic glycolysis (Warburg effect), which enables them to adapt to the tumor microenvironment. However, increasing evidence suggests that mitochondrial metabolism, including the tricarboxylic acid (TCA) cycle, oxidative phosphorylation (OXPHOS), and glutaminolysis, is paradoxically activated in MYCN-amplified malignancies. Unlike non-amplified cells, MYCN-amplified cancer cells significantly promote OXPHOS-dependent ATP synthesis. Furthermore, tumor cells are differentially dependent on fatty acid \u03b2-oxidation (FAO) according to N-Myc status. Therefore, upregulation of FAO-associated enzymes is positively correlated with both N-Myc expression level and poor clinical outcome. This review explores therapeutic strategies targeting cancer stem-like cells for the treatment of tumors associated with MYCN amplification. N-Myc contains a C-terminal basic region that can bind to DNA and a basic-helix-loop-helix-leucine zipper domain that is responsible for the physical interaction with its counterpart MAX. Myc/MAX heterodimers bind to the DNA sequence 5\u2032-CACGTG-3\u2032, which is termed the consensus E-box , 2. N-Myin vivo takes up cystine in exchange for glutamine, which is used for the synthesis of reduced glutathione (GSH) , 21, wheRen et al. reportedAlptekin et al. proposed an alternative therapeutic strategy against MYCN-amplified neuroblastoma. These authors demonstrated that upregulation of genes associated with the serine-glycine-one-carbon (SGOC) metabolic pathway underlies the excessive dependence on glycine decarboxylase (GLDC) . N-Myc a2O2 inactivates PTEN, a widely-known tumor suppressor, by oxidizing cysteine residues in the active site; this results in the formation of a disulfide bond, which prevents PTEN from inactivating the phosphatidylinositol-3-kinase (PI3K) signaling pathway (MYCN-amplified neuroblastoma cells exhibit enhanced expression of genes and proteins involved in aerobic glycolysis (Warburg effect), oxidative phosphorylation (OXPHOS), detoxification of reactive oxygen species (ROS), and FAO . In MYCN pathway . Therefo pathway , the mem pathway . Cancer pathway , 48. How pathway , 49. The pathway , 51. Can pathway demonstr pathway . Accordi pathway . This fiHigh expression levels of CPT1C, a brain-specific metabolic enzyme, in N-Myc-positive neuroblastoma cells suggest that increased FAO might be an important metabolic feature in this malignancy . While C+ levels following changes in NAD+-dependent deacetylase sirtuin-1 activity and epigenetic alterations characterized by histone H3 residue 9 acetylation and methylation status can partially rescue the synthetic lethal effect of N-Myc overexpressing cancer cells lacking N-Myc and MondoA strongly suggests the pivotal role of lipogenesis. Inhibitors of fatty acid synthesis are toxic to N-Myc overexpressing tumor cells (Deregulated N-Myc requires MondoA for lipid metabolic reprogramming in Myc-driven tumors . MondoA ynthesis . De novoor cells . Taken tde novo hepatocellular carcinoma (HCC). N-Myc is highly expressed in hepatic CSCs compared with non-CSCs. Furthermore, MYCN amplification occurs in at most 2.5% of HCC patients , epithelial cell adhesion molecule (EpCAM), CD90, CD133, delta-like 1 homolog, and glypican 3 \u201364, and patients . In de nas EpCAM , suggesthe liver . HCC celty acids . Lipid dty acids . The genty acids . In addity acids .Qin et al. investigated the therapeutic effect of acyclic retinoid (ACR), a synthetic retinoid X receptor \u03b1-ligand, against hepatic CSCs expressing high levels of N-Myc. Hepatic CSCs with high expression levels of both EpCAM and N-Myc are more susceptible to ACR than non-CSCs negative for N-Myc expression . In genede novo HCC recurrence. Such CSC population is enriched in enzymes necessary for lipid desaturation including FADS1/2 and SCD1. Considering the complexity of mitochondrial metabolism, further investigation is warranted to design novel therapeutic strategies targeting metabolic reprogramming triggered by N-Myc.N-Myc enables metabolic reprogramming of cancer cells, which cannot be simply explained by constitutive aerobic glycolysis (Warburg effect). However, MYCN-amplified cells depend on the TCA cycle and OXPHOS as well as lipid metabolism, rather than the Warburg effect. N-Myc upregulates ASCT2, the amino acid transporter contributing to glutamine addiction. MondoA, a nutrient-sensing transcription factor associated with Myc signaling, plays an important role in maintaining N-Myc-induced glutaminolysis and glutamine-derived lipid biosynthesis. ACR, a leading compound of vitamin A, was recently shown to specifically kill EpCAM-positive liver CSCs expressing high levels of N-Myc. ACR holds much promise for preventing GY searched the articles, wrote the manuscript, and submitted the paper to the journal.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "From 1 to 27 March 2020, we performed 180 examinations of patients with confirmed SARS-CoV-2 infection using a dedicated CT scanner. On 27 March 2020, this CT gantry was opened and sampled in each of the following components: (a) gantry case; (b) inward airflow filter; (c) gantry motor; (d) x-ray tube; (e) outflow fan; (f) fan grid; (g) detectors; and (h) x-ray tube filter. To detect SARS-CoV-2 RNA, samples were analysed using reverse transcriptase-polymerase chain reaction (RT-PCR). To detect bacterial or fungal agents, samples have been collected using \u201creplicate organism detection and counting\u201d contact plates of 24 cm2, containing tryptic soy agar, and subsequently cultured. RT-PCR detected SARS-CoV-2 RNA in the inward airflow filter sample. RT-PCR of remaining gantry samples did not reveal the presence of SARS-CoV-2 RNA. Neither bacterial nor fungal agents grew in the agar-based growth medium after the incubation period. Our data showed that SARS-Cov-2 RNA can be found inside the CT gantry only in the inward airflow filter. All remaining CT gantry components were devoid of SARS-CoV-2 RNA.We investigated whether the internal gantry components of our computed tomography (CT) scanner contain severe acute respiratory syndrome 2 (SARS-CoV-2) ribonucleic acid (RNA), bacterial or fungal agents Severe acute respiratory coronavirus 2 (SARS-CoV-2) ribonucleic acid (RNA) was found inside the computed tomograohy (CT) gantry.SARS-CoV-2 RNA was found solely in the inward airflow filter sample.This filter could be a barrier to SARS-CoV-2 dissemination.Neither bacteria nor fungi were cultured from CT gantry samplings.3 air/h) [Many computed tomography (CT) scanners are equipped with potent air-cooling systems to accomplish x-ray tube heat control. The drawbacks associated with air cooling include the potential contamination of both CT gantry and examination room that comes with the required substantial airflow . During 3 air/h) , as we aWe analysed a 16-slice CT scanner . From 1 to 27 March 2020 (when sampling was conducted), this CT scanner performed 180 consecutive examinations of patients with confirmed SARS-CoV-2 infection. No patients undergoing invasive ventilation were scanned. After each study, we used surface disinfection with 62\u201371% ethanol or 0.1% sodium hypochlorite. Passive air exchange was performed for 30\u201360 min, as well as a professional cleansing of the dedicated CT suite after each 8-h shift. During the aforementioned period, the CT gantry was not opened or suctioned, and none of the sampled internal components were disinfected.2. The sampling procedure is illustrated in Fig. To test the internal microbiologic contamination, the CT gantry was opened and sampled in each of the following zones: (a) gantry case; (b) inward airflow filter; (c) gantry motor; (d) x-ray tube; (e) outflow fan; (f) fan grid; (g) detectors; and (h) x-ray tube filter. Guided by dust deposition sites, swabs have been rotated slightly in such a way as to utilise every part available for sampling and have been swept in close parallel lines. Each sample covered a total area of 100 cm2, containing tryptic soy agar. Aliquots have been cultured, and analysis has been carried out to measure total microbial counts at 22 \u00b0C and 37 \u00b0C, total mycotic count, and to identify specific microorganisms.To detect SARS-CoV-2 RNA, each sample was inserted into a collection vial containing 1\u20133 mL of viral transport media and subsequently underwent reverse transcriptase-polymerase chain reaction (RT-PCR) assay . Besides, samples from each site have been collected using \u201creplicate organism detection and counting\u201d contact plates of 24 cmi.e., the inward airflow filter. RT-PCR of remaining gantry zone samples did not reveal the presence of SARS-CoV-2 RNA. The fluorescence curves for each sampling zone is shown in Fig. RT-PCR detected SARS-CoV-2 RNA in the zone (b) sample, In this brief study, we sampled different zones in a CT scanner that was dedicated and heavily used during the SARS-CoV-2 outbreak at our institution.There has been much concern regarding the survival of SARS-CoV-2 on inanimate surfaces. Earlier SARS-CoV research concluded that coronaviruses might persist in inanimate surfaces for up to 9 days. It was also found that commonly used disinfectants are effective in inactivating the virus . NotablyOur study found SARS-CoV-2 RNA only in the inward airflow filter. All other gantry sampling sites were free of SARS-CoV-2 RNA. These results are encouraging since this filter may act as a partial barrier to the virus. No viral RNA was detected in the dust of internal case and particularly on the outflow fan system (propeller and grid), meaning the absence of contamination in both the internal components of the CT gantry and the CT suite room. Besides, no bacterial or fungal agents were cultured. Even after 26 days of intensive use, conventional sanitisation measures were most probably successful in preventing large-scale contamination of the CT scanner.The study had some limitations. First, the analysis was performed at only one time point. Second, we did not perform exhaustive sampling of the CT gantry; though, the sampled areas were the ones most likely to have microbial agents since it was where dust deposited. Finally, RT-PCR has shown suboptimal sensitivities in previous studies, and therefore some areas containing SARS-CoV-2 RNA could have been missed.In conclusion, SARS-Cov-2 RNA was found internally in the CT gantry only in the inward airflow filter. All remaining CT gantry zones were devoid of SARS-CoV-2 RNA. No bacterial or fungal cultures were obtained from internal CT gantry sampling zones."} +{"text": "In patients presenting with nasal septum perforation, the differential diagnosis between ANCA-associated vasculitis and cocaine-induced midline destruction (CIMD) can be challenging. We describe the case of a 28-year old man who presented with a nasal septum perforation. He admitted the use of cocaine and showed no other symptoms of systemic inflammation. Perinuclear anti-neutrophilic cytoplasmatic antibodies (p-ANCAs) came back positive, as did anti-proteinase 3-antibodies. Further testing revealed antibodies to human neutrophil elastase (HNE), typically found in CIMD but rarely in ANCA-associated vasculitis. The combination of an atypical ANCA-pattern and the detection of HNE-antibodies led to the diagnosis of CIMD. In conclusion, HNE antibodies can be used to distinguish between CIMD and ANCA-associated vasculitis. Rhinoscopy showed a large nasal septum perforation. Nasal endoscopy revealed extensive necrosis and scabs. A computed tomography sinuses confirmed the presence of a nasal septum perforation without improvement. Laboratory analysis demonstrated inflammation with a C-reactive protein of 77\u00a0mg/L and mild leukocytosis granulomatosis with polyangiitis (GPA), anti-neutrophilic cytoplasmatic antibodies (ANCAs) were measured. Perinuclear ANCAs (p-ANCAs) came back positive (titer 1/80), as did anti-proteinase 3 (anti-PR3) antibodies . Myeloperoxidase antibodies were negative. Further testing revealed antibodies to human neutrophil elastase (HNE).Literature data show that patients with CIMD often have positive ANCAs, which makes it difficult to differentiate between CIMD and ANCA-associated vasculitis , 2. HoweThere is no conflict of interest.There was no funding for this publication.No ethical approval required.Informed consent was obtained from the patient.Dr Veerle Ide is the guarantor for this publication."} +{"text": "MicroRNAs are tiny but powerful regulators of gene expression at the post-transcriptional level. Aberrant expression of oncogenic and tumor-suppressor microRNAs has been recognized as a common feature of human cancers. Colorectal cancer represents a major clinical challenge in the developed world and the design of innovative therapeutic approaches relies on the identification of novel biological targets. Here, we perform a functional screening in colorectal cancer cells using a library of locked nucleic acid (LNA)-modified anti-miRs in order to unveil putative oncogenic microRNAs whose inhibition yields a cytotoxic effect. We identify miR-1285-3p and further explore the effect of its targeting in both commercial cell lines and primary colorectal cancer stem cells, finding induction of cell cycle arrest and apoptosis. We show that DAPK2, a known tumor-suppressor, is a novel miR-1285 target and mediates both the anti-proliferative and the pro-apoptotic effects of miR-1285 depletion. Altogether, our findings uncover a novel oncogenic microRNA in colorectal cancer and lay the foundation for further studies aiming at the development of possible therapeutic strategies based on miR-1285 targeting. Colorectal cancer (CRC) is the third leading cause of cancer death in developed countries . AlthougMicroRNAs (miRNAs) are small evolutionarily conserved non-coding RNAs (~22 nt in length) that act as post-transcriptional regulators of gene expression . Aberran2+/Calmodulin (CaM)-regulated serine threonine kinase belonging to the DAP-kinase family of proteins, which function as positive mediators of apoptosis and autophagy anti-Cyclin B1 . Monoclonal antibodies anti-Actin , anti-\u03b1-Tubulin and anti-Nucleolin or polyclonal antibody anti-GAPDH were used as loading controls. Bands were visualized and quantified with FluorChem E System . 3 cells/well) and transfected with 40 ng of Firefly luciferase vectors (empty psiCHECK-2 or psiCHECK-2-DAPK2 3\u2019UTR wt) and 4 ng of Renilla luciferase vector together with 50 nM LNAs. Lipofectamine 2000 (Invitrogen) was used as a transfection reagent (0.2 \u00b5L/well). The activity of both Firefly and Renilla luciferases was measured 48\u201372 h post-transfection using the Dual Luciferase Assay kit (Promega) and the luminescence plate reader Victor-X3 . Transfection efficiency was normalized by calculating the ratio firefly/Renilla. The experiment was performed 3 times in quadruplicate.In luciferase experiments, HeLa cells were seeded in 96-well plates and reverse-transcribed with random primers and M-MLV RT (Invitrogen) after DNase-I treatment . Real-time PCR was performed with SensiMix SYBR Hi-ROX using a gene-specific Taqman probe assay for human DAPK2 . Samples were run on a StepOne Real-Time PCR System according to standard procedures. Human GAPDH was used as endogenous control."} +{"text": "Scientific Reports 10.1038/s41598-020-76776-x, published online 12 November 2020Correction to: The original version of this Article contained a typographical error in the jointly supervising statement.\u201cThe author jointly supervised this work: Bowen Lan, Li Li, and Chun-Quan Ou.\u201dnow reads:\u201cThese authors jointly supervised this work: Bowen Lan, Li Li, and Chun-Quan Ou.\u201dThis error has now been corrected in the PDF and HTML versions of the Article."} +{"text": "Older adults in residential care settings are four times more likely than those not living in care facilities to experience falls. Yet, fall prevention efforts at long-term care settings are under-resourced, under-regulated, and under-studied. To address this gap, we developed and studied the impact of a specialty clinical, Fall Prevention Care Management (FPCM), for nursing students to decrease older adults\u2019 fall risks. We enrolled assisted living residents that facility liaison identified as being high fall risk and MOCA \u226515, in 2 assisted living facilities in Northwest USA. Participants received weekly, 1-hour, individual, semi-structured, Motivational Interviewing-based care management visits by same students over 6 visits. Changes in fall risks were measured by the CDC STEADI assessment (unsteadiness & worry), Falls Self-Efficacy Scale International-Short (FESI-S), and Falls Behavioral Scale (FAB). Twenty-five residents completed the study. Students addressed the following (multiple responses possible): emotional needs (n=23), improved motivation to prevent falls (n=21), and individualized education/coaching (n=10-17). FESI-S score improved from 16.0 to 14.4 . Frequency of those who felt steady while standing or walking increased and those who did not worry about falling increased . FPCM clinical offered valuable opportunity to address unmet care needs of older adults to reduce fall risks."} +{"text": "BamHI-A rightward fragment-derived microRNAs (BART miRNAs) or BamHI-H rightward fragment 1-derived miRNAs (BHRF1 miRNA) in EBV-infected cells have been recently reported. Host miRNAs are also upregulated upon EBV infection. Viral and host miRNAs are important in maintaining viral infection and evasion of host immunity. Although miRNAs in EBV-infected cells often promote cell proliferation by targeting apoptosis or cell cycle, this review focuses on the regulation of the recognition of the host immune system. This review firstly describes the location and organization of two clusters of viral miRNAs, then describes evasion from host immune surveillance systems by modulating viral gene expression or inhibiting innate and acquired immunity by viral miRNAs as well as host miRNAs. Another topic is the enigmatic depletion of viral miRNAs in several types of EBV-infected tumor cells. Finally, this review introduces the strong correlation of nasopharyngeal cancer cases with a newly identified single nucleotide polymorphism that enhances BART miRNA promoter activity.Epstein-Barr virus (EBV) is an oncogenic human herpes virus that was discovered in 1964. Viral non-coding RNAs, such as Epstein-Barr virus (EBV) is a double-stranded DNA virus that belongs to the Gammaherpesvirus subfamily and was discovered in a Burkitt's lymphoma (BL) cell . EBV priThe two infection cycles that enable successful propagation of the EBV progeny viruses are lytic and latent infection. During lytic infection, all the viral genes are expressed and the viral genome is rapidly replicated. In contrast, latent infection involves the restricted expression of a number of viral genes. Here, EBV evades host immune surveillance and the copy number of DNA in the viral daughter cells are maintained by synchronous duplication of viral and host genomes. A small subset of viral genes and microRNAs (miRNAs) expressed during the latent infection maintain viral episomes and stimulate host cell proliferation. EBV propagates viral genomes together with host cells during latent infection.Host cell proliferation associated with latent EBV infection induces malignancies, such as BL, Hodgkin's lymphoma (HL), EBV-positive diffuse large B-cell lymphoma (DLBCL), extranodal NK/T-cell lymphoma-nasal type (ENKL), nasopharyngeal carcinoma (NPC), and EBV-associated gastric carcinoma. EBV also causes the severe infectious disease called chronic active EBV infection \u20136.A miRNA is a non-coding single-stranded RNA comprising 20\u201322 bases that regulates post-transcriptional gene expression. More than 60% of protein-coding genes are regulated by miRNAs in mammals . miRNAs Here we discuss the role of EBV-encoded miRNAs in maintaining latent and lytic infection along with the function of host and viral miRNAs in regulating immune responses in EBV-associated diseases.BamH I-A rightward transcripts (BARTs) are alternatively spliced non-coding RNAs abundantly expressed during latent infection encodes for three pri-miRNAs called pri-miR-BHRF1-1, -BHRF1-2, and -BHRF1-3. BHRF1 miRNAs are expressed during lytic infection, inhibit apoptosis, and favor proliferation of infected cells to enable the early phase of viral propagation and BamH I-R leftward reading frame 1 that enable switching between latent and lytic EBV infection ..10).During lytic infection, miR-BHRF1-2-5p targets the 3\u2032 untranslated region of the interleukin-1 receptor 1 (IL-1R1) and suppresses IL-1 signaling . miR-BHRmiR-BART6-3p targets the retinoic acid-inducible gene-I (RIG-I) (an intracellular receptor for double-stranded RNA), thereby suppressing host innate immune responses . miR-BARThe BART miRNA coding sequence from the Akata strain was inserted into the B95-8 strain to restore the deleted region . As comp+ T cells into T helper 1 (Th1) cells, thereby abrogating host immune response. Thus, there is a reduction in cytotoxic T cells specific for the EBV antigens . BA. BA10). antigens , 32, 38.+ T cells (+ T cells (miR-BHRF1-3-5p and miR-BART17-5p target transporter associated with antigen processing 2 (TAP2) that transports antigenic peptides to MHC class I molecules, thus, viral antigen presentation is impaired in CD8 T cells . EBV miR T cells .The B cell receptor (BCR) that mediates adaptive immunity as well as lytic infection in EBV-infected B lymphocytes is inhibited by miR-BHRF1-2-5p and miR-BART2-5p . miR-BARLymphocyte antigen 75 (LY75) is a membrane protein that is expressed on dendritic cells and induces differentiation of Th0 to Th1 cells. miR-BART1-5p (transferred by exosomes) targets LY75 in dendritic cells suppressing Th1 cell differentiation .The roles of EBV miRNAs in suppressing innate and adaptive immunity has been summarized in EBV exploits host miRNAs to escape from the immune system. EBNA2 is a viral protein that expressed during type III latency and upregulates miR-21, that subsequently downregulates myeloid differentiation factor 88 (MyD88) and IL-1 receptor-associated kinase 1 (IRAK1) . The miRIn EBV-infected B lymphocytes, viral LMP1 activates NF-\u03baB signaling and host miR-155. But miR-155 attenuates NF-\u03baB signaling to stabilize persistent infection . The miRIn EBV-infected epithelial tumor cells, BART miRNAs are highly expressed and help in evading immune recognition . HoweverSimilarly, LMP1 is expressed in all the early NPC tumor cells and contributes to pleiotropy in NPCs . HoweverAs mutations and/or promoter methylation accumulate in the host genome, the presence of the viral genome may no longer be required for the growth of the tumor cell. In such a situation, carrying large EBV genomes may be a burden for host cells; thus, cells harboring the defective, but oncogenic, EBV genome may proliferate faster than cells infected with EBV having the complete genome. Alternatively, the increased levels of BART miRNAs may repress the expression of genes important for survival of EBV-positive cells. Therefore, further investigation is necessary to discern the physiological significance of BART miRNAs in EBV-positive tumor cells.BART miRNAs are important in evading the immune system and inhibiting apoptosis. However, multiple BART miRNAs frequently target the same gene to induce a high level of repression , 17. ThiEBV uses miRNAs to switch between lytic and latent infection. This helps maintain EBV infection and evade recognition of EBV by the host immune system by reducing viral gene (antigenic) expression. EBV miRNAs also target and suppress genes involved with host immunity. This oncogenic virus also exploits miRNAs for malignant transformation. Exosomes secreted from EBV-infected B lymphocytes contain a large amount of host and viral miRNAs that are transferred to epithelial cells . TherefoHI wrote the manuscript. HK, AK, and YK prepared the table and figures. HY complied the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "In the recent era, antimicrobial resistance has been identified as one of the most important threats to human health worldwide. The rapid emergence of antibiotic-resistant pathogens (ABRP) in the modern intensive care unit (ICU) also represents a \u201cnightmare scenario\u201d with unknown clinical consequences. In the Greek ICU, in particular, gram negative ABRPs are now considered endemic. However, the possible longitudinal impact of ABRPs on long-term outcomes of ICU patients has not yet been determined.In this two-year (January 2014-December 2015) single-centre observational longitudinal study, 351 non-neurocritical ICU patients\u2009\u2265\u200918\u00a0year-old were enrolled. Patients\u2019 demographic, clinical and outcome data were prospectively collected. Quality-adjusted life years (QALY) were calculated at 6, 12, 18 and 24\u00a0months after ICU admission.Fifty-eight patients developed infections due to ABRP (ABRP group), 57 due to non-ABRP (non-ABRP group), and 236 demonstrated no infection (no-infection group) while in ICU. Multiple regression analysis revealed that multiple organ dysfunction syndrome score and continuous renal replacement therapy were the only independent determinants for ABRP infections in ICU. Intra-ICU, 90-day and 2-year mortality was 27.9%, 52.4% and 61.5%, respectively. Compared to the non-ABRP and no-infection group, the ABRP group demonstrated increased intra-ICU, 90-day and 2-year mortality (P\u2009\u2264\u20090.022), worse 2-year survival rates in ICU patients overall and ICU survivor subset , and poorer progress over time in 2-year QALY kinetics in ICU population overall, ICU survivor and 2-year survivor subgroups (P\u2009\u2264\u20090.013). ABRP group was further divided into multi-drug and extensively-drug resistant subgroups . Compared to MDR subgroup, the XDR subgroup demonstrated increased ICU, 90-day and 2-year mortality (P\u2009\u2264\u20090.031), but similar 90-day and 2-year QALYs (P\u2009\u2265\u20090.549). ABRP infections overall , as well as XDR [HR\u2009=\u20091.889, 95% CI 1.075\u20133.320; P\u2009=\u20090.027) but not MDR pathogens, were independently associated with 2-year mortality, after adjusting for several covariates of critical illness.The present study may suggest a significant association between ABRP infections in ICU and increased mortality and inability rates for a prolonged period post-discharge that requires further attention in larger-scale studies. Intensive Care Unit (ICU) survivors may suffer tremendous changes in lifestyle post-discharge . CriticaAntibiotic resistant pathogens (ABRP) have been identified as one of the most important threats for the modern ICU, limiting treatment options and resulting in adverse clinical outcomes and excessive cost of care \u20138. In thThe complex consequences of critical illness are usually referred to as \u201cquality of life\u201d indices, on the basis of several different instruments which were developed in order to assess outcomes . These mThe aim of our study was to examine whether antimicrobial resistance in ICU is associated with increased long-term mortality and problematic quality of life for a prolonged time period post-ICU admission. For this purpose, ICU patients were divided into three subgroups: patients having suffered ABRP-induced ICU infection(s) (ABRP-group), non-ABRP infection(s) (non-ABRP-group) and patients who manifested no infection at all while in ICU (\u201cno-infection\u201d group). Survival data and QALY measurements were prospectively recorded over a two-year follow-up period.All consecutive adult patients admitted in the eight-bed medical-surgical ICU of a Greek regional community hospital between January 1, 2014, and December 31, 2015, were prospectively included. The study was approved by the Institutional Review Board and Ethics Committee of General Hospital of Trikala -ID: 123/October 15th/2013.Written informed consent was obtained from each patient or his/her legal representative.All patients\u2009\u2265\u200918\u00a0years old were considered as eligible for the study. Post-operative patients included both elective and emergency surgical patients. Exclusion criteria were: (1) Patients confined to Hospital and/or to bed prior to ICU admission, and (2) neurosurgical patients requiring advanced neurocritical support (transmission to a tertiary center). Patients who refused to cooperate or were not found at follow-ups were also excluded from the study.Demographic data, pre-hospitalization clinical status and comorbidities, admitting etiology, Acute Physiology and Chronic Health Evaluation II (APACHE II) on admission, clinical data including ICU infections/microbiology, treatment data including colistin administration, intra-ICU outcomes and survival data post-discharge were prospectively collected. The severity of multiple organ dysfunction syndrome (MODS) was assessed upon admission and during ICU infection(s), by using the MODS score as previously described ; the maxInfections were defined according to criteria of Centers for Disease Control and prevention . Both ICICU costs for each patient according to the national legislation were collected from the Economic Department of the Hospital were calculated by using the 5-level EuroQol-5-dimensional (EQ-5D-5L) questionnaire \u201331. The Patients with ICU infections due to ABRP were included in the \u201cABRP group\u201d, patients with ICU infections due to non-ABRP were included in the \u201cnon-ABRP group\u201d whilst the ones who manifested no infection nominated the \u201cno-infection\u201d group . PatientABRPs included MDR and XDR pathogens according to previously established criteria . Hence, The primary goal of our study was to examine whether antimicrobial resistance in ICU is associated with (1) increased intermediate (90-day) and longt-test or Man-Whitney U test to compare continuous variables as appropriate. One-way analysis of variance (ANOVA) was used for multiple comparisons between categories. Multiple regression and multivariate linear regression analyses were used to evaluate the independent clinical determinants of antimicrobial resistance, ICU costs and 2-year QALY score, as well as to exclude co-linearity among the univariate variables included in the models. To assess differences in 2-year QALY kinetics among subgroups, mean regression lines were created and compared by using linear mixed model analysis. Kaplan Meier 2-year analysis of survival was used to assess the association of ABRP, MDR/XDR pathogens and colistin administration with patients\u2019 long-term survival. Survival curves were compared by using Log-rank test (which performs better towards the right side of the curves), Breslow test (which focuses mainly on the left side of the curves), and Tarone-Ware test (which functions better in the middle part of the curves). A Cox-proportional hazard model was constructed to ascertain whether ABRP (and XDR) infections are independent predictors of outcome or rather a function of prolonged/intensive organ support. The statistical package SPSS 17.0 was used.Data is expressed as means\u2009\u00b1\u2009standard deviation (SD), otherwise as indicated. Kolmogorov\u2013Smirnov test was used for normality assessment. Chi-square or Fisher\u2019s exact test were used to compare categorical variables and A total of 373 ICU patients were assessed for eligibility to participate in the study during the study period . Twenty-two (22) of them were excluded for several reasons ; thus, 351 patients were finally enrolled and analyzed.Fifty-eight patients 16.52%) demonstrated infection(s) due to at least one (\u2265\u20091) ABRP (ABRP group), 57 (16.24%) manifested infection(s) due to\u2009\u2265\u20091 non-ABRP (non-ABRP group), while 236 (67.24%) manifested no infection at all (\u201cno-infection\u201d group). Eleven patients of the ABRP group demonstrated microbiological results of both ABRP and non-ABRP and higher MODS score , yet similar APACHE II on admission compared to MDR subset.Interestingly, patients who developed ICU infections due to XDR pathogens were of similar age , predominantly males , and demonstrated higher APACHE II on admission and MODS score , compared to the rest ICU population.In our study population, intra-ICU, 90-day and 2-year overall mortality were 27.9% 98/351), 52.4% (184/351) and 61.5% (216/351), respectively. ABRP infections not only were associated with increased ICU mortality, but also with enhanced 90-day and 2-year mortality rates due to ABRP(s) were associated with worse QALY kinetics not only in our ICU population overall Fig.\u00a0, but alsPatients\u2019 ABRP specific microbiology showed no association with either mortality or QALY values should be validated in larger-scale studies in the future. Second, most of our infections were non-catheter-related blood stream infections, while respiratory infections were far less common in our cohort. One could argue that the synthesis of our ICU infections and its specific microbiology (mainly gram negative bacteria) may have interfered with our findings. Certainly, genetic and geographic differences may exert diverse influence on the clinical phenotypes of special diseases in different populations. Furthermore, it has been shown that somThe present prospective study demonstrated a significant association between ABRP infections in ICU and increased mortality/inability rates for a prolonged period post-discharge. This relationship is likely to mainly concern patients with more severe critical illness, who manage to survive ICU though; in this respect, a history remarkable of antimicrobial resistance while in ICU should raise awareness for increased risk post-discharge and reconsider the anticipated benefits in long-term longevity and well-being, especially in patients with more severe critical illness and/or complex course of hospitalization.Additional file 1. Details in methodology used, results obtained.Additional file 2: Supplemental Figure 1 Examples of calculation of QALY-gained in our study. A. QALYs-gained by ICU treatment in a patient having survived the 2-year follow-up (QALYs gained= area A + area B + area C + area D). B. QALYs-gained by ICU treatment in the theoretical patient who died between the 12-month and the 18-month follow-up (QALYs gained= area A+ area B + area C). HRQoL= health-related quality of life; HRQoL1,2,3,4= utility index values at 6,12,18 and 24-month follow-up, respectively; ICU= intensive care unit; QALYs= quality-adjusted life years. Additional file 3: Supplemental Figure 2. Long-term outcomes in the MDR/XDR subgroups of our 58 patients with antibiotic resistant pathogens. In the upper panel, bars represent the number of 90-day (A) and 2-year (B) survivors and non-survivors with MDR and XDR infections; percentages within grey bars represent mortality in each category. In the lower panel, bars and vertical lines indicate mean QALY values and standard errors in 90-day and 2-year survivors, respectively. MDR= multi-drug resistant pathogen; XDR= extensively-drug resistant pathogen; QALY= quality-adjusted life years.Additional file 4: Supplemental Figure 3. Kaplan-Meier 2-year survival curves examining the effect of ABRP infections on long-term mortality post-discharge. Analysis was performed in our 351 ICU patients overall (panel A), and in the subset of 253 ICU survivors (panel B). Patients were divided into four groups according to having demonstrated MDR infection(s) (black dotted line), XDR infection(s) (continuous black line), infections due to non-ABRPs (red line) or \u201cno-infection\u201d at all (green line) while in ICU. ABRP= antibiotic resistant pathogen; MDR= multi-drug resistant; XDR= extensively-drug resistant; ICU= intensive care unit.Additional file 5: Supplemental Figure 4. Two-year QALY kinetics in ABRP, non-ABRP and \u201cno-infection\u201d subgroups of our ICU survivors (N=253). Bars and vertical lines indicate mean QALY values and standard errors, respectively. ABRP group demonstrates lower increase in QALYs over time (markedly depressed slope of the corresponding mean regression line) compared to its counterparts. QALY= quality-adjusted life years; ABRP= antibiotic resistant pathogen; ICU= intensive care unit; SE= standard error; Intercept of the regression line= the QALY value where the regression line crosses the y-axis at the theoretical day=0; Slope of the regression line= the rate at which QALY values change between two-consequent follow-up examinations. *P<0.001.Additional file 6: Supplemental Figure 5. Two-year QALY kinetics in ABRP, non-ABRP and \u201cno-infection\u201d subgroups of our 2-year survivors (N=135). Bars and vertical lines indicate mean QALY values and standard errors, respectively. ABRP group demonstrates lower increase in QALYs over time (lower slope of the corresponding mean regression line) compared to its counterparts. QALY= quality-adjusted life years; ABRP= antibiotic resistant pathogen; SE= standard error; Intercept of the regression line= the QALY value where the regression line crosses the y-axis at the theoretical day=0; Slope of the regression line= the rate at which QALY values change between two-consequent follow-up examinations.*P<0.05.Additional file 7: Supplemental Figure 6. Long-term outcomes in our 58 ABRP patients according to underlying pathogen. In the upper panel, bars represent number of 90-day (A) and 2-year (B) survivors and non-survivors with respect to underlying pathogen(s). In the lower panel, bars and vertical lines indicate mean QALY values and standard errors in 90-day and 2-year survivors regarding the underlying pathogen(s), respectively. ABRP= antibiotic resistant pathogens; QALY= quality-adjusted life years; Ab=Acinetobacter baumannii; Ent/ter=Enterobacter cloacae; KPC= Klebsiella pneumonia producing carbapenemases; Pa= Pseudomonas aeruginosa; Sa= staphylococcus aureus.Additional file 8: Supplemental Figure 7. Kaplan-Meier survival curves examining the effect of colistin administration in ICU on long-term mortality post-discharge. Analysis was performed in our 351 ICU patients overall (panel A), and in the subset of 253 ICU survivors (panel B). ICU= intensive care unit.Additional file 9: Supplemental Figure 8. Kaplan-Meier survival curves examining the effect of XDR versus non-XDR (MDR plus non-ABRP) infections on 2-year mortality in our 78 patients treated with colistin. XDR= extensively-drug resistant pathogens; MDR= multidrug resistant pathogens; ABRP= antibiotic resistant pathogens."} +{"text": "Scientific Reports 10.1038/srep42690, published online 17 February 2017Correction to: This Article contains a typographical error in the spelling of the author Yu-Chi Chen, which is incorrectly given as Yi-Chi Chen."} +{"text": "Currently, myelin oligodendrocyte glycoprotein (MOG)-IgG-associated encephalomyelitis (MOG-EM) is regarded as an independent inflammatory demyelinating disease. Magnetic resonance imaging (MRI) abnormalities occur in 44.4% of patients with MOG-EM. However, symmetrical deep gray matter involvement with leptomeningeal enhancement is rarely described in the literature.A 3-year-old boy was admitted to our hospital because of acute onset fever, headache, vomiting and disturbance of consciousness. Neurological examination showed somnolence, neck stiffness and positive Kernig\u2019s sign. Brain MRI demonstrated bilateral symmetrical lesions in the basal ganglia and thalamus as well as diffuse leptomeningeal enhancement along the sulci of bilateral hemisphere. Cerebrospinal fluid analysis demonstrated increased cell count and protein (1.17\u2009g/L) without glucose and chloride abnormality. Work-up for infectious and autoimmune causes, serum MOG IgG was positive by cell based assay. Therefore, a diagnosis of MOG-EM was established according to the international recommendatory criteria in 2018. He was administrated with intravenous methylprednisolone followed by oral corticosteroids and had recovered completely within 1\u2009week.In the setting of meningoencephalitis-like clinical presentation with bilateral symmetrical deep gray matter involvement, MOG-EM should be distinguished from other infectious and autoimmune disorders, such as Epstein-Barr virus (EBV) encephalitis, Japanese encephalitis and Anti-NMDA receptor (NMDAR) encephalitis. Besides, aseptic meningitis associated with leptomeningeal enhancement may be an atypical phenotype of MOG-EM. Myelin oligodendrocyte glycoprotein (MOG) is a glycoprotein localized on the outer surface of the myelin sheath and oligodendrocytes. The MOG antibody has been identified in inflammatory demyelinating diseases (IDDs), including acute disseminated encephalomyelitis (ADEM), neuromyelitis optica spectrum disorders (NMOSDs), optic neuritis (ON), transverse myelitis (TM), clinically isolated syndrome, and multiple sclerosis (MS). Nowadays, most experts regard MOG-IgG-associated encephalomyelitis (MOG-EM) as an independent entity, immunopathogenetically distinct from other IDDs. It is estimated that the incidence of magnetic resonance imaging (MRI) abnormalities in patients with MOG-EM is 44.4% .. HoweveBorrelia burgdorferi, were negative in CSF and serum. Moreover, he was routinely tested for human immunodeficiency virus (HIV) and syphilis and found to be negative for both. Autoimmune encephalitis-related autoantibodies, including N-methyl-D-aspartate-receptor (NMDAR) antibodies, contactin associated protein 2 (CASPR2) antibodies, leucine-rich glioma inactivated 1 (LGI1) antibodies, \u03b1-amino-3-hydroxy-5-methyl-isoxazolepropionic acid receptor (AMPAR) antibodies, and gamma-aminobutyric acid (GABA) receptor antibodies, were negative both in serum and CSF. On day 6 after admission, established cell-based immunoassays revealed negative serum anti-AQP4 antibodies but positive anti-MOG antibodies with a titer of 1:100. The patient completed 3\u2009days of intravenous methylprednisolone (30\u2009mg/kg) followed by oral prednisolone (2\u2009mg/kg). He had recovered completely within 1\u2009week after initiation of steroid treatment. On day 17 after admission, A follow-up MRI examination demonstrated no residual lesions and slightly increased heart rate (104 beats/min) with normal rhythm. Neurological examination revealed somnolence, neck stiffness and positive Kernig\u2019s sign without any other focal neurological deficit. Deep tendon reflexes were present and symmetrical. There was no change in bladder and bowel habits. A increase of leukocyte cell counts (14.000 cells/\u03bcL with 78% neutrophils), erythrocyte sedimentation rate (30\u2009mm/h) and C-reactive protein (17\u2009mg/l) was noted on routine laboratory investigations. On day 2 after admission, brain MRI demonstrated bilateral symmetrical lesions in the basal ganglia and thalamus, hyperintense on T2-weighted image and hypointense on T1-weighted image without restricted diffusion transmission occurs round the year whereas seasonal epidemics begin during the rainy seasons when the mosquito density is maximum. Likewise, bilateral symmetrical deep gray nuclei involvement is the characteristic imaging finding in Japanese encephalitis. Although the child live in South China, there is insufficient evidence for the diagnosis of Japanese encephalitis due to a lack of history of mosquito bites and the onset time of illness being out of epidemic seasons. Moreover, Japanese encephalitis has a poorer prognosis than MOG-EM. Twenty to 40 % of patients with Japanese encephalitis die during the acute stage and about 50% of the survivors have severe neurological sequelae [In the setting of meningoencephalitis-like clinical presentation with bilateral symmetrical deep gray matter involvement, MOG-EM should be distinguished from other infectious and autoimmune disorders, such as Epstein-Barr Virus (EBV) encephalitis, Japanese encephalitis and anti-NMDA receptor (NMDAR) encephalitis . Of all sequelae . Anti-NMsequelae . Moreovesequelae . When clIn conclusion, aseptic meningitis associated with leptomeningeal enhancement may be an atypical phenotype of MOG-EM. MOG-EM should be considered for the etiological spectrum of bilateral symmetrical deep gray matter involvement."} +{"text": "Still, a significant proportion of patients suffer from disease progression. A better understanding of resistance mechanisms depicts a central goal to avoid or overcome IO resistance and to improve patient outcome.Immunotherapy (IO) has revolutionized the therapy landscape of non-small cell lung cancer (NSCLC), significantly prolonging the overall survival (OS) of advanced stage patients. Over the recent years IO therapy has been broadly integrated into the first-line setting of non-oncogene driven NSCLC, either in combination with chemotherapy, or in selected patients with PD-L1We here review major cellular and molecular pathways within the tumor microenvironment (TME) that may impact the evolution of IO resistance. We summarize upcoming treatment options after IO resistance including novel IO targets as well as interesting combinational approaches such as IO combined with anti-angiogenic agents or metabolic targets . By discussing the fundamental mode of action of IO within the TME, we aim to understand and manage IO resistance and to seed new ideas for effective therapeutic IO concepts. Previously unanticipated long-term responses in advanced stage disease have been accomplished, with a 5 year overall survival (OS) of 20% in unselected and up to 40% in PD-L1Despite the striking clinical improvements, the majority of patients eventually fails to respond to ICI therapy due to the evolution of primary or secondary resistance. Prospective clinical studies to demonstrate treatment strategies following progression on IO therapy are still lacking.Various IO resistance mechanisms have been characterized, involving tumor cell intrinsic as well as environmental resistance patterns. The tumor microenvironment (TME) plays a critical role by influencing both extrinsic and intrinsic resistance pathways. A better understanding of the heterogenous TME will set stage for further optimizing strategies and guide new avenues in future IO treatment stratification.This review discusses the multitude of novel preclinical and clinical treatment approaches that aim to overcome IO resistance in NSCLC. The complexity of cellular and molecular alterations within the immunosuppressive TME build the fundament for designing rational and synergistic combination therapies that lower the risk of resistance and prolong benefit from IO therapy.IO resistance mechanisms result from the constantly evolving interactions between cancer cells and the surrounding cell populations within the TME, including immune cells, cancer-associated fibroblasts (CAF) and tumor endothelial cells (TEC) Fig.\u00a0. The fol+ T lymphocytes, regulatory T cells (Treg) and natural killer (NK) cells and modulates T cell activity via interaction with its ligand (PD-L1) in the TME play a central role in negative regulation of T cell reactivity and their inhibition via monoclonal antibodies can unleash T cell-triggered antitumor immune responses. The best studied IC are PD-1 and cytotoxic T lymphocyte antigen 4 (CTLA-4). PD-1 is broadly expressed on CD8TME Fig.\u00a0. CTLA-4 d organs , 4. OtheTumor infiltrating T lymphocytes (TIL) play a major role in antitumor immune responses within the TME . The pheIn chronically inflamed areas such as tumors, B and T lymphocytes are frequently organized in ectopic lymphoid aggregates, so-called tertiary lymphoid structures (TLS), where they convert to effector cells upon antigen presentation. The cellular organization ranges from simple lymphocyte clusters (immature TLS) to highly sophisticated structures (mature TLS) , 7. HighTumor infiltrating B cells harbor both immunostimulatory and immuSomatic mutations in the cancer genome, such as in DNA repair genes including mismatch repair (MMR), homologous recombination (HR) or polymerase epsilon (POLE) increase tumor mutational and neoantigen burden, which has been linked to greater TIL density and enhanced ICI efficacy \u201319. Thishigh advanced solid cancers (\u226510 mutations/megabase) in response to results from KEYNOTE-158. In contrast, in the complex multi-arm CheckMate227 trial testing ipilimumab plus nivolumab versus chemotherapy or nivolumab plus chemotherapy in NSCLC, neither TMB nor PD-L1 expression could segregate therapy responsiveness [Concerning TMB as predictive biomarker of ICI response, clinical trials report divergent results, possibly due to technical issues with TMB assessment . On the siveness , 29, howPdcd1 gene effectively suppressed tumor growth in several tumor models by mediating antitumor immunity (enhanced T effector memory cells) despite preserved T cell-specific PD-1 expression. These data underline the important role of myeloid-intrinsic effects in regulating anti-tumor immunity [Cancer cells can overexpress PD-L1 upon type I interferon (IFN I) stimulation to evadeimmunity .Clearly, PD-L1 expression is necessary to achieve adequate responses to PD-1/PD-L1 blockade and numerous studies associated high tumor cell PD-L1 expression with better outcomes to anti-PD-1/PD-L1 monotherapy in NSCLC. Controversially, some patients with very low or even absent PD-L1 expression show durable responses , an obseSo far, clinical trials considered tumor PD-L1 expression as the most robust and reproducible biomarker, and clinical NSCLC guidelines are based on this. However, PD-L1 immunohistochemistry (IHC) has several limitations and this may contribute to the above-mentioned controversial observations. Moreover, the TME is highly heterogenous and a single core biopsy only depicts one spatial tumor component, hence some patients may be PD-L1 negative in one biopsy and PD-L1 positive in other tumor areas. This also explains quantification errors in tissue-based biomarkers. One approach to resolve the limitation of spatial resolution involves PET-based PD-L1 imaging with zirconium-89-labeled atezolizumab. Interestingly, pre-treatment tumor PET signal was shown to better correlate with clinical treatment responses than IHC or RNA-sequencing based predictive biomarker-detection .Tumor-associated macrophages (TAM) are an abundant cell type within the TME and despite growing research, their role in cancer progression remains ambiguous. Along a functional scale, TAM polarize to either M1 or M2 phenotypes in response to environmental cues, including metabolic changes , 37. TheCancer associated fibroblasts (CAF) constitute one of the most prominent, yet highly heterogenous components of the TME. They express a variety of molecular markers, e.g. \u03b1-SMA, S100A4, FAP, PDGFR\u03b1/\u03b2, none of which, however, is unique for the fibroblast lineage. Next to immune cells CAFs have emerged as important mediators of the complex stroma-tumor interactions, promoting local immunosuppression and orchestrating immune cell trafficking . CAFs maTumor endothelial cells (TEC) have immunomodulatory functions by controlling immune cell transmigration, lymphocyte activation and function. They hold a \u201csentinel\u201d role in detecting foreign antigens as antigen (cross)-presenting cells, though this has been studied extensively in non-malignant inflammation and less in TEC , 45. TECIt remains to be answered why some patients attain sustained durable IO therapy response while others evolve primary or secondary resistance. The mode of action is definitely multifactorial and includes intrinsic and extrinsic mechanisms . The fol. Genetic instability due to impaired DNA repair can enhance tumor immunogenicity, which is the target of later discussed PARP inhibitors [Neo-antigen burden of cancer cells markedly determines tumor immunogenicity, which enhances ICI efficacy. Hence, low tumor immunogenicity may cause primary IO resistance. Immune-cancer cell interactions can promote the evolution of low-immunogenic and low-antigenic tumor subclones, a process named immuno-editing . Genetichibitors .In response to PD-1/PD-L1/CTLA-4 inhibition, T cells can upregulate alternative ICs, including T cell immunoglobin mucin-3 (TIM-3) or lymphocyte activation gene 3 (LAG-3), as adaptive resistance mechanism , 50. Co-An immunosuppressive TME facilitates tumor cell growth and tumor infiltrating Treg and MDSC are key players in sustaining this immunosuppression . IO effiChemokines mediate immune cell recruitment into the TME and directly impact cancer and endothelial cells to regulate tumor cell proliferation, neo-angiogenesis and hence cancer progression. Multiple chemokines have been identified with multi-faceted roles, acting both pro- or anti-cancerogenic in different tumor entities. Their impact on IO resistance and efficacy remains unclear .Vascular endothelial growth factor (VEGF) expression within the TME is heterogenous Fig. and mainThe treatment landscape of non-oncogene driven NSCLC has changed dramatically in recent years and IO is an important cornerstone of front- and later-line therapies . YeMultiple clinical trials in different cancer types are based on an exploding number of preclinical studies using novel IO combinations or targeted therapies. The following section will discuss the background, mode of action and clinical update of the most relevant up-coming treatment options in IO-refractory NSCLC.IC co-inhibition, by expanding the anti-PD-1 or PD-L1 backbone with a second ICI has been one of the first strategies to overcome IO resistance and most clinical experience has been gathered with combinational CTLA-4 inhibitor. The observed synergistic effect of PD-1/CTLA-4 inhibitors likely depends on the distinct patterns of PD-1 and CTLA4 in immune activation, as PD1 blockade inhibits peripheral and CTLA4-blockade central tolerance see 2.1, 3.plus nivolumab in advanced-stage disease ), independent of TMB or PD-L1 expression. Intriguingly, the OS effect was most prominent in PD-L1low patients. Treatment-related serious adverse events (AE) of any grade were more frequent with ipilimumab plus nivolumab than with chemotherapy (24.5% vs. 13.9%) [The combination of CTLA-4 and PD-1 inhibitors is effective in melanoma and rena. 13.9%) .plus atezolizumab compared to atezolizumab monotherapy in PD-L1 positive metastatic NSCLC patients. Particularly, a meaningful ORR improvement was seen in PD-L1high (TPS\u2009>\u200950%) expressing patients (55.2% vs 17.2%) [Recent results from the phase II CITYSCAPE trial showed a significant PFS and ORR benefit for the first-line combination of the TIGIT-inhibitor see 3.1.4 tiragolumab s 17.2%) , while tThese data emphasize the potency of IO combination, but optimal patient selection criteria are still lacking.In recent years, the dogma of disease progression being synonymous for drug resistance has been questioned , therefoRetrospective studies have investigated IO re-challenge in a small number of NSCLC patients with clinical benefit in only a minority of them \u201369. Recen\u2009=\u200917) could not show a substantial benefit of nivolumab plus ipilimumab after progression on first-line nivolumab [n\u2009=\u2009207) showed a significant ORR benefit for the \u201cimmunotherapeutic boost\u201d with 2\u20134\u2009cycles of nivolumab plus ipilimumab in the first-line as compared to nivolumab monotherapy [The question of dual ICI following IO progression has currently been investigated in two RCC studies. A small retrospective study and hence IO continuation should only be considered in patients with clinical benefit and lack of severe AE . Some NSApart from PD-1/PD-L1/CTLA-4, other inhibitory IC regulate T cell response and might influence IO resistance mechanism. Blocking these additional IC has proven highly efficient in preclinical and clinical studies as monotherapy or in combination with PD-1/PD-L1 inhibitors. The following IC have been investigated:+\u2009T cells. Hence, LAG-3 synergizes with other IC, particularly PD-1, and dual IC blockage with an anti-LAG3 antibody plus a PD-1/PD-L1 inhibitor has revealed promising preclinical results in different tumor entities and numerous clinical phase I/II trials are currently ongoing [plus nivolumab in LAG-3 positive tumors after progression on PD-1/PD-L1 inhibitors. Further phase I/II studies in NSCLC are ongoing as upfront IO combination or in the resistance situation .Lymphocyte activation gene 3 (LAG-3 or CD223) is expressed on various immune cells negatively regulates T cell activation Fig. . Even thl anergy , 81. Basl anergy : PrelimiLastly, T cell immunoglobulin (Ig) and immunoreceptor tyrosine-based inhibitory motif (ITIM) domains (TIGIT) is a lymphocyte-specific transmembrane glycoprotein receptor Fig. . As a coVEGF is the key promoter of hypoxia-driven neo-angiogenesis in the TME and also serves as important immunosuppressive molecule. Furthermore, VEGF inhibition has the ability to normalize tumor vasculature and restore chaotic blood flow, thus reducing tumor hypoxia and facilitating immune cell infiltration . These mTherapeutic combinations of AAD and IO have already been approved for RCC and endometrial cancer. In non-squamous NSCLC, the IMpower150 trial showed an OS benefit for the first-line quadruple (atezolizumab/bevacicumab/carboplatin/paclitaxel) therapy versus AAD/doublet-chemotherapy with a particular benefit in patients with EGFR-mutant/ALK-positive tumors or baseline hepatic metastases . The obsplus PD-L1 inhibitor enhanced radiation effects [Radiation acts cytotoxic by inducing caspase-driven genomic and mitochondrial DNA fragmentation in tumor cells, promoting the release of cytochrome c from mitochondria to activate caspase 9 (CASP9) to ultimately initiate intrinsic apoptosis. Also, radiation alters the inflammatory TME by activating cytosolic DNA sensing pathways in DC , possibl effects .The additive effect of radiotherapy and IO was investigated in the phase III PACIFIC trial. A long-term survival benefit was seen with PD-L1 inhibitor durvalumab versus placebo when used as consolidation therapy in patients with stage III unresectable NSCLC, who did not have disease progression after concurrent chemoradiotherapy .DNA damage occurs frequently during cell replication and cells have evolved various DNA Damage Response (DDR) pathways to repair damaged DNA, which when accumulating would lead to cell cycle arrest or apoptosis . One DDRPARP inhibitors (PARPi) are well established in the treatment of BRCA-mutated breast and ovarian cancer independent of HRD status , being highly associated with sensitivity to platinum-based chemotherapy .The BRCA-proficient NSCLC is not clinically responsive to PARPi monotherapy. However, numerous clinical studies showed synergistic effects of PARPi and IO in several solid BRCA-proficient malignancies . As obseplus olaparib in PD-1/PDL-1 refractory patients. The phase II Jasper trial (NCT03308942) studies first-line Niraparib plus a PD-1 inhibitor in PD-L1 positive patients progressive on chemotherapy. Results have not been released, however preliminary data from other tumor entities are promising [plus chemotherapy versus placebo plus chemotherapy in advanced or metastatic NSCLC patients.Following these encouraging investigations, combinational IO/PARPi NSCLC studies are ongoing: The phase II Hudson umbrella trial (NCT03334617) investigates durvalumab romising , 102. LaAltogether, combining PD-1/PD-L1 inhibitors with PARPi is preclinically active in BRCA-proficient tumors and numerous clinical investigations in NSCLC are ongoing.cGAS-STING pathway has been identified as key intracellular pathway bridging anti-cancer innate and adaptive immunity [+T cells and promoting DC migration and maturation, thus enhancing anti-tumor immune responses [The immunity . Stimulaesponses , 104. Caesponses .+ T cells at the tumor site can enhance concomitant anti-PD-1 therapy effect [ADU-S100 is currently under investigation in clinical phase I/II trials as i.t. monotherapy or in combination with ICI in advanced solid tumors or lymphoma. A first-in-human study (NCT03010176) of STING agonist MK1454 as i.t. monotherapy or together with pembrolizumab in advanced solid tumors or lymphomas showed encouraging results with PR in 24% of patients and substantial tumor size reduction (83% of both injected and non-injected target lesions).Based on this understanding, STING agonists, including STING-binding molecules and CDN derivatives, are being developed as novel cancer therapeutics. Preclinical studies showed dramatic anti-cancer effects of intratumorally (i.t.) applied STING agonist , 106\u2013108y effect , 110. ThIn conclusion, i.t. STING agonists may evolve as potent combination to ICI treatment by \u201cboosting\u201d cancer-directed immune responses and sensitizing tumor cells to ICI.eff proliferation and activation [Tryptophan catabolism, involving the key enzymes indoleamine 2,3-dioxygenase 1 and 2 (IDO1 and 2) and tryptophan-2,3-dioxygenase (TDO2) is a critical metabolic pathway in cancer progression. IDO is IFN-induced in cancer, stromal non-immune and immune cells that metabolizes tryptophan to kynurenine. Its overexpression has immunosuppressive functions by depleting tryptophan and increasing kynurenine in the TME. Indeed, kynurenine accumulation and tryptophan depletion promotes the generation of Tregs and MDSCs, and inhibits Ttivation . IDO1 uptivation . Varioustivation ). Althoutivation .IDO1 inhibitors (IDO1i) have been tested in multiple phase I/II trials in combination with PD-1/PD-L1/CTLA-4 inhibitors with promising results (reviewed in ). HoweveArginine is a semi-essential amino acid critical for lymphocyte proliferation and function. The enzymes arginase 1 and 2 (ARG1/2) regulate extracellular arginine availability by converting arginine to ornithine and urea. High ARG1/2 expression and activity has been shown in various cancer types including NSCLC and assoARG inhibitors have entered clinical trials and most substances competitively target ARG1 and ARG2. In advanced or metastatic solid cancers including NSCLC a phase I/II study (NCT02903914) investigates the small molecule INCB001158 alone or in combination with pembrolizumab. First results from CRC show manageable AEs and clinical responses. The substance OATD-02 is a selective ARG1/2 inhibitor and has shown significant anti-tumor immunity in preclinical tumor models alone or in combination with PD-1 or IDO1i.Epigenetic-modulating drugs like 5-azacitidin (DNA hypomethylating agent) and entinostat (class I HDAC inhibitor) are well established in hematology. In addition to reactivating expression of epigenetically silenced tumor suppressor genes in cancer cells, these drugs may also selectively inhibit MDSC by induction of viral mimicry via inducing retrotransposon-derived dsRNA. This increases tumor foreignness through enhanced neoepitope expression, as well as it upregulates genes related to immune-evasion, such as B2M. In preclinical models, the combination of epigenetic modulators and PD-1 inhibitors has shown major therapeutic effects , 118.Based on these investigations, numerous phase I/II clinical trials in various solid tumor entities have been initiated, including NSCLC. Though interim analysis showed promising results, most of these studies are currently still ongoing .eff and NK cell inhibition or CAF proliferation, thereby fostering a tumorigenic TME. CD73 expression and consequently adenosine generation is regulated via complex molecular pathways, including HIF-1alpha, MAPK, mTOR, TGF-beta [+ and CD8+ T cell activation and lower PD-L1 expression [Adenosine is an effective endogenous immunosuppressive mediator in normal and cancerous tissues. It gets either excreted by stressed or injured cells or generated via a multi-staged pathway from extracellular adenosine-triphosphate (ATP) through dephosphorylation of adenosine-monophosphate (AMP) by the enzyme CD73 . In the TGF-beta . Some tuTGF-beta , and in TGF-beta . In NSCLpression .plus durvalumab is being tested in phase II studies in locally advanced or metastatic ICI-refractory or as neo-adjuvant therapy in resectable NSCLC. Concerning A2aR antagonists the two oral small molecules cifroadenant (CPI-444) and AZD4635 are currently under investigation in phase I studies alone or in combination with PD-L1 inhibitors. NSCLC-regarding results of both studies have not been released yet.Therapeutic attempts have focused on inhibiting adenosine production by targeting CD73 or interfering with adenosine signaling by targeting A2aR. Different anti-CD73 antibodies have entered clinical trials as monotherapy or in combination with ICI: The anti-CD73 antibody oleclumab The CC chemokine receptor type 4 (CCR4) is expressed on Treg and other circulating/tumor-infiltrating T cells and binding of TME-derived ligands to CCR4 promotes recruitment of immunosuppressive Treg. Therapeutic Treg depletion may alleviate the suppression of anti-tumor immunity and hence synergize with PD-1 inhibition, as also suggested by a preclinical study . FurtherThe monoclonal anti-CCR4 antibody mogamulizumab exerts Treg-depleting effects and is FDA-approved for refractory T cell lymphoma. First results from phase I solid tumor trials in combination with PD-1/PD-L1/CTLA-4 inhibitors suggest an acceptable safety profile , 128 andPolarization of TAM to the pro-tumorigenic M2 phenotype is promoted by binding of tumor cell-derived M-CSF to CSF1R on TAM. Anti-CSF1R antibodies can deplete TAM, however clinical studies failed to show potent anti-tumor effects of the monotherapy (e.g. NCT01494688). A study by Kumar et al. showed that CSF downregulates granulocytic chemokine (e.g. CXCL1/2) production by CAF and that anti-CSF1 antibodies hence promote TME infiltration by immunosuppressive MDSC. Inhibition of both CSF1R and CXCR2 decreased TME infiltration by TAM and MDSC, significantly reduced tumor growth and enhanced the effect of PD-1 inhibitor .plus nivolumab in advanced pancreatic cancer failed its primary endpoint.Numerous ongoing preclinical studies are testing CSF1R antagonists with different IO partners. In advanced NSCLC, two phase I trials are currently investigating the CSF1R antagonist cabiralizumab in combination with an anti-CD40 mAb or nivolumab, respectively. Unfortunately, a recent phase II trial (NCT03336216) testing cabiralizumab Retinoic acid Inducible Gene 1 (RIG-I) is a cytosolic RNA receptor ubiquitously expressed in most human body cells and is known for its major role in antiviral immune defense by inducing pyroptosis. RIG-I is also expressed in cancer cells, acting pro-inflammatory by expressing INF I and other cytokines . In precn\u2009=\u200915). There were no dose-limiting toxicities, especially as only minimal systemic exposure was found after i.t. application. Interestingly, systemic chemokine elevation and INF-associated gene expression were detected. RIG-I agonists are only at the starting point of clinical applicability. Therapeutic challenges include the development of highly selective agonists due to ubiquitous RIG-I expression and to avoid uncontrolled cytokine release.Intratumoral application of the selective RIG-I agonist RGT100 was investigated in a small phase I/II first-in-human study (NCT03065023) in advanced or recurrent cancer (The immunosuppressive activity of CAF can be hampered by blocking cell surface markers and most experience has been gathered with fibroblast-activation protein \u03b1 (FAP\u03b1), a common but non-selective CAF marker in many cancer types . In a moA recent pioneer study investigated the use of a bispecific antibody (RO6874281) consisting of an interleukin-2 variant (IL-2v) domain that binds the IL-2 receptor on immune cells and a FAP\u03b1-specific domain, which tracks the antibody-drug conjugate inside the tumor and reduces efflux. RO6874281 showed an acceptable safety profile and displayed monotherapy activity in tumor types not previously reported to respond to IL-2 A phase In this article, we discussed relevant immunomodulatory pathways imprinted within the TME that fundamentally impact the evolution of IO resistance in NSCLC and summarized novel therapy approaches targeting many of these alterations. Considering that the majority of NSCLC patients eventually progress on IO therapy, combinational or multimodal treatment approaches are an unmet medical need.The mechanisms underlying IO efficacy are still incompletely understood. Factors such as the dynamic cellular composition and heterogeneity of immunogenic and metabolic pathways within the TME, as well as the mutational load driving tumor immunogenicity, all contribute to IO effectiveness and evolution of resistance mechanisms.The hallmarks of carcinogenesis are significantly influenced not only by cancer cell-intrinsic mechanisms but also by the different stromal cell populations . The hetMany of the discussed novel treatment approaches either aim to inhibit intrinsic immunosuppressive or promote proinflammatory/immunogenic pathways. Combinations of these targeted approaches with different ICI are often synergistic and may evolve as promising strategies to overcome IO resistance. Moreover, dual ICI therapy with PD-1/CTLA-4 antibodies may boost intrinsic anticancer immunity and has previously been translated into clinical OS benefit (see CheckMate227). Combinations of PD-L1 and alternative IC have shown promising results in phase I trials.Concerning biomarkers, PD-L1 is still considered the most robust biomarker in NSCLC, even though in many cases its predictive power is insufficient. Thus, the need for further, more complex biomarker-signatures that help to optimize patient selection for the different IO strategies is immense. A priori identification of resistance mechanisms in order to initiate targeted therapies upfront will depict a major challenge. In-depth tumor analysis including whole-genome sequencing, single cell RNA-sequencing, multidimensional flow cytometry or epigenetics might be implemented in the future as to find individualized treatment strategies.IO therapy induces a wide range of cellular and molecular alterations in the TME and resistance mechanisms are only partially understood. However, as research is rapidly growing, numerous targets have been identified that may inhibit or override IO resistance. With positive results from many clinical trials, these novel IO combinational approaches pose a promising outlook for future therapies that improve clinical outcome and patient survival."} +{"text": "Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a systemic necrotizing inflammation of the small vessels.\u00a0Central nervous system (CNS) ANCA-associated vasculitis is a rare manifestation of AAV.\u00a0Three mechanisms of AAV affecting the CNS have been reported which include contiguous granulomatous invasion from nasal and paranasal sinuses, remote granulomatous lesions, and vasculitis of small vessels. Chronic hypertrophic pachymeningitis (CHP) is the meningeal-site involvement in AAV caused by granulomatous inflammation in the dura mater. We present a case of\u00a0pachymeningitis manifested with slowly progressive cognitive dysfunction, leptomeningeal enhancement on MRI, and necrotic vessels with surrounding inflammation on biopsy. This case represents a rare development of subsequent CNS AAV in a patient with ANCA-associated interstitial lung disease treated with rituximab with a resolution of leptomeningeal enhancement on a follow-up magnetic resonance imaging (MRI). Pachymeningitis is fibrous inflammatory changes involving the central nervous system (CNS) dura matter. Various etiologies for pachymeningitis were identified including infectious diseases such as tuberculosis, syphilis, cryptococcal infection, and Lyme disease; autoimmune or inflammatory diseases such as granulomatosis with polyangiitis (GPA), sarcoidosis, and immunoglobulin G4(IgG4)-related disease; and malignancies, in particular, lymphoma . ClinicaChronic hypertrophic pachymeningitis (CHP) is the meningeal involvement in CNS antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). We present a case highlighting\u00a0the rare development of AAV-associated pachymeningitis in a patient with prior ANCA-associated interstitial lung disease, treated with rituximab with a resolution of leptomeningeal enhancement on follow-up magnetic resonance imaging (MRI).A 76-year-old Hispanic female presented with progressive forgetfulness, depression and personality changes over a few weeks. She was subsequently admitted to the hospital after the MRI brain showed leptomeningeal enhancement and multifocal white matter fluid-attenuated inversion recovery (FLAIR) hyperintensities. She also has interstitial lung disease ) with positive\u00a0perinuclear ANCA (p-ANCA) treated with rituximab three years prior to presentation. Serial pulmonary function tests (PFTs) and a follow-up chest computed tomography (CT) were stable. Her physical exam was notable for known basilar dry rales in both lungs, newly noticed low cognitive testing and flat affect.Laboratory evaluation revealed hemoglobin 11.1 , white blood cell count (WBC) 10.7 , platelet 365,000 , erythrocyte sedimentation rate 96 mm/hr and C-reactive protein 3.05 mg/dL . Urinalysis showed no proteinuria or hematuria. Antinuclear antibodies by enzyme-linked immunosorbent assay (ELISA) was positive with a titer of 1:160, homogeneous pattern . Anti-double-stranded deoxyribonucleic acid (anti-dsDNA) antibody, anti-Sj\u00f6gren's syndrome-related antigen A/antigen B (SSA/SSB) antibody, anti-ribonucleoprotein (RNP), and anti-Smith antibodies were negative. Anti-cyclic citrullinated peptide antibodies were negative, but rheumatoid factor (RF) was 115 . Repeat ANCA by immunofluorescence and ELISA showed positive p-ANCA/myeloperoxidase antibody (more than eight), negative cytoplasmic-ANCA (c-ANCA) and normal serum complement levels. Lumbar puncture revealed WBC 15 cells/\u00b5L with 58% lymphocytes , protein 141 mg/dL and glucose 57 mg/dL . Serology for Epstein-Barr virus, herpes simplex virus, enteroviruses, cytomegalovirus, and varicella zoster virus, bacterial and fungal cultures, were negative.\u00a0Magnetic resonance angiography (MRA) showed a patent intracranial vasculature without evidence of luminal irregularities.\u00a0A leptomeningeal biopsy was performed, with the surgeon commenting on grossly abnormal white-yellow thickened leptomeningeal tissue Figure . The bioAAV is a systemic vasculitis characterized by necrotizing inflammation of small-sized blood vessels with few or no immune deposits .\u00a0Major vThere are three pathogenic mechanisms described: first, granulomatous contiguous invasion from nasal and paranasal sinuses causing cranial nerve palsies, meningeal involvement and pituitary gland hormonal dysfunction; second, remote granulomatous lesions, less common than contiguous invasion, causing intracerebral granuloma and cranial nerve involvement; and third, vasculitis of small blood vessels causing ischemic and/or hemorrhagic complications .Our patient had a leptomeningeal enhancement which is a representation of CHP. CHP is the meningeal-site involvement in AAV and caused by granulomatous inflammation at the dura mater . The preOur patient presented with interstitial lung disease three years after diagnosis with cognitive impairment and leptomeningeal enhancement on MRI without another active organ involvement. She was diagnosed with ANCA-associated CNS vasculitis. There is evidence that a significant number of patients with interstitial lung disease and positive ANCA - especially anti-MPO ANCA - will go on to develop microscopic polyangiitis . InteresCHP is a rare disease resulting from meningeal involvement in CNS AAV. ANCA serology, MRI findings, and leptomeningeal biopsy help in making the diagnosis. Early detection of pachymeningitis is crucial in preventing permanent neurological damage. CNS AAV is typically treated similarly to AAV with other systemic involvement, with remission-induction therapy consisting of high-dose corticosteroids and rituximab. More studies are needed to assess the most effective and safest immunosuppressive therapy for ANCA-associated CNS vasculitis."} +{"text": "Respiratory syncytial virus (RSV) is the primary cause for acute lower respiratory syndrome in children younger than 5 years. Research on B cell repertoires and antibodies binding the RSV fusion protein (RSV F) is of major interest in the development of potential vaccine candidates and therapies. B cell receptors (BCRs) which have higher affinities for a specific antigen are preferentially selected for B cell clonal expansion in germinal center reactions. Consequently, antigen-specific BCR repertoires share common features, as for instance preferential variable gene usage, variable region mutation levels or lengths of the heavy chain complementarity-determining region 3. Since RSV repeatedly infects every person throughout life, memory B cells (MBC) expressing RSV F-binding BCRs circulate in the blood of healthy adults. This dataset of BCR variable region sequence features was derived from single cell-sorted RSV F-directed MBCs of a healthy adult blood donor Specifications Table\u2022The data enables characterization of a memory B cell receptor (BCR) repertoire directed against the fusion protein of RSV.\u2022The data can be used in different studies on RSV F-specific BCR repertoires. For example, comparison of the dataset with vaccine-induced RSV F-directed BCR repertoires may provide insights on how a certain vaccine reshapes RSV F-binding BCR repertoires, which were previously induced by natural infections.\u2022The data can be integrated and compared with any dataset of BCR sequence features, which was derived with the same or comparable means of antibody sequence analysis.\u2022The methods applied to acquire this dataset are applicable for BCR repertoire analyses in the context of different antigens and pathogens.1H, V\u03ba and V\u03bb) sequence features characterizing each single BCR in the repertoire was acquired using the bioinformatics software tools described in the methods sections, and is provided in tabular form in the Supplementary Table 1 (VH), Supplementary Table 2 (V\u03ba), and Supplementary Table 3 (V\u03bb). The isotypes of each BCR, which are listed in the Supplementary Table 4 (isotypes), were identified by the first four codons of the constant regions B cell receptor (BCR) sequences binding the pre-fusion (pre F) or post-fusion (post F) conformation of RSV F were derived from single cell-sorted memory B cells (MBCs) of the healthy adult blood donor BD09 \u03ba or V\u03bb sequences are listed in the color legend on the top right in H region mutation percentages of pre F- or post F-isolated IgMs, IgGs and IgAs, while L region mutation percentages of pre F- or post F-isolated V\u03ba or V\u03bb sequences.The numbers of analyzed pre F- and post F-specific IgM, IgG, IgA, Vp-values), are provided in the Supplementary Data .The posterior probability density functions of selection strengths (\u03a3) on replacement mutations in the heavy chain complementarity-determining regions (CDRH) and the heavy chain framework regions (FWRH) of the pre F- or post F-isolated IgMs, IgGs or IgAs are visualized in H and JH or VL and JL genes in the pre F- or post F-isolated BCR repertoires.The CDRH3 amino acid length distributions in the pre F or post F BCR repertoires are shown in The clonal relatedness data of the pre F- or post F-isolated BCR repertoires is visualized in H region mutation percentages of pre F-, post F- or pre/post F cross-binding clonotype sequences.The bar charts in p-values), are provided in the Supplementary Data (Supplementary Table 8 and PDF-files).The plot in H-VL sequence pairs.The CIRCOS plots in 3http://www.bu.edu/computationalimmunology/research/software/) for implementation of a Bayesian method [After isolating RSV pre or post F-binding BCR sequences from single cell-sorted MBCs of the healthy blood donor BD09 n method , 3. Sequhttp://selection.med.yale.edu/baseline/Archive/) to measure selection strengths on replacement mutations CDR, RCDR, SFWR, RFWR). Then, it calculates expected mutation frequencies based on an underlying mutability model to account for hotspot and coldspot motifs [Data on mutation selection strength was acquired using Bayesian estimation of Antigen-driven SELectIoN .BCR variable region sequence feature datasets were managed with Excel 2010 (Microsoft Corporation), and visualized with GraphPad Prism Version 7.00 (GraphPad Software) or R scripts .The authors have declared the following potential conflicts of interest: Simona Tavarini, Chiara Sammicheli, Silvia Guidotti, Giulia Torricelli, Ugo D'Oro, Oretta Finco and Monia Bardelli are employees of the GSK group of companies. Gerald Schneikart participated in a post-graduate studentship at GSK."} +{"text": "To monitor patients after CAR T cell treatment, measuring frequencies of chimeric antigen receptor (CAR) T cells is crucial. However, experimental assays to quantify CAR T cells are lacking. Here, we describe a quantitative single copy gene-based PCR approach to measure frequencies of CAR T cells based on the FMC63 single chain variable fragment (scFv) including commercially available CAR T cell products. Besides enabling to monitor development of CAR T cells after treatment and guide further therapeutic decisions, this quantification assay proved highly useful for diagnosis of CAR T cell associated neurotoxic side effects. Overall, this quantification approach contributes significantly to the better monitoring and safety of treatment of patients with CAR T cells.Chimeric antigen receptor (CAR) T cell (CART) therapy has been established as a treatment option for patients with CD19-positive lymphoid malignancies in both the refractory and the relapsed setting. Displaying significant responses in clinical trials, two second-generation CART products directed against CD19, axicabtagene ciloleucel (axi-cel) and tisagenlecleucel (tisa-cel), have been approved and integrated into the clinical routine. However, experimental assay for quantitative monitoring of both of these CART products in treated patients in the open domain are lacking. To address this issue, we established and validated a quantitative single copy gene (SCG)-based duplex (DP)-PCR assay (SCG-DP-PCR) to quantify CARTs based on the FMC63 single chain variable fragment (scFv), i.e., axi-cel and tisa-cel. This quantitative PCR (qPCR) approach operates without standard curves or calibrator samples, offers a tool to assess cellular kinetics of FMC63 CARTs and allows direct comparison of CART-copies in axi-cel versus tisa-cel patient samples. For treating physicians, SCG-DP-PCR is an important tool to monitor CARTs and guide clinical decisions regarding CART effects in respective patients. Treatment with chimeric antigen receptor (CAR) T cells (CARTs) is altering the landscape of immunotherapy for patients with relapsed and/or refractory (r/r) B cell malignancies including pediatric ,2 and adCARTs are personalized living drugs with variable pharmacokinetic and pharmacodynamic profiles that depend not only on patient-specific characteristics, but also on the administered CART dose, lymphodepletion strategy and targeted disease . ResponsInformed consent was obtained from all patients prior to treatment. Axi-cel and tisa-cel were administered as per clinical routine. Peripheral blood (PB) samples were collected weekly within the first two to three weeks following CAR T cell administration and at different timepoints thereafter. Cerebrospinal fluid (CSF) samples were collected in case of neurological alterations. RPPH1; in the following referred to as RNaseP) as human SCG and the FMC63 sequence of the CAR was performed on genomic DNA (100 ng) derived from PB mononuclear cells (PBMCs) and isolated cells from CSF or CART product samples. Sample processing, assay preparation, general PCR procedure as well as analyzing strategies were performed as described [Single copy gene (SCG)-based duplex (DP)-qPCR assay (SCG-DP-PCR) simultaneously amplifying the ribonuclease (RNase) P RNA component H1 (escribed .(1)FMC63 forward primer (FP): TGAAACTGCAGGAGTCAGGA, reverse primer (RP): CTGAGACAGTGCATGTGACG, probe: FAM-CTGGCCTGGTGGCGCCCTCA-MGB/NFQ. All oligonucleotides bind within the FMC63 sequence of the CAR constructs.(2)RNaseP primer probe reaction mix was used as described .The following primer and probe sets were used:2O. Although for SCG-DP-PCR no standard curves are required, validation was based on the use of standard curves for qPCR reactions targeting FMC63 as well as RNaseP. On one hand, the standard curve stock sample of genomic DNA isolated from an axi-cel product was used. In line with Fehse et al., who measured high transduction rates (83\u201399%) in axi-cel products via digital PCR , a CART 2) \u2265 0.98) of PCR reactions targeting FMC63 and RNaseP were assessed. Constancy of PCR efficiencies across a wide target concentration range (0.1% to 100%) was tested, i.e., similarity of \u0394(Ct FMC63\u2013Ct RNaseP) in all standards via a relative efficiency plot was confirmed. Three validation runs were performed.Method validation and evaluation of SCG-DP-PCR was performed as follows: using generated standard curves; efficiencies (100% \u00b1 10%) and linearities . For this, CART-copies were quantified in the PB sample of a patient after axi-cel treatment using the patient-specific FMC63 standard curve generated from the patient\u2019s axi-cel product (see above). The result was normalized to RNaseP as previously reported . Determi\u2212\u0394Ct calculation as previously described [After validation, SCG-DP-PCR was applied on PB and CSF samples of patients treated with axi-cel or tisa-cel and copy numbers assessed using the 2escribed , i.e., aFor calculation of the copy number/cell in a CART product this formula was modified :copy\u00a0nuEfficiencies and linearities of standard curves were within accepted ranges for all validation runs. PCRs targeting FMC63 and RNaseP in axi-cel and tisa-cel standard samples displayed similar efficiencies of 96.5% with slightly differing standard deviations of 2.8% and 1.4%. For all validation experiments, relative efficiencies were similar for defined target concentration ranges. Exemplary data from one of three validation experiments are displayed in \u2212\u0394(Ct FMC63 \u2212 Ct RNaseP) \u00d7 2) [Differences in results between SCG-DP-PCR and patient-specific ACM were within an acceptable range A: A CARTeP) \u00d7 2) , correspAfter validation, SCG-DP-PCR was used on samples of patients treated with axi-cel or tisa-cel. Results from six patients are displayed in Quantification of CARTs after patient treatment is of crucial importance to monitor CART expansion after treatment. SCG-DP-PCR as a FMC63-universal PCR approach for CART quantification described here enabled us to accurately monitor CART kinetics, but was also useful to diagnose ICANS and differentiate CART-associated neurotoxicity from other neurologic etiologies. Consequently, SCG-DP-PCR provides a tool to guide clinical decisions as high CART frequencies require careful monitoring of CART side effects, whereas vanishing CART levels might indicate the need for a potential second CART administration or T cell stimulating agents such as checkpoint inhibitors . Recently, a digital PCR (dPCR) to assess axi-cel CART copy numbers has been described . This apAlternatively, flow cytometry (FC) can be used to quantify CARTs. However, depending on the target population size and total event count, FC-based approaches can be less sensitive. Additionally, cytopenia that occurs frequently after CART treatment ,23,24,25Here, we describe a FMC63-universal PCR approach for quantification of CARTs including commercially available CART products. Besides monitoring CART frequencies in patients after CART treatment, SCG-DP-PCR proved useful in the detection and differentiation of CART-associated side effects. Overall, this quantification approach significantly contributes to improve clinical treatment with CARTs."} +{"text": "We demonstrated that tumor cell-intrinsic activation of the cytosolic innate immunoreceptor RIG-I by its synthetic ligand 3pRNA overcomes transcriptional HLA-I APM suppression in patient-derived IFN-resistant melanoma cells. De novo HLA-I APM expression is IRF1/IRF3-dependent and re-sensitizes melanoma cells to autologous cytotoxic CD8+ T cells. Notably, synthetic RIG-I ligands and ICB synergize in T cell activation, suggesting combinational therapy could be an efficient strategy to improve patient outcomes in melanoma.In recent years, therapy with immune modulating antibodies, termed immune checkpoint blockade (ICB), has revolutionized the treatment of advanced metastatic melanoma, yielding long-lasting clinical responses in a subgroup of patients. But despite this remarkable progress, resistance to therapy represents a major clinical challenge. ICB efficacy is critically dependent on cytotoxic CD8+ T cells targeting tumor cells in an HLA class I (HLA-I) antigen-dependent manner. Transcriptional suppression of the HLA-I antigen processing and presentation machinery (HLA-I APM) in melanoma cells leads to HLA-I-low/-negative tumor cell phenotypes escaping CD8+ T cell recognition and contributing to ICB resistance. In general, HLA-I-low/-negative tumor cells can be re-sensitized to T cells by interferons (IFN), augmenting HLA-I APM expression. However, this mechanism fails when melanoma cells acquire resistance to IFN, which recently turned out as a key resistance mechanism in ICB, besides HLA-I APM suppression. Seeking for a strategy to overcome these barriers, we identified a novel mechanism that restores HLA-I antigen presentation in tumor cells independent of IFN (Such Melanoma immunotherapy exploits the capability of cytotoxic CD8+ T cells to selectively kill tumor cells. This selectivity is achieved by the T cell receptor binding to specific HLA class I (HLA-I) antigen complexes on melanoma cells. However, in the tumor microenvironment T cell activity is blocked by the inhibitory co-receptor PD-1 (immune checkpoint), signaling upon engagement of its ligand PD-L1 on melanoma cells. Therapeutic antibodies have been developed that disturb the inhibitory PD-1/PD-L1 axis and release T cells from suppression. Remarkably, antibody-based therapy, termed immune checkpoint blockade (ICB), induces clinical responses in 40-50% of melanoma patients with advanced metastatic disease. But despite this tremendous clinical progress, still the majority of patients does not respond at all to ICB (primary resistance) or relapse after initial therapy response (acquired resistance).HLA-A, HLA-B, HLA-C, B2M, TAP1, TAP2, TABP, LMP7 and LMP9 genes, gives rise to those phenotypes that escape recognition by cytotoxic CD8+ T cells. Analyzing distinct melanoma transcriptomic data sets and annotated clinical data, we found low HLA-I APM expression levels associated with ICB resistance. Type I (IFN\u03b1/\u03b2) and type II (IFN\u03b3) interferons (IFN) are well defined for their capability to counteract HLA-I downregulation by activation of JAK/STAT signaling pathways . However, mutational inactivation of JAK1, a kinase involved in IFN-I/-II signaling, enables melanoma cells to preserve their immune-evasive HLA-I-low/-negative phenotype in an IFN-rich microenvironment. Defective IFN signaling was recently defined as a key resistance mechanism in ICB which led us to seek for strategies enhancing HLA-I antigen processing and presentation by IFN-dependent and IFN-independent mechanisms.We aimed to elucidate resistance mechanisms in ICB in order to improve patient outcome and identified the development of poorly immunogenic HLA-I-low and HLA-I-negative tumor phenotypes as a barrier to effective immunotherapy. Transcriptional suppression of the HLA-I antigen processing and presentation machinery (HLA-I APM), including de novo HLA-I APM expression in patient-derived JAK1 mutant melanoma cells and restored their T cell sensitivity, demonstrating that activated RIG-I triggered an IFN-independent salvage pathway capable of overcoming tumor cell-intrinsic T cell resistance. Mechanistic studies revealed that this process was dependent on the transcriptional activators IRF1 and IRF3 . In addition to its effects on antigen presentation, RIG-I activation also induced de novo expression of several T cell-attracting chemokines in IFN-sensitive and -resistant melanoma cells. Pronounced effects on tumor antigen presentation and T cell attraction were observed also upon intratumoral 3pRNA application in different xenotransplant melanoma models. Consistent with our in vitro and in vivo studies we found expression of HLA-I APM genes, chemokines, and markers of T cell activation enhanced in RIG-I (DDX58)-high tumors of the TCGA melanoma cohort. A strong correlation between expression of RIG-I (DDX58) pathway genes and HLA-I APM genes, was also detected in melanoma biopsies from ICB-treated patients. This prompted us to study the combination of 3pRNA and immune checkpoint blocking antibodies for its capacity to synergize in T cell activation. To this end, we took advantage of an autologous melanoma model from a patient with primary ICB resistance, consisting of CD8+ tumor infiltrating lymphocytes (TILs) showing positivity for PD-1 and TIGIT, another inhibitory co-receptor, and melanoma cells expressing the corresponding receptor ligands. In this model we demonstrated that HLA-I APM upregulation by 3pRNA and anti-PD-1/anti-TIGIT antibodies synergistically enhanced the reactivity of CD8+ TILs towards autologous melanoma cells.We speculated that innate pattern recognition receptors, controlling responses to viral infections, could be a mechanistic tool to efficiently induce HLA-I APM expression. As such, the cytosolic RNA helicase RIG-I responds to short 5\u2032-triphosphorylated double-stranded viral RNA and triggers expression of a gene set involved in detection and elimination of virus-infected cells. In fact, in distinct patient models we observed that activation of RIG-I by its ligand 3pRNA, a synthetic viral RNA mimetic, strongly enhanced HLA-I APM expression in melanoma cells and increased their sensitivity towards autologous CD8+ T cells. RIG-I activation also triggered the expression of IFN\u03b2, suggesting that autocrine and paracrine IFN-I signaling in melanoma cells elicited upregulation of the distinct HLA-I APM components. Strikingly, we detected similar effects in IFN-I-sensitive and IFN-I-resistant tumor cells. 3pRNA treatment even forced Overall, our study linked transcriptional HLA-I APM suppression in melanoma to ICB resistance and nominated RIG-I as a druggable therapeutic target to restore antigen processing and presentation and overcome T cell resistance of IFN-sensitive and IFN-resistant tumor cells. The observed synergistic effects of combined RIG-I activation and ICB in T cell activation provide a strong rational for ongoing clinical trials combining intratumoral injection of synthetic RIG-I ligands with anti-PD-1 or anti-PD-L1 antibodies. So far, this application is limited to accessible melanoma skin or lymph node lesions, indicating that development of specific transport systems allowing for tumor-specific drug delivery to distant metastases is urgently needed. Researchers should be encouraged to develop such carrier systems by the fact that, HLA-I APM-low phenotypes are involved not only in resistance to ICB but also other types of immunotherapy and can be detected also in other tumor entities besides melanoma."} +{"text": "The junctional adhesion molecule-A (JAM-A) is a cell surface adhesion molecule expressed on platelets, epithelial cells, endothelial cells and leukocytes (e. g. monocytes and dendritic cells). JAM-A plays a relevant role in leukocyte trafficking and its therapeutic potential has been studied in several pathological conditions due to its capacity to induce leukocyte migration out of inflamed sites or infiltration into tumor sites. However, disruption of JAM-A pathways may worsen clinical pathology in some cases. As such, the effects of JAM-A manipulation on modulating immune responses in the context of different diseases must be better understood. In this mini-review, we discuss the potential of JAM-A as a therapeutic target, summarizing findings from studies manipulating JAM-A in the context of inflammatory diseases (e.g. autoimmune diseases) and cancer and highlighting described mechanisms. The junctional adhesion molecule-A (JAM-A), also called junctional adhesion molecule-1 (JAM-1), is a member of the immunoglobulin superfamily and received its first denomination as F11 receptor (F11R), a molecule expressed on the surface of human platelets . JAM-A icis interactions). JAM-A dimers can then interact with other JAM-A dimers (trans interactions) to bridge epithelial cells in tight junctions and a membrane-proximal domain D2) Figure 1 Figure 1cis or trans interactions with other extracellular ligands (2 chain of the lymphocyte function-associated antigen 1 (LFA-1), but not of the macrophage-1 antigen (MAC-1), via the JAM-A D2 domain (3 integrin (CD61) aggregates in endothelial cell lysates (3 integrin (IIb integrin (CD41) , as wellntegrins , which mntegrins , and canntegrins , 20\u201322. 1 , as weigration , 23, traThe first report of a role for JAM-A in leukocyte migration comes from Mart\u00ecn-Padura et\u00a0al. , in whicIn vivo, treatment with anti-JAM-A mAb decreased leukocyte transendothelial migration through cremaster venules induced by IL-1\u03b2, but not by chemoattractants, leukotriene B4 (LTB4) or platelet-activating factor (PAF) . This sain vitro compared to their respective controls show an increased propensity to cross lymphatic endothelial cells when JAM-A activity is lacking . While mcontrols . These JA (cDNA) . These sDue to its expression in different cell types , its capacity to modulate cell adhesion and migration and its upregulation in inflamed tissues, JAM-A has been studied as a therapeutic target in a number of disease models.Inflammatory skin sites are characterized by proliferation of dermal cells, dilated blood vessels and accumulation of immune cells . In a moAdhesion molecules have been implicated in vascular wall integrity and play an important role in vascular diseases . JAM-A aIn models of ischemia-reperfusion (I/R) injury, both JAM-A genetic depletion and blockade with anti-JAM-A mAb suppressed leukocyte infiltration in response to cremaster muscle and livef11r messenger ribonucleic acid (mRNA) expression was found in both early atherosclerotic endothelium of carotid arteries (ex vivo perfusion model to demonstrate JAM-A role in early atherosclerosis. Treatment with this fusion protein inhibited the arrest of human monocytes and memory CD4+ T cells in murine atherosclerotic endothelium under blockade of very late antigen 4 (VLA-4), an intercellular adhesion molecule 1 (ICAM-1) ligand ligand . These rIn vitro, treatment with a JAM-A antagonistic peptide capable of blocking homophilic binding (peptide 4D), decreased platelet adhesion to inflamed endothelial cells, whereas agonistic reagents promoted platelet aggregation knockout animals lacking B and T cell development were also investigated in colonic tissues pointed to a possible role of this growth factor in the compensatory mechanism. Treatment with anti-TGF-\u03b21 mAb decreased body weight and increased disease activity index scores in JAM-A-deficient mice in comparison to anti-TGF-\u03b21-treated WT mice and to isotype-treated JAM-A-/- mice, demonstrating a protective role of TGF-\u03b21 in JAM-A-deficient mice from developing a more severe acute colonic inflammation.The intestinal mucosa forms an important barrier against potential hostile microorganisms and other foreign antigens. The permeability of the intestinal epithelium is mediated by tight junctions, formed of molecular components, such as adhesion molecules, that link intestinal epithelial cells and exert a control on the passage of environmental molecules. Intestinal barrier defects have been associated with chronic mucosal inflammation and inflammatory bowel diseases (IBDs) . A few s colitis . To addrstigated . Lack of-/- mice stimulated by these inflammatory mediators. Mice with selective loss of JAM-A on intestinal epithelial cells demonstrated increased intestinal permeability and reduced peritoneal neutrophil migration and macrophage chemokine production. These findings suggest that JAM-A expression in the epithelium is fundamental for JAM-A -mediated intestinal inflammation.Mice with selective loss of JAM-A in myelomonocytic cells, progenitors that can differentiate into monocytes, macrophages and subtypes of conventional DCs , were usExposure of human intestinal epithelial cells to cytokines was found to induce JAM-A cytoplasmatic tail tyrosine Y280 phosphorylation . ElevateDisruption of the blood-brain barrier of the central nervous system (CNS) leads to leukocyte accumulation in the cerebrospinal fluid, a main component of brain disorders such as meningitis and multf11r mRNA on PMBCs of RA patients (Rheumatoid arthritis (RA) is a chronic systemic inflammatory autoimmune disease that mainly affects the joints, with an essential participation of the adaptive immune system in its induction phase . A breacpatients . Neverthpatients , animalspatients .In a model of RA in which autoantibodies from arthritogenic K/BxN drive inflammation and tissue destruction in serum-recipient mice, treatment with anti-JAM-A mAb delayed the disease onset and partially ameliorated overall disease . SimilarThe six hallmarks of cancer are sustained proliferative signaling, resistance to cell death, replicative immortality, induction of angiogenesis, evasion of growth suppressors and activation of invasion and metastasis mechanisms . InterveIn vitro studies using human triple negative breast cancer or thyroid carcinoma cell lines showed increased tumor cell proliferation and migration under JAM-A gene silencing caused by JAM-A targeting suggests precaution in the interpretation of results from preclinical model studies. As such, studies with cell-selective JAM-A disruption that aim to distinguish pathway-specific effects in different pathological conditions will further our understanding of JAM-A role in autoimmunity and cancer and may highlight JAM-A as a potential therapeutic target for human disease.All authors listed have made substantial, direct, and intellectual contribution to the work and approved it for publication.CB was supported by CAPES Foundation, an agency under the Ministry of Education of Brazil (grant number 88881.129556/2016-01). CB, RB, JB, and PG were supported by the Arthritis Research UK (ARUK) programme grant number 19788 and the Innovative Medicines Initiative EU-funded project Be The Cure (BTCURE) [grant number 115142-2]. JB and PG were also supported by the Research into Inflammatory Arthritis Centre Versus Arthritis (RACE) (grant number 22072).Author RB was employed by the company Antibody Analytics Ltd.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "In the \u201cStudy of (E7080) Lenvatinib in Differentiated Cancer of the Thyroid (SELECT)\u201d, Lenvatinib significantly prolonged the progression-free survival, resulting in a more frequent use in clinical practice for this patient group. Due to considerable side effects, an accurate assessment of response to treatment is crucial in these patients. Therefore, we aimed to improve treatment individualization and reduce unnecessary therapies by selecting patients who will most likely benefit from Lenvatinib treatment using 2-deoxy-2-[18F] fluoro-D-glucose positron-emission-tomography/computed-tomography (18F-FDG-PET/CT) in the monitoring of functional tumor response compared to morphological response. Methods: In 22 patients, a modified Positron Emission Tomography Response Criteria In Solid Tumors (mPERCIST) evaluation before treatment with Lenvatinib and at 3 and 6 month follow up was performed. Further PET-parameters and morphologic tumor response using Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 were assessed and their prediction of progression-free survival (PFS) and disease-specific survival (DSS) was evaluated. Results: Most patients were rated stable in morphological evaluation and progressive using a metabolic response. All patients who responded to therapy through RECIST showed a decline in nearly all Positron Emission Tomography (PET)-parameters. For both time-points, non-responders according to mPERCIST showed significantly lower median PFS and DSS, whereas according to RECIST, only DSS was significantly lower. Conclusion: Tumor response assessment by 18F-FDG-PET outperforms morphological response assessment by CT in patients with advanced radioiodine refractory DTC treated with Lenvatinib, which seems to be correlated with clinical outcomes.Background: The tyrosine kinase inhibitor (TKI) Lenvatinib represents one of the most effective therapeutic options in patients with advanced radioiodine refractory differentiated thyroid carcinoma (DTC). We aimed to assess the role of 2-deoxy-2-[ Differentiated thyroid cancer (DTC) is the most frequent endocrine malignancy comprising the papillary (PTC), follicular (FTC) and poorly differentiated (PDTC) histological subtypes . AlthougTyrosine kinase inhibitors (TKIs), which inhibit VEGF receptor signaling and tumor angiogenesis, improve progression-free survival (PFS) in patients with structurally progressive, radioiodine refractory DTC ,8,9,10. 18F] fluoro-D-glucose positron emission tomography/computed tomography (18F-FDG-PET/CT) has the potential to improve diagnostic accuracy and prediction of the course of tumor development ..mean/max18F-FDG-PET/CT until time of death. The observation period ended on 26th of May 2020.PFS was defined as the time between treatment start and disease progression according to RECIST 1.1. Disease-specific survival (DSS) was calculated from the time of baseline p-values \u2264 0.05 were considered to indicate statistical significance. All analyses were performed using SPSS computer software .Ordinal and continuous variables are presented as median or mean \u00b1 standard deviation (SD). Change in % was calculated using the following formula: / \u2212 1) \u00d7 100. The Mann-Whitney u test was used to compare mean percentage changes between DC and PD patients. Survival analysis using Kaplan-Meier analysis was performed for PFS and DSS according to the mPERCIST and RECIST 1.1 criteria for 3 month and 6 month follow-ups. Quantitative survival data are given as median in months. Log rank test was used to compare survival rates between subgroups. 18F-FDG-PET/CT outperforms morphological response evaluation using CT and furthermore appears to be stronger correlated with outcome analysis. Monitoring tumor response with 18F-FDG-PET/CT in these patients has the potential to improve treatment individualization and avoid ineffective therapies by selecting patients who will most likely benefit from Lenvatinib treatment. Therefore, tumor response assessed by 18F-FDG-PET/CT is a highly promising modality in order to increase diagnostic accuracy in these patients and should be further investigated.In conclusion, our study suggests that in patients with advanced radioiodine refractory DTC undergoing Lenvatinib treatment, tumor response evaluation by"} +{"text": "COVID-19 (SARS-CoV-2) disease severity and stages varies from asymptomatic, mild flu-like symptoms, moderate, severe, critical, and chronic disease. COVID-19 disease progression include lymphopenia, elevated proinflammatory cytokines and chemokines, accumulation of macrophages and neutrophils in lungs, immune dysregulation, cytokine storms, acute respiratory distress syndrome (ARDS), etc. Development of vaccines to severe acute respiratory syndrome (SARS), Middle East Respiratory Syndrome coronavirus (MERS-CoV), and other coronavirus has been difficult to create due to vaccine induced enhanced disease responses in animal models. Multiple betacoronaviruses including SARS-CoV-2 and SARS-CoV-1 expand cellular tropism by infecting some phagocytic cells (immature macrophages and dendritic cells) via antibody bound Fc receptor uptake of virus. Antibody-dependent enhancement (ADE) may be involved in the clinical observation of increased severity of symptoms associated with early high levels of SARS-CoV-2 antibodies in patients. Infants with multisystem inflammatory syndrome in children (MIS-C) associated with COVID-19 may also have ADE caused by maternally acquired SARS-CoV-2 antibodies bound to mast cells. ADE risks associated with SARS-CoV-2 has implications for COVID-19 and MIS-C treatments, B-cell vaccines, SARS-CoV-2 antibody therapy, and convalescent plasma therapy for patients. SARS-CoV-2 antibodies bound to mast cells may be involved in MIS-C and multisystem inflammatory syndrome in adults (MIS-A) following initial COVID-19 infection. SARS-CoV-2 antibodies bound to Fc receptors on macrophages and mast cells may represent two different mechanisms for ADE in patients. These two different ADE risks have possible implications for SARS-CoV-2 B-cell vaccines for subsets of populations based on age, cross-reactive antibodies, variabilities in antibody levels over time, and pregnancy. These models place increased emphasis on the importance of developing safe SARS-CoV-2 T cell vaccines that are not dependent upon antibodies. In a T cells , 14. MER T cells , 16. Inf T cells and some T cells . The pos T cells . Also, n T cells .Characterizing variability of viral proteins can inform designing medical countermeasures (MCMs). For viral progeny, deleterious mutations are selected against . NeutralSARS-CoV-2 spike protein sequence from GenBank entry MN908947.3 was searched against the non-redundant (nr) and PDB database using the NCBI BLASTP web interface. Hit protein sequences were downloaded. Protein multiple sequence alignments were created with the Dawn program . The SpiDawn variation (V ) results for SARS-CoV-2 amino acid residues were classified as 650 V1 residues\u2014dark green, 263 V2 residues\u2014light green, 123 V3 residues\u2014yellow, 107 V4 residues\u2014light blue, and 152 V5+ residues\u2014dark blue . The darThe observed amino acid variations in SARS-CoV-2 proteins are consistent with expected natural variations in the context of random mutations and selection in the context of host immune responses. The Spike protein S1 extended domain shows the highest number of exposed surface highly variable residues . These sCoronaviruses have multiple approaches for infecting cells by direct receptor binding and by indirect antibody Fc uptake. The SARS-CoV-2 Spike protein contains receptor-binding domains (RBD) targeting human angiotensin I converting enzyme 2 (ACE2) , 33; thiin vitro human activated macrophages Fc\u03b3R reduced proinflammatory cytokine production may develop via more than one molecular mechanism. One model suggestions that antibody/Fc-receptor complex functionally mimics viral receptor enabling expanded host cell trophism of some phagocytic cells . Wan et 2) in COVID-19 patients virus, protein subunit, messenger ribonucleic acid (mRNA), or deoxyribonucleic acid (DNA) vaccine. Antibodies induced by vaccines can be neutralizing or non-neutralizing. Non-neutralizing antibodies can contribute to anti-viral activities with mechanisms including antibody-medicated complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) [reviewed ]. The yeMany of the viruses associated with ADE have cell membrane fusion mechanisms . For infp = 0.30) was observed in a randomized trial glycoproteins, Yuan et al. suggest B cell vaccines that target the Spike protein cell fusion mechanisms have the highest chance of raising neutralizing antibodies with minimal or no ADE risk due to antibody binding sterically blocking cell fusion. Antibodies targeting other portions of the Spike protein or other SARS-CoV-2 exposed proteins may enable infection of phagocytic immune cells even if they are neutralizing.T cell vaccines that target SARS-CoV-2 replicase proteins have the highest change of avoiding viral escape by antigenic variation and accumulation of mutations in variable residues. Lisziewicz and Lori describeGiven past data on multiple SARS-CoV-1 and MERS-CoV vaccine efforts have failed due to ADE in animal models , 81, it The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/DR conceived of the presented ideas, analyzed the data, and wrote the manuscript.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "As shown in c-e, a highly increased RV tracer uptake with only moderately increased LV tracer uptake was found . Tracer uptake co-localized with the echocardiographic finding of RV hypertrophy, suggesting cardiac involvement of AA amyloidosis with predominant right-sided amyloid deposition.A 62-year-old female with known systemic serum amyloid A (AA) amyloidosis presented with signs and symptoms of new-onset heart failure. Echocardiography demonstrated mild left ventricular (LV) dilation, preserved ejection fraction, and grade II diastolic dysfunction without typical signs of cardiac amyloidosis (CA) but a profound focal hypertrophy of the free right ventricular (RV) wall . Positron emission tomography/computed tomography (PET/CT) with the amyloid-binding tracer 18F-florbetaben PET/CT to detect AA-CA. PET/CT provides high image quality and quantitative measures of tracer uptake, thus making detection, localization, and absolute quantification of amyloid feasible and has the potential to substitute or even outperform endomyocardial biopsy, particularly in the case of focal or early-stage disease.Amyloid-binding radiotracers have already received approval for beta-amyloid brain imaging. Previous exploratory studies demonstrated their high diagnostic accuracy for CA of transthyretin or light-chain type \u20133, yet n"} +{"text": "Background: Little is known about the 2019 novel coronavirus disease (COVID-19) course and outcomes in patients receiving immunotherapy. Here we describe a metastatic Merkel cell carcinoma patient with a\u00a0severe acute respiratory syndrome coronavirus 2 infection while receiving pembrolizumab. Case presentation: A 66-year-old man, with a metastatic Merkel cell carcinoma receiving pembrolizumab, presented with fever. Chest computed tomography (CT) showed pulmonary ground-glass opacities, suggesting viral or immuno-related etiology. On day 7, the patient was hospitalized due to dyspnea and worsening of the radiological findings. Real time polymerase chain reaction\u00a0(RT-PCR) testing confirmed COVID-19. The patient developed acute respiratory distress syndrome and acute kidney injury. Hydroxychloroquine was administered for 5 days, but discontinued after supraventricular extrasystoles. Clinical improvement allowed the patient\u2019s discharge after 81 days of hospitalization. Conclusion: A careful evaluation of oncologic patients receiving immunotherapy during the COVID-19 pandemic is of utmost importance. The 2019 novel coronavirus disease (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged in Wuhan city and rapidly spread throughout China, causing a wide spectrum of severity and being classified by the WHO as a public health emergency of international concern. To date, WHO estimates that this pandemic has affected 20 million people and taken the lives of more than 750,000 individuals worldwide, not including the unreported cases; and its reach and severity continue to spread and 13 with severe disease [SD]), the total NK and CD8nfection . These rfections , also de[in vivo . However[in vivo . Therefoet al., reporting that hypermetabolic ground glass opacities with peripheral distribution by 18F-FDG PET/CT may appear even in SARS-Cov-2 infection or irAE induced by anti-PD-1 therapy [In addition, the clinical and radiological similarities between COVID-19 and ICI-induced pneumonitis may represent a pitfall in clinical practice. Pneumonitis as an immune-related adverse event (irAE) affects almost 5% of patients receiving anti-PD-1/PD-L1 in monotherapy and approximately 10% in anti-CTLA-4/anti-PD-1 combination therapy , rarely therapy . TherefoGuidelines have been published to guide and allow safe patient care, without jeopardizing the ongoing oncological treatment , includiTesting for the novel coronavirus is a top priority in response to the global SARS-CoV-2 pandemic, avoiding equivocal diagnosis or treatment delays. A careful evaluation and a rapid assessment of oncologic patients receiving ICI during the COVID-19 pandemic are essential. Gathering evidences to better characterize the spectrum of manifestations of COVID-19 and to guide tailored management algorithms in patients receiving ICI remains of utmost importance.The 2019 novel coronavirus disease (COVID-19) is a public health emergency of international concern.The role of immune checkpoint inhibitors in severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2) infection has not been established yet in prospective trials, although retrospective data suggest that chemo-immunotherapy can be safely delivered, eventually requiring regimen modifications or alternative schedules determined on an individual basis.NK cells and T lymphocytes exhaustion during the immune response against the SARS-Cov-2 infection is associated with PD-1 upregulation, viral disease progression and clinical decompensation.+ functions, which improves cellular immunity.Anti-PD-1 blockade can restore T CD8Immune overactivation, however, can lead to acute respiratory distress syndrome related to COVID-19.Suspected SARS-Cov-2 infection may mimic immune-related adverse events in those patients receiving immunotherapy, especially pneumonitis, which deserves a dedicated workup.Testing for the novel coronavirus is a top priority in response to the global SARS-CoV-2 pandemic, avoiding equivocal diagnosis or treatment delays.Evidences are needed to guide tailored management algorithms in patients receiving immune checkpoint inhibitor during the COVID-19 pandemic."} +{"text": "PTEN mutational loss has been associated with reduced RAD51 expression and homologous recombination deficiency (HRD), however; recent studies have failed to recapitulate these findings. Here, we show that RAD51 expression, foci formation and homologous recombination repair activity are unaltered in normal and tumorigenic PTEN-deficient cells and patient samples. Furthermore, we show that PTEN-deficient tumor cell lines do not synergize with the clinical PARP inhibitor olaparib, underscoring a need to discontinue its use in treating patients with PTEN-deficient tumors that do not otherwise exhibit HRD.PTEN is an important tumor suppressor that is frequently mutated in malignancy. PTEN to reduced RAD51 expression and function, a key factor involved in the homologous recombination (HR) pathway. However, these studies remain controversial, as they fail to establish a definitive causal link to RAD51 expression that is PTEN-dependent, while other studies have not been able to recapitulate the relationship between the PTEN expression and the RAD51/HR function. Resolution of this apparent conundrum is essential due to the clinically-significant implication that PTEN-deficient tumors may be sensitive to poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi) commonly used in the clinical management of BRCA-mutated and other HR-deficient (HRD) tumors. Methods: Primary Pten-deficient (and corresponding wild-type) mouse embryonic fibroblasts (MEFs) and astrocytes and PTEN-null human tumor cell lines and primary cells were assessed for RAD51 expression and DNA damage repair analyses . RAD51 foci analysis was used to measure HR-dependent DNA repair. Xrcc2-deficient MEFs served as an HR-deficient control, while the stable knockdown of RAD51 (shRAD51) served to control for the relative RAD51/HR-mediated repair and the phospho-53BP1 foci analysis served to confirm and measure non-homologous end joining (NHEJ) activity in PTEN-deficient and shRAD51-expressing (HRD) lines. Cell proliferation studies were used to measure any potential added sensitivity of PTEN-null cells to the clinically-relevant PARPi, olaparib. RAD51 levels and DNA damage response signaling were assessed in PTEN-mutant brain tumor initiating cells (BTICs) derived from primary and recurrent glioblastoma multiforme (GBM) patients, while expression of RAD51 and its paralogs were examined as a function of the PTEN status in the RNA expression datasets isolated from primary GBM tumor specimens and BTICs. Results: Pten knockout primary murine cells display unaltered RAD51 expression, endogenous and DNA strand break-induced RAD51 foci and robust DNA repair activity. Defective HR was only observed in the cells lacking Xrcc2. Likewise, human glioblastoma multiforme (GBM) cell lines with known PTEN deficiency show apparent expression of RAD51 and display efficient DNA repair activity. Only GBM cells stably expressing shRNAs against RAD51 (shRAD51) display dysfunctional DNA repair activity and reduced proliferative capacity, which is exacerbated by PARPi treatment. Furthermore, GBM patient-derived BTICs displayed robust RAD51 expression and intact DNA damage response signaling in spite of PTEN-inactivating mutations. RNA expression analysis of primary GBM tissue specimens and BTICs demonstrate stable levels of RAD51 and its paralogs , regardless of the PTEN mutational status. Conclusions: Our findings demonstrate definitively that PTEN loss does not alter the RAD51 expression, its paralogs, or the HR activity. Furthermore, deficiency in PTEN alone is not sufficient to impart enhanced sensitivity to PARPi associated with HRD. This study is the first to unequivocally demonstrate that PTEN deficiency is not linked to the RAD51 expression or the HR activity amongst primary neural and non-neural Pten-null cells, PTEN-deficient tumor cell lines, and primary PTEN-mutant GBM patient-derived tissue specimens and BTICs.PTEN mutation occurs in a variety of aggressive cancers and is associated with poor patient outcomes. Recent studies have linked mutational loss of PTEN encodes a phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase containing a tensin-like domain and a catalytic domain typical of those of the dual-specificity protein tyrosine phosphatases . Co. CoPTEN Poly ADP-Ribosylation (PARylation) A, olaparylation) A 55]..Poly ADPshSCM- and shRAD51-GBM lines was used to compare the specific requirement of RAD51 expression in PARPi-mediated growth suppression. The three GBM cell lines showed varying sensitivity to olaparib, which was not related to endogenous RAD51 levels. Interestingly, expression of shRAD51 and the resultant loss of the RAD51 protein dataset (PTEN-mutated or wild-type samples. A minor decrease in RAD51 expression was observed in PTEN-mutant samples from the TCGA dataset as compared to the wild type (p = 0.012), although this difference was nominal (<7%). Furthermore, we performed protein expression and DNA damage response signaling analyses on 2 primary (treatment-na\u00efve) and 2 recurring (treatment-resistant) GBM patient-derived BTIC lines expression of PTEN has no bearing on the expression of RAD51 or its paralogs, (ii) PTEN loss does not impart cellular HRD, (iii) PTEN expression has no effect on cellular DNA strand break repair rates, and (iv) PTEN loss does not synergize with PARPi. Consistent with our findings, studies have demonstrated that RAD51 expression and foci formation remain unchanged in other PTEN-deficient tumor cell lines , primary astrocytes, RNAi studies (shPTEN-expressing H1299), comparative prostatic tumor xenograft analyses , and human tumor tissue microarrays [Pten wild-type, heterozygote, and knockout astrocytes and MEF cells all display robust RAD51 expression and HR-dependent RAD51 foci formation in response to DNA damage regardless of the Pten status. Similarly, we find that PTEN-deficient glioblastoma lines show pronounced RAD51 expression and form HR-responsive RAD51-positive and NHEJ-responsive 53BP1-positive DNA damage-induced foci. Consistent with our data, Miyasaka and associates found that RAD51 levels were expressed broadly and IR-induced \u03b3H2AX and RAD51 foci formation was uniform amongst a set of 16 PTEN-proficient and deficient endometrial cell lines [\u201cSynthetic lethality\u201d (and the related \u201csynthetic sickness\u201d) in anti-cancer therapy arose from success in killing cancer cells by specifically targeting compensatory mechanisms, pathways, or activities that usually allow for tumor cell survival and growth despite the loss of the functionally important parallel cell growth/survival pathways ,60. Perh pathway ,61,62,63 pathway ,65. As a pathway ,68,69,70 pathway ,37,69. F pathway ,71. Amon complex . Subsequ complex ,33,72,73n cancer , with 40zygosity ,75, use roarrays ,35,76. Nll lines .RAD51B, RAD51C, and RAD51D [Pten-null astrocytes, our RNA expression analysis in human GBM patient-derived primary tumors and outgrowing BTICs clearly demonstrates that the expression of RAD51 and its paralogs remain unchanged regardless of PTEN status.It has been suggested that the apparent discordance in co-expression amongst these findings and other reports may reflect cell type-specific differences involving PTEN/RAD51 . Our stud RAD51D . AlthougshRAD51 co-expression does olaparib treatment enhance DNA damage and completely ablate PTEN-deficient tumor cell survival. Similarly, Turchick et al. [Although two studies show that PTEN-deficient endometrial tumor cells are not sensitive to PARPi monotherapy ,77, we ok et al. showed tPten-null MEFs [-/-Pten MEFs and astrocytes suggest that this divergence is likely due to the inherent differences amongst the two genetic Pten mouse models. It is noteworthy that cre recombinase expression in our conditional Pten (LoxP/LoxPPten) astrocyte and MEF cell lines results in deletion of exons 4 and 5, while other murine Pten-Rad51 analyses utilized a conditional Pten mouse line with loxP sites flanking only exon 5. Furthermore, McEllin and associates utilized a compound Pten-Ink4a/Arf double knockout line [de novo DNA damage induced in Ink4a/Arf single knock-out mice promotes DNA double-strand break-mediated high-grade gliomas [Pten lines may be required to resolve these issues.Our study reinforces the apparently autonomous nature of RAD51 expression and functional HR activity within PTEN-deficient cells, which clearly diverges with the previous studies that originally suggested a PTEN/RAD51 relationship ,32. One ull MEFs ,41,79. Hout line , which c gliomas , which m-/-PTEN HCT116: MLH1-deficiency) [S383Y) [Tyr-240 phosphorylation and co-recruitment of Ki-67 and RAD51 to chromatin in PTEN-proficient GBM, thus enhancing DSBR activity and radioresistance in GBM patient-derived stem-like cells (GSCs). Although conflicting, their study suggests that contextual modifications of the PTEN protein in tumors may regulate RAD51-mediated HR activity, possibly via FGFR-TACC fusions that accumulate in subsets of PTEN-positive/WT tumors [PTEN-null/mutated primary murine cells (MEFs and astrocytes), human tumor cell lines , and numerous patient-derived glioblastoma tumor specimens and BTICs.The conflicting PTEN-deficient tumor cell data in these studies may be due to the inherently complex genomic and biochemical landscape of tumors, which can accumulate multiple genetic lesions (in addition to PTEN loss), and heterogeneity, but also multiple genetic vulnerabilities, pleotropic roles, and microenvironmental changes throughout tumorigenesis, anti-tumor therapy, and recurrence ,82. The iciency) and Shen [S383Y) have sin [S383Y) ,85. Furt [S383Y) . However [S383Y) . FurtherT tumors . Our stuRB loss, TMPRSS2-ERG rearrangement, and TP53 alterations were also noted in this patient\u2019s tumor. A more detailed delineation of additional genetic vulnerabilities amongst individual PARPi-responsive PTEN-deficient tumors may reveal additional mechanistic interactions with the PARP-mediated DNA repair [PTEN status. In the context of our findings and the apparent lack of clinical evidence, future clinical use of PARPi in the treatment of tumors should be discontinued absent a clear indication of HRD independently of PTEN mutational status.Our findings are further reinforced by the striking lack of clinical evidence supporting therapeutic use of PARPis in the treatment of PTEN-deficient tumors despite the wide breadth of clinical trials ,28. In aA repair , thus inA repair . In factA repair , while cA repair , furtherPTEN loss does not synergize with PARPi. Our findings underscore the functional independence of the PTEN mutational status in RAD51-mediated HR and advocate discontinuance of PARPi treatments in PTEN-deficient tumors that do not feature HRD.The identification of HRD mutations in tumors has heralded widespread use of PARPis in a targeted therapeutic approach. Recent findings indicating that PTEN-deficient tumors suppress RAD51 expression and HR activity have been met with controversy, as recent studies have failed to recapitulate these results. Resolving this conflict is paramount due to the ensuing impacts to clinical care and patient trials. Our study demonstrates that RAD51 expression, HR DNA repair activity, and RAD51 foci formation remain intact and unaffected in PTEN-deficient normal and tumorigenic cells. We further show that"} +{"text": "The life expectancy of extensive-stage small cell lung (ES-SCLC) cancer patients has not improved in the last 2\u20133 decades until two recent trials (CASPIAN and IMpower133) showing the addition of anti-programmed death ligand (PD-L1) therapy to chemotherapy has survival benefit over chemotherapy alone. However, such benefit is relatively small and was not even observed in some other trials using immunotherapy, raising the question of optimal chemoimmunotherapy combination in the 1st-line setting for ES-SCLC. Here, we discussed several thought-provoking questions with the focus on IMpower133 and CASPIAN trials. The life expectancy of extensive-stage small cell lung cancer (ES-SCLC) patients has not improved in the last 2\u20133 decades until two recent trials benefit. In both studies, the median OS (mOS) was significantly longer in the immunotherapy plus platinum-etoposide group compared to the platinum-etoposide alone group (Table cis (same cell) to CD80 [ClinicalTrials.gov Identifier: NCT04256421), especially considering its ligand CD155 (or poliovirus receptor (PVR)) is broadly expressed in both the SCLC cell lines and patient tumor tissue [Despite the survival benefit observed in both studies, the absolute improvement in OS remains quite small, and not even statistically significant in the recent KEYNOTE-604 using anti-PD-1 agent pembrolizuamb in combination with platinum-etoposide (not yet published, from Merck\u2019s press release , suggest to CD80 , which i to CD80 . In cons to CD80 . Furtherr tissue , and co-r tissue . Finallyr tissue , and CTRr tissue . It is hr tissue , therefoIn summary, these two studies provided strong evidence to support the use of immune checkpoint blockade in ES-SCLC. However, questions remain regarding whether anti-PD-1/L1 in combination with other immune checkpoint inhibitors could further enhance the overall survival, and whether radiotherapy should be combined with chemoimmunotherapy in ES-SCLC."} +{"text": "Although 68Ga-PSMA-11 PET is widely used in research and clinical practice, full kinetic modeling has not yet been reported nor have simplified methods for quantification been validated. The aims of our study were to quantify 68Ga-PSMA-11 uptake in primary prostate cancer patients using compartmental modeling with arterial blood sampling and to validate the use of standardized uptake values (SUV) and image-derived blood for quantification.The positron emission tomography (PET) ligand 68Ga-PSMA-11 PET scan of the pelvis with axial T1 Dixon, T2, and diffusion-weighted magnetic resonance (MR) images acquired simultaneously. Time-activity curves were derived from volumes of interest in lesions, normal prostate, and muscle, and mean SUV calculated. In total, 18 positive lesions were identified on both PET and MR. Arterial blood activity was measured by automatic arterial blood sampling and manual blood samples were collected for plasma-to-blood ratio correction and for metabolite analysis. The analysis showed that 68Ga-PSMA-11 was stable in vivo. Based on the Akaike information criterion, 68Ga-PSMA-11 kinetics were best described by an irreversible two-tissue compartment model. The rate constants K1 and k3 and the net influx rate constants Ki were all significantly higher in lesions compared to normal tissue (p < 0.05). Ki derived using image-derived blood from an MR-guided method showed excellent agreement with Ki derived using arterial blood sampling (intraclass correlation coefficient = 0.99). SUV correlated significantly with Ki with the strongest correlation of scan time-window 30\u201345 min . Both Ki and SUV correlated significantly with serum prostate specific antigen (PSA) level and PSA density.Fifteen patients with histologically proven primary prostate cancer underwent a 60-min dynamic 68Ga-PSMA-11 kinetics can be described by an irreversible two-tissue compartment model. An MR-guided method for image-derived blood provides a non-invasive alternative to blood sampling for kinetic modeling studies. SUV showed strong correlation with Ki and can be used in routine clinical settings to quantify 68Ga-PSMA-11 uptake. PSMA-11 targets the prostate-specific membrane antigen (PSMA), which is overexpressed in most prostate cancer cells fluorocholine concluded that SUV could not be used to quantify [18F]fluorocholine uptake due to poor correlation between SUV and Ki [The strong correlation between V and Ki , demonstKi and SUV was not seen for the latest time-window, but rather the 30\u201345 min time-window. This result contrasts from current guidelines and publications, where a standard uptake time of around 60 min, with an acceptable range of 50 and 100 min, is recommended [Interestingly, a comparison of the SUV between time-windows 15\u201330 min, 30\u201345 min, and 45\u201360 min showed that the strongest correlation between ommended , 25\u201327. 68Ga-PSMA-11 are best described by an irreversible two-tissue compartment model. 68Ga-PSMA-11 is stable in vivo, which excludes the need for metabolite correction. The net influx rate Ki, estimated using an MR-guided image-derived input function showed excellent agreement with the arterial input function Ki. Image-derived blood can therefore be used as an alternative to arterial blood sampling in kinetic modeling studies. SUV correlated significantly with Ki and can be used in routine clinical settings to quantify 68Ga-PSMA-11 uptake.The kinetics of"} +{"text": "C. elegans neuroscience since the early days of its research (Ward 1973). Lewis and Hodgkin (1977) systematically isolated more than ten abnormal chemotaxis (che) mutants that showed defective chemotaxis to sodium (Na+) and chloride (Cl\u2013) ions (Lewis and Hodgkin 1977), whose responsible genes have already been molecularly characterized except for che-5(e1073). We here show that che-5(e1073) is a missense allele of gcy-22, which encodes a receptor guanylyl cyclase (rGC) specifically expressed in the ASE-right (ASER) gustatory neuron and is essential for chemosensation through the neuron.Mechanisms of chemotactic behaviors have been of great interest in C. elegans is attracted to the NaCl concentration at which it has experienced with food, while avoid the concentration at which it has experienced starvation. ASER plays a major role in food-associated salt concentration chemotaxis; input of salt information into ASER is required and sufficient for chemotaxis to the salt concentration associated with food . ASE neurons, consisting of bilaterally symmetrical ASE-left (ASEL) and ASER, are the major sensory neuron for water-soluble attractants (Bargmann and Horvitz 1991). They respectively sense different sets of ions such as Na+ and Cl\u2013 . A cyclic GMP (cGMP) signaling pathway consisting of rGCs and TAX-4/TAX-2 cyclic nucleotide-gated ion channels mediates sensory transduction in ASE . ASEL and ASER express distinct sets of rGCs . Of these, gcy-22 is essential for ASER to respond to multiple ion species; therefore it is proposed as a common component of chemoreceptor complexes . To further elucidate the mechanisms of chemosensation through ASER, we characterized as yet uncloned che-5. We focused on che-5 because CB1073 che-5(e1073) mutant, the unique strain/allele of the gene, showed chemotaxis defects characteristic of ASER-specific malfunction; a severe defect in attraction to Cl\u2013, whereas relatively moderate defect in Na+ chemotaxis (Lewis and Hodgkin 1977).che-5(e1073) responsible for salt chemotaxis defect between genetic positions 24.52 (single nucleotide polymorphism (SNP): WBVar00240760) and 25.54 (SNP: WBVar00053592) on chromosome V by using SNPs between N2 and CB4856 . The mapped region contained an ASER-specific chemotaxis gene, gcy-22 (genetic position: 25.28). This result was unexpected from the initial report that mapped e1073 on chromosome IV (Lewis and Hodgkin1977), but consistent with a recent observation in which whole-genome sequencing failed to identify a mutation corresponding to che-5(e1073) on chromosome IV .We mapped the mutation of gcy-22(tm2364) harbors a deletion in the middle of gcy-22 coding region that results in a frame shift and therefore is a putative null allele . Salt concentration chemotaxis of the animals heterozygous for e1073 and tm2364 showed that the two alleles failed to complement each other, indicating that these alleles affect the same locus . Nucleotide sequencing of the gcy-22 locus revealed that e1073 carried ACG GCA CCA AAG CAC ATT AGA in which the adenine residue in bold letter was guanine in wild type . This transition results in a missense change E697K in GCY-22 isoform a. The glutamate residue is located in the kinase-like domain and well conserved in rGC proteins. In addition, CB1073 carried another nucleotide substitution within the first intron of gcy-22, TAAACTATGTAAGAAATTTCC, in which the adenine residue in bold letter was guanine in wild type . Furthermore, expression of a cDNA for gcy-22a in CB1073 in ASER-specific manner completely rescued the salt chemotaxis defect of the mutant . These results strongly indicate that che-5 is allelic to gcy-22 and the chemotaxis defect of e1073 is due to the mutations of gcy-22 locus.et al. 2013). A chemotaxis index was calculated to quantify the behavior as follows. Chemotaxis index = {(N at high NaCl-region) \u2013 (N at low NaCl-region)} / { \u2013 (N that did not move from the origin)}, in which N represents number of animals. For complementation tests, males of PD4792 (mIs11 with gcy-22(+) background) or JN2608 (mIs11 with gcy-22(tm2364) background) were mated with CB1073 hermaphrodites. F1 progenies were tested for salt concentration chemotaxis, and crossed progeny hermaphrodites that carried mIs11 were separately counted from self-progeny hermaphrodites that did not carry the marker. For rescue experiments, 5 ng/microL gcy-5p::gcy-22a construct was introduced into CB1073 with 15 ng/microL myo-3p::venus as a transformation marker.Salt concentration chemotaxis was evaluated as described (Kunitomo Strains. The JN strains are available upon request. Others are available at Caenorhabditis Genetic Center (CGC).Bristol N2: wild typeCB4856: wild typeche-5(e1073) V.CB1073: gcy-22(tm2364) V.JN967: che-5(e1073) V; peEx2606[myo-3p::venus].JN2606: che-5(e1073) V; peEx2607[gcy-5p::gcy-22a myo-3p::venus].JN2607: mIs11[myo-2p::GFP pes-10p::GFP gut-promoter::GFP] IV; gcy-22(tm2364) V.JN2608: mIs11[myo-2p::GFP pes-10p::GFP gut-promoter::GFP] IV.PD4792:"} +{"text": "Scientific Reports 10.1038/s41598-020-63234-x, published online 07 April 2020Correction to: The Acknowledgements section in this Article was omitted. The Acknowledgments section should read:\u201cThis work has been supported by the project financed by the MIUR Progetti di Ricerca di Rilevante Interesse Nazionale (PRIN) Bando 2017 - grant 2017MHJJ55.\u201d"} +{"text": "Tuberculosis (TB) is the leading cause of adrenal insufficiency in resource-limited settings. The adrenal gland is the most commonly affected endocrine organ in TB infection. We assessed factors associated with functional adrenal insufficiency (FAI) among TB-HIV patients with and without drug-resistance in Uganda. Patients with drug-sensitive and drug-resistant TB were enrolled and examined for clinical signs and symptoms of FAI with an early morning serum cortisol level obtained. FAI was defined as early morning serum cortisol\u2009<\u2009414 nmol//L. Associations with FAI were modeled using multivariable logistic regression.). In multivariable analyses, drug-resistant TB , treatment duration\u2009>\u20091 month and abdominal pain were significantly associated with FAI. Early morning serum cortisol levels should be quantified in TB-HIV co-infected patients with drug-resistant TB.We screened 311 TB patients and enrolled 272. Of these, 117 (43%) had drug-resistant TB. Median age was 32 years (IQR 18\u201366) and 66% were men. The proportion with FAI was 59.8%. Mean cortisol levels were lower in participants with drug-resistant than susceptible TB (317.4 versus 488.5 nmol/L; p\u2009<\u20090.001 Mycobacterium tuberculosis and cytomegalovirus (CMV) infection 18\u201366) and 180 (66%) were men. Baseline characteristics were comparable between DR-TB and DS-TB participants (Table Of the 311 participants (213 in-patients and 98 out-patients) screened, 272 were enrolled. Of these, 155 (57%) had drug-susceptible TB (DS-TB) and 117 had DR-TB . DR-TB participants were more likely to have low basal morning [AM] serum cortisol (\u2264\u2009414 nmol/L) levels compared to DS-TB . Similarly, mean cortisol levels were significantly lower in DR-TB participants than DS-TB . Cortisol levels remained lower among DR-TB participants irrespective of treatment duration , but lower odds of skin hyperpigmentation . Being male was not associated with FAI . Subgroup analyses revealed that DS-TB patients with FAI had higher odds of longer treatment duration and 70% lower odds of skin hyperpigmentation [Additional file In this cross-sectional study of 272 HIV-TB co-infected adults in Uganda, approximately two-thirds had FAI. Mean serum cortisol levels were significantly lower among DR-TB participants, who had five times higher odds of FAI compared with DS-TB patients. The odds of FAI were higher with DR-TB co-infection, history of abdominal pain and treatment duration >\u20091 month, but lower among men and participants with skin hyperpigmentation.We found that a higher proportion of participants in our study had FAI than previously reported in India , NigeriaDR-TB participants had lower mean cortisol levels and higher odds of FAI than DS-TB. Prevalence of DR-TB is higher in previously treated TB patients , and theInfectious adrenalitis can be difficult to recognize clinically . In low-In agreement with prior work , we founFAI was common among TB-HIV co-infected adults as assessed by early morning cortisol levels. Presence of DR-TB, abdominal pain and longer treatment duration were associated with FAI. These factors may help clinicians in high TB-HIV burden and resource-limited settings to identify FAI and should be confirmed with early morning cortisol to guide clinical management.\u00ae MTB/RIF or DST results or those unable to consent, probably removed sicker participants with FAI. We did not have access to abdominal CT scan to assess adrenal morphology. Finally, the gold-standard ACTH stimulation test was not available, and we may have misclassified FAI status in some participants.A strength of our study is the inclusion of DR-TB and DS-TB participants, and the high prevalence of TB-HIV co-infection (50%). A limitation of this cross-sectional study is the inability to determine temporal relationships of TB and HIV infection in dually infected individuals thus limiting our analysis of clinical correlates of FAI. We were unable to perform sensitivity analyses to evaluate rifampicin-induced adrenal insufficiency due to the small number of TB treatment-na\u00efve participants. Exclusion of patients without XpertAdditional file 1: Figure S1. Study flow diagram. Study diagram describing the number of participants screened and enrolled and the reasons for screen-out. This to be inserted at end of line 127 on page 6.Additional file 2: Table S1. Associations with Functional Adrenal Insufficiency. Factors associated with functional adrenal insufficiency. This is to be inserted under results section at end of line 147 on page 7.Additional file 3: Table S2. Associations with functional adrenal insufficiency among DS-TB patients. Factors associated with FAI among drug-susceptible TB patients. This is to be inserted under results section at end of line 153 on page 7.Additional file 4: Table S3. Associations with functional adrenal insufficiency among DR-TB patients. Factors associated with FAI among drug-resistant TB patients. This to be inserted at end of line 153 on page 7."} +{"text": "Excess lipids trigger CD36 translocation through inhibition of v-ATPase function. Conversely, in yeast, glucose availability is known to enhance v-ATPase function, allowing us to hypothesize that glucose availability, via v-ATPase, may internalize CD36 and restore contractile function in lipid-overloaded cardiomyocytes. Increased glucose availability was achieved through (a) high glucose (25 mM) addition to the culture medium or (b) adenoviral overexpression of protein kinase-D1 (a kinase mediating GLUT4 translocation). In HL-1 cardiomyocytes, adult rat and human cardiomyocytes cultured under high-lipid conditions, each treatment stimulated v-ATPase re-assembly, endosomal acidification, endosomal CD36 retention and prevented myocellular lipid accumulation. Additionally, these treatments preserved insulin-stimulated GLUT4 translocation and glucose uptake as well as contractile force. The present findings reveal v-ATPase functions as a key regulator of cardiomyocyte substrate preference and as a novel potential treatment approach for the diabetic heart.The diabetic heart is characterized by a shift in substrate utilization from glucose to lipids, which may ultimately lead to contractile dysfunction. This substrate shift is facilitated by increased translocation of lipid transporter CD36 (SR-B2) from endosomes to the sarcolemma resulting in increased lipid uptake. We previously showed that endosomal retention of CD36 is dependent on the proper functioning of vacuolar H Pharmacological inhibition of v-ATPase by bafilomycin-A (BafA) caused >80% decrease of v-ATPase function in both aRCMs (To further investigate whether forced glucose influx (via high glucose or AdPKD overexpression) can restore proper endosomal acidification, we measured deoxyglucose) was used to investigate whether both treatments could preserve insulin-stimulated glucose uptake in lipid-overloaded cardiomyocytes. PKD overexpression caused an increase in glucose uptake (1.8 fold in aRCMs and 1.4-fold in HL-1 cells), which is due to increased insulin-independent GLUT4 translocation Deoxyglucose uptake into suspensions of cardiomyocytes was measured as previously described [[escribed . Uptake escribed .Contractile properties of aRCMs were assessed at 1 Hz field stimulation using a video-based cell geometry system to measure sarcomere dynamics . From the digitized recordings acquired with IonWizard acquisition software, the following parameters were calculated: Sarcomere shortening, time to peak, and decay time. This was conducted as previously described .P-values of less than 0.05 are considered statistically significant.All data are presented as means \u00b1 SEM. Statistical analysis was performed by using a two-sided Student\u2019s t-test with GraphPad Prism 5 software . In summary, the overall findings provide solid evidence that v-ATPase is both a lipid sensor and a glucose sensor in the heart, making v-ATPase a key regulator of cardiac substrate preference. The present findings as observed in rodents also extend to the human setting, given that the enforcement of glucose uptake also preserves v-ATPase activity and insulin-stimulated glucose uptake treatment in hiPSC-CMs upon over-exposure of these cells to lipids. Hence, the regulation of v-ATPase assembly/disassembly may offer a suitable target to combat cardiac dysfunction elicited by lipotoxic conditions."} +{"text": "An 82-year-old man suffering from prostate cancer that was scheduled for a radioreceptor-ligand therapy (RLT) presented with jaundice to our service. An abdominal ultrasound (US) revealed obstructive extrahepatic cholestasis due to a solid lesion located in the uncinate process of the pancreas. The Prostate Specific Membrane Antigen (PSMA) PET/CT prior to RLT showed multilocular PSMA positive tumor lesions in the lymph nodes, the lung and the pancreas. On request of the cancer board, an Endoscopic Ultrasound (EUS)-guided Fine-Needle Aspiration (FNA) of the pancreatic mass was performed revealing invasive pancreatic ductal adenocarcinoma incompatible with a prostate cancer metastasis leading to the diagnosis of a PSMA positive pancreatic ductal adenocarcinoma. An 82-year-old man suffering from prostate cancer was planned for Prostate Specific Membrane Antigen (PMSA) radioligand therapy. The 18F-PSMA PET/CT scan revNumerous solid tumor entities including renal cell carcinoma, thyroid cancer, or Hepatocellular Carcinoma (HCC) showed an increased uptake on 18F-PSMA PET/CT ,4,5. The"} +{"text": "Major Histocompatibility Complex (MHC) and Human Leukocyte Antigen (HLA) loci have strong genetic linkage with type 1 diabetes (T1D) in mice and humans, respectively. The diabetes-prone non-obese diabetic (NOD) strain of mice have a unique MHC-II locus with a distinct MHC-II A molecule (Ag7) and lack expression of the MHC-II E molecule. Expression of this MHC-II A molecule is associated with development of T1D, whereas transgenic restoration of the MHC-II E molecule dominantly protects against T1D. The Silverman laboratory recently demonstrated that MHC-II E molecule expression selects for a diabetes-protective intestinal microbiota in early life though a key knowledge gap remains: how do these two factors \u2014 MHC-II molecules and commensal microbiota \u2014 work together during a critical early-life period of microbiome and immune system ontogeny to prevent T1D? To address this question, the Silverman laboratory developed a gnotobiotic mouse model by designing a microbial community consisting of 9 intestinal microbes cultured from these diabetes-protected (Ea16/NOD) mice \u2013 called \u201cPedsCom\u201d. This gnotobiotic model allows for mechanistic, well-controlled studies of interactions between commensal microbes and the developing immune system.We used flow cytometry to sort commensal bacteria from PedsCom-colonized NOD and Ea16/NOD mice into IgA-coated and IgA-uncoated populations. We employed species-specific multiplex qPCR to quantify relative abundance of each PedsCom microbe in these sorted populations.K. cowanii and L. murinus, were preferentially IgA bound in both NOD and Ea16/NOD mice. L. johnsonii, A. caccae, and S. xylosus were preferentially IgA bound only in the presence of MHC-II E molecule expression. Many of the highly IgA coated microbes translocate to the mesenteric lymph nodes.Two microbes, Conclusion: We propose that MHC-II expression facilitates specific mucosal IgA responses, that MHC-II E expression allows for additional epitope recognition amongst PedsCom members which may potentiate this effect, and that preferential IgA coating may promote contact with mucosa-associated lymphoid tissues as early steps in tolerogenic immune system ontogeny that protects against development of T1D.All Authors: No reported disclosures"} +{"text": "Gallbladder stones after bariatric surgery are not uncommon. Roux-en-Y gastric bypass (RYGB) renders standard endoscopic retrograde cholangiopancreatography (ERCP) unfeasible. Endoscopic ultrasound (EUS)-directed transgastric ERCP (EDGE) using a lumen-apposing metal stent (LAMS) is gaining ground in this settingA 79-year-old woman presented with severe acute biliary cholangitis suspected to be due to ampulloma with biliary stones. She had a history of sleeve gastrectomy, which was converted to RYGB with further surgical gastrectomy of the excluded stomach. EUS was first used to attempt a one-shot EDGE procedure; however, it was difficult to locate the excluded duodenum and the one-shot procedure through a jejuno-duodenal anastomosis was considered risky. Thus, percutaneous transhepatic biliary drainage (PTBD) was planned.At 48 hours, EUS-guided jejuno-duodenostomy was performed, assisted by PTBD connected to a water pump . A freeVideo\u20061\u2002Endoscopic ultrasound-guided jejuno-duodenal anastomosis assisted by percutaneous transhepatic biliary drainage connected to a water-pump and further transjejunal endoscopic retrograde cholangiopancreatography in a patient with an atypical gastric bypass presenting with acute cholangitis.Endoscopy_UCTN_Code_TTT_1AS_2AD"} +{"text": "The Serious Communicable Diseases Unit (SCDU) at Emory University Hospital (EUH) is a special pathogen treatment biocontainment unit. Medical care is led by Infectious Disease (ID) specialists with an interdisciplinary group of nurses, infection preventionists, laboratorians, and consultants. In the 2014 Ebola Virus Disease (EVD) outbreak, the SCDU treated four EVD patients, one critically-ill requiring mechanical ventilation and dialysis. Critical care (CC) services were provided through consultation by CC staff, requiring just-in-time (JIT) training in high-level personal protective equipment (PPE) and real-time adaptation of standard operating procedures (SOPs) to meet biocontainment needs. The care provided was successful: all patients survived and no serious PPE breaches or provider exposures occurred. However, these events revealed the need for a comprehensive plan for SCDU CC services.SCDU-CC physician liaison and lead advance practice provider (APP) positions were created to implement SCDU CC in conjunction with the Emory Critical Care Center (ECCC). Previous CC services were reviewed and revised.An integrated staffing model of ID-CC co-management was proposed. CC provider roles were codified: participate in regular high-level PPE training/verification and procedural simulation; attend quarterly interdisciplinary trainings and monthly provider meetings; maintain high quality patient care per ECCC standards. Following unit activation, the SCDU-CC liaisons and lead APP construct a JIT CC schedule. APPs provide 24hour in-house coverage in 12hour day/night shifts. Depending on patient number/acuity, physician coverage consists of in-house day coverage with night home call or 12hour day/night shifts. CC co-manages with the ID physician.CC delivery in the SCDU has been improved after 2014. CC services were previously deployed ad hoc, with simultaneous need for JIT high-level PPE training of new personnel, urgent patient assessment, and modification of SOPs for biocontainment. We developed an integrated staffing model to provide ID-CC co-management within the SCDU.All Authors: No reported disclosures"} +{"text": "TMPRSS2-ERG fusion) have been identified as oncogenic drivers with the potential for patient stratification and as targets for effective prevention/intervention strategies in drug efficacy trials. In the present study, employing relevant TMPRSS2-ERG (fusion)-driven and non-TMPRSS2-ERG-driven mouse models of PCa, we report the potential usefulness of the non-steroidal anti-inflammatory drugs (NSAIDs) aspirin and naproxen specifically against TMPSS2-ERG fusion-driven prostate tumorigenesis. These findings are consistent with the clinical observations and warrant further investigation of the molecular mechanisms and utility of NSAID interventions for precision cancer prevention.Disparity in clinical outcome data due to the biological heterogeneity of prostate cancer (PCa) has drawn attention to approaches that stratify homogeneous subsets of patients. In recent years, PCa fusion genes -positive prostate cancer (PCa) compared to fusion-negative PCa in population-based case\u2013control studies; however, no extensive preclinical studies have been conducted to investigate and confirm these protective benefits. Thus, the focus of this study was to determine the potential usefulness of aspirin and another NSAID, naproxen, in PCa prevention, employing preclinical models of both TMPRSS2-ERG (fusion)-driven and non-TMPRSS2-ERG-driven (Hi-Myc+/\u2212 mice) PCa. Male mice (n = 25 mice/group) were fed aspirin- (700 and 1400 ppm) and naproxen- (200 and 400 ppm) supplemented diets from (a) 6 weeks until 32 weeks of Hi-Myc+/\u2212 mice age; and (b) 1 week until 20 weeks post-Cre induction in the fusion model. In all NSAID-fed groups, compared to no-drug controls, there was a significant decrease in higher-grade adenocarcinoma incidence in the TMPRSS2-ERG (fusion)-driven PCa model. Notably, there were no moderately differentiated (MD) adenocarcinomas in the dorsolateral prostate of naproxen groups, and its incidence also decreased by ~79\u201391% in the aspirin cohorts. In contrast, NSAIDs showed little protective effect against prostate tumorigenesis in Hi-Myc+/\u2212 mice, suggesting that NSAIDs exert a specific protective effect against TMPRSS2-ERG (fusion)-driven PCa.The consumption of the non-steroidal anti-inflammatory drug (NSAID) aspirin is associated with a significant reduction in the risk of developing TMPRSS2-ERG] fusion-positive PCa were used as a non-TMPRSS2-ERG fusion-driven PCa model. This mouse model is a spontaneous model of PCa, where tumorigenesis is Myc-driven ; this model is extensively used in pre-clinical anti-PCa efficacy studies . For H-score calculations, the % proportion area was given arbitrary scores . Variations among the groups were ascertained through either ANOVA or unpaired +/\u2212 mice]. There was no difference in LUT weights of flox/floxTMPRSS2-ERG Pten (+TAM) mice fed with control diet and NSAID-supplemented diets (+/\u2212 control mice were similar to mice fed with naproxen (200 and 400 ppm) and aspirin 700 ppm diets, a slight increase in the LUT weight of aspirin 1400 ppm-fed mice was observed compared to Hi-Myc+/\u2212 controls (No significant difference in body weight gain (LUT weights subtracted) between the efficacy study mice on control diets and NSAID-supplemented diets was observed. The overall control groups (no-TAM and WT-FVB mice), either fed with control diet or NSAID-supplemented diets, showed significantly lower LUT weights compared to their respective positive controls resulted in significant reduction of c-Myc expression in flox/floxTMPRSS2-ERG. Pten (+TAM) mouse prostate. In Hi-Myc+/\u2212 mice, both doses of naproxen (200 and 400 ppm) substantially reduced (p \u2264 0.01\u2013p \u2264 0.05) the expression of c-Myc in the prostate tissue, whereas aspirin treatments had no effect mice PCa tissues was reduced considerably after treatment with NSAIDs . Treatment with aspirin 1400 ppm dose did not result in any significant decrease in the ERG-positive population (TMPRSS2/ERG fusion and naproxen were associated with the decreased expression of COX-2. IHC analysis of COX-1 expression indicated that NSAIDs (both drugs/doses) significantly decreased the expression (p \u2264 0.01- p \u2264 0.001) only in flox/floxTMPRSS2-ERG. Pten (+TAM) prostate but not in the Hi-Myc+/\u2212 mice (NSAID treatments (both drugs/doses) significantly decreased and Hi-Myc+/\u2212 mouse prostate. Results indicated that the treatment with NSAIDs (both drugs/doses) significantly decreased (p \u2264 0.001) the expression of mesenchymal marker vimentin in flox/floxTMPRSS2-ERG. Pten (+TAM) mouse prostate. Concomitantly, there was an increase in the expression of epithelial marker E-cadherin in flox/floxTMPRSS2-ERG. Pten (+TAM) by aspirin and naproxen . Treatment with aspirin 700 ppm dose also increased the expression of E-cadherin, although it did not reach statistical significance. In the Hi-Myc+/\u2212 mouse prostate, vimentin expression was only decreased by aspirin 1400 ppm dose (p \u2264 0.01), while all NSAIDs (both drugs/doses) significantly increased (p \u2264 0.05\u2013p \u2264 0.001) E-cadherin expression fused with the coding sequence of Ets gene family members as oncogenic drivers ,37,38. TCa cases ,37,38. U factors . Thus, ty trials ,37. Thisy trials ,39.For the past several years, pre-clinical studies have strongly supported the protective benefits of NSAIDs for PCa prevention . HoweverTMPRSS2-ERG-positive PCa, and that the risk reduction was stronger with a longer duration of aspirin use [TMPRSS2-ERG-negative PCa [TMPRSS2-ERG fusion positivity, which was not known earlier and thus was not a selection criterion in patient accrual in the previous trials or case studies looking into NSAID efficacy against PCa. In light of this background, we recently characterized and compared the stage-specific progression of PCa under both TMPRSS2-ERG fusion-driven and non-fusion-driven states [TMPRSS2-ERG fusion-positive tumors, including a stage-specific increase, compared to fusion-negative tumors in preclinical PCa mouse models [TMPRSS2-ERG gene rearrangements in TMPRSS2-ERG fusion-negative PCa cells by stress, such as exposure to inflammatory cytokines, oxidative stress, and ionizing radiation [TMPRSS2-ERG fusion-positive PCa. This hypothesis also strongly supports the previous clinical observations that NSAIDs have protective benefits against PCa associated with prostatitis.Interestingly and highly relevant to the present study are the outcomes from a population-based case\u2013control study which reported that aspirin users showed a ~37% reduction in the risk of developing n states so that n states . Anothere models . This obadiation ,42,43. GTMPRSS2-ERG-driven PCa model , but not in the non-TMPRSS2-ERG-driven (Hi-Myc+/\u2212 mouse) PCa model. The lack of dose-dependent effect indicates that an optimum effect against fusion-driven PCa is achievable with lower doses of both NSAIDs and that increasing the doses may not necessarily increase efficacy. From the molecular analysis of the prostate tissue samples, differential molecular effects of NSAIDs treatment in both mouse models were observed; however, the biological basis of the strong efficacy of both aspirin and naproxen treatments in reducing prostate tumorigenesis in only the TMPSS2-ERG fusion-driven PCa model could not be ascertained. Given that the promoter elements of TMPRSS2 are androgen-sensitive and that as a result of TMPRSS2-ERG fusion, the androgen-bound androgen receptor binds to TMPRSS2 regions, and the downstream cascade of events is initiated for ERG overexpression [TMPRSS2-ERG-fusion state. Still, the main factor defining their anti-PCa effects, relative to TMPRSS2-ERG-fusion positivity, appears to be the ability of the NSAIDs to significantly affect the proliferative growth phase of the fusion-positive tumors. There is also a possibility that the lack of efficacy in the Hi-Myc model may be due to the inability of NSAIDs to interfere with the molecular mechanisms underlying PCa carcinogenesis in that model. However, the possibility that the very strong tumorigenic stimulus of the overexpression of Myc in Hi-Myc+/\u2212 mouse could result in insensitivity to NSAID-inhibition of PCa formation is negated by the fact that there is also increased expression of c-Myc in TMPRSS2-ERG-driven PCa model [TMPRSS2-ERG fusion-positive tumors by the NSAIDs did not involve apoptosis.Notably, in the present study, treatment with both NSAIDs, aspirin and naproxen, showed strong efficacy in reducing prostate tumorigenesis in the pression ; thus, tCa model . ResultsTMPSS2-ERG fusion-driven prostate tumorigenesis. This could be attributed, at least in part, to its more significant inhibitory effect on the inflammatory trigger molecule (COX-2), as COX-2 has been reported to be overexpressed in PCa, and its higher/aberrant expression has been implicated in PCa growth and progression as well as poor prognosis [TMPSS2-ERG-driven PCa model, but not in the non-TMPRSS2-ERG-driven PCa model.Importantly, compared to aspirin, naproxen showed relatively more protective benefits against rognosis ,7. AmongTMPSS2-ERG fusion-driven prostate tumorigenesis, which could help in patient stratification in future PCa clinical trials. Although there were some differential molecular effects of NSAID treatments in both mouse models, these molecular changes could not collectively establish the biological basis of the strong efficacy of both aspirin and naproxen NSAID treatments in reducing PCa tumorigenesis in only the TMPSS2-ERG fusion-driven prostate cancer model and not in the non-fusion-driven PCa model.Overall, the study outcomes from the present study are the first of its kind to indicate the benefits of NSAIDs, specifically against"} +{"text": "Multiple recent studies suggest a possible protective effect of the influenza vaccine against severe acute respiratory coronavirus 2 (SARS-CoV-2). This effect has yet to be evaluated in surgical patients. This study utilizes a continuously updated federated electronic medical record (EMR) network to analyze the influence of the influenza vaccine against post-operative complications in SARS-CoV-2-positive patients.The de-identified records of 73,341,020 patients globally were retrospectively screened. Two balanced cohorts totaling 43,580 surgical patients were assessed from January 2020-January 2021. Cohort One received the influenza vaccine six months-two weeks prior to SARS-CoV-2-positive diagnosis, while Cohort Two did not. Post-operative complications within 30, 60, 90, and 120 days of undergoing surgery were analyzed using common procedural terminology(CPT) codes. Outcomes were propensity score matched for characteristics including age, race, gender, diabetes, obesity, and smoking.SARS-CoV-2-positive patients receiving the influenza vaccine experienced significantly decreased risks of sepsis, deep vein thrombosis, dehiscence, acute myocardial infarction, surgical site infections, and death across multiple time points. Number needed to vaccinate (NNV) was calculated for all significant and nominally significant findings.Our analysis examines the potential protective effect of influenza vaccination in SARS-CoV-2-positive surgical patients. Limitations include this study\u2019s retrospective nature and reliance on accuracy of medical coding. Future prospective studies are warranted to confirm our findings. With over 279 million cases and 5.3 million deaths globally, the current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic continues to alter daily life . Given tThe COVID-Surg Collaborative has published an international cohort study of 1128 SARS-CoV-2 positive patients who underwent surgery, of whom 82% of deaths were attributed to post-operative pulmonary complications; significantly higher than surgical patients negative for SARS-CoV-2 , significantly decreased risk of acute MI within 60\u2013120 days post-operatively with nominal significance within 30 days [ NNV: 250\u2013715], significantly decreased risk of dehiscence within 90\u2013120 days post-operatively with nominal significance within 30 and 60 days [ NNV: 715\u20131000], and nominally significant decreased risk of DVT within 30\u2013120 days post-operatively [ NNV: 476\u20131000], and NNV: 256\u2013682] and had significantly fewer SSIs within 120 days post-operatively with nominal significance within 60 and 90 days [ NNV: 500\u2013833] receptors . DownregTwo final postulates are notable in the context of the present study. Firstly, influenza vaccination stimulates an immunologic cascade leading to plaque stabilization, thus, decreasing risk of cardiovascular events. Vaccine-induced antibodies interact with bradykinin-2 receptors leading to increased nitric oxide production and an anti-inflammatory effect . Lastly,Regardless of mechanism, the strong association between influenza vaccination and decreased risk of adverse post-operative outcomes in SARS-CoV-2-positive patients observed by this study merits further investigation. The hypothesis that individuals current on their influenza immunization have less baseline medical co-morbidities and risk factors for poor post-operative outcomes is supported by the \u201cBefore Matching\u201d column in By contextualizing study findings on a global scale, NNV has been an extremely valuable tool for effect size analysis throughout the SARS-CoV-2 pandemic to measure the protective effects of both influenza and coronavirus vaccines , 44. SpeEven with the unprecedented fast-tracking of multiple vaccines, the fact remains that a majority of the world is not fully vaccinated against SARS-CoV-2 . FurtherThis study is limited by its retrospective nature and reliance on the accuracy of medical coding. This study is also limited by its time window of January 2020-January 2021, prior to the widespread availability of SARS-CoV-2 vaccination, thereby prohibiting analysis of any potential synergistic or interactive effects between the influenza and COVID-19 vaccines in this patient population. Federated EMR networks lend themselves to measures of association, but not causation, thus future prospective studies are warranted to validate this study\u2019s finding that an emphasis on influenza vaccination will improve post-operative outcomes in COVID-positive surgery patients.Using a federated EMR network of over 73 million patients globally, this analysis examines the potential protective effect of influenza vaccination against adverse post-operative outcomes within 30, 60, 90, and 120 days of SARS-CoV-2-positive surgery patients. Significant findings in favor of the influenza vaccine in mitigating the risks of sepsis, acute MI, and dehiscence across all multiple time points while decreasing the risk of SSI and death by 120 days suggest a potential protective effect that merits further investigation and validation with prospective studies, such as randomized control trials."} +{"text": "To the editor,Aging is related to cancer, and an increasing incidence of malignant tumors among the older population is observed . UrinaryThe standard of care for the treatment of metastatic UC for more than 20 years has been cisplatin-based combinations . HoweverIn terms of presented novel agents and trials for UC in this congress, PD-1/PD-L1-based ICIs , platinum-based chemotherapy (PCT), antibody-drug conjugates (ADCs), (Enfortumab vedotin (EV) and Sacituzumab govitecan (SG)), and fibroblast growth factor receptor kinase inhibitors (Erdafitinib and Derazantinib) were most presented . UC patiEV-103 Cohort K (NCT03288545) evaluated first-line EV and Pembrolizumab or EV alone in patients with la/mUC who were cisplatin-ineligible. EV and Pembrolizumab led to EV dose reduction and showed a clinically meaningful objective response rate with a manageable safety profile compared to EV alone . Moreover, EV and Pembrolizumab contributed to clinically meaningful reductions in quality of life (QoL). Based on these results, three Phase 3 trials are being conducted for EV and Pembrolizumab in the first-line la/mUC and MIBC patients.IMVIGOR 130 phase III trial (NCT02807636) reported final overall survival (OS) data, showing that improved OS with Atezolizumab plus platinum/ gemcitabine vs placebo plus platinum/gemcitabine did not reach statistical significance in patients with la-/mUC. Two large Cohort trials (Javelin Bladder100 (NCT02603432) and NCT04822350) supported the usage of Avelumab as maintenance therapy in la/mUC patients without progression after PCT due to benefits of OS. In this meeting, CheckMate 274 trial (NCT02632409) reported extended follow-up data, indicating that Nivolumab continued to show disease-free survival (DFS), non-urothelial tract recurrence-free survival, and distant metastasis-free survival benefits versus placebo.Cohort B of KEYNOTE-057 (NCT02625961) reported that Pembrolizumab monotherapy showed notable antitumor activity in patients with BCG-unresponsive non-CIS papillary high-risk (HR) NMIBC after ~45 months of follow-up (12-month DFS rate: 43.5 (34.9-51.9); 12-month OS rate: 96.2 (91.1-98.4)). FIDES-02 study (NCT04045613) explored the activity of Derazantinib in patients with mUC and FGFR1-3 genetic aberrations. Confirmed ORR was 8.2% and the disease control rate was 28.6% . Other emerging agents showed various promising results and deserve expectation in the future. The drugs and trials were presented in For trials in progress, researchers still mainly concentrate on patients with mUC, HR BCG-unresponsive NMIBC or MIBC. What's different from the published trials is that researchers are commencing on investigating the effect of ADCs with or without immunotherapy on such patients, suggesting that ADCs are receiving more attention. The ongoing trials are summarized in Median OS for advanced UC patients undergoing PCT followed by immunotherapy is still smaller than 1 year and there is an urgent need for alternative therapies. With thBased on the current evidence and the advancements in this meeting, we proposed the following management strategy of UC. PCT is the first-line standard therapy for all patients who are candidates for either cisplatin or carboplatin. Patients positive for PD-L1 and ineligible for cisplatin may receive immunotherapy (Atezolizumab or Pembrolizumab) or EV and Pembrolizumab, among which Erdafitinib could be considered in la/mUC patients with FGFR alterations. If the disease does not progress after PCT, maintenance immunotherapy (Avelumab) is suggested. Immuno-therapy (Pembrolizumab) is the recommended second-line therapy for patients who do not have maintenance therapy. Later-line treatments like Derazantinib, EZH2 inhibitor (Tazemetostat), T-cell therapy (ADP-A2M 4CD8) and non-viral gene therapy (EG-70) could be tried in specific cases with the patient\u2019s consent. Pembrolizumab monotherapy might be recommended in patients with HR NMIBC unresponsive to BCG who declined or were ineligible to undergo radical resection. In future trials, oncogenic alterations can be considered in trial designs and PCT-based target therapy or ADCs with immunotherapy should be conducted in more mUC patients or MIBC candidates for RC.www.aginganddisease.org/EN/10.14336/AD.2023.0502.The Supplementary data can be found online at:"} +{"text": "A 69-year-old male patient with Machado-Joseph disease (MJD) presented with a mild cerebellar ataxia, global areflexia, and nystagmus. Magnetic resonance imaging showed cerebellar atrophy; brainstem atrophy, mainly pontine, and a linear abnormal bilateral hyperintense along the medial aspect of the globus pallidus internus on T2-weighted sequence and fluid-attenuated inversion recovery (FLAIR) . This r"} +{"text": "Nucleotide-binding and leucine-rich repeat receptors (NLRs) are a diverse family of intracellular immune receptors that play crucial roles in recognizing and responding to pathogen invasion in plants. This review discusses the overall model of NLR activation and provides an in-depth analysis of the different NLR domains, including N-terminal executioner domains, the nucleotide-binding oligomerization domain (NOD) module, and the leucine-rich repeat (LRR) domain. Understanding the structure-function relationship of these domains is essential for developing effective strategies to improve plant disease resistance and agricultural productivity. Plants have an innate immune system that can effectively defend them against pathogens. This system depends on the activation of various immune receptors that recognize pathogen molecules, such as pathogen-secreted proteins called effectors. Effectors manipulate the host by either suppressing its immune responses or by promoting its nutrient supply to increase the pathogen's fitness. Some effectors function in the extracellular apoplastic space, while others are secreted within the host nucleocytoplasm .To combat pathogens and their diverse effector repertoire, plants have evolved a large array of intracellular immune receptors, which primarily belong to the nucleotide-binding and leucine-rich repeat receptor (NLR) family . Upon paR gene products, and CED-4) involved in intramolecular regulation of the NLR, a C-terminal leucine-rich repeat (LRR) domain involved in ligand binding and intramolecular regulation, and an N-terminal domain responsible for initiating downstream signalling. The most common N-terminal domains are either Toll/Interleukin-1 Receptor (TIR) domains in TIR-NLRs or one of three sequence-unrelated but structurally and functionally conserved coiled-coil domains: the Rx-type coiled-coil (CC)-type NLRs (CC-NLRs), RESISTANCE TO POWDERY MILDEW 8 (RPW8)-type CC-NLRs (CCR-NLRs), and the G10-type CC-NLRs (CCG10-NLRs) .,12.+ cleomodimer 15,16].,16.+ cles active . Insteads active . These ns active . These Ts active ,22. The echanism . The exaechanism . Regardl 3\u2032cADPR , while H 3\u2032cADPR . Both ef 3\u2032cADPR ,24. This 3\u2032cADPR domain, resembling domains found in the N-terminus of mixed lineage kinase domain-like (MLKL) proteins, Casitas B-lineage lymphoma (Cbl) proteins, heterokaryon incompatibility (HET) proteins, and the HeLo domain (for HET-s/loss-of-pathogenicity (LOP-B) protein) found in fungi . Be. Be28]. 2+) permeable cation channels [2+ channel activity of certain, but not all, CC-NLRs [Both the ZAR1 resistosome and the Sr35 resistosome function as non-selective calcium of CC-NLRs contains a conserved and acidic EDVID motif, named after the most prominent amino acid residues found at each position in the motif, and which interacts with basic arginine residues in the LRR to stabilize both the active and inactive form of CC-NLRs via several salt bridges . CCR-NLR channel ,37. Simirization ,38.R-NLRs are important for immune signalling, proper localization, and specific phospholipid binding likely determined through their surface exposure in the inactive conformation of the NLR [G10 domain shares a similar four-helix bundle with CC/CCR-NLRs. Some CCG10 domains possess an N-terminal extension, which can be modified by the covalent attachment of fatty acids and which is required for localization to the plasma membrane and immune signalling [G10-NLRs is essential for cell death-inducing activity, and this cell death-inducing activity can be blocked by Ca2+-channel inhibitors, suggesting CCG10-NLRs like CC/CCR-NLRs also act as calcium-permeable channels [The second (\u03b12) and fourth \u03b1-helix (\u03b14) of both CC- and CC the NLR . In the the NLR . However the NLR . Similar the NLR . Structugnalling . As is tchannels ,43.Finally, non-flowering plant NLRs exhibit a greater diversity of N-terminal domains, including protein kinase domains and \u03b1/\u03b2 hydrolase domains ,45. WhetThe NB-ARC domain is a highly conserved feature in plant NLRs, providing a basis for their classification and function . The NB-R149 within the NBD thereby exposing the oligomerization interface. This configuration enables the NBD-HD1 surface of a protomer to interact with the NBD-WHD surface of its neighbour. The major interactions involve an HD1-WHD interface and an NBD-NBD interface. ATP/dATP, which is nested in the P-loop and a pocket in the NBD, stabilizes the active conformation of ZAR1 through direct interactions of the gamma phosphate group of ATP with ZAR1R149 [R149 and the MHD motif for instance, have facilitated mutation-based inactivation and autoactivation of NLRs, respectively \u2013RESISTANT TO P. SYRINGAE 4 (RPS4) TIR-NLR pairs, interact prior to activation . HoweverCED4 NLR , transieThe mechanism of pathogen detection by NLRs can be either direct or indirect. In direct recognition, NLR proteins recognize effectors through direct physical interaction without the need for additional host proteins. Indirect detection follows the guard and decoy models, where NLRs monitor the status of a host component, known as a guardee or decoy .Most plant NLRs possess an LRR domain that has a distinct horseshoe shape, which differs from the elongated and sometimes twisted shape of LRR domains found in many plant cell surface receptors. This distinct architectural difference could be the result of evolutionary constraints arising from the principle of \u2018form follows function\u2019, and these constraints could be a direct consequence of the NLR activation mechanism. Both direct and indirect ligand recognition by plant NLRs appear to employ an allosteric mechanism involving a \u2018steric clash\u2019 with the NBD . In thisThe RPP1 and Roq1 structures revealed a previously unobserved domain directly following the LRR domain termed the C-terminal jelly roll/Ig-like domain (C-JID) ,12. The Finally, plant NLRs can also possess other ligand-binding domains. For instance, the N-terminal late-blight resistance protein R1 domain occurs exclusively with the NB-ARC domain and might be involved in ligand-binding ,58. AcroIn summary, the N-terminal executioner domains, the NOD module responsible for the switch between inactive and active states, and the ligand-binding domain that facilitates NLR activation all contribute to the dynamic and versatile nature of these immune receptors. A deeper understanding of these molecular mechanisms and how they evolved will pave the way for the development of novel strategies to enhance plant resistance against pathogens and improve agricultural productivity.Understanding the structure-function relationship of plant NLRs is essential for devising efficient strategies to enhance plant disease resistance and boost agricultural productivity.R-NLRs to create similar pores at the plasma membrane.Current understanding indicates that ligand-binding by the C-terminal LRR domain of plant NLRs induces structural rearrangements in the nucleotide-binding oligomerization domain (NOD) module, which results in resistosome formation and activation of the N-terminal executioner domains. CC-NLRs create non-selective calcium-permeable pores at the plasma membrane, while TIR-NLRs form enzymatically active resistosome complexes that indirectly activate CCFuture directions in the field will likely focus on increasing our ability to predict the specific contributions of individual amino acids to NLR function, obtaining structural information on resistosome complexes within a membrane context, and investigating the activation mechanisms of paired and networked NLRs."} +{"text": "This article discusses the use of peptide receptor radionuclide therapy (PRRT) as a key treatment method for advanced, unresectable neuroendocrine tumors. It covers the multidisciplinary theranostic approach, treatment effectiveness, patient outcomes, and toxicity of PRRT for neuroendocrine neoplasms. We will also examine important research, and explore new radiopharmaceuticals for the treatment of these patients.Neuroendocrine neoplasms (NENs) are tumors originating from neuroendocrine cells distributed throughout the human body. With an increasing incidence over the past few decades, they represent a highly heterogeneous group of neoplasms, mostly expressing somatostatin receptors (SSTRs) on their cell surface. Peptide receptor radionuclide therapy (PRRT) has emerged as a crucial strategy for treating advanced, unresectable neuroendocrine tumors by administering radiolabeled somatostatin analogs intravenously to target SSTRs. This article will focus on the multidisciplinary theranostic approach, treatment effectiveness (such as response rates and symptom relief), patient outcomes, and toxicity profile of PRRT for NEN patients. We will review the most significant studies, such as the phase III NETTER-1 trial, and discuss promising new radiopharmaceuticals, including alpha-emitting radionuclide-labeled somatostatin analogs and SSTR antagonists. Neuroendocrine neoplasms (NENs) represent a highly heterogeneous group of neoplasms with varying biological behavior. In fact, some cases have a very malignant behavior, whereas in other patients, disease may be stable for a long time even without any treatment. NENs are characterized by a gap between the low incidence and the prevalence, as they are frequently slowly growing, and behave as chronic oncological diseases with a relatively long survival . Several111In]In-DTPA-Octreotide) is limited by a low accuracy in detecting lesions with size < 1 cm and by a difficult semiquantitative analysis. The subsequent development of different radiolabeled DOTA-conjugated peptides for positron emission tomography/computed tomography (PET/CT) has represented a relevant innovation, progressively showing the ability to detect at least 30% more lesions than [111In]In-DTPA-Octreotide and conventional CT Lu-DOTA-TATE can be easily monitored through whole-body scintigraphy performed the day after administration, using the gamma emission of lutetium is mandatory with the aim of obtaining a mapping of all SSTR-positive lesions. Candidates for PRRT should exhibit a strong SSTR expression, while diffuse hepatic and/or bone disease, as well as impaired renal function, may represent a limit to its indication. According to the ENETS Consensus Guidelines, \u201cPRRT is a therapeutic option in progressive SSTR-positive NET with homogenous SSTR expression \u201d Y-DOTA-TOC is currently used for locoregional treatments of liver metastases, due to its higher renal toxicity, or in some clinical trials. [177Lu]Lu-DOTA-TOC and [177Lu]Lu-DOTA-TATE are also used in PRRT, with the latter approved for gastro-entero-pancreatic (GEP-) NETs by the United States Food and Drug Administration (FDA) in 2018. The standard schedule for PRRT comprises four infusions of 7.4 GBq (200 mCi) [177Lu]Lu-DOTA-TATE every eight weeks, which may be extended up to 16 weeks if dose-modifying toxicity occurs [Several radiolabeled DOTA-derivatized are available. [y occurs .177Lu]Lu-DOTA-TATE, liver and kidney function, as well as blood-related measures, should be assessed as signs of toxicity may necessitate a longer treatment interval, reduced dosage, or even permanent cessation of the treatment Lu-DOTA-TATE and best supportive care (including Octreotide 30 mg) outperformed the monthly administration of Octreotide 60 mg alone. The progression-free survival (PFS) rates after 20 months of treatment were 65.2% and 10.8%, respectively. Following the publication of the preliminary results of NETTER-1 in the New England Journal of Medicine, the international scientific community began to recognize the potential of PRRT. Consequently, PRRT with [177Lu]Lu-DOTA-TATE has been approved by both the US FDA and the European Medicines Agency (EMA).PRRT has been studied in numerous retrospective studies and single-arm clinical trials in heterogeneous patient populations that have demonstrated that radiolabeled SSAs deliver targeted radiation with a high therapeutic index to tumors that express SSTRs, thus inhibiting tumor growth in 50\u201370% of GEP-NETs . HoweverThe final overall survival (OS) analysis was carried out five years after the last patient was randomized, with a median follow-up of 76 months for both groups . The PRR177Lu]Lu-DOTA-TATE was well-tolerated, safe, and provided significant quality-of-life benefits compared to high-dose octreotide Lu-DOTA-TATE/DOTATOC in 1758 advanced/inoperable NETs [These successful results were reinforced by a meta-analysis of 22 studies investigating the efficacy of [ble NETs . The poo68Ga]Ga-DOTA-SSA-uptake in all lesions, in NET G1\u2013G2 at disease progression, and in selected cases of NETs G3 with all lesions being positive at [68Ga]Ga-DOTA-PET/CT F-FDG) PET/CT may also aid in selecting patients for PRRT. As it documents the metabolic activity of tumoral lesions, and as many NENs present a low Ki-67, it has been initially reserved only for selected cases, mainly with poorly differentiated diseases. Recently, the International Consensus on the role of theranostics in NENs has expanded its application also to NECs, NETs G3, and even NETs G1\u2013G2, with the goal of identifying the mismatched lesions ([18F]F-FDG-PET/CT-positive/[68Ga]Ga-DOTA-SSA-negative) [18F]F-FDG-PET/CT could differentiate GEP-NETs G1\u2013G2 into low- and high-risk categories for poor response [18F]F-FDG-PET/CT, defined as the \u201cNETPET\u201d score F-FDG-PET/CT, while excluding cases with discordant ([18F]F-FDG-positive/SSTR-negative) lesions Lu-DOTA-TATE, a primary tumor was resectable in 26.3% of cases. The estimated 2-year PFS rate was 90\u201395%, while OS was 92.1%. Better responses were achieved in duodenal NETs, cases without regional lymph node metastases, primary tumor < 5 cm, liver lesions with size \u2264 1.5 cm and \u22643 in number, as well as an [18F]F-FDG uptake with a maximum standard uptake value < 5.Neoadjuvant PRRT for disease downstaging has been explored in a retrospective series, with the largest including 57 GEP-NETs with unresectable primary tumors, with or without liver metastases . After rThe NeoLuPaNET trial (NCT04385992) will assess the role of neoadjuvant PRRT in resectable Pan-NETs at a high risk of disease recurrence. The study endpoints include post-operative mortality rates and objective response rates. The NeoNet Trial (NCT05568017) will explore the role of neoadjuvant PRRT in unresectable or borderline resectable PanNETs G1\u2013G2.The feasibility of re-treatment with PRRT as a salvage therapy is still under investigation. 90Y]Y-PRRT [177Lu]Lu-PRRT in four or five cycles. The DCR was 84.6%, the median PFS was 22 months and the toxicity was mild.Severi et al. described a population of 26 progressive NETs previously treated with [Y]Y-PRRT . All patVan der Zwan et al. reported a larger experience with 181 patients affected by progressive bronchial or GEP-NETs receiving a re-(re)treatment with PRRT . Median A meta-analysis of seven studies and 414 patients affected by advanced NETs showed a median PFS of 12.52 months, and safety similar to the initial PRRT treatment . These e177Lu]Lu-DOTA-TATE is expected to be less effective in large bulky lesions characterized by heterogeneous SSTR distribution, due to lower energy and a shorter particle penetration range. In contrast, yttrium-90 offers potential advantages because of its longer beta particle penetration range Lu-PRRT or for neoadjuvant purposes [90Y]Y-DOTA-TATE imaging demonstrated excellent radiopharmaceutical uptake in nearly all patients. PRRT with Lu-DOTA-TATE was combined with capecitabine and temozolomide (CAPTEM) in advanced low-grade NETs, observing a DCR of 71%, a median PFS of 31 months, while median OS was not reached Lu-DOTA-TATE was combined with metronomic capecitabine as a radiosensitizing agent in advanced, progressive [18F]F-FDG-positive GEP NETs with Ki-67 < 55% Lu-Edotreotide vs. everolimus (NCT03049189).The COMPETE trial is currently recruiting unresectable, progressive GEP-NETs G1\u2013G2 to be treated with PRRT with [177Lu]Lu-DOTA-TATE in conjunction with a mixed amino acid solution, following the standard clinical practice. Treatment cycles were repeated until the absorbed dose to the kidneys reached 23 Gy, or until other reasons necessitated stopping the therapy. In 68.5% of patients, the targeted absorbed dose of 23 Gy to the kidneys was attained after more than four cycles, resulting in significantly longer PFS and OS rates compared to patients who had to discontinue therapy before reaching 23 Gy . No major radiation-induced nephrotoxicity or bone marrow irradiation exceeding the 2 Gy threshold was observed. Given these findings and the observed intra- and interindividual variation in absorbed radiation doses for the same administered activity, we believe that the incorporation of individualized dosimetry into clinical practice may enhance the total administered activity and the number of therapy cycles, thereby significantly improving upon the standard established by the NETTER-1 trial Pb-DOTAM-TATE in treatment-na\u00efve NETs (NCT03466216). Additionally, the outcomes of a first-in-human study on eight patients with progressive NETs resistant to SSAs and tandem therapy with [90Y]Y-/[177Lu]Lu-DOTA-TOC, who were treated with [213Bi]Bi-DOTA-TOC via either intra-arterial administration into the common hepatic artery (n = 7) or systemic administration (n = 1), can be reported. In these patients, 213Bi-DOTA-TOC was able to induce long-term tumor remission (overcoming resistance to beta-PRRT) while keeping nephrotoxicity and acute hematotoxicity within acceptable ranges Lu-DOTA-TOC or [177Lu]Lu-DOTA-TATE would not be feasible in these patients. Nonetheless, theranostic imaging with [68Ga]Ga-NODAGA-LM3 demonstrated tumor uptake greater than normal liver parenchyma in all patients. Despite the low internalization of antagonist-receptor complexes into tumor cells, SSTR antagonists have shown favorable pharmacokinetic characteristics compared to SSTR agonists, particularly higher tumor uptake values (due to the ability to occupy more binding sites with a lower dissociation rate), longer retention times in tumor tissue, and shorter retention times in healthy organs . The moss higher ,50. LastLastly, numerous clinical trials have attempted to merge these two hopeful approaches by utilizing SSTR antagonists marked with alpha-emitting radioisotopes; however, the results are still in the initial stages.It is evident that while the preliminary results of PRRT with alpha-emitting radioisotopes and/or SSTR antagonists are promising, more comprehensive studies involving larger patient cohorts and longer follow-up periods are necessary to validate these findings.In summary, NENs represent a highly heterogeneous disease treated with therapeutic protocols that are not fully standardized. Based on this observation, a multidisciplinary approach is recommended for their management. Based on the available data in the literature, PRRT represents a valid and effective therapeutic option for advanced NETs, especially G1\u2013G2 cases after SSA failure. Significant progress has been made in the past decade regarding treatments. Ongoing trials will help address the unresolved questions concerning PRRT for these patients, presenting new insights in terms of novel radiopeptides, therapy sequence and therapy combination. These findings, in conjunction with molecular profiling and the application of radiomics to understand tumor characteristics and behavior, will play a critical role in advancing precision medicine within this oncological domain."} +{"text": "In this commentary, we explore the pioneering implementation of 3D-printed thin liquid sheet devices for advanced X-ray scattering and spectroscopy experiments at high-repetition rate XFELs. This has enabled the development of novel methods, such as serial femtosecond crystallography (SFX), fluctuation X-ray scattering (FXS), and several X-ray spectroscopies, which have transformed atomic resolution molecular imaging and ultrafast materials characterization is predominantly utilized for liquid sample injection in XFELs X-ray spectroscopies (Ekimova"} +{"text": "HGF/c-MET signaling is a significant driver of glioblastoma (GBM) growth and disease progression. Unfortunately, c-MET targeted therapies have been found to be largely ineffective suggesting additional redundant mechanisms of c-MET activation.circ-HGF (hsa_circ_0080914) was identified as markedly upregulated in primary GBM and found to potentially encode an HGF protein variant (C-HGF) 119 amino acids in length. This candidate HGF variant was characterized and evaluated for its ability to mediate c-MET activation and regulate PDX GBM cell growth, motility and invasive potential in vitro\u00a0and tumor burden in intracranial xenografts in mice.Utilizing RNA-sequencing (RNA-seq) and ribosome profiling analyses of circular RNAs, circ-HGF RNA which mediated translation of the cross-junctional ORF encoding C-HGF and was observed to be highly expressed in GBM relative to normal brain tissue. C-HGF was also found to be secreted from GBM cells and concentrated cell culture supernatants or recombinant C-HGF activated known signaling cascades downstream of c-MET. C-HGF was shown to interact directly with the c-MET receptor resulting in its autophosphorylation and activation in PDX GBM lines. Knockdown of C-HGF resulted in suppression of c-MET signaling and marked inhibition of cell growth, motility and invasiveness, whereas overexpression of C-HGF displayed the opposite effects. Additionally, modulation of C-HGF expression regulated tumor growth in intracranial xenografted PDX GBM models.An internal ribosome entry site (IRES) was identified within the These results reveal an alternative mechanism of c-MET activation via a circular RNA encoded HGF protein variant which is relevant in GBM biology. Targeting C-HGF may offer a promising approach for GBM clinical management.The online version contains supplementary material available at 10.1007/s11060-023-04331-5. Glioblastoma is a highly lethal CNS cancer with a median survival of only 12\u201317\u00a0month , 4. SeveCircular RNAs (circRNAs) are covalently closed RNA transcripts which are generally expressed at reduced levels relative to their linear cognate mRNAs. Several circRNAs have been shown to be translated and the protein products demonstrated to have important roles in cancer . In factcirc-HGF RNA. This protein variant (C-HGF) is secreted from GBM cells and stimulates c-MET activity and its downstream signaling effectors. Modulation of C-HGF expression in PDX cell line models demonstrated regulation of cell growth, motility and invasive characteristics and markedly affected in vitro tumor growth in xenograft experiments.Here we report the discovery of a circRNA-templated HGF protein variant derived from IRES-mediated translation of the Details regarding cell cultures, reagents, in vitro and \u00a0in vivo protocols and data analyses are described in Online Resource 1 Supplemental Materials and Methods.circ-HGF (hsa_circ_0080914) as one of the most differentially upregulated circRNAs which we selected for further characterization as HGF/c-MET activation is a known driver of GBM [circ-HGF is generated by the backsplicing of exons 6\u201311 of HGF located on chromosome 7 in GBM . We identified a potential IRES within the circ-HGF RNA 211 nucleotides upstream of the cross-junction within the RNA circle . This IRES was validated in reporter assays utilizing a dual luciferase-split nanoluciferase construct in which nanoluciferase expression and activity is dependent on an IRES driving nanoluciferase translation [circ-HGF encodes a 119 amino acid protein whose translation is mediated via an IRES and is highly expressed in GBM.les Fig.\u00a0A, top. Wnslation resulted in significant activation of c-MET as monitored by P-Yignaling \u201321. Morecirc-HGF RNA or the C-HGF ORF, these xenografts displayed increased tumor growth and shortened the time of tumor onset relative to controls requirements for IRES-mediated translation of circRNAs have not been explored in depth. The expression of mRNAs bearing IRES elements is controlled by multiple mechanisms and enhanced when canonical cap-dependent initiation is compromised [The IRES-promised . Additiopromised \u201332. Whil6A modification within DRACH motifs [6A-induced ribosome engagement sites (MIRESs) have been reported to function as IRESs to drive circRNA translation [6A modifications can be enriched in circRNAs and a single m6A may be sufficient to initiate translation. Furthermore, circRNA translation has been reported to be stimulated by overexpression of the major methyltransferase complex METTL3/4 and inhibited by the m6A demethylase FTO [6A-dependent circRNA translation initiation [circ-HGF, however we were unable to detect m6A methylation of circ-HGF at these motifs via anti-m6A antibody immunoprecipitation and subsequent qRT-PCR analysis in GBM PDX lines (not shown). This suggests that C-HGFs primary mechanism of translation initiation is IRES-dependent.Translation on circRNAs can also be initiated as a result of mH motifs . These snslation , 35. m6Aitiation . We idencirc-HGF derived protein variant of HGF which is secreted by GBM cells and stimulates c-MET signaling leading to enhanced growth, motility and invasive characteristics. C-HGF was found to be highly expressed in GBM patient samples and promoted PDX cell growth In vitro and in xenografts. C-HGF was found to be translated via an IRES-dependent mechanism and contains 49 unique c-terminal residues which may serve as an effective anticancer target.In conclusion, these studies identified a novel Supplementary file1 (PDF 124 KB)Supplementary file2 (PDF 82 KB)Supplementary file3 (PDF 99 KB)Supplementary file4 (PDF 191 KB)Supplementary file5 (XLSX 82 KB)Supplementary file6 (PDF 101 KB)Supplementary file7 (PDF 268 KB)Below is the link to the electronic supplementary material."} +{"text": "Limited data and resources drove collaboration among healthcare providers treating COVID-19. In July 2021 a multi-disciplinary committee representing all facilities in Veterans Integrated Service Network (VISN) 9 developed COVID-19 guidelines for bacterial co-infection in hospitalized COVID-19 patients. We assessed the impact of these guidelines on appropriate antibiotic utilization (AU) in admissions for COVID-19.AU in COVID-19 admissions to VISN 9 facilities from 08/01/20 to 08/31/21 (pre-guidelines) were reviewed and compared to 11/01/21 to 11/30/22 (post-guidelines). COVID-19 positivity, hospitalization, and AU data were pulled from the Corporate Data Warehouse. COVID-19-specific antibiotic stewardship (AS) interventions and guideline distribution plans were collected for each facility. A weighted random sample of patients from each facility who received antibiotics was chart reviewed for appropriateness of AU. AU for non-respiratory indications were excluded from analysis (Table 1). AU was considered appropriate if at least one guideline criteria was met (Table 2). Antibiotic appropriateness was compared before and after guideline distribution for each site using t-test. The odds of appropriate antibiotic prescribing in the VISN after guidance distribution was determined by logistic regression adjusted for site.Initial rates of antibiotic use varied among facilities both pre and post guideline distribution (Table 1). Facility-specific interventions and guideline distribution also varied (Table 3). There was no significant change in the frequency of appropriateness for any individual site. Similarly, the odds of AU appropriateness for the VISN was not significantly changed in the post-guideline period (p=0.7) (Table 4).AU appropriateness did not change significantly after guidance distribution, likely due to a myriad of reasons. Reprioritization of AS programs\u2019 focus in the post-guidance period may have shifted resources away from initiatives targeting AU in COVID-19 patients. Additionally, guidance was distributed mid-2021 potentially missing the window for largest impact. Collaboration can fill in gaps for many ASPs; but the success of any intervention is multifactorial.Milner Staub, MD, MPH, Gilead: Stocks/Bonds|Johnson & Johnson: Stocks/Bonds"} +{"text": "Correction: Malaria Journal (2023) 22:147 https://doi.org/10.1186/s12936-023-04546-xPlasmodium falciparum strains with deleted Pfhrp2 gene, which is less detectable by many currently available RDTs, would then have an advantage over the P. vivax strains in the face of control strategies based increasingly on RDTs and post-diagnostic treatment only policy\".Following publication of the original article , the autP. falciparum strains\" instead of \"P. vivax strains\".Namely, the sentence previously (incorrectly) referred to \"\u2018wild-type\u2019 The authors thank you for reading this corrections and apologize for any inconvenience caused."} +{"text": "Correction:BMC Musculoskelet Disord24, 608 (2023)10.1186/s12891-023-06743-wFollowing publication of the original article , the autThe correct author names are :Leena RistolainenJyrki KettunenJouni LohikoskiHannu KautiainenMikko ManninenThe author group has been updated above and the original article has been"} +{"text": "Molecular Psychiatry 10.1038/s41380-023-02129-5, published online 26 June 2023Correction to: Data availabilityRNA-Seq results from Adnp-HT mice were deposited in the GEO (Gene expression Omnibus) database at NCBI under the accession number of GSE213354. Total proteomics data were deposited to the ProteomeXchange database under the accession number of PXD036544 and PXD037325 (PTM).The Data availability statement was incorrectly given as \u201cRNA-Seq results from Adnp-HT mice were deposited in the GEO (Gene expression Omnibus) database at NCBI under the accession number of GSE213354 (reviewer token: mbcjwieophmdhit).\u201d but should have been \u201cRNA-Seq results from Adnp-HT mice were deposited in the GEO (Gene expression Omnibus) database at NCBI under the accession number of GSE213354.\u201dThe original article has been corrected."} +{"text": "Frequent outbreaks of emerging and re-emerging pathogenic viruses have become one of the major challenges for global public health. As the first line of defense, the innate immune system plays a vital role in fighting the invasion of pathogenic microorganisms. In response to viral entry into the host cell, pattern recognition receptors (PRRs) recognize the pathogen-associated molecular patterns (PAMPs) of viruses and then activate innate immune signaling pathways, which subsequently trigger the expression of numerous interferon-stimulated genes (ISGs) to exert direct antiviral effects ,2. MeanwPorcine epidemic diarrhea virus (PEDV) is a positive-sense single-stranded RNA virus that belongs to a coronavirus family. Many studies have shown that several PEDV proteins, including nsp1, nsp3, nsp5, nsp8, nsp14, nsp15, nsp16, E, M, and N, can restrict host IFN signaling. The research article by Zhang et al. investigated multiple PRR-mediated signaling pathways involved in the anti-PEDV responses. The innate immune signaling adaptors TRIF, MAVS, and STING exhibit blatant anti-PEDV activity, according to the authors\u2019 screening of porcine innate immune signaling adaptors\u2019 antiviral activity using transfected Vero cells. To further confirm it, knockdown or knockout of endogenous TRIF, MAVS, and STING promoted PEDV replication via siRNA and CRISPR approaches [Recently, it has been important to study how the noncanonical NF-\u03baB pathway participates in innate immunity. Bisom et al. conducted research to investigate the function of RIOK3 during Rift Valley Fever virus (RVFV) infection. They found that RVFV infection activated the noncanonical NF-\u03baB pathway to weaken the antiviral IFN signaling response due to the production of the alternatively spliced RIOK3 X2 isoform, which encodes a truncated RIOK3 . This fiYao et al. reported their data on the crucial role of pulmonary microvascular endothelial cells (MVECs) in regulating inflammation during highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) infections. They reported that HP-PRRSV primarily induced virus-associated innate immune responses, whereas bacterial lipopolysaccharide (LPS) was responsible for the inflammatory response. HP-PRRSV infection exacerbated the inflammatory response due to secondary bacterial infections . These rWen et al. focused on fusing the autophagosome-associated LC3b protein to the nucleocapsid (N) antigen, which is expected to improve the SARS-CoV-2-specific T cell functionality for developing the next-generation vaccine against SARS-CoV-2 variants. They concluded that the N-LC3b protein group can simultaneously secrete multiple cytokines (IFN-\u03b3+/IL-2+/TNF-\u03b1+), improving T cell proliferation, especially for CD8+ T cell responses. In addition, their strategy was also induced a robust humoral immune response against the N antigen .The role of IFITM3 in the SARS-CoV-2 pandemic is still controversial. Xu et al. reported their data on the association between IFITM3 and the risk of acquiring a SARS-CoV-2 infection. They demonstrated that IFITM3 inhibited SARS-CoV-2 infection by preventing virus entry, which is dependent on the first 21 amino acids of IFITM3. In addition, they also found that the rs12252 CC genotype of IFITM3 increased the risk of acquiring a SARS-CoV-2 infection and the decreased level of neutralizing antibodies against SARS-CoV-2 .Another five review publications summarized the state-of-the-art research on the interplay between viruses and host innate immunity. Alves et al. summarized how placental cells engaged in innate immune responses play roles in response to Dengue virus (DENV) and chikungunya (CHIKV) infections . Li et aIn conclusion, these ten articles published in this Special Issue should improve our understanding on the complex interactions between viral infections and host innate immune responses. These findings provide a summary of the most updated findings on PEDV, RVFV, PRRSV, SARS-CoV-2, DENV, CHIKV, and WNV, which are crucial for the subsequent development of novel approaches to prevent and control viral infections."} +{"text": "SARS-CoV-2 infections have been associated with self-reported impaired cognitive function, but research examining objective cognitive assessments is scant. Given the potential impact of long-term cognitive impairment, it is important to characterize this post-infection phenotype.The Epidemiology, Immunology, and Clinical Characteristics of Emerging Infectious Diseases with Pandemic Potential (EPICC) study is a longitudinal cohort assessing the impact of SARS-CoV-2 infection in Military Health System (MHS) beneficiaries. A subset of EPICC enrollees consented to cognitive assessment using the Brain-Baseline Assessment of Cognition and Everyday Functioning app and completed 4 tasks: Trails Making Tests A and B, Stroop task, and Visuospatial Short-term Memory task. Participants completed the tasks in August-September 2022 and were categorized as impaired if their mean completion time was >1 SD above the control sample mean.A total of 482 participants completed the cognitive assessments, 71% of whom had a known history of SARS-CoV-2 infection. Among those with a history of SARS-CoV-2 infection, the mean time between first positive SARS-CoV-2 test and module completion was 9 months (SD=5). Participants were primarily active duty service members (80%), male (65%), and non-Hispanic white (70%). SARS-CoV-2 infections were primarily mild or asymptomatic with only 14 (4.1%) hospitalized. Logistic regression models adjusted for sex, race/ethnicity, age, and education showed no difference in impairment in any of the BRACE tests comparing those with and without a history of SARS-CoV-2 infection . Age was a risk factor for impairment across all tests with each additional year increasing risk of impairment by 6-8% (95% CI: 1.04 \u2013 1.11).MHS beneficiaries with a history of SARS-CoV-2 infection did not demonstrate a long-term higher prevalence of objectively measured cognitive impairment compared to participants without SARS-CoV-2 infection after adjusting for demographic variables. Further study is needed to understand the incongruence between reported cognitive symptoms and objectively measured cognitive performance.Julia Rozman, BS, AstraZeneca: TBD Mark P. Simons, PhD, AstraZeneca: The IDCRP and HJF were funded to conduct an unrelated phase III COVID-19 monoclonal antibody immunoprophylaxis trial as part of US Govt COVID Response Timothy Burgess, MD, MPH, AstraZeneca: The IDCRP and the Henry M. Jackson Foundation (HJF) were funded to conduct an unrelated phase III COVID-19 monoclonal antibody immunoprophylaxis trial Simon Pollett, MBBS, AstraZeneca: The IDCRP and the Henry M. Jackson Foundation (HJF) were funded to conduct an unrelated phase III COVID-19 monoclonal antibody immunoprophylaxis trial"} +{"text": "TARDBPare associated with frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS) and other degenerative diseases. The predictedC. elegansortholog ofTARDBPis encoded by tdp-1, but functional orthology has not been demonstratedin vivo.We undertook CRISPR/Cas9-based genome editing of thetdp-1locus to create a complete loss of function allele; alltdp-1 exons and introns were deleted, creatingtdp-1(tgx58), which resulted in neurodegeneration after oxidative stress. Next, we undertook CRISPR-based genome editing to replacetdp-1exons with human TARDBP coding sequences, creating humanized (hTARDBP)C. elegans expressing TDP-43.Based on the efficiency of this genome editing, we suggest that iterative genome editing of thetdp-1target locus using linked coCRISPR markers, likedpy-10, would be a more efficient strategy for sequential assembly of the large engineered transgenes.hTARDBPdecreased the neurodegeneration defect oftdp-1(tgx58), demonstrating functional cross-species orthology. To developC. elegansmodels of FTD and ALS, we inserted five different patientTARDBP variants in theC. eleganshTARDBPlocus. Only one clinical variant increased stress-induced neurodegeneration; other variants caused inconsistent or negligible defects under these conditions. Combined, this work yielded an unambiguous null allele fortdp-1, a validated, humanizedhTARDBP,and multiple ALS/FTD patient-associated variant models that can be used for future studies.Clinical variants of TARDBP, which encodes the RNA-binding protein TDP-43, are associated with a wide variety of degenerative disorders. These include frontotemporal dementia (FTD) which leads to degeneration and death of cortical neurons, as well as amyotrophic lateral sclerosis (ALS), which leads to degeneration and death of spinal and cortical motor neurons. TheC. elegansortholog ofTARDBPisTAR DNA-binding Protein homolog-1, known astdp-1. To enable studies of TDP-1 function, and to demonstrate functional homology between TDP-1 and TARDBP, as well as to examine how patient-associated missense variants alter protein function in live animals, we undertook several rounds of CRISPR/Cas9-based genome deletion, humanization, and variant generation , with a focus on generating reagents and assessing neurodegeneration.Patient variants inKnock out.First, we created atdp-1loss of function allele by precise excision using CRISPR-editing. Using CRISPR/Cas9-mediated homologous recombination anddpy-10 co-conversiontdp-1 coding exons, as well as introns (Panel B) resulting in an unequivocal complete loss of function allele. When generatingtdp-1 KOs, we observed robust co-conversion of thedpy-10 andtdp-1 loci on the same maternal chromosome (Panel D), as expected from previous work tdp-1(tgx58)deletion as homozygote, free of the Dumpy phenotype marker.tdp-1(lof). Assess neurodegeneration forTo determine if loss oftdp-1function inC. elegansleads to degeneration and/or loss of neurons, we examined stress-induced degeneration of glutamatergic sensory neurons. PHA and PHB neurons are bilaterally-symmetric phasmid neurons in theC. eleganstail that have exposed sensory endings (Panel F). Degenerative retraction of phasmid neuron sensory processes or phasmid neuron death can prevent fluorescent dye back-filling of the entire neuron. This assay has been used previously inC. elegans models of neurodegenerative diseaseC. elegansmodels of neurodegenerative diseasetdp-1function causes hypersensitivity to oxidative stresstdp-1(tgx58)animals at two concentrations of paraquat. Mild paraquat stress overnight had no impact on neurodegeneration in the dye filling assay in either wild type ortdp-1(tgx58)young adult animals . However, 10mM paraquat-treatment overnight led to modest dye filling defects in wild type with more dramatic defects intdp-1(tgx58)animals . These results confirm thattdp-1 loss of function increases neurodegeneration under moderate oxidative stress.Humanize and insert clinical variants. With a KO phenotype established, we used two serial rounds of co-CRISPR editingTARDBPas a replacement of thetdp-1coding sequence (Panel C). For this humanized construct, we focused on expressing the most abundant isoform of TARDBP (UNIPROT Q13148-1) re-coded forC. eleganscodon bias and with the insertion of three synthetic introns. A full length plasmid containing a synthetic TARDBP sequence could not be generated by our suppliers. As an alternative, we elected to consecutively insert two fragments of TARDBP using co-CRISPR-editing. Insertion of the first fragment of recoded TARDBP resulted only in cis co-editing events ; all animals homozygous for the intendedtdp-1edit were also Dumpy (homozygous fordpy-10(cn64)). To movehTARDBP to a wild type background, we undertook an additional round of CRISPR-editing to restore the original N2 wild typedpy-10sequence (dpy-10(wt)). Another round of co-CRISPR to insert the second fragment of recoded TARDBP also resulted in only cis co-editing and all animals homozygous for full length TARDBP were also Dumpy. Again, a round of repair CRISPR was used to restore thedpy-10locus back to nonDumpy .dpy-10alleles, followed by a need to repair to get back to wildtype phenotype, leads us to suggest that future studies should use a serial-editing strategy to build up a large gene with a \u201cflip-flop approach\u201d that alternates between Dumpy and nonDumpy animals (Panel E).The observation of cis-only editing with the co-generation ofhTARDBPwas confirmed by rtPCR and the LOF defect oftdp-1(-)was partially rescued in thehTARDBPanimals. (Panel H). We used co-CRISPR editing to insert each of the five ALS/FTD clinical variants into the humanized locus . Over-expression of TARDBP A315T is toxic in two differentC. elegansmodels and TARDBP M337V is toxic in two different C. elegansmodelsC. elegans models for the other alleles have not been reported previously.Expression of full lengthAssess neurodegeneration for clinical variants. Only insertion ofhTARDBP-G295Syielded a significant defect versus hTARDBP-WT(Panel I). Future work will likely require confirmation of the G295S defect with the generation and testing of another independently-derived allele. For the remaining alleles , no significant increases in stress-induced neurodegeneration were observed, compared tohTARDBP-WT, using this assay.Neurodegeneration: L4 stage animals were moved to NGM/OP50 plates containing 10mM (or 2.5mM) paraquat for 22 hours and neurodegeneration was examined the next day in adults based on backfilling of phasmids neurons with DiI (Molecular Probes). Animals were scored as affected if any of the four phasmid neurons failed to backfill with fluorescent dye, detected at a magnification level of 12.5x with moving animals on culture dishes; note that loss of a single neuron will be missed in some animals and we may underestimate neurodegeneration. Animals were scored blinded as to genotype.tdp-1(tgx58)KO allele was generated by deletion of the coding sequence using CRISPR/Cas9-mediated homologous recombination. The dpy-10co-conversion strategy was used to identify candidate lines after injectiontdp-1coding exon and the other after the stop codon creating a 1787bp deletion. All sgRNAs were synthesized by Synthego Corporation . The donor homology sequence was a single stranded oligonucleotide (ssODN) containing 35bp homology arms, a 3-frame stop sequence, and an XhoI restriction site (reagent table). Injections of the Cas9 protein, sgRNAs, and donor homology template mix were performed with N2 young adults. Genome edit candidates were selected from the F1 population based on a visual screen for the co-conversion Rol phenotype and screened by PCR for the deletion. Homozygous deletion lines were confirmed by sequencing.Genome editing: ThehTARDBP-WTinsertion was generated using a gene swap method dpy-10co-conversion CRISPR/Cas9 strategy to insert 725bp of thehTARDBPsequence. The injection mix included Cas9 protein, the 795bp dsDNA donor homology template, sgRNAs, and co-CRISPR reagents. This was injected into the gonads of adult hermaphrodite worms and the F1 animals displaying the co-CRISPR phenotype were isolated. Homozygous animals containing the insertion were identified; however, they also contained thedpy-10(cn64) mutation. An edit to correct this mutation was performed and animals with wild type sequence at thedpy-10 locus, but containing the partialhTARDBP-WT were identified. This strain was injected with the 756bp PCR product donor homology template containing the second half of thehTARDBPsequence and homology arms, along with the Cas9 protein, sgRNAs, and thedpy-10 co-CRISPR reagents. Animals homozygous for the insertion as well as thedpy-10(cn64)mutation were isolated and the corrective edit fordpy-10 was performed. Integration of the complete sequence ofhTARDBP was confirmed by Sanger sequence analysis. When complete, thehTARDBP-WTstrain contained a codon optimized versionhTARDBPwith 3 synthetic introns. Note thathTARDBPwas introduced into the nativetdp-1exon 1 such that the first 5 nativeC. elegans amino acids were preserved (i.e. MADET). VerificationhTARDBPmRNA expression was undertaken. RNA was extracted from thehTARDBP-WTstrain and N2 using TRI Reagent and the Direct-zol RNA kit (Zymo Research) and cDNA was synthesized using iScript Reverse Transcription Supermix for RT-qPCR (Bio-Rad Laboratories). To amplify the whole coding sequence, PCR was performed, see reagent table for primers, on the cDNA and visualized on an agarose gel. The full lengthhTARDBPsequence amplified from the humanized strain at a comparable level totdp-1in N2. The amplicons were also used as a template for DNA sequencing by Sequetech Corporation. Sequences were aligned and analyzed using Benchling and full-lengthhTARDBPwas observed.ThehTARDBPvariants were inserted using CRISPR/Cas9-mediated homologous recombination. For each variant, two sgRNAs flanking the desired edit were selected. The donor homology was designed with 35bp homology arms and re-coding to eliminate recutting of the repaired template and to make identification of the edits easier. The injection mixes included Cas9 protein, each donor homology single-stranded oligonucleotide (ssODN), the sgRNAs, and thedpy-10co-CRISPR reagents. Animals displaying the co-CRISPR phenotype were selected and High Resolution Melt Analysis was performed to identify animals containing the edit of interest. Homozygous animals were isolated and confirmed by sequencing of both the target locus and thedpy-10 locus. Original, edited strains were backcrossed to N2 four times to create outcrossed strains, which were used to generate results presented herein.TheStatistical analysis: Student\u2019s t-test used for comparison of neurodegeneration (Microsoft Excel)."} +{"text": "BRCA-mutated breast cancer (BC), including triple-negative BC (TNBC) and ovarian cancer (OvCa). A key challenge is to identify the factors associated with PARPi resistance; although, previous studies suggest that platinum-based agents and PARPi share similar resistance mechanisms.Poly (ADP-ribose) polymerase inhibitors (PARPi) are approved for the treatment of BRCA-mutated TNBC cell lines. In-silico analysis were performed using multiple databases including The Cancer Genome Atlas, the Genotype-Tissue Expression, The Cancer Cell Line Encyclopedia, Genomics of Drug Sensitivity in Cancer, and Gene Omnibus Expression.Olaparib-resistant (OlaR) cell lines were analyzed using HTG EdgeSeq\u00a0miRNA Whole Transcriptomic Analysis (WTA). Functional assays were performed in three p\u2009=\u20090.001) as well as in tumor tissues from TNBC patients (p\u2009=\u20090.001). We hypothesized that miR-181a downregulates the stimulator of interferon genes (STING) and the downstream proinflammatory cytokines to mediate PARPi resistance. BRCA1 mutated TNBC cell lines with miR-181a-overexpression were more resistant to olaparib and showed downregulation in STING and the downstream genes controlled by STING. Extracellular vesicles derived from PARPi-resistant TNBC cell lines horizontally transferred miR-181a to parental cells which conferred PARPi-resistance and targeted STING. In clinical settings, STING levels were positively correlated with interferon gamma (IFNG) response scores (p\u2009=\u20090.01). In addition, low IFNG response scores were associated with worse response to neoadjuvant treatment including PARPi for high-risk HER2 negative BC patients (p\u2009=\u20090.001). OlaR TNBC cell lines showed resistance to platinum-based drugs. OvCa cell lines resistant to platinum showed resistance to olaparib. Knockout of miR-181a significantly improved olaparib sensitivity in OvCa cell lines (p\u2009=\u20090.001).High miR-181a levels were identified in OlaR TNBC cell lines (miR-181a is a key factor controlling the STING pathway and driving PARPi and platinum-based drug resistance in TNBC and OvCa. The miR-181a-STING axis can be used as a potential marker for predicting PARPi responses in TNBC and OvCa tumors.The online version contains supplementary material available at 10.1186/s13578-023-01151-y. BRCA-mutated (gBRCA mt) or HER2-negative metastatic breast cancer (BC) who have been treated with chemotherapy either in the neoadjuvant, adjuvant, or metastatic settings [Poly (ADP\u2011ribose) polymerase inhibitors (PARPi) promote DNA damage accumulation in tumor cells with homologous recombination (HR) deficiency, leading to the concept of synthetic lethality . As a resettings . PARPi asettings . Severalsettings , 7. Althsettings . TherefoBRCA mt ovarian cancer (OvCa) [Three large clinical studies, using PARPi treatment for patients with r (OvCa) , 10, conr (OvCa) ; althougRecently, we reported that the downregulation of the cytosolic DNA sensor stimulator of interferon genes (STING) leads to cisplatin resistance by decreasing the expression of downstream proinflammatory cytokines in triple-negative breast cancer (TNBC) . STING iHomo sapiens (hsa)-miR-181a-5p (or miR-181a-5p from this point on) downregulates STING and thereby allows fallopian tube secretory epithelial cells (FTSEC) to bypass interferon-mediated cell death leading to cancer cell transformation and development of high-grade serous ovarian cancer (HGSOC) [hypothesized that the factors regulating STING levels modulate the downstream signaling in TNBC and OvCa and correlate with PARPi sensitivity as well as cross-resistance to platinum-based drugs.Previously, we reported that tumor tissue (HGSOC) . High tu (HGSOC) . Thus, win-vitro findings were validated using clinical specimens from TNBC and OvCa patients. This study describes a novel mechanism of PARPi resistance, as well as platinum-based drug cross-resistance; and suggests that the upregulation of miR-181a is a significant factor predicting PARPi responses in TNBC and OvCa.In this study, we obtained the miR profiles of PARPi resistant cell lines using HTG EdgeSeq miRNA Whole Transcriptome Assay (HTG miR WTA) and identified that miR-181a is significantly upregulated in PARPi-resistant TNBC cell lines. Functional assays were then performed to characterize the role of miR-181a in controlling STING pathways as a mechanism of PARPi resistance. Furthermore, the The study was conducted following the Declaration of Helsinki and was approved by the Ethics Committee at Saint John\u2019s Cancer Institute (SJCI) and WIRB: MORD-RTPCR-0995. Six formalin-fixed paraffin-embedded (FFPE) OvCa tissues surgically resected were obtained from patients at the Department of Obstetrics and Gynecology, Perelman School of Medicine, U. of Pennsylvania, PA. Three tissues were treatment na\u00efve at the time of surgery and three tissues were from a second debulking surgery after patients relapsed to adjuvant chemotherapy (Carboplatin/Paclitaxel). All the patient specimens were de-identified. Furthermore, a clinically annotated tissue microarray (TMA) for TNBC (#BR1301) was obtained from US Biomax .BRCA mt TNBC cell lines MDA-MB-436 (BRCA1 mt), HCC1395 (BRCA1 mt), and HCC1937 (BRCA1 mt) were obtained from the American Type Culture Collection and were cultured as recommended. The establishment of HCC1937 and HCC1395 cell lines olaparib-resistant (OlaR) are described in Additional file Three established human -436 BRCA mt, HCC1Total RNA from cell lines was extracted by the Direct-zol RNA miniprep kit according to the manufacturer\u2019s instructions. RNA isolation from EV is described in Additional file The HTG miR WTA was utilized to assess the difference in human miR transcripts between parental and OlaR cell lines using direct next-generation sequencing (NGS) as previously described . HTG miRPurified lentiviral particles for STING (LPP-E1218-Lv103-050), miR-181a (LPP-HmiR0023-MR03-050-S), and their respective controls were transduced into TNBC cell lines MDA-MB-436, HCC1395, and HCC1937 as previously described [Knockdown experiments were performed as previously described . TNBC ceOlaparib was dissolved in molecular-grade water at a concentration of 10\u00a0mM. For cell viability assays, the measurements were performed after treatment with different concentrations of olaparib for 48\u00a0h, as previously described , 18. ForBC FFPE TMA sections were stained with STING antibody by immunohistochemistry (IHC) as previously described [In-situ hybridization (ISH) for miR-181a was performed on a FFPE TNBC TMA section and FFPE OvCa sections using the miRNAscope Assay according to the manufacturer\u2019s instructions and as previously described [escribed . MiR-1813) were seeded in a 96-well culture plate. The number of viable cells was assessed using a Cell Titer-Glo Luminescent Cell Viability assay according to the manufacturer\u2019s instructions as previously described [TNBC cell lines (1\u2009\u00d7\u200910escribed . For asshttp://imagej.nih.gov/ij/). Western blotting was performed for CD9, CD63, and CD81 in EV derived from HCC1937 and HCC1395 parental cell lines.Traditional western blot was performed as previously described , except Automated western blotting was performed according to the manufacturer\u2019s protocol , and quantified as previously described . ProteinFor a detailed explanation of EV isolation please refer to Additional file https://www.R-project.org/.) in a two-tailed way. The distribution and variation within each group were assessed before statistical analysis. Two groups were compared using Student\u2019s t-test. Multiple groups were analyzed by One-way or Two-way ANOVA followed by a post-hoc Tukey\u2019s multiple comparisons test. The correlation between variables was determined using Pearson\u2019s correlation test. Overall survival (OS), recurrence-free survival (RFS), and distant metastasis-free survival (DMFS) were calculated from the time of taking the first specimen until death/recurrence/distant metastasis or last contact and were analyzed using the Log-rank test. For miRNA analysis, DESeq2 normalization and statistical comparisons were performed using the HTG REVEAL software version 2.0.1. Differentially expressed miRNAs were screened using Log2 fold change (FC)\u2009>\u20091.5 or\u2009<\u2009\u2212\u00a01.5, median normalized counts\u2009>\u20091000. MiR expression (counts per million) was logarithmically scaled (Log10) for volcano plot data visualization. A two-sided p\u2009<\u20090.05 was considered statistically significant: p\u2009<\u20090.05, **p\u2009<\u20090.01, ***p\u2009<\u20090.001, and ns\u2009=\u2009not significant. All figures were unified using Adobe Illustrator CC or CorelDraw graphics suite 8X .Statistical analyses were performed using the GraphPad Prism 8 or R version 4.1.2 (BRCA1 mt olaparib-resistant (OlaR) TNBC cell line was initially established as described in Additional File HCC1937 In-silico analysis using the TCGA BRCA dataset showed that miR-181a levels were significantly higher in primary BC compared to adjacent normal breast tissues with miR-181a overexpression but increased PARPi sensitivity in BRCA1 mt TNBC cell lines significantly was performed in the three TNBC cell lines using lentiviral transduction compared to normal breast and adjacent normal breast tissues , IFNB, interleukin-12A\u00a0(IL12A), and interleukin-12\u00a0(IL12B) compared to respective control cell lines in RT-qPCR analysis as the vehicle for miR-181a delivery to non-resistant cell lines. EVs derived from HCC1937 TNBC parental and OlaR (OlaR-EV) cell lines were isolated using differential ultracentrifugation (DUC) from culture supernatants. EVs were characterized using Western blotting, NTA, FL-NTA, and ACE. By western blot, the three EVs standard markers were detected, indicating the purity and characteristics of the EV fractions isolated in both BC and OvCa cell lines and BRCA1 promoter methylation predicted response to rucaparib [The early detection of drug resistance as well as the understanding of the intrinsic mechanisms that induced PARPi resistance are important for both TNBC and OvCa patients. Thus, the prospects for developing effective strategies for the early detection of PARPi resistance are a growing field. Previous studies have shown that germline and somatic and OvCa , 3. Alteand OvCa \u201329. A reucaparib .Tumors that are sensitive to platinum-based drugs are also sensitive to PARPi treatment , 32. OurThe present study offers additional insight into miR-181a regulatory functions controlling STING mRNA and protein levels. MiR-181a targets STING and promotes metastasis as well as recurrence in advanced stage HGSOC . Our stuSeveral studies have shown that the activation of STING and the stimulation of type I IFN production are critical for anticancer immune responses. Activation of STING signaling induces the production of type I IFN, which plays critical roles in activating both innate and adaptive immune responses . A studySeveral clinical trials have been conducted to test the efficacy of PARPi plus immune checkpoint inhibitors (ICI) in ovarian, breast, prostate, lung, bladder, gastric cancers, and other solid tumors . MiR-181PIAS3) [Recently, EVs have attracted extensive attention for their role in drug resistance , 38. ThePIAS3) , which tPIAS3) . This stTNBC cell lines resistant to PARPi showed enhanced levels of miR-181a HCC1937 cell line treated with different concentrations of olaparib (B) or cisplatin (C) (Two-way ANOVA and Sidak\u2019s multiple comparisons test). D Cell viability assays comparing parental and OlaR HCC1937 cell line (Two-way ANOVA and Sidak\u2019s multiple comparisons test). E Representative images for GFP positive cells with miR-181a-OV in MDA-MB-436, HCC1395, and HCC1937 cell lines using fluorescence microscopy. Scale bars: 100 \u00b5m. F\u2013H Cell viability assays comparing miR-181a-OV and empty vector (CTRL) in MDA-MB-436 (E), HCC1395 (F), and HCC1937 (G) cell lines (two-way ANOVA and Sidak\u2019s multiple comparisons test). I Cell viability assays comparing miR-181a-OV and empty vector (CTRL) HCC1937 cell lines treated with different concentrations of cisplatin (two-way ANOVA and Sidak\u2019s multiple comparisons test). Cell viability assays were performed in triplicates. Figure S2. STING overexpression or downregulation does not affect cell proliferation. A Representative images for STING-OV (GFP) in MDA-MB-436, HCC1395, and HCC1937 cell lines using fluorescence microscopy. Scale bars: 100 \u00b5m. B\u2013D Cell viability assays comparing STING-OV and empty vector (CTRL) in MDA-MB-436 (B), HCC1395 (C), and HCC1937 (D) cell lines (two-way ANOVA and Sidak\u2019s multiple comparisons test). E\u2013G Cell viability assays comparing si-STING and si-CTRL in MDA-MB-436 (E), HCC1395 (F), and HCC1937 (G) (two-way ANOVA and Sidak\u2019s multiple comparisons test). Figure S3. STING is downregulated in TNBC and relates to outcomes. A STING mRNA levels in normal breast and primary BC (Primary) tissues in the TCGA and GTEx databases (Student\u2019s t-test). BSTING mRNA levels in tissues from tumor-adjacent normal breast , Luminal (Lum), Luminal-HER2 (Lum-HER2), HER2, and TNBC in the TCGA BRCA dataset (One-way ANOVA and Tukey\u2019s multiple comparisons test). CSTING mRNA levels in tissues from the tumor-adjacent normal breast , Normal-like (Norm-like), Luminal-A (LumA), Luminal-B (LumB), HER2-enriched, and basal-like breast cancer (BLBC) in the TCGA BRCA dataset (One-way ANOVA and Tukey\u2019s multiple comparisons test). D\u2013F Survival analysis of RFS (D), OS (E), and DMFS (F) for patients with TNBC in the TCGA, GEO, and EGA databases combined (Log-rank test). G\u2013I Survival analysis of RFS (G), OS (H), and DMFS (I) for patients with BLBC in the TCGA, GEO, and EGA database combined (Log-rank test). *p<0.05, **p <0.01, ***p <0.001. Figure S4. Analysis of the mRNA levels of the downstream components of the STING pathways. A\u2013E Quantification by RT-qPCR of STING (A), IL6 (B), IFNB (C), IL12A (D), and IL12B (E) mRNA levels in empty vector (CTRL) and STING-OV HCC1395 cell line (Student\u2019s t-test). F\u2013J Quantification by RT-qPCR of STING (F), IL6 (G), IFNB (H), IL12A (I), and IL12B (J) mRNA levels in empty vector (CTRL) and STING-OV MDA-MB-436 cell line (Student\u2019s t-test). K\u2013O Quantification by RT-qPCR of STING (K), IL6 (L), IFNB (M), IL12A (N), and IL12B (O) mRNA levels in miR-181a-OV and CTRL in HCC1395 cell line (Student\u2019s t-test). P-T Quantification by RT-qPCR of STING (P), IL6 (Q), IFNB (R), IL12A (S), and IL12B (T) mRNA levels in miR-181a-OV and empty vector (CTRL) in MDA-MB-436 cell line (Student\u2019s t-test). *p<0.05, **p <0.01, ***p <0.001. Figure S5. Correlation analysis between miR-181a levels and drug activities. A\u2013D Correlation between miR-181a levels and cisplatin (A), rucaparib (B), olaparib (C), talazoparib (D) drug activity in the BC cell lines obtained from CCLE and GDSC BRCA datasets (Pearson\u2019s correlation coefficient). F\u2013I Correlation between miR-181a levels and cisplatin (F), rucaparib (G), olaparib (H), talazoparib (I) drug activity in the OvCa cell lines obtained from CCLE and GDSC BRCA datasets (Pearson\u2019s correlation coefficient). Figure S6. Uncropped western blotting images. A-C Uncropped western blotting images for Figures 3D (A), H (B), and I (C) are shown. Figure S7. Uncropped western blotting images. A\u2013D. Uncropped western blotting images for Figures 3J (A), K (B), 4A (C), and E (D) are shown. Figure S8. Uncropped western blotting images. A Uncropped western blotting images for Figure 6A (A) and G (B) are shown.Additional file 2: Table S1. Reagents and resources utilized in the study."} +{"text": "Plasmodium falciparum gametocytes within erythrocytes (P. falciparum antigen in the patient\u2019s blood. Abdominal ultrasonography identified a patchy hypoechoic lesion in the spleen (,On the final day of a 3-day oral artemether-lumefantrine combined malaria treatment, a 30-year-old man in the infectious diseases ward began experiencing pain in the left hypochondriac region. A peripheral smear examination revealed the presence of banana-shaped hrocytes . Additioe spleen , which we spleen . Observa"} +{"text": "Numerous novel and effective therapeutic agents and clinical trials addressing castration-resistant prostate cancer (CRPC) were reported during the 2023 American Society of Clinical Oncology-Genitourinary (ASCO-GU) Cancers Symposium. Notably, radionuclide drug conjugates (RDC), specifically 177Lu/111In-J591 and 225Ac-J591, exhibited enhanced therapeutic efficacy in treating patients with CRPC. Furthermore, promising treatment approaches for CRPC included dual anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and anti-programmed death-1 (PD-1) blockade in rare tumors (DART)-Lorigerlimab, prostate stem cell antigen (PSCA)-directed chimeric antigen receptor (CAR)-T cell immunotherapy-BPX-601, and protein kinase inhibitor (AKTi)-CAPltello-280. We have summarized the latest CRPC treatment strategies presented at the 2023 ASCO-GU Cancers Symposium, along with recent advances in CRPC clinical trials. Each year, the ASCO-GU Cancers Symposium showcases noteworthy developments and innovations in genitourinary oncology. We have comprehensively reviewed such notable advancements in drugs and novel therapies targeting CRPC, as presented at the 2023 ASCO-GU Cancers Symposium.RDC drugs utilize antibodies or small molecules to modulate specific targets and deliver cytotoxic or imaging agents to the target location, resulting in localized radiation from the radioisotope on the target tissue for efficient and precise treatment while minimizing systemic exposure and radiation-induced toxicity to other tissues .In a randomized, double-blinded phase II study, radioactive 177Lu and 111In, combined with ketoconazole or hydrocortisone, was used to label the anti-prostate-specific membrane antigen (PSMA) monoclonal J591 (NCT00859781). The results indicated a significant reduction in prostate-specific antigen (PSA) levels in most patients with non-metastatic CRPC (M0CRPC) treated with radiolabeled J591 and ketone/HC (PSA decline ratio\u2009>\u200950% (PSA50): 82% and 71% in the177Lu and 111In groups, respectively; PSA decline ratio\u2009>\u200990% (PSA90): 50% and 35% in the 177Lu and 111In groups, respectively). Biochemical progression-free survival (bPFS) was 18.67 and 8.87 months in the 177Lu and 111In groups, respectively, with a significantly higher 18-month PFS in the 177Lu group; however, hematologic toxicity was more common in this group [Another new triple therapy involving 225Ac-J591 (a PSMA-targeted radionuclide therapy), pembrolizumab, and an androgen receptor pathway inhibitor (ARPI) demonstrated a significant PSA response in both phase I/II trials NCT04946370). After six months of follow-up, 33% (4/12) of the patients remained progression-free. However, 58% (7/12) of the patients developed unexpected cytokine release syndrome (CRS) 7\u201314 days after treatment, characterized by a morbid rash, fever, and low blood cell count. Nevertheless, patient responses typically improved within one week after discontinuing ARPI. Additionally, typical immune-related adverse events (irAEs) occurred in 33% (4/12) of the patients, all of which were manageable [70. AfterA study reported data on lorigerlimab (a DART molecule that enhIn a phase III study, the efficacy of AKTi-CAPltello-280 (effective selective inhibition .Overall, the 2023 ASCO-GU Cancer Symposium showcased significant advancements in the therapeutic area of CRPC, as evidenced by the findings presented in Tables"} +{"text": "Further study showed that lncRNA-MEG3 regulates skeletal muscle regeneration via sponging miR-133a-3p to regulate proline-rich transmembrane protein 2 (PRRT2) expression level. These results suggested that lncRNA-MEG3 might be a potential target for skeletal muscle diseases.Skeletal muscle is the largest motor and metabolic organ of the body, which has a robust capacity for regeneration following injury or disease. Delayed regeneration after skeletal muscle injury reduces muscle contractility and leads to dysfunction of innervation. Therefore, identifying the regulation components in skeletal muscle regeneration and determining their molecular mechanisms are important to discover novel therapeutic markers for muscular diseases. Long non-coding RNA (LncRNA) has been implicated in skeletal muscle regeneration. Recent developed single-cell RNA sequencing (scRNA-seq) provides a higher resolution of cellular differences than bulk RNA-seq. Here, we re-analyzed single-cell transcriptomes data of skeletal muscle regeneration and identified lncRNA maternally expressed gene 3 muscle injection was used to determine the role of in vivo . In the ol group . Single-t injury further lncRNA-MEG3 on primary myoblasts -qPCR -qPCR H. This wIP)-qPCR I. SubseqRNA-MEG3 . Furtherneration . To furtntiation .lncRNA-MEG3 knockdown C2C12 and skeletal muscle at postnatal day 0 and day 65 declare that they have no conflict of interest.The National Natural Science Foundation of China (No. 31830090); The Basic and Applied Basic Research Foundation of Guangdong province, China (No. 2019B1515120059); The Agricultural Science and Technology Innovation Program, China (No. CAAS-ZDRW202006).This work was supported by All authors have agreed to publish this manuscript."} +{"text": "Many patients with hematological malignancies treated with stem cell transplantation (SCT) experience cognitive dysfunction. However, few studies have investigated treatment-related neurotoxicity in older adults with multiple myeloma (MM) treated with high dose chemotherapy (HDC) and autologous SCT (HDC/ASCT). In this study, we examined gray matter (GM) volume, resting state functional connectivity (RSFC), neurocognitive function (NF), and proinflammatory cytokines (PCy) in older patients with MM pre- and post-HDC/ASCT.Eighteen MM patients underwent magnetic resonance imaging, neurocognitive tests, and serum PCy measurement prior to HDC/ASCT, and fifteen patients completed follow ups an average of five months post-HDC/ASCT.left dorsolateral prefrontal cortex and right posterior parietal cortex (p = 0.022), and (2) the CEN involving the right posterior parietal cortex and the salience network involving the right dorsal anterior cingulate cortex (p = 0.029); these comparisons were no longer significant after multiple comparisons correction. There were no significant changes in GM volumes or NF, except for improvement in attention . There were significant increases in several PCy post-HDC/ASCT (p \u2264 0.05).There were significant decreases in RSFC from pre- to post-HDC/ASCT in (1) the central executive network (CEN) involving the This pilot study showed decreased RSFC involving the left frontal, right posterior parietal and right anterior cingulate cortices in MM patients post-HDC/ASCT, relatively stable NF, and increases in PCy. These findings are congruent with studies in patients with hematological malignancies and other cancers and provide supporting evidence for the vulnerability of frontoparietal regions to chemotherapy adverse effects. There is compelling evidence that chemotherapy is associated with neurotoxicity , with suNeurocognitive dysfunction has been documented in multiple myeloma (MM) patients after HDC and preautologous SCT (ASCT) with declines post-HDC/ASCT ; howeverolderMM patients undergoing HDC/ASCT, even though this intervention has been used more often in the elderly . Eligibility criteria: (1) MM diagnosis, (2) complete, partial, or very good partial disease remission at enrollment, as per standard International Myeloma Working Group Criteria, (3) age 60\u201375 at enrollment, (4) fluent in English. Exclusionary criteria: (1) disease progression during the study period, (2) CNS disease, or (3) history of neurological, psychiatric, or substance abuse disorders.StructuralImaging:T1-weighted anatomical images with whole brain coverage were obtained with spoiled gradient-recalled and high-resolution three-dimensional magnetization-prepared rapid acquisition with gradient-echo sequences. Functional imaging. For rsfMRI, T2*-weighted images were acquired with a single-shot gradient echo-planar imaging (EPI) sequence . For the rsfMRI, patients were instructed to keep the eyes open and fixated on a crosshair.Patients were imaged in Tesla scanners at MSK; five patients were imaged in two different Tesla scanners at each timepoint using the same parameters. structural image processing, VBM analysis was performed using the longitudinal processing stream in the VBM8 toolbox (http://dbm.neuro.uni-jena.de/vbm/) under the SPM8 software package within MATLAB . Following reconstruction, follow-up MPRAGE structural images were registered to baseline MPRAGE images for each subject, bias corrected, segmented into GM, WM, and cerebrospinal fluid compartments using the Montreal Neurologic Institute (MNI) T1 weighted template and tissue probability maps, linear and non-linear registered to MNI space, and the resulting GM tissue class smoothed using an isotropic Gaussian spatial filter (FWHM=8 mm). For rsfMRI pre-processing, a data pre-processing scheme was implemented according to published methods ; Brief TTrail Making Test Parts A & B (TMTA & TMTB) ; ControlHopkins Verbal Learning Test-Revised: Total, Delayed Recall, Discrimination Index . .Center for Epidemiological Study-Depression (CES-D) ;Functional Assessment of Chronic Illness Therapy-Fatigue Subscale, Version 4 (FACIT-FS V-4). .Blood samples were collected pre- and post-HDC/ASCT on the same day as the neurocognitive assessment and delivered to the Immune Monitoring Core Facility at MSK for plasma isolation and frozen storage until ready for batch analysis. PCy were quantitated from thawed plasma samples following manufacturer instructions for the V-PLEX Human Proinflammatory Panel 10-plex kit , which included interleukin Ibeta (IL-1 \u03b2), interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 6 (IL-6), interleukin 8 (IL-8), interleukin 10 (IL-10), interleukin 12 (IL-12), interleukin 13 (IL-13), interferon gamma (IFN\u03b3), and tumor necrosis factor alpha (TNF-\u03b1).Following omnibus testing, pairwise t-tests were performed at the group level to analyze within group changes from pre- to post-HDC/ASCT. For the structural contrast, initial uncorrected voxel-wise threshold was p\u2264 0.001 with resulting maps family-wise error corrected over the whole brain at p\u22640.05.Resting State Functional Connectivity Analysis (RSFC) was performed using region-of-interest based correlation as described previously . Three r(r) between each ROI pair in the CEN, SN, and DMN. Each of the pairwise ROI correlations were Fisher z-transformed for further statistical analysis. Changes in RSFC z-scores from pre- to post-HDC/ASCT were assessed using linear mixed models, adjusting for scanner .Correlation matrices were produced by extracting the time course from each of the ROIs and computing the Pearson correlation coefficient d) were calculated to quantify the magnitude of score changes over time. False discovery rate (FDR) was used to adjust p-values for multiple comparisons. The Reliable Change Index (RCI), which represents the change in scores divided by the standard error of measurement, was used to identify patients whose raw scores improved or declined beyond expected due to practice effects and measurement error. For each test score, we calculated the proportion of patients with RCI-indicated reliable decline.Raw neurocognitive test scores were transformed into z-scores based on age-adjusted normative values. Neurocognitive test z-scores and self-report scale scores were summarized at each timepoint using descriptive statistics, and differences in scores from pre- to post-HDC/ASCT were compared using Wilcoxon signed rank tests. Standardized effect sizes fit calibration curve was generated for each analyte using the standards to calculate the concentration of each analyte. Upper and lower limits of quantitation for each PCy were established as the highest and lowest points of the standard curve on each plate whose back calculated values were within 80\u2013120% of the expected values of the 4PL regression fit and exhibited less than 20% CV across the duplicate standard wells.Cytokine data was analyzed using the MSD Discovery WorkbenchSpearman correlations were calculated to assess the association of RSN z-scores, neurocognitive test z-scores, self-report scale scores, and PCy levels separately for each timepoint.Eighteen MM patients completed a neurocognitive assessment and a brain MRI pre-HDC/ASCT, and fifteen patients were available for follow up an average of five months post-HDC/ASCT. One patient was excluded from the imaging analysis due to scan misregistration at follow up. Thirteen patients provided blood samples for PCy analysis at each timepoint. Descriptive statistics for demographic and disease variables are presented in left dorsolateral prefrontal cortex (L-DLPFC) and right posterior parietal cortex (R-PPC) (p=0.022), and (2) the CEN ROI involving the R-PPC and the SN ROI involving the right dorsal anterior cingulate cortex (R-dACC) (p=0.029) from pre- to post-HDC/ASCT (The results showed significant decreases in RSFC in (1) CEN ROIs involving the HDC/ASCT ; these cThe neurocognitive tests z-scores and self-report scales scores are presented in The RCI results showed that most patients (>67%) had no reliable change on the neurocognitive tests from pre- to post-HDC/ASCT. Patients had reliable declines in the HVLT-R-D (7%), TMT A (34%), TMT B (13%), and BTA (7%). Reliable improvements were seen in the HVLT-R-T (13%), HVLT-R-D (7%), TMT A (34%), TMT B (20%), and BTA (27%).PCy levels were generally low in most patients at both timepoints, and pre-HDC/ASCT values were below the limits of quantitation for IL-1 b, IL-2, IL-4, IL-10, IL-12, and IL-13. There were significant increases in PCy post-HDC/ASCT, including IL-1 b, IL-2, IL-4, IL-8, IL-12, IL-13, and TNFa . There were no significant correlations among RSN z-scores, neurocognitive test z-scores, self-report scale raw scores, and PCy levels either pre- or post-HDC/ASCT.This is the first study describing alterations in RSFC in older MM patients treated with HDC/ASCT. The results showed decreased connectivity in the CEN involving the L-DLPF and R-PPC and in the CEN R-PPC and the SN R-dACC from pre- to an average of five months post-HDC/ASCT, suggesting that adverse effects of HDC may be of concern in this population. The CEN has been described as a frontoparietal network involved in attention control, working memory, and processing speed . The SN A structural neuroimaging literature review in non-CNS cancer patients describeIt is estimated that at least 50% of patients with hematological malignancies experience neurocognitive dysfunction prior to SCT, with either stable performance or declines months to years post-SCT . In thisCytokine dysregulation is common in MM and may influence the development of adverse effects . High PCIn this pilot study, changes in RSFC were more pronounced than in NF, possibly reflecting the use of compensatory mechanisms to maintain cognitive performance in the context of decreased CEN and SE connectivity , and the greater sensitivity of advanced neuroimaging tools to detect subtle alterations in functional connectivity in vulnerable regions. However, the RSFC results were no longer significant after multiple comparisons correction, and there were no significant associations between NF, RSFC, and PCy levels. These findings may be in part related to reduced power to detect small changes and associations beyond what was reported, and to MM patients receiving fewer lines of prior chemotherapy and less neurotoxic agents and conditioning regimens, compared to other hematologic malignancies .Decreased RSFC in older MM patients following HDC/ASCT provides further evidence for the prevailing notion that frontal-parietal regions may be vulnerable to chemotherapy adverse effects. Longitudinal studies with larger sample sizes are needed to further investigate the neural correlates of chemotherapy-related neurotoxicity and the role of PCy in older MM patients, with the goal of developing targeted therapeutic interventions."} +{"text": "Correction: Trials 23, 1041 (2022)https://doi.org/10.1186/s13063-022-06995-2The original publication of this article containeIncorrectIn males diagnosed with low- and intermediate-risk prostate cancer, the daily addition of 200 mg of phytoestrogen-rich foods to the diet for 6 weeks reduces prostate tumor proliferation compared to no addition of phytoestrogen-rich foods to the diet during the same period.CorrectIn males diagnosed with low- and intermediate-risk prostate cancer, the daily addition of phytoestrogen-rich foods (~200 mg phytoestrogens) to the diet for 6 weeks reduces prostate tumor proliferation compared to no addition of phytoestrogen-rich foods to the diet during the same period."} +{"text": "Existing research on predicting biochemical recurrence after prostate surgery has been insufficient. Here, we aimed to predict biochemical recurrence after radical prostatectomy leveraging recent advances in deep learning. We combined clinical variables with multiparametric magnetic resonance imaging using deep learning methods. Our method performed better than existing methods. Our method could direct patients to individualized care using routine medical imaging and could achieve better patient care.Radical prostatectomy (RP) is the main treatment of prostate cancer (PCa). Biochemical recurrence (BCR) following RP remains the first sign of aggressive disease; hence, better assessment of potential long-term post-RP BCR-free survival is crucial. Our study aimed to evaluate a combined clinical-deep learning (DL) model using multiparametric magnetic resonance imaging (mpMRI) for predicting long-term post-RP BCR-free survival in PCa. A total of 437 patients with PCa who underwent mpMRI followed by RP between 2008 and 2009 were enrolled; radiomics features were extracted from T2-weighted imaging, apparent diffusion coefficient maps, and contrast-enhanced sequences by manually delineating the index tumors. Deep features from the same set of imaging were extracted using a deep neural network based on pretrained EfficentNet-B0. Here, we present a clinical model , radiomics model, DL model (DLM-Deep feature), combined clinical\u2013radiomics model (CRM-Multi), and combined clinical\u2013DL model (CDLM-Deep feature) that were built using Cox models regularized with the least absolute shrinkage and selection operator. We compared their prognostic performances using stratified fivefold cross-validation. In a median follow-up of 61 months, 110/437 patients experienced BCR. CDLM-Deep feature achieved the best performance (hazard ratio [HR] = 7.72), followed by DLM-Deep feature (HR = 4.37) or RM-Multi (HR = 2.67). CRM-Multi performed moderately. Our results confirm the superior performance of our mpMRI-derived DL algorithm over conventional radiomics. Radical prostatectomy (RP), the first-line treatment for organ-confined or locally advanced prostate cancer (PCa), yields excellent long-term survival outcomes ,2,3. PosGenerally, multiparametric magnetic resonance imaging (mpMRI) integrates conventional anatomic sequences (T1- and T2-weighted imaging) with functional sequences, such as diffusion-weighted MRI (DWI), including the calculation of apparent diffusion coefficient (ADC) and dynamic contrast-enhanced (DCE) maps, and optionally, MR spectroscopy ,10,11,12The extraction of handcrafted radiomics features based on labeled PCa regions of interest (ROIs) on mpMRI allow objective qualitative and quantitative characterization of tumor phenotypes ,18,19,20Recently, deep learning (DL) models, which have yielded excellent results for the analysis of mpMRI frequently outperform radiomics studies ,28,29,30Accurate prediction of post-RP BCR requires long-term follow-up (>10 years) owing to the high variability of BCR events and PCa\u2019s slow-growing and often indolent nature ,38. MoreThis monocentric study was approved by the Institutional Review Board of Samsung Medical Center (SMC) (no. 2022-01-134), which waived the requirement for written informed consent owing to the retrospective study design. The study protocols were conducted in accordance with the tenets of the Declaration of Helsinki. The inclusion criteria were as follows: (1) primary prostate acinar adenocarcinoma confirmed by RP; (2) undergoing RP after 3 T prostate mpMRI; (3) mpMRI images of adequate quality available in the Picture Archiving Communication System ; (4) no history of neoadjuvant or adjuvant therapies for PCa; 5) available complete pathological and follow-up information. The exclusion criteria were as follows: (1) incomplete mpMRI or poor-quality MRI scans; (2) PSA persistence characterized by a postoperative PSA nadir >0.04 ng/mL 3 months post RP; (3) missing clinical variables; (4) tumors of other pathological types or mixed pathology. The primary endpoint was the prediction of post-RP BCR-free survival. Post-RP BCR was defined as an initial PSA value \u2265 0.2 ng/mL, confirmed by a subsequent PSA value of \u22650.2 ng/mL availabl. The timInitially, we retrospectively reviewed the baseline clinicopathological data of 512 patients with pathologically confirmed PCa who underwent RP, with or without lymphadenectomy, at the SMC between 2008 and 2009. RP was performed by five surgeons with varying surgical experience at our institution. The decision to perform lymphadenectomy was based on the preoperative lymph node involvement risk assessment and surgeon\u2019s discretion. Participants were followed up every 3 months during the first 2 years, every 6 months between the second and fifth years, and annually thereafter. Finally, a total of 437 patients were analyzed. The study flowchart is depicted in All the patients underwent prostate mpMRI using a 3 T MRI scanner equipped with a phased-array coil. Routine mpMRI protocols included T1WI, T2WI, DWI, ADC maps, and DCE sequences, acquired following intravenous injection of gadolinium diethylenetriamine penta-acetic acid ,48. TablFor each patient, tumor delineation was performed by manually placing the ROI on T2WI, ADC map, and DCE images, according to the histopathological results . Tumor RWe extracted radiomics features from the ROIs drawn on the T2WI, ADC, and DCE sequences of each patient using the open-source software, Python package \u201cPyradiomics\u201d (version 3.0.1) . SeventyIn this novel study, we developed a deep neural network for predicting post-RP BCR-free survival following the process outlined in Thereafter, we constructed a neural network to predict the binary BCR status, not BCR-free survival. The CNN architecture was characterized by connected nonlinear functions that learn multiple levels of representations of the input data, thereby yielding millions of possible features using a raw image ,31,32,51t-test and the chi-square or Fisher\u2019s exact test for the numerical and categorical variables, respectively, to compare the clinical characteristics of the patients with and without BCR. Quantitative variables are presented as the median (interquartile range [IQR]) or mean (standard deviation [SD]), whereas qualitative variables are presented as absolute values (percentages). We performed a univariate analysis to confirm the relevance of each clinical variable for BCR. Furthermore, Kaplan\u2013Meier (K\u2013M) curves were utilized to compare the ability of the risk models to assign a high and low risk of 10-year BCR-free survival using the median risk score as threshold. We used the hazard ratio (HR) with 95% confidence interval (CI) and log-rank tests to compare the two risk groups. The C-index measured the rank correlation between the risk score and BCR-free survival. The C-index value was higher if the time to BCR was shorter in patients at a higher risk than in patients at lower risk. Lastly, we calculated the integrated time-dependent area under the curve (iAUC), a time-wise AUC average for predicting events.We performed Student\u2019s p-values <0.05 were considered statistically significant. All statistical analyses were performed using Python .We split our data into two groups, the training and test sets, in a fivefold cross-validation manner. Thus, our data were split into five parts, where four parts were used for training, while the remaining part was used for testing. By repeating the procedure for the five different test sets and retaining the same ratio of BCR to non-BCR cases (1:3) across all test sets, potential overfitting caused by the relatively small cohort was reduced . We compp < 0.001) were observed between the BCR and non-BCR groups, which was consistent with previous research DCFPyL PET metrics could predict LNI and high-risk pathological tumor features in patients with intermediate- and high-risk primary PCa scheduled for robot-assisted RP with extended PLND [68Ga]Ga-PSMA-11 PET/MRI radiomics and ML equally compared to preoperative invasive biopsy [Prostate-specific membrane antigen (PSMA) is an oncogenic transmembrane glycoprotein overexpressed on PCa cells, and higher degrees of PSMA expression are associated with higher aggressive biology associated with PCa progression and recurrence ,84. Quanded PLND . Furtherded PLND ,93,96,97e biopsy . TherefoThis study had several limitations. Firstly, single-center mpMRI imaging data performed 14\u201315 years ago were retrospectively evaluated. Secondly, tumor ROI delineation was manually performed by one expert. Thirdly, the lack of generalizability of the model predictions prevented adequate external and prospective validation of our prediction models. Thus, future research should focus on multicenter, prospective multifaceted studies combined with multi-omics in large samples to assist the development of reproducible and interpretable DL risk models.Collectively, the effective integration of deep features from presurgical prostate mpMRI with clinicopathological parameters was the most powerful prognostic tool for long-term post-RP BCR-free survival. Thus, our novel DL risk model could facilitate prognostication based on routine prostate mpMRI, facilitating patient stratification following RP and individualized postoperative management."} +{"text": "The renin-angiotensin system (RAS) is a central modulator of cardiovascular physiology. Pathophysiology of hypertension is commonly accompanied by hyper-activation of RAS. Angiotensin II receptor blockers (ARBs) and Angiotensin-converting enzyme (ACE) inhibitors are the gold standard treatment for hypertension. Recently, several studies highlighted the crucial role of immune system in hypertension. Angiotensin-II-induced hypertension is associated with low grade inflammation characterized by innate and adaptive immune system dysfunction. Throughout the progression of hypertension, monocyte/macrophage cells appear to have a crucial role in vascular inflammation and interaction with the arterial wall. Since myelomonocytic cells potentially play a key role in angiotensin-II-induced hypertension and organ damage, pharmacological targeting of RAS components in monocyte/macrophages may possibly present an innovative strategy for treatment of hypertension and related pathology. More than 1 billion people worldwide are affected by hypertension . Hyperte+ Th1/Th17 and CD8+ Th17 inflammatory-like-phenotypes (Renin-angiotensin system (RAS) has two major axes. The classical axis ACE/Ang II/angiotensin II type 1 receptor (AT1R), and the counter-regulatory axes ACE-2/Angiotensin 1\u20137 (Ang 1\u20137) Mas receptor (MasR) and ACE2/Ang 1\u20139/AT2R . The Masenotypes . Yet, acenotypes and innaenotypes . Presencenotypes . Recentlenotypes . Throughenotypes . Furtherenotypes .ACE-2 plays a crucial role in cleaving Ang II to Ang 1\u20137. Ang 1-7 cleaved by ACE-2 from Ang II counteracts the deleterious effects of the activated RAS and play a protective role in control blood pressure-rise . During Circulating monocytes with different phenotypes indicate a clear status of the systemic immune functions which reflect the effects of infection and inflammatory responses on severity and possibly lethal complications . ActivatThe role of inflammation in vascular injury was extensively studied in atherosclerosis, including infiltration of leucocytes and monocyte/macrophage differentiation, as well as cytokine release and systemic and local innate immune responses . These iEndothelial dysfunction, oxidative stress and inflammation associated with hypertension induced by Ang-II or DOCA/salt, are prevented in models deficient in macrophage colony stimulating factor (M-CSF or CSF-1) inducing decrease of monocyte/macrophage cell profile . AdditioDue to the difficulty to study organ macrophage infiltration in hypertensive patients, most human studies focus on peripheral blood monocytes. Circulating monocytes are activated and display more adherence to endothelial cells during hypertension thus, ex++CD16+ intermediate) with increase in expression of IL-23, IL-1\u03b2 and TNF\u03b1. Interestingly, the activation of monocytes is stimulated by endothelial dysfunction probably due to Ang-II induced ROS generation and IL-6 release and also due to monocyte STAT activation monocytes regulates Ang-II-induced hypertension and vascular injury . DepletiC+Ly6G\u2212) and thatC+Ly6G\u2212) . ChemokiC+Ly6G\u2212) . AdditioC+Ly6G\u2212) . During C+Ly6G\u2212) which exHeart: Angiotensin II (AngII) signaling and effects are modulated mainly through the ATR1 and ATR2. The presence of ATR in different tissues and organs such as heart, kidney, aorta and brain explain end-organ injury during RAS activation. We previously demonstrated that 2 weeks of infusion with angiotensin II in mice induced hypertension and cardiac hypertrophy associated with fibrosis and massive monocyte/macrophage infiltration. Local infiltration of myeloid cells mediated partially Ang-II-induced cardiac cells proliferation such as myocytes , fibroblBrain: The synthesis of RAS components by nervous system cells point out the possible relevant role of this system and suggest that the interaction with glia and neuron may regulate blood pressure in brain diseases . It was Inflammation and innate immunity play a major role on angiotensin-II-induced hypertension and vascular damage. Activation of RAS components expressed in monocyte/macrophage modulates cell function and interaction with vascular endothelium. Hyper-activation of ACE/Ang II/AT1 axis or dysregulation of ACE-2/Angiotensin 1\u20137 (Ang 1\u20137) MasR axis potentially aggravates endothelial dysfunction, fibrosis, oxidative stress, monocyte/macrophage cell infiltration to perivascular tissue, in the kidney and heart, leading to blood pressure rise. Nevertheless, the signaling pathways by which angiotensin-II interacts with the monocyte/macrophage cells during hypertension and the specific role of ACE and ACE-2 remain unrevealed, hence more studies are necessary to clarify the mechanisms of Ang-II activating monocyte-macrophage cells during hypertension, and to highlight potential target treatments."} +{"text": "A novel type of efficient broadband pulse, called second-order phase dispersion by optimised rotation (SORDOR), has recently been introduced. In contrast to adiabatic excitation, SORDOR-90 pulses provide effective transverse 90 With the advent of point-to-point (PP) pulses, provide very efficient solutions universal rotation (UR) pulses can be used as full replacements of conventional, bandwidth-limited, hard pulses. UR pulses, however, are especially demanding regarding both rf amplitude and rf energy A multitude of composite and shaped pulses has been designed to cope with the bandwidth problem To reduce such demands, several concepts involving matching pulse shapes have been developed. Possibly the first such concept based on adiabatic pulses has been reported by B\u00f6hlen and Bodenhausen, requiring matched, linear-frequency-swept excitation and inversion pulses second-order phase dispersion by optimised rotation (SORDOR) pulses of Here we propose the recently introduced 2SORDOR pulses used here have been optimised as described in the reference B1 pulses . Essentially only unavoidable artefacts for spin systems with close chemical shifts and second-order contributions must be taken into account.It should be noted that the proposed approach considers that resonance frequencies during delays stay the same. Exchange effects and radiation damping will lead to offset changes and because of the offset-dependent rotation axes finally to distorted phases. Such distortions may be undesired or even helpful in the identification of e.g. exchange processes.Finally, we would like to make the remark that the relatively short SORDOR pulses used in this study are compensated for 5An implementation of the B\u00f6hlen and Bodenhausen concept using matched SORDOR-90 and SORDOR-180 pulse pairs is introduced. While SORDOR-180 pulses are equivalent to broadband inversion pulses with a defined, matched phase behaviour, corresponding SORDOR-90 pulses as introduced in SORDOR-180 pulses afford the same rf energy as time-optimal inversion pulses like BIP 10.5194/mr-3-53-2022-supplementThe supplement related to this article is available online at:\u00a0https://doi.org/10.5194/mr-3-53-2022-supplement."} +{"text": "Respiratory syncytial virus (RSV) is an important cause of respiratory illness and hospitalization in older adults and adults with certain underlying medical conditions. With novel RSV vaccines in development, it is critical to identify adults at increased risk of severe illness.Population-based surveillance was conducted through the RSV Hospitalization Surveillance Network (RSV-NET) over 8 seasons (2014\u20132022) across 75 counties in 12 states. We included non-pregnant adults (\u2265 18 years) residing in the RSV-NET catchment area who were hospitalized with laboratory-confirmed RSV infection (clinician-directed testing) during each season . Demographic and outcomes data and underlying medical conditions (October 2014\u2013April 2018) were abstracted from medical records. We calculated percentages of adults with intensive care unit (ICU) admission, mechanical ventilation (MV), and in-hospital death, stratified by demographic characteristics. We calculated age-adjusted percentages with these outcomes, stratified by underlying conditions.Table 1). ICU admission was recorded in 18.6%, MV in 7.2%, and in-hospital death in 4.2%. Most in-hospital deaths (54.1%) occurred among adults aged \u2265 75 years. Among adults 18\u201349 years, immune compromise (34.8%) and asthma (29.8%) were the most frequent underlying conditions . Adults with non-asthma chronic lung disease (CLD) and with cardiovascular diseases had the highest age-adjusted percentages of in-hospital death (Table 2). Adults with non-asthma CLD had the highest age-adjusted percentages of ICU admission and MV.We identified 13,080 RSV-associated hospitalizations among adults; 61.9% were aged \u2265 65 years (Among adults with RSV-associated hospitalization, older age and cardiopulmonary conditions were associated with severe illness. Hospitalized young adults more likely had immune compromise or asthma. This study was limited by clinician-driven testing, which likely under-detected RSV-associated hospitalizations. Older adults and adults with cardiopulmonary and immune compromising conditions may benefit from RSV vaccination when licensed products become available.All Authors: No reported disclosures"} +{"text": "Endoscopic retrograde cholangiopancreatography (ERCP) is technically challenging after Roux-en-Y gastric bypass (RYGB)Video\u20061\u2002Endoscopic ultrasound-directed transgastric ERCP (EDGE) used to successfully treat bile duct stones after Roux-en-Y gastric bypass.A 62-year-old man was admitted with fever and abdominal pain. Past medical history was relevant for RYGB and small bowel resection due to mesenteric ischemia. Abdominal computed tomography (CT) was consistent with choledocholithiasis and acute cholecystitis. For biliary drainage the patient underwent ERCP using a pediatric colonoscope, but selective biliary cannulation was not achieved with a forward-viewing instrument. Considering the altered anatomy, EDGE was proposed.Using a linear echoendoscope in the gastric pouch, EUS-guided puncture of the excluded stomach was accomplished with a 19G needle. Saline, methylene blue, and iodate contrast were injected allowing gastric fold visualization and lumen distension. A 20-mm lumen-apposing metal stent (LAMS) was successfully deployed creating a gastro-gastrostomy .After 7 days, anterograde progression to the papilla with a duodenoscope was posThe advantages of EDGE include its higher success rate and lower invasiveness, shortening hospitalization compared with endoscopy- and laparoscopy-assisted ERCPEndoscopy_UCTN_Code_TTT_1AS_2AD"} +{"text": "Speed is key during infectious disease outbreaks. Itis essential,for example, to identify critical host binding factors to pathogensas fast as possible. The complexity of host plasma membrane is oftena limiting factor hindering fast and accurate determination of hostbinding factors as well as high-throughput screening for neutralizingantimicrobial drug targets. Here, we describe a multiparametric andhigh-throughput platform tackling this bottleneck and enabling fastscreens for host binding factors as well as new antiviral drug targets.The sensitivity and robustness of our platform were validated by blockingSARS-CoV-2 particles with nanobodies and IgGs from human serum samples. Especiallyflow cytometry, enabling fast and high-throughput measurements ofcomplex mixtures, is widely used in clinics for immunophenotypingand would be an attractive and broadly available technique for suchpurposes.2 For example, previous work showedthat combining flow cytometry with Jurkat T-cells stably expressingSARS-CoV-2 Spike can be a sensitive tool to detect neutralizing antibodiesin human serum samples.3Emerging microbial pathogens,such as bacteria, fungi, and viruses, tremendously challenge humanhealth and cause significant economical and societal burden worldwide.Therefore, tools facilitating and improving pandemic preparednessare of uttermost importance to minimize these negative effects. Currentstate-of-the-art methods, such as enzyme-linked immunosorbent assay(ELISA), reverse transcription-polymerase chain reaction (RT-PCR),and RT loop-mediated isothermal amplification (RT-LAMP) usually relyon bulk measurements resulting in a single readout-value.4 but do not account for cells\u2019 three-dimensional nature. Three-dimensionalmodel systems such as large unilamellar vesicles (LUVs), giant unilamellarvesicles (GUVs), and cell-derived giant plasma membrane vesicles (GPMVs)help to recreate cellular curvature but are challenging to use inhigh-throughput flow cytometry because of their fragility and size-inhomogeneity.To complementexisting bulk measurement methods, we aimed to developa fast and high-throughput platform to study host\u2013pathogeninteractions. The system reconstitutes host cell proteins as wellas lipid bilayer, which is mostly neglected in current molecular interactionmethods but often hosts important attachment factors. The complexityof the mammalian plasma membrane consisting of thousands of differentlipids and proteins embedded between an outer glycocalyx and innercortical cytoskeleton is overwhelming. This complexity hampers ourefforts for the fast identification of important interaction partners.To overcome this bottleneck and reduce complexity, bottom-up modelmembrane systems are attractive alternatives which allow for precisecontrol over composition and properties. Among these, planar supportedlipid bilayer systems (SLBs) were widely used5 In this study, we show that fBSLBs carrying differenthost cell receptors, such as angiotensin-converting enzyme 2 (ACE2),can serve as a highly diverse platform to screen for molecules influencinghost\u2013pathogen interactions and the blocking efficiency of neutralizingantibodies present in human serum samples. Its fast implementation,easy adaptability of multiple parameters, and high-throughput capabilitypropel our method as an important platform to study host\u2013pathogeninteractions.For this reason, we coated cell-sized 5 \u03bcm silica beads witha lipid bilayer consisting of 98 mol % 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine(POPC) doped with 2 mol % of a nickelated anchoring lipid (18:1 DGS-NTA(Ni)).Next, we attached His-tagged host-cell proteins of interest (via NTA(Ni)-Hiscoupling) to membrane-coated beads to generate functionalized bead-supportedlipid bilayers (fBSLBs) serving as minimal synthetic host-cells 1A. In coFigure S1), which matched with previous data.7 We generatedfBSLBs ) carryingACE2 and studied their interaction with SARS-CoV-2 Spike expressingvirus-like particles using confocal microscopy . While there was strong interaction between ACE2-fBSLBsand Sp+VLPs, no interaction was observed with VLPs withno Spike (Sp\u2013VLPs) and Sp+VLPs pretreated with SARS-CoV-2neutralizing Spike nanobodies which were shown to be potent toolsto neutralize SARS-CoV-2 by blocking the interaction between Spikereceptor-binding domain (RBD) and its host receptor ACE2.10 Thus, fBSLBs can serve as powerful screening platform to identifyefficient inhibitors with therapeutic potential.Upon coating of 5 \u03bcm silica beads with POPC:DGS-NTA(Ni)98:2mol % of liposome solution, we first verified proper bilayer formationby measuring diffusion of a fluorescent lipid analogue using fluorescencecorrelation spectroscopy (FCS) S1, which+VLPs to ACE2-fBSLBs, we observed a strongincrease of fluorescence intensity per bead both in virus (green)and in membrane (red) channels .To increasethe number of data points and decrease acquisitiontime, we performed fast, quantitative, 3D lattice light-sheet microscopy(LLSM) and quantified viral loads per fBSLB 2A,B whicchannels 2C. Moreo+VLPs withreported host-cell binding partners neuropilin-1 (CD304),12 basigin (CD147),13 DPP4 (CD26),15 and TMPRSS217 using fBSLBs in combination withflow cytometry . As expected, Sp+VLPs showed strongest interaction with ACE2-fBSLBs also bound to CD304-fBSLBseffectively (and TMPRSS2-fBSLBs slightly) while they did not bindthe other proteins we tested or without any viral protein . These results highlight the need for additional high-affinityhost-cell binding factors for efficient virus\u2013host interaction.fBSLBs allow tight control on the composition ofnot only surfaceproteins but also lipids. We made use of this and screened for reportedlipid co-receptors for Spike, such as GM1 gangliosides.20 Moreover, it is very importantto understand whether antiviral IgGs in prevalent serum samples stillprotect from upcoming new variants to decide for vaccine-adjustmentsand therapeutic treatment options. To show the potential of our methodto answer these questions, we first determined the amount of Spike-IgGsin three human serum samples using a bead-based assay in combinationwith flow cytometry and pathogens makingit a valuable tool for future pandemic preparedness."} +{"text": "A switch from tenofovir disoproxil fumarate (TDF) to abacavir (ABC)-based therapy is one of treatment options for HIV-infected patients with decreased glomerular filtration rate (eGFR) after receiving a TDF-based regimen. However, ABC may not be available in some hospitals in Thailand. TDF dose reduction showed a significant improvement of eGFR without virological failure. This study aimed to compare the efficacy and safety of TDF dose reduction versus ABC switching for the treatment of patients with decreased eGFR after receiving TDF.This open-label, randomized, non-inferiority study was conducted at Siriraj Hospital, Thailand, during March 2019 to May 2021. Eligible participants were HIV-suppressed patients aged \u226418 years with decreased eGFR (eGFR 30-60 mL/min by Cockcroft-Gault equation) after receiving TDF-based therapy. Patients were randomized (1:1 by block of four) to a TDF dose-adjusted regimen or an ABC-based regimen. The primary outcome of study was the change in eGFR at 24 weeks after regimen modification. The non-inferiority margin was -12 mL/min. Other outcomes were the virological efficacy and safety at 24 weeks after regimen modification.30 patients were enrolled (15 patients in the TDF dose-adjusted regimen group and 15 patients in the ABC-based regimen group). The distribution of age, sex, CD4 count, serum creatinine, eGFR and duration of TDF treatment were similar between both groups. The mean change of eGFR (SD) at 24 weeks after regimen modification were +1.87 (7.90) mL/min in the TDF dose-adjusted regimen group and +15.64 (6.53) mL/min in the ABC-based regimen group (P< 0.001). The difference of mean change of eGFR between both groups at 24 weeks was -13.77 mL/min (P< 0.001). The sustained viral suppression was observed in both groups. Two patients in the TDF dose-adjusted regimen group developed progressive kidney impairment and one of them developed Fanconi syndrome.Baseline CharacteristicsOutcomesThe TDF dose-adjusted regimen demonstrated the efficacy in improving eGFR but this regimen did not prove non-inferiority over the ABC-based regimen. The progression of kidney dysfunction should be cautioned in patients who received the adjusted renal dosing of TDF.Eakkawit Yamasmith, Police General Hospital: Advisor/Consultant"} +{"text": "People with burn injury often experience long-term social participation challenges. A previous study explored demographic and injury variables predicting social participation outcomes after burn injury. This study aims to further examine post-traumatic growth, and physical and psychological symptoms as predictors of social participation.This is a multi-center Burn Model System (BMS) study with a prospective cohort design. Adult BMS Database participants from July 2018 to April 2022 were included. Predictors were measured at 12- and 24-month after injury including Post-Traumatic Growth Inventory Short Form (PTGI-SF), Post-Traumatic Stress Disorder Checklist Civilian Version (PCL-C), Patient-Reported Outcomes Measurement Information System (PROMIS-29) Depression, Anxiety, Fatigue, Sleep Disturbance, Pain Interference and Heat Intolerance. Social participation outcomes were measured with Life Impact Burn Recovery Evaluation (LIBRE) Social Interactions and Social Activities at 24-month after injury. Multivariable regression analyses were conducted to assess the association between predictors and outcomes.A total of 158 burn survivors were included with an average age of 47 years and a mean burn size of 19.5% total body surface area. Significant predictors included PCL-C and PROMIS-29 Depression for LIBRE Social Interactions, and Heat Intolerance, PROMIS-29 Pain Interference and Depression, and PCL-C for LIBRE Social Activities. See Table 1 for details of regression results.Overall, self-reported post-traumatic stress and depression are associated with limited social interactions while heat intolerance, pain interference, depression, and post-traumatic stress are related to fewer social activities.This study improves our understanding of the multiple and modifiable factors which influence social participation after burn injury."} +{"text": "Maternal antibodies against SARS-CoV-2 actively transfer across placenta providing passive immunity to neonates. However, levels of cord blood anti-SARS-CoV2 antibodies and protective effects are still unclear. The objectives of the study was to investigate level of cord blood SARs-CoV-2 antibody of infants born to the mothers who received at least 1 dose of COVID-19 vaccine during pregnancy or within 3 months before pregnancy, as well as having SARS-CoV-2 infection during pregnancy.Prospective observational study was conducted in Bangkok between March 2022 to March 2023. Cord blood samples were tested for anti-receptor binding protein IgG (anti-RBD IgG). Medical records were review for history of SARS-CoV-2 infections in the mothers.A total of 154 neonates were enrolled. The highest cord blood geometric mean concentration (GMC) of anti-RBD IgG was observed in infants who was born from the mother who received COVID-19 vaccine and were diagnosed with COVID-19 during pregnancy 4215.9-14007.8] AU/ml), while the lowest GMC was found in the infants born from mothers who received COVID-19 vaccination before pregnancy and did not have COVID-19 during pregnancy .Figure of Anti-RBD IgG comparison at birthVaccinating and having SARs-CoV-2 infection during pregnancy induced several folds higher antibody level in cord blood that could effectively provide protection in infants.All Authors: No reported disclosures"} +{"text": "Correction to: International Orthopaedics10.1007/s00264-023-05899-3Edoardo VigliettaLeonardo PreviVeronica GiulianiGiulia RescignoYuri GugliottaAndrea RedlerRaffaele IorioAll the author names are inverted in the original published paper, the correct names are given below:The original article has been corrected."} +{"text": "Patients with hematologic malignancies remain at higher risk for developing severe SARS-CoV-2 infections and have reduced immune responses to vaccines.A prospective observational study was conducted at Brigham and Women\u2019s Hospital and Dana-Farber Cancer Institute from January 2021 to May 2023. Adult participants were included if they had a history of lymphoid malignancy (LM) or had received hematopoietic stem cell transplantation (HSCT). SARS-CoV-2 antibodies were measured before vaccination and every 2-3 months thereafter. COVID-19 vaccination history and SARS-CoV-2 infections (by report or positive PCR) were recorded longitudinally. Our primary outcome for this study was to evaluate the incidence of breakthrough SARS-CoV-2 infections after receiving a bivalent COVID-19 vaccine booster (Omicron/wildtype).A total of 144 participants were included in the analysis. Of those, 98 (68%) received a bivalent COVID-19 booster. The median age was 68 (IQR 61 \u2013 73) and 51 (52%) were male. The majority of participants 61 (62%) had LM and 37 (38%) underwent HSCT with a median time from transplant to first vaccine of 317 days (IQR 171 \u2013 742). Overall, the incidence of breakthrough SARS-CoV-2 infections was 13 (13%) with a median time from vaccination to breakthrough infection of 72 days (IQR 54 \u2013 112). There were no SARS-CoV-2 related mortality or severe disease leading to hospitalization in our cohort.FigureOur analysis demonstrates a low incidence of breakthrough SARS-CoV-2 infections and no severe disease among those who received a bivalent mRNA vaccine, highlighting the potential protection of the bivalent booster against SARS-CoV-2 in patients with hematologic malignancies who had previously been vaccinated or boosted. Further studies are warranted to determine the optimal number and type of booster vaccinations needed to protect this vulnerable population.Nicolas C. Issa, MD, AiCuris: Grant/Research Support|Astellas: Grant/Research Support|Boehringer Ingelheim: Advisor/Consultant|Fujifilm: Grant/Research Support|GSK: Grant/Research Support|Merck: Grant/Research Support|Moderna: Grant/Research Support"} +{"text": "HLA-B*13:01 (Odds ratio (OR) = 45), dapsone-DRESS and HLA-B*13:01 (OR = 122.1), vancomycin-DRESS and HLA-A*32:01 (OR = 403), clindamycin-DHRs and HLA-B*15:27 (OR = 55.6), and strontium ranelate (SR)-SJS/TEN and HLA-A*33:03 (OR = 25.97) are listed. We summarized the immune mechanism of SCARs, update the latest knowledge of pharmacogenomics of antibiotics- and AOD-induced SCARs, and indicate the potential clinical use of these genetic markers for SCARs prevention in this mini review article.Drug-induced delayed hypersensitivity reactions (DHRs) is still a clinical and healthcare burden in every country. Increasing reports of DHRs have caught our attention to explore the genetic relationship, especially life-threatening severe cutaneous adverse drug reactions (SCARs), including acute generalized exanthematous pustulosis (AGEP), drug reactions with eosinophilia and systemic symptoms (DRESS), Stevens\u2013Johnson syndrome (SJS), and toxic epidermal necrolysis (TEN). In recent years, many studies have investigated the immune mechanism and genetic markers of DHRs. Besides, several studies have stated the associations between antibiotics-as well as anti-osteoporotic drugs (AOD)-induced SCARs and specific human leukocyte antigens (HLA) alleles. Strong associations between drugs and HLA alleles such as co-trimoxazole-induced DRESS and Adverse drug reactions (ADRs) are one of the general causes of death worldwide . In AmerAGEP, DRESS, and SJS/TEN are three important phenotypes of SCARs that we will review in this article . Over 90The pathogenesis of SCARs is strongly associated with specific human leukocyte antigens (HLA), T cell receptor (TCR), drug or its metabolites and further T cell-mediated immune response . In humaAGEP has been characterized as T cell-mediated neutrophilic inflammatory reaction . After eSJS and TEN are rare but life-threating dermatologic diseases and represented as T cell-mediated keratinocyte death . TEN is DRESS is a rare but severe cutaneous and systemic drug-delayed hypersensitivity reaction, mediated by T cell activation . Drugs, Several antibiotics- and strontium ranelate (SR)-induced SCARs have been proposed in recent 2\u00a0decades , and we Co-trimoxazole is an antibiotics indicated for urinary tract infection and Pneumocystis jiroveci pneumonia . The prePenicillin and its derivatives are antimicrobial agents frequently used to treat a variety of bacterial infection in the world . Zhou etPseudomonas aeruginosa , the frequency of HLA-B*13:01 is 8%\u201315% in Taiwan and China. Co-trimoxazole-induced DRESS can be avoided after HLA-B*13:01 testing in Asian population. Although the frequency of HLA-A*11:01 allele is lower in Japanese population compared to Chinese and Thai population , HLA-A*1With the increasing number of published studies regarding genetic polymorphisms associated with drug delayed hypersensitivity reaction, HLA alleles of SCARs could be drug-specific, ethnicity-specific and phenotypic-specific . Conside"} +{"text": "JC polyomavirus (JCPyV) infects oligodendrocytes and causes progressive multifocal leukoencephalopathy (PML), a potentially fatal complication of monoclonal antibody treatments for cancer and autoimmune diseases, and a rare but often-fatal disease in people living with HIV. There is currently no effective treatment against JCPyV and novel immunotherapies for PML are urgently needed. Natural killer (NK) cells play critical roles in defense against viral infections, yet NK cell responses to JCPyV remain largely unexplored.Intracellular cytokine staining (ICS) of NK and T cells stimulated with overlapping peptide pools covering the JCPyV capsid VP1 was performed. A novel flow cytometry-based assay was created to determine NK cell killing efficiency of infected SVG-A cells. Blocking antibodies were used to determine NK cell receptors responsible for immune recognition of JCPyV-infected cells. Single cell cloning was used to expand a homogenous population of NK cells.Using ICS, about 40% of healthy donors demonstrated NK cell responses, with CD107a upregulation and robust IFNy production by NK cells extending beyond T cell responses. Next, we showed that co-culture of NK cells and JCPyV-infected SVG-A cells leads to, on average, a 65% reduction of infected cells . Expression of ligands for the activating NK cell receptor NKG2D was modulated in JCPyV-infected cells, with overall enhanced expression of ULBP-2. Accordingly, NKG2D blockade resulted in decreased NK cell degranulation. Interestingly, we also identified JCPyV-derived peptides that elicit dominant responses by NK cells and can stabilize HLA-E, the ligand for the inhibitory NKG2A and the activating NKG2C receptors. Blockade of NKG2A enhanced NK cell response to JCPyV-infected targets. Finally, we have isolated and expanded single NK cell clones with potent cytotoxic functions against autologous target cells pulsed with JCPyV-derived peptides and JCPyV-infected SVG-A cells.Altogether, these findings suggest NKG2D-mediated activation and the NKG2/HLA-E axis may play key roles in controlling JCPyV replication and targeting these receptors and/or expansion of JCPyV-specific NK cells may be promising NK cell-based immunotherapeutic for PML.All Authors: No reported disclosures"} +{"text": "DSM-5\u2019s framing of Obsessive-Compulsive and Related Disorders (OCRDs) paved the way for the increasingly structured definition of obsessive-compulsive spectrum disorders. The spectrum would include, among others, body dysmorphia, hair-pulling, skin-picking, obsessional jealousy, and olfactory reference syndrome (ORS). ORS \u2013 i.e., persistent concern about emitting a foul or offensive body odor \u2013 causes clinically significant distress or impairment in several areas of functioning.This study aimed to investigate the relationship between obsessive traits and self-odor concern in a clinical sample that did not meet the diagnostic criteria for either OCRDs or ORS.In a sample of 220 adults referring to an outpatient Mental Health Service in Bologna, Northern Italy, we measured (1) self-odor concern through two specific items \u2013 sweat hatred (SH) and body odor hatred (BOH) \u2013 on the Body Uneasiness Test (BUT) and (2) obsessive traits through the total score of the Obsessive-Compulsive Inventory-Revised (OCI-R). Therefore, we performed correlation and regression analysis to examine the relationship between obsessive-compulsive traits and self-odor concern.2 = 0.109, p < 0.001] and BOH score , highlighting that obsessive-compulsive traits predict both sweat and body odor hatred.We found a positive correlation between OCI-R and SH scores (r = 0.330) and OCI-R and BOH scores (r = 0.188). Linear regression analysis demonstrated that OCI-R score significantly predicted SH score [F = 26.455, RThese results demonstrate that obsessive traits and self-odor concern are strictly connected. This knowledge may allow us, even in the absence of an overt diagnosis of OCRDs or ORS, to better identify an at-risk population before it suffers impairment in functioning. Overall, further research is needed to help characterize obsessive-compulsive spectrum disorders before symptom exacerbation.None Declared"} +{"text": "Kras proto-oncogene can remain phenotypically normal until an inflammatory event, which drives cellular plasticity and tissue remodeling. The inflammation-driven molecular, cellular, and tissue changes that precede and direct tumor formation remain poorly understood.Virtually all cancers begin with genetic alteration in healthy cells, yet mounting evidence suggests that non-genetic events such as environmental signaling play a crucial role in unleashing tumorigenesis. In the pancreas, epithelial cells harboring an activating mutation in the Understanding tumorigenesis requires a high-resolution view of events spanning cancer progression. We leveraged genetically engineered mouse models (GEMMs), single-cell genomic (RNA-seq and ATAC-seq) and imaging technologies to measure pancreatic epithelial cell-states across physiological, premalignant, and malignant stages. To analyze this rich and complex dataset, we developed computational approaches to characterize epigenetic plasticity and to infer cell-cell communication impacts on tissue remodeling.Kras-mutant cells are capable of acquiring multiple highly reproducible cell-states that are undetectable in normal or regenerating pancreata. Several such states align with experimentally validated cells-of-origin of neoplastic lesions, some of which display a high degree of plasticity upon inflammatory insult. These diverse Kras-mutant cell populations are defined by distinct chromatin accessibility patterns and undergo inflammation-driven cell fate transitions that precede pre-neoplastic and premalignant lesion formation. Furthermore, a subset of early Kras-mutant cell-states exhibit marked similarity to either the benign or malignant fates that emerge weeks to months later; for instance, Kras-mutant Nestin-positive progenitor-like cells display accessible chromatin near genes active in malignant tumors.Our data revealed that early in tumorigenesis, We defined and quantified epigenetic plasticity as the diversity in transcriptional phenotypes that is enabled or restricted by a given epigenetic accessibility landscape. Intriguingly, these plastic cell-states are enriched for open chromatin near cell-cell communication genes encoding ligands and cell-surface receptors, suggesting an increased propensity to communicate with the microenvironment. Given the rapid remodeling of both the epithelial and immune compartments during inflammation, we hypothesize that this epigenetically enabled communication is a major driver of tumorigenesis. We found that the premalignant epithelium displays extraordinary modularity with respect to communication gene co-expression patterns; distinct cell subpopulations each express a unique set of receptors and ligands that define the nature of incoming and outgoing signals that they can receive and send.Il33 expression during Kras-initiated neoplasia, we functionally demonstrated that the loop initiated by epithelial IL-33 directs exit from a highly plastic inflammation-induced epithelial state, enabling progression towards typical neoplasia.Through the development of Calligraphy, an algorithm that utilizes this receptor-ligand modularity to robustly infer the cell-cell communication underlying tissue remodeling, we showed that the enhanced signaling repertoire of early neoplastic tissue specifically endows plastic epithelial populations with greater capability for crosstalk, including numerous communication routes with immune populations. As one example, we identified a feedback loop between inflammation-driven epithelial and immune cell-states involving IL-33, previously implicated in pancreatic tumorigenesis. Using a new GEMM that enables spatiotemporally controlled suppression of epithelial Kras-mutant subpopulations markedly increase epigenetic plasticity upon inflammation, reshaping their communication potential with immune cells, and establishing aberrant cell-cell communication loops that drive their progression towards neoplastic lesions.Multimodal single-cell profiling of tumorigenesis in mouse models identified the cellular and tissue determinants of pancreatic cancer initiation, and a rigorous quantification of plasticity enabled the discovery of plasticity-associated gene programs. We found that The response to tumor-initiating inflammatory and genetic insults can vary amongst morphologically indistinguishable cells, suggesting yet uncharacterized roles for epigenetic plasticity during early neoplasia. To investigate the origins and impact of such plasticity, we performed single-cell analyses on normal, inflamed, pre-malignant, and malignant tissues in autochthonous models of pancreatic cancer. We reproducibly identified heterogeneous cell-states that are primed for diverse late-emerging neoplastic fates and linked these to chromatin remodeling at cell-cell communication loci. Using an inference approach, we revealed signaling gene modules and tissue-level crosstalk, including a neoplasia-driving feedback loop between discrete epithelial and immune cell populations that was validated in mice. Our results uncover a neoplasia-specific tissue remodeling program that may be exploited for pancreas cancer interception. Single-cell analysis reveals that enhanced epigenetic plasticity drives pro-neoplastic crosstalk in early pancreatic tumors. The initial events by which tissues diverge from normalcy to form benign neoplasms and malignant tumors remain poorly understood. It is well established that this process is driven by genetic mutations ; howeverDevelopmental, regenerative, and pathologic plasticity is largely determined at the chromatin level as increases or decreases in the repertoire of transcriptional programs that can be accessed by a given cell , 14. CelKRAS. However, KRAS-mutant epithelia can remain phenotypically normal and depend on inflammatory stimuli (pancreatitis) to transform into pre-neoplastic and neoplastic lesions is typically diagnosed too late for curative treatment and arises from cooperativity between genetic and epigenetic reprogramming events . Unlike lesions \u201322. We that accurately recapitulate many aspects of the human disease. Beyond providing a comprehensive charting of epithelial dynamics from normal metaplasia through malignant tissue states, our approach allowed us to expose, quantify, and perturb early plasticity traits endowed by oncogene-environment interaction, and define molecular, cellular, and tissue-level principles of pre-malignant tumor evolution.Ptf1a-Cre-dependent mKate2 fluorescent reporter to enrich pancreatic epithelial cells (Kras (K1\u2192K6) (Kras-mutant metaplasia is accelerated by an inflammatory insult (pancreatitis) , proceeds to benign pancreatic intraepithelial neoplasia, , and ultimately, malignant PDAC (K5) and distal metastases atlas of healthy, regenerating, benign neoplastic, and malignant epithelia using GEMMs that faithfully model cancer from initiation to metastasis. Our GEMMs incorporate a al cells , 26, 27, (K1\u2192K6) . In this (K1\u2192K6) , Kras-muases K6; and S1A.+) cells, we captured both abundant and rare constituents of normal, regenerating, and Kras-mutant epithelia, such as progenitor-like tuft , EMT-like (Zeb1+), neuroendocrine (Syp+Chga+), and other previously reported subpopulations and reactivation of developmental (Clu) or oncogenic programs ,D. Howevb1, Vim; , 36) , which emphasizes cell-state transitions along axes toward malignancy. As expected, we found that the Kras-mutant pancreatic epithelium undergoes progressive gene expression changes that activate metaplastic , neopla, Tff1+; ) and ult program identified multiple states that potentially act as distinct origins for the observed heterogeneity and three de-differentiated populations. Most of these states align with independent genetic lineage tracing studies that demonstrate their \u2018cell-of-origin\u2019 potential individually (Kras-mutant tissue (K2), which revealed clear transitional states (Anxa10+Nes+Msn+) in lesions containing both apex cells (Nes+Msn+) and the gastric-like cells (Anxa10+) predominant in neoplastic tissue weeks later and tumorigenesis-associated (S100a6) programs within 24 hpi (ADM-PDAC \u201cBridge\u201d) (Nes+ progenitor-like cells shift into a state showing reduced activation of tumor suppressive programs (Cdkn2a). Our findings thus suggest that oncogenic Kras enables the emergence of diverse high-potential states . For instance, during pancreatitis, well-differentiated acinar cells generate a metaplastic population with transcriptional features that are intermediate for acinar (Bridge\u201d) , and Neseata see and S2),eata see .Kras-mutant apex states and their injury-driven progeny arises through a diversification of permissive chromatin states. To determine how chromatin dynamics correspond to changes observed in our longitudinal scRNA-seq atlas, we first analyzed bulk ATAC (assay for transposase-accessible chromatin) sequencing data matching the above tissue stages (Kras-mutant epithelia (K1-K2) reproducibly shift toward states acquired in early neoplasia and sustained in advanced disease (K3-K6). Nevertheless, we observed that the chromatin landscapes of benign neoplastic lesions (K3-K4) and malignant tumors (K5-K6) are highly divergent. Consistent with this, large sets of regulatory elements (\u201cchromatin modules\u201d) exhibit mutually exclusive accessibility patterns across benign and malignant stages, with one set of ATAC-seq peaks showing increased accessibility in benign lesions but not in malignant counterparts (Benign Neoplasia chromatin module) and another set behaving opposite and genes proximal to Malignant module loci increase in malignant disease (K5) . Chromat (N1-N2) and S4A.Kras-mutant cells may eventually acquire as \u2018cell-fates\u2019\u2014those associated with benign neoplasia (K3-K4) or malignancy (K5-K6). We postulated that pre-neoplastic Kras-mutant cells expressing programs associated with the chromatin landscape of a single distinct fate may be epigenetically primed toward that fate, conferring greater propensity to acquire its phenotype over time or in response to certain exogenous triggers. We further reasoned that similarities between the transcriptomes of Kras-mutant cells from pre-neoplastic and later neoplastic stages (K3\u2013K6) would indicate such fate potential. We therefore developed a classification-based approach that first identifies gene expression patterns that accurately discriminate between cell populations in benign lesions or cancer, and then uses these patterns to assign cell-fate probabilities to pre-neoplastic cells based on the activation of fate-associated genes. Specifically, we trained a logistic-regression classifier to distinguish between benign neoplasia and malignancy , and used it to classify pre-neoplastic cells (The early establishment of a permissive chromatin landscape (K1-K2) that is later specified into a restricted, distinct set of accessible regulatory elements (K3-K6) is reminiscent of cell-fate determination occurring in developmental systems . We thus2) cells . This cl2) cells .Kras-mutant cells that are strongly skewed toward one or the other fate (Kras-mutant cells that are not well classified (Ptf1a+ acinar and Nes+ progenitor) captured independently by CellRank but impacted by inflammatory signals that epigenetically prime them towards diverse fates that can be predicted early in disease progression.Collectively, our results imply that tumorigenesis can proceed from multiple well-differentiated or progenitor-like states, and that their neoplastic progression is not dictated solely by cell intrinsic determinants profiles of pre-neoplastic (K1), pre-neoplastic inflamed (K2), benign neoplastic (K3), and adenocarcinoma (K5) epithelia. Consistent with an epigenetic basis for the observed pre-malignant diversity see , we founch stage and S5A.ch stage and S5B.ch stage , with Kr modules . These dNr5a2+ acinar, Neurod1+ neuroendocrine, Pou2f3+ tuft, and Nes+ progenitor cells . . 27). Thed cells , 26. In ulations and S5B.ulations suggestssion see .Nes+ progenitors, Tff2+ gastric cells) and experimentally validated cells-of-origin from lineage tracing studies by a given chromatin accessibility landscape. To first determine these potential transcriptional phenotypes, we used a simple classifier to identify gene expression patterns that discriminate cell-states. Assuming that proximal open chromatin conveys the potential for a gene\u2019s activation, we then applied the classifier to predict cell-states based on accessibility proximal to genes, rather than gene expression . We reast cells; , 42\u201344) t cells; . Some oft cells; , S2A,B, Csf2, Cxcl1, and Cxcr2 (Kras mutation (K2 vs. K1) to identnd Cxcr2 ) (Table nd Cxcr2 . Accordi vs. K1) , suggest vs. K1) . TogetheKras-mutant pancreatic epithelium. We hypothesized that chromatin remodeling of receptor and ligand gene loci contributes to plasticity in pre-neoplasia by enabling cells to respond to inductive signals from the environment.The dominance of the association between plasticity and cell-cell communication drove us to investigate how heterotypic interactions may result from plasticity or enhance it in the The delineation of communication events requires an assessment of the communication propensities of each cell-state (defined by its expressed receptors and ligands), which may then be used to link interacting cell-states based on prior knowledge of receptor-ligand binding partners. As a first step, we characterized communication gene accessibility and expression across cell-states of the pre-malignant epithelium using the scATAC-seq and scRNAseq datasets generated above. Each plastic cell-state reveals substantial variability in chromatin accessibility near communication genes, consistent with distinct molecular repertoires for potential communication . To idenWe next sought to infer actual cell-cell signaling interactions that may occur between cells expressing different communication modules. Although several methods have been developed to predict cellular interactions from single-cell data , their iin situ in smFISH data cell-state, likely reflecting the expected trans/dedifferentiation of acinar cells under inflammatory conditions showed much less diversity in communication module expression compared to their pression . Among cnditions . These cignature , implyinKras in adult acinar cells , with most cells expressing at least one of the Gastric (E6), Progenitor (E7), or Bridge (E3) modules . These mar cells from Kras-mutant tissues, before and after induction of pancreatitis (K1\u2013K3), identified all expected immune subtypes, including both abundant (macrophage) and rare types types 27). As. AsKras-C) types .Il1rl1/Il1rap), a ligand that accelerates the formation of mucinous PanIN lesions relative to Kras-wild type (N2) epithelial cells . To. To27). al cells . This fial cells predictsWithin Calligraphy\u2019s context-specific network were apparent \u2018master communication hubs\u2019 that participate in numerous interactions. We calculated a receiving score (ability to sense the environment via expressed receptors) and a transmission score (ability to remodel the environment via expressed ligands) based on the number of Calligraphy\u2019s statistically significant incoming and outgoing edges for each module . The twoAnxa10+Il18+Spp1+) and module E7-expressing progenitor cells (Nes+Il18rl+Cd44+) and S7B +Cd44+) .One of our most striking observations is the dramatic remodeling of epithelial and immune compartments within 24 to 48 hpi see and S10AIl33 is expressed during pancreatitis by a small subset (4%) of TFF1/ANXA10+Kras-mutant Gastric module-expressing epithelial cells and is predicted to initiate signaling to Tregs and ILCs (Module T8) by binding with its cognate receptor Il1rl1 and co-receptor Il1rap (+ mKate2+) and rare Tregs (Foxp3+) are in close spatial proximity in Kras-mutant pancreata under injury conditions (Il1rl1 (Module T8) also express the Th2 cytokine gene Il4 , consistent with the known role of IL-33 in triggering Th2-type immune responses (Il4) back to the Gastric Module (E6) via the IL-4 receptor (Il4ra), thereby closing the loop and potentially propagating signals to other modules in both immune and epithelial compartments (r Il1rap \u2013D. Suppont mice) . Subsequesponses . Module artments .Kras-mutant epithelial cell-states, including gastric, tuft cell and Nes+ progenitor populations (Il33) and receiving (Il4ra) factors, and do so at low levels, even during injury-induced regeneration -inducible GFP-coupled short hairpin RNA (shRNA) capable of suppressing Il33 (KC-shIl33), allowing potent Il33 suppression in the epithelial compartment following dox administration (Il33+Vim+ or Il33+aSMA+) \u2013D in shId ANXA10 ,B.Il33 on-dox mice show that IL-33 perturbation profoundly shifted the observed cell-states within both epithelial and immune compartments. We applied the Milo algorithm and other genes associated with a plastic state ,G. AlthoIl33 perturbation on Progenitor and Bridge modules, both of which are predicted to be downstream of IL-33 and express IL-4 receptor (Il4ra) see ,E; where X 10\u22127) . These rKras-mutant cells diversify the communication programs available to pre-neoplastic tissue, expanding downstream crosstalk throughout the tumor microenvironment. Moreover, in the Kras-mutant context, epigenetic reprogramming and the emergence of cancer-driving populations is remarkably dynamic, occurring within two days of insult by inflammation.While much is known about the molecular processes affecting tumor progression to advanced PDAC, pancreatic cancer is diagnosed late, and the paucity in molecular studies of early neoplasia has left us with little knowledge of how it emerges from a relatively homogeneous epithelium. By combining single-cell sequencing of mouse models with computational analysis, we found that permissive chromatin states in Mutation is known to drive plasticity in lung cancer via the loss of AT1 or AT2 lineage identity and acquisition of a phenotype intermediate between these states , 18. In Kras in all acinar cells, allowing us to comprehensively explore which states can initiate tumorigenesis. Using CellRank , which itself maps to a CellRank-predicted apex state. While non-acinar lineage cells can also undergo neoplastic transformation in mice . These findings link previous results on the relevance of Th2 signaling in PDAC tumorigenesis to thoseenotypes , 59. FurPDAC is frequently detected too late for curative intervention, a detailed understanding of early neoplastic events may enable the development of rational strategies to prevent, detect, and intercept tumors before they progress to an intractable stage. Our results show that GEMMs can be used to study and perturb early events, revealing epigenetically plastic cell-states in neoplasia that are not observed in the normal or regenerative pancreas. Further efforts to understand neoplasia-specific communication networks driving PDAC initiation hold promise for the development of therapeutics that block early cancer progression, and may also be effective against advanced disease.Kras-mutant cells. Samples from GEMMs were collected to span the entire range of PDAC progression (K1-K6) as well as regenerating pancreata (N1-N2). Additional samples were collected from GEMMs enabling selective genetic perturbation of pre-malignant Tissue dissociation and cell preparations for bulk and single-cell ATAC-seq were performed as previously described . For scRTissues were processed and stained for imaging or FACS analyses . Tables scRNA-seq data were processed with SEQC , filtereTableS1TableS3TableS4TableS5TableS6TableS10TableS14TableS18TableS7Supp Text"} +{"text": "Clin Sci (Lond) (2023) 137 (9): 727-753, would like to correct their paper. The monoclonal antibody depemokimab described as anti-IL-5R, have been corrected to anti-IL5 in the following statement and in the revised The authors of the original article, NCT04719832).Furthermore, benralizumab showed efficacy in patients with severe CRSwNP by decreasing nasal blockage score in a randomized placebo controlled study [229] Depemokimab, a long-acting anti-IL-5 monoclonal antibody, is currently being evaluated in phase III clinical trials in severe uncontrolled asthma with an eosinophilic phenotype ("} +{"text": "The three-dimensional chemical exchange saturation transfer (3D CEST) technique is a novel and promising magnetic resonance sequence; however, its application in nasopharyngeal carcinoma (NPC) lacks sufficient evaluation. This study aimed to assess the feasibility of the 3D CEST technique in predicting the short-term treatment outcomes for chemoradiotherapy (CRT) in NPC patients.Forty NPC patients and fourteen healthy volunteers were enrolled and underwent the pre-treatment 3D CEST magnetic resonance imaging and diffusion-weighted imaging (DWI). The reliability of 3D CEST was assessed in healthy volunteers by calculating the intra- and inter-observer correlation coefficient (ICC) for amide proton transfer weighted-signal intensity (APTw-SI) and magnetization transfer ratio (MTR) values. NPC patients were divided into residual and non-residual groups based on short-term treatment outcomes after CRT. Whole-tumor regions of interest (ROIs) were manually drawn to measure APTw-SI, MTR and apparent diffusion coefficient (ADC) values. Multivariate analysis and the receiver operating characteristic curve (ROC) were used to evaluate the prediction performance of clinical characteristics, APTw-SI, MTR, ADC values, and combined models in predicting short-term treatment outcomes in NPC patients.For the healthy volunteer group, all APTw-SI and MTR values exhibited good to excellent intra- and inter-observer agreements . For NPC patients, MTR values showed a significant difference between the non-residual and residual groups while no significant differences were observed for APTw-SI and ADC values (P\u2009>\u20090.05). Moreover, the diagnostic power of MTR value was superior to APTw-SI and comparable to ADC values in predicting short-term treatment outcomes for NPC patients. The prediction performance did not improve even when combining MTR values with APTw-SI and/or ADC values (P\u2009>\u20090.05).The pre-treatment MTR value acquired through 3D CEST demonstrated superior predictive performance for short-term treatment outcomes compared to APTw-SI and ADC values in NPC patients after CRT.The online version contains supplementary material available at 10.1186/s40644-023-00602-6. Nasopharyngeal carcinoma (NPC) patients have seen improved survival rate with the widespread application of intensity-modulated radiotherapy and optimized of chemotherapy strategies . Despitee) from pre-treatment DCE-MRI and total lesion glycolysis (TLG) obtained by 18\u00a0F-FDG PET/CT, have been utilized to predict survival odds in patients with advanced NPC , establishing a cutoff value of 34.66, , MTR\u2009+\u2009APTw-SI model: AUC:0.839), MTR\u2009+\u2009ADC model: AUC:0.827) and MTR\u2009+\u2009APTw-SI\u2009+\u2009ADC model: (AUC:0.830)], there was no significant distinction between the MTR value and any of the models . A non-selective saturation pulse in MT imaging can cause the interaction between semi-solid macromolecular protons (bound water pool) and free water protons in the tissues [In contrast, our study indicates that NPC patients with lower MTR value demonstrated better treatment outcome, as the pre-treatment MTR value showed a higher prediction efficacy in determining the short-term treatment outcome of NPC patients after CRT is commonly used to diagnose NPC recurrence, our founding of no significantly different mean ADC values between residual and non-residual groups was consistent with previous reports where no clear and definitive pre- and post-treatment ADC cutoff values have been established for clinical practice , 14. TheThe combination model using MTR with APTw-SI and ADC values did not significantly improve predictive efficacy . Therefore, pre-treatment MTR values remain a convenient and more effective predictor of the short-term treatment outcomes for NPC patients after CRT in clinical settings.Older age, advanced TNM stage, and invasion of adjacent structures are well-known features for patients with worse prognosis . The comDespite the valuable insights provided by our study, several limitations must be acknowledged. The relatively small sample size of forty patients with nasopharyngeal carcinoma, though strictly controlled for confounding factors, may introduce selection bias. Additionally, the relatively short average follow-up time limits the evaluation of long-term outcomes after chemoradiotherapy. Future research will aim to analyze the feasibility of APTw-SI and MTR values using 3D CEST technology in predicting long-term outcomes. Finally, the variation in dosages and time points of chemotherapy and radiotherapy among patients could impact the results, though the basic drugs for induction chemotherapy were consistent (GP regimens: gemcitabine\u2009+\u2009cisplatin) to ensure treatment uniformity.The pre-treatment MTR value had better prediction performance than ADC values and the APTw-SI acquired by a 3D CEST MR imaging, and it is more likely to predict short-term treatment outcomes of NPC patients after chemoradiotherapy. This finding holds promise for clinical applications, but further research and larger studies are needed to validate and establish the significance of these findings in the prediction of long-term treatment responses and patient outcomes.Below is the link to the electronic supplementary material.Supplementary Material 1"} +{"text": "Long-term care (LTC) facility residents are vulnerable to SARS-CoV-2 infection because of their advanced age, medical complexity, and congregate living situation; vaccination is an effective means for reducing COVID-19 incidence in this population.COVID-19 vaccination coverage among residents of participating LTC facilities within the National Healthcare Safety Network differed by race and geography. Bivalent COVID-19 vaccination rates were lowest among LTC facility residents in the South and Southeast U.S. regions and among Black or African American and multiracial residents.targeted strategies to reduce inequities in COVID-19 morbidity and vaccination coverage. Strategies aimed at increasing COVID-19 vaccination coverage should consider these demographic disparities to develop and implement Residents of long-term care (LTC) facilities constitute a population that is vulnerable to SARS-CoV-2 infection; COVID-19 vaccination effectively reduces severe COVID-19 in these settings. To examine demographic differences in primary and up-to-date vaccination status against COVID-19 among LTC facility residents, a descriptive analysis of COVID-19 vaccination data from the National Healthcare Safety Network (NHSN) COVID-19 vaccination data from October 31, 2022, to May 7, 2023, were analyzed. Being up to date was defined as having received a bivalent COVID-19 vaccine dose or having completed a primary vaccination series <2 months earlier. Geographic disparities in vaccination coverage were identified, with substantially lower prevalences of up-to-date status among LTC facility residents in the South (Region 6) (37.7%) and Southeast (Region 4) (36.5%) than among those in the Pacific Northwest (Region 10) (53.3%) and Mountain West (Region 8) (59.6%) U.S. Department of Health and Human Services regions. Up-to-date status was lowest among Black or African American (39.9%) and multiracial (42.2%) LTC facility residents. Strategies to increase up-to-date COVID-19 vaccination among LTC facility residents could include and address these geographic and racial differences.Long-term care (LTC) facility residents are vulnerable to SARS-CoV-2 infection because of their often advanced age, medical complexity, and congregate setting were eligible for inclusion, and >99% of records were reported by nursing homes . COVID-1Results from this analysis, the first to assess demographic and geographic disparities in up-to-date COVID-19 vaccination coverage among LTC facility residents using person-level data reported to NHSN, highlight geographic and racial and ethnic disparities in coverage among residents. These findings underscore the importance of improving the understanding of factors contributing to these geographic and demographic differences to guide public health practice and resource allocation Surveillance data reported to NHSN are an important tool to effectively monitor vaccination coverage among LTC facility residents as part of the COVID-19 public health response. As COVID-19 vaccination guidance evolves, strategic planning to increase vaccination coverage should include considerations to target and reduce demographic disparities.Residents of LTC facilities should receive COVID-19 vaccination irrespective of their demographic characteristics to protect them from COVID-19 in congregate living environments. Most recent guidance indicates that persons who are aged \u226565 years or who are immunocompromised should consider additional bivalent vaccine doses. ("} +{"text": "An hexaALS/FTD) . One of poly-PR) .C9orf72 HRE on the VAPB-PTPIP51 tethers in rodent and cell culture models of C9orf72-linked ALS/FTD. To quantify the interaction between endogenous VAPB and PTPIP51 proteins in situ we performed proximity ligation assays (PLA). The PLA assays are suitable for quantifying inter-organelle communication contacts and PLAs, including ones for VAPB and PTPIP51, have already been used to quantify ER-mitochondria contacts [Our recent study, published in Aging Cell , exploreC9orf72 expansion. C9orf72 HRE iPSc-neurons demonstrated reduced VAPB-PTPIP51 PLA signals compared to controls. A key function of the VAPB-PTPIP51 tethers is to facilitate ER-to-mitochondria Ca2+ delivery [2+ channel and the OMM located voltage-dependent anion-selective channel-1 (VDAC1), was also reduced in these neurons.First, we investigated the VAPB-PTPIP51 interaction in patient-derived induced pluripotent stem cell (iPSc) cortical neurons carrying the pathogenic delivery consistein vivo setting, a C9orf72 transgenic mouse that carries a bacterial artificial chromosome (BAC) containing a human pathogenic 450 GGGGCC repeat expansion. These mice develop age-dependent accumulation of RNA foci and DPRs, a phenotype that is accompanied by loss of hippocampal neurons and impaired cognitive function at 12 months [C9orf72 BAC transgenics compared to non-transgenic controls. These findings indicated that changes in VAPB-PTPIP51 binding are an early feature of disease in this mouse model, supporting the notion that disruption of the VAPB-PTPIP51 tethers might contribute to disease.The reduction in VAPB-PTPIP51 contact observed in iPSc-derived neurons was reproduced in an C9orf72 HRE involves production of toxic DPR polypeptides poly-GA, poly-GR and poly-PR, we enquired whether these DPRs might disrupt VAPB-PTPIP51 and IP3R-VDAC1 interactions in cultured rat cortical neurons. For these experiments, we used DPRs plasmids that utilise alternative codon sequences that preclude formation of RNA foci. Expression of the toxic DPRs reduced both PLA signals and the co-localisation ER and mitochondrial markers assessed using super resolution structured illumination microscopy (SIM). When addressing ER-mitochondria Ca2+ transfer in a cell line commonly used as a neuronal model, we confirmed that pathogenic C9orf72-derived DPRs were also associated with a disruption to this major ER-mitochondria signalling function.As a major pathogenic mechanism for C9orf72-derived DPRs or the precise mechanism by which GSK-3\u03b2 might influence VAPB-PTPIP51 binding still remains unknown.On a molecular level, our study placed activation of glycogen synthase kinase 3\u03b2 (GSK-3\u03b2), a previously known inhibitor of ER-mitochondria communication, as the responsible for the DPRs-mediated effects on the VAPB-PTPIP51 interaction. GSK-3\u03b2 is a ubiquitously expressed and constitutively active serine/threonine protein kinase involved in diverse physiological pathways ranging from metabolism, cell cycle to neuroprotection [post mortem tissue as well [Damaged ER-mitochondria signalling is seen in other cell and transgenic mouse models of familial ALS/FTD, such as those involving TAR-DNA binding protein 43kDa (TARDBP), VAPB, Fused in Sarcoma (FUS), superoxide dismutase 1 (SOD1), sigma non-opioid intracellular receptor 1 (SIGMAR1) or TANK binding kinase 1 (TBK1) [ as well .C9orf72 repeat expansion account for around 40% and 25% of familial ALS or FTD respectively, and for around 6% of sporadic cases in both as well. Our work also determined that changes in this signalling are an early feature of disease in a C9orf72-mouse model, representing a key pathogenic event prior to the onset of symptoms. Furthermore, this work also revealed important new information about the role of DPRs species in C9orf72-mediated toxicity.There is an emergence of effective therapies for ALS/FTD patients. ER-mitochondria contacts sites pose a pivotal molecular target. Our study highlighted this emerging research avenue in drug discovery for these devastating diseases consolidating ER-mitochondria signalling as a common pathogenic mechanism, affecting to the most frequent familial form of ALS/FTD; pathogenic Although research in this area is currently very active, important gaps persist. For example, despite the implications of ER-mitochondria signalling contributing to age-related disorders, little is known about ER-mitochondria contacts regulation throughout an organism\u2019s lifespan. In our study, we noticed age-related changes in ER-mitochondria contacts; we observed that in control non-transgenic mice, the numbers of VAPB-PTPIP51 PLA signals were fewer in 12-month compared to 6-month-old mice, while the molecular mechanisms underlying this age-dependent reduction are not clear at this stage.The aging process in the brain associates with a loss of homeostasis and impaired energy metabolism, with mitochondrial dysfunction as a major hallmark of aging. Whether ER-mitochondria associations might drive a role in these age-related processes constitute an exciting new area of research."} +{"text": "We aim to examine the prognostic value of major pathologic response in metastatic lymph nodes (mLN-MPR) after immunochemotherapy in non-small cell lung cancer (NSCLC), and demonstrate the pathological characteristic of regression in mLN. Adult patients consecutively undergone neoadjuvant immunochemotherapy and radical-intent surgery for initial stage cIII NSCLC between 2020 and 2021 were included. Hematoxylin- and eosin-stained slides of paraffinembedded sections of the degree of pathologic response in the primary tumor (PT) and its paired involved LNs were reviewed. Imaging mass cytometry was conducted to quantify the immunological status. With 10% as residual viable tumor (RVT) cutoff, mLN-MPR ) showed more significant correlation with DFS than ypN0 . And mLN-MPR combined with PT-MPR, compared with ypN stage combined with PT-MPR , can better distinguished the DFS curves of the 4 subgroups of patients. mLN-MPR(+)/PT-MPR(+) patients had the best prognosis compared with other subgroups. Pathologic responses of RVT in PT and paired regional LNs [MPR inconsistency rate: 21/53 (39.6%)], and across different LNs could be inconsistent, especially in squamous cell carcinoma. RVT% in mLNs after immunochemotherapy appeared to be polarized . Partial regression of LN metastasis could present with distinct immune subtypes: immune-inflamed or immune-evacuation subtype, and the former presented with higher CD3, CD8, and PD-1 expression in the invasive margin. mLN-MPR demonstrated a potential prognostic value in predicting DFS in patients treated with neoadjuvant immunochemotherapy, but further research is needed to validate its usefulness for other survival outcomes, including OS.The online version contains supplementary material available at 10.1186/s40164-023-00401-6. To the editorsNeoadjuvant immunotherapy has beenAdult patients consecutively undergoing neoadjuvant immunochemotherapy between January 2020 to January 2021 and radical surgery for initial stage cIII NSCLC were included. In total, 53 patients were included. All cases had initial LN metastasis diseases, and most of the patients were with squamous cell carcinoma (LUSQ) [n\u2009=\u200935 (66.0%)] (Supplementary Table\u00a01). Hematoxylin- and eosin-stained slides of paraffinembedded sections of the degree of pathologic response in the PT and its paired involved LNs were reviewed by two experienced pathologists. Calculation of RVT% was scored according to immune-related pathologic response criteria (irPRC) . ypN0 waTo determine the prognostic implications of mLN-MPR, univariable Cox model analyses for DFS were conducted Table\u00a0. CumulatFollowing a rigorous histopathological assessment procedure for slide handling, we found that the pathologic responses of RVT in PT, paired regional LNs, and across different LNs could be inconsistent (PT/mLN-MPR inconsistency rate: 39.6%). Nine out of thirty-one patients (29.0%) had PT-MPR(+) while RVT in mLN\u2009>\u200910%; and twelve out of twenty-two patients (54.5%) had PT-MPR(-) but reached mLN-MPR(+). Representative two cases of discordance pathological responses of RVT in PT and paired regional LNs, and across different LN stations were shown in Fig.\u00a0We also found that partial regression RVT\u2009>\u200910%, \u2264 50%) in mLN can present distinct pathological immune subtypes: immune-inflamed subtype (Fig.\u00a00%, \u2264 50%In the current ypTNM staging system , the ypNBelow is the link to the electronic supplementary material.Supplementary Table S1. Demographic characteristics, clinical-pathological characteristics and survival outcomes of 53\u00a0study participants."} +{"text": "Data regarding response to SARS-CoV-2 immunization in pediatric patients with predominantly antibody deficiency (PAD) is limited. We evaluated SARS-CoV-2 immunization response by anti-SARS-CoV-2-spike antibody level in 15 pediatric PAD patients. These data were compared to a published cohort of adult PAD patients (n=62) previously analyzed following SARS-CoV-2 immunization at our single center institution. We evaluated demographics, clinical characteristics, immunophenotype, infection history, and past medication use by chart review. Following a two-dose monovalent initial series SARS-CoV-2 immunization, mean anti-SARS-CoV-2-spike antibody levels were significantly higher in pediatric PAD patients compared to adult PAD patients . Pediatric PAD patients with low class-switched memory B-cells, defined as <2% of total CD19+ B-cells, had significantly lower mean anti-SARS-CoV-2-spike antibody levels than those without (p=0.02). Following a third-dose monovalent SARS-CoV-2 immunization, the mean anti-SARS-CoV-2-spike antibody levels in pediatric PAD patients significantly increased . These data support Centers for Disease Control guidelines regarding three-part SARS-CoV-2 vaccine series, including in the pediatric PAD patient demographic. Predominantly antibody deficiency (PAD) is the most commonly diagnosed inborn error of immunity (IEI) and the most frequent symptomatic primary immunodeficiency disorder world-wide. PAD encompasses a heterogeneous collection of disorders characterized by increased susceptibility to infections, low immunoglobulin levels, and impaired vaccine responses . The cliStudies have demonstrated that children produce long-term antibody responses to COVID-19 infection . HealthyThere have been several studies investigating COVID-19 infection outcomes in patients with IEI. A review of the Italian Primary Immunodeficiency Network found that there were no deaths among 33 COVID-19 positive children with an underlying IEI condition .The trials evaluating the safety and effectiveness of the SARS-CoV-2 vaccine in children and adolescents excluded those who had immunodeficiency diseases including PAD , 12. SubTo date, most studies regarding the efficacy of the SARS-CoV-2 vaccine in immunocompromised children are limited to those receiving immunosuppressants for a medical condition such as renal disease, malignancy, or organ transplant , 17. AmoThe aim of this study was to assess the efficacy of the SARS-CoV-2 vaccine, following two-dose initial series and third-dose boost, in pediatric patients of all ages with PAD.This study was performed at Mass General Brigham under an institutional review board\u2013approved protocol No. 2021P002414). Antibody response to the SARS-CoV-2 vaccine in patients with known PAD was evaluated. Pediatric PAD patients who received an initial two-dose SARS-CoV-2 vaccine series between May 2021 and September 2022 were included. The PAD diagnoses were confirmed by manual chart review by a clinical immunologist and met consensus definitions for PAD \u201321. PatiP002414. We evaluated demographic information and clinical characteristics including the type of PAD, vaccine type received, and prior immunophenotyping, including native immunoglobulin levels, native vaccine titers , and peripheral blood lymphocyte counts as available. We evaluated previous and current use of immunoglobulin replacement and other immunosuppressants or biologics received in the past or in close proximity to vaccination (defined as 6 months before to 1 month after immunization). We also evaluated infection history, outcome, treatment, and tixagevimab/cilgavimab use.Serologic assays were performed using the Roche Elecsys Anti-SARS-CoV-2 S-antibody test protein receptor binding domain; anti-spike antibody). This test is semiquantitative and has been correlated with neutralizing immunity , 24. TheWe used the Student t-test to compare mean anti-spike antibody values and the Mann-Whitney U-Test for comparing median values. All antibody responses to SARS-CoV-2 vaccine were reported as geometric means . Statistical analyses were completed with SAS software and Prism software ; two-tailed P-value less than.05 was considered significant.15 pediatric patients with PAD were included Table\u00a01.SARS-CoV-2 antibody levels were measured after pediatric PAD patients received their initial two-dose SARS-CoV-2 vaccine series (n=13), and after the third-dose vaccination (n=8). Median time from vaccination to SARS-CoV-2 antibody level testing was 85 days from initial series and 119 days from third-dose vaccine for pediatric PAD patients. In contrast, median time from vaccination to SARS-CoV-2 antibody level testing for adult PAD patients was 31 days from initial series, which was a statistically shorter time interval when compared to the pediatric cohort (p=0.0002). Two of 15 PAD patients had SARS-CoV-2 infections after their third-dose booster, but before their blood draw. These patients had SARS-CoV-2 antibody level testing performed after they cleared their infections (at a median time of 71.5 days post-positive polymerase chain reaction [PCR] test) to our previously published adult PAD cohort (n=62) to our pFigure\u00a01Age-related declines in humoral immune function have been widely described \u201332. We hPreviously, severity of PAD, secondary PAD, and a high risk underlying immunophenotype, specifically <2% class-switched memory B-cells, were all shown to be risk factors among PAD adults for lower mean anti-spike antibody levels following SARS-CoV-2 vaccination . We sougPAD adults have a significant increase in anti-spike antibody levels following a third monovalent dose of SARS-CoV-2 vaccine . We hypoTo determine whether passive antibody transfer could be confounding the anti-spike antibody analysis, we compared anti-spike antibody levels between patients that did (n=8) or did not (n=7) receive immunoglobulin replacement therapy (IgRT). After a second dose of SARS-CoV-2 vaccine, there was no significant difference in anti-spike antibody levels between pediatric patients who did or did not receive IgRT . After a third-dose of SARS-CoV-2 vaccine, anti-spike antibody levels in patients receiving IgRT trended lower than those that did not receive IgRT, although this did not reach statistical significance to those who had never had a COVID-19 infection at the time of their third-dose blood draw (n=5), and found no statistically significant difference in mean anti-spike antibody levels (Eight pediatric patients (53.3%) had naturally acquired SARS-CoV-2 infections during the study. Two patients recovered without treatment. Six patients received either anti-viral (n=4) or corticosteroid (n=2) treatment for their infections. Three patients who received anti-viral therapy received remdesivir, and one patient received ritonavir-boosted nirmatrelvir. There was no rebound COVID-19 in the patient who received ritonavir-boosted nirmatrelvir. No patients died from COVID-19 infection. There were no urgent care visits, ED visits, or hospitalizations in our cohort associated with COVID-19 infection. There were no secondary infections associated with COVID-19 infection.To our knowledge, this is the first study to assess the efficacy of the SARS-CoV-2 vaccine two-dose series and third additional dose vaccine in pediatric patients ages 4-16 years with PAD. In comparison to adult PAD patients, pediatric PAD patients had significantly higher mean anti-spike antibody levels in response to SARS-CoV-2 vaccination. This may be due, in part, to the composition of the cohorts, with the pediatric cohort being enriched for mild disease and the adult cohort being enriched for moderate and severe disease. However, even when stratified to severe PAD, pediatric patients had significantly higher mean anti-spike antibody levels compared to adult patients. These data were consistent with lower risk immunophenotypic markers in the severe pediatric PAD cohort, including higher absolute counts of CD3+ T-cells, CD4+ T-cells, CD19+ B-cells, and CD19+CD27+IgM-IgD- class-switched memory B-cells.The anti-spike antibody levels increased significantly after a third monovalent vaccine dose in pediatric PAD patients. This aligns with prior findings in adult PAD patients and prior CDC guidelines recommending three monovalent mRNA SARS-CoV-2 vaccines in pediatric patients , 33. CurIn our study, eight patients had naturally acquired SARS-CoV-2 infection. There were no deaths, and patients did not require ED visits or hospitalization for severe infection. We did not find a statistically significant difference in mean anti-spike antibody levels in those who did or did not have a COVID-19 infection. These data suggest limited confounding by naturally acquired COVID-19 infection to our SARS-CoV-2 vaccine response analysis. Additionally, we did not find a significant difference in anti-spike antibody levels between those receiving IgRT or not, suggesting that there is not a significant effect from passive antibody transfer from immunoglobulin replacement. This highlights the importance of vaccination, even in patients receiving immunoglobulin replacement.This work is subject to several limitations. The sample size was small, which limits the power of our analysis. In addition, there may be sampling bias in that differences may have existed between patients who consented to be a part of this study and those who did not. This analysis was performed using data from a large but single health care system, so these findings may not be generalizable to other settings. Our cohort was predominantly male and non-Hispanic white, which may also limit generalizability. We analyzed immunogenicity using the Roche Elecsys Anti-SARS-CoV-2 S-antibody test, which may not be generalizable to other assay testing platforms. We were unable to assess anti-spike antibody levels prior to initial vaccination in our cohort, which limits our ability to analyze seroconversion following initial series immunization. There was a difference in dose-to-draw times between dose 2 (85 days) and dose 3 (119 days) in the pediatric PAD cohort, which could skew towards relatively lower anti-spike antibody levels at the post-dose 3 timepoint as compared to the post-dose 2 timepoint. Despite this potential skewing to the null hypothesis, we still observed a significant boost in SARS-CoV-2 anti-spike antibody levels between dose 2 and dose 3 timepoints. Finally, we did not sub-stratify the anti-spike antibody analysis by isotype , as this isotype analysis was comparable when evaluated for the adult PAD cohort , which cAdditional studies are needed in larger cohorts of patients to further understand the immune response to SARS-CoV-2 vaccination in pediatric PAD patients, including assessing the benefits of the bivalent vaccine and/or future vaccine platforms. In addition to the antibody responses analyzed here, T-cell response to vaccination provides important cellular immune protection against severe infection, which we are unable to assess with these serologic data and future research regarding this response would be beneficial. Longitudinal studies are needed to evaluate the duration of response to vaccine to determine the optimal vaccination strategy, given that antibody responses can wane over time.In conclusion, pediatric PAD patients, specifically those with severe PAD, had significantly higher antibody response to SARS-CoV-2 vaccination when compared to adults with severe PAD. Pediatric PAD patients with low class-switched memory B-cells had significantly lower response to SARS-CoV-2 vaccination compared to those without. Anti-spike antibody levels increased significantly after receiving a third monovalent SARS-CoV-2 vaccine. These data support CDC guidelines regarding three-dose SARS-CoV-2 vaccine series, including in the pediatric PAD patient demographic.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by Mass General Brigham IRB. Written informed consent to participate in this study was provided by the participants\u2019 legal guardian/next of kin.JRF and SB conceived of the project. DD provided expertise in immunology. MT performed chart review, and wrote the manuscript with aid from JRF and SB. ZB performed statistics herein described. JRF, SB, DD, PH, and CR contributed patients."} +{"text": "Sluggish uptake of novel gram-negative antibiotics has jeopardized their future development and supply potentially risking patient lives. Understanding underutilization patterns might better inform unmet needs and market entry rewards. We studied patterns and drivers of use of recently U.S. FDA-approved novel gram-negative agents in patients with gram-negative infections (GNI) displaying difficult-to-treat resistance .Using the all-payer PINC-AI database, quarterly percent change in the post-approval use of seven gram-negative agents and secular trends in colistin use were calculated. For inpatients with DTR GNI at microbiology reporting hospitals, we compared novel vs older/reserve agent use by infection site and pathogen. At hospitals that use novel agents, generalized linear mixed models with weighting identified patient and hospital factors associated with the use of any novel (vs older/reserve) agents as targeted therapy against DTR GNI.Klebsiella pneumoniae carbapenemase (KPC) agent ceftazidime-avibactam (approved 2015), usage was relatively sluggish for subsequently approved anti-KPC-agents meropenem-vaborbactam and imipenem-cilastatin-relebactam and zero for plazomicin. At 302 microbiology reporting hospitals, 44.4% of 2830 overall DTR cases including 70.2% of 627 DTR-A. baumannii cases received only older/reserve agents . The odds of use of novel (vs older/novel) agents were greater in patients displaying hemodynamic instability and higher comorbidity burden, but similar across race categories and insurance status, and lower at Western(vs Midwestern) hospitals (Table1).At 619 hospitals, while colistin use declined between 2016Q1 and 2021Q2, post-approval use grew variably for each novel agent ; compared to the first novel anti\u2013A. baumannii are needed to bridge the utilization gap.Nearly half the patients in US hospital with DTR-GNIs are still receiving targeted therapy with older/reserve agents. While the value of novel agents is indicated by clinicians preferring them over older/reserve agents in sicker patients, future agents with distinctly novel mechanisms and activity against DTR-Morgan Walker, MD, Cytovale: Advisor/Consultant"} +{"text": "ALK fusion mutations harbored approximately 90 distinct fusion partners. Patients with different ALK fusions might respond distinctly to different-generation ALK inhibitors. In this case report, we identified a novel non-reciprocal ALK fusion, ALK-C2orf91(intergenic) (A19: intergenic) and PPFIA1-ALK (P2:A20), by next-generation DNA sequencing in an advanced lung adenocarcinoma patient. After 2\u2009months of alectinib, the targeted lung lesion regressed significantly, and evaluation of therapeutic efficiency indicated partial response. To date, the patient had achieved 12\u2009months of progression-free survival from alectinib treatment. Our study extended the spectrum of ALK fusion partners in ALK-positive NSCLC, and we reported a new ALK fusion, PPFIA1-ALK and ALK-C2orf91(intergenic), and its sensitivity to alectinib firstly in lung cancer. We believe that this case report has an important clinical reference.Non-small cell lung cancers (NSCLCs) with anaplastic lymphoma kinase (ALK)-rearrangement have favorable responses to ALK inhibitors. However, ALK gene rearrangement is one of the most important driver mutations in non-small cell lung cancer (NSCLC) occurring in 3%\u20137% of the cases (EML4)-ALK is the most common fusion variant, accounting for over 80% of fusion partners. With the increasing coverage of next-generation DNA sequencing, more than 90 rare ALK fusion subtypes have been discovered in NSCLC, such as kinesin family member 5B (KIF5B)-ALK and striatin gene (STRN)-ALK double-fusion variants is sensitive to first-line alectinib.he cases . EchinodTRN)-ALK . First- PPFIA1-ALK (P2:A20) and ALK-C2orf91 was identified in tissue scan revealed hydropneumothorax, compression atelectasis (approximately 80% collapse) and tumor mass of the right lung, and multiple nodules in the left lung double-fusion lung adenocarcinoma patient who is sensitive to alectinib. Multiple ALK fusion types have been reported in NSCLC patients, among which different fusion partners and different breakpoint variants may affect the response to ALK-TKIs (HIP1-ALK (H21:A20) and HIP1-ALK (H30:A20) responded well to crizotinib (HIP1-ALK (H19:A20) showed resistance to crizotinib with progression of pleural effusion as 5\u2032-ALK and PPFIA1-ALK (P2:A20) as 3\u2032-ALK in our case were defined together as a non-reciprocal ALK fusion. Zeng reported an NSCLC patient with non-reciprocal ALK fusion after resistance to first-line gefitinib who responded to alectinib and had a PFS of more than 26 months. Similar to the previous report, the patient in the present case was also sensitive to alectinib and had been receiving alectinib for 12 months.According to the global ALEX study, alectinib showed superior progression-free survival (PFS) versus crizotinib in untreated variant . For patizotinib . TherefoPPFIA1-ALK and ALK-C2orf91 (intergenic) double-fusion mutation sensitive to first-line alectinib.In summary, this case presented a rare novel non-reciprocal The original contributions presented in the study are included in the article/The studies involving humans were approved by Ethics Committee of First Affiliated Hospital of Guangxi Medical University. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.LY: Data curation, Writing \u2013 original draft, Writing \u2013 review & editing. JZ: Data curation, Writing \u2013 review & editing. QP: Data curation, Writing \u2013 review & editing. YL: Data curation, Writing \u2013 review & editing. PY: Investigation, Writing \u2013 original draft, Writing \u2013 review & editing. QC: Funding acquisition, Supervision, Writing \u2013 review & editing."} +{"text": "Dear Sir,European Journal of Nuclear Medicine and Molecular Imaging entitled \u201cCross-reactivity to glutamate carboxypeptidase III causes undesired salivary gland and kidney uptake of PSMA-targeted small-molecule radionuclide therapeutics,\u201d by Lucaroni et al. [177Lu]PSMA-617) with PSMA isozymes might represent the underlying cause of unwanted accumulation in healthy salivary glands and kidney. They based this on cross-reactivity of Glu-ureido-based inhibitors to proteins with high similarity to PSMA such as GCP III. There are, however, existing evidences that may not support their postulation.With great interest, we read a short communication in a recent issue of the i et al. . In thisS)-1-carboxy-3-methylbutyl]amino]carbonyl]-L-glutamic acid (ZJ-24) 160-fold higher [68Ga]PSMA-11 in the salivary glands and kidneys.Firstly, the inhibition constants of PSMA-targeted ligands (inhibitors) against GCP II (NAALAD or PSMA) and GCP III NAALAD2) are well documented and much stronger for GCP II than GCP III PSMA-11 showed no uptake in the salivary glands and kidneys of the PSMA null mice compared to wild-type control mice with Compound 1 were in line with similar fold difference (0.15 vs. 0.9) although the values of the constants were not the same, which might be explained by the use of recombinant expression discussed above.Staring from Fig.\u00a0https://www.proteinatlas.org/ENSG00000086205-FOLH1/tissue/salivary+gland)? Is HPA060802 specific for GCP III without cross-over to GCP II? Antibody specificity in differentiating GCP II vs. GCP II has been a problem [In Fig.\u00a02 (the antibodies): there are a few anti-PSMA antibodies (from Leica or Dako) used by many either clinically or experimentally, but why the authors used HPA010593, which did not detect any PSMA in the salivary glands according to Protein Atlas ( problem .Interestingly, a prior publication listed a"} +{"text": "This prospective study aimed to examine changes in mental health and differences due to educational status (ES) and country among young adults aged 20-40 from four countries during the COVID-19 pandemic in a three-month period.n = 321) and non-students (n = 519) aged 20-30, educated (n = 388), and non-educated (n = 486) adults aged 31-40 from Poland (n = 445), Slovenia (n = 430), Germany (n = 417), and Israel (n = 422) responded to online survey in February 2021 and May\u2013June 2021. The used measurements were: Perceived Stress Scale (PSS-10), Generalized Anxiety Disorder (GAD-7), and Patient Health Questionnaire (PHQ-8).The total of 1714 participants (932 women): students , and across countries for mental health indicators. The results showed stability over time in anxiety and depression while a small decrease in stress. Students scored significantly higher in stress, anxiety, and depression compared to non-/educated adults and in depression compared to non-studying peers. Participants from Poland and Germany scored higher in anxiety and depression than from Slovenia and Israel. Moreover, Polish participants reported the highest stress among all countries.The student population is more vulnerable to mental health issues than non-studying peers and adults with and without an academic degree, particularly in Poland and Germany.None Declared"} +{"text": "Objectives: The number of lung transplants is increasing year by year in China and globally. With the widespread use of donation after brainstem death (DBD) donor lungs and donation after circulatory death (DCD) donor lungs, donor-derived infection (DDI) poses a major challenge in lung transplantation. Using donor lungs infected or colonized with carbapenem-resistant Enterobacteriaceae (CRE) may have serious implications in lung-transplant recipients. Currently, traditional microbial culture along with antimicrobial susceptibility testing cannot fully meet the need for rapid and accurate diagnosis of CRE infection in a donor before organ harvest. Methods: The Xpert Carba-R device was used to detect and differentiate Klebsiella pneumoniae carbapenemase (KPC), New Delhi metallo-\u03b2-lactamase (NDM), Verona integron-encoded metallo-\u03b2-lactamase (VIM), active-on-imipenem (IMP), and OXA-48 carbapenemase genotypes in bronchial lavage fluid from donor lungs before organ harvest. Positive detection of 1 or more of these genotypes indicated a potentially CRE-infected donor lung, and these organs were removed from the lung transplantation cohort. Donor lungs negative for all KPC, NDM, VIM, IMP, and OXA-48 genotypes determined by the Xpert Carba-R device were used for lung transplantation. The incidence of CRE-associated DDI and infection-related complications were compared in the Xpert Carba-R screening group and an historic control group. Results: In this study, 21 donor lungs were tested with the Xpert Carba-R device to detect and differentiate carbapenemase genotypes. Among them, 4 were positive for 1 or more carbapenemase genotypes and were discarded, and the remaining 17 donor lungs showing no carbapenemase gene presence were used for lung transplantation. No CRE-associated DDI occurred in these 17 lung-transplant recipients. Conclusions: Rapid and accurate detection of the carbapenemase gene in donor lungs at the point of care before transplantation using the Xpert Carba-R device reduced the risk of CRE-associated DDI in lung-transplant recipients."} +{"text": "The two strains were mated to generate bi-transgenic Cyp19-Cre;ROSAmT/mG mice following a standard transgenic breeding scheme, and placental and fetal tissues were analyzed on embryonic day 17.5. Both maternal and paternal Cre inheritance were analyzed by mating the respective Cyp19-Cre and ROSAmT/mG males and females. The genotype results showed the expected percentage of Cyp19-Cre;ROSAmT/mG fetuses (73%) and Cre mRNA was expressed in all of the Cyp19-Cre placentas. However, surprisingly, only about 50% of the Cyp19-Cre;ROSAmT/mG placentas showed Cre-mediated recombinase activity as demonstrated by placental enhanced green fluorescent protein (EGFP) expression. Further genetic excision analysis of the placentas revealed consistent results showing the absence of excision of the tdTomato in all of the Cyp19-Cre;ROSAmT/mG placentas lacking EGFP expression. Moreover, among the EGFP-expressing placentas, there was wide variability in recombination efficiency, even in placentas from the same litter, leading to a mosaic pattern of EGFP expression in different zones and cell types of the placentas. In addition, we observed a significantly higher percentage of Cre recombination activity in placentas with maternal Cre inheritance. Our results show frequent mosaicism, inconsistent recombination activity, and parent-of-origin effects in placentas from Cyp19-Cre;ROSAmT/mG mice, suggesting that tail-biopsy genotype results may not necessarily indicate the excision of floxed genes in Cyp19-Cre positive placentas. Thus, placenta-specific mutagenesis studies using the Cyp19-Cre model require extensive characterization and careful interpretation of the placental phenotypes for each floxed allele.The aromatase-Cre recombinase (Cyp19-Cre) transgenic mouse model has been extensively used for placenta-specific gene inactivation. In a pilot study, we observed unexpected phenotypes using this mouse strain, which prompted an extensive characterization of Cyp19-Cre placental phenotypes using ROSA The Cre/loxP system is an integral experimental tool that permits spatial and temporal genetic manipulation during mouse development. The success of conditional gene targeting largely relies on the choice of Cre driver strains, but in the placenta, its use has been limited due to the lack of well-characterized placenta-specific promoters. To date, only a few Cre recombinase-expressing mouse lines have been developed with the ability to target trophoblast lineages in the placenta. Trophoblast-specific Cyp19-Cre was generated by making use of a 501 bp regulatory region within the first exon (I.1) encoding the 5\u2032-untranslated region of human aromatase P450 (CYP19) to drive the expression of the Cre recombinase in the mouse placenta ,3. The Cyp19-Cre mouse strain has been extensively used for trophoblast-specific disruption of genes to determine gene functions in the placenta. This apparent success has resulted in the determination of the Cyp19-Cre mouse strain as the model of choice for placenta-specific mutagenesis ,11,12,13mT/mG reporter mice, which harbor a membrane-targeted tandem dimer Tomato (tdTomato) gene flanked by loxP sites followed by a membrane-targeted enhanced green fluorescent protein (EGFP) cassette [mT/mG mouse is a well-characterized double-fluorescent Cre reporter strain known for its robust activity, reliable ubiquitous expression, and breeding performance. Our findings demonstrated dramatic inconsistencies in Cre-mediated loxP-dependent DNA recombination even in the placentas of littermates from pregnant Cyp19-Cre;ROSAmT/mG transgenic mice. In this study, we therefore sought to further characterize the Cyp19-Cre strain. We determined the efficiency of Cyp19-Cre activity in the placenta using ROSAcassette . The ROScassette . The ROSmT/mG, were obtained from The Jackson Laboratory . To determine the efficiency of Cre activity in placentas, Cyp19-Cre animals were bred with the ROSAmT/mG reporter strain, which constitutively expresses tdTomato (mT) in all cells that are replaced by EGFP after Cre-mediated recombination. For maternal inheritance, Cyp19-Cre females (n = 4) were mated with ROSAmT/mG males, while paternal Cre inheritance involved crossing Cyp19-Cre males with ROSAmT/mG females (n = 6). Pregnancies were identified by the presence of a vaginal plug and considered as embryonic day (E) 0.5.All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at Wayne State University (WSU) and University of Missouri\u2013Kansas City. Mice were housed in a facility controlled for temperature and humidity with a 12 h light\u2013dark cycle. Cyp19-Cre (Tg (Cyp19a1-cre)5912Gle; herein termed as Cyp19-Cre) mice were obtained from Dr. Gustavo Leone [Timed pregnant dams were sacrificed at E17.5. The number of implantation sites and resorptions was recorded. Placentas were hemisected in the transverse plane and one half of each placenta was fixed in 4% paraformaldehyde (PFA) for 5 h at 4 \u00b0C, cryopreserved in 10\u201330% sucrose gradient (for 72 h at 4 \u00b0C), embedded in OCT , and snap frozen on dry ice. The other half of each harvested placenta was minced, flash frozen, and stored at \u221280 \u00b0C for subsequent RNA, DNA, and protein analysis. Genomic DNA was extracted from fetal tail biopsies and genotyped by PCR using the following primers: CYP19-Cre forward primer 5\u2032-GACCTTGCTGAGATTAGATC-3\u2032, reverse primer 5\u2032-GAGAGAGAAGCATGTTTAGCTGGCC-3\u2032; mT/mG wild-type forward primer 5\u2032-AGGGAGCTGCAGTGGAGTAG-3\u2032, mutant forward primer 5\u2032-TAGAGCTTGCGGAACCCTTC-3\u2032, reverse primer 5\u2032-CTTTAAGCCTGCCCAGAAGA-3\u2032; sex-determining gene on the Y chromosome (Sry) forward primer 5\u2032-CCTATTGCATGGACAGCAGCTTATG-3\u2032, and reverse primer 5\u2032-GACTAGACATGTCTTAACATCTGTCC-3\u2032. Cyp19-Cre mediated recombination in placentas was detected in PFA-fixed 10 \u00b5m thick frozen sections via the presence of EGFP-associated fluorescence. Images were captured using a Zeiss fluorescence microscope. Positivity was assessed based on the presence of a green signal in the tissue. Each section was examined/scored by two individuals and grouped either as <30%, 30\u201370% or >70% Cre activity based on the EGFP signal observed across the placenta.Genomic DNA was extracted from frozen placentas using the DNeasy Blood & Tissue Kit according to the manufacturer\u2019s instructions. For PCR amplification, 60 ng of total template DNA was amplified using forward primer 5\u2032-GTGCTGTCTCATCATTTTGGCA-3\u2032 and reverse primer 5\u2032-TTGCTCACGGATCCTACCTTC-3\u2032. The presence of a 275 bp amplicon indicated Cre-mediated tdTomato gene excision and intra-chromosomal recombination between loxP sites, allowing for EGFP expression. Recombination efficiency was calculated as the percentage of positive placentas (containing the 275 bp amplicon) among the total placentas positive for Cyp19-Cre genotyping.\u2212\u0394\u0394CT method. The primer sequences were as follows: Cre forward primer 5\u2032-ACGAGTGATGAGGTTCGCAAG-3\u2032 and reverse primer 5\u2032-GGTTATTCAACTTGCACCATGCC-3\u2032; Gapdh forward primer 5\u2032-CTTTGGCATTGTGGAAGGGC-3\u2032 and reverse primer 5\u2032-GTGGATGCAGGGATGATGTTC-3\u2032.Total RNA was extracted from frozen placentas using TRIzol . RNA (2 \u03bcg) was reverse transcribed into cDNA using an oligo dT primer, and quantitative PCR was performed with the StepOne Plus Real-Time PCR system using a fast SYBR Green PCR Master Mix . Each RT-qPCR reaction was performed in duplicate and the relative expression of Cre mRNA levels (fold change) normalized to Gapdh was calculated using the 2t-test or one-way ANOVA followed by post hoc analysis. p < 0.05 was considered statistically significant.Data are represented as mean \u00b1 standard error of the mean (SEM). Analyses were performed using GraphPad Prism version 9.0 . Statistical comparisons were carried out using the Student mT/mG transgenic mice. We used the ROSAmT/mG Cre reporter mouse because of its known robust activity, reliable ubiquitous expression, and breeding performance, as demonstrated in previous research [mT/mG transgenic mice, we observed the expected and highly consistent EGFP expression in macrophages at the maternal\u2013fetal interface , Cyp19-Cre;ROSAmT/mG presented a robust, positive EGFP signal in the labyrinth and junctional zones of some placentas from 16 different litters, irrespective of maternal or paternal Cre inheritance. Unexpectedly, we observed an extreme mosaic pattern in some placentas and a complete lack of EGFP expression in other Cyp19-Cre;ROSAmT/mG genotype-positive placentas, irrespective of the mating scheme. These findings prompted us to examine the Cre activity in all 99 Cyp19-Cre;ROSAConsistent with our findings, it has been previously reported that the labyrinth zone trophoblast cells of E16 placentas exhibit mosaic Cyp19-Cre recombinase activity using the Rosa26 tdTomato reporter mouse strain . In addimT/mG placentas could suggest that the loxP sites in the tdTomato gene were not being excised, resulting in intra-chromosomal recombination failure. To provide a readout of recombination efficiency, we verified genetic excision by designing primers that bind regions outside the loxP sites (mT/mG placentas (n = 99) isolated from 16 pregnant females. The ROSAmT/mG transgenic mice were identified by PCR genotyping (primer sets recommended by the Jackson Laboratory), where either a double band for heterozygotes (WT and mT/mG transgene) or a single band for homozygotes (mT/mG transgene) was observed of Cyp19-Cre;ROSAmT/mG placentas showed excision of the tdTomato gene, while the rest were negative for recombination (mT/mG (Cre negative) placentas, Cre mRNA levels were found to be similar between recombination-positive and negative placentas, suggesting that Cre inactivation is not occurring at the transcriptional level had more than an 80% deletion of the Igf2 floxed allele [We also analyzed the intensity of the EGFP signal in all recombination-positive Cyp19-Cre;ROSArescence B. A majorescence B. This id allele . Howeverd allele . When pad allele reportinmT/mG reporter mice, we found that the Cyp19-Cre driver strain exhibits inconsistent Cre recombination activity and varying levels of mosaicism and parent-of-origin effects in the placenta, even among littermates. Although Cre-mediated recombination efficiency is influenced by both the Cre driver and the floxed target allele, our work provides compelling evidence of the extensive variability of Cyp19-Cre-mediated recombination activity in the placenta, which was not clearly delineated in previous reports. Our findings also indicate that relying solely on the genotyping analysis to interpret results from the Cyp19-Cre model can lead to highly misleading conclusions. Therefore, it is crucial for future studies to thoroughly characterize the Cyp19-Cre model for each floxed allele and carefully interpret the placental phenotypes.In our study using ROSA"} +{"text": "Background: Data regarding the effects of the SARS-COV-2 (COVID-19) pandemic on healthcare-associated infections (HAIs) in Canadian acute-care hospitals are limited. We examined the impact of the COVID-19 pandemic on HAIs and antimicrobial resistant organisms in hospitals participating in the Canadian Nosocomial Infection Surveillance Program. Methods: We analyzed 13,406 HAIs including adult mixed intensive care unit (ICU) central-line\u2013associated bloodstream infections (CLABSIs), and healthcare-associated (HA) Clostridioides difficile infection (CDI), methicillin-resistant Staphylococcus aureus (MRSA) bloodstream infections (BSI), vancomycin-resistant Enterococcus (VRE) BSI, and carbapenemase-producing Enterobacterales (CPE) infections collected using standardized case definitions and questionnaires from 29\u201364 hospitals participating in the Canadian Nosocomial Infection Surveillance Program (CNISP) from January 2018 to December 2021. We used a generalized linear mixed model with quasi-Poisson distribution to assess step and slope changes in monthly HAI rates between the pre\u2013COVID-19 pandemic period and the COVID-19 pandemic period . Results were reported as incidence rate ratios (IRRs) with 95% confidence intervals (CIs) and adjusted for seasonality, hospital clustering, and hospital characteristics of interest. Results: In the CNISP network, 7,352 (55%) HAIs were reported in the prepandemic period and 6,054 (45%) in the pandemic period. Median age was significantly younger during the pandemic period compared to the prepandemic period among patients with HA-CDI, HA-MRSA BSI, and adult mixed ICU CLABSIs, and more than half of cases among all reported HAIs were male . The 30-day all-cause in-hospital mortality rate did not significantly change between the prepandemic and pandemic periods for all reported HAIs and was highest among HA-VRE BSIs (34%). Modeling results indicated that the COVID-19 pandemic was associated with an immediate increase in HA-CDI and adult mixed ICU CLABSI rates whereas HA-MRSA BSI, HA-CPE and HA-VRE BSI rates immediately decreased. However, pandemic status did not have a statistically significant lasting impact on monthly rate trends for all reported HAIs after adjusting for seasonality, clustering, and hospital covariates . Adjusted IRRs for all HAIs ranged from 1.00 to 1.01 .Conclusions: Although the COVID-19 pandemic placed a significant burden on the Canadian healthcare system, the immediate impact on monthly rates of HAIs in Canadian acute-care hospitals was not sustained over time. Understanding the epidemiological effects of the COVID-19 pandemic in the context of changing patient populations, and clinical and infection control practices, are essential to inform the continued management and prevention of HAIs in Canadian acute-care settings.Disclosures: None"} +{"text": "CNP in OLs, as evidenced by downregulated expression of CNP in mental disorders and animal models. Little is known about how CNP expression is regulated in OLs. Especially, OL enhancers that govern CNP remain elusive. We have recently developed a powerful method that links OL enhancers to target genes in a principled manner. Here, we applied it to Cnp, uncovering two OL enhancers for it (termed Cnp-E1 and Cnp-E2). Epigenome editing analysis revealed that Cnp-E1 and Cnp-E2 are dedicated to Cnp. ATAC-seq and ChIP-seq data show that Cnp-E1 and Cnp-E2 are conserved OL-specific enhancers. Single cell multi-omics data that jointly profile gene expression and chromatin accessibility suggest that Cnp-E2 plays an important role in Cnp expression in the early stage of OL differentiation while Cnp-E1 sustains it in mature OLs.Oligodendrocytes (OLs) produce myelin sheaths around axons in the central nervous system (CNS). Myelin accelerates the propagation of action potentials along axons and supports the integrity of axons. Impaired myelination has been linked to neurological and neuropsychiatric disorders. As a major component of CNS myelin, 2\u2032,3\u2032-cyclic nucleotide 3\u2032-phosphodiesterase (CNP) plays an indispensable role in the axon-supportive function of myelin. Notably, this function requires a high-level expression of Oligodendrocytes (OLs) form myelin sheaths around axons in the central nervous system (CNS) . TraditiCnp knockout mice (Cnp\u2212/\u2212), axoglial interactions, which underlie the domain-specific clustering of ion channels and cell adhesion molecules around the node of Ranvier, were disrupted .2023-05-30_Supplemental_Data_ddad141Click here for additional data file."} +{"text": "Background: Throughout the COVID-19 pandemic, increased inappropriate antibiotic use (AU) drove concern for antimicrobial resistance. Antimicrobial stewardship efforts are critical for combatting antimicrobial resistance. Our objective was to compare AU between SARS-CoV-2 delta and omicron variant surge periods in COVID-19 patients hospitalized at Tufts Medical Center (TMC) in Boston. Infectious diseases consultation (IDC) was mandatory for patients diagnosed with COVID-19 throughout the SARS-CoV-2 delta variant surge. During the SARS-CoV-2 omicron variant surge, IDC was optional for certain patient populations. Instead, the antibiotic stewardship program (ASP) reviewed these patients for appropriate medical management. We hypothesized that AU would increase during the SARS-CoV-2 omicron variant surge compared to the delta variant surge due to optional IDC because IDC would reduce inappropriate AU for suspected viral pneumonia. Methods: Retrospective medical record review of patients hospitalized with COVID-19 during the SARS-CoV-2 delta and omicron variant surges was conducted. We collected data regarding vital signs, white blood cell count (WBC), length of stay (LOS), steroid use, IDC, and AU (defined as percentage of patients receiving at least 1 antibiotic dose), with a separate category for antibiotics commonly used for bacterial pneumonia . We determined that 71 patients from each group were needed to detect an absolute difference of 20% in AU between surges with 75% power, based on the CDC estimate that 80% of patients hospitalized with COVID-19 receive an antibiotic. Unpaired t tests and \u03c72 analyses were conducted on demographic data. Inferential statistics assessed for differences between the 2 SARS-CoV-2 variant surges in AU and days of therapy (DOT), supplemental oxygen (SaO2), steroid use, and IDC utilizing a Wilcoxon rank-sum test and logistic regression analyses. Results: Results showed no significant differences in AU between surges . Disease severity was not different between surges as measured by steroid use, initial WBC, and SaO2. WBC was a predictor for AU in both surges . Average LOS was higher throughout the SARS-CoV-2 delta variant surge for all patients and those who received antibiotics . Total DOT was significantly longer during the SARS-CoV-2 delta variant surge for all antibiotics and antibiotics commonly used for bacterial pneumonia . Conclusions: Making IDC optional for certain patient populations diagnosed with COVID-19 did not affect AU in a large, urban academic medical center with a comprehensive ASP.Disclosures: None"} +{"text": "Chimeric antigen receptor (CAR)-NK cell therapy has the advantages of a low incidence of side effects and a low cost. However, the clinical outcomes are not satisfactory due to limited antitumor effects and a limited proliferative capacity. Recently, progress in CAR-NK cell therapy has been made in NK cell engineering, target design and combination with other agents to treat relapsed or refractory hematological malignancies, especially acute myeloid leukemia and multiple myeloma. This correspondence summarizes the preclinical and clinical updates for universal CAR-NK cell therapy reported at the ASH 2022 annual meeting. To the editor:Chimeric antigen receptor (CAR)-T cell therapy has substantially improved the outcomes of patients with hematological\u00a0malignancies. However, insufficient autologous T cells, an extensive manufacturing time, severe side effects and a high price have restricted the clinical use of CAR-T cells . CAR-NK A new CAR screening platform was established to select an appropriate CAR transmembrane domain and endo-domain from 44 CAR constructs containing NK cell activating receptors, cytokine receptors and integrins to match NK cells through coculture target cell killing assays. The selected structure was tested in several cell lines, including SUP-B15, MOLT-4, and Raji, with different binding antigens and showed improved (more than 20%) antitumor efficacy in all killing assays compared with the reported NKG2D-2B4-CD3\u03b6 CAR structure (Abstract 1983) . The comIn a preclinical study, CD123 CAR-NK cells (5-day OS: 100%) also showed less acute toxicity than CD123 CAR-T cells (5-day OS: 0%) in a mouse model engrafted with human hematopoietic cells, while the antileukemia efficacy was comparable in acute myeloid leukemia (AML) mouse models (Abstract 3279) . AnotherHematopoietic stem cell-derived lymphoid progenitors with Bcl11b inhibition directly differentiate into NK cells rather than T cells, which contributes to the production of stable CAR-NK cells (Abstract 1220) (Table 1A Chinese team presented the initial results of a phase I clinical trial of human umbilical cord-derived CD33 CAR-NK cells for patients with relapsed or refractory AML Table . In 10 eExpansion condition, transduction efficiency, and anti-tumor efficacy are the most significant obstacles for CAR-NK cell therapy. In the future, genetically modified methods, preconditioning regimen, cell dose, and combined immunotherapies or hematopoietic stem cell transplant need to be optimized to improve CAR-NK cell therapy, which requires more study to promote the clinical translation of CAR-NK cells."} +{"text": "Haploidentical donor hematopoietic stem cell transplantation (Haplo-HSCT) has the potential to cure patients with refractory and relapsed (r/r) B and T cell non-Hodgkin lymphoma (NHL). In this matched-pair study, we compared intermediate and long-term outcomes between Haplo-HSCT and HLA-matched related donor (MRD) and unrelated donor (URD) transplantations in patients with r/r lymphoma using age, disease status, lymphoma classification and performance status as matching criteria. While we found comparable outcome and relapse rates among the three groups after >10 years of median follow-up, patients undergoing Haplo-HSCT exhibited lower acute and chronic graft-versus-host disease incidences. Given the rapid and nearly universal donor availability, we propose that Haplo-HSCT is an effective alternative to URD-HSCT in patients with non-remission disease.p = 0.03/0.03) and URD-HSCT , resulting in slightly higher 10-year GvHD-free and relapse-free survival (25%) and chronic GvHD-free and relapse-free survival (25%) in the Haplo-HSCT group. In conclusion, Haplo-HSCT is an effective treatment in patients with non-remission NHL. Given its advantage of immediate availability, haploidentical donors should be preferably used in patients with progressive disease lacking an HLA-matched related donor.Allogeneic hematopoietic stem cell transplantation has demonstrated its potential as a curative option for patients with r/r lymphoma. With the introduction of post-transplant cyclophosphamide-based (PTCY) graft-versus-host disease (GvHD) prophylaxis, allo-HCT using haploidentical related donors (Haplo-HSCT) has emerged as a valuable alternative for patients without an available HLA-matched donor. In this study, we compared intermediate and long-term outcomes between Haplo-HSCT and HLA-matched related donor (MRD) and unrelated donor (URD) transplantations in 16 matched pairs using age, disease status, lymphoma classification and performance status as matching criteria. Of note, 88% of patients in each group presented with active disease at the time of conditioning. After a median follow-up of >10 years, 10-year overall and progression-free survival and non-relapse mortality incidence after Haplo-HSCT were 31%, 25% and 38%, respectively, and did not differ compared to the values observed in MRD-HSCT and URD-HSCT. A remarkable lower incidence of acute GvHD \u2265 II and moderate and severe chronic GvHD was observed after Haplo-HSCT compared to MRD-HSCT (50%/50%, Novel immunotherapeutic drugs, in particular chimeric antigen receptor (CAR) T cells, have emerged as highly promising treatment options for patients with refractory and relapsed (r/r) non-Hodgkin lymphoma (NHL) ,2,3, butFurthermore, retrospective studies have demonstrated similar outcomes, with lower incidence of chronic GvHD in patients with r/r lymphoma grafted from haploidentical donors (Haplo) compared to patients grafted from HLA-related donors (MRD) or unrelated donors (URD) ,22,23,24Eligible were patients (\u226518 years) with r/r NHL undergoing non-myeloablative (NMC) or RIC allo-HSCT between 2000 and 2017 at our institution. Patients with r/r B-NHL who did not receive an anti-CD20 antibody prior to allo-HSCT initiation and/or all patients who underwent a previous allo-HSCT were excluded. Eligible donors included MRD, 8/8 or 10/10 HLA-matched URD or Haplo (related and mismatched \u2265 2 HLA-loci). All patients receiving Haplo-HSCT either lacked HLA-matched donors or required a donor search that was not performable within a reasonable timeframe with respect to the aggressive disease course. Donor-specific antibody (DSA) screening and cytotoxic crossmatch were uniformly performed when patients were evaluated for Haplo-HSCT, whereby Haplo-HSCT donors were considered ineligible if DSAs were found.2) for five days, as previously described [5 CD3+ cells/kg for Haplo-HSCT, 5 \u00d7 105 CD3+ cells/kg for URD-HSCT and 1 \u00d7 106 CD3+ cells/kg for MRD-HSCT and subsequent escalations.Recipients of Haplo-HSCT were limited to those undergoing our center-specific sequential cytoreductive chemotherapy prior to RIC Haplo-HSCT consisting of clofarabine , disease status prior to HSCT initiation, lymphoma classification and patient performance status served as matching criteria between the three groups .r/r NHL was defined as disease relapse/progression after \u22651 cycle of chemotherapy following a previous autologous HSCT or unresponsive to \u22652 cycles of chemotherapy. Active disease was defined when less than a CR was achieved by the most recent treatment prior to HSCT initiation. The Disease Risk Index (DRI) was defined as reported previously . ConsensAntimicrobial prophylaxis and infection surveillance were applied according to local center strategies . ProphylThe Haplo-HSCT group was compared against (1) the MRD-HSCT and (2) URD-HSCT groups. Overall survival (OS) was defined as the time from allo-HSCT without death from any cause. Progression-free survival (PFS) was defined as the time from transplantation without progression of disease/relapse or death. Cumulative incidences (CIs) for relapse/progression and non-relapse mortality (NRM) were calculated considering both events as competing risks. Death and relapse/progression were considered as competing events for acute and chronic GvHD. GvHD-free and relapse-free survival (GRFS) was defined as the time from transplantation without the development of grade III\u2013IV acute GvHD or chronic GvHD requiring systemic treatment, relapse or death, and chronic GvHD-free and relapse-free survival (CRFS) was defined as the time from transplantation without the development of moderate or severe chronic GvHD , relapse or death, as previously described . ProbabiAmong thirty-six recipients of Haplo-HSCT, sixteen patients could be successfully pair-matched with sixteen MRD-HSCT and sixteen URD-HSCT recipients using patient age at HSCT (\u00b15 years), disease status prior to HSCT initiation, lymphoma classification and patient performance status as matching criteria between the three groups .All patients were heavily pretreated, and most patients presented with a high to very high modified DRI score and a high HCT-CI score. Moreover, 88% of patients in each group presented with active disease at the start of conditioning. Patient characteristics are summarized in p < 0.001). There was no significant difference in terms of lymphoma histology and ABO incompatibility between the three groups. Detailed transplantation characteristics are provided in Haplo donors were children (n = 10), siblings (n = 4), parents (n = 1) and other relatives (n = 1) and the median donor age was 27 y (range: 19\u201368 y). Bone marrow (BM) was the preferred graft source in the Haplo-HSCT group, according to the original Baltimore protocol, while most of the MRD/URD transplants were performed with peripheral blood stem cell grafts (p = 0.002) and 14 days (range: 9\u201341) in the URD-HSCT group . Using BM as the predominant graft source in the Haplo-HSCT group, median time to neutrophil engraftment was 18 days (range 13\u201334) in the Haplo-HSCT group compared to 14 days (range: 10\u201322) in the MRD-HSCT group (p = 0.76), with mucositis (25%), creatinine elevation (19%) and transient elevation of liver enzymes (19%) as the most common ones in the Haplo-HSCT group (p = 0.03) and URD-HSCT in the MRD-HSCT group and 0/8 in the URD-HSCT group (p > 0.99) and in four patients of the MRD-HSCT group (p = 0.14). No CMV-associated pneumonia or diarrhea and no post-transplantation lymphoproliferative disorder (PTLD) were diagnosed in the entire cohort. JC and/or BK infections occurred in five patients (31%) in the Haplo-HSCT group, in five patients (31%) in the URD-HSCT group (p > 0.99) and in two patients (13%) in the MRD-HSCT group (p = 0.17). Furthermore, 10 patients were diagnosed with invasive fungal infections, including two (13%) proven and four (25%) possible cases in the Haplo-HSCT group, one (6%) proven case in the MRD-HSCT group (p = 0.07) and one (6%) proven and two (13%) possible cases in URD-HSCT group (p = 0.50).Severe non-hematological related toxicities (grade III\u2013IV) in the Haplo-HSCT group (50%) were comparable to those of the MRD-HSCT (44%) and URD-HSCT groups and URD-HSCT groups and the URD-HSCT group compared to the MRD-HSCT for the MRD-HSCT group and 44% for the URD-HSCT group (p = 0.92); URD, 44% (95% CI: 25\u201376); p = 0.87). Similarly, there was no significant difference in the 5- and 10-year PFS among the three groups for the Haplo-HSCT group compared to 44% comparable OS, PFS and NRM among the three groups, (ii) a trend towards higher GRFS and CRFS in the Haplo-HSCT group, (iii) remarkably lower acute and chronic GvHD incidences in the Haplo-HSCT group and, in particular, (iv) rapid donor availability, our data suggest that Haplo-HSCT is not only an effective treatment in patients with high-risk r/r NHL, but also should be favored over URD-HSCT in patients presenting with active disease requiring urgent treatment. To further elucidate optimal donor strategies including Haplo-HSCT as a first-choice decision, future prospective and controlled randomized studies are needed."} +{"text": "Although pulmonary co-infections are common in patients (pts) with hematological malignancies (HM) and invasive pulmonary aspergillosis (IPA), there is a paucity of recent studies on the type and the impact of co-infections on outcome in contemporary pts with HM and culture-documented IPA.We retrospectively reviewed the records of pts with HM at MD Anderson Cancer Center (January 2016 and October 2021) who had microbiologically-documented IPA (based on sputum and bronchoscopy lavage culture) to identify risk factors, clinical features, evidence of co-infections, and outcome. Independent risk factors for 42-day mortality from IPA diagnosis were assessed using a binary multivariable logistic regression model.Aspergillus fumigatus was the commonest agent of IPA (41%), Fig. 1). Potential co-pathogens were present in 79 pts (62%) at the time of IPA diagnosis. Bacteria (most commonly Gram-negative rods) and less commonly other fungi were isolated in 32 (25%) and 7 pts (5%), respectively . In addition, a variety of upper respiratory viruses and cytomegalovirus were recovered. 42-day mortality after IPA diagnosis was 48%, with no significant difference between pts with and without lung co-infections . Acute kidney injury , Sequential Organ Failure Assessment score \u2265 8 , and recovery from neutropenia were independent predictors of 42-day mortality.Among 128 IPA pts , 48 pts (38%) had neutropenia (< 500/\u00b5L) and 38 (30%) had prior allogeneic hematopoietic cell transplant. Type of Aspergillus spp (number of isolates)Figure 1.Isolated co-pathogens (number of isolates)Figure 2.Co-pathogens and 42-day mortalityTable 1.A quarter of patients with culture-documented IPA had concurrent lung infections with bacteria and less commonly with fungi. In addition, co-culture of upper respiratory viruses or CMV was common. In a background of high mortality of culture-documented IPA, there was no excess mortality if a lung co-infection was present. Further studies are needed regarding the impact of inter-kingdom interactions in pts with earlier, galactomannan-based (but BAL culture negative) IPA diagnosis.Dimitrios P. Kontoyiannis, MD, MS, ScD, PhD, AbbVie: Board Member|Astellas: Grant/Research Support|Cidara: Board Member|Gilead: Grant/Research Support|Merck: Advisor/Consultant|Scynexis/MSGERC: Board Member"} +{"text": "OPAT decreases length of stay/inpatient costs while benefiting patients. However, OPAT costs incurred in the ambulatory setting are poorly quantified. We evaluated unaccounted costs and potential savings from OPAT delivered via patient self-administration (S-OPAT), home care agencies/hemodialysis centers (HH-OPAT), and skilled nursing facilities (SNF-OPAT).The electronic health record (EHR) for all adult patients discharged on OPAT from Parkland Hospital (PH), a 900-bed safety-net hospital, during April - June 2021 and January - March 2022 was reviewed for the number and duration of antibiotics administered and post-discharge non-billable encounters (defined as an encounter on any day without a corresponding billable visit). Encounters on the day of a billable visit were excluded. Inpatient days avoided were defined as days post-discharge on which the patient received OPAT. An average daily hospital adjusted expense per inpatient day of $3,764 for Texas state hospitals and wholesale acquisition cost data from the Texas Department of State Health Services were used to estimate cost savings from OPAT. 340B pricing was used to calculate the cost of drugs provided to S-OPAT patients by PH. Antibiotics with different formulations were converted to equivalent daily doses and costs were averaged to estimate a daily antibiotic cost. The institutional IRB approved this study.Of 340 patients, 84 (25%) received SNF-OPAT, 115 (34%) HH-OPAT, and 141 (41%) S-OPAT. There were 255 non-billable encounters in the S-OPAT group, 242 in the HH-OPAT, and 220 in the SNF-OPAT group, with the highest rate per 100 person-days in SNF-OPAT recipients (Table 1), nearly twice that of S-OPAT. Antibiotic costs avoided by discharging patients on OPAT were $383,240 for HH-OPAT patients and $505,588 for SNF-OPAT patients (Table 2). For S-OPAT patients, costs incurred by PH based on 340B pricing were $33,785.All OPAT care models saved costs compared to hospitalization. Our study likely underestimates the unaccounted costs associated with OPAT; however, costs incurred may be significant and differ between care models. Personnel time and non-billable work should additionally be considered in determining true OPAT cost.All Authors: No reported disclosures"} +{"text": "To the Editor,I read with interest the paper by Saleemi et al., reporting on two women, 80- and 65-year old, who suffered both spontaneous coronary artery dissection (SCAD) and takotsubo syndrome (TTS) . Althoug"} +{"text": "BMC Infectious Diseases (2023) 23:26710.1186/s12879-023-08152-9The original publication of this article contained an incomplete authors name. The incorrect and correct information is listed in this correction article, the original article has been updated.IncorrectSafina Razzak and Farah QamarCorrectSafina Abdul Razzak and Farah N Qamar"} +{"text": "The COVID-19 pandemic has strongly impacted mental health outcomes of healthcare workers (HWs). In spite of the large literature reporting on Post-Traumatic Stress Disorder (PTSD) symptoms, only a few studies focused on potential positive aspects that may follow the exposure to the COVID-19 pandemic, namely post-traumatic growth (PTG) among HWs.In a large sample of Italian HWs, we aimed to investigate the prevalence of PTSD, its correlates and whether PTG dimensions independently affect the risk of PTSD during the first COVID-19wave.An online self-report survey was submitted to HWs throughout physicians\u2019 and nurses\u2019 associations, social networks and researchers\u2019 direct contacts, between April 4th and May 13th, 2020. Sociodemographic data, information about possible COVID-19 related stressful events, Impact of Event Scale-Revised (IES-R) and PTG Inventory-Short Form (PTGI-SF) scores were collected. IES-R and PTGI-SF scores were compared between subjects based on main sociodemographic, work- and COVID-19-related variables using the Student T-test or the one-way ANOVA where appropriate. Post-hoc comparisons were conducted using the Tukey test. Participants with total IES-R score >32 were assigned a provisional PTSD diagnosis and binary logistic regression analysis was conducted to investigate the contribution of each variable to the provisional PTSD diagnosis.Out of 930 respondents, 256 reported a provisional PTSD diagnosis. Female sex (p<.001), separation from cohabiting family (p<.001), family members infected with (p<.05) or deceased due to (p<.05) COVID-19, increased workload (p<.05), relocation to a different work unit (p<.05) and unusual exposure to sufferance (p<.001) were significantly associated with higher IES-R mean scores. The median PTGI-SF score was 24. Factors associated with greater mean PTGI-SF scores were female gender (p<.001), being a nurse (p<.05), being older than 40 years (p<.05), and increased workload (p<.05). The logistic regression model showed that previous mental disorders working in medical , or service units (compared to frontline unit), relocation to a COVID-19 unit , unusual exposure to sufferance and exposure to a traumatic event implying threat to self (compared to other work-related events) significantly increase the risk of receiving a provisional diagnosis of PTSD, while the availability of personal protective equipment and moderate or greater scores on PTGI-SF, particularly in the spiritual change domain , were found to be protective factors in relation to the PTSD diagnosis.Our results shed light on possible protective factors against PTSD symptoms in HWs facing COVID-19 pandemic.None Declared"} +{"text": "Anemia is a common complication of chronic kidney disease (CKD), and its prevalence rises as the disease progresses. Intravenous or subcutaneous erythropoiesis-stimulating agents (ESAs) are advised to treat CKD-associated anemia, since shortage of erythropoietin (EPO) and iron is the main cause of anemia. However, ESA resistance and safety have spurred a lot of interest in the development of alternate anemia therapies. Roxadustat, an orally administered hypoxia-inducible factor prolyl hydroxylase inhibitor (HIF-PHI), which increases erythropoiesis and may modulate iron metabolism, was recently licensed in China, Chile, South Korea, Japan, and the European Union for the treatment of CKD-related anemia. Despite this, clinical trials have shown a number of adverse effects, including cardiovascular disease, hyperkalemia, and infections. Roxadustat\u2019s potential effects on multiple organs and systems are also of concern. In this review, based on clinical evidence, we discuss the potentially detrimental effects of roxadustat to the known biology on systems other than kidney, and the need for long-term follow-up in order for roxadustat to be approved in more countries in the future. Erythropoietin (EPO) deficiency is a predominant cause of anemia in chronic kidney disease (CKD) , 2, but The kidney, the main organ that senses changes in systemic oxygen tension, is the main source of EPO. EPO synthesis is regulated by hypoxia-inducible factor (HIF) in an oxygen-sensitive manner in the kidney , 8. HIF Roxadustat (FG-4592) is the first-in-class of small molecule HIF-PHI. By mimicking \u03b1-ketoglutarate, one of PHD\u2019s substrates, Roxadustat, inhibits PHD and suppresses PHD\u2019s role in regulating the balance between HIF synthesis and degradation rates, thereby correcting anemia. Roxadustat achieved its first approval in China for adults with DD-CKD in December 2018. Next year, it was approved for the treatment of renal anemia for NDD and DD patients in China. Now, roxadustat has also been approved in Japan, Chile, South Korea, and the European Union for the treatment of anemia in CKD in NDD and DD adult patients.Because of the numerous downstream pathways involved, roxadustat may have multiple effects on various biological and physiological processes. Here, we review the function of roxadustat on multiple organs and some safety concerns it brings, chiefly focusing on long-term administration in patients with renal anemia and other renal diseases.Roxadustat is confirmed to improve renal anemia through increasing EPO expression within a physiological range. It reduces elevated hepcidin levels in CKD patients. It also promotes iron release from intestinal cells into the blood and Fe2+ absorption, as well as improves iron transport with roxadustat\u2019s target genes, divalent metal transporter-1 (DMT-1), duodenal cytochrome B (DCytB) . A cliniSimilar to the NDD-CKD clinical trial, hemoglobin response and the increase in the hemoglobin level was more efficient in the roxadustat group compared with the epoetin alfa group (88.4% vs 88.2%) among the 1043 incidences of dialysis for CKD patients. Hepcidin was always at a lower level in the roxadustat group compared with the epoetin alfa group . Other cAnemia is also common in kidney transplant recipients. Early-onset anemia within six months post-transplantation mostly results from operative blood loss, repeated blood sample tests, and rejection, while late-onset anemia is mostly caused by chronic inflammation, allograft renal function reduction, and immunosuppressive therapies. Roxadustat treatment is also confirmed to have a therapeutic effect (improve Hb level and iron metabolism disorder) on post-transplant patients until treatment lasts for 2\u20134 weeks. Even though about 71.4% of patients are responsive, the responsive rate decreased in EPO-resistant patients and in patients with inflammation and impaired renal function , 19. HowAs reported, the dose of roxadustat was not associated with the hs-CRP level in patients with inflammation, while darbepoetin alfa was positively related . UnfortuThe cardiovascular effects of roxadustat are of much attention. No significance of adverse events was found between roxadustat and placebo treatment. However, roxdustat seems not to decrease the incidence of cardiovascular diseases and fatal events compared to the ESA group. Serious adverse events are commonly seen in the roxadustat group vs the Epoetin Alfa group (14.2% vs 10%), among which cardiac and vascular disorders account for about 3.5% , 15, 16.In addition, the incidence of arteriovenous fistula thrombosis seems to be greater in the roxadustat treatment group compared with the epoetin alfa group (9.0% vs 7.3%), even though roxadustat could not enhance platelet activation, production, and function in vivo and in vitro , 24, 25.Based on the protective role exerted by roxadustat in anemia, roxadustat has been proven to be beneficial in anemia patients with lower-risk myelodysplastic syndrome who has low EPO level (<400 m IU/mL) and receive 1\u20134 RBC units infusion per eight weeks NCT03263091). Transfusion independence (TI) was significantly effective in the roxadustat-treated group in pure 91. TransRoxadustat can specifically combine with the hydrophobic space of thyroid hormone receptor-beta (THR\u03b2) through the hydrophobic phenyl extension through a similar structure to T3, and activate the THR\u03b2 transcription, but without THR\u03b1 overstimulation . RoxadusAs reported, the integrity of the intestinal barrier may be mediated by HIF-1/2\u03b1 stability with the barrier protective protein and anti-microbial protein expression . HoweverDue to the multiple target genes and pathways of HIFs, it is understandable that roxadustat can have impacts on many other diseases. Thus, to better use of roxadustat in the clinic, it is important to acknowledge the potential effects of roxadustat on multiple systems and diseases.The pathophysiology of acute kidney injury (AKI) occurrence and AKI to CKD transition is a result of pyroptosis, ferroptosis, and necroptosis . PretreaFurthermore, HIF-1\u03b1 stability is associated with the initiation and expansion of renal cyst through primary cilium loss and endothelial dysfunction in polycystic kidney disease (PKD) . TherefoIt has been demonstrated that HIF-1\u03b1 and the expression level of its target genes increased in heart samples undergoing acute ischemia and early infarction in 2000 . Pre- anPrevious studies have shown that the production of cardiac stem cells doubled at 5% O2 conditions, compared to 20% O2, with less senescent cells, better antioxidant stress resistance, and better recovery . It is kIn the vascular system, roxadustat seems to dilate vascular smooth muscles and alleviates hypertension induced by Ang-II via promoting the release of NO and angiotensin receptor type 1 (AGTR1/2) expression with HIF-1\u03b1 accumulation . In contThe lung is an organ that exchanges oxygen and carbon dioxide. Both hyperoxia and hypoxia may result in lung injury. In preterm, roxadustat can prevent bronchopulmonary dysplasia (BPD) by promoting pulmonary angiogenesis and pulmonary alveolarization . CompareAlzheimer\u2019s disease (AD) accounts for the majority of neurodegenerative diseases. It is characterized by amyloid beta-peptide accumulation in the brain and neurofibrillary tangles composed of hyperphosphorylated protein with an HIF decrease, which can be reversed by roxadustat . HoweverIn contrast to other organs, the effect of Roxadustat on hypoxic-ischemic brain injury remains unknown. HIF-1 reduces hypoxia-induced brain damage when administered prior to ischemia or after four days of hypoxia (>4 days), but it may promote cell death within 24 h of ischemia . The effTraumatic spinal cord injury (SCI) is a devastating disease with no specific effective treatments. Roxadustat treatment inhibits tert-butyl hydroperoxide-induced apoptosis, increases nerve cell survival, and promotes neuronal repair in SCI recovery . RoxadusRoxadustat is also involved in the treatment of depression, which is caused by neurological dysfunction. Roxadustat improves memory impairment by reversing the decline in memory-associated cAMP response element-binding protein (CREB)/brain-derived neurotrophic factor (BDNF) signals in the hippocampus. Similarly, the reduction in synaptic density proteins caused by chronic unpredictable mild stress can also be reversed by roxadustat . SynaptiRoxadustat is also useful in the treatment of many ophthalmic diseases. Meibomian gland dysfunction (MGD) is the major reason for dry eye disease and has no specific treatment. Roxadustat may be a novel method of treatment for MGD because it facilitates immortalized human meibomian gland epithelial cell differentiation and causes acidic conditions that activate DNase II, which is linked to programmed cell death and holocrine secretion. HIF can also boost the content of microbodies and neutral lipids, which eventually improve MGD . CompareEndothelial keratoplasty is the main type of optical surgery conducted for a corneal endothelial disease that can aid in the recovery of the wound and restore vision quickly, but the pressure exerted during surgery may lead to the loss of corneal cells from the donor endothelium. Pretreatment with roxadustat can effectively prevent mechanical stress induced by sonication and diminish endothelial cell loss by up to 23%, protecting endothelial cells from oxidative stress and apoptosis in vitro . HIF-1\u03b1 Mitochondrial respiratory chain monogenic disease, which is characterized by oxygen poisoning caused by impaired oxygen utilization, continuous oxygen transport, and utilization mismatch, is rare but is the largest category of congenital metabolic disorders without an effective method of treatment. Jain et al. found thIn Wilson\u2019s disease, roxadustat can decrease the level of hepatic Cu, improve hepatic steatosis and neurological symptoms by upregulating the expression levels of copper transporters, as well as improving hepatic metabolism by decreasing the expression of lipogenic genes , 82.Roxadustat may be effective for diabetes and diabetic nephropathy through its target genes, glucose transporter (GLUT), and EPO. GLUT-1 improves the absorption of glucose, while GLUT4 reinforces the sensitivity of insulin. Moreover, EPO is involved in restraining inflammation and gluconeogenesis. HIF-2\u03b1 stability decreases postprandial glucagon signaling and gluconeogenesis through ERK 1/2-dependent-phosphodiesterases .The small lumen of tumor vasculature and poor tight junctions result in hypoxia of the tumor microenvironment, which is positively associated with tumor progression and metastasis associated with high interstitial pressure. Roxadustat can normalize tumor microenvironment (TME) and restore tumor blood supply, which may restrain the progression and metastasis of the tumor. Furthermore, roxadustat can directly regulate Ly6Clo macrophages and transform them into the phagocytic phenotype, which can improve the tumor vessel lumen area and activate the phagocytic ability for tumor defense . Even thExcept for the effect on the tumor itself, roxadustat can enhance the antitumor effects of doxorubicin . RoxadusRoxadustat therapy reduces liver I/R injury through improving hepatic cell ballooning and steatosis while also promoting revascularization stability , 90. It Roxadustat administration promotes mature osteoclast bone resorption and new bone formation following a fracture, as well as bone marrow stem cells (BMSCs) proliferation and migration to fracture sites . OtherwiOrally accessible roxadustat appears to open a new door for the treatment of renal anemia in patients with acute or chronic inflammation, even in a dose-dependent manner, based on its positive effects on renal anemia. In addition to its more convenient form, roxadustat is more cost-effective than placebo . CompareAlthough roxadustat\u2019s effects on renal anemia are well-established, its effects on post-transplant anemia remain restricted. There have been very few trials , 19. WhiSome clinical trials suggest that roxadustat is beneficial for treating renal anemia in people with inflammation, however, the sample sizes are insufficient to give conclusive evidence. In addition, larger clinical trials must concentrate on the initial intensity of inflammation and the evolution of inflammation following treatment. Overall, we anticipate more applications for roxadustat in anemia with inflammation, EPO-resistant anemia, and post-transplant anemia.Every coin has two sides to it. Roxadustat\u2019s multi-targeting properties raise some safety concerns. The most common issue is cardiovascular risk, which is related to the rate of Hb improvement and the highest level of Hb. It is critical to understand ways to reduce cardiovascular risk. Although standard-dose treatment may be more successful in increasing Hb, it may be associated with a higher risk of unexpected adverse effects . A lesseIn addition to the recognized effects, hypothetical impacts are combined. Exogenous transplantation of HIF-PHIs may upset the intrinsic equilibrium, leading to the disruption of other pathways and the dysfunction of other organs, including the exacerbation of diabetic retinopathy, the growth of malignancies, and the enlargement of cysts. Candidates for roxadustat treatment should be concerned. Targeted therapy may provide a solution. However, we are still cautious when diabetic nephropathy is the underlying cause of renal anemia. In addition, tests for heart illness, cyst disease, and malignancies are still necessary even when focused therapy is utilized in the clinic.Additionally, Daprodustat, Vadadustat, Enarodustat, and Molidustat are the subject of extensive preclinical and clinical studies. Different drugs inhibit certain PHDs and produce distinct effects. For instance, Daprodustat, which inhibits PHD1 and PHD3 preferentially, demonstrated a risk of cancer in addition to cardiovascular problems. Hypertension is comparatively common in vadadustat. Although several prior publications have outlined the roles of HIF-PHIs in diverse systems , this arAbbreviations

ACE2 angiotensin-converting enzyme-2

AD Alzheimer’s disease

angiotensin receptor type 1/2

AKI acute kidney injury

ALI acute lung injury

AMD age-related macular degeneration

alpha-smooth muscle actin

BMSC bone marrow stem cell

BPD bronchopulmonary dysplasia

CKD chronic kidney disease

cAMP response element-binding protein/brain-derived neurotrophic factor

CRP C-reactive protein

DcytB duodenal cytochrome B

DD dialysis-dependent

DMOG dimethyloxallyl glycine

divalent metal transporter-1

EGFR epidermal growth factor receptor

EPO erythropoietin

EPOR erythropoietin receptor

ESA erythropoiesis-stimulating agent

ESRD end-stage renal disease

GLUT glucose transporter

hypoxia-inducible factor prolyl hydroxylase inhibitor

heme oxygenase-1

ischaemia-reperfusion

IBD inflammatory bowel disease

lectin-like oxidized low-density lipoprotein receptor 1

MGD meibomian gland dysfunction

NDD non-dialysis-dependent

Nrf2 erythroid 2-related factor 2

PASMC pulmonary arterial smooth muscle cell

PD Parkinson’s disease

PDK4 pyruvate dehydrogenase kinase-4

PGF placental growth factor

PHD prolyl hydroxylase

PKD polycystic kidney disease

PRCA pure red cell aplasia

ROP retinopathy of prematurity

ROS reactive oxygen species

SCI spinal cord injury

TH thyroid hormone

THR thyroid hormone receptor

TME tumor microenvironment

TMPRSS2 transmembrane protease serine 2

TSH thyroid-stimulating hormone

VEGF vascular endothelial growth factor

VHL von Hippel–Lindau protein

VSMC vascular smooth muscle cell

"} +{"text": "Acinetobacter baumannii (CRAB) produce carbapenem-hydrolyzing class D \u03b2-lactamases (OXA); other carbapenemase classes have historically been uncommon. We describe the epidemiology and molecular characteristics of New Delhi Metallo-\u03b2-lactamase (NDM)-producing CRAB reported to CDC since October 2013.In the United States, most carbapenem-resistant OX) and core genome (cg) MLST. We compared publicly available sequences of US NDM-CRAB to NDM-CRAB from non-US locations.We included a patient\u2019s first NDM-CRAB isolate reported to CDC through reference antimicrobial susceptibility testing, outbreak response, or Antimicrobial Resistance Laboratory Network, with specimen collection dates during 10/1/2013\u20133/31/2022. Two or more NDM-CRAB linked in space and time were classified as cluster-associated; other NDM-CRAB as sporadic. We described patient demographics and clinical characteristics and analyzed whole genome sequence (WGS) data for relatedness using both traditional multilocus sequence typing (MLST) with the Oxford scheme ; three were closely related to NDM-CRAB isolates from outside the US.CDC received reports of 263 NDM-CRAB from unique patients in 21 states. Patients had a median age of 65 years (IQR: 55-74) (Table 1). NDM-CRAB were from clinical cultures of respiratory (60), wound (44), urine (32), blood (11), and other sites (11) and from rectal or axilla/groin cultures (96). Eleven of 103 patients with information available were hospitalized outside the US \u226412 months prior to NDM-CRAB specimen collection, 82% were sporadic cases and 19% were a part of clusters. Overall, 191 were from cluster-associated cases in four distinct geographic regions that reported multifacility outbreaks; all cluster-associated cases were from cultures collected after June 15, 2019. WGS data were available for 208 (79%) isolates. The four regional outbreaks identified in the epidemiologic investigation were different STNDM-CRAB reported to CDC appears to reflect domestic acquisition rather than importation. ST differences across the regional outbreaks indicate they arose from unique introductions rather than inter-regional spread or dissemination of a successful clone.All Authors: No reported disclosures"} +{"text": "B cells play essential roles in immunity, mainly through the production of high affinity plasma cells (PCs) and memory B (Bmem) cells. The affinity maturation and differentiation of B cells rely on the integration of B-cell receptor (BCR) intrinsic and extrinsic signals provided by antigen binding and the microenvironment, respectively. In recent years, tumor infiltrating B (TIL-B) cells and PCs (TIL-PCs) have been revealed as important players in antitumor responses in human cancers, but their interplay and dynamics remain largely unknown. In lymphoid organs, B-cell responses involve both germinal center (GC)-dependent and GC-independent pathways for Bmem cell and PC production. Affinity maturation of BCR repertoires occurs in GC reactions with specific spatiotemporal dynamics of signal integration by B cells. In general, the reactivation of high-affinity Bmem cells by antigens triggers GC-independent production of large numbers of PC without BCR rediversification. Understanding B-cell dynamics in immune responses requires the integration of multiple tools and readouts such as single-cell phenotyping and RNA-seq,\u00a0in situ analyses, BCR repertoire analysis, BCR specificity and affinity assays, and functional tests. Here, we review how those tools have recently been applied to study TIL-B cells and TIL-PC in different types of solid tumors. We assessed the published evidence for different models of TIL-B-cell dynamics involving GC-dependent or GC-independent local responses and the resulting production of antigen-specific PCs. Altogether, we highlight the need for more integrative B-cell immunology studies to rationally investigate TIL-B cells as a leverage for antitumor therapies. Immuno-oncology research aims to deepen the understanding of the tumor microenvironment (TME) and immune cell interactions and functions to improve cancer patients\u2019 standard of care. Tumor-infiltrating T cells have been the major focus of research and development for immunotherapies. Despite remarkable results, there is still room for improving the response rates of patients treated by T-cell targeting treatments. Tumor-infiltrating T lymphocytes (TIL-T) are not the only immune cells in tumors. B cells also frequently infiltrate solid tumors, either isolated or grouped in ectopic lymphoid formations named tertiary lymphoid structures (TLS). B-cell subsets found in tumors are named tumor-infiltrating B cells (TIL-B). In the last 5 years, numerous publications have demonstrated the favorable prognostic impact of TIL-B cells in several indications and in response to immune checkpoint inhibitors such as anti-PD(L)1 antibodies.Despite their positive prognostic impact, the functions, interplay and dynamics of TIL-B cells in tumors remain unclear. Since B cells are fundamentally important for protective immune responses, the physiological mechanisms leading to the production of plasma cells (PCs), memory B (Bmems) cells and antibodies in secondary lymphoid organs (SLOs) have been under study for several decades. In response to activation by antigens, B cells diversify their BCR repertoires through the mechanism of affinity maturation, which occurs in germinal centers (GCs) and is a tightly regulated spatiotemporal process. Upon reinfection or reimmunization, Bmem cells can quickly differentiate into PCs in GC-independent reactions without further BCR diversification. Little is known, however, if these complex and dynamic processes actually occur within tumors.Here, we review the essential modern knowledge about B-cell physiological dynamics in SLOs and the recent comprehension of TIL-B complex reactivities in tumors elucidated by sophisticated studies that used human samples and murine models. We highlight the different state-of-the-art methods that allow integrative TIL-B characterization in human samples and mouse models. Based on the current knowledge on B-cell responses in SLOs and on the recent TIL-B literature, we propose theoretical models of TIL-B immune responses within tumors and discuss their cellular mechanisms.TIL-B abbreviations: For the sake of clarity, tumor infiltrating B cells are named \u201cTIL-B\u201d. More precisely, among TIL-B cells, tumor-infiltrating memory B cells, plasma cells, germinal centers, and na\u00efve B cells are named TIL-Bmem, TIL-PC, TIL-GC, and TIL-Bnaive, respectively. Tertiary lymphoid structures are named TLSs. Germinal center structures in TLS-positive tumors are named GC-TLS. Conventional germinal center structures found in SLOs are named GC-SLO.B cells play essential roles in immunity, in most cases through the production of high-affinity PC and Bmem cells. We and others have recently reviewed the main mechanisms by which B cells are activated, proliferate and differentiate upon antigen exposure in SLOs \u20133. Here,Na\u00efve B cells are formed in the bone marrow, where they undergo V(D)J recombination of immunoglobulin heavy and light chain genes to express a functional B-cell receptor (BCR) of the IgM isotype on their cell surface. Na\u00efve B cells go through a selection process where their BCR functionality and tolerance toward self-antigens are tested, demonstrating an effective additional step of BCR editing. Newly released na\u00efve B cells develop other peripheral tolerance mechanisms while circulating in the blood and traveling throughout the body to search for antigens. The encounter of a na\u00efve B-cell with a cognate antigen is favored in specialized secondary lymphoid tissues, which are able to attract lymphocytes and concentrate antigens mainly by using antigen-presenting cells such as follicular dendritic cells (FDCs). Na\u00efve B cells are attracted to follicles through their receptor CXCR5, which binds to the CXCL13 chemokine produced by FDCs. Na\u00efve B cells may encounter their cognate antigen at the surface of an FDC. Upon BCR engagement with an antigen, activated B cells migrate to the T-B border of a B-cell follicle to interact and present antigens in the form of peptide-major histocompatibility complex II (pMHC-II) to recruit cognate T follicular helper (TFH) cells.The activation of antigen-specific B cells through BCR signaling and the recruitment of cognate T-cell help may lead to three distinct fates Fig.\u00a0: further+ and IgM+ favoring the PC and Bmem fates, respectively [GCs are microanatomical structures where antigen-specific B cells undergo affinity maturation Fig.\u00a0. This dyectively . Due to Plasmablasts and PCs exit SLOs through the lymphatics and are transiently found in blood but establish long-term residency in specific regions, mostly in the bone marrow, where they are retained through the CXCR4-CXCL12 signaling axis. After being exported from GC reactions, Bmem circulate through the blood and migrate to other SLOs, where they will be ready to detect antigens upon secondary exposure in the future. Upon reactivation, high-affinity isotype-switched Bmem cells may undergo the same three fates as na\u00efve B cells Fig.\u00a0. HoweverAltogether, B-cell responses in SLOs involve GC-dependent and GC-independent pathways yielding short-lived and long-lived Bmem and PC progeny. Expression of a somatically mutated BCR implies affinity maturation through a GC reaction at some stage during the clonal history of the Bmem cell or PC. The pattern of intraclonal BCR variable sequence diversity discriminates recent GC-dependent versus GC-independent activation and differentiation.In recent years, TIL-B cells have been revealed as important players in antitumor responses in human cancers. Several reviews have extensively described their positive prognostic value in several conditions \u201315.+ T cells, suggesting that TIL-B cells could play a role in the response to immune checkpoint inhibitor therapies. A recent large-scale retrospective analysis of three independent cohorts also revealed that the presence of TLS containing germinal centers (GC-TLSs), named mature TLS in this study, was predictive of checkpoint inhibitor efficacy independently of PD-L1 expression or CD8+ T-cell densities [A recent clinical trial studying the impact of pembrolizumab (anti-PD1) combined with low-dose cyclophosphamide in patients with advanced soft tissue sarcoma (STS) demonstrated the clinical value of assessing TIL-B infiltration for patient prognosis . The 30 ensities .Beyond the prognostic importance of GC-TLS, a transcriptional analysis in large cohorts of lung cancer patients revealed that the dominant signature associated with improved overall survival upon anti-PD-L1 treatment was a TIL-PC signature. In this study, the presence of TIL-PCs was also associated with the presence of lymphoid aggregates and TLS .TLS and TIL-Bs are now in the spotlight of immuno-oncology as potential key actors for patient survival and response to ICIs. Nonetheless, their interplay and dynamics within tumors remain largely unknown compared to our deep understanding of B-cell dynamics in SLOs in the context of infectious diseases or vaccination.+CD20+IgD+CD27\u2212CD38\u2212), Bmem (CD19+CD20+IgD\u2212CD27+CD38\u2212), GC\u00a0(CD19+CD20+IgD\u2212CD10+CD38+), plasmablasts (CD19+CD20\u2212IgD\u2212CD27+CD38hi) and PC (CD19loCD20-IgD\u2212CD27+CD38hiCD138+) may be discriminated based on their expression of specific surface markers [The same methods that are used for studying B-cell responses in SLOs are used for studying B-cell responses in tumors Fig.\u00a0. Althoug markers , 22. Amo markers .Fig. 2InMS4A1, CD19, CD79A, CD79B), and PC lineage cells are identified by the high expression of IGH, IGK and IGL transcripts and other genes related to antibody production [IG transcripts than non-PCs and are clustered based on heavy chain and light chain isotypes when IG genes are retained in the highly variable genes computed before dimensionality reduction and clustering; this process leads to different clusters of PC that share the vast majority of their marker genes but differ based on their isotype [IGH and all IGK or IGL transcripts into unique pseudogenes enables us to focus the analysis on gene expression differences that are independent to the BCR isotype class [MKI67 and STMN1). Among non-PC cells, the expression of characteristic marker genes discriminates na\u00efve B cells , Bmem cells , and occasionally GC B cells . Finer subsets and activated states represented by minor populations of cells can be discriminated when large numbers of cells are analyzed [In single-cell RNA-seq, non-PC B-lineage cells are identified by the expression of several B-cell-specific transcripts \u201326. PCs isotype , 27. Colpe class . Among aanalyzed , 29. Sin+ FDCs and/or Ki67+ proliferating B cells [Fresh resection specimens may also be divided into different samples for archiving as fresh-frozen and FFPE blocks. Biopsy specimens are much smaller and usually only conserved as FFPE blocks. Recent imaging and genomics techniques enable the collection of large amounts of descriptive data on small amounts of tissue materials. Compared with single-marker immunohistochemistry (IHC), multiplex immunofluorescence (IF) assays allow the analysis of several markers on a single tissue slide, thereby saving tissue material and enabling the characterization of complex cellular phenotypes in situ. Multiplex IF has already been widely used for the characterization and quantification of TLS in large cohorts of patients , 30, 31. B cells . Bulk RN B cells , 34. If B cells , 36. Spa B cells . The mos B cells but yiel B cells , 40. In Single-cell RNA-seq or flow cytometry studies of fresh tumor samples have detailed TIL-B subset proportions and frequencies in several scenarios.+ cells in fresh tumor samples from NSCLC patients by flow cytometry. Single-cell studies performed by Lambrechts et al. [MS4A1, CXCR4, and HLA-DR, alongside IgG+ and IgA+ TIL-PCs, the latter being named MALT-B cells by the authors. Laughney et al. [+ TIL-Bmems. IgG+ and IgA+ TIL-PCs were equally represented in lung and ovarian tumors and more abundant in CRC samples (more than 50%) because of a higher proportion of IgA+ TIL-PCs. In a head and neck cancer sample, Wieland et al. [+ B cells in a head and neck cancer sample. Interestingly, the frequency of mature TLS containing TIL-GC cells is higher in human papillomavirus (HPV)-positive head and neck tumors [Germain et al. have ides et al. compariny et al. also repy et al. studied y et al. profiledd et al. identifid et al. identifik tumors , and TILk tumors , suggestIL10 transcript to detectable levels were not enriched in a particular cluster (subset) in unsupervised analyses [In inflammatory conditions, subsets of immunoregulatory B cells, mostly characterized by their capacity to produce IL-10 and/or IL-35 and collectively named Breg cells, have been described as critical negative regulators of immune responses , 46. Breanalyses , 50, whianalyses .In summary, flow cytometry and scRNA-seq analyses have revealed that TIL-B subsets are enriched in human solid tumors compared to their nontumoral tissue counterparts, with the most abundant subsets being TIL-PCs and TIL-Bmems cells. TIL-GC was detected in most studies but was often the rarest TIL-B subset. Breg cells may play important regulatory functions but do not correspond to a phenotypically distinguishable subset of TIL-B cells.+) and B (CD20+) cell aggregates, primary-follicle-like TLS (PFL-TLS) containing FDC (CD21+) networks to support TLS organization, and secondary-follicle-like TLS (SFL-TLS) containing GC-like structures (GC-TLSs) with CD23+ TIL-GCs. GC-TLS are also identified by the expression of BCL6 [TIL-B subsets are frequently organized using TLS. TLS organization and the mechanisms driving their formation in tumors have been extensively reviewed elsewhere , 52. TLS of BCL6 , a masteng FDC CD+ network+ CD20+ CD27-), Bmem cells (DAPI+ CD20+ CD27+) and IgA+ or IgG+ PC (DAPI+ CD20- IgA+ or IgG+) in both samples. In rare GC-TLS, we identified germinal center B cells (DAPI+ CD20+ BCL6+), but GC-TLS sizes were much smaller than those of GC-SLOs. Additionally, BCL6+ cell density was lower in GC-TLS. Ruffin et al. [Using multiplex IF imaging Fig.\u00a0, we compn et al. studied n et al. identifiThus, tumor TLS have different degrees of maturity, the most mature TLS presenting GC-like structures and TLS-GC cells with true phenotypic and transcriptional GC markers. However, GC-TLS structures are usually small and seem to lack the DZ-LZ polarization that is characteristic of GC-SLO and required for the cyclic process of affinity maturation.+ cytotoxic T cells) in tumor microenvironments. B cells themselves may express PD-1 in certain activation or inflammatory conditions [hi B cells have been described as playing an immunoregulatory role in hepatomas [Tumor infiltration with B cells is associated with a favorable response to immune checkpoint inhibitor (ICI) anti-PD(L)1 treatment that is designed to reinvigorate exhausted T cells in their study. Indeed, patients with poor CD8+ T-cell infiltration in their tumors had poorer outcomes regardless of TLS status, suggesting that CD8+ T-cell presence was necessary but not sufficient to generate a robust antitumor response. In NSCLC, Thommen et al. [hi CD8+ T cells to recruit B cells via CXCL13 secretion upon anti-PD-1 treatment. The presence of those PD-1hiCD8+ T cells was predictive of patient response to anti-PD-1 immunotherapy.Cabrita et al. showed il et al. revealedl et al. suggesten et al. highligh+ T cells were involved in the initial aggregation and reticular network formation of cancer-associated fibroblasts (CAFs), while B cells recruited via CXCL13 drove CAF proliferation and the expansion of TLS through lymphotoxin beta/lymphotoxin beta receptor (LTBR) crosstalk. In a murine breast cancer model with high mutational burden, Hollern et al. [These descriptive studies in human tumor samples shed light on potential B-T-cell collaboration in antitumor responses upon ICI treatment. Studies in murine models have explored the possible mechanisms that may be involved. Rodriguez et al. used a mn et al. showed t+ T cells are likely involved in the response to ICI treatment.Therefore, functional collaborations between TIL-B and CD8+ T-cell cytotoxicity via granzyme B upregulation, enhancing antitumor responses. In a mouse model of breast cancer treated by chemotherapy, B-cell-specific deletion revealed that ICOSL expression in B cells is important for B-T-cell interactions in TLS [+ B cells led to a higher Teffector/TREG ratio and improved antitumor immunity.Regardless of ICI treatments, several studies based on murine models have shed light on potential T-B cell interactions providing antitumor responses using systemic T-B-cell depletion and adoptive transfer manipulation. Cui et al. studied s in TLS . ICOSL eThese studies highlight B-TFH interactions that are beneficial to the antitumor immune response. It is still unclear whether beneficial B-TFH interactions occur primarily within tumor TLS or in tumor-draining SLOs.There is currently little evidence from studies on human tumors that GC-TLS reactions are productive. The frequent high density of TIL-PCs contrasts with the rare occurrence of low numbers of small GC-TLSs and questions the ability of GC-TLSs to generate such a high number of TIL-PCs. Could the majority of TIL-PCs be generated in GC-independent responses? Or could they be recruited from SLOs in permissive tumor microenvironments?+ fibroblast reticular tracks. The CXCR4-CXCL12 axis is well known for regulating long-lived PCs in the bone marrow. CXCL12+ stromal cells in the medullary area of SLOs are also known to provide signals such as BAFF or APRIL to promote B-cell survival [+ stromal cells in tumors function as a niche to host TIL-PCs that are either generated locally or recruited from the periphery. Meylan et al. [+ tracks extend from TLS areas within the tumor. They also associated the presence of TLS and TIL-PCs with IgG deposits on tumor cells, suggesting that TIL-PCs secrete tumor antigen-specific antibodies. In ovarian cancer, Biswas et al. [In a spatial transcriptomic analysis of human renal cell carcinoma (RCC) samples, Meylan et al. showed tsurvival . Thus, in et al. favor ths et al. showed tAs shown in Fig.\u00a0In conventional GC-SLO reactions generated by primary immunization, the SHM load in Bmem and GC B cells rarely exceeds ten nucleotide mutations in the heavy and light chain variable genes . Higher In all cases, the SHM levels reported are consistent with most TIL-PCs being derived from Bmem cell reactivation and differentiation into PCs and seem to imply a long history of affinity maturation rather than recent output from a primary GC. Bmem cells are able to re-enter ongoing GC under certain conditions but moreWhat antigens are recognized by TIL-B cells? Many tumor-specific antigens were historically identified by screening serum antibodies against tumor cell-derived cDNA expression libraries with the serological identification of antigens by the recombinant expression cloning (SEREX) method . Serum r+ TIL-PCs in the tumor samples. This finding is consistent with the observations from Meylan et al. [Recent reports in mice and human tumor samples , 66, 71 n et al. investign et al. also ideThus, TIL-PC-derived antibodies may target antigens expressed by tumor cells. However, it is unclear what proportion of TIL-B and TIL-PC express tumor-specific antibodies and what antigens are targeted.+ TIL-PCs targeted one of those HPV antigens in tumors and metastatic lymph nodes, with frequencies reaching 10% in some tumors. TIL-PC viral antigen specificity was correlated with serum titers against viral antigens in HPV-positive patients. Overacre-Delgoffe et al. [Helicobacter hepaticus (Hhep), an immunogenic intestinal bacterium, in a mouse model of colorectal cancer. Hhep colonization favored the antitumor immune response by inducing Hhep-specific TFH cells and mature TLS, demonstrating that TLS-GC-dependent B-cell responses targeting nontumor foreign antigens could also benefit the antitumor immune response.Wieland et al. identifie et al. studied HSPA4 protein was identified by Gu et al. as a tumIn ovarian cancer, Biswas et al. used humDespite interesting recent findings, a substantial amount of research is still needed to understand the antigen specificities of TIL-B subsets. To date, there is no way to computationally infer the epitope structure or sequence from the BCR sequence, although that challenge may be solvable by new technologies . PairingRecent evidence has put TIL-B in the spotlight of cancer immunotherapy research. Their organization in mature TLS or the high density of TIL-PCs within tumors are indicators of good prognosis for patient survival and response to ICI treatment. Building on the well-studied B-cell dynamics in SLOs in response to infection or vaccination, the remaining gaps in our current understanding of TIL-B responses are being filled.TIL-B organization as TLS in tumors is heterogeneous. Different degrees of TLS organization are found in tumors, with frequent immature TLS and rare GC-TLS. TIL-B dynamics and clonal diversification in tumors rely either on rare intratumoral GC-dependent reactions and more frequently on GC-independent TIL-Bmem reactivation and differentiation. In both cases, the resulting TIL-B activation relies on the assistance of T cells, and the differentiated TIL-PCs are supported by a supportive stroma, providing a survival niche for local antibody production Fig.\u00a0. In tumoDespite recent progress in our understanding of TIL-B responses, there are still many pending questions regarding the orchestration of B-cell responses within tumors.Since in most cases GC-TLS lack key GC-SLO features such as a DZ-LZ organization, we postulate that even in the rare occasions when GC-TLS are found in tumors, they do not effectively facilitate the differentiation of numerous high-affinity antigen-specific TIL-Bmem and TIL-PC. We propose that some circumstances induce the formation of \u201cperfect\u201d GC-TLS that adopt the spatial organization of GC-SLO to sustain affinity maturation and generation of long-lived TIL-Bmems and high-affinity TIL-PCs from de novo activated Bnaive cells (low levels of SHM) and reactivated Bmem cells (high levels of SHM).How can perfect GC-TLS be generated? Denton et al. studied ICI treatment most likely induces direct and indirect activation of TIL-B, but the precise mechanism and impact on TIL-B and TLS are still unclear. Are ICIs triggering the development of perfect GC-TLS?ICI treatment induces lymphocyte recruitment into tumors in a murine melanoma model, as reported by Asrir et al. . LymphocPerfect GC-TLS require the presence of functional TFH cells for affinity-based selection of TIL-GC B cells. TFH cells express high levels of PD-1 and are The systemic repercussions of TIL-B responses can be tracked by studying the antigen reactivity of serum antibodies in cancer patients. How stable are those systemic antibody responses? Lee et al. studied In the future, next-generation integrative B-cell immunology methods will yield a precise understanding of TIL-B organization as GC-TLS, antigen-specific clonal expansions and differentiation into TIL-PCs before and after treatment with ICIs. Such knowledge will be a remarkable asset to design innovative immunotherapy strategies specifically targeting TIL-B cells, with the potential to durably eliminate cancer."} +{"text": "Endoscopic ultrasonography (EUS)-guided transgastric drainage of pancreatic fluid collections (PFCs) using a lumen-apposing metal stent (LAMS) is an effective treatment for walled-off necrosis (WON)EUS-guided transgastric drainage with a LAMS was performed in a 29-year-old woman, for a WON induced by severe idiopathic acute pancreatitis . A convAn additional LAMS was successfully placed in order to remove the migrated LAMS.\u200aAfter balloon dilation of the LAMS, a 9.8-mm-diameter forward-viewing endoscope was inserted into the WON cavity through this additional LAMS and the migrated LAMS was observed . The miVideo\u20061\u2002Endoscopic retrieval of a migrated lumen-apposing metal stent (LAMS) with a forward-viewing endoscope from a walled-off necrosis cavity, following endoscopic ultrasonography (EUS)-guided placement of an additional LAMS.The use of an additional LAMS was an effective salvage procedure to endoscopically remove a LAMS that had migrated into a WON cavity during an EUS-guided attempt at transgastric drainage of the WON.Endoscopy_UCTN_Code_CPL_1AL_2AD"} +{"text": "Many wastewater-based surveillance (WBS) programs for COVID-19 sample from distal endpoints of sewage treatment systems . SARS-CoV-2 RNA measurements from these endpoints represent highly aggregated cross-sections from entire sewage systems, and are inherently unable to capture local variation in COVID-19 disease activity. Importantly, using highly aggregated samples from sewage system endpoints may limit the utility of SARS-CoV-2 RNA measurements as a leading indicator for forecasting applications. Geographically-focused, hyperlocal sampling in communities with more intense and/or earlier COVID-19 epidemic activity is a promising strategy for enhancing the public health value of WBS.Chelsea versus Boston-wide SARS-CoV-2 wastewater RNA concentration over timeWe examined SARS-CoV-2 RNA data in Chelsea, Massachusetts, a Boston-area community that has experienced disproportionately high COVID-19 disease burden across all phases of the epidemic thus far. Between November 2020 and April 2023, we collected biweekly samples from four local sewershed locations in Chelsea, which were analyzed for SARS-CoV-2 RNA concentration by BioBot Analytics. We compared these local measurements to those for identically analyzed samples collected at a distal sewer system endpoint (Massachusetts Water Resource Authority Deer Island Treatment Plant), for the same time points over the same collection period.Local sewershed SARS-CoV-2 RNA concentrations in Chelsea were consistently higher across all three major epidemic phases analyzed in our dataset. During the onset of the Delta wave, SARS-CoV-2 RNA concentrations in Chelsea increased earlier than Boston-wide levels measured at the sewer system endpoint.Local wastewater sampling can facilitate pre-emptive and tailored public health responses to more effectively counter the burden of SARS-CoV-2 in disproportionately affected communities. Sampling in these \u201csentinel\u201d communities may improve the utility of SARS-CoV-2 wastewater RNA concentrations as a leading indicator for COVID-19 forecasting applications.Julie H. Levison, MD, MPhil, MPH, eMED, LLC.: Advisor/Consultant"} +{"text": "Exploring the regulation of co-inhibitory and co-stimulatory (CD28) genes by chemotherapeutic drugs is important for combined immune checkpoint blockade (ICB) therapy. ICB interferes with T-cell receptor and major histocompatibility complex (MHC) signaling by antibody drugs directed against the co-inhibitors. Here, we analyzed urothelial (T24) cell line with respect to cytokine signaling by interferon \u03b3 (IFNG) and the leukemia lymphocyte (Jurkat) cell line with respect to T-cell activation as mimicked by phorbolester and calcium ionophore (pma/iono). Alongside, we considered possible intervention with the chemotherapeutics gemcitabine, cisplatin and vinflunine. Noteworthy, cisplatin significantly induced PD-L1-mRNA in na\u00efve and IFNG treated cells whereas gemcitabine and vinflunine had no effect on PD-L1-mRNA. At the protein level, PD-L1 showed typical induction in IFNG treated cells. In Jurkat cells, cisplatin significantly induced PD-1-mRNA and PD-L1-mRNA. Pma/iono administration did not alter PD-1-mRNA and PD-L1-mRNA but significantly increased CTLA-4-mRNA and CD28-mRNA levels where vinflunine suppressed the CD28-mRNA induction. In sum, we demonstrated that certain cytostatic drugs being relevant for the therapy of urothelial cancer, affect co-inhibitory and co-stimulatory modulators of immune signaling with potential impact for perspective combined ICB therapy of patients. The immune checkpoint blockade therapy (ICB) targets signaling between T-cell receptor (TCR) and major histocompatibility complex (MHC) by antibody drugs and is applied for the treatment of a growing number of malignancies including urothelial carcinoma . The immIn this scenario, we focused on chemotherapeutic drugs that are recommended for the treatment of urothelial cancer such as cisplatin, vinflunine and gemcitabine . As cellAdherent urothelial cell lines and the suspension T-cell derived Jurkat cell line Jurkat, (DSMZ No. ACC282) were cultured according to protocols by DSMZ, Braunschweig, Germany and as decribed . In subsScreening of several urothelial cell lines displayed different levels of PD-L1-mRNA each with typical induction by IFNG Fig. . For furNext, we analyzed Jurkat cells treated by gemcitabine, cisplatin and vinflunine. We tested control cells and pma/iono treated cells for mimicking T-cell activation. Cisplatin exerted significant induction of PD-1-mRNA and PD-L1-mRNA whereas Pma-iono treatment did not result in changes of PD-1-mRNA and PD-L1-mRNA Fig. . In addiThis study investigated combined effects of IFNG signaling and T-cell activation with cytostatic drugs for gene regulation of immune checkpoint modulators.The cisplatin effects on PD-L1 observed here add to related studies performed on various malignancies. Cisplatin induced PD-L1-mRNA in lung cancer cells and in tumor tissue of cisplatin treated patients . As a reApart from IFNG signaling, several other pathways have been demonstrated to induce PD-L1. The cGAS/STING pathway has been assigned a critical role for cisplatin- induced PD-L1 in ovarian cancer . A downs++ accumulation supporting this pharmacologic intervention as relevant trigger. The cisplatin-dependent upregulation of PD-1-mRNA along with PD-L1-mRNA in Jurkat cells, defines targets related to different signaling pathways. For PD-1 induction, IL-2 and TGF-\u03b21 signaling [In the Jurkat T-cell model, the induction of CD28 and CTLA4 during T-cell activation could beignaling were demignaling were demOf note, a recent experimental study compared combined therapy of anti-PD-1 therapy with either cisplatin or gemcitabine in lung and pancreatic cancer models and patients tissue samples . InteresAs a platinum-based drug, we focused on cisplatin that is the most common applied drug member from the first generation. Alternatively, carboplatin is employed for advanced urothelial cancer patients who are ineligible for cisplatin. Carboplatin displays less systemic toxicity and is administered in patients with poor performance status such as those with restricted renal function. Pharmacologically, cisplatin and carboplatin bind to DNA via intra- and interstranded crosslinks causing DNA damage thereby triggering cell cycle block and apoptosis. Chemically, carboplatin has a \u2018slower leaving group release\u2019 when reacting with nucleophiles such as N7-guanine in DNA and this is attributed to less myelosuppressive related side effects. The common pharmacologic action of cisplatin and carboplatin suggests similar regulation of PD-L1 and PD-1. Variations in \u201cnon-canonical\u201d actions targeting molecules beyond DNA, on the other hand, could differentially affect PD-L1 and PD-1 expression, an as yet undefined and speculative mechanism .In a phase 3 trial of metastatic urothelial cancer (IMvigor130) , combineThe presented experimental data from cancer and immune cells may provide a hint as to how chemotherapeutic drugs can interfere with ICB. Most strikingly, cisplatin interfered with gene regulatory pathways that target immune checkpoints in cancer or immune cells and thereby is connected with ICB.Restrictively, the time period considered in the cell culture studies (24\u2009h) with combined addition of chemotherapeutics, interferon \u03b3 or pharmacologic T-cell activation is shorter than the time period (>weeks) of chemotherapeutic treatment of patients. Our cell experimental strategy aimed to simulate the tumor microenvironment. During tumor progression, intercellular signaling between tumor cells and infiltrating immune cells occurs dynamically and over an extended period of time. These apparent differences point to the limitations of this cell biology study.In conclusion, we demonstrated that certain chemotherapeutic drugs that are relevant for the therapy of urothelial cancer can affect distinct co-inhibitory and co-stimulatory mediators of immune cell signaling . Strikin"} +{"text": "We studied cognitive (thinking) abilities and brain structure and function in older adults with multiple myeloma\u2014a cancer of plasma cells\u2014treated with high-dose chemotherapy and a stem cell transplant. The initial results suggested that after the chemotherapy and transplant, functional connectivity was diminished in regions involving the frontal and parietal lobes of the brain, while brain structure and cognitive function remained relatively stable. We also found increases in markers of inflammation after the transplant. The findings provide supporting evidence for the vulnerability of frontal and parietal brain regions to the side effects of chemotherapy. These preliminary findings would support the design of large future studies with the goal of developing therapeutic interventions.left dorsolateral prefrontal cortex and right posterior parietal cortex (p = 0.022) and (2) the CEN involving the right posterior parietal cortex and the salience network involving the right dorsal anterior cingulate cortex (p = 0.029). There were no significant changes in GM or NF, except for improvements in attention . There were significant increases in several PCy post-HDC/ASCT (p \u2264 0.05). In conclusion, RSFC decreased in frontal, parietal, and cingulate cortices post-HDC/ASCT, NF was relatively stable, and several PCy increased. These findings are congruent with other studies in cancer patients and provide supporting evidence for the vulnerability of frontoparietal regions to chemotherapy\u2019s adverse effects.There is a paucity of research on treatment-related neurotoxicity in older adults with multiple myeloma (MM) treated with high-dose chemotherapy (HDC) and autologous SCT (HDC/ASCT), despite the increasing use of this regimen. We examined resting state functional connectivity (RSFC), gray matter (GM) volume, neurocognitive function (NF), and proinflammatory cytokines (PCy) in older patients with MM pre- and post-HDC/ASCT. Eighteen patients underwent MRI, NF tests, and serum PCy measurements prior to HDC/ASCT, and fifteen patients completed a follow up five-months post-HDC/ASCT. There were significant decreases in RSFC post-HDC/ASCT in (1) the central executive network (CEN) involving the There is compelling evidence that chemotherapy is associated with neurotoxicity , with suNeurocognitive dysfunction has been documented in multiple myeloma (MM) patients after HDC and pre-autologous SCT (ASCT), with declines post-HDC/ASCT ; howeverThere is a paucity of research investigating neurotoxicity in older MM patients undergoing HDC/ASCT, even though this intervention has been used more often in the elderly, and NF has been recognized as a critical dimension of survivorship in older cancer patients . In thisMM patients scheduled for conditioning HDC/ASCT were recruited through the Adult Bone Marrow Transplant Service at Memorial Sloan Kettering Cancer Center (MSK). Eligibility criteria: (1) MM diagnosis, (2) complete, partial, or very good partial disease remission at enrollment, as per the standard International Myeloma Working Group Criteria, (3) aged 60\u201375 at enrollment, and (4) fluent in English. Exclusionary criteria: (1) disease progression during the study period, (2) CNS disease, or (3) history of neurological, psychiatric, or substance abuse disorders.Structural and Functional Imaging. Patients were imaged in Tesla scanners at MSK; five patients were imaged in two different Tesla scanners at each timepoint using the same parameters. Structural Imaging: T1-weighted anatomical images with whole-brain coverage were obtained with spoiled gradient-recalled and high-resolution three-dimensional magnetization-prepared rapid acquisition with gradient-echo sequences. Functional Imaging: For rsfMRI, T2*-weighted images were acquired with a single-shot gradient echo-planar imaging (EPI) sequence . For the rsfMRI, patients were instructed to keep the eyes open and fixated on a crosshair.structural image processing, VBM analysis was performed using the longitudinal processing stream in the VBM8 toolbox under the SPM8 software package within MATLAB . Following reconstruction, follow-up MPRAGE structural images were registered to baseline MPRAGE images for each subject, bias corrected, segmented into GM, WM, and cerebrospinal fluid compartments using the Montreal Neurologic Institute (MNI) T1-weighted template and tissue probability maps\u2014linear and non-linear registered to MNI space\u2014and the resulting GM tissue class smoothed using an isotropic Gaussian spatial filter (FWHM = 8 mm). For rsfMRI pre-processing, a data pre-processing scheme was implemented according to published methods [Image Processing. For methods . Briefly methods and five methods , tempora methods .Neurocognitive Tests and Self-Report Scales. Patients completed standardized neurocognitive tests [ve tests in domaive tests , includiAttention and Working Memory: Longest Digit Span Forward-LDSF; Longest Digit Span Backward-LDSB; and Longest Number Sequencing-LSS (WAIS-IV) evaluate auditory attention and involve the repetition of a series of numbers forwards, backwards, and from lowest to highest; the Brief Test of Attention (BTA) assesses auditory selective attention; the Auditory Consonant Trigrams Test (ACT) assesses auditory attention and susceptibility to the effects of interference.Executive Functions: Trail Making Test Parts A and B (TMTA and TMTB) assess timed visual scanning, graphomotor speed, and set-shifting; the Controlled Oral Word Association Test (COWA) assesses timed phonemic verbal fluency.Verbal Memory: The Hopkins Verbal Learning Test\u2014Revised: Total, Delayed Recall, Discrimination Index is a test of verbal memory. It requires the learning and recall of a word list over three trials and after a delay, and recognizing the words in a forced-choice format.Self-Report Scales: The Center for Epidemiological Study-Depression (CES-D) [ (CES-D) test inv (CES-D) involvesMultiplex Cytokine Panel. Blood samples were collected pre- and post-HDC/ASCT on the same day as the neurocognitive assessment and delivered to the Immune Monitoring Core Facility at MSK for plasma isolation and frozen storage until ready for batch analysis. PCy were quantitated from thawed plasma samples following the manufacturer\u2019s instructions for the V-PLEX Human Proinflammatory Panel 10-plex kit , which included interleukin 1beta (IL-1\u03b2), interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 6 (IL-6), interleukin 8 (IL-8), interleukin 10 (IL-10), interleukin 12 (IL-12), interleukin 13 (IL-13), interferon gamma (IFN\u03b3), and tumor necrosis factor alpha (TNF-\u03b1).Voxel-Based Morphometry (VBM). Following omnibus testing, pairwise t-tests were performed at the group level to analyze within-group changes from pre- to post-HDC/ASCT. For the structural contrast, the initial uncorrected voxel-wise threshold was p \u2264 0.001, with resulting maps family-wise errors corrected over the whole brain at p \u2264 0.05.Resting State Functional Connectivity Analysis (RSFC) was performed using region-of-interest-based correlations, as described previously [eviously . Three reviously and Yangeviously , respecteviously with ther) between each ROI pair in the CEN, SN, and DMN. Each of the pair-wise ROI correlations were Fisher z-transformed for further statistical analysis. Changes in RSFC z-scores from pre- to post-HDC/ASCT were assessed using linear mixed models, adjusting for the scanner .Correlation matrices were produced by extracting the time course from each of the ROIs and computing the Pearson correlation coefficient were calculated to quantify the magnitude of score changes over time. The false discovery rate (FDR) was used to adjust p-values for multiple comparisons. The Reliable Change Index (RCI), which represents the change in scores divided by the standard error of measurement, was used to identify patients whose raw scores improved or declined beyond expected levels due to practice effects and measurement errors. For each test score, we calculated the proportion of patients with RCI-indicated reliable decline.\u00ae software to measure the levels of a ten-PCy panel at each timepoint. A four-parameter logistic (4PL) fit calibration curve was generated for each analyte using the standards to calculate the concentration of each analyte. The upper and lower limits of quantitation for each PCy were established as the highest and lowest points of the standard curve on each plate whose back calculated values were within 80\u2013120% of the expected values of the 4PL regression fit and exhibited less than 20% CV across the duplicate standard wells.Cytokine data was analyzed using the MSD Discovery WorkbenchCorrelations. Spearman correlations were calculated to assess the association of RSN z-scores, neurocognitive test z-scores, self-report scale scores, and PCy levels separately for each timepoint.Eighteen MM patients completed a neurocognitive assessment and a brain MRI pre-HDC/ASCT, and fifteen patients were available for follow up for an average of five months post-HDC/ASCT. One patient was excluded from the imaging analysis due to scan misregistration at follow up. Thirteen patients provided blood samples for PCy analysis at each timepoint. Descriptive statistics for demographic and disease variables are presented in left dorsolateral prefrontal cortex (L-DLPFC) and right posterior parietal cortex , and (2) the CEN ROI involving the R-PPC and the SN ROI involving the right dorsal anterior cingulate cortex from pre- to post-HDC/ASCT (p > 0.05).The results showed significant decreases in RSFC in (1) CEN ROIs involving the HDC/ASCT ; these cp = 0.03); this comparison was no longer significant after multiple comparison correction. There were no significant changes in the CES-D and FACIT-FS scores, and scores were within normal limits at each timepoint [The neurocognitive test z-scores and self-report scales scores are presented in imepoint ,28. TherThe RCI results showed that most patients (>67%) had no reliable change in neurocognitive tests from pre- to post-HDC/ASCT. Patients showed reliable declines on the HVLT-R-D (7%), TMT A (34%), TMT B (13%), and BTA (7%). Reliable improvements were seen with the HVLT-R-T (13%), HVLT-R-D (7%), TMT A (34%), TMT B (20%), and BTA (27%).p \u2264 0.05, FDR-corrected). PCy levels were generally low in most patients at both timepoints, and pre-HDC/ASCT values were below the limits of quantitation for IL-1b, IL-2, IL-4, IL-10, IL-12, and IL-13. There were significant increases in PCy post-HDC/ASCT, including IL-1b, IL-2, IL-4, IL-8, IL-12, IL-13, and TNF\u03b1 (Correlations. There were no significant correlations among RSN z-scores, neurocognitive test z-scores, self-report scale raw scores, and PCy levels either pre- or post-HDC/ASCT.This is the first pilot study describing alterations in RSFC in older MM patients treated with HDC/ASCT. The preliminary results showed decreased connectivity in the CEN involving the L-DLPF and R-PPC and in the CEN R-PPC and the SN R-dACC from pre- to an average of five months post-HDC/ASCT, suggesting that the adverse effects of HDC may be of concern in this population. The CEN has been described as a frontoparietal network involved in attention control, working memory, and processing speed ,30. The A structural neuroimaging literature review on non-CNS cancer patients describeIt is estimated that at least 50% of patients with hematological malignancies experience neurocognitive dysfunction prior to SCT, with either stable performance or declines in the months to years post-SCT ,37. In tCytokine dysregulation is common in MM and may influence the development of adverse effects . High PCIn this pilot study, changes in RSFC were more pronounced than in NF\u2014possibly reflecting the use of compensatory mechanisms to maintain cognitive performance in the context of decreased CEN and SE connectivity , as wellDecreased RSFC in older MM patients following HDC/ASCT provides further evidence for the prevailing notion that frontal-parietal regions may be vulnerable to chemotherapy adverse effects. Longitudinal studies with larger sample sizes and longer follow-up periods are needed to further investigate the neural correlates and trajectory of chemotherapy-related neurotoxicity, as well as the role of PCy in older MM patients. The increasing number of older patients treated with stem cell transplant underscores the importance of investigating treatment adverse effects and identifying patients at increased risk. These studies will guide the development and implementation of targeted interventions to prevent or minimize neurocognitive dysfunction and its impact on quality of life."} +{"text": "Within the generated ST-seq datasets, immune cell types and states, termed here as exhausted/pro-tumour state or non-exhausted/anti-tumour state, were identified using multiple publicly available single-cell RNA and T-cell receptor sequencing datasets as references. HG TMEs revealed abundant exhausted/pro-tumour immune cells with no consistent increase in expression of PD-1, PD-L1 and CTLA4 checkpoints and angiogenic genes. Additional HG TME immunophenotype characteristics included: pro-tumour tissue-resident monocytes with consistently increased expression of HAVCR2 and LAG3 checkpoints; an exhausted CD8+ T cells sub-population with stem-like progenitor gene expression; and pro-tumour tumour-associated macrophages and monocytes within the recurrent TME with the expression of TREM2. Whilst limited by a modest sample size, this study represents the largest ST-seq dataset on human ccRCC. Our study reveals that high-risk ccRCC TMEs are infiltrated by exhausted/pro-tumour immunophenotypes lacking specific checkpoint gene expression confirming that HG ccRCC TME are immunogenic but not ICI favourable.Perioperative immune checkpoint inhibitor (ICI) trials for intermediate high-risk clear cell renal cell carcinoma (ccRCC) have failed to consistently demonstrate improved patient outcomes. These unsuccessful ICI trials suggest that the tumour infiltrating immunophenotypes, termed here as the immune cell types, states and their spatial location within the tumour microenvironment (TME), were unfavourable for ICI treatment. Defining the tumour infiltrating immune cells may assist with the identification of predictive immunophenotypes within the TME that are favourable for ICI treatment. To define the immunophenotypes within the ccRCC TME, fresh para-tumour (pTME, Approved first-line systemic therapy for metastatic ccRCC includes immunotherapies such as immune checkpoint inhibitors (ICIs) that serve to reactivate anti-tumour immune responses10. However, the final results from adjuvant ICI trials for localised ccRCC have failed to demonstrate an improvement in survival outcomes13. The efficacy of perioperative ICIs within intermediate high-risk ccRCC patients has been assessed in randomised phase III clinical trials17. These perioperative (or neoadjuvant) ICIs are aimed at priming the anti-tumour immune response without delaying surgical intervention17. A recent neoadjuvant nivolumab trial for localised ccRCC reported no delays in surgery16. However, due to the short-term follow-up in this trial, it is uncertain if an anti-tumour immune response was initiated by earlier ICI treatment.Clear cell renal cell carcinoma (ccRCC) accounts for the majority of kidney cancer-related deaths due to the presence or development of metastatic disease+ T cells, tumour associate macrophages (TAM) and monocytes may clarify the precise immunophenotypes within the intermediate high-risk TME. The advancement within transcriptomics methodologies makes it feasible to profile the ccRCC tumour infiltrating immunophenotypes\u2014defined here as the immune cell types, exhausted/pro-tumour or non-exhausted/anti-tumour states, and their spatial location within an individual patient\u2019s TME is an urgent unmet need for personalising oncology in high-risk ccRCC patients. Defining the TME-specific CD8ME Table . This stME Table . We hypop-value 2.9e-10) from para-tumour (pTME), low-grade (LG) and high-grade (HG) TMEs and macrophages within LG TMEs . Conversely, the highest exhausted CD8+ proliferative cells were found within HG TMEs . TAM and monocyte sub-typing within the LG TMEs . However, the highest tissue-resident monocytes were found mainly within HG TMEs . Interestingly, the CD8+ T cells, TAM and tissue-resident monocytes identified within the LG TMEs were mirrored within the HG_2 TME. The proportion of immune cell sub-types was based on the immune cell types and immunomodulatory marker ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1). Nonetheless, the TCF7 revealed the highest relative expression within sub-populations of CD8+ exhausted immediate-early genes (CD8+ exhausted IEG) and CD8+ exhausted T cells in HG_1.2 and HG_1.3 TMEs , a known marker for recurrence, was identified within a larger proportion of pro-tumour TAM sub-types in HG_3 TME; whilst an absence of immunosuppression markers human leucocyte antigen G (HLA-G) and cysteine protease cathepsin S (CTSS)19 was observed within TAM ISG high expression (TAM ISGhi) in HG_3 and HG_2 TME, respectively and novel IC genes. We determined the relative expression of targetable and novel IC genes within grouped CD8+ T cells and novel angiogenic genes within grouped CD8+ T cells , we show predominantly exhausted CD8+ T cells and pro-tumour tissue-resident monocytes within the tumour/immune regions. In line with previous studies, we show a sub-population of exhausted CD8+ T-cell sub-types expressing stem-like progenitor (TCF7). Both sub-populations of exhausted and non-exhausted CD8+ T-cell sub-types express immunomodulatory (ENTPD1) genes29. The pro-tumour TAM and monocyte sub-types express an anti-inflammatory gene signature30. Similarly, TREM2, an established marker for post-surgical recurrence of ccRCC30, shows higher expression in a large proportion of the pro-tumour TAM and tissue-resident monocytes for patient HG_3 with recurrent ccRCC. Furthermore, within distinct tumour/immune admixed regions, the tissue-resident monocytes surround the exhausted CD8+ T cells, suggestive of anti-inflammatory activity and/or an exclusion zone along the tumour margins in HG TME, as identified in other studies32. Importantly, we demonstrate a singular exhausted/pro-tumour or non-exhausted/anti-tumour immune cell states within individual specimens, suggesting that lymphoid and myeloid cells behave in unison, possibly through cellular interactions between the immune cell sub-types, as identified in previous studies33.In this study, we present the largest ST profile of immunophenotypes within para, low- and HG ccRCC TMEs. Consistent with previous bulk RNA-seq, single-cell RNA-seq (scRNA-seq) and single-nuclei RNA-seq (snRNA-seq) studies, we show abundant immune cell infiltration within the TME, dominated by T cells and monocytes+ T cells of the HG ccRCC TME, we show sub-populations expressing stem-like progenitor TCF7, suggestive of heterogeneity within the exhausted cell states and a capability of T cells to differentiate. It will be of interest to investigate within ex vivo studies, if these exhausted stem-like progenitors CD8+ T cells can be switched to an anti-tumour mode with ICI, as reported in other cancers39. The effectiveness of ICI is based on the expression of IC genes by infiltrating immune cells, as such we examined the expression of targetable and novel IC genes in grouped CD8+ T cells, TAM and monocytes across all samples. Targetable IC genes show no increase in expression within CD8+ T cells, TAM and monocytes, potentially explaining the limited efficacy of ICI40 observed in three adjuvant ICI trials13. However, these trial findings are in contrast with the Keynote-564 trial41. These conflicting trial outcomes of ICIs within intermediate high-risk ccRCC do not diminish the reported improved survival outcomes within the metastatic setting44. This further highlights a need for a precision oncological approach that integrates traditional histopathological and clinical information with systematic transcriptomics profiling.Within the exhausted CD8HAVCR2 (or TIM3) shows consistently increased expression within the tissue-resident monocytes which dominated the HG TMEs. Traditionally, tissue-resident monocyte populations are considered reservoirs for macrophages that play key roles in immune defence and inflammatory events45. However, it is uncertain if tissue-resident monocytes have the plasticity to differentiate into macrophages and later TAM within the ccRCC TME. Mouse model studies have demonstrated that tissue-resident monocyte- and macrophage-derived TAM respond very differently to cancer treatment46. Further exploration of the fate of these tissue-resident monocytes in the HG ccRCC TME is needed to elucidate their response to ICI treatment. As such, we are interested in the results of the phase I and II anti-HAVCR2 trial (NCT02817633) in advanced solid tumours. Looking beyond potential IC targets, we extended our investigation to also include the expression of targetable and novel angiogenic genes. However, immune cells show no consistent increased expression of angiogenic genes, suggesting that the promotion of angiogenesis within the ccRCC TME may involve non-immune cells.Novel IC Limitations in this study include minimal multi-region sampling at a single timepoint from a modest cohort of consenting ccRCC patients and variable tumour stages within both the LG and HG ccRCC groups. These limits may have restricted complete molecular profiling of the heterogeneity and complexity of the intermediate high-risk ccRCC TME. Nonetheless, we show intra- and inter-patient heterogeneity within the identified immunophenotypes of the limited TME regions presenting at variable time points . Therefore, our decision to use minimal multi-region sampling from six ccRCC patients for ST-seq preserves surgical margins, delivers uncompromised patient management, and still captures transcriptomic heterogeneity within the immunophenotypes present in the ccRCC TME. In future, the use of non-clinical mouse models of ccRCC can overcome these limits by allowing sampling from multi-regions and timepoints at similar tumour stage or a large clinical human cohort.In summary, unbiased transcriptomic profiling identified heterogeneous immunophenotypes, without a consistent targetable IC expression profile, within intermediate high-risk ccRCC TMEs. Translating these findings in the future might result in identified immunophenotype characteristics in ccRCC that are favourable for ICI treatment and deliver precision oncology.+ T cells, TAM and monocytes within the TME due to their recognised effector and tolerogenic response, defined here as non-exhausted/anti-tumour and exhausted/pro-tumour states, respectively presenting at a tertiary referral hospital from June 2021 to January 2022 on ten fresh frozen tissue sections collected in the operating theatre from six patients with ccRCC and imaged on an Axio Z1 slide scanner (Zeiss). Afterwards, the same tissue sections were permeabilised to release mRNA. These mRNA were captured by underlying ST-spots, and complimentary DNA libraries incorporating the spatial barcodes were synthesised. All libraries were loaded at 1.8 pM and sequenced using a Mid output reagent kit (Illumina) on a NextSeq500 (Illumina) instrument. Sequencing was performed using the following protocol: Read1 - 28\u2009bp, Index1 - 10\u2009bp, Index2 - 10\u2009bp, Read2 - 120\u2009bp. After sequencing the genes were mapped to the H&E images to generate spatially resolved transcriptional profiles involved the use of microarrayed glass slides with 55\u2009\u00b5m spots (or ST-spots) containing oligonucleotides with a sequence of deoxythymine (oligo-dT) and unique spatial barcodes printed within capture arrays. Thin 8\u2009\u00b5m cryosections were placed within a capture array overlaying the ST-spotsles Fig. 54.55 and Seurat (v4.1.0) R packages57. We confirmed the quality of the captured transcriptome using the following cut-offs: >50 genes per ST-spot, >100 unique molecular identifiers (UMI) per gene, \u2265500 nCount per ST-spot, >500 nFeature per ST-spot and <30% mitochondrial genes per ST-spot. Then, we merged all the individual ST-seq datasets and removed batch effects due to individual patient samples using the SCTransformfunction in Seurat58. With this merged ccRCC ST-seq dataset, Louvain clustering was performed with the most stable cluster resolution (res 0.4). These clusters were then annotated as immune and non-immune using published kidney and tumour immune atlas52 scRNA-seq datasets.The generated ST-seq datasets were processed and analysed using STUtility (v0.1.0)26 sequencing dataset from ccRCC patients that were ICI na\u00efve or exposed. In brief, the scRNA/TCR-seq dataset contained curated transcriptome signatures for exhausted or non-exhausted lymphoid and pro- or anti-tumour myeloid cell sub-types. Based on these signatures, we annotated the cell states of the T cells, macrophages and monocytes using the Semi-supervised Category Identification and Assignment (SCINA) algorithm59. In brief, this method leverages previously established gene signatures in a semi-supervised model using an expectation\u2013maximisation (EM) algorithm. Next, we focused on the CD8+ T cells, TAM and tissue-resident monocytes ; and non-exhausted state are: CD8+ tissue-resident and CD8+ NK-like. Myeloid cell sub-types with a pro-tumour state are: TAM with human leucocyte antigen DR (HLA-DR) high expression (TAM HLAhi), TAM with HLA-DR intermediate expression (TAM HLAint), TAM with interferon signalling genes high expression (TAM ISGhi) and CD14+/16+ monocytes termed tissue-resident monocytes; and anti-tumour state TAM ISG intermediate expression (TAM ISGint). This classification of exhausted/pro-tumour or non-exhausted/anti-tumour immune cell states is not static. Indeed, a spectrum of immune cell states is being recognised60. However, for brevity, here we utilised exhausted or non-exhausted states for five CD8+ T-cell sub-types and pro- or anti-tumour states for five TAM and tissue-resident monocyte sub-types identified within our ST-seq datasets es Table . CD8+ T-Further information on research design is available in the Supplementary MaterialReporting Summary"} +{"text": "A 35-year-old man presented with a 1-month history of bilateral lower limb weakness and numbness. On examination, he had paraparesis with lower limbs hyperreflexia, and T5 sensory level. Magnetic resonance imaging (MRI) of the spine revealed a longitudinally extensive myelitis (LETM) with a diffuse leptomeningeal enhancement . ThoraxLeptomeningeal involvement is a remarkable finding in neurosarcoidosis."} +{"text": "However, on 68Ga-Pentixafor PET/CT, all the myeloma lesions showed significantly lower tracer uptake in comparison with 18F-FDG PET. This false-negative result of recurrent multiple myeloma with extramedullary disease may be a potential limitation of 68Ga-Pentixafor in assessing multiple myeloma.Two patients with a history of multiple myeloma experienced a recurrence of the disease."} +{"text": "Pharmacy antimicrobial stewardship services were extended to cover the weekends, incorporating a clinical staff pharmacist with infectious disease (ID) training to cover antimicrobial stewardship program (ASP) activities on weekends is an opportunity. The aim of this study is to assess the impact of integration ASP clinical staff pharmacist into an ASP on weekends by collecting and analyzing the documented pharmacists\u2019 ASP interventions before and after implantation of the service.A single center, pre-post quasi-experimental study. Data were collected retrospectively from the electronic medical record (EMR). The study included at 2 sets of data: pre-implementation (2020) and post-implementation (2021) of an ASP weekend pharmacist,Clostridioides difficile infection (CDI) and infection-related readmission.The primary outcome is to evaluate pharmacist ASP Interventions through prospective audit and feedback review analysis. Secondary outcomes include antibiotics days of therapy (DOT), length of hospital stay (LOS), healthcare associated During the study, an increase in the number of documented interventions was observed with 452 interventions were documented on 362 patients during the post-implementation period compared to 114 interventions were documented on 108 patients during the pre-implementation period (p = 0.04).Clostridioides difficile infection (CDI) and infection-related readmission.A reduction in the LOS was observed with a median (IQR) of 16 days (8-34) during the post-implementation period compared to 27.5 days (10-56) during the pre-implementation period (p = 0.001), while the median (IQR) total DOT was increased during the post-implementation period 8 (6-11) versus 7 (4-11) during the pre-implementation period (p = < 0.001). No differences were observed in healthcare associated The pharmacists driven weekend antimicrobial stewardship is an opportunity for the pharmacists to intervene and optimize patients\u2019 treatment plan and contributed towards a significant shortened length of hospital stay. Therefore, healthcare facilities should prioritize the involvement of pharmacists in weekend ASPs to ensure consistent, high-quality care throughout the week.All Authors: No reported disclosures"} +{"text": "Objectives: Data comparing the immunogenicity of Sputnik-V and Sinopharm vaccines in seropositive and seronegative groups are lacking. We compared the immunogenicity of Sputnik-V (Gam-COVID-Vac) and Sinopharm (BBIBP-CorV) vaccines in seronegative and seropositive groups. Methods: In total, 60 adults participated the study. The immune response after vaccination was assessed using enzyme immunoassay. IgG levels were measured in all participants at 3 time points: before vaccination, 42 days after the first vaccine dose, and 6 months after the first vaccine dose. The results of the SARS-CoV-2 antibody test were quantified according to the WHO First International Standard and expressed in international units (BAU per mL). Results: The study participants were divided into 2 groups: 30 people (50%) were vaccinated with Sputnik-V (Gam-COVID-Vac) and 30 people (50%) were vaccinated with Sinopharm (BBIBP-CorV). The groups had no difference in sex composition. The highest antibody levels were observed 42 days after vaccination in both the seronegative group (P = .006) and the seropositive group (P < .001). At 6 months after vaccination, the IgG value declined much farther among the seronegative group (P = .003) compared to those who had recovered from COVID-19 before vaccination. However, the \u201chybrid immunity\u201d generated by the Sputnik-V vaccine had greater strength and duration (P < .001). Conclusions: This study showed that IgG levels in vaccinated individuals who previously recovered from SARS-CoV-2 infection (\u201chybrid immunity\u201d) were higher than in SARS-CoV-2\u2013na\u00efve individuals. In a comparative part of the study, the Sputnik-V vaccine had greater strength and duration of immune response across the 6-month observation period (P < .001)."} +{"text": "Dianthus caryophyllus (carnation) leaves. The CarSV-1 genome has notable sequence similarity (62%) to the well-studied CarSV viroid-like RNA and comprises the complete hammerhead consensus sequences involved in self-cleavage. CarSV-1 co-occurs with carnation viruses, such as CarMV.Here, we report the genome sequence of a new circular viroid-like RNA (CarSV-1) derived from Carnation mottle virus (CarMV+), and two plants were uninfected , without a protein coat . Small vinfected . The preinfected . All invinfected and siRN2 leaf fragments into powder using a mortar and pestle and dissolving the powder in QIAzol lysis reagent (Qiagen) for RNA isolation and small RNA enrichment using the miRNeasy minikit (Qiagen). Barcoded small RNA transcriptome sequencing (RNA-seq) libraries were generated using the Ion total RNA-seq kit v2 and Ion Xpress RNA-seq barcoding kit (Thermo Fisher Scientific) and sequenced . Adapter and quality trimming were performed using Torrent Suite v5.8.0 software (default parameters), resulting in 3.3 to 5.3 million reads per sample to the CarSV sequence and had an identical GC content (51%). Furthermore, it contained the complete hammerhead consensus sequences for both the plus and minus RNA strand forms 47. This snd forms ; 8034 47. GTGTAGATTCCAGGCTACGGC; reverse primer, TGCCACGTAGCCTCAAGG; probe, TTGGGTCCTCGATGATGGGA) confirmed the presence of CarSV-1 RNA in the CarMV+ samples, with threshold cycle (TC) values of 16 to 18 cycles, as has been observed for CarSV (Carnation etched ring virus (CERV) (Alternanthera mosaic virus (AltMV), based on the observed co-occurrence in this study.Reverse transcriptase quantitative PCR (RT-qPCR) analysis using a CarSV-1-specific primer/probe combination , 9, CarSPRJNA885393. The genome nucleotide sequence of CarSV-1 has been deposited under GenBank accession number OP506135.The raw sequence reads have been deposited in the NCBI Sequence Read Archive under BioProject accession number"} +{"text": "This is due to the strong correlation between the efficacy of PRRT and an SUVmax TLR\u2009>\u20092.2 [max TLR\u2009\u2265\u20098.1 has been associated with extended progression-free survival (PFS) in individuals with NET who are undergoing treatment with SSAs [68Ga]Ga-DOTA-TATE in patients receiving SSAs compared to those not receiving SSAs [The use of the positron emission tomography (PET) tracer [s (NETs) . PET-SSTs (NETs) . Somatoss (NETs) . These Ss (NETs) . The maxLR\u2009>\u20092.2 . Furtherith SSAs . Howevering SSAs . Consequ68Ga]Ga-DOTA-TATE varied between 10 and 60%, but the precise mechanism underlying this phenomenon remains unclear [The reduction in liver uptake of [ unclear . It has unclear . On the 68Ga]Ga-DOTA-TATE may be influenced by its metabolism in the liver [68Ga]Ga-DOTA-TATE, thereby affecting its decreased hepatic uptake. Based on these findings, our objective is to investigate the impact of CYP3A4 inhibitors on [68Ga]Ga-DOTA-TATE liver uptake.Another interesting hypothesis proposes that liver uptake of Ga-DOTA-TATE PET/CT scan between July 2016 and May 2023. As the administration of SSAs is known to be associated with a decreased [68Ga]Ga-DOTA-TATE hepatic uptake [68Ga]Ga-DOTA-TATE PET/CT imaging Ga-DOTA-TATE, treatment with [177Lu]Lu-DOTA-TATE, and medication use Ga-DOTA-TATE, conducting the PET/CT procedure, and performing image analysis have been extensively described elsewhere [max) of [68Ga]Ga-DOTA-TATE was calculated on standard iterative reconstructions with a spherical volume of interest (VOI) tool in the primary tumour, the liver and the left psoas major muscle (i.e. background). The following parameters were determined:max tumour-to-background ratio (SUVmax TBR)\u2009=\u2009SUVmax primary tumour/SUVmax psoas major muscleSUVmax liver-to-background ratio (SUVmax LBR)\u2009=\u2009SUVmax liver/SUVmax psoas major muscleSUVmax tumour-to-liver ratio (SUVmax TLR)\u2009=\u2009(SUVmax primary tumour)/(SUVmax liver)SUVThe procedures for labelling Ga-DOTA-TATE PET/CT. To the best of our knowledge, patients included in the study were taking the following medication, including the averaged prescribed dosages, at the time PET/CT imaging: amiodarone (200\u00a0mg/day), verapamil (180\u00a0mg/day), clarithromycin (750\u00a0mg/day), and ketoconazole (1200\u00a0mg/day). Among the CYP3A4 users group, a total of 10 patients exhibited no tumour. No significant differences were observed between the two groups in terms of primary tumour location, primary tumour resection, metastasis, WHO NETs grade, tumour stage, administered [68Ga]Ga-DOTA-TATE activity (MBq/kg), and [68Ga]Ga-DOTA-TATE peptide amount (ng/kg). These findings, as shown in Table A cohort of 70 patients underwent PET/CT scans that met the inclusion criteria and were consequently enrolled in the study. The characteristics of these patients during the PET/CT scan are summarized in Table erapamil 80\u00a0mg/daymax TBR between patients who used CYP3A4 inhibiting medication during the [68Ga]Ga-DOTA-TATE PET/CT and patients without CYP3A4 inhibitors (Table max LBR between patients who did not use CYP3A4 inhibiting medication and those who used CYP3A4 inhibitors. Furthermore, the median SUVmax TLR also showed no significant difference between the two groups under study.There were no significant differences in the median SUVrs Table . Similar68Ga]Ga-DOTA-TATE uptake. Our findings revealed no significant difference in SUVmax LBR between patients on CYP3A4 inhibitors and patients without CYP3A4 inhibiting medication at the time of [68Ga]Ga-DOTA-TATE PET/CT imaging. Additionally, there were no significant differences observed in SUVmax TBR and SUVmax TLR between these two groups.To the best of our knowledge, this study is the first study investigating the effect of CYP3A4 inhibiting medication on [68Ga]Ga-DOTA-TATE remains largely unknown [177Lu]Lu-DOTA-TATE does not undergo hepatic metabolism. Instead, it is primarily excreted as an intact compound through the renal route [68Ga]Ga-DOTA-TOC also follows a similar pattern of being excreted unchanged via the kidneys Ga-DOTA-TATE in the liver, leading to lower hepatic absorption of the tracer. However, our findings indicate that the observed percentage decrease in liver uptake of [68Ga]Ga-DOTA-TATE in the literature cannot be explained by the mechanism of CYP3A4 inhibiting. In the literature, the mean percentage decrease in liver uptake of [68Ga]Ga-DOTA-TATE after use of SSAs is reported to range from 10 to 60% [Our initial hypothesis suggested that the use of CYP3A4 inhibitors would result in decreased metabolism of [0 to 60% . If this68Ga]Ga-DOTA-TATE before and after the administration of CYP3A4 inhibitors within the same patient. Furthermore, the utilization of various PET/CT scanners throughout the study duration adds complexity to the comparison of SUVmax values. Nevertheless, the variation in PET/CT scanners is representative of real-world practice and is commonly acknowledged as a limitation in multicentre clinical trials. To address this issue, we used both tumour-to-background and tumour-to-liver ratios, which minimize discrepancies between different imaging systems and potentially facilitate the generalization of our findings to diverse clinical settings Ga-DOTA-TATE liver uptake. Therefore, we can conclude that CYP3A4 inhibition is an unlikely explanation for the observed decrease in hepatic uptake of [68Ga]Ga-DOTA-TATE in patients with SSAs.In conclusion, our study demonstrates that there is no significant effect of CYP3A4 inhibitors on ["} +{"text": "GBA1, LRRK2, and Parkin genes. In this review, we will highlight the contributions of various neuroimaging modalities, including positron emission tomography (PET), single-photon emission computed tomography (SPECT), and magnetic resonance imaging (MRI), in elucidating the underlying pathophysiological mechanisms and potentially identifying candidate biomarkers for genetic forms of PD.Parkinson\u2019s disease (PD) is a complex neurodegenerative disorder characterized by motor symptoms such as bradykinesia, rigidity, and resting tremor. While the majority of PD cases are sporadic, approximately 15\u201320% of cases have a genetic component. Advances in neuroimaging techniques have provided valuable insights into the pathophysiology of PD, including the different genetic forms of the disease. This literature review aims to summarize the current state of knowledge regarding neuroimaging findings in genetic PD, focusing on the most prevalent known genetic forms: mutations in the Parkinson\u2019s disease (PD) is a multifactorial disorder influenced by both genetic and environmental factors. PD is considered the second most common neurodegenerative disorder . While tPARK2), PINK1 (PARK6), and DJ-1 (PARK7) gene mutations are frequent forms of autosomal recessive genes associated with the risk for PD. Clinically, patients harboring these mutations manifest with earlier age of disease onset and milder disease course with higher rates of responsiveness to levodopa treatment ; [(MRI) AND (LRRK2)AND(parkinson\u2019s disease)]; [(nuclear imaging) AND (Genetics)AND(parkinson\u2019s disease)]; [(nuclear imaging) AND (GBA)AND(parkinson\u2019s disease)]; [(nuclear imaging) AND (LRRK2)AND(parkinson\u2019s disease)]; [(nuclear imaging) AND (PARKIN)AND(parkinson\u2019s disease)]; [((Neuroimaging) AND ) AND (Parkinson\u2019s disease)]; [imaging biomarkers in prodromal PD]; [(DTI) AND (genetic Parkinson\u2019s disease)]; [(DTI) AND ]; [(DAT SPECT) AND (Parkinson\u2019s disease genetics)]; [((MRI) AND (LRRK2)) AND (non-manifesting carriers)]; [((MRI) AND (GBA)) AND (non-manifesting carriers)]; [(FDG-PET) AND (genetic Parkinson\u2019s disease)]; [(FDG-PET) AND (genetic Parkinson\u2019s disease)]; [(FDG-PET) AND ]; [((DAT SPECT) AND (GBA)) AND (Parkinson\u2019s disease)]; [((DAT SPECT) AND (LRRK2)) AND (Parkinson\u2019s disease)]; [((DAT SPECT) AND (PARKIN)) AND (Parkinson\u2019s disease)]; [((FDG-PET) AND (PARKIN)) AND (Parkinson\u2019s disease)]; [((FDG-PET) AND (PARKIN)) AND (Parkinson\u2019s disease)]; [((FDG-PET) AND (LRRK2)) AND (Parkinson\u2019s disease)]; [((FDG-PET) AND (GBA)) AND (Parkinson\u2019s disease)]; [ AND (GBA Parkinson\u2019s disease)]; [ AND (LRRK2) AND (Parkinson\u2019s disease)]; [ AND (GBA) AND (Parkinson\u2019s disease)]; [(neuromelanin MRI) AND (Parkinson\u2019s disease)]; [(neuromelanin) AND (GBA) AND (Parkinson\u2019s disease)]; [(neuromelanin MRI) AND (LRRK2) AND (Parkinson\u2019s disease)]; [(neuromelanin MRI) AND (LRRK2) AND (Parkinson\u2019s disease)]; [\u2018neuroimaging biomarkers in prodromal PD\u2019]. The search yielded 1049 results in total. These were further filtered based on date of publications (articles published from 2000\u20132023 were considered), duplicates, case studies, being written in English, whether reporting findings in overall PD, and other conditions and/or diseases, yielding 94 articles on which this literature review is based on.We conducted a literature review using PubMedPositron emission tomography or single-positron emission tomography (SPECT)-based molecular imaging approaches are used to depict nigrostriatal dopaminergic neuronal dysfunction . The imaGBA1, LRRK2, SNCA, PINK1, or Parkin reported greater asymmetrical striatal dopaminergic uptake in GBA1 and LRRK2 carriers compared with SNCA, PINK1, and Parkin carriers [18F]-FP-CIT PET (DAT) study by Kim et al. examined striatal dopamine and cerebral perfusion in GBA1-PD compared to sporadic PD (sPD). Decreased perfusion in cerebral subregions including the parietal and occipital regions in GBA1-PD compared to sPD was detected. These differences coincided with higher frequencies of non-motor symptoms including REM sleep behavior disorder (RBD) and psychiatric symptoms in GBA1-PD -fluorodeoxyglucose (FDG)-PET studies in genetic PD patients have documented widespread metabolic alterations in GBA1-PD patients with N370S/R496H mutations encompassing frontal, parietal, striatal, and thalamic brain regions [GBA1-PDs, N = 14 LRRK2-PDs, N = 14 sporadic PDs (sPDs), and N = 14 healthy controls using FDG-PET. While all three PD groups showed similar disease networks, the location and modular distribution of the network connections differed across groups. For example, LRRK2-PDs gained connections within the network core, with the formation of distinct functional pathways between the cerebellum and putamen. In GBA1-PD, the majority of functional connections were formed outside the core of the network, involving cortico-cortical pathways at the network periphery. The authors concluded that these distinct connectivity patterns might give rise to the less aggressive disease course in LRRK2-PDs compared to that observed in GBA1-PDs [[ regions . SchindlGBA1-PDs .Volumetric measures: A study comparing N = 9 Parkin-PDs with N = 14 sPDs detected increased gray matter (GM) volume in the right globus pallidus externus, the head of the left caudate, and right putamen in Parkin-PD compared with sPD [Parkin-PD patients and reported reduced bilateral caudate volumes compared to age-matched sPD individuals. These changes were not found to be associated with clinical or behavioral differences between the study groups [with sPD . A diffey groups .LRRK2 gene, N = 10 LRRK2-non-manifesting carriers (NMC), N = 24 sPD, and N = 12 non-manifesting non-carrier (NMNC) healthy individuals. Higher GM volumes in the cerebellum and left precentral gyrus was observed in the LRRK2-PD group compared to age- and-sex matched sPD and healthy controls, as well as a significant GM volume decrease in bilateral putaminal volumes when comparing LRRK2-PDs vs. age-matched healthy controls. The authors argued that these observed patterns of GM volume alterations might be indicative of prominent compensatory mechanisms within motor networks of the brain in LRRK2-PD, related to the more benign disease course [LRRK2-PDs, and GBA1-PDs) compared to three groups of unaffected healthy participants . No structural differences were observed among the different groups of NMCs, and no differences in cortical thickness and deep gray matter (DGM) volumes were observed between the genetic PD groups and sPD [Voxel-based morphometry (VBM) was used to examine structural changes in N = 10 PD patients with mutations in the e course . Our gro and sPD . However and sPD . A recen and sPD . Neuromelanin-sensitive (NM) MRI: NM-MRI enables the assessment of the extent of dopaminergic neuronal damage in the SN. In these neurons, neuromelanin plays a protective role by chelating excess iron [PARK2-PD patients reported reduced NM-SN in the PARK2-PD group compared to sPDs, however, no significant correlations between the measured NM-SN signal and clinical motor scores were reported [LRRK2-PD and sPD participants compared to healthy controls, the authors investigated NM within brainstem structures including the SN, locus coerluleus (LC), and red nucleus (RN). These authors reported NM loss in the ventrolateral tier of the SN of both PD groups. Furthermore, while the measured NM-SN was a good discriminator between PD patients and controls, the NM-LC was found to best discriminate among PD subgroups with LRRK2-PD showing preserved NM-LC content compared to sPD. Based on these observations, the authors speculated that NM-based markers can serve as candidate markers for phenotypic PD features [LRRK2-PD, and GBA1-PD patients. The findings indicated genotype-specific differences in NM-based radiomic features only. Specifically, significant differences in the left and NM-SN skewness radiomic feature between sPD were observed, possibly indicating greater dopaminergic loss in the LRRK2-PD group [ess iron . Changesess iron ,39,40. Ireported . In a refeatures . In a rePD group .Diffusion tensor imaging (DTI): DTI has become one of the most popular MRI techniques in brain research, as well as in clinical practice, and is being constantly validated and developed in terms of acquisition schemes, image processing, analysis, and interpretation [retation . DTI proretation . The metGBA1-PD an N = 16 sPD patients. The authors identified reduced FA in the inter-hemispheric and intra-hemispheric bundles including the corpus callosum, olfactory tract, cingulum, and internal and external capsules in the GBA1-PD group. The measured mean FA along the corpus callosum, external capsule, and the olfactory tract was found to be associated with verbal fluency in the overall patient group. No significant correlations between further clinical and DTI scores were obtained in the overall group as well as in the GAB1-PD subgroup [PARKIN-PD using tract-based spatial statistics (TBSS) in N = 12 PARKIN-PDs, N = 14 sPDs, N= 15 PARKIN-NMCs, and N = 13 NMNCs. Compared to sPD, PARKIN-PD demonstrated significantly lower FA in the cortico-spinal tract, bi-lateral external capsules, genu of the corpus callosum, and the middle cerebellar peduncle and higher MD values in the left interior limb of internal capsule, right cortico-spinal tract, and the left externa capsule. Interestingly, no significant WM differences were detected between PARKIN-NMCs and NMNCs [PARKIN-PD manifests more severe WM damage encompassing fibers implicated in motor and cognitive functioning [In an earlier study, WM microstructural differences were investigated in N = 15 subgroup . These rsubgroup . A recennd NMNCs . While nctioning .Functional MRI (fMRI): fMRI is a non-invasive imaging technique enabling the assessment of brain activity. In many neurological diseases, including PD, fMRI experiments have contributed greatly to the understanding of functional plasticity mechanisms taking place parallel to the neurodegenerative process [PARKIN-PD and N = 11 sPD patients while \u201cON\u201d medication were investigated using a simple motor sequential finger task. The researchers of this study reported no between-group differences in task performance or cerebral activation patterns, possibly indicating equal response to dopaminergic therapy in the study groups [ process ,48. Braiy groups .Resting-state fMRI: Resting-state fMRI (rs-fMRI) studies mainly aim to investigate functional connectivity (FC) patterns within major functional networks of the brain [he brain ,51. Rs-fhe brain ,48.PINK1 and PARKIN-PD patients compared to N = 12 NMC and N = 22 NMNCs. The authors reported lower FC among both PD and NMC compared to NMNCs. PD patients demonstrated lower FC compared with NMCs in the right fronto-parietal network and the executive network, which was associated with their worse cognitive performance [Using rs-fMRI, FC levels within major brain functional networks were examined in N= 8 formance .LRRK2-PD patients (R1628P and G2385R mutations carriers) compared to N = 11 drug-na\u00efve sPD patients and N = 22 age-matched healthy controls using a seed-based approach. LRRK2-PD demonstrated reduced FC between the left anterior putamen and the right calcarine gyrus, the right anterior putamen and bilateral calcarine gyri, left posterior putamen and bilateral superior frontal gyri, and right posterior putamen and bilateral precuneus when compared with the sPD group. Moreover, LRRK2-PD showed lower FC levels between the left posterior putamen and the sensorimotor cortices compared to healthy controls [LRRK2-PD group could be a distinct clinical phenotype in these patients. It is worthwhile to point out that the investigated patients in this study were of a relatively younger age, averaging less than 50 years, and had short disease duration.FC within striatal regions was investigated in N = 11 drug-na\u00efve controls . The autGBA1 variants. The findings indicated reduced F-DOPA uptake in the bilateral caudate nuclei, the ipsilateral antero-medial putamen, and the contra-lateral nucleus accumbens relative to the affected side in GBA1-PD (p.E326K and p.T408M) carriers compared to sPD. Meanwhile, only minor clinical differences were observed between GBA1-PD and sPD. On FDG-PET, GBA1-PD showed significant reduction in the bilateral medial and lateral parietal lobe compared to sPD. On rs-fMRI, reduced FC was observed between left and right caudate and the bilateral occipital cortex and between the right nucleus accumbens and the left superior parietal cortex and right fusiform gyrus in GBA1-PD vs. sPD [GBA1-PD patients and their higher susceptibility for executive function decline, dementia, and visual hallucinations [GBA1 gene.A multimodal imaging study explored imaging differences in PD patients with different vs. sPD . The autinations . NonetheGBA1- and LRRK2-PD patients.See GBA1- and LRRK2-NMCs are summarized in Key findings from nuclear imaging and MRI studies in 11C-2\u03b2-carbomethoxy-3\u03b2-(4-fluorophenyl) tropane (CFT), cerebral metabolism and dopaminergic neuronal activity were assessed in N = 6 GBA1 mutation carriers with and without Parkinsonism [GBA1-PD showed significant reduction in cerebral glucose metabolism encompassing the medial surface of the frontal cortex and extending to the supplemental motor area (SMA) and cerebral hypo metabolism in the parieto-occipital cortices. Asymptomatic GBA1 carriers (N = 3) demonstrated increased dopaminergic activity in the caudate nucleus based on 11C-CFT imaging. While no associations between the measured FDG-PET binding and clinical measures in the asymptomatic carriers were reported, the authors argued that observed cerebral hypo-metabolism patterns might underlie certain motor symptoms such as akinesia and the increased dopaminergic activity in the caudate might reflect presynaptic dopamine transporter upregulation or increased endogenous dopamine secretion in GBA1 carriers [Combining FDG-PET and insonism . GBA1-PDcarriers .18F-flurodopa (FDOPA) binding and reduced DAT-SBRs were observed in N = 25 LRRK2-NMCs compared to N = 35 healthy controls; however, these observed levels in the NMC groups were significantly higher than those of LRRK2-PD [LRRK2-NMCs compared to NMNCs, indicating that this group might reflect an endophenotype where several of these NMCs would convert to definite PD [GBA1-NMCs compared with LRRK2-NMCs and healthy controls [Comparable striatal LRRK2-PD The authLRRK2-PD . SimilarLRRK2-PD , which rinite PD ,57. Rececontrols . The dircontrols ,58.Parkin-NMCs and PINK1-NMCs compared to age- and-sex matched NMNCs. The authors reported bilateral GM increase in the posterior putamen and the internal globus pallidum among NMCs irrespective of genotype. The authors concluded that this observed striatal volumetric increase might be related to excessive neuronal activity or reflect an adaption mechanism to compensate for dopaminergic activity in these individuals [SNCA-NMCs before and after development of PD compared the participants with N = 10 healthy controls. At baseline, SNCA-NMCs did not differ significantly from controls in caudate volume. However, at follow-up, SNCA-PDs demonstrated significantly lower caudate volumes compared to controls. Conversion to definite PD was not associated with cortical atrophy [SNCA gene to N = 13 non-carriers, no significant differences in measured cortical thickness were detected between the investigated genetic groups [Subtle structural alterations in non-manifesting carriers of PD-related mutations were explored in many studies ,59,60,61 atrophy . In a dic groups .PINK1 demonstrating psychiatric symptoms, compared to age- and gender-matched healthy individuals were investigated [LRRK2-NMCs and N = 30 NMNCs. No significant GM differences between the study groups were detected. In DTI, LRRK2-NMC showed higher FA and lower AD and MD in the substantia nigra, as well as lower AD and MD in the nucleus accumbens. However, these observed differences did not survive correction for multiple comparisons. Despite the small effect size of the study, the authors argued that these findings might be indicative of subtle structural reorganization in LRRK2-NMC, possibly reflecting structural compensation [In another study, brain patterns of volumetric brain alterations in N = 14 non-manifesting family members, all carriers of stigated . The autstigated . In a coensation .The microstructural changes responsible for the observed patterns of GM volume increases in mutation carriers with PD remain not fully clear . Such GMNM-MRI: To our knowledge, only one study has examined SN-NM in asymptomatic LRRK2 and GBA1 carriers. In this study, the authors compared the measured NM within the SN in N = 34 NMNCs, N = 24 LRRK2-NMCs, and N = 23 GBA1-NMCs. The obtained results indicated no significant differences between these study groups. Moreover, this study reported reduced NM in PDs compared to asymptomatic individuals, further highlighting that NM-based MRI markers can serve as sensitive tools for identifying the symptomatic phase of the disease [ disease ,38,40.fMRI: Functional imaging tools are useful for elucidating functional compensation coinciding with subtle dopaminergic decline and the appearance of non-motor symptoms in the prodromal phases of the disease or in individuals \u201cat risk\u201d. The occurrence of such functional changes in the prodromal phase was documented in numerous fMRI studies [LRRK2 NMCs and N = 32 NMNCs explored corticospinal connectivity patterns using rs-fMRI. The study detected connectivity changes among the LRRK2-NMCs with reduced functional connectivity between the right inferior parietal cortex and the dorsoposterior putamen but increased connectivity with the ventroanterior putamen, as compared with non-carriers. This shift in functional connectivity increased with age in LRRK2-NMC [LRRK2-NMCs and NMNCs. LRRK2-NMCs demonstrated decreased network organization compared with NMCs, however, no differences between both groups were observed within the motor network [GBA1-NMCs and LRRK2-NMCs compared to NMNCs. The findings of the study indicated distinct group-specific patterns, in which LRRK2-NMCs showed significantly reduced SBRs in the right putamen compared to NMNCs, while no difference in measured SBRs was observed between GBA1-NMCs and NMNCs. In rs-fMRI, the opposite pattern was observed; namely, LRRK2-NMCs showed higher intra-striatal FC within the putamen compared to GBA1-NMCs. Furthermore, these measured DAT-SPECT SBRs were found to inversely correlate with the MDS research criteria for prodromal disease likelihood ratio (LR) scores [GBA1-NMC group; whereas rs-fMRI FC levels were associated positively with LR scores in the LRRK2-NMC group. Based on these findings, we concluded that differential DAT-FC patterns could be observed among the different genotypic groups in the prodromal phase of the disease, paving the way for future longitudinal investigations to further examine the potential role of FC-based measures for risk prediction in the early stages [ studies ,77,78,79RRK2-NMC . SimilarRRK2-NMC . A graph network ,69,73,77) scores ,81 in thParkin-NMCs and PARK1-NMCs were found to recruit additional motor regions compared with NMNCs while performing a motor task, coinciding with comparable task performance. This might indicate that the recruitment of cortical motor areas might have a compensatory role in maintaining normal task performance indicative of early reorganization processes taking place in the \u201cat risk\u201d group [LRRK2-NMCs. While error rates and reaction times did not differ between the groups, LRRK2-NMCs showed reduced motor imagery-related activity in the right caudate, indicating functional impairment in the striatum and increased activity in the right dorsal premotor cortex, which was counteracted by increased FC between this region and the right extra-striate body [LRRK2-NMCs showed recruitment of three additional right-sided brain structures, inferior parietal lobe, precuneus, and the fusiform gyrus, compared to NMNCs while performing an attention interference (Stroop) task. The authors proposed that this observed right lateralized activation increase might be reflective of ventral attention network recruitment in these individuals [GBA1-NMCs compared to LRRK2-NMCs and NMNCs. In line with previous findings, no performance differences were observed between the three study groups. Nevertheless, right medial frontal gyrus activation was observed in the GBA1-NMC group, where this region showed a task-negative activation pattern in both LRRK2-NMC and NMNC groups [Numerous task-fMRI studies were carried out on NMCs of PD-related mutations aimed at exploring specific functional and cognitive aspects associated with the genetic status of risk for PD. For instance, k\u201d group . This noate body . A studyividuals ,78. AnotC groups .PD is among the leading neurological diseases with highly effective symptomatic therapies available, yet none of these has proven to have modifying effects on the disease time course or progression . This caGBA1- and LRRK2-NMCs believed to be at risk demonstrate striatal DAT increases compared to NMNCs. Moreover, higher striatal within-network FC in LRRK2-NMCs is also observed. Upon disease manifestation, distinct patterns of brain changes are observed where both genetic PD groups show higher DAT binding compared to sPD. Functionally, GBA1-PDs manifest with lower striatal within-network connectivity and cortico-cortical FC gain. LRRK2-PDs, on the other hand, show higher striatal within-network FC and reduced FC between striatal and higher cortical regions. Additionally, LRRK2-PDs show reduced putaminal volumes as well as higher precentral and cerebellar GM volumes. Nevertheless, major limitations of these studies may be related to several factors such as the heterogeneity of clinical symptoms of the enrolled study samples, pooling together of different mutation carriers into one group, small sample sizes, and the effects of administered treatments, rendering the generalizability of the obtained findings from such study designs limited [Despite its high spatial resolution, no MRI-based marker has been established as a \u201cgold standard\u201d for PD diagnosis to date. A growing mass of imaging studies, both structural and functional, over recent years have contributed to further understanding of the ongoing degenerative and compensatory processes co-occurring in the brain of these individuals. However, only limited knowledge regarding the natural history of PD and the underlying processes leading to the pathology spread can be obtained from cross-sectional imaging studies enrolling PD patients ,80. A su limited ,84,85. ANevertheless, the accuracy of various MRI methods was examined in detecting PD-specific changes related to disease over recent years ,85. AdvaPINK, GBA1, and LRRK2 that are associated with increased risk for developing PD paved the way for a paradigm shift in PD diagnosis and the prodromal phase prior to the appearance of clinical symptoms [The detection of genetic mutations such as symptoms ,92; provsymptoms ,92. Sevesymptoms ,79,93,94SNCA) can also offer the opportunity to assess candidate neural markers in these individuals. However, its prevalence is relatively rare and nine families with SNCA have been identified worldwide so far [In addition to these abovementioned genetic forms of PD, mutations in the alpha-synuclein (\u03b1-syn) gene (e so far . Hence, e so far . Poolinge so far . These ae so far . To thatFuture research utilizing advanced imaging methods together with longitudinal study designs over longer follow-up periods can capture the ongoing pathological and compensatory processes, paving the way for the development of highly sensitive mechanism-oriented diagnostic markers, as well as targeted interventions and therapeutic options that can be tailored to the specific subtypes\u2019 symptom profiles."} +{"text": "Correction: Journal of Medical Case Reports (2017) 11:334 10.1186/s13256-017-1489-7Following publication of the original article , the autThe incorrect author name is: Andi Praja Wira Yudha LutfiThe correct author name is: Andi Praja Wira Yudha LuthfiThe author group has been updated above and the original article has been"} +{"text": "The impact of corticosteroids on patients with severe coronavirus disease 2019 (COVID-19)/chronic hepatitis B virus (HBV) co-infection is currently unknown. We aimed to investigate the association of corticosteroids with these patients.2 \u2264 93% on room air; or oxygen index < 300 mmHg) with COVID-19/HBV co-infection were identified. The bias of confounding variables on corticosteroid effects was minimized using a multivariable logistic regression model and inverse probability of treatment weighting (IPTW) based on the propensity score.This retrospective multicenter study screened 5447 confirmed COVID-19 patients hospitalized between Jan 1, 2021, to December 1, 2022, in three centers in India, where the prevalence of chronic HBV infection is moderate to high. Severe patients who had chronic HBV and acute SARS-CoV-2 infection were potentially eligible. The diagnosis of chronic HBV infection was based on positive testing for hepatitis B surface antigen (HBsAg) or HBV DNA during hospitalization and a medical history of chronic HBV infection. Severe patients . Fifty-five patients received corticosteroid treatment and 50 patients did not. In the multivariable analysis, corticosteroid therapy was identified as an independent risk factor for 28-day mortality. With IPTW analysis, corticosteroid treatment was associated with delayed SARS-CoV-2 viral RNA clearance , increased risk of 28-day and in-hospital mortality , and acute liver injury . Methylprednisolone dose per day and cumulative dose in non-survivors were significantly higher than in survivors.In patients with severe COVID-19/HBV co-infection, corticosteroid treatment may be associated with an increased risk of 28-day and in-hospital mortality.All Authors: No reported disclosures"} +{"text": "Background: SARS-CoV-2 viral load decreases over time after illness onset. However, immunocompromised patients may take longer for viral load decrease or have a more erratic viral-load trajectory. We used strand-specific assay data from admitted patients to evaluate viral-load trajectories after illness onset. Methods: We reviewed records of hospitalized patients with a positive SARS-CoV-2 PCR and tested using the strand-specific SARS-CoV-2 PCR during July 2020\u2013April 2022. At Stanford Healthcare, we use a 2-step reverse real-time polymerase chain reaction (rRT-PCR) assay specific to the minus strand of the SARS-CoV-2 envelope gene to assess infectivity. Restricting our analysis to each patient\u2019s first strand-specific assay, we used logistic regression models to compare patients with single versus multiple assays. Among patients with multiple tests, we compared those who had an upward trajectory in cycle threshold (Ct) values versus those who did not. We analyzed presence of symptoms, immunocompromised state, immunosuppression reason, and severe COVID-19 leading to ICU care in univariate and multivariate models that further adjust for additional covariates. Significant differences were assessed using logistic regression odds ratios and an \u03b1 level of 0.05. Results: In total, 848 inpatients were included. Among them, 703 were tested only once and 145 were tested 2\u20136 times. The longest duration of minus-strand detection was 163 days. In univariate analyses, patients with a single minus-strand assay had lower odds of being symptomatic , of being immunocompromised , and of being admitted to the ICU with severe COVID-19 . In the multivariate analysis, being admitted to the ICU with severe COVID-19 was the only significant variable associated with having >1 test . Among patients who had multiple strand-specific SARS-CoV-2 assays, 119 had upward minus-strand trends of Ct values (as expected) and 26 did not. Being immunocompromised was associated with nonrising minus-strand CT values when holding all other covariates in the model constant. Conclusions: Immunocompromised patients with COVID-19 tend to actively replicate for longer and have unexpected viral trajectories compared to immunocompetent patients. Among immunocompromised patients, suspension of transmission-based precautions may require a case-by-case evaluation.Disclosures: None"} +{"text": "This review aims to highlight our current understanding of the roles of sRNAs including miRNAs, heterochromatic siRNAs (hc-siRNAs), phased, secondary siRNAs (phasiRNAs) and natural antisense siRNAs (nat-siRNAs) in disease resistance, and sRNAs-mediated trade-offs between defense and growth in crops. In particular, we focus on the diverse functions of sRNAs in defense responses to bacterial\u00a0and fungal\u00a0pathogens, oomycete and virus in crops. Further, we highlight the application of sRNA-based technologies in protecting crops from pathogens. Further research perspectives are proposed to develop new sRNAs-based efficient strategies to breed non-genetically modified (GMO), disease-tolerant crops for sustainable agriculture.Small RNAs (sRNAs) are a class of short, non-coding regulatory RNAs that have emerged as critical components of defense regulatory networks across plant kingdoms. Many sRNA-based technologies, such as host-induced gene silencing (HIGS), spray-induced gene silencing (SIGS), virus-induced gene silencing (VIGS), artificial microRNA (amiRNA) and synthetic In nature, plants constantly face diverse biotic stresses, including bacteria, fungi, oomycetes, nematodes and viruses. Pathogen infection causes approximately 30% of global crop losses annually worldwide. Therefore, disease control is vital for assuring food security worldwide. During long coevolution with pathogens, plants have armed with various defense tools to prevent pathogens, which constitute a two-tiered immune machinery to detect and prevent pathogen invasion. The first layer is pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) governed by cell surface pattern recognition receptors (PRRs). In order to circumvent PTI, pathogens evolve effector proteins to suppress host PTI response, which is known as effector triggered susceptibility (ETS). As a counter-defense to prevent further infection, plants have then evolved highly polymorphic nucleotide-binding site (NBS) and leucine-rich repeat (LRR) domain-containing (NLR) resistance (R) proteins that directly or indirectly recognize pathogen effectors and form homo- or hetero-NLR complexes or resistosomes to trigger effector-triggered immunity (ETI) and double-stranded RNAs (dsRNAs) by the cleavage activity of Dicer-like (DCL) proteins, respectively (Borges and Martienssen MIRs by RNA polymerase II (Pol II) and trimmed into precursor miRNAs (pre-miRNAs) by DCL1, and pre-miRNAs are further processed by DCL1 to generate mature miRNAs (Kurihara and Watanabe PHAS loci). After cleavage, the transcripts are converted to dsRNAs, which produce 21- or 24-nt phasiRNAs by the activity of DCLs that is converted into dsRNAs by RDR6 and then dsRNAs are cleaved into 21-nt siRNAs by DCL4 are a class of 18\u201330\u2009nt, non-coding RNAs, which play vital roles in regulating gene expression and maintaining the genome stability. Based on the different biogenesis pathway and the divergent modes of action, plant sRNAs can be cataloged into two major classes, microRNAs (miRNAs) and small interfering RNAs (siRNAs). MiRNAs and siRNAs are generated from miRNA genes to regulate auxin signaling gene, is induced upon infection by P. syringae carrying effector avrRpt2, and silences PPRL that negatively regulates RPS2-mediated disease resistance causes rice (Oryza sativa) blast and is the most destructive fungal pathogen worldwide levels genes. The miR396-OsGRF module plays a vital role in balancing growth and immunity against the blast fungus. Overexpressing of a miR396-resistant version of OsGRF or blocking miR396 expression not only enhances rice resistance to M. oryzae but also improves yield traits improves yield, flowering time and immunity to M. oryzae, which suggest the potential application of miRNAs in coordinating plant immunity with growth and development , Superoxide Dismutase X (SODX), and Copper Chaperone for Superoxide Dismutase (CCSD). csd1/2 and sodx mutants display increased H2O2 accumulation and enhanced blast resistance, while ccsd mutants show enhanced blast susceptibility with lower levels of H2O2 , a Fe transporter. Interestingly, Osa-miR7695 appears to be subjected to subspecies-specific selection. Overexpression of Osa-miR162a induces defense genes expression and the accumulation of H2O2, and increases blast resistance in transgenic rice\u00a0 region of PigmS promoter and subsequently facilitates PigmS expression level represses the expression of siR109944 in rice. One candidate target of siR109944 is F-Box domain and LRR-containing protein 55 (FBL55), which is a transport inhibitor response 1 (TIR1)-like protein. Transgenic plants disrupting siR109944 biogenesis or overexpressing FBL55 display enhanced resistance to R. solani, probably attributing to the altered auxin homeostasis by FBL55 , miR408 positively regulates plant resistance to wheat stripe rust fungus by guiding the cleavage of TaCLP1 mRNA , a novel miR9863 family triggers the biogenesis of 21-nt phasiRNAs and miR9863-phasiRNA cascade forms a feed-forward regulatory machinery to suppress the immune signaling mediated by group I Mildew resistance locus a (Mla) alleles in response to barley powdery mildew fungus. Overexpression of miR9863 members specifically attenuates disease resistance and cell death triggered by MLA1 but not MLA10 negatively regulate accumulation levels of hvu-miR398 which represses HvSOD1 accumulation and influences ETI in response to the powdery mildew fungus , the ghr-miR477-silencing lines display decreased resistance to Verticillium dahlia, while knockdown of its target CaM-binding protein GhCBP60A increases plant resistance by up-regulating isochorismate synthase GhICS1 expression to increase salicylic acid (SA) level and isotrichodermin C-15 hydroxylase (HiC-15), respectively , peroxiredoxin (PRXIIF) and cell wall-associated kinase (WAK) mRNAs, are targeted by Bc-siR3.2, Bc-siR3.1 and Bc-siR5, respectively. B. cinerea dcl1 dcl2 double mutant that cannot produce Bc-sRNAs displays reduced pathogenicity , which is exported into tomato cells after infection and sequentially loaded onto tomato ARGONAUTE 4a (SlyAGO4a), targets the CBL-interacting protein kinase SlyFRG4. slyfrg4 mutant plants exhibit enhanced disease susceptibility to Fol, while slyago4a knock-down plants display enhanced resistance to Fol and Phytophthora sojae (P. sojae) Fig. a.Solanum chacoense and Solanum tuberosum cv. D\u00e9sir\u00e9e) plants infected by P. infestans, miR160 is induced in both local and systemic leaves. miR160 knockdown plants fail to elicit systemic acquired resistance (SAR). miR160 targets and mediates the cleavage of StARF10 mRNA. StARF10 protein can bind to the promoter of StGH3.6, a key hub in SA-auxin cross-talk, suggesting the important roles of miR160-StARF10-StGH3.6 module in the antagonistic cross-talk between SA-mediated pathogen defense processes and auxin-mediated growth , the most devasting vascular diseases in rice, causes severe crop loss each year. Downregulation of Osa-miR156 or overexpression of its targets, Ideal Plant Architecture1 (IPA1) and transcription factor OsSPL7, lead to enhanced resistance against Xoo at the expense of rice yield effectors bind to specific motifs in host gene promoters and activate the expression of host genes treated by Bacillus velezensis FZB42, four miRNAs, Zma-miR169a-5p, Zma-miR169c-5p, Zma-miR169i-5p and Zma-miR395b-5p, are repressed, which might fine-tune the activity of NF-Y transcription factors to activate the induced systemic resistance (ISR) to enhance plant defense response against pathogen infections as well as plant endogenous virus-activated miRNAs or siRNAs are loaded onto antiviral AGOs to inhibit the invasion of DNA or RNA viruses. These siRNAs may repress viral RNA through mRNA cleavage or translational inhibition, silence viral DNA through RdDM pathway, or affect host resistance (Carbonell and Carrington L-Ascorbate Oxidase (AO) mRNA in rice. Loss-of-function of spl9 causes decreased Osa-miR528 accumulation and a substantial increase of AO, resulting in enhanced plant resistance to Rice stripe virus (RSV) due to the repression of auxin signaling , silencing of BraCP24 mediated by miR1885 is enhanced to initiate precocious flowering, whereas the repression of R gene BraTNL1 by miR1885-phasiRNAs cascade is antagonized by TuMV-induced BraTNL1 expression by repressing the N gene expression under normal circumstances. TMV infection diminishes the accumulation levels of miRNA-6019/6020 and phasiRNAs, thus releasing the repression of N gene and limiting the virus spread infection, the expression of RDR6 is downregulated. Besides, the accumulation of RDV vsiRNAs is reduced in the osrdr6 knockdown transgenic plants, which results in increased susceptibility in rice to enhance the degradation of multiple AGOs by 26S proteasome system and autophagy pathways before RISC assembly suppressor coat protein (CP) binds to AGO1, suppresses its translational inhibitory activity and further enhances AGO1 degradation through autophagy (Karran and Sanfa\u00e7on Sweet potato chlorotic stunt crinivirus (SPCSV), cuts 21-24-nt vsiRNAs into 14\u2009bp inactive products, thus effectively precluding the formation of antiviral RISC gene which negatively regulates the production of ROS -mediated RNAi, host-induced gene silencing (HIGS), spray-induced gene silencing (SIGS) and virus-induced gene silencing (VIGS) and synthetic GS) Fig.\u00a0a-d. ThesA. thaliana transgenic plants which express modified miR159 precursor-based amiRNAs targeting two viral suppressors P69 and Hc-Pro simultaneously, exhibit resistance against both Turnip yellow mosaic virus (TYMV) and Turnip mosaic virus (TuMV) in N. benthamiana enhance plant defense even 21\u2009days post viral inoculation infection efficiency in soybean that target multiple viruses at diverse genomic positions . Transgenic Arabidopsis plants expressing these syn-tasiRNAs show elevated resistance to both viruses enhance plant antiviral resistance , TMV, and Tobacco rattle virus (TRV) (Purkayastha and Dasgupta Barley stripe mosaic virus (BSMV), a tripartite RNA virus, which can infect many important crops like barley, wheat, rice and maize, is also modified for VIGS resistance is validated by VIGS in wheat . As reviewed above, small RNAs mediate multilayer regulation in crop immune responses against pathogens, including post-transcriptional gene silencing by guiding mRNA cleavage/degradation or translational repression, transcriptional gene silencing by direct DNA methylation or chromatin modification Fig. . A serieDespite the advantages of simplicity, high specificity, flexibility and stability, there are some limitations of RNAi-based approaches. For example, a major hurdle in the practical application of SIGS is the rapid degradation of naked RNAs. To overcome this problem, nanomaterials such as chitosan-complexed single-walled carbon nanotubes , disease-tolerant crops, SIGS will be a promising technology to enhance crop resistance to disease. sRNAs-based strategies may be synergistically applied with other molecular approaches. Combined with the use of newly developed multi-transgene stacking toolkit containing marker/marker-excision cassette (Zhu et al."} +{"text": "Various endoscopic defect closure methods following endoscopic full-thickness resection (EFTR) of submucosal tumors have been developedVideo\u20061\u2002Gastric full-thickness defect closure using a reopenable-clip over-the-line method with an omental patch.The patient had a 26-mm submucosal tumor on the anterior side of the antrum that was endoscopically resected via full-thickness resection with laparoscopic assistance . The diFluoroscopy, at 3 days post procedure, revealed no leakage, and the patient was allowed a liquid diet. Endoscopic follow-up 7 days later showed all clips still in place and complete closure of the full-thickness defect. The patient was discharged without experiencing any adverse events. Therefore, ROLM-OP appears to be a novel and feasible technique for full-thickness defect closure.Endoscopy_UCTN_Code_TTT_1AO_2AG"} +{"text": "N-acetylgalactosaminedid not impact furin activity in synthetic O-glycopeptides, but thepresence of sialic acid reduced the furin rate by up to 65%. Similarly,O-glycosylation with a sialylated trisaccharide had a negative impacton TMPRSS2 cleavage. With a chemistry-centered approach, we substantiateO-glycosylation as a major determinant of spike maturation and proposedisruption of O-glycosylation as a substantial driving force for VOCevolution.The emergence of a polybasic cleavage motif for the proteasefurinin SARS-CoV-2 spike has been established as a major factor for humanviral transmission. The region N-terminal to that motif is extensivelymutated in variants of concern (VOCs). Besides furin, spikes fromthese variants appear to rely on other proteases for maturation, includingTMPRSS2. Glycans near the cleavage site have raised questions aboutproteolytic processing and the consequences of variant-borne mutations.Here, we identify that sialic acid-containing O-linked glycans onThr678 of SARS-CoV-2 spike influence furin and TMPRSS2 cleavage andposit O-linked glycosylation as a likely driving force for the emergenceof VOC mutations. We provide direct evidence that the glycosyltransferaseGalNAc-T1 primes glycosylation at Thr678 in the living cell, an eventthat is suppressed by mutations in the VOCs Alpha, Delta, and Omicron.We found that the sole incorporation of Proteolytic cleavage of S protein iscritical in SARS-CoV-2infection. GalNAc-T1-derived sialoglycans on S confer furin resistanceand may be an evolutionary driver for COVID-19 variants of concern. Most VOCs and many minor circulating variants carryat least one mutation in that peptide region: Alpha (B.1.1.7) andDelta (B.1.617.2) display mutations at Pro681 to His and Arg, respectively,whereas all Omicron sublineages including BA.1, BA.2, and BA.5 combineP681H with the mutation N679K. Mutations in this region appear tohave arisen more than once independently: Delta (P681R), Alpha (P681H),and Omicron lineages (P681H) are suggested to be on different armsof the evolutionary tree with common ancestors that do not containmutations around the FCS, indicating that some selection pressureon this sequence must have been present during their evolution.48 Lower-prominence variants have featured substitutions at Gln675and Gln677,53 usually to amino acids with basic functionalities displayincreased proteolyticprocessing of spike into S1/S2.nalities 1A. In li28 Among these, Asn (N)-linked glycosylation is straightforward topredict due to the existence of a peptide consensus sequence . In contrast, the predictionof Ser/Thr-linked N-acetylgalactosamine glycosylation, which also greatly impacts viral biology,62 is an analytical challenge due to its greater biosynthetic complexityand the lack of a peptide consensus sequence.73 Notably, the peptide region between Gln675 and Pro681 of SARS-CoV-2spike harbors multiple Ser/Thr residues that may carry O-GalNAc glycans.27 Despite the analytical difficultiesin understanding O-glycan biology, emerging data suggests that O-GalNAcglycosylation impacts furin-mediated spike cleavage.74 Due to the relevance of furin cleavage for viral infectivity,understanding the role of glycosylation in this process is essential.Like most surface proteins on animal viruses, SARS-CoV-2 spikeis extensively coated with glycans that impact infectivity and immunogenicityof the mature virus.N-acetylgalactosamine fromthe activated substrate uridine diphosphate (UDP)-GalNAc on to Ser/Thrside chains by a family of 20 GalNAc transferase isoenzymes. GalNAc-Ts are often associated with isoenzyme-specific,decisive roles in physiological processes that are beginning to beunraveled.84 Understanding the substrate profiles of individual GalNAc-T isoenzymesyields insight into the regulation of such processes and can be thebasis for the development of tools, diagnostics, and therapeutics.However, assigning glycosylation sites to individual GalNAc-Ts ischallenging due to their complex and often overlapping interplay inthe secretory pathway.85 Additionally,the initial GalNAc residue is often further elaborated, generatingmature glycans containing galactose , N-acetylglucosamine(GlcNAc), and the acidic monosaccharide N-acetylneuraminicacid (Neu5Ac) as a capping structure, further complicating the analyticalprofiling of O-GalNAc glycoproteins by mass spectrometry (MS) glycoproteomics.Indirect methods are thus employed to establish links between GalNAc-Tisoenzymes and the glycosylation sites they modify to yield insightsinto O-glycan biology.89 Through co-expression of theindividual human GalNAc-Ts with spike in insect cells and lectin staining,Ten Hagen and colleagues found that GalNAc-T1 introduces GalNAc intorecombinant spike, resulting in reduced proteolytic processing ofS to S1/S2 and decreasing syncytia formation in a cellular infectionmodel.74 Mutations at P681, including themutation P681H, led to increased S processing related to WT spike.74 These findings are in line with earlier reportsthat furin cleavage of other secreted proteins can be impacted byO-glycosylation.94 However, the biosynthetic complexity and technical challenges associatedwith O-glycoproteome analysis have thus far hindered closer investigationinto the molecular details behind these observations. Specifically,we currently lack knowledge on the precise glycan attachment site(s)and the impact of glycan structure on proteolysis. We also do notknow yet which VOC mutations impact either O-GalNAc glycosylation,direct proteolysis, or both.The biosynthesis of O-GalNAc glycans is initiated by the introductionof the sugar 96 Through structure-based design, the active site of a GalNAc-T isoenzymewas expanded by mutagenesis to contain a \u201chole\u201d, whichis complementary to a \u201cbump\u201d in a chemically modifiedanalogue of the substrate UDP-GalNAc.96 The bumped substrate \u201cUDP-GalN6yne\u201d contained an alkynemoiety that enabled the bioorthogonal ligation of fluorophores orbiotin after transfer to a glycoprotein, allowing the profiling ofthe substrates of individual GalNAc-Ts.98 Recently, we introduceda clickable, positively charged imidazolium tag (termed ITag) thatenhances MS-based analysis by increasing the charge state and improvingthe fragmentation-based sequencing of glycopeptides.99 Importantly, UDP-GalN6yne can be biosynthesized in theliving cell through the introduction of an artificial metabolic pathwayand feeding with a membrane-permeable peracetylated GalN6yne precursor, allowing for the installation of a fullyfunctional GalNAc-T bump-and-hole system.100 Building on the power of our chemical tools to dissect the roleof O-GalNAc glycosylation, we sought to map the molecular detailsof glycan-mediated modulation of spike processing.Chemical tools have provided aninsight into glycobiology thatis orthogonal to classical methods of molecular biology. For example,by using a tactic termed \u201cbump-and-hole engineering\u201d,we have developed a chemical reporter strategy for the activitiesof individual GalNAc-T isoenzymes in the living cell 1B.95,96 Here, withaid from this repertoire of chemical biology tools,we spotlight O-linked glycosylation as a major determinant of SARS-CoV-2spike cleavage by the host proteases furin and TMPRSS2. We providedirect evidence by MS-glycoproteomics that identifies GalNAc-T1 asthe glycosyltransferase initiating Thr678 glycosylation in the livingcell. We demonstrate that the presence of elaborated glycans on Thr678reduce proteolytic cleavage by TMPRSS2 and that a negative charge on Thr678-containing glycopeptides completely abrogatesfurin activity. We further confirm that mutations on Pro681 impair glycosylation of Thr678and may therefore promote proteolytic processing of spike. By emphasisingO-glycosylation as a major determinant of SARS-CoV-2 spike maturation,we propose disruption of O-GalNAc glycosylation as a considerableevolutionary driver for the emergence of SARS-CoV-2 VOCs.101 While generally powerful, identifying the modifiedglycosylation sites is often challenging by these methods due to theinterplay and ensuing compensatory effects between GalNAc-T isoenzymes.Bump-and-hole engineering enables a direct relation to the engineeredGalNAc-T isoenzyme by introduction of a GalNAc analogue which canbe bioorthgonally tagged and detected by various analytical techniques(95 We then tagged the glycosylated proteins withbiotin picolyl azide by Cu(I)-catalyzed azide\u2013alkyne cycloaddition(CuAAC) and visualized glycosylation via streptavidin blot fragmentationrevealed that monoglycopeptides are exclusively GalNAc-modified onThr678, while diglycopeptides are modified at Thr676 and Thr678, indicatinga hierarchy of sites where Thr678 is glycosylated first . When BH-engineered GalNAc-T1and UDP-GalN6yne were used in an in vitro glycosylationassay with WT spike-derived peptides, we observed a similar trendof glycosylating Thr678 and Thr676 sequentially, confirming that BH-T1recapitulates the substrate specificity of WT-T1 .We then used a panel of synthetic peptides to studythe effectof spike mutations on GalNAc-T1-mediated glycosylation. The peptidepanel included variant-related mutations at the major hotspots: Gln675,Gln677, Asn679, and Pro681 3A. GalNAn = 48 SARS-CoV-2 vaccinated individuals for T cell interferon-gamma(IFN-g) secretion using an enzyme-linked immunosorbent spot (ELISpot)assay.103 As shown in Supporting Figure 2, the median [IQR] response to the spikeprotein was 33.5 [16.7\u201369] spot forming cells (SFC) per millionPBMC and to the combined pool of M and N proteins was 10 [0\u201329]SFC/million PBMC. The median [IQR] response to the peptide WT-GalNAcwas 0 [0\u20133.7] in n = 44 individuals, comparedto the peptide P681H 0 [0\u20133.5] in n = 21 andWT 10 [0\u201327] in n = 3 individuals. These resultsindicate that neither (glyco-)peptide is a T cell target in vaccinatedindividuals.Having established a link between VOC mutationsand glycosylation,we sought to rule out an immunological implication of the corresponding(glyco-)peptides that might impact any mechanistic deductions. PeptidesWT, P681H, and WT-GalNAc that was introduced via cell feeding .106 This treatment introduced GalN6yne in anisoenzyme-specific fashion while endowing glycopeptides with an additionalpositive charge that facilitates MS analysis.99 The separated FL-S and S1/S2 fractions were subjected to in-geldigestion and analyzed by tandem MS. While collisional fragmentation allows for the determinationof monosaccharide compositions and naked peptide backbone sequences,this technique does not allow for the localization of O-glycans totheir glycosites. The energy associated with collisional dissociationmethods breaks the most labile bonds, which in the case of glycopeptides,are the glycosidic linkages between monosaccharides and the connectionof the glycan to the peptide itself. To resolve site information inO-glycopeptides, electron-based dissociation methods must be employed;commonly, this involves electron-transfer dissociation (ETD).107 Thus, we used high intensity collision-induced dissociation (HCD)to obtain naked peptide sequences and glycan compositions and thenused the ITag-containing GalN6yne diagnostic ion to trigger ETD fragmentationof the peptide backbone and manual validation, we found that Thr678 carriedITag-modified GalN6yne in both FL-S and S1 samples exclusively incells expressing BH-T1, but not BH-T2 or any WT-GalNAc-Ts (Supplementary Data 1). The additional positivecharge of the ligated ITag permitted straightforward ETD fragmentationof a 21-amino-acid glycopeptide. In contrast, the corresponding glycopeptidein samples expressing WT-T1 could not be unambiguously sequenced,highlighting the ability of chemical tools to help advance site-specificO-glycoproteomics. We further found that both BH-T1 and BH-T2 glycosylatedThr323, a previously detected glycosylation site that had thus farnot been associated with any GalNAc-T isoenzyme . These glycosylation annotations were recapitulatedthrough in vitro glycosylation of recombinantlyexpressed spike with recombinantly expressed soluble constructs ofBH-GalNAc-T1 and BH-GalNAc-T2, followed by CuAAC ligation of ITag-azideand MS-glycoproteomics analysis . Our data directly proves that GalNAc-T1 glycosylatesThr678 in the living cell.The complex dynamics of GalNAc-T isoenzymesin the secretory pathway requires new approaches to assigning theiractivities to specific glycosylation sites in living cells. Furthermore,the FCS-adjacent region lacks cleavage sites of the proteases mostcommonly used in MS sample preparation, resulting in large glycopeptidesthat hamper MS analyses. The use of specialized chemical tools canaddress these shortcomings and report on GalNAc-T activity in thesecretory pathway while offering a bioorthogonal handle to aid MSanalysis. We stably transfected Expi293F cells with constructs forboth SARS-CoV-2 spike (Wuhan) and either WT- or BH-versions of GalNAc-T1or T2, along with the biosynthetic machinery to generate UDP-GalN6ynein the cell from a membrane-permeable precursor -active substrate peptidesto assess proteolytic activity. Peptides spanning residues 672 to689 contained N-terminal 2-aminobenzoyl (Abz) and C-terminal 3-nitrotyrosine(3-NO2Tyr) as fluorescence donor and quencher moieties,respectively. An increase in fluorescence intensity indicated proteolyticcleavage or P681H mutant spike (FRET-2). The P681Hmutation had no discernible effect on the rate of furin-mediated cleavage,confirming the data by Whittaker and colleagues that the additionof a basic amino acid is not by itself a defining characteristic ofspike furin cleavage enhancement in existing VOCs.57Glycosylation has the potentialto modulate the proteolytic processing of a peptide depending on thedistance to the cleavage site and glycan composition, as previouslyproposed for spike upon co-expression with GalNAc-T1.cleavage 5A.110 ToFRET-3) or its alkyne-containinganalogue GalN6yne (FRET-4) were generated by chemicaland chemoenzymatic synthesis, respectively. Glycosylation with thesingle monosaccharides alone did not substantially impact the furincleavage rate compared to the WT peptide FRET-1 . We speculated that elaborationof GalNAc to larger or charged glycans might introduce additionalstructural constraints on furin recognition. To test this notion,the alkyne tag present on GalN6yne gave an opportunity to modify thebiophysical properties of glycopeptides in a straightforward fashion,enabling synthetic efforts to furnish glycopeptides with specificadditional groups or functionalities. We reacted the alkyne-equippedglycopeptide FRET-4 with two organic azides under CuAACconditions: to evaluate the impact of a larger glycan, we used 6-azido-6-deoxy-glucoseyielding pseudodisaccharide FRET-5, while 3-azido-propionicacid introduced an additional acidic functionality to investigatethe impact of a negative charge on furin cleavage in glycopeptide FRET-6. Both click-elaborated glycopeptides displayed a significantreduction in furin cleavage . FRET-5 exhibited an 80% decrease in the rate of furincleavage with respect to FRET-1, which is attributableto the relative steric expansion. Strikingly, FRET-6,which carried a smaller, negatively charged modification, resultedin a 93% rate reduction, almost completely abrogating furin activity.While these modifications are not naturally occurring, we concludedthat the elaboration of O-glycans on Thr678, especially with negativelycharged modifications, severely impedes the activity of furin.We hypothesized that an increase of furin processingin VOC mutantspike may not stem directly from recognition of the bare peptide sequencebut rather a decreased capacity of GalNAc-T1 to introduce O-GalNAcglycans to peptides with mutations proximal to the furin recognitionsite. We thus tested whether glycosylation of furin substrate peptidesimpacts proteolytic cleavage. FRET reporter peptides carrying GalNAc,the simplest O-glycan, effect on the furin rate comparedto the parental peptide FRET-1, the presence of a sialicacid led to a striking 45% reduction of furin rate in glycopeptide FRET-8 and a 65% reduction in glycopeptide FRET-9 peptide FRET substrates FRET-1, FRET-3, and FRET-7 to FRET-9 impededcleavage more drastically than all other (glyco-)peptides.To ourknowledge, in contrast to furin,o FRET-9 5E. Whilein vitro glycosylation experiments suggestedthat S1 contains the only available GalNAc-T1 substrate on WT spikeafter secretion from human cell culture and uncleaved(FL-S) fractions from the same recombinant WT-spike preparation. Wesubjected the FL-S and S1/S2 gel bands to MS-glycoproteomics analysis,searching for both simple and elaborated O-GalNAc glycans in eachof the fractions. By calculating the intact masses of various expectedO-glycopeptides in recombinant spike and then obtaining the associatedextracted ion chromatograms (XICs), we found that over 5-fold higherglycopeptide signal is present in FL-S when compared to cleaved S1/S2fractions from the same spike preparation (Supplementary Data 2). Furthermore, when accounting for sialic-acid-containingglycopeptides only, an \u223c8-fold higher abundance was observedfor FL-S relative to S1/S2 (Supplementary Data 2).Our culture 2B. Such 74 suggestingthat glycosylation is a physiologically relevant modification thatcould restrict the maturation (by proteolysis) of spike in WT SARS-CoV-2.113 The propensity of SARS-CoV-2 variants of concernto outcompete each other has been linked to both increased infectivityand immune escape. Within the evolutionary trajectory to the Alpha,Delta, and Omicron variants, notable changes in the amino acid sequenceproximal to the FCS indicated that proteolytic cleavage is graduallyenhanced, congruent with their increased infectivity. Mutations ofPro681 have been detected in early variants such as Alpha (P681H).We found that this mutation did not intrinsically increase the rateof cleavage by both furin and TMPRSS2 but impacted O-glycosylationas a restricting factor for spike processing.45 Notably, the analogous mutation found on the more transmissibleDelta variant (P681R) has been linked to an increase in furin cleavage,45 suggesting an evolutionary trajectory that convolvessuppression of O-glycosylation with increasing intrinsic furin recognition.This trend is further underlined by the mutations found in Omicron,which features both the P681H and the N679K mutations: in contrastto P681H and consistent with mapped amino acid preferences of GalNAc-T1,114 the N679K mutation does not substantially impactglycosylation, but it leads to enhanced furin cleavage of syntheticpeptides.45 These Omicron mutations thereforeact synergistically and have likely evolved to both suppress glycosylationand intrinsically enhance furin cleavage. Since the closest relativesto SARS-CoV-2, the strains RaTG13 Bat-CoV and GD Pangolin-CoV, sharethe exact same peptide sequence with WT (Wuhan) spike but withoutan FCS (116Our data strongly indicate that elaborated,negatively chargedO-GalNAc glycans on Thr678 of SARS-CoV-2 spike have a supressing effecton proteolytic cleavage. Such glycans are produced on lung epithelialcells which express GalNAc-T1,tan FCS 1,we spe94 The glycosylation site at Thr678 of spike is not in direct proximityof the FCS, potentially explaining why a single GalNAc residue isnot sufficient to modulate furin activity and only minimally impactsTMPRSS2 activity. The necessity for the glycan to be elaborated orsialylated to reveal the suppressing effect upon the rates of cleavagefurther highlights the need for accurate glycan tracing techniques.115 Tuning the chemical properties of glycopeptidesin a straightforward fashion by CuAAC was pivotal in enabling an initialunderstanding of the substrate-activity relationship of furin. Thisstrategy informed the targeted synthesis of elaborated O-glycopeptidesubstrates, providing a convenient method to fine-tune substrate scopein a time- and resource-efficient manner. Chemical tools thus yieldedinsights into glycosyltransferase specificity, improved the efficacyof detection by detectability by MS, and allowed the exploration ofprotease substrate specificity, showcasing the power of such toolsin biomedical discovery.The presence of O-GalNAc glycans adjacentto proteolytic cleavagesites has been found to impact processing of secreted proteins."} +{"text": "Human T lymphotropic virus-1 (HTLV-1) was the first identified oncoretrovirus, which infects and establishes a persistent infection in approximately 10\u201320 million people worldwide. Although only ~5% of infected individuals develop pathologies such as adult T-cell leukemia/lymphoma (ATLL) or a neuroinflammatory disorder termed HTLV-1-asssociated myelopathy/tropical spastic paraparesis (HAM/TSP), asymptomatic carriers are more susceptible to opportunistic infections. Furthermore, ATLL patients are severely immunosuppressed and prone to other malignancies and other infections. The HTLV-1 replication cycle provides ligands, mainly nucleic acids , that are sensed by different pattern recognition receptors (PRRs) to trigger immune responses. However, the mechanisms of innate immune detection and immune responses to HTLV-1 infection are not well understood. In this review, we highlight the functional roles of different immune sensors in recognizing HTLV-1 infection in multiple cell types and the antiviral roles of host restriction factors in limiting persistent infection of HTLV-1. We also provide a comprehensive overview of intricate strategies employed by HTLV-1 to subvert the host innate immune response that may contribute to the development of HTLV-1-associated diseases. A more detailed understanding of HTLV-1-host pathogen interactions may inform novel strategies for HTLV-1 antivirals, vaccines, and treatments for ATLL or HAM/TSP. The human T lymphotropic virus type 1 (HTLV-1) was discovered as the first human oncogenic retrovirus and belongs to the family Retroviridae and genus Deltaretrovirus . HTLV-1 HTLV-1 is a highly oncogenic virus with regulatory proteins that can modulate host cellular signaling pathways and cell cycle checkpoints to enhance the clonal proliferation and long-term persistence of infected cells that can eventually lead to leukemia/lymphoma. In addition, HTLV-1 evades immune surveillance through limiting its replication via tight control of viral gene expression. Although HTLV-1 was discovered more than 40 years ago, the innate immune responses generated during the different stages of HTLV-1 infection remain poorly understood, likely due to the lack of a tractable infection model and unique infection mechanisms utilized by HTLV-1. However, there is accumulating research evidence describing immune escape strategies employed by HTLV-1 to counteract the host immune surveillance in infected individuals. In this review, we will discuss the current knowledge of innate immune sensing of HTLV-1 and the restriction factors involved to counteract viral replication at multiple levels. Additionally, this review also discusses the HTLV-1-mediated immune evasion strategies to prevent recognition and destruction of infected cells by the innate immune system.HTLV-1 is a spherical enveloped virus approximately 100 nm in diameter and contains linear, dimeric positive-sense and single-strand genomic RNA inside an envelope embedded with protruding viral glycoproteins (gp46 and gp21) . The icoThe life cycle of HTLV-1 is analogous to other retroviruses; however, HTLV-1 cell-free virions are poorly infectious, and cell-to-cell spread between infected cells and target cells via the virological synapse is crucial for the transmission of the virus ,16. The Pathogen recognition receptors (PRRs) recognize pathogen-associated molecular patterns (PAMPs) in viruses, typically nucleic acids, to trigger an immune response through the secretion of type I IFN and proinflammatory cytokines . PRRs arTLR3 is widely expressed in the endosomal compartment of innate immune cells and recognizes retroviral double-stranded RNA, a viral replication intermediate, to trigger antiviral activities through secreting IFNs and inflammatory cytokines . Single The immune response triggered by professional \u2018sentinel\u2019 or plasmacytoid dendritic cells (pDCs) is largely induced through TLR7- or TLR9-mediated recognition of RNA from retroviruses ,35. ColiIFI16 is a critical intracellular DNA sensor that interacts with dsDNA through direct binding and is characterized as an IFN-inducible protein in antiviral immune responses . IFI16 iSTING is an ER-localized adaptor that plays a critical role in the cGAS DNA sensing pathway. cGAS senses viral or host dsDNAs and generates the cyclic dinucleotide 2\u20323\u2032-cGAMP (cyclic GMP-AMP) that binds to and activates STING. STING then traffics to the Golgi where it recruits TBK1 for downstream activation of IRF3 and NF-\u03baB to induce type I IFNs and proinflammatory cytokines to limit viral replication ,49. STINKu70 is well studied for its crucial role in the dsDNA break repair pathway through non-homologous end joining in a complex with Ku80 and the catalytic subunit DNA-PKcs ,52. ReceInnate and adaptive immunity cooperate in multiple ways to counteract viral infection. In addition, several host intrinsic cellular proteins termed restriction factors (RFs) contribute to the frontline defense to impede viral infections. Restriction factors are either constitutively expressed or induced by mediators of innate immunity such as type I IFN (interferon-stimulated genes or ISGs) to recognize and inhibit viral replication at multiple steps of the viral infection cycle, in a cell-autonomous manner ,62. RFs Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3) catalyzes the deamination of cytidine to uridine in single-stranded DNA substrates resulting in G-to-A mutations . APOBEC3Although HTLV-1 predominantly infects T-cells, myeloid cells are also potential targets of HTLV-1 infection ,76,77. TTetherin, also known as bone marrow stromal cell antigen 2 or BST-2, functions as a restriction factor through inhibiting the release of viral particles from infected cells; however, HIV-1 Vpu antagonizes the action of tetherin in retaining viral particles at the cell surface through depleting its expression ,83,84. IThe MHC class II transcriptional activator (CIITA) plays a critical role in triggering adaptive immune responses through efficient control of antigen presentation to CD4+ helper (TH) cells and can also initiate direct antiviral activity through inhibiting viral expression in a cell-autonomous manner ,87. CIITThe CCCH-type zinc finger antiviral protein, also known as ZAP, successfully restricts retroviral infections such as HIV-1, murine leukemia virus (MLV), and avian leukosis virus (ALV) as well as several other viruses including Hepatitis B virus, influenza A virus, and flaviviruses ,94,95,96The tripartite motif (TRIM) family comprises several interferon-induced proteins that carry out key roles in restricting viral infections . TRIM5\u03b1 Another TRIM family member, TRIM19 ), also functions as a restriction factor against HTLV-1 infection . NuclearAdenosine deaminase acting on RNA (ADAR) is a cellular RNA-editing protein involved in post-transcriptional processing of dsRNA and comprises three members: ADAR1, ADAR2, and ADAR3 . Type I Micro RNAs (miRNAs) can also act as restriction factors to defend against retroviral infections, and recent studies have highlighted the importance of certain miRNAs in HTLV-1-associated disease pathogenesis ,114,115.Innate immune-mediated inflammation plays a critical role in inhibiting pathogenic viruses. Innate immune receptors trigger the production of pro-inflammatory cytokines and interferons (IFNs), as well as signals that recruit and activate cells that orchestrate inflammation and the induction of adaptive immunity. Likewise, cellular immune responses have been implicated in the control of HTLV-1 infection as well as the development of inflammatory disorders in patients . MembersAlthough the induction of innate immunity and inflammation in response to viral infection is important to control virus replication, several oncogenic viruses, including HTLV-1, modulate inflammatory effector molecules that promote an environment conducive to cancer development. Mounting evidence suggests that acute inflammation is mostly associated with an antipathogenic role, whereas chronic inflammation favors cancer development . MoreoveHTLV-1 infection triggers the stimulation of multiple PRRs and activation of immune pathways and ISGs to counteract viral infection at multiple steps during the HTLV-1 replication cycle. To establish successful infection, replication, and persistence, HTLV-1 employs numerous immune evasion strategies to counteract innate immune responses targeting the virus. HTLV-1 targets and manipulates different immune sensors, restriction factors, transcription factors, and downstream signaling cascades for IFN production. These immune escape mechanisms disrupt antiviral immune signaling to prevent host-mediated viral clearance and facilitate HTLV-1 persistence and potential development of HTLV-1-associated pathogenesis. Understanding the underlying immune escape mechanisms of HTLV-1 is essential to design and develop novel antiviral therapeutics. Below, we have highlighted known strategies utilized by HTLV-1 to evade host antiviral defense programs .The pX region of the HTLV-1 genome encodes a regulatory protein Tax which acts as a transcriptional activator that promotes viral gene expression through the recruitment of cellular transcription factors such as CREB and CBP/p300 to the viral 5\u2032 LTR promoter . Tax is Intriguingly, Tax interacts with and inhibits the adaptor TIR-domain-containing adapter-inducing interferon-\u03b2 (TRIF), likely to impair endosomal sensing of dsRNA through TLR3 signaling and downstream innate immune signaling . MoreoveThe cGAS-STING pathway recognizes dsDNA and triggers innate immune responses through IRF3-mediated IFN-\u03b2 production. HTLV-1 circumvents the antiviral action of type I IFN through Tax-driven suppression of IRF3 activity . Tax intIFNs trigger ISG expression through Janus kinase (JAK)-dependent phosphorylation of signal transducer and activator of transcription (STAT) 1 and STAT2 to amplify IFN-mediated antiviral immune responses and block viral replication at multiple steps. Tax prevents IFN-induced JAK-STAT signaling through competing with STAT2 for CBP/p300 coactivators . MoreoveThe minus strand of the HTLV-1 provirus encodes a bZIP (basic leucine zipper factor) nuclear factor, termed HBZ. Unlike Tax, HBZ is steadily expressed at low levels in vivo because of an intact 3\u2032 LTR and the absence of abortive epigenetic or genetic changes; HBZ promotes the persistent proliferation of HTLV-1-infected T-cells . InteresThe HTLV-1 doubly spliced mRNA containing open reading frame II (orf-II) encodes a nuclear/nucleolar protein, p30, with both transcriptional and post-transcriptional activity. The p30 protein attenuates active HTLV-1 replication and promotes viral latency through retaining the doubly spliced mRNA encoding Tax and Rex proteins in the nucleus and disrupting the CREB-Tax-p300 complex essential for 5\u2032 LTR activation ,177. HTLHTLV-1 open reading frame I (orf-I) encodes a small hydrophobic protein, p12, which can be further processed into p8 through proteolytic cleavage. p12/p8 are not essential for HTLV-1 replication and transformation of infected cells; however, the binding of p12/p8 to IL-2R results in increased phosphorylation of STAT5 that allows T-cell proliferation in the absence of IL-2 and with suboptimal antigen stimulation, thus providing a growth advantage for HTLV-1-infected T-cells . p12 is HTLV-1 infection is prevalent in different parts of the world with increasing spread of HTLV-1 to non-endemic areas. Although a minority of infected individuals develop HTLV-1-associated diseases later in life, immunosuppression commonly occurs in ATLL which allows for opportunistic infections. There is clearly an unmet need for effective HTLV-1 vaccines, antivirals, and novel therapeutic approaches for ATLL and HAM/TSP. The major obstacle in designing vaccines and effective antivirals stems from an inadequate understanding of the early events of HTLV-1 infection and the host response to infection. Additionally, immune evasion strategies of HTLV-1 and its interactions with the innate immune system are not well understood. Interestingly, HTLV-1 does not express any specific proteins dedicated for immune evasion seen with other retroviruses; also, the functional roles of HTLV-1-encoded proteins in immune modulation only are partially explored. Thus, further studies focusing on the role of HTLV-1-encoded structural proteins along with regulatory or accessory proteins may provide novel therapeutic targets for viral intervention. Of note, targeting molecular events to counteract host-mediated antiviral immunity during the progression from asymptomatic infection to ATLL or HAM/TSP may provide promising strategies to develop novel antivirals against HTLV-1 infection. Several innate immune sensors have been reported to interact with HTLV-1 RNA or DNA at multiple stages of the viral life cycle in different cell types. Interestingly, HTLV-1 infection in vitro involves broad tropism with restricted viral replication in certain cell types in vivo, thus raising the possibility of specific restriction factors or immune mediators triggering IFN production for the prevention of productive replication to generate mature virions. Likewise, cell types susceptible to HTLV-1 infection display minimal or undetectable IFN levels, implying successful immune evasion by HTLV-1 to induce clonal proliferation for long-term persistence. Restriction factors with antiviral action against HTLV-1 are very limited as the majority of investigations are inspired by the HIV field. However, the structural and functional similarity of Tax with foamy virus transactivator protein, Tas, may be instructive in discovering new restriction factors such as TRIM28 and Pirh2 that restrict foamy virus replication through proteasomal degradation of Tas ,193. TheIn conclusion, increased mechanistic insight into HTLV-1-triggered immune responses during early infection and the corresponding viral immune evasion strategies may inform new approaches to modulate antiviral innate pathways and restrict the proviral load and possibly prevent HTLV-1-associated diseases."} +{"text": "SMARCA4-deficient non-small cell carcinoma is an aggressive neoplasm with poor outcome. Several studies have highlighted its immunochemistry, pathophysiology, and underlying mechanisms, but studies of its definite treatment are few. Here, we report on a 69-year-old male with heterogenous pathological presentations of SMARCA4-deficient non-small cell carcinoma. He initially presented with neck lymphadenopathies. Immunohistochemistry staining and genomic profiling confirmed the diagnosis of SMARCA4-deficient non-small cell carcinoma. The patient responded well to immune checkpoint inhibitors with nivolumab. However, new lesions with various pathological presentations and various responses to nivolumab appeared during the treatment course. The patient survived more than 3 years from the initial diagnosis. This case shows the efficacy of nivolumab to treat SMARCA4-deficient non-small cell lung carcinoma. SMARCth edition of the WHO classification of thoracic tumors classified this entity into SMARCA-4 deficient undifferentiated thoracic tumors (SMARCA-4 DUT) and SMARCA4-deficient non-small cell lung carcinomas (SMARCA4-dNSCLC). Both are associated with smoking and male preponderance. In non-small cell lung carcinomas (NSCLCs), we usually assess molecular markers to determine our clinical practice. However, SMARCA4-dNSCLC lacks alterations in currently targetable oncogenic drivers, such as epidermal growth factor receptor (EGFR), anaplastic lymphoma kinase (ALK), and c-ros oncogene 1 (ROS1) . Current2via mediastinotomy was performed and pathology demonstrated metastatic carcinoma of 440 cancer-related genes. Genomic profiling showed a high tumor mutational burden with 40 nonsynonymous mutations identified. Among them, biallelic loss-of-function mutation in SMARCA4 along with 4 other mutations were considered clinically relevant variants analysis. H-CW: offered the case and treated the patient. Y-HJ: provided NGS analysis. Y-HK: offered the case, treated the patient, wrote the manuscript, searched the literature, and revised the manuscript. All authors contributed to the article and approved the submitted version."} +{"text": "Extended spectrum cephalosporin-resistant Enterobacterales (ESCR-E) are increasingly implicated in community-onset urinary tract infections (UTIs). In this study, we assessed risk factors for recurrence among patients with community-onset UTI caused by ESCR-E.This retrospective cohort study included adult patients evaluated April 2018 \u2013 December 2021 in the Duke University Health System with community-onset ESCR-E UTI, defined as (1) ESCR-E in a urine culture obtained in an outpatient clinic, emergency department, or within 48 hours of hospital admission; (2) \u226510 leukocytes per high-power field on urine microscopy or urine dipstick positive for leukocyte esterase; and (3) new antibiotic administration or prescription. ESCR-E UTI recurrence was assessed 14 to 180 days after completion of antibiotic treatment for the index UTI. Patients were right censored at end of follow up period or upon death. Univariate Cox proportional hazards regression was performed to evaluate the relationships between candidate risk factors and time to recurrence.Klebsiella pneumoniae UTI had a 53% greater hazard of recurrence compared to patients with ESCR E. coli UTI .1428 patients were included; 207 (14.5%) experienced recurrence. In unadjusted analyses, risk factors for recurrent ESCR-E UTI included diabetes mellitus , chronic renal insufficiency , neurogenic bladder , previous UTI diagnosis within one year , fluoroquinolone non-susceptibility , and trimethoprim-sulfamethoxazole non-susceptibility . Patients with ESCR ESCR-E UTI recurrence was common, and several clinical and microbiologic characteristics were associated with recurrence. Patients with these characteristics should receive particular consideration for aggressive UTI risk factor modification and other non-antibiotic prevention strategies. Future studies should evaluate strategies to reduce the risk of recurrence among patients with ESCR-E UTI.All Authors: No reported disclosures"} +{"text": "Arabidopsis thaliana.Ultraviolet-B (UV-B) light is an intrinsic part of sunlight that reaches the earth\u2019s surface, and affects plant survival and adaptation. How plants respond to UV-B light is regulated by the wavelength, intensity and duration of UV-B radiation, and is also regulated by photosynthetically active radiation perceived by phytochrome and cryptochrome photoreceptors. Non-damaging UV-B light promotes plant photomorphogenesis and UV-B acclimation which enhances plant tolerance against UV-B stress. However, high-level UV-B radiation induces DNA damage, generates reactive oxygen species (ROS) and impairs photosynthesis. Plants have evolved efficient mechanisms to utilize informational UV-B signal, and protect themselves from UV-B stress. UV RESISTANCE LOCUS8 (UVR8) is a conserved plant-specific UV-B photoreceptor. It interacts with CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1) to initiate UV-B-specific light signaling and regulate UV-B responsive gene expression. A set of transcription factors such as ELONGATED HYPOCOTYL5 (HY5) function downstream of the UVR8-COP1 module to promote seedling de-etiolation for photomorphogenic development and biosynthesis of sunscreen flavonoids for UV-B stress tolerance. In addition to UVR8 signaling pathways, plants subjected to damaging UV-B radiation initiate stress protection and repair mechanisms through UVR8-independent pathways. In this review, we summarize the emerging mechanisms underlying UV-B stress acclimation and protection in plants, primarily revealed in the model plant Arabidopsis thaliana about UVR8-dependent and -independent pathways that contribute to UV-B stress tolerance.Light provides plants with the energy source needed for photosynthesis and acts as an important environmental cue to regulate plant survival and development. However, light can also function as an abiotic stress factor for plants , which induces oxidative stress and oxidizes DNA, RNA, proteins, lipids and many small molecules in plant cells suffer from increased sensitivity to UV-B radiation gene is induced by UV-B light dependent on UVR8 signaling pathway, and is also induced by blue and UV-A light mutant showed enhanced DNA repair activity can be repaired efficiently by photolyases. Pyrimidine dimers can be repaired by nucleotide excision repair (NER), or bypassed by replicative polymerases Britt . The expArabidopsis mutants deficient in ascorbic acid biosynthesis (vitamin c defective 1 [vtc1] and vtc2) and in tocopherol cyclase activity (vitamin e deficient 1 [vte1]) exhibit oxidative damage in response to light-stimulated stress in plants and BIC2 which inhibit cryptochrome dimerization to repress their activation are upregulated by UV-B signaling , through the \u03b2-propeller domain and the C-terminal Val-Pro (VP) motif of UVR8 and the C-terminal WD40 domain of COP1 transcription factor HY5 plays a central role in UV-B light signaling, along with HY5-HOMOLOG (HYH) , CHALCONE ISOMERASE (CHI), and FLAVONOL SYNTHASE1 (FLS1), so as to play positive roles in UV-B stress tolerance in Arabidopsis family transcription factors play important roles in UV-B signaling and stress tolerance through functional connection with HY5. UV-B light induces BBX24/SALT TOLERANCE (STO) expression and stabilizes its protein accumulation. BBX24 negatively regulates UV-B-induced photomorphogenesis by interacting with both COP1 and HY5, and repressing HY5 activity signaling pathway is activated as a complementary strategy for stress tolerance , ATAXIA TELANGIECTASIA MUTATED (ATM) and ATM-AND RAD3-RELATED (ATR) mediate plant tolerance against double-strand breaks (DSBs) and DNA replication stress respectively , an indolamine hormone in plants, acts as an antioxidant that plays important roles in plant defense against a variety of biotic and abiotic stresses, including UV-B stress , N-acetylserotonin methyltransferase (ASMT), and caffeate O-methyltransferase (COMT) and jasmonic acid (JA) which mediate pathogen defense in plants (Vandenbussche et al. BES1 independent of UVR8, upstream signaling factors that initiate this pathway awaits to be identified. How does UV-B signaling regulate photosynthetic performance and photoprotection? How do plants integrate UV-B stress and other biotic and abiotic stimuli? Further investigation of these and related questions will develop our understanding on plant responses to UV-B stress, and shed light on the strategy of UV-B utilization in crop production and environmental preservation.To date, accumulating evidence has illustrated key factors and molecular framework in plant UV-B stress tolerance Fig.\u00a0. UVR8 is"} +{"text": "Klebsiella pneumoniae beyond hospital settings is a global critical issue within a public health and One Health perspective. Another worrisome concern is the convergence of virulence and resistance in healthcare-associated lineages of K. pneumoniae leading to unfavorable clinical outcomes. During a surveillance study of WHO critical priority pathogens circulating in an impacted urban river in S\u00e3o Paulo, Brazil, we isolate two hypermucoviscous and multidrug-resistant K. pneumoniae strains (PINH-4250 and PINH-4900) from two different locations near to medical centers. Genomic investigation revealed that both strains belonged to the global high-risk sequence type (ST) ST11, carrying the blaKPC-2 carbapenemase gene, besides other medically important antimicrobial resistance determinants. A broad virulome was predicted and associated with hypervirulent behavior in the Galleria mellonella infection model. Comparative phylogenomic analysis of PINH-4250 and PINH-4900 along to an international collection of publicly available genomes of K. pneumoniae ST11 revealed that both environmental strains were closely related to hospital-associated K. pneumoniae strains recovered from clinical samples between 2006 and 2018, in S\u00e3o Paulo city. Our findings support that healthcare-associated KPC-2-positive K. pneumoniae of ST11 clone has successfully expanded beyond hospital settings. In summary, aquatic environments can become potential sources of international clones of K. pneumoniae displaying carbapenem resistance and hypervirulent behaviors, which is a critical issue within a One Health perspective.The spread of carbapenemase-producing \u2022K. pneumoniae strains were recovered from urban rivers.WHO critical-priority \u2022Genomic data revealed presence of global hospital-associated clones KPC-2/ST11.\u2022Environmental hypermucoviscous KPC-2/ST11 carried broad resistomes/virulomes.\u2022Phylogenomics revealed clonal relatedness with human and environmental lineages.\u2022Expansion of KPC-2/ST11 clones beyond hospital walls is a critical One Health issue. On the other hand, the tetA gene, associated with tetracycline resistance, was exclusively detected in the PINH-4900 strain. Moreover, genes associated with resistance to silver (silABCEFRS), chlorhexidine (smvR) and quaternary ammonium compounds (oqxAB) were also detected, in both PINH-4250 and PINH-4900 environmental strains. Furthermore, plasmidome analysis demonstrated the presence of IncN1, IncFIB, and ColRNAI plasmids ; Fig. 1Bplasmids ; Fig. 1Birp-1-2, the operon ybtAEPQSTUX (yersiniabactin siderophore synthesis), fyuA (yersiniabactin receptor), iutA (iron uptake), clb genes(colibactin genotoxin synthesis), and the mrkBCDFHIJ cluster ; Fig. 1CgalF to wzc genes), at the 5\u2032 end of the cps locus; whereas the wzc-gnd region was consist to genes associated with flippase (wzx), piruvyl tranferase (wcoV), polymerase (wzy), non-initial and initial (wcaJ) glycosyltransferase, as previously reported [gnd-ugd region encompassed manB and manC genes, which are associated with the biosynthesis of GDP-D-mannose; as well as rmlA, rmlB, rmlC and rmlD genes, that are responsible for deoxythymidine diphosphate dTDP-L-rhamnose synthesis were detected in the genome of PINH-4250 and PINH-4900 . In accordance with the grant conditions of the Foundation, the author accepted manuscript version resulting from this submission is subject to a CC BY or equivalent license. Furthermore, the study was supported by the 10.13039/501100001807FAPESP (2020/08224-9 and 2019/15578-4) and 10.13039/501100003593CNPq (88882.333054/2019-01). FE was a FAPESP research fellow (2019/15578-4). BC and HF were CAPES research fellows (88882.333054/2019-01 and 88887.506496/2020-00), while BF was a PNPD/CAPES research fellow (88887.358057/2019-00). NL is a CNPq research fellow (314336/2021-4).This study received financial support from the All authors declare no conflicts of interest."} +{"text": "Global proteomic data generated by advanced mass spectrometry (MS) technologies can help bridge the gap between genome/transcriptome and functions and hold great potential in elucidating unbiased functional models of pro-tumorigenic pathways. To this end, we collected the high-throughput, whole-genome MS data and conducted integrative proteomic network analyses of 687 cases across 7 cancer types including breast carcinoma , clear cell renal carcinoma , colorectal cancer , hepatocellular carcinoma , lung adenocarcinoma , stomach adenocarcinoma , and uterine corpus endometrial carcinoma UCEC . Through the protein\u00a0co-expression network analysis, we identified co-expressed protein modules enriched for differentially expressed proteins in tumor as disease-associated pathways. Comparison with the respective\u00a0transcriptome network models revealed proteome-specific cancer subnetworks associated with heme metabolism, DNA repair, spliceosome, oxidative phosphorylation and several oncogenic signaling pathways. Cross-cancer comparison identified highly preserved protein modules showing robust pan-cancer interactions and identified endoplasmic reticulum-associated degradation (ERAD) and N-acetyltransferase activity as the central functional axes. We further\u00a0utilized these network models to predict pan-cancer protein regulators of disease-associated pathways. The top\u00a0predicted pan-cancer regulators\u00a0including RSL1D1, DDX21 and SMC2, were experimentally validated in lung, colon, breast cancer and fetal kidney cells. In summary, this study has developed interpretable network models of cancer proteomes, showcasing their potential in unveiling novel oncogenic regulators, elucidating underlying mechanisms, and identifying new\u00a0therapeutic targets.The online version contains supplementary material available at 10.1186/s13045-023-01517-2. To the EditorDysregulated proteins play a critical role in the development of tumors, but many large-scale -omics studies predominantly centered around transcriptomics which has some substantial discordance with proteomics \u20133. HenceUsing the matched adjacent normal samples of the same organs from the Clinical Proteomic Tumor Analysis Consortium (CPTAC), we first identified differentially expressed proteins (DEP) in all the cancer types except STAD for which there are no matched adjacent normal samples Fig.\u00a0C includiThrough the protein co-expression network analysis , HCT116 (colon), MDA-MB-231 (breast cancer), and HEK293T significantly reduced cell growth analysis; Fig. S8.. Most enriched pathways in PCPIC cores; Fig. S9.. Cross-talk across distinct PCPICs; Fig. S10.. Top hub genes in protein co-expression networks; Fig. S11.. Enrichment of various protein signatures in the cancer essential genes identified by the in vitro screening in the Archilles database; Fig. S12.. Comparison of proteome (PR) and transcriptome (TX) network connectivity in each cancer type; Fig. S13.. Enriched hallmark pathways in proteome- (PR) or transcriptome-(TX) specific hub genes, or shared hub genes in PR and TX; Fig. S14.. Validated drivers by gene perturbations signatures in cancer cells from LINCS database; Fig. S15.. Evaluation of TCGA Pan-cancer atlas (PanCanAtlas) and CPTAC transcriptome (TX) cohorts for proteome module preservation analysis; Supplemental Table Legend; Table S1. Description of the cancer proteome datasets; Table S2. Summary of differentially expressed protein (DEP) signatures; Table S3. The number of differentially expressed proteins (DEPs) in each cancer type and the number of proteome specific DEPs, i.e., DEPs without differential expression at the mRNA level in the respective tumor transcriptome; Table S4. Numbers of protein and mRNA modules by MEGENA; Table S5. List of core proteins in Pan-cancer protein interaction communities (PCPICs)"} +{"text": "A 75-year-old female patient was referred to our hospital by an otolaryngologist with a history of voice hoarseness resulting from left-sided vocal cord paralysis (VCP) and exertional dyspnea. Chest X-ray and chest computed tomography (CT) revealedEnglish_editing_certificate_omad108Click here for additional data file."} +{"text": "The COVID-19 pandemic heightened concerns about people experiencing more depressive symptoms. Among people with HIV (PWH), who have higher rates of depression, these symptoms may lead to adverse HIV-related outcomes. This study sought to characterize the effects of the COVID-19 pandemic on depression severity and to investigate the association between depression trajectories and viral load (VL) non-suppression among PWH enrolled in HIV care.The study sample was PWH in the Johns Hopkins HIV Clinical Cohort who reported depression symptoms on the Patient Health Questionnaire 8 (PHQ-8) via a self-administered survey pre-pandemic and during the COVID-era . Depression severity was categorized using standard PHQ-8 cutoffs ranging from normal (0-4) to severe (20-24). Depression severity categories pre-pandemic (last survey) and COVID-era (earliest survey) were compared, and trajectories were classified as: 1) remained depressed (PHQ-8 > 4 and no change in severity category) or worsened (change to a higher severity category) and 2) remained non-depressed (PHQ-8 \u2264 4 and no change in severity category) or improved (change to a lower severity category). The association between depression trajectories and VL non-suppression (HIV RNA > 200 copies/ml on the first measurement after a COVID-era survey) was assessed using logistic regression adjusting for age, gender, pre-pandemic VL, clinical diagnosis of mood and substance use disorders.Among 793 PWH in this study, 60% were male, 88% were Black and the mean age was 56 years. Approximately 24% of PWH remained depressed (9%) or worsened (15%), while 76% remained non-depressed (60%) or improved (16%). PWH who remained depressed or worsened were more likely to be virally unsuppressed compared to those who remained non-depressed or improved.In our cohort of PWH, a quarter either remained consistently depressed or experienced worsening depression in the COVID-era. Depression was significantly associated with VL non-suppression during the pandemic. Our findings suggest that strategies to monitor and address depression symptoms among PWH may contribute to reduced risk of VL non-suppression.Oluwaseun Falade-Nwulia, MBBS ,MPH, Abbvie Inc: Grant/Research Support|Gilead Sciences: Advisor/Consultant"} +{"text": "Colorectal cancer (CRC) is the second most common cause of cancer mortality, with mismatch repair proficient (pMMR) and/or microsatellite stable (MSS) CRC making up more than 80% of metastatic CRC. Programmed death-ligand 1 (PD-L1) and programmed death 1 (PD-1) immune checkpoint inhibitors (ICIs) are approved as monotherapy in many cancers including a subset of advanced or metastatic colorectal cancer (CRC) with deficiency in mismatch repair (dMMR) and/or high microsatellite instability (MSI-H). However, proficient mismatch repair and microsatellite stable (pMMR/MSS) cold CRCs have not shown clinical response to ICIs alone. To potentiate the anti-tumor response of PD-L1/PD-1 inhibitors in patients with MSS cold cancer, combination strategies currently being investigated include dual ICI, and PD-L1/PD-1 inhibitors in combination with chemotherapy, radiotherapy, vascular endothelial growth factor (VEGF) /VEGF receptor (VEGFR) inhibitors, mitogen-activated protein kinase (MEK) inhibitors, and signal transducer and activation of transcription 3 (STAT3) inhibitors. This paper will review the mechanisms of PD-1/PD-L1 ICI resistance in pMMR/MSS CRC and potential combination strategies to overcome this resistance, summarize the published clinical experience with different combination therapies, and make recommendations for future avenues of research.The online version contains supplementary material available at 10.1007/s00262-023-03520-5. The development of immunotherapeutic drugs has led to significant improvements in overall and progression-free survival for many patients with cancer \u20136. For cTo overcome the hyporesponsiveness to PD-1/PD-L1 inhibitors, recent preclinical studies and clinical trials have demonstrated combination strategies to potentiate the effectiveness of anti-PD-1 and anti-PD-L1 immunotherapy in patients with cold CRC. The FDA has approved combination use of PD-1/PD-L1 inhibitors and other therapy/inhibitors for treatment of patients with cold metastatic cancer. For example, combination of cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) inhibitor, tremelimumab, and PD-1 inhibitor, durvalumab, was approved for treating patients with unresectable hepatocellular carcinoma in 2022 , 22. ThiGenomic instability is a trademark of tumor cells. There are two different types of genomic instability: (1) chromosomal instability, which is the consequence of the loss or gain of chromosomes or large chromosomal fragments and is associated with the majority of CRCs, and (2) microsatellite instability (MSI) which is observed in a small fraction of CRCs. MicrosatdMMR/MSI-H CRCs generally have a higher tumor mutational burden (TMB). TMB directly correlates to tumor\u2019s ability to harbor a plethora of neoantigens . ImmunogTo date, many anti-PD-1 antibodies (Abs) and anti-PD-L1 Abs have been developed to block PD-1/PD-L1 signaling. Table Most Abs are genetically engineered for high binding specificity and low off-target adverse effects (AEs) , 37, 38.So far, anti-PD-1 and anti-PD-L1 mAb therapies confer significant clinic benefit only in specific patient populations. Specifically, there are almost no objective responses to anti-PD-1 and anti-PD-L1 therapies observed for patients with \u2018cold\u2019 tumors such as MSS mCRC. Combatting resistance mechanisms or hyporesponse of the anti-PD-1/PD-L1 therapy remains a challenge.The low immunogenic properties of MSS cancer lead to resistance to PD-L1/PD-1 blockade. To enhance clinical response to the PD-1/PD-L1 inhibitors in pMMR/MSS cancer, one promising strategy is to combine with other anti-tumor agents that target different pathways and increase the immunogenicity of the TME, converting cold tumors to hot tumors. It has been demonstrated that inhibition of CTLA-4, vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR), mitogen-activated protein kinase (MEK), and signal transducer and activator of transcription 3 (STAT3), or treatment with cytotoxic chemotherapy and radiotherapy increases tumor neoantigens, upregulates MHC-1 expression, enhances dendritic cell (DC) antigen presentation and the release of proinflammatory cytokines, increases the activation, infiltration, and killing activities of T cells, and decreases immunosuppressive cells and cytokines. , 42\u201356 ACTLA-4 is an immunoglobulin cell surface receptor constitutively expressed on FoxP3\u2009+\u2009Treg as well as conventional T cells following activation by T cell receptor (TCR) signaling , 84, 85.PD-1 and CTLA-4 function on different subsets of T cells, and on T cells at distinct locations and timing during the cancer-immune response , 90. PD-A phase II randomized clinical trial (NCT02870920) studying anti-PD-1 and anti-CTLA-4 (tremelimumab) combination in patients with pMMR/MSS reported that the dual ICI achieves a prolonged median overall survival (mOS) of 6.6\u00a0months in pMMR/MSS mCRC patients, but without objective response (OR) and significant improvement in median progression-free survival (mPFS) , 96. FurVEGF/VEGFR signaling plays a vital role in forming the immune-suppressive TME in CRC through indirect and direct pathways Fig.\u00a0. OverexpTumor cells increase the release of VEGF, which binds to its receptor (VEGFR) to induce angiogenesis. Angiogenesis in turn increases interstitial pressure and hypoxia at the tumor site, which inhibits cytotoxic T cells (CTL) and promotes regulatory T cell (Treg) infiltration. The neovasculature formed via angiogenesis also has higher expression of immunosuppressive molecules PD-L1 and FasL on the vascular endothelial cells (VECs) and lower expression of adhesion molecules. FasL selectively induces CTL apoptosis and PD-L1 inactivates T cells within the tumor vasculature. VEGF/VEGFR also directly modulates immune cell abundance and function. The binding of VEGF to VEGFR inhibits the differentiation and maturation of DCs, which results in reduced T cell activation in the priming phase. It also promotes the proliferation and activation of Tregs and myeloid-derived suppressor cells (MDSCs) and enhances the polarization of tumor-associated macrophages (TAMs) to an M2 phenotype. These immunoregulatory effects reduce CTL function. VEGF also increases the expression TOX in CTL, which in turn upregulates its PD-1 expression and promotes immune exhaustion. Drugs that inhibit VEGF/VEGFR signaling inhibit VEGF/VEGFR-mediated immunosuppression to increase the abundance and function of CTL at the tumor site. Drugs that inhibit PD-L1/PD-1 signaling would block the binding of PD-L1 on CTL to PD-1 on tumor cells and decrease Treg proliferation and function. In combination, anti-VEGF/VEGFR and anti-PD-L1/PD-1 induces a synergistic anti-tumor response immunosurveillance) and overcame PD-L1-induced T cell suppression \u2013122. In (NCT02982694) was terminated because the efficacy in the MSS subgroup (MSI like) did not meet the expectation [The combination of atezolizumab (anti-PD-L1 Ab) and bevacizumab (VEGF inhibitor) was studied in patients with MSI-H mCRC pretreated with chemotherapy (NCT01633970) and resulted in an objective response rate (ORR) of 30% and a disease control rate of 90%. One clinectation . SubsequAtezoTRIBE (NCT03721653) is a multicenter phase II randomized study for the combination of atezolizumab, bevacizumab, and chemotherapy (FOLFOIXIR) as first-line treatment in patients with unresectable mCRC without prior treatment with chemotherapy . ResultsBACCI (NCT02873195) is a multicenter randomized phase II placebo-controlled clinical trial comparing capecitabine (chemotherapy) and bevacizumab with or without atezolizumab in patients with refractory MSS mCRC . The triNCT03396926 is a recent phase II clinical trial evaluating the safety and efficacy of combination capecitabine, bevacizumab and pembrolizumab (anti-PD-1) in locally advanced and metastatic unresectable MSS mCRC patients , 130. ToOverall, anti-PD-1, atezolizumab, or pembrolizumab, in combination with bevacizumab and chemotherapy, has demonstrated promising results across multiple clinical studies. Exploratory analysis within studies demonstrated that besides MMR status, TMB, Immunoscore-IC, and the presence of liver metastasis are important predictors of treatment outcome. AtezoTRIBE demonstrated improved clinical benefit in patients with high TMB and high Immunoscore-IC, both of which are associated with MSI-H tumor , 132. CoVEGFR inhibitors such as regorafenib, lenvatinib, apatinib, and fruquintinib are studied in combination with PD-L1/PD-1 blockade in patients with pMMR/MSS CRC. The efficacy varied across clinical trials and retrospective studies. Most clinical trials studied the combination of regorafenib and anti-PD-1 mAbs since regorafenib could enhance T cell activation and increase M1/M2 macrophage ratio compared to inhibitors selective for VEGFR-2 , 133.REGONIVO is a phase Ib/II trial to evaluate regorafenib in combination with anti-PD-1 antibodies nivolumab and toripalimab, respectively, for patients with advanced or metastatic pMMR CRC refractory or intolerant to standard chemotherapy . The resRecent clinical studies with similar combination strategies continued to confer variable results. NCT03946917 is a phase 1b/II study that demonstrated promising results in a subset of unselected pMMR/MSS mCRC patients treated with regorafenib and toripalimab (anti-PD-1) who had progressed or were intolerant to at least 2 prior line of chemotherapy . NCT0371REGOMUNE (NCT03475953) is the first phase II study that evaluated the efficacy and safety of regorafenib in combination with avelumab (anti-PD-L1) in patients with MSS advanced or metastatic CRC refractory to at least one prior standard therapy . The comLEAP-005 (NCT03797326) is a recent phase II study evaluating the effectiveness of pembrolizumab and lenvatinib in selected refractory solid tumors including the pMMR/non-MSI-H metastatic and/or unresectable CRC cohort . PromisiNCT03912857 is a phase II trial of the anti-PD-1 mAb, camrelizumab, in combination with apatinib (a selective tyrosine kinase inhibitor for VEGFR-2) for the treatment of advanced or metastatic MSS CRC refractory to two or more prior lines of standard therapy. ObjectivDespite the glimpse of a new treatment opportunity for pMMR/MSS mCRC patients brought forward by the REGONIVO study, the results were not replicated in other clinical studies. Nonetheless, the studies suggest the potential use of CD8\u2009+\u2009T cell infiltration and low-level TAM2 as a positive predictor for treatment efficacy of regorafenib plus avelumab on the TME . The useMitogen-activated protein kinase (MAPK) cascades are universally conserved transduction pathways that permit extracellular signals to regulate a range of complex physiological cellular programs including cellular proliferation, development, differentiation, migration, survival, and apoptosis . It is wNCT01988896 is a phase Ib clinical study that evaluated the efficacy of cobimetinib (a MEK inhibitor) and atezolizumab in patients with solid tumors, 84 of whom have mCRC . The advSimilarly, another phase Ib clinical study (NCT02876224) of atezolizumab and bevacizumab in combination with cobimetinib (MEKi) was conducted in patients with mCRC refractory to one or more lines of prior chemotherapy . They foThe addition of cobimetinib was insufficient to overcome MSS mCRC resistance to atezolizumab. However, potential synergistic activity between MEKi, anti-VEGF, and anti-PD-L1 therapy was observed in the primary analysis of a clinical study described above. Although it is difficult to draw conclusions as to whether the effects were due to the addition of anti-VEGF and/or MEKi, the lack of therapeutic options available for patients with chemo-refractory pMMR/MSS mCRC suggests that a three agent combination strategy is worth exploring.In addition to MAPK signaling, PI3K/AKT/mTOR signaling is associated with cell survival, migration, division, and other activities. A phase I/II clinical trial (NCT03711058) is currently studying the combination of nivolumab with copanlisib (PI3K inhibitor) in relapsed/refractory pMSS CRC .STAT3 is an intracellular signaling molecule and transcription factor shown to regulate an array of specific target genes involved in key cellular processes . SustainNapabucasin is a STAT3 inhibitor studied in combination with anti-PD-1 pembrolizumab in a multicenter phase II clinical trial (NCT02851004) in patients with mCRC refractory or intolerant to at least one regimen of standard chemotherapy . AdverseAlthough primary end point was not met in this clinical trial, napabucasin with pembrolizumab showed greater anti-tumor activity compared to both agents alone. Future studies in a targeted population based on related biomarkers should be further investigated to identify the subset of MSS CRC patients that may receive clinical benefits from the combination therapy.Cytotoxic chemotherapy is a fundamental part of treatment for patients with mCRC . CurrentA multicenter phase II study (NCT02860546) was conducted in combination of FTD/TPI and nivolumab in patients with chemotherapy-refractory MSS mCRC . The addAs discussed in Sect.\u00a04.2, clinical trials are investigating the potentiation of anti-PD-L1/anti-PD-1 by combining chemotherapy and anti-VEGF inhibitor (bevacizumab). Promising results from the triple agent regimen have suggested that chemotherapy and anti-VEGF can synergistically modulate the TME to make PD-L1/PD-1 ICI more effective against cold pMMR/MSS CRC , 61, 123Radiation therapy has been shown to exhibit immune stimulatory effects on the TME via three distinct and overlapping mechanisms: (1) induction of immunogenic cell death (ICD) of tumor cells; (2) upregulation of neoantigen presentation on MHC-1; and (3) direct alteration of the TME at the site of radiation . The ICDTo date, no significant clinical responses have been observed across four clinical studies in combination with PD-1 inhibitors , 80, 82.One potential approach to improve the efficacy of anti-PD-1 plus radiotherapy in patients with MSS mCRC relies on the use of multiple nonredundant ICIs. In a phase II clinical trial (NCT03104439), MSS mCRC patients refractory to two or more lines of prior therapy received a combination treatment with ipilimumab (anti-CTLA-4) and nivolumab in conjunction with 8\u00a0Gy of radiotherapy . The comDespite the theoretical framework obtained from preclinical studies of pMMR/MSS cold CRC, limited success was observed across clinical studies for the different combination strategies. Small sample sizes and heterogeneity of tumors or TME in each trial could explain this finding. Comparisons between molecular and cellular phenotypes of common mouse syngeneic models and human tumors may increase our understanding of the mismatched results drawn from preclinical and clinical experiences. Better biomarker detection and patient classification prior to treatment is critical to improve outcomes of combination therapies. Furthermore, it is important to note that oncological signaling pathways have broad biological functions that could be difficult to target specifically or selectively in MSS CRC cells. There are other immune-suppressive molecules or pathways in TME; multiple signaling pathways participate in tumor development and progression. New combinations with other signaling inhibitors or reagents such as temozolomide, which can induce mutation in tumor cells, need to be investigated. We recognize the complexity of the TME; therefore, we suggest future studies to focus on identifying better preclinical models that closely mimic the TME of MSS CRC and efficacy biomarkers in the pMMR/MSS CRC population.168Oncologic outcomes are improving with acceptably safe use of aggressive surgical and local therapy for colorectal liver metastases in carefully selected patients. Evaluating the benefit of systemic immunotherapy either in conjunction with those therapies or following them will be an important avenue for future study. An active multicenter early phase II study is currently investigating the effectiveness of local tumor ablation (radiofrequency ablation or stereotactic body radiation therapy) in combination with durvalumab (Anti-PD-1) and tremelimumab (anti-CTLA-4) in ICI na\u00efve patients with unresectable colorectal liver metastases (NCT03101475). Combination strategies with other anti-tumor agents to potentiate the efficacy of anti-PD-L1/anti-PD-1 in patients with pMMR/MSS advanced or metastatic CRC has become a major research interest as it provides new therapeutic opportunities. In general, combination treatment is safe without significant AEs compared with monotherapy. Preliminary analyses of combination anti-PD-1/PD-L1 inhibitors and other anti-cancer therapies revealed potential clinical benefits in certain subgroups of patients with pMMR/MSS mCRC. Focused approaches to studying these combination regimens will improve outcome of PD-1/PD-L1 combination treatment. We believe that combination strategies involving PD-L1/PD-1 blockade remain a priority for future research as it has the potential to elicit benefits that will revolutionize the clinical landscape for patients with pMMR/MSS cold CRC.Supplementary file1 (DOCX 22 kb)Below is the link to the electronic supplementary material."} +{"text": "Staphylococcus aureus and Candida sp. bloodstream infections (BSI) are associated with considerable mortality. The COVID-19 pandemic presented new challenges in the acute care setting which may affect outcomes in high-risk populations.S. aureus or Candida sp. BSI between pre-pandemic (January 2017-February 2020) and pandemic (March 2020- February 2023). The primary clinical outcome was all-cause mortality. Secondary outcomes include length of stay (LOS) and 30-day readmission. Data was stratified across the pre-pandemic and pandemic period, COVID-19 positive and negative patients, and community and hospital-onset BSI.Retrospective cross-sectional analysis across a large healthcare system of all admitted patients aged 18 years or older with S. aureus and Candida sp. BSI patients.A total of 17,730 patients were included in the analysis. Baseline characteristics were similar between pre-pandemic and pandemic groups aside from higher hospital-onset infection rates in the pandemic COVID-19 positive population [Table 1]. No significant differences were found in mortality between COVID-19 negative groups [Table 2]. Concomitant COVID-19 infection was associated with increased mortality in both the S. aureus and Candida sp. community-onset and hospital-onset BSI did not change during the pandemic compared to pre-pandemic for patients without concomitant COVID-19 infection.While patients with concomitant COVID-19 infection had a substantially higher mortality compared to those without infection, the mortality in patients with Reese Cosimi, PharmD, Allergen: Advisor/Consultant"} +{"text": "Bdnf in the microglia accompanied by reduced adulthood sociability. Additionally, transgenic mice overexpressing microglia Bdnf\u2014regulated using doxycycline at different time points\u2014underwent behavioral, electrophysiological, and gene expression analyses. In these mice, long-term overexpression of microglia BDNF impaired sociability and excessive mPFC inhibitory neuronal circuit activity. However, administration of doxycycline to normalize BDNF from p21 normalized sociability and electrophysiological functions; this was not observed when BDNF was normalized from a later age (p45\u2013p50). To evaluate the possible role of BDNF in human sociability, we analyzed the relationship between adverse childhood experiences and BDNF expression in human macrophages, a possible substitute for microglia. Results show that adverse childhood experiences positively correlated with BDNF expression in M2 but not M1 macrophages. Thus, microglia BDNF might regulate sociability and mPFC maturation in mice during the juvenile period. Furthermore, childhood experiences in humans may be related to BDNF secretion from macrophages.Microglia and brain-derived neurotrophic factor (BDNF) are essential for the neuroplasticity that characterizes critical developmental periods. The experience-dependent development of social behaviors\u2014associated with the medial prefrontal cortex (mPFC)\u2014has a critical period during the juvenile period in mice. However, whether microglia and BDNF affect social development remains unclear. Herein, we aimed to elucidate the effects of microglia-derived BDNF on social behaviors and mPFC development. Mice that underwent social isolation during p21\u2013p35 had increased Microglia refine synapses and form developing brain circuits in an activity-dependent manner , 2. ThisFurthermore, similar to sensory functions, social behaviors develop via experience-dependent brain maturation during a limited postnatal window , 12. ForBrain-derived neurotrophic factor (BDNF) contributes to maturing inhibitory interneurons and closing critical periods . Microglad libitum access to food and water throughout the study. C57BL/6J mice isolated from the weaning age (p21) for two weeks were regrouped with age-, sex-, and strain-matched mice at p35 (3\u20135 mice per cage), labeled juvenile social isolation (j-SI) mice. Control C57BL6/J mice were group-housed following weaning at p21 and labeled Group-Housed (GH) mice. We crossed ionized calcium-binding adapter molecule 1(Iba1) promoter driving tetracycline transactivator (Iba1-tTA) transgenic mice (line 75) [Bdnf knock-in homozygous (BdnftetO/tetO) mice [Bdnf-overexpressing mice (Iba1-tTA::BdnftetO/+ mice) and control mice (BdnftetO/+ mice). Both Iba1-tTA and BdnftetO/tetO mice strains had a mixed C57BL/6J and 129/SvEv background; only F1 male mice were used for all experiments [Bdnf overexpression, F1 mice were fed doxycycline (DOX)-containing chow : from weaning (p21) or adulthood (p45\u2013p50). Groups of mice administered DOX from adulthood were provided chow with DOX ad libitum for at least two weeks before the experiment.All study protocols were approved by the Animal Care Committee of Nara Medical University in accordance with the policies established in the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The animals were housed in a temperature- and humidity-controlled animal facility under a standard 12-h light-dark cycle (lights on 08:00\u201320:00) with line 75) with tettO) mice , 33 and eriments . Genotyperiments , 32. To t-test. Even if the sample data passed the Shapiro-Wilk test for normality, the nonparametric Mann\u2013Whitney U-test was used if the F-test indicated unequal variances. When the sample data significantly differed from normal distribution as assessed by the Shapiro-Wilk test, the nonparametric Mann\u2013Whitney U-test was used. Values with right-skewed distribution were log-transformed for statistical analyses. The Kruskal-Wallis test was used to validate microglia-specific BDNF-overexpressing levels. Two-way ANOVA followed by a Bonferroni post hoc test analyzed the time spent in each zone in the three-chamber social preference test [Bdnf expression in peripheral blood mononuclear cells (PBMC) and microglia in mice, as well as between child abuse and trauma scale (CATS) scores and BDNF in macrophages in humans. The false discovery rate-controlling Benjamini\u2013Hochberg procedure was used to control for multiple comparisons [All statistical analyses were performed using Prism v.9 . When the sample data were normally distributed and had equal variances, as determined using the Shapiro-Wilk test and the subsequent F-test, significant differences between groups were assessed using an unpaired two-tailed Student\u2019s nce test . Two-wayparisons . All datDetailed methodological information is provided in the Supplementary materials and methods.Bdnf mRNA expression in microglia from the cerebral and prefrontal cortexes was higher in j-SI mice than GH mice . These findings indicate that microglial Bdnf expression changes are specific to microglia [Bdnf expression in the cerebral cortex, including the mPFC.Our single housing condition started at P21 and lasted for two weeks, followed by re-grouped housing with age-, sex-, and strain-matched mice . While s GH mice , d. Howeicroglia . MoreoveBdnf overexpression observed in j-SI and examine the causal relationship between microglia-specific Bdnf gene upregulation and social impairment, we exploited a tet-off system [Bdnf mRNA expression (Iba1-tTA::Bdnf(tetO/+) mice without DOX and MG-BDNF overexpression in Iba1-tTA::Bdnf(tetO/+) mice was normalized from day 5 after orally administering DOX and control mice, suggesting that genetically-induced MG-Bdnf overexpression in Iba1-tTA::Bdnf(tetO/+) mice does not significantly alter total Bdnf mRNA expression levels in the cerebral cortex or mPFC .To recapitulate the microglia-specific and sustained f system to inducpression . MG-BDNFring DOX . Bdnf mRIba1-tTA::Bdnf(tetO/+) mice had a lower social interaction score than the control mice and spent less time around the novel mice (Iba1-tTA::Bdnf(tetO/+) and control mice . However, considering that the three-chamber apparatus has certain interpretative issues [Iba1-tTA::Bdnf(tetO/+) mice made fewer approaches to other mice (Iba1-tTA::Bdnf(tetO/+) and control mice during the 1-h recording time mice in terms of spike frequency and amplitude (Iba1-tTA::Bdnf(tetO/+) mice compared with the control mice. In contrast, sEPSC and sIPSC amplitudes did not significantly differ (Iba1-tTA::Bdnf(tetO/+) mice, miniature EPSC (mEPSC) frequency was significantly lower mice (Iba1-tTA::Bdnf(tetO/+) mice revealed that sustained MG-BDNF overexpression altered gene expression related to Wnt-activated receptor activity , Wnt-protein binding , active borate transmembrane transporter activity , and complement C3a receptor activity mice. In particular, the Iba1-tTA::Bdnf(tetO/+) mice had significantly decreased C1qa and C3ar1 expression . These results suggest that MG-BDNF overexpression decreases the complement cascade functional status in the mPFC.RNA-sequencing and principal component analysis revealed that MG-BDNF overexpression during the juvenile period altered mPFC distribution at two months of age , b. Comp/+) mice , d. GeneIba1-tTA::Bdnf(tetO/+). Similarly, the control mice were also fed DOX from p21 (Iba1-tTA::Bdnf(tetO/+) and control mice at two months of age (Iba1-tTA::Bdnf(tetO/+) and control mice (Iba1-tTA::Bdnf(tetO/+) from the juvenile period leads to comparable excitability of mPFC layer V pyramidal cells and its inhibitory inputs to the control mice in adulthood.Given that BDNF is known to be associated with the critical period of experience-dependent neural plasticity , we manifrom p21 . In the s of age , indicats of age , d. Simis of age . In addirol mice , h, k, lIba1-tTA::Bdnf(tetO/+) and control mice from p45\u2013p50 . The Iba1-tTA::Bdnf(tetO/+) and control mice exhibited comparable social interaction scores and time spent around the novel mice at two months of age . Similarly, no differences existed in locomotion or anxiety during the open field test . In contrast, normalizing MG-BDNF during adulthood did not improve the electrophysiological abnormalities of mPFC layer V pyramidal cells in the Iba1-tTA::Bdnf(tetO/+) mice at two months of age. The spike frequency of excitability remained significantly reduced . In addition, sIPSC and mIPSC frequencies remained significantly increased in the Iba1-tTA::Bdnf(tetO/+) mice compared with control mice . Furthermore, the sIPSC amplitude was significantly reduced in the Iba1-tTA::Bdnf(tetO/+) mice compared with control mice . No significant differences existed in the frequency or amplitude of sEPSCs or mEPSCs . These results suggest that MG-BDNF overexpression during the juvenile period may be critical for forming inhibitory synapses in the mPFC. However, even the MG-BDNF intervention during adulthood can improve social behavior.Next, we assessed the effect of delayed normalization of MG-BDNF overexpression after p45 on social behavior and mPFC layer V pyramidal cell function. DOX was orally administered to the Bdnf expression between microglia from the brain and peripheral blood mononuclear cells in mice , we measured BDNF expression in human peripheral macrophages, which share properties with microglia [Bdnf in mice. CD14-positive monocytes were collected from the peripheral blood of participants and differentiated into M1/M2 macrophages; subsequently, BDNF mRNA expression was measured . The Japanese version of the CATS was used to assess adverse childhood experiences [BDNF expression in M2 macrophages. In the CATS sub-items, neglect and punishment, among other variables, positively correlated with BDNF expression in M2 macrophages to adulthood (p45\u201350). This is consistent with the critical period for social ability acquisition in mice, which is from p21 to p35 . In contThe excitability of layer V pyramidal cells in the mPFC of MG-BDNF-overexpressing mice is similar to that observed in j-SI mice , 18. ThiIn this study, we performed RNA-seq of the mPFC; our findings suggested the involvement of the complement system as a mechanism of MG-BDNF-induced reduction of sociability. The relationship between BDNF and the complement system has not previously been reported; nevertheless, complement C3 signaling starting at C1q is crucial for the experience-dependent synaptic pruning of microglia , 7. ThusBDNF expression in human peripheral M2 macrophages in this study. Microglia and macrophages should be considered separately [BDNF expression in macrophages that share similarities in CD11b expression and phagocytic capacity with microglia [BDNF in M2 macrophages are associated with reduced sociability remains unclear; however, BDNF abnormalities have been identified in humans with autism spectrum and posttraumatic stress disorders [Childhood experiences were also associated with parately as primiparately . Thus, ricroglia , 49 (theicroglia ). M1 macicroglia \u201380. M2 microglia \u201383. Whetisorders \u201386. Theyisorders , 88.In conclusion, these findings indicate that MG-BDNF is critical in developing social behaviors and mPFC function in a time-specific manner, potentially related to juvenile social experience-dependent social development. Our results provide new insights into experience-dependent social behavior formation and mPFC development."} +{"text": "Likewise, degradation (hydrolytic and enzymatic) over 21 days for all DBM-NP-MA content groups was significantly decreased, \u223c45% less in PBS and collagenase-containing PBS, in UV-crosslinked versus uncrosslinked groups. The incorporation of DBM-NP-MAs into scaffolds decreased mass loss compared to GNP-MA-only scaffolds during collagenase degradation. An in vitro osteogenic study with bone marrow-derived mesenchymal stem cells demonstrated osteoconductive properties of 3DP scaffolds for the DBM-NP-MA contents examined. The creation of photoreactive DBM-NP-MAs and their application in 3DP provide a platform for the development of ECM-derived colloidal materials and tailored control of biochemical cue presentation with broad tissue engineering applications.Demineralized bone matrix (DBM) has been widely used clinically for dental, craniofacial and skeletal bone repair, as an osteoinductive and osteoconductive material. 3D printing (3DP) enables the creation of bone tissue engineering scaffolds with complex geometries and porosity. Photoreactive methacryloylated gelatin nanoparticles (GNP-MAs) 3DP inks have been developed, which display gel-like behavior for high print fidelity and are capable of post-printing photocrosslinking for control of scaffold swelling and degradation. Here, novel DBM nanoparticles were fabricated and characterized prior to incorporation in 3DP inks. The objectives of this study were to determine how these DBM-NPs would influence the printability of composite colloidal 3DP inks, assess the impact of ultraviolet (UV) crosslinking on 3DP scaffold swelling and degradation and evaluate the osteogenic potential of DBM-NP-containing composite colloidal scaffolds. The addition of methacryloylated DBM-NPs (DBM-NP-MAs) to composite colloidal inks did not significantly impact the rheological properties associated with printability, such as viscosity and shear recovery or photocrosslinking. UV crosslinking with a UV dosage of 3 J/cm Craniofacial bone augmentation is often necessary as a result of tooth extraction, disease, trauma or tumor resection. Specifically, alveolar bone augmentation may be used to ensure sufficient bone depth and volume for dental implantation and assist in cleft palate repair . The cur3D printing (3DP) is a fabrication technique that has gained interest for tissue engineering and regenerative medicine because it allows for precise control over scaffold size, shape and microarchitecture, key for the complex geometries encountered in craniofacial and orthopedic bone regeneration. 3DP has been used previously to print DBM in microparticle form within a poly(lactic-co-glycolic acid) ink . Howeverin vitro. Furthermore, the osteoinductive and osteoconductive effects of DBM-NP-MA content in UV-crosslinked 3DP scaffolds were evaluated in a 21-day in vitro study of mesenchymal stem cell osteogenesis. This study presents the creation of novel DBM-NP colloidal components for 3DP inks and confirms the osteoconductive properties of DBM-incorporating colloidal composite 3DP scaffolds.In this study, DBM nanoparticles (DBM-NPs) were synthesized and methacryloylated [methacryloylated DBM-NPs (DBM-NP-MAs)] for use in colloidal composite 3DP inks with photoreactive methacryloylated GNPs (GNP-MAs). These inks combine the intrinsic bone regenerative cues found within DBM in photoreactive colloidal building blocks blended with GNP-MAs, formulated from ECM-derived gelatin, for increased printability and improved post-printing scaffold integrity. Here, we sought to determine the impact of DBM-NP inclusion on colloidal composite 3DP ink printability and the dose-dependent influence of DBM-NPs and ultraviolet (UV) crosslinking on 3DP scaffold swelling, degradation and osteogenic properties. To that end, DBM-NPs were synthesized and biochemically characterized. The rheological properties of GNP-MA and DBM-NP-MA composite colloidal inks were assessed as a function of incorporated DBM-NP-MA content. The physicochemical properties of GNP-MA:DBM-NP-MA scaffolds (henceforth referred to as GNP:DBM-NP scaffolds for the sake of simplicity since only methacryloylated nanoparticles were leveraged for 3DP) relative to DBM-NP-MA content and UV crosslinking were then analyzed via swelling and degradation studies, where changes in scaffold morphology and mass were evaluated over 21\u2009days The legs of 12- to 16-week-old female Sprague-Dawley SASCO rats were donated from Rice University in accordance with protocols approved by the Rice University Institutional Animal Care and Use Committee and the tibiae and femora harvested. Bone demineralization was adapted from previously established protocols . BrieflyGNPs were synthesized using a two-step desolvation method according to previously published methods . An amouDBM-NPs were synthesized using a two-step desolvation method modified from that for the GNPs described above . To soluScanning electron microscopy (SEM) was performed to quantify nanoparticle diameter using a Quanta 400 system . GNPs, GNP-MAs, DBM-NPs and DBM-NP-MAs were dispersed in Milli-Q water. An SEM imaging stand was coated with 20\u2009nm of gold and the nanoparticles were placed on top, flash frozen and then lyophilized. Thereafter, a 5-nm layer of gold was deposited on the lyophilized nanoparticles prior to imaging. This method produced a monolayer of particles to facilitate more representative estimation of size. ImageJ (version 1.53f) was used to quantify the dried diameters of GNPs, GNP-MAs, DBM-NPs and DBM-NP-MAs by manually measuring 100 nanoparticles per batch for three batches of each.Native bone, DBM and DBM-NP-MA samples (10\u2009mg/each) were solubilized in 1\u2009ml of 0.5 M HCl solution containing 1\u2009mg/ml pepsin for the analysis of DNA, total protein, collagen and sulfated glycosaminoglycan (sGAG) content and 1\u2009ml of 0.25 M acetic acid solution containing 1\u2009mg/ml of pepsin for calcium content by gently shaking at 37\u00b0C for 2 days , 25. The1H nuclear magnetic resonance (1H NMR) spectrometer to identify the methacryloyl group content of each material and confirm the methacryloylation of DBM-NPs and GNPs. The samples were prepared as reported previously and methacrylamide modification for DBM-NP-MAs versus DBM-NPs. These peaks were also observed in GNP-MAs after methacryloylation compared to gelatin and GNPs were evaluated. All inks showed shear-thinning behavior with similar decreases in ink viscosity in response to increasing shear rate, confirming extrudability . Of noteThe UV crosslinking GNP:DBM-NP composite colloidal inks was evaluated using photorheometry. First, frequency sweeps of inks were recorded before and after exposure to UV light. As seen in P\u2009<\u20090.05). Swelling over the first day, shown via changes in non-porous area and average pore area is found in ous area and averous area , demonstAdditional effects of DBM-NP-MAs and UV crosslinking on swelling over the full 21 days are presented in P\u2009<\u20090.05). In PBS and high (75:25 GNP:DBM-NP) DBM-NP-MA content scaffolds and evaluated over 21 days for cell proliferation and differentiation. On PicoGreen analysis of DNA content, higher DBM-NP content scaffolds generally facilitated higher cell proliferation . On Day An additional smaller study comparing DNA content and calcium deposition on 100:0 GNP:DBM-NP and 0:100 GNP:DBM-NP scaffolds see showed sThis study details the fabrication, characterization and application of DBM-NPs for 3DP aimed at bone regeneration. DBM has been widely clinically applied in bone augmentation and regeneration, including dental, craniofacial and spinal fusion applications , 6, 10. Following methacryloylation of the nanoparticles, biochemical analysis of the DBM-NPs confirmed successful demineralization and decellularization with retention of collagen. The level of DNA present in DBM-NP-MAs was confirmed to be below the maximum acceptable level of 50\u2009ng/mg for decellularization, above which clinical concern for an inflammatory reaction is increased . DespiteG\u2032 and G\u2033 following high shear replicates how these properties will adapt following extrusion, crucial for maintaining the shape fidelity of the printed fibers [DBM-NP-MAs were then incorporated into colloidal composite inks in varying ratios with GNP-MAs. A previous study demonstrated the ability to create stable, photoreactive extrusion 3DP inks using GNP-MAs . Here, Dd fibers , 36, 37.d fibers , 38. Thed fibers . The simd fibers . The retLikewise, DBM-NP-MA incorporation into GNP-MA inks did not reduce the photoreactivity of the colloidal composite inks. Methacryloylation has been widely explored for bioinks including decellularized ECMs as a means of rapid crosslinking with UV exposure and has been shown to have no significant impact on ECM-based material bioactivity . NMR datThe stability of these networks was further physicochemically evaluated relative to DBM-NP-MA content and UV crosslinking with 3DP scaffold swelling and degradation. Of note, uncrosslinked GNP-MA scaffolds were not stable in PBS over the full 21\u2009days of the study, with complete scaffold dissolution occurring between 3 and 7\u2009days. In contrast, both low and high DBM-NP-MA-incorporating uncrosslinked scaffolds persisted throughout the entire length of the swelling study. This rapid degradation of uncrosslinked GNP-MA scaffolds has been previously attributed to the partial degradation of glutaraldehyde-based covalent intraparticle crosslinking by the acidic environment generated by the byproduct methacrylic acid during the methacryloylation reaction . The resThe degradation of these scaffolds followed a similar pattern. In PBS, by the end of 21 days, UV-crosslinked scaffolds proved to have significantly less mass loss than uncrosslinked groups. Interestingly, UV-crosslinked 75:25 GNP:DBM-NP scaffolds also demonstrated significantly less mass loss compared to 100:0 GNP:DBM-NP scaffolds. In collagenase, again, uncrosslinked scaffolds demonstrated increased mass loss compared to UV-crosslinked scaffolds. In this case, the UV-crosslinked 95:5 GNP:DBM-NP scaffolds demonstrated less mass loss compared to UV-crosslinked 100:0 GNP:DBM-NP scaffolds. The persistence of these DBM-NP-MA groups again demonstrates that the incorporation of DBM-NP-MAs, although seen here in varying amounts, may increase the aqueous stability of 3DP colloidal composite scaffolds and reinforce the UV-generated covalent interparticle network. This study also demonstrates the potential for controlled degradation of ECM-incorporating materials in regenerative medicine applications, allowing for temporal precision in biochemical cue presentation and release.The osteoconductivity and osteoinductivity of low and high DBM-NP-MA-content scaffolds were then evaluated. Increased DBM-NP content supported increased cell proliferation at earlier timepoints in both media types, with the 75:25 GNP:DBM-NP group having higher DNA values than 95:5 GNP:DBM-NP scaffolds at 21\u2009days in basal media. This difference in proliferation may be attributed to the trophic factors found within the DBM-NP-MAs that increase their bioactivity compared to gelatin alone , 10. Whein vitro human MSC osteogenesis and in vivo bone formation, suggesting that the addition of this nanomaterial and its mineral cues may be beneficial within a colloidal composite system [in vitro osteoinductivity of DBM for human MSCs [Partially DBM has also been shown to significantly increase osteogenic differentiation of bone marrow-derived stem cells, indicating that the provision of the mineral component within these colloidal composite systems might improve osteoinductivity . Recent e system , 59. Likman MSCs . The DBMThe data here suggest that novel ECM-based nanoparticles may be fabricated and integrated into colloidal composite systems without significantly impacting properties such as viscosity and recovery behavior that determine extrudability and 3DP print fidelity. Additionally, the functionalization of these colloidal building blocks enables photocrosslinking. This study demonstrates that this process results in control over hydrogel swelling and degradation. These results show the potential for the development of ECM-based nanoparticles to expand to a broad range of tissues. ECM-based nanoparticles could then be incorporated within colloidal composite systems for 3DP or within injectable platforms. The generation of photoreactive ECM-based nanoparticles for 3DP in composite inks, therefore, represents a versatile tool for regenerative medicine, offering a system for controlled swelling and degradation as well as the modular introduction of additional materials or biomolecules.in vitro osteogenic study with bone marrow-derived MSCs, both 95:5 and 75:25 GNP:DBM-NP scaffolds showed similar osteoconductivity, demonstrated by calcium deposition in osteogenic media. These photocrosslinkable DBM-NP-MA and GNP-MA colloidal composite inks demonstrate a platform for developing ECM-derived colloidal materials and controlling the temporal presentation of biochemical cues for tissue engineering.In this study, novel photoreactive DBM-NP-MAs were developed and introduced into colloidal composite 3DP inks in different ratios with GNP-MAs . These inks demonstrated high printability with no disruption in the colloidal noncovalent network upon incorporation of DBM-NP-MAs demonstrated through the retention of shear-thinning and recovery behavior. Additionally, due to the photocrosslinkable nature of the colloidal components, physicochemical properties such as scaffold swelling and degradation were controlled with UV crosslinking. Uncrosslinked scaffolds for all DBM-NP-MA content levels demonstrated increased changes in scaffold area and average pore size compared to UV-crosslinked scaffolds. Likewise, significantly less mass loss was observed over 21\u2009days in both PBS and collagenase for scaffolds that were photocrosslinked. Uncrosslinked DBM-NP-MA-incorporating scaffolds demonstrated greater stability while swelling than 100:0 GNP:DBM-NP scaffolds, thought to be the result of additional noncovalent interactions with the complexity of DBM composition and DBM-NP-MA size. In an rbad090_Supplementary_DataClick here for additional data file."} +{"text": "Lung transplant (LTx) recipients universally receive peri- and post-transplant antibiotics. Approaches to antibiotics for candidates with cystic fibrosis (CF) are institution specific. Little has been published about these practices. Routine LTx perioperative prophylaxis at our institution is 96 hours of vancomycin and cefepime with antibiotics then adjusted for positive intraoperative donor and recipient cultures. In recipients with CF, perioperative prophylaxis is determined pre-LTx to cover colonizing organisms isolated in culture from the 2 years before LTx, adjusted for intraoperative cultures, and administered for 14-21 days post-LTx. Our study aims to add to the current literature by describing our center\u2019s experience with antibiotic management and outcomes in lung transplant recipients with CF to help guide best practices for prophylaxis strategies for this patient population.Pseudomonas aeruginosa, or Burkholderia cepacia complex. Peri- and post-operative antibiotic regimens, donor and recipient culture results, changes in antibiotic therapy post-LTx, and duration of antibiotic therapy were documented. Outcomes included rehospitalization, isolation in culture of the prophylaxis-targeted organisms post-LTx, and mortality, within 6 months of LTx.We performed a retrospective chart review of patients with CF who underwent LTx from 3/1/2015-11/18/21 at our center and were colonized pre-LTx with MRSA, Clostridioides difficile infection. One (5.3%) recipient died within 6 months post-LTx.Nineteen incidents of LTx were included in this study. Pre-LTx colonizing organisms and antibiotics administered are outlined in table 1. Prophylaxis-targeted organisms were infrequently isolated in culture after LTx (table 2), with 7/19 (36.8%) developing post-transplant infection with prior colonizing organisms. Four (21.1%) recipients developed post-LTx LTx recipients with CF who received targeted perioperative prophylaxis for pre-LTx colonizing organisms infrequently developed post-LTx culture positivity with the same bacteria including none during the antimicrobial prophylaxis period. Future prospective studies are needed to determine optimal antibiotic duration of peri-LTx antibiotics in this patient population.All Authors: No reported disclosures"} +{"text": "SARS-CoV-2 has caused more than 1 million deaths worldwide. Several studies showed that People with HIV had higher rates of hospitalization and mortality with COVID-19 compared with people without HIV but data on serologic response to COVID-19 vaccine among People Living with HIV (PLHIV) is still limited.nd dose of vaccination.A prospective cohort study on determination of serologic response to COVID-19 vaccine among completely vaccinated PLHIV enrolled in a treatment hub in the Philippines until December 31, 2021 was done. Baseline demographics were collected via chart review. History of COVID-19 infection was also asked during enrollment, on follow-up and at the end of 6 months after the 2Blood Extraction were done on enrollment and on follow-up visit to the treatment hub. All the specimen were processed using the SARS-CoV2 ( RocheElecsys Anti-SARS-CoV2 spike (S) protein assays.A total of 261 PLHIV who received vaccination from February 11, 2021 to December 29, 2021 were enrolled in the study. The participants received any of the following vaccine brands: CoronoVac/Sinovac (41%), Oxford-AstraZeneca (20.7%), Pfizer-BioNTech (21.5%), Moderna (14.2% and Janseen (2.7%). Univariate analysis showed that presence of opportunistic infection, HIV Viral load , Antiretroviral(ARV) Status, COVID-19 Vaccine brand were all significant predictors of antibody response. On multiple regression analysis, only the Vaccine Brand and Viral load remained significant. Sinovac gave a lower median antibody level and seroconversion rate as compared to other vaccine brands. HIV Viral load > 1000 copies/ml was predictive of lower antibody response.In this study among PLHIV, the type of vaccine and HIV viral load were significant predictors of antibody response to COVID-19 Vaccine. Specifically, Sinovac COVID-19 Vaccine gave a lower Median SARS-CoV-2 IgG(S) Antibody and Seroconversion rate as compared to other Vaccine brands. HIV Viral load > 1000 copies/ml was predictive of lower antibody response to COVID-19 Vaccine . Overall, the antibody response peaked at 2-4 weeks after vaccination and decreases thereafter.All Authors: No reported disclosures"} +{"text": "Scientific Reports 10.1038/s41598-019-55403-4, published online 12 Dec 2019Correction to: This Article contains an error in the Methodology, under the subheading \u2018Transesterification process\u2019, where the scheme incorrectly illustrates the transesterification process due to an incorrect double bond structure for the Triglyceride (Oil) and Methyl Ester (Biodiesel) parts.The correct scheme appears below:"} +{"text": "C9orf72 or a mutation in progranulin (PRGN) or have a sporadic cause with no family history of dementia [C9orf72 mutation, dipeptide pathology was present in the retina in the absence of TDP-43 pathology [Frontotemporal dementia (FTD) is a clinically heterogeneous disease, characterized by behavioural, language and movement symptoms. The underlying pathology is termed frontotemporal lobar degeneration (FTLD) and consists of distinct underlying pathologies including aggregation of transactive response DNA-binding protein 43 (TDP-43), tau and fused-in-sarcoma (FUS) . Approxidementia , 10, 12.PRGN mutation, retinal changes have been observed and include thinning and formation of fluorescent lesions, which precede symptoms of FTD [PRGN mouse model, neurodegeneration and nuclear depletion of TDP-43 was reported without TDP-43 aggregation [There are limited data on retinal changes in FTLD. In donors with FTLD due to s of FTD , 14. In C9orf72 repeat expansion, one PRGN mutation and two were genetically sporadic . Pathologically, donors presented with FTLD-TDP types A (PRGN), B (C9orf72), C (sporadic) and E (sporadic) [n\u2009=\u20095), FTLD-FUS (n\u2009=\u20091), AD with limbic-predominant age-related TDP-43 depositions (LATE) (n\u2009=\u20092) and without (n\u2009=\u20091), donors with ALS due to TDP-43 (ALS-TDP (n\u2009=\u20092)) and neurologically healthy controls (n\u2009=\u20096). Retinal TDP-43 pathological burden in FTLD-TDP donors was scored into none, few, frequent and abundant inclusions. Details and pTDP-43 scores of the donors are listed in Supplementary File 1. Immunohistochemical stainings were performed for (pan)TDP-43, phosphorylated TDP-43 (pTDP-43), p62 (SQSTM1), and the dipeptide repeat proteins (polyGA and polyGP). Double-fluorescence with calbindin D28K and calretinin was performed to identify horizontal cells and amacrine cells, respectively (for detailed procedures see Supplementary File 1).Post-mortem retina tissue was obtained from 7 donors with FTLD-TDP, of which four had a poradic) . We alsoC9orf72 and PRGN mutation carriers, as well as the sporadic FTLD-TDP donors, TDP-43 aggregates were observed in the outer plexiform layer of the retina. No colocalization was found with markers for horizontal or amacrine cells. No TDP-43 inclusions were observed in the retina of FTLD-tau, ALS, AD, and neurologically healthy control donors. Interestingly, all C9orf72 mutation carriers showed abundant presence of p62 and display aggregation of dipeptides linked to the hexanucleotide repeat expansion in the inner nuclear layer of the retina [In e retina . The FTLC9orf72 hexanucleotide repeat expansion and showed no retinal or cortical TDP-43/dipeptide pathology. Recently, an increased signal for pTDP-43 in the retina was reported in 6 ALS donors [C9orf72 mutation carriers. This suggests that genetic factors play a role in the occurrence of TDP-43 aggregates. On the other hand, we also observed TDP-43 aggregation in sporadic FTLD-TDP cases. Interestingly, TDP-43 inclusions in the retinal internuclear layer have been reported in subjects with chronic traumatic encephalopathy (CTE) [The ALS patients included in the current study did not carry the S donors . We obsehy (CTE) , 4. AddiC9orf72 mutations, dipeptide pathology is also present in the retina. In this study, we observed no TDP-43 inclusions in the retina of 2 ALS donors without cortical TDP-43 depositions or AD patients with limbic TDP-43 pathology, suggesting that retinal TDP-43 pathology reflects the cortical involvement of TDP-43 aggregation. These findings provide opportunities for retinal TDP-43 aggregation as a biomarker for FTLD-TDP, using non-invasive retinal imaging techniques with the purpose of diagnosing and monitoring progression of FTLD-TDP.To summarize, we report that manifestations of pathological TDP-43 are present in the retina of donors with FTLD-TDP. In donors with Supplementary file1 (DOCX 25 KB)Below is the link to the electronic supplementary material."} +{"text": "Circular RNA (circRNA), a newly identified important component of the transcriptome, is formed by covalently bonded single-stranded RNA through back splicing or other2\u2013\u2212) (\u2212-infected cells and virion-containing supernatants. Currently used methods of identifying circRNA are established on the examination of the back-splicing junction (BSJ) (Murine hepatitis virus (MHV), a betacoronavirus, has been used in a mouse model to study human coronaviruses . Gribble\u2212) . We hypoon (BSJ) .ViReMa is one such tool that can quickly and sensitively identify viral RNA splicing junctions, including forward-splicing junctions (FSJs) and BSJs from next-generation sequencing data . Therefo\u2212-infected cell monolayers and and MHV-WT and MHV-ExoN\u2212 viral supernatant, respectively. By normalizing the genome coverage, the number of MHV circRNAs in MHV-ExoN\u2212-infected cells . circRNAs have a covalently closed configuration and are hence more resistant to exoribonuclease RNase R than linear RNAs . Thus, wBombyx mori nucleopolyhedrovirus (Our overarching findings of this study include that MHV encodes circRNAs and nsp14-ExoN is important for the biogenesis of MHV circRNAs. Since nsp14 is required for RNA recombination , we specdrovirus . Howeverhttps://data.mendeley.com/datasets/kw453xnjkh/draft?a=c3c9ee47-38f6-43f3-84bd-41b4d2378281.Extended data are available from Mendeley Data:"} +{"text": "Androgen receptor (AR) expression is absent in 40\u201390% of estrogen receptor (ER)-negative breast cancers. The prognostic value of AR in ER-negative patients and therapeutic targets for patients absent in AR remains poorly explored.We used an RNA-based multigene classifier to identify AR-low and AR-high ER-negative participants in the Carolina Breast Cancer Study and The Cancer Genome Atlas . We compared AR-defined subgroups by demographics, tumor characteristics, and established molecular signatures .AR-low tumors were more prevalent among Black (relative frequency difference (RFD) = +7%, 95% CI = 1% to 14%) and younger participants in CBCS and were associated with HER2-negativity , higher grade , and higher risk of recurrence scores , with similar results in TCGA. The AR-low subgroup was strongly associated with HRD in CBCS and TCGA . In CBCS, AR-low tumors had high adaptive immune marker expression.Multigene, RNA-based low AR expression is associated with aggressive disease characteristics as well as DNA repair defects and immune phenotypes, suggesting plausible precision therapies for AR-low, ER-negative patients. Androgen receptor (AR), which is expressed in approximately 30\u201360% of estrogen receptor (ER)-/progesterone receptor(PR)-/human epidermal growth factor receptor 2(HER2) + breast cancers and 10\u201353% of triple negative breast cancers , has emeSome of the technical discrepancies in protein-based AR-staining could be overcome with RNA-based measures of AR-dependent expression. Several groups have reported that non-luminal androgen receptor TNBC molecular subtypes that express AR at low levels demonstrate AR-dependence for tumor cell growth or viability\u201321. HoweThe Carolina Breast Cancer Study is a population-based study of breast cancer that oversampled Black and younger women. Using gene expression data for 1202 CBCS participants, we trained a pathway-based classifier to identify AR-low patients and to examine the relationship between AR status and tumor aggressiveness among ER-negative participants. Results in CBCS were validated in TCGA.The Carolina Breast Cancer Study (CBCS) is a population-based study , 23 of whttps://gdc.cancer.gov/).The Cancer Genome Atlas (TCGA) is a large, publicly available data source containing extensive genomic data on over 30 cancer types. Study details are described elsewhere. For ourCharacterization of Androgen Receptor (AR) ExpressionOf the 4806 CBCS participants, 967 were excluded because they had no or too few available slides of FFPE cores for analysis, and 192 were excluded because they failed RNA extraction. Of these 3647 eligible samples, 2734 were selected for sequencing based on adequate coverage of additional sociological and pathological data by the CBCS bioinformaticist. Samples were split into 9 custom codesets, each containing a different mixture of genes from relevant biologic panels; some samples as well as some genes were included on multiple panels. In total, 1649 samples were included on a panel that profiled AR, 1202 (72%) of which passed quality control. Thus, AR RNA expression was profiled in 1202 CBCS samples (472 ER-negatives), using a custom NanoString protocol optimized for formalin-fixed paraffin-embedded (FFPE) samples\u201327. To aTo identify features of AR-low tumors, we calculated relative frequency differences (RFDs) and 95% confidence intervals by fitting a generalized linear model with binomial distribution and identity link where AR status was the outcome and the variable of interest was the predictor. Because triple negative status may confound associations between AR and clinical presentation, we also computed models adjusted for triple negative status.To understand overlap between AR-phenotype and deficiencies in immune and DNA repair processes, we analyzed CBCS samples that had RNA expression data on both AR and immune or AR and DNA repair classes. However, because a relatively small number of samples profiled for AR also included information on RNA expression of DNA repair genes , we developed a predictor of AR phenotype to identify additional samples with low AR expression. To do this, we split the 472 ER-negative samples with measured AR expression into five groups (\u201cfolds\u201d) using stratified random sampling, therefore ensuring consistent distribution of AR-high and AR-low samples within each fold. In each of five iterations of testing, we retained four of the folds for training and omitted the last for validation , then repeatedly fit Classification to Nearest Centroid (ClaNC) models that used between two and 150 genes to distinguish AR-low and AR-high tumors in the training set. For each of these 75 models, we estimated sensitivity, specificity, and the Youden\u2019s index (sensitivity + specificity \u2212 1) in the training and test sets. We selected the final number of genes to use in the classifier by finding the maximum Youden\u2019s index, averaged across the five folds, among the training sets. From this final model, we predicted the AR status of all ER-negative samples, using predictions to calculate a final sensitivity and specificity. Finally, we applied the AR classifier to all ER-negative CBCS samples assayed for selected genes (N = 669) and proceeded to compare AR phenotypes to other molecular indicators, described below.Using custom panels of 50 immune-related and 51 DNA repair-related genes, we classified samples with respect to three immune classes and two DNA repair classes (recombination/Fanconi anemia (HR/FA), and non-HR/FA) according to published methods.). As abWe used TCGA to validate associations between AR status and molecular tumor characteristics. After applying our ClaNC classifier of AR to ER-negative samples from TCGA, we used the composite Homologous Recombination Deficiency (HRD) Scoring method from Knijenburg et al to assesWe detected associations between single-gene AR RNA expression classes (high vs. low) and aggressive clinical features. RNA levels of AR were strongly correlated with AR protein as measured by RPPA tumors with AR-low phenotypes. Again, AR-low tumors were more likely to be younger, Black, HER2-negative, grade III, stages II-IV, and have high ROR-PT scores (Supplementary Table 1) as also observed in the TCGA cohort (Supplementary Table 2). ER/AR-low tumors also showed strong evidence of aggressive molecular phenotypes, with 47.0% having enrichment for adaptive immune tumor subtypes, and 85.6% showing enrichment for homologous recombination-related genes with AR-low phenotypes. AR-low tumors showed higher frequency of homologous recombination deficiency (HRD), with 81 (39.3%) having HRD scores above the clinical cutoff of 42 as compared to 11 (5.3%) of AR-high tumors. We did not find strong evidence of an association between AR and immune expression phenotypes in TCGA , although the TCGA lacks evidence of the immune quiet phenotype due to different selection factors for inclusion in TCGA (cite Hamilton). However, AR-low tumors again had higher proportions of expression-based HRD phenotypes than AR-high tumors .Unique disease features that distinguish ER-negative breast cancer patients by AR status for successful therapeutic targeting remains poorly understood as a result of limitations and inconsistencies with protein-based AR assessment approaches. Using CBCS expression data, we designed and validated a multigene classifier that distinguishes AR-low versus AR-high ER-negative breast cancers. AR-low status in ER-negative breast cancer was significantly associated with younger age at diagnosis, Black race, HER2-negativity, high-grade, and higher ROR; these associations remained significant after adjusting for TNBC status. These findings suggest that in ER-negative breast cancers, low AR expression is associated with aggressive disease. AR-low/ER-negative tumors occur more frequently in Black (versus white) women; consequently AR-low/ER-negative breast cancer may be important for understanding racial disparities in survival. Considering other biological phenotypes, AR-low cancers in the CBCS cohort exhibited adaptive immunity enrichment and both CBCS and TCGA data sets displayed significantly greater homologous recombination repair deficiency among AR-low cancers.et al. reported that women, under the age of 35 years, were diagnosed with AR-negative/ER-negative breast cancer more frequently than women over the age of 35 [Our findings are consistent with what has been previously reported for demographic factors. Park ectively). Severalectively), 32\u201334. ectively).et al. reported that AR-positivity is associated with longer relapse-free survival among HER2-negative patients[The prognostic role of low AR expression in TNBC remains controversial. Our findings provide evidence that AR-negative TNBC is associated with aggressive disease features such as advanced stage and high histological grade, 10, 11. patients. Wang an patients. Hence, Another distinction observed herein between AR-negative and AR-positive TNBC was with respect to immune profiles. Consistent with our findings, Davis and colleagues previously reported that AR-negative tumors are upregulated in T cell marker (CD4 and CD8), immune checkpoint , and immune cell signaling pathway marker RNA expression compared to AR-positive tumors in TNBC. These fPrevious studies have not evaluated associations between AR and specific DNA repair pathways. We observed a higher prevalence of homologous recombination deficiency in AR-low (versus AR-positive) ER-negative-breast cancer. This finding is consistent with previous studies showing increased genomic instability in AR-negative (versus AR-positive) TNBC. It has been discovered that AR-negative TNBCs have 1) increased epidermal growth factor receptor, cyclin-dependent kinase 6, Ki-67, and topoisomerase 2a but 2) downregulated PTEN and HER4\u201353. It wA limitation of this study was the small number of ER-negative breast cancers, particularly TNBC. We also did not evaluate protein-level AR localization compared to our multigene classifier. However previous studies show that AR RNA-based signatures expression correlate with AR protein expression, 56. How"} +{"text": "We present two case reports of women with estrogen-receptor (ER)-positive cancer of the breast who were vaccinated for COVID-19 in the deltoid muscle. [18F]FDG positron emission tomography (PET) demonstrated primary breast cancer and multiple axillary lymph nodes with increased [18F]FDG uptake, diagnosed as vaccine-associated [18F]FDG-avid lymph nodes. Subsequent [18F]FES PET revealed single axillary lymph node metastasis in the vaccine-associated [18F]FDG-avid lymph nodes. To the best of our knowledge, this is the first study showing the usefulness of [18F]FES PET in diagnosing axillary lymph node metastasis in COVID-19-vaccinated patients harboring ER-positive breast cancer. Thus, [18F]FES PET has potential applications in the detection of true-positive metastatic lymph nodes in patients with ER-positive breast cancer regardless of the ipsilateral or contralateral side, who have received COVID-19 vaccination.Coronavirus disease (COVID-19) vaccination is known to cause a diagnostic dilemma due to false-positive findings on [ To the best of our knowledge, this is the first study to evaluate the usefulness of [18F]FES PET in diagnosing axillary lymph node metastasis in COVID-19-vaccinated patients with ER-positive breast cancer.Coronavirus disease COVID-19) vaccination has caused a diagnostic dilemma due to false-positive findings on positron emission tomography (PET) with 2-[9 vaccinat cancer . 18F]FDvaccines . Patientph nodes . The Soct cancer ,7,8. In FFDvaccinThe study was approved by the institutional review board , and the requirement for informed patient consent was waived due to the retrospective nature of the study.18F]FDG PET/magnetic resonance imaging (MRI) for preoperative staging. The primary lesion in the breast, as well as multiple level I and II left axillary lymph nodes, showed significant FDG uptake staining and immunohistochemistry (IHC) She underwent FDG PET/CT for staging. In addition to intense [18F]FDG uptake in the primary breast tumor . She underwent pretreatment [18F]FDG PET plays an important role in breast cancer staging [18F]FDG PET [18F]FES PET can differentiate inflammatory lymph node enlargement after COVID-19 vaccine administration from lymph node metastasis, a difference which is difficult to distinguish using [18F]FDG PET. In addition, we could accurately identify CAM, which is even more complex.Although FDG PET ,15. In t11C] Choline [18F]Choline [18F] prostate-specific membrane antigen (PSMA) [68Ga]DOTA-peptide (DOTATATE) [177Lu] Lu-DOTATATE PRRT post-therapy planar scintigraphy, as well as single-photon emission computed tomography with computed tomography (SPECT/CT) [68Ga] fibroblast-activation protein inhibitor (FAPI) was reported to show no accumulation in the enlarged axillary lymph nodes after vaccine administration, similar to our report on [18F]FES, which may be useful in distinguishing lymph node metastasis FDG PET/CT could not distinguish between them. In contrast, [18F]FES PET clearly delineated ER-positive synchronous CAM from vaccine-associated hypermetabolic lymphadenopathy.CAM is a rare finding in patients with breast cancer, with an incidence rate between 1.9 and 6% . This coThis study has certain limitations: it is a case report of only two cases, and we cannot draw conclusions with a high level of clinical evidence. However, breast cancer cases in which COVID-19 vaccine was administered followed by both FDG-PET and FES-PET examinations are rare, and we believe that accumulating findings from these kinds of case reports will be useful for the diagnosis and treatment of breast cancer patients in the future.18F]FES PET combined with [18F]FDG characterized the distinct phenotypic features of the lesions. [18F]FES PET can be more specific than [18F]FDG for lymph node staging in patients with ER-positive breast cancer, independent of the cause of lymph node uptake.In conclusion, ["} +{"text": "Correction: Natural Products and Bioprospecting (2023) 13:11 10.1007/s13659-023-00375-2Following publication of the original article , the autThe correct title should read:Protective effect and mechanism insight of purified Antarctic krill phospholipids against mice ulcerative colitis combined with bioinformaticsThe original article has been"} +{"text": "This review summarizes the significant advancements in prophylaxis against graft-versus-host disease (GvHD) presented at the 2022 ASH Annual Meeting. The use of innovative agents and regimens, along with the conventional prophylactic approach of combining post-transplant cyclophosphamide and anti-thymocyte globulin, were discussed. The innovative agents and regimens highlighted in this review include abatacept, the first FDA-approved drug for acute GvHD prophylaxis; RGI-2001, which promotes the expansion of regulatory T cells; and cell therapies such as Orca-T and Orca-Q. These advancements provide promising strategies and options for GvHD prevention, offering hope for improved post-transplant patient outcomes in terms of survival rates. Hematopoietic stem cell transplantation (HSCT) can lead to a severe multisystem disorder known as graft-versus-host disease (GvHD), resulting in considerable morbidity and non-relapse mortality (NRM). We reviewed several latest reports on GvHD prophylaxis from the 2022 ASH Annual Meeting (ASH2022).Abatacept (ABA), a CD28:CD80/86 co-stimulation blockade, has shown potential in controlling early T cell allo-proliferative escape, a major contributor to breakthrough acute GvHD (aGvHD) following calcineurin inhibitor/methotrexate (CNI/MTX) prophylaxis . ABA\u2009+\u2009CThe augmentation of regulatory T cells (Tregs) has gained significant attention as an effective strategy for preventing GvHD while preserving the graft-versus-leukemia (GvL) effect. RGI-2001, a liposomal glycolipid, expands Tregs by activating invariant natural killer cells through CD1d receptor on antigen-presenting cells, thereby modulating GvHD pathogenesis. RGI-2001 administered intravenously with tacrolimus/MTX was effective in preventing aGvHD in individuals receiving grafts from peripheral blood stem cell (PBSC) and bone marrow sources, with a favorable safety profile. Promising results justify the planning of a phase 3 clinical trial to assess the efficacy of the regimen . Orca-T Combining anti-thymocyte globulin (ATG) and PTCy as prophylaxis against GvHD is a promising strategy, surpassing the limitations of individual administration. ATG-based regimens pose viral infection risks, while PTCy-based prophylaxis is less effective with PBSC grafts. A retrospective study showed tSalas et al. conducted trials investigating the use of reduced-dose ATG in various transplantation settings. In Abstract 2127 , a compaLow-dose PTCy has been investigated in elderly patients and those with cardiac comorbidities undergoing haplo-PBSCT . The stuThe emergence of novel agents and regimens has expanded the donor pool and allowed the use of different graft sources, including mismatched donors and PBSC, to address patient needs while minimizing the risk of GvHD. For PTCy and ATG combination prophylaxis, future endeavors could focus on investigating lower-dose combination of these agents to achieve the dual goals of minimizing adverse events and maintaining prophylactic efficacy."} +{"text": "Natural killer (NK) cells can both amplify and diminish immune responses to vaccination. Studies in humans and animals have observed NK cell activation within days after mRNA vaccination. In this study, we sought to determine if baseline NK cell frequencies, phenotype, or function correlate with antibody responses or inflammatory side effects induced by the Pfizer-BioNTech COVID-19 vaccine (BNT162b2).st and 2nd doses of BNT162b2. Inflammatory side effects were assessed by structured symptom questionnaires, and baseline NK cell functionality was quantified by an in vitro killing assay on participants that reported high or low post-vaccination symptom scores.We analyzed serum and peripheral blood mononuclear cells (PBMCs) from 188 participants in the Prospective Assessment of SARS-CoV-2 Seroconversion study, an observational study evaluating immune responses in healthcare workers. Baseline serum samples and PBMCs were collected from all participants prior to any SARS-CoV-2 infection or vaccination. Spike-specific IgG antibodies were quantified at one and six months post-vaccination by microsphere-based multiplex immunoassay. NK cell frequencies and phenotypes were assessed on pre-vaccination PBMCs from all participants by multi-color flow cytometry, and on a subset of participants at time points after the 1st vaccination had higher pre-vaccination NK cytotoxicity indices compared to individuals with low symptoms scores , and 3) pre-vaccination NK cell numbers were negatively correlated with spike-specific IgG levels six months after two BNT162b2 doses .Key observations include: 1) circulating NK cells exhibit an increase in CD56dim CD16- NK cells compared to baseline levels, providing evidence of NK cell activation in the week following vaccination, 2) individuals with high symptom scores after 1These results suggest that NK cell activation by BNT162b2 vaccination may contribute to vaccine-induced inflammatory symptoms and reduce durability of vaccine-induced antibody responses.David Tribble, MD, DrPH, AstraZeneca: The IDCRP and HJF were funded to conduct an unrelated phase III COVID-19 monoclonal antibody immunoprophylaxis trial as part of US Govt COVID response Timothy Burgess, MD, MPH, AstraZeneca: The IDCRP and the Henry M. Jackson Foundation (HJF) were funded to conduct an unrelated phase III COVID-19 monoclonal antibody immunoprophylaxis trial Simon Pollett, MBBS, AstraZeneca: The IDCRP and the Henry M. Jackson Foundation (HJF) were funded to conduct an unrelated phase III COVID-19 monoclonal antibody immunoprophylaxis trial"} +{"text": "Endoscopic ultrasound-guided hepaticogastrostomy (EUS-HGS) has been indicated after failed endoscopic retrograde cholangiopancreatography (ERCP). Critical adverse events such as stent migration or dislocation can occur during EUS-HGS;A 57-year-old man was admitted with obstructive jaundice due to inoperable pancreatic cancer. Because the second part of the duodenum was obstructed, EUS-HGS was performed using a partially covered self-expandable metal stent. However, abdominal pain and recurrence of obstructive jaundice were noted 5 days later, and CT revealed dislocation of the stent . After Novel endoscopic suture using an endoloop and endoclips successfully deployed for endoscopic treatment after dislocation of an endoscopic ultrasound-guided hepaticogastrostomy stent.Video 1In conclusion, the present technique may be useful for troubleshooting during EUS-HGS, particularly in the case of stent dislocation.Endoscopy_UCTN_Code_CPL_1AL_2AD"} +{"text": "Cancer remains a significant global health challenge, necessitating the exploration of novel and more precise therapeutic options beyond conventional treatments. In this regard, clustered regularly interspaced short palindromic repeats (CRISPR) systems have emerged as highly promising tools for clinical gene editing applications. The CRISPR family encompasses diverse CRISPR-associated (Cas) proteins that possess the ability to recognize specific target sequences. The initial CRISPR system consisted of the Cas9 protein and a single-guide RNA, which guide Cas9 to the desired target sequence, facilitating precise double-stranded cleavage. In addition to the traditional cis-cleavage activity, the more recently discovered Cas12 and Cas13 proteins exhibit trans-cleavage activity, which expands their potential applications in cancer diagnosis. In this review, we provide an overview of the functional characteristics of Cas9, Cas12, and Cas13. Furthermore, we highlight the latest advancements and applications of these CRISPR systems in cancer gene therapy and molecular diagnosis. We also emphasize the importance of understanding the strengths and limitations of each CRISPR system to maximize their clinical utility. By providing a comprehensive overview of the current state of CRISPR technology in cancer research, we aim to inspire further exploration and innovation in this rapidly evolving field. Despite being the cornerstone of cancer treatment, traditional therapies such as surgery [Cancer, characterized by an unrestrained and invasive proliferation of abnormal cells , has lon surgery , chemoth surgery , and rad surgery have lim surgery . Therefo surgery . With th surgery .The discovery of clustered regularly interspaced palindromic repeats (CRISPR) in Escherichia coli in 1987 laid the foundation for understanding its functional role in bacteria . The turCRISPR-Cas systems are classified into two major classes . Class 1Among CRISPR-based editing systems, CRISPR-Cas9 has been extensively investigated both in vitro and in vivo. Over the years, researchers have made various modifications to improve its specificity and efficiency, leading to promising preliminary results in ongoing clinical trials . RecentlThis review provides a comprehensive summary of the structural and functional characteristics of signature Class 2 Cas proteins, including Cas9, Cas12, and Cas13, as well as their applications in cancer gene therapy and molecular diagnosis. The inclusion of up-to-date research and clinical trials highlights the current progress in the field, while also addressing the key limitations of different CRISPR tools. Although CRISPR-based therapies and diagnostics offer great promise, further rigorous trials and long-term follow-up are needed to establish their safety and efficacy.CRISPR-Cas9 is a genome editing system composed of two essential components: guide RNA (gRNA) and Cas9. The Cas9 protein exhibits a bi-lobed structure, comprising the recognition lobe (REC) with three Helical domains and a Bridge Helix, and the nuclease lobe (NUC) containing a split RuvC domain, an HNH domain, a Topo domain, and a C-terminal domain (CTD) Fig.\u00a0A. These Upon DSB formation, DNA repair mechanisms are triggered, primarily via two pathways: homology-directed repair (HDR) and nonhomologous end joining (NHEJ) . HDR reqHaving elucidated the mechanism of CRISPR-Cas9, researchers have explored its diverse applications across various fields, including disease model generation, gene therapy, and high-throughput screening platforms for comprehensive gene analysis . Additio. established a Cas9 knock-in mouse model by delivering the CRISPR system to the lung via adeno-associated virus (AAV). They established adenocarcinoma pathology through targeted mutations in frequently altered genes such as Tumor Protein P53 (TP53), Liver kinase B1 (LKB1), or Kirsten ratsarcoma viral oncogene homolog (KRAS) [The CRISPR-Cas9 system has played a pivotal role in the development of disease models, particularly for cancer and genetic diseases , 37. Forg (KRAS) .. used CRISPR/Cas9 to generate a knockout (KO) of the tumor suppressor gene AT-rich interaction domain 1A (ARID1A) in primary TP53(\u2212/\u2212) human gastric organoids. This led to morphologic dysplasia, tumorigenicity, and mucinous differentiation [. reported the generation of a Cas9-edited TP53 and cyclin-dependent kinase inhibitor 2A (CDKN2A) KO gastro-esophageal junction (GEJ) organoid model. This study revealed the important role of TP53/CDKN2A inactivation in early GEJ neoplasia, which may aid in early diagnosis and prevention [In addition to mouse models, the advent of organoids has expanded the scope of gene therapy research. Lo et alntiation . Zhao etevention . The eff. developed a genome-scale CRISPR-Cas9 knockout (GeCKO) library and identified that numerous genes, both previously validated and newly discovered, that contribute to vemurafenib resistance in a melanoma model [. utilized the CRISPR-Cas9 screening tool to identify phosphoglycerate dehydrogenase (PHGDH) as a driver of resistance in hepatocellular carcinoma (HCC) treatment. The use of the PHGDH inhibitor NCT-503 successfully overcame the resistance, resulting in the abolition of in vivo HCC growth [. identified Haspin (GSG2) as targetable kinases that displayed synthetic lethal interactions with selective Aurora-A inhibitor alisertib (MLN8237) in breast cancer cells. They also discovered the Haspin inhibitor CHR-6494, which synergistically reduced tumor growth, enhancing the therapeutic effects of MLN8237 in a combinational therapy [Moreover, the CRISPR-Cas9 system has revolutionized high-throughput screening of gene activities in various biological processes, including tumor growth, metastasis, therapeutic resistance, and response to immunotherapy , 42. Forma model . SimilarC growth . Additio therapy . These f. first introduced the concept of dCas9, which refers to an endonuclease-deficient mutant Cas9 that retains its target recognition capability but lacks nuclease activity [. established a dual CRISPR interference and activation (CRISPRi/a) system that simultaneously silenced the X-inactive specific transcript (XIST) and activated Forkhead box P3 (FOXP3) in breast cancer cells. This manipulation led to increased H4 acetylation and decreased DNA methylation, highlighting the potential of CRISPRi/a for transcriptional and epigenetic modifications [In addition to its role in gene editing, extensive research has explored the transcriptional regulatory capabilities of CRISPR-Cas9. Qi et alactivity . By fusiactivity \u201349. Thesications .. utilized this screening strategy in human lung adenocarcinoma (LUAD) and identified CASP8AP2 as a key regulator influencing the viability of all eight examined LUAD cell lines [The regulatory capacity of CRISPR-Cas9 has sparked the development of screening platforms based on CRISPRa and CRISPRi for functional studies \u201353. For ll lines . Anotherll lines . The devCRISPR-Cas9 has been extensively investigated for its potential in gene therapy, particularly in the context of cancer treatment, which presents complex challenges and high mortality rates. The application of CRISPR-Cas9-mediated knock-out or knock-in strategies offers a valuable approach to interfere with oncogene expression and evaluate its impact on tumor progression . Encoura. reported improvements in median progression-free survival and median overall survival in advanced non-small-cell lung cancer (NSCLC) patients treated with PD-1 edited T cells (NCT02793856) . This approach successfully reduced TCR mispairing and improved antitumor immunity [. administered anti-CD19 CAR-T cells with integrated PD-1 into patients with relapsed/refractory aggressive B cell non-Hodgkin lymphoma (NCT04213469) and TCR \u03b2 constant (TRBC), and the insertion of two chains of a patient-specific neoantigen-specific TCR (neoTCR) into the TRAC locus. Five out of the sixteen participants demonstrated stable disease, while the others experienced disease progression [Recent clinical trials utilizing CAR-T cells have shown promising outcomes in cancer therapy. For instance, You et alimmunity . Zhang elocation . Trial fgression . These c. observed off-target editing in approximately 16% of human embryonic cells [. detected megabase-scale losses of heterozygosity (LOH) [. found unwanted repairing byproducts such as deletions, vector integrations, and chromosomal translocations [. noticed the production of functional foreign mRNAs and aberrant proteins in approximately 50% of CRISPR-Cas9 edited cell lines [While CRISPR-Cas9 editing holds great potential for clinical research, it faces challenges that impact its specificity and efficiency. Alanis-Lobato et alic cells , while Bty (LOH) . Liu et ocations , and Tulll lines . Until tTo address some of the limitations of CRISPR-Cas9 editing, novel CRISPR-based tools such as base editors and prime editors have been developed. These tools aim to avoid the induction of double-stranded breaks (DSBs) and the risk of deletions, insertions, and frameshift mutations during DNA repair processes . Base ed. in 2016 [. constructed a new transformer base editing (tBE) system that increases on-target specificity while reducing unintended mutations [. developed a dual adenine and cytosine base editor system by combining both deaminases with a Cas9 nickase, thereby expanding the scope of base editors to enable C-to-T and A-to-G conversions at the same target [The first CRISPR-based base editor was developed by Komor et al in 2016 . They fu in 2016 . Subsequ in 2016 , and C-t in 2016 were dev in 2016 , 72\u201374. utations . Later, e target , 76, dra. revealed the involvement of P21-activated Kinase 4/Extracellular Regulated Protein Kinases (PAK4/ERK) and Ribosomal S6 Kinase 2/Recombinant Tumor Protein p53 Binding Protein 1/ Phosphorylated H2A Histone family member X (RSK2/TP53BP1/\u03b3-H2AX) signaling pathways in anti-chemoresistance [Base editors have proven to be highly valuable in large-scale genome screening to identify gain- and loss-of-function variants , 78, as sistance .. employed CBEs to modify oncogenic mutations in a triple-negative breast cancer model [. used ABEs to correct KRAS and TP53 mutations in cancer organoids from patients [. achieved promising results by developing a CD7-directed allogeneic CAR-T (7CAR8) using CBEs, which showed high effectiveness against T-cell acute lymphoblastic leukemia (T-ALL) in various in vitro and in vivo models [Base editors have shown significant therapeutic potential in directly modifying oncogenes or tumor suppressor genes. Annunziato et aler model . Sayed epatients . Both stpatients . Diorio o models . These aAnother breakthrough in precise gene editing is prime editing, which incorporates an engineered reverse transcriptase (RT) with Cas9 nickase . TogethePrime editing represents a revolutionary advancement in precise gene editing. The initial version, Prime Editor 1 (PE1), integrated features of both CRISPR and reverse transcriptase but displayed suboptimal efficiency for mass application . Subsequ. utilized prime editing to model lung and pancreatic cancer. This system introduced clinically relevant mutations, such as KRAS mutations associated with drug resistance and TP53 mutations in pancreatic cancer [While the clinical safety of prime editors is still being evaluated, current applications have mainly focused on cancer modeling. Ely et alc cancer . Anotherc cancer . The estIn recent years, extensive research has been conducted on the Cas12 and Cas13 systems, complementing the well-established Cas9 system. Cas12 has demonstrated high specificity in recognizing and cleaving double-stranded DNA , 92. In Cas12 protein shares similar functions with Cas9 in recognizing and editing double-stranded DNA, but it relies solely on CRISPR RNA (crRNA) for guidance. Unlike the Cas9 system, which requires the host endonuclease RNase III for processing pre-crRNA into mature crRNA, Cas12 can independently carry out this step . The CasPrevious knowledge on Cas9 has emphasized the significance of the PAM region, which is also crucial for Cas12a. The PI, REC1, and WED domains of Cas12a enzymes collectively facilitate PAM recognition . Cas12a Extensive investigations have been carried out to explore the application of Cas12a in human cells for genetic editing and diagnostics. Studies evaluating various Cas12a homologs have demonstrated their effective genome editing capabilities. In comparison to the well-established Cas9 editing system, Cas12a has exhibited slightly reduced efficiency but improved accuracy in editing . This ca. leveraged the multiplex gene targeting capability of Cas12a by designing systems that target three frequently mutated genes , and Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha gene (PIK3CA)), in colorectal cancer patients [Cas12a has emerged as a powerful tool for genome editing that could be applied in cancer therapy. Specifically, it has been designed to disrupt commonly mutated genes such as vrafmurine sarcoma viral oncogene homolog B (BRAF)-V600E , 109. Inpatients . These sApart from its potential in gene therapy, Cas12a is widely employed as a diagnostic tool. This is accomplished through its collateral cleavage activity upon binding to the target. Upon binding, Cas12a undergoes a conformational change in the lid region, exposing the catalytic residue and enabling indiscriminate cleavage of single-stranded DNA (ssDNA) . This coThese features underscore the feasibility of Cas12a as an excellent diagnostic tool. Ongoing clinical trials primarily focus on genetic diseases or infections, with a single trial aiming to test the detection of Epstein-Barr virus (EBV) DNA in nasopharyngeal brushing and patient plasma as an indicator for nasopharyngeal carcinoma (NCT05447169) L858R, T790M and BRAF V600E, in samples from patients with non-small cell lung cancer (NSCLC) [Previous studies have demonstrated the attomolar sensitivity of Cas13a in detecting low-frequency cancer mutations in cell-free DNA fragments (cfDNA), comparable to the sensitivity by ddPCR and quantitative PCR (qPCR) . Gootenb (NSCLC) . More re (NSCLC) . miRNAs (NSCLC) , while m (NSCLC) . Other t (NSCLC) .Furthermore, research groups have designed more complexed circuits that allow Cas13a to detect protein markers, such as Programmed cell death 1 ligand 1 (PD-L1), Interleukin-6 (IL-6), and Vascular endothelial growth factor (VEGF), in patient serum , 133. ThCas-based detection methods offer several advantages compared to conventional detection methods such as real-time PCR (RT-PCR) or qPCR. RT-PCR and qPCR are labor-intensive, prone to human error, time-consuming, and expensive . They reIn 2020, the introduction of Combinatorial Arrayed Reactions for Multiplexed Evaluation of Nucleic acids (CARMEN) further expanded the detection range and efficiency of the SHERLOCK by incorporating numerous optimized crRNAs . CARMEN Over the years, the landscape of cancer treatment has witnessed remarkable progress, transitioning from traditional interventions to more innovative approaches such as immunotherapy and gene therapy . TraditiThe past decade has witnessed significant advancements in the understanding and application of CRISPR systems. Their simplicity, robustness, and high efficiency have opened up possibilities for the treatment and diagnosis of various diseases, including cancer . This reDespite the rapid development of CRISPR, which provides new hope for once-believed incurable diseases, there are still significant hurdles that limit its widespread application. These challenges include unstable editing efficiency, off-target editing concerns, and limitations in vivo delivery."} +{"text": "Numerous studies have developed strategies to utilize anti-cytomegalovirus (CMV) T cells for cancer treatment, as they have many beneficial characteristics including extraordinarily high numbers and frequent presence in cancer tissues. In this review, we present multiple strategies that exploit anti-CMV T cells for cancer (immuno)therapy in various ways. We aim to advance the understanding of how anti-CMV T cells can be applied best to further improve treatment outcomes for cancer patients. For this purpose, we identify similarities and discuss benefits, disadvantages, and challenges of each strategy. Finally, we comment on the future directions of this new promising field of cancer (immuno)therapy.pos T cells. Intriguingly, anti-CMV T cells accumulate over time to extraordinarily high numbers, are frequently present as tumor-resident \u2018bystander\u2019 T cells, and remain functional in cancer patients. Consequently, various strategies for redirecting anti-CMV CD8pos T cells to eliminate cancer cells are currently being developed. Here, we provide an overview of these strategies including immunogenic CMV peptide-loading onto endogenous HLA complexes on cancer cells and the use of tumor-directed fusion proteins containing a preassembled CMV peptide/HLA-I complex. Additionally, we discuss conveying the advantageous characteristics of anti-CMV T cells in adoptive cell therapy. Utilization of anti-CMV CD8pos T cells to generate CAR T cells promotes their in vivo persistence and expansion due to appropriate co-stimulation through the endogenous (CMV-)TCR signaling complex. Designing TCR-engineered T cells is more challenging, as the artificial and endogenous TCR compete for expression. Moreover, the use of expanded/reactivated anti-CMV T cells to target CMV peptide-expressing glioblastomas is discussed. This review highlights the most important findings and compares the benefits, disadvantages, and challenges of each strategy. Finally, we discuss how anti-CMV T cell therapies can be further improved to enhance treatment efficacy.Infection with cytomegalovirus (CMV) is highly prevalent in the general population and largely controlled by CD8 Notably, an extensive number of studies have observed exceptionally high frequencies of anti-CMV CD8pos T cells in (primarily elderly) individuals [The cytomegalovirus (CMV) is a herpesvirus with an estimated global seroprevalence of 83% . Howeverividuals ,6,7,8,9.In clinical practice, treatment with anti-CMV T cells is mainly used to prevent CMV disease after hematopoietic stem cell transplantation. Currently, an increasing number of studies are investigating alternative approaches for utilizing anti-CMV T cells in cancer (immuno)therapy. Here, we discuss various strategies to exploit anti-CMV T cells for the treatment of solid and non-solid malignancies.pos T cells are anergic [pos and CD8pos T cells, respectively. Remarkably, in CMV-seropositive cancer patients, the frequency of anti-CMV CD8pos T cells can be even higher than in healthy individuals, reaching up to 20% [Researchers have provided compelling lines of evidence that anti-CMV T cells may be particularly suitable for use in cancer (immuno)therapy. Firstly, CMV infection is controlled lifelong in both healthy individuals and cancer patients. The latter indicates that anti-CMV T cell responses remain functional and potent in cancer patients. Indeed, anti-CMV T cell responses appear to be intact even in cancer patients with malignancies such as chronic lymphocytic leukemia (CLL), in which the majority of CD8 anergic . Secondl anergic ,12. Thirp to 20% . These epos hematopoietic stem and progenitor cells are a critical reservoir of latent human CMV (HCMV) infection, which is maintained during differentiation into CD14pos monocytes [T cell responses to CMV are driven by the lifelong presence of the virus in the host. Clearance of infected and virus-producing cells resolves the primary infection, but in some cells viral genomes remain in a quiescent state. In particular, bone marrow-resident CD34onocytes ,14,15. Vonocytes ,17. Repeonocytes and has onocytes ,20,21,22onocytes ,23,24.pos T cells are repeatedly exposed to CMV antigens, they remain polyfunctional [pos T cells expose low levels of inhibitory markers such as PD-1 and retain their capacity to proliferate, secrete cytokines, and induce cytotoxicity [pos T cells have an effector\u2013memory phenotype and accumulate in non-lymphoid peripheral organs [pos T cells maintain a functional \u2018ready-to-go state\u2019 by exploiting alternative, rather than classical, co-stimulatory signals [pos T cells express the chemokine receptors CX3CR1 and CXCR3, which enables them to infiltrate inflamed tissues [pos T cells is highly diverse, and responses to the immunodominant viral proteins pp65 and IE-1 are the best studied [Although inflationary anti-CMV CD8nctional ,25,26 annctional or humannctional . In parttoxicity . Mouse al organs ,21,30,31 signals ,33. More tissues ,34. In h studied ,36.Notably, memory T cell inflation is not exclusive to CMV, as it is also observed for other latent virus types such as herpes simplex virus (HSV)-1 and Epstein\u2013Barr virus (EBV) .pos T cells, such as their abundance and constant renewability, various strategies have been developed to exploit these potent cytolytic effector cells for cancer immunotherapy. In contrast to na\u00efve T cells, antigen-experienced inflationary anti-CMV CD8pos T cells do not require classical priming through presentation of peptides by dendritic cells or other professional antigen-presenting cells [Due to the above-mentioned beneficial characteristics of anti-CMV CD8ng cells .pos T cells can recognize and eliminate cancer cells.The strategies described in this section all aim to mimic CMV infection by (re)decorating malignant cells with viral antigens on (endogenous or exogenous) human leukocyte antigen class I (HLA-I) complexes. In this way, anti-CMV CD8pos T cells. Among the examples are the so-called T cell epitope-delivering antibodies (TEDbodies), which can be used to selectively deliver CMV peptides into the cytosol of cancer cells. Once in the cytosol, the CMV peptide moiety of the TEDbody enters the natural antigen-processing and -presentation machinery of the targeted cancer cells and is ultimately presented by endogenous HLA complexes. In contrast, so-called antibody\u2013peptide epitope conjugates (APECs) do not enter the cytosol but are externally loaded into \u2018empty\u2019 HLA-I molecules on the surface of cancer cells. Moreover, anti-CMV T cell responses can be activated locally by intratumoral (i.t.) injection of CMV peptide epitopes, which are also externally loaded into cell surface-expressed \u2018empty\u2019 HLA-I (and HLA-II).The strategies described in the following paragraphs aim to load CMV peptides into endogenous HLA complexes on cancer cells to render these cells recognizable by anti-CMV CD8pos T cell epitope-delivering antibodies (TEDbodies) [pos T cells described CD8Dbodies) . TEDbodi T cells .pos T cells into NSG mice bearing pre-established orthotopic MDA-MB-231 xenografts or subcutaneous HCT116 colorectal cancers. Treatment of mice with TEDbodies reduced tumor volumes by 40% and 70%, respectively, and increased numbers of tumor-infiltrating anti-CMV CD8pos T cells by 15-fold compared to controls. Additionally, TEDbody treatment was combined with the agonistic OX40 antibody or the antagonistic PD-1 antibody pembrolizumab. Compared with monotherapy, co-administration of TEDbodies and agonistic OX40 antibody reduced the tumor volume by ~45% and increased the number of tumor-infiltrating anti-CMV CD8pos T cells. Intriguingly, despite cell surface expression of PD-1, pembrolizumab did not enhance the efficacy of TEDbody treatment.In their study, Jung et al. injected TEDbodies and anti-CMV CD8TEDbodies smartly take advantage of the natural antigen-processing and -presentation machinery of cells to mimic CMV infection by presenting CMV peptide\u2013HLA-I complexes on the surface of cancer cells. However, to evade immune recognition, both antigen-processing and -presentation are frequently modified or defective in cancer cells . As TEDbpos T cells developed an alternative strategy which circumvents the need for intracellular CMV peptide processing and loading. Antibody\u2013peptide epitope conjugates (APECs) consist of CMV-derived peptide epitopes conjugated to tumor-directed antibodies via a linker . This li T cells . In normpos T cells. In the breast cancer mouse model, MMP14\u2013cetuximab APEC-treated mice showed a ~60% reduction in tumor volume compared to the control groups. Median survival in the breast and liver cancer mouse models was prolonged by 14 and 6 days, respectively. In the lung cancer mouse model, MMP14\u2013cetuximab APEC-treatment did not prolong median survival, while overall survival was significantly improved in the intervention group despite the presence of right censoring. The study was terminated 44 days after the control group reached 0% survival. At this point, the survival rate of the intervention group was still 50%.Using a library of 96 APECs containing short protease recognition sequences for cancer-associated proteases, MMP2 and MMP14 were identified to have the most suitable protease sites for generating immunogenic CMV peptide epitopes. The HLA-A*02:01-restricted CMV pp65 peptide epitope NLV was predominantly used in APECs, but HLA-A*01:01- and HLA-B*08:01-restricted CMV peptide epitopes were also found to be functional. The tumor-directed domains of APECs were based on the clinically approved antibodies cetuximab (anti-EGFR) and rituximab (anti-CD20). MMP14\u2013cetuximab APEC was evaluated in NOD/SCID mice xenografted with breast (MDA-MB-231), liver (SNU-475), or lung cancer cells (MGH-1088). Mice were reconstituted by adoptive transfer of human anti-CMV CD8pp65 CD8pos T cells into the tumor mass and conveys robust cancer cell elimination. Additionally, a xenograft tumor model using NSG mice showed a ~50% decrease in tumor burden and 23 days prolonged median survival in animals treated with EpCAM-MMP7-CMV APEC.This proof-of-concept study was followed up by Zhang et al. (2022), who established an experimental pipeline to create patient-specific APECs for ovarian carcinoma treatment . APECs wIn contrast to TEDbodies, APECs bind directly to empty HLA-I complexes on the surface of cancer cells and hereby circumvent potential defects in the intracellular antigen-processing and -presentation machinery. However, APECs rely on the presence of empty HLA-I complexes on the surface of cancer cells. Unfortunately, in a subset of patients, cancer cells do not express (empty) HLA-I complexes . MoreoveAnother approach utilizing the presence of endogenous (empty) HLA/MHC complexes was examined by \u00c7uburu et al. (2022) . They oppos T cells. Immune infiltration was even more prominent in tumors than in secondary lymphoid organs. A large fraction of tumor-infiltrating MCMV-specific CD8pos T cells expressed CD69, indicating their differentiation into tumor-resident memory T cells.C57BL/6 mice were infected with MCMV. Of note, MCMV and HCMV infections are similar in terms of memory T cell inflation and broad T cell reactivity also see . Six monpos T cells. Moreover, i.t. injection of virus-derived peptide epitopes induced immune infiltration of tumors, as demonstrated by the increased numbers of CD8pos- and Th1-T cells, including T cells that were not targeted by the injected peptides. Increased numbers of bystander NK cells, B cells, neutrophils, and macrophages were also observed in the tumor bed. Apparently, injection of MCMV-derived epitopes changed both the cellular composition and activation status of immune cells in the tumor microenvironment (TME). Injection of MHC-I peptide epitopes (without pI:C) reduced tumor growth by ~95% and prolonged median survival by 21 days. Remarkably, complete tumor regression was observed only in mice that received MCMV-derived MHC-II peptide epitopes alone or in combination with MHC-I peptide epitopes. Tumor-free mice were rechallenged with TC-1 cancer cells after 4 months and did not develop any palpable tumors (compared to age-matched na\u00efve mice used as controls), suggesting that long-term antitumor immunity had been established.Subsequently, mice were injected with MCMV-derived MHC-I and MHC-II (m139) peptide epitopes in the presence (or absence) of the innate immune modifier polyinosinic: polycytidylic acid (pI:C). Already in the absence of pI:C, MCMV-derived MHC-I peptide epitopes induced necrotic tissue formation, demonstrating the powerful cytotoxic function of tumor-infiltrating MCMV-specific CD8pos T cells (>4-fold increase).Even treatment of immunologically \u2018cold\u2019 (B16-F10) melanoma xenografts with MCMV-derived MHC-I and MHC-II peptide epitopes reduced tumor volume by >80% and prolonged median survival by 18 days compared to the control group. Additionally, administration of MCMV-derived peptide epitopes caused significant activation of the TME, as was evident from a distinct increase in cancer-protective immune cells, particularly MHC-I peptide-specific CD8Finally, treatment with MCMV-derived peptide epitopes demonstrated high efficacy in a colon carcinoma model with a high mutation burden (MC-38 cancer cells xenografted into MCMV-infected C57BL/6 mice).pos T cells remain functional in both immunologically \u2018hot\u2019 and \u2018cold\u2019 murine tumor models and could overcome the immunosuppressive TME, although immune checkpoint inhibitors were not co-administered.In conclusion, anti-MCMV CD8pos T cell responses. Simultaneous mobilization of antiviral CD4pos and CD8pos T cells appears to be favorable, as it leads to the production of multiple cytokines and chemokines, which activate several immune pathways and promote immune cell infiltration of tumors [In contrast to TEDbodies and APECs, i.t. injection of CMV peptide epitopes involves MHC-II peptide epitopes and CD4f tumors . The prapos T cells towards cancer cells are equipped with a CMV peptide-loaded HLA-I/\u03b22M complex fused to an antibody (fragment) moiety directed towards a tumor-associated cancer cell surface antigen (To avoid the requirement for endogenous (empty) HLA-I complexes and bypass potential deficiencies in the antigen-presentation pathway, fusion proteins comprising CMV peptide\u2013HLA-I complexes have been developed. These fusion proteins that redirect anti-CMV CD8 antigen .pos T cells to B-CLL cells [pos) B-CLL cells, after which it recruits the biotinylated CMV\u2013HLA-I complex to the surface of cancer cells. TC-treated B-CLL cells were lysed in vitro by autologous anti-CMV CD8pos T cells with an efficiency similar to that of peptide-loaded B-CLL cells. Moreover, anti-CMV CD8pos T cells proliferated and produced proinflammatory cytokines in response to TC treatment.Mous et al. (2006) redirected anti-CMV CD8LL cells . They depos T cells, CMV-scHLA-A2-SS1(scFv) induced elimination of various cancer cell lines. Antigen-specific activation of anti-CMV CD8pos T cells (CD25pos/ CD107pos) and cytokine secretion (IL-2 and IFN\u03b3) occurred only in the presence of mesothelin-positive cancer cells. The in vivo efficacy was examined in BALB/c nude mice bearing human mesothelin-expressing N87 xenografts. Gastric carcinoma cells were subcutaneously injected into mice treated with CMV-scHLA-A2-SS1(scFv) in the presence of adoptively transferred human anti-CMV CD8pos T cells. This treatment inhibited tumor growth by ~50% compared to the control group.Noy et al. (2015) constructed a recombinant molecule, designated CMV-scHLA-A2-SS1(scFv), by genetic fusion of the CMV pp65 peptide epitope NLV to the single-chain (sc)HLA-A*02:01 molecule and scFv anti-mesothelin antibody fragment SS1 . In the pos T cells, pMHCI-IgGs facilitated the elimination of various tumor antigen-expressing cancer cell lines. Anti-CMV CD8pos T cells from multiple healthy donors responded to pMHCI-IgG-loaded cancer cells without prior expansion. Importantly, the anticancer activity of anti-MCSP pMHCI-IgG was compared to an analogous anti-MCSP bispecific T cell engager (BiTE). Treatment of cancer cells with conventional BiTEs aims to activate and redirect all patients\u2019 CD3pos T cells towards cancer cells, irrespective of T cell subtype and/or intrinsic T cell receptor (TCR) specificity. Consequently, BiTE-based therapies are typically associated with massive secretion of proinflammatory cytokines, including IFN\u03b3. High levels of proinflammatory cytokines may result in considerable in vivo toxicity, usually manifested as cytokine release syndrome [pos T cells and PBMCs. In this model, treatment reduced tumor volume and weight by ~40% and ~60%, respectively.Schmittnaegel et al. (2015), generated fusion proteins, designated pMHCI-IgG, composed of a full tumor antigen-specific immunoglobulin fused to a single HLA-A*02:01 domain bearing the covalently linked CMV pp65 peptide epitope NLV . pMHCI-Isyndrome . IntriguIn a follow-up study, Schmittnaegel et al. (2016) aimed to identify the best-suited format of their fusion protein by comparing \u2018single\u2019 and \u2018double\u2019 pMHCI-IgGs . Single pos T cell response, immunocompetent C57BL/6N mice were vaccinated with a synthetic M38 peptide coupled to a DC-targeted XCR1 monoclonal antibody and received a booster injection with the peptide and cytokines. At the peak of the immune response, mice were i.v. injected with melanoma cells (B16-FAP) and pMHCI-IgGs were administered (i.v.) before or after. Pretreatment of mice with pMHCI-IgGs prevented tumor engraftment in the lungs, indicating that pMHCI-IgGs are effective in vivo when cancer cells are still in the blood pool. Delaying pMHCI-IgG treatment reduced tumor growth by ~60% but did not abolish tumor growth in the lungs. BiTEs and pMHCI-IgG were almost equally potent in eliminating melanoma cells. When using an advanced solid (MC38-FAP) colon carcinoma model, neither BiTEs nor pMHCI-IgGs protected mice from the tumor. The observed therapy resistance was not due to antigen loss, hampered penetration of pMHCI-IgG into the tumor mass, or lack of effector T cells recruited to the tumor. Both treatments triggered PD-L1 expression and increased the number of Tregs. Apparently, immune suppression mechanisms can easily shut off the functionality of CD8pos T cells that have been retargeted to tumors by BiTEs or pMHCI-IgGs.Fischer et al. (2020) suggested that treatment with pMHCI-IgG complexes could also be applicable to CMV-seronegative patients with a matching HLA-I haplotype, if they would receive a vaccination against the CMV peptide contained in the particular pMHCI-IgG complex . To invepos T cell numbers specific for peptide TPR [pos T cells preferentially expand compared to HLA-A*02:01-restricted NLV-specific CD8pos T cells [pp65, in which an HLA-matched CMV pp65 peptide, a single-chain soluble HLA-B*07:02/\u03b22M molecule, and an EpCAM-directed Fab antibody fragment were fused in tandem [pos T cells. Comparison of EpCAM-ReTARGpp65 with the EpCAM-directed BiTE solitomab indicated a similar cancer cell elimination capacity but at ~55% decreased levels of T cell-secreted proinflammatory cytokines. Moreover, we reported for the first time a combinatorial treatment approach in which two fusion proteins recruiting anti-CMV CD8pos T cells with distinct HLA/peptide-specificities were exploited. Combinatorial treatment with EpCAM-ReTARGpp65 and EGFR-ReTARGIE\u22121, which additionally recruits HLA-C*07:02-restricted anti-CMVIE-1 CD8pos T cells, strongly potentiated selective cancer cell elimination compared to single-agent treatment, likely due to the concurrent cytolytic action of both cognate anti-CMV CD8pos T cell clones.We recently investigated whether CMV peptide epitopes other than HLA-A*02:01-restricted CMV pp65 peptide epitope NLV, could elicit similar or even more potent antitumor responses. Previously, it was reported that individuals with both HLA-A*02:01 and HLA-B*07:02 alleles show dominance of anti-CMV CD8 T cells . We desin tandem . We obseCUE Biopharma developed Immuno-Selective Targeting and Alteration of T cells (Immuno-STATs), which were originally designed to prime and expand na\u00efve and pre-existing anti-cancer T cell repertoires by co-delivering an engineered IL-2 variant (IL-2v) . Immuno-6E7 CD8pos T cells (CUE-101) [pos T cells with Immuno-STAT was preliminarily examined using the CMV pp65 peptide epitope NLV presented on HLA-A*02:01. The interaction of CMV pp65 Immuno-STAT with the TCR of anti-CMV CD8pos T cells in absence of co-stimulatory signals was sufficient to drive the proliferation of these cells in vitro and in a mouse model [Currently, an ongoing clinical trial is investigating Immuno-STATs to expand anti-HPV-1CUE-101) . The expse model . Recentlse model .pos T cells to eliminate cancer cells. Although IL-2v is affinity-attenuated, it may also activate T cells in the absence of cancer cells. Thus, possible in vivo toxicities must be thoroughly investigated.The introduction of IL-2v into fusion proteins comprising CMV peptide\u2013HLA-I complexes could improve the capacity of anti-CMV CD8pos T cells for cancer cell elimination. Targeted delivery of exogenous peptide-loaded HLA-I molecules renders these approaches independent of endogenous HLA expression levels in cancer cells. Several fusion protein formats have been evaluated, which consist of either one or two (streptavidin/biotin-coupled) parts and utilize whole IgG or only Fab/scFv antibody fragments., Multiple aspects must be considered when designing such molecules, including avidity, stability/in vivo half-life, and tissue-penetrating capacity. Usually, whole monoclonal antibodies have higher avidity than monovalent antibody fragments. High-molecular-weight protein drugs tend to be more stable and have a longer in vivo half-life but are limited in their capacity for extravasation and tissue penetration. Moreover, large CMV peptide\u2013HLA-I-equipped fusion proteins may disturb the intermembrane separation distance of the immunological synaptic cleft and potentially diminish the potency of T cell-mediated killing. Despite their larger size, two-part fusion proteins utilizing streptavidin/biotin-coupling provide more flexibility to readily adjust them to fit patients with other HLA haplotypes [In conclusion, several studies have underlined the functionality and efficacy of fusion proteins comprising CMV peptide\u2013HLA-I complexes in recruiting inflationary anti-CMV CD8plotypes . A fusioplotypes .pos T cells to improve the in vivo persistence of CAR/TCR-engineered T cells. Moreover, anti-CMV T cells were activated ex vivo, expanded, and reinfused into glioblastoma patients to directly target CMV peptide epitopes expressed in a subset of glioblastomas.Expanded and reinfused anti-CMV T cells are commonly used to prevent CMV disease after hematopoietic stem cell transplantation (HSCT). Recently, anti-CMV T cells were evaluated in additional (new) forms of adoptive cell therapy (ACT). Chimeric antigen receptors (CARs) and TCRs were transduced into anti-CMV CD8Although remarkable treatment results have been achieved with CAR T cell therapy, lack of long-term persistence and poor expansion of transferred tumor-specific T cells remain major challenges ,58. evaluated autologous CMV-Her2-CAR T cells in a Phase 1 dose-escalation study in patients with progressive Her2-positive glioblastoma . InfusioCruz et al. (2013) generated allogeneic CMV-CD19-CAR T cells to treat patients with residual B cell malignancies after HSCT . AllogenTo promote the engraftment of CMV-CAR T cells, Caruana et al. (2015) created a whole-cell CMV vaccine to be administered to patients who were infused with CMV-GD2-CAR T cells . The antWang et al. (2015) transferred CMV-CD19-CAR T cells into immunodeficient mice bearing human CD19-positive lymphomas . The antTaken together, CMV-CAR T cells demonstrated superior proliferation, survival, and antitumor activity in vivo compared to generic CAR T cells. It is well known that supraphysiological activation via CD3/CD28 drives T cell differentiation and exhaustion . Replacipos T cells for other forms of ACT, such as TCR-engineered T cells, has also been investigated. Heemskerk et al. (2004) retrovirally transduced a leukemia-reactive TCR directed against minor histocompatibility antigen (mHag) HA-2 into anti-CMV CD8pos T cells [Given the improved survival capacity and antitumor activity of CMV-CAR T cells in vivo, it is not surprising that the use of anti-CMV CD8 T cells . These T T cells . Althoug T cells . When ap T cells . Only twIn conclusion, it appears to be more challenging to create TCR-engineered CMV T cells compared to CMV-CAR T cells, as forced expression of the artificial TCR downregulates the expression of the endogenous TCR. Additionally, artificial and endogenous TCR chains can pair, leading to the formation of mixed TCR complexes with undesired, possibly harmful specificities .An alternative approach for using anti-CMV T cells in cancer therapy is to directly target CMV peptide epitopes expressed in approximately 40% of glioblastoma multiforme (GBM) patients . Intrigupos T cells in GBM patients was similar to that observed in healthy CMV-seropositive individuals. However, terminally differentiated (CD27neg/CD57pos) anti-CMV CD8pos T cells were more frequent in GBM patients [pos T cells in GBM patients [pos T cells isolated from resected GBM tumors lacked expression of CD103 and had augmented levels of the inhibitory receptors PD-1, TIM-3, and CTLA-4 [pos T cells (60\u201370%), the expression of TNF\u03b1, IFN\u03b3, MIP-1b, and CD107a was impaired [Unfortunately, anti-CMV T cells present in GBM tumors appear to be incapable of eliciting effective antitumor responses. In particular, Crough et al. (2012) showed that the frequency of precursor anti-CMV CD8patients . The lacpatients . Moreoved CTLA-4 . In a suimpaired .pos T cells are becoming exhausted and senescent, which is in sharp contrast to inflationary (effector\u2013memory) anti-CMV CD8pos T cells that can be found in other parts of the body of healthy individuals and cancer patients. Additional research is required to unravel why anti-CMV T cells in GBM patients are phenotypically distinct; however, the immunosuppressive nature of GBM is likely to be a contributing factor [Apparently, in the TME of GBM, anti-CMV CD8g factor .pos T cell function in GBM patients. Phase I trials have assessed the treatment of either newly diagnosed or recurrent GBMs. ACT protocols vary across trials but basically consist of the following steps: harvesting of PBMCs from patients through leukapheresis, culturing in growth medium supplemented with HLA-I- and HLA-II-restricted peptide epitopes from CMV, and subsequent stimulation with recombinant cytokines, such as IL-2. The reinfused anti-CMV CD8pos T cells are expected to migrate to the tumor site and recognize and eliminate CMV-positive cancer cells [Multiple clinical trials have been performed to determine whether expansion/in vitro stimulation and subsequent ACT could reconstitute anti-CMV CD8er cells trials showed that anti-CMV ACT enhanced progression-free survival (PFS) as well as overall survival (OS) and was associated with a reduced risk for side effects. The outcomes of these trials have been comprehensively reviewed by Sorkhabi et al. (2022) . In shorxpansion . Smith expansion . Reap et T cells .pos T cells are present in the TME. However, despite the correct antigen specificity, anti-CMV CD8pos T cells appear to be dysfunctional in GBMs, a phenomenon not observed in other cancers and likely related to the immunosuppressive nature of this tumor type. Multiple clinical trials have shown that it is possible to restore the function of exhausted autologous anti-CMV CD8pos T cells in vitro and reinfuse them into patients. Clinical benefits included enhanced PFS and OS.In conclusion, a subset of GBMs express CMV proteins, and cognate anti-CMV CD8pos T cell responses for cancer (immuno)therapy. In short, several studies aimed to mimic CMV infection by (re)decorating malignant cells with viral antigens on endogenous or exogenous HLA-I complexes. APECs, TEDbodies, and i.t. injection of CMV peptide epitopes facilitate recognition by anti-CMV CD8pos T cells by loading CMV peptides into HLA-I complexes expressed on cancer cells. All these strategies enhanced immune cell infiltration into the tumor, reduced tumor volumes, and prolonged survival in in vivo mouse models. Findings in tumor-directed fusion proteins comprising CMV peptide\u2013HLA-I complexes include efficient cancer cell lysis, T cell activation and proliferation in vitro, and reduced tumor growth in vivo. Adoptive cell therapy (ACT) of engineered CMV T cells aimed to provide optimal co-stimulation and improved in vivo persistence for CAR/TCR-engineered T cells following endogenous TCR interactions with latent virus antigens. Indeed, CMV-CAR T cells demonstrated superior proliferation, survival, and antitumor activity in vivo compared to generic CAR T cells. However, attempts to design TCR-engineered CMV T cells have not been very successful thus far and the competition for surface expression between the endogenous and artificial TCR remains a major challenge. Ex vivo reactivation and ACT of dysfunctional anti-CMV CD8pos T cells in GBM patients proved to be feasible and prolonged PFS and OS.Anti-CMV T cells are of particular interest for cancer (immuno)therapy because they are abundantly present, constantly renewable, and dominate the T-cell pool of the elderly, who are more often affected by cancer. In this review, we discuss various strategies for harnessing anti-CMV CD8pos T cells to eliminate cancer cells could be an alternative next-generation approach in (immuno)therapy for both solid and non-solid cancers.Taken together, a multitude of promising in vitro and in vivo data suggest that utilizing anti-CMV CD8pos T cells appear to have the following features in common:All treatment strategies utilizing anti-CMV CD8pos T cells do not require ex vivo manipulation or restimulation but display cytotoxicity directly after isolation from peripheral blood [pos T cells is necessary, for example due to insufficient T cell numbers, clinical-grade expansion protocols exist, and are safe and widely used. Obviously, ex vivo manipulation/expansion remains necessary for ACT-based strategies.Firstly, anti-CMV CD8al blood ,51. If epos T cells in GBM was more effective when initiated before recurrence of the tumor was evident [pos T cells) as high as possible to facilitate optimal cancer cell elimination with anti-CMV CD8pos T cells. To this end, pretreatment with cytoreductive agents could potentially improve the effectiveness of anti-CMV CD8pos T cell-based strategies.Secondly, the timing of the treatment appears to be crucial for obtaining optimal results. Treatment with CMV-CAR T cells worked better shortly after lymphodepletion, when the CMV viral load was high . Adminis evident . Pretrea evident . Accordipos T cells, non-viral T cells targeting tumor-associated antigens are activated and appear to contribute to the anti-tumor response [Thirdly, after initial cancer cell lysis by anti-CMV CD8response ,87, a phresponse ,90. Morepos T cell treatments can be boosted by vaccination [pos T cell responses [pos T cells in CMV-seronegative subjects as in CMV-seropositive subjects where they accumulated through memory T cell inflation.Fourthly, several studies have shown that anti-CMV CD8cination ,68,69,70cination . Additioesponses . Howeverpos T cells to attack cancer cells, such as TEDbodies, APECs, and fusion proteins comprising CMV peptide\u2013HLA-I complexes antiviral T cells against common viruses, such as EBV and Influenza. For example, EBV/influenza-CAR T cells ,67,94,95pos T cells, require determination and adjustment of treatment to match the patients\u2019 HLA-I haplotype. Multiple versions of APECs, TEDbodies, and fusion proteins comprising various CMV-specific peptides presented on corresponding common HLA haplotypes, such as HLA-A*02:01, HLA-C*07:02, and HLA-B*07:02, should be designed to make these strategies accessible to as many patients as possible. Given that CMV seroprevalence is highest in South America, Afrika, and Asia [All strategies involving anti-CMV T cells naturally depend on the CMV infection status of patients. Approximately 83% of the general population is CMV-seropositive and is therefore eligible for anti-CMV T cell-based therapies . Many stpos T cells had insufficient capacity to survive and engraft in mice. Using NOG mice, which are not only lymphocyte-deficient but also lack functional macrophages, engraftment was improved [pos T cells. Similarly, in NSG mice, co-administration of IL15/IL-R\u03b1-Fc was required to maintain anti-CMV CD8pos T cell persistence [pos T cells for cancer (immuno)therapy need to be identified and/or designed.The evaluation of anti-tumor effects in immunocompromised in vivo models seems to be a major challenge, as injected anti-CMV CD8improved . Howeversistence . In the pos T cells that are retargeted to tumors [pos T cells responded to PD-1 treatment in patients with melanoma [pos T cells. Combinatorial treatment with TEDbodies and agonistic OX40 antibody had beneficial outcomes and warrants further investigations [pos T cell recruitment and CD47-blocking antibodies should be considered to improve their efficacy.As the surface expression of immune checkpoints limits T cell efficacy, combinatorial treatment approaches with immune checkpoint inhibitors, such as PD-1 blocking agents, should be more thoroughly investigated. PD-1 appears to have an inhibitory role in advanced solid cancer mouse models and can shut off functional anti-CMV CD8o tumors . Circulamelanoma ,103. In igations . Recentligations . Therefopos T cells in vitro before CAR transduction or reinfusion for GBM treatment. Moreover, CMV-CAR T cells and tumor-directed fusion proteins comprising CMV peptide\u2013HLA-I complexes could be co-administered, thereby recruiting expanded and reinfused CMV-CAR T cells via their tumor-associated antigen (TAA)-targeting CARs as well as their TCRs to cancer cells. Thus, depending on the antibody (fragment) moiety of the fusion protein, multiple distinct TAAs could be targeted. Combinatorial treatment with CMV-CAR T cells and fusion proteins comprising CMV peptide\u2013HLA-I complexes may be of particular interest for heterogeneous tumors and for treatments in which therapy resistance is acquired due to (CAR) target antigen loss in cancer cells.The diversity of approaches utilizing anti-CMV T cells for cancer (immuno)therapy may also open new avenues to improve outcomes by combining multiple strategies. For example, Immuno-STATs could be used to expand anti-CMV CD8In conclusion, the use of anti-CMV T cells for cancer (immuno)therapy has been rapidly growing, advancing, and diversifying in recent years. Although many aspects remain to be elucidated before anti-CMV T cell-based therapies can enter the clinical practice, these therapeutic approaches are highly promising, as they convey the natural potency of anti-CMV T cells to cancer cells and can be easily adapted for patient-tailored treatment."} +{"text": "Cancer immunotherapy has become an established therapeuticparadigmin oncologic therapy, but its therapeutic efficacy remains unsatisfactoryin the majority of cancer patients. Accumulating evidence demonstratesthat the metabolically hostile tumor microenvironment (TME), characterizedby acidity, deprivation of oxygen and nutrients, and accumulationof immunosuppressive metabolites, promotes the dysfunction of tumor-infiltratingimmune cells (TIICs) and thereby compromises the effectiveness ofimmunotherapy. This indicates the potential role of tumor metabolicintervention in the reinvigoration of antitumor immunity. With themerits of multiple drug codelivery, cell and organelle-specific targeting,controlled drug release, and multimodal therapy, tumor metabolism-rewritingnanomedicines have recently emerged as an attractive strategy to strengthenantitumor immune responses. This review summarizes the current progressin the development of multifunctional tumor metabolism-rewriting nanomedicinesfor evoking antitumor immunity. A special focus is placed on how thesenanomedicines reinvigorate innate or adaptive antitumor immunity byregulating glucose metabolism, amino acid metabolism, lipid metabolism,and nucleotide metabolism at the tumor site. Finally, the prospectsand challenges in this emerging field are discussed. A summary of the progress of nanoparticle-based therapeuticstrategies that reinvigorate antitumor immunity by regulating metabolismof glucose, amino acids, lipids, and nucleotides at the tumor site. In summary, rewriting the metabolically hostile tumor microenvironment (TME) holds promisefor reinvigorating antitumor immunity.37 Encouragingly, metabolic interventions combined withICB-based tumor immunotherapies have entered clinical trials , which impedes the efficacy of cancer immunotherapiesby suppressing a variety of antitumor immune responses.l trials 1.35,3842 Nanosized materials that preferentiallyaccumulate in solid tumor tissues can improve the in vivo biodistributionand pharmacodynamic performance of macromolecular and small-moleculedrugs possessing immunomodulatory or tumor metabolic-rewriting properties.The blood\u2013brain barrier (BBB) penetration and cell-/organelle-specifictargeting capabilities of nanoparticles (NPs) have the potential toenhance the regulatory effects of drugs at the TMME.47 Significantly, nanomedicines can be customized to achieve controlledand spatiotemporal drug release in response to physical or biological stimuli , glutathione (GSH), etc.) that are present in the TME.51 This approach enhanced the therapeutic efficacy while minimizingadverse effects. Moreover, tumor metabolism-rewriting nanomedicines,when used in combination with minimally invasive therapeutic modalitieslike chemodynamic therapy (CDT), photodynamic therapy (PDT), photothermaltherapy (PTT), and sonodynamic therapy (SDT), have the potential toincrease tumor immunogenicity and promote the formation of an inflammatoryTME, resulting in synergistic immunotherapeutic effects.55 Taken together, the multifunctional features of nanomedicines offera range of benefits in regulating cancer metabolism and potentiallyenhancing antitumor immunity in a synergistic manner ,however, is a far less efficient method to generate ATP than oxidativephosphorylation (OXPHOS), which normal cells prefer to use.59 From this perspective, the suppression of tumor glycolysis may serveas a targeted strategy to selectively eliminate tumor cells. Consequently,this intervention would have low off-target effects, thereby offeringa benefit over the nonselective cytotoxicity frequently encounteredwith traditional chemotherapy and radiation therapy. Importantly,the increase in glucose uptake by tumor cells leads to intratumoralglucose scarcity, which not only restricts T cell activation and differentiation,but also limits proinflammatory cytokines secretionfrom Teff cells.62 Moreover, tumor cells with high glycolytic activitiesfacilitate intratumoral lactate production, which contributes to tumorimmune tolerance by activating a series of immunosuppressive pathways.63 Thus, the inhibition of tumor glucose metabolismand the removal of immunosuppressive lactate in the TME can serveas effective treatment strategies for boosting antitumor immune responses.Notably, clinical trials have been initiated to explore the efficacy of a combinedtherapeutic approach involving glucose metabolism intervention andimmune-checkpoint inhibitors (ICIs) , and the high glycolytic rate providesmore building blocks for anabolism in response to oncogenic signaling.s (ICIs) 1. In thi2.166 Hence, inhibitionof tumor glycolysis presents a viable strategy to disrupt lactate-mediatedimmune suppression and promote antitumor immunotherapy. Recently,Li et al. developed a pH-sensitive nanomedicine (SK/siR-NPs) by encapsulatinga glycolysis inhibitor (shikonin) and a PD-L1 small interfering RNA(siRNA) in folic acid (FA)-modified polymeric nanoassemblies (67 Due to the specific recognition of FA and folate receptor(FR), the resultant nanomedicine exhibited excellent targeting abilitytoward FR-positive cancer cells. Upon internalization by tumor cells,the shikonin released from this nanomedicine successfully inhibitedpyruvate kinase isozyme type M2 and reduced lactate production, ultimately promoting the polarizationof M2-like TAMs toward the M1-like phenotype. In addition, PD-L1 siRNA-mediatedPD-L1 downregulation helped to restore the tumoricidal function ofTeff cells. Likewise, a mannosylated lactoferrin nanocomposite (Man-LFNPs) was constructed to alleviate the immunosuppressive TMME by dual-targetedcodelivery of PD-L1 inhibitor (JQ1) and shikonin of the resultantnanomedicine selectively bound to the overexpressed low-density lipoproteinreceptor-related protein 1 (LRP-1) on tumor vascular endothelial cellsand tumor cells. In addition, the interaction between mannose andthe mannose receptor (MR) allowed Man-LF NPs to target M2-like TAMswith MR overexpression. However, these approaches for inhibiting tumorglycolysis did not consider the potential beneficial impact of mitochondrialOXPHOS. Increasing mitochondrial glucose metabolism in cancer cellshas been demonstrated to facilitate the infiltration and antigen presentationof Teff cells in the tumor site.69 Therefore,the concurrent upregulation of mitochondrial OXPHOS and downregulationof glycolysis in tumor cells provides a promising strategy to mitigatethe immunosuppressive TMME. In this regard, Jia et al. developed GSHand pH dual-responsive mPEG-PLA-PHis-ss-PEI polyplexes (DRP/Res/siP)encapsulating PD-L1 siRNA and resveratrol -activated proteinkinase (AMPK). In vivo antitumor experiments demonstrated that upregulationof mitochondrial OXPHOS pathways not only promoted the tumor infiltrationof CD8+/CD4+ T lymphocytes and IFN-\u03b3 secretionbut also suppressed Treg cells and MDSCs. This nanomedicine reprogrammedthe glucose metabolism in tumor cells and, in conjunction with PD-L1silencing, promoted the formation of a less immune-suppressive TME.However, the efficacy of this nanomedicine would be influenced bythe heterogenic GSH levels and the tumor acidity.Accumulating evidence reveals that the lactate produced from tumorglycolysis can promote the polarization of tolerogenic M2-like TAMsand thereby inhibit the activation and proliferation of TIICs, suchas Teff cells, DCs, and natural killer (NK) cells.semblies 2.67 Due shikonin 2.68 Notaveratrol 2.70 This71 Therefore, therapeuticstrategies that combine the manipulation of TMME and depletion ofstromal desmoplasia hold promise to enhance antitumor immune responses.For this purpose, Xiao et al. designed a pH-activatable nanomedicine(M-s/W-NP) to boost antitumor immunity against orthotopic pancreaticcancer (72 The M-s/W-NP was constructed by coself-assembly of C-X-Cmotif chemokine ligand 1 (CXCL1) siRNA, PI3K signaling inhibitor (wortmannin),and an amphiphilic block copolymer, followed by surface modificationwith anti-mesothelin antibody in mitochondriaplays a crucial role in regulating tumor glycolysis, representinga promising target for glycolysis inhibition in cancer cells.76 The therapeutic efficacy of dichloroacetate was hindered by limited cellular uptake andinadequate mitochondrial localization. To circumvent these limitations,a triphenylphosphonium (TPP) cation-modified nanoassembly (T-Mito-DCA-NP)was developed to achieve mitochondrion-targeted delivery of DCA, leadingto a reprogrammed immunosuppressive TMME components within theTME could also exert physical barriers to restrict the tumor infiltrationof immune cells.ccancer 2.72 The antibody 2A. The rantibody 2A. In adantibody 2A. In vir growth 2B and prr growth 2C. Theseive TMME 2.77 T-Mi78 This nanosystem possessed good BBB-penetrating abilitydue to the binding property of albumin to albumin-binding proteins overexpressed in cells residing in the BBB. In addition,the binding of lactoferrin to LRP-1 expressed on tumor cells alsodramatically increased the cellular uptake of this nanomedicine. Uponsuccessful entry into tumor cells, released shikonin downregulatedglycolysis and reduced the production of lactate through PKM2 inhibition.Importantly, reduced lactate levels in tumor tissues improved theantitumor immunity by facilitating the differentiation and proliferationof CD8+ Teff cells, while concurrently decreasing the proportionof Tregs. In addition, disulfiram inhibited ALDH1L1 to suppress thealternative energy metabolic pathway (NADH-ATP), thus synergizingwith shikonin to enhance ATP depletion in tumor cells. Suppressionof tumor energy metabolism, however, can activate cancer autophagyto maintain cell homeostasis by degrading their misfolded proteinsand damaged organelles.79 To overcome thisobstacle, Luo and co-workers developed a metabolic nanoregulator (D/B/CQ@ZIF-8@CS)by encapsulating 2-deoxy-d-glucose ,BAY-876 (a GLUT1 inhibitor), and chloroquine into a chondroitin sulfate (CS)-coated zeolitic imidazolate framework-8(ZIF-8)-based nanocarrier to ATP for an alternative energy supply when glycolysis is inhibited.To circumvent this problem, Zhao et al. described a hybrid albumin/lactoferrinnanoplatform (BSA/LF NP) to regulate TMME by codelivering PKM2 inhibitor(shikonin) and aldehyde dehydrogenase 1 family member L1 (ALDH1L1)inhibitor (disulfiram) to glioma cells in a fixed-dose ratio 2.78 Thisocarrier 2.80 The 2.281 The TerBio was constructedby self-assembly of Chlorin e6 (Ce6), transforming growth factor (TGF)-\u03b2receptor-I inhibitor SB505124 (SB) and lactate efflux inhibitor lonidamine(LND) at a suitable feed ratio without additional excipients were constructed for hydroxycamptothecin (HCPT) loading for decreasingthe intratumoral lactate levels and performing photodynamic amplifiedimmunotherapy against colorectal cancer 2.81 The cipients 3A. TerBicipients 3A. In vicipients 2.82 To p loading 3B. Subse2O2 and pyruvate, offers a potentialstrategy for removing intratumoral lactate.83 Recently, He et al. designed a \u201cnanofactory\u201d for synergisticCDT and lactate-trapping-mediated tumor metabolic reprogramming (84 The nanofactory (PLNPCu) was constructed byencapsulating LOX and Cu2+ in a cationic PEI-based nanoassemblythrough electrostatic interaction and coordination. The surface ofthe nanocarrier was cross-linked with pH-responsive detachable polyethyleneglycol (OHC-PEG-CHO) to improve its stability and biocompatibility.The obtained nanofactory was able to actively trap its surroundinglactate to promote lactate degradation due to the exposure of theprimary amine on PEI. Subsequently, LOX-mediated lactate catabolismpromoted H2O2 generation, which was furthercatalyzed by Cu2+ to generate strongly oxidizing hydroxylradicals (\u2022OH) through a Fenton-like reaction. The \u2022OH-induced ICD plus mitigation of lactate-mediatedimmunosuppressive TMME synergistically elicited a strong antitumorimmunity in 4T1 tumor-bearing mice. Of note, the byproduct H2O2 produced by LOX-mediated lactate metabolism can reactwith a chemiluminescence reagent phenyl]oxalate,CPPO), subsequently releasing energy capable of activating the photosensitizer.Therefore, Lu et al. constructed a glioma cell-biomimetic nanomedicine(M@HLPC) to achieve a synergistic combination of lactate metabolictherapy and chemiexcited PDT while decreasingthe level of lactic acid (LA) in tumor tissues. The produced PA suppressedtumor growth by downregulating histone expression and inducing cellcycle arrest. Moreover, the reaction of H2O2 with CPPO formed H2O2\u2013CPPO, which subsequentlytransferred energy to Ce6, thus achieving chemically excited PDT inthe presence of oxygen. Importantly, hemoglobin worked as an oxygenresource to facilitate lactate catabolism and improve the PDT efficiency.In contrast to exogenous laser-source-dependent PDT, this approachcircumvents the attenuated PDT efficacy resulting from inadequatelaser penetration within tumor tissues. Two-dimensional (2D) SnSenanosheets (NSs) are known to have a lactate dehydrogenase (LDH)-likeactivity, capable of converting lactate into pyruvate.87 Ling et al. developed a NSs-based nanosystem (SnSe@ABS NSs) by encapsulating4-(2-aminoethyl)benzenesulfonamide ) in order to enhance photothermal immunotherapy throughmitigating the tumor acidification-induced immunosuppression , a catalytic enzyme capableof convertinglactate into Hgramming 2.84 The ited PDT 2.85 Thisoma area 3D. As shpression 2.88 Nota2.389 Proinflammatory M1-likeTAMs mainly rely on aerobic glycolysis for energy supply, while tumorprogression-promoting M2-like TAMs are highly dependent on OXPHOSto sustain their protumoral activities.90 Notably, cancer cells within hypoxic TME can inhibit glycolysisin TAMs to reduce glucose competition and promote metastasis development.91 Therefore, regulating the metabolic phenotypeof TAMs is a potential strategy to enhance the antitumor immunity.Recent studies have revealed that the inhibition of phosphatidylinositol3-kinase gamma (PI3K\u03b3) and activation of nuclear factor kappa-B(NF-\u03baB) could shift M2-like TAMs toward M1-like phenotypes undernormoxia.94 However, M2-like TAMs often reside in the tumor hypoxicarea, which is unfavorable for the polarization of M1-like TAMs.96 To overcome this obstacle, Jiang et al. designed a nanoemulsionsystem (\u03b1-T-K) that encapsulated an oxidative stress inhibitor(\u03b1-tocopherol) and an ER stress inhibitor (KIRA6) oxidation (FAO) in cancer cells. This modulation of cellularmetabolism significantly reduced the level of M2-like TAMs and enhancedthe infiltration of cytotoxic CD8+ Teff cells in tumortissues 2.97 Thistissues 4B.In vitissues 2.98 The p-STAT3and IL-10.100 Moreover, angiogenesis playsa critical role in regulating tumor metabolism and antitumor immuneresponses as dysregulated vasculature results in tumor hypoxia andacidosis. This leads to the upregulation of immunosuppressive factors(such as VEGF and TGF-\u03b2) in TME, promoting tumor immunosuppressionand the development of metastasis.101 Hence,the simultaneous inhibition of the mTOR signaling pathway and angiogenesisholds great promise to exert active effects on antitumor immunity.In pursuit of this objective, Chen et al. developed a dual-targetingnanosystem (t-LRR) by encapsulating an mTOR inhibitor (rapamycin)and an antiangiogenic drug (regorafenib) in liposomes, followed bysurface modification of a PD-L1 nanobody and mannose compared to regorafenib(42.0%) and rapamycin (47.8%) in the CT26 subcutaneous colon carcinomamodel. Augmentation of cellular glycolysis levels represents an alternativeapproach to induce the transformation of M2-like TAMs toward M1-likephenotypes. However, it remains a challenge to selectively delivermetabolic regulators to TAMs while avoiding favorable metabolic regulationin malignant cells that utilize glycolysis as an energy source. Theoverexpression of MR (CD206) in TAMs offers potential for the developmentof TAMs-targeted drug delivery systems.103 Based on this concept, bacterial outer membrane vesicles (OMVs)-basednanosystem possessing sequential drug release capabilities were developedfor selective upregulation of glycolysis in M2-like TAMs and suppressionof tumor metastasis siRNA (Redd1 siRNA) was encapsulated into Gram-negativebacteria-derived OMVs by electroporation, followed by insertion ofDSPE-PEG-mannose and DSPE-PEG-CA-PTX into the phospholipid bilayer signaling pathway not only stimulates the uptake of glucoseand promotes glycolysis but also facilitates the polarization of TAMstoward M2-like phenotypes by upregulating mannose 2.102 Thetastasis 4C.104 To bilayer4D. This bilayer4E. Redd12.4106 In 2021 alone, there were morethan two hundred reported clinical trials with NK cell-based cancerimmunotherapy.107 NK cells can exert tumoricidalfunction by releasing cytolytic granules and cytotoxic cytokines,which are controlled by a complex array of activating and inhibitoryreceptors. However, the metabolic impairment promotes the dysfunctionof NK cells within TME.109 To enhance the NKcell-mediated innate antitumor immunity by reprogramming its cellularmetabolism, a immunosensitizer (BPQDs@HSA) was constructed by encapsulatingblack phosphorus (BP) quantum dots (QDs) in human serum albumin (HSA)2.110 In NK cells5B. NotabNK cells5C. SigniNK cells5D and OXNK cells5E in NK NK cells5F.330 High metabolic activities of tumor cells promote the deprivationof intratumoral amino acids and consequently impede activation anddifferentiation of T cells.112 Furthermore, manyenzymes involved in amino acid metabolism, e.g., 2,3-dioxygenase (IDO)and tryptophan 2,3-dioxygenase (TDO), can increase the intratumoralproduction and accumulation of immunosuppressive amino acid metabolites, which diminishes the effector function and memory formationof T cells.30 Therefore, targeting aminoacid metabolism in tumors could create an immune-stimulatory TME toenhance tumor immunotherapy. In this section, we summarize the recentdevelopment of nanomedicines capable of synergistically enhancingantitumor immunity by regulating the metabolism of glutamine, tryptophan,and l-arginine in TME.In addition to supporting biosynthesis, energy metabolism, andcellular redox homeostasis, amino acid metabolism is extensively involvedin the manipulation of antitumor immunity.3.1113 Rapidly proliferating cancer cells, however, use glutamine as animportant source of energy, thus causing intratumoral glutamine scarcity,which subsequently promotes differentiation of CD4+ T cellstoward immunosuppressive Tregs.115 Furthermore,a limited amount of glutamine in TME can severely inhibit CD8+ Teff cells which exploit glutamine metabolism to fuel ATPproduction.117 Therefore, promising approachesto reinvigorate antitumor immune responses could be achieved by targetingthe glutamine metabolism in tumor cells. Recently, Tang et al. constructeda two-dimensional molybdenum disulfide (MoS2)-based nanomedicine(MoS2-aPDL1-V9302) for tumor-targeted delivery of glutaminemetabolism inhibitor (V9302) and anti-PD-L1 . As expected, this nanomedicine-mediated tumor glutaminemetabolic intervention was shown to enhance the therapeutic effectof PD-L1-based immunotherapy against breast cancer. In a differentstudy, a cancer cell membrane-cloaked nanosystem (CTTPA-G) encapsulatingtype I aggregation-induced emission (AIE) photosensitizer (PS) andglutamine antagonist was developed to achieve synergistic PDT andtumor metabolic reprogramming (119 The obtained CTTPA-G generated high-performancesinglet oxygen (1O2) that triggered a robustICD even in hypoxic tumors, due to the use of O2-independenttype I PS. The glutamine antagonist released in TME further inhibitedtumor glutamine metabolism and overcame intratumoral glutamine deficiency.This intervention consequently provides the sufficient glutamine requiredby Teff cells to restore their antitumor functions. The CTTPA-G plusanti-PD-1 therapy inhibited the primary for synergistically enhancing PDT-induced tumor immunogenicity andreprogramming immunosuppressive TMME without excipient was developed to enhance chemoimmunotherapy by improving antigen presentationefficiency of mature DCs -loadedheavy chain ferritin cage (DOX@HFn) and l-methionine sulfoximine(MSO)-encapsulated galactose-modified zwitterionic liposomes (MSO@GZL)were released from this nanomedicine . The surface of NH2-MONs wasthen coated with cationic PEI and poly-citraconicanhydride (PAH-cit), which not only facilitated loading of antiglutaminasesiRNA (siGLS) via electrostatic interaction but also prevented thepremature release of LND. Upon arrival at tumor tissues, the GSH-triggereddegradation of NH2-MONs and pH-driven PAH-cit charge-reversalfacilitated the release of LND and siGLS. Subsequent inhibition ofglycolysis by LND and downregulation of glutaminase expression bysiGLS enabled the proper maintenance of nutrients necessary for Tcells, by reducing the consumption of glucose and glutamine from tumorcells.The upregulated activity of glutaminase (GLS) in cancercells promotesde novo synthesis of GSH by facilitating the production of essentialraw material (glutamate) of GSH.ive TMME 3.122 C9Sxcipient 6F. The 1ture DCs 3.124 Duemedicine 6G. DOX@Hmedicine 6G. MSO@Gmedicine 3.125 DMN3.2126 Of note, IDO plays a significantrole in the continuous intratumoral accumulation of Kyn, an immunosuppressorwhich has been clinically validated to facilitate tumor immune escape.127 Specifically, Kyn can impair tumor infiltration,proliferation, and activation of Teff cells while promoting the recruitmentof immunosuppressive peripheral Tregs and MDSCs.129 Therefore, inhibition of IDO has been suggested as a potential strategyto overcome tumor immunosuppression.130 Recently, IDO inhibitors have been shown to synergize with ICIsin several clinical trials . Upon light irradiation, the 1O2 generated by iPSs not only destroyed the primary tumors butalso produced neoantigens to elicit ICD-mediated antitumor immuneresponses. Significantly, the released NLG919 was able to reversethe immunosuppressive TMME by inhibiting IDO and tumor infiltrationof Tregs, eventually inducing both primary and distant tumor regressionthrough PDT-sensitized immunotherapy.Elevated tryptophan-degrading enzymes in tumor cells and other types of cells can accelerate tryptophan consumption at the tumortissue.ti-PD-L1 3.131 Then of DCs 7A. The sn of DCs 3.132 Ce6T) composed of a SP backbone and IDO1 inhibitor (M-Trp)-conjugatedhydrophilic side chain (PEG) for synergistic photodynamic immunotherapyof cancer photoirradiation inducedICD to improve tumor immunogenicity. The upregulation of the apoptoticbiomarker (caspase-3) during PDT triggered the specific release ofM-Trp, causing inhibition of IDO activity in TME (134 SPNK was constructedby attaching kynureninase (KYNase) to the surface of the SP core usinga PEGylated 1O2-cleavable linker. The 1O2 generated by SPNK under NIR photoirradiationexerted PDT and induced ICD, causing the release of tumor-associatedantigens. Concurrently, the 1O2-triggered releaseof KYNase also decreased intratumoral Kyn to facilitate the proliferationand infiltration of CD3+CD8+ Teff cells in boththe spleen and blood of B16F10 tumor-bearing mice. In another study,Wang et al. developed a pH-sensitive nanomodulator (AIM) to potentiatethe outcome of radiotherapy by inhibiting IDO NPswith organic polymers derived from the coordination reaction betweenthe IDO inhibitor (4-phenylimidazole (4PI)) and zinc ions. Upon tumoraccumulation, the pH-dependent dissociation of CaCO3 reversedacidity-induced radioresistance and promoted the release of 4PI toinhibit tryptophan metabolism. As a result, AIM NPs treatment plusradiotherapy elicited a robust antitumor immune response to inhibitthe growth of murine CT26 and 4T1 tumors. The concurrent inhibitionof cancer cell energy metabolism and mitigation of Kyn-mediated tumorimmune suppression presents a synergistic strategy to enhance antitumorimmunity. In this regard, a nanoassembly, utilizing an F127-coatedprodrug dimer (LSD), was developed to concurrently inhibit glycolysisand IDO within tumor cells was developed to improve ICB-based tumor immunotherapyby using tumor arginine intervention by forming acid-sensitive imine bonds,thus increasing its hydrophobicity (anti-PD-L1 with ArgNP promoted infiltration of CD8+ Teff cells and elevated the ratio of CD8+/CD4+ T cells in tumor tissues (Arginine (Arg) availability within TME playsa critical role in supporting antitumor immune responses, especiallyin the differentiation and activation of Teff cells.rvention 3.142 To hobicity 8A. Due t tissues 8B. Besid tissues 8C.In vi tissues 8D.4144 It should be noted that lipidmetabolism can also impact the phenotype and antitumor function ofTIICs.145 Bioactive lipids can promote tumor immune escape by affectingcancer immunity cycles, such as the formation and presentation oftumor-associated antigens, priming, and activation of Teff cells.146 Therefore, regulating lipid metabolism in theTME could be an effective way to restore antitumor immunity. In thissection, we summarize recent advances in the development of nanomedicinesthat can synergize with tumor immunotherapy by regulating lipid metabolismin TME.Under hypoxic and nutrient-poor conditions, cancer cells can useFAO-based lipid metabolism for supplying energy and maintaining theirsurvival and growth.4.1148 Inhibition of COX has been incorporated into clinical trials toassess its impact on the effectiveness of ICIs-based tumor immunotherapies, a PD-1/PD-L1 inhibitor (BMS-202), and a GSH-activatableprodrug (PEI-SS-Cele) containing the COX-2 inhibitor celecoxib (Cele).Upon arrival at the tumor site, the hydrophobic backbone in psHSAbecame hydrophilic in response to tumor acidity, leading to disassemblyof Cele-BMS-NPs and subsequent extracellular release of BMS-202 andPEI-SS-Cele. Furthermore, the cationic PEI-SS-Cele with excellentcell-penetrating ability could be activated upon disulfide bond cleavageby intracellular GSH. The released celecoxib suppressed Tregs andM2-like TAMs while inhibiting the intracellular function of COX-2and extracellular secretion of PGE2. In addition, BMS-202 releasedin TME blocked PD-1/PD-L1 interaction to achieve ICB-based immunotherapy.As expected, Cele-BMS-NP treatment promoted tumor infiltration ofCD8+ Teff cells significantly and decreased intratumoralimmunosuppressive M2-like TAMs, Tregs, and PGE2, thus eliciting anefficient antitumor immunity to inhibit the growth of poorly immunogenic4T1 breast tumors. Likewise, Sun and co-workers developed a biodegradablecatalytic cascade nanoreactor (PEG-Au@HMnMSNs) for enhanced antitumorimmunity by improving chemoimmunotherapy-induced ICD and maturationof DCs (150 The GSH-responsive nanoreactor was constructedby doping Mn2+ into the silicon shells of hollow mesoporoussilica-coated gold NPs (Au@MSNs), followed by surface PEGylation.After that, ASA (a COX-2 inhibitor) and DOX were coloaded into thenanoreactor through strong \u03c0\u2013\u03c0 stacking interactionsand physical absorption. High intracellular levels of GSH triggeredthe degradation of the nanoreactor, leading to rapid release of Mn2+ in cancer cells. Significantly, Mn2+ producedhighly toxic \u2022OH via a Fenton-like reaction whilefacilitating the consumption of GSH (an antioxidant). The gold NPswith glucose oxidase (GOX)-like catalytic activity decomposed glucoseto generate H2O2, thus synergistically enhancingthe production of \u2022OH. Furthermore, the above core\u2013shellstructure prevented the inactivation of gold NPs by inhibiting strongprotein adsorption, which consequently promoted H2O2 catalysis. Combinatorial DOX-mediated chemotherapy and enhancedCDT induced sufficient ICD to elicit systemic antitumor immunity.Moreover, the released ASA facilitated tumor infiltration of DCs andTeff cells while decreasing the number of immunosuppressive cellswithin the TME.Overexpression of cyclooxygenase (COX)-2 in tumor cells leads tointratumoral production of the immunosuppressive bioactive lipid metabolitePGE2, which not only promotes accumulation of MDSCs and M2-like TAMspolarization but also restricts tumor infiltration and cytokine secretion in Teff cells.nof DCs 4.150 The151 Due to its long-lasting and recyclable effectson the degradation of the targeted protein, PROTAC offers numerousbenefits over conventional inhibitors or genetic tools, such as loweradministration dose and higher therapeutic efficacy.152 Zhang et al. developed cathepsin B (CatB)-activatable nanoproteolysistargeting chimeras (nano-PROTACs) for synergistic phototherapeuticablation and cancer-specific COX-1/2 degradation was constructed by attaching the COX-1/2-targetingPROTAC peptide (CPP) to an SP backbone by using a CatB-cleavable segmentas a linker ,PVP), significantly inhibited tumor growth under multimodal imaging-guidedPTT. However, tumor recurrence occurred a few days after treatment,presumably due to PTT-induced inflammation. The deferoxamine mesylate(DFO)-induced disassembly of FeLPNPs triggered the release of luteolin,mitigated the inflammatory responses induced by PTT and inhibitedtumor recurrence by reducing TNF-\u03b1 and IL-6 secretion. Furthermore,luteolin also regulated immunosuppressive TME by inhibiting COX-2-mediatedPGE2 production and PD-L1 expression in tumor cells. As expected,FeLPNP treatment significantly inhibited tumor recurrence and metastasisin 4T1 tumor-bearing mice.Proteolysis targeting chimera (PROTAC) can promotepost-translationalknockdown of a targeted protein by concurrently binding to the E3ligase and the targeted protein via two covalently linked moieties.radation 4.153 Thea linker 9A. Overea linker 9B and C.ing mice 9D and E.ing mice 4.154 The4.2156 Blocking monoacylglycerol lipase(MGLL), a key lipolytic enzyme for FA generation, can inhibit themetabolism of lipids in tumor cells. However, MGLL downregulationcan promote the polarization of M2-like TAMs via 2-arachidonoylglycerol(2-AG)-mediated endocannabinoid receptor-2 (CB-2) activation.157 To overcome this obstacle, a GSH-activatablenanomedicine was developed to concurrently inhibit tumor lipid metabolismand M2-like TAMs polarization (158 This nanomedicinewas constructed by encapsulating MGLL siRNA (siMGLL) and CB-2 siRNA(siCB-2) in poly(disulfide amide) (PDSA)-based NPs. Highly abundantintracellular GSH facilitated the release of siMGLL and siCB-2 toinhibit MGLL activity and CB-2 expression, respectively. The MGLLblockade in tumor cells suppressed FAO pathways, leading to inhibitionof tumor growth, and the downregulation of CB-2 promoted the conversionof M2-like TAMs toward M1-like phenotypes. This nanomedicine was shownto significantly inhibit tumor progression in both xenograft and cholesterolavidity, which can facilitate their rapid growth by FAO pathway-mediatedATP production.rization 4.158 Thienograft 10B and oenograft 10C pancr4.3159 However, it remains a major hurdle to achieving directpharmacological inhibition of nuclear XBP1. To overcome this obstacle,Lu et al. developed a KIRA6-loaded \u03b1-tocopherol nanomedicine(KT-NE) to reinvigorate DCs-dependent antitumor immunity by scavengingintracellular ROS and inhibiting the upstream activator of XBP1 within TIDCs , functioning as an ROS scavenger, effectivelyrelieved oxidative stress in TIDCs, thereby inhibiting lipid oxidationand reducing the subsequent production of 4-hydroxynonenal (4-HNE).The decreased 4-HNE levels indirectly reduced the level of promotionof XBP 1s protein production. In vivo studies showed that the KT-NE-treatedDCs elicited a strong antitumor immunity by stimulating tumor-specificT cells in ovarian cancer-bearing mice. The highly active state ofmevalonate (MVA) signaling pathway associated with cholesterol metabolismin TIDCs could undermine their antigen presentation ability and ultimatelyimpair Teff cell priming.162 This phenomenon wasattributed to the ability of geranylgeranyl diphosphate (GGPP), ametabolite of MVA pathway, to promote lysosomal degradation of antigen,which consequently restricted antigen presentation by inducing excessivegeranylgeranylation of small GTPases in TIDCs.163 In this context, Shen et al. reported a versatile metabolism nanointervenor(Man-OVA(RSV) NPs)-loaded hydrogel delivery system (Gel@NPs) to interferewith cholesterol metabolism of TIDCs and restore their antigen presentationability -conjugated ovalbumin (Man-OVA)and rosuvastatin (RSV), exhibited a precise DC-targeting ability dueto the overexpression of MR on TIDCs. To achieve durable release ofthis nanointervenor, Man-OVA(RSV) NPs were encapsulated in a hydrogelformed by gelation action between Met and graphene oxide (GO). UponNIR photoirradiation, GO-mediated PTT induced ICD and promoted therelease of the tumor antigen, which was processed by TIDCs. Notably,RSV released from Man-OVA(RSV) NPs enhanced the antigen-presentationefficiency by preventing MVA pathway-mediated rapid antigen degradationin TIDCs. As expected, Man-OVA(RSV) NPs plus Met-induced PD-L1 blockadeinduced a robust antitumor immune response in the murine melanomamodel.Hyperactivated X-box binding protein 1 (XBP1) and a highintracellular ROS concentration within tumor-infiltrating DCs (TIDCs)can promote intracellular accumulation of peroxidized lipids, whichresult in poor T-cell priming and activation.in TIDCs 4.160 Actability 4.164 The4.4165 Thus, modulation of cholesterolmetabolism holds promise for improving T-cell-based antitumor efficiency.The suppression of acetyl-CoA acetyltransferase 1 with avasimibe (AVA) can enhance the tumoricidal activityof Teff by promoting TCR clustering.166 However, the efficacy of AVA was constrained by the distinct pharmacokineticsand biodistribution profiles between AVA and T cells. To overcomethis obstacle, a \u201cbackpacking strategy\u201d was developedto regulate cholesterol metabolism in T cells and boost T-cell-basedimmunotherapy against melanoma bearing liposomal Ava exhibited excellent antitumor efficiency inmelanoma and glioblastoma mouse models while producing no obvioussystemic side effects. The limited penetration of tumor metabolism-regulatingnanomedicine within tumor tissues significantly undermines their therapeuticefficacy. To overcome this problem, Liu et al. developed a matrixmetalloproteinase-2 (MMP-2) responsive tumor-penetrable nanosystem(EALP) to enhance photodynamic cancer immunotherapy by interveningthe cholesterol metabolism in T cells and cancer cells (168 The EALP was fabricated by loading AVA and the MMP-2-activatablepeptide (PPa-PLGLAG-iRGD) into liposomes. Upon accumulation in theTME, the MMP-2-triggered release of iRGD facilitated the deep penetrationof nanomedicines in tumor tissues. The AVA-mediated ACAT-1 inhibitionprevented cellular cholesterol esterification and improved cholesterollevels on the T cell membrane, thus enhancing antitumor immunity.In addition, inhibition of ACAT-1 in tumor cells limited the migrationof tumor cells and synergized with PPa-mediated PDT to elicit a strongantitumor immune response in melanoma and breast tumor models.Cholesterol on T-cell membranes is an important factor that can affectthe formation of T cell receptor (TCR) clustering and immunologicalsynapses.melanoma 4.167 Lipunctions 11A. The unctions 11B. Thisstrategy 11C. The er cells 4.168 The+ Teff cells as well as the differentiationof helper CD4+ T cells.169 Blockadeof FA catabolism in tumor-infiltrating Teff cells impairs their tumoricidalfunction.171 Peroxisome proliferator-activatedreceptor alpha (PPAR-\u03b1) agonist-induced upregulation of FA catabolismin Teff cells can facilitate their tumor infiltration capability andantitumor function under conditions of hypoxia and low-glucose.171 Nevertheless, the challenge persists in achievingthe upregulation of FA catabolism in TIICs while simultaneously avoidingfavorable metabolic regulation in cancer cells. To circumvent thisproblem, a T-cell-targeting nanomedicine (aCD3/F/ANs) was fabricatedby encapsulating fenofibrate (a lipid metabolism-activating drug)in anti-CD3 ef(ab\u2032)2 fragment-modified NPs (172 Upon anti-CD3ef(ab\u2032)2-mediated specific internalization by exhausted T cells,fenofibrate was released to improve the expression of PPAR-\u03b1and downstream FA metabolism-related genes. Through restoration ofmitochondrial functions, the activation of FAO metabolic pathwayssupported the survival and proliferation of Teff cells by mitigatingtheir metabolic stress within the TME. As expected, this nanosystemnot only improved the tumor infiltration of Teff cells but also enhancedtheir cancer cell-killing activity against B16F10 melanoma by improvingsecretion of cytokines . The developmentof T memory cells is a prerequisite for achieving effective and long-lastingantitumor immunity. In this regard, Luo et al. developed a nanovaccine(TA-Met@MS) incorporating tumor antigen (TA), metformin (Met), andhollow gold nanospheres (HauNS) 4.173 Thi5174 Significantly,nucleotides and their metabolites from cancer cells can promote orinhibit antitumor immune responses by activating several receptors.The accumulation of extracellular ATP (eATP), a strong pro-inflammatorysignal, can cause extensive antitumor immune responses by activatingToll-like receptors (TLRs).175 However,eATP can be metabolized to immunosuppressive adenosine in the presenceof ectonucleotidases CD39 (NTPDase 1) and CD73 (5\u2032-NT) in TME.177 The binding of adenosine to its receptors promotes the development of tumor-infiltrating immunosuppressivecells while undermining the infiltration and proliferation of Teffcells.28 Furthermore, adenosine causestumor immune suppression by inhibiting the expression of proinflammatorycytokine receptors and inducing PD-L1 expression on tumor cells, DCs,and TAMs.178 Therefore, inhibiting the adenosinergicpathway represents an attractive therapeutic strategy for improvedtumor immunotherapy.180 Several related clinical trialsare currently being tested already, by using anti-CD73 antibodies, anti-CD39 antibodies and adenosine receptorinhibitors to enhanceICB-based tumor immunotherapy was fabricatedby coself-assembly of boronic acid (BA)-containing cationic polymer,CD39/CD73 inhibitor (ARL67156), and PS (IR700)-containing lipid polymer.In this nanocomposite, the anionic nucleotide ARL67156 was linkedwith BA to form an ROS-labile covalent conjugate through electronicinteractions and phenylboronic ester. Upon NIR irradiation at thetumor site, ROS generated by PS not only induced robust ICD to releaseATP but also triggered the cleavage of phenylboronic ester that facilitatedthe release of ARL67156. ARL67156-mediated inhibition of ATP-adenosineaxis decreased the production of adenosine within TME. As expected,this nanosystem elicited a robust antitumor immune response to inhibittumor growth and conferred a long-term survival in mouse models oforal and 4T1 breast cancers. Similarly, Wu et al. described a cancercell-derived exosome-based nanodelivery system (C-PMet) to boost innateand adaptive tumor immunity by inhibiting the ATP\u2013adenosineaxis (183 This nanosystem achieved tumor-selective deliveryof CD39 antagonist (POM1) and AMPK agonist (Met), and prevented severeimmune-related adverse events. The Met-mediated AMPK activation improvedintratumoral levels of pro-inflammatory eATP, which potentiated T-cell-mediatedimmune responses by promoting the maturation of DCs and antigen presentationefficiency. Of note, the POM1-mediated CD39 blockade significantlydecreased the intratumoral adenosine production, thus overcoming immunosuppressionof Teff and NK cells. As expected, this nanosystem achieved synergisticantitumor immunity, thus inhibiting tumor growth and distant metastasis.Additionally, this nanosystem demonstrated the capability of inducinglong-term immune memory protection.Anticancertherapies, such as phototherapy and chemotherapy, can elicit a strongantitumor immunity by facilitating the accumulation of eATP in TME.pression5.182 In ineaxis 5.183 Thi2O4 NP (SMDNs) to overcome immunosuppressive TMME by simultaneouslyinhibiting lactate production and ATP catabolism . The alteration of tumor energy metabolismsignificantly decreased the intratumoral lactate production and \u03b1,\u03b2-methylene adenosine 5\u2032-diphosphate by using a disulfide-bond cross-linker(DBHD). Notably, the high abundance of extracellular GSH within TMEcleaved DBHD and resulted in the disassembly of AOZN. This triggeredthe rapid release of Orz and AMPCP, which led to the upregulationof gasdermin D (GSDMD) expression and the conversion of procaspase-1to active caspase-1, respectively. The activated caspase-1-mediatedGSDMD cleavage induced pyroptosis of tumor cells and promoted tumorimmunogenicity by increasing the secretion of high mobility group1 (HMGB1). Notably, the released Orz sensitized tumor cells toward anti-PD-L1 treatment through increasing their PD-L1 expression.Moreover, the AMPCP-mediated inhibition of CD73 decreased the levelof adenosine to overcome immunosuppressive TMME.In a recent study, Dai et al. developed an ultrasmall(<6 nm)dichloroacetic acid (DCA)-conjugated MnFetabolism 5.184 Upooduction 12C. Mostmor site 12D. Flowmor site 12E. As emor site 12F.In amor site 5.185 AOZ5.2186 To circumvent this issue, they developed apH-activatable nanomedicine (PPDAIn) for synergistic photothermalimmunometabolic therapy (186 PPDAIn was constructed byloading the A2AR inhibitor (SCH58261) into polydopamine-based nanocarriers,followed by masking with a pH-sheddable PEG layer through reversibleconjugation of BA and catechol. Upon arrival at the TME, the tumoracidity triggered the detachment of PEG, leading to exposure of theadhesive polydopamine layer and subsequent release of SCH58261. Notably,the exposed dopamine served as an anchor to the tumor tissue due toits mussel-mimicking adhesive properties, thus improving its tumorretention and accumulation capacity. Under laser irradiation, PPDAIn-inducedPTT resulted in the initiation of ICD and subsequently enhanced therelease of tumor neoantigens to promote DCs maturation. Significantly,SCH58261 reversed the metabolically suppressive effect of adenosineto strengthen the efficacy of ICD by increasing the tumor infiltrationof CD8+ T lymphocytes and reducing the population of MDSCs.This nanomedicine inhibited not only primary and abscopal tumors butalso distant tumor metastases in 4T1 tumor-bearing mice.Thestrategic utilization of an adenosine receptor blockade has also arisenas a promising strategy to enhance the therapeutic efficacy of cancerimmunotherapy. Recently, Liu et al. found that the negative feedbackpathway involving adenosine and adenosine 2A receptors (A2AR) wassignificantly enhanced during photothermal-induced ICD. therapy 5.186 PPD187 Upon NIR-II photoirradiationat the tumor site, ASPA exerted PTT effects to promote the rapid releaseof VIPA by inducing cleavage of the thermolabile linker (+/GrB+ primed T cells(2O2-responsive coassembled nanomedicine (CAT-NP) that encapsulateda camptothecin (CPT) prodrug (CPT-S-PEG), A2AR antagonist (AZD4635),and NIR-II molecule (TST) with aggregation-induced emission (AIE)properties , HMGB1, and eATP. Furthermore, the AZD4635-mediated A2AR pathwayblockade improved the proportion of Teff cells and concurrently decreasedthe number of MDSCs in tumor tissues.Inrecent years, there has been growing interest in utilizing the secondnear-infrared (NIR-II) light (1000\u20131500 nm) for PTT due tothe enhanced tissue penetration capacity and higher permissible energyfor skin irradiation. Therefore, a NIR-II light-activatable nanomedicine(ASPA) was prepared by encapsulating a fluorescent dye (NIR775) inNIR-II light-absorbing SP backbone-based NPs (ASPA), followed by surfacemodification with an A2AR antagonist through anazo-based thermosensitive linker 5.187 Upoe linker 13A. PTT-e linker 13B,tumo T cells13C and Ioperties 5.188 In 189 Due to the presence of E-selectin,ES-DSMs specifically adhered to the surface of leukocytes and subsequentlyrode leukocytes across biological barriers, thus improving their tumoraccumulation. After the accumulation of ES-DSMs at tumor tissues,microwave irradiation was administered to induce local hyperthermia,causing a rapid release of DOX and SCH58261. DOX-induced ICD triggeredthe release of tumor neoantigens to improve tumor immunogenicity.Significantly, by blocking the binding of adenosine to A2AR on thesurface of various TIICs , SCH58261 reversed the immunosuppressiveTMME to enhance DOX-induced ICD. It has been observed that blockingthe A1 adenosine receptor (A1AR) can destroy cancer cells and induceICD, but it can also undermine antitumor efficacy by increasing PD-L1expression.190 To overcome this issue,Guo et al. developed a core\u2013shell structured nanomedicine (DPCPX@Dz)for simultaneous inhibition of PD-L1 and A1AR in poly (PLGA)-based nanocore, followed by shell layer coating of metal-phenolicnetworks (MPNs) via coordination between Fe3+/Mn2+ ions and tannic acid (TA). Subsequently, the anti-PD-L1 DNAzyme (Dz) was encapsulated in the MPNs shell layer. Uponsuccessful entry into cancer cells, the released Mn2+ fromthis nanomedicine activated the metal-dependent Dz to cleave PD-L1mRNA. As a result, downregulation of PD-L1 plus DPCPX-mediated inhibitionof A1AR significantly elicited strong antitumor immune responses bypromoting maturation of DCs, infiltration, and activation of Teffcells in B16F10 tumor-bearing mice.The utilization of leukocytes as carriers to improvethe accumulationof nanomedicine at the tumor site has garnered increasing attention.In a recent study, Qi et al. developed a nanomedicine (ES-DSM) byencapsulating DOX and SCH58261 (an A2AR antagonist) in E-selectin-modifiedthermal-sensitive micelles 5.189 Dueand A1AR 5.191 Thi6Nanotechnology-assistedtumor metabolic intervention has emergedas an attractive and efficacious strategy to enhance antitumor immuneresponses. In this review article, we summarize the recent progressin the engineering of nanomedicines that can synergistically enhanceantitumor immunity by rewriting the metabolism of glucose, amino acids,lipids, and nucleotides at the tumor site 1. The co192 Therefore, simultaneouslytargeting multiple cellular metabolisms in cancer cells presents arobust approach to accomplishing efficacious intervention in tumormetabolism. Second, it is still difficult to develop a nanomedicinethat can selectively target tumor metabolic activities while minimizingunintended effects on TIICs. This is primarily attributed to the substantialsimilarities in cellular metabolic processes between cancer cellsand TIICs.193 The endeavor to identifyspecific cell surface markers that can differentiate cancer cellsfrom TIICs will contribute to the design of precision nanomedicinesthat can selectively target cancer cells while sparing TIICs. Additionally,endogenous stimulus-responsive strategies can be implemented to selectivelyliberate metabolic regulators exclusively within malignant cancercells. Third, the spatial\u2013temporal heterogeneity of tumorsimpedes the effectiveness of tumor metabolism intervention. Differentregions of tumors may have distinct metabolic characteristics, andthe cellular metabolic pathways in the TME are dynamic and constantlyevolving at different stages of tumor development and progression.195 Combination therapies that integrate nanoenabled metabolic interventionswith other treatment modalities, such as molecular-targeted therapy,immunotherapies, chemotherapy, and irradiation, offer potential solutionsto address these challenges. Fourthly, restricted accumulation andpenetration of nanomedicines196 in tumortissues may compromise the therapeutic effectiveness of tumor metabolism-regulatingnanomedicines. The therapeutic strategy combining stromal desmoplasiadepletion with TMME manipulation has been proposed to improve thespatial distribution of tumor metabolism-regulating nanomedicine.168 However, the suboptimal spatial distributionof these nanomedicines within tumor sites arises from the collectiveinfluence of multiple factors, such as the interplay of interstitialfluid pressure (IFP), dysregulated vasculature networks, dense ECM,and solid stress within the TME.197 Toovercome these obstacles, more efforts are needed to develop tumormetabolism-regulating nanomedicines that could simultaneously regulatethe tumor metabolism and the physically complex TME.Despite the fact that this emerging field hasachieved significantadvances, several major challenges must be overcome before successfulclinical translation can occur. First, increasing evidence demonstratesthat tumor cells can develop compensatory metabolic pathways, enablingthem to acquire resistance against the single intervention targetingtumor metabolism.7198 A better and in-depth understandingof these intricate tumor metabolic profiles and molecular immune networkswould identify vulnerabilities that can be targeted for therapeuticinterventions and promote the rational design of nanotherapeuticsfor tumor immunometabolic therapy. (2) Current nanotherapeutics havebeen developed to improve antitumor immunity by regulating the metabolismof cancer cell and TIICs, but have rarely considered cancer stem cells(CSCs). CSCs not only promote tumor recurrence and metastasis butalso affect antitumor immune responses.199 However, in comparison to high-glycolytic cancer cells, CSCs commonlyexploit the OXPHOS for energy supply. Therefore, developing nanodrugsfor simultaneously regulating the metabolic properties of CSCs andcancer cells would be a promising strategy. (3) Cancer cells dynamicallyalter their metabolism to facilitate metastasis, leading to the variationsin cellular metabolism between primary tumor and metastatic sites.200 For example, single-cell gene sequencing ofbreast cancer patient-derived xenografts indicates that micrometastasesexhibit higher expression of OXPHOS pathways than the primary tumorswhich have increased levels of glycolytic enzymes.201 Therefore, developing nanomedicine that can simultaneouslyregulate cancer metabolism in primary tumors and micrometastases hasthe potential to enhance antitumor immunity against both primary andmetastatic cancer.To makefurther progress in the development of nanomedicine forregulating tumor metabolism, it is crucial to consider various aspectsbeyond the aforementioned challenges. (1) TMME emerges as a complicatedsystem. The metabolic interdependence of TIICs and cancer cells, aswell as the interdependence between different types of immune cellswithin the TME remains an underexplored area. In addition, there isa complex crosstalk between the metabolism of glucose, amino acids,lipids, and nucleotides in the TME. Typically, the metabolites fromtumor glycolysis can serve as a prominent carbon source for lipidsynthesis."} +{"text": "This cross-sectional study analyzes Medicare Advantage surveys to compare Medicare and Medicaid dual-eligible individuals\u2019 experiences with care across 3 established categories of plans. Coordination-only D-SNPs (60.6% of D-SNPs in 2023) provide limited care coordination but do not manage Medicaid spending. Fully integrated D-SNPs (FIDE-SNPs) (8.0% of D-SNPs) have capitation contracts to manage Medicaid long-term care and behavioral health care spending.We analyzed MA Consumer Assessment of Healthcare Providers and Systems (CAHPS) surveys to compare dual-eligible individuals\u2019 experiences with care across 3 categories of plans: coordination-only D-SNPs, FIDE-SNPs, and non\u2013D-SNP MA plans.STROBE reporting guideline and was deemed exempt with a waiver of informed consent from the Brown University Institutional Review Board for its use of secondary data.We analyzed respondent-level MA-CAHPS data from 2015-2018 for dual-eligible individuals with full Medicaid . MA-CAHPS assesses respondent ratings of health plans and patient experiences with care in domains such as coordination. This cross-sectional study met the We used administrative data linked to MA-CAHPS respondents to identify enrollees in coordination-only D-SNPs, FIDE-SNPs, and non\u2013D-SNP MA plans in the survey month. Respondents with more than 100 days of nursing home care in the survey year and in institutional and chronic condition SNPs were excluded.Outcomes were 6 composite and 3 single-item measures from MA-CAHPS following CMS protocol for performance measurement and getting needed prescription drugs but higher ratings of their plan , prescription drug coverage , getting appointments and care quickly , customer service , and care quality rating .Respondents in FIDE-SNPs reported significantly higher ratings than those in coordination-only D-SNPs for plan rating and health care quality rating . Differences between FIDE-SNPs and coordination-only D-SNPs in other outcomes were not statistically significant.6 As D-SNPs evolve, continued monitoring is needed to ensure these plans provide increased value for dual-eligible individuals.In this cross-sectional study, we found that FIDE-SNPs performed better than non\u2013D-SNP MA plans in some domains but worse on domains including care coordination. Furthermore, FIDE-SNPs generally did not perform better than coordination-only D-SNP plans. The findings highlight some benefits in patient experience associated with enrollment in FIDE-SNPs and an opportunity to improve patient experience in these plans. This is particularly salient in areas such as care coordination, where integration could be improved. Study limitations included potential confounding from unmeasured enrollee differences across plans and analysis of the years preceding stronger federal regulations governing D-SNPs."} +{"text": "The evolution of classification of the idiopathic inflammatory myopathies (IIM) is fueled by myositis-specific antibodies (MSA), clinicopathological features, and discoveries in the \u201c-omics\u201d fields such as proteomics and transcriptomics. The major IIM in the current classification include dermatomyositis (DM), immune-mediated necrotizing myopathy (IMNM), antisynthetase syndrome (ASS), and inclusion body myositis (IBM) , 2. SARS18F)-labeled fluorodeoxyglucose PET (18FDG-PET) can identify patterns of muscle involvement, and detect concurrent interstitial lung disease and malignancy in IIM via glucose metabolism (11C-labeled Pittsburgh compound B-PET (PIB-PET) , ultrasonography, and positron emission tomography (PET) can identify typical patterns of individual skeletal muscle involvement and disease activity \u201316. Patttabolism . 11C-labPIB-PET) and 18F-a in IBM . The usea in IBM . Electria in IBM . The artWang and Liang raises awareness of and discusses current information on juvenile IMNM.The most prevalent IIM in juvenile patients is juvenile DM followed by IMNM (2.9\u201321% of juvenile IIM) and rarely ASS; IBM is not present in this age group. Without serological information, juvenile IMNM risks being misdiagnosed as jDM due to its association with skin lesions or as inherited myopathy due to insidious muscle weakness and refractoriness to steroid treatment. Misdiagnosis and delayed proper treatment in IMNM likely lead to unreversible muscle damage. A recent article by MuRF1, a muscle-specific E3 ubiquitin ligase-associated muscle atrophy gene) is upregulated in atrophic myotubes induced by in vitro anti-SRP and anti-HMGCR antibodies treatment expression; the finding is currently recognized as one of the diagnostic criteria for DM . The IFNreatment . Althoug atrophy , 29, YanNiedzielska et al.. expands the spectrum of concurrent anti-Mi-2 DM in patients with SARS-CoV-2 infection whose condition improved after corticosteroid therapy. Whether SARS-CoV-2 infection directly induced myositis or triggered pre-existing asymptomatic conditions in these single case reports are debatable.COVID-19-associated myopathy is likely caused by an immune-mediated mechanism rather than direct SAR-CoV-2 infection. This speculation is supported by autopsy studies in critically ill COVID-19 patients with myositis symptoms showing prominent myopathology without viral protein expression , 31 and iR-myositis is often associated with oculobulbar weakness, myocarditis, anti-acetylcholine receptor (anti-AChR) antibody, anti-striational antibodies, and MSA \u201335. ThisIIM consists of evolving heterogeneous entities which require multimodality approaches for diagnosis and appropriate management.JT: Conceptualization, Writing\u2014original draft. MN: Writing\u2014review and editing. TM: Writing\u2014review and editing. WS: Writing\u2014review and editing. IN: Writing\u2014review and editing."} +{"text": "Global health efforts have increased against infectious diseases, but issues persist with pathogens like Group B Streptococcus (GBS). Preclinical studies have elaborated on the mechanistic process of GBS-induced chorioamnionitis and its impact on the fetal programming of chronic neuropsychiatric diseases. GBS inoculation in rodents demonstrated the following: (i) silent and self-limited placental infection, similar to human chorioamnionitis; (ii) placental expression of chemokines attracting polymorphonuclear (PMN) cells; (iii) in vitro cytokine production; (iv) PMN infiltration in the placenta , linked to neurobehavioral impairments like cerebral palsy and autism spectrum disorders (ASD); (v) upregulation of interleukin-1\u03b2 (IL-1\u03b2) in the placenta and fetal blood, associated with higher ASD risk in humans; (vi) sex-specific effects, with higher IL-1\u03b2 release and PMN recruitment in male placenta; (vii) male offspring exhibiting ASD-like traits, while female offspring displayed attention deficit and hyperactivity disorder (ADHD)-like traits; (viii) IL-1 and/or NF-kB blockade alleviate placental and fetal inflammation, as well as subsequent neurobehavioral impairments. These findings offer potential therapeutic avenues, including sex-adapted anti-inflammatory treatment treatment). Blocking the IL-1 pathway offers therapeutic potential to alleviate chorioamnionitis-related disabilities, presenting an opportunity for a human phase II RCT that uses IL-1 blockade added to the classic antibiotic treatment of chorioamnionitis. Streptococcus (GBS) and neurobehavioral disorders in the offspring , a synthetic analog of viral ribonucleic acid (double-stranded RNA), acting through TLR-3 ,8,386,8,Beyond GBS-induced MIA, fetal brain injuries have been studied using different infectious causes of chorioamnionitis, as well as different rodent species. For instance, rat models of LPS-induced chorioamnionitis show the activation of the maternal pro-inflammatory cytokine profile ,58,59. CThe studies profiling the neurobehavioral impact of GBS-induced maternofetal inflammation were summarized in Changes at the levels of neural structure and function have behavioral implications. Decreases in volumes of periventricular WM, including the corpus callosum and external capsule, were found in the rat offspring exposed to GBS . There wMagnetic resonance imaging (MRI) and in situ analysis revealed a significant enlargement of the lateral ventricles in male rat offspring following in utero exposure to formaldehyde-killed GBS . Data frIn addition, as shown after in utero exposure to inactivated GBS in a preclinical model, the fronto-temporal circuits, located within the abnormally thinner external capsule adjacent to the lateral ventricles, likely contribute to their enlargement . Notably0) and 276 will receive Anakinra over the first 21 days of birth, and the frequency of adverse outcomes/events will be monitored [The identification of TLR2/6 and \u03b2-hemolysin/(NLR)-P3 pathways, as well as IL-1, as key mediators in the inflammatory response triggered by GBS-induced sepsis has prompted clinical trials of anti-inflammatory interventions to protect maternofetal organs. In preclinical models of chorioamnionitis triggered by GBS and LPS, the IL-1 blockade has already demonstrated placenta- and feto-protective effects ,73. Of ponitored ,75. A syonitored . While t"} +{"text": "Soc Psychiatry Psychiatr Epidemiol 2020; 55(5):645-657). However, identifying the genetic variants involved and how they interact with environmental risk factors underlying psychosis remains challenging.Gene-environment interactions increase psychosis risk (Gayer-Anderson DRD2), N-methyl-d-aspartate receptor and cannabinoid receptor type 1 (CB1R: CNR1) with psychosis.To investigate whether there are gene-environment interactions in the relationships of childhood trauma, lifetime cannabis use, and single nucleotide variants (SNVs) of dopamine D2 receptor :726-729), part of the EU-GEI consortium :645-657), 143 first-episode psychosis patients and 286 community-based controls of both sexes aged between 16 and 64 years were included over a period of 3 years. Twenty-three SNVs of D2R , NMDAR , and CB1R genes , were genotyped from peripheral blood DNA using a custom Illumina HumanCoreExome-24 BeadChip. Environmental adversities were evaluated using the Cannabis Experience Questionnaire :427\u2013436) and the Childhood Trauma Questionnaire :249-55). Associations between SNVs and environmental risk factors were performed using the nonparametric multifactor dimensionality reduction software (version 3.0.2).In a population-based case-control study nested in an incident study (Del-Ben CNR1. The best association models were the two-factor representing by the combination of CNR1 rs12720071 with lifetime cannabis use (p<0.001), and CNR1 rs12720071 with childhood trauma (p<0.05), both suggesting an increased risk of psychosis. Additionally, when considering the interaction of both environmental factors in the same model, we found CNR1 rs7766029 to be associated with psychosis (p<0.001).Single locus analysis showed no association among the 23 SNVs with psychosis; however, gene-environment analysis was significant for the polymorphic loci rs12720071 and rs7766029 in CNR1 SNVs (rs12720071 and rs7766029), childhood trauma and lifetime cannabis use in psychosis.Our study supports the hypothesis of gene-environment interactions for psychosis involving the T allele carriers of None Declared"} +{"text": "Oesophageal adenocarcinoma (OAC) is now the predominant subtype of oesophageal cancer in Western countries and its incidence is rapidly increasing due to rising levels of obesity . OAC privia tumor cell-intrinsic signaling these inhibitory immune checkpoint receptors can promote various hallmarks of cancer in OAC cells such as a cancer stem-like phenotype, DNA damage repair or 6 weeks post-surgery. The use of PD-1 blockade treatment enhanced lymphocyte-mediated killing of OE33 cells and increased production of anti-tumor cytokines in lymphocytes, overcoming the surgery-mediated suppression of lymphocyte cytotoxicity. This phenomenon was similarly observed in renal cell carcinoma patients who received adjuvant pembrolizumab or placebo and the progression-free survival at 24 months was 77.3% vs. 68.1% , pre-malignant Barrett\u2019s oesophagus (BO) cells (QH cells) and OAC cells (OE33 and OE19 cells) were examined . Proteinin vitro . It is vin vitro and cervin vitro , wherebyin vitro or dendrin vitro promotesin vitro . Strategin vitro . If TIGI+CTLA-4+ cells correlated with advanced stage disease and a poor response to neoadjuvant chemo(radio)therapy regimens in OAC patients to mimic the inhospitable tumor microenvironment (OE33 and OE19 cells were cultured under glucose deprivation and hypoxic conditions (0.5% Oironment . Under sironment . To testironment . These pironment .Like TIGIT, PD-1 expression on OE33 and OE19 cells reportedly increased under glucose deprived hypoxic conditions . ConsideEmerging evidence in cancer types outside of OAC suggest that the incidence of immune-related adverse events following treatment with ICB might be associated with clinical outcomes, this has yet to be elucidated in OAC . HoweverSeveral trials testing the effectiveness of anti-PD-1 as a dual checkpoint approach with novel ICBs such as anti-TIGIT, anti-LAG-3 or anti-TIM-3 are ongoing. The results are eagerly awaited with anticipation that these novel ICB combinations will represent the next generation of immunotherapies to benefit OAC patients.MD and NED conceptualized and wrote the manuscript. All authors contributed to the article and approved the submitted version."} +{"text": "The pathology of persistent post-COVID-19 symptoms remains poorly understood. We examined radiographic and physiological correlates in those with ongoing post-COVID-19 dyspnea compared to those with resolved dyspnea.The Epidemiology, Immunology, and Clinical Characteristics of Emerging Infectious Diseases with Pandemic Potential (EPICC) is a COVID-19 cohort study of Military Health System (MHS) beneficiaries. Study participants aged 18-65, with no pre-existing significant cardiopulmonary disease, and respiratory symptoms \u2265 3 months after COVID-19 onset were enrolled into the ChIPS sub-study as cases. Controls with resolved post-COVID-19 symptoms were also enrolled. Each participant underwent high resolution chest CT (HRCT), transthoracic echocardiography (TTE), electrocardiogram (ECG), full pulmonary function testing (PFT), impulse oscillometry (IOS), and a six minute walk test (6MWT) with Borg dyspnea scale.1/FVC, FVC and FEV1 was within normal ranges for both cases and controls, though mean FEV1/FVC was higher in those with persistent dyspnea (Table 2). DLCO and IOS were similar between groups (Table 2). Cases had a decreased 6MWT distance and higher post-6MWT Borg scores compared to controls (Table 2). There were no significant differences in TTE results between groups. Variable ECG changes were seen in both groups with no statistically significant differences. HRCT findings are currently being analyzed.There were 115 participants enrolled in the ChIPS sub-study, of whom 39 had persistent dyspnea (cases) and 76 had resolved dyspnea (controls) (Table 1). There was no statistically significant difference in age, sex, comorbidity index, or infecting variant between cases and controls. Cases were more likely to be unvaccinated at the time of initial infection. The average FEVWe noted a small decrease in 6MWT distance and increased post-exertional Borg scores in those with persistent post-COVID symptoms; these should be explored as interventional study endpoints. PFT, IOS, TTE, and ECG findings were similar between groups. Those with vaccine-breakthrough infections were less likely to have persistent dyspnea.Mark P. Simons, PhD, AstraZeneca: The IDCRP and HJF were funded to conduct an unrelated phase III COVID-19 monoclonal antibody immunoprophylaxis trial as part of US Govt COVID Response David Tribble, MD, DrPH, AstraZeneca: The IDCRP and HJF were funded to conduct an unrelated phase III COVID-19 monoclonal antibody immunoprophylaxis trial as part of US Govt COVID response Timothy Burgess, MD, MPH, AstraZeneca: The IDCRP and the Henry M. Jackson Foundation (HJF) were funded to conduct an unrelated phase III COVID-19 monoclonal antibody immunoprophylaxis trial Simon Pollett, MBBS, AstraZeneca: The IDCRP and the Henry M. Jackson Foundation (HJF) were funded to conduct an unrelated phase III COVID-19 monoclonal antibody immunoprophylaxis trial Michael Morris, MD, Janssen Pharmaceuticals: Paid speaker"} +{"text": "Administration of a dual mammary tumor-targeting piRNA delivery system in mice reduced tumor growth in vivo. RNA-seq, ChIP-seq and luciferase reporter assays demonstrated piR-2158 as a transcriptional repressor of IL11 by competing with AP-1 transcription factor subunit FOSL1 to bind the promoter of IL11. STAT3 signaling mediated piR-2158-IL11 regulation of cancer cell stemness and tumor growth. Moreover, by co-culturing of MDA-MB-231 and HUVECs in vitro and CD31 staining of tumor endothelial cells in vivo, we demonstrated inhibition of angiogenesis by piR-2158-IL11 in breast cancer. In conclusion, the current study not only reveals a novel mechanism through which piR-2158 inhibits mammary gland tumorigenesis via regulating cancer stem cells and tumor angiogenesis, but also provides a novel therapeutic strategy in treatment of breast cancer.Emerging evidence has indicated the aberrant expression of PIWI-interacting RNAs (piRNAs) in human cancer cells to regulate tumor development and progression by governing cancer cell stemness. Herein, we identified downregulation of piR-2158 in human breast cancer tumors, especially in ALDH+ breast cancer stem cells (BCSCs) from patients and cell lines, which was further validated in two types of genetically engineered mouse models of breast cancer (MMTV-Wnt and MMTV-PyMT). Enforced overexpression of piR-2158 in basal-like or luminal subtypes of breast cancer cells suppressed cell proliferation, migration, epithelial-mesenchymal transition (EMT) and stemness P-element-induced wimpy testis (PIWI)-interacting RNAs (piRNAs) are a class of small non-coding RNAs with ~30 nt in length, which were highly enriched and first discovered in germ cells Our previous small RNA-seq screening study identified a group of piRNAs in breast cancer cells, and demonstrated their involvement in regulation of breast cancer stem cell (CSCs). For example, we demonstrated the regulation of CSCs by piR-016658 in the estrogen receptor (ER) negative basal-like subtype of breast cancer lowCD44high and/or aldehyde dehydrogenase 1 (ADLH1) positive CSCs are a small population of cancer cells with high heterogeneity and strong ability to regenerate tumors. In breast cancer, CSCs were identified using biomarkers CD24Angiogenesis is an essential step in tumor development and progression, and is required for invasive tumor growth and distant metastasis in vitro and suppressed tumorigenicity and angiogenesis in vivo. The mechanism study demonstrated significant inhibition of IL11 expression by piR-2158 via competing with AP-1 transcription factor subunit FOSL1 to bind the promoter of IL11. Downregulation of IL-11 inactivated the JAK-STAT signaling pathway, leading to suppression of tumorigenesis. These findings not only reveal the regulatory mechanism of piR-2158 in suppressing breast cancer stem cells, but also suggest a novel therapeutic strategy in treatment of breast cancer.Our current study confirmed downregulation of piR-2158 in breast cancer. Overexpression of piR-2158 inhibited cell proliferation, migration, invasion and stemness Human breast tumor samples. Human breast cancer tumor samples and adjacent normal tissues were collected from Tongji University Shanghai East Hospital. All the procedures were approved by the Institutional Review Board (IRB) of Shanghai East Hospital. All patients were provided with written informed consent form.Animals. 6-week-old female nude mice and BALB/c mice were purchased from the Silaike Animal Company for in vivo assays. Mammary tumors from MMTV-Wnt or MMTV-PyMT transgenic mice were prepared by Dr. Suling Liu's lab. All animal studies were approved by the Institutional Animal Care and Use Committee of the Tongji University School of Medicine.Cell lines and cell culture. Human breast cancer cell line MDA-MB-231, mouse breast cancer cell line 4T1, human umbilical vascular endothelial cells (HUVECs) and human embryonic kidney 293T (HEK293T) cells were originally purchased from ATCC and maintained in our lab, and cultured in dulbecco's modified eagle medium (DMEM) or endothelial cell medium (ECM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin and 1% endothelial cell growth when needed. Lung metastatic MDA-MB-231 sublines 4173 and 4175 were presented by Dr. Guohong Hu at the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All of these cells were cultured at 37 \u2103 in a humidified environment with 5% CO2.Oligos and vectors. All oligos for small RNAs and DNA primers were synthesized by GenScript . Oligo transfection was performed using RNAiMAX (Invitrogen) following the manufacturer's instructions with a final concentration of 30 nM. Lentivirus vector LV3(H1/GFP&Puro)-piR-2158 was purchased from GenePharma , using a sequence not homology to any known mammalian gene as negative control (NC). The FOSL1 coding sequence was amplified from the cDNA of MDA-MB-231 cells and ligated into pcDNA3.1(+) at the BamHI and HindIII enzyme digestion sites. All target sequences are listed in ALDH assay. Single cell suspensions were prepared from breast cancer tumor following a protocol described in our previous publication Cell Counting Kit-8 (CCK-8) assay. 1x103 cells/well were seeded into 96-well plates in eight repeats. After incubation for the indicated time, the cells were stained with 10 \u00b5L CCK-8 solution per well for 3 h under cell culture conditions, followed by the OD measurement at 450 nm.Wound healing assay. 2x104 cells/well were seeded into 12-well plates in triplicates. After the cell density reached 95%, FBS-reduced DMEM medium (0.1% FBS) was applied to starve the cells for 24 h, followed by creating a vertical wound in each well using a 10 \u00b5L pipette tip. The wound widths were photographed and quantified under microscope (Zeiss) at the indicated time points. Six fields were randomly selected for statistical analysis using Image J software.Cell invasion assay. Transwell chambers with 8-\u03bcm pores were pre-coated with Matrigel , and placed in a 24-well plate containing cell culture medium. 2 x 104 pre-starved cells were seeded in the chambers with serum-free medium, followed by 6 hours' incubation at 37 \u00b0C and 5% CO2. Cells adherent to the upper surface of chambers were removed using cotton swabs. Chambers containing invaded cells were stained with 0.4% violet crystal acetate overnight. Six fields were randomly selected for photography using a microscope (Zeiss). The number of invaded cells was counted for statistical analysis.Quantitative real-time PCR (qRT-PCR) analysis of piRNA and mRNA. Quantitative analysis of piRNAs and mRNAs were performed following our previous publication Western blot assay. The following primary antibodies were used for western blot : anti-Slug , anti-Vimentin , anti-Fibronectin , anti-ZEB1 , anti-KLF4 , anti-NANOG , anti-SOX2 , anti-OCT4 , anti-IL-11 , anti-STAT3 , anti-Phospho-STAT3 (Tyr705) , anti-GAPDH and anti-\u03b2-actin . HRP-linked anti-rabbit IgG and HRP-linked anti-mouse IgG were used as secondary antibodies . Three independent experiments were performed for statistical analysis.Differently expressed genes (DEGs) screening and pathway enrichment analysis. MDA-MB-231 cells with or without overexpression of piR-2158 (n = 3 in each group) were applied RNA-seq analysis using BGISEQ sequencing platform. The abundance of each gene was quantified as TPM (Transcripts per million) value for differential analysis. 107 differently expressed genes (DEGs) were identified using the absolute value of fold change (FC) greater than 2 and p-value less than 0.05 as cutoff values.Functional enrichment analysis of 107 differently expressed genes (DEGs) was performed by Over-Representation Analysis (ORA) with Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Gene Set Enrichment Analysis (GSEA) with WikiPathway using web-based gene set analysis toolkit .Enzyme linked immunosorbent assay (ELISA) for secretory IL-11. Quantitative analysis of IL-11 in the supernatants of breast cancer cells was performed using the Human IL-11 ELISA Kit following the manufacturer's instruction.Immunohistochemistry (IHC) & Immunofluorescence (IF) staining. IHC and IF staining were performed following our previous publication HUVECs tube formation assay. HUVECs were cultured using the conditioned medium from breast cancer cells MDA-MB-231 cells with or without overexpression of piR-2158. Exogenous recombinant human protein IL-11 were added into the medium at a final concentration of 30 ng/mL as indicated. For the tube formation assay, precooled 96-well plates were coated with matrigel for 60 min in a cell culture incubator, then seeded with 3 \u00d7 104 HUVECs in each well and cultured in the conditioned medium with or without addition of exogenous rHu IL-11. Tube formation was photographed using a microscope at 12 h intervals. Tube formation ability was quantified by measuring the cumulative tube length in five random fields under a microscope. IL11 Promoter activity.Luciferase reporter assay for theIL11 promoter was amplified from the genomic DNA of MDA-MB-231 cells and ligated into pGL3-promoter vector at the KpnI and XhoI enzyme digestion sites. 1 x 104 HEK-293T cells were seeded in 24-well plates. When the cell density reached 80%, co-transfection was performed using 1 \u00b5g pGL3-promoter-IL-11, 1 \u00b5g pcDNA3.1(+)-FOSL1 and 20 pmol piR-2158 mimic per well. Lipofectamine 2000 was used as the transfection reagent. In 24 h after transfection, luciferase activities were measured using the Luciferase Reporter Assay Kit .Preparation of mammary tumor mice. 1 x 106 4T1 or MDA-MB-231 cells with or without overexpression of piR-2158 were mixed with matrigel and injected into the fat pad of the fourth mammary gland of BALB/c or nude mice (n = 10 for each group), respectively. From one week after cell transplantation on, the tumor volumes were measured every 2 days until the mice were sacrificed at the indicated timepoint. The tumor growth curves were plotted. All tumors were weighted and applied for further analysis.Administration of magnetic nanoparticles pre-loaded with piR-2158 or NC mimic (piR-2158/NC-MNPs). Zn0.4Fe2.6O4@SiO2 nanoparticles were coated with hyaluronic acid (HA), pre-loaded with piR-2158 or NC mimic, and self-assembled following the published procedure described in our previous publication Public data analysis. TCGA database were used to analyze the gene expression levels and survivals of breast cancer patients using R packages through Xiantao online tools (https://www.xiantao.love/). For IL11 survival curve analysis, surv_cutpoint function in survminer package was first used for optimal group cut-off screening. Then survival package was used to test the proportional risk hypothesis and fitted survival regression. The results were visualized using survminer package and ggplot2 package. ChIP-seq data of transcription factor FOSL1 in MDA-MB-231 and BT549 was obtained from Cistrome DB database (http://cistrome.org/db/#/) and analyzed using the online website .Statistical analysis. Data are presented as mean \u00b1 SEM unless otherwise stated. The two-tailed student's t test was used in statistical comparisons. p < 0.05 was considered statistically significant.Downregulation of piR-2158 in human and rodent breast cancer tumors. Our previous small RNA screening study indicated involvement of a group of piRNAs in regulation of breast cancer, including piR-2158 were end linked with adapters for amplification. RNA-seq analyses were applied using BGISEQ sequencing platform. The abundance of each gene was quantified as TPM (Transcripts per million) value. The absolute value of fold change (FC) greater than 2 and p-value less than 0.05 were set for cutoff. 107 differently expressed genes (DEGs) were identified including interleukin 11 (IL11) A. Suppres Figure B, 4C. Ens Figure . The xenIL11 transcription by competing with FOSL1.piR-2158 inhibited To determine the mechanism for piR-2158 to suppress IL11, the Basic Local Alignment Search Tool (BLAST) was applied. Two potential binding sites of piR-2158 were identified in the promoter region of IL11 or the two bindings sites (F2) -coated and piR-2158-preloaded magnetic nanoparticles (MNPs) Figure A. It was) Figure B, tumor ) Figure C and tum) Figure D. Highers Figure E and 7F.in vitro and in vivo assays demonstrated the promise of piR-2158 as a therapeutic target in breast cancer.The major clinical challenges for breast cancer include tumor relapse, distant metastasis and drug resistance, which were all related with CSCs. Development of effective therapeutic strategies targeting CSCs will lead to a new era in the fight against cancer. Involvement of non-coding RNAs in regulation of breast cancer and breast CSCs has been well demonstrated, including miRNAs piR-2158, also known as DQ572892, piR-2980, piRNA-21067, piR-41004, piR-3200, piR-34003 by different databases, is located at the minus chain of chromosome 16. piR-2158 regulation of stem cells has been reported in germline via recruiting DNA methyltransferase 1 (DNMT1) to its promoter region adenomatous polyposis coli (APC) ARHGAP11AIL11 in human breast cancer cells.Epigenetic regulation is a common mechanism for piRNAs in germ cells, and tumors as well. For example, piR-651 promoted cell proliferation and migration in breast cancer by suppressing Pten via FOSL1 signaling has been reported by Nishina et alFOSL1 gene expression, thereby promoting the expression of IL11 in ulcerative colitis patients IL11 inhibition by piR-2158 via competing with FOSL1 in breast cancer cells. The literature has demonstrated activation of STAT3 by IL-11 in carcinogenesis. IL-11 regulates endometrial cancer cell adhesion and migration via upregulating the phosphorylated (p)-STAT3 protein abundance FOSL1 is one of the main AP-1 family transcription factors with diverse functions. FOSL1 induces EMT and carcinogenesis et al. fabricated Poly- (PLGA)-based nanoparticles coated with human cancer cell membrane fractions to target cancer cells by interacting with membrane-associated receptors CXCR4 and CD44 Chemo therapy and immunotherapy, as two most popular approaches in treatment of cancer patients The uptake of nanoparticles by breast CSCs is both HA and CD44 dependent. HA specially binds and interacts with the cell surface receptor CD44, leading to cancer cell growth and survival Supplementary figures and tables.Click here for additional data file."} +{"text": "When the U.S. COVID-19 public health emergency declaration expires on May 11, 2023, national reporting of certain categories of COVID-19 public health surveillance data will be transitioned to other data sources or will be discontinued; COVID-19 hospitalization data will be the only data source available at the county level Figure).\u00b6https://stacks.cdc.gov/view/cdc/127731). Most discordant levels were reported during periods of high COVID-19 incidence during February and March 2022. When the levels were discordant, CCLs exceeded the hospital admission levels.A comparison of CCL and COVID-19 hospital admission level designations by week during February 2022\u2013March 2023 demonstrated >99% concordance among 3,220 counties and reporting delays. Further, retrospective findings do not account for reporting lags affecting recent data or potential future changes to reporting cadence , including for hospitalization data (COVID-19 hospital admission rates from NHSN are a timely and suitable primary indicator for monitoring trends in COVID-19 activity. Using the percentage of COVID-19 deaths from NVSS will allow more timely monitoring of COVID-19 severity and mortality trends. The percentage of COVID-19 ED visits and percentage of positive test results can serve as early indicators for COVID-19 trend monitoring. Collectively, these surveillance data sources and indicators can support monitoring of the impact of COVID-19 and related prevention and control strategies as ongoing public health priorities.COVID-19 monitoring will remain a public health priority after the U.S. public health emergency declaration expires on May 11, 2023.Assessment of available surveillance indicators found that COVID-19 hospital admission levels were concordant with COVID-19 Community Levels. COVID-19\u2013associated hospital admission rates lagged 1 day behind case rates and 4 days behind percentages of COVID-19 emergency department visits and positive SARS-CoV-2 test results. National Vital Statistics System trends in the percentage of COVID-19 deaths strongly correlated with, and were 13 days timelier, than aggregate death count data.Rates of COVID-19\u2013associated hospital admission and the percentages of positive test results, COVID-19 emergency department visits, and COVID-19 deaths are suitable and timely indicators of trends in COVID-19 activity and severity."} +{"text": "Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) matAbs are efficiently transferred during pregnancy and protect infants against subsequent SARS-CoV-2 infections. It is unknown if matAbs inhibit immune responses elicited by different types of SARS-CoV-2 vaccines.Maternal antibodies (matAbs) protect against a myriad of pathogens early in life; however, these antibodies can also inhibit We established a mouse model to determine if SARS-CoV-2 spike (S)-specific matAbs inhibit immune responses elicited by recombinant protein and nucleoside-modified mRNA-lipid nanoparticle (mRNA-LNP) vaccines. Mouse dams were vaccinated with SARS-CoV-2 S protein-encoding mRNA-LNP vaccine (or vehicle) in early pregnancy and pups were born and matAbs quantitated in pup serum by ELISA. Pups with and without matAbs were vaccinated at weaning with recombinant S protein (adjuvanted with Addavax or empty LNP) or mRNA-LNP and serum S-specific IgG quantitated over time.de novo antibody responses in mouse pups in the presence of matAbs. Recombinant protein vaccines were also able to circumvent the inhibitory effects of matAbs when co-administered with Addavax or empty LNP as adjuvants.S-specific matAbs were transferred to pups and decayed over time as expected. We found that SARS-CoV-2 mRNA-LNP vaccines elicited robust While additional studies need to be completed in humans, our results raise the possibility that mRNA-LNP-based and adjuvanted protein-based SARS-CoV-2 vaccines have the potential to be effective when delivered in the presence of matAbs.Reihaneh Hosseinzadeh, MASc, Acuitas Therapeutics: Employee Drew Weissman, MD, PhD, Intellectual Property/Patents: D.W. is named on the first patent describing nucleoside-modified mRNA and on patents describing its use as a treatment and vaccine platform. Scott Hensley, PhD, Intellectual Property/Patents: SEH is named on patents that describe the use of nucleoside-modified mRNA as a platform to deliver therapeutic proteins and as a vaccine platform"} +{"text": "Data supporting high tumor mutational burden (TMB-H) as a lone biomarker for an immune-responsive tumor microenvironment (TME) in metastatic breast cancer (MBC) are weak, yet tumor agnostic approval in TMB-H advanced tumors provides immune checkpoint inhibition (ICI) as a clinical option. We evaluated concurrent predictors of immune-responsive and non-responsive TME within MBC.Tumor samples from patients with MBC (N=5621) were analyzed by next-generation sequencing of DNA (592-gene panel or whole exome) and RNA (whole transcriptome) at Caris Life Sciences . TMB-H threshold was set to \u2265 10 muts/Mb. PDL-1 was evaluated using SP142 antibody. Gene expression profiling and RNA deconvolution were used to estimate immune and stromal cell population abundance in the TME, and transcriptomic signature of immunotherapy response (T cell-inflamed score).B2M mutations and CD274 amplifications were positively associated with T-cell inflamed scores in TMB-H tumors; CDH1 and ERBB2 mutations were negatively associated.461 (8.2%) TMB-H MBC samples were identified. Consistent with prior studies, TMB-H tumors exhibited significant dMMR/MSI-H enrichment and PD-L1+ expression compared to TMB-L. Across all samples, T cell-inflamed scores were weakly correlated with TMB. TMB-H was not associated with significantly increased immune responsive cell types or immune response gene signatures (e.g. antigen presentation), yet positive trends were observed, while immunosuppressive fibroblasts were significantly decreased in TMB-H tumors . HR+/HER2- breast cancer was the only subtype in which TMB-H tumors exhibited increased T cell-inflamed scores vs. TMB-L. Concurrent PD-L1+ or dMMR/MSI-H with TMB-H was associated with high T cell-inflamed scores in both HR+/HER2- and TNBC. Among several associated biomarkers, B2M mutation and CD274 amplification, may help predict ICI benefit amongst TMB-H tumors. Co-occurring biomarkers within TMB-H breast cancer warrant evaluation in larger cohorts for response or resistance to ICI to develop composite predictive biomarkers in MBC.High TMB alone does not strongly correlate with immune infiltrate or immune-related gene signatures in MBC. TMB-H predicts T-cell inflamed signature compared to TMB-L in HR+/HER2- tumors only. Along with MSI-H and PD-L1+, several biomarkers, including The U.S. Food and Drug Administration (FDA) approved pembrolizumab on June 16, 2020, for the advanced TMB-H (\u226510 mutations/megabase (mut/Mb), as determined by an FDA-approved test) solid tumors that have progressed following prior treatment with no satisfactory alternative treatment options. This approval was based on an overall response rate of 29% in the subset of patients with TMB-H solid tumors samples from patients with breast cancer (n=5621) were submitted by various academic and community cancer institutes, predominately in the United States, to a commercial CLIA-certified laboratory for molecular profiling . The present study was conducted in accordance with the guidelines of the Declaration of Helsinki, Belmont Report, and U.S. Common Rule. In compliance with policy 45 CFR 46.101(b), this study was conducted using retrospective, de-identified clinical data, patient consent was not required, and the study was considered IRB exempt.NGS of 592 cancer-relevant genes was performed on genomic DNA isolated from formalin-fixed paraffin-embedded (FFPE) tumor samples using the NextSeq platform . Matched normal tissue or germline DNA was not sequenced. A custom-designed SureSelect XT assay was used to enrich exonic regions of 592 whole-gene targets . All variants were detected with >99% confidence based on allele frequency and amplicon coverage, with an average sequencing depth of coverage of >500 and an analytic sensitivity threshold established of 5% for variant calling. Prior to molecular testing, tumor enrichment was achieved by harvesting targeted tissue using manual microdissection techniques. Genomic variants were classified by board-certified molecular geneticists according to criteria established by the American College of Medical Genetics and Genomics (ACMG). When assessing mutation frequencies of individual genes, \u2018pathogenic\u2019 and \u2018likely pathogenic\u2019 were counted as mutations, while \u2018benign\u2019, \u2018likely benign\u2019 variants, and \u2018variants of unknown significance\u2019 were excluded.RNA Whole Transcriptome Sequencing (WTS) uses a hybrid-capture method to pull down the full transcriptome from FFPE tumor samples and the Illumina NovaSeq platform . FFPE specimens underwent pathology review to discern the percent tumor content and tumor size; a minimum of 20% tumor content in the area for microdissection was required to enable enrichment and extraction of tumor-specific RNA. A Qiagen RNA FFPE tissue extraction kit was used for extraction, and the RNA quality and quantity were determined using the Agilent TapeStation. Biotinylated RNA baits were hybridized to the synthesized and purified cDNA targets, and the bait-target complexes were amplified in a post-capture PCR reaction. The resultant libraries were quantified and normalized, and the pooled libraries were denatured, diluted, and sequenced. Raw data were demultiplexed using the Illumina DRAGEN FFPE accelerator. FASTQ files were aligned with STAR aligner . A full 22,948-gene dataset of expression data was produced by the Salmon, which provided fast and bias-aware quantification of transcript expression BAM files from STAR aligner automated platform and staining techniques, per the manufacturer\u2019s instructions, and were optimized and validated per CLIA/CAP and ISO requirements. Staining was scored for intensity and staining percentage (0\u2013100%). Positive expression of immune cell (IC) PD-L1 (SP142), tumor cell ESTROGEN RECEPTOR (ER), and tumor cell PROGESTERONE RECEPTOR (PR) was defined as \u22651+ stain intensity and \u22651% of cells stained. Positive HER2 expression was determined according to the 2018 ASCO-CAP guidelines databases, or benign variants identified by Caris\u2019s geneticists. TMB-H was defined by a threshold of \u226510 mutations per megabase (mut/MB) based on the KEYNOTE-158 pembrolizumab trial (Deficient mismatch repair/microsatellite instability-high (dMMR/MSI-H) was determined by a combination of IHC and NGS Genome Browser database). The platforms generated highly concordant results as previously reported . Continuous data were assessed using a Mann-Whitney U test, and categorical data were evaluated using Chi-square or Fisher\u2019s exact test, where appropriate. P-values were adjusted for multiple hypothesis testing using the Benjamini-Hochberg procedure, unless noted as exploratory (not adjusted).Comprehensive molecular profiles of breast cancer patient samples (N=5621) were analyzed from various cancer institutes, predominantly within the United States. Samples were stratified into TMB-H and TMB-Low cohorts based on a threshold of \u226510 mut/MB \u201313. PD-L).Among breast cancer receptor subtypes, median T cell-inflamed scores were highest among TNBC samples, which were significantly increased compared to HR+/HER2- samples that exhibited the lowest median score , a scaffolding protein essential for MHC-I complex formation and peptide presentation. CD274 (PDL-1) amplification was also associated with T cell-inflamed score in TMB-H tumors.To identify new predictive biomarkers of inflamed tumor microenvironments, we compared T cell-inflamed scores in TMB-H and TMB-L cohorts stratified by biomarker status . Consistent with our initial analysis, PD-L1+ IC was associated with higher T cell-inflamed scores in TMB-H tumors, while scores associated with many of the most commonly altered biomarkers were much more variable and the median PFS was 10.6 weeks . HoweverIn a large cohort of 5621 breast cancer tumors, we identified 461 (8.2%) TMB-H tumors and examined concurrent predictive biomarkers of an immune-inflamed TME to assess predictors of immune checkpoint blockade (ICB) response. RNA signatures hold promise as biomarkers of immunotherapy response across solid tumor malignancies. We used a well validated T cell-inflamed scores defined by an 18-gene signature to select tumors with an immune responsive TME within this cohort , 16. OurWe then assessed the impact of known biomarkers of immune response in breast cancer and solid tumors within TMB-H breast cancer and found that PD-L1 positivity and microsatellite instability were enriched in TMB-H tumors and predicted inflamed TMEs. This finding was true regardless of tumor subtype (HR+ and TNBC) and histology . These findings are particularly clinically relevant as commercially available next generation sequencing tests routinely report PD-L1 and dMMR/MSI-H status along with TMB. A logical next step to this analysis would be to assess ICI responses in patients with TMB-H and PDL-1+ tumors in prospective or retrospective cohorts. One limitation of this study is the use of PD-L1 testing using the Ventana SP142 assay on tumor immune cells, which is no longer used in United States clinical practice. These findings should be repeated using diverse PD-L1 assays.CDH1 mutations are found in 53% of lobular breast cancers in the literature evaluated patients with metastatic lobular breast cancer treated with induction carboplatin followed by atezolizumab (PD-L1 inhibitor). Four (4/21) patients with triple negative disease had a partial response to treatment (Lobular breast cancer encompasses about 10% of all breast tumors with increasing incidence in recent decades . Severalterature and haveterature . In thisreatment without B2M mutations and CD274 amplifications were associated with a strong T-cell inflamed score within TMB-H tumors and not TMB-Low tumors, which was also observed in TNBC but not HR+/HER2- subgroups. Recent data suggest that somatic B2M mutations are associated with a higher load of neoantigens for MHC-I presentation (CD274 gene is a target for both PDL-1 and PD-1 inhibitors. Although PDL-1/CD274 amplification in solid tumors is rare, it has been linked to ICI response in small series (Lastly, our analysis showed that entation , which cl series . Furtherl series .There are several limitations of this analysis. The lack of matched treatment and response data limits our ability to determine potential therapy-induced effects on the TME signatures evaluated, as well as limiting the evaluation of immune-related signatures and co-alterations as predictive biomarkers of response to therapy. Additionally, as bulk tumor sequencing approaches do not allow for robust characterization of cell type-specific molecular features or signals, future studies utilizing single-cell sequencing may provide novel insights of breast cancer TMEs.B2M mutation and CD274 amplification may help predict benefit to ICI within TMB-H MBC. Co-occurring biomarkers within TMB-H breast cancer warrant further evaluation in larger cohorts for response or resistance to ICI to help develop composite predictive biomarkers in MBC.In conclusion, high TMB alone does not strongly correlate with immune infiltrate or immune-related gene signatures in further unselected MBC. In our dataset, TMB-H predicted a more immune responsive microenvironment compared to TMB-L in HR+, HER2- tumors which could further be enhanced when selecting PD-L1+ tumors. This subset of patients would be relatively rare, though a small prospective trial assessing immunotherapy strategies in this population would be warranted. aelliott@carisls.com of Caris Life Sciences.The datasets presented in this article are not readily available because the raw data is protected proprietary information. Requests to access the datasets should be directed to Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.SS and ET: Conception, design, data analysis, manuscript writing and editing. AE: Conception, design, data analysis, biostatistical analysis. RB-S and ST: data analysis, manuscript editing. SC, AT, and GS: data analysis, manuscript editing. All authors contributed to the article and approved the submitted version."} +{"text": "Such observation allowed177Lu-DOTATATE PRRT to be considered for an advanced, symptomatic, and multiple treatment-resistant patient with limited palliative treatment options left.A 50-year-old female patient of heavily pre-treated (chemotherapy and multiple treatment-resistant) and progressive intermediate-grade metastatic neuroendocrine neoplasm is presented, wherein the lesions showed mixed response following topotecan treatment and multiple hepatic metastasis showed increase in the SSTR expression and decrease in FDG concentration on dual-tracer PET/CT ( However, some intermediate-grade NENs showing minimal/low-grade SSTR positivity makes them unsuitable for PRRT. It has been observed and reported that certain forms of chemotherapy may induce a (i) radiosensitizing effect in tumor cells, which eventually increases the tumor response after PRRT, and also (ii) may enhance tracer uptake on SSTR-based PET-CT.The heterogeneity of the neuroendocrine neoplasms (NENs) varies between the complete spectrum of well-differentiated to poorly differentiated tumors/carcinomas. The recently adopted 2021 WHO classification gave two separate categories of high-grade grade 3) NENs as well-differentiated NEN and poorly differentiated NEN/neuroendocrine carcinoma (NEC). This new concept replaced the previous definition of grade-3 NEN based purely on the K NENs as 99mTc-HYNIC-TOC scan revealed low-moderate grade uptake in the left-sided pelvic mass, and histopathology and IHC review suggested a Mib-1 labeling index of 10%. Considered for chemo-PRRT, she received a total of 10 cycles of oral chemotherapy with capecitabine-temozolomide (CAPTEM) and 4 cycles PRRT (cumulative activity of177Lu-DOTATATE received was 705 mCi/26.08 Gbq), with no significant change in size and intensity of SSTR expression in rectal primary, left external iliac nodal mass, and left pelvic nodes on68Ga-DOTATATE-PET/CT. Surgical opinion was declined in view of inability to achieve R0 resection. Following a stable disease for the next 1.5 years, she developed a solitary liver lesion, which on USG-guided biopsy revealed it to be NET-grade II (Mib-1 index: 9\u201310%). The patient underwent lipiodol-TACE of the right lobe liver lesion (segment-V) using 50\u2009mg doxorubicin with 10\u2009mL of lipiodol. Follow-up liver MRI showed post-TACE changes in the lesion but also revealed a few other tiny lesions in the liver, for which multiple TACE and CT-guided RFAs were undertaken over next 2 years along with octreotide LAR, but recurrence developed in a very short period. The pelvic lesion was stable during this time while persistent progression of the liver lesions with an increase in FDG concentration in most lesions but very few lesions were adequately SSTR expressing on68Ga-DOTATATE PET/CT. The patient experienced significant weight loss, constipation, and occasional abdominal pain. Also noted was obstructive uropathy due to left-sided iliac mass with left-sided kidney showing a serial reduction in cortical function. of uptake . She exTopotecan is an inhibitor of topoisomerase I and acts by forming a stable covalent complex with the DNA/topoisomerase I aggregate, leading to breaks in the DNA strand, apoptosis, and cell death. It has been tried successfully in multiple tumors, especially in chemotherapy-resistant and heavily pre-treated patients with non-small cell lung cancer, ovarian, colon, and other solid tumors. Multiple groups have tried it in poorly differentiated and advanced heavily pre-treated NENs.68Ga-DOTATATE-PET/CT is not elucidated in the literature and this would be first such literature-report in advanced, treatment-resistant NENs. In the previously reported short case series, similar such observation was described in the context of CAPTEM and everolimus.There is paucity of literature regarding response evaluation post-topotecan therapy in heavily pre-treated NEN patients and such chemotherapy-induced increased SSTR expression on SSTR-based PET/CT. To our knowledge, post-topotecan increase in SSTR expression in lesions on68Ga-DOTATATE and18F-FDGPET/CT), wherein the observation of increased uptake and SSTR expression on tumor cells could open up suitable therapeutic avenues such as PRRT in such scenarios.In conclusion, heavily pre-treated and treatment resistant NENs may show a response to topotecan and could be better evaluated with dual tracer PET ("} +{"text": "The approach serves to restrict transgene expression to a tissue of interest - the nervous system in the example provided here - thereby promoting specific/exclusive targeting of discrete cellular subtypes. Recent innovations are bringing us closer to understanding how the brain is organized, how neural circuits function, and how neurons can be regenerated. Fluorescent proteins enable mapping of the 'connectome', optogenetic tools allow excitable cells to be short-circuited or hyperactivated, and targeted ablation of neuronal subtypes facilitates investigations of circuit function and neuronal regeneration. Optimally, such toolsets need to be expressed solely within the cell types of interest as off-site expression makes establishing causal relationships difficult. To address this, we have exploited a gene 'silencing' system that promotes neuronal specificity by repressing expression in non-neural tissues. This methodology solves non-specific background issues that plague large-scale enhancer trap efforts and may provide a means of leveraging promoters/enhancers that otherwise express too broadly to be of value for We show that a conserved neuron-restrictive silencer element (NRSE) can function to restrict transgene expression to the nervous system. The neuron-restrictive silencing factor/repressor element 1 silencing transcription factor (NRSF/REST) transcriptional repressor binds NRSE/repressor element 1 (RE1) sites and silences gene expression in non-neuronal cells. Inserting NRSE sites into transgenes strongly biased expression to neural tissues. NRSE sequences were effective in restricting expression of bipartite Gal4-based 'driver' transgenes within the context of an enhancer trap and when associated with a defined promoter and enhancer. However, NRSE sequences did not serve to restrict expression of an upstream activating sequence (UAS)-based reporter/effector transgene when associated solely with the UAS element. Morpholino knockdown assays showed that NRSF/REST expression is required for NRSE-based transgene silencing.Our findings demonstrate that the addition of NRSE sequences to transgenes can provide useful new tools for functional studies of the nervous system. However, the general approach may be more broadly applicable; tissue-specific silencer elements are operable in tissues other than the nervous system, suggesting this approach can be similarly applied to other paradigms. Thus, creating synthetic associations between endogenous regulatory elements and tissue-specific silencers may facilitate targeting of cellular subtypes for which defined promoters/enhancers are lacking. Accurate characterization of neurons and neural circuits requires that neuronal subtypes be unambiguously identified and indeDanio rerio) and many of the resultant transgenic lines show expression in the nervous system. However, despite the general success of this approach, so-called basal or background expression in heart, skeletal muscle, etc., can compromise the usefulness of these resources [Drosophila system [Drosophila community [et al. [Tg lines show previously uncharacterized expression within eye muscles and retinal cells). Regardless of the underlying mechanism, Gal4/UAS lines in which background expression is reduced or eliminated would provide improved resources for functional studies of the nervous system.Gene and/or enhancer trap screens, whereby endogenous cis-regulatory elements are co-opted to regulate transgene expression, eliminate the need to identify cell-specific promoters by allowing visual selection of expression patterns of interest. Transposons, such as Tol2, have been used extensively for gene/enhancer trapping in zebrafish to delimit transgene expression exclusively to neuronal cells.Intersectional and subtractive methods, whereby transgenes are restricted to cells expressing two or more patterning genes, have been developed to promote cell-specific expression . AlternaEvaluation of regulatory mechanisms underlying neural specificity of the synaptic protein stathmin-like 2 gene and a voltage-dependent sodium channel revealed that active repression of expression in non-neuronal cells played a central role -19. An NACGGACAGCGCC, is a canonical NRSE site that is highly conserved across species, and is composed of two non-palindromic half-sites separated by a non-conserved 2 bp spacer (underlined). We placed a tandem set of NRSE sites in upstream regulatory regions of several transgenic constructs and compared resulting expression patterns to non-NRSE parental plasmids. In all, this strategy was applied within the context of enhancer trap constructs [To test this idea, we integrated a pair of consensus NRSE sites into sevnstructs , definednstructs , definednstructs and UAS-nstructs .The data indicate that transgene expression was strongly biased to the nervous system when NRSE sequences were included in enhancer trap and defined enhancer constructs, thereby effective in delimiting the expression of the driver element of a bipartite expression system . However, expression biases were not evident when NRSE sequences were added to UAS-based reporter transgenes. Nevertheless, due to the bipartite nature of such systems, delimiting the expression of Gal4-VP16 drivers sufficed to restrict UAS reporters to the nervous system as well - because drivers are required to activate expression of reporters. Morpholino knockdown (this study) and zinc finger nuclease mediated gene disruptions verifiedcfos minimal promoter [cfos:KalTA4; see construct diagrams in Additional file 2xNRSE-cfos:KalTA4). CK or NRCK transgenes were injected into fertilized eggs from an established UAS effector-reporter line, c264 [cis). In the NRSE version, a loxP flanked 5xUAS:YFP reporter was placed downstream of NRCK . The control version consisted of the CK driver element upstream of tandem 5xUAS:nfsB effector and 14xUAS:YFP reporter components , as previously reported [To test whether associating NRSE sites with a minimal promoter would serve to restrict transgene expression to the nervous system, Gal4-VP16-based enhancer trap constructs were assembled with and without NRSE sites. Plasmids were composed of a mouse promoter upstreampromoter , and asspromoter . Initialrry)c264 ) and resreported .Et), 25 NRCK ) and 26 NRCK-5xMY ), as well as 37 CK-5xN-14xY ). These lines have been propagated to the F3 to F5 generation and 93% currently produce inheritance patterns consistent with a single insertion site . In addition to the data presented here, we have established a website for the dissemination of high-resolution imaging data and insertion site sequence information to the research community [To more stringently test the effects of NRSE sites on transgene expression patterns, CK, NRCK and NRCK-5xMY plasmids were used to establish a series of stable enhancer trap lines Figure . Individommunity . We contTg(14xUAS-E1b:nfsB-mCherry)c264 line is susceptible to methylation-based silencing [Tggmc930, see Additional file gmc930) line has shown no evidence of silencing over three generations, possibly due to our inclusion of 'barrier' insulator sequences [Initial evaluations of transgene expression patterns were promising. However, we noted that mCherry patterns often seemed unstable, that is, expression domains would differ slightly from one generation to the next. It was subsequently reported that the ilencing ,49. To aequences or the requences . Either gmc694 line provides a typical example, with expression in tectal neurons minimal promoters. By contrast, non-NRSE self-reporting CK-5xN-14xY controls [Phenotypic characterizations of KalTA4 driver lines at 6 to 7 dpf revealed clear differences between control (CK) and NRSE-containing (NRCK) expression patterns. CK-derived lines tended to have mixed expression patterns, with multiple cell types and tissues labeled exhibit a clear bias toward neural-restricted expression compared with controls Figure , Table 1s Figure or cis . Both were constructed in the miniTol2 vector [Etgmc680; Tg(14xUAS-E1b:nfsB-mCherry)c264) was crossed to an NR5xMY-HMY line gmc932) to create triple transgenic offspring , this would serve to restrict effectors/reporters to the nervous system regardless of the Gal4-VP16-specified pattern. In turn, this would provide a means of eliminating the background issues of previously derived Gal4-VP16 expressing transgenic lines - a possibility worth exploring given the numerous intriguing neural expression patterns characterized in previous gene/enhancer traps -10. Acco2 vector and usedg Figure . The CK-s Figure . We rease Figure . Insteade Figure , arrow. Etgmc607 line provides an example of this phenomenon knu3 (formerly HuC:GFP [gmc930). This analysis showed that the delay in non-neuronal repression we observed with the Cherry line was also evident when such lines were crossed with the YFP reporter line . In addition to revealing lines that express reporters only after terminal neuronal differentiation has begun (3 to 5 dpf), we also observed that some NRSE-delimited lines exhibited expression in muscle cells early on, which then faded over time - i.e., a 'delayed non-neuronal repression' phenotype. The HuC:GFP ) was usee Figure . In keepNRSF/REST expression is complex, displaying stage- and neuronal cell-type specific expression patterns and splice variants that reflect a diversity of roles in regulating gene expression. In zebrafish, NRSF/REST expression has only been characterized through early embryonic stages where it is expressed fairly ubiquitously throughout the nervous system until downregulated in differentiating ventrolateral domains of the central nervous system . This parest mRNA [gmc607). Comparisons among uninjected control, control MO and rest MO-injected larvae showed clear differences in reporter expression . The number of larvae expressing the mCherry reporter in muscle cells at 6 dpf was quantified across all three conditions in the uninjected and 8% (3 out of 37) in control MO groups did so. Furthermore, when the number of muscle cells expressing reporter protein at 3, 6 and 9 dpf was quantified from all imaged larvae, rest MO-injected larvae showed clear increases in muscle cell expression compared to controls against zebrafish est mRNA was injen Figure and contn Figure larvae dO Figure . In addif Figure , well afs Figure . A totals Figure . These rckground . TogetheWe have explored the use of tissue-specific silencer elements to delimit transgene expression to a region of interest by repressing transcription elsewhere. This strategy provides a potential means of eliminating unintended expression and/or undesirable expression . In particular, we tested whether inserting NRSE sites into upstream regulatory regions of Gal4-VP16 driver and/or UAS-based reporter constructs lines [It remains unclear whether the background patterns evident in Gal4-VP16 lines are predominantly an artifact , promiscuous position effects, Gal4-VP16 based amplification of previously undetectable gene/enhancer activity , or a co6) lines . Combine6) lines ,70, the The addition of NRSE sites to UAS reporter lines was not sufficient to restrict reporter expression to neural tissues Figures , Table 1Our findings indicate that new NRSE -delimited Gal4-VP16 driver lines will need to be derived to take advantage of the neural expression bias provided by this approach. Accordingly, we have begun a large-scale NRSE-delimited enhancer trap screen to create new Gal4-VP16 lines useful for dissecting neural circuit functions. To date, 62 NRSE-delimited KalTA4-expressing lines have been created. In a related screen, we are creating a series of NRSE-delimited LexA-based driver lines (manuscript in preparation). The use of two bipartite transgene expression systems would allow two neuronal subpopulations to be independently manipulated. Optimally, complementary platforms of this nature could be used to differentially modulate pre- and post-synaptic elements of discrete subcircuits - a possibility that improved trans-synaptic transporters would facilitate. In addition, the use of an inducible LexA-based transactivation system in transgenic zebrafish providesThe mechanism behind NRSE-delimited transgene expression is likely due to a repressive action of NRSF/REST in non-neural tissues ,4. Our det al., showing that early neural patterning is largely unaltered in rest mutants [REST is expressed nearly ubiquitously during early zebrafish development, including within the nervous system. Yet, early reporter expression is observed in the majority of NRSE-containing Gal4-VP16 driver lines and beyond - for instance, to assay behavioral consequences of altering neuronal activity - remains viable.rest MO-injected NRSE-containing Gal4-VP16 driver lines , generated by zinc finger nuclease targeting [gmc606, 607, 632 and 641) [sbu29/sbu99 restmutants suggests REST can repress expression in the nervous system as well. This is in keeping with studies suggesting that REST may act to repress gene expression in neuronal subsets [To further test whether REST was required for NRSE-delimited expression patterns, we knocked down REST expression in transgenic NRCK zebrafish embryos and larvae using a previously characterized morpholino. The data showed clear evidence that when REST function is disrupted, NRSE-mediated neural expression biases are lost, with spatially expanded and temporally extended strong skeletal muscle expression seen in argeting ,79, showand 641) . Interes subsets and that subsets . More re subsets . Future Cell-type specific lineages are often defined by multifactorial 'codes' of overlapping subsets of transcription factors . Thus, iThese studies validate the use of tissue-specific silencer elements to promote enhanced transgene expression specificity. NRSE sites served to bias the expression of trapped and defined DNA regulatory elements to the nervous system, providing a means of targeting neuronal cell subtypes by silencing expression in non-neural tissues. Transgene silencing effects were dependent on the expression of REST, in keeping with a well-characterized role of this NRSE-binding transcriptional repressor in maintaining neural-specific gene expression. Using the strategy, promoter/enhancer elements that would otherwise be too broadly expressed can be harnessed for functional assays. This approach also affords a solution to non-specific background expression issues that can compromise large-scale enhancer trap screens, as has been the case in the zebrafish field. NRSE-delimited transgenes can provide useful new tools for functional studies of the nervous system. Inclusion of bipartite expression systems, such as Gal4/UAS, ensures that a multitude of functional assays can be performed with NRSE-delimited transgenic resources. For instance, integrating new toolsets for manipulating neuronal activity or targeted cellular ablation systems into bipartite effectors will provide a versatile platform for the genetic dissection of neural circuit function. More broadly, similar genetic mechanisms may be used to reinforce expression specificity in other tissues. Thus, creating synthetic associations between endogenous regulatory sequences and tissue-specific silencer elements could provide a means of targeting unique cellular subsets for which cell-specific regulatory elements are lacking.This study was carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. An animal use protocol was approved by the Institutional Animal Care and Use Committee of Georgia Health Sciences University, which has an Animal Welfare Assurance on file in the Office of Laboratory Animal Welfare (Assurance Number: A3307-01). Using approved anesthetics, all efforts were made to minimize discomfort and suffering during experimental procedures.Tg(elavl3:EGFP)knu3 [Tg(14xUAS-E1b:nfsB-mCherry)c264 [Zebrafish were maintained using established temperature (28.5\u00b0C) and light cycle conditions . Embryos and larvae were cultured in standard growth media supplemented with paramecia and Sera micron flake starting at 5 dpf. Previously described transgenic zebrafish strains used in this study included GFP)knu3 and Tg(1rry)c264 .Et) and CK-5xN-14xY ). Two NRSE-containing transgenes were used to create new NRSE-delimited enhancer trap lines, designated NRCK ) and NRCK-5xMY ). CREST1 enhancer containing transgenes were used to make lines designated C1CK-5xY2N ) and NR C1CK-5xY2N ). In the CREST1 lines, a 'self-cleaving' viral peptide sequence, derived from porcine teschovirus-1 (P2A [YFP-2A-nfsB). A non-NRSE 5xUAS-based reporter transgene was used to make a reporter line designated 5xMY-HMY gmc830). In addition, a NRSE-containing 5xUAS-based reporter transgene was used to make a reporter line designated NR5xMY-HMY gmc835). All transgenes were assembled in the miniTol2 background to facilitate transgenesis efficiency [Tg(14xUAS:nfsb-mCherry)c264 line [he1a), which contained three regions highly conserved between he1a, he1b and he2, was used to drive expression of membrane-tagged YFP in the hatching gland, a set of cells located within the yolk sac that are resorbed after hatching. This allows UAS reporter carriers to be identified by a 'temporary tracer' that fades by 4 dpf, thus does not impinge on late larval imaging experiments pigmentation mutant background with Tol2-based transgenesis methods.Transgenes used to establish new transgenic lines during the course of these studies are diagrammed in Additional file s-1 (P2A ), was uss-1 according to the manufacturer's protocol and treated with DNase to remove genomic DNA contamination. The first-strand cDNA synthesis was performed using the SuperScript II First-Strand System . qRT-PCR reactions were carried out as described previously ,91. In brest exon 3 (5'-GGCCTTTCACCTGTAAAATACAGAA-3') was used. The control morpholino was the standard provided by GeneTools . Morpholinos were diluted to 0.1, 0.25, 0.5, 1 or 2 \u03bcM and a 1- to 2-nL volume injected into eggs as previously described [rest locus was as previously described [To knock down REST production, a previously characterized splice inhibiting morpholino targeted to the intron-exon boundary of zebrafish escribed . Images escribed .All single time point and time lapse confocal imaging of transgenic zebrafish larvae was performed as previously described .t-test to compare across treatment conditions, or a repeated measures t-test for time series data; i.e., when data from individuals were compared across time. Where symbols are present in figures, P-values were minimally \u2264 0.05.Statistical comparisons were performed using an independent sample Tg(loxP-5xUAS-E1b:gap43-YFP-loxP: he1a:gap43-YFP) transgene or transgenic reporter line ; 5xUAS: transgene sequence composed of five serial repeats of UAS binding sites; 14xNTR-Ch: 14xUAS-E1b:nfsB-mCherry)c264 transgenic line; bp: base pairs; C1CK-5xY2N: Tg transgene or transgenic line ; cfos: minimal promoter element from mouse cFos gene; CK: Tg transgene or transgenic lines ; CK-5xN-14xY: Et transgene or enhancer trap transgenic lines ; CREST1: highly conserved enhancer element from the zebrafish Islet-1 gene; dpf: days post-fertilization; E1b: basal promoter from carp beta-actin; Gal4: yeast transcription activator protein; Gal4/UAS: a bipartite transgene expression amplification system; Gal4-VP16: fusion protein linking the DNA binding domain of Gal4 and transcriptional activation domain of VP16; KalTA4: Gal4-VP16 fusion variant optimized for expression in zebrafish; lox: loxP recombination site; MO: morpholino-modified oligonucleotide; M-YFP: membrane-tagged yellow fluorescent protein; nfsB: bacterial gene encoding nitroreductase B; NR5xMY-HMY: Tg(2xNRSE-loxP-5xUAS-E1b:gap43-YFP-loxP: he1a:gap43-YFP) transgene or transgenic reporter line ; NRC1CK-5xY2N: Tg transgene or transgenic line ; NRCK: Et transgene or enhancer trap transgenic lines ; NRCK-5xMY: Et transgene or enhancer trap transgenic lines ; NRSE: neuron-restrictive silencer element; NRSF: neuron-restrictive silencing factor; qRT-PCR: quantitative reverse transcriptase polymerase chain reaction; RE1: restriction element 1; REST: RE1-silencing transcription factor; STMN2: stathmin-like 2 gene (aka SCG10); Tol2: a member of the hAT family of transposons; UAS: upstream activating sequence; VP16: viral protein 16, a strong transcriptional activator; YFP: yellow fluorescent protein.2A: a porcine 2A viral peptide sequence; 2xNRSE: a tandem repeat of a 21 bp consensus NRSE site; 5xMY-HMY: JSM and MTS have a financial interest in Luminomics, Inc., a small biotechnology company that uses the nitroreductase transgene-based system of targeted cellular ablation as a platform for studying cell-specific regeneration using models of degenerative and autoimmune diseases. JRM, M-AS and MTS are salaried employed of Luminomics. JSM receives consulting fees from Luminomics. XX, SLW, YT, MD, RWK and HIS confirm that they fdo not have any competing interests.XX, JRM, M-AS, and MTS created transgenic lines, and collected, analyzed and assembled the expression data. XX and SLW performed, analyzed and assembled the morpholino experiments. YT performed the qRT-PCR analysis. MD, RWK and HIS provided research materials. JSM conceived the study, and HIS, MTS and JSM participated in the design of the study. JSM drafted the manuscript with assistance from all other authors. All authors read and approved the final manuscript.Diagram of transgene constructs. Schematics showing pertinent details of the transgenes tested and corresponding acronyms. Core elements include: cfos - minimal promoter [KalTA4 - an optimized Gal4-VP16 fusion protein [GI - rabbit beta-globin intron to promote mRNA stability [pA - SV40 or bovine growth hormone polyadenylation sequences; 2xNRSE - a tandem repeat of a 21 bp consensus NRSE site [lox - loxP recombination sites to allow transgene cassette swapping [UAS - 17 bp upstream activator sequence [E1b - a basal promoter from carp beta-actin [CREST1 - a 800-bp enhancer element characterized as a cranial motor neuron-specific element [2A - a porcine 2A viral peptide sequence [nfsB - Escherichia coli gene encoding the prodrug converting bacterial enzyme nitroreductase (Ntr) which promotes chemically-induced cell ablation [HE - a 365-bp promoter element from the zebrafish hatching enzyme 1a locus (he1a) that allows facile detection of UAS reporter lines in the absence of Gal4-VP16 driver elements monomeric 'tag' yellow fluorescent protein (tagYFP); mCherry - a monomeric red fluorescent protein [promoter ; KalTA4 protein ; GI - ratability ; pA - SVRSE site , lox - lswapping ; UAS - 1sequence specificta-actin ; CREST1 element ; 2A - a sequence promotinsequence ; nfsB - ablation ,53,88; H43 locus 'enhance protein .Click here for fileEnhancer trap comparisons \u00b1NRSE. Confocal images of an additional 12 NRCK (left box) and 6 CK (right box) lines are shown in support of the phenotypic data summarized in Figure Click here for fileHigh-resolution imaging of branchiomotor neuron labeling. (A-E) Confocal images of 6-dpf NRC1CK-5xY2N transgenic line lmc003) showing specific labeling of branchiomotor neuron ganglia. When NRSE sites were placed upstream of CREST1-cfos, expression became restricted to cranial motor neuron subpopulations; the expression pattern originally characterized as CREST1-specified [pecified . Click here for fileEarly neuronal expression of NRSE Gal4 driver transgenes. Confocal images of 2-dpf triple transgenic line gmc607; Tg(14xUAS:nfsB-mCherry)c264; Tg(elavl3:EGFP)knu3) showing typical early neural expression of NRCK lines .Click here for fileHatching enzyme promoter-based transgene 'tracer'. Stereoscope micrograph shows expression of he1a:YFP 'tracer' transgene in 1-dpf embryos. This element allows transgenic UAS reporter lines gmc830, shown here) to be visually sorted from non-transgenic siblings (asterisks) at embryonic to early larval stages in the absence of Gal4 driver expression. The 365 bp he1a promoter is robustly active (arrow) from 1 to 3 dpf, after which expression rapidly fades. Inclusion of this element in UAS reporter lines has greatly simplified maintenance of our stocks.Click here for file"} +{"text": "Cucumber mosaic virus (CMV) Y-satellite RNA (Y-Sat) has asmall non-protein-coding RNA genome that induces yellowing symptoms in infectedNicotiana tabacum (tobacco). How this RNA pathogen inducessuch symptoms has been a longstanding question. We show that the yellowingsymptoms are a result of small interfering RNA (siRNA)-directed RNA silencing ofthe chlorophyll biosynthetic gene, CHLI. The CHLI mRNA contains a 22-nucleotide(nt) complementary sequence to the Y-Sat genome, and in Y-Sat-infected plants,CHLI expression is dramatically down-regulated. Small RNA sequencing and5\u2032 RACE analyses confirmed that this 22-nt sequence was targeted for mRNAcleavage by Y-Sat-derived siRNAs. Transformation of tobacco with a RNAinterference (RNAi) vector targeting CHLI induced Y-Sat-like symptoms. Inaddition, the symptoms of Y-Sat infection can be completely prevented bytransforming tobacco with a silencing-resistant variant of the CHLI gene. Theseresults suggest that siRNA-directed silencing of CHLI is solely responsible forthe Y-Sat-induced symptoms. Furthermore, we demonstrate that twoNicotiana species, which do not develop yellowing symptomsupon Y-Sat infection, contain a single nucleotide polymorphism within thesiRNA-targeted CHLI sequence. This suggests that the previously observed speciesspecificity of Y-Sat-induced symptoms is due to natural sequence variation inthe CHLI gene, preventing CHLI silencing in species with a mismatch to the Y-SatsiRNA. Taken together, these findings provide the first demonstration of smallRNA-mediated viral disease symptom production and offer an explanation of thespecies specificity of the viral disease.The Viral infections result in a variety of disease symptoms that vary in characterand severity depending on the type of viral infection and individual hostfactors. Despite extensive research, the molecular basis of viral diseasedevelopment has remained poorly understood. Both plant and animal virusesexpress 20\u201325 nucleotide siRNAs or microRNAs (miRNAs) in their host. Thesevirus-specific small RNAs (sRNAs) direct RNA silencing of homologous viral genesto restrict virus replication forming part of the host's antiviral defensemechanism. Using a plant viral satellite RNA as a model system, we demonstratehere a new function for virus-derived sRNAs: induction of disease symptoms bysilencing of a physiologically important host gene. Furthermore, we demonstratethat such viral-derived sRNA-induced disease symptoms can be prevented by theexpression of a either naturally evolved, or artificially introduced,silencing-resistant sequence variant of the viral siRNA-targeted gene. Thesefindings not only provide the first demonstration of a sRNA-mediated viraldisease mechanism, but also offer an alternate strategy to prevent the onset ofsuch viral diseases. Cucumber mosaic virus (CMV Y-Sat). The CMV Y-Sat consists of a369-nt single-stranded RNA genome and induces distinct yellowing symptoms in anumber of Nicotiana species including N. tabacum(tobacco) Plant viruses are often accompanied by small parasitic RNAs termed satellite RNAs.Satellite RNAs range in size from \u223c220 to 1400 nucleotides (nt) in length anddepend on their associated viruses (known as the helper virus) for replication,encapsidation, movement and transmission, but share little or no sequence homologyto the helper virus itself N.bigelovii and the disease-resistant species N.clevelandii, suggested that the yellowing symptoms induced upon CMVY-Sat infection are associated with a single, incompletely dominant gene in theNicotiana species N. tabacum sequence complementary to 22 nt of theY-Sat yellow domain vector targeting CHLI resulted in a dramatic decrease in CHLI expression andsevere chlorosis of the transgenic plants, which ranged from yellowing tocomplete bleaching , and transformed tobacco plantswith the mutated version of the CHLI gene (mtCHLI). The modified CHLI transgenecontains 10 nucleotide changes within the 22-nt region complementary to Y-Satyellow domain siRNAs, bringing nine mismatches to this region ofcomplementarity, rending it resistant to cleavage by these Y-Sat siRNAs A\u2013B. StrNicotiana species are susceptible to Y-Sat-inducedyellowing symptoms Nicotianaspecies, rendering these species \u2018resistant\u2019 or free of Y-SatsiRNA-induced CHLI silencing. We sequenced the CHLI transcript from fivedifferent Nicotiana species, including threedisease-susceptible and two resistant (N.clevelandii and N. debneyi) species , with the predominant species containing an A to G change at the targetedCHLI sequence, converting tobacco's A:U base pairing to a weaker G:U wobblepair near the mRNA cleavage site , a 359-ntnon-protein-coding RNA pathogen, has been associated with two \u223c20-nt partiallycomplementary regions of the PSTVd RNA genome known as virulence-modulating regionsAs observed for the yellowing symptoms associated with CMV Y-Sat infection, severalother disease symptoms induced by viral satellite RNAs have also been associatedwith a short sequence within the respective satellite RNA genomes. For instance, thechlorotic phenotypes induced by the B2 and WL3 satellite RNAs of CMV in tobacco andtomato are determined by a \u223c26-nt region (nt. 141\u2013166) of the satellitegenome The siRNA-mediated disease mechanism reported here is consistent with the previousobservation that disease induction by satellite RNAs involves the interaction ofsatellite RNAs, their helper viruses, and the host plant RNA silencing has been suggested to be a driving force for the evolution of subviralRNAs including viral satellite RNAs As discussed for satellites and viroids, infection of plants with both RNA and DNAviruses is also associated with the accumulation of virus-derived siRNAs We have demonstrated here that transformation of tobacco with a silencing-resistantversion of the CHLI gene completely prevented the yellowing symptoms associated withY-Sat infection. This finding offers a potential new strategy for preventing viralsiRNA-mediated diseases in plants and animals. However, viral replication hasrelatively high error rates and viruses often exist as quasispecies (mixtures ofminor sequence variants) Nicotiana species were grown in a 25\u00b0C glasshouse withnatural light. Infection of tobacco plants with Cucumber mosaicvirus plus Y-Sat, total RNA extraction and northern blothybridization were performed as previously described N. tobacum CHLI cDNA (accessions: U67064 and AF014053) asthe \u201cbest\u201d match with the Y-Sat sequence.All 5\u2032ATCTGGTACCAAAATGGCTTCACTACTAGGAACTTCC 3\u2032) and reverseprimer (5\u2032TCTAGTCTAGAAGCTTAAAACAGCTTAGGCGAAAACCTC 3\u2032) were usedfor both the PCR and RT-PCR reactions. PCR products were purified using aQIAquick PCR purification kit (Qiagen), cloned into the pGem-T Easy cloningvector (Promega) and sequenced. The full-length genomic and cDNA sequences (seeKpnI/XbaIand cloned into the intermediate vector pBC (Strategene).Full-length genomic and cDNA sequences of the CHLI gene were amplified from totalDNA and RNA using the NEB Long Amp Taq kit and Qiagen One Step RT-PCR kitrespectively according to the manufacturer's instructions. The CHLI forwardprimer (nces see were dig5\u2032 TGGCACAATCGACATTGAGAAAGC3\u2032) and reverse primers that spanned the 22-nt CHLI target siteand allowed for the introduction of modified nucleotides, and ii) the 3\u2032segment was amplified with a forward primer that also contained modified nucleotides spanningthe 22-nt CHLI target sequence overlapping with MT-R1 and a reverse primer. The two amplified productswere joined together using overlapping PCR with Pfu polymerase (Promega) togenerate a 630 bp fragment with a modified Y-satellite target sequence. A 223 bpfragment, containing the modified target sequence, was released byPstI and EcoRI digestion and used toreplace the PstI-EcoRI fragment of the wildtype sequence in the CHLI cDNA, giving rise to the modified sequence mtCHLI.Digestion of the mtCHLI sequence with PstI andBamHI released the modified region which was used toreplace the corresponding region in the wild-type CHLI genomic sequence, givingrise to the modified genomic clone gmtCHLI.To create the modified CHLI sequence (mtCHLI and gmtCHLI), a 630 bp sequencecontaining the Y-Sat targeted 22-nt sequence was amplified as two halves; i) the5\u2032 half was amplified with forward using T4 RNA ligase (Promega) at room temperaturefor 2 hours in 50 mM HEPES pH 7.5, 0.1 mg/mL BSA, 8% glycerol, 2units/\u00b5L RNasein RNase inhibitor (Promega) and 0.5 unit/\u00b5L T4 RNAligase (Promega). The ligation was purified by phenol-chloroform extraction andethanol precipitation. The purified product was reverse-transcribed using aCHLI-specific reverse primer (5\u2032AGCAGTTGGGAATGACAGTGGC 3\u2032). Primary PCR was thenperformed using a forward primer matching the RNA adaptor (5\u2032 AACAGACGCGTGGTTACAGTC 3\u2032)and the CHLI reverse primer (as above). The RT-PCR product was then amplifiedusing the same forward primer with a nested CHLI reverse primer (5\u2032 ATATCTTCCGGAGTTACCTTATC3\u2032). The nested PCR product was separated on a2% agarose gel, purified with the Ultra Clean-15 DNA purification kit (MoBio Laboratories), and ligated into the pGEM-T Easy vector (Promega) forsequencing.Total RNA (2 \u00b5g) was ligated to a 24-nt RNA adaptor at a wavelength of 663 nm and 645 nm respectively.Chlorophyll concentration was measured as nmol per gram of fresh weight.Figure S1N. tabacum with the CMV helper virus alone doesnot induce silencing of the CHLI gene. Approximately 10 \u00b5g of totalRNA from uninfected (lanes 1\u20132) and CMV-infected (lanes 3\u20134)were separated in formaldehyde-agarose gel, transferred to Hybdond-Nmembrane, and hybridized with 32P-labelled antisense RNA of theCMV coat protein (CMV-CP) sequence.Infection of (TIF)Click here for additional data file.Figure S2Distribution of plus (+) strand-specific siRNAs along the Y-Sat genome.A total of 1 million 21 to 22-nt (+) strand Y-Sat siRNAs were obtainedfrom the total sRNA sequencing population of approximately 4 million sRNAreads. Each point represents the number of reads (the Y-axis), and theposition of 5\u2032 terminal nucleotide of each detected siRNA along theY-Sat genome (the X-axis). The black line on the bottom represents thefull-length Y-Sat genome, in which the \u201cyellow domain\u201d is drawnin orange. Note that the \u201cyellow domain\u201d corresponds to a siRNAhot spot.(TIF)Click here for additional data file.Figure S3et al. and Kuwata etal. are consistentwith CHLI silencing being the cause of Y-Sat-induced yellowing symptoms. Asshown by the sequence alignment, Y-Sat mutants capable of causing theyellowing symptoms have a higher level of sequence complementaritywith the CHLI target sequence than those that do not inducethe yellowing symptoms. For instance, the Y5 sequence has strongercomplementarity with the CHLI sequence than the original Y-Sat sequence. Y7contains an introduced C-A mismatch, but this is compensated by thesubstitution of a G:U wobble pair with a strong G:C pair. All threenon-disease-causing mutants have more mismatches, or G:Uwobble pairs, with respect to the CHLI sequence, than the original Y-Satsequence. Underlined letters are the modified nucleotides in the Y-Satmutants. Perfectly matched nucleotides are shown in blue and mis-matchedones in red. Green letters indicate nucleotides that can form G:U wobblepairs with the CHLI sequence. The Y-Sat \u2018yellow domain\u2019 sequenceis boxed. The CHLI sequence matching the original Y-Sat sequence is shown inred. The \u2018+\u2019 and \u2018\u2212\u2019 symbols respectivelyindicate the presence and absence of Y-Sat yellowing symptoms uponinfection.Results from the mutagenesis studies by Jaegle (TIF)Click here for additional data file.Table S1Phenotypes of independent wtCHLI and mtCHLI transgenic tobacco lines inresponse to Y-Sat infection.(DOC)Click here for additional data file.Text S1Sequences of the tobacco CHLI gene.(DOC)Click here for additional data file."} +{"text": "Out-of-hospital cardiac arrest (OOHCA) still has a low survival rate, despite considerable efforts including early applications of basic life support and defibrillation in the pre-hospital setting. Post-resuscitation care after hospitalization, influencing the final outcome, may be less available during nights and weekends because of hospital, staffing, and response factors. We sought to determine whether outcomes after OOHCA differ during nights and weekends (off-hours) compared with daytimes of weekdays (on-hours).We performed a retrospective analysis of 4-year data collected prospectively in a single institute. Adults with witnessed OOHCA of cardiac origin were recruited. The therapeutic strategy after hospitalization, including extracorporeal cardiopulmonary resuscitation (ECPR), therapeutic hypothermia (TH) and primary percutaneous coronary intervention (PCI), was dependent on the critical care physicians in charge. We used a propensity-score matching to reduce the differences of pre-hospital variables between patients arriving during off-hours and on-hours. Primary endpoint was 90-day survival after cardiac arrest. We evaluated the survival difference using the log-rank test and identified the significant interventions affecting outcome using the Cox regression model.P = 0.025). Multivariate Cox regression analysis showed that TH was associated with 90-day survival after cardiac arrest (adjusted hazard ratio (HR), 0.43; 95% CI, 0.23 to 0.79), but there were no significant associations of ECPR and primary PCI .Of 185 patients, 131 arrived during off-hours (the off-hours group) and 54 arrived during on-hours (the on-hours group). The matching process selected 37 patients each from both groups. The matched off-hours group had a lower survival rate than the matched on-hours group (10.8% vs. 37.8%; log-rank Lower survival rates after OOHCA during nights and weekends were seen at our institute. TH was more likely to be induced in patients arrived during daytimes of weekdays, and independently associated with survival benefit."} +{"text": "This study integrates two lines of research on the AUDIT-C: a study which estimated average alcohol consumption at each AUDIT-C score using national U.S. population data, and a set of studies which evaluated the association of AUDIT-C scores with alcohol-related health outcomes. This presentation synthesizes results from these studies to depict the association between 1) AUDIT-C scores, and 2) mean daily alcohol consumption as well as adverse alcohol-related health outcomes.Mean daily alcohol consumption (drinks/day) at each AUDIT-C score (0-12 points) was evaluated in U.S. adults who provided detailed reports of consumption on the 2000-2001 National Epidemiologic Survey on Alcohol and Related Conditions (NESARC) and reported past-year drinking . Analyses were limited to NESARC participants age 45 and over to parallel the age of the samples in the health outcomes studies (below). The associations between AUDIT-C scores and subsequent risk for the following alcohol-related health outcomes were assessed in U.S. Veterans Health Administration (VA) patients: patient self-management of hypertension and diabetes; medication adherence; surgical complications; hospitalizations for upper GI bleeding, liver disease, and pancreatitis; trauma; inpatient health care utilization, and mortality.The presentation will graphically depict the relationship between AUDIT-C scores and mean daily alcohol consumption and identify AUDIT-C scores and mean daily alcohol consumption associated with each adverse health outcome. For example, patients with AUDIT-C scores of 7 drink an average of 2.7 drinks daily and are at increased risk for medication non-adherence, postoperative complications, and GI hospitalizations.This information may help primary care clinicians recognize the value of a scaled alcohol screen, routinely used in some primary care settings, including its utility for providing patients with feedback on their alcohol-related risks during brief alcohol interventions."} +{"text": "Non-transformed mammary epithelial cell lines such as MCF-10A recapitulate epithelial morphogenesis in three-dimensional (3D) tissue culture by forming acinar structures. They represent an important tool to characterize the biological properties of oncogenes and to model early carcinogenic events. So far, however, these approaches were restricted to cells with constitutive oncogene expression prior to the set-up of 3D cultures. Although very informative, this experimental setting has precluded the analysis of effects caused by sudden oncoprotein expression or withdrawal in established epithelial cultures. Here, we report the establishment and use of a stable MCF-10A cell line (MCF-10Atet) fitted with a novel and improved doxycycline (dox)-regulated expression system allowing the conditional expression of any transgene.BRAF-IRES-GFP, which responds to dox treatment with the production of a bi-cistronic transcript encoding hemagglutinin-tagged B-Raf and green fluorescent protein (GFP). This improved conditional expression system was then characterized in detail in terms of its response to various dox concentrations and exposure times. The plasticity of the phenotype provoked by oncogenic B-RafV600E in MCF-10Atet cells was analyzed in 3D cultures by dox exposure and subsequent wash-out.MCF-10Atet cells were generated by stable transfection with pWHE644, a vector expressing a second generation tetracycline-regulated transactivator and a novel transcriptional silencer. In order to test the properties of this new repressor/activator switch, MCF-10Atet cells were transfected with a second plasmid, pTET-HAV600E as a model oncoprotein, we show that its sudden expression in established 3D cultures results in the loss of acinar organization, the induction of an invasive phenotype and hallmarks of epithelial-to-mesenchymal transition (EMT). Importantly, we show for the first time that this severe transformed phenotype can be reversed by dox wash-out and concomitant termination of oncogene expression.MCF-10Atet cells represent a tightly controlled, conditional gene expression system. Using B-RafTaken together, we have generated a stable MCF-10A subline allowing tight dox-controlled and reversible expression of any transgene without the need to modify its product by introducing artificial dimerization or ligand-binding domains. This system will be very valuable to address phenomena such as EMT, oncogene addiction, oncogene-induced senescence and drug resistance. Notably, not more than a decade ago, several laboratories studying epithelial cells and their transformed counterparts began to grow epithelial cells in three-dimensional (3D) culture systems, which recapitulate many facets of epithelial tissues in vivo . Th. ThS-M2)onstruct . A direconstruct revealedonstruct , which ir Figure and their Figure . The latRAS or BRAF genes are rarely observed in the overall majority of breast cancers, they occur more frequently in cell lines derived from basal type breast cancers, a rare, but difficult to treat subtype [BRAF-IRES-GFP-bsr or pTET/HAhBRAFV600E-IRES-GFP-bsr . Given the potency of BRAFV600E to transform MCF-10A cells in 2D and 3D culture [BRAF-IRES-GFP-bsr constructs, as we have shown previously that the expression of B-Rafwt and GFP is well-tolerated in MCF-10A cells and does not induce dramatic phenotypic alterations as B-RafV600E [BRAF-IRES-GFP cassette is efficiently repressed in the absence of dox. This was also confirmed using highly sensitive anti-HA and GFP antibodies in Western blot analyses . However, dox concentrations as little as 0.02 \u03bcg/ml induce transgene expression, while concentrations from 0.2 \u03bcg/ml induce an almost complete induction of transgene expression. We also noticed that a higher dox concentration did not further increase transgene expression levels, which is in agreement with dose-response curves established in HeLa cell lines stably transfected with rtTA2S-M2 . One poM2 of MCF-10A cells and therefore, if expressed a priori, prevents acinar morphogenesis [V600E would impinge on later stages of acinar morphogenesis and homeostasis. Therefore, MCF-10Atet cells stably transfected with pTET/HAhBRAFV600E-IRES-GFP-bsr plasmids were seeded into matrigel as described previously [V600E expressing cells also protrude from the former acinus into the matrigel indicating that evading cells have destroyed the basal lamina . In this experimental setting, RTKs were fused with dimerization domains that can be cross-linked by an artificial ligand. However, this system relies strongly on the principle that RTKs often use dimerization as a mechanism for activation ,38. SimiV600E as a model oncogene, we have further substantiated that aberrant Raf-signaling counteracts lumen formation and confers invasive properties to MECs grown in 3D culture. We provide evidence that the impaired lumen formation in B-RafV600E expressing cells results most likely from the combination of a lack of proliferative suppression in late stage cultures and the failure to induce systematic luminal apoptosis. Our observations are in full agreement with previous studies showing that increased MEK/ERK signaling in 3D cultures of MCF-10A cells blocks luminal clearance by preventing the function and expression of the pro-apoptotic BIM protein, a critical regulator of acinar morphogenesis [V600E is a potent suppressor of BIM in colorectal cancer models [V600E is more drastic than that induced by \u0394Raf-1:ER. Firstly, this oncogenic Raf protein induces a complete EMT as judged by the loss of E-Cadherin and the gain of vimentin expression in hypo-osmolar electroporation buffer (Eppendorf) with 30 \u03bcg AhdI-linearized plasmid DNA by three electroporation pulses, each separated by a 1 min interval, using an Eppendorf multiporator set at 750 V and 70 \u03bcS. However, we have recently noticed that nucleofection of 2 \u03bcg AhdI-linearized plasmid DNA and program T-024 using solution T (Amaxa) gave better transfection efficiencies than conventional electroporation (data not shown). Regardless of the transfection method, the cells were placed under selection using the appropriate antibiotics (puromycin (Sigma): 1.5 \u03bcg/ml; blasticidin S (Roth): 6 \u03bcg/ml) following a recovery period for 24 h in conventional growth medium. MCF-10A cells were set up for 3D culture experiments as described previously [Parental MCF-10A cells were obtained from the stock collection at the Garvan Institute of Medical Research, Sydney, and represent an early passage population from an import from ATCC. Its subline, MCF-10AecoR , has been described by various assays and in detail before ,44,46-48promoter to exprence gene . Detailseviously ,25. Fixaeviously ,25.wt were plated and allowed to adhere for 24 h. Subsequently, the cells were incubated with the indicated dox concentrations. After 24 h, they were harvested by trypsinization and subjected to FACS analysis using a CyAN cytometer (Beckman-Coulter) and standard gating procedures to exclude non-viable cells. Time-course experiments were set up as described for dose-response experiments using a dox concentration of 2 \u03bcg/ml. At the indicated time points, the cells were harvested and analyzed by FACS.Dose-response experiments as shown in Figure BRAF-IRES-GFP cassettes were amplified by PCR from the retroviral pMIG/HAhBRAFwt or pMIG/HAhBRAFV600E expression vectors [NotI site into the PCR amplicon. The PCR amplicons were then subcloned into the cloning vectors pBSSK+ or pSCA-Amp/Kan (Stratagene) for further propagation. The subcloned amplicon was then recovered by NotI digestion and subcloned into NotI-linearized pTET-bsr. This plasmid is derived from pWHE636 and contains as novel features a NotI site in its multicloning site and a loxP-flanked blasticidin S resistance (bsr) cassette derived from pAloxP-bsr [PTRE, the second generation promoter PSG-TRE and green fluorescent protein (GFP), the HAh vectors using olloxP-bsr . pWHE636loxP-bsr ) and conRE (Ref. ) to drivRE (Ref. ). DetailloxP-bsr by BamHI\u00ae 546 goat anti-rabbit IgG, Alexa Fluor\u00ae 488 goat-anti rabbit IgG and Cy3 goat anti-mouse IgG . The polyclonal antibody against the Tet-transregulators was generated in-house in the laboratory of Dr. Christian Berens.Two D and 3D cultures of MCF-10A cells were processed to total cellular lysates and analyzed by Western blotting as described previously . LysatesFACS-analysis was performed to measure GFP expression in cells transfected with IRES-based bicistronic vectors. Cells were harvested by standard trypsinization as descr3D: Three dimensional; 4-HT: 4-hydroxy-tamoxifen; BSR: Blasticidin S resistance, CMV: Cytomegalovirus; DCIS: Ductal carcinoma in situ; Dox: Doxycycline; EGFP: Enhanced green fluorescent protein; EMT: Epithelial-to-mesenchymal transition; ER: Estrogen receptor; ERK: Extracellular signal-regulated kinase; FACS: Fluorescence activated cell sorter; GFP: Green fluorescent protein; HA: Hemagglutinin; IRES: Internal ribosomal entry site, MECs: Mammary epithelial cells; MMP: Matrix metalloproteinase; Raf: rapidly growing fibrosarcoma; Ras: Rat sarcoma; rtTA: Reverse tetracycline-dependent trans-activator; PCR: Polymerase chain reaction; RTK: Receptor tyrosine kinase; Tet: Tetracycline; tTS: Tetracycline-dependent trans-silencer; MEK: Mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase.The authors declare that they have no competing interests.RH performed all the functional assays in the manuscript and generated the MCF-10Atet cell line. RH, FUW, CD, CB and TB generated the novel expression constructs and/or were involved in their design. RH und FUW performed confocal microscopy. All authors contributed to data analysis. The manuscript was written by TB with input from RH, FUW and CB. All authors read and approved the manuscript.SM2K and Jr2SM2PPRegulatory properties of the cell lines Jr2. Jurkat cells were stably transfected with pWHE459 or pWHE644 resulting in the regulator lines Jr2SM2K and Jr2SM2PP, both expressing the transactivator variant rtTA2S-M2 and either the transrepressor tTSD-KRAB or tTSD-PP, respectively. To test their respective regulatory capacity, single-cell clones of each cell line were transiently transfected with 800 ng pUHC13-3, coding for firefly luciferase under Tet-control. For each cell line, three well-regulating clones are depicted above. The basal luciferase activity obtained in likewise transfected Jurkat cells not containing any transregulators is shown on the right and serves as control for repression by tTS and activation of transgene expression by rtTA.Click here for fileQuantification of normal and aberrant MCF-10A acini. Cells harboring dox-inducible B-RafWT, B-RafV600E and vector control constructs were seeded into matrigel, induced at day 17 and subjected to dox withdrawal at day 29. Representative micrographs were taken at day 59. At least 34 acini were counted and categorized into non-invasive phenotype and invasive phenotype (minimum three cells protruding from the acinus).Click here for file"} +{"text": "Genetic mosaic techniques have been used to visualize and/or genetically modify a neuronal subpopulation within complex neural circuits in various animals. Neural populations available for mosaic analysis, however, are limited in the vertebrate brain.HSP:Cre) and Tg (HuC:loxP-DsRed-loxP-GFP). We confirmed medaka HuC promoter-derived expression of the reporter gene in juvenile medaka whole brain, and in neuronal precursor cells in the adult brain. We then demonstrated that stochastic recombination can be induced by micro-injection of Cre mRNA into Tg (HuC:loxP-DsRed-loxP-GFP) embryos at the 1-cell stage, which allowed us to visualize some subpopulations of GFP-positive cells in compartmentalized regions of the telencephalon in the adult medaka brain. This finding suggested that the distribution of clonally-related cells derived from single or a few progenitor cells was restricted to a compartmentalized region. Heat treatment of Tg(HSP:Cre x HuC:loxP-DsRed-loxP-GFP) embryos (0\u20131 day post fertilization [dpf]) in a thermalcycler (39\u00b0C) led to Cre/loxP recombination in the whole brain. The recombination efficiency was notably low when using 2\u20133 dpf embyos compared with 0\u20131 dpf embryos, indicating the possibility of stage-dependent sensitivity of heat-inducible recombination. Finally, using an infrared laser-evoked gene operator (IR-LEGO) system, heat shock induced in a micro area in the developing brains led to visualization of clonally-related cells in both juvenile and adult medaka fish.To establish methodology to genetically manipulate neural circuits in medaka, we first created two transgenic (Tg) medaka lines, Tg (We established a noninvasive method to control Cre/loxP site-specific recombination in the developing nervous system in medaka fish. This method will broaden the neural population available for mosaic analyses and allow for lineage tracing of the vertebrate nervous system in both juvenile and adult stages. Cre induction can be spatially controlled by cell type-specific promoters/enhancers and site-specific viral infection Cre induction. Local heat treatment using a metal probe and an infrared laser results in ectopic Cre induction in a small number of cells in various tissues such as the gonads and epidermal tissues in medaka fish Genetic mosaic analysis is a powerful tool in the fields of neuroscience and developmental biology for labeling a subset of neurons, tracing cell-lineage, and modulating neuronal function Cre expression in neural precursor cells of medaka embryos Here we used an infrared laser-evoked gene operator (IR-LEGO) system to induce highly regulated spatiotemporal HuC. HuC belongs to a family of vertebrate neuronal-specific genes homologous to the Drosophila elav and serves as an early marker of differentiating neurons HuC is expressed in neuronal precursor cells during embryogenesis, and then high expression levels persist in most regions of post-hatching and larval brain HuC promoter is widely used for visualizing and/or modifying the function of neural circuits in juvenile fish HuC promoter is also applicable for visualizing the differentiation process during adult neurogenesis, as HuC expression is restricted to newborn and differentiating neurons in the adult zebrafish brain To examine whether a heat-inducible Cre/loxP gene induction system works in the medaka nervous system, we generated transgenic (Tg) medaka lines for the detection of Cre/loxP recombination with the promoter regions of medaka The work in this paper was conducted using protocols approved by the Animal Care and Use Committee of the University of Tokyo (permit number: 12\u201307). All surgery was performed under cold anesthesia, and all efforts were made to minimize suffering.Oryzias latipes, drR strain) and all Tg lines, Tg (HuC:loxP-DsRed-loxP-GFP) and Tg (HSP:Cre), were maintained in their respective groups in plastic aquariums (12 cm \u00d713 cm \u00d719 cm). All fish were hatched and bred in our laboratory. The water temperature was maintained at \u223c28\u00b0C and the light was provided by fluorescent lamps for 14 h per day (08\u223600 to 22\u223600).Medaka fish with KOD DNA polymerase (TOYOBO) and the following sets of primers carrying the indicated restriction enzyme sites (underlined): 5'-CCGCTCGAGCGGTTTTGTTGCACCGCTAATGTTAGG-3' and 5'-TCCCCGCGGGGAGTACAATGAAAGAAATCTAGGTCC-3', which contain the recognition sites for XhoI and SacII, respectively. ploxP-DsRed-loxP-EGFP plasmid was obtained from Prof. Tanaka The medaka mgfc:TagBFP-8xHSE:Cre containing fused gene of Cre recombinase with N-terminal nuclear localization sequence and the red fluorescent protein mCherry under the control of artificial heat shock inducible promoter gamma F crystalline promoter HSP:Cre transgene by blue fluorescence of their eyes without heat treatment. Insulators were inserted between the polyA sequence under the control of HSP promoter and TR3, and also inserted into the boundary region of the two promoters, HSP and Crystalline to prevent position-effects of the transgenes. The F1 embryos from wild type and vector-injected F0 were raised to sexual maturity and screened for germline transmission by a fluorescent microscopy examining BFP expression.The dual expression vector, pPBIS19-/eA2 solution glycerol and 0.1% (wt/vol) Triton X-100 http://www.fluorender.com).Fixed brains were washed in phosphate buffered saline (PBS) containing 0.5% Triton (PBST) and incubated in ScaImmunostaining was performed on 14-\u00b5m cryosections. Whole brains were embedded in OCT compound (Sakura Tissue Tek) and cut using Cryostat . The sections were blocked with 0.2%Triton, 1% dimethylsulfoxide, and 2% bovine serum albumin in PBS at room temperature for 1 h, then incubated in the primary antibodies diluted in the blocking buffer at 4\u00b0C overnight. Primary antibodies used in this study were mouse anti-HuC/D , rabbit anti-DsRed . Primary antibodies were detected by subclass-specific secondary antibodies labeled with Alexa 488/548 , respectively.in vitro synthesis of mRNAs were performed as described previously http://www.fluorender.com).Cre-SV40 was cloned into a pGEM-T easy vector . Template preparation and Ten embryos were placed into a tube with 200 \u00b5l medaka hatching buffer Embryos were mounted at stage 24, dorsal side up, in a drop of 2% methylcellulose and observed using the 20\u00d7 custom made objective lens for an Olympus epifluorescence microscope with IR-LEGO unit . Heat was induced in the telencephalon by 1480-nm light generated by a high-power single mode CW Raman fiber laser , as previously described DsRed-loxP-GFP under the control of a 3.3-kb medaka HuC promoter (HuC:loxP-DsRed-loxP-GFP). DsRed expression in the Tg embryo was first detected as early as stage 27 in the anterior brain vesicle-intermediate brain vesicle labeled the neural progenitor cells with huC expression. Furthermore, to confirm that DsRed is expressed in HuC-positive neural progenitors, we performed co-staining with antibody against HuC/D and DsRed by immunohistochemistry. The pattern of HuC/D expression overlapped with that of DsRed, including the habenular nucleus under the control of a ubiquitous beta-actin promoter HuC:loxP-DsRed-loxP-GFP) at the 1-cell stage (30 min after fertilization). Recombination by Cre mRNA injection led to changes in the expression of DsRed to that of GFP into Tg and generated three-dimensional images of the telencephalon of the Cre mRNA (20 ng/\u00b5l)-injected fish . InteresHSP:Cre/Cry:BFP) with an artificial HSP promoter containing eight consecutive artificial heat-shock response elements and beta-globin minimum promoter derived from Xenopus laevis. Expression of Cre in the Tg (HSP:Cre) is reported to be inducible by a brief heat shock treatment. The construct expressed blue fluorescent protein (BFP) under the control of the mouse Crystallin (Cry) promoter as a screening marker, and an insulator sequence was inserted between HSP:Cre and Cry:BFP (HSP and Cry promoters which are located in the same construct of the vector. We generated Tg (HuC:loxP-DsRed-loxP-GFP/HSP:Cre/Cry:BFP) by genetic crossing.To induce the recombination by heat treatment, we established a Cre-inducible Tg ( Cry:BFP [29] to HuC:loxP-DsRed-loxP-GFP/HSP:Cre/Cry:BFP) embryos (HuC:loxP-DsRed-loxP-GFP) (HuC:loxP-DsRed-loxP-GFP/HSP:Cre/Cry:BFP) embryos at the early blastula stage . GFP fluorescence was detected at the time of hatching in the whole brain of Tg ( embryos .Cre expression by laser irradiation HuC:loxP-DsRed-loxP-GFP/HSP:Cre/Cry:BFP) embryos juvenile and adult fish only in the right side (HuC:loxP-DsRed-loxP-GFP) or non-treated Tg (HuC:loxP-DsRed-loxP-GFP/HSP:Cre/Cry:BFP) fish system to inducs 2 dpf, by irradght side . The juvFP) fish and alsoThe present study demonstrated that injection of a low concentration of Cre mRNA induces stochastic Cre/loxP recombination. There are two possible reasons for the stochastic Cre/loxP recombination. The first is that the injected mRNA may be sparsely distributed. The second is that a low concentration in the cells may cause stochastic recombination, which is consistent with previous reports showing that low transcriptional Cre activity leads to stochastic recombination Next, we established a noninvasive method for controlled Cre/loxP site-specific recombination in the nervous system during medaka embryogenesis, which allowed us to visualize and/or modify the formation of a lineage-dependent structure. Recent studies with an advanced Gal4-UAS system in zebrafish promoted the genetic dissection of neural circuits HuC promoter activity is restricted to newborn and differentiating neurons. Thus, heat-inducible Cre/loxP site-specific recombination is also applicable for investigating adult neurogenesis in medaka fish. In contrast to mammals with limited neurogenesis in the adult brains, teleost fish such as medaka fish and zebrafish constitutively generate newborn neurons in numerous proliferating zones across the whole brain throughout life Furthermore, in the adult brain, medaka Figure S1DsRed expression in HuC: loxP-DsRed-loxP-GFP Tg medaka. (A\u2013C) Onset of DsRed expression in the brain from the lateral view at Stages 27 (A), 34 (B), and 40 (C). (D\u2013G) Photomicrographs depicting DsRed (red), HuC/D (green), DAPI (blue), and their merged image immunofluorescence in the HuC: loxP-DsRed-loxP-GFP Tg brain in the preoptic area (D), optic tectum and hypothalamus , and cerebellum (G). Scale bar, 100 \u00b5m.(TIFF)Click here for additional data file.Figure S2Schematic drawing of HuC-expressing neurons. Magenta dots indicate the position of the HuC-expressing neurons. A, nucleus anterioris of diencephalon; AOT, tractus opticus (optic tract) axialis; Cb, corpus cerebelli; CbSg, stratum granulare of corpus cerebelli; CbSm, stratum moleculare of corpus cerebelli; CbSp, stratum Purkinje of corpus cerebelli; CM, corpus mamillare; D, area dorsalis telencephali; Dc, area centralis of D; Dl, area lateroposterioris of D; Dld, area laterodorsalis of D; Dlp, posterior subdivision of dorsolateral telencephalon; Dlv, area lateroventralis of D; Dm, area medialis of D; DOT, tractus opticus (optic tract) dorsalis; Dp, dorsal posterior telencephalon; DT, nucleus tegmentalis dorsalis; ECL, external cell layer of olfactory bulb; EP, epiphysis; ep, ependyme; Fll, fasciculus longitudinalis lateralis; Flm, fasciculus longitudinalis medialis; Flt, fasciculus longitudinalis lateralis telencephali; GL, glomerular layer of olfactory bulb; gc, griseum central; Hc, hypothalamus caudalis; Hd, nucleus dorsalis of habenula; HD, hypothalamus periventricularis dorsalis; Hv, nucleus ventralis of habenula; IQ, inferior oblique of nucleus of nervus oculomotorius; IR, inferior rectus of nucleus of nervus oculomotorius; MC, commissural minor; MOT, tractus opticus (optic tract) medialis; MR, medial rectus of nucleus of nervus oculomotorius; NCILP, nucleus centralis posterioris of lobus inferiosis; NDIL, nucleus diffusus of lobus inferioris; NDTL, nucleus diffusus of torus lateralis; NGp, nucleus glomerulosus medialis; ON, nervus olfactorius; OT, optic tectum; Pc, nucleus pretectalis centralis; PGc, nucleus preglomerulosus centralis; PGm, nucleus preglomerulosus medialis; PGZ, periventricular grey zone; PMp, nucleus preopticus magnocellularis pars parvocellularis; PPa, nucleus preopticus periventricularis, anterioris; PPp, nucleus preopticus parvocellularis posterioris; PSi, nucleus pretectalis superficialis pars intermedialis; PSm, nucleus pretectalis superficialis pars medialis; rp/V3, recessus preopticus of ventriculus tertius; RT, nucleus tegmentalis rostralis; SC, nucleus suprachiasmaticus; SR, superior rectus of nucleus of nervus oculomotorius; TA, nucleus tuberis posterioris; TCT, tractus cerebellotectalis; TIT, tractus isthmotectalis; TP, nucleus tuberis posterioris; TS, torus semicircularis; v3, ventriculus mesencephali; v4, ventriculus quartus; V, area ventralis of the telencephalon; VC, valvula cerebelli; Vd, area dorsalis of V; Vi, area intermedialis of V; Vl, ventral telencephalon, lateral subdivision; VL, nucleus ventrolateralis; VM, nucleus ventromedialis; vm, ventriculus mesencephali; VOT, tractus opticus (optic tract) ventralis; Vp, ventral telencephalon, posterior subdivision; Vv, area ventralis of V.(TIFF)Click here for additional data file.Figure S3Cre/loxP recombination by Cre mRNA injection. (A) Schematic drawing of Cre/loxP recombination in Tg (beta actin:loxP-DsRed-loxP-GFP). (B\u2013F) Prominent GFP expression was observed in the whole body of Cre mRNA-injected embryos. Cre mRNA-injected embryos and negative control embryos are shown from dorsal and lateral views. Scale bar, 100 \u00b5m. (F) Mosaic pattern of GFP fluorescence in the Cre mRNA-injected Tg (HuC: loxP-DsRed-loxP-GFP) adult medaka brain. Different GFP patterns were observed in individual brains. Scale bar, 1 mm.(TIFF)Click here for additional data file.Movie S1Serial optical sections corresponding to (AVI)Click here for additional data file."} +{"text": "Drosophila melanogaster and its native Spiroplasma symbiont strain MSRO were investigated as to how the host's molecular, cellular and morphogenetic pathways are involved in the symbiont-induced male-killing during embryogenesis. TUNEL staining, anti-cleaved-Caspase-3 antibody staining, and apoptosis-deficient mutant analysis unequivocally demonstrated that the host's apoptotic pathway is involved in Spiroplasma-induced male-specific embryonic cell death. Double-staining with TUNEL and an antibody recognizing epidermal marker showed that embryonic epithelium is the main target of Spiroplasma-induced male-specific apoptosis. Immunostaining with antibodies against markers of differentiated and precursor neural cells visualized severe neural defects specifically in Spiroplasma-infected male embryos as reported in previous studies. However, few TUNEL signals were detected in the degenerate nervous tissues of male embryos, and the Spiroplasma-induced neural defects in male embryos were not suppressed in an apoptosis-deficient host mutant. These results suggest the possibility that the apoptosis-dependent epidermal cell death and the apoptosis-independent neural malformation may represent different mechanisms underlying the Spiroplasma-induced male-killing. Despite the male-specific progressive embryonic abnormality, Spiroplasma titers remained almost constant throughout the observed stages of embryonic development and across male and female embryos. Strikingly, a few Spiroplasma-infected embryos exhibited gynandromorphism, wherein apoptotic cell death was restricted to male cells. These observations suggest that neither quantity nor proliferation of Spiroplasma cells but some Spiroplasma-derived factor(s) may be responsible for the expression of the male-killing phenotype.Some symbiotic bacteria cause remarkable reproductive phenotypes like cytoplasmic incompatibility and male-killing in their host insects. Molecular and cellular mechanisms underlying these symbiont-induced reproductive pathologies are of great interest but poorly understood. In this study, Drosophila fruit flies, infection with Spiroplasma symbionts often causes male-specific embryonic mortality, resulting in the production of all-female offspring. This striking phenotype is called \u201cmale-killing\u201d, whose underlying mechanisms are of great interest. Here we investigated Drosophila melanogaster and its native Spiroplasma symbiont strain to understand how the host's molecular, cellular and morphogenetic pathways are involved in the symbiont-induced male-killing. Specifically in Spiroplasma-infected male embryos, pathogenic phenotypes including massive cell death throughout the body and neural malformation were observed. We unequivocally identified that the male-specific cell death preferentially occurs in the embryonic epithelium via the host's apoptotic pathway. Meanwhile, we found that, unexpectedly, the male-specific neural defects occur independently of host's apoptosis, suggesting that at least two different mechanisms may be involved in the Spiroplasma-induced male-killing. Also unexpected was the finding that Spiroplasma titers are almost constant throughout embryogenesis irrespective of sex despite the male-specific severe apoptosis. We serendipitously found Spiroplasma-infected sexual mosaic embryos, wherein apoptosis was associated with male cells, which suggests that some Spiroplasma-derived factor(s) may selectively act on male cells and cause male-killing.Symbiotic bacteria are ubiquitously associated with diverse insects, and affect their host biology in a variety of ways. In Wolbachia, Cardinium and Spiroplasma are generally parasitic rather than beneficial to their hosts, often causing negative fitness effects and also inducing reproductive phenotypes like cytoplasmic incompatibility, male-killing, parthenogenesis or feminization, by which these symbionts are able to spread their own infections into the host populations in a selfish manner Symbiotic microorganisms are ubiquitously associated with diverse insects, and affect their host biology in a variety of ways Spiroplasma, belonging to the class Mollicutes, are wall-less bacteria associated with diverse arthropods and plants Spiroplasma species and strains are known to cause male-killing phenotypes in fruit flies, ladybird beetles and butterflies, wherein infected females produce all-female or female-biased offspring due to male-specific mortality during embryogenesis and/or larval development Spiroplasma poulsoniiDrosophila, which are represented by the strains WSRO from D. willistoni, NSRO from D. nebulosa, MSRO from D. melanogaster and others Members of the genus Drosophila-Wolbachia symbiosis represents one of the best-studied model symbiotic systems Drosophila-Spiroplasma symbiosis has also been well-studied as another model system of infection dynamics Spiroplasma-induced male-specific embryonic pathology are still not well understood. Histological observations, mosaic analysis and in vitro culturing have suggested that nervous system is among the major target sites of Spiroplasma-induced male-killing in Drosophila embryos D. melanogaster, Spiroplasma-infected mutants deficient in dosage compensation complex genes fail to show male-killing phenotype, indicating that a functional dosage compensation complex is required for expression of the Spiroplasma-induced make-killing D. nebulosa infected with its native Spiroplasma strain NSRO, dying male embryos exhibit widespread TUNEL signals, suggesting possible involvement of host's pathway of programmed cell death or apoptosis While the D. melanogaster infected with its native Spiroplasma strain MSRO. In particular, we focused on host's molecular, cellular and morphogenetic pathways that may potentially be involved in the male-killing phenotype by utilizing the wealth of genetic resources available in D. melanogaster. Our observations unveiled several previously unknown aspects of Spiroplasma-induced male-killing, which include unequivocal demonstration of male-specific up-regulation of apoptotic pathway, identification of embryonic epithelium as the main target of male-specific apoptosis, male-specific malformation of embryonic nervous system independent of apoptosis, and specific killing of male cells in gynandromorphic embryos.In this study, we performed detailed investigation of the male-killing process during embryogenesis of Spiroplasma-infected female embryos Spiroplasma-infected male embryos exhibited more immunopositive signals than Spiroplasma-infected female embryos as well as uninfected male and female embryos, and the spatiotemporal patterns of the signals looked similar to those of the TUNEL signals , Spiroplasma titers per embryo remained almost constant, exhibiting no significant differences between male embryos and female embryos (Spiroplasma titers per host elongation factor 1\u03b1 100E (EF1a) gene copy exhibited higher values in male embryos than in female embryos , head involution defective (hid) and grim, which are collectively termed RHG genes Drosophila mutant H99 deficient in all these genes, apoptotic cell death is almost completely blocked during embryogenesis Drosophila inhibitor of apoptosis protein 1 (DIAP1) and disrupt its ability to inhibit caspase activity, by which apoptosis is triggered Spiroplasma-infected H99 mutant embryos were subjected to the TUNEL assay, TUNEL-positive cells were observed neither in female embryos nor in male embryos at stages 11 and 12 , some TUNEL-positive cells were detected specifically in embryos . The resDrosophila development is the expression control of RHG genes rpr by binding to its enhancer elements Spiroplasma-infected male embryos and female embryos, no sex-related differences were observed in their localization patterns localizes to the subapical region (SAR) in the epithelial junctions, thereby establishing apical-basal cell polarity protein is specifically expressed in differentiated neural cells Spiroplasma-infected male embryos, both CNS and PNS were disorganized . GMCs divide once to give rise to two neurons . Alternatively, the defects in neural tissues may somehow influence the organization of adjacent epithelial cells, thereby causing the male-specific epithelial apoptosis secondarily, or vise versa.In the light of these results, it is of focal interest how the epithelial apoptosis and the neural malformation in the normally , while iobserved . These rSpiroplasma-infected embryos, we occasionally identified gynandromorphic embryos with mosaic expression of Sxl host's apoptotic pathway is up-regulated in a male-specific manner, (ii) the male-specific apoptosis mainly targets embryonic epithelial cells, (iii) as previously reported, remarkable neural malformation is observed in male embryos, (iv) however, neither differentiated neural cells nor precursor neural cells exhibit apoptosis in male embryos, (v) the male-specific neural malformation occurs even when host's apoptotic pathway is disrupted, and (vi) therefore, the apoptosis-dependent epidermal cell death and the apoptosis-independent neural malformation may represent different mechanisms underlying the Spiroplasma-induced lethality in male embryos. We also found that (vii) Spiroplasma titers remain almost constant throughout the embryonic development and across male and female embryos, (viii) although at a low frequency (\u223c2%), gynandromorphic embryos are found in the Spiroplasma-infected embryos, (ix) in these embryos, apoptotic cell death is preferentially observed in male cells, and (x) therefore, neither quantity nor proliferation of Spiroplasma but some Spiroplasma-derived factor(s) selectively acting on host's male cells may be responsible for the expression of male-killing phenotype. These findings highlight complex molecular and cellular interactions in the Spiroplasma-Drosophila symbiosis, and provide invaluable clues to our deeper understanding of the symbiont-induced manipulation of host's development and reproduction.In conclusion, our study unveiled previously unknown molecular and cellular aspects underlying the D. melanogaster were raised at 25\u00b0C on a standard cornmeal diet in plastic tubes unless otherwise indicated. Oregon-R (wild-type strain) was provided by Takehide Murata . Sxl-Pe-EGFP G78b ri-1Df(3L)H99, kni, pp/TM3, 1SbDrosophila Genetic Resource Center (DGRC) at Kyoto Institute of Technology, Japan, respectively. After tetracycline treatment for curing bacterial infections as described Spiroplasma strain MSRO by hemolymph injection as described D. melanogaster strain Ug-SR derived from Uganda H99 mutant strain was re-balanced with GFP-tagged balancer and homozygous mutant individuals were identified by immunostaining with anti-GFP antibody.The following laboratory strains of Spiroplasma-infected female flies within three days after eclosion were allowed to mate with male flies for three days in plastic tubes. These insects were kept with grape juice agar plates for embryo collection. Embryos at different developmental stages were dechorionated, fixed in 4% formaldehyde and heptane for 20 min, and devitellinized by vigorously shaking in heptane and methanol. In this study, female-specific expression of Sxl, the master regulator of the sex determination system in DrosophilaD\u03b1-Catenin , rabbit anti-PKC\u03b6 , mouse anti-Elav , rat anti-Elav , chicken anti-GFP , mouse anti-Wingless , mouse anti-Engrailed/Invected , mouse anti-Antp , mouse anti-Ubx , mouse anti-Abd-B , and guinea pig anti-Kr\u00fcppel Sxl-Pe-EGFP embryos were determined visually under a stereoscopic fluorescent microscope (Leica M165 FC). Each of 12 embryos of both sexes, which were collected at stage 10, 11, 12 or 13, was individually subjected to DNA extraction using QIAamp DNA mini kit (Qiagen). The DNA samples were subjected to real-time quantitative PCR using SYBR Green (Takara) and Mx3000P qPCR system (Stratagene) essentially as described Spiroplasma titers in terms of dnaA gene copies were quantified using the primers \u2032-TGA AAA AAA CAA ACA AAT TGT TAT TAC TTC-3\u20325 and \u2032-TTA AGA GCA GTT TCA AAA TCG GG-3\u20325. The copy numbers of the host EF1\u03b1 gene were also quantified using the primers \u2032-TTA ACA TTG TGG TCA TTG GCC A-3\u20325 and \u2032-CTT CTC AAT CGT ACG CTT GTC G-3\u20325. The reaction mixture consisted of 1\u00d7 AmpliTaq Gold buffer, 1.5 mM MgCl2, 0.2 mM each of dATP, dGTP, dCTP and dUTP, 0.3 \u00b5M each of the forward and reverse primers, 1/100,000 SYBR green, and 0.02 U/\u00b5l AmpliTaq Gold DNA polymerase (Applied Biosystems). PCR was performed under a temperature profile of 95\u00b0C for 10 min followed by 38 cycles of 95\u00b0C for 1 min, 60\u00b0C for 1 min and 72\u00b0C for 1 min. The data were statistically analyzed using the software R version 2.15.0 . Multiple comparison was performed using non-parametric Kruskal-Wallis test followed by Scheffe test.After dechorionization, developmental stages and sexes of Quantitative analyses of TUNEL signals were performed by custom R scripts with EBImage package for image processing Figure S1Sxl-based molecular sexing and TUNEL-based detection of apoptosis in control embryos of D. melanogaster. (A and B) Female-specific EGFP signals in stage 12 embryos of the transgenic strain Sxl-Pe-EGFP, in which EGFP is expressed under the control of Sxl early promoter (Pe). (A\u2032 and B\u2032) Female-specific immunostaining of stage 12 embryos with anti-Sxl antibody. (A\u2033 and B\u2033) Merged images. (C and D) TUNEL staining of stage 11 control embryos. (C\u2032 and D\u2032) Double-staining of stage 11 control embryos with TUNEL and anti-DCAT-1 antibody. (E) Comparison of TUNEL-positive areas between control female embryos (red) and male embryos (blue) at stage 11. Medians and interquartile ranges are shown with sample sizes. No significant difference is detected between female embryos and male embryos . (F) Infection dynamics of the male-killing Spiroplasma in developing female embryos (red) and male embryos (blue) from stage 10 to stage 13 in terms of symbiont dnaA gene copies per host EF1\u03b1 gene copy. (G) Dynamics of host EF1\u03b1 gene copies in developing female embryos (red) and male embryos (blue) from stage 10 to stage 13. Medians and interquartile ranges of 12 measurements are shown. Different characters show significant statistical differences .(TIF)Click here for additional data file.Figure S2Expression patterns of Hox proteins and segment polarity proteins in Spiroplasma-infected embryos of D. melanogaster.Spiroplasma-infected female embryos and male embryos are stained with antibodies against Hox proteins Antennapedia , Ultrabithorax and Abdominal B , and segment polarity proteins Wingless and Engrailed . These embryos are counter-stained for nuclear DNA (magenta in merged images).(TIF)Click here for additional data file."} +{"text": "T cell immunity is characterized by striking tissue specialization. Tissue-specificity imprinting starts during priming by tissue-derived migratory dendritic cells in the non-random, specialized micro-anatomical area of the draining lymph node and is influenced by constitutive and induced cues from local environment. Besides tissue-specific effectors, memory cells also exhibit a tissue-specificity. Long-lived tissue-resident memory T cells likely play a considerable role in preventing pathogen invasion. Understanding of the mechanisms of tissue specialization of T cells is of major importance for the design of optimal vaccination strategies and therapeutic interventions in tissue/organ-specific inflammatory diseases. The present review summarizes our current knowledge and hypothesis about tissue-specificity imprinting and tissue residency of T cells. CM) T cells. Indeed, both populations share high expression of the LN-homing molecules (CCR7 and CD62L) and recirculation marker S1P1 and generate tissue-specific short-lived effector cells (SLECs), the major pool of terminally differentiated T cells highly active in pathogen clearance. Although the role of CD8\u03b1+ resident DCs in generation of tissue-specific SLECs in some types of infections has been documented likely represent major players involved in processing and transfer of skin-specific information during priming and cross-priming processes -oriented trafficking of recirculating na\u00efve T cells switches dramatically to peripheral tissue-oriented after antigen (Ag) exposure and priming in the T cell zone environment of draining lymph node (DLN) . The rol+ dermal DCs within interfollicular and outer paracortex area close to B cell zone and preferential positioning of dermal CD207+CD103+ DCs and LCs within deeper T cell zone as preferential sites of both, positioning following CD8+ T cell priming and constitutive prepositioning of CD8+ TCM cells (SCM) , 11, 12.s) (SCM) . Whether2D3-dependent manner and thereby program generated SLECs to home to the skin fucosyltransferases FucT-IV and FucT-VII glycosylation enzymes -dependent manner. This involves induction of a small intestine-specific zip code consisting of strong integrin \u03b14\u03b27 and chemokine receptor CCR9 expression . Likewisthe skin . The skithe skin . De novo enzymes , 18. Of and CCR9 . The mol artifacts related to high number transfer of Ag-specific transgenic T cells (iii) low level of induction of broad-spectrum homing molecules such as mucosal T cells-associated marker \u03b14\u03b27 integrin or inflammation-related molecules (iv) immunization route (v) priming such as Ag load, priming threshold, adjuvants and costimulation, polarization, or regulatory signals, and (vi) target tissue-analyzed , among others T cells. The simultaneous presence of memory T cells with different anatomical distributions (systemic and tissue/organ-specific) is likely to reflect the role of compartmentalization in the global strategy used by the immune system to ensure optimal surveillance and protection such as thymic leukemia antigen (TL) for their differentiation and survival, as described for intestinal intraepithelial lymphocytes (IELs) and with organs such as the brain, kidney, and pancreas, thus providing global organ/area-targeted protection , 37, 38.nfection . Severald for LC . Accordiomodimer . A possis (IELs) .RM pool results from seeding by blood-borne TCM-derived progeny. In contrast, it has been shown that TEM generated following skin infection accumulate as TRM in a organ-specific manner both, at site of infection and at distant sites , and CD4er S1P1) , 48. Hig subsets , 49. Theal input , 49, theal input , a theraRCM) CD4 T cells which phenotypically differ from TEM, as they are CCR7pos, CD62Lint, CD103\u00b1 in addition to the expression of the peripheral homing receptor E-selectin ligand on brainRM and blood memory T cells. Unraveling those mechanisms is of major importance for the design of new vaccination strategies capable of inducing strong cell-mediated organ/tissue-specific immunity and protection. Molecules involved in tissue/organ-specific positioning of memory T cells might also represent interesting therapeutic targets for a variety of tissue/organ-specific inflammatory diseases.We are still at the early beginning of the story and many questions remain unanswered about the mechanisms of tissue-specific imprinting of effector and memory T cells and the maintenance of resident T cell memory as well as the lineage relationships between TThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "In particular Env-APRIL induced higher anti-Env antibody responses in rabbits. Env-APRIL was also more efficient at inducing neutralizing responses against various tier 1 viruses. Second, we embedded immunostimulatory proteins within the Env sequence. We replaced the V1V2 domain of Env with granulocyte-macrophage colony-stimulating factor (GM-CSF). Probing with neutralizing antibodies showed that both the Env and GM-CSF components of the chimeric protein were folded correctly. Furthermore, the embedded GM-CSF domain was functional as a cytokine in vitro. Mouse immunization studies demonstrated that chimeric Env-GM-CSF enhanced Env-specific antibody and T cell responses compared to wild-type Env. Collectively, these results show that targeting and activation of immune cells using engineered cytokine domains within the protein can improve the immunogenicity of Env subunit vaccines.An HIV-1 vaccine that induces protective antibodies remains elusive because a number of factors limit the quantity and quality of the antibodies raised against the HIV-1 envelope glycoprotein complex (Env). We hypothesized that targeting Env vaccines directly to immune cells would improve Env-specific antibody responses. To this end we explored two approaches. First, we fused trimeric Env gp140 at the C-terminus to proteins that can target and activate B cells: CD40 ligand (CD40L), B-cell Activating Factor (BAFF), and A PRoliferation-lnducing Ligand (APRIL). Trimeric Env fused to APRIL, BAFF or CD40L triggered the secretion of IgM, IgG and IgA from human B cells"} +{"text": "Using transgenic Drosophila expressing human tau, we found that RNAi\u2013mediated knockdown of milton or Miro, an adaptor protein essential for axonal transport of mitochondria, enhanced human tau-induced neurodegeneration. Tau phosphorylation at an AD\u2013related site Ser262 increased with knockdown of milton or Miro; and partitioning defective-1 (PAR-1), the Drosophila homolog of mammalian microtubule affinity-regulating kinase, mediated this increase of tau phosphorylation. Tau phosphorylation at Ser262 has been reported to promote tau detachment from microtubules, and we found that the levels of microtubule-unbound free tau increased by milton knockdown. Blocking tau phosphorylation at Ser262 site by PAR-1 knockdown or by mutating the Ser262 site to unphosphorylatable alanine suppressed the enhancement of tau-induced neurodegeneration caused by milton knockdown. Furthermore, knockdown of milton or Miro increased the levels of active PAR-1. These results suggest that an increase in tau phosphorylation at Ser262 through PAR-1 contributes to tau-mediated neurodegeneration under a pathological condition in which axonal mitochondria is depleted. Intriguingly, we found that knockdown of milton or Miro alone caused late-onset neurodegeneration in the fly brain, and this neurodegeneration could be suppressed by knockdown of Drosophila tau or PAR-1. Our results suggest that loss of axonal mitochondria may play an important role in tau phosphorylation and toxicity in the pathogenesis of AD.Abnormal phosphorylation and toxicity of a microtubule-associated protein tau are involved in the pathogenesis of Alzheimer's disease (AD); however, what pathological conditions trigger tau abnormality in AD is not fully understood. A reduction in the number of mitochondria in the axon has been implicated in AD. In this study, we investigated whether and how loss of axonal mitochondria promotes tau phosphorylation and toxicity Abnormal phosphorylation and toxicity of a microtubule-associated protein tau are involved in the pathogenesis of Alzheimer's disease (AD). Tau is phosphorylated at multiple sites, and phosphorylation of tau regulates its microtubule binding and physiological functions such as regulation of microtubule stability. Abnormal phosphorylation of tau occurs in the AD brains and is thought to cause tau toxicity; however, what pathological conditions trigger abnormal phosphorylation and toxicity of tau in AD is not fully understood. Since a reduction in the number of mitochondria in the axon has been observed in the AD brains, we investigated whether and how loss of axonal mitochondria promotes tau phosphorylation and toxicity. Using transgenic flies expressing human tau, we found that knockdown of milton or Miro, an adaptor protein essential for axonal transport of mitochondria, enhanced human tau-induced neurodegeneration. This study demonstrates that loss of axonal mitochondria caused by milton knockdown increases tau phosphorylation at an AD\u2013related site through partitioning defective-1 (PAR-1), promotes detachment of tau from microtubules, and enhances tau-mediated neurodegeneration. Our results suggest that loss of axonal mitochondria may play an important role in tau phosphorylation and toxicity in the pathogenesis of AD. In neuronal axons, these requirements need to be addressed locally, and the proper distribution of mitochondria is essential for neuronal functions and survival Mitochondria are principal mediators of local ATP supply and CaTau is a microtubule-associated protein that is expressed in neurons and localizes predominantly in the axons, where it regulates microtubule dynamics. Tau is phosphorylated at a number of sites, and a fine-tuned balance between phosphorylation and dephosphorylation of tau is critical for its physiological functions, such as microtubule stabilization, in the axons Hyperphosphorylated tau is found in neurofibrillary tangles, the intracellular protein inclusions that are associated with a range of neurodegenerative diseases including AD Drosophila, mitochondrial transport is facilitated by milton and Miro, which regulate mitochondrial attachment to microtubules via kinesin heavy chain Drosophila, in the absence of milton or Miro, synaptic terminals and axons lack mitochondria, although mitochondria are numerous in the neuronal cell body Mitochondrial transport is regulated by a series of molecular adaptors that mediate the attachment of mitochondria to molecular motors Drosophila as a model system, we investigated the effects of knockdown of milton or Miro, an adaptor protein essential for axonal transport of mitochondria, on tau phosphorylation and toxicity. We demonstrate that loss of axonal mitochondria caused by milton knockdown increases tau phosphorylation at Ser262 through PAR-1, promotes detachment of tau from microtubules, and enhances tau-mediated neurodegeneration.In this study, using in vivo, we used transgenic Drosophila expressing human tau To test whether loss of axonal mitochondria enhances human tau toxicity It has been reported that overexpression of tau alone can reduce anterograde transport of a variety of kinesin cargos, including mitochondria Milton is a component of an adaptor complex that links mitochondria to kinesin heavy chain and is essential for axonal transport of mitochondria [19]. PrGD) dramatically enhanced neurodegeneration in the lamina at 3-day-old compared to fly eyes expressing tau alone (TRiP) was used. Expression of this RNAi in neurons reduced milton mRNA levels in the fly brain iai) that targets a different region of Miro. Expression of Miro RNAiiai reduced Miro mRNA levels and the mammalian homolog of PAR-1, microtubule affinity-regulating kinase (MARK), are reported to phosphorylate tau at Ser262 in vivoWe investigated the role of tau phosphorylation at Ser262 in the enhancement of tau-induced axon degeneration caused by milton knockdown. We first examined whether PAR-1 knockdown enhances or suppresses tau-induced axon degeneration in the milton knockdown background. RNAi-mediated knockdown of PAR-1 significantly suppressed neurodegeneration in the lamina of flies expressing human tau and milton RNAi expressed at the levels similar to the expression of wild-type tau were useOur results demonstrate that knockdown of milton or Miro enhances tau-induced neurodegeneration and increases tau phosphorylation at Ser262. PAR-1 mediates the increase in tau phosphorylation at Ser262, and tau phosphorylation site Ser262 and PAR-1 are critical for the enhancement of tau-induced neurodegeneration caused by milton knockdown. To further investigate the relationship between loss of axonal mitochondria and PAR-1, the effect of knockdown of milton or Miro on PAR-1 activity was examined.TRiP) . FurtherTRiP) . These eTRiP) .DrosophilaA previous report showed that total PAR-1 level increased when it was phosphorylated at Thr408 in Milton knockdown did not cause non-specific activation of kinases, since it did not increase the level of phosphorylated, active p44mapk in fly eyes . MoreoveWe also observed that the phenotype induced by PAR-1 overexpression was enhanced by milton knockdown. Overexpression of PAR-1 in fly eyes has been reported to cause eye degeneration Although knockdown of milton or Miro without human tau overexpression did not cause neurodegeneration in the young flies , we founWe quantified age-dependent progression of neurodegeneration caused by milton knockdown in the lamina, where neurodegeneration was the most prominent. Degeneration in the lamina is undetectable at 3-day-old, which is in line with the previous observation in milton mutant flies TRiP) was used. Expression of milton RNAiTRiP also caused age-dependent neurodegeneration in the lamina . Accumulation of amyloid-\u03b2 peptides is thought to be causative for AD and has been suggested to cause tau abnormality It has been reported that, in non-neuronal cultured cells or primary-cultured hippocampal neurons with virus-mediated overexpression of human tau, an excess of microtubule-bound tau blocks microtubule-dependent transport of vesicles and organelle including mitochondria and causes synaptic degeneration in vivo, we used a Drosophila model of human tau toxicity In contrast, this study examined whether and how specific loss of axonal mitochondria promotes tau phosphorylation and toxicity. To address this question Using this model system, we found that milton knockdown significantly enhanced tau-mediated neurodegeneration. Milton knockdown increased the levels of active PAR-1 and tau phosphorylation at Ser262, and promoted detachment of tau from microtubules. If the enhancement of tau toxicity caused by milton knockdown in our model is due to an additive reduction in the number of axonal mitochondria, blocking tau phosphorylation at Ser262, which increases tau binding to microtubules and blocks microtubule-dependent transport, would enhance neurodegeneration. However, our results have shown that blocking tau phosphorylation at Ser262 by PAR-1 knockdown rescues the enhancement of tau-mediated neurodegeneration in the milton knockdown background. These results suggest that the enhancement of tau toxicity in the milton knockdown background is not likely to be due to an additive reduction of axonal transport of mitochondria caused by an excess of microtubule-bound tau. Rather, this study suggests that, when axonal mitochondria are chronically depleted, increased free, microtubule-unbound, Ser262-phosphorylated tau promotes neurodegeneration.A fine-tuned balance of microtubule-binding of tau is critical for its physiological functions. It has been suggested that both an excess of microtubule-bound tau and an excess of free, microtubule-unbound tau can cause toxicity DrosophilaDrosophila homolog of adducin, a cytoskeletal protein involved in regulating actin filament dynamics We found that the levels of active PAR-1 are increased by milton knockdown . PAR-1 iDetachment of tau from microtubules has been suggested to initiate its abnormal metabolism and toxicity of tau in AD pathogenesis In summary, this study highlights a potential role of loss of axonal mitochondria in tau phosphorylation and toxicity in AD pathogenesis. Reductions in the function and number of mitochondria in the axon have also been implicated in several neurodegenerative diseases such as Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis 5\u2032-CCGGAATTCGATATGGGCTGAATACAAATCACAGAATCG-3\u2032, rev, 5\u2032-CTAGTCTAGATTCATTAAAACCGGGAGGTAGATGAGATGT-3\u2032 ), and the resulting constructs were subcloned into the pUAST Drosophila transformation vector and microinjected into fly embryos of the 1118w genotype. Transgenic fly lines carrying UAS-Miro RNAi were established by microinjecting the Miro RNAi construct ) into fly embryos of the 1118w genotype. The transgenic fly lines carrying S262A mutant tau was described previously Transgenic fly lines carrying UAS-luciferase RNAi were established following the method described previously en bloc with 0.5% aqueous uranyl acetate for 1 hr, dehydrated with ethanol and embedded in Epon. Thin-sections (70 nm) of laminas, in which photoreceptor axons were cut longitudinally, were collected on copper grids. The sections were stained with 2% uranyl acetate in 70% ethanol and Reynolds' lead citrate solution. Electron micrographs were obtained with a VELETA CCD Camera (Olympus Soft Imaging Solutions GMBH) mounted on a JEM-1010 electron microscope (Jeol Ltd.).Probosces were removed from decapitated heads, which were then immersion-fixed overnight in 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M sodium cacodylate buffer at 4\u00b0C. Samples were post-fixed 1 hr in 1% osmium tetroxide in 0.1 M sodium cacodylate buffer on ice. After washing, samples were stained Preparation of paraffin sections, hematoxylin and eosin staining, and analysis of neurodegeneration were described previously Twenty fly heads for each genotype were homogenized in SDS-Tris-Glycine sample buffer, and the same amount of the lysate was loaded to each lane of multiple 10% Tris-Glycine gels and transferred to nitrocellulose membrane. The membranes were blocked with 5% milk (Nestle), blotted with the antibodies described below, incubated with appropriate secondary antibody and developed using ECL plus Western Blotting Detection Reagents or imaging with an Odyssey system. One of the membranes was probed with anti-tubulin, and used as the loading control for other blots in each experiment. Anti-tau monoclonal antibody , and anti-tau pSer262 , phospho-Thr231 , anti-HA (Santa Cruz), anti-myc (Millipore), anti-active p44mapk (Promega), anti-tubulin (Sigma), anti-GFP (Clontech) were purchased. Anti-tau pS202 (CP13) was a kind gift from Dr. Peter Davis (Albert Einstein College of Medicine). Anti-PAR-1 pT408 was described previously Quantitative real-time PCR analysis was performed as described previously 5\u2032-GGCTTCAGGGCCAGGTATCT-3\u2032milton for 5\u2032-GCCGAACTTGGCTGACTTTG-3\u2032milton rev 5\u2032-AAAAGCACCTCATTCTGCGT-3\u2032Miro for 5\u2032-CCTCAGGTGAGGAAACGCT-3\u2032Miro rev 5\u2032-AAGCCCGGTGGCGGTGAGAA-3\u2032dTau for 5\u2032-GCGCCAGAAGCCGTCATGGA-3\u2032dTau rev 5\u2032-TGCACCGCAAGTGCTTCTAA-3\u2032Actin for 5\u2032-TGCTGCACTCCAAACTTCCA-3\u2032Actin rev 5\u2032-GCTAAGCTGTCGCACAAATG-3\u2032rp49 for 5\u2032- GTTCGATCCGTAACCGATGT-3\u2032rp49 rev 5\u2032- GCGGCTGTGATTATGCGAAT-3\u2032TBP for 5\u2032-AGGGAAACCGAGCTTTTGGA-3\u2032TBP rev Microtubule binding assay was performed using a previously reported procedure with a modification Statistics was done with the JMP software (SAS) with Student's t or Tukey-Kramer HSD. Values are given as mean \u00b1 standard deviation or standard error.Figure S1Expression of human 0N4R wild-type tau causes late-onset, progressive neurodegeneration in the lamina. The lamina expressing tau at 3-day-old (A) or 10-day-old (B), or the lamina of control flies (gmr-GAL4 driver only) at 10-day-old (C). Compare vacuoles indicated by arrows in A (3-day-old) and B (10-day-old). (D) Quantification of the area of vacuoles in the lamina (arrows in A and B), mean \u00b1 SEM, n\u200a=\u200a14\u201320. *, p<0.05, Student's t-test. Genotypes are as follows: (tau) +/+;gmr-GAL4/+;UAS-tau/+ and (control) +/+;gmr-GAL4/+;+/+.(TIF)Click here for additional data file.Figure S2Mitochondria are observed in the presynaptic terminals of photoreceptor neurons expressing tau. Transmission electron micrographs of presynaptic terminals in the lamina of flies expressing human tau. Presynaptic terminals are colored green to accentuate the structures. Arrows indicate mitochondria in synaptic terminals. Flies were 3 days-after-eclosion (day-old). Genotype is: +/+;gmr-GAL4/+;UAS-tau/+.(TIF)Click here for additional data file.Figure S3GD). Flies were 3 days-after-eclosion (day-old). Genotypes are as follows: (control) +/+;gmr-GAL4/+;+/+ and (milton RNAiGD) UAS-milton RNAiGD/+;gmr-GAL4/+;+/+.Presynaptic terminals in milton knockdown flies have larger vesicles. Transmission electron micrographs of presynaptic terminals in the lamina of control flies bearing the gmr-GAL4 driver only (control) and flies with milton knockdown (milton RNAi(TIF)Click here for additional data file.Figure S4GD/+;gmr-GAL4/+;UAS-tau/+.Milton knockdown causes loss of axonal mitochondria in the photoreceptor neurons expressing human tau in the fly brain. A representative transmission electron micrograph of a presynaptic terminal of a photoreceptor neuron in the lamina of the fly co-expressing human tau and milton RNAi. The presynaptic terminal is colored green to accentuate the structures. Note that mitochondria are not observed (compare to (TIF)Click here for additional data file.Figure S5TRiP reduces milton mRNA levels and causes mislocalization of mitochondria in the fly brain. (A) Reduction in milton mRNA levels by the expression of milton RNAiTRiP in eyes and neurons. Expression of UAS-luciferase (control) or UAS-milton RNAiTRiP (milton RNAiTRiP) was driven by a combination of two drivers, the pan-retinal gmr-GAL4 driver and pan-neuronal elav-GAL4 driver. More than thirty flies for each genotype were collected and frozen. Heads were mechanically isolated, and total RNA was extracted. Milton RNA levels were quantified by qRT-PCR . Note that milton RNAi is only expressed in eyes and neurons, while endogenous milton is ubiquitously expressed. Genotypes are as follows: (control) elav-GAL4/Y;gmr-GAL4/+;UAS-luciferase/+ and (milton RNAiTRiP) elav-GAL4/Y;gmr-GAL4/+;UAS-milton RNAiTRiP/+. (B) Milton RNAiTRiP reduces axonal mitochondrial in the fly brain. (Top) A schematic view of the mushroom body structure, where axons (orange) can be easily identified in the fly brain. (Bottom) Mito-GFP signal in the lobe tips. Ratios relative to control are shown . The mito-GFP signal in the axons was significantly decreased in the milton RNAiTRiP fly brains. Representative images are shown. The method for mito-GFP analysis in the brain is described in TRiP) elav-GAL4/Y;mito-GFP/+; milton RNAiTRiP/+.Milton RNAi(TIF)Click here for additional data file.Figure S6KK reduces axonal mitochondria in the fly brain. Mito-GFP signal in the lobe tips (axons) of the mushroom body structure in the brains of control and Miro RNAiKK flies. Representative images are shown at the top, and the ratio of mito-GFP signal in the lobe tips relative to control are shown at the bottom . Genotypes are as follows: (control) elav-GAL4/Y;mito-GFP/+;+/+ and (Miro RNAiKK) elav-GAL4/Y;mito-GFP/UAS- Miro RNAiKK;+/+.Miro RNAi(TIF)Click here for additional data file.Figure S7iai causes a reduction in Miro mRNA levels in the fly brain. Expression of UAS-Miro RNAiiai (Miro RNAiiai) was driven by the pan-neuronal elav-GAL4 driver. More than thirty flies for control flies (the elav-GAL4 driver only) or Miro RNAi flies were collected and frozen. Heads were mechanically isolated, and total RNA was extracted. Miro mRNA levels were quantified by qRT-PCR . Note that Miro RNAiiai is only expressed in neurons, while endogenous Miro is ubiquitously expressed. Genotypes are as follows: (control) elav-GAL4/Y;+/+;+/+ and (Miro RNAi) elav-GAL4/Y;+/+;UAS-Miro RNAiiai/+.Miro RNAi(TIF)Click here for additional data file.Figure S8Tau-mediated neurodegeneration in the lamina is not enhanced by RNAi targeting CG30106, CG4395, CG6064, or CG30340. Quantification of neurodegeneration measured by the area of vacuoles in the lamina, presented as mean \u00b1 SEM, n\u200a=\u200a10\u201312. The asterisk indicates significant difference between tau and tau+milton RNAi . Flies were 3 days-after-eclosion (day-old).(TIF)Click here for additional data file.Figure S9GD) (C), co-expressing human tau and Miro RNAiiai (tau+Miro RNAi) (D), or expressing milton RNAi alone (milton RNAiGD) (E). Genotypes are as follows: (control) +/+;gmr-GAL4/+;+/+, (tau) +/+;gmr-GAL4/+;UAS-tau/+, (tau+milton RNAiGD) UAS-milton RNAiGD/+;gmr-GAL4/+;UAS-tau/+, (tau+Miro RNAi) +/+;gmr-GAL4/+;UAS-tau/UAS-Miro RNAiiai and (milton RNAiGD) UAS-Milton RNAiGD/+;gmr-GAL4/+;+/+.Knockdown of milton or Miro does not enhance tau-mediated reduction in the external eye size. Eyes from flies carrying the pan-retinal gmr-GAL4 driver only (control) (A), expressing human tau alone (tau) (B), co-expressing human tau and milton RNAi (tau+milton RNAi(TIF)Click here for additional data file.Figure S10Expression of S262A tau causes late-onset, progressive neurodegeneration in the lamina. The lamina expressing S262A tau at 3-day-old (A) or 10-day-old (B). Vacuoles in A are indicated by arrows. (C) Quantification of the area of vacuoles in the lamina, mean \u00b1 SEM, n\u200a=\u200a10\u201312. *, p<0.05, Student's t-test. Genotype is: +/+;gmr-GAL4/+;UAS-S262Atau/+.(TIF)Click here for additional data file.Figure S11GD. Blots were probed with anti-phospho-mapk (P-p44mapk) or anti-tubulin. No significant differences were found . The asterisk indicates non-specific bands. (B) Western blots of eyes from flies expressing HA-tagged p44mapk alone or flies co-expressing p44mapk and milton RNAiGD. Blots were probed with anti-HA (p44mapk) or anti-tubulin. No significant differences were found . (C) Western blots of eyes from flies expressing GFP alone or flies co-expressing GFP and milton RNAiGD. Blots were probed with anti-GFP or anti-tubulin. No significant differences were found . (D) Western blots of eyes from flies expressing myc-tagged APP alone or flies co-expressing APP and milton RNAiGD. Blots were probed with anti-myc (APP) or anti-tubulin. No significant differences were found . All flies were 3 days-after-eclosion (day-old). Genotypes are as follows: (p44mapk) +/+;gmr-GAL4/+;UAS-p44mapk-HA/+, (p44mapk+milton RNAi) UAS-Milton RNAiGD/+;gmr-GAL4/+;UAS-p44mapk-HA/+, (GFP) +/+;gmr-GAL4/UAS-GFP;+/+, (GFP+milton RNAi) UAS-Milton RNAiGD/+;gmr-GAL4/UAS-GFP;+/+, (APP) +/+;gmr-GAL4/UAS-APP-myc;+/+ and (APP+milton RNAi) UAS-Milton RNAiGD/+;gmr-GAL4/UAS-APP-myc;+/+.Milton knockdown does not cause non-specific activation of kinases, non-specific accumulation of overexpressed proteins, or an increase in the expression of proteins under the control of GAL4/UAS system. (A) Western blots of eyes from flies expressing HA-tagged p44mapk alone or flies co-expressing p44mapk and milton RNAi(TIF)Click here for additional data file.Figure S12GD (F), and expressing milton RNAiGD alone (G) are shown. (H) Quantification of neurodegeneration (arrows in F), mean \u00b1 SEM, n\u200a=\u200a10\u201312. *, significant difference between PAR-1 and PAR-1+milton RNAiGD . Genotypes are as follows: (control) +/+;gmr-GAL4/+;+/+, (PAR-1) +/+;gmr-GAL4/+;UAS-PAR-1-myc/+, (PAR-1+milton RNAiGD) UAS-Milton RNAiGD/+;gmr-GAL4/+;UAS-PAR-1-myc/+ and (milton RNAiGD) UAS-Milton RNAiGD/+;gmr-GAL4/+;+/+.Milton knockdown enhances neurodegeneration caused by PAR-1 overexpression. (A\u2013D) PAR-1 overexpression causes late-onset, progressive neurodegeneration in the lamina. The lamina expressing PAR-1 at 3 days-after-eclosion (day-old) (A) or 10-day-old (B), or the lamina of control flies at 10-day-old (C). (D) Quantification of the area of vacuoles in the lamina (arrows in B), mean \u00b1 SEM, n\u200a=\u200a10\u201312. *, p<0.05, Student's t-test. (E\u2013H) Milton knockdown enhances PAR-1-induced lamina degeneration. The lamina of flies expressing PAR-1 alone (E), co-expressing PAR-1 and milton RNAi(TIF)Click here for additional data file.Figure S13Tau RNAi causes a reduction in tau mRNA levels in the fly brain. Expression of UAS-luciferase (control) or UAS-tau RNAi (tau RNAi) was driven by a combination of two drivers, the pan-retinal gmr-GAL4 driver and pan-neuronal elav-GAL4 driver. More than thirty flies for each genotype were collected and frozen. Heads were mechanically isolated, and total RNA was extracted. Tau mRNA levels were quantified by qRT-PCR . Genotypes are as follows: (control) elav-GAL4/Y;gmr-GAL4/+;UAS-luciferase/+, and (tau RNAi) elav-GAL4/Y;gmr-GAL4/+;UAS-tau RNAi/+.(TIF)Click here for additional data file.Table S1Fly stocks.(DOC)Click here for additional data file.Table S2Genotypes of the flies that were used in each experiment.(DOC)Click here for additional data file.Text S1(DOC)Click here for additional data file."} +{"text": "Adherence is one of the main determinants of PrEP efficacy. Most PrEP studies applied subjective adherence measures, which often produce overestimates and problematic efficacy data interpretation; creating a need for more objective measures. This study examines self-reported adherence to oral PrEP compared to Medical Events Monitoring System (MEMS).Seventy-two HIV-uninfected partners (50% women) in Uganda were randomized to daily or intermittent oral emtricitabine/tenofovir or placebo in a 2:1:2:1 ratio for four months. Adherence was assessed monthly by MEMS and self-reported taken or missed doses by timeline follow-back calendar. MEMS data was adjusted for extra openings without pill removal and removal of multiple pills. Non-fixed days within intermittent regimen were classified as adherent/non-adherent based on self-reported sex by SMS. Adherence rates by taken/missed doses were compared to raw MEMS data using Spearman correlation.Treatment and placebo groups were combined since adherence rates were similar. Daily raw MEMS adherence rate was significantly higher than fixed Intermittent rate (p=0.04) and post-coital dosing rate (p<0.0001). Raw MEMS data for daily and fixed intermittent dosing, poorly correlated with self-reported taken doses and missed doses . Self-reported daily adherence had high sensitivity but only fair positive predictive value (PPV) and very poor specificity. Self-reported adherence to intermittent fixed dosing had fair sensitivity, PPV and negative predictive value (NPV), but poor specificity. Self-reported adherence to post-coital dosing had very good sensitivity and NPV but poor specificity.Median adherence for daily and intermittent fixed PrEP was high by objective and subjective measures, but poorly correlated. Adherence to post-coital dosing was poor and likely overestimated by self-report . Self-reported adherence measures were highly sensitive but poorly specific."} +{"text": "Hyperactivation of the mTORC2 signaling pathway has been shown to contribute to the oncogenic properties of gliomas. Moreover, overexpression of the mTORC2 regulatory subunit Rictor has been associated with increased proliferation and invasive character of these tumor cells.ROSA26 locus. This floxed Rictor strain was crossed with mice expressing the Cre recombinase driven from the glial fibrillary acidic protein (GFAP) promoter whose expression is limited to the glial cell compartment. Double transgenic loxP/loxPGFAP-Cre/Rictor mice developed multifocal infiltrating glioma containing elevated mTORC2 activity and typically involved the subventricular zone (SVZ) and lateral ventricle. Analysis of Rictor-dependent signaling in these tumors demonstrated that in addition to elevated mTORC2 activity, an mTORC2-independent marker of cortical actin network function, was also elevated. Upon histological examination of the neoplasms, many displayed oligodendroglioma-like phenotypes and expressed markers associated with oligodendroglial lineage tumors. To determine whether upstream oncogenic EGFRvIII signaling would alter tumor phenotypes observed in the loxP/loxPGFAP-Cre/Rictor mice, transgenic loxP/loxPGFAP-EGFRvIII; GFAP-Cre/Rictor mice were generated. These mice developed mixed astrocytic-oligodendroglial tumors, however glioma formation was accelerated and correlated with increased mTORC2 activity. Additionally, the subventricular zone within the loxP/loxPGFAP-Cre/Rictor mouse brain was markedly expanded, and a further proliferation within this compartment of the brain was observed in transgenic loxP/loxPGFAP-EGFRvIII; GFAP-Cre/Rictor mice.To determine whether Rictor overexpression was sufficient to induce glioma formation in mice, we inserted a Cre-lox-regulated human Rictor transgene into the murine in vivo proliferative capacity of loxP/loxPGFAP-Cre/Rictor gliomas.These data collectively establish Rictor as a novel oncoprotein and support the role of dysregulated Rictor expression in gliomagenesis via mTOR-dependent and mTOR-independent mechanisms. Furthermore, oncogenic EGFRvIII signaling appears to potentiate the Moreover, we demonstrate that cooperative signaling between GFAP-EGFRvIII and loxP/loxPGFAP-Cre/Rictor mice leads to hyperactivated mTORC2 signaling and results in high-grade gliomas.Several reports have shown that the mTORC2 scaffolding protein Rictor is overexpressed in cancers including gliomas and has been shown to contribute to the oncogenic properties of these tumors, however, a causal role for Rictor in gliomagenesis has not been demonstrated in vivo we generated a mouse model in which a myc-tagged human Rictor transgene was conditionally expressed in glial cells. Using homologous recombination in embryonic stem cells, the Rictor transgene was inserted into the ROSA26 locus downstream of a floxed PGK-neo cassette containing a strong transcriptional termination stop sequence (To examine whether Rictor overexpression would induce tumor formation sequence [27]. Thsequence and Cre-sequence . HeterozloxP/+) and homozygous (RictorloxP/loxP) crosses to the GFAP-Cre line were born in the expected Mendelian ratios and appeared normal at birth. Upon examination of the brain at the time of euthanasia no macroscopic abnormalities were found, however, in GFAP-Cre/RictorloxP/loxP animals histological examination revealed bilateral, multifocal infiltrating glioma with nearly complete penetrance. In several cases, tumors involved the amygdalohippocampal region or surrounding cortex and were also found within close proximity to the subventricular zone (SVZ). The gliomas were characterized at low magnification by foci of hypercellularity on H & E stain with histological characteristics similar to the original gliomas from the respective transgenic mice are the actual primary progenitors within this compartment which give rise to actively proliferating transit amplifying, which give rise to immature neuroblasts (TuJ1 positive) +/G12DK-ras as well as in wild-type mice receiving intraventricular infusions of EGF or PDGF We observed significant expansion of the SVZ in Our results are also consistent with the observation that AKT activity is critical initiator of gliomagenesis. Previous reports have demonstrated that pharmacological inhibition of the PI3K/AKT axis reduces the number of GFAP-expressing cells in the olfactory bulb loxP/loxPGFAP-Cre/Rictor tumors harbor elevated mTORC2 activity which is further induced in tumors from loxP/loxPGFAP-EGFRvIII; GFAP-Cre/Rictor mice. Finally, hyperactive mTORC2 signaling in cells of the SVZ results in marked expansion of this stem cell compartment suggesting that these cells may constitute the glioma cell of origin in these models. These models may provide future insights into the mechanistic basis of gliomagenesis and resistance to chemotherapeutic intervention.In summary, our results demonstrate that Rictor overexpression alone is sufficient to produce gliomas which resemble human oligodendroglial tumors in the mouse. Moreover, oncogenic EGFRvIII signaling cooperates with Rictor overexpression to induce more aggressive mixed astrocytic-oligodendroglial tumors. ROSA26PA The human myc-tagged Rictor was obtained from Dr. David Sabatini and cloned into the shuttling vector pBigT and subsequently subcloned into loxP/wtRosa26hRictor mice were generated following a protocol similar to that described previously GFAP-EGFRvIII mice, originally on an outbred ICR background were backcrossed at least eight generations into FVB/N inbred mice. Genotyping of the +GFAP-Cre and GFAP-EGFRvIII mice was performed as previously described loxP/wtRosa26hRictor mice was as described for tm1SorGt(ROSA)26Sor with minor modification 5\u2032- GTGGTGGTGGGTCGACGATGG -3\u2032; RR39, 5\u2032-AAGCGCTCGTAGCCCTGCTG -3\u2032. All protocols conformed to the guidelines established by the Association for the Assessment and Accreditation of Laboratory Animal Care and were approved by the UCLA-VA Animal Care and Use Committees under protocol numbers 0010-11036 and 0009-09029.346- NDRG1, phospho-S657-PCK\u03b1, phospho-S473-AKT , rabbit anti-phospho-Y118-paxillin , Ki-67 , TUJ1 . Quantification of immunopositive cell numbers was performed on three representative sections from three mice for each genotype.Tissue for histological analysis was immersion-fixed in buffered formalin, paraffin-embedded, sectioned and stained with H&E according to established protocols. Immunohistochemical analysis was performed according to standard procedures with antigen retrieval performed as previously described 6 cells were injected in the flanks of SCID mice and growth assessed by direct caliper measurements.Oligodendrocytes were purified from postnatal mice as described 32/36-IkB\u03b1, IkB\u03b1, paxillin, phospho-Y1068-EGFR, EGFR , EGFR/EGFRvIII cocktail antibody (Upstate), anti-Myo1C . Detection was performed using Amersham ECL . Immunoprecipitations were performed as previously described Total protein from whole brain homogenates or cultured lines were extracted and subjected to SDS-PAGE, followed by transfer to PVDF membranes, blocked with 2.5% nonfat milk and incubated with primary antibody overnight at 4\u00b0C. The following primary antibodies were used in addition to those described above: Myc tag clone 9E10 (Millipore), Rictor, mTOR , phospho-SInk4a/Arf-null MEF focus-forming assays were performed by plating 1\u00d7103 cells per well in 24-well plates in a volume of 400 \u00b5L using a two-layered soft agar system as previously described 12, Rictor and Myc expression vectors utilized in these assays have been previously described 5 cells in the top well of Boyden chambers that contained growth factor-reduced Matrigel over a polyethylene terephthalate membrane with 8-mm pores . Cells were allowed to invade and Matrigel removed prior to fixation and staining. Cells adhering to the bottom surface of the membrane were counted.XTT assays were used to assess cell proliferation in vitro. Cells were plated into 96-well plates at 1,000 per well and incubated for various time points. Cell numbers were subsequently determined by the addition of 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt and assaying relative absorbance at 490 nm as described by the manufacturer. NIH3T3 and loxP/loxP and GFAP-EGFRvIII; GFAP-Cre/RictorloxP/loxPGFAP-Cre/Rictor mice, as well as, age matched littermate controls were aligned according to anatomical position based on hippocampal configuration and size. In this orientation a horizontal segment of the SVZ is displayed subjacent to the corpus callosum and an oblique segment connecting to a vertical section. The width of the SVZ at its widest point in the vertical section was measured perpendicular to the ependymal layer at x400 using a Nikon Optiphot-2\u00ae microscope equipped with a Optronics Microfire\u00ae digital camera. Optronics PictureFrame\u00ae imaging processing suite was used containing a calibrated scale bar for measurements. Mean + S.D. are shown and relative significant differences were determined using Student's t test.Parasagittal sections from Figure S1Rictor overexpression results in increased carcinogenesis. (A) NIH3T3 focus forming assay in which cells were transfected with empty expression vector (vec), Rictor or c-myc containing expression vector DNA. (B) Cooperative increase in foci formation between Rictor and H-rasV12 in Ink4a/Arf-deficient MEFs. Data shown are mean + S.D. of three independent experiments.(TIFF)Click here for additional data file.Figure S2A) PCR amplification from tail clipped DNA showing the appropriate size bands for both transgenes of interest (GFAP-EGFRvIII and loxP/loxPGFAP-Cre/Rictor) in loxP/loxPGFAP-EGFRvIII; GFAP-Cre/Rictor transgenic mice. (B) Immunoblot analysis comparing EGFRvIII-Y1068-phosphorylation, Rictor and Myo1C expression in NMO, Ric0 and R0E3 cells.((TIFF)Click here for additional data file.Figure S3A) H & E stained sections of xenografted Ric0 and R0E3 tumors from SCID mice. 1\u00d7106 cells were injected into the flanks of recipient mice and tumors harvested and sectioned for histological analysis. Arrows show oligodendroglial morphology of transplanted glioma cells derived from loxP/loxPGFAP-Cre/Rictor and loxP/loxPGFAP-EGFRvIII; GFAP-Cre/Rictor transgenic mice. Scale bar, 20 \u00b5m. (B) Cell extracts from NMO or Ric0 cells were immunoprecipitated with anti-mTOR antibodies and precipitates were immunobloted for the indicated proteins. Rictor demonstrates increased association with mTOR in Ric0 cells as compared to NMOs.((TIFF)Click here for additional data file."} +{"text": "In patients with congenital heart disease (CHD), it is desirable to accurately measure peak velocity (Vmax). Unfortunately, phase-contrast MR (PCMR) tends to underestimate peak velocities. Fourier Velocity Encoding (FVE) can measure peak velocities in MRI, but is not commonly used due to long acquisition times.Therefore, we have developed a FVE sequence that combines spiral trajectories with parallel imaging (SENSE), partial-Fourier acquisition and a novel velocity-unwrap technique. The aim of this study is to validate this sequence.FVE sequence: FVE was performed using a spiral trajectory table . Spiral In-vitro: A pulsatile flow pump was connected to a tube phantom (13mm diameter) with a stenosis (6mm diameter). Peak velocity measurements using the following techniques were compared at 15 different flow rates; 1) US doppler, 2) low-resolution PCMR (lr-PCMR), 3) high-resolution PCMR (hr-PCMR), 4) FVE.In-vivo: 12 CHD patients with stenoses were assessed. Peak velocity measurements were compared between; 1) lr-PCMR, 2) hr-PCMR, and 3) FVE.In-vitro: There were no statistically significant differences between Vmax measured using US and FVE table . HoweverFourier velocity encoding allows accurate assessment of peak velocities as it measures a velocity spectrum per pixel, rather than the average velocity. However this extra encoding takes time, which has reduced its clinical effectiveness. We have shown that it possible to achieve high resolution FVE within a short breath-hold by combining spiral trajectories, parallel imaging, partial Fourier and velocity-unwrap. This sequence was shown to be significantly more accurate than PCMR in-vitro, and also to provide higher peak velocities than PCMR in-vivo. Thus, the sequence should be able to replace Doppler echocardiography making CMR a true one-stop-shop in assessing congenital heart disease.JAS: EPSRC PhD+.VM: BHF."} +{"text": "C. elegans. We also reported the formation of enlarged lipid droplets in a class of peroxisomal fatty acid \u03b2-oxidation mutants. In the present study, we seek to provide further evidence on the organelle nature and biophysical properties of fat storage structures in wild-type and mutant C. elegans.Lipid droplets are a class of eukaryotic cell organelles for storage of neutral fat such as triacylglycerol (TAG) and cholesterol ester (CE). We and others have recently reported that lysosome-related organelles (LROs) are not fat storage structures in the nematode C. elegans that lack lysosome related organelles (LROs). The formation of lipid droplets and the targeting of BODIPY fatty acid analogs to lipid droplets in live animals are not dependent on lysosomal trafficking or peroxisome dysfunction. However, the targeting of Nile Red to lipid droplets in live animals occurs only in mutants with defective peroxisomes. Nile Red labelled-lipid droplets are characterized by a fluorescence emission spectrum distinct from that of Nile Red labelled-LROs. Moreover, we show that the recently developed post-fix Nile Red staining method labels lipid droplets exclusively.In this study, we provide biochemical, histological and ultrastructural evidence of lipid droplets in wild-type and mutant C. elegans. These results have important applications to the studies of fat storage and lipid droplet regulation in the powerful genetic system, C. elegans.Our results demonstrate lipid droplets as ubiquitous fat storage organelles and provide a unified explanation for previous studies on fat labelling methods in Lipid droplets are defined as a class of organelles for storing neutral fat such as triacylglycerol (TAG) and cholesterol ester (CE) in eukaryotes ,2. LipidC. elegans has emerged as an important model to study fat metabolism. In C. elegans, the majority of fat is stored in gut epithelial cells. However, the organelle nature and biophysical properties of fat storage structures are not fully defined. The putative fat storage structures have been given different names such as gut granules or lysosome-related organelles (LROs) (hjIs9), dhs-28(hj8);Is[ges-1p::glo-1::gfp](hjIs9), glo-4(ok623);Is[ges-1p::glo-1::gfp](hjIs9).The wild-type strain was N2 Bristol. The following mutant and transgenic strains were used: Bacteria culture and seeding of 6-cm and 10-cm NGM plates were the same as previously described .BODIPY and Nile Red vital staining in live animals was essentially the same as previously described . 100 \u03bcL glo-4, and daf-22;glo-4 animals, we used imaging parameters that detected fluorescence of wild-type animals within the linear range. In order to carry out emission spectrum analyses of vital Nile Red fluorescence, lambda mode confocal imaging and image processing by linear un-mixing were conducted , and daf-22;glo-4 animals with the same parameter settings. Spec-1s were pseudo-colored red. Spec-2s, green. No apparent co-localization of red and green structures and minimal un-assigned pixels indicated reliable spectral classification and un-mixing. Lambda imaging of red BODIPY fluorescence in dhs-28;Is[ges-1p::glo-1::gfp] animals and in wild-type animals were essentially the same as that of Nile Red fluorescence except that the emission spectrum spanned 566-738 nm at 10 nm per channel. Only one emission spectrum was identified in Red BODIPY-stained dhs-28 and wild-type animals. For each imaging experiment, 10-30 animals were imaged.Channel mode confocal images of BODIPY, Nile Red, and GFP fluorescence in late stage L4 animals were acquired essentially the same as previously described . To componducted . A Zeissglo-4 animals were fixed and stained essentially as previously described L1s were loaded onto a 10-cm NGM/OP50 plate. Animals were harvested at late L4 stage. One plate of animals was treated as one sample. The sample was subjected to lipid droplet isolation with the same procedure, except that the extraction volume was scaled down from 10 mL to 1 mL and the lipid droplet fraction volume was scaled down from 1 mL to 0.1 mL. The 0.1 mL final lipid droplet fraction and the 0.9 mL post-nuclear cytoplasm fraction generated during the first centrifugation step were concentrated and extracted for protein in an equal final volume of 60 \u03bcL. 7.5 \u03bcL of each protein sample was loaded onto a SDS-PAGE gel and was subjected to western blot using a custom made rabbit anti-GFP antibody (Y2769). Wild-type lipid droplet fraction and cytoplasm fraction were prepared in the same way and were loaded alongside as a negative control. Experiments were conducted two times. Three samples each time.To prepare lipid droplet fraction and cytoplasm fraction for western blot experiment, 4000 freshly hatched Electron microscopy was conducted the same as previously described .SOZ and HYM designed research; SOZ, RT and FG performed research; SOZ and HYM wrote the paper. All authors read and approved the final version of the manuscript.Dual labelling of BODIPY in live animals. In live wild-type animals, red BODIPY labelled GLO-1::GFP-encircled LROs with high intensity (arrowheads). It also labelled non-GLO-1::GFP-encircled structures with low intensity (arrows). Images were single confocal slices.Click here for fileIsolated lipid droplets free of LRO marker protein GLO-1::GFP. Lipid droplet fraction and cytoplasm fraction were prepared from stage L4 dhs-28;glo-1::gfp animals. Triplicates. Fractions were probed for the presence of GLO-1::GFP protein using a GFP antibody. GLO-1::GFP was detected in cytoplasm fractions but not in lipid droplet fractions. Asterisk denotes a non-specific protein recognized by the GFP antibody that is present in wild-type control (WT).Click here for filedhs-28 animals that were vital-labelled by Nile RedDistinct emission spectra of lipid droplets and membrane pellets isolated from . (A) In stage L4 dhs-28 animals that were grown and stained on 10-cm NGM/OP50/Nile Red plates, LROs displayed Spec-1 emission spectrum (pseudo-colored in red) and lipid droplets displayed Spec-2 (pseudo-colored in green). Similarly, membrane pellets (B) and lipid droplets (C) isolated biochemically from these dhs-28 animals displayed Spec-1 and Spec-2 respectively. (D) Emission spectrum profiles of Spec-1s (red line) and Spec-2s (green line) of intact dhs-28 animals and Spec-1s (dashed red line) and Spec-2s (dashed green line) of isolated membrane pellets and lipid droplets. Intensity values were normalized.Click here for fileThe same fluorescence emission spectrum of BODIPY-labelled LROs and lipid droplets. In live dhs-28 mutants, red BODIPY labelled both lipid droplets and LROs (marked by GLO-1::GFP). However, red BODIPY displayed the same emission spectrum (colored in gold) in lipid droplets and LROs. LD, lipid droplet. Images were single confocal slices.Click here for file"} +{"text": "Optimization of antigens as well as delivery system is crucial for development of an effective T-cell based AIDS vaccine. Our recent results suggested higher anti-viral efficacy of Vif- and Nef-specific CTLs as well as Gag-specific ones . Here, we examined efficacy of Gag-specific or Vif/Nef-specific CTL induction by vaccination against SIV infection.All 17 animals used in this study were Burmese rhesus macaques sharing MHC-I haplotype 90-010-Ie, which mostly show typical AIDS progression after SIVmac239 challenge . These animals were divided into three groups consisting of unvaccinated (n = 6), Gag-vaccinated (n = 5), and Vif/Nef-vaccinated (n = 6); the latter two were subjected to DNA-prime/Sendai virus vector-boost vaccination. We compared these three groups after an intravenous SIVmac239 challenge.After challenge, 3 out of 5 Gag-vaccinated and 3 out of 6 Vif/Nef-vaccinated animals controlled SIV replication. The SIV control was associated with Gag-specific CTL responses in the former and Vif-specific CTL responses in the latter.This is the first report indicating efficacy of vaccine-induced Vif-specific CTL responses against SIV replication. Our results imply that not only Gag but also Vif may be a promising antigen for T-cell based AIDS vaccines."} +{"text": "Most people infected with Human T-cell Lymphotropic Virus Type 1 (HTLV-1) remain clinically asymptomatic; however, a minority develops the debilitating myelopathy HAM/TSP. Current treatment of HAM/TSP is limited by our partial understanding of the protective immune response to HTLV-1 and the pathogenesis of HAM/TSP.We wished to test the hypothesis that a gene expression signature in peripheral blood distinguishes between patients with HAM/TSP and ACs. We investigated genome-wide transcription patterns in whole blood from HTLV-1 asymptomatic carriers , patients with HAM/TSP (n=20) and uninfected control subjects (n=17). We identified a 542-gene signature that was deregulated in all HTLV-1+ individuals and predominantly comprised transcripts involved in p53-mediated DNA damage responses (p=0.00489). An 80-gene signature distinguished patients with HAM/TSP from those with the clinically similar disease multiple sclerosis. Paradoxically, at a given proviral load patients with HAM/TSP, but not ACs, over-expressed antiviral interferon-stimulated genes .Expression of these ISGs (assessed by quantitative PCR and flow cytometry) was not limited to HTLV-1-infected CD4+ T cells, suggesting that all peripheral blood immune cells were exposed to interferons (IFN) in vivo. Neither elevated IFN plasma levels nor an abnormal capacity for IFN production was detected in patients with HAM/TSP. However, peripheral immune cells in patients with HAM/TSP were more sensitive to IFN-alpha and IFN-gamma stimulation.These findings suggest that chronic over-expression of a specific subset of ISGs is ineffective in containing HTLV-1 and may instead contribute to the pathogenesis of HTLV-1-associated myelopathy."} +{"text": "Rheumatoid arthritis (RA) is one of the most common articular diseases with a prevalence of 1% worldwide . The cliHuman embryonic kidney (HEK)-293 cells, HEK-293T cells, NIH3T3 cells and synovial cells were cultured in DMEM medium. Transient transfection assays were performed in HEK-293 cells and HEK-293T cells. HEK-293 cells transfected with NF-\u03baB-Luc were treated with 100 ng/ml of phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), or 10 ng/ml of TNF-\u03b1 for 24 h, and luciferase activities were measured. siRNAs with 21 nucleotides for human GCIP were chemically synthesized. Transfection with siRNAs and cell survival assay were carried out.Grap2 cyclin D interacting protein (GCIP), Id like HLH protein, was down-regulated in the rheumatoid synovial cells. Introduction of GCIP into mouse fibroblast NIH3T3 cells resulted in growth suppression, whereas knockdown with siRNAs in synovial cells enhanced cell growth. GCIP associated with CBP and repressed transcription of CREB-target genes such as cyclin D1 by inhibition of interaction between CBP and RNA polymerase II complexes. Binding assays revealed that GCIP bound to CBP via acidic region, not HLH domain, and this interaction was regulated by phosphorylation of GCIP in a cell cycle-dependent manner. Therefore, GCIP has inhibitory effect on cell proliferation via interference with CBP-mediated transcription.We propose the novel inhibitory mechanisms of Id protein family; the coactivator CBP is a functional target. Furthermore, down-regulation of GCIP may be a key factor in rheumatoid synovial cell outgrowth."} +{"text": "We previously showed that long-lasting complete remission (CR) non-Hodgkin lymphoma (NHL) patients treated with rituximab-containing chemotherapy have an attenuated antibody response to virosomal or MF-59 adjuvanted seasonal (or pandemic) influenza vaccine , associated with persistent CD27+ Memory B cell depletion and hypogammaglobulinemia. Here, we evaluated humoral and innate response to trivalent intradermal vaccination in NHL in CR previously treated with rituximab-containing chemotherapy (at least one year before vaccine administration), RIT group, and in CR cancer patients treated with chemotherapy without rituximab (at least one year before vaccine administration), Non-RIT group. Intradermal administration was chosen considering its promising data, compared to conventional intramuscular route, in terms of immunogenicity and safety. Humoral response was assessed by hemagglutinin inhibition assay on sera collected at time 0 (just before vaccination) and at time 28 (four weeks after vaccination). Innate response was assessed by whole-genome gene expression analysis (Affymetrix Humane Gene ST 1.0) on PBMC collected at time 0 and at time 1 (24 hours after vaccine administration). Patients treated with rituximab-containing chemotherapy had, overall, a lower antibody response, compared to patients treated with chemotherapy alone. Overall, intradermal vaccination induced dramatic changes in gene-expression profile already one day after vaccination. These changes underline the activation of IFN stimulated genes and modulation of NK-associated transcripts. In addition, pathway and gene-enrichment analysis show that RIT and non-RIT groups have different quantitative and qualitative transcriptomic changes 24 hours after vaccination administration. Concordantly with antibody-titer, the innate response was more intense in Non-RIT group compared with RIT group."} +{"text": "Despite more advanced T cell differentiation, patients T cells showed clonal composition comparable to healthy individuals, sharing a preference for TRBV20 and TRBV29 gene segment usage and several co-dominant public TCR clonotypes. Moreover, our data revealed the presence of relatively few dominant EBV antigen-specific T cell clonotypes, which mostly persisted following transient lympho-depletion (TLD) and lymphocyte recovery, likely related to absence of EBV reactivation and de novo T cell priming in these patients. Interestingly, persisting clonotypes frequently co-expressed memory/homing-associated genes supporting the notion that they are particularly important for long-lasting CD8 T cell responses. Nevertheless, the clonal composition of EBV-specific CD8 T cells was preserved over time with the presence of the same dominant clonotypes after non-myeloablative chemotherapy. The observed clonotype persistence demonstrates high robustness of CD8 T cell homeostasis and reconstitution.Persistent viruses are kept in check by specific lymphocytes. The clonal T cell receptor (TCR) repertoire against Epstein-Barr virus (EBV), once established following primary infection, exhibits a robust stability over time. However, the determinants contributing to this long-term persistence are still poorly characterized. Taking advantage of an Primary Epstein-Barr virus (EBV) infection is associated with massive viral replication. In spite of the generation of a robust T cell specific immune response, EBV establishes a latent infection in B cells . NeverthThe antigen-primed CD8 T cell pool is highly heterogeneous and comprises T cell subpopulations at varying degrees of cellular differentiation based on their phenotype, function and anatomic location, thus making the distinction between \u201ceffector\u201d and \u201cmemory\u201d cytolytic T cells challenging -6. GenerWhether memory cell responses are maintained for prolonged periods or periodically \u201crenewed\u201d remains a matter of debate. It is a particularly difficult question to address in humans since it requires in-depth analysis of protective immune responses over extended periods of time. In this regard, the T cell receptor (TCR) is an excellent marker that allows antigen-specific responses to be followed along T cell differentiation and over time . Thorougin vivo setting where the balance between virus and immune response may be temporarily compromised following transient lympho-depletion (TLD). Specifically, we evaluated the EBV antigen-specific CD8 T cell clonotype composition and persistence in melanoma patients who were treated with non-myeloablative chemotherapy regimen, followed by adoptive cell transfer (ACT) of autologous peripheral blood mononuclear cells (PBMCs) . EBV. EBV33].ex vivo assessment of individual TCR BV-CDR3beta (TRBV) gene segment usage [ex vivo sorted 5-cell of EBV-specific T cells with that of single T cells generated by in vitro limiting dilution cultures. Comparable proportions of individual TCR clonotype signatures were found when using the ex vivo sorted 5-cell and the in vitro T cell cloning approaches . We could observe the appearance of new clonotypes over time, however these newly detected clonotypes were of low frequencies . Together, these results are in line with those previously reported by our group and otheWe further assessed the EBV antigen-specific TCR repertoire in five melanoma patients to determine the recovery potential of dominant T cell clonotypes following TLD and ACT . Similarin vivo selection of dominant clonotypes with TRBV chains belonging to the TRBV14, TRBV20 and TRBV29 families . In lineex vivo single cell gene-expression profiling [CD27, IL7R, EOMES, CD62L/SELL and CCR5), as well as effector-related genes known to be upregulated along T cell differentiation . The hig, ANOVA) . pos subsets . No major difference in the overall gene expression of effector mediators could be detected induced by non-myeloablative chemotherapy has become of high interest in the field of cancer immunotherapy, as it strongly favors ed cells ,36-38. HIn the present study, we examined the robustness of the EBV-specific T cell response following TLD in comparison to steady-state conditions over a period of four years. We found that, similarly to the total CD8 population , EBV antpos healthy donors under steady-state conditions as well as EBVpos patients undergoing short-term chemotherapy. We found that the T cell clonotype selection and composition of the CD8 T cell response to an immunodominant epitope among patients closely resembled that of healthy individuals [Owing to a high fidelity DNA polymerase, viruses belonging to the herpesviridae family are genetically stable , therebyividuals . SpecifiTGNGYTF) . These fTGNGYTF) -44. In pTGNGYTF) ,45. T ceTGNGYTF) ,44, provex vivo new generation-sequencing, Klarenbeek and coworkers [in vivo BMFL1-specific T cell repertoire was also stable following transient immunosuppression, with the frequent TCR clonotypes remaining dominant despite fluctuations in frequencies of low-dominant clonotypes clonotypes, presumably because of increased competition for survival factors. Moreover, the immune reconstitution period following TLD is generally associated with a skewing of the global CD8 T cell repertoire, in part due to early preferential expansion of antigen-primed T cells, followed by the slow repopulation with new thymic emigrants that eventually re-establish a high TCR repertoire diversity . Furtheiewed in . Since tNarrowing of the CD8 TCR repertoire can be observed in old age and/or by CMV infection, leading to increased susceptibility to infection . In the ex vivo [Technological advancements in the characterization of T lymphocyte responses reveal an unforeseen degree of heterogeneity of the antigen-primed T cell pool. Moreover, the level of polyfunctionality manifested by individual T cells can often be under-estimated when studying defined cellular populations by flow cytometry. In contrast, single cell analyses allow the accurate characterization of the functional phenotype of individual T cells. Our group and others have developed sophisticated laboratory techniques to analyze the gene expression signatures in individual T cells directly ex vivo ,50,51. Sex vivo ,28. In tex vivo .ex vivo single T cell gene expression analyses allows the accurate determination of the genes expressed by (multiple representatives of) individual TCR clonotypes. As the generation of clonotypic-specific mAbs to identify such individual clonotypes by flow cytometry still remains technically challenging, the identification of single T cell clonotypes is currently most reliably performed at the gene expression level. We have previously shown strong concordance between TCR repertoire analysis performed on ex vivo sorted cells and in vitro generated T cell clones [ex vivo single-cell sorted experiments and IL-7Ra display improved survival compared with subsets lacking the expression of these markers ,53. Resutransfer ,54,55. Wpression . Thus, wreatment . ConversIL7R and CD27 even after two rounds of lympho-depletion treatment. Collectively, these data provide further support for the crucial role of IL-7 in memory T cell homeostasis and persistence, and the potential clinical benefits of including IL-7 administration in future vaccination and ACT strategies.CD27 expression on T cells is associated with resistance to apoptosis, high IL-2 production, and increased T cell expansion , while Iin vivo setting studied here differs from the well-established allogeneic bone marrow transplantation in which the immunological perturbations are likely more profound. It also differs from the phase I clinical trials, which involve the same non-myeloablative lympho-depleting chemotherapy regimens, but with the transfer of either tumor-infiltrating lymphocyte cultures [in vivo settings on the dynamics of anti-viral T cell responses. Furthermore, direct ex vivo high-resolution molecular characterization of individual T cells at the clonotype level, as shown here, provides enhanced insights in the processes shaping the in vivo long-term survival of anti-viral specific T cells. In conclusion, detailed analyses of phenotype, gene expression and TCR clonal repertoire of EBV antigen-specific CD8 T cells in healthy donors and melanoma patients revealed that the anti-viral specific clonotypes persisted over time and showed the ability to resist cytotoxic chemotherapy. Importantly, the cultures or autolcultures ,60. FutuFigure S1Exvivo phenotype analysis of HLA-A*0201/BMFL1-specific CD8 T cells in melanoma patients before and after transient lympho-depletion. Representative exvivo flow cytometry analysis of EBV antigen-specific CD8 T cells in patient LAU672 at time-points before (Leuka) and after TLC (post-ACT). Total CD8 T cells (R1 gated top panels) and EBV-multimerpos T cells were characterized for the cell surface expression of CCR7 and CD45RA (middle panels). Double staining for CD45RA and CD28 is shown for CCR7 negative gated populations. Quadrant percentages are depicted for each subset.(TIF)Click here for additional data file.Figure S2Reproducibility of TCR BV clonotype analysis.A. Positive correlation of clonotype frequencies obtained between direct exvivo 5-cell sample sorting (n = 162) and invitro generated single-cell cloning (n = 676) by Spearman\u2019s correlation. Plot shows all TCR clonotypes identified in blood samples at leukapheresis time-points from four melanoma patients with inset showing the correlation between TCR clonotypes with frequencies below 20%. B. Analysis of the TCR repertoire diversity of EBV-specific CD8 T cells from patient LAU 1013 at leuka II (n = 107) obtained from single-cell samples directly exvivo sorted from two separate experiments. Each dominant clonotype is indicated and color-coded. Non-dominant clonotypes are designed as \u201cBV others\u201d and are composed of non-clonotypic sequences. (TIF)Click here for additional data file.Figure S3Co-expression of effector-related genes by dominant EBV antigen-specific TCR clonotypes before and following transient lympho-depletion.A. Cumulative effector-gene expression profile of single cell samples for each of the four dominant clonotypes at Leuka I time-point. Individual EBV antigen-specific CD8 T cell clonotypes from patient LAU 1013 were sorted from the early-differentiated EM28pos subset (n = 94). B. Gene co-expression polyfunctionality was determined on single cell samples representing individual TCR clonotypes from EM28pos EBV antigen-specific CD8 T cell subset at Leuka I (n = 94) and Leuka II (n = 83) from patient LAU 1013. Colors of the pie arcs depict the co-expression of individual effector genes , whereas the color in the pie depicts the number of co-expressed effector-related genes, as determined by SPICE 5.2. Increased polyfunctional gene co-expression (from 0 to 4) is shown as progressive grey gradients (from white to black). P-values of the permutation test are shown.(TIF)Click here for additional data file."} +{"text": "Lateral wall infarction on DE-CMR, independent of papillary muscle involvement, confers increased risk for ischemic mitral regurgitation.The mitral apparatus contains two myocardial components - papillary muscles and the adjacent left ventricular (LV) wall. Delayed enhancement CMR (DE-CMR) enables in-vivo study of potential contributions of LV wall and papillary muscle infarction (PMI) to MR. This study examined the relative impact of papillary muscle and LV wall infarction on mitral regurgitation (MR) following ST elevation MI (STEMI).Multimodality imaging was prospectively performed among patients with first STEMI: DE-CMR was used to assess LV infarct pattern - including PMI and LV wall infarction . Cine-CMR (SSFP) was analyzed for cardiac function and geometry - including LV chamber size, regional wall motion (5 point score/segment), and mitral annular diameter. Echocardiography (echo) was used to quantify MR (0-4+ grade) using established consensus criteria. Each imaging modality was read independently.153 patients with first STEMI were studied. Echo and CMR (1.5T) were performed within 1 day in all patients (27\u00b18 days post STEMI). 42% had MR . MR severity was similar among patients with and without PMI (p=0.35; Figure Lateral wall injury - whether assessed by infarct size or contractile dysfunction - confers increased risk for MR following STEMI, even after controlling for both mitral annular and LV chamber geometry. Neither presence nor location of PMI independently impacts severity of post-STEMI MR.K23 HL102249-01, Lantheus Medical Imaging, Doris Duke Clinical Scientist Development Award."} +{"text": "Alternative splicing of genes generates novel mRNAs, leading to the evolution of new functional proteins. Cholecystokinin (CCK) induces the release of pancreatic enzymes and the contraction of the gallbladder to promote the digestion of fat and proteins. CCK activates two G-protein-coupled receptors, CCKA and CCKB. Here, we showed that a CCKsv (splicing variant), originated de novo during Catarrhini evolution by including a portion of intronic sequence of the CCK gene, encodes novel C-terminal peptide sequence followed by a new poly-adenylation signal. CCKsv is expressed in many human tissues and likely a secreted peptide retaining the original signal peptide and the N-terminal proteolytic processing signal, together with novel C-terminal sequences. Although CCKsv cannot activate CCK receptors, it partially inhibits the CRE- or SRF-driven reporter activities stimulated by wide type CCK-8 mediated by both CCK receptors. Co-treatment with CCKsv also partially antagonizes Ewing tumor cell growth stimulated by CCK-8. Our study provides an example of new peptide hormone antagonist evolution in primates. Alternative splicing allows the generation of various gene products with different functions from a single gene and is a major mechanism of generating protein diversity in eukaryotes HEK293T, KATO III, SK-PN-DW, and SK-N-MC cell lines were purchased from American Type Culture Collection. CCK-8 peptide was purchased from Phoenix Pharmaceuticals Inc. whereas CCKsv (GKNAASPSLTSALVPRLPMLTLFSSASLMGMTSL-amidated) was synthesized by the PAN facility at Stanford University. CRE, SRE, NFAT, SRF-luciferase reporter plasmids and the pSV-\u03b2-galactosidase control vector were purchased from the Promega.Total RNAs from different cells were isolated using a RNA extraction kit (Qiagen) and eluted with RNase-free, DEPC-treated water before treatment with DNase. After reverse transcription using Sensiscript RT kit (Qiagen), the expression of CCK and CCKsv were analyzed using specific primers using primer sets shown in Supplementary V5-tagged CCKsv was cloned using specific primers (Supplementary HEK293T cells seeded in 24-well plates were co-transfected by various luciferase reporters (30 ng), the pSV-\u03b2-Gal plasmid (3 ng), and the CCKA or CCKB receptor (30 ng). After 1 day, cells were treated in serum-free media for another 18 h with increasing doses of the CCK-8 or CCKsv peptide. For the estimation of antagonistic activities, cells were pre-incubated (30 min.) with the CCKsv peptide before treatment with CCK8 for 18 h. Luciferase activities were determined using luciferase assay kits (Promega) and normalized using \u03b2-galactosidase activities. All experiments were performed at least three times in triplicates. Data were analyzed using Graphpad Prism 5.0.5) were plated in 12-well plates and allowed to attach for 24\u201336 h. Cells were then cultured in serum-free media with 0.1 uM of the CCK-8 peptide with or without the CCKsv peptide. Fresh media containing peptides were replaced every two days. To demonstrate CCKsv antagonism, cells were pre-treated with different dose of CCKsv for 30 min. before adding the CCK-8 peptide. Cell growth was determined by crystal violet staining SK-N-MC and SK-PN-DW cells and P value <0.01(**).Experiments were repeated independently at least 3 times. Calculations were done with a standard statistical package . Statistical significance was defined as a One complete cDNA (GenBank sequence ID: AK300784) representing an alternative splicing form of the CCK mRNA was found in a cDNA library of a human neuronal epithelioma Ewing tumor cell line. Based on the analysis of genome sequences of the human CCK gene , wild tyTo trace the origination of CCKsv, the syntenic chromosome locations of orthologous CCK genes in diverse primate species were identified, followed by the alignment of DNA sequences and predThe expression pattern of human CCKsv in diverse human tissues was analyzed by quantitative RT-PCR using specific primers Click here for additional data file.Figure S2CCKsv expression analyses in cell lines. Total RNAs isolated from different cells were used as PCR templates to avoid genomic DNA contamination in total RNA. Each band for CCK and CCKsv were sub-cloned and sequenced for confirmation.(PDF)Click here for additional data file.Figure S3PCR primers sets.(PDF)Click here for additional data file.Figure S4Protein sequence of V5 tagged CCKsv. The V5 tag was inserted into the CCKsv mature region.(PDF)Click here for additional data file.Figure S5CCKsv does not stimulate diverse G protein signaling in CCKA or CCKB receptor expressing HEK293T cells. (A) CRE-luciferase for Gs activity, (B) SRE-luciferase for Gi and Gq activities, (C) NFAT-luciferase for Gq activity, (D) SRF-RE-luciferase for G12 activity.(PDF)Click here for additional data file.Figure S6CCKsv cannot alter the CRE-luciferase activity stimulated by relaxin in LGR7-expressing cells.(PDF)Click here for additional data file.Figure S7Alignment of CCKsv read though sequences in primates. The putative poly-adenylation signal is shown in bold and underlined.(PDF)Click here for additional data file."} +{"text": "Significant progress has been made over the last decade towards realizing the potential of natural killer (NK) cells for cancer immunotherapy. NK cells can respond rapidly to transformed and stressed cells, and have the intrinsic potential to extravasate and reach their targets in almost all body tissues. In addition to donor-derived primary NK cells, also continuously expanding cytotoxic cell lines such as NK-92 are being considered for adoptive cancer immunotherapy. High cytotoxicity of NK-92 has previously been shown against malignant cells of hematologic origin in preclinical studies, and general safety of infusion of NK-92 cells has been established in phase I clinical trials. To enhance their therapeutic utility, we genetically modified NK-92 cells to express chimeric antigen receptors (CAR) specific for tumor-associated surface antigens. Such CAR were composed of a tumor-specific scFv antibody fragment fused via hinge and transmembrane domains to intracellular signaling moieties such as CD3 zeta chain, or composite fusion molecules also containing a costimulatory protein domain in addition to CD3 zeta. For development towards clinical applications, here a codon-optimized second generation CAR was constructed that consists of an ErbB2-specific scFv antibody domain fused via a linker to a composite CD28-CD3 zeta signaling domain. GMP-compliant protocols for vector production, lentiviral transduction and expansion of a genetically modified NK-92 single cell clone (NK-92/5.28.z) were established. Functional analysis of NK-92/5.28.z cells revealed high and stable CAR expression, selective cytotoxicity against ErbB2-expressing but otherwise NK-resistant tumor cells of different origins in vitro, as well as homing to ErbB2-expressing tumors in vivo. Furthermore, antigen specificity and selective cytotoxicity of these cells were retained in vivo, resulting in antitumoral activity against subcutaneous and intracranial glioblastoma xenografts in NSG mice. Ongoing work now focuses on the development of these cells for adoptive immunotherapy of ErbB2-positive glioblastoma."} +{"text": "Human T-cell leukemia virus type-1 (HTLV-1) infection causes adult T-cell leukemia (ATL). Recently, a novel viral protein HTLV-1 bZIP factor (HBZ), which is encoded an antisense viral gene, has been identified. HBZ may have a functional key player in cellular leukemogenesis, but the function in cells is poorly understood. To characterize the function(s) of HBZ, we performed yeast two-hybrid screen using HBZ as bait and identified growth arrest and DNA damage gene 34 (GADD34). GADD34 is induced by endoplasmic reticulum (ER) stress and the impairment of DNA. Both endogenous HBZ and GADD34 could interact in HTLV-1 infected cells. HBZ interacts with C-terminal region of GADD34 via its N-terminal region in mammalian cells. HBZ and GADD34 showed the same subcellular localization. GADD34 promotes the dephosphorylation of the factors which are downstream of mammalian target of rapamycin (mTOR), therefore, GADD34 represses mTOR activity. Our current study is focused on understanding the molecular mechanisms by which the interaction between GADD34 and HBZ regulates the mTOR signaling pathway."} +{"text": "The purpose of this study was to evaluate Time-resolved MR venography (TR-MRV) of the pulmonary venous circulation using the time-resolved angiography with interleaved stochastic trajectories (TWIST) method of time-resolved MRA (TR-MRA) and compare it with the more commonly used conventional Contrast enhanced magnetic resonance angiography (CE-MRA) approach in atrial fibrillation patients referred for pre-ablation pulmonary vein mapping.1,2. CE-MRA depicts the left atrium and pulmonary veins with high spatial resolution, enabling accurate measurement of pulmonary vein ostia to be made with depiction of their relationship to other structures.1,3 Conventional CE-MRA however requires timing of contrast enhancement and produces images with overlap of venous and arterial structures, potentially obscuring pulmonary vein ostia. TR-MRA is an alternative to conventional CE-MRA and has been used successfully in other vascular territories.4 Such an approach may be particularly advantageous in the pulmonary circulation with its rapid arteriovenous transit time, allowing acquisition of pure pulmonary venous phase images with a simpler imaging protocol.Catheter-based ablation of the pulmonary veins prevents recurrence of atrial fibrillation in 70-80% of patients during the first year of follow-upQuantitative Analysis: Pulmonary vein ostium orthogonal dimensions were measured using double oblique multiplanar reformatting. Qualitative Analysis: For qualitative analysis, both source partition images and MIP images were assessed by two observers. Pulmonary vein conspicuity was scored on a scale of 1-4 . The number of pulmonary veins was recorded.26 patients referred for pre-ablation pulmonary vein mapping underwent both conventional CE-MRA and TR-MRV with TWIST. Imaging was performed on a 1.5 Tesla MRI scanner. Source partition and MIP images were evaluated. \u00b1 0.37 vs 1.38cm \u00b1 0.36, respectively); see Table Orthogonal venous diameters were comparable for both TR-MRV and conventional CEMRA (1.34cm We have demonstrated that TR-MRV using TWIST produces comparable anatomic images and pulmonary venous dimensions to the more widely used CEMRA technique. TR-MRV improves arterio-venous separation producing high resolution pulmonary venous phase images without arterial overlap."} +{"text": "Thus, inoculation into neonates and the resultant induction of immune tolerance are required for the development of murine gammaretrovirus-induced leukemia/lymphoma. On the other hand, most complex retroviruses establish persistent infection in immunocompetent adult animals and induce pathologies after a prolonged latent period. Friend virus (FV) composed of two simple gammaretroviruses, replication-competent Friend murine leukemia virus (F-MuLV) and acutely growth-inducing but defective spleen focus-forming virus, is a prominent exception to the above general rules and can induce a rapid development of fatal leukemia when inoculated into immunocompetent adult mice of susceptible strains . Virus aed cells , resulti1 (B6AF1) mice develop acute splenomegaly, but most recover and survive after FV infection. When injected with FV-induced FBL-3 leukemia cells, uninfected B6AF1 mice reject the tumor; however, in FV-infected and recovered B6AF1 mice, injected FBL-3 cells continue to grow and cause death within 5 weeks. Syngeneic EL-4 tumor cells irrelevant of FV grow rapidly and cause death, while E.G7 cells expressing an ovalbumin (OVA) epitope are partially rejected, in both uninfected and FV-infected mice. Tetramer staining revealed the expansion of OVA-or influenza NP-reactive CD8+ T cells in both FV-infected and -uninfected animals upon E.G7 injection or influenza infection, respectively, while FV antigen-reactive CD8 T cells were not detected in FV-infected mice after FBL-3 injection. Infectious center assays demonstrated the presence of FV-producing cells in the thymus at 1 week after infection, peaking in their numbers at 2 weeks post-infection (pi), and a gradual decrease towards 10 weeks pi. Double negative, double positive, and single positive populations of thymocytes were infected in the thymus. More importantly, thymic dendritic cells and both cortical and medullary epithelial cells expressed F-MuLV envelope protein on the surfaces in infected animals. Transplantation of the thymus from FV-infected mice into thymectomized recipients, but not the transplantation of an uninfected thymus, resulted in a lack of expansion of FV-reactive T cells upon FBL-3 injection, indicating a deletion of the FV antigen-reactive repertoire in the process of T cell development.(C57BL/6 \u00d7 A)FThus, FV induces virus-specific central tolerance through the infection of thymic antigen presenting cells. This process, along with the early induction of effector T cell exhaustion may cont"} +{"text": "Leprb+/+ mice and in Leprbdb/db mice expressing HA-LepRb in a neuron specific manner. We did not find evidence of LepRb localization or STAT3-signaling in axon-fibers or nerve-terminals of POMC and AgRP/NPY/GABA neurons. Three-dimensional serial EM-reconstruction of dendritic segments from POMC and AgRP/NPY/GABA neurons indicates a high density of shaft synapses. In addition, we found that the leptin activates STAT3 signaling in proximity to synapses on POMC and AgRP/NPY/GABA dendritic shafts. Taken together, these data suggest that the signaling-form of the leptin receptor exhibits a somato-dendritic expression pattern in POMC and AgRP/NPY/GABA neurons. Dendritic LepRb signaling may therefore play an important role in leptin\u2019s central effects on energy balance, possibly through modulation of synaptic activity via post-synaptic mechanisms.Leptin acts via neuronal leptin receptors to control energy balance. Hypothalamic pro-opiomelanocortin (POMC) and agouti-related peptide (AgRP)/Neuropeptide Y (NPY)/GABA neurons produce anorexigenic and orexigenic neuropeptides and neurotransmitters, and express the long signaling form of the leptin receptor (LepRb). Despite progress in the understanding of LepRb signaling and function, the sub-cellular localization of LepRb in target neurons has not been determined, primarily due to lack of sensitive anti-LepRb antibodies. Here we applied light microscopy (LM), confocal-laser scanning microscopy (CLSM), and electron microscopy (EM) to investigate LepRb localization and signaling in mice expressing a HA-tagged LepRb selectively in POMC or AgRP/NPY/GABA neurons. We report that LepRb receptors exhibit a somato-dendritic expression pattern. We further show that LepRb activates STAT3 phosphorylation in neuronal fibers within several hypothalamic and hindbrain nuclei of wild-type mice and rats, and specifically in dendrites of arcuate POMC and AgRP/NPY/GABA neurons of Amongbehavior -10. Leptbehavior ,12. In cbehavior ,14. Consbehavior ,16. Micebehavior ,18. In abehavior -21.in vivo [Leptin has structural homology to cytokines and the leptin receptor bears strong sequence similarity to the class I cytokine receptor super family ,23. Morein vivo ,30 and Sin vivo ,32. 2+-currents via post-synaptic LepRb-dependent mechanisms [LepRb is required for leptin-mediated cell-autonomous effects on membrane potentials and axonal firing of hypothalamic neurons ,34. Furtchanisms ,36. Yet chanisms and inflchanisms . Despite progress in the understanding of neuronal leptin receptor signaling and of leptin\u2019s metabolic actions via arcuate POMC and AgRP/NPY/GABA neurons, direct evidence of the sub-cellular localization of LepRb within soma and neuronal fiber compartments is not known. We show here that LepRb is expressed and signals in somato-dendritic compartments of both groups of neurons. In addition, we present evidence of LepRb signaling in close proximity to post-synaptic structures on dendritic shafts. These studies suggest that leptin receptor signaling in dendrites may be important for leptin\u2019s effect on energy balance, possibly by modulating neuronal excitability through post-synaptic signaling mechanisms. Animal care and procedures were approved by the Institutional Animal Care and Use Committee at Beth Israel Deaconess Medical Center. HA-Leprb flox)(prior name: HA-ObRb STOP) were described previously [Pomc-cre [Agrp-ires-cre mice [Pomc-EGFP mice were described earlier [EGFP flox (Z/EG) reporter mice (B6.129(Cg)-Tg(CAG-Bgeo/GFP)21Lbe/J) were purchased from Jackson Laboratories [Leprdb/db;Pomc-cre;HA-Leprb flox and Leprdb/db;Agrp-ires-cre;HA-Leprb flox mice that selectively express HA-LepRb in POMC or AgRP neurons respectively, were generated by crossing the appropriate cre/lox lines with Leprdb/+ mice, as we have described previously [Mice and rats were housed at 22\u201324\u00b0C using a 14 hr light/10 hr dark cycle with standard chow diet . Transgenic mice with cre-dependent activation of HA-tagged LepRb expression . Leprdbeviously . The aboMurine leptin was purchased from Dr. A.F. Parlow . Supplies for immunohistochemistry (IHC) were purchased from Sigma-Aldrich , apart from the ABC Vectastain Elite kit which was purchased from Vector Laboratories and the tyramide-signal-amplification kit (TSA) which was from Perkin Elmer . The phospho-specific-(Y705)-STAT3 rabbit antibody was from New England Biolabs and the anti-HA mouse antibody from Covance Inc. . The rabbit anti-POMC precursor (against amino residues 27-52) antiserum was obtained from Phoenix Pharmaceuticals , the rabbit anti-\u03b2-endorphin antibody was a kind gift from Dr. Ronnekleiv and the Transcardiac perfusion and fixation with paraformaldehyde, removal of brains, postfixation, and cryoprotection were performed as we have generally described previously ,21,38. B2O2, glycine and SDS, blocked with goat serum, and incubated with anti-pSTAT3 antibodies overnight at room temperature, as described [For transmission EM, mouse brains were first fixed via transcardiac perfusion with saline followed by 1.0% formaldehyde and 1.25% EM grade glutaraldehyde in phosphate buffer, pH 7.4 (PB). Brains were removed from the skull and post-fixed in 2.0% formaldehyde and 2.5% glutaraldehyde in PB at 4\u00b0C overnight. Following embedding in 3% LMP agarose, 100 \u00b5m thick coronal sections were cut on a vibratome and stored in 2.0% formaldehyde and 2.5% glutaraldehyde in PB at 4\u00b0C until further use (method modified from ). For prescribed . For DABhttp://synapses.clm.utexas.edu/tools/reconstruct/reconstruct.stm) [Immuno-stained tissue sections were silver enhanced with the InteSE kit (Amersham Biosciences), then postfixed in 0.5 % osmium tetroxide for 15 minutes, rinsed in water, dehydrated in graded ethanol solutions, transferred to propylene oxide, and embedded in epoxy resin between two layers of Aclar plastic .\u00a0After the plastic was\u00a0polymerized at 60\u00b0C\u00a0for 48 hrs,\u00a0the area of the arcuate hypothalamic nucleus was\u00a0dissected\u00a0out and re-mounted on a plastic block for sectioning. Ultrathin 50 nm thick sections were then cut on an Ultracut-S ultramicrotome and mounted on copper grids. Before examination, grids with DAB stained sections were negatively stained with 1% uranyl acetate, while grids with IgG gold labeled sections were negatively stained with both uranyl acetate and lead citrate. Finally, grids were examined in a TecnaiG2 Spirit BioTWIN and photomicrograph images were recorded with an AMT 2k CCD camera at the Electron Microscope Facility, Harvard Medical School. For serial EM, consecutive sections were aligned and selected structures were reconstructed using the 3D reconstruction software, Reconstruct (uct.stm) ,48. Ultrafine structures in the central nervous system were identified as described by Peters et al. and StuaNeuroPure E18 primary rat hypothalamus cells (#N400200) and Neuropure E18 primary rat hippocampal cells (#N100200) were purchased from Genlantis and were dissociated, plated and grown in 24 well dishes on poly-D-lysine-coated coverslips as generally directed by the manufacturer. After 4-7 days in vitro (DIV), neurons were transfected with LepRb expression vectors, or co-transfected with HA-LepRb and EGFP expression vectors using NeuroFECT (Genlantis) or LipoFectamine 2000 (Invitrogen) according to the manufacturer\u2019s instructions. One to two days later, neurons were serum-deprived for 3-12 hours and treated with 100 nM recombinant leptin or vehicle control solution. Cells were then fixed and the coverslips were subjected to pSTAT3- or HA-immunocytochemistry (ICC) as we have generally described previously ,38. in situ as described earlier [in vitro RNA synthesis of sense or antisense RNA\u2019s using appropriate SP6 or T7 polymerases and DIG labeling mix. Briefly, sections were mounted on Superfrost Plus slides and hybridized overnight with the DIG-labeled mouse Pomc anti-sense RNA probe or with the DIG-labeled mouse Npy anti-sense RNA probe, both at 0.6 \u00b5g/ml at 60\u00b0C. All brain sections were washed twice in 0.2X SSC at 60\u00b0C, blocked in PBS with 10% bovine serum, and reacted with anti-DIG antibodies fused to alkaline phosphatase . Sections were washed and incubated with alkaline phosphatase substrate producing a color precipitate. The reaction was stopped by addition of EDTA.Free-floating brain sections were generated as described for IHC (above) and processed for earlier . BrieflyWhen we first developed the immunohistochemical (IHC) assay for phosphorylated STAT3 (pSTAT3) in brain sections from leptin-treated rodents ,30 we noPomc-EGFP mice with leptin and applied fluorescent-IHC and confocal laser scanning microscopy (CLSM) to hypothalamic brain sections. As shown in Npy-hrGFP mice (not shown). POMC (and AgRP/NPY/GABA) neurons might therefore serve as a suitable model system to investigate the sub-cellular fiber distribution of LepRb.To investigate whether STAT3 is activated in fiber structures of known LepRb-expressing hypothalamic neurons such as the POMC neurons, we next treated loxP sites flanking a transcriptional blocking sequence upstream of the HA-LepRb cDNA to allow HA-LepRb expression in a cre-dependent manner (e.g. expression of HA-LepRb in POMC neurons by crossing HA-LepRb flox mice with Pomc-cre mice). The detailed strategy was described earlier [Pomc-cre;HA-Leprb flox mice is shown by immunofluorescence-IHC and CLSM in To date, sufficiently sensitive and specific anti-LepRb antibodies capable of detecting endogenous LepRb proteins in the rodent brain have yet to be reported. We therefore employed a genetic strategy to express HA-tagged LepRb in mice thus facilitating detection with well characterized sensitive monoclonal anti-HA antibodies. The genetic design included introduction of two earlier . SuccessPomc-cre;HA-Leprb flox;EGFP flox). To enable investigation of possible LepRb localization in neuronal fibers, we created mice expressing HA-LepRb and EGFP in POMC soma and fiber processes , as we h nuclei) and in pGFP mice .Leprdb/db;Pomc-cre;HA-Leprb flox mice we next applied fluorescent-IHC and CLSM to investigate fiber localization of pSTAT3 and the POMC-polypeptide-derived axonal marker, \u03b1-melanocyte stimulating hormone (\u03b1-MSH)[ In brain sections from leptin-treated e (\u03b1-MSH),55,57,58Leprdb/db;Pomc-cre;HA-Leprb flox mice and Leprdb/db;AgRP-ires-cre;HA-Leprb flox mice were subjected to pSTAT3 immuno-EM. Validation of proper anatomical localization of pSTAT3 IR consistent with the known location of AgRP neurons in Leprdb/db;AgRP-ires-cre;HA-Leprb flox mice is shown by IHC and LM in Leprdb/db;AgRP-ires-cre;HA-Leprb flox mice (6-9 weeks old) are nearly as obese and hyperleptinemic as Leprdb/db controls (not shown). As in POMC fibers of Leprdb/db;Pomc-cre;HA-Leprb flox mice, pSTAT3 IR was also observed in AgRP neuronal fibers of Leprdb/db;AgRP-ires-cre;HA-Leprb flox mice at the LM level methodology. Multiple attempts unfortunately failed to detect specific signals for HA-LepRb by immuno-HA EM, possibly due to antigen interference by glutaraldehyde in the EM fixative. We therefore focused these studies on investigations of the ultrastructural sub-cellular localization of leptin-induced STAT3 phosphorylation, as pSTAT3 immunostaining was not negatively affected by glutaraldehyde. These analyses were expanded to include investigations of the localization of leptin-induced pSTAT3 in AgRP neurons. To this end, brain sections from leptin-treated LM level .Leprdb/db;Pomc-cre;HA-Leprb flox mouse. In the left image, pSTAT3 IR neuronal nuclei (black arrow) and non-pSTAT3 IR nuclei (white arrow) can be seen. At higher magnifications, pSTAT3 IR dendritic shaft structures are identified, including dendrites in the photographic plane (top right). An example of a cross-section of a large dendrite with an array of microtubules (MT) and one mitochondrion (M) is presented at bottom right. In several brain sections from several grids, we identified random pSTAT3 IR structures and counted a majority of neuronal nuclei (N=49) and dendrites (N=70), and no axonal fibers or presynaptic structures (N=0). At this time-point after leptin treatment, immunostaining was rarely seen in the cytoplasm (soma) of neurons with pSTAT3 IR nuclei. Similarly, shown in Leprdb/db;AgRP-ires-cre;HA-Leprb flox mouse. Many pSTAT3 IR neuronal nuclei and dendritic structures are identified. Analysis of randomly selected pSTAT3 IR structures identified neuronal nuclei (N=16), dendrites (N=358) and axons (N=1). Because these above studies were done in genetically modified and obese Leprdb/db mice, we also investigated pSTAT3 IR cellular structures in the medial arcuate nucleus of leptin-treated wild type C57BL/6J mice dendritic structures in brains of s et al. and Liu s et al. showing o-GFP EM . In AgRPo-GFP EM . HoweverLeprdb/db;Pomc-cre;HA-Leprb flox and Leprdb/db;AgRP-ires-cre;HA-Leprb flox mice. To further examine LepRb-dependent STAT3 phosphorylation in POMC and AgRP dendrites, we performed serial-EM 3D-reconstruction of pSTAT3 IR dendritic segments from leptin-treated Leprdb/db;Pomc-cre;HA-Leprb flox mouse. One mitochondrion is present. The dendrite was followed through 160 adjacent 50 nm thick EM sections and reconstructed in 3-dimensions using the Reconstruct software. Liu et al [Leprdb/db;Pomc-cre;HA-Leprb flox mouse. a and b). The 3D-reconstruction of the ~12.5 \u03bcm long segment including its multiple mitochondria (green) and one spine-like structure is presented in We similarly reconstructed a pSTAT3 IR dendritic shaft-segment from a leptin-treated Combined, these ultrastructural data show leptin-dependent STAT3 phosphorylation within POMC and AgRP/NPY dendrites in close proximity to both symmetrical and asymmetrical shaft synapses. in vivo metabolic actions.The principal finding of these studies is that the long signaling form of the leptin receptor, LepRb, exhibits a somato-dendritic expression pattern in hypothalamic arcuate POMC and AgRP/NPY neurons. In addition, we show evidence of leptin-dependent LepRb signaling in close proximity to synaptic structures on dendritic shafts. Combined, these results suggest that leptin has dendritic actions that may involve modulation of synaptic function and be important for mediating leptin\u2019s in vivo leptin administration.The dendritic localization of LepRb is supported by the following findings: 1) Leptin-dependent STAT3 phosphorylation is found in relatively short neuronal fibers that do not extend significantly beyond each brain nucleus; 2) HA-LepRb is directly localized to proximal and distal dendritic fibers of POMC neurons; and 3) ultrastructual studies show STAT3 activation by leptin in dendrites in POMC and AgRP neurons. The lack of axonal LepRb expression and signaling is attributed to: 1) HA-LepRb immuno-reactivity is not found in POMC IR or \u03b2-Endorphin IR fibers; 2) leptin-activated pSTAT3 is not detected in \u03b1-MSH processes; 3) leptin-stimulated pSTAT3 is lacking in known axonal terminal beds of POMC and AgRP neurons, such as the PVH; 4) EM analyses did not show evidence of pSTAT3 in axon fibers of POMC and AgRP/NPY neurons in transgenic mice selectively expressing LepRb only in those neurons; and 5) EM analyses show negative results for pSTAT3 in axon fibers within the arcuate nucleus of wild-type animals. Despite this evidence, we cannot rule out the possibility that low levels of LepRb are present in axons and/or that STAT3 is activated in axons at either longer or shorter time points following Somato-dendritic expression and signaling by LepRb is consistent with a recent study reporting effects of leptin on translation of brain-derived neurotrophic factor (BDNF) mRNA in dendritic fibers of VMH neurons . In addi2+ influx in dissociated hippocampal and cerebellar neuronal cultures and in Xenopus oocytes transfected with LepRb- and NMDAR-encoding plasmids [ Relative to the soma, dendrites contain the vast majority of synapses on a given neuron. Our ultrastructural experiments showing leptin-dependent STAT3 phosphorylation in proximity to symmetrical and asymmetrical synapses, suggest that LepRb signaling might influence post-synaptic responses to GABA and glutamate. Indeed, electrophysiological slice studies of hippocampal neurons show that leptin can enhance the amplitude of excitatory post synaptic currents (EPSCs) via N-methyl-D-aspartate receptors (NMDAR) and \u03b1-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPAR),65. Leptplasmids ,36. CombAn early study of leptin\u2019s electrophysiological effects suggested that LepRb is localized to pre-synaptic axon-terminal structures of hypothalamic NPY (AgRP) neurons serving to influence GABA release onto POMC neurons . HoweverA more recent study reported inhibition of glutamate release by leptin onto neurons in the ventral tegmental area (VTA) and similarly to Cowley et al. , also shWe also show evidence that STAT3, a transcription factor, is activated in distal dendrites. However its function in this neuronal compartment is unknown. One possibility is that STAT3 is eventually transported to the soma/nucleus to influence gene expression. Consistent with this possibility we found that pSTAT3 is associated with microtubules, the major transportation machinery in dendrites . EarlierLeprbdb/db; Agrp-ires-cre; HA-Leprb flox mice, or indirectly because of the obesity or other abnormalities of these animals. Alternatively, STAT3 may activated by leptin in regions of AgRP/NPY/GABA dendrites that have a low density of spines. Our 3D-reconstructions further indicate that the number of shaft synapses may greatly out-number spine synapses on both POMC and AgRP/NPY dendrites. We also show evidence that many of the shaft synapses can be categorized as either symmetrical or asymmetrical, which typically represents GABAergic and glutamatergic synapses, respectively [Consistent with the recent studies by Liu et al. , we founectively ,84. The ectively ,16,85. Further implications of our finding of dendritic LepRb localization include the possibility that leptin-resistant obesity may be caused, at least in part, by altered LepRb expression or signaling in this neuronal compartment. In addition, it will be important to determine if LepRb itself is localized and signals directly within the post-synaptic density to affect synaptic activity.Figure S1Leptin activates STAT3 phosphorylation in neuronal fiber processes within the NTS of C57BL/6J mice.Shown are light microscopy (LM) images of phospho-STAT3IR (DAB) in coronal brain sections of the hindbrain from 8 weeks old wild type C57BL/6J male mice. Animals were given leptin and sacrificed after 30 minutes. Top: pSTAT3 IR is found within the NTS at the level of the area postrema (AP). The bottom image shows high magnification of stippled box from top. Arrows identify some of many pSTAT3 IR neuronal processes. XII: Hypoglossal nerve; NTS: nucleus of the solitary tract; DMX: dorsal motor nucleus of the vagus nerve; cc: central canal, AP: area postrema.(TIF)Click here for additional data file.Figure S2Leptin does not induce STAT3 phosphorylation within the PVH, a major axonal target zone of leptin-responsive POMC neurons. Left: pSTAT3 IR is present in the RCH/Arc region of the anterior hypothalamus, but importantly, not in the PVH of leptin-treated C57BL/6J mice. Bottom: High-magnification microphotograph demonstrating lack of pSTAT3 IR fibers (and nuclei) in the PVH. Right: Many POMC IR nuclei are found in the RCH/Arc. Bottom: Dense networks of neuronal POMC fibers are observed in PVH. RCH: retrochiasmatic area; Arc: arcuate; 3v: 3rd ventricle; PVH: paraventricular hypothalamic nucleus.(TIF)Click here for additional data file.Figure S3Leptin activates STAT3 phosphorylation in neuronal fiber processes in hypothalamic nuclei of Sprague Dawley rats. Left: Shown are light microscopy (LM) images of phospho-STAT3 IR (DAB) in coronal brain sections of the mediobasal hypothalamus from Sprague Dawley rats. Rats were given leptin for 45 minutes. . Right: High-magnification microphotographs of boxes in left column. Robust pSTAT3 IR is found in fibers within the VMH and PMN.(TIF)Click here for additional data file.Figure S4Expression of HA-tagged LepRb in hypothalamic POMC neurons.A. CLSM of POMC neurons (red (POMC-polypeptide IR)) and HA-LepRb (green (HA IR)) in a hypothalamic brain section from a Pomc-cre;HA-Leprbflox mouse (top row) and a negative control section from a HA-Leprbflox mouse (bottom row). Some non-specific HA IR (green) is observed along the lining of the 3rd ventricle and at the base of the Arc in the control section. Shown are single confocal planes. B. Top row: Example of a POMC soma (red) co-expressing HA-LepRb (green) in a Pomc-cre;HA-Leprbflox mouse. DAPI fluorescence (blue) identifies the nucleus. Bottom row: Example of a POMC neuron that does not express HA-LepRb in a HA-Leprbflox control mouse. Shown are single confocal planes.(TIF)Click here for additional data file.Figure S5Localization of leptin receptors and activation of STAT3 in neuronal fibers of transfected primary neurons.A. Primary hypothalamic neurons were co-transfected with plasmids encoding HA-tagged LepRb and GFP, and subjected to immunocytochemistry (ICC) for HA (green) and GFP (red). Leptin receptors are expressed in the soma and in fibers. Shown are collapsed confocal Z-stack sections B. As in A., neurons were transfected with plasmids encoding HA-LepRb. At DIV 12, cells were treated 100 nM leptin for 20 min and fixed. Slides were then subjected to ICC for pSTAT3 (green) and PSD95, a dendritic protein marker (red). DAPI (blue) was included to label nuclei. Top: Two neurons exhibit pSTAT3 IR in the soma and fibers. Bottom: Enlargement of box in top image showing punctate pSTAT3 staining in PSD95 positive fibers. Shown are single confocal planes. DIV: days in vitro.(TIF)Click here for additional data file.Figure S6db/dbLepr;Pomc-cre;HA-LepRbflox mice express pSTAT3 in neuronal processes of POMC neurons. Leprdb/db control mouse. Bottom Left: pSTAT3 IR in the arcuate (ARC), but not the VMH or LHA of obese db/dbLepr;Pomc-cre;HA-LepRbflox mice consistent with the targeting of HA-LepRb to POMC neurons. Insert: For comparison, the anatomical localization of POMC neurons is shown by insitu hybridization for Pomc mRNA. Right: Enlargement of box. Many positive pSTAT3 fibers are shown (arrows). 3v: 3rd ventricle; Arcuate: hypothalamic arcuate nucleus; VMH: ventromedial hypothalamic nucleus; LHA: lateral hypothalamic area.Top Left: LM shows lack of pSTAT3 IR (DAB) in the mediobasal hypothalamus of a leptin-treated obese (TIF)Click here for additional data file.Figure S7db/dbLepr;Pomc-cre;HA-LepRbflox mice express pSTAT3 in neuronal processes of AgRP neurons. db/dbLepr;Pomc-cre;HA-LepRbflox mouse. pSTAT3 IR is found in the medial arcuate (ARC), but not in the VMH or LHA, consistent with targeting of HA-LepRb expression to AgRP neurons. Insert: For comparison, the anatomical localization of AgRP/NPY neurons is shown by insitu hybridization for Npy mRNA. Right: Enlargement of box. Several pSTAT3 IR fibers are visible (arrows). 3v: 3rd ventricle; VMH: ventromedial hypothalamic nucleus; LHA: lateral hypothalamic area.Left: LM shows pSTAT3 IR (DAB) in the mediobasal hypothalamus of a leptin-treated obese (TIF)Click here for additional data file.Figure S8Activation of STAT3 phosphorylation by leptin in dendrites of arcuate hypothalamic neurons of wild type C57BL/6J mice. Immuno-EM for pSTAT3 (DAB) in the arcuate hypothalamic nucleus of a leptin-treated wild type C57BL/6J mouse. Top: A pSTAT3 IR neuronal nucleus is labeled with \u201cNu\u201d (black text) and several pSTAT3 IR dendrites are indicated with black arrows. A pSTAT3 IR negative neuronal nucleus is labeled \u201cNu\u201d in white text. Insert: A cross sections of a pSTAT3 IR dendritic shaft. Bottom: pSTAT3 IR dendrite in the photographic plane. Nu; nucleus; D: dendrite; bv: blood microvessel.(TIF)Click here for additional data file.Figure S9Activation of STAT3 phosphorylation by leptin in dendritic spines of POMC neurons. A Pomc-EGFP mouse was given leptin . IHC and CLSM was applied to visualize pSTAT3 IR (red) and EGFP-epifluorescence (green) in a brain section of the arcuate nucleus of the hypothalamus. All images are single confocal planes. The arrow depicts a dendritic spine-like structure.(TIF)Click here for additional data file."} +{"text": "Cell transfer therapy for cancer has made a rapid progress recently and the immunotherapy has been recognized as the fourth anticancer modality after operation, chemotherapy, and radiotherapy. Lymphocytes used for cell transfer therapy include dendritic cells, natural killer (NK) cells, and T lymphocytes such as tumor-infiltrating lymphocytes (TILs) and cytotoxic T lymphocytes (CTLs). In vitro activated or engineered immune cells can traffic to cancer tissues to elicit persistent antitumor immune response which is very important especially after immunosuppressive treatments such as chemotherapy. In this review, we overviewed recent advances in the exploration of dendritic cells, NK cells, and T cells for the treatment of human cancer cells. Inspired by the observation of complete tumor regression in a male patient with recurrent sarcoma after a postoperative infection of erysipelas, Coley treated advanced sarcoma patients with mixed toxins of streptococcus erysipelas and bacillus prodigiosus in 1891 , thus st\u03b1, and interferons (IFNs) are secreted by immune cells and play pivotal roles in the active immunotherapy. IL-2 is an important growth factor for lymphocytes. It has been proved by FDA to treat advanced melanoma and renal carcinoma. However, the serious systemic toxicities of high-dose IL-2 restrict its wide application [There are two types of immunotherapy for cancer, active immunotherapy and passive immunotherapy. The active immunotherapy mainly refers to vaccines, immune adjuvants, and cytokines, while the passive therapy consists of immune modulating antibody-based therapy and adoptive immunotherapy. Active immunotherapies can activate endogenous immune system and passive immunotherapies provide or strengthen immune reaction in cancer patients by infusing antibodies or effector cells produced in vitro. Among the active immunotherapy, cytokines including interleukin-2 (IL-2), interleukin-12 (IL-12), granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor (TNF)-lication .Depending on the different immunocytes transferred, cell transfer therapy (CTT) includes active immunotherapy and passive immunotherapy or ACT. Adoptive cell transfer therapy uses patient-specific autologous or HLA-matched allogeneic lymphocytes activated in vitro with growth factors while dendritic cells were frequently used in active cell transfer therapy to elicit antitumor immune response. The immunocytes used in ACT can be divided into at least two types. The first type includes lymphokine-activated killer (LAK) cells, CIK (cytokine-induced killer) cells, and NK cells, all of which can mediate cancer regression with non MHC restriction. Another type of effector cells refers to the tumor-infiltrating lymphocytes (TILs) and cytotoxic T lymphocytes (CTL), both of which recognize specific tumor antigens presented by MHC molecules.After the identification of dendritic cells (DC) as the most efficient antigen presenting cells (APC) in vivo , DC-baseP = 0.001). However, time for disease progression was not prolonged . Remarkably, no serious side effect was reported. Compared to the control group, the pooled relative risks (RR) of all adverse events , grade 3 to 5 adverse events , and cerebrovascular events were not significantly higher for men treated with sipuleucel-T. There are more reports from phase II/III trials showing promising clinical outcomes of DC-based vaccines and the outcomes are related with the vaccine-induced expansion of tumor-specific effector T cells [Sipuleucel-T (Provenge) is the first therapeutic cancer vaccine approved by the US Food and Drug Administration (FDA) for the treatment of castration-resistant minimally symptomatic prostate cancer in April 2010. Sipuleucel-T is a DC-based vaccine which is expanded ex vivo with a fusion protein (PA2024) comprising of granulocyte-macrophage colony-stimulating factor (GM-CSF) and prostatic acid phosphatase . Three i T cells .\u03b1 induced the greatest differentiation and maturation for DCs in patients with bone and soft tissue tumors in contrast with a combination of IL-4/GM-CSF/TL and a combination of IL-4/GM-CSF/OK-432 [Immature DCs not only function poorly in antigen presentation but also induce immune tolerance . TherefoF/OK-432 . DCs genF/OK-432 . SimilarF/OK-432 \u201317. FurtF/OK-432 . AlternaF/OK-432 .\u2212CD56+ lymphocytes, can rapidly lyse certain target cells without MHC restriction. The NK cell cytotoxicity is mainly dependent on the balance between activating and inhibitory signals [NK cells, phenotypically defined as CD3 signals \u201322. The signals . NK cell signals . Certain signals . Therefo signals . Another signals . On the signals , 29.The therapeutic NK cells can be derived from several sources including autologous NK cells, allogeneic NK cells, NK cell lines, genetic modified NK cells, hematopoietic stem cell (HSC), and induced pluripotent stem cells (iPS) . By cytoCompared with autologous or allogeneic NK cells, the NK cell lines expanded under good manufacturing practice (GMP) conditions can supply sufficient quantity for clinical adoptive therapy with easier and simpler procedures. Among NK cell lines, NK-92 cells are the only ones approved by FDA for investigational treatment of patients with advanced malignant melanoma and renal cell carcinoma \u201343. HoweStrong evidence supports that genetic modification of NK cells is another effective strategy to increased tumor-cell killing efficiency. The genetic manipulation approaches include the transgenic cytokine expression, upregulation of activating receptors, silencing inhibitory receptors, and redirecting NK cells via chimeric tumor-antigen specific receptors . Recentl\u03b3 (IFN \u03b3), and induced pronounced tumor regression [So far, ACT with NK cells has shown promising results mainly in hematological cancer patients , 48, 49.gression .Taken together, NK cell-based therapies have only achieved modest clinical success in cancer patients and more developments are necessary to improve the clinical efficacy of NK cell-based cancer treatment such as the in vitro activation of NK cells.\u03b3 [Cytokine-activated T cells are a heterogeneous population of CD3- and CD56-positive, nonmajor histocompatibility complex (MHC)-restricted, natural killer (NK)-like T lymphocytes derived from peripheral blood lymphocytes (PBL), and expanded in vitro by anti-CD3 monoclonal antibody and various cytokines such as IL-2, IL-1, and IFN \u03b3 . Cytokin\u03b3 , gastroi\u03b3 , advance\u03b3 and hepa\u03b3 . These c\u03b3 or engin\u03b3 or IL-2 \u03b3 .Tumor-infiltrating lymphocytes (TILs), isolated form freshly resected tumors, can mediate lysis of tumor cells after in vitro incubation with IL-2. The main effective cells of TILs are CD8+ T cells, whose antitumor function can be boosted by IL-2. Rosenberg and his colleagues treated patients with metastatic melanoma by adoptive transfer of TILs for the first time in 1988, and the efficacy turned out to be not satisfied . Until 2In addition to cytokines, chemicals in culture medium can also influence the function of T cells after transplantation. For instance, rapamycin-treated T cells acquire an antiapoptotic and rapamycin-resistant phenotype, resulting in better in vivo persistence after transplantation .To overcome the above-mentioned limitations, another approach was developed based on the genetic modification of autologous lymphocytes from cancer patients. T-cell functions can be significantly improved by genetic modifications such as downregulation of BH3-interacting domain death agonist (BID) , 73 or u\u03b1- and \u03b2-chains that recognize antigenic peptides presented by MHC molecules while CAR target antigens in non-MHC-restricted manner through single-chain variable fragments (scFv) [There are two types of antitumor receptors, one is the naturally occurring T-cell receptors (TCRs) and the other one is chimeric antigen receptors (CARs). TCR is a heterodimer of s (scFv) .Lentivirus vector containing TCR genes for recognition of TAAs. Morgan and his colleagues achieved the first success by applying T cells transduced with TCR that targeted the MART-1 (melanocyte/melanoma differentiation antigen) to 15 progressive metastatic melanoma patients. Among them, 2 patients showed objective response with sustained levels of engineered T cells (between 20% and 70%) at 1 year after infusion [T-cell receptor genes can be derived from effective TILs in melaninfusion . Althouginfusion , the appinfusion , 84, 85.The selection of tumor-specific antigens seems to be crucial. A follow-up study with TCRs recognizing MART-1 and gp100h antigens on melanomas reported on-target toxicities such as vitiligo, uveitis, and auditory toxicity because of low-level expression of MART-1 and gp100h on normal melanocytes . In addi\u03b1- and \u03b2-chains of introduced TCR can mispair with the corresponding chains of endogenous TCRs, which remarkably reduce the expression of TCRs on the surface of transduced T cells, lending to reduce antitumor efficiency. A creative approach to decrease mispairing is the introduction of transgene encoding small interfering RNA or zinc finger nucleases to downregulate endogenous TCR [However, there are some drawbacks of TCR gene-modified T cells. The nous TCR , 88. Besnous TCR . For the\u03b6, CD28/OX-40/CD3\u03b6), which further improve the full signaling capabilities of T cells [In contrast to TCRs, CARs can overcome these barriers. CARs are genetically engineered immunity receptors that bind specific antigens expressed on the surface of cancer cells and then activate T cells to eliminate tumor cells . General T cells , 104.The first exciting clinical trial using CARs was carried out by Pule in 2008. They treated 11 neuroblastoma patients with engineered Epstein-Barr virus (EBV)-specific CTLs expressing a chimeric antigen receptor targeted against the disialoganglioside GD2. Tumor necrosis or regressions even sustained complete remission were observed in 4 of 8 patients with evaluable tumors . However\u03b1 and IFN-\u03b3, causing pulmonary toxicity and multiple organ dysfunction syndrome [Unfortunately, there are several side effects to use CAR-engineered T cells. Morgan reported a patient with metastatic colon cancer experienced acute respiratory failure after infusion of anti-ErbB2 chimeric-antigen-receptor-transduced T cells and died five days later. The reason suspected was that modified T cells recognized ERBB2 expressed by normal lung cells and secreted inflammatory cytokines such as TNF-syndrome .The differentiation status of T cells is a great concern to maximize antitumor effect of T cells. Gattinoni and his colleagues discovered a human memory T-cell subset with stem cell-like properties, termed memory stem cells (TSCM) which can mediate superior antitumor responses than central memory T cells (TCM) and effector memory T cells (TEM) in a humanized mouse model . The morCompared with other standard therapies like chemotherapy, radiotherapy, and surgery, immunotherapy can provide highly targeted treatment owing to the highly specific protein/receptor protein interaction. Activated or engineered immune cells can traffic to cancer tissues to elicit persistent antitumor immune response. Most of current clinical trials confirmed promising albeit moderate clinical efficacy. However, combining immunotherapies or with other traditional therapies such as chemotherapy and radiation has shown potential by preclinical investigations suggesting synergistic effects on tumor response and overall survival \u2013122. In"} +{"text": "Gossypium herbaceum that differed in their drought tolerance adaptability. Three different methodologies, namely, microarray, pyrosequencing, and qRT\u2013PCR, were used for transcriptome analysis and validation.Root length and its architecture govern the adaptability of plants to various stress conditions, including drought stress. Genetic variations in root growth, length, and architecture are genotypes dependent. In this study, we compared the drought-induced transcriptome of four genotypes of G.herbaceum when exposed to mannitol-induced osmotic stress. Under osmotic stress, the drought tolerant genotypes Vagad and GujCot-21 showed a longer root length than did by drought sensitive RAHS-14 and RAHS-IPS-187. Further, the gene expression patterns in the root tissue of all genotypes were analyzed. We obtained a total of 794 differentially expressed genes by microarray and 104928 high-quality reads representing 53195 unigenes from the root transcriptome. The Vagad and GujCot-21 respond to water stress by inducing various genes and pathways such as response to stresses, response to water deprivation, and flavonoid pathways. Some key regulatory genes involved in abiotic stress such as AP2 EREBP, MYB, WRKY, ERF, ERD9, and LEA were highly expressed in Vagad and GujCot-21. The genes RHD3, NAP1, LBD, and transcription factor WRKY75, known for root development under various stress conditions, were expressed specifically in Vagad and GujCot-21. The genes related to peroxidases, transporters, cell wall-modifying enzymes, and compatible solutes were also highly expressed in Vagad and Gujcot-21.The variations in root length and growth were found among four genotypes of Gossypium herbaceum, including a comparative transcriptome analysis and the selection of genes for root traits and drought tolerance.Our analysis highlights changes in the expression pattern of genes and depicts a small but highly specific set of drought responsive genes induced in response to drought stress. Some of these genes were very likely to be involved in drought stress signaling and adaptation, such as transmembrane nitrate transporter, alcohol dehydrogenase, pyruvate decarboxylase, sucrose synthase, and LEA. These results might serve as the basis for an in-depth genomics study of Oryza sativa, and their differences were calculated for the fold change between GujCot-21 and RAHS-IPS-187 . The most significant categories, namely response to water deprivation (24%), response to jasmonic acid stimulus (21%), ethylene-mediated signaling pathway (10%), hyperosmotic response (7%), hyperosmotic salinity response (6%), water transmembrane transporter activity (6%), and so on, were the major abiotic stress-related GO categories exclusively present in GujCot-21 and, hence, gained attention. Other GO terms exclusively present in GujCot-21 include response to inorganic substance (30%), methionine metabolic process (9%), anatomical structure arrangement (8%), response to brassinosteroid stimulus (6%), negative regulation of signal transduction (5%), and so on. Also noteworthy were the categories of response to heat (22%), water-soluble vitamin biosynthetic process (8%), response to sucrose stimulus (8%), starch biosynthetic process (5%), regulation of stomatal movement (5%), and so on, which were exclusively present in RAHS-IPS-187 with interesting GO terms. The genes associated with 4 common metabolic pathways include response to reactive oxygen species (12% and 9% of associated genes), response to hydrogen peroxide (8% and 6% of genes), response to high light intensity (7% and 9% of genes), and lipid localization (5% and 7% of genes) in GujCot-21 and RAHS-IPS-187, respectively and RAHS-IPS-187 (1161 genes involved) were identified. The significant GO terms were characterized into biological processes and molecular functions by agriGO (2O2) and superoxide are the major reactive oxygen species (ROS) that are produced in plant cells under biotic or abiotic stresses [2O2[We identified the transcripts that encode various antioxidant enzymes which were predominantly expressed in GujCot-21, and, hence, these genes could be considered probable components of the complex gene networks involved in the drought condition. Hydrogen peroxide . A total of 138 genes encoding various related transcription factors belonging to 30 different families were affected under the drought condition. The genes encoding WRKY , C2C2-CO-like , ARID , MADS , EIL , BZR , and RAV TFs were exclusively affected in sensitive genotypes and -sensitive genotypes (RAHS-14 and RAHS-IPS-187) showed significant variations in root structure and their length under control and mannitol stress of up regulated unique genes in RAHS-14 under control condition. Description: Excel file containing the list of unique up regulated genes and their annotation of RAHS-14 under control condition.Click here for fileAnnotation and fold change (fold change\u2009\u2265\u20092) of up regulated unique genes in RAHS-14 under drought stress. Description: Excel file containing the list of unique up regulated genes and their annotation of RAHS-14 under drought stress.Click here for fileAnnotation and fold change (fold change \u22652) of up regulated genes (probe sets) in Vagad under control condition. Description: Excel file containing the list of unique up regulated genes and their annotation of Vagad under control condition.Click here for fileAnnotation and fold change (fold change\u2009\u2265\u20092) of up regulated unique genes in Vagad under drought stress. Description: Excel file containing the list of unique up regulated genes and their annotation of Vagad under drought stress.Click here for fileContigs analysis by R-statistics of GujCot-21 and RAHS-IPS-187 genotypes. Description: Excel file containing detail analysis of contigs in both genotypes. Worksheet 1 containing total differential contigs, worksheet 2 is up-regulated contigs in RAHS-IPS-187, worksheet 3 is up-regulated contigs in GujCot-21, worksheet 4and 5 are agriGO of RAHS-IPS-187 and GUjCot-21 respectively. Worksheet 6 and 7 are KOBAS analysis of RAHS-IPS-187 and GUjCot-21 respectively.Click here for fileBLASTX analysis of contigs and singlets against NCBI NR database. Description: Excel file containing BLASTX (TAIR database) results of supercontigs (worksheet1), super singleton (worksheet2), contigs of GujCot-21 (worksheet3), singletone of GujCot-21 (worksheet4), contigs of RAHS-IPS-187 (worksheet 5) and singletone of RAHS-IPS-187 (worksheet 6).Click here for fileBLASTX analysis of both transcriptome contigs and singlets against TAIR database. Description: Excel file containing BLASTX (NCBI NR database) results of supercontigs (worksheet1), super singleton (worksheet2), contigs of GujCot-21 (worksheet3), singletone of GujCot-21 (worksheet4), contigs of RAHS-IPS-187 (worksheet 5) and singletone of RAHS-IPS-187 (worksheet 6).Click here for fileBLASTN analysis of both transcriptome contigs and singlets against publicly available cotton EST sequences. Description: Excel file containing BLASTN (Cotton EST) results of supercontigs (worksheet1), super singleton (worksheet2), contigs of GujCot-21 (worksheet3), singletone of GujCot-21 (worksheet4), contigs of RAHS-IPS-187 (worksheet 5) and singletone of RAHS-IPS-187 (worksheet 6).Click here for fileVenn diagram of contigs obtained after BLASTX in NCBI-NR, TAIR and Cotton EST database (BLASTN). Contigs of (A) Drought tolerant genotype (GujCot-21) (B) Sensitive genotype (RAHS-IPS-187) (C) Supercontigs Description: PPT contains a figures of venn diagram representing unique and common contigs in different database.Click here for file"} +{"text": "MYC and phosphoinositide 3-kinase (PI3K)-pathway deregulation are common in human prostate cancer. Through examination of 194 human prostate tumors, we observed statistically significant co-occurrence of MYC amplification and PI3K-pathway alteration, raising the possibility that these two lesions cooperate in prostate cancer progression. To investigate this, we generated bigenic mice in which both activated human AKT1 and human MYC are expressed in the prostate (MPAKT/Hi-MYC model). In contrast to mice expressing AKT1 alone (MPAKT model) or MYC alone (Hi-MYC model), the bigenic phenotype demonstrates accelerated progression of mouse prostate intraepithelial neoplasia (mPIN) to microinvasive disease with disruption of basement membrane, significant stromal remodeling and infiltration of macrophages, B- and T-lymphocytes, similar to inflammation observed in human prostate tumors. In contrast to the reversibility of mPIN lesions in young MPAKT mice after treatment with mTOR inhibitors, Hi-MYC and bigenic MPAKT/Hi-MYC mice were resistant. Additionally, older MPAKT mice showed reduced sensitivity to mTOR inhibition, suggesting that additional genetic events may dampen mTOR dependence. Since increased MYC expression is an early feature of many human prostate cancers, these data have implications for treatment of human prostate cancers with PI3K-pathway alterations using mTOR inhibitors. TMPRSS2-ERG gene fusion PTEN) tumor suppressor gene leading to accumulation of its substrate phosphatidylinositol 3,4,5-triphosphate (PIP3) and constitutive PI3K-pathway up-regulation MYC oncogene Prostate cancer is the second most common cause of cancer-related deaths in American men, who carry a 16% lifetime risk of developing invasive prostate cancer. Effective treatment of early-stage localized disease involves active surveillance, surgery or radiation therapy; however, recurrent and/or metastatic disease is incurable and androgen deprivation therapy is the primary treatment modality The PI3K-pathway activates multiple targets including AKT and its downstream effector mammalian target of rapamycin (mTOR) via allosteric inhibition of mTORC1 via release of the negative feedback on AKT that is potentiated by activated S6K in the absence of rapamycin, or via mTORC2 signaling, which is largely insensitive to rapamycin via an S6K-PI3K-Ras-dependent pathway PI3K-pathway inhibitors are undergoing clinical evaluation in multiple tumor types MYC oncogene has not yet been documented in human prostate cancer, although pathway-interaction has been suggested by several in vitro and in vivo models The MYC transcription factor directly regulates expression of the translational machinery for protein synthesis, as well as genes controlling cell cycle progression, metabolism, mitochondrial number and function and stem cell self renewal MYC amplification in a cohort of primary and metastatic human prostate cancer samples. To explore a cooperative role for the PI3K-pathway with the MYC oncogene in human prostate cancer, we used existing murine models of human prostate cancer harboring prostate-specific homozygous deletion of PTEN (PTENpc\u2212/\u2212 model) AKT1 (MPAKT model) pc\u2212/\u2212/Hi-MYC bigenic cross was used to validate results of a related study PTEN and MYC signaling using prostate-specific deletion of PTEN with concurrent Cre-induced focal MYC expression to induce high-grade mPIN (HG-mPIN) lesions and invasive adenocarcinoma. To address whether AKT downstream of PTEN might be the key mediator, we further explored the cooperation between these pathways using a bigenic mouse cross, MPAKT/Hi-MYC. Treatment with an mTOR inhibitor allowed direct assessment of the impact of MYC expression on the well-documented sensitivity of prostate lesions in the activated AKT model PTEN-deficient human cancers, as compared to single-lesion transgenic mouse models, may arise from secondary genetic alterations in human tumors.We identified an association between PI3K-pathway alteration and Detailed methods are provided as supplemental information .Human prostate tissues analyzed in this study were from patients treated at Memorial Sloan-Kettering Cancer Center (MSKCC), all of whom provided written informed consent. The study was approved by the MSKCC Institutional Review Board and the MSKCC Human Tissue Utilization Committee. Animal studies were carried out under protocol 06-07-012 approved by the MSKCC Institutional Animal Care and Use Committee. Institutional guidelines for the proper, humane use of animals in research were followed.MYC, PIK3CA, AKT1, AKT2 and AKT3, and for PTEN-loss have been described MYC-PAI transgene) loxP/loxP mice loxP/loxP/Hi-Myc offspring (F2) crossed with PTENloxP/wt/Pb-Cre4 males pc\u2212/\u2212/Hi-MYC mice (F3). MPAKT (rPb-myr-HA-AKT1 transgene) http://cbio.mskcc.org/Public/Sawyers_Clegg_AktMyc_2010. Gene and protein expression were assessed by quantitative real-time RT-PCR and immunoblot.PTENPTEN inactivation, and amplification of MYC are common genetic alterations in prostate cancer that correlate with high histological grade and poor prognosis MYC oncogene amplification co-occur in human prostate cancer, we examined oligonucleotide array CGH data from 194 prostate tumors, including 37 metastases. PI3K-pathway activation rarely occurred through point mutation of PTEN or PIK3CA in this dataset: exon-resequencing of 80 tumors revealed only 2 tumors with PIK3CA mutation and none with PTEN mutation Activation of the PI3K signaling pathway, often via PTEN, PIK3CA, AKT1, AKT2 and AKT3 (single- or multi-copy), was found in 27% of all samples and 70% of metastases. MYC multi-copy gain was identified in 6% of all samples and 24% of metastases, increasing to 20% of all samples and 51% of metastases when both single- and/or multi-copy MYC gain are considered (MYC copy-number gain (single-copy or greater) and found a positive association models. The role of PI3K signaling in prostate cancer has been modeled in mice by deletion of PTEN or by transgenic expression of activated AKT, while the role of MYC has been investigated by transgenic expression of MYC. A recent study demonstrated interaction between PTEN and MYC signaling using prostate-specific hetero- or homozygous deletion of PTEN with concurrent focal probasin-Cre-driven MYC overexpression pc\u2212/\u2212 conditional knockout mouse (C57BL/6J strain) pc\u2212/\u2212/Hi-MYC mice.To assess the functional implications of the association between PI3K-pathway alteration and 2PB) probasin promoter-driven expression of human MYC in the prostate results in murine prostate intraepithelial neoplasia (mPIN) in the lateral prostate (LP) by 4 weeks of age that progresses to adenocarcinoma in all mice by 6\u20139 months. The ventral prostate (VP), dorsal prostate (DP) and anterior prostate (AP) are affected to a lesser extent. The PTENpc\u2212/\u2212 model expresses probasin-Cre4 (Pb-Cre4) PTEN alleles in the VP, LP, DP and AP. PTENpc\u2212/\u2212 mice develop HG-mPIN that progresses to invasive adenocarcinoma after \u223c6 months of age In the Hi-MYC model pc\u2212/\u2212/Hi-MYC bigenic mice have large prostatic adenocarcinomas at 3 months (PTEN) and MYC in the PTENpc\u2212/\u2212/Hi-MYC prostatic epithelium revealed a subpopulation of cells expressing both proteins at high levels in areas of invasion (pAKT) confirmed AKT activation in MPAKT and, at lower levels, in bigenic MPAKT/Hi-MYC mice , S3A, S4MYC mice , S3A. BiMYC mice . By 5\u20139 MYC mice . AlthougMYC mice . At thisMYC mice , 4. This73 (pAKT)MYC mice , 4, S6 [MYC mice , S6.Progression to adenocarcinoma was accelerated in the MPAKT/Hi-MYC model with evidence of invasion in 8% of mice at 5\u20139 weeks, and in 67% mice at 16\u201320 weeks, compared respectively with 0% and 25% of Hi-MYC mice . In moreAKT1 and MYC in the mouse prostate is associated with an infiltration of T- and B-lymphocytes, as well as macrophages.The tumor microenvironment can significantly influence tumorigenesis, and cells from the stromal compartment such as fibroblasts and inflammatory cells can exert effects on adjacent epithelial cells via paracrine signals and extracellular matrix components To explore the cellular mechanism of AKT-MYC cooperativity, we examined the prostates of bigenic mice and their littermates, using markers of proliferation and apoptosis. As expected The AKT-induced mPIN phenotype in young MPAKT mice is dependent on mTOR To confirm that mTOR was inhibited in RAD001-treated mice, we examined the phosphorylation status of the downstream mTOR substrate ribosomal-S6 protein by immunohistochemistry with a widely-used phosphospecific antibody to Ser235/236 (pS6). In all vehicle-treated MPAKT mice, pS6 in the regions of mPIN was similarly high, and treatment with RAD001 led to dramatically reduced pS6 staining , indicatIn summary, mPIN lesions in young MPAKT mice were fully reverted upon RAD001-treatment; however, mPIN lesions in Hi-MYC and MPAKT/Hi-MYC bigenic mice did not respond to RAD001 despite effective mTORC1 inhibition. We conclude that transgenic MYC expression is sufficient to override the mTOR dependence of lesions arising from constitutive AKT activation. RAD001 treatment did not affect intensity or composition of the inflammatory infiltrate in prostates of bigenic mice.The mTOR dependence of the activated AKT-driven mPIN phenotype has been demonstrated only in young MPAKT mice increased by RAD001 treatment and MPAKT/Hi-MYC (n\u200a=\u200a5) mice all showed enotype) , S10. Thenotype) , S10. ThMYC amplification and PI3K-pathway disruption in 194 human prostate tumors, including 37 metastatic tumors. To investigate the potential functional interaction between the MYC and PI3K-pathways in the prostate, we first generated a PTENpc\u2212/\u2212/Hi-MYC bigenic mouse that confirmed a prior model of cooperativity between these two pathways MYC, we crossbred previously characterized mice expressing activated human AKT1 (MPAKT model) PI3K-pathway upregulation in primary and metastatic prostate cancers provides the rationale for clinical evaluation of PI3K-pathway inhibitors . Here we demonstrate a statistically significant co-occurrence of H17 and/or Treg (FoxP3+) T-cells in development or progression of human prostate cancer The prostate glands of MPAKT/Hi-MYC mice are characterized by significant stromal reaction and infiltration of B- and T-lymphocytes, as well as macrophages early in development of mPIN and persisting throughout tumorigenesis. This inflammatory response is of particular interest because of possible roles for the immune system in tumor growth regulation. In the prostate, inflammation is commonly observed in cancer precursor lesions increased, possibly due to feedback activation of the MEK/ERK-pathway Due to growing interest in evaluating PI3K-pathway inhibitors in prostate cancer patients, we explored the activity of the rapamycin analog RAD001 in the MPAKT/Hi-MYC model. In contrast to the exquisite sensitivity of young MPAKT mice to this compound Rapalogs, which selectively inhibit the TORC1 complex, can paradoxically activate AKT through loss of S6 kinase-mediated negative feedback at the level of PI3K Another potential mechanism for rapalog-resistance may be the documented mitigation of cellular senescence upon mTOR inhibition in tumors with activated senescence programs Rapalogs have been explored in pilot studies in prostate cancer, and PI3K and mTORC1/2 kinase inhibitors are now in early-stage clinical trials across tumor types. In this context, our demonstration that MYC overexpression can convert AKT-activated mouse prostate tumors from rapalog-sensitive to rapalog-resistant has implications for clinical studies of PI3K-pathway inhibitors in men whose prostate cancers also harbor increased AKT signaling. As is clear with other tumor types such as glioblastoma and breast cancer, secondary genetic alterations such as PTEN loss can mitigate the response to EGFR or HER2 inhibitors Figure S1MYC amplification co-occurs with AKT pathway activation in human prostate tumor samples. Contingency tables for co-occurrence of MYC and PI3K-pathway copy-number alterations (defined by aCGH) from (A) 157 primary and 37 metastatic prostate tumor samples , or from 37 metastatic samples alone. The tables indicate in red the proportion of tumors with PIK3CA amplification or general PI3K pathway gain, that also exhibit MYC amplification. (A), (B) The association between multi-copy MYC amplification and single- or multi-copy PI3K-pathway alterations is shown. (C) The association between single- or multi-copy MYC gain and PI3K pathway alterations is shown. The statistical significance of each association is reported as a P-value determined by 2-tailed Fisher's exact test.(TIF)Click here for additional data file.Figure S2MYC amplification co-occurs with AKT pathway activation in human prostate tumor samples. Graphic representation of contingency tables from (A) 157 primary and 37 metastatic prostate tumor samples , or from (B) 37 metastatic samples alone. The graphs indicate the proportion of tumors with PIK3CA amplification or general PI3K pathway gain, that also exhibit MYC amplification. The statistical significance of each association is reported as a P-value determined by 2-tailed Fisher's exact test.y tables , Fig. S1(TIF)Click here for additional data file.Figure S3The AKT and MYC transgenes are expressed in prostates of bigenic MPAKT/Hi-MYC mice, albeit at lower levels than in the single transgenic mice. (A) Immunohistochemistry using antibodies for pAKT (Ser473) and MYC on ventral prostates from mice aged 30\u201333 weeks. Note the high levels of pAKT membrane staining associated with regions of mPIN in MPAKT/Hi-MYC mice. pAKT staining is absent in Hi-MYC mice. Nuclear MYC staining is evident in Hi-MYC and MPAKT/Hi-MYC, but absent in MPAKT mice. Scale-bars: 50 \u00b5m (black), 30 \u00b5m (red). (B) qRT-PCR analysis of the myr-HA-AKT1 and MYC transgenes in prostates from 7 week-old MPAKT, MPAKT/Hi-MYC and Hi-MYC mice . P < 0.01 (determined by two-way ANOVA with Bonferroni post-test) for Tg-AKT expression in MPAKT/Hi-MYC vs MPAKT.(TIF)Click here for additional data file.Figure S4pAKT is expressed in cells near areas of invasion in MPAKT/Hi-MYC mice. Invasive area in lateral prostate of MPAKT/Hi-MYC mouse aged 21 weeks (upper panels: hematoxylin & eosin). Lower panels: Immunohistochemistry using an antibody for pAKT (Ser473) indicates lower but detectable pAKT expression in tumor cells compared to PIN lesions. Scale bars: 200 \u00b5m (black), 100 \u00b5m (red).(TIF)Click here for additional data file.Figure S5Prostate epithelial cells display a higher degree of nuclear atypia in PIN lesions from MPAKT/Hi-MYC mice than from Hi-MYC mice. mPIN lesions in 8-week-old lateral prostates from Hi-MYC and MPAKT/Hi-MYC mice, indicating a greater degree of nuclear atypia in the MPAKT/Hi-MYC cells with larger nuclei and more open chromatin (H&E). Scale bars: 20 \u00b5m.(TIF)Click here for additional data file.Figure S6The MPAKT/Hi-MYC ventral prostate displays areas of stromal remodeling characteristic of microinvasive foci. Immunohistochemistry for smooth muscle actin and collagen IV providing additional examples (as in middle and right columns). Note (left column), an example of minimally attenuated smooth muscle sheath and collagen IV around a gland with no evidence of microinvasion. Scale bars: 100 \u00b5m.es as in of disru(TIF)Click here for additional data file.Figure S7The MPAKT/Hi-MYC phenotype is characterized by an increase in cell proliferation and apoptosis compared to MPAKT mice, and the pro-apoptotic effects of MYC are not rescued by AKT expression. Mouse prostates from animals aged 5\u20139 weeks were stained with TUNEL or antibody to Ki67. Results were quantified as the mean percentage (\u00b1 SD) of positive cells, counting at least 300 cells from representative areas from each of 9 mice per transgenic genotype and 4 wild-type mice. P < 0.05 for MPAKT/Hi-MYC vs MPAKT, MPAKT/Hi-MYC vs WT, Hi-MYC vs MPAKT, Hi-MYC vs WT.(TIF)Click here for additional data file.Figure S8mPIN lesions of older MPAKT mouse prostates are variably resistant to RAD001. Additional examples (as in es as in showing (TIF)Click here for additional data file.Figure S9Uncropped immunoblots. Immunoblots from which portions were cropped for display in (TIF)Click here for additional data file.Figure S10Tumors in prostates of RAD001-treated Hi-MYC and MPAKT/Hi-MYC mice display lower levels of apoptosis and similar levels of proliferation compared with vehicle-treated mice. Lower magnification views of images displayed in (TIF)Click here for additional data file.Table S1Primary copy number alternation data from array CGH analysis.(DOC)Click here for additional data file.Text S1Supplemental Materials and Methods.(PDF)Click here for additional data file."} +{"text": "Lateolabrax japonicus using RNA-seq and DGE in an attempt to gain insights into the immunogenetics of marine fish.Systematic research on fish immunogenetics is indispensable in understanding the origin and evolution of immune systems. This has long been a challenging task because of the limited number of deep sequencing technologies and genome backgrounds of non-model fish available. The newly developed Solexa/Illumina RNA-seq and Digital gene expression (DGE) are high-throughput sequencing approaches and are powerful tools for genomic studies at the transcriptome level. This study reports the transcriptome profiling analysis of bacteria-challenged Vibrio harveyi-challenged L. japonicus is considerably altered, as indicated by the significant up- or down-regulation of 1,224 strong infection-responsive transcripts. Results indicated an overall conservation of the components and transcriptome alterations underlying innate and adaptive immunity in fish and other vertebrate models. Analysis suggested the acquisition of numerous fish-specific immune system components during early vertebrate evolution.RNA-seq analysis generated 169,950 non-redundant consensus sequences, among which 48,987 functional transcripts with complete or various length encoding regions were identified. More than 52% of these transcripts are possibly involved in approximately 219 known metabolic or signalling pathways, while 2,673 transcripts were associated with immune-relevant genes. In addition, approximately 8% of the transcripts appeared to be fish-specific genes that have never been described before. DGE analysis revealed that the host transcriptome profile of This study provided a global survey of host defence gene activities against bacterial challenge in a non-model marine fish. Results can contribute to the in-depth study of candidate genes in marine fish immunity, and help improve current understanding of host-pathogen interactions and evolutionary history of immunogenetics from fish to mammals. Homo sapiens), mouse (Mus musculus), frog (Xenopus laevis), chicken , and zebrafish (Danio rerio), has led to the emergence of studies focusing on the identification and characterization of immune-related genes in teleost fish based on comparative genomics. These have provided preliminary observations on fish immunogenetics and evolutionary history of immune systems from lower vertebrates to mammals [Danio rerio) due to the inadequate number of high-throughput deep sequencing technologies available [Since it is a representative population of lower vertebrates serving as an essential link to early vertebrate evolution, fish is believed to be an important model in various developmental and comparative evolutionary studies -3. Fish mammals ,8. Howevvailable ,10. ThisRecently developed RNA deep sequencing technologies, such as Solexa/Illumina RNA-seq and Digital gene expression (DGE), have dramatically changed the way immune-related genes in fish are identified because these technologies facilitate the investigation of the functional complexity of transcriptomes ,12. RNA-Lateolabrax japonicus) is an economically important marine species widely cultured in fisheries worldwide. Various diseases caused by bacterial and viral pathogens plague this species [Vibrio harveyi, a typical gram-negative pathogen of a wide range of marine animals. Infection results in a variety of vibriosis, a common aquatic animal disease associated with high mortality throughout the world [L. japonicus, V. harveyi infection leads to bacterial septicaemia with muscle ulcer as well as subcutaneous and gastroenteritic haemorrhage.Japanese sea bass raw reads from the head kidney and spleen tissues of bacteria- and mock-challenged fish, respectively, were generated using Solexa/Illumina RNA-seq deep sequencing analysis. Repetitive, low-complexity, and low-quality reads were filtered out prior to assembly of sequence reads for non-redundant consensus. Using Grape software, reliable reads were assembled into contigs, which were then compared with all PE reads. Overlap of PE reads with two contigs was taken to indicate that the contigs are short segments of a scaffold. Reads were used for gap-filling of these scaffolds to generate final scaffold sequences. Using tgicl and cap3 software programs, scaffold sequences were assembled into clusters that were then analysed for consensus. A total of 150,125 and 140,330 non-redundant consensus sequences, ranging from 100 to 2,000-bp, were generated from each group. Then, consensus sequences were merged for DGE analysis. Removal of partial overlapping sequences yielded 169,950 non-redundant consensus sequences ,33. CD-cL. japonicus, the immune-relevant genes, metabolic and signalling pathways were analysed. Approximately 2,673 consensus sequences were found to be homologous to known immune-relevant genes in other species and corresponding adaptors in mammals and in other fish species , while 848 consensus sequences were significantly down-regulated (including 10 most down-regulated sequences that decreased by 10-100-times) , TCR signalling pathways , antigen presenting and processing relevant pathways , TGF-\u03b2 signalling pathway , and various inflammatory cytokines and receptor relevant pathways . There were annotations in several other biological processes that may indirectly participate in immune response, such as the cell cycle; DNA replication, transcription, and translation; metabolisms of carbohydrates, amino acids, and lipids; and activation of ATPase family members, transcription elongation factor B, membrane transport protein, NADH dehydrogenase, NAD kinase, nucleolar protein 6, tyrosine protein kinase, ribosomal protein L32, nuclear receptor, and replication initiator 1. Among the 61 most over-expressed transcripts and the 10 most down-regulated transcripts, enrichments of factors involved in metabolic or signalling pathways that have not been linked to immune responses previously, such as cytoskeleton regulation, calcium signalling pathway, MAPK signalling pathway, aimnocyl-tRNA biosynthesis, and methionine, glutamate, and aminosugar metabolism, were detected. Highly responsive consensus sequences are shown in Table L. japonicus involved in the response to bacterial challenge. Among these novel sequences, 13 were differentially regulated by more than 100-fold, implying that they were strongly infection-responsive genes. ProDom analysis identified one HSP domain- and one protein kinase domain-containing sequence [Among the differentially expressed transcripts, more than 1,183 transcripts were well annotated, whereas approximately 41 transcripts had low sequence homology to known sequences in public databases, suggesting that they might be putative novel immune-relevant genes in sequence . SignalPsequence ,44. Obse46-C48-C74-C91) that are the hallmarks of IL-8 CXC chemokines and can be found throughout the vertebrate IL-8 family , serial analysis of gene expression (SAGE), and SAGE-derived technologies, which include massively parallel signature sequencing (MPSS) and polony multiplex analysis of gene expression. However, these approaches have several inherent limitations. For example, the array-based approaches allow detection of specific sequences only and capture the transcriptome while ignoring splice-junction information or alternative splicing events. The EST approach provides only partial sequences of individual cDNA clones, is sensitive to cloning biases, and is associated with high costs and difficulties in data analysis. SAGE and MPSS are also costly and cannot be used for splicing events . Thus, tpurposes ,10,18-29L. japonicus was conducted through these two approaches in an attempt to gain deep insights into the immunogenetics of a marine species. As expected, a large set of transcriptional sequences with complete or differing lengths of encoding regions was generated. KEGG analysis showed that more than 52% of transcripts are enrichment factors involved in approximately 219 known metabolic or signalling pathways, including cellular growth, differentiation, apoptosis, migration, endocrine, and immune system processes. Further, more than 8% of transcripts represent novel fish-specific genes that have never been described previously. Detailed analysis of immune-relevant genes and pathways showed that more than 2,673 transcripts are homologous to known immune-relevant genes, whereas approximately 2,082 transcripts can be enriched in various immune-relevant metabolic or signalling pathways. Challenging the fish with V. harveyi resulted in large alterations of the host transcriptome profile, including significant up- or down-regulation of 1,224 transcripts, among which 41 sequences might be novel immune-relevant genes in fish. In addition, several other biological processes that have not been linked to host immunity before, such as the metabolism of carbohydrates, amino acids, and lipids; activation of ATPase, NADH dehydrogenase, NAD kinase, and tyrosine protein kinase; and up-regulation of nuclear receptors, replication initiators, and ribosomal proteins, were found to be dramatically involved in host immune response. These significantly regulated transcripts might represent strong infection-responsive genes in L. japonicus, and reflect a number of immune activities during fish defence against bacterial challenge. The transcriptome profiling data sets obtained in this study provide strong basis for future genetic research in marine fish and support further in-depth genome annotation in vertebrates. Future molecular and functional characterisation of infection-responsive genes could lead to global identification of immune-relevant genes and infection markers in marine fish.In this study, the transcriptome profile analysis of bacteria-challenged Oncorhynchus mykiss), Atlantic salmon , medaka (Oryzias latipes), and zebrafish (Danio rerio), the immune-relevant transcriptional profiling data sets obtained from fish are still insufficient. Recently, DGE- and microarray-based transcriptome profiling studies performed in zebrafish revealed that zebrafish and its developing embryo are useful in vivo models for the identification of host determinants of responses to bacterial infection [L. japonicus presented in this study may largely improve knowledge on fish immunogenetics in other analytical systems. The present study also demonstrates the advantages of new deep sequencing approaches for gene discovery, thus providing new leads for functional studies of candidate genes involved in host-bacteria interactions. The RNA-Seq and DGE analyses conducted in this study were found to complement each other well. RNA-Seq was very effective in unravelling transcriptome complexity, and can detect a large set of genes, including numerous low-expressing genes or novel genes. DEG data can be merged with RNA-Seq data sets, indicating an affordable method for comparative gene expression study. Thus, RNA-Seq was initially performed in this study to provide strong reference transcriptome database for subsequent DGE analysis.At present, transcriptome analysis in fish relies mainly on the EST approach ,46. Althnfection ,10. HoweL. japonicus may contribute greatly to better understanding of the molecular and cellular activities involved in fish immunity. Results unexpectedly showed that the fish immune system is more complex than previously thought. On one hand, the substantial amount of immune-relevant genes involved in metabolic and signalling pathways and the induction of genes encoding cell surface receptors, signalling intermediates, transcription factors, and inflammatory mediators show a clear conservation of mechanisms detected in other vertebrate models, including humans. On the other hand, a large set of novel immune response genes and infection markers that have never been linked previously to immune responses in other vertebrate systems was identified in L. japonicus, indicating the existence of numerous fish-specific immune activities during early vertebrate evolution.Emerging hallmark components and the cells necessary for innate and adaptive immunity in higher vertebrates have been identified in fish ,48. ThisL. japonicus. The identified TLRs include the majority seen in mammals and humans (TLR1-13), and four TLRs seen in fish species. Adaptor proteins and downstream effectors identified include the majority known in mammals and humans, including MYD88, BTK, TOLLIP, FADD, HMGB1, HRAS, HSPD1, CASP8, MAPK8IP3, PELI1, RIPK2, SARM1, TICAM2, TIRAP, EIF2AK2, IRAK1, IRAK2, MAP3K7, MAP3K7IP1, NR2C2, PPARA, PRKRA, TRAF6, UBE2N, and UBE2V1. These adaptor proteins and downstream effectors have been found to be well enriched in various known TLR signalling pathways. Downstream transcriptional factors and pro-inflammatory cytokines mediated by these pathways, including NF-\u03baB, JNK/p38, NF/IL6, IRF, IFN-\u03b1/\u03b2, TNF-\u03b1, IL-2, IL-6, IL-8, and IL-10, was also be identified successfully. These suggest that TLR mechanisms are conserved from fish to mammals throughout vertebrate evolution. A putative draft of TLR signalling pathways in L. japonicus based on knowledge of TLR signalling in mammalian species was constructed . These pathway members largely contribute to the proliferation and activation of T cells in mammals, thus suggesting that TCR signalling mechanisms underlying the T cell activation might be conserved between teleost fish and mammals. A putative draft of TCR signalling pathways based on knowledge of pathways known in mammals was constructed at 25\u00b0C and fed with commercial pellet food at a daily ration of 0.7% body weight. All fish were maintained in the laboratory for at least two weeks prior to experimental use to allow for acclimatisation and evaluation of overall fish health. Only healthy fish, as determined by general appearance and level of activity, were used in the experiment.V harveyi strain (96-915), a pathogen for bacterial septicaemia in L. japonicas, was maintained in the laboratory. It was cultured in Thiosulfate Citrate Bile Salts Sucrose at 27\u00b0C overnight. The desired number of cells was adjusted to 5 \u00d7 108 CFU/ml. Cells were inactivated with 5% formalin at 27\u00b0C overnight before thorough washing with sterile PBS (pH 7.0). They were re-suspended in PBS prior to use.Wild-type marine fish-virulent V harveyi at 1 \u00d7 108 CFU per fish. In parallel, fish in the control groups were administrated with 0.2 ml of mock PBS. Both groups were kept under conditions as described above. At seven days post-challenge, fish were sacrificed after anaesthesia, and tissues from the head kidney and spleen were collected. Tissue samples from 15 fishes were mixed for RNA preparation. Total RNA was isolated using a TRIzol reagent (Gibco BRL) following the manufacturer's instructions and treated with RNase free DNase I (Qiagen). RNA concentrations were measured using a spectrophotometer and integrity was ensured through analysis on a 1.5% (w/v) agarose gel.Fish in the experimental groups were inoculated intraperitoneally with 0.2 ml of After RNA extraction, poly-A-containing mRNAs were purified using oligo-dT-attached magnetic beads and fragmented into small pieces using divalent cations under elevated temperature. Cleaved RNA fragments were copied into first strand cDNA using reverse transcriptase and random primers. This was followed by second strand cDNA synthesis using DNA polymerase I and RNase H. These cDNA fragments underwent end repair process, addition of a single 'A' base, and ligation of adapters. Products were subsequently purified and amplified through PCR to create the final cDNA libraries.http://www.phrap.org/[Transcriptome sequencing was conducted using Solexa/Illumina RNA-seq. Four fluorescently labelled nucleotides and a specialised polymerase were used to determine the clusters base by base in parallel. The 75-bp raw PE reads were generated by the Illumina Genome Analyzer II system. Raw reads were then assembled into non-redundant consensus sequences using Grape, tgicl, and CAP3 softwares ,53. All hrap.org/. Short shrap.org/. The resDGE analysis included sample preparation and sequencing. Sequence tag preparation was performed using the Digital Gene Expression Tag Profile Kit (Illumina) according to the manufacturer's instructions. Briefly, 6 \u03bcg total RNA was used for mRNA purification using oligo-dT magnetic bead adsorption and oligo-dT was used to guide reverse transcription for double-stranded cDNA synthesis. The generation of 5' ends of tags was done using endonuclease NlaIII, which recognizes and cuts off the CATG sites on cDNA. cDNA fragments with 3' ends were purified through magnetic bead precipitation, and Illumina adapter 1 was added to the 5' ends. The junction of Illumina adapter 1 and CATG site was the recognition site of MmeI, which cuts 17 bp downstream of the CATG site, producing tags with adapter 1. After removal of 3' fragments with magnetic bead precipitation, the 21-bp unique tags with adaptor 1 were purified and ligated to adaptor 2 to form a cDNA tag library. These adapter-ligated cDNA tags were enriched after 15 cycles of linear PCR amplification. The resulting 85-bp fragments were purified by 6% TBE polyacrylamide gel electrophoresis. Fragments were then digested and the single-chain molecules were fixed onto the Solexa Sequencing Chip (flowcell). Sequencing by synthesis was performed using the Illumina Genome Analyzer II system according to the manufacturer's protocols. Image analysis, base calling, generation of raw 17-bp tags, and tag counting were performed using the Illumina pipeline. Raw data (tag sequences) were deposited in the GEO database under submission number GSE21712.Clean tags and count number of DGE libraries from bacteria- and mock-challenged groups were collected and summarised using custom Bio-perl scripts. All tags were mapped to the reference transcriptome generated by RNA-seq. To monitor mapping events on both strands, both sense and complementary antisense sequences were included in the mapping process. Only perfect matches over the entire 21-bp length of the 17-bp tag plus the 4-bp NlaIII recognition site were allowed. This study was limited to tags that mapped to ORFs only and cannot show tags that mapped to mRNA with long 3'UTRs.Rigorous algorithms were developed to identify differentially expressed genes between two samples. The correlation of the detected count numbers between parallel libraries was assessed statistically by calculating the Pearson correlation. In addition to the P value, FDR was manipulated to determine differentially expressed genes . AssuminRepresentative consensus sequences with complete ORFs generated by RNA-seq were selected for experimental cloning and sequencing validation. The cDNAs of these genes were amplified by RT-PCR using the primers shown in Supplemental Table 6. All PCR products were purified using Gel Extraction Kit (Qiagen), cloned into pUCm-T vector (TaKaRa), and sequenced on MegaBACE 1000 Sequencer (GE) using the DYEnamic ET Dye Terminator Cycle Sequencing Kit (Pharmacia). Protein sequence alignments were generated using the Cluster W program (version 1.83). The phylogenies of protein sequences were estimated using MEGA 3.0 with the neighbour-joining method.Most used abbreviationsABCF: ATP-binding cassette subfamily F; AICDA: Activation-induced cytidine deaminase; AP-1: Activator protein 1; ASB: Ankyrin repeat and SOCS box protein; ASP: Apoptosis-stimulating of p53 protein; BTK: Bruton's tyrosine kinase; CAMP: Calmodulin-regulated spectrin-associated protein; CARD: Caspase recruitment domain; CBLB: E3 ubiquitin-protein ligase CBL-B; CDC: Cell division cycle; CDK: Cyclin-dependent kinase; CEBP: CCAAT/enhancer-binding protein; CFLAR: CASP8 and FADD-like apoptosis regulator; c-FOS: Cellular Proto-oncogene; CHUK: Conserved helix-loop-helix ubiquitous kinase; CNTFR: Ciliary neurotrophic factor receptor; CRADD: Death domain-containing protein CRADD; CRLF: Cytokine receptor-like factor; CSF: Macrophage/Granulocyte Colony-stimulating factor; CUB: First found in C1r/uEGF/bone morphogenetic protein; CYBB: Cytochrome b-245 heavy chain; CYFIP: Cytoplasmic FMR1 interacting protein; DEDD: Death effector domain-containing protein; DFD: Death-fold domain including CARD/DED/DEATH; DGCR: DiGeorge syndrome critical region; DMBT: Deleted in malignant brain tumors; DOCK: Dedicator of cytokinesis; DRAM: Damage-regulated autophagy modulator; Dscam: Down syndrome cell adhesion molecule; EBI: Epstein-Barr virus induced G-protein coupled receptor; EGF: Epidermal growth factor; EGR: Early growth response protein; EIF: Eukaryotic translation initiation factor; ELK: ETS domain-containing protein; EMAP: Echinoderm microtubule-associated protein; EMILIN: Elastin microfibril interfase located proteIN; ERCC: DNA-repair protein complementing XP-G cells homolog; FADD: Fas-associating death domain-containing protein; FAM: Family with sequence similarity; FGF: Fibroblast growth factor; FIMAC: Factor I membrane attack complex; FLT: Fms-related tyrosine kinase; FND: Fibronectin type III domain-containing protein; GALNAC4S-6ST: N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase GALNAC4S6ST; GATA: Trans-acting T-cell specific transcription factor; GDF: Growth/differentiation factor; GFI: Growth factor independent; GPR: G-protein coupled receptor; HELLS: Lymphoid-specific helicase encoded; HLA: Human leukocyte antigen; HMG: High mobility group protein; HSP: Heat shock protein; HTR: HEAT repeat-containing protein; ICAM: Intercellular adhesion molecule; ICE: Interleukin Catalytic Enzyme; IFRD: Interferon-related developmental regulator; IGBP: Immunoglobulin-binding protein; IKKA: Inhibitor of nuclear factor \u03ba-B kinase subunit \u03b1; IMPDH/IMDH: Inosine-5'-monophosphate dehydrogenase; IRAK: Interleukin-1 receptor-associated kinase; IRG: Immune-responsive gene; ITCH: Itchy homolog E3; JAK: Janus kinase; JAG: Protein jagged-1; JNK: c-Jun N-terminal kinase; KI13B: Kinesin-like protein KIF13B; KLF: Krueppel-like factor; LAG: Lymphocyte activation gene; LIFR: Leukemia inhibitory factor receptor; LRP: Prolow-density lipoprotein receptor-related protein; LRR: Leucine-rich repeat; MACPE: Membrane attack complex/perforin; MAF: Macrophage activating factor; MALT: Mucosa-associated lymphoma translocation protein; MAPK: Mitogen-activated protein kinase; MASP: Mannose binding lectin associated serine protease; MBL: Mannose binding lectin; MCA: Multisynthetase complex auxiliary component; MHC: Major histocompatibility complex; MIF: Macrophage migration inhibitory factor; Myd88: Myeloid differentiation primary response gene 88; NALP: NACHT; LRR and PYD domains-containing protein; NCK: Cytoplasmic protein; NF \u03ba: Nuclear factor \u03ba; NFAT: Nuclear factors of activated T cells; NOD: Nucleotide-binding oligomerization domain; NOS: Nitric oxide synthase; NR2C: Nuclear receptor subfamily 2 group C; OSGI: Oxidative stress-induced growth inhibitor; PAMP: Pathogen-associated molecular pattern; PARC: p53-associated parkin-like cytoplasmic protein; PAWR: PRKC apoptosis WT1 regulator protein; PCGF: Polycomb group RING finger protein; PEA15: Phosphoprotein enriched in astrocytes 15 gene; PGRP: Peptidoglycan recognition protein; PHLA: Pleckstrin homology-like domain family A; PIGR: Polymeric immunoglobulin receptor; PPARA: Peroxisome proliferator-activated receptor \u03b1; PRR: PAMP recognition receptor; PSMA: Proteasome subunit \u03b1 type; PSMB: Proteasome subunit \u03b2 type; PSMC: Protease 26 S subunit; ATPase; PSMD: Proteasome 26 S subunit; non-ATPase; PSME: Proteasome activator complex subunit; PTAFR: Platelet-activating factor receptor; PYCARD: PYD and CARD domain containing protein; PYD: Domain in pyrin; RAC: Ras-related C3 botulinum toxin substrate; REL: C-Rel proto-oncogene protein; RFX5: Regulatory factor \u00d7 5; RGC: Response gene to complement protein; RGS: Regulator of G-protein signaling; RHOA: Ras homolog gene family A; RIPK: Receptor-interacting serine/threonine-protein kinase; SARM: SAM and TIR containing; SCYE: Small inducible cytokine subfamily E; SFTPD/SP-D: Surfactant; pulmonary-associated protein D; SIGIRR: Single Ig IL-1-related receptor; SLC20A-1/2: Sodium-dependent phosphate transporter 1/2; SMAD: Mothers against decapentaplegic; SNED: Sushi nidogen and EGF-like domain-containing protein; SOCS: Suppressor of cytokine signaling; TANK: TRAF associated NF-\u03ba-B activator; TAPBP: TAP binding protein; TBX: T-box transcription factor; TGFA: Transforming growth factor -associated protein; TICAM: Toll-like receptor adapter molecule; TIRAP: Toll/interleukin-1 receptor domain-containing adapter protein; TMED: Transmembrane emp24 domain-containing protein; TNF(TNR): Tumor necrosis factor (receptor); TOLLIP: Toll-interacting protein; TPR: Tetratrico peptide repeat; TRADD: TNFRSF1A-associated death domain protein; TRAF: TNF-receptor-associated factor; TRIM: Tripartite motif-containing protein; TSP1: Thrombospondin type 1 repeats; TYK: Tyrosine-protein kinase; UBE: Ubiquitin-conjugating enzyme; UCRP/CRP: Ubiquitin cross-reactive protein; VEGF: Vascular endothelial growth factor; WAS: Wiskott-Aldrich syndrome; WSC: Cell wall integrity and stress response component; WWP: WW domain-containing proteins; XPA: DNA-repair protein complementing XP-A cells.LXX and DH conceived and designed the study, participated in the bioinformatics analysis, and drafted the manuscript. WRD and YWZ performed the experiments and designed the tables. JZS conceptualized the project, reviewed the manuscript, and provided guidance. All authors read and approved the final manuscript.Vibrio harveyi culture. We would also like to thank Guang-ping Liu, Hui-hui Liu, and Jian-qiu Zou for their help in data and figure processing. This work was supported by grants from Hi-Tech Research and Development Program of China (863) (2008AA09Z409), the National Basic Research Program of China (973) (2006CB101805), the National Natural Science Foundation of China , and the Science and Technology Foundation of Zhejiang Province .We acknowledge the Beijing Genomics Institute at Shenzhen for its assistance in original data processing and related bioinformatics analysis. We are thankful to professor Guo-liang Wang of Ningbo University for providing us with L. japonicasTable S1: Details on immune-relevant genes/homologues in .Click here for fileTable S2: Summary of tag mapping in DGE analysis for experimental and control groups.Click here for fileFigure S1: Tag abundance for mock- (A) and bacteria- (B) challenged group. Normalised tag copy number was calculated by dividing tag counts for each gene with the total number of tags generated for each library and are presented per one million transcripts. PM and 1 MM stand for perfect match and 1 miss match, respectively.Click here for fileTable S3: Summary of differentially expressed CD-containing consensus sequences.Click here for fileTable S4: Summary of putative novel immune/stress response consensus. Each consensus was analyzed using ProDom, SignalP, and TMHMM programs.Click here for fileTable S5: Primers used for validation analysis.Click here for fileFigure S2: Clustal W analysis for all IL-8-like CXC chemokines across all vertebrates.Click here for fileFigure S3: Phylogenetic analysis for all IL-8-like CXC chemokines across all vertebrates. A phylogenetic tree was constructed using the maximum likelihood method to show the relationship between L. japonicus IL-8 and other known vertebrate IL-8-like CXC-chemokines. Local bootstrap percentages were obtained after 10000 replications.Click here for file"} +{"text": "We previously demonstrated that vaccination of lactating rhesus monkeys with a DNA prime/ vector boost strategy induces strong SIV-specific cellular immune responses, but limited Envelope-specific humoral responses in breast milk. Therefore, we sought to improve vaccine-elicited Envelope-specific antibody responses in the milk compartment by using a transmitted/founder (T/F) HIV Envelope immunogen in a prime-boost strategy modeled after that of the moderately-successful RV144 HIV vaccine trial.Eight female, hormone-induced lactating rhesus monkeys were intramuscularly primed with either recombinant DNA (n = 4) or MVA pox virus vector (n = 4) expressing the T/F clade C HIV Envelope 1086C. All animals were intramuscularly boosted twice with the 1086C gp120 protein and the adjuvant MF59. Milk, vaginal, rectal and plasma samples were assessed for HIV Envelope-binding IgG and IgA responses. Anti-V1V2 antibodies and neutralization responses were also measured in milk and plasma.Envelope 1086C-binding IgG responses were detected in plasma, milk, and vaginal samples of all vaccinated animals and two of four rectal samples from MVA-vaccinated animals. Moreover, anti-V1V2 IgG antibodies were detected in all plasma, but only one milk sample. Low magnitude Envelope 1086C-specific IgA responses were detected in milk of two of four DNA-primed and three of four MVA-primed animals, but in none of the rectal samples. In contrast, all vaginal samples from MVA-primed, but none from DNA-primed, animals had detectable Envelope-specific IgA. Remarkably, strong tier 1 and low to moderate tier 2 neutralization was detected in plasma and milk of each group. The plasma neutralization titers against MW965 and CAP45 were significantly higher in MVA-primed than DNA-primed animals.MVA prime/ T/F Envelope protein boost strategy appears to induce stronger systemic and mucosal binding and neutralizing antibody responses than the DNA prime/protein boost regimen in lactating rhesus monkeys."} +{"text": "The regulation mechanism for intracellular 8-nitro-cGMP formation and its metabolic fate still remain unclear, however.We have discovered earlier 8-nitroguanosine 3Here we identified that hydrogen sulfide anion (SH-) rather than H2S gas per se is a potent negative regulator for endogenous 8-nitro-cGMP. SH- or free thiolate anion, effectively reacts with various 8-nitro-cGMP in cells to primarily generate each SH substituted, or sulfhydrated derivative via their direct chemical sulfhydration with SH-. This electrophile sulfhydration can therefore produce a new nucleophilic derivative, i.e., electrophile to nucleophile bioconversion to be caused by hydrogen sulfide. We also clarified that sulfhydration is functionally active in regulation of electrophilic cellular signaling, mediated by H-Ras oncogene. One of the major pharmacological effects of SH- was brought about by suppression of cellular senescence, caused by 8-nitro-cGMP-dependent Ras activation, involving downstream signaling molecules including Raf/Mek/Erk pathway finally activating p53 in cells.This may thus indicate an entirely novel aspect of biological functions of hydrogen sulfide in that it eventually behaves as an anion form, rather than a gaseous molecule, and interacts 8-nitro-cGMP to modulate its signaling functions. Moreover, we now consider that 8-nitro-cGMP is a major second messenger regulating electrophilic cellular signaling occurring widely in different species of biota including plants. The discovery of 8-nitro-cGMP and its signal modulator (i.e. hydrogen sulfide) may thus not only open a new era of research on the nucleotide cell signaling but also promote better understanding of the NO-mediated signaling mechanism in both eukaryotic and prokaryotic cells in general."} +{"text": "Atrial natriuretic peptide (ANP), via its guanylyl cyclase A (GC-A) receptor and cyclic GMP formation, exerts cardiac antihypertrophic and antifibrotic actions. Conversely, aldosterone promotes pathological cardiac remodeling via the mineralocorticoid receptor (MR). To investigate whether local cardiac ANP/GC-A signaling counteracts the effects of aldosterone during experimental hypertensive cardiac remodeling.We studied the impact of the MR antagonist eplerenone (100 mg/kg/day) on cardiac remodeling after transverse aortic constriction (TAC) in mice with conditional, cardiomyocyte-restricted deletion of GC-A (CM GC-A KO) and respANP, via GC-A/cGMP/cGKI signaling in cardiac myocytes, attenuates hypertensive cardiac remodeling and dysfunction. These protective ANP effects seem to be mediated at least in part by counterregulation of the deleterious genomic (MR-mediated) cardiac actions of aldosterone."} +{"text": "Cine steady-state free precession (SSFP) is the preferred sequence for ventricular function evaluation. However, SSFP demands uninterrupted RF excitation to maintain steady-state (SS) during suspended respiration. This is feasible in adults who can perform breath-holding (BH), but is difficult to accomplish in sedated or uncooperative children. To overcome this, many pediatric groups routinely perform multi-NSA acquisitions (MN) during free breathing. In this work, we validate a respiratory triggered (RT) SSFP sequence that drives the magnetization to steady-state before commencing cardiac gated cine acquisition in sedated pediatric population.This prospective study was performed on 12 sedated children with congenital heart disease (age: 7\u00b13 yrs) with IRB approval.All imaging was performed on a commercial MR scanner . Identical imaging parameters were used for MN and RT cine SSFP sequences [1] covering both the ventricles in short-axis (SA) orientation .Image quality assessment Figure and quanThe clinical scores for RT-SSFP were consistently better than MN-SSFP Figure . BA analThe free breathing RT-SSFP sequence allows clinically diagnostic images in sedated children without any penalty for total scan time, and offers improved myocardial blood-pool contrast, and edge definition when compared to MN-SSFP.None."} +{"text": "Drosophila have been shown to function as neoplastic tumor suppressor genes (nTSGs), including Endosomal Sorting Complex Required for Transport-II (ESCRT-II) components vacuolar protein sorting 22 (vps22), vps25, and vps36. However, most studies of endocytic nTSGs have been done in mosaic tissues containing both mutant and non-mutant populations of cells, and interactions among mutant and non-mutant cells greatly influence the final phenotype. Thus, the true autonomous phenotype of tissues mutant for endocytic nTSGs remains unclear. Here, we show that tissues predominantly mutant for ESCRT-II components display characteristics of neoplastic transformation and then undergo apoptosis. These neoplastic tissues show upregulation of c-Jun N-terminal Kinase (JNK), Notch, and Janus Kinase (JAK)/Signal Transducer and Activator of Transcription (STAT) signaling. Significantly, while inhibition of JNK signaling in mutant tissues partially inhibits proliferation, inhibition of JAK/STAT signaling rescues other aspects of the neoplastic phenotype. This is the first rigorous study of tissues predominantly mutant for endocytic nTSGs and provides clear evidence for cooperation among de-regulated signaling pathways leading to tumorigenesis.Multiple genes involved in endocytosis and endosomal protein trafficking in Drosophila, as well as in other organisms, genes that control endocytosis and endosomal protein sorting behave as nTSGs. Such endocytic nTSGs include avalanche (avl) Rab5vps45Rabenosyn (Rbsn) tumor suppressor protein 101 (tsg101 aka erupted (ept) or vps23) vps28vps25vps22 (aka larsen (lsn)) vps20shrub (shrb) vps2vps4Tumor development involves destabilization of the well-controlled processes of cell proliferation, cell polarization, and programmed cell death that are tightly regulated by widely conserved signaling pathways. Therefore, genes that act as regulators of these signaling pathways may behave as nTSGs. In vps22, vps25, and vps36. The products of these genes mediate the transfer of cargo from ESCRT-I to ESCRT-III The ESCRT machinery promotes the maturation of early endosomes into multi-vesicular bodies (MVBs) ESCRT-II components and other endocytic nTSGs focused on their mosaic phenotype, when mutant clones are surrounded by wild-type cells. Thus, the complex mosaic phenotype of endocytic nTSGs has been well characterized. Epithelial polarity and proliferation control are disrupted in mutant clones tsg101, vps22, or vps25vps25 mutant clones also promote non-cell autonomous cell survival through upregulation of the apoptosis inhibitor Diap1 Previous studies of the mutant phenotypes of vps22, vps25, and tsg101 mosaic tissues through non-cell autonomous upregulation of JAK/STAT and Yorkie signaling In mutant clones of endocytic nTSGs, endosomal trafficking is blocked and membrane proteins accumulate in abnormal endosomal compartments tsg101 and vps25 are apoptotic vps25 mutant clones unleashes a strong neoplastic phenotype characterized by massive tumorous overgrowth, loss of cell polarity, and invasive properties In mosaic tissues, mutant clones of tsg101 and vps25 mutant clones, Yorkie signaling is up-regulated vps25 mosaic discs, Yorkie signaling is only detectable non-cell autonomously in non-mutant neighboring cells From these studies, it is clear that the interactions between the mutant and non-mutant populations of cells greatly influence the final phenotype. However, while the non-cell autonomous mechanisms that cause hyperplastic overgrowth are well characterized, the mechanisms that cause autonomous neoplastic transformation of tissue mutant for endocytic nTSGs are poorly understood. Because endocytic trafficking controls multiple signaling pathways, it is likely that tumors caused by mutations in endocytic nTSGs acquire their neoplastic characteristics through the de-regulation of numerous signaling pathways. In hypomorphic Drosophila STAT, Stat92E, are increased, leading to increased JAK/STAT signaling activity Stat92E condition throughout the disc that affects both autonomous and non-cell autonomous phenotypes In endocytic nTSG mutant tissues, the protein levels of the JAK/STAT ligand Unpaired (Upd), the JAK/STAT receptor Domeless (Dome), and the ey-FLP/cell lethal (cl) system vps22, vps25 and vps36. These overgrown, neoplastic tumors display disorganized cellular architecture and disrupted epithelial structures with expanded apical-basal domains. Additionally, these tissues are unable to terminally differentiate and are invasive. Unexpectedly, although competitive cellular interactions have been largely eliminated by the ey-FLP/cl method, these predominantly mutant tissues are also very apoptotic. Within mutant tissues, JNK, Notch, and JAK/STAT signaling are up-regulated. Reducing JNK activity in ESCRT-II mutant tissue partially blocks the overproliferation phenotype and apoptosis but does not otherwise affect neoplastic transformation. In addition, complete loss of JAK/STAT signaling strongly rescues the neoplastic phenotype. Thus, this study supports the idea that de-regulation of signaling pathways, especially JNK and JAK/STAT signaling, in vps22, vps25, and vps36 mutant tissues leads to neoplasia.Here, in order to understand the cause of the neoplastic transformation of these mutant clones, we employed the 5F3-8vps22N55vps25\u039469vps36(this study), H16ark397Stat92Epuc-lacZGbe-Su(H)-lacZE(spl)m8 2.61-lacZ10X-STAT-GFPDNUAS-bskey-Gal4\u039469vps36 is a null allele generated by imprecise excision of the P-element transposon inserted in the first exon 29 base pairs upstream of the initiator ATG in the L5212vps36 allele.The following mutants and transgenic lines were used: vps22, vps25, or vps36, we used the ey-FLP/cl technique cl indicates an anonymous cell lethal mutation that kills cells when homozygous ESCRT-II mutant alleles were crossed to ey-FLP; FRT cl flies. The use of the FRT depended on the location of the ESCRT-II gene in the genome. The complete genotypes are indicated in the legends to the figures.To generate imaginal discs predominantly mutant for intra ; mouse \u03b1-BrdU ; rabbit \u03b1-cleaved Caspase-3 ; mouse \u03b1-\u03b2-gal and rabbit \u03b1-pJNK ; and rabbit \u03b1-aPKC . AF488-phalloidin and AF546-phalloidin were obtained from Sigma Aldrich. Cy-3 and Cy-5 fluorescently-conjugated secondary antibodies were obtained from Jackson ImmunoResearch. Vectashield with DAPI was obtained from Vector Laboratories. TUNEL kit was obtained from Roche Diagnostics. Images were captured using Olympus Optical FV500 or FV1000 confocal microscopes and processed using Adobe Photoshop CS4.Imaginal discs were dissected from third instar larvae and stained using standard protocols. The following antibodies were used: mouse \u03b1-Dlg (1\u223620), rat \u03b1-ELAV (1\u223640), mouse \u03b1-Mmp1 , and mouse \u03b1-Notchey-FLP/cl method generates eye-antennal imaginal discs that are almost entirely composed of mutant tissue in otherwise heterozygous animals (see Methods) ey-FLP-induced mitotic recombination by a cell lethal (cl) mutation that is present on the homologous chromosome arm. The use of the ey-FLP ensures high FLP activity such that most cells undergo mitotic recombination and only a few heterozygous cells remain. Thus, eye-antennal discs generated by this method are almost entirely mutant for the gene of interest.The ey-FLP/cl system to generate tissues predominantly mutant for ESCRT-II components vps22, vps25, or vps36. These predominantly mutant epithelial tissues have a very striking phenotype: unlike wild-type single-layered eye-antennal imaginal discs, they overgrow into multi-layered, dense \u201cballs\u201d of cells labeling to mark cells in S-phase. Control discs show the normal BrdU pattern in eye-antennal discs . Of noteire disc . Post-miESCRT-II components, we first labeled discs with phalloidin. Phalloidin recognizes cortical actin and thus highlights cellular architecture and organization throughout tissues. Control discs stained with phalloidin show a consistent shape characteristic of Drosophila eye-antennal imaginal discs . However, these studies in mosaic tissues fail to answer two important questions: (1) What signaling pathways are de-regulated in predominantly mutant tissues completely independent from interactions with non-mutant populations of cells? (2) Does this autonomous de-regulation of signaling contribute to the autonomous neoplastic phenotype? To answer the first question, we examined levels of Notch, JAK/STAT, and JNK signaling in discs predominantly mutant for ESCRT-II components.Due to the endosomal sorting defect in ESCRT components ESCRT-II components. To assess Notch signaling, we used two Notch reporters, the Gbe-Su(H)-lacZ reporter E(spl)m8 2.61-lacZ reporter Gbe-Su(H)-lacZ reporter in vps25 mutant discs showed that Notch signaling is very high throughout the entire disc (E(spl)-lacZ reporter to examine Notch activity in vps22 and vps36 mutant tissues and found that Notch signaling is indeed very high but only in about half of each mutant disc (Gbe-Su(H)-lacZ reporter. From these data, we clearly see that Notch signaling is up-regulated in tissues predominantly mutant for ESCRT-II components.Multiple studies have shown that Notch signaling is up-regulated in tissues mosaic for ire disc . We usedant disc . To furtant disc -D, suppotsg101 and vps25 mutant clones, and Notch-induced upregulation of the JAK/STAT ligand Upd has been shown to contribute to the non-cell autonomous increase of proliferation in neighboring non-mutant cells ESCRT-II mutant tissues. To assess levels of JAK/STAT signaling, we used the well-characterized 10X-STAT-GFP reporter ESCRT-II mutant discs and TUNEL labeling in predominantly mutant discs. In control discs, a few Cas-3* positive cells are scattered throughout the tissue, but most cells are not apoptotic , DNbskey-Gal4. In control discs, overexpression of DNbsk in otherwise wild-type discs has no apparent effect on architecture, polarity, differentiation, and Mmp1 expression , allowing a convenient double mutant analysis. It was recently shown that Stat92E mutant clones are eliminated by cell competition Stat92E in which competitive interactions are eliminated reveal only weak abnormalities strongly rescues the neoplastic characteristics seen in vps22 single mutant tissues. The disorganization of cellular architecture observed in vps22 mutant discs is significantly rescued by removal of JAK/STAT signaling. Labeling with phalloidin shows that double mutant discs retain their characteristic eye-antennal imaginal disc shape .Overall, it appears that the same signaling pathways that are induced in mosaic clones are also activated in predominantly mutant tissues. However, two results of this study are noteworthy. First, it is surprising that JNK activity is strongly induced in tissues predominantly mutant for ESCRT-II mutant tissues undergo neoplastic transformation, they also show high levels of apoptosis. Animals with predominantly mutant eye-antennal imaginal discs die as headless pharate pupae, a phenotype likely caused by the apoptosis of the imaginal discs before the adult stage. Reduction of JNK signaling in vps22, vps25, or vps36 mutant discs leads to lower levels of apoptosis, supporting a role for JNK signaling in the cell death of the predominantly mutant tissues. More excitingly, JNK also controls proliferation in these tissues, as shown by the reduction of proliferation seen when JNK signaling was down-regulated. This observation is consistent with previous findings that JNK can induce non-cell autonomous proliferation ESCRT-II mutant discs, it does not affect other aspects of the neoplastic phenotype.Interestingly, although ESCRT-II mosaic discs, Notch-induced secretion of the JAK/STAT ligand Upd triggers non-cell autonomous proliferation vps22 mutants. In vps22 Stat92E double mutant discs, organization of cellular architecture is definitively rescued with the layout of the tissue closely resembling that of a wild-type eye-antennal imaginal disc. In addition, apical-basal polarity markers are localized more-or-less correctly in these tissues, indicating that epithelial polarity is more intact. Finally, differentiation in the posterior portion of the eye disc is preserved when JAK/STAT signaling is inhibited. Thus, de-regulation of JAK/STAT signaling in vps22 mutant discs contributes to the cellular disorganization and the lack of differentiation seen in the tissues, which is consistent with a previous study that implicated JAK/STAT signaling in cell cycle control, cell size, and epithelial organization in tsg101 mutant tissues The role of JAK/STAT signaling in these mutants is complex. In mutant clones of ESCRT-II mutant cells has not been observed. In fact, these mutant cells are eliminated by apoptosis. Only if apoptosis is blocked in these cells, is a strong overgrowth phenotype with neoplastic characteristics observed It was recently shown that cells with strong gain of JAK/STAT activity transform into supercompetitors and eliminate neighboring cells with normal JAK/STAT activity by cell competition Notch function both in wild-type and mutant tissue causes general problems in tissue growth) prevented us from examining this possibility.How endosomal trafficking specifically regulates JAK/STAT signaling and, thus, how blocking trafficking leads to increases in signaling pathway activity are interesting questions to answer in the future. It is possible that, like endocytic regulation of the Notch receptor, the endosomal pathway tightly regulates Domeless (Dome), the JAK/STAT pathway receptor. It has been shown previously that Dome is trafficked through the endocytic machinery and that this trafficking of Dome can affect the downstream output of the JAK/STAT signaling pathway ESCRT-II mutants causes neoplastic transformation. JAK/STAT signaling is known to be an oncogenic pathway in Drosophiladpp signaling It will be important to examine how de-regulated JAK/STAT signaling in Tsg101, as early studies showed that downregulation of Tsg101 promotes the growth of mouse 3T3 fibroblasts in soft agar Tsg101 functions as a tumor suppressor in metazoans. Importantly, a number of studies have shown changes in expression of ESCRT components in human cancer cells, including changes in expression of ESCRT-I components Tsg101 . Since the primary proteins that function in endocytosis and endosomal trafficking are conserved from yeast to humans, it is likely that our findings in Drosophila may have important implications for human disease.A number of studies have implicated genes that function in endocytosis and endosomal protein sorting as tumor suppressors in human cancers. Most well known is Figure S1Notch protein levels are upregulated in vps22, vps25, and vps36 mutant tissues. Shown are predominantly mutant eye-antennal imaginal discs. Scale bars represent 50 \u00b5m. Phalloidin (green) is used to mark the overall shape of the tissue. Notch protein levels are shown by staining with an antibody recognizing the intracellular domain of the protein (\u03b1-Nintra). Notch protein levels are increased in imaginal discs predominantly mutant for vps22 (B), vps25 (C), or vps36 (D), as compared to Notch protein levels in control discs . Genotypes: (A) eyFLP;; FRT82B/FRT82B cl. (B) 5F3-8/FRT82B cl.eyFLP;; FRT82B vps22 (C) N55 y+/FRT42D cl.eyFLP; FRT42D vps25 (D) \u039469 FRT80B/cl FRT80BeyFLP;; vps36.(TIF)Click here for additional data file.Figure S2JNK signaling is upregulated in tissues predominantly mutant for vps25. Shown are predominantly mutant eye-antennal imaginal discs. Phalloidin (green) is used to mark the overall shape of the tissue. puc-lacZ is detected by \u03b2-gal labeling . (A-B) Imaginal discs predominantly mutant for vps25 induce high levels of puc-lacZ (B), as compared to puc-lacZ expression in control discs . Scale bars represent 50 \u00b5m. Genotypes: (A) eyFLP; FRT42D/FRT42D cl; puc-LacZ/+. (B) N55 y+/FRT42D cl; puc-LacZ/+eyFLP; FRT42D vps25.(TIF)Click here for additional data file.Figure S3Inhibition of JNK signaling does not affect the disorganization of cellular architecture, the failure of differentiation, and the invasive potential of ESCRT-II mutant tissue. Shown are predominantly mutant eye-antennal imaginal discs. JNK signaling is inhibited by expression of the DNUAS-bsk transgene using ey-Gal4. Phalloidin (green) is used to mark the overall shape of the tissue. Scale bars represent 50 \u00b5m. aPKC ) and Dlg ) labelings of discs predominantly mutant for vps25 in which JNK signaling is inhibited show that cellular architecture is disrupted (B-B\u2019\u2019\u2019). Cellular architecture is not disrupted in control discs in which JNK signaling is inhibited (A-A\u2019\u2019\u2019). ELAV labelings of discs predominantly mutant for vps25 in which JNK signaling is inhibited show that very few cells in the mutant discs differentiate normally . Differentiation occurs normally in control discs in which JNK signaling is inhibited . Mmp1 labelings of discs predominantly mutant for vps25 in which JNK signaling is inhibited show that levels of this protein are increased . Mmp1 levels are not affected in control discs in which JNK signaling is inhibited . Genotypes: DN; FRT42D y+/FRT42D cl; ey-Gal4/+.eyFLP/UAS-bsk DN; FRT42D vps25N55 y+/FRT42D cl; ey-Gal4/+eyFLP/UAS-bsk.(TIF)Click here for additional data file."} +{"text": "In OVA-challenged mice, we demonstrated that IVIg markedly attenuates airway hyperresponsiveness (AHR) and abrogates airways inflammation, accompanied by substantial induction of antigen-specific Foxp3+CD25+Foxp3+Treg was determined by flow-cytometry. AHR was measured, using a flexiVent small animal ventilator. Phenotypic properties of dendritic cells (DC) from various experimental groups were assessed by flow-cytometry. Expression of DCIR on DC was evaluated by flowcytometry and ICC. Adoptive transfer of DC was carried out to show the tolerogenic activity of IVIg-primed DC.Mice were sensitized (i.n.) with OVA and then received IVIg or sialic acid enriched IVIg (SA-IVIg) fragments (i.p.), and then underwent challenge (i.n.). The induction of CD4IVIg and the SA-IVIg fraction induced Treg and abrogated AHR in OVA-challenged mice comparably. It followed by tolerogenic predisposition of DC (decrease of CD80/CD86 expression and IFN-\u03b3 production and increased level of IL-10). Adoptive transfer of DC from IVIg treated mice to OVA-challenged WT syngeneic mice has the similar anti-inflammatory activity of IVIg/SA-IVIg. Expression of DCIR (Inhibitory C-type lectin receptors) on DC of IVIg and SA-IVIg treated mice increased significantly.IVIg induces Treg likely via conferring tolerogenic activities to DC. This mechanism is dependent on sialylated fraction of IVIg. DCIR is an inhibitory C-type lectin receptor that can be targeted by SA-IVIg and induce an inhibitory signal into the ligated cells. More dissection is required to confirming the role of DCIR in this model."} +{"text": "Hepatitis D virus (HDV) infection is considered to cause more severe hepatitis than hepatitis B virus (HBV) monoinfection. With more than 9.5 million HBV-infected people, Vietnam will face an enormous health burden. The prevalence of HDV in Vietnamese HBsAg-positive patients is speculative. Therefore, we assessed the prevalence of HDV in Vietnamese patients, determined the HDV-genotype distribution and compared the findings with the clinical outcome.266 sera of well-characterized HBsAg-positive patients in Northern Vietnam were analysed for the presence of HDV using newly developed HDV-specific RT-PCRs. Sequencing and phylogenetic analysis were performed for HDV-genotyping.The HDV-genome prevalence observed in the Vietnamese HBsAg-positive patients was high with 15.4% while patients with acute hepatitis showed 43.3%. Phylogenetic analysis demonstrated a predominance of HDV-genotype 1 clustering in an Asian clade while HDV-genotype 2 could be also detected. The serum aminotransferase levels as well as total and direct bilirubin were significantly elevated in HDV-positive individuals (p<0.05). HDV loads were mainly low (<300 to 4.108 HDV-copies/ml). Of note, higher HDV loads were mainly found in HBV-genotype mix samples in contrast to single HBV-infections. In HBV/HDV-coinfections, HBV loads were significantly higher in HBV-genotype C in comparison to HBV-genotype A samples (p<0.05).HDV prevalence is high in Vietnamese individuals, especially in patients with acute hepatitis B. HDV replication activity showed a HBV-genotype dependency and could be associated with elevated liver parameters. Besides serological assays molecular tests are recommended for diagnosis of HDV. Finally, the high prevalence of HBV and HDV prompts the urgent need for HBV-vaccination coverage. Hepatitis D virus (HDV) infection is considered to account for more severe complications of viral hepatitis with rapid progression to cirrhosis, increased risk of hepatic decompensation and death compared to hepatitis B virus (HBV) monoinfection ,2. HepatHDAg) molecules and a single-stranded, circular RNA-genome of approximately 1.7 kb . A serial dilution of pSVL-HDV plasmids with concentrations from 3.04 log10 copies/\u00b5l to 5.04 log10 copies/\u00b5l was used as standard and to calibrate the system as described . In. In53]. 7 copies/ml] versus 1.6 x 107 copies/ml ) ) . Analysipatients ,38 probaNearly exclusively in the AHB group, HDV-loads of 609 to 4.108 HDV-copies/ml could be detected demonstrating active HDV-replication. Interestingly, the higher HDV-loads were secondary only detectable in HBV-genotype mix infections. Interviral interference between HBV-genotypes in genotype mix infections can lead to suppression of one of the participating HBV-genotypes . TherefoDetermination of circulating HDV genotypes in Vietnam has not been performed to date. The phylogenetic analysis of the present study showed that the predominant HDV-genotype was HDV-genotype 1 (90.5%) which mainly clustered to the Asian HDV-genotype 1 branch. One HDV strain (B171) was closely related with the HDV-genotype 1 strain of Central Africa (AJ000558) showing a 91% bootstrap value .Our results suggest a wider distribution of HDV genotype 1 and did not support a narrow geographical spread of distinct HDV subtypes. Furthermore, HDV-genotype 2 was also detected in our patient cohort which was commonly found in Taiwan , Japan and RussIn conclusion, the results of the present study showed a high prevalence of HDV (15.4%) and were most prevalent in the acute hepatitis group (43.3%) while HDV-genotype 1 is the predominant virus in the Vietnamese HBsAg-positive patients. HBV/HDV coinfection could be associated with constantly elevated liver parameters, a finding which is mainly found in the chronically infected patient groups and not in acute infections, if compared HDV-positive patients with HDV-negative patients. HDV replication activity seemed to favor HBV genotype mixes and distinct HBV genotypes (HBV-A) revealing that HDV may have a HBV-genotype dependency. However, the cross-sectional study design with the bias of hospitalized HBsAg-positive patients prevents us from drawing more definitive conclusions which should be evaluated in further studies. We conclude that molecular tests should be used in routine diagnostics to detect HDV in HBsAg-positive patients even when elevated liver parameters are presented."} +{"text": "Drosophila oocyte uses mRNA localization to establish polarity and hence provides a genetically tractable model in which to study this process. The spatial restriction of oskar mRNA and its subsequent protein product is necessary for embryonic patterning. The localization of oskar mRNA requires microtubules and microtubule-based motor proteins. Null mutants in Kinesin heavy chain (Khc), the motor subunit of the plus end-directed Kinesin-1, result in oskar mRNA delocalization. Although the majority of oskar particles are non-motile in khc nulls, a small fraction of particles display active motility. Thus, a motor other than Kinesin-1 could conceivably also participate in oskar mRNA localization. Here we show that Dynein heavy chain (Dhc), the motor subunit of the minus end-directed Dynein complex, extensively co-localizes with Khc and oskar mRNA. In addition, immunoprecipitation of the Dynein complex specifically co-precipitated oskar mRNA and Khc. Lastly, germline-specific depletion of Dhc resulted in oskar mRNA and Khc delocalization. Our results therefore suggest that efficient posterior localization of oskar mRNA requires the concerted activities of both Dynein and Kinesin-1.In order for eukaryotic cells to function properly, they must establish polarity. The Many cellular processes such as endocytosis, cell division, and cell migration require the specific and active transport of molecules to defined cellular sites . This trMost members of the Kinesin family of motor proteins transport cargo toward the plus end of microtubules, whereas cytoplasmic Dynein delivers cargo to the microtubule minus end ,3. Over Drosophila oocyte is an excellent model in which to examine the in vivo function and regulation of microtubule motors. Within the oocyte, oskar mRNA is specifically localized at the posterior pole ; P{w[+mC]=matalpha-GAL4-VP16}V2H ; P{w[+mC]=UASp-Act5C.mRFP}13, w[*] . UASp-Dic-GFP; GFP-Stau (St Johnston lab); par1w3 ; par1632w3 [6323 ,38; grk2323 [2B6 ,36; grk2B6 [2E12 ; dhc shRPlease note that an addition maternal alpha-tubulin driver is available from the Bloomington stock center (Stock #7063). However, this driver is active in early-stage egg chambers in addition to mid and late-stage egg chambers.Drosophila Genome Resource Center) into a UASp vector which allows C-terminal tagging with 3 concatemeric copies of the eGFP sequence [The Dic-GFP transgene was constructed by cloning the dic coding sequence and 248 nt of 5\u2019UTR upstream of the initiation codon . The ovaries were homogenized in lysis buffer A , and Halt protease inhibitor cocktail (Pierce)). The lysates were cleared by centrifugation at 10,000g at 4\u00b0C for 10 min. For each immunoprecipitation, 600ug of lysate was incubated with the respective antibodies at 4\u00b0C for 1.5hrs. The complexes were isolated using proteinA coupled agarose beads (Pierce). The beads were washed four times for 5 min. each with gentle shaking at 4\u00b0C using wash buffer A . The bound complexes were eluted in 100ul of elution buffer at 68\u00b0C for 10min. RNA was extracted from the eluate by phenol chloroform extraction. The RNA pellet was resuspended in 20\u03bcl of RNAsecure (Life Technologies). Two \u03bcl of each sample was reverse transcribed using SuperscriptIII (Life Technologies) and random hexamers. Ovaries from well-fed females were dissected as described. The ovaries were homogenized in lysis buffer B ). The lysates were cleared by centrifugation at 10,000g at 4\u00b0C for 10min. Dic-GFP was immunoprecipitated from 600ug of total lysate using GFP-trap beads (Chromotek). The lysates were incubated with the beads for 1.5hrs. The beads were subsequently washed three times (using lysis buffer B) to eliminate non-specifically bound proteins. The bound complexes were eluted by boiling in Laemmli buffer, run on a gel, and analyzed by western blotting.oskar, bicoid and gurken mRNAs were visualized as previously described with a few modifications [ications . In brieUnless specifically stated, the indicated dilutions are for immunofluorescence. The following antibodies were used: Mouse anti-Dhc ; rabbit anti-Khc ; rabbit anti-GFP ; rat anti-GFP ; rabbit anti-Lis-1 ; mouse anti-Dic ; mouse anti-Orb ; mouse anti-LaminDmO ; rabbit anti-Staufen ; rabbit anti-EB1 , rabbit anti-CLIP-190 , rabbit anti-Glued mouse anti-gamma-tubulin ; rabbit anti-Osk ; goat anti-rabbit Alexa 594 and 488 ; goat anti-mouse Alexa 594 ; goat anti-mouse HRP and goat anti-rabbit HRP .Figure S1oskar and bicoid mRNAs.Dic-GFP associates specifically with Ovarian lysates from flies expressing Act5c-GFP (lane 1) and Dic-GFP (lane 2) were subjected to immunoprecipitation using GFP antibody beads. The co-precipitating RNAs were extracted and analyzed using RT-PCR. oskar and bicoid mRNAs were enriched in the Dic-GFP pellet. By contrast, vasa and smb mRNAs were present at background levels in both pellets.(EPS)Click here for additional data file.Figure S2Expression profile of matalpha-Gal4.Flies containing a UASp-Act5c-mRFP transgene were crossed to the matalpha-Gal4 driver (Bloomington stock 7062). Female progeny from this cross were dissected, fixed and stained with DAPI (blue) to visualize nuclei. Act5c-RFP (red) is strongly expressed in mid to late-stage egg chambers. Upon increasing the gain, Act5c-RFP signal could also observed at lower levels in earlier stage egg chambers.(EPS)Click here for additional data file.Figure S3oskar mRNA levels are unaffected in Dhc depleted ovaries.Ovaries from flies expressing shRNA targeting either luciferase or different regions of dhc (dhc shRNA A and B) were dissected. Total RNA was extracted, and the level of oskar and gamma-tubulin mRNAs were determined using RT-PCR. Depletion of Dhc has no effect on the level of oskar mRNA.(EPS)Click here for additional data file.Figure S4oskar mRNA localization.Depletion of EB1 does not affect Dhc and (A-B) Ovaries from flies expressing shRNA targeting either luciferase or eb1 were dissected and processed for immunofluorescence using an antibody against EB1. EB1 was abundantly expressed in the germline of control oocytes, but was significantly depleted in egg chambers expressing shRNA targeting eb1.eb1 were processed for in situ hybridization using probes against oskar mRNA. No defects were observed in the localization of oskar mRNA.(C) Ovaries from flies expressing shRNA targeting eb1 were processed for immunofluorescence using an antibody against Dhc. Dhc was enriched at the posterior pole in EB1 depleted oocytes.(D) Ovaries from flies expressing shRNA targeting (EPS)Click here for additional data file.Figure S5oskar mRNA.Over-expression of p50/Dynamitin delocalizes (A) Ovaries from wild-type flies were processed for in situ hybridization using anti-sense probes against oskar mRNA. oskar mRNA. The level of posteriorly localized oskar mRNA was decreased upon p50 over-expression (B). In parallel, the level of delocalized oskar mRNA within the oocyte was increased in these egg chambers (C). Panel C represents the same egg chamber depicted in \u2018B\u2019 imaged using increased gain.(B-C) Ovaries from flies expressing UASp-p50/Dmn driven using the maternal alpha-tubulin driver, were processed for in situ hybridization using anti-sense probes against (EPS)Click here for additional data file."} +{"text": "Siglec-F and Siglec-8 are functional paralog proapoptotic cell surface receptors expressed on mouse and human eosinophils, respectively. Whereas Siglec-8 mediated death involves caspases and/or reactive oxygen species (ROS) generation and mitochondrial injury, very little is known about Siglec-F-mediated signaling and apoptosis. Therefore the objective of the current experiments was to better define apoptosis pathways mediated by Siglec-F and Siglec-8. Given that Siglec-F-induced apoptosis is much less robust than Siglec-8-induced apoptosis, we hypothesized that mechanisms involved in cell death via these receptors would differ.Consequences of engagement of Siglec-F on mouse eosinophils were studied by measuring ROS production, and by performing apoptosis assays using eosinophils from normal, hypereosinophilic, NADPH oxidase-deficient, src homology domain-containing protein tyrosine phosphatase (SHP)-1-deficient, and Lyn kinase-deficient mice. Inhibitors of caspase and Src family kinase activity were also used.Engagement of Siglec-F induced mouse eosinophil apoptosis that was modest in magnitude and dependent on caspase activity. There was no detectable ROS generation, or any role for ROS, NADPH oxidase, SHP-1, or Src family kinases in this apoptotic process.in vivo in mice may not provide identical mechanistic predictions for consequences of Siglec-8 targeting in vivo in humans.These data suggest that Siglec-F-mediated apoptosis is different in both magnitude and mechanisms when compared to published data on Siglec-8-mediated human eosinophil apoptosis. One likely implication of this work is that models targeting Siglec-F Eosinophils have been implicated in a variety of disorders ranging from asthma and allergic diseases to helminth parasite immunity to hematologic disorders X) Siglecs are a family of single-pass transmembrane cell surface proteins found predominantly on leukocytes Mechanistic studies with Siglec-8 implicated both caspases and reactive oxygen species (ROS) generation resulting in mitochondrial injury in eosinophil death Most siglecs, including Siglec-8 and Siglec-F, have immuno-receptor tyrosine-based inhibitory motifs (ITIMs) in their intracellular domain, suggesting an inhibitory role for signaling Conclusions from studies of Siglec-F have generally paralleled those of Siglec-8. For example, antibody cross-linking of Siglec-F on mouse eosinophils causes apoptosis Given the potential similarities and differences involved in Siglec-8 and Siglec-F mediated apoptosis, the goal of this work was to define apoptosis pathways mediated by Siglec-F and explore the role of ROS, NADPH oxidase activity, SHP-1 and caspases involved in Siglec-F-induced cell death. Our data suggest that at least for studies of eosinophil apoptosis, examination of Siglec-F does not always provide the same mechanistic conclusions as for Siglec-8.+/+ mice) displaying splenomegaly and marked blood eosinophilia m1JNcf1/J mice with defective NCF1/p47phox protein, resulting in reduced NADPH oxidase activity and ROS production (Ncf\u2212/\u2212 mice), viable motheaten mice deficient in Src-homology 2-domain phosphatase (SHP)-1 (mev (Ptpn6me-v) mice) and wild-type mice were obtained from The Jackson Laboratory . For some experiments, (CD3d-IL5)NJ.1638Nal transgenic mice were backcrossed with B6(Cg)-m1JNcf1/J mice and then interbred to create IL-5 transgenic mice with defective NCF1/p47phox protein (Ncf/IL-50/+ mice). Lyn-deficient mice were obtained from Dr. Juan Rivera and wild type littermates were used as control. Depending on the purpose of the experiment, cells from 8\u201322 week old mice were used.All experiments were performed in accordance with the National Institutes of Health guidelines for the humane treatment of animals, and were approved by the Johns Hopkins Institutional Animal Care and Use Committee under protocol #MO10M417 and the Cincinnati Children\u2019s Hospital Medical Center Institutional Animal Care and Use Committee under protocol #OD11085. All mice were of the C57BL/6 background strain. (CD3d-IL5)NJ.1638Nal transgenic mice , Siglec-F , CCR3 , and irrelevant isotype-matched rat mAb (BD Biosciences) were purchased from the sources indicated.+ and Ncf/IL-50/+mice. Spleens were also simultaneously obtained and single cell suspensions were generated. Erythrocytes were then lysed hypotonically. This yielded eosinophils of purity ranging from 28% to 55%, with all contaminating cells being mononuclear cells. In some experiments, immunomagnetic negative selection of EDTA-anticoagulated whole blood buffy coats was performed to generate blood eosinophils of >96% purity by depleting mononuclear cells with a mixture of CD90.2 and CD45R coated beads as previously described Blood was obtained via cardiac puncture from IL-5\u2212/\u2212 and mev mice by flushing the bone marrow cavity with RPM 1640 medium . After red cell lysis, the bone marrow cells were cultured at 106/ml in medium containing RPMI 1640 with 20% fetal bovine serum , 100 IU/ml penicillin, 10 \u00b5g/ml streptomycin, nonessential amino acids, 1 mM sodium pyruvate , and 50 \u00b5M 2-ME supplemented with 100 ng/ml stem cell factor (SCF) and 100 ng/ml FLT3 ligand (FLT3-L) from days 0 to 4. On day 4, the cells were moved to new flasks and the medium containing SCF and FLT3-L was replaced with medium containing 10 ng/ml recombinant mouse IL-5 . Every other day, from this point forward until cells were used, one-half of the media was replaced with fresh media containing IL-5, and the concentration of the cells was adjusted each time to 106/ml. Cells were enumerated in a hemocytometer and viability (consistently >90%) and purity were determined as mentioned above for human eosinophils. These methods yield eosinophils of normal morphology expressing proteins seen in mature eosinophils, including normal cell surface levels of Siglec-F Ex vivo generation by culture of mouse bone marrow-derived eosinophils was performed essentially as previously described by Dyer et al + granulocytes) employing a FASCalibur flow cytometer (BD Biosciences) as described previously Binding of antibodies and appropriate isotype controls was determined on eosinophils by use of single or dual color immunofluorescence and flow cytometry with appropriate gating . Furthermore, eosinophils isolated from the blood or the spleen of IL-5+ mice crossbred with the Ncf\u2212/\u2212 mice produced very little ROS, even in response to PMA treatment (panel C). This was not due to differences in Siglec-F surface expression among cell populations, because eosinophils from all sources used expressed similar surface levels of Siglec-F, and use of eosinophils cultured for up to 24 hours in the absence of IL-5 yielded similar results (data not shown). Furthermore, this was not due to an effect of contaminating cells, because other preparations containing blood eosinophils from IL-5 transgenic mice enriched to >96% purity also failed to produce any detectable ROS in response to anti-Siglec-F antibody, even though similar levels of ROS to those made by impure cells were detected in response to stimulation with PMA (data not shown).Previous studies of Siglec-8-induced apoptosis using inhibitors of ROS generation in human eosinophils suggested that this pathway is particularly important in IL-5-activated human eosinophils \u2212/\u2212 mice. Note that the magnitude of this modest apoptotic response was remarkably similar to that seen with identical methods employed with higher purity eosinophils derived from IL-5 transgenic mice as we and others have previously published, even when a secondary polyclonal anti-rat secondary antibody was used Despite the lack of detectable ROS production following Siglec-F engagement, eosinophils grown from bone marrow-derived precursors , underwe\u2212/\u2212 and mev mice, the latter used to explore the role of SHP-1 in Siglec-F-mediated apoptosis. Apoptosis was determined in the presence or absence of Siglec-F mAb and the pan-caspase inhibitor Z-VAD-FMK. As shown in \u2212/\u2212 and mev mice, and significant inhibition by Z-VAD-FMK of Siglec-F-induced apoptosis in all three types of mouse eosinophils was also seen, demonstrating that Siglec-F-induced apoptosis does not require NADPH oxidase activity or SHP-1 but is dependent on caspase activation. These results are in contrast to published work showing that human eosinophil apoptosis induced by Siglec-8 is only partially caspase-dependent, and after exposure to IL-5 or IL-33 primarily involves mitochondrial pathways and the generation of ROS The next set of experiments was designed to directly explore the role of NADPH oxidase, SHP-1, Src family kinases and caspases in Siglec-F-induced eosinophil apoptosis. For these experiments, eosinophils were derived from the bone marrow of wild type, NcfBecause the ITIM and ITIM-like domains may have functions independent of SHP-1 activation, we tested the role of Src family kinases that have been shown to phosphorylate ITIM/ITIM-like domains of inhibitory receptors, including siglecs. As seen in Lastly, mouse eosinophils were tested to determine whether Siglec-F engagement might trigger generation of ROS within mitochondria (perhaps at low-levels), and whether mitochondria-derived ROS might be necessary or sufficient for Siglec-F-induced apoptosis. For these experiments, a pharmacologic inhibitor of ROS production was employed. As shown in \u2212/\u2212 mice deficient in NADPH oxidase activity. Instead, Siglec-F-mediated apoptosis was completely blocked by the addition of a broad-spectrum caspase inhibitor. While the latter has also been reported to play a role in Siglec-8 function in non-cytokine primed human eosinophils In this paper, we have found that there is no detectable role for ROS in Siglec-F-mediated apoptosis. This conclusion was based on an inability to detect any ROS production after Siglec-F mAb exposure in normal or IL-5 primed mouse eosinophils derived from blood, spleen or culture, and similar rates of apoptosis using eosinophils derived from Ncfv mice deficient in SHP-1 function, it was observed that SHP-1 was not required for Siglec-F-induced eosinophil apoptosis. While one cannot rule out the possibility that other tyrosine phosphatases may be involved in Siglec-F or Siglec-8-mediated signaling, most prior reports of signaling via CD33-family siglecs have implicated SHP-1 In additional experiments to explore the potential involvement of the Siglec-F cytoplasmic ITIM domain using eosinophils derived from meIn conclusion, the work reported here has uncovered potential differences between Siglec-F and Siglec-8 signaling and its consequences. Despite the remarkably consistent benefits of targeting Siglec-F in mouse models of hypereosinophilia, asthma and gastrointestinal eosinophilia and the exaggerated eosinophilic responses seen in mice deficient in Siglec-F when put through various models of allergic inflammation"} +{"text": "Blood is rapidly ejected from the left ventricle during early systole, travels in the aortic arch perpendicular to the MRI\u2019s main field, thus generating a Magnetohydrodynamic (MHD) voltage which is larger than the real ECG QRS complex in high field MRI. The MHD voltage (VMHD) is severally irregular in patients with arrhythmia, since arrhythmic ECG beats are interleaved between successive sinus rhythm (SR) beats. The VMHD overlay in ECG traces can result in intermittent QRS detection, leading to blurred images and longer scan times. Since accurate gating is essential for successful cardiac imaging, we developed a \u201c3D-QRS\u201d method for real-time detection of the QRS complex based on 12-lead ECG traces acquired inside the MRI. We validated this method at 3T in patients with Premature Ventricular Contractions (PVCs), Atrial Fibrillation (AF) and in an exercising athlete with time-varying heart rate.An MRI-compatible ECG system was consFig. In high-field MRI, the 3D-QRS method allowed accurate detection of the QRS, and beat-type separation in arrhythmia patients, which allowed for real-time scanner gating.NIH U41-RR019703, R43 HL110427-01, AHA 10SDG261039"} +{"text": "Cucumber mosaic virus(CMV) Y satellite RNA (Y-sat) is a non-coding subviral RNA and modifies thetypical symptom induced by CMV in specific hosts; Y-sat causes a bright yellowmosaic on its natural host Nicotiana tabacum. The Y-sat-inducedyellow mosaic failed to develop in the infected Arabidopsis andtomato plants suggesting a very specific interaction between Y-sat and its host.In this study, we revealed that Y-sat produces specific short interfering RNAs(siRNAs), which interfere with a host gene, thus inducing the specific symptom.We found that the mRNA of tobacco magnesium protoporphyrin chelatase subunit I hada 22-nt sequence that was complementary to the Y-sat sequence, including fourG-U pairs, and that the Y-sat-derived siRNAs in the virus-infected plantdownregulate the mRNA of ChlI by targeting the complementarysequence. ChlI mRNA was also downregulated in the transgeniclines that express Y-sat inverted repeats. Strikingly, modifying the Y-satsequence in order to restore the 22-nt complementarity toArabidopsis and tomato ChlI mRNA resultedin yellowing symptoms in Y-sat-infected Arabidopsis and tomato,respectively. In 5\u2032-RACE experiments, the ChlI transcriptwas cleaved at the expected middle position of the 22-nt complementary sequence.In GFP sensor experiments using agroinfiltration, we further demonstrated thatY-sat specifically targeted the sensor mRNA containing the 22-nt complementarysequence of ChlI. Our findings provide direct evidence that theidentified siRNAs derived from viral satellite RNA directly modulate the viraldisease symptom by RNA silencing-based regulation of a host gene.Symptoms on virus-infected plants are often very specific to the given virus. Themolecular mechanisms involved in viral symptom induction have been extensivelystudied, but are still poorly understood. Cucumber mosaic virus (CMV) Y satellite RNA (Y-sat) is aninteresting subviral RNA because it changes the green mosaic induced by CMV intoa bright yellow mosaic in Nicotiana tabacum. The molecularbasis underlying the induction of symptoms by viruses is not well understood,and this Y-sat-mediated modification of symptoms has been a long-standingmystery. In this study, we discovered the molecular mechanism involved in theY-sat-induced yellowing. First, we showed that transgenic N.benthamiana plants that expressed the inverted-repeatsequence of Y-sat also developed a yellow phenotype, similar to theY-sat-infected plants. Then, we found that tobacco magnesium protoporphyrinchelatase subunit I gene was downregulated in the transgenic plants and in theY-sat-infected plants. We then identified a 22-nt long sequence that iscomplementary to the Y-sat including four G-U pairs in the ChlImRNA. Finally, we demonstrated that a short interfering RNA (siRNA) derived fromY-sat specifically targeted and downregulated the ChlI mRNA,thus impairing the chlorophyll biosynthesis pathway. This discovery of themolecular basis of the symptom modification induced by Y-sat is the firstdemonstration that a subviral RNA can induce disease symptoms by regulating hostgene expression through the RNA silencing machinery. Plants infected with viruses often display various symptoms, which can be veryspecific to given viruses. Despite past efforts, the molecular bases underlyingvirus-induced diseases symptoms are still poorly understood. Subviral non-coding RNAmolecules such as satellite RNAs (satRNAs) or defective interfering (DI) RNAs areoften associated with plant viruses and can modify the symptoms induced by helperviruses Cucumber mosaic virus (CMV) are dependent on helperviruses for their replication and encapsidation and often attenuate the diseasesymptoms induced by CMV. Specifically, Y-satellite RNA (Y-sat) modifies the symptomsand exacerbates the pathogenicity of CMV in specific hosts; Y-sat induces a brightyellowing of leaves of Nicotiana tabacum andother related species ,which is yellower than a typical chlorosis, whereas it induces systemic necrosis ontomato SatRNAs of RNA silencing is a conserved, sequence-specific gene regulation system, which has anessential role in development and maintenance of genome integrity. RNA silencingrelies on short RNA (sRNA) molecules (21\u201324 nt), which are the key mediatorsof RNA silencing-related pathways in almost all eukaryotic organisms Subviral RNAs such as satRNA and DI RNA of tombusvirus have been also used tounderstand the roles of RNA silencing in viral replication and in symptomdevelopment. The DI RNA-induced RNA silencing response is known to control the levelof helper virus, facilitating the long-term co-existence of the host and the viralpathogen ChlI have the yellow phenotype ChlI gene of tobacco or cotton was targeted by virus-inducedgene silencing (VIGS) Arabidopsis mutantdefective for ChlI also had pale-green to yellow leaves ChlI is downregulated by Y-sat in thevirus-infected plants.Magnesium (Mg)-chelatase is the key enzyme in chlorophyll biosynthesis, and threesubunits of the tobacco magnesium protoporphyrin chelatase arerequired for the proper function of the enzyme N.benthamiana plants develop a yellow phenotype when expressingthe inverted-repeat sequence of Y-sat, similar to the symptoms of the Y-sat-infectedplants. Moreover, we provided evidence that Y-sat targets the ChlIgene using the host RNA silencing machinery in such a way that Y-sat-derived siRNAsefficiently downregulate ChlI mRNA through RNA silencing-mediatedcleavage. Our findings strongly suggest that this yellow phenotype is the result ofa disorder in chlorophyll synthesis caused by the downregulation of theChlI gene.In this study, we show that transgenic N. benthamiana plants that express the Y-satsequence, expecting the yellow phenotype to be induced without CMV as a helpervirus. We have used this strategy to avoid any effect of virus replication onhost gene expression, because virus infection itself has been shown to regulatethe expression of numerous genes N.benthamiana plants, we observed that the transgenicN. benthamiana lines (16c:YsatIR) had ayellow phenotype (N. benthamiana that expressed dsRNA of GUS(16c:GUSIR), demonstrating that the expression of dsRNA of an unrelated sequencein the same Y-sat IR transformation cassette does not cause a yellow symptom .Among tana 16c) , furtherana 16c) . More inana 16c) , indicatChlI) gene (accession AF014053). Because ChlI is a componentof the primary enzyme that catalyzes the first step in chlorophyll synthesis viathe tetrapyrrole biosynthesis pathway ChlI gene of N.benthamiana. We then found that both theChlI genes from N.tabacum and N.benthamiana had the 22-nt sequence complementary to theY-sat sequence (ChlI gene and the Y-sat sequence as the yellow region (YR)and satellite yellow region (SYR), respectively between the yellow-inducing domain of Y-satsequence .Hereaftectively . We thenh plants andconftoplasts , confirmChlI gene using VIGScan induce similar yellow symptoms in the absence of Y-sat. The 150-bp ofChlI (817 to 966) was inserted into the two CMV vectors,CMV-A1 and CMV-H1; CMV-A1 lacks the C-terminal one-third of the intact 2bprotein N. benthamiana plants infected with either of the viralvectors had systemic yellow symptoms similar to those induced by the replicatingY-sat in the presence of the helper virus (ChlI mRNA was downregulated in both CMV-A1:ChlI150- andCMV-H1:ChlI150-infected N. benthamiana plants compared tocontrol plants infected with one of the empty vectors (ChlI sequence replicated and accumulated to a similarlevel in the systemic leaves at 14 days post-inoculation (dpi) (We next examined whether silencing of the er virus . Althoug vectors . Using eon (dpi) .ChlI genes of pepper, tomato and Arabidopsisthaliana were obtained from the gene database, and the 22-ntcomplementary sequences of the ChlI genes and Y-sat werealigned genes genes . We nextctively) . When tothamiana . Quantited plant . There wthamiana , confirmChlI gene seemed to have a specificinteraction through their sequence complementarity, we then examined thepossible involvement of RNA silencing in the Y-sat-mediated yellow phenotype.First, we tested whether the Y-sat-derived siRNAs can be hybridized and detectedby ChlI mRNA probe. As shown in ChlI sense RNAprobe. On the other hand, we failed to detect antisense siRNAs from Y-sat byNorthern blots using the ChlI antisense RNA probe. This resultseems reasonable because the YR and SYR sequences do not share complementarityin the antisense orientation , which agrees with the expected cleavage site(s) driven by the21-nt and 22-nt siRNAs (N. benthamiana leaves that had bright yellowsymptoms (GFP mRNA (To clarify whether the t siRNAs . To verit siRNAs . The consymptoms . GFPaccGFP mRNA . The accGFP mRNA . These rRCC1 gene in Arabidopsisinfected with Cauliflower mosaic virus (CaMV) was downregulated byvirus-derived siRNAs, but contrary to expectations, the decrease in gene expressiondid not affect either viral accumulation or symptoms. It is, in fact, quitedifficult to clarify the relationship between such small RNAs and viralpathogenicity although the idea that host gene silencing against a particular genemight contribute to the specific expression of symptoms is very attractive.Plant RNA silencing has often been implicated as a molecular mechanism for symptominduction caused by viruses or viral subviral agents. Viral suppressors of RNAsilencing (VSRs) are able to compromise the endogenous RNA silencing pathways ChlI gene, resulting in the inhibition of chlorophyllbiosynthesis. Here we provide several lines of evidence that Y-sat-induced brightyellow mosaics are the outcome of specific interference between the pathogen-derivedsiRNAs and a host gene. First, the 22-nt long region of Y-sat (SYR) producesspecific siRNAs that were complementary, including four G-U pairs, to the 22-nt longregion of tobacco ChlI mRNA (YR). Second, the ChlImRNA could detect Y-sat-derived siRNAs in Northern blots. Third, 5\u2032-RACEexperiments revealed that the ChlI mRNA was cleaved exactly in theexpected middle of the YR. Fourth, the levels of the ChlItranscript significantly decreased in both Y-sat-infected plants and the transgenicplants expressing Y-sat dsRNA. Fifth, the Y-sat mutants that had the modified SYR toeither Arabidopsis ChlI1 mRNA or tomato ChlI mRNAwere able to induce yellow symptoms in these host plants. In contrast, thesemodified Y-sat lost the ability to induce yellow symptoms on tobacco. Sixth, the GFPsensor construct carrying the YR sequence was specifically targeted inY-sat-infected plants. Considering all these results, we propose a model thatexplains that the Y-sat-mediated yellow symptom results from the cleavage of hostChlI mRNA by RNA silencing machinery of satRNAs, their evolutionary strategy andbiological significance have long been intriguing topics. Since the originalisolation of Y-sat in Japan more than 30 years ago Nicotiana benthamiana, Capsicum annuum,Solanum lycopersicum and Arabidopsisthaliana were used as host plants for the analysis.Nicotiana benthamiana line 16c having a single copy of theGFP transgene N. benthamiana lines expressing the inverted repeat(IR) of Y-sat were generated by transforming N. benthamiana 16cwith the binary vector pIG121-Hm carrying the IR of Y-sat under the CaMV 35Spromoter. In the sense and antisense orientations, the 317-bp (53 to 369) Y-satsequence (GenBank accession D00542) was inserted in the pJM007 vector Transgenic ChlI gene, we used two CMV-based vectors,CMV-A1 and CMV-H1. CMV-A1 and CMV-H1 are derived from RNA2 of CMV-Y, and CMV-A1lacks the C-terminal one-third of the intact 2b protein as a consequence ofintroducing a multiple cloning site ChlI gene (817 to966) was inserted into the CMV vectors to create CMV-A1:ChlI150 andCMV-H1:ChlI150, respectively. To avoid severe mosaic symptom induction by CMV-Y,we used a pseudorecombinant virus that contains RNA components derived from RNA1and RNA3 of CMV strain L together with RNA2 of the vector. Each plasmidcontaining a full-length cDNA clone of RNA1 to RNA3 was transcribed in vitroafter linearization with a restriction enzyme N. benthamiana weredusted with carborundum and rub-inoculated with the RNA transcripts. Forinoculation of tomato plants, leaves of young plants were rub-inoculated withthe sap from virus-infected tissues of N.benthamiana. Successful systemic infection with the viruscontaining the full insert sequence was confirmed by RT-PCRs. Viral accumulationwas examined by conventional ELISA CMV strain Y (CMV-Y) was used as a helper virus for satellite RNA. To induce genesilencing to the N. benthamiana ChlI cloneincluding the entire ORF was amplified by RT-PCR using the primer pair designedfrom the tobacco ChlI sequence . Quantitative real-time RT-PCR was performed essentially asdescribed before N. benthamiana ChlI gene were as follows:5\u2032-CTTATTGGTTCGGGTAATCCTG-3\u2032 for forward primerand 5\u2032-GCTGAGTCGATTTGGTTCTG-3\u2032 for reverse primer.The N. benthamiana actin gene was amplified using5\u2032-GCGGGAAATTGTTAGGGATGT-3\u2032 for forward primerand 5\u2032-CCATCAGGCAGCTCGTAGCT-3\u2032 for reverse primerand used for data normalization. Northern blot hybridization was performedessentially as previously described ChlI gene was generated by PCR with the PCR DIG ProbeSynthesis Kit (Roche Diagnostics) to amplify the 371 bp (634 to 1004) of3\u2032-terminal regions of the ChlI gene using the primerpair ChlI-634F (5\u2032-GAGCCTGGTCTTCTTGCTAAAGC-3\u2032) and ChlI-1004R(5\u2032-GCTGAGTCGATTTGGTTCTG-3\u2032). In the Northernblots of the small RNAs corresponding to the 22-nt complementary sequence region, the SYRs were detected by using32P-labeled locked nucleic acid (LNA) oligonucleotide probesdescribed previously Total RNAs were extracted by either a conventional phenol/chloroform method ChlI mRNA cleavage sites were analyzed by modifiedRNA-ligase mediated 5\u2032-RACE + mRNA was ligated to the GeneRacer RNA Oligo adaptorusing the GeneRacer Kit (Invitrogen). Ligated RNAs were reverse transcribedusing the gene-specific reverse primer for the ChlI gene,ChlI-1004R (5\u2032-GCTGAGTCGATTTGGTTCTG-3\u2032). The 5\u2032end ofthe cDNA was then amplified by PCR using the GeneRacer 5\u2032 primer and thegene-specific reverse primer used for the reverse transcription for the firstPCR. The GeneRacer 5\u2032 nested primer was also used for the subsequentnested PCR. The amplified product from the nested PCR was excised from1.2% agarose gel and cloned into pGEM-T Easy (Promega) forsequencing.The N.benthamiana as described before ChlI and CAB gene were measuredby quantitative real-time RT-PCR . Primers for quantitative real-timeRT-PCR for the CAB gene were 5\u2032-CGGCCGATCCAGAAACTTT-3\u2032 for forward primerand 5\u2032-GCCCATCTGCAGTGAATAACC-3\u2032 for reverseprimer.Protoplasts were prepared from leaves of N.benthamiana plants. Small RNAs were isolated essentially asdescribed Total RNA was extracted from CMV and Y-sat-infected BamHI andSacI sites in the pBE2113 vector. The Ti-plasmid constructwas then introduced into Agrobacterium tumefaciens KYRT1strain, which was supplied by Dr. G. B. Collins .Agrobacterium infiltration was carried out essentially as described The GFP-YR sensor gene was inserted between the Total proteins were extracted from the sample tissues by grinding in Laemmlibuffer, separated by SDS-PAGE, and /transferred onto a PVDF membrane . Anti-GFP antibodies were purchased from Roche and used at a1\u22361000 dilution. For immunostaining, an alkaline phosphatase-conjugatedgoat anti-rabbit antibody was added to the blots at a 1\u22363000 dilutionfollowed by colorimetric development with BCIP and NBT.Figure S1Nicotianabenthamiana 16c and N.benthamiana 16c:YsatIR . Redcircles in the gel of 16c indicate the spots that decreased in 16c:YsatIRcompared to 16c. Blue circles in the gel of 16c:YsatIR indicate the spotsthat increased in 16c:YsatIR compared to 16c. Among these spots, we selectedfive spots (M1-M5) that had markedly changed between 16c and 16c:YsatIR forLC-MSMS analysis. The analyzed proteins were identified as follows: M1,ribulose bisphosphate carboxylase large chain (RuBisCo large subunit); M2,ribulose bisphosphate carboxylase activase; M3, glyceraldehyde-3-phosphatedehydrogenase A ; M4, ribulose bisphosphate carboxylase small chain 1 ; M5, ribulose bisphosphate carboxylase small chain 1. This proteome analysis revealed thatchloroplast-related proteins were significantly altered in 16c:YsatIR, andthat the mobility of the RuBisCo small subunit had shifted in atwo-dimensional gel, suggesting that RuBisCo small subunit in 16c:YsatIR wasmodified at the posttranslational level.Two-dimensional electrophoresis of extracted proteins from (TIF)Click here for additional data file.Figure S2ChlI gene probe inNorthern blots. RNAs were prepared from 16c:YsatIR, 16c and CMV-infectedN. benthamiana with or without Y-sat.Left panel, detection of sense small RNAs of Y-sat by the hybridization withthe ChlI sense RNA (mRNA) probe. Right panel, detection ofantisense small RNAs of Y-sat by the hybridization with theChlI antisense RNA probe. For the RNA probe, theamplified ChlI fragments werecloned downstream of the T7 promoter in the pGEM-T easy vector (Promega).The sense and antisense RNA probes specific to the ChlIwere prepared using DIG RNA Labeling Mix (Roche). Arrows indicate small RNAsof Y-sat. The 22-nt sequence complementarity between theChlI and Y-sat is shown below each panel. A continuous22-nt complementary sequence including G-U pairs is formed between theChlI sense RNA and Y-sat sense RNA, but there are fourmismatches in the region between the ChlI antisense RNA andY-sat antisense RNA.Detection of Y-sat small RNAs by the (TIF)Click here for additional data file.Figure S3Arabidopsis. RNAs were prepared fromArabidopsis leaves infected with CMV or CMV+Y-satmut-Ara1. For the RNA probe, the amplified ChlI1 fragments were cloned downstream of the T7 promoter in thepGEM-T easy vector (Promega). The sense RNA probe specific to theArabidopsis ChlI1 was prepared using DIG RNA LabelingMix (Roche). Note that Arabidopsis ChlI1 sense RNA probedetected small RNAs from Y-sat mut-Ara1 (shown by an arrow) in the lane forY-sat mut-Ara1-infected leaves.Northern blots of Y-sat mut-Ara1 small RNAs in Y-sat mut-Ara1-infected(TIF)Click here for additional data file.Figure S4Confirmation of the relative abundance of Y-sat small RNAs by Northern blothybridization. The small RNAs derived from the hot spots that were observedin the Y-sat small RNA profiles were det(TIF)Click here for additional data file.Figure S5ChlI small RNAs inN. benthamiana plants infected withCMV-Y and Y-sat. Location and frequency of the ChlI-derivedsmall RNAs (21- to 24-nt) were mapped to the ChlI sequencein either sense (above the x-axis) or antisense (below thex-axis) orientation. Data from 21-, 22-, 24-nt smallRNA are color-coded in green (21 nt), red (22 nt), and yellow (24 nt). Tablein graph gives the number of small RNA reads and percentage of each size.Note that the small RNAs are mostly generated from the 3\u2032 regiondownstream of the cleavage site indicated by an arrow.Deep-sequencing analysis of the (TIF)Click here for additional data file.Table S1Genes downregulated in 16c:YsatIR 40% less than in 16c plants inmicroarray analysis. Among the 134 genes, 31 genes were chloroplast-relatedgenes.(DOC)Click here for additional data file.Text S1Supplementary materials and methods for the DNA microarray and two-(DOC)Click here for additional data file."} +{"text": "Myocardial perfusion MRI with sliding-window conjugate-gradient HYPR (SW-CG-HYPR) allows increased spatial coverage (whole left ventricular coverage), resolution, signal-to-noise ratio and reduced motion artifacts. The accuracy of this technique for detecting coronary artery disease (CAD) has not been determined in a large number of patients.The purpose of this study was to prospectively evaluate the diagnostic performance of adenosine-induced stress myocardial perfusion MRI with SW-CG-HYPR in patients with suspected CAD.Forty consecutive patients with suspected CAD who were scheduled for coronary angiography underwent myocardial adenosine stress perfusion MRI with SW-CG-HYPR at 3.0T. Perfusion defects were interpreted visually by 2 blinded observers and were correlated to x-ray angiographic stenoses \u2265 50%.The prevalence of CAD was 55%. In the per-patient analysis, the sensitivity, specificity and accuracy of SW-CG-HYPR myocardial perfusion imaging were 95%, 83% and 90%, respectively. In the per-vessel analysis, these values were 98%, 89% and 93%, respectively. Figure Adenosine-Induced stress myocardial perfusion MRI using SW-CG-HYPR allows whole left ventricular coverage and has high diagnostic accuracy in patients with suspected CAD."} +{"text": "Immunologically humanized mice are promising tools to analyze in vivo human anit-tumor immune responses. When human PBMCs are transferred into NOG (NOD/Shi-scid IL2r\u03b3null) mice, severe GVHD developed in a couple of weeks hinders long term detailed analysis of human immune responses. In this study, we developed novel MHC class I and class II-deficient NOG mice, and characterized the reconstituted human immune systems in the mice after inoculation of human PBMC. NOG mice with no murine MHC class I and class II were generated by knockout of \u03b22-microglobulin gene, a component of MHC class I molecule, and IA\u03b2, the light chain of the class II IA . Administration of human PBMC into NOG-dKO (huNOG-dKO) mice induced mild GVHD, but its severity was much less than that occurred in control NOG mice in spite of sufficient engraftment of human immune cells, when evaluated by decrease of body weight and survival as well as human T cell infiltration in various organs. Various types of human immune cells were detected in the peripheral blood of NOG-dKO until day14 (DC and na\u00efve T cells), day21 (NKT cells), day35 (NK cells), or more than day100 (T cells and B cells). Immunization with an inactivated influenza vaccine resulted in the increase of serum influenza-specific human IgG Ab along with increases of influenza-specific Ab producing B cells in the spleens, indicating the induction of antigen specific B cells in the huNOG-dKO. Immunization with human monocyte-derived dendritic cells (DC) pulsed with HLA-A2 restricted CMV peptide (CMVpp65) induced CMVpp65-specific CTLs in spleens, bone marrows, and peripheral blood, indicating the induction of antigen specific T cells in the huNOG-dKO. When activated human peripheral blood T cells transduced with TCR specific for melanoma antigen MART-1, were transferred into NOG-dKO and NOG mice implanted with human melanoma cell lines, stronger tumor growth inhibition was observed in the NOG mice along with more increased transferred T cells due to xenogenic GVHD responses than the NOG-dKO mice, and the NOG mice died in 4-8 weeks. However, administration of human recombinant IL2 into the NOG-dKO mice resulted in enhanced anti-tumor effects without any sign of GVHD accompanied by prolonged survival of T cells containing higher MART-1/HLA-A2 tetramer positive cells, indicating more detailed analysis of anti-tumor immune responses is possible using NOG-dKO mice. Therefore, NOG-dKO mice are useful tools for more detailed analysis of both induction and effector phases of T cell and B cell responses for human tumor immunology research."} +{"text": "Immunohistochemistry (IHC) for detecting key signal molecules involved in programmed cell death (PCD) in archival human pathology specimens is fairly well established. Detection of cleaved caspase-3 in lymphocytes in rheumatoid arthritis (RA) and gastric surface foveolar glandular epithelia but not in synoviocytes in RA, gastric fundic glandular epithelia, or nasal NK/T-cell lymphoma (NKTCL) cells suggests anti-apoptotic mechanisms in cell differentiation and in oncogenesis such as the induction of survivin. Enzymatically pretreated and ultra-super sensitive detection of beclin-1 in synoviocytes in RA and gastric fundic glandular epithelia suggests enhanced autophagy. The deposition of beclin-1 in fibrinoid necrosis in RA and expression of beclin-1 in detached gastric fundic glandular cells suggest that enhanced autophagy undergoes autophagic cell death (ACD). NKTCL exhibited enhanced autophagy through LC3 labeling and showed densely LC3 labeled cell-debris in regions of peculiar necrosis without deposition of beclin-1, indicating massive ACD in NKTCL and the alternative pathway enhancing autophagy following autophagic vesicle nucleation. Autophagy progression was monitored by labeling aggregated mitochondria and cathepsin D. The cell-debris in massive ACD in NKTCL were positive for 8-hydroxydeoxyguanosine, suggesting DNA oxidation occurred in ACD. Immunohistochemical autophagy and PCD analysis in archival human pathology specimens may offer new insights into autophagy in humans. ARantigen retrievalIHCimmunohistochemistryPDCprogrammed cell deathACDautophagic cell deathRArheumatoid arthritisHPHelicobacter pyloriEBVEpstein-Barr virusNKnatural killersynoviocytessynovial fibroblastsEDTAethylendiaminetetraacetic acidHRPhorse radish peroxidaseCSAcatalyzed signal amplificationnsCSA systemnew simplified CSA systemAtgautophagy-related proteinVpsvascular protein sorting-associated proteinVps15serine/threonine-protein kinase Vps15Vps34phosphatylinositol 3-kinaseFlipFlice (caspase-8)-like inhibitory proteinTIA-1T-cell restricted intracellular antigen-1Bcl-2B-cell lymphoma 2LC3mammalian Atg8 homologue light chain 38-OHdG8-hydroxydeoxyguanosineTGthymidine glycoliNOSinducible nitric oxide synthaseNGnitroguanosineNKTCLNK/T-cell lymphomasEBER-1EBV-encoded small RNA-1ROSreactive oxygen speciesBcl-XBcl-2-like 1Recent molecular and cell biological research efforts have been rewarded by fruitful results and many antibodies against signal transduction molecules and their antagonists have been supplied commercially. Through developed antigen retrieval and immunohistochemistry (AR-IHC) using these antibodies, it becomes possible to label these signal transduction molecules and antagonists in archival formalin-fixed and paraffin-embedded human pathology specimens, a huge number of which have been collected in the so-called Department of Human Pathology. Comparative AR-IHC using antibodies against the representative signal transduction molecules and antagonists in the archival human pathology specimens with and without various diseases is expected to be informative about differences in features of signal transduction between normal and diseased human cells in the tissues and to contribute to the medicine for the diseases and probably to further molecular and cell biological research.Apoptosis, a form of programmed cell death (PCD) ,2, has bMolecular mechanisms of autophagy and autophagic cell death (ACD), known as type II PCD ,6,7,8, hHelicobacter pylori (HP) [In the present report, we aimed to differentiate apoptosis, autophagic cell death (ACD) and other types of PCD using archival human pathology specimens. We describe our investigations involving AR-HC detecting apoptosis and autophagy in inflamed hyperplastic and degenerative lesions in rheumatoid arthritis (RA). This paper also reports on homeostatic mass control in the gastric fundic glandular mucosa with and without ori (HP) and pecuori (HP) ,16, and Synovial tissue in RA exhibits hyperplastic and degenerative synovial cells along with fibrinoid necrosis and chronic inflammatory cell infiltration with lymph follicle formation. Synovial fibroblasts (synoviocytes) and macrophages activate molecular mechanisms against apoptosis ; their dTo investigate antigen retrieval (AR), we heated archival formalin-fixed paraffin-embedded RA synovial specimen-deparaffinized sections in EDTA solution with pH > 9 for 5 min at 120 \u00b0C by autoclaving (heating-AR at high pH), and utilized an extremely sensitive indirect enzyme-labeled antibody method, the horse radish peroxidase (HRP)-labeled and secondary antibody-labeled polymer reagent method , ACD (PCD II) and coagulation necrosis (PCD III) in normal cells under homeostatic mass control and in neoplastic cells . Cleaved caspase-3 can be detected by using a polymer method involving heating-AR at high pH or heating-AR independent of pH. Since nuclear melting is occasionally observed when heating-AR at high pH with AR independent from the pH of AR solution ,17, it wTM FLEX) and ultra-sensitive IHC through CARD reaction are rea8-OHdG, a well-known marker of oxidative stress, is usually excreted into the urine following DNA repair. As shown in The use of IHC for detecting PCD in the archival human pathology specimens is expected to extend to the field of apoptosis, autophagy and DNA oxidation and nitration. Characterization of the differences between the signal molecules involved in the PCD of neoplastic and non-neoplastic cells might be useful in the differential diagnosis of neoplastic cells in surgical pathology as well as AR-IHC of survivin . Further"} +{"text": "Preclinical studies have demonstrated that intraprostatic/intratumoral (IT) administration of vaccine in combination with systemic vaccination (SC) may improve antitumor efficacy. In this study we assessed the differential effects of IT/SC vaccination in peripheral blood mononuclear cells (PBMCs) and tumor-infiltrating lymphocytes (TILs).+ TILs positive for FoxP3 (putative Tregs) were counted manually.Measurements were performed before and near day 113 post IT/SC administration of PSA-TRICOM vaccine in 21 patients (pts) with locally recurrent or progressive prostate cancer (PC) enrolled in a dose-escalation phase 1 trial. Pts received initial SC vaccination with recombinant vaccinia-PSA-TRICOM and IT boosts with recombinant fowlpox (rF)-PSA-TRICOM. Cohort 3-5 received intraprostatic rF-GM-CSF. Cohort 5 received SC boosts with rF-PSA-TRICOM and rF-GM-CSF. We evaluated Treg suppression, NK function, and frequencies of immune cell subsets from PBMC by flow-cytometry. TILs were measured by digital immunohistochemistry (IHC). Prostate cores were obtained by transrectal ultrasound-guided biopsies. Prostate sections were stained for CD4, CD8, FoxP3, or isotype controls. Digital IHC images were acquired with an Aperio ScanScope AT Turbo and analyzed with the Aperio ImageScope membrane algorithm for cell membrane analysis. CD4+PBMCs and CD4+ TILs post-vaccine . CD4+ and CD8+ TILs increased post-vaccine (p<0.001 for both) independently of hormone status and GM-CSF treatment. Pts with low CD4+ TILs pre-vaccine showed a greater increase in CD4+ TILs post-vaccine . Tregs (as % of CD4+ TILs) decreased post-vaccine (p<0.001). There was also a correlation between CD8+ TILs increase post-vaccine and decreases in serum PSA .The function of Treg and NK cells isolated from PBMC, as well as circulating immune cell subsets, did not change after vaccine. A lower percentage of circulating Tregs post-vaccine correlated with decreases in serum PSA . There was an inverse correlation between CD4The use of digital IHC after IT/SC vaccination can help to document variations of immune infiltration and clinical correlates in the absence of relevant changes in PBMC immune cell subsets."} +{"text": "Clinical trials evaluating anti-CD20-mediated B-cell depletion in multiple sclerosis (MS) and neuromyelitis optica (NMO) generated encouraging results. Our recent studies in the MS model experimental autoimmune encephalomyelitis (EAE) attributed clinical benefit to extinction of activated B-cells, but cautioned that depletion of na\u00efve B-cells may be undesirable. We elucidated the regulatory role of un-activated B-cells in EAE and investigated whether anti-CD20 may collaterally diminish regulatory B-cell properties in treatment of neuroimmunological disorders.+ antigen presenting cells (APC) were assessed. Peripheral blood mononuclear cells from 22 patients receiving anti-CD20 and 23 untreated neuroimmunological patients were evaluated for frequencies of B-cells, T-cells and monocytes; monocytic reactivity was determined by TNF-production and expression of signalling lymphocytic activation molecule (SLAM).Myelin oligodendrocyte glycoprotein (MOG) peptide-immunized C57Bl/6 mice were depleted of B-cells. Functional consequences for regulatory T-cells (Treg) and cytokine production of CD11b+ APC. Paralleling this pre-clinical finding, anti-CD20 treatment of human neuroimmunological disorders increased the relative frequency of monocytes and accentuated pro-inflammatory monocyte function; when reactivated ex vivo, a higher frequency of monocytes from B-cell depleted patients produced TNF and expressed the activation marker SLAM.We observed that EAE-exacerbation upon depletion of un-activated B-cells closely correlated with an enhanced production of pro-inflammatory TNF by CD11bThese data suggest that in neuroimmunological disorders, pro-inflammatory APC activity is controlled by a subset of B-cells which is eliminated concomitantly upon anti-CD20 treatment. While this observation does not conflict with the general concept of B-cell depletion in human autoimmunity, it implies that its safety and effectiveness may further advance by selectively targeting pathogenic B-cell function. Accumulating evidence suggests that in the pathogenesis of multiple sclerosis (MS) and neuromyelitis optica (NMO), B-cells, plasma cells and self-reactive antibodies play an essential pathogenic role. In MS, an oligoclonal antibody response generated by a limited repertoire of activated B-cells remains a hallmark diagnostic finding in the cerebrospinal fluid (CSF). While t+ AQP4-specific plasma cell precursors provides the sole and entire basis for therapeutic benefit of anti-CD20 in NMO [Based on these pathogenic B-cell properties, substantial interest has developed for testing anti-CD20 antibodies in MS and NMO. These antibodies deplete immature and mature B-cells, but spare CD20-negative plasma cells. The retrospective analysis of 25 NMO patients receiving rituximab demonstrated a reduction in attack frequency with subsequent clinical stabilization , it is u0 in NMO . Clinica0 in NMO ,8; in tr0 in NMO . Immunol0 in NMO ; further0 in NMO . Togethe+ B-cells may actively contribute to progression of autoimmune disease. Animal models of human autoimmunity suggest that through provision of anti-inflammatory IL-10, na\u00efve B-cells in contrast regulate autoimmune responses [Notwithstanding these encouraging results, not all CD20esponses and contesponses . Accumulesponses . In a reesponses . Functio+ APC. In light of these preclinical findings and the newly established role of B-cell subsets in regulation of human autoimmunity, we further investigated whether anti-CD20 treatment may collaterally abolish B-cell regulatory properties in human neuroimmunological disorders. Paralleling our findings in EAE, we report that anti-CD20 treatment of MS and NMO is associated with an accentuation of pro-inflammatory monocyte function, providing the first evidence that besides abrogation of pathogenic B-cell function, anti-CD20 diminishes B-cell regulation of myeloid APC.Our recent study testing anti-CD20 treatment in an animal model of MS, revealed that B-cell depletion exacerbated experimental autoimmune encephalomyelitis (EAE) induced by the short T-cell determinant myelin-oligodendrocyte glycoprotein (MOG) peptide (p)35-55, a setting in which B-cells are not required or involved in a pathogenic manner . One aimThis study was approved by the local ethics committee of the Technische Universit\u00e4t M\u00fcnchen. After informed consent, subjects were enrolled in four groups: rituximab-treated patients with neuroimmunological disorders, untreated patients with neuroimmunological disorders, rituximab-treated B-cell lymphoma patients and untreated patients with other non-inflammatory disorders table . Patient+ monocytes was evaluated 24 hours thereafter. Frequency of CD14+ monocytes expressing SLAM was determined as shown in additional file PBMCs were stained for CD19, CD4, CD14, CD25, CD127, SLAM/CD150 or CD8a (eBioscience). FACS staining was analyzed on a Cyan ADP9C using software Summit 4.3 (Beckmann Coulter). PBMCs were stimulated with lipopolysaccharid (LPS) and SLAM-expression of CD14Magnetically activated cell sorting (MACS)-separated monocytes were plated in TNF capture antibody-precoated Multi-Screen Filter Plates (Millipore) in triplicates and stimulated with LPS for 18 hours. Plates were washed and incubated successively with TNF detection antibody, streptavidin-alkaline phosphatase and BCIP/NBT substrate. Plates were analyzed with an automated imaging system and software .+ cells in na\u00efve mice. Results are representative of 3 separate experiments.All murine experiments were carried out as approved by the government of Upper Bavaria (protocol number 55.2-1-54-2531-67-09). C57BL/6 female mice were immunized with 100 \u03bcg MOG p35-55 in Complete Freund's Adjuvant (CFA) followed by 200 ng of pertussis toxin (PTX) i.p. at the day of immunization and 2 days thereafter. Mice were assessed for signs of EAE as described previously . Mice re12 days after immunization, MACS-purified splenic monocytes were stimulated with the indicated concentrations of LPS. After 24 hours, supernatants were collected and analyzed for murine TNF by ELISA (R&D Systems). Plates were read at 450 nm wavelength by a Tecan Genios plate reader and analyzed using Magellan6 software.As frequency of regulatory T-cells followed a skewed distribution, the Mann-Whitney U-Test was used for comparisons. Frequency of monocytes was distributed normally and analyzed by t-Test. Variability of monocytic SLAM expression was compared using the Siegel-Tukey test, capable to deal with non-normal data. Variability of TNF-producing monocytes in anti-CD20 treated vs. untreated patients was compared using the F-Test based on a normal distribution of values. All statistical tests were two-sided and conducted in an explorative manner on a 5% level of significance. Descriptive statistics for continuous, normally distributed data are given by the mean, its standard error (SEM) or the range (min. - max.). Skewed data is presented by the median as well as 20% and 80% percentiles. Categorical data is summarized by absolute and relative frequencies.+ APC. In order to dissect the relative responsibility of either effect for clinical deterioration, we utilized an anti-CD25 Treg-depleting antibody to neutralize for alterations in Treg frequency. Prior to disease induction, mice were injected with anti-CD25, anti-CD20 or a combination of both antibodies. As expected, anti-CD20-mediated B-cell depletion exacerbated disease severity and a pronounced pro-inflammatory differentiation of myeloid CD11by Figure . Depleti+ APC were isolated from all four groups of mice and evaluated for production of the pro-inflammatory hallmark cytokine TNF. As indicated in Figure + cells produced increased levels of pro-inflammatory TNF. This effect was further accelerated when mice were in addition depleted of Treg, resulting in a close correlation between the relative increase in monocytic TNF release and the extent of clinical deterioration. In our previous study, elevated TNF production by CD11b+ cells resulted in an enhanced ability of these APC to generate encephalitogenic Th1 and Th17 cells [+ APC and highlight an enhanced pro-inflammatory APC function as explanation for exacerbation of CNS autoimmune disease upon depletion of na\u00efve B-cells.We investigated next whether alternatively, elimination of B-cell-mediated regulation of APC activity may account for anti-CD20-associated worsening of peptide-induced EAE. CD11b17 cells . TNF was17 cells . Collect+ B-cells whereas PBMCs from control patients contained a mean frequency of 7.9 \u00b1 1.1% B-cells were isolated from 22 rituximab-treated patients with MS, NMO, myasthenia gravis or autoimmune neuropathy and compared to PBMCs from 23 age- and sex-matched untreated patients . In order to compare pro-inflammatory monocyte reactivity, we evaluated LPS-induced release of TNF and expression of signaling lymphocytic activation molecule (SLAM), an activation marker which physiologically serves as a co-stimulatory molecule promoting development of pro-inflammatory T-cells [The main purpose of this translational approach was to investigate whether anti-CD20 treatment of human neuroimmunological disorders may concomitantly abrogate B-cell regulation of other APC. As shown in table T-cells . As showWhile the majority of patients with neuroimmunological disorders clearly benefit from anti-CD20 treatment ,7,8, fewIn conclusion, we herein provide novel evidence that besides abrogation of pathogenic B-cell function, anti-CD20 treatment eliminates preexisting B-cell regulation in human autoimmunity. In treatment of NMO and MS, this observation in conjunction with our EAE findings could indicate that individual patients with minor counter-balancing pathogenic B-cell involvement may not benefit or even deteriorate upon pan-B-cell depletion via CD20. Whereas our study does not conflict with the projected general potential of B-cell depletion in treatment of autoimmune disorders, it cautions that its indication should be assessed individually and supports further development of this therapeutic approach to selectively target pathogenic B-cell function.APC: antigen presenting cell; AQP-4: aquaporin-4; CFA: Complete Freund's Adjuvant; CIS: clinically isolated syndrome; CNS: central nervous system; CSF: cerebrospinal fluid; EAE: experimental autoimmune encephalomyelitis; ELISA: enzyme linked immunosorbent assay; ELISPOT: enzyme linked immuno spot technique; FACS: fluorescence activated cell sorting; IL-10: interleukin 10; LPS: lipopolysaccharid; MACS: magnetically activated cell sorting; MAG: myelin associated glycoproteins; MOG: myelin oligodendrocyte glycoprotein; MS: multiple sclerosis; NMO: neuromyelitis optica; PBMC: Peripheral blood mononuclear cell; RR-MS: relapsing-remitting multiple sclerosis; SEM: standard error of the mean; SLAM: signalling lymphocytic activation molecule; TNF: tumor necrosis factor.The authors declare that they have no competing interests.KL-H performed experiments, interpreted data and contributed in drafting the manuscript, ES performed experiments and interpreted data; DH, AH and TK interpreted the data; NvB contributed to conception and design of the study; RH and AB have been involved in drafting the manuscript; BH revised the manuscript critically for important intellectual content; MSW performed experiments, designed the research, interpreted the data and wrote the manuscript. All authors have given final approval of the version to be published.Characteristics of patients with B-cell lymphoma or various non-inflammatory neurological disorders and analysis of peripheral blood mononuclear cells. Anti-CD20 treated B-cell lymphoma and untreated non-inflammatory (control) patients were age- and sex-matched. Frequencies of leucocyte subpopulations are indicated as percentage of all peripheral blood mononuclear cells (PBMCs) and as percentage of CD4+, CD4+/CD8+ or CD14+CD4+/CD8+ PBMCs to \"normalize\" for treatment-related absence of B-cells.Click here for filesignalling lymphocytic activation molecule (SLAM)Activation-induced monocytic expression of . PBMCs were stimulated with increasing concentrations of LPS. Expression of SLAM was evaluated by FACS (gated on CD14+ monocytes); non-stimulated PBMCs served as base value and gates were set accordingly.Click here for fileIn treatment of B-cell lymphoma, anti-CD20-mediated B-cell depletion is associated with an increased frequency of regulatory T-cells but not with an enhanced pro-inflammatory activity of monocytes. Peripheral blood mononuclear cells (PBMCs) were isolated from anti-CD20-treated patients with B-cell lymphoma or untreated control patients with non-inflammatory neurological disorders The frequency of regulatory T-cells is indicated as percentage of CD4+CD25+CD127- within all CD4+ T-cells . b) The frequency of monocytes is indicated as the percentage of CD14+ cells within the pool of PBMCs expressing CD4+/CD8+/CD14+ . c) MACS-separated monocytes were stimulated with the indicated concentrations of LPS; secretion of TNF was evaluated by ELISPOT. Shown is the number of TNF-producing cells/3,000 monocytes (black lines represent the mean of each group). d) PBMCs were stimulated with the indicated concentrations of LPS and monocytic expression of signalling lymphocytic activation molecule (SLAM) was evaluated by FACS. Indicated is the percentage of SLAM+ cells within all CD14+ monocytes (black lines represent the median of each group).Click here for file"} +{"text": "Staphylococcus aureus, Uruguay\u201d by Xiao Xue Ma et al., errors occurred on pages 973 and 974.In \u201cCommunity-acquired Methicillin-resistant Staphylococcus aureus clone (Uruguay clone) with a non\u2013multidrug-resistant phenotype caused a large outbreak, including 7 deaths, in Montevideo, Uruguay.The first sentence of the abstract should read as follows: A novel, methicillin-resistant Staphylococcus aureus (MRSA) infections have been increasingly recognized in the community, and MRSA strains isolated from patients with community-associated cases have been called community-associated MRSA (CA-MRSA).The first sentence of the article should read as follows: Since the 1990s, methicillin-resistant The first sentence of Figure 1 legend (p. 974) should read as follows:The monthly accumulation of cases of infections due to non\u2013multidrugresistant MRSA strains from January 2002 to October 2003.http://wwwnc.cdc.gov/eid/article/11/6/04-1059_article.htm.The corrected article appears online at We regret any confusion these errors may have caused."} +{"text": "Tuberculosis (TB) is a major public health disease, affecting one third of the world\u2019s population and killing approximately two million people yearly. The emergence of resistance to anti-tuberculosis drugs, particularly MDR-TB.To determine prevalence of multidrug-resistant tuberculosis (MDR-TB) and associated risk factors among adult (\u226518 years) HIV positive patients registered at Mpilo Opportunistic Infection (OI) clinic. To assess the association of CD4 count and MDR-TB.A health facility based cross-sectional study was carried out at Mpilo OI Clinic between 01 March and 31 July 2012. Convenience sampling was used to recruit 275 adult HIV positive patients into the study on a daily basis. A single sample for MDR-TB was collected from each one of these participants. A total of 275 sputum and aspirate samples were collected and cultured for MDR-TB using both the Liquid using BACTEC Mycobacterium Growth Indicator Tube 960 (MGIT) and the Conventional Solid Lowenstein Jensen (LJ) culture methods. Whole blood for CD4 count was collected from each participant and tested using BD FACS Calibur Flow Cytometry CD4 count machine. Logistic regression was used to determine predictors of MDR-TB prevalence.The prevalence of MDR-TB was 2.6% among adult HIV patients registered at Mpilo OI Clinic and attended the clinic between 01 March and 31July 2012.In the multivariate analysis, MDR-TB prevalence was associated with CD4 count (OR 0.14 p=0.043).A prevalence of 2.6% of MDR-TB among HIV positive patients was found. This is very high considering this high MDR-TB risk group. A CD4 count of >200 cells/ul was found to be protective of high MDR-TB prevalence. Targeted interventions of MDR-TB are necessary to reduce incident MDR-TB cases among HIV positive patients. Increased MDR-TB case finding through culture and Drug Susceptibility testing before initiation of First line drugs is necessary to reduce mistreatment. Infection control measures need to be put in place to reduce transmission of MDR-TB.None declared."} +{"text": "In vitro, ventricular myocytes (VMs) and sympathetic neurons (SNs) isolated from neonatal rat ventricles and superior cervical ganglia were cultured at a close distance. Then, morphological and functional coupling between SNs and VMs was assessed in response to GDNF (10 ng/ml) or nerve growth factor (50 ng/ml). As a result, fractions of neurofilament-M-positive axons and synapsin-I-positive area over the surface of VMs were markedly increased with GDNF by 9-fold and 25-fold, respectively, compared to control without neurotrophic factors. Pre- and post-synaptic stimulation of \u03b21-adrenergic receptors (BAR) with nicotine and noradrenaline, respectively, resulted in an increase of the spontaneous beating rate of VMs co-cultured with SNs in the presence of GDNF. GDNF overexpressing VMs by adenovirus vector (AdGDNF-VMs) attracted more axons from SNs compared with mock-transfected VMs. In vivo, axon outgrowth toward the denervated myocardium in adult rat hearts after cryoinjury was also enhanced significantly by adenovirus-mediated GDNF overexpression. GDNF acts as a potent chemoattractant for sympathetic innervation of ventricular myocytes, and is a promising molecular target for regulation of cardiac function in diseased hearts.Molecular signaling of cardiac autonomic innervation is an unresolved issue. Here, we show that glial cell line-derived neurotrophic factor (GDNF) promotes cardiac sympathetic innervation in vitro and in vivo. Sympathetic nervous system plays a critical role in regulating cardiac function in balance with parasympathetic activity. However, in diseased hearts, nonuniform myocardial innervation in association with enhanced sympathetic activity is supposed to create a substrate for life-threatening arrhythmias Trypanozoma cruzi infection (Chagas disease) causing sympathetic as well as parasympathetic denervation A plethora of neurotrophic factors is involved in sympathetic innervation and nerve sprouting. Several experimental studies have shown that nerve growth factor (NGF) facilitates sympathetic innervation in vitro proximity co-cultures of rat neonatal ventricular myocytes (VMs) and sympathetic neurons (SNs) obtained from the superior cervical ganglia showed: (i) GDNF is more potent than NGF in stimulating sympathetic axon growth and enhancing functional coupling between SNs and VMs; (ii) using VMs overexpressing GDNF (AdGDNF-VMs), we have shown a potent action of endogeneous GDNF for sympathetic \u201caxon guidance\u201d. Using the adult rat hearts in vivo, we also demonstrate that: (iii) axon outgrowth toward the denervated myocardium after cryoinjury is enhanced by GDNF overexpression. GDNF may become a novel target in restoring impaired sympathetic innervation of the diseased heart.Here, we present that All procedures were conducted in accordance with a protocol approved by the Animal Experimentation Committee, Research Institute of Environmental Medicine, Nagoya University.proximity co-culture system (a random co-culture system used in our previous study proximity co-culture: SNs (4\u00d7105/ml) and the VMs (2\u00d7106/ml) were seeded separately on a gelatin-coated cover slip or 64-electrode array at a close distance (1 mm) to observe the outgrowth of axons toward VMs. A glass ring frame was placed initially (15 hours) for their separation (VMs inside and SNs outside). -transgenic 1-day-old neonatal Wistar rats through enzymatic digestione system which isutside). \u201342 and 98% humidity. The culture medium was supplemented either with NGF 50 ng/ml (Sigma-Aldrich), GDNF 10 ng/ml (R&D systems). Cell cultures were continued for 5 days to observe the axon outgrowth of SNs towards the VMs.SNs/VMs were cultured in Dulbecco's Modified Eagle's Medium containing 10% (vol/vol) fetal bovine serum , insulin, transferrin, selenite liquid media supplement (Sigma-Aldrich) and cytosine arabinofuranosid (1 \u00b5mol/L: Sigma-Aldrich), at 37\u00b0C in an incubator with 5% CO6 viral particles) was injected into the inner area of the injured ring. In the control group, phosphate-buffered saline (PBS) containing recombinant adenoviruses encoding GFP (AdGFP) was injected into the inner area of the injured ring. After the injection, chest walls were closed and rats were transferred to animal cages to recover from anesthesia. Five days later, the rats were anesthetized with pentobarbital. Then, the hearts were extracted and fixed for the consequent immunohistochemistry to examine the axon growth to the cryoinjured area. Whole-mount immunolabeling for NFM was carried out with the use of 3,3\u2032-diaminobenzidine, tetrahydrochloride (DAB) as the secondary antibody.8 weeks-old male Wistar rats were anesthetized with pentobarbital (50 mg/kg) and mechanically ventilated. After midline excision, the heart was exposed and a ring-shaped cryo-injury was induced in epicardial surface of the left ventricular free wall with a glass ring frozen by liquid nitrogen. Then, the rats were divided into two groups: (1) GDNF and (2) Control groups. In the GDNF group, PBS containing recombinant adenoviruses encoding GDNF antibody , anti-neurofilament M (NFM) , anti-synapsin I (SynI) , anti-\u03b21-adrenergic receptors (BAR) antibody , anti-GDNF antibody . The samples were then incubated for 1 hour at room temperature with a 1\u2236200 (v/v) dilution of appropriate secondary antibodies: Alexa Fluor 488-conjugated goat anti-mouse immunoglobulin G (IgG) , Alexa Fluor 568-conjugated goat anti-rabbit IgG , and Alexa Fluor 633-conjugated donkey anti-goat IgG . Immunofluorescence images were acquired using a confocal laser-scanning microscope . The fractions of NFM-positive axon, SynI and BAR over VMs were calculated using Image-pro Plus software . The hearts excised from adult rats after cryodenervation were immunolabeled similarly for NFM, AA and GDNF.4 in 0.1M phosphate buffer containing 4.5% sucrose for 1 hour. They were embedded with epoxy resin. Ultra-thin sections were prepared and examined with a JEPL1210 electron microscope . The images were taken at 10,000 power and scanned into a computer .Co-cultures of CMs/SNs were fixed with 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1M phosphate buffer for 15 min, and then post-fixed with 1.0% OsO1-adrenergic receptors (BAR), noradrenaline was used as an agonist. Average spontaneous beating rates of VMs during each 3 min were obtained.Spontaneous beating activity of VMs co-cultured with SNs was assessed by recording extracellular potentials of VMs by using a 64-electrode array system . To evaluate the presynaptic functional coupling, 1 \u00b5M nicotine was added to stimulate SNs Recombinant adenoviruses encoding GDNF (AdGDNF) were prepared as described previously Detailed information for experimental procedures for the culture of iPS cells has been shown in We examined the effects of GDNF and NGF on the axon outgrowth toward VMs using the proximity co-culture system . Time-laWe next investigated synapse formation between sympathetic axons and VMs in the proximity co-cultures of GFP-knocked-in VMs and SNs . Figure The ultrastructure of the contact area between nerve terminals (from SNs) and VMs was examined by electron microscopy. 1-adrenergic receptors (BARs) in VMs Catecholamines (norepinephrine and epinephrine) are the major neurotransmitters released from SNs acting primarily on \u03b2We examined the effects of presynaptic and postsynaptic stimulation by nicotine and noradrenaline, respectively, in the absence and presence of GDNF or NGF to assess functional coupling between SNs and VMs. The left panels of Our results show that exogenously-applied GDNF has a potent effect on cardiac sympathetic innervation. It is conceivable to assume that endogenous GDNF released from VMs has a similar effect. To address this issue, we prepared VMs overexpressing GDNF by using adenoviral vectors. Co-cultures of AdGDNF-VMs and mock-transfected VMs (Mock-VMs) with SNs in close proximity (1 mm) are shown . When thThe axons growth toward the two different groups of VMs was quantified by immunolabeling. The axons were labeled by anti-neurofilament-M antibody (red), whereas cardiomyocytes and GDNF were labeled by anti-\u03b1-actinin and anti-GDNF antibodies, respectively (green and blue). Representative fluorescence images are shown in Effects of GDNF expression on axon guidance on iPS cell-derived cardiomyocytes (iPSCMs) were also examined using close proximity (1 mm) co-cultures of AdGDNF-iPSCMs and mock-transfected iPSCMs (Mock-iPSCMs) with SNs. As a result, AdGDNF-iPSCMs attracted significantly more abundant axons than Mock-iPSCMs. .in vivo model of adult rats in order to obtain more insight into its pathophysiological role. A ring-shaped epicardial surface of the left ventricular free wall was cryoinjured and cardiomyocytes that sympathetic innervation and synaptogenesis influence the structure of the sarcolemmal membrane and the organization and distribution of \u03b2et al. demonstrated that in a rat model of chemical sympathectomy by 6-hydroxydopamine, the GDNF protein level increased transiently prior to axonal regrowth in a cardiac tissue homogenate Trypanosoma cruzi (Chagas disease) anti-GDNF gold particles in atrial granules increased transiently at the time of maximal autonomic denervation Our experiments using exogenously-applied GDNF suggested that this molecule acts as a potent chemoattractant if it were excreted by VMs. To verify this assumption, we created VMs and iPSCMs overexpressing GDNF by adenovirus infection . In the proximity co-culture experiments separately placed AdGDNF-VMs, Mock-VMs and SNs, axon growth from SNs indeed occurs predominantly toward AdGDNF-CMs, confirming the above hypothesis. Previously, Martinelli in vivo adult rat heart model of denervation created by cryoinjury, no substantial GDNF upregulation was recognized in the denervated regions of the control hearts (without AdGDNF injection), and this was associated with moderate axon growth toward the denervated region. In contrast, in the rat hearts with AdGDNF injection inside the denervated region considerable GDNF expression and prominent axon growth was observed in the denervated region. These results do not support a role for endogenous GDNF per se in the reinnervation of adult heart following injury, but they do suggest that GDNF can potentially be used to promote reinnervation.In our in vivo, appropriate autonomic reinnervation will be required for the transplanted cardiac myocytes to function in response to a variety of physical demands. Our experiments using proximity co-cultures of iPS-derived CMs overexpressing GDNF (AdGDNF-iPSCMs), mock-transfected iPSCMs and SNs have revealed that GDNF was expressed in iPSCMs and certainly acts a potent chemoattractant for the sympathetic axon guidance.Myocardial regeneration therapy by transplantation of stem cell-derived cardiac tissues attracts a great deal of attention as an innovation for the treatment of heart failure refractory to conventional therapies. To obtain functional regenerated myocardium The present study demonstrated that GDNF promotes sympathetic innervation in both native cardiac cells and stem cell-derived cardiac cells. The results implicated that local delivery of GDNF would exert strong effects to navigate sympathetic axons to target cardiac tissues in cases of myocardial injury or transplantation of regenerated myocardial tissue.Figure S1iPS cell-derived cardiomyocytes (iPSCMs). A) A representative image of a spontaneous beating mass of iPSCMs microdissected from iPS cell-derived EBs. B) Extracellular potentials recorded in spontaneous beating mass shown in (A). Each trace was obtained from corresponding electrodes shown in (A). Potentials were recorded with MED64 system. C,D) Effects of isoproterenol (1 \u00b5M) (C) and carbamyl choline (10 \u00b5M) (D) on iPSCMs. Values are means\u00b1SD. *p<0.05 vs baseline. E) Representative immunolabeling images of nanog (green) and actin (red) in spontaneous beating iPSCMs. Note most of the cells are positive for actin. Bar indicate: 50 \u00b5m.(EPS)Click here for additional data file.Figure S2Axon growth of sympathetic neurons toward GDNF-expressing iPSCMs. GDNF- or mock-transfected iPSCMs were prepared by using adenovirus vectors (AdGDNF-iPSCMs and Mock-iPSCMs) and co-cultured with SNs at a close distance (\u223c1 mm). A) A bright field image of the triangular co-culture (day 5) (left) and its schematic illustration (right). The axons from SN grow predominantly toward AdGDNF-iPSCMs. Bar indicate 1 mm. Video images (B) Sequential time-lapsed images. The pictures were taken at 0, 20, 40, 80 and 140 hours after removal of the glass-ring separation. Bar indicate 1 mm. C) Representative immunolabeling images of neurofilament-M , \u03b1-actinine and nanog-GFP (green). Left: AdGDNF-iPSCMs close to SNs, Right: Mock-iPSCMs close to SNs. The NFM-positive axons (blue fibrous structure) distribute abundantly on AdGDNF-iPSCMs, whereas only scarcely on Mock-iPSCMs. Bars indicate 20 \u00b5m. D) The fraction of axon (NFM-positive area) distributing on iPSCMs (AA-positive area). Values are means\u00b1SD of 5 co-cultures in each group. *p<0.05 vs mock-iPSCMs.o images are avai(EPS)Click here for additional data file.Text S1Cultures of induced-pluripotent stem (iPS) cells. A cell line of Nanog-GFP knock-in iPS cells generated from Fbx15 iPS cells were provided by Dr. Shinya Yamanaka (Kyoto University) (DOCX)Click here for additional data file.Video S1Axon growth of sympathetic neurons toward cardiomyocytes. Sequential time-lapsed microscopic images from 0 to 50 hours after the proximity (\u223c1 mm)co-cultures of SNs (right) and CMs (left) with the exogenous application of NGF (50 ng/ml).Note that axons from SNs are growing as if they are searching for \u201ctarget\u201d cells. See (MPG)Click here for additional data file.Video S2Axon guidance of sympathetic neurons by GDNF-overexpressing VMs. Sequential time-lapsed microscopic images from 0 to 160 hours after the proximity (\u223c1 mm) co-cultures of SNs and AdGDNF-VMs (left upper) and Mock-VMs (right upper). Note axons from SNs are grown predominantly toward AdGDNF-VMs (left upper). See (AVI)Click here for additional data file.Video S3Axon guidance of sympathetic neurons by GDNF-overexpressing iPSCMs. Sequential time-lapsed microscopic images from 0 to 144 hours after the proximity (\u223c1 mm) co-cultures of SNs and AdGDNF-iPSCMs (left upper) and Mock-iPSCMs (right upper). Note axons from SNs are grown predominantly toward AdGDNF-iPSCMs (left upper). See (WMV)Click here for additional data file."} +{"text": "Carbon monoxide-releasing molecules (CO-RMs) are a class of organometallo compounds capable of delivering controlled quantities of CO gas to cells and tissues thus exerting a broad spectrum of pharmacological effects. CO-RMs containing transition metal carbonyls were initially implemented to mimic the function of heme oxygenase-1 (HMOX1), a stress inducible defensive protein that degrades heme to CO and biliverdin leading to anti-oxidant and anti-inflammatory actions. Ten years after their discovery, the research on the chemistry and biological activities of CO-RMs has greatly intensified indicating that their potential use as CO delivering agents for the treatment of several pathological conditions is feasible. Although CO-RMs are a class of compounds that structurally diverge from traditional organic-like pharmaceuticals, their behaviour in the biological environments is progressively being elucidated revealing interesting features of metal-carbonyl chemistry towards cellular targets. Specifically, the presence of carbonyl groups bound to transition metals such as ruthenium, iron or manganese appears to make CO-RMs unique in their ability to transfer CO intracellularly and amplify the mechanisms of signal transduction mediated by CO. In addition to their well-established vasodilatory activities and protective effects against organ ischemic damage, CO-RMs are emerging for their striking anti-inflammatory properties which may be the result of the multiple activities of metal carbonyls in the control of redox signaling, oxidative stress and cellular respiration. Here, we review evidence on the pharmacological effects of CO-RMs in models of acute and chronic inflammation elaborating on some emerging concepts that may help to explain the chemical reactivity and mechanism(s) of action of this distinctive class of compounds in biological systems. The heme oxygenase enzymes (HMOX1 and HMOX2) generate, among other interesting molecules, the gas carbon monoxide (CO) ,4. Reseahttp://www.clinicaltrials.gov). In parallel and as an alternative to this experimental approach, we have focused our strategy on utilizing chemicals that could bind and carry CO stably but deliver the gas when used in biological systems. We have identified and termed these compounds CO-releasing molecules (CO-RMs) 2) also known as tricarbonyldichlororuthenium(II) dimer; CORM-3: Ru(CO)3Cl(glycinate) also known as tricarbonylchloro(glycinato)ruthenium(II); CORM-371: [Me4N][Mn(CO)4(thioacetate)2]; CORM-A1: NaH3BCOOH, also known as sodium boranocarbonates; COX-2: Cyclooxygenase-2; Hb: Hemoglobin; (HMOX1): Heme oxygenase-1; ICAM-1: Intercellular adhesion molecule 1; IL: Interleukin; IL-1\u03b2: Interleukin 1 beta; iNOS: Inducible nitric oxide synthase or NOS(III); LPS: Lypopolysaccharide; MAPKs: Mitogen activated protein kinases; Mb: Myoglobin; MbCO: Carbon monoxy myoglobin; MMP: Matrix metallo proteinase; MPO: Myeloperoxidase; NO: Nitric oxide; Nfk\u03b2: Nuclear factor kappa beta; O2\u2212: Superoxide anion; PGE2: Prostaglandin E2; PMNs: Polymorphonuclear neutrophils; ROS: Reactive oxygen species; TNF-\u03b1: Tumor necrosis factor alpha.CLP: Cecal ligation and puncture; CO: Carbon monoxide; CO-RMs: Carbon monoxide-releasing molecules; CO: Carbon monoxide; CORM-2: [Ru(CO)Dr. Roberto Motterlini was founder and scientific director of hemoCORM (2004-2008), holds patents on the CO-RMs technology and is a shareholder of Alfama.RM and RF wrote the manuscript. BH drafted an earlier version of the manuscript and helped with the outline of Table 1. All authors have read and approved the manuscript."} +{"text": "H-Ras, -Her2/neu, -c-Myc, -PymT, -Wnt1 and C3(1)/SV40 T/t-antigen transgenic mice, fl/flBRCA1;p53+/-;MMTV-cre knock-out mice and the fl/flp53;MMTV-cre transplant model.MicroRNAs (miRNAs) are small, non-coding, endogenous RNAs involved in regulating gene expression and protein translation. miRNA expression profiling of human breast cancers has identified miRNAs related to the clinical diversity of the disease and potentially provides novel diagnostic and prognostic tools for breast cancer therapy. In order to further understand the associations between oncogenic drivers and miRNA expression in sub-types of breast cancer, we performed miRNA expression profiling on mammary tumors from eight well-characterized genetically engineered mouse (GEM) models of human breast cancer, including MMTV-in silico.miRNA expression patterns classified mouse mammary tumors according to luminal or basal tumor subtypes. Many miRNAs found in luminal tumors are expressed during normal mammary development. miR-135b, miR-505 and miR-155 are expressed in both basal human and mouse mammary tumors and many basal-associated miRNAs have not been previously characterized. miRNAs associated with the initiating oncogenic event driving tumorigenesis were also identified. miR-10b, -148a, -150, -199a and -486 were only expressed in normal mammary epithelium and not tumors, suggesting that they may have tumor suppressor activities. Integrated miRNA and mRNA gene expression analyses greatly improved the identification of miRNA targets from potential targets identified This is the first large-scale miRNA gene expression study across a variety of relevant GEM models of human breast cancer demonstrating that miRNA expression is highly associated with mammary tumor lineage, differentiation and oncogenic pathways. Caenorhabditis elegans during genetic screens for regulators of developmental timing ; miRNA gene expression raw data of normal mammary gland tissues from different mouse genetic background [GSE23977]; mRNA gene expression raw data [GSE23938].EMT: epithelial-to-mesenchymal transition; ER: estrogen receptor; FACS: fluorescence activated cell sorting; FDR: false discovery rate; GEM: genetically engineered mouse; LTR: long terminal repeat; miRNA: microRNA; MMTV: mouse mammary tumor virus; PCR: polymerase chain reaction; PyMT: polyoma middle T antigen; RFP: red fluorescence protein; RT-PCR: reverse transcription PCR;UTR: untranslated region.The authors declare that they have no competing interests.MZ contributed to the design and conception of the experiments, conducted molecular biology experiments, analyzed and interpreted data and drafted the manuscript. CHK performed the microarray experiments and helped with quality control and analysis. MY and RS normalized the data and performed all statistical analyses. CD and DM provided tumor tissue samples that they had characterized. JEG conceived of the project and participated in its design, helped to analyze and interpret the data and draft the manuscript. All authors have read and approved the manuscript for publication.Figure S1 - miRNA gene expression profile of normal mammary gland tissues from different mouse genetic backgrounds. The miRNAs of the normal mammary glands are compared to those of the C3(1)/Tag mammary tumors as a control.Click here for fileFigure S2 - unsupervised hierarchical clustering of the 22 differentially expressed miRNA genes identified in Additional file 1over 41 mammary tumors derived from 8 genetically engineered mouse models and 5 normal mammary tissues. The heatmap shows the expression of miRNAs at the probe level. Heatmap colors represent relative miRNA expression as indicated in the color key.Click here for fileFigure S3 - double-immunofluorescence staining of mouse samples for basal/myoepithelial and luminal cytokeratins. Normal mammary gland and mammary tumors from the indicated mouse models are stained for cytokeratin 18 and cytokeratin 14 .Click here for fileFigure S4 - correlation of miRNA microarray data with quantitative RT-PCR miRNA expression data. Shown are the pairwise scatter plots for individual miRNAs. The y-axis of the plot shows the log2 intensity of the microarray data, whereas the x-axis shows the -delta cycle threshold (CT) value of the RT-PCR results. Each dot in the plot represents one sample from individual tumor models or normal mammary tissues. Person correlation coefficients (r) and P-values are calculated.Click here for filemiRNAs that are highly associated with basal- and luminal- mammary tumor subtypes.Click here for filemiRNAs that are highly associated with individual genetically engineered mouse models and normal mammary tissues.Click here for fileGenetically engineered mouse model-specific miRNAs and their potential mRNA targets.Click here for fileBasal- or luminal-like miRNAs and their potential mRNA targets.Click here for fileFigure S5 - analysis of the inverse relationship between transcript levels of miRNAs and their putative target mRNAs in mouse mammary tissues. Global distribution of the Pearson correlation coefficients between mRNAs and (a) miR-10b, (b) miR-412 and (c) miR-494. The dotted curves show the distribution of the correlation coefficients for all mRNAs. The solid curves show the correlation coefficients for only those mRNAs that are predicted targets of miR-10b, miR-412 or miR-494.Click here for fileFigure S6 - Ingenuity Pathway Analysis\u2122 of the potential target genes of miR-494. Twelve of the mRNA target genes of miR-494 from Table 3 were input into Ingenuity , and core analysis was then performed to retrieve the target genes' association with cancer and disease.Click here for fileFigure S7 - overexpression of miR-494 in M6 cells as determined by quantitative real-time RT-PCR. M6 cells were transduced with plemiR lentivirus expressing miR-494. Control cells were M6 cells and M6 cells transduced with plemiR lentivirus vector. Following infection, cells were FACS sorted for RFP and RNA was extracted. Real-time RT-PCR was then performed to examine the expression of miR-494 in these cells.Click here for fileBmi1 or Ptpn12 determined by quantitative real-time RT-PCRFigure S8 - overexpression of miR-494 in M6 cells does not alter expression of . M6 cells were transduced with plemiR lentivirus expressing miR-494. Control cells were M6 cells, M6 cells transduced with plemiR lentivirus vector, and M6 cells transduced with lentivirus expressing scrambled miRNA. Following infection, cells were FACS sorted for RFP and RNA was extracted. Real-time RT-PCR was then performed to examine the expression of Bmi1 (top) and Ptpn12 (bottom) in these cells. P-value (Bmi1: M6_miR494 versus M6_scramble) = 0.06; P-value (Ptpn12: M6_miR494 versus M6_scramble) = 0.0502.Click here for fileFoxo3a and Spry4 by miR-412 in DB7 cellsFigure S9 - increased expression of . DB7 cells were transduced with plemiR lentivirus expressing miR-412. Control cells were DB7 cells, DB7 cells transduced with plemiR lentivirus vector, and DB7 cells transduced with lentivirus expressing scrambled miRNA. Following infection, cells were FACS sorted for RFP and RNA was extracted. Real-time RT-PCR was then performed to examine the expression of Foxo3a and Spry4 in these cells. P-value (Foxo3a: DB7_miR412 versus DB7_scramble) = 0.125; P-value (Spry4: DB7_miR412 versus DB7_scramble) = 2.75E-06.Click here for file"} +{"text": "Neuroimaging studies report cerebellar activation during both motor and non-motor paradigms, and suggest a functional topography within the cerebellum. Sensorimotor tasks activate the anterior lobe, parts of lobule VI, and lobule VIII, whereas higher-level tasks activate lobules VI and VII in the posterior lobe. To determine whether these activation patterns are evident at a single-subject level, we conducted functional magnetic resonance imaging (fMRI) during five tasks investigating sensorimotor (finger tapping), language (verb generation), spatial , working memory (N-back), and emotional processing . Finger tapping activated the ipsilateral anterior lobe (lobules IV-V) as well as lobules VI and VIII. Activation during verb generation was found in right lobules VII and VIIIA. Mental rotation activated left-lateralized clusters in lobules VII-VIIIA, VI-Crus I, and midline VIIAt. The N-back task showed bilateral activation in right lobules VI-Crus I and left lobules VIIB-VIIIA. Cerebellar activation was evident bilaterally in lobule VI while viewing arousing vs. neutral images. This fMRI study provides the first proof of principle demonstration that there is topographic organization of motor execution vs. cognitive/emotional domains within the cerebellum of a single individual, likely reflecting the anatomical specificity of cerebro-cerebellar circuits underlying different task domains. Inter-subject variability of motor and non-motor topography remains to be determined."} +{"text": "The current BNP immunoassay cross-reacts with glycosylated proBNP, and the NT-proBNP assay underestimates glycosylated NT-proBNP. In addition, the recently developed high performance dialyzer removes medium-sized molecular solutes such as \u03b22-microgloburin. We therefore investigated the effects of high performance dialysis on measured levels of glycosylated proBNP, glycosylated NT-proBNP and other BNP-related peptides in end-stage renal disease (ESRD) patients on hemodialysis.O-glycosydase. We also measured plasma ANP and cGMP using radioimmunoassays.We used our newly developed immunoassay to measure plasma total BNP, proBNP and mature BNP in 36 ESRD patients before and after hemodialysis. Plasma glycosylated NT-proBNP and nonglycosylated NT-proBNP were measured using Elecsys II after treatment with the deglycosylating enzymes neuramoinidase and Total BNP (-38.9%), proBNP (-29.7%), mature BNP (-54%), glycosylated NT-proBNP(-45.5%), nonglycosylated NT-proBNP(-53.4%), ANP(-50.4%) and cGMP(-72.1%) were all significantly reduced after hemodialysis. The relative magnitudes of the reductions did not correlate with any indices of plasma volume, but instead appeared to be molecular weight dependent. The proBNP/total BNP and glycosylated NT-proBNP/nonglycosylated NT-proBNP ratios were increased after hemodialysis. The proBNP/total BNP ratio correlated positively with hemodialysis vintage and negatively with left atrial diameter and systolic blood pressure, whereas glycosylated/nonglycosylated NT-proBNP ratios correlated positively with parathyroid hormone levels.These results suggest that plasma BNP and its related peptides measured immediately after hemodialysis may not be good indices of body fluid status in ESRD patients undergoing hemodialysis using a high performance dialyzer. ProBNP/total BNP may be influenced by hemodialysis vintage, cardiac afterload and diastolic function."} +{"text": "Tumor necrosis factor related apoptosis-inducing ligand (TRAIL) induced apoptosis specifically in tumor cells. However, with approximately half of all known tumor lines being resistant to TRAIL, the identification of TRAIL sensitizers and their mechanism of action become critical to broadly use TRAIL as a therapeutic agent. In this study, we explored whether c-Met protein contributes to TRAIL sensitivity. We found a direct correlation between the c-Met expression level and TRAIL resistance. We show that the knock down c-Met protein, but not inhibition, sensitized brain tumor cells to TRAIL-mediated apoptosis by interrupting the interaction between c-Met and TRAIL cognate death receptor (DR) 5. This interruption greatly induces the formation of death-inducing signaling complex (DISC) and subsequent downstream apoptosis signaling. Using intracranially implanted brain tumor cells and stem cell (SC) lines engineered with different combinations of fluorescent and bioluminescent proteins, we show that SC expressing a potent and secretable TRAIL (S-TRAIL) have a significant anti-tumor effect in mice bearing c-Met knock down of TRAIL-resistant brain tumors. To our best knowledge, this is the first study that demonstrates c-Met contributes to TRAIL sensitivity of brain tumor cells and has implications for developing effective therapies for brain tumor patients. In an effort to develop anti-brain tumor therapies that overcome TRAIL resistance, we explored the interaction between c-Met and DRs and their contribution in TRAIL resistance in brain tumors. Furthermore, we addressed the effect of targeting c-Met by lentivirial-mediated expression of shRNA in combination with mesenchymal stem cell (MSC)-delivered a secretable form of TRAIL (S-TRAIL) in MSC-tumor cell co-cultures Medulloblastoma (MB) cell lines from American Type Culture Collection (ATCC) 4/well) were seeded in 96-well plates and incubated with varying concentrations (0\u223c1000 ng/ml) of S-TRAIL obtained from transduced 293T cells for 24 h. Cell viability was measured by a quantitative luminescence assays using an ATP-dependent luminescent reagent according to the manufacturer's instructions. Based on our previously published screening of glioma cells to TRAIL mediated apoptosis, we chose TRAIL sensitive and resistant glioma lines Human 293T cells were transduced with lentiviral vector LV-S-TRAIL Following treatment, MB cells were lysed with NP40 buffer supplemented with a protease inhibitor mixture and phosphatase inhibitors II/III . 30 \u00b5g of harvested proteins from each lysate were resolved on 10% SDS-PAGE, and immunoblotted with antibodies against DR5, DR4 , c-Met, caspase 8, cleaved caspase 3, cleaved poly (ADP-ribose) polymerase (PARP), FLICE inhibitory protein (FLIP) , or \u03b1-tubulin (Sigma); and detected by chemiluminescence after incubation with HRP-conjugated secondary antibodies (Santa Cruz). Quantification of western blot signals was performed using Image J. The data was normalized to \u03b1-tubulin expression. For immunoprecipitation, whole cell lysates were pre-incubated with Protein G Resin for 3 h at 4\u00b0C to exclude nonspecific binding to Protein G-Sepharose. After centrifugation, supernatants containing proteins were added with IgG (Santa Cruz) or indicated antibodies, rotated over night at 4\u00b0C, and then added with Protein G-Sepharose for 3 h. The immune complex was spun down, washed, resuspended in SDS-gel sample buffer and boiled at 100\u00b0C for 5 min. Immunoprecipitates were separated by SDS-PAGE and immunoblotted with indicated antibodies.For co-culture experiments, UW473scr-Fmc or UW473shMet-Fmc cells were plated with varying numbers of MSC-GFP or MSC-S-TRAIL (10\u22361 or 5\u22361) in a 96-well plate, incubated for 36 hours and then cell viability was measured by a quantitative luminescence assays using an ATP-dependent luminescent reagent , according to the manufacturer's instructions.5 tumor cells/mouse and 6\u00d7104 MSCs/mouse) in the following co-ordinates: 2.2 mm lateral from bregma, 2.5 mm ventral from dura on the cranial suture. Mice were imaged for Fluc activity on 1, 3 and 7 days after implantations as previously described in vivo procedures were approved by the Subcommittee on Research Animal Care at Massachusetts General Hospital.UW473scr-Fmc, UW473shMet-Fmc, MSC-GFP, and MSC-S-TRAIL cells were harvested at 80%\u201390% confluence and implanted in various combinations as the following experimental groups: UW473scr-Fmc with MSC-GFP, UW473scr-Fmc with MSC-S-TRAIL, UW473shMet-Fmc with MSC-GFP, UW473shMet-Fmc with MSC-S-TRAIL. These combinations were implanted stereotactically into nude mice brains directly into the heart and the brains were fixed in 4% PFA and frozen sections were obtained for immunohistochemistry. For cleaved caspase 3 staining, sections were incubated for 1 hour in a blocking solution at room temperature, followed by incubation at 4\u00b0C overnight with anti-cleaved caspase 3 antibody diluted in blocking solution. Sections were incubated in Alexa Fluor 647 goat anti-rabbit secondary antibody (Invitrogen), and visualized using confocal microscope . The number of cleaved caspase 3 positive cells was calculated by counting positive cells in randomly selected field of views under a microscope (20\u00d7).t-test when comparing two groups and by ANOVA when comparing more than two groups. Data are expressed as mean \u00b1 s.e.m., and differences were considered significant at P<0.05.Data were analyzed by Student's In order to investigate the relationship between c-Met protein and TRAIL resistance in brain tumor cells, we first tested a panel of human medulloblastoma (MB) cell lines for their sensitivity to S-TRAIL treatment. While UW473 and DAOY lines demonstrated resistance to various concentrations of S-TRAIL ranging from 100\u223c1000 ng/ml, UW426 line exhibited most sensitivity to these concentrations . PI/AnneWe further explored the mechanism underlying the role of c-Met protein in TRAIL-resistance of brain tumor cells. As the UW473 line is fully resistant to TRAIL, we utilized this line for further studies. Western blot analysis showed that UW473shMet line had a stable knock-down of c-Met levels compared to UW473scr or UW473 lines . Next, win vivo, we first engineered in vivo imageable UW473scr and UW473shMet cells by transducing them with LV-Fluc-mCherry (Fmc). A direct correlation between Fluc signal and cell number was seen within the ranges tested in both cell lines and affecting DISC formation and subsequently TRAIL-mediated apoptosis execution both Figure S1Induction of apoptosis by S-TRAIL treatment in MB cell lines. Effects of S-TRAIL treatment on UW426, DAOY and UW473 lines. Numbers in the respective quadrants indicate the percentage of cells presents in this area. In UW426 cells, the Annexin V positive cell population was significantly increased upon S-TRAIL treatment. * P<0.05, ** P<0.01.(TIF)Click here for additional data file.Figure S2Characterization of modified UW473 lines. (A) Cell viability analysis showing the growth rate of both UW473scr-Fmc and UW473shMet-Fmc cells. (B) Top, representative fluorescent images of both UW473scr-Fmc and UW473shMet-Fmc cells. Bottom, plots showing the Fluc intensities of modified tumor cells with different cell numbers.(TIF)Click here for additional data file.Figure S3The c-Met protein levels in UW473 lines stably transduced with LV-scrambled or LV-shMet. Western blot analysis of c-Met and tubulin levels in UW473 cells stably transduced with LV-scrambled (UW473scr) or LV-shMet (UW473shMet).(TIF)Click here for additional data file."} +{"text": "Mature Schwann cells have been considered a promising candidate for cell-transplantation therapies to repair the damaged nervous system, whereas these \u201cSchwann-spheres\" would provide a more potential autologous cell source for such transplantation.Multipotent somatic stem cells have been identified in various adult tissues. However, the stem/progenitor cells of the peripheral nerves have been isolated only from fetal tissues. Here, we isolated Schwann-cell precursors/immature Schwann cells from the injured peripheral nerves of adult mice using a floating culture technique that we call \u201cSchwann-spheres.\" The Schwann-spheres were derived from de-differentiated mature Schwann cells harvested 24 hours to 6 weeks after peripheral nerve injury. They had extensive self-renewal and differentiation capabilities. They strongly expressed the immature-Schwann-cell marker p75, and differentiated only into the Schwann-cell lineage. The spheres showed enhanced myelin formation and neurite growth compared to mature Schwann cells In recent years, multipotent somatic stem cells have been identified in various adult tissues. In the peripheral nerves, stem/progenitor cells that are self-renewing and multipotent, with the potential to differentiate into neurons, glial cells, and myofibroblast, have been detected and isolated from fetal After peripheral nerve injury, mature Schwann cells undergo a reversion in their molecular phenotype, and come to resemble those observed in fetal immature nerves in vitro suggested their potential use in cell transplantation therapy for the damaged nervous system.Here, we cultured the dedifferentiated Schwann cells obtained from the injured peripheral nerves of adult mice at the specific time-point under the floating culture condition and isolated Schwann-cell precursors/immature Schwann cells, as spheres, which we called \u201cSchwann-spheres.\" This is the first report showing that \u201cSchwann-spheres\" can be obtained from adult peripheral nerves. Moreover, their differentiation, myelination, and neurite growth promoting properties loxP/loxP-EGFP) Normal, specific pathogen-free, adult C57BL/6J mice were purchased from CLEA Japan, Inc., Tokyo, Japan. Nestin-EGFP transgenic mice carry enhanced green fluorescent protein (EGFP) under the control of the second intronic enhancer of the nestin gene, which acts selectively in stem/progenitor cells The adult C57BL/6J mice, Nestin-EGFP mice, and MBP-Cre/Floxed-CAG-EGFP mice were anesthetized using an intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg). The animals were housed in groups under a 12-hour light/dark cycle, with access to food and water ad libitum. The sciatic nerve was exposed through a dorsal gluteal muscle-splitting approach. The nerve was subjected to a contusive injury at the sciatic notch using a brain aneurysm clip . The clip was closed and left in place for 5 min with a holding force of approximately 50 g. All interventions and animal care procedures were performed in accordance with the Laboratory Animal Welfare Act, the Guide for the Care and Use of Laboratory Animals , and the Guidelines and Policies for Animal Surgery provided by the Animal Study Committee of Keio University, and were approved by the Ethics Committee of Keio University.5 cells/ml) were transferred to a sphere-forming floating medium consisting of DMEM/F-12 (1:1) supplemented with insulin (25 mg/ml), transferrin (100 mg/ml), progesterone (20 nM), sodium selenate (30 nM), putrescine (60 nM) , recombinant human EGF (100 ng/ml) , human FGF-basic (100 ng/ml) (Pepro Tech), B27 (20 ng/ml) , soaked overnight in 10% sucrose followed by 30% sucrose, and embedded in cryomolds for sectioning at 6\u20138 \u00b5m. The spheres were immunostained using the anti-undifferentiated-cell marker, Nestin ; Schwann-cell markers, P0 and p75 , or the proliferative-cell marker, PCNA , to define the cell population. Immunoreactivity was visualized using secondary antibodies conjugated with Alexa 488 or Alexa 555 (Molecular Probes). Nuclear counterstaining was performed with Hoechst 33342 . The samples were observed with a universal fluorescence microscope .Spheres were plated on poly-L-lysine (PLL) (Sigma)-coated 8-well chamber slides and cultured for 7 days in the following differentiation medium: DMEM/F12 (1\u22361) supplemented with 10% FBS, without any growth factors The distal portions of intact or injured sciatic nerves (day 7 after the injury) from MBP-Cre/Floxed-EGFP mice were postfixed for 24 hours in 4% PFA, soaked overnight in 10% sucrose followed by 30% sucrose, and embedded in a cryomold for sectioning at 10 \u00b5m. The primary antibodies were anti-GFP and anti-p75 . The secondary antibodies were anti-goat IgG (Alexa 488) and anti-rabbit IgG (Alexa 555). Nuclear counterstaining was performed with Hoechst 33342 . The samples were observed with a confocal laser scanning microscope .To examine the tri-lineage differentiation potential, spheres derived from injured adult sciatic nerves from MBP-Cre/Floxed-CAG-EGFP mice were plated on PLL-coated 8-well chamber slides and cultured for 7 days in the above-described differentiation medium. For immunocytochemistry, the cells were fixed in 4% PFA and stained with the following primary antibodies: anti-GFP , anti-P0 , anti-S100 , anti-\u03b2-III tubulin , and anti-\u03b1SMA . Secondary antibodies were the following: anti-goat IgG (Alexa 488), anti-rabbit IgG (Alexa 555), anti-chick IgG (Alexa 568), anti-mouse IgG2b (Alexa 488), and anti-mouse IgG2a (Alexa 647). The samples were observed with a universal fluorescence microscope .\u03b2-actin mRNA was used as a reference. PCR was performed with KOD plus DNA polymerase according to the manufacturer's instructions. The PCR products were resolved by electrophoresis in 1-3% agarose gels, and the bands were visualized with ethidium bromide under UV light Total RNA was isolated from each sample with Trizol reagent and DNase I treatment. Total RNA (1 \u00b5g) was used to synthesize cDNA with oligo-d(T) primers. The cDNA synthesis was performed at 42\u00b0C for 50 min in a final volume of 20 \u00b5l, according to the manufacturer's instructions for Superscript III reverse transcriptase (Invitrogen). To normalize the template cDNA, the ubiquitously expressed 2 field by surveying six fields. The average was calculated.DRG neurons were co-cultured with cells derived from intact sciatic nerves or Schwann-spheres using the modified method of Hoshikawa et al p<0.05 using one-factor ANOVA and the Tukey-Kramer test for the primary and secondary sphere-forming assays. Student's t-test was used to compare the data between groups for the myelination and neurite outgrowth assays.All values are presented as the mean \u00b1 standard error of the mean (SEM). Statistical significance was determined as nestin-EGFP mice nestin-EGFP mice were positive for EGFP, suggesting that they were Nestin-positive immature cells analysis was conducted to evaluate the mRNA expression of various stem-cell and Schwann-cell markers in the injured adult sciatic nerve-derived spheres and fetal neural crest-derived spheres . The sphin vitroin vitro.To examine the Schwann-spheres' therapeutic potential, we performed myelination and neurite growth assays This is the first report that Schwann-cell precursors/immature Schwann cells, in the form of cultured \u201cSchwann-spheres,\" can be isolated from adult peripheral nerves. Mature myelinating and non-myelinating cells respond to nerve injury by reverting to a molecular phenotype similar to that of immature Schwann cells, to provide essential support for axonal regrowth Schwann cells are considered a promising candidate for cellular transplantation therapies to repair the injured central or peripheral nervous system Recently, Agudo et al. reported the novel and potentially useful properties of an early cell in the Schwann-cell lineage, the Schwann-cell precursor in vitro. Skin-derived precursor (SKP)-derived Schwann cells can myelinate axons In the present study, we also demonstrated that the Schwann-spheres derived from injured adult sciatic nerves demonstrated much higher potentials for myelin formation and neurite-growth enhancement than mature Schwann cells isolated from intact sciatic nerves Many investigators have studied stem-cell transplantation therapies for regenerating the central nervous system. Although the transplantation of fetal neural stem/progenitor cells into the injured spinal cord can promote functional recovery in adult mice Figure S1Trilineage differentiation potential of the spheres derived from the injured adult sciatic nerves of MBP-Cre/Floxed-EGFP mice. EGFP+ spheres derived from the injured adult sciatic nerves differentiated into glial cells C, but n(TIF)Click here for additional data file."} +{"text": "Antibodies (Abs) that mediate antibody-dependent cellular cytotoxicity (ADCC) activity against HIV-1 are of major interest. Considerable evidence supports a role for ADCC activity in the control of HIV-1 infection and in the context of vaccination. One method widely used to assess the role of ADCC is the rapid and fluorometric antibody-dependent cellular cytotoxicity (RFADCC) assay. In the RFADCC assay specific killing of target cells by PBMC is assessed by loss of intracellular CFSE but retention of membrane dye PKH26 (CFSE-PKH26+), which is assumed to be derived from CFSE+PKH26+ target cells killed by NK cells. We have revisited this assay to assess the role of effector cells in mediating ADCC.Multi-color flow cytometry was used to analyse gp140-pulsed, CFSE and PKH26 double labeled CEM.NKr-CCR5 cells incubated with HIV+ plasma or purified IgG samples (n=57) and co-cultured with PBMC, purified NK cells, or monocytes prepared from healthy donor blood. Effector/target cell interaction was visualized using image stream flow cytometry and live cell imaging.Backgating analysis and phenotyping of CFSE-PKH26+ cells identified CD3-CD14+ monocytes as the major effector cell type. This was confirmed for all 57 HIV+ plasma samples tested. Emergence of the CFSE-PKH26+ cell population was observed following co-culture with purified monocytes but not purified NK cells. No significant IFN\u03b3 production or CD107a degranulation was detected in NK cells in this assay. Image flow cytometry and microscopy confirmed a monocyte-specific interaction with target cells. Monocytes acquire PKH26+ cell membrane presumably derived from killed target cells without typical morphological changes associated with phagocytosis, suggesting monocyte-mediated ADCC.Our studies advance the understanding of the cellular events underlying HIV-specific ADCC. The RFADCC assay primarily reflects Ab-mediated monocyte function and has to be treated with caution in regard to NK cell-mediated ADCC. Further studies on the biological importance of HIV-specific monocyte-mediated ADCC are warranted."} +{"text": "Drosophila heart model to examine its role in cardiac physiology, in conjunction with RNAi-mediated gene silencing specifically in the heart in vivo. Analysis of cardiac physiology was carried out using high-speed video recording in conjunction with movement analysis algorithms. unc-45 knockdown resulted in severely compromised cardiac function in adults as evidenced by prolonged diastolic and systolic intervals, and increased incidence of arrhythmias and extreme dilation; the latter was accompanied by a significant reduction in muscle contractility. Structural analysis showed reduced myofibrils, myofibrillar disarray, and greatly decreased cardiac myosin accumulation. Cardiac unc-45 silencing also dramatically reduced life-span. In contrast, third instar larval and young pupal hearts showed mild cardiac abnormalities, as severe cardiac defects only developed during metamorphosis. Furthermore, cardiac unc-45 silencing in the adult heart (after metamorphosis) led to less severe phenotypes. This suggests that UNC-45 is mostly required for myosin accumulation/folding during remodeling of the forming adult heart. The cardiac defects, myosin deficit and decreased life-span in flies upon heart-specific unc-45 knockdown were significantly rescued by UNC-45 over-expression. Our results are the first to demonstrate a cardiac-specific requirement of a chaperone in Drosophila, suggestive of a critical role of UNC-45 in cardiomyopathies, including those associated with unfolded proteins in the failing human heart. The dilated cardiomyopathy phenotype associated with UNC-45 deficiency is mimicked by myosin knockdown suggesting that UNC-45 plays a crucial role in stabilizing myosin and possibly preventing human cardiomyopathies associated with functional deficiencies of myosin.UNC-45 is a UCS (UNC-45/CRO1/She4P) class chaperone necessary for myosin folding and/or accumulation, but its requirement for maintaining cardiac contractility has not been explored. Given the prevalence of myosin mutations in eliciting cardiomyopathy, chaperones like UNC-45 are likely to be equally critical in provoking or modulating myosin-associated cardiomyopathy. Here, we used the These motors are critical for cellular processes such as cytokinesis, vesicle transport, cell motility, and muscle movement Drosophila UNC-45 and the yeast UCS protein She4p have been resolved; these proteins mainly consist of armadillo-like helical repeats elegans and Drosophila each contain a single unc-45 gene. C. elegans unc-45 mutants are characterized by uncoordinated movement and abnormal muscle structure Drosophila UNC-45 possesses chaperone activity that is required for myosin accumulation, and a null mutation leads to embryonic lethality C. elegans and Drosophila, vertebrates have two unc-45 genes UNC-45B, whereas the other is expressed in multiple cell types and is designated UNC-45AXenopus, UNC-45B is required for cardiac and skeletal muscle function and a deficit or mutation leads to striated muscle dysfunction, paralysis and embryonic lethality Structurally, UNC-45 is composed of three domains. The N-terminal tetratricopeptide repeat (TPR) domain is necessary for binding to HSP-90 Drosophila model to investigate UNC-45 function during metamorphosis and in adult cardiac tissue. Drosophila is an advantageous model for studying cardiac development and function since, unlike vertebrates, heart function can be significantly compromised without causing immediate death Drosophila heart is a tubular structure consisting of a single layer of contractile cardiomyocytes and non-contractile pericardial cells that align along each side of the heart wall. The heart is supported by alary muscles and, in adults, by a layer of ventral longitudinal muscle cells de novo in the first and second abdominal segments Although cardiac dysfunction and lethality are associated with inhibition of expression of the UNC-45B isoform Drosophila to study the establishment and maintenance of cardiac function associated with a myosin chaperone encoded by unc-45. Our genetic, structural and functional approaches demonstrate that UNC-45 is crucial for cardiac morphology, physiology and myosin accumulation in the myocardium. We show that unc-45 silencing in third instar larval and young pupal hearts results in mild cardiac abnormalities. However, major cardiac defects appear as a result of knockdown during metamorphosis, indicating that UNC-45 participates in the process of remodeling of the adult heart to ensure normal sarcomeric structures and contractility. Cardiac unc-45 silencing in the adult heart (after metamorphosis) leads to a less severe phenotype. Our demonstration that UNC-45 deficiency is mimicked by myosin knockdown suggests that UNC-45 plays a crucial role in stabilizing myosin and is likely involved in preventing human cardiomyopathy.Here, we describe the use of unc-45 in the Drosophila heart was carried out using the UAS-Gal4 system Hand-Gal4 driver unc-45-RNAi transgenes Hand-Gal4 mediated KD of unc-45 in hearts of 1 week old flies (Hand>UAS-unc-45-RNAi) reduced levels of UNC-45 by \u223c80% compared to age-matched controls . Analysis of heart function in 1 week old Hand>UAS-unc-45 RNAi flies (referred to hereafter as unc-45 KD) revealed severe heart defects compared to controls approach. Silencing of controls : unc-45 ife-span . Interesunc-45 caused a significant prolongation of the heartbeat length, as manifested in an increase of both systolic and diastolic intervals . This waunc-45, control and KD hearts were probed with an antibody against myosin. Myofibrillar disarray was assessed with immunofluorescence microscopy. As shown in unc-45 KD hearts showed severe reduction in myosin content . As shown in unc-45 KD hearts compared to those in control hearts. However, actin expression remained unchanged. This is consistent with our observation with intact hearts probed with anti-myosin and phalloidin upon unc-45 KD (unc-45 KD hearts was confirmed by probing an immunoblot with myosin antibody (not shown). However, myosin expression in indirect flight muscles was similar in both control and unc-45 KD flies (not shown) confirming that the KD was specific to heart muscle.The lack of patterned myosin immunoreactivity in the myofibrils of nc-45 KD . The redunc-45 KD suggested that the ensuing heart structure and function defects were mainly the result of myosin deficiency. Therefore, we tested if KD of myosin heavy chain (Mhc) itself can reproduce an unc-45 KD-like phenotype. We evaluated flies with cardiac-specific Mhc KD using the Hand-Gal4 driver. As for cardiac unc-45 silencing, Mhc KD hearts showed prolonged systolic and diastolic intervals . The finding that cardiac Mhc or unc-45 KD produce a similar reduction in myosin abundance as well as similar cardiac structure/function phenotypes suggests that UNC-45 may be not only critical for myosin accumulation in the myocardium but also for establishing or maintaining normal myosin-dependent cardiac functionality.The decrease in myosin levels due to ntervals and incrntervals resultinntervals . In addintervals . In addiHand-Gal4 driver used to KD unc-45 is expressed in the heart from embryogenesis through adulthood, results of manipulations using this driver do not allow us to distinguish whether unc-45 is required during development for establishing normal heart function, or whether it is needed to maintain cardiac integrity during adulthood. Previous analyses demonstrated that during metamorphosis the Drosophila heart undergoes significant remodeling with major morphological and structural transformations unc-45 mRNA expression is progressively up-regulated in the remodeling heart during metamorphosis Hand>unc-45-RNAi hearts 1-4 days after eclosion and found that, as for hearts from 1-week or older unc-45 KD flies, cardiac function was significantly compromised compared to age-matched controls pupae regarding cardiac contractility compared to wild type. Cardiac contractility of control and unc-45 KD young pupae is shown in unc-45 KD in third instar larvae and young pupae are shown in unc-45 KD hearts showed a small but significant increase in heart rate (tachycardia) compared to age-matched control was depressed compared to control third instar larvae or in young adults (after metamorphosis). Control flies were also exposed to the same temperature conditions. Conditional cardiac KD of unc-45 with the TinC\u03944-Gal4; Gal80ts starting prior to metamorphosis resulted in a severe cardiac phenotype in adult hearts showed prolonged systolic and diastolic intervals compared to controls , dilation of the heart did not occur . This differential expression may be true for the heart as well unc-45 is not null) are sufficient to maintain cardiac contractility in the larval or young pupal hearts. Based upon these data and our cardiac phenotype, we speculate that the continued requirement for UNC-45 during later stage metamorphosis results in more severe cardiac deficits that may not permit compensatory changes in heart rate seen in larvae and young pupae. Thus, UNC-45 is critical for folding and assembly of myosin in an almost complete remodeling from the larval heart to the adult heart.The Drosophila cardiac structure and function. UNC-45 appears to be critical for myosin incorporation into sacomeres/myofibrils of the myocardium, as it is in skeletal muscles. Since this myosin chaperone is critical for maintaining the structural integrity of the cardiac contractile apparatus, it is possibly essential for human cardiac function and survival as well. Indeed, proteomic analysis of hearts from patients suffering from ischemic heart failure has detected increased levels of UNC-45, supporting the hypothesis that this chaperone may be important during human cardiac arrest Our study represents the first evidence of a role for a molecular chaperone in unc-45 (CG2708) gene were obtained from the Vienna Drosophila RNAi Center (VDRC). Each RNAi transgene was made with inverted repeat of an unc-45 fragment, driven by the UAS-promoter as previously reported nd and 3rd chromosome (stock IDs 2708R-1 and 2708R-2) for unc-45 were also obtained from the National Institute of Genetics Fly Stock Center (NIG), Tokyo, Japan. Myosin RNAi transgenes (CG17927) were also obtained from VDRC and NIG (construct IDs 1485 and 102402 from VDRC and stock ID 17927 R-1 from NIG). The cardiac tissue-specific Hand-Gal4 driver was gift from Eric Olsen unc-45 lines were generated as recently described Two UAS-RNAi fly lines (construct IDs 9815 and 101311) for the unc-45 RNAi males or virgin females were crossed to Hand-Gal4 flies and incubated at 25\u00b0C throughout development. Male and female F-1 progeny were separated and allowed to develop with food changes every third day. The temperature-sensitive Gal4\u2013Gal80 system was used to control timing of RNAi KD as previously reported w1118 flies crossed with Hand-Gal4 or tub-Gal80-ts; TinC\u03944-Gal4 or w1118 flies crossed with each of the UNC-45 RNAi transgenic lines. Results from female progeny are reported here.For RNAi silencing, Drosophila heart tubes with myosin antibody or fluorescently-labeled phalloidin was carried out using an Apotome Imager Z1 (Zeiss) and an AxioCam MRm (Zeiss) microscope as previously described Semi-intact hearts were prepared as described previously Drosophila UNC-45 antibody as recently described For analysis of UNC-45 and myosin expression, specimens were collected and protein was extracted with Laemmli sample buffer containing 200 mM \u03b2-mercaptoethanol unc-45 KD was carried out using standard genetic techniques as outlined in unc-45 mutant unc-45 RNAi stocks were rescued with transgenic over-expression of UNC-45. Cardiac physiological, structural and biochemical analyses of the rescued hearts were carried out as reported for control and unc-45 KD hearts.Transgenic rescue of the Figure S1RNAi KD of unc-45 with the 24B-Gal4 driver and its impact on myosin expression. (A) Immunoblot analysis of unc-45 expression (top) in 20 h old embryos from control and unc-45 KD and 24B-Gal4 > unc45 RNAi-2 (VDRC)) flies. UNC-45 expression was reduced significantly (\u223c70-80%) in the KD embryos. (B) Myosin content was reduced significantly in the unc-45 KD embryos , however actin content appears to be similar for all groups. Each lane represents the total extracted protein from 20 embryos.(TIF)Click here for additional data file.Figure S2Knock down of unc-45 results in significant reduction in both myosin accumulation and myofibrillar organization. Immunofluorescence micrographs of cardiac tubes from 1 week old flies are shown. Hearts from controls and unc-45 KD flies were probed with antibody against muscle myosin and phalloidin respectively as described in the main test. Control cardiac tubes show typical spiral myofibrillar arrangements within the cardiomyocytes . Myofibrillar organization is completely disrupted in unc-45 KD with loss of most myosin-containing myofibrils and significant dilation (C). Remarkably, even with minimal myosin present, myofibrils still form, albeit in a considerably disorganized fashion, as seen by probing with labeled phalloidin, which binds to filamentous actin (D). Myofibrils within cardiomyocytes are shown with blue arrows in A, B and D. KD of unc-45 leads to loss of most myosin-containing myofibrils in cardiac muscle whereas longitudinal ventral muscle myofibrils (white arrow in C) remain unaffected. All images were taken at 25X magnification.(TIF)Click here for additional data file.Figure S3Cardiac defects associated with unc-45 KD in 4-day old adults. (A) Hearts (4-day old) from unc-45 KD flies show prolonged diastolic and systolic intervals compared to control hearts. (B) Diastolic and systolic diameters of the KD hearts were significantly higher compared to age-matched control hearts. (C) Cardiac contractility of the unc-45 KD hearts was significantly reduced and significant cardiac arrhythmia was observed. Mean values \u00b1 SD are shown. Statistical differences between control and unc-45 KD hearts were determined using an unpaired Student's t test (***\u200a=\u200a p<0.001).(TIF)Click here for additional data file.Figure S4Cardiac defects associated with unc-45 KD in third instar larvae. Comparison of cardiac diameter (diastolic and systolic (A) and (B), respectively) and cardiac efficiency ) in control and unc-45 KD hearts from third instar larvae. Both diastolic and systolic cardiac diameters were significantly increased in unc-45 KD larvae hearts. Cardiac performance of unc-45 KD third instar larvae were significantly reduced compared to age matched controls. Statistical differences between control and unc-45 KD hearts were determined using an unpaired Student's t test (*\u200a=\u200a p<0.05).(TIF)Click here for additional data file.Figure S5Scheme for transgenic over-expression of unc-45 to rescue defects associated with unc-45 KD. Genetic crosses using multiple balancers were carried out to rescue cardiac phenotypes associated with unc-45 KD. unc-45 RNAi and a cardiac driver transgenes are inserted in the second chromosome and the transgenic unc-45 gene described in the scheme is inserted in the fourth chromosome. Transgenic unc-45 inserted in the X-chromosome was also used to rescue the cardiac phenotype associated with unc-45 KD.(TIF)Click here for additional data file.Figure S6Transgenic over-expression of unc-45 partially rescues defects associated with unc-45 KD. As for one week-old flies .(TIF)Click here for additional data file.Figure S7Transgenic over-expression of unc-45 partially rescues lethality associated with unc-45 KD. Cardiac-specific KD of unc-45 results in a decrease in mean life span . This reduced lifespan was partially rescued by transgenic over-expression of UNC-45, as only \u223c15% of flies are dead in 3 weeks. The average of a total of 250 flies from three experiments was determined for each group.(TIF)Click here for additional data file.Movie S1Control and unc-45 KD adult beating hearts. Representative movies of 1 week old Drosophila hearts; the first clip shows a typical regularly beating heart. The following clip shows the irregular beating pattern observed in response to unc-45 KD in the heart. This unc-45 KD heart also exhibits the significant dilation and the reduction in contractility that is typical of these KD hearts.(MOV)Click here for additional data file.Movie S2Control and unc-45 KD white pupae beating hearts. Movie clips of white pupae (young) showing contractions in control hearts and unc-45 KD hearts . Note increased heart rate in response to unc-45 KD.(MOV)Click here for additional data file.Movie S3Adult beating hearts that had unc-45 KD before or after metamorphosis. Movie clip of 1 week old adult fly hearts where unc-45 KD was carried out either before or after metamorphosis (following 10s) using the TARGET system and the heart specific TinC\u03944-Gal4 driver.(MOV)Click here for additional data file.Movie S4Control, unc-45 KD and rescued adult beating hearts. Movies of control , unc-45 KD and unc-45 KD rescued with transgenic over-expression of UNC-45 . The compromised beating pattern and dilation seen in the unc-45 KD are greatly reduced in the rescued heart.(MOV)Click here for additional data file."} +{"text": "Cytotoxic T lymphocytes (CTLs) play an important role in the control of HIV-1. CTL responses to HIV-1 have been well studied in HIV-1 clade B-infected and clade C-infected individuals. However cross-clade CTL recognitions have not been well analyzed. In this study, we analyzed cross-clade CTL recognition for clade B and A/E viruses in A/E virus-infected Japanese individuals.PBMC samples were collected from chronically HIV-1 infected Japanese cohort in NCGM. Twenty-six clade A/E-infected individuals were analyzed by ELISPOT assay using the 11-mer overlapping peptides and then the responses of CTLs to these peptides was compared to those from 402 clade B-infected Japanese individuals. Thereafter CTL responses to each single peptide and to truncated peptides were evaluated by ELISPOT assay and intracellular cytokine staining (ICC) assay, respectively.Similar level of CTL responses to Gag, Pol and Nef were found in clade A/E-infected individuals as compared to that in clade B-infected ones. We identified 15 cross-clade CTL epitopes from 14 cocktails where the frequency of responders was high in clade A/E infected samples. The sequences of 7 epitopes were conserved between clade B and clade A/E viruses, whereas 8 epitopes showed different amino acid sequences between two viruses. In these 8 epitope regions, we confirmed cross-clade CTL recognition by ICC assay using clade A/E consensus sequence peptide.Cross-clade CTLs were predominantly induced in clade A/E-infected individuals by clade B consensus sequence peptides in this study. Moreover, CTL responses were induced not only in conserved region but also in different sequence region between the 2 viruses, indicating that polymorphic sequence epitopes among clades can be also candidate for the target of CTL-based vaccines. Further analysis of cross-clade CTL recognition is needed for the widely applicable vaccine development."} +{"text": "This reference should have been numbered 12 and as a result of this error all subsequent references and citations were incorrectly labeled. Please refer to this document for correct in-text citations and updated references: The additional reference is: Yazdanbakhsh M (1999) Common features of T cell reactivity in persistent helminth infections: lymphatic filariasis and schistosomiasis. Immunol Lett 65: 109-115. S0165-2478(98)00133-3 [pii]."} +{"text": "Breakpoint cluster region (Bcr) is a multi-domain protein that contains a C-terminal GTPase activating protein (GAP) domain for Rac. Transglutaminase 2 (TG2) regulates Bcr by direct binding to its GAP domain. Since TG2 has transglutaminase activity that has been implicated in the response to extreme stress, we investigated if Bcr can also act as a substrate for TG2.We here report that activation of TG2 by calcium caused the formation of covalently cross-linked Bcr. Abr, a protein related to Bcr but lacking its N-terminal oligomerization domain, was not cross-linked by TG2 even though it forms a complex with it. A Bcr mutant missing the first 62 amino acid residues remained monomeric in the presence of activated TG2, showing that this specific domain is necessary for the cross-linking reaction. Calcium influx induced by a calcium ionophore in primary human endothelial cells caused cross-linking of endogenous Bcr, which was inhibited by the TG2 inhibitor cystamine. Treatment of cells with cobalt chloride, a hypoxia-mimetic that causes cellular stress, also generated high molecular weight Bcr complexes. Cross-linked Bcr protein appeared in the TritonX-100-insoluble cell fraction and further accumulated in cells treated with a proteasome inhibitor.Bcr thus represents both an interacting partner under non-stressed conditions and a target of transglutaminase activity for TG2 during extreme stress. The breakpoint cluster region (Bcr) protein was originally identified as the amino-terminal part of a fusion protein including the Abl tyrosine kinase, which causes chronic myeloid leukemia and Ph-chromosome-positive acute lymphoblastic leukemia. The fusion of Bcr to Abl deregulates the tyrosine kinase activity of Abl . AlthougBCR gene encodes a multidomain protein. Apart from the oligomerization domain, it additionally contains serine/threonine protein kinase, tandem DH-PH, C2 and GTPase activating protein (GAP) domains. The latter domain has a relatively well-described function: it down-regulates the activated GTP-bound conformation of the small G-protein Rac in vitro ), followed by permeabilization in 0.2% Triton X-100 . Cells were blocked in 1% bovine serum albumin in PBS and stained with TG2 antibodies and Bcr antibodies in blocking solution overnight at 4\u00b0C, followed by incubation with Cy3-conjugated anti-mouse IgG and FITC-conjugated anti-rabbit IgG antibodies . After mounting in Vectashield containing 4',6'-diamidino-2-phenylindole , cell images were acquired with a Zeiss 710 confocal microscope.HPAECs were grown on fibronectin-coated coverslips (Fisher Scientific) for 1-2 days and then treated with A23187 or CoClBcr: breakpoint cluster region; TG2: transglutaminase 2; GAP: GTPase activating protein; HPAECs: human pulmonary artery endothelial cells; PMSF: phenylmethylsulfonyl fluoride; RT: room temperature; DAPI: 4',6'-diamidino-2-phenylindole; DMSO: dimethyl sulfoxide.SY participated in the study design, performed all experiments described here and wrote a draft of the manuscript. JG contributed ideas for experiments; NH participated in study design, provided ideas for experiments and wrote the manuscript. All authors read and approved the final manuscript."} +{"text": "Aim of this study was to identify new miR-221 gene targets to improve our understanding on the molecular tumor-promoting mechanisms affected by miR-221. Gene expression profiling of miR-221-transfected-SNU-398 cells was analyzed by the Sylamer algorithm to verify the enrichment of miR-221 targets among down-modulated genes. This analysis revealed that enforced expression of miR-221 in SNU-398 cells caused the down-regulation of 602 mRNAs carrying sequences homologous to miR-221 seed sequence within their 3\u2032UTRs. Pathways analysis performed on these genes revealed their prominent involvement in cell proliferation and apoptosis. Activation of E2F, MYC, NFkB, and \u03b2-catenin pathways was experimentally proven. Some of the new miR-221 target genes, including RB1, WEE1 (cell cycle inhibitors), APAF1 (pro-apoptotic), ANXA1, CTCF , were individually validated as miR-221 targets in SNU-398, HepG2, and HEK293 cell lines. By identifying a large set of miR-221 gene targets, this study improves our knowledge about miR-221 molecular mechanisms involved in tumorigenesis. The modulation of mRNA level of 602 genes confirms the ability of miR-221 to promote cancer by affecting multiple oncogenic pathways. In vitro studies showed that miR-221 caused an increase in cell proliferation rate and invasion capability, while anti-miR-221 induced a decrease in cell growth and promoted apoptosis . Genes were ordered according to fold-change, from the most down-regulated to the most up-regulated in miR-221 transfected cells and the ordered gene list was analyzed with the Sylamer algorithm (EMBL-EBI), through the web-interface SylArray (http://www.ebi.ac.uk/enright-srv/sylarray) , Diana MicroT v.3 (http://diana.cslab.ece.ntua.gr/microT/) treated SNU-398 samples were performed using Agilent Whole Human Genome Oligo Microarray platform (Agilent Technologies), following manufacturer's procedures, as previously described of putative target genes into psiCheck-2 vector (Promega), downstream of the renilla luciferase gene, using XhoI and PmeI restriction sites. The primers used to amplify 3\u2032UTR regions and the lengths of cloned regions are listed in Table pMIF-GFP-miR-221 was prepared by cloning hsa-miR-221 gene in the pMIF-GFP-Zeo plasmid (System Biosciences), using the SNU-398, HepG2, and Hek293, from American Type Culture Collection, were cultured in Iscove Modified Dulbecco's Medium (IMDM) supplemented with 10% fetal calf serum (Sigma-Aldrich) and 0.1% Gentamicin (Sigma-Aldrich).miR-221 precursor (AM17100\u2014PM10337) and negative control #2 (NC2\u2014AM17111) were obtained from Ambion and were transfected at the final concentration of 100 nM. The anti-miR-221 oligonucleotide was synthetized by Integrated DNA Technology (IDT) and it has the following sequence: 5\u2032-mG*mA*mA mAmCmC mCmAmG mCmAmG mAmCmA mAmUmG mU*mA*mG* mC*mU-3\u2032 . It was transfected at the final concentration of 100 nM.For miRNA precursor transfection, cells were seeded in 24-well plates at a density of 100,000 cells/well, 24 h before transfection. Transfection was performed according to Lipofectamine2000 protocol (Invitrogen), using Optimem medium (Invitrogen-Gibco). Luciferase vectors (psiCheck-based vectors) were transfected at the final concentration of 800 ng/ml. Each transfection was performed in triplicate. For RNA extraction, cells were collected 48 h after transfection and the RNA was extracted following Trizol protocol (Invitrogen). For luciferase assays, firefly and renilla luciferase activity were measured using the Dual-Luciferase Reporter Assay (Promega), 24 h after transfection. The firefly luciferase activity was used to normalize the reporter renilla luciferase signal.To experimentally investigate the involvement of miR-221 in cancer processes, we used the Cignal Finder 10-Pathway Reporter Arrays kit (SABiosciences), a commercial reporter array that allows for simultaneous evaluation of 10 cancer-related signaling pathways activation. It is a reverse transfection system that makes use of specific pathway-focused transcription factor-responsive firefly luciferase vectors. Ten thousand cells were seeded in wells containing the responsive and normalization vectors and luciferase activities were measured after 48 h. Data normalization was based on an included vector that expresses the renilla luciferase gene under the control of the strong cytomegalovirus (CMV) promoter.SNU-398 cells were transfected with pMIF-GFP-miR-221 plasmid. After 24 h from transfection cells were diluted and cultured in IMDM Medium supplemented with 10% fetal calf serum (Sigma-Aldrich), 0.1% Gentamycin (Sigma-Aldrich) and 400 \u03bcg/ml of zeocin. After 4 weeks, single colonies were picked up and miR-221 expression was evaluated using Real Time PCR.miR-221 probe (Applied Biosystems). Five nanogram of purified RNA were retro-transcribed using TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems) and mature miR-221 MicroRNA Assay , following manufacturer's protocol. Real Time quantitative PCR was performed using TaqMan MicroRNA Assay specific for hsa-miR-221 and for hsa-miR-222 . The reaction was carried out in a 96-well PCR plate at 95\u00b0C for 10 min followed by 40 cycles of 95\u00b0C for 15 s and 60\u00b0C for 1 min on Biorad-Chromo4 thermal cycler real-time PCR instrument. Each sample was analyzed in triplicate. The level of miRNA was measured using Ct (threshold cycle) and the amount of target was calculated using 2\u2212\u0394\u0394Ct method. To normalize the expression levels of miR-221, TaqMan endogenous control RNU6B was used. For gene expression analysis we performed Real Time EvaGreen PCR. Five hundred nanogram of total RNA were retro-transcribed using random examers and oligo dT. Diluted cDNAs were amplified in Real Time PCR using Qiagen Taq DNA Polymerase for EvaGreen detection. The following primers were used: RB1_2612F (TCA GAA GGT CTG CCA ACA CCA ACA), RB1_2744R (TGA GCA CAC GGT CGC TGT TAC ATA); APAF1_1046F (GGC TGT GGG AAG TCT GTA TTA G), APAF1_1195R (CAA CCG TGT GCA AAG ATT CTG); ANXA1_835F (TGG AGT TGA AAG GTG ACA TTG), ANXA1_982R (CGG GAA ACC ATA ATC CTG ATC); WEE1_1994F (CGA ATA GAA TTG AAT GCC GAA AAG), WEE1_2142R (GAT GTT CTA TTA CTC TGG GTG G); CTCF_1499F (AGT GTT CCA TGT GCG ATT AC), CTCF_1654R (GGG TTC TCA TGT GCC TTT TC); GAPDH_1107F (CTA TAA ATT GAG CCC GCA GCC), GAPDH_1257R (CCC AAT ACG ACC AAA TCC GT); 18S_616F (AGC AGC CGC GGT AAT TCC AGC T), 18S_784R (CGG GAC ACT CAG CTA AGA GCA TC). The reactions were incubated in a 96-well PCR plate at 95\u00b0C for 15 min followed by 40 cycles of 95\u00b0C for 30 s and 58\u00b0C for 30 s. Each sample was analyzed in triplicate. Fluorescence measurements were completed using a Biorad-Chromo4 thermal cycler real-time PCR instrument. The level of each mRNA was measured using Ct (threshold cycle) and the amount of target was calculated using 2\u2212\u0394\u0394Ct method. Gene expression levels were normalized using either 18S or GAPDH expression.The RNA purification by Trizol was performed according to manufacturer's indications (Invitrogen). For mature microRNA quantification we performed a Taqman Real time PCR, using http://rsbweb.nih.gov/ij/) and protein expression levels were normalized according to \u03b2-actin expression.Cells were collected by trypsin-EDTA and dissolved in RIPA buffer (Radio-Immunoprecipitation Assay) (Sigma-Aldrich), supplemented with Protease inhibitors (Sigma-Aldrich), according to Manufacturer's protocol. The following antibodies were used: anti-human Retinoblastoma Protein , anti-APAF1 , anti-ANXA1 , anti-WEE1, , monoclonal anti-actin , HRP-linked anti-mouse antibody , HRP-linked anti-rabbit antibody . For signal detection LiteAblot Turbo Extra-Sensitive Chemiluminescent Substrate (Euroclone) was used. Signals were quantified by the ImageJ software . This cell line was chosen because it expresses low level of miR-221 enrichment for miR-221 target sequences was detected in the 3\u2032UTRs of many of the down-regulated genes , Figure . All inchttp://www.genego.com/, August 2012), 10 could not be evaluated because their expression was undetectable in SNU-398 cells. Among the remaining 25 genes, 14 were identified by Sylamer within the list of the 1800 most down-regulated genes; five , although still down-regulated, were outside the selected cut-off and six were found slightly up-regulated Table . We cannThe published gene targets detected by Sylamer contained at least 1 or 2 complementary sites for the 7-mer seed within their 3\u2032UTRs. As expected, with the exception of RALGAPA1 (or GARNL1), THRB and Connexin43, the remaining 10 proven gene targets were predicted by at least one of the online programs MicroCosm, Targetscan or Diana microT, indicating that presently known gene targets were largely identified through an initial scanning using available online predictions.We intersected the 602 genes that emerged from Sylamer analysis with genes predicted by three online algorithms . Overall, 125 genes identified by Sylamer (19.9%) were also predicted by online algorithms Table . This seWe investigated the biological processes associated with the genes that emerged from Sylamer analysis: the 602 genes with at least one site matching the seed sequence of miR-221 were analysed for detecting their association with molecular pathways through the use of Genespring GX 11 (Agilent Technologies) and GeneGO (Thomson Reuters) programs.p-value cut-off of 0.05, Genespring and GeneGO found several pathways that were enriched in genes targeted by miR-221 , a commercial reporter array that allows for simultaneous evaluation of 10 cancer-related signaling pathways activation levels in cells, using specific pathway-focused transcription factor-responsive firefly luciferase constructs. This kit was used to evaluate the ability of miR-221 to induce cancer-related pathways in the SNU-398/miR221 clone 2 cells, engineered to stably express increased levels of miR-221 Figure . We founFrom the list of genes identified by Sylamer and the list of pathways significantly affected by miR-221, we focused our attention on genes involved in cell proliferation and apoptosis regulation to individually validate them as miR-221 targets. They included the cell cycle regulators retinoblastoma 1 (RB1) and WEE1, the pro-apoptotic gene Apoptotic Peptidase Activating Factor 1 (APAF1), CCCTC-binding factor (CTCF), a transcriptional repressor and Annexin A1 (ANXA1). In addition, we also investigated Fas Ligand (FASLG), which contains a miR-221 target region in its 3\u2032UTR, but was not within the Sylamer-derived-602 genes list. The genes studied are listed in Table To validate potential miR-221 target genes, we first performed luciferase assays. We cloned a portion of the 3\u2032UTRs containing miR-221 target sequences into a psiCheck-2 reporter vector, downstream the luciferase reporter gene. We assayed the luciferase activity in the presence or absence of added miR-221 mimics in three different cell lines: HEK-293 (embryonic kidney derived cells) and two hepatocarcinoma derived cells . The reporter vector constructs were transfected into cells together with miR-221 precursor or negative control (NC2). We found that miR-221 induced a significant decrease in luciferase activity of all the vectors containing 3\u2032UTRs of genes identified through the Sylamer approach. Instead, no change in luciferase activity could be detected for the psiCheck-FASLG 3\u2032UTR vector Figure . In the p-value <0.003) of RB1 mRNAs in both cell lines transfected with miR-221 (\u221264% in SNU-398 and \u221272% in HepG2); similarly, we found 26 and 42% decrease in APAF1 mRNA level, respectively, in SNU-398 and HepG2 ; miR-221 caused a decrease of 38% (SNU-398) and 77% (HepG2) of ANXA1 mRNA ; WEE1 mRNA was 44% (SNU-398) and 61% (HepG2) less expressed in presence of microRNA and CTCF mRNA showed a 38% decrease in miR-221 transfected SNU-398 cells , while in HepG2 we found a slight (but not significant) decrease , further confirming Anxa1 as target of miR-221 regulation. APAF1 was recently independently demonstrated as miR-221 target in lung cancer derived-cell lines (Quintavalle et al., Pathway analysis of the 602 putative miR-221 target genes revealed that these genes are involved in cellular processes related to cell cycle regulation and apoptosis. Experimental tests revealed that various pathways, which included WNT/\u03b2-catenin, E2F/RB cell cycle, MYC and NFkB, may be promoted by miR-221 over-expression.in vitro (Galardi et al., in vivo models (Pineau et al., in vivo models also by showing the anti-tumor effect achieved by anti-miR-221 oligonucleotides: anti-miR-221 could significantly inhibit tumor cell proliferation of human HCC xenografts (Park et al., Previous experiments demonstrated that miR-221 was able to induce tumor cells proliferation, both This work, by showing that the Sylamer algorithm could reveal the enrichment for a miRNA seed sequence among a group of down-regulated mRNAs, represents a general approach that could be applied to any miRNA for the experimental identification of a wide range of gene targets. In the case of miR-221, the list of modulated mRNAs was used to improve our understanding of its role in tumor pathways. However, these results may potentially be useful for defining the molecular mechanisms that are influenced in any other physiological or pathological condition in which miR-221 could be eventually implicated.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "TRE-controlled transgenes. We show that these transgenic strains can be effectively combined with established mouse models of disease, including both Cre/LoxP-based approaches and non Cre-dependent disease models. The integration of these new tools with established mouse models promises the development of more flexible genetic systems to uncover the mechanisms of development and disease pathogenesis.Tetracycline or doxycycline (dox)-regulated control of genetic elements allows inducible, reversible and tissue specific regulation of gene expression in mice. This approach provides a means to investigate protein function in specific cell lineages and at defined periods of development and disease. Efficient and stable regulation of cDNAs or non-coding elements (e.g. shRNAs) downstream of the tetracycline-regulated element (TRE) requires the robust expression of a tet-transactivator protein, commonly the reverse tet-transactivator, rtTA. Most rtTA strains rely on tissue specific promoters that often do not provide sufficient rtTA levels for optimal inducible expression. Here we describe the generation of two mouse strains that enable Cre-dependent, robust expression of rtTA3, providing tissue-restricted and consistent induction of Genetically engineered mouse models (GEMMs) are an invaluable tool to investigate the biology of development and disease in a mammalian organism. Since the development of the first knockout mouse almost 25 years ago, a wide variety of knockout, knock-in and conditional mutant strains have been developed to interrogate gene function TRE) that regulates gene or shRNA expression, and a trans-acting, tet-sensitive, tet-transactivator (tTA) or reverse tet-transactivator (rtTA) protein TRE promoters, but is inhibited in the presence of tet, or its more common analog, doxycycline (dox). Conversely, rtTA promotes dox-dependent gene induction. Early versions of the rtTA protein showed \u2018leaky\u2019 gene expression in the absence of dox, but newer variants such as rtTAM2 and rtTA3 http://www.tetsystems.com/fileadmin/tettransgenicrodents.pdf). As expression of the TRE-regulated cassette is dependent on both the presence of tet-transactivator and dox, induction can be ubiquitous or tissue specific (by controlling tTA/rtTA expression), inducible and reversible (by controlling dox exposure). Tissue specific TRE gene regulation is usually achieved by restricting the expression of tTA or rtTA to defined cell lineages using a tissue-specific promoter. Though convenient, this approach is absolutely dependent on robust expression of the tTA/rtTA. Moreover, cellular response downstream of transgene/shRNA induction may alter cell fate and compromise sustained TRE-regulated control. We recently described a new reverse tet-transactivator strain, CAGs-rtTA3, that shows stronger and more ubiquitous induction of TRE-regulated elements than any other existing strains we have tested TRE-induction seen with CAGs-rtTA3 and enabling tissue specific TRE-regulation.The tetracycline (tet) system is, by far, the most widely used inducible model in mice. It consists of two essential components: a tetracycline-responsive element cassette (CAGs-LSL-rtTA3). In addition, we cloned a variant that also carried the mKate2 far-red fluorescent gene Internal ribosomal entry site (IRES) (CAGs-LSL-rtTA3-IRES-mKate2 \u2013 CAGs-LSL-RIK) , which showed homozygous targeting to the Rosa26 locus. We further confirmed targeting on both copies of chromosome 6 in this ESC line by fluorescence in-situ hybridization (FISH) . In thisn (FISH) and lateCAGs-LSL-rtTA3 (designated Y1) and CAGs-LSL-RIK (D34) cells showed robust expression of rtTA3 protein and therefore strong induction of TRE-regulated transgenes. To do this we took advantage of a recombinase-mediated cassette exchange (RMCE) \u2018landing pad\u2019 downstream of the col1a1 locus in C10 ESCs TRE-regulated (and other) transgenes, including cDNAs col1a1 RMCE targeting vector we previously described that expresses GFP and an shRNA directed against Renilla luciferase (Ren.713) downstream of TRE (TG-Ren.713). Throughout this study we use the TG-Ren.713 transgene as a neutral fluorescent marker of dox-mediated induction. Following hygromycin selection, individual clones were expanded and transduced with a limiting titer of adenovirus expressing Cre recombinase to achieve recombination in 10\u201325% of cells. As expected, adenovirus transduced cells treated with doxycycline showed strong induction of GFP , demonstrating that both Y1 and D34 are robust ESC lines that can serve as a platform for the production of conditional, TRE-inducible mice for analysis.To confirm the quality of Y1 and D34 ESCs for animal production, we generated wholly ESC-derived mice by tetraploid embryo complementation and bred multiple founder animals. Each cell line produced numerous viable and fertile mice that showed expected Mendelian transmission of the d allele . MoreoveRosa26 promoter (R26-rtTA) results in restricted induction of TRE-driven GFP in adult mouse tissues CAGs-rtTA3 transgenes targeted to the Rosa26 locus, we crossed each strain to a CAGs-Cre \u2018deletor\u2019 mouse that induces LoxP recombination at or before the two-cell stage LSL cassette in the F1 progeny from each strain was confirmed by PCR and each of the recombined alleles could be propagated through breeding at Mendelian ratios, indicating there was no appreciable toxicity from the CAGs-driven expression of rtTA3 or mKate2 in vivo can drive strong and widespread expression of rtTA3, allowing dox-dependent TRE induction in almost all tissues. Importantly, this implies that in combination with an appropriate Cre driver, both CAGs-LSL-rtTA3 and CAGs-LSL-RIK strains can provide robust, tissue and/or cell-type specific TRE-mediated gene expression.Importantly, both CAGs-driven strains showed almost identical GFP expression indicating that the presence of mKate2 in the CAGs-LSL-rtTA3 and CAGs-LSL-RIK mice with Adenoviral-Cre (AdenoCre) and treated mice with dox for one week. As expected, AdenoCre, dox-treated CAGs-LSL-rtTA3 and CAGs-LSL-RIK mice showed mosaic expression of GFP in the liver (Tissue restricted Cre recombinase expression can be achieved in mice through the delivery of virus (adeno- or lentivirus) to specific organs. Intravenous (tail-vein) injection of adenovirus results in almost exclusive transduction of liver hepatocytes. As a first step to evaluate Cre-mediated, tissue specific TRE-induction we injected he liver . ImportaG12DG12D expressing lung epithelium we treated G12D/+;TG-Ren.713/+;CAGs-LSL-RIK/+LSL-Kras mice with AdenoCre (5\u00d7106 PFU) via intratracheal injection and treated with dox for one week prior to analysis. Three months following AdenoCre treatment animals showed small KrasG12D-driven adenomas throughout the lung epithelium mutation is considered an initiating and driving event in most pancreatic ductal adenocarcinomas. In the mouse, pancreatic restricted expression of KrasERBB2 gene) is a common feature of human breast cancer. In the mouse, this event can be mimicked via expression of a mutant rat ortholog V664Eneu allele downstream of a mouse mammary tumor virus (MMTV) promoter V664EMMTV-neu model has been used extensively to investigate the genetics and physiology of breast cancer progression. Expression of neuV664E in this model is not dependent on the activity of Cre recombinase, therefore we asked whether our Cre-dependent CAGs-LSL-RIK allele could be used effectively in combination with V664EMMTV-neu to express inducible shRNAs in the mammary gland and drive tumorigenesis. For this, we generated female V664E/+;CAGs-LSL-RIK/+;TG-Ren.713/+MMTV-neu animals also carrying the murine whey acidic protein gene promoter (WAP)-driven Cre transgene which responds to lactogenic hormones WAP-Cre and were treated with doxycycline to induce shRNA expression. These mice showed luminal epithelial expression of mKate2 and GFP .Overexpression of HER2/Neu and thus, tissue-restricted induction of TRE-controlled transgenes. Further, we show that these transgenic strains can be effectively combined with established mouse models of disease, including Cre/LoxP-based approaches and Cre-independent model systems. The integration of established models of disease with the flexibility of inducible and reversible gene regulation will allow a more detailed interrogation of the underpinnings of disease pathogenesis and evolution. For instance, model systems that incorporate constitutive and inducible genetic alterations with inducible and reversible gene silencing (or overexpression) offer the unique opportunity to investigate how the temporal order of events determines disease progression and whether those events are required for disease maintenance. Such work will ultimately lead to better understanding of disease and the development of more accurate and effective preclinical models.http://www.tetsystems.com/fileadmin/tettransgenicrodents.pdf) our approach integrates the use of established tissue-restricted Cre recombinase strains to initiate TRE induction. Previous studies have reported the generation of Cre-dependent tTA and rtTA strains driven by the endogenous Rosa26 promoter Rosa26 promoter activity can vary significantly in different tissues of adult mice, resulting in sub-optimal TRE induction in a range of cell types CAGs promoter provides robust TRE induction in most adult tissues in individual crypts and villi of the small and large intestine .In contrast to strategies that rely on tet-transactivators driven by tissue specific promoters , which did not show expression of mKate2, suggesting Cre-induced recombination of only a subset of \u2018floxed\u2019 genes in the genome. Because KrasG12D-driven phenotypes are not 100% penetrant in either tissue, we have not been able to measure the frequency of cells that show recombination only at the CAG-LSL-RIK allele and not G12DKras. The reasons behind this incomplete recombination are not clear but may reflect the regions surrounding the LoxP sites. Of note, we have observed increased recombination efficiency of the CAGs-LSL-rtTA3 allele compared to the CAGs-LSL-RIK strain in cases of low or transient Cre expression; the two alleles vary slightly in sequences close to the 3\u2032 LoxP site due to alternate cloning strategies. It is likely that incomplete LoxP recombination is a feature of many complex, Cre-dependent models, but it goes undetected due to a lack of reporter-based approaches. The presence of mKate2 as a Cre reporter in CAGs-LSL-RIK provides a means to clearly identify cells and tissues that express rtTA3 and are capable of inducing shRNA or transgene expression, irrespective of fluorescent tags linked to such transgenes (e.g. GFP). Thus, it enables tracking and/or prospective isolation of Cre-recombined cells prior to induction of, and post-withdrawal of, TRE-driven transgene/shRNA expression.During our analysis of multi-allelic animals carrying CAGs-LSL-rtTA3 (Y1) or CAGs-LSL-RIK (D34) as well as the col1a1 homing cassette for RMCE. Thus Y1 and D34 cells could be employed by investigators wanting to fast track analysis of a gene or genes in a setting where Cre recombinase is delivered extrinsically or used as a base ESC line for the introduction of additional genetic manipulations, such as the incorporation of tissue specific Cre knock-in alleles. Alternately these alleles could be incorporated into ESC-GEMMs through re-derivation of new ESC lines. In fact, we have recently validated the use of this approach by producing a number of KrasG12D-based pancreatic cancer models, using CAGs-LSL-RIK to drive pancreas-specific expression of positive and negative regulators of tumor initiation and progression We and others recently described a conceptually new approach to complex mouse modeling, based on the derivation and manipulation of conditional, multi-allelic embryonic stem cells (ESCs), which we use to generate tailored genetic models for analysis in vivo, tet-based systems for manipulation of gene expression by providing robust tools for tissue-restricted induction of transgenes and shRNAs. Integrating these new strains with existing mouse modeling platforms promises to provide a wealth of new discoveries by unearthing the details of gene function in all stages of development and throughout the pathogenesis of disease.Together, the ESCs and mouse strains described here bolster the already impressive arsenal of 7) were electroporated with 50 ug linearized targeting vector using a BioRad Gene Pulser and plated in M15 media as previously described All ES cells were maintained on irradiated feeders in M15 media containing LIF as previously outlined in vivo, CAGs-LSL-rtTA3 and CAGs-LSL-RIK (tm1(CAGs-LSL-rtTA3)Slo and R26Sortm2(CAGs-LSL-RIK)SloR26Sor) mice were crossed to the CAGs-Cre transgenic strain and F1 progeny were genotyped for LoxP recombination using specific primers . For removal of the \u2018LSL cassette\u2019 mers see . TG-Ren.Production of mice and all treatments described were approved by the Institutional Animal Care and Use Committee (IACUC) at McGill University or Memorial Sloan Kettering Cancer Center (NY) under protocol numbers: 2001\u20134751 (McGill), 11-06-012, 11-06-015 and 12-04-006 (MSKCC).For adenoviral delivery to the liver: 5\u00d7108 PFU AdenoCre/mouse was injected intravenously via the tail vein. For adenoviral delivery to the lung: 6\u201310 week-old mice were anesthetized by i.p. injection of ketamine 80 mg/kg, xylazine 10 mg/kg 6 PFU AdenoCre/mouse. Three months following AdenoCre treatment, lungs were collected, fixed and analyzed by immunofluorescence or immunohistochemistry.Doxycycline was administered via food pellets (625 mg/kg) (Harlan Teklad). Adenovirus expressing Cre recombinase (AdenoCre) was purchased from The University of Iowa Gene transfer Core. rtTA3: F: 5\u2032-CAATGGTGTCGGTATCGAAG-3\u2032, R: 5\u2032-CTTGTTCTTCACGTGCCAGT-3\u2032; mKate2: F: GGTGAGCGAGCTGATTAAGG-3\u2032 and R: 5\u2032-TTTTGCTGCCGTACATGAAG-3\u2032; and GFP: F: 5\u2032-ATCGACTTCAAGGAGGACGGCA-3\u2032 and R: 5\u2032-CGTTCTTCTGCTTGTCGGCCAT-3\u2032.Protein lysates were prepared in RIPA buffer and quantified by Lowry assay (BioRad). Western blots were probed with antibodies against: TetR (rtTA3) , GFP , tRFP (mKate2) and \u03b2-Actin-HRP . RNA was prepared from sorted cells by Trizol extraction and column purification. cDNA was prepared from 1 \u00b5g total RNA using Taqman reverse transcription kit with random hexamers. Quantitative PCR detection was performed using SYBR green reagents (Applied Biosystems) using primers specific to Immunostaining and FACS analysis for blood lineages were performed as previously described 2O2 for 10 mins and blocked in TBS containing 1% BSA. For immunofluorescence, sections were not treated with peroxidase. Primary antibodies, incubated at 4C overnight in blocking buffer, were: chicken anti-GFP , rabbit anti-tRFP and rabbit anti-ki67 . For immunohistochemistry, sections were incubated with anti-rabbit ImmPRESS reagent and developed using ImmPACT DAB according to the manufacturer instructions. For immunofluorescent stains, secondary antibodies were applied for 1 hour at room temp in TBS in the dark, washed twice with TBS, counterstained for 5 mins with DAPI and mounted in ProLong Gold . Secondary antibodies used were: anti-chicken 488 and anti-rabbit 568 . Images of fluorescent and IHC stained sections were acquired on a Zeiss Axioscope Imager Z.1 using a 10x (Zeiss NA 0.3) or 20x (Zeiss NA 0.17) objective and an ORCA/ER CCD camera . Raw.tif files were processed using Photoshop CS5 software to adjust levels and/or apply false coloring.Tissue, fixed in 10% neutral buffered formalin for 24 hours, was embedded in paraffin and sectioned by IDEXX RADIL . Sections were rehydrated and unmasked by heat treatment for 5 mins in a pressure cooker in 10 mM Tris/1 mM EDTA buffer (pH 9) containing 0.05% Tween 20. For immunohistochemistry, sections were treated with 3% HFigure S1Targeting CAGs-LSL-rtTA3 to the Rosa26 locus.A. Schematic of the Rosa26 locus before and after recombination of the CAGs-LSL-rtTA3 or CAGs-LSL-RIK targeting vector. Key restriction sites used for clone identification by Southern blot are indicated. Sizes of each predicted fragment are also shown and a solid black line highlights the position of the Southern probe. B. Southern blot images showing identification of Y1 (2.3 kb band) and D34 (4.8 kb band) clones, following EcoRV/BglII and EcoRI digests, respectively. C. Fluorescence in situ hybridization on a metaphase spread from D34 ES cells using the CAGs-LSL-RIK fragment as a probe, showing homozygous targeting of CAGs-LSL-RIK to Chromosome 6.(TIF)Click here for additional data file.Figure S2GFP induction following Adenoviral Cre transduction in targeted Y1 and D34 ESCs. Y1 and D34 ESCs carrying TG-Ren.713 at the col1a1 locus were transduced with adenovirus expressing Cre or not transduced , treated with doxycycline (1 ug/ml) for 2 days and analyzed by flow cytometry. Graphs represent bulk population of transduced cells (not single clones). Bulk populations were single cell cloned to assess the uniformity of GFP induction in the presence of constitutive rtTA3 expression (see sion see .(TIF)Click here for additional data file.Figure S3GFP induction and mKate2 expression in large intestine and liver. Immunofluorescence stains for GFP and mKate2 in the large intestine and liver of \u2018no rtTA\u2019, R26-rtTA, CAGs-rtTA3 and CAGs-RIK mice following 1 week of doxycycline treatment. All rtTA strains show strong GFP induction in large intestine (A), but only CAGs-rtTA3 and CAGs-RIK show robust and uniform GFP expression (and mKate2 for RIK) in the liver tissue (B).(TIF)Click here for additional data file.Figure S4Mosaic mKate2 expression in a proportion of lung adenomas. Immunohistochemical stains for mKate2 and Ki67 in lung sections of double transgenic mice treated with intratracheal Adenoviral Cre (AdenoCre) or vehicle (Tris-HCl). 12 weeks following Cre delivery LSL-KrasG12D mice show small, moderately proliferative adenomas. Some adenomas show uniform mKate2 staining (top panel of \u2018AdenoCre\u2019), while a subset showed both positive and negative mKate2 cells (arrows) suggesting Cre-driven activated KrasG12D but not rtTA3-IRES-mKate2. Adenoma area highlighted by dotted line.(TIF)Click here for additional data file.Table S1Genotyping primers. Primer sequences and expected PCR product sizes for genotyping Rosa26-targeted CAGs-rtTA3 strains.(DOCX)Click here for additional data file."} +{"text": "CAPS is a rare autoinflammatory syndrome caused by autosomal dominant mutations in the NLRP3/CIAS 1 gene on chromosome 1q44 encoding for the cryopyrin protein, an important component of the inflammasome, leading to excessive production of interleukin-1beta (IL-1\u00df). CAPS encompasses three different entities of variable clinical severity: familial cold auto-inflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS) and chronic infantile neurological cutaneous articular syndrome (CINCA)/ neonatal \u2013 onset multisystem inflammatory disease (NOMID). They are all characterised by recurrent episodes of systemic inflammation involving particularly skin, joints, central nervous system and eyes.To analyse and quantify various cytokines in sera and cerebrospinal fluid (CSF) in five patients with central nervous system (CNS) manifestations of cryopyrin-associated periodic syndromes (CAPS) carrying the Q703K mutation in heterozygosity.Five Caucasian patients with CNS manifestations including optic nerve inflammation, recurrent cranial nerve palsy, migraine, fatigue, and recurrent meningitis were identified as heterozygous carriers of the cryopyrin Q703K substitution. CSF investigations were performed for diagnostic purposes in all patients and showed pleocytosis in 3 patients. In addition, concentrations of the proinflammatory cytokines interleukin beta (IL-1), interleukin-6 (IL-6), interleukin 17 (IL-17), tumor necrosis factor alpha and FGF (Fibroblast growth factor) were determined in the CSF and sera using a multiplex assay.IL-6 concentrations in the CSF were clearly elevated in two patients during acute attacks of CAPS-associated CNS manifestations. The other three patients were investigated during remission and showed no IL-6 elevations in the CSF. The other serum cytokine levels were increased in one patient.Our results show a correlation between CSF IL-6 concentrations and CAPS-associated disease activity in the CNS. IL-6 levels in the CSF therefore may serve as a marker of disease activity in CAPS patients with CNS manifestations.E. Schuh: None declared, P. Lohse: None declared, M. Frankenberger: None declared, I. Meinl: None declared, T. Kuempfel Grant / Research Support from: Novartis"} +{"text": "Compared to the nonadjuvant group , pSV-OVA-CTB intranasal priming/rTTV-OVA-CTB intramuscular boosting group significantly improved the magnitudes of T-cell responses at spleen , mesenteric LN , draining LNs of respiratory tract and female genital tract . These results collectively demonstrated that fusion-expressed CTB could act as a potent adjuvant to improve both systemic and mucosal T-cell responses.Previous study showed that CTB (Cholera toxin subunit B) can be used as a genetic adjuvant to enhance the systemic immune responses. To further investigate whether it can also be used as a genetic adjuvant to improve mucosal immune responses, we constructed DNA and recombinant Tiantan vaccinia (rTTV) vaccines expressing OVA-CTB fusion antigen. Female C57BL/6 mice were immunized with an intranasal DNA priming/intramuscular rTTV boosting regimen. OVA specific T-cell responses were measured by IFN- DNA vaccines are insufficient to stimulate strong mucosal and systemic immunity when inoculated alone . VariousCholera toxin (CT) is a strong mucosal immunogen as well as an effective adjuvant ; both thAll DNA and recombinant vaccinia virus vaccines were constructed in our previous work. The 6\u20138-week-old female C57BL/6 mice were bred and maintained under specific pathogen-free condition. All animal experiments were reviewed by the Institutional Animal Care and Use Committee (IACUC) of Shanghai Public Health Clinical Center.\u03bcg/mouse) mixed with Turbofect was given intranasally on weeks 0, 2, and 4. And on week 7, mice were boosted intramuscularly with recombinant Tiantan vaccinia vaccine (1 \u00d7 106\u2009pfu/mouse). Two weeks after the final vaccination, mice were euthanized. Vaginal lavage, bronchial alveolar lavage, and serum were collected for the detection of specific antibody response. Spleen, cervical, axillary, iliac, inguinal, and mesenteric lymph nodes were isolated for T-cell response assay.DNA vaccine coated with anti-mouse IFN-\u03b3 antibody at 50\u2009\u03bcL/well (2 \u00d7 105\u2009cells/well). The splenocytes were stimulated with OVA peptide (amino acids 257\u2013264) at the final concentration of 5\u2009\u03bcg/mL. After incubation at 37\u00b0C with 5% CO2 for 20 hours, the ELISPOT plates were developed according to the manufacturer's manual and read with Immunospot Reader .Freshly isolated mouse splenocytes were adjusted to the concentration of 4 \u00d7 10\u03bcg/mL OVA were used for the detection of anti-OVA antibodies (Abs). Serum, bronchial lavage, or vaginal lavage samples were 2-fold serially diluted in PBS containing 5% skimmed milk and 0.5% TWEEN-20. OVA specific IgG and IgA were detected by peroxidase conjugated anti-mouse IgG and anti-mouse IgA, respectively. End point titers were determined by the last dilution, whose OD was beyond or equal to 2-fold that of the corresponding dilution of mice sera immunized with mock control.ELISA plates coated with 2\u2009t-test and comparisons among three or more groups were done by using the method of one-way ANOVA . Significant difference was defined as P \u2264 0.05.Comparisons between two groups were done by the method of unpaired \u03b3 ELISPOT assay. Specific binding antibody in serum was detected by ELISA.Mice were immunized according to the schedule shown in 6 splenocytes for pSV-OVA intranasal priming/rTTV-OVA-CTB intramuscular boosting group and 1562 \u00b1 567\u2009SFCs/106 splenocytes for pSV-OVA-CTB intranasal priming/rTTV-OVA-CTB intramuscular boosting group) than rTTV-OVA boosting groups . OVA speg group) .6 lymphocytes) and pSV-OVA-CTB intranasal priming/rTTV-OVA-CTB intramuscular boosting group was the second (109 \u00b1 60\u2009SFCs/106 lymphocytes). Both were significantly higher than the nonadjuvant group .The mean titer of OVA specific IgA in bronchi alveolar lavage induced by adjuvant groups was lower than the nonadjuvant group. Significant difference was observed between pSV-OVA intranasal priming/rTTV-OVA intramuscular boosting group and pSV-OVA intranasal priming/rTTV-OVA-CTB intramuscular boosting group .6 lymphocytes) were significantly higher than rTTV-OVA boosting groups .No significant difference was observed among the OVA specific IgG titers in vaginal lavage of all groups . OVA spe6 lymphocytes for pSV-OVA intranasal priming/rTTV-OVA-CTB intramuscular boosting group and 96 \u00b1 84\u2009SFCs/106 lymphocytes for pSV-OVA-CTB intranasal priming/rTTV-OVA-CTB intramuscular boosting group) elicited significantly higher T-cell responses than rTTV-OVA boosting groups . in vivo. Majority of previous studies seek to induce mucosal immune responses by mucosal immunization [The mucosae constitute the major portal of entry of infectious agents. An ideal vaccine should induce both systemic and mucosal immune responses in order to block the entry of pathogens and contain infectionsnization \u201311; howenization .In this study, we constructed DNA and recombinant Tiantan vaccinia (rTTV) vaccines encoding OVA-CTB fusion antigen and immunized C57/BL mice in an intranasal DNA priming/intramuscular rTTV boosting regimen. Our data showed that pSV-OVA-CTB priming (i.n.)/rTTV-OVA-CTB boosting (i.m.) elicited the highest magnitude of T-cell responses in spleen (system level). And the genetic adjuvant effect of CTB was more significant for recombinant vaccinia vaccine than for DNA vaccine, since the T-cell responses induced by pSV-OVA priming (i.n.)/rTTV-OVA-CTB boosting (i.m.) were significantly higher than pSV-OVA priming (i.n.)/rTTV-OVA boosting (i.m.) and the T-cell responses elicited by pSV-OVA-CTB priming (i.n.)/rTTV-OVA boosting (i.m.) were only slightly higher than the nonadjuvant group. In contrast, OVA specific IgG responses elicited by adjuvant groups tended to be lower than the nonadjuvant group although no statistical significance was reached.We further tested specific immune responses at different mucosal sites and found that pSV-OVA-CTB priming (i.n.)/rTTV-OVA-CTB boosting (i.m.) consistently raised significant higher cellular immune response than the nonadjuvant group at respiratory, intestinal, and female genital tract, which indicated that the fused-expression of CTB in both DNA and rTTV vaccines is essential for eliciting robust T-cell responses at mucosal sites. Very interestingly, we found that rTTV-OVA-CTB boosting was especially efficient at improving specific T-cell responses in mesenteric lymph node. Besides, similar to the observations of antibody responses in serum, we found that the adjuvant groups tended to induce lower specific IgA titer in both bronchi alveolar lavage and vaginal lavage. in vivo, which may facilitate the uptake and presentation of OVA. This hypothesis was supported by our previous work, in which we found that the adjuvant effect decreased when separating CTB and the antigen into two plasmids . Further experiments will be conducted to confirm the proposed mechanisms and clarify the missing details.When being used as an adjuvant of protein vaccine, CTB can enhance specific immune response through either GM1 receptor-mediated antigen uptake or stimuTaken together, in spite of being short of mechanistic explanation, our data clearly showed that fusion-expressed CTB could serve as a potent adjuvant to enhance both systemic and mucosal T-cell response, not only for DNA vaccine but also for viral vectored vaccine.The fusion expressed CTB could improve specific T cell responses elicited by TRIVN . However, when separating TRIVN and CTB into two DNA vaccines, the genetic adjuvant effect decreased.Click here for additional data file."} +{"text": "O), with the presence of anti-site P on Zn sites (PZn). P-doped ZnO nanowires were high resistance and the related P-doping mechanism was discussed by combining EELS results with electrical measurements, structure characterization and photoluminescence measurements. Our method provides an efficient way of synthesizing P-doped ZnO nanowires and the results help to understand the P-doping mechanism.We developed a novel approach to synthesize phosphorus (P)-doped ZnO nanowires by directly decomposing zinc phosphate powder. The samples were demonstrated to be P-doped ZnO nanowires by using scanning electron microscopy, high-resolution transmission electron microscopy, X-ray diffraction spectra, X-ray photoelectron spectroscopy, energy dispersive spectrum, Raman spectra and photoluminescence measurements. The chemical state of P was investigated by electron energy loss spectroscopy (EELS) analyses in individual ZnO nanowires. P was found to substitute at oxygen sites (P Zn + 2VZn) formed by combination between antisite substitutional P (PZn) and two zinc vacancies (VZn); substitutional P at oxygen sites (PO) direction. Phosphorus incorporated into individual ZnO nanowires, which were directly confirmed by EELS analyses. EELS analyses demonstrated that P incorporated substitutionally at O sites in the as-grown P-doped ZnO nanowires with the presence of P"} +{"text": "Gene expression profile (transcriptome) and glycan profile (glycome) of HTLV-1-infected CD4+T cells may reflect the pathologic cellular mechanism and intercellular recognition, respectively in HAM/TSP though these profiles are still not obtained. To identify responsible cellular genes and relevant glycans of HAM/TSP, we performed experiments with microarray on RNAs and and lectin array on proteins extracted from CD4+Tcells from each four subjects of three goups including HAM/TSP, asymptomatic carriers (AC), and HTLV-1 negative controls (NC). In transcriptome analysis, transcripts of 177 genes were found up-regulated only in HAM/TSP, and those may possibly be causative or resultant genes. In glycome analysis with lectin array carrying 45 species of lectins, standardized signals of UDA and STL which recognize N-glycan (GlcNAc)n were significantly high in samples from HTLV-1 infected groups (HAM/TSP and AC) compared with NC. Interestingly, UDA has been recently reported to inhibit cell-to-cell transmission of HTLV-1 in vitro. These genes and glycans may play roles in the pathogenesis of HAM/TSP."} +{"text": "While passively administered broadly neutralizing monoclonal antibodies (bnmAbs) prevented SHIV acquisition, polyclonal Abs with high neutralizing titers provided only moderate protection in primates.We tested whether passive immunization with polyclonal IgG raised in rhesus monkeys (RMs) with chronic clade C SHIV infection, termed SHIVIG, could protect RMs against multiple low-dose intrarectal challenges with the R5 tier-2 SHIV-2873Nip carrying an HIV clade C envelope heterologous to the viruses/envelopes against which the IgG responses had been elicited. We compared in vitro SHIVIG characteristics with in vivo protection.In vitro, SHIVIG demonstrated binding to SIV Gag, HIV Tat and Env of different clades, contained b12 and 4E10-like Abs and neutralized tier-1 and 2 viruses, including SHIV-2873Nip. NK-cell depletion decreased neutralizing activity in PBMC assays 20-fold. SHIVIG completely inhibited viral replication by ADCVI assay, but showed only 35% target-cell killing by ADCC assay.Four groups of RMs were given SHIVIG at different doses: Group 1 (400 mg/kg), Group 2 (675 mg/kg), Group 3 (25 mg/kg) and Group 4 followed by weekly low-dose challenges with SHIV-2873Nip. All controls and all SHIVIG-treated animals became systemically infected. RMs given 400 mg/kg of SHIVIG showed significantly lower peak viral RNA loads compared to controls. Surprisingly, single-genome analysis revealed a significant increase in the number of transmitted variants in Group 3 compared to controls (P=0.032), suggesting increased acquisition. Complement-mediated Ab-dependent enhancement of infection (C\u2019-ADE) at low SHIVIG concentrations was observed in vitro.Lack of protection and possibly increased acquisition has been reported for a passive immunization study that tested the efficacy of HIV hyperimmune globulin in preventing infection in Ugandan infants born to HIV-positive women . Thus, our primate model data paralleled clinical phase III results and suggest that polyclonal anti-HIV-1 Abs play a dual role upon virus encounter."} +{"text": "Serum autoantibodies against the water channel aquaporin-4 (AQP4) are important diagnostic biomarkers and pathogenic factors for neuromyelitis optica (NMO). However, AQP4-IgG are absent in 5-40% of all NMO patients and the target of the autoimmune response in these patients is unknown. Since recent studies indicate that autoimmune responses to myelin oligodendrocyte glycoprotein (MOG) can induce an NMO-like disease in experimental animal models, we speculate that MOG might be an autoantigen in AQP4-IgG seronegative NMO. Although high-titer autoantibodies to human native MOG were mainly detected in a subgroup of pediatric acute disseminated encephalomyelitis (ADEM) and multiple sclerosis (MS) patients, their role in NMO and High-risk NMO remains unresolved.We analyzed patients with definite NMO (n = 45), HR-NMO (n = 53), ADEM (n = 33), clinically isolated syndromes presenting with myelitis or optic neuritis , MS (n = 71) and controls for serum IgG to MOG and AQP4. Furthermore, we investigated whether these antibodies can mediate complement dependent cytotoxicity (CDC). AQP4-IgG was found in patients with NMO , HR-NMO and in one CIS patient (3%), but was absent in ADEM, MS and controls. High-titer MOG-IgG was found in patients with ADEM , NMO , HR-NMO , CIS , MS and controls . Two of the three MOG-IgG positive NMO patients and all seven MOG-IgG positive HR-NMO patients were negative for AQP4-IgG. Thus, MOG-IgG were found in both AQP4-IgG seronegative NMO patients and seven of 21 (33%) AQP4-IgG negative HR-NMO patients. Antibodies to MOG and AQP4 were predominantly of the IgG1 subtype, and were able to mediate CDC at high-titer levels.We could show for the first time that a subset of AQP4-IgG seronegative patients with NMO and HR-NMO exhibit a MOG-IgG mediated immune response, whereas MOG is not a target antigen in cases with an AQP4-directed humoral immune response. Neuromyelitis optica (NMO), a severe inflammatory demyelinating disorder, has gained increasing interest since the discovery of serum NMO-IgG autoantibodies targeting the aquaporin-4 (AQP4) water channel protein ,2. The din vivo studies demonstrated spontaneous development of NMO-like symptoms with severe opticospinal experimental autoimmune encephalomyelitis (EAE) in a double-transgenic opticospinal EAE (OSE) mouse model expressing T cell and B cell receptors specific for MOG [Recent experimental studies indicated that myelin oligodendrocyte glycoprotein (MOG), a glycoprotein localized on the outer surface of the myelin sheath and oligodendrocytes , might b for MOG ,20. This for MOG . Additio for MOG -23.Whereas in humans anti-MOG antibodies in MS have been extensively investigated, their role in NMO has not been adressed so far. High-titer IgG autoantibodies to conformational epitopes of MOG (MOG-IgG) were detected in a subgroup of pediatric patients with acute disseminated encephalomyelitis (ADEM) and MS, but rarely in adult-onset MS -29. A poTherefore, we decided to investigate the frequency and titer levels of IgG antibodies to MOG and AQP4 in a multicenter study of patients with CNS demyelinating diseases using a live cell staining immunofluorescence assay with HEK-293A cells transfected with either AQP4 or MOG . In addiUsing our assay with M23 AQP4 transfected HEK-293A cells, we detected significantly increased frequencies of serum AQP4-IgG in NMO and HR-NMO in two of 71 MS patients (secondary progressive MS and pediatric MS). Within the CTRL cohort, MOG-IgG was observed in two of 27 SLE patients (1:320 and 1:160) and one of 24 OND patients , while two patients presented with IgG1 and IgG3 antibodies (13%). In contrast to anti-AQP4 autoantibodies, analysis of IgG1-IgG4 isotypes revealed that human serum MOG-IgG antibodies of 15 investigated patients consisted only of the IgG1 isotype.Using our live cell staining immunofluorescence assay (IF) assay, we found that human AQP4-IgG are able to activate the complement cascade at high-titers, leading to the formation of the terminal complement complex (TCC). The resultant TCC was exclusively detected on the surface of AQP4-EmGFP transfected cells Figure . Furtherin vitro in the same manner as shown for AQP4-IgG antibodies. Using MOG transfected cells with and without EmGFP fusion protein, we could clearly show a co-localization of the TCC with MOG-EmGFP , HR-NMO (n = 33), ADEM (n = 19), CIS (n = 14), MS (n = 10) and CTRL (n = 14) based on their ability to initiate MOG-IgG or AQP4-IgG dependent complement activation , as shown in Table In this multicenter study we describe for the first time the presence of serum high-titer MOG-IgG antibodies in patients with NMO and HR-NMO. Our data confirm several studies demonstrating the presence of MOG-IgG in a subgroup of patients with ADEM -29, as wHowever, it has been speculated whether different pathomechanisms are involved in AQP4-IgG seronegative NMO and HR-NMO patients compared to subjects with \"AQP4 autoimmune channelopathies\". This assumption is supported by findings showing no development of NMO-like symptoms in animals immunized with purified antibodies from AQP4-IgG seronegative NMO patients . In contin vivo studies demonstrating the spontaneous development of human NMO-like symptoms in a double-transgenic mouse strain with opticospinal EAE [An involvement of antibodies directed against MOG in NMO and HR-NMO is encouraged by inal EAE ,20. Exprinal EAE . In addiinal EAE . Severalinal EAE -23. Howeinal EAE . Howeverinal EAE -27,37. Hinal EAE -40. Therin vitro studies have demonstrated the pathogenic effect of AQP4-IgG in the presence of active complement [in vitro, resulting in the formation of the TCC on living MOG transfected HEK-293A cells. To our knowledge, these observations are novel and might provide a deeper insight into the role of high-titer serum anti-MOG antibodies. Thus, the detection of high-titer MOG-IgG might not only serve as a valuable biomarker in AQP4-IgG negative NMO and HR-NMO patients, but possibly play a role as pathogenic factor in human demyelinating diseases, although this needs to be further investigated.Several mplement ,41,42, wThere are two limitations that need to be addressed regarding our study. The first limitation concerns the usage of an immunofluorescence assay to measure AQP4-IgG and MOG-IgG antibodies and TCC formation. This is often criticised by other researchers using automated assays like flow cytometry or immunoprecipitation for the measurement of specific antibodies. However, experiences from the last decades have strongly emphasized that immunofluorescence assays are the gold standard for the detection of several autoantibodies, such as anti-nuclear antibodies. Furthermore, immunofluorescence assays were shown to yield the highest sensitivity for the detection of AQP4-IgG ,14,43-45We could show for the first time that AQP4-IgG antibody seronegative patients with NMO and HR-NMO harbor a MOG-IgG directed immune response. MOG is not a target antigen in \"AQP4 channelopathies\", raising the question of whether MOG-IgG positive NMO and HR-NMO patients share a possible disease overlap with MOG-IgG positive ADEM. Overall, these results are highly relevant for clinical practice in order to optimize patients' treatment, and might help to elucidate the disease pathomechanisms of these rare CNS demyelinating diseases.The following patients were recruited from Austria (n = 295), Germany n = 19), Slovakia (n = 2) and Serbia (n = 19) and all Austrian patients or parents/legal guardians gave written informed consent to the study protocol. All Serbian and Slovakian patients gave their informed consent for serum sampling and this study was approved by the Institutional Review Board of the Clinic of Neurology, Clinical Center of Serbia, Belgrade. The Slovakian patients signed the translated informed consent form of the Innsbruck Medical University. All German samples were tested anonymously as requested by the institutional review board of the University of Heidelberg.Analysis of M23 AQP4-IgG was performed using a live cell staining IF assay as recently described ,33,48.HEK-293A cells were transiently transfected using the Vivid Colours\u2122 pcDNA\u2122 6.2C-EmGFP-GW/TOPO plasmid , expressing M23 AQP4 fused C-terminally to an emerald green fluorescence protein (EmGFP). The AQP4-IgG IF assay was performed by blocking the transfected cells with 4 \u03bcg/ml goat IgG diluted in PBS/10% FCS (Sigma-Aldrich), subsequently incubating the cells with pre-absorbed serum samples at a 1:20 and 1:40 dilution for one hour at 4\u00b0C. Bound antibodies were detected using Cy\u21223-conjugated goat anti-human IgG antibody for 30 minutes at room temperature. Dead cells were excluded by DAPI staining (Sigma-Aldrich). The AQP4-IgG status was determined using a fluorescence microscope . Each serum sample was individually evaluated by three independent, clinically blinded investigators , yielding a concordance rate of 100%.In order to determine AQP4-IgG titer levels, AQP4-IgG seropositive samples were further diluted until loss of specific antibody staining. AQP4-IgG was purified from a NMO patient's plasma exchange material as recently described and servSerum MOG-IgG was determined in pre-absorbed samples using HEK-293A cells transiently transfected with human MOG cloned into the mammalian expression vector Vivid Colours\u2122 pcDNA\u2122 6.2 C-EmGFP/TOPO (Invitrogen), expressing MOG fused C-terminally to EmGFP as previously reported . Serum MIn order to exclude unspecific background staining, we additionally performed serum antibody stainings using untransfected HEK-293A cells for both IF assays, transfected cells expressing the fusion protein (EmGFP) as a control for the AQP4-IgG assay as well as CD2-EmGFP transfected cells (another protein of the immunoglobulin superfamily) for the MOG-IgG assay. Non-specific background binding was clearly distinguishable from a specific antibody staining in our immunofluorescence setting.Furthermore, MOG-IgG and AQP4-IgG seropositive and seronegative control samples are regularly retested for antibody titer levels to ensure the quality of the testing system. Titer levels remain constant in the serum samples which are stored at -20\u00b0C, even two years after first analysis.\u00ae 546 goat anti-mouse IgG (Invitrogen) for 30 minutes. Dead cells were excluded by DAPI staining and analysis was performed by three independent investigators .Serum antibodies to MOG and AQP4 were analyzed in a subgroup of 15 patients for IgG1-IgG4 isotypes via our live cell staining IF assay using MOG or AQP4 transfected cells. After blocking with goat IgG, the transfected cells were incubated with the pre-absorbed serum samples (1:20 and 1:40 dilution) for one hour. Subsequently, cells were washed and incubated with mouse monoclonal anti-human IgG1-IgG4 isotype antibodies for 30 minutes , followed by detection using Alexa Fluor\u00ae 546 goat anti-mouse IgG antibody , cells were washed with PBS/10% FCS and dead cells were visualized by DAPI staining. All samples were assessed for the presence of the surface membrane attack complex by three independent investigators blinded for clinical information as well as the design of the assay concerning usage of active/inactive complement .Antibody mediated complement activation was investigated in 23 NMO, 33 HR-NMO, 19 ADEM, 14 CIS, 10 MS and 14 CTRL. The selection of patients for the analysis of complement-mediated cytotoxicity was based on the availability of serum samples and the use of samples which are representative for our entire study population. Briefly, serum samples and human complement (Sigma-Aldrich) were heat-inactivated at 56\u00b0C for 45 minutes. Inactivated serum samples were diluted 1:10 in serum-free X-VIVO 15 medium and pre-absorbed with rabbit liver powder. Cells expressing either MOG or AQP4 were washed three times with X-VIVO 15 medium and subsequently incubated with heat-inactivated, pre-absorbed serum samples and 20% active versus 20% heat-inactivated human complement for 90 minutes at 37\u00b0C. After washing the cells three times with 100 \u03bcl X-VIVO 15 medium, detection of TCC formation was performed by adding the murine-monoclonal anti-human SC5b-9 for one hour at 4\u00b0C. Following a 30 minutes incubation with the fluorescence labelled Alexa FluorTo analyze the co-localization of the TCC and serum MOG-IgG or AQP4-IgG, we used HEK-293A cells expressing MOG or AQP4 without EmGFP fusion protein.To obtain M23 AQP4 without EmGFP fusion protein, the M23 AQP4 isoform was cloned into the pcDNA3.1 Directional TOPO Expression vector (Invitrogen) . In orde\u00ae 488 goat anti-mouse IgG antibody and Cy\u21223-conjugated goat anti-human IgG antibody were diluted in X-VIVO 15 medium and incubated for 30 minutes. Co-staining of AQP4-IgG and MOG-IgG antibodies (red) and TCC (green) was investigated in a blinded fashion , and dead cells were visualized by DAPI staining. Control experiments using active complement in the absence of serum showed no TCC formation on MOG or AQP4 expressing cells , the cells were washed X-VIVO 15) and stained with the murine-monoclonal anti-human SC5b-9 as described above. Following three washing steps, the Alexa Fluor5 and staAQP4-IgG mediated complement activation was confirmed via SEM. Briefly, HEK-293A cells were seeded on poly-L-lysine (Sigma-Aldrich) coated glass slides and transiently transfected with the AQP4-EmGFP vector. Thereafter, serum samples supplemented with either active or inactive complement were added to the cells. Following incubation at 37\u00b0C for 90 minutes, the cells were washed with PBS and fixed in glutaraldehyde . After incubation for 30 minutes at room temperature, the fixative was replaced by fresh fixative, and incubated for another two hours at room temperature. Subsequently, complement activation was investigated via SEM according to standard procedures . SamplesU test, Fisher's exact test and Chi-square test as appropriate. Correlation of parameters was analyzed with Spearman's non-parametric correlation. Statistical significance was defined as two-sided p-value less than 0.05 and Bonferroni's correction was applied for multiple comparisons when appropriate.Statistical analysis and significance of group differences were done using IBM SPSS software or GraphPad Prism 5 . Between-group comparisons were performed with Kruskal-Wallis test, Dunn's multiple comparison post-hoc test, Mann-Whitney The authors declare that they have no competing interests.SM, VG, KS and MR conceived and designed the experiments. SM, VG and KS carried out all experiments. SM and MR analysed and interpreted the data. KP performed the scanning electron microscopy. KR, ID, AL, SJ, FDP, BK, RE, FD, FAE, MS, PK, JD, WK, TB and MR participated in serum and data collection. SM, VG, KS and MR wrote the initial manuscript. All authors have read and approved the final version of the manuscript.Complement dependent cytotoxicity on the surface of AQP4 transfected cells occurs exclusively in AQP4-IgG positive serum samples. Heat incativated serum samples of patients with NMO (AQP4-IgG positive), LETM (MOG-IgG positive) and ADEM (MOG-IgG positive and negative) were incubated on AQP4-EmGFP (green) expressing cells in the presence of active complement, and were analysed for AQP4-IgG mediated complement activation . The serum of an AQP4-IgG positive NMO patient together with active complement resulted in TCC formation, and an increased number of dead cells . Additionally, we observed a co-localization of the TCC (red) with the AQP4-EmGFP transfected cells (green), which is shown in the merged picture of the NMO patient. In contrast to the NMO patient, AQP4-IgG negative serum samples of patients with LETM or ADEM did not result in TCC formation in the presence of active complement. As an additional control, active complement without serum samples was added, showing no AQP4-IgG mediated complement activation.Click here for fileComplement dependent cytotoxicity on the surface of MOG transfected cells is restricted to the presence of serum high-titer MOG-IgG. Heat inactivated serum samples of patients with NMO (AQP4-IgG positive), LETM (MOG-IgG positive), ADEM (MOG-IgG positive and negative) were incubated on MOG-EmGFP (green) transfected cells supplemented with human active complement. MOG-IgG specific complement activation was observed using high-titer MOG-IgG positive sera of a patients with LETM and ADEM. Furthermore, the TCC co-localized with the MOG-EmGFP transfected cells (merged), resulting in an increased number of dead cells . Serum MOG-IgG negative patients (NMO and ADEM), as well as active complement (without serum) did not result in TCC formation.Click here for fileAQP4-IgG and MOG-IgG serostatus of the patients (Table 3) investigated for antibody mediated complement activation. AQP4-IgG or MOG-IgG TCC formation in patients with NMO, HR-NMO, ADEM, CIS, MS and CTRL, which were subdivided according to their antibody serostatus: AQP4-IgG positive and MOG-IgG negative (AQP4+MOG-), AQP4-IgG negative and MOG-IgG seropositive (AQP4-MOG+) or double negative for AQP4-IgG and MOG-IgG (AQP4-MOG-). * = The AQP4-MOG- as well as AQP4+MOG- cohort includes patients with MOG-IgG titer levels below the threshold of 1:160 (cut-off), which are defined in our study population as MOG-IgG negative. Therefore, MOG-IgG titer levels below the threshold level are indicated as MOG titer (1:) *. Antibody titer levels are shown as median titer level (range). Abbreviation: TCC = terminal complement complex.Click here for file"} +{"text": "Ink4a, Arf and Pten. Yet, gliomas are cellularly heterogeneous; they recruit and trap normal cells during infiltration.Gliomas are thought to form by clonal expansion from a single cell-of-origin, and progression-associated mutations to occur in its progeny cells. Glioma progression is associated with elevated growth factor signaling and loss of function of tumor suppressors We performed lineage tracing in a retrovirally mediated, molecularly and histologically accurate mouse model of hPDGFb-driven gliomagenesis. We were able to distinguish cells in the tumor that were derived from the cell-of-origin from those that were not. Phenotypic, tumorigenic and expression analyses were performed on both populations of these cells. Here we show that during progression of hPDGFb-induced murine gliomas, tumor suppressor loss can expand the recruited cell population not derived from the cell-of-origin within glioma microenvironment to dominate regions of the tumor, with essentially no contribution from the progeny of glioma cell-of-origin. Moreover, the recruited cells can give rise to gliomas upon transplantation and passaging, acquire polysomal expression profiles and genetic aberrations typically present in glioma cells rather than normal progenitors, aid progeny cells in glioma initiation upon transplantation, and become independent of PDGFR signaling.bona fide tumor, and deviate from the generally established view of gliomagenesis.These results indicate that non-cell-of-origin derived cells within glioma environment in the mouse can be corrupted to become Moreover, identifying and distinguishing GBM cells from the surrounding stroma is not a trivial task - glioma cells are often defined histologically, demonstrating high mitotic indices, expression of stem or progenitor cell markers, abnormal global gene expression patterns, presence of genetic alterations, and the ability to serially transplant the disease bona fide tumor cells, have not been addressed. In order to study this phenomenon of cellular contribution to glioma heterogeneity, we employed lineage tracing, molecular analysis and functional characterization of non-cell-of-origin derived cells using two different mouse models of hPDGFb-driven gliomas.It has been recently shown that gliomas induced in adult or neonatal rats by hPDGFb-expressing retroviruses contain stem or progenitor-like cells expressing neural markers, that are contributing to glioma mass and are induced to proliferate by glioma environment Nestin-tv-a (Ntv-a) mice with an RCAS vector expressing hPDGFb and eGFP . Many but not all of these cells were derived from the cell-of-origin and expressed viral eGFP (\u201cprogeny cells\u201d), while others appeared to not be infected with the RCAS-PSG virus (\u201crecruited cells\u201d), as they did not express eGFP or hPDGFb, with hPDGFb expression determined by immunostaining for hemagglutinin epitope tag included in RCAS-PSG vector data not shown) ; Figure S1e,f). In these tumors, histologically-defined pathognomic glioma structures . We refer to these tumors as \u201cvirally-induced gliomas\u201d.We first infected bacTRAP olig2 RP-eGFP reporter mice, expressing a fusion between ribosomal protein L10a and eGFP (RP-eGFP) under the Olig2 promoter bacTRAP system is that olig2 RP-eGFP expression in the adult murine brain labels not only progenitor cells, but also more differentiated oligodendrocytes Modeling systems employing intracranial transplantation of glioma cells are frequently used in glioma research. To rule out the possibility of spontaneous proviral excision or loss of eGFP expression in the originally infected cells in the virally induced gliomas, we performed transplantations of acutely isolated non-fluorescent hPDGFb-driven mouse glioma cells derived from Ntv-a gliomas induced by the non-fluorescent RCAS-hPDGFb vector into the Ntv-a mice with germline deletions of Ink4a, Arf and/or Pten, the latter achieved by Cre-mediated recombination of the floxed Pten alleles by RCAS-Cre Arf and Pten, but not Ink4a, shortened tumor latency and increased incidence of murine GBMs, histologically defined by the presence of microvascular proliferation and pseudopalisading necrosis Ntv-a Ink4a/Arf-/-, Pten-/- tumors , injections of RCAS-RSR and RCAS-Cre into -/-Ptenfl/flNtv-a Ink4a/Arf mice could give rise to high-grade gliomas that did not express PDGFR\u03b1 and contained regions of proliferating cells that did not express mRFP .We then determined whether such robust expansion of the recruited cell population upon tumor suppressor loss is limited to modeling systems characterized by paracrine growth factor signaling, or whether it could apply to murine gliomas with cell-autonomous activation of oncogenes. While mutations in bacTRAP system. Transplantation of non-fluorescent hPDGFb-driven glioma cells into -/-Ptenfl/fl bacTRAP olig2 RP-eGFPInk4a/Arf reporter mice resulted in formation of high-grade glioma structures comprised of both progeny and recruited cells . Transplanted gliomas induced by hPDGFb-driven glioma cells in hosts with altered tumor suppressor function showed higher percentages of the overall recruitment than in the wild-type hosts, indicating that complete or partial tumor suppressor loss may enhance the ability of the cells to be recruited or epigenetic events. The rarity of such events would in turn result in the expansion of an altered cell population from a single cell, be it progeny or recruited. Although neither +/-Pten+/flNtv-a Ink4a/Arf nor -/-Ptenfl/flNtv-a Ink4a/Arf mice generate spontaneous gliomas, it is likely that experimentally provided genetic hits may represent the rate limiting step for transformation and thus, transformation of recruited cells may be less common in human gliomas.The above data indicates that murine glioma cells, especially those driven by PDGF signaling, recruit proliferating olig2-expressing progenitors, and their contribution to high-grade glioma structures is enhanced upon homozygous loss of Ntv-a Ink4a/Arf+/-Pten+/fl mice by RCAS-PSG and RCAS-Cre was intermediate between the wild type and Ink4a/Arf-/-Ptenfl/fl mice, the distribution of glioma grades at the point when the tumors caused mice to become moribund was similar to that of gliomas characterized by homozygous tumor suppressor loss at initiation . However, unlike the mixed progeny-recruited cell composition seen in gliomas null for Ink4a, Arf and Pten at tumor initiation, gliomas arising in mice initially heterozygous for these tumor suppressors frequently showed regional domination by either progeny or recruited cells . Of note, these regions dominated by recruited cells showed loss of function of the remaining wild type tumor suppressor alleles by LOH (in the case of Ink4a/Arf), or loss of expression at the protein level with or without LOH (in the case of Pten), as in human gliomas . While Ink4a/Arf loci were most frequently lost at the genetic level as determined by PCR, of 40 hPDGFb-driven gliomas arising in -/-Ptenfl/flNtv-a Ink4a/Arf or +/-Pten+/flNtv-a Ink4a/Arf injected with RCAS-Cre, 28 did not express Pten; of these, 21 retained Pten allele by real-time PCR, while 7 lost it . Thus, while loss of Pten expression occurred in \u223c70% of all cases, loss of Pten at the DNA level occurred in 17.5% cases. There was a small but statistically significant difference with respect to latency medians depending on whether a given glioma was predominantly derived from progeny or recruited cells . However, their variances largely overlapped (F test), and GBMs containing large areas with pseudopalisades predominantly comprised of recruited cells represented \u223c30% of all mouse GBM cases ; ). The primary mechanism for EGFR and IGFR expression in recruited regions is unclear; however, low-level amplification of these genes was seen in some cases. FISH analysis for mouse EGFR of eleven murine gliomas containing large regions of recruited cells with high expression of EGFR and/or IGFR showed >2 copies of EGFR in 5 of 11 cases, while 1 of 11 showed >2 copies of IGFR . qPCR analysis of a panel of these and 15 other murine gliomas containing regions comprised of recruited cells was consistent with amplification of EGFR at the DNA level occurring at most in 6 of 18 (\u223c30%) of all cases, while IGFR was only amplified at most in 2 of 19 (\u223c10.5%) .We then characterized tumors with regions dominated by recruited cells for expression of growth factor receptors commonly activated in human gliomas, including PDGFR, EGFR and IGFR Ntv-a Ink4a/Arf+/-Pten+/fl glioma-bearing mice with PTK787 for a week at the onset of symptoms; eight mice survived the full course of treatment. Of these, three mice had gliomas that expressed PDGFR\u03b1 and eGFP in high-grade tumor areas, concomitant with reduced proliferation rates (p<0.0001) . Thus, regions dominated by recruited cells became independent of PDGFR signaling during glioma progression, and the response to PTK787 treatment was dependent on the cellular composition of a given glioma. Although PTK787 also targets VEGFR, the conclusion that recruited glioma regions are independent of PDGFR activity is not confounded by this fact, since our readout is the relative lack of cell cycle arrest in the recruited glioma regions.In hPDGFb-induced gliomas, pharmacologic blockade by the PDGFR/VEGFR inhibitor PTK787 that crosses the blood-brain barrier results in cell cycle arrest of the tumor cells; thus, these gliomas retain their dependence on PDGFR signaling Ntv-a Ink4a/Arf-/-Ptenfl/fl or Ink4a/Arf+/-Pten+/fl mice injected with RCAS-PSG and RCAS-Cre . Single sorting of recruited cells (\u226598.5% purity) gave rise to cell fractions that formed tumors with latency, grade and histology similar to gliomas induced by eGFP-positive progeny, but showed variable contamination with progeny cells in the resulting lesions with up to 10% progeny cells . Double sorting of eGFP-negative cells gave a population that was \u226599.5% recruited and formed high-grade gliomas not contaminated with progeny cells upon transplantation, albeit at a low frequency and requiring large numbers of transplanted cells . While large numbers of recruited cells were necessary to induce gliomas upon transplantation, it is important to remember that non-eGFP-expressing cells in virally-induced gliomas include normal stromal cells types . Transplanted gliomas arising from stringently gated double-sorted eGFP-negative recruited cells did not express viral eGFP and had no detectable provirus by real-time PCR (Table S1). In addition to EGFR expression and unlike gliomas with the typical oligodendroglial morphology induced by transplantation of progeny cells, these tumors showed regionally high expression of nestin, GFAP, CD44, vimentin and VEGFR, while expression of olig2 was variable in some tumor areas and absent in most Although glioma patients do not die from glioma cells transplanted into them but rather from the massive expansion/infiltration by the glioma cells, tumor cells in rodent systems are most stringently defined by their ability to form secondary tumors upon transplantation into recipient mice -/-Ptenfl/flNtv-a Ink4a/Arf or +/-Pten+/flNtv-a Ink4a/Arf mice injected with RCAS-PSG and RCAS-Cre were not derived from progeny cells and were all recruited, as confirmed by RT-PCR, qPCR and FISH on FACS-sorted cells . Moreover, while transplantation of single-sorted CD133-positive cells resulted in gliomas contaminated by the eGFP-positive progeny, without the expansion of progeny cells in some cases .We further addressed whether the CD133-positive subset of recruited cells showed functional importance for initiation or progression of transplanted hPDGFb-induced murine gliomas by cell-mixing experiments. While purified CD133-positive recruited cells could not give rise to transplanted gliomas at 10,000 cells (0/24 cases), and progeny cells did not robustly form tumors with cell numbers less than 3,000 (3/20 cases), addition of 10,000 CD133-positive recruited cells to 1,000 eGFP-positive progeny resulted in robust gliomagenesis . Unsupervised hierarchical mRNA clustering likewise indicated that recruited olig2 cells clustered more closely to glioma olig2 cells . Of 500 mRNAs most different between glioma and normal olig2 cells, 490 mRNAs were changed in the same direction but smaller magnitude in recruited olig2 cells, 440 of which were at least two-fold different, and 183 were five-fold different or more . From the above data, the expression profile of recruited cells closely resembled that of tumor cells, placing the two closely together in the continuum of change towards the \u201cmalignant state\u201d, and further confirming that recruited olig2 expressing cells are tumor. While only \u223c300 mRNAs were the same between normal and either glioma or recruited olig2 cells, the number of mRNAs that were the same between glioma and recruited olig2 cells was at least three-fold more, amounting to 1,047 (data not shown).Pair-wise comparisons indicated that out of 2,593 mRNAs differentially present on the polysomes, 2,310 mRNAs were differentially present in glioma vs. normal olig2 cells and 2,262 mRNAs in recruited vs. normal olig2 cells ; of note, 86% of these differentially represented mRNAs were the same mRNA was 5.3 fold higher in the recruited than tumor olig2 cells. mRNA populations corresponding to genes defining oligodendrocytic character were reduced in olig2 recruited cells, while mRNAs associated with astroglial and endothelial characteristics were upregulated in the recruited population. In addition, 41 mRNAs were increased on recruited cell polysomes by 10-fold or more, as compared to the tumor olig2 cells and differentially from normal olig2 cells .RCAS-hPDGFb-HA-SV40-eGFP (RCAS-PSG) containing human PDGFb with partially deleted 5\u2032UTR, RCAS-hPDGFb-HA, RCAS-Cre and RCAS-mRFP-SV40-Ras (RCAS-RSR) vectors were transfected into DF-1 chicken cells (ATCC) 2, their brain tissue extracted and fixed for 3 days in 4% paraformaldehyde at 4\u00b0C, and subsequently processed using standard paraffin embedding and processing techniques.Experiments involving mice were performed in accordance with the regulations set by the MSKCC IACUC committee. Upon the appearance of brain tumor symptoms , mice were anesthetized by an intraperitoneal injection of 1 mg/kg Nembutal solution or ketamine (150 mg/kg)/xylazine (15 mg/kg) cocktail and underwent trans-cardiac perfusion with 10 ml ice-cold heparin normal saline, followed by 10 ml of ice-cold 4% paraformaldehyde. Brain tissue was extracted and post-fixed for 30 minutes in ice-cold 4% paraformaldehyde, transferred into 30% sucrose at 4\u00b0C for cryoprotection, embedded using the OCT compound, frozen on dry ice and stored at \u221280\u00b0C until sectioned. A small subset of animals were sacrificed using COolig2 RP-eGFP bacTRAP reporter mice. Mouse gliomas induced by injection of a non-fluorescent RCAS-hPDGFb Ntv-a olig2 RP-eGFP bacTRAP reporter mice and immunoprecipitated; each tumor was processed as a separate sample. Briefly, mouse tissues were collected into ice-cold cyclohexamide-containing buffer, homogenized, cells were lysed in NP-40 and DHPC-containing buffer, centrifuged at 20,000 g for 15 min, supernatant incubated with anti-eGFP-conjugated protein G beads 30 min at 4C, washed and RNA collected using Trizol reagent as per manufacturer's instructions Normal olig2 progenitors were collected from three replicates of pooled cortices of three http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE30016).Data was analyzed using Genespring GX10 software, as described in the text and in Glioma-bearing brains were extracted from anesthetized mice, gliomas excised from surrounding brain parenchyma, dissociated using a mix of collagenases as previously described, and sorted using MoFlo FACS sorting. Various numbers of cells resuspended in sterile 1X PBS were injected into brain parenchyma of neonatal immunonaive mice of various genetic backgrounds, or stereotactically transplanted into anesthetized adult SCID mice.Pten, and the Ink4a/Arf locus were performed using previously published primers. Control GAPDH reactions were performed in the same PCR mix. Data was obtained using GelDoc Software.Mouse glioma sections were scraped from slides and lyzed with 0.05% SDS buffer. Genomic DNA was extracted using phenol-chloroform according to standard techniques, and precipitated at \u221220\u00b0C overnight. Prior to PCR for hPDGFb, DNA was heated at 56\u00b0C for 2 hours or for 5 min at 100\u00b0C, concentration measured, and equal amounts of DNA added to PCR reactions. PCR reactions for human PDGFB, EGFR or mouse IGFR . Pten qPCR was performed using previously published primers DNA was extracted using 0.05% SDS buffer as above; Roche primers were used to detect amplifications of mouse Total RNA from sorted cells was extracted using the Qiagen RNeasy kit and resuspended in 30-50 ul of DEPC-treated water. About 5 ug of total RNA was reverse-transcribed using the SuperScriptIII Reverse strand synthesis kit from Invitrogen. Quantitative RT-PCR was performed using primers for human PDGFb and mouse actin as a control.Cryogenically processed murine glioma tissue was sectioned at 7 microns, washed with PBS, blocked with 10% serum in PBS using 0\u20130.3% TX-100 for polyclonal rabbit/goat or MOM kit for monoclonal mouse and rat antibodies, and incubated with primary antibodies for 1 hour at room temperature or overnight at 4C. The primary antibodies included PDGFR\u03b1 and PTEN from Cell Signaling; olig2, Ki67, hNA and EGFR from Abcam; NeuN from Chemicon; HA from Santa Cruz; and eGFP from Molecular Probes. Secondary fluorochrome-conjugated antibodies were applied for 1 hr at room temperature, slides washed several times, counterstained with DAPI (1\u22363000 of a 1 mg/ml 1X PBS solution), mounted in 70% glycerol, coverslipped and photographed with inverted fluorescent or confocal Leica microscopes.Glioma-bearing brains were extracted from anesthetized mice, gliomas excised from surrounding brain parenchyma, dissociated using a mix of collagenases as previously described, and sorted using DAKO-Cytomation MoFlo FACS sorters for eGFP expression. Samples were gated to exclude debree, dead cells and doublets. Various numbers of cells resuspended in sterile 1X PBS were injected into the brain parenchyma of neonatal immunonaive mice of various Ntv-a genetic backgrounds, or stereotactically transplanted into anesthetized adult SCID mice, using Hamilton syringe. CD133 (eBioscience) staining of acutely dissociated murine glioma cells was done as per manufacturer's instructions; CD133 antibody was used at a 10-fold lower concentration based on titration. Single- or double-sorted CD133-positive cells were transplanted as described above. Cell-mixing experiments were performed using double-sorted eGFP-positive progeny and double-sorted CD133-positive recruited cells, which were mixed just prior to transplantation, and injected using Hamilton syringe.RCAS DNA was fragmented using nick translation kit, labeled with biotin, and digested to 500 bp-2 kb probe size. Resulting probes were denatured at 80\u00b0C and applied to tumor sections blocked with salmon sperm DNA for an overnight incubation at 37\u00b0C. Slides were washed with 2X SSC-50% formamide solution at 45\u00b0C, signal amplified according to standard techniques using TSA kit, and revealed with Alexa488 and Alexa555. Slides were counterstained with DAPI (1\u22363000 of 1 mg/ml PBS solution), mounted, coverslipped and photographed using inverted fluorescent or confocal Leica microscopes.Ntv-a wild-type vs. -/-Ntv-a Arf mice injected with RCAS-PSG vs. -/- Ptenfl/flNtv-a Ink4a/Arf mice injected with RCAS-PSG and RCAS-Cre. Survival curves for Ntv-a wild-type vs. -/-Ntv-a Ink4a, or for -/-Ntv-a Ink4a/Arf vs. -/-Ntv-a Arf, were not statistically different from each other. Statistical analysis of other data, including percentages of eGFP-positive cells in different murine Ntv-a backgrounds and percentages of Ki67-positive cells in PTK787-treated murine gliomas, was performed using unpaired two-tailed t-tests and F-tests in Prism4.Kaplan-Meyer curves for mouse glioma latency were made using Prism4 software and analyzed with standard log rank test, or using R. The p value was determined to be less than 0.001 for survival curves of Figure S1Background information for RCAS-PSG-induced Ntv-a gliomas. (a\u2013d) H&E-stained paraffin sections of low-grade Ntv-a gliomas containing secondary structures of Scherer characteristic of human gliomas: subpial accumulations of tumor cells (a), white matter tracking (b), perivascular (c) and perineuronal satellitosis (d). Red arrowheads indicate glioma cells. Expression of eGFP in high-grade -/-Ntv-a Arf murine gliomas is detectable by FACS (e), and correlates with RCAS-hPDGFb infection. eGFP+ and eGFP- glioma cells were sorted and used for real-time PCR. (-/-Ntv-a Arf high-grade gliomas were used in lieu of wild-type because wild-type Ntv-a gliomas contained very few recruited cells.) (f) shows normal murine Ntv-a brain, total mixed tumor cell population, and sorted progeny or recruited cell fractions for Ntv-a gliomas shown in (e).(TIF)Click here for additional data file.Figure S2Expression of eGFP in progeny cells of low- and high-grade Ntv-a gliomas correlates with hPDGFb expression. Images (a\u2013d) show native eGFP, anti-HA staining for the hemagglutinin tag on viral hPDGFb and corresponding composites in low-grade and high-grade Ntv-a gliomas. Overall expression of eGFP and hPDGFb correlates on a cell-to-cell basis, but relative amounts of expression of eGFP and hPDGFb proteins may differ. High-magnification composites showing correlation of hPDGFb and eGFP expression in pseudopalisade regions of high-grade Ntv-a gliomas. Asterisks in indicate pseudopalisade lumens.(TIF)Click here for additional data file.Figure S3Validation of the bacTRAP olig2 RP-eGFP system in Ntv-a gliomas. (a) olig2 (red) and nestin (green) immunostaining of Ntv-a wild-type mouse RCAS-hPDGFb-induced gliomas, showing expression of olig2 in most tumor cells. (b) Experimental design to label and isolate olig2 tumor cells and olig2 recruited cells. DAPI-stained frozen sections of murine gliomas induced in bacTRAP olig2 RP-eGFP reporter mice, with olig2 and eGFP stains shown in red. bacTRAP olig2 RP-eGFP mice accurately recapitulate expression of olig2 and eGFP.(TIF)Click here for additional data file.Figure S4Tumor suppressor loss in hPDGFb-induced Ntv-a gliomas results in shorter glioma latency, increased grade and larger numbers of recruited cells. (a) H&E-stained section of an Ntv-a glioma containing pseudopalisading necrosis and microvascular proliferation, red arrowheads. (b) Tumor incidence and presence of low- and high-grade glioma structures in gliomas of various Ntv-a mouse backgrounds, induced with RCAS-PSG with or without RCAS-Cre. X-axes color-coding corresponds to color-coding of Ntv-a mouse strains in the Kaplan-Meyer analysis in c) Correlations between PTEN immunostaining (IHC) and Pten LOH during glioma progression in Ntv-a gliomas heterozygous targeted for Ink4a, Arf and Pten tumor suppressor loss at glioma initiation. DNA concentrations were measured, and equal amounts of DNA were loaded per tumor sample. Normal, uninjected murine Ntv-a brain; PDGF and PDGF/Cre, murine gliomas induced in Ntv-a mice homozygous targeted for Ink4a, Arf and Pten loss at glioma initiation. All other samples are derived from +/-Pten+/flNtv-a Ink4a/Arf mice injected with RCAS-PSG and RCAS-Cre. Note lack of Pten expression in some murine gliomas that retain Pten at the genetic level. PTEN expression is absent in Ntv-a GBMs heterozygous targeted for Ink4a, Arf and Pten tumor suppressor loss at glioma initiation.(TIF)Click here for additional data file.Figure S5The recruitment phenomenon is not limited to murine gliomas characterized by paracrine hPDGFb signaling: K-Ras-driven Ntv-a gliomas with tumor suppressor loss contain recruited cells.Quantification of recruited cells and comparison of glioma latency in GBMs with large regions of recruitment or progeny cell contribution. (a) Construction of RCAS-mRFP-SV40-KRasG12D vector. mRFP expression DF1 cells transfected with RCAS-mRFP-SV40-KRasG12D. Expression of mRFP in frozen sections of an Ntv-a GBMs induced by injection of RCAS-mRFP-SV40-KRasG12D and RCAS-Cre, mRFP immunostaining. Note the presence of cells not expressing mRFP. (f) Immunostaining for RP-eGFP expression (red) in recruited olig2 cells in transplanted gliomas arising in bacTRAP olig2 RP-eGFP mice induced by transplantation of Ras-driven Pten-deleted murine glioma cells. (g) PDGFR\u03b1 stain (red) of an Ntv-a glioma induced by RCAS-K-RasG12D and RCAS-Cre. Note absence of PDGFR\u03b1 expression in tumor cells. (h) RP-eGFP is expressed in olig2 tumor cells contributing to pseudopalisades of transplanted gliomas induced by the hPDGFb-expressing murine glioma cells and arising in bacTRAP olig2 RP-eGFP mice. (i) Tumor latency for Ntv-a gliomas containing large regions of progeny or recruited cells induced in Ntv-a mice heterozygous targeted for loss of Ink4a, Arf and Pten by RCAS-PSG and RCAS-Cre. While there is a small but statistically significant difference with respect to latency medians , latency variances largely overlap (F test). (j) FACS plots of 4 Ntv-a gliomas heterozygous targeted for tumor suppressor loss of Ink4a, Arf and Pten at glioma initiation, containing large numbers of recruited cell pseudopalisades. Percentages of recruited cells vary from 0.91% to 36.8%. (k) Graph shows variability of contribution from the eGFP-expressing progeny and the eGFP-negative recruited cells across various Ntv-a mouse strains, including Ntv-a gliomas containing large regions of recruitment.(TIF)Click here for additional data file.Figure S6hPDGFb-induced Ntv-a gliomas show regional expression of growth factor receptors important in human glioma biology, expression of which in recruited cells may be associated with low level amplifications in some cases. (a\u2013d) Ntv-a gliomas induced in +/-Pten+/flNtv-a Ink4a/Arf mice injected with RCAS-PSG and RCAS-Cre stained with IGFR (red), EGFR (pink) or PDGFR\u03b1 (red) . Glioma regions predominantly derived from progeny cells express PDGFR\u03b1; expression of EGFR and IGFR is limited to perivascular areas. Adjacent sections stained with PDGFR\u03b1 and EGFR (red) show regional expression of growth factor receptors; eGFP omitted for easier view. (e) FISH for IGFR (green) and EGFR (red) on Ntv-a GBMs with large areas of recruitment and high EGFR expression. Panels show a neuron, EGFR-expressing non-amplified GBM with extensive recruitment, and EGFR-expressing GBM with EGFR amplification in the recruited cells. (f) Summary of mEGFR FISH performed on EGFR-expressing GBMs with large areas of recruitment. Gliomas containing more than 15% of cells with \u22653 mEGFR signals are marked with an asterisk (amplified). real-time qPCR on DNA extracted from sections of mouse Ntv-a GBMs containing large areas of recruitment (blue squares) highly expressing EGFR. Primers for mEGFR kinase domain (g) or intron region of mEGFR (h) were designed by Roche; graphs show fold increase over normal EGFR copy number (\u2205); gliomas with large areas composed of progeny cells (green squares) were used to establish range of variability of primer noise. Gliomas marked with asterisks were considered amplified, and gliomas marked with arrows appeared amplified by both primer pairs, potentially representing tumors with larger chromosomal amplifications.(TIF)Click here for additional data file.Figure S7Recruited cells can initiate gliomas upon transplantation and can be serially passaged in mouse hosts. (a\u2013d) H&E-stained sections depicting histology of mouse gliomas induced by transplanting recruited cells. (e\u2013g) Purification of recruited cells from +/-Pten+/flNtv-a Ink4a/Arf mice injected with RCAS-PSG and RCAS-Cre; (h) DAPI-stained frozen section of a glioma induced by recruited cells; note absence of cells with virally encoded eGFP. (i\u2013l) Gliomas induced by the recruited cells could be serially passaged in mice. (i\u2013k), FACS plots of gliomas induced by serial transplantation of recruited cells into mouse hosts at passaging; recruited glioma cells were single purified between passages. (l) DAPI-stained section of a transplanted glioma induced by recruited cells, showing absence of progeny cells with virally encoded eGFP.(TIF)Click here for additional data file.Figure S8Immunohistochemical analysis of murine gliomas induced by transplanting recruited cells. Images (a\u2013l) show native eGFP and immunostaining for Ki-67 , olig2 , GFAP , nestin , VEGFR (i), YKL40 (j) and CD44 .(TIF)Click here for additional data file.Figure S9Single-sorted CD133-positive cells are contaminated by low numbers of progeny cells and give rise to gliomas containing small numbers of progeny. FACS plot of an Ntv-a GBM induced by RCAS-PSG and RCAS-Cre, stained with CD133. Single-purified CD133-positive cells are recruited, but sorted fractions contain low numbers of progeny cells. Double-purified CD133 cells are recruited (d). (b) PCR and qPCR analysis of sorted CD133-positive recruited cells and eGFP-positive progeny, showing absence of viral integration. (e\u2013j) H&E and DAPI-stained sections of transplanted gliomas induced by single-sorted eGFP-positive or CD133-positive cells. Note the presence of contaminating progeny in transplanted gliomas induced by single-sorted CD133-positive cell fractions.(TIF)Click here for additional data file.Figure S10Microarray analysis of normal olig2, recruited olig2 and glioma olig2 cells indicates that recruited cells are similar to glioma cells and differ from normal progenitors. (a) Glioma induced by transplantation of non-fluorescent hPDGFb-driven mouse glioma cells into wild-type olig2 RP-eGFP bacTRAP reporters, showing transgenic RP-eGFP expression and anti-eGFP stain (red). Note large numbers of RP-eGFP-positive recruited cells. Quality Controls Metrics plot and Pearson's correlation plot for Affymetrix arrays for normal (N), tumor (T) or recruited (R) olig2 cells. (d) Genespring GX10-generated profile plots showing averaged normalized intensity values for 500 mRNAs most different between glioma and normal olig2 cells (first plot), changing at least two-fold (second plot) or at least five-fold (third plot) between recruited olig2 cells and normal olig2 progenitor cells. (e) Principal component analysis on mRNAs with ANOVA-tested mRNA set removed. Note that all samples do not cluster well when the ANOVA-tested set of mRNAs specifically changed in tumor olig2 cells versus normal olig2 cells is removed.(TIF)Click here for additional data file.Table S1The dilution table for transplanted double-sorted recruited eGFP-negative cells. Incidence of gliomas induced by transplanting double-purified eGFP-negative recruited cell fractions derived from Ntv-a RCAS-PSG-induced mouse gliomas homozygous or heterozygous deleted for Ink4a, Arf and Pten at tumor initiation. Shown in the Table are numbers of large tumors and smaller proliferative lesions arising in recipient mice.(TIF)Click here for additional data file."} +{"text": "Together, the phosphorylation of PRAS40 is critical for the activation of mTOR in CNI-induced VEGF overexpression and renal cancer progression.Malignancy is a major problem in patients treated with immunosuppressive agents. We have demonstrated that treatment with calcineurin inhibitors (CNIs) can induce the activation of proto-oncogenic Ras, and may promote a rapid progression of human renal cancer through the overexpression of vascular endothelial growth factor (VEGF). Interestingly, we found that CNI-induced VEGF overexpression and cancer cell proliferation was inhibited by rapamycin treatment, indicating potential involvement of the mammalian target of rapamycin (mTOR) pathway in this tumorigenic process. Here, we examined the role of mTOR pathway in mediating CNI- and Ras-induced overexpression of VEGF in human renal cancer cells (786-0 and Caki-1). We found that the knockdown of raptor (using siRNA) significantly decreased CNI-induced VEGF promoter activity as observed by promoter-luciferase assay, suggesting the role of mTOR complex1 (mTORC1) in CNI-induced VEGF transcription. It is known that mTOR becomes activated following phosphorylation of its negative regulator PRAS40, which is a part of mTORC1. We observed that CNI treatment and activation of H-Ras (through transfection of an active H-Ras plasmid) markedly increased the phosphorylation of PRAS40, and the transfection of cells using a dominant-negative plasmid of Ras, significantly decreased PRAS40 phosphorylation. Protein kinase C (PKC)-\u03b6 and PKC-\u03b4, which are critical intermediary signaling molecules for CNI-induced tumorigenic pathway, formed complex with PRAS40; and we found that the CNI treatment increased the complex formation between PRAS40 and PKC, particularly (PKC)-\u03b6. Inhibition of PKC activity using pharmacological inhibitor markedly decreased H-Ras-induced phosphorylation of PRAS40. The overexpression of PRAS40 in renal cancer cells significantly down-regulated CNI- and H-Ras-induced VEGF transcriptional activation. Finally, it was observed that CNI treatment increased the expression of phosho-PRAS40 in renal tumor tissues Recent improvements in immunosuppressive therapies have significantly reduced the incidence of acute rejection of allografts, and increased the survival of transplant patients et al.et al.et al.The immunosuppressive agents are thought to compromise immune surveillance mechanism(s) of tumor cells and/or interfere with normal DNA repair mechanisms Vascular endothelial growth factor (VEGF) is one of the most potent angiogenic cytokines that plays important role in tumor growth In contrast to CNIs, the mammalian target of rapamycin (mTOR) inhibitor rapamycin (RAPA) may have a completely opposite effect in terms of tumor development The mTOR pathway plays a key role in cell survival, growth, protein synthesis, cellular metabolism, and angiogenesis In this study, we show that CNI-induced and Ras-PKC-mediated VEGF overexpression can be channeled through the mTORC1 signaling pathway, and this is mediated through the regulation of PRAS40. We demonstrate that CNI treatment and activation of H-Ras and PKC can lead to the phosphorylation of PRAS40; and overexpression of PRAS40 leads to the down-regulation of CNI- and Ras-induced VEGF transcriptional activation. The results of our study suggest a novel cross-talk among Ras, PKC and mTOR in regulating CNI-induced VEGF overexpression.We have recently demonstrated that treatment with calcineurin inhibitors (CNIs) can promote VEGF overexpression in human renal cancer cells through both transcriptional and post-transcriptional regulations We next examined the role of mTOR complex1 (mTORC1) in CNI-induced VEGF transcription. As discussed earlier, raptor is a part of mTORC1 upper panel); however, there was no significant change in the expression of total PRAS40 in these cells (lower panel). This observation suggests that the mTOR pathway is active in renal cancer cells.We have recently shown that CNI treatment can induce activation of H-Ras in renal cancer cells upper panel); however, there was no significant change in the expression of total PRAS40 following CsA treatment (lower panel).We next determined the effect of CsA treatment on PRAS40 phosphorylation. 786-0 cells were treated with either increasing concentrations of CsA or the vehicle alone; and the expression of phospho-PRAS40 was examined by Western blot analysis. As shown in first panel); there was no significant change in total PRAS40 following H-Ras activation (second panel). The overexpression of H-Ras(12V) in these cells was confirmed by Western blot analysis (third panel).Next, we sought to evaluate the effect of H-Ras activation on PRAS40 phosphorylation. To this end, Caki-1 cells were transfected with either increasing concentrations of the plasmid expressing activated form of H-Ras, H-Ras(12V), or the empty expression vector. Following transfection, the expression of phospho-PRAS40 and total PRAS40 was measured. We found that the activation of H-Ras significantly increased the level of phosho-PRAS40 compared with vector-transfected control (upper panel); however, there was no significant change in the expression of total PRAS40 (lower panel). Together, these observations suggest that CNI treatment and activation of the H-Ras pathway in human renal cancer cells can induce mTOR through increased phosphorylation of PRAS40.Finally, we checked the effect of the inhibition of endogenous Ras on PRAS40 phosphorylation. The Caki-1 cells were transfected either with the dominant-negative mutant of Ras, Ras(17N), or the empty vector, and the expression of phospho-PRAS40 was measured. As shown in In our previous report upper panel); however, there was no significant change in the expression of total PRAS40 (lower panel). Together, these observations suggest that PKC can associate with PRAS40, and regulate its phosphorylation. However, it cannot be concluded if there is a direct complex formation between PKC and PRAS40, and whether some other associated molecules are involved in PKC-mediated PRAS40 phosphorylation.Next, we tested if inhibition of PKC through the treatment of pharmacological inhibitor could decrease the phosphorylation of PRAS40. 786-0 cells were treated with either increasing concentrations of calphostin C or the vehicle alone. Following treatment, the expression of phospho-PRAS40 and total PRAS40 was measured by Western blot analysis. As shown in upper panel), activation of H-Ras induced the phosphorylation of PRAS40 compared with vector-transfected control; and the inhibition of PKC significantly down-regulated H-Ras-induced PRAS40 phosphorylation. There was no significant change in the expression of total PRAS40 following H-Ras activation and PKC inhibition (lower panel). These observations clearly suggest that the Ras-PKC pathway, which is critical for CNI-induced tumorigenic signaling events, can phosphorylate PRAS40, and may thus activate mTOR.In our earlier experiments, we have demonstrated that H-Ras activation could induce PRAS40 phosphorylation. Here, we wished to explore if the inhibition of PKC could down-regulate H-Ras-induced phosphorylation of PRAS40 in renal cancer cells. To this end, Caki-1 cells were transfected with either H-Ras(12V) or the empty expression vector in absence or presence of the PKC inhibitor calphostin C. Following transfection, the expression of phospho-PRAS40 and total PRAS40 was measured. As shown in lower panel).Our previous experiments suggested that CNI-induced and Ras-mediated signaling pathways could inactivate PRAS40 through its increased phosphorylation. Here, we first examined if the overexpression of PRAS40 could inhibit CNI-induced overexpression of VEGF in renal cancer cells. 786-0 cells were co-transfected with the VEGF promoter-luciferase construct and either PRAS40 overexpression plasmid or the empty expression vector. Cells were treated with either CsA or the vehicle alone. As shown in Next, we determined if the overexpression of PRAS40 could inhibit H-Ras-induced VEGF transcription. Caki-1 cells were co-transfected with the VEGF promoter-luciferase construct and H-Ras(12V) in absence or presence of the PRAS40 overexpression plasmid. Control cells were transfected with empty expression vectors. As shown in nu/nu) mice, CNI (CsA) treatment significantly accelerated the growth of human renal tumors (786-0) through VEGF-induced angiogenesis, compared with vehicle-treated controls top right panel), compared with tumor tissues from the vehicle-treated control group (top left panel). However, there was no significant change in the expression of total PRAS40 in tumor tissues obtained from CsA-treated (middle right panel) or vehicle-treated group (middle left panel). Our in vivo data is similar to our in vitro findings, and it suggests that CNI-mediated and VEGF-induced accelerated growth of human renal tumors may involve increased phosphorylation of PRAS40, which may lead to the activation of mTOR pathway.We have recently demonstrated that in immunodeficient following CNI treatment can promote the phosphorylation of PRAS40, and thereby may relieve the inhibition of mTORC1. Thus, targeting this pro-tumorigenic pathway may serve as novel therapeutics for the prevention and treatment of renal cancer, particularly in CNI-treated patients.CsA (Novartis) was purchased from Children's Hospital Boston pharmacy, and RAPA was purchased from LC laboratories. The PKC inhibitor calphostin C was obtained from Calbiochem. The small interfering RNA (siRNA) for raptor and its control were purchased from Qiagen. The transfection of siRNA was performed using Lipofectamine 2000 (Invitrogen).The human renal cancer cell lines (786-0 and Caki-1) were obtained from American Type Culture Collection. 786-0 cells were grown in RPMI 1640, and Caki-1 cells were grown in McCoy's medium supplemented with 10% fetal bovine serum (GIBCO). Human normal renal proximal tubular epithelial cells (RPTEC) were purchased from Clonetics and were grown in complete epithelial medium (REGM BulletKit).ras(12V) overexpression plasmid encodes active human H-Ras, in which expression is under the control of the cytomegalovirus promoter A 2.6-kb VEGF promoter-luciferase construct in pGL2 basic vector (Promega), containing full-length VEGF promoter sequence (\u22122361 to +298 bp relative to the transcription start site) was used in transient transfection assay 5 cells) were transfected with the Ras expression plasmids, PRAS40 expression plasmid, or the VEGF promoter-luciferase plasmid using Effectene Transfection Reagent (Qiagen), according to the manufacturer's protocol. The total amount of transfected plasmid DNA was normalized using a control empty expression vector. For luciferase assay, cells were harvested 48 hours after transfection, and luciferase activity was measured using a standard assay kit (Promega) in a luminometer. Transfection efficiency was determined by co-transfection of the \u03b2-galactosidase gene under control of cytomegalovirus immediate early promoter and by measurement of \u03b2-galactosidase activity using standard assay system (Promega).786-0 or Caki-1 using anti-PRAS40 (Invitrogen). Immunocomplexes were captured with protein A-Sepharose beads , and bead-bound proteins were subjected to Western blot analysis using either anti-PKC-\u03b6 or anti-PKC-\u03b4 (Santa Cruz Biotechnology).Protein samples were run on SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride membrane (Millipore Corporation). The membranes were incubated with anti-PRAS40 (Invitrogen), anti-phospho-PRAS40 (Invitrogen), anti-Ras (BD Transduction laboratories), anti-VEGF (Santa Cruz Biotechnology), anti-raptor or anti-\u03b2-actin (Sigma-Aldrich), and subsequently incubated with peroxidase-linked secondary antibody (Santa Cruz Biotechnology). All primary antibodies were diluted at 0.5 \u00b5g/ml; secondary antibodies were diluted at 0.2 \u00b5g/ml. The reactive bands were detected by using chemiluminescent substrate (Pierce). Expression was quantified by densitometry using the software Quantity One (version 4.6.2).2 x bV \u200a=\u200a \u03c0/6 x a, wherein a is the short axis and b is the long tumor axis. Mice were sacrificed at designated times after injection or if complications occurred, which included signs of inactivity, cachexia, or decreased responsiveness. The protocol (# 09-03-1298) for animal studies was approved by the review board of Children's Hospital Boston.Human renal cancer cells (786-0) were injected s.c. in immunodeficient (nu/nu) mice. The tumor volume was measured by following standard method Tissue sections were incubated first with either rabbit anti-human phospho-PRAS40 (Invitrogen) or mouse anti-human PRAS40 (Invitrogen), and then with a species-specific horseradish peroxidase-conjugated secondary antibody. Specimens were washed thoroughly in between incubations, developed in 3-aminoethylcarbazole, and counterstained with Gill's hematoxylin.t test. Differences with P<0.05 were considered statistically significant.Statistical evaluation for data analysis was determined by Student's"} +{"text": "The role of anti-cyclic citrullinated peptide (anti-CCP) antibodies in juvenile idiopathic arthritis (JIA) has become better understood; however, the identity of the target proteins of this modification remains elusive. We evaluated serum from patients with various subtypes of JIA to investigate the presence of anti-citrullinated fibrinogen and anti-citrullinated \u03b1-enolase antibodies, and their association with rheumatoid factor (RF) and anti-CCP antibodies.Sera were obtained from 96 JIA patients, 19 systemic lupus erythematosus (SLE) patients, and 10 healthy children. All sera were measured for antibodies against citrullinated and native fibrinogen and \u03b1-enolase by enzyme-linked immunosorbent assay. All results were correlated with clinical and laboratory parameters.Thirty-one (32%) JIA patients demonstrated reactivity to citrullinated fibrinogen and 9 (9%) to citrullinated \u03b1-enolase. Reactivity to citrullinated fibrinogen and \u03b1-enolase was predominantly found in IgM RF-positive polyarthritis patients . Anti-citrullinated fibrinogen antibodies were significantly elevated in JIA patients when compared to the healthy and SLE control groups (p<0.05). Antibody reactivity patterns showed that the largest group of JIA patients reacted only with fibrinogen (17%). Ninety-three percent of JIA patients positive for IgG anti-CCP antibodies also reacted with citrullinated fibrinogen, making up 10% of the JIA population. Anti-citrullinated fibrinogen antibodies correlated significantly with IgG and IgA anti-CCP antibodies and IgA and IgM RF (p<0.05). Significantly elevated levels of IgG, IgM, and IgA anti-CCP antibodies, and IgA and IgM RF were noted in JIA patients who were also positive for anti-citrullinated fibrinogen antibodies. IgM RF and anti-citrullinated fibrinogen antibodies demonstrated the highest sensitivity for IgM RF-positive polyarthritis and JIA overall (43.4% and 32.3% respectively). IgG and IgA anti-CCP antibodies and anti-citrullinated fibrinogen antibodies exhibited the highest specificity for JIA .Of the antibodies measured in this study, anti-citrullinated fibrinogen antibodies showed the strongest association with JIA when compared to healthy and SLE control groups. Additionally, anti-citrullinated fibrinogen antibodies demonstrated high sensitivity and specificity for IgM RF-positive polyarthritis patients, along with IgG anti-CCP antibodies and IgM RF. Our data would suggest that measuring anti-citrullinated fibrinogen antibodies, in addition to anti-CCP antibody isotypes and IgM RF, may be beneficial in identifying patients that will develop more aggressive disease. For the first time, we have identified fibrinogen as a potential target for citrullination in JIA, particularly in patients with IgM RF-positive polyarticular JIA.Brooke E. Gilliam: None; Melinda R. Reed: None; Anil K. Chauhan: None; Amanda Dehlendorf: None; Peri H. Pepmueller: None; Terry L. Moore: None."} +{"text": "Cells respond to misfolded and trafficking-deficient proteins by eliciting the unfolded protein response (UPR) and Activating Transcription Factor (ATF6) has been identified as a key regulator of the mammalian UPR. In this study, we investigated the role of ER chaperone proteins in the processing of G572R-hERG and E637K-hERG mutant proteins.Long QT syndrome type 2 (LQT2) is the second most common type of all long QT syndromes. It is well-known that trafficking deficient mutant human pcDNA3-WT-hERG, pcDNA3-G572R-hERG and pcDNA3-E637K-hERG plasmids were transfected into U2OS and HEK293 cells. Confocal microscopy and western blotting were used to analyze subcellular localization and protein expression. Interaction between WT or mutant hERGs and Calnexin/Calreticulin was tested by coimmunoprecipitation. To assess the role of the ubiquitin proteasome pathway in the degradation of mutant hERG proteins, transfected HEK293 cells were treated with proteasome inhibitors and their effects on the steady state protein levels of WT and mutant hERGs were examined.Our results showed that levels of core-glycosylated immature forms of G572R-hERG and E637K-hERG in association with Calnexin and Calreticulin were higher than that in WT-hERG. Both mutant hERG proteins could activate the UPR by upregulating levels of active ATF6. Furthermore, proteasome inhibition increased the levels of core-glycosylated immature forms of WT and mutant hERGs. In addition, interaction between mutant hERGs and Calnexin/Calreticulin was stronger after proteasome inhibition, compared to WT-hERG. These results suggest that trafficking-deficient G572R-hERG and E637K-hERG mutant proteins can activate ER stress pathways and are targeted to the proteasome for degradation. Calnexin and Calreticulin play important roles in these processes. It is characterized by delayed ventricular repolarization, QT prolongation on ECG, development of ventricular arrhythmias (torsades de pointes) and sudden deaths, particularly in children and teenagers Cells respond to the expression of misfolded and trafficking-deficient transmembrane proteins by eliciting the unfolded protein response (UPR), an ER stress pathway that increases the synthesis of chaperones proteins Although hERG channels have been studied extensively, little is known about the exact mechanism underlying the maturation and processing of trafficking-deficient hERG mutant proteins. G572R-hERG and E637K-hERG are two mutant forms of the hERG channel that have been previously reported as trafficking-deficient 2 incubator at 37\u00b0C. Cells were not used beyond 30 passages.The WT-hERG was cloned into pcDNA3 vector as described previously HEK293 cells expressing WT and mutant hERGs in 100-mm-diameter culture dishes were harvested 48 hours after transfection. Proteins were separated on SDS polyacrylamide gels and then transferred to polyvinylidene difluoride membranes . Primary antibodies used included rabbit polyclonal anti-hERG , rabbit polyclonal anti-ATF6 , mouse monoclonal anti-Calnexin and anti-Calreticulin . Blots were visualized with SuperSignal West Pico Chemiluminescent Substrate and developed using Syngene Chemi-Genius imaging system .U2OS cells seeded on coverslip in 6-well plate were transiently transfected with pcDNA3-WT-hERG, pcDNA3-G572R-hERG or pcDNA3-E637K-hERG plasmid. At 48 hours post-transfection, cells were fixed with paraformaldehyde, permeabilized with 0.1% Triton X-100 and blocked with 5% goat serum at room temperature (RT). Cells were then labeled with rabbit polyclonal anti-hERG (1\u223625 dilution) and mouse monoclonal anti-Calnexin or anti-Calreticulin (1\u223625 dilution) at 4\u00b0C overnight, followed by incubation with FITC-conjugated goat anti-rabbit IgG secondary antibody and TRITC-conjugated goat anti-mouse IgG secondary antibody at RT for 2 hours. Signals were captured with a Leica TCS SP2 confocal laser scanning microscope.HEK293 cells were transiently transfected with pcDNA3-WT-hERG, pcDNA3-G572R-hERG or pcDNA3-E637K-hERG plasmid and then lysed in 500 \u00b5l of immunoprecipitation buffer with protease inhibitors . Cell lysates were pre-cleared by incubation with protein G plus-agarose beads and incubated with 3 \u00b5g of antibody against Calnexin or Calreticulin at 4 \u00b0C overnight. The antigen-antibody complexes were isolated with protein G plus-agarose beads and washed with the immunoprecipitation buffer. The bound antigens were eluted from the protein G plus-agarose beads by 2\u00d7sample buffer and analyzed by immunoblotting with anti-hERG, anti-Calnexin and anti-Calreticulin antibodies.Transiently transfected HEK293 cells were treated with 20 \u00b5M lactacystin, 50 \u00b5M ALLN or 100 \u00b5M leupeptin for 24 hours. Part of the cell lysates were immunoblotted with anti-hERG, anti-Calnexin and anti-Calreticulin antibodies. The remaining lysates were immunoprecipitated with anti-ubiquitin , anti-hERG, anti-Calnexin or anti-Calreticulin antibodies.HEK293 cells were transiently transfected with G572R-hERG and E637K-hERG plasmids. At 24-hrs post- transfection, cells were treated with 100 \u00b5g/ml of cycloheximide and harvested at 0, 4, 8, and 12 hr post-treatment.The subcellular localization and protein trafficking of WT-hERG, G572R-hERG and E637K-hERG proteins were examined by immunostaining and confocal imaging Fig. 1. To better understand the maturation and trafficking of wild-type and mutant hERG proteins, HEK293 cells were transiently transfected with pcDNA3-WT-hERG, pcDNA3-G572R-hERG or pcDNA3-E637K-hERG plasmid and analyzed by Western blot. In To assess the physical interaction between hERG and Calnexin/Calreticulin, immunoprecipitation was done with anti-Calnexin or anti-Calreticulin antibody, followed by Western blot analysis with anti-hERG antibody. As shown in Since Calnexin/Calreticulin are chaperone proteins with important regulatory roles in the ER quality control pathways, we want to test whether the trafficking-deficient G572R-hERG and E637K-hERG mutants can activate ER stress responses. Given that ATF6 has been identified as a key regulator of transcriptional control during the mammalian UPR, HEK293 cells were transfected with wild-type and mutant hERG protein plasmids and ATF6 protein expression was analyzed by Western blot. The cells were harvested and lysed at 48-hrs post-transfection. As shown in , treatment with lactacystin or ALLN but not leupeptin (a lysosome inhibitor) results in an increase in the levels of core-glycosylated, immature form of hERG, Calnexin and Calreticulin. To further validate that the immature forms of wild-type and mutant hERG proteins are degraded through the ubiquitination-mediated proteasome pathway, transiently transfected HEK293 cells were treated with lactacystin, ALLN or leupeptin and lysates were subjected to immunoprecipitation with anti-hERG antibody. Precipitates were then immunoblotted with antibody against poly-ubiquitinated proteins. We found that proteasome inhibition increases levels of hERG poly-ubiquitination and to a greater extent for G572R-hERG or E637K-hERG than WT-hERG on the steady state protein levels of WT-hERG, G572R-hERG and E637K-hERG. As shown in WT-hERG Fig. 5b.To further examine the role of proteasome in the degradation of hERG proteins, coimmunoprecipitation analysis was performed. Similarly, transiently transfected HEK293 cells were treated with lactacystin or ALLN and cells were harvested at 24-hours post-treatment. The cell lysates were then immunoprecipitated with anti-Calnexin or anti-Calreticulin antibody and the precipitates were immunoblotted with anti-hERG antibody. As shown in Previous studies have shown that misfolded, incompletely folded and unassembled proteins are retained in the ER. This process is under stringent surveillance by the ER quality control system, which involves molecular chaperones that transiently associate with newly synthesized proteins to promote their proper folding and assembly Our results show that the trafficking-deficient G572R-hERG and E637K-hERG mutant proteins are mainly expressed in the cytoplasm and retained in the ER in association with Calnexin/Calreticulin. Furthermore, the proportion of Calnexin/Calreticulin in association with the core-glycoyslated, immature forms of G572R-hERG and E637K-hERG mutants is higher than that for WT-hERG. This indicates that the hERG mutant proteins fail to fold properly and thereby, accumulate in the ER in prolonged association with Calnexin/Calreticulin. Similarly, Gong et al has shown that the N470D-hERG mutant results in protein folding-deficiency and has prolonged association with Calnexin but not Calreticulin It is well-established that the ER quality control system operates efficiently such that only correctly folded molecules can exit the ER while misfolded proteins accumulate in the ER and activate the UPR Misfolded proteins retained in the ER are degraded by the ERAD process, which targets its substrates for ubiquitin-mediated proteasome degradation pathway. After treatment with proteasome inhibitors (lactacystin and ALLN), our results show that there is a notable increase in levels of core-glycosylated, immature forms of G572R-hERG and E637K-hERG proteins as well as accumulation of poly-ubiquitinated mutant hERG proteins. Furthermore, proteasome inhibition increases the association of Calnexin/Calreticulin with G572R-hERG and E637K-hERG. Previous study has also shown that lactacystin or ALLN treatment results in the accumulation of poly-ubiquitinated Y611H-hERG proteins in the ER and prolonged association with molecular chaperone compared to WT-hERG In conclusion, our findings provide evidence that the trafficking-deficient E637K-hERG and G572R-hERG mutants are retained in the ER and targeted to the proteasome for degradation. In addition, Calnexin/Calreticulin have prolonged association with E637K-hERG and G572R-hERG mutant proteins and may contribute to their degradation. Therefore, elucidating the exact mechanism by which Calnexin/Calreticulin interact with the mutant hERG channels will provide further insights into the pathophysiology underlying LQT2 and also facilitate the search for new pharmacological methods to restore proper trafficking of mutant hERG channels."} +{"text": "Replication-defective SIV elicited protective immunity in animals. In this first-in-human therapeutic vaccination study, a replication-defective HIV-1 vaccine was tested in HIV-1 infected subjects under antiretroviral therapy.A010 is an ongoing randomized, placebo-controlled dose-escalation clinical trial to evaluate the safety and the immunogenicity of two doses of a replication defective HIV-1 vaccine (HIVAX\u2122) in subjects receiving stable highly active antiretroviral therapy (HAART) who have an HIV-1 RNA <50 copies/ml and CD4 cell count >500 cells/mm3. Following the randomized placebo-controlled vaccination phase subjects who received active vaccine and who meet eligibility will undergo a 12-week analytical antiretroviral treatment interruption.HIVAX\u2122 is well tolerated in HIV infected subjects. Only mild injection site reaction occurred with transient duration. No medical treatment is necessary. High level of cell-mediated immune responses measured by ELISPOT assay was noticed after vaccination.The replication defective HIV vaccine appears no severe adverse effect in HIV-1 infected subjects. High level of cell-mediated immune response was elicited in the vaccinees. HIVAX\u2122 is worth for further evaluation of protective efficacy."} +{"text": "Hedgehog signalling has been implicated in prostate tumorigenesis in human subjects and mouse models, but its effects on transforming normal basal/stem cells toward malignant cancer stem cells remain poorly understood.We produced pCX-shh-IG mice that overexpress Hedgehog protein persistently in adult prostates, allowing for elucidation of the mechanism during prostate cancer initiation and progression. Various markers were used to characterize and confirm the transformation of normal prostate basal/stem cells into malignant cancer stem cells under the influence of Hedgehog overexpression.+ basal/stem cells along with simultaneous activation of Hedgehog signalling members, suggesting that P63+/Patch1+ and P63+/Smo+ cells may serve as cancer-initiating cells and progress into malignant prostate cancer stem cells (PCSCs). In the hyperplastic lesions and tumors, the progeny of PCSCs differentiated into cells of basal-intermediate and intermediate-luminal characteristics, whereas rare ChgA+ neuroendocrine differentiation was seen. Furthermore, in the metastatic loci within lymph nodes, kidneys, and lungs, the P63+ PCSCs formed prostate-like glandular structures, characteristic of the primitive structures during early prostate development. Besides, androgen receptor (AR) expression was detected heterogeneously during tumor progression. The existence of P63+/AR-, CK14+/AR- and CD44+/AR- progeny indicates direct procurement of AR- malignant cancer trait.The pCX-shh-IG mice developed prostatic intraepithelial neoplasia (PIN) that led to invasive and metastatic prostate cancers within 90 days. The prostate cancer was initiated through activation of P63These data support a cancer stem cell scenario in which Hedgehog signalling plays important roles in transforming normal prostate basal/stem cells into PCSCs and in the progression of PCSCs into metastatic tumor cells. Adult prostate epithelial stem cells reside within the basal cell layer and possess high self-renewal capacity, leading to the generation of intermediate, luminal, and neuroendocrine cell lineages ,2. Norma+ luminal cell population [(- or low) [Like many other cancer stem cells, a hypothesis of prostate cancer stem cells (PCSCs) originated from normal stem cells has been proposed based on their highly tumorigenic trait and basal/stem cell-like properties of self-renew and differentiation -9. The hpulation . The ent or low) , likely + basal cells in human specimens and these cells are capable of differentiation into multiple lineages, suggesting that Hh signalling may promote primary prostatic cancer stem cells [Hedgehog (Hh) signalling plays a key role in stem cell plasticity and in many developmental, physiological, and pathogenetic processes . Bindingem cells . Howeverem cells , taking em cells ,24.+ basal/stem cells and to examine whether these cancer cells can maintain stem cell characteristics after metastasis. More importantly, we intended to elucidate whether P63+ basal/stem cells can be directly transformed into AR- cancer cells. We demonstrated that Hh overexpression initiated malignant transformation of P63+ basal/stem cells that subsequently differentiated into both AR+ and AR- progeny of the basal-intermediate and intermediate-luminal progeny, but rare ChgA+ neuroendocrine cells. The Hh-initiated P63+ basal/stem cells were characteristic of PCSCs, as they were able to form primitive prostate-like glandular structures in the metastatic loci. Besides, androgen receptor (AR) expression was detected heterogeneously in PCSCs when they were differentiated into intermediate and luminal cells, indicating that androgen were not necessary for these PCSCs (AR-). These data challenge the model of AR+ transition into androgen-independent PCSCs and suggest a potentially better treatment strategy by inhibition of Hedgehog signalling prior to androgen-deprivation therapy.In this study, we used the Hh overexpression mouse model to elucidate whether the PCSCs arise from P63Mouse Shh-expressing pCX-shh-IG vector and pCX-IG vehicle control vectors were kindly provided by Dr. Kerby C. Oberg, Loma Linda University . The pCXICR strain male mice aged 8-10 weeks were purchased from National Laboratory Animal Center, Academia Sinica, Taipei for use in this study. The mice were anesthetized and exposed of their prostate glands by surgery, followed by intraprostatic injection and electroporation to introduce the pCX-shh-IG or pCX-IG vectors as described in our previous study . All aniStandard procedures were followed to prepare prostate tissue sections for immunohistochemistry. Antigen retrieval was achieved by boiling tissue in citrate buffer (pH 6.0) for 20 min. Primary antibodies were goat anti-Shh antibody (N-19), rabbit anti-Patch1 (H-267), goat anti-Patch1 (G-19), rabbit anti-Smo (H-300), rabbit anti-Gli1 (H-300), goat anti-Gli2 (N-20), goat anti-Gli3 (N-19), goat anti-CK14 (C-14), and mouse anti-CD44 (DF-1485); all were purchased from Santa Cruz Biotechnology . The mouse anti-p63 (4A4), mouse anti-CK8 (TS1), rabbit anti-AR (RB-9030), mouse anti-PCNA (MS-106), rabbit anti-ChgA (RB-9003) and mouse anti-tubulin (MS-581) were purchased from Lab Vision Corporation . The secondary antibodies were horseradish peroxidase- conjugated anti-mouse, anti-goat, and anti-rabbit IgG purchased from Jackson ImmunoResearch Laboratories, Inc., PA. For double-immunofluorescence detection, primary antibodies were applied simultaneously, followed by incubation with donkey anti-mouse rhodamine Red-X and FITC, anti-rabbit rhodamine Red-X and FITC anti-goat FITC (1:50) and counterstained with DAPI. Standard brightfield and immunofluorescence microscopy were performed for photography using a Zeiss Axioskop2 Plus microscope and SoftWoRx software. In situ apoptosis assay (TUNEL assay) was processed following the manufacturer's instruction .Standard procedures were performed for western blot analysis. Briefly, protein extract (150 \u03bcg) was fractionated on SDS-polyacrylamide electrophoresis gel and transferred to a polyvinylidine difluoride membrane . The membrane was then incubated with primary antibody (described above) overnight at 4\u00b0C, followed by incubation with secondary antibody for 1 hour. The immune complexes on membranes were detected by chemiluminescence methods .2 boxes showing the normal, PIN or the CaP sites were used for quantification. The results were presented as the average percentage of double-positive cells over total counted cells. The difference between the normal, PIN, and the CaP sites was analyzed by Student's t test (significant when p < 0.001).Tissue sections from five prostates of the pCX-IG-injected vehicle controls and five pCX-shh-IG-injected mouse prostates were used. Each prostate specimen was from an individual mouse. In each specimen, three randomly picked 1000 \u03bcm+ . The tumors, found exclusively in the prostates, showed characteristics of progressive tumorigenesis through stages of prostatic gland hyperplasia, prostatic intraepithelial neoplasia (PIN), and prostate cancer (CaP) Figure . The pCX+ Figure . The Pat+ Figure to 2L, w+ Figure to 2O. M+ Figure to 2J, i+ Figure to 2E. T+ Figure . Patch1,+ Figure . Activat+ basal/stem cells were activated under the influence of Hedgehog overexpression, we examined the pCX-shh-IG-injected prostates during the progression of PIN toward CaP to gain further insights indicated P63+/Smo+ cells). In contrast, only very limited Patch1 or Smo expression was detected in the quiescent P63+ basal cells in the vehicle controls indicated P63+/Smo+ cells). These data support Hedgehog involvement in promotion of basal cell hyperplasia toward malignant transformation.Since prostate tumorigenesis was induced and P63rostates , it is t+ cancer cells induced by Hedgehog overexpression were characteristic of PCSCs, we examined their stemness property and capacity of metastasis. Since pCX-shh-IG vector was only introduced in the prostates, the GFP signals were expected to be detected only within the prostates indicated AR- basal cells). This finding was confirmed by western blot analysis at day 90 after the injection , CK14 , CD44 (intermediate cells), and CK8 . In the normal vehicle control prostates, fewer CK14+ basal cells lay flat along the basement membrane cells in the normal prostates cells appeared to be differentiated into P63+/CK14+ /CK14+ /CD44(low or -) cells in the normal prostates cells /CD44+ cells /CK8- cells in the normal prostates progeny Figure and inte) Figure progeny + cells, we detected P63+/AR-, CK14+/AR-, CD44+/AR- and even the CK8+/AR- cells in the CaP and P63+/Smo (Low or -) quiescent basal cells into the P63+/Patch1+ and P63+/Smo+ hyperplastic basal cells, comparable to the human BCH condition that had been previously observed [+ cells showed major cancer cell and stem cell peculiarity on the metastatic sites. The P63+ cells were not only present within various metastatic loci, but also differentiated into prostate-like glandular structures where they were located within the basal compartment. Thirdly, the P63+ basal/stem cells, after being transformed into malignant cells, were capable of differentiation into the basal-intermediate (P63+/CK14+) and intermediate-luminal (CK14+/CD44+ and CK14+/CK8+) progeny, and rarely the ChgA+ neuroendocrine lineage.In this study, we provide the evidence to further support PCSCs derivation from normal prostate stem cells, based on several lines of observations. Firstly, the entire process of prostate tumorigenesis was reconstituted in vivo and the effects of Hh overexpression on the normal prostate stem cells was shown by the transformation of P63observed . Secondl+ and CD49f+ cells from the mouse prostates [+, CD133+, and \u03b12/\u03b21 integrin+ cells from the human prostates [- P63-CD44+Nestin+ HPET-5 cells were purified from prostate cancer cell lines [+ cancer cells in the metastatic loci and generation of prostate-like glandular structures. Our data support the prostate cancer stem cell characteristics observed in previous studies and, to our knowledge, these are the first data to confirm that PCSCs metastasis occurs under in vivo conditions.PCSCs derivation from normal prostate stem cells has also been supported by several lines of evidence. Purified cells such as Sca-1rostates ,30 or CDrostates ,32 were ll lines . Despite- cancer cells found in many high grade prostate cancers [+ and AR- cells were capable of forming androgen-independent prostate cancer cells as the tumorigenesis progresses to the more advanced stages. In this study, both the AR+ and AR- prostate cancer cells were generated under the effects of persistent Hedgehog signalling activation in the mouse model . Here, the key findings in this study have confirmed that overexpression of Hedgehog can transform prostate basal cells in vivo and lead the transformed cells to progress into aggressive AR- PCSCs progeny. Although the nuclear AR+ (active form) and cytoplasmic AR+ (inactive form) cells were both observed in the aggressive tumors in this in vivo model and our data showed that Hedgehog signalling activation may substitute the androgen-AR axis for tumor survival or malignant transformation, the underlying mechanisms remain to be further investigated by using in vitro studies.Clinically, it is known that advanced metastatic androgen-independent prostate cancers exhibit more basal/stem cell-like differentiation although the underlying mechanism remains unclear ,10. The cancers -40 and t cancers ,41. Therl Figure . Our dat+ hyperplastic basal cells targeted by Hh overexpression may be the true cellular origin of primary prostate cancer. This study also supports that inhibition of Hedgehog signalling may be a better treatment strategy for androgen-independent tumors prior to androgen-deprivation therapy.Our data support the hypothesis that P63The authors declare that they have no competing interests.HHC, BYC, and DPL designed the study, carried out production of pCX-shh-IG and pCX-IG mice, and contributed to the writing of manuscript. CYW helped with animal maintenance, plasmid vector preparation, immunohistochemical and double-immunofluorescence staining. ZJT performed TUNEL assay and western blot analysis. CPC and CRY carried out the quantification of positive and double-positive cells."} +{"text": "Ferroportin (FPN) is the only known cellular iron exporter in mammalian cells and plays a critical role in the maintenance of both cellular and systemic iron balance. During iron deprivation, the translation of FPN is repressed by iron regulatory proteins (IRPs), which bind to the 5\u2032 untranslated region (UTR), to reduce iron export and preserve cellular iron. Here, we report a novel iron-responsive mechanism for the post-transcriptional regulation of FPN, mediated by miR-485-3p, which is induced during iron deficiency and represses FPN expression by directly targeting the FPN 3\u2032UTR. The overexpression of miR-485-3p represses FPN expression and leads to increased cellular ferritin levels, consistent with increased cellular iron. Conversely, both inhibition of miR-485-3p activity and mutation of the miR-485-3p target sites on the FPN 3\u2032UTR are able to relieve FPN repression and lead to decreased cellular iron levels. Together, these findings support a model that includes both IRPs and microRNAs as iron-responsive post-transcriptional regulators of FPN. The involvement of microRNA in the iron-responsive regulation of FPN offers additional stability and fine-tuning of iron homeostasis within different cellular contexts. MiR-485-3p-mediated repression of FPN may also offer a novel potential therapeutic mechanism for circumventing hepcidin-resistant mechanisms responsible for some iron overload diseases. Cellular iron homeostasis is maintained by a sophisticated system that responds to iron levels and coordinates the expression of targets important for balancing iron export and uptake with intracellular storage and utilization. Ferroportin is the only known cellular iron exporter in mammalian cells and plays a critical role in both cellular and systemic iron balance. Thus the ability to regulate cellular iron export is of great interest in the search for therapeutic strategies to control dysregulated iron homeostasis, iron overload disorders, and conditions affected by cellular iron concentrations such as antimicrobial resistance. During iron deprivation, repression of ferroportin levels reduces iron export and preserves cellular iron. Ferroportin translation is known to be repressed by iron regulatory proteins that bind to the 5\u2032UTR, yet alternative mechanisms that can post-transcriptionally regulate ferroportin have not been previously reported. Here, we find that miR-485-3p is induced during iron deficiency and represses ferroportin by directly targeting its 3\u2032UTR, and further experimental evidence supports a model that includes both iron regulatory proteins and microRNAs as post-transcriptional regulators of ferroportin. These findings demonstrate a novel role for microRNAs in the cellular response to iron deficiency and can have therapeutic implications for various diseases of iron homeostasis. While iron is an essential nutrient for all cells, high levels of iron can lead to toxicity. Therefore, cellular iron homeostasis is carefully maintained by an exquisite system of iron regulatory proteins (IRPs) that respond to iron levels and coordinate the expression of targets important for balancing iron export and uptake with intracellular storage and utilization Given the important regulatory role of FPN, it is not surprising that FPN is regulated at multiple levels\u2013transcriptionally by heme Hfe and Hjv, which encode proteins important for the hepcidin hormone response to systemic iron availability Although there is a greater understanding of transcriptional and IRP-mediated regulation of FPN under various stresses such as heme, nitric oxide, oxidative stress, and hypoxia In this study, we examine the potential role of microRNAs in the IRP/IRE-independent post-transcriptional regulation of FPN. We identify microRNAs with altered expression under cellular iron deprivation and find that the microRNA miR-485-3p directly targets the 3\u2032 UTR of FPN. Through gain-of-function and loss-of-function studies, we provide compelling evidence to support a role for miR-485-3p as an important post-transcriptional regulator of endogenous FPN expression and modulator of cellular iron homeostasis.To investigate the iron-responsive regulation of the FPN expression, we first demonstrated the known iron-dependent IRP-mediated regulation of the FPN 5\u2032UTR using a luciferase reporter construct with the full FPN 5\u2032UTR placed upstream of luciferase . The iroTo determine whether the FPN 3\u2032UTR, which lacks IRE, could also be a target of iron-dependent regulation, we used a reporter construct with the full FPN 3\u2032UTR placed downstream of luciferase and analyzed reporter activity in HepG2 cells under different iron conditions. Surprisingly, we found that iron depletion led to significant inhibition of FPN 3\u2032UTR reporter activity . AdditioBoth RNA-binding proteins and microRNAs are known to function as post-transcriptional regulators via the 3\u2032UTR. To identify microRNAs that could play a role in this regulation, we performed microRNA profiling to identify iron-responsive microRNAs in the K562 erythroid cell line, a well-characterized model for the study of cellular iron metabolism To prioritize the iron-responsive microRNAs with potential regulatory roles in iron homeostasis, we first analyzed the microRNAs \u2013S2B withTo date, miR-485-3p has shown only one confirmed target, which is involved in the expression of DNA topoisomerase II in human lymphoblastic leukemia cells The FPN 3\u2032UTR has predicted target sites for both miR-194 and miR-485-3p . To deteNext, we mutated the sequence of the only predicted canonical 8mer miR-485-3p binding site on the FPN 3\u2032UTR, given the high confidence for microRNA-mediated repression with this predicted seed match type Next, we assessed the effect of miR-485-3p on endogenous FPN protein expression and intracellular iron regulation. A previously published FPN antibody The specific inhibition of miR-485-3p activity in HepG2 cells by AMOs led to significant and reproducible increase in FPN protein levels in response to iron depletion \u2013S3J. We Finally we sought to mimic the regulation of endogenous FPN mRNA by constructing a luciferase reporter (FPN-5UTR-LUC-3UTR) containing both the FPN 5\u2032UTR and FPN 3\u2032UTR placed upstream and downstream of luciferase, respectively . We measWe used the FPN-5UTR-LUC-3UTR, FPN-5\u2032UTR, and FPN 3\u2032UTR reporters to further characterize the effect of both regulatory regions on post-transcriptional regulation of FPN in response to miR-485 overexpression or inhibIn summary, we demonstrate the post-transcriptional regulation of FPN during the iron-deficient condition by miR-485-3p via the 3\u2032UTR in addition to the well-recognized regulation by IRPs via the 5\u2032UTR . These fGiven the crucial role of FPN in iron metabolism, extensive regulation of FPN occurs at multiple levels, including the transcriptional in vitro. The use of primary macrophages and several cell types studied, and their longstanding use in this field offers a broad baseline and physiologically relevant context to indicate the potential relevance of individual microRNAs and their functional target(s) in cellular iron regulation. But it will be important to further establish the in vivo relevance of these findings using clinical samples from individuals with iron overload and iron deficiency conditions or receiving treatments to correct conditions of iron deficiency or overload.The discovery of iron-responsive microRNAs and microRNA-mediated regulation of FPN in several cell lines and primary macrophages illustrates the complexity of regulatory mechanisms for the precise and dynamic regulation of cellular iron. However, these findings were mainly obtained from the cellular response to varying iron levels Hjv and Hfe, both important for hepcidin expression, and has been shown to play an important role in the control of murine systemic iron homeostasis Several microRNAs have been found to regulate targets with key roles in iron homeostasis. The hypoxia-induced miR-210 is known to directly target ISCU1/2, which play a role in the biogenesis and integrity of iron-sulfur clusters in vivo target mRNAs associated with Ago2 during different iron states. Such exploration of iron-responsive microRNAs and their respective targets will lead to a more comprehensive pathway demonstrating an integrated role for microRNAs in the regulation of cellular iron homeostasis.The regulation of FPN by microRNAs is likely to be distinct from other well-established mechanisms in several important ways. Unlike the systemic regulation of FPN by circulating hormone hepcidin, the monitoring of local iron levels by cellular microRNAs can lead to a more dynamic response to spatial and temporal fluctuations. While both microRNAs and IRPs are iron-responsive and target a group of mRNAs, they may also respond to different sets of non-iron environmental conditions and regulate distinct sets of target mRNAs to allow for diversity and fine-tuning of gene regulation. The targeting of FPN by microRNAs in the 3\u2032 UTR allows for the possibility of iron-dependent regulation of subsets of FPN mRNAs known to lack the 5\u2032 UTR Since FPN is known to be repressed by the IRP/IRE system under the iron-deficient condition, our findings suggest a potential cooperative relationship between RNA-binding proteins (RBPs) and microRNAs in the regulation of FPN. The cooperative contribution of RBPs, including both IRPs and the microRNA-guided RISC, to the post-transcriptional regulation of target RNAs constitutes a major regulatory layer of gene expression SalmonellaFinally, microRNA-mediated regulation by miR-485-3p may offer a novel alternative means to target intracellular FPN and alter cellular iron status. Successful proof-of-concept studies supporting the use of therapeutic microRNA mimics have been demonstrated with microRNAs identified as functional tumor suppressors in mouse models of cancer K562 cells were maintained in RPMI; 293 and HepG2 cells were maintained in DMEM. All cells were incubated in humidified atmosphere of 5% CO2 at 37\u00b0C and supplemented with 10% fetal bovine serum (HyClone) and 1% penicillin and streptomycin. Ferric ammonium citrate (FAC) and deferoxamine (DFE) were purchased from Sigma. For iron depletion, cells were treated with DFE (100 \u00b5M unless otherwise indicated) diluted in PBS and added to the media for indicated time intervals (16\u201324 hours). For iron supplementation, cells were treated with FAC (500 nM) diluted in PBS and added to the media for indicated time intervals (16\u201324 hours).Human peripheral blood mononuclear cells (PBMC) were isolated by Ficoll Paque density centrifugation from whole blood. Monocytes were enriched from freshly isolated PBMC through plastic adherence for 1\u20132 hours. To differentiate monocytes into macrophages, cells were plated into RPMI 1640 media with 2 mM glutamine (Gibco) containing 10% fetal bovine serum (Hyclone), 100 \u00b5g/ml streptomycin, 100 U/ml penicillin, 1% Na-pyruvate, 1% NEAA and 50 ng/ml rHu M-CSF. Cells were allowed to differentiate over a course of seven days, and then treated.CAGCTAGCCCGACTCGGTATAAGAGCTG; reverse: CAGCTAGCAACAGGAGTGCAAGGAACTG) and cloned upstream of Renilla luciferase to form FPN-5UTR-LUC. The 3\u2032UTR of FPN amplified using primers and cloned into the MCS downstream of Renilla luciferase (FPN-3UTR-LUC). The 3\u2032UTR of TFRC amplified using primers and cloned into the MCS downstream of Renilla luciferase (TFRC-3UTR-LUC).Luciferase reporters were constructed using the psi-CHECK2 vector (Promega). The 5\u2032UTR of FPN was amplified using primers . Reporter assays were conducted using the Dual Luciferase Reporter Assay System (Promega) and the Tecan Infinite F200 reader according to manufacturer's protocol.Mutant reporters were constructed using primer-based overlapping PCR with the following primers: FPN3UTRmt448-F- AAAAGAAGTCAGCCATGTGT; pc-miR-194-forward: GAATTCCCATGATGAGCAAAAGGAATC; pc-miR-194-reverse: CTCGAGATCAAAAGTAACAGCATCTC; Antisense-2\u2032O-methyl-modified nucleotides were purchased from (Dharmacon). AMO-485-3p: AGAGAGGAGAGCCGTGTATGAC; AMO-CNTL1: AAGGCAAGCUGACCCUGAAGU; AMO-CNTL2: CCAUCUUUACCAGACAGUGUUA. Control (Ambion) and FPN siRNA (SMARTpool from Dharmacon) were transfected into K562 and HepG2 cells using nucleofection (Amaxa) and lipofection (Invitrogen) methods.Expression constructs encoding miR-485 (pc-miR-485) and miR-194 (pc-miR-194) were created by insertion into a cytomegalovirus-based pcDNA3 cloning vector (Invitrogen) using the following primers: pc-miR-485-forward:TCATGTGTGGTACTTGGAGA; pc-miR-485-reverse: Quantitative real-time RT-PCR (qRT-PCR) analysis of microRNA expression using TaqMan Low Density Arrays (TLDA) Human microRNA Panel (Applied Biosystems) was conducted according to manufacturer's instructions using the ABI 7500 real-time PCR system (Applied Biosystems). The Ct data were obtained by the RQ Manager v1.2 software using automatic threshold settings and normalized to RNU48 endogenous control. MicroRNAs with a log2 expression change of at least 0.5 in either the iron-rich or iron-deficient condition when compared to baseline were considered to be iron-responsive. Individual qRT-PCR Taqman mature microRNA assays (Applied Biosystems) were used for validation of results from TLDA. qRT-PCR analysis of mRNA expression using Power SYBR Green (Applied Biosystems) was conducted as described previously Western blots were performed as described in t test. Results were considered statistically significant at a p-value <0.05 (*), <0.01 (**), or <.0001 (***). n.s\u200a=\u200anonsignificant. Graphs were generated using Prism 5 software Statistical analyses were performed using the Student's GCACATAGTAGGCGCTCAATQPCR-Beta tubulin-F- ATCTGGAGACCCAGCTTCTTQPCR-Beta tubulin-R- GACATGAGCAAGAGCCTACQPCR-FPN-F- AGGCTGGTTGTAGTAGGAGAQPCR-FPN-R- AAAATCCGGTCTAGGCACAGQPCR-TFRC-F- CCTTTAAATGCAGGGACGAAQPCR-TFRC-R- GGGTCTGTCTCTTGCTTCAACQPCR-FTL-F- GGTTGGCAAGAAGGAGCTAAQPCR-FTL-R- AGCAAGAGCACAAGAGGAAGQPCR-GAPDH-F- GGTTGAGCACAGGGTACTTTQPCR-GAPDH-R- Figure S1Activity of FPN 5\u2032UTR and 3\u2032UTR luciferase reporters in K562 cells following iron-supplementation (FAC) or iron-depletion (DFE). Data expressed as fold change in luminescence \u00b1 SEM relative to baseline condition, normalized to empty reporter control (n\u200a=\u200a3). * Significantly different by Student's t-test: **p<0.01, ***p<0.0001.(PDF)Click here for additional data file.Figure S2(A\u2013B) Predicted iron-relevant mRNA targets of significantly upregulated (A) and downregulated. (B) microRNAs (listed in (PDF)Click here for additional data file.Figure S3(A) Western blot confirmation of antibody specificity by silencing of FPN with siRNAs. HepG2 lysate from cells transfected with control or FPN-targeting siRNAs were analyzed by PAGE and probed with FPN antibody. One indicated \u223c68 KDa band was recognized as FPN protein by its reduction in intensity following the silencing of FPN. (B) Western blot analysis of FPN in K562 cells transfected with miR-485 (pc-miR-485) or vector control (pc-vector), normalized to tubulin levels. (C) Ferritin protein levels from corresponding samples in (PDF)Click here for additional data file.Table S1List of genes involved in cellular iron ion homeostasis from gene ontology (GO: 006879) analysis, using the microRNA.org and TargetscanHumanv6.0 databases.(PDF)Click here for additional data file.Table S2A list of miR-485-3p target mRNAs predicted by Targetscan.(PDF)Click here for additional data file."} +{"text": "Interferon-beta (IFN-\u03b2) has both immuno-modulating and anti-viral effects. In a longitudinal study of multiple sclerosis (MS) patients undergoing interferon-beta therapy, we have performed a comprehensive study of factors in the innate and adaptive immune response to the two types of virus associated with MS: human endogenous retroviruses (HERVs), and herpesviruses.Anti-viral antibodies towards HERVs and herpesviruses were assayed using TRIFMA or ELISA. Cytokine profiling was performed using the Luminex-system. Factors in the lectin complement activation pathway were assayed using TRIFMA.We demonstrate significant decreases in anti-Envelope antibody reactivity for the two closely related Gammaretroviral HERVs, HERV-H and HERV-W, as a consequence of IFN-\u03b2 therapy, closely linked to efficacy of therapy/low disease activity. We also found strong indications of a protective effect of high levels of two components in the innate pathogen-associated molecular pattern recognition: mannan-binding lectin (MBL), and MASP-3.Serum levels of typical Th1- and Th2- related, MS-relevant cytokines were also monitored. We found no overall changes in Th1/Th2 ratios.Our results support that IFN-\u03b2 exerts effects on immune response to HERV-H/HERV-W, and that this antiviral response may play a role in MS development. Components in the immune response to HERVs have potential as biomarkers for disease activity."} +{"text": "PTHLH feedback-mediated cell adhesion gene ontology (GO) network of human hepatocellular carcinoma (HCC) compared with the corresponding low expression activated GO network of no-tumor hepatitis/cirrhotic tissues (HBV or HCV infection). Activated PTHLH feedback-mediated cell adhesion network consisted of anaphase-promoting complex-dependent proteasomal ubiquitin-dependent protein catabolism, cell adhesion, cell differentiation, cell-cell signaling, G-protein-coupled receptor protein signaling pathway, intracellular transport, metabolism, phosphoinositide-mediated signaling, positive regulation of transcription, regulation of cyclin-dependent protein kinase activity, regulation of transcription, signal transduction, transcription, and transport in HCC. We proposed activated PTHLH coupling feedback phosphoinositide to G-protein receptor signal-induced cell adhesion network. Our hypothesis was verified by the different activated PTHLH feedback-mediated cell adhesion GO network of HCC compared with the corresponding inhibited GO network of no-tumor hepatitis/cirrhotic tissues, or the same compared with the corresponding inhibited GO network of HCC. Activated PTHLH coupling feedback phosphoinositide to G-protein receptor signal-induced cell adhesion network included BUB1B, GNG10, PTHR2, GNAZ, RFC4, UBE2C, NRXN3, BAP1, PVRL2, TROAP, and VCAN in HCC from GEO dataset using gene regulatory network inference method and our programming.Studies were done on analysis of biological processes in the same high expression (fold change \u22652) activated PTHLH is one of our identified significant high expression (fold change \u22652) genes in human hepatocellular carcinoma (HCC) compared with low expression no-tumor hepatitis/cirrhotic tissues (HBV or HCV infection) from GEO data set GSE10140-10141 [ PTHLH is presented in some papers, such as Mouse pthlh gene-specific expression profiles distinguish among functional allelic variants in transfected human cancer cells [ PTHLH feedback-mediated cell adhesion mechanism in HCC is not clear and remains to be elucidated.Study ofer cells ; parathyer cells ; parathyer cells ; parathyer cells ; parathyer cells ; dysreguer cells ; all majer cells ; parathyer cells . Yet thePTHLH feedback-mediated cell adhesion GO network in HCC were identified and computed compared with the corresponding low expression activated GO network of no-tumor hepatitis/cirrhotic tissues (HBV or HCV infection), the different compared with the corresponding inhibited PTHLH feedback-mediated cell adhesion GO network of no-tumor hepatitis/cirrhotic tissues, and the same compared with the corresponding inhibited GO network of HCC, respectively. Simultaneous occurrence of biological processes was identified between the same activated PTHLH feedback-mediated cell adhesion GO network of HCC (compared with the corresponding activated GO network of no-tumor hepatitis/cirrhotic tissues) and the different (compared with the corresponding inhibited PTHLH feedback-mediated cell adhesion GO network of no-tumor hepatitis/cirrhotic tissues), or the same (compared with the corresponding inhibited GO network of HCC) for putting forward hypothesis of activated PTHLH coupling feedback phosphoinositide to G-protein receptor signal-induced cell adhesion network. Activated PTHLH feedback-mediated cell adhesion molecular network and numbers in HCC were extracted and computed from the same activated PTHLH GO-molecular network of HCC compared with the corresponding activated GO-molecular network of no-tumor hepatitis/cirrhotic tissues. PTHLH coupling feedback phosphoinositide to G-protein receptor signal-induced cell adhesion molecular relationship in HCC was identified including different molecules but same GO term and same molecule but different GO terms from the same activated PTHLH GO-molecular network of HCC compared with the corresponding activated GO-molecular network of no-tumor hepatitis/cirrhotic tissues.In this study, biological processes and occurrence numbers of the same activated high expression (fold change \u22652) PTHLH feedback-mediated cell adhesion mechanism of HCC based on GEO data set GSE10140-10141 . The raw microarray data was preprocessed by log base 2. Microarray 6,144 genes were used for analyzing activatedhttp://www-stat.stanford.edu/~tibs/SAM/) [225 significant high expression (fold change \u22652) molecules in HCC compared with no-tumor hepatitis/cirrhotic tissues (HBV or HCV infection) were identified using significant analysis of microarrays (SAM) (bs/SAM/) . We sele PTHLH feedback-mediated cell adhesion mechanism of HCC was analyzed by using Molecule Annotation System, MAS . The primary databases of MAS integrated various well-known biological resources, such as Gene Ontology (http://www.geneontology.org/), KEGG (http://www.genome.jp/kegg/), BioCarta (http://www.biocarta.com/), GenMapp (http://www.genmapp.org/), HPRD (http://www.hprd.org/), MINT (http://mint.bio.uniroma2.it/mint/Welcome.do), BIND (http://www.blueprint.org/), Intact (http://www.ebi.ac.uk/intact/), UniGene (http://www.ncbi.nlm.nih.gov/unigene), OMIM (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIM), and disease . ActivatedPTHLH feedback-mediated cell adhesion GO network in HCC were identified and computed compared with the corresponding low expression activated GO network of no-tumor hepatitis/cirrhotic tissues (HBV or HCV infection), the different compared with the corresponding inhibited PTHLH feedback-mediated cell adhesion GO network of no-tumor hepatitis/cirrhotic tissues, and the same compared with the corresponding inhibited GO network of HCC by our programming, respectively.Biological processes and occurrence numbers of the same activated high expression (fold change \u22652) PTHLH feedback-mediated cell adhesion GO network of HCC (compared with the corresponding activated GO network of no-tumor hepatitis/cirrhotic tissues) and the different (compared with the corresponding inhibited PTHLH feedback-mediated cell adhesion GO network of no-tumor hepatitis/cirrhotic tissues), or the same (compared with the corresponding inhibited GO network of HCC) for putting forward hypothesis of activated PTHLH coupling feedback phosphoinositide to G-protein receptor signal-induced cell adhesion network by our programming, respectively.Simultaneous occurrence of biological processes was identified between the same activated PTHLH feedback-mediated cell adhesion molecular network and numbers in HCC were extracted and computed from the same activated PTHLH GO-molecular network of HCC compared with the corresponding activated GO-molecular network of no-tumor hepatitis/cirrhotic tissues by our programming, respectively. ActivatedPTHLH coupling feedback phosphoinositide to G-protein receptor signal-induced cell adhesion molecular relationship in HCC was identified including different molecules but same GO term and same molecule but different GO terms from the same activated PTHLH GO-molecular network of HCC compared with the corresponding activated GO-molecular network of no-tumor hepatitis/cirrhotic tissues, and constructed network by GRNInfer [At last, GRNInfer and our GRNInfer \u201325 and iPTHLH feedback-mediated cell adhesion GO network in HCC were identified and computed compared with the corresponding low expression activated GO network of no-tumor hepatitis/cirrhotic tissues (HBV or HCV infection), the different compared with the corresponding inhibited PTHLH feedback-mediated cell adhesion GO network of no-tumor hepatitis/cirrhotic tissues, and the same compared with the corresponding inhibited GO network of HCC, respectively.Biological processes and occurrence numbers of the same activated high expression (fold change \u22652) PTHLH feedback-mediated cell adhesion GO network in HCC included anaphase-promoting complex-dependent proteasomal ubiquitin-dependent protein catabolism, cell adhesion, cell differentiation, cell-cell signaling, endothelial cell migration, G-protein-coupled receptor protein signaling pathway, G-protein signaling, intracellular transport, metabolism, phosphoinositide-mediated signaling, positive regulation of transcription, protein amino acid phosphorylation, regulation of cyclin-dependent protein kinase activity, regulation of transcription, signal transduction, transcription, and transport compared with the corresponding activated GO network of no-tumor hepatitis/cirrhotic tissues. The same biological processes of activated PTHLH feedback-mediated cell adhesion GO network in HCC contained integrin-mediated signaling pathway, intracellular transport, microtubule cytoskeleton organization and biogenesis, regulation of cell growth, regulation of cyclin-dependent protein kinase activity compared with the corresponding inhibited GO network of no-tumor hepatitis/cirrhotic tissues.The different biological processes of activated PTHLH feedback-mediated cell adhesion GO network in HCC included anaphase-promoting complex-dependent proteasomal ubiquitin-dependent protein catabolism, cell adhesion, cell differentiation, cell-cell signaling, DNA repair, G-protein-coupled receptor protein signaling pathway, integrin-mediated signaling pathway, metabolism, nucleotide and nucleic acid metabolism, oxidation reduction, phosphoinositide-mediated signaling, positive regulation of transcription, protein modification, proteolysis, regulation of cyclin-dependent protein kinase activity, regulation of transcription, signal transduction, and transcription, transport compared with the corresponding inhibited GO network of HCC, as shown in The same biological processes of activatedPTHLH feedback-mediated cell adhesion molecular network and numbers in HCC were extracted and computed from the same activated PTHLH GO-molecular network of HCC compared with the corresponding activated GO-molecular network of no-tumor hepatitis/cirrhotic tissues. Our result showed that PTHLH feedback-mediated cell adhesion molecular network consisted of BUB1B, GNG10, PTHR2, GNAZ, RFC4, UBE2C, NRXN3, BAP1, PVRL2, TROAP, VCAN, CCNA2, CDC6, CDKN2C, and ENAH in HCC, as shown in Activated PTHLH feedback-mediated cell adhesion mechanism in HCC. In this study, biological processes and occurrence numbers of the same activated high expression (fold change \u22652) PTHLH feedback-mediated cell adhesion GO network in HCC were identified and computed compared with the corresponding low expression activated GO network of no-tumor hepatitis/cirrhotic tissues (HBV or HCV infection), the different compared with the corresponding inhibited PTHLH feedback-mediated cell adhesion GO network of no-tumor hepatitis/cirrhotic tissues, and the same compared with the corresponding inhibited GO network of HCC, respectively ( Our aim is to study novel high expression-activatedectively . PTHLH feedback-mediated cell adhesion GO network of HCC (compared with the corresponding activated GO network of no-tumor hepatitis/cirrhotic tissues) and the different (compared with the corresponding inhibited PTHLH feedback-mediated cell adhesion GO network of no-tumor hepatitis/cirrhotic tissues), or the same (compared with the corresponding inhibited GO network of HCC) for putting forward hypothesis of activated PTHLH coupling feedback phosphoinositide to G-protein receptor signal-induced cell adhesion network, respectively.Simultaneous occurrence of biological processes was identified between the same activated PTHLH feedback-mediated cell adhesion GO network of HCC (compared with the corresponding activated GO network of no-tumor hepatitis/cirrhotic tissues) and the different (compared with the corresponding inhibited PTHLH feedback-mediated cell adhesion GO network of no-tumor hepatitis/cirrhotic tissues). Simultaneous occurrence of biological processes consisted of intracellular transport, regulation of cyclin-dependent protein kinase activity between the same activated PTHLH feedback-mediated cell adhesion GO network of HCC (compared with the corresponding activated GO network of no-tumor hepatitis/cirrhotic tissues), and the same (compared with the corresponding inhibited GO network of HCC).Simultaneous occurrence of biological processes included anaphase-promoting complex-dependent proteasomal ubiquitin-dependent protein catabolism, cell adhesion, cell differentiation, cell-cell signaling, G-protein-coupled receptor protein signaling pathway, metabolism, phosphoinositide-mediated signaling, positive regulation of transcription, regulation of cyclin-dependent protein kinase activity, regulation of transcription, signal transduction, transcription, transport between the same activated PTHLH coupling feedback phosphoinositide to G-protein receptor signal-induced cell adhesion network in HCC.The studies of phosphoinositide with adhesion are presented as follows. Phosphoinositide lipid phosphatase SHIP1 and PTEN coordinate to regulate cell migration and adhesion , TAPP2 lPTHLH feedback-mediated cell adhesion molecular network and numbers in HCC were extracted and computed from the same activated PTHLH GO-molecular network of HCC compared with the corresponding activated GO-molecular network of no-tumor hepatitis/cirrhotic tissues . Activated PTHLH feedback-mediated cell adhesion network consisted of anaphase-promoting complex-dependent proteasomal ubiquitin-dependent protein catabolism, cell adhesion, cell differentiation, cell-cell signaling, G-protein-coupled receptor protein signaling pathway, intracellular transport, metabolism, phosphoinositide-mediated signaling, positive regulation of transcription, regulation of cyclin-dependent protein kinase activity, regulation of transcription, signal transduction, transcription, and transport in HCC. We proposed activated PTHLH coupling feedback phosphoinositide to G-protein receptor signal-induced cell adhesion network. Our hypothesis was verified by the different activated PTHLH feedback-mediated cell adhesion GO network of HCC compared with the corresponding inhibited GO network of no-tumor hepatitis/cirrhotic tissues, or the same compared with the corresponding inhibited GO network of HCC. Activated PTHLH coupling feedback phosphoinositide to G-protein receptor signal-induced cell adhesion network included BUB1B, GNG10, PTHR2, GNAZ, RFC4, UBE2C, NRXN3, BAP1, PVRL2, TROAP, and VCAN in HCC from GEO data set using gene regulatory network inference method and our programming. In summary, studies were done on analysis of biological processes in the same high expression (fold change \u22652) activated"} +{"text": "In this study, we synthesized an organic electrochemical transistor (OECT) using dielectrophoresis of a carbon nanotube-Nafion (CNT-Nafion) suspension. Dielectrophoretically aligned nanowires formed a one-dimensional submicron bundle between triangular electrodes. The CNT-Nafion composite nanowire bundles showed p-type semiconductor characteristics. The drain-source current decreased with increasing gate voltage. The nanowire bundles showed potential as pH sensor because the drain-source current ratio varied linearly according to the gate voltage in pH buffers. Recently, there has been significant research in the area of organic thin-film transistors (OTFTs), because of the many benefits of organic semiconductors, such as structural flexibility, low temperature processing, and low cost -7. Organp-type semiconductor properties in a phosphate buffer solution at pH 5.6 using blank electrodes of the cantilever array as gate electrodes [Recently, we have developed a real-time, label-free, step-wise, and target-specific aptasensor for protein molecules using dielectrophoretically aligned single-walled carbon nanotube (SWNT) films between patterned cantilever electrodes. We used the SWNT film as a two-terminal resistive sensor and demonstrated its excellent performance for detecting thrombin and vascular endothelial growth factor (VEGF). We verified that the SWNT film had ectrodes . The strectrodes -12. Thisectrodes -20. Our In this article, we report the fabrication of CNT composite nanowires with Nafion, a well-known proton conductor ,22 and tFigure Figure IDS) versus drain voltage (VDS) curves at different gate voltages (VG) in 5 \u03bcL of a phosphate-buffered saline (PBS) droplet for CNT-Nafion nanowires and blank electrodes, respectively. Figure IG) versus VDS for CNT-Nafion nanowires under the same conditions. The maximum value of IDS for the nanowire transistor was approximately 700 \u03bcA at VG = 0.5 V. The leakage current, IDS at the blank electrodes and IG were at the most 0.2 \u03bcA. The leakage current through the electrolyte was negligible because the IDS value at the blank electrode and IG were approximately one thousand times smaller than the current through the CNT-Nafion nanowires. The value of IDS decreased with increasing electrolyte gate bias . The normalized drain current to gate voltage ratio was linearly dependent on the buffer pH was deposited with a chrome layer (200 \u00c5) as an adhesion layer using an e-beam evaporator on a silicon substrate covered with 1 \u03bcm of low-stress silicon nitride using low-pressure chemical vapor deposition (LPCVD). For the cantilever structure, the silicon nitride was etched using standard reactive ion etching (RIE), and the silicon was etched using isotropic wet etching using RSE-200 etchant. The SWNTs with 1.0-1.2 nm diameters and lengths of 5-20 \u03bcm were purchased from Ilgin Nanotech, and a SWNT-COOH suspension was prepared by oxidizing the CNTs in a strong acid with sonication . Nafion The CNT-Nafion solution was placed on the cantilever electrodes, and an AC voltage of 1 MHz and 10 V peak-to-peak was applied. The SWNTs and monomers were aligned between the cantilever electrodes by the dielectrophoretic force. The SWNT-Nafion solution was removed partially while maintaining the AC electric field and the SWNT-Nafion nanowire bundles were synthesized as the remaining solution evaporated.Figure CNT-Nafion: carbon nanotube-Nafion; EDS: energy dispersive X-ray spectroscopy; LPCVD: low-pressure chemical vapor deposition; OECT: organic electrochemical transistor; OTFTs: organic thin film transistors; PBS: phosphate-buffered saline; RIE: reactive ion etching; SEM: scanning electron microscope; SWNT: single-walled carbon nanotube; VEGF: vascular endothelial growth factor.The authors declare that they have no competing interests.WSC and GL conceived of the study, and participated in its design and coordination. WSC and TA carried out the experiments. WSC drafted the manuscript. All authors read and approved the final manuscript."} +{"text": "Evaluate the application of temporal filtering via the Karhunen-Loeve Transform for increasing signal-to-noise ratio in real-time blood flow quantification.Real-time blood flow velocity quantification requires fast acquisition time and both high spatial and temporal resolution in order to capture clinically relevant features. Multi-coil parallel imaging techniques trade fast real-time cine acquisition with high temporal resolution for lower signal quality. Methods for increasing signal-to-noise ratio (SNR) will allow higher acceleration rates in parallel imaging, leading to faster acquisition times in the context of exercise stress while maintaining signal quality.Increased SNR via the Karhunen-Loeve Transform (KLT) isKLT was applied retrospectively to ten through-plane real-time velocity data sets of the aorta at rest acquired at 1.5T . Optimal identification of noise-only eigenmodes was determined using the Marcenko-Pastur Law , and SNROptimal selection of SNR gain via the Marcenko-Pastur Law results in SNR increase of 61.5 \u00b1 3.7% (mean \u00b1 standard deviation) with no statistically significant difference in peak velocity measurement . Figure Temporal filtering of real-time velocity images via KLT results in significant gains in SNR with minimal velocity profile smoothing and minimal effect on peak velocity measurement and may be useful in improved blood flow velocity quantification."} +{"text": "A 3D coronary black-blood approach, however, has been shown to provide better coverage of the coronary tree . Furthermore, uptake of gadolinium contrast agent in coronary walls may indicate on-going inflammation or vessel wall fibrosis .To investigate whether in-plane 3D coronary vessel wall imaging utilizing a local-inversion technique and spiral image acquisition, in combination with contrast-enhanced inversion-recovery (IR) imaging allows more comprehensive assessment of KD.Patients with previous coronary aneurysms due to KD undergoing routine CMR evaluation had additional vessel wall imaging before and after contrast administration (0.2 mmol/kg gadopentetate dimeglumine). All examinations were performed on a 1.5T MR-system and the coil was selected according to patient size (two-element/five-element). Coronary MRA was followed by a targeted, free-breathing, ECG-triggered 3D vessel wall sequence using local inversion and spiral image acquisition . Areas of vessel wall thickening on in-plane imaging were then further evaluated with through plane 2D DIR vessel wall technique.Post-contrast imaging was performed using a free-breathing, ECG-triggered, 3D IR segmented gradient-echo (TFE) sequence .13 CMR examinations were performed in 12 children . 6 cases showed persistent aneurysms; 4 cases had coronary ectasia and the remaining resolved aneurysms had a normal lumen. One case showed a mild LAD stenosis shown on CMRA and confirmed on conventional angiography. Complete black blood coverage of the affected portions of the coronary arteries was achieved using the new 3D in-plane local-inversion technique. Targeted 2D cross-sectional imaging showed that aneurismal segments had greater wall thickness (mean 0.87mm) than ectatic segments (0.42mm) or resolved aneurysms (0.31mm). Delayed enhancement of the coronary vessel wall was only present in three cases . Figures Our data suggests that inflammation of the coronary vessel wall in Kawasaki disease may persist for up to 2 years post-acute insult. Vessel wall remodeling seems to have occurred effectively in the resolved aneurysms studied here. 3D in-plane local-inversion black-blood imaging gives more complete coverage of affected segments and allows targeting of cross-sectional imaging of thickened areas. The protocol outlined here gives a more comprehensive assessment of affected coronaries after KD."} +{"text": "The liver secretes very-low-density lipoproteins (VLDLs) and plays a key role in lipid metabolism. Plasma total triglyceride (TG) level variations have been studied in patients with hepatitis C virus (HCV)-related chronic hepatitis (CH-C). However, the results of these studies are variable. A homogenous assay protocol was recently proposed to directly measure the TG content in VLDL (VLDL-TG) and VLDL remnants.Using the assay protocol, we determined serum VLDL-TG levels in 69 fasting patients with biopsy-proven HCV-related chronic liver disease and 50 healthy subjects. Patients were classified into stages F0\u2013F4 using the 5-point Desmet scale. Serum total TG levels in patients with non-cirrhotic (F1\u2013F3) CH-C did not demonstrate significant differences compared with healthy subjects, but serum VLDL-TG levels did demonstrate significant differences. Mean serum VLDL-TG levels tended to decrease with disease progression from F1 to F4 (cirrhosis). Compared with healthy subjects, serum non-VLDL-TG levels significantly increased in patients with stages F2 and F3 CH-C; however, we observed no significant difference in patients with liver cirrhosis. Furthermore, the serum VLDL-TG/non-VLDL-TG ratio, when taken, demonstrated a significant decrease in patients with CH-C from the mildest stage F1 onward.The decrease in serum VLDL-TG levels was attenuated by increase in non-VLDL-TG levels in patients with non-cirrhotic CH-C, resulting in comparable total TG levels. Results of previous studies though variable, were confirmed to have a logical basis. The decrease in the serum VLDL-TG/non-VLDL-TG ratio as early as stage F1 demonstrated TG metabolic alterations in early stages of CH-C for the first time. The involvement of TG metabolism in CH-C pathogenesis has been established in experimental animals, while conventional TG measurements are generally considered as poor indicators of CH-C progression in clinical practice. The serum VLDL-TG/non-VLDL-TG ratio, which focuses on TG metabolic alterations, may be an early indicator of CH-C. Plasma triglyceride (TG)-rich lipoproteins comprise a mixture of lipoprotein species that are characterized by differences in density and apolipoprotein composition. In the fasting state, plasma TGs are predominantly transported by very-low-density lipoproteins (VLDLs) secreted by the liver In many countries, chronic hepatitis C virus (HCV) infection is the leading cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Interactions between chronic HCV infection and lipid metabolism have been suggested. Some studies indicate a higher prevalence of hypocholesterolemia Furthermore, plasma TG level variations have been reported in patients with HCV-related chronic hepatitis (CH-C) Because TGs in plasma are transported by a mixture of different lipoprotein species, such as CMs, VLDLs, and VLDL remnants [also called intermediate-density lipoproteins (IDLs)], selective measurement of these TG-rich lipoproteins is essential to obtain further insight into TG metabolic alterations under pathological conditions.Compared to low-density lipoprotein (LDL) and high-density lipoprotein (HDL), technological difficulties have hampered specific measurement of VLDL although TGs are transported mainly by VLDL in the fasting state; these conditions differ from those surrounding cholesterols. Cholesterols are transported mainly by LDL To overcome these difficulties, Okada et al. The study was approved by the Human Ethics Committee of Chiba University. Written informed consent was obtained from all the subjects. The Human Ethics Committee of Chiba University also approved the use of residual sera collected during routine laboratory sampling from the patients for the study, provided that strict anonymity was maintained. The patient sera were irrevocably anonymized and unlinked from the persons who provided the sample.As mentioned in the This study included 69 patients with untreated HCV-related chronic liver disease who had been hospitalized for liver biopsy at the Chiba University Hospital. The control group consisted of 50 apparently healthy, fasting subjects who had sought an annual medical check-up at the Kashiwado Clinic. All subjects were negative for hepatitis B surface antigen, and none had a history of excessive alcohol drinking or drug abuse. In control subjects, anti-HCV antibody test results were all negative and significant hepatic steatosis was not found by ultrasonography. The characteristics of patients and healthy subjects are summarized in Liver biopsies were performed with a Tru-Cut needle (14G) under ultrasound guidance At the time of the liver biopsy, venous blood was drawn after a 12-h overnight fast to determine blood chemistry values. Following routine laboratory tests, serum total TG and VLDL-TG levels were determined using residual sera. Serum total TG levels were measured by a conventional enzymatic assay .Serum VLDL-TG levels were determined using the surfactant-based homogenous assay proposed by Okada et al. U-test for comparisons between groups to determine their significance using the 4-Step Excel Statistics software application . Significance levels were set at p<0.05.Values are presented as mean \u00b1 SD (standard deviation). The results were analyzed using the Mann-Whitney When comparisons were made between healthy subjects and patients with non-cirrhotic CH-C (stages F1\u2013F3), total TG levels were similar , but serum VLDL-TG levels were significantly lower (p\u200a=\u200a0.044) in patients with non-cirrhotic CH-C (59.8\u00b129.4 mg/dl) than in healthy subjects (78.5\u00b148.9 mg/dl). In contrast, serum non-VLDL-TG levels were significantly higher (p<0.0001) in patients with non-cirrhotic CH-C (36.6\u00b113.1 mg/dl) than in healthy subjects (27.8\u00b110.6 mg/dl). The differences were more remarkable when serum VLDL-TG/non-VLDL-TG ratios were compared .With regard to the disease stages classified with the 5-point Desmet scale, total TG levels in patients with CH-C did not demonstrate any significant differences at any stage compared with those in healthy subjects; however, a significant difference was observed only when the liver disease progressed to liver cirrhosis (stage F4) . In contMean serum VLDL-TG levels demonstrated a tendency to decrease with progression of liver disease from F1 to F4 , while nAs a result, serum VLDL-TG/non-VLDL-TG ratios were significantly decreased in both early and advanced stages of HCV-related chronic liver disease. Even in patients with the mildest form of CH-C (F1), serum VLDL-TG/non-VLDL-TG ratios alone were significantly different from those in healthy subjects .In the present study, the changes in serum VLDL-TG and non-VLDL-TG levels were comparable between patients with HCV genotype 1 and HCV genotype 2.The results of this study indicate that serum TG alterations, evaluated as decreases in the serum VLDL-TG/non-VLDL-TG ratio, occur as early as the stage F1 of CH-C. In contrast, no statistically significant differences were observed in serum total TG levels between healthy subjects and patients with stage F1 of CH-C as well as between healthy subjects and patients with non-cirrhotic CH-C (F1\u2013F3).Although it is known that the serum total TG level is decreased in advanced chronic liver disease Several possible mechanisms could lower the serum VLDL-TG/non-VLDL-TG ratio from the earliest stages of CH-C. Liver disease resulting from HCV infection might impair VLDL synthesis in hepatocytes. Previous studies with transgenic mice revealed that the HCV core protein inhibits microsomal transfer protein activity and causes impaired VLDL secretion The liver uptakes non-VLDL-TG in the form of free fatty acids and glycerols produced by peripheral lipases and hepatic triglyceride lipase In view of the above findings, the premise that \u201cit remains to be established whether or not the serum total TG levels in patients with CH-C are significantly lower than those in healthy subjects\u201d is confirmed to have a logical basis. Furthermore, because of these changes in serum VLDL-TG and non-VLDL-TG levels, serum VLDL-TG/non-VLDL-TG ratios might indicate the clinical stage of HCV-related chronic liver disease more accurately than serum total TG or VLDL-TG levels alone.When HCV-related liver disease progressed to stage F4, serum non-VLDL-TG levels, being different from those in patients with non-cirrhotic CH-C, did not demonstrate any significant difference when compared with those in healthy subjects . This maThe involvement of TG metabolism in CH-C pathogenesis has been established in experimental animals and cultured cells It should be noted that hepatic biopsy was not performed for the healthy subjects, and thus their livers were not confirmed to be completely normal. However, in control subjects, hepatitis B surface antigen and anti-HCV test results were all negative and their serum transaminase levels were within the reference intervals . In cont"} +{"text": "N-methyl-D-aspartate receptor (NMDAR) encephalitis, a newly recognized anti-neuronal antibody-mediated inflammatory brain disease, seen by the Rheumatology service over an 18 month period.Inflammatory brain disease causes severe psychiatric and neurological deficits in previously healthy children. The aim of this study was to report characteristic clinical features and outcomes of children diagnosed with Anti-Over an 18-month period (July 2009-Dec 2010), consecutive children presenting with newly acquired psychiatric and/or neurologic deficits consistent with anti-NMDAR encephalitis and evidence of central nervous system (CNS) inflammation were screened. Serum and cerebrospinal fluid (CSF) samples were obtained and sent for anti-NMDAR antibody testing. Children were included in the study if they had confirmatory evidence of these antibodies in the serum and/or CSF. Details of clinical presentation and results of investigations, including inflammatory markers, CSF studies and neuroimaging, were documented. Type and duration of treatment and outcomes at last follow-up were evaluated.Over the study period a total of 18 children presented with clinical features compatible with anti-NMDAR encephalitis including psychiatric manifestations, seizures and/or movement disorders. Four children (22%) had positive anti-NMDAR antibodies and were diagnosed with anti-NMDAR encephalitis. These included one male and three females, with a median age of 12.8 years (range 3-16 years). The remaining 14 children were subsequently diagnosed with 1) primary CNS vasculitis (4); 2) post-infectious inflammatory brain disease (2); 3) channelopathy (1); 4) reversible splenial lesions syndrome (1); 5) epilepsy (1); and 6) inflammatory brain disease NYD (5). All children with anti-NMDAR encephalitis presented with neuropsychiatric deficits including, seizures, speech disorder, sleep disturbance, and fluctuating level of consciousness. The three older patients also had prominent psychiatric features, while the younger child had significant autonomic instability and prominent involuntary movement disorder. None had an underlying tumor. Immunosuppressive therapy with intravenous immunoglobulin, steroids and/or rituximab, resulted in near or complete recovery; however, two of the patients had early relapse requiring re-treatment.Anti-NMDAR encephalitis is an important, reversible cause of neuropsychiatric deficits in children that must be included in the differential diagnosis of CNS vasculitis and other inflammatory brain diseases. Early diagnosis and treatment are essential for neurologic recovery.Nadia J.C. Luca: None; Tassalapa Daengsuwan: None; Josep Dalmau: Euroimmun, 2, 9, Individual patent, 2, 9; Kevin Jones: None; Gabrielle deVeber: None; Jeff Kobayashi: None; Ronald M. Laxer: None; Susanne M. Benseler: None."} +{"text": "Naturally infected sooty mangabeys (SM) do not progress to AIDS unlike SIV-infected non-natural hosts including rhesus macaques (RM). Differences in the innate immune response to SIV may be critical in avoidance of disease progression in SIV-infected SM. In this study we investigated innate and adaptive immune responses to recombinant HSV vectors expressing SIV Gag, Env, and a Rev-Tat-Nef fusion protein in six SIV-negative SM and four RM following single intramuscular inoculation.Plasma samples were analyzed using Luminex 28-plex assay. Innate immune cells and SIV-specific cellular immune responses were investigated by flow cytometry and IFN-\u03b3 ELISPOT assay.A significant transient decline of circulating Natural Killer T (NKT) cells, pDCs, and NK cells was observed in SM on day one post vaccination, with a return to baseline levels by day 3-7. Also an increased activation of SM NK cells was observed that returned to baseline by day 9. In contrast, RM displayed no significant change in circulating NKT cell and NK cell frequencies. Both RM and SM exhibited similar transient increases in plasma levels of several inflammatory cytokines/chemokines including IL-1RA, IFN-\u03b3, IL-6, IL-8, macrophage migration inhibitory factor (MIF), monocyte chemotactic protein 1 (MCP-1), vascular endothelial growth factor (VEGF), and interferon-inducible T cell alpha-chemoattractant (I-TAC) by day one, suggesting activation of multiple immune cells including macrophages and DCs. By day 14, plasma levels of analytes such as IL-6 and Eotaxin persisted at elevated levels in RM, but not SM, suggesting delayed resolution of immune activation in RM. SIV-specific cellular immune responses were detected in both species.These data confirm that SM mount a robust innate immune response and do not have blunted immune activation. Further investigation of the mechanism by which SM resolve the transient but higher immune activation would be helpful in improving vaccine strategy."} +{"text": "Pseudomonas putida KT2440 which can accumulate large amounts of polymer from cheap substrates such as glucose. Current research focuses on enhancing the strain production capacity and synthesizing polymers with novel material properties. Many of the corresponding protocols for strain engineering rely on the rifampicin-resistant variant, P. putida KT2442. However, it remains unclear whether these two strains can be treated as equivalent in terms of mcl-PHA production, as the underlying antibiotic resistance mechanism involves a modification in the RNA polymerase and thus has ample potential for interfering with global transcription.The substitution of plastics based on fossil raw material by biodegradable plastics produced from renewable resources is of crucial importance in a context of oil scarcity and overflowing plastic landfills. One of the most promising organisms for the manufacturing of medium-chain-length polyhydroxyalkanoates (mcl-PHA) is P. putida KT2440 and KT2442, we characterized the growth and PHA accumulation on three categories of substrate: PHA-related (octanoate), PHA-unrelated (gluconate) and poor PHA substrate (citrate). The strains showed clear differences of growth rate on gluconate and citrate but not on octanoate. In addition, P. putida KT2442 PHA-free biomass significantly decreased after nitrogen depletion on gluconate. In an attempt to narrow down the range of possible reasons for this different behavior, the uptake of gluconate and extracellular release of the oxidized product 2-ketogluconate were measured. The results suggested that the reason has to be an inefficient transport or metabolization of 2-ketogluconate while an alteration of gluconate uptake and conversion to 2-ketogluconate could be excluded.To assess PHA production in P. putida KT2440 and KT2442 two different strains in terms of mcl-PHA production. The differences include the onset of mcl-PHA production (nitrogen limitation) and the resulting strain performance (growth rate). It remains difficult to predict a prioriwhere such major changes might occur, as illustrated by the comparable behavior on octanoate. Consequently, experimental data on mcl-PHA production acquired for P. putida KT2442 cannot always be extrapolated to KT2440 and vice versa, which potentially reduces the body of available knowledge for each of these two model strains for mcl-PHA production substantially.The study illustrates that the recruitment of a pleiotropic mutation, whose effects might reach deep into physiological regulation, effectively makes Pseudomonas putida KT2440 and KT2442 belong to the best-known producers of mcl-PHA. They accumulate polymer from both PHA-related carbon sources (e. g. fatty acids) via \u03b2-oxidation and from PHA-unrelated carbon sources (e. g. sugars) via de novo fatty acid synthesis ; C(2-KGln): 2-ketogluconate-based carbon concentration [g L-1]; CDW: cell dry weight [g L-1]; CPR: carbon dioxide production rate [mol L-1 h-1]; FG: gas flow [L h-1]; Gln: gluconate; 2-KGln: 2-ketogluconate; \u03bcmax: maximum specific growth rate [h-1]; NH4-N: ammonium nitrogen [g L-1]; OD600: optical density (of a cell culture) at 600 nm [-]; OUR: oxygen uptake rate [mol L-1 h-1]; mcl-PHA: medium-chain-length polyhydroxyalkanoate; scl-PHA: short-chain-length polyhydroxyalkanoate; q: specific uptake/production rate [g g-1 h-1]; TOC: total organic carbon [g L-1]; RQ: respiratory quotient [mol mol-1]; Vm: molar volume [L mol-1]; Vw: working volume [L]; Xr: PHA-free biomass [g L-1]; y: molar fraction; YXr/C: growth yield for carbon [g g-1]; YXr/N: growth yield for nitrogen [g g-1]; YPHA/C: PHA yield for carbon [g g-1].C(Gln): gluconate-based carbon concentration [g L0: initial; C*: carbon effectively consumed by the cells for respiration and: biomass production; C(Gln): carbon present in gluconate; C(2-KGln): carbon present in 2-ketogluconate; e: end of exponential phase; f: final; in: inlet gas to the bioreactor; out: outlet gas from the bioreactor.The authors declare that they have no competing interests.SF performed the laboratory experiments and drafted the manuscript. MZ advised the experimental design and together with SP revised the manuscript. All authors read and approved the final manuscript."} +{"text": "AID misregulation can result in genomic instability and oncogenic transformation. This is classically illustrated in Burkitt's lymphoma, which is characterized by AID-induced mutation and reciprocal translocation of the c-MYC oncogene with the IgH loci. Originally thought to be B cell-specific, AID now appears to be misexpressed in several epithelial cancers, raising the specter that AID may also participate in non-B cell carcinogenesis.Activation Induced cytidine Deaminase (AID) targets the immunoglobulin genes of activated B cells, where it converts cytidine to uracil to induce mutagenesis and recombination. While essential for immunoglobulin gene diversification, AID expression. MicroRNAs (miRs) have this capacity, and we have examined the publically available human AID EST dataset for miR complementarities to the human AID 3'UTR. In this work, we have evaluated the capacity of two candidate miRs to repress human AID expression in MCF-7 breast carcinoma cells.The mutagenic potential of AID argues for the existence of cellular regulators capable of repressing inappropriate AID mRNA. Luciferase reporter assays indicate that both miR-93 and miR-155 can interact with the 3'UTR of AID to block expression. In addition, over-expression of either miR in MCF-7 cells reduces endogenous AID protein, but not mRNA, levels. Similarly indicative of AID translational regulation, depletion of either miR in MCF-7 cells increases AID protein levels without concurrent increases in AID mRNA.We have discovered moderate miR-155 and pronounced miR-93 complementary target sites encoded within the human AID translation in MCF-7 cells, suggesting widespread roles for these miRs in preventing genome cytidine deaminations, mutagenesis, and oncogenic transformation. In addition, our characterization of an obscured miR-93 target site located within the AID 3'UTR supports the recent suggestion that many miR regulations have been overlooked due to the prevalence of truncated 3'UTR annotations.Together, our findings demonstrate that miR-93 and miR-155 constitutively suppress Activation Induced cytidine Deaminase (AID) protein is required for initiating mutagenesis and recombination of the immunoglobulin (Ig) genes to promote immunity. AID is a cytidine deaminase that converts single-stranded genomic cytidine into uracil, and its activity is pronounced at the Ig variable (V) and switch (S) regions .Click here for fileAID 3'UTR is recognized by miRs -93 and -155Figure S1. The . (A) The AID 3'UTR recognition by miRs -93 and -155 is specific. (A) Diagrams of luciferase reporters. The AID 3'UTR luciferase reporter (TS Intact) and control 3'UTR luciferase reporter (TS Scram) were generated respectively by placing the AID 3'UTR miR -93 and -155 target sites (TS Intact)(with intervening sequences deleted) or an identical sequence in which the target site nucleotides had been scrambled (TS Scram) into a multiple cloning site in the Renilla luciferase 3'UTR [B) TS Intact is specifically repressed by both miR-93 and miR-155 in 293 transient transfections. Luciferase assays (n = 9) of HEK293 lysates after transfection of TS Intact and either pAL-1, pAL-155 or pAL-93. Transfections were normalized to TS Intact alone. RLU, relative light units. (C) TS Scram is unaffected by co-transfection of pAL-1, pAL-155 or pAL-93 in 293 transient transfections. Cotransfections done as in B, except TS Scram replaced TS Intact.se 3'UTR . HSV-TKpClick here for fileFigure S2. MicroRNAs miR-93 and miR-155 are widely expressed. RT-PCR of pre-miR template in three different cell lines, HEK293 embryonic kidney cells, MCF-7 breast cancer line, and Ramos Burkitt's lymphoma line. (top) RT-PCR products of the miR-93 locus amplified from indicated cellular RNAs. m, miR-93; \u03b2, Beta actin positive control; - Ctl, no template. (bottom) RT-PCR products of the miR-155 locus amplified from indicated cellular RNAs. Annotations are same as above except (m) denotes pre-miR-155 amplicon. PCR of source RNAs failed, indicating above RT-PCRs templated from the generated cDNA.Click here for fileFigure S3. MiR-93 represses endogenous AID in a dose responsive manner. (A) Representative western demonstrating that transient transfection of pAL-93 expression constructs repress endogenous AID protein levels in MCF-7 cells in a dose responsive manner. (B) Relative AID mRNA levels corresponding to replicate transfections performed as in A (n = 2) determined by quantitative PCR.Click here for file"} +{"text": "Simultaneous acquisition PET-MRI is a new technology that has the potential to significantly impact diagnostic patient care. Cardiac imaging using PET-MRI offers high signal resolution MRI images superimposed on PET metabolic functional assessment. Specifically, 18F-fluorodeoxyglucose (FDG) PET-MR has the potential to provide both anatomic scar tissue evaluation and information regarding myocardial glucose metabolism. While early brain and soft tissue data have demonstrated that PET specific uptake values (SUVs) obtained using MRI for attenuation correction (AC) are comparable to SUVs obtained using CT AC, SUV measurements of myocardial tissue have not been compared. The objective of this pilot study is to determine the reproducibility of SUVs obtained by PET imaging using an AC \u00b5-map comprised of a dual echo VIBE Dixon MRI sequence instead of CT.30 patients with no known cardiac history underwent full body PET-CT imaging (Siemens Biograph 40), followed by full-body simultaneous PET-MRI imaging (Siemens Biograph mMR). A single dose (10-15 mCi) of 18F-FDG radiotracer was injected on average 59 +/- 7 minutes prior to PET-CT image acquisition, and127 +/- 23 minutes prior to PET-MRI image acquisition. Images were whole body images acquired without cardiac gating. For PET-MR the AC \u00b5-map was a dual echo VIBE Dixon sequence that separates water and fat . Average SUVs were obtained by tracing the entire cross section of the left ventricular myocardium in the short axis of the heart at the papillary muscle level, using the syngo TRUE-D computer software (Siemens).There is no statistically significant difference between the average SUVs obtained by PET-CT and PET-MRI . Although FDG uptake rate and the SUVs are highly variable among this study group, there is excellent per patient correlation between the values acquired by PET-CT and PET-MRI .Myocardial PET SUVs measured using MRI as AC show excellent correlation to those obtained by standard PET-CT imaging. Future studies will focus on the optimization of cardiac-gated simultaneous PET-MRI image acquisition.Mallinckrodt Institute of Radiology Departmental Funds."} +{"text": "Progressive pseudorheumatoid arthropathy of childhood (PPAC) is a specific subtype of spondyloepiphyseal dysplasie (SED) tarda. The skeletal disorder and is characterized by polyarthropathy of large and small joints. Typical signs are prominent epiphysis, progressive joint stiffness, muscle weakness and early fatigue . OptimizQuantification of pathologic motion patterns during walking.In a retrospective study eight adolescents suffering from PPAC were compared with 20 healthy young person (cg) . 3D-gaitanalysis was performed with infrared cameras and the Plug-in-Gait Model. Analyses focused spatio-temporal and kinematic parameters in the sagittal plane. Mann-Whitney-U-Tests (p<0.05) and correlation calculations (Pearson) between age, body-mass-index (BMI), kinematic and spatio-temporal-parameters were used for statistical analysis.Patients with PPAC walk very slow (p<0.001) with short step length (p<0.001) and broadened step width (p<0.001). The foot off occurs noticeable late (p<0.001). The kinematic data are highly significant different to the cg in pelvis, hip, knee and ankle. Especially the range of motion (ROM) in the hip, in the knee flexion (loading response) and extension (single support phase) as well as the ankle ROM (plantar flexion while push off) are decreased. Within the PPAC-group high negative correlations appear between BMI and ankle ROM (plantar flexion (push off)) (r=-.860).The results are determined by very small ROM in the lower limb due to distinctive joint stiffness and muscle weakness. The effects of muscular weakness are intensified by high body weight.The use of 3D-gaitanalysis is a helpful tool to individualize functional treatment to decelerate the progressive joint destruction in the lower limb."} +{"text": "Patients with anterior MI experience worse LV remodeling and dysfunction than non-anterior MI patients. It remains unclear whether this difference is due to larger MI size or whether infarct location plays a role beyond MI size.To assess the relationship between myocardial infarction (MI) location and size and their reciprocal influences on post-infarction left ventricular (LV) remodeling.A cohort of 260 reperfused ST-segment elevation MI patients was studied with cardiovascular magnetic resonance (CMR) at 1-week (baseline) and 4-month (follow-up). Area at risk (AAR) and MI size were quantified by T2-weighted and late gadolinium enhancement imaging, respectively. Adverse LV remodeling was defined as increase in LV end-systolic volume \u226515% at follow-up.One-hundred twenty-seven(49%) patients had anterior MI and 133(51%) patients had non-anterior MI. Although the degree of myocardial salvage was similar between groups (p=0.74), anterior MI patients had larger AAR and MI size than non-anterior MI patients yielding worse regional and global LV function at baseline and follow-up. At univariable analysis, anterior MI was associated with increased risk of adverse LV remodeling (p=0.017) and lower LV ejection-fraction at follow-up (p=0.001), but not when accounted for baseline MI size. Accordingly, at multivariable analysis baseline MI size but not its location was an independent predictor of adverse LV remodeling and ejection-fraction at follow-up .Anterior MI patients experience more pronounced post-infarction LV remodeling and dysfunction than non-anterior MI patients due to greater magnitude of irreversible ischemic LV damage without any independent contribution of MI location."} +{"text": "Statistical quantification and classification of heart disease, using clinical indices such as ejection fraction (EF) or left-ventricular mass (LVM), are used routinely in clinical practice for both diagnosis and prognosis. However, 3D CMR of the left ventricle (LV) provides a wealth of shape features which can maximise the classification power and accuracy of such indices. To exploit these, we propose a framework whereby shape parameters of a 3D LV finite element atlas are used to optimally classify patients according to linear statistical decompositions Fig. .Multi-Ethnic Study of Atherosclerosis (MESA) cohort (1) and 300 patients with myocardial infarction from the Defibrillators To Reduce Risk By Magnetic Resonance Imaging Evaluation (DETERMINE) cohort (2) made available through the Cardiac Atlas Project (3). Bias due to scan protocol differences between cohorts was corrected (4). Shape classifiers were constructed to optimally detect which cohort each case belonged. A comparison between principal component analysis (PCA) and information preserving component analysis (IPCA) (5) was performed, using shapes at end-diastole (ED), end-systole (ES) and the difference in shape between ED and ES (ED-ES) which included information on regional wall motion. Traditional clinical classifiers of EF, end-diastolic/end-systolic volume (EDV/ESV) and LVM were also included for comparison. Ten-fold cross-validation experiments were performed in which 90% of the cases were used for training and 10% for validation, repeated 10 times with different training/validation cases.Finite-element shape models of the LV were customised to 600 cardiac MRI volumes with previously standardised and validated software . The dataset comprised 300 community-based participants without a history of cardiovascular disease, aged 45-84 from 4 ethnic groups from the Classification results showed that this framework was able to determine clinically relevant modes and that IPCA achieved the lowest error rates using the ED-ES shape difference, with a single classifier number. This classifier can also be used to quantify severity of disease (degree of match with each group). Ten-fold cross-validation experiments corroborated the robustness of this approach which averaged 100% specificity and 99% sensitivity for IPCA versus 83% and 93% respectively when compared to ejection fraction and St. Jude Medical provided grant support for the DETERMINE trial. MESA was supported by contracts N01-HC-95159 through N01-HC-95169 from the NHLBI and by grants UL1-RR-024156 and UL1-RR-025005 from NCRR."} +{"text": "However, no genome-wide analysis of MYC-binding sites by chromatin immunoprecipitation (ChIP) followed by next generation sequencing (ChIP-Seq) has been conducted in BL so far.MYC is a key transcription factor involved in central cellular processes such as regulation of the cell cycle, histone acetylation and ribosomal biogenesis. It is overexpressed in the majority of human tumors including aggressive B-cell lymphoma. Especially Burkitt lymphoma (BL) is a highlight example for MYC overexpression due to a chromosomal translocation involving the ChIP-Seq was performed on 5 BL cell lines with a MYC-specific antibody giving rise to 7,054 MYC-binding sites after bioinformatics analysis of a total of approx. 19 million sequence reads. In line with previous findings, binding sites accumulate in gene sets known to be involved in the cell cycle, ribosomal biogenesis, histone acetyltransferase and methyltransferase complexes demonstrating a regulatory role of MYC in these processes. Unexpectedly, MYC-binding sites also accumulate in many B-cell relevant genes. To assess the functional consequences of MYC binding, the ChIP-Seq data were supplemented with siRNA- mediated knock-downs of MYC in BL cell lines followed by gene expression profiling. Interestingly, amongst others, genes involved in the B-cell function were up-regulated in response to MYC silencing.The 7,054 MYC-binding sites identified by our ChIP-Seq approach greatly extend the knowledge regarding MYC binding in BL and shed further light on the enormous complexity of the MYC regulatory network. Especially our observations that (i) many B-cell relevant genes are targeted by MYC and (ii) that MYC down-regulation leads to an up-regulation of B-cell genes highlight an interesting aspect of BL biology. Once bound to its target promoter, MYC can interact with histone modifiers, such as histone acetylases (HATs), GCN5/PCAF, P300/CBP, TIP60 or HAT-associated proteins (e.g. TRRAP), resulting in local hyper-acetylation of histones P15, P21, P27) by blocking the respective activation factors such as SMAD, YY-1, SP1, MIZ-1, TFII-I and NF-Y MYC can also inhibit transcription of genes (e.g. MYC. This translocation leads to a fusion of MYC to one of the three immunoglobulin (Ig) loci MYC was first discovered in Burkitt lymphoma (BL), which harbors a chromosomal translocation of MYC-binding sites were previously analyzed by ChIP-on-chip (Chromatin immunoprecipitation in combination with promoter tiling arrays) and by ChIP-PET (Paired end sequencing of the precipitated DNA fragments) analysis, in a single BL cell line and one MYC-inducible lymphoblastoid cell line (P493-6), respectively, latter serving as BL model The primary goal of the present study thus was the generation of a genome-wide map of MYC-binding sites in BL genomes. To this end, we carried out ChIP in 5 human BL cell lines, employing a MYC-specific antibody followed by next generation sequencing of the precipitated DNA fragments. We identified 7,054 MYC-binding sites and used the associated genes to detect functionally relevant gene sets. In addition we performed MYC knock-down experiments in BL cell lines followed by gene expression profiling. Similar functional groups of genes were found by ChIP-Seq and by MYC silencing. Of special interest was, however, our finding that \u2013 in addition to already established MYC target genes \u2013 genes important for the function and immunogenicity of B-cells are targeted by MYC binding and are up-regulated by MYC inhibition.2 in RPMI 1640 supplemented with 10% Sera Plus at 37\u00b0C.Five human Burkitt lymphoma (BL) cell lines were purchased from the German Collection of Microorganisms and Cell Cultures and cultured with 5% COA polyclonal MYC antibody (N-262: LOT E2308) Santa Cruz, CA, USA) was employed for ChIP. The specificity and suitability of this antibody for ChIP has already been shown by previously published work NPM1, an already well defined MYC target gene Successful enrichment (threshold >20-fold) of MYC-bound DNA fragments was determined by real-time DNA-PCR with primers for PRAME gene were employed, with the exception of the Ramos cell line where the PRAME 3\u2032- end could not be amplified, and therefore primers annealing to the ACTIN gene were used for normalization. Furthermore, selected MYC-binding sites discovered by the ChIP-Seq analysis described below were validated by real-time DNA-PCR. All primers employed were tested to display an efficiency of approximately 100% (+/\u221210%). Primer sequences are available from Relative quantification of real-time DNA-PCR results was calculated using the comparative \u0394\u0394CT method Approximately 200 ng of ChIP-DNA was used as template for generating an Illumina sequence library . The DNA was not further size fractionated and directly taken for adaptor ligation, using a standard Illumina genomic library preparation kit. Briefly, DNA was end-repaired using a mix of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment and dATP to yield a protruding 3\u2032-\u2018A\u2019 base for ligation of Illumina adapters which have a single \u2018T\u2019 base overhang at the 3\u2032- end. After adapter ligation fragments of \u223c250\u2013350 bp (insert plus adaptor sequences) were isolated from an agarose gel and were PCR amplified with Illumina primers for 15 cycles. The purified DNA was captured on an Illumina flow cell for cluster generation. These libraries were submitted to high-throughput sequencing on the Illumina Genome Analyzer II (GAII).http://bowtie-bio.sourceforge.net/manual.shtml#the-n-alignment-mode) for mismatches were considered for further analysis (Bowtie option \u2013m 1).The resulting sequence reads were mapped to the human reference genome using Bowtie The detection of genomic regions enriched by ChIP versus input control was conducted with HOMER (v2.6) for each experiment individually All discovered putative peaks were merged into one list of putative peak regions that were detected in at least one experiment. A matrix containing the number of reads for every experiment in each putative peak region was assembled. DESeq (v1.4.0) was employed to test the number of reads for being differential over all ChIP versus input samples http://aug2010.archive.ensembl.org/index.html) www.ncbi.nlm.nih.gov/geo/) under the accession number GSE30726.The remaining peaks were ranked by their FDR and annotated with ChIPpeakAnno using Ensembl Biomart employing the Ensembl Genes 59 database and the GCRh37 (hg19) dataset (http://genome.ucsc.edu/cgi-bin/hgLiftOver).In order to compare our ChIP-Seq data to previously published MYC-binding sites, we additionally analyzed the data provided by Zeller et al. http://fantom3.gsc.riken.jp/) using the oligo capping method, (ii) to determine the occurrence of E-boxes, (iii) to perform an orthologous region search in 6 placental mammalian (Eutheria) species and (iv) to determine the overrepresentation of the MYC motive as discovered by CoreSearch.The overlap of genomic intervals determined by ChIP-Seq with genomic features, such as transcriptional start sites or exons, was calculated utilizing RegionMiner de novo calculation of a MYC-binding motive using the 100 highest ranked genomic intervals CoreSearch (Genomatix) with default settings was employed for a MYC smart pool siRNA and control siRNA , respectively. Resulting down-regulation of MYC protein expression was monitored by immunoblot analysis SiRNA-mediated knock-down of MYC was performed employing the BL cell lines Raji, BL41 and Blue1 in order to detect changes of MYC-driven gene expression. For this purpose, the cells were Amaxa-transfected using MYC siRNA-treated and control siRNA-treated cells was extracted employing the RNeasy Midi kit according to standard protocols . Affymetrix GeneChip analysis (HG-U133A) was performed according to the manufacturer's recommendations, starting with 5 \u00b5g total RNA. CEL files were generated with the help of the GCOS 1.3 software (Affymetrix). The CEL files of all experiments are available via the Gene Expression Omnibus (GEO) of the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov/geo/) under the accession number GSE30726.Total RNA of MYC siRNA treatment using an empirical Bayesian method. P-values were corrected for multiple testing by the method proposed by Benjamini and Hochberg For analysis of Affymetrix data the expression levels were normalized using VSN The differential expression of selected genes was confirmed by Western blotting as previously described http://www.bioconductor.org/packages/2.5/bioc/html/ChIPpeakAnno.html). The nearest transcription start region (TSR) was used as reference point. By default, the distance is calculated as the distance between the start of the binding site and the TSR that is the gene start for genes located on the forward strand and the gene end for genes located on the reverse strand.Ensembl annotations closest to or overlapping with the 7,054 genomic intervals were determined employing the Bioconductor package ChiPpeakAnno (MYC siRNA experiments (http://david.abcc.ncifcrf.gov/) Ensembl annotations next to significant MYC-binding sites and the eriments were uplIG-MYC translocation positive BL cell lines were analyzed after MYC chromatin immunoprecipitation (MYC-ChIP) by next generation sequencing, leading to a total number of more than 16 million and 12 million sequence reads in the MYC immunoprecipitated (ChIP) and input samples, respectively. Alignment to the human reference genome using Bowtie default settings mapped 10,882,577 ChIP reads and 8,495,924 input reads uniquely, which were used for further analysis. More details for read numbers and identified peaks at different stages of data processing are given in DNA-fragments obtained from five The software package HOMER (v2.6) NME1 (rank position 5) and NPM1 (rank position 47) revealed a higher enrichment as compared to lower ranked genes such as BOB1 or MS4A1 (CD20) , thereby confirming the reliability of our ChIP-Seq data of the identified MYC-binding sites were located in close proximity to transcriptional start sites within +/\u22121000 bp . AdditioMoreover, we found 2,320 exact matches for the canonical E-box sequence CACGTG in our 7,054 genomic intervals . This corresponds to a 4.6 fold higher enrichment as compared to the 194 occurrences in 590 intervals described by Zeller et al. de-novo from our ChIP-Seq data and is shown in The typical MYC-binding E-box motive could also be calculated Pan troglodytes, Macaca mulatta, Bos tarus, Canis familaris, Mus musculus and Rattus norvegicus; 5,558 of the 7,054 (78.8%) binding sites are evolutionary conserved with orthologous regions in at least 4 out of 6 placental mammalian genomes displayed MYC binding in BL. 10 of these (71.4%) had orthologous regions in at least 4 of 6 placental mammals , CD21 (CR2), CD22, CD79a/b, SYK, LYN, BLK, BLNK, BOB1 (POU2AF1) and PAX5) were identified as active MYC-binding sites with MYC-specific siRNAs resulted in a reduction of the MYC protein of approx. 75% , as well as for NF-kB associated proteins , which are down-stream components of the B-cell receptor pathway .The understanding of MYC biology is of paramount importance to elucidate its role in the pathogenesis of Burkitt lymphoma (BL), a disease characterized by a consistent high MYC protein expression due to a genomic translocation. Therefore we undertook a comprehensive approach to identify MYC-binding sites by chromatin immunoprecipitation with subsequent deep sequencing (ChIP-Seq). This led to a ranked list of 7,054 MYC-binding genomic intervals in BL .NPM1, NME1) and identification of MYC-binding sites associated with miRNAs known to be regulated by MYC Confirmation of the exact MYC-binding position in the promoters of previously published MYC target genes (e.g. r-29b-2) [24], [4r-29b-2) . (iii) Tr-29b-2) .Our genome-wide approach largely extends previous MYC ChIP-analyses from one BL cell line using promoter tiling arrays (ChIP-on-chip) covering only 4,839 proximal promoters myc gene along with the E-box containing RiBi core-promoter signature MYC is a prerequisite for maintenance of this precise cell number, most likely by a lack of MYC-driven cell proliferation.Gene set enrichment analysis using the Ensembl annotations related to MYC binding revealedHox genes BMI1 as MYC-collaborating oncogene EZH1/2, EED and BMI1. Given the important role of PcG in B-cell development, lymphomagenesis, and tumor stem cell development Another important finding of our study is the binding of MYC to many Polycomb group (PcG) genes in BL. PcG proteins were identified in Drosophila more than 30 years ago as regulators of anterior-posterior body patterning through the repression of CD19, CD20 (MS4A1), CD21 (CR2), CD22, PAX5, SYK, LYN, BLK, BLNK, BOB1 (POU2AF1), CD79a/b) as active MYC target genes are illustrated for the 5\u2032- ends of the NME1 gene by using the UCSC genome browser (http://genome.ucsc.edu/). Reads in red map to the forward strand and blue reads to the reverse strand. The location of real-time DNA-PCR ( DNA-PCR is schem DNA-PCR .(PDF)Click here for additional data file.Figure S2MYC-binding sites in the NPM1 gene. ChIP-Seq reads obtained after MYC ChIP-Seq and from input controls analyzing 5 BL cell lines are illustrated for the 5\u2032- ends of the NPM1 gene by using the UCSC genome browser (http://genome.ucsc.edu/). Reads in red map to the forward strand and blue reads to the reverse strand. The location of real-time DNA-PCR ( DNA-PCR is schem DNA-PCR .(PDF)Click here for additional data file.Figure S3MYC-binding sites in the BYSL gene. ChIP-Seq reads obtained after MYC ChIP-Seq and from input controls analyzing 5 BL cell lines are illustrated for the 5\u2032- ends of the BYSL gene by using the UCSC genome browser (http://genome.ucsc.edu/). Reads in red map to the forward strand and blue reads to the reverse strand. The location of real-time DNA-PCR ( DNA-PCR is schem DNA-PCR .(PDF)Click here for additional data file.Figure S4MYC-binding sites in the PAX5 gene. ChIP-Seq reads obtained after MYC ChIP-Seq and from input controls analyzing 5 BL cell lines are illustrated for the 5\u2032- ends of the PAX5 gene by using the UCSC genome browser (http://genome.ucsc.edu/). Reads in red map to the forward strand and blue reads to the reverse strand. The location of real-time DNA-PCR ( DNA-PCR is schem DNA-PCR .(PDF)Click here for additional data file.Figure S5MYC-binding sites in the MS4A1 gene. ChIP-Seq reads obtained after MYC ChIP-Seq and from input controls analyzing 5 BL cell lines are illustrated for the 5\u2032- ends of the MS4A1 gene by using the UCSC genome browser (http://genome.ucsc.edu/). Reads in red map to the forward strand and blue reads to the reverse strand. The location of real-time DNA-PCR ( DNA-PCR is schem DNA-PCR .(PDF)Click here for additional data file.Figure S6NOP56 intron 8 (negative control). ChIP-Seq reads obtained after MYC ChIP-Seq and from input controls analyzing 5 BL cell lines are illustrated by using the UCSC genome browser (http://genome.ucsc.edu/). Reads in red map to the forward strand and blue reads to the reverse strand. The location of real-time DNA-PCR ( DNA-PCR is schem(PDF)Click here for additional data file.Figure S7CCR7 (negative control). ChIP-Seq reads obtained after MYC ChIP-Seq and from input controls analyzing 5 BL cell lines are illustrated by using the UCSC genome browser (http://genome.ucsc.edu/). Reads in red map to the forward strand and blue reads to the reverse strand. The location of real-time DNA-PCR ( DNA-PCR is schem(PDF)Click here for additional data file.Figure S8SEMA4C (negative control). ChIP-Seq reads obtained after MYC ChIP-Seq and from input controls analyzing 5 BL cell lines are illustrated by using the UCSC genome browser (http://genome.ucsc.edu/). Reads in red map to the forward strand and blue reads to the reverse strand. The location of real-time DNA-PCR ( DNA-PCR is schem(PDF)Click here for additional data file.Figure S9Significantly enriched KEGG pathways detected by ChIP-Seq analysis in relat(TIF)Click here for additional data file.Table S1Primer sequences for real-time DNA-PCRs.(XLS)Click here for additional data file.Table S2Complete list of Ensembl annotations annotated with the number of detected MYC-binding sites for each Ensembl annotation.(XLS)Click here for additional data file.Table S3Differential gene expression after siRNA mediated knock-down of MYC. Three BL cell lines were analyzed employing U133A Affymetrix GeneChip hybridization.(XLS)Click here for additional data file.Table S4Number of reads in the Fastq files for all MYC ChIP-Seq and input samples mapped to the human hg19 reference genome. The number of reads within the putative peaks discovered by HOMER and within the final list of 7,054 merged peaks (DESeq) is shown in the far right column.(DOC)Click here for additional data file.Table S57,054 peak regions (i.e. MYC-binding sites) with a FDR below 1e-4 are ranked by their FDR. Their closest or overlapping gene annotation is given.(XLS)Click here for additional data file.Table S6MYC.01 Transcription factor binding matrix. The MYC.01 binding matrix was generated de novo by CoreSearch using the top 100 sequences in (XLS)Click here for additional data file.Table S7Biological annotations associated with genes next to the 7,054 genomic intervals (i.e. MYC-binding sites). Interesting terms are highlighted in red.(XLS)Click here for additional data file.Table S8Biological annotations associated with 5,558 of the 7,054 genomic intervals with orthologous regions in at least 4 of 6 mammalian species. Genomatix RegionMiner with default parameters was employed for the search of orthologous genomic regions. Interesting terms are highlighted in red.(XLS)Click here for additional data file.Table S9Biological annotations associated with genes differentially expressed after siRNA-mediated MYC knock-down in 3 BL cell lines . Interesting terms are highlighted in red.(XLS)Click here for additional data file."} +{"text": "Many factors influence breast cancer progression, including the ability of progenitor cells to sustain or increase net tumour cell numbers. Our aim was to define whether alterations in putative progenitor populations could predict clinicopathological factors of prognostic importance for cancer progression.Primary cultures were established from human breast tumour and adjacent non-tumour tissue. Putative progenitor cell populations were isolated based on co-expression or concomitant absence of the epithelial and myoepithelial markers EPCAM and CALLA respectively.Significant reductions in cellular senescence were observed in tumour versus non-tumour cultures, accompanied by a stepwise increase in proliferation:senescence ratios. A novel correlation between tumour aggressiveness and an imbalance of putative progenitor subpopulations was also observed. Specifically, an increased double-negative (DN) to double-positive (DP) ratio distinguished aggressive tumours of high grade, estrogen receptor-negativity or HER2-positivity. The DN:DP ratio was also higher in malignant MDA-MB-231 cells relative to non-tumourogenic MCF-10A cells. Ultrastructural analysis of the DN subpopulation in an invasive tumour culture revealed enrichment in lipofuscin bodies, markers of ageing or senescent cells.Our results suggest that an imbalance in tumour progenitor subpopulations imbalances the functional relationship between proliferation and senescence, creating a microenvironment favouring tumour progression. Significant interest surrounds the question whether cancer stem/progenitor cells drive tumour formation ,2, howevBreast progenitor cells are isolated based on expression of markers suggesting capabilities to generate cells of mixed myoepithelial and luminal epithelial lineages ,4. OtherUsing primary cultures from human breast tumour and non-tumour tissue, we sought to define correlations between progenitor cell numbers and clinicopathological or functional indicators of cancer aggressiveness. Our results demonstrate an imbalance between two putative progenitor cell populations in clinicopathologically-aggressive tumours, in conjunction with functional alterations promoting increased proliferation or reduced growth arrest. Taken together, full investigations of progenitor populations in relation to clinicopathological parameters could make an important contribution towards a better understanding of breast cancer progression.Suppliers: trypsin-EDTA, penicillin/streptomycin, penicillin/streptomycin/neomycin, fungizone, Cyquant, X-gal, Alexa-Fluor antibodies (Invitrogen); soybean trypsin inhibitor, collagenase I, hyaluronidase 1-S, DMEM/Ham's F12, bovine insulin, peroxidase-labelled secondary antibodies (Sigma); HMEC, mammary epithelial growth medium (MEGM) kits, foetal bovine serum ; glutaraldehyde (Fluka); osmium tetroxide (Electron Microscopy Services). Antibody suppliers: actin, ESA and SMA (Sigma); cytokeratin-19, PE-conjugated CALLA, FITC-conjugated EPCAM, FITC- or PE-conjugated IgG controls (Dako); cytokeratin-18 (Abcam); cytokeratin-14 (Millipore); vimentin and p63 (BD Biosciences).Breast primary cultures were generated from patient lumpectomy/mastectomy samples with informed consent as approved by the Medical Ethics committees of Beaumont Hospital and the Mater Misericordiae Hospital, in accordance with the Declaration of Helsinki. One piece each of tumour tissue and non-tumour margins grown normally in DMEM-F12, 5% horse serum, 0.5 \u03bcg/ml hydrocortisone, 10 \u03bcg/ml insulin, 100 ng/ml cholera toxin, 20 ng/ml human recombinant EGF (MCF10A) or DMEM, 10% FBS, 2 mM L-glutamine(MDA-MB-231) were conditioned in MEGM for 2-3 weeks and used in flow cytometry experiments as controls for normal and tumourogenic phenotypes respectively.3) were plated in triplicate and harvested after 0, 3 or 6 days. Cyquant solution was incubated on freeze-thawed cells (5 min), and emitted fluorescence detected at 520 nm on a Wallac plate-reader. Fluorescence readings of unknown samples were translated into cell numbers by referring to two separate fluorescence standard curves - one for non-tumour and one for tumour cultures- constructed from known cell numbers were plated in duplicate, and stained for senescence-associated \u03b2-galactosidase activity [Primary cells (5 \u00d7 10activity . Three bPrimary cells (passage 1-2) grown in chamber slides were fixed in 3.7% paraformaldehyde and immunostained for epithelial or myoepithelial markers using DAPI as a nuclear counter-stain. Primary antibodies were omitted in negative controls, and slides visualized on a Zeiss LSM510-meta confocal microscope.Confluent primary cultures were harvested in RIPA containing protease and phosphatase inhibitors. Lysates were dounced and 25 \u03bcg supernatant subjected to SDS-PAGE and Western blot analysis for K19, K18, VIM and p63.+/EPCAM+, CALLA+/EPCAM-, CALLA-/EPCAM- or CALLA-/EPCAM+ populations on a BD FACSAria cell sorter. Some passage 0 cells were analyzed for activity of the stem cell marker ALDH by Aldefluor assay [5 cells were resuspended in assay buffer and incubated with activated substrate or the negative control reagent before analysis.Confluent passage 0 primary cells (T25 flask/condition) were trypsinized, blocked in human serum and co-incubated with FITC-conjugated mouse anti-human EPCAM and PE-conjugated mouse anti-human CALLA (4\u00b0C/30 min). Negative controls were unlabelled or single-stained with FITC-EPCAM, PE-CALLA, FITC-IgG or PE-IgG. Cells were analyzed on a Beckman Coulter Cyan-ADP and/or an Accuri-C6 flow cytometer. Cells were sorted into CALLAPassage 0 primary cultures or HMECs were fixed with 2.5% glutaraldehyde, processed as described and analt-tests. Population variances were first compared using Instat-3.3.6 to inform the choice of equal/unequal variance between populations. The proliferation:senescence ratio was calculated based upon the data shown in Figure Data are expressed as mean \u00b1 standard error of the mean. Non-tumour versus tumour results were compared using non-parametric tests and one-tailed unpaired left). Two cellular populations were observed - large polygonal cells in colony centres , and small polygonal cells (spc) at the peripheries. Since spc and lpc resembled respectively myoepithelial and luminal epithelial cells, expression of epithelial and myoepithelial markers was examined by immunofluorescence microscopy and tumour (T) human breast tissue yielded adherent organoids with outwardly-proliferating colonies Figure , left. Ty Figure . In compy Figure detectioy Figure also revNT versus VT). Multi-nucleation of tumour cells was frequently observed, in parallel with compromised nuclear membranes . Furthermore, tumour cell mitochondria were abnormal, elongated and occasionally fused . Finally, non-tumour cells displayed a well-differentiated rough endoplasmic reticulum (RER) while that in tumour cells was fragmented and dispersed .Ultrastructural analysis of matched cultures was undertaken to confirm differences between tumour and non-tumour specimens Figure . Firstlyleft). Since Cyquant proliferation assays quantify all cells rather than just actively-proliferating cells, we performed senescence-associated (SA) \u03b2-galactosidase assays [right). Non-tumour cultures had two-fold higher SA-\u03b2-galactosidase staining than that in tumour cultures. This was independent of the grade of the originating tumour, and did not reflect an impaired capacity to senesce in response to exogenous stimulation (data not shown).We next investigated if morphological differences were accompanied by cell fate differences Figure . Prolifee assays to estimAs the balance between proliferation and senescence is more important than either parameter alone, we examined whether altered proliferation:senescence ratios in breast primary cultures could identify aggressive tumours. The proliferation:senescence relationship was estimated based on proliferation graph slopes and senescence values Figure . Our datarrow) resembling those described as progenitor/stem cells in mammary duct basal laminae [3b, top arrow). Layering of dark filament-rich cells and EPCAM markers ,12. All Given DN differences in aggressive HG or ER-negative tumours versus aggressive HER2-positive tumours, we performed ultrastructural analysis on DN populations from one non-tumour and one tumour culture . Although both populations had many similarities (data not shown), unique to the tumour DN population was the presence of abundant lipofuscin bodies Figure , arrows.Since both DN and DP populations are putative progenitor/stem cells ,4, we quin vivo.Intriguing recent work has suggested that immunohistochemical profiling of breast tumours for cancer stem cell populations may have prognostic value . To probMyoepithelial marker expression was found to dominate over luminal epithelial expression, consistent with observations in HMEC ,18. ExprMesenchymal/basal-like phenotypes also promote progenitor growth and tissue regeneration . The expWe sought to correlate subpopulation changes with tumour clinicopathological parameters, and observed decreased DP populations in aggressive tumours of high grade or ER negativity. ALDH activity was also reduced in HG tumours, an interesting fact since ALDH expression has been correlated with poor prognosis in breast cancer ,24 - altDN and DP populations have been described as slightly different putative progenitor/stem cell populations; with DN representing an undifferentiated population while DP represents a multipotent population ,12. SincInterestingly, we did not observe DN reductions in HER2-positive cultures. However elevated HER2 can drive premature senescence , and higWe have reported reduced senescence in tumour versus non-tumour breast primary cultures, and stepwise increases in the proliferation:senescence ratio with increasing tumour grade. Isolation of putative progenitor subpopulations revealed a novel correlation between increased DN:DP ratios and clinicopathological indicators of aggressive tumours . Our data suggest that progenitor population imbalance could promote tumour progression by altering the relationship between proliferation and senescence Figure . Future in situ; IDC: invasive ductal carcinoma; LC: lobular carcinoma; ITLC: invasive tubular lobular carcinoma; SA-\u03b2-gal: senescence-associated \u03b2-galactosidase; ER: estrogen receptor; PR: progesterone receptor; ESA: epithelial-specific antigen; SMA: smooth muscle actin; VIM: vimentin; CALLA: common acute lymphoblastic leukaemia antigen; EPCAM: epithelial cell adhesion molecule; DP: CALLA & EPCAM double-positive; DN: CALLA & EPCAM double-negative; HG: high grade; LG: low grade; ALDH: aldehyde dehydrogenase; TEM: transmission electron microscopy; K14: cytokeratin-14; K18: cytokeratin-18; K19: cytokeratin-19.MEGM: mammary epithelial growth medium; HMEC: human mammary epithelial cells; DCIS: ductal carcinoma The authors declare that they have no competing interests.SD and AMH conceived and designed the study, analyzed and interpreted the data, drafted the manuscript and revised it. SD performed most of the experimental work, with assistance from LH (primary culture generation), IA (senescence assay set-up), DCC (electron microscopy) and AB (cell sorting). DCC, AB and ADKH contributed to the interpretation of the results. ADKH, PAD, MJS, MS and MRK contributed to patient selection, sample acquisition and clinical interpretation. All authors read and approved the final manuscript.Primary culture patient information.Click here for fileProliferation assay standard curves for tumour and non-tumour cultures. Two non-tumour and two tumour cultures were used to generate standard curves to calculate numbers of cells from fluorescence values obtained at different time points of the Cyquant proliferation assays.Click here for fileMEGM medium does not alter the morphology of MCF-10A and MDA-MB-231 cells. MCF-10A and MDA-MB-231 cells were cultured for 15 days in MEGM or their standard serum-positive media, and imaged by phase contrast microscopy. No overt morphological differences were observed in either cell type after the media was switched.Click here for file"} +{"text": "Heart failure results in 280,000 deaths each year in the US and post-myocardial infarction (MI) remodeling is among the leading causes. An unresolved question is the integrity of the molecular contractile apparatus in the peri-infarct or borderzone (BZ) myocardium and the resulting effect on infarct stretching and thinning and BZ expansion. A difficulty is the lack of available methods to noninvasively measure the BZ stress-strain relation under varying loading conditions in vivo. Our hypothesis is that the BZ myocardium has significantly reduced end-systolic stiffness as measured by MRI-derived regional preload recruitable stroke work (rPRSW). To test this hypothesis, we transiently varied loading conditions in post-infarct swine and measured real time LV apparent fiber length using nonlinear image reconstruction and sub-Nyquist golden angle MRI.Yorkshire male swine were used in an IACUC approved posterobasal MI study. At 1 week post-MI, a hydraulic balloon catheter device was positioned in the IVC. Real time golden angle radial bSSFP MRI synchronized with LV pressure during IVC occlusion (N=2) and dobutamine stress test (N=1) was performed. Images were reconstructed using Gadgetron framework using an iterative SENSE-based algorithm with k-space weighted imaging contrast (KWIC) temporal filtering . The ventricular cavity and myocardium was segmented using a level-set algorithm and endocardial and epicardial points wall thickness (WT) was tracked using a non-rigid intensity based algorithm from wall contours throughout inflow occlusion. rPRSW was quantified by the area of the Pressure- Relative Fiber Length loop.We found a decrease in relative myofiber length and stroke work in the healthy myocardium during inflow occlusion as shown in all animals. Data from one representative animal is shown in Figure Regional variations in local work performed were observed during transient preload reduction using a real time magnetic resonance imaging method. These findings provide insight about load-dependent changes in contractile function in post-infarction LV remodeling.R01-HL103723, R01-HL63954, R01-HL73021, and T32-EB009384."} +{"text": "Featuring the modified MR compatible stent valve delivery device, real-time MR guided TAVI has been performed in vivo in eight swine.Transcatheter, transarterial aortic valve implantation (TAVI) is rapidly emerging as a promising new treatment option for patients with severe symptomatic aortic valve stenosis who are considered at high or prohibitive surgical risk. Envisioning real-time MR guidance of the TAVI procedure, the objective of this study was the systematical The self-expandable Medtronic CoreValve\u00ae aortic bioprosthesis is composed of a nitinol stent frame with an integrated trileaflet porcine pericardial tissue valve and is either implanted via the femoral or subclavian artery. Its delivery catheter has a 12Fr shaft with 18Fr distal end comprising the crimped prosthesis which can be released stepwise. The original catheter shaft revealed ferromagnetic attraction thus potentially compromizing MR imaging and safety. The delivery device consequently was re-designed obviating any metal braiding resulting in full MR compatibility of the delivery device. MR-guided TAVI was performed on 8 farm pigs (75-85 kg) via transfemoral (2/8) and via subclavian (6/8) access on a 1.5 T MRI system equipped with an interventional in-room monitor. Catheter placement and stent release was performed under real-time MR-guidance with a rt-TrueFISP sequence providing 5 fps.The nitinol-based self-expandable stent-valve was excellently visualized with delineation even of small details. The commercial delivery catheter shaft of this device revealed strong ferromagnetic artifacts, thus also raising concerns regarding RF-related device heating and ferromagnetic attraction. Replacement of the commercial delivery device by the re-designed delivery device resulted in artifact elimination and excellent real-time visualization of catheter movement and valve deployment. MR-guided TAVI was successful in 6/8 swine Figure . Post-inThe self-expandable CoreValve aortic stent-valve is potentially suited for real-time MRI-guided placement after suggested design modifications of the delivery-system. MR imaging in this interventional setup provided excellent pre-interventional anatomic and functional evaluation of the native aortic valve, precise real-time instrument guidance allowing accurate placement of the stent-valve within the native aortic annulus, and finally detailed post-interventional evaluation of therapeutic success."} +{"text": "Growing evidence suggests that DNA methylation plays a role in tissue-specific differentiation. Current approaches to methylome analysis using enrichment with the methyl-binding domain protein (MBD) are restricted to large (\u22651 \u03bcg) DNA samples, limiting the analysis of small tissue samples. Here we present a technique that enables characterization of genome-wide tissue-specific methylation patterns from nanogram quantities of DNA.Rgs20, Hes2, Nfic, Cckbr and Six3os1) were validated by pyrosequencing.We have developed a methodology utilizing MBD2b/MBD3L1 enrichment for methylated DNA, kinase pre-treated ligation-mediated PCR amplification (MeKL) and hybridization to the comprehensive high-throughput array for relative methylation (CHARM) customized tiling arrays, which we termed MeKL-chip. Kinase modification in combination with the addition of PEG has increased ligation-mediated PCR amplification over 20-fold, enabling >400-fold amplification of starting DNA. We have shown that MeKL-chip can be applied to as little as 20 ng of DNA, enabling comprehensive analysis of small DNA samples. Applying MeKL-chip to the mouse retina (a limited tissue source) and brain, 2,498 tissue-specific differentially methylated regions (T-DMRs) were characterized. The top five T-DMRs (MeKL-chip enables genome-wide methylation analysis of nanogram quantities of DNA with a wide range of observed-to-expected CpG ratios due to the binding properties of the MBD2b/MBD3L1 protein complex. This methodology enabled the first analysis of genome-wide methylation in the mouse retina, characterizing novel T-DMRs. DNA methylation is an epigenetic modification known to be important in many cellular processes, including tissue-specific gene expression. There is no standardized method for DNA methylation analysis and most array-based methods for genome-wide profiling require input DNA in microgram quantities, making them impractical for small tissue samples Table\u00a0. Sodium Methylation data obtained by hybridization to promoter, CpG island or CpG site-specific microarrays is biased and restricted by the design of the array platform. To overcome this bias, the comprehensive high-throughput array for relative methylation (CHARM) platform was developed to interrogate CpG sites genome-wide, irrespective of proximity to genes or CpG islands. As the While MIRA can enrich DNA samples in the nanogram range, hybridiCurrently, no protocols are available for MBD-chip with low amounts of starting DNA. With the goal of establishing a user-friendly method for assessing genome-wide DNA methylation in small (nanogram) DNA samples, we developed a new protocol based upon MBD2b/MBD3L1 enrichment followedUsing the published LM-PCR protocol as a starting point, we explretinol-binding protein 3 (Rbp3) and rhodopsin (Rho) genes are specifically expressed in retinal photoreceptors and have been shown to be hypomethylated in a cell-specific manner in the retina compared to non-retinal tissues (14955). QPCR wa (14955)2O. Modified linkers [Rbp3) primer pair as a loading control for DNA in each reaction. The cycling conditions were 72\u00b0C for 3 min for initial fill-in, 94\u00b0C for 2 min for initial denaturation, followed by 35 cycles of and a disassociation curve. The relative fold-change between the various ligation conditions were calculated and plotted for five replicate experiments, along with the standard error of the mean unless otherwise stated. Based on the LM-PCR protocol developed by the Ren laboratory, 5 \u03bcg ofA TC 3\u00b4) includedA TC 3\u00b4), ligatioFragmented (input) DNA was labeled with Cy3, and enriched (methylated) DNA was labeled with Cy5, using the NimbleGen Dual-Color DNA Labeling kit (Roche) according to the manufacturers\u2019 instructions. Samples were hybridized to the custom NimbleGen 2.1M feature mouse CHARM microarray at the Johns Hopkins Medical Institutions Deep Sequencing & Microarray Core Facility or the Johns Hopkins Bloomberg School of Public Health Genomic Analysis and Sequencing Core Facility.P < 0.005. Consequently, T-DMRs were constituted by neighboring differentially methylated probes. T-DMRs with less than three probes were excluded from further analysis.Analysis of the MeKL-chip data was performed using the R/Bioconductor software for CHARM as previously published,31. In bNCpG, NC and NG are the number of CpGs, and nucleotide Cs and Gs, respectively, and L is the length of the T-DMR sequence.The definition of local CpG density by Pelizzola et al. was adopRgs20 (Gene ID: 58175), Hes2 (15206), Nfic (18029), Cckbr (12426) and Six3os1 (100043902). Pyrosequencing primers were designed , 1 \u03bcg of genomic DNA was bisulfite converted. Bisulfite-converted DNA was eluted twice in 10 \u03bcL M-Elution buffer and stored as 5 \u03bcL aliquots at \u221280\u00b0C. Genomic sequences surrounding the RefSeq genes were obtained using the UCSC Genome Browser for c.c: Correlation coefficient; CHARM: Comprehensive high-throughput array for relative methylation; CpGO/E: Observed-to-expected CpG ratio; Ct: Threshold cycle; DMR: Differentially methylated region; dNTP: Deoxyribonucleotide triphosphates; HBSS: Hanks\u2019 balanced salt solution; KLM-PCR: Kinase-modified ligation-mediated PCR; LM-PCR: Ligation-mediated PCR; MBD: Methyl-binding domain protein; MeDIP: Methylated DNA immunoprecipitation; MeKL: MBD2b/MBD3L1 enrichment of DNA followed by KLM-PCR; MIRA: Methylated-CpG island recovery assay; MRE: Methylation-sensitive restriction enzyme; PCR: Polymerase chain reaction; PEG: Polyethylene glycol; QPCR: Quantitative PCR; RRBS: Reduced-representation bisulfite sequencing; T-DMR: Tissue-specific differentially methylated region; TSS: Transcription start site; WGA: Whole genome amplification; WGBS: Whole genome bisulfite sequencing.The authors declare that they have no competing interests.SLM, DJZ, VFO and SA designed the study. VFO performed the experiments. JW and JQ designed and performed the bioinformatics analysis. VFO, JW, SA, DJZ, JQ and SLM wrote the manuscript. DJZ, JQ and SLM supervised the project. All authors read and approved the final manuscript.(A) Modification of the universal adapter oligo sequence. The original LM-PCR oligo contained a palindrome at the 3\u2032 end[(B) NanoDrop quantification of the mean micrograms of DNA produced after KLM-PCR. Bars represent the mean of duplicate experiments from amplification of 10, 25, 50 and 250 ng amounts of pre-enriched starting DNA, or 10 ng of unenriched (UE) DNA. Error bars show standard deviation (n = 2).The KLM-PCR protocol. e 3\u2032 end. PreventClick here for fileTable S2. PCR amplification primers used for pyrosequencing the top five DMRs identified using MeKL-chip. Table S3. Sequencing primers used for pyrosequencing validation of the top five DMRs identified using MeKL-chip.Top 20 differentially methylated regions identified between mouse retina and brain from 250 ng of starting DNA. Click here for fileP < 0.001, Student\u2019s two-tailed, paired t-test) between the retina (red bars) and brain (blue bars) in a second cohort of mice. Error bars show the 95% CI (n = 5).Relative CpG methylation in the retina (red) and brain (blue) of the four other top T-DMRs (black boxes) evaluated using MeKL-chip (top plots) and pyrosequencing validation of the differential methylation (bottom graphs). See Figure\u00a0Click here for file(A) Post-enrichment, Rbp3 (grey bars) and Rho (black bars) were enriched for methylated DNA in the brain samples for all amounts of starting DNA, whereas no enrichment was observed in unenriched (UE) DNA. (B) Post-KLM-PCR, QPCR showed maintenance of the enrichment pattern for methylation in the brain samples for Rho (black bars) and Rbp3 (grey bars) and no enrichment of the UE samples.Pre-hybridization validation of enrichment from low-input enriched DNA from retina and brain samples. The mixed effects regression model with a random intercept for the measures from triplicate QPCR of two PCR amplifications of the same sample were used to calculate the mean difference and standard error in fold enrichment. Click here for fileRgs20 region identified as a T-DMR for the 250-ng high-input samples in the brain (blue) and retina (red) (top plot) as previously shown in Figure\u00a0Rgs20 is shown for direct comparison (lower plot) in brain (blue) and retina (red). Each point is the relative percentage methylation for 1 probe in 1 sample. The 250 ng plot contains biological triplicates and blue and red lines show the average methylation. The 10 ng plot contains one biological sample. The T-DMRs are still detectable in the 10 ng low-input sample.MeKL-chip CpG site methylation profiles of the Click here for file"} +{"text": "The FHSS (a points-based scoring system) was previously used to identify families at high risk of an inherited colorectal cancer syndrome. AM-I (Amsterdam-I) families scored high \u226412), but some scored low (\u22658) when scored from unaffected relative\u2019s perspective [2, but soFamily members in Lynch and Type X families identified from the Jagelman Registry database were scored according to the score sheet described in Table They were scored from perspective of an affected proband (AP) or unaffected proband (UP), affected (AS) or unaffected (US) sibling or a child of each sibling (Child).91 probands in 48 AM-I, 14 Amsterdam-II (AM-II), 6 Amsterdam-Like (AM-Like), 10 Familial Colon Cancer (FCC), 6 no syndrome (syn) families and 7 Type X were scored. 197 relatives were scored . AM-I and Type X median scores were higher than other syndromes (<12) and suggestive of HNPCC when scored from the perspective of the proband, sibling or child of AS. The median scores were lower in AM-II, FCC, AM-Like, and no syn families with fewer colon cancers (Table Findings were similar to validation study conducted on AM-I families. The FHSS is a reliable tool to determine familial risk of colorectal cancer especially in AM-I families and depends on the perspective of person being scored. In small families, those with predominantly extra colonic cancers or high-risk polyps, or if affected relatives are more than a generation from an individual, the FHHS will not always identify high risk or Lynch families."} +{"text": "Human T-cell Leukemia Virus type I (HTLV-I) infects an estimated 15-20 million persons worldwide. A number of diseases have been associated with this virus including adult T-cell leukemia/lymphoma (ATLL), HTLV-associated myelopathy/tropical spastic paraparesis (HAM/TSP), HLTV-I uveitis, and HTLV-I\u2013associated infective dermatitis. The screening process for HTLV-I among blood banks includes detection by an enzyme immunoassay (EIA) followed by a confirmatory western blot (WB). Seropositive results are defined by the presence of all core bands as well as the band specific for recombinant HTLV-I Env glycoprotein. However, numerous cases have been documented in which a positive response was detected by ELISA, but WB displayed an incomplete banding pattern. These samples were then categorized as seroindeterminates. Using the LIPS assay, 60% of the 53 seroindeterminate samples previously screened by ELISA and WB displayed anti-HTLV-I Gag, Env or Tax antibody responses . The LIPS assay was also able to detect anti-HTLV-I antibody responses in 6% of the 167 samples determined to be HTLV-I ELISA negative. A number of these samples were subsequently found to have an indeterminate WB pattern. Although the significance of these HTLV-I/II seroindeterminates is unclear, it may suggest a much higher prevalence of exposure to HTLV-I/II than previously estimated, as seroindeterminate samples may indicate exposure to the virus. The LIPS assay is a sensitive and high throughput antibody detection assay which may prove to be a useful tool in HTLV-I related clinical investigations."} +{"text": "Drosophila melanogaster, specification of wing vein cells and sensory organ precursor (SOP) cells, which later give rise to a bristle, requires EGFR signaling. Here, we show that Pumilio (Pum), an RNA-binding translational repressor, negatively regulates EGFR signaling in wing vein and bristle development. We observed that loss of Pum function yielded extra wing veins and additional bristles. Conversely, overexpression of Pum eliminated wing veins and bristles. Heterozygotes for Pum produced no phenotype on their own, but greatly enhanced phenotypes caused by the enhancement of EGFR signaling. Conversely, over-expression of Pum suppressed the effects of ectopic EGFR signaling. Components of the EGFR signaling pathway are encoded by mRNAs that have Nanos Response Element (NRE)\u2013like sequences in their 3\u2019UTRs; NREs are known to bind Pum to confer regulation in other mRNAs. We show that these NRE-like sequences bind Pum and confer repression on a luciferase reporter in heterologous cells. Taken together, our evidence suggests that Pum functions as a negative regulator of EGFR signaling by directly targeting components of the pathway in Drosophila.In Upon activation, the signaling proceeds through Drk, Sos, and Ras activation, to a phosphorylation cascade involving Raf (MAPKKK) and Dsor1 (MEK). The pathway culminates in activation of Drosophila melanogaster, adult wing blade has five wing veins, which are differentiated in the wing imaginal disc during larval and pupal stages. EGFR signaling during the larval period promotes wing vein cell differentiation. Enhanced EGFR signaling results in the development of extra-wing veins, whereas reduced signaling results in wing vein loss In achaete (ac) and scute (sc) Large bristles (macrochaetes) on the notum of adult flies arise from a single sensory organ precursor (SOP) cell in the wing imaginal disc during larval development. Each SOP cell is selected from a group of equipotent cells in a proneural cluster that is specified by high level expression of proneural genes such as pum mutants, which is reminiscent of phenotypes associated with up-regulation of EGFR signaling. Our genetic interaction analysis suggests that Pum functions as a negative regulator of EGFR signaling. Pum is a translational repressor that binds to the Nanos Responsive Element (NRE) sequence at the 3\u2019UTR of its target mRNAs rl) 3\u2019UTRs and represses reporters containing these NRE sequences. This study revealed a role for Pum in formation of wing veins and bristles by negatively regulating EGFR signaling.In our study, we observed extra wing veins and thoracic macrochaetes in 7pum/7pum and 1688pum/1688pum adult \u201cescapers\u201d have rare extra wing veins , 3/Mscpum , 3/1688pum , 1688/Mscpum . Extra wing veins also arose where pum function is reduced in wing imaginal disc via RNAi of pum ; these flies displayed extra-wing veins only in the posterior compartment, where en-GAL4 is active (arrowhead in [Sem]rl heterozygotes ([Sem]rl/+) with elevated MAPK activity en-Gal4 . ConversUAS-Ras) or Pum iUAS-Ras) .pum+ on the wing vein phenotypes associated with [Sem]rl. Eliminating one copy of pum (pum/+) by itself does not produce ectopic wing vein (data not shown). However, eliminating one copy of pum greatly enhanced the extra wing vein phenotype in [Sem]rl flies resulted in extra wing veins around the wing boundary (If ng notch . Co-overAS-EGFR) suppress-Gal4/+) resultedGal4/+) . Thus, bpum homozygote mutants and transheterozygotes of pum alleles have extra macrochaetae , 1688/Mscpum , 7/1688pum . In contrast, ectopic expression of pum in the SOP cells with the sca-Gal4 driver UAS-EGFR) by sca-Gal4 induced extra bristles did not affect the number of bristles by itself (data not shown), but greatly enhanced bristle number induced by ectopic expression of EGFR and found that co-expression of Pum and EGFR resulted in the elimination of EGFR-induced extra bristles, phenotypes similar to Pum over-expression ; Table 1pression ; Table 1HD) mediates Pum binding to NRE and Nos and possesses translational repressor activity HD) compartment boundary . To distinguish uniform expression from uniform background, we performed two additional experiments. Using the boundary , we overdpp-GAL4 regulates accumulation of GFP encoded by this reporter AUA, where N is any nucleotide) as a first step in determining whether regulation by Pum might be direct luciferase (luc) gene, using wild type and mutant hunchback (hb) NRE sequences as controls luc containing EGFR NRE1, Rl NRE, Sos NRE, and Drk NRE1; the putative Raf NRE1, which does not bind appreciably to Pum, does not mediate repression can drive expression of the RNA binding region of Pum (1092\u20131427) UAS-Pum-IR, UAS-EGFR-IR, UAS-Ras-IR and UAS-Raf-IR (in vivo RNA interference) lines were obtained from VDRC RNAi library HAUAS-Nos line was obtained from H. Lin. EGFR pathway alleles and transgenes were used as follows: [Sem]rlUAS-EGFR, [Sem]UAS-rlUAS-Rassca-Gal4 and C253-Gal4 drive expression in proneural clusters disk-Gal4dppser-Gal4, and en-Gal4 drive expression in a stripe at the anterior-posterior boundary, dorso-ventral boundary, and in the posterior compartment of wing pouch, respectively. They are described in detail at FlyBase (http://flybase.bio.indiana.edu). UAS-Pum lines were constructed as follows; the full-length Pum coding sequence was removed from pOT2-pum (LD44635) via digestion with EcoRI, XbaI digestion. The ends were filled using Klenow fragment. The pum sequence was then cloned into the XhoI site (blunted with Klenow) of pINDY5 UAS-Pum. The construct was verified by DNA sequencing. Five independent transgenic flies were produced and used in this study.The Drosophila Pum was generated from the LD44635 by polymerase chain reaction (PCR) and cloned into EcoRI/XhoI sites of pACT2 AD vector (Clontech) to produce Gal4 transcriptional activation domain (GAD)-Puf (GAD-Puf). The NRE and its mutant fragments were generated by oligomer dimerization, and inserted into the SmaI/SpeI sites of pIIIA/MS2-2 to express NRE-MS2 RNA for three-hybrid test hb NRE and pIII/MS2/hb NRE mt produce hb NRE-MS2 RNA and hb NREmt-MS2 RNA, respectively. Yeast strain, YPH-500 , was used to analyze RNA-protein interaction and three-hybrid assays were performed as described \u03b2-galactosidase activity of three or more transformants were carried out as described The C-terminal Puf region (1093\u223c 1427) of the EcoRI/XhoI sites of pcDNA3 (Invitrogen) by PCR. The same NRE fragments used for the yeast three-hybrid assay were inserted into the BamHI/XhoI sites of pcDNA3-LUC LacZ expression from pSV-\u03b2-gal (Promega). The plasmid DNAs used for transfection included the reporter plasmid having NRE wild-type or its mutant, the pSV-\u03b2-gal control plasmid, and pcDNA3-pum.A full-length Pum from LD44635 was cloned into the The 3rd instar larval wing discs were dissected in PBS. The tissues were fixed for 15 minutes with gentle rocking in 4% formaldehyde in PBS. After fixation, the tissues were washed three times in PBT at RT for 15 minutes. The tissues were then blocked for 1 hour by 5% normal Goat serum in PBT. Primary antibodies were incubated overnight at 4\u00b0C. The wing discs were then washed four times with PBT for 10 minutes, and incubated for two hours with secondary antibodies, then washed and mounted in a VectaShield Mounting medium(Vector Laboratories). The following antibodies were used (dilutions noted in parentheses): Rabbit anti-GFP (1:1000) (Molecular Probes); rat anti-Pum ; and monoclonal anti-HA (Roche). Fluorescence-conjugated secondary antibodies were used as follows: Anti-rabbit Alexa 488 conjugated (1:200); anti-rat Rhodamine conjugated (1:200); and anti-mouse Alexa 568 conjugated (1:200) (molecular Probes). The stained images were processed via the LSM 510 confocal microscope (Zeiss).Figure S1Reduction of EGFR signaling causes loss of wing veins. Wing veins are lost by the reduction of EGFR signaling (en-GAL4/UAS-EGFR-IR (A), en-GAL4/+; UAS-Ras-IR/+ (C), en-GAL4/UAS-Raf-IR (E)). Concomitant reduction of Pum does not overrule vein loss by reduced EGFR signaling , en-GAL4/+; UAS-Pum-IR/UAS-Ras-IR (D), en-GAL4/UAS-Raf-IR; pum-IR/+ (F)).(TIF)Click here for additional data file."} +{"text": "Caenorhabditis elegans chromosome V. Examining this region for multiple crossover events in heteroallelic configurations with limited dimorphism, we observed high levels of crossover interference in oocytes with only partial interference in spermatocytes.In certain organisms, numbers of crossover events for any single chromosome are limited (\u201ccrossover interference\u201d) so that double crossover events are obtained at much lower frequencies than would be expected from the simple product of independent single-crossover events. We present a number of observations during which we examined interference over a large region of Caenorhabditis elegans provides an outstanding model for meiotic regulation and a system of choice to study detailed genetic properties of meiosis , dpy-11(e224), rol-9(sc148) Used for production of feminized animals LGII: tra-2(q122gf) Other mutations present (in post-recombination tester strains) LGI: ccIs9753 LGIII: pha-1(e2123ts) LGV: rde-1(ne300).Mapping markers LGV: unc-60(e723), dpy-11(e224), and rol-9(sc148) mutations on LGV were generated dpy-11(e224) rol-9(sc148) V heterozygous triple-mutant hermaphrodites. Progeny were singled onto separate plates. Genotypes of the animals resulting from this cross were determined through subsequent self-fertilization and examination of subsequent generations for appearance of Unc, Dpy, and Rol phenotypes. Selection of cross progeny in the first generation cross is critical for interpretation of the gametic source of recombination.To measure recombination frequency in oogenesis, we performed two parallel experiments with a similar experimental outline. First, a cross was performed between males with a wild-type chromosome V and unc-60(e723) dpy-11(e224) rol-9(sc148)/+++ were crossed with males having integrated GFP markers on chromosome I, allowing specific selection of the cross progeny. Cross-progeny animals were singled onto separate plates and their progeny were used to quantify different crossover events. Note in this case that introduction of the transgene postdates the inferred recombination events.In a first set of experiments, the heterozygous triple-mutant tra-2(q122gf) males resulted in a population of heterozygous XX females carrying the feminizing mutation tra-2(q122gf). Because these hermaphrodites are not able to self-fertilize because of the tra-2(q122gf) mutation, a subsequent cross with wild-type (N2) males resulted in only cross-progeny animals. Cross-progeny animals were singled onto separate plates and their progeny were used to identify parental genotypes and thus to quantify recombination frequencies. Singled animals carrying the tra-2(q122gf) mutation on chromosome II were discarded because of their inability to self-fertilize.The second set of experiments initiated from a cross of homozygous triple-mutants to a unc-60(e723) dpy-11(e224) rol-9(sc148)/+++ heterozygous triple-mutant males to pha-1(e2123ts) hermaphrodites at the nonpermissive temperature. Homozygous pha-1(e2123ts) embryos failed to survive growth at temperatures more than 23\u00b0, ensuring survival of only cross progeny. Cross-progeny animals were singled onto separate plates, with occurrence of phenotypes in subsequent generations used to determine genotypes and to detect crossover events.To quantify double-crossover frequency in male spermatogenesis, we crossed unc-60(e723) dpy-11(e224) rol-9(sc148)/+++ heterozygous triple-mutant animals to self-fertilize and then singled to separate plates their phenotypically wild-type progeny. Although not all double-crossover events can be detected , one of the genotypes (dpy-11(e224)/+) can only be produced through a double-crossover\u2013type event.To quantify double crossovers in self-fertilization, we allowed The confidence intervals (C.I.s) were calculated using the Clopper and Pearson exact method for binomial proportion .C. elegans is a hermaphroditic species, we assayed frequencies in hermaphrodite oogenesis, male spermatogenesis, and in an aggregate measure that combines hermaphrodite spermatogenesis and oogenesis -to-dpy-11(e224) and dpy-11(e224)-to-rol-9(sc148) intervals (ctively) . In the This analysis provides evidence for gender-specific interference effects. Despite comparable levels of total recombination , we found an extreme difference in proportion of double-crossover events with double-crossover products not detected in oocyte meiosis while accounting for 3.4% of total crossover products from male spermatocyte meiosis. The difference between oocytes and male spermatocytes is statistically significant, with p-value of 0.002 .In these experiments, the interrogated oocytes and sperm differ in both cell biology and chromosome content . An additional self-fertilization experiment was performed to assess double crossovers in XX sperm. As diagrammed in The use of distant genetic markers on LGV allowed us to monitor double crossovers and interference intensity in oogenesis and spermatogenesis. We found interference for LGV during oogenesis to be very high (with the observation of zero detected double recombinants among 495 sampled meioses compared to an expectation of 24 for no interference), suggesting complete or at least near-complete interference. Crossover interference during male spermatogenesis was not complete, with 12 double-recombinant animals detected among 687 sampled . The cytological mechanism underlying the dichotomy of high interference in oogenesis and lower interference in spermatogenesis remains a question for future work."} +{"text": "Cardiac Glycosides (CGs) are commonly used to treat congestive heart failure. CGs inhibit the Sodium Potassium ATPase (Na+/K+ ATPase) pump. Interestingly, CGs have been suggested to inhibit proliferation and migration of breast cancer cells. A pool of non-pumping Na+/K+ ATPase reportedly localizes in specific membrane organelles, caveolae, by interacting with the structural protein caveolin-1. It has been postulated that Na+/K+ ATPase forms a complex with caveolin-1 and the tyrosine kinase Src, and that binding of CGs to Na+/K+ ATPase activates Src-dependent signalling cascades. In this project we explored whether CGs reduce proliferation in breast cancer cells and whether these effects might involve Src and ERK .MDA-MB 231 cells [highly-invasive breast cancer cells] were transfected with siRNA to knock down caveolin-1 expression. MDA-MB-231 cells in which caveolin-1 was knocked down and MCF 10A cells (non-invasive breast cancer cells) were treated with CGs and subjected to MTT proliferation assays. MCF 7 cells (weakly-invasive breast cancer cells) were treated with the Src inhibitor PP2 in the presence or absence of CGs and analysed by western blotting for phosphorylated Src and phosphorylated ERK.High dose CGs reduced proliferation in MCF 10A cells over 72 hrs. Caveolin-1 was successfully knocked-down in MDA-MB 231 cells, but this did not appear to abrogate the anti-proliferative effects of CGs. Early results suggest that phospho-Src and phospho-ERK expression were increased in MCF 7 cells treated with Digoxin. Interestingly, this was not abrogated by pre-treatment with the Src inhibitor PP2.This project has demonstrated that CGs exert anti-proliferative effects on a range of breast cancer cell lines. Early results suggest that the effects of CGs may not be directly linked to Src and ERK signalling. Ongoing work is determining whether caveolin-1 knockdown alters the anti-proliferative response to CG treatment. We suggest that further exploration of the mechanisms whereby CGs inhibit proliferation may reveal potential uses for CGs as anti-cancer drugs in the future."} +{"text": "Perivascular adipose tissue (PVAT) has been recognized as an important factor in vascular biology due to its ability to produce a variety of vasoactive substances. In addition, it is regarded as an important source of proinflammatory mediators and reactive oxygen species (ROS). Experiments from our laboratory demonstrated that mice lacking adipose triglyceride lipase (ATGL), a crucial enzyme of triglyceride catabolism, suffer from severe micro- and macrovascular endothelial dysfunction. Since blood vessels of ATGL knockout mice (ATGL(\u2212/\u2212) mice) are surrounded by large amounts of PVAT, we investigated its potential contribution to the observed endothelial dysfunction.phox complex was significantly upregulated at protein level. Heme oxygenase-1, which has been described protective against oxidative and inflammatory stress, was increased about 5-fold in ATGL deficiency. To distinguish between direct PVAT-mediated effects and those originating from the cardiac dysfunctional phenotype of the animals, we additionally analyzed tissue isolated from ATGL(\u2212/\u2212) mice with cardiomyocyte-specific overexpression of ATGL (rescued cardiac phenotype). Interestingly, the effect of ATGL knockout on TNF-\u03b1 and leptin expression was reversible. By contrast, increased adipose NOX2/p67phox, MCP-1 and IL-6 expression persisted even upon restoration of cardiac function.PVAT encompassing thoracic aortas of wild-type (WT) and ATGL(\u2212/\u2212) mice was isolated, characterized, and analyzed for protein and mRNA expression of different adipokines, inflammation markers, and sources of oxidative stress using real-time PCR and Western blot analysis, respectively. Knockout of ATGL caused a 7-fold increase in PVAT wet weight. While mRNA expression of adiponectin was reduced to about 50%, leptin mRNA was increased about 4-fold in ATGL deficiency. Adipose mRNA levels of the inflammation markers tumor necrosis factor alpha (TNF-\u03b1), monocyte chemoattractant protein 1 (MCP-1), and interleukin-6 (IL-6) were about 5-fold higher in ATGL-deficient PVAT. In addition, the NOX2/p67Our data indicate that PVAT-derived inflammatory and NADPH oxidase-mediated oxidative stress might contribute to endothelial dysfunction in ATGL deficiency. The functional consequences of these findings are currently being investigated in our laboratory."} +{"text": "Pam3Cys (Toll-like receptor-1/2 agonist) decorated chitosan DNA nanoparticles (NP) were explored by using a three-dimensional (3D) cell culture model of the human epithelial barrier. Pam3Cys functionalised and non-functionalised chitosan DNA NP were sprayed by a microsprayer onto the surface of 3D cell cultures and uptake of NP by epithelial and immune cells (blood monocyte-derived dendritic cells (MDDC) and macrophages (MDM)) was visualised by confocal laser scanning microscopy. In addition, immune activation by TLR pathway was monitored by analysis of interleukin-8 and tumor necrosis factor-\u03b1 secretions (ELISA).Plasmid DNA vaccination is a promising approach, but studies in non-human primates and humans failed to achieve protective immunity. To optimise this technology further with focus on pulmonary administration, we developed and evaluated an adjuvant-equipped DNA carrier system based on the biopolymer chitosan. In more detail, the uptake and accompanying immune response of adjuvant Pam3Cys adjuvant functionalised and non-functionalised DNA NP, ELISA of interleukin-8 and tumor necrosis factor-\u03b1 demonstrated clearly that Pam3Cys functionalisation elicited an overall higher immune response with the ranking of Pam3Cys chitosan DNA NP\u2009>\u2009chitosan DNA NP\u2009=\u2009DNA unloaded chitosan NP\u2009>\u2009control (culture medium).At first, a high uptake rate into antigen-presenting cells was obtained. Although no significant difference in uptake patterns was observed for Pam3Cys adjuvant functionalisation of chitosan DNA NP enhances significantly an environment favoring recruitment of immune cells together with a Th1 associated (cellular) immune response due to elevated IL-8 and TNF-\u03b1 levels. The latter renders this DNA delivery approach attractive for potential DNA vaccination against intracellular pathogens in the lung .Chitosan-based DNA delivery enables uptake into abluminal MDDC, which are the most immune competent cells in the human lung for the induction of antigen-specific immunity. In addition, Escherichia coli), purified and injected into the host-[R-cysteinyl]-\u03c9-amido-graft-6-0-carboxymethyl-N,N,N-trimethylchitosan; CLSM: Confocal laser scanning microscopy; DC: Dendritic cells; EC: Epithelial cells; pGFP: Plasmid DNA expressing green fluorescence protein; IL-8: Interleukin-8; MDDC: Blood monocyte-derived dendritic cells; MDM: Blood monocyte-derived macrophages; NH2-PEG-Pam3Cys: \u03c9-amido--[R]\u2013cysteinyl]-\u03b1-amino poly(ethylene glycol); NP: Nanoparticles; PDI: Polydispersity index; pDNA: Plasmid DNA; PRR: Pattern recognition receptors; TLR: Toll-like receptor; TNF-\u03b1: Tumor necrosis factor-\u03b1; TPP: Pentasodium tripolyphosphate.APC: Antigen-presenting cells; BSA: Bovine serum albumin; CTC: The authors declare that they have no competing interests.SH performed the experiments, analysed results and drafted the manuscript. DR, BR were involved in the analysis of results. BR, PG and GB oversaw the experimental work and participated in drafting of the manuscript. All authors have read and approved the manuscript."} +{"text": "A key feature of innate immunity is the ability to recognize and respond to potential pathogens in a highly sensitive and specific manner. In plants, the first layer of defense is induced after recognition by pattern recognition receptors of microbe-associated molecular patterns. This recognition elicits a defense program known as pattern-triggered immunity. Pathogen entry into host tissue is a critical early step in causing infection. For foliar bacterial pathogens, natural surface openings such as stomata, are important entry sites. Stomata in contact with bacteria rapidly close and can thus restrict bacterial entry into leaves. The molecular mechanisms regulating stomatal closure upon pathogen perception are not yet well-understood. Plant lectin receptor kinases are thought to play crucial roles during development and in the adaptive response to various stresses. Although the function of most plant lectin receptor kinases is still not clear, a role for this kinase family in plant innate immunity is emerging. Here, we summarize recent progresses in the identification of lectin receptor kinases involved in plant innate immunity. We also discuss the role of lectin receptor kinases in stomatal innate immunity signaling. Plants face threats from various pathogenic microbes and resist attacking pathogens through both constitutive and inducible defenses . The patArabidopsis stomata close when in contact with bacteria, thus functioning as innate immunity gates to actively prevent bacteria entry into plants (Pseudomonas syringae pv. tomato strain DC3000 (Pst DC3000) bacteria, Arabidopsis stomata close as a result of stomatal innate immunity activation. Virulent bacteria such as Pst DC3000 can re-open Arabidopsis Col-0 stomata 3\u20134 h after infection through the action of the chemical effector coronatine (COR) suggesting that plant pathogens have evolved virulence factors to suppress innate immunity functions of stomata (COI1 gene (Pst DC3000 and Pectobacterium carotovorum ssp. carotovorum (Pcc) infection that are characterized by an extracellular legume lectin-like domain, a transmembrane domain and an intracellular kinase domain , LecRK-I.9 mediates cell wall\u2013plasma membrane (CW\u2013PM) continuum (in planta induced-O), a secreted effector protein of the oomycete pathogen Phytophthora infestans, disrupts CW\u2013PM adhesions upon interaction with a variety of cellular proteins, including LecRKs , WAK and CrRLK and MPK6 (Mitogen-activated protein kinase 6) activities, PTI-responsive gene expression, and callose deposition is important during herbivory by ttenuata . Importafolivory . The inscontrols . Inhibiterbivory . More rebidopsis .Arabidopsis stomatal innate immunity (fls2 (lecrk-VI.2-1 mutants demonstrate a high sensitivity to Pst DC3000 COR- deficient bacterial mutants that cannot re-open stomata upon infection. Since Arabidopsis is resistant to these bacterial mutants (lecrk-VI.2-1 (LecRK-VI.2 demonstrate constitutive stomatal closure, further suggesting a positive role for LecRK-VI.2 in stomatal innate immunity (lecrk-VI.2-1 demonstrates wild-type stomatal closure levels in response to ABA indicating that LecRK-VI.2 acts upstream or independently of ABA signaling during stomatal closure (In addition to positively regulating apoplastic PTI, LecRK-VI.2 is also critical for immunity . Notablyty (fls2 , lecrk-V mutants , LecRK-V mutants . Consistk-VI.2-1 . This suimmunity . The mut closure .Arabidopsis stomatal innate immunity is LecRK-V.5. However, in contrary to LecRK-VI.2 that positively regulates stomatal innate immunity, LecRK-V.5 negatively regulates stomatal closure upon bacterial infection. Plants lacking a functional LecRK-V.5 are resistant to Pst DC3000 and Pcc surface inoculation, but are normally sensitive to infiltration inoculation (Arabidopsis defenses by restricting bacterial entry into leaves and point to a role of LecRK-V.5 in stomatal innate immunity (lecrk-V.5 indeed revealed that this mutant possesses constitutively closed stomata (LecRK-V.5 are less resistant to Pst DC3000 COR- and this is correlated with a re-opening of stomata in LecRK-V.5 over-expression lines even in the absence of COR. These observations suggest the existence of a stomatal re-opening mechanism positively modulated by LecRK-V.5 (Arabidopsis resistance to bacteria through fine-tuning of stomatal innate immunity (scord5 mutant that shows a defective stomatal innate immunity but exhibits wild-type apoplastic immunity (lecrk-V.5 mutants. COR treatments re-open closed stomata in lecrk-V.5 mutants (lecrk-V.5 mutants accumulate high levels of ROS in guard cells and chemical inhibition of ROS accumulation in lecrk-V.5 guard cells re-opens closed stomata (Arabidopsis over-expressing LecRK-V.5 (lecrk-V.5 mutants and deficient stomatal closure in LecRK-V.5 over-expression lines, respectively. In addition, lines over-expressing LecRK-V.5 demonstrate a compromised ABA-mediated stomatal closure (2 availability following prolonged stomatal closure.Another LecRK involved in culation . These oimmunity . Analyse stomata . TransgeecRK-V.5 . Interesimmunity . Localizimmunity . Similarimmunity , apoplas mutants , suggest stomata . By contecRK-V.5 . Since R closure , thus Le closure . NegativAlthough new knowledge about lectin receptor kinases function and signaling has emerged recently, many questions still remain unanswered. For example, what are the potential ligands and downstream partners that modulate lectin receptor kinase-dependent innate immunity responses are critical points that need to be solved. Importantly, the unraveling of the mechanisms modulating ligands perception by lectin receptor kinases will provide further insights into how LecRKs affect the plant response to pathogens. This may clarify whether these receptor kinases function as PRRs. Knowledge derived from such studies could lead to novel methods for managing plant disease resistance.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Mycobacterium tuberculosis (Mtb) persists within lung granulomas, despite being subjected to diverse stress conditions, including hypoxia. We hypothesized that the response of host phagocytes to Mtb experiencing hypoxia is radically altered and designed in vitro experiment to study this phenomenon. Hypoxia-stressed (Mtb-H) and aerobically grown Mtb (Mtb-A) were used to infect Rhesus Macaque Bone Marrow Derived Macrophages (Rh-BMDMs) and the comparative host response to Mtb infection studied. Mechanistic insights were gained by employing RNAi. Mtb-H accumulated significantly lower bacterial burden during growth in Rh-BMDMs, concomitantly generating a drastically different host transcriptional profile . A key component of this signature was significantly higher TNF and apopotosis in Mtb-H- compared to Mtb-A-infected Rh-BMDMs. Silencing of TNF by RNAi reversed the significant control of Mtb replication. These results indicate a potential mechanism for the rapid clearance of hypoxia-conditioned bacilli by phagocytes. In conclusion, hypoxia-conditioned Mtb undergo significantly different interactions with host macrophages compared to Mtb grown in normoxia. These interactions result in the induction of the TNF signaling pathway, activation of apoptosis, and DNA-damage stress response. Our results show that Mtb-H bacilli are particularly susceptible to killing governed by TNF. Mtb primarily infects pulmonary alveolar macrophages. This causes a robust response that culminates in the formation of the lung granuloma Mtb is arrested due to the direct action of the activated immune cells, as well as due to nutritional restrictions in a granulomatous environment Mtb must contend with inside granulomatous lesions Tuberculosis (TB) is responsible for over \u223c8.7 million new cases and 1.4 million deaths every year Mtb has developed a specialized transcriptional program regulated by the dosR (devR)-encoded protein that enables it to persist in hypoxic conditions Mtb transcriptional and metabolic profile is assumed to undergo a rapid and significant reprogramming in response to hypoxia and other stress conditions prevalent within human granulomas. Clearly, this would cause major changes in the antigenic profile presented by the pathogen and may influence and modulate host-pathogen interaction. Macrophage-Mtb interactions induce the transcriptional machinery resulting in the secretion of several proinflammatory cytokines, chemokine, expression of costimulatory molecules and effector molecules, which provide host defense to MtbMtb. Thus, we tested our hypothesis that tubercle bacilli grown in-vitro in anaerobic or hypoxic conditions exhibit markedly different interactions with host macrophages relative to actively growing Mtb. Rhesus macaque bone marrow derived macrophages (Rh-BMDMs) were infected with Mtb strain H37Rv that was either cultured by aerobic shaking (Mtb-A) or subjected to prolonged hypoxia (Mtb-H). Data suggest that infection with Mtb-H causes extensive transcriptional changes relative to infection with Mtb-A to modulate the host immune response. Our results indicate that host response to Mtb-H is radically different than Mtb-A and Mtb-H is rapidly killed within host macrophages due to coordination between TNF and apopotosis. Unsurprisingly, RNAi mediated silencing of TNF abrogated the clearance of Mtb-H by Rh-BMDMs.Our current study characterized the impact of hypoxia on the response(s) mounted by host macrophages against Mtb H37Rv cultured in Middlebrooks\u2019s 7H9 media supplemented with ADC to logarithmic phase (A595 \u223c0.3) with shaking (aerobic), henceforth referred to as Mtb-A. To subject Mtb to hypoxia, Mtb-A (from above) were dispensed into screw-capped air tight 15 ml tubes (head-space 0.6), tightly sealed, wrapped with parafilm and left standing at 37\u00b0C for 30 days (Mtb-H) with control tubes containing methylene blue (1.5 \u00b5g/ml) as an indicator of oxygen depletion in parallel Mtb-A and Mtb-H were enumerated by CFU assay on 7H10 plates prior to infection. To disrupt bacterial clumps, all Mtb cultures were repeated passage 10 times (using 27/28 gauge needles) prior to infection. As a control, Mtb-H was heat-killed (90\u00b0C/45 min) and used to infect Rh-BMDMs.Mtb and CFU analysis have been described earlier 2 incubator. Rh-BMDMs were infected at multiplicity of infection (MOI) of 10\u22361 (10 bacteria per 1 cell) in all experiments. After 3-hour cells were washed with PBS and further incubated for 1-hour in IMDM complete containing amikacin (200 \u00b5g/ml) (zero hr time-point). The infected cells were lysed (0.1% saponin) for CFU assay or added with 1 mL Trizol for RNA isolation. When macrophages were adherent, both floater as well as adherent macrophages were lysed and plated at 10-fold dilutions on 7H10 agar for CFU counts. The infected Rh-BMDMs in remaining wells were further incubated for 4, 24 and 72 hr.Procedures for the generation and maintenance of Rh-BMDMs as well as their infection with Mtb antibody for detection of Mtb is well established and has been used earlier For confocal microscopy, Rh-BMDMs were grown and infected in chamber slides , as described earlier Mtb-A or Mtb-H suspended in IMDM (Cy5). Rhesus macaque whole genome 4\u00d744k arrays (Agilent Technologies) were used for profiling. Data was analyzed as described earlier + Array Analyzer locally weighted scatterplot smoothing. Database for Annotation, Visualization and Integrated Discovery (DAVID) was used to analyze gene-ontologies differentially included in the transcriptomics experiments as described earlier http://omics.pnl.gov/software/VennDiagramPlotter.php. Microarray data including raw data and images (accession number GSE47163) of the present study can be accessed from Gene Expression Omnibus (GEO).DNA Microarray studies were performed as described earlier Mtb dosR regulon genes that were induced during the 30 days hypoxia experiment in Mtb-H cultures; 16S rRNA gene was used as an invariant normalization control. Detailed information about oligonucleotides used in RT-qPCR assays is available in Quantitative RT-PCR (RT-qPCR) was used to validate microarray results for a subset of genes. RT-qPCR was performed with cDNA corresponding to 1000 ng DNA-free RNA, using the SYBR green Supermix (Applied Biosystems). A RT-negative (devoid of reverse transcriptase) reaction was used to account for residual DNA if any and relative expression levels were normalized using 18S rRNA as an invariant transcript and data was analyzed using the \u0394\u0394Ct method . AdditioMtb-A or Mtb-H or IMDM complete medium alone for 24 hours were used for quantification of secreted TNF and IL8 using nonhuman primate (NHP) cytokine-23-milliplex kit (Millipore) according to the manufacturer\u2019s directions.Supernatants collected from Rh-BMDMs infected with either in situ cell death detection kit, fluorescein as previously described 2) using a TCS-SP2 confocal microscope (Leica Microsystems).Tunel assay was performed using 5 Rh-BMDMs pre-infected with Mtb-H. Finally, all transfections were undertaken in a final volume of 1000 \u00b5L, with a final siRNA concentration of 25 nM. The Rh-BMDMs infected with Mtb-H were incubated in the absence or presence of small interfering RNA (siRNA) for TNF gene or positive control or negative controls , for 24 hours. Percentage of Rh-BMDMs undergoing apoptosis after 24 hours of exposure to Mtb-H alone (no siRNA) or Mtb-H with siRNA quantified by in-situ Tunel assay.Transfection was performed as per manufacturer\u2019s instructions (Thermo-Scientific). Briefly, a dose curve 5\u201325 nM TNF siRNA was combined with appropriate volume of transfection reagent for 20 minutes at RT and added to 12-well plate with 1\u00d710Statistical analysis was performed with One-way ANOVA or Student\u2019s t-test with Graphpad prism 6.0b and SAS9.2.dosR and DosR-dependent Mtb genes was measured by RT-qPCR on cDNA made from RNA isolated from Mtb-A and Mtb-H cultures. Induction of dosR regulon genes unambiguously shows that true hypoxia was established in Mtb-H cultures and were used to infect Rh-BMDMs (107 viable Mtb to 106 Rh-BMDMs). The extent of Mtb replication was measured by viable cfu count at the end of 4 hr infection (time 0 h) and subsequently 4, 24 and 72 hrs later Mtb-H was susceptible to killing by Rh-BMDMs and their numbers progressively declined with time. Thus, at the 4 hr time-point, a half-log reduction in bacillary load was observed during infection with Mtb-H. By 24 hrs, this difference increased to one-log and by 72 hrs (3 days), the difference in the persistence levels of Mtb-A relative to Mtb-H was greater than one and a half-log time-points was viable at beginning of infection and able to successfully infect Rh-BMDMs. Again, this result clearly shows that significantly reduced persistence of Mtb-H in Rh-BMDMs during 3 days of infection is not due to any loss in viability at the beginning of the infection.To ensure that the significantly reduced persistence of hypoxia-conditioned Mtb-H and Mtb-A infected Rh-BMDMs at 24 hr time-point were used to perform rhesus macaque whole-genome transcriptomics as described earlier In this study, we utilized global host transcriptomics, wherein significant and crucial observations were confirmed at the level of the protein. Host RNA isolated from uninfected (cells cultured in IMDM complete), Mtb-A, comparable to the number of host macrophage genes those were significantly perturbed upon infection with Mtb in an earlier study Mtb-H. Thus, Mtb subjected to hypoxia elicited an extremely different response from Rh-BMDMs than Mtb grown aerobically, both in breadth and in magnitude.The expression of 226 genes was perturbed in Rh-BMDMs upon infection with Mtb-A and Mtb-H infected Rh-BMDMs, we employed Gene Ontology and KEGG pathways biological processes using DAVID and cytokine induced genes allowed us to identify an IFN gamma-inducible signature or induced to significantly higher magnitude during Mtb-A infection . The expression of several pro-apoptotic genes was induced to high-levels only in Mtb-H-infected Rh-BMDMs , were surprisingly induced to higher levels only in Rh-BMDMs infected with Mtb-H . The up croscopy . Higher Rh-BMDMs . These rMtb-H but not Mtb-A were induced in Rh-BMDMs infected with ot Mtb-A . These rMtb-H relative to Mtb-A, Confocal microscopy based Tunel assay was performed to examine Rh-BMDM apoptosis , and treated with either TNF-specific or negative non-target control siRNA as described earlier Mtb-H, we employed RT-qPCR. PPIB and non-target siRNA were used as the silencing reference standard. The difference between PPIB-transfected Rh-BMDMs and the corresponding negative control was used to calculate the percentage of PPIB mRNA that remained in Rh-BMDMs. The PPIB siRNA knocked down the PPIB mRNA by >80% in Rh-BMDMS infected with Mtb-H (data not shown). Similarly, TNF siRNA induced the reduction of TNF mRNA and protein levels, by 52% and 60% respectively in Rh-BMDMs infected with Mtb-H . Our results are consistent with a previous study Mtb subjected to prolonged hypoxia may interact differently with host phagocytes relative to aerobic-Mtb. However, the mechanism(s) involved in regulation of these variations in response to the pathogen are not totally understood.TNF is known to activate JNK pathway and subsequently the apoptosis loop Mtb are rapidly killed by activated host macrophages. This is concomitant with a highly altered phagocyte response elicited by Mtb-H, characterized primarily by high levels of TNF and apoptosis, a critical determinant for containing Mtb infection. Our results suggest a correlation of possible mechanism of Mtb containment invivo and provide new insights on host-elicited variations in gene ontology to two different physiological forms of Mtb. Our current study did not examine the effects of hypoxia on either host macrophage function or their ability to kill Mtb. These experiments constitute future studies underway in our laboratory. We also hope to unravel the specific pathways utilized by TNF to preferentially kill Mtb-H.In conclusion our data indicates that hypoxia-conditioned but not aerobically-grown Supplement S1Primers used in this study. The nucleotide sequence of forward and reverse primers used in this study is shown in 5\u2032-3\u2032 orientation. \u2018Mtb\u2019 means, primers derived from Mycobacterium tuberculosis genome and \u2018Rh\u2019 means, primers derived from Rhesus Macaque genome.(DOCX)Click here for additional data file.Supplement S2Gene/Gene ontology in Rh-BMDMs infected with Mtb. Various gene ontology identified in Rh-BMDMs infected with Mtb-A (excel sheet 1); Mtb-H (excel sheet 2); comparison list of genes (excel sheet 3), common genes, (excel sheet 4) identified in Rh-BMDMs, Mtb-H vs. Mtb-A.(XLS)Click here for additional data file."} +{"text": "Enhancer elements determine the level of target gene transcription in a tissue-specific manner, providing for individual patterns of gene expression in different cells. Knowledge of the mechanisms controlling enhancer action is crucial for understanding global regulation of transcription. In particular, enhancers are often localized within transcribed regions of the genome. A number of experiments suggest that transcription can have both positive and negative effects on regulatory elements. In this study, we performed direct tests for the effect of transcription on enhancer activity.Drosophila enhancers controlling the expression of the white and yellow genes. The results show that transcription from different promoters affects the activity of enhancers, counteracting their ability to activate the target genes. As expected, the presence of a transcriptional terminator between the inhibiting promoter and the affected enhancer strongly reduces the suppression. Moreover, transcription leads to dislodging of the Zeste protein that is responsible for the enhancer-dependent regulation of the white gene, suggesting a 'transcription interference\u2019 mechanism for this regulation.Using a transgenic reporter system, we investigated the relationship between the presence of pass-through transcription and the activity of Our findings suggest a role for pass-through transcription in negative regulation of enhancer activity. The development of multicellular organisms involves differentiation of various cell types, which is achieved by the establishment of requisite spatial and temporal patterns of gene expression. Regulation of transcription is a highly complex process involving different regulatory DNA elements, enhancers in particular. Enhancers are positive DNA sequences containing multiple binding sites for a variety of transcription factors. These regulatory elements can activate genes over long distances, up to several tens of thousands of base pairs, and act independently of the distance and orientation with respect to the promoters of target genes,2.Drosophila117/TM3,Sb line (Bloomington Stock Center #5138), in which the marker mini-white gene was deleted as described. The HindIII-EcoRI fragment containing the minimal hsp70 promoter with five GAL4 binding sites upstream of it was excised from the pUAST vector (26) and cloned into the pBluescript SK + vector between lox sites to produce the lox(UAS) plasmid.The constructs were made on the basis of the CaSpeR vector. The 5-k plasmid cleaved eviously. The DNA0 vector [\u2206prw-pCXbaI-XbaI fragment of the lox(UAS) plasmid was inserted into \u2206prw-C2\u2206700-y(-893) cleaved with XbaI.The white gene regulatory sequences from position\u20131180 to -1849 bp relative to the transcription start site (23) was cloned into pBluescript SK + between frt sites to produce the frt(Ee) plasmid. The HincII-BamHI fragment (containing the eye enhancer) of the frt(Ee) plasmid was inserted in direct orientation into the yr-pGEM7 plasmid cleaved with BglII [yr-frt(Ee)]. The XbaI-BamHI fragment of the yr-frt(Ee) plasmid was cloned into the yc-C2 plasmid cleaved with XbaI and BamHI [yr-frt(Ee)-yc-C2]. The XbaI-XbaI fragment of the lox(UAS) plasmid was inserted into the yr-frt(Ee)-yc-C2 plasmid cleaved with XbaI.The eye enhancer (Ee) corresponding to the BglII (yr-EeR). The XbaI-BamHI fragment from the yr-EeR plasmid was cloned into the yc-C2 plasmid cleaved with XbaI and BamHI (yr-EeR-yc-C2). The XbaI-XbaI fragment of the lox(UAS) plasmid was inserted into the yr-EeR-yc-C2 plasmid cleaved with XbaI.The eye enhancer without flanking frt sites was cut out of the Ee-pBluescript SK + plasmid and cloned in reverse orientation into the yr plasmid cleaved with BglII (yr-Ee). The XbaI-BamHI fragment from the yr-Ee plasmid containing enhancers was cloned into the yc-C2 plasmid cleaved with XbaI and BamHI (yr-Ee-yc-C2). The XbaI-XbaI fragment of the lox(UAS) plasmid was inserted into the yr-Ee-yc-C2 plasmid cleaved with XbaI.The eye enhancer without flanking frt sites was cut out of the Ee- pBluescript SK + plasmid and cloned in direct orientation into the yr plasmid cleaved with EcoRV [SV40(s)-pSK]. The XhoI-BamHI fragment of the SV40(s)-pSK was cloned into yc-C2 cleaved with BglII [yc-SV40(s)-C2]. The SpeI-KpnI fragment of the (UAS)Ey(e)YW construct was inserted into yc-SV40(s)-C2 cleaved with BamHI.The 222-bp SV40 terminator from the pGL3basic vector (Promega) was inserted into the pBluescript SK + plasmid cleaved with yellow translation start containing AflII-AflII fragment was cut out of the C\u2206-y plasmid and inserted into the pBluescript SK + plasmid cleaved with EcoRV [y(ATG)-pSK]. The XbaI-XbaI fragment of the lox(UAS) plasmid was inserted into the y(ATG)-pSK plasmid cleaved with SmaI. The BamHI-BamHI fragment corresponding to the lox(UAS)-y(ATG) was cloned into the yc-C2 cleaved with BamHI.The XbaI-AorI fragment containing the yellow gene enhancers was cut out of the yr plasmid and inserted between frt sites in pGEM-7zf [frt(yr)]. The lox(UAS) sequence was inserted into the frt(yr) plasmid cleaved with XbaI [lox(UAS)-frt(yr)]. The KpnI\u2013NotI fragment of the lox(UAS)-frt(yr) plasmid was cloned into \u2206prw-C2\u2206700-y(-893) cleaved with XbaI.The SalI-BamHI fragment of the frt(yr) plasmid was inserted into the lox(UAS) plasmid cleaved with BamHI [lox(U)R-frt(yr)]. The sequence corresponding to the lox(UAS)R-frt(yr) was cloned into \u2206prw-C2\u2206700-y(-893) cleaved with XbaI.The Elongation factor 1\u03b148D gene was PCR-amplified with primers 5\u2019-attgttaactgatttcgcaagc-3\u2019 and 5\u2019-tggatgattacactatggctgtt-3\u2019. The PCR product was inserted into pBluescript SK + between lox sites [lox(prEf1)]. The resulting lox(prEf1) plasmid was sequenced to confirm that no unwanted changes had been introduced into the promoter sequence. The XbaI-XbaI fragment of the lox(prEF1) plasmid was inserted into the yr-frt(Ee)-yc-C2 plasmid cleaved with XbaI.The promoter of SalI\u2013SacII fragment of the frt(yr) plasmid was inserted into \u2206prw-pCaSpeR\u2206700-y(-893) cleaved with XbaI [frt(yr)-\u2206prw-C2\u2206700-y(-893)]. The XbaI-XbaI fragment of the lox(prEf1) plasmid was inserted into frt(yr)-\u2206prw-C2\u2206700-y(-893) cleaved with XbaI.The HindIII-EcoRI fragment of the pUAST vector was cloned into pBluescript SK + [UAS-pSK]. The 717-bp fragment consisting of the GFP coding region (used as a spacer) was cloned into the UAS-pSK plasmid cleaved with HincII [UAS-gfp]. The XbaI-BamHI fragment of the pUAST vector containing the 702-bp SV40 terminator was inserted into pBluescript SK + between lox sites [lox(SV40b)-pSK]. The XbaI\u2013XbaI fragment of lox(SV40b)-pSK was cloned into the UAS-gfp plasmid cleaved with XhoI [UAS-gfp-lox(SV40b)]. The XbaI\u2013KpnI fragment containing the yellow and white enhancers was cut out of the yr-frt(Ee) plasmid and cloned into UAS-gfp-lox(SV40b) cleaved by BamHI [UAS-gfp-lox(SV40b)-yr-frt(Ee)]. The SpeI\u2013SpeI fragment of UAS-gfp-lox(SV40b)-yr-frt(Ee) was inserted into yc-SV40(s)-C2 cleaved with BamHI.The XbaI\u2013BamHI fragment of the pUAST vector containing the 702-bp SV40 terminator was inserted into the lox(UAS) plasmid cleaved with ApaI [lox(UAS)-SV40b]. The XbaI-XbaI fragment of the lox(UAS)-SV40b plasmid was inserted into \u2206prw-C2\u2206700-y(-893) cleaved with XbaI.The ras64B were used to standardize the overall amount of cDNA used in PCR assays. Primers used for Q-PCR are given in Additional fileRNA was isolated from 20 mid-late pupae with TRI reagent (Ambion) according to the manufacturer\u2019s instructions. Purified RNA pools were digested by RNase-free DNase I (BioLabs) and re-purified using the RNeasy Mini kit (Quagen). For reverse transcription, 3 \u03bcg of the generated RNA was incubated with ArrayScript Reverse Transcriptase (Ambion) in the presence of dNTPs, Oligo(dT) (Fermentas) and RNase inhibitor (Ambion) in the supplied reaction buffer at 42\u00b0C for 1.5 h, according to the manufacturer\u2019s instructions. The reverse transcriptase was inactivated by heating at 95\u00b0C for 5 min. To control DNA digestion by DNase I, additional negative control experiments were performed without reverse transcriptase in the reaction mixture. The generated cDNA pools were used as templates in real-time qPCR using a C1000\u2122 Thermal Cycler with the CFX96 real-time PCR detection module (Bio-Rad). Each PCR was performed in triplicate; cDNA pools were obtained in technical duplicate. Relative levels of mRNA expression were calculated in the linear amplification range by calibration to a DNA fragment standard curve (for genomic DNA) to account for differences in primer efficiency. The results of RT-PCR detection of 2, 0.5% Triton X-100, 0.5 mM DTT) supplemented with the EDTA-free protease inhibitor cocktail and formaldehyde as a crosslinking agent . The reaction was stopped by adding glycine . The homogenate was cleared by passing through 100-\u03bcm nylon cell strainer and pelleted by centrifugation at 4,000 g, 4\u00b0C for 5 min. After washing in three 3-ml portions of buffer A1 at 4\u00b0C (5 min each) and 3 ml of lysis buffer without SDS, the pellet was treated with 0.5 ml of complete lysis buffer and sonicated to break chromatin into fragments with an average length of 700 bp. The material was pelleted by centrifugation at 18,000 g for 5 min, and the supernatant fluid was transferred to a new tube. The pellet was treated with the second 0.5-ml portion of lysis buffer, and the preparation was centrifuged at 18,000 g for 5 min. The two portions of the supernatant fluid were pooled, cleared by centrifuging twice at 18,000 g for 10 min, and the resultant chromatin extract (1 ml) was used in four ChIP experiments after preincubation with A-Sepharose or G-Sepharose (see below). One aliquot (1/10 volume) of chromatin extract after preincubation with Sepharose was kept as a control sample (Input).For each experiment, 200 heads from 2-to 5-day-old flies were collected. The material was homogenized in 5 ml of buffer A1 or G-Sepharose (Zeste) beads (Thermo Scientific). The enrichment of specific DNA fragments was analyzed by real-time qPCR, using a C1000\u2122 Thermal Cycler with CFX96 real-time PCR detection module (Bio-Rad).Primers used in ChIP/real-time PCR analyses are listed in Additional fileEscherichia coli, affinity purified on Ni Sepharose 6 Fast Flow according to the manufacturer\u2019s protocol and injected into rats/rabbits following the standard immunization procedure. Antibodies were affinity-purified on the same epitope as was used for immunization and tested by Western blotting from wild-type and null material or by IP to confirm their specificity were raised in rats. Antibodies against Sfmbt (1-348 aa of Sfmbt protein isoform B) and PH (87-521 aa of Ph-p protein isoform A) were raised in rabbits. In all cases, epitopes for antibody production were expressed as 6 \u00d7 His-tagged fusion proteins in ChIP: Chromatin immunoprecipitation; DTT: Dithiothreitol; PcG: Polycomb group; PCR: Polymerase chain reaction; qPCR: Quantitative polymerase chain reaction; EDTA: Ethylenediaminetetraacetic acid; EGTA: Ethylene glycol-bis(2-aminoethylether)-N,N,N\u2019,N\u2019-tetraacetic acid; SDS: Sodium dodecyl sulfate.The authors declare that they have no competing interests.ME, VS, PG and DC conceived and designed the experiments. ME, AD and DC performed cloning, fly crosses and analysis, and antigen expression. ME performed affinity purification of antibodies and ChIP analysis. ME and DC performed RT-PCR experiments. AP performed embryo injections. ME, VS, PG and DC analyzed the data and wrote the manuscript. All authors read and approved the final manuscript.R)EyeYW transgenic lines.Summarized results of wing and body phenotype analysis in (a) (UAS)Ey(e)YW and (b) was subtracted from normalized specific ChIP signals (obtained with specific antibodies) at each position.Results of ChIP with antibodies to (a) Ph and (b) Sfmbt. Diagrams summarize the results of ChIP with specific antibodies followed by real-time PCR. The ordinate shows the percentage of target sequences in the immunoprecipitated material relative to the input , with the genome regions for which DNA enrichment was tested being indicated on the abscissa: Click here for filePrimers used for RT-qPCR analysis of transcripts from transgenic flies.Click here for filePrimers used for PCR in X-ChIP experiments with DNA fragments from the genome or transgenic constructs.Click here for filezv77h larvae. Upper panel, antibodies against Zeste; lower panel, control anti-tubulin antibodies. (B) Western blot analysis of nuclear extracts (Input line), PH immunoprecipitates (PH line) and control IgG immunoprecipitates (IgG line) from Sg4 cells with antibodies against PH. (C) Western blot analysis of nuclear extracts (Input line), Sfmbt immunoprecipitates (Sfmbt line) and control IgG immunoprecipitates (IgG line) from Sg4 cells with antibodies against Sfmbt.(A) Testing of Zeste antibodies by Western blot. Protein extract was prepared from wild-type (WT) or Click here for fileWestern blotting, preparation of the nuclear extracts and immunoprecipitation.Click here for file"} +{"text": "In this study, we analyzed the changes of protein expression in trachea and kidney tissues from chicken embryos, following IBV infection in ovo, using two-dimensional gel electrophoresis (2-DE) coupled with matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF-TOF MS).Avian infectious bronchitis (IB) is one of the most serious diseases of economic importance in chickens; it is caused by the avian infectious coronavirus (IBV). Information remains limited about the comparative protein expression profiles of chicken embryonic tissues in response to IBV infection 17 differentially expressed proteins from tracheal tissues and 19 differentially expressed proteins from kidney tissues were identified. These proteins mostly related to the cytoskeleton, binding of calcium ions, the stress response, anti-oxidative, and macromolecular metabolism. Some of these altered proteins were confirmed further at the mRNA level using real-time RT-PCR. Moreover, western blotting analysis further confirmed the changes of annexin A5 and HSPB1 during IBV infection.in ovo. The data obtained should facilitate a better understanding of the pathogenesis of IBV infection.To the best of our knowledge, we have performed the first analysis of the proteomic changes in chicken embryonic trachea and kidney tissues during IBV infection Gamma coronavirus of the coronavirus genus and replicates primarily in the upper respiratory tract, kidney, and oviduct of chickens -1-propanesulfonate; DTT: dithiothreitol; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; IBV: Infectious bronchitis coronavirus; IEF: isoelectric focusing; IPG: immobilized pH gradient; MALDI-TOF-TOF/MS: matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry; PMF: peptide mass fingerprinting; RT-PCR: reverse transcriptase-polymerase chain reaction;SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis; SPF: specific pathogen free; TFA: trifluoroacetic acid; 2-DE: two-dimensional gel electrophoresis.The authors declare that they have no competing interests.SL designed the study. SL and ZC drafted the manuscript. ZC, ZH and YS carried out virus infection and test for the presence of IBV. ZC and HG carried out the 2-DE experiments, image analysis, excised the protein spots, data analysis and interpretation, and confirmed the differential expression by real-time RT-PCR and Western blotting analysis. SL wrote the manuscript. XK revised the manuscript. All authors read and approved the final manuscript.Additional_file_1.doc containing the MALDI-TOF spectrum and MALDI-TOF-TOF spectrum of differentially expressed protein spots in IBV-infected chicken embryo tracheal tissues.Click here for fileAdditional_file_2.doc containing the PMF spectrum and Mascot database search results of differentially expressed protein spots in IBV-infected chicken embryo tracheal tissues.Click here for fileAdditional_file_3.doc containing the MALDI-TOF spectrum and MALDI-TOF-TOF spectrum of differentially expressed protein spots in IBV-infected chicken embryo kidney tissues.Click here for fileAdditional_file_4.doc containing the PMF spectrum and Mascot database search results of differentially expressed protein spots in IBV-infected chicken embryo kidney tissues.Click here for file"} +{"text": "Hibiscus rosa-sinensis is one of the most prevalent ornamental plants grown in private and public gardens. Hibiscus chlorotic ringspot virus (HCRSV) is a member of the Carmovirus genus, with a positive single-strand RNA that putatively encodes seven proteins. The complete genome of the first Israeli isolate of HCRSV, HCRSV-IL, comprises 3,908 nucleotides and shows 93% nucleotide sequence identity to the Singapore isolate and 87% identity to the Taiwanese isolate. Hibiscus rosa-sinensis is one of the most prevalent ornamental plants grown in private and public gardens. Since 2010, we have identified unfamiliar symptoms on hibiscus plants growing in public gardens across Israel. The disease symptoms include chlorotic spots, yellow rings, and vein banding.2; FEI-Philips, Eindhoven, The Netherlands). Purified virus preparations served as a source material for virion RNA extractions (17) primers and later with the sequence-specific reverse complement primers R-HC-424 (5\u2032-TGTCAACCAACCTCCTTTCC-3\u2032) and R-HC-1785 (5\u2032-AAACACCGGCTTCATTTGAC-3\u2032). The obtained cDNA was used to produce double-stranded cDNA (ds-cDNA) fragments that were cloned into pUC19 (which was predigested by SmaI and dephosphorylated) (Fermentas), and both strands of each recombinant plasmid were sequenced . The obtained nucleotide sequences were subjected to a BLASTn search against the GenBank database and revealed high sequence identity to hibiscus chlorotic ringspot virus (HCRSV), a member of the Carmovirus genus with a positive single-strand RNA that putatively encodes seven proteins . Symptoms of HCRSV infection appeared 2\u00a0weeks after inoculation, producing typical chlorotic spots on C. quinoa, while the inoculated H.\u00a0trionum showed chlorotic vein banding. The infections were confirmed by RT-PCR using the HCRSV-specific primers F-HC-3247 (5\u2032-GGCCAATCTGAAGGGGAT-3\u2032) and R-HC-3908 (5\u2032-GGGCTGCCTCACAACTATG-3\u2032). The 647-bp amplicons we obtained were cloned and sequenced to validate the authenticity of the isolate's identification as HCRSV-IL.Symptomatic leafs were collected and used as a source material for viral purification as described previously . The preKC876666.The genome sequence of the Israeli isolate of HCRSV has been deposited at GenBank under the accession no."} +{"text": "Cladosiphon navae-caledoniae Kylin was prepared by enzymatic digestion. We investigated whether a combination of FE with chemotherapeutic agents had the potential to improve the therapeutic efficacy of cancer treatment.Fucoidan, a fucose-rich polysaccharide isolated from brown alga, is currently under investigation as a new anti-cancer compound -4. In th2 at 37 \u00b0C. The abalone glycosidase-digested fucoidan extract (FE) was obtained from Daiichi Sangyo Corporation . The cells were treated with FE and chemotherapeutic agents like cisplatin, tamoxifen or paclitaxel. The cell growth was determined by MTT assay. Apoptosis was evaluated using annexin V binding assay and flow cytometry analysis. Signaling proteins were analyzed by western blot. Intracellular reactive oxygen species (ROS) were determined using DCFH-DA and determined using IN Cell Analyzer 1000. The reduced glutathione (GSH) concentration was measured by the GSH assay kit.Estrogen receptor (ER)-positive MCF-7 and ER-negative MDA-MB-231 breast cancer cells were cultured in DME medium supplemented with 10% fetal bovine serum in a humidified atmosphere of 5% COThe co-treatments significantly induced cell growth inhibition, apoptosis, as well as cell cycle modifications in MDA-MB-231 and MCF-7 cells. FE enhanced apoptosis in cancer cells that responded to treatment with cisplatin, tamoxifen, or paclitaxel after 48 h of treatment (Figure FE protected normal human fibroblast TIG-1 cells from apoptosis by cisplatin and tamoxifen, suggesting its favorable characteristic for application to cancer therapy.\u2022 Combination of FE and three chemotherapeutic agents exhibit highly synergistic inhibitory effects on the growth of breast cancer cells.\u2022 Combination treatments induced modifications in cell cycle distribution.\u2022 Combination treatments modified the Bcl-2 expression, and ERK and Akt phosphorylation induced by FE, demonstrating different effects on apoptotic pathways in MDA-MB-231 cells and MCF-7 cells.\u2022 Generation of intracellular ROS and depletion of GSH are related to the cell death in combination treated -breast cancer cells."} +{"text": "Human herpes virus 8-unrelated primary effusion lymphoma (PEL)-like lymphoma is a rare type of large B cell lymphoma. This report presents the case of a male with abdominal pain and distension who was found to have massive ascites and enhanced peritoneum, mesenterium and greater omentum on enhanced computer tomography (CT) scan with negative ascitic cytology. The diagnosis of PEL-like lymphoma was established by fluorodeoxyglucose (FDG) positron emission tomography (PET)/CT and laparoscopic biopsy of the greater omentum. To the best of our knowledge, this is only the second case report to describe FDG PET/CT presentations of PEL-like lymphoma, and the first case report to use laparoscopy for diagnosis. Primary effusion lymphoma (PEL) is a rare type of large B-cell lymphoma characterized by lymphomatous effusion of body cavities without lymphadenopathy or organomegaly. PEL often occurs in patients with human immunodeficiency virus (HIV) and/or human herpes virus type 8 (HHV-8) infections . However9 cells/l and hemoglobin levels of 12.8 g/dl. Aspartate aminotransferase levels were 14 U/l and alanine aminotransferase levels were 70 U/l. Tests for hepatitis markers, Epstein-Barr virus (EBV), HHV-8, HIV and tumor markers were negative.A 39-year-old male was referred to the First Affiliated Hospital of Wenzhou Medical College with a one-week history of abdominal pain and distension. Laboratory tests revealed a white blood cell (WBC) count of 10.1\u00d710The WBC count of the ascitic fluid was 640,000 cells/ml: 5% polymorphonuclear leukocytes, 71% lymphocytes and 24% abdominal cells. Bacterial cultures were negative. The ascitic effusion test for HHV-8 using polymerase chain reaction was also negative. Ascitic cytology was performed three times but no malignant cells were found. Gastroscopy and colonoscopy were also normal.Enhanced abdominal CT scan showed massive ascites and enhanced peritoneum, mesenterium and greater omentum but no detectable mass or lymphadenopathy. Therefore, the patient underwent FDG PET/CT examination. FDG PET/CT showed FDG uptake in the peritoneum, mesenterium and greater omentum . No massA laparoscopic biopsy of the greater omentum was performed, revealing lymphoma cells with large nuclei and abundant cytoplasm which exhibited a B-cell phenotype . ImmunohThe patient was diagnosed with HHV-8-unrelated HIV-negative PEL-like lymphoma (indeterminate phenotype). The patient and his relatives refused chemotherapy and the patient succumbed to PEL-like lymphoma one month later.PEL is often associated with HHV-8 and occurs most frequently in immunodeficient states . Howeveret al or rituximab-containing regimen (2) have frequently been administered in these cases. Although the prognosis of HIV-negative HHV-8-unrelated PEL-like lymphoma patients is better than the HIV-positive PEL group ( or rituxIn conclusion, PET/CT and laparoscopic biopsy may be useful diagnostic tools for PEL-like lymphoma when the origins of ascites cannot be determined by general ascitic examination or conventional imaging tests, such as CT scans."} +{"text": "Deep-brain stimulation (DBS) is used to treat medication-refractory Parkinson's disease (PD). However, tuning stimulation parameters requires time intensive visits with a clinician using a trial-and-error process until therapy is achieved with minimal side effects . There i"} +{"text": "A very low calorie diet (VLCD) induces considerable weight loss in patients with type 2 diabetes mellitus, associated with improved insulin sensitivity and decreased triglyceride (TG) stores in (non)adipose tissues. Long-term effects of a VLCD on tissue-specific TG accumulation, including pericardial fat, have not been documented.To assess the effects of a 16-week VLCD and of subsequent 14 months of follow-up on a regular diet on tissue-specific TG stores in obese type 2 diabetes patients.2). (Non)adipose TG stores were measured using magnetic resonance (MR) imaging and MR proton spectroscopy before and after a 16-week VLCD and after a 14-month follow-up without dietary interventions.We included 14 obese patients with insulin treated type 2 diabetes . After an additional 14 months of follow-up on a regular diet, weight and hepatic TG content increased significantly to 90 and 73% of baseline values (P<0.05). After these 14 months the preferential loss in visceral fat compared to subcutaneous abdominal fat was lost. In contrast, pericardial fat volume remained reduced to the same extent.VLCD-induced weight loss and subsequent regain of weight during regular diet induces tissue-specific variations in (non)adipose TG stores in obese type 2 diabetes patients."} +{"text": "The NO redox sibling nitroxyl (HNO) elicits soluble guanylyl cyclase (sGC)-dependent vasodilatation. HNO has high reactivity with thiols (unlike NO), which is attributed with HNO-enhanced left ventricular (LV) function. The present study tested the hypothesis that the concomitant vasodilatation and inotropic actions induced by the HNO donor, Angeli\u2019s salt (sodium trioxodinitrate), are sGC-dependent and sGC-independent, respectively.8-37, 0.1\u03bcM) or voltage-dependent potassium channels were determined in isolated adult male rat hearts.Haemodynamic responses to bolus doses of Angeli\u2019s salt (10pmol - 10\u03bcmol), alone and in the presence of selective scavengers of HNO or NO or selective inhibitors of sGC , calcitonin gene-related peptide (CGRP) receptors (CGRP8-37 nor 4-AP affected Angeli\u2019s salt actions.Angeli\u2019s salt elicited concomitant, potent dose-dependent increases in coronary flow and LV systolic and diastolic function. Both L-cysteine and ODQ caused a rightward shift in the dose-response curve of each of these effects, implicating HNO and sGC in both the vasodilator and inotropic actions of Angeli\u2019s salt. In contrast, neither HXC, CGRPThese data suggest that each of the vasodilator, inotropic and lusitropic actions of Angeli\u2019s salt are mediated by L-cysteine-sensitive, HNO/sGC-dependent mechanisms. Our findings represent the first evidence that sGC specifically contributes a significant component of the inotropic and lusitropic actions of an HNO donor in the intact heart. Thus, HNO acutely enhances LV contractile function and LV relaxation, whilst concomitantly unloading the heart, potentially favourable properties for the failing heart."} +{"text": "Delayed enhancement (DE) CMR is the gold standard for detecting irreversibly damaged myocardial tissue (scar) . Yet, direct impact of scar on regional systolic and diastolic left ventricular (LV) function is not well understood. Standard tools for LV velocities (Tissue Doppler Imaging) are limited by poor reproducibility and incomplete assessment of all regions and motion directions. Myocardial CMR velocity mapping (MVM) is reproducible, non-invasive, and allows direct measure of myocardial velocities of all LV motion components in all regions. Here, we analyze effects of LV scar burden on myocardial motion.CMR with DE-CMR (inversion recovery PSIR) and MVM in 49 patients (age=54\u00b116 years) with cardiomyopathy (n=16), normal myocardium (n=10), inflammatory disease (n=4) ischemic heart disease (n=3), heart failure (n=3), and other (n=13) were acquired in basal, mid-ventricular, and apical short axis orientation during breath hold. DE-CMR: A radiologist assessed whether scar was present in segments from the AHA 16-segment model and classified based on number of segments with scar . MVM: Consisted of black-blood prepared CINE phase-contrast sequence with 3-directional velocity encoding . Data analysis included manual segmentation of LV contours and conversion of measured velocities into radial, rotational, and long-axis velocities. Systolic and diastolic radial and long-axis velocities were derived and mapped on the AHA 16-segment model. Global radial and long-axis velocities were analyzed using t-test.Patients with high scar showed reduced regional peak velocities compared to patients with no and low scar figure . SystoliDE-CMR and MVM, measuring scar burden and regional myocardial velocities, were co-registered to detect impaired regional myocardial structure and function. Segmental analysis showed significant decreases in mean velocities in patients with high scar indicating a direct relationship between structural damage to the LV and impaired myocardial function. Future work will analyze specific patient groups and assess other changes in velocity markers.AHA 12GRNT 12080032"} +{"text": "We have previously engineered computationally-designed \u2018epitope-scaffold\u2019 constructs for the broadly neutralizing, MPER-specific antibody 4E10, consisting of the epitope grafted as a structural unit onto non-HIV scaffold proteins for optimal presentation during immunization. 4E10 epitope-scaffolds display dissociation constants for mature 4E10 ranging down to picomolar values and can elicit epitope-specific responses during immunization. Sera from immunized animals failed to potently neutralize HIV, at least partially due to differences between human and non-human germline repertoires. Successful use of epitope-scaffolds as vaccine immunogens will require optimizing interactions with both the mature antibody target and appropriate precursors, while preserving or generating neutralization potency during maturation.Since a unique germline-encoded precursor sequence for 4E10 cannot be unambiguously assigned, we have generated an ensemble of the twelve likeliest candidates in order to study their functional and recognition properties. Interaction parameters between mature and candidate germline-encoded precursor (CGP) antibodies for engineered epitope-scaffolds, peptides, membrane components and HIV proteins were determined in surface plasmon resonance analyses and three-dimensional structures of free and ligand-complexed forms were determined by x-ray crystallography. Neutralization potencies were determined and polyspecificity and autoreactivity were analyzed with large-scale peptide arrays.Unlike other anti-HIV CGPs, which display negligible affinities for HIV-related ligands, 4E10 CGPs display affinities for epitope-scaffolds which, while 1,000- to 100,000-fold weaker, still reach into the nanomolar range. Structural studies show remarkable conservation of recognition mechanisms while functional studies show retention of anti-HIV activity. Polyspecificity of both mature and CGP forms is very limited, but the significant autoreactivity of mature 4E10 appears directed to specific targets.The functional gap between mature 4E10 and its CGPs is narrower than in other HIV-related systems. Strategies based on our results can be proposed to generate mature- and CGP-specific epitope-scaffolds for use in prime-boost vaccinations to target specific CGPs with desirable functional properties while potentially avoiding autoreactivity."} +{"text": "Drosophila as a model, in which long-range BMP transport from the longitudinal veins plays a critical role during the pupal stages. Here, we show that RhoGAP Crossveinless-C (Cv-C) is induced at the PCV primordial cells by BMP signaling and mediates PCV morphogenesis cell-autonomously by inactivating members of the Rho-type small GTPases. Intriguingly, we find that Cv-C is also required non-cell-autonomously for BMP transport into the PCV region, while a long-range BMP transport is guided toward ectopic wing vein regions by loss of the Rho-type small GTPases. We present evidence that low level of \u00df-integrin accumulation at the basal side of PCV epithelial cells regulated by Cv-C provides an optimal extracellular environment for guiding BMP transport. These data suggest that BMP transport and PCV morphogenesis are tightly coupled. Our study reveals a feed-forward mechanism that coordinates the spatial distribution of extracellular instructive cues and morphogenesis. The coupling mechanism may be widely utilized to achieve precise morphogenesis during development and homeostasis.A variety of extracellular factors regulate morphogenesis during development. However, coordination between extracellular signaling and dynamic morphogenesis is largely unexplored. We address the fundamental question by studying posterior crossvein (PCV) development in in vivo. We addressed the fundamental question by studying posterior crossvein (PCV) development in Drosophila as a model, in which a long-range transport of bone morphogenetic protein (BMP) type ligands from adjacent longitudinal veins plays a critical role during the pupal stages. Here, we first showed that RhoGAP Crossveinless-C (Cv-C) is induced at the PCV region by BMP signal and mediates PCV morphogenesis. By modulating wing vein morphogenesis, we then found that PCV morphogenesis is required for BMP transport, while ectopic wing vein morphogenesis sufficiently guides a long-range BMP transport. These data suggest a feed-forward mechanism that coordinates the spatial distribution of extracellular instructive cues and morphogenesis. The coupling mechanism may be widely utilized to achieve precise tissue morphogenesis and tissue homeostasis.It has been extensively studied how tissue morphogenesis is regulated by a variety of extracellular cues. Given that dynamic morphogenesis coincides with arrival of extracellular factors, there must be also mechanisms that coordinate extracellular signaling and intracellular morphogenesis. However, the coordination is largely unknown, due to the complexity of morphogenesis This includes identification of such extracellular signaling molecules and intracellular mechanisms that trigger morphogenesis. Since arrival of extracellular factors coincides with dynamic morphogenesis, there must be mechanisms to coordinate signaling and morphogenesis. The coordination can be achieved by an instructive role of morphogenesis in determining the regions where extracellular signals arrive or are activated. However, this is largely unknown, due to the complexity of morphogenesis Drosophila, Decapentaplegic (Dpp), a homologue of BMP2/4, is secreted either as a homodimer or a heterodimer with another BMP-type ligand (Glass bottom boat (Gbb) or Screw (Scw)). The ligands bind to the type I receptor Thickveins (Tkv) and type II receptor Punt and phosphorylate the transcription factor Mad. Then phosphorylated Mad (pMad), together with Medea, translocates into the nucleus for transcriptional regulation of various genes The bone morphogenetic proteins (BMPs) are extracellular factors that regulate morphogenesis as well as growth and patterning The Rho-type small GTPases, including Rho, Rac, and Cdc42, play critical roles in actin cytoskeleton organization, cell-extracellular matrix (ECM) adhesion, cell polarity, cell cycle progression, and cell migration dpp is initially transcribed at the prospective longitudinal veins (LVs) (sog transcription at the PCV region about 20 hr AP Posterior crossvein (PCV) development mediated by BMP signaling during the pupal stages provides an excellent system for understanding how the long-range BMP signal regulates morphogenesis . dpp is ns (LVs) , then lans (LVs) \u2032. It hasns (LVs) [13]. Clns (LVs) [12]. InIn contrast with the extracellular regulation of BMP transport, little is known about how the BMP signal regulates wing vein morphogenesis recognized by apposition at the basal side of two wing epithelial layers cv-c was identified as RhoGAP88C required for a variety of embryo morphogenesis cv-c is induced at the PCV region by the BMP signal and mediates PCV morphogenesis by inactivating various members of the Rho-type small GTPases. Intriguingly, we found that loss of cv-c inhibited Sog-Cv dependent BMP transport into the PCV region, while an ectopic Sog-Cv-dependent BMP signal was induced toward ectopic wing veins by loss of the Rho-type small GTPases. Taken together, our data suggest that Cv-C mediates a feed-forward loop coupling BMP transport and PCV morphogenesis. We also provide evidence that the initial PCV morphogenesis precedes BMP signaling and sog transcription, highlighting an instructive role of morphogenesis in guiding BMP transport.A candidate for mediating PCV formation is Crossveinless-C (Cv-C), whose viable mutant allele displays a PCV-less phenotype To investigate how the PCV region undergoes morphogenesis, we analyzed optical cross-sections in the prospective PCV region, marked by pMad accumulation. The tissue architecture and the cell-extracellular matrix (ECM) adhesion were visualized by phalloidin staining of F-actin and immunostaining for \u00df-integrin 70cv, a null allele of cv, in which pMad accumulation was absent due to lack of BMP transport (shvdpp mutant (s4/dpps11dpp) s4/dpps11dpp, BMP signal was severely affected both in the LVs and CVs during pupal stages (20\u201326 hr AP), and consequently, distal parts of L4, L5 and PCV were not formed in the adult wing , which facilitates ligand-receptor binding in a short-range manner and cv-2 . The genckground . When GFckground . Taken tcv-c is required for PCV morphogenesis, we hypothesized that BMP transport is coupled with wing vein morphogenesis. To test this, we analyzed 2cdc42, in which ectopic CVs were frequently observed in the adult wings mutants, 1/mysnj42mysor nj42mys , act as tumour suppressors in a variety of contexts 70cv, P129Dsog, KO1cv2, 3Kpnshv-Gal4, and UAS-GFP-Dpp flies were described previously 1cv-cand c524cv-c flies were obtained from H. Skaer UAS-PNKG58AeGFP flies were kindly provided by J.C. Hombria nj42 flies were obtained from F. Schoeck. The shv-lacZdpp, s11dpp, s4dpp, 2cdc42, 72ORho1, \u0394Rac2, J11Rac1, UAS-GFP, P11885sog, UAS-Rho1RNAi, BS1348-Gal4, and 1mys flies were obtained from Bloomington Drosophila Stock Center. Vkg-GFP flies were obtained from Fly Trap stock collection. cv-c MARCM clones were generated using y w hs-FLP; tubP-GAL4 UAS-mCD8::GFP; c524FRT82B cv-c/FRT82B tubP-GAL80.The Drosophila pupal wings were fixed at 4\u00b0C overnight and then dissected from the pupae. All immunohistochemistry and in situ hybridizations were performed, as previously described To analyze the number of GFP-Dpp dots, approximately 10 confocal sections were taken at app. 1-\u00b5m intervals to cover a single cell layer and the images were processed by maximum intensity profile and quantified using analyze particle command in ImageJ software . To analyze GFP-Dpp distribution along apical-basal axis, relative position of GFP-Dpp dots from basal surface of the PCV region was measured individually in a single confocal image using Image J. The heatmap of the intensity of pMad signal was produced using the \u201cHeatMap Histogram\u201d plugin of ImageJ.Figure S1in situ hybridization of \u00df-integrin at 20 hr AP and at 24 hr AP . Antisense and sense probes of myospheroid (mys) encoding \u00df-integrin.\u00df-integrin expression in the pupal wing. (A\u2014D) (TIF)Click here for additional data file.Figure S2s4/dpps11dpp. (A) Adult wing of s4/dpps11dpp. pMad staining at 24 hr AP in control yw (B) and in s4/dpps11dpp (C). (D\u2013F) Optical cross-sections of the PCV region. F-actin staining at 22 hr AP (D), 24 hr AP (E), 26 hr AP (F) in s4/dpps11dpp. A number of hemocytes were observed in the lumen at 22 hr AP. Slight delay of apposition of the two wing layers may reflect loss of the LVs fates in s4/dpps11dpp. (G\u2013G\u2033) Optical cross-sections of the PCV region. pMad (G), F-actin (G\u2032), and \u00df-integrin (G\u2033) staining at 24 hr AP in s4/dpps11dpp.The initial PCV morphogenesis in (TIF)Click here for additional data file.Figure S3ap>PKNG58AeGFP. (B) GFP signal in the dorsal layer of shv>GFP. (C) Rho1 activity in the dorsal wing layer at 20 hr AP in 70cv, ap>PKNG58AeGFP. The basal side of the invervein regions is indicated by arrows. The PCV region is marked by parentheses. ap-Gal4 is induced in the dorsal layer of the wing epithelial cells. (D) Rho1 (D), pMad (D\u2032) staining, and merge (D\u2033) in wild-type yw. All the images are optical cross-sections of the PCV region.Rho1 activity and protein localization around the PCV region. (A) Rho1 activity in the dorsal layer at 24 hr AP in (TIF)Click here for additional data file.Figure S4c524cv-c clones. A sagittal section of the PCV region, containing cv-c null clones. (A) c524cv-c clones (green), (A\u2032) F-actin (white), and (A\u2033) pMad (purple). F-actin accumulation at the basal side of the PCV is indicated by arrow. Apical-basal cell lengths of wild-type and cv-c mutant are indicated by double-headed arrows.Cell-autonomous F-actin accumulation at the basal side of the PCV region in (TIF)Click here for additional data file.Figure S5yw , 70cv/+, BS1348>Rho1 RNAi , and 70cv, BS1348>Rho1 RNAi. . The PCV position is indicated by arrows . Ectopic pMad accumulation is marked by arrowhead (C). All the pupal wings were fixed at 24 hr AP. Images were processed by maximum intensity profile from a single wing layer.Loss of Rho1 by RNAi induces Sog-Cv-dependent BMP signaling. pMad , \u00df-integrin staining, merged images , and adult wings in wild-type (TIF)Click here for additional data file.Figure S6Vkg-GFP wing. (B) Vkg-GFP signal (B) and F-actin staining (B\u2032) in Vkg-GFP pupal wing. (C) GFP signal (C) and F-actin staining (C\u2032) in control yw pupal wing. In addition to few punctuate signal, Collagen IV was also weakly and uniformly distributed in the lumen. (D) Vkg-GFP signal (D) and F-actin staining (D\u2032) in Vkg-GFP larval wing imaginal disc. (E) Optical cross-sections of the PCV region at 24 hr AP. Representative images of GFP-Dpp distribution along apical-basal axis. GFP-Dpp dots (E), F-actin staining (E\u2032), and schematic position of GFP-Dpp dots (E\u2033) in shv>GFP-Dpp.Apical-basal polarity in the PCV region and intervein region. (A\u2013C) Optical cross-sections of the PCV region at 24 hr AP. (A) aPKC staining (A), Dlg staining (A\u2032), Vkg-GFP signal (A\u2033), and merged image (A\u2034) in (TIF)Click here for additional data file."} +{"text": "Cannabis sativa has been cultivated throughout human history as a source of fiber, oil and food, and for its medicinal and intoxicating properties. Selective breeding has produced cannabis plants for specific uses, including high-potency marijuana strains and hemp cultivars for fiber and seed production. The molecular biology underlying cannabinoid biosynthesis and other traits of interest is largely unexplored.9-tetrahydrocannabinolic acid synthase in the Purple Kush transcriptome, and its replacement by cannabidiolic acid synthase in 'Finola', may explain why the psychoactive cannabinoid \u03949-tetrahydrocannabinol (THC) is produced in marijuana but not in hemp. Resequencing the hemp cultivars 'Finola' and 'USO-31' showed little difference in gene copy numbers of cannabinoid pathway enzymes. However, single nucleotide variant analysis uncovered a relatively high level of variation among four cannabis types, and supported a separation of marijuana and hemp.We sequenced genomic DNA and RNA from the marijuana strain Purple Kush using shortread approaches. We report a draft haploid genome sequence of 534 Mb and a transcriptome of 30,000 genes. Comparison of the transcriptome of Purple Kush with that of the hemp cultivar 'Finola' revealed that many genes encoding proteins involved in cannabinoid and precursor pathways are more highly expressed in Purple Kush than in 'Finola'. The exclusive occurrence of \u0394Cannabis sativa genome enables the study of a multifunctional plant that occupies a unique role in human culture. Its availability will aid the development of therapeutic marijuana strains with tailored cannabinoid profiles and provide a basis for the breeding of hemp with improved agronomic characteristics.The availability of the Cannabis sativa L. has been used for millennia as a source of fibre, oil- and protein-rich achenes (\"seeds\") and for its medicinal and psychoactive properties. From its site of domestication in Central Asia, the cultivation of cannabis spread in ancient times throughout Asia and Europe and is now one of the most widely distributed cultivated plants that has been shown to activate medium- and long-chain fatty acids including hexanoate and scaffold29030 [genbank:JH245535]. OLS, which encodes the putative cannabinoid pathway polyketide synthase [JH226441] and scaffold16618 [genbank:JH237993].The PK genome contains two copies of two genes involved in cannabinoid biosynthesis. Copies of synthase , was fouC. sativa, we estimated the frequency of SNVs in four taxa. In addition to PK and 'Finola', our analysis included the Illumina reads we generated from the hemp cultivar 'USO-31', as well as the reads from the marijuana strain Chemdawg, which were released by Medical Genomics, LLC [JH239911]) was identified that contained the THCAS gene as a single 1638 bp exon with 99% nucleotide identity to the published THCAS sequence. Querying the PK transcriptome returned the same THCAS transcript that was found to be expressed at high abundance in female flowers was also identified. We used the CBDAS sequence [genbank:AB292682] to querys Figure . A THCASB292682] to queryK Figure . A 347-bTHCAS and CBDAS sequences. In total, 23 candidates were identified that had greater than 65% nucleotide identity with these sequences. These include four genes that we designated THCAS-like1 to THCAS-like4, which encode proteins that are 89%, 64%, 68%, and 59% identical to THCAS at the amino acid level, respectively. We also identified transcripts corresponding to CBDAS2 and CBDAS3, which are closely related to CBDAS but do not encode enzymes with CBDAS activity . The version described in this paper is the first version, [genbank:AGQN01000000], and corresponds to the canSat3 assembly in the Cannabis Genome Browser. Assembled transcripts \u2265 200 nt have been deposited in the NCBI Transcriptome Shotgun Assembly (TSA) sequence database with accession numbers between [genbank:JP449145] - [genbank:JP482359]. Raw sequence read data have been deposited in the NCBI Sequence Read Archive with the following study identifiers: PK genomic DNA - [SRA:SRP008673]; PK RNA-Seq - [SRA:SRP008726]; 'Finola' genomic DNA - [SRA:SRP008728]; 'Finola' RNA-Seq - [SRA:SRP008729]; 'USO-31' genomic DNA - [SRA:SRP008730].The PK Whole Genome Shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession [genbank:C-methyl-D-erythritol 4-phosphate; ORF: open reading frame; OLS: olivetol synthase; PK: Purple Kush; PT: prenyltransferase; QC: quality control; RPKM: reads per kb per million reads; SNV: single nucleotide variants; THC: \u03949-tetrahydrocannabinol; THCA: \u03949-tetrahydrocannabinolic acid; THCAS: \u03949-tetrahydrocannabinolic acid synthase; THCV: \u03949-tetrahydrocannabivarin.AAE: acyl-activating enzyme; bp: base pair; BP: before present; CBC: cannabichromene; CBCA: cannabichromenic acid; CBCAS: cannabichromenic acid synthase; CBD: cannabidiol; CBDA: cannabidiolic acid; CBDAS: cannabidiolic acid synthase; CBGA: cannabigerolic acid; CMK: 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase; CTAB: cetyl trimethylammonium bromide; DXR: 1-deoxy-D-xylulose 5-phosphate reductoisomerase; DXS: 1-deoxyxylulose-5-phosphate synthase; EST: expressed sequence tag; FIGE: Field inversion gel electrophoresis; FISH: fluorescence in situ hybridization; Gb: giga base pair; GPP: geranyl diphosphate; GPP synthase lsu: GPP synthase large subunit; GPP synthase ssu: GPP synthase small subunit; HDR: 4-hydroxy-3-methylbut-2-enyl diphosphate reductase; HDS: 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase; HPL: hydroperoxide lyase; kb: kilo base pair; LOX: lipoxygenase; Mb: mega base pair; MCT: 4-diphosphocytidyl-methylerythritol 2-phosphate synthase; MDS: 2-C-methyl-D-erythritol 2:4-cyclodiphosphate synthase; MEP: 2-The authors declare that they have no competing interests.TRH and JEP conceived of the project. HvB performed the genome and transcriptome assembly and generated the figures. JMS and JEP extracted the nucleic acids. AGC prepared the Illumina sequencing libraries. CMT prepared and sequenced the 454 libraries under the direction of AGS. TRH, JEP, HvB and JMS wrote the manuscript. All authors have read and approved the manuscript for publication."} +{"text": "Left atrial (LA) function is strongly related to left ventricular (LV) filling pressures and has shown association to cardiovascular outcomes. A recently developed speckle tracking technique can assess LA deformation using CMR cine sequences. Myocardial scar assessed by late gadolinium enhancement (LGE-CMR) relates to cardiac remodeling, but its association to LA function is unknown. We explored the relationship of LA function with the amount of myocardial scar.A total of 1666 participants from the MESA, age range 55-94 yrs, underwent LGE-CMR using 1.5T scanners at six field centers. Myocardial scar was visually detected, classified (ischemic/non-ischemic), and quantified using semi-automated methods in 136 participants. The amount of scar was quantified as the ratio of scar mass over total LV mass and values greater than 5% were defined as clinically significant. LA function was evaluated using multimodality tissue tracking (MTT) from SSFP 2- and 4-chamber long-axis cine CMR images in all participants with myocardial scar and in an age and gender matched control group of 136 participants without scar. LA function was assessed using peak longitudinal strain (Smax), diastolic longitudinal strain rate (SRdia), ejection fraction (LAEF), emptying fraction (LAEmF), and LA maximum volume (Vmax). Wilcoxon rank-sum test was used to evaluate differences between groups: control, scar>5%, and scar<5%. Pearson's correlation assessed the relationship between the amount of scar and LA function parameters.From the total of 136 participants with myocardial scar , 43 participants had scars that were clinically significant . No significant difference was found between the groups for Vmax and LAEF. LAEmF, Smax, and SRdia were the most robust parameters comparing the groups (Table LA parameters are associated with cardiac remodeling in clinically significant myocardial scar. LAEmF, Smax and SRdia are more sensitive to changes in cardiac function resulting from the presence of myocardial scar than traditional LA functional parameters Vmax and LAEF.NHLBI N01-HC-95159NHLBI N01-HC-95168NCRR UL1-RR-024156NCRR UL1-RR-025005"} +{"text": "Conventional harm reduction (HR) projects overlook IDUs\u2019 capabilities in offering them services but no active roles to play in preventing HIV. In contrast, \u201cpeer-driven interventions\u201d (PDIs) offer IDUs rewards to educate and recruit peers for services. All IDU-recruits receive the opportunity to play both roles.In 2010, the International HIV/AIDS Alliance-Ukraine implemented PDIs in 12 Ukrainian cities that relied entirely on IDUs to access and teach IDUs who either had: (a.) never received HR services, or (b.) former PDI/HR-respondents eligible for a one-year follow-up (FU) intervention. IDU-recruiters were trained to administer two completely different bodies of prevention information: one to new recruits, the other to FU-recruits. Recruits from both groups were administered an 8-point knowledge test (KT) at their appointment that measured how well their recruiters educated them.In 6 months of operation, the 12 PDIs recruited 8,115 IDUs:2,782 (34%) new recruits5,333 (66%) PDI-FU-recruits- New recruits: 87% scored 7 or higher on the first KT- FU-recruits were administered both KTs at the follow-up appointment.86.4% scored 7 or higher on the 1st KT89.6% scored 7 or higher on the 2nd KTThe PDIs documented that IDUs can play active roles in preventing HIV by recruiting IDU-peers who have never received HR services, or IDUs eligible for FU intervention. They are also able to deliver two entirely different bodies of prevention information. Compared to HR projects that relied on traditional staffs of salaried outreach workers, the PDI proved to be far more powerful and cost-effective model. Alliance-Ukraine now plans to broaden its investment in PDIs even more heavily by further expanding projects targeting IDUs, but also female sex workers and homeless/runaway street children."} +{"text": "In vivo, LOX inhibition attenuated LPS-induced expression of GEF-H1 and lung dysfunction. These findings present a novel mechanism of stiffness-dependent exacerbation of vascular inflammation and escalation of ALI via stimulation of GEF-H1 - Rho pathway. This pathway represents a fundamental mechanism of positive feedback regulation of inflammation.Acute lung injury (ALI) is accompanied by decreased lung compliance. However, a role of tissue mechanics in modulation of inflammation remains unclear. We hypothesized that bacterial lipopolysacharide (LPS) stimulates extracellular matrix (ECM) production and vascular stiffening leading to stiffness-dependent exacerbation of endothelial cell (EC) inflammatory activation and lung barrier dysfunction. Expression of GEF-H1, ICAM-1, VCAM-1, ECM proteins fibronectin and collagen, lysyl oxidase (LOX) activity, interleukin-8 and activation of Rho signaling were analyzed in lung samples and pulmonary EC grown on soft (1.5 or 2.8 kPa) and stiff (40 kPa) substrates. LPS induced EC inflammatory activation accompanied by expression of ECM proteins, increase in LOX activity, and activation of Rho signaling. These effects were augmented in EC grown on stiff substrate. Stiffness-dependent enhancement of inflammation was associated with increased expression of Rho activator, GEF-H1. Inhibition of ECM crosslinking and stiffening by LOX suppression reduced EC inflammatory activation and GEF-H1 expression in response to LPS. Exacerbated inflammation and lung barrier dysfunction are hallmarks of acute respiratory distress syndrome (ARDS), a condition with dangerously high rates of morbidity and mortality. Along with acute alterations in blood-gas barrier and inflammatory activation of lung cells, lung injury also stimulates provisional extracellular matrix formation which persists during the fibroproliferative phase in ARDS. These matrices further emit signals to activate an additional inflammatory response, but may also lead to permanent matrix remodeling Due to its functional requirements, the lung is a biomechanically sensitive organ which is particularly dependent on the composition and architectural organization of ECM components. Lysyl oxidase (LOX) is an extracellular enzyme that catalyzes oxidative deamination of peptidyl lysine and hydroxylysine residues in secreted collagen precursors, and lysine residues in elastin leading to ECM fibers cross-linking. Excess LOX-dependent cross-linking contributes to excess ECM accumulation and stiffening in fibrotic diseases Our recent study demonstrated significant increase in thrombin-induced actin stress fiber formation and signaling in human pulmonary endothelial cells grown on stiff (42 kPa) substrate as compared to cells grown on matrices of more physiological stiffness Tissue inflammation resulting from bacterial infections or sepsis is triggered by Toll-like receptor (TLR)-mediated stimulation of inflammatory signaling cascades including TLR4-MyD88-IRAK-TRAF6 cascade, p38 MAPK, Erk-1,2, JNK stress kinase and NFkB activation in vitro as well as increases vascular leak and lung inflammation in vivo. Such disruptive effects were linked to activation of Rho signaling caused by LPS-induced MT disassembly and release of Rho-specific GEF-H1 from MTs Guanine nucleotide exchange factor H1 (GEF-H1) is a Rho-specific GEF, which localizes on microtubules (MT). In MT-bound state, the guanine-exchange activity of GEF-H1 is suppressed but activated upon GEF-H1 release caused by MT disassembly Based on these findings, we hypothesized that LPS-induced inflammation, increased ECM synthesis and activation of Rho signaling may interplay via stiffness-dependent mechanisms. We examined LPS effects on expression of ECM proteins and ECM-modifying enzyme LOX, evaluated effects of substrate stiffness on LPS-induced EC inflammatory activation and tested role of GEF-H1 as potential mechanism of stiffness-dependent stimulation of Rho signaling and exacerbation of LPS-induced inflammation.Human pulmonary artery endothelial cells (HPAEC) and human lung microvascular endothelial cells (HLMVEC) were obtained from Lonza , maintained in a complete culture medium according to the manufacturer's recommendations and used for experiments at passages 5\u20137. Unless specified, biochemical reagents were obtained from Sigma . TNF\u03b1 was purchased from R&D Systems ; \u03b2-aminopropyl nitrile (BAPN) was from Sigma. Antibodies to fibronectin, LOX, VCAM1, and ICAM1 were obtained from Santa Cruz Biotechnology ; antibpdies to phosphorylated myosin light chain phosphatase (MYPT), diphospho-myosin light chain (MLC), GEF-H1 were from Cell Signaling . All reagents for immunofluorescence were purchased from Molecular Probes . LOX activity and interleukin-8 (IL-8) production in conditioned medium was measured using LOX activity assay and IL-8 ELISA kit , respectively, according to the manufacturers' instructions.Human donor lungs that could not be transplanted were obtained from deceased donors through Gift of Hope/Regional Organ Bank of Illinois and were stored at 4\u00b0C for up to 2 days before use. Precision cut lung slices were obtained and cultured as previously described PAA substrates were prepared on glass coverslips with an acrylamide/bis-acrylamide ratio to obtain gels with shear elastic moduli of 1.5 kPa, 2.8 kPa, and 40 kPa and coated with collagen as characterized previously For GEF-H1 or LOX knockdown, pre-designed ON-TARGET plus SMARTpool human GEF-H1- or LOX-specific siRNA sets were ordered from Dharmacon . Transfection of EC with siRNA was performed as previously described Protein extracts from lung or EC homogenates were separated by SDS-PAGE, transferred to polyvinylidene difluoride membranes, and the membranes were incubated with specific antibodies of interest. Equal protein loading was verified by reprobing membranes with \u03b2-actin antibodies. Immunoreactive proteins were detected with the enhanced chemiluminescent detection system according to the manufacturer's protocol .Endothelial monolayers plated on glass cover slips were subjected to immunofluorescence staining as described previously 5\u2032-CTTTGGTGCAGCACAACTTC-3\u2032 (forward) and 5\u2032-TGGAATTTCCTCCTCGAGTC-3\u2032 (reverse); collagen type I A1: 5\u2032-AAGAGGAAGGCCAAGTCGAG-3\u2032 (forward) and 5\u2032-AGATCACGTCATCGACAAC-3\u2032 (reverse); LOX: 5\u2032-CATCATGCGTATGCCTCAG-3\u2032 (forward) and 5\u2032-TTCCCACTTCAGAACACCAG-3\u2032 (reverse); GAPDH: 5\u2032-AGGTGAAGGTCGGAGTCAAC-3\u2032 (forward) and 5\u2032-AGTTGAGGTCAATGAAGGGG-3\u2032 (reverse).Reverse transcription (RT) was performed with 1 \u00b5g of total RNA isolated from EC. RT-PCR reactions were performed as previously described and gene expression fold changes were calculated according to the \u0394\u0394Ct method Escherichia coli O55:B5) and then injected daily. After 72 hours of LPS challenge, animals were sacrificed by exsanguination under anesthesia. Measurements of cell count, protein concentration, Evans blue extravasation and histological assessment of lung injury were conducted as described All experimental protocols involving the use of animals were approved by the University of Chicago Institutional Animal Care & Use Committee for the humane treatment of experimental animals. C57BL/6J mice were randomized to concurrently receive sterile saline solution or BAPN (100 mg/kg) by intraperitoneal injection the day before intratracheal LPS administration and lysyl oxidase (LOX), the enzyme involved in ECM crosslinking and stiffening of extracellular matrix Pulmonary EC grown on polyacrylamide hydrogels of physiologically relevant stiffness (2.8 kPa) were stimulated with LPS. LPS induced time-dependent phosphorylation of MYPT and MLC or stiff (40 kPa) polyacrylamide hydrogels and stimulated with LPS or another ALI-relevant inflammatory mediator, TNF\u03b1 for 6 hrs. Both mediators induced significantly higher levels of ICAM-1 and VCAM-1 protein expression and increased FN mRNA expression by EC grown on stiffer substrate Figure 4Because LPS stimulated Rho signaling and stiffness-dependent EC inflammatory activation, we next tested whether these events can be linked via GEF-H1. LPS-stimulation of pulmonary EC grown on 2.8 kPa substrate markedly increased GEF-H1 protein expression after 24\u201348 hrs . Histological analysis of lung tissue sections stained with hematoxylin and eosin revealed that in contrast to control animals, intratracheal LPS injection induced neutrophil infiltration in the lung parenchyma and accumulation of protein-rich fluid in alveolar space indicative of alveolar-capillary barrier dysfunction. In consistence with results of BAL analysis and Evans Blue extravasation assay, BAPN administration suppressed LPS effects and elevated stiffness (40 kPa). This stiffness range was detected in perivascular regions of LPS-challenged lungs by AFM measurements in precision cut lung slices . LPS caused more pronounced ICAM-1 expression and IL-8 production in EC grown on stiff substrate, thus supporting the stiffness dependent mechanism of inflammatory propagation.in vivoIn agreement with proposed hypothesis considering the role of lung stiffening in exacerbation of LPS-induced inflammation, the pretreatment of mice with BAPN prior to intratracheal LPS administration significantly attenuated parameters of lung injury and inflammation. Previous study shows the two-fold increase in LOX activity in the lungs of LPS-stimulated mice Previous studies by our group and others showed requirement of the Rho signaling for the full activation of LPS-induced lung inflammation in vitro are currently unavailable, we focused on ECM deposition by cultured EC and cell plating on polyacrylamide hydrogels of different stiffness. Our other unpublished study using atomic force microscopy probing the crossection area of lung microvessel in precision cut live lung slices shows five-fold increase of local stiffness in the microvascular region after 48 hrs of LPS intratracheal instillation in mouse lungs. These findings support our hypothesis about local micromechanical changes in lung microvasculature of inflamed lungs.Besides pro-fibrotic mechanisms including matrix deposition at later phase of inflammation, local tissue stiffness in the inflamed organ may change due to other factors, for example tissue swelling. This event develops relatively fast and is consistent with the time frame of GEF-H1 activation, Rho signaling and expression of inflammatory markers including ECM protein fibronectin described in our study, which exacerbate cell inflammatory response to pro-inflammatory mediator. Stiffness effects on GEF-H1 expression were seen as early as 6 hrs after cell plating , and werOne potential mechanism of stiffness-dependent regulation of inflammation may include activation of Rho-Rho kinase signaling which can be activated by cyclic stretch or pulling single cells by micropipette or electromagnetic field applied to cell-attached magnetic beads Based on these data, we propose that GEF-H1 plays an essential role in stiffness-dependent enhancement of LPS-induced inflammation and forms a positive feedback loop of inflammation Figure 8"} +{"text": "Aortic pulse wave velocity sampled in the descending aorta is associated with maximal stenosis severity, visually scored on CE-MRA in patients with PAOD whereas stenosis severity is correlated to a lesser extent with carotid vessel wall.In atherosclerosis, arterial wall thickening and stiffening precede luminal narrowing. MRI is well-validated for imaging vessel wall thickness (VWT) and stiffness expressed in pulse wave velocity . Contrast-enhanced MR angiography (CE-MRA) has evolved into a reliable tool for stenosis detection in peripheral arterial occlusive disease (PAOD). The purpose of this study was to use a comprehensive 3T MRI-approach for comparing stenosis severity on CE-MRA with VWT, sampled in the common carotid artery, and PWV, sampled in the descending aorta.Forty-two patients with clinically suspected PAOD were included. Standardized single-injection 3-station moving-table CE-MRA, carotid vessel wall imaging and PWV-assessment were performed at 3T MRI (Philips). With CE-MRA, the arterial tree was evaluated from infrarenal aorta down to the tibial and peroneal arteries Figure . Visual Four transverse images of the common carotid artery were obtained by multi-slice 2D dual inversion recovery black-blood (DIR) fast gradient-echo Figure with spePWV was assessed for the descending aorta by applying two one-directional through-plane velocity-encoded MRI acquisitions, planned perpendicular to the aorta and transecting the proximal and abdominal descending aorta, respectively Figure . PWV wasPWV was compared with carotid VWA indexed for body surface area and maximal stenosis severity class detected with CE-MRA.Mean Fontaine class was 2.3\u00b10.6. Maximal stenosis class per patient presented on CE-MRA was as follows: 2 patients without stenosis (class 1), 1 patient with class 2, 2 patients with class 3, 9 patients with class 4 and 28 patients with class 5. PWV in the descending aorta was well-correlated with maximal stenosis class (Spearman correlation 0.63 (p<0.001), Figure PWV in the descending aorta is associated with maximal stenosis severity, visually scored on CE-MRA in patients with PAOD whereas stenosis severity is correlated to a lesser extent with carotid vessel wall.None."} +{"text": "The present approach aimed at the identification and annotation of osmoprotectant-related sequences applied to short transcripts from a soybean HT-SuperSAGE database, and also its comparison with other transcriptomic and genomic data available from different sources.Despite the importance of osmoprotectants, no previous A curated set of osmoprotectants related sequences was generated using text mining and selected seed sequences for identification of the respective transcripts and proteins in higher plants. To test the efficiency of the seed sequences, these were aligned against four HT-SuperSAGE contrasting libraries generated by our group using soybean tolerant and sensible plants against water deficit, considering only differentially expressed transcripts (p \u2264 0.05). Identified transcripts from soybean and their respective tags were aligned and anchored against the soybean virtual genome.P5CS: 4, P5CR: 2), Trehalose , Glycine betaine (BADH: 4) and Myo-inositol ], also mapped in silico in the soybean genome (25 loci). Another approach considered matches using Arabidopsis full length sequences as seed sequences, and allowed the identification of 124 osmoprotectant-related sequences, matching ~10.500 tags anchored in the soybean virtual chromosomes. Osmoprotectant-related genes appeared clustered in all soybean chromosomes, with higher density in some subterminal regions and synteny among some chromosome pairs.The workflow applied resulted in a set including 1,996 seed sequences that allowed the identification of 36 differentially expressed genes related to the biosynthesis of osmoprotectants [Proline , allowing the identification of interesting candidates for biotechnological inferences. The identified tags aligned to corresponding genes that matched 19 soybean chromosomes. Osmoprotectant-related genes are not regularly distributed in the soybean genome, but clustered in some regions near the chromosome terminals, with some redundant clusters in different chromosomes indicating their involvement in previous duplication and rearrangements events. The seed sequences, transcripts and map represent the first transversal evaluation for osmoprotectant-related genes and may be easily applied to other plants of interest.Soybean presents all searched osmoprotectant categories with some important members differentially expressed among the comparisons considered (drought tolerant or sensible Osmoprotectants figure among the most fundamental solutes in living organisms, being present from bacteria and fungi to higher plants and animals . Main plMyo-inositol), complex sugars and charged metabolites regarding genomic and transcriptomic libraries.Considering the potential of these molecules for plant biotechnological approaches, the present work generated a curated list of osmoprotectants, osmoprotectant-related sequences and important regulatory elements, indicating most adequate tools for their identification and annotation. To evaluate the sensitivity of the proposed approach, the generated seed sequences and the proposed workflow were used to search of osmoprotectant-related sequences in short sequences (26 bp) generated from HT-SuperSAGE depositeet al. for each compared pair of libraries are presented in the Table The carried approach was very successful, allowing the identification of 36 differentially expressed HT-SuperSAGE tags associated to 65 osmoprotectant-related sequences anchored in 25 loci to G. max sequences from four loci coding BADHs and annotated as Aldehyde Dehydrogenase Family 10A gene superfamily here identified in soybean genome were also categorized by Kotchoni et al. that proet al. also obs , suggesting PvP5CS2 represented a soybean P5CS homolog gene. Indels (insertion and deletion events) and SNPs (single nucleotide polymorphisms) were found in the cloned PvP5CS2 genome sequence when the authors compared different accessions, helping in the development of a molecular marker in the chromosome b01. The association of molecular markers and phenotypes, in this case Pro accumulation is highly applicable for genetic improvement of plants and germplasm screening.Significant upregulation (RTqPCR) in leaves of PvP5CS transformed with P5CR in sense and antisense directions. The most rapid increase in Pro content was found in the sense transformants that exhibited the least water loss, while the slowest elevation of Pro levels was detected in the antisense transformants that exhibited the greatest water loss during stress. Correspondingly, the level of the Pro precursors Glu and Arg was higher in sense transformants and lower in antisense ones compared to the wild type plants during the initial exposure to stress (drought and heat) . For this purpose a initial list was generated based on well known data from Arabidopsis thaliana were adjusted to allow the anchoring of soybean sequences position along the soybean virtual chromosomes. Afterwards the identified anchoring positions were submitted to the Circos program and so eMyo-inositol 1-phosphate phosphatise; MIPS: L-Myo-inositol 1-phosphate synthase; NGS: next generation sequencing; OAS: O-acetyl-L-serine; OAS-TL: O-acetyl-L-serine thiol lyase; OAT: ornithine d-aminotransferase; OsTPS: Oryza sativa Trehalose-6-phosphate synthase; P5CR: delta(1)-pyrroline-5-carboxylate reductase; P5CS: delta(1)-pyrroline-5-carboxylate synthase; PEG: polyethylene glycol; Pro: Proline; PtdInsP: phosphatidylinositol phosphate; QTLs: quantitative trait loci; RT-qPCR: real-time quantitative PCR; SAL1: myo- inositol polyphosphate 1-phosphatase; SAT: serine acetyltransferase; SNP: single nucleotide polymorphism; T6P: trehalose-6-phosphate; TPP: trehalose-6-phosphate phosphatise; TPPB: Trehalose 6- phosphate phosphatase B; TPS: Trehalose-6-phosphate synthase; TSM: tolerating a single mismatch; UDP: Glycosyltransferase/trehalose-phosphatase family protein; UR: up-regulated; UTRs: untranslated region.ABA: abscisic acid; ALDH: aldehyde dehydrogenase; Arg: arginine; BACs: bacterial artificial chromosome; BADH: betaine aldehyde dehydrogenase; CDS: coding sequence; CMO: choline monooxygenase; DR: down-regulated; FC: fold change value; GB: glycine betaine; GENOSOJA: Brazilian Soybean Genome Consortium; Glu: Glutamic acid; Glucose-6-P: glucose 6-phosphate; HP-SA: high performance sequencing approaches; HSP70: 70 kilodalton heat shock proteins; HT-SuperSAGE: High Throughput Super Serial Analysis of Gene Expression; IMP: L-The authors declare that they have no competing interests.EAK, VP and ALN generated the HT-SuperSAGE libraries. EAK and JRCFN carried out the identification of tags and differential expression analysis. RLOS, LCB, JPBN, NMSC and MDS generated and curated the seed sequence bank, while JPBN and AMBI generated the data for genome anchoring. AMBI coordinated the research.The publication costs for this article were funded by the first author's institution.BMC Bioinformatics Volume 14 Supplement 1, 2013: Computational Intelligence in Bioinformatics and Biostatistics: new trends from the CIBB conference series. The full contents of the supplement are available online at http://www.biomedcentral.com/bmcbioinformatics/supplements/14/S1.This article has been published as part of Table S1. BLASTx results regarding nucleotide sequences involved in the biosynthesis pathway and corresponding best hit (cut-off e-10) in the Uniprot-SwissProt and Phytozome (v.8 Glycine max) databases, represented by the respective loci/transcripts in soybean (Glyma) as well as their annotation.Click here for fileTable S2. BLASTn results using HT-SuperSAGE 26-bp tags against Glycine max seed sequences given in Table S1. Libraries consisted of soybean cultivar Embrapa 48, tolerant (T) against drought, with roots submitted to water deficit after 1-6 h of stress submission (TS) as compared with the tolerant non stressed control (TC). The same treatment was given to the drought sensible cultivar (BR-16) considering stressed (SS) and its respective control (SC). Tag annotation occurred against transcripts on the soybean Phytozome database. For each library normalized frequencies were considered (tpm: tags per million). Regarding comparisons among different treatments, fold change (FC), regulation (reg) are given, as well as the tag-mapping position in the respective soybean transcript (Glyma).Click here for fileTable S3. Main soybean transcripts similar to known osmoprotectants-genes (I.) from Arabidopsis thaliana (used as seed sequences). II. tBLASTn results and sequence evaluation of soybean Osmoprotectants-genes. Information available include number of hits, best match of each gene class and features of hits: size range in nucleotides (n), ORF (Open Reading Frame) size range in amino-acids (aa), e-value range based on SoyBase web resource, as well as number of matching HT-SuperSAGE tags.Click here for file"} +{"text": "As recently shown, non-invasive late gadolinium contrast enhanced MRI (LGE-CMR) is able to detect myocardial infarction (MI) typical patterns in patients after heart transplant (HTX). Patho-physiologically, the underlying reason is an accelerated vascular immuno-atherosclerosis, the so-called transplant coronary artery disease (TCAD) which limits long-term survival of HTX pts. To date, there is only scarce data on the detection of early infarctions after HTX, the time course and the effects on myocardial function.We hypothesized that LGE-CMR could detect TCAD-related MI in pts already early after the HTX procedure.123 patients (pts) were divided into group I and group II . LGE-CMR (Gadolinium:0.2mmol/kg bw) was performed on a 1.5T Whole Body MRI scanner and analysed blindly by two experienced observers. For anatomic LGE description, hearts were divided according to the 17-segment model. Areas of infarct-typical LGE patterns were defined as sub-endocardial LGE patterns of various transmurality and were quantified by delineation of hyper-enhanced areas related to the myocardial mass on LGE images (relative infarct size). Groups were compared using ANOVA. P-values \u2264 0.05 were considered statistically significant.In group I, already 21% of patients and 18 of 1054 segments (1.7%) showed infarct-typical LGE patterns. In contrast, significantly more patients (33%) of group II and 34 of 1037 segments were affected . These findings could have potential impact on a modified HTX patient risk stratification including LGE-CMR for tissue characterisation and detection of unknown MI.None."} +{"text": "LINE-1 elements make up the most abundant retrotransposon family in the human genome. Full-length LINE-1 elements encode a reverse transcriptase (RT) activity required for their own retrotranpsosition as well as that of non-autonomous Alu elements. LINE-1 are poorly expressed in normal cells and abundantly in cancer cells. Decreasing RT activity in cancer cells, by either LINE-1-specific RNA interference, or by RT inhibitory drugs, was previously found to reduce proliferation and promote differentiation and to antagonize tumor growth in animal models. Here we have investigated how RT exerts these global regulatory functions.We report that the RT inhibitor efavirenz (EFV) selectively downregulates proliferation of transformed cell lines, while exerting only mild effects on non-transformed cells; this differential sensitivity matches a differential RT abundance, which is high in the former and undetectable in the latter. Using CsCl density gradients, we selectively identify Alu and LINE-1 containing DNA:RNA hybrid molecules in cancer but not in normal cells. Remarkably, hybrid molecules fail to form in tumor cells treated with EFV under the same conditions that repress proliferation and induce the reprogramming of expression profiles of coding genes, microRNAs (miRNAs) and ultraconserved regions (UCRs). The RT-sensitive miRNAs and UCRs are significantly associated with Alu sequences.The results suggest that LINE-1-encoded RT governs the balance between single-stranded and double-stranded RNA production. In cancer cells the abundant RT reverse-transcribes retroelement-derived mRNAs forming RNA:DNA hybrids. We propose that this impairs the formation of double-stranded RNAs and the ensuing production of small regulatory RNAs, with a direct impact on gene expression. RT inhibition restores the \u2018normal\u2019 small RNA profile and the regulatory networks that depend on them. Thus, the retrotransposon-encoded RT drives a previously unrecognized mechanism crucial to the transformed state in tumor cells. Evidence that the genome is pervasively transcribed, including in its non-coding component, is now an established finding and is cMobile retroelements, including retrotransposons and endogenous retroviruses, make up as much as 45% of the human genome . In contAlu and LINE-1 families display differentially modulated patterns of expression ,11. OtheLINE-1 elements are transcribed by Pol II. They provide a source of small RNAs derived from bidirectional transcripts and endowed with regulatory functions . They acclinicaltrials.gov/ct2/show/NCT00964002?term=NCT00964002&rank=1). In contrast with the empirical efficacy of RT inhibitors, however, the molecular role of RT in cancer genomes is still elusive.Previous work in our laboratory showed that LINE-1-encoded RT activity is crucial for control of cell proliferation, differentiation and tumor progression end-labelled polyU (100 nt) and [3H] end-labelled poly dA:U were RNA and DNA:RNA hybrid density markers, respectively; densities of these latters were determined by mixing aliquots of gradient fractions with scintillation cocktail and counting in a Beckman scintillation counter. To determine the product sizes, fractions 8 (bulk) and 21 (hybrid) from control and EFV-treated A375 cells were digested with DNAse I or RNAse H/ DNAse I, labelled with alpha-32P dCTP using the Invitrogen Random Primers DNA Labeling System kit (18187-013) and fractionated through 1.7% agarose gels; the gels were then dried and exposed to autoradiographic films.Where indicated, fractions 8 and 21 collected from the gradient were digested with either DNAse I alone, or simultaneously with RNAseH/DNAse I and subjected to PCR amplification. Circular and Bam H1-linearized pCMV-EGFP plasmid DNA (Clontech) were used as DNA density markers while [www.repeatmasker.org], we first annotated from the human Reference sequence all REs flanking (+/\u2212 100 Kbp) each individual sequence of the three analyzed classes and non-modulated elements. Using RepeatMasker [Smit, Hubley and Green. RepeatMasker Open-3.0 1996-2010, sses see . Gene-resses see . Statistserpin-1, tachykinin (precursor 1), sox-11 and hmox1 genes using the 2-DDCt method [beta-actin. The following primers were designed using the Primer Express\u2122 software v2.0 (Applied Biosystems):serp1: Fw-CCCGGATCGTCTTTGAGAAG; Rev- TCCAGAGGTGCCACAAAGCTtac1: Fw-AATTACTGGTCCGACTGGTACGA; Rev- AAAGGGCTCCGGCAGTTCsox-11: FW-ACATGGTATTCTTGCCACTGGA; Rev- CCAAAATGCCATCAGAGTCTGTb-actin: Fw-GCCGGGACCTGACTGACTA; Rev- TGGTGATGACCTGGCCGThmox1 was analyzed using the PrimePCR\u2122 SYBR\u2122 Green Assay (BIORAD cat. 100-25636)Quantitative PCR was performed using a 7300 Real-Time PCR System (Applied Biosystems) and SYBR\u2122 Green JumpStart\u2122 Taq ReadyMix\u2122 (Sigma-Aldrich Co). 50 ng of cDNA from EFV-treated and control (DMSO-treated) (4 days) A-375 cells were used. The relative amounts of transcript were analyzed for t method and norm"} +{"text": "Chronic hypoxia is associated with remodeling in various organs. Reactive oxygen species (ROS) derived from NADPH oxidases (Nox), and TGF-beta1 have been implicated in the pathogenesis of hypoxia-induced remodeling. The aims of this study were to determine in hypoxia-stimulated nasal polyp-derived fibroblasts (NPDF) the effect of hypoxia on the differentiation of myofibroblasts, the role of ROS, the major Nox homolog mediating myofibroblast differentiation, and the role of TGF-beta1.alpha-SMA, Nox1, Nox3, Nox4, Nox5, and fibronectin mRNA was performed. Western blotting for alpha-SMA and fibronectin was done. ROS production was detected using a fluorometer. NPDF were pretreated with ROS scavengers and transfected with siNox4. The TGF-beta1 protein level was measured by ELISA. The effect of treatment with TGF-beta1 type I tyrosine kinase inhibitor SB431542 on myofibroblast differentiation was ob-served.Eight primary cultures of NPDF were established from nasal polyps, which were incubated under hypoxic conditions. Reverse transcription polymerase chain reaction for Hypoxic stimulation of NPDF significantly increased alpha-SMA and fibronectin mRNA and protein expression. ROS production was increased by hypoxia, and ROS scavengers inhibited myofibroblast differentiation. Nox4 mRNA was the only Nox homolog increased by hypoxia. Transfection with siNox4 inhibited myofibroblast differentiation. TGF-beta1 was secreted endogenously by hypoxic NPDF. SB431542 significantly inhibited myofibroblast differentiation.Hypoxia induces myofibroblast differentiation of NPDF through a signaling pathway involving Nox4-dependent ROS generation and TGF-beta1. Therapies targetingNox4 may be effective against remodeling of nasal polyps."} +{"text": "In vivo bioluminescence imaging (BLI) is a powerful method for the analysis of host-pathogen interactions in small animal models. The commercially available bioluminescent Listeria monocytogenes strain Xen32 is commonly used to analyse immune functions in knockout mice and pathomechanisms of listeriosis.L. monocytogenes strains EGDe-lux and murinised EGDe-mur-lux to characterise bacterial dissemination in orally inoculated BALB/cJ mice. After four days of infection, Xen32 and Xen32-mur infected mice displayed consistently higher rates of bioluminescence compared to EGDe-lux and EGDe-mur-lux infected animals. However, surprisingly both Xen32 strains showed attenuated virulence in orally infected BALB/c mice that correlated with lower bacterial burden in internal organs at day 5 post infection, smaller losses in body weights and increased survival compared to EGDe-lux or EGDe-mur-lux inoculated animals. The Xen32 strain was made bioluminescent by integration of a lux-kan transposon cassette into the listerial flaA locus. We show here that this integration results in Xen32 in a flaA frameshift mutation which makes this strain flagella deficient.To analyse and image listerial dissemination after oral infection we have generated a murinised Xen32 strain (Xen32-mur) which expresses a previously described mouse-adapted internalin A. This strain was used alongside the Xen32 wild type strain and the bioluminescent L. monocytogenes strain Xen32 is deficient in flagella expression and highly attenuated in orally infected BALB/c mice. As this listerial strain has been used in many BLI studies of murine listeriosis, it is important that the scientific community is aware of its reduced virulence in vivo.The bioluminescent For example, the now commercially available Listeria monocytogenes strain Xen32 was first used to demonstrate that the gallbladder is an important organ reservoir of listerial replication and pathogen shedding[Listeria directed immune mechanisms in knockout mice[L. monocytogenes dissemination to target organs of listeriosis such as the bone marrow[L. monocytogenes in fetal listeriosis[Bioluminescent shedding-3. Sinceout mice and kinee marrow. More reteriosis,7.L. monocytogenes strain Xen32 in an oral mouse listeriosis model to analyse the dissemination of the pathogen from the intestine to internal organs. To enable efficient transmission of this listerial strain through the murine gut mucosa we used our previous approach of murinisation to optimise the binding of the listerial surface protein internalin A (InlA) to the murine E-cadherin host receptor[The aim of this study was to use the bioluminescent receptor.Listeria monocytogenes strains EGDe-lux (Lmo-EGDe-lux) and L. monocytogenes EGDe-InlA-mur-lux (Lmo-EGDe-mur-lux) have been described previously[. L. monocytogenes Xen32 was purchased from Perkin Elmer and genetically modified for expression of a mouse-adapted InlA as previously described[L. monocytogenes strains, growth curves were performed as previously described[L. monocytogenes culture at indicated timepoints on the IVIS 200 imaging system . Genomic flaA fragments from L. monocytogenes Xen32 were amplified with flaA forward primer (5\u2032-AGAGAAGTCTTTTCTAAACCGAATGTAGGA-3\u2032) and flaA reverse primer (5\u2032-CTAAGGGTAAACAATGTTCGATAAATG-3\u2032), sequenced and analysed with MacVector 11.0.2 . For analysis of flagella expression, listerial strains were grown overnight in BHI medium at 24\u00b0C and negatively stained with 2% uranyl acetate and examined in a Zeiss TEM910 at 80\u00a0kV. Cell invasion assays with the human colorectal epithelial cell line Caco-is deficient in flagella expression 2 (ATCC HTB-37) and the murine colon carcinoma cell line CT26 (ATCC CRL-2639) were performed as previously described[For oral inoculation of female, 9-10\u00a0weeks old BALB/cJ mice we used our previously published mouse infection model[escribed. Analysiescribed. LuminesL. monocytogenes strain Xen32 was originally generated by screening a lux-kan transposon integration library of the parental strain L. monocytogenes strain 10403S for high levels of bioluminescence[in vivo evaluation of strong photon emission in infected BALB/c mice[inlA with inlAS192N-Y369S as previously described[L. monocytogenes strain was called Xen32-mur and the introduced mutations in the inlA locus validated by sequencing and Western blot analysis of InlA and internalin B protein expression (data not shown). All L. monocytogenes strains were profiled for in vitro growth in BHI media and emitted levels of luminescence. While no differences in in vitro growth rates were found between the listerial strains, both Xen32 strains showed higher levels of bioluminescence compared to the EGDe strains Xen32, Lmo-Xen32-mur, Lmo-EGDe-lux, or Lmo-EGDe-mur-lux and analysed by BLI for a time period of 9\u00a0days post infection (d.p.i.). After 4\u00a0days of infection, Lmo-Xen32 and Lmo-Xen32-mur infected mice displayed high levels of light emission in abdominal regions 80\u00a0bp downstream of the methionin start codon . However, the scientific community should be aware that infections with L. monocytogenes Xen32 of wild type, immunocompetent mouse strains might result in smaller effects on host responses due to its in vivo virulence attenuation.The bioluminescent The authors declare that they have no competing interests.L. monocytogenes strains. KS contributed to the study design and coordination of experiments. AL designed experiments, analysed data and drafted the manuscript. All authors read and approved the final manuscript.SB conducted all mouse infection experiments. MR did the transmission electron microscopy analysis of the different In vitro growth and luminescence profiles of Lmo-Xen32, Lmo-Xen32-mur, Lmo-EGDe-lux and Lmo-EGDe-mur-lux. Lmo-Xen32, Lmo-Xen32-mur, Lmo-EGDe-lux and Lmo-EGDe-mur-lux were grown in triplicates in BHI media and their growth curves and emitted levels of luminescence measured as described in Material and Methods. No major differences were detected in strain growth rates but Xen32 strains emitted higher levels of luminescence at indicated timepoints.Click here for file10\u00a0CFU Lmo-Xen32, Lmo-Xen32-mur, Lmo-EGDe-lux and Lmo-EGDe-mur-lux. BALB/cJ mice were intragastrically infected with 1\u2009\u00d7\u20091010\u00a0CFU Lmo-Xen32, Lmo-Xen32-mur, Lmo-EGDe-lux or Lmo-EGDe-mur-lux and the progress of infection was assessed by BLI for 9\u00a0days as described in Methods. Serial BLI data are shown for a set of 5 representative mice out of 10 for a time period of 9\u00a0days p.i.. Empty spaces indicate mice that succumbed to the infection or were euthanized for ethical reasons. The colour bar indicates photon emission with 3 or 4\u00a0min integration time in photons/s/cm2/sr.Bioluminescence Imaging of orally infected mice with 1\u2009\u00d7\u200910Click here for filelux-kan transposon integration site in the flaA locus of Listeria monocytogenes strain Xen32. Shown are the nucleotide and translated protein sequences. The insertion of the lux-kan transposon cassette results in a frameshift mutation with the generation of an amber stop codon (TAG) after 80\u00a0bp. Translated protein sequences of flaA are shown in blue, the frameshift protein translation is shown in red. The amber stop codon is underlined and depicted in bold red.Sequence of the Click here for fileU-test).Invasion and intracellular growth of Lmo-Xen32, Lmo-Xen32-mur, Lmo-EGDe-lux and Lmo-EGDe-mur-lux. Confluent layers of Caco2 and CT26 cells were infected for 60\u00a0min with Lmo-Xen32, Lmo-Xen32-mur, Lmo-EGDe-lux and Lmo-EGDe-mur-lux. Extracellular bacteria were killed by gentamycin treatment (100\u00a0\u03bcg/ml). At indicated timepoints cells were washed with PBS and lysed with sterile water containing 0,2% Triton X-100. Intracellular bacteria were quantified by plating serial dilutions of cell lysates on BHI agar plates. Graphs demonstrated mean CFU values of triplicate growth assays for each strain with standard error. Statistical significance between strains is indicated (*p\u2009<\u20090.05, ***p\u2009<\u20090.001, non-parametric Mann\u2013Whitney-Click here for file"} +{"text": "This approach implicated 43 genes in regulation of embryonic myogenesis, including a transcriptional repressor, the zinc-finger protein RP58 [We created a whole-mount in situ hybridization database, termed EMBRYS Knockout and knockdown approaches confirmed an essential role for RP58 in skeletal myogenesis. Cell-based high-throughput transfection screening revealed that RP58 is a direct MyoD target. Microarray analysis identified two inhibitors of skeletal myogenesis, Id2 and Id3, as targets for RP58-mediated repression. Consistently, MyoD-dependent activation of the myogenic program is impaired in RP58 null fibroblasts and downregulation of Id2 and Id3 rescues MyoD's ability to promote myogenesis in these cells.Our combined, multi-system approach reveals a MyoD-activated regulatory loop relying on RP58-mediated repression of muscle regulatory factor inhibitors. We applied our systems approaches to other locomotive tissues research including cartilage and tendon, and revealed novel molecular network regulating joint cartilage development and homeostasis via microRNA-140 ,3 and te"} +{"text": "This dates back to 1950; and, although there is at present no single antiviral drug specifically licensed for the chemotherapy or -prophylaxis of poxvirus infections, numerous candidate compounds have been described over the past 50 years. These compounds include interferon and inducers thereof ( Th. Thi.e.,inducers , carbocyinducers , 3\u2019-fluoinducers , (S)-HPMS)-HPMPA , EICAR describon range , but theon range , but whion range .supra) in immunocompetent mice, and in a lethal model for VV infection in SCID (severe combined immune deficiency) mice that had been infected with VV either intranasally, intraperitoneally or intravenously [S)-HPMPC], the latter proved more efficacious at a lower dose (25 mg/kg/day) in its protective activity against intranasal VV infection in SCID mice . From the beginning, it was immediately clear that (S)-HPMPA and especially (S)-HPMPC (because it seemed to be less toxic in mice) had therapeutic potential as antiviral drugs , and one)-HPMPC] prodrugs of cidofovir (HDP-CDV and ODE-CDV) were developed and molluscipoxviruses (such as molluscum contagiosum), which may both lead to serious infections in immunocompromised patients . eveloped , intentieveloped . Esterifeveloped , and thueveloped . In moleeveloped . This rein vitro ,82. Incrrus (CV) , or ectrrus (CV) .i.e., HDP and ODE) derivatives have also been prepared from (S)-HPMPA, the prototype of the acyclic nucleoside phosphonates }pyrimidine [(R)-HPMPO-DAPy] and 1-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]-5-azacytosine (HPMP-5-azaC) or the related acyclic nucleoside phosphonate analog (R)-HPMPO-DAPy, at 24 hours after lethal intratracheal infection of cynomolgus monkeys (Macaca fascicularis) with monkeypox virus. This study was actually planned after I had met Koert Stittelaar at a meeting in London . This study, published in 2006 in Nature [R)-HPMPO-DAPy at 24 hours after monkeypox virus infection resulted in significantly reduced mortality wanted to compare the effectiveness of post-exposure smallpox vaccination and antiviral treatment with either cidofovir [(n Nature clearly ortality . In cont"} +{"text": "PML-RARA-induced acute promyelocytic leukemia (APL) is a morphologically differentiated leukemia, many groups have speculated about whether its leukemic cell of origin is a committed myeloid precursor (e.g. a promyelocyte) versus an hematopoietic stem/progenitor cell (HSPC). We originally targeted PML-RARA expression with CTSG regulatory elements, based on the early observation that this gene was maximally expressed in cells with promyelocyte morphology. Here, we show that both Ctsg, and PML-RARA targeted to the Ctsg locus (in Ctsg-PML-RARA mice), are expressed in the purified KLS cells of these mice , and this expression results in biological effects in multi-lineage competitive repopulation assays. Further, we demonstrate the transcriptional consequences of PML-RARA expression in Ctsg-PML-RARA mice in early myeloid development in other myeloid progenitor compartments [common myeloid progenitors (CMPs) and granulocyte/monocyte progenitors (GMPs)], which have a distinct gene expression signature compared to wild-type (WT) mice. Although PML-RARA is indeed expressed at high levels in the promyelocytes of Ctsg-PML-RARA mice and alters the transcriptional signature of these cells, it does not induce their self-renewal. In sum, these results demonstrate that in the Ctsg-PML-RARA mouse model of APL, PML-RARA is expressed in and affects the function of multipotent progenitor cells. Finally, since PML/Pml is normally expressed in the HSPCs of both humans and mice, and since some human APL samples contain TCR rearrangements and express T lineage genes, we suggest that the very early hematopoietic expression of PML-RARA in this mouse model may closely mimic the physiologic expression pattern of PML-RARA in human APL patients.Because PML-RARA is produced by t, and is found only in the hematopoietic cells of patients with acute promyelocytic leukemia (APL). When PML-RARA is expressed in mice using regulatory elements from the human or mouse cathepsin G gene (CTSG/Ctsg) or the human S100A8 (MRP8) promoter/enhancer, it can initiate APL; when RARA or PML-RARA are expressed in mouse bone marrow cells via retroviral transduction, both can decrease myeloid maturation and increase self-renewal PML-RARA expression to myeloid-restricted cells, we and others have suggested that myeloid-restricted disease might result from targeted expression of PML-RARA to the promyelocyte compartment PML and murine Pml are expressed in early CD34+ hematopoietic progenitor cells, and human PML-RARA expression may not be limited to committed myeloid progenitors and promyelocytes The fusion gene PML-RARA expression in flow-sorted CD34+/CD38\u2212 cells , but did detect PML-RARA expression in CD34+/CD38+ cells from two APL patients, using semi-quantitative RT-PCR +/CD38\u2212 AML cells into NOD/SCID mice, but no engraftment of similarly sorted CD34+/CD38\u2212 cells from APL patients, suggesting that these were not the initiating cells for this subtype of AML PML-RARA under the control of Ctsg or MRP-8 regulatory elements has led to myeloid leukemia et al. recently reported expression of T-lineage transcripts and TCR rearrangements in 60% of hypogranular t APL cases, suggesting the translocation may affect HSPCs with the capacity to differentiate into both myeloid and lymphoid lineages PML-RARA may initiate disease (in human patients) within a multipotent progenitor compartment Several studies have suggested that in APL, the leukemic cell of origin must be a committed myeloid progenitor Ctsg and PML-RARA during early hematopoietic development in Ctsg-PML-RARA mice. We found that Ctsg mRNA is expressed not only in the KLS (Kit+/Lin\u2212/Sca+) compartment, but also in SLAM cells (CD150+/CD41\u2212/CD48\u2212 KLS), which are even more primitive. We observed striking changes in the gene expression profile of flow-sorted common myeloid progenitors (CMPs) and granulocyte/monocyte progenitors (GMPs) derived from Ctsg-PML-RARA mice, which suggests that PML-RARA has important transcriptional consequences in early myeloid progenitor cells. We extend these findings with functional validation of PML-RARA effects on lymphoid and erythroid lineages, which confirm that PML-RARA is expressed at a very early stage in the hematopoietic development of Ctsg-PML-RARA mice. We observed that in flow-sorted Ctsg-PML-RARA promyelocytes, PML-RARA expression does result in significant gene expression changes but does not result in distinct gene expression signature, nor does it promote self-renewal. These results change our understanding of the cellular compartments in which PML-RARA initiates leukemia in the Ctsg-PML-RARA mouse model of APL.In this study, we use state-of-the-art flow-sorting, mRNA amplification, and expression profiling strategies to carefully define the timing of activation of +Lin\u2212Sca+CD150+CD41\u2212CD48\u2212), KLS cells (cKit+Lin\u2212Sca+), CMPs (Lin\u2212Sca-1\u2212cKit+CD34+Fc\u03b3RII/IIIlo), GMPs (Lin\u2212Sca-1\u2212cKit+CD34+Fc\u03b3RII/IIIhi), megakaryocyte-erythrocyte progenitors , \u201cpromyelocytes\"/early myeloid cells (Ly6gintSCCintB220\u2212CD115\u2212Ter119\u2212), and neutrophils (Ly6g+SCChighB220\u2212CD115\u2212Ter119\u2212) from 2\u20136 individual Ctsg-PML-RARA or WT mice . Ctsg expression increases massively in the CMP compartment; the high level of expression persists in the GMP compartment and in promyelocytes, and declines in neutrophils. Ctsg is minimally expressed in the MEP compartment.In our mouse model of APL, ARA mice . Ctsg exCtsg activation, we found that Ctsg mRNA could be detected not only in the KLS compartment but also in SLAM cells expression was limited to promyelocytes and neutrophils, and that Kit and Flt3 expression declined during myeloid maturation, as expected . Promyelocytes had relatively low but detectable expression of Cd34 with an average expression intensity of 785.912\u00b172.575 in Ctsg-PML-RARA promyelocytes (n\u200a=\u200a3) and 467.255\u00b1189.475 in WT promyelocytes (n\u200a=\u200a6) with much higher Cd34 expression in the earlier myeloid compartments, CMPs and GMPs, as expected , elastase (Ela2), and proteinase3 (Prtn3) are expressed early in myeloid development in primary granules; lactoferrin (Ltf) is expressed later in secondary granules; and matrix metallopeptidase 9 (Mmp9) found in tertiary granules is a marker of mature neutrophils (Fpr1) and lysozyme-2 (Lyz2) are appropriately expressed in our sorted cell populations 5\u2032 flanking region) resulted in myeloid leukemia, while two others (using Fes and Itgam (CD11b) promoters) did not Fes and Itgam are both expressed at much lower levels in KLS cells than Ctsg, with expression peaking in neutrophils, rather than promyelocytes samples segregated by genotype in an unsupervised clustering analysis, suggesting that expression of PML-RARA within the KLS compartment significantly alters the expression of a specific set of genes analysis to define significantly dysregulated genes in each subset (Notch1 (Cadherin 1 (Cdh1), a tumor suppressor in epithelial cancers, which was strikingly up-regulated in both the CMP and GMP compartments of Ctsg-PML-RARA mice (cyclin H (Ccnh) expression is down-regulated in the CMP compartment of Ctsg-PML-RARA mice in both the CMP and GMP ANOVA results (the supervised heatmap was created by plotting the relative expression of each of these genes to each other) . InteresPML-RARA in Ctsg-PML-RARA mice has clear transcriptional consequences that occur early in myeloid development and are specific for the CMP and GMP cell populations. These transcriptional changes, in turn, may influence the gene expression profile of more mature myeloid cells, just as gene expression changes driven by the presence of PML-RARA expression in earlier hematopoietic precursors may shape the distinct expression signature seen in the CMP and GMP compartments.To further validate the changes in gene expression in the CMP and GMP compartments, we performed supervised clustering analyses of all the significantly dysregulated genes by ANOVA from the GMP and CMP compartments individually . As expected, the clustering with genes dysregulated in either the CMP or GMP compartment segregated both the GMP and CMP populations, but not the MEP populations, by genotype . Thus, tCtsg-PML-RARA bone marrow cells after competitive transplantation. We transplanted total bone marrow from healthy 6-week-old (i.e. non-leukemic) Ctsg-PML-RARA mice (CD45.2+) at ratios of 1\u22369, 1\u22361, and 9\u22361 with competitor Ly5.1/Ly5.2 bone marrow (CD45.1+/CD45.2+) into Ly5.1 recipients (CD45.1+). Peripheral blood was assessed at 6 weeks, 3 months, and 6 months post-transplant. Four mice developed leukemia between 3 and 6 months, and could not be analyzed further. As expected, we noted a consistent expansion of Ctsg-PML-RARA+ cells within the Ly6g+ (myeloid) compartment at all time points tested revealed normal hemoglobin levels and red cell size in all mice PML-RARA is inserted into the murine Ctsg locus, it begins to be expressed in KLS cells, where it alters myeloid, lymphoid, and erythroid hematopoiesis . It is massively upregulated in CMP and GMP cells, where it significantly alters the expression of a number of genes. In contrast, PML-RARA expression in promyelocytes is not associated with a striking change in gene expression or inappropriate self-renewal. The studies described here define the hematopoietic compartments that are initially exposed to (and perturbed by) PML-RARA in healthy, pre-leukemic Ctsg-PML-RARA mice; the immunophenotype and cytomorphology of leukemia initiating cells within the subsequent leukemias have been defined elsewhere, and are not addressed in this analysis The fusion protein CTSG, Ctsg, and S100A8 loci, which were thought to target PML-RARA to the promyelocyte compartment Ctsg-PML-RARA mice, PML-RARA is consistently expressed in cells within the KLS compartment, PML-RARA expression in mice: PML-RARA directed from the CTSG, Ctsg and S100A8 (MRP8) loci cause APL; expression directed by regulatory elements from the cFes and Itgam (CD11b) loci do not Ctsg, CTSG, S100a8, and S100A8 are all expressed in multipotent progenitor compartments, while cFes and Itgam display low to absent levels of expression in KLS cells, with maximal expression in neutrophils. This suggests that APL development may require PML-RARA expression in an early hematopoietic compartment, and that induction of expression at the promyelocyte stage is insufficient to initiate leukemia, perhaps because PML-RARA cannot reverse the commitment of terminally differentiated neutrophils to die.One of the important arguments used to support a committed progenitor as the leukemic cell of origin has been the successful generation of leukemia using PML-RARA and MLL fusion transcripts using viral transduction PML-RARA, which is variable among cell types Other authors have evaluated the cell-specific effects of +CD34+Fc\u03b3RII/III+Ly6gint) as the leukemia-initiating cell in the hMRP8-PML-RARA transgenic mouse model of APL. Here we show the transcriptional and functional consequences of PML-RARA expression in Ctsg-PML-RARA pre-leukemic cells. These findings are not mutually exclusive, since the hematopoietic compartment susceptible to PML-RARA transformation may not be at the same developmental stage as the resultant leukemia Importantly, this work addresses a different question than the work of Guibal et al., which defined the immunophenotype of a committed myeloid progenitor in sub-lethally irradiated recipient B6 mice. However, this strategy does not determine whether the engrafted cells are myeloid-restricted (i.e. derived from promyelocytes) or whether they are multi-lineage (i.e. arising from stem/progenitor cells that contaminated the purified \u201cpromyelocytes\"). In contrast, we used Ly5.1 mice as the recipients for our purified promyelocyte populations, and found that even large numbers of promyelocytes were insufficient to lead to long-term engraftment of myeloid restricted cells. Our data therefore do not support the idea that the promyelocytes from Ctsg-PML-RARA mice have altered self-renewal properties that contribute to the expansion of these cells in vivo.Our work clarifies and expands the findings of Wojiski et al. There are no standard immunophenotypic markers for defining murine promyelocytes, and our flow-sorting strategy for promyelocytes was different than that used by Wojiski et al.; however, we extensively validated our approach using both expression data from genes known to be developmentally-regulated during myeloid development as well as traditional cytomorphology, in which a trained hematopathologist performed blinded differentials [8], [34Ctsg-PML-RARA mice, PML-RARA is first expressed in very early hematopoietic stem/[progenitor cells (KLS cells), and that its expression continues throughout myeloid development . Biological effects of PML-RARA can be seen in pre-leukemic KLS cells, CMPs and GMPs. Thus, in this model system, it is highly likely that PML-RARA initiates leukemia in very early hematopoietic cells, and not in promyelocytes, as originally predicted. Although we cannot formally exclude the possibility that pre-leukemic promyelocytes acquire self-renewal properties, it seems very unlikely, based on recent data that some human APL cells have TCR rearrangements, and express T lineage genes Ctsg-PML-RARA mouse model may recapitulate the physiologic timing of PML-RARA activation in very primitive human HPSCs that are capable of giving rise to both lymphoid and myeloid cells.Nonetheless, our data demonstrate that in healthy, pre-leukemic PML-RARA does not appear to alter normal hematopoietic homeostatic feedback or cause increasing cell numbers within an immunophenotypically-defined multipotent progenitor compartment Ctsg-PML-RARA mice. Since Ctsg-PML-RARA mice virtually never develop lymphocytic leukemia or erythroleukemia, our data suggests that the myeloid restriction of leukemia must be explained by myeloid-restricted genes or proteins that cooperate with PML-RARA in leukemic cells (ic cells . AlthougPML-RARA from the murine Ctsg locus (Ctsg-PML-RARA) and Ctsg deficient mice (CG-KO) have been previously reported Ctsg-PML-RARA transgenic mice used in this study were backcrossed more than 12 generations into the C57Bl/6 Taconic background Mice expressing 4Cl, 10 mM KHC03, 0.1 mM Na2EDTA) on ice for 10 minutes. Non-specific staining was blocked with Miltenyi FcR Blocking Reagent for mouse . Isotype-matched antibodies were used as negative controls and the Fluorescence Minus One (FMO) strategy was used to set appropriate gates. Flow sorting was performed on a Reflection high-speed cell sorter . Cells were sorted directly into Trizol . Cells were stained by standard protocols with the following antibodies (eBioscience unless otherwise noted): KLS (c-Kit+/lineage negative/Sca-1+) cells were assessed using the following lineage markers: FITC-conjugated \u03b1Gr-1, \u03b1Ter119, \u03b1CD3, \u03b1CD4, \u03b1CD8, \u03b1B220, \u03b1CD19, and \u03b1CD127; APC-\u03b1c-Kit, and PE-\u03b1Sca-1. CMPs (Lin\u2212Sca-1\u2212cKit+CD34+Fc\u03b3RII/IIIlo), GMPs (Lin\u2212Sca-1\u2212cKit+CD34+Fc\u03b3RII/IIIhi), megakaryocyte-erythrocyte progenitors were done with the same antibody cocktail as KLS with addition of APC-conjugated \u03b1Fc\u03b3RII/III (clone 93). SLAM (CD150+/CD41\u2212/CD48\u2212 KLS) cells were assessed using KLS staining as above, except for PerCP-Cy5.5\u2013conjugated \u03b1Sca-1, with the addition of FITC-\u03b1CD41, FITC-\u03b1CD48 and PE-\u03b1CD150 antibodies. Promyelocyte (Gr1intSCCintB220\u2212CD115\u2212Ter119\u2212) and neutrophil (Gr1+SCChighB220\u2212CD115\u2212Ter119\u2212) sorting was done using APC-\u03b1Gr-1, PE-\u03b1CD115, FITC-\u03b1B220 and PE-Cy7-\u03b1Ter119. Additional cells from both the promyelocyte and neutrophils fraction were sorted into FACS buffer for morphological validation of cytospins. Samples analyzed in expression array profiling were generated during five separate flow-sorting experiments to limit technical bias. 15 mouse Ctsg-PML-RARA leukemia samples were prepared as previously described Bone marrow cells from individual mice were harvested from both femurs and tibia of 6-week-old and 13-week-old mice and prepared previously described http://pathology.wustl.edu/research/cores/lcg/index.php). Affymetrix Expression Console software was used to process array images, export signal data, and evaluate image and data quality relative to standard Affymetrix quality control metrics. Exon array data for all samples used in this study have been deposited on GEO .For expression array profiling, total cellular RNA was purified using TRIzol reagent (Invitrogen), quantified using UV spectroscopy (Nanodrop Technologies), and qualitatively assessed using an Experion Bioanalyzer. Amplified cDNA was prepared from 20 ng total RNA using the whole transcript WT-Ovation RNA Amplification System and biotin-labeled using the Encore Biotin Module, both from NuGen Technologies, according to the manufacturer's instructions. Labeled targets were then hybridized to Mouse Exon 1.0 ST arrays (Affymetrix), washed, stained, and scanned using standard protocols from the Siteman Cancer Center, Molecular and Genomic Analysis Core Facility . For PML-RARA qRT-PCR, we also used the Qiagen RT-PCR kit with the following primers: Forward TCTTCCTGCCCAACAGCAA, Reverse GCTTGTAGATGCGGGGTAGAG. We used GAPDH as the PCR control with the following primers: Forward TGCACCACCAACTGCTTAG, Reverse GGATGCAGGGATGATGTTC.For Cells used for competitive repopulation studies were injected retroorbitally, as previously described 6 total bone marrow cells, 3,000 KLS cells or 50,000 promyelocytes/mouse. At least 20,000 events and 40,000 events (bone marrow) per recipient mouse were collected for analysis.Determination of engraftment of flow sorted bone marrow cells is described in Ctsg-PML-RARA mice and littermate controls were collected and plated (in duplicate) in 1.1 ml of methylcellulose medium at 41.5\u00d7103 cells/ml or 8.3\u00d7103/ml (MethoCult 3434) Bone marrow cells from 6-week-old and 8-week-old, healthy Ly6g, whose 2 probesets are within the extended probesets group. Probesets having an expression signal less than 200 in all samples were removed and an unsupervised hierarchical clustering analysis was performed using z-score normalized expression values in SPOTFIRE or with the Partek Genomics Suite with the expression of each gene standardized to a mean of 0 and standard deviation of 1 and using the Pearson dissimilarity as a distance measure. http://www-stat.stanford.edu/~tibs/SAM/, Palo Alto, CA) as an Excel add-in performing two-class unpaired analyses with unlogged data, 100 permutations, standard regression and fold change \u22652 with false discovery rates and q-values noted in the text. Competitive repopulation analysis: a one sample, two-tailed t-test with alpha set at 0.05 compared outcomes with expected values of 10%, 50%, and 90% . CFU-E analysis was done with a paired t-test .Expression array analysis: exon-level summary was generated using the RMA algorithm in the Affymetrix Expression Console . Only core probesets were used in order to limit the analysis within well-annotated exons with the exception of Figure S1Murine promyelocyte and neutrophil flow sorting strategy. A. Total bone marrow cells were labeled with B220, Ter119, CD115 and Gr1 and early myeloid cells/promyelocytes (Pros) and neutrophils (PMNs) were identified as B220\u2212, Ter119\u2212, CD115\u2212 cells that are either BSCintGr1int (Pros), or BSChighGr1high (PMNs). B. Results of 200 cell differential counts by a blinded hematopathologist from 4 Pros (2 WT and 2 Ctsg-PML-RARA) and 5 PMNs (3 WT and 2 Ctsg-PML-RARA) separate sorted samples. C. Representative cytomorphology of WT and Ctsg-PML-RARA (labeled PR) Pros samples counted in B . D. Representative cytomorphology of WT and Ctsg-PML-RARA (labeled PR) PMNs samples counted in B .(EPS)Click here for additional data file.Figure S2Validation of Ctsg and PML-RARA expression using quantitative reverse-transcriptase PCR. A. Ctsg expression was validated with quantitative RT-PCR normalized to Gapdh. B. Quantitative RT-PCR using PML-RARA specific primers normalized to Gapdh in the indicated mice and Nugen amplified mRNA. C. Agarose gel of PCR products from Panel B. PML-RARA expected size is 145 bp. Markers are 100 and 200 base-pairs.(EPS)Click here for additional data file.Figure S3Expression of Ly6g, Kit, Flt3 and Cd34 in flow-sorted bone marrow cells and mouse leukemia samples. Expression profile in indicated WT and Ctsg-PML-RARA (labeled mCG-PR) flow-sorted bone marrow cells and 15 Ctsg-PML-RARA leukemia samples (labeled Mouse APL) using Nugen amplified mRNA and Affymetrix Mouse Exon 1.0ST arrays. We plotted Ly6g, Kit, Flt3 and Cd34 expression using representative probesets.(EPS)Click here for additional data file.Figure S4Expression of 7 developmentally-regulated myeloid genes in flow-sorted bone marrow cells. Expression profile in indicated WT and Ctsg-PML-RARA (labeled mCG-PR) flow-sorted bone marrow cells and 15 Ctsg-PML-RARA leukemia samples (labeled Mouse APL) using Nugen amplified mRNA and Affymetrix Mouse Exon 1.0ST arrays. We plotted Elane, Prtn3, Mpo, Ltf, Mmp9, Lyz2, and Fpr1 expression using representative probesets.(EPS)Click here for additional data file.Figure S5Expression profile of flow-sorted Ctsg-PML-RARA and WT bone marrow cells by unsupervised clustering analyses. Bone marrow cells were flow sorted as described in (EPS)Click here for additional data file.Figure S6Unsupervised heatmap clustering analyses for WT vs. Ctsg-PML-RARA CMP, GMP and MEP samples. Bone marrow cells were flow sorted as described in the Ctsg-PML-RARA samples. B. Unsupervised heatmap clustering analyses for GMP WT vs. Ctsg-PML-RARA samples. C. Unsupervised heatmap clustering analyses for MEP WT vs. Ctsg-PML-RARA samples.(EPS)Click here for additional data file.Figure S7Individual supervised clustering analyses for the significantly dysregulated genes by ANOVA from the GMP and CMP comparisons between WT and Ctsg-PML-RARA samples. These heatmaps were created by traditional z-score scaling using SPOTFIRE. The clustering with genes dysregulated in either the CMP or GMP compartment by ANOVA segregated both the GMP and CMP populations, but not the MEP populations, by genotype. The legend is shown below the heatmaps with downregulated genes in green and upregulated genes in red. A. Supervised clustering heatmap using genes dysregulated by ANOVA comparison between WT and Ctsg-PML-RARA CMP populations. B. Supervised clustering heatmap using genes dysregulated by ANOVA comparison between WT and Ctsg-PML-RARA GMP populations.(EPS)Click here for additional data file.Figure S8Erythroid colony formation (CFU-E) in Ctsg-PML-RARA vs. WT mice. Bone marrow cells from 6-week-old and 8-week-old, healthy mice were plated in methylcellulose containing erythropoietin (Methocult 3334). Results of paired t-tests are shown in each panel. A. Immunophenotypes of cells in these colonies were assessed as indicated: A. Ter119+CD71high (mature erythrocytes) and B. CD11b+ (myelomonocytic/monocytic cells). C. and D. Hemoglobin and mean corpuscular volume (MCV) values were determined in a cohort of healthy 4-month-old mice with the indicated genotypes.(EPS)Click here for additional data file.Figure S9Examples of bone marrow immunophenotypes following transplantation of flow sorted KLS and promyelocytes. Bone marrow KLS and promyelocytes were sorted from littermate Ctsg-PML-RARA (mCG-PR) and WT mice and transplanted into sub-lethally irradiated Ly5.1 recipients (cipients . Twelve (EPS)Click here for additional data file.Data S1Comparative gene expression analysis between cell populations derived from Ctsg-PML-RARA versus WT mice. The supplementary data includes a summary of gene expression differences for comparisons between Ctsg-PML-RARA and WT SLAM, KLS, CMP, GMP, MEP and promyelocyte cell populations. The analysis includes a comparison of dysregulated genes to previously identified PML-RARA binding sites and to known dysregulated mRNA abundance identified in primary human APL samples (DOCX)Click here for additional data file.Table S1Ctsg-PML-RARA versus WT SLAM ANOVA Results.(XLSX)Click here for additional data file.Table S2Ctsg-PML-RARA versus WT KLS ANOVA Results.(XLSX)Click here for additional data file.Table S3Ctsg-PML-RARA versus WT CMP ANOVA Results.(XLSX)Click here for additional data file.Table S4Ctsg-PML-RARA versus WT GMP ANOVA Results.(XLSX)Click here for additional data file.Table S5Ctsg-PML-RARA versus WT MEP ANOVA Results.(XLSX)Click here for additional data file.Table S6Ctsg-PML-RARA versus WT Promyelocyte ANOVA Results.(XLSX)Click here for additional data file.Table S7Concordant Dysregulated Genes in both the CMP and GMP Compartments by ANOVA.(XLSX)Click here for additional data file.Table S8Ctsg-PML-RARA versus WT SLAM SAM Results.(XLSX)Click here for additional data file.Table S9Ctsg-PML-RARA versus WT KLS SAM Results.(XLSX)Click here for additional data file.Table S10Ctsg-PML-RARA versus WT CMP SAM Results.(XLSX)Click here for additional data file.Table S11Ctsg-PML-RARA versus WT GMP SAM Results.(XLSX)Click here for additional data file.Table S12Ctsg-PML-RARA versus WT MEP SAM Results.(XLSX)Click here for additional data file.Table S13Ctsg-PML-RARA versus WT Promyelocyte SAM Results.(XLSX)Click here for additional data file."} +{"text": "The identification of optimal antigen(s) and adjuvant combination(s) to elicit potent, protective, and long-lasting immunity has been a major challenge for the development of effective vaccines against HIV-1.Here, we designed disulfide-stabilized recombinant HIV-1 subtype B (SF162) envelope glycoproteins (Env), gp120 and gp140, by insertion of site-specific cysteine pairs between two layers (layer 1 and 2) in inner domain of gp120. In addition, we identified a novel adjuvant approach using Carbopol 971P, a cross-linked polyanionic carbomer, in combination with the Novartis proprietary oil-in water adjuvant, MF59, to augment humoral immune responses to the Env glycoprotein. We performed thorough in vitro analysis of the disulfide-stabilized Env glycoprotein followed by in vivo evaluations of the adjuvanted-Env glycoprotein boost in rabbits.Intramuscular immunization of rabbits with disulfide-stabilized Env glycoproteins formulated in Carbopol 971P plus MF59 gave significantly higher titers of binding and virus neutralizing antibodies as compared to immunization using Env glycoprotein with either MF59 or Carbopol 971P alone. In addition, the antibodies generated were of higher avidity. Mapping of serum antibodies to determine epitope specificities showed that the disulfide-stabilized gp140 proteins elicited broader Env glycoprotein-specific antibody responses directed against epitopes that included the CD4-binding site, CD4-induced site and V1V2-loop. Importantly, the use of the novel adjuvant, Carbopol plus MF59, did not appear to present any obvious tolerability issues in animals upon intramuscular administration.Hence, the use of rationally stabilized Env-antigens in potent Carbopol 971P plus MF59 adjuvant may provide a benefit for evaluations of future vaccine against HIV-1."} +{"text": "A determinant of HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) development is HTLV-1-infected cell burden. Viral proteins Tax and HBZ, encoded by the positive and negative strands of the pX region respectively, play a key role in HTLV-1 persistence. Tax drives CD4+ T-cell clonal expansion but is the immunodominant antigen. Valproate (VPA), an histone deacetylase inhibitor, has been proposed to trigger Tax expression and expose latent HTLV-1 reservoir to immune destruction. We evaluated the impact of VPA treatment on Tax, Gag and HBZ expression in cultured lymphocytes from asymptomatic HTLV-1 carriers and HAM/TSP patients. Around one-fifth of provirus-positive CD4+ T cells spontaneously became Tax-positive. The estimation rose up to two-thirds of Tax-positive infected cells when VPA was added. VPA enhanced Gag p19 protein release. Tax and Gag mRNA levels spontaneously peaked, before a decline concomitant to HBZ mRNA increase. VPA treatment enhanced and prolonged Tax mRNA expression, while it blocked HBZ expression. This is the first ex vivo study on the balance between Tax and HBZ expression (i.e. sense and antisense transcription). Our data suggest that besides modulation of the expression of Tax, another mechanism involving repression of HBZ may determine the outcome of VPA treatment on HTLV-1-infected cell proliferation and survival."} +{"text": "H but more importantly it can hamper the activation of immune cells which is a hallmark of stable ICs. The natural role of ICs is enhancing uptake by DCs, DC activation, induction of antigen presentation and induction of T cell responses. Furthermore, ICs are captured by follicular DCs that activate the B cells for Ab production, Ab affinity maturation and \u00edsotype switching. We explore stable Env-ICs as a vaccine candidate. To form stable Env-ICs we fused the Fc-region of immunoglobulins to trimeric gp140. Env-IC maintained a native Env conformation which was evaluated by ELISA with Env-specific Abs. Native PAGE analyses and size exclusion chromatography showed that Env-ICs formed trimers, but hexamers consisting of 2 Env trimers and 3 dimeric Fc-tails were also observed. The functionality of the Fc-tail was evaluated by immuno-precipitation of the Env-IC with protein-G couple beads. Capture of Env-IC by DCs was enhanced with 50% compared to wild-type Env. Moreover, Env-IC captured by DCs more efficiently activated gp120-specificT helper cells.The development of an HIV-1 vaccine that elicits strong neutralizing antibody (nAb) and T cell responses is challenging. Classical vaccine strategies such as live attenuated vaccines are considered unsafe whereas envelope glycoprotein (Env)subunit vaccines induce low nAb titers that do not protect against HIV-1 infection. We showed previously that most HIV-1-antibody immune complexes (HIV-ICs) formed with either broadly nAbs or Abs derived from patient sera dissociate into free HIV-1 virions and Ab when captured by dendritic cells (DCs). Dissociation of HIV-ICs allows for transmission from DCs to CD4"} +{"text": "To compare left atrial (LA) late gadolinium enhancement (LGE) imaging using bellows- and NAV-gating.The LGE method can visualize post-ablation scar in the LA, using increased spatial resolution (1-2). However, the inversion pulse required for LGE also nulls the liver signal at the time of data acquisition , so that NAV-gating, used for respiratory compensation, becomes impossible. The NAV-restore (3), which reinverts the region of the liver immediately after the initial inversion pulse, permits gating. However, pulmonary vein (PV) blood magnetization is also reinverted, and flows into the left atrium, generating inflow artifacts. These artifacts have been addressed by 1) a NAV with a smaller diameter, 2) monitoring applied earlier before data acquisition (4), or directly after data acquisition (5), when the liver magnetization will be non-zero due to a shorter or longer effective TI (with no NAV restore needed). However, optimal NAV-gating demands monitoring coincident with acquisition of central k-space. Bellows-gating is an early respiratory compensation method, with recent data suggesting a strong correlation with the superior-inferior diaphragmatic motion (6-9). Here we present the results of bellows-gated LGE.Nine healthy subjects were imaged in random order 15-25 minutes post contrast with identical 3D LGE sequences, using NAV-gating and commercially provided bellows-gating. The LA was imaged using an axial (N=6), or LV short-axis (N=3) orientation with parameters as previously described (1). Image sharpness and ghosting were evaluated on scale of 1-4 ; the appearance of inflow artifacts in the right PVs were noted .Figure For 3D LGE LA imaging bellows-gating can provide similar respiratory compensation as the NAV, without inflow artifacts."} +{"text": "The wavefront phenomenon describes the transmural progression of myocardial infarction (MI) from endocardium to epicardium with increasing ischemia duration. A corollary is once subendocardial MI has developed, the infarct lateral border (InfarctLatBor) delineates the Area-at-risk (AAR) lateral border, and thus, can be used to measure overall AAR size. However, with short ischemia time a confluent subendocardial layer of infarction may not develop, and InfarctLatBor may underestimate AAR size. The transmural extent of infarction necessary for InfarctLatBor to accurately reflect AAR size is unknown.In-vivo assessment of InfarctLatBor with delayed-enhancement-CMR (DE-CMR) has been compared with surrogates of the AAR . However, no comparison exists with a pathology-based truth standard of the AAR (i.e microspheres). We sought to examine: (1) on pathology studies, the threshold of infarct transmurality necessary for the InfarctLatBor to accurately delineate the AAR, and (2) the ability of in-vivo DE-CMR (via InfarctlatBor assessment) to quantify the AAR in comparison with pathology.PATH). After TTC-staining the infarct lateral border was used to estimate AAR size (InfarctLabBorPATH).In 15 canines, MI with various infarct transmuralities was produced by temporary occlusion (50-120mins) of the LAD or LCx. A complete LV short-axis stack of DE-CMR images were obtained following gadoversetamide administration (0.2mmol/kg). Prior to sacrifice, the infarct-related-artery was reoccluded at the same site (same suture) and microspheres were injected into the left atrium to determine AAR size , though correlation was excellent (r=0.994). In slices with mean infarct transmurality <10% InfarctlatBorPATH underestimated AARPATH, whereas no systematic under- or overestimation occurred when infarct transmurality was >10% , and again the correlation was excellent (r=0.979). The greatest underestimation (-8.4%ofLV) was found in the subject with lowest mean infarct transmurality (11%) and highest number of slices (N=4) with infarct transmurality <10%. Excluding this subject, the maximum bias was lower than -4%ofLV for all other subjects.Comparing pathology-based measurements per-slice N=114), InfarctLatBor14, InfarThe lateral border of infarction allows for precise quantification of true AAR size unless a subendocardial layer of infarction less than 10% transmural is present. In-vivo DE-CMR assessment of the infarct lateral border can be used to accurately estimate AAR size, however, underestimation may occur if mean infarct transmurality is near 10%.This study was funded in part by following NIH-grant: 5R01HL064726-07."} +{"text": "Bovine leukemia virus (BLV) proviral latency represents a viral strategy to escape the host immune system and allow tumor development. Besides the previously demonstrated role of histone deacetylation in the epigenetic repression of BLV expression, we showed here that BLV promoter activity was induced by several DNA methylation inhibitors (such as 5-aza-2'-deoxycytidine) and that overexpressed DNMT1 and DNMT3A, but not DNMT3B, down-regulated BLV promoter activity. Importantly, cytosine hypermethylation in the 5'-long terminal repeat (LTR) U3 and R regions was associated with true latency in the lymphoma-derived B-cell line L267 but not with defective latency in YR2 cells. Moreover, the virus-encoded transactivator Tax(BLV) decreased DNA methyltransferase expression levels, which could explain the lower level of cytosine methylation observed in the L267(LTaxSN) 5'-LTR compared with the L267 5'-LTR. Interestingly, DNA methylation inhibitors and Tax(BLV) synergistically activated BLV promoter transcriptional activity in a cAMP-responsive element (CRE)-dependent manner. Mechanistically, methylation at the -154 or -129 CpG position (relative to the transcription start site) impaired in vitro binding of CRE-binding protein (CREB) transcription factors to their respective CRE sites. Methylation at -129 CpG alone was sufficient to decrease BLV promoter-driven reporter gene expression by 2-fold. We demonstrated in vivo the recruitment of CREB/CRE modulator (CREM) and to a lesser extent activating transcription factor-1 (ATF-1) to the hypomethylated CRE region of the YR2 5'-LTR, whereas we detected no CREB/CREM/ATF recruitment to the hypermethylated corresponding region in the L267 cells. Altogether, these findings suggest that site-specific DNA methylation of the BLV promoter represses viral transcription by directly inhibiting transcription factor binding, thereby contributing to true proviral latency."} +{"text": "The pool of transcriptionally silent proviruses is established early in infection and persists for a lifetime, even when viral loads are suppressed below detection levels using anti-retroviral therapy. Upon therapy interruption the reservoir can re-establish systemic infection. Different cellular reservoirs that harbor latent provirus have been described. In this study we demonstrate that HIV-1 can also establish a silent integration in actively proliferating primary T lymphocytes. Co-culturing of these proliferating T lymphocytes with dendritic cells (DCs) activated the provirus from latency. Activation did not involve DC-mediated C-type lectin DC-SIGN signaling or TCR-stimulation but was mediated by DC-secreted component(s) and cell-cell interaction between DC and T lymphocyte that could be inhibited by blocking ICAM-1 dependent adhesion. These results imply that circulating DCs could purge HIV-1 from latency and re-initiate virus replication. Moreover, our data show that viral latency can be established early after infection and supports the idea that actively proliferating T lymphocytes with an effector phenotype contribute to the latent viral reservoir. Unraveling this physiologically relevant purging mechanism could provide useful information"} +{"text": "Radix Angelica Sinensis, the dried root of Angelica sinensis (Danggui), is a herb used in Chinese medicine to enrich blood, promote blood circulation and modulate the immune system. It is also used to treat chronic constipation of the elderly and debilitated as well as menstrual disorders. Research has demonstrated that Danggui and its active ingredients, as anti-arthrosclerotic, anti-hypertensive, antioxidant anti-inflammatory agents which would limit platelet aggregation, are effective in reducing the size of cerebral infarction and improving neurological deficit scores. Danggui, the dried root of Angelica Sinensis (Radix Angelica Sinensis), is a commonly used Chinese medicinal herb to enrich blood, promote blood circulation and treat blood deficiency pattern and menstrual disorders such as dysmenorrhea and irregular menstrual cycle [et al. [Danggui (105 \u03bcg/ml) plays an immunostimulatory role in mitogen-stimulated murine lymphocytes in vitro. Angelan, a purified polysaccharide component of Angelica nakai thought to improve immune function, increases the expression of cytokines in splenocytes as Angelan enhances and the production of interleukin-6 (IL-6) and interferon-\u03b3(IFN-\u03b3) of activated macrophages, helper T cells and natural killer cells [Danggui extract are classified into essential oil and water soluble parts including lipid compounds, phenolic compounds, carbohydrates, organic acids and other constituents [al cycle . Wilasru [et al. reporteder cells . The chetituents . The mos[Danggui 05 \u03bcg/ml Danggui in reducing the size of cerebral infarction and improving neurological deficit scores.This article aims to provide an overview of the pharmacological effects of Angelica sinensis', 'Danggui', 'Angelica polysaccharides', 'Z-Ligustilide', 'Ferulic acid' and 'Ischemic stroke' as keywords.We searched Medline, PubMed, Cochrane Library and the China National Knowledge Infrastructure (Chinese language database) between 1990 and 2010, using '2+ for binding calmodulin [Danggui increases NO formation and relaxes the endothelium [Danggui, increases the generation of NO, thereby inhibiting platelet aggregation on endothelium and proliferation of smooth muscles and preventing leucocytes adhering to the endothelium [Nitric oxide (NO) is synthesized with nitric oxide synthase (NOS) which includes three different isoforms, namely endothelial NOS (eNOS), neuronal NOS (nNOS) and inducible NOS (iNOS) . While nlmodulin ,6. Due tlmodulin . Moreovelmodulin . Therefolmodulin . It is pothelium , therebyothelium .Danggui, inhibits (4-8 \u03bcg/ml) the spontaneous contraction of isolated rat uterus in a dose-dependent manner [Z-Ligustilide , a component of t manner . Moreovet manner . Z-Ligust manner .Danggui. Ferulic acid (10-3 mol/L) relaxes the phenylephrine-induced contraction of aorta ring in spontaneous on rat (SHR) whereas the effects of ferulic acid may be partially blocked by pretreatment of the aorta with NG-nitro-L-arginine methyl ester which inhibits the production of NO from L-arginine [-3 mol/L) reduces the production of thromboxane B2 in the aorta ring of SHR [-4 mol/L) also significantly reduces the generation of NADPH-dependent production of the superoxide anion [via effects on (1) eNOS; (2) the inhibition of thromboxane B2 to relax aorta ring; (3) reactive oxygen species (ROS) scavenging activity to increase the availability of NO in endothelial cell of aorta [Ferulic acid is the main organic acid component of arginine . Ferulicg of SHR . Ferulicde anion and enhade anion . Taken tof aorta .Stroke is divided into two major groups according to the cerebral damage, cerebral infarction and cerebral hemorrhage. Eighty percent (80%) of stroke patient suffer from cerebral infarction . Cerebravia the inhibition of TGF-\u03b21 and basic fibroblast growth factor (bFGF). The alteration of TGB-\u03b2 activity leads to the atherosclerotic change of vessel wall and increased TGB-\u03b2 signaling plays a protective role of atherosclerosis [via the regulation of interstitial collagenase expression in the early stages of atherosclerosis. Wang et al. [Danggui (20 mg/ml) and its component of sodium ferulate (0.3 mg/ml). These results indicate that both Danggui and sodium ferulate have anti-atherogenic effects [et al. [vs. 11.79 mmol/L), triglyceride (TG) and high density lipoprotein cholesterol (HDLC) and low density lipoprotein cholesterol (LDLC) increased in rabbits on a high-lipid diet compared to the control group which was on a normal diet. After 25% Danggui was administered (i.v.) for four weeks, the levels of TG decreased from 3.52 mmol/L to 1.68 mmol/L. The plaque area of thoracic aorta was also reduced after Danggui treatment, from 63.31% to 35.58% [Danggui reduced the increase of the serum malonyldialdehyde (MDA) levels caused by the high-lipid diet [Danggui and sodium ferulate inhibit the formation of atherosclerosis because Danggui reduces the TG and lipid peroxidation levels or increases NO production, or both.A principal contributor to cerebral infarction and atherosclerosis is believed to be initiated by an excessive inflammatory-fibro-proliferative response . Atherosclerosis . A studyclerosis reportedg et al. found th effects . Yu et a [et al. found thAnti-platelet aggregation agents such as aspirin, ticlopidine and clopidogrel are widely used to prevent secondary ischemic stroke ,26. A clDanggui dose-dependently inhibited adenosine diphosphate (ADP)-induced platelet aggregation and collagen-induced platelet [Danggui (20 g/kg) reduced platelet aggregation by 87.9% in rats with ADP-induced platelet aggregation and 33.0% in rats with collagen-induced platelet aggregation [ex vivo [Danggui and Z-Ligustilide exert anti-platelet aggregation effects.platelet . Dangguiregation . Adminisregation . In an aregation . The maxregation . Compare[ex vivo . DangguiSophora japonica L. and paeoniflorin inhibit IL-1\u03b2 secretion, thereby reducing cerebral infarction size and neurological deficit [Pro-inflammatory cytokines, such as IL-1\u03b2 and TNF-\u03b1, are increased in the brain tissue of transient middle cerebral artery occlusion (MCAo) rats ,30. IL-1 deficit . Moreoveeg 80 and 100 mg/kg, i.v.) reduces the size of cerebral infarction and neurological deficit scores and inhibits ICAM-1 and NF-\u03baB expression in transient MCAo rats [eg 100 mg/kg, i.v.) exerts anti-inflammatory action by reducing the generation of 4-hydroxy-2-nonenal (4-HNE), 8-hydroxy-2'-deoxyguanosine (8-OHdG) and apoptosis in the reperfusion period after cerebral ischemia, thus providing neuroprotection [via enhancing gamma-aminobutyric acid type B1 (GABAB1) receptor expression to against p38 mitogen activated protein kinase (MAPK)-mediated NO-induced apoptosis [Danggui reduces inflammatory cell infiltration and TNF-\u03b1 and TGF-\u00df1 mRNA expression; it also reduces TNF-\u03b1 and TGF-\u00df1 positive cells in radiation-induced pneumonitis in mice [Danggui polysaccharides reduce TNF-\u03b1 levels in the colon mucosa during intra-colon enema with 2,4,6-trinitrobenzene sulfonic acid (TNBS) and ethanol in rats [Danggui and ferulic acid exert anti-inflammatory effects.Ferulic acid in mice [Ginkgo biloba extract reduce cerebral damage in rodent models of ischemia and reperfusion [B receptor agonist baclofen may be neuro-protective via the inhibition of N-methyl-D-asparate (NMDA) receptor-mediated NO production in brain ischemic injury [B1 in the reperfusion period (three and 24 hours after ischemia) in rats [Danggui or its components or both demonstrate anti-oxidant activity in ischemia-reperfusion injury models.Reactive oxygen species (ROS) including the superoxide anion, hydrogen peroxide and hydroxyl radical are generated after cerebral ischemia. The ROS affect mitochondrial function, DNA repair and transcription factors, leading to apoptosis ,6. Super in mice . Anti-oxerfusion . GABAB rc injury . Ferulic in rats . Z-ligus in rats . Z-ligus in rats . DangguiDanggui increased blood circulation and neuronal metabolism in an MCAo rat model [Danggui reduced the size of cerebral infarction, neurological deficit scores and increased blood flow and SOD activity in the MCAo rat model [et al. [Danggui administered to 1040 patients with acute cerebral infarction improved neuro-function scores and the Barthel index score more than Salvia miltiorrhiza (78.7% vs. 59.3%). Danggui reduces the size of cerebral infraction and improves neurological deficit scores.at model . Dangguiat model . Z-ligusat model . Moreoveat model . Our preat model found th [et al. reportedDanggui on cerebral infarction are through multiple pathways, including anti-arthrosclerosis, improving microcirculation, anti-platelet aggregation, anti-inflammatory and anti-oxidative effects (Table Danggui may be useful in treating the cerebral infarction type of stroke.The effects of ts Table . Danggui4: carbon tetrachloride; ASP: water-soluble polysaccharide; NO: nitric oxide; eNOS: endothelial nitric oxide synthase; nNOS: neuronal nitric oxide synthase;iNOS: inducible nitric oxide synthase; MCAo: middle cerebral artery occlusion; L-NAME: NG-nitro-L-arginine methyl ester; SHR: spontaneous hypertensive rat; NADPH:nicotinamide adenine dinucleotide phosphate; HHQ: hydroxyhydroquinone; ROS: reactive oxygen species; VEGF: vascular endothelial growth factor; FGH: fibroblast growth factor; TGF-\u03b2: transforming growth factor-\u03b2; TNF-\u03b1: tumor necrosis factor -\u03b1; MCP-1: monocyte chemoattractant protein-1; bFGF: basic fibroblast growth factor; TC: total cholesterol; TG: triglyceride; HDLC: high density lipoprotein cholesterol; LDLC: low density lipoprotein cholesterol; ADP: adenosine diphosphate; APTT: activated partial thromboplastin time; PT: prothrombin time; ICAM-1: intracellular adhesion molecule-1; NF-\u03baB: nuclear factor-\u03baB; 4-HNE: 4-hydroxy-2-nonenal; 8-OHdG: 8-hydroxy-2'-deoxyguanosine; GABAB1: gamma-aminobutyric acid type B1; MAPK: mitogen activated protein kinase; mRNA: messenger ribonucleic acid; SOD: superoxide dismutase; NMDA: N-methyl-D-asparate; GSH-Px: glutathione peroxidase; BCCAo: bilateral carotid artery occlusionIL: interleukin; IFN-\u03b3: interferon-\u03b3; AP: polysaccharide component; ALT: serum alanine transferase; MDA: malondialdehyde; NOS: nitric oxide synthase; CCLThe authors declare that they have no competing interests.YCW searched the literature, organized the information and wrote the manuscript. CLH analyzed the information and revised the manuscript. Both authors read and approved the final version of the manuscript."} +{"text": "AGS-003 is an autologous dendritic cell immunotherapy consisting of matured DC co-electroporated with amplified autologous tumor RNA and synthetic CD40L RNA. A Phase 2 trial evaluated AGS-003-induced T cell immunity in RCC patients in combination with sunitinib. A comprehensive immune analysis using an unbiased bioinformatics approach to analyze multi-color flow cytometry data on longitudinal blood draws identified statistically significant correlates between T cell responses and overall survival.Immune monitoring using multi-color flow cytometry was performed on subjects receiving at least five doses of AGS-003 (n=14). The large data sets where analyzed using a novel bioinformatics methodology consisting of an adaptation of binary tree-structured vector quantization (BTSVQ). BTSVQ implements two-way unsupervised clustering to partition cytotoxic T cell (CTL) subsets into related groups without consideration of clinical outcomes. This approach identified an AGS-003-induced central/memory CTL signature defined by the co-expression of CD28, CD27, and CCR7 receptors in the absence of CD45RA expression. Furthermore, increases in absolute numbers of antigen-reactive CD28+CCR7+CD27+CD45RA- CTL after five doses of AGS-003 correlated with survival. Importantly, these CTL consist of proliferating CTL secreting multiple cytokines with lytic activity. We further identified a pre-treatment CTL signature and post-treatment CTL signature, with both subsets lacking the CD28 receptor, as inversely correlated with survival. One patient treated long term with AGS-003 maintained multi-functional CD28+CCR7+CD45RA- CTL outwards to 3 years post-treatment resulting in patient survival in excess of 30 months.These observations confirm the in vivo mechanism of action of AGS-003 and provide early biomarkers that could predict long-term clinical outcome. BTSVQ analysis identified multi-functional CTL correlating with clinical outcome. To the best of our knowledge, this is the first report correlating changes in the magnitude of an adaptive immune response post DC immunotherapy with clinical outcome. Data presented herein shows the durability of the AGS-003 induced immune response, present after quarterly dosing with AGS-003. Furthermore, our immune monitoring platform will serve as the basis to correlate immune responses and clinical efficacy in RCC patients receiving AGS-003 in the ongoing randomized Phase 3 ADAPT study using AGS-003 in combination with standard treatment."} +{"text": "Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. Annually, about 200,000 patients died of HCC in China. Liver transplantation (LT) holds great theoretical appeal in treating HCC. However, the high recurrence rate after transplantation is the most important limiting factor for long-term survival.To assess the value of alpha-fetoprotein (AFP) messenger RNA (mRNA), Glypican-3 (GPC3) mRNA-expressing cells in the peripheral blood (PB) for prediction of HCC recurrence following orthotopic liver transplantation (OLT).29 patients with HCC who underwent OLT with a minimum clinical follow-up of 12 months were included in this retrospective study. We detected AFP mRNA, GPC3 mRNA-expressing cells in the PB by TaqMan real-time reverse transcriptase-polymerase chain reaction (RT-PCR), pre-, intra- and post-operatively. The early recurrence of patients was evaluated.8 (28%), 15 (52%), and 9 (31%) patients had AFP mRNA detected pre-, intra-, and post-operatively, respectively. With 12 months of follow-up, HCC recurred in 7 (24%) patients. Univariate analysis revealed that positive pre- and post-operative AFP mRNA, TNM stage as well as vascular invasion were significant predictors for the HCC recurrence. Multivariate analysis revealed that being positive for AFP mRNA pre-operatively remained a significant risk factor for HCC recurrence after OLT. GPC3 mRNA was expressed in all PB samples. There was no significant difference in the expression levels of GPC3 mRNA between the HCC and control groups. There were no significant differences in GPC3 mRNA expression values between those patients with and without tumor recurrence.The pre-operative detection of circulating AFP mRNA-expressing cells could be a useful predictor for HCC recurrence following OLT. GPC3 mRNA-expressing cells in PB seem to have no diagnostic value. Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide, with a global incidence of 500,000 new cases per year; 82% of cases (and deaths) occur in developing countries (with 55% in China) [2]. HCC In the present study, we applied TaqMan real-time RT-PCR to assess the usefulness of detecting AFP mRNA and GPC3 mRNA-expressing cells in the PB for predicting the risk of HCC recurrence after orthotopic liver transplantation (OLT).Twenty-nine liver transplant recipients with HCC who underwent OLT in Orient Organ Transplant Center during 2008 were included in this retrospective study. The diagnosis of HCC was confirmed by pathologic study by experienced pathologists. The criteria for OLT in patients with HCC are absence of extrahepatic malignancies, macroscopic tumor thrombosis, or extrahepatic metastasis of HCC. Of the 29 studied patients, 27 had hepatitis B virus (HBV)-induced cirrhosis, and two patients had hepatitis C virus (HCV)-induced cirrhosis. Twelve patients had received treatment for HCC; treatments included resection, radiotherapy or transcatheter hepatic arterial chemoembolization (TACE) prior to transplantation. The remaining 17 patients had not received any treatments. Clinical characteristics of HCC patients are summarized in The present study was approved by the Institutional Ethics Committee of our institute. The informed consent was obtained from each patient. The procedure met all applicable institutional guidelines of our institute, and governmental regulations concerning the ethical use of donated organs. PB (15 mL) was drawn with a sterile syringe containing ethylenediaminetetraacetic acid (EDTA). PB samples were obtained at the following time points: pre-operatively (just before the skin incision), intra-operatively (during the anhepatic period), and post-operatively from all 29 patients. AFP mRNA detectable each time after operation was considered as \"real post-operative AFP mRNA positivity.\" In the meantime, the control group who provided informed consent were also tested for AFP mRNA, and GPC3 mRNA.Total RNA was extracted with PB mononuclear cells (PBMCs) with TRIzol reagent . The real-time RT-PCR used for the detection of AFP mRNA was performed as described previously . The seqRelative expression = copy number of target molecule/Copy number of \u00df-zactinThe RT-PCR assay was repeated twice and performed with an ABI Prism GeneAmp 7300 Sequence Detection System Kaplan-Meier survival analysis and log-rank test were used to derive the survival curve. To assess the risk factors, univariate and multivariate analyses were performed using the Kaplan-Meier method (long-rank test) and Cox's proportional hazards model. Comparison between two groups was made by independent Student's t test for numerical variables, and x(2) test for categorical variables. One-way ANOVA with Bonferroni post hoc test was used for comparisons between more than two groups. A p value < 0.05 was considered statistically significant. Analyses were made using SPSS\u00ae software ver 12 for Windows\u00ae .All patients had at least one year of follow-up. HCC recurred in 7 (24%) of 29 patients at 4-11 (median 6.8) months after OLT during the follow-up period. AFP mRNA was positive in 8 (28%) patients pre-operatively, in 15 (52%) intra-operatively, and in 9 (31%) post-operatively. AFP mRNA was positive pre-operatively in 6 (21%) patients-pre-, intra- and post-operatively. None of healthy volunteers had AFP mRNA-expressing cells in their PB, and one (5%) of patients with chronic HBV or HCV cirrhosis without HCC, had AFP mRNA-expressing cells in his PB pre-operatively; these frequencies were significantly different from pre-operative patients with HCC . Of the nine variables assessed, four were significantly related to recurrence in univariate analysis. These were TNM stages, vascular invasion, pre-operative AFP mRNA, and post-operative AFP mRNA .The presence of pre-operative AFP mRNA-positive cells in PB was in favor of a lower recurrence-free survival rate . Pre-opeIt is a well-developed and mature technique in recent years that marked RNA of tumor cells in PB was amplified using RT-PCR to confirm the presence of tumor cells in blood10]. Pre. Pre10].Operative manipulation would enhance the dissemination of HCC cells into PB; this cell spread could be partly responsible for tumor recurrence after OLT, although this is an intermittent and transient phenomenon. However, it has been shown that surgical intervention induces a release of non-neoplastic liver cells into the circulation and increases false-positive signals of AFP mRNA. To reduce the likelihood of false-positive results, the post-operative blood samples were taken one week after transplantation, as liberated non-neoplastic cells are presumably filtered from the PB within one week, as previously proposed by Ijichi, et al. Our stu"} +{"text": "Adiposity greatly increases the risk of atherothrombotic events, a pathological condition where a chronic state of oxidative stress is reported to play a major role. This study aimed to investigate the involvement of (NO)-soluble guanylyl cyclase (sGC) signaling pathway in the platelet dysfunction from high fat-fed (HFF) rats.Male Wistar rats were fed for 10 weeks with standard chow (SCD) or high-fat diet (HFD). ADP (10 \u03bcM)- and thrombin (100 mU/ml)-induced washed platelet aggregation were evaluated. Measurement of intracellular levels of ROS levels was carried out using flow cytometry. Cyclic GMP levels were evaluated using ELISA kits.n = 8) and thrombin from HFD rats (n = 8) were significantly greater (P < 0.05) compared with SCD group. Platelet activation with ADP increased by 54% the intraplatelet ROS production in HFD group, as measured by flow cytometry (n = 6). N-acetylcysteine and PEG-catalase (1000 U/ml) fully prevented the increased ROS production and platelet hyperaggregability in HFD group. The NO donors sodium nitroprusside and SNAP (10 \u03bcM), as well as the NO-independent soluble guanylyl cyclase stimulator BAY 41-2272 (10 \u03bcM) inhibited the platelet aggregation in HFD group with lower efficacy (P < 0.05) compared with SCD group. The cGMP levels in response to these agents were also markedly lower in HFD group (P < 0.05). The prostacyclin analogue iloprost (1 \u03bcM) reduced platelet aggregation in HFD and SCD rats in a similar fashion (n = 4).High-fat fed rats exhibited significant increases in body weight, epididymal fat, fasting glucose levels and glucose intolerance compared with SCD group. Platelet aggregation induced by ADP (Metabolic abnormalities as consequence of HFD cause platelet hyperaggregability involving enhanced intraplatelet ROS production and decreased NO bioavailability that appear to be accompanied by potential defects in the prosthetic haem group of soluble guanylyl cyclase. Iloprost was supplied by Schering (Germany).n rats. The statistical significance between groups was determined by using one-way ANOVA followed by the Bonferroni test. Where appropriate, unpaired Student's t test was used to compare specific groups. Significance was established at P < 0.05.Data are expressed as means \u00b1 SEM of P < 0.05), respectively (Table P < 0.05). The OGTT showed that glucose levels remained increased after glucose consumption in HFD in all evaluated time-points in comparison with SCD group . Insulin sensitivity was markedly reduced in HFD compared with SCD rats (P < 0.05), as assessed by the ITT exhibited a significant increase in body weight and epididymal fat compared with animals receiving standard-chow diet (SCD), approximately 20% and 135% in washed platelets obtained from HFD compared with SCD group . Similarly, platelet aggregation induced by thrombin (100 mU/ml) was significantly greater (P < 0.05) in washed platelets obtained from HFD compared with SCD group .Platelet aggregation induced by ADP (10 \u03bcM) was significantly greater or PEG-catalase did not significantly affect thrombin- or ADP-induced platelet aggregation in SCD rats ( min or Pn = 4) and SNAP , as well as by the NO-independent soluble guanylyl cyclase stimulator BAY 41-2272 , as shown in Figure P < 0.001) in the amounts of intracellular cGMP levels, respectively .In SCD rats, ADP-induced platelet aggregation was largely reduced by prior incubation with the NO donors SNP , a prostacyclin analogue that acts directly in cAMP/PKA signaling pathway, nearly abolished the ADP-induced platelet aggregation, as observed in both SCD and HFD rats (The present study shows that rats fed with high-fat diet (HFD) exhibit ex-vivo platelet hyperaggregability to ADP and thrombin, which is accompanied by higher intraplatelet ROS production. The platelet hyperaggregability was prevented by the antioxidant compounds PEG-catalase and NAC in HFD group indicating a critical role for intracellular ROS in this phenomenon. Moreover, the NO donors SNP and SNAP, as well as the soluble guanylyl cyclase stimulator BAY 41-2272 showed a lower efficacy in inhibiting the platelet aggregation in HFD rats, possibly as a consequence of lower platelet cGMP productions in this diet-induced obesity model.2- production [Evidences have shown that persistent hyperglycemia can activate alternative glucose metabolism pathways, which in turn result in the formation of deleterious products derived from protein or lipid structure alterations named advanced glycation end products (AGEs), which can deeply affect the function of the cardiovascular system . In vascoduction . Acute hoduction ,23.2-) is considered the primary ROS, and it can further interact with other molecules, either directly or through enzyme- or metal-catalyzed processes, to generate other physiological relevant ROS such as hydrogen peroxyde (H2O2) and -OH, as well as peroxynitrite (ONOO-) [2- including the NADPH-oxidase, xanthine oxidase and arachidonate-derived prostaglandin-like metabolites [Radicals derived from oxygen represent the most important class of ROS generated in living systems. Superoxide anion (O (ONOO-) . Adiposi (ONOO-) . Differeabolites -29. Howeabolites . Moreoveabolites , signifiabolites .2- production in platelets of HFD rats inactivates SNP- and SNAP-derived NO. This is consistent with studies performed in obese subjects and type 2 diabetic obese patients where platelets are resistant to glyceryl nitrate and SNP [Increased oxidative stress may also influence platelet function by decreasing NO bioavailability . Nitric and SNP ,34.- is able to oxidize the prosthetic haem group of sGC to its NO-insensitive Fe3+ state [The soluble guanylyl cyclase (sGC) is a widely distributed signal transduction enzyme that, under activation by NO, converts GTP into the second messenger cGMP which in turn affects various downstream targets such as protein kinases, cyclic nucleotide-gated channels or phosphodiesterases . One of 3+ state -43. If t3+ state . Interes3+ state .Besides the NO - cGMP - PDE5 pathway, the activation of platelets is inhibited by cAMP-elevating agents . ElevatiOur findings clearly show that metabolic abnormalities as consequence of HFD in rats cause platelet hyperaggregability involving enhanced intraplatelet ROS production and decreased NO bioavailability accompanied by potential defects in the prosthetic haem group of sGC.ADP: adenosine 5'diphosphate; HFD: high fat diet; HFF: high fat-fed; H2O2: hydrogen peroxyde; IBMX: 3-isobutyl-l-methyl-xanthine; ITT: insulin tolerance test; NAC: N-acetylcysteine; NO: nitric oxide; OGTT: oral glucose tolerance test; ONOO-: peroxynitrite; PRP: Platelet-rich plasma; ROS: reactive-oxygen species; sGC: soluble guanylyl cyclase; SNAP: S-nitroso-N-acetylpenicillamine; O2-: superoxide anion; TXA2: thromboxane A2.The authors declare that they have no competing interests.AZ and EA carried out interpreting the data and writing the manuscript; PFM, RPM, MAD, MEC, MELP and SM carried out data acquisition and reviewing statistical analysis. All authors read and approved the final manuscript."} +{"text": "Diastolic dysfunction prominently contributes to heart failure with preserved ejection fraction (HFpEF). Owing partly to inadequate understanding, HFpEF does not have any effective treatments. Cardiac myosin-binding protein-C (cMyBP-C), a component of the thick filament of heart muscle that can modulate cross-bridge attachment/detachment cycling process by its phosphorylation status, appears to be involved in the diastolic dysfunction associated with HFpEF. In patients, cMyBP-C mutations are associated with diastolic dysfunction even in the absence of hypertrophy. cMyBP-C deletion mouse models recapitulate diastolic dysfunction despite in vitro evidence of uninhibited cross-bridge cycling. Reduced phosphorylation of cMyBP-C is also associated with diastolic dysfunction in patients. Mouse models of reduced cMyBP-C phosphorylation exhibit diastolic dysfunction while cMyBP-C phosphorylation mimetic mouse models show enhanced diastolic function. Thus, cMyBP-C phosphorylation mediates diastolic function. Experimental results of both cMyBP-C deletion and reduced cMyBP-C phosphorylation causing diastolic dysfunction suggest that cMyBP-C phosphorylation level modulates cross-bridge detachment rate in relation to ongoing attachment rate to mediate relaxation. Consequently, alteration in cMyBP-C regulation of cross-bridge detachment is a key mechanism that causes diastolic dysfunction. Regardless of the exact molecular mechanism, ample clinical and experimental data show that cMyBP-C is a critical mediator of diastolic function. Furthermore, targeting cMyBP-C phosphorylation holds potential as a future treatment for diastolic dysfunction. Heart failure occurs when cardiac output cannot meet the body\u2019s demand. It has an estimated global prevalence of 23\u00a0M . Lifetimmin, pressure/volume relationship during diastolic filling) and monotonically decreases with worsening diastolic dysfunction i, demonstrating that impaired relaxation is caused by myofilament dysfunction, not by slowed calcium handling i decay times in all the mouse models (Fig.\u00a0cMyBP-C phosphorylation may mediate diastolic function by modulating the relative cross-bridge detachment rate with respect to cross-bridge attachment rate Fig.\u00a0. Myocardectively , 42. Sined Ea/Sa , 46. Inc-C-/-, Ex-10 and c-C-/-, Ex-10 and c-C-/-, Ex-10 and c-C-/-, Ex-10 and cClinical evidence and animal models demonstrate that cMyBP-C mediates diastolic function. The correlation of intact papillary muscle experiments and in vivo TD measurements suggests that cMyBP-C phosphorylation modulates relative cross-bridge detachment rate with respect to attachment rate to mediate diastolic function. Thus, targeting cMyBP-C phosphorylation holds great potential for the treatment of diastolic dysfunction."} +{"text": "The equivalency of continuous venovenous hemofiltration and intermittent hemodialysis (2B) was described as a key recommendation of the Surviving Sepsis Campaign guidelines in 2008. However, there are some discrepancies associated with the evaluation of blood purification in severe sepsis and septic shock in Japan. Direct hemoperfusion with polymyxin-B immobilized fiber (PMX-DHP), developed and currently in use in Japan, has not yet been evaluated abroad. We performed a retrospective study to re-evaluate PMX-DHP for severe sepsis or septic shock patients in our ICU.2/FiO2 ratio, catecholamine requirement), inflammatory mediators , endothelial-related markers and procalcitonin levels were examined in each group.We enrolled 302 patients group: 201, nonsurvival (NS) group: 101) in whom PMX-DHP had been performed for severe sepsis and septic shock from 1994 to 2010. These patients were allocated into two groups: those who survived for at least 28 days after the start of PMX-DHP therapy (S group: 201 patients) and those who did not (NS group: 101 patients). Background factors score), hemodynamics (blood pressure, PaO2/FIO2 ratio both improved markedly immediately after PMX-DHP. Also, the average required amount of catecholamine decreased after PMX-DHP. IL-6 and IL-1ra levels decreased immediately after PMX-DHP in both groups, but these values before PMX-DHP did not show any statistically significant difference between the groups. PAI-1 levels showed a significant decrease after PMX-DHP in both groups.On background factors, only the Goris MOF score showed a statistically significant difference among the groups. Blood pressure and the PaOWe confirmed an improvement in pulmonary oxygenation and hemodynamic parameters using PMX-DHP for severe sepsis and septic shock patients. The levels of various inflammatory mediators decreased using PMX-DHP, but we did not find any correlation between these changes and outcome."} +{"text": "Hepatitis B virus (HBV) infection in patients with end-stage renal disease (ESRD) on renal replacement therapy-hemodialysis and/or renal transplantation-usually has an unfavorable course with a tendency towards chronicity leading to increased morbidity and mortality . Further"} +{"text": "The transcription factor MYB46, together with its redundant paralog MYB83, regulates expression of secondary cell wall biosynthesis genes in Arabidopsis [reviewed in 1].We isolated the Populus MYB46 ortholog, PtxtMYB021 from hybrid aspen (Populus tremula x tremuloides) [To establish whether the AC-type motif in CAZyme promoters is important for their function, we analyzed loss-of-function and gain-of-function GUS reporter constructs of the GT43A promoter in a transient transcription assay, by co-infiltration with a 35S:MYB021 effector in Nicotiana benthamiana leaves.We show that mutation of the AC-type element in the GT43A promoter abolishes transactivation by MYB021, whereas an AC multimer confers MYB021 transactivation to a minimal 35S promoter. Others have shown that the recombinant Eucalyptus grandii MYB46/MYB83 ortholog, EgMYB2, directly binds the AC-motif variant CCACCTACC found in the EgCCR gene promoter [Interestingly, the downstream MYB regulators of lignin biosynthesis MYB58 and MYB63 bind the AC element but reportedly do not regulate CAZyme-mediated secondary cell wall polysaccharide biosynthesis . EstabliThe AC regulatory element, here preliminarily redefined as the sequence CCACCAAC, is a true \u201clignocellulose synthesis response element\u201d, mediating MYB46-dependent transactivation of the whole secondary cell wall gene program. This knowledge will enable inference of MYB46-centered regulatory networks in different species, through bioinformatic analysis."} +{"text": "Although crucial roles of microRNA have begun to emerge, detailed studies with ATL patients have not been achieved. Using 40 primary ATL samples and 22 samples of normal CD4+ T-cells, we determined the microRNA signatures of ATL and revealed loss of miR-31, which has recently been reported as a metastasis-associated miRNA. All ATL cases invariably showed undetectable or very low levels of miR-31, clearly implying that miR-31 loss is involved in ATL development.As a novel miR-31 target gene, we identified NF-\u03baB inducing kinase (NIK) that plays central roles in noncanonical signaling and constitutive activation of NF-\u03baB in various cancers, including ATL. Restoration of miR-31 downregulated the levels of NIK and NF-\u03baB activity, resulting in reduction of malignant phenotypes, containing proliferative index, anti-apoptosis, and chemotaxis in ATL cells. Furthermore, lentivirus-introduced miR-31 could induce strong apoptosis in primary tumor cells freshly isolated from ATL patients, indicating pivotal functions of miR-31 as a tumor suppressor.Global copy number profiling demonstrated that 21 cases out of 168 (12.5%) have genomic loss of 9p21 containing miR-31 region. Furthermore, expression profiling and ChIP assay showed requirement of overexpression of histone methyltransferase in epigenetic suppression of miR-31 and aberrant NF-\u03baB activation in primary ATL cells. Knockdown of methyltransferase complex restored the miR-31 expression and consequently inhibited NIK-dependent NF-\u03baB cascade. These findings illustrated that genetic and epigenetic abnormalities link to NF-\u03baB activation through the loss of miR-31. Considering aberrant epigenomics associated with cancers, the emerging relationship provides us a conceptual advance in understanding the broad-acting oncogenic signaling."} +{"text": "Coitus in snakes may last up to 28 hours; however, the mechanisms involved are unknown.To evaluate the relevance of the nitric oxide (NO)-cyclic guanosine monophosphate (cGMP)-phosphodiesterase type 5 (PDE5) system in snake corpus cavernosum reactivity.Crotalus durissus terrificus) and studied by light and scanning electronic microscopy. Isolated Crotalus corpora cavernosa (CCC) were dissected from the non-spiny region of the hemipenises, and tissue reactivity was assessed in organ baths.Hemipenes were removed from anesthetized South American rattlesnakes (Cumulative concentration-response curves were constructed for acetylcholine (ACh), sodium nitroprusside (SNP), 5-cyclopropyl-2-pyridine-3-yl]pyrimidin-4-ylamine (BAY 41-2272), and tadalafil in CCC precontracted with phenylephrine. Relaxation induced by electrical field stimulation (EFS) was also done in the absence and presence of Nw nitro-L-arginine methyl ester , 1H- oxadiazoloquinoxalin-1-one and tetrodotoxin .+0.17 and 5.10 + 0.08 (N = 3\u20134), respectively. In precontracted CCC, EFS caused frequency-dependent relaxations that lasted three times longer than those in mammalian CC. Although these relaxations were almost abolished by either L-NAME or ODQ, they were unaffected by TTX. In contrast, EFS-induced relaxations in marmoset CC were abolished by TTX.The hemipenes consisted of two functionally concentric corpora cavernosa, one of them containing radiating bundles of smooth muscle fibers (confirmed by a-actin immunostaining). Endothelial and neural nitric oxide synthases were present in the endothelium and neural structures, respectively; whereas soluble guanylate cyclase and PDE5 were expressed in trabecular smooth muscle. ACh and SNP relaxed isolated CCC, with the relaxations being markedly reduced by L-NAME and ODQ, respectively. BAY 41-2272 and tadalafil caused sustained relaxations with potency (pEC50) values of 5.84 Rattlesnake CC relaxation is mediated by the NO-cGMP-PDE5 pathway in a manner similar to mammals. The novel TTX-resistant Na channel identified here may be responsible for the slow response of smooth muscle following nerve stimulation and could explain the extraordinary duration of snake coitus."} +{"text": "The model has generated a sizable literature confirming the ego-depletion effect in multiple spheres. Our meta-analysis of published ego-depletion studies computed a medium-sized effect (d = 0.62) across 198 tests against the against the study precision (1/standard error).Click here for additional data file.Figure S2Funnel plot of the ego-depletion effect size against the standard error of the effect size.Click here for additional data file.Click here for additional data file."} +{"text": "Our system uses the human insulin promoter to initiate a Cre-mediated shift in reporter color within a single transgene construct and is useful for FACS selection of cells from single cultures for further analysis. Application of our reporter to presumably clonal HIT-T15 insulinoma cells, as well as other presumably clonal lines, indicates that these cultures are in fact heterogeneous with respect to INS+ phenotype. Our strategy could be easily applied to other cell- or tissue-specific promoters. We anticipate its utility for FACS purification of INS+ and glucose-responsive beta-like-cells from primary human islet cell isolates or in vitro differentiated pluripotent stem cells.Many research studies use immortalized cell lines as surrogates for primary beta- cells. We describe the production and use of a novel \u201cindirect\" dual-fluorescent reporter system that leads to mutually exclusive expression of EGFP in insulin-producing (INS Cell replacement strategies are thus being developed to treat this metabolic disease. A key treatment step will be production of sufficient quantities of fully functional pancreatic beta- or beta-like-cells suitable for replacing missing or defective beta-cells. This goal has stimulated renewed interest in understanding human islet cell biology. However, because of the difficulty and high cost associated with isolating human islets, most studies focus on Rodent insulinoma cell lines derived from cancers arising after radiation treatment or viral transformation have been especially useful models of beta-cell biology; at the time of their establishment, they exhibit high levels of insulin production and glucose responsiveness +) beta- and non-insulin-producing (INS-) cells. Our reporter contains a single transgene with two expression cassettes. The first is a fragment of the human insulin gene promoter (phINS) that drives expression of Cre recombinase protein exclusively in INS+ cells. The second contains the CMV promoter and an mCherry coding region flanked with LoxP (L) sites, followed by an EGFP coding region. In cells with active insulin promoter activity, the Cre protein excises the mCherry coding region, and the cells exhibit green fluorescence. In cells with no insulin promoter activity, the mCherry coding region is not excised, so the cells exhibit red fluorescence. This new \u201cindirect\" reporter strategy uses mutually exclusive expression of green or red fluorescence to eliminate ambiguity observed when a human insulin promoter directly drives expression of EGFP in combination with a CMV-regulated mCherry. Distinguishing INS+ from INS- cells with the \u201cdirect\" strategy depends on identifying cells that are doubly fluorescent and often leads to ambiguous results\u2014the relative levels of fluorescence for the two reporter colors can be highly variable . Our \u201cindirect\" dual-color system, in contrast, reports all cells that have been transduced or transfected, so efficiency of transduction/transfection is easily calculated.In this study, we describe the development and application of a new dual-color fluorescent reporter system for identifying insulin-producing fluorescence. Transient (data not shown) and stable transfection of phINS-EGFP-CMV-dsRed , while MB-231 cells did not show Cre expression (+ (red), but not all red cells were green. This suggests that not all cells were transfected with both plasmids and motivated us to use a single construct containing a combination of both cassettes.To confirm that insulin promoter activity led to expression of Cre recombinase, we constructed a plasmid containing the Cre gene under the human insulin promoter . This plrescence .+ cells should activate Cre recombinase expression and excise the mCherry reporter by Cre-LoxP recombination, thus converting red fluorescence to green. In contrast, transfected INS\u2013 cells should only exhibit red fluorescence. We refer to this reporter as \u201cindirect,\" since phINS does not directly drive EGFP expression, but rather changes the color of the reporter being expressed under control of the ubiquitous CMV promoter.We next constructed [pA-Cre-phINS(reverse orientation)]-pCMV-L-mCherry-L-EGFP by inserting a phINS-Cre-pA cassette fragment upstreamTransiently transfected fibroblasts showed only red fluorescence , indicatWe expected all stably transfected cells to exhibit green fluorescence, since HIT-T15 is believed to be a clonal model of beta-like-cells and all should have activated the insulin promoter and expressed Cre + cells relative to mCherry+ cells expression , or perhaps cells change from INS+ to GCG+ in culture by overexpression of aristaless-related homeobox (Arx) but almost totally eliminated the capacitance increase in green cells rise in response to increased extracellular glucose 2+]i. The other colored lines in 2+]i in both basal and stimulating glucose concentrations , and it also leads to mutually exclusive marking of cells with one or the other fluorescent protein. Regardless of which fluorescent protein gets expressed, expression is under control of the same strong and ubiquitous CMV promoter. We thus observe only a single color for each phenotype that reports successful INS gene expression by a discrete change in color from red to green .+ cells to serve as an appropriate control for non-insulin-positive cell phenotypes. A very similar dual-color system to ours was reported Another advantage of our indirect dual-color reporter relates to possible off-target effects of the introduced plasmid or viral vector. Introduction of plasmid or viral vectors into cells may alter cellular phenotype + beta-like-cells, while roughly 30% were INS\u2212, non-beta-like-cells (possibly of mixed cell phenotypes). This result might be due to unanticipated differentiation of the cells into different phenotypic classes during culture or could possibly indicate a non-clonal nature of the reference HIT-T15 cell line we obtained from ATCC. It was recently reported that pancreatic beta-cell identity is maintained by DNA methylation-mediated repression of Arx + beta-cells converts them into GCG+ alpha-cells by derepression of Arx transcription repressor in beta-cells Using our new reporter, we found that presumably clonal HIT-T15 cells + beta-like-cells in a mixed population with a significant proportion of non-beta-like-cells .Our fluorescent reporter enables rapid identification and FACS purification of a small percentage of unambiguously identified INSke-cells . We founglucagon . Furtherecretion . These cHuman islet transplantation using the Edmonton protocol is an effective treatment for type I diabetes 2 in a humidified incubator. For all transfection experiments, unless otherwise specified, we used Lipofectamine 2000 (Invitrogen) using the manufacturer's instructions. For making stable HIT-T15 cells, cells were transiently transfected with our reporter (pA-Cre-pINS-pCMV-loxP-mCherry-pA-loxP-EGFP-pA). Two days after transfection, cells were split at a 1\u223610 ratio with fresh growth medium into 10 cm tissue culture dishes and selected with 800 \u00b5g/ml G418 until colonies were visible. The whole populations of stable transfectants or individual colonies were screened for expression of fluorescent proteins.HIT-T15 Expression of fluorescent proteins was examined with an Olympus IX70 fluorescence microscope or a Leica TCS SP5 confocal microscope . Images were analyzed and edited in MetaMorph software.+ and mCherry+ cells. FACS was performed by the USC FACS Core Facility, using a BD FACSAria cell sorter. After cell sorting, total RNAs were isolated from GFP+ or mCherry+ cells using Trizol (Invitrogen). After digestion of total RNAs with Turbo DNase (Ambion), cDNAs were made from the total RNAs with an iScript cDNA synthesis kit (Bio-Rad), according to the manufacturer's instructions. The cDNA was diluted three-fold with water prior to quantitative PCR (q-PCR) analysis. Gene-specific q-PCR primer/probe sets for hamster INS, GCG, GAPDH, Cyclophillin, and human Pdx-1 and equivalent amounts of cDNA generated as a template were used for q-PCR. Reactions were performed in for each sample using TaqMan Universal PCR Master Mix with a CFX-96 system (Bio-Rad). The q-PCR was performed using the manufacturer's instructions. For each sample, expression of marker genes was normalized to GAPDH or Cyclophillin. Data are expressed as a relative expression level. For detection of Arx that is not well studied in the hamster system, we performed q-PCR using mouse primers . Cells were blocked overnight at 4\u00b0C with 5% fetal goat serum, 1% bovine serum albumin, and 0.1% Triton X-100, and incubated for 1 hour with the primary mouse monoclonal anti-Cre antibody diluted in blocking solution (1\u2236500). Cells were then washed and incubated for 1 hour with the secondary anti-mouse Cy-2 or Cy-3 conjugated antibody . Images were taken using a Leica TCS SP5 confocal microscope. EGFP and mCherry were visualized by endogenous fluorescence.Fluorescent signal of EGFP and mCherry in yellow cells were visualized by a Leica TCS SP2 AOBS confocal microscope system using 10\u00d7 and 63\u00d7 magnifications. A Leica DM IRE2 inverted microscope was powered by Argon and HeNe lasers for the detection of EGFP and mCherry fluorescence. Images were collected in xyz series and analyzed by Leica LCS imaging software .SalI and blunted at both sites with Klenow DNA polymerase (NEB), obtaining phINS-EGFP-pA fragment. The fragment was then subcloned upstream of CMV promoter in pDsRed2-N1 (Clontech) digested using AseI and blunted at both sites with Klenow.All constructs were made using standard molecular cloning methods. To construct phINS-EGFP-pA-pCMV-DsRed2-pA, plasmid phINS-EGFP HindIII and XhoI restriction enzyme sites at each 5\u2032 and 3\u2032 ends, respectively, was obtained by polymerase chain reaction (PCR) using a template and reverse (ctcgagataacttcgtataatgtatgctatacgaagttatAGATC-TGTCGAGGCCGCGAATTAAAAAACC5\u2032-ggaattc) primers. Underlined sequences are the LoxP site and its reverse complement, and the bold capitalized ATG is the start codon of mCherry. The bold fonts are the restriction enzyme sites for digestion. The PCR product was cut by HindIII plus XhoI and blunted at the XhoI site with Klenow. The HindIII-XhoI (blunt) fragment was inserted into pEGFP-N1 (Clontech) digested with HindIII plus SalI and blunted at SalI site with Klenow.For constructing pCMV-L-mCherry-pA-L-EGFP-pA, a fragment of L-mCherry-pA-L containing ATGccgcgg CCCAAGAAGAAGAGGAAGGTGTC5\u2032-ggaattc- , reverse (cttaaggagctc GAACAAACGACCCAACACCCGTG5\u2032-gaattc) primers. After digesting with SacII and AflII, the PCR product was inserted downstream of human INS promoter in phINS-EGFP that was digested with SacII and AflII and removed EGFP-pA by gel extraction, generating plasmid phINS-Cre-pA. To combine phINS-Cre-pA with pCMV-L-mCherry-pA-L-EGFP-pA, phINS-Cre-pA fragment was obtained from plasmid phINS-Cre-pA by digesting with SalI plus AflII and blunted at both sites with Klenow. The fragment was inserted in the reverse orientation upstream of CMV promoter in pCMV-L-mCherry-pA-L-EGFP-pA digested with AseI and blunted at both sites with Klenow. DNA sequences were confirmed using standard sequencing protocols (USC DNA Sequencing Core Facility).To obtain plasmid phINS-Cre-pA, a Cre-pA fragment was generated by PCR using pCMV-Cre-pA (a gift from Li Zhang) as a template, and forward: 2, 2 mM CaCl2, 10 mM glucose, pH adjusted to 7.2\u20137.4, and osmolarity adjusted to 300\u2013310. The pipette solutions contained 10 mM NaCl, 145 mM cesium glutamate, 10 mM HEPES, 2 mM CaCl2, 1 mM MgCl2, 0.1 mM EGTA, 2 mM ATP, 0.3 mM GTP, titrated with 5 M CsOH to pH 7.2\u20137.4, and osmolarity adjusted to 290\u2013300.Cells were patch-clamped 2\u20134 days after plating. Current recordings were obtained in conventional whole cell patch-clamp configuration with an Olympus IX70 inverted microscope, EPC-9 amplifier, and Pulse software (HEKA Electronics). The glass-bottomed chambers with adherent cells were washed with PBS and then filled with standard extracellular solution consisting of 140 mM NaCl, 2.8 mM KCl, 10 mM HEPES, 1 mM MgCl2+ concentration was determined by the ratio of the excitation of the ratiometric calcium dye Fura2-AM (Invitrogen) at 350 and 380 nm using the Polychrom V by Till Photonics. Emission was measured at 500\u2013520 nm using a Photometric Cascade CCD camera and Metafluor Software.In order to observe calcium changes associated with glucose stimulation, we used the ratiometric calcium dye Fura2-AM (Invitrogen). The intracellular CaCells were incubated at room temperature for 1 hour in standard patch-clamp external solution with a modified glucose concentration of 3 mM and supplemented with 4 \u00b5M Fura2-AM. The cells were washed three times with 3 mM glucose external solution before recording. Fura-2 fluorescence was detected with a timelapse of 300 ms at 500\u2013520 nm wavelength following excitation at 350 nm (F350) and 380 nm (F380). The ratio, F350/F380, was calculated after subtracting background from each wavelength using Igor programming software. After recording fluorescence measurements in 3 mM glucose, high-glucose extracellular solution was added for a final concentration of 14 mM glucose to stimulate glucose response. The ratio, F350/F380, was again obtained using Igor software.Figure S1Transfection efficiency in MB-231 and HIT-T15 cells by pEGFP-N1.(DOC)Click here for additional data file.Figure S2Transiently transfected HIT-T15 and beta-TC-6 cells with the indirect dual-color reporter. Confocal images of HIT-T15 (a) and beta-TC-6 (b) cells. Green and red fluorescence is colocalized in one cell.(DOC)Click here for additional data file."} +{"text": "A question still remains regarding whether a long thymine-nucleotide stretch (T-stretch) involved in the recognition of the replication origin exists in the control region (CR) of Orthoptera mitochondrial DNA (mtDNA). Herein, we completed the sequencing of whole mitogenomes of two congeners , which possess overlapping distribution areas. Additionally, we performed comparative mitogenomic analysis to depict evolutionary trends of Orthoptera mitogenomes.Orthoptera, the largest polyneopteran insect order, contains 2 suborders and 235 subfamilies. Orthoptera mitochondrial genomes (mitogenomes) follow the ancestral insect gene order, with the exception of a Sinochlora mitogenomes possess 37 genes and one CR, a common gene orientation, normal structures of transfer RNA and ribosomal RNA genes, rather low A+T bias, and significant C skew in the majority strand (J-strand), resembling all the other sequenced ensiferans. Both mitogenomes are characterized by (1) a large size resulting from multiple copies of an approximately 175 bp GC-rich tandem repeat within CR; (2) a novel gene order (rrnS-trnI-trnM-nad2-CR-trnQ-trnW), compared to the ancestral order (rrnS-CR-trnI-trnQ-trnM-nad2-trnW); and (3) redundant trnS(UCN) pseudogenes located between trnS(UCN) and nad1. Multiple independent duplication events followed by random and/or non-random loss occurred during Sinochlora mtDNA evolution. The Orthoptera mtDNA recognition sequence of the replication origin may be one of two kinds: a long T-stretch situated in or adjacent to a possible stem-loop structure or a variant of a long T-stretch located within a potential stem-loop structure.Both Sinochlora mitogenomes reveal that the mtDNA architecture within Orthoptera is more variable than previously thought, enriching our knowledge on mitogenomic genetic diversities. The novel genome rearrangements shed light on mtDNA evolutionary patterns. The two kinds of recognition sequences of replication origin suggest that the regulatory sequences involved in the replication initiation process of mtDNA have diverged through Orthoptera evolution.The unique Insect mitogenomes are generally compact with few intergenic spacers and possess stable gene content and organization. They are usually about 16 kb in size and bear 13 protein-coding genes (PCGs), 2 ribosomal RNA genes (rRNAs), 22 transfer RNA genes (tRNAs), and one control region (CR) that includes replication and transcription origins is indicated. Large arrowheads indicate the sites where major signals were observed and small arrowheads show the sites where minor signals were observed. Arrows indicate the direction of replication. The nucleotide sequence of L. migratoria, which potentially forms the stem-loop structure upstream of the ON, is underlined. The nucleotides highlighted in red represent the location of T-stretch or T-stretch variant.)Click here for fileThe potential stem-loop structure involving a T-stretch or T-stretch variant in cockroaches (A) and termites (B). The nucleotides highlighted in green represent the T-stretch variant.Click here for filePCR primers used in the present study.Click here for fileList of taxa used in the present analysis.Click here for file"} +{"text": "Infection with the human T-cell leukemia virus-1 (HTLV-1) is associated with a range of outcomes ranging from asymptomatic infection, to the development of HAM/TSP and adult T cell leukemia/lymphoma. Pathogenesis of these disorders involves complex interactions with the host immune system. Our previous studies showed that phorbol ester (PMA) and T cell receptor (TCR)-mediated activation of chronically infected CD4 T cells increased expression of HTLV-1 gene products . TCR stiWe also investigated mechanisms of HTLV-1 activation following PMA stimulation. Surprisingly, PMA treatment was associated with a rapid, marked stabilization of HTLV-1 tax/rex mRNA. Increased RNA stability represents a novel mechanism for increasing HTLV-1 gene expression in chronically infected cells."} +{"text": "Adult T cell leukemia/lymphoma (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) are caused by HTLV-I infection. In these patients, the antibody titer and provirus load are elevated compared to levels in asymptomatic carriers (AC). These methods that determine HTLV-I infection do not differentiate between AC, HAM/TSP patients, and ATL patients. We have reported on a Luciferase Immunoprecipitation System (LIPS), a highly sensitive, quantitative technology that can efficiently detect HTLV-I antibody responses in serum of infected individuals ["} +{"text": "In vitro, exposure of cultured BMCs to apelin led to a gradual increase in SDF-1\u00e1 and CXCR4 expression. Intramyocardial delivery of apelin-BMCs in post-MI mice resulted in a significant increase number of APJ+/c-kit+/Sca1+ cells in the injected area compared to GFP-BMCs treated post-MI mice. Treatment with apelin-BMCs increased expression of VEGF, Ang-1 and Tie-2 in post-MI mice. Apelin-BMCs treatment also significantly increased angiogenesis and attenuated cardiac fibrosis formation in post-MI mice. Most importantly, treatment with apelin-BMCs significantly improved left ventricular (LV) systolic function in post-MI mice. Mechanistically, Apelin-BMCs treatment led to a significant increase in Sirtuin3 (Sirt3) expression and reduction of reactive oxygen species (ROS) formation. Treatment of cultured BMCs with apelin also increased Notch3 expression and Akt phosphorylation. Apelin treatment further attenuated stress-induced apoptosis whereas knockout of Sirt3 abolished anti-apoptotic effect of apelin in cultured BMCs. Moreover, knockout of Sirt3 significantly attenuated apelin-BMCs-induced VEGF expression and angiogenesis in post-MI mice. Knockout of Sirt3 further blunted apelin-BMCs-mediated improvement of cardiac repair and systolic functional recovery in post-MI mice. These data suggest that apelin improves BMCs therapy on cardiac repair and systolic function in post-MI mice. Upregulation of Sirt3 may contribute to the protective effect of apelin-BMCs therapy.Our previous study shows that treatment with apelin increases bone marrow cells (BMCs) recruitment and promotes cardiac repair after myocardial infarction (MI). The objective of this study was to investigate whether overexpression of apelin in BMCs improved cell therapy and accelerated cardiac repair and functional recovery in post-MI mice. Mouse myocardial infarction was achieved by coronary artery ligation and BMCs overexpressing apelin (apelin-BMCs) or GFP (GFP-BMCs) were injected into ischemic area immediately after surgery. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health .in vivo, the experimental mice received intraperitoneal (i.p.) apelin daily for 14 days after myocardial ischemia. At 24 hours and 14 days of myocardial ischemia, BM\u2013derived mononuclear cells were obtained by flushing the tibias and femurs with 10% FBS EGM. Immediately after isolation, 105 BM\u2013derived mononuclear cells were plated into 6-well culture plates. After 4 days of culture, the nonadherent cells were removed and the adherent cells were washed three times with phosphate-buffered saline solution (PBS). BMC isolated from post-MI mice were cultured and harvested for the western blot analysis To assess the role of apelin on BMCs activity of post-MI mice 5 cells per dish) were then seeded in 2% methylcellulose medium. After 7 days of incubation, BMCs colony formation and colony number were scored under phase-contrast microscopy BM\u2013derived mononuclear cells were isolated from sham control, post-MI 14 days, apelin treated sham control and apelin treated post-MI mice. BMCs (109 PFU) or Ad-GFP (1\u00d7109 PFU) The donor mice received intravenous tail vein injection of Ad-apelin (1\u00d7106 cells) either overexpressing GFP or apelin were anesthetized with ketamine (100 mg/kg) plus xylazine (15 mg/kg), intubated, and artificially ventilated with room air. Adequate anesthesia was monitored by toe pinch. Myocardial infarction was achieved by ligation of the left anterior descending coronary artery (LAD). Sham controls underwent surgery without the LAD r apelin .phox and gp91phox, APJ, VEGF, Jagged1, Notch3, CXCR-4 and SDF-1\u03b1 , and apelin antibodies. The densitometric analysis was carried out using image acquisition and analysis software (TINA 2.0).The hearts or cultured BMCs were harvested and homogenized in lysis buffer for Western blot analysis. The membranes were immunoblotted with Ang-1 , beclin-1, LC3-I/II, Sirt3, Tie-2, Akt and eNOS, , p47+/Sca1+ and c-kit+/APJ+ cells in the injected area were assessed by counting the number of positive cells per 100 nuclei Heart tissue sections (8 \u00b5m) from injected area of ischemia were incubated with APJ, Sca1 and c-kit antibodies overnight. Sca1 and c-kit were visualized using FITC labeled goat anti-mouse IgG antibodies; APJ was visualized with Fluorolink\u2122 Cy\u21223 labeled goat anti-mouse IgG antibodies. Myocardial APJ2) of tissue. Myocardial arteriole density was measured using image analysis software Eight-micrometer heart tissue sections were cut and incubated with fluorescerin-labeled Isolectin B4 and Cy3-conjugated anti-\u03b1 smooth muscle actin (SMA) . The number of capillaries (IB4-positive EC) was counted and expressed as capillary density per square millimeter (mmHeart tissue sections were stained with transferase deoxyuridine nick end labeling (TUNEL) following the manufacturer's instructions . Apoptosis was indexed by counting TUNEL positive cells per 100 nuclei in the infarcted tissue Experimental mice were anesthetized with ketamine (100 mg/kg) plus xylazine (15 mg/kg), intubated and artificially ventilated with room air. A 1.4-Fr pressure\u2013conductance catheter was inserted into the left ventricle (LV) to record baseline cardiac hemodynamics of the hearts Cardiac hypertrophy was assessed by measuring heart-to-body weight ratio at 14 days of post-MI. Cardiac \u03b2-myosin heavy chain (\u03b2-MHC) and atrial natriuretic peptide (ANP) expression were examined by western blot analysis. Cardiac fibrosis was stained with Masson's trichrome and quantified by measuring the blue fibrotic area P<0.05.The results are expressed as the mean \u00b1SD. Statistical analysis was performed using one way ANOVA followed by post hoc multiple comparisons test. Significance was set at Our recent study demonstrates that treatment of ischemic hearts with apelin increased the recruitment of BM derived progenitor cell into ischemia area +/c-kit+/Sca1+ cells in the injected area was evaluated after BMCs intramyocardial injection. As shown in +/c-kit+/Sca1+ cells in comparison with GFP-BMCs treatment. Next, we investigated whether intramyocardial injection with apelin-overexpressing BMCs affected angiogenic growth factors expression and angiogenesis in the ischemic border zone of post-MI mice. Treatment with GFP-BMCs significantly increased expression of VEGF, Ang-1 and Tie-2 as well as phosphorylation of Akt-473 and eNOS-1177. The expression of VEGF, Ang-1 and Tie-2 and phosphorylation of Akt-473 and eNOS-1177 were significantly higher in the border zone of apelin-BMCs treated mice compared to GFP-BMCs treated mice and +dP/dtmax pressure and a higher end-systolic volume (ESV) compared to non-ischemic sham controls for 0.5, 1, 2 and 24 hours, pro-survival and angiogenic growth factors were measured. Exposure of BMCs to apelin led to a gradual increase in SDF-1\u03b1 and CXCR4 expression at 0.5 hours and up to 24 hours . TreatmeTo further confirm the histological findings and functional effect of Sirt3 observed in the apelin-BMCs treated post-MI mice, parallel studies were performed in cultured BMCs exposed to serum free medium (starvation) to induce cell apoptosis. Exposure of BMCs to apelin (5 \u00b5M) for 48 hours significantly attenuated starvation-induced apoptosis compared to BMCs without apelin treatment. Knockout of Sirt3 significantly increased starvation-induced cell apoptosis in cultured BMCs. Furthermore, knockout of Sirt3 completely abolished the anti-apoptotic effect of apelin treatment .Similarly, transfection of WT-BMCs with Ad-apelin significantly attenuated starvation-induced apoptosis compared to GFP transfected BMCs. Transfection of Ad-apelin in Sirt3KO-BMCs failed to inhibit cell apoptosis A. FurthIn the present study, we have demonstrated that treatment of post-MI mice with overexpressing apelin-BMCs upregulates expression of Sirt3 and VEGF as compare to BMCs treatment. This is accompanied by a significant increase in myocardial angiogenesis. Apelin-BMCs treatment significantly increases LC3 and beclin-1 expression and attenuates myocardial apoptosis as compare to BMCs treatment. Apelin-BMCs treatment also significantly improves cardiac function in post-MI mice. Intriguingly, knockout of Sirt3 in cultured BMCs completely abolished apelin-mediated anti-apoptotic effect. Moreover, apelin-mediated cardiac repair and functional recovery are significantly blunted in Sirt3KO-BMCs. Our study suggests that the protective mechanism of apelin-BMCs may be due, in part, to upregulation of Sirt3 signaling pathway.phox and gp91phox expression as well as ROS formation in post-MI mice. Treatment with apelin-BMCs also increased autophagy gene beclin-1 and LC3-I/II expression. These are accompanied by dramatic reduction of myocardial apoptosis. Apelin-BMCs treatment further upregulated expression of angiogenic growth factors and dramatically increased angiogenesis in the injected area. Surprisingly, no GFP+-BMCs or GFP+-apelin-BMCs were found in hearts of post-MI mice after 14 and 28 days of BMCs injection, suggesting that apelin-overexpressed BMCs had not differentiated into neovessels in ischemic hearts. These data are consistent with a recent study revealed that apelin treatment improves cardiac repair by stimulation of myocardial angiogenesis but not through BMCs transdifferentaiton +/Sca1+/c-kit+ cells at ischemic area in post-MI mice. Moreover, the number of APJ+/Sca1+/c-kit+ cells was significantly reduced in Sirt3KO-apelin-BMCs accompanied by a significant decline of cardiac function in post-MI mice. Taken together, these data suggested that increased number of APJ+ stem cell at ischemia area via upregulation of SDF-1\u03b1/CXCR-4 pathway maybe responsible for apelin-BMCs therapy-mediated cardiac repair. It is unknown whether APJ+ stem cell releases more proangiogenic and anti-apoptotic factors than other stem cells during ischemia. Therefore more studies are needed to investigate the functional roles of APJ+ stem cell on cardiac functional recovery after MI.Acute myocardial ischemia has been shown to induce rapid mobilization of BMCs in vitro. Treatment of cultured BMCs with apelin or overexpression of apelin in BMCs significantly reduces stress-induced cell apoptosis. Our data also showed that treatment with apelin led to a greater phosphorylation of eNOS and upregulation of Notch3 expression in the BMCs isolated from 14 days of post-MI mice. These data suggest that overexpression of apelin in BMC may boost BMCs therapy via secretion of VEGF, activation of Notch3 and Akt/eNOS signaling pathway, which resulting in an improvement of cell survival and angiogenesis in post-MI mice. Our data further showed that injection of GFP-BMCs into ischemic hearts significantly ameliorated LV dilation, but it failed to improve LV systolic function in post-MI mice. Most intriguingly, injection of apelin-BMCs significantly improved each of these variables compared to GFP-BMCs treatment, indicating that apelin enhances cardiac systolic functional recovery of BMCs therapy in post-MI. Taken together, the present data implicate a therapeutic potential of apelin in the BMCs therapy during cardiac repair and functional recovery of post-MI.Our previous study indicates that apelin may promote cardiac repair and heart functional recovery of post-MI by increasing BM derived vascular progenitor cell homing and stimulating angiogenesis via a paracrine mechanism Sirtuins are a highly conserved family of histone/protein deacetylases whose activity can prolong the lifespan of organisms Autophagy has been reported to have a protective role in the heart following myocardial ischemia/reperfusion In summary, the current study provides evidence that overexpression of apelin in BMCs enhances the therapeutic efficiency of BMCs and improves systolic function in post-MI mice. Our data suggest that modification of BMCs with apelin could use as a novel cell-based therapy for the treatment of patients with myocardial ischemia and heart failure. Our findings further suggest that upregulation of Sirt3 may improve BMCs therapeutic effect for cardiac repair after MI.Figure S1A. Representative images and quantification of serum-free (starvation) induced cell apoptosis in cultured BMCs isolated from WT and Sirt3 KO mice transfected with Ad-apelin and Ad-GFP. Transfected of BMCs with Ad-apelin attenuated starvation-induced cell apoptosis. Starvation-induced cell apoptosis was significantly increased in cultured BMCs of Sirt3KO mice transfected with Ad-apelin compared to that of WT mice transfected with Ad-apelin . B. Transfected of BMCs with Ad-apelin significantly increased cell proliferation compared to GFP transfected BMCs. The proliferative rate of BMCs was significantly reduced in cultured BMCs of Sirt3KO mice compared to that of WT mice transfected with Ad-apelin as measured by MTT method .Overexpression of apelin reduces apoptosis and increases proliferation of cultured BMCs. (DOCX)Click here for additional data file."} +{"text": "Pharmacological inhibition of the MAPK pathway with MEK or BRAF antagonists has proved successful in inducing regression of melanoma tumors bearing the targeted activating mutations. Moreover, antibodies targeting T-cell immune checkpoint inhibitors CTLA-4 or PD-L1/PD-1 have demonstrated the capacity to generate durable responses in patients with multiple cancer types. Thus, combining MAPK pathway-targeted agents with antibodies that enhance anti-tumor immunity represents an increasingly attractive treatment paradigm for cancer. However, little is known about the impact of tumor-targeted agents on immune function as similar signaling pathways drive both T-cell activation and cancer cell proliferation. Accordingly, agents targeting MAPK-dependent tumor growth would be predicted to also inhibit T-cell immunity. Here we show that, unexpectedly, potent suppression of T-cell receptor (TCR) function by MEK inhibition can be largely overcome in the presence of co-stimulation by anti-CD28 in vitro or blockade of the inhibitory PD-L1/PD-1 pathway in T cells in vivo. The ability of anti-CD28 to override suppression of T-cell activation by MEK inhibitors was dependent on the PI3K/mTOR pathway. Enhanced anti-tumor activity was also observed combining MEK inhibition with PD-L1 blockade, which was likely potentiated by upregulation of tumor MHC Class I expression through inhibition of MEK. Interestingly, inhibitors targeting BRAF V600E mutations actually augmented TCR-driven proliferation in vitro and T-cell function in vivo when combined with a vaccine or blockade of PD-L1 exclusively in the context of a wildtype BRAF background. These data demonstrate that targeting the MAPK pathway can be compatible with or even enhance T-cell function and provide rationale for combining these inhibitors with immunotherapy in clinical trials."} +{"text": "The heart responds to myriad stresses by well-described transcriptional responses that involve long-term changes in gene expression as well as more immediate, transient adaptations. MicroRNAs quantitatively regulate mRNAs and thus may affect the cardiac transcriptional output and cardiac function. Here we investigate miR-499, a microRNA embedded within a ventricular-specific myosin heavy chain gene, which is expressed in heart and skeletal muscle.Egr1, Egr2 and Fos), \u00df-myosin heavy chain (Myh7), and skeletal muscle actin (Acta1). We verified the effect of miR-499 on the immediate early response genes by miR-499 gain- and loss-of-function in vitro. Consistent with a role for miR-499 in blunting the response to cardiac stress, asymptomatic miR-499-expressing mice had an impaired response to pressure overload and accentuated cardiac dysfunction.We assessed miR-499 expression in human tissue to confirm its potential relevance to human cardiac gene regulation. Using a transgenic mouse model, we found that elevated miR-499 levels caused cellular hypertrophy and cardiac dysfunction in a dose-dependent manner. Global gene expression profiling revealed altered levels of the immediate early stress response genes (Elevated miR-499 levels affect cardiac gene expression and predispose to cardiac stress-induced dysfunction. miR-499 may titrate the cardiac response to stress in part by regulating the immediate early gene response. Egr1 and the protooncogene Fos, both of which are involved in the cardiac stress response Heart failure affects over five million individuals in the United States and is characterized by progressive cardiac dysfunction. Such cardiomyopathies are characterized by significant changes in gene expression MicroRNAs are small non-coding RNAs that play an important role in gene regulation. Each of the over 650 known human microRNAs likely regulates hundreds of mRNAs and thus has the potential to affect many biologic processes, although the effect on rapid transcriptional responses has not been extensively studied. Some microRNAs are expressed ubiquitously while others display tissue-specific expression patterns, suggesting unique functions within tissues. Several microRNAs, including miR-1, miR-133, miR-206 and miR-208 Myh7B) and is highly enriched in the cardiac ventricles. miR-499 plays a role in myosin gene regulation In screening for microRNAs enriched in the human heart, we identified an abundant microRNA, miR-499, which has been the subject of several recent studies. Global microRNA expression profiling studies have identified miR-499 in the heart , Table S1), along with the well-studied microRNAs, miR-1 and miR-133. miR-499 is distinct from miR-1 and miR-133 in that it is encoded in only one genomic locus. By genomic sequence alignment, miR-499 is completely conserved throughout evolution with the exception of a single nucleotide change in chicken , had hearts that appeared unremarkable under basal conditions Fig. 3A.in vitro target of miR-499 by cloning the Sox6 3\u2032-UTR and then tested for microRNA repression by luciferase assays. We transfected increasing amounts of miR-499 into 293T cells in culture and found dose-dependent inhibition of the Sox6 UTR-luciferase construct, however another cardiac microRNA, miR-133, had no effect regardless of the dose (Myh7 (\u00df-MyHC) transcript, while Sox6 mRNA was unaltered, similar to its protein levels that did not display overt cardiac dysfunction. RNA was prepared from the ventricles of postnatal day 17 miR-499 transgenic mice or littermate controls, and gene expression was compared by microarray analysis Fig. 5A.natriuretic peptide precursor type B (Nppb), \u00df-myosin heavy chain (Myh7), and alpha 1 skeletal muscle actin (Acta1), genes that are known to be upregulated in hypertrophy, were increased 2.0, 1.9, and 1.6 fold, respectively . We noted that the most downregulated transcripts were the immediate early response genes, early growth response 1 (Egr1) and FBJ osteoscarcoma oncogene (Fos) the immediate early response genes are rapidly altered in response to cardiac stress by serum Fig. 6A,in vivoWe next investigated whether elevated miR-499 levels affected the epidermal growth factor (EGF)-induced immediate early gene response We assessed whether serum response factor (SRF), which is upstream of the immediate early genes, was altered in the miR-499 transgenics. When we compared SRF protein levels in WT and TG mice, there was no difference Fig. 6D,, Given the effect of miR-499 levels on the immediate early gene response, which is known to be involved in cardiac stress and hypertrophy Table S2). None of the genes were predicted by Targetscan. Importantly, pathway analysis revealed enrichment for the GO terms sarcomere (P\u200a=\u200a0.0074), contractile fiber part (P\u200a=\u200a0.0085), myofibril (P\u200a=\u200a0.0095), and contractile fiber (P\u200a=\u200a0.010), further highlighting the potential role of miR-499 in regulating sarcomeric function in the heart.To determine the dose-responsiveness of miR-499 sensitive genes, we analyzed gene expression in the higher miR-499 expressing TG-9 line at 17 days of age. Thirty-two common transcripts were altered in both TG-17 and TG-9, and thirty of these were greater in magnitude in TG-9 than TG-17 , \u00df-MyHC (Myh7) and alpha 1 skeletal muscle actin (Acta1). This suggests gene expression changes due to miR-499 that favor hypertrophy. These changes were confirmed in the second transgenic line. The limited magnitude of transcript changes by miR-499 suggests that the hypertrophic phenotype observed was not due to gross gene expression disruption. Although the Sox6 3\u2032UTR could be targeted by miR-499, as we and others have demonstrated in vivo, or that additional compensatory mechanisms may be involved in vivo. It is possible that targets seen in luciferase assays may not necessarily translate into targeting in vivo.We identified a discrete set of altered transcripts in unmanipulated, normally functioning, miR-499 transgenic hearts, including natriuretic peptide precursor type B . Second, inhibition of miR-499 in culture led to increases in induced Egr1 and Fos levels, which demonstrates that this regulation is present even outside of the context of the cardiac transgene. Pressure-induced stress, which is known to invoke the immediate early gene response, dramatically altered cardiac function in transgenic mouse hearts. We therefore hypothesize that miR-499 alters the cardiac response to stress in part by modulating the immediate early gene response.The connection between miR-499 and the immediate early gene response is important since activation of this pathway is thought to precede further transcriptional responses to stress. Similar to TG-17 mice expressing miR-499, mice deficient in Egr1 are normal at baseline, but have an impaired response to cardiac stress Care and handling of all experimental animals used in this study were in accordance with the University of California San Francisco's Institutional Animal Care and Use Committee policies and approved under protocol #AN080001.32P-labelled locked nucleic acid probe (Exiqon) or DNA oligonucleotide probe complementary to mature miR-499 or to U6. We transfected 293T cells (ATCC) with miR-499-expression construct or vector alone for RNA for positive and negative controls. Signals were detected by phosphoimaging.Total RNA (12 mcg) was separated on 12% polyacrylamide/urea gels and transferred to nitrocellulose membranes. Membranes were probed with RNA samples were reverse transcribed for microRNA detection with microRNA-specific primers (Applied Biosystems) or for mRNAs with oligo-dT (Invitrogen). miR-499 levels and mRNAs were quantified using RNA from transgenic mouse hearts and littermate controls. Quantitative real-time PCR (qPCR) was performed in an ABI HT7900 with Taqman primers (Applied Biosystems). Taqman primers used: miR-499 (001352), miR-16 (000391), \u00df-myosin heavy chain , Sox6 (Mm01274768_m1), Egr1 (Mm00656724_m1), Egr2 (Mm00456650_m1), Fos (Mm01302932_g1).We amplified fragments containing miR-499 from genomic DNA using high-fidelity PCR and directional cloning into pcDNA3.1-TOPO (Invitrogen) for expression studies in cell culture. The UCSC genome browser and Sanger miRBase were used to analyze cloned products, to assess microRNA conservation, and for primer design.We surveyed mutliple bioinformatics prediction programs that predict potential microRNA targets. MiRanda, TargetScan, and Mirtarget in vivo, we amplified DNA surrounding the mouse pre-miR-499 region and cloned the 574-bp fragment into the alpha-myosin heavy chain promoter vector using the following primers and SalI linkers: 5\u2032-CGT GTC GAC CAA GTC TGG GGT GAA AGA GAA G-3\u2032 (forward), 5\u2032-TGT GTC GAC GGT CAT GAG CTT GTT GAG GTT C-3\u2032 (reverse) and injected into one-cell FVB/NCrl embryos before implantation in pseudopregnant females. Phenotyping was performed by echocardiography with standard measurement techniques or vehicle was injected intraperitoneally into control or transgenic mice, and hearts were collected after 1.5 hours for analysis of immediate early gene expression.To express miR-499 Protein lysates were run on SDS-PAGE gels, transferred to PVDF membranes, and probed with antibodies to Sox6 , slow myosin , SRF , or Gapdh (Santa Cruz).http://www.ncbi.nlm.nih.gov/geo/) series accession number GSE21104 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=nrqpfuaaqeacsxo&acc=GSE21104). Total RNA was isolated from ventricular tissue from three miR-499-transgenic mice and three littermate controls at postnatal day 17. Gene expression was assessed using Mouse Gene 1.0 ST Array as recommended by the manufacturer (Affymetrix). Differentially expressed genes were identified using the limma package. Adjustment for multiple testing was performed as described in the text. Further validation was performed using qPCR as described above. Human heart and liver RNA samples (Virogen), gestational age 6\u20138 weeks, were labeled and run for microRNA expression as described by the manufacturer (Exiqon). Gene ontology analysis was performed using DAVID Bioinformatics Resources 6.7 Data have been deposited in The National Center for Biotechnology Information (NCBI) Gene Expression Omnibus or a morpholino directed against the miR-499 precursor or a scrambled control morpholino (GeneTools) was electroporated (Amaxa) into H9c2 and allowed to recover overnight. After serum starvation in 1% fetal bovine serum, 10% serum was applied for designated times and RNA collected for RT-PCR analysis. Sequence of morpholino directed against the miR-499 precursor Total RNA was isolated using guanidine thiocyanate and phenol and following manufacturer's instructions for chloroform extraction, isopropanol precipitation, and 75% ethanol washing. For tissue RNA extraction, tissues were processed using a bead homogenizer (WisBiomed).5\u2032-CAC CCA AGT CTG GGG TGA AAG AGA AG-3\u2032 (forward) and 5\u2032-GGT CAT CAG CTT GTT GAG GTT C-3\u2032 (reverse). 3\u2032UTR sequence primers: Egr1 5\u2032-GGC AGG AAA GAC ATA AAA GCA C-3\u2032 (forward) and 5\u2032-ACA TAT CCC ATG GGC AAT AGA G-3\u2032 (reverse), Egr2 5\u2032-AAC ACT ACC ACC CTT CCC TGT T-3\u2032 (forward) and 5\u2032-AGC CAT CCA TTA TCT GAA CTC C-3\u2032 (reverse), Fos 5\u2032-TGG TGC ATT ACA GAG AGG AGA A-3\u2032 (forward) and 5\u2032-TGG AAC AAT AAG CAA ACA ATG C-3\u2032 (reverse), Sox6 5\u2032-CTC ACT AGT TGG CTC CAC AAT ACA TCA GC-3\u2032 (forward) and 5\u2032-CTC AAG CTT CCA AGT GAC AAA ATG GCT CA-3\u2032 (reverse).Genomic amplification primers for human miR-499: Table S1Enrichment of microRNAs in human heart versus liver. Log ratio for each microRNA and standard deviation are shown. Positive ratios indicate enrichment in heart compared to the liver.(DOC)Click here for additional data file.Table S2Common genes altered in miR-499 transgenic lines, TG-17 and TG-9. For each gene, the gene symbol, genomic location, log ratios and fold change of TG versus WT are shown.(XLS)Click here for additional data file."} +{"text": "The use of cytokine inhibitors has been a major progress in the treatment of chronic inflammation. However, not all patients respond and response will be often lost when treatment is stopped. These clinical aspects indicate that other cytokines might be involved and we focus here on the role of IL-17. In addition, the chronic nature of joint inflammation may contribute to reduced response and enhanced chronicity. We had previously observed that patients not responding well to TNF inhibition had higher blood expression of synoviolin, an E3 ubiquitin ligase previously shown to be implicated in synovial hyperplasia in human and mouse rheumatoid arthritis (RA). Therefore we studied the capacity of IL-17 to regulate synoviolin in human RA synoviocytes and in chronic reactivated streptococcal cell wall (SCW)-induced arthritis.Chronic reactivated SCW-induced arthritis was examined in IL-17R deficient and wild-type mice. Synoviolin expression was analysed by real-time RT-PCR, Western Blot or immunostaining in RA synoviocytes and tissue, and p53 assessed by Western Blot. Apoptosis was detected by annexin V/ propidium iodide staining, SS DNA apoptosis ELISA kit or TUNEL staining and proliferation by PCNA staining. IL-17 receptor A (IL-17RA), IL-17 receptor C (IL-17-RC) or synoviolin inhibition were achieved by small interfering RNA (siRNA) or neutralizing antibodies.IL-17 induced sustained synoviolin expression in RA synoviocytes. Sodium nitroprusside (SNP)-induced RA synoviocyte apoptosis was associated with reduced synoviolin expression and was rescued by IL-17 treatment with a corresponding increase in synoviolin expression. IL-17RC or IL-17RA RNA interference increased SNP-induced apoptosis, and decreased IL-17-induced synoviolin. IL-17 rescued RA synoviocytes from apoptosis induced by synoviolin knockdown. IL-17 and TNF had additive effects on synoviolin expression and protection against apoptosis induced by synoviolin knowndown. In IL-17R deficient mice, a decrease in arthritis severity was characterized by increased synovial apoptosis, reduced proliferation and a marked reduction in synoviolin expression. A distinct absence of synoviolin expressing germinal centres in IL-17R deficient mice contrasted with synoviolin positive B cells and Th17 cells in synovial germinal centre-like structures.IL-17 induction of synoviolin may contribute in part to RA chronicity by prolonging the survival of RA synoviocytes and immune cells in germinal centre reactions. These results extend the role of IL-17 to synovial hyperplasia."} +{"text": "Cine SSFP sequences have become the gold standard for assessment of myocardial function at 1.5T. However, cine imaging at higher-field strengths is hampered by SAR limitations, the increased inhomogeneity of the B1 (RF) and B0 field and the high sensitivity of SSFP sequences to off-resonance artefacts. The effect is even more pronounced in HDDS studies, which are associated with a significant increase in heart rate and consequently more complex and faster blood flow. Previously, spoiled gradient-echo sequences have been proposed as an alternative for stress studies at 3T. Recently, the introduction of a dual-source RF transmission system with patient-adaptive local RF-shimming has lead to a significant improvement of image quality of SSFP imaging at 3T during resting conditions and may thus allow for reliable imaging of cardiac function even at high heart rates.To investigate the feasibility of high-dose dobutamine stress (HDDS) imaging using SSFP sequences at 3T employing patient-adaptive local RF-shimming using a dual-source RF transmission system.B-SIM)/(SIB+SIM)].8 Patients with suspected CAD underwent a HDDS protocol on a 3T MRI scanner , equipped with a dual-source RF transmission system. SSFP cine sequences at every stress level were performed in the short (SA), vertical (VLA) and horizontal long axis (HLA) using patient-adaptive local RF-shimming (RF-S) and compared to cine images acquired with identical sequence parameters at rest and maximal stress, without additional shimming. Overall image quality was evaluated on a 4-point grading scale and number of non-diagnostic segments assessed. Contrast between interventricular septum and blood pool was calculated [ and in both long axis at rest compared to sequences without RF-S. The amount of non-diagnostic segments was significantly reduced when using RF-S at rest and maximum stress with RF-S, the difference was not statistically relevant.Patient-adaptive local RF-shimming using a dual-source RF transmission MRI system allows for reliable SSFP imaging in a clinical high-dose dobutamine stress protocol. RF-S significantly improves image quality and reduces the number of non-diagnostic myocardial segments."} +{"text": "BSI-surveillance is thought to be useful in monitoring trends of healthcare-associated infections (HAI) including outbreaks, emerging multiresistant pathogens, and effects of HAI intervention programmes.Healthcare-associated bloodstream infection (BSI) is the 4Introduced 1995 in acute care, prospective and systematic BSI surveillance at the University of Geneva hospitals was extended to a hospital-wide survey in 2003. Positive blood culture results identified by an electronic automated alert system were individually investigated and allocated to the following categories: contamination, secondary BSI (sBSI), primary BSI (pBSI) and catheter-related BSI (CRBSI).A non-significant trend was detected for BSI to increase between 2003 (0.15 BSI/1000 patient-days) and 2009 (0.28/1000 patient-days) . This was predominantly due to rising rates of pBSI from 0.24/1000 patient-days in 2003 to 0.47/1000 patient-days in 2009 . No change was detected for sBSI and contaminations while CRBSI episodes increased between 2006 (0.17/1000 patient-days) and 2007 (0.27/1000 patient-days). All BSI-outcomes decreased in 2010 following a hospital-wide training in catheter care. No major outbreak was detected.Eight years BSI-surveillance did not show significant variation making this mode of surveillance less likely to be affected by confounding factors. Whether improved catheter care contributed to the reduced BSI-rates in 2010 needs to be confirmed.None declared."} +{"text": "To identify the genetic defects underlying retinitis pigmentosa (RP) in Pakistani families.Genome-wide high-density single-nucleotide-polymorphism microarray analysis was performed using the DNA of nine affected individuals from two large families with multiple consanguineous marriages. Data were analyzed to identify homozygous regions that are shared by affected sibs in each family. Sanger sequencing was performed for genes previously implicated in autosomal recessive RP and allied retinal dystrophies that resided in the identified homozygous regions. Probands from both families underwent fundus examination and electroretinogram measurements.TULP1) was present in the largest homozygous region in both families. Sequence analysis identified a previously reported mutation in one family and a novel pathogenic variant in the other family. Both variants were found to be present in a homozygous state in all affected individuals, were heterozygous present in the unaffected parents, and heterozygous present or absent in normal individuals. Affected individuals of both families showed an early-onset form of RP.The tubby-like protein 1 gene (TULP1 in two large Pakistani families with early-onset retinitis pigmentosa.Homozygosity mapping, combined with candidate-gene analysis, successfully identified genetic defects in The clinical symptoms of RP are the loss of night vision in the early phase of disease, later followed by peripheral vision loss, tunnel vision, and sometimes complete blindness -G-R-V-[ST]-x-A-S-V-K-N-F-Q) of the Tubby family of proteins, and this signature sequence contains 11 invariant amino acids that are highly conserved (Prosite) . ReplaceTULP1 involvement [In family A, the presence of a typical yellow-colored perifoveal annular ring was also indicative of olvement ; whereasThe previously identified mutation p.Thr380Ala has only been reported in two unrelated Pakistani families ,15. Our TULP1 mutations have been identified in 33 families (TULP1 mutations were found in 4.3% (10/231) of LCA families [TULP1 mutations causing arRP or LCA include two nonsense, two frame-shift, and seven splice-site mutations; a six-base-pair duplication; and 14 missense mutations and one previously identified disease-causing mutation in"} +{"text": "Nicotiana benthamiana plants in which alternative respiratory pathway capacity was either increased by constitutive expression of AOX, or decreased by expression of a dominant-negative mutant protein (AOX-E). N. benthamiana was used because it is a natural mutant that does not express a functional RNA-dependent RNA polymerase 1.Salicylic acid (SA) regulates multiple anti-viral mechanisms, including mechanism(s) that may be negatively regulated by the mitochondrial enzyme, alternative oxidase (AOX), the sole component of the alternative respiratory pathway. However, studies of this mechanism can be confounded by SA-mediated induction of RNA-dependent RNA polymerase 1, a component of the antiviral RNA silencing pathway. We made transgenic Aox transgenic plants while SA-induced resistance to this virus appeared to be stronger in Aox-E transgenic plants. These effects, which were limited to directly inoculated leaves, were not affected by the presence or absence of a transgene constitutively expressing a functional RNA-dependent RNA polymerase (MtRDR1). Unexpectedly, Aox-transgenic plants infected with potato virus X (PVX) showed markedly increased susceptibility to systemic disease induction and virus accumulation in inoculated and systemically infected leaves. SA-induced resistance to PVX was compromised in Aox-transgenic plants but plants expressing AOX-E exhibited enhanced SA-induced resistance to this virus.Antimycin A and SA triggered resistance to tobacco mosaic virus (TMV). Resistance to TMV induced by antimycin A, but not by SA, was inhibited in We conclude that AOX-regulated mechanisms not only play a role in SA-induced resistance but also make an important contribution to basal resistance against certain viruses such as PVX. Salicylic acid (SA) is an important defensive signal in plants that is required for elicitor-triggered immunity and the establishment of systemic acquired resistance (SAR)-8. PlantRNA silencing is thought likely to contribute to SA-induced virus resistance although it is unlikely to be the only mechanism involved . RNA silThere is evidence that mitochondrial signaling processes regulate some aspects of SA-induced virus resistance . ReacAox1a gene expression or 1 mM SA dissolved in 0.05% (w/v) ethanol before rub inoculation with PVX or TMV on one or two lower leaves as previously described for tobacco [For whole-plant treatments with SA, five-to-six week old tobacco ,20. ChemWSL and FSF carried out the experimental procedures, with additional experiments by JVL on PVX accumulation. JPC initiated and directed the research and wrote the manuscript. All authors read and approved the final manuscript.Nicotiana benthamiana plant lines used in this studyDetection of AOX-E protein expression and over-expression of AOX protein in transgenic . Immunoblot detection of AOX (A) or mutant AOX-E (B) present in non-transgenic (NT) N. benthamiana plants and T2 generation transformed plants belonging to various independent lines (numbered) harboring AOX or AOX-E transgenes expressed under the control of the 35S constitutive promoter. Equal amounts of Triton X-100 soluble proteins were denatured in the presence of 0.1 M dithiothreitol and subjected to immunoblot analysis using an anti-AOX monoclonal antibody. Anti-AOX binding was detected using anti-mouse immunoglobulin conjugated to horseradish peroxidase and a chemiluminescent substrate. A protein sample extracted from a plant of the Sn6 transgenic tobacco plant line, which over-expresses AOX , served as the Positive Control. The major cross-reacting band in all cases corresponded in size (apparent Mr c.35kDa) to the reduced form of AOX. Pre-stained Mr markers were not visible on the X-ray film. All lines express AOX or AOX-E at much higher levels than the native AOX protein which is not detectable on this western blot.Click here for fileN. benthamianaImage showing TMV-induced systemic symptoms of TMV on non-transgenic, control transgenic and MtRDR1-transgenic . Non-transgenic (NT) Nicotiana benthamiana plants, plants transformed with an 'empty' transformation vector (Control) and transgenic lines constitutively expressing MtRDR1 (35S:MtRDR1: Reference 22) were inoculated with TMV U1 strain (0.05 ug/ml) and photographed four weeks later. Mock-inoculated NT plants are shown for comparison. The scale bar is 8 cm.Click here for file"} +{"text": "Drosophila appendage-patterning genes encode DNA-binding proteins, whose cross-regulatory interactions remain to be better characterized at the molecular level, notably by studying their direct binding to tissue-specific transcriptional enhancers. A fine-tuned spatio-temporal expression of bric-a-brac2 (bab2) along concentric rings is essential for proper proximo-distal (P-D) differentiation of legs and antennae. However, within the genetic interaction landscape governing limb development, no transcription factor directly controlling bab2 expression has been identified to date. Using site-targeted GFP reporter assay and BAC recombineering, we show here that restricted bab2 expression in leg and antennal imaginal discs relies on a single 567-bp-long cis-regulatory module (CRM), termed LAE . We show that this CRM (i) is necessary and sufficient to ensure normal bab2 activity in developing leg and antenna, and (ii) is structurally and functionally conserved among Drosophilidae. Through deletion and site-directed mutagenesis approaches, we identified within the LAE essential sequence motifs required in both leg and antennal tissues. Using genetic and biochemical tests, we establish that in the LAE (i) a key TAAT-rich activator motif interacts with the homeodomain P-D protein Distal-less (Dll) and (ii) a single T-rich activator motif binds the C2H2 zinc-finger P-D protein Rotund (Rn), leading to bab2 up-regulation respectively in all or specifically in the proximal-most ring(s), both in leg and antenna. Joint ectopic expression of Dll and Rn is sufficient to cell-autonomously activate endogenous bab2 and LAE-driven reporter expression in wing and haltere cells. Our findings indicate that accuracy, reliability and robustness of developmental gene expression do not necessarily require cis-regulatory information redundancy.Most identified Drosophila, leg and antennal development along the proximo-distal (P-D) axis relies on relatively-well known genetic cascades, in which most appendage-patterning genes encode transcription factors (TF). However, their cross-regulatory interactions remain to be better characterized at the molecular level. A fine-tuned expression of the bric-a-brac2 (bab2) gene is essential for normal leg and antennal segmentation. However, within the genetic cascades governing P-D limb development, no TF directly controlling bab2 expression has been identified to date. We show here that restricted bab2 expression in developing leg and antenna is governed by a single enhancer, termed LAE, which is necessary and sufficient in-vivo to ensure bab2 functions there. We show that leg and antennal cis-regulatory elements are closely associated and that essential LAE sites interact with Distal-less (Dll) and Rotund (Rn) TFs, leading to bab2 activation in all or specifically in the proximal-most expressing cells, respectively. Finally, joint ectopic expression of Dll and Rn is sufficient to instruct wing and haltere cells to up-regulate bab2. Taken together, our work indicates that a single enhancer is necessary and sufficient to reliably govern bab2 expression in distinct morphogenetic fields.In insects, leg and antenna are homologous limbs, though derive from a single ancestral appendage. In Starting from a previous systematic identification of tissue-specific enhancers within the 150-kilobase (kb)-long bab locus performed by Williams et al. bab2 endogenous gene, both in leg and antennal tissues. This CRM is physically and functionally conserved between D. melanogaster and D. virilis. We find that the LAE is both necessary and sufficient in-vivo to ensure proper bab2 expression in leg and antennal imaginal discs, and for normal segmentation of the mature appendices. Using targeted deletions and site-directed mutagenesis, we show that leg and antennal cis-regulatory elements are closely associated. Furthermore, activation of bab2 expression in proximal- and distal-most rings is dependent on separate DNA elements. Moreover, we show that discrete essential LAE sites interact with Distal-less and Rotund transcription factors leading to bab2 activation in all or specifically in the proximal-most expressing cells, respectively. Finally, ectopic co-expression of Dll and Rn is sufficient to instruct wing and haltere cells to up-regulate bab2. Taken together, our work indicates that a single enhancer, under the direct control of the P-D proteins Dll and Rn, is necessary and sufficient to reliably govern Drosophila bab2 expression in distinct limb morphogenetic fields.Although several bab locus identified leg-specific cis-regulatory elements within a 11 kb region encompassing two overlapping genomic fragments (termed BP42 and BP47) localized between the bab1 and bab2 transcription units bab2 transcription unit and the LAE-containing intergenic region to rescue AR07bab phenotypes. Normal leg segmentation and limb-specific Bab2 expression were restored by a single copy of the LAE-bab2cDNA construct , that removed the CR3 motif, led to a modest decrease in GFP expression without affecting expression in the right cells (S5 construct) further reduced the level of expression (35 and 32% in leg vs. antenna) (S1 construct), removing the CR2 sequence, led to a drastic reduction of the signal strength (10 and 3%), with a nearly-complete loss of GFP expression in the distal-most bab2-expressing cells, in either leg or antennal tissues led to complete loss of limb-specific GFP expression did not drive either leg- or antennal expression , we conclude that CR2 is also not sufficient for LAE activity in the distal-most bab2-expressing ring.Conversely, a 100-bp 5\u2032 deletion removing the CR1 sequence (pression . This inS3 construct) led to reduced expression levels in the two proximal-most leg rings (spanning tarsi ts1\u20132 and ts2\u20133) and the proximal antennal ring (spanning a3\u20134 segments) leg and antennal expression employ shared regulatory information; (iii) the 337 bp 3\u2032-part is only required for signal strength; and finally (iv) the 230 bp 5\u2032-part is critical for signal specificity, with a key role of the CR1 sequence, while the 32 bp 5\u2032-end and CR2 sequences are required quantitatively for normal expression levels in the proximal- and distal-most rings, respectively.Taken together these data indicate that (i) the entire LAE is required to recapitulate normal spatial, temporal and quantitative (LS1-8) detectably affected GFP expression qualitatively and/or quantitatively displayed strong defects. LS2 showed almost complete loss of GFP expression in both leg and antennal tissues , but more pronounced in the proximal-most leg and antennal ring LS7 showed a partial inter-ring de-repression in leg tissues; (ii) whereas LS3 and LS6 exhibited a slight GFP intensity decrease specifically in antennal tissues (50% each) .Taken together these site-directed mutagenesis data indicate (i) tightly-associated leg and antennal CR1 regulatory information, and (ii) that the LS2 sub-sequence is indispensible for LAE activity.cis-regulatory DNA elements, we next sought positively acting factors that directly bind there. We noticed that the critical LS2 motif is embedded within an A/T-rich sequence (ATTAATGGTAATAAAAA), including three potential homeodomain (HD)-binding sites (TAAT or ATTA motifs) of which two were disrupted in the LS2 mutant. Given that HD-containing Dll protein is cell-autonomously required for bab2 expression LAE-GFP reporter also requires Dll activity, using clonal analysis. As for endogenous bab2, GFP expression was abolished in mutant leg and antennal clones for SA1Dll, a protein-null allele . All LAE fragments including one to six of the 11 TAAT/ATTA motifs bound in-vitro translated Dll, albeit with distinct affinities , when tested individually. We therefore examined whether Dll binds with a higher efficiently to a larger DNA fragment including the three TAAT/ATTA motifs present in CR1 . As a matter of fact, Dll strongly bound this extended fragment (#6) (TAAT/ATTA motifs. Whereas singly-mutated (TAAT vs. CCCC) fragments bound Dll with lower affinity, the mutation of the double overlapping TAAT/ATTA sites showed a stronger effect, while Dll binding was abolished when all three TAAT/ATTA motifs were mutated (probe #7).In addition to the 3 present in CR1, the entire LAE comprises 8 additional putative HD-binding sites of which 7 are clustered within the CR2 and CR3 sequences and S2. finities . Unexpecent (#6) . Furtherin-vivo regulation by Dll, we then introduced the same TAAT-mutated sequence into the LAE-GFP reporter (H4 construct). The H4 construct no longer expressed GFP either in leg or antennal tissues has been fortuitously created in LS3 rotund (rn), a spineless target gene rn-Gal4 and UAS-GFP constructs. GFP expression takes place in the right cells and at the right time rn-Gal4 expression is only detected in the proximal-most Bab2-expressing tissues, and extends more proximally than bab2, particularly in antennal tissues . To analyse the role of rn on bab2 and LAE-driven expression, we generated mitotic clones (GFP deficient) of cells homozygous for the 16rn null allele. Strong cell-autonomous reduction of endogenous bab2 (in blue) and LAE-RFP reporter (in red) expression was then observed for leg and antennal clones overlapping the proximal-most bab2-expressing rings , we thenbab2 expression via the R32 sequence at the LAE 5\u2032end, specifically required for GFP reporter expression in the proximal-most tarsal and antennal bab2-expressing ring(s). To further confirm its functional requirement, we deleted the R32 sequence in the context of the 230-bp S5-GFP construct drove relatively strong GFP expression in the distal-most ring but only very weakly in the proximal-most ring(s), particularly in antennal tissues , that resembles binding sites for the vertebrate Rn homolog in-vitro translated Rn bound both probes, the R32 fragment was bound about 5-fold more than T13 embedded within a 13-bp-long T-rich (T13) sequence experiments, using the S5 and S10 reporter constructs. Ectopically-expressed Rn (Dll>Rn) activated endogenous bab2 and S5-GFP expression throughout the Dll-expressing leg and antennal cells this regulation is direct, via Rn binding to the LAE T13 sequence.Taken together with the clonal analyses, we deduce that (i) Rn activity is necessary and sufficient for bab2 and LAE-dependent reporter activation. This raised the possibility that Dll and Rn together can instruct cells to up-regulate the LAE. To investigate their joint instructive properties, we mis-expressed Dll, Rn or Dll+Rn in wing, haltere and eye tissues using flip-out GOF experiments bab2 is weakly expressed in restricted domains in developing dorsal appendages (bab2 (not shown). In equivalent analyses with mis-expressed Rn, eye, wing and haltere clones induced neither endogenous bab2 nor LAE-RFP expression (n>50 for each tissue) cell-autonomously activated both bab2 and LAE-RFP expression . Significantly, eye clones co-expressing Dll+Rn failed to activate endogenous bab2 and LAE-driven reporter genes (not shown), indicating tissue specificity for their joint instructive properties. These data establish that ectopic co-expression of Dll and Rn is sufficient in instructing dorsal appendage cells to activate endogenous bab2 and LAE-driven reporter expression, presumably adopting a \u201cproximal-ring transcriptional mode\u201d, supporting (i) their direct binding to the LAE and (ii) a tissue-specific functional synergy involving these two transcription factors.Sustained ectopic expression of Dll protein in developing wing disc induces the entire leg P-D differentiation program including bab2 expression in distal leg and antennal epithelial cells, from larval to adult stages. In contrast to the view that enhancer redundancy be a rule of thumb for developmental genes bric-a-brac locus is both necessary and sufficient to reliably govern gene expression in distinct limb morphogenetic fields. Further, we have shown that (i) the LAE regulatory activity is under the direct control of Dll, (ii) full expression in proximal-most rings depends on the direct binding of Rn, and (iii) each transcription factor interacts with a single critical binding site. Lastly, joint mis-expression of both Dll and Rn is sufficient to ectopically up-regulate endogenous bab2 as well as LAE-RFP expression in wing and haltere cells.In this report, we have characterized a 567-bp-long evolutionarily-conserved enhancer element ensuring Drosophila leg and antenna are thought to be homologous structures evolved from a common ancestral appendage, as shown by leg-to-antenna or antenna-to-leg transformations caused by mis-expression of P-D patterning or homeotic genes bab2 is the first example of a developmental gene for which a single transcriptional enhancer is shown to be both necessary and sufficient to accurately ensure a complex gene expression pattern in distinct limb morphogenetic fields. As none of our mutated LAE constructs specifically affected antennal or leg expression, our findings thus support the idea that an ancestral P-D genetic cascade emerged before limb diversification in insects.The bab2 expression in developing leg and antennal discs is dynamic and complex, going from broad regional expression at early L3 stage to precisely positioned rings, and later on, to graded expression at pupal and adult stages 26B15 BAC construct partially rescues the bab mutant abdominal pigmentation defects and \u201cmaster\u201d (limb) tissue-specific transcriptional enhancers with distinct functional constraints. defects , suggestbab2 is a direct target of the Dll homeodomain-containing transcription factor, acting through at least the critical AAATTAATGGTAAT composite binding site present in the CR1 sequence , two other direct Dll target genes bab2 expression starts only at the early-mid L3 stage, in the form of a circular domain within the Dll-expressing distal territory . For the first time, we report that ectopically-expressed Rn protein is sufficient to activate and maintain bab2 throughout the Dll expression domain, in both developing leg and antenna expression in wing and haltere but not in eye tissues, thus forming a context-specific instructive couple. The molecular basis of their functional synergy remains to be deciphered. Furthermore, in the proximal bab2-expressing domain, Dll and Rn are likely to function together with additional, still-unknown activators binding to LS4\u20135 and LS8 sequences expression is transient in leg tissues. In addition to their previously described roles in ts1\u20133 growth ss and rn counteract repressive activities of dac and/or bowl, both of which are dynamically expressed during the critical L3 stage bab2 activator should be present to relieve transient repression by bowl. Consistent with this view, ss activity represses bowl (and dac) expression rn activation. As bowl (and dac) expression has decayed in the tarsal cells which earlier transiently expressed ss and rn, maintenance of bab2 expression would no longer require Rn activity. Maintenance of antennal Rn (and Ss) expression may in fact counteract additional bab2 repressors whose expression persists throughout development, such as hth and spalt after egg laying (AEL) as a circular domain nested within the earlier-initiated Dll expression domain bab2. We suppose that a hypothetical distal activator (X) binding to CR2 , giving rise to a single large ring , a second ring emerges proximally, both in the antenna and the leg tissues , depending on transient Rn activity, and the two distal-most rings (dark green) emerge both by tarsal growth and inter-ring down-regulation (through at least the LS1-binding repressor). Of note, rotund activity is indirectly required for the appearance of the two distal-most rings, due to its role in ts3 growth Our findings, coupled with results of previous studies gulation . bab2 exg to CR2 may contbab1 and bab2 transcription units of 22 Drosophilidae species for which the entire bab locus sequence is available (not shown). This suggests strong topological constraints during evolution. As (i) the duplicated bab genes are merely co-expressed during leg and antennal development bab2 expression (this study) and (iii) no other limb-specific cis-regulatory regions could be identified within the 150-kb bab locus bab1 expression as well. Consistent with our assumption that strong functional constraints have operated during evolution, a LAE-like sequence with partially conserved CR1-2 sequences is even present between paralogous bab1 and bab2 genes of the tsetse fly Glossina morsitans , the T13 Rn-binding site is poorly conserved in D. virilis and related Drosophila subgenus species heat shocks at 38\u00b0C, in early first- to late second-instar larvae of genotypes: (i) y w hsFlp; FRT82B Ub-GFP/16FRT82B rn and (ii) y w hsFlp; SA1 FRT42Darm-lacZ FRT42D/Dll, respectively. Flip-out clones over-expressing either Dll, Rn or both Dll plus Rn were generated by 40mn heat shocks at 38\u00b0C, in second- or third-instar larvae of genotypes: (i) y w LAE-RFP hsFlp; Pact>y+>Gal4, UAS-GFP/UAS-Dll, (ii) y w LAE-RFP hsFlp; Pact>y+>Gal4, UAS-GFP/1UAS-Rn and (iii) y w LAE-RFP hsFlp; Pact>y+>Gal4, UAS-GFP/1UAS-Dll UAS-Rn, respectively. UAS-GFP.[S65T], 1 and rn-Gal4UAS-Rn lines were obtained from the Bloomington stock center. UAS-Dll and EM212-Gal4Dll line were provided by S. Cohen and M. Suzanne, respectively.D. melanogaster or D. virilis bab locus were amplified by standard PCR , cloned into pBP-S3aG, a home-made derivative of the attB-containing pS3aG plasmid obtained from T.M. Williams and S. Carroll bab2 minimal promoter. The pLAE-RFP and pLAE-Bab2 plasmid constructs were made by substituting the GFP insert of the pLAE-GFP construct by a pH2B-RFP insert (obtained from A. Vincent) or a bab2 full-length cDNA (from pNB-bab2), respectively. Site-directed mutagenesis (including linker scanning) was performed by PCR, using the overlap extension method attP genomic sites, including the 2A platform on the X chromosome that was used for all constructs reported in this study.Genomic DNA fragments from the D. melanogaster LAE were recovered by Blat analysis at the UCSC Genome Browser website (http://genome.ucsc.edu/cgi-bin/hgBlat?command=start) and aligned with MAFFT . The multiple alignment was then shaded with Boxshade (http://www.ch.embnet.org/software/BOX_form.html).The genomic sequences homologous to the Third-instar larval imaginal discs were prepared and stained using standard procedures. Confocal analyses were done with a LEICA TCS SP5 microscope. Rat anti-Bab2 in-vitro transcription/translation with T7 RNA polymerase and rabbit reticulocyte lysate . pET-Dll and pCS2-MycRn plasmid constructs were obtained from S. Cohen and P. Couso, respectively. EMSA experiments were performed mainly as described E. coli DNA polymerase I in presence of [\u03b1-32P]dCTP, and purified on mini Quick spin columns (Roche). Specific activities were roughly similar for all tested probes. Free and shifted probes were revealed with a PhosphorImager.Dll and Rn proteins were synthesized by coupled Figure S1bab2 expression. Leg (A\u2013F) and antennal (ant) (G\u2013L) imaginal discs from late third-instar larvae expressing GFP-reporter constructs (depicted in the left side) shown in bab2 expression.GFP-reporter constructs recapitulating leg and antennal (TIF)Click here for additional data file.Figure S2Drosophila species. The genomic sequences homologous to the D. melanogaster LAE were recovered by Blat analysis at the UCSC Genome Browser website (http://genome.ucsc.edu/cgi-bin/hgBlat?command=start) and aligned with MAFFT . The multiple alignment was then shaded with Boxshade (http://www.ch.embnet.org/software/BOX_form.html). The large (>20 bp) highly-conserved regions (CR1\u20133) are framed. Notice that D. mojavensis and D. willistoni LAE sequences have long inserts (1\u20132 kb) between the CR2 and CR3 regions.LAE sequence alignment from 22 (PDF)Click here for additional data file.Figure S3bab mutant abdominal phenotypes, independently of the LAE. (A) The 150-kb bab locus is shown (see bab2-containing 26B15 BAC (in blue) used for phenotypic rescue experiments is indicated, with the internal LAE depicted as a red box. The positions of the two abdominal-specific cis-regulatory elements, within the bab1 transcription unit, are indicated as yellow boxes. (B) Dorsal views of female abdomens from the wild-type (C) and AR07bab heterozygous, carrying none (D) or a 26B15 BAC construct copy either unmodified (E) or LAE-deleted (F), are shown. Whereas pigmentation of wild-type abdominal tergites on segment A2\u20136 is limited to posterior stripes, females carrying a single AR07bab allele display nearly fully-pigmented A5\u20136 segments, evoking male-specific pigmentation. For the A5\u20136 segments of each genotype, pigmentation extends toward the anterior are indicated by solid bars. The bab mutant pigmentation defects are partially rescued in females carrying either an unmodified or a LAE-deleted 26B15 BAC construct.The 26B15 BAC partially rescues hown see . The pos(TIF)Click here for additional data file.Figure S4LAE-GFP construct or one out of its four LS-mutated derivatives not shown in A linker-scanning mutagenesis of the critical CR1 region reveals functionally relevant motifs. (A) The sequences of the wild-type and of each mutated CR1 sub-region are shown in beneath the entire LAE structural organization, as determined by evolutionary conservation among Drosophilidae . For eac(TIF)Click here for additional data file.Figure S5Glossina morsitans. G. morsitans LAE-like sequence was identified though blast analyses using the Trace archive nucleotide blast server at NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and aligned with LAE sequences from representative Drosophilidae species, using MAFFT . Homology shading was made using BoxShade (http://www.ch.embnet.org/software/BOX_form.html). The CR1-2 sequences are framed and locations of the Rn and Dll binding sites are indicated above the alignment. Note that the Rn binding site is poorly conserved. (B) Sequence conservation of the CR1-encompassing LAE portion among Sophophora and Drosophila subgenera. Sequences were aligned and processed as above. The structural organization scheme of the entire D. melanogaster (Sophophora subgenus) and D. virilis (Drosophila subgenus) LAE sequences are shown in the middle part, with the aligned portions depicted with broken lines. The T13 Rn-binding site of D. melanogaster and the T13-like sequence of D. virilis are well conserved among Sophophora and Drosophila subgenus species, respectively (T-rich sequences are underlined). Note that the T13-like D. virilis sequence is located (i) in a 3\u2032-end extended CR1 and (ii) in an inverted orientation.LAE sequences have been conserved among Dipterans. (A) Sequence conservation of the limb-specificity LAE portion among Drosophilidae and in the Glossinidae (TIF)Click here for additional data file."} +{"text": "Metastasis via the lymphatic system is promoted by lymphangiogenesis. Alterations of the lymphatic channels during the progression of metastasis to regional lymph nodes (LNs) remain unexplored. To examine whether tumor-induced LN lymphangiogenesis controls metastasis to regional LNs, we investigated cervical LN metastasis in a mouse model of oral melanoma.Injection of B16F10 melanoma cells into mouse tongues replicated spontaneous cervical LN metastasis. We performed histological, immunofluorescent, and histomorphometric analyses of tumor-reactive lymphadenopathy and lymphangiogenesis in tumor-associated LNs. We investigated the expression of vascular endothelial growth factor (VEGF)-C and its receptor, VEGF receptor-3 (VEGFR-3), in tumor cells and tissues, and LNs by reverse transcription polymerase chain reaction and immunofluorescence.Tumor-associated LNs comprised sentinel LNs (SLNs) before and after tumor cell invasion (tumor-bearing SLNs), and LNs adjacent or contralateral to tumor-bearing SLNs. Extensive lymphangiogenesis appeared in SLNs before evidence of metastasis. After metastasis was established in SLNs, both LNs adjacent and contralateral to tumor-bearing SLNs demonstrated lymphangiogenesis. Interaction between VEGF-C-positive melanoma cells and VEGFR-3-positive lymphatic vessels was evident in tumor-associated LNs.LN lymphangiogenesis contributes a progression of tumor metastasis from SLNs to other regional LNs. The lymphatic system functions in regulating tissue fluid balance and immune cell trafficking, and it is involved in the pathogenesis of edema and metastasis. Tumor cell dissemination to lymph nodes (LNs) through the lymphatic system is common and early event in human malignant tumors. LN metastasis is the first sign of tumor progression in most malignant tumors, and is a crucial determinant in their staging, prognosis, and treatment . LymphatChanges in LNs begin before metastasis, a process termed tumor-reactive lymphadenopathy . RegionaLymphangiogenic factors promoting formation of tumor lymphatics and metastasis of tumor cells to LNs have been identified ,14. ThesAppropriate animal models are necessary to study detailed changes to regional LNs during lymphatic metastasis. To characterize LN metastasis, we established a mouse model of spontaneous LN metastasis according to Iwahashi et al. in which injection of B16 melanoma cells into mouse tongues is known to replicate spontaneous cervical LN metastasis . Althoug1. To histologically characterize regional LNs proximal to tumors.2. To investigate increased lymphangiogenesis in LNs by histomorphometric analysis of lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) -positive areas.3. To examine an interaction of VEGF-C with VEGFR-3 in LN lymphangiogenesis using dual immunofluorescence.Our results indicate that tumor-associated LNs show extensive lymphangiogenesis, which may facilitate further metastasis.in vitro until confluent and were detached with 0.25% trypsin/0.02% ethylenediaminetetraacetic acid (EDTA) solution. These cells were then used for the metastatic model, cell immunostaining, and total RNA extraction.The mouse melanoma cell line, B16/F10 (RCB2630), was provided by the RIKEN BRC through the National BioResource Center through the National Bio-Resource Project of the Ministry of Education, Culture, Sports and Technology . Cells were maintained in Dulbecco\u2019s modified Eagle\u2019s medium supplemented with 10% fetal calf serum and penicillin/streptomycin. Cells were cultured 5 in 50 \u03bcl DMEM) were injected submucosally into the left border of the tongue [Female C57BL/6 mice (6\u20138 weeks old) were purchased from Kyudo Co., Ltd. . All animal studies were conducted using protocols approved by the Animal Care and Use Committee, Fukuoka Dental College. For the spontaneous LN metastasis model, tumor cells in phosphate-buffered saline (PBS) was injected into sites of melanoma cell inoculation 15 min before sacrifice.Cervical LNs were excised 1\u201321 days after injection from three animals in each treatment group. On the terminal day, the weight of each LN was measured, and the specimens immediately frozen in liquid nitrogen. Frozen specimens were cut into sections of 6-\u03bcm thickness and stained with hematoxylin and eosin (HE) to visualize histopathological changes. Frozen sections were also used for immunofluorescence and extraction of total RNA.Tissue sections and B16F10 cells were fixed with 4% paraformaldehyde in PBS for 15 min at 4\u00b0C, then washed in PBS. To evaluate lymphangiogenesis in tumor-associated LNs, we simultaneously performed three types of double immunofluorescent staining on frozen sections comprising two mixtures of two primary antibodies, goat anti-mouse/rat tyrosinase-related protein 1 and biotinylated anti-mouse LYVE-1 and rat anti-mouse CD45RB and biotinylated anti-mouse LYVE-1 and a mixture of rat anti-mouse CD31 and biotinylated anti-mouse LYVE-1 for 2 h at room temperature. After washing with PBS, sections were incubated in a mixture of anti-goat immunoglobulin G (IgG) antibody conjugated with Alexa Fluor 488 or anti-rat IgG antibody conjugated with Alexa Flour 488 , and streptavidin conjugated with Alexa Fluor 568 for 30 min at room temperature. These two simultaneously incubated double immunofluorescence stainings were applied to examine the codistribution of VEGF-C and Fms-related tyrosine kinase 4 in tumor-associated LNs. A mixture of anti-rabbit IgG conjugated with Alexa Flour 488 and anti-rat IgG conjugated with Alexa Flour 568 was overlaid on tissue sections for 45 min at room temperature, followed by preincubation with mixture of rabbit anti-mouse VEGF-C and rat anti-mouse VEGFR-3 for 2 h. For immunofluorescent staining of B16F10 cells, paraformaldehyde-fixed cells were incubated with VEGF-C antibody for 1 h and were then visualized with anti-rabbit IgG conjugated with Alexa Flour 488 for 30 min at room temperature. Immunostained sections and cells were then counterstained with 4, 6-diamidino- 2-phenylindole .t-test. Data were presented as the mean \u00b1standard error and P values of < 0.05 were considered statistically significant.Lymphatic vessel area was measured in 616 x 484-mm LYVE-1-stained LN section images at 100x magnification using ImageJ . Statistical analysis was performed with the two-tailed Student\u2019s TTACAGACGGCCATGTACGA-3\u2019 (forward) and 5\u2019-TTTGTTAGCATGGACCCACA-3\u2019 (reverse: product size 288 bp), and human glyceraldehyde-3-phosphate dehydrogenase (G3PDH), 5\u2019-TCCACCACCCTGTTGCTGTA-3\u2019 (forward) and 5\u2019-ACCACAGTCCATGCCAT-3\u2019 (reverse: product size 450 bp). Amplification was performed by a thermal cycler for 35 cycles as follows: 30s of denaturation at 94\u00b0C, 30 s of annealing at 60\u00b0C, and 1 min of extension at 72\u00b0C for all primers. Amplified products were resolved on 1.2% agarose/Tris-acetate EDTA gels electrophoresed at 100 mV, and then visualized with ethidium bromide.Total RNA was isolated from B16F10 cells and serial frozen sections of tumor-bearing LNs by acid guanidiniumthiocyanate-phenol-chloroform extraction using an ISOGEN kit . Isolates were quantified, and their purity evaluated spectrophotometrically. Reverse transcription PCR (RT-PCR) was performed using the Access RT-PCR System according to the manufacturer\u2019s instructions. We used the following primers: human VEGF-C, 5\u2019-B16F10 melanoma cells reliably underwent metastasis to the tumor-draining cervical LNs following their injection into the tongues of syngeneic C57BL/6 mice . Conversely, LNs adjacent and contralateral to tumor-bearing SLNs showed a remarkable increase in LYVE-1-positive sinuses Figures A and B. Recent studies demonstrated that VEGF-C/VEGFR-3 signaling promotes tumor lymphangiogenesis and contributes to the promotion of metastasis ,14. We eDespite increasing evidence supporting involvement of the lymphatic system in the metastasis of various malignant tumors, little is known about the mechanism of continuous spreading of tumors via regional LNs. In this study, we established an experimental model of cervical LN metastasis to investigate changes in tumor-associated LNs such as SLNs before metastasis, tumor-bearing SLNs, and LNs adjacent or contralateral to tumor-bearing SLNs. We present three lines of evidence to support the conclusion that lymphangiogenesis is evident in tumor-associated regional LNs. First, all tumor-associated LNs exhibited tumor-reactive lymphadenopathy. Second, measurement of the LYVE-1-positive areas in tumor-associated LNs indicated extensive lymphangiogenesis. Third, immunohistochemical interaction of VEGF-C with VEGFR-3 was examined in LN lymphangiogenesis.Both macroscopic and microscopic observations indicate that LNs proximate to oral melanoma show tumor-reactive lymphadenopathy regardless of the presence of tumor cells. The dilated lymphatic sinuses evident in tumor-associated LNs differ from those evident in inflammatory lymphadenopathy, which are full of lymphocytes . These dImmunohistochemical quantification of the LYVE-1-positive area revealed lymphangiogenesis in all tumor-associated LNs. These results indicate that extensive lymphangiogenesis is significantly correlated with tumor-reactive lymphadenopathy in these LNs. In this study, tumor-induced lymphangiogenesis was evident in tumor-draining SLNs before tumor cell invasion. This supports recent observations that SLN lymphangiogenesis precedes tumor metastasis ,11. SLN VEGF-C mRNA and VEGF-C protein in cultured B16F10 cells and melanoma-bearing tissues. These results suggest that tumor cells are actively responsible for lymphangiogenesis by producing of VEGF-C. Double immunofluorescent staining showed that VEGF-C in tumor cells promotes increased expression of its receptor, Flt-4, on lymphatic endothelia. In both primary tongue tumors and tumor-bearing SLNs, lymphatic vessels close to tumor cells expressed Flt-4. Interestingly, an increase in Flt-4-positive LN sinuses was observed in all tumor-associated LNs. A recent study proposed that VEGF-C-induced lymphangiogenesis in SLNs promotes tumor metastasis to distant sites [In this study, we considered tumor-reactive lymphadenopathy to result from extensive lymphangiogenesis, suggesting that tumor-derived signals are transported via the lymphatic system to tumor-associated LNs where they induce lymphangiogenesis. Recent studies reported that VEGF-C activates lymphatic vessel growth by stimulating VEGFR-3 expressed on lymphatic endothelium ,14. RT-Pnt sites . In our In conclusions, our findings demonstrate that all tumor-associated LNs exhibit tumor-reactive lymphadenopathy, histologically characterized by extensive lymphangiogenesis. These data suggest that LN lymphangiogenesis is premetastatic condition in regional LNs and contributes to metastasis from SLN to remote LNs.LN: Lymph node; SLN: Sentinel lymph node; VEGF: Vascular endothelial growth factor; VEGFR: VEGF receptor; LYVE-1: Lymphatic Vessel Endothelial hyaluronan receptor 1; DMEM: Dulbecoo\u2019s Modified Eagle\u2019s medium; PBS: Phosphate-buffered Saline; HE: Hematoxylin and Eosin; TRP-1: Tyrosinase-related Protein 1; IgG: Immunoglobulin G Flt-4: Fms-related tyrosine kinase 4; RT-PCR: Reverse Transcription-polymerase Chain Reaction.The authors declare that they have no competing interests.RO and TI performed experiments, participated in the immunostaining, and prepared the manuscript. JO performed experiments, analyzed the data, and prepared the manuscript. KT participated in performing pathological examinations. All authors have read and approved the final manuscript."} +{"text": "Cardiac atrial natriuretic peptide (ANP) is an essential physiological regulator of arterial blood pressure and intravascular volume. Unlike other \"natriuretics\", which reduce interstitial fluid volume with little change in plasma volume, ANP has important extrarenal, endothelial actions that enable it to reduce plasma volume preferentially. Thus ANP, via its guanylyl cyclase-A (GC-A) receptor and cyclic GMP formation, enhances microvascular endothelial permeability to albumin, ultimately moving plasma fluid into interstitial pools. However, the cellular pathways mediating this effect are unknown.v Here we investigated the role of caveolae-mediated endothelial albumin transcytosis in the mechanism of increased permeability induced by ANP.-/- mice. In vitro, ANP increased fluorescent albumin uptake and transcytosis in cultured microvascular endothelial cells without affecting transendothelial electrical resistance. ANP stimulation of albumin uptake was abolished in GC-A- or cav-1-deficient endothelia. Lastly, enrichment of caveolae from mouse lung together with western blot analyses demonstrated that a population of GC-A receptors colocalizes with Cav-1 in caveolae microdomains.Intravital microscopy showed that the vasodilating effect of ANP on cremaster precapillary arterioles was similar in mice with endothelial-restricted deletion of GC-A (EC GC-A KO) and in control mice. In controls, ANP stimulated the extravasation of fluorescent albumin from cremaster postcapillary venules. This permeability effect was abolished in EC GC-A KO mice and in mice with ablated caveolin-1 (cav-1), the caveolae scaffold protein. Concomitantly, the ANP-induced acute systemic loss of intravascular fluid volume was abolished in EC GC-A KO and Cav-1ANP exerts two types of microvascular effects. It acts as endothelium-independent vasodilatator. In addition the peptide mildly stimulates transendothelial caveolae-mediated vesicular albumin transport through GC-A localized in membrane rafts and caveolae. This effect is independent from the vasodilating action. The ANP-mediated communication between the heart and the microcirculatory endothelium is essential for the maintenance of intravascular volume homeostasis."} +{"text": "Human Amnion-derived Mesenchymal Stem Cells (hAMSCs) were transplanted into the infarct and peri-infarct regions of a pig ischemia-reperfusion model. The hAMSC therapy improved cardiac systolic function post-MI, compared to control animals, and Cardiac MRI with Manganese-Enhanced MRI (MEMRI) was able to detect increased CNR from live populations of hAMSCs within infarct and peri-infarct zones, as confirmed by human nuclear antigen (hNA) immunostaining.Stem cell-mediated restoration of cardiac function after myocardial infarction (MI) has been reported; however, it remains unclear whether transplanted stem cells survive and engraft in the heart following transplantation. To investigate stem cell viability in vivo, our laboratory previously validated a Manganese-Enhanced MRI (MEMRI) contrast agent, EVP-1001-1 , that specifically enters live cardiac tissue in a pig ischemia-reperfusion (IR) injury model. T1-weighted MRI imaging following EVP-1001 injection delineates infarct from remote and border zones. EVP-1001-1 is also taken up strongly by live stem cells. In this study, EVP-1001-1 was used to track human amnion-derived mesenchymal stem cells (hAMSCs) after transplantation into pig hearts post-IR.Five adult farm pigs underwent one-hour IR to the mid LAD coronary artery. One week post-IR, pigs hearts were injected with either hAMSCs or normal saline into ~8 peri-infarct and infarct zones, using fluoroscopic guidance and a BioCardia catheter injection system . Cardiac MRI was performed to assess ventricular function , infarct % by Delayed Gadolinium Enhancement MRI (DEMRI), and evidence for cell survival using MEMRI at serial timepoints post-IR.The average EF was similar at baseline (57\u00b14% (n=5)) and 1 week post-IR (pre-injection EF: 24\u00b16%). DEMRI infarct size was also similar between the groups Figure . hAMSC shAMSC delivery to the peri-infarct and infarct zones post-IR improves left ventricular systolic function compared to saline-injected or control animals. Increased MEMRI CNR of the infarct zone is associated with positive hNA staining in hAMSC-treated hearts, providing evidence for live hAMSC populations weeks after cell delivery that may be contribute to improved ventricular function. MEMRI of transplanted hAMSCs may allow longitudinal tracking of transplanted cells in vivo.NIH-NHLBI: K08 (RD), R01 (PY)."} +{"text": "Self-amplifying RNAs (replicons) of positive-strand viruses such as alphaviruses are potentially safe and useful vectors for delivering vaccine antigens. Recombinant alphavirus replicon particles (VRP), carrying the self-amplifying RNA, protects rhesus macaques against SHIVSF162P4 challenge when used in a prime-boost regimen.Novartis VRPs are being further tested using a current state-of-the art physiologically relevant low-dose SIV virus swarm challenge. To meet the need for the large numbers of VRP an alphavirus packaging cell line (PCL) was used for VRP production. We manufactured, characterized, stability and small animal potency tested VRPs expressing SIVmac239 envelope (env) and gag/pol fusion proteins for a large macaque vaccine study. Macaques were co-immunized with both VRPs thrice followed by two boosts with an MF59-adjuvanted CHO cell-derived SIVmac239 trimeric env protein.Here we show that three VRP priming immunizations induce both env- and gag-specific IgG and T-cell responses, robustly. Binding env-specific IgG titers were demonstrable in 100% of animals with titers ranging from ~10000-400000. T-cell responses to env and gag developed in 80% of macaques when assayed using an IFN\u03b3 T-cell ELISpot. The MF59-adjuvanted Env protein by itself was also robustly immunogenic with Env-specific IgG titers and T-cell responses ranging from ~100000-1700000 (100% response) and ~100-1000 SFC/106 PBMCs (90% response), respectively. No adverse events were reported upon immunization of either the VRP or the MF59-adjuvanted env vaccines.We provide further details on the currently ongoing efficacy evaluations of these safe and immunogenic vaccines in a prime-boost regimen using repeated low-dose heterologous SIVsmE660 intra-rectal challenges. NIH Grant N01-AI-50007."} +{"text": "Although they share certain biological properties with nucleic acid based infectious agents, prions, the causative agents of invariably fatal, transmissible neurodegenerative disorders such as bovine spongiform encephalopathy, sheep scrapie, and human Creutzfeldt Jakob disease, propagate by conformational templating of host encoded proteins. Once thought to be unique to these diseases, this mechanism is now recognized as a ubiquitous means of information transfer in biological systems, including other protein misfolding disorders such as those causing Alzheimer's and Parkinson's diseases. To address the poorly understood mechanism by which host prion protein (PrP) primary structures interact with distinct prion conformations to influence pathogenesis, we produced transgenic (Tg) mice expressing different sheep scrapie susceptibility alleles, varying only at a single amino acid at PrP residue 136. Tg mice expressing ovine PrP with alanine (A) at (OvPrP-A136) infected with SSBP/1 scrapie prions propagated a relatively stable (S) prion conformation, which accumulated as punctate aggregates in the brain, and produced prolonged incubation times. In contrast, Tg mice expressing OvPrP with valine (V) at 136 (OvPrP-V136) infected with the same prions developed disease rapidly, and the converted prion was comprised of an unstable (U), diffusely distributed conformer. Infected Tg mice co-expressing both alleles manifested properties consistent with the U conformer, suggesting a dominant effect resulting from exclusive conversion of OvPrP-V136 but not OvPrP-A136. Surprisingly, however, studies with monoclonal antibody (mAb) PRC5, which discriminates OvPrP-A136 from OvPrP-V136, revealed substantial conversion of OvPrP-A136. Moreover, the resulting OvPrP-A136 prion acquired the characteristics of the U conformer. These results, substantiated by in vitro analyses, indicated that co-expression of OvPrP-V136 altered the conversion potential of OvPrP-A136 from the S to the otherwise unfavorable U conformer. This epigenetic mechanism thus expands the range of selectable conformations that can be adopted by PrP, and therefore the variety of options for strain propagation. Prions are infectious proteins, originally discovered as the cause of a group of transmissible, fatal mammalian neurodegenerative diseases. Propagation results from conversion of the host-encoded cellular form of the prion protein to a self-propagating disease-associated conformation. It is believed that the self-propagating pathogenic form exists in a variety of subtly different conformations that encipher prion strain information. Here we explored the mechanism by which prion protein primary structural variants, differing at only a single amino acid residue, interact with prion strain conformations to control disease phenotype. We show that under conditions of co-expression, a susceptible prion protein variant influences the ability of an otherwise resistant variant to propagate an otherwise unfavorable prion strain. While this phenomenon is analogous to the expression of genetically-determined phenotypes, our results support a mechanism whereby dominant and recessive prion traits are epigenetically controlled by means of protein-mediated conformational templating. Aplysia, antiviral innate immune responses Prion-mediated phenotypes and diseases result from the conformationally protean characteristics of particular amyloidogenic proteins. The prion state has the property of interacting with proteins in their non-prion conformation, thus inducing further prion conversion. The prion phenomenon has been described for a variety of different proteins involved in diverse biological processes ranging from translation termination in yeast, memory in In the case of mammalian neurodegenerative diseases the prion state is pathogenic as well as transmissible. A hallmark of such conditions is the inexorable progression of pathology between synaptically connected regions of the central nervous system (CNS), consistent with advancing cell-to-cell prion spread. Experimental transmission in several settings has been convincingly demonstrated in the case of the amyloid beta (A\u03b2) peptide which features prominently in Alzheimer's disease (AD), the intracytoplasmic protein tau, also involved in AD as well as various neurodegenerative diseases referred to as taopathies, and \u03b1-synuclein, the primary constituent of Lewy bodies found in Parkinson's disease (PD) C, to the corresponding prion, or scrapie form, PrPSc. Since TSEs share numerous properties with nucleic acid-based pathogens, including agent host-range, stable strain properties, and the ability to mutate and respond to selective pressure, early researchers assumed a viral etiology for these diseases. While this is not the case, the unequivocal infectivity of TSEs set these prions apart. Their singular capacity to cause fatal neurodegeneration in genetically tractable animal models, and the ability to propagate and quantify infectivity, in vivo, in cell culture or cell-free conditions, provide unparalleled settings to elucidate general mechanisms and devise integrated therapeutic approaches for all diseases involving conformational templating The prototypic and best-characterized prion diseases are the transmissible spongiform encephalopathies (TSEs) of animals and humans, including sheep scrapie, bovine spongiform encephalopathy (BSE), chronic wasting disease (CWD) of cervids, and human Creutzfeldt-Jakob disease (CJD). TSEs result from conformational conversion of the host-encoded cellular form of the prion protein, PrPTSEs have long incubation periods ranging from months to years, are invariably fatal, and currently incurable. While a variant of CJD (vCJD) is unequivocally linked to prions causing BSE Sc constituting the infectious prion, and substrate PrPC expressed in the host are closely related PRNP polymorphisms in humans and animals. For example, a strong association between susceptibility/resistance to natural scrapie is associated with the valine (V)/alanine (A) dimorphism at PrP residue 136 Sc. While this remains the favored explanation for prion strain diversity, the mechanism by which primary and higher order PrPC and PrPSc structures interact to influence pathogenesis are not understood.Prion strain properties and the primary structure of PrP are the two major elements controlling prion transmission. Optimal disease progression appears to occur when the primary structures of PrPOur previous studies demonstrated that A at ovine PrP residue 136 is a component of the monoclonal antibody (mAb) PRC5 epitope +/\u2212 and Tg(OvPrP-V136)4166+/\u2212 mice were close to that of PrP expressed in the CNS of wild type mice (We created Tg mice expressing OvPrP encoding either A or V at residue 136. Using semi-quantitative Western and immuno dot blotting we ascertained that levels of expression in the CNS of Tg(OvPrP-A136)3533ype mice .+/\u2212 mice with SSBP/1 prions +/\u2212 mice, mean incubation times were \u223c230 to 280 d longer 4166+/\u2212 mice after >560 d. These distinct transmission profiles are consistent with previously recognized strain differences between SSBP/1 and CH1641 scrapie prions +/\u2212 mice confirmed that the molecular profiles which distinguish PrPSc constituting SSBP/1 and CH1641 prions +/\u2212 and Tg(OvPrP-V136)4166+/\u2212 mice are capable of distinguishing scrapie strain-specific transmission patterns, and in turn that these properties are influenced by the A/V136 dimorphism.Both lines of Tg mice tolerated these levels of expression without spontaneously developing recognizable signs of disease . In contd longer . In contSc conformational stability and the incubation times of mouse and cervid prions Sc in brain extracts of SSBP/1 infected Tg(OvPrP-V136)4166+/\u2212 mice with short incubation times and SSBP/1 infected Tg(OvPrP-A136)3533+/\u2212 mice with long incubation times. Analyses using mAb 6H4 revealed distinct stability curves for OvPrPSc-V136 and OvPrPSc-A136. The conformational stability of OvPrPSc-V136 was lower than OvPrPSc-A136 in the range of GdnHCl concentrations between 1 and 2 M, 4166+/\u2212 mice with rapid incubation times was less stable than OvPrPSc-A136 produced in Tg(OvPrP-A136)3533+/\u2212 mice with longer incubation times. We refer to these conformations as unstable (U) and stable (S), and to the rapidly and slowly propagating prions composed of these conformers as SSBP/1-V136(U), and SSBP/1-A136(S).Previous studies revealed a positive correlation between PrPand 2 M, and GdnHSc distribution Sc-A136(S) and OvPrPSc-V136(U) deposition in the CNS. While OvPrPSc-A136(S) had a punctate pattern of accumulation throughout the midbrain, pons, and oblongata of slow incubation time Tg(OvPrP-A136)3533+/\u2212 mice (Sc-V136(U) in the same sections of rapid incubation time Tg(OvPrP-V136)4166+/\u2212 mice was distinctly different, being more intense and diffusely deposited than OvPrPSc-A136(S) (+/\u2212 and Tg(OvPrP-V136)4166+/\u2212 mice were both engineered using the same cosSHa.Tet cosmid vector which drives expression from the PrP gene promoter, we conclude that these differences are not the result of expression OvPrPC-A136 and OvPrPC-V136 in different neuronal populations.We then used histoblotting +/\u2212 mice , the neu-A136(S) . Since T+/\u2212 or Tg(OvPrP-V136)4166+/\u2212 mice as sources of OvPrPC-A136 and OvPrPC-V136 substrates for protein misfolding amplification (PMCA) Sc in the absence of seeded prions 3533+/\u2212 or Tg(OvPrP-V136)4166+/\u2212 mouse brains using sheep SSBP/1 as seed. Apart from a slight but consistent decrease between rounds two and five, OvPrPSc-V136 production, detected using mAb 6H4, was sustained throughout rounds one to 10 3533d prions , SSBP/1 of PMCA . In conttemplate . We therne to 10 . As expe(Sc-V136 . In conte and 10 .C-V136 and OvPrPC-A136 propagate SSBP/1-V136(U) and SSBP/1-A136(S) prions with relatively rapid and slow incubation times respectively, we produced Tg(OvPrP-A/V136) mice expressing both OvPrPC-A136 and OvPrPC-V136 and inoculated them with SSBP/1 to examine whether disease developed with fast, slow or intermediate kinetics. Although more rapid than the \u223c130 d onset of disease in Tg(OvPrP-V136)4166+/\u2212 mice (P\u200a=\u200a0.0094), the mean 105\u00b15 d onset of disease contrasted with the >400 d SSBP/1 incubation times observed in Tg(OvPrP-A136)3533+/\u2212 mice (Having established that Tg mice expressing OvPrP+/\u2212 mice .Sc produced in the brains of diseased Tg(OvPrP-V136)4166+/\u2212 and Tg(OvPrP-A/V136) mice were superimposable over most of the range of GdnHCl concentrations (Sc produced in Tg(OvPrP-A/V) mice shared the conformation of OvPrPSc-V136(U) produced in SSBP/1 infected Tg(OvPrP-V136)4166+/\u2212 mice. In accordance with this notion, histoblotting using mAb 6H4 showed that the neuroanatomical distribution of OvPrPSc(U) in the brains of diseased Tg(OvPrP-A/V) mice mirrored the diffuse deposition of the OvPrPSc-V136(U) conformer located in similar sections of rapid incubation time Tg(OvPrP-V136)4166+/\u2212 mice were consistent with propagation of SSBP/1-V136(U) prions, remarkably, western blotting of diseased Tg(OvPrP-A/V136) brain extracts with mAb PRC5 revealed substantial conversion of OvPrC-A136 to OvPrPSc-A136 mice.While the rapid SSBP/1 incubation times, and properties of the converted PrPPSc-A136 . DensitomAbs 6H4 and PRC5mAbs 6H4 allowed Sc-A136 among total OvPrPSc produced in the brains of diseased Tg(OvPrP-A/V136) mice. The 1.64 GdnHCl1/2 value of OvPrPSc-A136 produced under these conditions was distinct from that of OvPrPSc-A136(S) produced in long incubation time Tg(OvPrP-A136)3533+/\u2212 mice (GdnHCl1/2\u200a=\u200a2.11), and their non-superimposable PRC5 denaturation curves were significantly different in the range of 1.5\u20132.5 M GdnHCl (Sc-A136 in rapid incubation time Tg(OvPrP-A/V) mice was distinct from OvPrPSc-A136(S) produced in long incubation time Tg(OvPrP-A136)3533+/\u2212 mice. We refer to this novel conformation as OvPrPSc-A136(U), and to the resulting prions as SSBP/1-A136(U).We then used mAb PRC5 to determine the conformation of OvPrPM GdnHCl . These fSc-A136(S) in the CNS of long incubation time Tg(OvPrP-A136)3533+/\u2212 mice (Sc-V136(U) in the CNS of diseased Tg(OvPrP-V136)4166+/\u2212 mice was refractory to detection by mAb PRC5 (Sc-A136(U). In contrast to the punctate deposits of OvPrPSc-A136(S) in long incubation time Tg(OvPrP-A136)3533+/\u2212 mice (Sc-A136(U) in Tg(OvPrP-A/V136) mice (Sc-V136(U) in Tg(OvPrP-V136)4166+/\u2212 mice mice acquired+/\u2212 mice . Consist+/\u2212 mice .C-A136 and OvPrPC-V136 in PMCA reactions seeded with SSBP/1. Under these conditions, similar to when OvPrPC-V136 was present in isolation mice in vitro, we mixed equal quantities of OvPrPsolation , we obseound one . Probingmaterial . Thus, six to 10 .+/\u2212 or Tg(OvPrP-V136)4166+/\u2212 mice. We monitored conversion of OvPrPC-A136 or OvPrPC-V136 templates every two hours for a total of 12 h of PMCA. SSBP/1-V136(U) had the same PMCA properties as SSBP/1: both SSBP/1 and SSBP/1-V136(U) prions efficiently converted OvPrPC-V136 in isolation, but not OvPrPC-A136 in isolation; when both templates were present in the PMCA reaction, the presence of OvPrPC-V136 facilitated conversion of OvPrPC-A136 to OvPrPSc-A136 by SSBP/1 or SSBP/1-V136(U) prions , such prions failed to facilitate conversion of OvPrPC-A136 in the presence of OvPrPC-V136 Cheviot sheep flock, and has subsequently been passaged as a pool. We next compared the seeding properties of SSBP/1 with those of SSBP/1-A136(S) or SSBP/1-V136(U) prions derived from SSBP/1-infected Tg(OvPrP-A136)3533) prions . In contmAb PRC5 ; howevermAb PRC5 . The prorPC-V136 .Previous studies described the production of Tg mice expressing OvPrP, and reported their susceptibility to scrapie prions +/\u2212 and Tg(OvPrP-V136)4166+/\u2212 mice express transgene-encoded PrP, either slightly lower, or slightly higher than PrP levels normally expressed in the CNS of wild type mice. Since Tg(OvPrP-A136)3533+/\u2212 and Tg(OvPrP-V136)4166+/\u2212 lines were produced using the cosSHa.Tet cosmid vector which drives expression from the PrP gene promoter C-A136 and OvPrPC-V136 in identical neuronal populations, and therefore that both alleles are co-expressed in the same cells of Tg(OvPrP-A/V) mice. Finally, other than variable transgene insertion loci, both lines are otherwise sygeneic on an inbred Prnp0/0/FVB background.Tg(OvPrP-A136)3533+/\u2212 mice, that were also produced in a Prnp0/0/FVB background using the cosSHa.Tet cosmid vector +/\u2212mice were 75 d, and this line expresses OvPrP at levels only slightly higher than Tg(OvPrP-V136)4166+/\u2212 mice. While we exercise caution when comparing results from mice produced by different groups, the otherwise similar properties of Tg(OvPrP)14882+/\u2212 and Tg(OvPrP-V136)4166+/\u2212 mice suggest that even slight differences in the levels of transgene expression can have significant effects on prion incubation time.Previous studies reported on Tg mice expressing OvPrP with V at 136, referred to as Tg(OvPrP)14882+/\u2212 and Tg(OvPrP-V136)4166+/\u2212 mice is in accordance with the properties of these isolates in sheep of various genotypes +/\u2212 mice with incubation times exceeding 400 days, the general effects of the A/V136 dimorphism on SSBP/1 transmission observed in sheep are recapitulated in Tg(OvPrP-A136)3533+/\u2212 and Tg(OvPrP-V136)4166+/\u2212 mice 3533+/\u2212 mice . Similar+/\u2212 mice . In prev+/\u2212 mice. While the condition of A/V136 heterozygosity has not been previously modeled in Tg mice, this difference may result from double the levels of transgene expression in Tg(OvPrP-A/V) mice compared to Tg(OvPrP-A136)3533+/\u2212 and Tg(OvPrP-V136)4166+/\u2212 mice. Tg(OvPrP-A/V136) mice were derived by mating Tg(OvPrP-A136)3533+/+ with Tg(OvPrP-V136)4166+/+ mice, and therefore express greater total levels of OvPrP than Tg(OvPrP-A136)3533+/\u2212 and Tg(OvPrP-V136)4166+/\u2212 mice (+/\u2212 and Tg(OvPrP-A/V) mice, and we show that OvPrP-A136 also becomes available for conversion, this situation results in more available substrate for conversion. While previous studies revealed an inverse correlation between transgene expression levels and prion incubation times in Tg mice +/\u2212 mice reflect overall differences in PrPC expression levels remains uncertain. Differences in scrapie pathogenesis between mice and sheep may also reflect the influence of additional factors on disease in the natural host including other PRNP polymorphisms Although SSBP/1 incubation times are prolonged in A/V136 compared to V/V 136 sheep +/\u2212 mice . Since t+/\u2212 and Tg(OvPrP-A136)3533+/\u2212 mice respectively, reflected preferential conversion by SSBP/1 prions of OvPrPC-V136, rapidly producing a relatively unstable OvPrPSc-V136(U) conformation that was diffusely deposited in the CNS, compared to the slower conversion of OvPrPC-A136 to the more stable OvPrPSc-A136(S) conformer which accumulated in the CNS with a punctate pattern (+/\u2212 and Tg(OvPrP-V136)4166+/\u2212 mice respectively. We also show that PMCA recapitulates the influence of the A/V136 polymorphism on the kinetics of SSBP/1 propagation observed in Tg mice. The general conclusions from these studies agree with previously published assessments of the mechanism of conformational selection by distinct PrP primary structures Our observations in Tg mice expressing individual allele products suggested that rapid or prolonged SSBP/1 incubation times in TgOvPrP-V136)4166 pattern \u20133. Our r64166 patSc produced under these conditions with OvPrPSc-V136(U) in Tg(OvPrP-V136)4166+/\u2212 mice, we speculated that OvPrP-A136 played no part during the propagation of SSBP/1 prions in Tg(OvPrP-A/V) mice. To address this we used mAb PRC5 to exclusively monitor conversion of OvPrPC-A136. Surprisingly, in contrast to its relatively slow conversion when OvPrPC-A136 is expressed in isolation, co-expression with OvPrPC-V136 in Tg(OvPrP-A/V136) mice facilitated rapid conversion of OvPrPC-A136 to OvPrPSc-A136. The conformation and diffuse CNS distribution of the resulting OvPrPSc-A136(U) were equivalent to that of OvPrPSc-V136(U) and not OvPrPSc-A136(S). Collectively, these results lead us to conclude that once OvPrPSc-V136(U) is formed by conversion of OvPrPC-V136 by SSBP/1 prions, the resulting unstable conformation induces rapid conversion of OvPrPC-A136 to OvPrPSc-A136(U). That this outcome is dependent on allele co-expression within the host is demonstrated by the inability of the OvPrPSc-V136(U) conformer to template OvPrPC-A136 when it is expressed in isolation.Based on the rapid SSBP/1 incubation times in Tg(OvPrP-A/V136) mice, and shared conformational and distribution properties of OvPrPSc-A136, OvPrPSc-V136, or mixtures of both, and to draw additional conclusions about the effects of the A/V136 dimorphism of prion propagation using PMCA. Similar to our observations in Tg mice, SSBP/1 failed to convert OvPrPC-A136 to OvPrPSc-A136 by PMCA, except in the presence of OvPrPC-V136 (C-V136 and OvPrPC-A136. These results suggest that the SSBP/1-V136(U) is the dominant strain in the natural SSBP/1 isolate. Multiple parameters could account for this, including, but not restricted to, the effects of OvPrP genotype, for example as a result of exclusive propagation in sheep of the V136/V136 genotype, route of transmission in the infected sheep, and differential/selective prion replication in the lymphoreticular or central nervous systems of sheep. While our analyses indicate the presence of both OvPrP-V136 and OvPrP-A136 alleles in SSBB/1 prions, the presence of OvPrPC-V136 inhibited PMCA of OvPrPC-A136 PMCA conversion of PrPSc by SSBP/1 with either OvPrPC-V136 or mixtures of OvPrPC-V136 and OvPrPC-A136 correlates with early onset of disease following SSBP/1 infection of both Tg(OvPrP-V136) and Tg(OvPrP-A/V136) mice, the subsequent inhibitory effects of OvPrPSc-A136 observed in PMCA would be impossible to detect in vivo, since Tg(OvPrP-A/V) mice succumb to the lethal effects of early PrPSc accumulation. Consistent with an inhibitory effect of OvPrPSc-A136(U), prions from Tg(OvPrP-A/V) mice, while they converted OvPrPC-V136 in isolation, failed to convert OvPrPC-A136 to PrPSc in the presence of OvPrPC-V136 4166+/\u2212 mice are consistent with this notion, that is selection of the U conformer by OvPrP-V136, and the S conformer by OvPrP-A136, our unprecedented ability to analyze allele specific conversion in infected Tg(OvPrP-A/A136) mice reveals a more complex mechanism where mixtures of PrP variants may assist or inhibit the propagation of strains under various conditions. For example, SSBP/1 or SSBP/1-V136(U) prions facilitate conversion of OvPrPC-A136 to OvPrPSc-A136(U) only in the presence of OvPrPC-V136. Expressed in isolation, conversion of OvPrPC-A136 is favored by the OvPrPSc(S) conformer. Our results demonstrate that co-expression of different polymorphic forms of PrP, which would be the norm in humans and animals, have profound effects on conformational selection of prion strains.The inter-related effects of PrP primary and higher order structures on prion transmission were addressed in the Conformational Selection Model, which proposed that strains are composed of a range of PrPSc(U), which, we speculate, involves physical association of otherwise \u201csusceptible\u201d and \u201cresistant\u201d allele products. Consistent with the observations reported here, prion strain interference may also utilize similar mechanisms of conformational selection in a host expressing different PrP allele products infected with long and short incubation period strains with different PrPSc conformational stabilities The results reported here address the molecular mechanisms associated with the phenomenon of prion strain over-dominance first observed by Dickinson and Outram PRNP polymorphisms, and combinations thereof, is likely to have a significant influence on strain diversity.In conclusion, we have used a combination of transgenic, immunologic, and in vitro approaches to explore the mechanism by which PrP primary structure variations and the conformations enciphered by different prion strains interact to control TSE propagation. While our results support previous studies indicating that PrP susceptibility polymorphisms, expressed in isolation, act to restrict or promote the propagation of particular prion conformers, we now show that under conditions of allele co-expression a dominant conformer may alter the conversion potential of an otherwise resistant PrP polymorphic variant to an unfavorable prion strain. While such responses are analogous to the phenotypic expression of genetically determined heritable traits, dominant prion conformers act epigenetically by means of protein-mediated conformational templating. By expanding the range of possible conformations adoptable by a particular prion protein primary structure, such interactive effects provide a mechanism for promoting strain fitness, and, we speculate, strain diversification. While the precise number scrapie strains in sheep and goats remains uncertain, the description of at least 24 additional major sheep All animal work was conducted according to the National Institutes of Health guidelines for housing and care of laboratory animals, and performed under protocols approved by the Colorado State University Institutional Animal Care and Use Committee, with approval number 11-2996A.Prnp0/0/FVB mice. Tg founders were identified by PCR screening of genomic DNA isolated from tail snips. Founder mice were mated with inbred Prnp0/0/FVB mice, and generally maintained with the transgene in the hemizygous state, with Tg mice identified by PCR screening of genomic DNA from weanlings. It was also possible to generate homozygous counterparts of each line, and Tg(OvPrP-A/V136) mice were generated by crossing homozygous Tg(OvPrP-V136)4166+/+ mice with homozygous Tg(OvPrP-A136)3533+/+ mice. We used immuno-dot blotting and Western blotting with mAb 6H4 to estimate the levels of OvPrP expression. Tg mice subsequently shipped to and maintained in Edinburgh were crossed onto the Prnp0/0/129Ola background Sequences upstream of codon 44 of the OvPrP-A136 and V136 coding sequences were replaced with the corresponding sequence from mouse PrP. The resulting constructs contained the OvPrP coding sequence, except for addition of an extra residue for glycine at codon 31, and the mouse PrP N-terminal signal peptide instead of OvPrP signal peptide. Tg mice were generated by cloning the OvPrP-A136 and OvPrP-V136 expression constructs into the cosSHa.Tet cosmid vector PRNP coding sequences.SSBP/1 originated as a homogenate of three natural scrapie brains that were subsequently passaged mostly through Cheviot sheep at the Neuropathogenesis Unit (NPU), Edinburgh UK Ten % mouse brain homogenates (w/v) were prepared in phosphate-buffered saline (PBS) lacking calcium and magnesium ions by repeated extrusion through 18- and 21-gauge needles. Sheep brain homogenates (10%) in PBS were prepared by repeated extrusion through 14-gauge, followed by 18- to 28-gauge needles in PBS. Total protein content was determined by bicinchonic acid (BCA) assay .Anesthetized mice were inoculated intracerebrally with 30 \u00b5l of 1% (w/v) brain extracts prepared and diluted in PBS.General health was monitored daily. Onset of prion disease was determined by observation of the progressive development of at least three of the following clinical signs: truncal ataxia, loss of extensor reflex, difficulty righting from a supine position, plastic tail, head bobbing or tilting, kyphotic posture, circling and paresis/paralysis. Animals were diagnosed when at least two investigators agreed with the manifestation of these signs. Incubation time is defined as the period between the time of inoculation to the day on which subsequently progressive clinical signs were initially recorded.5\u2032-GGACAGGGCAGTCCTGGA-3\u2032, 5\u2032-GTGATGCACATTTGCTCCACCACT-3\u2032. PCR products were purified with QIAquick Gel Extraction kit , digested with BspH I that only recognizes the OvPrP-V136 allele, and the products were resolved on a 1.2% agarose gel.Brain homogenates containing 500 \u00b5g protein were digested with 400 \u00b5g/ml proteinase K (PK) in 0.4 M NaCl, 10 mM Tris\u2013HCl, pH 8.0, 2 mM EDTA, pH 8.0, and 2% SDS at 55\u00b0C overnight. Genomic DNA was precipitated with isopropanol. The partial OvPrP coding sequence was amplified by PCR with the forward and reverse primers: Tg mice were perfused with PBS/5 mM EDTA. Ten % brain homogenates (w/v) were prepared in PBS containing 150 mM NaCl, 1.0% Triton X-100, and the complete TM cocktail of protease inhibitors . Samples were clarified by brief, low-speed centrifugation. Protein concentrations of brain homogenates used as substrates for PMCA were adjusted to contain equivalent amounts of OvPrP-A136 or OvPrP-V136, based on the estimated relative levels of transgene expression. Substrates in which OvPrP-A136 and OvPrP-V136 were mixed were adjusted based on the estimated relative levels of transgene expression, so that approximately equal amounts of each allele product were present in the PMCA reaction. PMCA reactions were performed as described previously Brain homogenates and cell lysates were digested with 100 \u00b5g/ml or 30 \u00b5g/ml of PK respectively in cold lysis buffer for 1 h at 37\u00b0C. Digestion was terminated with phenylmethylsulfonyl fluoride at a final concentration of 2 \u00b5M. Samples were boiled for 10 min in the absence of \u03b2-meracaptoethanol Brain homogenates containing 5 \u00b5g protein were incubated with various concentrations of guanidine hydrochloride (GdnHCl) in 96-well plates for 1 h at room temperature. Samples were adjusted with PBS to a final of concentration of GdnHCl of 0.5 M and transferred onto nitrocellulose using a dot blot apparatus. After two PBS washes, the membrane was air-dried for 1 h, then incubated with 5 \u00b5g/mL PK in 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.5% sodium deoxycholate, 0.5% Igepal CA-630 for 1 h at 37\u00b0C. PK was inactivated with 2 mM PMSF. The membrane was incubated in 3 M guanidine thiocyanate in Tris-HCl, pH 7.8 for 10 min at room temperature. After four washes with PBS, the membrane was blocked with 5% nonfat milk in TBST for 1 h, and probed with mAbs 6H4 or PRC5 (1\u22365000) overnight at 4\u00b0C, followed by HRP-conjugated goat anti-mouse IgG secondary antibody. The membrane was developed with ECL Plus and scanned with GE image quant 4000. The signal was analyzed with ImageQuant TL 7.0 software.Histoblots were produced and analyzed according to previously described protocols"} +{"text": "The neurofilament light subunit (NF-L) binds to myosin Va (Myo Va) in neurons but the sites of interaction and functional significance are not clear. We show by deletion analysis that motor domain of Myo Va binds to the NF-L rod domain that forms the NF backbone. Loss of NF-L and Myo Va binding from axons significantly reduces the axonal content of ER, and redistributes ER to the periphery of axon. Our data are consistent with a novel function for NFs as a scaffold in axons for maintaining the content and proper distribution of vesicular organelles, mediated in part by Myo Va. Based on observations that the Myo Va motor domain binds to intermediate filament (IF) proteins of several classes, Myo Va interactions with IFs may serve similar roles in organizing organelle topography in different cell types. Cellular transport is mediated by molecular motor proteins including kinesin, dynein/dynactin complex, and myosins. Kinesin and dynein/dynactin motors are powered by microtubule-dependent mechanisms, whereas myosin motor proteins move their cargoes by a \u201chand-over hand\u201d mechanism along actin filaments dilute-lethal [dl] phenotype Myo Va in neurons is believed to transport synaptic vesicles, ER, mitochondria and membrane bound vesicles along axons and within synaptic terminals, and to facilitate the accumulation of mRNA/protein complexes in dendritic spines More recently, Myo Va has been shown to bind to neurofilaments (NFs), and other intermediate filament (IF) proteins from different cell types In this study, we demonstrate that NF-L, rod domain, binds directly the N-terminal motor domain of Myo Va. It is well-established that the Myo Va cargo domain binds vesicular organelles (7\u20139), and our morphological and fractionation data (this study) demonstrate association among vesicular organelles, Myo Va, and NF-L. We showed that loss of NF-L and Myo Va leads to reduction in axonal levels of organelle markers and increased peripheral distribution of ER toward the actin-rich subaxolemmal region. Collectively, our studies provide evidence that Myo Va binding to NF-L modulates the distribution of vesicular organelles in axons. The binding of various IFs to the Myo Va head domain raises the possibility that IFs may facilitate Myo Va-mediated distribution of organelles along multiple IF systems in different cell types.All the animal protocols described in the paper were approved by the Nathan Kline Institute Animal Care and Use Committee Protocol AP2005-155.NF-L null mice are provided by Dr. Jean-Pierre Julien, Laval University, Canada 5\u2032-GCA TGA ATT CCA GAA AGA TGT CCT TAG T-3\u2032 and 5\u2032-GCA AGC TTC TGA ATG GTG ATG GCA GC-3\u2032; a 800\u2013881 construct was PCR amplified with primer sets of 5\u2032-GCA TGA ATT CAG ATA TGT CAG AGG GCA C-3\u2032 and 5\u2032-GCA AGC TTT CGA GCC AGC CAG CCT CTC-3\u2032 and chicken Myo Va as a template Myo Va head, neck and tail regions tagged with maltose binding protein (MBP) were described previously A full-length NF-L-myc tagged construct MBP tagged-Myo Va constructs and NF-L-Myc tagged constructs were transformed into the BL21-DE bacterial strain. Cultures were grown to 0.6 OD and induced with IPTG to overexpress proteins for 1 hr at 37\u00b0C. Cultures were centrifuged and the pellets were suspended in a 50 mM sodium phosphate buffer pH 7.0 containing 1% SDS and 1 mM EDTA. Cells were subjected to freeze-thaw in a dry ice-ethanol bath (3 times), sonicated and centrifuged. Clear supernatants were assayed for protein, immunoblotted with either MBP or NF-L (NR-4 or 21.4) or Myc (9E10) antibodies. Some of the MBP-Myo Va constructs were affinity purified on amylose columns . Bacterial cells after induction were lysed in a column buffer containing 20 mM Tris-HCl, pH 7.4, 200 mM NaCl and 1 mM EDTA by freeze thawing in a dry ice ethanol bath (3X). The lysates were centrifuged and the pellets were dissolved in the column buffer with 6M-guanidine hydrochloride, dialyzed in TBS buffer over night at 4\u00b0C, and centrifuged. Clear supernatants were then loaded on a 5 ml amylose column (30 ml/hr), washed and the proteins were eluted with 10 mM maltose in column buffer.NF-L was purified from mouse spinal cord NF pellet as described earlier Brain, optic nerve, spinal cord, and sciatic nerves from wild type (WT) and NF-L null mice were dissected and cytoskeletal preparations enriched for NFs were made according to Optic nerves were homogenized in a buffer containing 50 mM Tris-HCl, pH 7.4, 0.25 M sucrose, 1 mM each of EDTA, EGTA and DTT Bacterially expressed Myo Va mutant proteins, and NF-L mutant proteins, purified or cytoskeletal preparations containing NF-L, and total tissue extracts from mouse neuronal tissues were prepared according to Using purified MBP as a standard , we quantified the amount of MBP-tagged Myo Va mutant proteins for each expressed construct on immunoblots with anti-MBP antibody. Equal amounts of protein corresponding to each MBP-Myo Va construct (0.5\u20132 \u00b5g) and purified or Myc-tagged NF-L (0.5\u20132 \u00b5g) were mixed for 1 hr in 1X RIPA buffer for co-IP assays. Antibody directed against NF-L (NR-4 or 21.4 or 9E10 for Myc tag) was then added and the mixture was incubated for 1 hr at room temperature (RT). Protein A/G Sepharose beads were added and incubated for another 1 hr at RT. Immunocomplexes were centrifuged and the precipitates were washed (3X) with high salt solution , dissolved in Laemmli buffer, fractionated on polyacrylamide gels along with 5\u201310% of the supernatant from the IPs and the input. Gels were transferred to nitrocellulose membranes and immunoblotted with MBP and NF-L antibodies as indicated above. To take equimolar concentrations of the protein, bacterial extracts containing NF-L and MBP-tagged Myo Va proteins were fractionated on gels along with known concentrations of BSA. The Coomassie stained gels were scanned and protein bands of NF-L, MBP-Myo Va and BSA were quantified using the multigauge program. To compare two proteins based on the number of amino acids each protein has, we calculated the molecular weight (MW) of the proteins and the MW of the bigger protein was divided by that of the smaller protein. The resultant value was multiplied by the amount of protein taken for the smaller construct to obtain the equimolar concentration of the larger protein. To achieve an excess amount of NF-L for IPs, we took a 2\u20133 fold greater amount of NF-L relative to the MBP-Myo Va constructs.Myo Va enriched from vesicles was prepared from mouse brain as described previously Blot overlay assays were conducted as described previously Mice were perfused and optic nerves were fixed and embedded for electron microscopy as described previously To identify which of the Myo Va mutants bind to full length NF-L, we co-immunoprecipitated (co-IP) each of these Myo Va mutant proteins in equimThe Myo Va cargo binding region is present in the tail region where it binds to melanosomes We confirmed the specificity of Myo Va head binding to NF-L, by co-immunoprecipitating NF-L with either MBP-Myo Va-Head (5-752), purified MBP alone, or bovine serum albumin (BSA). MBP-Head did co-immunoprecipitate with NF-L, while MBP alone or BSA (data not shown) showed no binding activity, indicating that the MBP-Myo Va head interaction with NF-L is specific.We next used spinal cord cytoskeletal fractions in blot overlay assays In similar blot overlay analyses, we confirmed that NF-L is a major ligand for the Myo Va head domain (5-752) in optic and sciatic nerves and spinal cords of WT mice . Upon loTo further demonstrate the specificity of Myo Va head binding to NF-L, we subjected cytoskeletal fractions of spinal cord lane 4; In the above studies, the Myo Va head domain construct used spans amino acids 5-752. We constructed 3 additional mutants of the head region , which were tagged with MBP to furthBecause our studies previously demonstrated binding activity of a Myo Va construct containing the neck region (654-1402) To identify the site on NF-L to which the Myo Va motor domain binds, we constructed four Myc-tagged NF-L deletion mutants , we performed co-IP assays. Our data indicate that Myo Va bound to all the NF-L constructs containing amino acids 94-243 in the rod domain , lane 5.All IF proteins have a common structural core of 300\u2013330 amino acid residues within the rod domain, which are flanked by variable amino- and carboxy-terminal regions in vivo conditions, Myo Va, NF-L and actin holoproteins form complexes and these were identified by co-immunoprecipitation assays in mouse optic axons The actin and NF-L binding sites are about 300 amino acids apart on the Myo Va head domain . BecauseTo examine whether NF-L would activate Myo Va-ATPase activity, we incubated fractionated Myo Va in NF-L null optic axons. Moreover, Myo Va is involved in the transport of synaptic vesicles, ER, and other membrane-bound organelles If NF-L-Myo Va binding cage and/or anchor organelles to maintain a relatively uniform organelle distribution throughout the axoplasm, we hypothesized that loss of the NF-L or Myo Va would reduce the content of organelles and possibly alter their distribution within NF-L and Myo Va mutant axons. To test this possibility, we performed electron microscopy of fixed optic axons of NF-L null and DL20J mice, and further morphometric analysis by counting clear ER vesicles in these mutant axons and calculating the density of ER see . Our morMoreover, we observed that clear vesicular organelles normally distribute uniformly throughout the three-dimensional space of axons in WT mice . Consist35S]-methionine as previously described To investigate whether reduced axonal Myo Va levels in NF-L null mice Our studies identify a novel role for the binding between Myo Va and NFs as a mechanism to anchor organelles within the axoplasm of large caliber axons. We demonstrate that NF-L, and specifically the NF-L rod domain, binds directly the N-terminal motor domain of Myo Va. It is well-established that the MyoVa cargo domain binds vesicular organelles (7\u20139), and our morphological and fractionation data . Our previous studies also demonstrated that depletion of NFs and axonal Myo Va had no effect on slow axonal transport rates of NF-M or actin Several lines of evidence from our studies indicate that Myo Va binding to NF-L regulates organelle distribution in the axon but loss of this interaction does not affect the slow or fast axonal transport cargoes Xenopus melanophores indicate that IFs significantly slow down the run lengths of the motor on microtubules. This particular study suggests that IFs hinder Myo Va dependent organelle transport in melanophores Our studies confirm and extend our previous observations that Myo Va binds selectively not only to NF-L but also to other IF proteins of several classes, including \u03b1-internexin, GFAP, peripherin, vimentin, and desmin, but not keratin. A Blast analysis of the IF proteins identified a highly conserved (EREGLEETLRNL) sequence within the rod domain of IF proteins that bind Myo Va compared to IFs that do not bind Myo Va . These observations suggest that IF networks may serve as linear arrays for Myo Va-mediated organelle distribution in a variety of cell types. In this regard, dynamic rearrangements of vimentin have been shown to be important in modulating the centrifugal movements of melanosomes in B16 cells Figure S1Immunoblot analyses of Myo Va and NF-L constructs. Bacterially expressed Myo Va head , neck and tail domains Coomassie stained (A) and immunoblotted with anti-MBP antibody (B). Myosin Va head region of Myo Va was further deleted to make 41-505 (95-kDa), 41-326 (75-kDa), and 327-505 (52-kDa) constructs (C), and the neck mutants 800-881 (50-kDa), 604-881 (85-kDa), 717-799 (65-kDa) (D) were immunoblotted with MBP antibody. His-tagged tail domain deletion mutants were immunoblotted with hexa-His antibody . (F) Purified , and bacterially expressed Myc tagged NF-L , immunoblotted with NF-L specific and anti-Myc (9E10) antibodies (lanes 7&8). (G). Bacterial expression of Myc-tagged NF-L mutants. Lane 1: full length NF-L ; lane 2: C-terminal deletion mutant 1-369 (37-kDa); lane 3: 1-243 (27-kDa); lane 4: N-terminal rod domain 94-243 (20-kDa), and lane 5: 1-93 (17-kDa) were immunoblotted with anti-Myc antibody. The positions of the protein bands on all membranes are indicated with arrows.(TIF)Click here for additional data file."} +{"text": "Purpose. To report B-scan and \u201cEn-face\u201d spectral-domain optical coherence tomography (SD-OCT) findings in acute retinal pigment epitheliitis (ARPE). Methods. Two patients (3 eyes) with ARPE were examined. Fluorescein and indocyanine green (ICGA) angiography, B-scan, and \u201cEn-face\u201d SD-OCT were performed in each patient at initial and follow-up visits. Results. Both patients presented with acute onset of blurred vision, and one with bilateral involvement. B-can OCT revealed disruption of the macular retinal pigment epithelial (RPE) inner band layer and photoreceptors' inner and outer segment (IS-OS) junction. Hyperreflective dots were observed in the outer nuclear layer (ONL) above the RPE/IS-OS disruption. Just around these hyperreflective dots, slight thickening of the hyperreflective IS/OS junction was observed. During the late phase, indocyanine green angiography (ICGA) showed a macular cockade-like hyperfluorescent halo. \u201cEn-face\u201d OCT showed the same cockade-like appearance with a hyporeflective center and a hyperreflective border matching the pattern observed on ICGA. At followup, as vision improved without treatment, B-scan OCT demonstrated progressive resolution of the hyperreflective and disrupted lesions; \u201cen-face\u201d OCT also showed disappearance of the macular cockade-like halo with a transient discrete hyperreflective macular star at the RPE level in one eye. Conclusion. \u201cEn-face\u201d OCT associated with B-scan SD-OCT analysis appears to be very helpful in the diagnosis and followup of ARPE. The pathophysiology of ARPE remains complex and still poorly understood. These techniques help define the location and extent of structural damage occurring in this disease. Acute retinal pigment epitheliitis (ARPE), or Krill's disease, named after its first description by Krill in 1974, is a benign, self-limited, bilateral yet asymmetrical pathology affecting adults between 10 and 40 years of age , 3. In addition to these clinical parameters, fluorescein (FA) and indocyanine green angiography (ICGA) help in identifying the diagnosis. The high resolution of the scans generated by spectral-domain optical coherence tomography (SD-OCT) offers a helpful tool in the management of ARPE. SD-OCT allows a direct, noncontact visualization of the involved retinal layers: inner segment-outer segment (IS-OS) junction and inner band of the retinal pigment epithelium (RPE).\u201cEn-face\u201d OCT is a novel imaging technique generating frontal scans derived from SD-OCT . In contTwo patients with ARPE lesions underwent complete ocular examination comprising best-corrected visual acuity (BCVA), biomicroscopy, fundoscopy, autofluorescence, fluorescein angiography (FA), and indocyanine green angiography (ICGA). SD-OCT B-scans and \u201cen-face\u201d OCT were performed at the initial visit and during the follow-up period with a Spectralis-HRA and a Cirrus OCT . A 17-year-old woman was referred with a 7-day history of sudden bilateral blurred vision. She reported being ill for 15 days with fever associated with Marshall syndrome . Medical and ocular history were unremarkable. She had not inhaled poppers. Best-corrected visual acuity was 20/40 in both eyes. Fundus examination showed a yellowish foveolar hypopigmented lesion without vitritis in both eyes. Autofluorescence measurement and fluorescein angiography were normal Figures . The latFive days later, visual acuity progressively recovered to 20/20 in both eyes correlating with an improvement in retinal morphology on B-scan SD-OCT. Three weeks later, SD-OCT (B-scan and \u201cen-face\u201d) showed a restored and continuous normal IS/OS layer and RPE inner band. A 25-year-old woman presented with a central scotoma in the left eye that had appeared 5 days ago. She had a flu-like syndrome 10 days preceding the visual blurring and she had already undergone an extensive neurologic examination that was negative. She had not inhaled poppers. Best-corrected visual acuity in the left eye was 20/32. Color fundus photography of the left eye revealed a yellowish hypopigmented area in the macula. Autofluorescence measurement and fluorescein angiography were normal Figures . B-scan One month later, SD-OCT B-scan demonstrated a return to normality while \u201cen-face\u201d scans showed an incomplete recovery of the disrupted central IS/OS junction Figures . A transThe pathophysiology of ARPE is not completely understood. Funduscopy shows a yellowish halo around the fovea, without vitritis. MEWDS (Multiple Evanescent White Dots Syndrome) may have a similar presentation. In both pathologies, the natural history leads usually to complete and spontaneous resolution within weeks. In MEWDS, fundus examination shows differences with discrete perifoveolar dots, very mild vitritis, and, in some cases, papillitis. On fluorescein angiography these dots appear hyperfluorescent and are hypofluorescent on ICGA. There is no cockade-like foveolar lesion as in ARPE. Moreover, EDI-OCT frequently demonstrates choroidal thickening associated with MEWDS lesions , 8.Also, it is important to ask if the patient repeatedly inhaled poppers which may be associated with vision loss due to the disruption of foveal cone outer segments . These lC-scan or \u201cen-face\u201d OCT is an interesting technique in order to more precisely characterize the anatomical changes in ARPE \u201315. The Acute retinal pigment epitheliitis refers to a complex disease that resolves quickly without treatment. Outer retinal and pigment epithelial changes observed with multimodal imaging are transient and may disappear quickly in some cases. \u201cEn-face\u201d OCT imaging enables the assessment of the extent of structural damage occurring in ARPE. \u201cEn-face\u201d OCT enhances its sensitivity, allowing earlier diagnosis of retinal changes and a more reliable followup. Further prospective studies, including more patients, will be necessary to confirm these results."} +{"text": "We demonstrate that projection imaging of first-pass myocardial perfusion is robust to a major cause of the so-called subendocardial dark-rim artifact (DRA); thereby proposing radial k-space sampling as the preferred acquisition scheme for DRA-free perfusion imaging.Current methods for clinical myocardial perfusion (MP) imaging suffer from image artifacts, specifically the so-called subendocardial dark-rim artifact (DRA) -3. DRAs In Cartesian MP MR, Gibbs ringing along the phase-encode (PE) direction leads to DRAs due to an inherent property of the Fourier transform (FT) at sharp signal intensity transitions . HoweverFig. Recently, a major approach for eliminating the DRAs has been to improve the spatial resolution and thereby reducing Gibbs ringing ,6. In thGrant sponsors: American Heart Association Postdoctoral Fellowship Award 11POST7390063 (PI: B. Sharif); National Institutes of Health grants nos. NHLBI HL38698 (PI: D. Li) and NHLBI HL091989 (PI: R. Dharmakumar)."} +{"text": "Regulatory T cells (Treg) play a role in suppression of anti-melanoma immunity; however, the exact mechanism is poorly understood. Through intravital two photon microscopy, we found that Pmel-1 effectors engage in cell-cell interactions with tumor resident Tregs. To determine if contact between Tregs and T effectors (Teff) hinders killing of tumor cells in vivo, we utilized ex-vivo three-dimensional collagen-fibrin gel cultures of B16 melanoma cells. Collagen-fibrin gel cultures recapitulated the in vivo suppression, rendering the dissociated tumor resistant to killing by in vitro activated antigen specific Teff. In vivo depletion of Tregs in foxp3-DTR mice prior to tumor excision reversed the suppression. Additionally, In vivo modulation of intra-tumor Tregs suppressive function by GITR ligation had a similar effect, leading to ex-vivo tumor killing. Using neutralizing antibodies, we found that blocking TGF-\u03b2 reversed the suppression. In addition, soluble factors from collagen-fibrin gel tumors do not inhibit killing suggesting that suppression is contact or proximity dependent. The CD8 Teff recovered from these gels exhibit a decrease in Granzyme B expression and an increase in expression of T cell exhaustion marker PD-1. These findings support the conclusion that intra-tumor contact with Tregs during the effector phase of the immune response is responsible for inhibiting anti-melanoma immunity in a TGF-\u03b2 dependent manner, elucidating a novel way to target intratumoral Tregs."} +{"text": "ATP-binding cassette transporter A1 (ABCA1) mediates the lipidation of exchangeable apolipoproteins, the rate-limiting step in the formation of high density lipoproteins (HDL). We previously demonstrated that HDL oxidized ex vivo by peroxidase-generated tyrosyl radical increases the availability of cellular cholesterol for efflux and reduces the development of atherosclerosis when administered to apolipoprotein E-deficient mice as compared to treatment with control HDL.In the current study we determined that tyrHDL requires functional ABCA1 for this enhanced activity. Like lipid-free apolipoprotein A-I (apoA-I), tyrHDL increases total and cell surface ABCA1, inhibits calpain-dependent and -independent proteolysis of ABCA1, and can be bound by cell surface ABCA1 in human skin fibroblasts. Additionally, tyrHDL apoproteins are susceptible to digestion by enteropeptidase like lipid-free apoA-I, but unlike lipid-bound apoA-I on HDL, which is resistant to proteolysis.These results provide the first evidence that lipid-bound apolipoproteins on the surface of spherical HDL particles can behave like lipid-free apoA-I to increase ABCA1 protein levels and activity. High density lipoprotein cholesterol (HDL-C) levels in human plasma correlate generally with protection against coronary heart disease, an effect believed to be due to multiple protective actions of HDL including stimulating the removal of excess cholesterol from cells and reducing inflammation in the artery wall . The iniIn addition to increasing ABCA1 expression transcriptionally, therapies that increase ABCA1 cell surface stability represent a potential means to increase HDL production. We previously showed that oxidation of HDL ex vivo with peroxidase-generated tyrosyl radical (tyrosylated HDL or tyrHDL) increases the ability of HDL to deplete the regulatory pool of intracellular cholesterol , increas14C]Oleate (55 mCi/mmol) was from GE Healthcare. Dulbecco's modified Eagle's medium (DMEM) was purchased from Hyclone. PE-SIL G plastic backed flexible plates used for thin-layer chromatography analysis were from Whatman. Nitrocellulose membranes, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) reagents, pre-stained protein molecular mass markers and Chelex 100 resin were from Bio-Rad. \u03bc-Calpain was from Calbiochem, and Trizol from Invitrogen. Cross-linking agent dithiobis(succinimidyl) propionate (DSP) was from Pierce.Cholesterol, L-tyrosine, horseradish peroxidase, hydrogen peroxide , dietheylenetriaminepentaacetic acid , essentially fatty acid-free bovine serum albumin (BSA), N-acetyl-Leu-Leu-norleucinal (ALLN), LXR agonist T0901317 and fetal bovine serum were purchased from Sigma. oleate bound to 3 mM BSA oleate quantified. As reported previously [Lipid-free apoA-I depletes the regulatory pool of cholesterol available for esterification by acyl-CoA:cholesterol acyltransferase (ACAT) much more readily than HDL , due to eviously , tyrHDL To test whether tyrHDL has an effect on ABCA1 transcription, cholesterol-loaded fibroblasts were incubated with either fatty acid-free bovine serum albumin (BSA) alone or with the addition of 10 \u03bcg/mL apoA-I, HDL, or tyrHDL for 2 h or 16 h prior to determination of ABCA1 mRNA level by real-time PCR. At 2 h, ABCA1 mRNA level was reduced similarly by all treatments when compared to cholesterol-loaded cells at 0 h Figure . FollowiApoA-I has previously been shown to increase ABCA1 protein levels in cultured cells and in the liver of apoA-I-injected mice ,4. ConsiCalpain-dependent proteolysis of ABCA1 is mediated through a proline, glutamic acid, serine, and threonine-rich (PEST) sequence , and is Lipid-free apoA-I also increases ABCA1 protein levels by interfering with calpain-independent proteolysis of ABCA1 . IncreasApoA-I-dependent reduction of ABCA1 proteolysis results in increased cell surface as well as total cellular ABCA1 . To deteSeveral studies using the chemical cross-linker dithiobis(succinimidyl) propionate (DSP) have suggested apoA-I can directly bind to ABCA1, whereas HDL does not ,19,20. T188 to form a major fragment of about 22 kDa, but has no ability to cleave lipid bound apoA-I present either on reconstituted or native HDL (15). Enteropeptidase treatment of lipid-free apoA-I converted a majority of the protein to a lower molecular weight fragment (~22 kDa), while HDL was resistant to proteolysis (Figure To investigate the possibility that lipid-bound apos on tyrHDL can assume a lipid-free conformation, the susceptibility of tyrHDL to enteropeptidase digestion was compared to unmodified HDL and apoA-I. Enteropeptidase cleaves lipid-free apoA-I at Args Figure . Exposurs Figure and 7B. The current study provides several lines of evidence that oxidation of HDL by peroxidase-generated tyrosyl radical induces conformational changes in lipid-bound HDL apolipoproteins that allows them to interact with ABCA1 like lipid-free apoA-I. Incubation of ABCA1-expressing human fibroblasts with tyrHDL increases total cellular and cell surface ABCA1 and inhibits calpain-dependent and -independent proteolysis of ABCA1, as also mediated by lipid-free apoA-I but not HDL ,4. Additlower ability to deplete ACAT-accessible cholesterol than equivalent concentrations of lipid-free proteins from native HDL [Our findings indicate that tyrHDL increases cholesterol mobilization from cells by interacting directly with ABCA1 and enhancing ABCA1 protein stability. Like lipid-free apoA-I, no effect was seen by tyrHDL on ABCA1 mRNA levels. Consistent with this, previous studies demonstrated the lipid extract of tyrHDL had no ability to enhance cholesterol mobilization from cells when reconstituted with control HDL apoproteins, suggesting oxysterols present in tyrHDL lipids were not driving ABCA1 expression at a transcriptional level . Those stive HDL . tyrHDL tive HDL . These rThe combined results suggest HDL apoproteins oxidized by tyrosyl radical and specifically the apoAI-AII heterodimer created by this oxidation binds to the surface of the lipoprotein differently than apoproteins on HDL, and that the modifications induced by tyrosyl radical oxidation produce a partially lipid-free conformation in lipid-bound apoA-I. We propose a possible model wherein the more lipophilic apoA-II component of the apoAI-AII heterodimers on tyrHDL tethers The nature of the conformation of tyrHDL apoAI-AII heterodimers on the surface of tyrHDL requires further investigation. Particular regions of apoA-I thought to be critical to the apoA-I-ABCA1 interaction have been suggested but not yet confirmed -23. The Whether or not peroxidase-generated tyrosyl radical oxidation of HDL represents a significant mechanism of HDL oxidation in vivo is an unanswered question, and is not the purpose of these studies. Myeloperoxidase is known to generate tyrosyl radical as one of its oxidizing products in addition to hypochlorous acid , and LDLThe results presented here provide the first demonstration of lipid-bound HDL apolipoproteins capable of enhancing ABCA1 protein level and activity like lipid-free apoA-I, and an explanation for the previously identified salutary effects of tyrHDL.The abbreviations used are: HDL: high density lipoproteins; apo: apolipoprotein; ABCA1: ATP-binding cassette transporter A1; tyrHDL: tyrosyl radical-oxidized HDL; BSA: fatty acid-free bovine serum albumin; DMEM: Dulbecco's modified Eagle's medium; ALLN: N-acetyl-Leu-Leu-norleucinal; ACAT: acyl-CoA:cholesterol acyltransferase.MAH participated in the design of experiments and writing of the manuscript, and performed the majority of the experiments. SN performed the initial experiments suggesting an interaction of tyrHDL with ABCA1. TC performed experiments for the study and assisted with figure generation and writing of the manuscript. MNO provided intellectual input and assistance with the writing of the manuscript. GAF designed the experiments, oversaw the project and data analysis, and wrote most of the manuscript. All authors read and approved the final manuscript."} +{"text": "Innate responses are key determinants of the outcome of HIV infection, influencing critical events in the earliest stages of infection. Innate antiviral immune defenses are triggered through the recognition of conserved pathogen associated molecular pattern (PAMP) motifs within viral products by intracellular pattern recognition receptor (PRR) proteins in infected cells. Type I interferons (IFN \u03b1 and beta) are induced directly in response to viral infection, resulting in an antiviral state for the cell.A ligand independent constitutively active form of PRR adaptor protein (\u2018super-PRR\u2019) was constructed and tested for antiviral properties. NF-kb and IFN production by construct was tested by Luciferase reporter assays. The construct was transfected into RAW 264.7 cells, analyzed for surface markers and inflammatory cytokines. RT-PCR was performed to analyze the expression of cytokine/chemokine genes. TZM-bl cells were transfected with plasmid expressing \u2018super-PRR\u2019, infected with HIV-BaL virus and beta-galactosidase activity was measured.The constitutively active \u2018super-PRR\u2019 generated 50-fold and 10-fold increase in NF-kb and IFN-beta production respectively as compared to vector alone. There was significant increased expression of CD80, CD86, CD40, CCR7, HLA-DR, secretion of IL-1beta, IL-6, TNF-\u03b1 and expression of RANTES, MIP-1beta, IP-10 on transfected RAW cells. TZM-bl cells transfected with \u2018super-PRR\u2019 after infection with HIV-Bal virus showed significantly reduced replication of virus. In a transwell experiment, 293T cells transfected with the \u2018super-PRR\u2019 was sufficient to significantly reduce HIV-1 infection of TZM-bl cells, suggesting soluble factors are involved.This constitutively active form of PRR adapter protein induced potent anti-viral innate immune response and prevented infection with HIV. The modulation of innate immunity has potential as a powerful strategy to complement traditional approaches to HIV therapy by protecting cells from viral infection. We are currently constructing lentiviral vector vaccines encoding this \u2018super-PRR\u2019 as a method to target this anti-viral response to the site of HIV-1 Infection."} +{"text": "Patients often report reductions in negative affect in response to biofield and touch interventions. While reasons for improvement remain unclear, current theory and evidence suggest the need to examine oxytocin as well as placebo mechanisms. We investigated changes in negative affect (NA) during an acute session of verum or touch-based healing, and related NA changes to placebo elements, plasma oxytocin changes, and the oxytocin receptor (OXTR SNP rs53576) polymorphism .Thirty-two fatigued breast cancer survivors received 60-minute touch-based verum or mock-healing intervention. After receiving the first session, patients immediately rated their beliefs on treatment helpfulness in terms of fatigue, well-being, and immune function. Within a week, patients received a second session, during which pre-post session NA (Profile of Mood States) and blood draws were obtained and assayed for plasma oxytocin levels (ELISA) and OXTR genotyping. Data were analyzed using Pearson correlations and general linear models.Both verum and sham groups showed significant pre-post session decreases in NA (p=.005) with no group differences (p=.31). Pre-post session, NA decreases were marginally associated with increases in plasma oxytocin . Pre-session NA and plasma oxytocin did not differ by OXTR genotype (p>.33). However, A-allele carriers showed significantly greater reductions in pre-post NA than G/G carriers (t(30)=2.702, p=.011, n=19). A-allele carriers also uniquely showed significant associations with belief and NA decrease, such that for A-allele carriers only, prior-rated belief in treatment helpfulness was associated with greater decreases in negative affect .OXTR rs53576 A-allele carriers show greater reductions in NA and unique associations with belief and NA reductions in response to touch-based interventions. Results suggest that psychological and placebo conditioning responses to touch-based interventions may be predicted by biological sensitivity to social context."} +{"text": "Metabolic syndrome (MetS) is associated with cardiovascular mortality and increased risk of type 2 diabetes.We examine the prevalence of MetS in a cohort of Caucasian women with previous gestational diabetes (GDM) (n=116), and those with normal glucose tolerance (NGT) during pregnancy (n=51). Fasting glucose alone (known DM/pre-diabetes post-partum patients) or 75g OGTT (other patients), lipid profile, insulin and c-peptide were performed. We calculated insulin resistance using the HOMA2-IR computer model.Metabolic syndrome and insulin resistance are significantly more prevalent in Caucasian patients with GDM progressing to post-partum DM/pre-diabetes than those who do not (p<0.01), suggesting a need to target lifestyle changes in the early post-partum period to help prevent progression to T2DM."} +{"text": "Graft-versus-host disease is one of the major transplant-related complications in allogeneic hematopoietic stem cell transplantation. Continued efforts have been made to prevent the occurrence of severe graft-versus-host disease by eliminating or suppressing donor-derived effector T cells. Conventional immunosuppression does not adequately prevent graft-versus-host disease, especially in mismatched transplants. Unfortunately, elimination of donor-derived T cells impairs stem cell engraftment, and delays immunologic reconstitution, rendering the recipient susceptible to post-transplant infections and disease relapse, with potentially lethal consequences. In this review, we discuss the role of dynamic immune regulation in controlling graft-versus-host disease, and how cell-based therapies are being developed using regulatory T cells and other tolerogenic cells for the prevention and treatment of graft-versus-host disease. In addition, advances in the design of cytoreductive conditioning regimens to selectively target graft-versus-host disease-inducing donor-derived T cells that have improved the safety of allogeneic stem cell transplantation are reviewed. Finally, we discuss advances in our understanding of the tolerogenic facilitating cell population, a phenotypically and functionally distinct population of bone marrow-derived cells which promote hematopoietic stem cell engraftment while reducing the risk of graft-versus-host disease. Graft-versus-host disease (GVHD) remains a major obstacle for the clinical application of hematopoietic stem cell (HSC) transplantation . GVHD is+ T cell and CD8+ T cell)-mediated GVHD are multifactorial and include activation of macrophages and antigen-presenting cells (APC) by transplantation conditioning regimens to damage host tissue, releasing soluble cytokines such as TNF-\u03b1 and IL-1; alloreactive T cell activation, proliferation and differentiation in response to host or donor APC; and alloreactive T cell infiltration and release of pro-inflammatory cytokines which leads to damage of the target tissue [+/CD25+/forkhead/winged helix transcription factor + (FoxP3+), CD8+/CD28-, T/natural killer (NK) cells, and TCR+/CD4-/CD8-, most studies have concentrated on CD4+/CD25+/FoxP3+ T cells [+ T cell population, CD4+/CD25+/FoxP3+ regulatory T cells (Treg) have been demonstrated to suppress a variety of immune responses dependent on effector T cells.The mechanisms of donor T cell production, direct cell-cell contact has also been postulated to be required as a mechanism of action. Natural Treg are generated in the thymus and are nonspecific in their suppressive capability [reg must encounter antigens to exert their suppressive effects, once activated they suppress in an antigen-nonspecific manner, presumably through the release of immunosuppressive cytokines such as IL-10 and TGF-\u03b2 [reg are inducible and need to be activated through their TCR in order to mediate their suppressive activities. The expression of receptors of chemokines, such as C-C chemokine receptor type 5 (CCR5) and CXC chemokine receptor 3 (CXCR3), on antigen-specific Treg support a role for proper trafficking of Treg to target tissue in the prevention of acute GVHD in murine models [reg to secondary lymphoid tissues and sites of inflammation may play an important role in the control of GVHD.CD4 of GVHD and, to of GVHD -8. Althopability ,9. Althoe models ,12. A rereg can suppress proliferative activity of conventional CD4+ T cells and CD8+ T cells to alloantigenic stimulation in vitro and induce transplantation tolerance and reduce acute GVHD occurrence in vivo [+/CD25+ Treg isolated from the spleen or bone marrow of C57BL/6 mice can suppress lethal GVHD induced by transplanted donor CD4+/CD25- T effector cells after allogeneic T cell-depleted bone marrow transplantation (BMT) [+/CD25+ Treg does not abrogate the beneficial effect of donor T cell-mediated GVL [et al. recently evaluated the impact of early infusion of freshly isolated human donor CD4+/CD25+ Treg followed by conventional T cells in human leukocyte antigen (HLA)-haploidentical HSC transplantation [reg prevented acute as well as chronic GVHD in the absence of immunosuppressive drug therapy after transplantation. Moreover, Treg promoted immune reconstitution of CD4+ T cells, CD8+ T cells, B cells, and NK cells, and improved protective immunity against cytomegalovirus infection.Several studies in experimental murine models have shown that T in vivo -15. Donoon (BMT) . Importaated GVL . Ianni eantation . Adoptivreg-based therapy to prevent GVHD is obtaining sufficient numbers of antigen-specific Treg and maintaining their regulatory properties after infusion [ex vivo expansion of mouse recipient-specific CD4+/CD25+ Treg can be obtained using donor Treg stimulation with allogeneic recipient APC [Ex vivo expansion of Treg resulted in specific tolerance for recipient-type alloantigens, but not for third-party antigens. Moreover, the Treg effectively controlled GVHD while favoring immune reconstitution and maintaining GVL in a murine model [reg from human CD4+/CD25-/CD45RA+ cells exhibit a potent suppressive function in vitro compared with natural Treg, and suppress acute GVHD in a xenogeneic NOD/SCID mouse model [reg for the prevention and/or treatment of GVHD have recently been described. Ianni et al. reported that the adoptive transfer of freshly isolated, donor-derived natural Treg followed by conventional donor-derived T cells (Tcon) at the time of full-haplotype-mismatched adult HSC transplantation in patients being treated for hematologic malignancies prevented acute or chronic GVHD while favoring Tcon-mediated post-transplantation immune reconstitution [et al. detailed a 'first-in-human' phase 1 clinical trial of nonspecific (CD3/CD28 bead)-based ex vivo expanded umbilical cord blood-derived Treg in patients after umbilical cord blood transplantation and noted a reduced incidence of severe acute GVHD [The major limitation for the clinical application of Tinfusion . Studiesient APC ,19. Ex vne model . Anotherse model . Clinicatitution . Brunsteute GVHD .reg as diagnostic and prognostic cellular biomarkers for acute GVHD have been investigated in mouse and rat models and human HSC transplantation [+ Treg in skin biopsies from GVHD patients was significantly higher in patients responding to GVHD treatment and with a lower grade of GVHD compared with those with more severe GVHD [reg can regulate immune responses and induce tolerance to alloantigen after HSC transplantation. These findings provide evidence for the use of Treg as a useful biologic to improve GVHD diagnosis and treatment.Tantation -25. Studantation , suggestere GVHD . Taken tIn GVHD, functional immune cells in the donor bone marrow recognize the recipient as foreign and mount an immunologic attack on the patient's organs and tissues, impairing their ability to function . Strict et al. recently reported the outcomes of 68 patients with poor-risk hematologic malignancies who were conditioned with fludarabine, cyclophosphamide and 2 Gy total body irradiation prior to receiving T cell-replete bone marrow from HLA-haploidentical, first-degree relatives [The major clinical rationale for HLA-haploidentical BMT is to extend the potential benefits of HSC transplantation and the graft-versus-tumor effect to patients who lack an HLA-matched donor. The major challenge is to reduce the incidence of fatal graft rejection, severe GVHD and treatment-related mortality while promoting engraftment. Luznik elatives . Donors elatives . Recoverelatives . One yeaBased upon the results of Luznik and Fuchs ,36,39, w+/TCR- facilitating cells (FC) capable of promoting allogeneic HSC engraftment and establishment of mixed chimerism [T cell depletion (TCD) of allogeneic bone marrow is extremely effective at reducing the risk of GVHD, but greatly increases the risk of engraftment failure. Three possible reasons for TCD-associated-engraftment failure have been hypothesized. First, HSC can engraft across MHC-matched but minor disparate barriers, but fail to engraft in allogeneic recipients because they are harmed or removed during TCD. Second, T cells act as essential partner cells to promote allogeneic HSC engraftment. Third, an additional bone marrow population that shares some T cell markers protects the HSC from rejection or 'facilitates' HSC engraftment, but is inadvertently removed as an 'innocent bystander' during TCD. Investigations in different laboratories have provided strong evidence in support of the latter hypothesis. Initial studies in mice using different cell subsets sorted from donor bone marrow identified a distinct population of CD8himerism . The addhimerism . Transpl+/TCR- FC population shares phenotypic characteristics with CD8\u03b1 plasmacytoid precursor dendritic cells (p-preDC) and that total FC are heterogeneous in morphology [et al. [+/TCR- FC were positive for CD11c+ (65% to 70%) and B220+ (75% to 88%) expression. While only 15% of the B220+ population was composed of B cells, 55% co-expressed CD11c (CD11c+/B220+), suggestive of a dendritic cell component. Further analysis into this CD11c+ dendritic subset revealed that a p-preDC phenotype (CD11dim/B220+/CD11b-) is the predominant cell type (93% to 95%) within this CD11c+ subset , and secondary transplantation of the Treg with donor HSC plus third-party HSC resulted in engraftment of only the donor HSC, demonstrating antigen-specificity.It was believed that p-preDC were the primary activators of naive CD4 as well . The Tay T cells . The las T cells . Recent sources . The B7 ficiency . CD86 isntiation . More reIn recent years there have been significant advances in understanding the mechanism of FC function, which have provided additional support for the promising potential for FC to be used in the clinic to generate tolerance after HSC transplantation. Data collected from recent studies have helped to distinguish FC from other hematopoietic cells such as p-preDC. However, there are questions that remain to be answered regarding the role of FC in inducing tolerance, preventing GVHD and promoting chimerism following BMT. A phase 2 clinical study approved by the Food and Drug Administration is currently underway to test the use of FC-based HSC transplantation in recipients of living donor renal allografts. Preliminary findings have demonstrated that high levels of donor macrochimerism without GVHD can be established in mismatched recipients conditioned with fludarabine, cyclophosphamide and 200 cGy total body irradiation .The use of cell-based therapies in the prevention and treatment of GVHD is now being applied in a number of clinical protocols, with promising results. Cell-based therapies have the potential to promote engraftment, preserve immunocompetence and prevent GVHD, thereby avoiding the toxicity of broad acting nonspecific immunosuppressive agents.reg: regulatory T cell; TNF-\u03b1: tumor necrosis factors-\u03b1; UCB: umbilical cord blood.APC: antigen-presenting cells; BMT: bone marrow transplantation; CCR5: C-C chemokine receptor; CXCR3: CXC chemokine receptor 3; DC: dendritic cells; FC: facilitating cells; FoxP3: forkhead/winged helix transcription factor; GVHD: graft-versus-host disease; GVL: graft-versus-leukemia; HLA: human leukocyte antigen; HSC: hematopoietic stem cell; IL-1: interleukin-1; MHC: major histocompatibility complex; NK: natural killer; p-preDC: plasmacytoid precursor dendritic cells; TCD: T cell depletion; Tcon: conventional donor-derived T cells; TCR: T cell receptor; TGF-\u03b2: transforming growth factor beta; TSTI has significant equity interest in Regenerex, LLC, a start-up biotechnology company based on the facilitating cell technology. All other authors have no conflict of interest to declare.reg cells in GVHD prevention; HX: studies on role of cyclophosphamide; IG: review of FC mechanism of action; STI: overall editing and preparation of review. All authors have read and approved the final manuscript.JL: overall preparation and editing of review; YH: studies on role of TThe pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1741-7015/10/48/prepub"} +{"text": "Drosophila humoral innate immune response fights infection by producing antimicrobial peptides (AMPs) through the microbe-specific activation of the Toll or the Imd signaling pathway. Upon systemic infection, the production of AMPs is both positively and negatively regulated to reach a balanced immune response required for survival. Here, we report the function of the dRYBP (drosophila Ring and YY1 Binding Protein) protein, which contains a ubiquitin-binding domain, in the Imd pathway. We have found that dRYBP contributes to the negative regulation of AMP production: upon systemic infection with Gram-negative bacteria, Diptericin expression is up-regulated in the absence of dRYBP and down-regulated in the presence of high levels of dRYBP. Epistatic analyses using gain and loss of function alleles of imd, Relish, or skpA and dRYBP suggest that dRYBP functions upstream or together with SKPA, a member of the SCF-E3-ubiquitin ligase complex, to repress the Imd signaling cascade. We propose that the role of dRYBP in the regulation of the Imd signaling pathway is to function as a ubiquitin adaptor protein together with SKPA to promote SCF-dependent proteasomal degradation of Relish. Beyond the identification of dRYBP as a novel component of Imd pathway regulation, our results also suggest that the evolutionarily conserved RYBP protein may be involved in the human innate immune response.The Drosophila uses the evolutionarily conserved host defense of innate immunity to protect against microbial infection and relies mainly on the Toll and Imd pathways to regulate the expression of different AMP genes Regulation of NF-\u03baB pathway activity in both invertebrates and vertebrates is achieved at multiple levels through ubiquitin-mediated post-translational modification of signaling components dRYBP (drosophila Ring and YY1 Binding Protein) gene Nucleoporin Zinc Finger) type Drosophila have described the phenotypic effects of high and low levels of dRYBP expression and also its interactions with several proteins involved in a range of biological processes, including epigenetic transcriptional regulation mediated by the Polycomb and trithorax groups of proteins Death Effector Domain) containing proteins Drosophila, high levels of dRYBP induces apoptosis in imaginal disc cells and this apoptosis is dependent on dFADD and DREDD DrosophilaThe In this work we show that dRYBP contributes to the negative regulation of the Imd signaling pathway. Notably, we have found that dRYBP is required for the inhibition of the production of AMPs upon systemic infection. We propose that dRYBP functions in the immune response to promote ubiquitin-dependent degradation of IMD-pathway components, among those the Relish protein.SCanton were used as control flies. dRYBP null allele stocks were 1/CyO GFPdRYBP and \u039455/ CyO GFPdRYBPdRYBP genomic region were Df(2R)BSC598 and Df(2R)BSC787 (http://flybase.bio.indiana.edu). The GAL4 lines used were c564-Gal4, en-Gal4, hs-Gal4 (http://flybase.bio.indiana.edu). The UAS lines used were: RNAiUAS-dRYBP, UAS-dRYBPUAS-imdRNAiUAS-skpA (http://www.flyrnai.org/TRiP-HOME.html), UAS-Relish-His-6pUASt-Venus-dRYBP (this work). The RNAiUAS-VDRC lines http://stockcenter.vdrc.at.Erwinia carotovora carotovora 15 (Ecc15), and the Gram-negative Escherichia coli (E. coli) were grown overnight as shaking cultures in LB medium at 29\u00b0C (Ecc15) and 37\u00b0C (E. coli) and pelleted to an OD600 of 200 (Ecc15) or to an OD600 of 400 (E. coli). Systemic infection of flies was done by pricking a needle dipped in a bacterial pellet into the thorax of 2\u20135-day old adult females. Where appropriate, pathogens were heat-killed at 95\u00b0C during 15 min.The Gram-negative entomopathogen dRYBP (BDGP DGC clone LD18758) without start codon was cloned into pENTR-D-TOPO (Life Technologies) using the following primers: Forward: 5\u2032-CACCGACAAGAAATCCTCGCCG- 3\u2032, Reverse: 5\u2032-CTAACTCCGGCTGTCGTTG-3\u2032. The Gateway system (Life Technologies) was used to generate the expression vector pUASt-Venus-dRYBP. Transgenic flies were obtained following standard procedures using 1118white flies as host.The coding sequence of RpL32. The sequences of the primers and their efficiencies (in brackets) are the following:RNA was isolated from 10\u201315 adult female flies of appropriate genotype. RNA extraction and RT reactions were performed as previously described RpL32 Forward: 5\u2032-GACGCTTCAAGGGACAGTATCTG-3\u2032, Reverse: 5\u2032-AAACGCGGTTCTGCATGAG-3\u2032 ; dRYBP Forward: 5\u2032-CATGTTGACACCTGGCTCCTG-3\u2032, Reverse: 5\u2032-CGAAGGTGATCGAGGAGAAC-3\u2032 ; Dpt: Forward: 5\u2032-GCTGCGCAATCGTTCTACT-3\u2032, Reverse: 5\u2032-TGGTGGAGTGGGCTTCATG-3\u2032 ; AttB Forward: 5\u2032-CCTACAACAATGCTGGTCATGGT-3\u2032, Reverse: 5\u2032-CCTACAACAATGCTGGTCATGGT-3\u2032 ; Relish Forward: 5\u2032-TTAGCGTGGCCAACACAATG-3\u2032, Reverse: 5\u2032-GAACTGCCATGTGGAGTGCAT-3\u2032 ; PGRP-LC Forward: 5\u2032-GCATTCAATGGTGGTCCCA-3\u2032 Reverse: 5\u2032-CCGGATCTTCGTGTTTGGAG-3\u2032 ; imd Forward: 5\u2032-TTCGGCTCCGTCTACAACTT-3\u2032, Reverse: 5\u2032-GTGATCGATTATGGCCTGGT-3\u2032 ; Dredd F: 5\u2032-CAAAAGGTGGGCCTCTGCT Reverse: 5\u2032-GTAGGTGGCATCCGAGTGGT-3\u2032 ; Tab-2 Forward: 5\u2032-TGTCATGGAGGAATGCGATC-3\u2032, Reverse: 5\u2032-GCTTCTGACGCTCGATAGTGG-3\u2032 ; TAK1 Forward: 5\u2032-GATCTGAGTCCCAGCGAAAGC-3\u2032, Reverse: 5\u2032-CATCGCTCTTTGCGTTCGT-3\u2032 ; ird-5 Forward: 5\u2032-TAGTGATCCATTGGCGAAACC-3\u2032, Reverse: 5\u2032-GCTTGGTGGCAATTTCACG-3\u2032 ; skpA Forward: 5\u2032-CTCCCGAGGAAATACGCAAG-3\u2032, Reverse: 5\u2032-CGGGCGAAAAGTCCTTCTTA-3\u2032 ; dIAP2 Forward: 5\u2032-ATGCAAGGTATGCTTGGACGA-3\u2032, Reverse: 5\u2032-TGATTGCAGGTGGCCAAGT-3\u2032 The data from all the qRT-PCR experiments represent the mean + SEM of three biological repeats with 10\u201315 individuals per sample. For inter-assay comparability, values within each experiment were routinely normalized to the wild type or relevant other control at a given time point. Data were analyzed using ANOVA with Bonferroni post-test.Fat bodies from adult females were dissected in PBS, fixed in 4% paraformaldehyde and stained with either anti-dRYBP antibody (1\u2236100) dRYBP indicate that this gene plays a role in diverse biological processes dRYBP interacting genes and found a number of components of the Imd pathway , which are always induced upon activation of the immune response. mRNA expression levels of Diptericin (Dpt), an AMP normally induced upon activation of the Imd pathway dRYBP null adult flies dRYBP mutant flies, Dpt expression was not significantly altered compared to the wild-type control (Erwinia carotovora carotovora 15 (Ecc15), Dpt expression was significantly increased beyond wild type levels after 8 h of infection in 1dRYBPand \u039455dRYBP homozygous mutant flies, as well as in flies of 1dRYBP or \u039455dRYBP genotype over dRYBP genomic deficiencies excluding background effects . Since the Imd pathway includes both cytosolic and nuclear steps, we asked whether dRYBP localized to any cellular compartment in particular during infection. To this aim we constructed UAS-Venus-dRYBP flies and used the c564-Gal4 line to drive Venus-dRYBP expression specifically in the fat body where AMPs are highly up-regulated in response to systemic infection Ecc15, as shown by fluorescence intensity profiles across cells. This suggests that either the putative dRYBP expression in the cytoplasm is too low to be detected using this approach or that dRYBP acts on nuclear Imd pathway components rather than upstream cytosolic adaptors.We have previously shown that dRYBP is ubiquitously expressed in the embryo and the imaginal discs of the larva and localizes to the nuclei of cells Dpt expression when the pathway was activated by forced expression of IMD or Relish, both of them activators of the Imd cascade skpA, a known repressor of the Imd cascade To delineate the hierarchical positioning of dRYBP in the Imd signaling cascade, we studied whether overexpression of dRYBP could repress hs-Gal4,UAS-imd flies resulted in increased production of Dpt hs-Gal4,UAS-imd/UAS-dRYBP flies, Dpt expression was repressed . This inhibition is not due to dilution of the GAL4 protein as the expression levels of both dRYBP and imd were very similar in flies overexpressing a single gene versus flies overexpressing both genes concomitantly Dpt expression was still diminished in hs-Gal4/UAS-Relish;UAS-dRYBP flies dRYBP in these conditions did not affect the expression of Dpt containing proteins Drosophila RYBP protein and the vertebrate RYBP/DEDAF/YAF2 protein have been shown to interact with Death Effector Domain (DED) proteins, a subfamily of the DD proteins www.deathdomain.org) The vertebrate homolog of Relish, the NF-\u03baB subunit p105 protein, has been demonstrated both to be ubiquitylated and to belong to the superfamily of DD Crossing scheme. The engrailed-Gal4 driver was used to express UAS-dRYBPRNAi and UAS-VDRCRNAi in the wing. Female virgins en-Gal4/RNAi /MKRSCyO;UAS-dRYBP were crossed with males RNAiUAS-VDRC and maintained at 29\u00b0C. From these crosses, the RNAien-Gal4;UAS-VDRC progeny were analyzed for wing phenotypes associated with the particular RNAiVDRC line under study and the RNAi,/UAS-VDRCRNAien-Gal4;UAS-dRYBP progeny were analyzed for the penetrance of the wing blister phenotype. (B) Wild-type wing. (C) RNAien-Gal4>dRYBP wing showing a blister (arrow) in the posterior compartment. (D) Quantification of flies with wing blisters of the indicated genotypes. RNAi/UAS-GFPen-Gal4>dRYBP was used as a control. Importantly, this screen merely shows genetic interaction between dRYBP and Imd pathway components. It is not yet clear why the penetrance of blister phenotype in our screen can be modulated by mutations in genes involved in the innate immune response or why the penetrance is modulated by mutations in both activators and repressors of the Imd pathway. The wing phenotypes are probably due to these factors involved in other biological processes The (TIF)Click here for additional data file.Figure S2. (A) Mass spectrometry results showing the scores for the indicated dRYBP interacting proteins. (B) Alignment of Human p105 and Drosophila Relish protein sequence. Indicated in green is the predicted Death Domain (DD) sequence (www.deathdomain.org). (C) Magnification of the predicted DD domain. (D) Alignment of other DD containing proteins with the predicted DD domain in the Relish proteins.Mass spectrometry data and localization of a Death Domain in the Relish protein(TIF)Click here for additional data file."} +{"text": "A subsequent, left axillary lymph node biopsy revealed PTGC. PTGC can present as a false positive finding on FDG-PET/CT in lymphoma patients and biopsy should be done in HD patients in clinical remission but have a positive FDG-PET/CT scan.FDG-PET/CT is a widely established imaging modality for staging, restaging and monitoring therapy response in lymphoma patients. Progressive transformation of germinal centres (PTGC) is a benign condition presenting characteristically as asymptomatic lymphadenopathy. This paper presents a case of a 53-year-old man with a history of Hodgkin\u2019s disease (HD) whose F A 53-year-old man underwent FDG-PET/CT scanning for detection of recurrent disease. He was diagnosed with Hodgkin\u2019s disease (HD) five years ago and successfully treated with chemotherapy. The patient was asymptomatic. 12.6 mCi FDG was injected and images acquired using a Siemens Biograph 6 PET-CT scanner . The PETLeft axillary lymph node biopsy was performed and revealed progressive transformation of germinal centres (PTGC). PTGC was initially described by Lennert and Muller-Hermerlink as large follicles composed predominantly of diffuse small lymphocytes and an obscured mantle zone . PTGC isThe causes of false positive FDG-PET/CT include infection, inflammation, granulomatous disease ,7 and im"} +{"text": "A priori, if EPC aging plays a \u2018master-switch JPF-role\u2019 in stroke pathogenesis, juvenile EPC therapy alone should delay stroke-onset. Using a hypertensive, transgenic-hyperlipidemic rat model of spontaneous ischemic-hemorrhagic stroke, spTg25, we tested the hypothesis that freshly isolated juvenile EPCs are JPFs that can attenuate stroke progression and delay stroke onset.Identification of juvenile protective factors (JPFs) which are altered with age and contribute to adult-onset diseases could identify novel pathways for reversing the effects of age, an accepted non-modifiable risk factor to adult-onset diseases. Since endothelial progenitor cells (EPCs) have been observed to be altered in stroke, hypertension and hypercholesterolemia, said EPCs are candidate JPFs for adult-onset stroke. cd45- [cd34+/kdr+] EPCs decrease with progression to stroke in spTg25 rats, exhibit differential expression of the dual endodthelin-1/VEGFsp receptor (DEspR) and undergo differential DEspR-subtype specific changes in number and in vitro angiogenic tube-incorporation. In vivo EPC infusion of male, juvenile non-expanded cd45-[cd34+/kdr+] EPCs into female stroke-prone rats prior to stroke attenuated progression and delayed stroke onset (P<0.003). Detection of Y-chromosome DNA in brain microvessels of EPC-treated female spTg25 rats indicates integration of male EPCs into female rat brain microvessels. Gradient-echo MRI showed delay of ischemic-hemorrhagic lesions in EPC-treated rats. Real-time RT-PCR pathway-specific array-analysis revealed age-associated gene expression changes in cd45-[cd34+/kdr]EPC subtypes, which were accelerated in stroke-prone rats. Pro-angiogenic genes implicated in intimal hyperplasia were increased in stroke-prone rat EPCs (P<0.0001), suggesting a maladaptive endothelial repair system which acts like a double-edged sword repairing while predisposing to age-associated intimal hyperplasia.FACS analysis revealed that cd45-[cd34/kdr+]EPCs are juvenile protective factors for ischemic hemorrhagic stroke as modeled in the spTg25-rat model. The ability to delay stroke onset emphasizes the importance of EPC-mediated roles in vascular health for ischemic-hemorrhagic stroke, a high unmet need.Altogether, the data demonstrate that A priori, targeting the maintenance of juvenile protective factors comprises a mechanism-based paradigm for the prevention and intervention of diseases associated with aging, such as stroke, neurodegenerative and/or cardiovascular diseases. In this way, instead of aging being a non-modifiable risk factor, JPFs become a translational pathway to modifying the contribution of aging to disease pathogenesis.Although much research has focused on environmental risk factors for adult-onset diseases as a paradigm for prevention and intervention, the counterpart concept of \u201cjuvenile protective factors\u201d is less studied but equally important. As a conceptual construct, juvenile protective factors (JPFs) are endogenous physiological factors \u2013 be it cells, proteins, nucleic acids, or biochemical equivalent \u2013 which prevent disease in the young but which undergo age-associated cumulative changes which contribute to adult-onset diseases. a priori putative juvenile protective factors for vascular health. Although there are controversies regarding definition and methods of isolation, as a concept EPCs are circulating cells involved in endothelial repair, maintenance of capillary network, and postnatal vascularization and angiogenesis in both physiological and pathological conditions As part of the endothelial repair mechanism, endothelial progenitor cells (EPCs) are ex vivo culture-expanded EPCs that are cd45+ [cd34+/kdr+]EPCs since cd45- [cd34+/kdr+]EPCs do not undergo ex vivo expansion cd45+ [cd34+/kdr+]EPCs are pro-angiogenic in vitro and in vivo in contrast to freshly isolated cd45+ [cd34+/kdr+]EPCs which are not ex vivo culture expansion is, a priori not a physiological paradigm, and since freshly isolated (non-expanded) cd45+ [cd34+/kdr+]EPCs have been observed to not induce revascularization cd45- [cd34+/kdr+]EPCs underlie in vivo real-time endothelial repair for the maintenance of endothelial integrity and microvascular networks, and hence are critical juvenile protective factors for microvascular health. To test this, we focused on ischemic-hemorrhagic stroke because of the key role of the microcirculation in cerebral ischemia and hemorrhagic transformation.Currently, most pre-clinical EPC studies focus on Ischemic-hemorrhagic strokes and hemorrhagic transformation after an ischemic stroke are major causes of morbidity and mortality and remain without major treatment breakthroughs. Based on the successful modeling of the clinical spectrum of cerebral chronic low-flow ischemia, microvascular paucity, microhemorrhages and subsequent ischemic-hemorrhagic strokes in a genetically hypertensive/transgenic-hyperlipidemic rat model, spTg25 rat model cd45- [cd34+/kdr+]EPCs exhibit subset-specific functions and pathway-specific profile changes with age, and that cell-therapy with freshly isolated juvenile cd45- [cd34+/kdr+]EPCs delays the onset of ischemic-hemorrhagic strokes in the spTg25 rat model.Here we report that EPC subtype-specific changes occur in a non-linear fashion in response to developmental programming of increased stroke susceptibility, that fresh All rat studies were done in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Boston University School of Medicine Institutional Animal Care and Use Committee (IACUC Protocol Number AN14055). The inbred, transgenic[hCETP]25 Dahl Salt-sensitive rat line (Tg25) was maintained in-house as heterozygous in pathogen-free conditions, and stroke-prone Tg25+ rats were bred on Purina 5001 regular rat chow with 0.4% NaCl as described To determine rat EPC subtypes by FACS analysis, we performed FACS analysis on the buffy coat from 1 ml of whole blood in citrate dextrose buffer from each rat per study group. Following standard FACS protocols Immunophenotyping was performed using fluorescently labeled primary antibodies: AF647-anti-rat KDR, AF488 anti-ratCD34 , and PE anti-ratCD45 , and PerCP Cy5.5-anti-ratDEspR antibodies at 1 ug/ml. Cells were washed and fixed in Cytofix buffer and filtered . Differential immunophenotyping-fluorescence intensities were read in a BD FACS scanner and analyzed using Summit version 4.0 .4Cl2 rbc lysis buffer for FACs analysis (BD Sciences). Immunophenotyping was performed using AF647-anti-rat KDR, AF488 anti-ratCD34 , and PE anti-ratCD45 , and PerCP Cy5.5-anti-ratDEspR antibodies at 1 ug/ml in 200 ul ice-cold staining buffer for 1 hour in the dark, followed by 2 washes in 2% FBS, 1\u00d7PBS solution. Cells were then resuspended in 500 ul wash buffer and cd45- [cd34+/kdr+]EPCs were isolated using MoFlo high-speed cell sorter with gating for cd45-, cd34+ and kdr+ EPCs for EPC-infusion therapy experiments. For tube incorporation experiments and nano-RT2 pathway-specific PCR array analysis, anti-DEspR immunophenotyping was added, and the additional DEspR+ vs. DEspR- gating performed during MoFlo high-speed cell sorting. MoFlo high-speed cell sorting was done after parameters were adjusted using control beads, prepared single-color control samples, and a universal negative control. Gated EPCs were collected in 200 ul sterile phosphate buffered saline with 2% fetal bovine serum for EPC therapy or in endothelial complete medium for tube incorporation assays. For RNA isolation for array analysis, EPCs were immediately subjected to quick-freeze after collection.In order to minimize \u201ctime-out-of-body\u201d and processing steps, EPCs were isolated directly from the \u201cbuffy coat\u201d mononuclear cell layer from 10-mls of rat whole blood in citrate phosphate dextrose buffer after removal of contaminating red blood cells (rbc) using NHEPCs were isolated from 8-week old donor non-stroke prone male inbred-Dahl S rats on the day of infusion and infused immediately via tail vein into recipient inbred-Dahl S stroke prone Tg25+ rat females at 3-months and 4-months of age prior to any stroke signs. Vehicle 1\u00d7PBS was injected into controls at identical time points. Stroke onset was determined by the detection of neurologic deficits such as seizures, paresis, paralysis, athetoid movements. Ex vivo 11.7T MR-imaging was performed on brains from control and stroke-prone rats as described To confirm EPC integration, another set of recipient spTg25+ female rats were infused with male donor EPCs and analyzed at 1-, 2- and 3-weeks after infusion to test for Y-DNA presence. Y-chromosome DNA testing was done on DNA isolated from kidney, half-brain and microvessels isolated from the other half-brain as described pkh26 red fluorescent cell linker after MoFlo high speed cell sorting isolation as described above. Isolated pkh26-labeled EPCs in 200 ul complete medium was mixed with 5,000 HUVECs in 300 \u00b5l of complete medium and then layered onto matrigel-coated culture slide system . After 16\u201318 hours to allow tube formation, EPC-incorporation into HUVEC-tubes was viewed and photographed using a Nikon Epifluorescence microscope using identical exposures.Tube incorporation with human umbilical vein cells (HUVECs) undergoing tubulogenesis was analyzed using standard angiogenesis conditions with Matrigel basement matrix mix . To distinguish EPCs from HUVECs, EPCs were labeled with deltaCT where deltaCT\u200a=\u200aaverage CT of gene-X of test group \u2013 average CT of gene-X of reference group. Two-fold or greater vectorial change in gene expression between test groups were cataloged per pathway and analyzed for statistical significance.Total cellular RNA with no contaminating DNA was isolated from subtype-EPCs from 6 rats per study group. Per study group, EPC-RNA samples from 2 rats were pooled for a total of 3 independent biological replicates. Six arrays were used to obtain 2 technical replicates per RNA pool. Pathway-specific array analysis was done using RT2 nano-preAmp cDNA synthesis kit, RT2 Profiler PCR arrays, and Rat Pathway Finder array with angiogenesis, apoptosis, stem cell, and cell senescence pathways represented , and run on an ABI StepOnePlus instrument. Quality controls comprised of equivalent positive PCR and reverse transcription controls among samples, and absence of reverse transcription inhibitors and DNA contamination. Genes with cycle threshold (CT) values >35 were considered not-expressed as recommended. The average of CT values were obtained only when variation among the 6 replicates was <10%. Per gene, the ratio of gene expression between different test groups to a common reference group was calculated as delta CT, and the fold change calculated as 2Data were assessed for normality and presented as means \u00b1 sd. One-way ANOVA and Tukey\u2019s multiple comparison tests were used to compare differences in EPC subtype-specific levels and functional parameters among different study groups. Kaplan Meier survival curve analysis and Holm-Sidak test for P values were used to determine efficacy of EPC-therapy. Two-way (gene\u00d7study group) ANOVA and Holm-Sidak multiple pairwise comparisons were done to assess statistically significant gene expression changes. GraphPad Prism and SigmaStat software programs were used for statistical analyses.cd45- [cd34+/kdr+]EPCs. The focus on non-expanded EPCs is based on the logic that real time in vivo surveillance and repair of endothelial cell turnover or injury would require circulating \u201con-the-ready\u201d EPCs to detect, home to, and repair endothelial turnover and injury, prior to platelet adhesion and thrombosis. Experiments were therefore designed to test whether cd45- [cd34+/kdr+]EPCs quantitatively and/or qualitatively decline with age, and test the converse whether juvenile EPCs contribute to \u201con-the-ready\u201d real-time surveillance and endothelial repair critical for the maintenance of endothelial integrity, thus capable of delaying stroke onset.To determine whether EPCs are juvenile protective factors for microvascular health, we performed a series of quantitative and qualitative analyses of the effects of age, sex and stroke susceptibility on freshly isolated, non-\u201cculture-expanded\u201d cd34+/kdr+] EPCs to cd45+ white blood cell count, did not exhibit aging-associated changes in both males and females of juvenile and 17 wk-old male and female rats old rats Dahl S rats. The 17-wk time point was selected to be able to study stroke-prone Tg25 rats prior to strokes, since spTg25 female rats start exhibiting stroke signs around this age To specifically test the role of EPCs as juvenile protective factors in the context of aging and stroke susceptibility, we first studied putative changes in EPC subtype-specific numbers in stroke prone rats compared to control non-stroke prone rats that were genetically identical with the exception of developmental programming for stroke induced by gestational exposure to increased NaCl cd45- [cd34+/kdr+]EPCs/ml than 4 m-old rats regardless of transgenic expression of human cholesteryl ester transfer protein, which induces significant hypercholesterolemia on regular rat chow cd45- [cd34+/kdr+]EPCs by 4-label LSRII-FACS analysis. DEspR was selected as a previously untried EPC surface marker detected on some embryonic hemangioblasts and involved in developmental vasculogenesis and angiogenesis leading to embryonic lethality by E10.5\u201312.5 embryonic day cd45- [cd34+/kdr+]EPCs exhibit differential modulation patterns with age, stroke susceptibility and transgenic[hCETP]-induced combined hyperlipidemia spTg25+ rat EPCs exhibit gene expression profiles similar to old (4 m-old) non-spTg25 rat controls suggesting that accelerated EPC-aging is associated with stroke-susceptibility. This observation is seen in both DEspR+ and DEspR- EPCs, although differential DEspR-subtype specific expression patterns are observed compared to 4 m stroke-prone spTg25+ female rats could alleviate the course of microvascular paucity and ensuing chronic low flow ischemia, thereby delaying the onset of ischemic hemorrhagic stroke in the most susceptible subgroup, spTg25+ female rats. Littermate spTg25+ female rats were randomly assigned to EPC therapy at 3- and 4-months of age (n\u200a=\u200a8) or mock-therapy infused rats (n\u200a=\u200a7). Stroke onset was defined by the onset of neurological deficits such as seizure, paresis/paralysis, or athetoid movements as described P<0.003) Table 2.To prove that indeed EPCs are integrated into microvessels, and determine how long donor-EPCs are detectable in recipient rat tissues by PCR-amplification analysis, we performed Y-chromosome DNA analysis on recipient rat tissues after EPC infusions from 1\u20134 weeks post-infusion. We observed that Y-chromosome DNA was detected only in brain microvessels but not in kidney or whole brain, and only at 1- and 2-weeks post infusion timepoints of EPCs infused systemically Click here for additional data file."} +{"text": "However, few functional studies are available for miR-21, and few targets have been identified. In this study, we explored the role of miR-21 in human breast cancer cells and tissues, and searched for miR-21 targets.MicroRNAs (miRNAs) are a class of small non-coding RNAs (20 to 24 nucleotides) that post-transcriptionally modulate gene expression. A key oncomir in carcinogenesis is in vitro and in vivo assays to explore the role of miR-21 in the malignant progression of human breast cancer, using miR-21 knockdown. Using LNA silencing combined to microarray technology and target prediction, we screened for potential targets of miR-21 and validated direct targets by using luciferase reporter assay and Western blot. Two candidate target genes (EIF4A2 and ANKRD46) were selected for analysis of correlation with clinicopathological characteristics and prognosis using immunohistochemistry on cancer tissue microrrays.We used in vitro, and tumor growth in nude mice. Knockdown of miR-21 significantly increased the expression of ANKRD46 at both mRNA and protein levels. Luciferase assays using a reporter carrying a putative target site in the 3' untranslated region of ANKRD46 revealed that miR-21 directly targeted ANKRD46. miR-21 and EIF4A2 protein were inversely expressed in breast cancers .Anti-miR-21 inhibited growth and migration of MCF-7 and MDA-MB-231 cells miR-21 in MCF-7 and MDA-MB-231 cells inhibits in vitro and in vivo growth as well as in vitro migration. ANKRD46 is newly identified as a direct target of miR-21 in BC. These results suggest that inhibitory strategies against miR-21 using peptide nucleic acids (PNAs)-antimiR-21 may provide potential therapeutic applications in breast cancer treatment.Knockdown of Breast cancer (BC) is by far the most frequent cancer of women , with an estimated 1.15 million new cases worldwide in 2002 [miR-21 [miRBase: MIMAT0000076] has emerged as a key oncomir, since it is the most consistently up-regulated miRNA in a wide range of cancers and ankyrin repeat domain 46 (ANKRD46) [NCBI: NM198401] were selected for correlation analysis between protein levels and clinicopathological characteristics as well as prognosis using immunohistochemistry (IHC) on cancer tissue microrrays (TMAs).In this study, we explored the role of In situ hybridization analysis was performed on fresh samples from BC or fibroadenoma (FA) tissues with paired normal adjacent tissues obtained from Sun Yat-sen University Cancer Center (SYSUCC) between January and March 2009. For IHC staining of miR-21 predicted target genes, formalin-fixed paraffin-embedded tissues were obtained from 99 randomly selected BC patients without neoadjuvant therapy at SYSUCC from January 2000 to November 2004, from whom informed consent and agreement, and clinicopathological information was available. A pathologist reviewed slides from all blocks, selecting representative areas of invasive tumor tissue to be cored. Selected cores were analyzed in duplicate using a MiniCore Tissue Arrayer with a 1-mm needle. The diagnosis and histological grade of each case were independently confirmed by two pathologists based on World Health Organization classification and EIF4A2 [NCBI: NP001958] protein levels by IHC on TMAs constructed by the BC cases described in Materials and Methods. EIF4A2 was found in the cytoplasm, and ANKRD46 was seen in both the cytoplasm and nucleus Figure . ANKRD46P = 0.019), and ANKRD46 protein correlated with ER (P = 0.021) and PR , significantly higher than those with a low EIF4A2 protein level , which was not statistically different from those with low ANKRD46 protein . Nonetheless, treatment with anti-miR-21 reduced MCF-7 xenograft growth by approximately 68% for up to nine days. In vivo results suggested that the PNA-based miR-21 inhibitor had a subtle yet reproducible inhibitory effect on tumor growth. MCF-7 xenograft tumor sections demonstrated that miR-21 inhibition induced apoptosis of MCF-7 cells, confirming a previous study [MCF-7 cells are hormone-sensitive and difficult to culture -7 cells , althoug-7 cells . In our miR-21 as an important regulator of ANKRD46 mRNA and protein levels in BC cells. Our data showed that miR-21 directly interacted with the ANKRD46 3' UTR and inhibited ANKRD46 expression, though there was no significant association between miR-21 and ANKRD46 in resected patient tumors. This discrepancy may be due to three reasons. First, the artificial luciferase reporter assays do not fully recapitulate miRNA regulation in vivo [in vitro subsequent increase of ANKRD46 protein at the time point of observation (the indicated hours after transfection); third, immunohistochemistry (IHC) is conventional a semi-quantitative method with relatively limited sensitivity. IHC may not be sensitive enough to observe the down-regulation of ANKRD46 by miR-21. Functional study of ANKRD46 is required in the future to determine weather ANKRD46 is a functional target of miR-21 in BC progression as demonstrated in this study.ANKRD46, also known as ankyrin repeat small protein (ANK-S), is a 228-amino acid single-pass membrane protein, of unclear function. For the first time, we identify in vivo ; second,EIF4A2, an ATP-dependent RNA helicase, is expressed widely in human tissues [miR-21 and EIF4A2 protein were inversely expressed in resected BC patient tumors. But we did not find miR-21 binding sites in the EIF4A2 3' UTR and found no significant increase of EIF4A2 protein upon miR-21 knockdown in MCF-7 and MDA-MB-231 cells, although EIF4A2 mRNA increased after anti-miR-21 transfection. Taken together, the data reported here suggest that there maybe unknown indirect interactions between miR-21 and EIF4A2 in BC progression. In adult mice, the expression of the two EIF4A isoforms is dependent on cell growth status, with EIF4A1 expressed in all tissues, while EIF4A2 is expressed only in tissues with a low rate of cell proliferation [ tissues . In thisferation , indicatin vitro and in vivo, as well as cell migration in vitro. In addition, ANKRD46 is newly identified as a direct target of miR-21 in BC. These results suggest that miR-21 inhibitory strategies using PNA-antimiR-21 may have potential for therapeutic applications in BC treatment.We demonstrate that MCF-7 and MDA-MB-231 cells transfected with anti-miR-21 show growth inhibition in situ hybridization; DAPI: 4',6-diamidino-2-phenylindole; EIF4A2: eukaryotic initiation factor 4A: isoform 2; ER: estrogen receptor; F: forward primer; FA: fibroadenoma; FISH: fluorescein in situ hybridization; FITC: fluorescein isothiocyanate; GEO: Gene Expression Omnibus; IHC: immunohistochemistry; L: length; LNA: Locked nucleic acid; miRNAs: microRNAs; MTT: 3--2,5-diphenyltetrazolium bromide; Mut: mutation; NATs: normal adjacent tissues; NBT/BCIP: nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate; NCBI: National Center for Biotechnology Information; OS: overall survival; PNAs: peptide nucleic acids; PR: progesterone receptor; qRT-PCR: quantitative reverse transcription-polymerase chain reaction; R: reverse primer; RQ: relative quantification; RT: reverse transcription primer; SABC-AP: alkaline phosphatase-conjugated strept-avidin-biotin complex; SD: standard deviation: SYSUCC: Sun Yat-sen University Cancer Center; TMAs: tissue microrrays; TNM: tumor-lymph node-metastasis; W: width; WT: wild-type.3' UTR: 3' untranslated region; AJCC: American Joint Committee on Cancer; ANK-S: ankyrin repeat small protein; ASOs: antisense oligonucleotides; BC: breast cancer; CerbB2: v-erb-b2 erythroblastic leukaemia viral oncogene homolog 2 receptors; CISH: chromogenic Miss Li Xu Yan and Mrs Yan Zhang are doctoral degree candidates; Miss Qi Nian Wu and Mrs Yang Yang Li are master's degree candidates at SYSUCC. Mr Ding Zhun Liao, Mr Jing Hui Hou and Mrs Jia Fu are technicians at SYSUCC. Dr Mu-Sheng Zeng, Jing Ping Yun, Qiu Liang Wu and Yi Xin Zeng are Professors at SYSUCC. Dr Shao is a Professor and Vice Director at Department of Pathology of SYSUCC. The authors declare that they have not received any reimbursements, fees, funding, or salary, nor hold any stocks or shares in an organization that may in any way gain or lose financially from the publication of this manuscript, either now or in the future. The authors do not hold or are not currently applying for any patents relating to the content of the manuscript. The authors declare that they do not have any other financial or non-financial competing interests.LXY, QNW and YZ carried out the substantial experiment work and drafted the manuscript. JYS designed and financially supported the study. QNW and DZL were responsible for patient samples and tissue array construction. JHH and JF supported the immunohistochemistry. YYL carried out the luciferase reporter assay. JPY, MSZ, QLW and YXZ helped carry out the research design and critically reviewed the final version of the manuscript for submission. All authors read and approved the final manuscript.Word document containing the protocol of LNA-based CISH and FISH for miRNA in the present study.Click here for filemiR-21 and putative target genesExcel file containing a table listing RT and PCR primers for .Click here for filemiR-21 in human BC tissues and FA tissues by FISHPDF file comprising a figure showing the expression of . Positive in situ hybridization signals are green (FITC). miR-21 expression levels in BC tissues are much higher than that in corresponding NATs.Click here for filemiR-21 induction by LNA-antimiR in BC cellsPDF file comprising a figure showing relative . Relative miR-21 induction in LNA-antimiR-21 transfected MCF-7 (a) and MDA-MB-231 cells (b). Cells were transfected either with LNA-antimiR-21 or with LNA-control using Lipofectamine 2000 (Invitrogen) at indicated doses, and harvested at 12 h, 24 h, 36 h, 48 h, 60 h and 72 h, respectively. Total RNAs were isolated to quantify miR-21 expression by relative qRT-PCR, normalizing on U6 RNA levels. The graph shows a log2-scale RQ calculated by normalizing the miR-21 expression values in the LNA-antimiR-21 transfected cells on that in the LNA-control independently for each time point and dose. 50 nM LNA reagents at 48 h for MCF-7 and 50 nM at 36 h for MDA-MB-231 cells showed the best inhibition effects. Data indicate the mean (+SD) of three independent transfection experiments.Click here for file"} +{"text": "The cholinergic anti-inflammatory pathway (CAP) is a physiological mechanism that inhibits cytokine production and minimizes tissue injury during inflammation. CAP-mediated anti-inflammatory signals in vagal efferent nerve fibers result in the release of acetylcholine, which interacts with innate immune cells that express the nicotinic acetylcholine receptor subunit \u03b17 (\u03b17nAChR).Endothelial dysfunction during sepsis is responsible for increased endothelial permeability, leukocyte-endothelial interaction and functional breakdown of microvascular perfusion. Endotoxemia-induced endothelial dysfunction can be reduced by cholinergic CAP activation . The aimUsing fluorescent intravital microscopy, we determined venular wall shear rate, macromolecular efflux and leukocyte adhesion in mesenteric postcapillary venules of male Wistar rats. Endotoxemia was induced over 120 minutes by intravenous infusion of lipopolysaccharide (LPS). Control groups received an equivalent volume of saline. Cdp-choline was applied as an i.v. bolus in treatment groups. Animals received either (i) saline alone, (ii) cdp-choline 10 minutes prior to saline administration, (iii) cdp-choline 10 minutes prior to LPS administration, (iv) cdp-choline 30 minutes after LPS administration or (v) LPS alone.There were no significant differences in venular wall shear rate between the groups after 120 minutes. There was no significant difference in the number of adhering leukocytes between the cdp-choline/LPS groups and the LPS group after 120 minutes. Macromolecular efflux significantly increased in all groups over 120 minutes. All groups showed a significantly reduced macromolecular efflux compared with the LPS group after 120 minutes.Cdp-choline has no effect on leukocyte-endothelial interaction and microhemodynamic alterations during endotoxemia. By activating the CAP, cdp-choline reduces capillary leakage. Thus cdp-choline might have a prophylactic and therapeutic anti-inflammatory effect on LPS-induced endothelial permeability. These findings identify the endothelium as a target of anti-inflammatory cholinergic mediators and cdp-choline as a potential therapeutic substance in sepsis treatment."} +{"text": "High-risk human papillomavirus (hrHPV) infections are causally related to cervical cancer development. The additional (epi)genetic alterations driving malignant transformation of hrHPV-infected cells however, are not yet fully elucidated. In this study we experimentally assessed the role of the PI3-kinase pathway and its regulator PIK3CA, which is frequently altered in cervical cancer, in HPV-induced transformation.in vitro model system of hrHPV-transfected keratinocytes, representing the immortal and anchorage independent phenotype, was assayed for PI3-kinase activation and function using chemical pathway inhibition i.e. LY294002 treatment, and PIK3CA RNA interference. Phenotypes examined included cellular viability, migration, anchorage independent growth and differentiation. mRNA expression of hTERT and HPV16 E6E7 were studied using quantitative RT-PCR and Northern blotting.Cervical carcinomas and ectocervical controls were assessed for PIK3CA mRNA and protein expression by quantitative RT-PCR and immunohistochemical staining, respectively. A longitudinal in vitro, expression of the catalytic subunit PIK3CA as well as activation of downstream effector PKB/AKT progressively increased in parallel. Inhibition of PI3-kinase signalling in HPV16-transfected keratinocytes by chemical interference or siRNA-mediated silencing of PIK3CA resulted in a decreased phosphorylation of PKB/AKT. Moreover, blockage of PI3-kinase resulted in reduced cellular viability, migration, and anchorage independent growth. These properties were accompanied with a downregulation of HPV16E7 and hTERT mRNA expression. In organotypic raft cultures of HPV16- and HPV18-immortalized cells, phosphorylated PKB/AKT was primarily seen in differentiated cells staining positive for cytokeratin 10 (CK10). Upon PI3-kinase signalling inhibition, there was a severe impairment in epithelial tissue development as well as a dramatic reduction in p-PKB/AKT and CK10.Cervical carcinomas showed significant overexpression of PIK3CA compared to controls. During HPV-induced transformation The present data indicate that activation of the PI3-kinase/PKB/AKT pathway through PIK3CA regulates various transformed phenotypes as well as growth and differentiation of HPV-immortalized cells and may therefore play a pivotal role in HPV-induced carcinogenesis. It has been well established that high-risk human papillomavirus (hrHPV) infections are causally related to cervical cancer development . While tin vitro transformation of primary keratinocytes mediated by full-length hrHPV was accompanied with a gain of chromosome 3q in immortalized descendants -Thymidine incorporation was also reduced in rat intestinal epithelial cells as a result of LY294002 treatment [Functionally, we showed that PIK3CA and concomitant PI3-kinase signalling is involved in the different stages of HPV-mediated transformation st in G1 or inducst in G1 . Prolifereatment . This laFunctional studies in cervical cells are restricted to the common hrHPV positive cervical cancer cell lines such as SiHa, HeLa and CaSki and did not include the explicit analysis of PIK3CA as a candidate oncogene. Treatment of SiHa with the PI3-kinase inhibitor LY294002 led to reduced proliferation and increased apoptotic DNA fragments . For HeLin vitro model system, which mimics the different stages of cervical carcinogenesis. We were able to show a progressive upregulation of PI3-kinase signalling during HPV-induced transformation by using the elevation of p-PKB/AKT as a reporter. Indeed, p-PBK/AKT was elevated in anchorage independent cells relative to HPV-immortalized cells. The expression levels of both PIK3CA and especially p-PKB/AKT in the latter cells were increased compared to primary keratinocytes (data not shown). This increased signalling functionally correlated with multiple attributes of transformed cells, such as cell growth, migration, and anchorage independent growth . Upper panel: cells transfected with non-targeting siRNA pool, lower panel: cells transfected with siRNA pool against PIK3CA. Pictures were taken immediately after scratch induction and 24 hours post-induction.Click here for fileFigure S4. Organotypic raft cultures of primary human foreskin keratinocytes without and with PIK3CA silencing. H&E staining showing raft culture morphology following transfection of primary human foreskin keratinocytes (p3) with non-targeting siRNAs (left) and siRNAs targeting PIK3CA (right).Click here for file"} +{"text": "Protease-activated receptors (PARs) are G protein-coupled receptors activated through proteolytic cleavage. They are localized on epithelial, endothelial and inflammatory cells, as well as on Transient Receptor Potential Vanilloid 1 (TRPV1) receptor-expressing capsaicin-sensitive sensory nerves. Tachykinins, such as substance P (SP) and neurokinin A (NKA) encoded by the TAC1 gene are released from these fibres and play an important role in inflammatory and nociceptive processes. We investigated the involvement of capsaicin-sensitive peptidergic afferents, TRPV1 ion channels and TAC1-encoded tachykinins in mast cell tryptase (MCT)-induced joint swelling, hyperalgesia and synovial microcirculation.1) gene-deficient animals were also studied compared to their wild-type (WT) C57Bl/6 counterparts. Knee diameter was measured with a digital micrometer, mechanonociceptive threshold with dynamic plantar aesthesiometry and spontaneous weight distribution with incapacitance tester throughout a 6-hour period. Synovial bloodflow in urethane-anaesthetized animals was determined by laser Doppler imaging. In these studies, MCT was applied topically on the joints.The natural PAR2 activator MCT was injected into the right tibiotarsal joint of mice. Pretreatment with high doses of the TRPV1 receptor agonist resiniferatoxin (RTX) was used to selectively inactivate capsaicin-sensitive peptidergic sensory nerves. TRPV1, TAC1 and neurokinin 1 receptor from the same capsaicin-sensitive fibres. In contrast, these afferents and the TRPV1 receptors are essential in acute synovial vasodilatation, but tachykinis are not involved in this response.These data provide evidence that MCT-evoked acute oedema and hyperalgesia are mediated by tachykinins through NK"} +{"text": "Spleen tyrosine kinase (Syk) is a cytoplasmic protein expressed mainly in immune cells including macrophages and neutrophils and is associated with receptors containing an immunoreceptor tyrosine-based activation motif (ITAM), such as Fc\u03b3 receptors. As Syk-mediated signaling plays an important role in activation of immune responses, to investigate whether specific interruption of Syk-mediated signaling can affect the development of rheumatoid arthritis (RA), we used tamoxifen-induced conditional Syk-KO mice (iSyk KO) to evaluate the importance of Syk on disease development. Using a collagen antibody-induced arthritis model (CAIA), iSyk KO mice showed significantly attenuated disease severity compared to Syk non-deleted mice Figure . Althoug"} +{"text": "Genetic aberrancies within epidermal growth factor receptor (EGFR) pathway are associated with therapeutic outcomes of EGFR-tyrosine kinase inhibitors (TKIs) in advanced non-small cell lung cancer (NSCLC). However, the impact of chemotherapy on EGFR-related genes alterations has not been defined in NSCLC. Our study aims to investigate the impact of neoadjuvant chemotherapy (Neoadj-Chemo) on EGFR activating mutations and associated EGFR-TKIs resistance-related genes.Matched tumor samples were obtained retrospectively from 66 NSCLC patients (stages IIb\u2013IIIb) corresponding to pre- and post- Neoadj-Chemo. EGFR mutations were detected by denaturing high performance liquid chromatography (DHPLC) and confirmed by Amplification Refractory Mutation System technology (ARMS), KRAS mutations, T790M mutation and c-MET amplification were identified using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP), ARMS, and real-time PCR, respectively.Before Neoadj-Chemo, EGFR mutations were identified in 33.3% (22/66) of NSCLC patients. Only 18.2% (12/66) of patients carried EGFR mutations after Neoadj-Chemo (p\u200a=\u200a0.013). The median peak value of EGFR 19 exon mutations decreased non-significantly after Neoadj-Chemo. KRAS mutation rate decreased from 4.6% (3/66) to 3.0% (2/66) with Neoadj-Chemo. Although the overall percentage of patients exhibiting c-MET amplifications (6.1% [4/66]) did not change with Neoadj-Chemo, two patients transitioned from negative to positive c-MET amplification, and two patients reversed these changes post-Neoadj-Chemo. T790M mutations were absent from all samples.Neoadjuvant chemotherapy tends to decrease the mutation frequency of EGFR mutation and downstream genes, which suggests that real-time samples analysis for genetic aberrancies within EGFR pathways have important value to delineate specific patient populations and facilitate individualized treatment. Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) represent revolutionary personalized therapies for NSCLC patient, a subset of who carry specific EGFR mutations that are predictive of a favorable clinical response to EGFR-TKIs The outcome of EGFR-TKI therapy is determined not only by the presence of EGFR sensitizing mutations, but also by EGFR resistant or its bypass or downstream related genes aberrances. Specifically, EGFR T790M mutation, was identified mechanism of both acquired and primary EGFR-TKI resistance i.e., KRAS mutations, T790M, or c-MET amplification) are consistent in pre- and post-chemotherapy samples. Therefore, it is necessary to evaluate the impact of chemotherapy on tumor molecular profiles. Chin et al reported that prior exposure to platinum agents may reduce the benefit from subsequent treatment with EGFR-TKI for an erlotinib-sensitive EGFR-mutant NSCLC cell via the phosphatidylinositol 3-kinase/AKT survival pathway GFR mutation frequencies in patients with NSCLC, a likely result of a preferential response of sub-clones with EGFR mutations in tumors with heterogeneous tumor cell populations The detection of EGFR mutations currently is recommended for the selection of patients who could benefit from first-line EGFR-TKI therapy. However, it is unknown whether the status of EGFR mutation and downstream resistance-related genes aberrances performance status of 0-2 and had received 2-4 cycles of Neoadj-Chemo without any previous chemotherapy or biologic/immunologic treatment. All patients provided matched tissue samples from biopsies performed pre- Neoadj-Chemo and from resections post-Neoadj-Chemo.e.g., cisplatin or carboplatin in combination with gemcitabine or taxanes). The radiographic response to Neoadj-Chemo was determined using the RECIST guidelines Sixty-six patients who were screened from the our database established in 1999, including more than 1,900 patients with clinical data met the above criteria and were treated in Beijing Cancer Hospital from September 2001 to June 2010 . The available chemotherapy regimens involved platinum-based drugs . To avoid the influence of Neoadj-Chemo-induced necrosis of tissues on the EGFR and related genes aberrance detections, post-operative samples were macro-dissected from paraffin-embedded surgical tissue sections to ensure that only tumor tissues were obtained. Tumor contents were recorded for each sample using immediately adjacent tissue sections.EGFR mutation detection by denaturing high performance liquid chromatography (DHPLC) and Amplification Refractory Mutation System (ARMS)We analyzed all matched samples in the same condition in order to equalize the detection conditions. The EGFR exon 19 deletion or exon 21 substituted mutations were detected according to the method reported by us previously ARMS, a more sensitive method, was used to re-evaluate the cases with EGFR mutation discrepancies pre- and post- neoadjuvant chemotherapy A semi-quantitative analysis of exon 19 mutation abundance was performed by calculating the ratio between the peak heights of mutant (M) and wild-type/normal (W) products . This analysis was not extended to exon 21 because the M and W peaks overlapped in this exon.PCR-RFLP was performed to analyze KRAS mutation status according to standard protocols Paired tumor samples were analyzed for c-MET gene copy number using real-time PCR detection according to standard protocols. A tumor sample was defined as amplification-positive if its ratio value exceeded the following: mean (M)+2\u00d7standard deviation (SD) Amplification Refractory Mutation System (ARMS) technology was used to detect the T790M mutation. The detection method and discrimination criteria were according to manufactory .i.e., EGFR [including T790M] mutations, KRAS mutation, and c-MET amplification) before and after treatment. Cochran-Armitage trend test was used to test whether the change in mutation status was associated with clinical outcome in terms of partial response, stable disease. The associations of unpaired categorical variables were analyzed using the chi-square test, except that Fisher's exact test was used for small sample sizes (n<5 in any cell of the contingency table). Wilcoxon rank sum test was applied to compare the mutant abundance of EGFR 19 exon between pre- and post Neoadj-chem. Statistical significance was set at a level of 0.05. Two-sided tests were performed in all settings. All calculations were performed using SAS Version 10.0 .Frequency tabulation and summary statistics were provided to characterize the data distribution. McNemar's test was applied to compare the change of mutation status , 27 patients were classified as stable disease (SD), and none exhibited progressive disease (PD).The baseline characteristics of the study population are presented in Among 66 NSCLC patients, the pre-Neoadj-Chemo EGFR mutation rate was 33.3% (22/66), including 12 patients carrying mutations in exon 19, 9 patients with mutations in exon 21, and 1 patient with mutations in both exons. Post-Neoadj-Chemo, the EGFR mutation rate decreased to 18.2% (12/66), including 7 patients with mutations in 19 exon and 5 with mutations in exon 21 in adenocarcinoma, which is higher than measured in patients with squamous cell carcinoma (17.1% [6/35]). However, the EGFR mutation rates in the patients who underwent Neoadj-Chemo dropped to 33.3% (9/27) in adenocarcinoma and 8.57% (3/35) in squamous cell carcinoma, with a non-significant difference (P>0.05). The four patients with adenosquamous carcinoma composed too small of a group for statistical analysis .To assess variations in mutation quantity, mutations in EGFR exon 19 were semi-quantitatively detected from 13 patients carrying mutations in this exon . The ratAll patients were evaluated for their response after Neoadj-Chemo. 39 patients (59.1%) achieved PR, 27 patients (40.9%) reached SD, and none exhibited PD . Among tThe KRAS mutation rate varied from 4.5% (3/66) pre-Neoadj-Chemo to 3.0% (2/66) after treatment . The conThe overall c-MET amplification ratio did not change with Neoadj-Chemo treatment (6.0% [4/66]) . HoweverT790M mutations were absent from all samples, as determined using ARMS technology.Previous studies have not evaluated whether the molecular profiles of tumor tissues change between the initial diagnosis and post-chemotherapy. Based on our prophase study results EGFR mutations are well-established predictors of the outcome to first-line EGFR-TKI therapy In our study, the overall EGFR mutation rate significantly decreased in post-Neoadj-Chemo tissue samples compared with pre-Neoadj-Chemo ones in patients with early stage NSCLC. We detected a discordant rate of 18.2% (12/66), which included patients who transitioned from EGFR mutation-positive to -negative (n\u200a=\u200a10) and the reverse (n\u200a=\u200a2). No significant differences have been identified regarding the incidence of EGFR mutations with early-to-advanced staging of NSCLC The mechanisms contributing to chemotherapy-related shifts in EGFR mutation status remain unclear. Heterogeneous intra-tumoral EGFR mutations and different sensitivities of EGFR mutant and wild-type tumor cells to chemotherapy may be associated with alterations in overall EGFR mutation status following chemotherapy Our previous study detected variations in EGFR mutation status through microdissection of different foci from 85 advanced NSCLC patients following palliative surgery resection. The results showed that approximately 30% of specimens contained both EGFR mutant and wild-type cells with the proportion of EGFR mutant cells ranging from 1% to 90% The discordance of EGFR mutation status between biopsy and surgically resected samples pre- and post-Neoadj-Chemo, respectively, also may be attributed to sampling differences resulting from intra-tumoral heterogeneity. Specifically, small biopsy samples with limited materials might not represent the complete biological features of the tumor, and miniscule proportions of mutant cells may be overlooked. However, the current study observed a higher incidence of EGFR-related gene aberrances in biopsy samples of pre-Neoadj-Chemo compared with surgery resected samples of post-Neoadj-Chemo. This suggests that the discordance of EGFR mutation status observed in the samples of pre- and post-Neoadj-Chemo didn't derive from sampling bias.We demonstrated that Neoadj-Chemo treatment of NSCLC patients impacted not only EGFR mutation status but also aberrancies in related downstream genes, including KRAS and c-MET. The KRAS mutation rate was 4.6% (3/66) pre-Neoadj-Chemo and decreased to 3.0% (2/66) following therapy. Although the c-MET amplification ratio pre- and post-Neoadj-Chemo did not change (6.1% [4/66]), 4 patients exhibited shifts. Specifically, two cases transitioned from amplification-negative to -positive with Neoadj-Chemo, and two patients exhibited the reverse change. Our results suggest that c-MET and KRAS gene aberrancies exist in untreated tissue samples at low frequencies; this is consistent with studies by Turke et al We cannot fully explain the potential mechanisms contributing to alterations in these resistance-related genes during the course of chemotherapy. Several studies have recently reported similar alterations in EGFR resistant genes We found that EGFR T790M mutation was absent in both pre- and post-Neoadj-Chemo samples. It's inconsistent with the findings of Maheswaran et al Limitations were that: this study is a small samples and retrospective study and the patients with squamous cell carcinoma (SQC) in this study accounted for 53%. Former study showed the mutation rate of SQC in Caucasians is no more than 3.6% We evaluated variations in EGFR mutations (including T790M), KRAS mutations, and c-MET amplification both pre- and post- Neoadj-Chemo treatment. Our findings supported that chemotherapy could affect molecular biomarker profiles and indirectly indicate the presence of tumor heterogeneity. Therefore, the effects of treatments on tumor profiling should be evaluated before making decisions regarding second- or third-line EGFR-TKI therapies for NSCLC patients. The preparation of real-time molecular biomarker profiles for EGFR-TKIs is suggested to delineate specific patient populations and facilitate individualized treatment."} +{"text": "Ericaulon buergerianum (Gujingcao) is an ophthalmic, anti-inflammatory and antimicrobial Chinese medicinal herb. This study aims to investigate the neuroprotective effects of Ericaulon buergerianum ethanol extract (EBE) and to elucidate its underlying action mechanism.The viability of dopaminergic (DA) neuron in zebrafish was examined by anti-tyrosine hydroxylase (TH) immunostaining. The locomotor activity of zebrafish was assessed with a digital video tracking system. The viability and cellular damage of the PC12 cells were determined by MTT and LDH assays respectively. The nuclear morphological changes in apoptotic cells were evaluated with DNA staining by Hoechst 33342 dye. Intracellular nitric oxide (NO) was quantified by DAF-FM diacetate staining. The expression of inducible nitric oxide synthase (iNOS) was determined by Western blot.in vitro) activities of EBE are related to its neuroprotective effects in 6-OHDA-induced DA neuron damage.EBE inhibited the 6-OHDA-induced decrease in total distance of movement in zebrafish. Pretreatments of EBE increased the viability of 6-OHDA-damaged PC12 cells in a dose dependent manner. Protection against 6-OHDA-induced nuclear fragmentation and accumulation of apoptotic bodies was also observed in EBE pretreated cells. Anti-oxidative (inhibition of NO production and iNOS expression in PC12 cells in vivo, inhibition of 6-OHDA-induced decrease of total distance in movement in zebrafish. The iNOS-NO pathway may be involved.EBE exhibited significant neuroprotective activities in zebrafish, including recovery of dopaminergic neuron loss caused by 6-OHDA in a dose-dependent manner The toxic effects of 6-OHDA are mainly attributed to the formation of free radicals, inflammatory processes and apoptosis -2, 5-diphenyl tetrazolium bromide; FBS: Fetal bovine serum; DMSO: dimethyl sulfoxide; PBS: phosphate-buffered saline; 6-OHDA: 6-hydroxydopamine; NO: Nitric oxide; L-NAME: L-nitroarginine methylester; SNP: sodium nitroprusside dehydrate; CNS: central nervous system; PD: Parkinson's disease; SNpc: substantia nigra pars compacta; dpf: day post fertilization.EBE: The authors declare that they have no competing interests.MWW and ZJZ performed the experiments. MWW wrote the manuscript. ZXL reviewed the literature and study design. LCVC revised the manuscript. SMYL supervised the study. All authors read and approved the final version of the manuscript."} +{"text": "In this review, the coherence of purine nucleoside-related pathways and MAPK activation in the endogenous neuroprotective regulation of the nervous system's development and neuroplasticity under hypoxic stress will be discussed.Even a short blockade of oxygen flow in brain may lead to the inhibition of oxidative phosphorylation and depletion of cellular ATP, which results in profound deficiencies in cellular function. Following ischemia, dying, injured, and hypoxic cells release soluble purine-nucleotide and -nucleoside pools. Growing evidence suggests that purine nucleosides might act as trophic factors in the CNS and PNS. In addition to equilibrative nucleoside transporters (ENTs) regulating purine nucleoside concentrations intra- and extracellularly, specific extracellular receptor subtypes for these compounds are expressed on neurons, glia, and endothelial cells, mediating stunningly diverse effects. Such effects range from induction of cell differentiation, apoptosis, mitogenesis, and morphogenetic changes, to stimulation of synthesis and/or release of cytokines and neurotrophic factors under both physiological and pathological conditions. Multiple signaling pathways regulate the critical balance between cell death and survival in hypoxia\u2013ischemia. A convergent pathway for the regulation of multiple modalities involved in O In acute neurological conditions such as stroke severe injuries to the CNS occur and stroFollowing hypoxia\u2013ischemia, dying, injured, and hypoxic cells release soluble purine- nucleotide and -nucleoside pools , normallS-adenosyl homocysteine and guanosine 5\u2032-monophosphate (GMP) (Gs) or inhibit (Gi) adenylate cyclase, the enzyme that catalyzes the formation of cAMP, whereby A1R and A3R interact with Gi/ Go proteins, and A2A and A2B with Gs (2+- and mitogen-activated protein kinases (MAPKs), are also relevant (Adenosine receptors (AR) belong to the superfamily of G-protein-coupled receptors characterized by seven transmembrane helices . There aGs, except in striatum, where A2AR interacts with Golf, whereby both result in coupling to its canonical protein kinase A (PKA)-activating pathway -PKA pathway, the MAPK pathway, the stress-activated protein kinase pathway, and the phosphatidylinositol 3-kinase-Akt pathway cells cells , which aensitive . Numerouensitive . Results2 receptor modulation of both voltage-sensitive potassium IK(V) and calcium ICa currents in PC12 cells (S-(4-nitrobenzyl)-6-thioinosine caused an increase in adenosine-mediated rescue of viability pathway, serine\u2013threonine kinases constitute a convergent pathway for the regulation of multiple modalities involved in O sensing and regu sensing . Emergins module . Along ts module , that MAs module and studs module . In cells module .MAPK was shown to positively modulate the hypoxia-inducible factor-1 alpha (HIF-1\u03b1) by phosphorylation control . Direct + channels and the activation of the phosphatidylinositol 3-kinase/Akt pathway (Next to the clear-cut effects of adenosine receptor-mediated activation of MAPK-HIF-1\u03b1, neuroprotection of hypoxic neuronal cells apparently does involve other pathways that deserve future attention. Amongst purine nucleosides guanosine attracted attention for its strong neurite-stimulating capacity ( pathway , made a pathway of poten pathway . Adenosi pathway .in vivo studies demonstrated inosine's ability to stimulate neurons to extend new projections to denervated areas in adult rats with unilateral cortical infarcts (In vitro studies confirmed the amazing neuroprotective capability of purine nucleosides in several neuronal hypoxia systems e.g PC12 cells and cerebellar granule neurons (2 sensing (Hypoxic\u2013ischemic brain injury starts with the insult but extends into a recovery\u2013reperfusion period . In para sensing . These r sensing . Amongst sensing . LikewisMany stroke patients fail clinical time windows for acute effective treatment, hence making approaches that promote repair and recovery essential for integrated stroke therapy . Thus, a"} +{"text": "An HIV-1 vaccine remains elusive, in part because various factors limit the quantity and quality of the antibodies raised against the viral envelope glycoprotein complex (Env). We hypothesized that targeting Env vaccines directly to B cells, by fusing them to molecules that bind and activate these cells, would improve Env-specific antibody responses.We fused trimeric Env gp140 to A PRoliferation-Inducing Ligand (APRIL), B-cell Activating Factor (BAFF), and CD40 Ligand (CD40L).The Env-APRIL, Env-BAFF and Env-CD40L gp140 trimers all enhanced the expression of activation-induced cytidine deaminase (AID) expression, the enzyme responsible for inducing somatic hypermutation, antibody affinity maturation and antibody class-switching. They also triggered IgM, IgG and IgA secretion from human B cells in vitro. The Env-APRIL trimers induced higher anti-Env antibody responses in rabbits, including neutralizing antibodies against Tier 1 viruses. The enhanced Env-specific responses were not associated with a general increase in total plasma antibody concentrations, indicating that the effect of APRIL was Env-specific. All the rabbit sera raised against gp140 trimers, irrespective of the presence of CD40L, BAFF or APRIL, recognized trimeric Env efficiently, while sera raised against gp120 monomers did not. The levels of trimer-binding and virus-neutralizing antibodies were strongly correlated, suggesting that gp140 trimers are superior immunogens to gp120 monomers.Targeting and activating B cells with a trimeric HIV-1 Env-APRIL fusion protein may improve the induction of humoral immunity against HIV-1. Targeting B cells directly may also be useful for other vaccines."} +{"text": "Major genetics factors for age-related macular degeneration (AMD) have recently identified as susceptibility risk factors, underlying the role of the vascular endothelial growth factor (VEGF) system in AMD -4.Our purpose was to determine whether (VEGF) gene polymorphisms play a role in either susceptibility risk for age-related macular degeneration (AMD) serum VEGF levels (s-VEGF) variations and treatment with intravitreal bevacizumab in Tunisians.The case-control study included 157 patients with AMD and 207 age-matched controls. In all patients, ophthalmological examinations, visual acuity, optical coherence tomography (OCT), fundus photography and fluorescein angiography were performed. Sixty-two patients were treated with intravitreal bevacizumab. Single nucleotide polymorphism (SNP) genotyping were performed using direct sequencing. The serum VEGF was assayed by ELISA (R&D).p=0.018 and p<10-3, respectively). Haplotype analysis of SNP+936, +405 and -2578 revealed that TGA was associated to exudative form of disease (p<0.0001). However, single allele, genotype and haplotype association analyses showed no significant association with s-VEGF levels variations, clinical forms of AMD or better outcome for distance and reading visual acuity after three bevacizumab injections.The single SNP +936 TT and +405 CC genotypes were significantly higher in AMD patients than in controls (Our results show that VEGF variants do contribute to the susceptibility to AMD in Tunisian patients. Further expression studies are needed to investigate the potential pharmacologic role of these variants in antiangiogenesis therapy."} +{"text": "In liver, glucose utilization and lipid synthesis are inextricably intertwined. When glucose availability exceeds its utilization, lipogenesis increases, leading to increased intrahepatic lipid content and lipoprotein secretion. Although the fate of three-carbon metabolites is largely determined by flux rate through the relevant enzymes, insulin plays a permissive role in this process. But the mechanism integrating insulin receptor signaling to glucose utilization with lipogenesis is unknown. Forkhead box O1 (FoxO1), a downstream effector of insulin signaling, plays a central role in hepatic glucose metabolism through the regulation of hepatic glucose production. In this study, we investigated the mechanism by which FoxO1 integrates hepatic glucose utilization with lipid synthesis. We show that FoxO1 overexpression in hepatocytes reduces activity of carbohydrate response element binding protein (Chrebp), a key regulator of lipogenesis, by suppressing O-linked glycosylation and reducing the protein stability. FoxO1 inhibits high glucose- or O-GlcNAc transferase (OGT)-induced liver-pyruvate kinase (L-PK) promoter activity by decreasing Chrebp recruitment to the L-PK promoter. Conversely, FoxO1 ablation in liver leads to the enhanced O-glycosylation and increased protein level of Chrebp owing to decreased its ubiquitination. We propose that FoxO1 regulation of Chrebp O-glycosylation is a mechanism linking hepatic glucose utilization with lipid synthesis. The liver plays a central role in integrating glucose and lipid metabolism, effectively exchanging carbons from one energy source to the other for storage and utilization Lpk), one of the rate-limiting enzymes of glycolysis Acaca) and fatty acid synthase (Fasn) Key transcriptional mediators of insulin signaling and glucose signaling are FoxO1 and Chrebp. FoxO1 is an Akt substrate and regulates glucose production and bile acid synthesis Impetus for the present studies came from prior observations that genetic ablation of FoxO1 in liver increases systemic insulin sensitivity, and results in lower hepatic glucose production, increased glycogen storage, and increased lipogenesis We purchased antibodies against Chrebp from Novus Biologicals, O-GlcNAc from Covance, OGT (DM-17) from Sigma, FoxO1 (9462) from Cell Signaling, FoxO1 (H-128), Ubiquitin (P4D1), Tubulin (B\u20137) from Santa-Cruz, HA (12CA5) from Roche. We used these antibodies for immunoprecipitation or immunoblotting according to manufacturer's protocol.We have previously described expression vectors encoding Flag-tagged FoxO1-ADA and His-HA-Ubiquitin 5\u2032-ACGGAGGATTGAACCAGTATA-3\u2032. The OGT specific siRNA sequence is 5\u2032-CGACATGCCTTGCGGCTGA -3\u2032. siRNA was transfected using DharmaFECT Duo (Dharmacon). In some experiments, we infected primary hepatocytes with adenovirus at MOI of 10 or 30, 5 hrs before treatments with high glucose. All experiments were repeated at least three times.We purchased primary culture of mouse hepatocyte from Primary Cell Co., Ltd and cultured the cells in DMEM supplemented with 10% FCS. The FoxO1-specific siRNA sequence is 5\u2032-GATTTGAGCCTTTGATCCAGGCTC-3\u2032 and 5\u2032-AAGTTCCCTCCATCTATACAGTGC-3\u2032 according to the previously described methods We performed luciferase assays as previously described 2), immunoprecipitated and analyzed by immunoblotting.We lysed cultured cells in RIPA buffer containing protease inhibitors (Roche). After centrifugation, cell extracts were diluted with Co-IP buffer . We performed real-time RT-PCR using ImProm-II\u2122 Reverse Transcription System (Promega) and LightCycler System (Roche). Primer sequences used for real-time PCR are as follows, for Chrebp; 5\u2032-We performed metabolic labeling of Chrebp with tetraacetylated azide-modified N-acetylglucosamine (GlcNAz) in mouse primary hepatocytes. After immunoprecipitation with anti-Chrebp antibody, we detected O-glycosylation modification using biothin-avidin system.floxFoxo1 alleles were detected using PCR with primers 5\u2032-GCT TAG AGC AGA GAT GTT CTC ACA TT-3\u2032, 5\u2032-CCA GAG TCT TTG TAT CAG GCA AAT AA-3\u2032 and 5\u2032-CAA GTC CAT TAA TTC AGC ACA TTG A-3\u2032. Individually caged mice were housed in a temperature-controlled facility. All animal care and experimental procedures were approved by the Institutional Animal Care and Experimentation Committee at Gunma University. H-E staining was performed using 4- \u00b5m-thick paraffin sections following the standard methods. Hepatic triglyceride (TG) contents were measured as described previously We generated liver specific FoxO1 knockout mice using FoxO1 flox/flox mice Lpk, a target of Chrebp, was significantly decreased, despite unchanged levels of Chrebp mRNA ebp mRNA . Therefoebp mRNA . ConversLpk promoter by crossing Albumin-Cre mice with FoxO1 flox mice controls , consistWe next investigated the physiological consequences of FoxO1 ablation in the liver. Because we showed FoxO1 ablation in the liver enhanced Chrebp protein stability and Chrebp recruitment to its target gene promoter, we predicted that hepatic lipid contents should be increased in L-FoxO1-KO mice. However, histological analysis using the liver sections showed no morphological difference between L-FoxO1-KO and control mice . Further2-terminal domain and nuclear translocation Our studies identify a direct molecular link between insulin signaling pathways regulating hepatic glucose production and those regulating glycolysis and lipogenesis. In addition to Chrebp, another critical transcription factor for lipogenesis is sterol regulatory element-binding protein 1c (Srebp-1c) Glucose taken into hepatocytes is mainly converted to pyruvate or glycogen to produce or store energy. However, excess glucose enters into hexosamine biosynthetic pathway (HBP), leading to the production of UDP-N-acetylglucosamine (UDP-GlcNAc). By using UDP-GlcNAc as the donor substrate, O-GlcNAc transferase (OGT) catalyzes O-glycosylation modification of proteins on Ser/Thr residues. Although only \u223c2\u20133% of intracellular glucose enters the HBP Fasn and Lpk, two critical Chrebp targets, without affecting Chrebp mRNA levels FoxO1 is a member of the forkhead box containing protein of the O subfamily, which regulates metabolism as well as cellular proliferation, apoptosis, differentiation and stress resistance in vivo.In this study, we showed FoxO1 ablation in the liver enhanced Chrebp protein stability and Chrebp recruitment to its target gene promoter. Therefore we predicted that hepatic lipid synthesis should be increased in L-FoxO1-KO mice. However, hepatic lipid contents were unchanged in these mice . One posvs. ubiquitination (Lys), it remains unclear how increased O-glycosylation is associated with decreased ubiquitination of Chrebp. However, one possible mechanism is that O-glycosylation may change protein structure, affecting the susceptibility of ubiquitination and subsequent protein degradation Considering that different amino acid residues are targeted by O-glycosylation (Ser/Thr)"} +{"text": "The wound-healing process induced by chronic hepatitis C virus (HCV) infection triggers liver damage characterized by fibrosis development and finally cirrhosis. Liver Transplantation (LT) is the optimal surgical treatment for HCV-cirrhotic patients at end-stage liver disease. However, acute cellular rejection (ACR) and HCV recurrence disease represent two devastating complications post-LT. The accurate differential diagnosis between both conditions is critical for treatment choice, and similar histological features represent a challenge for pathologists. Moreover, the HCV recurrence disease severity is highly variable post-LT. HCV recurrence disease progression is characterized by an accelerated fibrogenesis process, and almost 30% of those patients develop cirrhosis at 5-years of follow-up. Whole-genome gene expression (WGE) analyses through well-defined oligonucleotide microarray platforms represent a powerful tool for the molecular characterization of biological process. In the present manuscript, the utility of microarray technology is applied for the ACR and HCV-recurrence biological characterization in post-LT liver biopsy samples. Moreover, WGE analysis was performed to identify predictive biomarkers of HCV recurrence severity in formalin-fixed paraffin-embedded liver biopsies prospectively collected. Hepatic fibrogenesis is considered a model of the wound-healing response to chronic liver injury. This process is characterized by extra-cellular matrix (ECM) proteins accumulation as consequence of an imbalance between the deposition and degradation of ECM components -4. DiffeHepatotropic viruses, especially hepatitis C (HCV) virus, constitute the principal chronic liver injury etiology . After 1Unfortunately, HCV recurrence is universal. HCV RNA serum load dramatically decreases until almost undetectable level within 24-48 hours post-LT. However, it increments few days post-LT with a peak at 1-3 months, and reaching a 1-2 logs plateau higher than the pre-LT viral load after the first year post-LT . Of thosThe discovery of reliable biomarkers for differential diagnosis of both complications, and also for HCV recurrence disease surveillance constitutes the research endeavor. The implementation of whole-genome gene expression (WGE) studies using microarray technology represents an outstanding opportunity for biomarkers discovery. The present article is focused on performed WGE analysis aimed to differentiate ACR in the setting of HCV recurrence disease, and to predict liver fibrosis progression in HCV-infected recipients.gold standard\" for proper differentiation between both post-LT conditions. However, subtle similarities in the histopathology and clinical course turn difficult and uncertain the pathological differentiation, even among experiences hepatologists [The differential diagnosis of ACR in the setting of HCV recurrence disease remains an important cause of morbidity and late graft failure in liver-transplant recipients. The assessment of liver allograft biopsy is still considered to be the \"ologists ,19. HCV ologists . ACR occologists ,21.The implementation of large-scale genomic analyses strategies has provided new insights into various disease processes and had assisted on elucidating genomic patterns for mechanism, diagnosis, prognosis, and treatment selection of complex and multi-factorial diseases . For ins\u00ae . The study was approved by the Institutional Review Board at Virginia Commonwealth University, and informed consents were obtained from all patients. The patients cohort included liver biopsy samples from 24 HCV-recipients with histological diagnosis of HCV recurrence disease (n = 13) and ACR in the setting of HCV . Liver tissue from chronic HCV-infected patients without LT were included for comparison analysis proposes. Tissue collection, total RNA isolation and quality control criteria, cDNA synthesis, in vitro transcription for labeled cRNA probe, and microarray hybridization and analysis were performed as described previously [http://www.ingenuity.com. Those differentially expressed genes in ACR biopsy samples were associated with pathways involved in immune and inflammatory responses, apoptosis, complement system, and growth factor receptors. HCV recurrence samples revealed predominantly gene expression patterns associated with cell cycle and cell division, cell proliferation and blood coagulation. The supervised hierarchical clustering analysis including specific probesets differentially expressed for HCV-ACR vs. HCV recurrence clustered all samples in two well-differentiated groups characterizing both different post-LT conditions , were evaluated using microarray techniques. From the analysis, 179 probesets were found significantly differentially expressed among HCV-ACR, no-HCV-ACR, and HCV recurrence biopsy samples. A total of 71 exclusive genes were identified for HCV-ACR vs. HCV recurrence pairwise comparison. Interestingly, no significant probesets were found differentially expressed between ACR samples from HCV and no-HCV infected recipients, which suggest a particular ACR molecular nature independent of the HCV infection. Gene ontology analysis identified canonical pathways specifically associated to a cytotoxic T cell profile in for HCV recurrence samples, and inflammatory response related genes as the ACR profile. The supervised clustering analysis including probesets (n = 80) representing those 71 specific genes displayed two well-differentiated groups for ACR and HCV recurrence samples. Interestingly, no-HCV-ACR samples were clustered within the HCV-ACR group .\u00ae microarrays clearly demonstrate a differential gene expression signature between both conditions. Indeed, HCV recurrence disease is characterized by an adhesion and apoptosis of cytotoxic T cells profile regulated by canonical pathways related to IFN-\u03b3 and NF\u03baB, while ACR is related to genes associated with an immediate hypersensitivity reaction [+ and CD8+ T cells together with macrophages and eosinophil cells for ACR [The concluding results from the analysis of both experimental approaches using GeneChipreaction . The his for ACR ,24,25. T for ACR ,23. MoreChronic HCV-related hepatic insufficiency is associated with decreased rates of patient and allograft survival in comparison with other indications for liver transplantation . Differe\u00ae Assay following manual instructions . HumanRef-8 Expression BeadChips hybridization and followed analyses were described in Mas et al 2011 [p \u2264 0.001) and differentially expressed after a moderate F test statistical assay. By linear contrast examination, fourteen beads were found between G1 vs. G2, five beads between G2 vs. G3, and fifty beads between G1 vs. G3. Agglomerative hierarchical clustering analysis was performed by incorporating only the 50 beads differentially expressed between G1 vs. G3. The analysis displayed two independent groups composed by G1 or G3 samples. Interestingly, G2 samples were found to be randomly associated within both clusters may be reflecting a pathology miss-classification component liver biopsies at the time of HCV recurrence diagnosis ,28. HCV t Figure . Gene onYet, the mechanisms involved in HCV recurrence development and progression are largely unknown. WGE analyses demonstrated to be a useful tool to either reveal the HCV recurrence molecular biology, and to encourage the identification of potential biomarkers to predict disease severity. A set of no invasive biomarkers have been proposed with promising results for the hepatic fibrosis progression assessment using specific peripheral blood serum proteins as biomarkers -32. Up tThe accurate follow-up and the differential diagnosis of post-transplant complications have been further struggled by no reliable and difficult pathology reports, essentially in post-transplanted HCV-infected patients . HoweverACR: Acute cellular rejection; ALT: Alanine aminotransferase; cDNA: copy Deoxyribonucleotide acid; CLIP4: CAP-GLY domain containing linker protein 4; cRNA: copy Ribonucleotide acid; DASL: cDNA-mediated Annealing, Selection, Extension, and Ligation; ECM: Extra-cellular matrix; FFPE: Formalin-fixed paraffin embedded; HCC: Hepatocellular carcinoma; HCV: Hepatitis C virus; IFN-\u03b3: Interferon-gamma; IL-28: Interleukin-28; IL-28RA: Interleukin-28 receptor, alpha ; LASSO: Least absolute shrinkage and selection operator; LT: Liver transplant; MELD: Model of end-stage liver disease; NF\u03baB: Nuclear factor kappa-B; RNA: Ribonucleotide acid; ROC: Reactive oxygen species; STS1: Suppressor of T cell receptor signaling 1; WGE: Whole-genome gene expression.The authors declare that they have no completing interests, or other interests that might be perceived to influence the content included into this article."} +{"text": "Human hepatitis B virus (HBV) causes chronic hepatitis and is associated with the development of hepatocellular carcinoma. HBV infection alters mitochondrial metabolism. The selective removal of damaged mitochondria is essential for the maintenance of mitochondrial and cellular homeostasis. Here, we report that HBV shifts the balance of mitochondrial dynamics toward fission and mitophagy to attenuate the virus-induced apoptosis. HBV induced perinuclear clustering of mitochondria and triggered mitochondrial translocation of the dynamin-related protein (Drp1) by stimulating its phosphorylation at Ser616, leading to mitochondrial fission. HBV also stimulated the gene expression of Parkin, PINK1, and LC3B and induced Parkin recruitment to the mitochondria. Upon translocation to mitochondria, Parkin, an E3 ubiquitin ligase, underwent self-ubiquitination and facilitated the ubiquitination and degradation of its substrate Mitofusin 2 (Mfn2), a mediator of mitochondrial fusion. In addition to conventional immunofluorescence, a sensitive dual fluorescence reporter expressing mito-mRFP-EGFP fused in-frame to a mitochondrial targeting sequence was employed to observe the completion of the mitophagic process by delivery of the engulfed mitochondria to lysosomes for degradation. Furthermore, we demonstrate that viral HBx protein plays a central role in promoting aberrant mitochondrial dynamics either when expressed alone or in the context of viral genome. Perturbing mitophagy by silencing Parkin led to enhanced apoptotic signaling, suggesting that HBV-induced mitochondrial fission and mitophagy promote cell survival and possibly viral persistence. Altered mitochondrial dynamics associated with HBV infection may contribute to mitochondrial injury and liver disease pathogenesis. Hepatitis B virus (HBV) chronic infections represent the common cause for the development of hepatocellular carcinoma. Mitochondrial liver injury has been long recognized as one of the consequences of HBV infection during chronic hepatitis. Mitochondria are dynamic organelles that undergo fission, fusion, and selective-autophagic removal (mitophagy), in their pursuit to maintain mitochondrial homeostasis and meet cellular energy requirements. The clearance of damaged mitochondria is essential for the maintenance of mitochondrial and cellular homeostasis. We observed that HBV and its encoded HBx protein promoted mitochondrial fragmentation (fission) and mitophagy. HBV/HBx induced the expression and Ser616 phosphorylation of dynamin-related protein 1 (Drp1) and its subsequent translocation to the mitochondria, resulting in enhanced mitochondrial fragmentation. HBV also promoted the mitochondrial translocation of Parkin, a cytosolic E3 ubiquitin ligase, and subsequent mitophagy. Perturbation of mitophagy in HBV-infected cells resulted in enhanced mitochondrial apoptotic signaling. This shift of the mitochondrial dynamics towards enhanced fission and mitophagy is essential for the clearance of damaged mitochondria and serves to prevent apoptotic cell death of HBV-infected cells to facilitate persistent infection. Hepadnavirus family. HBV DNA genome encodes four overlapping open reading frames designated as pre-S/S , C , P , and X (HBx) Hepatitis B virus (HBV) infection affects nearly 350 million people worldwide and leads to chronic liver disease, liver failure, and hepatocellular carcinoma (HCC) 2+ signaling, mitochondrial depolarization and dysfunction and reactive oxygen species (ROS) generation Mitochondrial injury and oxidative stress are prominent features of chronic Hepatitis B and C in vivo conditions.HBV and in particular, HBx have been shown to induce bulk autophagy We investigated the HBV-induced morphological changes of mitochondria in the human hepatoma Huh7 cells transiently expressing wild-type 1.3mer HBV genome (hereafter referred to as HBV). As shown in When mitochondria are depolarized, PTEN-induced putative kinase 1 (PINK1), a mitochondrial Ser/Thr kinase, accumulates on the outer mitochondrial membrane and recruits Parkin to the mitochondria We then investigated whether HBV stimulates the expression of mitophagy-related genes. As shown in Next, we analyzed the formation of mitophagosome in Huh7 cells co-expressing HBV and GFP-LC3 protein by confocal microscopy, as we demonstrated the HBV-induced mitochondrial fission and stimulation of Parkin and LC3B expression in Huh7 cells and 2G. The final step of mitophagy is the fusion of mitophagosomes with lysosomes where the cargo is delivered, degraded and recycled As damaged mitochondria are eliminated via mitophagy, we observed a decline in mitochondrial number in HBV-expressing cells, but not in those treated with BafA1 or untransfected cells . Parkin Mitochondrial dynamics is integrally linked to apoptosis Mitochondria are dynamic organelles that undergo fission (fragmented mitochondria), fusion , and trafficking Many DNA and RNA viruses have been shown to tightly modulate the autophagy process to prevent their clearance by autophagy, to inhibit host immune response, or to favor viral replication and maturation events in vivo studies, it is inferred that HBV-infected hepatocytes maintain a persistent phenotype and that HBV replicates within infected hepatocytes noncytopathically in vivo conditions. Currently, the most convenient way to test our hypothesis in vivo settings is the use of animal models of HBV infection such as Woodchucks, Ducks, or Tupaia to determine if abrogation of Parkin-mediated mitophagy pathway promotes specific death of HBV-infected hepatocytes and alleviates persistent HBV infection in these animals.Although the role of HBV and HBx in regulating apoptotic signaling has long been debated It is believed that HBx sensitizes and indirectly participates in the onset of liver oncogenesis In summary, this study provides a unique insight into the probable involvement of HBV-induced altered mitochondrial dynamics and mitophagy in possibly facilitating the persistence of infection and pathogenesis of liver disease associated with infection thus unraveling potential newer avenues for design of novel therapeutics against chronic HBV infection.Human hepatoma cell line Huh7 was grown in high-glucose DMEM (Gibco) supplemented with 10% fetal bovine serum (Hyclone), 1% MEM non-essential amino acid (Gibco), 100 units/ml penicillin (Gibco), and 100 \u00b5g/ml streptomycin (Gibco). Human hepatoma HepG2 and HepAD38 cells harboring HBV full-length genome were maintained in RPMI 1640 (Gibco) supplemented with 20% fetal bovine serum, 1% MEM non-essential amino acid, 100 units/ml penicillin, and 100 \u00b5g/ml streptomycin. In addition, HepAD38 cells were grown in the presence of 0.5 mg/ml G418 (Invitrogen) and 1 \u00b5g/ml tetracycline. NT-KD (expressing non-target shRNA) and P-KD (expressing Parkin shRNA) cells used in this study were maintained in the presence of 2.5 \u00b5g/ml of puromycin, as described previously and knockdown level of Parkin gene was shown in the previous study Bgl2 and BamHI to remove LC3 coding sequences, resulting in plasmid p-mRFP-EGFP production and then, polymerase chain reaction product for mitochondrial targeting signal sequences of human cytochrome c oxidase subunit VIII amplified from pEYFP-mito (Clontech) was inserted N-terminally in frame into p-mRFP-EGFP.The pHBV1.3mer and pHBV-\u0394X plasmid DNAs encoding wild-type HBV genome and HBx-deficient HBV genome, respectively, were a kind gift from Dr. Jing-hsiung James Ou . The pHBx-flag plasmid DNA was described previously To conduct laser scanning confocal microscopy, the cells grown on coverslips were transfected with the indicated plasmid DNAs followed by immunofluorescence assay, as described previously Chemical reagents used in this study were Bafilomycin A1 (Enzo Life Sciences) and 3-Methyladenine (Sigma). Primary antibodies used in this study include the following: rabbit monoclonal anti-Drp1 ; rabbit monoclonal anti-phospho-Drp1 (S616) ; rabbit monoclonal anti-ATF4 ; rabbit polyclonal anti-Parkin (Abcam); rabbit monoclonal anti-LC3B ; rabbit polyclonal anti-PINK1 (Abcam); rabbit polyclonal anti-VDAC1 ; mouse monoclonal anti-Mfn2 (Abcam); goat polyclonal anti-VDAC3 (Santa Cruz); rabbit polyclonal anti-GAPDH (Santa Cruz); goat polyclonal anti-\u03b2-actin (Santa Cruz); mouse monoclonal anti-TOM20 (BD); rabbit polyclonal anti-TOM20 (Abcam); mouse monoclonal anti-Flag M2 (Sigma); rabbit polyclonal anti-DTKDDDDK-tag (GenScript); goat polyclonal anti-DDDDK (Abcam); mouse monoclonal anti-HBsAg (Thermo Scientific); mouse monoclonal anti-Ubiquitin ; rabbit monoclonal anti-cleaved PARP ; rabbit monoclonal anti-cleaved caspase-3 ; rabbit polyclonal anti-cytochrome c ; normal rabbit IgG ; normal mouse IgG (Santa Cruz). The secondary antibodies used for immunofluorescence were Alexa Fluor 350, 488, 594, or 647 donkey anti-mouse, rabbit, or goat IgG (Molecular Probe). The secondary antibodies used for Western blot analysis were HRP-conjugated anti-mouse IgG , HRP-conjugated anti-rabbit IgG , and HRP-conjugated anti-goat IgG (Jackson Laboratories).Small interfering RNA (siRNA) pools used in this study were siGENOME SMARTpool for Parkin (NM_004562) and non-targeting #1 control (NT) (Dharmacon). The cells were transfected with siRNA (50 nM) for the indicated times using DharmaFECT 4 transfection reagent according to the manufacturer's instructions (Dharmacon).5\u2032-TACGTGCACAGACGTCAGGAG; Parkin reverse, 5\u2032-GACAGCCAGCCACACAAGGC; PINK1 forward, 5\u2032-GGGGAGTATGGAGCAGTCAC; PINK1 reverse, 5\u2032-CATCAGGGTAGTCGACCAGG; LC3B forward, 5\u2032- GAGAAGACCTTCAAGCAGCG; LC3B reverse, 5\u2032- AAGCTGCTTCTCACCCTTGT; GAPDH forward, 5\u2032-GCCATCAATGACCCCTTCATT; and GAPDH reverse, 5\u2032-TTGACGGTGCCATGGAATTT. Real-time qPCR was conducted by using an ABI PRISM 7000 Sequence Detection System (Applied Biosystems).To analyze the expression levels of Parkin, PINK1, and LC3B genes, total cellular RNA and subsequent complementary DNAs were prepared, as described previously For Western blot analysis, whole cell lysates (WCL) were extracted from cells, subjected to SDS-PAGE, transferred to nitrocellulose membrane (Thermo Scientific), and Western blot analyzed with antibodies against the indicated proteins, as described previously To isolate pure cytosolic and mitochondrial fraction, HepAD38 cells were homogenized and isolated, as described previously The activity of caspase-3/7 in HepG2 and HepAD38 cells transfected with siRNA were measured by using Caspase-Glo 3/7 assay kit according to the manufacturer's instructions (Promega).Apoptotic cells death in HepG2 and HepAD38 cells transfected with siRNA were measured by using Click-iT TUNEL Alexa Fluor 488 imaging assay kit according to the manufacturer's instructions (Invitrogen). For quantitative analysis, at least 1,000 cells on immunofluorescence image were counted.Statistical analyses using Student's t-test were performed by using Sigma Plot software .Figure S1HBx stimulates Parkin gene expression, triggers Parkin recruitment to mitochondria, and physically interacts with Parkin and VDAC3. (A) Confocal microscopy of Parkin aggregates on the mitochondria in HBx-expressing cells. Huh7 cells transfected with HBx-flag construct were prestained with MitoTracker and immunostained with anti-Parkin (green) and anti-flag (white) antibodies. Nuclei are demarcated with white dot circles. Transfected (+) and untransfected (\u2212) cells are marked. The zoom images display the accumulation of endogenous Parkin on the mitochondria. (B) Quantification of fluorescence intensity of Parkin aggregates on the mitochondria . (C) Whole cell lysates extracted from Huh7 cells transfected with HBx-flag construct for 48 h were analyzed by Western blotting with antibodies specific for the indicated proteins. (D) HBx protein in whole cell lysates extracted from Huh7 cells co-transfected with HBx-flag and mCherry-Parkin constructs was immunoprecipitated by anti-flag antibody, followed by Western blotting with antibodies specific for the indicated proteins. Normal mouse IgG was used as a negative control (Ctrl Ab) for co-immunoprecipitation (co-IP).(PDF)Click here for additional data file.Figure S2HBx induces Parkin-mediated mitophagosome formation. (A) Huh7 cells transiently expressing GFP-LC3 protein were transfected with HBx-flag construct in the absence or presence of 3-MA (10 mM) and BafA1 (100 nM), respectively, for 8 h before fixation. At 2 days post-transfection, cells were immunostained with antibodies specific to flag (white), TOM20 (blue), and Parkin (red). (B) GFP-LC3-expressing Huh7 cells were transfected with HBV-\u0394X construct. At 36 hours post-transfection, cells prestained with MitoTracker were immunostained with anti-Parkin (green) and anti-HBsAg (white) antibodies. Nuclei are demarcated with white dot circles. Transfected (+) and untransfected (\u2212) cells are marked. (A and B) The arrows (white puncta) in the zoom images display the merge of GFP-LC3 puncta (green), TOM20 (A)/Mito (B), and Parkin. (C and D) Quantification of the number of total GFP-LC3 puncta (C) and GFP-LC3 puncta colocalized with TOM20 (D) in the panels (A) and (B) . (E) Quantitative analysis of the area of GFP-LC3 puncta (white) representing merge of GFP-LC3 puncta, TOM20/Mito, and Parkin in the panels (A) and (B) .(PDF)Click here for additional data file.Figure S3HBx induces complete mitophagy. (A and B) Confocal microscopy showing complete mitophagic process in HBx-expressing cells. (A) Huh7 cells transiently expressing GFP-LC3 protein were transfected with HBx-flag DNA construct in the absence or presence of 3-MA (10 mM) and BafA1 (100 nM), respectively, for 8 h before fixation. At 2 days post-transfection, cells prestained with LysoTracker were immunostained with anti-flag (white) and TOM20 (blue) antibodies. Nuclei are demarcated with white dot circles. Transfected (+) and untransfected (\u2212) cells are marked. In the zoom images, the arrows (white puncta) indicate GFP-LC3 puncta (green) colocalized with TOM20 and lysosome. (B) Quantification of the colocalization of GFP-LC3 puncta containing lysosome with mitochondria in the panel (A) . (C and D) Rescue effect of BafA1 in HBV-induced decline of mitochondria. Huh7 cells transfected with HBV construct were treated BafA1 (50 nM) for 12 h before fixation. At 36 hours post-transfection, cells prestained with MitoTracker were immunostained with anti-HBsAg antibody (green) (C and E). (E and F) Inhibitory effect of Parkin silencing in HBV-induced decline of mitochondria. Stable cells expressing Parkin-specific shRNA (P-KD) were transfected with HBV DNA construct. Stable cells expressing non-targeting shRNA (NT-KD) were used as a negative control. Nuclei are demarcated with white dot circles (C) or were stained with DAPI (blue) (E). Transfected (+) and untransfected (\u2212) cells are marked. The zoomed images indicate mitochondrial morphology in transfected and untransfected cells, respectively. (D and F) Quantitative analysis of mitochondria-specific fluorescence intensity in the panels (C) and (E) .(PDF)Click here for additional data file."} +{"text": "Here, we performed post-infection passive immunization of SIV-infected rhesus macaques with polyclonal SIV-specific, antibody-dependent cell-mediated viral inhibition (ADCVI)-competent non-NAbs.Antiviral antibodies, especially those with neutralizing activity against the incoming strain, are potentially important immunological effectors to control human immunodeficiency virus (HIV) infection. While neutralizing activity appears to be central in sterile protection against HIV infection, the entity of inhibitory mechanisms via HIV and simian immunodeficiency virus (SIV)-specific antibodies remains elusive. The recent HIV vaccine trial RV144 and studies in nonhuman primate models have indicated controversial protective efficacy of HIV/SIV-specific non-neutralizing binding antibodies (non-NAbs). While reports on HIV-specific non-NAbs have demonstrated virus inhibitory activity mac239-infected, NAb-negative rhesus macaques, respectively. Their binding capacity to whole SIVmac239 virions showed a propensity similar to ADCVI activity. A cocktail of three non-NAb lots showing high virion-binding capacity and ADCVI activity was administered to rhesus macaques at day 7 post-SIVmac239 challenge. This resulted in an infection course comparable with control animals, with no significant difference in set point plasma viral loads or immune parameters.Ten lots of polyclonal immunoglobulin G (IgG) were prepared from plasma of ten chronically SIVin vitro, their passive immunization post-infection did not result in SIV control in vivo. Virion binding and ADCVI activity with lack of virus neutralizing activity were indicated to be insufficient for antibody-triggered non-sterile SIV control. More diverse effector functions or sophisticated localization may be required for non-NAbs to impact HIV/SIV replication in vivo.Despite virus-specific suppressive activity of the non-NAbs having been observed Development of a successful vaccine is crucial for global human immunodeficiency virus (HIV) control. A recent clinical trial in Thailand has shown partial efficacy of an HIV vaccine regimen, RV144 ,3. Thus,in vitro suppressive effects against virus replication, such as ADCC (antibody-dependent cellular cytotoxicity) and ADCVI (antibody-dependent cell-mediated virus inhibition) ); where p27p and p27c are the average p27 concentrations in wells with anti-SIV and control antibodies, respectively. Experiments were performed twice in duplicate.HSC-F cells and concentrated by Amicon Ultra 4, MW50000 (Millipore) to 30 mg/ml. Purified IgG solutions were confirmed negative for SIVmac239-specific neutralizing activity. Three lots prepared from three macaques were mixed to obtain the IgG inoculums for passive non-NAb immunization. Five lots of IgG solutions were also prepared from five chronically SIVmac239-infected rhesus macaques with detectable SIVmac239-specific NAb responses, respectively. Control IgG (CAb) was prepared from pooled plasma of non-infected rhesus macaques.Ten lots of IgG solutions were prepared from ten chronically SIVMacaca mulatta) were challenged intravenously with 1,000 TCID50 of SIVmac239. For passive immunization, animals were intravenously administered with 300 mg of anti-SIV non-NAb IgG or control IgG at day 7 post-challenge. The determination of major histocompatibility complex class I (MHC-I) haplotypes was based on the family study in combination with the reference strand-mediated conformation analysis (RSCA) of Mamu-A and Mamu-B genes and detection of major Mamu-A and Mamu-B alleles by cloning the reverse transcription (RT)-PCR products as described previously [Burmese rhesus macaques induction as described previously [mac239 Gag, Pol, Vif, Vpx, Vpr, Tat, Rev, Env, and Nef amino acid sequence. Intracellular IFN-\u03b3 staining was performed using CytofixCytoperm kit (Becton Dickinson). Fluorescein isothiocianate-conjugated anti-human CD4, Peridinin chlorophyll protein-conjugated anti-human CD8, allophycocyanin-conjugated anti-human CD3 and phycoerythrin-conjugated anti-human IFN-\u03b3 antibodies (Becton Dickinson) were used. Specific T-cell levels were calculated by subtracting non-specific IFN-\u03b3+ T-cell frequencies from those after SIV-specific stimulation. Specific T-cell levels less than 100 cells per million PBMCs are considered negative.Virus-specific CD8eviously . PBMCs wmac239 Env were amplified by nested RT-PCR from plasma RNAs and subjected to direct sequencing by using dye terminator chemistry and an automated DNA sequencer (Applied Biosystems). Predominant non-synonymous mutations were determined.Viral RNAs were extracted using High Pure Viral RNA kit (Roche Diagnostics) from macaque plasma obtained at around 1 year after challenge. Fragments of cDNAs encoding SIV+ T-cell counts, peripheral blood central memory CD4+ T-cell frequencies, and the number of non-synonymous mutations in Env-coding regions between non-NAb-infused and control animals was performed by nonparametric Mann\u2013Whitney U test with significance levels set at p < 0.05. Statistical analysis was performed by Prism software version 4.03 . Comparison of viral loads, peripheral blood CD4mac239-infected, NAb-negative rhesus macaques, respectively. SIVmac239-binding capacity was screened by whole virus ELISA using virions purified from culture supernatants of SIVmac239-infected HSC-F cells (a macaque T-cell line) . The meall line) . Polycloll line) .in vitro virus-suppressive activity of the non-NAb cocktail, ADCVI activity was evaluated using PBMCs as effectors and MHC-mismatched macaque HSC-F cells as infected targets (in vivo antibody concentration immediately after passive immunization (300 mg IgG in 300 ml body fluid), implying that the observed ADCVI activity is likely to occur in vivo after passive immunization.To examine the targets . IgG lotin vitro anti-viral property of the non-NAb cocktail, we performed the post-infection passive immunization. Five rhesus macaques were challenged intravenously with SIVmac239 followed by passive immunization with the non-NAb cocktail (300 mg IgG) at day 7 post-challenge. When we previously passively immunized rhesus macaques with polyclonal antibodies having anti-SIV neutralizing activity by this regimen (300 mg IgG i.v. at day 7), enhanced virus uptake by DCs, subsequent augmentation of SIV-specific CD4+ T-cell responses, enhancement of in vitro virus-suppressive activity in CD8+ cells, and set-point viremia control were observed [mac239 challenge were used as controls.Having confirmed the observed ,21. The cocktail 0 mg IgG observed . Six anide novo virus-specific antibody induction, plasma reactivity against SIV antigens was measured by immunoblotting .All five macaques infused with the non-NAbs failed to contain set-point viremia, similar to the six control animals . The nono groups . Periphe+ T-cell responses in the chronic phase (p = 0.08 by Mann\u2013Whitney U test). Thus, the passive non-NAb immunization at day 7 post-challenge showed no significant impact on SIV replication in vivo. Considering our previous study of NAb-triggered SIV control and facilitation of T-cell responses ,21, we eic phase . Neitheric phase . Analysiin vivo was a major interest in this study. Our results indicate that passive non-NAb immunization does not influence primary SIV replication when administered at early post-infection. In agreement with the limited protective effect observed when non-NAbs were administered locally on mucosa before virus challenge [in vitro.Whether augmentation of ADCVI without virus neutralizing activity may influence SIV replication control hallenge , systemiin vivo, which consequently proposes the following notions. For assessment of antibody binding affinity to antigens on virions, we utilized purified SIV virions instead of recombinant Env proteins as the ain vivo in HIV/SIV infections, unlike in other chronic viral infection models [First, this study indicates that augmentation of non-NAb-derived virus-suppressive activity does not alter SIV control course once infection is achieved. While previous studies on immunized macaques indicated inverse correlation between ADCC or ADCVI activity at virus challenge and acute plasma viremia \u201314, the n models .+ T-cell preservation is important for any immune augmentation [Second, this study indicates the requirement of neutralizing activity of antibody for the suppression of primary SIV replication by passive NAb immunization post-infection, as observed in our previous study . The NAbentation ,37,38.in vivo. In conclusion, the post-infection passive non-NAb immunization did not result in primary SIV control in a rhesus macaque AIDS model. Our results suggest that virion binding and ADCVI activity with lack of virus neutralizing activity in the acute phase are insufficient for giving an impact on primary HIV/SIV replication. Further sophistication of local and targeted induction of functional non-NAb responses may be required to impact HIV/SIV replication"} +{"text": "MYCN oncogene amplification and N-Myc oncoprotein over-expression. Long noncoding RNAs (lncRNAs) are emerging as critical regulators of gene expression and tumourigenesis. While Myc oncoproteins are well-known to exert tumourigenic effects by regulating the expression of protein-coding genes and microRNAs, little is known about which lncRNAs are Myc targets and whether the Myc target lncRNAs play a role in Myc-induced oncogenesis. Here we performed differential gene expression studies using lncRNA microarray in neuroblastoma cells after transfection with control or N-Myc-specific small interfering RNA (siRNA), and identified N-Myc target lncRNAs including the novel lncRNA linc00467, the expression and function of which were completely unknown. RT-PCR, chromatin immunoprecipitation and luciferase assays showed that N-Myc suppressed linc00467 gene expression through direct binding to the linc00467 gene promoter and reducing linc00467 promoter activity. While N-Myc suppressed the expression of RD3, the protein-coding gene immediately down-stream of linc00467 gene, through direct binding to the RD3 gene promoter and reducing RD3 promoter activity, linc00467 reduced RD3 mRNA expression. Moreover, Affymetrix microarray analysis revealed that one of genes significantly up-regulated by linc00467 siRNA was the tumour suppressor gene DKK1. Importantly, knocking-down linc00467 expression with siRNA in neuroblastoma cells reduced the number of viable cells and increased the percentage of apoptotic cells, and co-transfection with DKK1 siRNA blocked the effects. These findings therefore demonstrate that N-Myc-mediated suppression of linc00467 gene transcription counterintuitively blocks N-Myc-mediated reduction in RD3 mRNA expression, and reduces neuroblastoma cell survival by inducing DKK1 expression.The worst subtype of neuroblastoma is caused by MYCN oncogene and consequent N-Myc oncoprotein over-expression occur in approximately 40% of high risk neuroblastoma, and is clinically associated with cancer metastasis, resistance to therapies and poor patient outcome Neuroblastoma is a solid extracranial paediatric cancer that arises from neural crest cells, and accounts for 15% of cancer-related death in children Myc oncoproteins, including N-Myc and c-Myc, exert biological effects through modulating gene transcription. After Myc oncoproteins dimerize with Max, the Myc-MAX complex binds to Myc-responsive element E-boxes at target gene promoters, leading to transcriptional activation Long noncoding RNAs (lncRNAs) are transcripts longer than 200 nucleotides without a functional open reading frame, and can be divided into five different types: sense, antisense, bidirectional, intronic and intergenic (lincRNA) Myc oncoproteins have been extensively shown to modulate the expression of microRNAs, and targeting the microRNAs is a promising approach for treating Myc-induced cancers according to published DNA and cDNA sequencing data Myc oncoproteins exert biological effects by modulating gene transcription. However, it is unknown whether N-Myc modulates the transcription of lncRNAs. We therefore performed differential gene expression analysis using NCode\u2122 Human Non-coding RNA Microarray in BE(2)-C neuroblastoma cells 30 hours after transfection with control siRNA or N-Myc siRNA No. 1 (N-Myc siRNA-1). As shown in MYCN oncogene amplified human neuroblastoma cell lines, BE(2)-C and Kelly, followed by real-time RT-PCR study of linc00467. As shown in To validate the microarray data, we performed siRNA transfections with control siRNA, N-Myc siRNA-1 or N-Myc siRNA-2 for 48 hours in two linc00467 gene transcription, we firstly analysed transcription factor binding sites at the linc00467 gene promoter with Gene-Regulation software . Results showed that Sp1-binding sites were enriched at \u2212176 bp to \u221214 bp upstream of linc00467 gene transcription start site as well as +7 bp to +426 bp in intron 1 -C neuroblastoma cells -C cells after transfection with control siRNA or N-Myc siRNA-1, followed by transfection with a pLightSwitch_Prom construct expressing empty vector or the linc00467 promoter region. Results showed that knocking-down N-Myc expression significantly up-regulated luciferase activity of the pLightSwitch_Prom construct expressing the linc00467 promoter region Results showed that Sp1-binding sites were enriched at +475 bp to +731 bp of RD3 gene intron 1, relative to intron 1 start site -C neuroblastoma cells -C cells after transfection with control siRNA or N-Myc siRNA-1, followed by transfection with a pLightSwitch_Prom construct expressing empty vector or the RD3 intron 1 region. Results showed that knocking-down N-Myc expression significantly up-regulated luciferase activity of the pLightSwitch_Prom construct expressing the RD3 intron 1 region -C and Kelly cells with control siRNA or linc00467 siRNA for 48 hours, followed by Alamar blue assays. As shown in To understand the mechanism through which linc00467 promotes neuroblastoma cell survival, we performed differential gene expression study of linc00467 target genes in BE(2)-C cells 48 hours after transfection with control siRNA or linc00467 siRNA-1. As shown in To examine whether up-regulation of the tumour suppressor gene DKK1 contributed to linc00467 siRNA-mediated apoptosis, we transfected BE(2)-C cells with control siRNA, linc00467 siRNA-1, DKK1 siRNA, or combination of linc00467 siRNA-1 and DKK1 siRNA. RT-PCR analysis showed that DKK1 siRNA reduced DKK1 gene expression, and blocked linc00467 siRNA-mediated DKK1 gene up-regulation . Alamar lncRNAs are emerging as critical regulators of gene transcription, tumour initiation, progression and metastasis linc00467 gene core promoter is enriched in Sp1-binding sites, and that c-Myc binds to the Sp1-binding site-enriched region of the lin00467 gene core promoter in K562 leukemia cells according to a publically available ChIP-Seq dataset. Moreover, our own ChIP assays have confirmed that N-Myc indeed binds to the Sp1-binding site-enriched region of the lin00467 gene core promoter, luciferase assays show that N-Myc siRNA enhances linc00467 gene promoter activity, and RT-PCR data demonstrate that linc00467 gene expression is reduced by N-Myc and up-regulated by N-Myc siRNAs. As N-Myc is well-known to repress gene transcription by direct binding to target gene promoter regions enriched in Sp1-binding sites linc00467 gene expression by direct binding to the linc00467 gene promoter region enriched in Sp1-binidng sites and suppresses linc00467 gene promoter activity.linc00467 was identified by Human Genome Organisation Gene Nomenclature Committee (HGNC) based on published DNA and cDNA sequencing data in cis and in trans regulation of RNA expression at both transcriptional and post-transcriptional levels. For examples, a number of lncRNAs have been shown to up- or down-regulate the expression of their neighboring protein-coding genes through modulating chromatin structure and gene transcription PTEN gene transcription and PTEN mRNA stability RD3, which encodes a retinal protein responsible for the retinal degeneration disorder Leber congenital amaurosis type 12 RD3 gene expression through direct binding to the RD3 gene promoter region enriched in Sp1-binding sites and reducing RD3 gene promoter activity. These data indicate that linc00467 reduces RD3 mRNA expression most likely through an in cis mechanism, and that N-Myc-mediated suppression of linc00467 gene transcription counterintuitively blocks N-Myc-mediated reduction in RD3 mRNA expression. In addition, our differential gene expression study with Affymetrix microarray has identified DKK1 as one of the genes significantly up-regulated by linc00467 siRNA, suggesting that linc00467 is also likely to modulate gene expression through in trans mechanisms.lncRNAs exert biological effects through linc00467 gene transcription counterintuitively blocks N-Myc-mediated cell survival.While the biological function of linc00467 is completely unknown in the literature, the Wnt antagonist DKK1 is well-known to induce cancer cell apoptosis and function as a tumour suppressor gene linc00467 gene transcription through direct binding to the linc00467 gene promoter. linc00467 reduces the expression of its neighbouring protein-coding gene RD3, while N-Myc suppresses RD3 gene transcription through direct binding to the RD3 gene promoter. Importantly, linc00467 enhances neuroblastoma cell survival through reducing DKK1 expression. These findings therefore demonstrate that N-Myc-mediated suppression of linc00467 gene transcription counterintuitively blocks N-Myc-mediated reduction in RD3 mRNA expression, and reduces neuroblastoma cell survival by inducing DKK1 expression.In summary, this study identifies lncRNAs as targets of N-Myc in neuroblastoma cells through genome-wide differential gene expression study, and demonstrates that N-Myc suppresses Neuroblastoma BE(2)-C cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum Cells were transfected with AllStars negative control siRNA, N-Myc siRNA, linc00467 siRNA or DKK1 siRNA (Qiagen) using Lipofectamine 2000 (Life Technologies) as previously described Following siRNA transfections, RNA was extracted from cells using PureLink RNA Mini kit (Life Technologies) according to the manufacturer's instructions. RNA samples were then quantified using Nanodrop spectrophotometer and treated with DNAse 1 (Life Technologies) to remove remaining genomic DNA. Synthesis of cDNA from RNA samples was carried out using M-MLV Reverse Transcriptase (Invitrogen). Real-time RT PCR was performed in Applied Biosystems 7900 using SYBR green PCR Master Mix (Life Technologies) as previously described For the analysis of protein expression by immunoblot, cells were lysed, protein extracted and separated by gel electrophoresis. After western transfer, membranes were probed with mouse anti-N-Myc antibody (1\u22361000) (Santa Cruz Biotech), followed by horseradish peroxidase-conjugated anti-mouse (1\u223610000) antiserum (Santa Cruz Biotech). Protein bands were visualized with SuperSignal (Pierce). The membranes were lastly re-probed with an anti-actin antibody (Sigma) as loading controls.Neuroblastoma BE(2)-C cells were transfected with scrambled control siRNA or N-Myc siRNA-1. Thirty hours after siRNA transfection, RNA was extracted from the cells with RNeasy mini kit and treated with DNAse 1. Differential gene expression was examined with NCode\u2122 Human Non-coding RNA Microarray (Invitrogene), according to the manufacturer's instructions. Results from the microarray hybridization were analysed with GeneSpring software (GeneSpring), and deposited at Gene Expression Omnibus website (accession number: GSE52984)http://www.r-project.org/) with bioconductor package (http://www.bioconductor.org/), and deposited at Gene Expression Omnibus website (accession number: GSE52985).Neuroblastoma BE(2)-C cells were transfected with scrambled control siRNA or linc00467 siRNA-1. Fourty-eight hours after siRNA transfection, RNA was extracted from the cells with RNeasy mini kit. Differential gene expression was examined with Affymetrix HuGene-2_0-st Arrays (Affymetrix), according to the manufacturer's instructions. Results from the microarray hybridization were analysed in R or a control antibody and PCR with primers targeting negative control region or Sp1-binding site-enriched region of the linc00467 gene promoter region (\u2212248 bp upstream of linc00467 gene transcription start site to +567 bp of intron 1) was custom-cloned into the pLightSwitch_Prom construct by SwitchGear Genomics. Sp1-binding site enriched intron 1 region of RD3 (0 bp to +1043 bp) was also custom-cloned into the pLightSwitch_Prom construct by SwitchGear Genomics. BE(2)-C neuroblastoma cells were transfected with control siRNA or N-Myc siRNA-1, followed by co-transfection with Cypridina TK control construct plus empty vector, linc00467 or RD3 gene promoter pLightSwitch_Prom construct. Luciferase activities were measured with a LightSwitch Dual Assay System kit (SwitchGear Genomics) according to the manufacturer's instructions, and expressed as percentage changes relative to control siRNA transfected samples.Sp1-binding site enriched Alamar blue assays were performed as previously described For the analysis of cells at sub-G1 phase, seventy-two hours after siRNA transfection, cells were harvested, fixed in 80% ethanol, washed and then stained with 50 \u00b5g/ml propidium iodide (Sigma) in solution containing 2 \u00b5g/ml RNase (Roche). Flow cytometric analysis of the cells was performed using FACS Calibur machine and FACS Diva software (BD Biosciences). The percentage of cells at sub-G1 phase of the cell cycle was analyzed.For the analysis of apoptosis, seventy-two hours after siRNA transfection, cells were incubated with FITC-conjugated Annexin V (BD Biosciences), and then subjected to flow cytometric analysis of FITC-positive cells using FACS Calibur machine and FACS Diva software.Experiments were conducted 3 times in duplicates. Data were analysed on Prism 6 software (GraphPad) and presented as mean \u00b1 standard error. Differences were analyzed for significance using ANOVA among groups or two-sided unpaired t-test for two groups. A p value of less than 0.05 was considered statistically significant.Figure S1P<0. 01.N-Myc directly binds to the ODC1 gene promoter. ChIP assays were performed with a control or anti-N-Myc antibody (Ab) and primers targeting a negative control region or the ODC1 gene core promoter region. Fold enrichment was calculated by dividing PCR products from DNA samples immunoprecipitated with the anti-N-Myc Ab by PCR products from DNA samples immunoprecipitated with the control Ab, relative to input. Fold enrichment at the negative control region was artificially set as 1.0. Error bars represented standard error. ** indicated (PDF)Click here for additional data file.Figure S2P<0. 01, compared with control siRNA-transfected samples.N-Myc and linc00467 do not have a co-operative effect on RD3 expression in neuroblastoma cells. BE(2)-C cells were transfected with scrambled control siRNA, N-Myc siRNA-1, N-Myc siRNA-2, linc00467 siRNA-1, linc00467 siRNA-2, combination of N-Myc siRNA-1 and linc00467 siRNA-1, or combination of N-Myc siRNA-2 and linc00467 siRNA-2 for 48 hours, followed by RT-PCR analysis of RD3 expression. Error bars represented standard error. * indicated (PDF)Click here for additional data file.Table S1Modulation of target gene expression by linc00467 siRNA-1 by more than 1.8 fold, as identified by Affymetrix microarray, in BE(2)-C cells 48 hours after transfection with control siRNA or linc00467 siRNA-1. The cut-off was set at 1.80 fold, as linc00467 siRNA-1 reduced the expression of linc00467 by 1.841 fold.(DOCX)Click here for additional data file."} +{"text": "ATP was first shown to enhance the activity of natriuretic peptide (NP)-stimulated guanylyl cyclase (GC)-A in 1987 . BeginniRecent studies have determined how ATP allosterically activates GC-A and GC-B . In the GC-A and GC-B are asymmetric homodimers with distinct and reciprocally regulated catalytic and allosteric sites that bind GTP and ATP, respectively."} +{"text": "Some of these undesired actions appear after repeated administration and are related to adaptive changes directed at counteracting the consequences of sustained opioid receptor activation. Here we will discuss adaptations that contribute to the development of tolerance. The focus of the first part of the review is set on molecular mechanisms involved in the regulation of opioid receptor signalling in heterologous expression systems and neurons. In the second part we assess how adaptations that take place Opiates are among the most effective analgesics known but their clinical use is limited by severe side effects. Some of these undesired actions including tolerance, dependence and abuse usually appear after repeated opioid administration, and have been linked to adaptations that take place in order to counteract prolonged opioid receptor activation ,2. Adaptper se warrants neither resensitization nor absence of tolerance. For example although SNC-80 produces rapid internalization of DORs -N, N-diethyl-benzamide); (CFA): complete Freund's adjuvant; (DAMGO): -enkephalin; (DORs): delta opioid receptors; (DRG): dorsal root ganglion; (ERK1/2): extracellular signalregulated kinases; (GASP): G protein coupled receptor associated sorting protein; (GIRK): G protein activated inward rectifier K+; (GPCRs): G protein-coupled receptors; (LC): locus coeruleus; (MORs): mu opioid receptors; (AR-M1000390): N, NDiethyl- 4-(phenylpiperidin-4-ylidenemethyl) benzamide.The authors declare that they have no competing interests.KN conducted reference research, contributed discussion and figure concerning signalling complexes. GP conducted reference research and wrote the review. All authors read and approved the final manuscript."} +{"text": "Ewing sarcoma patients have a poor prognosis despite multimodal therapy. Integration of combination immunotherapeutic strategies into first-/second-line regimens represents promising treatment options, particularly for patients with intrinsic or acquired resistance to conventional therapies. We evaluated the susceptibility of Ewing sarcoma to natural killer cell-based combination immunotherapy, by assessing the capacity of histone deacetylase inhibitors to improve immune recognition and sensitize for natural killer cell cytotoxicity.Using flow cytometry, ELISA and immunohistochemistry, expression of natural killer cell receptor ligands was assessed in chemotherapy-sensitive/-resistant Ewing sarcoma cell lines, plasma and tumours. Natural killer cell cytotoxicity was evaluated in Chromium release assays. Using ATM/ATR inhibitor caffeine, the contribution of the DNA damage response pathway to histone deacetylase inhibitor-induced ligand expression was assessed.in vivo, regardless of chemotherapy-response and disease stage. Soluble NKG2D-ligand plasma concentrations did not differ between patients and controls.Despite comparable expression of natural killer cell receptor ligands, chemotherapy-resistant Ewing sarcoma exhibited reduced susceptibility to resting natural killer cells. Interleukin-15-activation of natural killer cells overcame this reduced sensitivity. Histone deacetylase inhibitor-pretreatment induced NKG2D-ligand expression in an ATM/ATR-dependent manner and sensitized for NKG2D-dependent cytotoxicity (2/4 cell lines). NKG2D-ligands were expressed Our data provide a rationale for combination immunotherapy involving immune effector and target cell manipulation in first-/second-line treatment regimens for Ewing sarcoma. TET gene family products, though rarely other activating transcription factors [Ewing sarcoma is an aggressive round cell sarcoma characterized by specific gene fusions most commonly involving factors -3. It us factors -6, which factors ,5.x vivo activation of immune cells) and/or sensitization of target cells for immune-mediated killing by combination immunotherapy may improve immunotherapeutic efficacy. For example, pre-conditioning of various cancer cell types by agents that activate the DNA damage response pathway may sensitize for natural killer cell cytotoxicity via induction of activating natural killer cell receptor ligands and/or death receptor expression. Comparable results have been observed for histone deacetylase inhibitors (HDI), which are currently emerging as potent anti-tumour agents [Natural killer (NK) cells are the main cytotoxic effector cells of the innate immune system, contributing to host anti-microbial and anti-tumour immune responses. In contrast to T-lymphocytes, these cells lack antigen-specific receptors. Instead, recognition of target cells and subsequent triggering of effector functions is regulated by integration of signals delivered from inhibitory and activating , DNAX accessory molecule 1 (DNAM1), natural cytotoxicity receptor (NCR)) cell surface receptors ,8. Naturr agents -19.in vitro as well as in vivo and to sensitize for both conventional and more experimental treatment modalities [Pre-clinical studies show that Ewing sarcoma can be targeted by (cytokine-activated) natural killer cells in a NKG2D-, DNAM1- and, as recently demonstrated, NCR-dependent manner -24. Moredalities -32, the Peripheral blood samples from newly diagnosed Ewing sarcoma patients 2004-2009) were collected prior to start of chemotherapy (n = 27) and, in case of complete remission, six months after completion of therapy (n = 7) and results were analyzed using Cellquest software, as previously described . Ligand 4-\u03bcm sections containing representative tumour were deparaffinized and citrate antigen retrieval was performed. Subsequent immunohistochemical stainings were performed and (semi-quantitatively) scored as previously described .Concentrations of soluble MICA in plasma samples were determined by sandwich-ELISA using the MICA (human) detection set, according to the manufacturer's protocol . In short, plates were coated overnight at 4\u00b0C with 5 \u03bcg/ml of the anti-MICA antibody (AMO1). After blocking with 15% BSA/PBS for 15 minutes at 37\u00b0C, plates were washed with 0.05% Tween-20/PBS. Samples or recombinant MICA*04, serving as a standard, were added in 7.5% BSA/PBS. After incubation for two hours at 37\u00b0C and washing, detection antibody (anti-MICA/B (BAMO3)) was added at a concentration of 1 \u03bcg/ml . Plates were washed and incubated with HRP-conjugated anti-mouse IgG2a antibody for one hour at 37\u00b0C. After extensive washing, TMB peroxidase substrate was added and plates were incubated for 35 minutes at room temperature in the dark. HRP activity was stopped by addition of 1 M phosphoric acid and absorbance was measured at 450 nm wavelength.6 cells/ml in RPMI-1640 supplemented with streptomycin/penicillin and 10% FBS. As determined by flow cytometric analysis, natural killer cell purity always exceeded 88% and T-cell contamination > 0.2% was never detected. For cytokine activation, natural killer cells were cultured in AIM-V medium (Invitrogen), supplemented with 10% pooled human AB-serum and streptomycin/penicillin and stimulated with 10 ng/ml recombinant human interleukin-15 (IL-15) . Activated natural killer cells were used for Chromium (51Cr) release assays within two-four weeks of culture.Peripheral blood mononuclear cells were obtained from healthy blood bank donors after informed consent and were isolated using Ficoll density gradient separation. Isolation of natural killer cells was performed using the MACS NK enrichment kit and LS columns, according to the manufacturer's protocol and isolated cells were plated for overnight recovery at 1-2 \u00d7 1051Cr release assays as previously described [51Cr release assay. We previously demonstrated that pre-incubation of natural killer cells with control isotype (IgG1)-matched antibody (anti-CD56) did not affect cytotoxicity [Cytotoxicity was determined in standard 4-hour escribed . For spetoxicity . Moreove3 cells/well. Following overnight attachment, cells were incubated for 24 hours with increasing concentrations of specific HDI. Cell viability was measured by 3--5-(3-carboxymethoxy-phenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) cell viability assay . The cytotoxic effect of HDI was quantified by determining IC50 values, as defined by the concentration of drug at which 50% of the cells were still metabolically active t-tests or one-way ANOVA tests were used for comparison of means within or between samples or groups of samples. P < 0.05 was considered statistically significant. Artwork was created using Graphpad Prism 5.0 .51Cr release assays revealed significantly reduced sensitivity of chemotherapy-resistant cell lines to resting natural killer cell-mediated cytolysis, at effector-to-target ratio's \u2265 2.5:1 .To assess possible cross-resistance of chemotherapy-resistant Ewing sarcoma to natural killer cell cytotoxicity, a panel of both chemotherapy-sensitive and -resistant cell lines was evaluated for susceptibility to lysis by resting natural killer cells obtained from 4-8 healthy donors. As illustrated in Figure 51Cr release assays reduced natural killer cell-mediated cytolysis of both chemotherapy-sensitive and -resistant cell lines to a similar degree (data not shown).Natural killer cell cytotoxicity to Ewing sarcoma cells critically depends on combined NKG2D and DNAM1 signaling ,23. Therin vitro as well as in vivo, natural killer cell receptor ligands are expressed regardless of chemotherapy-sensitivity.Immunohistochemical staining for expression of NKG2D-ligands MICA and ULBP1 in sequential primary Ewing sarcoma tumour samples obtained from patients with different histological responses to chemotherapy demonstrin situ expression of MICA in Ewing sarcoma tumours, soluble MICA expression levels were measured in plasma samples from Ewing sarcoma patients. Results were compared to those obtained in plasma samples from age-comparable healthy controls. The majority of plasma samples from healthy controls contained levels of soluble MICA close to the lower detection limit (10 pg/ml) of the assay . Plasma concentrations of soluble MICA in patients prior to treatment or after completion of therapy did not significantly differ from those observed in healthy controls Figure .Pre-activation of natural killer cells by either recombinant human IL-15 or co-culture with genetically modified IL-15/4-1BBL expressing K562 feeder cells results in more efficient recognition and lysis of Ewing sarcoma cells ,23. Ther50 values concentrations of HDI revealed heterogeneous but consistent induction of several activating NKG2D ligands, in particular MICB, in all cell lines evaluated. The most pronounced effects were observed upon pre-treatment with NaB and MS-275, resulting in up to five-fold induction of MICB , p < 0.05 at effector-to-target ratio's \u2265 5:1). Similarly, pre-treatment of SK-ES-1 cells with SAHA , but not NaB or MS-275, resulted in significantly increased cytolysis (data not shown). In cell lines TC71 and STA-ET2.1, despite induction of activating NKG2D ligands, no sensitization for natural killer cell cytotoxicity was detectable dependence on signaling via this activating natural killer cell receptor since blocking reduced lysis of both untreated and HDI-treated cells with differential expression of genes involved in apoptosis, including caspase-8 and p53 pathways ,40,41. BImportantly, the relative resistance of chemotherapy-resistant Ewing sarcoma cells to lysis by resting natural killer cells could be overcome by pre-activation of natural killer cells with IL-15. Although the exact mechanisms underlying cross-resistance to chemotherapeutic drugs and immune-mediated cytotoxicity have not been fully elucidated, utilization of complementary (apoptotic) pathways or stronger pro-apoptotic signals by cytokine-activated natural killer cells may contin vivo immune recognition of Ewing sarcoma tumours, however, was provided by immunohistochemical analysis of sequential Ewing sarcoma tumour samples demonstrating expression of (at least one of the) activating natural killer cell receptor ligands MICA and ULBP1 throughout all stages of disease and regardless of the histological response to chemotherapy. Moreover, and similar to previous observations in leukemia [in situ expression of NKG2D ligands, tumour cells may escape from NKG2D immunosurveillance by (enhanced) shedding of these ligands from their cell surfaces. Subsequently, circulating tumour-derived soluble ligands may cause downregulation of NKG2D and, in turn, severely impair cytotoxic effector functions of both T- and natural killer cells [in situ expression of ligands for activating natural killer cell receptors throughout all stages of disease, including (chemotherapy-resistant) metastatic disease, 2) the absence of (enhanced) shedding of these activating natural killer cell receptor ligands, 3) the above mentioned reduced expression of inhibitory HLA class I molecules in advanced-stage Ewing sarcoma cases and 4) the observed susceptibility of (chemotherapy-resistant) Ewing sarcoma to IL-15-activated natural killer cells further support the potential of Ewing sarcoma as an attractive target for natural killer cell-based immunotherapy.Due to the nature of this (bone) tumour, attempts to establish primary tumour cell cultures from therapy-naive biopsies for evaluation of natural killer cell cytotoxicity towards these targets have so far been unsuccessful. Support for potential leukemia , a potenleukemia . In addileukemia . We receleukemia . Despiteer cells -39. Baseer cells , we do ner cells . Our curin vitro and in vivo and sensitization for both conventional and more experimental treatment modalities has been suggested [in vitro were below or within the range of the maximum tolerated dose as determined in recent clinical trials (in children) [Several phase I-III clinical trials now prove HDI-treatment to be safe and effective in both hematological and solid tumours (as reviewed by ) and recuggested ,31,32. Hhildren) ,60. NatuCollectively, our data provide a rationale for combination immunotherapy involving immune effector cell and target cell (HDI) manipulation in first- and/or second-line treatment regimens for Ewing sarcoma.Ex vivo immune cell activation and/or (simultaneous) sensitization of target cells for immune-mediated killing by combination immunotherapy may overcome intrinsic/acquired resistance to conventional therapies and improve (immuno)therapeutic efficacy. Here, we provide the first evidence that combination immunotherapy using histone deacetylase inhibitors and (interleukin-15-activated) NK cells improves immune recognition of both therapy-sensitive and -resistant EWS and sensitizes for NK cell cytotoxicity. In vivo expression of activating NK cell receptor ligands throughout all disease-stages, regardless of chemotherapy-response, supports their potential in vivo role in immune recognition of EWS. Our data provide a rationale for combination immunotherapy involving simultaneous immune cell (interleukin-15-activated NK cells) and target cell (histone deacetylase inhibitors) manipulation in first-/second-line treatment regimens for EWS.Patients with Ewing sarcoma (EWS) have a poor prognosis, despite current multimodal therapy. Integration of immunotherapeutic strategies, including natural killer (NK) cell-based therapies, into first-line treatment regimens or introduction of these approaches as second-line therapy may represent promising treatment options. The authors declare that they have no competing interests.All authors contributed to conception and/or design of the study. DB, HIV, SJS and SK conducted experiments and performed data analyses. DB, MWS, EPB, PCWH and ACL were involved in interpretation of data. All authors were involved in drafting and/or critical revision of the manuscript and approved the final submitted version.Antibodies used for flow cytometry, NK cell receptor (ligand) blocking, immunohistochemistry and ELISA.Click here for fileA. Constitutive surface expression of inhibitory (HLA class I) or activating natural killer cell receptor ligands in chemotherapy-sensitive (grey) and -resistant (black) Ewing sarcoma cell lines, as assessed by flow cytometry. Results are expressed as the mean \u00b1 SD MFI-ratio, obtained in at least two independent experiments. Statistical analysis (t-test) was performed on mean MFI-ratio's (for each ligand) of chemotherapy-sensitive versus -resistant cell lines, revealing no significant differences in expression levels of these ligands (p > 0.05). B. Cytotoxic activity of resting natural killer cells was evaluated in 51Cr release assays using chemotherapy-sensitive (TC71 (blue), SK-ES-1 (purple), SK-N-MC (pink)) and -resistant (STA-ET-2.1 (black), CADO-ES (dark grey), IOR/BER (light grey)) Ewing sarcoma cell lines as target cells. Ewing sarcoma cells were either left untreated (solid bars) or pre-incubated with HLA class I blocking antibody DX17 (checked bars). Results are expressed as the mean \u00b1 SD percentage of specific lysis obtained in at least two independent experiments using different healthy donors.Click here for file51Cr release assays using chemotherapy-sensitive (TC71 (blue), SK-ES-1 (purple)) and -resistant (STA-ET-2.1 (black), CADO-ES (grey) Ewing sarcoma cell lines as target cellsCytotoxic activity of IL-15-activated natural killer cells was evaluated in . Ewing sarcoma cells were either left untreated (solid bars) or pre-incubated with NKG2D and DNAM-1 blocking antibodies (checked bars). Results are expressed as the mean \u00b1 SD percentage of specific lysis obtained in at least two independent experiments using different healthy donors.Click here for file51Cr release assays using MS-275-pretreated TC71 and STA-ET2.1 cellsA. Cytotoxicity of resting natural killer cells was evaluated in . Despite induction of activating NKG2D ligands, no sensitization for natural killer cell cytotoxicity was detectable . Similar results were observed for both cell lines upon pre-treatment with NaB and SAHA (not shown). Results are expressed as the mean \u00b1 SEM percentages of specific lysis obtained in at least two independent experiments using different healthy donors. B. Upon HDI-pretreatment, persistent dependency of resting natural killer cell-mediated cytotoxicity on signaling via activating receptor NKG2D was demonstrated when 51Cr release assays were performed in the presence of a blocking antibody against NKG2D. Blocking reduced resting natural killer cell-mediated lysis of both untreated and HDI pre-treated cells to similar levels, as demonstrated for CADO-ES upon pre-treatment with NaB. Similar results were obtained for CADO-ES with MS-275 and SAHA, as well as for SK-ES-1 with SAHA (not shown). K562 and EBV B-LCL cell line 107 were used as positive and negative control respectively (not shown). Results are expressed as the mean \u00b1 SEM percentages of specific lysis obtained in at least two independent experiments using different healthy donors.Click here for file"} +{"text": "Evidence suggests that HTLV-I-induced immune activation plays a key role in the pathogenesis of HAM/TSP. The HTLV-I-encoded transactivating protein Tax is known to activate nuclear factor kappa B (NFkB), a key host signaling pathway regulating immune response, but the contribution of the NFkB pathway to the immune activation associated with HAM/TSP has yet to be fully defined. We examined NFkB activation in peripheral-blood mononuclear cells (PBMC) from subjects with HAM/TSP, and tested the effect of NFkB inhibition on key ex vivo correlates of immune activation in HAM/TSP.We examined the role of NFkB activation during immune activation associated with HAM/TSP by using small molecule NFkB inhibitors, including a newly developed selective inhibitor of NFkB, PBS-1086.NFkB activation was assessed in peripheral-blood mononuclear cells (PBMC) from subjects with HAM/TSP and in healthy donors (HD). Nuclear translocation of the NFkB RelA was significantly higher in PBMC from subjects with HAM/TSP compared to HD (p=0.032) following short-term (20 h) culture, indicating increased activation of the NFkB pathway in HAM/TSP. Treatment with the small molecule inhibitor PBS-1086 reduced NFkB activation (p<0.01). PBS-1086 reduced expression of CD25 and CD69 in HAM/TSP PBMC as well as phosphorylation of STAT5 in a dose-dependent manner . PBS-1086 also inhibited spontaneous lymphoproliferation of HAM/TSP PBMC in a dose-dependent manner (p=0.0286). PBS-1086 treatment resulted in a mean proviral load reduction of 20% compared to untreated PBMC in a 72 h cultureThese results indicate that NFkB activation plays a critical upstream role in the immune activation associated with HAM/TSP, and identify the NFkB pathway as a potential therapeutic target for immune modulation in HAM/TSP."} +{"text": "Clinical-grade T cells are genetically modified ex vivo to express chimeric antigen receptors (CARs) to redirect their specificity to target tumor-associated antigens in vivo. We have developed gene therapy approach to render T cells specific for invasive fungal infections (IFI) due to Aspergillus. We adapted the pattern-recognition receptor Dectin-1 to activate T cells via chimeric CD28 and CD3-zeta (designated D-CAR) upon binding with carbohydrate cell wall in Aspergillus germlings. T cells genetically modified with Sleeping Beauty system to stably express D-CAR were selectively propagated on artificial antigen presenting cells using an approach that is approved by FDA to develop CAR T cells for clinical trials. The D-CAR+ T cells exhibited specificity for beta-1,3-gucan and damaged and thus inhibited hyphal growth of Aspergillus. Treatment of D-CAR+ T cells with steroids did not compromise anti-fungal activity. Thus, we report a clinically-appealing strategy to transfer innate immunity for mycology to cytotoxic T cells."} +{"text": "The calcification ofaortic valves is a common diseaseand valve replacement is the only established treatment. Herewith, we use infrared (FT-IR) spectroscopyto investigate and characterize the mineral deposits in order to understand the mechanism of aortic valve calcification and stenosis.30 aortic valves of patients (65-80 years), who underwent surgical aortic valve replacement due to aortic stenosis, were used. The ATR-FT-IR spectra were recorded with a Nicolet 6700 thermoscientific spectrometer. SEM-EDX and XRD were from Fei Co, the Netherlands and Simens D-500 X-Ray diffractometer, respectively.-1resulted from hyperoxidation of lipids due to oxidative stress. The characteristic bands at the spectral regions 1200-900 cm-1 and 700-400 cm-1showed the formation of low crystallinity biological hydroxyapatite (Ca10(PO4)6(OH)2) and calcium monophosphate (CaHPO4) salts. The results were confirmed using SEM-\u0395DAX and XRD analysis. The observed cross-linking bonds between collagen and elastin were the principal sites for calcium deposition and progression. The findings confirmed the hypothesis that hydroxyapatite is formed predominantly due to ATP cycle, where the release of phosphate anions take place in ischemic pathways.The changes of FT-IR spectraat 1743 cm2HPO4 and calcium phosphates. SEM-EDAX data showed substitution of calcium cations from magnesium cations leading to amorphous salts, preventing thus the aortic valve stenosis. Treatment of these patients with magnesium salts maybe could reduce the progress of aortic valve stenosis after valve replacement.The characteristic FT-IR absorption bands of calcified aortic valves showed hyperoxidation of membranes (a pro-inflammation stage), while the mineral deposits were consistent of low crystallinity biological hydroxyapatite, Ca"} +{"text": "Dear Editor,In their recent interesting published manuscript, Ahmadi et al. showed that in predialysis patients with chronic renal failure (CRF) doubling the dose of Hepatitis B virus (HBV) vaccine from 20mcg to 40mcg was not benefit regarding the seroconvertion rate . AlthougIn CRF patients the impaired immune function is multifactorial. The impaired interaction between antigen presenting cells (APCs) and T-cells is indicated by the decreased T-cell proliferation and \u03b6-chain phosphorylation after a major histocompatibility complex (MHC) dependent T-cell receptor (TCR) stimulation and certainly contributes to the impaired immune function. Decreased MHC and TCR expression, altered expression of the adhesion molecules lymphocyte function antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1), as well as reduced expression of the co-receptors CD80/CD86 in APCs and CD28 in T-cells may be responsible for it. Besides the impaired APC-T-cell interaction; other mechanisms have been also proposed for the acquired immunity disturbances in CRF patients. Increased soluble CD40 molecules, which inhibit the interaction between T-cells and B-cells, decreased TCR complex \u03b6-chain expression, altered cytokine status, which affects T-cell differentiation and finally increased apoptosis of T-cells and B-cells have all been incriminated for the impaired adaptive immune response . The aboThe solution could be in the development of new vaccines with adjuvants other than the aluminum hydroxide, which is generally used. For example, an approved vaccine against HBV for CRF patients contains mono phosphoryl lipid A (MPL). MPL is a low-toxicity derivative of lipopolysaccharide that interacts with Toll-like receptors and the mentioned vaccine showed improved immunogenicity when administered in CRF patients . Recent"} +{"text": "The reactive stromal phenotype in the prostate is mechanistically important for understanding prostate cancer progression and may be a target for prevention. We mimicked the interaction of endocrine, paracrine, and immune factors on induced androgen metabolism in prostate stroma by coculturing human primary prostate stromal cells and LAPC-4 prostatic adenocarcinoma cells. The aim of the study was to use this model to investigate dose dependent effects of hop-derived polyphenols xanthohumol (Xan), isoxanthohumol (iXan), 8-prenylnaringenin (8-PN) and structurally-related compounds quercetin (Que), naringenin (Nar), and 6-dimethylallylnaringenin (6-DMAN) on PSA secretion.Conversion of DHEA (D) by the reactive stroma to androgens following TGF-beta (T) stimulation was assessed by induced PSA secretion in LAPC-4 cells. Direct epithelial PSA secretion was induced using the non-metabolizable androgen R1881. The natural compounds were used in doses of 0.01-10 \u00b5M or 0.1-10 \u00b5M .All compounds tested dose-dependently attenuated epithelial PSA production resulting from stromal androgen metabolite production following D+T treatment . Following direct epithelial stimulation by R1881 treatment , 8-PN and 6-DMAN also showed a dose-dependent response pattern. All other compounds inhibited this epithelial response only at the highest dose (10 \u00b5M). Comparative studies with pure estrogen receptor agonists demonstrated the involvement of these receptors in mediating the response, however the high inhibition of the stromal response by Que, Xan, iXan and Nar cannot be explained by ER-dependent mechanisms only, as they are very weak estrogens.In conclusion, hop-derived polyphenols, as well as structurally related compounds are very potent inhibitors of stromal conversion of androgenic prohormones. Only the two most potent estrogenic compounds showed the same inhibition of PSA induction in response to R1881 treatment. More studies are needed to examine the value of these compounds in prevention of prostate cancer progression."} +{"text": "Familial Mediterranean fever (FMF) is a hereditary autoinflammatory disease that affects the populations with certain ethnic backgrounds. It is characterized by self-limiting febrile attacks of polyserositis. In recent years, some studies reported that FMF patients had increased vascular wall alterations and damage which may be another clinical phenotype of the disease.In the present study, we extensively evaluated biomarkers related with endothelial damage in regularly treated and attack-free FMF patients.Forty FMF patients and eighteen healthy controls with no known cardiovascular risk factors were included. All patients were receiving regular colchicine treatment and examinations were performed during attack-free periods. Serum samples were used for the determination of high sensitive C-reactive protein (hs-CRP), tissue factor (TF), tissue plasminogen activator (t-PA) and osteoprotegerin (OPG). Plasma samples were used for the determination of asymmetric dimethylarginine (ADMA) and thrombomodulin (TM).There were 40 FMF patients and18 healthy subjects . The median disease duration was 15 (0.6-45) years. Age, sex distribution, waist circumference, body mass index, smoking status and serum lipids were similar between the patients and controls (P > 0.05). The concentrations of high sensitive C-reactive protein (hs-CRP) was significantly higher in FMF patients compared to controls . Asymmetric dimethylarginine (ADMA), osteoprotegerin (OPG) and thrombomodulin (TM) concentrations were significantly lower in the patients\u2019 group compared to those of controls . However, von Willebrand factor (vWF), tissue factor (TF) and tissue plasminogen activator (t-PA) levels were similar between the groups (P > 0.05).In this study we showed that markers related with endothelial injury including ADMA, OPG and TM were significantly down-regulated in FMF patients who were on regular colchicine treatment during attack-free disease state.None declared"} +{"text": "We investigated the influence of IM and p210BCR-ABL on Separase as a potential driver of centrosomal amplification in CML. Short-term cell cultures of p210BCR-ABL-negative , positive and inducible (U937p210BCR-ABL/c6 (Tet-ON)) human cell lines were treated with therapeutic doses of IM and analyzed by qRT-PCR, Western blot analysis and quantitative Separase activity assays. Decreased Separase protein levels were observed in all cells treated with IM in a dose dependent manner. Accordingly, in all p210BCR-ABL-negative cell lines, decreased proteolytic activity of Separase was found. In contrast, p210BCR-ABL-positive cells showed increased Separase proteolytic activity. This activation of Separase was consistent with changes in the expression levels of Separase regulators . Our data suggest that regulation of Separase in IM-treated BCR-ABL-positive cells occurs on both the protein expression and the proteolytic activity levels. Activation of Separase proteolytic activity exclusively in p210BCR-ABL-positive cells during IM treatment may act as a driving force for centrosomal amplification, contributing to genomic instability, clonal evolution and resistance in CML.Separase, an endopeptidase required for the separation of sister-chromatides in mitotic anaphase, triggers centriole disengagement during centrosome duplication. In cancer, separase is frequently overexpressed, pointing to a functional role as an aneuploidy promoter associated with centrosomal amplification and genomic instability. Recently, we have shown that centrosomal amplification and subsequent chromosomal aberrations are a hallmark of chronic myeloid leukemia (CML), increasing from chronic phase (CP) toward blast crisis (BC). Moreover, a functional linkage of p210BCR-ABL tyrosine kinase activity with centrosomal amplification and clonal evolution has been established in long-term cell culture experiments. Unexpectedly, therapeutic doses of imatinib (IM) did not counteract; instead induced similar centrosomal alterations The BCR-ABL tyrosine kinase (TK) formed by the balanced translocation t is the key player in the pathogenesis of chronic myeloid leukemia (CML). Its characteristic deregulated TK activity affects various downstream signaling pathways and results in reprogramming of the prior lineage commitment of hematopoietic stem and early progenitor cells Imatinib (IM) is a selective TK inhibitor (TKI) and presents the current first line treatment for CML Centrosome amplification, in particular, the accumulation of additional centrosomes, is frequently detected in solid and hematological human cancers in vitro study on a CML CP model we have established a functional link of p210BCR-ABL TK activity with centrosome amplification and clonal evolution bcr-abl-negative cell line models and in vivoRecently, we have shown that centrosome amplification is an early event in the transformation process of CML and occurs at the earliest identifiable step in CML development The maintenance of constant centriole numbers in normal proliferating cells is tightly linked to the cell cycle In this study, we set out to analyze the short-term effects of IM on the \u201concogene\u201d separase in BCR-ABL-positive and -negative cells. We employed a panel of human cell lines varying in p210BCR-ABL expression levels that served as models for different stages of CML. We report on separase transcription, protein expression, and Separase proteolytic activity. Furthermore, proteins of the corresponding master regulatory pathways were analyzed. We observed a post-translational activation of Separase proteolytic activity in BCR-ABL-positive cells after treatment with therapeutic IM doses. The potential clinical impact was discussed.bcr-abl-negative malignant cells, K562 and LAMA-84 are well established model systems for CML BC. U937p210BCR-ABL/c6 cells with inducible p210BCR-ABL expression (Tet-On) display one single bcr-abl transgene with moderate p210BCR-ABL expression in the (Doxycycline-) induced state and served as a model of CML CP To analyze the conditional context between p210BCR-ABL, separase activity and IM treatment, we performed cell culture experiments using a panel of six well established human cell lines . Primarybcr-abl-negative cell lines, we performed short-term cell culture experiments to assess the impact of therapeutic doses of IM on expression and proteolytic activity of Separase. Focusing on changes occurring within the first few rounds of the cell cycle after IM administration, our experimental setting should provide insight into the post-translational regulatory mechanisms elapsing before any phenotypic alterations in centrosomal or cytogenetic status may become detectable. Since the proteolytic activity of Separase is regulated in a tight cell cycle-dependent manner, treatment periods were chosen with respect to the respective cell doubling times so that less than two cell cycle rounds were completed under IM treatment (As a continuation of our previous studies on long-term cell cultures reatment and lessreatment .All cell lines were treated with therapeutic doses of IM (range: 0.5 to 10 \u00b5M) as performed in our previous studies bcr-abl-positive cell lines , karyotype and centrosome status, and proliferation rate . Proteinll lines . LAMA-84ll lines revealedSeparase protein level analysis revealed a general overexpression (range 27- to 151-fold) in all BCR-ABL-positive cells when compared to NHDF cells . This isFor all BCR-ABL-negative cells a dose-dependent decrease in Separase protein levels was observed after IM exposure , Table 2Separase proteolytic activity seems tightly linked to protein levels as dose-dependent decreases in proteolytic activity were found in all IM-treated cell lines . RelativOne might assume that the observed effect could be due to IM-related delay in the cell cycle, i.e. decreased proportion of cells entering anaphase, where separase activation occurs. However, FACS analysis of NHDF, UROtsa, HL-60 and U937 cells revealed no significant decreases of G2/M cell proportion under IM treatment . Rather,The corresponding separase transcript levels as analyzed by qRT-PCR , Table 2Analogous experiments were performed with the BCR-ABL-positive cell lines . CompareUnexpectedly, despite the observed decrease in separase transcript and Separase protein levels, increased levels of Separase proteolytic activity were measured . IncreasAs a result, about 25% of the residual Separase protein perform about 130% proteolytic activity in LAMA-84 cells Treatment of BCR-ABL-negative cells with IM strongly pointed to a regulation of Separase protein expression on levels of translation and/or protein stability rather than transcription, as transcript and protein level changes did not coincide upon IM application . This maii) Post-translational regulation on the proteolytic activity level becomes evident when all untreated cell lines under investigation were compared with respect to BCR-ABL TK activity, Separase protein levels and Separase proteolytic activity . While SDecreased Separase protein levels were observed in all investigated cell lines after IM application. This effect is BCR-ABL-independent as it was equally observed in both BCR-ABL-positive and negative cells. Except for BCR-ABL-positive cells, decreased Separase proteolytic activity levels were observed in all p210BCR-ABL-negative cell lines. FACS analyses revealed that the parallel changes in Separase protein and activity levels are not associated with changes in the proportion of G2/M cells. Decreased Separase protein level may be related to decreased translation and/or enhanced degradation of Separase protein. Reduced Separase proteolytic activity may be best explained by a reduced proportion of cells entering mitotic anaphase, where the protease is regularly activated by the anaphase-promoting complex/cyclosome (APC/C) The second level of regulation (proteolytic activity level) was exclusively affected by IM in p210BCR-ABL-positive cells . We observed increased Separase proteolytic activities despite lowered Separase protein levels after IM application. This unexpected activation were analyzed and 6 d . Untreated cells served as controls.Cells were treated with IM in concentrations of 0.25 to 10 \u00b5M for 24 h , 48 h , 2% complete protease inhibitor mix , 1% phosphatase inhibitor cocktails I and II (Sigma-Aldrich). Aliquots of clarified lysates were used for Bradford protein assays . About 50\u2013100 \u00b5g protein per lane were resolved by SDS-PAGE on BIORAD PreCast TGX 4\u201315% gradient gels, transferred to Immobilon-P membrane followed by blocking with 5% dry milk powder for 1 h and immunostaining with the respective primary antibody dilution for 1 to 4 h at RT or over night at 4\u00b0C. Primary antibodies (1\u22361000 dilutions): anti-Separase rabbit polyclonal antibody or mouse monoclonal antibody XJ11-1B12 detecting the 220 KDa full length separase; anti-CyclinB1 monoclonal mouse antibody ; anti-phospho-Separase-S1126 rabbit polyclonal antibody ; anti-phospho-CrkL (Tyr207) polyclonal rabbit antibody ; anti-ABL1 monoclonal mouse antibody ; anti-Securin monoclonal mouse antibody ; anti-PP2A A subunit rabbit mAb (81G5) . Anti-Actin pan Ab-5 mouse monoclonal antibody or anti-Actin (13E5) rabbit mAb HRP Conjugate were diluted 1\u223610000. Signals were visualized with a ChemiDoc\u2122 XRS+ System after secondary antibody staining utilizing SuperSignal\u00aeWest Maximum Sensitivity Substrate . Image acquisition and densitometric analysis was performed using Image Lab\u2122 Software . All values were normalized with Actin as loading control. Image cropping and tonal adjustments across the entire image were performed with Adobe Photoshop CS4 Approximately 1\u00d710PLUS SYBR Green I ready-to-use hot-start PCR mix with Taq DNA polymerase (Roche Diagnostics) diluted with purified water according to the manufacturer's protocol. Relative transcript levels calculated from triplicate measurements were expressed as ratio separase/g6pd.Total RNA was extracted using RNeasy kit and reverse transcribed using Superscript II kit (Gibco/Invitrogen). For quantification of separase (ESPL1) transcript levels, the commercial Hs_ESPL1_1_SG QuantiTect Primer Assay (QIAGEN) was employed according to the instructions (two-step Light Cycler 480 protocol) of the manufacturer. For normalization, the housekeeping gene glucose-6-phosphate dehydrogenase was amplified. QRT-PCR was performed with the Roche LightCycler 480 System, using LC480 DNA Master SYBR Green and the standard LightCycler protocol . In brief, 2 \u00b5l of cDNA were added to 18 \u00b5l of reaction mix containing primers at 0.2 \u00b5M for the separase target and at 0.25 \u00b5M for G6PD in LightCycler\u00ae FastStart DNA MasterSubconfluent cells were harvested and washed in 1\u00d7phosphate buffered saline (PBS), subsequently fixed in icecold 75% ethanol and stained with propidium iodide (10 \u00b5g/ml). DNA content was measured by fluorescence-activated cell sorting (FACS) using a flow cytometer FACScalibur .was performed as described previously Cellular distribution of Separase and centrosomal status was analyzed by immunfluorescence microscopy as described previously About 60 \u00b5g cleared native protein lysate was analyzed in a quantitative fluorogenic assay according to Basu et al. Statistical significance of unpaired data was analyzed by the Student's t-test using the GraphPad Prism software version 5.0 . Values of p<0.05 were considered significant."} +{"text": "It is controversial whether all critically ill patients with RIFLE-F class acute kidney injury (AKI) should receive renal replacement therapy (RRT). We reviewed the outcome of open-heart surgery patients with severe AKI who did not receive RRT.We identified all patients who developed AKI after cardiac surgery during a 4-year period, and obtained baseline characteristics, intraoperative details and in-hospital outcomes. We analyzed physiological and biochemical features at the time of RRT initiation or at peak creatinine if no RRT was provided.2/FIO2 ratio were all significantly higher. Their serum creatinine was also higher . Only three died in-hospital. Detailed review of cause and mode of death was consistent with non-RRT-preventable deaths. In contrast, 27 patients with RIFLE-R or RIFLE-I class received RRT. Compared with RRT-treated RIFLE-F patients, they had a trend towards a more severe presentation and a higher mortality . See Figure We reviewed 1,504 patients. Of these, 137 (9.1%) developed postoperative AKI with 71 meeting RIFLE-F criteria and 23 (32.4% of RIFLE-F cases) not receiving RRT. Compared with RRT-treated RIFLE-F patients, no-RRT patients had lower APACHE III scores, less intraaortic balloon pump requirements, shorter intensive care stay and a trend toward lower mortality. At peak creatinine, their urinary output, arterial pH and PaOAfter cardiac surgery, RRT is typically applied to patients with the most severe clinical presentation irrespective of creatinine levels. A RIFLE score-based trigger for RRT is unlikely to improve patient survival."} +{"text": "CV) and mean primary osteon diameters (POD), were evaluated in a phylogenetic framework and assessed for correlations with other biologically significant variables . Changes in %CV and POD correlated strongly with evolutionary changes in body size . Bone wall thickness tended to be high in early therocephalians and lower in the gracile-limbed baurioids, but showed no general correlation with cross-sectional area or degree of vascularity . Clade-level patterns, however, deviated from previously studied within-lineage patterns. For example, Moschorhinus, one of few therapsid genera to have survived the extinction boundary, demonstrated higher %CV in the Triassic than in the Permian despite its smaller size in the extinction aftermath. Results support a synergistic model of size reductions for Triassic therocephalians, influenced both by within-lineage heterochronic shifts in survivor taxa (as reported in Moschorhinus and the dicynodont Lystrosaurus) and phylogenetically inferred survival of small-bodied taxa that had evolved short growth durations . These findings mirror the multi-causal Lilliput patterns described in marine faunas, but contrast with skeletochronologic studies that suggest slow, prolonged shell secretion over several years in marine benthos. Applications of phylogenetic comparative methods to new histologic data will continue to improve our understanding of the evolutionary dynamics of growth and body size shifts during mass extinctions and recoveries.Therocephalians were a speciose clade of nonmammalian therapsids whose ecological diversity and survivorship of the end-Permian mass extinction offer the potential to investigate the evolution of growth patterns across the clade and their underlying influences on post-extinction body size reductions, or \u2018Lilliput effects\u2019. We present a phylogenetic survey of limb bone histology and growth patterns in therocephalians from the Middle Permian through Middle Triassic of the Karoo Basin, South Africa. Histologic sections were prepared from 80 limb bones representing 11 genera of therocephalians. Histologic indicators of skeletal growth, including cortical vascularity (% Lystrosaurus Assemblage Zone in the Karoo Basin of South Africa (ca. 252.3 Ma), but growth dynamics underlying these patterns are not fully understood. Therocephalians represent an exemplary clade of nonmammalian therapsids that thrived from the Middle Permian to Middle Triassic, and survived the end-Permian extinction as important components of Triassic survivor and recovery faunas in the Karoo Basin have long ghost lineages extending into the Permian, indicating that they too survived the extinction but lack a Permian record within the depositional basin and in fast-growing portions of the skeleton . The abundance of dicynodont fossils in Permian and Triassic rocks and recent advances in their systematic relationships have permitted more detailed comparisons of growth patterns in this diverse subclade (Lystrosaurus), and determinate growth patterns with peripheral rest lines and systematic cortical remodeling in large kannemeyeriiforms . Howeversubclade . Phylogeriiforms .Notosollasia\u2019 (= Theriognathus). Given the comparatively more vascularized cortical bone in the radius of the whaitsiid (1969: plate IV), Ricql\u00e8s suggested that therocephalians might have exhibited accelerated growth rates later in their evolutionary history, paralleling the aforementioned temporal pattern of increasing growth rates in some dicynodonts. More recently, Pristerognathus\u2019), in addition to other therapsid material, and argued for similar \u2018flexible\u2019 growth patterns in gorgonopsians, basal therocephalians, and most early cynodonts. The authors suggested that more rigorous taxonomic sampling would better substantiate parallel trends toward a loss in developmental plasticity and acceleration of growth rates as in dicynodonts. Inadequate sampling of eutherocephalians before and after the end-Permian mass extinction limits our understanding of evolutionary patterns in therapsid histomorphology and skeletal growth during this important geologic transition.The diversity of growth patterns in other nonmammalian therapsid groups, as well as their phylogenetic and temporal distributions, is incompletely known. A body of literature on nonmammalian cynodont histology has accrued in recent years e.g., , but samTetracynodon: Moschorhinus: Although eutherocephalians have not been sampled histologically for such comparisons, recent revisions to Permo-Triassic boundary-crossing taxa have necessitated cursory descriptions of eutherocephalian histology for its ontogenetic and paleobiological implications or clade specific histomorphology. Specimens that were semi-articulated and included diagnostic cranial material were preferred for accuracy of taxonomic identifications. Some specimens were not diagnosable to genus, but were resolved to their respective higher taxon as in the case of five indeterminate scylacosaurids described here. Scylacosaurids are generally difficult to identify unless a complete and accurate antecanine tooth count can be made, and some authors have suggested that the diversity of scylacosaurids is over-split because variations in tooth count may be ontogenetically variable e.g., . The samomphodon , plus adomphodon . Moschorlsewhere , but datanalyses .Bone tissue texture exhibits marked variation in therocephalians and other therapsids, varying from highly organized and lamellar to disorganized and woven. Only a few recent studies have integrated qualitative and quantitative assessments of tissue texture and vascular proxies of growth in Permo-Triassic therapsids . GeneralCV, the relative area of the cortex that is occupied by porous, vascular spaces) follows POD) by measuring in microns the transverse (or minimum) widths of 15 primary osteons visible in the subsampled regions and averaging them across all regions within a given midshaft cross-section.For quantitative histomorphometric analysis, two vascular proxies of skeletal growth were selected: cortical vascularity and mean primary osteon diameter. These proxies were selected in order to evaluate the extent to which histological correlatives of growth varied across phylogeny, and whether their evolution was tied to body size or other biological factors. Vascular proxies have offered useful indicators of skeletal growth in extant and extinct tetrapods , and theK CV, POD, RBT) and midshaft cross-sectional area were recorded for each sectioned limb bone. The data were organized into propodial, epipodial, and pooled subsets to control for the effects of increased variance from pooling limb bones of different types . For each data partition, Pearson\u2019s product-moment correlation tests were performed between histomorphometric variables and the natural log of midshaft cross-sectional area. Vascular growth proxies were also tested for correlations with bone robusticity independent of size, and with each other to assess whether %CV and POD provided comparable estimates of vascularization.We performed a series of correlation tests in order to evaluate the extent to which variations in vascular growth proxies were dependent upon size and robusticity, which bear a strong influence on many aspects of organismal biology . For insCV%, POD, RBT and the natural log of midshaft area of the propodials and epipodials separately (limb bones were not pooled for independent contrasts). Second, ancestral character states (estimated using squared-change parsimony) were checked for the assumption of Brownian motion evolution that governs independent contrasts . By contrast, some smaller-bodied taxa in both the Permian and Triassic tended to incorporate more parallel-fibered bone or showed evidence of increased parallel-fibered and lamellar bone deposition with less vasculature toward the outer cortex, indicative of growth attenuation and attainment of somatic maturity in some elements . This condition, however, was lost in baurioids, which were instead characterized by a much thinner cortical bone wall and more slender limb bones.Due to space limitations, the present description is accompanied by a more detailed account in the maturity . This paPermian basal therocephalians and eutherocephalians ; 3A, 3B Lycosuchus, scylacosaurids, Moschorhinus)] .Results of raw and phylogeny-corrected correlation tests are presented in rhinus)] ; Table 2CV and POD. Eutherocephalians and particularly baurioids demonstrated a noteworthy pattern in which reconstructed ancestor-descendant size reductions of Late Permian and Triassic lineages were associated with decreases in the average level of tissue vascularization. Patterns in limb bone robusticity were less clear. As in the analyses of the raw data, phylogeny-corrected correlations between RBT and size or growth proxies were non-significant.Results of phylogeny-corrected correlations further indicated a strong relationship between size and some histometrics (but not with limb bone robusticity) . EvolutiLycosuchus, scylacosaurids, Moschorhinus). Previous tests incorporating a large histologic sample of dicynodonts and other therapsids found similar correlations between raw vascular growth proxies and size (estimated from skull lengths), although tests on a subset of dicynodonts were only marginally significant and non-significant when independent contrasts were evaluated (CV and POD) even when corrected for phylogeny. Prior analyses implementing phylogeny-independent contrasts on dicynodonts were unable to identify similar patterns, despite a significant correlation between the raw data (RBT) in dicynodonts also showed no clear association with size or degree of vascularization as in the present study, even though a positive association with size was discovered when corrected for phylogeny. No correlation was observed between bone robusticity (RBT) and size or degree of vascularization in therocephalians (raw or phylogeny-corrected), suggesting that size and rate of growth may have had limited influence over bone robusticity as compared to other aspects of organismal biology such as mechanical regime .Previous interpretations of growth patterns in early therocephalians were based on limited information from incomplete specimens . Additio5 \u00b5m/day . It is niognathus and someCynognathus and some large gorgonopsians showed modest cortical vascularity and smaller mean primary osteon diameters than in basal lycosuchids, scylacosaurids or akidnognathids, but the baurioids had the least vascularized bone tissues have indicated slow growth rates with frequent interruptions to growth in earliest Triassic shells by the early-Late Permian. Medium-to-large Late Permian dicynodonts continued to show patterns of increased cortical vascularity, especially within the Permo-Triassic boundary-crossing genus Lystrosaurus, which demonstrated some of the highest levels of tissue vascularity (\u223c20%). Triassic specimens of Lystrosaurus showed relatively higher vascularity and fewer growth marks. In the therocephalian Moschorhinus, the only large therapsid predator to cross the Permian-Triassic boundary, within-lineage size reduction was associated with the maintenance of rapid but attenuating growth over a short period .New data on growth patterns in nonmammalian therapsids, as well as other Permo-Triassic tetrapods, offer the potential to further evaluate patterns of selectivity during mass extinctions . Bone hit period . This paLystrosaurus and Moschorhinus is suggestive of within-lineage heterochronic shifts, clade-level patterns introduce a more complex explanation for observed body size reductions in therocephalians. In particular, body size reductions occurred early during the evolution of eutherocephalians and were associated with a lesser degree of cortical vascularity in medium-to-small-bodied Permian and Triassic forms . The two major subclades of therocephalians that persisted into the earliest Triassic, Akidnognathidae and Baurioidea, revealed distinctly different growth patterns from each other as interpreted through their tissue texture and degree of vascularity. Their histology suggests a bimodality of life history strategies in earliest Triassic therocephalians: small-to-medium akidnognathids with well-vascularized (fast-growing) bone and smaller-bodied baurioids with less vascularized (slower-growing) bone. However, both groups shared a reduced number of growth marks compared to their Permian relatives in addition to their generally smaller sizes. This nuance is not evident from the quantitative analysis based on vascular proxies of growth rate alone, but is evident from growth mark counts in surveyed specimens. Permian theriodonts that have been sampled histologically, including some gorgonopsians, the cynodont Procynosuchus, basal therocephalians, Permian (but not Triassic) specimens of Moschorhinus, hofmeyriids, and Theriognathus, typically showed evidence of prolonged, multi-year growth often to larger body sizes, a pattern that is not represented in earliest Triassic therapsids sampled to date . For example, the degree of development of the secondary palate has been linked anecdotally to new environmental conditions with the onset of the Early Triassic, particularly a dramatic decline in atmospheric pOTriassic . HoweverTriassic . SimilarTriassic . No cleaTriassic .Tetracynodon; Scaloposaurus, is distinguished from other small Triassic baurioids in its retention of the parietal foramen and pineal body).Similar studies addressing the possible effects of hypoxia on cortical tissue vascularity have found few differences between Permian and Triassic therapsids, instead demonstrating that highly vascularized tissues with enlarged canals evolved early in bidentalian dicynodonts . In therLystrosaurus and Moschorhinus). A synergistic combination of local within-lineage effects and differential extinction patterns strongly influenced Triassic Lilliput faunas, weakening the hypothesized role of rapid adaptive evolution of new small-bodied forms. Similar within-lineage size decreases and size selective extinctions contributed strongly to Lilliput patterns in marine gastropods, foraminifera, and brachiopods, although all three mechanisms have been invoked to explain Early Triassic foraminifera size distributions globally ecological removal of large-bodied taxa having prolonged, multi-year growth patterns; (2) cladistically inferred survival of small-bodied taxa with modest skeletal apposition rates, but truncated growth durations ; and (3) within-lineage shifts in growth patterns observed in boundary-crossing genera in the Karoo . Future applications of phylogenetic comparative methods to studies of body size and growth during the Permo-Triassic will enhance our understanding of interplay between macroevolution and extinctions, and will identify areas of phylogeny that correspond to shifts in trait evolution that conferred success on lineages.Although some effects on size and growth are observable in Triassic therocephalians , it is important to note that much of the diversity observed in the earliest Triassic 10.7717/peerj.325/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.325/supp-2Supplemental Information 2(A) SAM-PK-9084, radius midshaft, cortical fibrolamellar bone viewed at high magnification (crossed-nicols with wave plate). (B) Same as (A) viewed under normal polarized light without wave plate. (C) SAM-PK-9084, ulna midshaft, cortex showing growth marks and well-vascularized fibrolamellar bone viewed at low magnification (non-polarized light). Note the thick bone wall and inner coarse cancellous structure. (D) SAM-PK-K9012, femur midshaft, dorsal cortex showing subplexiform fibrolamellar bone viewed under non-polarized light. (E) SAM-PK-K9012, femur midshaft, posterior region of cortex showing three bands of parallel-fibered bone (blue bands denoted by arrows) representing possible growth marks, viewed at low magnification (crossed-nicols with wave plate). Arrows denote growth marks. Abbreviations: po, primary osteon.Click here for additional data file.10.7717/peerj.325/supp-3Supplemental Information 3Glanosuchus macrops, BP/1/6228, ulna midshaft, cortical fibrolamellar bone viewed at low magnification (crossed-nicols with wave plate). (B) Scylacosauridae indet., SAM-PK-5018, fibula midshaft close-up of secondary osteon in deep cortex (crossed-nicols with wave plate). (C) Scylacosauridae indet., CGS R300, humerus midshaft cortex viewed at low magnification showing growth marks (crossed-nicols with wave plate). (D) Scylacosauridae indet., BP/1/5576, ulna midshaft cortex viewed at low magnification showing growth marks (crossed-nicols with wave plate). (E) Scylacosauridae indet., BP/1/5587, ulna midshaft cortex viewed at low magnification showing growth marks (crossed-nicols with wave plate). Arrows denote growth marks. Abbreviations: cl, cement line; Hc, Haversian canal; po, primary osteon.(A) Click here for additional data file.10.7717/peerj.325/supp-4Supplemental Information 4Moschorhinus (SAM-PK-K118) humerus midshaft shown at same scale as (C) for comparison (crossed-nicols with wave plate). Note the densely packed reticular and radial primary osteons and globular osteocyte lacunae. (E) SAM-PK-K10617, femur midshaft, cortical fibrolamellar bone viewed at low magnification (crossed-nicols with wave plate). (F) SAM-PK-K10617, femur midshaft, close-up of primary osteons and interstitial bone matrix . Arrows denote growth marks. Abbreviations: pfb, parallel-fibered bone; po, primary osteon.(A) NMQR 3605, cross-sectional profile of humerus midshaft viewed at low magnification (crossed-nicols with wave plate). Note the occluded medullary region and relatively thick cortical bone wall. (B) NMQR 3605, humerus midshaft, cortical fibrolamellar bone showing large primary osteons preceding a thin zone of parallel-fibered bone near a LAG (crossed-nicols with wave plate). (C) NMQR 3605, humerus midshaft, cortex showing thick zone of reticular fibrolamellar bone followed by parallel-fibered bone and a LAG (crossed-nicols with wave plate). (D) Triassic Click here for additional data file.10.7717/peerj.325/supp-5Supplemental Information 5(A) BP/1/4404, cortex of humerus midshaft viewed at low magnification (crossed-nicols with wave plate). (B) BP/1/4404, cross-sectional profile of radius midshaft viewed at low magnification (crossed-nicols with wave plate). (C) Same as (B) close-up of cortex showing growth marks and lamellar bone in outer cortex (crossed-nicols with wave plate). (D) Same as (B) close-up of cortex showing longitudinal primary osteons and outer lamellar bone (crossed-nicols with wave plate). (E) BP/1/4404, ulna midshaft cross-section viewed at low magnification (crossed-nicols with wave plate). (F) BP/1/4404, ulna midshaft cortex viewed at high magnification, showing sharp transition to lamellar bone in outer cortex (crossed-nicols with wave plate). Brackets denote outer zone of lamellar bone with simple canals, indicating marked decrease in bone apposition. Abbreviations: pfb, parallel-fibered bone; po, primary osteon.Click here for additional data file.10.7717/peerj.325/supp-6Supplemental Information 6(A) SAM-PK-K6511, humerus midshaft cross-section viewed at low magnification (crossed-nicols with wave plate). (B) SAM-PK-K6511, radius midshaft cortex (crossed-nicols with wave plate). (C) SAM-PK-K6511, ulna midshaft cortex (crossed-nicols with wave plate). (D) SAM-PK-K6511, femur midshaft cortex showing extensive parallel-fibered and lamellar bone . (E) SAM-PK-K6511, tibia midshaft cross-section viewed at low magnification (non-polarized light). (F) SAM-PK-K6511, fibula midshaft cross-section viewed at low magnification (non-polarized light). Bracket indicates avascular outer zone. Abbreviations: pfb, parallel-fibered bone; po, primary osteon.Click here for additional data file.10.7717/peerj.325/supp-7Supplemental Information 7(A) NMQR 3375, femur midshaft cross-section viewed at low magnification (non-polarized light). (B) NMQR 3375, femur midshaft cortical bone viewed at medium magnification showing growth marks (arrows) (non-polarized light). (C) same as \u2018B,\u2019 viewed at high magnification with wave plate, showing close-up of outer zone of well-vascularized fibrolamellar bone (bracket). (D) BP/1/719, femur midshaft cortex (crossed-nicols with wave plate). Arrows denote growth marks. Abbreviations: flb, fibrolamellar bone; rc, radial canals.Click here for additional data file.10.7717/peerj.325/supp-8Supplemental Information 8(A) SAM-PK-K8659, humerus midshaft cross-section viewed at low magnification . (B) SAM-PK-K8659, radius midshaft cortex and inner cancellous bony scaffold (crossed-nicols with wave plate). (C) SAM-PK-K8659, tibia midshaft cortex and inner cancellous bony scaffold (crossed-nicols with wave plate). (D) SAM-PK-K8659, femur midshaft showing inner cancellous bone and outer bone compacta with a woven-fibered matrix (crossed-nicols with wave plate). (E) SAM-PK-K10423, femur distal shaft cortex (crossed-nicols with wave plate). (F) SAM-PK-K10423, fibula proximal shaft cortex (crossed-nicols with wave plate). (G) BP/1/75, humerus midshaft cortex (crossed-nicols with wave plate). (H) BP/1/4092, midshaft cross-section of large humerus viewed at low magnification (crossed-nicols with wave plate). (I) BP/1/4092, radius close-up showing growth mark (annulus) in outer cortex (non-polarized light). Arrows denote growth marks. Abbreviations: nc, nutrient canal; pfb, parallel-fibered bone; spc, subplexiform canals; wb, woven-fibered bone.Click here for additional data file.10.7717/peerj.325/supp-9Supplemental Information 9(A) NMQR 3745, humerus midshaft cross-sectional profile viewed at low magnification (non-polarized light). (B) UCMP 78396, humerus midshaft cortex . Bracket denotes outer zone of parallel-fibered and lamellar bone. (C) UCMP 78396, humerus midshaft cortex showing outer line of arrested growth (demarcated by cement line at arrow) (non-polarized light). (D) UCMP 78395, radius midshaft cortex and perimedullary region viewed at low magnification (crossed-nicols with wave plate). (E) UCMP 78396, femur midshaft cortex and perimedullary region showing woven- and parallel-fibered bone (crossed-nicols with wave plate). (F) UCMP 78396, femur midshaft cortex close-up . (G) UCMP 78396, fibula midshaft cortical bone packed with longitudinal primary osteons (non-polarized light). Arrows denote growth marks. Abbreviations: elb, endosteal lamellar bone; pfb, parallel-fibered bone; po, primary osteon; Sf, Sharpey\u2019s fibers; wb, woven-fibered bone.Click here for additional data file.10.7717/peerj.325/supp-10Supplemental Information 10(A) Humerus midshaft cross-section showing fibrolamellar bone deposition followed by a thin collar of lamellar bone in the subperiosteal region . (B) Humerus midshaft cortex close-up showing longitudinal primary osteons and a large nutrient canal within a woven-fibered bone matrix (crossed-nicols with wave plate). Abbreviations: lb, lamellar bone; nc, nutrient canal; po, primary osteon.Click here for additional data file.10.7717/peerj.325/supp-11Supplemental Information 11(A) NMQR 3605, humerus midshaft cross-section viewed at low magnification (crossed-nicols with wave plate). Note local histovariation in the arrangements of vascular canals. (B) NMQR 3605, femur midshaft cross-sectional profile viewed at low magnification (crossed-nicols with wave plate). (C) Close-up of \u2018B\u2019 showing abundant reticular canals in cortex and lack of growth marks. (D) NMQR 3605, tibia midshaft cross-sectional profile viewed at low magnification (crossed-nicols with wave plate). (E) NMQR 3605, fibula midshaft cortex (crossed-nicols with wave plate). Abbreviations: cc, circular canal; rc, reticular canal; po, primary osteon; Sf, Sharpey\u2019s fibers.Click here for additional data file."} +{"text": "Our article did not acknowledge that conflicting data have been published examining the ability of rat brain endothelial cells (EC) in presenting antigen to antigen-specific T cells, although the general consensus is that rat brain EC are able to present antigen but are poor inducers of T cell proliferation. This weak antigen presenting capacity may be due to species differences between the rodent and the human model used in this article. We would like to acknowledge this discrepancy as well as the previous publications below:Pryce G, Male D, Sedgwick J (1989) Antigen presentation in brain: brain endothelial cells are poor stimulators of T-cell proliferation. Immunology 66: 207-212.Fabry Z, Waldschmidt MM, Moore SA, Hart MN (1990) Antigen presentation by brain microvessel smooth muscle and endothelium. J Neuroimmunol 28: 63-71.Risau W, Engelhardt B, Wekerle H (1990) Immune function of the blood-brain barrier: incomplete presentation of protein (auto-)antigens by rat brain microvascular endothelium in vitro. J Cell Biol 110: 1757-1766."} +{"text": "Although the use of recombinant hepatitis B virus surface (HBsAg) protein vaccine has successfully reduced global hepatitis B infection, there are still a number of vaccine recipients who do not develop detectable antibody responses. Various novel vaccination approaches, including DNA vaccines, have been used to further improve the coverage of vaccine protection. Our previous studies demonstrated that HBsAg-based DNA vaccines could induce both humoral and CMI responses in experimental animal models. However, one form of the the HBsAg antigen, the large S antigen (HBs-L), expressed by DNA vaccine, was not sufficiently immunogenic in eliciting antibody responses. In the current study, we produced a modified large S antigen DNA vaccine, HBs-L(T), which has a truncated N-terminal sequence in the pre-S1 region. Compared to the original HBs-L DNA vaccine, the HBs-L(T) DNA vaccine improved secretion in cultured mammalian cells and generated significantly enhanced HBsAg-specific antibody and B cell responses. Furthermore, this improved HBsL DNA vaccine, along with other HBsAg-expressing DNA vaccines, was able to maintain predominantly Th1 type antibody responses while recombinant HBsAg protein vaccines produced in either yeast or CHO cells elicited mostly Th2 type antibody responses. Our data indicate that HBsAg DNA vaccines with improved immunogenicity offer a useful alternative choice to recombinant protein-based HBV vaccines, particularly for therapeutic purposes against chronic hepatitis infection where immune tolerance led to poor antibody responses to S antigens. Hepatitis B virus (HBV), a member of the Hepadnavirus family, is the main pathogen for human viral hepatitis; chronic infection can lead to liver cirrhosis and hepatocellular carcinoma The HBV vaccine has proven effective in preventing HBV infection. The hepatitis surface protein antigen (HBsAg) is the target for protective antibody responses for HBV vaccines HBsAg is composed of three related co-carboxyl terminal surface proteins created by different translational initiation sites in the viral S gene: the small (HBs-S), middle (HBs-M), and large (HBs-L) proteins. The HBs-S, consisting of 226 amino acid (aa), is a common region of the three HBsAg; HBs-M has an addition of a pre-S2 domain (55 aa) to the N-terminus of HBs-S; and the HBs-L possess another N-terminal addition of a pre-S1 domain . Although the HBs-S based recombinant protein vaccination has been very successful, HBsAg mutations capable of escaping diagnostic detection and antibodies elicited by vaccination have been reported. Universal vaccination has actually accelerated wider epitope range of vaccine-resistant mutants Over the past few years, our group has investigated the relative immunogenicity among the three natural forms of HBV S protein. Our results have demonstrated that the DNA vaccine expressing the middle antigen (HBs-M) induced the highest levels of antibody and CMI responses in mice. Our results also showed that the HBs-S DNA vaccine was also highly immunogenic. However, DNA vaccine expressing the large antigen (HBs-L) was not very immunogenic but the mechanism is not clear In our previous study, we found that the DNA vaccine expressing the full length HBsAg large antigen (HBs-L) was not immunogenic in eliciting either antibody or cell-mediated immune (CMI) responses in mice Expression of the HBs-L(T) DNA vaccine was examined in transiently transfected 293T cells. Both cell lysate and supernatant collected at 72 hours after transfection were measured by ELISA using monoclonal antibodies that are either specific for the small surface antigen anti-S, or specird DNA immunization were measured by ELISA against two different types of S antigen preparations: 1) the commercial HBsAg produced from HBV infected patient plasma (The relative immunogenicity of DNA vaccines expressing HBs-L and HBs-L(T) were examined in Balb/C mice by electroporation as previous described t plasma , and 2) t plasma . The rest plasma . While trd DNA immunization and B cell ELISPOT was performed to investigate HBsAg-specific antibody secreting cells (ASC) in immunized mice against the commercial HBsAg DNA vaccines were the result of a difference at the B cell level. Mouse splenocytes were harvested at 4 weeks after the 3al HBsAg . ConsistAt the same time, we examined whether there is any difference in T cell responses between HBs-L and HBs-L(T) DNA vaccines. IFN-\u03b3 and IL-4 were used in this study, representing Th1 and Th2 T cell immune responses, respectively. ELISPOTs and intracellular cytokine straining studies were conducted with a well-characterized T-cell epitope in S antigen The immunogenicity of HBV DNA vaccines has been well established in small animals including previous studies from our group showing the high immunogenicity of HBs-S and HBs-M antigens We next examined the relative antibody responses elicited by different designs of HBs DNA vaccines and compared these responses to those elicited by two HBV protein-based vaccines commonly used in China. One commercially available HBV vaccine in China was produced in CHO cells and the other one was produced in a yeast expression system . The expression of DNA vaccines expressing HBs-L(T), HBs-M, and HBs-S were examined by Western blot using 293T cell samples transiently transfected with these HBsAg DNA vaccines. CHO-HBs and Yeast-HBs vaccines were also included in the analysis. Anti-S mAb was able to recognize HBs-L(T), HBs-M, and HBs-S proteins with expected molecular weights, along with CHO-HBs and Yeast-HBs, which had the expected size of a small antigen (\u223c27 kD) . In contnd immunization and S antigen-specific antibody responses were measured against either the commercial HBsAg produced from HBV infected patient plasma or the HBs-M protein produced by transiently transfected 293T cell supernatant, as previous described.Balb/C mice were immunized individually with each of the three DNA and two recombinant protein vaccines at Weeks 0 and 4. For HBs-L(T), HBs-M, or HBs-S DNA vaccines, eletroporation was used to deliver 100 \u00b5g DNA vaccines at each immunization while CHO-HBs and Yeast-HBs vaccines were used at 1/10 of human dose by intramuscular needle injection. Immunized mouse sera were collected at 2 weeks after the 2The HBs-S DNA vaccine and two recombinant protein vaccines (CHO-HBs and Yeast-HBs) elicited high level antibody responses against commercial HBsAg , which were significantly higher than those induced by HBs-L(T) or HBs-M DNA vaccines (p<0.05) . InteresThe quality of antibody responses elicited by HBs DNA vaccines was further analyzed. The HBsAg-specific IgG1 and IgG2a isotypes in animal immune sera were determined by ELISA . The recin vivo and in vitroThe small subunit of HBsAg (HBs-S) has been used as the HBV vaccine immunogen for most of the current recombinant protein-based HBV vaccines on the global market. Although, overall, such HBV vaccines are immunogenic, there are \u201cnon-responders\u201d to this type of vaccine DNA vaccination is a useful tool to test the immunogenicity of different immunogen designs by antigen engineering. In our previous studies, we investigated the antibody, B cell, and T cell responses in mice receiving DNA vaccines expressing either the large antigen (HBs-L), middle antigen (HBs-M) or small antigen (HBs-S) to compare three forms of HBsAg The results demonstrated that the N-terminal truncated HBs-L(T) DNA vaccine could express the antigen in both supernatant and the cell lysate and were detectable by both anti-S or anti-preS1 monoclonal antibodies, while the original HBs-L DNA could only express the antigen in the cell lysate. The truncated large antigen, HBs-L(T), had lower reactivity to the preS1 mAb compared to the wild type HBs-L while both HBs-L and HBs-L(T) had similar reactivity to anti-S mAb, confirming that a deletion in the preS1 region may have affected the epitope recognized by the anti-preS1 mAb. The HBs-L(T) DNA vaccine generated significantly higher levels of HBs-specific antibody responses in mice compared with the wild type HBs-L DNA vaccine, against two different antigens: 1) HBsAg purified from HBV infected patient plasma, and 2) the middle antigen expressed by the HBs-M DNA vaccine. More significantly, HBs-L(T) DNA vaccine elicited higher levels of HBsAg-specific antibody secreting cells, which may account for the higher levels of overall HBsAg-specific antibody responses.\u03b3 specific T-cell immune responses, as shown by ELISPOT, and IFN-g specific CD8+ T cell responses, as shown by intracellular staining (ICS), were similar in the HBs-L immunized group when compared to levels observed in the HBs-L DNA vaccine group. Slightly higher HBs-specific IL-4 T cell responses were observed in the HBs-L(T) DNA vaccine group when compared with the HBs-L group, however, this difference was not statistically significant.At the same time, the HBs-L(T) DNA vaccine was able to maintain its immunogenicity for eliciting T-cell immune responses as the original HBs-L DNA vaccine. Overall IFN-The above findings imply that the N-terminal sequence-truncated HBs-L(T) antigen enhanced HBs-specific antibody responses and possible Th2 cytokine production by T cells without compromising production of Th1 cytokines, including IFN-\u03b3 production from CD8+ T cells. Our results support the use of the HBs-L(T) DNA vaccine as a candidate HBV DNA vaccine since it covers the preS1, preS2 and S antigen regions in one single DNA vaccine.Once we have established the immunogenicity of HBV large, middle, and small antigens, as tested by DNA vaccination, a side-by-side comparison was conducted with recombinant protein-based HBV vaccines. In the current study, two commercially available recombinant protein HBV vaccines, HBV vaccines expressed by the yeast expression system (Yeast-HBs) and HBV vaccines expressed by the CHO expression system (CHO-HBs), were tested for their immunogenicity in the mouse and compared against the immunogenicity observed for the three forms of HBs DNA vaccines (HBs-L(T), HBs-M and HBs-M).Although both yeast-HBs and CHO-HBs and different forms of HBs DNA vaccines induced positive HBs-specific antibody responses against either HBsAg purified from HBV patient plasma or HBs-M expressed by DNA vaccine, differences were observed. Antibody responses induced by recombinant protein-based yeast-HBs and CHO-HBs vaccines were predominantly Th2 type antibody responses while the DNA vaccines mainly induced Th1 type antibody responses. Literature has suggested that the Th1 rather than the Th2 antibody responses might be more relevant to protective immunity against viral infections, such as HSV-2 CTGCAG PstIATGAACCCCCTGGGCTTCTTC 3\u2032) and HBV L(T)-Adr-opt-2 (5\u2032GAGCTCGGATCCBamHITCAGATGTACACCCAC 3\u2032), and then subcloned into pSW3891. Each individual DNA vaccine plasmid was prepared in large amounts from Escherichia coli (HB101 strain) with a Mega purification kit for both in vitro transfection and in vivo animal immunization studies.The codon optimized large HBsAg (HBs-L) gene coding for the large HBsAg protein, including pre-S1, pre-S2, and S domains, was chemically synthesized by Geneart , with added restriction enzyme sites of PstI and BamHI for subcloning purposes immediately upstream of the start codon and downstream of the stop codon, respectively The expression of the HBs DNA vaccine constructs was examined by transient transfection of 293T cells as previously reported The S antigens produced in transiently transfected 293T cells were analyzed by Western blot. Samples were subjected to denaturing SDS-PAGE and blotted onto PVDF membrane (BioRad). Blocking was done with 5% non-fat dry milk with 0.1% Tween-20 in PBS. Commercial anti-HBs-S mouse monoclonal antibody 1023 and anti-PreS1 monoclonal antibody AP1 from Santa Cruz Biotechnology, Inc. were used as detecting antibodies at 1\u2236500 dilution. The membranes were washed with blocking buffer and reacted with AP-conjugated goat anti-rabbit (Tropix) at a 1\u22365000 dilution 6 cells/ml in R10 medium .BALB/c mice (6\u20138 weeks old), purchased from Shanghai Animal Center, Chinese Academy of Science, were used for immunogenicity studies. The mice were housed in the Department of Animal Medicine at the Nanjing Medical University in accordance with approved protocol. Animals were sedated with Ketamine (0.1 mg/g of body weight) for DNA immunizations. Electroporation was used for DNA vaccine delivery as previous described This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, USA. The protocol was approved by the review of Institutional Animal Use Committee at Nanjing Medical University, China.2SO4, and the plates were read at an OD of 450 nm. The end titration titer was determined as the highest serum dilution that has an OD reading above twice of that from the negative control serum.An ELISA was conducted to measure HBs-specific antibody responses in immunized mice, as previously described HBs-specific antibody secreting cells (ASCs) in immune mouse splenocytes were detected, as previous described Gamma interferon (IFN-\u03b3) and interleukin 4 (IL-4) ELISPOT assays were performed to detect the HBs peptide-specific T cell responses in mouse splenocytes, as previously described The freshly isolated splenocytes (106 cells in 200 \u00b5l) were cultured at 37\u00b0C for 5 h in 96-well round-bottom plates in completed R10 medium supplemented with 0.5 mg/ml Brefelding A. Stimulatory conditions included 5 \u00b5g/ml of the given HBs peptide or 4 \u00b5g/ml of HBsAg, as described above Student's t-test was used to analyze the differences in antibody, ASC, ICS, and T-cell responses between different animal immunization groups.Figure S1Western blot analysis of HBs-L(T) expression using various anti-S antibodies. A: anti-HBs-M mouse sera; B: anti-HBs mAb; and C: anti-Pre-S1 mAb, respectively. Transfected 293T cell lysates of HBs-L(T) DNA vaccine or empty vector were loaded as indicated.(TIF)Click here for additional data file."} +{"text": "ARTEMIN (ARTN) is an estrogen regulated growth factor, the expression of which promotes resistance to antiestrogen therapies and predicts poorer survival outcome of patients with estrogen receptor (ER) positive mammary carcinoma (ER+MC) treated with tamoxifen. ARTN is also expressed in ER negative mammary carcinoma (ER-MC). Herein, we determined the role of ARTN in ER-MC and defined the mechanism of action producing poor patient prognosis.in vitro measures of oncogenicity, including the expression of TWIST1 messenger RNA (mRNA) and protein. In vitro results were correlated to xenograft studies in immunodeficient mice. Co-expression of ARTN and TWIST1 and their association to poor survival outcome were examined in a cohort of patients with ER-MC. Pathway analysis was performed by pharmacological inhibition of phosphorylation of AKT (pAKT-Ser 473) or modulation of TWIST1 expression.We modulated the expression of ARTN in two ER- mammary carcinoma cell lines (BT549 and MDA-MB-231) by forced expression or small interfering RNA (siRNA) mediated depletion. The effects of modulation of ARTN expression were examined by various ARTN expression resulted in ER-MC cells with enhanced mesenchymal characteristics, including increased invasion and a gene expression profile consistent with enhanced mesenchymal phenotype. ARTN stimulated ER-MC cell anchorage independent and 3D matrigel growth, endothelial cell adhesion and transmigration of ER-MC cells through an endothelial cell barrier. Forced expression of ARTN produced a larger, locally invasive tumour mass with tumour emboli that produced distant metastasis. ARTN regulated TWIST1 expression in ER-MC cells and ARTN expression was significantly correlated to TWIST1 expression in a panel of mammary carcinoma cell lines and in a cohort of patients with ER-MC. Low expression of both ARTN and TWIST1 predicted 100% relapse free and overall survival in patients with ER-MC, whereas high expression of both ARTN and TWIST1 was associated with a poor survival outcome. ARTN stimulated an increase in TWIST1 expression via increased AKT activity. siRNA mediated depletion of TWIST1 abrogated ARTN stimulated cellular behaviour associated with metastasis, and forced expression of TWIST1 abrogated the functional effects of ARTN depletion.ARTN and TWIST1 synergize to produce a worse outcome in ER-MC and combined inhibition of ARTN and phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) may therefore provide a novel therapeutic strategy in this subtype of mammary carcinoma. SNAI1 and TWIST1, and cancer stem cell-like features [Progression of mammary carcinoma is a complex process that involves aberrant regulation of multiple signalling pathways . Determifeatures ,5. Compafeatures . Clinicofeatures . IdentifARTN is an estrogen-regulated gene and it has been demonstrated that ARTN reduces the efficacy of anti-estrogens in ER+MC [ARTEMIN (ARTN) is a member of the glial-cell line-derived neurotrophic factor (GDNF) family of ligands . ARTN hain ER+MC . Furtherin ER+MC .TWIST1 belongs to the family of basic helix-loop-helix transcription factors originally found to modulate the expression of various target genes through canonical E-box responsive elements ,13. IncrProgression from estrogen dependence to estrogen independence (anti-estrogen resistance) in mammary carcinoma involves the altered expression of one or more estrogen-regulated gene networks ,20. In aWe report herein that ARTN stimulates oncogenicity and metastasis of ER-MC cells via expression of TWIST1 and that functional interaction between ARTN and TWIST1 promote a worse survival outcome in ER-MC.Cell lines used in this study were obtained from the American Type Culture Collection and cultured as recommended. BT549-ARTN and BT549-VEC cells have been previously described . MDA-MB-Tetramethylrhodamine isothiocyanate phalloidin was purchased from Sigma . AKT inhibitor IV was purchased from Calbiochem .SNAI2 primers used for qPCR were; forward 5'-TGTTTGCAAGATCTGCGGCAAG-3' and reverse 5'-TGACCTGTCTGCAAATGCTC-3'. SPARC primers used for qPCR were forward 5'-AGCACCCCATTGACGGGTA-3' and reverse: 5'-GGTCACAGGTCTCGAAAAAGC-3'. For the in vivo xenograft, the following primers were used: hHPRT forward 5'-TTCCTTGGTCAGGCAGTATAATCC-3' and reverse 5'-AGTCTGGCTTATATCCAACACTTCG-3' mgapdh forward 5'-CTCACTCAAGATTGTCAGCAATG-3' and reverse 5'-CACATTGGGGGTAGGAACAC-3'.Quantitative PCR (qPCR) was performed as described earlier ,22,23. IWestern blot analysis was performed as described earlier were washed and harvested in PBS and subsequently injected into the lateral tail vein in a volume of 0.1 ml. A 3 \u00d7 106 sample of viable MDA-MB-231-VEC and MDA-MB-231-ARTN cells were injected subcutaneously into the mammary fat pad of immunodeficient nude mice (n = 6 for each group). After four weeks, mice were euthanized and lungs and livers were surgically resected for histology. Tissue samples were either fixed in 4% paraformaldehyde-PBS (pH = 7.4), embedded in paraffin and 6 \u03bcm-thick sections cut or were frozen at -80\u00b0C in RNALater for RNA extraction and qPCR analysis.All animal work was conducted in accordance with a protocol approved by the institutional animal care and ethics committee. Tumor growth and metastasis assays were performed as mentioned earlier ,26,27. F2 iv bolus, d1,8; methotrexate, 40 mg/m2 iv bolus, d1,8; 5-fluorouracil, 600 mg/m2 iv infusion, d1,8; every four weeks for six cycles). Patients who had undergone chemotherapy or radiation therapy before surgery were excluded from this study. Of 94 patients, 76 possessed full follow-up data ranging from 5 to 64 months, with a median time of 60.0 months and a mean time of 44.7 months.Tissue samples were collected from 94 ER-negative female mammary carcinoma patients attending the First Affiliated Hospital of Anhui Medical University presenting between 2001 and 2002. Institutional ethics committee approval for the project was obtained before commencement of the study and was in compliance with the Helsinki Declaration. Written informed consent was obtained from all patients. Of 94 patients, 33 were HER-2 positive and the remaining 61 were HER-2 negative as described in the additional file Immunohistochemical (IHC) analysis of paraffin-embedded specimens was performed as described previously . In briet-test (P < 0.05 was considered as significant) using Microsoft Excel XP (bar graphs) unless otherwise indicated (chi-squared test). Cox regression analysis was performed to determine the association of ARTN and TWIST1 expression to the risk of relapse and death.All numerical data are expressed as mean \u00b1 standard error of the mean from a representative experiment performed in triplicate, and statistical significance was assessed by Student's To determine the functional consequences of ARTN expression in ER-MC, we generated stable cell clones with forced expression of ARTN in BT549 and in MDA-MB-231 cells as previously described see add. Forced CDH1), \u03b3-CATENIN (CTNNG), and \u03b2-CATENIN (CTNNB1). Decreased OCCLUDIN (OCLN) mRNA expression was also observed in BT549-ARTN cells but not in MDA-MB-231-ARTN cells. Increased mRNA expression of mesenchymal markers TWIST1 and SPARC was observed in BT549-ARTN and MDA-MB-231 cells, whereas VIMENTIN (VIM) and SLUG (SNAI2) mRNA expression was increased in BT549-ARTN cells and NFKB1 in both cell pairs. Increased expression of MTA2 leads to estrogen independent growth of human mammary carcinoma cells [NFKB1 expression increases the invasive potential of carcinoma cells [NME1, MMP9 and MYC was observed in BT549-ARTN cells and MMP2 mRNA expression was increased in MDA-MB-231-ARTN cells, as compared with the respective control VEC cells. Thus, autocrine production of ARTN in ER-MC cells resulted in decreased expression of epithelial markers and increased expression of mesenchymal and metastatic related markers.During the progression of carcinoma toward a less differentiated and more metastatic state, cells lose their epithelial characteristics and acquire a mesenchymal morphology, together with concomitant changes in gene expression . Such a ma cells ,29. Forcma cells and incrWe next depleted BT549 and MDA-MB-231 cells of endogenous ARTN with small interfering RNA (siRNA) see add and examIncrease in cellular invasiveness and resistance to anoikis are key components of metastasis . Forced Concordantly, BT549-siARTN and MDA-MB-231-siARTN cells significantly inhibited monolayer proliferation by 23% and 17%, soft agar colony formation by 48% and 38% [see additional files n = 7), four weeks after injection with BT549-ARTN cells, whereas pulmonary metastases were detected in only three of seven mice injected with BT549-VEC cells. The nodules formed in the lungs of animals injected with BT549-ARTN cells gave rise to, on average, more than three nodules per lung as compared with less than one nodule per lung on average in animals with BT549-VEC cells. A higher frequency of liver metastasis was also observed in mice injected with BT549-ARTN cells (n = 4) compared with mice injected with BT549-VEC cells (n = 1) [see additional file In our xenograft model, BT549 cells failed to form consistent tumor masses. Therefore, to determine whether ARTN contributes to the metastatic ability of ER-MC cells, we injected BT549-ARTN cells into the tail vein of immunodeficient nude mice and examined their ability to form pulmonary and hepatic metastasis compared with the control VEC cell line. Metastatic nodules were readily detectable by histology in the lungs of all mice (HPRT (hHPRT) mRNA in the respective organs. We observed that the level of hHPRT mRNA expression was increased three-fold in lung and 1.3-fold in liver in animals injected with BT549-ARTN cells compared with BT549-VEC cells. Mouse gapdh (mgapdh) was used as an internal control four weeks after injection with MDA-MB-231-ARTN cells and in only five of the six mice injected with MDA-MB-231-VEC cells and overall survival (OS)). IHC analysis showed that both ARTN and TWIST1 protein were highly expressed in ER-MC Figure . All 94 P = 0.01) and OS (P = 0.03) of patients was observed and OS (P = 0.004) and OS : P = 0.02) at five years compared with low ARTN-low TWIST1 expression (Table P = 0.002) and OS (P = 0.005) and OS 100% (P = 0.005)), compared with low TWIST1 expression alone (RFS 78.3% (P = 0.01) and OS 78.3% (P = 0.03)) and OS : P = 0.01) at five years compared with low ARTN-low TWIST1 expression Figure [see add) Figure [see add) Figure [see addTo determine the functional significance of ARTN-stimulated TWIST1 expression we employed siRNA to selectively reduce TWIST1 expression in both BT549 and MDA-MB-231 cells with or without forced expression of ARTN. The siRNA targeting TWIST1 efficiently depleted TWIST1 protein expression in both cell pairs Figure whereas As observed in Figure It has been previously reported that ARTN activates AKT to mediate its oncogenic effects in endometrial carcinoma cells . The AKTWe next evaluated whether signalling through AKT was required for ARTN stimulated oncogenicity and invasion in ER-MC cells. Treatment of BT549-VEC cells with AKT Inhibitor IV decreased the basal levels of pAKT but did not affect the total AKT level. ARTN stimulated activation of AKT was also decreased by AKT inhibitor IV without altering the expression level of total AKT Figure . AKT inhWe determined if increased TWIST1 expression could substitute for the functional effects of ARTN in ER-MC cells. We generated stable cell clones with forced expression of TWIST1 in BT549 cells Figure using a We next analyzed the functional effects of siRNA mediated depletion of ARTN in BT549-TWIST1 cells. Forced expression of TWIST1 significantly promoted colony formation in soft agar, 3D matrigel growth, migration and invasion in transwell assays compared with the control BT549-VEC cells. BT549-TWIST1 cells exhibited a 60% increase in soft agar colony formation, 90% increase in 3D matrigel growth, 95% increase in cell migration and 110% increase in cell invasion Figures to 9e asMammary carcinoma is a heterogeneous disease, which comprises a significant fraction of ER negative subclasses . Given tARTN expression has previously been correlated with poorer survival outcome in mammary carcinoma overall and in Ein vivo tumor initiating capability (manuscript in preparation). ARTN and TWIST1 may therefore functionally synergize in aspects of mammary carcinoma cell behavior other than EMT and metastasis reported herein.The prometastatic potential of TWIST1 in mammary carcinoma cells is well established ,29,33. PAlthough ARTN is an estrogen-regulated gene we have demonstrated that it also mediates oncogenic effects in ER-MC including cell invasion and metastasis. Functional involvement of GDNF family ligands has not been previously reported in ER-MC and the identified interactions with TWIST1 provide a novel therapeutic opportunity for this MC subtype. Furthermore, examination of the combined expression of ARTN and TWIST1 in ER-MC provides a powerful predictor of survival in these patients. It is therefore warranted that combinatorial therapeutic approaches to inhibit both ARTN signalling and PI3K/AKT, leading to reduced TWIST1, be considered to improve prognosis in patients with ER-MC.ARTN: artemin; CFMDA: 5-chloromethylfluorescein diacetate; EMT: epithelial-mesenchymal transition; ER: estrogen receptor; ER+MC: estrogen positive mammary carcinoma; ER-MC: estrogen negative mammary carcinoma; F-actin: filamentous actin; FBS: fetal bovine serum; GDNF: glial-cell line-derived neurotrophic factor; HER2: human epidermal growth factor receptor; IF: immunofluorescence; IHC: immunohistochemistry; MTA: metastasis-associated protein family member; OS: overall survival; PBS: phosphate-buffered saline; PR: progesterone receptor; qPCR: quantitative real time PCR; RFS: relapse free survival; siRNA: small interfering RNA; TGF: transforming growth factor; TMA: tissue microarray.ZT and PEL received consultancies from Saratan Therapeutics Ltd . Salaries of DXL and AB were part supported by Saratan Therapeutics Ltd. DXL and PEL are inventors on PCT/NZ2008/000152. PEL is an inventor on PCT/NZ2010/000207.PEL designed research, wrote the paper and analyzed the data. AB designed research, wrote the paper, performed research and analyzed the data. ZSW, PXQ performed research and analyzed data. JK and VP contributed to the execution of experiments. DXL, TZ analyzed data. All authors have read and approved the manuscript for publication.Histopathological Scoring [ Scoring -53.Click here for fileARTN modulates the morphology of estrogen receptor-negative mammary carcinoma (ER-MC) cells.Click here for fileARTN modulates anchorage independent growth of estrogen receptor-negative mammary carcinoma (ER-MC) cells.Click here for fileXenograft growth of MDA-MB-231 cells with forced expression of ARTN in immunodeficient mice.Click here for fileAssociation of ARTN and TWIST1 expression with overall survival outcome in estrogen receptor-negative mammary carcinoma (ER-MC) cells.Click here for file"} +{"text": "A vaccine for HIV-1 would ideally block HIV-1 acquisition as well as durably control viral replication in breakthrough infections. A recently published study showed that optimal SIV vaccines can reduce SIV infection risk and setpoint viral loads following SIV challenges in rhesus monkeys, and that immunization with SIV Env was required for blocking acquisition of infection (Nature 2012 482:89-93). Here we investigate whether CD8+ T lymphocytes from these vaccinated rhesus monkeys mediate viral inhibition in vitro and whether these responses predict virologic control following SIV challenge.PBMC from 23 monkeys that received DNA/MVA, MVA/MVA, Ad26/MVA, or MVA/Ad26 vaccines expressing SIVsmE543 Gag/Pol/Env were used in CD8+ T-cell-mediated in vitro viral inhibition assays. CD8-depleted PBMC were infected with SIVmac251, and viral inhibition was defined as the log reduction in p27 of cultures of CD8-depleted PBMC with and without CD8+ T lymphocytes. Viral inhibition was correlated with cellular immune responses and setpoint viral loads using Spearman rank-correlation tests.In vitro CD8+ T-cell-mediated viral inhibition prior to challenge correlated with Gag-specific ELISPOT (P=.002), total and central memory CD8+ (P<.001 for both), and total and central memory CD4+ responses . A trend was observed with Gag-specific effector memory CD8+ T-cell responses . Viral inhibition did not correlate with Pol- or Env-specific cellular immune responses. Moreover, in vitro viral inhibition prior to challenge inversely correlated with in vivo setpoint viral loads following challenge (P=.014).These data demonstrate for the first time that the in vitro viral inhibition assay following vaccination is a predictor of in vivo virologic control following infection. Furthermore, in vitro viral inhibition correlated with Gag-specific, but not Pol- or Env-specific, cellular immune responses. These data suggest the importance of including Gag in an HIV-1 vaccine in which virologic control is desired."} +{"text": "Inflammation plays a critical role in plaque initiation, progression, and disruption. As such, inflammation represents an emerging target for the treatment of atherosclerosis.We investigated the contributing factors for plaque enhancement and examined the relationships between regional contrast enhancement and the inflammatory activity of atherosclerotic plaques in an experimental rabbit model using contrast-enhanced high-resolution 3D black-blood magnetic resonance imaging (MRI) in comparison with histopathology.Ten atherosclerotic rabbits and three normal control rabbits underwent high-resolution 3D contrast-enhanced black-blood MRI. MR images and the corresponding histopathological sections were divided into four quadrants. Plaque composition was analyzed for each quadrant according to histopathological and imaging criteria (enhancement ratio (ER), ER=SIpost/SIpre).A total of 62 non-calcified plaques were identified based on histopathology. Mean ER values were significantly higher in atherosclerotic vessel walls than in normal vessel walls . Mean ER values were significantly higher in macrophage-rich plaques compared to the macrophage-poor plaques . Using multiple regression analysis, macrophage area and microvessel density were independently associated with ER values that reflected plaque enhancement (p <0.001).Contrast-enhanced high-resolution 3D black-blood MRI may be an efficient method to predict plaque inflammation."} +{"text": "Myocilin is a gene linked directly to juvenile- and adult-onset open angle glaucoma. Mutations including Gln368stop (Q368X) and Pro370Leu (P370L) have been identified in patients. The exact role of myocilin and its functional association with glaucoma are still unclear. In the present study, we established tetracycline-inducible (Tet-on) wild type and mutant myocilin-green fluorescence protein (GFP) expressing RGC5 stable cell lines and studied the changes in cell migration and barrier function upon induction.After several rounds of selection, clones that displayed low, moderate, or high expression of wild type, Q368X or P370L myocilin-GFP upon doxycycline (Dox) induction were obtained. The levels of wild type and mutant myocilin-GFP in various clones were confirmed by Western blotting. Compared to non-induced controls, the cell migration was retarded, the actin stress fibers were fewer and shorter, and the trypsinization time needed for cells to round up was reduced when wild type or mutant myocilin was expressed. The barrier function was in addition aberrant following induced expression of wild type, Q368X or P370L myocilin. Immunoblotting further showed that tight junction protein occludin was downregulated in induced cells.Tet-on inducible, stable RGC5 cell lines were established. These cell lines, expressing wild type or mutant (Q368X or P370L) myocilin-GFP upon Dox induction, are valuable in facilitating studies such as proteomics, as well as functional and pathogenesis investigations of disease-associated myocilin mutants. The barrier function was found impaired and the migration of cells was hindered with induced expression of wild type and mutant myocilin in RGC5 cell lines. The reduction in barrier function might be related to the declined level of occludin. The retarded cell migration was consistent with demonstrated myocilin phenotypes including the loss of actin stress fibers, lowered RhoA activities and compromised cell-matrix adhesiveness. Glaucoma is a major blinding disease characterized by progressive loss of retinal ganglion cells (RGCs) and their axons, as well as cupping of the optic nerve head. The most common form of this disease, primary open angle glaucoma (POAG), is highly heterogeneous, caused by several susceptibility genes http://www.sciencedirect.com/science/article/pii/S0002944010603501 - ref_bib13Myocilin, the first candidate gene linked to juvenile- and adult-onset POAG, was originally cloned from cultured human trabecular meshwork (TM) cells after prolonged treatment of dexamethasone Myocilin protein is detected in eye tissues including the TM, the Schlemm's canal, the sclera, the ciliary body, the retina and the optic nerve head To facilitate studies of myocilin and its mutants, we established tetracycline-inducible (Tet-on) RGC5 stable cell lines that would express, upon induction, green fluorescence protein (GFP)-tagged wild type and mutant (Q368X and P370L) myocilin. These cell models allowed studies that require confluent myocilin-expressing cell cultures such as migration and barrier functions. Our results disclosed that when the expression of wild type or mutant myocilin was induced, the actin stress fibers were lost, RhoA activity was reduced and cell migration was blocked. In addition, the trypsinization sensitivity was heightened and the barrier function was impaired. The expression level of tight junction protein occludin was also lowered which may contribute to the reduced barrier function.WT-GFP or MYOCWT-GFP) expressing RGC5 stable cell lines were established using a single plasmid vector pTRE-MYOC-EGFP-INS-rtTA-IRES-hyg-pcDNA3.1z, which contains both tetracycline regulatory and responsive components based on Clontech's Tet-on advance system (WT-GFP was expressed following doxycycline (Dox) induction.The inducible Tet-on wild type myocilin-GFP -GFP were additionally prepared using a similar strategy. Clones with different expression levels were also obtained and banked in liquid nitrogen. MyocilinQ368X-GFP in the low expressers displayed a diffuse cytoplasmic distribution pattern shorter for myocilinWT-, myocilinQ368X- and myocilinP370L-GFP-expressing cells than their respective non-induced counterparts , indicating increased trypsin sensitivity or reduced cell-matrix adhesiveness.To further examine the cell-matrix adhesiveness, RGC5 inducible cells were subjected to trypsin sensitivity tests. The time needed for cells to round up was recorded using a confocal live cell imaging system and analyzed. It was found that RGC5 cells became more sensitive to trypsinization when induced . The tryG-LISA RhoA activation assays were carried out to measure the amounts of active RhoA in the cells. The level of GTP-bound or active RhoA in wild type or mutant myocilin-GFP-expressing RGC5 cells was approximately 35\u201350% lower , P<0.01 WT-, myocilinQ368X- or myocilinP370L\u2013GFP showed lower TER values than non-induced controls using an electrical cell-substrate impedance sensing (ECIS) system. Dox-treated RGC5 cells that expressed myocilincontrols , signify370L-GFP . The sta370L-GFP .Glaucoma is a group of chronic, degenerative optic neuropathies. Myocilin is one of the candidate genes linked to POAG, the most common form of glaucoma Myocilin mutations are typically associated with high IOP cases Our laboratory has performed studies on myocilin using cultured cells derived from normal human TM tissues Progressive loss of RGCs and their exons is one of the hallmarks in POAG. In light of the potential impact of myocilin mutations on neuronal cells, and also due to the difficulties associated with cultured human TM cells, we undertook the current study, establishing inducible cell lines and extending our efforts to examine the consequences of myocilin and Q368X and P370L mutants on neuronal cells. RGC5, an immortalized RGC cell line used widely for various biological investigations Q368X- and myocilinP370L-GFP also showed prominent cytoplasmic aggregates , in agreement with previous reports that dexamethasone treatment enhanced the tight junction protein ZO-1 expression level, reduced the permeability and increased fluid flow resistance in human TM cells In the retina, the retinal pigment epithelium (RPE) forms the outer blood retinal barrier and the endothelium of the retinal vessels constitutes the inner blood retinal barrier Signal molecules such as small guanosine triphosphatases (GTPases) of the Rho family are known to regulate cell adhesion, and cell-cell junctional assembly and disassembly In summary, the Tet-on inducible RGC5 cells provide a new tool for exploring the effects of myocilin upregulation and mutations at cellular and molecular levels. Since the percentage of cells on the plate that would express the transgene upon induction approaches 100, studies including genomics, proteomics, and signal transduction are made possible. The consequences of a combination of myocilin mutation and stress such as hypoxia can also be examined utilizing the inducible cells.EcoR I and BamH I linearized pTRE-tight vector (Clontech). TRE-MYOC-EGFP was then shuttled to pBluescriptII-SK(+) vector and the insulator (INS) fragment was inserted to generate pBS-TRE-OPTN-EGFP-INS. Finally, rtTA-IRES-hygromycin fragment was digested from construct rtTA-IRES-hyg-pcDNA3.1z and ligated into pBS-TRE-OPTN-EGFP-INS A plasmid vector pTRE-MYOC-EGFP-INS-rtTA-IRES-hyg-pcDNA3.1z which contains both tetracycline regulatory and responsive components based on the Clontech's Tet-on advance system was constructed . The proRabbit anti-GFP and anti-RhoA antibodies were from Santa Cruz Biotechnology . Horseradish peroxidase (HRP)- or Cy3-conjugated goat anti-rabbit and goat anti-mouse secondary antibodies were from Jackson ImmunoResearch . Anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody was obtained from Trevigen . Rabbit polyclonal anti-occludin antibody was from Invitrogen . Monoclonal anti-myocilin antibody Rat retinal ganglion RGC5 cells were obtained from the departmental core facility, deposited by Dr. Paul Knepper RGC5 cells transfected with above-mentioned plasmid vectors were selected in hygromycin (100 \u00b5g/ml)-containing medium for approximately 2 weeks until colonies grew out. The cells were then trypsinized and induced with 1 \u00b5g/ml of Dox for 48 h. GFP positive cells that were sorted using DakoCytomation MoFlo into 96 well plates (1 cell/well) were incubated with maintenance medium (containing 50 \u00b5g/ml of hygromycin but without Dox) for another 2 weeks. Cells were screened for low, moderate, or high myocilin-GFP expressers after Dox induction by fluorescence microscopy. These expresser clones were allowed to multiply and were banked in liquid nitrogen.WT-, myocilinQ368X-, or myocilinP370L-GFP, or non-induced as controls. Cells were harvested and lysed in the Cellytic buffer . Protein concentration was determined by Bradford assay . Proteins in total cell lysates and culture media were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and were subsequently transferred to nitrocellulose membranes for immunoblotting. Membranes were blocked with 5% non-fat milk in Tris-buffered saline with Tween 20 (TBST) for 1 h and incubated overnight with rabbit anti-GFP or mouse anti-myocilin to detect the expression level of myocilin-GFP fusion proteins, or with rabbit-occludin antibody to detect the level of occludin. Membranes were washed with TBST buffer 3 times and blotted with HRP-conjugated goat anti-rabbit or goat anti-mouse secondary antibody for 1 h. After further washing, the membranes were allowed to react with ECL and the chemiluminescence signal was detected using the ImageQuant LAS4000 digital image system .RGC5 inducible cells plated in T25 culture flasks were either induced for 48 h with Dox (1 \u00b5g/ml) to express myocilinRGC5 inducible cells were plated in 8-well chamber slides at 5000 cells/well. They were either induced for 48 h with Dox (1 \u00b5g/ml) or non-induced as controls. Actin filaments were stained with rhodamine-phalloidin per manufacturer's protocol. GFP fluorescence and actin staining in the cells were examined under a confocal laser scanning microscope . To further study the dose-dependence of the actin phenotype, low expresser was mix-cultured with moderate or high expressers of wild type myocilin-GFP, treated with Dox, and stained with rhodamine-phalloidin. Actin staining was also performed in parental (native) RGC5 cells following Dox treatment for 48 h. Untreated cells were used as controls. For occludin staining, non-induced cells, induced low expressers, and induced cells in a mixed culture of low, moderate, and high expressers were fixed in 4% paraformaldehyde and incubated with rabbit anti-occludin and Cy3-goat anti-rabbit IgG.RGC5 inducible cells from moderate clones were plated in 6 well plates and induced for 48 h. Cells were serum starved for 2 h and treated with 5 \u00b5g/ml of mitomycin C for 2 h RGC5 inducible cells (moderate expressers) were plated in poly-D-lysine coated 35 mm glass bottom dishes . After Dox induction, the cells were washed with PBS and Versene. Following addition of 0.125% trypsin-EDTA solution, morphologic changes were monitored and recorded every 10 s for a total of 5 min using a confocal spinning disc live cell imaging system (Carl Zeiss). The time needed for the cells to round up was analyzed RGC5 inducible cells either induced with Dox (1 \u00b5g/ml) for 48 h or non-induced were serum-starved for 18 h and lysed. G-LISA\u00ae RhoA activation assay Biochem Kit\u2122 (absorbance based) was used to determine the active, GTP-bound RhoA per manufacturer's instruction. The absorbance at 490 nm from wild type or mutant myocilin-GFP-expressing cells (mean \u00b1 SEM) was compared with their respective non-induced controls. Experiments were repeated 2 times.Active RhoA was also measured with pull down assay as previously described The cell barrier properties were measured using an ECIS system Figure S1Immunostaining of rat retinal section for occludin. The section was stained with polyclonal rabbit anti-occludin antibody . Hematoxylin and eosin staining (right panel) was performed on a serial section to demonstrate retinal layers. Retinal pigment epithelial (RPE) and retinal ganglion cell (RGC) layers showed strong staining of occludin. Scale bar, 100 \u00b5m.(TIF)Click here for additional data file."} +{"text": "To assess the occurrence of diagnostic cross-over in Eating Disorders (EDs) and assess its relationship with psychopathology, environmental risk factors, personality and genes.Participants were 316 ED patients. The EATATE part 1 (a semi-structured interview) was used to examine diagnostic cross-over in EDs. The Eating Disorder Inventory (EDI-2), Temperament and Character Inventory (TCI-R), Oxford Risk Factor Interview (ORFI), the EATATE part 2 [used to assess obsessive-compulsive personality (OCPD) traits and impulsive behaviours] and four candidate genes were used to assess differences in cross-over patterns.p<.05). No significant associations were found for environmental risk factors, the four candidate genes and diagnostic cross-over.The majority of ED patients (65%) presented with diagnostic instability. The most commom cross-over change (23.42%) was from Anorexia Nervosa Restrictive (AN-R) subtype to a bulimic disorder. Significant differences across four ED cross-over groups , a stable group (34.5%) and a remitted group (18.67%) were obtained the EDI bulimia, asceticism and impulse regulation subscales, the TCI-R self-directideness and cooperativeness subscales, childhood OCPD traits and impulsive behaviours (The findngs of the current study indicate that diagnostic instability is very common in EDs and that especially psychopathological and personality correlates should be taken into consideration when treating patients with cross-over patterns.Anorexia Nervosa \u2013 Characteristics and Treatment stream of the 2013 ANZAED Conference.This abstract was presented in the"} +{"text": "E.coli. The resulting fusion proteins were found to assemble into chimeric VLPs. Both unmodified HBcAg and chimeric VLPs induced HBcAg-specific antibody responses in mice, however, only chimeric VLP-immunized sera possessed EV71 epitope-specific IgG antibodies and efficiently neutralized different EV71 strains. Collectively, our results indicate that the chimeric VLP is capable of eliciting broadly neutralizing antibody responses and is therefore a promising EV71 vaccine candidate.To develop an effective vaccine against multiple genotypes of enterovirus 71 (EV71), we generated chimeric virus-like particles (VLPs) repetitively displaying the common neutralizing epitopes of EV71 and evaluated their immunogenicities in mice. The two conserved epitopes, encompassing amino acids 163-177 and 208-222 of VP1 of EV71, were fused to hepatitis B core antigen (HBcAg) and expressed in"} +{"text": "To assess and compare acute treatment optimization as measured by the Migraine Treatment Optimization Questionnaire (M-TOQ) within a population-based sample of persons with migraine.AMPP is a longitudinal, US-population-based study for which questionnaires were mailed to 24,000 severe headache sufferers and followed annually. Respondents with ICHD-2 migraine were stratified as either CM (>15 headache-days/month) or EM (<15 headache-days/month). Acute-treatment optimization was measured with M-TOQ, a valid/reliable patient-report tool assessing 5 domains: functioning, rapid relief, relief consistency, recurrence risk, tolerability over preceding 4 weeks. Respondents rated statements in each area as either occurring: never, rarely, < or > half the time. An item response theory (IRT) model used to define scaled treatment optimization scores as function of M-TOQ item set: lower scores=less/problematic optimization; higher scores=greater optimization. The model was expanded to incorporate persons with CM/EM on scaled scores and explored demographic adjustments for age and gender.8612 persons met criteria for migraine and completed M-TOQ. IRT model parameters indicated excellent M-TOQ psychometric properties. Scaled treatment optimization scores were significantly lower for persons with CM (3.25) vs EM , corresponding to a 0.5 standard deviation (SD) difference between CM and EM. After adjustment, mean difference on scaled-optimization score remained significantly lower (worse) for CM .Treatment regimens were less well-optimized and more lacking in domains measured by M-TOQ among persons with CM vs EM. Funding: The AMPP study was funded through a research grant to the NHF from Ortho-McNeil Neurologics. Additional analyses were supported by Allergan, Inc."} +{"text": "Electrophysiological and behavioral experiments in mice reveal that a cGMP-dependent kinase amplifies neurotransmitter release from peripheral pain sensors, potentiates spinal synapses, and leads to exaggerated pain. \u2212/\u2212 mice). Patch clamp recordings showed that activity-induced LTP at identified synapses between nociceptors and spinal neurons projecting to the periaqueductal grey (PAG) was completely abolished in SNS-PKG-I\u2212/\u2212 mice, although basal synaptic transmission was not affected. Analyses of synaptic failure rates and paired-pulse ratios indicated a role for presynaptic PKG-I in regulating the probability of neurotransmitter release. Inositol 1,4,5-triphosphate receptor 1 and myosin light chain kinase were recruited as key phosphorylation targets of presynaptic PKG-I in nociceptive neurons. Finally, behavioural analyses in vivo showed marked defects in SNS-PKG-I\u2212/\u2212 mice in several models of activity-induced nociceptive hypersensitivity, and pharmacological studies identified a clear contribution of PKG-I expressed in spinal terminals of nociceptors. Our results thus indicate that presynaptic mechanisms involving an increase in release probability from nociceptors are operational in the expression of synaptic LTP on spinal-PAG projection neurons and that PKG-I localized in presynaptic nociceptor terminals plays an essential role in this process to regulate pain sensitivity.Synaptic long-term potentiation (LTP) at spinal neurons directly communicating pain-specific inputs from the periphery to the brain has been proposed to serve as a trigger for pain hypersensitivity in pathological states. Previous studies have functionally implicated the NMDA receptor-NO pathway and the downstream second messenger, cGMP, in these processes. Because cGMP can broadly influence diverse ion-channels, kinases, and phosphodiesterases, pre- as well as post-synaptically, the precise identity of cGMP targets mediating spinal LTP, their mechanisms of action, and their locus in the spinal circuitry are still unclear. Here, we found that Protein Kinase G1 (PKG-I) localized presynaptically in nociceptor terminals plays an essential role in the expression of spinal LTP. Using the Cre-lox P system, we generated nociceptor-specific knockout mice lacking PKG-I specifically in presynaptic terminals of nociceptors in the spinal cord, but not in post-synaptic neurons or elsewhere . In this study, we examine the molecular and cellular mechanisms of LTP at synapses from nociceptors onto spinal neurons. We use multiple experimental approaches in mice, from genetic to behavioural, to show that this form of LTP involves presynaptic events that unfold in nociceptors when they are repetitively activated. In particular, an enzyme activated by the second messenger cGMP, referred to as Protein Kinase G-I, phosphorylates presynaptic proteins and increases the release of neurotransmitters from nociceptor endings in the spinal cord. When we genetically silence Protein Kinase G-I or block its activation in nociceptors, inflammatory pain is markedly reduced at the behavioural level. These results clarify basic mechanisms of pathological pain and pave the way for new therapeutic approaches. Plasticity in peripheral nociceptors and their synapses with spinal neurons has been proposed as a cellular basis for the development and maintenance of pain hypersensitivity following peripheral inflammation or nerve injury Synaptic LTP evoked by natural, asynchronous low-rate discharges in C-nociceptors on spino-PAG neurons was recently shown to constitute a very fitting correlate of spinal amplification phenomena underlying inflammatory pain Studies on several different biological systems have shown that cGMP regulates multiple cellular targets, including diverse cGMP-gated ion channels, such as cyclic nucleotide-gated (CNG) and hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, the cGMP-dependent protein kinases, PKG-I/cGK-I and PKG-II/cGK-II, as well as diverse phosphodiesterases (PDEs) Based upon this background, this study was designed with two goals in mind. First, it addressed the potential involvement of presynaptic mechanisms in the expression of synaptic potentiation on spinal projection neurons, which has not been explored or described previously. Second, it aimed to explore a potential role for PKG-I localized presynaptically in the spinal terminals of nociceptors in spinal potentiation and to clarify cellular and molecular mechanisms underlying these processes. We reasoned that the use of a conditional, region-specific gene deletion strategy to specifically manipulate presynaptic mechanisms might constitute an unambiguous approach towards addressing the above questions. Our results show that spinal synaptic potentiation triggered by nociceptor activation is associated with a long-lasting change in the probability of neurotransmitter release from spinal terminals of nociceptors. Using viable, developmentally normal transgenic mice lacking the PKG-I specifically in nociceptors with preserved expression in spinal neurons, brain, and all other organs, we demonstrate here that PKG-I localised in nociceptor terminals constitutes a key mediator of synaptic LTP and that its activation is functionally associated with pain hypersensitivity in vivo.\u2212/\u2212) were generated via Cre/loxP-mediated recombination by mating mice carrying the floxed prkg1 allele (PKG-Ifl/fl) v1.8 promoter (SNS-Cre) v1.8-expressing) sensory neurons, without affecting gene expression in the spinal cord, brain, or any other organs in the body \u2212/\u2212 mice \u2212/\u2212 mice (\u2212/\u2212 mice (p<0.001). In contrast, a few large-diameter neurons showed low levels of anti-PKG-I immunoreactivity in DRGs of PKG-Ifl/fl, which was entirely retained in SNS-PKG-I\u2212/\u2212 mice (4 (IB4)-labelled non-peptidergic nociceptors and substance P-expressing peptidergic nociceptors in PKG-Ifl/fl mice, both of which are selectively lost in SNS-PKG-I\u2212/\u2212 mice , which were not significantly different from each other . Moreover, the level of substance P immunoreactivity was similar in the superficial spinal dorsal horn across genotypes . Importantly, confocal microscopy revealed normal density of synapses between substance P-containing nociceptive afferents and PSD-95-positive puncta (representing postsynaptic aspects of glutamatergic synapses) in the spinal dorsal horns of SNS-PKG-I\u2212/\u2212 mice as compared to PKG-Ifl/fl mice , which were retrogradely labelled upon stereotactic injection of DiI in the PAG .To assess the role of PKG-I localized presynaptically in spinal nociceptor terminals, we then analysed PKG-I\u2212/\u2212 mice. Analysis of EPSC magnitude evoked by the first and the last pulse of the conditioning train revealed short-term depression of evoked EPSCs during the conditioning train, which was equivalent in PKG-Ifl/fl mice and SNS-PKG-I\u2212/\u2212 mice (p\u200a=\u200a0.95), showing that the conditioning stimulus was equally effective in mice from both groups. Furthermore, basal C-fiber-evoked EPSCs were comparable between PKG-Ifl/fl and SNS-PKG-I\u2212/\u2212 mice. We also established detailed input-output curves representing the relationship between the intensity of dorsal root stimulation and evoked EPSCs in the absence of a conditioning stimulus and found no differences between PKG-Ifl/fl and SNS-PKG-I\u2212/\u2212 mice (p\u200a=\u200a0.74). Furthermore, the intensity of dorsal root stimulation required to elicit an action potential in post-synaptic spinal-PAG projection neurons was identical in PKG-Ifl/fl and SNS-PKG-I\u2212/\u2212 mice . Finally, as an additional indicator of the number of fibers activated during electrical stimulation, we recorded fiber volleys in input/output measurements. Recording C-fiber volleys in the L4 and L5 dorsal roots derived from PKG-Ifl/fl and SNS-PKG-I\u2212/\u2212 mice revealed typical responses, which increased in amplitude with increasing stimulus intensity , demonstrating directly that the number of fibers activated upon electrical stimulation was comparable between genotypes and could therefore not explain the failure to evoke synaptic potentiation in SNS-PKG-I\u2212/\u2212 mice. These comprehensive analyses show that a presynaptic loss of PKG-I was specifically linked to a failure of activity-dependent potentiation of transmission at synapses between nociceptors and spinal-PAG projection neurons, but not to modulation of basal synaptic transmission.For a clear interpretation of these data, it is imperative to address how basal nociceptive transmission at these synapses is affected in SNS-PKG-I\u2212/\u2212 mice since the molecular perturbation is specific to nociceptor terminals. Thus, the net contribution of C-fibers to the population of mEPSCs is difficult to assess because it is unclear which fraction of mEPSCs can be attributed to C-fibers, which then makes detection of potentially small presynaptic changes highly unlikely when performing mini-analysis. To study synaptic events which could be clearly assigned to activation of presynaptic primary afferent fibers alone, we employed a protocol of minimal stimulation, setting the dorsal root stimulation parameters such that a synaptic failure rate of approximately 60% was achieved in recording solution containing 1 mM Ca2+ and 5 mM Mg2+. The failure rate remained constant over a period of at least 30 min upon repetitive test stimulation in the absence of a conditioning stimulus (55.9%\u00b13.9% pre- and 57.1%\u00b18.1% at 30 min). However, upon application of the conditioning stimulus, minimal stimulation using the same parameters evoked a decrease in the frequency of synaptic failures within a few minutes in slices derived from PKG-Ifl/fl mice, indicating a change in the probability of neurotransmitter release; decrease in synaptic failures was accompanied by a corresponding rise in the magnitude of C-fiber-evoked EPSCs . In contrast, the rate of synaptic failures did not change significantly following conditioning stimulus in slices derived from SNS-PKG-I\u2212/\u2212 mice . EPSC values at the end of the recording were not significantly elevated as compared to basal values in SNS-PKG-I\u2212/\u2212 mice .Following our observation that a specific presynaptic alteration in PKG-I expression perturbed spinal LTP, we then addressed potential contributions of presynaptic mechanisms at synapses between nociceptors and spinal projection neurons fl/fl mice demonstrated a clear change in PPF or PPD following conditioning stimulus, neurons derived from SNS-PKG-I\u2212/\u2212 mice did not . This was markedly reduced in formalin-injected SNS-PKG-I\u2212/\u2212 mice . These results show that persistent activation of nociceptors leads to rapid signalling via PKG-I in DRG neurons in vivo, which is lost in SNS-PKG-I\u2212/\u2212 mice, as expected.In an effort to understand the underlying molecular mechanisms, we then addressed potential substrates for the kinase activity of PKG-I in nociceptors. In particular, we reviewed known substrates of PKG-I in other biological systems and focussed on those for which we hypothesized a role in synaptic transmission. We first set up an assay system for testing involvement of PKG-I substrates in the DRG selectively upon persistent nociceptive stimulation in vivo, using Vasodilator-stimulated phosphoprotein (VASP), a classical target of PKG-I, as an indicator of PKG-I activity pression , basal. fl/fl mice, which was found to be lacking in formalin-treated SNS-PKG-I\u2212/\u2212 mice \u2212/\u2212 mice . These d/fl mice . This fi/fl mice .p\u200a=\u200a0.004 as compared to vehicle-treated control slices). Furthermore, consistent with our observations in SNS-PKG-I\u2212/\u2212 mice, inhibition of MLC phosphorylation did not affect basal transmission at this synapse (p\u200a=\u200a0.761). Thus, the PKG-I target, pMLC, is functionally linked to potentiation of synaptic transmission in nociceptive laminae.Interestingly, synaptic potentiation induced by a conditioning stimulus on spinal-PAG projection neurons was abolished in the presence of ML-7, an inhibitor of MLCK for live identification of small-diameter nociceptive neurons . Stimulation of calcium release via activation of Gq/11-phospholipase C-IP3R1 pathway by addition of ligands, such as bradykinin (BK) and the P2Y-receptor ligand, UTP, led to typical increases in the ratio of Fura-2 fluorescence at 340/380 nm in neurons from PKG-Ifl/fl mice, which were markedly reduced in neurons from SNS-PKG-I\u2212/\u2212 mice (see p<0.001 with respect to BK and UTP). In contrast, Fura2-labelled neurons with large-diameter somata, which were IB4-negative, did not show differences in calcium responses between PKG-Ifl/fl mice and SNS-PKG-I\u2212/\u2212 mice (an example is indicated by arrow in \u2212/\u2212 mice and PKG-Ifl/fl mice (p>0.05), showing thereby that a loss of PKG-I in nociceptive neurons is specifically linked to defects in IP3R-mediated calcium release from intracellular stores.PKG-I-mediated phosphorylation of serine 1755 in IP/fl mice ; p>0.05,3R1 activity and MLC phosphorylation in sensory neurons, which is functionally linked to synaptic LTP at synapses between C-nociceptors and spinal-PAG projection neurons.Taken together, these biochemical and functional experiments suggest that following persistent nociceptive stimulation, PKG-I mediates potentiation of IPp<0.01 for 2-APB and ML-7 in comparison to vehicle control, respectively), implicating involvement of IP3R function and MLC phosphorylation, respectively. Similarly, SNS-PKG-I\u2212/\u2212 mice showed markedly reduced phase II responses than PKG-Ifl/fl mice (p<0.001 as compared to PKG-Ifl/fl mice).We then went on to address whether these findings bear relevance to pain-related behaviour in vivo and found a functional role for PKG-I and its substrates in behavioural paradigms for spinal sensitization. As a test system, we studied the Phase II of formalin-induced nocifensive behavioural responses, which are manifest at 10\u201360 min after intraplantar formalin injection, for two reasons: one, this represents a widely used paradigm for studying central changes in pain processing caused by a persistent activation of nociceptors \u2212/\u2212 mice and their control littermates (p>0.05). Furthermore, motor performance on a Rotarod was unaffected in SNS-PKG-I\u2212/\u2212 mice (p\u200a=\u200a0.20). We have previously shown in details that SNS-Cre mice show no alterations in the processing of acute pain or chronic inflammatory or neuropathic pain Basal withdrawal thresholds and response latencies to acute application of paw pressure . Finally, SNS-PKG-I\u2212/\u2212 mice showed a significantly lower magnitude of thermal hyperalgesia than PKG-Ifl/fl mice at 6 h after CFA and did not show hyperalgesia at all from 12 h onwards after CFA injection, whereas PKG-Ifl/fl mice continued to show thermal hyperalgesia all the way up to the latest time point tested, namely 96 h following CFA injection . We infer from the above that the development of primary hyperalgesia and mechanical allodynia following somatic inflammation is impaired by a loss of PKG-I in nociceptors.In the context of studying disease-induced pain hypersensitivity, we first focussed on a model of inflammatory pain which is associated with primary hyperalgesia in the inflamed area and ongoing nociceptive inputs from the periphery throughout the time of testing, namely unilateral hindpaw inflammation induced by injection of Complete Freund's Adjuvant (CFA) /fl mice and hype/fl mice or to pl/fl mice applied \u2212/\u2212 mice, it is conceivable that a peripheral role for PKG-I in nociceptors may at least partially account for changes in primary hyperalgesia. To address functional changes in nociceptor sensitivity in the inflamed tissue, we utilised the skin-nerve preparation fl/fl mice as well as outside of the flare . In PKG-I/fl mice . It is w/fl mice ; in cont/fl mice , indicat/fl mice ; in cont/fl mice , indicat\u2212/\u2212 mice , demonstfl/fl mice demonstrated a pronounced leftward and upward shift in the stimulus-response curve to von Frey hairs applied to the plantar paw surface , SNS-PKG-I\u2212/\u2212 mice completely failed to develop hyperalgesia following intrathecal NMDA delivery represent a key molecular link between NO and activation of PKG-I, the above results imply that NO activates sGC in spinal presynaptic terminals of nociceptors. While some studies report a lack of sGC expression in DRG neurons fl/fl mice also contribute to cGMP production in some organs /fl mice . Interes\u2212/\u2212 mice . These r\u2212/\u2212 mice. We constructed chimeric Adeno-associated virions of the serotypes AAV1 and AAV2 expressing an C-terminally GFP-tagged version of the murine PKG-I cDNA fl/fl mice and SNS-PKG-I\u2212/\u2212 mice expressing GFP-tagged PKG-I or GFP alone showed normal basal sensitivity to graded von Frey stimuli ; in contrast, a presynaptic change involving cGMP- and PKG-I-dependent increase in neurotransmitter release may mediate the expression of LTP at synapses between nociceptors and spinal-PAG projection neurons.Previous studies have shown that the NMDA receptor-NO-cGMP pathway is important in the induction of spinal LTP 3R1 and MLC, and observed that PKG-I modulates intracellular calcium release as well as MLC phosphorylation differently in DRG neurons as compared to other biological systems, such as the smooth muscle. For example, in some biological systems, PKG-I has been reported to negatively modulate calcium signals via its interaction with IRAG 3R1 at serine 1755, a site associated with positive functional modulation, implicate PKG-I as a positive modulator of calcium signalling in nociceptive neurons. In light of electrophysiological analyses reported here, this raises the possibility that calcium released from IP3R1-gated stores may participate in modulating presynaptic function. Although a few studies at hippocampal synapses have proposed an involvement of calcium stores in modulation of presynaptic release Mechanistically, this may come about via involvement of multiple phosphorylation targets of PKG-I. While some targets have been identified in heterologous systems, very little is known about the nature and functional role of PKG-I targets in vivo. Here, we identified and validated two primary targets in DRG neurons, namely the IP3R, its substrates involved in LTP, inhibited phase II behavioural responses. However, a contribution of central mechanisms could not be inferred from the formalin data due to several reasons: Although MLCK/IP3R inhibitors were administered spinally, the genetically induced loss of PKG-I in SNS mice occurred throughout the nociceptor, including peripheral terminals. Moreover, there is still some ongoing activation of peripheral nociceptors in the phase II of the formalin response The possibility of functionally linking synaptic changes described here to changes in nociceptive behaviour simultaneously represents a good opportunity and a major challenge. As the first parameter to test this relationship, we focused on the phase II responses in the intraplantar formalin test, which has been attributed to spinal nociceptive sensitization triggered by an initial barrage of C-fiber inputs \u2212/\u2212 mice. Our results indicated that capsaicin-evoked mechanical hypersensitivity is neither dependent on peripheral PKG-I function nor does it require ongoing peripheral nociceptor sensitisation. Moreover, they revealed that PKG-I expressed in central terminals of nociceptors plays a decisive role in the induction of mechanical hypersensitivity after persistent C-fiber stimulation via capsaicin. Finally, reinstating PKG-I expression in the DRG in adult SNS-PKG mice fully restored capsaicin-evoked mechanical hypersensitivity, indicating that PKG-I directly, and not some factor affected by a genetic loss of PKG-I, is a functional determinant of C-fiber-evoked mechanical hypersensitivity.Therefore, we focused on pain models in which nociceptive hypersensitivity is triggered by peripheral nociceptors, but maintained via central mechanisms that outlast and are independent of peripheral inputs. One of these is the chronic muscle pain model in which injections of dilute acidic saline in the gastronemius muscle evokes a long-lasting secondary mechanical hyperalgesia in the ipsilateral and contralateral paws, which lasts for several weeks, is unrelated to muscle damage and is not maintained by continued primary afferent input from the site of injury, as shown by experiments involving dorsal rhizotomy and lidocaine injections in the muscle In summary, this study shows that PKG-I expressed in nociceptors terminals is the principal target of cGMP at nociceptive synapses. Furthermore, it suggests that PKG-I-mediated presynaptic facilitation and LTP in spinal projection neurons is functionally involved in activity-dependent centrally mediated nociceptive hypersensitivity.Additional details on methods are provided in prkg1 gene, which encodes the cGMP dependent kinase 1 (PKG-Ifl/fl) fl/fl mice were crossed with SNS-Cre mice fl/fl;SNS-Cre+ mice (referred to as SNS-PKG-I\u2212/\u2212 mice in this article) and PKG-Ifl/fl mice (control littermates). Mice were crossed into the C57BL6 background for more than 8 generations. Mice lacking PKG-I globally (PKG-I\u2212/\u2212 mice) have been described before. Only littermates were used in all experiments to control for background effects.Homozygous mice carrying the flox allele of the mouse 2PO4, 1.2; Ca2+ 1.0; Mg2+, 5; NaHCO3, 26; glucose, 15; oxygenated with 95% O2, 5% CO2; pH 7.4). Dorsal root was stimulated at intensity of threshold to evoke EPSCs on DiI-labelled spino-PAG projection neuron. Under these conditions, the failure rate was 60.9%\u00b16.3% (n\u200a=\u200a10). To induce synaptic potentiation, low frequency stimulation was applied to dorsal root as a conditioning stimulus with the same intensity as the test stimulus Mice (14\u201318 d old) were anesthetized with a mixture of Dormitor, Dormicum, and Fentanyl, and stereotactic injections of DiI into the PAG were carried out were used (see pS1755 IP3R1 (kind gift from Prof. Richard Wojcikiewicz), anti-VASP , anti-MLC, anti-alpha tubulin (Sigma), anti-pSer239 VASP, anti-pThr18/pSer19 MLC , anti-PKG-I antibody The following antibodies were used for Western blots and biochemical analyses: anti-IP3R1, anti-The following antibodies were used for immunohistochemistry: phospho-ERK1/2 antibody , anti-Fos antibody (Chemicon), anti-Isolectin B4 antibody (vector laboratories), anti-Calcitonin gene related peptide antibody (Immunostar), anti-Neurofilament 200 antibody (Chemicon), anti-Substance P antibody (Chemicon), anti-PKG-I antibody 3R , NMDA , the NO donor, NOC-12 , atrial natriuretic peptide, brain natriuretic peptide and c-type natriuretic peptide , and the PKG-I inhibitor KT5283 (200 pmoles). See The soluble guanylyl cyclase inhibitor, ODQ , and the membrane guanylyl cyclase inhibitor, LY83583 , were dissolved in 50% DMSO and injected in a volume of 250 \u00b5l intraperitoneally. The following drugs were administered intrathecally in vivo: an inhibitor of MLCK , an inhibitor of IPfl/fl and 15 SNS-PKG-I\u2212/\u2212 mice were used in the electrophysiological recordings of nerve activity. An ex vivo skin-nerve preparation was used to study the properties of mechanosensitive C- and A-\u03b4 afferent fibres which innervate the skin in the inflamed area 24 h following CFA inoculation (20 \u00b5l) as described previously . All behavioural measurements were done in awake, unrestrained, age-matched mice of both sexes that were more than 3 mo old by individuals who were blinded to the genotype of the mice being analyzed (see 7 transfection units per DRG) in deeply anesthetized mice as described in detail previously The open reading frame of mouse PKG-I fused C-terminally with GFP t test was employed. Unless otherwise specified, the p values shown in the figures and text are derived from ANOVA and post hoc Fisher's test. p<0.05 was considered significant.All data are presented as mean \u00b1 standard error of the mean (S.E.M.). For comparisons of multiple groups, analysis of variance (ANOVA) for random measures was performed followed by post hoc Fisher's test to determine statistically significant differences. When comparing two groups that were studied in parallel, Student's Figure S1\u2212/\u2212 mice as compared to their PKG-Ifl/fl littermates. Typical examples (B) and quantitative summary (C) from three independent Western blot experiments; Tubulin expression serves as a control. * p<0.001, ANOVA, post hoc Fisher's test.Further evidence for specific deletion of PKG-I in nociceptors, but not in spinal neurons. (A) Anti-Cre immunohistochemistry on SNS-Cre mice demonstrating Cre expression in small-diameter neurons of the DRG, but not in spinal cord. Scale bars represent 50 \u00b5m for DRG and 100 \u00b5m for spinal cord. (B & C) Western blot analysis reveals intact expression of PKG-I in the spinal cord and brain and reduced expression in the DRG of SNS-PKG-I(PDF)Click here for additional data file.Figure S2\u2212/\u2212 mice. (A) Adult PKG-Ifl/fl mice and SNS-PKG-I\u2212/\u2212 mice show similar patterns of targeting nociceptors in the spinal cord (upper panels) and the skin (lower panels) as shown by binding to TRITC-labelled Isolectin-B4 (red) and immunoreactivity for Substance P (Sub P) or CGRP (green). Level of immunoreactivity for Sub P in spinal superficial laminae was similar across genotypes The(PDF)Click here for additional data file.Figure S3n\u200a=\u200a12 slices each. (C) Blockade of PKG-I specifically in the postsynaptic neuron via application of KT5823 in the patch pipette did not affect LTP; n\u200a=\u200a6 slices each. (D) Quantitative summary of the magnitude of increase in c-EPSCs in the above groups at 30 min after the conditioning stimulus (LFS) over basal vales . RKRARKE represents a distinct PKG-I inhibitor, which was tested in parallel experiments.Further validation of C-fiber-evoked synaptic long-term potentiation (LTP) at spinal synapses in wild-type mice. (A) Typical examples of retrogradely labelled spinal lamina I projection neurons (lower panels) following DiI injections into the PAG (upper panel). (B) LTP was preserved upon spinal blockade of inhibitory neurotransmission with a combination of bicuculline and strychnine; (PDF)Click here for additional data file.Figure S4\u2212/\u2212 mice and their PKG-Ifl/fl littermates. It is noteworthy that in PKG-Ifl/fl mice, synapses showing a large LTP show a decrease in PPF, indicating an increase in the release probability. An increase in PPF is only seen with a few synapses that do not show a robust LTP. These changes do not come about in SNS-PKG-I\u2212/\u2212 mice.Magnitude of LTP at synapses between C-fibers and spino-PAG neurons is plotted as a function of change in PPF or PPD at the same synapse in SNS-PKG-I(PDF)Click here for additional data file.Figure S5\u2212/\u2212 mice and PKG-Ifl/fl littermates in the na\u00efve state or following formalin injection in the hindpaws. (C) Expression levels of total VASP or MLC do not vary between the above experimental groups. (D) Expression levels of total IP3R1 do not vary between the above experimental groups. In all panels, quantitative summaries are derived from three such independent experiments, each involving eight mice/group. * p<0.05, ANOVA of random measures followed by post hoc Fisher's test. Data are represented as mean \u00b1 S.E.M.Nociceptive activity-driven phosphorylation of PKG-I substrates in DRG and spinal cord in vivo. A typical example (A) and quantitative summary (B) of levels of VASP phosphorylated on serine 239 in L4-L5 DRGs of SNS-PKG-I(PDF)Click here for additional data file.Figure S6\u2212/\u2212 mice and their PKG-Ifl/fl littermates. (A) In comparison with PKG-Ifl/fl mice (n\u200a=\u200a8), SNS-PKG-I\u2212/\u2212 mice (n\u200a=\u200a8) show comparable paw withdrawal latency to radiant heat and paw withdrawal threshold to punctuate pressure. (B) Latency to fall from a rotating rod was similar in SNS-PKG-I\u2212/\u2212 mice (n\u200a=\u200a6) and PKG-Ifl/fl mice (n\u200a=\u200a6). (C) The magnitude of paw edema was similar between SNS-PKG-I\u2212/\u2212 mice and their PKG-Ifl/fl littermates following CFA-induced paw inflammation. Shown are complementary analyses of paw size in terms of paw volume and volume of water displaced by paws (in ml). All data points represent mean \u00b1 S.E.M. n\u200a=\u200a8\u201310 mice per genotype or treatment group.Nociceptive withdrawal responses and development of paw inflammation in SNS-PKG-I(PDF)Click here for additional data file.Text S1Supplementary Methods and Materials.(DOCX)Click here for additional data file."} +{"text": "We then used our model of pulmonary infection to investigate the use of an inhibitor of WPB exocytosis to prevent systemic inflammation and hemodynamic instability.Gram-positive bacterial lung infection is commonly associated with sepsis, and particularly important in older individuals. Systemic vascular dysfunction associated with sepsis is characterized by vascular permeability that can lead to tissue hypoxia. Our studies focus on angiopoietin-2 (Ang-2), whose increased plasma concentration is associated with severity of lung injury and with mortality. Ang-2 is stored in the Weibel-Palade body (WPB), an endothelial-specific secretory organelle. Here, we examine the release of Ang-2 from primary human pulmonary microvascular endothelial cells (HPMECs) n = 6/group), were pretreated with TAT-NSF700 (0.5 mg/kg) or saline intraperitoneally. Thirty minutes later, LTA (150 \u03bcg) and PGN (500 \u03bcg) or saline alone were instilled intratracheally. Pulse oximetry was assessed in awake mice prior to and 6 hours post instillation. The mice were then euthanized by exsanguination under anesthesia and bronchoalveolar lavage (BAL) was performed. BAL fluid total and differential counts were evaluated and protein and cytokine concentrations in plasma and BAL were assessed using commercial assays.HPMECs were treated with lipoteichoic acid and peptidoglycan in the presence or absence of TAT-NSF700 fusion peptide (10 \u03bcM) to inhibit WPB exocytosis. Ang-2 in culture medium was measured by ELISA, and WPB exocytosis assessed using immunofluorescent (IF) staining of Ang-2. HPMEC monolayer permeability was measured using FITC-dextran. Male C57/Bl6 mice, 8 to 10 weeks old (P = 0.003) in HPMEC culture medium within 30 minutes, which was blocked by TAT-NSF700 pretreatment. IF staining showed aggregation and localization of Ang-2 in the cytoplasm suggesting WPB exocytosis within 30 minutes. LTA-PGN also induced HPMEC permeability. LTA-PGN induced both local and systemic inflammation resulting in decreased heart and breath rates and oxygen saturation at 6 hours. These physiological changes were prevented in mice pretreated with TAT-NSF700.LTA-PGN increased Ang-2 levels dose dependently (up to ninefold, In vivo, LTA-PGN induces significant changes in heart and breath rates and oxygen saturation that was prevented by inhibition of WPB exocytosis. Thus, we have defined WPB, a storage organelle for multiple proinflammatory mediators, as a potential target to control overwhelming inflammation in Gram-positive sepsis and improve tissue oxygenation and hemodynamics.LTA-PGN induced rapid Ang-2 secretion via WPB exocytosis and this release is associated with significant changes in permeability."} +{"text": "RanBPM (Ran-binding protein in the microtubule-organizing centre) was originally reported as a centrosome-associated protein in human cells. However, RanBPM protein containing highly conserved SPRY, LisH, CTLH and CRA domains is currently considered as a scaffolding protein with multiple cellular functions. A plant homologue of RanBPM has not yet been characterized.Arabidopsis thaliana. AtRanBPM protein has highly conserved SPRY, LisH, CTLH and CRA domains. Cell fractionation showed that endogenous AtRanBPM or expressed GFP-AtRanBPM are mainly cytoplasmic proteins with only a minor portion detectable in microsomal fractions. AtRanBPM was identified predominantly in the form of soluble cytoplasmic complexes ~230 \u2013 500\u2009kDa in size. Immunopurification of AtRanBPM followed by mass spectrometric analysis identified proteins containing LisH and CRA domains; LisH, CRA, RING-U-box domains and a transducin/WD40 repeats in a complex with AtRanBPM. Homologues of identified proteins are known to be components of the C-terminal to the LisH motif (CTLH) complexes in humans and budding yeast. Microscopic analysis of GFP-AtRanBPM in vivo and immunofluorescence localization of endogenous AtRanBPM protein in cultured cells and seedlings of Arabidopsis showed mainly cytoplasmic and nuclear localization. Absence of colocalization with \u03b3-tubulin was consistent with the biochemical data and suggests another than a centrosomal role of the AtRanBPM protein.Based on sequence similarity, we identified a homologue of the human RanBPM in Arabidopsis RanBPM protein physically interacts with LisH-CTLH domain-containing proteins. The newly identified high molecular weight cytoplasmic protein complexes of AtRanBPM showed homology with CTLH types of complexes described in mammals and budding yeast. Although the exact functions of the CTLH complexes in scaffolding of protein degradation, in protein interactions and in signalling from the periphery to the cell centre are not yet fully understood, structural conservation of the complexes across eukaryotes suggests their important biological role.We showed that as yet uncharacterized Arabidopsis genome contains three genes encoding AtRan was cloned into Gateway-compatible plant binary vectors for N-terminal GFP and C-terminal GFP fusion. r et al. . Transfo799 was cArabidopsis extracts, the antibody recognized a band of 52\u2009kDa, the predicted molecular mass (MW) of AtRanBPM, and a band corresponding to the MW of GFP-AtRanBPM while the truncated version of human RanBPM is present in centrosomal and ectopic microtubule nucleation sites , AtRanBPRanBPM belong to genes with unknown functions. We found that the product of AtRanBPM is predominantly a cytoplasmic protein that is a part of protein complexes with a molecular mass of approximately 230 \u2013 500\u2009kDa. A large protein complex of RanBPM was described by Nishitani et al. was obtained by PCR amplification using P fusion were useArabidopsis suspension cultures of Landsberg erecta ecotype stably expressing GFP-AtRanBPM or transiently expressing AtRanBPM-GFP were prepared according to the protocol of Mathur et al. for V. vinifera, [Swiss-Prot:B9MWC1] for P. trichocarpa, [Swiss-Prot:C5XUT1] for S. bicolor, [Swiss-Prot:Q6ZI83] for O. sativa, [Swiss-Prot:B6UAR9] for Z. mays, [Swiss-prot: Q6VN20] for human RanBP10, [Swiss-prot: A3KMV8] for RanBP10 from Bos taurus, [Swiss-prot: B5LX41] for RanBP10 from Felis catus, [Swiss-prot: Q6VN19] for RanBP10 from Mus musculus, [Swiss-prot: Q1LUS8] for RanBP10 from Danio rerio, [Swiss-prot: Q9PTY5] for RanBP9 from Xenopus laevis, [Swiss-prot: Q96S59] for human RanBP9, [Swiss-prot: P69566] for RanBP9 from Mus musculus, [Swiss-prot: Q4Z8K6] for RanBP9/10 from Drosophila melanogaster and [Swiss-prot: P53076] for Gid1/Vid30 homologue from Saccharomyces cerevisiae. Distance bars are given bottom left and bootstrap values are indicated at the nodes. software . Branch Click here for fileImmunopurification of GFP-AtRanBPM protein. Immunopurification of GFP-AtRanBPM from extracts of GFP-AtRanBPM expressing cell cultures (IP GFP-RanBPM). GFP immunopurification from extracts of wild type Ler Arabidopsis cells (IP WT) was used as a negative control. A- Proteins were silver stained after separation on SDS-PAGE. Bands corresponding to MW similar of the proteins copurified with GFP-AtRanBPM (IP GFP-RanBPM) were not present in the negative control (IP WT). B- Signal for AtRanBPM was absent in the negative control (IP WT) after detection with anti-AtRanBPM antibody on Western blots. C- Proteins identified by MALDI-MS in negative control (IP WT in A) were background contamination. Click here for fileIdentities and similarities between proteins copurifying with AtRanBPM and human CTLH complex members. Identities and similarities between Arabidopsis and human proteins were analysed in WU-BLAST. Click here for fileAdditional proteins copurified with AtRanBPM. The proteins were identified by LC-MALDI-MS/MS and the identity of the matched peptides was confirmed by high-resolution MALDI-FTMS with mass accuracy below 1\u2009ppm. Click here for fileGFP-AtRanBPM inArabidopsisroot cells. AtRanBPM-GFP signal is dynamic and moving with the cytoplasmic stream. Click here for fileCellular localization of C-terminal GFP and N-terminal GFP AtRanBPM fusion proteins. Cells of Arabidopsis expressing C-terminal GFP AtRanBPM (AtRanBPM-GFP) showed weak cytoplasmic and nuclear signal and accumulation of perinuclear GFP signal similarly as observed for N-terminal GFP AtRanBPM (GFP-AtRanBPM). Click here for file"} +{"text": "L/BCL-2 (inhibiting the intrinsic pathway) or FLIPL (inhibiting the extrinsic pathway), in hematopoietic stems cells (HSCs). Transplantation of HSCs expressing MYC into syngeneic recipient mice resulted in development of AML and T-cell lymphomas within 7\u20139 weeks as expected. Importantly, coexpression of MYC together with BCL-XL/BCL-2 resulted in strongly accelerated kinetics and favored tumor development towards aggressive AML. In contrast, coexpression of MYC and FLIPL did neither accelerate tumorigenesis nor change the ratio of AML versus T-cell lymphoma. However, a change in distribution of immature CD4+CD8+ versus mature CD4+ T-cell lymphoma was observed in MYC/FLIPL mice, possibly as a result of increased survival of the CD4+ population, but this did not significantly affect the outcome of the disease. In conclusion, our findings provide direct evidence that BCL-XL and BCL-2 but not FLIPL acts in synergy with MYC to drive AML development.Myc plays an important role in tumor development, including acute myeloid leukemia (AML). However, MYC is also a powerful inducer of apoptosis, which is one of the major failsafe programs to prevent cancer development. To clarify the relative importance of the extrinsic (death receptor-mediated) versus the intrinsic pathway of apoptosis in MYC-driven AML, we coexpressed MYC together with anti-apoptotic proteins of relevance for AML; BCL-X MYC family genes are common in a number of tumor types and are also found in AML although at a low frequency MYC resides) is common in AML, and the resulting increased MYC gene dosage has been suggested as a leukemogenesis mechanism MYC and MYCN are frequently overexpressed in AML as a result of oncogenic events such as the class I and II mutations mentioned above Acute myeloid leukemia (AML) represents a clonal expansion of hematopoietic stem and myeloid progenitor cells that have undergone malignant transformation myc transgenic mouse model L and BCL-2, which regulate the intrinsic pathway. This mechanism is often bypassed during lymphoma progression of E\u03bc-myc mice L in these mice L act in synergy with MYC in tumorigenesis. Moreover, Myc induces expression of pro-apoptotic proteins such as Bim and Bax MYCN-amplified neuroblastoma myc lymphoma model in vivoL, thereby triggering the death receptor pathway L in these cells could completely rescue the cells from apoptosis L expression in a number if cells types both in culture and in vivo by binding to the LFLIP gene promoter region, resulting in increased sensitivity to TRAIL-induced apoptosis L and/or MCL-1 and reduced expression of Bax is frequently observed in AML. AML cells are often resistant to TRAIL- and Fas-induced apoptosis, which has been attributed to death receptor or FADD downregulation, caspase 8 mutations or overexpression of FLIPLOverexpression of MYC in cells in culture or in animal models fuels cell growth and proliferation but is also a potent trigger of the apoptotic machinery, which acts as a failsafe mechanism to suppress tumorigenesis Although the contribution of the extrinsic pathway to MYC-induced apoptosis has been studied extensively in different cell types in culture, only a few studies of the importance of this pathway for MYC-driven tumorigenesis in vivo have been performed , all using B- or T-lymphoma models. The potential impact of this pathway on MYC-induced AML is therefore unknown. In particular, the relative importance of the intrinsic and the extrinsic anti-apoptotic pathways in tumorigenesis fueled by MYC in vivo, including AML, remains to be determined.in vivo-impact of these two apoptotic pathways during MYC-induced malignant transformation of hematopoietic stem cells. We therefore coexpressed MYC together with anti-apoptotic gene products of either the extrinsic death receptor-mediated pathway (FLIPL) or the intrinsic pathway of apoptosis (BCL-2 or BCL-XL) in hematopoietic stem cells (HSC) followed by transplantation of the cells into syngeneic recipient mice. Expression of MYC alone in HSC resulted in development of both myeloid and T-lymphoid tumors within two months after transplantation. Expression of MYC together with BCL-XL or BCL-2 resulted in almost immediate development of AML-like disease. Surprisingly and contrary to our expectations, expression of MYC together with FLIPL did not accelerate tumorigenesis. However, while the ratio of AML versus T-cell lymphoma was not altered, the incidence of mature CD4+ T-cell lymphoma increased at the expense of immature CD4+/CD8+ T-cell lymphoma in MYC/FLIPL mice. In conclusion, anti-apoptotic proteins of the intrinsic pathway accelerate the development of MYC-driven hematopoietic tumors to a larger extent than those of the extrinsic pathway of apoptosis.The aim of this work was to assess the The human retroviral packaging cell line Phoenix-Eco was grown as described NcoI and NotI sites. The pMSCV-BCL-XL-IRES-EGFP vector was obtained by subcloning the EcoRI fragment containing human BCL-XL from the pLXIN-BCL-XL expression vector L-IRES-EGFP vector was generated as described previously 5\u2032-acgtgaattccaccatgcccctcaacgttagcttc and 5\u2032-tacgtctcgagcttacgcacaagagttccgtag and subsequently cloned into the EcoRI and XhoI sites of pMSCV-IRES-EYFP to obtain the pMSCV-MYC-IRES-EYFP expression vector. pMSCV-BCL-2-IRES-EGFP was obtained by subcloning human BCL-2 from the cDNA clone MGC:21366 into the EcoRI site of pMSCV-IRES-EGFP.The pMSCV-IRES-EYFP vector was produced by replacing the enhanced green fluorescent protein (EGFP) of the pMSCV-IRES-EGFP vector with the enhanced yellow fluorescent protein (EYFP) of pEYFP using the L-IRES-EGFP (BCL-XL-GFP), pMSCV-BCL-2-IRES-EGFP (BCL-2-GFP), pMSCV-FLIPL-IRES-EGFP (FLIPL-GFP) or pMSCV-MYC-IRES-EYFP (MYC-YFP) expression vectors were used to transiently transfect Phoenix-Eco packaging cell line using Lipofectamine 2000 Reagent . Supernatants containing recombinant viral particles were harvested 48 and 72 hours after transfection, passed through a 0.45 \u00b5 M filter, and kept in aliquots at \u221280\u00b0C until used for viral transduction.Ten micrograms of the plasmids pMSCV-IRES-EGFP (GFP), pMSCV-IRES-EYFP (YFP), pMSCV-BCL-Xfemur and tibia of mice 48 h after i.p. injection of 150 mg/kg of 5-fluorouracil . Bone marrow cells were enriched for hematopoietic progenitors and stem cells by negative selection using the StemSep kit according to the manufacturer's specifications. Cells were cultured for 24 hours in OPTIMEM (Invitrogen) supplemented with 10% FCS, 2 mM L-glutamine, 1 mM sodium pyruvate, 50 U/ml penicillin, 50 \u00b5g/ml streptomycin, IL-6, IL-3 and SCF containing supernatants L-GFP, BCL-2-GFP, FLIPL-GFP and MYC-YFP in the presence of 8 \u00b5g/ml polybrene . This procedure was repeated for three consecutive days and the cells were then cultured for another three days. The percentage of GFP and YFP positive cells were measured by flow cytometry prior to transplantation. Non-transduced cells were sometimes used to dilute GFP+/YFP+ cells to frequencies below 5%. Transplantation was performed by intravenous injection of 1\u00d7106 cells into lethally irradiated (800rad for BALB/c or 960 rad for DBA/2) syngeneic recipient mice. The mice were given 8 \u00b5g/ml of doxycycline in the drinking water and kept in filter top cages.Bone marrow was extracted from the Mice were monitored three times a week for signs of disease by palpation and observation and were judged as terminally ill when they displayed signs of paralysis in limbs or persistently hunched posture and slow movements. Mononuclear cell suspensions of spleen, thymus, liver, bone marrow and lymph nodes were obtained by passing tissues through nylon mesh cell strainers . Tissues from the mice were fixed in buffered 4% formaldehyde.Single cell suspensions from spleen, thymus, femoral bone marrow and liver were incubated with mAbs including anti-CD4-APC, anti-Gr1-APC, anti-CD19-bio, and anti-CD71-bio . Pacific blue-labeled anti-CD8 (clone 2.43), anti-CD11b (clone M1/70.15) anti-Ter119 and anti-IgM (clone M41) MAb were prepared in our laboratory according to standard procedures. Biotinylated samples were then incubated with streptavidin-APC (BD PharMingen) before being analyzed on a Cyan ADP flow cytometer . EGFP and EYFP were detected using 510/21-nm and 550/30-nm bandpass filters separated by a dichroic 525-nm mirror. Flow cytometric data were analyzed with FlowJo .\u2212) hematopoietic stem cells (HSC) were enriched from bone marrow and transduced with replication incompetent retroviral expression vectors, followed by transplantation to lethally irradiated syngeneic recipient mice. Expression of the gene of interest is in this system driven by the retroviral LTR promoter that gives expression in all lineages of hematopoietic cells L (regulating the intrinsic apoptotic pathway) or FLIPL (regulating the extrinsic pathway) linked to GFP. As controls the same GFP and YFP vectors lacking other genes were used. Previously, using vectors expressing GFP or YFP, we have shown that this results in a near to random frequency of single and double expressing hematopoietic cells after transduction and transplantation in vivo. The expression of YFP and GFP as determined by flow cytometry after transduction of various combinations of MYC, BCL-XL, FLIPL or control vector into Lin\u2212 bone marrow cells prior to transplantation is shown in L and FLIPL were all expressed in cells transduced with the respective retroviral constructs were recorded at 7, 14, 35 and 49 days after transplantation. antation .L/MYC mice appeared to have a latency period before development of splenomegaly (L/MYC mice at later time points (+7 weeks) had grossly enlarged spleens (average 579 mg), to a similar extent as Mock/MYC mice (average 816 mg).As in Mock/Myc mice, FLIPnt mice) . MoribunL construct was functionally active, Fas-sensitive A20 cells were transduced with the FLIPL-GFP construct and thereafter incubated with or without agonistic anti-Fas mAb for 20 hours and thereafter analyzed by flow cytometry. L efficiently inhibited Fas-induced apoptosis in this system. This suggested that inhibition of the intrinsic but not the extrinsic pathway of apoptosis synergized with MYC to promote tumorigenesis of HSC.To ensure that the FLIPL or FLIPL, respectively, using flow cytometry. Single cell suspensions from femoral bone marrow, spleen, thymus, liver and enlarged lymph nodes of mesenteric or cervical origin were stained with the myeloid markers anti-CD11b and anti-Gr1, the T-cell markers anti-CD4 and anti-CD8, the B-cell markers anti-CD19 and anti-IgM, and the erythroid markers anti-CD71 and Ter119. Mock/Mock animals all showed similar staining patterns with normal frequencies of myeloid as well as lymphoid cells and often myeloid cells . Analyses of spleen cells from a few representative Mock/MYC mice are shown in \u2212YFP+) and Mock/MYC (GFP+YFP+) cells as exemplified by MYC/Mock #176 spleen or T-cell lineage origin (CD4+). FLIPL/MYC spleen and infiltrating cells of the liver frequently showed a mixed population of both myeloid and lymphoid origin as in Mock/MYC mice See . ExtensiO+ cells . No infiO+ cells . In MYC/ markers and S7. Mock/MYC .L, MYC/BCL-2 and MYC/FLIPL recipient mice. These cells were further characterized by flow cytometry and all MYC-YFP+ appeared with similar phenotypes as the primary tumors (data not shown).Tumor cell lines could be established from all moribund Mock/MYC, MYC/BCL-XL or BCL-2 strongly favored tumor development towards myeloid leukemia, whereas coexpression of MYC and FLIPL did not affect the distribution of myeloid versus lymphoid tumor cells, but changed the ratio between single CD4+ and double positive CD4+CD8+ T-cell lymphomas.These results are summarized in L expression, hematopoietic development was studied in detail at 7, 14 and 35 days after transplantation of Mock/MYC- or MYC/BCL-XL-transduced HSC into recipient mice. in vivo. Mock-GFP/Mock-YFP-expressing cells, on the other hand, did not expand and were kept at constant relative numbers reflecting the proportions of transduced cells before transplantation in forward scatter were analyzed in CD11be levels . In summMYC oncogene in development of AML L, BCL-2 and FLIPL. The transduced HSC were transplanted to lethally irradiated syngeneic recipient mice after which tumor development was monitored.Several lines of evidence from the human disease and mouse models have implicated the L or BLC-2 strongly accelerated MYC-induced tumorigenesis and all mice died or were moribund within 2 weeks after transplantation. Co-expression of MYC with BCL-XL or BCL-2 also influenced the tumor phenotype; Rather than a mixed phenotype of myeloid and T lymphoid tumors, development of an aggressive AML-like disease was favored , under other conditions.In contrast to the effects of BCL-XAlthough MYC was over expressed in cells of all hematopoietic lineages and resulted in blast transformation, morbidity was primarily due to development of myeloid and/or T-cell tumors.+ or CD4+/CD8+) and latency (7\u201310 weeks) to those of VavP-MYC17 mice \u2212 bone marrow cells. In another transplantation model, overexpression of MYC resulted in development of aggressive pre-B cell lymphomas, with low penetrance and after long latency (>100 days) T-cell lymphomas were similar both in phenotype for 20 hours and thereafter analyzed by flow cytometry using propidium iodide (PI) to discriminate between live and dead cells.(TIF)Click here for additional data file.Figure S2L-GFP/Myc-YFP expressing HSCs.Early hematopoietic development of DBA/2 mice reconstituted with Mock-GFP/Mock-YFP, Mock-GFP/MYC-YFP or BCL-X Phenotypic analysis of bone marrow (left) and spleen (right) at 7, 14 and 35 days after transplantation of Mock-GFP/Mock-YFP, Mock-GFP/MYC-YFP or BCL-XL-GFP/MYC-YFP expressing HSCs into DBA/2 mice was performed with FACS. The horizontal axis indicates days after transplantation and the vertical axis represents percentage of cells expressing the marker indicated to the left. Black triangles: GFP+YFP\u2212 cells, black squares; GFP\u2212YFP+ cells and white circles; GFP+YFP+ cells.(TIF)Click here for additional data file.Figure S3L recipient mice.Appearance of leukemic blasts in the blood of MYC/BCL-X Blood smears stained with May-Grunewald-Giemsa. Blood smear from a Mock/Mock mouse shows a normal picture with few leukocytes (A) as compared to the leukocytosis seen in MYC/BCL-XL mice (B) (primary magnification 20\u00d7). Higher magnification shows a dominance of blast-like cells with only few maturing granulocytes in MYC/BCL-XL mice (C and D) (primary magnification 60\u00d7).(TIF)Click here for additional data file.Figure S4Immunohistochemical staining of sections of spleen and liver of Mock/MYC mice. The section slides were stained with antibodies directed against the T-cell markers CD45R and CD3 and the myeloid marker myeloperoxidase (MPO) and analyzed by immunohistochemistry as described in (TIF)Click here for additional data file.Figure S5Immunohistochemical staining of sections of spleen of Mock/Mock mice. The analysis was performed as described in the legend to (TIF)Click here for additional data file.Figure S6L mice.Immunohistochemical staining of sections of spleen and liver of MYC/BCL-X The analysis was performed as described in the legend to (TIF)Click here for additional data file.Figure S7Immunohistochemical staining of sections of spleen and liver of MYC/BCL-2 mice. The analysis was performed as described in the legend to (TIF)Click here for additional data file.Figure S8L mice.Immunohistochemical staining of sections of spleen, liver and thymus of MYC/FLIP The analysis was performed as described in the legend to (TIF)Click here for additional data file.Materials and Methods S1Immunohistochemistry.(DOCX)Click here for additional data file.Table S1L,, BCL-2, FLIPL or control virus recipient mice.Flow cytometric analysis of bone marrow, thymus, spleen and liver in MYC, BCL-X(DOCX)Click here for additional data file.Table S2Differential count of nucleated cells in peripheral blood smears in MYC/BCL-XL and control mice.(DOCX)Click here for additional data file."} +{"text": "We have earlier shown that protein levels of vascular endothelial growth factor-A (VEGF-A) and chemokine ligand-2 (CCL2) were elevated in Indian amyotrophic lateral sclerosis (ALS) patients. Here, we report the mRNA levels of VEGF-A and CCL2 in Indian ALS patients since they display extended survival after disease onset.VEGF-A and CCL2 mRNA levels were measured in peripheral blood mononuclear cells (PBMCs) of 50 sporadic Indian ALS patients using Real Time Polymerase Chain Reaction (PCR) and compared with normal controls (n = 50). Their levels were adjusted for possible confounders like cigarette smoking, alcohol and meat consumption.VEGF-A and CCL2 mRNA levels were found to be significantly elevated in PBMCs in ALS patients as compared to controls. PBMCs from definite ALS revealed higher VEGF-A mRNA expression as compared to probable and possible ALS. CCL2 mRNA levels were found to be unaltered when definite, probable and possible ALS were compared. PBMCs from patients with respiratory dysfunction showed much higher VEGF-A and CCL2 elevation when compared to patients without respiratory dysfunction. No association of smoking, alcohol and meat consumption with VEGF-A and CCL2 was observed after analyzing the data with univariate and multivariate analysis.VEGF-A and CCL2 mRNA upregulation in PBMCs may have a clinico-pathological/etiological/epidemiological association with ALS pathogenesis. The cross-cultural and cross-ethnic investigations of these molecules could determine if they have any role in enhancing the mean survival time unique to Indian ALS patients. Vascular endothelial growth factor-A (VEGF-A) is a dimeric secreted polypeptide that was discovered first in the VEGF family which also includes placental growth factor (PLGF), VEGF-B, VEGF-C, VEGF-D and VEGF-E. VEGF-A stimulates growth of blood vessels during embryonic development and helps in proliferation of blood collaterals in diseased conditions including ALS through a tyrosine kinase dependent VEGF receptor-2 (VEGFR2) . Apart fical ALS and convical ALS . On the ical ALS .Indian ALS patients are known to exhibit significantly extended survival duration after disease onset as compared to Western ALS patients -8. We re50 patients, born in North India and diagnosed with ALS were included from a convenience sample of Neurology outpatient, post graduate institute of medical education and research (PGIMER), Chandigarh after obtaining informed consent as a part of research protocol as per institute ethical committee guidelines (No. 7055-PG-1Tg-05/4348-50). Based on the \"El Escorial criteria\", there were 25 definite ALS patients, 15 individuals were probable ALS and remaining 10 were possible ALS at the time of sample collection. ALS-functional rating score-revised (ALSFRS-R) revealed that 11 patients had respiratory dysfunction such as orthopnea and dyspnea accompanied with other respiratory insufficiencies, although none of the patients needed respiratory support have alsPBMCs were isolated as per Histopaque-1077 datasheet. Briefly, 6.0 ml blood was collected from each subject and layered on equal volume of Histopaque-1077. It was then centrifuged at 1800 rpm for 30.0 mins at room temperature and PBMCs were collected from plasma/Histopaque-1077 interface and preserved in RNA later at -80\u00b0C until used.Total RNA was extracted from PBMCs using RNAeasy columns . RNA concentration was measured by taking absorbance at 260.0 nm. About 500.0 ng - 5000.0 ng total RNA was used to synthesize cDNA according to RevertAid\u2122 first strand cDNA kit .Real Time Polymerase Chain Reaction (PCR) was used to quantitate expression of VEGF-A and CCL2 mRNA using published primers -14. Metht test and one-way analysis of variance (ANOVA) followed by Fisher's least significant difference (LSD) post hoc analysis was applied for statistical comparisons. Crude and adjusted odds ratio (OR) was evaluated by univariate and multivariate logistic regression respectively to check any possible influence of smoking, alcohol and meat consumption on VEGF-A and CCL2 mRNA levels and \u03c72 (chi square) test was performed to find significance level.Because the data was normally distributed as indicated by quintile-quintile (Q-Q) plot, unpaired, independent, 2-tailed student p-value was considered significant at \u22640.05. Statistical analysis was performed by statistical package and service solutions (SPSS) 16 software. Results were analyzed by two independent and masked researchers.Real Time PCR indicates that VEGF-A expression is 77-fold higher in ALS than controls VEGF-A in ALS patients was observed possibly because of genetic changes in promoter regions -17. IncrAs VEGF-A and CCL2 are neurotrophic, Indian ALS patients may enhance VEGF-A and CCL2 expression in an attempt to ameliorate excitotoxicity through upregulation of glutamate receptor as reported earlier ,23. IncrSince elevated CCL2 initiates inflammatory reaction by increasing production of nitric oxide and other inflammatory chemokines from unregulated monocytes/macrophages and VEGFAt this moment, we are not able to state whether the increased VEGF-A and CCL2 mRNA is a consequence of genetic and/or epigenetic changes of upstream regulatory sequences, altered transcriptional regulation or amyotrophy and thus the present report lays the foundation for future studies to screen promoter elements of VEGF-A and CCL2 in Indian ALS population for subtle genetic differences. The stress conditions, like respiratory problems, may also modify transcriptional gene regulation as indicated by increased VEGF-A and CCL2 mRNA expression in the 11 ALS patients with respiratory dysfunction and signifies a possible association with hypoxia Figure .Based on existing literature ,30, elevAlthough it can not be concluded that increased VEGF-A and CCL2 expression contributes towards enhanced survival yet the importance of clinico-pathological, etiological and epidemiological association of increased VEGF-A and CCL2 with survival of Indian ALS patients may not be underestimated and needs further investigations.ALS: amyotrophic lateral sclerosis; ALSFRS-R: ALS functional rating score-revised; ANOVA: analysis of variance; CCL2: chemokine ligand-1; CCR2: chemokine receptor-2; CNS: central nervous system; CSF: cerebrospinal fluid; EDTA: ethylene diamine tetraacetate; HRE: hypoxia response element; LSD: least significant difference; mRNA: messenger ribonucleic acid; NMDA: N-Methyl-D-aspartate; NP-1: neuropilin-1; OR: odds ratio; PBMCs: peripheral blood mononuclear cells; PCR: polymerase chain reaction; PI3-K: phosphatidylinositol 3-kinases; SOD1: superoxide dismutase 1; VEGF: vascular endothelial growth factor; VEGFR1: VEGF receptor-1; VEGFR2: VEGF receptor-2.Ethical approval was obtained by institute ethical committee, PGIMER, Chandigarh, India - 160012 (No. 7055-PG-1Tg-05/4348-50).The authors declare that they have no competing interests.PKG Acquisition of data and writing of manuscript. SP inclusion of patients, grant PI and clinical scoring. CA Acquisition of data. NKS Statistical analysis. AA Interpretation and analysis of data, grant co PI and writing and editing of manuscript. All authors read and approved the final manuscript.Real Time Polymerase Chain reaction (PCR). Methodology of Real Time PCR; PCR cycling conditions and amplicon size of VEGF-A and CCL2; sequences and references of primers used.Click here for file"} +{"text": "Tumor-associated myeloid cells play a critical role in supporting tumor progression and metastasis. However, molecular mechanisms by which tumor cells interact with myeloid cells to modify therapeutic responses to anticancer treatments remain largely unknown. We identified several key factors affecting antitumor effects of chemotherapy by manipulating the modes of interaction between tumor cells and myeloid cells within tumor microenvironments. Tumor-derived factors such as VEGF-A and arginase-I upregulate TIM-3 on dendritic cells. TIM-3 interacts with HMGB1 released from chemotherapy-treated tumor cells, and suppresses innate pattern recognition signals mediated by nucleic acids from dying tumor cells. The inhibition of TIM-3 augments antitumor effects of cytotoxic chemotherapy against established tumors. Moreover, danger-associated molecular patterns (DAMPs) released from chemotherapy-damaged tumor cells induced TIM-4 on TAMs recruited from bone marrow-derived precursors. TIM-4 directly interacted with AMPK-\u03b11 at phagosome and activated autophagy-mediated lysosomal degradation of ingested tumors, leading to reduced antigen presentation and impaired CTL responses. Consistently, blockade of the TIM-4/AMPK-\u03b11/ autophagy pathway augmented antitumor effect of chemotherapeutics by enhancing tumor-specific CTL responses. Taken together, our findings provide new evidences that targeting of key factors regulating interplay between tumor cells and myeloid cells provides new therapeutic strategy to eradicate chemoresistant tumors."} +{"text": "Intramyocardial hemorrhage commonly occurs in large reperfused myocardial infarctions. However, its long-term fate remains unexplored. We hypothesized that acute reperfusion intramyocardial hemorrhage leads to chronic iron deposition.Fifteen patients , who underwent successful angioplasty for first STEMI, were recruited following informed consent. Cardiovascular Magnetic Resonance (CMR) imaging (1.5T) was performed on day 3 and month 6 post-angioplasty. 2D T2* maps and Late Gadolinium Enhancement (LGE) images of the entire left ventricle (LV) were acquired. Threshold-based image analysis was performed to identify remote, hemorrhagic (Hemo+) and non-hemorrhagic (Hemo-) myocardium.Fourteen canines, subjected to ischemia-reperfusion (I-R) injury (3 hours of LAD occlusion followed by reperfusion), underwent CMR (1.5T) on days 3 and 56 post-I-R injury. Three sham-operated animals (Shams) were also studied using CMR at similar time points. 2D T2* maps and LGE images of the entire LV were acquired. Threshold-based image analysis was performed to identify remote, Hemo+ and Hemo- myocardium. Subsequently, animals were euthanized (day 56), hearts were excised and imaged ex-vivo. Sections of Hemo+, Hemo-, remote and Sham myocardium were isolated and histology was performed. The concentration of iron ([Fe]) within each type of tissue was measured using mass spectrometry.Six months post-angioplasty, mean T2* of the scar tissue in patients with Hemo+ infarctions at both 3 days and 56 days post-I-R injury Figure . SimilarAcute reperfusion intramyocardial hemorrhage leads to regional chronic iron deposition within the infarct zones. T2* CMR can accurately characterize localized chronic iron deposition following reperfusion-induced myocardial hemorrhage. The clinical significance of this finding remains to be investigated.This work was supported in part by grants from American Heart Association (SDG 0735099N) and National Heart, Lung, And Blood Institute (HL091989)."} +{"text": "In this study, we used two-dimensional gel electrophoresis (2-DE) and two-dimensional fluorescence difference gel electrophoresis (2-DIGE), coupled with matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF-MS), to explore the global proteome profiles of trachea and kidney tissues from chicken at different stages infected 5 strain and those given the embryo-passaged, attenuated P115 stain. In addition, the dynamic transcriptional alterations of 12 selected proteins were analyzed by the real-time RT-PCR, and western blot analysis confirmed the change in abundance of heat shock proteins (HSP) beta-1, annexin A2, and annexin A5.Fifty-eight differentially expressed proteins were identified. Results demonstrated that some proteins which had functions in cytoskeleton organization, anti-oxidative stress, and stress response, showed different change patterns in abundance from chicken infected with the highly virulent ck/CH/LDL/97I PThe proteomic alterations described here may suggest that these changes to protein expression correlate with IBV virus' virulence in chicken, hence provides valuable insights into the interactions of IBV with its host and may also assist with investigations of the pathogenesis of IBV and other coronavirus infections. Coronaviridae in the order Nidovirales. They are able to infect humans as well as other animals, including cows, pigs, mice, and chickens, they generally cause respiratory infection, gastrointestinal, and neurological disorders of varying severity. Infectious bronchitis virus (IBV) was the first coronavirus to be discovered, and is classed among the Gamma coronaviruses on the basis of antigenic and genetic relatedness -1-propanesulfonate; DTT: Dithiothreitol; IBV: Infectious bronchitis coronavirus; IEF: Isoelectric focusing; IPG: Immobilized pH gradient; MALDI-TOF-TOF/MS: Matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry; PMF: Peptide mass fingerprinting; SDS-PAGE: Sodium dodecyl sulfate polyacrylamide gel electrophoresis; SPF: Specific pathogen free; TFA: Trifluoroacetic acid; 2-DE: Two-dimensional gel electrophoresis; 2-DIGE: Two-dimensional fluorescence difference gel electrophoresis.The authors declare that they have no competing interests.SL designed the study. SL and ZC drafted the manuscript. ZC, ZH, YS, and XL carried out virus infection, serum antibody detection, and quantitative analysis of IBV. ZC, JS and DY carried out the 2-DE experiments, image analysis, excised the protein spots, data analysis and interpretation, confirmed the differential expression by real-time RT-PCR and Western blotting analysis. SL wrote the manuscript. XK revised the manuscript. All authors read and approved the final manuscript.5 and ck/CH/LDL/97I P115Table S1 Serological results post inoculation with IBV ck/CH/LDL/97I P.Click here for file5 and P115Figure S1 Summary of changes in protein levels over time following infection with IBV ck/CH/LDL/97I P. The y axis shows the number of differentially expressed protein spots; individual spots can be found in Tables Click here for fileThis includes the PMF spectrum and confirmed MALDI-TOF-TOF spectrum of differentially expressed protein spots in IBV-infected chick tracheal tissues.Click here for fileThis includes the PMF spectrum and confirmed MALDI-TOF-TOF spectrum of differentially expressed protein spots in IBV-infected chick kidney tissues.Click here for fileTable S2 Biological function of differentially expressed proteins reported in viral infection.Click here for fileTable S3 Comparison of the fold changes for protein abundance observed by 2-DE gel analysis and mRNA level obtained by real-time RT-PCR in trachea tissues.Click here for fileTable S4 Comparison of the fold changes for protein abundance observed by 2-DIGE gel analysis and mRNA expression obtained by real-time RT-PCR in kidney tissues.Click here for fileTable S5 Experiment design of the different fluorescent dye labeling.Click here for fileTable S6 The primers of Real-time RT-PCR.Click here for file"} +{"text": "Enteral administration of lipid-enriched nutrition was previously shown to attenuate inflammation and organ damage via a cholecystokinin-mediated vagovagal reflex in animal studies. The current proof-of-principle study investigates the immunomodulatory potential of enteral lipid-enriched and protein-enriched nutrition during experimental human endotoxemia.Escherichia coli lipopolysaccharide . Subjects in the fasted group (n = 6) were deprived of food throughout the study, while subjects in the intervention groups were fed either enriched (n = 6) or isocaloric control nutrition (n = 6) via a nasojejunal tube, starting 1 hour prior to LPS administration until 6 hours afterwards.After an overnight fast, 18 healthy male subjects received an intravenous bolus of P < 0.01) and fasted subjects . Additionally, enriched nutrition augmented the anti-inflammatory response, reflected by increased IL-10 release compared with fasted subjects (P < 0.0001). See Figure LPS administration resulted in a marked inflammatory response. Continuous postpyloric administration of nutrition increased plasma cholecystokinin levels. Enriched nutrition attenuated circulating levels of the proinflammatory cytokines TNF\u03b1 and IL-6 and the IL-1 receptor antagonist compared with control nutrition (all: The current study establishes the anti-inflammatory potential of enriched nutrition in humans. The immediate anti-inflammatory effect of enriched nutrition suggests that the beneficial effects are mediated via a cholecystokinin-dependent vagovagal reflex. Enteral administration of enriched nutrition is a promising intervention to modulate the immune response in the early course of systemic inflammation."} +{"text": "Cre and other site-specific DNA recombinases have come to play a central role in achieving temporally regulated and cell type-specific genetic manipulation. In zebrafish, both Cre and Flp recombinases have been applied for inducible activation, inactivation and inversion of inserted genomic elements. Here we describe the addition of Dre, a heterospecific Cre-related site-specific recombinase, to the zebrafish genomic toolbox. Combining Dre-based recombination in zebrafish with established Cre/lox technology, we have established an effective strategy for transgene activation and inactivation using lox and rox (TAILOR). Using stable transgenic lines expressing tamoxifen-inducible T2CreER and RU486-inducible DrePR fusions, we demonstrate that Cre and Dre retain non-overlapping specificities for their respective lox and rox target sites in larval zebrafish, and that their combinatorial and sequential activation can achieve precisely timed transgene activation and inactivation. In addition to TAILOR, the successful application of Dre/rox technology in zebrafish will facilitate a variety of additional downstream genetic applications, including sequential lineage labeling, complex genomic rearrangements and the precise temporal and spatial control of gene expression through the intersection of partially overlapping promoter activities.The ability to achieve precisely tailored activation and inactivation of gene expression represents a critical utility for vertebrate model organisms. In this regard, Cre/lox and Flp/frt systems have been utilized for a variety of applications, including transgene activation, transgene excision, and transgene inversion Cre-like site-specific recombinase known as Dre was identified by sequencing homologous genomic regions of D6, a transducing bacteriophage related to the P1 phage from which Cre was isolated Cre, Dre is a site-specific tyrosine recombinase capable of catalyzing both excision and integration events, with no requirement for other phage-encoded or bacterial proteins. While Cre displays specificity for lox target sites, Dre recognizes a different target sequence known as rox. While lox and rox sites are similar in size (34 bp vs. 32 bp), sequence (21 nucleotides in common) and structure (inverted repeats flanking an asymmetric spacer), Cre has no ability to recombine rox sites and Dre similarly has no activity at lox sites, either in bacteria or in mammalian cells Site-specific DNA recombinases have become critical components of genome manipulation strategies in vertebrates. In zebrafish, both the Dre/rox system in mice Based on the recent successful application of the Tg (herein ubb-Dre), Tg (herein ubb-Cre), Tg(ubb:lox-Nuc-mCherry-stop-lox-eGFP) (herein Lox-Nuc-mCherry-Lox-eGFP), Tg(ubb:rox-Nuc-mCherry-stop-rox-eGFP) (herein Rox-Nuc-mCherry-Rox-eGFP), Tg (herein ubb-DrePR), T2;cmlc2:eGFP)Tg(ubb:CreER (herein T2CreER) Tg (herein LSL-Rox-Nuc-mCherry-Rox-eGFP). Dre and DrePR cDNAs were kindly provided by Dr. Francis Stewart. All new transgenic lines were generated using the backbone of T2KXIG\u0394IN All experiments involving zebrafish were approved by the Johns Hopkins University Institutional Animal Care and Use Committee. Fish were raised and maintained under standard laboratory conditions. The following strains were established and/or utilized: Cre activity in T2CreER-expressing embryos, 25\u201330 stage-matched embryos were incubated with E3 medium freshly mixed with 5 \u00b5M 4-OHT. To induce Dre activity in DrePR-expressing embryos, 25\u201330 stage-matched embryos were incubated with E3 medium freshly mixed with 4 \u00b5M RU486. The treated embryos were immediately put into a closed and dark 28.5\u00b0C incubator for 24 hrs. The embryos were subsequently placed in a fresh E3 medium and grown as described previously.4-Hydroxytamoxifen and RU486 were dissolved in ethanol at a final stock concentration of 10 mM and kept in single-use aliquots in the dark at \u201320\u00b0C. To induce DrePR recombination in a RU486-dose dependent manner, 25\u201330 stage-matched embryos (n\u200a=\u200a5) were treated with E3 medium freshly mixed with 0 \u00b5M, 1 \u00b5M, 2 \u00b5M, 4 \u00b5M, and 10 \u00b5M of RU486 between 24 and 48 hpf. Two days later, 4 dpf the larval zebrafish were fixed overnight in 4% PFA and the intestine was dissected and mounted in the mounting media . To quantify the efficiency of sequential T2CreER- and DrePR-mediated recombination, larval zebrafish (n\u200a=\u200a5) were treated sequentially with 4-OHT at 24 hpf and RU486 at 48 hpf, and fixed overnight in 4% PFA at 7 dpf. Following dissection, sections of intestine, liver, and pancreas were prepared. The total number of DAPI-labeled cells also labeled by either mCherry or eGFP was counted, with a minimum of 400 cells counted for each tissue.Tissue dissection and confocal microscopy were performed as described previously Dre-based recombination in zebrafish, we utilized standard Tol2-mediated transgenesis to establish Dre-driver and a Dre-responder lines in which relevant elements are expressed under the control of zebrafish ubiquitin b (ubb) promoter also incorporated a crystalline aa:Venus cassette ubb-Dre transgene integration. The Dre responder line contained a Nuc-mCherry-stop cassette flanked by 32 bp rox sites and followed by eGFP . Stable Dre responder lines were selected based on ubiquitous expression of ubb promoter-driven nuclear mCherry, evident shortly following the mid-blastula transition.As an initial evaluation of promoter . The ubbDre/rox and Cre/lox systems in larval zebrafish, we also established Cre-driver and Cre-responder lines under the control of the ubb promoter (To confirm non-overlapping specificities for the promoter . As in tox-eGFP) .Dre was active in larval zebrafish, we crossed male ubb-Dre fish to female Rox-Nuc-mCherry-Rox-eGFP fish. These experiments confirmed efficient Dre-based deletion of the Rox-Nuc-mCherry-Rox cassette, as evidenced by activation of eGFP expression ligand binding domain engineered to be selectively responsive to the synthetic ligand RU486. First, we established a DrePR-driver line under control of the ubb promoter. To facilitate the identification of DrePR-expressing embryos, this transgene also incorporated a crystalline aa:eCFP cassette ubb promoter were treated with E3 medium freshly mixed with RU486-dose dependent manner, 0 \u00b5M, 1 \u00b5M, 2 \u00b5M, 4 \u00b5M, and 10 \u00b5M. To quantify the efficiency of Dre recombination in Dre-expressing embryos and DrePR recombination in DrePR-expressing embryos, the intestine of larval zebrafish (4 dpf) was dissected and DAPI-labeled cells also labeled by either nucleus mCherry or cytoplasmic eGFP was counted. A dose-response curve revealed that the efficiency of RU486-induced DrePR activation became saturated at between 2 and 4 \u00b5M of RU486. No cytoplasmic eGFP-labeled cells were observd in DrePR embryos in the absence of RU486 (DrePR (68.9% at 4 \u00b5M of RU486) was similar to that of non-inducible Dre lacking the PR fusion (76.5%), indicating that C-terminal fusion of Dre with the PR does not interfere greatly with recombinase functionality also incorporated a crystalline aa:mCherry cassette ubb promoter. To generate a stable, temporally-regulated Cre driver line, we used a 4-OHT inducible CreERT2 element under the control of ubb promoter T2;cmlc2:eGFP,ubb:CreER subsequently referred to as T2ubb-CreER) also incorporated cmlc2:eGFP cassette to facilitate the identification of transgene-expressing embryos based upon cardiac expression of eGFP. Following the creation of double-transgenic T2ubb-CreER; ubb-DrePR fish, additional crosses were completed to assemble these T2CreER and DrePR driver alleles together with the LSL-Rox-Nuc-mCherry-Rox-eGFP reporter and after (96 hpf) treatment with RU486 showed no transgene-specific fluorescence besides that provided by the ocular mCherry, ocular eCFP and cardiac eGFP markers , 30\u201340% of cells undergoing single recombination mediated by CreERT2 (red mCherry fluorescence only), and 40\u201350% of cells undergoing sequential transgene activation and inactivation mediated by both T2CreER and DrePR (green eGFP fluorescence only).Not unexpectedly, embryos treated sequentially with 4-OHT and RU486 and harvested at 96 hpf continued to have a fraction of cells displaying ongoing red nuclear fluorescence .c.2, potDre/rox-based recombination as a new and effective method for temporally- and spatially-regulated genetic manipulation in zebrafish. In addition to this general utility, we present a new strategy for sequential activation and inactivation of inserted transgenes, referred to as TAILOR. Prior methods for temporally regulated transgene expression in zebrafish have included the use of thermally-inducible heat shock promoters Together, these studies establish Dre/rox to the zebrafish genetic toolbox will enable a variety of experimental strategies requiring precise temporal and spatial control of transgene expression. New TAILOR transgenes employing additional \u201cLSL-R-cDNA-R\u201d cassettes will facilitate a wide variety of experiments requiring sequential and cell type-specific transgene activation and inactivation. As in the case of our ubb:lox-stop-lox-rox-mCherry-stop-rox-eGFP allele, recombinase-mediated activation and inactivation of any LSL-Rox-cDNA-Rox cassette is likely to be mosaic. However the incorporation of fluorescent proteins into LSL-Rox-cDNA-Rox alleles, either by in-frame fusion or by 2A peptide-based independent translation Cre- and Dre-mediated recombination events, will allow ever more precise temporal and spatial regulation of transgene expression in highly selective cell types characterized by intersectional expression of both Cre and Dre. Dre/rox-based recombination will now also allow independent genetic manipulation of separate transgenes flanked by lox and rox sites, respectively. For example, Dre/rox-based recombination could be used to activate transgene expression in one cell population, with Cre/lox-mediated activation of a different transgene occurring in neighboring cells. As capabilities for gene targeting by endonuclease-facilitated homologous recombination Cre/lox and Dre/rox technology will also facilitate temporally regulated and cell type-specific manipulation of endogenous genetic loci.The current addition of"} +{"text": "Drosophila have contributed extensively to our understanding of nervous system development, physiology and behavior in addition to being valuable models of human neurological disease. Here, we have generated a novel series of modular transgenic vectors designed to optimize and accelerate the production and analysis of transgenes in Drosophila. We constructed a novel vector backbone, pBID, that allows both phiC31 targeted transgene integration and incorporates insulator sequences to ensure specific and uniform transgene expression. Upon this framework, we have built a series of constructs that are either backwards compatible with existing restriction enzyme based vectors or utilize Gateway recombination technology for high-throughput cloning. These vectors allow for endogenous promoter or Gal4 targeted expression of transgenic proteins with or without fluorescent protein or epitope tags. In addition, we have generated constructs that facilitate transgenic splice isoform specific RNA inhibition of gene expression. We demonstrate the utility of these constructs to analyze proteins involved in nervous system development, physiology and neurodegenerative disease. We expect that these reagents will facilitate the proficiency and sophistication of Drosophila genetic analysis in both the nervous system and other tissues.Transgenic Drosophila genetics combined with the advent of transformation with transposable elements vectors Drosophila a powerful model system in which to address a multitude of biological problems including the development and function of the nervous system Drosophila transgenic technology has been further improved by the ability to reproducibly target transgenes to specific genomic loci using \u03d5C31 phage integrase based DNA recombination attP) and a bacterial attachment site (attB) Drosophila transgenesis, an attP site is introduced into the genome using conventional transposon techniques attB site can then integrate at this \u2018landing\u2019 or \u2018docking\u2019 site when \u03a6C31 integrase is provided from either a co-injected mRNA The ability to express an exogenous gene in a transgenic animal with control over the timing, level and pattern of expression is essential for many types of experimental analysis. The sophistication of One drawback of the \u03c6C31 transgenesis technique is that the level of expression of integrated transgenes can differ depending upon the genomic location of the landing site. This can be somewhat mitigated by screening for landing sites that allow high levels of expression Drosophila \u03d5C31 transgenesis compatible vectors designed to allow specific and uniform transgene expression from different attP integration sites. Modifying a minimal attB containing vector that carries a mini-white gene B, Insulated, Drosophila), we have built new GAL4 inducible vectors where we have replaced Hsp70 promoter sequences with a Drosophila Synthetic Core Promoter (DSCP) attP landing sites and facilitate transgenic toxin generation. Building upon these constructs, we also generated novel UAS vectors that allow simple N or C-terminal fusion of transgenes to bright fast folding variants of Yellow or Red Fluorescent Proteins for in vivo imaging or protein epitopes for immunohistochemical or biochemical experiments. We find no evidence of unwanted expression from transgenes generated with these vectors in the absence of Gal4. Finally, we have used the pBID backbone to generate a vector that allows the rapid construction of UAS driven inverted hairpin sequences and show that these can be used for transgenic RNA inhibition (RNAi) of alternative splice isoforms of endogenous genes as a complement to existing whole genome libraries Drosophila.Here we have constructed a new series of Drosophila transgenes, including constructs introduced by \u03d5C31 transgenesis attP18 on the site on the X chromosome, attP40 site on chromosome 2 and the attP2 site on chromosome 3 B, Insulated, Drosophila) . The fincaz gene Drosophila ortholog of the ALS-associated gene FUS/TLS attP2 landing site and successfully identified integrants using white selection. We then determined if the caz genomic insert in pBID was functional by using these transgenes to rescue caz mutants, the majority of which die before adult eclosion caz genomic transgene could fully rescue caz mutant adult eclosion to control levels . We also introduced a Gateway cloning cassette into this vector to generate pBID-UASC-G.We next designed UAS versions of the pBID vector. As insulator elements have been associated with GAL4 independent expression from UAS vectors that employ attP landing sites using these vectors. To do this we used pBID-UASC-G to generate transgenes with a destabilized form of EGFP (dsEGFP) which has a protein half-life of approximately two hours providing a sensitive measure of transgene expression attP18 (X) and attP40 (2) landing sites. When we examined by western blot analysis the level of dsEGFP protein expressed from either line when driven in eye tissues with GMR-Gal4, we found similar levels of dsEGFP protein expression were produced by either insert or attP2 (3) landing sites. Expression of TeTxLC from these lines completely inhibited neurotransmitter release when expressed in motor neurons similar to existing p-element based lines (data not shown). Similar to dsEGFP, when driven with GMR-Gal4, we also did not observe any difference in the level TeTxLC protein expressed from inserts at either attP40 or attP2 variant mVenus expected . Using aexpected . Finallyexpected and puriexpected . TherefoattP2. We observed similar results with fluorescent fusion transgenes inserted at attP40 or attP18 (data not shown). To examine if leaky expression could be occurring below our detection threshold by light microscopy, we homogenized and generated protein extracts from whole larvae with UASC-MyctRFP-Caz transgenes in attP2 with or without G14 Gal4 fasII gene generates at least four protein isoforms by alternative splicing of several 3\u2032 exons fasII exons produce one of either of two FasII-A transmembrane isoforms (designated PEST+ or PEST-), a putative GPI linked isoform (FasII-C) and a fourth isoform (FasII-B) that remains poorly characterized. All isoforms of FasII protein are detectible with the monoclonal antibody 34B3 while only FasII-A isoforms are detected by the monoclonal antibody 1D4 To test the functionality of this vector, we inhibited alternative splice isoforms of Fasciclin II (FasII), the fasII exon 12, common to both FasII-A isoforms but not FasII-B or FasII-C, into pBID-UAS-GGi to generate UAS FasII-A RNAi and for Caenorhabditis elegans, where a modular restriction enzyme based vector series Analysis of transgenic genes and proteins in attP landing site. For example, reporter constructs lacking insulators exhibit large differences in expression levels from attP18, attP40 and attP2 landing sites attP landing sites, we cannot exclude the possibility that expression at other landing sites may not be similar due to incomplete protection by the insulating elements. Also for transgenic animal identification, our constructs use the mini-white gene, a common and easy to use selectable marker white containing transgenic constructs can cause aberrant behavior ZH-attP landing sites white gene to be flanked by both a loxP site derived from the pBID vector and loxP site present in these landing sites. Transgenic Cre can then be used delete the region between the loxP sites removing the mini-white selectable marker and other plasmid sequences while leaving the remainder of transgenic construct intact Our vectors offer a number of advantages but also some limitations over existing reagents. First, by design, our vectors incorporate insulator sequences to allow equivalent transgene expression independent of the Drosophila Synthetic Core Promoter as the basal promoter pers.comm) consistent with little or no Gal4 independent expression. However, as described in other studies hsp70 based basal promoters. This is a particular disadvantage for transgenic RNAi constructs where high levels of hairpin production are desirable Second, for all UAS vectors apart from pBID-UAS-GGi, we used a in vitro recombination based system allows robust high throughput parallel cloning of inserts into multiple vectors, easier cloning of large or complex inserts that may be refractory to restriction enzyme based cloning and simplifies the generation of fusion proteins Drosophila S2 or mammalian cell expression that can aid in protein analysis and compatible cDNA libraries exist for many mammalian genes. It is also noteworthy that as part of our construction process, we generated several intermediate vectors (pMartini-Gateway series) that can facilitate the subcloning of Gateway cassettes and the generation of new \u2018destination\u2019 vectors , even though it was expressed at similar levels to untagged constructs and similar fusions of TeTxLC to fluorescent proteins have been employed in hippocampal neurons and transgenic zebrafish Drosophila tissues Drosophila community will take advantage of the modularity of our constructs to expand upon and improve these vectors with further innovations.Finally, we have generated constructs that allow fusion to the yellow fluorescent protein mVenus http://www.addgene.org/a non-profit plasmid repository.All vector constructs have been deposited at Addgene Drosophila wild type Canton S genomic DNA with MCS-Gypsy and Gypsy-BamHI primers and cloned in pCR8GW-TOPO vector, resulting in pCR8GW-MCS-Gypsy-BamHI. The pUASTattB white, loxP, attB, AmpR and bacterial replication regions while removing other sequences. A 460-bp gypsy fragment released from pCR8GW-BamHI-Gypsy-MCS with BamHI/NotI and a 468-bp gypsy insulator released from pCR8GW-MCS-Gypsy-BamHI with BamHI/NotI were ligated into BamHI fragment to generate the pBID vector.A three-fragment ligation was used to generate the pBID vector. A 473-bp DNA fragment containing the Gypsy insulator sequence The 1711-bp Gateway Cassette Reading Frame A (Invitrogen) was blunt end cloned into the EcoRV site of pMartini , resulting in pMartini Gate A R1-R2 and in the opposite orientation pMartini Gate A R2-R1. The 1713-bp Gateway Cassette Reading Frame B (Invitrogen) was blunt end cloned into the EcoRV site of pMartini, resulting in pMartini Gate B R1-R2 and in the opposite orientation pMartini Gate B R2-R1. The 1714 bp Gateway Cassette Reading Frame C.1 (Invitrogen) was blunt end cloned into the EcoRV site of pMartini, resulting in pMartini Gate C R1-R2 and in the opposite orientation pMartini Gate C R2-R1.A 1763-bp Gateway cassette was excised from pMartini Gate A R2-R1 with XhoI/XbaI and cloned into XhoI/XbaI sites of pBID vector to generate pBID-G.. The 526-bp containing the 10X UAS region was then ligated into this vector. The resulting p10xUASTattB was digested with BamHI, and a 1264-bp BamHI fragment was inserted into BglII site of pBID to generate pBID-UAS.The 10X UAS region of pGD264 was excised with NotI and modified with Klenow, subsequently digested with BglII and the 526-bp fragment was gel purified. pUASTattB was treated sequentially with HindIII, Klenow and BglII to remove UAS sequencesDrosophila Synthetic Core Promoter) sequence was PCR amplified from pBPGUw with Gateway attR2 and EcoRI-DSCP reverse primers, and a 151-bp fragment released from the 344-bp DSCP fragment with SacI/EcoRI was cloned into SacI/EcoRI sites of pBluescript SK(+), resulting in pBS-DSCP. A 132-bp HindIII/SacI 5xUAS fragment of pCR8GW-5xUAS and a 159-bp SacI/EcoRI DSCP fragment of pBS-DSCP were inserted in HindIII/EcoRI sites of pBID-UAS, giving rise to pBID-UASC.pBID-UASC was constructed by three-fragment ligation. To this end, 255-bp 5xUAS sequence was PCR amplified from pUAST A 1763-bp Gateway cassette was released from pMartini Gate A R2-R1 with XhoI/XbaI and cloned into XhoI/XbaI sites of pBID-UASC, giving rise to pBID-UASC-G.Drosophila Kozak sequence added prior to start codon was PCR amplified from pSK2691 with HindIII-BglII-mVenus and mVenus-no stop-SphI primers, digested with HindIII/SphI and cloned into HindIII/SphI sites of pMartini Gate A R1-R2. The resulting pMartini-mVenus-G was digested with BglII/XhoI and the released 2480-bp fragment was inserted into BglII/XhoI sites of pBID-UASC, giving rise to pBID-UASC-VG.To construct a mVenus-Gateway cassette fusion, a 739-bp mVenus fragment with a To construct a Gateway-mVenus fusion, a 719-bp mVenus fragment was PCR amplified from pSK2691 with SphI-mVenus and mVenus-stop-XbaI primers, digested with SphI/XbaI and cloned into SphI/XbaI sites of pMartini Gate B R2-R1. The resulting pMartini-G-mVenus was digested with XhoI/XbaI and the released 2476-bp fragment was inserted into XhoI/XbaI sites of pBID-UASC, giving rise to pBID-UASC-GV.Drosophila Kozak sequence added prior to the start codon was PCR amplified from pCMV-3Tag-4C (Stratagene) with fly HindIII-3xMyc FWD and 3xMyc-XbaI REV primers. TagRFP-T was PCR amplified as 749-bp XbaI-TagRFP2-SphI fragment from pSK3007 with XbaI-TagRFP2 and TagRFP2-no stop-SphI primers. The 3x Myc fragment was cut with HindIII/XbaI, the TagRFP-T fragment with XbaI/SphI, and both were simultaneously ligated into pMartini Gate A R1-R2 prepared with HindIII/SphI. The resulting pMartini-d3myc-TagRFP2-G was cut with XhoI and released a 2626-bp d3myc-TagRFP2-G fragment, inserted into the XhoI site of pBID-UASC, giving rise to pBID-UASC-MRG.119-bp 3xMyc tag with a A 113-bp 3x Myc tag was PCR amplified from pCMV-3Tag-4C (Stratagene) with XbaI-3xMyc FWD and 3xMyc-stop-HindIII REV primers. TagRFP-T was PCR amplified as 749-bp SphI-TagRFP2-XbaI fragment from pSK3007 with SphI-TagRFP2, TagRFP2-no stop-XbaI primers. The 99-bp 3x Myc fragment was cut with XbaI/HindIII, the 731-bp TagRFP-T fragment with SphI/XbaI, and both were simultaneously ligated into pMartini Gate B R2-R1 prepared with SphI/HindIII, creating pMartini-G-TagRFP2\u20133Myc. A 2606-bp G-TagRFP2\u20133Myc EcoRI/XhoI fragment derived from pMartini-G-TagRFP2\u20133Myc was cloned into EcoRI/XhoI site of pBID-UASC, giving rise to pBID-UASC-GRM.Drosophila Genomics Resource Center (DGRC)) with XhoI/NheI and cloned into XhoI/XbaI sites of pBID-UASC, resulting in pBID-UASC-FG.A 3xFLAG tag followed by Gateway cassette was excised from pAFW . White and attB primers were used for sequencing the insertion in pBID vectors. The DSCP primer was used for sequencing inserts after a DSCP. The SV40 pA primer was used for sequencing inserts in front of SV40 polyA. All Primers are described in PCR was performed using AccuPrime To generate pBID-UASC-dsEGFP, dsEGFP was PCR-amplified from pd2EGFP-N1 (Clontech) using dsEGFP F and R primers. By PCR, an NLS sequence was added the N-terminus and NotI site inserted between amino acids 154 and 155 to facilitate future reporters. The construct was inserted into pBID-UASC using BglII and XhoI.Tetanus toxin light chain (TeTxLC) construct pBID-UASC-TeTxLC was derived by an LR reaction between pBID-UASC-G and entry plasmid pCR8GW-TeTxLC .The TeTxLC with a C-terminal mVenus fusion gene construct pBID-UASC-TeTxLC-mVenus was derived by an LR reaction between pBID-UASC-GV and the entry plasmid pCR8GW-TeTxLC-no-stop .Drosophila TBPH Drosophila Caz with an N-terminal MyctRFP fusion was generated by an LR reaction between pBID-UASC-MRG and the Caz entry plasmid pCR8GW-Caz Drosophila Caz with an N-terminal Flag fusion was derived by an LR reaction between pBID-UASC-FG and the Caz entry plasmid pCR8GW-Caz . The PCR product was TOPO cloned into the pCR8GW-TOPO vector (Invitrogen) to generate pCR8GW-FasII-A. An LR reaction between pCR8GW-FasII-A and pBID-UAS-GGi was used to generate pBID-UAS-FasII-A RNAi.UAS-FasIIRNAi-A The GAL4 lines used were the eye driver GMR-GAL4 attP18 (X chromosome), attP40 (chromosome II), or attP2 (chromosome III) landing sites Transgenes were inserted into the Dissection and immunohistochemistry was performed as previously described Adult fly heads were directly homogenized in sample loading buffer. The heads extract was centrifuged at 16,000 rpm for 5 minutes, subjected to 10% or 12% SDS-PAGE, transferred to Protran\u00ae nitrocellulose membrane (Whatman), and detected with Pierce\u00ae ECL Western Blotting Substrate (Thermo Scientific). The primary antibodies used were: mouse anti-tetanus toxin light chain , rabbit anti-GFP , mouse anti-\u03b2-tubulin .Table S1Sequence of PCR primers employed.(PDF)Click here for additional data file."} +{"text": "Serum response factor (SRF) acts as a multifunctional transcription factor regulated by mutually exclusive interactions with ternary complex factors (TCFs) or myocardin-related transcription factors (MRTFs). Binding of Rho- and actin-regulated MRTF:SRF complexes to target gene promoters requires an SRF-binding site only, whereas MAPK-regulated TCF:SRF complexes in addition rely on flanking sequences present in the serum response element (SRE). Here, we report on the activation of an SRE luciferase reporter by Tip, the viral oncoprotein essentially contributing to human T-cell transformation by Herpesvirus saimiri. SRE activation in Tip-expressing Jurkat T cells could not be attributed to triggering of the MAPK pathway. Therefore, we further analyzed the contribution of MRTF complexes. Indeed, Tip also activated a reporter construct responsive to MRTF:SRF. Activation of this reporter was abrogated by overexpression of a dominant negative mutant of the MRTF-family member MAL. Moreover, enrichment of monomeric actin suppressed the Tip-induced reporter activity. Further upstream, the Rho-family GTPase Rac, was found to be required for MRTF:SRF reporter activation by Tip. Initiation of this pathway was strictly dependent on Tip's ability to interact with Lck and on the activity of this Src-family kinase. Independent of Tip, T-cell stimulation orchestrates Src-family kinase, MAPK and actin pathways to induce SRF. These findings establish actin-regulated transcription in human T cells and suggest its role in viral oncogenesis. Serum response factor (SRF) is widely expressed in both invertebrates and vertebrates. SRF plays an essential role in embryogenesis, but is also involved in multiple processes in developed organisms including neuronal and muscle cell function.SRF binds as a dimer to a specific DNA sequence known as the CArG box in the promoter of hundreds of target genes. Selective binding is determined by interactions with more than 60 different cofactors, which turn SRF into a versatile transcription factor translating cell- and stimulus-specific signaling into selective target gene expression ,2.c-fos promoter, contains an Ets motif adjacent to the CArG box . Results were assigned to the categories p > 0.05 , p < 0.05 (*), p < 0.01 (**), p < 0.001 (***).Jurkat T cells were transfected with 20 \u03bcg of the individual effector plasmids and 10 \u03bcg of the reporter plasmid pSRE-luc containing five SRE of the c-fos promoter (Stratagene) or p3D.A-Luc comprisi6 cells/ml. The SFK inhibitor PP2 and the MAPK inhibitors U0126 and PD0325901 (1 \u03bcM) were added 8 h post transfection and remained in the cultures until harvesting of the cells. 12-O-tetradecanoylphorbol-13-acetate , combined with MAPK inhibitors if applicable, was added for 15 h. To modulate actin polymerization, cells were treated with Latrunculin B , Cytochalasin D for 24 h. Under these conditions all inhibitors were not toxic to Jurkat T cells as measured by propidiumiodide staining and flow cytometry. T cell-receptor stimulation of transfected Jurkat T cells was carried out for 14 h in a 6-well plate at a density of approximately 1 \u00d7 106 cells/ml previously coated with antibodies against CD3 and CD28 .For inhibitor treatment, transfected Jurkat T cells were seeded in a 12-well plate at a density of approximately 0.5 \u00d710O-tetradecanoylphorbol-13-acetate; RhoGTPases: Guanosine triphosphatases of the Rho family; SFK: Src-family kinase; SH3: Src homology domain 3 binding sequence; SRE: Serum response element; SRF: Serum response factor; STAT: Signal transducer and activator of transcription; StpC: Saimiri transformation-associated protein of subgroup C; TCF: Ternary complex factor; Tip: Tyrosine interacting protein.CSKH: Sequence homologous to the C-terminus of Src-family kinase domains; ERK: Extracellular signal-regulated kinase; G-actin: Globular actin; GEF: Guanine nucleotide exchange factor; GTP: Guanosine triphosphate; HVS: Herpesvirus saimiri; MAPK: Mitogen-activated protein kinase; MRTFs: Myocardin-related transcription factors; MYH9: Myosin heavy chain 9: non muscle; MYL9: Myosin light chain 9: regulatory; PMA: 12-The authors declare that they have no competing interests.All authors have read and approved the final manuscript. BB conceived and approved the experiments. KK and JS performed the experiments. KK, SJdJ, JCA, JS, HG, GP and BB analyzed and interpreted the data. BB and KK drafted the manuscript."} +{"text": "In prokaryotes, the Shine-Dalgarno (SD) sequence in the 5\u2019 untranslated region (UTR) of mRNA forms a duplex with the anti-Shine-Dalgarno (anti-SD) sequence to initiate translation . The mecBy comparison analysis of 1,182 completely sequenced eubacterial genomes, we found fifteen genomes in which no 16S rRNA genes carry the anti-SD motif. Loss of the anti-SD motif in these \u2018non-anti-SD\u2019 genomes is always accompanied by loss of SD-like sequences in 5\u2019 UTR. These non-anti-SD genomes belong to either the phylum Proteobacteria (n=3), the class Flavobacteria (n=9), or the genus Mycoplasma (n=3) (Figure The non-anti-SD genomes we reported here represent an extreme aspect of the dynamic evolution in the translation initiation mechanisms, strongly indicating extinction of SD/anti-SD interaction in some bacterial lineages. Attentive analysis of such peculiar genomes led us to suggest likely factors that gave rise to the loss. First, advanced association with eukaryotic hosts and subsequent immense genomic/functional reduction. Second, intra-genomic dominance of SD-independent translation initiation mechanisms. Finally, hemotropic environment.Our study described here has been published as an original research article ."} +{"text": "To evaluate the ability of velocity encoded (VENC) magnetic resonance imaging (MRI) to identify patients with diastolic dysfunction in comparison with Doppler-echocardiography.Diastolic dysfunction was recently recognized as an important cause for heart failure in patients with preserved ejection fraction. Diastolic dysfunction is typically assessed in clinical routine using transmitral and pulmonary-venous flow characteristics by Doppler-echocardiography. We hypothesized that VENC-MRI has a similar ability to identify patients with diastolic dysfunction compared to Doppler-echocardiography.The study included 76 patients with normal systolic left-ventricular function and a high probability of diastolic dysfunction. Transmitral flow and pulmonary venous flow profiles were obtained by VENC-MRI and Doppler-echocardiography. Measurements included maximal early- and late-diastolic transmitral velocities (E- and A-waves) as well as maximal systolic and diastolic pulmonary venous velocities (S- and D-wave). Furthermore, VENC-MRI was used to assess total diastolic left ventricular (LV) filling as well as the early and late portions of diastolic LV filling.Doppler-echocardiography identified 38 patients with diastolic dysfunction. 24 patients had grade I diastolic dysfunction, 8 patients hat grade II diastolic dysfunction and 3 patients had grade 3 diastolic dysfunction. There was a very good correlation between VENC-MRI and Doppler-echocardiography for the E/A-ratio using peak velocities . A moderate correlation was found for the S/D-ratio between VENC-MRI and Doppler-echocardiography using peak velocities . In grade I diastolic dysfunction, the contribution of early LV filling to total LV filling was significantly lower compared to patients with normal diastolic function or higher grades of diastolic dysfunction (Figure VENC-MRI has the ability to identify diastolic dysfunction by measurements of transmitral flow velocities. Furthermore, the assessment of early and late LV diastolic filling volumes may provide additional information for the grading of diastolic dysfunction."} +{"text": "Dendritic cells (DCs) are promising mediators of anti-tumor immune responses due to their potent antigen-presentation capacity. Unfortunately, cancer cells can often disarm differentiated DCs by rendering them incapable of maturation or by promoting their apoptosis. DC vaccine regimens attempt to generate functional DCs and preload them with Tumor-Associated Antigens (TAAs) to target various malignancies. Despite these efforts, the efficacy of DC vaccines in clinical trials is still rather disappointing to date. In addition to undergoing cancer-induced apoptosis, it is well established that DCs are intrinsically short-lived cell types. It is likely that a significant portion of infused DCs undergo apoptosis prior to locating and activating na\u00efve TAA-reactive T cells., rat HER-2/neu (rHER-2), along with five candidate mouse DC survival factors that operate in both the extrinsic and intrinsic cycles of apoptosis. The murine DC cell line, DC2.4 was transduced separately with each novel LV construct. Infected cells were enriched via flow cytometric methods based on rHER-2 expression. Transduced DC2.4 cell lines were then exposed to Fetal Calf Serum (FCS) withdrawal and to specific pharmacological apoptosis-inducing agents. DC2.4 cell death was assayed based on Annexin V and PI double-positive staining via flow cytometry. The phenotype and function of transduced DC2.4 cells and primary bone marrow-derived DCs were then assessed via expression and secretion of DC markers and cytokines, respectively.In our current study, we constructed and investigated novel bicistronic lentivectors (LVs) encoding the cDNA for the xeno-TAAS, c-FLIPL, Bcl-XL, and M11L were protected from apoptosis when exposed to low FCS-containing culture media. When treated with an anti-CD95 antibody, only DC2.4 cells transduced with LVs encoding c-FLIPS and c-FLIPL were protected from apoptosis. In contrast, only DC2.4 cells transduced with LVs encoding Bcl-XL and M11L were protected from effects of staurosporine (STS) treatment. Also, LV-modified DCs maintained their original phenotype and function.DC2.4 cells transduced with LVs encoding cDNAs for c-FLIPWe present evidence that by employing novel recombinant bicistronic LVs we can simultaneously load DCs with a relevant TAA and block apoptosis; thereby confirming the usage of such LVs in the modulation of DC lifespan and function. Bcl-2, Bcl-XL, and Bfl-1 were then amplified by PCR from the respective subcloned pDIG/EMCV IRES vectors using primers designed to be flanked by either SalI or PspXI restriction enzyme sites for subsequent non-directional molecular subcloning steps. The primers used for PCR amplification were as follows: Table\u00a0The cDNAs for c-FLIPS/c-FLIPL/Bcl-XL/M11L/AKT-1] nucleotide fragments were then subcloned into the previously engineered lentivirus SIN transfer vector pCCL-rHER-2/neu using the restriction enzyme sites SalI or the SalI-compatible restriction enzyme site, PspXI Clone AF6-120.1 (BD Biosciences), Mouse IgG2a\u03ba isotype Clone G155-178 (BD Biosciences). rHER-2 staining was performed as described above for DC2.4 cells.DC2.4 cell lines were cultured as described above and matured using recombinant murine IFN-\u03b3 at a concentration of 100 ng/mL for 24 hours and subsequently harvested for flow cytometry analyses. BMDCs were cultured, differentiated, transduced, and matured as previously described . On day Twenty-four hours after maturation, culture supernatants were collected for cytokine secretion quantification. Murine IL-6 and murine TNF-\u03b1 were detected using the BD OptEIA ELISA kits (BD Biosciences) as per the manufacturer\u2019s instructions.p values < 0.05 were designated as statistically significant (*).Experiments were performed at least twice independently unless otherwise stated and representative data are shown with error bars representing standard deviations. For statistical comparison of treatment groups, two-tailed unpaired Student\u2019s t-tests were performed. BMDC: Bone marrow-derived dendritic cells; DC: Dendritic Cell; c-FLIP: Cellular FLICE Inhibitory Protein; EMCV: Encephalomyocarditis Virus; FCS: Fetal Calf Serum; FADD: Fas-Associated Death Domain; IRES: Internal Ribosomal Entry Site; LV: Lentivector; MOI: Multiplicity of Infection; NT: Non-Transduced; rHER-2: rat Human Epidermal growth factor Receptor 2; STS: Staurosporine; TAA: Tumor-Associated Antigen; TRAIL: TNF Related Apoptosis-Inducing Ligand; WPRE: Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element.The authors declare no potential conflicts of interest.JCMW, TCF, and BCYA performed experiments. JCMW, TCF, DHF, GAD, and JAM participated in the design of the study. JCMW and JAM wrote the manuscript. All authors read, edited, and approved the final manuscript."} +{"text": "The ability of HIV-1 to rapidly accumulate mutations provides the virus with an effective means of escaping CD8+ cytotoxic T lymphocyte (CTL) responses. Here we describe how subtle alterations in CTL epitopes expressed by naturally occurring HIV-1 variants can result in an incomplete escape from pre-existing CTL recognition to create a pro-inflammatory environment, providing the virus with a selective advantage.We developed a dendritic cell (DC)-based in vitro model to induce primary CTLs specific against naturally occuring HIV-1 Gag epitope variants identified through sequencing of virus obtained in a longitudunal study from a subject with chronic HIV-1 infection. CTL function was assessed by ELISPOT, flow cytometry, and 51Cr-release cytoxicity assays.The HIV-1 specific CTLs generated proved to be broadly cross-reactive. While the magnitudes of the responses between the variant and priming peptides were often similar, important qualitative differences were found. Most notably, epitope variants preferentially induced the \"helper\" activity of the CTLs while inhibiting their killing capacity. Importantly, instead of eliminating variant antigen-expressing immature DC as a negative immune feedback mechanism, the cross-reactive CTLs promoted the differentiation of DC into highly reactive mature DC capable of producing enhanced levels of IL-12 and IL-6, as well as the T cell chemoattractants CXCL10 and CCL5. These CTL-matured DC also develped long interconnected nanotubule extensions capable of facilitating intracellular transfer of HIV-1.The selective induction of pre-existing CTL \"helper\" activity in the absence of killing, induced by altered peptide presentation, adds a novel dimension to the concept of \u201coriginal antigenic sin\u201d. This phenomenon likely contributes to the chronic immune activation associated with HIV-1 infection and could be utilized by HIV-1 to promote spread and persistence. Developing a means to specifically interrupt the described CTL-DC positive immune feedback loop could prove critical for the effectiveness of future anti-HIV-1 vaccine therapies."} +{"text": "TAR-DNA binding protein 43 (TDP-43) is a heterogeneous ribonucleoprotein (hnRNP) identified as a major protein constituent of cytosolic inclusions in spinal cord of patients with motor neuron disease. The inclusions are formed by movement of TDP-43 from its predominantly nuclear localization to the cytosol, followed by accumulation of ubiquitinated and phosphorylated C-terminal TDP-43 fragments. The mechanisms controlling TDP-43 trafficking and accumulation are not well known, however, it has been demonstrated previously that kinases control movement and accumulation of hnRNPs. Therefore, we investigated whether kinases also controlled TDP-43 accumulation during cell stress.Neuronal cultures were treated with the mitochondrial inhibitors paraquat or sodium arsenite, or transfected with C-terminal TDP-43 or full length TDP-43 constructs and examined for kinase control of TDP-43 localization.We found that stress induction induced robust cytosolic accumulation of C-terminal TDP-43 into RNA stress granules, some of which progress to ubiquitinated inclusions. Inhibitors of c-Jun N-terminal kinase (JNK) and cyclin-dependent kinase 2 (CDK2) specifically inhibit and/or reverse the cytosolic accumulation of TDP-43 with little effect on ubiquitous stress granule formation. Our studies have shown active CDK2 co-localizes with accumulated TDP-43 and heterogeneous ribonucleoprotein K (hnRNP K). CDK2 inhibition blocks phosphorylation of hnRNP K, preventing its incorporation into stress granules. Due to Interaction between hnRNP K with TDP-43, the loss of hnRNP K from stress granules prevents accumulation of TDP-43. We have also determined that specific C-terminal mutations to TDP-43 alter expression of hnRNP K during cell stress. Preliminary studies indicate that this may involve altered hnRNP K phosphorylation.Due to the recently identified role for hnRNPs in motor neuron disease and frontotemporal dementia, further investigation of the association between hnRNP K and TDP-43 is warranted. Understanding how kinase activity modulates TDP-43 accumulation may provide new pharmacological targets for disease intervention."} +{"text": "Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV). A sequence of the CBSV coat protein (CP) highly conserved between the two virus species was used to demonstrate that a CBSV-CP hairpin construct sufficed to generate immunity against both viral species in the cassava model cultivar (cv. 60444). Most of the transgenic lines showed high levels of resistance under increasing viral loads using a stringent top-grafting method of inoculation. No viral replication was observed in the resistant transgenic lines and they remained free of typical CBSD root symptoms 7 month post-infection. To generate transgenic cassava lines combining resistance to both CBSD and CMD the hairpin construct was transferred to a CMD-resistant farmer-preferred Nigerian landrace TME 7 (Oko-Iyawo). An adapted protocol allowed the efficient Agrobacterium-based transformation of TME 7 and the regeneration of transgenic lines with high levels of CBSV-CP hairpin-derived small RNAs. All transgenic TME 7 lines were immune to both CBSV and UCBSV infections. Further evaluation of the transgenic TME 7 lines revealed that CBSD resistance was maintained when plants were co-inoculated with East African cassava mosaic virus (EACMV), a geminivirus causing CMD. The innovative combination of natural and engineered virus resistance in farmer-preferred landraces will be particularly important to reducing the increasing impact of cassava viral diseases in Africa.Cassava brown streak disease (CBSD) and cassava mosaic disease (CMD) are currently two major viral diseases that severely reduce cassava production in large areas of Sub-Saharan Africa. Natural resistance has so far only been reported for CMD in cassava. CBSD is caused by two virus species, Manihot esculenta Crantz) production in large areas of Africa is severely affected by two major viral diseases, cassava brown streak disease (CBSD) and cassava mosaic disease (CMD). CBSD is caused by two virus species, Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV) (picorna-like (+) ssRNA viruses, genus Ipomovirus, family Potyviridae) Nicotiana benthamianaCassava (Manihot glaziovii Muell.-Arg.) CMD2 resistance gene. CMD resistance markers also allowed the introgression of the CMD resistance trait into farmer-preferred cassava cultivars Because of the occurrence of CMD pandemics in Africa the cassava research community has traditionally focused its activities on CMD resistance. Breeding programs were conducted by several national programs and later at CGIAR centers to exploit natural CMD resistance using locally grown cultivars and a wild-relative of cassava (Exploitation of the plant immune system against viruses In vitro culture of the cv. 60444 and the Nigerian landrace TME 7 (Oko-Iyawo) were used for cassava transformation. CBSV-[TAZ:DES:01] (GenBank JN091565.1) and UCBSV-[UG:Kab4\u223607] (GenBank HM346952) isolates were initially propagated in AR34 and Ebwanateraka cassava cultivars, respectively. The CBSV isolate was transferred to cv. 60444 by grafting. CBSV-infected cv. 60444 scions were grafted onto EACMV-Ug (GenBank Z83257)-infected Ebwanateraka rootstocks. Upon development of typical CMD symptoms scions were subsequently multiplied to generate cv. 60444 rootstock plants co-infected with CBSV and EACMV-Ug. All cassava plants were grown under standard greenhouse conditions .Agrobacterium tumefaciens LBA4404.The CBSV coat protein (CP) sequence from position 538 to 1063 (GenBank JN091565.1) was amplified from CBSV-infected cassava following reverse transcription. Inverted sequences of the binary expression vector pRNAi-dsAC1 cv. 60444 and TME 7 were transformed following an improved transformation protocol \u22121 were considered as potential target sites by CBSV-CP hairpin-derived small RNAs.CBSV and UCBSV sequences were aligned using the Clustal W method (DNASTAR Lasergene). A virtual repertoire of all possible 21nt small RNA sequences derived from the CBSV-CP hairpin was generated and used to predict targets on the UCBSV sequence using RNAduplex (Vienna package) HindIII enzyme and blotted onto Hybond membrane . Membranes were hybridized with DIG-labeled (Roche Diagnostics) hptII probe amplified using hptII specific primers (hptII-F:5\u2032-TCTCGATGAGCTGATGCTTTGG and hptII-R:5\u2032-AGTACTTCTACACAGCCATCGG).Twenty \u00b5g of genomic DNA were digested with 32P-labelled oligonucleotide probe complementary to the CBSV-CP sequence (5\u2032-ATCAGAATAGTGTGTCTGCTGG) and visualized using a phosphorimager (Bio-Rad Laboratories).Small RNA detection from selected independent transgenic lines was performed according to a previously established protocol Scions from three-month old transgenic and wild-type cassava plants were grafted onto three-month-old wild-type rootstocks multiplied from virus-infected stock plants. EACMV-Ug and CBSV were co-inoculated in cassava by grafting. A standard top grafting procedure was used for cassava inoculation with CBSV PP2A gene as internal control CBSV-CPF: 5\u2032-GAAGTTGAGAATTGGGCCATC and CBSV-CPR: 5\u2032-CAGCTCTCCACGATTTCTCATT) and for UCBSV (UCBSV-CPF: 5\u2032-CCATTTGAGGCTAGGAGATTGA and UCBSV-CPR: 5\u2032-ACTTCCCCATCATCTGGTTCTC) were designed and validated for qPCR.A standard leaf sampling procedure was performed on infected rootstocks and inoculated scions. In the standard sampling procedure three leaves below the point of grafting were sampled and pooled for RNA extraction. Similarly, three fully expanded leaves above the point of grafting were sampled and pooled to quantify viral load in the inoculated scions. Viral loads were quantitated by RT-qPCR based on a method for precise CBSV quantitation in cassava using the in silico analysis to produce the majority of the small interfering RNAs (siRNAs) with high similarity to both CBSV- and UCBSV-CP sequences in silico analysis that the UCBSV-CP sequence is a potential target of a large number of hairpin-derived small RNAs based on the free energy of siRNA-target duplexes was optimized using the improved transformation protocol Nicotiana benthamianaViruses develop various types of interactions when co-inoculated in a host plant Selected transgenic TME 7\u2013Hp (TME 7-Hp 11 & TME 7-Hp 12) scions were grafted on cv. 60444 rootstocks co-inoculated with CBSV and EACMV-Ug. Rootstock plants infected with the two viral species developed typical CMD symptoms. Sporadic CBSV-associated symptoms were observed on leaves with reduced CMD-associated symptoms . CBSV anThe emergence of new viral diseases based on the rapid evolution of existing viruses is threatening and particularly damaging crop production in many tropical countries Papaya ringspot virus in transgenic papaya has so far provided the most durable resistance against a plant RNA virus Our objective was to generate cassava plants resistant to both CMD and CBSD. Post-transcriptional gene silencing (PTGS) of the CP gene of in silico analysis using free energy of siRNA-target duplexes showed that our CBSV-CP hairpin could generate many small RNAs potentially targeting virus isolates from both CBSV and UCBSV species.Similarity between hairpins and their targeted viral sequences has previously been hypothesized to restrict the hairpin-based resistance in transgenic plants to virus species and isolates sharing more than 90% sequence similarity In our study the transgenic cassava lines expressing hairpin RNA fully homologous to CBSV were consistently resistant to both CBSV and UCBSV using grafting as a stringent inoculation procedure. The precise virus quantitation method In addition, our evaluation of engineered CBSD resistance using co-infection of viruses causing CMD and CBSD demonstrated that replicating EACMV-Ug show no or limited interference with the hairpin-based RNAi resistance strategy against CBSD when engineered in a CMD-resistant landrace.Engineering resistance to the many viral species infecting cassava in the field is a difficult and lengthy task that can be facilitated by exploiting naturally occurring virus resistance in cassava germplasm. Optimization of the transformation protocol for TME cassava landraces Figure S1Alignment of selected CBSV-CP and UCBSV-CP sequences . Alignment was performed using the Clustal W method. (DNASTAR Lasergene). Similarity with the CBSV-TAZ-DES01 is highlighted with black boxes. Sequence used for CBSV-CP hairpin construction is indicated by red arrows.(TIFF)Click here for additional data file.Figure S2Alignment of UCBSV-[UG:Kab4\u223607]-CP and CBSV-[TAZ:DES:01]-CP partial sequences. Short sequences (21nt) derived from CBSV-[TAZ:DES:01]-CP forming RNA duplexes with UCBSV-CP sequence are plotted below the alignment. Detailed information about RNA duplexes is reported in (EPS)Click here for additional data file.Figure S3Southern blot analysis of transgenic cv. 60444 (A) and TME 7 (B) plantlets using the hptII probe.(TIF)Click here for additional data file.Figure S4Detection of hairpin-derived small RNAs in selected independent transgenic lines. Hairpin-derived small RNAs in transgenic cv. 60444 (A) and TME 7 (B) lines were detected with a CBSV-CP specific oligoprobe.(TIF)Click here for additional data file.Figure S5CBSV and UCBSV quantitation in AR34 rootstocks and cv. 60444 scions. CBSV quantitation by qPCR in CBSV-infected AR34 roostock plants and corresponding grafted scions from transgenic, wild-type and control lines. Numbers following the cassava line identifiers indicate the biological replicates.(TIFF)Click here for additional data file.Figure S6Symptoms associated with CBSV and EACMV-Ug co-infection in cassava leaves. A. Fully expanded leaf of wild-type cv. 60444 rootstock co-inoculated with CBSV and EACMV-Ug. B. Fully expanded leaf of wild-type TME 7 scion grafted on a CBSV and EACMV-Ug co-inoculated rootstock.(TIF)Click here for additional data file.Table S1Summary table of small RNA duplexes between 21nt CBSV-CP hairpin-derived small RNA and UCBSV-CP target sequence.(XLSX)Click here for additional data file.Table S2Summary table of phenotypic and molecular data of the 60444 - Hp transgenic scions. Transgenic 60444-Hp scions were grafted on CBSV-infected cv. 60444 rootstocks.(DOCX)Click here for additional data file.Table S3Summary table of phenotypic and molecular data of the 60444 - Hp transgenic scions. Transgenic 60444-Hp scions were grafted on CBSV-infected AR34 rootstocks.(DOCX)Click here for additional data file.Table S4Summary table of phenotypic and molecular data of the 60444 - Hp and TME 7 - Hp transgenicscions. Transgenic 60444-Hp and TME 7-Hp scions grafted on UCBSV-infected Ebwanateraka rootstocks.(DOCX)Click here for additional data file.Table S5Summary table of phenotypic and molecular data of the TME 7 - Hp transgenic scions. Transgenic TME 7-Hp scions were grafted on CBSV-infected AR34 rootstocks.(DOCX)Click here for additional data file."} +{"text": "Drosophila, RNAi targeting of either dGyk or dGK can result in two alternative phenotypes: adult glycerol hypersensitivity or larval lethality. Here we compare these two phenotypes at the level of glycerol kinase (GK) phosphorylation activity, dGyk and dGK-RNA expression, and glycerol levels. We found both phenotypes exhibit reduced but similar levels of GK phosphorylation activity. Reduced RNA expression levels of dGyk and dGK corresponded with RNAi progeny that developed into glycerol hypersensitive adult flies. However, quantification of dGyk/dGK expression levels for the larval lethality phenotype revealed unexpected levels possibly due to a compensatory mechanism between dGyk and dGK or RNAi inhibition. The enzymatic role of glycerol kinase converts glycerol to glycerol 3-phosphate. As expected, elevated glycerol levels were observed in larvae that went on to develop into glycerol hypersensitive adults. Interestingly, larvae that died before eclosion revealed extremely low glycerol levels. Further characterization identified a wing phenotype that is enhanced by a dGpdh null mutation, indicating disrupted glycerol metabolism underlies the wing phenotype. In humans, glycerol kinase deficiency (GKD) exhibits a wide range of phenotypic variation with no obvious genotype-phenotype correlations. Additionally, disease severity often does not correlate with GK phosphorylation activity. It is intriguing that both human GKD patients and our GKD Drosophila model show a range of phenotype severity. Additionally, the lack of correlation between GK phosphorylation and dGyk/dGK-RNA expression with phenotypic severity suggests further study including understanding the alternative functions of the GK protein, could provide insights into the complex pathogenic mechanism observed in human GKD patients.In Drosophila GKD model Glycerol kinase (GK) is an enzyme that catalyzes the conversion of glycerol to glycerol 3-phosphate in an ATP dependent reaction Drosophila glycerol kinase genes dGyk (CG18374) or dGK (CG7995) results in two alternative phenotypes: larval lethality or glycerol hypersensitive adult flies rd instar larvae that developed into glycerol hypersensitive adults revealed successful targeting of dGyk and dGK that correlated with reduced glycerol kinase phosphorylation activity and elevated glycerol levels. Glycerol hypersensitive flies die rapidly when placed on a food source supplemented with glycerol, and sensitivity is enhanced by null mutations in eye pigmentation genes in vivo glycerol has been shown to play an important role in the control of water balance and insect desiccation resistance RNAi targeting of the dGyk- and dGK-RNA expression and glycerol levels in 3rd instar larvae for both glycerol hypersensitive and larval lethality phenotypes. This analysis revealed GK phosphorylation levels were reduced but similar for both phenotypes. Further analysis detected distinct dGyk and dGK expression patterns between the two phenotypes. As expected, elevated glycerol levels were detected in 3rd instar larvae that went on to develop into glycerol hypersensitive flies. However, 3rd instar larvae that died before eclosion had below normal levels of glycerol, suggesting the existence of a deleterious metabolic pathway. Additionally, a crumpled wing phenotype was produced by RNAi targeting of dGyk, the severity of which was enhanced by a null mutation of the glycerol 3-phosphate dehydrogenase (dGpdh) gene, the next step in the glycerol metabolism pathway, indicating that this wing phenotype was caused by disrupted glycerol metabolism.We hypothesized that phenotypic severity would correlate with glycerol kinase phosphorylation activity and expression level of the RNAi target gene. Therefore, we compared glycerol kinase phosphorylation, dGyk- and dGK-RNA expression levels, and glycerol levels is similar to the complexity observed in GKD clinical studies. Therefore further study of this Drosophila model for GKD could provide powerful insight into the complex pathogenic mechanism that underlies the wide range of phenotype severity observed in human GKD patients.We propose that the lack of correlation between RNAi phenotype severity with glycerol kinase phosphorylation activity, dGyk or dGK expression, named dGyk-IR and dGK-IR respectively (IR: inverse repeat) was initially performed using a Tubulin-GAL4 (Tub-GAL4) driver for ubiquitous expression of the inserted construct. Each RNAi fly line was crossed to the Tub-GAL4 driver fly line and the progeny examined for physical phenotypes dGyk-IR-sur and dGK-IR-sur) or lethality during larval development (named dGyk-IR-let and dGK-IR-let). Adult flies were subsequently found to be hypersensitive to glycerol Analysis of RNAi fly lines targeting pUdsGFP RNAi vector co-expresses GFP) are greater in dGyk-IR-let/Tub-GAL4 3rd instar larvae as compared to dGyk-IR-sur; Tub-GAL4 3rd instar larvae dGK-IR-let/Tub-GAL4 compared to dGK-IR-sur; Tub-GAL4 3rd instar larvae. This indirect measure of the inverse repeat (IR) expression levels suggested that the larval lethality phenotype was due to greater expression of the IR expression construct and consequently lower levels of dGyk or dGK. Here we characterize the larval lethality phenotype and perform a comparison of the larval lethality phenotype to the glycerol hypersensitive phenotype at the level of GK phosphorylation activity, dGyk/dGK-RNA expression, and glycerol levels.We have previously shown that GFP levels (the dGyk or dGK should result in decreased GK activity. Using radiolabelled 14C glycerol to assay for glycerol kinase (GK) phosphorylation activity, we found decreased but similar levels of GK activity for dGyk-IR-sur; Tub-GAL4, dGyk-IR-let/Tub-GAL4, dGK-IR-sur; Tub-GAL4, and dGK-IR-let/Tub-GAL4 3rd instar RNAi progeny (Glycerol kinase (GK) phosphorylates glycerol to glycerol 3-phosphate. Therefore successful targeting of progeny . This redGyk and dGK in RNAi progeny from Tub-GAL4 crosses . Data for GK activity, RNA expression, glycerol levels are summarized in Glycerol kinase phosphorylates glycerol to glycerol 3-phosphate in an ATP dependent reaction. Therefore, with decreased GK activity (as defined as glycerol phosphorylation) we would anticipate elevated glycerol levels. As expected, we found increased levels of glycerol in r larvae . These lc564-GAL4 driver that has previously been shown to drive expression of GAL4 in the larval fat body c564-GAL4; dGyk-IR-let; and c564-GAL4; dGK-IR-let 3rd instar larvae but unchanged levels in c564-GAL4; dGyk-IR-sur and c564-GAL4; dGK-IR-sur 3rd instar larvae . In humans, expression of glycerol kinase is highest in the liver r larvae .dGyk-IR-sur, dGK-IR-sur, dGyk-IR-let, and dGK-IR-let fly lines. GAL4 drivers tested included c564 , 24B (embryonic mesoderm and muscle), Elav (nervous system), and GMR (eye). In addition to the larval lethality phenotype obtained in progeny from dGyk-IR-let and dGK-IR-let with the Tub-GAL4 driver, we found lethality at larval and pupal stages of development for RNAi progeny from dGyk-IR-let and dGK-IR-let fly lines using c564-GAL4 and 24B-GAL4 driver crosses often exhibited melanotic masses before lethality at the pupal stage of development. The majority of c564-GAL4; dGyk-IR-let progeny die as pharate adults , with escapers exhibiting a curled or crumpled wing phenotype . As both dGyk and dGpdh play important enzymatic roles in glycerol metabolism, we would predict that the n1-4dGpdh mutation would enhance the c564-GAL4; dGyk-IR-let wing phenotype if the phenotype was caused by disrupted glycerol metabolism. Crosses were performed between c564-GAL4; dGyk-IR-let and n1-4dGpdh flies and progeny examined for the crumpled wing phenotype. Wings of c564-GAL4/n1-4dGpdh; dGyk-IR-let flies were found to have a more severe wing phenotype compared to c564-GAL4; dGyk-IR-let flies to be elevated in larvae that die before eclosion compared to the glycerol hypersensitive adults OB1 gene (homolog of the human gene encoding leptin) in relation to obesity dGyk or dGK to be lower for the lethality phenotype as compared to the glycerol hypersensitive phenotype. However, here we show that the underlying molecular basis has a greater level of complexity, a characteristic shared with GKD patients.In humans, GKD patients show a range of phenotypic severity with no correlation with GK glycerol phosphorylation activity. This has led to the hypothesis of an important role for modifier loci and/or alternative protein functions of glycerol kinase in determining phenotype severity. Remarkably, our dGyk can partially rescue lethality of c564-GAL4; dGK-IR-let. Future studies correlating phenotype to dosage levels between dGyk and dGK could provide an interesting insight into the individual functions of dGyk and dGK. The presence of other distinct protein domains within the dGyk and dGK amino acid sequence, e.g., domains for protein interaction and mitochondrial apoptosis At the amino acid level, dGyk and dGK are 46% identical (67% similar if including conservative substitutions) and share the \u201cFGGY\u201d domain responsible for glycerol phosphorylation dGyk and dGK glycerol hypersensitive and larval lethality phenotypes, glycerol kinase activities showed a trend toward reduction. However, distinct dGyk- and dGK-RNA expression profiles were found between the glycerol hypersensitive and larval lethality phenotypes. One notable feature was a compensatory mechanism between dGyk and dGK. We observed that the dGyk-IR-sur; Tub-GAL4 flies had reduced levels of dGyk and elevated levels of dGK, whereas dGK-IR-let; Tub-GAL4 showed reduced levels of dGK and elevated levels of dGyk. This compensatory mechanism was at the level of RNA expression and did not restore GK activity to normal levels. These results indicate that both dGyk and dGK are required for normal levels of GK activity. In bacteria, the glycerol kinase protein can exist as a dimer or tetramer with each state affecting the protein conformation and glycerol kinase activity Drosophila.For the Tub-GAL4; dGyk-IR and Tub-GAL4; dGK-IR flies had decreased levels of GK phosphorylation activity. However, RNA expression analysis of dGyk-IR-let; Tub-GAL4 3rd instar larvae unexpectedly revealed levels of dGyk that were not statistically different as compared to controls. We hypothesize that this is due to inhibition of the RNAi mechanism. Recent studies have shown that RNAi constructs that trigger apoptotic cell death can result in RNAi inhibition in adjacent cells dGyk-IR-let; Tub-GAL4 flies, cell specific RNAi inhibition could mask RNAi knockdown of dGyk-RNA levels in other cells. However without experimental confirmation this remains speculation.All the In silico analysis of the dGyk-IR and dGK-IR construct sequences did not identify any potential off-targets in the Drosophila genome . Additionally, dGyk-IR does not target the dGK transcript and the dGK-IR does not target the dGyk transcript. However, without a dGyk-specific antibody to perform immunohistochemistry, we have been unable to confirm dGyk knockdown at the protein level in the dGyk-IR-let; Tub-GAL4 flies . The fact that dGyk-IR-let; Tub-GAL4 flies had reduced GK activity and a phenotype resembling that of the dGK-IR-let; Tub-GAL4 flies suggests that total dGyk protein levels are reduced.dGyk-IR-sur; Tub-GAL4 and dGK-IR-sur; Tub-GAL4 flies. Interestingly, both dGyk-IR-let; Tub-GAL4 and dGK-IR-let; Tub-GAL4 flies had glycerol levels that were lower than control levels. Further evidence for altered metabolite levels was obtained by quantitation hemolymph trehalose. Decreased trehalose levels were found in both c564-GAL4; dGyk-IR-let; and c564-GAL4; dGK-IR-let 3rd instar larvae whereas trehalose levels were unchanged in c564-GAL4; dGyk-IR-sur and c564-GAL4; dGK-IR-sur 3rd instar larvae. We hypothesize that reduced glycerol and trehalose is part of the pathogenic mechanism in which disrupted metabolism contributes to larval lethality. Future comprehensive metabolic profiling could reveal clues to the underlying pathogenic mechanism.The metabolic role of glycerol kinase is to phosphorylate glycerol to glycerol 3-phosphate in an ATP dependent reaction. Therefore, with decreased GK activity we would anticipate elevated glycerol levels. As expected, elevated glycerol levels were found in Drosophila GKD phenotypes.As all the knockdown flies had normal triglyceride levels, we also predict that glycerol utilization through an alternative metabolic pathway could contribute toward the deleterious outcome of larval lethality. For example, future studies are required to determine whether reduced glycerol kinase activity alters di-acylglycerol (DAG) levels. DAG can bind a number of signaling proteins that affect a variety of cellular processes such as cytoskeletal reorganization, membrane trafficking, exocytosis, immune synapse formation, synaptic transmission and phagocytosis c564-GAL4; dGyk-IR-let flies could be as a result of altered cell signaling pathways. Wing phenotypes in Drosophila can arise when cell signaling pathways such as Notch signaling pathway are affected The larval lethality and crumpled wing phenotypes also sugc564-GAL4; dGyk-IR-let escaper flies in addition to larval lethality shows glycerol kinase also plays an important role in Drosophila development. To determine whether the crumpled wing phenotype was due to disrupted glycerol metabolism or due to loss of an alternative function of dGyk, we used another Drosophila mutant with disrupted glycerol metabolism. Using a loss of function allele in the glycerol 3-phosphate dehydrogenase 1 gene (n1-4dGpdh) we were able to show that c564-GAL4/n1-4dGpdh; dGyk-IR-let flies had a more severe wing phenotype than c564-GAL4; dGyk-IR-let flies. Therefore we conclude that the wing phenotype is due to disruption of glycerol metabolism.In humans, the study of GKD patients clearly demonstrates an important role for glycerol kinase in development GPDH1) result in transient infantile hypertriglyceridemia, fatty liver, and hepatic fibrosis dGyk and dGpdh1 expression levels is required to determine how glycerol metabolism is affected and whether this could provide clues to the pathogenic mechanism underlying this crumpled wing phenotype.Both glycerol kinase and glycerol 3-phosphate dehydrogenase control levels of glycerol 3-phosphate, a precursor for phospholipid biosynthesis. Interestingly, mutations in glycerol 3-phosphate dehydrogenase into the pUDsGFP vector pUDsGFP construct co-expresses GFP with the inverted repeat, allowing easy recognition of GFP-positive larvae that possess both the RNAi construct and the GAL4 driver. Primer pairs for PCR amplification were as follows: dGyk-IR-for d5\u2032- AGTTGGATCCGAAATAATCACGATTGGAA -3\u2032 and dGyk-IR-rev d5\u2032- AGTTGGTACCTAGTAATCCGTGCGTTGAG-3\u2032; dGK-IR-for d5\u2032- AGTTGGATCCCTGCTCAAGACGTTCGGTA -3\u2032 and dGK-IR-rev d5\u2032- AGTTGGTACCTCGAACTGGCAGAGATTGA -3\u2032. Evaluation of the inverse repeat sequences using the online web application E-RNAi version 3.2: http://www.dkfz.de/signaling/e-rnai3//Drosophila genome. Additionally, dGyk-dsRNA does not target the dGK transcript and the dGK-dsRNA does not target the dGyk transcript.Using the dGyk and dGK were PCR amplified and subcloned into the pEx-UAS vector dGyk and dGK were as follows: dGyk-for d5\u2032ATTGCGGCCGCAAAAAAAATGGATTCTCCC3\u2032 and dGyk-rev d5\u2032ATTTCTAGATGATCACGCTCCGTCAAAGGC3\u2032; dGK-for d5\u2032ATTGCGGCCGCAAGCAGCATGACCGAGGGC3\u2032 and dGK-rev d5\u2032AGCTCTAGATATTTACTGGCCACTCGCAGC3\u2032. Microinjection of DNA constructs, identification of transformants and balancing were performed by BestGene Inc .For over-expression constructs, the complete coding regions for dGyk-IR x Tub-GAL4 crosses revealed 3 lines that resulted in viable adult flies and 6 lines that resulted in progeny that died during larval development. For dGK-IR x Tub-GAL4 crosses, 7 lines resulted in viable adults flies and 2 lines resulted in progeny that died during larval development. Neither \u201clet\u201d nor \u201csur\u201d transgenes were homozygous lethal. For all subsequent experiments, 2 fly lines for each RNAi phenotype were chosen for analysis (results are shown for single fly lines).As described previously P{TubP-GAL4}P{GawB}c564P{GawB}how[24B]P{GawB}Elav[C155]P{GMR-GAL4}1n1-4Gpdh/SM1 All GAL4 driver fly stocks were obtained from the BDSC: rd instar larvae and assayed in duplicate using 4 \u00b5g of total cellular protein for 20 min using assay conditions and reaction mix previously determined to be optimal for 3rd instar larvae protein extracts (data not shown). Incorporation of 14C-glycerol into glycerol 3-phosphate was measured using a scintillation counter and GK activity of test samples calculated by comparison to a standard curve.Glycerol kinase activity was determined using a radiolabelled assay as previously reported rd instar larvae using the RNAeasy\u00ae mini kit according to manufacturer's instructions. Total RNA (1 \u00b5g) was used for first-strand cDNA synthesis using the SuperScript\u00ae III reverse transcriptase and random primers (Invitrogen). Quantitative real-time PCR (qRT-PCR) was performed using PerfecCTa\u2122 SYBR\u00ae Green FastMix\u2122 ROX on a StepOne\u2122 real time PCR machine . Fold differences for each of the genes tested were calculated using the 2[Delta][Delta]CT method dGyk and dGK were normalized to RpII. Primers were designed using Primer3 software dGykd5\u2032TAGGCATAACATCGGTTCTGG3\u2032 and d5\u2032GCCTTCCGTCCTAGTTGGTAG-3\u2032; dGKd5\u2032AGACGACAATCGTCTGGGATG3\u2032 and d5\u2032CACGATCTGCTCCACTGTAG3\u2032; RpIId5\u2032AAGGCTATGGTGGTGTCTGG3\u2032 and d5\u2032GCTTACCCTCCACGTTCTGT3\u2032.RNA was extracted from ten 3rd instar larvae were homogenized in 250 \u00b5l homogenization buffer including Complete protease inhibitor (Roche). Next, 14 \u00b5l of 20% triton X-100 was added to 186 \u00b5l of the sample. After heating at 70\u00b0C (5 mins) to inactivate endogenous enzymes, samples were centrifuged at 13000 rpm (5 mins) and the supernatant transferred to a new tube (after homogenizing the white lipid ring with the tip of the pipette). Glycerol levels were measured using Free Glycerol Reagent (Sigma-Aldrich). Values were normalized against protein concentration using the Micro BCA\u2122 Protein Assay Kit and experiments were performed in triplicate for each genotype.For glycerol and triglyceride measurements, batches of three 3One way ANOVA with post-hoc pair wise multiple comparison procedures (Tukey Test) were applied to qRT-PCR and biochemical data where stated. Error bars represent SEM.Figure S1Control RNA expression data for . Relative RNA expression levels of dGyk and dGK were quantitated for parental fly lines used to generate RNAi knockdown flies (A and B). For each group, values were not found to be statistically different. Statistical analysis using ANOVA was performed by comparison to GAL4 fly line.(TIF)Click here for additional data file.Figure S2Hemolymph trehalose measurements.Relative hemolymph trehalose levels in 3rd instar larvae were determined for the following genotypes: c564-GAL4; dGyk-IR-sur, c564-GAL4; dGyk-IR-let, c564-GAL4; dGK-IR-sur, and c564-GAL4; dGK-IR-let. The control genotype was 1118w; c564-GAL4. Both c564-GAL4; dGyk-IR-let and c564-GAL4; dGK-IR-let had decreased trehalose levels whereas trehalose levels were unchanged in c564-GAL4; dGyk-IR-sur and c564-GAL4; dGK-IR-sur 3rd instar larvae. Statistical analysis using ANOVA was performed by comparison to the control *P<0.05, **P<0.01.(TIF)Click here for additional data file.Methods S1Trehalose assay.(DOC)Click here for additional data file.Table S1Initial phenotypic characterization of RNAi fly lines using a Tub-GAL4 driver for ubiquitous expression.(DOCX)Click here for additional data file."} +{"text": "Left bundle branch block (LBBB) is a marker of increased delay between septal and left ventricular (LV) lateral wall electrical activation, and is a predictor of which patients will benefit from cardiac resynchronization therapy (CRT). Recent analysis has suggested that one third of patients meeting conventional ECG criteria for LBBB are misdiagnosed and new strict LBBB criteria have been proposed. We tested the hypothesis that strict LBBB patients have greater LV mechanical dyssynchrony than patients only meeting conventional LBBB criteria, while there is no difference between patients with conventional-only LBBB and LV conduction delay with QRS duration 110-119 ms.Sixty-four cardiomyopathy patients referred for a primary prevention implantable cardioverter defibrillator (ICD) underwent 12-lead ECG and cardiac magnetic resonance (CMR) myocardial tagging. The patients were classified as strict LBBB, conventional-only LBBB or non-LBBB, indicating nonspecific LV conduction delay with QRS duration 110-119 ms. The time delay between septal and lateral LV wall peak circumferential strain, septal-to-lateral wall delay, was measured by CMR.Patients with strict LBBB (n=31) had a greater septal-to-lateral wall delay, compared to patients with conventional-only LBBB (n=19) . There was no significant difference between conventional-only LBBB and non-LBBB (n=14) septal-to-lateral wall delay . The results are presented in the figure below.Strict LBBB ECG criteria identify patients with greater mechanical dyssynchrony compared to patients only meeting conventional-only LBBB criteria, while there was no significant difference between conventional-only and non-LBBB patients. The greater observed LV dyssynchrony may explain why strict-LBBB patients have better response to CRT.The study was supported by the National Heart, Lung, and Blood Institute, National Institutes of Health , the DW"} +{"text": "MMPs are a broad family of peptidases, which proteolysethe extracellular matrix and have an important role in inflammation. Verapamil is acalcium channel blocker extensively used in the treatment of numerous cardiovasculardiseases such as arrhythmia and hypertension. The anti-tumor and anti-inflammatoryeffects of verapamil have also been shown. In this study, the effect of verapamil ongelatinase activity in human peripheral blood mononuclear cells (PBMCs) has beenassessed In this experimental study, PBMCs from healthy adultvolunteers were isolated by ficoll-hypaque-gradient centrifugation. The cells werethen cultured in complete RPMI-1640 medium and after that incubated with differentconcentrations of verapamil (0\u2013200 \u03bcM) in the presence or absence of phytoheamagglutinin(PHA) (10 \u03bcg/ml) for 48 hours. The gelatinase A (MMP-2)/gelatinaseB (MMP-9) activity in cell-conditioned media was then evaluated by gelatinzymography. Statistical comparisons between groups were made by analysis ofvariance (ANOVA).Verapamil significantly decreased the MMP-2/MMP-9 activity in human PBMCsafter 48 hours incubation time compared with untreated control cells. The associationwas dose-dependent.In this study verapamil exhibited a dose-dependent inhibitory effecton gelatinase A and gelatinase B activity in human PBMCs. It seems that theanti-inflammatory properties of verapamil may be in part due to its inhibitory effectson gelatinase activity. Regarding the beneficial effects of MMPs- inhibitorsin the treatment of some cardiovascular diseases, the positive effect of verapamilon such diseases may be in part due to its anti-MMP activity. Verapamil with itsinhibitory effects on gelatinases activity may be a useful MMP-inhibitor. Given thebeneficial effect of MMP-inhibitors in some cancerous, inflammatory and autoimmunedisorders, it seems likely that verapamil could also be used to treat thesediseases. MMPs arin vivo , 15, andin vivo . In addiin vivo and the in vivo .Mononuclear cells play an important role in inflammation, 18 throThis experimental study was approved by TheDeputy Director of Research in the Faculty ofMedicine at Shahed University.RPMI-1640 medium, penicillin, streptomycin,PHA (phytoheamagglutinin) and trypan blue (TB)were obtained from Sigma (USA). MTT . The cells were then resuspended in RPMI-1640 medium supplemented with 10% FCS andwere incubated in 5% CO2. The cells were seededat a density of 1\u00d7106 cells/ml and then treatedwith different concentrations of Verapamil (0-200 \u03bcM) in the presence of PHA (10 \u03bcg/ml)for 48 hours. Afterward the supernatants fromthe cell cultures were collected, centrifuged andstored at -20\u02daC for subsequent tests. All experimentswere done in triplicate.The method used for cell culture and treatmenthas been described in detail previously .BrieflyMMP-2 and MMP-9 activity in cell-conditionedmedia were evaluated using the gelatin zymographytechnique according to the modified Kleinerand Stetler-Stevenson method 1994, as previThe relative intensity of the gelatin lysis bandscompared to the control was measured using UVIPro gel documentation system and expressedas relative gelatinolytic activity.MMP-2 and MMP-9 activity measurement in cellconditionedmedia was performed in three independentexperiments and the results were expressed asmean \u00b1 SEM. Statistical comparisons between groupswere made by analysis of variance (ANOVA). P<0.05was considered significant. Multiple comparisonswere tested using the Tukey method (5%) for statisticallysignificant differences. The software SPSS 11.5and Excel 2003 were used for statistical analysis andgraph making respectively.Effect of verapamil on gelatinase-A (MMP-2)and gelatinase-B (MMP-9) activity in human PBMCsin different concentrations are shown in figuresVerapamil significantly decreased the gelatinase-A (MMP-2) activity in PHA-stimulatedhuman PBMCs in a dose-dependent fashion after48 hours incubation compared with untreatedcontrol cells . The decVerapamil significantly decreased the gelatinase-B (MMP-9) activity in PHA-stimulated humanPBMCs in a dose-dependent fashion after 48 hoursincubation time compared with untreated controlcells . The decIn this study, a 200 \u03bcM concentration of verapamilinhibited the gelatinase A (MMP-2) andgelatinase B (MMP-9) activity in human PBMCs.These results are consistent with the study byFar\u00edas, et al. in whichin vivo activity inhuman PBMCs. Thus verapamil may be of potentialuse in the preparation of MMP-inhibitors.MMPs have important role in inflammation, so verapamil,along with its long-term usage in cardiovasculardisease, may be a good candidate for thedevelopment of anti- inflammatory agents."} +{"text": "In patients with prior myocardial infarction, intra-scar surviving fibers may create conducting channels (CC) which are the substrate of most sustained monomorphic ventricular tachycardias (VT). These channels can be identified by endocardial voltage mapping. Recent studies show high correlation between information from endocardial voltage mapping and delayed-enhanced MRI (DE-MRI) images.DE-MRI and left ventricular electroanatomic voltage maps (CARTO\u00ae) were obtained in 18 patients with chronic myocardial infarction referred for VT ablation. DE-MRI studies were performed with a 1.5 T Unit (Philips Intera\u00ae) in standard views, 10 min after injecting 0.02 mmol/kg of gadolinium contrast (Omniscan\u00ae), with 3D T1-TFE acquisition.The 3D endocardial representation was computed off-line using proprietary software developed in the MATLAB environment (Mathworks). Starting with the short-axis view of the DE-MRI study Figure , left veThe resulting color-coded endocardial shell was assessed by a cardiac electrophysiologist for comparison with electroanatomic voltage maps Fig . A CC waGiven the agreement with electroanatomic voltage maps for identification of CC inside the scar tissue, we conclude that the proposed color-coded endocardial shell might be a useful tool for non-invasive location of such channels and for both pre-planning and guidance of ablation procedures."} +{"text": "Bovine Leukemia Virus (BLV), a delta-retrovirus related to humanT-cell leukemia virus-1 (HTLV-1), is associated with the development of B-cell leukemia in experimentally-infected sheep. Using this outbred animal model of B-cell transformation, oncogenic modifications reflected in altered microRNA expression can be identified and compared as the disease progresses. We have analyzed the miRNome of transformed B-cells isolated from leukemic sheep. Using Taqman Low Density Array (TLDA) assays and High-Throughput (HT) sequencing of small RNA libraries, we identified differentially-expressed microRNAs associated with B-cell transformation. For miRBase database-matched sheep orthologs there was a good overall quantitative correlation between data generated with both techniques. Furthermore, deep sequencing identified variants of mature microRNA transcripts, indicating that isomir distribution might be of biological significance. Finally, HT sequencing revealed unknown candidate microRNAs which were confirmed both in silico using miRDeep and experimentally using stem-loop RT-QPCR methods. Target prediction tools suggest that these microRNAs might target both cellular and viral mRNAs. Down-regulation of viral mRNAs might contribute to tumor-associated virus silencing and play a role in immune escape mechanisms. Ongoing work aims at the validation of bioinformatics predictions of microRNA targets. Altogether, this work should lead to a better understanding of the microRNA-mRNA regulation network associated with leukemia progression."} +{"text": "Non-invasive MRI tissue characterisation after HTX could potentially replace the hazardous myocardial biopsy for tissue staging and detection of transplant rejection. Recently, late gadolinium contrast enhanced cardiac MRI (LGE-CMR) showed infarct-typical patterns already early after HTX. Additionally, infarct-atypical LGE patterns were also shown in general but neither morphological differentiation nor its time course after HTX have been investigated.We hypothesized that MRI could detect and differentiate different forms of infarct-atypical LGE patterns and show differences in their morphological appearance during the time course after HTX.123 patients (pts) were divided into group I and group II . LGE-CMR (Gadolinium:0.2mmol/kg bw) was performed on a 1.5T Whole Body MRI scanner and analysed blindly by two experienced observers. For anatomic LGE description, hearts were divided according to the 17-segment model. Areas of infarct-atypical LGE patterns were classified into four types as a) LGE at the RV-insertion , b) intramural, c) nodular and d) diffuse. Groups were compared using ANOVA. P-values \u2264 0.05 were considered statistically significant.In group I, 65% of patients and 210 of 1054 segments (20%) showed infarct-atypical LGE patterns. In contrast, significantly less patients (41%) of group II and only 54 of 1037 (both p<0.01) segments were affected (figure LGE-CMR is a novel and sensitive imaging technique for myocardial tissue characterisation after HTX. Unfortunately, the exact patho-mechanism for the occurrence of infarct-atypical CE-MRI patterns is illusive but could potentially include the amount of organ rejections, myocarditis or other immune processes. Further studies need to clarify the correlation between myocardial biopsies and the various patterns on MRI images. Nevertheless, LGE-CMR in HTX could be a valuable tool for non-invasive tissue characterisation in pts after HTX and could have the potential to replace invasive biopsy procedures in the future.None."} +{"text": "Salvia sclarea) and extraction of the plant material. In clary sage, sclareol mainly accumulates in essential oil-producing trichomes that densely cover flower calices. Manool also is a minor diterpene of this species and the main diterpene of related Salvia species.Sclareol is a diterpene natural product of high value for the fragrance industry. Its labdane carbon skeleton and its two hydroxyl groups also make it a valued starting material for semisynthesis of numerous commercial substances, including production of Ambrox\u00ae and related ambergris substitutes used in the formulation of high end perfumes. Most of the commercially-produced sclareol is derived from cultivated clary sage (SsLPPS) produced labda-13-en-8-ol diphosphate as major product from geranylgeranyl diphosphate (GGPP) with some minor quantities of its non-hydroxylated analogue, -copalyl diphosphate. A class I diTPS (SsSS) then transformed these intermediates into sclareol and manool, respectively. The production of sclareol was reconstructed in vitro by combining the two recombinant diTPS enzymes with the GGPP starting substrate and in vivo by co-expression of the two proteins in yeast (Saccharomyces cerevisiae). Tobacco-based transient expression assays of green fluorescent protein-fusion constructs revealed that both enzymes possess an N-terminal signal sequence that actively targets SsLPPS and SsSS to the chloroplast, a major site of GGPP and diterpene production in plants.Based on previous general knowledge of diterpene biosynthesis in angiosperms, and based on mining of our recently published transcriptome database obtained by deep 454-sequencing of cDNA from clary sage calices, we cloned and functionally characterized two new diterpene synthase (diTPS) enzymes for the complete biosynthesis of sclareol in clary sage. A class II diTPS (SsLPPS and SsSS are two monofunctional diTPSs which, together, produce the diterpenoid specialized metabolite sclareol in a two-step process. They represent two of the first characterized hydroxylating diTPSs in angiosperms and generate the dihydroxylated labdane sclareol without requirement for additional enzymatic oxidation by activities such as cytochrome P450 monoxygenases. Yeast-based production of sclareol by co-expresssion of SsLPPS and SsSS was efficient enough to warrant the development and use of such technology for the biotechnological production of scareol and other oxygenated diterpenes. Diterpenoids constitute a large class of chemically diverse metabolites that is widely distributed throughout the plant kingdom with more than 12,000 known compounds, the majority of which derives from bicyclic \u2018labdane-related\u2019 diterpene intermediates and a plSalvia sclarea (Lamiaceae) and SsSS were amplified with gene-specific primers and cloned into pENTR/D-TOPO (Invitrogen). The resulting clones were transferred by Gateway LR reactions as recommended by the manufacturer (Invitrogen) into the destination vector pMDC83, in-frame with a C-terminal green fluorescent protein (GFP).The GFP fusion constructs for transient expression in N. benthamiana seeds were disinfected with chlorine fumes for 6 h and placed on Murashige and Skoog basal salt medium (Sigma) containing 0.7% agar. After incubation for 2 weeks at 24\u00b0C, seedlings were transferred to soil and the plants were grown in a chamber with a 16 h photoperiod and a temperature range of 22\u00b0C (night time low) to 25\u00b0C (day time high). Plants that were 4 to 6 weeks old were used for transformation.Agrobacterium tumefaciens strain C58 (pMP90) by chemical transformation as reported elsewhere and SsSS and SsdiTPS3 [B] were generated in CLC bio as compared to representative class II and class I diterpene synthases. Grey shading indicates strictly conserved residues. The catalytically relevant aspartate-rich motifs are highlighted and predicted plastidial transit peptides are underlined. N-terminal truncations for expression of recombinant proteins in E.coli and yeast and protein fragments used for subcellular localization studies are marked as red and green arrows, respectively. Abbreviations: SsLPPS, Salvia sclarea labda-13-en-8-ol diphosphate synthase [GenBank: JQ478434]; NtCPSL, Nicotiana tabacum 8-hydroxy copalyl diphosphate synthase [GenBank: CCD33018]; CcCLS, Cistus creticus copal-8-ol synthase [GenBank: ADJ93862]; SmCPSL, Salvia miltiorhizza copalyl diphosphate synthase-like [GenBank: EU003997]; AtCPSL, Arabidopsis thaliana ent-copalyl diphosphate synthase [GenBank: AAA53632]; SsSS, S. sclarea sclareol synthase [GenBank: JQ478435]; SsdiTPS3, S. sclarea diterpene synthase-3[GenBank: JQ478436]; SmKSL, S. miltiorhizza kaurene synthase-like [GenBank: EF635966]; AtKS, A. thaliana ent-kaurene synthase [GenBank: AF034774]; SrKS, Stevia rebaudiana kaurene synthase [GenBank: AAD34295]; NtKSL, N. tabacum kaurene synthase-like [GenBank: CCD33019].Click here for fileFigure S2. Mass spectra of assay products as compared to reference spectra of authentic standards and relevant databases. Illustrated are characteristic mass spectra of enzymatic reaction products as compared to authentic standards or reference mass spectra from the National Institute of Standards and Technology MS library searches (Wiley W9N08L): peak a, 13-epi-manoyl oxide 8; peak b, manoyl oxide 7; peak c, putative 13(16)-14-labdien-8-ol; peak d, putative copalol; peak e, sclareol 4; peak f, unknown compound; peak g, labda-13-en-8,15-diol; peak i, manool 6.Click here for fileTable S1. Oligonucleotides used for the amplification of cDNA sequences.Click here for fileFigure S3. Codon optimized sequences of SsSS and SsdiTPS3. Codon optimized sequences of SsSS and SsdiTPS3 that have been used for Escherichia coli and yeast-based heterologous protein expressions are shown in FASTA format.Click here for file"} +{"text": "In patients with stenoses, it is desirable to accurately measure peak velocity (Vmax). Unfortunately, phase-contrast MR (PCMR) tends to underestimate peak velocities. Fourier Velocity Encoding (FVE) can measure peak velocities in MRI, but is not commonly used due to long acquisition times.We have developed a FVE sequence that combines spiral trajectories with parallel imaging (SENSE), partial-Fourier acquisition and a novel velocity-unwrap technique. The aim of this study is to validate this sequence.FVE was performed using a uniform-density spiral trajectory with 16 interleaves . This allows accurate unfolding of velocity data.In-vitro: A pulsatile flow pump was connected to a tube phantom (diameter 13mm) with a stenosis of 6mm. At 15 different flow rates, Vmax was measured using; 1) ultrasound (US), 2) low-resolution PCMR (lr-PCMR), 3) high-resolution PCMR (hr-PCMR), 4) FVE with SENSE and partial-Fourier with 21 reconstructed velocities (FVE21) and 5) FVE with SENSE and partial-Fourier, plus velocity-unwrap giving 41 reconstructed velocities (FVE41). SNR estimates were compared between FVE21 and FVE41.In-vivo: Six patients with stenoses were also assessed .In-vitro: There were no statistically significant differences between Vmax measured using US and FVE table . HoweverIn-vivo: As in-vitro, PCMR underestimated Vmax. There were no statistical differences between Vmax measured using US and FVE sequences. However there was a trend towards FVE21 overestimating Vmax.FVE allows more accurate assessment of Vmax than PCMR as it measures a velocity spectrum per pixel, rather than the average velocity. We have demonstrated that it is possible to achieve high resolution FVE within a short breath-hold by combining spiral trajectories, parallel imaging, partial-Fourier and velocity-unwrap. This sequence was shown to be significantly more accurate than PCMR in-vitro and in-vivo. Furthermore using the novel velocity-unwrap technique there was a trend towards higher accuracy due to higher velocity resolution. Thus, the sequence may be able to replace US in assessment of Vmax.JAS: EPSRC PhD+.VM: BHF."} +{"text": "Disruption of the ubiquitin-proteasome system, which normally identifies and degrades unwanted intracellular proteins, is thought to underlie neurodegeneration. Supporting this, mutations of Parkin, a ubiquitin ligase, are associated with autosomal recessive parkinsonism. Remarkably, Parkin can protect neurons against a wide spectrum of stress, including those that promote proteasome dysfunction. Although the mechanism underlying the preservation of proteasome function by Parkin is hitherto unclear, we have previously proposed that Parkin-mediated K63-linked ubiquitination may serve to mitigate proteasomal stress by diverting the substrate load away from the machinery. By means of linkage-specific antibodies, we demonstrated here that proteasome inhibition indeed promotes K63-linked ubiquitination of proteins especially in Parkin-expressing cells. Importantly, we further demonstrated that the recruitment of Ubc13 (an E2 that mediates K63-linked polyubiquitin chain formation exclusively) by Parkin is selectively enhanced under conditions of proteasomal stress, thus identifying a mechanism by which Parkin could promote K63-linked ubiquitin modification in cells undergoing proteolytic stress. This mode of ubiquitination appears to facilitate the subsequent clearance of Parkin substrates via autophagy. Consistent with the proposed protective role of K63-linked ubiquitination in times of proteolytic stress, we found that Ubc13-deficient cells are significantly more susceptible to cell death induced by proteasome inhibitors compared to their wild type counterparts. Taken together, our study suggests a role for Parkin-mediated K63 ubiquitination in maintaining cellular protein homeostasis, especially during periods when the proteasome is burdened or impaired. The proteasome is a major intracellular proteolytic machinery that plays a vital role in maintaining cellular protein homeostasis through its ability to destroy unwanted proteins rapidly . ProteinConceivably, the potential ability of the cell to promote K63-linked polyubiquitination during proteasomal stress would involve a dynamic partnership between relevant E3 members and Ubc13 - the only E2 known to date to mediate the formation of K63-linked ubiquitin chains . Consist6-Ubc13 as template and cloned into pCMV-myc vector via EcoRI and XhoI restriction sites. Untagged full length parkin was generously provided by H. Walden . YFP-Mitofusin2 was a gift from R. Youle (Addgene plasmid #28010). Control and Ubc13 shRNA (V2LHS_171792 and_220048) were purchased from Thermoscientific. The following mouse monoclonal antibodies were used: anti-c-myc (clone 9E10) , anti-FLAG , anti-HA (Sigma), anti-\u03b2-actin (Sigma), anti-Ubc13 , anti-parkin clone PRK8 (Covance) and anti-ubiquitin clone FK1 . Linkage-specific K48 and K63 antibodies were purchased from Millipore and BIOMOL respectively. Rabbit anti-GFP was purchased from Abcam . All other reagents were purchased from Sigma, except clasto-lactacystin \u03b2-lactone and MG-132 .Plasmids expressing, HA- or myc-tagged synphilin-1, myc-tagged Siah-1 and -2, HA-tagged wild type or mutant ubiquitin, FLAG-tagged wild type or mutant parkin have been described previously ,11,18. T2 atmosphere. Cells were transiently transfected with the desired plasmid(s) using the LipofectAMINE PLUS reagent according to the manufacturer\u2019s instructions. For proteasome inhibition studies, cells were treated at 24 h post-transfection with 1 (or 2) \u00b5M MG132 for 16 h before harvesting. Sequential fractionation of transfected cell lysates into Triton-X-soluble (S) and SDS-soluble (P) fractions was carried out as previously described [Human embryonic kidney (HEK) 293 cells were grown in DMEM with 10% FBS in a 5% COescribed . Immunopescribed . Primaryescribed were genescribed .AAGATATCTAGCGTTTAAA- CGGGC 3\u2019) and a MunI restriction site-containing reverse primer (5\u2019 AGCCAATTG- GCGGCCGCTCGAG 3\u2019). Amplified product was digested with EcoRV and MunI and inserted into EcoRV and EcoRI sites of pL6mCWmIRESCherry. The lentivector pL6mCWmIRESCherry was modified from pLenti6/V5-D-TOPO (Invitrogen) by reengineering of the multiple cloning site, insertion of the cPPT and WPRE elements, and insertion of the IRESmCherry reporter cassette. Lentivirus packaging was performed in 293FT cells according to the protocol provided with the ViraPower\u2122 Lentiviral Directional TOPO\u00ae Expression Kit (Invitrogen). Lentivirus particles were concentrated from cell culture supernatant according to the protocol of Deiseroth Lab (http://www.stanford.edu/group/dlab/resources/lvprotocol.pdf). Lentivirus carrying the ubiquitin expression constructs was used to transduce wild type or Ubc13 knockout MEFs. Prior to transduction, cells were cultured to ~90% confluence. Concentrated virus particles were added to cell culture medium containing 6 \u00b5g/ml of Polybrene. Long term transgene expression was maintained by selecting for resistance to Blasticidin S at a final concentration of 10 \u00b5g/ml. Transgene expression was detected by mCherry epifluorescence.cDNA sequences encoding myc-tagged UbcH7 and Ubc13 were PCR-amplified from their respective pcDNA3.1 plasmid clones using an EcoRV restriction site-containing forward primer unless otherwise stated.The autophagic clearance of inclusions formed under conditions of proteasomal impairment was investigated using a method originally described by Fortun et al . Cells wRecently, K63-specific antibodies have become available from commercial sources. Although we have independently confirmed its linkage specificity in the present study , we founTo test our hypothesis that parkin-mediated K63 ubiquitination may be enhanced in cells undergoing proteasomal stress, we next examined the immunoreactivity of anti-UbK63 in Triton-X-100-soluble (S) and -insoluble (P) lysates sequentially prepared from parkin-expressing cells in the presence or absence of proteasome inhibition. We detected a modest but significant increase in the levels of K63-linked polyubiquitination specifically in the P fraction in untreated cells expressing parkin compared to control cells . ImportaGiven that Ubc13 is uniquely associated with K63-linked ubiquitination, and our observation above that parkin-mediated K63-linked ubiquitination is promoted by proteasome inhibition, we surmised that the binding affinity between Ubc13 and parkin may be influenced by the functional status of the proteasome. To address this, we carried out co-immunoprecipation experiments with cells transfected with myc-tagged Ubc13 and FLAG-tagged parkin in the presence or absence of MG132 treatment. As suspected, the amount of parkin that co-immunoprecipitated with Ubc13 is significantly enhanced in the presence of proteasome inhibition & S2C. NIn view of the recent demonstration by Chaugule and colleagues that parkin activity is normally repressed by its ubiquitin-like (Ubl) domain , we examNotwithstanding the above, how proteasome inhibition increases the affinity between parkin and Ubc13 remains unclear, although a recent study by Sha and colleagues have demonstrated that parkin phosphorylation by PINK1 promotes its interaction with Ubc13/Uev1a and concomitantly activates its K63-linked ubiquitination activity . We therPreviously, we have demonstrated that synphilin-1 ubiquitination by parkin and Siah-1 occurs via K63 and K48 respectively . ConsistGiven the recent finding by our group and others that K63 polyubiquitin may act as a signal to target proteins to the aggresome-autophagy pathway , it is tWe surmised that the upregulation of K63-linked ubiquitination in the presence of proteasome inhibition may be a cellular protective response. To address this, we subjected wild type and Ubc13-/- MEFs to MG132 treatment to examine their relative susceptibility to proteasome inhibition-induced cell death. As per our speculated protective role of K63-linked ubiquitination, we observed that a significant population of the MG132-treated Ubc13-/- MEFs became rounded and reflective, i.e. indicative of dying cells whereas wild type MEFs treated with MG132 were relatively spared of these features . QuantitIn essence, the main finding of our current study is that proteasome inhibition promotes parkin-Ubc13 interaction and concomitantly enhances parkin-mediated K63-linked ubiqiuitination. Our study thus identifies a mechanism by which parkin could promote K63-linked ubiquitin modification in cells undergoing proteolytic stress, which appears to facilitate the subsequent clearance of selected parkin substrates via autophagy. How parkin is modified such that its affinity for Ubc13 is increased in the presence of proteasome inhibition however remains elusive. Nonetheless, our study suggests a role for parkin-mediated K63 ubiquitination in maintaining cellular protein homeostasis, especially during periods when the proteasome is heavily burdened or impaired.Parkin was originally identified as a gene whose mutations are causative of autosomal recessive parkinsonism [insonism . . The dinsonism ,28, inclinsonism \u201331. Howeinsonism . Notablyinsonism . ConsistMechanistically, how proteasome inhibition promotes the affinity between parkin and Ubc13 is currently unclear to us. Although parkin phosphorylation by PINK1 was previously demonstrated by Sha et al to promote its interaction with Ubc13/Uev1a , we founInterestingly, parkin-related cases are frequently devoid of Lewy bodies (LB), the classic histological hallmark of Parkinson\u2019s disease (PD), suggesting that the catalytic activity of parkin may play a role in LB biogenesis . We founvia the autophagic route, their result suggests that parkin may facilitate the clearance of proteins by autophagy. We have subsequently extended the study by Olzmann et al by showing that K63-linked polyubiquitin acts as a novel cargo selection signal for the autophagy apparatus [Given that K63-linked polyubiquitination of proteins is generally uncoupled from the proteasome, it is conceivable that enhanced cellular ubiquitin modification of proteins via K63 would promote their accumulation and subsequent aggregation in the cell. Indeed, we have demonstrated previously with ubiquitin mutant over-expression and in tpparatus ,6. Furthpparatus , facilitpparatus \u201340. ThusNotably, a recent study by Paine et al that involved the use of linkage-specific antibody demonstrated that K63-linked ubiquitin pathology accompanies proteasome impairment in a mouse model of proteasome dysfunction . HoweverFigure S1K63 polyubiquitinated proteins reside in detergent-insoluble fractions of cell lysates.(A) Representative anti-ubiquitin (FK1), anti-K48 or -K63 immunoblots of chemically synthesized K48 or K63 polyubiquitin chains (BIOMOL), as indicated. (B) Representative anti-HA and anti-K63 immunoblots of cell extracts sequentially prepared with Triton-X 100 (S) and SDS (P)-containing buffer from HEK cells transfected with various ubiquitin species, as indicated. The blots above were stripped and reprobed with anti-actin antibody to reflect loading variations. (C) Representative anti-HA and anti-K63 immunoblots of S and P fractions of HEK cells transfected with HA-tagged wild type ubiquitin and various myc-tagged E2 species, as indicated. Top and bottom arrows point to Uev1a and Ubc13 respectively.(PDF)Click here for additional data file.Figure S2K63-polyubiquitination is enhanced in parkin-expressing cells in the presence of proteasome inhibition.(A) Representative anti-K63 and anti-FLAG immunoblots of cell extracts sequentially prepared with Triton-X 100 (S) and SDS (P)-containing buffer from control HEK cells or those transfected with HA-Ubiquitin alone or with FLAG-tagged parkin in the absence or presence of various proteasome inhibitors, as indicated. The blots above were stripped and reprobed with anti-actin antibody to reflect loading variations. These experiments were duplicated with similar results. (B) Bar graph showing the chymotrypsin-like proteasome activities of lysates prepared from untreated cells or those treated with various proteasome inhibitors, as indicated . Control refers to lysates added with MG132 in vitro (C) Left, A portion of Triton-X-soluble lysates prepared from untreated or MG132-treated HEK293 cells expressing FLAG tagged parkin alone or with myc-tagged Ubc13 were subjected to anti-myc immunoprecipitation followed by anti-FLAG and anti-myc immunoblotting (IPmyc). The remainder lysates prepared from these variously transfected cells (INPUT) were subjected to anti-FLAG and anti-myc immunoblotting to show the expression levels of FLAG-parkin and myc-Ubc13 respectively. These experiments were replicated at least three times. (D) Anti-K63 immunoblot of lysates prepared from FLAG-parkin transfected MG132-treated cells in the absence or presence of 2 shRNA species to Ubc13 or control shRNA. The blots above were stripped and reprobed with anti-actin antibody to reflect loading variations. The efficiency of the Ubc13 knockdown is shown in the anti-Ubc13 blot. Notice that the level of parkin as revealed by anti-parkin blot is reduced in the presence of Ubc13 silencing. (E) Anti-parkin immunoblot of lysates prepared from FLAG-parkin transduced WT or Ubc13-/- MEFs in the absence or presence of MG132 treatment, as indicated. The blot was stripped and reprobed with anti-actin antibody to reflect loading variations. (F) Anti-K63 and anti-parkin immunoblot of lysates prepared from wild type (WT) and parkin KO MEFs in the absence or presence of MG132 treatment, as indicated. The blots above were stripped and reprobed with anti-actin antibody to reflect loading variations. These experiments were duplicated.(PDF)Click here for additional data file.Figure S3Parkin does not appear to be phosphorylated in the presence of MG132.(A) Anti-phosphoserine and anti-phosphothreonine immunoblots showing the absence of serine/threonine phosphorylation of immunoprecipitated FLAG-tagged parkin in the absence or presence of MG132 treatment or PINK1 co-expression. (B) Anti-phosphoserine and anti-phosphothreonine immunoblots of lysates prepared from cells treated with DMSO or Calyculin a, a potent protein phosphatase inhibitor, shows that the antibodies work fine. (C) Anti-FLAG immunoblotting of 2D gel fractionated cell lysate prepared from FLAG-tagged parkin transfected cells in the absence or presence of MG132 treatment. Note that the parkin-positive spot remains unmodified in both cases (top and middle panels). As a control, when a phospho-mimetic parkin T175D mutant is co-transfected with wild type (WT) parkin, a more acidic parkin-positive spot (arrowhead) can be observed alongside the unmodified one (bottom panel).(PDF)Click here for additional data file.Figure S4Accumulation of synphilin-1 in cells expressing K63 mutant ubiquitin.(A) Cell extracts sequentially prepared with Triton-X 100 (S) and SDS (P)-containing buffer from HEK cells transfected with HA-synphilin alone or with FLAG-parkin, myc-Siah-1 or -2, myc-CHIP or GFP dorfin were subjected to immunoblotting with various antibodies, as indicated. Asterisk denotes non-specific bands. Equal loading of the different cell lysates was verified by anti-actin immunoblotting. (B) Bar graphs showing the steady state levels of HA-synphilin in S and P fractions of cell lysate after normalization to their respective loading controls . (C\u2013D) Representative anti-myc and anti-HA immunoblots of cell extracts sequentially prepared with Triton-X 100 (S) and SDS (P)-containing buffer from HEK cells transfected with myc-tagged synphilin-1 (Myc-SP) and various ubiquitin species, as indicated. Equal loading of the different cell lysates was verified by anti-actin immunoblotting.(PDF)Click here for additional data file.Figure S5Anti-K63 antibody does not cross react with K0 ubiquitinated proteins.Representative anti-myc, anti-K63 and anti-HA immunoblots of cell extracts sequentially prepared with Triton-X 100 (S) and SDS (P)-containing buffer from HEK cells transfected with myc-tagged synphilin-1 (Myc-SP) and K0, K63 or K6 ubiquitin mutant, as indicated. Equal loading of the different cell lysates was verified by anti-actin immunoblotting.(PDF)Click here for additional data file."} +{"text": "The Tax oncoprotein promotes T-cell transformation, in part via constitutive activation of the NF-\u03baB transcription factor; however, the underlying mechanisms remain unknown. Ubiquitination is a type of post-translational modification that occurs in a three-step enzymatic cascade mediated by E1, E2 and E3 enzymes and regulates protein stability as well as signal transduction, protein trafficking and the DNA damage response. Emerging studies indicate that Tax hijacks the ubiquitin machinery to activate ubiquitin-dependent kinases and downstream NF-\u03baB signaling. Tax interacts with the E2 conjugating enzyme Ubc13 and is conjugated on C-terminal lysine residues with lysine 63-linked polyubiquitin chains. Tax K63-linked polyubiquitination may serve as a platform for signaling complexes since this modification is critical for interactions with NEMO and IKK. In addition to NF-\u03baB signaling, mono- and polyubiquitination of Tax also regulate its subcellular trafficking and stability. Here, we review recent advances in the diverse roles of ubiquitin in Tax function and how Tax usurps the ubiquitin-proteasome pathway to promote oncogenesis.Human T-cell leukemia virus type 1 (HTLV-1) is a complex retrovirus that infects CD4+ T cells and causes adult T-cell leukemia/lymphoma (ATLL) in 3%\u20135% of infected individuals after a long latent period. HTLV-1 Tax is a Human T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia/lymphoma (ATLL) and the neurodegenerative disorder HTLV-1 associated myelopathy/tropical spastic paraparesis. It is estimated that 20 million people worldwide are infected with HTLV-1 with 3%\u20135% of infected individuals developing ATLL after a prolonged latent period (40\u201360 years) . Th. Th92]. is-Golgi . Interesis-Golgi . In addiis-Golgi . Taken tis-Golgi . Althougis-Golgi . WhetherTax also undergoes SUMOylation at sites that overlap the known ubiquitin sites although its role in NF-\u03baB activation remains controversial . SUMOylaTax ubiquitination can be counteracted by host DUBs that cleave polyubiquitin chains to regulate Tax stability and localization. A recent study showed that CYLD, a DUB and tumor suppressor involved in NF-\u03baB signaling pathways, interacts with Tax and removes ubiquitin chains that results in impaired Tax interaction with NEMO . In HTLVAnother important DUB that plays a prominent role in the negative regulation of key NF-\u03baB pathways is A20 ,106. A20Tax contains NLS and NES sequences which regulate the nucleo-cytoplasmic shuttling of Tax. A fine regulation of Tax cellular distribution is controlled by specific PTMs, leading to conformational changes and inducible protein-protein interactions. The balance between Tax ubiquitination and SUMOylation may potentially regulate Tax shuttling between different cellular compartments and activation of NF-\u03baB . The E3 CDC20 E3 ligase complex ahead of schedule leading to proteasomal degradation of cyclin B1 and securin . B. BC-termo system , we haveo system . It remaIL-17RB gene may potentially compensate for the loss of Tax-induced NF-\u03baB in ATLL. It is also plausible that distinct receptors may be amplified or overexpressed by other mechanisms to drive NF-\u03baB signaling in IL-17RB-negative ATLL cases.To gain more insight into the mechanisms of HTLV-1 T-cell transformation, we recently conducted a next-generation RNA sequencing study to identify genes aberrantly expressed in T cells immortalized by HTLV-1. This effort identified the IL-25 receptor subunit IL-17RB as an aberrantly overexpressed gene in HTLV-1 immortalized T cells . Tax indcis-Golgi where Tax activates IKK [In this review, we have highlighted some of the mechanisms used by the multifunctional HTLV-1 Tax oncoprotein to hijack the cellular ubiquitin-proteasome machinery to promote aberrant signaling linked to cell survival and proliferation. Ubiquitination serves as a versatile tool that can modulate Tax function including nuclear export (mono-ubiquitination), degradation in the nuclear matrix (K48-linked polyubiquitination) or NEMO binding and IKK/NF-\u03baB activation (K63-linked polyubiquitination). Tax hijacks the host ubiquitin machinery to promote its K63-linked polyubiquitination, likely to enhance NEMO binding and other protein-protein interactions in the ates IKK ,93. Inteates IKK . These rTax also activates host E3 ligases (TRAF6) to stabilize MCL-1 and mitigate cell death triggered by genotoxic stress agents and chemotherapy drugs such as etoposide . Convers"} +{"text": "In vitro, miR-218 over-expression decreased clonogenicity, migration, and invasion. Survivin (BIRC5) was subsequently identified as an important cervical cancer target of miR-218 using in silico prediction, mRNA profiling, and quantitative real-time PCR (qRT-PCR). Concordant with miR-218 over-expression, survivin knockdown by siRNA decreased clonogenicity, migration, and invasion. YM155, a small molecule survivin inhibitor, significantly suppressed tumor growth and lymph node metastasis in vivo. Our findings demonstrate that the miR-218~survivin axis inhibits cervical cancer progression by regulating clonogenicity, migration, and invasion, and suggest that the inhibition of survivin could be a potential therapeutic strategy to improve outcome in this disease.Cervical cancer is the third most common cancer in women worldwide. In the present study, global microRNA profiling for 79 cervical cancer patient samples led to the identification of miR-218 down-regulation in cervical cancer tissues compared to normal cervical tissues. Lower miR-218 expression was associated significantly with worse overall survival (OS), disease-free survival (DFS), and pelvic/aortic lymph node recurrence. Cervical cancer is the third most common cancer in women globally . In patiMicroRNAs are small, non-coding RNAs that post-transcriptionally down-regulate the expression of multiple target genes . MicroRNMicroRNA-218 (miR-218) down-regulation has been reported in several human malignancies, including head and neck squamous cell carcinoma (HNSCC), non-small cell lung cancer (NSCLC), pancreatic ductal adenocarcinoma (PDAC), and gastric cancer -15. In cin vitro and in vivo using a small molecule survivin suppressant (YM155), and provide data in support of targeting the miR-218~survivin axis in cancer therapy and preventing metastasis.In the current study, new miR-218-related associations were identified in clinically annotated cervical cancer samples. Furthermore, the cellular and molecular functions of miR-218 and one of its key targets, survivin (BIRC5), were elucidated. Lastly, we validated these observations et al. [; in brief, these patients have all been treated for cure (radiation and chemotherapy) with a median follow-up time of 6 years. We therefore investigated the association between miR-218 expression with patient survival. Initially, the median miR-218 expression value was utilized to divide the 79 cervical cancer patients into high vs. low expression groups . The miR-218 low expression group experienced a worse overall survival (OS), and disease-free survival (DFS) data determined that expression of miR-218 was significantly reduced in 79 cervical cancer tissues compared to 11 normal cervix tissues positive cervical squamous cell carcinoma lines, were transfected with pre-miR negative control (miR-NC) or pre-miR-218 (miR-218). Forty-eight hours post-transfection, miR-218 was over-expressed by more than 200-fold in SiHa and ME-180 cells P<0.01, . In bothBecause miR-218 down-regulation was observed to be associated with lymph node metastasis and recurrence in our patients, we performed migration and invasion assays. Consistent with the clinical data, miR-218 over-expression reduced migration and invasion capacities of both SiHa and ME-180 cells [in silico predicted targets and mRNAs that were up-regulated by greater than 2 fold were 35 candidate targets , 20. The/miRDB/) . At the s Figure . For thenal rank .Survivin was the most consistently and significantly reduced target after miR-218 transfection in both cell lines (The top 10 candidate target genes from survivin 3\u2032-untranslated region (3\u2032-UTR), we cloned the survivin 3\u2032-UTR (which included a miR-218 predicted binding site) into the pMIR-REPORT luciferase vector showed a significant reduction in luciferase activity in both SiHa and ME-180 cells , thereby confirming specific and direct survivin 3\u2032-UTR targeting by miR-218.In order to confirm direct targeting and binding between miR-218 and the r Figure . Cells tSurvivin is the smallest member of the inhibitor of apoptosis (IAP) family, and is mainly associated with the regulation of mitosis and inhibition of apoptosis . Some fuIn general, survivin is absent in normal adult cells and only expressed in cancer cells; thus, survivin might serve as a useful drug target . Furtherin vivo anti-tumor activity. Mice treated with YM155 had significantly reduced tumor growth compared to control mice treated with saline and metastasis (in vivo), in cooperation with XIAP, another IAP family member [Our data showed that survivin knockdown by siRNA significantly reduced clonogenicity, migration, and invasion in SiHa and ME-180 cells, phenocopying the results of miR-218 over-expression. Survivin is known to promote tumor cell invasion , and/or the microenvironment might well account for this discrepant observation. Other survivin-inhibiting compounds are currently in development and might be even more effective inhibitors [Survivin is typically absent in normal adult cells (except for germ cells), and is highly over-expressed in cancer cells, thereby serving as a drug target. YM155 leads to the repression of complex . Phase I complex . Our dathibitors . NonetheThe role of the mir-218~survivin axis in potentially promoting nodal metastasis may suggest a therapeutic opportunity for survivin inhibitors in treating node-positive, locally advanced cancers, and/or cancers with occult nodal involvement. Survivin inhibition may be particularly helpful for adjuvant therapy used to inhibit lymph node metastasis after primary tumor resection. Further work will be required to investigate the use of survivin inhibitors with existing standard treatments for such disease. Moreover, whether this therapeutic opportunity exists in other cancers remains to be investigated.In conclusion, the miR-218~survivin axis is pivotal, both on a clinical and basic cellular level, in regulating clonogenicity, migration, and invasion in cervical cancer. Anti-survivin therapy might provide a potentially useful strategy in partially restoring this axis, and thereby improve outcome for patients with cervical cancer.Written informed consent was obtained from patients according to a protocol approved by the University Health Network (UHN) Research Ethics Board. Animal experiments were performed in strict accordance with the protocols approved by the Animal Care Committee (ACC) of the Ontario Cancer Institute (OCI), UHN.Seventy-nine cervical cancer tissues and 11 normal cervix tissues were collected from fresh frozen punch biopsies. Total RNA was isolated using the Total RNA Purification Kit (Norgen Biotek), according to the manufacturer's protocol. Global microRNA and mRNA profiles were analyzed with the TLDA Human MicroRNA A Array V2.0 (Life Technologies), and GeneChip Human Genome U133 Plus 2.0 Array (Affymetrix), respectively.https://www.broadinstitute.org/software/igv) [Level 3 segmented copy number data for 105 cervical squamous cell carcinoma samples generated by TCGA using SNP 6.0 arrays were downloaded from the Broad Firehose website (2012_11_02 stdata Run). Copy number data for miR-218 encoding loci were then visualized using the integrated genome viewer .2. These cells were authenticated every six months at the Centre for Applied Genomics using the AmpF/STR Identifier PCR Amplification Kit (Applied Biosystems); as well, they were determined to be mycoplasma free every 3 months using the MycoAlert Mycoplasma Detection Kit (Lonza).Cervical cancer cell lines, SiHa and ME-180, were obtained from American Type Culture Collection (ATCC) and cultured in \u03b1-MEM with 10% Fetal Bovine Serum at 37\u00b0C, 5% COSiHa and ME-180 cells were transfected with Lipofectamine RNAiMAX (Life Technologies), according to the manufacturer's protocol. Pre-miR miRNA negative control, pre-miR-218 (Life Technologies), AllStars Negative Control siRNA, and two survivin siRNAs (Qiagen) were transfected at 10 nM.\u2212\u0394\u0394 Ct method was used to calculate relative miR-218 expression, using RNU44 as a reference gene.Total RNA was isolated using the Total RNA Purification Kit (Norgen Biotek), and miR-218 expression levels were measured using the TaqMan MicroRNA Assays (Life Technologies). The 2survivin specific primers . QRT-PCR was performed using SYBR Green PCR Master Mix (Life Technologies) with primers . All mRNTotal protein was extracted using RIPA buffer , then separated using a Novex 4-20% Tris-Glycine Gel (Life Technologies). Proteins were detected using survivin and \u03b1-Tubulin antibodies. Specific proteins were detected using SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific).Cell viability was examined using the CellTiter 96 Non-Radioactive Cell Proliferation Assay , according to the manufacturer's protocol. For clonogenic assays, transfected cells were re-seeded 48 hrs post-transfection at low density in 6-well plates. Cells were incubated for 10-14 days, then fixed and stained with 0.2% methylene blue in 50% methanol. The surviving fraction was calculated by comparison with control cells.5 cells/well) were seeded with medium containing low serum (1%) in the upper chamber. The lower chamber was filled with medium containing high serum (20%) as a chemoattractant. Cells were incubated for 48 hrs and then membranes were stained using Diff-Quick (Siemens). A light microscope was used to count the number of migrating and invading cells.Invasion and migration assays were performed using the BD BioCoat Matrigel Invasion Chambers and Control Inserts (BD Biosciences), respectively. Briefly, cells (1 \u00d7 10survivin 3\u2032-untranslated region (3\u2032UTR) containing the predicted miR-218 binding site were amplified by Platinum Taq DNA polymerase (Life Technologies). Primer sequences are listed in Wild-type (WT) or mutant (MT) fragments of the Luciferase containing lentivirus (Lenti-Luc) was generated by transient transfection of 293T cells with psPAX2, pCMV-VSVG (Addgene), and CSII-CMV-Bsd-Luciferase plasmids using Lipofectamine 2000 (Life Technologies). At 48 hrs after co-transfection, lentivirus-containing supernatant was collected, and then passed through a 0.45 \u03bcm filter. Cultured ME-180 cells were incubated for 48 hrs with Lenti-Luc, then cultured for 2 weeks in the presence of 4.0 \u03bcg/mL Blasticidin S (Life Technologies).7) cells were injected into the left flank or left gastrocnemius muscle subcutaneously or intramuscularly as indicated. Tumor growth was monitored by measuring tumor volume or tumor plus leg diameter (mm). YM155 treatment commenced once the tumor volume reached 50 mm3 or the leg diameter reached 8 mm. YM155 or saline control (mice randomized) was administered subcutaneously as a 3-day per week continuous infusion for 2 weeks using the Alzet Osmotic Pump\u00ae (Model 1003D).Six to eight week-old severe combined immunodeficient (SCID) female mice were utilized for all experiments. ME-180 or Luc-ME-180 (1 \u00d7 107) cells were injected into the left gastrocnemius muscle intramuscularly (donor mice). Once the leg diameter reached 9-12mm, tumors were excised and dissected. The tumors were then cut into 2-3 mm3 fragments in \u03b1-MEM media and placed on ice. Recipient mice were anesthetized and their uteruses exposed. A small incision was made in the cervix and a tumor fragment was sutured in place using a single 8-0 silk suture. The peritoneal membrane was closed in two layers using 8-0 silk sutures followed by skin closure using wound clips [For the orthotopic xenograft model, Luc-ME-180 . Images were analyzed with Living Image 4.1 Software .\u03a72 test. Overall survival curves were plotted according to the Kaplan-Meier method, with the log-rank test applied for comparison. Statistical analyses were performed using JMP5 (SAS Institute).All experiments have been performed at least three independent times, and the data are presented as the mean \u00b1 standard error of the mean (SEM). Statistical significance between treatment groups was determined using the Student's t-test or"} +{"text": "Neuroendocrine tumours (NETs) are a heterogeneous group with significant variation in morphological characteristics and functional behaviour. This poses challenges in terms of both biochemical and imaging assessment.2) is overexpressed in NETs [Somatostatin receptor (sst) overexpression is documented in several malignancies. The sst subtype 2 [In-111-octreotide (Octreoscan), a gamma imaging sst agent, binds with relatively high affinity to sst 80-100%) .PET agents such as Ga-68-DOTA-labelled somatostatin analogues have been developed with higher receptor affinity when compared to gamma-based agents. This leads to improved target-to-background ratio and provides the improved imaging characteristics inherent of PET tracers. A 2012 meta-analysis demonstrated pooled sensitivity and specificity in detecting NETs of 93% and 91%, respectively with higWhilst other PET tracers such as F-18-DOPA have shown utility and higher sensitivity than conventional sst scintigraphy, their role is limited to problem solving at present. F-18-FDG can, however, provide prognostic information with patients showing uptake having a progression free survival of 0% at 2 years compared with 75\u00b110% in those without uptake .Peptide receptor radionuclide therapy (PRRT) utilises primarily beta-emitting radioisotopes such as Lu-177 and Y-90 linked to somatostatin analogues such as DOTATATE and DOTATOC. Whilst phase 3 data comparing PRRT with other therapies is awaited, treatment with Y-90 and Lu-177 PRRT has been shown to have response rates of approximately 80% and to confer a survival advantage over historical controls [Due to the differences in beta particle energy and path length, it has been postulated that Lu-177 PRRT would be best suited to smaller tumour volumes compared with Y-90 which emits a more energetic particle with longer path length. Kunikowska and colleagues have now demonstrated that overall survival is significantly higher in patients treated with combination Y-90/Lu-177-DOTATATE compared with Y-90-DOTATATE alone [Research into the role of sst antagonists is relatively new. Several studies have shown a significantly greater number of binding sites for antagonists when compared to agonists such as Lu-177-DOTATATE. Pilot studies have shown 1.7-10.6 times higher tumour dose with the antagonist Lu-177-DOTA-JR11 when compared to Lu-177-DOTATATE ["} +{"text": "Immunotherapy for breast cancer is now being seriously considered, despite past beliefs that this cancer type was non-immunogenic. Immune profiling has been variably associated with outcome, but standard techniques have greatly limited interpretation. These studies examine immune profiling of breast cancer patients using high-sensitivity methods in relation to clinical outcome.High-sensitivity detection and analysis methods were used to determine Immune Profiles of female human breast cancer in Tumour Infiltrating Leukocytes (TIL) in longitudinal cohort comparative outcome studies.Immune profiles showed predominantly CD3, CD4, CD45RO TIL with low/absent IL-2a receptor expression. However, these were significantly correlated to 5- and 10-year survival times.Immune profiling of the TIL infiltrate in human breast carcinoma using high-sensitivity detection and analysis techniques showed predominance of CD3 cells, being ab-TCR, CD4 T cells of mainly memory phenotype. Importantly, these findings were strongly predictive of 5- and 10-year survival. This indicates the real possibility that TIL infiltration in breast cancer might be open to immunological manipulation therapeutically to improve clinical outcome."} +{"text": "T-MAE cells. Here, we demonstrate that CDV inhibits metastasis induced by FGF2-driven, virus-independent tumor cells. Pre-treatment of luciferase-expressing FGF2-T-MAE cells with CDV reduced single cell survival and anchorage-independent growth in vitro and lung metastasis formation upon intravenous inoculation into SCID mice. This occurred in the absence of any effect on homing of FGF2-T-MAE cells to the lungs and on the growth of subconfluent cell cultures or subcutaneous tumors in mice. Accordingly, CDV protected against lung metastasis when given systemically after tumor cell injection. Lung metastases in CDV-treated mice showed reduced Ki67 expression and increased nuclear accumulation of p53, indicating that CDV inhibits metastasis by affecting single cell survival properties. The anti-metastatic potential of CDV was confirmed on B16-F10 melanoma cells, both in zebrafish embryos and mice. These findings suggest that CDV may have therapeutic potential as an anti-metastatic agent and warrants further study to select those tumor types that are most likely to benefit from CDV therapy.The FDA-approved anti-DNA virus agent cidofovir (CDV) is being evaluated in phase II/III clinical trials for the treatment of human papillomavirus (HPV)-associated tumors. However, previous observations had shown that CDV also inhibits the growth of vascular tumors induced by fibroblast growth factor-2 (FGF2)-transformed FGF2- S)-1-(3-hydroxy-2-phosphonyl-methoxypropyl)cytosine, CDV] is a broad-spectrum antiviral agent approved (Vistide\u00ae) for the treatment of cytomegalovirus-induced retinitis in AIDS patients was obtained from Gilead Sciences Cidofovir . Lysates were cleared by centrifugation and the protein concentration was determined. SDS-PAGE gel electrophoresis of the cell lysates were performed as described . After eThe wild-type AB zebrafish line was maintained at the Zebrafish Facilities of the University of Brescia as described . B16-F10t test was used to determine the statistical significance of the data; p values < 0.05 were considered significant.Two-tailed Student's"} +{"text": "Allergic diseases are characterized by tissue eosinophilic and basophilic inflammation. There is substantial evidence that this particular inflammatory profile results from the migration to tissues of a common eosinophil-basophil (Eo/B) progenitor that undergoes a differentiative process regulated by local cytokines, termed in situ hemopoiesis. We therefore investigated the role and mechanisms of TSLP involvement in human Eo/B in situ hemopoiesis in relation to atopy. Also, since candidate gene and genome-wide association studies have identified \u2018protective\u2019 associations between the single nucleotide polymorphism (SNP) rs1837253 in the TSLP gene and risk for allergy, asthma and airway hyper-responsiveness, we evaluated the secretion of TSLP protein from primary nasal epithelial cells (NEC) in relation to rs1837253 genotype.Peripheral blood CD34+ cells derived from atopic and nonatopic individuals were stimulated with and/or IL-3, IL-5, or GM-CSF and assessed for Eo/B colony forming units (CFU) in both methylcellulose and nasal epithelial/CD34+ cell air-liquid interface (ALI) co-cultures, and cytokine surface receptor expression by flow cytometry. Genotyping was performed using a commercially available TaqMan\u00ae genotyping assay.\u2218 TSLP preferentially enhanced IL-3/TNF\u03b1-dependent Eo/B CFU from CD34+ cells;\u2218 Eo/B CFU and TSLPR expression were significantly increased in atopic-derived CD34+ cells post TSLP- (p<0.05) and IL-3/TSLP-stimulation (p<0.001), compared to non-atopic-derived CD34+ cells;\u2218 In progenitor/NEC ALI co-cultures, secreted TSLP was biologically active, and sufficient to induce the differentiation of CD34+ cells into Eo/B CFU;\u2218 There was decreased TSLP secretion by nasal epithelial cells obtained from heterozygous and homozygous rs1837253 TSLP minor allele individuals, compared to homozygous \u2018wild-type\u2019 individuals .These studies further support the concept of in situ hemopoiesis and point out a previously unrecognized, critical role for TSLP in the development of allergic upper airway inflammation and its role in Eo/B differentiation, a link between adaptive and innate immunity. Novel functional genomics of TSLP show that the rs1837253 polymorphism may be directly involved in the regulation of TSLP secretion, providing explanations for both genetic association studies and recently reported positive clinical trials targeting TSLP in allergic asthma."} +{"text": "Our group has developed an oncolytic poliovirus, PVSRIPO, capable of lysing malignant cells while generating inflammation at the site of the tumor. PVSRIPO is a recombinant polio:rhinovirus chimera, engineered to eliminate neuropathogenicity, that has cancer tropism due to ectopic expression of the poliovirus receptor, CD155, in solid cancers. Importantly, PVSRIPO therapy is effective in the presence of neutralizing antibodies and an innate antiviral response. A first-in-human Phase I study with PVSRIPO has shown remarkable promise in patients with recurrent glioblastoma (GBM), a uniformly lethal disease. PVSRIPO tumor cell killing is associated with the induction of danger- and pathogen-associated molecular patterns (DAMPs and PAMPs) via antiviral type I interferons (IFNs) and simultaneous non-lethal infection of antigen-presenting cells (APCs) such as monocytes and dendritic cells (DCs).in vitro human assay. Human DCs generated from HLA-A2+ donor cells were incubated with PVSRIPO-induced tumor cell lysate and then used to stimulate autologous T cells in vitro followed by a cytotoxic T lymphocyte (CTL) assay. The following HLA-A2 human cell lines were used for this study: DM6 (MART+) melanoma cell line, MDA-MB231 (CEA+) triple-negative breast cancer cell line, SUM149 (EGFR+) inflammatory breast cancer cell line and LNCaP (PSA+).To understand key immune events associated with poliovirus infection of APCs, we examined the effects of PVSRIPO treatment on the human macrophage cell line (THP1) and primary human monocyte-derived DCs. PVSRIPO-treated DCs were evaluated for expression of maturation/activation markers and compared to untreated immature and mature DCs. To determine whether DCs exposed to PVSRIPO-induced dying tumor cells present tumor antigens to T cells, we performed an in vitro, are capable of stimulating tumor antigen-specific T cells. Autologous DCs transfected with RNA that encodes for CEA, EGFR, MART or PSA were used to assess tumor antigen-specificity of T cells.PVSRIPO infection of THP1 macrophages and human DCs is sublethal; induces MHC class II and costimulatory molecule expression; and leads to IFN-\u03b2, IL-12, and TNF-\u03b1 production. Coculturing of DCs with PVSRIPO-induced tumor lysate stimulates DC activation and IL-12 production. Human DCs loaded with PVSRIPO-induced tumor cell lysate in vitro human immunotherapy assay. In ongoing studies we are analyzing oncolytic poliovirus mediated immune events in syngeneic, immunocompetent murine models.Our data suggests that along with destruction of the primary tumor, oncolytic poliovirus mediates immune events. We demonstrate that human DCs co-incubated with PVSRIPO-induced tumor cell lysate stimulate tumor antigen-specific T cell responses in an"} +{"text": "Bacterial cells sense their population density and respond accordingly by producing various signal molecules to the surrounding environments thereby trigger a plethora of gene expression. This regulatory pathway is termed quorum sensing (QS). Plenty of bacterial virulence factors are controlled by QS or QS-mediated regulatory systems and QS signal molecules (QSSMs) play crucial roles in bacterial signaling transduction. Moreover, bacterial QSSMs were shown to interfere with host cell signaling and modulate host immune responses. QSSMs not only regulate the expression of bacterial virulence factors but themselves act in the modulation of host biology that can be potential therapeutic targets. Quorum sensing (QS) is coined to describe the phenomenon of an intercellular co-operative behavior of bacteria used to coordinate the activities of individual cells. Diffusible QS signal molecules (QSSMs) play crucial roles in signal transduction of which, when QSSMs reach a threshold concentration, can coordinate multiple gene expression and a change in the behavior of bacterial population through the activation of sensor regulatory proteins . BacteriPseudomonas aeruginosa is an ubiquitous Gram-negative bacterium with remarkably large and complex genome and is capable of adapting to versatile environments. In human cystic fibrosis (CF) lungs where P. aeruginosa has evolved the ability to form biofilms which are difficult to be eradicated by antibiotics (P. aeruginosa are regulated by QS (P. aeruginosa QSSMs focusing on their roles in interference with host cells (Table 1) and the development of novel compounds that counteract the QSSMs activities.ibiotics . QS is red by QS . Here weAliivibrio fischeri . When an AHLs concentration of 10 nM is reached, AHLs interact with LuxR and form a complex which promotes the expression of target genes, luxICDABE for bioluminescence production and also the LuxI production (N-acyl homoserine lactone consists of a homoserine lactone ring from S-adenosylmethionine (SAM) and acyl chain from acyl acyl-carrier-protein (acyl-ACP) linked by an amide bond (A. fischeri N-(3-oxohexanoyl) homoserine lactone (3-oxo-C6-HSL) is produced for controlling bioluminescence production. In P. aeruginosa two AHL synthases, RhlI and LasI, produce a wide spectrum of AHLs including N-butanoyl-homoserine lactone (C4-HSL), N-hexanoyl-homoserine lactone (C6-HSL) by RhlI and N-(3-oxooctanoyl)-homoserine lactone (3-oxo-C8-HSL), N-(3-oxodecanoyl)-homoserine lactone (3-oxo-C10-HSL), N-(3-oxododecanoyl)-homoserine lactone (3-oxo-C12-HSL) and N-(3-oxotetradecanoyl)-homoserine lactone (3-oxo-C14-HSL) by LasI (N-(3-oxohexadecanoyl)-homoserine lactone (3-oxo-C16-HSL) secreted by an environmental Pseudomonas sp. from a diseased Tilapia fish suggests that 3-oxo-C16-HSL may contribute to the pathogenesis pathways . In addition to its cytotoxicity, the role of 3-oxo-C12-HSL in immunomodulation has been intensively investigated . In murine fibroblasts and human lung epithelial cells peroxisome proliferator-activated receptor beta/delta (PPAR\u03b2/\u03b4) and PPAR\u03b3 may be the 3-oxo-C12-HSL receptors for pro-inflammatory responses (N-tetradecanoyl-homoserine lactone) mediated apoptosis indicating that XBP1s is a critical host target in response of AHLs -based QS system and the signal molecule was termed Pseudomonas quinolone signal -quinolone (PQS) and its precursor molecular 2-heptyl-4(1H)-quinolone (HHQ) are major QSSMs that cooperates with the AHL-QS from anthraniloyl-coenzyme A and malonyl-CoA, decarboxylating coupling of 2-ABA to an octanoate group of octanoic acid that linked to PqsBC to produce HHQ -thiazole-4-carbaldehyde (IQS) encoded by the recently . IQS is function . Moreoveon model , suggestP. aeruginosa virulence was firstly described by phnAB operon and the production of elastase, 3-oxo-C12-HSL and PQS that promotes the production of numerous virulence determinants. The pqsR mutant was attenuated up to 320-fold in the Arabidopsis plant infection model and caused a 65% reduction of mortality in a murine burn wound model and mucopurulent fluid from distal airways of end-stage CF lungs removed for transplant and at different stages from asymptotic early stage to late progression, suggesting a potential role of PQS in coordinating virulence factors during the course of infections in plasma was suggested to be the biomarker for P. aeruginosa infection in CF lungs and pqsABCDE, were expressed 10-fold greater than the expression when P. aeruginosa was cultured in media containing glucose alone as the carbon source and TNF-\u03b1 in mitogen-stimulated human peripheral blood mononuclear cells pathways in murine macrophages and cells obtained from BAL (P. aeruginosa, did not interfere with neutrophils phagocytic capability and viability (P. aeruginosa with another strategy for bacterial survival via the interference in multiple aspects of host biological activities.s PBMCs; . PQS inhs PBMCs; . Additiofrom BAL . A receniability . Massiveiability . PQS mayP. aeruginosa infections in an acute murine lung infection model (P. aeruginosa infection had higher survival than those without immunization (in vivo dermal infection model (P. aeruginosa infection increased mice survival and lowered the bacterial dissemination to the organs (P. aeruginosa but also effectively reduced the formation of antibiotic-tolerant persisters (Due to the fact that QSSMs have been implicated in the involvement of pathogenesis, the search for inhibitors and the development of vaccines that antagonize QSSMs are currently intensively investigated. on model . In a bunization . A high-on model . Since ae organs . MvfR-rersisters . These sQuorum sensing-based bacterial communication links the individual bacterial cells to behave as multicellular organisms by employing signal molecules and to promote its population survival in the environment or hosts. QSSMs also interact with host cell signal pathways and the modulation of immune cell biology. For more than a decade strategies have been proposed from the use of inhibitors of QS for containing chronic infections to the aY-CL, K-GC, C-YC wrote the paper. Y-CL made the figure. Y-CL and C-YC made the table.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Receptor activator of nuclear factor kappa B ligand (RANKL) is a key regulator of bone metabolism. Anti-citrullinated protein antibodies (ACPA) have been suggested to cause bone destruction by osteoclast activation. We investigated the relationship between RANKL and ACPA in patients with early untreated rheumatoid arthritis (RA).Patients with newly diagnosed untreated RA (n = 183) were analyzed at baseline and 3 months after initiating methotrexate (MTX) treatment. Serum RANKL , ACPA (anti-CCP2) and ACPA specificities (anti-citrullinated (cit)-vimentin, anti-cit-enolase and anti-cit-fibrinogen) were determined by enzyme-linked immunosorbent assay (ELISA). Synovial RANKL expression was evaluated by immunohistochemistry in a small group of patients (n = 15). The relationship between anti-cit-vim antibodies and bone destruction was further validated in 1116 RA patients included in the EIRA cohort. Pearson\u2019s chi-square test, Wilcoxon rank sum test, Wilcoxon signed rank test and linear regression models were used.p <0.05) in ACPA-positive compared with ACPA-negative patients and this difference was also seen for synovial RANKL expression. Serum RANKL associated with ACPA (p <0.05) and bone erosions in rheumatoid factor (RF)-negative patients (n = 59). Among ACPA specificites, anti-cit-vimentin (amino acids 60\u201375) was associated with higher RANKL concentration and higher prevalence of bone erosion (p <0.05). Significant reductions in both serum RANKL and ACPA levels were observed after 3 months of MTX treatment (p <0.05).Serum RANKL concentration was significantly higher (RANKL was elevated in ACPA-positive and in anti-cit-vimentin-positive patients with early untreated RA and associated with bone erosions. These findings give further support for an early direct pathogenic link between ACPA and bone destruction in RA. Osteoimmunology is a conceptual and molecular understanding of how the immune system influences the bone metabolism in diseases such as rheumatoid arthritis (RA) , 2. RA iReceptor activator of nuclear factor kappa B ligand (RANKL) is in the concept of osteoimmunology; a key molecule in the regulation of bone metabolism and the linkage between immune and skeletal systems , 16. RANIn this study, we aimed to determine to what extent RANKL levels associate with presence of ACPA, bone erosions and MTX treatment in a cohort of patients with early untreated RA.In summary, we can report that RANKL was elevated in ACPA-positive and in anti-cit-vimentin-positive patients and associated with bone erosions in patients with early untreated RA.The study was performed in a cohort of 183 patients with early untreated RA with symptom onset <1 year prior to diagnosis, recruited at the Rheumatology Clinic at Karolinska University Hospital, Stockholm (during years 1996\u20132006) and part of the Epidemiological Investigation of Rheumatoid Arthritis (EIRA) study cohort . ClinicaSerum samples and DAS28 based on the erythrocyte sedimentation rate (ESR) were obtained at baseline and at clinical follow-up, which occurred after a median of 14 weeks (interquartile range 25\u221275 % (IQR) 13\u221215). Data on the presence of HLA-DRB1 shared epitope (SE) gene allele, protein tyrosine phosphatase gene allele (PTPN22 rs2476601) and body mass index (BMI) at inclusion were also available \u201328.Review of the medical records provided information on the presence or absence of at least one bone erosion at baseline, based on standard radiographs of hands and feet according to clinical routine at our unit. Seven patients were excluded from posttreatment analyses due to delayed MTX treatment initiation (>7 weeks after baseline) or secession of treatment; these patients were, however, included in pretreatment (baseline) analyses.Additionally, 1116 patients from the EIRA study cohort with available study data on standard radiograph changes in hands (at least one change according to 1987 RA criteria ) and serACPA were detected by anti-cyclic citrullinated peptide version 2 (anti-CCP2) test using the manufacturer\u2019s protocol. ACPA levels above the highest standard sample >3200 AU/ml) were reassessed after appropriate dilution. Antibodies (anti)-cit-enolase peptide 1 , anti-cit-vim peptide (amino acids 60\u201375), and anti-cit-fibrinogen (fib) peptide (amino acids 563\u2013583) were measured in sera by previously established enzyme-linked immunosorbent assay (ELISA). Native forms of the same peptides were used as controls . Serum c00 AU/ml Synovial biopsies obtained during arthroscopy were available from 15 patients with early untreated RA (including 7 of the 183 patients that donated blood). Patients had a median age of 56 years range 33\u201378); 10 out of 15 were females; 7 out of 15 were ACPA-positive and 2 out of 15 had bone erosion at baseline. Synovial expression of RANKL was detected by immunohistochemistry staining, using a monoclonal anti-human RANKL detection antibody at a final concentration of 5 \u03bcg/ml as previously described , 33. The\u201378; 10 oPearson\u2019s chi-square test, Wilcoxon rank sum test and Wilcoxon signed rank test were used for independent and paired comparisons as appropriate. To investigate the relationship between RANKL and ACPA independent of RF, the analysis of RANKL was performed both in the overall group (n = 183) and also in the subset of RF-negative patients (n = 59).p values <0.05 were considered statistically significant. No adjustments for multiple testing were performed in the explorative part of the study, but Bonferroni correction was applied for comparisons in the validation cohort.In order to investigate the association between RANKL concentration (logarithm-transformed concentration) and ACPA status, we also tested other possible predictors for RANKL: age, sex, smoking habits (history of ever or never smoking), BMI, DAS28-ESR, ESR, CRP, IL-6 serum levels, TNF-RI serum levels, health assessment questionnaire (HAQ) values, concurrent prednisolone usage, concurrent usage of antiporotic treatment, presence of HLA-DRB1 SE, presence of PTPN22 risk allele, one by one in univariate linear regression models. A multiple regression model for the association between RANKL and ACPA was then obtained by including the significant predictors from the univariate analyses. Statistical analyses were conducted using SAS 9.3 . Two tailed Of the 183 patients, 125 (68%) were ACPA-positive with a median concentration of 752 AU/ml (IQR 278\u20132174). Among these ACPA-positive patients 58% were also positive for anti-cit-enolase, 52% for anti-cit-vim, and 31% for anti-cit-fib (see Table\u00a0p <0.05). Similarly, median synovial expression of RANKL or anti-cit-vim negative , respectively.To further investigate the relationship between RANKL and ACPA independent of RF, we performed a separate analysis of RF-negative patients. Similar to the whole cohort, the median RANKL serum concentration remained significantly higher in RF-negative ACPA-positive as compared to RF-negative ACPA-negative patients Fig.\u00a0. SignifiUsing linear univariate regression models we identified significant association between serum RANKL and ACPA, age, DAS28-ESR and BMI, while all other tested variables were not significant predictors for RANKL. Only ACPA and BMI remained significant in the multivariate model. A mean of 232 pmol/l (95 % CI: 155\u2013346) RANKL in ACPA positive and 140 pmol/l (95 % CI: 114\u2013171) RANKL in ACPA negative was estimated after adjustments of age, DAS28-ESR and BMI (Table\u00a0p <0.05) in patients with evidence of bone erosions at baseline compared with those without bone erosions and significantly more prevalent in anti-cit-vim (amino acids 60\u201375) -positive than anti-cit-vim-negative patients Fig.\u00a0. Baselin05) Fig.\u00a0, while np value <0.05) and anti-cit-vim-negative patients , respectively antibodies and bone destruction finding was confirmed in the validation cohort (n = 1116) in which we found, that both ACPA-positive and anti-cit-vim (amino acids 60\u201375) -positive patients had a higher frequency of destructions in comparison to ACPA-negative and the changes were similar in the subset of RF-negative patients. Serum RANKL levels decreased in a large majority of the patients and RF-negative ACPA-negative patients . Interestingly, RANKL concentration after 3 months of MTX treatment remained higher in RF-negative ACPA-positive patients than in RF-negative ACPA-negative patients .Serum RANKL concentrations decreased significantly following MTX treatment from baseline to 3 months , for the patients that were ACPA-positive at baseline and/or at 3 months (n = 120) at baseline to 556 (IRQ 197\u20131920) at 3 months (20) Fig.\u00a0. ACPA le\u20132197 at p <0.05), anti-cit-vim (amino acids 60\u201375) and anti-cit-fib (amino acids 563\u2013583) , for patients positive either at baseline or at 3 months ACPA-positive, 4 of the 71 (5.6 %) anti-cit-enolase (amino acids 5\u201321) -positive, 21 of the 64 (33 %) anti-cit-vim (amino acids 60\u201375) -positive and 12 of the 40 (30 %) anti-cit-fib (amino acids 563\u2013583) -positive patients. Conversion from seronegative at baseline to seropositive at 3 months was observed infrequently: anti-cit-vim (amino acids 60\u201375) (3 out of 112) (2.7 %); anti-cit-fib (amino acids 563\u2013583) (2 out of 136) (1.5 %). No patients who were ACPA or anti-cit-enolase (amino acids 5\u201321) seronegative at baseline converted to seropositive.Bone destruction, essentially dependent on the effect of RANKL on osteoclast precursors, associates with the presence of ACPA in RA. ACPA directly affects osteoclast precursors, but the relationship between ACPA and RANKL has not yet been investigated. In this study, we report that serum and synovial RANKL levels, used as surrogate makers of local and systemic bone destruction, are higher in ACPA-positive than in ACPA-negative newly diagnosed untreated RA patients. Both ACPA and RANKL levels decrease following treatment with methotrexate (MTX),.We show that RANKL serum levels are higher in ACPA-positive as compared to ACPA-negative patients with early untreated RA, in contrast to one previous study were they found difference in a degradation product of type I collagen (CTX-I) but not RANKL . DetectiNone of the measured inflammatory parameters associated with ACPA and bone erosion in contrast to RANKL, which suggests an association between RANKL, ACPA and bone erosion that is at least partially uncoupled from inflammation. One previous investigation of serum pre-RA samples (n = 79) failed to detect differences in RANKL serum levels as compared to controls despite ACPA have previously been associated with presence of radiological bone destruction in early RA patients , 39\u201341 tValidation of our findings in other cohorts, testing for antibodies against more cit-peptides and investigation of the direct in vitro effect of different ACPAs specificities on osteoclastogenesis and bone metabolism are needed. We chose to define erosive disease as presence of at least one erosion on X-rays of hands and feet, allowing increased detection sensitivity in this cohort of patients already fulfilling the 1987 ACR classification criteria, in accordance with recently published recommendations from a EULAR task force .However, a future more detailed analysis of bone changes not available in the current study might reveal more specific associations.Lastly, serum RANKL, ACPA and ACPA specificities (anti-cit-enolase (amino acids 5\u201321), anti-cit-vim (amino acids 60\u201375), and anti-cit-fib (amino acids 563\u2013583)) levels significantly decreased after 3 months of treatment with MTX and a relative high proportion of patients converted from seropositive to seronegative. These results are consistent with previous studies showing that MTX decreased synovial expression of RANKL in vivo, and cellular expression of RANKL in vitro , 35, 36.In summary, serum RANKL in ACPA-positive early untreated RA associate with erosive disease and is modulated by MTX. Our findings give further support for an early direct pathogenic link between ACPA and bone destruction in RA."} +{"text": "Refractory headaches (RH) represent a difficult challenge, even for the most skilled practitioners.In-depth psychological and psychopathological evaluation of patients with RH.We enrolled 51 consecutive patients (age range 24-65 years) with a chronic headache who satisfied 1 or more of the following conditions: 1) failure of at least 2 prophylactic treatments; 2) medication overuse non-responsive to at least 2 properly conducted protocols of detoxification; 3) medication overuse relapsed at least twice after successful detoxification. Patients were evaluated by a clinical psychologist by the means of SCID-I (for psychiatric disorders), SWAP-200 and investigation of life/traumatic events.Ninety-six-percent of the patients showed at least one psychiatric disorders of the anxiety-depression spectrum: 65% at least one anxiety-disorder, 63% at least one mood-disorder and 50% one of somatoform-disorders with 67.65% showing more than one disorder. No patient showed Obsessive-Compulsive disorder or Headache Attributed to Psychiatric disorders (ICHD-III-\u03b2). As regards personality clinical characteristics, a relevant portion of subjects showed obsessive personality prototype (43.5%), dysphoric high-functioning personality prototypes was detected in 23% of subjects, while clinical traits of Histrionic and Avoidant personality clinical prototypes were present in 20.6% and 35%, respectively. None of the subjects showed Schizoid, Anti-Social, Emotional Dysregulated, Dependent or Hostile personality prototypes. Forty-seven percent showed some kind of infantile trauma/abuse and 88% referred at least one stressful event after headache onset.Psychological and psychopathological factors are of critical importance in RH, and should be taken into consideration for both diagnostic and therapeutic purposes.No conflict of interest."} +{"text": "TFR was significantly higher in CML patients with acRTL \u22640.09 . CML stem cells harboring longer telomeres possibly maintain a proliferative potential after treatment discontinuation.We studied telomere length in 32 CML patients who discontinued imatinib after achieving complete molecular remission and 32 age-sex-matched controls. The relative telomere length (RTL) was determined by q-PCR as the telomere to single copy gene (36B4) ratio normalized to a reference sample (K-562 DNA). Age-corrected RTL (acRTL) was also obtained. The 36-month probability of treatment-free remission (TFR) was 59.4\u00a0%. TFR patients showed shorter acRTL compared to relapsed contains supplementary material, which is available to authorized users. Telomeres are specialized nucleoprotein structures composed of long arrays of TTAGGG repeats localized at the ends of human chromosomes able to maintain genome stability and integrity and to protect the cell from progressive DNA shortening during repeated division . TelomerThirty-two chronic-phase CML patients discontinued TKI treatment after achieving complete molecular remission (CMR) for at least 18\u00a0months. All patients received imatinib therapy for more than 24\u00a0months. Two patients underwent second-line treatment with nilotinib because of molecular relapse. The median follow-up after discontinuation was 30\u00a0months (range 18\u201360). A complete molecular response was defined as undetectable breakpoint cluster region-Abelson (BCR/ABL1) by real-time quantitative polymerase chain reaction (qRT-PCR) with a sensitivity of the assay corresponding to molecular response (MR)4 and MR4.5. Peripheral blood samples from 32 age- and sex-matched healthy individuals were used for control purposes. The relative telomere length (RTL) was determined by q-PCR according to the technique described by Cawthon in 2002 [U test showed shorter acRTL in TFR patients compared to patients with molecular relapse progenitors would result in telomere shortening, leading to genetic instability. Later, CML cells would escape senescence and apoptosis through upregulation of telomerase and restored telomere length. This would promote the occurrence of genetically unstable CML subclones with a selective growth advantage . DiscontCML, chronic myeloid leukemia; TFR, treatment-free remission; CMR, complete molecular remission; MR, molecular response; qRT-PCR, real-time quantitative polymerase chain reaction; BCR/ABL1, breakpoint cluster region-Abelson; TKIs, tyrosine kinase inhibitors; RTL, relative telomere length; acRTL, age-corrected relative telomere length; WBC, white blood cell; PLT, platelets"} +{"text": "Hepatitis C virus (HCV) infection is a major risk factor for chronic hepatitis, cirrhosis and hepatocellular carcinoma (HCC). Our aim is to explore molecular changes that underlie HCV infection-associated HCC in a humanized mouse model, in order to identify markers of HCC progression.Liver proteins from human hepatocyte-engrafted and HCV-infected MUP-uPA/SCID/Bg mice were compared with either uninfected controls or HCV-infected but HCC-negative mice by Western blotting. MicroRNA markers of HCC positive or uninfected mouse liver were analyzed by RT-PCR.We describe the depletion of tumor suppressor proteins and induction of oncoproteins and oncogenic microRNAs (oncomiRs) in HCV-infection associated HCC. Similar depletion of PTEN protein in both HCC-positive and HCV-infected but HCC-negative liver suggests that PTEN depletion is an early, precancerous marker of HCC. By contrast, induction of oncoprotein cMyc, oncomiRs and inflammatory response proteins correspond to HCC progression.While the loss of PTEN is important for the initiation of HCV infection-associated HCC, PTEN depletion by itself is insufficient for tumor progression. Liver tumor progression requires induction of oncoproteins and oncomiRs. Overall, human hepatocyte-engrafted (MUP-uPA/SCID/Bg) mice provide a suitable small animal model for studying the effects of oncogenic changes that promote HCV infection associated HCC. Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide; its incidence is increasing because of the prevalence of chronic hepatitis C and B viral infections , 2. The ex vivo manipulation of progenitor cells followed by their transplantation into recipient mice [Earlier reports of molecular markers of liver cancer in a mouse model were examined by ent mice \u20136. WhileUnlike the expression of uPA transgene under albumin promoter (Alb-uPA) as originally described , MUP-uPATo identify molecular markers of HCV infection-associated HCC, we compared expression levels of oncoproteins and tumor suppressor proteins from liver tissues of HCV-infected HCC with the HCV-infected but HCC-negative mice. Results suggest that loss of PTEN tumor suppressor protein is an early indicator of HCV infection-associated HCC. By contrast, induction of c-Myc and inflammatory response molecules, correlate with HCC progression.Micro-RNAs have been studied as independent markers of oncogenic progression . In compProcedures for human hepatocyte engraftment and HCV infection of immune compromised (MUP-uPA/SCID/Bg) mice were described earlier . Eleven Liver tissues used for Western blot analysis were from human hepatocyte-engrafted MUP-uPA/SCID/Bg uninfected control, or HCV-infected but HCC-negative or HCV infection-induced liver tumors (HCC positive) mice.t-test for each subgroup compared with the reference (loading controls). Data shown is SE of the mean (SEM). Statistical significance is set to *p\u2009<\u20090.05.Liver samples were stored at -80\u00a0\u00b0C until analysis. Liver tissues were lysed with RIPA buffer supplemented with protease inhibitor cocktail (Roche) as described . Cell fr1ug of total RNA was added to QuantiMir RT Kit Small RNA Quantification System (System Biosciences) following the manufacturer\u2019s protocol. The reverse-transcribed product was then diluted 40 fold. Real time quantitative PCR was performed with iTaq SYBR Green Supermix with Rox (Bio-Rad) on ABI 7300 PCR system. Primers used for detecting hsa-miR-141, hsa-miR-23a, hsa-miR-122, hsa-miR-181, hsa-miR-21, hsa-miR-221, hsa-miR-26a and hsa-miR-16 respectively were as follows: 5\u2019-TAACA CTGTC TGGTA AAGAT GG-3\u2019, 5\u2019-ATCAC ATTGC CAGGG ATTTC C-3\u2019, 5\u2019-TGGAG TGTGA CAATG GTGTT TG-3\u2019, 5\u2019-AACAT TCAAC GCTGT CGGTG AGT-3\u2019, 5\u2019-TAGCT TATCA GACTG ATGTT GA-3\u2019, 5\u2019-AGCTA CATTG TCTGC TGGGT TTC-3\u2019, 5\u2019-TTCAA GTAAT CCAGG ATAGG CT-3\u2019 and 5\u2019-TAGCA GCACG TAAAT ATTGG CG-3\u2019Statistical analysis: All quantitative real-time PCR and densitometry data were analyzed using Microsoft Excel 2010, using miR-16 (which does not change following HCV infection) for normalization.Loss of tumor suppressor proteins and induction of oncogenic proteins are the initiating events that promote deregulation of molecular pathways underlying the development of hepatocellular carcinoma , 13. We Immunoblots of PTEN protein from liver tumors (T) of thirteen hepatocyte-engrafted and HCV-infected MUP-uPA/SCID/bg mice and twelve control (C) mice that were engrafted with human hepatocytes but not infected with HCV are shown Fig.\u00a0The next issue is whether the PTEN produced in chimeric mouse liver is functional. PTEN is a dual specificity phosphatase with lipid and protein phosphatase activities \u201319. CytoNucleus-localized PTEN protein has an essential role in cell cycle homeostasis and genomic stability , 19, 20.c-Myc is a constitutively induced transcription activator in a broad range of human cancers , 22. We DLC1 (Deleted in Liver Cancer 1) gene maps to chromosome 8 (8p21.3-22), a region that is frequently deleted in solid tumors. DLC1 encodes GTPase-activating protein (GAP) that acts as a negative regulator of the Rho family GTPases . Loss ofHCV structural and non-structural proteins have been shown to interact with and modulate transcriptional regulatory activity of p53 tumor suppressor protein \u201326. ModuPersistent viral infection is an underlying cause of inflammation-induced cancer, including HCC. More than 90\u00a0% of HCCs arise in the context of hepatic injury and inflammation. Inflammation-associated oncogenic response is mediated by STAT proteins; in particular, activated STAT3 , 28. To Next, we investigated whether the activation of STAT3 in liver tumors was coordinately regulated with other inflammatory response proteins, notably interleukin-6 (IL-6) and IL-6R \u201330, in HWnt signaling, are frequently mutated in liver cancer. Disruption of Wnt signaling results in \u03b2-Catenin stabilization and translocation to the nucleus where it activates cell survival and cell proliferation genes [Signal transduction pathways, in particular on genes . Activaton genes . ConsideMicroRNAs can function as tumor suppressors or oncogenes (oncomiRs) . AlteredMicroRNA 141 (miR-141) is induced in HCV-infected human primary hepatocytes. Importantly, miR-141 directly targets DLC1 tumor suppressor protein expression , attestiTumor suppressor microRNA 26a (miR-26a) is depleted in liver tumor as compared to the surrounding normal liver tissues . We thenAltered expression levels of miRNAs have also been reported from liver tumors of heterogeneous origin. To identify changes in miRNA expression that distinguish HCV infection-associated HCC, we examined selected examples of miRNAs that have been reported from studies of various liver tumors. Increase in miR-181 levels was reported in AFP-positive tumors and in embryonic liver cells . Our resContribution of miR-122 in HCV replication has been studied in a number of cell culture systems and in animal models; however, its role in HCV infection-related HCC in humans is less clear , 39. A rMicroRNA 23a (miR-23a) has been shown to target genes involved in gluconeogenesis at different stages of hepatocarcinogenesis in mouse liver as well as in primary human HCC , 42. In Our studies suggest that amplification of oncomiRs and the loss of tumor suppressor miR-26a are specific indicators of HCV infection-associated HCC. By contrast, altered expression of miR-181, miR-122 and miR-23a appears to be a less specific indicator of HCV infection-associated HCC.in vitro studies of HCV-infected human hepatocytes reported earlier [In this study of a mouse model of HCV infection-associated liver tumor, we have attempted to identify tumor suppressor genes and miRNAs whose expression defines HCV infection-associated HCC. PTEN is a haploinsufficient tumor suppressor gene ; single earlier . CompariInduction of inflammatory effector molecules in HCV infection-associated HCC described here support the notion that an inflammatory microenvironment is an essential factor in the development of HCC. Further studies with the humanized mouse model are needed to address the issue of whether the oncogenic changes induced early in HCV infection promote the sustained inflammatory microenvironment required for tumor progression . A summaInduction cMyc oncoprotein is more pronounced in HCV-infected liver tumors as compared to HCC-negative liver Fig.\u00a0 and d, sMicroRNAs (miRNAs) represent a substantial fraction of tissue-specific small non-protein coding RNA modulators of gene expression. MicroRNAs can function as gene silencers by blocking mRNA translation and destabilizing the target mRNA. Phenotypic consequences of miRNA-regulated genes evoke essential features of tumor biology, including modulation of apoptosis, cell proliferation, signal transduction and stress response. Dysregulated expression of miRNAs has proven valuable in tumor classification and prognosis , 34, 35.Tumor suppressor genes modulate signaling networks that protect against tumor initiation and progression. Loss of function of tumor suppressor genes (TSGs) is generally due to inactivation by deletions, point mutations or promoter hypermethylation. We present evidence of down-regulation of tumor suppressor proteins in a humanized mouse model of HCV infection-associated HCC. These studies represent the first example of functional insufficiency of tumor suppressor proteins induced by HCV infection. The studies with a humanized mouse model of HCC should encourage investigation of molecular pathways that are modulated by amplification of oncogenes and the inhibition of tumor suppressor genes."} +{"text": "Teratoma assay allows assessing invivo immunogenicity of iPS cells and of their differentiated progeny simultaneously. We observed that early-passage Ser-iPS cells formed more teratomas with less immune cell infiltration and tissue damage and necrosis than MEF-iPS cells. Differentiating Ser-iPS cells in embryoid bodies (EBs) showed reduced T cell activation potential compared to MEF-iPS cells, which was similar to syngeneic ES cells. However, Ser-iPS cells lost their reduced immunogenicity invivo after extended passaging invitro and late-passage Ser-iPS cells exhibited an immunogenicity similar to MEF-iPS cells. These findings indicate that early-passage Ser-iPS cells retain some somatic memory of Sertoli cells that impacts on immunogenicity of iPS cells and iPS cell-derived cells invivo and invitro. Our data suggest that immune-privileged Sertoli cells might represent a preferred source for iPS cell generation, if it comes to the use of iPS cell-derived cells for transplantation.Sertoli cells constitute the structural framework in testis and provide an immune-privileged environment for germ cells. Induced pluripotent stem cells (iPS cells) resemble embryonic stem cells (ES cells) and are generated from somatic cells by expression of specific reprogramming transcription factors. Here, we used C57BL/6 (B6) Sertoli cells to generate iPS cells (Ser-iPS cells) and compared the immunogenicity of Ser-iPS cells with iPS cells derived from mouse embryonic fibroblast (MEF-iPS cells). Ser-iPS cells were injected into syngeneic mice to test for their Induced pluripotent stem cells (iPS cells) resemble embryonic stem cells (ES cells) and can differentiate into all cell types of our body invitro caused significant levels of T cell infiltration after syngeneic transplantation iPS cells are believed to be particularly appealing as a cell source for personalized regenerative therapies, since autologous iPS cell-derived cells are expected to bypass immune rejection invitro and several questions remained: (i) Is low immunogenicity of these iPS cells also observed invivo? (ii) Does passage number affect iPS cell immunogenicity? (iii) Do other immune-privileged cells impact on iPS cell immunogenicity?An interesting question is what might cause immunogenicity of iPS cells and their differentiated progeny? The somatic cell type used for reprogramming might impact on the immunogenicity of iPS cells Testis is considered an immunologically privileged organ invivo and invitro.Here, we used Sertoli cells to generate iPS cells (referred to as Ser-iPS) and compared their immunogenicity with iPS cells derived from mouse embryonic fibroblasts (MEF-iPS). We systematically analyzed the immunogenicity of Ser-iPS cells and found that Ser-iPS cells indeed possess reduced immunogenicity both Sertoli cells from C57BL/6 (B6) mice (age 7\u201310 days) were obtained as described iPS cells from B6 Sertoli cells and B6 MEF were generated with retroviral vectors expressing Oct4, Sox2, Klf4 and c-Myc (OSK and OSKM) as described previously invitro, undifferentiated iPS cells and ES cells (JM8) were trypsinized into single cells and plated on gelatin-coated dish for 40 min to remove MEF feeder cells. Cells were transferred into differentiation medium (ES cell medium without LIF) and EBs were generated in hanging drops (500 cells per 20 \u00b5l drops) in an inverted bacterial Petri dish. EBs were collected 3 days later and kept in suspension culture for another 3 days. On day 6 of differentiation, EBs were plated on 0.1% gelatin-coated dishes for further differentiation (6 days) and used in T cell co-cultures.For spontaneous differentiation http://www.tm4.org) with fold change in gene expression normalized to \u03b2-actin.RNA was isolated using NucleoSpin RNA Kit and concentration was determined by NanoDrop 1000 . 1 \u00b5g RNA was used for reverse-transcription using High Capacity cDNA reverse transcription Kit . RT-PCR was performed in thermal cycling machine . For qRT-PCR 50 ng cDNA, fast SYBR Green PCR master mix and primers were used . PCR reaAP staining was done with Alkaline Phosphatase Staining Kit II according to the manufacture\u2019s instruction. For immunofluorescence staining, cells were grown on gelatin-coated cover-slips, fixed with 4% paraformaldehyde (PFA) and permeabilized with PBS containing 0.1% Triton X-100 . Samples were stained with first antibody , washed twice with PBS and incubated with secondary antibody . Cell nuclei were stained with DAPI . Samples were mounted with mounting solution and images were acquired with Axiovert 200 microscope . The following primary antibodies were used: Oct4 and SSEA1 . The secondary antibody was Alexa594 Goat anti-mouse Ig (H+L) .6 B6 Ser-iPS cells, MEF-iPS cells and ES cells (JM8) were injected subcutaneously into B6 mice. Four weeks later teratomas were excised, fixed in 4% PFA, embedded in paraffin and stained with rabbit polyclonal anti-CD3 antibody . The secondary antibody was VECTASTAIN\u00ae ABC Reagent . Hematoxylin and eosin (HE) staining was performed at IZKF Immunohistochemistry Core Facility . Frequency of teratoma formation was assessed as number of injections relative to teratomas formed. All experimental procedures involving mouse work were approved by the local authorities in compliance with the German animal protection law . All efforts were made to minimize animal suffering.1\u00d710CD4 T cells were obtained from spleen of B6 mice by MACS selection . For T cell proliferation assay, CD4 T cells were labeled with carboxyfluorescein succinimidyl ester (CFSE) and co-cultured with undifferentiated iPS cells or with EB iPS cells (day 12\u201317 of differentiation) at 2\u22361 ratio. T cells treated with phorbol myristate acetate and ionomycin (0.02 mM) were used as positive control. After 5 days of co-culture, proliferation of CD4 T cells was analyzed by assessing the dilution of CFSE signal using flow cytometry . The CD4 antibody used was PE anti-mouse CD4 .For regulatory T cell (Treg) assay, CD4 splenic T cells were activated with PMA and ionomycin (14\u201316 hours) and co-cultured with undifferentiated iPS cells or with EB iPS cells (day 12\u201317 of differentiation) at 2\u22361 ratio. After 5 days of co-culture, T cells were harvested and stained for surface markers CD4 and CD25 . Subsequently, cells were fixed and permeabilized using the Foxp3 fixation/permeabilization buffer and intracellular Foxp3 staining was carried out according to manufacturer\u2019s instruction. The percentage of Tregs was determined by flow cytometry based on the expression of CD4, CD25 and Foxp3.Results are given as the mean \u00b1 standard derivation. Statistical analysis was performed using unpaired Student\u2019s t-test. P values below 0.05 were considered to be statistically significant.Sertoli cells were obtained from testis of day 7\u201310 pups and showed the typical Sertoli cell morphology in culture invitro EB assay and invivo teratoma assay in NOD-SCID mice gene expression was lower in Ser-iPS cell teratomas compared to those of MEF-iPS cells . ExpressExpression of Zg16 and Hormad1 genes has been associated with immunogenicity of iPS cells invivo and showed lower immune cell infiltration, which is consistent with less tissue damage and necrosis. Additionally, there is apparently no correlation between Zg16 and Hormad1 expression and iPS cell immunogenicity.In summary, Ser-iPS cells have reduced immunogenicity compared to MEF-iPS cells invitro. EB assay represents the invitro equivalent of teratoma formation invivo. Therefore, Ser-iPS cells were subjected to EB assays (12 days), dissociated and co-cultured with CD4 T cells (Tissue destruction in teratomas is T cell dependent T cells . MEF-iPS T cells . In co-c T cells .Sertoli cells skew T cells towards a Treg profile and this represents one way how Sertoli cells generate an immune-privileged testicular environment All results obtained so far are based on early-passage iPS cells (p9\u201315). Therefore, we further investigated whether extended culture periods affect immunogenicity of Ser-iPS cells. We found that the frequency of teratoma formation and teratoma size of late-passage Ser-iPS cells (p35\u201338) is similar to MEF-iPS cells , indicatinvitro compared to MEF-iPS cells, which very much relates to the reduced immunogenicity observed in teratoma assays. Yet, Ser-iPS cells apparently do not possess the potential to convert T cells into a Treg phenotype.Collectively, EBs of Ser-iPS cells exhibit significantly lower T cell stimulation potential invivo and invitro. Ser-iPS cells formed more teratomas upon syngeneic transplantation with lower T cell, B cell and DC infiltration and less tissue damage and necrosis than MEF-iPS cells. In addition, EBs formed by Ser-iPS cells possessed lower T cell stimulation potential than MEF-iPS cells. Thus, Ser-iPS cells show reduced immunogenicity and this may be due to some residual somatic memory originating from Sertoli cells used for reprogramming.In this study we demonstrate that Ser-iPS cells exhibit low immunogenicity both invitro culture conditions, cell types used for transplantation and transplantation sites invivoPrevious studies investigated the immunogenicity of iPS cells and their derivatives following transplantation into syngeneic host. Yet, the immunogenicity of iPS cells and their derived cells has remained highly controversial invivo results, EBs of Ser-iPS cells are less potent in stimulating T cell proliferation compared to MEF-iPS cells in invitro co-culture experiments. However, in their pluripotent state both Ser-iPS cells and MEF-iPS cells showed T cell responses similar to ES cells. Thus, the immunogenicity of iPS cells was found to be related to the somatic cells used for reprogramming and to the differentiated state.In our study Ser-iPS cells have a stronger capacity to regulate the recipient\u2019s immune response compared to MEF-iPS cells. Ser-iPS cells and/or their differentiated progenies appear to prevent efficient infiltration of host derived immune cells into teratomas resulting in high teratoma formation frequency with less T cell, B cell and DC infiltration. This is consistent with less tissue damage and necrosis in Ser-iPS cell teratomas, since tissue destruction goes along with infiltration of activated T cells invitro and invivo appears to be unrelated to the Treg profile.The immune-privileged function of Sertoli cells includes the generation of Tregs Several factors have been implicated in the immune-privileged function of Sertoli cells and thus might be responsible for and/or contribute to the reduced immunogenicity of Ser-iPS cells. The anti-inflammatory cytokine transforming growth factor \u03b21 (TGF\u03b21) and immunosuppressive enzyme indoleamine 2,3-dioxygenase (IDO) produced by Sertoli cells have been reported to protect islet graft in mouse models Sertoli cell immune function comprises further factors, such as multiple cytokines, chemokines, anti-inflammatory modulators, complement activation inhibitors and adhesion molecules Two genes, zymogen granule protein 16 (Zg16) and Hormad1, which were expressed in regressing iPS cell teratomas but not in ES cell teratomas, were reported to contribute to iPS cell immunogenicity invitro culture The low immunogenicity of Ser-iPS cells was observed in early-passage iPS cells (p9\u201315). However, late-passage Ser-iPS cells (p35\u201338) exhibit an immunogenicity similar to the respective MEF-iPS cells. The low immunogenicity in early-passage Ser-iPS cells and its loss in late-passage Ser-iPS cells appear to be consistent with the presence of somatic memory in iPS cells. Somatic memory refers to some remnants of the somatic profile of the cell type used for reprogramming invitro culture. These findings indicate that the somatic cell type impacts on the immunogenicity of respective iPS cells. Our results reinforce the concept of using immune-privileged somatic cells for iPS cell generation and derivation of differentiated progeny for transplantation.In summary, we compared the immunogenicity of iPS cells derived from two different somatic cell types, immune-privileged Sertoli cells and MEF. Both Ser-iPS cells and MEF-iPS cells, and the cells derived thereof, showed immunogenicity. Additionally, Ser-iPS cells exhibited reduced immunogenicity compared to MEF-iPS cells, which however got lost upon extended Figure S1Ser-iPS cells are pluripotent. (A) Sertoli cells from day 7\u201310 B6 mice in culture. Phase contrast image at passage 1. Scale bar, 800 \u00b5m. (B) RT-PCR analysis of Sertoli cells in (A). Markers for germ cells, spermatogonial stem cells, Leydig cells and Sertoli cells are shown as indicated. Loading control, GAPDH. (C) RT-PCR analysis of pluripotency genes in Ser-iPS clones as in (TIF)Click here for additional data file.Figure S2Ser-iPS cell teratoma formation in B6 mice. (A) Teratomas of Ser-iPS cells in B6 mice. Controls, teratomas of MEF-iPS cells and ES cells. Representative images of tissue sections of ectoderm, mesoderm and endoderm from Ser-iPS cells , MEF-iPS cells are shown as in (TIF)Click here for additional data file.Figure S3+ cells followed by CD25+ cells and Foxp3+ cells. T cells without treatment were used as a negative control (T). MEF-iPS cells and ES cells were used as controls as in (A). Sertoli cells are shown as a positive control. Ser-iPS cells and MEF-iPS cells in A and B refer to average values as in T cell proliferation and Treg profile during co-culture of CD4 T cells with Ser-iPS cells. (A) Proliferation of CD4 T cells co-cultured with Ser-iPS cells (day 0\u20135) in T cell medium. MEF-iPS cells and ES cells were used as controls. PMA and ionomycin activated T cells, positive control. T cell proliferation refers to the percentage of dividing T cells after 5 days of co-culture (n\u200a=\u200a3) as in (TIF)Click here for additional data file.Table S1Primers of qRT-PCR and RT-PCR.(PDF)Click here for additional data file."} +{"text": "The RNA/DNA-binding protein, TDP-43, is the key component of ubiquitinated inclusions characteristic of amyotrophic lateral sclerosis (ALS) and the majority of frontotemporal lobar degeneration (FTLD-TDP) referred to collectively as TDP-43 proteinopathies. To further elucidate mechanisms of pathological TDP-43 processing and identify TDP-43 epitopes that could be useful as potential biomarkers of TDP-43 proteinopathies, we developed a panel of novel monoclonal antibodies (MAbs) directed at regions extending across the length of TDP-43. Here, we confirm previous observations that there is no or minimal accumulation of TDP-43\u00a0N-terminal domains in neocortical inclusions in human TDP-43 proteinopathy tissues and we identify a subset of these MAbs that are specific for human versus mouse TDP-43. Notably, one of these MAbs recognized an epitope that preferentially detected pathological TDP-43 inclusions with negligible reactivity for normal nuclear TDP-43 resembling anti-phospho-TDP-43 specific antibodies that only bind pathological TDP-43. Hence, we infer that this new MAb recognizes a phosphorylation independent but disease-specific pathologic conformation in abnormal TDP-43. These data suggest that the novel MAbs reported here will be useful for patient-oriented research as well as for studies of animal and cell-based models of TDP-43 proteinopathies including ALS and FTLD-TDP. Since the landmark discovery of the RNA/DNA binding protein, TDP-43, as the main constituent of ubiquitin-positive, tau/synuclein-negative inclusions in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) referred to as FTLD-TDP , there hE. coli cells using the pCOLD vector system . TDP-43 proteins were extracted from E. coli 16\u00a0h after IPTG induction, and the vast majority of the rTDP-43 proteins were present in inclusion bodies. The bacterial pellet was sequentially extracted with PBS and 1% Triton X-100 to remove soluble bacterial proteins. For immunization, the Triton X-100 insoluble rTDP-43 was solublized with 1% Sarkosyl buffer or 4\u00a0M urea. Sarkosyl was removed by exhaustive dialysis before use. For epitope mapping, the Triton X-100 insoluble rTDP-43 was solublized directly in Laemmli sample buffer, and immunoblot (IB) analysis was done as described previously [Human recombinant TDP-43 (rTDP-43) proteins including full length TDP-43 (FL-rTDP-43), i.e. amino acids (aa) 1\u2013414, N-terminally truncated TDP-43 (Nt-rTDP-43), aa 1\u2013261, and C-terminally truncated TDP-43 (Ct-rTDP-43), aa 182\u2013414 were expressed in BL21 (DE3) eviously ,14.Murine MAbs were raised against human rTDP-43 proteins using similar methods described previously -18. MiceThe TDP-43 domains harboring the epitopes recognized by these new MAbs were first estimated using FL-rTDP-43, Nt-rTDP-43 and Ct-rTDP-43 in IB studies similar to our characterization of MAbs we previously generated to tau and alpha-synuclein ,19. AfteThe 384-well format sandwich ELISA method used to evaluate the MAbs was similar to those described previously for a 96-well format, except the sample volumes used were reduced to 30 \u03bcL/well . BrieflyHuman CNS tissues from the superior temporal cortex (FTLD-TDP subtypes A-D) and spinal cord (ALS) (one patient each) were examined to evaluate the reactivity of TDP-43 MAbs in pathological aggregations and neuronal nuclei . Slides were coverslipped with CytoSeal 60 mounting medium (Thermo Scientific).IHC of mouse CNS tissues was performed in the same manner described above, and in addition, we used a mouse on mouse (MOM) immunodetection kit (Vector Laboratories) to minimize endogenous mouse IgG background. For all sections, labeling was detected using species-specific biotinylated secondary antibodies incubated for 1\u00a0h and the Vectastain ABC kit followed by the ImmPACTThe degree of non-pathological nuclear TDP-43 and pathological TDP-43 deposits in human lower motor neurons (LMN), white-matter glial cell inclusions (GCI), neuronal cytoplasmic inclusions (NCI), dystrophic neurites (DN) and neuronal nuclear inclusions (NII) were reviewed by a trained examiner (DJI) and rated on a semi-quantitative scale for each MAb. Digital images of IHC results were obtained using an Olympus BX 51 microscope equipped with a bright-field and fluorescence light source with a DP-71 digital camera and DP manager software (Olympus).To gain further insights into the molecular and neuropathological roles of TDP-43 in neurodegeneration, we generated new MAbs for future studies of TDP-proteinopathies. Approximately 4500 potential MAb clones from three hybridoma fusion procedures were screened by FL-rTDP-43 indirect ELISA of which 168 clones were positive on this initial screen and all were evaluated further in IB and IHC studies. Only 45 clones continued to show immunoreactivity toward TDP-43 in the secondary screens and all of these MAbs were studied further to determine the epitopes and their antibody isotype as described ,19. UsinTo determine whether or not the selected 18 MAbs detected both human and mouse TDP-43, IHC was performed using normal human and non-transgenic mouse brain sections. All 18 MAbs detected nuclear TDP-43 in sections of human hippocampus Figure\u00a0a,c, but As of now, there is no reliable method to quantify TDP-43 proteins in biofluids such as plasma or cerebrospinal fluid (CSF) samples which would be useful as a biomarker assay. In order to determine if the MAbs generated in this study might be useful for developing an assay to detect TDP-43 in CSF or plasma, we evaluated them in a sandwich ELISA using previously developed Nt- or Ct-TDP-43 pAbs as captuTDP-43 MAbs were screened for reactivity in human CNS tissues using IHC in the spinal cord of ALS and superior temporal cortex of morphological subtypes of TDP-43 (Type A-D) . Semi-quThe reactivity to neocortical TDP-43 pathology by MAbs specific for C-terminal TDP-43 epitopes was accompanied by a moderate to high reactivity for normal nuclear TDP-43 by all MAbs except MAb 138. MAb 138, which is specific for region C in the RRM2, displayed high reactivity to pathological TDP-43 deposits similar to other TDP-43 MAbs specific for Regions C-F, in the absence of significant normal nuclear TDP-43 staining showed robust reactivity for neocortical TDP-43 pathology across FTLD-TDP subtypes and ALS spinal cord inclusions. These MAbs directed against epitopes in the mid- and C-terminus of TDP-43 detected a moderate to abundant normal nuclear TDP-43 with the exception of MAb 138 (Region C) (Figure\u00a0There are several possible explanations for the unique IHC staining by MAb 138. First, there is a disease-specific pathological modification to one or more aa within the epitope recognized by MAb 138, but this seems unlikely as all these novel MAbs were generated using un-modified human rTDP-43. Second, a recent report suggests that the RRM2 region can undergo self-association when interacting with nucleic acids and this could alter the conformation of TDP-43 or the accessibility of the epitope recognized by MAb 138 . FinallyProtein misfolding and prion-like spread of neurodegenerative disease proteins has emerged in the forefront of research for drug-development in FTLD-TDP and ALS ,29. AlthThe authors declare that there are no competing interests to disclose.LKK, DJI, AKW, VML, and JQT designed the study and wrote the manuscript. DMR carried out the hybridoma fusion and subcloning as well as the ICC experiments. LKK, AKW, and YX performed the MAb characterizations and evaluations. DJI evaluated the human neuropathology. All authors read and approved the final manuscript.Figure S2. Serial Dilutions of MAb 138 show minimal nuclear reactivity and Table S1. Summary of patient demographics.Representative ELISA results for a newly developed MAb, Click here for file"} +{"text": "To the Editor: Two predominant community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) clones have been reported in South America: 1) sequence type 30 staphylococcal cassette chromosome mec IV (ST30-SCCmec IV) (USA 1100), first found in Uruguay (2002) and later in Brazil and Argentina (2005); and 2) ST8-SCCmec IVc/E (USA300\u2013Latin American variant), found predominantly in Ecuador and Colombia (2006\u20132008) (mec I) and active purulent secretion , panel AFor the past 2 years, the patient had lived in a remote, rural, jungle village in Peru. She was a housewife but also farmed in nearby areas. There were ducks, chickens, and guinea pigs on the farm where she lived. Her village had neither running water nor roads and almost no access to healthcare . She previously experienced several episodes of malaria (most recently in February 2014), for which she received antimalarial medication provided by a Brazilian military post at the border of Peru. She had never taken antimicrobial drugs and had not traveled in the past 2 years. She first noticed the skin lesion on the first day of an 8-day boat trip from her home village to Iquitos, the largest city in the Peruvian Amazon Basin. S. aureus resistant to oxacillin, tetracycline, and erythromycin and susceptible to ciprofloxacin, gentamicin, rifampin, and trimethoprim/sulfamethoxazole (susceptibility testing performed by an agar dilution method). D test showed inducible resistance to clindamycin. The presence of mecA and the genes encoding Panton-Valentine leucocidin (PVL) were confirmed by PCR.Cultures from wound exudate and skin biopsy samples yielded mecV. Whole-genome sequence analyses identified a predicted protein with 100% aa identity (98% coverage) to the truncated \u03b2-hemolysin of the reference genome of S. aureus USA300_FPR3757 (GenBank accession no. gb|ABD20946.1|) and the prophage groups 1, 2, and 3. Pulsed-field gel electrophoresis (PFGE) exhibited a pulsotype different from other typical CA-MRSA PFGE patterns found in MRSA from Latin America, labeled as CA-MRSA 120 and methicillin-sensitive S. aureus t701 from colonized inpatients has been well described (mec V PVL producer in this area of the Amazon Basin. This case of a skin and soft tissue infection caused by a CA-MRSA ST6-t701-SCCTechnical Appendix. Pulsed-field gel electrophoresis of known hospital-associated and community-associated methicillin-resistant Staphylococcus aureus isolates from Latin America."} +{"text": "Radiofrequency thermal ablation (RFA) of hepatic and renal tumors can be accompanied by non-desired tumorigenesis in residual, untreated tumor. Here, we studied the use of micelle-encapsulated siRNA to suppress IL-6-mediated local and systemic secondary effects of RFA.We compared standardized hepatic or renal RFA and sham procedures without and with administration of 150nm micelle-like nanoparticle (MNP) anti-IL6 siRNA , RFA/scrambled siRNA, and RFA/empty MNPs. Outcome measures included: local periablational cellular infiltration (\u03b1-SMA+ stellate cells), regional hepatocyte proliferation, serum/tissue IL-6 and VEGF levels at 6-72hr, and distant tumor growth, tumor proliferation (Ki-67) and microvascular density in subcutaneous R3230 and MATBIII breast adenocarcinoma models at 7 days.For liver RFA, adjuvant MNP anti-IL6 siRNA reduced RFA-induced increases in tissue IL-6 levels, \u03b1-SMA+ stellate cell infiltration, and regional hepatocyte proliferation to baseline . Moreover, adjuvant MNP anti-IL6- siRNA suppressed increased distant tumor growth and Ki-67 observed in R3230 and MATBIII tumors post hepatic RFA (p<0.01). Anti-IL6 siRNA also reduced RFA-induced elevation in VEGF and tumor MVD (p<0.01). Likewise, renal RFA-induced increases in serum IL-6 levels and distant R3230 tumor growth was suppressed with anti-IL6 siRNA (p<0.01).Adjuvant nanoparticle-encapsulated siRNA against IL-6 can be used to modulate local and regional effects of hepatic RFA to block potential unwanted pro-oncogenic effects of hepatic or renal RFA on distant tumor. Nuclease-free water was purchased from Qiagen . All siRNA duplexes are from Dharmacon . The synthesized sequences of siRNA targeting IL-6 were: 3\u2019(sense),33. A sc (sense) .in vivo. The hydrophobic interactions between the lipid moieties in DOPE-PEI and those in PEG-PE resulted in their self-assembly into micelle-like particles with a mean size of 150 nm and provide some steric stabilization of complexes by forming a protective barrier and promoting increased stability in vivo. The DOPE-PEI/siRNA complexes were prepared by mixing equal volumes of DOPE-PEI with siRNA-IL-6 (equimolar pool of 3 sequences) or scramble siRNA to a final N/P ratio of 16. The siRNA solution was transferred to the polymer solution, mixed by vigorous pipetting and incubated for 15 min. The polymer/siRNA ratio was expressed as the nitrogen/phosphate (N/P) ratio, and calculated assuming that 43 g/mol corresponds to each repeating unit of PEI containing one amine and 316 g/mol corresponds to each repeating unit of siRNA containing one phosphate. Then, a previously freeze-dried lipid film of PEG-PE was hydrated with the complexes and allowed to stand at room temperature for 1h with intermittent shaking The PEG-PE2kDa:DOPE-PEI weight ratio was 2:1 [Micelle-like nanoparticles were selected as the delivery vehicle based upon prior work demonstrating beneficial temporal delivery kinetics (6\u201312hr) and greater interstitial penetration in tissues surrounding the ablation zone . We cons was 2:1 . Empty fAnimals were sacrificed at specified times outlined above using carbon dioxide euthanasia. The target tissue was harvested, and sectioned perpendicularly to the direction of electrode insertion ,30. DistSerum and tissue levels of IL-6 and VEGF were determined using an enzyme-linked immunosorbent assay (ELISA) kit according to manufacturer\u2019s instructions. Briefly, flash-frozen liver tissue was homogenized in a cold lysis buffer consisting of a 0.1% proteinase inhibitor (Sigma-Aldrich). The homogenates were then centrifuged at 14,000 rpm for 20 min at 4\u00b0C, and the total protein concentration was determined using a bichinchoninic acid method (BCA) (Sigma-Aldrich). Undiluted serum was used. IL6 and VEGF values were then normalized to protein concentration. All samples and standards were measured in duplicate, and the average value was recorded as pg per mL ,38.Sections from ablated tissue and distant tumors were prepared and immunohistochemistry staining was performed for the following markers: \u03b1-SMA , CDC-47 ,23), Ki-P value of less than 0.05 was considered significant. Tumor growth curves after treatment were analyzed using goodness-of-fit characterization. Mean post-treatment growth curve slopes plus or minus SD were also calculated and compared using ANOVA and paired, two-tailed T-tests.The SPSS 13.0 software package was used for statistical analysis. All data were provided as mean plus or minus SD. Selected (Day 0 and at the time of sacrifice) mean tumor sizes, and immunohistochemical quantification were compared using analysis of variance (ANOVA). Additional post-hoc analysis was performed with paired, two-tailed Student\u2019s T-test, if and only if, the analysis of variance achieved statistical significance. A Initial hepatic RF ablation studies were performed in normal C57Bl mice using standardized protocols to allow comparison to prior studies in normal and IL-6 knockout mice . The folAs reported previously , standarAdjuvant MNP anti-IL6 siRNA also had organ-wide effects after hepatic thermal ablation. As shown by Rozenblum et al , thermalTo determine the impact on distant tumor growth to simulate the common condition of distant metastases, we used a subcutaneous breast tumor model (R3230) in which hepatic RF ablation has been associated with distant tumor growth . Here, tTumor proliferative index (Ki-67) in the distant R3230 subcutaneous tumor was measured for all treatment arms , Table 1Next, serum IL-6 levels were quantified by ELISA at 6hr after treatment for each of the six arms to determine the effect of adjuvant MNP anti-IL6 siRNA on ablation-induced systemic IL-6 levels. This time point was selected as serum IL-6 levels peaked at 6hr in this model post-hepatic RFA. Serum IL-6 levels were increased after thermal hepatic ablation (266\u00b19pg/ml) compared to sham treatment . The addFinally, timing of siRNA administration was compared, and MNP anti-IL6 siRNA administered immediately after hepatic RFA (Day 0) resulted in complete suppression of RFA-induced tumor growth compared to administration at a later time point (3d) after hepatic RFA , Table 1Periablational liver tissue levels of VEGF were increased after thermal ablation, peaking at 72hr. Here, tissue VEGF levels in the periablational rim were compared using ELISA at 72hr after treatment for the following arms: hepatic RF thermal ablation alone, sham procedure, RFA/MNP anti-IL6 siRNA , RFA/MNP scrambled siRNA, MNP anti-IL6 siRNA alone, RFA/empty vehicle . Hepatic ablation increased periablational tissue VEGF levels compared to sham treatment at 72hr . AdjuvanMicrovascular density (using CD34 staining) was then compared in distant subcutaneous tumors for each of the six treatment arms at 7d post-treatment , Table 1For tumor growth studies, kidney RF thermal ablation alone, sham procedure, RFA/MNP anti-IL6 siRNA , and MNP anti-IL6 siRNA alone were compared . Similarly, RF ablation of normal kidney increased distant R3230 tumor growth compared to sham treatment that was also suppressed with single-dose adjuvant MNP anti-IL6 siRNA (given at Day 0) , Table 1Next, serum IL6 levels were studied in the following treatment arms: kidney RF thermal ablation alone, sham procedure, RFA/MNP anti-IL6 siRNA , RFA/MNP scrambled siRNA, MNP anti-IL6 siRNA alone, RFA/empty vehicle . Serum IL-6 levels were increased after kidney thermal ablation (342\u00b132pg/ml) compared to sham treatment . The addNext, the effect of MNP anti-IL6 siRNA on hepatic thermal ablation was assessed in a second subcutaneous tumor model (MATBIII rat breast adenocarcinoma line) at 3.5d post-treatment . HepaticSeveral studies have reported that incomplete thermal tumor ablation of intrahepatic or renal tumors can have either stimulatory or suppressive effects on tumor cell growth in the partially injured residual cells present in periablational tissue or in intrahepatic/intra-organ tumor foci separate from the ablation site ,22,39,40Our study further demonstrates that adjuvant nanoparticle siRNA can be successfully used to target IL-6 mediated locoregional and systemic effects of thermal ablation. Specifically, adjuvant siRNA can be used to suppress thermal ablation-induced increases in local and serum IL-6 cytokine levels, alter cellular infiltration patterns surrounding local thermal ablation, affect liver physiology/homeostasis by reducing ablation-induced hepatocyte proliferation in untreated liver, and achieve systemic suppression of un-intended \u2018off-target\u2019 distant pro-oncogenic effects of thermal ablation. Furthermore, we demonstrate that these RF-induced effects occur in various settings , are IL6-mediated in these scenarios, and most importantly that they can be reproducibly modulated and attenuated with nano-delivered siRNA.Thermal ablation incites several additional local tissue reactions, including increased oxidative and nitrative stress-mediated apoptosis and production of local tissue factors such as heat shock proteins, hepatocyte growth factor, and pro-angiogenic VEGF, HIF-1\u03b1, and PDGF ,18,21,22Our successful \u2018proof-of-concept\u2019 study demonstrates that nano-delivered siRNA can be used to suppress the secondary local and systemic \u2018off-target\u2019 effects (such as IL6-mediated inflammation) occurring after at least one clinically common procedure. Yet, there is also broader potential clinical applicability of this approach, as thermal ablative techniques have been incorporated into the treatment of a large number of diseases, including therapies such as pulmonary vein ablation for cardiac arrhythmias, esophageal ablation of Barrett\u2019s esophagus, and in newer endovascular treatments for renovascular hypertension, mitral regurgitation, vascular stenosis, and venous reflux disease \u201348. For One of the main limitations of transitioning siRNA to therapeutic applications has been due to challenges in drug-delivery . In partWe acknowledge several limitations in this study. Although we have focused on the pro-oncogenic effects of tumor ablation-induced inflammation, several studies have reported upon the potential for ablation-induced anti-tumor immunity with resultant tumor growth suppression ,56\u201358. WIn conclusion, we demonstrate that thermal ablation of normal organ parenchyma can result in Interleukin-6-mediated locoregional and systemic effects (including distant tumor growth). We further show that the novel paradigm of combining adjuvant nanoparticle anti-IL6 siRNA can be successfully used to suppress potentially pro-oncogenic effects of thermal ablation."} +{"text": "Drosophila eye development, wingless (wg) is expressed at the lateral margins of the eye disc and serves to block retinal development. The T-box gene optomotor-blind (omb) is expressed in a similar pattern and is regulated by Wg. Omb mediates part of Wg activity in blocking eye development. Omb exerts its function primarily by blocking cell proliferation. These effects occur predominantly in the ventral margin. Our results suggest that the primary effect of Omb is the blocking of Jak/STAT signaling by repressing transcription of upd which encodes the Jak receptor ligand Unpaired.Organ formation requires a delicate balance of positive and negative regulators. In Drosophila compound eye originates from the eye-antenna anlage in the embryo. These cells proliferate and form the eye-antennal disc in the larva. In the mid-third instar eye disc, a wave of cell cycle coordination and apical cellular constriction, called the morphogenetic furrow (MF) forms at the posterior margin and progressively moves toward anterior. Posterior to the MF, retinal cell fates are specified by a series of cellular interactions )escribed . Anti-Brupd RNA in situ hybridization is executed as described [escribed .Semi-thin plastic eye sections were performed according to .Scanning electron micrographs of adult eyes were obtained as described . Due to omb mutations caused changes in eye size.In the eye imaginal disc, Omb is expressed in two cell types. Within the main epithelium, Omb is expressed at the dorsal and ventral margins ., arrow.omb hypomorphic allele combinations and omb knock-down, the Omb level was reduced in both margins and the eye disc was enlarged ombD4, l(1)omb282, and l(1)omb3198 [ombbi/l(1)omb282: N = 952, SD = 61, n = 9).In enlarged .. This womb3198 ,62. In tombP1 [omb hypomorphs (fng-lacZ expression [mirr-lacZ expression (l(1)ombD4/ombP7 hypomorphic larvae . This is consistent with omb expression not overlapping with the activity of the GMR-GAL4 driver, which is restricted to cells posterior to the MF [These loss-of-function effects were also observed when omb-RNAi in its oeye disc . and adueye disc . When omo the MF .omb caused reduction or elimination of the eye. In the regulatory dominant gain-of-function allele ombFor [ombFor larvae had smaller eye discs . Targeted mis-expression of omb in the lateral and posterior margins by dpp-GAL4 (dpp>omb) causes a strong reduction or total absence of the adult eye [omb at the lateral margins using 30A-GAL4 [UAS-omb line .In contrast to the loss-of-function effects, gain-of-function of ombFor , Omb wasye discs . and thedult eye . Specifi30A-GAL4 caused aomb indicate that omb is a negative regulator of eye development. The effect is stronger on the ventral side of the eye.In summary, the loss and gain-of-function phenotypes of omb on proliferation and cell death.Deviations from normal eye size can arise by several mechanisms. Changes in proliferation, cell death, morphogenetic furrow progression or retinal differentiation can all affect eye size. We tested the effect of loss and gain of omb hypomorphs, cell cycle activity was increased in the ventral eye disc, as monitored by histone H3 phosphorylation (pH3) . Therefore, omb appears to affect cell divisions in the undifferentiated region anterior to the MF.In tic wave , as expe mutants and by komb loss of function mutant clones on cell proliferation. The l(1)ombD4 null allele was used because it yielded stronger effects than the hypomorphic alleles In wild type eye discs, control clones (marked by lack of GFP) were of similar size relative to their twin spots, irrespective of location only in the ventral margin caused a strong reduction to total absence of the adult eye [dpp>omb+GFP eye disc (dpp>omb+p35) did not rescue adult eye size (data not shown) nor retinal differentiation in eye disc, although apoptosis was strongly reduced .Increase in ommatidial number in brissae) . This inTo understand the mechanism by which Omb impedes proliferation in the ventral anterior eye disc, we tested the effect of Omb on the Upd/Jak/STAT signaling pathway, which promotes cell proliferation ,10,18,24outstretched, os) of the Jak/STAT pathway is expressed in the ventral eye disc at first instar and in the posterior center of the eye disc at second and early third instar [wg transcription to promote MF initiation [Grh-STAT-lacZ and 10X-STAT-GFP reporter expression is high in the posterior region, consistent with STAT activity being induced by the Upd ligand [10X-STAT-GFP is expressed in the posterior part of the second instar eye disc, before MF initiation. In the third instar eye disc, the 10XSTAT-GFP signal is much reduced and represents perdurance from earlier expression [omb hypomorph ombP7, STAT activity was ectopically activated in the ventral margin (ombP3>omb-RNAi yielded similar results (not shown). A STAT-lacZ enhancer trap reporter, although not fully recapitulating the STAT92E mRNA pattern, is known to be negatively regulated by Jak/STAT activity [l(1)omb15 eye disc, STAT-lacZ expression was lost in the ventral region ombD4 mutant clones. We found that 10XSTAT-GFP-nls was nonautonomously induced in ventral clones with omb (dpp>omb+hop) (hop. dpp>hop (not shown) and dpp>hop+GFP , which caused attenuation of the ventral outgrowth of eye disc . Hop lar>hop+GFP caused aomb could suppress Jak/STAT activity by downregulating upd expression. Expression of omb along the posterior margin (in dpp>omb+GFP) suppressed upd-lacZ expression largely rescued retinal development in the eye disc , disc size and neuronal differentiation were partly recovered (dpp expression domain (dpp>omb-RNAi+GFP) caused no significant effect on retinal development (arm by ey-GAL4 (ey>arm) caused a reduction of adult eye size and retinal differentiation in eye disc (omb dosage was reduced (l(1)ombD4/+), the ey>arm phenotype was partially rescued with full penetrance could completely block retinal development blocked retinal development (hop (dpp>omb+hop) could nearly fully restore retinal development in the eye disc partially suppressed the ombP7 enlarged eye phenotype . This finding and the cell-autonomous repression of upd by Omb . Based nt were attenuated when the omb dosage was reduced. The partial rescue suggests that Omb mediates part of the Wg effect and that additional factor(s) are likely to be involved. Our analyses show that the effect of Omb is partly similar to that of Wg and partly different causes overgrowth [A clear difference was the opposite effect on cell proliferation. Omb repressed cell proliferation, through a block of Upd/Jak/STAT signaling. In contrast, enhanced Wg signaling [teashirt, for instance [Loss of x (Iro-C). Its actinstance ,80. The omb induces the dorsally restricted expression of homothorax (hth) which together with ubiquitous extradenticle (exd) allows the formation of Hth/Exd complexes which specify DR development [Iro-C genes causes DR fate also along the ventral margin. The monopolar Iro-C expression thus determines the different functional outcome of symmetrical omb expression at the two margins. Like in early eye development, loss of omb has little consequence for dorsal eye development during specification of DR ommatidia. The discrepancy between strong effect in omb gain-of-function and little effect in omb loss-of-function dorsal eye phenotypes has been attributed to a redundant function exerted by the related T-box genes Dorsocross (Doc) [During pupal eye development, a dorsal Wg gradient specifies three cell fates at the dorsal eye boundary: pigment rim, polarization-sensitive dorsal rim (DR) ommatidia, and bald ommatidia (lacking the mechanosensory hairs). Omb which is induced by Wg, is sufficient to induce the DR fate in the dorsal eye. Ectopic elopment . Dorsaliss (Doc) .omb-related vertebrate T-box genes Tbx2/3/5 are all expressed in a polar pattern, first in the dorsal eye cup and later in the dorsal retina [omb in eye development suggest conservation of ancient functions which were already present in an evolutionary precursor before the split into the protostome and deuterostome lineages [Intriguingly, the closely l retina ,84,85,86l retina ,88. The lineages , in spitlineages ,91.S1 FigombP3>omb-RNAi, (C) ombFor, (D) ombP3>GFP and (E) ombP7>GFP. In ombFor, Omb is increased in the dorsal and ventral margin and in the retinal basal glia. The disc size is reduced (C). The marginal expression of ombP7-GAL4 was broader than that of ombP3-GAL4. In (C) and (D) the retinal basal glial cells were below the focal plane.Eye-antennal discs stained with anti-Omb (green) and anti-Elav (red). Marginal expression is indicated by arrows, expression in retinal basal glial cells by arrowheads. (A) wild type, (B) (TIF)Click here for additional data file.S2 Figfng-lacZ . Dotted lines mark the MF and the projection from the optic stalk entry point onto the MF. In the dotted line also visualises the line of mirror symmetry in the Bar expression pattern. The BarH1 and BarH2 expression in photoreceptor cells R1 and R6 [fng-lacZ expression domain. (A) w1118/Y, (B) ombP7/Y, (C) fng-lacZ and (D) ombP7/Y; fng-lacZ. The D/V eye field was symmetrical in the third instar eye disc of wild type, but the ventral field was expanded in ombP7/Y. (E) The number of rows of ommatidia in each eye disc, and the numbers of ommatidia in the dorsal and ventral eye fields were counted at different stages of eye disc development. In wild type eye disc, the dorsal and ventral eye fields were always of equal size. In ombP7/Y, the ventral eye field was consistently larger than the dorsal field.(A-D) The boundary of dorsal/ventral fields in third instar eye discs was monitored by the position of the optic stalk (white arrowhead), anti-Bar antibody staining and the ventrally expressed 1 and R6 is mirro(TIF)Click here for additional data file.S3 Figdpp-GAL4 driven GFP (dpp>GFP) expression overlapped with the omb-lacZ expression (red) domain in the lateral margins. In early eye disc, dpp-GAL4 expression is similar to that of dpp-lacZ dpp>omb+GFP, as dpp>omb, completely blocked eye development in adult (B) and in late third instar eye disc (C). The eye disc has no neuronal differentiation but has elevated activated caspase 3 (red). (D) Blocking apoptosis by coexpression of p35 (dpp>omb+p35) significantly reduced the caspase 3 signal but did not rescue eye size or retinal differentiation. Scale bar: 50um.(A) dpp-lacZ . in the (TIF)Click here for additional data file.S4 FigombP7 eye discs. (A-A\u201d) 10XSTAT-GFPnls was found in the posterior eye field in the early third instar eye disc of ombP7. (B-B\u201d) Jak/STAT activity was activated in posterior eye field and ventral margin (arrow) of mid-third instar larvae. Jak/STAT activity was detected in the posterior eye field as well as ventral margin (arrow) in the late third eye field. Elav (red), GFP (green).10XSTAT-GFPnls is a Jak/STAT reporter. (A-C) Jak/STAT activity in (TIF)Click here for additional data file.S5 FigombP1-lacZ (red) and upd>GFP (green) did not overlap in late second (A), early third (B) and late-third instar eye discs (C). ombP1-lacZ is also expressed in the retinal basal glia which lies at the basal surface and does not overlap with the upd expressing cells in the neuroepithelial layer (not shown). (D) The expression pattern of upd-lacZ (red) in wild type. Elav (cyan). dpp>omb+GFP suppressed upd-lacZ expression (red) at the center of the posterior margin (arrow)(A-C\u2019) The expression pattern of (TIF)Click here for additional data file.S6 Figomb , (A\u2019-E\u2019) Wg , and (A\u201d-E\u201d) dpp were followed during eye-antennal disc development from early second instar to late third instar. (A\u201d\u2019-E\u201d\u2019) shows the merge images of ombP3>GFP, dpp-lacZ and Wg immunostaining.(A-E) The expression patterns of (TIF)Click here for additional data file."} +{"text": "The field of T cell therapy of cancer holds great promise, as demonstrated by results obtained in Chimeric Antigen Receptor (CAR) modified T cell therapy of CD19+ leukemia. However, these anti-tumor effects are often limited by T cell tolerance and the immunosuppressive tumor microenvironment. Oncolytic viruses armed with immunostimulatory molecules constitute a potent form of immunotherapy. In essence, the danger signal caused by virus replication, coupled with the actions of the transgene, and effective presentation of tumor epitopes by lysis of the cells, results in a personalized cancer vaccine, manufactured for each patient in their tumor.in vivo.Previously, we have identified interleukin 2 (IL-2) and Tumor Necrosis Factor alpha (TNF-\u03b1) as the most promising factors to stimulate the graft used in adoptive T cell therapy. Both cytokines are important activators of immune cells and also known for their direct anti-tumor properties. Therefore, we aimed to enhance the efficacy of ACT by coupling it with adenoviruses coding for immunostimulatory cytokines. Notably, these cytokines can cause severe side effects when administered systemically. Previously, we and others have achieved long lasting, high level cytokine expression locally but low level systemically when using armed oncolytic viruses Therefore, we developed oncolytic adenoviruses expressing one or both above mentioned human cytokines . For more detailed immunological studies in immune competent mouse models, we constructed non-replicative adenoviruses with murine cytokines (Ad5-CMV-mIL-2 and Ad5-CMV-mTNF-\u03b1). These viruses were studied in combination with adoptive transfer of OT-1 TCR transgenic T cells to treat C57BL/6 mice bearing B16-OVA melanoma tumors. The animals were administered intraperitoneally with CD8+-enriched OT-1 cells with or without intratumoral injections of cytokine-coding viruses.Combination treatment with Ad5-CMV-mIL-2 and OT-1 resulted in statistically significant anti-tumor efficacy when compared with either monotherapy or untreated control. In further experiments a triple combination of Ad5-CMV-mIL-2 + Ad5-CMV-mTNF-\u03b1 and OT-1 T cells improved anti-tumor efficacy was observed over dual agent therapies. Furthermore, in mice bearing two tumors we saw abscopal effect when only one of the tumors was injected with cytokine encoding viruses but reduction in tumor size was detected also in the non-injected tumor.In summary, these results support the further development of cytokine-armed adenoviruses as facilitators of adoptive T cell therapy."} +{"text": "Conventional breath-held gadolinium-based contrast-enhanced MR angiography (CE-MRA) provides excellent definition of extra-cardiac anatomy, but does not provide diagnostic image quality for intra-cardiac anatomy because it is not gated to ECG due to time constraints associated with breath-holding and the need to capture the first-pass of gadolinium. To address this issue, we propose a 4D non-breath-held multiphase steady-state imaging sequence (MUSIC) using ferumoxytol, which is an FDA approved iron-oxide particle for treating iron-deficiency anemia, as an intravascular contrast agent and demonstrate its feasibility in children with congenital heart disease (CHD).Eight pediatric CHD patients underwent cardiac MRI under general anesthesia and mechanical ventilation, which is our standard practice for young children who cannot cooperate with breath-holding instructions. The breath-held CE-MRA was first performed under ventilator-controlled breath-hold (VCBH) during the first-pass of a ferumoxytol bolus injection (4 mg-Fe/kg) and repeated 2-3 min later during the delayed-phase. Subsequently, the cardiac-phase-resolved 4D MUSIC acquisition was performed during the steady-state distribution phase of ferumoxytol without VCBH and the ventilator tubing air pressure signal was fed into the MR scanner for respiratory gating. Standard 2D cardiac cine images were also acquired under VCBH. Subjective image quality scores were visually assessed and the LV volumes were measured based on standard 2D cine and the 4D MUSIC data.As shown in Fig. Our study represents a potential new paradigm of cardiovascular MRI for young children. The use of ferumoxytol eliminates the time constraints of conventional CE-MRA and enables much higher quality 4D dynamic cardiac imaging with sub-millimeter isotropic resolution.NIH (NHLBI) 1R21 HL113427."} +{"text": "During 2D echocardiography, the septal leaflet-tricuspidannulus distance showed to be 25 mm, confirming EA. While the right ventricle (RV) wasenlarged with tricuspid annular plane systolic excursion > 23 mm and mitralregurgitation grade III, left ventricular (LV) size and function showed to be normalwith an ejection fraction of 56% without wall motion abnormalities. However, all LVregions moved in almost the same counterclockwise direction, confirming absence of LVtwist, called \"rigid body rotation\" (RBR) that had never undergonepalliation was assessed (the case originates from the MAGYAR-Path Study). Completetwo-dimensional (2D) Doppler and three-dimensional (3D) speckle-trackingechocardiography were carried out with commercially available Toshiba Artidan\" (RBR) . The mea"} +{"text": "A proportion of Alzheimer's disease cases displays inclusions of the RNA-binding protein, TDP-43. Considering the pathogenic role of tau mis-splicing, we compared tau isoform expression between Alzheimer's disease cases with or without TDP-43 inclusions. The average ratio of tau isoforms containing or lacking exon 10 (4R/3R ratio) or the total level of tau mRNA was not significantly different\u00a0between cases with or without TDP-43 pathology in any of the brain regions examined. Although TDP-43 functions may be affected, TDP-43 does not critically regulate expression or splicing of tau in Alzheimer's disease suggesting that TDP-43 contributes to Alzheimer's disease through mechanisms independent of tau. In addition, 20%\u201330% of AD cases display cytoplasmic inclusions of transactive response DNA-binding protein of 43 kDa (TDP-43). The presence of TDP-43 inclusions in the hippocampus and entorhinal cortex correlates with neuronal loss in late onset dementia and, furthermore, a strong correlation exists between the presence of TDP-43 pathology and specific clinical features of AD, especially impaired cognition and amnesia . TDP-43 2t test.Frozen brain tissue was obtained from individuals free from neurological disease and individuals with AD and controls. Cases were assessed for TDP-43 pathology by immunocytochemistry. RNA was extracted using the Qiagen RNeasy/lipid kit (Qiagen). Tau E10 splicing was analyzed by end-point reverse transcription polymerase chain reaction using a forward primer 5\u2032-labelled with an infrared fluorescent dye, yielding a signal independent of the length of the individual products. Gels were imaged, and polymerase chain reaction products were quantified using an Odyssey Infrared Imaging System (LI-COR Biosciences) . Statist3We analyzed tau splicing in a cohort of 14 AD cases with (ADTDP+) and 15 AD cases without (ADTDP\u2212) TDP-43 inclusions and 15 age-matched nondemented healthy controls . The amy4TARDBP gene, encoding TDP-43, have been linked to familial forms of amyotrophic lateral sclerosis (Mutations in the clerosis , thus arclerosis . Howeverclerosis . Whetherclerosis . ConsideThe authors declare that they have no conflict of interest."} +{"text": "Hypoxic-ischemic injury (HI) to the developing brain remains a major cause of morbidity. Hypothermia is currently the only established neuroprotective treatment available for term borns with hypoxic-ischemic encephalopathy, saving one in eight infants from developing severe neurological deficits. Therefore, additional treatments with clinically applicable drugs are indispensable. Furthermore, the pathophysiological mechanisms of hypothermia-induced recovery are not clearly understood. This study examines a potential additive neuroprotective effect of hypothermia combined with levetiracetam in neonatal mouse HI.9-days-old C57BL/6-mice were subjected either to a sham-operation or to HI (modified Rice-Vannucci-model). After HI, the pups were randomized into six groups: 1) no treatment, 2) hypothermia , 3) high-dose levetiracetam intraperitoneal (70 mg/kg body weight), 4) hypothermia combined with high-dose levetiracetam intraperitoneal, 5) low-dose levetiracetam intraperitoneal (7 mg/kg body weight), 6) hypothermia combined with low-dose levetiracetam intraperitoneal. Parameters of apoptosis and myelination (myelin basic protein) were analyzed 24 hours after HI by protein analysis and immunhistochemistry. From P28 to P60, cognitive and sensorimotor function was assessed via different tests.Hypothermia only and combined with low-dose levetiracetam was associated with a decrease of apoptosis and an increase of myelinated cells, but without additive effects. Intraperitoneal treatment with high-dose levetiracetam caused an increase of apoptotic factors. Behavioural testing demonstrated improved cognitive and sensorimotor outcome after treatment with hypothermia.Whole-body cooling provides neuroprotection in the neonatal mouse brain by reducing apoptosis and preservation of myelination. However, treatment with levetiracetam after hypoxic-ischemic injury has no additive effects."} +{"text": "Even in the era of successful combination antiretroviral therapy (cART), co-infection of Hepatitis C virus (HCV) remains one of the leading causes of non-AIDS-related mortality and morbidity among HIV-positive individuals as a consequence of accelerated liver fibrosis and end-stage liver disease (ESLD). The perturbed liver microenvironment and induction of host pro-inflammatory mediators in response to HIV and HCV infections, play a pivotal role in orchestrating the disease pathogenesis and clinical outcomes. How these viruses communicate each other via chemokine CCL2 and exploit the liver specific cellular environment to exacerbate liver fibrosis in HIV/HCV co-infection setting is a topic of intense discussion. Herein, we provide recent views and insights on potential mechanisms of CCL2 mediated immuno-pathogenesis, and HIV-HCV cross-talk in driving liver inflammation. We believe CCL2 may potentially serve an attractive target of anti-fibrotic intervention against HIV/HCV co-infection associated co-morbidities. Uncontrolled fibrosis may progress to severe forms of the disease, such as liver cirrhosis (CH) and hepato-cellular carcinoma (HCC). Globally, ~ 35 million people are infected with HIV out of which 20-30% individuals are co-infected with HCV . The preividuals ,5 but th+ T cells in HIV mono-infection but the data are conflicting for HIV/HCV co-infection. One study reported CD4+ T cell recovery following 4-years of HAART [+ T cell reconstitution following HAART [HCV is a hepatotropic RNA virus that causes hepatitis, CH and HCC . Given tof HAART while otof HAART ,11. Furtng HAART . TherefoCCL2, also known as monocyte chemo-attractant protein-1 (MCP-1), is a small molecular weight protein of C-C chemokine family with strong chemotactic behaviour toward monocytes, NK cells and CD4+ T cells ,14. ManyFibrosis is a key event associated with liver injury triggered by virus and other inflammatory agents. It is characterized by excessive deposition of extra-cellular matrix (ECM) components including collagens, fibronectin and proteoglycan into Desse and reduced levels of tissue inhibitor of metalloproteinase (TIMP-1), an ECM removing matrix metalloproteinase (MMP) . Human l++ CD16\u2212 and \u2018non-classical\u2019 CD14+ CD16+ subsets. The later frequency found to be preferentially higher in liver fibrosis and HIV infection [hi monocytes into the liver in response to CCL2 [+ inflammatory subset, an equevalent of human CD14+CD16+ cells.In addition to liver-resident macrophage (Kupffer cell), human hepatic macrophages can be divided into \u2018classical\u2019 CD14nfection ,29. The nfection . Functio to CCL2 and thatHCV-induced liver injury and subsequent progression to fibrosis is an immuno-pathological event governed by complex virus- host interactions ,32. ThouImmuno-pathology of HIV/HCV co-infection associated liver disease is multi-factorial . SeveralImmune activation is a hallmark of advanced HIV disease associated with chronic T cells activation and sustained plasma levels of pro-inflammatory cytokines. Systemic immune dysfunction and mucosal CD4+ T lymphocytes depletion from lymphoid tissues of gastrointestinal (GI) tract of HIV positive individuals are thought to be the major driving force behind this event. The mucosal permeability and subsequent microbial translocation leads to production of high levels of soluble(s) CD14, sCD163 and IL-6 by activated macrophages in response to microbial product, lipopolysaccharide (LPS) . In addiThe association of microbial translocation and contribution of LPS to liver inflammation in HIV/HCV co-infection setting has recently been recently appreciated -61. ThesHost chemokines induced by HIV may either support or inhibit virus replication. For example, C-C chemokine CCL3, CCL4, CCL5 and C-X-C chemokine CXCL12 (SDF-1\u03b1) has been described as potent inhibitors of R5 and R4 HIV strains by binding to their respective co-receptors CCR5 and CXCR4 -66. ThusA number of clinical data including ours have shown elevated CCL2 in the serum ,71 and c+CD16+ monocyte subset has been found to be the major source of CCL2 in HIV-infected individuals [Evidence of CCL2 production is also observed in non-human primate (NHP) model of simian human immunodeficiency virus (SHIV) infection , supportividuals as well ividuals .+ leukocytes including; monocytes, NK cells, CD4+ T cells into the liver to start inflammatory reactions [A series of inflammatory mediators are up-regulated during HCV infection and associated liver diseases such as CH and HCC -84. Someeactions ,86. Compeactions ,88 that eactions . The releactions , HCC [91eactions . Thus, aA detailed association between chemokines and liver diseases has been described elsewhere . Some of+ monocytes and CD4+ T cells from the hepatic portal blood to the site of infection in a feed-back loop manner, resulting in rapid HIV replication and subsequent increase in viremia. (3) CCL2 can also act on HSC in a positive feed-back loop [+ T cells [The natural infection of both HIV and HCV is affected by their presence in the same host. Compared to HCV, the effect of HIV on HCV infection is considered more deleterious (described in mechanisms of HIV/HCV immuno-pathogenesis section). Given that CCL2, an important factor associated with pathogenesis of both the viruses, it is important to understand the impact on liver cellular microenvironment and virus-triggered hepatic fibrosis. We herein summarize the potential mechanisms plus ribavirin (RBV) . A regimIn addition to antivirals, chemokine and chemokine receptor based blockers and antagonists could provide an attaractive immuno-therapeutic approach against HIV/HCV co-infection associated liver diseases. This startegy has proven success in HIV infection, where CCR5 antagonist, Maraviroc effectively suppress HIV replication in infected individuals . Given tThe majority of the literature supports the notion that mortality among HIV/HCV co-infected individuals occurs due to liver failure rather than AIDS-related complications. Since fibrosis is an immune-pathological event it would wise to target those factors which are induced by both the viruses and are critical in liver diseases severity. CCL2 is one such pro-inflammatory molecule that could be targeted for anti-inflammatory strategies since CCL2 blockade strategy has already been implemented clinically in breast and prostate cancer immune-therapy ,16. Furt"} +{"text": "In this study, we performed gain-of-function and loss-of-function analysis in Drosophila and identified flfl as a negative regulator of JNK pathway-mediated cell death. While ectopic expression of flfl suppresses TNF-triggered JNK-dependent cell death, loss of flfl promotes JNK activation and cell death in the developing eye and wing. These data report for the first time an essential physiological function of flfl in maintaining tissue homeostasis and organ development. As the JNK signaling pathway has been evolutionary conserved from fly to human, a similar role of PP4R3 in JNK-mediated physiological process is speculated. Drosophila protein phosphatase 4 (PP4) regulatory subunit 3 (PP4R3) [ in vitro studies have reported that PP4 is involved in a variety of molecular and cellular processes including regulation of c-Jun N-terminal kinase (JNK) pathway [ in vivo functions remain poorly understood. The human homolog of flfl is SMEK, which recruits PP4c to promote neuronal differentiation by dephosphorylating Par3 [ in vivo functions of SMEK remain largely elusive.falafel (flfl) is a PP4R3) , which s (PP4R3) . flfl bi , which pathway , hematop pathway , apoptos pathway , and cel pathway , flfl's ing Par3 . However Drosophila to mammal [ Drosophila has been used as an excellent genetic model to study tumor necrosis factor- (TNF-) induced cell death in development. In Drosophila, the TNF ortholog Eiger (Egr) triggers cell death through its receptor Grindelwald (Grnd) [Drosophila homologs of JNKKK-JNKK-JNK) kinase cascade [ GMR-Gal4 (GMR > Egr hereafter) induces JNK-dependent cell death and produces small eyes in adult [The JNK pathway is evolutionary conserved fromo mammal . As its d (Grnd) , the E2 d (Grnd) , 13, thed (Grnd) , the TAKd (Grnd) , and the cascade , 17. In in adult , 17. GMR > Egr small eye phenotype. From the screen, we found that expression of flfl suppresses Egr-triggered cell death. On the other hand, knocking down flfl induced JNK activation and JNK pathway-dependent cell death, suggesting a physiological function of flfl in animal development. To our knowledge, this is the first report that flfl negatively regulate TNF-JNK signaling-induced cell death in vivo.To identify additional factors that regulate Egr-triggered JNK-mediated cell death, we performed a genetic screen for dominant modifiers of the Drosophila media, and crosses were performed at 25\u00b0C. UAS-flfl-IR (V103793) was obtained from Vienna Drosophila Research Center, UAS-flfl-IR (31690), flflEY03585, UAS-GFP-IR, and ap-Gal4 were obtained from Bloomington Stock Center, and UAS-bsk-IR (5680R-2) was from Fly Stocks of National Institute of Genetics (NIG). pucE69 [ GMR-Gal4, en-Gal4, pnr-Gal4, UAS-GFP [ UAS-Egr [ UAS-Egrw [ UAS-Hep, and UAS-BskDN [All stocks were raised on standard pucE69 , GMR-Gal UAS-GFP , 20, UASflfl-IR 390, flflAS-BskDN were preEye discs from 3rd instar larvae were dissected in 1% PBS buffer. AO staining procedure was based on previous assay . Floresc3-day-old flies of each genotypes were collected and immediately frozen at \u221280\u00b0C. For the image, flies were mounted on 1% agarose plates. Light images of eye and thorax were documented with OLYMPUS stereo microscope SZX16.\u03b2-galactosidase at 37\u00b0C for 24 hours.X-Gal staining was performed as previously described with minor modification , 24. Winhttps://www.oncomine.org/).Invasive breast carcinoma stroma versus normal data was obtained from Oncomine database encoding the Drosophila JNK ortholog [ GMR > Egr small eye phenotype and identified Nopo, Ben, Wnd, and Wg signaling as essential regulator of Egr-JNK pathway induced cell death [As previous study showed, ectopic expression of Egr under the control ofhenotype . This phortholog , which iortholog . To idenll death , 26. GMR > Egr small eye phenotype driven by GMR-Gal4 resulted in a rough eye phenotype staining that specifically labels dying cell. As reported previously [ UAS-Egr transgene (UAS-Egrw) driven by GMR-Gal4 induced mild cell death in eye discs posterior to the morphogenetic furrow (MF), as revealed by AO staining . Expression of Hep, the Drosophila homolog of JNK, driven by pnr-Gal4 induced cell death and produced a small scutellum in adult fly and checked cell death with AO staining. We found that loss of flfl triggered extensive cell death in the posterior compartment of wing discs in human. Previous study has reported that JNK signaling is essential for cell migration and tumor invasion [https://www.oncomine.org/) that SMEK1 expression is indeed downregulated whereas DUSP1 is upregulated in invasive breast carcinoma stroma compared to normal tissue . We found that loss of flfl triggered rough eye phenotype ( GFP RNAi and loss of Bsk signaling produced no evident phenotype in adult eyes (Figures flfl induced cell death is JNK pathway-dependent.Knocking downye discs and prodye discs . These r Figures . To undehenotype and incrhenotype were sighenotype . As a co Figures . These r Drosophila. While ectopic expression of flfl impedes JNK signaling-induced cell death, loss of flfl induces JNK pathway activation and cell death in Drosophila eye and wing discs and produced morphological defects in the adult eye. These data suggest an important physiological function of flfl in maintaining tissue homeostasis in Drosophila organ development. flfl's ability to inhibit JNK signaling is likely retained by its human homolog SMEK1. Consistently, while activated JNK pathway promotes dermal fibroblasts cell migration in wound healing [In this study we have identified flfl as a negative regulator of TNF-trigger JNK-mediated cell death in healing , ectopic healing . In addi healing ."} +{"text": "After six months of selective culture, pEHZ-LDLR-LDLR was recovered from FH-iPSC-LDLR and transfected into Ldlr-deficient CHO-a7 cells, which then exhibited feedback-controlled LDLR-mediated endocytosis. To quantify endocytosis, FH-iPSC\u2009\u00b1\u2009LDLR were differentiated into mesenchymal cells (MC), pretreated with excess free sterols, Lovastatin, or ethanol (control), and exposed to DiI-LDL. FH-MC-LDLR demonstrated a physiological response, with virtually no DiI-LDL internalization with excess sterols and an ~2-fold increase in DiI-LDL internalization by Lovastatin compared to FH-MC. These findings demonstrate the feasibility of functionalizing genetically deficient iPSC using episomal plasmids to deliver physiologically responsive transgenes.Acquiring sufficient amounts of high-quality cells remains an impediment to cell-based therapies. Induced pluripotent stem cells (iPSC) may be an unparalleled source, but autologous iPSC likely retain deficiencies requiring correction. We present a strategy for restoring physiological function in genetically deficient iPSC utilizing the low-density lipoprotein receptor (LDLR) deficiency Familial Hypercholesterolemia (FH) as our model. FH fibroblasts were reprogrammed into iPSC using synthetic modified mRNA. FH-iPSC exhibited pluripotency and differentiated toward a hepatic lineage. To restore LDLR endocytosis, FH-iPSC were transfected with a 31\u2009kb plasmid ( Induced pluripotent stem cells (iPSC) derived from patients are a unique, potential alternative for replacing diseased tissue because of their virtually unlimited supply, differentiation potential, and tolerance by the immune systemFamilial Hypercholesterolemia (FH) is one example of a monogenic autosomal dominant disorder that affects low-density lipoprotein cholesterol (LDL) metabolism3LDLR physiologically controlled by 10\u2009kb upstream genomic regulatory control sequence and (b) the minimum number of required Epstein-Barr Virus (EBV) replication and retention sequences (Epstein-Barr Nuclear Association 1 (EBNA1) and origin of plasmid replication (oriP))We used FH as a model system for interrogating the LDLR functionalization of FH-iPSC. We restored FH-iPSC LDLR-mediated endocytosis using a novel 31\u2009kb plasmid containing (a) wild-type OCT4, SOX2, KLF4, c-MYC, and LIN28in vitro spontaneous differentiation and in vivo teratoma formation, with both assays demonstrating formation of the three germ layers . Karyotyping were examined to verify their inherent LDLR dysfunction using an LDL internalization assay . After pyotyping and DNA FH patients benefit from a variety of pharmacological and biomechanical interventions principally targeting liver hepatocytes. Thus, we wanted to determine if FH-iPSC could differentiate towards a hepatic lineage using a modified version of an established five-stage hepatic differentiation protocol that mimics key developmental embryologic cues 11. At thDuring each round of FH-HLC production, we noticed the presence of differentiating cells that took on an increasingly granular, vacuolar appearance that looked lipid-like \u2013 an observation consistent with that of Fattahi and colleaguesin vivo (\u2212/\u2212\u2009\u00d7\u2009LDLR\u2212/\u2212 double-knockout mice for 2 weeks. We labeled the explanted constructs with Albumin-488 and UEA-1-Cy5 (a lectin that binds to human endothelium). Fluorescence confocal microscopy showed that FH-HLC had engrafted and expressed Albumin in the presence of SVF, whereas FH-HLC did not survive in the absence of SVF. Together, these findings suggest that FH-iPSC can differentiate down a hepatic lineage and, given the proper stromal and vascular support, survive and engraft in vivo.We then asked if FH-HLC could engraft in vivo . We prevLDLR transgene expression for treating FH has not yet achieved a cure1719pEHZ-LDLR-LDLR to impart physiological responsiveness by 10\u2009kb of up-stream genomic control sequences821FH-iPSC retain the same genetic dysfunction as their source patient fibroblasts and therefore offer limited therapeutic value with regards to restoring LDLR-mediated endocytosis. Since constitutive pEHZ-LDLR-LDLR physiological restoration of receptor-mediated endocytosis in the Ldlr-deficient cell line CHO-a7 its function is independent of genomic integration.pEHZ-LDLR-LDLR had an effect on the inherent pluripotence of our FH-iPSC. Therefore, we assessed expression of TRA-1\u201360 on transfected cells cultured for over a year under antibiotic selection pressure. Like their non-transfected FH-iPSC counterparts , differences in internalization between the two groups (FH-MC: 188.57\u2009\u00b1\u200974.33\u2009vs. FH-MC-LDLR: 438.82\u2009\u00b1\u2009180.57 fluorescence units/mg protein (FU/mg); mean\u2009\u00b1\u2009SEM) reflecting the cellular lipid requirement due to the lipoprotein-deficient media environment. This difference was amplified when the cells were exposed to the additional stimulus of Lovastatin. Transgene-expressing cells displayed a statistically significant increase (p\u2009=\u20090.035) in specific internalization versus their non-transfected counterparts (FH-MC: 321\u2009\u00b1\u200961.7\u2009FU/mg vs. FH-MC-LDLR: 526.15\u2009\u00b1\u200921.18\u2009FU/mg). As expected, free sterols suppressed DiI-LDL internalization to a statistically equivalent level regardless of transgene presence since free sterol internalization is receptor independentp\u2009=\u20090.00004) and significant with regards to the ethanol control (p\u2009=\u20090.02). Presenting these differences another way, transfected FH-MC-LDLR demonstrated 1.63- and 2.33-fold increases in specific DiI-LDL internalization over FH-MC in the presence of Lovastatin and ethanol, respectively. These findings suggest that the pEHZ-LDLR-LDLR plasmid functionally restores LDL receptor-mediated endocytosis without direct genetic manipulation in FH-iPSC-derived progeny.To facilitate the quantification of Here, we present a proof-of-concept strategy with broad implications for generating and functionally restoring genetically deficient iPSC utilizing transient reprogramming and non-viral, episomal vectors with genomic transgene control, respectively. The significant findings of this project are that (a) an episomal transgene plasmid can be maintained in an extra-chromosomal position within iPSC, (b) the plasmid does not diminish iPSC pluripotency, and (c) genomic control of plasmid transgene expression can restore physiologic functionality to genetically deficient cells.We used modRNA to reprogram FH-fibroblasts into FH-iPSC. modRNA is reported to yield one of the highest reprogramming efficiencies, approaching 2% in normal fibroblasts1229LDLR in E1-deleted adenovirusLDLR gene therapy with constitutive transgene expression driven by hepatocyte-specific promoters (such as \u03b11-antitrypsin or thyroid binding globulin) did not detect cytotoxic lipid accumulations31LDLR expression with hepatocyte-specific promoters may provide some unidentified mechanism for protecting against lipid accumulationWith regards to genetic correction, attempts to resolve LDLR deficiency via gene therapy approaches have primarily relied on viral vectors3132333119LDLR genetic deficiency. The inclusion of genomic sequences allows for precise physiological control of the transgene and enhances long-term LDLR expressionpEHZ-LDLR-LDLR, is maintained as an episome by the inclusion of the EBV retention and replication sequences EBNA1 and oriPtrans regulatory protein is the only EBV protein required for retained episomal functionEpisomal plasmids containing genomic DNA expression control sequences are an alternative approach to addressing the LDLR expression is tightly controlled by a feedback mechanism within the cellpEHZ-LDLR-LDLR plasmid restored receptor-mediated endocytosis in FH fibroblasts and persisted long-term in non-dividing hepatocytes in vivopEHZ-LDLR-LDLR restores physiologically sensitive receptor-mediated endocytosis in FH-iPSC and that this function is retained after differentiation into various progeny.As previously mentioned, LDLR expression.We also demonstrate the effectiveness of large plasmid transfection into reprogrammed iPSC. Although transfection of the source FH fibroblasts before reprogramming is an option, it is unknown if a large 31\u2009kb episomal plasmid would affect reprogramming or if long-term plasmid retention would be compromised. Directly transfecting the FH-iPSC derivatives was also an option, but the cells would still require the time and culture for antibiotic selection, both of which are problematic with hepatocytesin vitro with antibiotic selection until a minimally proliferative state is reached prior to implantation, or (b) generate a liver-tissue mimic with stromal and vascular support that facilitates selection and phenotype stabilization prior to implantation.A major issue to address with our approach is plasmid retention, a common problem with using episomal viral vectors for direct gene therapy to the liverThough our approach specifically targets the LDLR dysfunction in FH, a strategy using episomes with genomic control of transgenes can be tailored to other biological conditions or clinical deficiency with potential for amelioration by iPSC intervention, further enhancing its clinical potential in tissue engineering applications.All animal procedures were conducted in compliance with University of Louisville School of Medicine IACUC-approved protocols and NIH guidelines. All recombinant DNA work was approved by the University of Louisville Institutional Biosafety Committee and performed in accordance with NIH guidelines.et al.FH-iPSC were plated to confluence in Matrigel-pretreated wells of a 6-well tissue culture plate or 6 Permanox chamber slides (Sigma-Aldrich). Each well of the 6-well plate and each Permanox slide (used for immunocytochemistry) corresponded with one of the six stages of differentiation . To generate HLC, we used a modified version of the protocols and medias proposed by Song FH-HLC derived from transfected FH-iPSC (FH-HLC-LDLR) were starved overnight in 5% lipoprotein-deficient serum supplemented with 2\u2009\u03bcM of Lovastatin, excess sterols (12\u2009\u03bcg/ml cholesterol and 0.6\u2009\u03bcg/ml 25-hydroxysterol), or ethanol (vehicle control). The following day, the starvation media was replaced with 5\u2009\u03bcg/mL DiI-LDL in DMEM-HG for 4.5\u2009hours. Cells were thoroughly washed with di-cationic PBS prior to imaging via an Olympus IX81 fluorescence microscope.Ldlr-deficient CHO-a7 cells via Lipofectamine 2000 to demonstrate non-integration and preservation of plasmid functionality after rescue.Plasmid rescue was performed on transfected FH-iPSC-LDLR using methods described by Lufino and colleaguesMesenchymal cells were generated as previously described125Iodinated-LDL. Specific receptor-mediated internalization was determined by subtracting the internalization of [DiI-LDL\u2009+\u200950-fold excess of unlabeled LDL (Alfa Aesar)] from the internalization quantified with DiI-LDL alone. Cells were then lysed with 250\u2009\u03bcl 0.1\u2009N NaOH . Fluorescence was quantified using a 96-well plate reader (BioTek), with the excitation and emission set to 514\u2009nm\u2009and 550\u2009nm, respectively. Fluorescence units were normalized to total protein content, which was determined via a DC Protein Assay kit (Bio-Rad).FH-MC\u00b1LDLR were plated in gelatin-coated wells of a 24-well tissue culture plate. We utilized an established protocolt-test and confirmed via one-way ANOVA using SigmaPlot (Systat Software).Experiments were conducted in triplicate with duplicated data points per experiment. Values are presented as means\u2009\u00b1\u2009S.E.M. Significance was determined using a Student\u2019s Additional procedures can be found in the How to cite this article: Ramakrishnan, V. M. et al. Restoration of Physiologically Responsive Low-Density Lipoprotein Receptor-Mediated Endocytosis in Genetically Deficient Induced Pluripotent Stem Cells. Sci. Rep.5, 13231; doi: 10.1038/srep13231 (2015)."} +{"text": "The present study was planned to investigate the chemopreventive efficacy of Chrysin (CH) against renal carcinogenesis in Wistar rats. Ferric nitrilotriacetate (Fe-NTA) is a potent nephrotoxicant and known renal carcinogen. CH is a natural flavonoid found in honey, propolis, blue passion flower. In vitro data suggests that it has anti-oxidative, anti-inflammatory, anti-apoptotic properties. Renal carcinogenesis is a multistep process and it originates from a series of molecular and histopathological alterations. Renal cancer was initiated by single intraperitoneal injection of N-nitrosodiethylamine and promoted chronically by twice weekly administration of Fe-NTA for 16 weeks and rats were sacrificed after 24 weeks. CH was administered at two doses daily. The possible mechanism could be induction of oxidative stress via upregulation of Lipid peroxidation (LPO), protein carbonyl (PC) and xanthine oxidase (XO); cellular proliferation via up regulation of Proliferative cell nuclear antigen (PCNA), Ki-67 and ornithine decarboxylase (ODC) along with reduction of inflammation by down regulating nuclear factor kappa B (NFkB), Cyclooxygenase-2 (Cox-2), inducible nitric oxide (iNOS), tumor necrosis factor-\u03b1 (TNF-\u03b1), interleukin-6 (IL-6) and prostaglandin E2 (PGE2). Prophylactic treatment of CH mitigated serum toxicity markers, oxidative stress markers, reduced tumor incidences, down regulated proliferation and inflammation. These results provide a powerful evidence for the chemo preventive efficacy of CH at pre-clinical stage."} +{"text": "The original version of this article unfortunThe correct text is provided below:The partial sequences of the 11 MLST loci used in this study have been deposited in the GenBank/EMBL databases under accession numbers KR078648-KR079455,KR079658-KR080061,KP224504-KP225109, KU512980 - KU513181 and KU513182 - KU513383."} +{"text": "PRKDC encodes for DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a kinase that forms part of a complex (DNA-PK) crucial for DNA double-strand break (DSB) repair and V(D)J recombination. In mice, DNA-PK also interacts with the transcription factor AIRE (autoimmune regulator) to promote central T cell tolerance.We sought to understand the causes of an inflammatory disease with granuloma and autoimmunity, associated to decreasing T and B cell counts over time diagnosed in two unrelated patients.Genetic, molecular, and functional analyses were performed to characterize an inflammatory disease evocative of a combined immunodeficiency.PRKDC mutations in both patients. These patients exhibited a defect in DNA DSB repair and V(D)J recombination. Circulating T cells had a skewed cytokine response typical of Th1 and Th2 profiles. Moreover, mutated DNA-PKcs failed to promote AIRE-dependent transcription of peripheral tissue antigens in vitro. The latter defect correlated in vivo, with the production of anti-Calcium Sensing Receptor (anti-CaSR) autoantibodies, which are usually found in AIRE-deficient patients.We identified Deficiency of DNA-PKcs, a key AIRE partner, can present as an inflammatory disease with organ-specific autoantibodies and these findings highlight the essential role of DNA-PKcs in regulating autoimmune responses and maintaining AIRE-dependent tolerance in human.None declared."} +{"text": "Schistosoma mansoni, a parasitic flatworm related to the free-living planarian. We find that the function of NF-YB in regulating male germ cell proliferation is conserved in schistosomes. This finding is especially significant because fecundity is the cause of pathogenesis of S. mansoni. Our findings can help elucidate the complex relationship between self-renewal and differentiation of SSCs, and may also have implications for understanding and controlling schistosomiasis.Gametes are the source and carrier of genetic information, essential for the propagation of all sexually reproducing organisms. Male gametes are derived from a progenitor stem cell population called spermatogonial stem cells (SSCs). SSCs give rise to male gametes through the coordination of two essential processes: self-renewal to produce more SSCs, and differentiation to produce mature sperm. Disruption of this equilibrium can lead to excessive proliferation of SSCs, causing tumorigenesis, or can result in aberrant differentiation, leading to infertility. Little is known about how SSCs achieve the fine balance between self-renewal and differentiation, which is necessary for their remarkable output and developmental potential. To understand the mechanisms of SSC maintenance, we examine the planarian homolog of Nuclear Factor Y-B (NF-YB), which is required for the maintenance of early planarian male germ cells. Here, we demonstrate that NF-YB plays a role in the self-renewal and proliferation of planarian SSCs, but not in their specification or differentiation. Furthermore, we characterize members of the NF-Y complex in Schmidtea mediterranea. Additionally, we extend our study to the parasitic flatworm Schistosoma mansoni, the causative agent of the major neglected tropical disease schistosomiasis and evolutionary cousin of the free-living planarian. We find that there is a loss of proliferating cells in the testes of the parasite when schistosome NF-Y components are inhibited. This observation is significant since the reproductive output of S. mansoni is the primary cause of the morbidity associated with schistosomiasis. Together, our results establish NF-YB as an important regulator of SSC maintenance, and may open avenues for combating schistosomiasis.Sexually reproducing organisms require gametes\u2013sperm and eggs\u2013for the perpetuation of life and transmission of genetic information between generations. Male gametes (sperm) arise from a dedicated population of stem cells known as spermatogonial stem cells (SSCs). Identification of factors involved in SSC maintenance has important biomedical implications, including deciphering the etiology of testicular tumors and optimizing fertility treatments. Here we show that a male germ cell-specific homolog of the ubiquitous Nuclear Factor-Y family of transcription factors, NF-YB, is required for the self-renewal and proliferation of SSCs in the freshwater planarian, Spermatogenesis is highly prolific, relying on SSCs for continual production of progeny. This prodigious output must employ multiple mechanisms to maintain the fine balance between SSC self-renewal and differentiation. Understanding the mechanisms of SSC maintenance is crucial for the treatment of several physiological and disease conditions. Self-renewal of SSCs without differentiation can result in tumor formation. For instance, seminoma-like growth of undifferentiated spermatogonia is seen upon expression of activated RAS, or overexpression of GDNF, or Cyclins D2 and E1, or BCL6B . In contSchistosoma mansoni, a causative agent of schistosomiasis, a disease affecting over 200 million people worldwide. The pathogenicity of schistosomiasis is due to the body\u2019s immune response to eggs laid by adult worms in their human hosts. The spermatogenic output of these parasites is clear from the observation that individuals with schistosomiasis can pass eggs over 30 years after initial infection [The maintenance of germline stem cells is also a key feature behind the fecundity of trematodes such as nfection ,6. Thus,D. melanogaster [C. elegans [D. rerio [Schmidtea mediterranea [S. mediterranea [NF-YB knockdown, animals initially lost their SSC pool followed by more differentiated male germ cells. After over a month of NF-YB(RNAi), mature sperm were seen in sperm ducts of sexual planarians, and some animals had small testes filled with mostly spermatids and some sperm. Thus, NF-YB(RNAi) animals appeared to complete the initial rounds of spermatogenesis, but failed to maintain sperm production over time, possibly due to the loss of SSCs. This phenotype is strikingly similar to that seen in Plzf and TAF4b mutant mice [One such molecule is a planarian homolog of Nuclear Factor-Y B (NF-YB), which belongs to the NF-Y family of transcription factors \u20139. The Nnogaster \u201312, C. e elegans , and D. D. rerio , and a fterranea . More reterranea . In the ant mice ,18. How NF-YB(RNAi) using new markers to track individual stages of spermatogenesis [S. mediterranea, NF-YB does not control germ cell specification or differentiation, but instead promotes self-renewal and proliferation of early germ cells. Interestingly, the NF-YB(RNAi) phenotype in the male germline is strikingly similar in both S. mediterranea and the trematode S. mansoni. Our findings provide mechanistic insight into the role of NF-YB, show the conserved function of this molecule in the testes of both free-living and parasitic flatworms, and may have implications for combating schistosomiasis.In this study, we provide a phenotypic characterization of planarian ogenesis . Our expNF-YB(RNAi) phenotype progression in the male germ cells of S. mediterranea (schematic nanos), spermatogonia , spermatocytes (= tektin1/tkn-1), and spermatids (= protein kinase A/pka) [NF-YB(RNAi), indicated by loss of nanos and gH4 labeling animals also show varying degrees of mature spermatozoa loss during the RNAi timecourse. At later time points the testes only contained clusters of spermatids, labeled with pka . Although all animals showed a progressive loss of male germ cells starting with the least differentiated cells (SSCs and spermatogonia), there was some variability both between samples and within samples in NF-YB(RNAi) animals. We hypothesize that this variability could be a reflection of the NF-YB mRNA/protein half-life in the system, or possibly reflect the variability of germ cell turnover among animals and between different testis lobes (To observe the different stages of e A/pka) \u201322. Lossing Figs and S1. een Figs and S1. pka Figs and S1 ais lobes .NF-YA (A1 and A2), two of NF-YB (B and B2) and one of NF-YC. ClustalW analyses showed a high degree of conservation between the histone-fold motifs of these proteins with their human counterparts . NF-YA2(RNAi) had no somatic or germ cell phenotype animals is reminiscent of the knockdown of planarian nanos [NF-YB(RNAi) and nanos(RNAi) led us to speculate that the two genes might act in concert to control germ cell development. The presence of a CCAAT box, the NF-Y DNA binding motif, -118bp upstream of the nanos transcription start site made NF-YB an attractive candidate regulator of nanos expression.The early germ cell loss seen in an nanos , a gene S. mediterranea sexual hatchlings, expression of nanos is detected within 3 days post hatching [nanos expression, we reasoned that NF-YB expression would precede that of nanos. In situ analyses showed that the NF-YB transcript was seen in the soma in early hatchlings. However, germline NF-YB transcript expression is observed only at later time points relative to nanos expression 2\u20133 times and amputated anterior to the ovaries. The resulting head fragments (lacking reproductive structures at the time of amputation) were monitored for the re-appearance and maintenance of nanos-expressing cells at various regeneration time points in sexually mature planarians (6 feedings over a month) until nanos+ cells were lost (NF-YB(RNAi) (n = 11/11) animals at this early time point animals persisted through regeneration for over a month animals had fewer SSC clusters and the nanos+ cells remained mostly as single cells in these clusters animals head fra a month . In late animals , consist effects Figs. ToNF-YB knockdown was through differentiation or apoptosis. We knocked down NF-YB in juvenile sexual planarians; in these animals, the testes contain clusters of SSCs and spermatogonia, but lack the more differentiated germ cells. Thus, aberrations in spermatogonial differentiation are more easily assayed in these animals compared to mature sexual animals that already possess differentiated male germ cells. The dsRNA-fed animals were processed at early and late knockdown timepoints, cryosectioned, and sections of the same animal were used for phospho-histone H3 (PH3S10) and TUNEL labeling animals showed a dramatic reduction in mitotic cell number following two feedings of dsRNA (NF-YB(RNAi) animals do not show a reduction in nanos+ SSCs or the nanos transcript at this RNAi timepoint animals. We found low levels of TUNEL labeling in NF-YB(RNAi) animals after two feedings of dsRNA animals in later stages of RNAi (after four feedings) still have differentiated male germ cells (spermatocytes and spermatids), indicating that the early germ cells are unlikely to be undergoing apoptosis themselves and are capable of differentiation. Conditional deletion of both NF-YB alleles in primary mouse embryonic fibroblasts causes a block in progression of the cell cycle and induction of apoptosis [We next tested whether germ cells were undergoing apoptosis in of dsRNA . Howeverrm cells . NF-YB animals, and surprisingly Sm-NF-YA(RNAi) and Sm-NF-YC(RNAi) animals, showed a loss of Sm-nanos-1 labeling and late (14 days) RNAi time points. After 7 days of knockdown, control (RNAi) animals showed a large number of EdU+ cells in the testes (Sm-NF-YB(RNAi) animals had fewer EdU+ male germ cells (Sm-nanos-1(RNAi) animals had fewer proliferating male germ cells but greater male germ cell loss compared to Sm-NF-YB(RNAi) animals (Sm-NF-YB(RNAi) and Sm-nanos-1(RNAi) animals phenotype in planarians is strikingly similar to those observed in Plzf [TAF4b [Plzf and TAF4b mutant mice are born with normal number of gonocytes/primordial germ cells, indicating proper specification of the germ cells; planarian tissue fragments lacking germ cells and NF-YB activity regenerate normal number of SSCs, consistent with proper specification of these cells. Plzf and TAF4b mutant mice complete the initial rounds of spermatogenesis but show decreasing fertility with age; in planarians, NF-YB(RNAi) SSCs and spermatogonia are able to differentiate initially but fail to do so over time. Plzf and TAF4b mutant mice show a decrease in proliferative spermatogonia over time; NF-YB(RNAi) animals also show reduced proliferation of male germ cells in both S. mediterranea and S. mansoni. Given the striking similarities between Plzf, TAF4b, and NF-YB functions in the male gonad, it is not unreasonable to speculate that these three genes might be controlling similar targets, or genes that perform similar functions.The in Plzf ,18 and Tf [TAF4b mutant mNF-YB(RNAi). NF-YB(RNAi) results in loss of nanos+ SSCs, but these cells do not show obvious TUNEL labeling. Several possibilities exist to explain this observation. SSCs may be undergoing apoptosis but the signal may be too weak or transient to be detected, or they may use a non-apoptotic mechanism of cell death. It is also possible that the early germ cells could be entering the differentiation pathway aberrantly, resulting in apoptosis of the differentiating cells. NF-YB(RNAi) results in loss of elongated spermatids and sperm in addition to early germ cells is the loss of early germ cells, the expression of NF-YB transcript in the more differentiated male germ cells leaves open the possibility that NF-YB regulates additional target(s) vital for the survival of these differentiated cells.We also observed an increase in apoptotic germ cells in later stages of lls Figs and S1. NF-YB is required for the maintenance of planarian SSCs, provides a valuable tool for understanding the dynamics of early, undifferentiated germ cells. Putative SSC-specific targets of NF-YB will help reveal the function of the NF-Y complex in the planarian, in which tissue-specific knockdown is not possible. Several genes required for the maintenance of various stages of planarian germ cell development have homologs in other species [rap55, ELAV, and MSY4 [NF-YB transcript is upregulated in mouse spermatogonia relative to other male germ cells [The identification of a testis-specific component of the planarian NF-Y complex, and our finding that species . Many ofand MSY4 \u201360, are rm cells , we predIn adherence to the Animal Welfare Act and the Public Health Service Policy on Humane Care and Use of Laboratory Animals, all experiments with and care of vertebrate animals were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Illinois at Urbana-Champaign .S. mediterranea [Clonal lines of hermaphroditic terranea were maiterranea . Clonal terranea were maiCoding DNA sequences of the NF-Y complex were obtained from SmedGD . Mixture\u03b2-tubulin according to manufacturer\u2019s instructions, DNase treated (Fisher Scientific) and purified using an RNA clean-up kit (Zymo) before reverse transcription . Prior to RNA extraction, animals were starved for 7 days after the last RNAi feeding to ensure that any remnant dsRNA was cleared from the system. qPCR was performed using GoTaq qPCR master mix (Promega) using Applied Biosystems StepOne Plus RT-PCR system. All experiments were done in biological and technical triplicates. Transcript levels were normalized to ccdb bacterial gene encoded in pJC53.2 [Planarian double-stranded RNA (dsRNA) synthesis and feeding were performed as previously described . Briefly pJC53.2 served aWhole-mount in situ hybridization of planarians was performed as previously described with modSamples developed through the NBT/BCIP colorimetric method were mounted in 80% glycerol and imaged using a Leica M205A stereomicroscope , equipped with Leica DFC420 camera. Whole-mount FISH animals were mounted in Vectashield and imaged on a Zeiss Stereo Lumar V12 . For confocal FISH images, samples were mounted in Vectashield and imaged using a Zeiss LSM710 confocal microscope running ZEN 2011. Images were processed using Adobe Photoshop CS5.Planarians were cut longitudinally and one half was killed with 2% HCl for 3 minutes on ice and fixed in Methacarn. Cryosectioning was performed as previously described . Anti-Ph2, 0.8 \u03bcl 1:50 DIG-dUTP in dATP, 14.7 \u03bcl water). After rinsing 3X with PBSTx, the sections were blocked with 5% Horse Serum (Sigma H1138) in PBSTx for 30 minutes. Block was replaced with 1:1000 anti-DIG-POD (Roche 11207733910) diluted in block solution. DAPI (1 \u03bcg/ml) was added at this step. Sections were covered with coverslips and incubated for 1 hour at RT. Signal was revealed using Cy3-tyramide (Perkin-Elmer). Slides were rinsed in PBSTx and mounted in Vectashield.A whole-mount TUNEL protocol was modiSchistosoma mansoni, Strain NMRI\u2014exposed Swiss Webster mice (NR-21963) were provided by the NIAID Schistosomiasis Resource Center at the Biomedical Research Institute through NIH-NIAID Contract HHSN272201000005I for distribution through BEI Resources. Mice were perfused with DMEM containing 10% heat-inactivated serum and schistosomes were cultured in vitro [in vitro . In situin vitro . For RNAin vitro ,71). Aniin vitro .Nucleotide sequences have been deposited in GenBank with the following accession numbers: NF-YB\u2014KU366699; NF-YB2\u2014KU366700; NF-YA1\u2014KU366701; NF-YA2\u2014KU366702; NF-YC\u2014KU366703.S1 FigNF-YB(RNAi) time points. nanos+ SSCs were the first cells to be lost in NF-YB(RNAi) animals, after 14 days of RNAi (4 feedings), closely followed by gH4+ SSCs and spermatogonia. At these early stages, very few testis lobes showed reduced tkn-1+ spermatocytes and pka+ spermatids. By 23 days of RNAi (6 feedings), more testis lobes showed reduced nanos and gH4 expression, and the number of testis lobes with reduced tkn-1 labeling increased slightly. 32 days after starting RNAi (8 feedings), all testis lobes examined lacked nanos and gH4 labeling, many testis lobes showed reduced tkn-1 expression and about half the lobes showed reduced pka expression. By 42 days (10 feedings) almost all germ cells were lost. Elongated spermatids and sperm were lost early and this loss was visualized using DAPI. Ten testis lobes per animal (n = 4\u20136) were counted for each testis marker per RNAi time point.Percentage of testis lobes showing normal expression of each male germ cell marker during different (TIF)Click here for additional data file.S2 Fig(A) ClustalW analysis of the human and planarian NF-Y complex members showing the highly conserved domains. (B)NF-YB2 transcript is expressed in somatic tissues. NF-YA1, NF-YA2, and NF-YC are expressed in both the testes and the soma. Scale bars, 1 mm. (C) RNAi of NF-YB2, NF-YA1, or NF-YC results in lesions, head regression (shown with arrows), and lethality after 5 feedings of dsRNA spaced 5 days apart. NF-YA2(RNAi) animals show no somatic phenotype. (D)NF-YA2(RNAi) animals show no loss of germ cells following 6 feedings of dsRNA. Scale bars, 50 \u03bcm.(TIF)Click here for additional data file.S3 FigNF-YB and nanos knockdown animals. In addition to the loss of early germ cells, NF-YB(RNAi) animals also show the loss of mature sperm to varying degrees. After 4 feedings of dsRNA, the most differentiated stage present in NF-YB(RNAi) animals is round spermatids. nanos(RNAi) animals do not show loss of spermatozoa during the initial stages of RNAi. The nanos(RNAi) phenotype also manifests faster. Scale bars, 50 \u03bcm.Animals show an initial loss of SSCs and spermatogonia followed by the more differentiated cells of the testes. Animals were fixed following 2, 4, 6, and 8 feedings, with 4\u20135 day intervals between feedings. There are subtle differences between (TIF)Click here for additional data file.S4 Fig(A) Following 6 feedings of dsRNA, nanos was not detected in the testes of NF-YB(RNAi) animals. (B)dmd1(RNAi) animals do not respecify their male germ cells. Scale bars, 50 \u03bcm. (C) qRT-PCR to measure the levels of the NF-YB transcript (to determine the efficiency of knockdown), NF-YB2 transcript (to ensure specificity of NF-YB knockdown), and smedwi1 transcript (to determine if the somatic stem cells/neoblasts are perturbed following NF-YB knockdown). RNA extraction was done immediately following amputation (Day 0), and at timepoints when head regenerates were fixed for nanos in situ hybridization . Unpaired, parametric two-tailed T-test with Welch\u2019s correction was performed on all samples. NF-YB(RNAi) animals showed significant reduction in NF-YB mRNA levels .(TIF)Click here for additional data file.S5 Fig(A) 15 days post amputation (p.a.) control and NF-YB(RNAi) animals showed 10.1 \u00b1 1.6 (n = 11/11) and 13.7 \u00b1 2.2 (n = 11/11) SSCs respectively. The difference was not significant. (B) 45 days p.a. control animals showed significantly (P<0.05) higher number of SSC clusters than NF-YB(RNAi) animals . (C) 45 days p.a., the number of nanos+ cells per SSC cluster was significantly (P<0.05) higher in control animals compared to NF-YB(RNAi) animals . Scatter plots show mean with SD. Unpaired parametric two-tailed T-test with Welch\u2019s correction was performed on all samples to determine significance .(TIF)Click here for additional data file.S6 FigNF-YB transcript can each knock down NF-YB mRNA and nanos+ SSCs are respecified in either knockdown experiment. (A) Experimental schematic. The experiment for de novo respecification of germ cells was repeated using dsRNA corresponding to the 5\u2019 end of the NF-YB coding sequence as template. In situ hybridization was used to detect NF-YB and nanos mRNAs. A riboprobe corresponding to the 3\u2019 end of NF-YB coding sequence was generated and used for FISH. (B) Control (RNAi) and NF-YB-5\u2019(RNAi) animals show nanos expression following regeneration. (C) Control (RNAi) animals show expression of NF-YB, NF-YB-5\u2019(RNAi) animals do not. Bottom panel\u2013low magnification view of the hatchling with additional exposure showing the inability to detect NF-YB transcript throughout the animal. (D-F) The above experiment was also performed using the 3\u2019 end of the NF-YB transcript. Scale bars, 50 \u03bcm.This experiment was performed to demonstrate that two halves of the (TIF)Click here for additional data file.S7 Fig(A) Following 2 feedings of dsRNA (n = 6/6), NF-YB(RNAi) animals exhibit robust nanos labeling. From multiple prior RNAi experiments, we know that loss of nanos+ cells in NF-YB(RNAi) animals occurs only following 4\u20136 feedings of dsRNA. Scale bars, 50 \u03bcm. (B) We quantified SSCs in control and NF-YB(RNAi) animals to ensure that the reduced PH3S10 labeling was not due to fewer nanos+ cells in NF-YB(RNAi) animals. Following 2 feedings of dsRNA , percentage of nanos+ cells per testis lobe in NF-YB(RNAi) animals was not significantly different (P<0.05) from control (RNAi) animals . Unpaired parametric T-test with Welch\u2019s correction was performed. Scatter plot shows mean with SD. (C) qRT-PCR assay showing that nanos mRNA levels were unaffected following 2 feedings of NF-YB dsRNA. Unpaired parametric two-tailed T-test with Welch\u2019s correction was performed to determine significance .(TIF)Click here for additional data file.S8 Fig(A) Illustration of male S. mansoni depicting the location of the testes and whole-mount in situ hybridization (WISH) in male schistosomes showing Sm-NF-YA and Sm-NF-YC expression in testes. Scale bars, 1 mm. (B) Magnified view of Sm-nanos-1 expression in control (RNAi), Sm-NF-YA(RNAi), and Sm-NF-YC(RNAi) animals. Sm-nanos-1 expression is not detected in Sm-NF-YA(RNAi), and Sm-NF-YC(RNAi) animals. Scale bars, 1 mm. (C) Left and middle panels show high magnification view of the testes in control (RNAi), Sm-NF-YA(RNAi), and Sm-NF-YC(RNAi) animal at early and late KD time points. Scale bars, 50 \u03bcm. Right panel shows whole-mount images showing reduction or loss of EdU labeling in the testes in Sm-NF-YA(RNAi) and Sm-NF-YC(RNAi) animals. Scale bars, 1 mm.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file."} +{"text": "Transthoracic Doppler-echocardiography (TTE) is the standard clinical method for diagnosis and staging of aortic stenosis (AS). AS staging is based on measurement of aortic peak velocity, transvalvular gradient, and calculation of aortic valve area. Unidirectional through-plane phase-contrast magnetic resonance imaging (1DPC-MRI) has been widely applied in clinical imaging to quantify aortic peak velocities and flow. Nonetheless, 1DPC-MRI requires accurate positioning of imaging planes perpendicular to flow direction in order to avoid peak velocity underestimation, which can be challenging in patients with multiple or eccentric jets. Therefore PC techniques with multi-directional velocity quantification would likely improve the accuracy of velocity determination, and allow for more accurate grading of AS severity. The aim of this study is to determine whether a rapid technique that is able to capture 3 directions of velocity in a 2D image plane in a single breath-hold (3DPC-MRI) provides more accurate estimation of diagnostic parameters compared with the traditional 1DPC-MRI, using TTE as the reference standard.We included 13 patients diagnosed with mild to severe AS by TTE . The average time elapsed between TTE and CMR was 24 days. Velocity-encoded CMR included breath-hold 1DPC-MRI and 3DPC-MRI. Acquisition parameters are listed in Table 3DPC-MRI peak velocities showed higher correlation with TTE , than 1DPC-MRI . 3DPC-MRI mean gradient estimation also showed better correlation with TTE results than 1DPC-MRI . Since peak gradient estimations derive from peak velocity estimations, 3DPC-MRI peak gradients again showed better correlation with TTE than 1DPC-MRI . Bland-Altman plots between TTE vs 1DPC-MRI (A-C), and TTE vs 3DPC-MRI parameters (D-F) are shown in Figure Initial results in a small patient cohort support the hypothesis that 3DPC-MRI provides better estimation of hemodynamic parameters in AS patients in comparison to 1DPC-MRI.Research grant from Siemens."} +{"text": "As a central mediator of the natriuretic peptide-cGMP signalling cascade, membrane bound type II cGMP dependent protein kinase (PKG II) is a key regulator of bone growth, renin secretion, and memory formation. It represents an important drug target for treating osteoporosis, cystic fibrosis, and memory loss -5. In spWe screened and identified CNB domains of PKG II that are suitable for our structural studies using a high throughput Ligation Independent Cloning method. Our affinity measurements of the resulting CNB domains showed that CNB-B binds cGMP with a higher affinity, providing almost 500-fold selectivity, while CNB-A only offers 10-fold selectivity . To undeOur structural comparison with cGMP selective PKG I CNB-B domain shows that it lacks cGMP specific hydrogen bonding contacts at the C-helix, which suggests a distinct cGMP selectivity mechanism for PKG II's CNB-B Figure . Cyclic"} +{"text": "On the contrary, down-regulation of miR-204 significantly increased cancer stemness and the lymph nodes incidence of orthotopic animal models. Furthermore, co-knockdown with sh-Slug and sh-Sox4 synergistically rescued miR-204-supressing cancer stemness and EMT properties. Clinical results further revealed that a miR-204lowSlughighSox4high signature predicted the worse survival prognosis of OSCC patients by Kaplan-Meier survival analyses. Up-regulated miR-204-targeting Slug and Sox4 by epigallocatechin-3-gallate (EGCG) treatment significantly inhibited the proliferation rate, self-renewal capacity, and the percentage of ALDH1+ and CD44+ cells in OSCC-CSCs Oral-feeding of EGCG effectively alleviated tumor-progression in OSCC-CSCs-xenotransplanted immunocompromised mice through miR-204 activation. In conclusion, miR-204-mediated suppression of cancer stemness and EMT properties could be partially augmented by the anti-CSCs effect of EGCG.The feature of oral squamous cell carcinomas (OSCC) is commonly metastasizing to locoreginal lymph nodes, and the involvement of lymph nodes metastasis represents the one of important prognostic factors of poor clinical outcome. MicroRNAs (miRNAs) have been shown to be key players of cancer-related hallmarks including cancer stemness, EMT , and metastaisis. Herein we showed that OSCC-derived ALDH1 Oral squamous cell carcinomas (OSCC) is the sixth most common cancer type worldwide with poor prognosis . UnfortuMicroRNAs (miRNAs), a class of small noncoding RNAs regulating the gene expression by binding to the 3\u2032 untranslated region (UTR) of target mRNAs, has been involved in many important biological processes by controlling gene expression at the post-transcriptional level \u201313. SeveIn this report, our studies illustrated an novel-regulatory role of the miR-204-targeting Sox4 and Slug co-expression in the regulation of cancer stemness, EMT, and lymph node metastasis of OSCC cells. Through the upregulation of miR-204 by EGCG appears to suppress cancer stemness in OSCC-CSCs.+, SP+, and sphere-forming OSCC cells but high in ALDH1\u2212, MP, and parental cells . A scrambled vector-transfected control (pLV/miR-Scr.) was also generated simultaneously. The effect of ectopic miR-204 over-expression in OSCC-CSCs was validated by quantitative miRNA RT-PCR analysis samples, local tumor (T) samples and metastatic lymph nodes (LN) samples from OSCC patients using miRNA real-time RT-PCR analysis. As shown in Figure w Figure . These f\u2212 cells isolated from SAS and OECM1 cells, and subjected these cells to functional and molecular analysis. The sphere formation ability , 26. Forhigh/CD24low population, and sphere-forming ability in breast cancer cells /2), and then analyzed using Image-Pro Plus software. Body weight was assessed daily after cell injection. After 26 days, the animals were euthanized, and the primary tumors were weighed and for miR-204 analysis [All procedures involving animals were in accordance with the institutional animal welfare guidelines of the Chung Shan Medical University. For the nude mice xenograft model, 5-6 weeks old immuno-deficient nude mice (BALB/c nu/nu mice) weighing 18-22 g were used. The mice were housed with a regular 12 h light/12 h dark cycle and ad libitum access to standard rodent chow diet and were kept in a pathogen-free environment at the Laboratory Animal Unit. OSCC-CSCs of the Chung Shan Medical University. Cells from each stable Spg-Ctrl. or Spg-miR-204 cells will be injected into tongues of BALB/c nude mice (6-8 weeks). Thirty days after tumor injection, all mice will be sacrificed and necropsy will be performed with removal of tongue tumors and cervical lymph nodes. All tumors and cervical lymph nodes were fixed in formalin and embedded in paraffin for H&E staining for evaluation of metastasis [Statistical Package of Social Sciences software (version 13.0) was used for statistical analysis. Student's t test was used to determine statistical significance of the differences between experimental groups; p values less than 0.05 were considered statistically significant. The level of statistical significance was set at 0.05 for all tests."} +{"text": "Tamoxifen, an agonist of estrogen receptor, is widely prescribed for the prevention and long-term treatment of breast cancer. A side effect of tamoxifen is fatty liver, which increases the risk for non-alcoholic fatty liver disease. Prevention of tamoxifen-induced fatty liver has the potential to improve the safety of long-term tamoxifen usage.UPase1\u2212/\u2212and UPase1-TG.Uridine, a pyrimidine nucleoside with reported protective effects against drug-induced fatty liver, was co-administered with tamoxifen in C57BL/6J mice. Liver lipid levels were evaluated with lipid visualization using coherent anti-Stokes Raman scatting (CARS) microscopy, biochemical assay measurement of triacylglyceride (TAG), and liquid chromatography coupled with mass spectrometry (LC-MS) measurement of membrane phospholipid. Blood TAG and cholesterol levels were measured. Mitochondrial respiration of primary hepatocytes in the presence of tamoxifen and/or uridine was evaluated by measuring oxygen consumption rate with an extracellular flux analyzer. Liver protein lysine acetylation profiles were evaluated with 1D and 2D Western blots. In addition, the relationship between endogenous uridine levels, fatty liver, and tamoxifen administration was evaluated in transgenic mice UPase1\u2212/\u2212with increased pyrimidine salvage activity were protected against tamoxifen-induced liver lipid droplet accumulation. In contrast, UPase1-TG mice with increased pyrimidine catabolism activity had intrinsic liver lipid droplet accumulation, which was aggravated following tamoxifen administration.Uridine co-administration prevented tamoxifen-induced liver lipid droplet accumulation in mice. The most prominent effect of uridine co-administration with tamoxifen was the stimulation of liver membrane phospholipid biosynthesis. Uridine had no protective effect against tamoxifen-induced impairment to mitochondrial respiration of primary hepatocytes or liver TAG and cholesterol export. Uridine had no effect on tamoxifen-induced changes to liver protein acetylation profile. Transgenic mice Uridine co-administration was effective at preventing tamoxifen-induced liver lipid droplet accumulation. The ability of uridine to prevent tamoxifen-induced fatty liver appeared to depend on the pyrimidine salvage pathway, which promotes biosynthesis of membrane phospholipid. Tamoxifen is an effective drug widely used for the treatment of estrogen receptor-positive breast cancer . Women tThe mechanism underlying tamoxifen-induced fatty liver is a topic of active investigation. Evidence from several independent research groups supports tamoxifen-induced impairment of mitochondrial fatty acid oxidation (FAO) as a primary cause of lipid accumulation in the liver -11. Co-aUPase1\u2212/\u2212mice elevates tissues and plasma levels of uridine . It. It33]. UPase1\u2212/\u2212mice with elevated endogenous uridine levels both had enhanced pyrimidine salvage activity [A previous study on the anti-proliferative effect of tamoxifen in human MCF-7 breast cancer cells proposed that tamoxifen prevented DNA synthesis by blocking uridine transport, thus, inhibiting the pyrimidine salvage pathway . In our activity . Both miactivity . It is uThe balance between purine and pyrimidine nucleotides is critical for the maintenance of genomic stability and regulation. A surge in uridine concentration subsequently leads to a rise in pyrimidine nucleotides and perturbs the balance of the nucleotide pool . TherefoCARS: Coherent anti-Stokes Raman scattering; CDPC: Cytidine diphosphocholine; DAG: Diacylglycerol; HDL: High-density lipoprotein; LC-MS: Liquid chromatography coupled with mass spectrometry; LDL: Low-density lipoprotein; OCR: Oxygen consumption rate; PC: Phosphatidylcholine; TAG: Triacylglyceride.The authors declare that they have no competing interest.TTL and GP designed experiments. TTL and GP contributed reagents, samples, and analytical tools. TTL and YU performed experiments and analyzed data. TTL prepared the manuscript. All authors read and approved final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/2050-6511/15/27/prepub"} +{"text": "Microtubules (MT) play a vital role in many cellular functions, but their role in peripheral actin cytoskeletal dynamics which is essential for control of endothelial barrier and monolayer integrity is less understood. We have previously described the enhancement of lung endothelial cell (EC) barrier by hepatocyte growth factor (HGF) which was associated with Rac1-mediated remodeling of actin cytoskeleton. This study investigated involvement of MT-dependent mechanisms in the HGF-induced enhancement of EC barrier. HGF-induced Rac1 activation was accompanied by phosphorylation of stathmin, a regulator of MT dynamics. HGF also stimulated MT peripheral growth monitored by time lapse imaging and tracking analysis of EB-1-decorated MT growing tips, and increased the pool of acetylated tubulin. These effects were abolished by EC pretreatment with HGF receptor inhibitor, downregulation of Rac1 pathway, or by expression of a stathmin-S63A phosphorylation deficient mutant. Expression of stathmin-S63A abolished the HGF protective effects against thrombin-induced activation of RhoA cascade, permeability increase, and EC barrier dysfunction. These results demonstrate a novel MT-dependent mechanism of HGF-induced EC barrier regulation via Rac1/PAK1/stathmin-dependent control of MT dynamics. Enhancement of the endothelial cell (EC) peripheral actin cytoskeleton and increased assembly of cell adhesive complexes by barrier protective agonists provide a structural basis for the maintenance of vascular barrier integrity and prevent catastrophic consequences of uncontrolled vascular leakiness in the lung or other organs caused by bacterial pathogens, cytokine storm accompanying sepsis, trauma, or excessive mechanical forces Hepatocyte growth factor (HGF) is a multifunctional mesenchyme-derived pleiotropic factor secreted by several cell types. Along with other bioactive substances it appears in lung circulation under pathological conditions, such as acute lung injury, sepsis, lung inflammation, and ventilator-induced lung injury (VILI), and regulates a number of biological events such as cell mitogenesis, morphogenesis, organogenesis, and cell survival 16, S25, S38, and S63) reduces its microtubule-destabilizing activity Increasing evidence suggests the role of microtubule (MT) dynamics in control of EC permeability. MT depolymerization by plant-derived MT poisons vinblastin and nocodazole induces robust activation of Rho signaling leading to Rho-kinase dependent microfilament reorganization, stress fiber formation and actomyosin contraction This study tested the hypothesis that, in contrast to effects of barrier-disruptive agonists, HGF stimulation promotes growth of peripheral MT growth in the vascular endothelial cells. We also examined involvement of microtubule-associated proteins stathmin and GEF-H1 in downregulation of barrier disruptive Rho signaling underlying barrier-protective effects of HGF. We evaluated a role of Rac/PAK-mediated stathmin phosphorylation as a potential mechanism of HGF-induced MT preservation and negative Rac-Rho crosstalk central to HGF-mediated EC barrier protection.421 cortactin were from Cell Signaling ; stathmin, and End-Binding protein-1 (EB1) were from BD Transduction Laboratories ; Rac1, RhoA, His-tag were from Santa Cruz Biotechnology . Stathmin phospho-S63 specific antibody, cat. #76583, was from Abcam . Unless otherwise specified, all biochemical reagents were obtained from Sigma .Human pulmonary artery endothelial cells (HPAEC) were obtained from Lonza . Human HGF was from R&D . c-Met kinase inhibitor, N-(3-Fluoro-4-(7-methoxy-4-quinolinyl)phenyl)-1-(2-hydroxy-2-methylpropyl)-5-methyl-3-oxo-phenyl-2,3-dihydro-1H-pyrazole carboxamide, was from Millipore . Reagents for immunofluorescence were purchased from Molecular Probes . Antibodies to phospho-MYPT, GEF-H1, PAK1, phospho-MLC, phospho-YPre-designed Rac1-specific siRNA of standard purity was ordered from Ambion . PAK1-specific set of three Stealth siRNA duplexes was purchased from Invitrogen . Transfection was performed as previously described Active RhoA was captured using GST-rhotekin beads as previously reported 2 chamber and a heated stage. Time lapse images were taken with 2 second intervals for 40 to 60 seconds. 20 consecutive images in each condition were used for projection analysis using ImageJ software. For tracking analysis, EB1 in the cell margin area (2\u201310 \u00b5m from cell border) was tracked with the Manual Tracking plug-in in ImageJ software. The median track length was calculated using Excel software.Endothelial monolayers plated on glass cover slips were subjected to immunofluorescence staining with Texas Red phalloidin to visualize F-actin MT-enriched fractions were isolated as previously described Measurements of transendothelial electrical resistance (TER) across HPAEC were performed using the electrical cell-substrate impedance sensing system (ECIS) as described t-tests. For multiple-group comparisons, a one-way variance analysis (ANOVA), followed by the post hoc Fisher's test, were used. P<0.05 was considered statistically significant.Results are expressed as means \u00b1 SD of three to five independent experiments. Stimulated samples were compared to controls by unpaired Student's s.To assess HGF effects on peripheral MT density and growth, control and HGF-stimulated EC were fixed with methanol and subjected to immunofluorescence staining with antibody to \u03b2-tubulin or End-Binding protein-1 (EB1) which tracks the growing plus end of microtubules. Analysis of MT structure showed expanded peripheral microtubule network in HGF-treated EC . These HGF effects were abolished by cell pretreatment with the c-Met inhibitor .Effects of HGF on microtubule dynamics were further examined using a live imaging approach. For this purpose, EC were transfected with GFP-tagged EB1 which tracks the growing plus end of microtubules. EB1 tracking in live cells was performed by live videomicroscopy, and projection images were generated as described in the Methods section. HGF stimulation increased the length of EB1 tracks, which represents episodes of uninterrupted microtubule growth . These events were also accompanied by an increase in acetylated tubulin. HGF-induced stathmin phosphorylation and increase in acetylated tubulin were abolished by cell pretreatment with c-Met inhibitor or by si-RNA-induced knockdown of Rac1 . Involvement of PAK1 kinase in the HGF-induced stathmin phosphorylation was directly tested in experiments with siRNA-induced PAK1 knockdown . The results showed that depletion of endogenous PAK1 abolished both HGF-induced stathmin phosphorylation and the increase in the pool of acetylated tubulin. These results demonstrate the role of the c-Met \u2013 Rac1 \u2013 PAK1 axis in the HGF-induced modulation of stathmin activity and MT dynamics.HGF treatment rapidly stimulated Rac1 within 2 min reduces its microtubule-destabilizing activity 63 in HGF-induced MT growth. To minimize adverse effects of stathmin-S63A overexpression on global MT arrangement, we used the EC cultures at earlier times after transfection with stathmin-S63A plasmid (16\u201318 hrs). This post-transfection time was sufficient to detect the changes in the HGF-stimulated cells expressing S63A mutant and Rho-kinase mediated phosphorylation of myosin-associated phosphatase (MYPT) and myosin light chain (MLC) induced by thrombin. Direct analysis of GEF-H1 activity showed that thrombin-induced GEF-H1 activation was attenuated by HGF. Expression of stathmin-S63A abolished HGF-mediated downregulation of GEF-H1 activity .Subcellular fractionation experiments showed that thrombin stimulation decreased the pool of insoluble tubulin with a concomitant increase in the soluble fraction which reflects thrombin-induced partial MT depolymerization. Thrombin-induced MT depolymerization was attenuated by cell co-treatment with HGF. Expression of stathmin-S63A abolished HGF's inhibitory effect on thrombin-induced MT depolymerization activity of MT associated Rho regulator GEF-H1 has been implicated in the regulation of Rho signaling by microtubules. In the microtubule-bound state, the GEF activity of GEF-H1 is suppressed, whereas GEF-H1 release caused by microtubule disruption stimulates its Rho-specific GEF activity On the other hand, a stathmin mutation at SInterestingly, despite the existence of multiple stathmin phosphorylation sites (at least four) regulating stathmin interaction with tubulin, the mutation of a single site was sufficient for attenuation of HGF-induced MT growth, effects on Rho signaling and EC permeability. These data suggest an allosteric mechanism of stathmin activity regulation by phosphorylation. An interesting implication of this observation is that MT stability may be regulated via stathmin phosphorylation by different signaling kinases which in turn may be stimulated by different barrier-protective agonists. Our data show that non-phosphorylatable stathmin-S63A mutant attenuates, but not completely blocks, the HGF-induced MT peripheral growth, the HGF-induced EC barrier enhancement response and the HGF-induced suppression of thrombin-induced Rho signaling. This result does not exclude a potential involvement of other MT-associated proteins regulating MT growth and stability in control of EC permeability. We speculate that the regulation of EC barrier by MT dynamics in the native environment may be a result of the superposition of the effects of these proteins on MT stability and peripheral growth, which ultimately contributes to the EC functional response to agonist stimulation.HGF-induced stathmin phosphorylation had a profound effect on increased EC barrier properties monitored by TER measurements. Activation of Rac1 results in activation of cytoskeletal effector proteins including cortactin, which triggers peripheral actin polymerization and the formation of the peripheral actin rim In summary, these results provide a functional link between microtubule dynamics and endothelial barrier enhancement by HGF. Based on previous reports and the results of this study, we propose a scheme of HGF-induced small GTPase regulation and vascular endothelial barrier protection Figure 8"} +{"text": "Reduced estimated glomerular filtration rate (eGFR) using the cystatin-C derived equations might be a better predictor of cardiovascular disease (CVD) mortality compared with the creatinine-derived equations, but this association remains unclear in elderly individuals.The aims of this study were to compare the predictive values of the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI)-creatinine, CKD-EPI-cystatin C and CKD-EPI-creatinine-cystatin C eGFR equations for all-cause mortality and CVD events .Prospective cohort study of 1165 elderly women aged>70 years. Associations between eGFR and outcomes were examined using Cox regression analysis. Test accuracy of eGFR equations for predicting outcomes was examined using Receiver Operating Characteristic (ROC) analysis and net reclassification improvement (NRI).Risk of all-cause mortality for every incremental reduction in eGFR determined using CKD-EPI-creatinine, CKD-EPI-cystatin C and the CKD-EPI-creatinine-cystatic C equations was similar. Areas under the ROC curves of CKD-EPI-creatinine, CKD-EPI-cystatin C and CKD-EPI-creatinine-cystatin C equations for all-cause mortality were 0.604 (95%CI 0.561\u20130.647), 0.606 and 0.606 respectively. For all-cause mortality, there was no improvement in the reclassification of eGFR categories using the CKD-EPI-cystatin C and CKD-EPI-creatinine-cystatin C compared with CKD-EPI-creatinine equation. Similar findings were observed for CVD events.eGFR derived from CKD-EPI cystatin C and CKD-EPI creatinine-cystatin C equations did not improve the accuracy or predictive ability for clinical events compared to CKD-EPI-creatinine equation in this cohort of elderly women. However, epidemiological studies have also shown that eGFR between 60\u201374.9 mL/min/1.73 m2 is associated with a higher risk of CVD-related death compared to eGFR of \u226575 mL/min/1.73 m2 in patients following myocardial infarction suggesting that the risk of adverse clinical events is not confined to those with eGFR of less than 60 mL/min/1.73 m2Chronic kidney disease (CKD) is a major public health burden worldwide. Patients with CKD, especially those on dialysis, suffer from reduced life expectancy and quality of life Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation 2.Several newly-derived eGFR equations such as the CKD-EPI cystatin C and CKD-EPI creatinine-cystatin C equations have shown improvement in the precision and accuracy of determining GFR compared to CKD-EPI creatinine equation, but uncertainties remain as to the clinical significance and cost-effectiveness of using cystatin C-derived eGFR estimations over creatinine-derived eGFR estimations in the general population, particularly in elderly individuals. A recent meta-analysis of sixteen population cohorts reported both CKD-EPI cystatin C and combined CKD-EPI creatinine-cystatin C equations improved the accuracy in predicting all-cause and CVD mortality compared to CKD-EPI creatinine equation, but the majority of the included population cohorts were younger individuals of mixed gender with dissimilar proportion of muscle mass One thousand five hundred women were recruited in 1998 to a five-year prospective, randomized, controlled trial of oral calcium supplements to prevent osteoporotic fractures, the Calcium Intake Fracture Outcome study Baseline medical history including the presence of diabetes, hypertension, smoking history (current/former smokers or non-smokers) and medications were obtained from all participants. Blood pressure was measured on the right arm with a mercury column manometer using an adult cuff after the participants have been seated in an upright position and had rested for 5 minutes. An average of three blood pressure readings was recorded.Inker et al and these are presented in Fasting blood samples were collected at baseline (i.e. at time of randomisation in 1998) with sera stored in \u221270\u00b0C freezer until analysis. Creatinine and cystatin C measurements were performed using stored sera after 2008 and results were available in 1165 women (77%). Serum creatinine was analysed using an isotope dilution mass spectrometry (IDMS) traceable Jaffe kinetic assay for creatinine on a Hitachi 917 analyser . Serum cystatin C was measured on the Siemens Dade Behring Nephelometer, traceable to the International Federation of Clinical Chemistry Working Group for Standardization of Serum cystatin C and the Institute for Reference Materials and Measurements certified reference materials. eGFR was estimated by three equations derived by Participants' general practitioners verified their medical histories and medications where possible, and were coded using the International Classification of Primary Care\u2013Plus (ICPC-Plus) method th Revision, Australian Modification (ICD-10-AM), I00-I99 The primary outcomes of the study were all-cause mortality and CVD hospitalizations and/or mortality retrieved from the Western Australian Data Linkage System (WADLS) for each of the study participants from 1998 until 10 years following their initial study visit. CVD hospitalizations and mortality were defined using primary diagnosis codes from ICD-9-CM, 390-459 Baseline characteristics were expressed as mean and standard deviation (SD) for continuous variables or as number and proportion for categorical variables. Association between eGFR and all-cause mortality and CVD hospitalization and/or mortality was examined using Cox proportional hazard regression model and results were expressed as hazard ratio (HR) with 95% confidence interval (CI) for every incremental reduction in eGFR to allow comparison between equations. The covariates included in the Cox regression models were age, smoking history, body mass index (BMI), diabetes, antihypertensive medications, systolic blood pressure, treatment code, prevalent renal and CVD.2), and then reclassified into new eGFR categories with CKD-EPI cystatin C equation and CKD-EPI creatinine-cystatin C equation as compared with CKD-EPI creatinine equation. P-values of less than 0.05 in two tailed testing were considered statistically significant. The data was analysed using SPSS and STATA .To assess performance of the different equations for estimating eGFR, we assessed the discrimination of the three different models using the Area Under Curve (AUC). Discrimination refers to how well the model distinguishes individuals with and without the outcomes of interests. To assess discrimination, we calculated the area under the receiver operating characteristic (ROC) curve (AUC). An area of 1 implies perfect discrimination, whereas an area of 0.5 represents random discrimination. The sidak option provides adjusted p-values comparing the ROC areas between eGFR equations, assuming a \u201cgold standard\u201d being the CKD-EPI creatinine equation. For net reclassification improvement (NRI), participants were classified into three eGFR categories for all-cause and CVD hospitalization and/or mortality , which was similar for CKD-EPI cystatin C and CKD-EPI creatinine-cystatin C equations.There was at least over 30% increase in CVD events between participants with eGFR of <60 mL/min/1.73 mquations . Howeverquations . For CKDFor prediction of all-cause mortality, the AUCs varied between 0.604 , 0.606 and 0.606 respectively using the CKD-EPI creatinine, CKD-EPI cystatin C and CKD-EPI creatinine-cystatin C equations adjusted for age, BMI, hypertension, diabetes, systolic blood pressure, prevalent renal disease and CVD, smoking history and treatment group . The corFor the prediction of CVD hospitalization and/or mortality, the AUCs varied between 0.660 , 0.659 and 0.660 respectively using the CKD-EPI creatinine, CKD-EPI cystatin C and CKD-EPI creatinine-cystatin C equations adjusted for age, BMI, hypertension, diabetes, systolic blood pressure, prevalent renal disease and CVD, smoking history and treatment group . The corThe reclassification of eGFR categories in predicting all-cause mortality and CVD hospitalization and/or mortality between CKD-EPI cystatin C and CKD-EPI creatinine-cystatin C equations compared with CKD-EPI creatinine equation is shown in In elderly individuals, the accurate evaluation of eGFR for CKD staging is critical to determine correct drug dosing and risk stratification for major clinical events including CVD and all-cause mortality. Our study findings suggest that the association between reduced GFR and clinical outcomes is similar for eGFR equations with and without cystatin C. In addition, the combined CKD-EPI creatinine-cystatin C eGFR or CKD-EPI cystatin C prediction equations were not superior in predicting or reclassifying CVD hospitalization and/or mortality or all-cause mortality over the CKD-EPI creatinine eGFR equation in a cohort of elderly women.Eriksen et al. has shown that cystatin C was not superior in estimating measured GFR compared to creatinine in the general population Cystatin C appears to be a superior GFR marker compared to creatinine 2. The use of the combined CKD-EPI creatinine-cystatin C equation was able to improve reclassification of individuals with creatinine-derived eGFR of 45\u201374 ml/min/1.73 m2 , and also 17% of individuals with creatinine-based eGFR of 45\u201359 ml/min/1.73 m2 to \u226560 ml/min/1.73 m2. In a recent meta-analysis of 11 general population studies comprising of 90,750 participants, there was a more consistent linear association between reduced eGFR derived from CKD-EPI cystatin C and CKD-EPI creatinine-cystatin C equations and increased risks of all-cause and CVD mortality for all eGFR values below 85 mL/min/1.73 m2 compared with CKD-EPI creatinine equation, well above the threshold of 60 mL/min/1.73 m2 for the detection of CKD with CKD-EPI creatinine-based eGFR Two recently developed CKD-EPI creatinine-cystatin C and CKD-EPI cystatin C equations were shown to perform better in predicting measured radionuclide GFR compared to CKD-EPI creatinine equation 2) and therefore the applicability of our study findings to males, other ethnic minorities or racial groups and those with poor nutrition and low muscle mass remains unclear.The strengths of this study include the use of a large prospective cohort of subjects with complete and accurate data collection over a 10-year period. We were able to accurately examine the association between estimates of GFR using the newly developed cystatin C equations and clinical outcomes in a population with a low prevalence of CVD and renal diseases, which further strengthens this association. However, the strengths of the study must be balanced against the limitations, which include a lack of radionuclide GFR measurements and availability of single time-point measurements of creatinine and cystatin C to estimate baseline GFR. In addition, our study cohort only included white female participants with presumed adequate nutrition and muscle mass Click here for additional data file."} +{"text": "Natriuretic peptides have become important tools for diagnosis, risk stratification and therapeutic decision making for patients with heart failure and hydrostatic pulmonary edema. However, the practical use of N-terminal pro-brain natriuretic peptide (NT-proBNP) in ARDS patients and its relationship with non-cardiogenic pulmonary edema remain controversial.To evaluate the relationship between plasma concentrations of NT-proBNP, hemodynamics and extravascular lung water and to assess the possibility of using NT-proBNP to diagnose pulmonary edema in ARDS.2, Pulsion Medical Systems, Germany) with measurement of mean arterial pressure, cardiac index (CI), central venous pressure, global end-diastolic volume index (GEDVI), systemic vascular resistance index and extravascular lung water index (EVLWI). Hemodynamic, respiratory parameters and plasma concentrations of NT-proBNP were assessed on the day of admission and on days 3 and 5 of hospitalization. Patients received goal-directed therapy using CI, GEDVI and EVLWI. The statistical analysis was performed using Mann-Whitney U-test and Spearman's correlation coefficient. The data are presented as median (25th-75th percentiles).Fifteen adult patients with mean age of 43 (27-50) years with diagnosed ARDS according to Berlin definition were enrolled into an observational one-center prospective pilot study. The patients received hemodynamic monitoring using transpulmonary thermodilution (PiCCO2. We did not find correlation of plasma NT-proBNP with CI and EVLW.The causes of ARDS included severe pneumonia (33%), sepsis (33%), multiple trauma (27%) and acute poisoning (7%). On admission, 8 patients had mild, 5 - moderate and 2 - severe ARDS. The ICU mortality was 27% (4 patients). The mean plasma concentrations of NT-proBNP in ARDS patients were higher than the normal values, with a trend for decrease during hospitalization: from 4706 (334-15173) pg/ml on admission to 1843 (118-10815) pg/ml and 1236 (332-11100) pg/ml on days 3 and 5, respectively . The plasma concentration of NT-proBNP on admission in patients with mild ARDS was 325 (107-4059) pg/ml vs. 17541 (3797-40795) pg/ml in the group of patients with moderate and severe ARDS . On admission, NT-proBNP correlated with GEDVI , which was 701 (652-910) ml/mIn ARDS patients, we observed increased plasma concentrations of NT-proBNP on admission with a trend for decrease over time. In moderate and severe ARDS, NT-proBNP concentrations are higher than in mild ARDS. The plasma concentrations of NT-proBNP correlate with GEDVI; however we observed no relationship of NT-proBNP with CI and EVLW. Thus, NT-proBNP has a limited value to diagnose pulmonary edema in ARDS."} +{"text": "Abacavir hypersensitivity reaction (ABC-HSR) is associated with the presence of HLA-B*5701 allele. Alternative tests for ABC-HSR associated allele determination are needed where sequence-based HLA typing is not available, particularly in resource-limited settings or developing countries. This study focused on the development and evaluation of two HLA-B*5701 tagging SNPs (single nucleotide polymorphism) HCP5 (HLA complex P5) rs2395029 and TNF (tumor necrosis factor) rs3093726 genotyping assays. Two hundred and thirteen purified genomic DNA samples were used to evaluate the performance characteristics of a HLA-B*5701 screening method based on allele-specific polymerase chain reaction (AS-PCR) with melting curve analysis. HCP5 rs2395029 and TNF rs3093726 were also genotyped using simple probe real-time PCR assay. All samples were successfully genotyped wherein AS-PCR genotyping provided 100% sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) when compared with specific HLA-B status by sequencing based assay. When comparing the AS-PCR screening method with the HCP5 rs2395029 and TNF rs3093726 genotyping method, the former had 100% sensitivity, 100% specificity, 100% PPV and 100% NPV using a simple probe; while the latter one had 95.24% sensitivity, 100% specificity, 100% PPV and 99.48% NPV, respectively. In conclusion, our study lends support on a molecular tool for pharmacogenetic screening in resource-limited settings. Thus, serious drug hypersensitivity associated with ABC may potentially be reduced in Thailand by further evaluation of the proposed assay in clinical practice."} +{"text": "We aim to investigate the effect of utilizing aortic valve timing in the measurement of cardiac magnetic resonance (CMR)-derived ejection fraction (EF). Although CMR-derived left ventricular (LV) EF is the gold-standard for volumetric evaluation of the heart, it always reports higher values when compared to other modalities such as echocardiography and radionuclide ventriculography. The higher EF has often been attributed to better endocardial definition by CMR. In the ACCF/ACR/AHA/NASCI/SCMR 2010 Expert Consensus Document on CMR, a 1987 paper is cited with utilizes the \"maximal and minimal left ventricular cross-sectional area at the mid-ventricle\" to identify end-systole and end-diastole. The 2013 CMR Guideline suggests phases with the largest and smallest global LV blood volume be utilized to identify end-diastole and end-systole. The same guideline also suggests aortic valve timing be utilized when arrhythmia or mitral regurgitation is encountered. We investigated the effect of different schemes on measured LVEF.Standard retrospectively EKG-gated bSSFP short axis and LVOT cine images in 46 clinical patients in normal sinus rhythm with a range of EF (5.8 - 75.8 %) were acquired and analyzed. Scan parameters were as follows: in-plane resolution: 1.25 - 2.08 mm, slice spacing: 8 mm with a gap of 2 mm, reconstructed temporal resolution: 18.2 - 58.8 ms. Aortic valve opening and closing as a percentage of cardiac cycle was identified on LVOT images. Volumetric evaluation of short axis images was performed via semi-automated segmentation for all phases and all left ventricular slices . The schemes (shown in Table Figure Aortic valve timing results in a small but consistent decrease in ejection fraction relative to current standard techniques. Furthermore, if real-time techniques which acquire slice-by-slice data are not synchronized with external timing information such as aortic valve timing or EKG, the stroke volume will be overestimated leading to an additional overestimation of EF.K99-HL108157, R01-HL103723, T32HL007954, T32-EB009384."} +{"text": "Plants respond to diverse environmental cues including microbial perturbations by coordinated regulation of thousands of genes. These intricate transcriptional regulatory interactions depend on the recognition of specific promoter sequences by regulatory transcription factors. The combinatorial and cooperative action of multiple transcription factors defines a regulatory network that enables plant cells to respond to distinct biological signals. The identification of immune-related modules in large-scale transcriptional regulatory networks can reveal the mechanisms by which exposure to a pathogen elicits a precise phenotypic immune response.Arabidopsis thaliana (hereafter Arabidopsis) transcriptomic data, which consists of a wide spectrum of immune responses to pathogens or pathogen-mimicking stimuli treatments. We employed both linear and non-linear models to generate Arabidopsis immune co-expression regulatory (AICR) network. We computed network topological properties and ascertained that this newly constructed immune network is densely connected, possesses hubs, exhibits high modularity, and displays hallmarks of a \u201creal\u201d biological network. We partitioned the network and identified 156 novel modules related to immune functions. Gene Ontology (GO) enrichment analyses provided insight into the key biological processes involved in determining finely tuned immune responses. We also developed novel software called OCCEAN to discover statistically enriched promoter elements in the upstream regulatory regions of Arabidopsis at a whole genome level. We demonstrated that OCCEAN exhibits higher precision than the existing promoter element discovery tools. In light of known and newly discovered cis-regulatory elements, we evaluated biological significance of two key immune-related functional modules and proposed mechanism(s) to explain how large sets of diverse GO genes coherently function to mount effective immune responses.We have generated a large-scale immune co-expression network using a comprehensive set of We used a network-based, top-down approach to discover immune-related modules from transcriptomic data in Arabidopsis. Detailed analyses of these functional modules reveal new insight into the topological properties of immune co-expression networks and a comprehensive understanding of multifaceted plant defense responses. We present evidence that our newly developed software, OCCEAN, could become a popular tool for the Arabidopsis research community as well as potentially expand to analyze other eukaryotic genomes.The online version of this article (doi:10.1186/1471-2164-15-421) contains supplementary material, which is available to authorized users. In lighinfinitum, 5. Our infinitum, 6. Bothinfinitum, 8. The infinitum\u201311. Howecis-regulatory elements (transcription factor binding sites) can aid in the identification of co-regulated gene clusters and characterization of transcriptional regulatory networks [In recent years, systems biology approaches, specifically network analyses that integrate experimentally derived information with computational modeling, have emerged as powerful tools for studying complex traits in diverse species , 12\u201315. networks , 14, 17.i.e. PCC (Pearson Correlation Coefficient), ARACNE multiplicative (Algorithm for the Reconstruction of Accurate Cellular Networks), ARACNE additive, CLR (Context Likelihood of Relatedness), and MRNET (Minimum Redundancy NETwork) with different thresholds, which yielded 15 pairs of experimental networks along with their respective random networks [cis-regulatory elements. First we developed a new, comprehensive software interface for cis-regulatory elements discovery in Arabidopsis . We demonstrated that OCCEAN boasts higher capacity and features higher precision than MEME (Multiple EM for Motif Elicitation). We identified several statistically enriched novel cis-regulatory elements in our dataset. Finally, we evaluated and discussed key immune-related functional modules.In the current study we employed a systems biology approach and report the construction of a large-scale Arabidopsis immune co-expression regulatory (AICR) network. We tested five diverse algorithms, networks \u201325. We ek-nearest neighbor (mKNN) to process the calculated PCC values [i.e., PCC (0.9), K3, K10, K20, K50, K100 and K250 . To characterize the significance of these highly connected proteins, we combined Hub50 of the AICR with biophysical interactions from plant-pathogen immune network, version 1\u2032 with the highest betweeness centrality Figure\u00a0. Among tAnother essential feature of a majority of biological networks is their ability to naturally organize into modules , 17, 18.cis-regulatory elements to define the potential functions of the immune-related modules (described below).We also investigated the enrichment of Gene Ontology (GO) terms , 58 in ti.e., are direct targets of a common transcription factor). Given that transcriptional regulatory networks are highly complex and that functional modules may display crosstalk among themselves, we can also expect that the same transcription factor can regulate co-expressed genes in multiple immune-related modules. Whereas experimentally verified plant cis-regulatory elements can be retrieved from PLACE (A Database of Plant Cis-acting Regulatory DNA Elements) [cis-regulatory elements are currently known in Arabidopsis, and a limited set of ~30 immune-related cis-elements have been described. In addition, the prediction of cis-elements from classical approaches is typically driven by a single experiment or dataset. Moreover, the currently available online motif discovery software has limited capacity to process a small number of gene entries. For example, frequently used software MEME (Multiple EM for Motif Elicitation) can only process up to 60,000 characters in a single run [cis-regulatory elements, AthaMap can only manage up to 200 gene entries [cis-regulatory elements. Thus, we first aimed to develop OCCEAN software that can process the promoter gene sequences at the entire genome scale (over 30 million characters) in a single run. OCCEAN identifies the statistically enriched/depleted cis-regulatory elements our newly developed BLASTN-based program to identify short sequences and (ii) an improved version of the bootstrapping tool POBO (a promoter bootstrapping program) [cis-regulatory elements in the gene\u2019s promoter as the output. OCCEAN is freely available online at http://occean.cis.uab.edu:8080/occean/. We employed OCCEAN to individually process the sequences of immune-related genes\u2019 promoters for our \u201ctop 10\u201d immune-related modules. The promoter sequences of the genome were used as background to compute the fold enrichments of putative cis-regulatory elements. We also analyzed the occurrences of all the putative cis-regulatory elements (cluster mean), performed 1,000 \u00d7 bootstrapping to determine the background mean and finally calculated fold enrichment ratios for all six-mer sequences. Three different fold enrichment ratios prioritized newly identified cis-regulatory elements in \u201ctop 10\u201d immune-related modules , false positives (nFPs) and precision for MEME as well as OCCEAN enrichment ratios\u2009\u2265\u20094, \u2265 3 and\u2009\u2265\u20092 , Athena lements) and AGRIlements) , 62, onl length) , 64. Ano entries . These ls Figure\u00a0 and inteTo fend off potential pathogens, plants employ two major types of immune responses: MAMPs-Triggered Immunity (MTI) and Effector Triggered Immunity (ETI) . ETI is Diverse kinases account for the major functional category of Module 1, encompassing 46 out of 178 proteins representing receptor-like kinases (RLKs), mitogen-activated protein kinases (MAPKs), calcium-dependent protein kinases (CDPKs), wall-associated kinases (WAKs), etc. Typically, RLKs perceive MAMPs such as flg22, elf18, chitin and OGs and trigger MTI . SubsequNLR genes in the kinase sub-cluster of Module 1. These include proteins conferring resistance to diverse pathogens such as Pseudomonas syringae, Albugo candida, Hyaloperonospora arabidopsidis and various Fusarium races.At the second, more powerful layer of the immune response, plants usually deploy NLR receptors . The NLR receptors directly recognize specific effectors or indirectly detect effector activities and trigger ETI , 2, 6, 7The second most abundant GO category in Module 1 was immune-related genes (count: 32). This functional sub-cluster contains various defense-related transcription factors such as WRKY15, WRKY33, MYB113 and ANAC061 , 47, fouCNI1 (Carbon-Nitrogen Insensitive 1) that is a key regulator of the Carbon/Nitrogen response for growth phase transition in Arabidopsis seedlings [Syntaxin 121/PEN1 and MLO2 genes, required for resistance against barley powdery mildew, Blumeria graminis sp. hordei and a fungal pathogen Colletotrichum higginsianum[Another enriched GO category in Module 1 was membrane-associated proteins including both plasma membrane- and organellar membrane-resident peptides; not surprisingly, the largest class of these membrane proteins is transporters. In plants, uptake and translocation of nutrients play essential roles in physiological processes including plant growth, nutrition, signal transduction, and development \u201379. Traneedlings , as welleedlings . Last buginsianum, 85.i.e., to determine presence of common cis-regulatory elements among genes of the same module. Several known cis-regulatory elements were discovered in the promoters of Module 1 genes [Another essential aspect of large-scale transcriptomic analyses is to link co-expression with co-regulation, promoter . APETALArocesses \u201389. Inhirocesses . Involve 1 (AS1) .ATCTTG that resembles the binding site for Ethylene-Insensitive 3 (EIN3) and three EIN3-LIKE (EIL) proteins [GTCGTC and CGTCGT, which overlap with the core binding site of JASE2 motif in two JA biosynthetic genes OPR1/OPR2[SID2[cis-regulatory elements were discovered in our study have extended list of binding specificities beyond the currently known cis-regulatory elements. We propose that the above described transcription factors and additional unknown regulatory proteins coordinate the gene regulation in this module.Jasmonic Acid (JA) and Ethylene (ET) are two plant hormones antagonistic to SA and downproteins as well OPR1/OPR2. RecentlOPR2[SID2. Given tOPR2[SID2. In addiOur data provide further insights into the transcriptional regulatory mechanisms repressing additional putative negative regulators of plant defense upon treatments with pathogen-mimicking stimuli.cis-regulatory elements using OCCEAN is an effective method of solving the issue of finding novel motifs within a sequence set. OCCEAN has advantages over other programs of the same purpose, such as APPLES and MEME [cis-regulatory elements (the putative transcription factor binding sites) in the promoters of the co-expressed genes made it possible to link co-expression to co-regulation of genes in the same module.In this study, we used a systems-level network biology approach to construct genome-wide Arabidopsis immune co-expression network and demonstrated that this network shares properties of a \u2018real-world network\u2019. Topological properties-based partitioning allowed us to unravel 156 distinct immune-related functional modules. We demonstrated that nodes in the same module are not only highly correlated at the expression level but also densely connected to each other. Detailed analyses of two key immune-related modules provided a systems-level understanding of how plant cells coordinate distinct immune signals to orchestrate fine-tuned and pathogen-specific defense responses. Our novel approach to discover and MEME , 97. APPhttp://www.arabidopsis.org[We utilized available transcriptomic data of transcriptional responses extracted from 271 microarray experiments representing nine major immune-related studies , false positives (nFPs) and precision for MEME as well as OCCEAN were calculated as described in [Putative transcription factor binding sites results from BLAST were analyzed for statistically significant over representation/under representation using the POBO (a promoter bootstrapping program) tool . POBO isribed in .The bulk of OCCEAN on the server-side was developed in the Java language (version 7) using Apache Tomcat on a Linux server. A Java Servlet interface was used to communicate with OCCEAN\u2019s web interface. OCCEAN automates and integrates the information obtained from BLAST and POBO analysis. This user friendly software requires promoter sequences in FASTA format. Background sequences of the species under study are imbedded in the software. OCCEAN is time-efficient and will return a link to the file containing the results the BLAST and POBO. OCCEAN\u2019s web interface was developed using HTML, CSS, Javascript, and AJAX for in-page update notifications.Additional file 1: Table S1: Differentially expressed (DE) genes that were determined from defense-related experiments. (XLS 26 KB)Supporting methods Transcriptomic data of transcriptional responses extracted from 271 microarray experiments representing nine major immune-related previous studies. (DOCX 38 KB)Additional file 2: Additional file 3: Table S2: AGIs of the up-regulated and down-regulated genes derived from Table S1 studies. (XLSX 253 KB)Additional file 4: Figure S1: Distribution of shortest paths in the AICR (blue circles) and random (red diamonds) networks. (TIFF 707 KB)Additional file 5: Figure S2: Closeness centrality property of the AICR network. Distribution of closeness centrality in the AICR (blue circles) and random (red diamonds) networks. (TIFF 1 MB)Additional file 6: Figure S3: Evaluation of frequency of number of shared neighbors in the AICR (blue circles) and random (red diamonds) networks. (TIFF 884 KB)Additional file 7: Figure S4: Determination of neighborhood connectivity frequency in the AICR (blue circles) and random (red diamonds) networks. (TIFF 774 KB)Additional file 8: Figure S5: Distribution of topological coefficients in the AICR (blue circles) and random (red diamonds) networks. (TIFF 2 MB)Additional file 9: Figure S6: Degree distributions of main components in the AICR (blue circles) and random (red diamonds) networks. (TIFF 56 KB)Additional file 10: Figure S7: Betweenness centrality of main components in the AICR (blue circles) and random (red diamonds) networks. (TIFF 57 KB)Additional file 11: Table S3: Identification of 156 immune-related modules. Size of each module is indicated. (XLS 146 KB)Additional file 12: Figure S8: Distribution of module size in the AICR network. Frequency of module size in the AICR (blue circles) network is shown in log scale. The AICR network exhibits a power law distribution, a network property shared by \u2018real-world networks\u2019. (TIFF 462 KB)Additional file 13: Table S4: GO enrichment in ten largest immune-related modules. (XLS 104 KB)cis-regulatory elements in ten largest immune-related modules using OCCEAN. (XLS 128 KB)Additional file 14: Table S5: Identification of Supporting results Module 8 in Arabidopsis immune co-expression regulatory (AICR) network. (DOCX 34 KB)Additional file 15:"} +{"text": "Although telomerase is an almost universal target for cancer therapy, there has been no effective telomerase targeted inhibitor that has progressed to late stage human clinical trials. Recently, we reported that a telomerase-mediated telomere-disrupting compound, 6-thio-2\u2032-deoxyguanosine (6-thio-dG), was very effective at targeting telomerase positive cancer cells while sparing telomerase silent normal cells. 6-thio-dG, a nucleoside analogue of the already-approved drug 6-thioguanine, is incorporated into telomeres by telomerase, resulting in disruption of the telomere-protecting shelterin complex. This disruption leads to Telomere dysfunction-Induced Foci (TIFs) formation and rapid cell death for the vast majority of cancer cells. Since most chemotherapies eventually fail due to drug acquired resistance, novel drugs such as 6-thio-dG, as a single first line agent or in the maintenance setting, may represent an effective new treatment for cancer patients. At the end of linear chromosomes, repetitive sequence structures called telomeres are protected from being recognized as random DNA double strand breaks, which would otherwise activate DNA damage signaling responses. These sequences, (TTAGGG)n in mammals, are responsible for genomic stability by preventing recombination, end-end fusions/degradation and are also important in completing DNA replication during each cell cycle . If teloWhile there have been many different approaches to directly or indirectly target telomerase, only Imetelstat GRN163L) has progressed to late stage human clinical trials. One concern with Imetelstat is the development of hematological toxicities requiring drug holidays that enable telomere re-elongation. An effective inhibitor would ideally permit long-term, robust (>99%) telomerase inhibition or telomere dysfunction and rapid tumor shrinkage. Direct telomerase inhibitors in clinical trials do not show rapid tumor shrinkage not has robust telomerase inhibition been demonstrated. This is important since we have shown that only one percent of telomerase activity in cancer cells is sufficient to maintain the shortest telomeres and permit cells to continue to divide 3L has pr.in situ in cells) may be a telomerase-directed telomere uncapping compound. 6-thio-dG is rapidly converted to telomerase substrate 6-thio-2\u2032-deoxyguanosine-5\u2032-triphosphate (6-thio-dGTP) and consequently uses telomerase for its incorporation into telomeres. The guiding concept for initial proof-of-principle studies was that, since 6-thio-dG is converted rapidly into 6-thio-dGTP, it is potentially incorporated into both genomic DNA (by DNA polymerases) and telomeric DNA (by telomerase). We therefore predicted that 6-thio-dG would be both a more effective agent compared to 6-thioguanine and also induce cancer cell killing much more rapidly than a \u201cclassic\u201d telomerase inhibitor. Once 6-thio-dG is incorporated into telomeres, the telomere sequence TTAGGG is modified at guanine bases, resulting in uncapping of telomeric DNA and likely loss of recognition and dissociation of the shelterin proteins from the de-novo formed modified telomeres. This leads to Telomere dysfunction Induced Foci (TIF) formation and rapid growth arrest or cell death of telomerase-positive cells. This relatively fast anti-cancer effect of 6-thio-dG has an important advantage compared to other direct telomerase inhibition approaches. Long and frequent treatment cycles of direct telomerase inhibition-based therapies can cause side effects, as evidenced in some anti-telomerase clinical trials [Hence, we and othel trials . ImportaIn summary, application of 6-thio-dG, with its proposed telomere-targeting mechanism of action, appears to be a promising anti-cancer treatment approach. As with any chemotherapy treatment, 6-thio-dG resistance mechanisms are expected to emerge. Understanding these resistance mechanisms may lead to more personalized based therapeutic regimens. In preliminary studies, we have tested a series of commonly-used multi-drug resistant cell lines and found 6-thio-dG to be active and effective in a significant fraction of these cell lines. Thus, 6-thio-dG may be effective in the maintenance setting after first line chemotherapy, with treatment yielding long-term durable responses for cancer patients."} +{"text": "The status of lung cancer surgery in UK has seen many changes over the last 20 years, with innovations in surgical technique and investigatory modalities together with significant organisational changes.To assess how these changes have impacted on an individual surgical practice spanning this era.We have retrospectively reviewed a single-surgeon practice from consultant appointment to present (1997-2015). We studied 1717 consecutive lung cancer operations: 962 lobectomy, 250 extended lobectomy, 57 pneumonectomy 296 sublobar, 43 open/close. Additionally, 710 surgical staging procedures were performed. We analysed trends with time in type of procedure; approach used (VATS/Open); open/close rates and in-hospital mortality.1566 anatomic resections were performed . The following trends were observed:1) Related to the disease itself- A significant decrease in pneumonectomy rates (p < 0.001)- An inversely proportional, increasing use of sleeve-resections (p = 0.088).2) Related to surgical technique- An increasing number of anatomical segmentectomies (p < 0.001)- Stable rates of non-anatomical wedge resections - An increasing proportion of VATS resection, both for lobectomies (p < 0.001) and segmentectomies .3) Related to healthcare system- A significant decrease in use of surgical mediastinal staging, particularly after 2010 (p < 0.001)- A significant decrease in-hospital mortality - A significant reduction of open/close rates, particularly after 2004 .There has been significant evolution in lung cancer surgery over the last two decades, which is reflected in this individual surgeon's practice. Whilst increased surgical experience may partly explain the changes, most important factors include: a change in the proportion of central squamous and peripheral adenocarcinomas; earlier tumour detection, facilitating more VATS and lung-sparing surgery; improved perioperative care and use of lesser resections, reducing mortality; new techniques in staging reducing the need for surgical staging and the number of futile thoracotomies."} +{"text": "Normal thyroid hormone secretion or optimal substitution of L-thyroxine is necessary for the proper functioning of the hypothalamic-pituitary-IGF-I axis. Over 30 years ago Cacciari et al. indicateThe rGH therapy might disclose previously unrecognised thyroid insufficiency rather than induce hypothyroidism ,3. The pIn GH-deficient children from the beginning of rhGH therapy - in euthyroid status - has not been recommended the obligatory L-T4 supplementation due a little evidence for the development of clinically significant hypothyroidism in most of previously euthyroid patients and spontaneous recovery to pre-treatment thyroid function in most patients . Maintai"} +{"text": "Drosophila visual system, two neuroepithelia, the outer (OPC) and inner (IPC) proliferation centers, generate neuron subtypes for four ganglia in several ways. Whereas neuroepithelial cells in the medial OPC directly convert into neuroblasts, in an IPC subdomain they generate migratory progenitors by epithelial-mesenchymal transition that mature into neuroblasts in a second proliferative zone. The molecular mechanisms that regulate the identity of these neuroepithelia, including their neurogenesis modes, remain poorly understood. Analysis of Polycomblike revealed that loss of Polycomb group-mediated repression of the Hox gene Abdominal-B (Abd-B) caused the transformation of OPC to IPC neuroepithelial identity. This suggests that the neuroepithelial default state is IPC-like, whereas OPC identity is derived. Ectopic Abd-B blocks expression of the highly conserved retinal determination gene network members Eyes absent (Eya), Sine oculis (So) and Homothorax (Hth). These factors are essential for OPC specification and neurogenesis control. Finally, eya and so are also sufficient to confer OPC-like identity, and, in parallel with hth, the OPC-specific neurogenesis mode on the IPC.Differences in neuroepithelial patterning and neurogenesis modes contribute to area-specific diversifications of neural circuits. In the Summary: Polycomb-mediated repression of the Abd-B Hox gene controls expression of retinal determination genes and hence identity of the Drosophila optic lobe neuroepithelia. Drosophila, several neurogenesis modes have been identified in the embryonic and postembryonic brain and ventral nerve cord (VNC) sheets. In an initial proliferative phase, NE cells expand by symmetric cell divisions and become patterned into discrete territories. During a subsequent neurogenic phase, NE cells convert into neural stem cells (NSCs), which undergo asymmetric self-renewing divisions to produce diverse neuronal and glial subtypes . Recent rd (VNC) . HoweverDrosophila visual system, photoreceptors (R-cells R1-R8) within the compound eye extend axons into the optic lobe consisting of four ganglia: the lamina, medulla, lobula plate and lobula that caused the formation of conspicuous ectopic NE cell clusters within the lobula plate area. Detailed analysis of observed phenotypes revealed that large clusters originated from the OPC and adopted IPC characteristics. Pcl is required for maintaining OPC identity by preventing ectopic expression of the Hox gene Abdominal-B (Abd-B) and thus interference with area-specific determinants. Our search for these factors uncovered that the optic lobe NE default state is IPC-like, whereas the RDGN members Eyes absent (Eya), Sine oculis (So) and Hth . This approach relies on three transgenes, 3.5-Gal80ey, lama-Gal4 and UAS-FLP, to generate homozygous mutant somatic clones in the optic lobe, while leaving wild-type activity in the eye .To uncover the molecular pathways that control NE patterning in the OPC and IPC, we performed an ethyl methane sulfonate (EMS)-based forward genetic mosaic screen for chromosome 2R using the ELF system ( the eye . Optic l the eye . One mutPcl (3-78*38Pcl. Pcl and its vertebrate homolog PHF1 belong to the highly conserved Polycomb group (PcG) family of chromatin-modifying proteins. These form two functionally distinct Polycomb repressive complexes, PRC1 and PRC2 , confirming Pcl as the responsible gene. Finally, expression of a reporter transgene for the IPC-specific NE cell marker hairy to facilitate tracing of epithelial membranes, revealed that ELF system-induced clusters were continuous with the OPC neuroepithelium (eyeless (ey)-FLP -FLP as recom(ey)-FLP E. These clusters F at simi3-78*38Pcl mutant ectopic clusters with wild-type OPC NE cells in wandering third instar larvae, we observed a slight nonsignificant decrease in mitotic activity. Moreover, the average number of mitotic cells in ectopic clusters in early and wandering third instar larvae remained constant . Absence of staining with the Nb-specific markers Miranda (Mira) and Deadpan (Dpn) around 3-78*38Pcl mutant clusters indicated that these did not generate Nbs ectopically expressed Fas3 . Mutant clusters were discernible from the early third instar larval stage onwards G-I\u2032. Comrate Nbs L,M. Finarate Nbs we foundsed Fas3 N,O. ThisFig.\u00a0S2A-H), only Abd-B was ectopically expressed in 3-78*38Pcl mutant clones in the optic lobe , consistent with a requirement of the TGF\u03b2 family member Dpp for EMT in IPC subdomains -expressing Nbs within cell streams , consistent with the previously described so requirement in embryonic optic placode formation . Furthermore, the Six protein Optix which defines regional compartments in the OPC .In eye-antennal disc epithelia, the RDGN bestows retinal identity . Some meormation and perdormation and Twinormation , as wellormation and a reormation were not the OPC , was notclift1eya and 3so clones, as well as hth, eya and so single or double knockdown experiments. Knockdown was achieved using validated UAS-RNA interference (RNAi) transgenes in combination with MH766-Gal4esg and 3.5-Gal80ey transgenes . These experiments revealed that in the OPC NE domain dedicated to generating medulla neurons, so expression depends on eya signaling maintains the NE state of the OPC, and that downregulation, in part mediated by the proneural factor Lethal of scute , is required for the timely conversion of medial NE cells into medulla Nbs . Although the simultaneous knockdown of eya, hth and Optix affected all four genes (because so depends on eya), it did not result in ectopic Fas3 expression , remained unaffected , co-expression of eya and so reduced Fas3 levels in p-IPC NE cells and, strikingly, induced the direct conversion of p-IPC NE cells into Dpn- and Mira-positive Nbs . Although ectopic Hth did not affect Fas3 levels . The proneural gene l'sc, the Notch target gene E(spl)m\u03b3HLH and the EGF receptor target gene pointed (pnt) serve as proneural wave markers in the OPC and therefore could not be used as OPC-specific readouts. However, eya and so overexpression induced ectopic Hth . Consistent with a progression through the OPC-specific temporal series, more Hth- than Ey-positive cells were found in the proximity of transformed OPC-like NE cells . Conversely, the d-IPC specific factors Atonal (Ato) and Dac failed to be expressed . Expression and functional studies were conducted using combinations of the Gal4/UAS, the FLP/FRT system-based ELF containing 0.05\u2005M sodium phosphate buffer (pH 7.4), and washed in PBS containing 0.5% Triton X-100 (Sigma-Aldrich) OPC volumes of wild-type and IRUAS-hth-expressing animals, (2) the numbers of PH3-positive cells in wild-type OPC NE cells or Fas3-positive 3-78*38Pcl mutant cell clusters, (3) the number of Pcl-deficient migratory progenitors that prematurely differentiate into Nbs, and (4) the numbers of Hth- and Ey-positive cells in eya/so gain-of-function experiments are described in detail in supplementary Materials and Methods. Calculations of 95% confidence interval error bars and unpaired two-tailed Student's t-test P values were performed using Microsoft Excel software [Confidence.T and T.Test ]. Prism 6 GraphPad was used to perform Shapiro\u2013Wilk and D'Agostino\u2013Pearson omnibus normality tests and to present quantifications as scatter plots and bar graphs. P<0.05 was considered to be statistically significant; ****P<0.0001.Sample numbers for each experiment in this study are provided in"} +{"text": "Small non-coding RNAs serve as specificity factors that guide effector proteins to ribonucleic acid targets via base-pairing interactions, to achieve transcriptional or post-transcriptional regulation. Because of the small sequence complementarity required for microRNA-dependent post-transcriptional regulation, thousands of microRNA (miRNA) putative targets have been annotated in Drosophila. In Drosophila somatic ovarian cells, genomic parasites, such as transposable elements (TEs), are transcriptionally repressed by chromatin changes induced by Piwi-interacting RNAs (piRNAs) that prevent them from invading the germinal genome. Here we show, for the first time, that a functional miRNA pathway is required for the piRNA-mediated transcriptional silencing of TEs in this tissue. Global miRNA depletion, caused by tissue- and stage-specific knock down of drosha (involved in miRNA biogenesis), AGO1 or gawky (both responsible for miRNA activity), resulted in loss of TE-derived piRNAs and chromatin-mediated transcriptional de-silencing of TEs. This specific TE de-repression was also observed upon individual titration (by expression of the complementary miRNA sponge) of two miRNAs (miR-14 and miR-34) as well as in a miR-14 loss-of-function mutant background. Interestingly, the miRNA defects differentially affected TE- and 3' UTR-derived piRNAs. To our knowledge, this is the first indication of possible differences in the biogenesis or stability of TE- and 3' UTR-derived piRNAs. This work is one of the examples of detectable phenotypes caused by loss of individual miRNAs in Drosophila and the first genetic evidence that miRNAs have a role in the maintenance of genome stability via piRNA-mediated TE repression.RNA interference-related silencing mechanisms concern very diverse and distinct biological processes, from gene regulation . MiRNAs can reduce the stability or the activity of the many cellular messenger RNAs that contain miRNA complementary sequences. In animal gonads, the harmful expression and proliferation of genomic parasites, such as transposable elements, is prevented by a similar, sequence homology-based silencing mechanism that involves a different class of small RNAs, the Piwi-interacting RNAs (piRNAs). We report here that, in Drosophila somatic ovarian tissues, two miRNAs, miR-14 and miR-34, are required for the accumulation of piRNAs that prevent the expression of transposable elements and, probably, the subsequent invasion of the germinal genome. On the other hand, we found that other sources of piRNA production, such as the 3' end of genes, are miRNA-independent, suggesting the existence of variations in the piRNA biogenesis pathways depending on the piRNA genomic origin. Our results therefore highlight a novel miRNA function in the maintenance of genome stability through piRNA-mediated TE repression. Each RISC contains one of three types of small regulatory RNAs that have different roles and mechanisms of action. Specifically, more than 230 AGO1-associated microRNAs regulate gene expression, during development . Partreviews: ,6. On threviews: \u201311.Argonaute-mediated silencing can occur at the transcriptional or post-transcriptional level. Most Argonaute proteins, such as siRNA-loaded AGO2 or piRNADrosophila adult ovarian somatic support cells (follicle cells), piRNA-mediated TE transcriptional repression is exclusively achieved by the loading onto Piwi of primary piRNAs generated by unidirectional transcription of heterochromatic loci, called piRNA clusters, such as flamenco onli onli72] et al. Embo J. 30:3977) and are available via the NCBI Gene Expression Omnibus (accession no. GSM767598 and GSM767599 respectively). Sequencing data concerning the small RNA and RNA-IP libraries generated in this study are available via the NCBI Gene Expression Omnibus (GEO) (http://www.ncbi.nlm.nih.gov/geo/) under accession no. GSE60974 Schematic representation of the domain organization of Flag-HA-tagged wild type (WT) and trans-dominant negative (TN) Drosha constructs. In the TN-Drosha construct, each RNAseIII catalytic domain was inactivated by substituting an essential Asp residue with an Ala residue. dsRBD: double-stranded RNA binding domain. (B) Co-immunoprecipitation of endogenous Pasha (a Dosha-binding protein) and tj-driven Flag-HA-tagged WT- and TN-Drosha proteins. Ovaries containing only the tj-Gal4 driver were used as controls of the anti-Flag immunoprecipitation (middle). (C) tj-driven expression of TN-Drosha or of the two long Drosha inverted repeats (drosha-IR) in follicle cells resulted in stabilization of the three tested pri-miRNAs. Quantification was done relative to RpL32 and normalized to the respective controls (\u00d8>drosha-IR for tj-GAL4>drosha-IR and tj-GAL4>\u00d8 for both tj-GAL4>WT-Drosha and tj-GAL4>TN-Drosha) . (D) tj-driven expression of TN-Drosha in follicle cells correlates with the loss of the three tested miRNAs. The miRNA level was assayed using follicle cell-enriched ovarian extracts. The miRNA level was expressed relatively to a follicle cell-specific RNA (tj) and normalized to control (tj-GAL4>WT-Drosha) .(EPS)Click here for additional data file.S2 Figtj-GAL4>WT-Drosha* (left panel) and tj-GAL4>TN-Drosha* (right panel) mapper reads normalized to one million of genome-unique 42AB mappers. (B) WebLogo on mapper reads of both tj-GAL4>WT-Drosha* (left panel) and tj-GAL4>TN-Drosha* (right panel) libraries. The logo was build using WebLogo software (http://weblogo.berkeley.edu/). The height of each letter represents the relative proportion of each nucleotide at each position.(A) Barplots displaying the length distributions of (EPS)Click here for additional data file.S3 Figtj-GAL4>WT-Drosha (upper panels) and tj-GAL4>TN-Drosha (lower panels) libraries. Libraries were normalized to 1 million of genome-unique 42AB mappers, a germline-specific piRNA cluster. The y axis indicates the number of piRNAs collapsed to their 5\u2019 end. (A) Profiles of piRNAs mapping to the F-element , or to ZAM (middle) and Tabor (right) (two follicle cell-specific TEs). (B) Profiles of piRNAs originating from a germline-specific piRNA cluster (80 E-F), two follicle cell-specific piRNA clusters (flamenco and cluster #17). Only genome-unique piRNAs are mapped.(A-B) Normalized profiles of ovarian piRNAs (sense: up (red); antisense: down (blue)) mapping to TE consensus sequences and to piRNA clusters in (EPS)Click here for additional data file.S4 FigZAM, Tabor and Rpt5 (negative control) induced by the \u0394miR-14 null mutation. Quantification was done relative to RpL32 and normalized to the heterozygous siblings . Note that, since the heterozygous \u0394miR-14/Cy control might contain more TE copies than the mutant , TE de-repression could have been under-estimated.Fold changes in the steady-state RNA levels of (EPS)Click here for additional data file.S5 Figflamenco piRNA cluster .Fold changes in the steady-state RNA levels of five short (region 1 to region 5: about 100 nt-long) and three long (extended region 1 to extended region 3: 300\u2013400 nt-long) fragments from the (EPS)Click here for additional data file.S6 Figtj-driven expression of either WT- or TN-Drosha. Blue arrows point towards the \u03b2-Gal staining of \"polar-like\" follicle cells facing a mislocalized oocyte, identified by its darker vitellus.Shown is the \u03b2-Gal staining of ovaries subjected to constitutive (EPS)Click here for additional data file.S1 Table(XLS)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLS)Click here for additional data file.S4 TableDrosophila melanogaster sequenced genome. miRNA reads were separated from the other small regulatory RNAs read based on their identity with miRBase (http://www.mirbase.org/). Then, piRNAs and siRNAs were identified based on their size (21 nt for siRNAs and 23 to 29 nt for piRNAs). The 42AB reads correspond to 42AB-derived genome-unique piRNAs and the flamenco reads correspond to flamenco-derived genome-unique piRNAs. (B) Sequencing counts for all generated RNA-IP samples. \u201cMappers\u201d correspond to reads perfectly mapping to the Drosophila melanogaster sequenced genome. Pri-miRNA reads were annotated based on their identity with miRBase (http://www.mirbase.org/). Flamenco mapped reads correspond to reads that can derive from flamenco whereas the flamenco uniquely mapped reads correspond to reads exclusively coming from flamenco.(A) Sequencing counts for all generated small RNA libraries. \u201cMappers\u201d correspond to reads perfectly mapping to the (XLS)Click here for additional data file.S5 Tabletj-GAL4> \u00d8, tj-GAL4>WT-Drosha and tj-GAL4>TN-Drosha)) and in the three IP experiments were normalized to one million genome-unique reads. ND indicates that these six miRNAs have not been tested in the miR-SP genetic screen.The number of RNA reads sequenced in the three inputs (input ((XLS)Click here for additional data file.S6 Table(XLS)Click here for additional data file.S7 TableThe number of reads in each input was normalized to one million genome-unique reads.(XLS)Click here for additional data file.S1 Text(DOC)Click here for additional data file."} +{"text": "However, sno-lncRNAs from other regions of the human genome or from other genomes have not yet been documented.Intron-derived long noncoding RNAs with snoRNA ends (sno-lncRNAs expressed in all three species. In total, using available data from a limited set of cell lines, 19 sno-lncRNAs have been identified with tissue- and species-specific expression patterns. Although primary sequence analysis revealed that snoRNAs themselves are conserved from human to mouse, sno-lncRNAs are not. PWS region sno-lncRNAs are highly expressed in human and rhesus monkey, but are undetectable in mouse. Importantly, the absence of PWS region sno-lncRNAs in mouse suggested a possible reason why current mouse models fail to fully recapitulate pathological features of human PWS. In addition, a RPL13A region sno-lncRNA was specifically revealed in mouse embryonic stem cells, and its snoRNA ends were reported to influence lipid metabolism. Interestingly, the RPL13A region sno-lncRNA is barely detectable in human. We further demonstrated that the formation of sno-lncRNAs is often associated with alternative splicing of exons within their parent genes, and species-specific alternative splicing leads to unique expression pattern of sno-lncRNAs in different animals.By exploring non-polyadenylated transcriptomes from human, rhesus and mouse, we have systematically annotated sno-lncRNAs is species-specific and that their processing is closely linked to alternative splicing of their parent genes. This study thus further demonstrates a complex regulatory network of coding and noncoding parts of the mammalian genome.Comparative transcriptomes of non-polyadenylated RNAs among human, rhesus and mouse revealed that the expression of Although only about 2% of the human genome encodes protein sequences ,2, recenX. tropicalis [sno-lncRNAs) [In addition to intergenic regions, introns account for over 20% of noncoding sequences in the human genome and provide yet another source to generate lncRNAs. By removal of redundant rRNAs and poly(A)+\u2009RNAs, a relatively pure population of non-polyadenylated and non-ribosomal (poly(A)-/ribo-) RNAs was obtained and subjected to high-throughput deep sequencing . This tyopicalis . What melncRNAs) . This fisno-lncRNAs derived from introns of the Prader-Willi syndrome (PWS) region (15q11-q13) were highly expressed in human embryonic stem cells and strongly associated with Fox family splicing regulators to alter patterns of splicing [sno-lncRNAs and their association with Fox family splicing regulators offers a functional connection of sno-lncRNAs in the molecular pathogenesis of PWS [sno-lncRNAs may exist in the genome. Given that the vast majority of snoRNAs are encoded in introns of protein-coding genes [sno-lncRNAs in a genomewide manner. Moreover, signals of poly(A)-\u2009transcripts from intronic regions have been widely detected in a variety of cultured cells [sno-lncRNAs from different cell lines.Strikingly, five ) region q11-q13 wsplicing ,19. Imposplicing -22. Alth) region q11-q13 wng genes , it was ed cells , which hsno-lncRNAs genome-widely from poly(A)-/ribo-\u2009transcriptomes of human, rhesus and mouse. In total, 19 sno-lncRNAs have been identified with tissue- and species-specific expression patterns from available species/cell lines. PWS region sno-lncRNAs are highly expressed in human, somewhat in rhesus, and none in mouse. In contrast, a RPL13A region sno-lncRNA is highly expressed in mouse, but almost absent in human. We further demonstrated that the formation of sno-lncRNAs often requires alternative splicing, indicating a complex regulatory network of coding and noncoding parts of the genome.Here, we applied computational pipelines to identify sno-lncRNAs depends on the snoRNA machinery at both ends [sno-lncRNAs are highly expressed in introns of PWS imprinted region of chr15 and are strongly associated with Fox family splicing regulators to alter patterns of splicing [sno-lncRNAs from other regions of the human genome or other species were less obvious. Since the vast majority of snoRNA genes are located in introns of protein-coding genes . In total, 400 annotated snoRNAs were downloaded from snoRNABase [http://www.ncbi.nlm.nih.gov/refseq/, downloaded on 2013/3/4), respectively. Since snoRNAs in rhesus are not well annotated, we transposed human and mouse snoRNA annotations over to the rhesus genome to generate 375 putative rhesus snoRNAs (Methods). We then analyzed the genomic locations of these snoRNAs in different species.The processing of intron-derived oth ends . Five suoRNABase for humasno-lncRNAs could be detected, including six reported sno-lncRNAs[C17orf76-AS1 region -/ribo-\u2009RNA-seq datasets. For example, in human pluripotent H9 cells, only seven o-lncRNAs and a nesno-lncRNA candidates, we developed a custom computational pipeline to predict sno-lncRNAs by integrating snoRNA annotations with poly(A)-/ribo-\u2009RNA-seq datasets from human [sno-lncRNAs were identified from different species and/or different cell lines . We exa lncRNAs and pred lncRNAs +\u2009RNA-seq dataset (GSE53942). Pair-wise sequence alignments between human is repeated at least eight times in the rhesus PWS region; however it is unclear whether this repetitive region occurs only in rhesus or has been lost in humans during evolution.We next inspected PWS region s Figure\u00a0A, and putaset GSE3942. Paitaset GSE3942. PaiSNURF-SNRPN exons revealed that two PWS region sno-lncRNAs in rhesus are similar to human PWS region sno-lncRNA3 and sno-lncRNA4, respectively. Although predicted rhesus SNORD116 snoRNAs are scattered among individual introns +\u2009RNA-seq revealed a variety of alternatively spliced SNURF-SNRPN transcripts in rhesus, thus leading to the formation of sno-lncRNAs in rhesus SNURF-SNRPN region .Sequence alignment of SNORD116 snoRNAs and their parent s Figure\u00a0A, de novsno-lncRNAs could function as molecular sponges by associating with Fox family splicing regulators and altering patterns of splicing [sno-lncRNAs with human in the genomic context, we reasoned that they might function similarly as well. We thus scanned the rhesus sno-lncRNA sequence for Fox binding motifs, and identified an enrichment of Fox binding sites gene +\u2009RNA-seq dataset, and successfully identified the alternative splicing event that results in the location of SNORD33 and SNORD34 into one intron -/ribo-) from human, rhesus and mouse, and systematically annotated https://www-snorna.biotoul.fr/) were downloaded from UCSC Genome Bioinformatics database . 132 mouse snoRNA annotations were downloaded from RefSeq database . 375 putative rhesus snoRNAs were transposed from human and mouse snoRNA annotations using liftOver (http://genome.ucsc.edu/cgi-bin/hgLiftOver) with minMatch\u2009=\u20090.95 and combined together to be used as rhesus snoRNA annotations. All these snoRNA annotations were overlapped respectively with relevant gene annotations according to their species-derivation to find snoRNA pairs (at least two snoRNAs in one intron) in introns, as indicated in Figure\u00a0sno-lncRNAs.Annotated human snoRNAs derived from snoRNABase . Cufflinks v2.0.2 [de novo RNA transcripts. All mapping results were normalized and uploaded to the UCSC Genome Browser (http://genome.ucsc.edu/) for visualization.The poly(A)+\u2009or poly(A)-/ribo-\u2009RNA-seq reads were uniquely aligned to relevant genomes using TopHat 2.0.8 , as indicated in Figure\u00a0et al., in preparation) from Bowtie mapped poly(A)-/ribo-\u2009RNA-seq reads. A putative sno-lncRNA was selected with 1) both snoRNA pairs are expressed with RPKM\u2009\u2265\u20091; 2) at least 80% of the internal region between snoRNA pairs have poly(A)-/ribo-\u2009RNA-seq signals by sliding window examination , ENCODE cell lines (GEO: GSE26284). RNA-seq files for rhesus ESCs, mouse ESCs and mouse hippocampus can be accessed from the NCBI Sequence Read Archive by Gene Expression Ominbus accession number (GEO:GSE53942).To systematically identify http://genome.lbl.gov/vista/) was employed to inspect the conservation landscape for a given region from different genomes, including human (Feb. 2009), Cow (Oct. 2011), Mouse (Dec. 2011 or Jul. 2007), Callithrix jacchus v.2.0.2 (Jun. 2007), Rhesus (Jan. 2006), Pongo pygmaeus abelii v.2.0.2 (Jul. 2007), Gorilla (Dec. 2009), Chimp (Mar. 2006), Rat (Nov. 2004), Dog (May 2005) and Horse (Jan. 2007).VISTA Browser (http://SNURF-SNRPN exons were defined according to rhesus poly(A)+\u2009RNA-seq mapping signals. Sequences of human . Pair-wise sequence alignments were carried out using T-Coffee [Putative rhesus snoRNA116s Figure\u00a0B were man Figure\u00a0A and rhen Figure\u00a0B SNURF-ST-Coffee .sno-lncRNAs and rhesus putative PWS sno-lncRNAs were extracted from UCSC Genome Bioinformatics database (http://genome.ucsc.edu/). All these sequences were scanned for Fox hexanucleotide motifs including UGCAUG, GCAUGU, GUGAUG, UGGUGA and GGUGGU [Sequences of human PWS d GGUGGU .http://hgdownload.cse.ucsc.edu/goldenpath/hg19/database/phastCons46wayPrimates.txt.gz, updated on 2009/12/6) were downloaded from UCSC and corresponding PhastCons scores for lncRNAs [sno-lncRNAs or with nucleofection (Lonza) according to the manufacturer\u2019s instructions. Rhesus rhesus RNAs were extracted from ESC line IVF3.2 . Mouse Rsno-lncRNA2 [sno-lncRNA2-5A . DIG-labeledRNA Molecular Wight Marker III is from Roche.Cultured cell lines or cells with different treatments were harvested in Trizol (Invitrogen) and RNAs were extracted according to the manufacturer\u2019s instruction, followed by DNase I treatment at 37\u00b0C for 30 mins . Poly(A)+\u2009and poly(A)-/ribo-\u2009RNA transcripts were fractionated and sequenced as previously described . Raw seqThe authors declare that they have no competing interests.LY and LLC designed the project. XOZ and LY performed bioinformatics analyses. QFY, HBW, YZ, TC, PZ and XL performed experiments. LY, LLC and XOZ analyzed the data. LY and LLC wrote the paper with inputs from other authors. All authors read and approved the final manuscript.Identification and validation of two novel human sno-lncRNAs. (A) Expression patterns of predicted sno-lncRNA in human cell lines. Normalized read densities of poly(A)-/ribo-\u2009RNA-seq (red) and poly(A)+\u2009RNA-seq (black) were indicated in H9 and HeLa, respectively. Red bar, NB probe for (B). (B) Northern blot validation of this novel sno-lncRNA (~598 nt) in H9, PA-1 and HeLa-J cell lines. (C) Expression patterns of predicted sno-lncRNA in human cell lines. Left, normalized read densities of poly(A)-/ribo-\u2009RNA-seq (red) and poly(A)+\u2009RNA-seq (black) were indicated in H9 and HeLa, respectively. Red bar, NB probe for (B). (D) NB validation of this novel sno-lncRNA (~585 nt) in H9, PA-1 and HeLa-J cell lines.Click here for fileNorthern blots of sno-lncRNAs with native agarose gel. (A) and (B) Northern bolts show that sno-lncRNAs can be recapitulated after replacing the snoRNA end from C/D box snoRNA to H/ACA box snoRNA (A) or vice versa (B). Top, a schematic drawing of wild-type sno-lncRNAs (sno-lncRNA2 and sno-lnc5AC) or modified sno-lncRNAs (sno-lncRNA2-5A and sno-lnc5C-14) in the expression vector. Black/grey boxes, exons; Black bars, NB probes; Blue circles, C/D snoRNAs; Yellow circles, H/ACA snoRNAs; Bottom, Northern blot validation. NT, no transfection; EV, empty vector. RNA marker III was used to indicate RNA sizes. Denatured RNAs were separated on 1% agarose gel. Note that similar RNA separations were obtained by both denatured PAGE gels exhibit a remarkably higher conservation across species than SNURF-SNRPN exons and introns. Y-axis, species selected for comparing (left panel) and conservation levels (right panel); Red bars, human PWS region sno-lncRNAs; Blue circles, human PWS region snoRNAs (SNORD116 cluster); Black bars, exons of human PWS region sno-lncRNA host gene (SNURF-SNRPN).Click here for fileExpression of PWS region in rhesus. Normalized read densities of poly(A)-/ribo-\u2009RNA-seq (red) and poly(A)+\u2009RNA-seq (black) in rhesus ESCs showed two highly expressed sno-lncRNAs in PWS region. Note that there is no RefGene annotation in rhesus, instead, homologues genes from other species are shown.Click here for fileExpression of PWS region in mouse. Normalized read densities of poly(A)-/ribo-\u2009RNA-seq (red) and poly(A)+\u2009RNA-seq (black) of PWS region in mouse ESC R1 and mouse hippocampus showed undetected expression of PWS region sno-lncRNAs. Note that mouse SNORD116 snoRNAs are over 50 kb away from mouse SNURF-SNRPN. These SNORD116 snoRNAs and their adjacent spliced ESTs are not expressed in mESCs, but are expressed in mouse hippocampus.Click here for filePair-wise sequence alignments of SNURF-SNRPN exons between human and rhesus .Click here for fileNorthern blot of mouse-specific sno-lncRNA in RPL13A region. Northern blot validation of mouse specific sno-lncRNA from multiple mouse cell lines. Total RNAs from ESC R1, NIH 3T3, VD\u03b2 and MEF were denatured and separated on 8% denatured PAGE gel. After separation, the gel was stained with ethidium bromide for rRNA/tRNA visualization (A), and transferred to membrane for Northern blot with probe for SNORD33 and conservation levels (right panel); Red bars, a non-human sno-lncRNA; Blue circles, mouse snoRNAs ; Black bars, exons of the non-human sno-lncRNA host gene (RPL13A).Click here for fileTransfection of species-specific sno-lncRNA into cell lines derived from different species. (A) Transfection of human sno-lncRNA ito mouse NIH 3T3 cell line generates the human sno-lncRNA as revealed by NB. NT, no transfection; EV, empty vector. (B) Transfection of mouse sno-lncRNA to human HeLa-J cell line generates the mouse sno-lncRNA as revealed by NB. NT, no transfection; EV, empty vector.Click here for fileSpecies-specific RPL13A region sno-lncRNA is derived from species-specific alternative spliced rpl13a transcripts in rhesus.De novo transcript assembly revealed previously uncharacterized alternative spliced rpl13a transcripts (rhesus_pAplus_cufflinks). One new rhesus rpl13a isoform (indicated by arrow) was identified to splice out a large intron containing SNORD33 and SNORD34. Y-axis, normalized read densities of poly(A)-/ribo-\u2009RNA-seq (red) and poly(A)+\u2009RNA-seq (black) of rhesus ESC transcriptomes.Click here for file"} +{"text": "Patients with advanced NSCLC are treated with first-line PT-DC, which is associated with a median OS of 8\u201310 months and 1-year and 2-year survival rates of 30\u201340% and 10\u201315%, respectively. Nivolumab (a fully human IgG4 anti-programmed death-1 immune checkpoint inhibitor antibody) alone and in combination with ipilimumab (a fully human IgG4 cytotoxic T-lymphocyte antigen-4 immune checkpoint inhibitor antibody) has demonstrated encouraging clinical benefit across multiple tumor types. Two randomized Phase III trials demonstrated superior survival with nivolumab vs docetaxel in previously-treated patients with advanced squamous (SQ) (CheckMate 017) and non-squamous (non-SQ) NSCLC (CheckMate 057). Preliminary results of a Phase I study (CheckMate 012) of nivolumab with or without ipilimumab demonstrate acceptable safety and encouraging activity in first-line metastatic NSCLC across histologies. This Phase III trial (CheckMate 227) evaluates nivolumab monotherapy and nivolumab plus ipilimumab combination regimens vs PT-DC in patients with chemotherapy-na\u00efve stage IV or recurrent SQ and non-SQ NSCLC.Adult patients with stage IV or recurrent NSCLC, ECOG performance status \u22641, no prior systemic anti-cancer therapy, and measureable disease per RECIST version 1.1 are eligible. Tissue will be evaluated for programmed death-ligand 1 (PD-L1) expression during screening. Patients who have untreated CNS metastases are ineligible. Patients will be randomized to nivolumab monotherapy, nivolumab plus ipilimumab combination regimens, or PT-DC. PT-DC will be administered according to histology (gemcitabine with cisplatin or carboplatin for SQ and pemetrexed with cisplatin or carboplatin for non-SQ). Patients will receive treatment until progression or unacceptable toxicity. The co-primary endpoints are overall survival and progression-free survival in patients receiving nivolumab monotherapy or nivolumab plus ipilimumab combination regimens vs patients receiving PT-DC. Secondary endpoints include objective response rate in nivolumab monotherapy or nivolumab plus ipilimumab combination regimens vs PT-DC and disease related symptom improvement measured by the Lung Cancer Symptom Scale in all patients.ClinicalTrials.gov identifier NCT02477826."} +{"text": "This issue is especially relevant to methicillin-resistant S. aureus (MRSA) strains in the context of invasive endovascular infections. In the current study, we used three well-characterized and clinically-derived DAP-susceptible (DAP-S) vs. resistant (DAP-R) MRSA strain-pairs to elucidate potential genotypic mechanisms of the DAP-R phenotype. In comparison to the DAP-S parental strains, DAP-R isolates demonstrated (i) altered expression of two key determinants of net positive surface charge, either during exponential or stationary growth phases , (ii) a significant increase in the D-alanylated wall teichoic acid (WTA) content in DAP-R strains, reflecting DltA gain-in-function; (iii) heightened elaboration of lysinylated-phosphatidylglyderol (L-PG) in DAP-R strains, reflecting MprF gain-in-function; (iv) increased cell membrane (CM) fluidity, and (v) significantly reduced susceptibility to prototypic cationic host defense peptides of platelet and leukocyte origins. In the tested DAP-R strains, genes conferring positive surface charge were dysregulated, and their functionality altered. However, there were no correlations between relative surface positive charge or cell wall thickness and the observed DAP-R phenotype. Thus, charge repulsion mechanisms via altered surface charge may not be sufficient to explain the DAP-R outcome. Instead, changes in the compositional or biophysical order of the DAP CM target of such DAP-R strains may be essential to this phenotype. Taken together, DAP-R in S. aureus appears to involve multi-factorial and strain-specific adaptive mechanisms.Development of DAP) is a calcium-dependent lipopeptide antibiotic with potent bactericidal effects against most Gram-positive pathogens Staphylococcus aureus bacteremia and right-sided endocarditis in 2006, DAP has become a key antibiotic for invasive staphylococcal infections. This is especially relevant to methicillin-resistant S. aureus (MRSA) infections in the era of rising vancomycin MICs associated with clinical treatment failures S. aureus (VISA)-related infections S. aureus and enterococcal DAP-resistant (DAP-R) strains emerging during DAP treatment failures S. aureus strains differ substantially from those defined among DAP-R enterococci CM), key impacts upon cell wall (CW) turnover processes seem to play a cardinal role in this regard i) increased positive surface charge (\u2018charge-repulsion hypothesis\u2019) ii) altered CM fatty acid composition resulting in altered CM fluidity iii) increased CM carotenoid pigment content yielding very rigid CMs iv) a combination of factors; for example, enhanced CM content of positively-charged phospholipids, as well as increased D-alanylation of CW teichoic acid, resulting in reduced affinity of DAP to the CM target v) augmented synthesis of CW teichoic acid, creating a thickened CW phenotype and a putative mechanical barrier to DAP penetration to reach its CM target Daptomycin and the dlt operon have received particular attention S. aureus strains, since a broad range of genotypic correlates with the DAP-R phenotype have emerged, including: i) acquisition of gain-in-function single nucleotide polymorphisms (SNPs) in the mprF ORF ii) over-expression of dlt genes iii) dysregulation of both mprF and dlt expression profiles dltA overexpression as potentially causal in the DAP-R phenotype. This intriguing investigation was, however, somewhat limited, in that within the DAP-S and DAP-R strain-sets: i) there was no growth phase-dependent gene expression profiling performed; ii) there were no phenotypic correlates of dlt and mprF over-expression studied, especially surface charge measurements; iii) specific phenotypic readouts of dlt and mprF over-expression were not quantified other parameters of CM physiology previously associated with DAP-R were not queried v) assessment of DAP-R isolates for in vitro \u2018cross-resistance\u2019 with several prototypical host defense cationic peptides (HDPs) involved in staphylococcal pathogenesis was not performed S. aureus strains The above phenotypic correlates of DAP-R in agr typing, multi-locus sequence typing and staphylococcal casette chromosome (SCCmec) typing. The three strain-pairs were previously characterized for mutations in the mprF genes. Two of the three DAP-R variants within each strain-pair exhibited a non-synonomous SNP in the mprF ORF amount of bound cytochrome c. At least three independent runs were performed on separate days.The cytochrome S. aureus CW and WTA were specifically isolated as described in detail before 2HPO4) containing 0.25% (wt/vol) glucose, washed twice in sodium acetate buffer and disrupted in the same buffer with glass beads for 1 h on ice in a cell disruptor (Euler). Protein-free CW was isolated, and WTA was released by treatment with 5% trichloroacetic acid in sodium acetate buffer for 4 h at 60\u00b0C. The CWs were removed by centrifugation and WTA was quantified by determining its inorganic phosphate (Pi) content as described before The D-alanylation of the WTA polymers was assayed and quantified as described before The CW thickness of study strains were measured by transmission electron microscopy TEM; . The meaBacillus subtilis bioassay Thrombin-induced platelet microbicidal proteins (tPMPs) were obtained from thrombin-stimulated rabbit platelets as previously described 3 stationary phase CFU was employed. The peptide concentrations used in the 2 h killing assays were: 1.5 or 2.0 \u00b5g/ml bioactivity equivalent for tPMPs; 5 or 10 for hNP-1; and 0.5 and 1 \u00b5g/ml for RP-1 and LL-37. After extensive pilot studies, these peptide concentrations were selected based on: (i) sub-lethality, with <50% reductions in counts of the parental DAP-susceptible (DAP-S) strain; and (ii) encompassing peptide concentrations used in prior investigations of HDP:S. aureus interactions bona fide \u201cresistance\u201d breakpoint for HDPs, the mean percent survival (\u00b1 SD) was statistically evaluated for potential correlates of HDP and DAP susceptibility profiles. Data included a minimum of three experiments performed on separate days.For tPMPs and RP-1, a microtiter bactericidal assay was carried out in minimal liquid nutrient medium in appropriate buffers mprF polymorphisms and CM features, PLs were extracted from study strains under specific test conditions as described S. aureus were separated by two-dimensional thin-layer chromatography (2-D TLC) using Silica 60 F254 HPTLC plates (Merck). Fluorescamine labeling , combined with ninhydrin staining localization, was used within the 2-D TLC plate assay to assess the translocation of L-PG between the inner-to-outer CM bilayer 660.To investigate potential correlates between Given the impact of fatty acid composition on CM adaptability to stress, the comparative fatty acid profiles of the DAP-S/DAP-R strain-pairs were determined. Approximately 20 mg of bacterial cells were harvested from late log phase growth preparations, and then saponified, methylated, and fatty acid esters extracted into hexane as described previously CM fluidity was determined by fluorescence polarization spectrofluorometry as detailed previously S. aureus using the method of Dyer and Iandolo mprF ORF was performed as we have previously described, using the primers, mprF-F-bam (5\u2032-CCCGGATCCAATTAGAATTGATGTGAAAAAATG-3\u2032) and mprF-R-sph (5\u2032-CCCGCATGCAGCGCTTCAGGCATAACTGT-3\u2032) Genomic DNA was isolated from dltA and dltB ORFs were performed as we have described previously with primer pairs dltA-ORF-F (5\u2032-CAGTGGCGACACACACAATA-3\u2032) and dltA-ORF-R (5\u2032-GACTGGTAATAATGCAATTAAAGCAA-3\u2032), dltB-ORF-F (5\u2032-TGGAACAATTGCCATTGACTT-3\u2032) and dltB-ORF-R (5\u2032-TCCAACTGTTTGGAAAGA ATCA-3\u2032), respectively mprF and dlt genes was kindly performed at City of Hope, Duarte, CA.PCR amplification of the S. aureus strains were used to inoculate NZY broth to an optical density at 600 nm (OD600) of 0.1. Cells were harvested during both exponential growth and stationary phase (12 h). Total RNA was isolated from the cell pellets by using the RNeasy kit and the FASTPREP FP120 instrument , according to the manufacturer's recommended protocols.For RNA isolation, fresh overnight cultures of mprF, dltA, and gyrB genes were detected using specific primers and computational methods as described before Quantitative real time PCR assay was carried out as detailed previously As anticipated, the peptides exerted distinct efficacies against the study strains Table 2.Outcomes of relative positive surface charge comparisons between DAP-S and DAP-R isolates varied substantially between strain-pairs. For example, for strain-pair 1, the DAP-R isolate (1C) exhibited a significantly more relative positive surface charge than its parental DAP-S isolate (1A). In contrast, the 3A/3B strain pair showed the exact opposite surface charge relationships; the DAP-S strain 3A bound more cytochrome C than did strain 3C. The 2A/2C strain set had virtually identical surface charge readouts.P<0.05). In contrast, in the strain-pairs 1A/1C and 3A/3B, no difference in WTA amount in the CWs of the respective DAP-R strains was detected.A significant increase in the amount of WTA in the CW of the DAP-S/DAP-R strain-pair 2A/2C was detected, with the DAP-R strain producing significantly more WTA than the respective DAP-S strain of the DAP-R strains that did not exhibit an increase in WTA content (1C and 3B) was also significantly higher than that observed in their respective DAP-S parental strains (1A and 3A). These latter data speak to a clear-cut gain-in-function of the DltA gene Table S1). In two of the three strain-pairs (1A\u20131C and 2A\u20132C), fatty acid composition exhibited a rather similar overall profile, including proportionality of iso- and ante-iso branched chain fatty acids, unsaturated fatty acids (UFAs), and acyl chain-length profiles. Thus, the increases in CM fluidity observed in the above two DAP-R S. aureus strains could not be directly correlated to changes in the proportion of key fatty acids known to impact CM fluidity; i.e, iso- vs anteiso branch chain fatty acids, and saturated vs unsaturated fatty acids). In contrast, in one DAP-R strain (3B), fatty acid analyses demonstrated a significant increase in the proportion of anteiso-branch chain species (vs iso-branch chain species) and decrease in saturated fatty acids as compared to its respective parental DAP-S strain (3A) (P<0.05). In this strain-pair, the total proportions of unsaturated fatty acids were not altered, although an additional fatty acid species was present in DAP-R strain 3B. This increase in anteiso-branch chain species appeared to correspond with a significant reduction in the major iso-branched chain and saturated fatty acid (SFAs) species, respectively, in this DAP-R strain (P<0.05). These proportionality perturbations in this latter DAP-R strain provided a potential explanation for the observed increment in CM fluidity in this latter strain (see below).Fatty acid compositional data for the three strain-pairs are shown in mprF regulates the lysinylation of PG to yield L-PG, as well as its subsequent outer CM translocation, we compared these parameters in the three DAP-S/DAP-R strain pairs. As noted in Since The polarization indices for all three DAP-R isolates were substantially lower than that of their respective DAP-S parental strains, indicating that the CMs of these DAP-R isolates were more fluid than their DAP-S counterparts. These differences reached statistical significance for strain-pairs 2A\u20132C and 3A\u20133B.mprF during exponential vs stationary growth phases in these strain-pairs. During exponential growth, the DAP-R strain 1C showed \u223c4-fold increase in mprF expression vs its DAP-S parental strain. Expression profiles for the other two strain pairs did not demonstrate such increases among the DAP-R isolates. In contrast, at stationary growth in the dltA gene as compared to its DAP-S parental strain. This mutation yields a phenylalanine-to-leucine (F255L) amino acid substitution (data not shown). Sequence analyses of dltB in the three pairs revealed no differences between the DAP-S and DAP-R pairs.Although a few nucleotide sequence polymorphisms were observed within the dlt operon in the DAP-R phenotype. These investigators documented the exponential growth-phase enhancement of dlt expression in three DAP-R MRSA strains which emerged during DAP therapy, in the presence or absence of mprF \u2018hot spot\u2019 SNPs. These authors proposed that dlt over-expression is a common pathway of DAP-R. Of note, their data regarding the potential role of dlt overexpression and DAP-R mirrors recent findings from our own laboratories dlt over-expression could be causal in DAP-R.Recently, Cafiso et al First, as has been seen previously in selected DAP-R MSSA and MRSA strains dltA expression is frequently dysregulated, either by enhanced exponential phase or increased stationary phase transcription profiles. Of interest, dltA expression is generally quite minimal at stationary phase in DAP-S S. aureus strains dltA transcription, only one of the three DAP-R isolates exhibited a non-synonymous SNP in dltA. Phenotypically, all three DAP-R strains demonstrated a significant increase in their WTA D-alanylation content, reflecting DltA gains in funtionality. Of interest, concomitant increases in CW thickness were not observed in any of the three DAP-R strains (data not shown).The current study, utilizing these same DAP-S/DAP-R strain-pairs sought to extend and adjudicate the studies of Cafiso et al Second, mprF expression profiles were remarkably similar to those of dltA above in all three strain-pairs, whether or not \u2018hot spot\u2019 non-synonymous mprF SNPs were identified in the DAP-R isolates. The observation that mprF transcriptional dysregulation occurred despite the absence of such SNPs supports the notion that regulatory genes outside of mprF can affect its expression profile. As expected, phenotypically, in the two DAP-R strains with mprF SNPs, enhanced amounts of L-PG synthesis were detected as compared to their respective DAP-S parental isolates. In the remaining DAP-R strain, despite overt mprF dysregulation, L-PG synthesis was not increased. This finding is reminiscent of data in selected MSSA DAP-R isolates Third, one prevailing concept that has been put forward to explain the DAP-R phenotype in S. aureus has been perturbations in relative cell surface positive charge, creating a \u201ccharge repulsion\u201d scenario against calcium complexed-DAP. However, despite the increases in dltA transcription, D-alanylation of WTA and L-PG synthesis among the three DAP-R strains, this did not translate into consistent enhancement of surface positive charge in these isolates. These data refute charge repulsion as a principal or comprehensive explanation for the DAP-R phenotype in these strain-pairs.Fourth, all three DAP-R strains demonstrated substantial increases in their CM fluidity properties as compared to their respective DAP-S parental strains. Altered CM fluidity has been closely linked to resistance to killing by cationic antimicrobial peptides in general S. aureus strains, making them more or less susceptible to cognate host defense peptides (HDPs) with specific structure-activity signatures. Thus, CMs that exhibit high degrees of rigidity or fluidity are relatively resistant to interactions with specific HDPs that likely rely most upon more intermediate degrees of CM order in vitro \u201ccross-resistance\u201d of the DAP-R strains to cationic HDPs which are structurally distinct from DAP, including prototypical peptides from mammalian platelets and leukocytes. Such cross-resistance between DAP and HDPs has been well-chronicled in recent studies amongst both staphylococci and enterococci Bacillus subtilis in response to cold shock S. aureus is highly likely to be multi-factorial and strain-specific. Although over-expression and dysregulation of dltA transcription may well be identifiable in selected DAP-R S. aureus strains and correlated with increased WTA D-alanylation, its subsequent potential phenotypic consequences were not sufficient to entirely explain the DAP-R phenotype in these strains.Thus, in summary, the ultimate mechanism(s) of DAP-R in Table S1Distinct fatty acid (FA) compositions of strain pairs.(DOC)Click here for additional data file."} +{"text": "Alterations in cerebral resting-state functional connectivity (RSFC) have been reported outside of migraine attacks. To date, no studies studied possible changes in RSFC during migraine attacks.To investigate resting-state functional connectivity in salience (SN), sensorimotor (SMN) and default mode networks (DMN) during the early phase of pituitary adenylate cyclase-activating polypeptide-38 (PACAP38)-induced migraine attacks.In a double-blind randomized study, 24 female migraine patients without aura received intravenous PACAP38 or vasoactive intestinal polypeptide (VIP) for 20 min. Both peptides are closely related and cause vasodilatation, but only PACAP38 induces migraine attacks. VIP was therefore used as an active placebo. Functional MRI was recorded before and during PACAP38-induced attacks (n=16) and before and after VIP infusion (n=15). Data were analyzed by SPM8 and the REST toolbox for Matlab in a seed-based fashion.During PACAP38-induced attacks, we found increased connectivity of the bilateral opercular part of the inferior frontal gyrus (Brodmann area 44) in the SN. In SMN, there was increased connectivity in the right premotor cortex and decreased activity in the left visual cortex. Several areas showed increased and decreased connectivity in DMN. We found no resting-state network changes after VIP.The early phases of PACAP38-induced migraine attacks are associated with altered connectivity of several large-scale functional networks of the brain."} +{"text": "B-Raf inhibitors have been used for the treatment of some B-Raf\u2013mutated cancers. They effectively inhibit B-Raf/MEK/ERK signaling in cancers harboring mutant B-Raf, but paradoxically activates MEK/ERK in Ras-mutated cancers. Death receptor 5 (DR5), a cell surface pro-apoptotic protein, triggers apoptosis upon ligation with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or aggregation. This study focused on determining the effects of B-Raf inhibition on DR5 expression and DR5 activation-induced apoptosis in Ras-mutant cancer cells. Using chemical and genetic approaches, we have demonstrated that the B-Raf inhibitor PLX4032 induces DR5 upregulation exclusively in Ras-mutant cancer cells; this effect is dependent on Ras/c-Raf/MEK/ERK signaling activation. PLX4032 induces DR5 expression at transcriptional levels, largely due to enhancing CHOP/Elk1-mediated DR5 transcription. Pre-exposure of Ras-mutated cancer cells to PLX4032 sensitizes them to TRAIL-induced apoptosis; this is also a c-Raf/MEK/ERK-dependent event. Collectively, our findings highlight a previously undiscovered effect of B-Raf inhibition on the induction of DR5 expression and the enhancement of DR5 activation-induced apoptosis in Ras-mutant cancer cells and hence may suggest a novel therapeutic strategy against Ras-mutated cancer cells by driving their death due to DR5-dependent apoptosis through B-Raf inhibition. B-Raf mutation, an oncogenic driver mutation, frequently occurs in certain types of cancers such as melanoma (50\u201380% of cases), papillary thyroid carcinoma ~45%), hepatocellular carcinoma (~40%) and colorectal cancer ~10%)0%12. The235%, hepat266791017Death receptor 5 is a cell surface receptor for the death ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Human cells have another homologue of TRAIL receptor known as death receptor 4 , whereas in mouse cells only one TRAIL receptor is present. Several types of immune cells including natural killer (NK) cells, T cells, natural killer T (NKT) cells, dendritic cells and macrophages expresses endogenous TRAIL13151417Our previous studies have demonstrated that Ras/Raf/MEK/ERK signaling increases CHOP- and Elk-dependent DR5 expression19We first compared the effects of PLX4032 with AZD6244 (a MEK inhibitor) on ERK activation and DR5 expression in various cancer cell lines with B-Raf mutation, Ras (K-Ras and N-Ras) mutation or wild-type (WT) B-Raf and Ras. As previously reportedTo determine whether PLX4032-induced DR5 expression is secondary to MEK/ERK activation, we further compared the effects of PLX4032 and AZD6244 in a detailed way using multiple concentrations in Ras-mutant A549 cells and confirmed their opposing effects on ERK phosphorylation and DR5 expression; i.e., PLX4032 increased ERK phosphorylation and DR5 expression, whereas AZD6244 suppressed ERK phosphorylation and DR5 expression . In the 7To determine whether the presence of mutant Ras is required for PLX4032 to activate ERK and increase DR5 expression, we genetically knocked down Ras expression in two Ras-mutant cell lines and then examined its impact on PLX4032-induced ERK activation and DR5 expression. As presented in Moreover, we found that enforced expression of mutant K-Ras (12V) gene in 293T cells enhanced the basal levels of ERK phosphorylation and DR5 expression in comparison with those in cells carrying vector or WT K-Ras, both of which were further increased upon treatment with PLX4032 . In agreIt is known that regulation of DR5 expression can occur at transcriptional level2319Given that DR5 is a receptor for TRAIL, we speculated that B-Raf inhibition in Ras-mutant cancer cells will sensitize them to TRAIL-induced apoptosis due to a MEK/ERK-dependent increase in DR5 expression. Therefore, we pre-treated Ras-mutant cancer cells with PLX4032 overnight and then switched to TRAIL treatment. We detected much higher levels of cleaved caspase-8, caspase-3 and PARP in A549 and H157 cells pre-exposed to PLX4032, than in cells without PLX4032 pre-treatment . In agreFinally, we determined whether B-Raf inhibition-induced sensitization of Ras-mutant cells to TRAIL-induced apoptosis is dependent on the activation of c-Raf/MEK/ERK signaling. Pre-exposure of A549 cells (approximately 18\u2009h) to PLX4032 enhanced TRAIL-induced apoptosis, as evidenced by decreased cell survival , elevateThis study demonstrates that B-Raf inhibition induces DR5 expression exclusively in Ras-mutant cancer cell lines. This effect is secondary to Ras/c-Raf-dependent activation of MEK/ERK signaling and eventual enhancement of CHOP and Elk1-mediated transcription of the DR5 gene. Hence the current results, together with our previously reported findings1920910In the study, we noted that dabrafenib at high concentration ranges failed to activate ERK and increase DR5 expression . This coc-Raf-dependent rebound activation of MEK/ERK signaling in Ras-mutant cancer cells by B-Raf inhibitors is generally thought to be a mechanism that underlies resistance or accounts for an increased risk of secondary primary tumors6872728293031Clinical success with recombinant TRAIL or an agonistic DR5 antibody has not been demonstrated although the induction of apoptosis with these agents has emerged as an attractive cancer therapeutic strategy1733Immunotherapy has become a very promising strategy in fight of cancers such as melanoma and lung cancer, and involves apoptotic death of cancer cells. Death ligand-induced apoptotic signaling, mainly by TRAIL from T cells, monocytes and dendritic cells, is one of the primary underlying mechanisms1215Induction of apoptosis by the interaction of endogenous TRAIL and DR5 is a critical immunosurveillance mechanism for our body to eliminate cancer cells12PLX4032 and AZD6244 (selumetinib) were purchased from Selleckchem . Dabrafenib was purchased from LC Laboratories . Human recombinant TRAIL was purchased from PeproTech, Inc. . c-Raf antibody was purchased from Cell Signaling . Other antibodies, WT and mutant K-Ras (12V) were described previously192.The B-Raf or Ras mutation status of the tested human cancer cell lines is summarized in Cells were seeded in 96-well cell culture plates and on the second day exposed to different agents. Viable cell numbers were then estimated with sulforhodamine B (SRB) assayPlus kit was used to measure a poptosis according to the manufacturer\u2019s instructions. Western blot analysis was used to detect cleavage of caspase and PARP for additional indications of apoptosis.A Cell Death Detection ELISAPreparation of whole-cell protein lysates and Western blotting were the same as described previouslyDetection of cell surface DR5 expression with flow cytometry was described previouslyRT-PCR as described previously39The reporter construct containing a 552-bp 5-flanking region of the DR5 gene, pGL3-DR5 (\u2212552)-luc was generously provided by Dr. H-G Wang 1920Gene knockdown was achieved by transfecting siRNA with HiPerFect transfection reagent following the manufacturer\u2019s instruction. Control , K-Ras, CHOP and Elk1 siRNAs were described in our previous studyHow to cite this article: Oh, Y.-T. et al. Paradoxical activation of MEK/ERK signaling induced by B-Raf inhibition enhances DR5 expression and DR5 activation-induced apoptosis in Ras-mutant cancer cells. Sci. Rep.6, 26803; doi: 10.1038/srep26803 (2016)."} +{"text": "The HTLV-1 Tax transactivator initiates transformation in adult T-cell leukemia/lymphoma (ATL), a highly aggressive chemotherapy-resistant malignancy. The arsenic/interferon combination, which triggers degradation of the Tax oncoprotein, selectively induces apoptosis of ATL cell lines and has significant clinical activity in Tax-driven murine ATL or patients. Yet, the role of Tax expression in maintaining the transformed phenotype and of Tax loss in ATL response is disputed and the molecular mechanisms driving degradation remain elusive. Here we demonstrate that ATL-derived or HTLV-1 transformed cells are addicted to continuous Tax expression, suggesting that Tax degradation underlies clinical responses to the arsenic/interferon combination. The latter enforces PML nuclear body (NB) formation and partner protein recruitment. In arsenic/interferon-treated ATL-derived cells, Tax is recruited onto NBs, undergoes PML-dependent hyper-sumoylation by SUMO2/3, but not SUMO1, ubiquitination by RNF4 and proteasome-dependent degradation. Thus, the arsenic/interferon combination clears ATL through degradation of its Tax driver and could have broader therapeutic value by promoting degradation of other pathogenic sumoylated proteins."} +{"text": "C. elegans. We show here that the guanylyl cyclase ODR-1 functions non-cell-autonomously to downregulate ASH-mediated aversive behaviors and that ectopic cGMP generation in ASH is sufficient to dampen ASH sensitivity. We define a gap junction neural network that regulates nociception and propose that decentralized regulation of ASH signaling can allow for rapid correlation between an animal\u2019s internal state and its behavioral output, lending modulatory flexibility to this hard-wired nociceptive neural circuit.All animals rely on their ability to sense and respond to their environment to survive. However, the suitability of a behavioral response is context-dependent, and must reflect both an animal\u2019s life history and its present internal state. Based on the integration of these variables, an animal\u2019s needs can be prioritized to optimize survival strategies. Nociceptive sensory systems detect harmful stimuli and allow for the initiation of protective behavioral responses. The polymodal ASH sensory neurons are the primary nociceptors in C. elegans serves as an excellent animal model to study circuit-level modulation of neuronal function. By employing a combination of genetic and behavioral approaches, we have identified a non-cell-autonomous role for the guanylyl cyclase ODR-1 in the regulation of nociceptive sensory behaviors, and we provide the first evidence for circuit-level modulation of neuronal activity by the second messenger cGMP. While revealing a new mechanism for the coordination and optimization of animal behavior, our work also supports a growing body of evidence that hard-wired neuronal circuitry can be dynamically repurposed in a context-dependent manner.Studying the logic of small neural circuits is an essential step toward understanding more complex circuits and, ultimately, the computational and integrative properties of whole nervous systems. With a compact nervous system (just 302 neurons) and a well-characterized behavioral repertoire, the small roundworm Chemical stimuli, including odorants and tastants, can provide information about food availability and quality to affect appetitive behaviors across species. While all sensory circuits use specialized cells in the periphery to detect environmental stimuli, how the sensitivity of sensory neurons is tuned and how chemical information is processed and relayed through downstream interneurons remains largely unknown. As ultrastructural analyses of simple brains and small brain regions are providing connectome data \u20137, the cChemical synapses allow neurons to communicate with each other through the vesicular release of neurotransmitters into synaptic clefts between the cells. These molecules then bind to receptors on the postsynaptic neurons. In contrast, gap junctions allow for direct cytoplasmic communication and electrical coupling between neurons. As such, gap junctions are often referred to as electrical synapses. Importantly, the presence of gap junctions in the nervous system allows for the establishment of even more complex circuits than can be generated by synaptic signaling alone.2+, IP3 and the cyclic nucleotides cAMP and cGMP [Vertebrate gap junctions are formed through the association of transmembrane connexin proteins. Within one cell, six connexin proteins assemble to form one connexon hemichannel. Two hemichannels on neighboring cells then dock which each other, allowing homotypic (consisting of a single protein species on both cell membranes), heterotypic (consisting of two different homomeric connexons on the two cell membranes) or heteromeric (consisting of a mixture of protein species on both cell membranes) gap junctions to be made ,23. Gap and cGMP \u201332. The invertebrate analogues of the connexins) [C. elegans genome encodes 25 members of this protein family [C. elegans innexins remain unknown, the characterized cases have shown that these gap junction components are involved in diverse processes ranging from embryonic development and cell fate determination to adult neural functions [The gap junction protein family consists of vertebrate connexins and invertebrate innexins (nnexins) ,34. The n family . Althougunctions \u201337.C. elegans is an ideal model system in which to study the link between hardwired connectivity and the functional circuitry usage that drives animal behavior. The serial electron micrographs that showed the anatomical positioning of each of the 302 C. elegans hermaphrodite neurons also revealed all of the morphologically identifiable connections between neurons and between neurons and muscles [C. elegans wiring diagram shows a total of 6393 chemical synapses, 890 gap junctions and 1410 neuromuscular junctions [C. elegans exploits a highly developed chemosensory system to detect olfactory and gustatory cues associated with food, danger and mating [ muscles \u201341. The unctions . It is td mating \u201345. In gC. elegans head chemosensory neurons extend their ciliated ends to the tip of the animal\u2019s nose, allowing for direct or indirect exposure to sensory stimuli in their environment [C. elegans are attracted to and chemotax towards. In addition, while the ASJ, ASI, ADF and ASG sensory neurons are primarily involved in regulating dauer formation, they do also play a role in other processes, including a minor role in chemotaxis. Conversely, the ASH, ADL, AWB and ASK chemosensory neurons detect aversive stimuli that animals avoid by initiating backward locomotion upon stimulus detection.The 11 pairs of ironment ,39,40. Cironment . For exaC. elegans bears resemblance to its mammalian counterparts, in which noxious stimuli are sensed predominantly by peripheral glutamatergic sensory neurons that synapse onto spinal dorsal horn neurons and, following further sensory processing in multiple regions of the brain, generate the perception of pain and aversive behavior [The two bilaterally symmetric ASH nociceptors are particularly important for the avoidance of noxious stimuli, as they are \u201cpolymodal.\u201d This neuron pair responds to a broad range of aversive stimuli, including not only soluble chemicals and odorants (e.g. octanol), but also ions (e.g. Na+), osmotic stress and mechanosensory stimulation (nose touch) \u201354. ASH behavior \u201357.C. elegans lacking EGL-4 function are hypersensitive in their response to a subset of ASH-detected stimuli; egl-4(lof) animals avoid dilute stimuli that wild-type animals do not respond to. Our data suggested that EGL-4 likely normally acts to dampen ASH sensitivity by phosphorylating and activating the GTPase activating proteins RGS-2 and RGS-3, which then downregulate G protein-coupled sensory signaling in the ASH nociceptors. Surprisingly, although EGL-4 requires cGMP binding to negatively regulate ASH sensitivity, no guanylyl cyclases are known to be expressed in ASH [We previously reported a role for the cGMP-dependent protein kinase EGL-4 in the modulation of ASH-evoked nociceptive behavioral responses . C. elegd in ASH .C. elegans transmembrane guanylyl cyclase ODR-1 functions in a non-cell-autonomous manner to provide cGMP to regulate EGL-4 function in ASH. Like egl-4(lof) animals, odr-1(lof) animals are hypersensitive in their avoidance of a subset of ASH-detected stimuli. However, while EGL-4 functions directly in the ASHs, ODR-1 expression in the AWB, AWC and ASI head sensory neurons is sufficient to restore normal behavioral sensitivity to odr-1(lof) animals. We further provide evidence that the pool of cGMP produced by ODR-1 flows through a gap junction network from its site of production to the ASH nociceptors. Taken together, our data reveal a new way by which an animal\u2019s nervous system can utilize information about the organism\u2019s well-being to set the threshold of nociceptor sensitivity and coordinate behavioral responses that are appropriate for its internal state.Herein we provide evidence that the odr-1 loss-of-function (lof) animals respond to dilute (1 mM) quinine than wild-type animals , ceh-36p3 (AWC), gpa-4p (ASI), trx-1p (ASJ) and srbc-66p (ASK) [odr-1(lof) quinine hypersensitivity phenotype animals returned quinine response to the level seen when odr-1 was expressed under the control of its own promoter (odr-1p::odr-1), consistent with ODR-1 functioning in multiple neurons to regulate quinine sensitivity \u201366. Whilhenotype . This susitivity .odr-1(lof) animals develop with altered membraneous structures at the distal segments of the AWB, but not ASI, dendritic cilia [odr-1 gene was placed under the control of a heat shock inducible promoter [odr-1(lof) animals. Induction of odr-1 expression by heat shock in adult animal stages returned the behavioral response to dilute quinine to wild-type levels when assayed four hours later (odr-1(lof) animals animals (odr-1p::odr-1(\u0394ECD) construct restored quinine sensitivity to wild-type levels, similar to the expression of the wild-type odr-1 construct (odr-1p::odr-1) (ODR-1 is a receptor-type guanylyl cyclase with an extracellular domain (ECD) and an intracellular catalytic domain that processes GTP into cGMP . To asse animals . Express::odr-1) . This suodr-1(lof) animals expressing odr-1p::odr-1(E874A) remained hypersensitive in their response to quinine quinine . Two innexin mutants, inx-4(lof) and inx-20(lof), responded better than wild-type animals to dilute quinine hypersensitive response, while simultaneous expression of INX-4 in ASH and ADF, using the osm-10 [srh-142 [inx-4 expression in ODR-1-expressing neurons [odr-1p::inx-4 construct did not rescue the quinine hypersensitivity of inx-4(lof) animals animals to determine when inx-4 function is required to modulate quinine sensitivity. Induction of inx-4 expression by heat shock in adult animal stages significantly dampened the behavioral hypersensitivity of inx-4(lof) animals to dilute quinine when assayed four hours later (inx-4(lof) animals animals are hypersensitive in their response to distinct ASH-detected stimuli and, although EGL-4 functions in the ASH sensory neurons to regulate these behaviors, this cGMP-dependent protein kinase does not regulate ASH sensitivity in general [egl-4(lof) animals respond normally to the bitter tastant primaquine, the detergent SDS and the heavy metal copper [odr-1 or inx-4 increased overall sensitivity of ASH, or also selectively affected sensory signaling, we assayed odr-1(lof) and inx-4(lof) animals for their response to primaquine, SDS and copper. For each of these stimuli, odr-1(lof) and inx-4(lof) animals responded similarly to wild-type animals ;inx-4(lof) double mutant animals should display a behavioral phenotype similar to odr-1(lof) animals and the hypersensitivity should not be additive. We found that the double mutant animals\u2019 response to dilute (1 mM) quinine was indistinguishable from animals lacking only ODR-1 function ;inx-4(lof) background, expression of only either odr-1 or inx-4 had no effect on the quinine hypersensitivity animals in response to 1 mM quinine or inx-4(lof);lite-1(lof) animals. After exposure to blue light, lite-1(lof) animals expressing BlgC in the ADFs displayed a diminished response to 5 mM (inx-4(lof);lite-1(lof) transgenic animals expressing BlgC in the ADFs did not display a diminished response following blue light exposure animals are hypersensitive in their response to dilute concentrations of the bitter tastant quinine, and ODR-1 function in three distinct pairs of sensory neurons appears to contribute to the modulation of ASH-mediated avoidance [C. elegans will populate areas of decaying plant matter [C. elegans in a given area could signify low or dwindling food availability there, which could in turn result in poor nutritional status. Thus, the AWBs, AWCs and ASIs detect distinct sets of environmental stimuli that can all provide the animal with information about potential food quality and availability.Another potential benefit of decentralized ASH regulation is that by utilizing multiple sensory neurons to regulate ASH function, the integration of diverse sets of environmental information can maximize the appropriateness of an animal\u2019s response to its surroundings. For example, in addition to detecting the aversive odorants 100% octanol and 2-nonanone ,89, whicrcescens . All of hiazole) ,99 are pt matter . This trFollowing the initial detection of an environmental stimulus, the AWB, AWC or ASI sensory neurons may then subsequently modulate ASH sensitivity to indirectly optimize nociceptive responses in a context-dependent manner. For example, when animals are well-fed, they are more sensitive to aversive stimuli, including quinine , than thC. elegans lack a nitric oxide synthase. Thus, the mechanistic link between an animal\u2019s feeding status and ODR-1 activity is unclear. Interestingly, calcium levels rise in the AWCs upon withdrawal of either odorant or bacterial-conditioned media [We speculate that food somehow suppresses ODR-1 activity in the upstream AWB, AWC and/or ASI sensory neurons, while removal of food allows for ODR-1 activation and cGMP accumulation . ODR-1 ied media . An attrCollectively, our data support a model wherein, upon food removal, the ODR-1-expressing neurons AWB, AWC and ASI provide a pool of cGMP that flows through a gap junction network from the site of its production in these sensory neurons, through ADF, AFD and AIA, to the ASH nociceptors . Howeverinx-4(lof) animals drove GFP expression in the AVBs and AVKs [inx-19 promoter did not express in these neurons but instead was seen to drive GFP expression in 18 other neuron pairs [C. elegans nervous system. While our combination of cell-specific rescue and ablation experiments strongly supports a role for the proposed gap junction network in the modulation of ASH activity, it is possible that extracellular cGMP could also enter ASH through an INX-4 hemichannel.INX-4 function in the ASHs is sufficient to rescue the quinine hypersensitivity of animals , suggestand AVKs , a 5.8 kand ASH) . Thus, fC. elegans lack a complex central nervous system with which to simultaneously process several sets of sensory information, a gap junction network may allow for the efficient integration of diverse signals representing the environmental landscape with an animal\u2019s experiences and internal state. This may allow for a more sophisticated modulation of behavior than could be achieved by expressing a guanylyl cyclase(s) directly in the ASHs themselves, especially given that the ASHs detect multiple forms of aversive stimuli [odr-1(lof) animals are hypersensitive in their response to some (e.g. quinine), but not all , ASH-detected stimuli [unc-13 in both the sense and antisense orientations of otherwise wild-type animals. RNAi knock-down of unc-13 in the AWB neurons resulted in diminished avoidance of a 1:10 dilution of 2-nonanone . (B) Loss of UNC-13-dependent synaptic signaling from the AWC head sensory neurons resulted in failure to chemotax towards benzadehyde, which is detected by the AWCs [ceh-36p3 (AWC) [gpa-4 (ASI) [str-1p (AWB) [unc-13 in both the sense and antisense orientations of otherwise wild-type animals. RNAi knock-down of unc-13 in the AWC neurons or in the AWB, AWC or ASI neurons in combination abolished chemotaxis to a 1:200 dilution of benzaldehyde. The combined data of \u2265 3 independent lines and n \u2265 120 transgenic animals is shown in each panel. Avoidance index = ((A + B)\u2013(E + F)) \u00f7 total number of animals on the assay plate [odr-1(n1936) loss-of-function and inx-4(ok2373) loss-of-function. WT = the N2 wild-type strain. lof = loss-of-function.(A) The loss of UNC-13-dependent synaptic signaling from the AWB head sensory neurons resulted in failure to avoid the aversive odorant 2-nonanone, which is detected by the AWBs . The str-1 (AWB) promoterthe AWCs . The cehp3 (AWC) , gpa-4 (-4 (ASI) and str-1p (AWB) promoteray plate . Chemotaay plate . Alleles(TIF)Click here for additional data file.S2 Figinx-4 promoter [inx-4p::gfp). Transgenic animals were incubated with the lipophilic dye DiD (shown in red), which is taken up by the ASH sensory neurons and was used to visualize the neuronal cell body. As previously reported [inx-4 expression was seen in the ASH sensory neurons of larval-stage animals, including the L2 stage (top panel). In addition, inx-4 expression was observed in the ASHs of adult animals (bottom panel).The promoter was usedreported , inx-4 e(TIF)Click here for additional data file.S3 FigC. elegans respond to aversive stimuli in addition to quinine. (A-C) odr-1(lof) and inx-4(lof) animals responded similarly to wild-type animals to the bitter tastant primaquine, the detergent SDS and the heavy metal copper, across a range of concentrations (p > 0.2 for each concentration). The percentage of animals responding is shown. n \u2265 60 for each. All tastants were dissolved in M13 buffer, pH 7.4. Alleles used: inx-4(ok2373) loss-of-function and odr-1(n1936) loss-of-function. WT = the N2 wild-type strain. lof = loss-of-function.(TIF)Click here for additional data file.S4 Figosm-10 [srb-6 [lite-1(lof) animals. osm-10p drives expression in ASH and weakly in ASI in the head, and in PHA and PHB in the tail. srb-6p drives expression in the ASH, ADL and ADF head sensory neurons, and in PHA and PHB in the tail. Adult lite-1(lof) animals expressing BlgC or BlaC were tested without blue light exposure (white bars) or after a 30 second exposure (grey bars). While lite-1(lof) animals responded robustly to 5 mM quinine, similar to WT animals (p > 0.2), transgenic animals expressing BlgC displayed a significantly diminished response following blue light exposure (p < 0.0001 for each ASH-selective promoter). Transgenic animals expressing BlaC remained sensitive following blue light exposure . The percentage of animals responding is shown. The combined data of \u2265 3 independent lines and n \u2265 120 transgenic animals is shown in each panel. Allele used: lite-1(ce314) loss-of-function. WT = the N2 wild-type strain. lof = loss-of-function.The ASH-selective osm-10 and srb-0 [srb-6 promoter0 [srb-6 in lite-(TIF)Click here for additional data file.S5 Figsrh-142 promoter [lite-1(lof) or inx-4(lof);lite-1(lof) animals. Adult lite-1(lof) or inx-4(lof);lite-1(lof) animals expressing BlgC or BlaC were tested without blue light exposure (white bars) or after a 30 second exposure and 30 second recovery (grey bars). While lite-1(lof) animals responded robustly to 10 mM quinine, similar to WT animals (p > 0.1), transgenic animals expressing BlgC in the ADFs displayed a significantly diminished response following blue light exposure (p < 0.001). Transgenic animals expressing BlaC did not display a diminished response following blue light exposure . inx-4(lof);lite-1(lof) transgenic animals expressing either BlgC or BlaC did not display a diminished response following blue light exposure . The percentage of animals responding is shown. The combined data of \u2265 3 independent lines and n \u2265 120 transgenic animals is shown in each panel. Alleles used: lite-1(ce314) loss-of-function and inx-4(ok2373) loss-of-function. WT = the N2 wild-type strain. lof = loss-of-function.The ADF-specific promoter was usedpromoter in lite-(TIF)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file."} +{"text": "Both tumor-associated neutrophils (TAN) and cancer-associated fibroblasts (CAFs) display specific phenotypic and functional features and contribute to tumor cell niche. However, their bidirectional crosstalk has been poorly studied, in particular in the context of hematological malignancies. Follicular lymphomas (FL) and diffuse large B-cell lymphomas (DLBCL) are two germinal center-derived lymphomas where various cell components of infiltrating microenvironment, including TAN and CAFs, have been demonstrated to favor directly and indirectly malignant B-cell survival, growth, and drug resistance. We show here that, besides a direct and contact-dependent supportive effect of neutrophils on DLBCL B-cell survival, mediated through the BAFF/APRIL pathway, neutrophils and stromal cells cooperate to sustain FL B-cell growth. This cooperation relies on an overexpression of IL-8 by lymphoma-infiltrating stromal cells that could thereafter efficiently promote neutrophil survival and prime them to neutrophil extracellular trap. Conversely, neutrophils are able to activate stromal cells in a NF-\u03baB-dependent manner, inducing their commitment towards an inflammatory lymphoid stroma phenotype associated with an increased capacity to trigger malignant B-cell survival, and to recruit additional monocytes and neutrophils through the release of CCL2 and IL-8, respectively. Altogether, a better understanding of the lymphoma-supporting effects of neutrophils could be helpful to design new anti-tumor therapeutic strategies. Strikinin vitro [BM-MSCs were recently reported to support neutrophil survival in vitro , 8. We vSince BM and tonsil-derived stromal cells triggered neutrophil recruitment and survival, we decided to test the tripartite co-culture between stromal cells, neutrophils, and B cells.in vitro but were found in close contact with FL lymphoid stromal cells in situ, we tested how neutrophil/stromal cell cooperation could favor primary FL B-cell survival. Importantly, neutrophils increased the anti-apoptotic activity of both resting and TNF/LT-pretreated stromal cells towards purified FL B cells , the neutrophil-primed Resto signature comprising 1152 PS, and the neutrophil-primed stroma signature obtained by comparing the 6 unprimed stromal cell samples to the 6 neutrophil-primed stromal cell samples and comprising 577 PS approach based on previously published signatures to highlight the major pathways activated by neutrophils in stromal cells. Interestingly, neutrophil-primed MSCs and Resto cells showed signs of lymphoid stroma polarization and inflammatory response, with a strong activation of TNF and NF-\u03baB signaling pathways Figure . Among tto cells . Accordito cells . They alto cells . Of notetrophils .We next sought to determine whether these neutrophil-primed FRC-like cells became fully competent not only as tumor B-cell feeders, but also as organizers of lymphoma B-cell niche. We decided to focus on monocyte and neutrophil migration since FL-MSCs have been shown to recruit both cell subsets more efficiently than HD-MSCs. We first revealed that supernatants of neutrophil-primed MSCs and Resto cells triggered an improved chemotaxis of monocytes and neutrophils, compared to resting stromal cells Figure . To asceTaken together, these results support the hypothesis that neutrophil-primed stromal cells were engaged toward lymphoid stroma differentiation, in association with an increased capacity to support malignant B-cell growth and to trigger monocyte and neutrophil recruitment.IL-8 and CCL2 induced by addition of exogeneous TNF (data not shown). Interestingly, inhibition of IKK by wedelolactone, unlike the specific blockade of TNFR1, strongly reduced the neutrophil-mediated induction of IL-8 and CCL2 in stromal cells expression was not modulated. Interestingly, CXCL5 is also upregulated during lymphoid stroma differentiation [Bidirectional crosstalk between stromal cells and neutrophils has several tumor-promoting functional consequences. First, neutrophil-primed stromal cells supported more efficiently the growth of malignant B cells, a property shared with stromal cells committed towards lymphoid stroma by TNF/LT or by co-culture with malignant B cells . We prev outcome and STAT outcome thus fav outcome , 44. Fin outcome . TGF-\u03b2 i outcome . Besidesntiation . These cOne of the most interesting feature of FL-TAM is their opposite predictive value depending on the treatment schedule. In fact, FL-TAM could favor tumor progression but also contribute to the clinical efficacy of antibody-based anti-lymphomadrugs . WhetherSamples came from subjects recruited under institutional review board approval and informed consent process according to the Declaration of Helsinki. BM aspirates were obtained from FL patients at diagnosis and age-matched patients undergoing cardiac surgery, tonsils from children undergoing routine tonsillectomy, LN biopsies from FL and DLBCL patients, and peripheral blood (PB) from adult healthy volunteers. BM plasma were collected by centrifugation and frozen until use. BM-MSCs and tonsil-derived stromal cells (Resto) were obtained as previously described , 17 and FITC-conjugated monoclonal antibodies (mAbs) to CD14, CD19, CD20, and CD66b; PE-conjugated mAbs to CD19, CD20, CD54, and CD105; PC7-conjugated mAbs to CD19, and CD20; and PB-conjugated mAbs to CD15 were provided by Beckman Coulter. Alexa-647-cojugated mAb to CD105 was provided by ebioscience. Appropriate isotype-matched mAbs were used as negative controls and all analyses were performed using a Gallios (Beckman Coulter) flow cytometer. Cell death was checked using TOPRO-3 or DAPI (Life Technologies) staining. Apoptosis evaluation was performed using PE-conjugated active caspase-3 apoptosis kit (Becton Dickinson).\u00ae, LFB Biomedicaments) and TACI-Fc inhibitor . Stromal cells were pretreated with TNF-\u03b1 and lymphotoxin-\u03b11\u03b22 and/or primed with neutrophils before washing and co-culture with B cells. For primary lymphoma samples, purified malignant B cells were seeded in the presence or not of stromal cells and/or neutrophils before analysis of B-cell death.After serum deprivation, RL and SUDHL4 were seeded with low serum concentration in the presence of purified neutrophils and/or on a confluent stromal cell monolayer. B-cell growth was evaluated by tritiated thymidine incorporation or by counting the number of viable B cells using FlowCounts beads (Beckman Coulter). B-cell apoptosis was analyzed by the use of active caspase-3 staining. When indicated, neutrophils and B cells were separated by a 0.4-\u03bcm pore Transwell filter or were co-cultured in the presence of polyclonal human immunoglobulin or wedelolactone . The dose of wedelolactone was chosen as efficient to inhibit NF-\u03baB signaling [IL-8 was quantified by ELISA (R&D Systems) in BM plasma, in the supernatants of HD-MSCs and FL-MSCs collected at the end of passage 1, and in the supernatants of HD-MSCs stimulated with TNF/LT or cocultured with RL or purified FL B cells for 3 days. In addition, we measured by ELISA IL-8, IL-6, CCL2 (R&D Systems) and PGE2 levels in the supernatants of stromal cells primed or not by neutrophils for 3 days before neutrophil removal and additional 3-day culture period. To confirm the stromal origin of secreted cytokines after priming with neutrophils, we checked by RQ-PCR that the mRNA level of ignaling without 5 cells/100\u03bcL) were added in RPMI-0.1% human serum albumin (neutrophil migration medium) or RPMI-1% FCS (monocyte migration medium) to the upper compartment of Transwell chambers with 5-\u03bcm pore filters. Lower chambers contained supernatants of stromal cells obtained after 3 days of priming by TNF/LT, RL, or neutrophils. For IL-8 and CCL2 depletion, 2.107 Dynabeads pan mouse IgG (Life Technologies) were conjugated to 10 \u03bcg anti-IL-8 or anti-CCL2 mAbs (R&D Systems) before incubation with supernatants. Supernatants were thereafter seeded inside a magnetic field to allow immune complex retention. We checked by ELISA that IL-8 or CCL2 were undetectable in corresponding depleted supernatants. Conversely, the use of an isotype-matched control mAb did not modify the concentration of IL-8 or CCL2. The absolute number of DAPInegCD66bpos viable neutrophils or DAPInegCD14pos viable monocytes was quantified using FlowCount beads in the lower chamber after 1 and 2 hours, respectively.Purified PB neutrophils or monocytes . Microarray data were deposited at NCBI GEO data set under accession number GSE62782. Data were analyzed with the Partek Genomics Suite (Partek). Expression signal values and P-values were obtained for each probeset (PS) using the Partek Genomics Suite software (Partek) after normalization by Robust Multichip Averaging algorithm with GC content adjustment (GC-RMA). Background noise was decreased by eliminating PS with a low standard deviation to mean ratio. To compare stromal cells primed or not by neutrophils, supervised analysis were carried out by paired Student t-test with Partek Genomics Suite software allowing the selection of PS with absolute log2 fold change >1.2 and an adjusted P-value less than 0.05. Gene Set Enrichment Analysis (GSEA) software was used to determine the specific enrichment of specific gene sets in the PMN-primed stroma signatures.GEP was performed on 3 HD-MSCs and 3 tonsil-derived Resto cells primed or not with neutrophils for 1 day (ratio 1:1.5). RNA was extracted after neutrophils removal by extensive washes, using RNeasy Mini Kit, (Qiagen) and the purity and integrity of RNA were checked with the Bioanalyzer 2100 (Agilent Technologies). We checked for the absence of residual neutrophils by RQ-PCR for t calculation method. PUM1 was determined as the appropriate internal standard gene using TaqMan Endogenous Control Assays (Life Technologies). Quantification of TNFRSF13 and TNFRSF13B was performed using appropriate assay-on-demand primers and probes and ABL1 as internal standard gene in GC B-cell lines, purified CD14pos monocytes, and purified CD15pos neutrophils. For each sample, the Ct value for the gene of interest was determined, normalized to its respective value for ABL1, and results were then standardized by comparison to gene expression of a pool of five whole tonsil cells.An additional set of neutrophil-stromal cell coculture experiments was performed for validation of GEP data and RNA was generated from resting and neutrophil-primed stromal cells. cDNA synthesis was performed with the Superscript II reverse transcriptase and random hexamers (Life Technologies). For quantitative RQ-PCR, we used assay-on-demand primers and probes, and Taqman Universal Master Mix (Life Technologies). Gene expression was measured using the StepOnePlus (Life Technologies) based on the \u0394CBM-MSCs and Resto cells were seeded on chamber coverslips and cultured with or without neutrophils for 3 days before neutrophil removal by extensive washes and staining for transglutaminase and podoplanin . Cells wFor NET formation, purified neutrophils were seeded onto slides coated with poly-D-lysine or HD-MSC monolayer for 12 hours before stimulation for 3 hours with 20 ng/ml PMA (Sigma-Aldrich). Cells were fixed with 4% paraformaldehyde-PBS and stained with Texas Red-X Phalloidin (Life Technologies). Coverslips were mounted with mowiol mounting medium including Sytox blue and examined using a confocal microscope.Statistical analyses were performed with GraphPad Prism 6.0 software using the non-parametric Wilcoxon test for matched pairs or the Mann-Whitney nonparametric U test as appropriate."} +{"text": "Measurement of 17-hydroxyprogesterone (17-OHP) in daily dried blood spot profiles via radioimmunoassay is a convenient and accepted method for monitoring of glucocorticoid therapy in Congenital Adrenal Hyperplasia. Characteristics of this method that serve to limit its clinical usefulness include its lack of specificity for 17-OHP, and a relatively high limit of detection. Mass Spectroscopy is the gold standard method for steroid quantification. We aim to establish a High Performance Liquid Chromatography-Tandem Mass Spectroscopy (HPLC-MS/MS) method for dried blood spot 17-OHP quantification and develop normative age- and tanner-specific, as well as Congenital Adrenal Hyperplasia genotype-phenotype correlated reference ranges to guide glucocorticoid therapy.Four 3mm blood spots were punched from patient dried blood spot filter paper specimens. 17-OHP was eluted into solvent and concentrated using liquid nitrogen. Steroids were separated using high performance liquid chromatography and quantitated by Tandem Mass Spectrometry. For the radioimmunassay / HPLC-MS/MS correlations, measurements were performed by both methods on 49 samples from children with glucocorticoid-dependent Congenital Adrenal Hyperplasia, as well as children undergoing dynamic endocrine function testing at The Mater Children\u2019s Hospital. Reference samples for HPLC-MS/MS calibration, and determination of sensitivity, precision, and recovery were prepared using whole blood samples spiked with 17-OHP spotted onto filter paper. Concentrations were expressed as mean (nmol/L), standard deviation, and coefficient of variation (%).2=0.9610). For the radioimmunoassay method, the lower limit of quantification has been established at <5 nmol/L while LC-MS/MS allows detection to 1.0 nmol/L with a proposed lower limit of quantification of 1.5 nmol/L .There was excellent correlation between HPLC-MS/MS and radioimmunoassay methods (rWe have established an accurate, reliable, and specific method for quantifying 17-OHP concentrations from dried blood spot using HPLC-MS/MS. This will allow for the establishment of clinically relevant and time-specific reference ranges to guide glucocorticoid dose adjustment in children with Congenital Adrenal Hyperplasia."} +{"text": "Intracellular cytokine staining of SIY-peptide restimulated spleen cells from DMXAA-treated animals revealed that the expanded SIY-specific CD8+ T cells produced high-levels of IFN-\u03b3. This enhanced immune response also translated to a benefit in survival. After C1498.SIY AML cell-challenge, 80% of DMXAA-treated mice survived long-term, while controls succumbed to AML within approximately 3-4 weeks, as expected. DMXAA-treated mice surviving a primary C1498.SIY cell challenge also rejected a secondary challenge with parental C1498 cells (SIY-negative), suggesting DMXAA-induced STING activation led to potent immunological memory against native C1498-expressed antigens. DMXAA treatment of mice following a systemic challenge with parental C1498 cells also led to significantly enhanced survival compared to control-treated animals. This effect was T and/or B cell-dependent, as DMXAA treatment of leukemia-bearing RAG-/- mice had no effect on survival. Collectively, these data suggest that DMXAA-mediated activation of the STING pathway enhances adaptive immunity to AML, culminating in prolonged survival and even disease cure. Human STING agonists are currently in development and, if STING pathway activation demonstrates efficacy in additional pre-clinical leukemia models, it may be of interest to target this pathway in AML patients.Type I interferon (IFN) production by innate immune cells is critical to prime spontaneous T cell responses against solid tumors. Emerging pre-clinical data suggests that tumor-derived DNA induces potent IFN-\u03b2 production by activating a cytosolic DNA sensing receptor called STING (Stimulator of Interferon Genes), ultimately resulting in tumor antigen-specific T cell priming and in some cases, tumor rejection. However, because of their disseminated growth pattern, hematological malignancies, such as acute myeloid leukemia (AML), may not release sufficient quantities of DNA during cell death to activate the STING pathway in innate immune cells, which contributes to the T cell tolerance observed in these hosts. Thus, we hypothesized that STING pathway activation would promote a host type I IFN response sufficient to facilitate leukemia-specific T cell priming and immune-mediated control of AML progression. Murine C1498 AML cells expressing the model SIY antigen (C1498.SIY) were inoculated intravenously into syngeneic C57BL/6 mice which then received a single dose of the murine STING agonist, DMXAA , or vehicle control 5 days later. DMXAA induced potent IFN-\u03b2 production by C57BL/6 spleen cells (90-fold induction over vehicle control). STING pathway activation via DMXAA treatment of C1498.SIY-bearing animals resulted in a massive expansion (10-fold over vehicle control) of endogenous SIY antigen-specific T cells as analyzed by SIY/K"} +{"text": "Herpesviridae; dsDNA genome) to Hantavirus ssRNA genome) to human immunodeficiency virus ssRNA genome) are associated with the rapid up-regulation of the NF-kB-sensitive pro-inflammatory microRNA-146a (miRNA-146a) in the host shortly after infection. This significant miRNA-146a up-regulation appears to be beneficial to the infecting virus as part of an immune-evasion strategy. Interestingly, miRNA-146a is also significantly up-regulated in several human central nervous system (CNS) disorders. These include Alzheimer's disease (AD) and prion disease where miRNA-146a participates in pro-inflammatory and innate-immune signaling. This opinion paper will comment on some recently clarified roles for the NF-kB-regulated, pro-inflammatory miRNA-146a in viral-induced cellular dysfunction, and how anti-miRNA-146a and/or related therapeutic strategies may be beneficial in the clinical management of a broad spectrum of viral-mediated CNS disease.A remarkably wide variety of human neurotrophic viruses\u2014ranging from herpes simplex 1 miRNA-146a lies at the crossroads of multiple biological processes involved in the innate-immune response, viral-infection and inflammatory disease ssRNA genome; Selvamani et al., Picornaviridae; (+)ssRNA genome; Ho et al., Herpesviridae; dsDNA genome; Jonigk et al., Bunyaviridae; (\u2212)ssRNA genome; Shin et al., Flaviviridae; (+)ssRNA genome; Joshi et al., Herpesviridae; dsDNA genome; Higaki et al., Paramyxoviridae; (\u2212)ssRNA genome; Stewart et al., Orthomyxoviridae; (+)ssRNA genome; Chen et al., Hepadnaviridae; dsDNA genome; Liu et al., Retroviridae; (+)ssRNA genome; Duskova et al., Retroviridae; (+)ssRNA genome; Pichler et al., Flaviviridae; (+)ssRNA genome; Pareek et al., each virus mentioned above is associated with progressive neuropathological change. Interestingly (i) miRNA-146a up-regulation has been associated with common age-related, human inflammatory degenerations such as sporadic Alzheimer's disease (AD), and the rare sporadic prion diseases Creutzfeldt-Jakob disease (sCJD) and Gerstmann-Straussler-Scheinker (GSS) syndrome; and (ii) the etiopathogenesis of AD has recently been associated with multiple viral infections, and most recently with latent HCV, HIV-1 or HSV-1 reactivation : (i) Chikungunya virus ssRNA genome] that causes a highly lethal hemorrhagic fever syndrome in humans rapidly induces 3 EBOV genome-derived miRNAs that subsequently target host mRNA (Liang et al., Out of about 24,000 miRNAs so far identified in all species, only about 300 are encoded by viruses (miRBase v.20; Liu, In summary, viruses have evolved multiple and complex strategies to subvert and evade the host immune-response to ensure their own replication and survival (Hill et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Nanoparticle (NP)-based materials are very promising agents for enhancing cancer diagnosis and treatment. Once functionalized for selective targeting of tumor expressed molecules, they can specifically deliver drugs and diagnostic molecules inside tumor cells.In the present work, we evaluated the in vivo melanoma-targeting ability of a nanovector (HFt-MSH-PEG) based on human protein ferritin (HFt), functionalized with both melanoma-targeting melanoma stimulating hormone (\u03b1-MSH) and stabilizing poly(ethylene glycol) (PEG) molecules. We used two independent and complementary techniques, such as whole-specimen confocal microscopy and magnetic resonance imaging, to detect the in vivo localization of NP constructs endowed with suitable tracers .Targeted HFt-MSH-PEG NPs were shown to accumulate persistently at the level of primary melanoma and with high selectivity with respect to other organs. Melanoma localization of untargeted HFt-PEG NPs, lacking the \u03b1-MSH moiety, was less pronounced and disappeared after a few days. Further, HFt-MSH-PEG NPs accumulated to a significantly lower extent and with a different distribution in a diverse type of tumor (TS/A adenocarcinoma), which does not express \u03b1-MSH receptors. Finally, in a spontaneous lung metastasis model, HFt-MSH-PEG NPs localized at the metastasis level as well.These results point at HFt-MSH-PEG NPs as suitable carriers for selective in vivo delivery of diagnostic or therapeutic agents to cutaneous melanoma."} +{"text": "Transthyretin Familial Amyloid Polyneuropathy (TTR-FAP) is a rare, progressive, debilitating and life-threatening neurodegenerative disease. The purpose of this study was to assess the health-related quality of life (HRQoL) impairment of TTR-FAP disease versus Portuguese general population and to identify individual patient characteristics - such as disease stage \u2013 that affects their HRQoL. Literature on TTR-FAP patients HRQoL is scarce at worldwide level and no evidence for Portugal has been published.HRQoL was measured using the validated EuroQoL five dimensions three levels (EQ5D-3L) questionnaire being the index score (utility) calculated trough the Portuguese scoring algorithm. The Portuguese general population reference set (n = 1500) was pooled with TTR-FAP patients specific data extracted from Transthyretin Amyloidosis Outcomes Survey (THAOS) registry. Ordinary Least Squares regression for utility was set to test if being asymptomatic carrier caused HRQoL impairment, conditional in other individual characteristics. Generalized linear models (GLM) were specified for disutility in order to disentangle individual patient characteristics that affect quality of life and quantify the impact. Demographic variables include gender and age. Clinical variables include disease onset (early/late), polyneuropathy disability (PND) score, liver transplant and pharmacologic treatment. Akaike information criteria were used to select the most adequate statistical model.In a scale from -0.50 to 1.00 the average utility score was 0.76 (0.25) for general population, 0.823 (0.24) for TTR-FAP asymptomatic carriers (n=525) and 0.50 (0.37) for symptomatic TTR-FAP patients (n = 566). Ordinary Least Squares regression indicated no significant statistical effect on utility for being a TTR-FAP asymptomatic carrier versus general population . The GLM was used to detect a significant statistical effect for gender, age and being symptomatic TTR-FAP patient versus general population. Average women aged 44 years and symptomatic TTR-FAP patient, has average 40% impairment on utility versus women aged 44 years from general population. Within TTR-FAP population, individual patient characteristics such as gender, age, disease onset (early/late), polyneuropathy disability (PND) score, liver transplant and pharmacologic treatment were tested for significant statistical effect .The preference-based utility measures used in this study adequately quantify the large impact of TTR-FAP disease on patient's health-related quality of life and allow discriminating across different TTR-FAP clinical stages, interventions and demographic characteristics. Assuming that these values represent the patients' preferences and the utility associated with their health state, the results presented in this study may be used in future health technologies cost-utility studies."} +{"text": "The article has since been corrected online.In the article \u201cEffect of 23-Valent Pneumococcal Polysaccharide Vaccine Inoculated During Anti-Cancer Treatment Period in Elderly Lung Cancer Patients on Community-Acquired Pneumonia Hospitalization: A Nationwide Population-Based Cohort Study\u201d,"} +{"text": "The HTLV-1 Tax transactivator initiates transformation in adult T-cell leukemia/lymphoma (ATL), a highly aggressive chemotherapy-resistant malignancy. The arsenic/interferon combination, which triggers degradation of the Tax on coprotein, selectively induces apoptosis of ATL cell lines and has significant clinical activity in Tax-driven murine ATL or patients. Yet, the role of Tax expression in maintaining the transformed phenotype and of Tax loss in ATL response is disputed and the molecular mechanisms driving degradation remain elusive. Here we demonstrate that ATL-derived or HTLV-1 transformed cells are addicted to continuous Tax expression, suggesting that Tax degradation underlies clinical responses to the arsenic/interferon combination. The latter enforces PML nuclear body (NB) formation and partner protein recruitment. In arsenic/interferon-treated ATL-derived cells, Tax is recruited onto NBs, undergoes PML-dependent hyper-sumoylation by SUMO2/3, but not SUMO1, ubiquitination by RNF4 and proteasome-dependent degradation. Thus, the arsenic/interferon combination clears ATL through degradation of its Tax driver and could have broader therapeutic value by promoting degradation of other pathogenic sumoylated proteins."} +{"text": "The kcc gene is the highly conserved Drosophila melanogaster ortholog of K+/Cl\u2212 cotransporter genes thought to be expressed in all animal cell types. Here, we examined the spatial and temporal requirements for kcc loss-of-function to modify seizure-susceptibility in flies. Targeted RNA interference (RNAi) of kcc in various sets of neurons was sufficient to induce severe seizure-sensitivity. Interestingly, kcc RNAi in glia was particularly effective in causing seizure-sensitivity. Knockdown of kcc in glia or neurons during development caused a reduction in seizure induction threshold, cell swelling, and brain volume increase in 24\u201348 hour old adult flies. Third instar larval peripheral nerves were enlarged when kcc RNAi was expressed in neurons or glia. Results suggest that a threshold of K+/Cl\u2212 cotransport dysfunction in the nervous system during development is an important determinant of seizure-susceptibility in Drosophila. The findings presented are the first attributing a causative role for glial cation-chloride cotransporters in seizures and epileptogenesis. The importance of elucidating glial cell contributions to seizure disorders and the utility of Drosophila models is discussed.Flies carrying a Drosophila.Glial cells are proposed to be important players in seizure disorders because of their critical role in maintaining extracellular ionic homeostasis in the nervous system \u2212 ions together with Na+ and/or K+ ions across the plasma membrane. The Na+-coupled CCCs utilize the large inwardly-directed electrochemical gradient for Na+ to transport Cl\u2212 into the cell. K+/Cl\u2212 cotransporters (SLC12A4-7 or KCC1-4 in vertebrates) mainly transport K+ and Cl\u2212 out of the cell utilizing the electrochemical gradient for K+.Nine CCCs comprise the SLC12 family of transmembrane proteins (SLC12A1\u2013A9). Evolutionarily ancient, these solute carriers are described for vertebrates, arthropods, worms, plants, fungi, and bacteria \u2212, K+, and water, mediated by KCC3 and ion channels \u2212, Na+, K+ and water, mediated by NKCC1 and ion exchangers CCCs are involved in a variety of physiological mechanisms in humans and experimental models Drosophila, there are five CCC genes: kcc (kazachoc), an ortholog of vertebrate KCC1-4; ncc69, an ortholog of vertebrate NKCC1 and NKCC2; and three less well-understood members kcc function causes seizure-sensitivity, in part via abnormal excitatory GABAergic signaling due to intracellular neuronal Cl\u2212 misregulation Drosophila resemble those of vertebrates that implicate excitatory GABAergic signaling in the genesis of neonatal seizures, temporal lobe epilepsy, and seizures occurring after ischemic-hypoxic insult kcc loss-of-function in Drosophila is more complex than previously described.In kcc loss-of-function in either glia or neurons. ncc69 loss-of-function in glia, but not neurons, caused seizure-sensitivity. These neurological phenotypes correlated with cell and tissue volume abnormalities in larvae and adults.In the present experiments, CCC loss-of-function flies were generated by ectopic spatial and temporal restriction of RNA interference (RNAi) transgene expression. Seizure-sensitivity was observed in animals with Drosophila strains used in this study and their sources are listed in DHS1kcc mutation is a 13 base pair insertion in intron 11 of kcc (kazachoc). The mutation leads to an approximate two-fold reduction in kcc transcript as determined previously by RT-PCR; and an approximate four-fold reduction in Kcc protein as determined by Western blot analysis DHS1kcc behaves as a hypomorph EY08304kcc embryonic lethal mutation is a P-element insertion at 2R:19,812,398, upstream of the sequence encoding the C-terminus tail predicted to be recognized by the Kcc antibody used in this study to yield a final measure of %BS paralysis at 24\u201348 h-old. Movies of BS paralysis were acquired using a mobile cellular device and edited in Windows Live Movie Maker. Flies kept at 30\u00b0C for Temporal and Regional Gene Expression Targeting Behavioral seizure-like activity, or bang-sensitive (BS) paralysis, was quantified as described previously in vivo electrophysiological assays were from the adult giant fiber system Drosophila embryos were stained following Bossing's whole-mount protocol (Abcam). Third instar larvae were filleted in hemolymph-like solution Drosophila melanogaster Kcc isoforms at their identical C-termini (RGGGREVITIYS) . Control experiments with kcc deletion mutations in embryos show antibody specificity to Kcc protein . Fluorescent micrographs were processed using ImageJ software . Embryo Kcc fluorescence intensities were averages of internal regions defined by perimeter signal for each genotype, after background subtraction. Calyx Kcc fluorescence intensities were averages of regions of interest defined by the GFP+ channel for each genotype in three slices , after background subtraction. Mean Kcc fluorescence intensities of embryos and adults were normalized to control genotype intensities. Adult brain size was approximated as the volume of an ellipsoid with semi-axes along the dorsal-ventral axis , the anterior-posterior axis , and the lateral-medial axis . Volume estimation of neuron somata was achieved by measuring the longest observable diameter for each cell. Quantification of larval peripheral nerve average cross-sectional area was performed by averaging fourteen evenly-distributed diameter measurements at the center-most GFP channel optical slice along 500 \u00b5m of each nerve beginning below the ventral nerve cord (assuming each nerve was a cylinder). Peripheral nerve sections with glial cell nuclei were excluded from measurements. Statistical tests were unpaired two-sample Student's Drosophila with Western blot analysis showing a loss of immunoreactivity in kcc mutants DHS1kcc, a loss of Kcc protein causes seizure-sensitivity DHS1kcc seizure-sensitivity can be rescued by supplying Kcc back to neurons or back to glia Kcc protein was shown previously to be expressed throughout the adult brain of wild-type In third instar larvae, prominent Kcc staining is present surrounding the ventral nerve cord and peripheral nerves, as well as in neuronal cell bodies of the ventral nerve cord . High-maIn the adult brain, Kcc immunoreactivity was present in glial, as well as neuronal cell types. For example, To explore nervous system Kcc expression further, specimens were prepared with triple labeling of glial cell membrane, neuronal membrane, and Kcc protein . ImagingDrosophila K+/Cl\u2212 cotransporter function, such as in the DHS1kcc hypomorphic mutant, raises seizure-susceptibility through a reduction in kcc expression. The DHS1kcc mutant displays seizure-sensitivity as evidenced by a lowered threshold to evoked electrophysiologically recorded seizure-like activity, and bang-sensitive behavioral seizure-like activity and paralysis kcc loss-of-function in subsets of cells kcc RNAi under control of Upstream Activation Sequence; UAS) to drive promoter-determined kcc knockdown in F1 progeny (i.e. promoter-GAL4>UAS-kcc-RNAi progeny). We tested a total of 64 different GAL4 drivers in combination with two different kcc RNAi transgenes for BS paralysis. We found that this was a robust method of inducing seizure-sensitivity, particularly using UAS-kcc-RNAi-B , which appeared to be more effective than UAS-kcc-RNAi-V (Loss of -RNAi-V) . Twenty-c155 with either kcc-RNAi transgene . Cholinergic neuron drivers caused semi-lethality and 100% BS paralysis in escapers with kcc-RNAi-V, and produced late pupal lethality with kcc-RNAi-B or nearly complete (>90%) BS paralysis, and c507 and OK6 drivers cause lethality. These data suggest that a threshold of kcc RNAi. Drivers expressing in all or many neurons induced lethality and/or extreme BS paralysis and were especially sensitive to behavioral seizure-like activity. Gentle handling of the vial was sufficient to trigger seizures; this appears to be the most severely seizure-sensitive genotype that we have observed for Drosophila to date. Substantial (90%) BS paralysis was also observed with NP2222-GAL4 driving kcc-RNAi-B was about one-fourth that of wild-type and control genotypes had a low threshold of 17.5\u00b11.5 V HFS. Thresholds were even lower for flies of the genotype Gli-GAL4>kcc-RNAi-B (9.9\u00b11.9 V HFS) expressing RNAi in the SPG subset of glia. Representative traces of seizure-like discharges recorded from dorsal longitudinal muscles of the aforementioned genotypes are presented in kcc by RNAi in either nerve cells or glial cells increases seizure-sensitivity.Reducing scharges , consistrepo-GAL4>kcc-RNAi-B,mCD8::GFP; volume: 5.49\u00d7106\u00b12.70\u00d7105 \u00b5m3, n\u200a=\u200a12) was significantly larger than for wild-type brains . ComparaCD8::GFP 2, and OKCD8::GFP , were enCD8::GFP .repo-GAL4>kcc-RNAi-B,mCD8::GFP). Whereas, in wild-type brains mushroom body neuronal cell bodies and calyces were wrapped and interlaced with glial membranes in genotypes with CD8::GFP , and theCD8::GFP calyces CD8::GFP . Kcc fluol calyx , howeverkcc RNAi transgenes and UAS-ncc69-RNAi-V or cell volume phenotypes Although a glial 7 V HFS) . A represitivity and cellncc69 expression in third instar larvae caused abnormalities in abdominal peripheral nerves +/K+/2Cl\u2212 transport function) show prominent localized swellings and defasciculation of neuronal processes, termed \u201cfraying\u201d kcc loss-of-function. repo-GAL4>kcc-RNAi-B,mCD8::GFP larvae displayed an increase in the average nerve cross-sectional area along the entire length of their nerves , albeit less severe than that of glial kcc knockdown . Thus, the questions related to CCCs in glia require more investigation to be answered.Excitatory GABA signaling is an unlikely mechanism for seizure-sensitivity following + stained with anti-GFP (A1) and anti-Kcc (A2). Faint auto-fluorescence in GFP-negative embryos arising from developing gut can be seen in the GFP channel confirming that they, and other similarly scored specimens, were not unfertilized eggs. GFP-negative embryos were presumably Ad4kcc/Ad4kcc. Marginal Kcc signal was detected surrounding all embryos of any genotype suggesting a negligible degree of non-specific staining or presence of persistent maternally-deposited Kcc. (A3) GFP-negative embryos were almost always Kcc-negative (n\u200a=\u200a42 out of 51). Thus, kccAd4 appears to be a null mutation. Representative images of 20\u201322 h-old F1 embryos of self-crossed EY08304kcc/Cyo,Act-GFP flies stained with anti-GFP (B1) and anti-Kcc (B2). GFP-negative embryos are presumably EY08304kcc/EY08304kcc. (B3) GFP-negative embryos were almost always Kcc-negative (n\u200a=\u200a28 out of 33). Thus, EY08304kcc appears to be a null mutation. (C) Quantification of highly significant Kcc loss in Ad4kcc/Ad4kcc mutant embryos compared to controls. (D) Quantification of highly significant Kcc loss in EY08304kcc/EY08304kcc mutant embryos compared to controls. Error bars are S.E.M. and significance for Student's t-tests is: *** \u200a=\u200ap<0.001. Scale bars are in microns.(TIF)Click here for additional data file.Table S1The genotypes and sources of the Drosophila strains used in this study.(DOC)Click here for additional data file.Table S2All results from the UAS-kcc-RNAi-V and UAS-kcc-RNAi-B screen for RNAi-induced behavioral seizure-like activity.(DOC)Click here for additional data file.Movie S1Behavioral seizure-like activity of three 24-48 h-old repo-GAL4>kcc-RNAi-B flies.(MP4)Click here for additional data file.Movie S2Behavioral seizure-like activity of eight 24-48 h-old Gli-GAL4>kcc-RNAi-B flies.(MP4)Click here for additional data file.Movie S3Behavioral seizure-like activity of six 24-48 h-old repo-GAL4>ncc69-RNAi-V flies.(MP4)Click here for additional data file."} +{"text": "Pseudoalteromonas sp. strains isolated from surface waters at Kabeltonne, Helgoland, a long-term ecological research station in the North Sea. These strains contribute knowledge of the genomic underpinnings of a developing model system to study phage-host dynamics of a particle-associated ocean copiotroph.Draft genomes are presented for 6 Pseudoalteromonas are Gram-negative aerobic chemoorganoheterotrophs of marine microbial communities to be further assembled with the original reads using ALLPATHS-LG (http://www.ncbi.nlm.nih.gov/books/NBK174280). Genome completeness was estimated with CheckM . Indexed paired-end sequencing libraries were prepared with an Illumina TruSeq DNA sample preparation kit and sequenced on an Illumina HiSeq2000 platform. The 100-bp reads were quality trimmed using sickle version 1.33 with no h CheckM .Pseudoalteromonas phage-host system, prophages were predicted with VirSorter and the RefSeq virus database , LODI00000000 (strain H71), LODK00000000 (strain H100), LOFG00000000 (strain H103), LOFH00000000 (strain H105), and LOFI00000000 (strain 10-33). This paper describes the first versions.This whole-genome shotgun project has been deposited in GenBank under the following accession numbers:"} +{"text": "Guidelines recommend screening of epidemiologically linked patients to contain the spread of NDM-producing enterobacteriaceae (NDM-EB).We assessed the effectiveness of this strategy in controlling the spread of NDM-EB using Epi-score and whole-genome sequencing (WGS).This study was conducted at Tan Tock Seng Hospital (TTSH) between September 2010 and December 2011. TTSH implemented a reactive infection control strategy which constituted pre-emptive cohorting and rectal surveillance of patients with epidemiological linkage (contacts) to patients with NDM-EB isolated from clinical cultures (index). A clinical transmission model was produced for NDM-EB patients both from clinical and surveillance cultures based on epidemiological relatedness. The Epi-score graded epidemiologic-relatedness from 0 (unrelated) to 4 (very related), based on spatiotemporal ward overlap (2 points), shared medical teams (1 point), and shared medical department (1 point). This was the compared with a molecular transmission model which was produced using WGS of all NDM-EB isolates with core-genome single-nucleotide polymorphism analysis after excluding recombinant sites.A total of six index clinical NDM-EB were detected (5 urine and 1 bile). Contact screening as part of reactive strategy involved 436 patients, of which 2 were newly-detected NDM-EB carriers. Epi-score distributions: 3 points ; 2 points , 1 point . Of the 3 point pairs, one (5-6) was confirmed direct transmission by whole-genome phylogenetic analysis (4 SNVs). None of the other patients were identified by WGS as direct transmission. In six (75%) isolates, NDM was carried on plasmid pTR3 which is unique to Singapore.Only one direct ward transmission pair (confirmed by whole-genome-sequencing) was detected by the reactive strategy. The Epi-score performed well in classifying the direct ward transmission pair in the highest relatedness category. Most (75%) NDM-producing EB originated in Singapore, with possibly 25% of NDM-producing EB from overseas.None declared."} +{"text": "Systemic siRNA administration to target and treat glioblastoma, one of the most deadly cancers, requires robust and efficient delivery platform without immunogenicity. Here we report newly emerged multivalent naked RNA nanoparticle (RNP) based on pRNA 3-way-junction (3WJ) from bacteriophage phi29 to target glioblastoma cells with folate (FA) ligand and deliver siRNA for gene silencing. Systemically injected FA-pRNA-3WJ RNPs successfully targeted and delivered siRNA into brain tumor cells in mice, and efficiently reduced luciferase reporter gene expression (4-fold lower than control). The FA-pRNA-3WJ RNP also can target human patient-derived glioblastoma stem cells, thought to be responsible for tumor initiation and deadly recurrence, without accumulation in adjacent normal brain cells, nor other major internal organs. This study provides possible application of pRNA-3WJ RNP for specific delivery of therapeutics such as siRNA, microRNA and/or chemotherapeutic drugs into glioblastoma cells without inflicting collateral damage to healthy tissues. PBS injected mice were used as fluorescence negative control. Major internal organs together with brain from the harvested mice were collected and subjected to fluorescence imaging for assessment of biodistribution profile study.To investigate the delivery of pRNA-3WJ RNPs in vivo, U87EGFRvIII-Luc cell-induced brain tumor was prepared into two groups of mice (n=5). At 5, 7 and 9 days post-surgery, 1 mg/kg of mouse body weight of FA-Alexa647-pRNA-3WJ-si(Luc) RNP (or siScrm as negative control) was injected through the mouse tail vein in 100 \u03bcL of PBS. After each injection, mice were subjected to bioluminescence whole body imaging to detect the endogenous luciferase expression level. Mice were injected with 75 mg/kg Luciferin , and anesthetized. Bioluminescence from the anesthetized mice was detected by ZFOV-24 zoom lens-installed IVIS Lumina Series III Pre-clinical In Vivo Imaging System . The luminescence intensity was expressed as Averaged Radiance , then normalized by tumor volume (mm3).To investigate the siRNA delivery and silencing effect of pRNA-3WJ RNPs p<0.05 was considered significant.All statistical analyses comparing groups of mice treated with test and control RNPs were performed by either ANOVA or student t-test."} +{"text": "The secondary messenger cGMP does not only play an important role in the cardiovascular system (vasodilation and inhibition of platelet aggregation), but also regulates the functions of numerous cell types like immune cells, adipocytes or neurons ,2. BesidAdditionally, extracellular cGMP could be degraded by ecto-phosphodiesterases and the degradation products (GMP and guanosine) may have first messenger functions of their own. In summary, the biological functions of cGMP are much more versatile than previously acknowledged. Therefore, our project aims at differentiating between first- and second messenger functions of cGMP and the other cyclic nucleotides (cNMPs) cAMP, cUMP and cCMP in various cellular systems and functional readouts.-), (2) flow cytometric cell cycle determination of HEL (human erythroleukemia) cells, (3) ELISA-based quantitation of T-cell receptor-mediated (anti-CD3 antibody stimulation) IL-2 production of HuT-78 lymphoma cells and (4) determination of chloride flux in Calu 3 lung epithelial cells with the chloride-sensitive dye MQAE (N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide).The second messenger functions of cGMP were imitated by the membrane-permeable acetoxymethyl ester analogue (cGMP-AM), which releases cGMP after intracellular hydrolysis. Unmodified cGMP was applied for the characterization of extracellular first messenger effects. In a similar way, the first- and second messenger effects of cAMP, cCMP and cUMP were characterized. As cellular models and functional readouts we used (1) flow cytometric determination of apoptosis (annexin V/propidium iodide staining) of wildtype S49 lymphoma cells (S49 wt) and their kinase-negative counterparts caused apoptosis in S49 wt or S49 kinThe T cell receptor-mediated IL-2 production of HuT-78 cells was inhibited by cGMP, but not by cGMP-AM. The CFTR-mediated chloride flux in Calu 3 cells was slightly stimulated by cAMP-, cGMP- and cUMP-AM, but cCMP-AM had no significant effect. No significant stimulation of chloride flux in Calu 3 cells was observed with cell-impermeable unmodified cNMPs.Our data suggest that first and second messenger functions of cGMP depend on cell type and functional readout. Thus we plan to perform further studies with a variety of model systems in order to generate a \u201cfunctional profile\u201d of first- and second messenger effects of cGMP. Moreover, the surprisingly strong pro-apoptotic effect of cCMP-AM on S49 cells warrants similar studies with cyclic pyrimidine nucleotides. cCMP and cUMP now fulfil most of the criteria for second messenger functions . Finally"} +{"text": "In contrast, relatively low levels of BLyS in the blood of HIV-uninfected CSWs coincided with a rather preserved B-cell compartment.We have previously shown that excess B lymphocyte Stimulator (BLyS)/BAFF in plasma and on surface of blood dendritic cells (DC) of HIV-infected progressors coincides with B-cell dysregulations and increased frequencies of \u201cprecursor\u201d innate marginal zone (MZ)-like B-cells. In contrast, both blood BLyS levels and frequencies of this population remained unaltered in HIV elite-controllers. Based on these observations, we hypothesized that control of BLyS and innate B-cell status could be associated with natural immunity against HIV infection. Therefore, we assessed blood BLyS levels and B-cell status in HIV highly-exposed commercial sex workers (CSWs) from Benin. We found blood BLyS levels of HIV-uninfected CSWs were lower than those observed in both HIV-infected CSW and HIV-uninfected non-CSW groups. Furthermore, levels of BLyS expression on blood T-cells and monocytes were lower in HIV-uninfected CSWs when compared to HIV-infected CSWs, but higher than those observed for HIV-uninfected non-CSWs. Concomitantly, HIV-infected CSWs presented a dysregulated blood B-cell compartment, characterized by increased total IgG1, increased frequencies of populations presenting immature and/or innate profiles and a higher ratio of IgG The detailed characterization of the Ig repertoire of cervical and systemic B-cells from a Kenyan HESN individual revealed that site-specific responses occur with unique regulation of tolerance and recruitment into local memory or blast B-cell compartments, and the infusion of systemic post-germinal center (GC) B-cells to the cervix seems to be a common eventAlthough the specific factors responsible for the natural immunity against HIV have yet to be fully unraveled, we believe that observations from HIV elite-controllers (EC) can shed some light. As such, our previous studies suggest that control of HIV disease progression may be linked to B lymphocyte Stimulator (BLyS)/BAFF expression status, and to its capacity of orchestrating B-cell population dynamics and responses1616The socio-demographic characteristics of female CSWs and non-CSWs are shown in - T-cells were increased in the blood of both HIV-uninfected and HIV-infected CSWs when compared to HIV-uninfected non-CSWs . Na\u00efve resting B-cell frequencies were higher in HIV-uninfected CSWs when compared to those in HIV-infected CSWs, but lower than those found in HIV-uninfected non-CSWs . Convers16ted CSWs . Altoget+ and IgG+ plasmablasts but higher percentages of IgA+ plasmablasts when compared to HIV-infected CSWs CSWs, may involve relatively high frequencies of BLyS expressing cells while preserving homeostatic regulation of its cell surface expression level and soluble release. Recent studies have shown that in contrast to mDCs, plasmacytoid DCs exposed to HIV The relatively higher levels of BLyS observed in the serum of HIV-infected CSWs is consistent with previous reports by us and others showing that BLyS expression is increased in the context of HIV disease, and not fully restored following therapy1625319+ plasmablasts recently described in the blood of HIV-infected individuals+, whereas IgA+ plasmablasts were found predominantly in HIV-uninfected CSWs and controls, likely reflecting homeostatic requirements at mucosal interfaces such as the GALT+ plasmablasts and IgG1 hyperglobulinemia in the blood of HIV-infected CSWs is consistent with polyclonal B-cell activation found in chronic viral infections. However, we noticed a relative reduction in serum IgG2 in these individuals when compared to HIV-uninfected CSWs, which could reflect an increased trigger/need for IgG1 or perhaps IgG2 class switch interference by HIV factors such as Nef\u200933In contrast to HIV-uninfected CSWs, and consistent with previous findings by us and others1530The fact that relative frequencies of \u201cmature\u201d MZ-like B-cells were reduced in the blood of both HIV-uninfected and HIV-infected CSW groups may reflect chronic exposure to HIV at peripheral mucosal entry and/or replication sites, and recruitment of this cell population and its involvement in immunity against HIV. We have previously observed the reduction of this population in the blood of HIV-infected slow progressors and ECThe B-cell phenotype we describe in the blood of HIV-uninfected CSWs likely reflects an active but regulated immune response against HIV, and is reminiscent of that we reported for EC16183739As such, the recent characterization of transient Gp41-specific IgA in mucosal genital fluids from patients within the first weeks after HIV transmission, suggest these Abs might have originated from first-line B-cell populations+ blood T-cell counts and HIV plasma viral loads are provided for HIV-infected CSWs in Female CSWs were recruited through a dedicated sex worker clinic in Cotonou, Benin. Non-CSW HIV negative community control subjects at low risk for exposure were enrolled from a separate general health clinic in Cotonou. Women were invited to participate in the study as they attended clinics. Women under 18 years old, pregnant or menstruating were excluded from the study. Recruited women were asked to answer a questionnaire about their age, duration of prostitution (if applicable), number of clients per week (if applicable), sexual behavior, vaginal douching practices, and use of condom or hormonal contraceptive. Subjects underwent vaginal examination by a physician and vaginal specimens were used for diagnosis of candidiasis, trichomoniasis and bacterial vaginosis by microscopic examination and Herpes simplex virus (HSV) infection by polymerase chain reaction (PCR). Endocervical swabs were obtained for the detection of Neisseria gonorrhea and Chlamydia trachomatis (BD ProbeTec Et system). Blood samples were collected and tested for HIV status, diagnosis of syphilis and progesterone levels. Subjects with fever, visible vaginal inflammation and infection other than HIV were excluded from the study. HIV-1 positivity was defined by the presence of HIV-1 specific IgG tested with Vironostika HIV Uni-Form II Ag/Ab . Non-reactive samples were considered HIV seronegative, whereas reactive samples were tested with Genie II HIV-1/HIV-2 . Genie II dually reactive samples (to HIV-1 and HIV-2) and discordant samples (Vironostika reactive/Genie II (non-reactive) were further tested by INNO-LIA HIV I/II Score . For the present study we selected samples from 10 HIV-1-uninfected and 10 treatment-na\u00efve HIV-1-infected CSWs, and 21 HIV-1-uninfected non-CSW control subjects from the general population. CD4Written informed consent was obtained from all subjects who participated in the study. The investigation and methods reported in this paper were approved and carried out in accordance with guidelines and regulations by the Comit\u00e9 National Provisoire d\u2019\u00c9thique de la Recherche en Sant\u00e9 in Cotonou and the CHUM Research Ethics Committee.Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood by centrifugation on Ficoll gradients, washed and suspended in freezing medium , 10% DMSO) and kept in liquid nitrogen until use. Plasma and serum were kept frozen at \u221280\u2009\u00b0C until use.Serum levels of lipopolysaccharide (LPS), lipopolysaccharide binding protein (LBP) and soluble CD14 (sCD14) were measured respectively with commercial Enzyme-linked immunosorbent assay (ELISA) kits by CUSABIO , Hycult Biotech , and R&D systems according to manufacturers\u2019 protocol. Serum levels of BLyS were determined by a commercial ELISA kit by R&D systems .Levels of total immunoglobulin and isotypes IgG1, IgG2, IgG3, IgG4, IgM and IgA were measured in serum using the multiplex bead assay Milliplex Map Kit with human immunoglobulin isotyping Magnetic Bead panel by EMD Millipore according to manufacturer\u2019s protocol. Analysis was performed on a Luminex 200 System .6 PBMCs per sample were used for staining. Live/Dead cell exclusion was performed using AQUA Live/Dead Fixable stain . Non-specific binding sites were blocked using a Fluorescence-activated cell sorting (FACS) buffer supplemented with 20% hi-FBS and 10ug mouse IgG per 106 cells. PBMCs were stained using the following fluorochrome conjugated mouse anti-human monoclonal antibodies: AlexaFluor 700-A anti-CD16, APC anti-CD11c, FITC anti-CD3, PE-Cy7 anti-HLA-DR, and PerCP-Cy5.5 anti-CD14 , PE anti-BLyS . PBMCs were fixed with 1.25% paraformaldehyde and kept at 4\u2009\u00b0C for a minimum of 12\u2009hours before flow cytometry analysis. A minimum of 105 events per sample were acquired with a LSRFortessa and analyzed with FlowJo7.6.3 software . Flow-cytometry data analysis quadrants were set based on the expression values obtained with fluorescence minus one (FMO) and isotype controls. Representative FMO staining controls can be viewed in PBMCs were thawed and washed with RPMI 1640 followed by 1X PBS. A maximum of 10PBMCs processing, staining and analysis were performed as mentioned above. PBMCs were stained using the following fluorochrome conjugated mouse anti-human monoclonal antibodies: APC-Cy7 anti-CD10, Pacific Blue anti-CD19 , Alexa Fluor700 anti-CD27, FITC anti-IgM, PE anti-CD21, BV650-anti-CD19, APC-H7-anti-IgG, BV605-anti-IgM , APC-anti-CD3, APC-anti-CD56, PE-Cy7-anti-CD20, PE-anti-CD38, PerCP efluor710 anti-CD1c , and FITC-anti-IgA . Flow-cytometry data analysis quadrants were set based on the expression values obtained with fluorescence minus one (FMO) and isotype controls. Representative FMO staining controls can be viewed in Data from HIV-uninfected CSWs were compared separately from those of HIV-infected CSWs and HIV-uninfected non-CSWs. The statistical significance of difference between groups was determined by Fisher\u2019s exact test for categorical variables and unpaired Student\u2019s T-test or one-way ANOVA analysis for variance when continuous variables were normally distributed or by Mann-Whitney U test otherwise. The D\u2019Agostino-Pearson normality test was used to determine whether the values were sampled from a Gaussian distribution. Analyses were performed using GraphPad Prism 5.00 for Windows .How to cite this article: Sabourin-Poirier, C. et al. Blood B Lymphocyte Stimulator (BLyS)/BAFF levels may reflect natural immunity to HIV in highly exposed uninfected Beninese Commercial Sex Workers. Sci. Rep.6, 32318; doi: 10.1038/srep32318 (2016)."} +{"text": "Novel motion-corrected single-shot phase sensitive inversion recovery (PSIR) late gadolinium enhancement (moco-LGE) cardiovascular MR in 3.0T system may have advantages over conventional segmented breath-held LGE (bh-LGE), especially for vulnerable patients with arrhythmia or respiratory motions.In a consecutive cohort of 58 patients referred for clinical enhanced cardiac MR, bh-LGE and moco-LGE were collected contemporarily with identical image parameters using a 3.0T scanner. The moco-LGE was acquired just after the bh-LGE while the patients were asked to breathe freely. Images were randomized and scored for image quality and diagnostic confidence for myocardial LGE separately base on the American Heart Association 17-segmented model. In patients with diagnostic image quality and definite LGE with identifiable margin, the myocardial LGE mass was quantified. Paired t test was used to compare the image quality, diagnostic confidence. Linear regression and correlation plots were used to compare LGE mass.2=0.95, P=0.000) without bias.55 patients had regular heart rate (HR), the mean HR was 78\u00b114 beats per minute (bpm). The other 3 patient had irregular HR including atrial fibrillation and atrial flutter. In all the patients, the moco-LGE with free-breathing had similarly high image quality , and diagnostic confidence compared with bh-LGE. A total of 16 patients with marked image artifacts in bh-LGE for arrhythmia or respiratory motion, moco-LGE had significantly higher image quality and confidence. The myocardial LGE mass was quantified and compared in 22 patients, the results correlated highly (RIn general, moco-LGE and bh-LGE have similar image quality and myocardial LGE quantification. In vulnerable patients with marked artifacts of bh-LGE, moco-LGE probably has higher image quality and diagnostic confidence.N/A."} +{"text": "The 48-week interim analysis of the MODAT study showed that confirmed virologic failure (CVF) was more frequent in patients simplifying to ATV/r monotherapy compared to maintaining ATV/r-based triple therapy. The DSMB recommended stopping study enrollment but continuing follow-up of enrolled patients. We present the 96-week efficacy analysis.Multicentre, randomized, open-label, non-inferiority trial (non-inferiority margin \u221210%). Treatment failure (TF) was defined as CVF (two consecutive HIV-RNA >50 cp/mL) or discontinuation for any cause. In the monotherapy arm, patients with CVF re-introduced their previous NRTIs and remained in the study if HIV-RNA <50 copies/mL within 12 weeks of re-intensification.101 patients evaluated : 85% malDespite the small sample size, the primary 96-week analysis showed that simplification to ATV/r monotherapy showed inferior efficacy to maintaining ATV/r triple-therapy but appeared to be superior when re-intensification was considered success."} +{"text": "AML patients are reported to have impairments of immune cells which contribute to leukemia progression. Tumor-derived exosomes (TEX) have recently emerged as carriers of the molecular and genetic cargo with potent immunosuppressive properties. We showed that plasma of newly-diagnosed AML patients prior to any therapy contained high levels of exosomal proteins relative to those in plasma of normal donors (NC). AML exosomes were enriched in membrane-associated TGF-\u03b21, MICA/MICB and markers of myeloid blasts. We hypothesize that these plasma-derived virus-size (30-100nm) membrane-bound vesicles operating in AML deliver suppressive signals to immune cells and thus may promote leukemia progression.Venous blood (20-50 mL) was obtained from patients newly diagnosed with AML prior to any treatment, from AML patients who achieved CR following initial therapy and from age-matched NC. Exosome fractions were isolated from plasma using mini-size exclusion chromatography (mini-SEC) with Sepharose 2A. Exosome protein levels, numbers and size of exosomes (qNano) and their morphology (TEM) were determined. Exosomes were characterized by Western blots for expression of exosome markers, Tsg101 and CD81, and myeloid cell-surface markers associated with AML, interleukin-3 receptor alpha chain (CD123) and C-type lectin-like molecule-1 (CLL-1), CD44 and CD96. Isolated normal human NK cells were co-incubated with AML exosomes and multiparameter flow cytometry was used to monitor changes in expression levels of NKG2D on NK cells.Exosome fractions isolated from AML patients' plasma at diagnosis (n=16) had higher mean protein content than fractions obtained from NC plasma . Exosomes were isolated from paired AML plasma samples obtained at diagnosis and in CR after chemotherapy (n=13). In the exosome fractions isolated at CR, at the time when leukemic blasts were undetectable in the bone marrow by conventional hematopathological methods, protein levels remained elevated . AML exosomes isolated at CR carried blast markers, CD123, CLL-1, CD44, CD96 and were enriched in the mature form of TGF-\u03b21. Co-incubation of these exosomes with activated normal human NK cells resulted in down-regulation of NKG2D expression with a concomitant reduction of NK-cell cytotoxicity. Ten/13 AML patients in CR whose plasma exosome levels remained elevated eventually relapsed.The exosome fractions in plasma of patients with AML are enriched in blast-derived exosomes carrying leukemia blast markers and biologically-active TGF-\u03b21. The persistently elevated levels of these exosomes in plasma of AML patients in CR contribute to impaired anti-leukemia immune responses and promote leukemia progression."} +{"text": "Infections caused by multi-drug resistant (MDR) Gram-negative (GN) organisms in critically ill patients are a therapeutic challenge. Few therapeutical options are available.The administration of high-dose aminoglycoside (HDA) therapy coupled with high-flow continuous veno-venous hemodiafiltration (CVVHDF) could allow required high drug peaks to be achieved with reduced toxicity.All adult patients present on the intensive care unit (ICU) between October 2009 and July 2014 who had MDR-GN sepsis were considered for HDA and high-flow (>45 mL/kg/h) CVVHDF when an isolated pathogen was at least partially sensitive to aminoglycosides and the patient's condition was not improving with conventional therapy. Optimal antibacterial activity was defined as a peak concentration of at least 8 times the minimal inhibitory concentration.Fifteen patients infected with MDR-GN pathogens were treated with amikacin (n = 11), gentamicin (n = 3) or tobramycin (n = 1) and high-flow CVVHDF. A favorable clinical response was observed in 8 (53%) patients, including 3 in whom microbial eradication was obtained. Six patients were discharged alive from the ICU, and five from the hospital. No renal toxicity was observed.In this cohort of septic patients with MDR-GN infections, HDA combined with high-flow CVVHDF represented a valuable therapeutic option. The effectiveness of this approach should be further evaluated in larger studies."} +{"text": "Borna disease virus (BDV), a non-segmented, negative-strand RNA virus, establishes a persistent infection without obvious cytopathic effects. Unique among animal non-retroviral RNA viruses, BDV persistently establishes a long-lasting persistent infection in the nucleus. These features make BDV ideal for RNA virus vector persistently expressing small RNAs. Here, we demonstrated that the recombinant BDV (rBDV) containing the miR-155 precursor, rBDV-miR-155, persistently expressed miR-155 and efficiently silenced its target gene. The stem region of the miR-155 precursor in rBDV-miR-155 was replaceable by any miRNA sequences of interest and that such rBDVs efficiently silence the expression of target genes. Collectively, BDV vector would be a novel RNA virus vector enabling the long-term expression of miRNAs for RNAi therapies.RNA interference (RNAi) has emerged as a promising technique for gene therapy. However, the safe and long-term expression of small RNA molecules is a major concern for the application of RNAi therapies Small RNA molecules, such as small interfering RNAs (siRNA), microRNAs (miRNA) and short-hairpin RNAs (shRNA), are central to RNA interference (RNAi), a naturally occurring regulatory mechanism causing posttranscriptional gene silencing in most eukaryotic cells1111in vivoPosttranscriptional gene regulation by RNAi is a promising approach to gene-specific suppression and new therapeutic strategies against various diseases. At present, however, RNAi-based therapies have largely not been practical, because of a lack of tools for delivering these small RNA molecules efficiently into the target cells. Thus, one of the major challenges of RNAi-based therapies is the delivery of the RNAi molecules to the correct cells at the right time. Furthermore, stable and secure expression of these micromolecules in the cells is required for the RNAi therapies to be effective Currently, viral vector systems are used widely for the expression of miRNAs and shRNAs. Adeno-associated virus (AAV), adenovirus and retrovirus vectors are commonly used as delivery systems for these small RNA molecules. Although these systems may express the micromolecules effectively, each vector has specific limitations that restrict its application. For example, it is known that AAV and adenovirus vectors are gradually diluted out in dividing cells and, therefore, are not suitable for the long-term expression of small RNAs678Vectors based on negative-strand RNA viruses (NSV) for expression of small RNAs have been reported rarely because almost all RNA viruses are transcribed in the cytoplasm, whereas the processing machinery of pri-miRNAs is in the nucleus. Recently, we established a novel RNA virus-based vector using Borna disease virus (BDV) reverse genetics1012121314Here, we report the establishment of a novel BDV vector carrying a pri-miRNA-cassette sequence in an intercistronic, noncoding region of the viral genome. The recombinant BDV (rBDV), rBDV-miR-155, containing the pri-miR-155 sequence, stably expressed miR-155 in cultured cells for a long period of time and efficiently silenced a reporter gene containing the miR-155 target sequence in the 3\u2032-untranslated region (UTR). Furthermore, downregulation of Dicer abrogated silencing of the target by rBDV-miR-155. We also demonstrate that the pre-miRNA sequence in the pri-miR-155-cassette is replaceable by any miRNA sequences of interest and that such BDV vectors, named miBDV vectors, can efficiently suppress the expression of endogenous and exogenous target genes. Our results show that the BDV vector can be used for the delivery and long-term expression of small RNA molecules and should provide a novel tool for RNAi therapies.Bst BI and Pac I restriction sites of the cassette region using a standard BDV reverse genetics protocol established previously To determine whether functional miRNAs are expressed from BDV vectors, we generated a luciferase reporter plasmid, pLuc-miR-155as, containing an artificial 22-nt sequence complementary to miR-155 inserted into the 3\u2032-UTR of the luciferase gene , and traTo determine whether the functional miRNA in the rBDV-miR-155-infected cells was expressed from antigenomic RNA or mRNA encoding the pri-miR-155 sequence, we measured the levels of BDV RNAs. If the mature miRNAs were produced directly from the pri-mRNA sequences in the antigenomic RNA by Drosha, the level of BDV antigenome RNA should be reduced in the rBDV-miR-155-infected cells. As shown in 16Bbs I sites into the pri-miR-155 region of pBDV-miR-155, which enable the replacement of pre-miR-155 with the pre-miRNA of any miRNA sequences of interest . As shown in in vivo for RNAi therapiesVirus vectors provide a promising approach to delivering small RNAs Recently, it has been reported that some RNA viruses, such as sindbis virus (SINV), vesicular stomatitis virus and influenza A virus (IAV), can be engineered to express miRNAs2021222420In this study, we used a BDV vector to develop a novel NSV vector able to express miRNAs efficiently and persistently. BDV possesses unique features suitable for the long-term expression of miRNA molecules. Firstly, BDV replicates in the nucleus12131412The use of BDV vector for humans may be a concern since BDV infection has been known to induce a fatal encephalitis in some animals, including horse and sheep2831In the miBDV vector system, viral mRNA, which encodes the pri-miRNA sequences, could be the source of mature miRNA in the target cells, consistent with the previous studies16We found that duplication of the pri-miRNA sequence in the miBDV vector system could enhance silencing activity on expression of the target gene. On the other hand, we could not detect significant differences in luciferase activity between rBDV-miR-155x2 and rBDV-155x4-infected cells, using the reporter system . We obseTo produce artificial miRNAs of interest from the miBDV vector, we replaced both the mature miR-155 and its complementary sequences within the stem of the pri-miR-155 sequence. We used the loop region of pre-miR-155 between the stem sequences, because the loop sequences are likely to be required for efficient nuclear export of pre-miRNAIn summary, we demonstrated the potential of the miBDV vector for the long-term expression of small RNAs. The miBDV vector may provide potential solutions that overcome several concerns underlying the existing vector systems for small RNA-delivery. Further improvement of the miBDV vector, such as increasing the efficiency of miRNA processing and reducing the pathogenicity of BDV, is necessary to expand the utility of this vector system for RNAi therapies.BstBI and PacI sites of pBDV. The reporter plasmid for miRNA was generated by subcloning the firefly luciferase gene of the pGL5 plasmid (Promega), with an artificial 22-nt sequence complementary to the miRNA in the 3\u2032UTR, into the EcoRI and XbaI sites of the pcDNA3 plasmid (Invitrogen). A pair of oligos (5\u2032-ACC GGC TCC TAC CTG TTA GCA TTA AGT TAT TCA AGA GAT AGC TTA ATG CTA ATT GTG ATA GGG GTT TTT TGG GCC C-3\u2032 and 5\u2032-CGA AGG GCC CAA AAA ACC CCT ATC ACA ATT AGC ATT AAG CTA TCT CTT GAA TAA CTT AAT GCT AAC AGG TAG GAG C-3\u2032) were annealed and inserted into the BbsI sites of pRSI9-U6-(sh)-UbiC-RFP-2A-Puro (Cellecta) (pRSI9-U6-miR-155-UbiC-RFP-2A-Puro).The BDV vector plasmid harboring an extra transcription cassette, pBDV, was generated by subcloning a cloned full-length cDNA of He/80, with the insertion of transcription initiation (S3) and termination (T2) signal sequences between the P and M genes, into pCAG-HRSV3, as described previouslyThe sequences of the primers used in this study are as follows:BDV genome-specific primer, 5\u2032-GTT GCG TTA ACA ACA AAC CAA TCA T-3\u2032BDV antigenome-specific primer, 5\u2032-TGC GCT ACA ACA AAG CAA CAA CC-3\u2032BDV-forward primer, 5\u2032-ATG CAT TGA CCC AAC CGG TA-3\u2032BDV-reverse primer, 5\u2032-ATC ATT CGA TAG CTG CTC CCT TC-3\u2032miR-Tubb3-specific primer, 5\u2032-TTA ACC TGG GAG CCC TAA TGA G-3\u2032miR-GAPDH-specific primer, 5\u2032-TTG ATG ACA AGC TTC CCA TTC T-3\u2032let-7a-specific primer, 5\u2032-TGA GGT AGT AGG TTG TAT AGT T-3\u2032Vero cells (a monkey kidney cell line) were cultured in Dulbecco\u2019s modified Eagle\u2019s medium (DMEM) supplemented with 2% fetal bovine serum (FBS). OL cells (a human oligodendrocyte cell line), 293T cells (a human embryonic kidney cell line) and 293LTV cells were cultured in DMEM supplemented with 5% FBS.rBDVs were produced by reverse genetics as reported previouslyVero cells were infected with rBDVs at an MOI of 0.01 at 37\u2009\u00b0C. After absorption for 1\u2009h, the cells were washed with phosphate-buffer saline (PBS) and passaged every 3 days. Virus propagation was detected by IFA. Primary hippocampal neuron cultures were infected with rBDV at an MOI of 0.03 on DIV 3. The cultures were fixed on DIV 10.Lentiviral vector expressing miR-155 was prepared by co-transfection of pRSI9-U6-miR-155-UbiC-RFP-2A-Puro together with psPAX2 (Cellecta) and pMD2.G (Cellecta) in 293LTV cells. Vero cells were infected with the lentivirus at an MOI of 5 at 37\u2009\u00b0C.rBDV-infected and lentivirus-infected Vero cells were transfected with pLuc-miR-155as, pLuc-miR-Tubb3as or pLuc-miR-GAPDHas with pRL-TK (Promega). For the Dicer-knockdown experiments, rBDV-infected OL cells were transfected with siDicer (Qiagen) using Hiperfect (Qiagen). The cells were incubated for 48\u2009h and further transfected with pLuc-miR-155as and pRL-TK. For the mouse miR-155 mimic experiments, rBDV-infected Vero cells were transfected with Syn-mmu-miR-155-5p miScript miRNA Mimic (Qiagen), pLuc-miR-155as and pRL-TK using Lipofectamine 2000 (Invitrogen). At 24 or 48\u2009h after reporter transfection, the luciferase activity of the cells was measured using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer\u2019s instruction.Total RNA was extracted from infected cells and reverse transcribed using a Verso cDNA Synthesis Kit (Thermo Scientific) with BDV genome-specific or BDV antigenome-specific primers. Quantitative real-time RT-PCR (qRT-PCR) assays were carried out using a gene-specific, double fluorescent dye-labeled probe with a Rotor-Gene Q System (Qiagen), as described previouslyCells were fixed for 20\u2009min in 4% paraformaldehyde and permeabilized by incubation in PBS containing 0.25% Triton X-100 for 10\u2009min. After permeabilization, the cells were incubated with a mouse anti-GAPDH (Millipore), a mouse anti-tubulin \u03b23 (Millipore), a rabbit anti-BDV N and a rabbit anti-M antibodies for 1\u2009h. This was followed by incubation with the appropriate Alexa Fluor-conjugated secondary antibodies (Invitrogen). The cells were counterstained with 4\u2032,6-diamidino-2-phenylindole (DAPI). A confocal laser-scanning microscope ECLIPSE Ti was used for cell immunofluorescence imaging and data collection.Vero cells infected with rBDV were lysed with SDS sample buffer. The total cell lysate was subjected to SDS-PAGE and transferred onto polyvinylidene difluoride membranes . The membranes were then blocked and incubated with the primary antibodies. Antibodies used in this study were as follows: a mouse anti-BDV N (HN321), a mouse anti-GFP (Clontech) and a mouse anti-Tubulin antibodies. After three washes with 0.05% Tween 20 in TBS, horseradish peroxidase-conjugated secondary antibodies (Invitrogen) were applied for 1\u2009h at 37\u2009\u00b0C. The bound antibodies were detected using an ECL prime Western blotting system (Amersham Pharmacia Biotech).Total RNA was extracted with TRIzol reagent (Invitrogen) from Vero cells infected with rBDVs. Aliquots of 10\u2009\u03bcg total RNA were separated on a 9% denaturing Tris-Urea gel and transferred onto nylon membranes (Roche). After UV cross-linking, the membrane was prehybridized in ULTRAhyb solution (Ambion) for 30\u2009min at 37\u2009\u00b0C, followed by hybridization overnight with a digoxigenin (DIG)-labeled locked nucleic acid (LNA) probe complementary to mouse miR-155 (Exiqon), miR-Tubb3 (5\u2032-DIG-CTC ATT AGG GCT CCC AGG TTA A-3\u2032), miR-GAPDH (5\u2032-DIG-AGA ATG GGA AGC TTG T-3\u2032) and human let-7a (Exiqon). The hybridized probe was detected with an alkaline phosphatase (AP)-conjugated anti-DIG antibody (Roche).2. Cultures were maintained in Neurobasal medium (Invitrogen) with 2% (vol/vol) B27 supplements (Invitrogen).Mouse hippocampal neuron cultures were prepared from embryonic day 18 C57Bl/6 mouse embryos, as previously described, with some modificationsAll procedures including animal studies were conducted in accordance with the guidelines for the Care and Use of Laboratory Animals of the Ministry of Education, Culture, Sports, Science and Technology, Japan. All experimental procedures were approved by the Institutional Animal Care and Use Committees (IACUC)/ethics committee of Kyoto University institutional review board (protocol number D12-11).How to cite this article: Honda, T. et al. Long-term expression of miRNA for RNA interference using a novel vector system based on a negative-strand RNA virus. Sci. Rep.6, 26154; doi: 10.1038/srep26154 (2016)."} +{"text": "Azospirillum brasilense is one of the most promising PGPB and wheat roots colonized by A. brasilense is a good model to investigate the molecular basis of plant-PGPB interaction including improvement in plant-NUE promoted by PGPB.The rapid growth of the world\u2019s population demands an increase in food production that no longer can be reached by increasing amounts of nitrogenous fertilizers. Plant growth promoting bacteria (PGPB) might be an alternative to increase nitrogenous use efficiency (NUE) in important crops such wheat. A. brasilense strain FP2. cDNA libraries from biological replicates of colonized and non-inoculated wheat roots were sequenced and mapped to wheat and A. brasilense reference sequences. The unmapped reads were assembled de novo. Overall, we identified 23,215 wheat expressed ESTs and 702 A. brasilense expressed transcripts. Bacterial colonization caused changes in the expression of 776 wheat ESTs belonging to various functional categories, ranging from transport activity to biological regulation as well as defense mechanism, production of phytohormones and phytochemicals. In addition, genes encoding proteins related to bacterial chemotaxi, biofilm formation and nitrogen fixation were highly expressed in the sub-set of A. brasilense expressed genes.We performed a dual RNA-Seq transcriptional profiling of wheat roots colonized by PGPB colonization enhanced the expression of plant genes related to nutrient up-take, nitrogen assimilation, DNA replication and regulation of cell division, which is consistent with a higher proportion of colonized root cells in the S-phase. Our data support the use of PGPB as an alternative to improve nutrient acquisition in important crops such as wheat, enhancing plant productivity and sustainability.The online version of this article (doi:10.1186/1471-2164-15-378) contains supplementary material, which is available to authorized users. Finally, the seeds were washed 4-times with ultrapure sterilized water and then germinated in water agar plates at 30\u00b0C for 12\u00a0h under darkness. Germinated seedlings were transferred to sterile glass tubes containing 25\u00a0mL Hoagland\u2019s nutrient solution . DEseq package was used to estimate differential gene expression, performing a negative binomial distribution and a shrinkage estimator for the distribution\u2019s variance and size-factor normalization [p-value\u2009<\u20090.05 and absolute fold-change \u2265\u20092-fold. Gene ontology analysis was performed by Blast2go software [The RNA-seq transcriptional analysis was carried out with two biological replicates of each treatment of colonized and non-inoculated software and A. bAdditional file 1: Figure S1: RNA-Seq and RT-qPCR experiments design. CWR: colonized wheat roots; N-IWR: non-inoculated wheat roots; each biological replicate was compound by five tubes with two seedlings per tube. (PDF 510 KB)Table S1B. RNA-seq: expressed and differentially expressed genes. a3\u00d7 or higher coverage; b1\u00d7 or higher coverage; cNot determined. (PDF 88 KB)Additional file 2: Table S1A: RNA-seq: mapping results. Triticum aestivum Expressed ESTs. aFold-change in red indicates down-regulation in colonized wheat roots (CWR); (+) ND not expressed in the N-IWR libraries; (\u2212) ND not expressed in the CWR libraries. (XLSX 1 MB)Additional file 3: Table S2: Triticum aestivum expressed micro-RNAs. aFold-change in red indicates lower level of expression in colonized wheat roots (CWR); bNot considered as expressed micro-RNA. (PDF 15 KB)Additional file 4: Table S3: Triticum aestivum-ESTs. aFold-change in red indicates down-regulation in colonized wheat roots (CWR); (+) ND not expressed in the N-IWR libraries; (\u2212) ND not expressed in the CWR libraries. (PDF 386 KB)Additional file 5: Table S4: Differentially expressed Triticum aestivum-ESTs in colonized roots. Table S5B. Overrepresented GO categories of down-regulated Triticum aestivum-ESTs in colonized roots. aFold-change in red indicates down-regulation in colonized wheat roots (CWR). (PDF 116 KB)Additional file 6: Table S5A: Overrepresented GO categories of up-regulated Triticum aestivum genes presented in the scatter plot: TaS_37759224**, TaS_52545880**, contig_4027***, TaS_52541981*, TaS_22370925*, TaS_37846208*, TaS_17890143*, TaS_26026646*, TaS552541449** and contig_2766**. RNA-seq data represent biological duplicates of colonized and non-inoculated wheat roots. RT-qPCR data represent biological duplicates of colonized and non-inoculated wheat roots, obtained independently from the RNA-seq samples, and each sample was run in triplicate. Significant differences in the RT-qPCR were determined using one-tailed t-test. Pearson coefficient correlation between RNA-seq and RT-qPCR was >\u20090.97, p-value\u2009<\u20090.0001. (PDF 8 KB)Additional file 7: Figure S2: Scatter plot: RNA-seq vs. RT-qPCR. RNA-seq and RT-qPCR relative level of expression are presented as log2 (fold-change). Triticum aestivum MADs-Box expressed-ESTs. aFold-change in red indicates lower level of expression in colonized wheat roots (CWR). (PDF 15 KB)Additional file 8: Table S6: Azospirillum brasilense strain FP2 colonizing Triticum aetivum seedling roots. (PDF 36 KB)Additional file 9: Table S7: Expressed ORFs of aFold-change in red indicates down-regulation in colonized wheat roots (CWR); (+) ND not expressed in the N-IWR libraries; (\u2212) ND not expressed in the CWR libraries. (PDF 30 KB)Additional file 10: Table S8: Host response found during plant-microbe interaction: Defense mechanism, hormone imbalances and secretion of phytochemicals. Triticum aestivum encoding transporters. aFold-change in red indicates lower level of expression in colonized wheat roots (CWR); (+) ND not expressed in the N-IWR libraries; Up-regulated, Down-regulated and Expressed ESTs are shading in red, blue and yellow respectively. (PDF 333 KB)Additional file 11: Table S9: ESTs of Triticum aestivum encoding proteins that were grouped in transport activity by GO analysis. aFold-change in red indicates lower level of expression in colonized wheat roots (CWR); (+) ND not expressed in the N-IWR libraries; Up-regulated, Down-regulated and Expressed ESTs are shading in red, blue and yellow respectively. (PDF 752 KB)Additional file 12: Table S10: ESTs of Triticum aestivum encoding proteins related to cell cycle regulation.aFold-change in red indicates down-regulation in colonized wheat roots (CWR). (PDF 17 KB)Additional file 13: Table S11: ESTs of"} +{"text": "TAMs, a unique and distinct M2-skewed myeloid population of tumor stroma, exhibiting pro-tumor functions is fast emerging as a potential target for anti-cancer immunotherapy. Macrophage-recruitment and M2-polarization represent key TAMs-related phenomenon that are amenable to therapeutic intervention. However successful translation of these approaches into effective therapeutic regimen requires better characterization of tumor-microenvironment derived signals that regulate macrophage recruitment and their polarization. Owing to hypoxic milieu being a persistent feature of tumor-microenvironment and a major contributor to malignancy and treatment resistance, the current study was planned with an aim to decipher tumor cell responses to hypoxia vis-a-vis macrophage homing and phenotype switching. Here, we show that hypoxia-primed cancer cells chemoattract and polarize macrophages to pro-angiogenic M2-polarized subtype via Eotaxin and Oncostatin M. Concordantly, hypoxic regions of human breast-cancer specimen exhibited elevated Eotaxin and Oncostatin M levels with concurrently elevated M2-macrophage content. Blockade of Eotaxin/Oncostatin M not only prevented hypoxic breast-cancer cells from recruiting and polarizing macrophages towards an M2-polarized phenotype and retarded tumor progression in 4T1/BALB/c-syngenic-mice-model of breast-cancer but also enhanced the efficacy of anti-angiogenic Bevacizumab. The findings established these two cytokines as novel targets for devising effective anticancer therapy particularly for tumors that are refractory or develop resistance to anti-angiogenic therapeutics. After the tumor attained a size of 6-9 mm3, the tumor bearing mice were randomized into four groups based on tumor volume with each group comprising of three-five mice. After observing tumor volume for four days, mice were treated with anti-Oncostatin M/anti-Eotaxin neutralizing antibody or isotype control antibody at tumor site on day 0, 3 and 6 at the dose of 5ug/mice. An additional group receiving equal amount of vehicle (PBS) served as control. At the end of experiment i.e. at day 7 or 8, animals were euthanatized under anesthesia and tumor was excised. The tumor samples were visualized and photographed using Leica M205FA stereo zoom microscope and processed further for flow immunohistochemistry.4T1/BALB/c tumors were initiated by injecting 1\u00d710Human breast tumor specimens were collected after informed consent from fourteen patients who underwent surgery for breast cancer at King George Medical University, Lucknow (India) The ethics committee at King George Medical University, Lucknow (India) approved the study protocol (#ECM IIB/P17), which followed the Declaration of Helsinki.Statistical evaluations were done using SPSS 17.0. Experimental data was analyzed using one way anova followed by Bonferroni post hoc test. For all tests, the level of significance was set at p<0.05. Error bars throughout the figures indicate standard error."} +{"text": "Unlike surface-associated fXII activation, platelet secretion inhibited activated fXII (fXIIa), particularly due to a released C1-inhibitor. Platelet surface-associated fXIIa formation triggered contact pathway-dependent clotting in recalcified plasma. Computer modelling suggests that fXIIa inactivation was greatly decreased in thrombi under high blood flow due to inhibitor washout. Combined, the surface-associated fXII activation and its inhibition in solution herein may be regarded as a flow-sensitive regulator that can shift the balance between surface-associated clotting and plasma-dependent inhibition, which may explain the role of fXII at high shear and why fXII is important for thrombosis but negligible in haemostasis.Coagulation factor XII (fXII) is important for arterial thrombosis, but its physiological activation mechanisms are unclear. In this study, we elucidated the role of platelets and platelet-derived material in fXII activation. FXII activation was only observed upon potent platelet stimulation accompanied by phosphatidylserine exposure and was localised to the platelet surface. Platelets from three patients with grey platelet syndrome did not activate fXII, which suggests that platelet-associated fXII-activating material might be released from \u03b1-granules. FXII was preferentially bound by phosphotidylserine-positive platelets and annexin V abrogated platelet-dependent fXII activation; however, artificial phosphotidylserine/phosphatidylcholine microvesicles did not support fXII activation under the conditions herein. Confocal microscopy using DAPI as a poly-phosphate marker did not reveal poly-phosphates associated with an activated platelet surface. Experimental data for fXII activation indicates an auto-inhibition mechanism ( Coagulation factor XII (fXII) is a serine protease zymogen that can undergo auto-activation in the presence of foreign surfaces. FXII is the main component of contact blood clotting initiation. However, fXII deficiency in humans and animals is not associated with a tendency for bleeding, which indicates that fXII is dispensable for haemostasis ; howeverInterest in fXII recently increased due to the findings that fXII-deficient mice were protected from thrombosis , 4], , and primGiven such discrepancies, we attempted to study fXII activation and answer the following questions. i) Can platelets activate fXII or simply accelerate its activation using other plasma proteins? ii) Does the platelet surface directly participate in fXII activation? iii) Do platelet subpopulations differ in fXII binding and activation? iv) Finally, which platelet component is most powerful for fXII activation: the platelet surface, MPs or released materials (including poly-P)?The results herein demonstrate preferential fXII binding by procoagulant phosphatidylserine (PS)-exposing platelets, which promote fXII activation through their surface, retain bound essential levels of fXIIa, and trigger contact pathway clotting in recalcified plasma regardless of the material secreted outside the platelet. Because \u2018grey\u2019 platelets (from grey platelet syndrome patient blood) cannot activate fXII, \u03b1-granule components are likely important for developing the fXII-activating capacity. Under the conditions herein, the platelet surface was the most powerful fXII activating component compared with platelet-derived microparticles and material secreted outside the platelet. Unexpectedly, the full effect of the material secreted outside the platelet was inhibitory, not activating. The C1-inhibitor (C1-INH) was released and specifically inhibited FXIIa. Based on the results and earlier published data, we constructed a computer model that demonstrates a potential role for filtration velocity in regulating thrombi contact pathway activation.The following materials were used: thrombin ; fXIIa and fXII ; AlignFlow flow cytometry alignment beads (2.5 \u03bcm for 488-nm excitation), fluorescein-5-isothiocyanate (FITC), and tetramethylrhodamine (TMRM) ; unlabelled and FITC-annexin V ; AlexaFluor-647-annexin V ; prostaglandin E1 (PGE1) ; PPACK ; the chromomeric substrates S2238 and S2302 ; HEPES, bovine serum albumin, Sepharose CL-2B, Protein G sepharose, apyrase grade VII, mepacrine (quinacrine), PBS, EDTA and DMSO ; calpeptin and MDL 28170 ; G1/C1-inhibitor and polyclonal goat anti-human serpin G1/C1-inhibitor antibody ; goat polyclonal anti-human factor XII antibody ; peroxidase-AffiniPure donkey anti-goat IgG ; egg phosphatidylcholine and egg phosphatidylserine ; DAPI ; and a kit to estimate fXII activation . Collagen-related peptide (CRP) was kindly provided by Prof. R.W. Farndale , and the FXIIa inhibitor corn trypsin inhibitor (CTI)) was kindly provided by Dr. Smolyaninov . Active site thrombin was titrated using PPACK and S2238 as previously described , , , 15]; h; h15]; hThese considerations may explain why fXIIa is important for thrombosis but negligibly important for haemostasis.S1 Fig(A) The effect of 200 mg/mL CTI on the rate of 200 M S2302 cleavage by fXIIa in buffer A. (B) The CTI addition timing and the level of its effect on platelet-associated fXIIa generation (S2302 cleavage). The platelets were activated with A23187 and applied to a reaction with 20% chelated plasma. We added CTI (200 mg/mL) to the reaction with platelets with simultaneously or 15 min thereafter (n = 3). (C) The effect of 50 nM fVIIai on 90% recalcified plasma clotting by 0.75 pM TF. (D) SDS PAGE analysis of the FITC-fXII preparation used in binding assays. The preparation was applied to 7.5% SDS PAGE, and the protein bands were developed using FITC fluorescence and Coomassie R250 (right image). The figures on the left indicate the percent FITC fluorescence for the corresponding bands. The figures between two images represent molecular mass markers (kDa).(TIF)Click here for additional data file.S2 FigMP formation was inhibited by 200 \u03bcM calpeptin.(TIF)Click here for additional data file.S3 Fig(A) A typical experiment where the platelet contributes to the total fXII activation through reacting with 20% plasma (the platelets were activated by 10 nM thrombin). The curve with grey squares corresponds to platelet independent fXII auto-activation; white triangles\u2014total fXII activation in the presence of platelets; asterisk\u2014the optical density decrease due to platelet aggregation; black circles\u2014calculated platelet-dependent fXII activation. (B) The effect of 200 mM S2302 on fXII activation. Factor XII activation was initiated by mixing A23187-activated platelets with plasma . S2302 was added to the different mixture aliquots with the platelets simultaneously or 5 and 20 min thereafter. We initiated the fXIIa activity measurements simultaneously for all aliquots 20 min after the reaction began. The curves correspond to the total reaction, including both platelet-dependent and platelet-independent fXIIa activity (the change in OD405 in the absence of S2302 was subtracted). (C) The dose-dependence of the S2302 conversion rate on the concentration of fXIIa added. The reaction was measured in buffer A with 200 mM S2302. Each point shows mean value (\u00b1SEM) for three experiments. The calibration curve generated was used to determine the correlation coefficient and convert the S2302 cleavage rate into the fXIIa concentration. (D) Determination of the platelet-dependent and platelet-independent fXIIa formation parameters upon reaction with purified fXII (n = 3). White circles correspond to platelet-independent fXII auto-activation; black circles correspond to the total reaction in the presence of platelets. The hyperbolic curves for the representative, typical experiment were fit using the given parameters. (E) The effect of 100-fold diluted platelet activators on platelet-independent S2302 cleavage in 20% plasma. We terminated 10 nM thrombin activation through adding 100 nM PPACK. Further, these concentrations were 100-fold lower upon platelets dilution for the reaction with plasma.(TIF)Click here for additional data file.S4 Fig(A) A histogram of FITC-fXII (450 nM) binding to platelets: 1\u2014control (platelets without fXII); 2\u2014non-activated platelets; 3\u2014activated platelets. (B) The effect of EDTA on 450 nM FITC-fXII binding to a non-activated or an activated platelet membrane. (C) Correlation between fXII binding and its added concentration for non-activated and activated PS-positive and PS-negative platelets (n = 3). (D) FITC-fXII (450 nM) binding to MPs compared with binding to PS-positive platelets. We identified the microparticles in flow-cytometry dot plots as shown in (TIF)Click here for additional data file.S5 Fig(A) The level of remaining platelets after centrifugation. (B) The level of remaining MPs after centrifugation. In panels (A and B), the mean values (\u00b1SD) were calculated for three independent experiments (n = 3). (C). The effects of calpeptin and MDL 28170 on the variation in the MP to platelets ratio (n = 3). (D) The inhibitors\u2019 effects on PS-positive platelet formation (n = 3).The microparticles were identified in flow-cytometry dot plots as shown in (TIF)Click here for additional data file.S6 Fig(A) Co-localisation of DAPI with mepacrine confirms dense granule staining. (B) Different localisation for DAPI and TMRM confirms that, under the conditions herein, DAPI does not stain mitochondria (n = 3). (C) Fluorescence spectra for 50 mM DAPI and DAPI-poly-P complex under the conditions used herein . The poly-P preparation with the chain length 1\u2013200 was kindly provided by Dr. Kulakovskaya T.V. .(TIF)Click here for additional data file.S7 Fig(A) SDS PAGE analysis (silver staining); (B) Western blot analysis using antihuman C1-INH antibody as the primary antibody; Western blot analysis using antihuman factor XII antibody as the primary antibody. Lanes 1 and 2 correspond to purified C1-INH preparation ; lanes 3\u2014molecular mass marker (the figures on the left depict the molecular masses in kDa); lanes 4 and 5\u2014immuno-precipitation results obtained in the absence or presence of added fXII, respectively; lane 6\u2014the control purified fXII immuno-precipitation performed in the absence of platelets. The arrows indicate the covalent complexes formed between C1-INH and fXII fragments under conditions herein.(TIF)Click here for additional data file."} +{"text": "Natriuretic peptides (ANP/BNP) increase cGMP and exert cardiovascular protective effects via guanylyl cyclase A (GC-A) receptor, which is distributed in many organs such as the heart, the vasculature and the brain . SympathTo investigate whether ANP/GC-A signaling inhibits SNS activity through the suppression of the brain MR, we examined urinary catecholamine secretion in global GC-A receptor KO mice and the effect of intracerebroventricular (ICV) infusion of MR blocker.norepinephrine (U-NE) secretion in wild type and global GC-A KO mice. Both BP and U-NE is significantly higher in GC-A KO than in wild type mice, indicating SNS is activated in GC-A KO mice. To study whether SNS activation is caused by the brain MR, we infused Eplerenone (MR blocker) into the ICV with osmotic mini pump for 2 weeks. Contrary to our hypothesis, both BP and U-NE did not change after 2 weeks ICV infusion, suggesting that activated SNS in GC-A KO is independent of MR. Furthermore, high sodium diet (NaCl 6%) for 2 weeks did not increase BP and U-NE in GC-A KO mice. MR protein expression in the hypothalamus was almost similar between GC-A KO and Wild type mice. These data suggest that SNS activity in GC-A KO mice is independent of MR and insensitive to sodium load. Unexpectedly, the most of GC-A KO mice died after ICV infusion of Losartan (AT1 receptor blocker), whereas wild type mice survived.We measured blood pressure (BP) and urinary Natriuretic peptides/GC-A signaling regulates SNS activity independent of both brain MR and sodium load. Brain AT1 receptor might be important in the regulation of cardiovascular system in global GC-A KO mice."} +{"text": "Different mechanisms in cancer cells become resistant to one or more chemotherapeutics is known as multidrug resistance (MDR) which hinders chemotherapy efficacy. Potential factors for MDR includes enhanced drug detoxification, decreased drug uptake, increased intracellular nucleophiles levels, enhanced repair of drug induced DNA damage, overexpression of drug transporter such as P-glycoprotein(P-gp), multidrug resistance-associated proteins , and breast cancer resistance protein (BCRP). Currently nanoassemblies such as polymeric/solid lipid/inorganic/metal nanoparticles, quantum dots, dendrimers, liposomes, micelles has emerged as an innovative, effective, and promising platforms for treatment of drug resistant cancer cells. Nanocarriers have potential to improve drug therapeutic index, ability for multifunctionality, divert ABC-transporter mediated drug efflux mechanism and selective targeting to tumor cells, cancer stem cells, tumor initiating cells, or cancer microenvironment. Selective nanocarrier targeting to tumor overcomes dose-limiting side effects, lack of selectivity, tissue toxicity, limited drug access to tumor tissues, high drug doses, and emergence of multiple drug resistance with conventional or combination chemotherapy. Current review highlights various nanodrug delivery systems to overcome mechanism of MDR by neutralizing, evading, or exploiting the drug efflux pumps and those independent of drug efflux pump mechanism by silencing Bcl-2 and HIF1\u03b1 gene expressions by siRNA and miRNA, modulating ceramide levels and targeting NF-\u03baB. \u201cTheragnostics\u201d combining a cytotoxic agent, targeting moiety, chemosensitizing agent, and diagnostic imaging aid are highlighted as effective and innovative systems for tumor localization and overcoming MDR. Physical approaches such as combination of drug with thermal/ultrasound/photodynamic therapies to overcome MDR are focused. The review focuses on newer drug delivery systems developed to overcome MDR in cancer cell. Cancer is a heterogeneous disease and use of multiple drugs simultaneously can result in drug resistance which is either intrinsic or acquired known as multidrug resistance (MDR). MDR renders cancer cells immune to standard treatments with many anticancer agents and is a major challenge in cancer therapy as it needs to address multiple phenotypes including MDR phenotypes. Tumor heterogeneity and tumor cell resistance to anticancer drugs thus remains key formidable challenges for effective targeting of drug delivery systems for successful chemotherapy. Drug resistance toward antineoplastic agents is a result of reduction in the effective concentration of drug in the cell prior to its interaction with the target or due to a combination of processes. The numerous mechanism of drug resistance reported includes (a) over expression of drug efflux pumps such as permeability glycoprotein (P-gp), multidrug resistance associated protein (MRP), and breast cancer resistance protein (BCRP) (b) alterations in lipid metabolism (ceramide pathway) (c) drug elimination by detoxification systems (d) drug test sequestration inside lysosomes and endosomes (e) reduced drug uptake due to altered surface receptors/carriers (f) inactivation of drugs via glutathione-mediated reduction (g) over expression of target enzymes such as up-regulated thymidylate synthase (h) altered drug targets such as topoisomerase II (i) increased DNA repair capacity (j) reduced ability to undergo apoptosis (k) hypoxia up-regulated expression of MDR-linked genes such as ABC transporters, Bcl-2 family genes, glutathione, metallothionein, etc. through activation of transcription factor HIF1 (l) chromosomal abnormalities in cancer cells lead to over-expression of anti-apoptotic genes (m) altered signal transduction pathways in cancer cells governed via integrin receptors, growth factor receptors etc. leads to blockage of apoptosis and expression of MDR-linked genes those involved in DNA repair and drug-efflux pumps or cluster of differentiation 243 (CD243) a ATP-binding cassette sub-family B member 1 encoded in human by ABCB1 gene (b) Multidrug Resistance Associated Protein 1 (MRP1) a ATP-binding cassette sub-family C member 1 encoded in human by ABCC1 gene, Multidrug Resistance Associated Protein 2 (MRP2) also called as canalicular multispecific organic anion transporter 1 (cMOAT) a ATP-binding cassette sub-family C member 2 encoded in human by ABCC2 gene (c) BCRP also known as cluster of differentiation (CDw338) a member of white sub-family and ATP-binding cassette G member 2 encoded in human by ABCG2 gene while class II isoforms export phosphatidylcholine into bile (MDR2/3/ABCB4) , and Biricodar citrate (VX-710).They have high potency and specificity for P-gp transporters over second generation agents. They do not interfere with cytochrome P450 3A4 unaffecting drug pharmacokinetics with no dose alterations in chemotherapy. They include Tariquidar-XR9576, Zosuquidar-LY335979, Laniquidar-R101933, ONT-093 (substituted diarylimidazole), Elacridar-GF120918, OC 144-093, Mitotane (NSC-38721), Annamycin, and R101933 Ozben, . Most prin vitro and greater tumor growth inhibition in vivo in drug-resistant tumor mouse model compared to paclitaxel nanoparticles alone with promising results in clinical trials levels below 10 mm-Hg where normal tissues range from 24 to 66 mm-Hg Breast cancer: AC , CAF , EC , FEC ; (ii) Colorectal cancer: FL , FOLFOX , FOLFIRI . Chemotherapy regimen is based on the assumption that the mutations conferring drug resistance will not convey resistance to all the agents in the regimen and high-dose chemotherapy regimens could be given to cancer patients. Such approach assumes that despite resistance to standard doses of anticancer drugs, a dose-response relationship exists for tumors and high doses of chemotherapy might overcome the resistance.In vitro and in vivo studies with biotin-functionalized nanoparticles co-encapsulating paclitaxel and P-gp targeted siRNA partially overcame tumor drug resistance or small interfering RNA (siRNA) has been explored. Transient RNAi mediated silencing can be achieved by siRNA or stable RNAi-mediated gene silencing through short hairpin RNA (shRNA) transfection. The siRNAs assembles into endoribonuclease inside the cells containing complexes known as RNA-Induced Silencing Complexes (RISCs) which guides the RISCs to complementary RNA molecules, cleaving and destroying the target RNA. Antisense oligonucleotides and catalytic RNAs have been successful in inhibiting P-gp, MRP, and BCRP expression and sensitized drug-resistant cells have potential for targeting P-gp and kill MDR tumor cells. Anti-P-gp MAbs such as MRK-16 and MRK-17 along with chemosensitizers reverses P-gp mediated MDR and conjugated MAbs such as bispecific antibody, immunotoxin and radioisotope conjugates enhance anti-tumor activity. Combination of MRK-16 with Cyclosporin-A or PSC-833 reversed Doxorubicin resistance in K562/ADM cells and inhibited tumor growth in athymic mice bearing HCT-15/ADM2-2 xenografts. MRK-16 increased Cyclosporine-A accumulation in MDR cells but not affected intracellular PSC-833 accumulation in MDR cells, instead Cyclosporin-A and PSC-833 increased MRK-16 binding to P-gp revealing a synergistic MDR reversal activity. MAbs with other anti-P-gp MAbs such as UIC2, 4E3, and series of HYB antibodies have potential to inhibit drug transport was 62-fold more resistant to paclitaxel and 15-fold resistant to BMS-184476. Also the human ovarian cancer cells with acquired taxane resistance expressed 9-fold resistance to BMS-184467 and 32-fold to paclitaxel. Studies of BMS-184476 against human tumor xenografts with both acquired and primary taxane resistance models revealed superiority of BMS-184476 , BMS-184476 (Phase I), RPR 109881A (Phase II), Ortataxel (Phase II), Trabectedin-ET-743 (Phase II and III) are not recognized by P-gp transporter and are evaluated in clinical trials for their broad spectrum activity in sensitive and resistant tumor cell lines to overcome MDR resistant cells showed dose-dependent inhibition of cell growth against K562/ADR cells as compared with Doxorubicin alone of Doxorubicin loaded-LNPs was 30-fold lower than free Doxorubicin in MCF-7/ADR, indicating intracellular retention of Doxorubicin and bypassing drug resistance loaded with plasmid iMDR1-pDNA for gene delivery revealed higher transfection efficiency and lower cytotoxicity than PEI/DNA nanoparticles against human breast cancer MCF-7 cells and drug-resistant MCF-7/ADR cells with Doxorubicin SLN compared to Doxorubicin solution. While comparable cell uptake and IC50 values were obtained with both Idarubicin SLN and free Idarubicin in P-gp over expressing P388/ADR and HCT-15 cells mouse tumor models. Study revealed the potential of Doxorubicin SLN in overcoming P-gp-mediated MDR both in-vitro in P388/ADR leukemia cells and in-vivo in murine leukemia mouse model and liposomal daunorubicin (DaunoXome\u00ae) which preferentially accumulates in tumor tissues via EPR effect to overcome drug resistance or accumulates within extracellular space of tumor stroma and leaks into tumor environment which provides pharmacologic advantage for liposomes over free drug to overcome drug resistance showed greater cytotoxicity, selective targeting and reversal of P-gp mediated drug resistance in resistant leukemia K562 cells than non-targeted co-loaded liposomes. Doxorubicin liposomes increased cytotoxicity on HL60 cells and Vincristine resistant HL60 cells due to rapid internalization and drug release inside the cells and A-549 (0.10 \u00b1 0.04 \u03bcg/ml) with Paclitaxel-Pluronic P123/F127 mixed polymeric micelles compared to Taxol and free Paclitaxel are versatile platform for cancer drug delivery , high surface to volume ratio, surface conjugation with multiple ligands and biocompatibility cells with reversal power of 31.3 and 2.2-fold for Paclitaxel NLCs and Doxorubicin NLCs respectively compared to Taxol and Doxorubicin solution cells. The reversal power of Paclitaxel and Doxorubicin NLCs were 34.3- and 6.4-folds, respectively on liposome significantly promoted gene transfection, Bcl-2 antisense phosphorothioate oligonucleotides complexed with cationic liposomes suppressed human cancer cell growth and induced apoptosis in human cervix epithelial carcinoma cell lines HeLa and mouse fibroblast NIH3T3 cells , Verapamil) in human CRC cell lines revealed synergistic effect of caspase dependent apoptosis, poly ADP ribose polymerase(PARP) cleavage, DNA fragmentation, cell cycle arrest, increased mitochondrial membrane permeability and enhanced protein expression of tumor suppressor p53 and least active C16-ceramide in MDA435/LCC6 human breast cancer and J774 mouse macrophage cell lines in poly(ethylene oxide)-modified poly(epsilon-caprolactone) nanoparticles revealed > 4.3- and 3-fold increase in tumor growth delay and 3.6- and 3-fold increase in tumor volume doubling time in wild-type SKOV-3 and multidrug resistant (MDR-1 positive) SKOV-3TR models respectively compared to individual agents nanoparticles was high compared to free drug in sensitive MCF-7 and multidrug resistant MCF-7TR (MDR-1 positive) human breast adenocarcinoma ] in Gemcitabine resistant human pancreatic cancer cell line revealed cytotoxicity and inhibited tumor growth plays vital role in cancer development and resistance. Degradation of inhibitor \u03baB after phosphorylation by inhibitor \u03baB kinases activates NF-\u03baB translocating into nucleus and initiating transcription contributing to tumor development, progression, chemoresistance, inflammation, and autoimmune diseases and independent (NF-\u03baB) drug resistance mechanisms enhances cell apoptosis and induce cancer cell death. Paclitaxel and Curcumin (NF-\u03baB and P-gp inhibitor) co-encapsulated in flaxseed oil nanoemulsion enhanced cancer cell sensitivity to Paclitaxel and cytotoxicity in SKOV3 and drug resistant SKOV350 in cervical carcinoma cells. Temperature sensitive poly(N-isopropylacrylamide) nanocarriers release anticancer drugs in presence of specific temperature triggers. Hyperthermia enables magnetic nanoparticles to enter tumor cells by generating heat in tissues/cells and is utilized for selective targeting through cancer-specific binding agents and controlled drug delivery over conventional hyperthermia resulting in activation and/or initiation of intracellular and extracellular degradation mechanisms like protein denaturation, protein folding, aggregation and DNA cross linking, changing tumor cell physiology and leading to apoptosis or making cancer cells more sensitive to anti-cancer drugs. Hyperthermia increases blood flow to the tumor cells and enhances delivery of nanocarriers and thus used as an adjunct treatment to increase efficacy of chemotherapy and enhance radiation induced tumor damage. Depending on the degree of temperature, hyperthermia is classified (i) in-thermo ablation; tumor subjected to >46\u00b0C (upto 56\u00b0C) causes cells to undergo direct tissue necrosis, coagulation or carbonization (ii) moderate hyperthermia (41\u201346\u00b0C) affects both cellular and tissue (iii) diathermia (<41\u00b0C) for rheumatic diseases. Cellular effects of moderate hyperthermia include induction and regulation of apoptosis, signal transduction and MDR. Super-paramagnetic iron oxide particles induced therapeutic hyperthermia; liposomal nanocarrier revealed high intra-tumoral accumulation of magnetic particles on application of magnetic field (100\u2013120 kHz) to attain temperatures 40\u201345\u00b0C. Folate receptor targeted Doxorubicin liposomes with hyperthermia reduced IC\u00ae P105 micelles with ultrasound resulted in high intracellular drug accumulation in promyelocytic leukemia HL-60 cells, ovarian carcinoma drug sensitive and multidrug resistant cells (A2780 and A2780/ADR) and breast cancer (MCF-7) cells -co-poly(\u03b2-benzyl-l-aspartate) loaded Doxorubicin with ultrasound treatment enhanced intracellular drug accumulation in ovarian carcinoma tumor model in nu/nu mice and inhibited tumor growth rate chlorin (Foscan) did not improved the photocytotoxicity or intracellular accumulation of Foscan in esophageal cancer cell line when compared to unmodified liposomes has wide applications in cancer therapy due to its specificity and selectivity. PDT involves three components light, oxygen, and a photosensitizer (non-toxic drug) to achieve photocytotoxicity. PDT includes administration of a photosensitizer which specifically accumulates in cancer cells and when illuminated with red visible light (620\u2013690 nm) generates reactive oxygen species in presence of tissue oxygen and causes cell death. Photofrin-2 (hematoporphyrin derivative) is the only PDT drug approved for clinical application in treatment of bladder, lung and esophageal cancer. Folic acid coated phospholipid-capped protoporphyrin IX (PpIX) loaded FITC-sensitized mesoporous silica nanocarriers (NanoPDT) effectively targeted receptors overexpressed on HeLa cells with high intracellular PpIX concentrations compared to free PpIX. NanoPDT on irradiation generated oxygen species, enhanced photocytotoxicity and inhibited 65% tumor growth in nude mice inoculated with B16F10 melanoma . PEGylated Doxorubicin gold nanoparticles increased intracellular uptake and nuclear localization significantly up to 6 h by endocytosis (Wang et al., in-vivo colon cancer imaging revealed high tumor uptake with application of an external magnetic field and MRI signal changes in human colon cancer xenograft mouse model (Cho et al., in-vivo in CT-26 cells with no toxicity (Schleich et al., Nanotheranostics or quadrugnostic are third generation integrated nanovehicles comprising of four elements for diagnosis of tumor location/s, specific targeting to cancer cells, eradication of malignant cells with cytotoxic drug/s and neutralize drug resistance mechanism Figure . NanotheNanodrug delivery systems are versatile platform for delivery of anticancer drugs and have been utilized successfully toward overcoming cancer drug resistance mechanisms, maximizing chemotherapeutic efficacy. Nanocarriers have effectively overcome challenges of limited aqueous solubility, low bioavailability, lack of targeting cancer tissues, increase drug therapeutic index, preferential accumulation by EPR effect and divert ABC-transporter mediated drug efflux MDR with potential to be multi-functionalized for cancer treatment. Nanocarriers promise to alleviate many challenges in clinical cancer therapy to benefit patients in future. Cancer science is progressing rapidly and understanding the molecular basis of drug resistance in cancer promises more effective treatments.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "The Peripheral Perfusion Index (PFI) has been utilized for early detection of impaired organ perfusion in order to avoid tissue hypoxia, which could lead to organ failure . A decreA GE digital-IT ECG recording system modified to be MRI-compatible was usedSP varied over different body segments, with major blood vessels corresponding to greater changes in SP Figure . FluctuaVMHD processing using this method exhibits characteristic SP patterns and perfusion levels for each body segment. Measured PFI levels were comparable to normal values. Future work includes comparison of the processing result with paired PO-based PFI measurements.NIH U41-RR019703, NIH R03 EB013873-01A1, SBIR-1 R43 HL110427-01."} +{"text": "Thus, we speculate that defective PI3K-dampening by Itpkb or other moderators contributes to the frequent HSC-defects in bone marrow (BM) failure syndromes [The Greek philosopher Plato emphasized the importance of moderating personal desires for the functioning of society. The virtue of moderation is also critical for the live-long function of hematopoietic stem cells (HSC), the origin of all blood cells. To prevent undue activation, which can deplete HSC and increase the risk of blood cancer, HSC need to restrain signaling by phosphoinositide 3-kinases (PI3K) - a pivotal pathway whose hyperactivation contributes to many diseases . Yet, th) in HSC . Itpkb\u2212/HSC are pluripotent and long-lived. Their longevity relies on their relative metabolic quiescence and only rare division for self-renewal. This dormancy is facilitated by HSC-residence in hypoxic BM niches. Metabolic adaptation and interactions with niche adhesion-molecules and cyto-/chemokines including stem-cell-factor (SCF) keep HSC quiescent and self-renewing , 4. Hema3), a recruiting and activating ligand for Akt and other effectors. To prevent excessive PI3K-signaling, PIP3-levels in many cell types are limited through its removal by the lipid-phosphatases PTEN and SHIP . But2+-mobilizing second-messenger IP3 into inositoltetrakisphosphate (IP4). We and others have identified receptor-induced IP4 production by Itpkb as essential for signaling in lymphocytes, granulocyte-monocyte-progenitors (GMP) and neutrophils activity. They had severely reduced competitive long-term-repopulating potential. Aging \u2212/\u2212ItpkB mice lost HSC and HPC and died with anemia.Itpkb phosphorylates the Catol1,3,4,tetrakisp\u2212/\u2212Itpkb HSC had elevated mTORC1 activity in vivo, and showed increased SCF-activation of Akt and mTORC1 in vitro. This was prevented by treatment with cell-permeable IP4 or an Akt-inhibitor. Transcriptome-analysis suggested Akt/mTORC1-hyperactivity and increased oxidative phosphorylation and protein biosynthesis in \u2212/\u2212Itpkb HSC. A recent study suggests that HSC-quiescence requires restrained protein biosynthesis and implies moderation of mTOR signaling [\u2212/\u2212Itpkb mice [Supporting an Itpkb-role in moderating PI3K-signaling in HSC, ignaling . Injecti\u2212/\u2212 mice . Thus, w\u2212/\u2212Itpkb mice resembles the phenotypes of PTEN-inactivation or dominant-active Akt expression. But \u2212/\u2212Itpkb mice have not yet shown T-ALL or AML. Possible reasons include differential effects of PTEN-loss, Akt-activation or Itpkb-loss on PI3K-signaling in HSC, or premature death of \u2212/\u2212Itpkb mice from anemia or infections [\u2212/\u2212Itpkb mice [The HSC transient expansion but later depletion in fections . Itpkb-l\u2212/\u2212 mice .\u2212/\u2212Itpkb mice but did not rescue their CFU-activity. This contrasts with \u2212/\u2212PTEN or myrAkt-expressing HSC and may suggest that mTORC1-unrelated mechanisms contribute to HSC-control by Itpkb [wt HSC CFU-activity, and genetic studies suggest mTORC1-requirements for HSC-regeneration and -function . This mnt aging might da4/PIP3-antagonism as a non-canonical mechanism regulating PI3K-function. It will now be important to assess the human relevance of this mechanism, and to determine if human BM failure patients show disease-driving mutations in Itpkb. Moreover, it will be interesting o explore if transient specific and selective Itpkb-inhibition can mobilize or expand human HSC without impairing their function.Wrapping up, Itpkb-identification as a moderator of cytokine- and PI3K-signaling in HSC advances our understanding of the HSC-intrinsic mechanisms balancing self-renewal with activation and broadens the importance of IP"} +{"text": "III(OH2), (dmb)CoII(OH2), and (dmb)CoIII(CH3) sites for solution Cbl and CoIII(OH2), CoII(OH2), and CoIII(CH3) sites in CoFeSP-Cbl were identified. Our data support binding of a serine residue from the reductive-activator protein (RACo) of CoFeSP to the cobalt ion in the CoFeSP-RACo protein complex that stabilizes Co(II). The absence of an \u03b1-ligand at cobalt not only tunes the redox potential of the cobalamin cofactor into the physiological range, but is also important for CoFeSP reactivation.A cobalamin (Cbl) cofactor in corrinoid iron-sulfur protein (CoFeSP) is the primary methyl group donor and acceptor in biological carbon oxide conversion along the reductive acetyl-CoA pathway. Changes of the axial coordination of the cobalt ion within the corrin macrocycle upon redox transitions in aqua-, methyl-, and cyano-Cbl bound to CoFeSP or in solution were studied using X-ray absorption spectroscopy (XAS) at the Co K-edge in combination with density functional theory (DFT) calculations, supported by metal content and cobalt redox level quantification with further spectroscopic methods. Calculation of the highly variable pre-edge X-ray absorption features due to core-to-valence (ctv) electronic transitions, XANES shape analysis, and cobalt-ligand bond lengths determination from EXAFS has yielded models for the molecular and electronic structures of the cobalt sites. This suggested the absence of a ligand at cobalt in CoFeSP in \u03b1-position where the dimethylbenzimidazole (dmb) base of the cofactor is bound in Cbl in solution. As main species, (dmb)Co K\u03b1-fluorescence-detected XAS spectra were collected using an energy-resolving 13-element germanium detector (Canberra) on samples held in a liquid-helium cryostat (Oxford) at 20 K. The detector was shielded against scattered X-rays by a 10 \u03bcm iron foil. The K-edge inflection point at 7709 eV of a simultaneously measured cobalt metal foil was used for calibration of the energy axis. Detector deadtime corrected XAS spectra (scan duration ~30 min) were averaged for signal-to-noise ratio improvement. No radiation induced spectral changes (i.e. in the XANES) were observed for increasing XAS scan numbers on single sample spots. XAS data processing was carried out as previously described to yield = 0.85) , 43. In am XANDA . Multiplam XANDA using mo-, H2O, CH3-, or CN- groups were added). The total charge and spin multiplicity of the models was set to the desired low-spin cobalt oxidation state cluster bound to the large subunit (CfsA) of CoFeSP [III(OH2) site as more likely. Direct binding of Thr374 at the \u03b1-position at Co(III) was seemingly excluded, corroborating the crystallographic assignment. Single-electron reduction of CoFeSP-AqCbl results in formation of a CoII(OH2) site with a weaker water-cobalt interaction. Cobalt sites lacking a \u03b1-ligand were clearly identified in CoFeSP-MeCbl containing Co(III) and in CoFeSP-AqCbl-RACo containing Co(II) [III(CH3) site in CoFeSP-MeCbl. In an earlier study of CoFeSP from another organism, a MeCbl species with a water (\u03b1) and a methyl (\u00df) ligand at Co(III) has been proposed; the water-cobalt bond, however, presumably was considerably elongated [III(CH3) site in the C. hydrogenoformans enzyme under our conditions. We cannot fully exclude a remote water ligand, which might have escaped detection in the XAS analysis, but consider it as unlikely.Our results suggest that oxidized CoFeSP-AqCbl contains mostly Cof CoFeSP \u201367. Takig Co(II) . Our intg Co(II) and prevg Co(II) . Serine g Co(II) , 14, as longated . Our datIII(CH3) and transfer of the methyl cation to acetyl-CoA synthase is facilitated by a more positive reduction potential, compared, e.g., to base-on MeCbl, of the CoIII(CH3) site in CoFeSP. Control of the axial cobalt ligation therefore may play an important role both in methyl group transfer and reductive activation of CoFeSP.Our DFT calculations suggest that Co(III) and Co(II) reduction in base-off CoFeSP-AqCbl presumably occurs at more positive potentials compared to base-on AqCbl or CoFeSP-AqCbl with two water ligands. The determined reduction potentials of the Co(II/III) and Co(I/II) couples in CoFeSP-AqCbl (about +350 mV and -500 mV) indeed are more positive compared to the values of AqCbl in solution (about +200 mV and -600 mV) , 66, 68.Analysis of cobalt K-edge XAS spectra in combination with DFT calculations of pre-edge absorption features facilitates determination of axial ligation and redox state of cobalt in solution Cbl and CoFeSP-Cbl. This supports the likely absence of a \u03b1-ligand in base-off CoFeSP-AqCbl and -MeCbl compared to base-on solution AqCbl and MeCbl, in agreement with earlier crystallographic and spectroscopic data. Coordination of a serine side chain from RACo to Co(II) in the CoFeSP-RACo protein complex is in agreement with our analysis. Control of the axial cobalt ligation may tune the redox potential of the cobalamin cofactor into the range of its electron transfer partners and likely is important for reductive activation of CoFeSP and methyl group shuttling.S1 Fileoptical absorption spectra of solution Cbl and CoFeSP-Cbl samples , EPR spectra of CoFeSP-Cbl samples , XANES spectra of cobalt reference compounds , multiple scattering calculations of cobalamin XANES spectra , K-edge energies from XANES simulations , correlation of EXAFS fit parameters , supporting references.(PDF)Click here for additional data file."} +{"text": "Additional Sanger sequencing confirmed co-segregation of the mutation with the disorder. IFIH1, encoding melanoma differentiation-associated protein 5 (MDA5), is a member of the RIG-I-like receptor family and functions as a cytoplasmic pattern recognition receptor recognizing viral double stranded RNA (dsRNA). Immunohistochemistry demonstrated the localization of MDA5 in all affected target tissues including heart, skin and cartilage. Functional analysis revealed that the IFIH1 c.2465G>A mutation enhanced MDA5 function in interferon beta induction after polyinosinic:polycytidylic acid (poly (I:C)) stimulation in vitro. This indicates that SMS-MDA5 is hyperactive to non-self dsRNA. According to additional in vitro studies, interferon signature genes including SIGLEC1 were upregulated in blood and dental cells derived from SMS individuals. Taken together, our data demonstrate that the MDA5 gain-of-function mutation p.Arg822Gln causes the autosomal dominant disorder SMS through dysregulation of the human innate immune response.Singleton-Merten Syndrome (SMS) is a rare autosomal-dominant disorder characterized by a variety of symptoms including extreme calcifications of the ascending aorta and cardiac valves, dental anomalies , psoriasis and widened medullary cavities of the phalanges with focal osteoporosis. Employing whole exome sequencing of five SMS individuals of three families we identified the missense mutation, c.2465G>A (p.Arg822Gln) in the"} +{"text": "Early re-admission after hospitalization is an increasing focus of health-care policy because it results in high costs and often reflects opportunities for improving treatment quality. The goal of this study is to examine the relationship between 30-day mental health/substance use disorder hospital re-admission for persons with schizophrenia, and patient characteristics, hospital utilization, and community treatment quality and capacity.Observational study of schizophrenia-diagnosed enrollees having \u2265 1 behavioral health hospitalization in 2005 from 18 state Medicaid programs (N = 28083). Regression models examined the relationship between 30-day behavioral health hospital re-admission, enrollee characteristics (demographic and comorbidity), and county-level indicators for: 1) quality of care ; 2) behavioral health hospitalization ; and 3) treatment capacity .Fifty-one percent of the study population had a co-occurring substance use disorder; nearly 47 percent had a co-occurring chronic general medical condition. Enrollee comorbidity was associated with higher predicted probability of 30-day behavioral health re-admission, particularly for enrollees with substance use disorders (predicted probability [95% CI] = 23.9% [21.5%\u201326.3%]) versus without (14.7% [13.9%\u201315.4%]). Chronic medical conditions were associated with increased re-admissions in a dose-response manner . Higher county rate of behavioral health visits within 7 days post-hospitalization was associated with lower re-admission for individual enrollees . In contrast, higher county rate of behavioral health hospitalization was associated with higher re-admission probability for individual enrollees .Efforts to reduce 30-day psychiatric re-admissions should focus on comorbid substance use and general medical care coordination, as well as factors that contribute to hospitalization in general and improving transitions to community care. Comorbid substance use disorders were particularly prominent in 30-day behavioral health re-admission\u2014patients with comorbid substance use disorders had a 63.7 percent higher predicted probability of 30-day re-admission compared to those without. These findings demonstrate the substantial role of comorbid substance use disorders in behavioral health 30-day re-admissions. They also highlight an opportunity for Medicaid policy to influence improved access to substance use disorder treatment, including its coordination with behavioral health and general medical care, in an effort to reduce 30-day re-admission for individuals with severe mental illness."} +{"text": "CCK-AR polymorphism is associated with a defective splicing of the primary transcript of CCK-AR mRNA, which may result in the lower expression of the CCK-AR. Therefore, we evaluated the role of genetic polymorphism of CCK-AR gene in FD.Functional dyspepsia (FD) is characterized by epigastric pain, burning, early satiety and post-prandial fullness in absence of organic or metabolic causes. Cholecystokinin receptor-A (CCK-AR) is known to modulate satiety signal and delay gastric emptying, which are associated with FD. CCK-AR gene polymorphism (PCR-RFLP). Patients with FD were sub-classified into epigastric pain syndrome (EPS), post-prandial distress syndrome (PDS) and EPSPDS overlap.237 consecutive patients with FD (Rome III) and 250 healthy controls (HC) were genotyped for CCK-AR (rs1800857) was infrequent among patients than HC . However, genotypes distribution was comparable among patients with different subtypes of FD (p=0.44).Patients with FD were comparable with HC with respect to age and gender. 26/237 (11%) had EPS, 55 (23.2%) PDS and 156 (65.8%) EPSPDS overlap. Among 237 patients with FD, CC (variant) genotype of CCK-AR polymorphism is protective for FD. EPSPDS overlap was common among patients with FD.CC genotype of"} +{"text": "Most current infection control (IC) culture-change programs are standardised and do not take into account possible differences between HCWs.1. Identify PP features among various HCW categories to inform the development of a personality-based educational culture-change \u201cblueprint\u201d to improve uptake of IC initiatives.2. Compare the accuracy of PPs derived from the Human Resources (HR) database at 5 Australian hospitals (HR-Ps) with those derived from direct participant surveys (PS-Ps) from these sites.3. Use findings to develop targeted marketing strategies for each HCW group.We used an innovative colour-based PP tool to identify PPs using: Basic HR data and ColourGrid surveys completed by HCWs at the 5 sites. HR-Ps and PS-Ps were compared for 3 HCW categories \u2013 Doctors (D), Nurses/Allied (N-A) and Support staff (SS). Among Ds, PS-Ps were compared for senior hospital clinicians (full-time [SMO] vs part-time [VMO]) and junior staff (interns/fellows [HMO]).vs VMO vs HMO suggesting targeted messaging strategies are critical.HR data was obtained for 34 243 HCWs, with 1045 completing a ColourGrid survey. HR-Ps suggested that HCWs are substantially different to the general Australian population, being more affluent; established; well informed; likely to adopt new technology and new experiences; often cynical about advertising messages; challenging to others who do not share their interests or concerns; want to make a difference and leave a heritage of success. HR-Ps and PS-Ps were highly concordant for all 3 HCW categories \u2013 with both suggesting a need for messaging differences. Overall, Ds exhibited more individualism, lower power distance and less uncertainty avoidance, but PS-Ps were different for SMO PP identified major differences among D, N-A and SS; and a need for targeted marketing strategies. Among Ds, subtle but important, differences also exist that need consideration if culture-change initiatives are to be successful.None declared."} +{"text": "Echocardiography is commonly used during both venoarterial (V-A) and venovenous extracorporeal membrane oxygenation (ECMO). In many circumstances, transoesophageal echocardiography (TOE) is the preferred monitoring tool. It can aid in cannula positioning, especially during double-lumen cannula placement for V-V ECMO, weaning of V-A ECMO and diagnose causes of high-access pressures and circuit flow problems. We use TOE as our preferred monitoring equipment before, during and after establishing ECMO. We sought to investigate how often information gained from TOE imaging had a major impact on management decisions.A single-centre observational study at a tertiary referral institution. All patients supported with V-A or V-V ECMO during an 18-month period were included. Routine procedures such as wire position checks during cannulation or information gained to assist weaning from V-A support were not included.n = 3), pericardial clot formation and tamponade (n = 1), persistent left superior vena cava making planned cannula placement into the right atrium impossible or potentially fatal, diagnosis of patent foraminae ovale providing left atrial decompression and appropriate drainage cannula positioning during liver transplantation on ECMO.Twenty patients were supported with either V-A or V-V ECMO during the observation period. In 12 patients (60%) TOE was instrumental in diagnosing potentially fatal complications or altered clinical management. In three patients on V-A support, afterload reduction and modulation of inotropic support was necessary due to extensive spontaneous echo contrast formation in the left ventricle and stagnant pulmonary blood flow; two out of these three patients, immediately after establishing support, required intra-aortic balloon counterpulsation to reverse clot formation around the aortic valve and root. Further diagnosis were Avalon cannula misplacement in the blind end of a piggyback cava following liver transplant, collapsing right atrial free wall with 'sucking down' into the access cannula (In our practice, TOE is an indispensable tool for safe ECMO practice, both during V-A and V-V support."} +{"text": "Military Medical Research would like to thank all our reviewers who have contributed to the journal in volume 1 (2014) and 2 (2015).The editors of Maria das Gra\u00e7as AngueraBrazilChristian BautistaUnited States of AmericaEric BernesSwitzerlandZhao-xiang BianHong KongTimothy BilliarUnited States of AmericaSelami \u00c7akmakTurkeyChuang CaiChinaGuangwen CaoChinaWu-Chun CaoChinaRoberto CasaleItalyKevin CaseyAfghanistanTheresa M. CaseyUnited States of AmericaAnne-Marie ChangUnited States of AmericaBohua ChenChinaYao-Long ChenChinaNing-Hai ChengChinaAngela ChowSingaporeAhmet Yilmaz CobanTurkeyLaura CoffeyIrelandLe CongUnited States of AmericaTravis CraddockUnited States of AmericaRobert DahnkeGermanyDomenico De BerardisItalyAndrew DworkUnited States of AmericaRoberto Augusto Estrada Casta\u00f1onMexicoChristiane Kruse F\u00e6steNorwayIbrahima Soce FallSenegalJie FanUnited States of AmericaYi-Qun FangChinaCheng FengChinaWolfgang FreundGermanyCarlo Fremd GermanyXiao-Bing FuChinaHui GaoChinaXu-Bing GaoChinaAngelo GemignaniItalyYan GengChinaAayush GuptaIndiaMarios HadjicharalambousCyprusJohn HarknessAustraliaRod HayUnited KingdomAntonia HildebrandGermanyHiroyuki HirasawaJapanYamashita HiromasaJapanLan HuangChinaSha HuangChinaSigurd HystadNorwayJian-Xin JiangChinaRonald KesslerUnited States of AmericaJae-Seok KimSouth KoreaYoung Wan KimSouth KoreaLuther C. KlothUnited States of America Yankaskas KurtUnited States of AmericaBas De LaatNetherlandsXi-Lan LaiChinaLi LiChinaJing LiChinaShao-hua LiUnited States of AmericaYou-Sheng LiChinaThomas LiesegangUnited States of AmericaJi-Long LiuUnited KingdomWei LiuChinaWei-Wei LiuChinaXiao-rong LiuChinaXin LuoChinaGang LvChinaVinay MahishaleIndiaLuis MassucaPortugalIng How MooSingaporeShawnda MorrisonSloveniaStephen MuzaUnited States Minor Outlying IslandsDavid NelsonUnited States of AmericaEisuke OchiJapanJohn ParkUnited States of AmericaDidier PayenFranceXuetao PeiChinaRui-yun PengChinaYang PengUnited States of AmericaSandeep PhatakUnited States of AmericaIvan PoonAustraliaGeorgi PopivanovBulgariaLarry PruittUnited States of AmericaXing-Shun QiChinaWei-Min QuChinaDaniel G. RemickUnited States of AmericaSamaneh RouhiIran Frank A. J. L. ScheerUnited States of AmericaAlexa Smith-OsborneUnited States of AmericaMohan L. SoporiUnited States of AmericaMichael SteketeeUnited States of AmericaLei SuChinaKentaro SuganoJapanXin SunChinaZachary TaylorUnited States of AmericaStefania TegliItalyMarcia TestaUnited States of AmericaGursimran ThandiUnited KingdomJames TsaiUnited States of AmericaJacob TurnerUnited States of AmericaBhan UrvashiUnited States of AmericaTimothy R. Varney United States of AmericaLakshmi VasIndiaLee VernonSingaporeMichael WallerAustraliaDe-yun WangSingaporeFu-Sheng WangChinaGuo-qiang WangChinaHong-ying WangChinaJiang Huai WangIrelandKuang-Te WangTaiwanYan-Jiang WangChinaYang WangUnited States of AmericaBryant WebberUnited States of AmericaRodney WilloughbyUnited States of AmericaMee Lian WongSingaporeMichael W\u00fcnningGermanyXuan XiaoChinaTai XieChinaNoriko Yaekashiwa JapanXiao YangChinaZheng-lin YangChinaYong-Ming YaoChinaXue YuUnited States of AmericaJing YuanChinaPaul ZarogoulidisGreeceLian-yang ZhangChinaMin ZhaoUnited States of AmericaFei-Hu ZhouChinaQi-Quan ZhouChinaJure ZupanSlovenia"} +{"text": "Accumulating evidence indicates that oxidative stress contributes to the pathogenesis of acute lung injury (ALI). Previous studies have demonstrated that LPS-induced reactive oxygen species (ROS) mediates lung epithelial cell apoptosis. Fas/FasL signaling is an important cellular pathway in the induction of lung epithelial cell apoptosis in ALI.To determine the interaction between ROS generation and Fas activation in regulation of LPS-induced lung epithelial A549 cell apoptosis.2\u00b7- scavenger), catalase (H2O2 scavenger), or sodium formate (OH- scavenger) was performed to determine the specificity of ROS detection. To determine the source of ROS generation induced by LPS, diphenylene iodonium (DPI), a known inhibitor of NADPH oxidase, or rotenone, an inhibitor of mitochondrial electron transport chain, were pretreated, and their effects on ROS generation was examined. LPS-induced cell apoptosis was analyzed by Annexin-V staining followed by flow cytometry in the presence or absence of ROS scavengers. Furthermore, Fas blocking antibody ZB4 and Fas siRNA were pretreated to verify the role of Fas signaling in LPS-induced ROS generation and cell apoptosis. To determine the effects of ROS on Fas signaling, Fas levels of A549 in response to LPS were measured by flow cytometry in the presence or absence of ROS scavengers. The downstream caspase-3 and -8 activity were examined, and their inhibitors were treated to confirm the effects of caspase-3 and -8 on LPS-induced apoptosis.Intracellular ROS was measured using 2',7'-dichlorofluorescein diacetate as fluorescent probes by flow cytometry in LPS-treated A549 cells. Addition of superoxide dismutase diminished LPS-induced intracellular ROS generation as well as inhibited A549 cell apoptosis. Similarly, the DPI and rotenone also prevented from LPS-induced apoptosis through inhibition of ROS generation. Furthermore, we found that inhibiting ROS generation attenuated LPS-induced Fas up-regulation and inhibited the activation of caspase-8 and -3. Addition of caspase-8 inhibitor Z-IETD-FMK or caspase-3 inhibitor Z-DQMD-FMK diminished LPS-induced cell apoptosis. Of note, blocking Fas by Fas antibody ZB4 or siRNA inhibited LPS-induced cell apoptosis, but was not associated with ROS production.Our data show that LPS induces superoxide, hydrogen peroxide and hydroxyl radical production either from NADPH oxidase or from mitochondrial electron transport chain in A549 cells. The LPS-induced ROS generation results in A549 apoptosis through Fas up-regulation and downstream caspase-8 and -3 activation. Blocking Fas signaling has no effect on regulation of LPS-induced ROS generation while inhibiting cell apoptosis.This work was supported by the Ministry of Science and Technology, Taiwan (MOST 103-2314-B-006 -063)."} +{"text": "This research was funded by an ANR Blanc \"Biopocket\", and by The Laboratoire d'Excellence (LabEx) ParaFrap ANR-11-LABX-0024 ("} +{"text": "Adult T-cell leukemia (ATL) develops in individuals infected with human T-cell lymphotropic virus-1 (HTLV-1). Presently there is no curative therapy for ATL. The HTLV-1-encoded protein Tax up-regulates Bcl-xL expression and constitutively activates interleukin-2 (IL-2), IL-9, and IL-15 autocrine/paracrine systems resulting in amplified JAK/STAT signaling. Consequently, inhibition of JAK signaling reduces ex vivo proliferation of PBMCs from ATL patients in smoldering and chronic stages. Currently, two JAK inhibitors are approved for human use. In this study, we examined activity of multiple JAK inhibitors in IL-2-dependent and IL-2-independent ATL cell lines. The highly selective JAK inhibitor ruxolitinib was examined in a high-throughput matrix screen and the Bcl-2/Bcl-xL inhibitor navitoclax was identified as a strong candidate for multicomponent therapy. An examination of the mechanistic underpinnings of this combination highlighted a stimulation of Bim and PUMA expression and reduced phosphorylation of BAD upon cellular exposure to ruxolitinib. The combination was noted to strongly activate BAX, effect mitochondrial depolarization and increase caspase 3/7 activity that leads to PARP and Mcl-1 cleavage. Ruxolitinib and navitoclax independently demonstrated modest antitumor efficacy while the combination dramatically lowered tumor burden and prolonged survival in an aggressive ATL murine model. Critically, this combination strongly blocked ex vivo proliferation of five ATL patients\u2019 PBMCs. These studies provide support for a therapeutic trial in patients with smoldering and chronic ATL using a drug combination that inhibits JAK signaling and anti-apoptotic protein Bcl-xL."} +{"text": "Exercise induced anaphylaxis and its subtype the Food-dependent exercise-induced anaphylaxis are uncommon and therefore under-diagnosed forms of physical allergy. Triggers include various degrees of exercise in combination with ingestion of specific food products. Treatment remains identical to that of Immunoglobulin E- mediated allergic reactions. The presentation is commonly under diagnosed and this case should raise the awareness of the attending Allergist/Physician.A 30 year old female was seen in the Allergy Clinic upon request of her General Practitioner. She reported an episode where after dinner she went into a local park to exercise. Shortly thereafter she collapsed with rash, lip swelling and breathing difficulties. Upon admission to hospital she was found to be hypotensive and required fluid resuscitation, systemic corticosteroids and adrenaline. She made a full and eventful recovery. Based on the clinical story and the subsequent specific allergy markers her presentation was attributed to food- induced exercise-induced anaphylaxis (FD-EIA).Skin testing:- Shellfish 4 mm- Wheatflour 4 mm- Milk 2 mm- Eggs 2 mm- positive control 5 mmwhich at that time offered the possible diagnosis of either allergic reaction to omega-5-gliadin (wheat) or prawns given the skin test was positive according to international standard norm (>3mm).Following that further serological tests were organised which revealed:- ImmunoCAP Specific IgE blood test- Omega-5-gliaidin allergen 0.03 kU/ml- Shellfish 2.00 kU/ml"} +{"text": "Left atrium (LA) volume and function are important markers of cardiovascular disease. LA volume can be assessed by several different methods. In clinical practice, the Simpson's method is well accepted as a reference standard, although there is no standardization for LA volume calculations. We aimed to compare the estimations of LA volume by the Simpson's method and the modified biplane Simpson's method; and to introduce Multimodality Tissue Tracking as a new semi-automated method for quantifying LA function based on tissue feature tracking.Thirty subjects including twenty subjects with cardiovascular events and ten healthy subjects, with CMR imaging were evaluated in the Multi-Ethnic Study of Atherosclerosis (MESA). LA volumes were measured using the modified biplane Simpson's method from 2- and 4-chamber projections and the original Simpson's method using short-axis slices. For the manual methods, LA endo- and epicardial boundaries were delineated at left-ventricular end-diastole (Vmin), end-systole (Vmax) and just before the pre-atrial contraction (VpreA). Using MTT, LA endocardial and epicardial borders were manually delineated at end-systole and the boundaries were propagated automatically throughout the cardiac cycle. LA total (LAEF = Vmax-Vmin/Vmax), active (LAAEF = VpreA-Vmin/VpreA) and passive ejection fraction (LAPEF =Vmax-VpreA/Vmax) were calculated (Figure LA parameter analysis using the two different computations was significantly different for functional LA parameters by both methods Table . LA funcMTT derived biplane LA structure and function is accurate, less time consuming, highly reproducible and could potentially be used in large studies.NHLBI N01-HC-95159 NHLBI N01-HC-95168 NCRR UL1-RR-024156 NCRR UL1-RR-025005."} +{"text": "These data relate to the differentiation of human dental pulp stem cells (DPSC) and DPSC immortalized by constitutively expressing human telomerase reverse transcriptase (hTERT) through both osteogenic and adipogenic lineages . The data augment another study to characterize immortalized DPSC for the study of neurogenetic \u201cCharacterization of neurons from immortalized dental pulp stem cells for the study of neurogenetic disorders\u201d Specifications Table\u2022The effects of hTERT immortalization on the ability of DPSC to differentiate into osteocytes and adipocytes was previously unknown.\u2022These data may assist researchers in the decision to use hTERT immortalization of a cell line depending on the desired lineage.\u2022These data support the use of DPSC for the generation of adipocytes differentiation.Value of the data11.1The data shown are microscopy images (Bright-field) and qRT-PCR of Non-immortalized DPSC (NI-DPSC) and Immortalized DPSC (I-DPSC) at passage 5.21.RUNX2: Forward: -TTT GCA CTG GGT CAT GTG TT-, Reverse: -TGG CTG CAT TGA AAA GAC TG-2.BSP: Forward: -TGAA ACG AGT CAG CTG GATG-; Reverse: -TGA AAT TCA TGG CTG TGG AA-3.WNT10B: Forward: -TGG GAT GTG TAG CCT TCT CC-; Reverse: -CCC AGC CAA AAG GAG TAT GA-4.PPAR\u03d2: Forward: -GTC GTG CAG GAG ATC ACA GA-; Reverse: -GGG CTC CAT AAA GT CAC CAA-5.FASN: Forward: -TCC TGA GCA TGC TGA ACG AC-; Reverse: -AGC AGA TGA ACC AGA GCG G-6.PPIA: Forward:-CAG ACA AGG TCC CAA AGA CAG-; Reverse: -TTG CCA TCC AAC CAC TCA GTC-Adipogenic and osteogenic differentiation of DPSC was as previously described for bone Fig. 13Teeth for the generation of DPSC were obtained through the Department of Pediatric Dentistry at the University of Tennessee Health Science Center (UTHSC). The UTHSC Institutional Review Board approved this study and informed consent was obtained from the parent or legal guardian of all participants."} +{"text": "Our results showed that up-regulation of circulating miR-148a, miR-1246 or miR-1290 at early-phase was significantly associated with HCC recurrence after liver transplantation. Among them, miR-148a (p=0.030) and miR-1246 (p=0.009) were significant predictors of HCC recurrence. MiR-1246 was an independent predictor of overall (p=0.023) and disease-free survival (p=0.020) of HCC recipients. The level of early-phase circulating miR-1246 was positively correlated with serum AST and ALT levels in HCC recipients after liver transplantation. The expression of hepatic miR-1246 was positively correlated with TNF\u03b1 mRNA. In vitro experiments indicated that injury-induced activation and differentiation of macrophages significantly elevated the expression and secretion of miR-1246. In conclusion, early-phase circulating miR-1246 is an indicator of hepatic injury and a novel prognostic biomarker for tumor recurrence and survival of HCC recipients after liver transplantation.Post-liver transplantation tumor recurrence is a major challenge for hepatocellular carcinoma (HCC) recipients. We aimed to identify early-phase circulating microRNAs after liver transplantation for predicting tumor recurrence and survival of HCC recipients. Circulating microRNA profiles at early-phase after liver transplantation were compared between HCC recipients with (n=4) and without tumor recurrence (n=8) by microarray analyses. Candidate microRNAs were validated in 62 HCC recipients by quantitative RT-PCR. The prognostic values of microRNAs for tumor recurrence and survival were examined. Simulated Liver transplantation is regarded as the best curative treatment for early stage hepatocellular carcinoma (HCC) patients under stringent selection criteria . ShortagMicroRNAs (miRNA) are small, approximately 20-30 nucleotides, single stranded non-coding molecules function at the post-transcriptional level. miRNAs suppress the expression of their target mRNAs by degradation of mRNAs or inhibition of translation process . Increasin vitro models to explore the possible reasons of alteration of miRNAs during early-phase of liver transplantation.In this study, we applied miRNA microarray analysis to identify early-phase circulating miRNAs aiming to predict HCC recurrence and survival of HCC recipients after liver transplantation. We also used simulated IRI-related In microRNA microarray analysis, after normalization with the expression level of miRNAs in healthy donors, 14 significantly upregulated miRNAs were identified in recurrent recipients at a false discovery rate (FDR) of 0% compared to non-recurrent recipients Figure . There wp=0.010), miR-1246 (p=0.004) or miR-1290 (p=0.031) was detected in HCC recipients with HCC recurrence after liver transplantation compared to that without tumor recurrence for tumor recurrence by ROC analysis. Among them, miR-148a (Table The High expression group (High group) and the Low expression group (Low group) for each miRNA were determined using optimal cut-off value from Youden index analysis . Because1] Table . Moreovep=0.023) and disease-free survival , 1 day (LT-Day1) and 1 week (LT-Day7/8) was significantly higher than the level in healthy donors and before liver transplantation Figure . The expTNF-a) mRNA at early-phase after liver transplantation is the major complication at early-phase after liver transplantation . ResearcNF\u03b1 gene , 35. OurNF\u03b1 gene , our stuIn conclusion, our study demonstrated that early-phase circulating miR-148a and miR-1246 were potential biomarkers in predicting HCC recurrence after liver transplantation. Early-phase circulating miR-1246 was not only a biomarker for acute hepatic injury but also a novel prognostic biomarker of poor overall and disease-free survival of HCC recipients after liver transplantation.Sixth-two HCC patients received liver transplantation from Oct 2003 to May 2010 in Queen Mary Hospital, Department of Surgery, the University of Hong Kong, were included in this study. The number of patients in this study represented about 54.4% of the total number of HCC patient (114) undergone liver transplantation during these years. The last follow-up date of the patients was in March 2015. Among them, 9 HCC recipients were found to have recurred tumor after liver transplantation. The clinical information between recurrent and non-recurrent patients was summarized in Human normal liver cell line (MIHA) and human monocytic cell line (THP-1) were purchased from American Type Cell Culture and maintained in the optimal medium according to the instruction.Total RNAs including small RNAs were extracted from the plasma of patients or the media of cells using miRNeasy Mini Kit (Qiagen) according to manufacturer's instruction. Total RNAs in liver tissues or cell lines were extracted by TriZol reagent (Life Technologies) according to manufacturer's instruction.MicroRNA microarray analyses were performed among the plasma of healthy donors (n=2) and early-phase (LT-2hr) plasma of HCC recipients with (n=4) and without (n=8) tumor recurrence after liver transplantation. Each of 100 ng of plasma RNA was labeled with Cy-3 fluorescent dye and performed miRNA profiling using Agilent Human miRNA Microarray Kit (V2) . The array contains 723 human and 76 viral miRNA probes. The slide was scanned by the Agilent Microarray Scanner. After filtering the non-expressed probes and normalization by 75% percentile shift method, the miRNA data was analyzed by GeneSpring software (Agilent Technologies).sample)= \u0394Ct-\u0394Ct(miRNAsample), where \u0394Ct=Ct-Ct; \u0394Ct(miRNAsample)=Ct(miRNAsample)-Ct(RNU6Bsample). The calibrator was defined as the sample with the highest Ct value of miRNA (sample with the lowest expression level of miRNA) among all samples [in vitro experiments, the expression level of miR-1246 was determined as fold difference to untreated cells using 2\u2212\u0394\u0394Ct method. The expression level of TNF\u03b1 mRNA in the samples was analyzed by quantitative RT-PCR (Life Technologies) as described previously [TNF\u03b1, forward:5\u2032-GCCCATGTTGTAGCAAACCC-3\u2032, reverse:5\u2032-GGTTATCTCTCAGCTCCACGC-3\u2032; for beta-actin, forward:5\u2032-CTCTTCCAGCCTTCCTTCCT-3\u2032, reverse:5\u2032-AGCACTGTGTTGGCGTACAG-3\u2032. All reactions were performed in duplicate.The expression levels of miRNAs were determined by TaqMan real-time quantitative PCR and normalized to the level of U6 Small Nuclear 2 using a 7900HT PCR machine (Life Technologies) according to the manufacturer's protocol. Probes for Let7C000379), miR-21(000397), miR-23b(000400), miR-27b(000409), miR-122(002245), miR-125b(000449), miR-148a(000470), miR-151-5p(002642), miR-192(000491), miR-195(000494), miR-199a-3p(002304), miR-215(000518), miR-1246(462575_mat) and miR-1290(002863) were purchased from Life Technologies company. The relative expression level of miRNA for each sample was calculated as: \u0394\u0394Ct for 2 hours. RNAs from media and cells were extracted for analysis of the expression of miR-1246 by real-time quantitative RT-PCR. The experiment was repeated for three times. P<0.05 was considered as statistical significance.To investigate whether accelerated oxidative stress during early-phase of liver transplantation might cause induction of miR-1246 from hepatocyte, we generated a short-term oxidative stress on the normal liver cell line, MIHA. Briefly, MIHA cells were incubated with different concentrations of Hin vitro monocyte-to-M1 macrophage model [P<0.05 was considered as statistical significance.To investigate whether increased inflammation response might increase the level of miR-1246, we performed an ge model , 39. BriP<0.05 was considered as statistically significant.The expression levels of circulating miRNAs in clinical samples were graphed by Prism Version 5.01 (Graphpad). Statistical analyses were performed by SPSS software version 20 . Categorical variables were analyzed by Chi-square test or Fisher's exact test, while continuous variables were analyzed by Mann-Whitney U test. The correlations of differential circulating miRNAs to clinical factors were analyzed by Pearson correlation analysis. To examine the prognostic value of miRNAs, Receiver Operating Characteristic (ROC) curve was generated for each miRNA in predicting tumor recurrence after liver transplantation. The optimal cut-off value for each miRNA was obtained from Youden index. The sensitivity and specificity of miRNA to predict HCC recurrence after liver transplantation was determined by ROC analysis. Kaplan-Meier analysis applying log-rank test was performed to analyze the prognostic value of each miRNA in predicting overall and disease-free survival of HCC recipients after liver transplantation. Univariate Cox-regression analysis was performed to examine the hazard ratio of miRNAs and clinical factors in predicting overall and disease-free survival of HCC recipients. Multivariate Cox-regression analysis was performed to compare the significant factors in univariate cox-regression analysis."} +{"text": "Ultimately, anti-ALK CART could provide a novel therapy for pediatric solid tumors expressing cell-surface ALK.Neuroblastoma is the most common non-CNS tumor in children. Poor prognosis for patients presenting with high-risk disease underlines the need for novel therapeutic strategies. Chimeric antigen receptor T cell (CART) therapies combine the epitope specificity of a monoclonal antibody with the cytotoxicity of an activated T cell, and have demonstrated clinical efficacy in pediatric leukemia. To expand this approach to neuroblastoma, we focused on anaplastic lymphoma kinase (ALK), which can exist in native, over-expressed, or mutated forms and may help drive disease progression. Single chain variable fragments (scFvs) derived from four different monoclonal antibodies against ALK were used to construct second-generation CAR vectors featuring either CD28/CD3-zeta or CD137/CD3-zeta signaling domains. Some CAR constructs also included an IgG1 CH2CH3 spacer domain between the scFv and transmembrane domain. Retroviral vectors expressing ALK-specific CARs were generated and used to transduce human peripheral blood mononuclear cells. Transduction efficiencies ranged from 8% to 99%, depending on specific characteristics of each CAR construct. The resulting CART lysed ALK-positive neuroblastoma and Ewing's sarcoma cell lines and had minimal activity against an ALK-negative control. The ability to lyse tumor cells varied across scFvs, and short CART (no Ig spacer) showed enhanced lysis compared to long CARs (with Ig spacer). Short CART also produced Th1 cytokines when co-incubated with ALK-positive tumor lines; however, long CARs did not. Additionally, short anti-ALK CART significantly delayed the growth of human neuroblastoma in an NSG xenograft model when tumor-bearing mice were preconditioned with cyclophosphamide. Ongoing studies aim to: 1) further assess anti-ALK CART therapy"} +{"text": "The vast majority of hepatocellular carcinoma (HCC) arise in the context of chronic inflammation, especially with hepatitis B or hepatitis C viral infection. Several studies have identified prognostic immune biomarkers in HCC tumors and peritumoral regions. Recently, a Phase 1 trial of a PD-1 inhibitor has demonstrated efficacy in HCC. In order to characterize the diversity of immune microenvironments in HCC, we investigated co-expression networks of immune lineage-specific genes.1 and Chiang interferon subclass2, and was less prevalent among HCC with CTNNB1 mutations.We conducted a meta-analysis of gene expression data from over 500 HCC tumors and matched adjacent liver specimens. PD-L1 and PD-L2 had higher RNA levels in adjacent liver, compared with tumors. Tumoral expression of PD-L1 and PD-L2 were correlated with macrophage lineage genes. We identified 3 major co-expression network modules that corresponded with different immune cell sub-types: (1) An infiltrating T cell module was enriched for TCR activation, recruitment chemokines and elevated immune checkpoints. (2) A hepatic stellate cell module was associated with extracellular matrix remodeling, epithelial-to-mesenchymal transition and TGF-beta signaling. (3) A macrophage module had elevated macrophage lineage genes. By integrating these co-expression modules with HCC molecular sub-classes, the infiltrating T cell signature was enriched in the Hoshida S1 subclassTranscriptomic analyses revealed immune cell types and potential regulators in HCC. The joint profiling of infiltrating immune sub-types and genetic alterations may guide the selection of combination therapies."} +{"text": "Cancer vaccines aim at inducing tumour-specific immune responses. However, in clinical studies so far these approaches have limited antitumour effect. Evidence is accumulating that MDSC (myeloid-derived suppressor cells) can suppress the antitumour immune response. Reversal of MDSC-mediated immune suppression by treatment with the tyrosine kinase inhibitor sunitinb can therefore possibly increase the efficacy of cancer vaccines.We developed a method to assess MDSC depletion in a preclinical model of HPV-induced neoplasia. TC-1 (cells expressing HPV16-E7) tumour-bearing mice were injected with different dosages of sunitinib daily, for 9 days, with and without immunisation with SFVeE6,7 . Intra-tumoral, intra-splenic and circulating MDSC and CD8 T cell levels were assessed after treatment.Upon sunitinib treatment, the absolute numbers of intra-tumoral, intra-splenic and circulating MDSC decreased in a dose-dependent manner. Combined sunitinib and immunisation therapy led to a marked decrease of intra-tumoral, intra-splenic and circulating MDSC levels as compared to non-treated control or immunisation alone. The bi-therapy regimen markedly enhanced intra-tumoral, intra-splenic and circulating levels of CD8 T cells. The highest number of circulating CD8 T cells undergoing degranulation (CD107ab+) was observed after combined treatment. Most importantly, this combined sunitinib and immunisation treatment regimen abrogated tumour growth.In summary, we demonstrated that sunitinib alone or in combination with immunisation can decrease intra-tumoral, intra-splenic and circulating MDSC levels. Also, combination of sunitinib treatment with immunisation enhanced levels and degranulating activity of CD8 T cells, thus resulting in reversal of tumour growth. This study indicates that SFV-based immunotherapy combined with sunitinib treatment could improve treatment outcome."} +{"text": "Drosophila melanogaster Hox developmental TFs and MED complex proteins. We find that the Med19 subunit directly binds Hox homeodomains, in vitro and in vivo. Loss-of-function Med19 mutations act as dose-sensitive genetic modifiers that synergistically modulate Hox-directed developmental outcomes. Using clonal analysis, we identify a role for Med19 in Hox-dependent target gene activation. We identify a conserved, animal-specific motif that is required for Med19 homeodomain binding, and for activation of a specific Ultrabithorax target. These results provide the first direct molecular link between Hox homeodomain proteins and the general PolII machinery. They support a role for Med19 as a PolII holoenzyme-embedded \u201cco-factor\u201d that acts together with Hox proteins through their homeodomains in regulated developmental transcription.Hox genes in species across the metazoa encode transcription factors (TFs) containing highly-conserved homeodomains that bind target DNA sequences to regulate batteries of developmental target genes. DNA-bound Hox proteins, together with other TF partners, induce an appropriate transcriptional response by RNA Polymerase II (PolII) and its associated general transcription factors. How the evolutionarily conserved Hox TFs interface with this general machinery to generate finely regulated transcriptional responses remains obscure. One major component of the PolII machinery, the Mediator (MED) transcription complex, is composed of roughly 30 protein subunits organized in modules that bridge the PolII enzyme to DNA-bound TFs. Here, we investigate the physical and functional interplay between Drosophila melanogaster may provoke spectacular changes in form: transformations of one body part into another, or loss of organs. This attribute identifies them as important developmental genes. Insect and vertebrate Hox proteins contain highly related homeodomain motifs used to bind to regulatory DNA and influence expression of developmental target genes. This occurs at the level of transcription of target gene DNA to messenger RNA by RNA polymerase II and its associated protein machinery (>50 proteins). How Hox homeodomain proteins induce fine-tuned transcription remains an open question. We provide an initial response, finding that Hox proteins also use their homeodomains to bind one machinery protein, Mediator complex subunit 19 (Med19) through a Med19 sequence that is highly conserved in animal phyla. Med19 mutants isolated in this work show that Med19 assists Hox protein functions. Further, they indicate that homeodomain binding to the Med19 motif is required for normal expression of a Hox target gene. Our work provides new clues for understanding how the specific transcriptional inputs of the highly conserved Hox class of transcription factors are integrated at the level of the whole transcription machinery.Mutations of Hox developmental genes in the fruit fly Drosophila melanogaster Bithorax and Antennapedia Complexes, play a central role in the development of a wide spectrum of animal species Drosophila proteins make use of their homeodomains to play widespread and crucial roles in differentiation programs yielding the very different forms of sea urchins, worms, flies or humans extradenticle (exd)/Pbx and homothorax (hth)/Meis, which assist Hox proteins to form stable ternary DNA-protein complexes with much-enhanced specificity. This involves contacts with the conserved Hox Hexapeptide (HX) motif near the HD N-terminus, or alternatively, with the paralog-specific UBD-A motif detected in Ubx and Abdominal-A (Abd-A) proteins Distal-lessThe finely regulated gene transcription permitting development of pluricellular organisms involves the action of transcription factors (TFs) that bind DNA targets and convey this information to RNA polymerase II (PolII). Hox TFs, discovered through iconic mutations of the Drosophila TFIID component BIP2 binds the Antp HX motif Contrasting with our knowledge of collaborations involving Hox and partner TFs, virtually nothing is known of what transpires at the interface with the RNA Polymerase II (PolII) machinery itself to generate an appropriate transcriptional response. The lone evidence directly linking Hox TFs to the PolII machine comes from the observation that the Another key component of the PolII machinery is the Mediator (MED) complex conserved from amoebae to man that serves as an interface between DNA-bound TFs and PolII. MED possesses a conserved, modular architecture characterized by the presence of head, middle, tail and optional CDK8 modules. Some of the 30 subunits composing MED appear to play a general structural role in the complex while others interact with DNA-bound TFs bridging them to PolII. Together, these subunits and the MED modules they form associate with PolII, TFs and chromatin to regulate PolII-dependent transcription Drosophila skuld/Med13 mutation isolated by dose-sensitive genetic interactions with homeotic proboscipedia (pb) and Sex combs reduced (Scr) genes led us to view MED as a Hox co-factor Med19 mutations isolated in this work reveal that Med19 affects Hox developmental activity and target gene regulation. Taken together, our results provide the first molecular link between Hox TFs and the general transcription machinery, showing how Med19 can act as an embedded functional partner, or \u201cco-factor\u201d, that directly links DNA-bound Hox homeoproteins to the PolII machinery.Our analysis of a in vitro GST-pulldown assay. GST fusions of the eight Drosophila Hox proteins , Deformed (Dfd), Scr, Ubx, Abd-A and Abdominal-B (Abd-B), or portions of Pb and Antp) were probed with 35S-labelled MED proteins in standard conditions for 11 MED subunits that are not required for cell viability in yeast and/or that are known to interact physically with TFs in mammalian cells assay. Auto-fluorescence from the Venus variant of Green Fluorescent Protein (GFP), abolished by truncating Venus protein into N- and C-terminal portions (VN and VC), can be effectively reconstituted when VC and VN fragments are coupled to interacting protein partners that reunite them in cultured cells Med19 mutants (described below). UAS-directed co-expression of Med19-VC with VN-Ubx, VN-Dfd or VN-AbdA in embryos under engrailed-Gal4 control (en>Med19-VC+VN-Hox) resulted in serial stripes of clear nuclear fluorescent signal for VN-Ubx and VN-Dfd common to all Hox proteins. Consistent with this possibility, GST fusions containing the HX-HD regions of Pb and Antp bound Med19 at levels 15- to 50-fold superior to Pb N-ter (a.a. 1\u2013158), Pb C-ter (a.a. 267\u2013782) and Antp N-ter (a.a. 1\u201390) peptides . On disslina Ubx . Tests wlina Ubx . Contrarlina Ubx . Since Min vivo, comparable signals were observed on co-expressing Med19-VC with full-length VN-Ubx, or its HD alone (VN-HDUbx), in the normal Ubx expression domain mutations, 1Med19 upstream deletion), and 2Med19 . In \u201ctwin spot\u201d experiments where \u2212/\u2212 and +/+ cells arise from mitotic recombination in a single \u2212/+ cell during mitosis, only +/+ cells were subsequently detected mutation \u2212/+M cells is associated with disorganization of the distinctive pedicellar sensillae in halteres If Hox/Med19 binding is functionally relevant to homeotic activity, Med19 mutants might provoke Hox-like phenocopies or modify Hox-induced homeotic defects. In light of the strong maternal contribution of Med19 present in embryos, we turned our attention to later developmental stages. Hypomorphic piracles . Rare adry palps . Tissue-Antp for the pupal anterior spiracles Dfd for the adult maxillary palps Ubx for haltere sensory organs Med19 mutants can act as dose-sensitive modifiers of Hox activity in genetic interaction tests. For Antp, the fully penetrant spiracle loss observed in 1/Med192Med19 pupae NsAntp allele directs a fully penetrant transformation of antenna toward leg was enhanced in 2Med19 heterozygotes (1Dfd (20% of adult heads) were fully suppressed in 1Dfd/2Med19 double heterozygotes , is induced by ectopic Ubx in vivobab2 in \u2212/\u2212Ubx haltere cells and found that Bab2 accumulation is cell-autonomously abolished expression in the wing pouch is absent from Ubx-expressing cells of the haltere pouch. sal is de-repressed in \u2212/\u2212Ubx haltere disc cells Ubx mutant cells .Binding of Hox proteins to Med19 specifically involves their conserved homeodomain. To identify Med19 sequences involved in HD binding, we used GST-pulldown to test full-length or deleted versions of Med19 with GST-HDAntp. Binding was retained on truncating the terminal regions of Med19 . As shown in the Western blot of Med19 is required for Ubx-mediated activation of specific target genes We next co-expressed these proteins with Ubx-HA and tested for their association in cellulo. As seen in in vivo, we generated transgenic lines containing the same UAS-Med19-VC, -Med19\u0394HIM-VC and -HIM-VC constructs used above, and tested each protein's ability to bind to VN-HDUbx in the BiFC assay. In control experiments, Med19-VC, HIM-VC and Med19\u0394HIM-VC accumulated at comparable levels in wing imaginal discs FLP/FRT-mediated mitotic recombination was used to generate cells devoid of wild-type protein, while (ii) UAS/Gal4-directed expression supplied normal or HIM-deleted Med19-VC, and (iii) the edge-GFP reporter was employed to assess Ubx-mediated activation of CG13222. En-Gal4-directed UAS-Flp expression in the posterior haltere disc compartment served to induce mitotic recombination there, while en-Gal4 simultaneously directed expression of Med19-VC or Med19\u0394HIM-VC in the posterior compartment were observed not only in Med19-VC but also in Med19\u0394HIM-VC expressing discs . The exiDrosophila Ultrabithorax-like mutant affecting the large subunit of RNA PolII provokes phenotypes reminiscent of Ubx mutants Drosophila Hox developmental TFs and the MED transcription complex. Our results unveil a novel aspect of the evolutionary Hox gene success story, extending the large repertory of proteins able to interact with the HD Drosophila MED subunit Med19. HD binding to Med19 via the conserved HIM suggests this subunit is an ancient Hox collaborator. Accordingly, our loss-of-function mutants reveal that Med19 contributes to normal Hox developmental function and does so at least in part via its HIM element. Thus this analysis reveals a previously unsuspected importance for Med19 in Hox-affiliated developmental functions.Hox homeodomain proteins are well-known for their roles in the control of transcription during development. Further, much is known about the composition and action of the PolII transcription machine. However, virtually nothing is known of how the information of DNA-bound Hox factors is conveyed to PolII in gene transcription. The S. cerevisiae places Med19 at the interfaces of the head, middle and CDK8 kinase modules A fundamental property of the modular MED complex is its great flexibility that allows it to wrap around PolII and to change form substantially in response to contact with specific TFs Med19 contributes to developmental processes with Antp (spiracle eversion), Dfd , and Ubx . Other phenotypes identified with our mutants indicate further, non-Hox related roles for Med19. As shown here, complete loss of Med19 function leads to cell lethality that can be conditionally alleviated when surrounded by weakened, Minute mutation-bearing cells. These observations, that uncouple HIM-dependent functions from the role of Med19 in cell survival/proliferation edge-GFP in Med19\u2212 cells expressing Med19\u0394HIM-VC was not altogether refractory to Ubx-activated edge-GFP expression bapression ; and with pActin-V5 ; or adding pUAS-Ubx-HA (Med19-Ubx co-IP). 107 cells were transfected with driver plasmid plus the UAS responder plasmid(s). After 72 hr, cells were harvested by scraping and pooled, collected by centrifuging then washed with 1x PBS. All subsequent steps until Western blotting were carried out at 4\u00b0C. Cell pellets were resuspended in IP buffer , lysed by four-fold passage through a 27G needle, then centrifuged for 10 min at 14,500 rpm. Immunoprecipitation from 1.5 mg of total protein extract (5 \u00b5g/\u00b5l in IP buffer) was performed with mouse anti-GFP (ROCHE 4 \u00b5g/IP), with gentle agitation overnight. 15 \u00b5l of G-protein-coupled Sepharose beads were added, then gently agitated for 2 hr. The non-bound fraction was discarded. Beads were washed 4 times with fresh IP buffer, taken up in 2X Laemmli buffer containing DTT and SDS, heated to 95\u00b0C, and centrifuged. Supernatants were then submitted to polyacrylamide gel electrophoresis. Med1 and Med19 were revealed using polyclonal sera from guinea-pig (diluted 1\u2236500), while Ubx-HA was detected with rabbit anti-HA (SIGMA) diluted 1\u22361000.Cultured Constructions corresponding to UAS-VN-Ubx, UAS-VN-AbdA and UAS-VN-HDAbdA transgenic lines are described in NsAntp, 1Dfd, Cbx1Ubx and Df(3L)BSC8 were from the Bloomington Drosophila Stock Collection. NsAntp+Rc3 was provided by R. Mann. Edge-GFP and vgQ-lacZ originate from the S. Carroll lab. The Ub-Med19 transgenic line expressing full-length Med19 cDNA under ubi73 control is a homozygous-viable insertion on the X chromosome at attP site ZH 2A. These transgenic elements carry the visible marker mini-white. UAS-RNAi against Dfd is from a non-directed insertion on chromosome 2 (Vienna Drosophila Research Collection stock 50110). UAS-RNAi lines against Med19 are stocks 27559 and 33710 from the Bloomington collection. Gal4-expressing driver lines used were: dpp-Gal4 , arm-Gal4 (ubiquitous), ptc-Gal4 , en-Gal4 (posterior compartment), ap-Gal4 , Ubx-Gal4 and abdA-Gal4 .Stocks and crosses were maintained at 25\u00b0C on standard yeast-agar-cornmeal medium. Mutant stocks harboring Med19 alleles were generated by imprecise excision, mobilizing the viable P{EPgy2}EY16159 insertion marked with mini-w+ (see Flybase). Among 154 white-eyed candidates, two 1Med19 and 2Med19 (described in Df(3L)BSC8.Loss-of-function The following stocks were employed for rescue tests:2Med19/TM6B, Hu Tb;Ub-Med19; 1Med19/TM6B, Hu Tb;2/Med19TM6B, Hu Tb;Arm-Gal4; 1Med19/TM6B, Hu Tb;2Med19/TM6B, Hu Tb;UAS-Med19-VC; 2Med19/TM6B, Hu Tb.UAS-Med19\u0394HIM-VC; en>Flp, ap>Flp). Clones were generated and identified in marked progeny from crosses using the following stocks:Mitotic clones were induced by Flp recombinase expressed from a hsp70-Flp transgene on heat induction (30\u2032 at 38\u00b0C), or from a UAS-Flp element under Gal4 control as indicated above on two successive days. Resulting y w hsp70-Flp/w; 2Med19 FRT-2A/P[D1ovo] FRT-2A adult females were crossed with 2Med19 FRT-2A/TM3, Ser twist>GFP males. Embryos resulting from germline clones were collected on egg lay plates, then analysed by confocal microscopy after mounting in DAPI-containing Vectashield medium. In positive controls where Med19+ replaced 2Med19, all expected zygotic classes were obtained as viable, fertile adults. In the absence of heat shock, no eggs were laid.After crossing Performed as described in E. coli and enriched by affinity chromatography. Anti-Med19 sera from terminal bleeds was used for immunocytology without purification at a 1\u2236500 dilution after prior pre-absorption on wild type larvae.Guinea pigs were immunized (Eurogentec) with GST-Med19 or GST-Med1 proteins extracted from Adult phenotypes were analyzed by light microscopy (Zeiss Axiophot) of dissected samples mounted in Hoyer's medium or by scanning electron microscopy (Hitachi TM-1000 Tabletop model) of frozen adultsThese were generated with the T-Coffee Program, employing the methodology described by Figure S1Drosophila embryo. Cuticles of wild-type embryos, or of embryos ectopically expressing Hox proteins. First line: left, wild-type embryo showing anterior cuticle from the head to abdominal segment 1 (A1). The three thoracic belts of fine denticles and the first band of denser abdominal denticles are indicated ; middle and right, similar transformations of T1, T2 and T3 denticle belts to A1 are induced by the ubiquitous expression of Ubx or of chimeric VN-Ubx, respectively, from UAS enhancers under arm-Gal4 control (arm>). Second line: left, wild-type embryonic head with cephalo-pharyngeal cuticle. Middle and right: arm-Gal4 driver-directed expression of Dfd or VN-Dfd from a UAS enhancer (arm>) results in similar, major defects of normal head structures, accompanied by the appearance of ectopic maxillary cirri (arrows and inset) typical of Dfd function.Venus fusion proteins VN-Ubx and VN-Dfd are functional in the (TIF)Click here for additional data file.Figure S2Drosophila Ubx, with its hexapeptide (HX), linker region and HD; (middle) the HD region of Drosophila Ubx, but with its hexapeptide mutated (HXm) as described in Hudry et al (34); (right) the HD region of crustacean Artemia Ubx, whose HD is identical to the Drosophila sequence but whose linker region is much shorter. On the right, GST pulldowns show similar binding to wild-type Ubx (7% of input), Ubx whose HX is mutated (5%), or Artemia Ubx with shortened linker (5%).Hexapeptide and linker region are dispensable for interaction with Med19. Bar drawings on the left represent (top) the HD region of (TIF)Click here for additional data file.Figure S3Strong maternal effect of Med19 mutant germline clones. The photos in A, B and C present the cellular progression of embryos lacking maternally contributed Med19 (as seen by DAPI staining of nuclear DNA). These embryos are pre-cellular, aged \u22481 hr and \u22482 hr, with the latter corresponding to the onset of zygotic transcription. (C) This embryo, seen shortly after cellularisation, shows massive disorganisation.(TIF)Click here for additional data file.Figure S4daughterless-Gal4 control (da>dsMed1927559). These photos reveal defects of posterior spiracles (above) or of larval mouthparts (bottom), resembling the embryonic defects of Hox mutants (noted to the right).Med19 dsRNA affects the differentiation of larval posterior spiracles and mouthparts. Left column photos: wild-type larval posterior spiracles (top) and mouthparts (bottom). Right column photos: L3 larvae expressing UAS-dsRNA directed against Med19 under (TIF)Click here for additional data file.Figure S52Med19 null clones can be rescued by UAS-Med19 transgene expression or in a Minute context. Mitotic clones homozygote for 2Med19 were induced in wing imaginal discs by en-Gal4 coupled with UAS-Flp (en>Flp) as described in text. : en-Gal4 also directed UAS-Med19-VC expression (en>Flp>Med19VC). A large \u2212/\u2212 clone is detected by the absence of green GFP (A); Med19 and Med19-VC proteins are both detected by anti-Med19 sera ; the merged image is shown in A\u2033. : Mitotic clones were induced as for A\u2013A\u2033. Rather than supply transgenic Med19, clones were induced in the presence of a Minute mutation on the homologous chromosome. (B) \u2212/\u2212 clones are detected on the right-hand side of this wing imaginal disc by the absence of green GFP marker. (B\u2032) Anti-Med19 sera (red) showed no signal in mutant cells. (B\u2033) Merged images confirm the absence of red signal in mutant cells.(TIF)Click here for additional data file.Figure S6Antp gof allele NsAntp; with the Antp lof allele NsAntp+RC3; with the Dfd gof allele 1Dfd; and with a lof combination for Dfd ). The heterozygous presence of 2Med19 significantly altered the phenotypic outcome in each case.Table 1, interaction data. Phenotypic analyses indicate interactions of Med19 lof mutations with the (TIF)Click here for additional data file.Figure S7Drosophila Med19 indicates the internal location of the HIM element. Sequence alignments are shown for the species listed at the bottom.The Med19 Hox \u201cHomeodomain Interacting Motif\u201d (HIM) is conserved across the animal kingdom. At top, a block representation of (TIF)Click here for additional data file.Figure S8Drosophila S2 cells transfected with act5C-Gal4 driver alone (control), with UAS-Med19-VC or - \u0394HIM-VC plasmid, were immunoprecipitated with anti-GFP directed against the VC tag. Western blots of these precipitates tested with anti-Med1 revealed association of the three known Med1 isoforms (Input) with both Med19-VC and \u0394HIM-VC in the presence of anti-GFP (IP GFP) but not in controls (IP). (B) Characterisation of expression levels and cellular localisation for Med19-VC, \u0394HIM-VC and HIM-VC. The three proteins are accumulated at similar levels when expressed under dpp-Gal4 control in wing imaginal discs, as seen with anti-GFP. (C) Med19-VC is expressed as a band in the wing imaginal disc under dpp-Gal4 control, detected here with anti-GFP. (F) Enlargement of the boxed region of C. reveals nuclear Med19-VC (arrow), that coincides with anti-Med19 staining (F\u2032) and DAPI staining of nuclear DNA (F\u2033). F\u2033\u2032 presents the merged signals. (D) \u0394HIM-VC expressed in the wing imaginal disc under dpp-Gal4 control is detected with anti-GFP. (G\u2013G\u2033\u2032) G, enlargement of the boxed region of D. A single representative cell (arrow) shows co-localisation for anti-GFP (G) and anti-Med19 (G\u2032). As shown by DAPI (G\u2033) and in the merged image (G\u2033\u2032), \u0394HIM-VC is present both in the nucleus and the cytoplasm. (E) Expression of HIM-VC under dpp-Gal4 control, visualised in a wing imaginal disc with anti-GFP. (H) Enlargement of the boxed region of E. Nuclear HIM localisation (arrow) is confirmed in H\u2032 (anti-Med19), H\u2033 (DAPI) and in the merged image (H\u2033\u2032).Med19 variant incorporation into MED, expression levels and nuclear location. (A) Co-immunoprecipitation experiment. Extracts of (TIF)Click here for additional data file.Figure S9arm-Gal4 driver directed expression of UAS-Med19-VC and -\u0394HIM-VC, in the pupal-lethal 1Med19/2Med19 context. Culture temperatures are noted. Adult viability was partially restored by Med19-VC, but not by \u0394HIM-VC. Spiracle eversion and maxillary formation were rescued to a greater extent by Med19-VC. Bottom: \u2212/\u2212 haltere clones were induced in the presence of a Minute mutation by apterous-Flp (ap-Flp), alone or in the presence of UAS-Med19-VC or -\u0394HIM-VC. Only Med19-VC yielded apparent rescue.Table 2, phenotypic rescue by Med19-VC and \u0394HIM-VC constructs. These forms of Med19 were employed to rescue effects of Med19 mutant combinations. Top: (TIF)Click here for additional data file."} +{"text": "Oxcarbazepine (OXC) is a structural analog of carbamazepine (CBZ). OXC is considered a promising alternative medication for patients who cannot tolerate CBZ because of its equivalent clinical effiecacy and fewer cutaneous adverse drug reactions (cADRs) compared to CBZ. HLA-B*15:02 allele was shown to strongly associate with carbamazepine (CBZ)-induced Stevens-Johnson syndrome (SJS)/toxic epidermal necrolysis (TEN) in Chinese and Southeast Asian populations.However,the epidemiological data about OXC-induced cADRs in recent years is limited. This study aims to investigate the clinical characteristics of OXC-induced cADRs and their genetic associations with HLA-B*15:02.We conducted a perspective study of patients with OXC-induced cADRs from Chang Gung Memorial Hospital Health System and Taiwan-SCAR consortium since 2006 to 2013. The diagnosis of OXC-cADRs were based on clinical features, exclusions of alternative causes, supported by ancillary investigations such as histological and laboratory findings. Clinical course, latent period, drug dosage, organ involvement, complications and the mortality were analyzed. We also examined the HLA-A and -B genotypes of all patients with OXC-induced cADRs comparing to OXC-tolerant controls.The study included 31 patients with OXC-cADRs, including 12 SJS (no TEN case), 4 drug rashes with eosinophilia and systemic symptoms (DRESS ), 13 maculopapular exanthema (MPE), 2 bullous fixed drug eruption (BFDE) and 101 OXC-tolerant controls. Comparing to CBZ, the clinical severity of OXC-induced SJS and DRESS were less severe. All cases of OXC-SJS presented with limited skin detachment (<5%) without mortality, although there were typical mucosal involvements and typical histopathologicl findings of epidermal necrosis. All four cases of OXC-DRESS also presented with less severe liver injuries. Interestingly, similar to CBZ-SJS, the HLA study showed HLA-B 15:02 allele was significantly associated with patients with OXC-SJS (P< 0.01), but was not associated with other phenotypes. However, HLA-A*3101 was not significantly associated with all phenotypes of OXC-cADRs.Our findings suggest that HLA-B*15:02 allele is significantly associated with OXC- SJS in Han Chinese. However, the genetic association is phenotype-specific (only for OXC-SJS) and the strength is less than CBZ-SJS/TEN. Furthermore, in comparison to CBZ, OXZ-SJS presented with less clinical severity and better clinical outcomes, suggesting a decrease of antigenicity for induction of SJS than CBZ."} +{"text": "Intermittent preventive treatment in pregnancy (IPTp) with sulfadoxine-pyrimethamine (SP) is recommended in HIV-negative women to avert malaria, while this relies on cotrimoxazole prophylaxis (CTXp) in HIV-positive women. Alternative antimalarials are required in areas where parasite resistance to antifolate drugs is high. The cost-effectiveness of IPTp with alternative drugs is needed to inform policy.The cost-effectiveness of 2-dose IPTp-mefloquine (MQ) was compared with IPTp-SP in HIV-negative women . In HIV-positive women the cost-effectiveness of 3-dose IPTp-MQ added to CTXp was compared with CTXp alone . The outcomes used were maternal clinical malaria, anaemia at delivery and non-obstetric hospital admissions. The poor tolerability to MQ was included as the value of women\u2019s loss of working days. Incremental cost-effectiveness ratios (ICERs) were calculated and threshold analysis undertaken.For HIV-negative women, the ICER for IPTp-MQ versus IPTp-SP was 136.30 US$ (2012 US$) per disability-adjusted life-year (DALY) averted, or 237.78 US$ , depending on whether estimates from Gabon were included or not. For HIV-positive women, the ICER per DALY averted for IPTp-MQ added to CTXp, versus CTXp alone was 6.96 US$ . In HIV-negative women, moderate shifts of variables such as malaria incidence, drug cost, and IPTp efficacy increased the ICERs above the cost-effectiveness threshold. In HIV-positive women the intervention remained cost-effective for a substantial (up to 21 times) increase in cost per tablet.Addition of IPTp with an effective antimalarial to CTXp was very cost-effective in HIV-positive women. IPTp with an efficacious antimalarial was more cost-effective than IPTp-SP in HIV-negative women. However, the poor tolerability of MQ does not favour its use as IPTp. Regardless of HIV status, prevention of malaria in pregnancy with a highly efficacious, well tolerated antimalarial would be cost-effective despite its high price.NCT 00811421; Pan African Trials Registry PACTR2010020001429343 and PACTR2010020001813440ClinicalTrials.gov Plasmodium falciparum, is likely to be significant ; 8 Ministry of Health- Republic of Kenya (2012). \"Early Infant Diagnosis Program. National AIDS and STI Control Program.\" www.nascop.org/eid [accessed February 2014]; 9 Moraleda C. et al. (2014) J Acquir Immune Defic Syndr; 10Ministry of Health and Social Welfare-The United Republic of Tanzania (2013). \"UNAIDS 2013 Global Report.\" http://pmtct.or.tz/pmtct-tanzania/pmtct-in-tanzania/ [accessed February 2014].(DOCX)Click here for additional data file.S2 Tablea Intention to treat; b Low birth weight < 2500 gr; c Proportional difference; d Arithmetic difference(DOCX)Click here for additional data file.S3 Tablea Intention to Treat (ITT) analysis adjusted by country.; b Risk ratio; c Mean difference; d Assessed by the Ballard score (excluding incomplete data).(DOCX)Click here for additional data file.S4 Tablea Intention to treat; b Episodes per person/year, adjusted by country.(DOCX)Click here for additional data file.S5 Tablea Intention to treat; b Episodes per person/year, adjusted by country.(DOCX)Click here for additional data file."} +{"text": "Genetic and environmental contributions to the global Eating Disorders Examination (EDE) scores over early and later adolescence were investigated to identify whether different sources influence disordered eating (DE). Specific sources of environmental risk were also examined for differences early and late in the adolescent developmental trajectory.Adolescent females from the Australian Twin Registry were interviewed by telephone, including the EDE and impairment/risk-related self-report measures. Data were collected at 12-15 and 16-19 years .Analyses involved bivariate Cholesky decomposition modelling of genetic and non-shared environmental influences. At 12-15 years, additive genetic and non-shared environmental sources significantly contributed to the DE phenotype, continuing to contribute at 16-19 years; additional independent additive genetic and non-shared environmental sources also conveyed risk at ages 16-19. Linear mixed models of environmental risk identified weight-related peer teasing in early-mid adolescence predicted DE later in adolescence.A second risk period for DE onset appears in late adolescence. The predominance of family relationships attenuates among teenagers synchronously with a strong surge in attention to, and investment in, peer-peer connectivity. This accords with our findings implicating weight-related peer teasing - a non-shared environmental risk factor - in risk of DE. Increased genetic risk and sensitivity to non-shared environmental influences are likely antecedents in development of DE in late teenagehood."} +{"text": "NLRP3 mutations. All these diseases are currently considered as different phenotypes of the cryopyrin-associated periodic syndromes (CAPS). A variable degree of somatic NLRP3 mosaicism has been recently detected in \u224835% of patients with CINCA. However, no data are currently available regarding the relevance of this genetic mechanism in other CAPS phenotypes.Familial cold autoinflammatory syndrome, Muckle-Wells syndrome (MWS), and chronic, infantile, neurological, cutaneous and articular (CINCA) syndrome are dominantly inherited autoinflammatory diseases associated to gain-of-function NLRP3 mosaicism as the disease-causing mechanism in patients with CAPS phenotypes other than CINCA and NLRP3 mutation-negative by conventional, Sanger-based genetic studies.To evaluate somatic NLRP3 analyses were performed by Sanger sequencing and by targeted deep sequencing. Apoptosis-associated Speck-like protein containing a CARD (ASC)-dependent nuclear factor kappa-light chain enhancer of activated B cells (NF-\u03baB) activation and transfection-induced THP-1 cell death assays determined the functional consequences of the detected variants.NLRP3 mosaicism was detected in 9.3% of enrolled patients (3/32). Their clinical phenotypes were identical to that seen in MWS. Three different missense variants were identified, being two novels (p.D303A and p.L411F). Bioinformatic and functional analyses confirmed that they were disease-causing, gain-of-function NLRP3 mutations. Treatment with anti-IL-1 drugs showed long-lasting and positive clinical and biochemical responses.32 Spanish patients fulfilling clinical inclusion criteria were enrolled. A variable degree (9.4-34.9%) of somatic NLRP3 mosaicism in MWS pathogenesis, which probably represents a shared genetic mechanism in CAPS not restricted to CINCA syndrome. The data here described allowed us to achieve the definitive diagnoses of these patients, which have had serious clinical implications such as gaining access to anti-IL-1 treatments under legal indication and genetic counseling. The detection of somatic gene mosaicism is difficult when using conventional methods. Potential candidates should benefit from the use of novel technologies such as targeted deep sequencing.We herein show novel evidence about the role of somatic"} +{"text": "In the current study, preparations of MRP2-expressing and control membrane vesicles, containing inside-out orientated vesicles, were used to directly characterise the membrane transport of oxaliplatin-derived platinum measured by inductively coupled plasma mass spectrometry. Oxaliplatin inhibited the ATP-dependent accumulation of the model MRP2 fluorescent probe, 5(6)-carboxy-2,'7'-dichlorofluorescein, in MRP2-expressing membrane vesicles. MRP2-expressing membrane vesicles accumulated up to 19-fold more platinum during their incubation with oxaliplatin and ATP as compared to control membrane vesicles and in the absence of ATP. The rate of ATP-dependent MRP2-mediated active transport of oxaliplatin-derived platinum increased non-linearly with increasing oxaliplatin exposure concentration, approaching a plateau value (Vmax) of 2680 pmol Pt/mg protein/10 minutes , with the half-maximal platinum accumulation rate (Km) at an oxaliplatin exposure concentration of 301 \u03bcM , in accordance with Michaelis-Menten kinetics (r2 = 0.954). MRP2 inhibitors (myricetin and MK571) reduced the ATP-dependent accumulation of oxaliplatin-derived platinum in MRP2-expressing membrane vesicles in a concentration-dependent manner. To identify whether oxaliplatin, or perhaps a degradation product, was the likely substrate for this active transport, HPLC studies were undertaken showing that oxaliplatin degraded slowly in membrane vesicle incubation buffer containing chloride ions and glutathione, with approximately 95% remaining intact after a 10 minute incubation time and a degradation half-life of 2.24 hours . In conclusion, MRP2 mediates the ATP-dependent active membrane transport of oxaliplatin-derived platinum. Intact oxaliplatin and its anionic monochloro oxalate ring-opened intermediate appear likely candidates as substrates for MRP2-mediated transport.The platinum-based anticancer drug oxaliplatin is important clinically in cancer treatment. However, the role of multidrug resistance-associated protein 2 (MRP2) in controlling oxaliplatin membrane transport, The platinum-based anticancer drug oxaliplatin, and its combination therapies, are clinically important for treating colorectal cancer and other gastrointestinal malignancies . HoweverABCC2 gene and also known as canalicular multispecific organic anion transport (cMOAT) leukotrieneC4 transport - -, which may not have been detected by the HPLC assay, is another candidate substrate as it is formed early during oxaliplatin degradation reactions with chloride ions - . Reactioalate)]- . As the DACH)Cl2 . These side ions and is aide ions . Furtherin vitro experimental findings is currently unclear and requires further study. MRP2 is expressed on the apical membranes of hepatocytes and proximal renal tubular cells, where it functions in the biliary and renal excretion of endogenous and exogenous compounds [The pharmacological and clinical significance of these ompounds . In thisompounds , 50 and ompounds \u201330. Furtompounds .MRP2 mediates the active transport of platinum derived from oxaliplatin in a manner depending upon ATP. Intact oxaliplatin and its anionic monochloro oxalate ring-opened early degradation product are likely candidate substrates for active transport mediated by MRP2."} +{"text": "B16-derived OVA-expressing melanomas resist curative immunotherapy with either adoptive transfer of activated anti-OVA OT1 cytotoxic T lymphocytes (CTLs) or agonist anti-CD137 (4-1BB) mAb. However when acting in synergistic combination, these treatments consistently achieve tumor eradication. Tumor-infiltrating lymphocytes that accomplish tumor rejection exhibit enhanced effector function in both transferred OT-1 and endogenous CTLs. This is consistent with higher levels of expression of eomesodermin in CTLs and with confocal microscopy evidence for more efficacious tumor-cell killing. Combined immunotherapy of tumors monitored by intravital live-cell two-photon microscopy reveals persistence of the OT1 CTL-effector phenotype over prolonged periods of time. Anti-CD137 mAb delayed loss of function with focused and confined interaction kinetics of OT-1 CTL with target cells lasting up to ten days post-transfer. The synergy of adoptive T cell therapy and anti-CD137 mAb thus results from in-vivo enhancement of effector functions."} +{"text": "Foxp3 mRNA up-regulation. Thus, our mouse model clearly reflects the \u201cdual-allergen-exposure hypothesis\u201d in which allergic sensitization results from cutaneous food antigen exposure before its consumption. When allergy is induced by epicutaneous sensitization then oral challenge, basophil-depleted or TSLP-receptor-deficient mice do not produce OVA-specific IgE and are protected from oral challenge-induced anaphylaxis. IL-33-deficient mice produce normal levels of OVA-specific IgE, however, IL-33-deficient mice and mice treated with recombinant soluble IL-33 receptor are protected from anaphylaxis. Thus, basophils and TSLP have pivotal roles in Th2 development in the skin during the sensitization phase of food allergy. In contrast, while IL-33 is dispensable for promoting cutaneous antigen sensitization, the cytokine is essential for inducing IgE-dependent anaphylaxis in the gut.Cutaneous sensitization with a food antigen before its consumption elicits the development of food allergy. Here we report the site and stage dependent roles of basophils and pro-allergic cytokines, thymic stromal lymphopoietin (TSLP) and IL-33, in a mouse model of food allergy initially sensitized cutaneously with the food antigen. Mice are epicutaneously sensitized with the food antigen ovalbumin (OVA) followed by oral challenge with OVA. Epicutaneously-sensitized mice produce OVA-specific IgE and develop IgE-dependent anaphylaxis after oral challenge. If OVA is given orally before epicutaneous administration, development of food allergy is prevented with"} +{"text": "In paired recordings, the release probability at synapses between TML-Cs was increased by the CB1R antagonist AM251, demonstrating baseline endocannabinoid regulation of TML-C synapses. Apart from defining the synaptic and neurochemical features of TML-Cs, our findings reveal the morphological identity of a class of dentate CB1R-positive neurons that do not express CCK. Our findings indicate that TML-Cs can mediate cannabinoid sensitive feed-forward and feedback inhibition of dentate perforant path inputs. \u00a9 2015 The Authors Hippocampus Published by Wiley Periodicals, Inc.Activity of the dentate gyrus, which gates information flow to the hippocampus, is under tight inhibitory regulation by interneurons with distinctive axonal projections, intrinsic and synaptic characteristics and neurochemical identities. Total molecular layer cells (TML-Cs), a class of morphologically distinct GABAergic neurons with axonal projections across the molecular layer, are among the most frequent interneuronal type in the dentate subgranular region. However, little is known about their synaptic and neurochemical properties. We demonstrate that synapses from morphologically identified TML-Cs to dentate interneurons are characterized by low release probability, facilitating short-term dynamics and asynchronous release. TML-Cs consistently show somatic and axonal labeling for the cannabinoid receptor type 1 (CB The dentate gyrus is known for its laminar inputs with commissural and associational fibers targeting the inner molecular layer (IML) while medial and lateral perforant path project to the middle and outer molecular layers in the dentate molecular layer. CB1R is present in certain glutamatergic and GABAergic terminals in the dentate IML or anti-PV antibody using previously described protocols were considered significant.Briefly, horizontal brain slices (300 \u03bcM) were prepared from male, Wistar rats >30 days old under protocols approved by Rutgers-NJMS, Newark, NJ, IACUC. Neurons in the subgranular hilus were patched using microelectrodes containing equal concentrations of KCl and K-gluconate and 0.2% biocytin compared to FS-BCs and aspiny dendrites extending into both the molecular layer and hilus. Molecular layer dendrites of TML-Cs extended to the hippocampal fissure (n = 14 cells) and higher input resistance (Rin) than FS-BCs during sustained depolarization and a greater membrane potential sag during hyperpolarization . Although TML-C intrinsic properties such as adapting firing pattern, high input resistance and presence of membrane sag resemble those of CCK expressing HICAP cells and FS-BCs . Compared to synapses between FS-BCs (7 pairs), unitary inhibitory postsynaptic currents (uIPSCs) between TML-Cs (5 pairs) were characterized by low release probability (P < 0.05) and amplitude (P < 0.05 by U test). TML-C uIPSC amplitude potency excluding failures was 19.5 \u00b1 3.9 pA. Similarly, 20\u201380% rise time and decay times were slower than in FS-BC synapses. TML-C synapses had longer latency (P < 0.05 by U test) and higher CV of latency than FS-BCs, which is similar to data from IPSCs between presumed HICAP cells indicating that inhibition was mediated by GABAA receptors. These data demonstrate that, like intrinsic properties, synaptic characteristics of morphologically identified TML-Cs are distinct from FS-BC and similar to dentate HICAP cells , in addition to IML axonal fibers co-labeled for CB1R and CCK. Since perforant path inputs do not exhibit cannabinoid modulation . However, TML-Cs lacked somatic or dendritic labeling for CCK (n = 8) despite prominent CCK immunoreactivity in adjacent neurons (1R in the CCK expressing cell). Since TML cells showed somatic labeling for CB1R, and neurons with IML axons recorded under similar conditions were co-labeled for CCK and CB1R (data not shown), it is unlikely that the recording conditions resulted in absence of CCK expression in TML-Cs. Thus, TML-Cs are morphologically distinct from HICAP cells and can be neurochemically distinguished from CCK- and CB1R-positive neurons with IML axons. In addition to the soma, molecular layer axon collaterals of TML-Cs were labeled with CB1R consistently and reversibly enhanced the synaptic success rate (1R agonist WIN-55212 (10 \u03bcM) reduced the probability of synaptic release suggesting that CB1R-modulation of TML-C synaptic release is bidirectional. Additionally, release probability at TML-C synapses on FS-BCs was also enhanced by AM251 . Together these data demonstrate functional, baseline endocannabinoid modulation of unitary TML-C synapses.Activation of CB Freund, . The cha1R-sensitive synapses between TML-Cs, release at TML-C synapses on granule cells and other interneurons are also likely modulated by CB1Rs. Thus, in addition to the commissural-associational inputs in the IML that are regulated by asynchronous inhibition from HICAP cells, perforant path inputs in the middle and outer molecular layers are potentially regulated by feed-forward and feedback inhibition from TML-Cs with facilitating synaptic dynamics.This study constitutes the first detailed physiological and neurochemical characterization of dentate TML cells, a class of GABAergic neurons in the hilar-granule cell layer border. Although TML-Cs were initially described over 20 years ago (Soriano and Frotscher, 1R. TML-C synapses exhibit low release probability that was enhanced by the CB1R inverse agonist AM251 (1R-dependent suppression of release as reported in cortical and hippocampal CB1R-expressing neurons (Losonczy et al., 1R and not CCK in TML-Cs is distinct from both HICAP cells expressing CCK designated as CCK-positive basket cells (Hefft and Jonas, 1R-modulation (Liu et al., 1R (Ali and Todorova, 1R and lack CCK. While earlier studies have suggested the presence of CB1R in dentate interneurons lacking CCK (Hajos et al., 1R and not CCK, our findings shed light on differential distribution of CCK- and CB1R-expressing inhibitory axonal collaterals across the dentate molecular layer (Hefft and Jonas, 1R-sensitive inhibition in the dentate cannot be considered synonymous with inhibition from CCK-expressing interneurons.A salient finding of this study is that TML-Cs, show somatic and axonal expression of CBst AM251 indicati"} +{"text": "Leukemia inhibitory factor (LIF) is a multi-function cytokine. Its role in cancer is not well-understood. Recent studies including ours show that LIF is frequently overexpressed in many types of human tumors and promotes the progression and metastasis of tumors. However, the underlying mechanism of LIF's promoting effects on tumor progression and metastasis is poorly defined. Epithelial-mesenchymal transition (EMT) plays an important role in tumor metastasis. This study reports that LIF promotes EMT in human tumor cells. Overexpression of LIF promotes tumor cells to acquire mesenchymal features, including morphological changes of cells from epithelial-like to mesenchymal-like, increased expression levels of mesenchymal markers and decreased expression of epithelial markers. Knockdown of endogenous LIF reverses EMT in cancer cells. We further identified that LIF induces the expression of microRNA-21 (miR-21), which in turn mediates the promoting effect of LIF on EMT. LIF induces miR-21 expression through the activation of STAT3. Importantly, blocking miR-21 function greatly abolished the promoting effect of LIF on EMT and the migration ability of cancer cells. Taken together, results from this study identified an important function and a novel underlying mechanism of LIF in EMT and tumor metastasis. Recent reports have indicated that EMT promotes the emergence of cancer stem cells (CSCs) , 39. CSCmiR-21 has been reported to promote the proliferation and growth of tumor cells, a phenotype that is also observed in cells with LIF overexpression , 31. OneIn addition to its role in cancer, EMT is also involved in normal physiological process of embryonic implantation and the initiation of placenta formation. LIF is highly induced at the implantation stage in uterine tissues and regulates several important steps during implantation, including the receptive state of endometrial, the interaction between endometrial and embryo, stromal decidualization, the invasion and development of blastocyst . It is uIn summary, this study demonstrates that LIF promotes EMT, which is a novel mechanism by which LIF promotes tumor progression and metastasis. This function of LIF is mainly mediated through the induction of miR-21 by STAT3. Results from this study suggest that targeting LIF/STAT3/miR-21 could be a potential therapeutic strategy for tumors with LIF overexpression.Human breast cancer cell lines MCF7, T47D, MDA-MB-231 and MDA-MB-468 and human colorectal cancer cell line HCT116 were purchased from ATCC. MCF7 and T47D cells were cultured in RPMI1640 with 10% FBS. MDA-MB-231, MDA-MB-468 and HCT116 cells were culture in DMEM with 10% FBS. The ectopic LIF stable cell lines MCF7-LIF and T47D-LIF and MDA-MB-231 cells with stable knockdown of endogenous LIF (MDA-MB-231-shLIF1 and MDA-MB-231-shLIF2) were established in our lab as previously described [The LIF expression vector pLPCX-LIF was constructed in our lab as previously described . The seqTotal miRNA and RNA were extracted from cells by using a miRNesay Mini Kit and an RNeasy Kit, respectively (Qiagen). cDNA was prepared by using a Taqman Reverse Transcription Kit (Life technology). The U6 and miR-21 probes were purchased from Life technology. The expression levels of EMT markers were detected by following primers: E-cadherin (E-cad) forward: 5\u2032-ATT TTT CCC TCG ACA CCC GAT-3\u2032, reverse: 5\u2032-TCC CAG GCG TAG ACC AAG A-3\u2032, N-cadherin (N-cad) forward: 5\u2032-TGC GGT ACA GTG TAA CTG GG-3\u2032, reverse: 5\u2032-GAA ACC GGG CTA TCT GCT CG-3\u2032 and Vimentin (VIM) forward: 5\u2032-AAT GGC TCG TCA CCT TCG TGA AT-3\u2032, reverse: 5\u2032-CAG ATT AGT TTC CCT CAG GTT CAG-3\u2032.Standard Western blot assays were used to analyze the levels of protein. The EMT markers were detected by the following antibodies: anti-E-cad , anti-N-cad and anti-VIM . LIF was detected by anti-LIF . Anti-p-STAT3 and anti-STAT3 antibodies were used to detect the activation of the STAT3 signaling pathway.For migration assays, the trans-well system was employed as we previously described . In brieRenilla as an internal control to normalize transfection efficiency by using Lipofectamine 2000 (Invitrogen). The luciferase activity was measured by using the Luciferase Assay Kit (Promega) and normalized with Renilla luciferase activity. To determine whether STAT3 transactivates the pGL2-miR-21 reporter vector, cells were co-transfected with pGL2-miR-21 reporter vector, pRL-null vector and STAT3 expression vector.Cells were transiently transfected with luciferase report vector pGL2 containing miR-21 promoter region together with pRL-null vector which expresses t-test was used to identify the difference between two groups. Values of p < 0.05 were considered to be statistically significant.The Student"} +{"text": "Legionella vacuole with lysosomes. Caspase-11, independently of the inflammasome, also promotes phagolysosomal fusion. However, it is unclear how these proteases alter intracellular trafficking. Here, we show that caspase-11 and caspase-1 function in opposing manners to phosphorylate and dephosphorylate cofilin, respectively upon infection with Legionella. Caspase-11 targets cofilin via the RhoA GTPase, whereas caspase-1 engages the Slingshot phosphatase. The absence of either caspase-11 or caspase-1 maintains actin in the polymerized or depolymerized form, respectively and averts the fusion of pathogen-containing vacuoles with lysosomes. Therefore, caspase-11 and caspase-1 converge on the actin machinery with opposing effects to promote vesicular trafficking.Inflammasomes are multiprotein complexes that include members of the NOD-like receptor family and caspase-1. Caspase-1 is required for the fusion of the Actin filaments play a role in cell cycle, cellular motility, and proper vesicle transport, and are critical for providing structure and subcellular organization5Legionella pneumophila (Legionella) is a facultative, intracellular pathogen that causes Legionnaire\u2019s disease (LD). The majority of people exposed to Legionella remain asymptomatic or suffer only mild self-limiting infection. Cigarette smoking, chronic lung disease, and immunosuppression have been consistently implicated as risk factors7Legionella reaches the alveoli where it encounters and multiplies in human alveolar macrophages at the site of infection, which is crucial to the pathogenesis of LD910Legionella infection, allowing bacterial growth121314Legionella1213Legionella replication via activation of caspase-1, caspase-7, and caspase-11 resulting in fusion of the containing phagosome with the lysosome leading to bacterial degradation and growth restriction1617181920Legionella infection contributing to bacterial clearance by promoting phagosome-lysosome fusionNlrc4\u2212/\u2212, Naip5\u2212/\u2212, Casp1\u2212/\u2212, Casp-7\u2212/\u2212, or Casp11\u2212/\u2212) are permissive to Legionella infection172223In contrast, macrophages from C57BL/6 mice (designated wild-type (WT) in the text) restrict Legionella infection in WT macrophages. Dephosphorylation of cofilin is accompanied by an increase in the filamentous actin (F-actin) and globular actin (G-actin) ratio and promotion of fusion of the Legionella-containing vacuole with the lysosome. We found that caspase-11 is required for the phosphorylation of RhoA and cofilin, whereas caspase-1 promotes the dephosphorylation of cofilin via Slingshot. In WT macrophages, where both caspases are active, the F/G ratio dynamically changes during Legionella infection, as it increases within 2\u2009hours of infection and the pathogen-containing vacuole fuses with the lysosome. When caspase-11 or caspase-1 is lacking, such as in their corresponding single knock outs, cofilin remains phosphorylated or dephosphorylated, respectively. The F/G-actin ratio remains unchanged and the fusion of the pathogen-containing vacuole with the lysosome is halted and Legionella replicates. Notably, the enzymatic activity of caspase-11 is required for its ability to modulate actin polymerization. Interestingly, the absence of either caspase-11 or caspase-1 does not influence the trafficking of E. coli-containing vacuoles. Our results identify new key players downstream of caspase-11 and caspase-1 that modulate the trafficking of phagosomes harboring pathogenic intracellular microbes.Here we demonstrate that cofilin loses its basal phosphorylation status early upon Legionella-containing vacuole (LCV) fails to fuse with the lysosome in macrophages believed to only lack caspase-117\u2212/\u2212 macrophages used in previous publications also lacked caspase-1125262728Casp-1\u2212/\u2212Casp-11Tg) during Legionella infection Legionella in Casp-1\u2212/\u2212Casp-11Tg bone marrow-derived macrophages (BMDMs). Fusion of the LCV with the lysosome was assessed by quantifying the number of GFP-expressing bacteria colocalized with LysoTracker\u2122 red, a dye that stains acidic vacuoles. In WT macrophages, intracellular bacteria readily trafficked to lysosomes (~38%), while in Casp-1\u2212/\u2212Casp-11Tg cells, significantly less bacteria (~20%) did so (Escherichia coli (E. coli) trafficked to the lysosome regardless of caspase-1 expression (~90%) (It has been previously shown that the ) did so . Conversn (~90%) .Legionella in macrophages (in vitro) and lungs (in vivo) of Casp-1\u2212/\u2212Casp-11Tg mice was assessed via colony forming units (CFUs). During in vitro infection, Casp-1\u2212/\u2212Casp-11Tg macrophages exhibited significantly more bacterial replication at 24, 48, and 72\u2009hrs after infection and insoluble (F-actin) cytoplasmic fractions were separated and analyzed by western blot. Restrictive WT macrophages exhibited dynamic reduction then increased ratios of F/G actin during Legionella infection. Comparatively, in permissive macrophages lacking caspases-11, the F/G ratio remained unchanged throughout infection and a flagellin (flaA\u2212) mutant of Legionella. After 1, 2, and 4\u2009hrs of infection, Western blot analysis showed that the dotA\u2212 and the flaA\u2212 mutants failed to modulate cofilin phosphorylation status when compared to the parental Legionella strain Nlrc4 and Naip5 form a multiprotein inflammasome complex that is able to detect intracellular bacteria, triggering canonical pyroptosis and non-pyroptotic clearance of pathogens161722WT cells . Since Na strain . To undeto do so . Togethe\u2212/\u2212 macrophages displayed similar levels of Rac activation in response to Legionella infection, and this was confirmed by Western blot using phospho-Rac/Cdc42 antibodies throughout the infection thus, extending host cell survivalLegionella vacuole and increases bacterial clearance irrespective of cell deathThe inducible caspase-11, like other caspases, executes apoptotic functions when strongly induced and activated and elicits non-apoptotic roles when moderately activated7285152\u2212/\u2212 macrophages is comparable, similar to that of Zamboni\u2019s workLegionella survival in vivo and in vitro was significantly increased in Casp-11\u2212/\u2212 mice and their derived macrophagesLegionella-containing vacuole (LCV) with lysosomes leading to bacterial degradation, whereas in macrophages lacking caspase-11, the trafficking of the LCV is stalled and the pathogen escapes degradation\u2212/\u2212 macrophages, the LCV also escapes fusion with the lysosome while the host cell survives Legionella is not solely dictated by the death of the host cell , in models where LPS is delivered to the cytoplasm or in cases where pathogens escape the intracellular vacuole5758Legionella LPS mutants still dephosphorylate cofilin in WT cells (data not shown). Convergence of caspase-11 and caspase-1 on cofilin is dependent on bacterial flagellin, as WT macrophages infected with either a flagellin (flaA\u2212) or T4SS, (dotA\u2212) mutant failed to dephosphorylate cofilin. These results indicate that access of flagellin to the cytosol is essential for the activation of caspase-11 and caspase-1 to alter the activation of cofilin.Notably, WT cells displayed dynamic changes in the F/G ratio during infection. However, vacuoles containing non-pathogenic bacteria such as \u2212/\u2212 exhibits defects in Rho-GTPase activation while Rho activity was similar in WT and Casp-1\u2212/\u2212Casp-11Tg cells. On the other hand, dephosphorylation (activation) of the Slingshot phosphatase does not occur in cells lacking caspase-1 but proceeds efficiently in WT and Casp-11\u2212/\u2212 macrophages during Legionella infection. These data demonstrate that both caspases modulate cofilin phosphorylation in an opposing mode by differentially regulating upstream RhoA and Slingshot , to promote lymphocyte migrationLegionella devotes several molecules secreted through its T4SS that alter the cytoskeletal network2676869S. typhimurium, L. donovani, or Mycobacteria spp. prevents the fusion of the lysosome37657273The importance of the cytoskeleton in host defense is supported by the fact that These data raise the intriguing possibility that host cells activate caspases to promote cellular immunity by engaging their alternative non-apoptotic functions to control intracellular replication of pathogenic microbes.Legionella pneumophila (Legionella) strain Lp02 is a streptomycin resistant (Smr) thymine-auxotroph derivative of Philadelphia-1. The flaA\u2212 and T4SS (dotA\u2212) mutants have been previously describeddotA\u2212 were complemented with a plasmid for green fluorescent protein (GFP). All in vitro infections were performed at an MOI of 0.5, unless otherwise stated, and were performed in the absence of thymidine, ferric nitrate, and L-cysteine to restrict extracellular growth. Non-fluorescent Escherichia coli (E. coli) strain BL21DE3 was grown overnight to Log phase as previously described14\u2212/\u2212 mice were generously donated by Dr. Yuan at Harvard Medical School\u2212/\u2212/Casp-11Tg mice were a gift from Dr. Vishva Dixit at Genentech\u2212/\u2212 mice were from Dr. Russell Vance at University of California BerkeleyWild-type (restrictive) C57BL/6 (B6) were purchased from The Jackson Laboratory (Bar Harbor ME). Casp-11Bone marrow-derived macrophages (BMDMs) were cultured as previously described2, 1\u2009mM EGTA, 142\u2009mM KCl with NP-40), in the presence of complete, EDTA-free, protease inhibitor cocktail (Roche). Samples were clarified, denatured with SDS buffer, and boiled. Proteins were separated by SDS-PAGE and then transferred to a polyvinylidene fluoride (PVDF) membrane (Biorad). Blots were probed with antibodies against caspase-11 (Sigma), caspase-1 (Genentech), phospho-cofilin, cofilin, Rac/Cdc42, and phospho-Rac , actin (Abcam), calreticulin (Stressgen). Detection was achieved using appropriate secondary antibodies conjugated with horseradish peroxidase (HRP), as previously described121422Cell lysates were prepared with an isotonic buffer and for studies examining colocalization of GFP-expressing bacteria (Legionella and E. coli) with the lysosome, Lysotracker\u2122 red was used to stain acidic vesicles of infected BMDMs, as previously described\u00ae viability/cytotoxicity kit (Molecular Probes), according to manufacturer\u2019s instructions. Images were captured using laser scanning confocal fluorescence microscope with a 60X objective (Olympus Fluoview FV10i).F- and G-actin were visualized from Casp-11\u2212/\u2212, and Casp-1\u2212/\u2212Casp-11Tg according to the manufacturer\u2019s recommendations . Briefly, macrophages were infected and lysed with F-actin stabilization buffer. Lysates were then ultracentrifuged and cytoplasmic fractions were separated and run out on an SDS-PAGE gel. Antibodies specific for actin were then used to visualize amounts of F- and G-actin.Amounts of G- and F-actin were assessed from lysates of WT, Legionella-infected macrophages were assayed for Rac1 and RhoA GTPase activity by G-LISA assay according to manufacturers recommendations . Briefly, cell lysates were incubated on a Rac1 or RhoA affinity plate and then this colorimetric assay was developed with HRP detection reagent. Samples were read on a Spectra Max M2 plate reader (Molecular Devices) and results were expressed as absorbance at 490\u2009nm.Lysates from Data were analyzed using GraphPad Prism software. Data in figures are presented as mean averages of at least 3 independent experiments and error bars represent SEM. Comparisons of groups for statistical significance were analyzed with Student\u2019s two-tailed t-test. p value \u22640.05 was considered significant.How to cite this article: Caution, K. et al. Caspase-11 and caspase-1 differentially modulate actin polymerization via RhoA and Slingshot proteins to promote bacterial clearance. Sci. Rep.5, 18479; doi: 10.1038/srep18479 (2015)."} +{"text": "Drosophila melanogaster wing disc epithelium, we demonstrate that disruption of the junctional vs. basal polarity complexes results in increased epithelial proliferation via distinct downstream signaling pathways. Disruption of the basal polarity complex results in JNK-dependent proliferation, while disruption of the junctional complex primarily results in p38-dependent proliferation. Surprisingly, the Rho-Rok-Myosin contractility apparatus appears to play opposite roles in the regulation of the proliferative phenotype based on which polarity complex is disrupted. In contrast, non-autonomous Tumor Necrosis Factor (TNF) signaling appears to suppress the proliferation that results from apical-basal polarity disruption, regardless of which complex is disrupted. Finally we demonstrate that disruption of the junctional polarity complex activates JNK via the Rho-Rok-Myosin contractility apparatus independent of the cortical actin regulator, Moesin.The establishment and maintenance of apical-basal polarity is a defining characteristic and essential feature of functioning epithelia. Apical-basal polarity (ABP) proteins are also tumor suppressors that are targeted for disruption by oncogenic viruses and are commonly mutated in human carcinomas. Disruption of these ABP proteins is an early event in cancer development that results in increased proliferation and epithelial disorganization through means not fully characterized. Using the proliferating A defining feature of epithelia is the presence of apical-basal polarity (ABP), or the asymmetric distribution of lipids, proteins, and mRNAs within the cell. This asymmetric distribution of subcellular components allows for specialized regional function. ABP is also required for the proper establishment and maintenance of intercellular junctions, normal mitoses, and vesicular trafficking. ABP influences cell proliferation and cell migration, and mutations in components of the various ABP complexes can occur early in carcinoma development and contribute to increased proliferation, invasion and metastasis ,2. DefecD. melanogaster imaginal disc epithelia contribute to dysplastic overgrowth [Genetic disruption of the basolateral Scribble (Scrib)/Discs Large (Dlg)/Lethal Giant Larvae (Lgl)-complex, or the junctional Cdc42/Par6/Par3/Atypical PKC (aPKC) complex, but not the apical Crumbs/Pars/Pals complex in ergrowth ,5, but tD. melanogaster) [D. melanogaster Wnt) and Decapentaplegic signaling in neighboring wild type cells, that contributes to the proliferative phenotypes [Disruption of the basolateral polarity complex in epithelial cells results in apoptosis. These dying cells can then secrete proliferative signals to neighboring epithelial cells, which proliferate to replace lost cells. If polarity disruption is combined with expression of the baculovirus caspase inhibitor P35 , apoptosogaster) ,5,10. Dienotypes .Drosophila melanogaster imaginal wing disc, a proliferating epithelium. Our results demonstrate that interruption of junctional or basolateral ABP regulation leads to distinct downstream signaling events.While some components required for proliferation downstream of basolateral and junctional ABP disruption are known, a full understanding of how this process occurs is lacking. Moreover, whether disruption of the different ABP complexes lead to proliferative phenotypes through shared or unique pathways is not known. Here we examine signals that result from, or are associated with, disruption of the junctional versus basolateral ABP complex in the developing w1118 was used as wild-type. Stocks are described in FlyBase (http://flybase.org/). pucE69, UAS-P35 (B#5073), UAS-Scrib-RNAi (B#29552), UAS-Bsk-RNAi (B#32977), UAS-Moesin-myc (B#8631), UAS-Moesin-RNAi (B#8629), UAS-Mekk1-RNAi (B#28587), UAS-Ask1-RNAi (B#32464), UAS-Hep-RNAi (B#28710), UAS-Mkk4-RNAi (B#35140), UAS-p38b-RNAi (B#35252), UAS-p38a-RNAi (B#34744), bsk1 (B#3088), UAS-Baz-RNAi (B#39072), UAS-aPKC-RNAi (B#25946) and \u201cDlg-RNAi #2\u201d (B#35286) were provided by the Bloomington Drosophila Stock Center (BDSC); Zip1 by T. Wolff (NIH Janelia Research Campus); patched-GAL4 by R. Cagan ; UAS-Cdc42- RNAi and UAS-Rho1-RNAi were described previously [All crosses and staging were performed at 25\u00b0C unless otherwise noted. eviously ; \u201cUAS-Dlrd instar larval wing discs were dissected in phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde diluted in PBS for 40 min, washed once for five minutes in PBX (PBS with 0.1% Triton X-100), twice for 20min in PAXD , and once for 20min in PAXDG , all on ice. Tissues were incubated overnight in primary antibody diluted in PAXDG at 4\u00b0C and washed three times in PBX at room temperature. After four or more hours incubated in secondary antibody diluted in PAXDG at 4\u00b0C, they were washed twice in PBX, and washed once in PBS, all at room temperature. Prepared tissues were mounted in Vectashield mounting media . Antibodies used were rat anti-DE-cadherin (1:20), mouse anti-Discs large (1:50), mouse anti-Matrix Metalloproteinase 1 (MMP1) (1:20), rabbit anti Activated Caspase 3 (AC3) , rabbit anti-b-galactosidase , guinea pig anti-Scrib . Secondary antibodies were Alexa 488 and 568 (Invitrogen) and Cy5 (Jackson ImmunoResearch). Immunofluorescence was analyzed on a Zeiss LSM700 confocal microscope using a 10X Neofluor (NA 0.3) air lens, 20X Apochromat (NA 0.8) air lens, or 63X Apochromat oil immersion lens (NA 1.4). Image J64 was used to adjust brightness and contrast of whole images.Wandering 3The degree of proliferation was quantified as the ratio of area of the GFP-positive area to total wing disk area to compensate for any effects that led to asynchrony in wing disc development. Total wing areas were determined by applying a Canny edge-detection algorithm to E-cadherin immunofluorescence images and filling in the resulting outlines. GFP-positive region areas were determined by applying a manually determined uniform brightness threshold to endogenous GFP fluorescence images. The proliferation index was computed as a ratio of GFP-positive area to total wing disc area. Image analysis and calculations were performed with a custom Matlab program, and code is available upon request to the corresponding author. P values were calculated via unpaired, two-sided Student\u2019s t test. *p < .05, **p < .01, ***p < .001.D. melanogaster imaginal disc epithelium, a proliferating epithelium, can result in JNK-dependent apoptosis and compensatory hyperproliferation when apoptosis execution is inhibited [D. melanogaster wing imaginal disc epithelium to quantify the extent of proliferation that occurred following disruption of either ABP polarity complex in the presence of the apoptosis inhibitor P35. The fly wing imaginal disc is a simple, two-layered, proliferating epithelium with an intact basement membrane. The wing disc undergoes a sequence of morphogenetic steps throughout development and to form the adult wing [D. melanogaster makes this a very useful system to interrogate signaling pathways downstream of polarity disruption in an intact epithelium. In quantifying proliferation, we controlled for possible asynchrony in development caused by genetic manipulation that might affect imaginal disc proliferation by quantifying a ratio of the GFP-positive proliferative area to the entire wing disc area. We developed custom image analysis software that calculated a ratio of GFP-positive patched (ptc) area to total wing disc area and used this as a measure of proliferation expression or removal of a genomic copy of Bsk only partially rescued the proliferative phenotype . Bsk JN). These Dlg-RNAi) + P35 did not require any single JNK-KK. Rather, Tak1, Hep, Mkk4, and Mekk1 all partially rescued the proliferative phenotype for the henotype , and nonhenotype .Bsk), we considered the possibility that Mekk1 may be activating other downstream MAPKs, in addition to JNK (Bsk), to promote proliferation. Mekk1 can act as an upstream activator of JNK or p38-MAPKinase [Since Mekk1 depletion uniquely rescued proliferation following junctional polarity complex disruption much more significantly than depletion of JNK resulted in increased proliferation of imaginal discs in which Cdc42-RNAi + P35 were present levels were reduced proteins are critical organizers of the cell cortex as they link cortical and transmembrane proteins to the actin cytoskeleton . Moesin activity S5 Fig)..D. melanactivity , we askeDisruption of the ABP signaling network is known to contribute to increased proliferation and invasion of epithelial cells. These phenotypes have been observed in epithelia following disruption of either the junctional or basolateral polarity complex. However, the cellular signaling pathways activated to achieve this behavior is dependent on whether the junctional or basolateral complex is disrupted. Here we show that hyperproliferation following disruption of the basolateral complex was solely dependent on JNK MAPK, while disruption of the junctional complex caused proliferation that requires both JNK and p38 MAPK. Non-autonomous TNF signaling suppressed the proliferation that results from disruption of either complex. This could represent an epithelial-intrinsic or immunologic defense against the expansion of polarity-deficient cells in an epithelium. Our data suggested that this TNF-mediated suppression is indifferent to the genetic perturbation that led to the dysfunction. On the other hand, the Rho-Rok-Myosin contractility apparatus appeared to play opposite roles depending on which polarity complex was disrupted. This indicated that epithelial cells respond to polarity disruption via overlapping, yet distinct pathways depending on which components of the ABP signaling network are perturbed.The fact that different MAPK signaling pathways are activated following disruption of the junctional versus basolateral polarity complex is, perhaps, not entirely surprising as these two complexes do serve unique functions. While the Par and Scrib complex have roles in regulating many shared processes, including intercellular junction homeostasis, cell polarity regulation, and RhoGTPase regulation, they are localized to different regions of the cell and therefore have unique suites of interacting proteins and signaling pathways.Furthermore, during cell migration, apical-basal polarity proteins are reoriented to aid in establishing polarity along front-rear axis of migrating cells and play distinct roles in this process as well. Cdc42 is localized at the front of the cell and is important for protrusion formation in the direction of migration , and othOur data demonstrated that disruption of ABP leads to JNK- or p38-dependent proliferation based on whether the junctional or basolateral complex is depleted. Why there is activation of one MAPK pathway versus the other in dysplastic polarity-deficient epithelial cells, and whether there are functional consequences, remains to be determined. This difference could result from distinct subcellular localization of JNK and p38 MAPKs, with JNK being localized more basolaterally and p38 more apically in the cell. An important function of the ABP network is to spatially segregate signaling networks such that cells can effectively integrate information from both the intracellular and extracellular environment, and transmit that information via appropriate signaling pathways \u201329. The Another contrast in the phenotypic response to junctional versus basolateral complex disruption was the opposite role played by the Rho/Rok/Myosin contractility apparatus. Disruption of the junctional complex leads to activation of JNK via Rho/Rok/Myosin, and depletion of any of these contractility components decreases the proliferative phenotype . In contS1 Figptc-GAL4 (green). (B) Identified regions: whole-wing (red outline) and Ptc region (green outline). (C) E-cadherin immunofluorescence with overlay of identified regions. (D) Ptc-GFP image with overlay of identified regions. Scale bars represent 100\u03bcm.(A) Full wing disk showing immunofluorescence staining of E-cadherin (red) and expression of GFP via (TIF)Click here for additional data file.S2 Figptc-GAL4, or in MARCM clones of Dlg1(Da and Db) or Cdc424(Ea and Eb). Scale bars represent 100\u03bcm in A-C and E. Scale bar represents 20\u03bcm in D.Confocal immunofluorescent localization of DE-cadherin (DE-cad), GFP , Dlg , MMP1 , and Activated Caspase 3 (AC3) (Eb) in larval wing discs expressing Dlg-RNAi #1 (Aa and Ab), Dlg-RNAi #2 (Ba and Bb), or the combination of Dlg-RNAi#1, Bsk-RNAi, and P35 (Ca and Cb), Baz-IR and P35 (Fa and Fb), aPKC-IR and P35 (Ga and Gb), or Par6-IR and P35 (Ha and Hb) via (TIF)Click here for additional data file.S3 Figgmr-GAL4 alone (A), or in combination with UAS-Egr (B), UAS-Egr and UAS-Egr-RNAi (C) UAS-Egr and UAS-Hep-RNAi (D), or UAS-Egr and UAS-Wgn-RNAi (E). Confocal immunofluorescent localization of DE-cadherin (DE-cad) (Fa) and Scrib (Fb) in larval wing disc expressing Scrib-RNAi, via ptc-GAL4. Scale bar represents 100\u03bcm.Adult eyes expressing (TIF)Click here for additional data file.S4 Figptc-GAL4. Scale bar represents 100\u03bcm.Confocal immunofluorescent localization of DE-cadherin (DE-cad) and GFP (Aa), and phospho-Myosin Light Chain (pMLC) (Ab) in larval wing disc expressing Rok-RNAi via (TIF)Click here for additional data file.S5 Figptc-GAL4. Scale bars represent 100\u03bcm.Confocal immunofluorescent localization of DE-cadherin (DE-cad) and GFP (Aa and Ba), cleaved caspase 3 (AC3) (Ab), and myc (Bb) in larval wing discs expressing UAS-Moe-RNAi (Aa and Ab) or UAS-Moe-myc (Ba and Bb) via (TIF)Click here for additional data file."} +{"text": "Low birth weight is associated with an increased adult metabolic disease risk. It is widely discussed that poor intra-uterine conditions could induce long-lasting epigenetic modifications, leading to systemic changes in regulation of metabolic genes. To address this, we acquire genome-wide DNA methylation profiles from saliva DNA in a unique cohort of 17 monozygotic monochorionic female twins very discordant for birth weight. We examine if adverse prenatal growth conditions experienced by the smaller co-twins lead to long-lasting DNA methylation changes.Overall, co-twins show very similar genome-wide DNA methylation profiles. Since observed differences are almost exclusively caused by variable cellular composition, an original marker-based adjustment strategy was developed to eliminate such variation at affected CpGs. Among adjusted and unchanged CpGs 3,153 are differentially methylated between the heavy and light co-twins at nominal significance, of which 45 show sensible absolute mean \u03b2-value differences. Deep bisulfite sequencing of eight such loci reveals that differences remain in the range of technical variation, arguing against a reproducible biological effect. Analysis of methylation in repetitive elements using methylation-dependent primer extension assays also indicates no significant intra-pair differences.Severe intra-uterine growth differences observed within these monozygotic twins are not associated with long-lasting DNA methylation differences in cells composing saliva, detectable with up-to-date technologies. Additionally, our results indicate that uneven cell type composition can lead to spurious results and should be addressed in epigenomic studies. Both observational human and experimental animal studies have confirmed that low birth weight is associated with an increased risk of metabolic diseases, like type 2 diabetes (T2D) -3. AlthoOne of the possible molecular mechanisms explaining this non-genetic association suggests that poor prenatal conditions induce epigenetic modifications . These eHNF4A gene, which is involved in monogenic diabetes.The notion that poor intra-uterine conditions cause epigenetic modifications during prenatal development is supported by data from animal studies, where dietary restriction or surgical interventions are used to induce fetal growth restriction, resulting in epigenetic modifications on metabolic disease-related genes (reviewed by ). The nucis- or trans-acting genetic variant . The DBS data are available in the Sequence Read Archive under accession number [SRA075928].BW-MVP: birth weight-associated methylation variable positions; DBS: deep bisulfite sequencing; DC: dichorionic; DZ: dizygotic; EFPTS: East Flanders Prospective Twin Survey; EWAS: epigenome-wide association study; GEO: Gene Expression Omnibus; HELP: HpaII tiny fragment enrichment by ligation-mediated PCR; IUGR: intra-uterine growth restriction; MC: monochorionic; MI: methylation index; MVP: methylation variable position; MZ: monozygotic; SIRPH: single-nucleotide primer extension assays in combination with ion-pair reversed-phase high-performance liquid chromatography separation techniques; SNP: single nucleotide polymorphism; SNuPE: single nucleotide primer extension; T2D: type 2 diabetes.The authors declare that they have no competing interests associated with this manuscript.NS designed the study, conducted the experimental work, participated in the statistical data analysis and wrote the manuscript. PL performed bioinformatical and statistical analyses and participated in writing the manuscript. ST and JG assisted in the DBS and methylation-dependent primer extension experiments. GG and MR generated the HumanMethylation450 profiles. CD, JF and MZ were involved in the study design, collecting the twin data and samples. JW supervised the study, participated in the study design, writing the manuscript and provided technical and material support. All authors read and approved the final manuscript.Supplemental methods, tables and figures. Supplemental methods include Infinium HumanMethylation450 data pre-processing, adjustment for cell type heterogeneity, and association analysis and candidate selection. Table S1: DNA methylation profiles used to create the cell-type reference data set. Table S2: cell type-specific quantitative markers used as explanatory variables in heterogeneity adjustment. Table S3: characteristics of the 45 CpG sites that are significantly differentially methylated between the heavy and light co-twins identified using the Infinium HumanMethylation450 BeadChip. Table S4: distribution of the samples across the bead chips, detected CpGs and the corresponding call rate per sample. Table S5: reaction conditions and primer sequences of the bisulfite-PCRs. Table S6: reaction conditions and primer sequences of the SIRPH analysis. Table S7: statistical power of the twin study. Figure S1: pair-wise correlations for each pair of samples, calculated from approximately 480,000 CpGs. Figure S2: sample-independent Infinium methylation controls. Figure S3: sample-dependent Infinium methylation controls. Figure S4: pair-wise correlations for each pair of samples, including the reference dataset for whole-blood and buccal (27k), calculated from approximately 25,978 CpGs. Figure S5: mixing experiment with KG1a and K562 cells profiled on the Infinium HumanMethylation450 BeadChip. Figure S6: distribution of the correlation coefficients of the methylation values of the approximately 480,000 CpGs to the methylation values of the PTPN7 CpG (cg18384097). Figure S7: pair-wise correlations for each pair of samples after adjusting for cell type composition using the PTPN7 CpG (cg18384097). Figure S8: examples of methylation profiles generated using the deep bisulfite sequencing data of the APPL2, PPARGC1B, PHKG2 and PTPN7 amplicons. Figure S9: correlation plots in which the (unadjusted) Infinium 450K data of the validated CpGs are plotted against the (unadjusted) deep bisulfite sequencing (DBS) data for every sample separately. Figure S10: box-plot of the correlation coefficients calculated between the Infinium 450K data and the deep bisulfite sequencing (DBS) data of the validated CpGs for every individual sample. Figure S11: correlation plots of the (unadjusted) Infinium 450K data and the (unadjusted) deep bisulfite sequencing (DBS) data of the 17 discordant MZ twin pairs for each validated CpG separately. Figure S12: continuation of Figure S11. Figure S13: box-plot of the intra-pair differences in \u03b2-values of the 64 SNPs present on the Infinium HumanMethylation450 BeadChip before and after normalisation using internal controls and background subtraction by the GenomeStudio software.Click here for file"} +{"text": "Some autistic children also have symptoms of depression, anxiety or obsessive-compulsive disorder (OCD). Aromatic anti-epileptic drugs (AEDs) may be used as a mood-stabilizing medication to treat these symptoms, including carbamazepine (CBZ), oxcarbazepine (OX-CBZ), and lamotrigene (LMG). Moreover, one in every four autistic spectrum disorder children have seizures, the use of AED becomes imperative in managing symptoms. Previous studies found a strong association between HLA-B*1502 and carbamazepine (CBZ)-induced cutaneous adverse reactions in Thai epileptic patients. This study aimed to identify whether HLA-B*1502 is associated with AEDs-induced hypersensitivity syndrome (HSS) reactions and also determine the frequency of HLA-B*15:02 in autistic spectrum disorder (ASD) in Thailand. In this study, patients that developed fever and cutaneous eruptions in the presence or absence of organ involvement with exposure to carbamazepine (CBZ), phenytoin (PHY), or lamotrigine (LTG) were enrolled. HLA-B* 15:02 was then determined in 21 patients with AED-induced HSS , phenytoin , and lamotrigine . In addition, the allele frequencies of HLA-B*15:02 in ASD was 14.72% (43/292). HSS-induced by CBZ, PHY and LMG is strongly, moderately and slightly associated with HLA-B*15:02 in Thai patients, respectively. Our findings suggest that HLA-B*15:02 testing before AED therapy would be effective at risk of hypersensitivity and applicable to ASD populations providing hope for prevention in the future."} +{"text": "C. elegans embryos that could regulate progenitor identity. In this study we identified a broad embryonic role for the C. elegans OTX transcription factor ceh-36, which was previously shown to be required for the terminal specification of four neurons. ceh-36 is expressed in progenitors of over 30% of embryonic cells, yet is not required for embryonic viability. Quantitative phenotyping by computational analysis of time-lapse movies of ceh-36 mutant embryos identified cell cycle or cell migration defects in over 100 of these cells, but most defects were low-penetrance, suggesting redundancy. Expression of ceh-36 partially overlaps with that of the PITX transcription factor unc-30. unc-30 single mutants are viable but loss of both ceh-36 and unc-30 causes 100% lethality, and double mutants have significantly higher frequencies of cellular developmental defects in the cells where their expression normally overlaps. These factors are also required for robust expression of the downstream developmental regulator mls-2/HMX. This work provides the first example of genetic redundancy between the related yet evolutionarily distant OTX and PITX families of bicoid class homeodomain factors and demonstrates the power of quantitative developmental phenotyping in C. elegans to identify developmental regulators acting in progenitor cells.While many transcriptional regulators of pluripotent and terminally differentiated states have been identified, regulation of intermediate progenitor states is less well understood. Previous high throughput cellular resolution expression studies identified dozens of transcription factors with lineage-specific expression patterns in Caenorhabditis elegans is a powerful system in which to identify developmental regulators because it has a rapid and reproducible development, yet it shares most of its developmental regulators with more complex organisms such as humans. We used state-of-the-art microscopy and computer-aided cell tracking methods to identify the developmental role of worm homologs of the OTX and PITX genes, whose human homologs play a role in the development of the brain, eye, and pituitary among other tissues. We identified broad roles for OTX in regulating development for many distinct cell types including muscles, neurons and skin, and found a redundant role for both OTX and PITX in a subset of cells. Future studies of these genes should address whether these genes also act redundantly in mammals.Animals develop as one initial cell, the fertilized egg, repeatedly divides and its progeny differentiate, ultimately producing diverse cell types. This occurs in large part by the expression of unique combinations of regulatory genes, such as transcription factors, in precursors of each cell type. These early factors are typically reused in precursors of different cell types. The nematode worm Caenorhabditis elegans, a simple model organism where lineage relationships are already understood, large-scale gene expression resources allow rapid identify patterns of TF overlap, and powerful tools exist for characterizing mutant phenotypes across all embryonic cells. Previous studies of genetic redundancy in C. elegans have prioritized gene pairs for synthetic lethality testing based on similar functional interactions . We. WeC. elyo Figs. , definedyo Figs. . In someyo Figs. . The CEHceh-36 mutant and wild-type embryos. We identified 124 cells whose deviation from expected position was at least 3.5 standard deviations greater than in the wild-type set and that had aberrant neighbors as defined by an empirical neighbor-distance score position defects examined were scored as likely migration defects. The migration defects include both cells that undergo novel migrations in the mutant , but we saw no defects of this class in the 22 wild-type control embryos, and all eight of these cells normally express ceh-36. This indicates that C. elegans cells\u2019 lateral position is not merely a result of their birth position but is regulated by factors that include ceh-36.We observed dramatic defects in eight laterally positioned cells that were born in the correct position but subsequently migrated across the midline to the opposite lateral side of the embryo, sometimes displacing the position of their bilateral counterpart e.g. . These lceh-36 mutant elongated (pretzel-stage) embryos have three FKH-4(+) cells, indicating that ceh-36 is not necessary for FKH-4 expression. However, one FKH-4(+) cell is laterally mispositioned in 14% of ceh-36(-) embryos , increasing confidence in the low-penetrance defects identified by lineage analysis.We determined that lateralization defects are maintained through embryonic elongation and not corrected by subsequent cell movements by examining worms expressing FKH-4::GFP, a marker of three visceral muscles embryos. We observed altered pharyngeal gland morphology in 20% of ceh-36(ok795) elongated embryos. An additional 9% of embryos were missing one or more hlh-6::GFP-positive cells of larvae with normal gland morphology arrested prior to the L4 stage, 92% (46/50) of larvae with abnormal gland morphology arrested. Thus, defects in pharyngeal gland morphology predict larval arrest in ceh-36 mutants.Multiple pharyngeal gland cell precursors had cell cycle and position defects in mutants . Precurs mutants , 4. Sincve cells , Table 2ok795 embryos. Most of the defective cells normally express CEH-36::GFP (77%), significantly more than the 30% fraction of all cells that express ceh-36 . Most of the defective cells that do not normally express ceh-36 were only called as defective in one embryo. Still, even defects seen in a single embryo were enriched in expressing cells (59% of such cells express CEH-36::GFP). While cells with prior cell cycle defects were 2.9-fold more likely to have position defects (p < 10-9), 90% of cells with position defects had no detectable cell cycle defect. Only 22 expressing cells had defects in at least 50% of analyzed embryos (e.g. ceh-36(-) embryos.Defects occurred in 223 unique cells, typically with low penetrance; only 82/223 (37%) cells were defective in two or more (of eight) yos e.g. . The lowceh-36 (MI(sams-5) and ASEL(gcy-5)) showed the expected terminal defect frequencies in the ceh-36 deletion. As described above, the visceral muscle reporter FKH-4::GFP and the pharyngeal gland reporter hlh-6p::GFP also showed terminal position defect frequencies consistent with the observed embryonic defects. Finally, a FLP-1::GFP reporter reported as expressed in the AVK neuron showed little or no terminal defects (~2%). Given that the mutant embryos hatch without major morphological defects despite an average 40 cells with position defects and 10 cells with altered division timing, development of C. elegans embryos must be robust to a substantial amount of developmental error.We determined whether cells with low penetrance defects have noticeable defective terminal positions or numbers by examining several fluorescent markers expressed in these cells . Reporteceh-36(-) may result from regulatory events occurring in mitotic progenitor cells. If this is true, partially penetrant defects should preferentially co-occur in closely related cells within a given embryo. We identified 71 examples of defective sister cell pairs in ceh-36-expressing cells. We found preferential co-occurrence of defects in sisters for seven embryos (p < 0.001) by using a bootstrap evaluation, and this co-occurrence was only significant in cells expressing ceh-36. This along with the early and dynamic CEH-36::GFP expression suggests that ceh-36 regulates development in part through its activity in progenitor cells, rather than the terminal cells that exhibit the defects.The rescuing CEH-36::GFP transgene expression is typically strongest several divisions before the birth of the terminal cells where most defects were identified, suggesting that defects in ceh-36(ok795) embryos result from loss of ceh-36, we specifically examined high-penetrance (\u22656 of 8 ok795 embryos) position defects in an embryo carrying a second predicted ceh-36 null mutation (ky646). We found four of the five cells examined had similar defects in this embryo. We examined these cells in two ceh-36(ok795) embryos expressing CEH-36::GFP, and one embryo with mosaic CEH-36::GFP expression, and found that these defects were rescued in all CEH-36::GFP expressing cells. Taken together, these results show that ceh-36 regulates the robustness of cell cycle and migration patterns in many cells. Our analysis did not explicitly test for changes in cell fate, but given the known role of ceh-36 in fate specification ..ceh-36 single mutant embryos (n = 6). Since MLS-2::GFP is expressed later and ~10-fold more strongly than CEH-36::GFP in the excretory duct and pore lineages and the ABpxp-specific functional interaction between unc-30 and ceh-36 are consistent with context-dependent roles for these factors. Each factor has distinct expression outside of the early ABpxp coexpression, suggesting that each may work with other factors in these other lineages; indeed, unc-30 is a well-established regulator of motor neuron differentiation later in development [bwIs2[su1006))]sams-5 3\u2032::4xNLS-GFP + lin-15(+)] V [nsIs396[15(+)] V lin-15(+); gcy-5::GFP][ntIs1[y-5::GFP]ceh-37::GFP][sEx14784[-37::GFP]+ unc-119(+)]ujEx173[CEH-36::GFP + myo-2::mCherry + myo-3::mCherry]ujEx130[CEH-36::GFP ceh-36 2KB promoter::GFP][oyIs48[ter::GFP]ceh-36 5KB promoter::HIS-24-mCherry]stIs10501 IIujIs113[+ unc-119(+)] I [wgIs108[FKH-4::GFP19(+)] I hlh-6::GFP + unc-119(+)] X [wwIs19[19(+)] X csIs55[MLS-2::GFP] X [wgIs395[UNC-30::GFP+unc-119(+)]All strains were grown as previously described . N2 was ceh-36(ok795) and unc-30(ok613) were outcrossed three times. VC579 ceh-36(ok795)/szT1 hermaphrodites were mated with males carrying an extrachromosomal copy of ceh-36(+)::GFP (ujEx173), and F2 progeny were tested for ok795, which deletes 406 base pairs of ceh-36, by PCR. Additional outcrossing of ceh-36(ok795) was with N2 males. unc-30(ok613) was outcrossed by mating unc-30(ok613) hermaphrodites with N2 males and picking F2 Unc progeny. Combinations of reporters with ceh-36(-) were created using a mating strategy that did not produce heterozygous ceh-36(-) hermaphrodites at any step or else were verified using PCR.Knockout consortium alleles unc-30(ok613) and ceh-36(ok795) were created using nT1[qIs51] to balance unc-30(ok613) while testing for ceh-36(ok795) by PCR. unc-30(ok613)/nT1[qIs51]; ceh-36(ok795) males were mated with unc-119(tm4063); ceh-36(ok795); ujEx173[ceh-36::GFP + unc-119(+)] hermaphrodites, and F2 Unc progeny with the genotype unc-30(ok613); ceh-36(ok795); ujEx173 were isolated. ujEx173 was generated by microparticle bombardment of the CEH-36::GFP Transgeneome fosmid young adult hermaphrodites were dissected and embryos counted as described above. Embryonic lethality was scored the next morning. Unhatched embryos were mounted in 20\u03bcm beads in egg buffer/methyl cellulose [ceh-36(ok795) worms carrying wwIs19(hlh-6::GFP).Lethality checks of ellulose and scorAll strains were grown at 20\u00b0C for over two generations before young adult hermaphrodites were dissected at room temperature in egg buffer and embryos with four or more cells were mounted into a solution of 20\u03bcm beads in egg buffer/methyl cellulose. Sealed slides containing 10\u201315 embryos were incubated overnight at 20\u00b0C and scored the following morning.unc-30(ok613); ceh-36(ok795) double mutant phenotypes followed the above protocol except that embryos were also scored for CEH-36::GFP expression in ASE and AWC neurons following DIC examination to exclude rescued animals.Examination of We acquired confocal images with a Leica TCS SP5 resonance scanning confocal microscope (67 z planes at 0.5 \u03bcm spacing and 1.5 minute time spacing) and generated lineages using StarryNite and AceTree as previously described ,21,72\u201375C. elegans embryogenesis through the 600-cell stage using eighteen embryos expressing fluorescently tagged histone by tracing four embryos to the comma stage. Deviation of cell-cycle length, division orientation, and anterior-posterior position for eight ujIs113; ceh-36(ok795) embryos, one ujIs113; ceh-36(ky646) and five ujIs113; unc-30(ok613); ceh-36(ok795); ujEx173(CEH-36(+)::GFP) embryos was calculated as previously described [ceh-36 expressing versus non-expressing). Cells born before the onset of ceh-36 expression were not considered. The number of defective sister cells expected by chance was determined by 100,000 iterations of counting sister pairs from (X) randomly picked cells from a defined subgroup. A p-value was calculated by dividing the total number of iterations equal to or greater than the observed value (Y) with 100,000.We updated the 4D reference model of wild-type escribed . Deviantceh-36(-) mutants. Positional defects and wild-type variation of fluorescent reporters were measured using LASAF software. Single-molecule RNA FISH was performed as previously described [Mixed-stage embryos were picked into a solution of 10mM sodium azide and 1% methyl cellulose in egg buffer with 25\u03bcm beads on top of a glass slide. Coverslips were sealed using petroleum jelly, and embryos became immobilized due to azide and hypoxia. All fluorescent reporters were scored by analyzing confocal GFP and DIC z-stacks of pretzel-stage embryos, which provided a more discrete developmental stage than possible in larvae due to the larval arrest of escribed ,76.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S1 FigThis shows all expressing sublineages. Some nonexpressing cells were not curated to the last time point and are not shown in this figure.(PNG)Click here for additional data file.S2 FigThis shows all expressing sublineages. Some nonexpressing cells were not curated to the last time point and are not shown in this figure.(PNG)Click here for additional data file.S3 Fig(PNG)Click here for additional data file.S4 Fig(PNG)Click here for additional data file.S5 FigThis shows all expressing sublineages. Some nonexpressing cells were not curated to the last time point and are not shown in this figure.(PNG)Click here for additional data file.S6 FigThis shows all expressing sublineages. Some nonexpressing cells were not curated to the last time point and are not shown in this figure.(PNG)Click here for additional data file.S7 FigDefects are displayed as in Figs (PDF)Click here for additional data file."} +{"text": "Adoptive immunotherapy, including transfer of activated NK cells, is currently under active investigation for patients with refractory and relapsed acute myeloid leukemia (AML). However, a highly immunosuppressive microenvironment sustained in part by tumor-derived exosomes, creates a major hurdle for adoptive immunotherapy. We recently reported that exosomes, virus-size (30-100nm) membrane-bound vesicles, represent one immunosuppressive mechanism operating in AML. We found high levels of exosomes in plasma of newly-diagnosed AML patients prior to any therapy. These exosomes carried an immunosuppressive cargo, including membrane-associated TGF-\u03b21, PD-1, MICA/MICB and markers of myeloid blasts. Infusion of activated NK cells in patients with AML did not result in the expected recovery of NK cell activity. We hypothesize that the presence of immunosuppressive plasma exosomes in refractory/relapsed AML patients may impair anti-tumor activity of adoptive cell therapies.Venous blood (20-50 mL) was obtained from patients with refractory/relapsed AML (n=7). Exosome fractions were isolated from the patients' plasma and plasma of normal controls by using mini-size exclusion chromatography with Sepharose 2A. Protein levels, numbers and the size of exosomes (qNano) and their morphology (transmission electron microscopy) were determined. Exosomes were characterized by Western blots for expression of exosome markers, Tsg101 and CD81, and myeloid cell-surface markers associated with AML, interleukin-3 receptor alpha chain (CD123) and C-type lectin-like molecule-1 (CLL-1), CD44, CD96 and TGF-\u03b21. Isolated normal human NK cells were co-incubated with AML exosomes and multiparameter flow cytometry was used to monitor changes in expression levels (mean fluorescence intensity) of NKG2D on NK cells.Exosome fractions isolated from AML patients' plasma with refractory/relapsed AML had mean protein content of 36 \u00b5g protein/mL plasma. AML exosomes contained blast markers, CD123, CLL-1, CD44, CD96 suggesting their leukemia origin. They were also enriched in TGF-\u03b21, which is known to interfere with NK cell activity and promote Treg expansion. Co-incubation of AML exosomes with activated NK cells resulted in down-regulation of NKG2D expression with a concomitant reduction of NK-cell cytotoxicity. Antibody neutralization of TGF-\u03b21 carried by AML exosomes significantly abrogated their immunosuppressive activity.The persistently elevated levels of biologically-active TGF-\u03b21+ exosomes carrying leukemia blast markers in plasma of refractory/relapsed AML patients indicate that existing immune suppression will interfere with anti-leukemia effects of adoptive cell therapies."} +{"text": "Des-\u03b3-carboxyprothrombin (DCP) has been used as a tumor marker for hepatocellular carcinoma (HCC). Recently the DCP/NX-DCP ratio, calculated by dividing DCP by NX-DCP, has been reported useful in detecting HCC. The purpose of this study is to clarify the significance of DCP and NX-DCP expression in HCC tissues.HCC and non-HCC tissue samples were obtained from 157 patients and were immunohistochemically examined for DCP and NX-DCP expression using anti-DCP antibody and anti-NX-DCP antibody. DCP and NX-DCP expression scores were calculated by multiplying staining intensity grade by percentage of stained area. Serum DCP and NX-DCP levels were determined in 89 patients. We evaluated the relationship between tumor expression, serum level, and pathomorphological findings.Intrahepatic metastasis (im) was significantly more frequent in cases with high DCP expression than in cases with low DCP expression. High NX-DCP expression was associated with significantly lower histological grade, and less frequent im or portal vein invasion (vp) than low NX-DCP expression. Serum DCP was correlated with DCP expression, but serum NX-DCP was not correlated with NX-DCP expression. DCP-positive (\u226540 mAU/L), NX-DCP-positive (\u226590 mAU/L), and DCP/NX-DCP ratio-positive (\u22651.5) cases were associated with significantly larger tumor size and more frequent vp than negative cases. DCP was rarely expressed, but NX-DCP was frequently expressed in non-cancerous liver tissues. Patients with NX-DCP expression-negative tumors showed a lower survival rate than those with NX-DCP expression-positive tumors (p = 0.04), whereas the survival in serum NX-DCP-positive cases was lower than that of serum negative cases (p = 0.02).DCP and NX-DCP were produced in HCC tissues, but differed in expression level and biological properties. DCP expression, serum DCP or NX-DCP level, and DCP/NX-DCP ratio were closely related to malignant properties of HCC. Hepatocellular carcinoma (HCC) is the third most common cause of cancer death in the world. Advances and improvements in the screening and treatment of patients at high risk for HCC have improved the prognosis of early-stage HCC . HoweverDes-\u03b3-carboxyprothrombin (DCP), also known as \u201cprotein induced by vitamin K absence or antagonist-II\u201d (PIVKA-II), is an abnormal prothrombin that has been widely used as a tumor marker for HCC, and could be predictive of worse tumor behavior and prognosis \u20135. FurthHCC tissue samples for immunohistochemistry were obtained from 157 patients who underwent surgical resection of single HCC nodules at Kurume University Hospital between 2007 and 2012. Of these cases, 6 patients had been taking warfarin. Non-cancerous liver tissues were available from 148 cases. None of the patients had previously received any treatments, including arterial embolization, chemotherapy, or radiofrequency ablation. Patients consisted of 117 men and 40 women aged from 32 to 84 years (median age 68 years). Eighty-nine cases were hepatitis C virus antibody-positive; 24 cases were hepatitis B surface antigen-positive; 3 cases were positive for both hepatitis B surface antigen and hepatitis C virus, and 41 cases were negative for both. Ninety-nine of the 157 cases were diagnosed with chronic hepatitis (CH), and 58 cases had liver cirrhosis (LC) based on histological examination. Pathological diagnosis was performed according to General Rules for the Clinical and Pathological Study of Primary Liver Cancer edited by Liver Cancer Study Group of Japan . Written informed consent was obtained from cases from 2009 to 2012 prior to participation. Our institutional review board waived the need for written informed consent from cases of 2007 and 2008 because the data for these patients were retrospectively analysed.DCP and NX-DCP expressions were found to be positive in 83 of 157 cases (53%) and 101 of 157 cases (64%), respectively, in HCC tissues. Fifty-five cases were positive for both DCP and NX-DCP , and 28 We calculated expression scores of DCP and NX-DCP in HCC tissues according to the method described above. A typical immunostaining photomicrograph of each grade is shown in Survival rate was significantly lower in the NX-DCP expression-negative group than in the low or high NX-DCP expression groups (p = 0.04) . OverallDCP expression in non-cancerous tissue was observed in only one case of obstructive jaundice. On the other hand, NX-DCP expression in non-cancerous tissue was detected in 115 of 141 (82%) HCC cases not taking warfarin and in aThere was a significant correlation between serum DCP level and DCP expression score in HCC patients . However, no significant correlation was observed between serum NX-DCP level and NX-DCP expression score . There was no significant difference in serum NX-DCP level between the cases with CH and LC when the cases taking warfarin were excluded.Serum DCP was positive (\u2265 40 mAU/L) in 50 of 89 cases (56%), and serum NX-DCP was positive (\u2265 90 mAU/L) in 28 cases (31%). DCP/NX-DCP ratio was positive (\u2265 1.5) in 39 of 89 cases (44%), and DCP/NX-DCP ratio of 4 cases taking warfarin ranged from 0.91 to 2.13. The relationship between clinicopathological features and serum DCP level, serum NX-DCP level, or DCP/NX-DCP ratio is shown in Previous studies on the relationship between tissue DCP expression, serum DCP level, and clinicopathological findings of HCC \u201321 reporIn the previous studies serum DCP level did not always correlate with DCP expression in HCC tissue , 21. HowSeveral studies reported that serum NX-DCP was useful for detection of HCC. Recently, Takeji et al. reported that high serum NX-DCP level was significantly associated with worse prognosis for patients with high stage HCC. They speculated NX-DCP levels reflected hepatic functional reserve of non-cancerous liver tissue of HCC patients . Tanaka DCP expression was observed in 11.8\u201325.7% of cases in non-cancerous tissue adjacent to HCC , 19. InaNX-DCP expression in non-cancerous liver tissue was found in 82% of cases in the present study. Tanaka et al. reportedMany studies have shown that the DCP/NX-DCP ratio is useful to detect HCC in warfarin-taking patients with positive serum DCP level , 13. We Alpha fetoprotein (AFP) and AFP-L3 also have served as a tumor marker of HCC, and these have been reported to be useful in early detection of HCC . HoweverTwo different types of abnormal prothrombin, DCP and NX-DCP, were produced in HCC tissues, but had different expression levels and exhibited different biological properties. Our data suggest that increases in DCP expression in HCC tissue, serum DCP or NX-DCP level, and DCP/NX-DCP ratio were closely related with malignant properties of HCC."} +{"text": "We should consider the possibility that Low- and High- spontaneous rate (SR) auditory nerve fibers (ANFs) constitu"} +{"text": "Diabetes mellitus is a chronic progressive metabolic disease, resulting from both insulin resistance and the dysfunction of beta-cells. Beta-cell apoptosis is a crucial pathophysiology leading to diabetes . Aberran KCNJ11 gene contributed to an increased risk for type 2 diabetes in school-aged child and adolescence. K23-allele-containing genotypes conferring increased plasma insulin level during OGTT in normal subjects. However, the diabetic subjects with the K23-allele-containing genotypes had lower fasting plasma insulin levels after adjustment of age and BMI percentiles.The past 15\u201320 years has seen a dramatic increase in the prevalence of T2D in children and adolescents \u20138. Type \u03b2-cell function and increased \u03b2-cell mass developed in response to the glucocorticoid-induced insulin resistance involve inhibition of the islet AS160 protein activity.T2DM is a multifactorial metabolic disease mainly characterized by hyperglycemia , but befRecently, human islet transplantation has achieved insulin independence in type 1 diabetes and the success rates have been markedly improved . Howevertrans-retinoid acid (ATRA) and exendin-4 treatment alone preserves pancreatic beta-cells; ATRA and ATRA plus exendin-4 treatment delays the onset of autoimmune diabetes. However, after the onset of autoimmune diabetes, ATRA and/or exendin-4 treatment is unable to reverse hyperglycemia or improve survival.Type 1 diabetes is characterized by the progressive loss of pancreatic beta-cells caused by autoimmune attack . AlthougTien-Jyun ChangTien-Jyun ChangGang XuGang XuJyuhn-Huarng JuangJyuhn-Huarng Juang"} +{"text": "A 64-year-old man suffering from an acute posterior wall myocardial infarction underwent primary percutaneous coronary intervention. After several aspiration attempts, tirofiban infusion and pre- and post-dilatation, a bare-metal stent was successfully implanted in the culprit right coronary artery. While the patient did not show any neurological symptoms before or during the procedure, he exhibited hemiplegia and loss of spontaneous speech. Additional magnetic resonance imaging showed an extensive brain stem infarction. This is the first report of a brain stem infarction as a complication of percutaneous coronary intervention.The online version of this article (doi:10.1007/s12471-015-0717-2) contains supplementary material, which is available to authorized users. Here we present a case of a 64-year-old man, without any history of cardiovascular risk factors or other comorbidities, who underwent acute percutaneous coronary intervention (PCI) for an acute posterior wall myocardial infarction. Coronary angiography revealed an occluded right coronary artery Fig.\u00a0. Thrombo"} +{"text": "REST-003. We show that processing of REST-003 into ncRNAs is controlled by an uncharacterized serine/arginine repeat-related protein, SRRM3. SRRM3 expression may be under REST-mediated transcriptional control, as it increases following REST downregulation. The SRRM3-dependent regulation of REST-003 processing into ncRNAs has many similarities to recently described promoter-associated small RNA-like processes. Targeting ncRNAs that control invasiveness could lead to new therapeutic approaches to limit breast cancer metastasis.RE1-Silencing Transcription factor (REST) has a well-established role in regulating transcription of genes important for neuronal development. Its role in cancer, though significant, is less well understood. We show that REST downregulation in weakly invasive MCF-7 breast cancer cells converts them to a more invasive phenotype, while REST overexpression in highly invasive MDA-MB-231 cells suppresses invasiveness. Surprisingly, the mechanism responsible for these phenotypic changes does not depend directly on the transcriptional function of REST protein. Instead, it is driven by previously unstudied mid-size (30\u2013200\u2009nt) non-coding RNAs (ncRNAs) derived from the first exon of an alternatively spliced REST transcript: REST-003 is a non-coding RNA that is processed into a complicated series of sense and antisense ncRNAs with sizes ranging from 30\u2013200\u2009nt. The alternative splicing of REST transcript in invasive cells appears to be controlled by a previously unstudied SR protein, SRRM3, as well as by the REST protein itself. REST expression results in downregulation of both SRRM3 and REST-003 transcript. REST-003 levels correlate positively with cancer cell invasiveness, and knockdown of REST-003 is sufficient to convert invasive cancer cells to a non-invasive phenotype. Our results reveal regulatory interactions among REST, REST-003, and a cancer cell-specific splicing activator (SRRM3) that may coordinate gene regulation required for development of the invasive cancer phenotype in breast cancer.Here, we used RNA deep sequencing to identify a previously uncharacterized alternatively spliced form of the REST gene, We explored the REST-dependent invasive phenotype in more detail using two breast cancer cell lines: MDA-MB-231, which is strongly invasive, and MCF-7, which is weakly invasive. We first confirmed the invasive potential of each cell line using Matrigel invasion chamber assays2REST in MCF-7 cells using two small interfering RNAs (siRNAs) of MCF-7 and MDA-MB-231 cells. RNA-seq data suggested downregulation of REST transcript levels in MCF-7 cells following treatment with 2 different siRNAs designed to target the protein coding region of REST. REST expression also increased when REST cDNA was transfected into MDA-MB-231 cells analysis . We confined our analysis to these forms , which are illustrated schematically in REST gene . REST-004 lacks the E2 initiation codon and may thus produce non-coding RNA (ncRNA). REST-003 contains the initiation codon but lacks other parts of the E2 coding sequence. Since no available data identify REST-003 as a protein-coding gene, we analyzed the structures of the REST ASPs with qRT-PCR, using specific primers to distinguish the presence or absence of the E2 initiation codon . When testing primers that exclude the initiation codon but are specific to REST-003 (3-B primer pair), we observed low expression in MCF-7 cells . These results support our RNA-seq data and suggest that only ncRNA derived from REST-003 correlates positively with invasiveness.Only four REST ASPs are catalogued in the Ensembl Human Genome Browser database following REST downregulation in MCF-7 cells decreased relative to control expression , a pattern similar to that of REST-001 (REST in MDA-MB-231 cells resulted in decreased REST-003 expression (REST-003 ncRNAs (following loss of REST) may mediate breast cancer cell invasiveness.We next established the effect of REST modulation on -7 cells , Fig. 2AREST-003 in controlling invasiveness, we knocked down its expression in MDA-MB-231 cells with siRNA (si-REST-003). The si-REST-003-treated MDA-MB-231 cells exhibited decreased REST-003 expression (>50%) and reduced Matrigel invasion (>50%) relative to control cells treated with scrambled RNA in MDA-MB-231 cells following REST overexpression , we observed lower SRRM3 and REST-003 expression positioned within the first exon of REST mRNA . Notably, we did not detect any ~21\u201323\u2009nt double-stranded RNAs potentially derived from Dicer processing of REST-003. Taken together, our data indicate at least eight RNA variants derived from REST primary transcript . Three appear to be ncRNAs that likely promote invasiveness of MDA-MB-231cells treated with si-REST-003 (targeting the E1-3 region). Fifty-six genes from DESeq were differentially expressed in the treated versus control samples (2930313233REST-003 ncRNAs. These results indicate that REST-003 ncRNAs play an important role in cancer cell invasion that is largely independent of REST and REST target gene function.We next sought a link between REST-003). We propose that these ncRNAs provide a key regulatory function by controlling expression of genes important for cancer cell invasiveness. They may thus represent a new functional example of a PASR-like process of genes have been described process .REST-003), which are apparently further processed into mid-size (30\u2013200\u2009nt) ncRNAs. As expected from alternative splicing, REST-003 levels correlate negatively with those of REST-001 (the protein coding form of the REST gene). More importantly, we found that REST-003 levels correlate positively with cancer cell invasiveness in several cancer cell lines.One key technical aspect of our approach was to perform RNA deep sequencing without first selecting polyadenylated RNA. This allowed identification of transcripts derived from the first exon of an alternatively spliced REST transcript \u2009=\u2009HER2+\u2009>\u2009luminal B \u2009>\u2009luminal A . We found that this order correlates well with levels of REST-003 . Low REST-003 levels were observed in MCF-7, T47D , BT474 , SKBR3 (HER2+) and MDA-MB-468 cell lines, all of which are non-invasive in a Matrigel assay . Conversely, high REST-003 levels were observed in basal-like triple negative and invasive MDA-MB-231 cells . These results suggest that REST-003 could be used as a prognostic marker to access invasive potential of breast and other types of cancer cells. Further studies will be required to establish this more firmly.Subtypes of breast cancer are often classified with markers such as estrogen receptor (ER), progesterone receptor (PR), and HER2 that relate to clinical outcomes. At least four subtypes are recognized based on different expression patterns for these markers363736REST-001 and REST-003. We found that REST itself negatively regulates expression of SRRM3, which suggests a positive association between the expression of SRRM3 and REST\u2010003 . These genes are known to be important not only for cell line invasiveness . Each of these genes may thus be regulated in different ways by the REST-003 mid-size ncRNAs. Our RNA-seq data place the focus on REST-003-derived mid-size ncRNAs.Following si-siveness , but alsREST-003 and SRRM3 in regulating cancer cell invasiveness suggests novel therapeutic approaches for limiting breast (and other types of) cancer invasion and metastasis.In conclusion, a mechanistic role for MDA-MB-231 and MCF-7 cancer cell lines were obtained and cultured as described previouslyFor qRT-PCR, cDNAs were made from the total RNAs treated with DNase as described in our previous studyInvasion was measured using BD BioCoat Matrigel Invasion Chambers (BD Biosciences) according to the manufacturer\u2019s instructions, as described in our previous studyRibosomal RNA (rRNA) was depleted from 100\u2009ng of total RNA using a Ribo-Zero Magnetic Gold kit (Epicentre) according to the manufacturer\u2019s protocol. Libraries from rRNA-depleted samples were prepared using a TruSeq RNA Sample Preparation kit v2 (Illumina) following the recommended protocol starting from the RNA fragmentation stage. Purification of polyadenylated RNA was omitted. Libraries were pooled (4 samples per pool), clustered on cBOT (Illumina), and sequenced on HiSeq2000, each pool in one lane. We performed single-end 100 bp reads. Reads were mapped using TopHat followed by data analysis using Cufflinks and Cuffdiff software, as well as our own pipeline22REST-003 treatment were averaged from TNBC and controls by our pipeline22For tissue data, expression reads of downregulated genes by si-Bar graphs show the mean\u2009\u00b1\u2009SEM of biological replicates (n). Detailed statistical methods are indicated in the figure legends.How to cite this article: Lee, N.S. et al. Non-coding RNAs derived from an alternatively spliced REST transcript (REST-003) regulate breast cancer invasiveness. Sci. Rep.5, 11207; doi: 10.1038/srep11207 (2015)."} +{"text": "Yersinia pestis. There are enhancing factors and inhibiting one (HmsP) for Yersinia pestis biofilm formation. The RcsAB regulatory complex acts as a repressor of Yesinia biofilm formation, and adaptive pseudogenization of rcsA promotes Y. pestis to evolve the ability of biofilm formation in fleas. In this study, we constructed a set of isogenic strains of Y. pestis biovar Microtus, namely WT (RscB+ and RcsA-), c-rcsA (RscB+ and RcsA+), \u0394rcsB (RscB- and RcsA-), and \u0394rcsB/c-rcsA (RscB- and RcsA+). The phenotypic assays confirmed that RcsB alone had an inhibiting effect on biofilm/c-di-GMP production whereas assistance of RcsA to RcsB greatly enhanced this inhibiting effect. Further gene regulation experiments showed that RcsB in assistance of RcsA tightly bound to corresponding promoter-proximal regions to achieve transcriptional repression of hmsCDE, hmsT and hmsHFRS and, meanwhile, RcsAB positively regulated hmsP most likely in an indirect manner. Data presented here disclose that pseudogenization of rcsA leads to dramatic remodeling of RcsAB-dependent hms gene expression between Y. pestis and its progenitor Y. pseudotuberculosis, enabling potent production of Y. pestis biofilms in fleas.Biofilm formation in flea gut is important for flea-borne transmission of Y. pestis is potent to synthesize biofilms, which are a population of bacterial colonies embedded in self-produced matrix12Y. pestis biofilms in flea gut is important for flea-borne transmission of this pathogen12YY. pestis biofilm matrix is primarily composed of poly-B-1,6-N-acetylglucosamine exopolysaccharide12hmsHFRS operon is responsible for biosynthesis and translocation of biofilm exopolysaccharide through cell envelope446\u03b2-barrel structure, and HmsF functions as a polysaccharide deacetylase; these two proteins form a complex for modification/export of partially deacetylated exopolysaccharide through outer membrane5Y. pestis produces a total of two diguanylate cyclases HmsT and HmsD, and both of them are required for c-di-GMP biosynthesis and biofilm formation10in vitro biofilm formationhmsD gene is a member of the three-gene operon hmsCDE. HmsD is a trans-inner-membrane protein composed of three distinct domains, namely a periplasmic sensor domain, an HAMP signal converter domain and a cytoplasmic output GGDEF domain1212Y. pestis expresses the sole c-di-GMP-specific phosphodiesterase HmsP, which is responsible for degradation of c-di-GMP and therefore has an inhibiting effect on biofilm formation7The 3\u2032,5\u2032-cyclic diguanosine monophosphate (c-di-GMP), a small-molecule second messenger promoting exopolysaccharide biosynthesis, is produced from guanosine triphosphate by GGDEF-domain-containing diguanylate cyclases and degraded by EAL-domain-containing phosphodiesteraseshmsHFRS orthologs can be found in several bacterial speciespgaABCD operon in Escherichia coliY. pestis might employ the conserved c-di-GMP-HmsRS association mechanism to control exopolysaccharide production.The Enterobacteriaceae Rcs phosphorelay system is an atypical two-component regulatory system composed of three proteins, RcsB, RcsC and RcsDThe Y. pestis and its genetically very closed progenitor Y. pseudotuberculosis is negatively regulated by the Rcs phosphorelay system19rcsA gene is inactivated in Y. pestis due to a 30\u2005bp duplication insertion in its coding region, and replacing the rcsA pseudogene with functional rcsA allele of Y. pseudotuberculosis strongly represses Y. pestis biofilm formation and essentially abolished flea blockage19rcsA to a pseudogene during evolution from Y. pseudotuberculosis to Y. pestis is most likely a case of positive Darwinian selection19The biofilm formation of Y. pestis biofilm formation through directly repressing transcription of hmsCDE, hmsT and hmsHFRS meanwhile positively regulating hmsP in an indirect manner. The above results denote dramatic remodeling of biofilm-related hms gene expression between and Y. pestis and its progenitor Y. pseudotuberculosis due to adaptive pseudogenization of a regulatory gene rcsA.The present work discloses that the RcsAB complex acts as a major repressor of rcsA into WT generated the rcsA-complemented strain c-rcsA (RscB+ and RcsA+), which led to a considerable decrease in c-di-GMP/biofilm production (Transformation of pACYC184-oduction .rcsB from WT generated the rcsB-null strain \u0394rcsB (RscB- and RcsA-), and further transformation of pACYC184-rcsA into \u0394rcsB generated another rcsA-complemented strain \u0394rcsB/c-rcsA (RscB- and RcsA+); compared to WT, both \u0394rcsB and \u0394rcsB/c-rcsA gave significantly enhanced c-di-GMP/biofilm production (Deletion of oduction .rcsB into \u0394rcsB generated the rcsB-complemented strain c-rcsB (RscB+ and RcsA-), which had a biofilm/c-di-GMP production phenotype very similar to WT has an inhibiting effect on biofilm/c-di-GMP production, whereas assistance of RcsA to RcsB greatly enhances this inhibiting effect.hmsCDE, hmsT and hmsHFRS, indicating that they might serve as direct RcsAB targets . However, LacZ fusion assay , c-rcsA (RscB+ and RcsA+), \u0394rcsB (RscB- and RcsA-), and \u0394rcsB/c-rcsA (RscB- and RcsA+). RcsAB acts as a major repressor of Y. pestis biofilm formation through directly repressing transcription of biofilm-enhancing genes hmsCDE, hmsT and hmsHFRS and meanwhile positively regulating biofilm-enhancing one hmsP in an indirect manner. RcsB in absence of RcsA does have residual regulatory effects on biofilm formation and hms gene expression and, moreover, RcsB-dependent regulation is greatly increased with assistance of RcsA, which was consistent with previous results1921hmsCDE, hmsT, hmsHFRS and hmsP in the above isogenic strains and thus distinct potencies of these strains to produce c-di-GMP/biofilm is avirulent to humans but highly virulent to miceY. pestis , and the resulting recombinant vector was transformed into each indicated Y. pestis strain lack of the corresponding functional gene, generating the corresponding complemented mutant (The wild-type . pestis . For in d mutant . All the620) of about 1.0 were diluted 1:50 into 18\u2005ml of fresh LB broth for further cultivation at 26\u00b0C with shaking at 230\u2005rpm to reach middle stationary phases (an OD620 of 0.8 to 1.2), followed by cell harvest for further gene regulation or phenotypic assays. Immediately before bacterial harvest for RNA isolation, double-volume of RNAprotect reagent (Qiagen) was mixed with one-volume of cell culture, and total RNA was extracted using TRIzol Reagent (Invitrogen). RNA quality was monitored by agarose gel electrophoresis, and RNA quantity was determined by spectrophotometry.Overnight cell cultures in the Luria-Bertani (LB) broth with an optical density . Radioactive species were detected by autoradiography. If different Y. pestis strains were involved in a single experiment, equal amounts of the total RNA samples were used as the starting materials. The relative mRNA level was determined with the observed band intensity of the primer extension product of each target gene. The 5\u2032-terminus of RNA transcript of each target gene was mapped according to the size of primer extension product.As described in our previous studies29lacZ reporter gene. Y. pestis strains transformed with the recombinant plasmid or the empty pRW50 (negative control) were grown to measure \u03b2-galactosidase activity in cellular extract using \u03b2-Galactosidase Enzyme Assay System (Promega)29A promoter-proximal DNA region of each indicated gene was cloned into the low-copy-number transcriptional fusion vector pRW50Y. pseudotuberculosis rcsA or Y. pestisrcsB was cloned into plasmid pMAL-c4X (Invitrogen)Y. pestis strain KIM6+ and the rcsB null mutant of KIM6+ were employed as host cells for expression of maltose-binding protein (MBP)-tagged RcsA (MBP-RcsA) and 6 \u00d7 His-tagged RcsB (His-RcsB), respectivelyThe entire coding region of 32P-labeled target DNA fragment was incubated with increasing amounts of purified His-RcsB, or with increasing amounts of purified His-RcsB with addition of 24\u2005pmol of purified MBP-RcsA, for 30\u2005min at room temperature in a binding buffer2929Each indicated 5\u2032-32P-labeled end was incubated with increasing amounts of purified His-RcsB-p with addition of 24\u2005pmol of purified MBP-RcsA, which was followed by partial digestion of RQ1 RNase-Free DNase I (Promega). The digested DNA samples were purified and analyzed in an 8\u2005M urea-6% polyacrylamide gel. Detection of sequencing and radioactive species was as above. Footprints were identified by comparison with sequence ladders.For DNase I footprinting29Y. pestis biofilms. First, in vitro biofilm masses, attached to well walls when bacteria were grown in polystyrene microtiter plates, were stained with crystal violet. Second, percentages of fourth-stage larvae and adults (L4/adult) of C. elegans after incubation of nematode eggs on Y. pestis lawns, negatively reflecting bacterial ability to produce biofilms, were determined. Third, rugose colony morphology of bacteria grown on LB agar plates, positively reflecting bacterial ability to synthesize exopolysaccharide, was observed. In addition, intracellular c-di-GMP levels were determined by a chromatography-coupled tandem mass spectrometry (HPLC-MS/MS) method as described in our previous studyAs described in our previous studyt-test was performed to determine statistically significant differences; P <0.01 was considered to indicate statistical significance. For primer extension and colony morphology observation, shown were representative data from at least two independent bacterial cultures.For LacZ fusion, crystal violet staining of biofilms, and determination of L4/adult nematodes or c-di-GMP, experiments were performed with at least three independent bacterial cultures/lawns, and values were expressed as mean \u00b1 standard deviation. Paired Student's Supplementary Information"} +{"text": "Chimeric read-through RNAs are transcripts originating from two directly adjacent genes (<10 kb) on the same DNA strand. Although they are found in next-generation whole transcriptome sequencing (RNA-Seq) data on a regular basis, investigating them further has usually been refrained from. Therefore, their expression patterns or functions in general, and in oncogenesis in particular, are poorly understood.We used paired-end RNA-Seq and a specifically designed computational data analysis pipeline (FusionSeq) to nominate read-through events in a small discovery set of renal cell carcinomas (RCC) and confirmed them in a larger validation cohort.BC039389-GATM was higher expressed in RCC compared to benign adjacent kidney; KLK4-KRSP1 was expressed in 46/169 (27%) RCCs, but rarely in normal tissue. KLK4-KRSP1 expression was associated with worse clinical outcome in the patient cohort. In cell lines, both read-throughs influenced molecular mechanisms (i.e. target gene expression or migration/invasion) in a way that counteracted the effect of the respective parent transcript GATM or KLK4.324 read-through events were called overall; 22/27 (81%) selected nominees passed validation with conventional PCR and were sequenced at the junction region. We frequently identified various isoforms of a given read-through event. 2/22 read-throughs were up-regulated: Our data suggests that the up-regulation of read-through RNA chimeras in tumors is not random but causes regulatory effects on cellular mechanisms and may impact patient survival.The online version of this article (doi:10.1186/s12864-015-1446-z) contains supplementary material, which is available to authorized users. SLC45A3-ELK4 as the prototype read-through in prostate cancer is not just a biomarker ."} +{"text": "Methylomonas methanica , Methylomonas koyamae , Methylomonas lenta (R-45370), and Methylosinus sp. (R-45379) were obtained. These aerobic methanotrophs were isolated from terrestrial ecosystems, and their distinct phenotypes related to nitrogen assimilation and dissimilation were previously reported.The genome sequences of Methylomonas methanica was acquired from NCIMB and was originally isolated from freshwater sediment (M.\u00a0methanica strains (R-45363 and R-45371) and Methylomonas lenta R-45370 were isolated from the top layer of a denitrification tank of a Belgian wastewater treatment plant and the PQQ-dependent methanol dehydrogenases (mxaFI). The genomes of strains R-45363, R-45371, R-45370, R-45383, and R-45379 also contain the genes for the soluble methane monooxygenase (mmoXYBZDC). As expected, the gene inventory for nitrogen metabolism was strain dependent and the periplasmic nitrate reductase (napCBADF), both the copper-dependent (nirK) and the cytochrome cd1-dependent nitrite reductase (nirS), and the cytochrome-dependent nitric oxide reductase. The other strains have various combinations of these nitrogen dissimilation genes, with M.\u00a0koyamae R-49807 also harboring the gene for quinol-dependent nitric oxide reductase, while R-45383 does not encode any nitric oxide reductase. Methylosinus sp. R-45379 seems limited to the assimilation of various nitrogen compounds.All eight sequenced terrestrial methanotrophs are obligate methane and methanol utilizers. All the genomes harbor genes typical for methanotrophs, including genes encoding particulate methane monooxygenase (ependent . Genes irificans . M.\u00a0methThese genomes provide a valuable resource to link previously observed phenotypes to genomic inventory, gather new insights into the redundancy and environmental controls of nitrogen metabolism in closely related methanotrophs and the interplay between nitrogen and one-carbon metabolism.The genome sequences have been deposited in GenBank under accession numbers listed in"} +{"text": "HMG-CoA-reductase-inhibitors (statins) have been shown to interfere with HCV replication in vitro. We investigated the mechanism, requirements and contribution of heme oxygenase-1(HO-1)-induction by statins to interference with HCV replication.HO-1-induction by fluva-, simva-, rosuva-, atorva- or pravastatin was correlated to HCV replication, using non-infectious replicon systems as well as the infectious cell culture system. The mechanism of HO-1-induction by statins as well as its relevance for interference with HCV replication was investigated using transient or permanent knockdown cell lines. Polyacrylamide(PAA) gels of different density degrees or the Rho-kinase-inhibitor Hydroxyfasudil were used in order to mimic matrix conditions corresponding to normal versus fibrotic liver tissue.in vitro. HO-1-induction was mediated by reduction of Bach1 expression and induction of the Nuclear factor (erythroid-derived 2)-like 2 (NRF2) cofactor Krueppel-like factor 2 (KLF2). Knockdown of KLF2 or HO-1 abrogated effects of statins on HCV replication. HO-1-induction and anti-viral effects of statins were more pronounced under cell culture conditions mimicking advanced stages of liver disease.All statins used, except pravastatin, decreased HCV replication and induced HO-1 expression, as well as interferon response Statin-mediated effects on HCV replication seem to require HO-1-induction, which is more pronounced in a microenvironment resembling fibrotic liver tissue. This implicates that certain statins might be especially useful to support HCV therapy of patients at advanced stages of liver disease. About 3% of the world's population are chronically infected with HCV in vitroStatins are among the most frequently prescribed medication worldwide. They interfere with endogenous cholesterol biosynthesis by competitively inhibiting its key enzyme HMG-CoA-reductase. Apart from this, statins have been described to possess other biological activities, e.g. exerting anti-proliferative effects in cancer in vitroIt has been reported that statins are able to induce expression of the heme degrading enzyme heme oxygenase 1 in endothelial cells in vitro. We found that all statins, except PRV, were able to induce HO-1 expression and reduce HCV replication. Anti-virally active statins interfered with the expression of the HO-1 transcriptional inhibitor Bach1 and induced expression of nuclear factor (erythroid-derived 2)-like 2 (NRF2) and the NRF2 co-factor Krueppel-like factor 2 (KLF2), which turned out to be crucial for HO-1-induction and anti-viral effects of statins. Induction of HO-1 by statins was sufficient to initiate interferon response in vitro and to support anti-viral effects of exogenous interferon or telaprevir incubation.In consequence we compared heme oxygenase 1 (HO-1)-induction and anti-viral activity of different statins like fluva- (FLV), simva- (SMV), rosuva- (ROV), atorva- (ATV) and pravastatin (PRV) Interestingly, statin-mediated effects were more pronounced when cells were growing in an environment mimicking conditions found during advanced stages of liver disease.HMG-CoA-reductase inhibitors fluva- (FLV), simva- (SMV), prava- (PRV) , atorva- (ATV) and rosuvastatin (ROV) as well as NS3/4A protease Inhibitor telaprevir were dissolved in DMSO . As a vehicle control, DMSO was diluted to the concentrations used on statin-incubated cells. The Rho-kinase inhibitor Hydroxyfasudil (HA1100) was dissolved in sterile water. Recombinant interferon alpha-2b (Intron A) was purchased from Essex Pharma, M\u00fcnchen, Germany.CTG CGG CAA GAC CTA CAC CAA ; siControl . ShRNA expressing vectors were based on the lentiviral pLKO.1 construct , and normalized to the protein content of the individual sample.To visualize HCV infection, E2 proteins were stained. Antibodies: human monoclonal A3R3 against E2 , chicken anti-human Alexa-488 . The procedure included fixation , permeabilization and blocking . Pictures were taken using an inverted microscope with an LCachN/20X/0.40 Phc/1/FN22 UIS objective.RT-qPCR was performed as described previously 50 \u00b5g of total protein were fractionated by 12% SDS-polyacrylamide gel electrophoresis and blotted onto nitrocellulose membranes as described previously Polyacrylamide (PAA) gel supports of various elasticity were prepared on glass cover slips as described previously t test, if two groups were compared and 1way ANOVA in combination with Bonferroni's Multiple Comparison Test if more than 2 groups were compared. All data are expressed as a mean \u00b1 SEM. * p\u22640.05; ** p\u22640.01; *** p\u22640.001.Results were analyzed using Student's Statins are widely used drugs to control biosynthesis of cholesterol and to reduce amounts of LDL-cholesterol by inducing LDL-receptor (LDLR) expression. Therefore, biological activity of statins used in our experiments was verified by measuring their ability to increase LDL-receptor (LDLR) expression by RT-qPCR . InvestiFurther investigations revealed that all statins, except PRV, significantly induced expression of the anti-viral enzyme HO-1 within 6\u20138 hours of incubation, shown by RT-qPCR and WestTo investigate the contribution of HO-1-induction to the anti-viral effects of statins, we established replicon cell lines with a stable knockdown of HO-1 (shHO1) or a control gene (shGFP). In the shHO-1 cell line we observed an about 50% decrease of the HO-1 background expression, compared to the control cell line containing shGFP, while FLV was not able to increase HO-1 expression in those cells . FurtherInvestigating the mechanism of HO-1-induction by statins, we detected significantly reduced expression of the HO-1 transcriptional repressor Bach1 in statin-incubated cells . Here PRViral infections of the liver frequently result in chronic inflammation and formation of fibrosis. In order to investigate how these conditions might influence anti-viral effects of statins we prepared Polyacrylamid (PAA) gels of different stiffness as supports for the replicon cell culture system, mimicking physiological (soft) or fibrotic (stiff) liver tissue. In fact, hepatocellular carcinoma (HCC) cell morphology as well as proliferation has been shown to be stiffness-dependent in vitro, and might reduce resistance development With increasing average body mass in western countries and the increased risk of developing cardiovascular diseases, HMG-CoA-reductase inhibitors have found their place among the most frequently prescribed medication. Although the primary indication for statin prescription is treatment of hypercholesteremia to prevent adjacent secondary events, e.g. coronary problems, evidence is increasing that statins might also display beneficial effects on other kinds of diseases These results are promising but do not generally reflect anti-viral statin effects in patients Statins have been shown to regulate gene expression. Deficiency in cholesterol leads to the activation of sterol regulatory element-binding proteins (SREBPs), transcription factors, which increase expression of LDL-receptor (LDLR), subsequently leading to reduction of LDL-cholesterol in vivo, the regular culture on plastic surfaces has to be considered a non-physiological stiff environment, resembling fibrotic or cirrhotic conditions. It has already been shown that environmental stiffness affects interferon treatment and thereby therapeutic responses in HCV infection Aiming to provide a possible explanation on inconsistent anti-viral effects of e.g. FLV in patients we investigated the role of matrix stiffness, corresponding to degrees of cirrhosis. Matrix stiffness has so far been mostly neglected in the study of therapeutic responses in cell culture. Since hepatocytes under physiological conditions reside in a soft environment R-diastereomer. Telaprevir is both a substrate and an inhibitor of the isoenzyme As an additional benefit we observed that statins were able to induce endogenous interferon response under cell culture conditions which are comparable cirrhotic tissue conditions. Since exogenous interferon treatment has been shown to display severe side effects, stimulation of endogenous interferon response might be an alternative attempt or at least help to reduce therapeutic doses of exogenous interferon. Hence, FLV might further support therapy especially under stiff matrix conditions by inducing endogenous interferon response. The direct antiviral acting (DAA) compound telaprevir is metabolized in the liver by cytochrome P-450 (CYP) isoenzyme 3A4 to the inactive (\u03b1-ketoamide bond) metabolites and the active Taken together our data provide evidence that HO-1-inducing statins might represent a beneficial therapeutic support for HCV patients with advanced liver disease."} +{"text": "Kinetic modelling in PET requires the arterial input function (AIF), defined as the time-activity curve (TAC) in plasma. This measure is challenging to obtain in mice due to low blood volumes, resulting in a reliance on image-based methods for AIF derivation. We present a comparison of PET- and MR-based region-of-interest (ROI) analysis to obtain image-derived AIFs from the left ventricle (LV) of a mouse model. ROI-based partial volume correction (PVC) was performed to improve quantification.18F-FDG bolus, 45 minute emission listmode acquisition reconstructed with 3DRP in four cardiac frames) on a split-magnet PET camera [MRI and dynamic PET images were obtained from a recent study investigating treatment effects in 12 mice following myocardial infarction [AIF extraction AIFs were obtained by taking mean time courses from LV Lumen ROIs, shown in Figure i values between the treated and untreated groups in KGTM-based PVC gives best results in mice when ROIs are based on MRI data, due to its high-resolution and excellent soft-tissue contrast."} +{"text": "Enhancing tumor-directed immune responses has emerged as an important therapeutic approach to many cancers. Th17/Tc17 cells can mediate robust anti-tumor responses in rodent models and are associated with improved prognosis in some human cancers. RORgt is the key transcription factor controlling the development and function of these cells by supporting the expression of pro-inflammatory cytokines and survival genes while reducing expression of co-inhibitory receptors such as PD-1.in vitro, increase production of IL-17A and other cytokines/chemokines, and improve cell survival after resting or re-activation.To enhance the function of anti-tumor Type 17 T cells, small molecule RORg agonists were designed. These synthetic agonists, when present during murine and human T cell activation This improved survival translates to a potent and durable anti-tumor response as adoptive cell therapy using RORg agonist-treated T cells significantly reduces growth of established B16F10 and EG.7 tumors compared to untreated cells. Up to 71 days post transfer, more donor T cells can be recovered from spleens and tumors of mice receiving RORg agonist-treated cells. These cells maintain elevated IL-17 production and reduced co-inhibitory receptor expression suggesting that RORg agonist-induced changes are long-lasting. Interestingly, tumor-specific T cells recovered from mice receiving agonist-treated cells expressed central memory (CD44+CD62L+) or stem-like (CD44-CD62L+) markers vs. the predominantly effector (CD44+CD62L-) cells recovered from animals receiving untreated T cells. In separate experiments, oral administration of RORg agonists significantly inhibits the growth of subcutaneous MC38 and 4T1 tumors in an immune-dependent manner with significantly increased RORg and IL-17 expression in tumors consistent with an increased survival or recruitment of type 17 cells.By enhancing cytokine production, decreasing co-inhibitory receptor expression while promoting long term survival and self-renewal of T cells, RORg agonists represent a promising immunotherapy approach for the treatment of cancer."} +{"text": "We sought to identify gene polymorphisms that confer susceptibility to in-stent restenosis after coronary artery bare-metal stenting in a Central European population.BCHE gene), rs529038 (ROS1), rs1050450 (GPX1), rs1800849 (UCP3), rs17216473 (ALOX5AP), rs7412, rs429358 (ApoE), rs2228570 (VDR), rs7041, rs4588 (GC), rs1799986 (LRP1) and rs2228671 (LDLR). Multivariable logistic regression was used to test for associations.160 controls without post\u2013percutaneous coronary intervention in-stent restenosis were matched for age, sex, vessel diameter, and diabetes to 160 consecutive cases involving in-stent restenosis of the target lesion within 12\u00a0months. Using real time polymerase chain reaction and melting-curve analysis, we detected 13 single-nucleotide polymorphisms in 11 candidate genes - rs1803274 . No association was found with the other studied SNPs.The rs1803274 polymorphism of The A allele of rs1803274 represents a risk factor for in-stent restenosis in Central European patients after percutaneous coronary intervention with bare-metal stent implantation. Coronary stent implantation has significantly improved percutaneous coronary intervention (PCI). It has enabled management of early complications of plane balloon angioplasty and prevention of elastic recoil and constrictive remodeling, decreasing the frequency of restenosis after PCI. However, with these improvements has come a new complication: in-stent restenosis (ISR) arising from neointimal hyperplasia. The clinical incidence of ISR after bare-metal stent (BMS) implantation is about 20\u201335\u00a0% and currently represents one of the main limitations of coronary angioplasty . The useBCHE - butyrylcholinesterase gene, ROS1 - the closest homolog of the v-Ros oncogene of the avian sarcoma, GPX1 - glutathione-peroxidase-1 gene, UCP3 - uncoupling protein 3 gene, and ALOX5AP - arachidonate 5-lipoxygenase-activating protein gene. Our aim was to assess associations in our Central European population ."} +{"text": "A previous large, multi-site trial showed that computer-facilitated screening and physician brief advice (cSBA) was an effective way to reduce adolescents' alcohol use. What isWe analyzed a subset of data from a quasi-experimental, asynchronous effectiveness trial of 12-18 y/o patients at primary care sites. An initial 18-month Treatment as Usual (TAU) phase was followed by a 1-hour physician training and an 18-month cSBA phase with computerized screening, immediate feedback and information on the health risks of drugs, follow by physicians brief advice. Adolescents rated their visit and physician immediately post-visit. Only data for physicians with >5 patients in each study arm were included. We conducted stratified multiple logistic regression modeling with adjustment for known covariates and within-site clustering. Endpoints were past 3- and 12-month alcohol at follow-ups.Subjects: 20 physicians and 1158 patients, mean age 15.6+2.0 yrs. Youth-provider connectedness was high (median score 32 [IQR 29-34] out of 35 max). However, female physicians' patients scored significantly higher on youth-provider connectedness than patients of male physicians (p<0.0001), regardless of patient gender. The 3-month effect of cSBA on adolescent alcohol use was stronger among girls and female physicians (Table Physician advice regarding alcohol use may be particularly effective for girls within the context of an ongoing relationship with their physician, and when delivered by female physicians whose care is associated with higher patient-provider connectedness."} +{"text": "Plastic debris pervades in our oceans and freshwater systems and the potential ecosystem-level impacts of this anthropogenic litter require urgent evaluation. Microbes readily colonize aquatic plastic debris and members of these biofilm communities are speculated to include pathogenic, toxic, invasive or plastic degrading-species. The influence of plastic-colonizing microorganisms on the fate of plastic debris is largely unknown, as is the role of plastic in selecting for unique microbial communities. This work aimed to characterize microbial biofilm communities colonizing single-use poly (PET) drinking bottles, determine their plastic-specificity in contrast with seawater and glass-colonizing communities, and identify seasonal and geographical influences on the communities. A substrate recruitment experiment was established in which PET bottles were deployed for 5\u20136 weeks at three stations in the North Sea in three different seasons. The structure and composition of the PET-colonizing bacterial/archaeal and eukaryotic communities varied with season and station. Abundant PET-colonizing taxa belonged to the phylum Bacteroidetes and diatoms . The PET-colonizing microbial communities differed significantly from free-living communities, but from particle-associated (>3 \u03bcm) communities or those inhabiting glass substrates. These data suggest that microbial community assembly on plastics is driven by conventional marine biofilm processes, with the plastic surface serving as raft for attachment, rather than selecting for recruitment of plastic-specific microbial colonizers. A small proportion of taxa, notably, members of the Cryomorphaceae and Alcanivoraceae, were significantly discriminant of PET but not glass surfaces, conjuring the possibility that these groups may directly interact with the PET substrate. Future research is required to investigate microscale functional interactions at the plastic surface. Plastic pollution was first reported in remote, offshore basins of the north Atlantic ocean over forty years ago , 2, twenAs the spatial distribution of marine plastic debris continues to be better resolved, research is increasingly focused on assessing its effects on environmental and public health. The impacts of plastic pollution range from organismal, such as morbidity and mortality due to entanglement and intestinal blockage to food Plastic surfaces in seawater can form microbial biofilms visible by eye within one week and cause a physical change, with a significant increase in plastic hydrophilicity and a shift from positive towards neutral buoyancy after 2 weeks . Yet, thArcobacter and Colwellia, which have previously been shown to affiliate with hydrocarbon contamination . Sterile PCR grade water served as negative control and known DNA as positive control , taxonomy assignment and picking of operational taxonomic units, OTUs, (label = 0.03) were carried out using Mothur . Chloropth percentile of reads were removed from analyses, proportions of OTUs per sample were calculated , proportions were square root-transformed, and scaled by the minimum library size per subset. This work was performed in R or calculating Monte-Carlo tests [\u2018p(MC)\u2019]. Unique perm, unique permutations. The results of the PERMDISP tests calculated to centroids are also provided.(XLSX)Click here for additional data file.S4 TableDiscriminant OTUs identified, using class and subclass distinctions, in comparisons of: (a) PET-associated, particle-attached (>3 \u03bcm) and free-living (0.2 \u03bcm-3 \u03bcm) seawater communities in summer (lefse class: treatment) across all stations (lefse subclass: station), (b) PET- and glass-attached communities in spring (lefse class: treatment) across all stations (lefse subclass: station), (c) PET-associated communities from summer, spring, winter (lefse class: season) across all stations (lefse subclass: station), (d) PET-associated communities from Dowsing, Warp, Gabbard (lefse class: station) across all seasons (lefse subclass: season), (e) attached (PET and >3\u03bcm seawater fraction) and free-living (3\u20130.2 \u03bcm seawater fraction) communities across all stations (lefse subclass: station).(XLSX)Click here for additional data file.S5 TableDiscriminant OTUs identified, using class and subclass distinctions, in comparisons of: (a) PET-associated, particle-attached (>3 \u03bcm) and free-living (0.2 \u03bcm-3 \u03bcm) seawater communities in summer (lefse class: treatment) across all stations (lefse subclass: station), (b) PET- and glass-attached communities in spring (lefse class: treatment) across all stations (lefse subclass: station), (c) PET-associated communities from summer, spring, winter (lefse class: season) across all stations (lefse subclass: station), (d) PET-associated communities from Dowsing, Warp, Gabbard (lefse class: station) across all seasons (lefse subclass: season), (e) attached (PET and >3\u03bcm seawater fraction) and free-living (3\u20130.2 \u03bcm seawater fraction) communities across all stations (lefse subclass: station).(XLSX)Click here for additional data file."} +{"text": "Chemotherapy remains the only available treatment for triple-negative (TN) breast cancer, and most patients exhibit an incomplete pathologic response. Half of patients exhibiting an incomplete pathologic response die within five years of treatment due to chemo-resistant, recurrent tumor growth. Defining molecules responsible for TN breast cancer chemo-resistance is crucial for developing effective combination therapies blocking tumor recurrence. Historically, chemo-resistance studies have relied on long-term chemotherapy selection models that drive genetic mutations conferring cell survival. Other models suggest that tumors are heterogeneous, being composed of both chemo-sensitive and chemo-resistant tumor cell populations. We previously described a short-term chemotherapy treatment model that enriches for chemo-residual TN tumor cells. In the current work, we use this enrichment strategy to identify a novel determinant of TN breast cancer chemotherapy resistance [a nuclear isoform of basic fibroblast growth factor (bFGF)].Studies are conducted using our in vitro model of chemotherapy resistance. Short-term chemotherapy treatment enriches for a chemo-residual TN subpopulation that over time resumes proliferation. By western blotting and real-time polymerase chain reaction, we show that this chemotherapy-enriched tumor cell subpopulation expresses nuclear bFGF. The importance of bFGF for survival of these chemo-residual cells is interrogated using short hairpin knockdown strategies. DNA repair capability is assessed by comet assay. Immunohistochemistry (IHC) is used to determine nuclear bFGF expression in TN breast cancer cases pre- and post- neoadjuvant chemotherapy.TN tumor cells surviving short-term chemotherapy treatment express increased nuclear bFGF. bFGF knockdown reduces the number of chemo-residual TN tumor cells. Adding back a nuclear bFGF construct to bFGF knockdown cells restores their chemo-resistance. Nuclear bFGF-mediated chemo-resistance is associated with increased DNA-dependent protein kinase (DNA-PK) expression and accelerated DNA repair. In fifty-six percent of matched TN breast cancer cases, percent nuclear bFGF-positive tumor cells either increases or remains the same post- neoadjuvant chemotherapy treatment (compared to pre-treatment). These data indicate that in a subset of TN breast cancers, chemotherapy enriches for nuclear bFGF-expressing tumor cells.These studies identify nuclear bFGF as a protein in a subset of TN breast cancers that likely contributes to drug resistance following standard chemotherapy treatment. Targeted therapies are not available for triple-negative (TN) breast cancer, which lacks estrogen receptor, progesterone receptor, and human epidermal growth factor receptor-2 (HER2) over-expression. Although TN breast tumors initially respond to chemotherapy, this response is incomplete in more than half of these patients , 2. NotaWe previously characterized an in vitro model of chemo-resistance/tumor recurrence . In thisThe basic fibroblast growth factor family (bFGF) consists of both cytosolic (secreted) and nuclear isoforms. Expression of these bFGF isoforms is regulated at the level of translation. Specifically, cytosolic forms are regulated by cap-dependent translation, whereas nuclear forms are regulated by cap-independent translation . These iCytosolic (secreted) isoforms of bFGF are implicated in tumor resistance to anti-angiogenic therapy \u201315. HoweCS) and a regulatory subunit (Ku70 and Ku80 heterodimer), which recruits DNA-PKCS to DNA. The status of the cell cycle determines whether DNA-PK or BRCA repairs DNA, with DNA-PK being responsible in growth-arrested cells -Thymidine for 16 h before harvesting onto glass-fiber filters. [3H]-Thymidine incorporation was measured as counts per minute (CPM) using a Tri-Carb 2100TR time-resolved liquid scintillation counter .Cells were plated in 96-well plates (3 \u00d7 103 cells/well) in 100 \u03bcl complete medium. After 4 h, 10 \u03bcl/well Alamar Blue (Life Technologies) reagent was added. After 2 h, fluorescence was measured using a Cytation3 plate reader (BioTek).Cells were plated in 96-well black, clear-bottom plates (2 \u00d7 10Cells were grown to 50 % confluence in a 10-cm dish. The transfection mixtures contained: 1) 2 \u03bcg bFGF shRNA or control shRNA plasmid + Optimem (Life Technologies) and 2) Lipofectamine 2000 (Life Technologies) + Optimem. These mixtures were incubated separately at room temperature for 5 minutes, combined, and incubated for 30 minutes at room temperature. Cells were washed twice with HBSS (Life Technologies). Optimem was then added to the RNA/Lipofectamine mixture, and the mix was added to the cells, which were incubated overnight at 37 \u00b0C. This medium was removed the next day and replaced with media containing puromycin . Cells were expanded in puromycin and tested for bFGF knockdown by western blotting. For bFGF addback, plasmids expressi4AC, 95 % EtOH) for 30 minutes, followed by 70 % ethanol for 30 minutes. Slides were then stained with a 1:500 dilution of Hoechst (Life Technologies) for 15 minutes and washed with PBS. Samples were examined using a fluorescence microscope, and the presence of comet tails was quantified using Gen5 image analysis software (BioTek). Cells from three fields were analyzed for each time point. Each field contained at least 50 cells.Cells were challenged with doxorubicin . Fresh medium was added after chemotherapy removal. Cells were harvested at sequential time points after chemotherapy, mixed with low-melting-point agarose, and spread on comet slides using a Trevigen CometAssay\u00ae Kit . After incubation with lysis solution and neutral solution, slides were subjected to electrophoresis at 19 V for 50 minutes under neutral conditions. Slides were incubated with DNA precipitation solution .Total RNA from SUM159 cells was extracted using PrepEase\u00ae RNA Spin Kit and treated with RNase-free DNase to remove residual genomic DNA. Single-stranded cDNAs were synthesized using the iScript cDNA synthesis kit . Human fibroblast growth factor-2 (FGF2) and human DNA-PKcs primers were purchased from realtimeprimers.com . Real-time PCR on the Mx3005P\u00ae QPCR System was performed in the presence of 12.5 \u03bcl VeriQuest\u2122 Fast SYBR Green qPCR Master mix (2\u00d7) , 2 \u03bcl cDNA, and H6 cells/flask) and, after 2 days, treated with 1 \u03bcg/ml doxorubicin (Sigma) plus dimethyl sulfoxide (DMSO) or NU7441 . The drug was removed after 2 days, and cells were fed with new medium every third day. Chemo-residual cells were harvested on day 7 with trypsin-EDTA, and re-plated in 6-well plates. Medium was changed every 3 to 4 days. Colonies evolving from chemo-residual cells were stained with crystal violet. Colonies containing >50 cells were counted.Cells were seeded in T225 cell culture flasks . Retrospectively collected tumor biopsies (obtained pre-chemotherapy) and biopsies/resections (obtained post-chemotherapy) from these patients were retrieved. Formalin-fixed, paraffin-embedded tissues were subjected to bFGF immunohistochemistry. Slides were baked at 60 \u00b0C for 1 h, and deparaffinized in xylene followed by 100 % ethanol. Antigen retrieval was performed in sub-boiling EDTA (pH 8) at 100 \u00b0C for 40 minutes. bFGF staining was performed in an autostainer according to the following program: Endogenous Peroxidase Quench 5 minutes; Protein block , 10 minutes; bFGF antibody , 1 h; secondary detection kit , 20 minutes; 3,3-diaminobenzidine (DAB), 5 minutes; hematoxylin, 1 minute; and Bluing, 1 minute. Slides were then placed in water and dehydrated in xylene before adding a coverslip.Two pathologists (blinded to patient samples) assigned scores for the percentage of nuclear bFGF-positive cells and percentage of cytosolic bFGF-positive cells. The scoring of all cases was confirmed by a board-certified pathologist (SVP).To enrich for chemo-resistant tumor cells, we treated SUM159 and BT549 TN breast tumor cells with doxorubicin at a clinically relevant concentration . DoxorubPrevious studies suggest that continuous chemotherapy selection models promote the growth of cancer stem-like cells \u201329. AccobFGF signaling has been implicated in tumor resistance to targeted therapies \u201315. AccoStudies in Fig.\u00a0To determine which bFGF isoform facilitates chemo-residual tumor cell survival and colony formation in our model, we transfected bFGF shRNA-expressing cells with a vector expressing 18-kDa rat bFGF, 23-kDa rat bFGF, or an empty control vector Fig.\u00a0. The 18-Elevated DNA repair activity is associated with chemo-resistance in many tumors \u201334. To cCS [CS is a downstream target of nuclear bFGF in our TN breast cancer chemo-resistance model, we determined the expression level of DNA-PKCS in chemotherapy-enriched TN tumor cells. Chemo-residual TN tumor cells expressed increased levels of both DNA-PKCS and phospho (Ser-2056)-DNA-PKCS, representing the activated form of DNA-PKCS [DNA-PK is the key protein responsible for non-homologous end joining (NHEJ) of DNA DSBs. Over-expression of bFGF in HeLa cells drives the expression and activation of DNA-PKCS . To deteDNA-PKCS protocol for detecting nuclear and cytosolic bFGF in formalin-fixed, paraffin embedded tissues. In a pilot study, we selected nine patients with TN breast cancer that exhibited an incomplete pathologic response to neoadjuvant chemotherapy treatment. Matched samples from patients with TN breast cancer Fig.\u00a0, obtainePrevious chemotherapy selection models have shown the relevance of cancer stem-like cells in TN breast cancer chemo-resistance , 28. In Novel markers of TN breast cancer chemo-resistance were recently identified by performing microarray on patient tumor samples obtained before and after neoadjuvant chemotherapy treatment . It is iExpression of nuclear versus cytosolic bFGF isoforms is determined by alternative translation pathways. Whereas cytosolic bFGF isoforms are regulated by cap-dependent translation, nuclear bFGF isoforms are regulated by cap-independent translation. Notably, we observed increased protein levels of nuclear but not cytosolic bFGF isoforms in chemo-residual tumor cells. Our data suggest that chemo-residual tumor cells may support cap-independent translation, driving expression of nuclear bFGF and DNA repair. We are currently addressing this important hypothesis. Ultimately, it may be possible to eliminate these chemo-residual tumor cells by targeting the cap-independent translation pathway.Previous studies indicate that tumor resistance to anti-angiogenic therapy is associated with increased expression of cytosolic FGF, which is able to restore tumor angiogenesis \u201315. AccoWe observed that chemo-residual cells, relative to parental SUM159 tumor cells, express increased levels of both the 22-kDa and the 24-kDa nuclear forms of bFGF Fig.\u00a0. In ordeWe also observed that bFGF RNA levels are increased in chemo-residual tumor cells relative to parental tumor cells Fig.\u00a0. IdentifCS as a downstream target of nuclear bFGF in chemo-residual TN tumor cells. DNA-PKCS has previously been implicated in therapy resistance [CS expression/activity [CS expression. Notably, nuclear bFGF did not increase DNA-PKcs mRNA levels, as assessed by real-time PCR (data not shown). Further work is needed to determine the mechanism by which nuclear bFGF increases DNA-PKcs expression/activity. In addition, we are investigating the ability of nuclear bFGF to activate other DNA repair pathways.Our work identifies DNA-PKsistance \u201334. Longactivity . The curCS reduced both the number of chemo-residual tumor cells (Fig.\u00a0We also found that a small molecule inhibitor of DNA-PKlls Fig.\u00a0 and the lls Fig.\u00a0 in our iCS expression/activity in a manner dependent on a nuclear bFGF receptor, a topic of current investigation. This possibility is supported by the literature, which demonstrates that nuclear FGF cooperates with a nuclear FGF receptor to drive gene transcription in neurons [Nuclear-localized EGF receptor is a central determinant of DNA-PK activity . Based o neurons . IdentifOur studies of TN breast cancer samples obtained before and after neoadjuvant chemotherapy treatment demonstrate that a subset of these patients exhibit an increased percentage of nuclear bFGF-positive cells post treatment. These data validate our in vitro findings, showing that chemotherapy enriches for nuclear bFGF-positive cells. All of these patients received either anthracycilne or anthracyline + taxane therapy, and exhibited an incomplete pathologic response. While 56 % of matched samples exhibited increased or sustained nuclear bFGF-positive cells, 44 % of matched samples exhibited reduced nuclear bFGF-positive cells post neoadjuvant chemotherapy. Considering that multiple TN breast cancer subtypes have been identified , we hypoUsing a short-term chemotherapy enrichment model, we demonstrated a critical role for nuclear bFGF in TN breast cancer chemotherapy resistance. Notably, previous continuous chemotherapy selection models did not identify nuclear bFGF as a driver of TN breast cancer chemo-resistance. To begin to demonstrate the relevance of our findings to the clinic, we showed that nuclear bFGF-positive tumor cells were increased or maintained in the majority of patients with TN breast cancer post neoadjuvant chemotherapy treatment. Follow-up studies are needed to determine if nuclear bFGF expression in TN breast cancer cells predicts an incomplete pathologic response and/or future tumor recurrence in patients with TN breast cancer.Chemotherapy remains the core therapy for TN breast cancer and studies of new agents will likely continue to be done in combination with chemotherapy for the foreseeable future. Our studies suggest that nuclear bFGF maintains survival of a chemo-resistant subpopulation that then drives metastatic recurrence. Developing therapies that target this mechanism may be essential for overcoming this chemo-resistance and reducing TN breast cancer-associated mortality."} +{"text": "Left atrial (LA) remodeling in response to cardiovascular and hemodynamic stress may precede atrial fibrillation (AF) and heart failure (HF). We hypothesized that LA systolic synchronous contraction as a functional measure of LA remodeling is deranged in patients with paroxysmal AF and HF.c) and Peak systolic strain rate (Peak Srac) and SD-Time to peak systolic strain rate (Peak-SraL). Wilcoxon-rank sum test (non-parametric) or two sample t-test (parametric) were used for comparison between the groups.We performed a nested case-control analysis with 1:2 matching for 39 cases of paroxysmal AF ) and HF and 78 controls with similar demographic and clinical characteristics at the baseline was significantly higher in the cases compared to controls . Similarly, case group had greater longitudinal dyssynchrony than controls; SD-TP PreA SrL and SD-TP-Peak SraL , systolic circumferential dyssynchrony (SD-TP-PreA SrPatients with paroxysmal atrial fibrillation and heart failure have significantly higher LA circumferential and longitudinal systolic dyssynchrony compared to normal controls.N/A."} +{"text": "Proximal tubule epithelial cells (PTEC) of the kidney line the proximal tubule downstream of the glomerulus and play a major role in the re-absorption of small molecular weight proteins that may pass through the glomerular filtration process. In the perturbed disease state PTEC also contribute to the inflammatory disease process via both positive and negative mechanisms via the production of inflammatory cytokines which chemo-attract leukocytes and the subsequent down-modulation of these cells to prevent uncontrolled inflammatory responses. It is well established that dendritic cells are responsible for the initiation and direction of adaptive immune responses. Both resident and infiltrating dendritic cells are localised within the tubulointerstitium of the renal cortex, in close apposition to PTEC, in inflammatory disease states. We previously demonstrated that inflammatory PTEC are able to modulate autologous human dendritic cell phenotype and functional responses. Here we extend these findings to characterise the mechanisms of this PTEC immune-modulation using primary human PTEC and autologous monocyte-derived dendritic cells (MoDC) as the model system. We demonstrate that PTEC express three inhibitory molecules: (i) cell surface PD-L1 that induces MoDC expression of PD-L1; (ii) intracellular IDO that maintains the expression of MoDC CD14, drives the expression of CD80, PD-L1 and IL-10 by MoDC and inhibits T cell stimulatory capacity; and (iii) soluble HLA-G (sHLA-G) that inhibits HLA-DR and induces IL-10 expression by MoDC. Collectively the results demonstrate that primary human PTEC are able to modulate autologous DC phenotype and function via multiple complex pathways. Further dissection of these pathways is essential to target therapeutic strategies in the treatment of inflammatory kidney disorders. Proximal tubule epithelial cells (PTEC) of the kidney line the proximal tubule downstream of the glomerulus where one of their major physiological roles is the re-absorption of small molecular weight proteins, peptides and glucose via receptors present on the villi of their luminal surface. This localisation and biological role exposes PTEC to numerous challenging stimuli in the event of any up-stream pathology, with excessive protein, glucose, toxins, advanced glycation end products (AGEs) and bacterial products all being able to perturb normal PTEC physiology \u20133. In thDendritic cells (DC) are a powerful population in the innate immune system. They are professional antigen presenting cells (APC) that, following activation in the presence of danger signals, induce and regulate adaptive immune responses. Triggering of DC leads to the up-regulation of co-stimulatory and inhibitory (PD-L1) molecules, production of pro-inflammatory (IL-12) and regulatory (IL-10) cytokines and priming of T cell responses .+ DC, a prominent subclass of the myeloid DC lineage [+ DC surface antigen (Ag) expression, Ag-uptake, cytokine expression and T cell stimulatory function. [In humans, two subclasses of DC that have been frequently reported within diseased kidney are (i) monocyte-derived DC (MoDC), an inflammatory DC population that rapidly develop from blood monocytes in response to inflammation and infection and (ii) CD1c lineage , 10\u201312. unction. .In this current report we extend these findings to characterise the mechanisms of PTEC modulation of DC phenotype and function using primary human PTEC and autologous MoDC as the model system. We focus, in particular, on cell surface-expressed PD-L1, soluble HLA-G (sHLA-G) and intracellular indoleamine-2,3-dioxygenase (IDO) molecules expressed by primary human PTEC, as we have recently demonstrated their immuno-regulatory effects on autologous T and B lymphocytes , 8.Kidney tissue from the healthy portion of malignant and non-malignant nephrectomies and peripheral blood from these same donors three to six months post-nephrectomy were obtained following written informed consent under approval by the Queensland Institute of Medical Research (P293) and Royal Brisbane and Women\u2019s Hospital (2002/011) Ethics Committees.Cortex tissue was dissected from macroscopically/microscopically normal portions of the kidney and processed for PTEC purification within one hour. PTEC were purified following the method of Glynne and Evans and cultPTEC were cultured in DM until 70\u201380% confluence. As previously established [+ immuno-magnetic selection (>95% purity) according to the manufacturer\u2019s instructions. For differentiation into MoDC, purified monocytes were cultured for 5 days in Complete Medium (CM) consisting of RPMI 1640, containing 10% heat-inactivated human AB serum, 100U/ml penicillin, 100\u03bcg/ml streptomycin, 2mM L-glutamine, 1mM sodium pyruvate, 0.1mM non-essential amino acids, 10mM HEPES buffer solution , and 50\u03bcM 2-mercaptoethanol and supplemented with 800U/ml GM-CSF (Miltenyi Biotec) and 1000U/ml IL-4 (Miltenyi Biotec). Cultures were established in either; standard 24-well plates for contact-dependant (CD) cultures or 0.4\u03bcM transwell 24 well plates for contact-independent (CI) cultures, alone (Ctrl-MoDC) or in the presence of IFN-\u03b3-activated, irradiated PTEC (PTEC-MoDC).Peripheral blood mononuclear cells (PBMC) were isolated using Ficoll-Paque Plus density gradient centrifugation . Monocytes were isolated from PBMC by CD14For programmed death ligand-1 (PD-L1) blocking, 10 \u03bcg/mL of anti-human PD-L1 blocking antibody was added to the surface of PTEC and incubated for 2 h. Following incubation, the PTEC wells were extensively washed with 5 changes of warm CM to remove all traces of unbound antibody and the co-cultures were established. To neutralise soluble human leukocyte antigen-G (sHLA-G) and IDO, 10 \u03bcg/mL of anti-human sHLA-G blocking antibody and 1500 \u03bcM/mL IDO inhibitor 1-methyl-tryptophan (1-MT) respectively was added into the PTEC or control wells immediately following co-culture establishment. The effective blocking concentration levels of 1-MT were determined via titration using cell viability and tryptophan-kynurenine ratios monitored by HPLC (data not shown).Cells were labelled with combinations of PE-, FITC-, PerCP-, APC-, PE-Cy7-, V450- and Pacific Orange-conjugated mouse anti-human CD14, CD80, CD86, DC-SIGN (CD209) , PD-L1 (CD274) (Biolegend) and HLA-DR (Invitrogen) antibodies and appropriate isotype controls, using 0.25 \u03bcg of each antibody per staining reaction. Acquisition was performed on a BD FACSCanto II flow cytometer (BD Biosciences) and analysis of flow data was performed using FlowJo 7.6.4 . For viability staining, cells were labelled with a Near-IR dead cell stain kit (Invitrogen) according to the manufacturer\u2019s instructions and analysed by flow cytometry.Culture supernatants were harvested and levels of IL-10 and IL-12p70 were determined using Flow Cytometric Bead Arrays (BD Biosciences) according to the manufacturer\u2019s instructions.+ T cells were isolated from PBMC by negative immuno-magnetic selection using the CD4+ T cell isolation kit II (>95% purity) (Miltenyi Biotec). MoDC separated from PTEC, were plated in triplicate in 96-well plates at 1:2 serial dilutions starting at 2.5x104 cells/well. Allogeneic CD4+ T cell responders were added at 1x105 cells/well. Cells were cultured for 5 days and proliferation was assessed by the addition of 1\u03bcCi 3H-thymidine/well for the last 8 hours of culture.Allogeneic CD4Comparisons between two groups were performed using a two-tailed Wilcoxon signed rank test. Statistical tests were performed using GraphPad Prism 5.0 analysis software . P values \u22640.05 were considered statistically significant.We first undertook contact-dependent and transwell contact-independent parallel co-culture experiments to establish whether PTEC modulation of autologous DC phenotype and function operated through contact or soluble mechanisms.+ monocyte differentiation into MoDC was confirmed by the expression of the DC marker DC-SIGN is one of the definitive features of DC. To identify the mechanism through which autologous PTEC modulate MoDC to down-regulate allogeneic CD4+ T cell responses, allo-MLR assays were established utilising Ctrl- and PTEC-MoDC obtained from CD and CI culture systems. PTEC-MoDC were less effective at stimulating CD4+ T cell responses when compared with their respective Ctrl-MoDC from both CD and CI culture systems. Although PTEC-MoDC from CI cultures stimulated higher levels of T cell proliferation compared to PTEC from CD co-cultures, suggesting a partially contact-mediated mechanism for PTEC suppression PD-L1 expression on PTEC-MoDC . The expFollowing differentiation, the Ctrl- and PTEC-MoDC were harvested and stained for surface antigen expression and the culture supernatants analysed for IL-10 levels. The blocking of sHLA-G reversed HLA-DR expression on PTEC-MoDC, where two out of three donors demonstrated a partial restoration in the expression of this molecule . The expThe levels of IL-10 were partly reduced in all donors in the presence of blocking antibody to sHLA-G . This reBlocking IDO activity in our co-cultures decreased the expression of CD14 in five out of seven donors , suggest+ T cell proliferation in four out of five donors . However, we observed partial restoration of CD4e donors when PTE+ monocytes were differentiated into MoDC in CD or CI culture conditions to identify whether autologous PTEC regulate MoDC differentiation and function by surface expressed molecules (CD mechanism) or by soluble factors (CI mechanism).As we have previously published , the preConfirming our previous findings , all MoD+ T cell responses were also investigated by using Ctrl- and PTEC-MoDC derived from both CD and CI culture systems. Autologous PTEC up-regulated the expression of IL-10 from MoDC through a CD mechanism. The allo-MLR assay showed that PTEC-MoDC from both CD and CI culture systems were less effective at stimulating allogeneic CD4+ T cell proliferation than Ctrl-MoDC. However, a partial restoration in T cell stimulation by PTEC-MoDC from CI cultures compared to PTEC-MoDC from CD cultures suggests that autologous PTEC inhibit the allogeneic-inducing function of MoDC through both CD and CI mechanisms.The mechanism/s of autologous PTEC regulation of MoDC function, including their cytokine profiles and their ability to stimulate allogeneic CD4in vivo. Our blocking studies indicate that a pivotal role of PTEC-expressed PD-L1 is to increase the expression of PD-L1 on autologous MoDC. As PD-L1 is surface expressed, it was logical to conclude that this was one of the primary CD mechanisms responsible for our observed low T cell stimulatory capacity of PTEC-MoDC. However, our functional PD-L1 blocking studies did not support this assumption.Following the identification of CD or CI regulatory mechanisms behind the expression of phenotypic markers and function of MoDC, molecules participating in these mechanisms were investigated. The up-regulation of immuno-regulatory molecule PD-L1 on autologous PTEC was reported in our previous publication . PD-L1 oOur subsequent functional blocking experiments identified IDO as the candidate molecule responsible for this mechanism. The IFN-\u03b3-inducible intracellular enzyme IDO catalyses the breakdown of essential amino acid tryptophan into kynurenine and creates a micro-environment devoid of tryptophan. Tryptophan depletion and the generated kynurenine bi-products have both been recognised as immune-regulators of antigen presenting cells \u201321. We hOur results contribute to this apparent dichotomy by demonstrating that PTEC-produced IDO both up-regulates costimulatory CD80 and inhibitory PD-L1 and regulatory IL-10 on APC. In addition, our results show that PTEC-expressed IDO plays a role in the retention of monocyte marker CD14 on developing MoDC, inhibiting complete monocyte-to-MoDC differentiation and contributing to our observed weak T cell stimulatory capacity of these cells. However, it should be noted from our transwell experiments that IL-10 expression from developing MoDC is contact-dependant, indicating that the soluble effects of tryptophan depletion upon IL-10 induction could be driving an autocrine up-regulation of another, as yet un-identified, immuno-regulatory surface molecule on PTEC.et al did not stimulate PTEC with IFN-\u03b3, which induces the expression of sHLA-G. Functionally, the role of sHLA-G has been demonstrated to be beneficial in various clinical conditions including pregnancy, transplantation and various inflammatory diseases [Our results also implicated another soluble inhibitory molecule, sHLA-G, in the decreased expression of HLA-DR and induction of IL-10 from MoDC. We have demonstrated HLA-G mRNA and intracellular protein up-regulation in our primary PTEC cultures in response to IFN-\u03b3 stimulation (data not shown), with concomitant increases in sHLA-G from the supernatants of these cells . This isdiseases , 26. In diseases and incrdiseases have botIn\u2013vivo these pathways may be cumulative, with the net outcome on DC functional response dependent upon multiple factors including the stage of the disease process, functional competency of the resident PTEC, the cytokine milieu present within the interstitium and the developmental/activation stage of responding DC. Overall however, we believe these mechanisms have evolved to enable PTEC to act as negative regulatory cells to down-modulate DC function within the early inflammatory setting and to control unrestrained inflammation.Collectively, our results confirm that primary human PTEC are able to modulate autologous APC phenotype and function via multiple complex interactive pathways including PTEC-PD-L1 induction of MoDC PD-L1; IDO maintenance of CD14 and induction of CD80, PD-L1 and IL-10 expression on MoDC; and sHLA-G inhibition of HLA-DR up-regulation and induction of IL-10 expression from MoDC. Further dissection of these pathways should enable the development of novel immunotherapy strategies for the treatment of inflammatory chronic kidney disease."} +{"text": "Children with single ventricle physiology require multiple open heart surgeries for palliation, including sternotomies and cardiopulmonary bypass. The reduced morbidity of a catheter-based approach is attractive. We hypothesize real-time multiplanar MRI guidance enables closed-chest percutaneous bidirectional Glenn shunt because of arbitrary-plane imaging capability.Ten swine underwent transcatheter bidirectional Glenn procedures under MRI at 1.5T. An MRI antenna-needle was advanced from the superior vena cava (SVC) into the target pulmonary artery (PA) bifurcation using real-time MRI guidance. A caval-pulmonary sheath introduced endografts. Balloon-expansion secured a proximal end-to-end caval anastomosis that also occluded the azygos, and a distal end-to-side pulmonary anastomosis that preserved blood flow to both branch pulmonary arteries.Real-time MRI needle access of adjacent vessels ."} +{"text": "We obtained the full genome of Middle East respiratory syndrome coronavirus (MERS-CoV) from a camel in Qatar. This virus is highly similar to the human England/Qatar 1 virus isolated in 2012. The MERS-CoV from the camel efficiently replicated in human cells, providing further evidence for the zoonotic potential of MERS-CoV from camels. Betacoronavirus; the closest relative to this virus is bat CoVs clade 2c (Middle East respiratory syndrome coronavirus (MERS-CoV) is a novel coronavirus that can cause severe lower respiratory tract infection in humans , and samples were tested for MERS-CoV by using 2 TaqMan assays: 1 for the envelope (upE) and 1 for the nucleocapsid gene (N), as described previously , eluted in 60 \u03bcL water, and reverse transcribed with the Superscript III First-Strand Synthesis System with random hexamers. The MERS-CoV genome was amplified by using MERS-CoV\u2013specific overlapping primer sets as described previously . Similar to the genome of human MERS-CoV isolates, the genome of camel MERS-CoV isolates contains 10 complete open reading frames (ORFs) , 8 transcription-regulatory sequences, and 2 terminal untranslated regions. The alignment of the camel MERS-CoV with known human MERS-CoVs, including 1 near-complete camel MERS-CoV (NRCE_HKU205) sequence, showed overall nucleotide identities of 99.5%\u201399.9% between camel and human MERS-CoV isolates from different geographic regions.wwwnc.cdc.gov/EID/article/20/8/14-0663-Techapp1.pdf).Phylogenetic analysis of the complete genome clearly showed that MERS-CoV camel/Qatar_2_2014 is highly similar to human MERS-CoV; the closest relative to camel MERS-CoV was England/Qatar1 2012 (99.9% identity) , and it In addition, most amino acid residues critical for receptor binding . After 48 hours, we observed cytopathic changes in cells (320 50% tissue culture infectious dose/mL). After isolation, the passage-3 virus stock was used for all subsequent experiments.To check for adaptive mutations obtained during cell culture, we used 454 deep-sequencing technology to analyze the full-genome sequence as described elsewhere cells with MERS-CoV camel/Qatar_2_2014. After 2 days, virus-induced cytopathic effects were observed in the inoculated cell cultures Figure. We isolated MERS-CoV from the nasal cavity of 1 dromedary camel and demonstrated its infectiousness. Further studies are needed to test whether camels infected at a young age are more likely than adult dromedary camels to excrete infectious virus, possibly because of the MERS-CoV seronegative status of the younger camels. In addition, our results add to recent findings that MERS-CoVs from camels and humans are nearly identical (Middle East respiratory syndrome coronavirus (MERS-CoV) in human hepatoma cells; variable amino acids in the MERS-CoV protein in different isolates."} +{"text": "In order to locate conserved regions in the hdcA gene of Gram-positive bacteria, this article provides a nucleotide sequence alignment of all the hdcA sequences from Gram-positive bacteria present in databases. For further utility and discussion, see \u2329http://dx.doi.org/ 10.1016/j.foodcont.2015.11.035\u232a\u232a The decarboxylation of histidine -carried out mainly by some gram-positive bacteria- yields the toxic dietary biogenic amine histamine (Ladero et al. 2010 \u2329 Specifications tableValue of the data\u2022hdcA gene present at databases.Identification of Gram-positive bacteria \u2022hdcA sequence.Sequence alignment defines conserved and variable regions within the \u2022hdcA conserved regions -flanking variable ones- that can be used to identify histamine-producing species in food matrices by PCR-DGGE.Obtained data allowed to design primers within 1hdcA sequences of the histamine-producing Gram-positive bacteria strains present in Genbank database were aligned using ClustalW software Full-length 2http://www.ncbi.nlm.nih.gov/) database. Full-length hdcA sequences of representative histamine-producing Gram-positive bacteria strains present in database were selected for the alignment (Sequence data were obtained from NCBI (lignment . The seq"} +{"text": "Listeria monocytogenes engineered to express the tumor-associated antigen mesothelin. CRS-207 boosts responses against mesothelin and is unique in its capacity to stimulate both innate and adaptive immunity by activating T cells and NK cells. In a recently completed Phase II study, the CY/GVAX plus CRS-207 combination resulted in statistically-significant improvement of overall survival (OS) compared to CY/GVAX alone .A heterologous prime-boost vaccination strategy using GVAX pancreas vaccine and CRS-207 is showing promise in patients with metastatic pancreatic adenocarcinoma (PDA). GVAX is composed of lethally-irradiated, allogeneic pancreatic cancer cells modified to express GM-CSF and induces a broad response against multiple tumor antigens. GVAX is given with low-dose cyclophosphamide (CY) to inhibit regulatory T cells. CRS-207 is live-attenuated rd+ line) and 90 subjects into an exploratory cohort of patients with only one prior treatment regimen for metastatic disease (2nd line). Subjects will be randomized in a 1:1:1 ratio to receive either 2 doses of CY/GVAX and 4 doses of CRS-207 (Arm A), 6 doses of CRS-207 (Arm B) or physician's choice of select single-agent chemotherapy (Arm C). The primary objective is to compare OS in the primary cohort between Arms A and C. Secondary/exploratory objectives include: comparison of OS in both primary and exploratory cohorts between all treatment arms, assessment of safety and clinical responses (tumor assessments and CA19-9 levels) and correlation of Lm- and mesothelin-specific T cell and other immunological responses with OS, progression-free survival and best overall response. .This is a Phase IIb study comparing CY/GVAX and CRS-207 to chemotherapy or to CRS-207 alone in adults with previously-treated metastatic PDA. Subjects will be enrolled in two cohorts: 150 subjects into a primary cohort of patients with at least two prior treatment regimens for metastatic disease (3"} +{"text": "Cryo plus anti-CTLA-4 therapy induces antigen-specific clonal T cell expansion, enhanced survival, and long-term anti-tumor immunity in mice . We rece\u00ae DNA deep sequencing platform and ImmunoSEQ\u2122 software. Clones comprising \u22650.01% of sample DNA were analyzed, and results are reported descriptively.In a pilot study, women with ESBC were treated with cryo 7-10d before mastectomy (6 pts), single-dose Ipi (10 mg/kg) 8-15d before mastectomy (6 pts), or cryo+Ipi (6 pts). Peripheral blood mononuclear cells (PBMCs) and tumor tissue were obtained pre-mastectomy (immediately preceding cryo and/or 1-5d after Ipi), and at mastectomy. T cell repertoire analysis was conducted on extracted DNA using an Illumina2-104 amplicons of cases. 21% of all TIL clones were detectable in time-matched PBMC, whereas 16% of expanding (\u2265102) TIL clones were detectable in time-matched PBMC.Cryo with or without Ipi was associated with decreases in absolute TIL count . However, cryo+Ipi was associated with the greatest expansion of TIL clones across the range of 102-104 range can be used to predict recurrence-free survival.Cryo plus Ipi expands more TIL clones than either strategy alone. Therapy-associated clonal expansion may be difficult to detect in PBMCs. These data highlight the potential importance of TIL repertoire analysis for the monitoring of pts treated with cryo and/or Ipi in the preoperative setting. In a follow-up randomized study, we will evaluate whether TIL clonal expansion across the 10"} +{"text": "We investigated a polyethylene glycol non-precipitable low-density lipoprotein (LDL) subfraction targeted by IgG and the influence of statin therapy on plasma levels of these small LDL-IgG-immune complexes (LDL-IgG-IC). LDL-subfractions were isolated from 6 atherosclerotic subjects and 3 healthy individuals utilizing iodixanol density gradient ultracentrifugation. Cholesterol, apoB and malondialdehyde (MDA) levels were determined in each fraction by enzymatic testing, dissociation-enhanced lanthanide fluorescence immunoassay and high-performance liquid chromatography, respectively. The levels of LDL-IgG-IC were quantified densitometrically following lipid electrophoresis, particle size distribution was assessed with dynamic light scattering and size exclusion chromatography. The influence of simvastatin (40 mg/day for three months) on small LDL-IgG-IC levels and their distribution among LDL-subfractions were investigated in 11 patients with confirmed coronary artery disease (CAD). We demonstrate that the investigated LDL-IgG-IC are small particles present in atherosclerotic patients and healthy subjects. In vitro assembly of LDL-IgG-IC resulted in particle density shifts indicating a composition of one single molecule of IgG per LDL particle. Normalization on cholesterol levels revealed MDA values twice as high for LDL-subfractions rich in small LDL-IgG-IC if compared to dominant LDL-subfractions. Reactivity of affinity purified small LDL-IgG-IC to monoclonal antibody OB/04 indicates a high degree of modified apoB and oxidative modification. Simvastatin therapy studied in the CAD patients significantly lowered LDL levels and to an even higher extent, small LDL-IgG-IC levels without affecting their distribution. In conclusion simvastatin lowers levels of small LDL-IgG-IC more effectively than LDL-cholesterol and LDL-apoB levels in atherosclerotic patients. This antiatherogenic effect may additionally contribute to the known beneficial effects of this drug in the treatment of atherosclerosis. Development and progression of atherosclerosis are associated with elevated levels of LDL and oxidized LDL (oxLDL) . A hallmIn autoimmune disease the removal of IC is dependent on size and structure of the complex. Very large and large IC are delivered mainly to the spleen and liver whereas small soluble IC persist in circulation and are likely to penetrate the endothelial barrier of blood vessels inducing atherogenic effects by triggering inflammatory processes . LDL autTo our knowledge only two studies report on the effect of statin treatment on PEG precipitable IC , 21. TheAnother approach to characterize circulating LDL-IgG-IC from patients with coronary atherosclerosis was to primary isolate the IC from serum by affinity chromatography using anti-human IgG-agarose. The isolated IC were further purified by salt gradient ultracentrifugation. The authors suggest that multiple-modified desialylated LDL is the circulating autoantigen for anti-LDL autoantibodies .Our novel approach in this study was to try to identify and characterize LDL-IgG-IC in LDL-subfractions after an initial purification step by ultracentrifugation. Ultracentrifugation separates LDL-IgG-IC from unbound proteins (free IgG) that would disturb subsequent analytical procedures. The use of single-step iodixanol gradient ultracentrifugation assures a very high level of preservation of particle integrity and enabled us to examine the presence of LDL-IgG-IC in supernatants from PEG precipitation experiments. In a second approach utilizing the classical two-step salt gradient ultracentrifugation we focused on the effect of statin therapy on the level and distribution of these specific LDL-IgG-IC in patients with CAD.The following questions were addressed in the present study: (i) Is there a particular LDL-subfraction which is preferentially targeted by IgG? (ii) Is there a correlation between LDL-IgG-IC levels and the MDA content of a given LDL-subfraction (indicating oxidative modification)? (iii) Do these LDL-IgG-IC have a defined size and antigen/antibody ratio? (iv) Are these LDL-IgG-IC precipitable with PEG? (v) How does simvastatin therapy affect the level and distribution of these LDL-IgG-IC?The study was approved by the local ethics committee (Ethikkommission der Medizinischen Universit\u00e4t Graz). Approval number: 19\u2013327 ex 07/08. The study subjects were enrolled at the Medical University of Graz . The study was approved by the local ethics committee and written informed consent was obtained from all study subjects.The study group included 3 female and 3 male patients (age range 60\u201392 years). The 6 patients suffered from peripheral artery occlusive disease (PAOD) with restenosis and were considered high-grade atherosclerotic. Blood collected from these patients and from 3 healthy controls was used for isolation of LDL-subfractions following single-step iodixanol gradient ultracentrifugation.This study group included 11 male statin-naive patients aged 35\u201375 years with confirmed CAD (assessed by coronary angiography). Subjects were treated with simvastatin (40 mg/day) for a period of three months. LDL-subfractions were isolated applying salt gradient ultracentrifugation.All materials were from Sigma-Aldrich; Austria, unless indicated otherwise.EDTA-plasma or serum was collected after an overnight fast. In group B blood was drawn at baseline and after 3 months of simvastatin therapy as described previously .A: Iodixanol density gradient ultracentrifugation: Lipoprotein subfractions were prepared from PAOD patients (group A) and healthy control subjects by self-generated iodixanol gradient single-step ultracentrifugation using a modification of the protocols of Davies et al. and Yee et al. [\u00ae density gradient medium) and centrifuged in open-top polyclear tubes at 16\u00b0C and 36,000 rpm for 67 h . A total of 25 fractions (the bottom fraction was excluded) were isolated in steps of 3.0 or 1.0 mm by a piston gradient fractionator . Lipoprotein fractions were analysed for density , total cholesterol , apoB, dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA) and LDL-IgG-IC content .e et al. , 27. In B: Salt density gradient ultracentrifugation: Lipoprotein fractions (very low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), LDL and high-density lipoprotein (HDL)) from CAD patients (group B) were prepared from 6 mL of plasma by preparative ultracentrifugation. Subsequently LDL (density: 1.019\u20131.065 g/mL) was further fractionated into six density subfractions by equilibrium density gradient centrifugation [fugation , 28. Denfugation , 29.MDA was determined according to a previously described high-performance liquid chromatography (HPLC) method after derivatization with 2,4-dinitrophenylhydrazine (DNPH) . For alk\u00ae West Femto Max. Sensitivity Substrate was used for blot development. Detection and analysis of signals were performed with the ChemiDoc MP System and software Image Lab 4.0.1 build 6 and TotalLab Array v2009 . Furthermore, we performed electrophoresis of LDL-subfractions rich in LDL-IgG-IC and the major LDL-subfraction in 3% polyacrylamide gels under conditions described elsewhere [Lipoprotein subfractions were separated on 1% agarose gels as described elsewhere . Lipoprolsewhere .\u00ae microfiltration apparatus was used to transfer lipoprotein subfractions onto nitrocellulose membranes. The samples were diluted in Tris-buffered saline and 100 \u03bcL were applied in triplicates. The blots were processed according to the protocol of the electrophoresis experiments.A Bio-DotIn a first approach to assemble small LDL-IgG-IC in vitro we used the most prominent LDL-subfraction (150 \u03bcL of fraction #8 (cholesterol concentration: 380 mg/dL) derived from the iodixanol gradient centrifugation. This LDL-subfraction did not contain detectable LDL-IgG-IC. The incubation was carried out using a biotinylated IgG antibody targeting human apoB-100 . Within 1 min the antibody solution was added dropwise to the LDL solution under gentle mixing. Further incubation was carried out at room temperature (RT) for 30 min on a roller mixer. Subsequently the whole mixture was applied to a second iodixanol gradient ultracentrifugation step. The harvested fractions were diluted 1:150 in 0.01 mol/L phosphate buffer, pH 7.4 and 100 \u03bcL were used to coat microtiter plates. After washing, blocking and incubation with Eu-labelled Streptavidin fluorescence counts were recorded by a DELFIA Research Fluorometer as described previously . Density2 fragment of human IgG in order to calculate the density shift resulting from binding of the antibody to the IgG of LDL-IgG-IC. Incubations were performed with different amounts of the HRP-antibody 2 fragment goat anti-human IgG, F(ab')2 fragment specific; Jackson ImmunoResearch Europe Ltd). The HRP-antibody concentration was adjusted to achieve an antibody:LDL particle ratio of 1:100, 1:1,000 and 1:10,000; no antibody was added in the control. After a second ultracentrifugation separation run the collected fractions (1.0 mm steps) were analysed for HRP activity, cholesterol content and the amount of LDL-IgG-IC . The density difference of subfractions representing peak levels of HRP activity and LDL-IgG-IC (control) was calculated.In a second approach we used the LDL-subfraction that showed the highest levels of LDL-IgG-IC. Approximately 5% of this LDL-subfraction represented LDL-IgG-IC and about 95% was uncomplexed LDL. This LDL-subfraction was incubated with an HRP-antibody directed against the F(ab')Precipitation of LDL-IC was performed by incubation of equal volumes of serum and PEG solution in accordance to frequently applied protocols , 32, 33.\u00ae 20, pH 7.4) using a microplate washer . Subsequently 300 \u03bcL I-Block\u2122 Blocking Reagent was applied for 1.5 h at RT. The dialysed LDL-subfractions were diluted according to the protein content to yield 2 \u03bcg LDL/200 \u03bcL , transferred to the wells and incubated for 2 h at RT . Subsequently, after washing 200 \u03bcL polyclonal biotinylated goat anti-human apoB-100 were added per well and the dishes were incubated for 1 h at RT (shaking at 300 rpm). Finally after appropriate washing 200 \u03bcL DELFIA Eu-labelled Streptavidin was added and incubated at RT for 1 h (shaking at 300 rpm). After 6 washing cycles the plates were incubated with 200 \u03bcL DELFIA enhancement solution for 5 min on a shaker . Fluorescence counts were recorded by a DELFIA Research Fluorometer as described previously [DELFIA plates were coated for 12 h (4\u00b0C) with 1 \u03bcg anti-human IgG \u03b3-chain antibody purified from rabbit antiserum diluted in 200 \u03bcL 0.01 mol/L phosphate buffered saline . The plates were rinsed three times with 500 \u03bcL wash buffer was used to detect oxidatively modified apoB in LDL-IgG-IC according to a procedure described elsewhere . An LDL-High performance gel permeation chromatography with a TSK 5000 PW column was performed to elucidate the size distribution of an LDL-IgG-IC enriched subfraction according to the method described by Carroll and Rudel . FractioDynamic light scattering was applied to assess particle sizes using a Zetasizer 3000 HS . An LDL-subfraction rich in LDL-IgG-IC (pooled LDL-IgG-IC peak fractions) was measured prior and after absorption of LDL-IgG-IC by beadBALL-Protein G according to the manufacturer's protocol. The complete removal of LDL-IgG-IC was confirmed by electrophoresis and immunodetection as described above.ANOVA with post-hoc comparisons performed according to Scheff\u00e9 was applied to assess significant differences of LDL-IgG-IC distribution (study group B) . Shapiro-Wilks test was used to confirm normal distribution and Levene's test was applied to assess the equality of variances. For analysis of the effect of simvastatin therapy on cholesterol or apoB levels and LDL-IgG immune complexes (two-sided test) the paired-samples T test was used. For estimation of differences of LDL-IgG-IC levels normalized to cholesterol or apoB levels a two-sided unpaired T test was used. A p-value < 0.05 was considered significant.The density gradient of the self-generated iodixanol single-step ultracentrifugation (range of LDL indicated) is shown in LDL-cholesterol concentration peaks in subfraction #8 (density: 1.028 g/mL). Interestingly, the peak of LDL-IgG-IC (LDL-subfraction #9 and #10) lags one or two subfractions behind the cholesterol peak fraction. The mean MDA concentration in the PAOD patients was 3.6 \u00b1 1.2 nmol/mL. The mean MDA level of control subjects (n = 10) was markedly lower (1.86 \u00b1 0.48 nmol/mL). In 3 out of 6 PAOD patients, MDA levels peaked, like LDL levels, in subfraction #8. Thus, the dominant LDL-subfraction apparently contains high or the highest (absolute) amounts of oxidatively modified LDL. Moreover, if normalized to cholesterol levels, the MDA concentrations of subfractions containing the majority of LDL-IgG-IC (combined subfractions #9 and #10) were 1.7 \u00b1 0.3 fold higher compared to that of combined subfractions #7 and #8. In addition, modifications of LDL from LDL-IgG-IC were detected with an oxidation-specific antibody (see below).2-antibody (MW: 190 kDa) targeting IgG of the small LDL-IgG-IC resulted in formation of LDL-IgG-F(ab')2-IC and a similar shift of ~6\u20137 mm in the centrifugation tube (fractionation step size: 1.0 mm), which corresponds to a similar density shift of ~15 mg/mL and the identical fraction after removal of small LDL-IgG-IC (22.3 \u00b1 6.2 nm). Approximately 5% of LDL represent small LDL-IgG-IC (enrichment factor ~5) in these fractions. These results indicate the absence of large (multimeric) LDL-IgG-IC.Agarose and polyacrylamide gel electrophoresis displayed migration of LDL-IgG-IC as distinct bands indicating a narrow particle size range. Using 3% polyacrylamide slab gels LDL-IgG-IC present in #11, #12, #13 and #7 (LDL peak fraction) showed a very similar electrophoretic mobility (indicating similar particle size) .During size exclusion chromatography of an LDL-IgG-IC enriched subfraction LDL and LDL-IgG-IC eluted from the column with a very similar distribution profile . Dot bloPurified small LDL-IgG-IC (retained by protein A/G beads) and residual LDL were prepared from an LDL-subfraction rich in small LDL-IgG-IC. We compared the levels of oxidatively modified LDL by means of the monoclonal antibody OB/04 (directed against oxidation-specific epitopes) in these fractions. The levels of oxidatively modified LDL were increased ~70-fold in small LDL-IgG-IC compared to unbound LDL . We therPEG 8000 did not precipitate small LDL-IgG-IC from serum samples. Small LDL-IgG-IC are still present in the respective supernatants . HoweverAgarose gel electrophoresis of the 6 isolated subfractions and immunodetection of LDL-IgG-IC showed an electrophoretic mobility similar to subfractions derived from the iodixanol gradient ultracentrifugation. Determination of small LDL-IgG-IC levels with DELFIA we calculated that ~0.1\u20131% (combined LDL-subfractions) represent small LDL-IgG-IC. Due to the very work-intensive analytical process and strong individual variations of small LDL-IgG-IC levels it was impossible to work out significant differences between study groups.The assessment of autoimmunogenicity of lipoproteins is complex and multi-faceted. However, it is generally accepted that LDL-IgG-IC bear atherogenic potential as the modification of LDL and the presence of the IgG-Fc region are both considered as driving factors behind foam cell formation , 9, 10. 2-IC results in density shifts that perfectly fit to an LDL/IgG ratio of 1:1. Upon binding of one single IgG the protein mass percentage of LDL (dominant fraction: 22.2%) is increased by 4.4%. Based on experimental and emulsion particle model data particle density would be raised by 13.2, 15.0 or 16.1 mg/mL [LDL-IgG-IC particle integrity (presence of both apoB and IgG) was verified by DELFIA and immunodetection following lipid electrophoresis. The distribution patterns of LDL-IgG-IC and cholesterol are very similar in shape but show an offset of ~2 fractions. A similar offset was described for affinity purified LDL-IgG-IC. Tertov et al. attributed the observed density shift to the binding of IgG and/or desialylation of LDL . To our .1 mg/mL , 38. TheBased on our results and published experimental data we conclude that the investigated subset of LDL-IgG-IC most likely originates from the dominant LDL-subfractions. A priori we expected a higher IC formation tendency of more dense LDL-subfractions as these fractions are considered to be highly atherogenic \u201343. Our The important question which modification of LDL is primarily responsible for IgG targeting and formation of IgG-IC is still unanswered. The investigated small LDL-IgG-IC contain increased amounts of structurally modified apoB due to chemical modification of the protein and/or the lipid components of LDL. However, IgG is detectable within the density range of VLDL, IDL, LDL and Lp(a) probably indicative of an immunogenic role of modified apoB. We assume that modified apoB is a recognized pathogen able to initiate an adaptive immune response. Due to its size and plasma half-life we consider oxLDL as a likely candidate to act as a trigger particle of the immune response against modified apoB. However, the IgG antibodies may result from a polyclonal response targeting a variety of LDL associated epitopes.+ T cells implies that regulatory T cells (Tregs) exist to control these apoB-reactive T cells. When the balance between apoB-reactive effector T cells and Tregs is shifted in favour of the effector T cells, a local loss of tolerance against LDL in the plaque could aggravate atherosclerosis [Several native and MDA-modified peptide sequences in apoB have been identified that were recognized by autoantibodies in human plasma. The immune responses against native apoB may be of equal importance as the presence of apoB reactive CD4clerosis .Migration of LDL-IgG-IC in 1% agarose and 3% polyacrylamide slab gels showed an electrophoretic mobility similar to uncomplexed LDL particles indicating the absence of multimeric structures. Gel permeation chromatography and DLS analysis confirmed our hypothesis about the small size and monomeric (non-latticed) structure of LDL-IgG-IC. As determined by Gutierrez at al. the intensity-averaged diameter of a dimeric LDL-IgG-IC is ~34 nm and ~23 nm for monomeric LDL . ParticlIn our PEG experiments the precipitated cholesterol amounts were in good agreement with published values \u201319. HoweOne major goal of this study was to examine if small LDL-IgG-IC present in LDL-subfractions would respond to statin treatment. In our setting simvastatin was highly effective in lowering small LDL-IgG-IC without affecting their distribution. Total LDL-IgG-IC levels were reduced by 62.7% whereas the reduction of LDL-cholesterol and LDL-apoB was 29.7% and 26.0%, respectively. So far it has not been shown that statin treatment decreases LDL-IgG-IC to a higher extent than LDL , 22, 23.Antibodies of IgM subclass to phosphorylcholine and oxLDL are protective factors for atherosclerosis in patients with hypertension . A cliniKobayashi et al. reported that IgM anti-oxLDL antibodies recognize the oxidized lipid moiety and IgG anticardiolipin antibodies recognize \u03b22-glycoprotein I (\u03b22GPI) complexed with oxLDL. Both types of antibodies bind to oxLDL/\u03b22GPI complexes, but their roles on atherogenesis are opposite. The anti-oxLDL antibodies are anti-atherogenic and anticardiolipin antibodies in antiphospholipid syndrome (APS) are atherogenic. The authors assume that IgG anti-\u03b22GPI antibodies contribute to lipid metabolism (housekeeping of oxLDL by macrophages) and that IgM anti-oxLDL antibodies are antiatherogenic. The oxLDL/\u03b22GPI immune complexes may be internalized via Fc\u03b3RI on macrophages .It has been shown that oxLDL forms a stable and non-dissociable complex with \u03b22GPI and that IgG anti-\u03b22GPI autoantibodies are able to recognize this complex, thus facilitating macrophage-derived foam cell formation in patients with APS. However, the immunopathological mechanisms of oxLDL/\u03b22GPI complexes in promoting foam cell formation are not fully understood. Zhang et al. demonstrated that toll-like receptor 4 plays an important role in the process of oxLDL/\u03b22GPI/anti-\u03b22GPI complex-induced transformation of macrophages to foam cells, which may accelerate the development of atherosclerosis in the setting of APS. It was also shown that \u03b22GPI alone functions as an antiatherogenic protein by preventing the foam cell formation induced by oxLDL .High levels of oxLDL/\u03b22GPI complexes and anti-complex IgG as well as IgM have been reported in systemic lupus erythematosus (SLE) . The titNowak et al. reported elevated serum concentration of IgG anti-oxLDL-\u03b22GPI antibodies and IgM anti-oxLDL-\u03b22GPI antibodies in the SLE group compared to the controls. There was a statistically significant positive correlation between LDL concentration and anti-oxLDL antibody concentration in the SLE group .+ T cells and the ensuing T cell activation leads to cytokine secretion that can promote macrophage activation and inflammation. Once T cells are activated toward native apoB epitopes, they may support B cells recognizing native LDL, apoB, or lipids such as phosphocholine (PC), but also oxidatively modified epitopes such as MDA-apoB or oxPC [According to Hansson and Hermansson antigen-presenting cells (APC) take up native and modified LDL by LDL receptors and scavenger receptors, respectively. Uptake of IgG-IC could also be mediated by Fc\u03b3-receptors. Presentation of peptides derived from apoB are recognized by CD4 or oxPC .Unexpectedly, Li et al. demonstrated that anti-oxLDL IgG immune therapy is capable of modulating macrophage pro-inflammatory activity through delivery of dominant inhibitory Fc\u03b3R-signaling in vitro, and that it reduces inflammation and improves insulin sensitivity. The inhibitory effect is mediated through LDL-IgG-IC formation and is dependent on the antibody Fc fragment and Fc\u03b3RII on responsive cells to transduce inhibitory intracellular signaling .There exists strong evidence that Fc\u03b3R play an important role regarding atherogenic potential of immune complexes. Fc\u03b3R bind IgG monomers or IgG-IC. The activating receptors, Fc\u03b3RI (CD64) and III (CD16), are expressed in both humans and mice, whereas Fc\u03b3RIIa and IV are expressed in humans and mice, respectively. Activating Fc\u03b3R contain cytosolic immunoreceptor tyrosine-based activation motifs that recruit kinases and phospholipases to promote inflammatory responses. The inhibitory receptor, Fc\u03b3RIIb (CD32), is expressed on B cells and phagocytes. Fc\u03b3RIIb has a cytosolic immunoreceptor tyrosine-based inhibition motif that recruits phosphatases for dampening of responses. It has been shown that, as IC levels rise, with the associated increase in activating Fc\u03b3R occupancy, macrophages shift from a pro- to anti-inflammatory phenotype, as evidenced by reduced IL-12 and increased IL-10 production .Zhu et al. showed that deficiency of CD16 in the hyperlipidemic apoE-knockout mouse model showed attenuated atherosclerotic lesions, reduced foam cell formation, without affecting the expression of other scavenger receptors. It was demonstrated that CD16 recognized MDA epitopes in MDA-LDL and CD16-MDA-LDL interaction resulted in induction of pro-inflammatory cytokine and chemokines .Asciutto et al. reported recently that low levels of IgG autoantibodies against the native or MDA-modified apoB peptide p210 are associated with an increased risk of cardiovascular death in patients undergoing carotid endarterectomy . The actOxLDL is poorly defined and neo-eptiopes are continually formed and degraded during the oxidation process so the density shift upon binding of \u03b22GPI to in vitro oxLDL is unpredictable. However, we cannot exclude the presence of \u03b22GPI in the investigated small LDL-IgG-IC. Theoretically, the density shift observed for one IgG molecule would be achieved upon binding of 3 \u03b22GPI molecules per LDL particle. As the binding of \u03b22GPI depends on negative charges of the binding partner LDL may appear as multiple target. Binding of IgG would shift the particle into small dense fraction or even out of LDL density range. However, we calculated that one single IgG would transport LDL particles from the dominant fraction exactly to the position where small LDL-IgG-IC have been detected. The presence of additional proteins in small LDL-IgG-IC would markedly affect its size and result in a more diffuse distribution due to compositional heterogeneity.Bancells et al. analysed the proteome of electropositive LDL and electronegative LDL in blood of healthy subjects. LC-ESI MS/MS analysis of both LDL fractions identified up to 28 different proteins. Immunoglobulin lambda chain was detected in electronegative LDL which showed a higher content of most minor proteins .Calculations based upon older data regarding the distribution of \u03b22GPI among density fractions revealed that ~5% of total LDL is bound with one molecule of \u03b22GPI or ~1% is bound by 5 molecule of \u03b22GPI. In the studied healthy subjects ~2% of total \u03b22GPI was found associated with the LDL fraction . In contSeveral studies have shown that IgG anti-oxLDL/\u03b22GPI antibody levels are higher in SLE patients than in healthy controls, and even higher in SLE patients with APS as compared to the ones without APS. Their correlation with atherosclerotic disease in patients with autoimmune diseases is still under investigation. The appearance of antibodies to \u03b22GPI is associated with both SLE and APS. Circulating oxLDL/\u03b22GPI IC and corresponding antibodies are found in sera of patients. OxLDL is an important factor in the acceleration of atherosclerosis in SLE and APS patients. The reason that it may be an accelerating factor is that while oxLDL is quickly removed from the circulation, oxLDL/\u03b22GPI IC that form in these diseases due to the increased production of the antibodies persist for prolonged period of time, significantly increasing macrophage activation and foam cell formation. Immunostaining showed co-localization of oxLDL and \u03b22GPI in atherosclerotic lesions supporting the notion that these complexes are deposited in the lesions and are atherogenic .Considering this overall context the physiological and/or pathophysiological role (atherogenicity) of small LDL-IgG-IC appears unpredictable since their plasma residence time and susceptibility to undergo further modifications are presently unknown and may depend on various interacting factors.Our results show that the analysed small LDL-IgG-IC consist of one single IgG molecule per LDL particle and are not precipitable with PEG. The levels of small LDL-IgG-IC are low if compared to PEG precipitated IC. A prevalence of more dense LDL particles to form LDL-IgG-IC was not observed. IgG-IC of VLDL, IDL and Lp(a) were also detectable in lipoprotein subfractions. The primary autoimmunogenic epitope of small LDL-IgG-IC may be related to oxidative modification of apoB similar to that seen in human atherosclerotic plaques. Simvastatin reduced small LDL-IgG-IC levels more effectively than LDL-cholesterol and LDL-apoB levels and may therefore display therapeutic qualities beyond the lipid-lowering effect.S1 FigA). DELFIA setup to determine apoB (B). Linearity and imprecision profile of the LDL-IgG-IC DELFIA assay. The assay system shows good linearity over a range from 0.1\u201350 \u03bcg/mL . The diagram displays sample counts per second (cps) vs. coefficient of variation (CV) (C).DELFIA setup to determine LDL-IgG-IC ((TIF)Click here for additional data file.S2 FigA), human plasma (B) and an HRP-conjugated antibody (C) were fractionated following ultracentrifugation. The isolated fractions were transferred to nitrocellulose (A) or microtiter plates (B and C). Human IgG was detected by a specific antibody (A and B) and HRP-activity was measured directly (C). The fractionation step size of the displayed fractions was 3.0 mm. The results indicate that free (unbound) IgG molecules are detectable in subfractions \u2265 #16. This observation ensures that the LDL-subfractions that generally contain the fraction of small LDL-IgG-IC are free from unbound human IgG.Purified human IgG ((TIF)Click here for additional data file.S3 FigA). Electrophoresis of LDL-IgG-IC and free (unbound) human IgG (B).A representative distribution pattern of human IgG among isolated subfractions (Std: control LDL-IgG-IC fraction) is shown ((TIF)Click here for additional data file.S4 FigAffinity purified small LDL-IgG-IC (bound) and residual LDL were prepared from an LDL-subfraction rich in small LDL-IgG-IC. DELFIA counts of apoB levels were determined (setup shown in (TIF)Click here for additional data file.S5 FigA). Immunodetection of LDL-IgG-IC. Representative blots (prepared from 1% agarose gels) from the same CAD patients illustrate that the major concentrations of small LDL-IgG-IC are located in the more dense LDL subfractions (LDL4\u2014LDL6) (B). Total amounts and particle ratio of small LDL-IgG-IC. Values represent the total amounts of small LDL-IgG-IC expressed as apoB mass in evaluable patients (n = 8) prior and after statin treatment (C). ApoB mass and particle ratio of small LDL-IgG-IC were calculated based on DELFIA data . An averaged apoB DELFIA calibration curve was used to convert fluorescence counts from the LDL-IgG-IC DELFIA into apoB mass. Detection antibodies used in the DELFIA procedures applied for small LDL-IgG-IC and apoB determination are identical (see: LDL (density: 1.019\u20131.065 g/mL) isolated from 6 mL plasma by preparative salt gradient ultracentrifugation was subsequently fractionated into six subfractions. DELFIA counts (representing LDL-IgG-IC) were converted into a percent value. For each subject and condition (pre-statin and post-statin) the fluorescence counts obtained for LDL 1\u20136 were summed and set to 100%. The distribution of small LDL-IgG-IC is presented as boxplot including all patients (n = 11) pre- and post-statin treatment. Boxplot displays minimum, lower quartile, median, upper quartile and maximum. ANOVA revealed significant differences ; \u00a7\u00a7p < 0.01 (respective LDL-subfraction vs. LDL-subfraction #5)) Click here for additional data file."} +{"text": "Stimulated CD4+ T cells act as helper cells for cytotoxic CD8+ T cells to kill tumor cells by mediating strong proliferation and licensing DCs, increasing their presentation capacity. Tumors bearing MHC-II molecules can also be directly destroyed by cytotoxic CD4+ T cells. As an efficient anti-tumor response strongly depends on the interplay of DCs, CD4+ and CD8+ T cells, these three cell types are considered to be essential for successful immunotherapy design.In recent years, activation of the patient's immune system to defend against tumors was demonstrated to be a promising alternative strategy to classical cancer treatments. Dendritic cells (DCs) can present antigens on MHC-II and -I leading to the activation of CD4in vitro transcribed (ivt) RNA. Subsequently, peripheral blood lymphocytes were primed by DCs expressing TAAs on MHC-I and -II enabling activation and interaction of CD4+ and CD8+ T cells. By this approach, we were able to induce and identify TAA-specific CD4+ and CD8+ T cells in the same experiment.In clinical trials, DCs were either endogenously or exogenously loaded with tumor antigens for the presentation on MHC-I rather MHC-II. As the antigen presentation pathways differ, a simultaneous loading of MHC-I and -II was suboptimal. To overcome this obstacle, we used a signaling sequence (CrossTAg) to force MHC-II cross-presentation of tumor-associated antigens (TAAs) encoded by ivtRNA compared to conventional TAA-ivtRNA was shown in the humanized NOD/scid IL2Rgnull (NSG) mouse model. NSG mice were engrafted with human peripheral blood mononuclear cells (PBMC) and vaccinated twice with DCs either transfected with CrossTAg-TAA-ivtRNA or conventional TAA-ivtRNA. Reisolated PBMC of mice vaccinated with CrossTAg-TAA-ivtRNA transfected DCs showed higher numbers of antigen-specific CD8+ T cells and stronger activation/cytotoxic activity against tumor cells.In addition, superior induction efficiency of DCs loaded with CrossTAg-TAA-+ and CD4+ T cells can be induced side by side allowing interactions and T cell help.These in vitro and in vivo data illustrate the benefits of loading DCs with CrossTAg-linked target antigens as CD8"} +{"text": "Higher-order chromatin structure is often perturbed in cancer and other pathological states. Although several genetic and epigenetic differences have been charted between normal and breast cancer tissues, changes in higher-order chromatin organization during tumorigenesis have not been fully explored. To probe the differences in higher-order chromatin structure between mammary epithelial and breast cancer cells, we performed Hi-C analysis on MCF-10A mammary epithelial and MCF-7 breast cancer cell lines.Our studies reveal that the small, gene-rich chromosomes chr16 through chr22 in the MCF-7 breast cancer genome display decreased interaction frequency with each other compared to the inter-chromosomal interaction frequency in the MCF-10A epithelial cells. Interestingly, this finding is associated with a higher occurrence of open compartments on chr16\u201322 in MCF-7 cells. Pathway analysis of the MCF-7 up-regulated genes located in altered compartment regions on chr16\u201322 reveals pathways related to repression of WNT signaling. There are also differences in intra-chromosomal interactions between the cell lines; telomeric and sub-telomeric regions in the MCF-10A cells display more frequent interactions than are observed in the MCF-7 cells.We show evidence of an intricate relationship between chromosomal organization and gene expression between epithelial and breast cancer cells. Importantly, this work provides a genome-wide view of higher-order chromatin dynamics and a resource for studying higher-order chromatin interactions in two cell lines commonly used to study the progression of breast cancer.The online version of this article (doi:10.1186/s13059-015-0768-0) contains supplementary material, which is available to authorized users."} +{"text": "The phospholipid lipid phosphatidylserine (PS) normally resides in the inner plasma membrane leaflet in most mammalian cells, including tumor and tumor associated vascular cells. Inducers of cellular stress, such as hypoxia and oxygen radicals encountered in tumors, and treatments by cytotoxic therapies promote PS relocation to the outer leaf of the plasma membrane. In tumors this re-localization allows PS recognition by a number of receptors on myeloid and lymphoid cells in the microenvironment, promoting tumor growth and metastatic disease through the development of an immunosuppressive environment. Currently the PS-targeting antibody bavituximab is being used to treat patients with solid tumors in multiple late-stage clinical trials. Bavituximab's anti-tumor properties are attributed in part through alleviating PS-receptor mediated immunosuppression and assisting in generating an Fc-FcR mediated pro-inflammatory response.Immune competent mice with E0771 induced tumors were administered either paclitaxel, or anti-PD-1/PD-L1 single agent therapy, or in triple combination with PS targeting antibody (ch1N11) to evaluate the efficacy of PS and PD-1/PD-L1 immune checkpoint inhibitors blockade in combination with cytotoxic chemotherapeutics. The levels of PS and PD-L1 expression were also evaluated on E0771 tumor cells following in vitro treatment via FACS analysis.in vitro treatment with paclitaxel, suggesting that combining paclitaxel with PS and PD-1 targeting antibodies will have greater anti-tumor activity in triple combination treatments. No in vivo adverse effects were observed in animals given repeated doses of single or triple combinations.Preliminary results in multiple studies demonstrated differential sensitivity to either paclitaxel, or anti-PD-1/PD-L1 single agent therapy while inclusion of ch1N11 triple combination significantly enhanced anti-tumor efficacy over either single agent therapy. Also, FACs analysis demonstrated induction of PS and PD-L1 levels on E0771 cells following These results support combination blockade of PS and PD-1/PD-L1 with paclitaxel to treat breast cancer."} +{"text": "Strigamia maritima possesses an XX/XY system of sex chromosomes, with males being the heterogametic sex. This is, to our knowledge, the first report of sex chromosomes in any geophilomorph centipede. Using the recently assembled Strigamia genome sequence, we identified a set of scaffolds differentially represented in male and female DNA sequence. Using quantitative real-time PCR, we confirmed that three candidate X chromosome-derived scaffolds are present at approximately twice the copy number in females as in males. Furthermore, we confirmed that six candidate Y chromosome-derived scaffolds contain male-specific sequences. Finally, using this molecular information, we designed an X chromosome-specific DNA probe and performed fluorescent in situ hybridization against mitotic and meiotic chromosome spreads to identify the Strigamia XY sex-chromosome pair cytologically. We found that the X and Y chromosomes are recognizably different in size during the early pachytene stage of meiosis, and exhibit incomplete and delayed pairing.We show that the geophilomorph centipede Strigamia maritima has emerged as a model system for genomic and developmental studies of myriapods were paMeiotic chromosomes were obtained from the testes of sub-adult males as described in . BrieflyGenomic probes were prepared from gDNA extracted separately from adult males and females by the standard phenol-chloroform procedure. The probes were labelled using a Nick Translation Kit (Abbott Molecular Inc.); male DNA with Cy3-dUTP and female DNA with fluorescein-12-dUTP (Invitrogen). Unlabelled female gDNA, used as a species-specific competitor, was sonicated using a Sonopuls HD 2070 (Bandelin Electric), with two cycles of five pulses at 70% power. CGH was performed essentially following the procedure described in . BrieflyFor FISH, we followed the procedure described in with somPreparations were observed in a Zeiss Axioplan 2 microscope equipped with appropriate fluorescence filter sets. Black-and-white images were taken either with a cooled F-View CCD camera (DAPI- and CGH-stained preparations) or an Olympus CCD monochrome camera XM10, and captured separately for each fluorescent dye with either AnalySIS software, version 3.2 (Soft Imaging System GmbH) or with cellSens 1.9 digital imaging software (Olympus Europa Holding), respectively. The images were pseudo-coloured and merged using Adobe Photoshop CS4 and CS5 (Adobe Systems Inc.).S1 FigA-D) show detailed analysis of the pachytene complement: (A) merged image; (B) DAPI image; (C) hybridization pattern of the female genomic probe; (D) hybridization pattern of the male genomic probe. Both probes highlighted one heterochromatic arm of the large metacentric bivalent (arrow), the nucleolus (N) associated with a middle-sized bivalent, and the centromeric heterochromatin of all chromosomes (asterisks), but did not differentiate a sex chromosome pair. Scale bar = 10 \u03bcm.Chromosomes were counterstained with DAPI (blue). Female-derived genomic probe was labelled with fluorescein-12-dUTP (green) and male-derived genomic probe with Cy3-dUTP (red). Panels ((TIF)Click here for additional data file.S2 FigA-F) correspond to the panels labelled 1 to 6 respectively, and (G) to the control panel, in Original, uncropped gels from (TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 TableCoverage and length of each genomic scaffold, and qPCR data to validate X-linked scaffolds.(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file."} +{"text": "Prostate cancer is the second most common male malignancy worldwide with a wide spectrum of biological behaviour ranging from fairly indolent disease to highly aggressive metastatic castrate resistant tumours. In the past decade there have been advances in therapies for treating advanced and recurrent prostate cancer and improved diagnostic/prognostic tools are required to refine the therapeutic approach at various stages in the management of prostate cancer patients.For tumour staging/lesion localisation multiparametric prostate MRI is the modality of choice. Several PET tracers are proving more sensitive than conventional imaging techniques for early detection of lymph node and bone metastases. However a general limitation of PET is the spatial resolution which limits the reliability for detection of small lesions.The use of F-18 fluoro-deoxyglucose (FDG) PET/CT in prostate cancer has been disappointing largely due to the fact that many prostate cancers have only low level glucose metabolism.C-11/F-18 choline, markers of cell membrane metabolism, are now widely used in Europe and in certain UK centres particularly in the scenario of biochemical relapse and high risk staging with equivocal findings on conventional work-up . HoweverProstate specific membrane antigen (PSMA), a cell surface membrane glycoprotein, is a promising target for the diagnosis and treatment of prostate cancer. Recently Ga-68 radiolabelled ligand targeted to PSMA has been introduced to a few European centres. Early data suggests this may be more sensitive than choline PET/CT in patients with biochemical failure and low PSA -7. In adF-18 sodium fluoride (NaF) is highly sensitive compared to bone scintigraphy with Tc99m labelled phosphates but only assess skeletal disease [Other PET tracers being explored include C-11 acetate targeting lipogenesis, tracers radiolabelling bombesin and targeting gastrin-releasing peptide receptor (GRPR), amino acid transport with anti-1-amino-3-F18-fluorocyclobutane-1-carboxylic acid (FACBC ) and F-18-fluoro-5\u03b1-dihydrotestosterone (FDHT) targeting the androgen receptor ,10.This talk reviews the diagnostic utility of PET in prostate cancer."} +{"text": "The prognosis of high-grade glioma (HGG) is poor despite advancements in neurosurgery, radio-and chemotherapy. DC-based immunotherapy has emerged as a promising and feasible treatment approach due to its high degree of selectivity and its ability to induce an antigen-specific immune-memory response. Our research group investigates the efficacy of vaccinating both primary and relapsed HGG patients with DC vaccines pulsed with autologous whole tumor lysate. The method of preparing autologous whole tumor lysate is, however, not standardized yet with most groups applying multiple freeze-thaw (FT) cycles to induce necrosis of tumor cells. As irradiation is known to induce oxidation-associated molecular patterns (OAMPs) like oxidized or carbonylated proteins, potent enablers of danger signaling, we hypothesized that irradiation could increase the immunogenicity of FT-treated necrotic tumor cells used to pulse DC vaccines in the context of HGG.All experiments were performed in the murine syngeneic GL261 orthotopic glioma model. Prophylactic DC immunotherapy was performed with murine bone marrow-derived DCs pulsed with total GL261 lysate. Total GL261 lysate was either prepared by subjecting GL261 cells to six FT cycles (GL261-FT) or by additionally applying 60 Gy X-irradiation after the six FT cycles (GL261-FT+IR).Protein oxidation/carbonylation, an indirect measure of proteotoxicity-based immunogenicity, was significantly increased in GL261-FT+IR as compared to GL261-FT. We observed no differences in the expression levels of DC maturation markers on matured DCs pulsed with either GL261-FT or GL261-FT+IR lysates. Flowcytometric analysis of brain-infiltrating immune cells revealed an increased infiltration of CD3+ T cells and a decreased infiltration of FoxP3+ Tregs, F4/80+ macrophages and Ly6C+ and Ly6G+ myeloid-derived suppressor cells in mice receiving prophylactic DC vaccines pulsed with GL261-FT+IR as compared to mice treated with DCs pulsed with GL261-FT. Moreover, vaccination of mice with DCs loaded with GL261-FT+IR resulted in a significant improvement of overall survival, compared to mice treated with DCs pulsed with GL261-FT.Collectively, these data suggest that irradiation of necrotic tumor cells used to pulse DC can further increase the DC vaccine-induced anti-tumor immunity in the context of glioma. We are currently evaluating the contribution of the irradiation-induced OAMPs to the increased immunogenicity of the GL261-FT+IR lysate."} +{"text": "AR-antagonist-induced neuronal network activity were affected following templated Tau-misfolding using synthetic preformed Tau fibrils in cultured primary neurons. Electrophysiological analysis in organotypic hippocampal slices of Tau transgenic mice demonstrated impaired synaptic transmission and impaired long-term potentiation following Tau-seed induced Tau-aggregation. Intracerebral injection of Tau-seeds in TauP301S mice, caused prion-like spreading of Tau-pathology through functionally connected neuroanatomical pathways. Electrophysiological analysis revealed impaired synaptic plasticity in hippocampal CA1 region 6\u00a0months after Tau-seeding in entorhinal cortex (EC). Furthermore, templated Tau aggregation impaired cognitive function, measured in the object recognition test 6\u00a0months post-seeding. In contrast, Tau-seeding in basal ganglia and subsequent spreading through functionally connected neuronal networks involved in motor control, resulted in motoric deficits reflected in clasping and impaired inverted grid hanging, not significantly affected following Tau-seeding in EC. Immunostaining, biochemical and electron microscopic analysis in the different models suggested early pathological forms of Tau, including Tau-oligomers, rather than fully mature neurofibrillary tangles (NFTs) as culprits of neuronal dysfunction. We here demonstrate for the first time using in vitro, ex vivo and in vivo models, that prion-like spreading of Tau-misfolding by Tau seeds, along unique neuronal connections, causes neuronal network dysfunction and associated behavioral dysfunction. Our data highlight the potential relevance of this mechanism in the symptomatic progression in Tauopathies. We furthermore demonstrate that the initial site of Tau-seeding thereby determines the behavioral outcome, potentially underlying the observed heterogeneity in Tauopathies, including in TauP301 mutants.Prion-like seeding and propagation of Tau-pathology have been demonstrated experimentally and may underlie the stereotyped progression of neurodegenerative Tauopathies. However, the involvement of templated misfolding of Tau in neuronal network dysfunction and behavioral outcomes remains to be explored in detail. Here we analyzed the repercussions of prion-like spreading of Tau-pathology via neuronal connections on neuronal network function in TauP301S transgenic mice. Spontaneous and GABAThe online version of this article (doi:10.1007/s00401-015-1413-4) contains supplementary material, which is available to authorized users. Tauopathies are a diverse group of neurodegenerative diseases, characterized by the presence of Tau aggregates composed of misfolded hyperphosphorylated Tau , 41, 62.Accumulating evidence indicates that Tau and related proteins linked to neurodegenerative proteinopathies display prion-like properties , 37, 72.In this work, we have addressed this question using in vitro, ex vivo and in vivo models with induction and spreading of Tau-pathology. Seeding of Tau pathology was shown to impair neuronal network function in primary neuronal cultures and in organotypic hippocampal slices. Furthermore, Tau-seeding caused prion-like spreading of Tau-aggregation through functionally connected neuronal networks and neuronal network dysfunction in TauP301S transgenic mice, leading to either cognitive or motoric deficits, depending on the initial site of Tau-seeding. Our data furthermore point to early pathological forms of Tau, including Tau oligomers, rather than somatic NFTs as culprits for the functional deficits.Transgenic TauP301S mice (PS19) expressiGeneration of Tau-PFFs from recombinant Tau Tau-PFFs (synthetic preformed fibrils) or Tau-seeds were generated as described FDG (FDG) as described in detail in supplemental data\u00a0 was used to measure nonspatial memory , increased the number of synchronously oscillating neurons and induced regularly shaped responses (characterized by a steep rise and slower decay), as reported previously [AR-antagonist, resulting in impaired GABA-ergic transmission. We further identified PTX-induced calcium oscillations as excitatory AMPA- and NMDA-dependent oscillations as they were completely blocked following application of CNQX and D-APV , as shown previously [To analyze the effect of Tau-seed induced Tau-aggregation on neuronal network function, we performed Tau-seeding in primary neurons derived from TauP301S transgenic mice , 63, 73.eviously Figs.\u00a0b, 4b. Tan\u00a0\u2265\u00a03 for each condition) Tau aggregation was detected in striatum, thalamus, brain stem and cortical regions including the motor cortex Fig.\u00a0c. To asson) Fig.\u00a0e in braiTaken together, our results indicate that depending on the initial site of injection of Tau-seeds, i.e. cortical/hippocampal, basal ganglia or entorhinal cortex Fig.\u00a0a\u2013d, diffDisease-specific patterns of glucose metabolism are generally considered to reflect neuronal dysfunction and have been demonstrated to correlate with high NFT load in different Tauopathies. To analyze the functional repercussions of spreading of Tau-aggregation through neuronal networks dependent on the site of initiation, we used in first instance FDG-PET. Mice injected with Tau-seeds and control mice were subjected to small-animal FDG-PET at 6\u00a0months post-injection. However, no significant differences were detected between Tau-seeded and non-seeded mice, despite the presence of robust Tau-pathology and Tau hyperphosphorylated at pathological epitopes S202/T205 (AT8) and T212/S214 (AT100) Fig.\u00a0a. The prWe next analyzed the presence of these different forms of Tau following Tau-seed induced Tau-aggregation in primary neurons (PNC) and organotypic cultures (OC) Fig.\u00a0. Tau-seeAccumulating evidence has demonstrated that Tau displays prion-like properties and that prion-like spreading of Tau-pathology occurs through connectivity rather than proximity . This prA receptor antagonist [We demonstrated that templated Tau-misfolding by Tau-seeds affected neuronal network activity in primary neuronal cultures. Primary cortical neurons display synchronized calcium oscillations in culture, which were previously shown to be modulated by PTX, a GABAtagonist . Exposurtagonist . A refratagonist , their rtagonist . In orgatagonist , 60, 70.Prion like induction of Tau-pathology\u2014a process which raised considerable scientific interest\u2014has been elegantly demonstrated and analyzed in exquisite detail for different aspects , 53, 72.Besides in AD patients, spreading of Tau-aggregation according to a characteristic pattern along functionally connected brain circuitries is also observed in argyrophilic grain disease, a different Tauopathy. Other Tauopathies are very heterogeneous between individuals with identical mutations, including for patients with Tau mutated at Proline 301 (P301L) . This heThe clearcut demonstration of impaired neuronal network function and behavior by prion-like seeding raises questions about the causal culprit, in terms of pathological forms of Tau. The combination of our functional analysis with biochemical and immunological analysis points to early pathological forms of Tau, including pathological hyperphosphorylated and misfolded Tau and Tau oligomers rather than fully mature NFTs as potential pathological culprits. Notably, strong somatic AT8 aggregation was only observed in 5\u201310\u00a0% of neurons following Tau-seeding in primary neurons. Furthermore, the presence of fully mature fibrils was below detection limit using electron microscopy and ThioS staining was not observed in PNC. It must be noted that Tau-seeding in primary neurons in this work was less robust than previously published , probablIn summary, our data indicate for the first time and unequivocally, using in vitro, ex vivo and in vivo approaches that (i) prion-like Tau-seed induced Tau-aggregation with NFT formation\u2014a mechanism under intensive investigation\u2014causes synaptic and neuronal network dysfunction, resulting in behavioral impairments. Our data thereby indicate that prion-like spreading of Tau-pathology may contribute to progression of disease symptoms in Tauopathies by affecting intrinsic functional critical networks. Furthermore, our data indicate unequivocally that (ii) behavioral outcomes following seeding in Tau P301 transgenic mice, are determined by the initial site of Tau seeding. Hence, motoric problems were demonstrated following Tau-seeding in basal ganglia (substantia nigra), while absent following injection in entorhinal cortex, resulting in cognitive defects. These findings are reminiscent of the heterogeneity of clinical symptoms in familial cases with P301 mutations, in which initial seed formation by environmental, genetic or accidental factors, may determine the behavioral outcomes. Finally, (iii) our results support a role of early pathological forms of Tau, including oligomeric Tau rather than fully mature NFTs as pathogenic culprits of prion-like induced spreading of neuronal dysfunction. Our findings provide a basis to further identify the molecular mechanisms involved in Tau-seed induced synaptic dysfunction and provide a model to develop novel therapeutic strategies targeting Tau-seed induced neuronal dysfunction, and to analyze repercussions on neuronal function induced by different Tau-\u201cstrains\u201d.Supplementary material 1 (TIFF 7636\u00a0kb)Supplementary material 2 (TIFF 4289\u00a0kb)Supplementary material 3 (TIFF 816\u00a0kb)Supplementary material 4 (TIFF 3810\u00a0kb)Supplementary material 5 (TIFF 23459\u00a0kb)Supplementary material 6 (TIFF 13614\u00a0kb)Supplementary material 7 (TIFF 5042\u00a0kb)Supplementary material 8 (DOCX 58\u00a0kb)Supplementary material 9 (DOCX 22\u00a0kb)"} +{"text": "Extracorporeal membrane oxygenation (ECMO) is increasingly used for the treatment of refractory but potentially reversible respiratory and/or cardiac failure. Data on perioperative support with veno-arterial (V-A) and veno-venous (V-V) ECMO for adult liver transplant recipients are scarce ,2.We repA retrospective study in a specialist tertiary referral ICU. Patients supported with V-V or V-A ECMO before, during, or after orthotopic liver transplant (OLT) were identified.In total, four patients were supported during a 12-month period. Two patients required V-V and two patients V-A support. Two patients with ALF were bridged to OLT, one patient V-V ECMO for refractory respiratory failure and the other patient required emergency V-A support for treatment of intraoperative arrest. Both patients were successfully transplanted but died subsequently on ECMO: disseminated aspergillosis and haemophagocytic syndrome, and anoxic brain injury respectively. Two patients received postoperative ECMO support. The first was treated with V-V ECMO for refractory persistent hypoxaemia following OLT for hepato-pulmonary syndrome, the second received emergency V-A support (eCPR) following cardiac tamponade and arrest on postoperative day 2. Both patients made a full recovery.Emergency ECMO support before and after liver transplant is feasible. Despite the poor outcome in patients with ALF, we consider ECMO a valuable option to bridge selected patients to transplant."} +{"text": "Mycobacterium abscessus infections are common but reliable diagnosis is hampered by non-specific clinical symptoms and insensitive mycobacterial culture. In the present study we established novel methods for rapid detection and immune characterization of Mycobacterium abscessus infection in cystic fibrosis patients. We performed Mycobacterium abscessus specific DNA-strip- and quantitative PCR-based analyses of non-cultured sputum samples to detect and characterize Mycobacterium abscessus infections. Concomitantly in vitro T-cell reactivation with purified protein derivatives (PPDs) from different mycobacterial species was used to determine Mycobacterium abscessus specific T-cell cytokine expression of infected cystic fibrosis patients. Four of 35 cystic fibrosis patients (11.4%) were Mycobacterium abscessus culture positive and showed concordant DNA-strip-test results. Quantitative PCR revealed marked differences of mycobacterial burden between cystic fibrosis patients and during disease course. Tandem-repeat analysis classified distinct Mycobacterium abscessus strains of infected cystic fibrosis patients and excluded patient-to-patient transmission. Mycobacterium abscessus specific T-cells were detected in the blood of cystic fibrosis patients with confirmed chronic infection and a subgroup of patients without evidence of Mycobacterium abscessus infection. Comparison of cytokine expression and phenotypic markers revealed increased proportions of CD40L positive T-cells that lack Interleukin-2 expression as a marker for chronic Mycobacterium abscessus infections in cystic fibrosis patients. Direct sputum examination enabled rapid diagnosis and quantification of Mycobacterium abscessus in cystic fibrosis patients. T-cell in vitro reactivation and cytokine expression analyses may contribute to diagnosis of chronic Mycobacterium abscessus infection.Cystic fibrosis patients are highly susceptible to infections with non-tuberculous mycobacteria. Especially Mutations on both alleles of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) are the genetic cause of Cystic Fibrosis (CF), the most common single-gene caused disease in Caucasians . CF pathMycobacterium (M.) avium complex (MAC) and M. abscessus complex (MABSC) are the non-tuberculous mycobacterial species most commonly detected in the sputum of CF patients were subtracted from each stimulus. Ratios of PPD specific T-cells were calculated by dividing the proportions of CD40L/IL-2 double positive T-cell proportions for abscessin/tuberculin and abscessin/sensitin. For calculation of normalized values, we set the sum of abscessin-, tuberculin-, and sensitin-specific CD40L/IL-2 double positive T-cells proportions to 1. For presentation of cytokine expression pattern (i.e. three cytokines and CD40L) graphical depiction is not possible because 4-dimensional presentation (followed by selection of subsets) is technically not feasible. As a consequence the events for each cytokine pattern expressing T-cell subpopulation (numbers of non-stimulated numbers were subtracted) were counted and the number of T cells expressing at least one cytokine per activation marker in response to M. abscessus was calculated. Calculated proportions of each subpopulation of all activated T cells are indicated for each individual donor and compared between study groups.Heparinized blood was processed freshly (within five hours of blood collection) to assure maximal sensitivity of this assay (own unpublished data). In brief, blood (100 \u03bcl) was diluted (1:1) in RPMI containing 1% L-Glutamine (Sigma Aldrich), 1% Penicillin/Streptomycin (Life Technologies) and supplemented with recombinant human (rh) IL-7 for overnight culture in 96-well round-bottomed microtiter plates as described . IL-7 haP-values < 0.05 were considered to be significantly different.Statistical calculations were performed using SigmaStat (Systat Software). Parametric or non-parametric tests were chosen according to Kolmogorov-Smirnow normality test. Accordingly, the student t-test or the Mann-Whitney Rank Sum Test was used and indicated in the text and figure legend. in vitro culture from all sputum expectorating CF patients (n = 23). DNA-strip test detected MABSC in the sputum of all culture positive CF patients at study onset. We performed a DNA-strip test on sputum without prior , CF022) , Table 3ion step . The rpo100 CFU) . AccordirpoB-qPCR was applied to determine MABSC concentrations of CF patient\u00b4s sputum samples. MABSC-confirmed CF patients had marked differences of MABSC sputum burden with concentrations ranging from 5.6x105 to 3.9x107 bacteria per ml sputum were unable to expectorate sputum and therefore mycobacterial culture or PCR-based analyses were not possible. Cross-reactive mycobacterial antigens may confound these results but concomitant testing of PPDs from different mycobacteria was shown to reveal the causative mycobacteria , a previous contact with atypical mycobacteria cannot be excluded. Therefore we assume that MABSC non-confirmed CF patients underwent asymptomatic infection (e.g. colonization) with different atypical mycobacteria in the past. This assumption is supported by the heterogeneity of PPD specificity in this group as compared to MABSC confirmed CF patients that suggested different causative mycobacteria. To avoid or at least minimize mycobacterial cross-reactivity for the detection of MABSC infection, identification of MABSC-specific immunogenic proteins would be prerequisite. Since such proteins are not identified so far, our comparative approach to concomitantly analyze abscessin-, tuberculin-, and sensitin-specific T-cells is a promising alternative. In addition, marked differences in the level of T-cell response against abscessin (as compared to tuberculin and sensitin) indicated that MABSC-specific immunogenic proteins with limited cross-reactivity exist. Identification of such proteins would be an important step towards the development of a specific in vitro T-cell test for MABSC infection (comparable to IGRAs used for detection of M. tuberculosis).In our study, all patients with current or past MABSC infection had MABSC-specific and-dominant T-cell responses. This finding indicated a high sensitivity of this assay, rendering it a putative strong tool for exclusion of MABSC infection in CF patients. However, MABSC-specific T-cell responses could be observed for patients without confirmed MABSC infection as well. Indeed, immunological detection of MABSC infection may always be confounded by several environmental and population specific factors that induce cross-reactive T-cells including i) Bacille-Calmette Gu\u00e9rin (BCG) vaccination; ii) latent et al. described increased proportions of M. tuberculosis specific T-cells expressing CD40L in tuberculosis patients although M. tuberculosis specific T-cells were generally diminished in patients. Notably, CD40L expressing T-cells were optimal for discrimination of tuberculosis patients from healthy latently M. tuberculosis infected contacts in this study [+ effector T-cells in tuberculosis [Our data indicated that besides antigen-specificity, cytokine expression differed between T-cells from MABSC confirmed and non-confirmed CF patients. Cytokine expression pattern analyses revealed increased CD40L-positive / IL-2-negative T-cell proportions in confirmed MABSC infected CF patients. Evidence for CD40L as a marker of acute mycobacterial disease is provided by studies on human tuberculosis . Streitzis study . IL-2 wais study , 24. We rculosis ) will harculosis , e.g. carculosis , 27.12 of 35 CF patients recruited in this study did not expectorate sputum samples. According to ATS criteria , none ofIn conclusion immunological and PCR-based assays may help to diagnose the NTM infection especially early during MABSC infection and exacerbation. This is of special importance for the large group of patients not able to expectorate sputum. Our novel tests may help to decide whether additional diagnostics should be performed. As a consequence the complicated treatment of MABSC infection can be started earlier with likely better efficacy and prognosis.S1 FigM. abscessus (MABSC), sensitin of M. avium (MAC), and tuberculin of M. tuberculosis (MTB), the non-clonal T-cell activator SEB (Staphylococcus enterotoxin B), and without stimulation (w/o) are shown.A representative analysis of flow cytometry results from a MABSC-negative CF patient is depicted. Results of stimulation with different mycobacterial antigens, i.e. abscessin of (EPS)Click here for additional data file.S2 FigMABSC) and non-confirmed MABSC T-cell responders (CFnon-confirmed) are shown. Memory T cells are gated as indicated in Flow cytometry dot plots indicating for confirmed MABSC infected CF patients (CF(EPS)Click here for additional data file."} +{"text": "Carbamazepine (CBZ) is associated with the severe cutaneous drug reaction Stevens-Johnson Syndrome/toxic epidermal necrolysis (SJS/TEN). CBZ-SJS/TEN has been associated with HLA-B*15:02 carriage and specific T-cell clonotypes. We aimed to characterize the interactions between specific T-cell receptor (TCR) clonotypes, HLA-B*15:02 and CBZ.In silico modelling was used to examine the interaction between the known specific TCR V and V chains, CBZ and HLA-B*15:02.Peripheral blood mononuclear cells (PBMCs) were isolated from patients with CBZ-SJS/TEN and healthy controls, stimulated with 10ug/mL CBZ and cultured over a 9-14 day period. RNA was isolated at various time points and TCR V subtypes were assessed using digital droplet PCR . Drug specific T-cell INF and granulysin responses were assessed by ELISpot and ICS. In silico modelling of the CBZ-HLA-B*15:02-TCR interaction suggests that CBZ binds non-covalently in the P4 binding pocket of the HLA-B*15:02 antigen binding cleft in a site that is typically occupied with bound peptide.CBZ specific T-cell INF and granulysin responses were detected many years following the original SJS/TEN reaction. Expansion of specific TCR CDR3 sequences was confirmed in CBZ SJS/TEN patient cultures. These findings raise two non-mutually exclusive possibilities: 1) CBZ may block peptide binding and be presented by HLA-B*15:02 in a solvent exposed manner available for direct recognition by the TCR, 2) CBZ binding the central portion of the HLA-B*15:02 antigen binding cleft may permit long peptides to bind conventionally at the peptide termini (in the A and F pockets) and bulge over the drug in the central residues to permit indirect recognition of CBZ (peptide mediated TCR contact)."} +{"text": "Immunotherapeutic approaches such as vaccination or immune checkpoint blockade have proven to be clinically active in prostate cancer, but only in fractions of treated patients; this calls for personalized application of these novel therapies based on predictive biomarkers.Our own research over the past years has focused on the clinical efficacy in patients with castration-resistant prostate cancer of the combination of an allogeneic cell line-based vaccine (Prostate GVAX) and an anti-CTLA4 checkpoint inhibitor (ipilimumab) in a Phase-I/II dose escalation/expansion trial. We carried out an extensive immune monitoring programme comprising flowcytometric profiling of lymphoid and myeloid subsets in peripheral blood (PB) and T cell and serological reactivity to a panel of known tumor antigens, all before and after treatment.On-treatment PSA declines of more than 50% were observed in 5, and PSA stabilizations in 12 of 28 patients. Regressing bone and lymph node metastases were observed in 2/5 responding patients. Significantly prolonged overall survival (OS) was observed for patients with high pre-treatment frequencies of CD4+CTLA-4+, CD4+PD-1+, or differentiated CD8+ T cells, or low pre-treatment frequencies of regulatory T cells. Treatment-induced activation of PB Dendritic Cell subsets was similarly associated with significantly prolonged OS. In contrast, high pre-treatment frequencies of monocytic Myeloid-Derived Suppressor Cells (MDSC) were associated with reduced OS. Th2/Th17 cytokine profiles were induced. Indeed, profound up-regulation of CD4+IL-5+ T cell frequencies was associated with improved OS (p=0.03) and correlated significantly with the breadth of the induced antibody response. IgG antibody responses against 11 (prostate) tumor-associated antigens were determined and increased seroreactivity to prostate-specific membrane antigen (PSMA), pyridoxamine 5'-phosphate oxidase (PNPO) and/or Neuropilin-2 (NRP2) was significantly correlated with improved OS . Finally, patients with pre-existing NY-ESO-1 T cell reactivity also demonstrated a significantly prolonged OS (p=0.044).Together these data provide an immune profile to predict clinical outcome. Importantly, cluster analysis revealed pre-treatment expression of CTLA-4 by circulating CD4+ T cells and an immune-stimulatory myeloid profile to be dominant predictors for OS after Prostate GVAX/ipilimumab therapy. These flowcytometry-based parameters may thus provide potentially useful and easy-to-use biomarkers for patient selection."} +{"text": "Persistent hypoxia stimulation, one of the most critical microenvironmental factors, accelerates the acquisition of epithelial\u2013mesenchymal transition (EMT) phenotypes in lung cancer cells. Loss of phosphatase and tensin homologue deleted from chromosome 10 (PTEN) expression might accelerate the development of lung cancer in vivo. Recent studies suggest that tumor microenvironmental factors might modulate the PTEN activity though a decrease in total PTEN expression and an increase in phosphorylation of the PTEN C-terminus (p-PTEN), resulting in the acquisition of the EMT phenotypes. Nevertheless, it is not known whether persistent hypoxia can modulate PTEN phosphatase activity or whether hypoxia-induced EMT phenotypes are negatively regulated by the PTEN phosphatase activity. We aimed to investigate hypoxia-induced modulation of PTEN activity and EMT phenotypes in lung cancers.Western blotting was performed in five lung cancer cell lines to evaluate total PTEN expression levels and the PTEN activation. In a xenograft model of lung cancer cells with endogenous PTEN expression, the PTEN expression was evaluated by immunohistochemistry. To examine the effect of hypoxia on phenotypic alterations in lung cancer cells in vitro, the cells were cultured under hypoxia. The effect of unphosphorylated PTEN (PTEN4A) induction on hypoxia-induced EMT phenotypes was evaluated, by using a Dox-dependent gene expression system.Lung cancer cells involving the EMT phenotypes showed a decrease in total PTEN expression and an increase in p-PTEN. In a xenograft model, loss of PTEN expression was observed in the tumor lesions showing tissue hypoxia. Persistent hypoxia yielded an approximately eight-fold increase in the p-PTEN/PTEN ratio in vitro. PTEN4A did not affect stabilization of hypoxia-inducible factor 1\u03b1. PTEN4A blunted hypoxia-induced EMT via inhibition of \u03b2-catenin translocation into the cytoplasm and nucleus.Our study strengthens the therapeutic possibility that compensatory induction of unphosphorylated PTEN may inhibit the acquisition of EMT phenotypes in lung cancer cells under persistent hypoxia. The tumor microenvironment, which involves activation of various signal pathways, accelerates the acquisition of epithelial-mesenchymal transition (EMT) in lung cancers cells , 2. PersBased on the knowledge that persistent hypoxia-induced aberrant signaling pathway should be therapeutic target for lung cancer , 19, we To evaluate total PTEN expression levels and the p-PTEN/PTEN ratio in lung cancer cells, western blotting was performed in the following five lung cancer cell lines: H441, H358, A549, H157 and H1299 . WesternNext, to evaluate the effect of persistent hypoxia on modulation of PTEN expression in lung cancers, H358 cells were treated under hypoxia in vitro. Under persistent hypoxia stimulation, total PTEN levels decreased in a time-dependent manner but p-PTEN levels remained steady in H358 cells, leading to an approximately eightfold increase in the p-PTEN/PTEN ratio 72\u00a0h after hypoxia stimulation antibody was from Novus biologics ; monoclonal mouse anti-PTEN antibody (clone 6H2.1) was from Cascade Bioscience ; anti-\u03b2-catenin antibody and purified and fluorescein isothiocyanate (FITC)-conjugated mouse anti-E-cadherin antibody were from BD Biosciences ; purified anti-fibronectin antibody was from Santa Cruz Biotechnology, Inc ; streptavidin (SAv)-Alexa 594 (SAv-594)-conjugated and SAv-Alexa 488 (SAv-488)-conjugated anti mouse antibody was from Invitrogen Life Technologies ; purified rabbit anti-phospho-PTEN (Ser380/Thr382/Thr383) antibody was from Cell Signaling Technology ; Affinity-isolated rabbit anti-actin antibody was from Sigma-Aldrich . Hoechst33342 was from Dojindo . The Hypoxyprobe-1 plus kit was purchased from Hypoxyprobe, Inc. . Horseradish peroxidase (HRP)-conjugated anti-mouse antibody, 3, 3\u2032-diaminobenzidine (DAB) Substrate Kit, and Hematoxylin QS were from Vector Laboratories . PhosSTOP was from Roche Applied Science . Can Get Signal was also purchased from Toyobo Co. . Doxycycline (Dox) was from Clontech . pTRE-Tight vector and pTet-On Advanced were from Clontech .A Dox-dependent gene expression system was applied to H358 cells, a human lung cancer cell line, carrying pTet-On Advanced (H358ON) . The H352) for the indicated periods using a hypoxic chamber for the indicated periods. For immunocytochemistry, some cells were cultured in an 8-well Lab-Tek Chamber Slide System . Pimonidazole hydrochloride at final concentration of 150\u00a0\u03bcM was added 90\u00a0min before immunostaining for pimonidazole [Human lung cell lines H441, H358, A549, H157 and H1299, were maintained in RPMI medium as previously described . To examnidazole .We performed real-time PCR, by using a TaqMan ABI 7300 Sequence Detection System . The following oligonucleotide primers and probe were used: twist (twist1: NM_000474) sense (5\u2032-CCAGCTATGTGGCTCACGAG-3\u2032) and antisense (5\u2032-CTAGTGGGACGCGGACATGG-3\u2032), internal fluorescence-labeled probe (FAM) (5\u2032-CCTCCATCCTCCAGACCGAGAAGGCG-3\u2032) . The twiProtein immunodetection was performed by electrophoretic transfer of SDS-PAGE, separation of proteins on nitrocellulose, incubation with primary antiboby, followed by the appropriate HRP-conjugate second antibody, and chemiluminescent second-step detection using ECL Plus . The amoImmunocytochemistry and immunohistochemistry was done as previously described , 42, 43.6) expressing Dox-dependent GFP were inoculated subcutaneously into the flank of 6-week-old female nude mice. The treated mice were maintained on water containing Dox at a final concentration of 2\u00a0mg/ml [H358ON cells for multiple comparisons. A p value of less than 0.05 was considered statistically significant."} +{"text": "JAM-C blockade in the recipients induced greater emigration of monocyte-derived cells and further diminished the size of atherosclerotic plaques. Our findings have shown that JAM-C forms a one-way vascular barrier for leukocyte transendothelial migration only when present at homeostatic copy numbers. We have also shown that blocking JAM-C can reduce the number of atherogenic monocytes/macrophages in plaques by emigration, providing a novel therapeutic strategy for chronic inflammatory pathologies.Atherosclerosis, caused in part by monocytes in plaques, continues to be a disease that afflicts the modern world. Whilst significant steps have been made in treating this chronic inflammatory disease, questions remain on how to prevent monocyte and macrophage accumulation in atherosclerotic plaques. Junctional Adhesion Molecule C (JAM-C) expressed by vascular endothelium directs monocyte transendothelial migration in a unidirectional manner leading to increased inflammation. Here we show that interfering with JAM-C allows reverse-transendothelial migration of monocyte-derived cells, opening the way back out of the inflamed environment. To study the role of JAM-C in plaque regression we used a mouse model of atherosclerosis, and tested the impact of vascular JAM-C expression levels on monocyte reverse transendothelial migration using human cells. Studies in-vitro under inflammatory conditions revealed that overexpression or gene silencing of JAM-C in human endothelium exposed to flow resulted in higher rates of monocyte reverse-transendothelial migration, similar to antibody blockade. We then transplanted atherosclerotic, plaque-containing aortic arches from hyperlipidemic ApoE It is driven by a chronic and maladaptive inflammatory response, wherein tissue resident macrophages derived from monocytes become engorged with cholesterol and persist in the lesion instead of being cleared through emigration or otherwise , 2. Impopositive . As recepositive .Recruitment of monocytes to sites of tissue injury/inflammation and atherosclerotic plaques, is tightly regulated by a series of events involving interactions between adhesion molecules. Monocyte capture and adhesion is followed rapidly by transendothelial migration (TEM) into the tissue, through intercellular endothelial junctions , 5. A maVascular JAM-C expression has been shown to play a critical role in inflammatory disease and metastasis \u201321. It h+) cells is through their increased egress from the plaques, which is consistent with the effects of JAM-C blockade on rTEM in-vitro.In the present study, we have examined the effect of blockade of JAM-C in a mouse model of atherosclerotic plaque regression, as well as increased and reduced endothelial JAM-C expression on monocyte rTEM in-vitro. Blocking JAM-C function through injection of antibody (Ab) was found to cause a greater reduction in the critical plaque area as well as in the relative inflammatory cell content of these lesions following the exposure of advanced plaques to a normolipidemic environment. This occurred without significant changes to either circulating leukocyte subpopulations or their recruitment into the plaques, consistent with our previous findings in-vitro of no effect of JAM-C blockade on the primary-TEM of monocytes through endothelial cells . NotablyUnless otherwise noted, reagents were purchased from Sigma-Aldrich Chemie GMBH . Antibodies (Abs) used for flow cytometry and/or immunofluorescence were anti- Vascular endothelial (VE)-Cadherin and Alexafluor 488 conjugated anti- Platelet/endothelial cell adhesion molecule-1 (PECAM-1)/CD31 , anti-JAM-B, anti-ICAM-1 , anti-zonula occludens-1 (ZO-1), anti-Occludin-1 and anti-claudin-5 . Rabbit serum prior to immunization was used as a control (day 0) for labeling with polyclonal to human JAM-C (day 74) . Alexafluor 488 conjugated goat anti-rabbit IgG (Invitrogen) and Fluorescein isothiocyanate (FITC) conjugated goat anti-mouse IgG . FITC conjugated Anti-CD62L, and anti-CD14 (BD Biosciences). For histology and flow cytometry on Human umbilical vein endothelial cells (HUVECs), JAM-C studies were conducted using antibody 225.3, or a mouse anti-human IgG1isotype control (both produced in-house). For flow cytometry studies on HUVECs transfected with Small interfering RNA (siRNA), an anti-JAM-C antibody H33 conjugated to Alexa-488 was used. Labelling was done with a goat anti-human FITC or PE-conjugated secondary antibody .-/- mice were perfused with Cryo-OCT compound and snap frozen in liquid nitrogen. Samples were cut into 10 \u03bcm sections and fixed in 4% paraformaldehyde. Samples were labelled with Alexafluor 488 conjugated anti-PECAM-1/CD31 and purified rabbit polyclonal anti-JAM-C followed by DyLight594 conjugated anti-rabbit secondary. Nuclear staining was performed using 4',6-diamidino-2-phenylindole (DAPI) and samples were visualized with confocal microscopy . JAM-C expression was evaluated using ImageJ software with pixel intensity being the readout of relative expression level. Fiji (ImageJ) software was used to quantify endothelial JAM-C expression on stained sections of carotid arteries. Measurements were conducted on three images from two independent experiments for each of two conditions (10-wks vs 6-mths). The maximal pixel intensity was measured for 6\u20139 region-of-interests (ROI) for each image, and the average maximal pixel intensity per condition was calculated.Carotid arteries were isolated from young (6-wks) and old (24-wks) ApoEHuman umbilical vein endothelial cells (HUVEC) were isolated by collagenase treatment of umbilical veins as previously described \u201331 and mHUVECs were transfected with 300 nM human JAM-C siRNAs1 and 2 using the Amaxa Nucleofector (Life Technologies) and cultured for 24-48-hrs before experimentation. Inhibition of JAM-C expression in HUVECs after transfection was compared with transfection using control siRNAs: siRNA non-homologous to any known human gene (ctrl siRNA) or mock (buffer only). Expression of target and reference genes were analyzed by real-time polymerase chain reaction (qPCR). The sequence for JAM-C-specific primers were as follows: Forward 5'- aag aac cca ggg aaa cca gat gga-3'; Reverse 5'- tcg ctg cct tga cag gag ttt cta-3\u2019. The values were normalized to the expression levels of human beta-actin, beta-tubulin, and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), according to the GeNorm method [Construction of lentivirus JAM-C-Enhanced green fluorescent protein (EGFP) (LV-JAM-C-EGFP) as well as control EGFP lentivirus (LV-EGFP) vectors has been previously described , 33. EGFSlides containing HUVEC monolayers were fixed in methanol (\u221220\u00b0C) for 5-mins and washed in Phosphate buffered saline (PBS) containing 0.5% BSA wash buffer. Monolayers expressing JAM-C-EGFP were fixed for 20-30-mins in methanol to inactivate EGFP or with 4% paraformaldehyde to maintain EGFP activity. Preincubation with human serum was conducted before immunostaining. Slides were mounted in Mowiol/DABCO for confocal microscopy .PBMCs were first isolated from blood of healthy donors followed by monocyte purification using a monocyte isolation kit . Purity and activity of monocytes were controlled by flow cytometry by labelling with anti-CD14 and anti-CD62L Abs (data not shown). Only populations of 90% or more double positive cells were used.5 cells/slide as previously described [6 cells/ml) using a calibrated pump . The flow rate was representative of shear rates in small venules/capillaries (0.05 Pa/0.5 Dynes). Flow assays were conducted as described in a previous study [HUVECs were cultured at 4 to 5 \u00d7 10us study , was obsInteractions studies were performed on a Biacore 2000.The human soluble JAM-C (up to the QEMEV sequence) protein flagged at the C terminus was produced in BOSC cells and purified through an anti flag column. The protein was immobilized at a concentration of 1 ug/ ml on a M5 sensor chip using the amine coupling kit (NHS-EDC) provided by the Biacore Supplier to the level of 155 resonance units. The background signal from a reference channel without soluble JAM-C was automatically subtracted. Protein G purified Abs (50 ul) were injected at a flow rate of 20-ul/min, in the provided running buffer at different concentrations: 100, 50, 10, 5 and 1 ug/ ml.All procedures described were approved by the Animal Use and Care Committee at the NYU School of Medicine and the Institutional Animal Care and Use Committee (IACUC). The model of aortic arch transplantation has been previously detailed \u201337. Brie-/- mice on a C57BL/6 background were weaned at 3-4-wks old onto a high-fat Western Diet . Mice were maintained on WD for 12-wks, after which a subset was used for baseline measurements (BL), and the remainder were used for transplantation procedures. 48-hrs prior to sacrifice, all donor mice were injected with 5-ethynyl-2\u2019-deoxyuridine (EdU) in order to allow EdU incorporation into DNA during active monocyte precursor proliferation in the bone marrow. The released, labeled monocytes will then enter various peripheral tissues, including the atherosclerotic plaques. After transfer of the aortic arches containing the labeled cells in the plaques, if some of them emigrate in the regression , the difference between the number of baseline lesional EdU+ cells and the recipient transplanted arch lesional EdU+ cells will reflect leukocyte egress in each group [ApoEch group .+ cell content of the plaques, frozen aortic arch segment sections were allowed to thaw to room temperature, and then fixed with 4% paraformaldehyde in PBS for 1-hr. After washing twice with a 3% BSA in PBS serum solution, the sections were permeabilized with 0.5% Triton X-100 in PBS for 1-hr. The sections were again washed twice with 3% BSA in PBS and then incubated with a Click-iT reaction cocktail containing Click-iT reaction buffer (Invitrogen), CuSO4, Alexa Fluor 647 Azide, and reaction buffer additive for 1-hr while protected from light. The sections were washed once more with 3% BSA in PBS. For subsequent nuclear staining, sections were washed once with PBS and then incubated with 5ug/ml DAPI solution for 15-mins. Finally the slides were washed twice with PBS and coverslipped with Prolong Gold Antifade Reagent (Life Technologies). Cells were imaged using confocal microscopy, with EdU+ cells identified by Alexa Fluor 647. Quantification of EdU+ cells/section was performed by counting AF647+ cells in the indexed fashion similar to morphometrics and bead assay. All age-matched recipient mice were WT on the C57BL/6 background. The recipient groups consisted of: 1) PBS treated controls (WT), 2) i.v. injected with 8mg/kg monoclonal JAM-C at 24-hrs prior to transplantation and 24 and 48-hrs afterwards (JAM-C), or 3) Mice injected similarly with a JAM-C isotype control . Recipients were maintained on chow diet following surgery and were sacrificed 4-days post-transplantation. 48-hrs before recipient sacrifice, all recipients were injected with fluorescent latex beads diluted in PBS 1:4 (Polysciences Inc.), which are taken up by circulating monocytes [2 euthanasia.For quantification of EdUonocytes . Since tonocytes , 39). In+ using primary rat anti-mouse CD68 (Serotec), followed by biotinylated anti-rat IgG secondary, and visualization using a Vectastain ABC kit (Vector Laboratories). Briefly, sections were blocked with 4% rabbit serum, stained for 1-hr with primary CD68, incubated 20-mins with biotinylated IgG, treated for 5-mins with alkaline phosphatase, and treated 8-10-mins with Vector Red substrate in Tris-HCl buffer. Vector red staining of sections was stopped with ddH20 and subsequently underwent a standard hematoxylin/bluing counterstaining, dehydrated to xylene and mounted with coverslips [+ total area, and CD68+ % of lesion area were used as four summary parameters of the lesion morphometrics.Following perfusion with 10% sucrose, baseline aortic arches and transplanted arch grafts were removed, embedded in OCT, and frozen. Serial sections of 6 \u03bcm were obtained and mounted on glass slides. For lesion immunohistochemistry (IHC), sections were fixed in 100% acetone and stained for CD68verslips , 40. Morhi staining (CD45: Phycoerythrin (PE)-Cy7), and from this monocyte populations were characterized as by CD115 and Ly6-C/G , such that Ly6Chi monocytes were CD115hiLy6-C/Ghi, Ly6Clo monocytes were CD115hiLy6-C/Glo, and neutrophils were CD115loLy6-C/Glo.Leukocytes were identified from collected blood using previously established methods . Briefly+ populations were similarly analyzed for FITC to check for non-specific labeling which might obscure results. Flow cytometry was performed using a LSRII analyzer (BD), and analysis was performed using FlowJo X software (Tree Star).For bead labeling normalization, the % of FITC-positive monocytes was analyzed 24-hrs after injection to determine relative degree of labeling when comparing across groups. Other CD45hi, and CD45+/CD115+/ly6clow groups by flow cytometry from a terminal bleed with a volume of 200 ul. A volume of 50 ul of blood was also taken for hemacytometer measurements, allowing the total number of white blood cells (CD45+) to be calculated. Using these measurements the percentage of each population was calculated (CD45+ number * % of each population).Total white blood cell count from collected mouse blood was performed using a hematology cell counter (Oxford Science Inc.). At sacrifice, relative populations were calculated as a percentage of total CD45, CD45+/CD115+/ly6cData presented is expressed as mean\u00b1standard error measurement (SEM) or as Median values. For all comparative analysis of morphometrics, leukocyte populations, and atherosclerosis, 1-way ANOVA was used with Tukey-Kramer post-hoc analysis when appropriate. All other analysis was conducted using the Student unpaired T-test unless otherwise stated. Significance was marked as follows: NS = Not Significant (P>0.05), * = p<0.05, ** = p<0.01, *** = p<0.001 and **** = p<0.0001.+ cells) from the plaques.In order to investigate the effect of JAM-C blockade on monocyte/macrophage trafficking in atherosclerosis, we used a mouse aortic transplantation model. Atherosclerotic plaques were induced by feeding ApoE-/- \u201cdonor\u201d mice a Western-type diet (WD) for 12-weeks (wks). It has been shown that transplantation of the atherosclerotic aortic arch into a normolipidemic recipient mouse resulted in dramatic plaque regression , 36, in + was used to determine cells that were presumably monocytes and inflammatory cholesterol-loaded macrophage foam cells, and quantification of atherosclerosis through CD68+ content has been extensively used in the past in this regard (+ images per group). Although neutrophils can also express CD68, there was only background staining for Ly6G (a neutrophil marker) in all groups. Some minor populations of cholesterol-loaded smooth muscle cells can also show CD68 expression, however their speed of migration is relatively low compared to monocytes, and there is no evidence of massive short-term immigration or emigration from plaques (see below) [2 compared to 0.11 \u00b10.017 mm2, P<0.05). This change in total area was attributed to a decrease in the plaque inflammatory cell content, taken as the area of CD68+ staining (2 in PBS buffer-control recipients vs. 0.036 \u00b10.003 mm2 in Basline (BL) donors (P<0.001). We measured the relative monocyte/macrophage content in the plaque .Therefore we transplanted the atherosclerotic aortic arch into WT recipient mice, and these were sacrificed 4-days later . Immunose below) , 43. Cone below) , 37, thestaining , which w2 compared to 0.016 \u00b10.001 mm2, P<0.01). The percentage of the inflammatory macrophage area of the total lesion of the anti-JAM-C antibody treated recipients was also reduced compared to the PBS buffer-control recipients 24-hrs pre-transplantation, and 24- and 72-hrs post-transplantation showed significantly greater regression of the inflammatory lesion . While tAs we have observed previously , 44, redTo test for the possibility that interference with JAM-C and monocyte TEM may elicit a response in circulating leukocytes, we evaluated leukocyte populations from mouse whole blood at sacrifice. As expected, hyperlipidemic baseline mice exhibited monocytosis and neutrophilia , and had+/CD115+ monocyte pool, Ly6Chi and Ly6Clo cell numbers were evaluated at sacrifice . Baseline ApoE-/- mice after WD had a significantly increased number of Ly6Chi monocytes as expected from their hypercholesterolemia [hi monocytes across the normolipidemic recipient WT groups. No differences between any groups were present in Ly6Clo monocytes. These data suggest that anti-JAM-C antibody treatment does not affect circulating leukocytes in a manner that would confound the interpretation of the results.In the CD45erolemia , but no + cells in our mouse model of atherosclerosis regression, we investigated the distribution of JAM-C within the atherosclerotic plaques. Previous studies from our group and others showed that JAM-C localization at the arterial endothelium could be observed during chronic inflammation [-/- mice and 6-month (mth) old ApoE-/- mice that developed spontaneous atherosclerotic lesions. As expected, we could not detect JAM-C expression in the arterial endothelium of plaque-free mice expression) . These rPrevious studies in humans have shown that disease onset and pathology are both marked by the accumulation of monocytic-derived cells . We therWe have previously shown that down-regulating JAM-C expression on HUVECs using siRNA or JAM-C blockade increased human monocyte rTEM. We conducted these studies using a cross-reactive anti-mouse JAM-C antibody . The draWe then recorded the time individual monocytes spend in the abluminal compartment of HUVECs before rTEM. Monocytes typically occupied the abluminal compartment for 40-mins under control conditions, which was significantly reduced to 8-mins after JAM-C blockade with 225.3 antibody (P<0.005) . HUVECs During the onset of atherosclerosis, we found increased JAM-C expression at endothelial junctions, while endothelium in healthy vessels show low or marginal expression levels. Furthermore, previous studies have indicated that oxidised low-density protein (oxLDL); a known contributor to atherosclerosis, can lead to increased JAM-C expression on endothelial cells . We therTo understand whether increased expression of JAM-C in HUVECs also had an effect on monocyte behaviour, we compared primary and rTEM using HUVECs transfected with JAM-C-EGFP (JAM-C-1.8x and JAM-C-6.6x) and a WT-control (JAM-C-WT). Rates of primary monocyte TEM under flow were comparable between JAM-C-WT and JAM-C-1.8x HUVECs, with levels of 98% \u00b11.4% and 95% \u00b11.4% respectively . HoweverHaving established similar rTEM patterns with higher and lower levels of vascular JAM-C compared to homeostatic controls, we were keen to identify parameters that could differentiate between these effects. To this end, we measured the velocity of monocytes in the luminal and abluminal compartment on HUVECs with JAM-C expression ranging from close to zero expression (JAM-C-neg), to above physiological levels (JAM-C-6.6x).This was done by calculating the average velocity of individual monocytes on activated HUVEC monolayers immediately after a capture or a TEM event . IndividAlmost identical observations were made for monocyte velocities in the abluminal compartment . No signIn summary, our in-vitro model using single cell analysis has shown that HUVECs with increased JAM-C expression have the capacity to support monocyte migration at higher velocities. This increased monocyte velocity was reduced to control levels when treated with a functional blocking of JAM-C. However, these observations were distinct from those using normal and reduced levels of JAM-C where a further cumulative reduction in monocyte velocity did not occur.Having established an effect of JAM-C blockade on human monocyte rTEM by the in-vitro model, we decided to investigate the effect of blocking JAM-C in-vivo using our inflammatory, atherosclerosis regression model. Specifically, we asked whether the elevated levels of junctional JAM-C in the atherosclerotic plaques would actually promote or inhibit emigration of recruited monocytes, and if JAM-C blockade with a blocking antibody would enhance this process. Using this model, the dynamics of the regressing plaque could be attributed broadly to the reduced recruitment of circulating monocytes to the inflammatory lesion or increased egress of tissue-resident macrophages from the plaque into the circulation/lymphatics , 49.To investigate these possibilities, we first utilized an in-vivo bead labeling technique to assess monocyte recruitment to plaque-bearing aortas after their transfer to a normolipidemic environment. Briefly, the WT mice that received the aortic transplant (recipient) were injected intravenously 2-days prior to sacrifice with fluorescent latex beads that then label circulating monocytes. These beads remain within the monocytes after entering tissues and therefore reflect their recruitment . The num-/- donor mice were injected with EdU to label monocytes in the bone marrow prior to transplantation of the aorta. These labeled monocytes then exit into the circulation and enter tissues as EdU+ cells. Plaques in aortic arches of donor atherosclerotic mice showed a robust incorporation of EdU+ cells within the lesions. Baseline colonization of the aorta before transplantation was determined as 26.0 \u00b12.4 EdU+ cells/section , and the in-vivo atherosclerosis model, where JAM-C blockade increased leukocyte emigration from plaques, which accelerated the regression of the inflammatory lesion.Since this increased emigration resulted in reduced plaque macrophage content, it suggests that the blockade of JAM-C function increases monocyte and macrophage rTEM, preventing the accumulation of these cells in plaques. With regard to the effects on monocytes, this would also prevent their subsequent differentiation into macrophages. As for macrophage rTEM, this effect of JAM-C functional blockade is reminiscent of our recent studies on dendritic cells, another monocyte-derived cell, in which inhibiting JAM-C promoted dendritic cell rTEM and increased antigen presentation to T cells .The role of JAM-C in leukocyte retention represents an extension in the functionality for the classical JAMs. A recent study has shown that endothelial deficiency in JAM-A, a close family member of JAM-C, did not affect vascular permeability, but profoundly affected leukocyte extravasation . HoweverSuch observations must be considered when evaluating JAM-C as a therapeutic target, where an increased vascular JAM-C expression is perceived to have the same effect as blockade. However, our studies have shown other variables play a role, which requires further investigation. As discussed, increasing interactions with monocytes and vascular JAM-C may well be a pivotal factor that promotes disease progression. Furthermore, similar tactile interactions with increased JAM-C on smooth muscle cells and fibroblasts may alsoThis study has demonstrated the potential for JAM-C as a target for therapy in pathologies where accumulating leukocytes play a major role in disease progression. In atherosclerosis this may be particularly effective, given that lipid lowering treatment appears to stimulate some degree of rTEM of monocytes/macrophages , 37, so In summary, our findings have described a mechanistic role for JAM-C in regulating inflammation during normal physiology as well as pathology. Moreover, this study has contributed to a model where inflammatory diseases may be profiled for chronicity based on vascular and tissue JAM-C expression.S1 Appendix(DOCX)Click here for additional data file.S1 Fig(A) Characterization of the novel anti-human JAM-C antibody 225.3. Biacore evaluation of anti-JAM-C Abs on immobilized soluble JAM-C comparing the anti-mouse JAM-C cross-reactive H36 Abs with the anti-human JAM-C 225.3. (B) Validation of JAM-C down-regulation on HUVECs after siRNA delivery by qPCR. Inhibition of JAM-C expression on cultured HUVECs after transfection with huJAM-C siRNA 1 and 2, when compared to control siRNA and mock control. All values were normalized to the expression levels of human beta-actin, beta-tubulin, and GAPDH. JAM-C siRNA 1 silenced > 75% of the huJAM-C mRNA in HUVECs after transfection and was used for all monocyte coculture experiments. Bars represent mean \u00b1 standard deviation (SD) . P values were calculated compared to control siRNA . (C) Validation of JAM-C down-regulation on HUVECs by flow cytometry. JAM-C expression on HUVECs was reduced after transfection with siRNA1 (pink) and 2 (blue), compared to JAM-C expression on non-transfected HUVECs (green). Expression of JAM-C on HUVECs transfected with control siRNA (blue) and non-transfected HUVECs remained comparable. An isotype control was included in all experiments (black). Histograms are representative of at least 2 experiments. (D) Overexpression of recombinant JAM-C does not affect distribution of other junctional proteins. Localization of the junctional proteins VE-Cadherin. PECAM-1. Occludin-1, Claudin-5, AF6 and ZO-1 were examined using confocal microscopy. No differences were observed between cells transfected with the control EGFP (marked as \u2018-\u2018) and JAM-C-EGFP constructs (marked as \u2018+\u2018). Images are representative of at least 4 independent experiments (N = 4). All antibody isotype controls included for immunofluorescence showed no staining (data not shown).(TIF)Click here for additional data file.S2 Fig(A) Cultured HUVEC monolayers were stimulated with TNF-alpha and fixed at set time-points of 0 (unstimulated), 1- and 4-hrs. Human HUVECs were stained for human JAM-C using antibody 225.3 or an isotype control. JAM-C distribution remained unchanged throughout the 4-hr time-course with JAM-C remaining mostly in the junctions. The isotype control antibody showed no staining (data not shown). (B) Cultured HUVEC monolayers were transfected with JAM-C-EGFP lentivirus and stimulated with TNF-alpha at 0 (unstimulated), 1- and 4-hrs. Distribution of JAM-C-EGFP was similar to endogenous JAM-C and localized to intercellular junctions but also showed accumulation intracellularly. (C) Analysis by flow cytometry established total JAM-C expression in 73\u201376% of HUVECs transfected with JAM-C-EGFP and this increased in a linear fashion when compared to total JAM-C. Identical profiles were seen at 0-, 1- and 4-hrs after stimulation with TNF-alpha. (D) Increased total JAM-C expression using the JAM-C-EGFP construct (squares) was typically ~2-times higher than normal endogenous JAM-C expression (circles). (E) Comparison of JAM-C expression to the starting level of expression (MFI) in the endogenous (circles), total (squares) and JAM-C-EGFP populations (triangles) confirmed expression levels in each population were stable and remained unchanged up to 24-hrs. (F) An example set of flow cytometry profiles illustrating how total JAM-C expression increases with LV-JAM-C-EGFP load (G) Titration of LV-JAM-C-EGFP on cultured HUVECs. Flow cytometry studies indicated lentivirus JAM-C-EGFP preparation on cultured HUVECs stimulated with TNF-alpha increased total surface JAM-C expression in a dose-dependent manner. Titrations tested in this experiment were 1:100 (white circles), 1:500 (grey circles), 1:1000 (white triangle), 1:2000 (grey triangle), 1:5000 (white square) and a no virus control (grey square). Profiles of JAM-C expression remained constant at all concentrations up to 24-hrs for each concentration. (H) The MFI of total JAM-C expression increased in a linear fashion with LV load. Dilution rates of 1/1000 and 1/100 were used to generate HUVECs with 1.8 (JAM-C-1.8x) and 6.6 fold increase (JAM-C-6.6x) above homeostatic JAM-C levels. (I) Increasing JAM-C expression had no effect on VE-cadherin expression. While JAM-C expression was increased on HUVECs, VE-cadherin remained unaffected. (J) VE-cadherin remained similarly unaffected on HUVECs after 4-hrs stimulation with TNF-alpha (conditions used in flow assay). Data shown is representative of two independent experiments (N = 2).(TIF)Click here for additional data file.S3 Fig Images are for two example monocytes (A and B) and contain representative cell tracking paths, plus a corresponding summary table detailing cell position and velocity analysis. The Capture point from flow, and cell position is represented by circles denoting the time (mins) and XY position. Circles with a black or red border denote a monocyte in the luminal and abluminal compartment respectively. The track direction is represented by green and red tracks for monocyte-A and -B respectively. An early timepoint of 15-mins was selected for the images in order to illustrate the tracking paths associated with each monocyte in different compartments. The extended track of Monocyte-A and\u2013B can be observed as Monocyte-2 and -3 respectively in (TIF)Click here for additional data file.S1 MovieAn adherent monocyte undergoing capture, firm adhesion and migration on luminal surfaces has a phase-grey appearance using phase contrast micrscopy (highlighted with intermittent red). As the monocyte transmigrates across the endothelial cell junction, there is change in phase appearance from grey to black (highlighted with intermittent green). Transmigrated monocytes remain phase-black for the duration of their occupancy until they undergo rTEM back onto luminal surfaces and revert to a phase-grey appearance.(MP4)Click here for additional data file.S2 MovieCell position and velocity were recorded for selected individual monocyte captured from flow onto activated HUVEC luminal surfaces. This was typically done for 25 monocytes in an individual field where monocyte position and XY coordinates were recorded at 1-min intervals. Circles with a black or red border denote a monocyte in the luminal and abluminal compartment respectively. Each colored track corresponds to an individual monocyte and a data summary table describing event, XY position and velocity, as described in (MP4)Click here for additional data file."} +{"text": "Staphylococcus\u00a0aureus (MRSA) causes serious infections that are even more difficult to treat when associated with a biofilm phenotype that facilitates evasion of the host immune system and antibiotics. As a first step toward understanding the mechanisms underlying biofilm formation, we sequenced the genomes of two prolific biofilm-forming strains belonging to the two most important globally disseminated clonal lineages, USA300 and EMRSA-15.Methicillin-resistant Staphylococcus aureus (MRSA) represents a major cause of hospital- and community-acquired infections ranging from minor skin infections to life-threatening diseases such as pneumonia, meningitis, osteomyelitis, endocarditis, and septicemia, as well as infections associated with medical implants and postsurgical wound infections. Prolonged persistence of MRSA infections is at least partly linked to the formation of biofilms in vivo (mec type IV (SCCmec IV)-harboring USA300 and EMRSA-15 . The UAS391 and H-EMRSA-15 strains belong to multilocus sequence types (ST) 80 and 22, respectively, with the former also harboring the pvl gene and an arginine catabolic mobile element (ACME) I element. This study reports the whole-genome sequencing of UAS391 and H-EMRSA-15, which is a prerequisite to understanding the molecular mechanisms underlining their pronounced clinical and biological phenotypes.Methicillin-resistant in vivo , with bi in vivo . Two of MRSA-15 35. We havin silico whole-genome maps generated from publicly available S.\u00a0aureus whole-genome sequences identified two strains as the closest homologs. UAS391 was found to have a whole-genome map identical to that of S.\u00a0aureus USA300_TCH1516 (GenBank accession no. NC_010079), whereas the map of H-EMRSA-15 showed 99.4% similarity to that of S.\u00a0aureus HO 5096 0412 (GenBank accession no. HE681097), as H-EMRSA-15 possesses an additional genomic insertion between nucleotide positions 409805 and 409733. The complete sequences of UAS391 and H-EMRSA-15 were then determined on an Illumina HiSeq2000 platform . Sequence data of UAS391 and H-EMRSA-15 were de novo assembled using Velvet and CLC Genomics Workbench 6.5.1 . Assembled contigs were ordered against the UAS391 and H-EMRSA-15 whole-genome maps using MapSolver software . De novo assembled sequences were further corroborated by reference assembly using genome sequences of TCH1516 and HO 5096 0412 as corresponding templates. Genome analysis of UAS391 and H-EMRSA-15 is in progress to reveal the genetic and genomic basis of biofilm formation in these two major clonal lineages. We used the NCBI Prokaryotic Genome Automatic Annotation Pipeline (PGAAP) for function-based annotation maps of UAS391 and H-EMRSA-15 were obtained using the Argus Optical Mapping System . Comparison with CP007690 and CP007659, respectively. The versions described in this paper are the first versions.The genome sequences for UAS391 and H-EMRSA-15 have been deposited at DDBJ/EMBL/GenBank under the accession numbers"} +{"text": "Intestinal monocytes/macrophages sustain the intestinal immune homeostasis and might be an attractive therapeutic target for the management of inflammatory bowel disease (IBD). Granulocyte macrophage colony-stimulating factor (GM-CSF) exerts beneficial effects in intestinal inflammation and promotes STAT3-mediated expansion of myeloid-derived suppressor cells (MDSCs). We explored whether GM-CSF mediates its beneficial effects in IBD via myeloid regulatory cells (Mreg).Rag1-/- mice with increased production of IL-4, IL-10, IL-13 and decreased production of IFN\u03b3 in LPMCs. Confirming this finding, GMaM attract T cells and shape their differentiation towards Th2 cells in vitro. In addition, GMaM induce regulatory (Foxp3+) T cells (Treg) in vitro and adoptive transfer of GMaM in chronic DSS-induced colitis ameliorates disease in vivo with accelerated gut homing of GMaM and induction of colonic Treg. Myeloid-cell specific STAT3 activation protects gp130757F/F mice from colitis via MDSC expansion and increased production of suppressive and protective cytokines. LysMcre/STAT3flox mice with myeloid-specific STAT3-deficiency show opposite effects and are not protected from colitis. Additionally, MDSCs of gp130757F/F mice produce significantly more IL-4, IL-10 and IL-13.Here we show that GM-CSF i) provokes non-classical monocyte activation; ii) drives monocytes towards an anti-inflammatory phenotype; iii) enhances innate immune functions; iv) primes monocyte responses to secondary microbial stimuli; and v) accelerates epithelial healing via monocytes. GM-CSF-activated monocytes (GMaM) show therapeutic activity in T cell-induced colitis in In summary, beneficial effects of GM-CSF in IBD may possibly be mediated through reprogramming of monocytes and MDSCs via enhanced innate immune functions as well as regulation of adaptive immunity. Our findings support the exploration of stimulating rather than suppressive therapies for patients with IBD and underpin that Mreg might become a promising novel cell-based therapeutic option."} +{"text": "This review is to explore whether potential gene interactions in the cell cycles of gametes, zygotes, and embryonic stem (ES) cells are associated with the development of cancer.MEDPILOT at the Central Library of the University of Cologne, Germany that covers 5,800 international medical journals and 4,300 E-journals was used to collect data. The initial searches were done in December 2012 and additional searches in October 2013\u2013May 2015. The search terms included \u201ccancer development,\u201d \u201cgene interaction,\u201d and \u201cES cells,\u201d and the time period was between 1998 and 2015. A total of 147 articles in English language only were included in this review.de novo gene synthesis and neofunctionalization. Post-translationally, mutated genes are preserved in pre-neoplastic ES cell subpopulations that can give rise to overt cancer stem cells. Thus, TFs operate as cell/disease-specific epigenetic messengers triggering clinical expression of neoplasms.Transgenerational gene translation is implemented in the zygote through interactions of epigenetic isoforms of transcription factors (TFs) from parental gametes, predominantly during the first two zygote cleavages. Pluripotent transcription factors may provide interacting links with mutated genes during zygote-to-ES cell switches. Translation of post-transcriptional carcinogenic genes is implemented by abnormally spliced, tumor-specific isoforms of gene-encoded mRNA/non-coding RNA variants of TFs employing Potential gene interactions in the cell cycle of gametes, zygotes, and ES cells may play some roles in the development of cancer. DNA-encoded gene replication is usually stable but not permanent during meiotic and mitotic cell cycles. Indeed, the genome can be interpreted as an evolutionary organ of epigenetic gene transcription/translation; its complex gene replication patterns affect normal, as well as neoplastic cells , 2. The Mutagenic and carcinogenic properties of somatic cells are acquired by gene neosynthesis and neofunctionalization through alternative splicing of enzymatic messenger-/non-coding RNAs (m-/ncRNA) to isoforms that produce neoplastic effector proteins for translation , 4. It iGene translation is the final step in phenotypic expression of post-transcriptional genes and is therefore important in cancer formation and prevention. It is implemented in loops of interacting networks of epigenetic TFs involving alternatively spliced mRNA/ncRNA isoforms and effector proteins through binding sites at cis-reacting imprinting control regions (ICRs) and UTR (untranslated gene regions). Research on potential interactions between/among genes involved in gene translation in normal and neoplastic cell replications may help elucidate the mechanisms of cancer etiology and treatment.Epigenetic TFs control mitotic cell cycles as drivers. They originate in parental gametes and are transmitted transgenerationally to the fertilized ovum. In the zygote, pronuclear TFs interact with, and translate, genes of their fusion partners, trigger cleavages of the zygote, cause zygote-to-ES cell switches, and induce proliferation/differentiation of diploid gamete-to-zygote-to-embryonic stem (GZES) cells. It is now well established that parental occupational exposure to environmental carcinogens causes dMemory is based on transcriptional decisions of parental genes on events of the past that are mitotically transmitted through TFs to ES cells. This requires erasure of preceding/ancestral DNA-CpG transcriptional patterns through excision-glycosylases converting DNA-CpG-5mC (5methyl-cytosine) to 5hmC (5hydroxy-methyl-cytosine), to 5cC (5carboxyl-cytosine) and, finally, to thymine , 17. ModMEGs) at the 4\u20138 cell stage is implemented through MEG-dominated alterations of the CpG histone methylation status by gene-encoded methyltransferase/demethylases (DNMT/DM) /ZAR-like (ZARL) proteins in translational control sequences (TCS) that bind to maternal mRNAs at 3\u2032UTR and Piwil2 (protein of the ARGONAUTE family) (Mil1/Blimp1 (transcription repressors of somatic genes Hox and Snail). Maternal modulating TFs include cis-reacting RNA-binding proteins (RBPs), cytoplasmic polyadenylation elements (CPEs), and \u201cdeleted-azoospermia-like\u201d (DAZL) family. CPEs mediate adaptation of gonadal homeostasis, metabolism, gene transcription of cell cycle cyclins, and apoptosis. DAZL is an mRNA translational component that controls RBP networks in the initial stages of zygote development until the embryonic genome is composed at the third post-fusion cleavage , Nlrp5 (Mater), subcortical maternal complexes (SCMC), Nlrp14 (nucleotide-binding oligomerization in early embryonic development), Hsf1 (heat shock), Npm2 (nucleoplasmin), Cdh1 (e-cadherin), Pms2 (mismatch repair gene2), Ezh2 , and Smarca 4 (Brg1).Transcriptional incompatibilities between parental pronuclei are silenced by histone-modulations during interactions in the zygote ensuring transcription stability in ES cell development. During the first two zygotic cleavages, reprograming is under signaling dominance of maternal gonad-specific factor (GSF)\u2013maternal effect genes (MEGs) . The firDNMT/DM) , 21. MEGat 3\u2032UTR . Mutant family) . Also incleavage , 25. In or sites and liceMEGs exert fundamental influences on the development of the ES cell genome. They transmit genetic/epigenetic maternal memory information to the early stages of zygote-to-ES cell switches , 36. IntGenes interact with enzymatic TFs at ICRs. ICRs are transcriptional/translational sites composed of repetitive, germline-derived, differentially methylated DNA sequences on chromosomes positioned between eu- and heterochromatin. They function as insulators in genomic reprograming by delimiting acetylation-mediated barriers against allele-specific interactions of somatic as well as imprinted genes . ICRs seGenes transcribed in mitoses become functional and exercise their properties through translation to effector proteins.A multifunctional mRNA-/polymerase A-binding protein (PABP1) serves as scaffold for protein\u2013protein interactions. Production of effector proteins is mediated/controlled by translational TFs-mRNA/ncRNA. Indeed, unless genes are translated, genes are silenced and stored in heterochromatin. Thus, gene transcription and translation are interdependent but distinct and consecutive processes that can be separated by the specificities of their enzymes. Translating TFs include lysyl-tRNA synthetase (lysRS), isoform1 of translation elongation factor eEF that adjusts transcriptional yields to translational needs by associating with elongating RNA Pol II at 3\u2032UTR, and cell fate effector DACHS. The latter mediates cytoplasmic oncogenic translation (for example in EMT) by affecting Y box-binding snail proteins. Clearly, it is translation that directs phenotypic expression of transcribed DNA sequences and therefore decides on the phenotypic, clinical expression of carcinogenic genes.Pre-mRNAs are formed from DNA templates at UTRs. They are specified by APA and RNA-recognition motifs (RRMs) for splicing into isoforms with chAn important group among TFs is non-coding RNAs (ncRNA). They enzymatically regulate pre-mRNA splicing and edit mRNA-mediated gene transcription/translation , 45. Edi(EZH1) pathways with functional overlaps in EMT-coding pathways; and, importantly, are involved in stem cell pluripotency (HOXA gene cluster) is a highly specific regulator for gene expression in switches from granulocytic proliferation to maturation phases in integrin-controlled cell cycles. Furthermore, lncRNAs control gene transcription by recruitment of silencing complexes to homology-containing loci of the genome. Thus, lncRNAs are important in embryonic development and in the pathogenesis of neoplastic diseases /small interfering RNAs (s/siRNAs) , 50, 51,ipotency . Secondadiseases .An additional group of indispensible TFs, characterized as being \u201cadjuvant,\u201d is shown in Figure TFs are enzymes that do not catalyze their targets directly. Rather, they control protein production indirectly through isoform copies that are produced by alternate splicing. In fact, 90% of gene-encoded mRNAs and ncRNAs undergo alternative splicing of their exons (the information-bearing parts of the gene) . APA is p53/p63/73 , 72. ForFBS/TSS) . Other iFBS/TSS) (a cell FBS/TSS) . This isModes of translation differ in different types of neoplasms. Some switch to translation by neoplasm tic splicing only during proliferation; others, mostly of poor clinical prognosis, periodically turn dysregulated gene-specific ncRNA/mRNA interactions on and off. Still others monitor translation of gene transcriptomes by spontaneous expression of their own disease-associated isoforms without epigenetic triggers , 76. ForClearly, carcinogenic isoform variants of TFs are of key importance in neoplastic translation and phenotypic expression. This complies with the concept of carcinomas being epigenetic diseases . It is aDe novo synthesis of genes has a major impact on evolutionary traits. Indeed, genomes adopt to new epigenetic situations, such as transgenerational information, exposure to environmental carcinogenic contaminants, and other forms of biotic stress that are known to be mutagenic in ES cells (MEG-derived ubiquitin-mediated proteolysins1 (ZAPAC-Ump1) tails and of co-translational proteasomal proteins. Complexes appear to bind to 3\u2032-UTR-mRNA elements via ncRNA predominAC-Ump1) , 81. IntAC-Ump1) . Also invailable . CCR4-NOia ncRNA . Interes (PEGCs) . Thus, i (PEGCs) , 86. Nov (PEGCs) , 87.GATA1-regulated complexes driven by context-specific nucleosomes that may determine the clinical outcome of cancer patients , have been used in anti-neoplastic therapy . The patand Zac1 , 106. Inand Zac1 . Imprintand Zac1 , 99, 106and Zac1 from addand Zac1 , 109.de novo synthesis of embryonic TF isoforms (\u03b2-cateninWNT/ (JAK/STAT (NOTCH (MAPK/ERK (PI3K/AKT (JAK (Janus activating tyrosine kinase) triggers the Wnt/\u03b2-catenin pathway (encoded by gene Ctnnb1) as tumorigenic gatekeeper in GZES cells for myeloproliferative diseases , reinforces network pluripotency, and promotes resistance to differentiation. Switches to homologous TF expression (Oct4+/+) induce GZES cell differentiation , Trp53 (tumor repressor), and Rb1 (retinoblastoma suppressor) , 136. Ovpathways .Akt signaling and binds to Fox01, a nuclear TF in metabolic and energy homeostasis , HDACi, H3 histone acetylator of tumor-suppressor gene PRDX2, and polo-like kinase SNK/PLK2 . Other approaches have re-enforced tumor suppressor genes by inhibition of lysine-specific LSD1 demethylase for H3K4me1, 2 in acute myelogenous leukemia and small cell lung cancer by activation of all-retinoic-acid differentiation pathways, or have altered DNMT-mediated DNA-CpG methylation patterns (Promising attempts of interfering with enzymatic reactions have so far mostly been limited to patterns . Indeed,MEGs in both pronuclei. Pre-/neoplastic DNA damage is modulated by chromatin histone H3 methylation/acetylation and interacts with its zygotic fusion partner (Figure Genetics and epigenetics constitute a functional entity in embryonic and post-natal cell proliferation. This review focuses on potential gene interactions in cell cycles of gametes, zygotes, and ES cells that may be related to neoplastic transformation. Figure r Figure C, predomr Figure D, and arr Figure E. ClearlIn clinically overt carcinogenesis Figure F, carcinThe author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Calpain is activated following myocardial infarction and ablation of calpastatin (CAST), an endogenous inhibitor of calpains, promotes left ventricular remodeling after myocardial infarction (MI). The present study aimed to investigate the effect of transgenic over-expression of CAST on the post-infarction myocardial remodeling process.We established transgenic mice (TG) ubiquitously over-expressing human CAST protein and produced MI in TG mice and C57BL/6J wild-type (WT) littermates.The CAST protein expression was profoundly upregulated in the myocardial tissue of TG mice compared with WT littermates (P < 0.01). Overexpression of CAST significantly reduced the infarct size (P < 0.01) and blunted MI-induced interventricular hypertrophy, global myocardial fibrosis and collagen I and collagen III deposition, hypotension and hemodynamic disturbances at 21 days after MI. Moreover, the MI-induced up-regulation and activation of calpains were obviously attenuated in CAST TG mice. MI-induced down-regulation of CAST was partially reversed in TG mice. Additionally, the MI-caused imbalance of matrix metalloproteinases and their inhibitors was improved in TG mice.Transgenic over-expression of CAST inhibits calpain activation and attenuates post-infarction myocardial remodeling. After an acute myocardial infarction (MI), the global heart undergoes a series of structural changes, termed post-infarction myocardial remodeling, leading to the incidence of heart failure . Heart fThe role of proteolytic systems in the development of myocardial remodeling has received much attention . The calin vivo model of MI to investigate the role of CAST over-expression in post-infarction myocardial remodeling.Therefore, the present study established transgenic mice ubiquitously over-expressing CAST and used cultured an This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Ethics Committee and the Institutional Animal Care and Use Committee of Chengdu Military General Hospital. All surgery was performed under anesthesia, and all efforts were made to minimize suffering.Transgenic founder mice were generated on a C57BL/6J genetic background. The cDNA from human CAST was cloned into the pDown-CAST vector and verified by sequencing . The linFasting plasma glucose (FPG), triglycerides (TG), total cholesterol (TC), low-density lipoprotein-cholesterol (LDL-C) and high-density lipoprotein-cholesterol (HDL-C) of the CAST transgenic (TG) and wild-type (WT) mice were measured using a commercially available kit in accordance with the manufacturer's instructions.ad libitum food and water. All surgical procedures were performed using aseptic techniques. MI model was constructed by left anterior descending (LAD) artery ligation as previous described [CAST TG and WT mice aged 6\u20138 weeks were housed under a 12 h/12 h day/night cycle, with For evaluation of left ventricular hemodynamics, mice were anesthetized by intraperitoneal injection of pentobarbital (75 mg/kg), then a 1.4-F microconductance pressure catheter was introduced through the right common carotid artery into the ascending aorta and then advanced into the left ventricle as described previously . Data weHearts were retrograde-perfused with phosphate-buffered saline and were fixed with 10% (v/v) formalin and embedded in paraffin. Paraffin sections (5 \u03bcm thickness) were stained with Masson\u2019s trichrome for assessments of infarct scar size and fibrotic area . The posThe paraffin heart sections (5 \u03bcm thickness) were treated with 3% hydrogen peroxide for 5 min. The sections then were incubated with the primary antibodies: anti-collagen I and anti-collagen III at 4\u00b0C overnight. After being washed, the paraffin sections were incubated with goat anti-rabbit immunoglobulin G biotinylated secondary antibody for 1 h at room temperature, and stained using a DAB detection kit .The proteins of heart were extracted using a protein extraction kit . The protein concentrations were determined with an enhanced BCA Protein Assay Kit . Forty micrograms of extracted protein were loaded onto 12% SDS polyacrylamide gels. The separated proteins were then transferred to PVDF membranes. Membranes were blocked with TBS-T containing 5% skim powdered milk for 1 hour and then incubated with anti-calpain-1 , anti-calpain-2 , anti-CAST , anti-matrix metalloproteinase (MMP)-2 , anti-MMP-9 , anti-tissue inhibitor of MMP (TIMP)-1 , anti-TIMP-2 , anti-TGF-\u03b2 and GAPDH antibodies overnight. Membranes were rinsed three times with TBS-T and incubated with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G for 1 hour. Membranes were rinsed three times with TBS-T. Chemiluminescence detection reagent were dropwise added on the membranes. The luminescent signal was detected by exposure to x-ray film.Calpain activity was determined by using a fluorescence substrate N-Succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin (Suc-LLVY-AMC) with a calpain activity assay kit as described in a previous study .\u2212\u0394\u0394CT and was normalized to a housekeeping gene 18s rRNA. Each sample was run and analyzed in triplicate. The average of the relative amount of each mRNA in WT group was defined as 1.0. PCR primer sequence is listed as follow: CAST: F, 5\u2019-GAG CAG TCA GCC TTC CAG AC-3\u2019; R, 5\u2019-TCT GTG GTA CTC ATG CTG GG-3\u2019; 18s rRNA: F, 5\u2019-CGC GGT TCT ATT TTG TTG GTT T-3\u2019; R: 5\u2019-GCG CCG GTC CAA GAA TTT-3\u2019.The mRNA expression of mouse endogenous CAST in the heart tissue of WT and TG mice was detected by RT-PCR. The RT-PCR was performed using One Step SYBR Prime Scrip RT-PCR Kit II . The relative amount of mRNA was calculated by 2post-hoc t-tests . Survival curves were created by the method of Kaplan and Meier, and compared by log-rank test. P < 0.05 was considered statistically significant.Continuous data are presented as mean \u00b1 standard error (SE). Comparisons between the groups were determined by ANOVA with The transgenic fragments containing the human CAST cDNA were microinjected into the male pronuclei of 150 fertilized oocytes of C57BL/6J mice. A total of 90 injected eggs were implanted into the oviducts of 3 pseudo-pregnant foster mothers, which gave birth to 23 offspring. Two offspring mice were identified to be carrying the CAST cDNA by PCR analysis . WesternThe infarct size at 24 hours after surgery has no difference between the TG and WT mice . No infaMI produced a hypotension status in WT mice, which was partially reserved by transgenic expressing CAST . Hemodynvs. 6.56 \u00b1 0.16 mg/g, P<0.01) and TG mice . However, the increase in HW/BW ratio was blunted in TG mice (P<0.01). Histological analysis revealed that MI caused significant cardiomyocytic hypertrophy of interventricular septum and remarkable cardiac fibrosis of infarct area, border zone, and even remote region Click here for additional data file."} +{"text": "The development of resistance against metronidazole , thrown a challenge to find out alternate medication.Trichomoniasis is the most common sexually transmitted To Design and synthesize novel agents to be effective against MTZ resistant trichomoniasis.Trichomonas.[Trichomonas activity.[14-28) as N-alkyl/aralkyl-4-(3-substituted-3-phenylpropyl)piperazine-1-carbodithioates have been designed, synthesized and evaluated for their anti-Trichomonas activity profile to be useful as vaginal microbicide. All compounds were tested for safety through cytotoxic assay against human cervical cell line (Hela) and compatibility with vaginal flora, Lactobacillus.Benzenepropanamines and selective serotonin reuptake inhibitor (SSRI) antidepressants viz. fluoxetine and paroxetine, possibly interacting with sulfhydryl groups present over chomonas. Alongsidactivity. In our oCompound 17) was the most promising compound with anti\u2013Trichomonas activity in comparison to MTZ . Six compounds were more active against resistant strain in comparison to Metronidazole. The extreme safety profile against vaginal epithelium (HeLa cells) and compatibility with vaginal flora (lactobacillus) supported its suitability for vaginal application.2-(pyrrolidin-1-yl)ethyl 4-(3-oxo-3-phenylpropyl)piperazine-1-carbodithioate (A novel molecule to be effective against resistant Trichomoniasis in comparison to MTZ has been identified to be developed for topical application emphasizing on improvement of women reproductive health.None declared."} +{"text": "Foot-and-mouth disease virus (FMDV) possess a positive sense, single stranded RNA genome. Internal ribosomal entry site (IRES) element exists within its 5\u2032 untranslated region (5\u2032UTR) of the viral RNA. Translation of the viral RNA is initiated by internal entry of the 40S ribosome within the IRES element. This process is facilitated by cellular factors known as IRES trans-acting factors (ITAFs).Foot-and-mouth disease (FMD) is host-restricted disease for cloven-hoofed animals such as cattle and pigs, but the factors determining the host range have not been identified yet. Although, ITAFs are known to promote IRES-mediated translation, these findings were confirmed only in cells derived from FMDV-insusceptible animals so far.Renilla and Firefly luciferase genes. Furthermore, we analyzed the effect of the cellular factors on IRES-mediated translation by silencing the cellular factors using siRNA in both FMDV-susceptible and -insusceptible animal cells.We evaluated and compared the IRES-mediated translation activities among cell lines derived from four different animal species using bicistronic luciferase reporter plasmid, which possesses an FMDV-IRES element between 45 promoted IRES-mediated translation in all cell lines, and the effects of poly-pyrimidine tract binding protein (PTB) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) were observed only in FMDV-susceptible cells. Thus, PTB and 4E-BP1 may influence the host range of FMDV.Our data indicated that IRES-mediated translational activity was not linked to FMDV host range. ITAF45 promoted IRES-mediated translation in all cells, and the effects of PTB and 4E-BP1 were observed only in FMDV-susceptible cells.IRES-mediated translation activity of FMDV was not predictive of its host range. ITAF Foot-and-mouth disease (FMD) is a highly contagious infectious disease in cloven-hoofed animals such as cattle, pigs, and other related species . It is cAphtovirus genus of the Picornaviridae family. FMDV possesses an internal ribosomal entry site (IRES) element within the 5\u2032 untranslated region (5\u2032UTR), and virus proteins are synthesized by IRES-mediated translation . The membranes were then washed 3 times with 0.05\u00a0% Tween- 20-TBS for 10\u00a0min. Secondary antibodies were subsequently added, and specific protein bands were visualized with enhanced chemical luminescence .t-test to evaluate significant differences .All data are presented as means\u2009\u00b1\u2009SEM from three independent experiments. Statistical analysis was performed using Student\u2019s All experiments were approved by the ethics committee of Kagoshima University. This study did not include animal experiments.Not applicable.Data and materials are available upon request."} +{"text": "The Dixon-Water-Fat segmentation (DWFS) method is a standard attenuation correction (AC) method in PET/MRI on the Siemens mMR and has demonstrated a systematic quantitative bias in [18F]-FDG-PET/MRI studies of the brain compared to PET/CT. The aim of this study was to evaluate the impact of DWFS-AC in a hybrid PET/MR scanner on regional and global quantitation of [11C]-PiB cerebral amyloid imaging of the brain and in the clinical reading.Twenty-eight healthy volunteers, 6 with mild cognitive impairment (MCI), 4 with Alzheimers disease (AD), and 6 other dementia cases underwent a simultaneous PET/MRI (Siemens mMR) acquisition 40 min pi of (170-709) MBq [11C]-PiB. A single 30 min frame was reconstructed twice with the only difference being the AC calculation. AC was performed using either the standard DWFS or a head low-dose CT scan acquired independently on the same day. Activity concentration (Bq/mL) was sampled on AC-PET from symmetrically delineated ROI\u2019s. Ratios of region-to-cerebellar grey matter (SUVr) were calculated. Amyloid uptake was considered abnormal at cortical SUVr >1.5 or increased amyloid uptake in two or more grey matter regions on visual evaluation.The average activity concentration in all ROI\u2019s was biased by -18 % in DWFS-AC compared to CT-AC. Although the visual categorization of both, amyloid-positive (n=11) and -negative (n=33) scans was unaffected by DWFS-AC, 3 healthy subjects were quantitatively reclassified as amyloid positive. The SUVr values were overestimated by 0.11 in the caudate nuclei and 0.04 in lateral cortical ROI\u2019s in DWFS-AC compared to CT-AC.The visual and quantitative consequences of MR-AC using DWFS in the brain with [11C]-PiB exhibit a noticeable radially variable bias. Although robust in visual evaluation, the quantitative diagnostic criteria (SUVr) using this biomarker with DWFS-AC may need to be modified."} +{"text": "Phleum pratense p 5], Merck/ALK-Abell\u00f3), a sublingual Timothy grass immunotherapy tablet, has been evaluated in several randomized, placebo-controlled, double-blind trials; three of these trials were conducted in adults and children in North America (the United States and Canada) who have allergic rhinitis with or without conjunctivitis (AR/C). We conducted a post-hoc analysis to investigate the effect of MK-7243 in Canadian subpopulations.The effect of MK-7243 ; P05239 ; and P08067 . Trial data from the same grass pollen seasons (GPS) were pooled. Subjects received once-daily MK-7243 or placebo starting \u226512 wk before and continuing throughout the GPS, for a mean total of \u226523 wk. The therapeutic effect of MK-7243 was evaluated for rhinoconjunctivitis symptoms and symptomatic medication use, measured as a total combined score averaged over the entire GPS. Safety was assessed by monitoring adverse events (AEs).Canadian subjects taking MK-7243 (n=42) in the pooled adult-pediatric 2009 trials showed a 38% mean TCS reduction versus placebo . Canadian subjects taking MK-7243 (n=122) in the adult-pediatric 2012 trial showed a 33% mean TCS reduction relative to placebo . Approximately 90% of treatment-related AEs were mild or moderate in severity. No serious or life-threatening treatment-related AEs occurred.MK-7243 Timothy grass sublingual tablet significantly improved AR/C induced by Timothy grass pollen in Canadian adults and children 5 y and older. Similar efficacy and safety results were obtained for the overall populations of the three trials.ClinicalTrials.gov Identifiers: NCT00562159; NCT00550550; NCT01385371"} +{"text": "Plasmodium falciparum and Plasmodium vivax infections compromise dendritic cell (DC) function and expand regulatory T (Treg) cells in both clinical disease and experimental human sub-microscopic infection. Conversely, in asymptomatic microscopy-positive (patent) P. falciparum or P. vivax infection in endemic areas, blood DC increase or retain HLA-DR expression and Treg cells exhibit reduced activation, suggesting that DC and Treg cells contribute to the control of patent asymptomatic infection. The effect of sub-microscopic (sub-patent) asymptomatic Plasmodium infection on DC and Treg cells in malaria-endemic area residents remains unclear.P. falciparum and 15 with sub-microscopic P. vivax infection. Flow cytometric data were re-analysed after re-grouping asymptomatic individuals according to PCR results into negative controls, sub-microscopic and microscopic parasitaemia to examine DC and Treg cell phenotype in sub-microscopic infection.In a cross-sectional household survey conducted in Papua, Indonesia, 162 asymptomatic adults were prospectively evaluated for DC and Treg cells using field-based flow cytometry. Of these, 161 individuals (99\u00a0%) were assessed retrospectively by polymerase chain reaction (PCR), 19 of whom had sub-microscopic infection with P. falciparum or P. vivax infection had DC HLA-DR expression and Treg cell activation comparable to PCR-negative controls. Sub-microscopic P. falciparum infection was associated with lower peripheral CD4+ T cells and lymphocytes, however sub-microscopic Plasmodium infection had no apparent effect on DC sub-set number or Treg cell frequency.Asymptomatic adults with sub-microscopic Plasmodium infection, no phenotypic evidence of dysregulation of DC and Treg cells was observed in asymptomatic sub-microscopic Plasmodium infection in Indonesian adults. This is consistent with DC and Treg cells retaining their functional capacity in sub-microscopic asymptomatic infection with P. falciparum or P. vivax in malaria-endemic areas.In contrast to the impairment of DC maturation/function and the activation of Treg cells seen with sub-microscopic parasitaemia in primary experimental human The online version of this article (doi:10.1186/s12936-016-1382-7) contains supplementary material, which is available to authorized users. Plasmodium parasitaemia without the presence of symptoms. This is thought to reflect either the early stages of infection or the acquisition of clinical immunity that restricts parasite expansion and prevents clinical disease positive) plasmacytoid DC (pDC), CD1c+ (BDCA-1 positive) mDC (CD1c+ mDC), and CD141+ (BDCA-3 positive) mDC (CD141+ mDC) as per flow cytometric gating of DC sub-sets [+ mDC, and CD141+ mDC in asymptomatic adults with either sub-microscopic, or patent P. falciparum or P. vivax infection were not significantly different from controls of participants and reported as the absolute number of CD303+CD25+CD127low lymphocytes, and sub-divided into CD45RA\u2212-activated and CD45RA+-resting Treg cells as per flow cytometric gating [P. falciparum or P. vivax infection, there were no changes in the absolute or relative number of Treg cells compared to controls and rTreg-to-aTreg ratios were comparable to aparasitaemic controls of participants and were identified by flow cytometry as CD4c gating are highly suppressive and proliferative Treg cells [+CD45RA+-resting Treg cells (rTreg) provide a reservoir capable of subsequent activation. Adults with asymptomatic sub-microscopic P. falciparum or P. vivax infection showed neither a reduced nor an over-activated Treg cell response, characterized by aTreg frequencies and rTreg-to-aTreg ratios that were comparable to uninfected adult controls. This contrasted with the reduced Treg cell response characterized by a relative decrease in aTreg frequency and increase in rTreg-to-aTreg ratio in asymptomatic patent Plasmodium infection [In contrast to DC activation, Treg cell activation may contribute to progression of disease by suppressing T cell responses and host immunity, favouring parasite growth , 21, 28.eg cells , while Cnfection . Furthernfection , 20. TheCorrelations between parasitaemia and Treg cells could not be examined since the degree of sub-microscopic parasitaemia was not quantified. Boyle and colleagues recentlyP. falciparum infection, peripheral lymphocytes and CD4+ T cells were reduced in asymptomatic adults, a finding similar to that observed in experimental sub-microscopic P. falciparum infection of na\u00efve volunteers [P. falciparum or P. vivax infection, suggesting that stable Treg cells potentially minimize immune suppression and thereby support immune activation. Although asymptomatic adults with sub-microscopic P. falciparum infection were lymphopenic, their lymphocyte and CD4+ T cell counts were still significantly higher compared to adults with species-matched acute malaria.In sub-microscopic lunteers . DespiteThe study had a number of limitations. As patent individuals were treated and sub-microscopic infections were only retrospectively identified, participants could not be longitudinally followed in this study, preventing the assessment of whether infected individuals remained asymptomatic, cleared the infection, or developed symptomatic malaria. It is also acknowledged that the sample sizes in the asymptomatic sub-microscopic groups with DC data were low due to poor sample quality and/or limitations in available blood volume. In addition, PCR was not performed on the sub-set of children in the original study because P. falciparum or P. vivax infection. DC in both sub-microscopic and patent infection expressed normal levels of HLA-DR; Treg cells exhibited reduced activation in patent infections and were unaltered in sub-microscopic infections. The lack of apparent phenotypic dysregulation in DC and Treg cells identified in asymptomatic sub-microscopic Indonesian adults contrasts with the compromised DC induced by comparable PCR-level parasitaemia in experimental infection of malaria-na\u00efve volunteers. The data extend the view that these immune cells may contribute to anti-parasitic immune mechanisms. Collectively, the findings suggest that malaria vaccines should aim to ensure appropriate DC and Treg cell responsiveness, a state observed in asymptomatic malaria-endemic area adults with sub-microscopic Plasmodium infection, but not found in adult volunteers experiencing first exposure to sub-microscopic Plasmodium infection.The study provides no phenotypic evidence of DC and Treg cell dysregulation in asymptomatic sub-microscopic"} +{"text": "Even though non-immediate type allergic reactions with beta-lactam antibiotics occur more frequently than immediate reactions little is known about the actual prevalence of non-immediate beta-lactam allergy in childhood.To determine the rate and risk factors for non-immediate type beta-lactam allergy in children with a history of allergic reaction occurring at least 1 hour after the drug intake.A prospective study was performed using ENDA questionnaire in 103 children with 124 non-immediate type hypersensitivity reactions. Prick and intradermal skin testing with major/minor determinant mixture, penicilin G, ampicillin, amoxicillin-clavulanate and the culprit drug were done. In patients with negative skin tests, patch and intradermal tests with late readings were done with the culprit drug. Drug provocation was performed in case of negative interventional tests.Fifteen children (14.6%) with 16 non-immediate reactions (12.9%) was diagnosed as beta-lactam allergic. The drugs responsible for non-immediate type beta-lactam allergy were amoxicillin-clavulanate (75%), sulbactam-ampicillin (6.3%), penicillin (6.3%), ceftriaxone (6.3%), cefaclor (6.3%).The diagnosis of non-immediate type beta-lactam allergy was determined by provocation tests (n=11) prick/epidermal tests (n=3), patch tests (n=2). Only angioedema was found to increase the risk for diagnosis of non-immediate type beta-lactam allergy .Diagnosis of drug allergy is also common in children with a history of nonimmediate reactions to beta-lactams. Classification due to timing of the reaction might not be appropriate to define the underlying pathogenesis of beta-lactam hypersensitivity since three patients had a positive skin test indicating IgE mediated beta-lactam allergy.Intradermal tests with late readings might not be practical in the diagnosis of non-immediate type beta-lactam allergy."} +{"text": "Mesenchymal stem cells (MSCs) have potent stabilizing effects on the vascular endothelium injury, inhibiting endothelial permeability in lung injury via paracrine hepatocyte growth factor (HGF). Recently, it has been indicated that MSCs secreted more factors by MSC-endothelial cell (MSC-EC) interaction. We hypothesized that MSC-EC interaction restored endothelial permeability induced by lipopolysaccharide (LPS) via paracrine HGF.We investigated the endothelial permeability induced by LPS under two co-culture conditions in transwells. HPMECs were added into the upper chambers of cell-culture inserts, while there two different co-culture conditions in the lower side of transwells as follows: MSC-EC interaction group: MSCs and HPMEC contact coculture in the lower chambers; and MSC groups: MSCs only in the lower chambers. The endothelial permeability in the upper side of transwells was detected. Then the concentration of HGF was measured in the culture medium using an enzyme-linked immunosorbent assay kit, following by neutralizing HGF with anti-HGF antibody in the co-culture medium. In addition, VE-cadherin protein expression were measured under the co-culture conditions by western blot, adherens junctions (AJs) protein including F-actin and VE-cadherin were detected by immunofluorescence technique as well.P < 0.01). Meanwhile, MSC-EC interaction more significantly decreased endothelial permeability induced by LPS (P < 0.05 or P < 0.01). Moreover, HGF levels in the MSC-EC interaction group were much higher than those of the MSC group (P < 0.01). However, neutralizing HGF with anti-HGF antibody inhibited the role of MSC-EC interaction in improving endothelial permeability (P < 0.05). Compared with the MSC group, MSC-EC interaction increased VE-cadherin protein expression (P < 0.01), and restored remodeling of F-actin and junctional localization of VEcadherin. However, the MSC effect was significantly blocked by anti-HGF antibody (P < 0.05 or P < 0.01).The permeability significantly increased after LPS stimulation in a dose-dependent and time-dependent manner (These data suggest that MSC-EC interaction decreased endothelial permeability induced by LPS, which was mainly attributed to HGF secreted by hMSCs. The main mechanisms of HGF restoring the integrity of EC monolayers are remodeling of endothelial intercellular AJs and decreasing caveolin-1 protein expression."} +{"text": "However, data are acquired in an ECG synchronized manner over multiple heart beats and the impact of cardiac arrhythmia, which can frequently be encountered in patients with atrial fibrillation (AF), on flow parameters is unclear. To assess the impact of arrhythmia and thus RR interval variability on ECG-gated PC MRI acquisitions we have performed a simulation study to systematically investigate the impact of beat-to-beat variations on ECG gated multi-beat flow imaging with MRI using real time in-vivo TEE data in 5 AF patients with known arrhythmia.Blood flow quantification using ECG gated phase contrast (PC) MRI has proven to be a useful clinical tool for the evaluation of cardiovascular pathologies such aortic valve disease or cardiac shuntsReal-time 2-dimensional transesophageal echocardiography (TEE) in color flow Doppler mode was performed in five patients in AF . TEE data provided real-time left atrial (LA) and left ventricular (LV) flow velocities in 2-4 consecutive cardiac cycles with different RR-interval durations. PC-MRI acquisitions were simulated using k-space data, simulated from the TEE velocity measures, to construct time-resolved PC-MRI k-space data for a segmented sampling scheme typically used for ECG gated 2D PC MRI. Each simulation was repeated 100 times to minimize effects from data that may be weighted to a particular beat in the center of k-space. The resulting LA and LV velocities were compared to the average TEE velocities and to the TEE velocity data from individual cardiac cycles. Average velocity measurements were quantitatively compared using regions of interest (ROIs) in the LA and LV.The average R-R interval duration for all five patients was 643 \u00b1 161ms, with a range of 364-1000ms. Results of the simulation study (see figure) demonstrated that ECG gated flow MRI in patients with cardiac arrhythmia can reliably assess patterns of blood flow velocities that are consistently present over multiple heart beats (persistent or average flow patterns). ECG gated flow imaging with MRI could reproduce persistent average LA and LV mean velocities within 7.0-7.4% compared to TEE. Average TEE and simulated MR velocity measurements within the ROIs were not significantly different.PC-MRI velocity measurements in patients with varying R-R interval durations are not significantly different from time-averaged real-time velocity data for a typical segmented k-space data acquisition schemes. Though beat-to-beat variations in atrial velocities that were observed with TEE cannot be detected with ECG gated multi-beat PC MRI, it can reliably assess average flow patterns across multiple beats.AHA (12GRNT12080032) and NIH-NHLBI (1R21HL113895)."} +{"text": "Among the spectrum of patients with coronary artery disease (CAD), those suffering from high-grade stenosis are at a significantly elevated risk for adverse events . Such hiwith 3-5 slice coverage and no time-gap in between consecutive slice-interleaved radial readouts, acquiring 5,000 projections per slice during the 40-second real-time FPP scan . Retrospective cardiac self-gating was performed automatically using a high temporal-resolution reconstruction of the mid slice at low spatial resolution. The self-gating information was then used to perform a cardiac phase-resolved reconstruction of the FPP data for all slices at high resolution employing a compressed sensing approach. WM for the cine perfusion images was scored on a standard 1-4 scale. To test the clinical feasibility of this approach, patients (n=3) with known high-grade (\u226590%) right coronary artery (RCA) stenosis were studied.Canines (n=9) with surgically implemented reversible stenosis below the first diagonal along the left anterior descending (LAD) artery (\u224890% stenosis) were studied at 3T. Real-time adenosine stress/rest cine perfusion data was acquired using an ungated continuously-sampled FPP sequence without saturation recovery preparation . The T1-Real-time stress FPP scans in stenotic dogs Fig. showed wcine FPP method capable of simultaneous detection of stress-induced perfusion defects and WMAs in a single ungated scan using continuous acquisition (<1 minute). Our initial results demonstrate that worsening of WM (compared to rest) in the perfusion defect territories seen in the real-time stress cine FPP scan may be a marker of severe CAD.We presented a multi-slice American Heart Association Scientist Development Grant 14SDG20480123; NIH National Heart, Lung and Blood Institute grant nos. K99 HL124323-01, R01 HL038698-18, R01 HL091989-05, R01 HL090957-01."} +{"text": "Penicillium digitatum is one of the most destructive postharvest pathogen of citrus fruits, causing fruit decay and economic loss. The emergence of fungicide-resistant strains made the control of P. digitatum more difficult. While the genome of P. digitatum is available, there has been few reports about its resistant mechanism from the transcriptome perspective and there has been no large-scale functional annotation of the genome using expressed genes derived from transcriptomes.\u00a0P. digitatum\u00a0strain HS-F6 (prochloraz-resistant strain) and HS-E3 (prochloraz-susceptible strain) before and after prochloraz-treatment were extracted and sequenced on an Illumina Hiseq 2000 platform. The transcriptome data of four samples were compared and analyzed using differential expression analysis, novel transcripts prediction and alternative splicing analysis, SNP analysis and quantitative real-time PCR. Total RNA ofP. digitatum. The whole RNA was extracted from a prochloraz-resistant strain (HS-F6) and a prochloraz-susceptible strain (HS-E3) before and after prochloraz-treatment and sequenced by Illumina technology. A total of more than 100 million reads were generated and de novo assembled into 9760 transcripts that contained annotated genes after quality control and sequence assembling. 6625 single nucleotide variations (SNVs) were identified from the sequences aligned against the reference genome. Gene expression profiling analysis was performed upon prochloraz treatment in HS-F6 and HS-E3, and differential expression analysis was used to identify genes related to prochloraz-response and drug-resistance: there are 224 differentially expressed genes in HS-E3 and 1100 differentially expressed genes in HS-F6 after prochloraz-treatment. Moreover, gene expression profile in prochloraz-resistant strain HS-F6 is quite different from that in HS-E3 before prochloraz-treatment, 1520 differential expression genes were identified between the two strains. Gene ontology (GO) term enrichment and KEGG enrichment were then performed to classify the differential expression genes. Among these genes, there are a lot of transporter encoding genes including 14 MFS (Major Facilitator Superfamily) transporters, 8 ABC (ATP-binding cassette transporter) and 3 MATE (multidrug and toxic compound extrusion family) transporters. Meanwhile, the roles of typical MFS, ABC and MATE proteins in prochloraz resistance were investigated using real-time quantitative PCR.We present a large scale analysis about the transcriptome data of P. digitatum demonstrate differences between prochloraz-resistant and prochloraz-susceptible strains with prochloraz-treatment. The differences existed in expressed transcripts, splice isoforms and GO categories, which would contribute to our knowledge on the molecular mechanisms involved in drug resistance of P. digitatum.The sequencing-based transcriptome data of The online version of this article (doi:10.1186/s12864-015-2043-x) contains supplementary material, which is available to authorized users. P. digitatum is the most destructive disease of citrus fruit which cause green mold and responsible for up to 90\u00a0% of total crop losses during postharvest packing, storage, transportation, and marketing .The datasets supporting the results of this article are available in the NCBI SRA repository ["} +{"text": "N-glycosylation profiles. In the present study, we outline an improved automated sample preparation method for N-glycan MALDI imaging, which uses in situ PNGase F-mediated release and measurement of N-linked glycans from sections of formalin-fixed murine kidney. The sum of the presented data indicated that N-glycans can be cleaved from proteins within formalin-fixed tissue and characterized using three strategies: (i) extraction and composition analysis through on-target MALDI MS and liquid chromatography coupled to electrospray ionization ion trap MS; (ii) MALDI profiling, where N-glycans are released and measured from large droplet arrays in situ; and (iii) MALDI imaging, which maps the tissue specificity of N-glycans at a higher resolution. Thus, we present a complete, straightforward method that combines MALDI imaging and characterization of tissue-specific N-glycans and complements existing strategies.Recent developments in spatial proteomics have paved the way for retrospective in situ mass spectrometry (MS) analyses of formalin-fixed\u00a0paraffin-embedded clinical tissue samples. This type of analysis is commonly referred to as matrix-assisted laser desorption/ionization (MALDI) imaging. Recently, formalin-fixed\u00a0paraffin-embedded MALDI imaging analyses were augmented to allow in situ analyses of tissue-specific The online version of this article (doi:10.1007/s00216-014-8293-7) contains supplementary material, which is available to authorized users. O-linked) or asparagine (N-linked) residues + values to their calculated values indicated that the automated sample preparation was sufficiently homogeneous across multiple analysis regions so as to minimize mass deviations caused by matrix heterogeneity. It should be noted that external calibration on an adjacent tissue is not ideal for precious tissues. As such, internal calibrants or a higher mass accuracy MS instrument would constitute important additions to any future N-glycan MALDI imaging workflow [Comparing the initial nine workflow , 19.+: 2304.909 for the two PNGase F-treated sections and buffer control section, respectively. As this example ion intensity map shows, the spatial distribution of N-glycans, in this case to the cortex, can be clearly visualized using this MALDI imaging method. Figure\u00a0Figure\u00a0+ values which correspond to (Hex)2 (HexNAc)2 + (Man)3(GlcNAc)2 3(GlcNAc)2 3(Deoxyhexose)3 + (Man)3(GlcNAc)2 4 (Deoxyhexose)4 + (Man)3(GlcNAc)2 could be employed to improve PNGase F enzyme efficiency [N-glycans in MALDI imaging [N-glycan analysis in MALDI imaging could also be attempted in the negative mode. Although the ionization intensity might be lower than positive mode, valuable MS/MS fragmentation data could be acquired similar to that observed in the LS-ESI-MS/MS analysis. Additionally, negative mode analysis of native N-glycan anion adducts [To further improve the results presented here and detect more of the LC-MS/MS-identified ficiency . Predige imaging . These k imaging Finally, adducts \u201333 can b adducts .N-glycans on FFPE tissue sections as well as straightforward in-solution N-glycan release for downstream characterization by LC-MS/MS. The use of automated sample preparation instruments for PNGase F printing and matrix spraying presents a clear advantage over previous methods [The sum of the presented data indicates that the methods developed in this study allow for spatial annotation of methods .N-glycan MALDI imaging data for murine kidney [N-glycan values from murine kidney to those available from the Consortium for Functional Glycomics (CFG) [N-glycan masses were characterized by LC-MS/MS and matching candidate masses were detected for 23 of\u00a0the 28 N-glycans [N-glycans which complements a previous work on kidney tissue.In the context of the work published during the preparation of the current manuscript, Powers et al. also presented e kidney . This wocs (CFG) . The metcs (CFG) . This ma-glycans . The worN-glycan MALDI imaging. Ultimately, the methods presented will be applied to N-glycan profiling in human cancer tissues [As mentioned above, further method development will aim to improve the sensitivity and mass accuracy for tissues . Ovarian tissues and, sec tissues .N-glycan MALDI imaging will initially focus solely on the identification of tissue regions where the number and/or type of detectable N-glycan species changes. The identification of these tissue regions will allow one of several additional experimental lines to be pursued. Firstly, the N-glycan profiles can be used to develop tissue-specific classifiers in a similar fashion to those developed for proteomic MALDI imaging profiles [N-glycans in the vast archives of human cancer tissue which are available globally and ultimately increase our understanding of cancer biology through large retrospective studies.Future analyses employing profiles . Secondlprofiles or spatiESM 1(PDF 6857\u00a0kb)Below is the link to the electronic supplementary material."} +{"text": "Drosophila sensory neurons, through complete pruning and regeneration, can elaborate two distinct dendritic trees, innervating independent sensory fields. An expression screen identified Cysteome proteinase-1 (Cp1) as a critical regulator of this process. Unlike known ecdysone effectors, Cp1-mutant ddaC neurons pruned larval dendrites normally but failed to regrow adult dendrites. Cp1 expression was upregulated/concentrated in the nucleus during metamorphosis, controlling production of a truncated Cut homeodomain transcription factor. This truncated Cut, but not the full-length protein, allowed Cp1-mutant ddaC neurons to regenerate higher-order adult dendrites. These results identify a molecular pathway needed for dendrite regrowth after pruning, which allows the same neuron to innervate distinct sensory fields.Dendrites often exhibit structural changes in response to local inputs. Although mechanisms that pattern and maintain dendritic arbors are becoming clearer, processes regulating regrowth, during context-dependent plasticity or after injury, remain poorly understood. We found that a class of Dendrites are the primary sites of information input for neurons. Their initiation, arborization, targeting, and function are regulated by a series of finely tuned cellular events . CriticaDrosophila peripheral nervous system dendritic arborization (da) neurons, classified into four classes (I\u2013IV) based on their location and dendritic arbor complexity, have served as a powerful model system for studying conserved pathways controlling dendrite morphogenesis and its critical role in regulating ddaC neuron dendrite regeneration to innervate the adult sensory fields.Here, we show that ddaC C4 da neurons regenerate adult dendritic arbors in a different manner after pruning than initially during development. Starting with an expression screen, we identified pickpocket (ppk)-EGFP reporter line to follow the abdominal segment ddaC neurons through metamorphosis, we found that their dendritic arbors changed into a different architecture after regrowth , ddaC neurons initiated dendrite regrowth, projecting primary dendrites along the wall from a lateral-to-medial direction . This inCp1 gene, that showed increased EGFP expression during ddaC neuron dendrite remodeling in ddaC neurons , we generated Cp1-mutant ddaC neuron clones. Unlike other signals downstream of ecdysone identified thus far during ddaC neuron dendrite remodeling , the mammalian homolog to Cp1, is homeodomain transcription factor Cut-like 1 (Cux1) . During 1 (Cux1) . Cux1 iselopment . We asken larvae , these nody wall . Quantifransgene .Cp1 and cut-mutant ddaC neurons led us to probe further their molecular connections. We tested whether a portion of Cp1's function regulating ddaC neuron dendrite regrowth is to produce a truncated Cut isoform. For this, we inserted a 3\u2019 HA tag to better resolve immunohistochemical (IHC) staining signals during nuclear localization and generated a UAS-cut-HA transgenic line. In vivo functionality of this tagged transgene was confirmed by successful repeat of rescue experiment in cut-mutant ddaC neurons . When expressed in Cp1-mutant ddaC clones, truncated Cut1176-2207-HA showed consistently diffuse nuclear localization at 24 hr APF together with either UAS-cut-HA or UAS-cut1176-2207-HA DNA constructs into Drosophila S2 cells. Western blotting of transfected S2 cell lysates with anti-HA antibody showed that the full-length Cut-HA protein was 250 kDa as predicted, and the truncated Cut1176-2207-HA was near 160 kDa in size and measured from the body wall using MATLAB script.For depth analyses, dendrites were traced using the Simple Neurite Tracer module in Fiji by a standard calcium phosphate method. Western analyses were performed using a standard protocol: mouse anti-Cut ; mouse anti-HA ; rabbit anti-Cp1 ; and mouse anti-actin . Further details are in Supplementary Figures and Methods"} +{"text": "Previously identified single nucleotide polymorphisms (SNPs) in genome wide association studies (GWAS) of cardiovascular disease (CVD) in participants of mostly European descent were tested for association with subclinical cardiovascular disease (sCVD), coronary artery calcium score (CAC) and carotid intima media thickness (CIMT) in the Multi-Ethnic Study of Atherosclerosis (MESA). The data in this data in brief article correspond to the article Common Genetic Variants and Subclinical Atherosclerosis: The Multi-Ethnic Study of Atherosclerosis Specifications TableValue of the data\u2022Genetic variations play an important role in the atherosclerotic process.\u2022The data shows novel associations between genetic variations and atherosclerosis.\u2022The data also shows that previously described genetic associations with atherosclerosis vary considerably depending on ethnicity.\u2022More research is needed to further elucidate the effect of ethnic-specific genetic variation in cardiovascular disease.1Previously identified single nucleotide polymorphisms (SNPs) in genome wide association studies (GWAS) of cardiovascular disease (CVD) in participants of mostly European descent were tested for association with subclinical cardiovascular disease (sCVD), coronary artery calcium score (CAC) and carotid intima media thickness (CIMT) in the Multi-Ethnic Study of Atherosclerosis (MESA).22.1The MESA study has been previously described and it was designed to investigate the impact of sCVD and CVD risk factors on the development of clinically overt CVD 2.2P-value\u226510\u22125.The 66 single nucleotide polymorphisms (SNPs) included in this study were obt2.3The imaging outcomes in the present study are coronary artery calcium and carotid artery intima-media thickness .CAC was measured by either electron-beam tomography or multi-detector computed tomography, as described previously CIMT measurements were performed by B-mode ultrasonography of the right and left, near and far walls, and images were recorded using a Logiq 700 ultrasound device . Maximal CIMT-i and CIMT-c was measured as the mean of the maximum values of the near and far wall of the right and left sides at a central ultrasound reading center as described previously 2.4Given skewed distributions, the common (CIMT-c) and internal (CIMT-i) IMT values were log normalized. CAC was analyzed as a continuous variable by obtaining the log of the raw CAC score plus one (CAC-c) or as a dichotomous variable (CAC-d) with CAC>0. Analyses were first performed stratified within each racial/ethnic group. For analysis involving EUA and CHN, an unrelated subset of individuals was constructed by selecting at most one individual from each pedigree. For analysis of phenotypes with a substantial familial component, among AFA and HIS, the analysis was performed using a linear mixed-effects model (continuous variables) and by generalized estimating equations (dichotomous variables). Associations between each SNP and each individual phenotype was determined using separate multiple linear regressions (continuous variables) or logistic regressions (dichotomous variables) assuming an additive model. Two models were used to analyze the data. Model 1 accounted for age, sex, site of ascertainment, and principal components. Model 2 included Model 1 plus HDL cholesterol (HDL-C), LDL cholesterol (LDL-C), triglycerides, body mass index (BMI), hypertension status , diabetes status (fasting blood glucose was 126\u00a0mg/dL or greater or use of diabetes medications), and current smoking use (self-reported current smoking use within the past 30 days). Fixed effect meta-analysis was used to combine results across all four race/ethnic groups, as implemented in METAL. Fine mapping of the 9p21 region was performed for each ethnic group by selecting all SNPs on the chromosome 9 imputation set (NCBI Build 37) between positions 21997022\u201322225503. A total of 3282 SNPs were identified . This list of SNPs was supplemented by adding novel SNPs identified by deep sequencing efforts in this region p<7.6\u00d710\u22124 given 66 SNPs tested (0.05/66) for the initial analysis, with greater number of SNPs used for the correction for the fine mapping effort). To assess genetic heterogeneity seen in stratified analyses of the four MESA race/ethnic groups, we used the I2 heterogeneity metric to quantify the proportion of total variation across studies attributable to heterogeneity rather than chance Significance was defined by Bonferroni correction by dividing an alpha of 0.05 by the number of SNPs tested (http://www.nhlbi.nih.gov). MESA Family is conducted and supported in collaboration with MESA investigators; support is provided by grants and contracts R01HL071051, R01HL071205, R01HL071250, R01HL071251, R01HL071252, R01HL071258, R01HL071259, M01-RR00425, UL1RR033176, and UL1TR000124. Funding for MESA SHARe genotyping was provided by NHLBI Contract N02\u2010HL\u20106\u20104278. The provision of genotyping data was supported in part by the National Center for Advancing Translational Sciences, CTSI grant UL1TR000124, and the National Institute of Diabetes and Digestive and Kidney Disease Diabetes Research Center (DRC) grant DK063491 to the Southern California Diabetes Endocrinology Research Center. This manuscript was approved for submission by the Presentations and Publications Committee.MESA and the MESA SHARe project are conducted and supported by contracts N01-HC-95159, N01-HC-95160, N01-HC-95161, N01-HC-95162, N01-HC-95163, N01-HC-95164, N01-HC-95165, N01-HC-95166, N01-HC-95167, N01-HC-95168, N01-HC-95169, UL1-TR-001079 and UL1-TR-000040 from the National Heart, Lung, and Blood Institute (NHLBI,"} +{"text": "G12D Lox-Stop-Lox murine lung cancer model in combination with a conditional RhoAflox/flox and RhoC-/- knockout mouse models. Deletion of the floxed Rhoa gene and expression of K-RasG12D was achieved by either CCSP-Cre or adenoviral Cre, resulting in simultaneous expression of K-RasG12D and deletion of RhoA from the murine lung. We found that deletion of RhoA, RhoC or both did not adversely affect normal lung development. Moreover, we found that deletion of either RhoA or RhoC alone did not suppress K-RasG12D induced lung adenoma initiation. Rather, deletion of RhoA alone exacerbated lung adenoma formation, whereas dual deletion of RhoA and RhoC together significantly reduced K-RasG12D induced adenoma formation. Deletion of RhoA appears to induce a compensatory mechanism that exacerbates adenoma formation. The compensatory mechanism is at least partly mediated by RhoC. This study suggests that targeting of RhoA alone may allow for compensation and a paradoxical exacerbation of neoplasia, while simultaneous targeting of both RhoA and RhoC is likely to lead to more favorable outcomes.Numerous cellular studies have indicated that RhoA signaling is required for oncogenic Ras-induced transformation, suggesting that RhoA is a useful target in Ras induced neoplasia. However, to date very limited data exist to genetically attribute RhoA function to Ras-mediated tumorigenesis in mammalian models. In order to assess whether RhoA is required for K-Ras-induced lung cancer initiation, we utilized the K-Ras In the United States, lung cancer kills more people each year than breast, prostate and colon cancer combined . Lung ad1-S phase: namely, increased RhoA activity inhibits p21Waf1/Cip1 and results in increased amounts of cyclin D1 and p27Kip1 [The mammalian Rho GTPase family includes over 20 members, of which RhoA, Rac1 and Cdc42 are among the best characterized. These Rho GTPases regulate the cell cycle and actin cytoskeleton and are thus critical regulators of processes such as cell shape, adhesion, migration, polarity and proliferation \u20138. Impor p27Kip1 \u201316. Inde p27Kip1 \u201322.More recently, greater attention has been paid to the role of Rac1 in tumorigenesis due to the availability of murine genetic models. These studies have confirmed a positive role of Rac1 in tumor initiation. Rac1 was found to be required for K-Ras-induced lung adenoma formation in mice and a Rac1 splicing variant with increased activity, Rac1b, appears to promote tumorigenesis in this context ,24. Two G12D knock-in mouse model of lung adenoma induction in combination with our RhoA conditional knockout, RhoAflox/flox, and RhoC-/- mouse models. Our investigation of these genetic studies produced surprising results that individually RhoA and RhoC are dispensable for the oncogenic K-Ras induced lung tumorigenesis and loss of RhoA alone exacerbates, rather than suppresses, tumor initiating activity. Our study implies that pharmacological targeting of multiple Rho family members, e.g. RhoA and RhoC in this context, is required to prevent tumorigenesis.In the present work, we have sought to investigate the role of RhoA and the closely related Rho GTPase, RhoC, in K-Ras-induced tumorigenesis using a well-established inducible K-Rasflox/flox) with mice harboring the Club Cell Secretory Promoter Cre (CCSP-Cre), as outlined in We first sought to determine whether mice would be viable without RhoA expression in their bronchiolar epithelium. We crossed a RhoA conditional mouse transgene into the CCSP-Cre;RhoAflox/flox mice. At least two different constructs of CCSP-Cre have been successfully used to induce lung tumors in mice [G12D mice develop a strong inflammatory component to their disease with abundant infiltration of alveolar macrophages and neutrophils [G12D;RhoAflox/flox mice to have grossly abnormal lungs with abundant infiltration and adenoma formation mice were born at the expected Mendelian ratio, did not display any signs of overt respiratory distress and their lungs were grossly normal staining demonstrated adenoma formation in RhoWT, RhoAcKO, RhoC-/- and DKO mice were ones in which RhoA was not fully deleted. Quantification of the number and size of tumors revealed similar numbers and sizes of tumors between RhoWT and RhoC-/- mice had increased tumor burden [G14V) hindered tumor formation whereas dominant-negative RhoA (RhoAT19N) accelerated tumorigenesis [Our counterintuitive findings are in line with several recent studies that have begun to challenge the conventional paradigm that RhoA is a simple proto-oncogene. Two whole-exome sequencing studies of human lymphomas found recurrent Gly17Val mutations in RhoA which confer a dominant negative or loss of function phenotype ,48. Anotr burden . Furtherigenesis . While tG12D is expressed, and thus our work does not address whether RhoA plays a role in tumor maintenance or progression. It is possible that once oncogenic K-Ras induced tumors have developed, they become addicted to permissive signaling from wild-type RhoA. In this case, RhoA signaling would be required for tumor maintenance. Indeed, an examination of human cancer cell lines treated with RhoA-specific siRNA or the Rho inhibitor Rhosin shows that they are dependent on RhoA function for proliferation and progression (data not shown). These findings pose an intriguing possibility that targeted inhibition of RhoA activity alone is useful for suppressing advanced tumors in certain instances, but may exacerbate preneoplastic lesions. Another caution interpreting our results is that both mouse cancer models are accompanied by significant inflammatory responses which could potentially be modulated by RhoA status, which in turn may be promoting the inflammatory component (i.e. effects through tumor microenvironment).A limitation of our mouse model in this study is that RhoA is deleted at the same time as K-Raset al. also found that whole organism deletion of both B-Raf and c-Raf resulted in no obvious phenotype in mice, also similar to our finding of overtly healthy mice with dual deletion of RhoA and RhoC from the bronchiolar epithelium [The interplay between RhoA and RhoC signaling is in some ways similar to that of B-Raf and c-Raf in oncogenic K-Ras-driven adenoma initiation. Recent studies have found that c-Raf, but not B-Raf, is required for K-Ras-driven adenoma formation, despite historical evidence in mouse embryonic tissues and fibroblasts suggesting the opposite, that B-Raf but not c-Raf is required for ERK phosphorylation \u201355. In fithelium .Our study of the relationship between RhoA, RhoC and K-Ras in murine lung adenoma formation sheds light on the broader subject of cellular signal transduction networks, and how signal redundancy and compensation in these networks can result in counterintuitive results when the network is modified. These insights are important considering the increased interest and emphasis on targeted therapeutics against individual pathways. The robustness of signal transduction networks paired with the evolutionary power of clonal selection in cancer suggests the best antineoplastic strategies may need to incorporate multiple drug targets with an anticipation of how cancer cells will adapt to and evade targeted therapies. Our study suggests that simultaneous targeting of both RhoA and RhoC signaling is critical, as targeting of RhoA alone may worsen clinical outcomes. Further study confirming our proposed compensatory mechanism is warranted, as is a deeper understanding of the overlapping and differential functions of RhoA, RhoC and other related Rho GTPases in cancer biology. Last, our study suggests that targeting ROCK signaling, which is a shared downstream mediator of both RhoA and RhoC, may be sufficient to block K-Ras-driven cancer as has been previously suggested ,59.flox/flox mice generated as previously described [\u03942\u20133, LSL-K-Ras, CCSP-Cre and mTmG mice have been previously characterized [in vivo experiments were performed using age-matched mice. Genotyping primers can be found in the supplementary information supplemented with protease inhibitors cocktail and phosphatase inhibitor cocktail consisting of sodium fluoride, sodium orthovanadate, sodium pyrophosphate, \u00df-glycerophosphate and sodium molybdate . Transfer was performed using the Trans-Blot Turbo Transfer System (Bio-Rad). Visualization was performed using fluorescently conjugated secondary antibodies and an infrared imaging System (LiCor Odyssey CLx).Briefly, tissues were fixed using 3.7% paraformaldehyde in PBS overnight and embedding to paraffin. Secondary antibodies and horse-radish peroxidase (HRP) used consisted of either Signal Stain Boost or anti-rabbit donkey secondary and Vectastain Elite ABC Kit (Vector Labs) avidin-biotin complex. The developing reagent was DAB HRP substrate (Vector Labs). Counterstaining was performed using Gill\u2019s hematoxylin or nuclear fast red. Quantification of stainings were made by serially sectioning lungs in 250\u03bcm step increments and counting a sample of evenly spaced sections for the number of tumors. Tumor size was expressed as tumor area as measured in ImageJ.Primary antibodies for immunoblotting or immunohistochemistry are as follows: RhoA (67B9) Rabbit mAb , Myosin Light Chain 2 (D18E2) Rabbit mAb (CST #8505), Phospho-Myosin Light Chain 2 (Ser19) Mouse mAb , Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) Rabbit mAb (CST #4370), Phospho-MEK1/2 (Ser221) (166F8) Rabbit mAb (CST #2338), GAPDH (D16H11) Rabbit mAb (CST #5174), Anti-Myosin Light Chain (phospho S20) antibody (Abcam ab2480), Ki67 Rabbit mAb, (790\u20134286 Ventana), cleaved-caspase 3 Rabbit Ab (orb137850 Biorbyt), N-Terminal Pro SPC Rabbit Ab (Seven Hills Bioreagents WRAB-9337), and CCSP Rabbit Ab (Seven Hills Bioreagents WRAB-3950). All antibodies were used at the manufacturers recommended dilutions unless otherwise stated.Data was gathered and analyzed in either ImageJ 1.47 (NIH), GraphPad Prism (GraphPad) or the GNU Image Manipulation Program 2.8 (GIMP). Analysis in GraphPad Prism comprised of unpaired two-way student T-tests or ANOVA.S1 FigWT (K-WT) or CCSP-Cre LSL-K-RasG12D mice (KRas). Pairs of K-RasWT and LSL-K-RasG12D mice for each Rho background are shown. Deletion bands for LSL-K-RasG12D mice demonstrate expression of K-RasG12D (deletion band is the larger band at 315bp). Deletion bands for RhoA demonstrate recombination of the RhoAflox/flox locus (deletion band at 667bp). Yellow arrows point to the 500bp band of a 100bp ladder.DNA was isolated from mouse lungs of different Rho background mice mated to either CCSP-Cre K-Ras(TIFF)Click here for additional data file.S2 Fig(A) RhoA immunohistochemistry (brown) of hyperplastic lesions with hematoxylin counterstain of CCSP-Cre LSL-K-RasG12D mice from different Rho backgrounds (bars represents 100\u03bcm). (B) Percentage of Ki67 positive cells in CCSP-Cre LSL-K-RasG12D driven adenomas . (C) Representative image of cleaved-caspase 3 (CC3) staining of CCSP-Cre LSL-K-RasG12D driven adenomas. Positive control is injured renal tubules (20X). Bars represent 100\u03bcm.(TIFF)Click here for additional data file.S3 FigG12D mice. Rows represent serial sections of the same adenoma from their respective Rho backgrounds. Columns are of RhoA, pMLCSer20, pMEKSer221 and pERKThr202/Tyr204 stainings respectively with hematoxylin counterstain (bars represents 100\u03bcm).Immunohistochemistry of serially sectioned adenomas from CCSP-Cre LSL-K-Ras(TIFF)Click here for additional data file.S4 Fig(A) RhoA immunohistochemistry (brown) with hematoxylin counterstain of Adeno-Cre induced LSL-K-RasG12D mice from different Rho backgrounds (bars represents 100\u03bcm). (B) Percentage of Ki67 positive cells in Adeno-Cre LSL-K-RasG12D driven adenomas . (C) Representative image of cleaved-caspase 3 (CC3) staining of Adeno-Cre LSL-K-RasG12D driven adenomas. Positive control is injured renal tubules (20X). Bars represent 100\u03bcm.(TIFF)Click here for additional data file.S5 Fig(TIFF)Click here for additional data file.S1 TableTable of genotyping and deletion primers used for PCR. Specific PCR protocols can be found in the references for transgenic mice under Materials and Methods.(DOCX)Click here for additional data file."} +{"text": "MicroRNAs (miRNAs) are small non-coding RNAs that regulate protein expression. HTLV-1 is able to promote oncogenesis in T cells by altering the expression of miRNAs involved in the control of cell-cycle. It is not known whether HTLV-1 deregulates miRNAs expression in cells of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients. To asses if HTLV-1 infection might alter the expression of miRNAs involved in inflammatory response, we evaluated the expression of 84 miRNAs involved in inflammatory process in asymptomatic HTLV-1 carriers (AC) and HAM/TSP patients using the miScript miRNA PCR Array Human Inflammatory Response & Autoimmunity (SABioscience). For this purpose, fourteen HTLV-1-positive individuals were selected and classified into three groups: five asymptomatic carriers (AC), 4 HAM/TSP patients with EDSS score of 1-5 (=mild HAM/TSP), and 5 HAM/TSP patients with EDSS score of 5.5-9 (=severe HAM/TSP). Total RNA was isolated from PBMCs and pooled according to the groups. qBase software was used for normalization, ANOVA was used for comparisons and False Dicosvery Rate to correct for multiple comparisons. We found nine differentially expressed miRNAs between AC and HAM/TSP patients (mild and severe HAM/TSP). Twelve miRNAs were differentially expressed among mild HAM/TSP, severe HAM/TSP and AC groups. These findings support results previously reported in Adult T-cell leukemia/lymphoma (ATLL) cells, in which hsa-miR-145, miR-130a, miR181a and miR101a were found to be down-regulated and miR-30d was found to be up-regulated in comparison to those of healthy donors. Further analysis to confirm these findings are needed."} +{"text": "Animal studies suggest that when PA-specific IgG has waned, survival after spore challenge correlates with an activation of PA-specific memory B cells. Here, we characterize the quantity and the longevity of AVA-induced memory B cell responses in humans. Peripheral blood mononuclear cells (PBMCs) from individuals vaccinated \u22653 times with AVA (n = 50) were collected early or late after their last vaccination , pan-stimulated, and assayed by ELISPOT for total and PA-specific memory B cells differentiated into antibody secreting cells (ASCs). PA-specific ASC percentages ranged from 0.02% to 6.25% (median: 1.57%) and did not differ between early and late post-vaccination individuals. PA-specific ASC percentages correlated with plasma PA-specific IgG and toxin neutralization early post vaccination. PA-specific ASC percentages correlated with supernatant anti-PA both early and late post vaccination . These data suggest PA-specific memory B cell responses are long-lived and can be estimated after recent vaccination by the magnitude and neutralization capacity of the humoral response.Anthrax Vaccine Adsorbed (AVA) generates short-lived protective antigen (PA) specific IgG that correlates with The generation of immunological memory to T cell-dependent antigens results in both humoral and cellular immunity. In this process, na\u00efve B cells enter the germinal center reaction and exit as antibody secreting cells or memory B cells . Togethein vitro lethal toxin (LT) neutralization activity (LTNA). In turn, LTNA has been demonstrated to be predictive of survival in several animal models, including a non-human primate Bacillus anthracis spore challenge model \u00d7 100.Memory B cells were quantified by ELISPOT as described . PBMCs wPA-specific antibodies are the strongest correlate of protection against anthrax infection and intoxication in most animal models; unfortunately, anti-PA levels decline rapidly after vaccination. We find that individuals sampled early and late post vaccination with similar levels of plasma anti-PA IgG have similar quantity and quality of PA-specific memory B cells. However, the quantity of circulating PA-specific memory B cells is highly variable, and humoral measures may only serve as a surrogate measure in the first year after vaccination. Indeed, the lack of correlation between memory B cells and antibody late post-vaccination suggests that antibody is maintained independent of memory B cells, likely by long-lived plasma cells. Further, longitudinal studies are necessary to describe the kinetics of the memory B and long-lived plasma cell response in AVA-vaccinated individuals. Regardless of specific kinetics, we demonstrate high levels of PA-specific ASCs exist in the absence of high levels of anti-PA in our late-vaccination group, suggesting that, upon exposure, rapid production of anti-PA IgG could be possible."} +{"text": "It is generally regarded that E-cadherin is downregulated during tumorigenesis via Snail/Slug-mediated E-cadherin transcriptional reduction. However, this transcriptional suppressive mechanism cannot explain the failure of producing E-cadherin protein in metastatic breast cancer cells after overexpressing E-cadherin mRNA. Here we reveal a novel mechanism that E-cadherin is post-transcriptionally regulated by Slug-promoted miR-221, which serves as an additional blocker for E-cadherin expression in metastatic tumor cells. Profiling the predicted E-cadherin-targeting miRNAs in breast cancer tissues and cells showed that miR-221 was abundantly expressed in breast tumor and metastatic MDA-MB-231 cells and its level was significantly higher in breast tumor or MDA-MB-231 cells than in distal non-tumor tissue and low-metastatic MCF-7 cells, respectively. MiR-221, which level inversely correlated with E-cadherin level in breast cancer cells, targeted E-cadherin mRNA open reading frame (ORF) and suppressed E-cadherin protein expression. Depleting or increasing miR-221 level in breast cancer cells induced or decreased E-cadherin protein level, leading to suppressing or promoting tumor cell progression, respectively. Moreover, miR-221 was specifically upregulated by Slug but not Snail. TGF-\u03b2 treatment enhanced Slug activity and thus increased miR-221 level in MCF-7 cells. In summary, our results provide the first evidence that Slug-upregulated miR-221 promotes breast cancer progression via reducing E-cadherin expression. Understanding the mechanisms that govern tumor metastasis, a main reason of tumor-related mortality6851011et al., unpublished). MicroRNAs (miRNAs), as a class of noncoding RNAs, that regulate gene expression at posttranscriptional level, resulting in either mRNA degradation or translational inhibition1618et al. reported that knockdown of miR-200a in mammary glands prevented increases in E-cadherin mRNA expression and thus decreased E-cadherin signalet al. reported that miR-9 could inhibit E-cadherin expression by binding to the 3\u2032-UTR of E-cadherin mRNAHowever, recent studies have shown that E-cadherin expression might be also modulated at a posttranscriptional level14in vitro and in vivo results showed that Slug-promoted miR-221 enhanced the breast tumor progression through reducing E-cadherin protein expression.In the present study, we determined a novel miRNA-based regulatory mechanism for E-cadherin expression in metastatic breast cancer cell. We demonstrated that Slug specifically promoted miR-221 expression, which in turn, directly targeted the E-cadherin mRNA transcript, leading to reduction of E-cadherin protein level. Furthermore, both First, we compared the protein expression and mRNA level of E-cadherin in breast tumor tissues and distal normal tissue, as well as metastatic breast cancer MDA-MB-231 cells and non-metastatic MCF-7 cancer cells. In the experiment, 8 paired breast tumor tissue and distal normal tissue samples were collected and tested. As shown in 5Since the repression of mRNA transcripts by miRNAs is one of important posttranscriptional regulation, in which miRNAs block mammalian cell protein translation by base pairing to 3\u2032-UTRs or ORF of target mRNA transcriptsBioinformatics analysis predicted one putative miR-221 binding site within the ORF region of E-cadherin transcript . The minThe correlation between miR-221 and E-cadherin expression was further examined by evaluating E-cadherin expression in MCF-7 and MDA-MB-231 cells after overexpression or knockdown of miR-221. In these experiments, miR-221 overexpression was achieved by transfecting cells with Pre-miR-221, whereas miR-221 knockdown was achieved by transfecting cells with anti-miR-221 oligonucleotide . Clearly, the expression of E-cadherin protein was significantly inhibited by the introduction of miR-221 in MCF-7 cells , while aTo confirm that miR-221 regulates the E-cadherin gene by binding to the predicted binding site of ORF, we constructed a mutant E-cadherin overexpression vector (MUT), in which the miR-221 binding sequence was mutated without alteration of amino acid sequence . In the Snail family of zinc-finger transcriptional factors, including Slug and Snail (SNAI1), are master regulators of epithelial-mesenchymal transition (EMT)24Given that TGF-\u03b2 has been reported to stimulate cell migration, invasion and metastasis of breast cancer cells by inducing the transcription factors SlugAs E-cadherin is a key adhesive molecule that prevents tumor cell metastasis, we determined whether targeting E-cadherin by miR-221 can promote breast cancer cell migration and invasion. The wound healing assays showed that the migration capacity of MCF-7 cells was significantly enhanced by overexpression of miR-221 . In contTranswell invasion assay showed that overexpression of miR-221 in MCF-7 cells enhanced cell invasion , whereasIn vitro wound healing and Transwell invasion assays showed that two modified MDA-MB-231 cell lines, E-cad (MUT) and E-cad (WT) plus anti-miR-221, displayed a decreased migration and invasion compared to control MDA-MB-231 cells and MDA-MB-231 cells stably transfected with E-cad (WT) , cells stably transfected with WT E-cadherin (E-cad (WT)), cells stably transfected with WT E-cadherin combined with anti-miR-221 (E-cad (WT) plus anti-miR-221), and cells stably transfected with MUT E-cadherin (E-cad (MUT)). The qRT-PCR assay and Western blot analysis confirmed that the designed four cell lines were successfully produced . In vitrcad (WT) .Subsequently, we injected these four modified MDA-MB-231 cell lines into female nude mice through the tail vein . After 8HE staining also showed a significant difference of tumor number and growth in mouse lungs among these modified MDA-MB-231 cells . In miceDownregulation of E-cadherin expression is a leading event in the progression of various tumors into the metastatic cascade22861012et al. showed that target of mdm2 by miR-221 could prevent the degradation of the SlugCompared to healthy cells, tumor cells often displayed abnormal expression profile of miRNAs1931Since E-cadherin is an important adhesive molecule preventing cell EMT and tumorigenesisAlthough the association of upregulated Slug with reduced E-cadherin expression has been shown in previous studies3610113038The human breast cancer cell lines MCF-7 and MDA-MB-231 was purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences . TGF-\u03b21 was purchased from R&D Systems . Antibodies were from various sources: anti-E-cadherin antibody , anti-SLUG antibody , anti-SNAIL1 and anti-\u03b1-tubulin antibodies . Antibody used for immunofluorescence staining in MCF-7 and MDA-MB-231 cells was anti-E-cadherin .Eight-paired breast tumor tissues and adjacent cancer tissues were collected from patients undergoing a surgical procedure at the Affiliated People\u2019s Hospital of Jiangsu University . Both tumors and noncancerous tissues were confirmed histologically. The pathological type of each cancer was determined to be infiltrating ductal carcinoma. All of the patients provided written consent, and the Ethics Committee from Nanjing University approved all aspects of this study, and the methods were carried out in accordance with the approved guidelines. Tissue fragments were immediately frozen in liquid nitrogen at the time of surgery and stored at \u221280\u2009\u00b0C. The clinical features of the patients are listed in \u2212\u0394\u0394CT, in which \u0394\u0394CT\u2009=\u2009(CT miRNA\u2009\u2212\u2009CT U6)target\u2009\u2212\u2009(CT miRNA\u2009\u2212\u2009CT U6)control. To quantify E-cadherin and \u03b2-actin mRNA, Real-time PCR was performed according to previous publicationsT values were determined by setting a fixed threshold. The amount of E-cadherin mRNA was normalized to \u03b2-actin.A TaqMan probe\u2013based qRT-PCR assay was performed to quantitative determination of serum miRNAs according to the manufacturer\u2019s instructions as described previouslymiRNA overexpression was achieved by transfecting cells with miRNA mimics, whereas knockdown was achieved by transfecting cells with a miRNA inhibitor according to previous publicationsThe mammalian expression plasmids designed to specially express ORF of human E-cadherin with either wild-type (WT) was purchased from GeneCopoeia and mutant (binding sites that interact with miR-221 were mutated) form of ORF was contructed in this study. The siRNA (sense: 5\u2032-TTAGAGTCCTGCAGCTCGCdTdT-3\u2032 and anti-sense: 5\u2032-GCGAGCTGCAGGACTCTAAdTdT-3\u2032) targeting human SLUG and were synthesized by GenePharma. Overexpression plasmid or siRNA were transfected cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer\u2019s instructions. To test the direct binding of miR-221 to the target gene E-cadherin, a luciferase reporter assay was performed according to previous publicationsE-cadherin, SLUG and SNAIL1 protein levels were quantified by western blot analysis of whole-cell extracts using antibodies. Normalization was performed by blotting the same samples with an antibody against \u03b1-tubulin. Protein bands were analyzed using Bandscan software (Image J). Immunofluorescence analysis was performed according to previous publications2 atmosphere saturated with H2O. After incubation, cells that had entered the lower surface of the filter membrane were fixed with 4% paraformaldehyde for 25\u2009minutes at room temperature, washed 3 times with distilled water, and stained with 0.1% crystal violet in 0.1\u2009M borate and 2% ethanol for 15\u2009minutes at room temperature. Cells remaining on the upper surface of the membranes were scraped off gently with a cotton swap. The upper surfaces with migrant cells were captured 5\u20136 fields per chamber by a photomicroscope , and the number of cells were counted blindly.The migrations of MCF-7 and MDA-MB-231 cells transfected with different miRNAs or plasmids were tested in a Transwell Boyden Chamber . The polycarbonate membranes (8-\u03bcm pore size) on the bottom of the upper compartment of the transwells were coated with 1% human fibronectin (R&D systems). Cells were harvested 48\u2009hours later after transfection, and suspended in FBS-free DMEM or L-15 culture medium. Then cells were added to the upper chamber (4\u2009\u00d7\u2009104 cells/well). At the same time, 0.5\u2009ml of DMEM or L-15 with 10% FBS was added to the lower compartment, and the plates were incubated for 8\u201312\u2009hours in a 5% COScratch wound healing assay was performed to assess cell migration. In brief, Cells were seeded into fibronectin coated 12-well microtiter plates at densities of 120000 cells per well and transfected with different miRNAs or plasmids the following day. Cells were wounded with a plastic pipette tip and the remaining cells were washed twice with fresh medium to remove cell debris the next day. After 16\u201324\u2009hours incubation, the migrant cells at the wound front were photographed with a microscope. Wound closure was calculated and expressed as a percentage of the initial wound width.Viruses produced using the vectors encoding E-cadherin (WT or Mutant) were packaged using HEK-293T cells and the viruses were used to infect the MDA-MB-231 cells as described previously44Animal maintenance and experimental procedures were carried out in accordance with the US National Institute of Health Guidelines for Use of Experimental Animals and approved by the Medicine Animal Care Committee of Nanjing University . Experiments were carried out using female severe combined immune deficiency (SCID) mice as previously reportedP\u2009<\u20090.05 using a Student\u2019s t-test.These above experiments were performed in triplicate, and each was repeated several times. The results are presented as the means\u2009\u00b1\u2009SEM of at least three independent experiments. The differences were considered statistically significant at How to cite this article: Pan, Y. et al. Slug-upregulated miR-221 promotes breast cancer progression through suppressing E-cadherin expression. Sci. Rep.6, 25798; doi: 10.1038/srep25798 (2016)."} +{"text": "RNA sequencing (RNA-Seq) is often used for transcriptome profiling as well as the identification of novel transcripts and alternative splicing events. Typically, RNA-Seq libraries are prepared from total RNA using poly(A) enrichment of the mRNA (mRNA-Seq) to remove ribosomal RNA (rRNA), however, this method fails to capture non-poly(A) transcripts or partially degraded mRNAs. Hence, a mRNA-Seq protocol will not be compatible for use with RNAs coming from Formalin-Fixed and Paraffin-Embedded (FFPE) samples.To address the desire to perform RNA-Seq on FFPE materials, we evaluated two different library preparation protocols that could be compatible for use with small RNA fragments. We obtained paired Fresh Frozen (FF) and FFPE RNAs from multiple tumors and subjected these to different gene expression profiling methods. We tested 11 human breast tumor samples using: (a) FF RNAs by microarray, mRNA-Seq, Ribo-Zero-Seq and DSN-Seq (Duplex-Specific Nuclease) and (b) FFPE RNAs by Ribo-Zero-Seq and DSN-Seq. We also performed these different RNA-Seq protocols using 10 TCGA tumors as a validation set.The data from paired RNA samples showed high concordance in transcript quantification across all protocols and between FF and FFPE RNAs. In both FF and FFPE, Ribo-Zero-Seq removed rRNA with comparable efficiency as mRNA-Seq, and it provided an equivalent or less biased coverage on gene 3\u2032 ends. Compared to mRNA-Seq where 69% of bases were mapped to the transcriptome, DSN-Seq and Ribo-Zero-Seq contained significantly fewer reads mapping to the transcriptome (20-30%); in these RNA-Seq protocols, many if not most reads mapped to intronic regions. Approximately 14 million reads in mRNA-Seq and 45\u201365 million reads in Ribo-Zero-Seq or DSN-Seq were required to achieve the same gene detection levels as a standard Agilent DNA microarray.Our results demonstrate that compared to mRNA-Seq and microarrays, Ribo-Zero-Seq provides equivalent rRNA removal efficiency, coverage uniformity, genome-based mapped reads, and consistently high quality quantification of transcripts. Moreover, Ribo-Zero-Seq and DSN-Seq have consistent transcript quantification using FFPE RNAs, suggesting that RNA-Seq can be used with FFPE-derived RNAs for gene expression profiling.The online version of this article (doi: 10.1186/1471-2164-15-419) contains supplementary material, which is available to authorized users. The development of massively parallel sequencing for use in gene expression profiling is known as RNA-sequencing (RNA-Seq). RNA-Seq has had an enormous impact on gene expression studies. Compared to hybridization-based technologies like DNA microarrays, it provides consistent quantification and manifests its superiority in terms of the dynamic range, sampling depth, and has independence from pre-existing sequence information , 2. RNA-0t-kinetics-based normalization method to deplete abundant sequences that reanneal quickly, such as those derived from the highly abundant rRNAs and tRNAs [To allow for mRNA/gene detection, highly abundant ribosomal RNAs (rRNAs) must be removed from total RNA before sequencing. One standard solution is to enrich for the polyadenylated (poly(A)) RNA transcripts with oligo (dT) primers, similar to how DNA microarrays are primed; however, this method eliminates all non-poly(A) RNAs in addition to rRNAs. Recent studies suggested that certain non-polyA RNAs, either non-coding or protein coding, are functionally important \u201315. Mored tRNAs . In thisTo rigorously evaluate the feasibility of reproducible gene expression profiling using RNA from clinically relevant FFPE materials, we collected FFPE and fresh-frozen (FF) tumor RNAs for matched sets of tumors from two different sources (UNC and TCGA). Most tumors were subjected to gene expression profiling using six different methods that included: 1) Agilent DNA microarrays using FF RNA, 2) mRNA-Seq using FF RNA, 3) Ribo-Zero-Seq using FF RNA, 4) DSN-Seq using FF RNA, 5) Ribo-Zero-Seq using FFPE RNA, and 6) DSN-Seq using FFPE RNA; see Figure\u00a0The efficiency of rRNA removal is a key factor to maximize reads mapping to transcripts, because if left alone, rRNAs make up >80-90% of the total RNA of an un-enriched sample . Due theThe precision of RNA-Seq gene quantification is directly dependent on the number of reads that are mapped to transcripts, thus we first assessed the fraction of reads aligning to the reference human genome UCSC hg19 relative coverage of exons, introns, and intergenic regions, and (b) uniformity of transcript coverage.In FF samples, bases mapping to transcripts (i.e. coding and UTR regions) constituted 62.3% total bases in mRNA-Seq, while a marked reduction was observed in the two rRNA-depletion protocols for the read coverage of the 1000 most highly expressed transcripts enrichment strategies yield an accurate and reproducible measurement of transcript abundance with a wide dynamic range , 20, 21.Additional quality assessments were made on the TCGA dataset, to account for the fact that a much smaller set of reads were mapped to transcriptome in RNA depletion protocols. We generated eight technical replicates with the Ribo-Zero-Seq-FFPE protocol to balance the total number of transcriptome reads for the comparison with FF mRNA-Seq. The assessment of technical reproducibility suggested that these FFPE replicates were indistinguishable (Pearson =0.991). The correlation between Ribo-Zero-Seq on FF and FFPE as well as between Ribo-Zero-Seq-FFPE replicate pairs has also been confirmed in Norton et al. .Lastly, Hierarchical clustering analysis provides a global examination whether biologically relevant expression signatures are consistently measured by distinct protocols. In this example, we tested whether the same sample assayed by different protocols \u201cpaired\u201d or \u201cpartnered\u201d together; if so, then this is a very high level of assay validation as not only are the overall subtype expression profiles maintained, but also the profiles that are unique to that sample are maintained. We performed hierarchical clustering analysis of the RNA-Seq data using a previously published \u2018intrinsic gene list\u2019 on three Basal-like and three Luminal samples of UNC dataset that were sequenced by mRNA-seq, Ribo-Zero-Seq and DSN-Seq respectively. Comparison of the top 500 most variably expressed genes between Basal-like and Luminal tumors revealed that each pair of RNA-Seq protocols shared about 350 differential expressed genes, and more than 300 genes were consistently identified by all the protocols . We identified 410 genes with a FDR of 0 that were differentially expressed between mRNA-Seq and Ribo-Zero-Seq includes 20,531 genes. To reduce the noise, we only counted genes as present if there were 3 or greater read counts. Using the average number of genes detected on our Agilent microarrays as the baseline , 13.5 million reads from FF mRNA-Seq libraries would allow detection of the same number of genes Figure\u00a0, which iThe growing popularity of RNA-Seq makes it one of the more desired methods to explore the transcriptome. Preparing RNA-Seq libraries with poly(A) enrichment provides an accurate method to characterize mRNAs, which is functionally equivalent to what DNA microarrays have been accomplishing for more than a decade. However, certain biologically relevant RNA species that do not possess poly(A) tails are largely undetected using a poly(A) selection protocol. In addition, FFPE samples, such as those collected as part of standard medical practice, also require library preparation methods that do not rely on the intact poly(A) structure due to the highly degraded nature of the FFPE RNA. In this study, we demonstrate that a Ribo-Zero-Seq protocol using either fresh-frozen (FF) or FFPE samples eliminates rRNA with good efficiency. In evaluation of a possible coverage bias, 5\u2032-to- 3\u2032 bias was reduced in FF Ribo-Zero-Seq libraries as it does not rely on poly(A) selection step.One major distinction across these various protocols is the coverage of the transcriptome. To more directly investigate the relationship between sequencing depth and transcriptome coverage, we performed a simulation approach where mRNA-Seq was the most cost effective strategy to equal a microarray in terms of total genes detected with a minimum of ~13.5 million reads needed. For the same transcriptome coverage, the reads required for Ribo-Zero-Seq in FF and FFPE and DSN-Seq in FF were 35-65\u00a0M reads. However, rRNA depletion protocols also appear to measure immature transcripts (pre-mRNAs) and therefore provide more information on splicing patterns and possible splice junctions. Thus to achieve the same level of exonic reads as FF mRNA-Seq, one needs to sequence 2\u20134 times the number of reads in rRNA-depletion on FFPE RNA libraries.Despite fewer of the total reads mapping to exonic regions and a greater number of transcripts being detected, we did not observe a marked decrease in the correlation between microarray and RNA-Seq in rRNA-depleted libraries, where RNA-zero-Seq and DSN-Seq were found to be highly consistent in gene quantification. Our evaluation of the quantitative consistency of RNA-Seq on FFPE with microarray may be limited in two aspects: (a) the quality of a few UNC FFPE samples was less satisfactory, and (b) not all the tumors have RNA-Seq data on matched FFPE samples that passed our quality control available for this analysis. Yet we still observed very good correlations with microarray data for those samples with complete FFPE data, which gave correlation values nearly identical to those seen when comparing an Agilent microarray versus an Affymetrix microarray .Given the consistent quantification, mRNA-Seq and rRNA depletion protocols exhibited their merits in different aspects. In the set of genes detected by all the protocols, mRNA-Seq provided the highest sensitivity in detecting differentially expressed genes, which was likely due to the greater fraction of reads mapping to the transcriptome. On the other hand, Ribo-Zero-Seq detected about 550 more annotated genes than mRNA-Seq RNA. Moreover, the two rRNA depletion methods have consistent transcript quantification using FFPE RNAs and show high reproducibility.We constructed RNA-Seq libraries using eleven UNC breast tumor samples using different sample preparation protocols including: (a) FF RNA samples by mRNA-Seq, Ribo-Zero-Seq and DSN-Seq and (b) FFPE samples by Ribo-Zero-Seq and DSN-Seq Figure\u00a0B. One ofhttp://supportres.illumina.com/documents/myillumina/7836bd3e-3358-4834-b2f7-80f80acb4e3f/dsn_normalization_sampleprep_application_note_15014673_c.pdf). Sequencing: All cDNA libraries were sequenced using an Illumina HiSeq2000, producing 48x7x48 bp paired-end reads with multiplexing.mRNA-Seq library: Illumina TruSeq\u2122 RNA Sample Prep Kit (Cat# RS-122-2001) was used with 1ug of total RNA for the construction of libraries according to the manufacturer\u2019s protocol. Ribo-Zero library: rRNA was removed from FF or FFPE total RNA using Epicentre\u2019s Ribo-Zero rRNA Removal kit (Cat# RZH11042). For FF samples, 30-100\u00a0ng Ribo-Zero RNA was used for the construction of the library using the Illumina TruSeq\u2122 RNA Sample Prep Kit (Cat# RS-122-2001) and followed the manufacturer\u2019s instruction, except for omitting the purification step before fragmentation. For FFPE samples, 30-100\u00a0ng Ribo-Zero RNA was then incubated with Random Primers at 65\u00b0C for 5\u00a0minutes then Illumina TruSeq\u2122 RNA Sample Prep Kit (Cat# RS-122-2001) was used to construct the library according to the manufacturer\u2019s protocol from the step of First Strand cDNA Synthesis. DSN library: Illumina TruSeq\u2122 RNA Sample Prep Kit (Cat# RS-122-2001) was used with 100\u00a0ng of total RNA for the construction of libraries following the manufacturer\u2019s protocol, except for omitting the purification of mRNA step in FF samples, and the purification and fragmentation step in FFPE samples. The total RNA libraries went through DSN treatment and PCR enrichment according to Illumina DSN Normalization Sample Preparation Guide (http://picard.sourceforge.net/). The aligned reads were sorted and indexed using SAMtools, and then translated to transcriptome coordinates and filtered for indels, large inserts, and zero mapping quality using UBU v1.0 (https://github.com/mozack/ubu). For the reference transcriptome, UCSC hg19 GAF2.1 for KnownGenes [All samples were processed and filtered as described in The Cancer Genome Atlas . Bases awnGenes was usedwnGenes v1.1.13.The RNA-Seq data was filtered by requiring the gross RSEM count to be \u22653 for each gene. For each protocol, the detected gene sets were defined as genes that were reported in >70% tumor lanes and with 3 or more reads. To determine the amount of input reads needed for sufficient transcriptome coverage, a simulation test was performed on the UNC data. A series of fixed number of reads were randomly selected from each protocol in a drawing without replacement method. For all the resampling levels, the simulated data followed the same alignment and filtering pipeline as described above. Gene sets detected were then identified for all the various levels.For all the FF tumors and the Common Reference Sample, Agilent 244,000 feature whole genome microarrays were hybridized with tumor RNAs (Cy5) and a human common reference (Cy3) and lowess normalized as described in Herschkowitz et al. . In the The RNA-Seq gene quantification data was next filtered by gene counts as above. The log2 transformed abundance of tumor samples was reported and was used to derive the correlation between RNA-Seq protocol pairs. Using R package MethComp, Deming regression was applied to compare the sensitivity in detecting differentially expressed genes. An unpaired two-class SAM analysis was used to identify genes that have differential expression level in a) mRNA-Seq versus Ribo-Zero-Seq, and b) Ribo-Zero-Seq versus DSN-Seq.https://cghub.ucsc.edu/) and DCC .Gene expression quantification by microarray and RNA-Seq for all samples new to this manuscript can be found in GEO database under accession GSE51783. Aligned BAM files are available at dbGaP under the series ID of phs000676.v1.p1. TCGA sample RNA-Seq data is available at cgHub Read pile-up plots of GATA3 in Sample 020578B showing data for five different RNA-Seq libraries. (B) Close-up of the read mapping identifying reads that span exon-intron boundaries, which identify unspliced mRNA species. (PDF 447 KB)Additional file 2: Figure S2: Intrinsic gene set clustering analysis. Hierarchical cluster using a breast cancer intrinsic gene set (~2000 genes) and 88 breast tumor samples prepared using the multiple protocols, with an additional 816 samples from the TCGA Breast Cancer Project . The rows above the heat map identify the 88 samples from this study, their RNA-Seq protocol type, and the red arrows show the location of the few mismatched samples. (PDF 1 MB)Additional file 3: Figure S3: Comparison of the top 500 differentially expressed genes between Basal and Luminal tumors detected by mRNA-Seq, Ribo-Zero-Seq and DSN-Seq. (PDF 194 KB)Additional file 4: Table S1: Comparison of the top 500 differentially expressed genes between Basal and Luminal tumors detected by mRNA-Seq, Ribo-Zero-Seq and DSN-Seq. The list of 307 differentially expressed genes that are identified SAM analysis in all the three protocols. (XLSX 55 KB)Additional file 5: Table S2: Differentially expressed gene list across RNA-Seq protocols obtained from Significance Analysis of Microarray. SAM analysis comparison of mRNA-Seq versus Ribo-Zero-Seq using an FDR\u2009=\u20090. (A) Uniquely expressed genes in Ribo-Zero-Seq. (B) Lowly expressed genes in Ribo-Zero-Seq. SAM analysis comparison of Ribo-Zero-Seq versus DSM-Seq using an FDR\u2009=\u20090. (C) Uniquely expressed genes in DSN-Seq. (D) Lowly expressed genes in DSN-Seq. (XLSX 64 KB)Additional file 6: Table S3: Comparison of genes detected by mRNA-Seq and Ribo-Zero-Seq in FF samples. (A) Genes detected only by mRNA-Seq and (B) genes detected only by Ribo-Zero-Seq. (XLSX 62 KB)"} +{"text": "Myeloid-differentiation factor 88 (MyD88) is shared between several TLRs and the Interleukin-1 receptor family. MyD88-deficiency protects mice from intestinal cancer formation in genetic models for colon cancer. The genetic mouse model presented here allows tissue-specific expression of MyD88, and thereby the dissection of the complex interaction between tumour and immune system during intestinal carcinogenesis.Pattern recognition receptors from the Toll-like receptor (TLR) family are pivotal components of innate immunity, and have been shown to contribute to colon cancer formation. However, the molecular and cellular mechanisms underlying TLR-signaling in colon cancer remain unclear. The adapter protein myD88 expression (MyD88LSL), faithfully phenotyping a global gene knock-out. Tissue-specific re-expression of MyD88 in mice is mediated based on the Cre-recombinase. Breeding of MyD88LSL mice with LysMCre or pvillin-Cre mice leads to tissue-specific excision of the 'intron-gene-trap', retaining endogenous regulation of gene expression. MyD88 expression and successful reconstitution of TLR-signaling was detected in either myeloid cells (MyD88MYEL) or intestinal epithelial cells (MyD88IEC). Subsequently, these animals were mated with Apc1638N/+ mice, an established genetic mouse model for human colon cancer.Insertion of an 'intron-gene-trap' flanked with loxP motifs into the first intron of the MyD88 gene locus leads to global inactivation of 1638N/+ mice. Re-expression of MyD88 in intestinal epithelial cells only partially restored tumor formation. On the other hand, reconstitution of MyD88 expression in myeloid cells triggered tumour development virtually indistinguishable from parental Apc1638N/+ mice. Activation of the canonical Wnt signaling pathway, induced by loss of function of Apc, was independent of MyD88. In contrast, MyD88 expression was required for full activation of MAPK/ERK signaling in intestinal epithelial cells. Furthermore, our results suggest a pro-tumorigenic function for the pro-inflammatory cytokines IL-1beta and IL-6, which were produced in a MyD88-dependent fashion by myeloid cells.Global MyD88 deficiency dramatically decreased tumour incidence and aggressiveness in ApcMyD88-mediated signaling has pro-tumorigenic effects in both IECs and in myeloid cells, but via different mechanisms. Moreover, MyD88 function in myeloid cells is crucial for intestinal tumour development, and its inhibition may form a promising therapeutic strategy."} +{"text": "However it is difficult to completely suppress residual luminal blood which may mimic plaque leading to difficulties in diagnosis [Fan. JMRI. 2010:31], especially after contrast enhancement (CE). Additionally, a more efficient imaging protocol is needed that covers both carotid and intracranial arteries as atherosclerosis is a systematic disease that usually affects multiple vascular beds. The purpose of this work is to develop a 3D combined carotid and intracranial vessel wall imaging protocol with high resolution . A coronal imaging slab slightly rotated transversely was oriented to cover both carotid and vertibrobasilar arterial systems with one scan. Healthy subjects (n=14) and symptomatic patients (n=6) were scanned with DANTE-SPACE, SPACE-only and T1w 2D TSE in a randomized order pre- and post-CE (0.1mmol/kg). DANTE-SPACE parameters include: FA = 10; train length = 100; GDANTE-SPACE improved both arterial and venous blood suppression compared with SPACE-only Figure . Five p. Also veDANTE-SPACE significantly improved blood suppression and vessel wall CNR both pre- and post-CE when SPACE-only resulted in residual blood. Combined high-resolution carotid and intracranial imaging using DANTE-SPACE was feasible and time-efficient (<6 min), with the potential ability to identify different plaque components including calcification, intra-plaque haemorrhage and fibrous cap.NIH/NHLBI: R01HL096119."} +{"text": "Moorea producens (Lyngbya majuscula). Kalkitoxin exhibited N-methyl-d-aspartate (NMDA)-mediated neurotoxicity and acted as an inhibitory ligand for voltage-sensitive sodium channels in cultured rat cerebellar granule neurons. Subsequent studies revealed that kalkitoxin generated a delayed form of colon tumor cell cytotoxicity in 7-day clonogenic cell survival assays. Cell line- and exposure time-dependent cytostatic/cytotoxic effects were previously observed with mitochondria-targeted inhibitors of hypoxia-inducible factor-1 (HIF-1). The transcription factor HIF-1 functions as a key regulator of oxygen homeostasis. Therefore, we investigated the ability of kalkitoxin to inhibit hypoxic signaling in human tumor cell lines. Kalkitoxin potently and selectively inhibited hypoxia-induced activation of HIF-1 in T47D breast tumor cells (IC50 5.6 nM). Mechanistic studies revealed that kalkitoxin inhibits HIF-1 activation by suppressing mitochondrial oxygen consumption at electron transport chain (ETC) complex I (NADH-ubiquinone oxidoreductase). Further studies indicate that kalkitoxin targets tumor angiogenesis by blocking the induction of angiogenic factors in tumor cells.The biologically active lipopeptide kalkitoxin was previously isolated from the marine cyanobacterium Moorea producens sp. nov. (Oscillatoriaceae), previously classified as Lyngbya majuscula Gomont, typically forms localized mini-blooms that may cover sections of a coral reef and vertebrate species . Thus, by simultaneously acting as both a voltage-gated Na+ channel blocker and a potent rotenone-like mitochondrial disruptor, kalkitoxin may be able to provide an additional level of grazing defense against a broad range of herbivore species.Sodium channel and mitochondria-associated neurotoxicity may limit the therapeutic potential of kalkitoxin as an antitumor chemotherapeutic agent. Mechanistically, kalkitoxin and other recently reported marine natural product HIF-1 inhibitors suppressiewed in ). This i"} +{"text": "MYC. Previous reports have characterized metabolic changes in proliferating cells upon MYC loss or gain of function. However, metabolic differences between MYC-dependent cancer cells and their isogenic differentiated counterparts have not been characterized upon MYC suppression in vitro.Cancer cells engage in aerobic glycolysis and glutaminolysis to fulfill their biosynthetic and energetic demands in part by activating MYC-dependent mouse osteogenic sarcomas and differentiated osteoid cells induced upon MYC suppression. While osteogenic sarcoma cells increased oxygen consumption and spare respiratory capacity upon MYC suppression, they displayed minimal changes in glucose and glutamine consumption as well as their respective contribution to the citrate pool. However, glutamine significantly induced oxygen consumption in the presence of MYC which was dependent on aminotransferases. Furthermore, inhibition of aminotransferases selectively diminished cell proliferation and survival of osteogenic sarcoma MYC-expressing cells. There were minimal changes in ROS levels and cell death sensitivity to reactive oxygen species (ROS)-inducing agents between osteoid cells and osteogenic sarcoma cells. Nevertheless, the mitochondrial-targeted antioxidant Mito-Vitamin E still diminished proliferation of MYC-dependent osteogenic sarcoma cells.Here we report metabolic changes between MYC-driven tumors.These data highlight that aminotransferases and mitochondrial ROS might be attractive targets for cancer therapy in Tumor cells display increased glucose metabolism to meet the anabolic demands required for cell proliferation . Glycolyc-MYC (herein termed MYC) glucose and 2 mM L-glutamine, or 2 mM L-[U-13C]glutamine and 10 mM D-glucose. After six hours, metabolites were extracted with 50% methanol and analyzed using an Agilent 6970 gas chromatograph and an Agilent 5973 mass selective detector. Analysis of 13C enrichment and mass isotopomer distribution was performed as previously described -glutamine and unlabeled glucose and examined 13C enrichment in citrate. Citrate m+4 is formed when glutamine-derived alpha-ketoglutarate is metabolized in the TCA cycle to generate OAA m+4; this can combine with an unlabeled acetyl-CoA to form citrate with four additional mass units. We observed a modest increase in the unlabeled fraction, and a decrease in the amount of glutamine-derived citrate m+4 in the osteocytes , indicative of an increase in cytosolic ROS levels convert glutamate into alpha-ketoglutarate to fuel the TCA cycle. To test whether aminotransferases are required for glutamine-dependent increase in OCR in MYC-dependent osteogenic sarcoma cells, we utilized AOA, an inhibitor of aminotransferase activity. Glutamine substantially stimulated OCR which was diminished by AOA and rescued by DMK -1-piperazineethanesulfonic acid; FCCP: Trifluorocarbonylcyanide phenylhydrazone; AOA: aminooxyacetic acid; NAC: N-acetylcysteine; PIETC: beta-phenylethyl isothiocyanate; BSO: buthionine sulfoximine; VDAC1: voltage dependent anion channel 1; GOT2: glutamic oxaloacetic transaminase 2; GPT2: glutamic pyruvate transaminase 2; ROS: reactive oxygen species; roGFP2: redox sensitive green fluorescent protein 2; mito-roGFP2: mitochondrial targeted roGFP2; OCR: oxygen consumption rate; GFP: green fluorescent protein; DTT: dithiothreitol; TCA: tricarboxylic acid cycle; TMRE: tetramethylrhodamine, ethyl ester; TFAM: mitochondrial transcription factor A; MVE: mitochondrial targeted vitamin E; OAA: oxaloacetate; GLSs: glutaminasesThe authors declare that they have no competing interests.MYC-dependent osteogenic sarcoma cells and carefully edited the manuscript. JMM provided the antibody against GLS1 and carefully edited the manuscript. RJD designed the experiments and edited the paper. NSC and EA designed the experiments and wrote the paper. All authors read and approved the final manuscript.EA technically performed the experiments in Figures\u00a01 and 2 and 4, 5, 6. ARM performed the experiments in Figure\u00a03. DWF provided the"} +{"text": "Apc) mutation. The levels of O-GlcNAcylation and O-GlcNAc cycling enzymes were simultaneously upregulated in intestinal adenomas from mice, and in human patients. In two independent microarray data sets, the expression of OGA and OGT was significantly associated with poor cancer-specific survival of colorectal cancer (CRC) patients. In addition, OGA heterozygosity, which results in increased levels of O-GlcNAcylation, attenuated intestinal tumor formation in the Apcmin/+ background. Apcmin/+OGA+/\u2212 mice exhibited a significantly increased survival rate compared with Apcmin/+ mice. Consistent with this, Apcmin/+OGA+/\u2212 mice expressed lower levels of Wnt target genes than Apcmin/+. However, the knockout of OGA did not affect Wnt/\u03b2-catenin signaling. Overall, these findings suggest that OGA is crucial for tumor growth in CRC independently of Wnt/\u03b2-catenin signaling.Emerging evidence suggests that aberrant O-GlcNAcylation is associated with tumorigenesis. Many oncogenic factors are O-GlcNAcylated, which modulates their functions. However, it remains unclear how O-GlcNAcylation and O-GlcNAc cycling enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), affect the development of cancer in animal models. In this study, we show that reduced level of OGA attenuates colorectal tumorigenesis induced by Adenomatous polyposis coli ( N-acetylglucosamine to the Ser and Thr residues (O-GlcNAc) of target proteins, respectively.1 Increasing evidence suggests that OGT and/or OGA expression and O-GlcNAc levels are altered in different types of cancer, including colon, lung,2 breast,4 liver,5 prostate,7 pancreas8 and bladder.9 Furthermore, tumor aggressiveness is closely associated with the levels of O-GlcNAcylation, OGT and/or OGA. Consistent with this, various O-GlcNAc-modified proteins are perturbed in tumorigenesis. For example, O-GlcNAc-modified proteins are involved in cancer-relevant processes such as transcriptional regulation, cell proliferation and the cell cycle.10 The activity of many oncogenic factors, including c-Myc,11 \u03b2-catenin,12 p5313 and FoxM1,14 is regulated by direct O-GlcNAcylation. In addition, O-GlcNAc cycling is required for the precise control of the cell cycle, suggesting an essential role for O-GlcNAc cycling enzymes in cell proliferation.16O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) are enzymes that regulate the addition and removal of monosaccharides of O-linked \u03b2-Apc) is a tumor suppressor gene that is mutated in \u223c80% of sporadic adenomatous polyps and CRCs.17 Mutant APC causes the oncogenic activation of \u03b2-catenin. Notably, \u03b2-catenin is directly O-GlcNAcylated. Several studies have suggested that the O-GlcNAcylation of \u03b2-catenin affects its transcriptional activity and subcellular localization.18 Moreover, higher levels of O-GlcNAcylation and OGT expression are found in colon tumors than in corresponding non-tumorous mucosal tissues.2 Although current evidence implicates O-GlcNAcylation in CRC, it remains unclear exactly how the levels of O-GlcNAcylation or O-GlcNAc cycling enzymes affect colorectal tumorigenesis. To evaluate the role of OGA in colorectal tumorigenesis, we investigated whether OGA heterozygosity could alter CRC susceptibility in Apcmin/+ mice.Colorectal cancer (CRC) is the third common cancer worldwide. Adenomatous polyposis coli in colonic adenomas compared with normal mucosa 20 were analyzed for the association between OGT and OGA expression and patient survival and cancer recurrence. The Moffitt Cancer Center data set reported the disease-specific survival times for 177 CRC patients. The gene expression levels of OGT and OGA had a strong positive correlation , supporting the upregulation of these genes in cancer (P=0.00245) and 2.42 (P=0.0155). Patients with reduced levels of OGT and OGA exhibited improved survival rates n cancer . Expressal rates .OGA+/\u2212 mice to explore the effect of increased OGT and OGA expression on colorectal tumorigenesis. Apcmin/+ mice were crossed with OGA+/\u2212 mice. The intestines of OGA+/\u2212 mice showed reduced levels of both OGA and OGT expression compared with control mice (OGA\u2212/\u2212 mice displayed perinatal lethality, and that OGA\u2212/\u2212 mouse embryonic fibroblasts downregulate OGT, which might compensate for the increased levels of O-GlcNAcylation.15 Therefore, OGA+/\u2212 mice are the correct model for assessing the effect of reduced OGT and OGA expression on colorectal tumorigenesis. A quantitative analysis of tumor burden revealed a significant twofold reduction in the number and size of adenomas in the small intestine of Apcmin/+OGA+/\u2212 compared with Apcmin/+ mice (Apcmin/+OGA+/\u2212 mice displayed fewer and smaller tumors along the intestinal tract (Apcmin/+OGA+/\u2212 mice exhibited a significantly increased survival rate compared with Apcmin/+ mice, and all Apcmin/+ mice died within 8 months. In contrast, Apcmin/+OGA+/\u2212 mice showed dramatically increased survival rates (We next used rol mice . We demon/+ mice . Apcmin/al tract . Apcmin/al rates .Apcmin/+OGA+/\u2212 mice, we next analyzed molecular changes. We examined whether OGA heterozygosity affects the expression of known Wnt/\u03b2-catenin target genes, which are upregulated in APC-mediated intestinal tumors. Nine genes were decreased in Apcmin/+OGA+/\u2212 intestines compared with Apcmin/+ intestines (Axin2 and Jun were not significantly changed in 8-week-old Apcmin/+OGA+/\u2212 mice compared with age-matched Apcmin/+ controls. However, dramatically reduced expression of Axin2 and Jun was observed in 16- and 24-week-old animals (15 In addition, OGT knockdown attenuates the growth of breast,14 lung and colon cancer cells.2 These results suggest that reduced OGT and OGA levels disrupt the dynamic regulation of O-GlcNAcylation, which attenuates cell growth.Based on the attenuated intestine tumor formation in testines . We obse animals . These rApcmin/+ mice.21 Importantly, \u03b2-catenin is directly O-GlcNAcylated, and we also observed that \u03b2-catenin is modified with O-GlcNAc (22 OGT interacts with \u03b2-catenin to regulate cyclin D1 synthesis upon serum stimulation.12 Therefore, we assessed whether OGA deficiency affects Wnt/\u03b2-catenin signaling. The deletion of OGA did not affect Wnt3-mediated \u03b2-catenin accumulation (Axin2 and Jun mRNA levels between OGA+/+ and OGA\u2212/\u2212 mouse embryonic fibroblasts after stimulation with Wnt3a (The activation of \u03b2-catenin is essential not only for tumor initiation but also for tumor progression in O-GlcNAc . The O-Gmulation . In addith Wnt3a . We alsoth Wnt3a . Increas23 However, it remains unclear whether the elevated O-GlcNAcylation caused by increased OGT activity promotes tumorigenesis in vivo. Here, we suggested a critical role for O-GlcNAc cycling enzymes in colorectal tumorigenesis. We observed that human colonic adenomas exhibit elevated O-GlcNAcylation, and increased levels of both OGT and OGA. Interestingly, an analysis of microarray data sets revealed that OGA and OGT expression was correlated. This supports the immunoblotting data, which showed simultaneously increased OGT and OGA in colonic adenomas. Importantly, OGT and OGA are both correlated with survival and cancer recurrence in patients with CRC. Our results demonstrate that OGA heterozygosity reduces APC-mediated colorectal tumorigenesis and enhances the survival of OGA heterozygous mice in the Apcmin/+ background. Wnt target genes were downregulated in Apcmin/+OGA+/\u2212 mice compared with Apcmin/+ controls. Although \u03b2-catenin is directly O-GlcNAcylated, elevated O-GlcNAcylation did not affect Wnt/\u03b2-catenin signaling in vitro. Although O-GlcNAcylation is elevated in colonic adenomas, we observed reduced tumorigenesis in OGA+/\u2212 mice, in which O-GlcNAcylation is constantly elevated. These results suggest that the levels of OGA and OGT are critical for colorectal tumorigenesis. These results support previous studies showing that the knockdown of OGA or OGT reduced cell growth in vitro.15 This suggests that reduced levels of OGT and OGA do not properly regulate dynamic O-GlcNAcylation, which influences the functions of O-GlcNAc target proteins. Nevertheless, the mechanism by which OGA heterozygosity attenuates tumorigenesis remains unclear. However, many studies have suggested that aberrant O-GlcNAcylated proteins contribute to the disruption of cancer cell growth. Notably, many transcription factors, including c-Myc, p53, NF-\u03baB, SP1, FoxM1 and HCF-1, are O-GlcNAcylated.24 O-GlcNAcylation modulates their activity or stability, which affects cancer cell growth. Phosphorylation is regulated by a variety of protein kinases and phosphatases. Although O-GlcNAcylation is similar to phosphorylation, unlike phosphorylation it is regulated by only OGT and OGA, which are encoded by single genes. Therefore, reduced levels of O-GlcNAc cycling enzymes cause various changes in the activity of entire target proteins. Furthermore, reduced levels of OGT and OGA might not normally regulate dynamic O-GlcNAcylation during the cell cycle, which is required for cell cycle control.16 We conclude that O-GlcNAc enzymes are essential for tumor growth in CRC, and that targeting OGA may help suppress the intestinal tumorigenesis initiated by Apc mutation.Many studies have suggested that elevated O-GlcNAcylation and/or O-GlcNAc cycling enzymes contribute to tumorigenesis. In colon cancer, the levels of O-GlcNAc and OGT are increased."} +{"text": "The clonal expansion and malignant transformation of HTLV-1 infected CD4+ T-cells have been linked to the reprogramming effects of HTLV-1 on host transcriptional profile. Coupled to transcription, alternative splicing (AS) is a post-transcriptional process that plays critical role in the complexity of transcriptome and splicing abnormalities frequently occur in cancer. To examine whether AS modifications associate with HTLV-1-associated leukemogenesis, we compared the exon expression profiles of ATL cells with that of CD4+ T-cell clones obtained by limited-dilution cloning of PBMC deriving from HTLV-1 carriers. 3 ATL cells and 12 untransformed infected clones clustering in infected, uninfected, PHA-stimulated or unstimulated CD4+ T cells were compared for exon RNA content using Exon Chip Human microarray. Hierarchical clustering analysis identified 12516 alternative spliced events (3642 genes) that clearly separated ATL samples from the 4 untransformed phenotypes mentioned above. In contrast, the exon content of 1539 genes differed between untransformed infected and uninfected T-CD4+ cells. Overall, less than 5% alternatively spliced genes were found differentially expressed at the transcriptional level. Microarray data were confirmed for 18 AS events using exon specific RT-PCR analysis. Pathway analysis of alternatively spliced genes (3642) in ATL cells revealed new AS-based pathways for p53 signaling, cell cycle and DNA replication while those of untransformed infected CD4+ T-cells were enriched in pathways for cellular movement and DNA repair. These findings unveil a new layer of complexity in the interplay between HTLV-1 and host cell gene expression machinery in which AS might play a central role in tumor initiation and promotion."} +{"text": "Studies using T-cell receptor (TCR) or chimeric antigen receptor (CAR) transduced T-cells have shown the effectiveness of adoptive immunotherapy to treat different malignancies. The efficacy and safety of such interventions greatly depends on good target selection to prevent on-target toxicity. Furthermore, the broad application of TCR-based adoptive immunotherapy is hampered by a lack of an effective immune response against self-antigens. Through self-tolerance, T-cells carrying high-affinity TCRs reactive to self-antigens are deleted during thymic selection. An attractive strategy is to exploit the immunogenicity of foreign human leukocyte antigen (HLA) molecules to generate an effective immune response against these antigens. Here, we describe a protocol to efficiently isolate high-avidity alloHLA-restricted T-cells targeting the B-cell compartment.From a B-cell peptide elution library 15 peptides derived from genes exhibiting B-cell restricted expression patterns were identified and peptide-MHC multimers (pMHC) of HLA-A*0201 were generated. Via MACSorting and FACSorting a plethora of pMHC-multimer binding T-cell clones from HLA-A*0201-negative individuals were isolated. Generated T-cell clones were selected based on peptide-specificity and avidity for further characterization.We successfully isolated two distinct T-cell clones carrying high-affinity TCRs specific for a CD20 peptide presented in HLA-A*0201. CD20 dependent recognition could be demonstrated by genetically engineering CD20-negative K562-A2 cells to express CD20. The isolated T-cell clones efficiently recognized CD20-expressing HLA-A*0201 primary chronic lymphocytic leukaemia (CLL), acute lymphoblastic leukaemia (ALL) and mantle cell lymphoma (MCL), while recognition of CD20-negative hematopoietic and non-hematopoietic cell-subsets was absent. In addition, the CD20-specific T-cell clones were able to more efficiently recognise ALL cell-lines than CD20 specific antibodies. We demonstrated that on ALL cell lines with only very low CD20 surface expression, the CD20-specific T cell clones could still efficiently recognise endogenously processed CD20-derived peptides in the context of HLA-A*0201.In conclusion, we developed a platform for the rapid identification of high-affinity TCRs of therapeutic relevance targeted to self-antigens by combining gene expression data with valuable information on peptide processing from peptide elution studies and exploiting the immunogenicity of foreign HLA. Using this platform we successfully isolated CD20-specific TCRs which could broaden the application of immunotherapies targeted to CD20 in cases were CD20-cell surface expression is low. Based on its general principle the developed platform could easily be adapted to target other malignancies."} +{"text": "In contrast, no such induction of the antigen-specific CD8+ T cells was observed, when mice were immunized with the peptide alone or peptide plus an aluminum adjuvant. These results suggest that the Tax peptide-carrying PIC nanoparticles are capable of inducing cellular immune responses and may have potential as an effective vaccine adjuvant for anti-HTLV-1 vaccines.Development of safe and effective vaccines is important for controlling a variety of infectious diseases, including retroviral infections. The induction of cytotoxic T lymphocytes (CTLs) is a promising strategy for elimination of infected cells. Polyion complex (PIC) nanoparticles have been created using anionic biodegradable poly(\u03b3-glutamic acid) (\u03b3-PGA) and cationic protamine. Amphiphilic graft copolymers, consisting of \u03b3-PGA and l-phenylalanine (l-Phe) hydrophobic side chain, were synthesized by grafting l-Phe to \u03b3-PGA. The PIC nanoparticles were prepared by mixing the graft copolymers with protamine in phosphate buffered saline. In this study, antigen peptide-carrying PIC nanoparticles were examined for their effect on the induction of antigen-specific cellular immunity in mice. The antigen-specific CTL response was evaluated by intracellular cytokine staining and IFN-\u03b3 ELISPOT assay. The immunization with PIC nanoparticles carrying HTLV-1 Tax peptide could induce the expansion of Tax-specific CD8"} +{"text": "Multiple studies have shown that dendritic cell (DC)-based vaccines can induce antitumor immunity. Previously, we reported that gemcitabine enhances the efficacy of DC vaccination in a mouse model of pancreatic carcinoma. The present study aimed at investigating the influence of gemcitabine on vaccine-induced anti-tumoral immune responses in a syngeneic pancreatic cancer model.+ T cells and antibody titers were monitored by FACS analysis and ELISA, respectively.Subcutaneous or orthotopic pancreatic tumours were induced in C57BL/6 mice using Panc02 cells expressing the model antigen OVA (PancOVA). Bone marrow-derived DC were loaded with soluble OVA protein (OVA-DC). Animals received gemcitabine twice weekly. OVA-specific CD8+ T cells and antibody titers. DC migration to draining lymph nodes and antigen cross-presentation were unaffected. Despite reduced numbers of tumour-reactive T cells in peripheral blood, in vivo cytotoxicity assays revealed that CTL-mediated killing was preserved. In vitro assays revealed sensitization of tumour cells to CTL-mediated lysis by gemcitabine. In addition, gemcitabine facilitated recruitment of CD8+ T cells into tumors in DC-vaccinated mice. T and B cell suppression by gemcitabine could be avoided by starting chemotherapy after two cycles of DC vaccination.Gemcitabine enhanced clinical efficacy of the OVA-DC vaccine. Interestingly, gemcitabine significantly suppressed the vaccine-induced frequency of antigen-specific CD8Gemcitabine enhances therapeutic efficacy of DC vaccination despite its negative influence on vaccine-induced T cell proliferation. Quantitative analysis of tumour-reactive T cells in peripheral blood may thus not predict vaccination success in the setting of concomitant chemotherapy."} +{"text": "Several monoclonal antibodies (MAbs) with demonstrated clinical anti-cancer activities have been engineered as fully human IgG1 entities to also encompass their potential to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) of human tumor cells. Avelumab is a fully human IgG1 MAb targeting the co-regulatory protein Programmed Death-Ligand 1 (PD-L1), and is thus distinct from other MAbs targeting the PD-L1/PD-1 axis currently being evaluated in clinical trials. Concern has been raised that an anti-PD-L1 antibody capable of inducing ADCC may negatively affect PD-L1 expressing immune cell subtypes. This work is intended to determine if there is any validity to this concern.The clinical activity of Avelumab, observed in several tumor types in ongoing clinical studies such as NCT01772004, has been and will be reported elsewhere. In the studies reported here, Avelumab is shown to mediate ADCC of several types of human tumor cell lines in vitro, with tumor cell lysis mediated mainly by human CD16+ monocytes and natural killer (NK) cells. Since some human immune cell subsets express PD-L1 on their surface, studies were undertaken to evaluate changes in the frequency of immune cell subsets in peripheral blood mononuclear cells (PBMC) from cancer patients pre- vs post-treatment with Avelumab. Immune cells evaluated were PD-L1 positive and PD-L1 negative subsets of the following: CD4+ T cells, CD8+ T cells, NK cells, regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSC), natural killer T cells (NKT), plasmacytoid dendritic cells (DC), conventional DC, and B cells.Forty-two post-treatment PBMC samples were evaluated as follows: pre vs 1 dose of Avelumab ; pre vs 3 doses of Avelumab ; and pre vs 9 doses of Avelumab . In all cases there were no statistical differences pre- vs post-treatment in any immune cell subset, and at any time point analyzed, regardless of whether the immune subset expressed PD-L1 or not. In addition, no changes were observed in absolute lymphocyte counts at any time point analyzed. Statistical analysis of all relevant immune cell subsets will be presented.While immune cell subsets pre- vs post-treatment continue to be analyzed in various patient cohorts, these studies provide evidence that Avelumab, a fully human IgG1 MAb, capable of mediating ADCC, can be administered safely to cancer patients without altering the balance of numerous PBMC immune cell subsets."} +{"text": "Drosophila hematopoietic progenitors (prohemocytes). However, many questions remain about how ROS alter the regulatory machinery to promote progenitor differentiation. Here, we provide evidence for the hypothesis that ROS reduce E-cadherin levels to promote Drosophila prohemocyte differentiation. Specifically, we show that knockdown of the antioxidants, Superoxide dismutatase 2 and Catalase reduce E-cadherin protein levels prior to the loss of Odd-skipped-expressing prohemocytes. Additionally, over-expression of E-cadherin limits prohemocyte differentiation resulting from paraquat-induced oxidative stress. Furthermore, two established targets of ROS, Enhancer of Polycomb and FOS, control the level of E-cadherin protein expression. Finally, we show that knockdown of either Superoxide dismutatase 2 or Catalase leads to an increase in the E-cadherin repressor, Serpent. As a result, antioxidants and targets of ROS can control E-cadherin protein levels, and over-expression of E-cadherin can ameliorate the prohemocyte response to oxidative stress. Collectively, these data strongly suggest that ROS promote differentiation by reducing E-cadherin levels. In mammalian systems, ROS promote embryonic stem cell differentiation, whereas E-cadherin blocks differentiation. However, it is not known if elevated ROS reduce E-cadherin to promote embryonic stem cell differentiation. Thus, our findings may have identified an important mechanism by which ROS promote stem/progenitor cell differentiation.Mitochondrial reactive oxygen species (ROS) regulate a variety of biological processes by networking with signal transduction pathways to maintain homeostasis and support adaptation to stress. In this capacity, ROS have been shown to promote the differentiation of progenitor cells, including mammalian embryonic and hematopoietic stem cells and Drosophila hematopoietic progenitors (prohemocytes) Drosophila hematopoietic system has facilitated the identification of causal links between wasp parasitization, ROS, and prohemocyte fate choice in vivoReactive oxygen species (ROS) are produced primarily in the mitochondria and increase in response to cellular stressors such as infection, starvation, or hypoxia. As a result, increased ROS levels alert the cell to changes in environmental conditions and the level of ROS correlates with the severity of stress. Consequently, high levels of ROS lead to loss of viability, whereas moderate increases promote cellular adaptation to stress Drosophila prohemocytes share key characteristics with mammalian hematopoietic stem cells, including quiescence, multipotency, and niche-dependence Prohemocytes are located within a specialized larval organ known as the lymph gland Drosophila primordial germline and in mammalian models of cancer Drosophila ovarium, over-expression of either E-cadherin or Superoxide dismutase can prolong the lifespan of germline stem cells. However, it is not known if these factors work together to control stem cell aging E-cadherin is the founding member of a large evolutionarily conserved family of calcium-dependent transmembrane proteins that are the principal components of adherens junctions Drosophila prohemocyte differentiation. In support of this hypothesis, we show that knockdown of Superoxide dismutatase 2 (SOD2) and Catalase (Cat) reduces E-cadherin levels. Importantly, this occurs prior to the loss of Odd-skipped- (Odd) expressing prohemocytes. Additionally, over-expression of E-cadherin limits prohemocyte differentiation resulting from paraquat-induced oxidative stress. Furthermore, FOS and the polycomb protein Enhancer of Polycomb (E(Pc)), both established targets of ROS In this study, we provide evidence that ROS reduce E-cadherin levels to promote 1118w or 67c23y w flies served as the wild-type stock for these studies. The following strains were generous gifts from colleagues: UAS-E-cadherin from G. Longmore (Washington University); domeless-Gal4 from M. Crozatier (University Paul Sabatier); Tep4-Gal4 from T. Tokusumi and R. A. Schulz (University of Notre Dame). The following strains were obtained from the Bloomington Stock Center: 1 shg2 bw1 sp1/CyOcn, 1 v1; UAS-Sod2RNAiy, UAS-Sod2; 1 w67c23; Sod2KG06854y, 1 v1; UAS-CatRNAiy, 1 v1; UAS-JafracRNAiy, 1 v1; UAS-fosRNAiy, 1 v1; UAS-E(Pc)RNAiy, 1 v1; UAS-basketRNAiy, 1 sc* v1; UAS-ND75RNAiy.Early-third instar larvae (collected 78 to 86 hours after egg laying) were placed on media containing either 0 or 10 mM paraquat for 6 hours. Larvae were then thoroughly washed, transferred to fresh media without paraquat, and allowed to recover from treatment for at least 18 hours prior to dissection.1118w or 67c23y w mates. In general, the dome-Gal4 driver was used for all transgene expression studies. However, the Tep-Gal4 driver was used in experiments involving paraquat treatment because dome-Gal4/+ and dome-Gal4/+; UAS-E-cadherin/+ animals died after treatment.Gene expression analyses were conducted using lymph glands from mid-third instar larvae (collected 96 to 104 hours after egg laying). However, as indicated in specific experiments, gene expression analyses were also conducted using either early-third instar larvae (collected 78 to 86 hours after egg laying) or late-third instar larvae (collected 112 to 120 hours after egg laying). All control and experimental samples were age matched and cultured on standard media at 23\u00b0C. The UAS/Gal4 binary system 2 or more than 5 individual lamellocytes were visible The dissection and fixation of larval lymph glands were performed as previously described Drosophila, plasmatocyte differentiation increases in Sod2/Sod2 hypomorphs during the late-third larval instar domeless-Gal4 driver to express the RNAiUAS-ND75 transgene in prohemocytes , and we observed a statistically significant increase in both plasmatocytes and crystal cells under these conditions and Tep-Gal4 heterozygotes (Tep/+) served as controls. We identified prohemocytes using the specific marker, Odd, and assessed aberrant prohemocyte differentiation by assaying for lamellocytes.Our results suggest that increased levels of superoxide promote prohemocyte differentiation by reducing E-cadherin levels. If this is the case, then over-expression of E-cadherin should limit superoxide-induced differentiation. Paraquat has been widely used to increase superoxide production First, we showed that paraquat treatment reduced prohemocyte number by comparing the results from Tep/+ treated and untreated animals. Under these circumstances, we observed a statistically significant decrease in the number of prohemocytes in treated animals compared to untreated controls . Next weDrosophila, excess hydrogen peroxide can lead to aberrant blood cell differentiation Superoxide is a precursor of hydrogen peroxide dome-Gal4 driver to express the RNAiUAS-Jafrac transgene in prohemocytes. Knockdown of Jafrac led to aberrant lamellocyte differentiation and concomitant loss of E-cadherin expression in late-third instar lymph glands signal transduction pathway is activated by a variety of environmental stress signals, including elevated levels of ROS Drosophila prohemocytes. Given that increased ROS activate JNK signaling, these findings are consistent with the hypothesis that elevated ROS repress E-cadherin.JNK signaling also downregulates polycomb activity GATA factors regulate gene expression during a variety of biological processes across taxa. Furthermore, GATA activity is modified through its interaction with the transcriptional co-factor, Friend of GATA (FOG). We recently showed that the GATA factor, Srp, and the FOG factor, U-shaped (Ush), regulate E-cadherin expression in prohemocytes. Srp represses E-cadherin to promote prohemocyte differentiation. However, when Srp is bound to Ush, the capacity to repress E-cadherin is diminished. Thus, with the downregulation of Ush, the amount of unbound Srp increases, which leads to the reduction of E-cadherin RNAiUAS-Sod2 transgene , and did not observe a significant change in the level of Srp , control the level of E-cadherin protein expression. Specifically, E(Pc) maintains E-cadherin, whereas FOS represses E-cadherin. Furthermore, these effects were detected prior to the onset of Odd-expressing prohemocyte loss. Our findings strongly suggest that FOS and E(Pc) control prohemocyte differentiation, at least in part, by regulating E-cadherin protein levels. As a result, our findings provide additional support for ROS-induced control of E-cadherin levels through networking with two established targets of ROS. Notably, all three factors, FOS, E(Pc), and E-cadherin, regulate the differentiation of lamellocytes. FOS is required for lamellocyte differentiation The results presented here extend the previous model of ROS-induced prohemocyte differentiation. In this new model, increased levels of ROS upregulate both Srp and JNK signaling to reduce E-cadherin and, thereby, promote differentiation . While oWe showed that Cat is required to maintain E-cadherin in early-third instar prohemocytes. In contrast, Jafrac is not required to maintain E-cadherin, either directly or indirectly, until at least after the mid-third instar. Notably, ROS levels increase in prohemocytes from the early- to late-third instar Drosophila E-cadherin protein expression in the lymph gland. Studies using mammalian systems have shown that FOS downregulates E-cadherin gene expression. In breast cancer cell lines, FOS has been shown to upregulate the E-cadherin transcriptional repressor, ZEB We showed that FOS, a downstream target of JNK signaling, represses The polycomb protein E(Pc), another downstream target of JNK signaling, most likely maintains E-cadherin by silencing genes involved in E-cadherin repression. Importantly, both polycomb activity and E-cadherin are required to establish and maintain mammalian pluripotent stem cells Drosophila, Srp performs the functions of all three GATA factors in that it is required to maintain the prohemocyte pool Caenorhabditis elegans and in tissue culture models of cardiomyocyte differentiation There are three mammalian hematopoietic GATA factors, GATA-1, -2 and -3 Drosophila, Srp binds to Ush to form a GATA\u2236FOG complex that regulates hematopoiesis ush gene expression during hematopoiesis ush gene expression driven by an increase in Srp activity. If this is the case, then Srp upregulation of ush could produce a negative feedback loop that promotes GATA\u2236FOG complex formation and thereby prevents excessive prohemocyte differentiation. This is supported by our previous work that showed when Srp binds Ush it cannot block E-cadherin expression or promote prohemocyte differentiation GATA factors interact with FOG proteins to regulate gene expression across tissues and taxa Drosophila prohemocytes and mammalian pluripotent stem cells In summary, we present evidence that reduction of E-cadherin is necessary to promote differentiation in response to oxidative stress. Furthermore, our studies suggest that both JNK signal transducers and GATA transcriptional activity mediate ROS-induced downregulation of E-cadherin. Given the conservation of E-cadherin function between Figure S1Knockdown of ND75 in prohemocytes reduces E-cadherin expression. E-cadherin expression is greater in (A) control than in (B) ND75 knockdown (ND75RNAi) lymph glands. dome-Gal4 females were crossed to UAS-RNAiND75 or wild-type (+) males. Yellow dotted lines delineate the entire lymph gland; white dotted lines delineate the prohemocyte pool. (C) Histogram showing the relative level of E-cadherin expression was significantly greater in control (+) lymph glands than in those with knockdown of ND75. (D) Histogram showing the percentage of E-cadherin-expressing prohemocytes was significantly reduced in ND75RNAi lymph glands compared to controls (+). Student's t-test; error bars show standard deviation; P values are as shown; n\u200a=\u200a10.(TIF)Click here for additional data file.Figure S2Knockdown of SOD2 reduces the level of E-cadherin. Histogram showing the relative level of E-cadherin expression was significantly greater in control (+) lymph glands than in those with knockdown of SOD2 during the early-third instar. Student's t-test; error bars show standard deviation; P values are as shown; n\u200a=\u200a14.(TIF)Click here for additional data file.Figure S3Loss of SOD2 reduces the number of Odd-expressing prohemocytes in late-third instar lymph glands. Odd-expressing prohemocytes in (A) control and (B) Sod2/Sod2 hypomorphic (Sod2) lymph glands from late-third instar larvae. White dotted lines delineate the entire lymph gland; yellow dotted lines delineate the prohemocyte pool. (C) Histogram showing the percentage of Odd-expressing prohemocytes was significantly reduced in Sod2 lymph glands compared to controls (+). Student's t-test; error bars show standard deviation; P values are as shown; n\u200a=\u200a14.(TIF)Click here for additional data file.Figure S4Paraquat treatment increases ROS levels and reduces E-cadherin expression in the lymph gland. ROS levels were measured using the superoxide specific dye, dihydroethdium (DHE). ROS levels increased in the lymph glands of (B) paraquat-treated (10 mM) compared to (A) untreated (0 mM) controls. E-cadherin expression in the lymph gland was assessed in paraquat treated larvae. (D) Paraquat treatment (10 mM) reduces the level of E-cadherin expression compared to (C) untreated (0 mM) controls. White dotted lines delineate the entire lymph gland; yellow dotted lines delineate the prohemocyte pool.(TIF)Click here for additional data file.Figure S5Knockdown of SOD2 results in increased levels of Srp expression. Histogram showing the relative levels of Srp expression in control (+) lymph glands and those with SOD2 knocked down (RNAiSod2) during the early-third instar. Student's t-test; error bars show standard deviation; P values are as shown; n\u200a=\u200a15.(TIF)Click here for additional data file."} +{"text": "The cAMP-dependent protein kinase (PKA) and the cGMP-dependent protein kinase (PKG) are highly homologous enzymes differing in their specificity for cAMP and cGMP, respectively. Recent structure-function studies led to the identification of key residues responsible for cGMP-specificity in PKG . IntroducGMP-specific threonine and arginine residues found in PKG I were introduced into both the N-terminal and C-terminal cyclic nucleotide binding domains (CNB-A and CNB-B) of the PKA regulatory subunit I\u03b1 (RI\u03b1) using site-directed mutagenesis. The four resulting constructs were: wild type, CNB-A mutant (T192R/A212T), CNB-B mutant (G316R/A336T) and CNB-A/B mutant which has all aforementioned mutations.While wild type RI\u03b1 showed a strong selectivity for cAMP vs. cGMP in both in vitro binding and kinase activation studies, either CNB-A or -B mutants bound both nucleotides with equal affinity, resulting in PKA holoenzymes that were activated by both nucleotides equally. The CNB-A/B mutant had a significantly higher affinity for cGMP, and the corresponding PKA holoenzyme was activated selectively by cGMP.Mouse embryonic fibroblasts derived from a PKA RI\u03b1 knockout mouse were contransfected with the RI\u03b1 constructs along with an AKAR to test their responsiveness for the cell-permeant analogs 8-CPT-cAMP and 8-CPT-cGMP, respectively . Our resCyclic nucleotide-dependent regulation of protein kinase activity is an important aspect of eukaryotic signal transduction. The vice versa specificity of PKA and PKG determines the fidelity of cAMP-PKA and cGMP-PKG pathways. We applied our understanding of cyclic nucleotide selectivity to cellular sensor design and showed that mutating four key residues within a cAMP-specific reporter switches it into a cGMP-specific reporter.Cyclic nucleotide selectivity has apparently evolved through mutations in the CNB. The identification of these key residues will improve the design of cyclic nucleotide-selective cellular reporters."} +{"text": "Multi-modality interrogation of myocardial health and disease using fused Positron Emission Tomography (PET) and magnetic resonance (MR) data has been limited by the need for separate scans, and complex and potentially imprecise co-registration of the individually-acquired PET and MR images. We sought to explore the potential of hybrid simultaneous PET/MR (hs-PET/MR) imaging to precisely localize changes in cardiac metabolism in areas of acute myocardial infarction (MI) and the area-at-risk (AAR) in acute ST-segment elevation myocardial infarction (STEMI) patients reperfused by primary percutaneous coronary intervention (PPCI).18F-fluorodeoxyglucose (FDG) PET metabolic imaging was acquired, following patient preparation . Matched short-axis slices of each modality: LGE, T2 maps, FDG, covering the entire left ventricle were analysed by 2 blinded investigators using 3 segmentation methods : manual; Otsu; 2 standard deviation (2SD) thresholding. 2 further investigators analysed respective coronary angiograms to derive BARI and APPROACH angiography jeopardy scores, both CMR-independent estimates of AAR size.Hs-PET/MR cardiac imaging was performed in 21 STEMI patients within 10 days of PPCI. A standard CMR protocol, included: localisers, functional cine imaging, with T2 mapping and late gadolinium enhancement to delineate the AAR, and area of infarction respectively. Simultaneously, The AAR delineated by T2-mapping correlated significantly with both the BARI and APPROACH scores, thereby independently validating this technique at 3T. In all patients myocardial FDG uptake was reduced in areas of MI (LGE). The total volume of myocardium with reduced FDG uptake was larger than the measured MI size , and was similar in size to the AAR measured by T2-mapping , the Rosetrees Trust, and was also supported by the National Institute for Health Research University College London Hospitals Biomedical Research Centre. SKW is supported by British Heart Foundation Clinical Research Training Fellowship (grant number FS/10/72/28568)."} +{"text": "Double inversion recovery (DIR) is the most commonly used black blood (BB) preparation method. However its single slice nature makes the acquisition inefficient and flow-dependence causes insufficient blood suppression when stagnant blood or in-plane blood flow is present. In this work, we propose to use ferumoxytol, an FDA approved iron oxide particle for treating iron deficiency anemia, as an MR contrast agent to achieve flow-independent BB imaging by taking advantage of its strong R2 relaxivity. As our technique eliminates the need for DIR preparation, we propose to achieve higher scan efficiency by interleaving the imaging slices.On seven healthy volunteers, we acquired 12 consecutive short axis (SA) slices covering the ventricles and 2 horizontal long axis (HLA) slices, both pre- and post- ferumoxytol injection. Conventional ECG gated single-slice breath-held DIR Turbo Spin Echo (TSE) sequence was used for pre-contrast acquisition, with TR=2 R-R interval, TE=37ms, echo spacing (ESP)=5.25 ms, echo train length (ETL)=15 and TI=600ms. Subsequently, ferumoxytol was injected (4 mg-Fe/kg) and the proposed BB imaging shown in Fig. In mid-ventricle SA slices (slice number 7-12), both pre- and post- contrast images offered satisfactory dark blood contrast. For apical slices (slice number 1-6), post-contrast images showed significant improvement in all 7 volunteers by eliminating stagnant blood signal. In HLA views (shown in Fig. Compared to conventional DIR BB imaging, the proposed ferumoxytol-enhanced slice-interleaved TSE technique provides improved blood signal suppression that does not depend on flow and is at least 3X faster than conventional DIR for the same anatomical coverage. It also provides higher SNR and CNR due to signal boost from ferumoxytol.N/A."} +{"text": "Currently, standard segmented cardiac cine and delayed enhancement images are acquired with independent sequences requiring multiple breath-holds (BH), ideal inversion time (TI) setting, and individual image analysis. We describe our initial experience with the use of a multi-contrast real-time cine (multi-TI cine) prototype sequence .2; slice thickness: 7 mm; temporal resol.: 40 ms; 7 heart beats (HB)/slice), modified Look-Locker inversion recovery post-contrast T1 mapping , and segmented spoiled gradient-echo late gadolinium enhancement (LGE) images . Followed by multi-TI real-time cine performed in the same cardiac planes . The multi-TI cine prototype has been described in detail elsewhere1, but briefly it consists of an inversion recovery highly accelerated SSFP 2D real-time cine sequence, featuring sparse sampling and k-t regularization. Using an offline reconstruction algorithm based on a registration and motion-propagation strategy, a full-length cine can be reconstructed for each acquired TI that included short- and long-axis steady-state free-precession (SSFP) segmented cine measurements were included. All sequences were successfully performed and reconstructed, rendering good-quality images on subjective analysis. In all patients, a multi-TI cine, with ideal myocardial nulling, could be produced for simultaneous cardiac function and LGE analysis. Figure Despite currently having inferior spatial and temporal resolution as compared to standard cine and LGE sequences, multi-TI cine seems to be able to detect myocardial tissue and functional abnormalities, with the advantage of shorter acquisition times, ideal myocardial nulling and combined functional and LGE assessment. Future developments and studies will be needed to adequately determine its accuracy and validate its clinical application.Internal."} +{"text": "We then harvested and characterized the intratumoral cellular infiltrate and demonstrated the presence of a PD-L1+ CD11b+ CSF1r+ inhibitory antigen-presenting cell (iAPC) population. Gene expression profiles of harvested iAPCs were assessed using the novel Nanostring nCounter analysis system. We found that treatment with DC vaccination and adjuvant PD-1 mAb blockade and PLX-3397 induced a highly significant therapeutic benefit to animals bearing well-established i.c. gliomas (and the inhibition of iAPC negative regulatory function.Glioma tumor lysate-pulsed dendritic cell (DC) vaccination is an effective treatment modality. However, cure rates in the established tumor setting are not therapeutically significant in our preclinical models. We inferred that immunosuppressive antigen presenting cells (iAPCs) present in the tumor environment acting via the PD-1/PD-L1 mechanism mediated immune suppression in malignant glioma. To test this hypothesis in our"} +{"text": "Drosophila JNK homolog) induced cell death is mediated by the canonical Wg signaling. First, loss of Wg signaling abrogates Bsk-mediated caspase-independent cell death. Second, activation of Wg signaling promotes cell death in a caspase-independent manner. Third, activation of Bsk signaling results in upregulated transcription of wingless (wg) gene. Finally, Wg pathway participates in the physiological function of Bsk signaling in development. These findings not only reveal a previously undiscovered role of Wg signaling in Bsk-mediated cell death, but also provide a novel mechanism for the interplay between the two important signaling pathways in development.Cell death is an essential regulatory mechanism for removing unneeded cells in animal development and tissue homeostasis. The c-Jun N-terminal kinase (JNK) pathway has pivotal roles in the regulation of cell death in response to various intrinsic and extrinsic stress signals. The canonical Wingless (Wg) signaling has been implicated in cell proliferation and cell fate decisions, whereas its role in cell death remains largely elusive. Here, we report that activated Bsk (the This pathway is initiated by various intrinsic and extrinsic signals including the tumor necrosis factor (TNF) family ligands, and is mediated through a mitogen-activated protein (MAP) kinase cascade.\u03b2-catenin pathway, the Wnt/Ca2+ pathway, and the Wnt/Frizzled (Fz)-Planar Cell Polarity (PCP) pathway.15 The canonical Wnt signaling represents one of the best investigated pathways and has crucial roles in regulating cell proliferation, cell migration, and cell fate decisions.17 Deregulated Wnt signaling is frequently associated with human diseases, especially cancers.20 Genetic studies of the Drosophila Wnt protein Wingless (Wg) have made great contributions to our understanding of Wnt signaling.23 In the absence of Wg signal, the \u03b2-catenin homolog Armadillo (Arm) is phosphorylated and degraded by a multi-protein 'destruction complex' that includes the Adenomatous Polyposis Coli protein (APC), Shaggy (Sgg)/Zeste-white-3, Axin (Axn), and Casein Kinase1 (CK1). In the presence of Wg signal, the receptor dFrizzled (dFz) and co-receptor Arrow (Arr) bind to Wg, recruit and phosphorylate the scaffold protein Dishevelled (Dsh), which antagonizes the 'destruction complex' and prevents Arm from degradation. As a consequence, accumulated Arm translocates to the nucleus and stimulates target genes expression by binding to the transcription factor Pangolin (Pan).25Wnt proteins are a family of highly conserved secreted molecules that regulate cell\u2013cell interactions through distinct intracellular signal transduction pathways, including the canonical Wnt/28 Yet little is known about the interaction between JNK and the canonical Wnt/Wg pathways in development. A collaboration between JNK and the canonical Wg signaling was reported to promote dorsal closure and ventral patterning during Drosophila embryogenesis, but the two pathways appear to function in parallel.29 An interaction between AP-1 and \u03b2-catenin/TCF complex was also reported to have a role in carcinogenesis, though the underlying mechanism remains elusive.30Crosstalk between different signaling pathways occurs extensively and forms a complex signaling network that enable cells to interpret multiple inputs and execute different responses in a context-dependent manner. The interplay between JNK and the non-canonical Wnt/Frizzled (Fz)-PCP pathways has been extensively studied, in which JNK signaling acts downstream of Dsh to promote PCP establishment.Drosophila. Loss of Wg signaling specifically suppresses Egr or Hep-induced Bsk-dependent cell death in eye, wing, and thorax. Stimulation of Wg signaling phenocopies Bsk activation and induces caspase-independent cell death. Furthermore, Wg signaling is both necessary and sufficient for the expression of Bsk signaling reporter puckered (puc). Epistasis experiments indicate that Wg signaling acts downstream of Bsk to regulate cell death and other physiological functions. Finally, activated Bsk signaling upregulates wg transcription, providing a molecular mechanism for the genetic interaction between Bsk and Wg pathways. These findings not only expand our existing knowledge about the molecular basis of complex signaling network in regulating cellular activities, but also provide additional strategy and approach for cancer therapy.In the present study, we have identified from a genetic screen that the canonical Wg signaling has a crucial role in Bsk-mediated cell death in GMR-Gal4 (GMR>Egr) triggers massive cell death and produces a Bsk-dependent small eye phenotype animals induces vast cell death and produces small and rough eyes with fused ommatidia encoding the Drosophila JNK kinase. Expression of HepCA in the eye (GMR>HepCA) induces Bsk-dependent cell death and generates a small eye phenotype triggers Bsk-dependent cell death in the thorax and produces a small scutellum phenotype compartment boundary driven by ptc-Gal4 results in elevated cell death in third instar wing discs as revealed by acridine orange (AO) staining and Bsk activation (puc-LacZ expression) than Hep in the wing disc (Df(3\u2009L)H99 , expression of the caspase inhibitor Diap1 . As a po (Cas3*) . Further (Cas3*) are not (3\u2009L)H99 that delor Diap1 , a domin1 .41 In wild-type wing discs of third instar larva, puc is only expressed in the dorsal tip presented only in the wing disc and salivary gland, but not in the eye disc, is required for Wg signaling to induce puc expression.To examine whether Wg signaling is sufficient to elicit ignaling . Consistignaling . Howeverye discs , suggestpuc expression in both the wing disc and the salivary gland.Together, the above data provide compelling evidence that the canonical Wg pathway is both necessary and sufficient for Bsk-induced DN boundary and in two concentric rings in the wing pouch that resulted in a dramatic reduction of the posterior compartment , lethal giant larvae (lgl) and disc large (dlg),43 results in Bsk-mediated cell death and elimination from the tissue.45 Knocking-down of lgl along the A/P compartment boundary (ptc>lgl-IR) in the wing pouch induces Bsk-dependent cell death by expressing P35, induce compensatory proliferation in neighbor cells by secreting growth factors like Wg, Dpp and Hh.50 Both secretion of growth factors and compensatory proliferation depends on Bsk signaling,52 yet the mechanism has not been fully illustrated. In mammal, a connection between c-Jun and TCF4, transcription factors of JNK and Wnt pathways respectively, is reported to have a role in intestinal tumorigenesis.30The interplay between JNK signaling and the non-canonical Wnt/Fz-PCP pathway has been extensively studied, in which JNK acts downstream of Dsh through Rho family small GTPase, and regulates the key effectors such as Myosin II and other cell adhesion components.Drosophila, and obtain the following results: (1) loss of Wg signaling suppresses Bsk-mediated caspase-independent cell death and puc expression; (2) activation of Wg signaling induces caspase-independent cell death and puc expression, which challenges the previous opinion that puc is a direct transcription target and readout of Bsk signaling. We cannot rule out the possibility that a context-dependent feed-back loop may exist between Wg and Bsk signaling; (3) Wg signaling acts downstream of Bsk in promoting cell death; (4) Wg pathway participates in the physiological function of Bsk signaling; (5) activated Bsk signaling results in upregulated wg transcription. Thus, we not only deliver compelling evidences for the conclusion that activated Bsk signaling promotes Wg pathway-dependent cell death, but also provide the underlying mechanism for the interplay of the two pathways that have crucial roles in development. However, since Wg signaling is necessary for cell proliferation and viability, complete loss of Wg signaling would result in developmental defect and animal lethality, we used heterozygous mutants or RNAi-mediated knock-down approach to examine the effect of loss-of-Wg signaling on Bsk-mediated cell death. In such backgrounds, Wg signaling is effectively reduced, but not completely abolished. Consequently, Bsk-mediated cell death and resulted phenotypes are significantly, but not fully, suppressed H99 and expression of Diap1, a dominant-negative form or knockdown of dronc, or knockdown of drice and dcp-1 . Constitutively active Wnt signaling has been associated with tumor progression in many cancers, yet in this study we characterize a role of Wg signaling in promoting cell death, which implies that Wnt signaling may also possess a tumor suppressor function. Meanwhile, a substantial body of evidence also suggests that JNK signaling is closely related with tumor formation and metastasis,64 thus the present study shed new light on the crosstalk and involvement of JNK and Wnt signaling in cell death and cancer development.Since the discovery of the first wg1\u201317;65Sp;34dsh6;66wgIG22, dshV26, armXM19;67arm1, pan13a;68 SggEP1576;69UAS-Wg, UAS-Dsh, UAS-wg-IR and UAS-arm-IR;70UAS-Pan;71bsk1; 72UAS-EgrRegg1;10UAS-HepCA, UAS-Egr, UAS-BskDN, UAS-Puc, Df(3\u2009L)H99, DIAP1, DRONCDN and GUS-Dp53259H;32UAS-HepWT;31pucE69, pnr-Gal4;12GMR-Gal4, ptc-Gal4 and sd-Gal4;73UAS-Dp53H159N.35 The wg-LacZ, UAS-Rpr, UAS-Dp53, UAS-Arm, UAS-Apc2, UAS-Axn, and UAS-dsh-IR lines were obtained from the Bloomington stock center. The UAS-pan-IR and UAS-lgl-IR lines were obtained from the Vienna Drosophila RNAi Center (VDRC). The UAS-wg-IR, UAS-arm-IR, UAS-dronc-IR, UAS-drice-IR, UAS-dcp-1-IR lines were got from Fly Stocks of National Institute of Genetics (NIG-FLY).The following stocks have been described previously: y w1118hs-Flp; act>CD2>Gal4 UAS-GFP/Cyo strain. Clones were induced by heat shock at 37\u2009\u00b0C for 10\u2009min, late third-instar larvae were dissected after recovering for 3 days.Fluorescently labelled clones were produced in larval imaginal discs using the \u03b2-Gal (DSHB). The second antibodies were used as follows: 1\u2009:\u20091000 anti-mouse CY3 (CST), 1\u2009:\u20091000 anti- rabbit CY3 (CST).The following primary antibodies were used: 1\u2009:\u2009400 rabbit-anti-Caspase 3 , 1\u2009:\u2009300 mouse-anti-Wingless , 1\u2009:\u2009500 mouse-anti-32The discs were dissected at the third instar larva stage, and stained for AO as described.The discs were dissected at the third instar larva stage, and stained for TUNEL using the Fluorescein Cell Death Kit produced by Boster Company. Imaging of prepared sample was conducted by a Leica confocal microscope .\u03b2-galactosidase as described.74The discs were dissected at the third instar larva stage, and stained for 75 primers pairs of rp4975 and puc76 were used as previously described.Wing discs were dissected at third instar larva, for each genotype, more than 200 discs were collected and total RNA was isolated using TRIzol . qRT-PCR was performed as previously described,P-value of <0.05 was considered as significant.For loss of ACV phenotype, statistics were analyzed using chi-square test. For AO stanining and area of disc clones, one-way analysis of variance test followed by the post Dunnett test or Kruskal\u2013Wallis text followed by the post Dunns text was used. A"} +{"text": "Adoptive transfer of tumor-infiltrating lymphocytes (TIL) can mediate complete regression of metastatic cervical cancer, but the immunological landscape of the anti-tumor T cell responses in these patients is not fully understood. Reactivity against the human papillomavirus (HPV)-derived antigens may contribute to the clinical responses, but whether other immunogenic tumor antigens are also targeted by these effective TIL is unknown. Tumor-specific neo-antigens arising from somatically mutated genes are thought to be important for the therapeutic efficacy of immunotherapies in a variety of solid tumors. Cervical cancers also harbor somatic mutations that may be the targets of tumor-specific T cells.Here we assessed whether mutated neo-antigens were the targets of clinically effective TIL in a patient with metastatic HPV16+ cervical cancer who experienced complete tumor regression, ongoing beyond 2 years, after TIL therapy.Whole-exome sequencing of a metastatic tumor was performed to identify tumor-specific non-synonymous somatic mutations. Subsequently, minigene constructs encoding 222 putative mutations were generated and transfected into autologous antigen presenting cells. Finally, reactivity of TIL against the transfected mutated minigene constructs was assessed by IFN-g ELISPOT, and up-regulation of T cell activation markers by flow cytometry.The patient's infused TIL were previously reported to contain T cell reactivity against the HPV E6 and E7 antigens. Here, we found that these TIL also contained CD8+ T cell reactivity against three mutated neo-antigens encoded by the SETDB1, METTL17 and ALDH1A1 genes. The mutation-reactive CD8+ T cells showed exclusive recognition of their mutant 25-mer peptide over the wild-type counterpart, suggesting bona fide mutation-driven T cell reactivities. Furthermore, mutated neo-antigen T cell reactivities comprised ~24% of the infused TIL as assessed by CD137 up-regulation assay, and were mono- to oligoclonal as suggested by T cell receptor V\u03b2 chain analysis of mutation-reactive bulk-isolated populations. Finally, no T cell reactivity against mutated antigens was detectable in peripheral blood pre-treatment, while mutation-reactive CD8+ T cells were present in peripheral blood at one month post-treatment. Analysis of mutation-derived neo-antigen T cell reactivities in additional HPV+ cancer patients treated with TIL therapy is currently ongoing.Our data reveal that mutated neo-antigens, in addition to previously reported HPV antigens, were the targets of clinically effective TIL in a patient with metastatic HPV+ cervical cancer who experienced complete tumor regression. This finding has important implications for future study and understanding of anti-tumor T cell responses in HPV+ cancers."} +{"text": "We showed in cross-sectional studies that tryptophan (Trp) catabolism into kynurenine (Kyn) by IDO enzyme expressed by dendritic cells (DC) contributes to regulatory T-cells (Tregs) expansion and immune suppression in chronic HIV infection. We prospectively assessed Trp catabolism and anti-inflammatory response following primary HIV infection (PHI).Plasma and Peripheral blood mononuclear cells (PBMCs) were longitudinally collected in 41 PHI patients (infection <90 days), 24 remained untreated (ART-naive) and 17 were ART-treated one year later. In addition, samples from elite controllers and healthy subjects were also assessed. IDO enzymatic activity marker (Kyn/Trp ratio) was measured by isotope dilution tandem mass spectrometry. IL-6, IL-18, TNF-\u03b1 and IP-10 plasma levels were assessed by Luminex. Frequency of Tregs (CD4+CD25highCD127lowFOXP3high), CD11c+ myeloid DC (mDC) and CD123+ plasmacytoid DC (pDC) as well as HLA-DR/CD38 co-expression of on T-cells were assessed.PHI patients had elevated Kyn/Trp ratio compared to HS and EC and further increased during the chronic phase, while normalized following ART. Accordingly, an increase of Treg frequency was observed at the baseline and continues to increase in the chronic phase only for those remaining untreated, when compared to HS and EC. Conversely, the frequency of mDC and pDC decreases over time only for those who remained untreated. Higher Kyn/Trp ratios were inversely correlated with the frequency of mDC and pDC at PHI and for those untreated. Importantly, the highest level of immune activation (HLA-DR+CD38+ CD8 T-cells) was observed during PHI followed by a decrease in chronic phase in ART-na\u00efve and became comparable to EC and HS when receiving ART. Importantly, Kyn/Trp ratio was correlated with level of CD8 T-cell activation during PHI and for those who remained untreated. In line with this, positive correlations were observed between Kyn/Trp ratio and levels of IL-18 and TNF-\u03b1 as well as markers of HIV disease progression IL-6 and IP-10.The progressive increase of Kyn/Trp ratio observed in the chronic phase of HIV infection in contrast to decreased viral load and T-cell activation, support the contribution of tissue damage and/or myeloid inflammatory syndrome in addition to viral replication for the development of immunosuppression."} +{"text": "Fluorescence-guided surgery (FGS) of cancer is an area of intense development. In the present report, we demonstrate that the telomerase-dependent green fluorescent protein (GFP)-containing adenovirus OBP-401 could label colon-cancer liver metastasis in situ in an orthotopic mouse model enabling successful FGS. OBP-401-GFP-labeled liver metastasis resulted in complete resection with FGS, in contrast, conventional bright-light surgery (BLS) did not result in complete resection of the metastasis. OBP-401-FGS reduced the recurrence rate and prolonged over-all survival compared with BLS. In conclusion, adenovirus OBP-401 is a powerful tool to label liver metastasis in situ with GFP which enables its complete resection, not possible with conventional BLS. Green fus OBP-40 that exper cells . Since rer cells , in contus OBP-40 that expWe have previously demonstrated OBP-401-based fluorescence-guided surgery is highly effective in various types of cancers including soft tissue sarcoma , pancreaRecently, GFP-expressing liver metastasis in an orthotopic mouse models was completely resected. In contrast, conventional bright-light surgery (BLS) could not fully resect the metastasis . Howeverin situ with GFP, enabling complete resection of liver metastasis by FGS and prolonged survival compared to BLS.In the present report, we demonstrate OBP-401 brightly labels colon cancer liver metastasis in an orthotopic mouse model 2 inhalation when they met the following humane endpoint criteria: prostration, skin lesions, significant body weight loss, difficulty breathing, epistaxis, rotational motion and body temperature drop. The use of animals was necessary to develop fluorescence-guided surgery of liver metastasis. Animals were housed with no more than 5 per cage. Animals were housed in a barrier facility on a high efficiency particulate arrestance (HEPA)-filtered rack under standard conditions of 12-hour light/dark cycles. The animals were fed an autoclaved laboratory rodent diet -protocol specifically approved for this study and in accordance with the principals and procedures outlined in the National Institutes of Health Guide for the Care and Use of Animals under Assurance Number A3873-1. In order to minimize any suffering of the animals, anesthesia and analgesics were used for all surgical experiments. Animals were anesthetized by intramuscular injection of a 0.02 ml solution of 20 mg/kg ketamine, 15.2 mg/kg xylazine, and 0.48 mg/kg acepromazine maleate. The response of animals during surgery was monitored to ensure adequate depth of anesthesia. Ibuprofen was used in order to provide analgesia post-operatively in the surgically-treated animals. The animals were observed on a daily basis and humanely sacrificed by COent diet .hTERT) gene which drives the expression of E1A and E1B genes linked to an internal ribosome entry site for selective replication only in cancer cells, and the GFP gene which is driven by the CMV promoter [The GFP-expressing adenovirus OBP-401 contains the promoter element of the human telomerase reverse transcriptase (promoter .The human colon cancer cell lines HCT-116 and HT-29 expressing RFP (HCT-116-RFP and HT29nu/nu) nude mice were kept in a barrier facility under HEPA filtration. Mice were fed with an autoclaved laboratory rodent diet . All animal studies were conducted in accordance with the principles and procedures outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals under Assurance Number A3873\u201301.Athymic (6) were injected into the spleen of female arthymic nude mice (5 weeks old). HCT-116-RFP cells produced experimental liver metastasis one month after injection. The experimental liver metastasis were harvested and re-injected into the spleen. High-metastatic colon cancer cells, termed HCT-116L3-RFP, were selected after three such cycles. An orthotopic solitary-metastatic model was developed with HCT-116-L3-RFP cells implanted under the serosa in the liver of nude mice. The orthotopic metastases readily grew in the liver. Tumor growth was followed by RFP fluorescence using noninvasive fluorescence imaging.For development of high liver-metastatic colon cancer cells, HCT-116-RFP cells . For who8 PFU), was imaged using the OV100 before surgery. FGS was performed under GFP guidance using either a hand-held DinoLite fluorescence imaging system [All animal procedures were done under anesthesia using s.c. administration of the ketamine mixture, described above. The experimental liver metastasis, labeled with GFP by OBP-401 (1 \u00d7 10 Taiwan) ,15 or th Taiwan) which enP values of < 0.05 were considered significant.Data are shown as means \u00b1 SD. For comparison between two groups, significant differences were determined using Student\u2019s t-test. Pearson chi-square analysis was used to compare the rate of recurrence between BLS and OBP-401-FGS. Statistical analysis for over-all survival was performed using the Kaplan-Meier test along with log-rank test. Time-course imaging showed that OBP-401 labeled RFP-expressing HCT-116 (HCT-116-RFP) and HT29 (HT29-RFP) colon cancer cells with GFP in a dos6) were injected into the spleen of female athymic nude mice (5 weeks old). HCT-116-RFP cells produced experimental liver metastasis within one month after injection. The experimental liver metastasis were harvested and re-injected into the spleen. High-metastatic colon cancer cells, termed HCT-116L3-RFP, were selected after three such cycles on orthotopic solitary liver metastases . The tum8 PFU) (Solitary metastases were resected 3 days after i.t. injection of OBP-401 (1 \u00d7 108 PFU) . OBP-4018 PFU) . Tumor i8 PFU) . OBP-4018 PFU) .GFP fluorescence of liver metastasis after OBP-401-GFP labeling was sufficiently bright to use the Dino-Lite hand-held fluorescence imager for FGS 3 days after OBP-401 infection . OBP-401OBP-401 was injected into the liver metastasis 3 days before surgical resection. After BLS, both RFP and GFP fluorescence were detected in the surgical bed . OBP-401OBP-401 was injected into the liver metastasis 3 days before surgical resection. OBP-401 GFP labeling enabled detection of a satellite as well as the main metastatic tumor . OBP-FGSFifteen of sixteen mice that underwent BLS for liver metastasis had a large local recurrence Table 1Table 1. Currently, single liver metastases are resected at some medical centers, but with frequent metastatic recurrence . A greatThe results of the present study demonstrate the power of OBP-401 to specifically label a liver metastasis in situ, enabling complete and precise resection by FGS, recurrence of 19% compared to 94% with BLS, and increased survival compared to BLS. The results of the present study suggest clinical promise for OBP-401 to improve outcome of liver metastasis, the often lethal aspect of colon and other cancers.The parent virus of OBP-401, OBP-301, has proven safe in clinical trials . AlthougS1 Checklist(PDF)Click here for additional data file.S1 MovieProcedure for OBP-401-FGS with the Dino-Lite hand-held fluorescence-scope.(MOV)Click here for additional data file."} +{"text": "Unfortunately, the original version of this article containeWe had compared Aro to two published methods for identifying smFISH transcripts \u2013 threshold-picking and FISHFigure 5b (below). Comparison of spot identification and classification methods. B. A plot of manually counted spot number (x-axis) and estimated spot number (y-axis) by Aro, threshold-picking, and FISH-Quant across 28 C. elegans embryos. Both FISH-Quant and threshold-picking tend to underestimate the true number of spots (particularly at higher spot counts) while our Aro machine learning method performs well across a range of spots numbers. Spearman correlations (r) between the true and estimated spot number are listed for each method. All three techniques perform significantly better than random on this dataset. Aro and FISH-quant results are highly correlated with the manual count, and FISH-quant undercounting could be easily corrected by an appropriate factor. Interval estimates are depicted for Aro. Neither FISH-Quant nor threshold-picking provides a way to estimate error."} +{"text": "Globodera rostochiensis. By contrast, heterologous expression of Gr-VAP1 and two other venom allergen-like proteins from the beet cyst nematode Heterodera schachtii in plants resulted in the loss of basal immunity to multiple unrelated pathogens. The modulation of basal immunity by ectopic venom allergen-like proteins in Arabidopsis thaliana involved extracellular protease-based host defenses and non-photochemical quenching in chloroplasts. Non-photochemical quenching regulates the initiation of the defense-related programmed cell death, the onset of which was commonly suppressed by venom allergen-like proteins from G. rostochiensis, H. schachtii, and the root-knot nematode Meloidogyne incognita. Surprisingly, these venom allergen-like proteins only affected the programmed cell death mediated by surface-localized immune receptors. Furthermore, the delivery of venom allergen-like proteins into host tissue coincides with the enzymatic breakdown of plant cell walls by migratory nematodes. We, therefore, conclude that parasitic nematodes most likely utilize venom allergen-like proteins to suppress the activation of defenses by immunogenic breakdown products in damaged host tissue.Despite causing considerable damage to host tissue during the onset of parasitism, nematodes establish remarkably persistent infections in both animals and plants. It is thought that an elaborate repertoire of effector proteins in nematode secretions suppresses damage-triggered immune responses of the host. However, the nature and mode of action of most immunomodulatory compounds in nematode secretions are not well understood. Here, we show that venom allergen-like proteins of plant-parasitic nematodes selectively suppress host immunity mediated by surface-localized immune receptors. Venom allergen-like proteins are uniquely conserved in secretions of all animal- and plant-parasitic nematodes studied to date, but their role during the onset of parasitism has thus far remained elusive. Knocking-down the expression of the venom allergen-like protein Gr-VAP1 severely hampered the infectivity of the potato cyst nematode Plant-parasitic nematodes have a major impact on global food security, as they reduce the annual yield of food crops by approximately 10 percent. For decades, the application of non-selective toxic chemicals to infested soils controlled outbreaks of plant-parasitic nematodes. The recent bans on most of these chemicals has redirected attention towards a wider use of basal, broad-spectrum immunity to nematodes in crop cultivars. However, it is currently not known if this most ancient layer of immunity affects host invasion by plant-parasitic nematodes at all. Basal immunity in plants relies on the detection of molecular patterns uniquely associated with infections in the apoplast by surface-localized receptors. Here, we demonstrate that venom allergen-like proteins in secretions of soil-borne cyst nematodes suppress immune responses mediated by surface-localized pattern recognition receptors. Migratory stages of cyst nematodes most likely deliver venom allergen-like proteins together with a range of plant cell wall-degrading enzymes into the apoplast of host cells. We therefore conclude that these nematodes most likely secrete venom allergen-like proteins to modulate host responses triggered by the release of immunogenic fragments of damaged plant cell walls. Soil-borne plant-parasitic nematodes are major constraints on global food security, as they reduce the annual yield of food crops by approximately 10 percent Plants utilize pattern recognition receptors belonging to the receptor-like kinase (RLK)/Pelle superfamily to detect extracellular microbes or their actions in the apoplast , or its interaction with Rcr3pim, in nematode virulence is not clear.Recently, we showed that the receptor-like protein Cf-2 in tomato mediates dual disease resistance by guarding a common virulence target of a nematode and a fungus Venom allergen-like proteins constitute a monophyletic clade of cysteine-rich secretory proteins within the Sperm Coating Protein/Tpx-1/Ag-5/Pr-1/Sc-7 (SCP/TAPS) superfamily . MembersGlobodera and Heterodera) and root-knot nematodes (genus Meloidogyne), deliver effectors into the apoplast and cytoplasm of host cells to induce the formation of a permanent feeding structure M. incognita suggest that sedentary plant-parasitic nematodes may have adapted to this by evolving a separate set of apoplastic effectors to further control host immunity Sedentary plant-parasitic nematodes, such as cyst nematodes were challenged with the dsRNA-treated infective juveniles, and monitored for nematode infections for 7 days post inoculation. Treatment with Gr-VAP1-specific dsRNA significantly reduced the number of nematodes inside tomato roots compared to the treatment with NAU-specific dsRNA (lyc from tomato (S. lycopersicum). For this experiment, Gr-VAP1 and C14tub/lyc were separately produced in the apoplast of agroinfiltrated leaves of Nicotiana benthamiana. Protease activity was subsequently determined by the binding of fluorescent DCG-04 to C14tub and C14lyc in the presence of Gr-VAP1 on gels of mixtures of isolated apoplastic fluids. The experiment was repeated three times, and each attempt resulted in significantly less binding of the fluorescent probe to C14tub, but not to C14lyc . By conto C14lyc . Only C1Arabidopsis thaliana is a far better model to study the molecular changes induced by venom allergen-like proteins in plants than either potato or tomato. However, G. rostochiensis is not able to establish infections in A. thaliana. We therefore continued our investigations with two homologous venom allergen-like proteins from the beet cyst nematode Heterodera schachtii, which is a parasite of A. thaliana. These two venom allergen-like proteins are formally designated as Nem-Hsc-SCP/TAPS-1A and Nem-Hsc-SCP/TAPS-2A Meloidogyne incognita only caused faster developing and larger lesions on transgenic Arabidopsis expressing Hs-VAP2 .Next, we reasoned that if the VAP-enhanced susceptibility of the transgenic Arabidopsis lines to cyst nematodes involves modulation of the innate immunity, these lines might also be more susceptible to entirely unrelated plant pathogens. To test this, we analyzed the transgenic Arabidopsis lines overexpressing . tomato . The oves plants . Similar. tomato . Only Arcumerina . By cont Hs-VAP2 . Surpris Hs-VAP2 . HoweverP. syringae pv. tomato harbor an immunogenic epitope of twenty two amino acids (flg22) that is recognized as a pathogen-associated molecular pattern in Arabidopsis Hs-VAP1 and Hs-VAP2 in Arabidopsis seedlings largely abrogated this growth inhibition by flg22 . More thmembrane . By contmembrane .Hs-VAP1 and Hs-VAP2 in A. thaliana, we subjected all differentially expressed genes to a KEGG pathway gene set enrichment analysis -12). The vast majority of these Arabidopsis genes currently assigned to this pathway have been associated with innate immunity to plant pathogens To resolve specific pathways particularly affected by the overexpression of Hs-VAP1 and Hs-VAP2, we first focused on the most down-regulated genes (To further investigate the expression of specific genes associated with the loss of immunity in the transgenic Arabidopsis plants overexpressing ed genes . The annHs-VAP1-overexpressing Arabidopsis plants (Log2 fold change \u200a=\u200a\u221232.0). Albeit less, AT4G21630 was also strongly down-regulated in the transgenic Arabidopsis lines overexpressing Hs-VAP2. AT4G21630 encodes a putative plant cell wall-associated subtilase-like serine protease . NPQ4 encodes the chlorophyll-associated Photosystem II subunit S protein (PsbS), which is involved in non-photochemical quenching of excess excitation energy Next, we focused on four of the most up-regulated transcripts in the transgenic Arabidopsis plant overexpressing npq4-1 lacking PsbS displays an enhanced response to flg22 npq4-1 knockout mutant is also less attractive to herbivorous insects than the corresponding wild type Arabidopsis plants npq4-1 knockout mutant to demonstrate that the lack of PsbS enhances immunity of Arabidopsis to H. schachtii suppresses immunity to H. schachtii . As our mutant analyses showed, each of these components separately has a major impact on immunity to cyst nematodes in Arabidopsis. An important question that needs further research is if all three components are part of a single signaling pathway that spans different subcellular compartments. This might not be the case, because promiscuous effectors like Gr-VAP and Avr2 can interact with multiple apoplastic papain-like cysteine proteases, each of which may feed into different signaling pathways.Gr-VAP1 expression in preparasitic second stage juveniles (ppJ2s) of G. rostochiensis was knocked-down by soaking nematodes in double-stranded (ds) RNA matching 820 base pairs of the Gr-VAP1 coding sequence as described by Chen et al Nautilus gene from Drosophila melanogaster (Genbank accession number M68897) was used as control treatment. RNA interference was induced in nematodes by soaking approximately 15,000 freshly hatched ppJ2s of G. rostochiensis Ro1 Mierenbos in a 1 mg/ml dsRNA solution, including 50 mM octopamine, 3 mM spermidine, and 0.05% gelatin. The treatments were done in duplo so that 15,000 juveniles could be processed further for infectivity assay on tomato seedlings and 15,000 juveniles could be used for semi-quantitative reverse transcription (RT)-PCR.G. rostochiensis, we inoculated plates with five two-week old tomato seedlings (cultivar Moneymaker) on Gamborg B5 medium with 400 dsRNA-treated ppJ2s To test the effect of RNA interference on infectivity of Gr-VAP1 expression after dsRNA treatment, total RNA was extracted from dsRNA-treated ppJ2s using the RNeasy Mini kit . RT-PCR was done following the protocol of the SuperScript\u2122 III One-Step RT-PCR System (Invitrogen) using the primer Gr-VAP1-sRTFw and Gr-VAP1-sRTRv , or 300 nM of Gr-VAP1 isolated from apoplastic fluids of agroinfiltrated N. benthamiana leaves (see below) in 50 mM sodium acetate (pH 5.5) and 100 \u00b5M DTT. To label the available active sites in these cysteine proteases, the proteins were subsequently incubated for 5 h with 1 \u00b5M of fluorescent DCG-04-TMR Fluorescent activity based protease profiling was used to test whether Gr-VAP1 perturbs the active site of cysteine proteases. The cysteine proteases Gr-VAP1 at different time points before and after inoculation, we used semi-quantitative RT-PCR as described above. Messenger RNA extraction and cDNA synthesis was conducted on parasitic second, third, and fourth stage juveniles and the adult males and females isolated from roots of susceptible potato (cultivar Bintje) at 13, 19, 23, 27, and 34 days post inoculation respectively. Gr-VAP1 expression in these developmental stages was examined by targeting a gene specific fragment of 146 base pairs of Gr-VAP1 with primers Gr-VAP1-RTFw and Gr-VAP1-RTRv was PCR amplified with the primers cAMP-RTFw and cAMP-RTRv . The expression of Gr-VAP1 was checked by quantitative PCR (qPCR) using the primers qGrVAP1-Fw and qGrVAP1-Rv overexpressing Gr-VAP1 in the apoplast were generated as described by Postma et al rVAP1-Rv on RNA erVAP1-Rv as a refG. rostochiensis pathotype Ro1-Mierenbos were soaked in potato root diffusate on a 100-\u00b5m sieve to collect ppJ2s in vitro-grown plants potato plant. Adult females per plant were counted 6 to 8 weeks after inoculation. Two independently transformed potato lines were used in these experiments. The infection assays were repeated at least 3 times.Dried cysts of H. schachtii, we first queried the expressed sequence tag database at Genbank using the sequence of Gr-VAP1 as query. Four cDNA library clones, from which matching expressed sequence tags derived, were acquired from \u201cThe Washington University Nematode EST Project\u201dTo identify and clone venom allergen-like proteins from the beet cyst nematode A. thaliana Columbia 0 with A. tumefaciens strain GV3101 carrying constructs of Gr-VAP1, Hs-VAP1, and Hs-VAP2 in pMDC32, and pMDC32 without insert, using the floral dip method Hs-VAP1 and Hs-VAP2 was checked by PCR on genomic DNA extracted from seedlings using the DNeasy Plant Mini Kit (Qiagen). The expression of the transgenes was checked by qPCR using the primers qHsVAP1-F, qHsVAP1-R, qHsVAP2-F, and qHsVAP2-R , PAP4 (At2g27420), PAP5 (At3g49340), the serine protease SBT3.13 (At4g21630), and the chlorophyll-associated Photosystem II subunit S (At1g44575) were obtained from the SALK homozygote T-DNA collection. The mutant plants were propagated under standard greenhouse conditions of a 16-h/8-h light/dark regime and 60% relative humidity.Seeds of the homozygous transgenic T-DNA insertion mutants of the cysteine proteases Arabidopsis and wild-type A. thaliana Col-0 were vapor sterilized and planted in 12-well cell culture plates (Greiner bio-one) containing modified Knop's medium H. schachtiiH. schachtii was counted by visual inspection. The statistical significance of the pairwise differences between plant genotypes and the empty pMDC32 vector control and the wild type Arabidopsis was assessed with a one-way ANOVA.Seeds from transgenic Arabidopsis plants to infections by B. cinerea, P. cucumerina, A. brassicicola, and P. brassicae was determined on 4-week-old soil-grown plants B. cinerea, P. cucumerina, A. brassicicola, plants were drop inoculated by placing two 4-\u00b5l drops of conidial suspension (5\u00d7105 conidia/ml) on each leaf. Plants were incubated at 20\u00b0C, 100% relative humidity, and a 16-h/8-h light/dark regime. Arabidopsis was inoculated with P. brassicae by placing 5-mm-diameter mycelial plugs of a 2-week-old P. brassicae agar plate culture onto leaves. Subsequently, the plants were incubated at 16\u00b0C, 100% relative humidity, and a 16-h/8-h light/dark regime. After two days the mycelial plugs were removed from the leaves. Disease progression for these pathogens was scored at regular intervals, and representative pictures were taken at 4 days after inoculation. The statistical significance of the pairwise differences between plant genotypes and the empty pMDC32 vector control was assessed with a one-way ANOVA.The susceptibility of the Arabidopsis with V. dahliae, 2-week-old soil-grown plants were uprooted and inoculated by dipping the roots for 2 min in a conidial suspension (106 conidia/ml). After replanting in soil, plants were incubated at standard greenhouse conditions of a 16-h/8-h light/dark regime and 60% relative humidity. Disease progression was monitored until 25 days after inoculation. The statistical significance of the pairwise differences between plant genotypes and the empty pMDC32 vector control was assessed with a one-way ANOVA. All infection assays were performed at least 2 times.For inoculation of Arabidopsis plants to infections by P. syringae pv. tomato DC3000 was determined on 2-week-old Arabidopsis seedlings as previously described Pst at 109 CFU/ml, in 10 mM MgSO4 supplemented with 0.01% (v/v) Silwet L77, was inoculated on the two most expanded and in the center of the leaf rosette. Inoculated plants were subsequently incubated at 21\u00b0C, 100% relative humidity, and a 16-h/8-h light/dark regime. Disease severity was scored 3 days after challenge inoculation. Colonization levels of the bacteria were determined with the method described by Pieterse et al. The susceptibility of the Arabidopsis growth inhibition assays were performed as described elsewhere Arabidopsis plants, grown under the same conditions as for the infection with H. schachtii, were collected, flash-frozen in liquid nitrogen and total RNA was extracted with the Maxwell\u00ae 16 LEV simplyRNA purification kit (Promega). cDNA synthesis, library preparation (200-bp inserts), and Illumina sequencing (90-bp paired-end reads) was performed at BGI (Hong-Kong). Reads were mapped to the Arabidopsis genome (tair10) using TopHat and transformed into a count per gene per sample by using the BEDTools suite (function coverageBed). The edgeR http://www.geneontology.org/), and gene numbers were calculated for every term using an ultra-geometric test to find significantly enriched GO terms in DEGs. Calculated p-value went through a Bonferroni Correction, taking corrected p-value \u22640.05 as a threshold. KEGG pathway enrichment analysis was used to identify significantly enriched metabolic pathways or signal transduction pathways in DEGs comparing with the whole genome background. Subcellular localization was determined for all DEGs using the SUBcellular localization database for Arabidopsis proteins Two-weeks-old transgenic N. benthamiana was assessed by using Gr-VAP1, Hs-VAP1, Hs-VAP2, and Mi-VAP1 subcloned into pGWB411 Agrobacterium tumefaciens GV3101, and used for agroinfiltration in leaves of the N. benthamiana. Empty pGWB411 and plasmids carrying GFP in pGWB411 N. benthamiana of 0.1 N. benthamiana using a 1 ml syringe. Co-infiltration of different constructs was performed by mixing equal volumes of the bacterial suspensions to a final optical density of 0.3. Agroinfiltrated leaves were monitored for up to 7 d for cell death symptoms.The suppression of programmed cell death in leaves of thamiana . A. tumeN. benthamiana, we isolated apoplastic fluids by vacuum-infiltrating ice-cold extraction buffer for 10 min. Infiltrated leaves were surface dried and placed in a 10-ml syringe hanging in a 50 ml tube and centrifuged at 2000 g for 10 min at 4\u00b0C. Apoplastic fluids were subsequently separated under reducing conditions by SDS-PAGE on a 12% Bis-Tris gel and transferred to an Invitrolon\u2122 PVDF membrane (Life Technologies). For visualization of VAPs on western blots, we used a mouse monoclonal ANTI-FLAG\u00ae M2-Peroxidase (HRP) antibody to detect the FLAG tag at the carboxyl terminus of the recombinant proteins. Pictures were taken using the G:BOX Chemi System device (SynGene).To assess whether the VAPs harboring their native signal peptide for secretion (in pGWB411) were indeed secreted to the apoplast of agroinfiltrated leaves of S1 Figuretub of potato (Apoplastic Gr-VAP1 perturbs the active site of the extracellular defense-related papain-like cysteine protease C14Solanum tuberosum). Labeling densities of the fluorescent activity-based probe DCG-04 to the active site of (A) C14tub and (B) C14lyc of tomato (S. lycopersicum) following treatment with Gr-VAP1 isolated from apoplastic fluids of agroinfiltrated leaves. Treatments with the Avr2, egg white cystatin, and apoplastic fluids from agroinfiltrations with the empty binary expression vector (EV), and with buffer alone (Buffer) were included as controls. Labeling densities were quantified in triplicates and statistical significance of differences was determined with an ANOVA. Different letters indicate significant differences when using of P-value <0.05 as threshold.(TIF)Click here for additional data file.S2 FigureProtein sequence variation in venom allergen-like proteins from cyst nematodes and root-knot nematodes. (A) Protein sequence alignment of venom allergen-like proteins from the potato cyst nematode Globodera rostochiensis (Gr-VAP1), the beet cyst nematode Heterodera schachtii (Hs-VAP1 and Hs-VAP2), and the root-knot nematode Meloidogyne incognita (Mi-VAP1). Colors indicate identity (black background) or similarity among the sequences (gray background). (B) Protein similarity matrix of venom allergen-like proteins. Numbers represent the percentage of amino acid residues that are similar (bottom left corner) and identical for any pair of proteins.(TIF)Click here for additional data file.S3 FigureEctopic venom allergen-like proteins enhance susceptibility to Pseudomonas syringae pv. tomato (Pst) in Arabidopsis. Heterologous expression of the venom allergen-like proteins Hs-VAP1 and Hs-VAP2 from Heterodera schachtii in the apoplast of transgenic Arabidopsis lines enhances their susceptibility to Pst DC3000. Two independent transgenic lines per construct (-A and -B) were compared with corresponding transgenic line harboring the T-DNA of the empty vector (EV) and wild type A. thaliana (Col-0). (A) Population densities were determined 4 days after inoculation with Pst. Bars represent colony forming units (cfu/mg of tissue) for three independent replicates of 8 plants each. (B) Disease incidence was evaluated 4 days after inoculation. Bars represent the mean percentage of leaves, which had developed chlorotic symptoms of 24 plants. Statistical significance of differences was determined with an ANOVA. Different letters indicate statistical significance when using P-value <0.05 as threshold. (C) Pictures show typical symptoms on Arabidopsis plants inoculated either with Pst, or mock inoculated.(TIF)Click here for additional data file.S4 FigureEctopic venom allergen-like proteins enhance development of disease symptoms of fungal and oomycete pathogens in Arabidopsis. Bars represent mean percentage infected leaf area of transgenic Arabidopsis line overexpressing Hs-VAP1 and Hs-VAP2 3 days after inoculation with (A) Botrytis cinerea, (B) Plectosphaerella cucumerina, and (C and D) two isolates of Phytophthora brassicae (CBS686.95 and HH). Statistical significance of differences with transgenic plants harboring the T-DNA of the corresponding empty expression vector (EV) and wild type Arabidopsis (Col-0) was determined with an ANOVA. Different letters indicate significant differences when using of P-value <0.05 as threshold.(TIF)Click here for additional data file.S5 FigureEctopic venom allergen-like proteins abrogate the inhibition of seedling growth by flg22 in Arabidopsis. Photograph of typical root length of transgenic Arabidopsis lines overexpressing Hs-VAP1 and Hs-VAP2 after 10 days of growth in the presence of 10 \u00b5M flg22. Transgenic plants harboring the T-DNA of the corresponding empty expression vector (EV) and wild type Arabidopsis (Col-0) were used to show the normal inhibition of root growth in the presence of flg22.(TIF)Click here for additional data file.S6 FigureEctopic venom allergen-like proteins regulate immunity related pathways in Arabidopsis. Global gene expression analysis as determined by RNA-seq in 2 weeks old Arabidopsis plants overexpressing Hs-VAP1 and Hs-VAP2 in the apoplast. Venn's diagrams depict the total number of significantly up- (A) and down-regulated (B) genes relative to transgenic Arabidopsis plants harboring T-DNA of the corresponding empty expression vector (EV) when using a false discovery rate of 0.05 as cutoff. (C) Pie charts depict percentage of products of genes significantly down-regulated by ectopic Hs-VAP1 and Hs-VAP2 in Arabidopsis, according to their predicted subcellular localization in the SUBA database.(TIF)Click here for additional data file.S7 FigureVAPs harboring their native signal peptide for secretion are secreted to the apoplast of agroinfiltrated leaves of Nicotiana benthamiana.Heterodera schachtii VAPs (Hs-VAP1 and \u2013VAP2), Globodera rostochiensis VAP1 (Gr-VAP1), and Meloidogyne incognita VAP1 (Mi-VAP1) were transiently expressed in N. benthamiana plants as recombinant carboxyl terminus FLAG tagged proteins together with empty vector (EV) controls. VAPs were detected in apoplastic fluids isolated from agroinfiltrated leaf segments at 5 days post infiltration on western blots using FLAG specific antibody.(TIF)Click here for additional data file.S1 TableMost down-regulated transcripts by ectopic venom allergen-like proteins in Arabidopsis. Differentially expressed genes in transgenic Arabidopsis thaliana overexpressing Hs-VAP1 relative to the corresponding transgenic empty vector control plants.(XLSX)Click here for additional data file.S2 TableMost down-regulated transcripts by ectopic venom allergen-like proteins in Arabidopsis. Differentially expressed genes in transgenic Arabidopsis thaliana overexpressing Hs-VAP2 relative to the corresponding transgenic empty vector control plants.(XLSX)Click here for additional data file.S3 TableMost up-regulated transcripts by ectopic venom allergen-like proteins in Arabidopsis. Differentially expressed genes in transgenic Arabidopsis thaliana overexpressing Hs-VAP1 relative to the corresponding transgenic empty vector control plants.(XLSX)Click here for additional data file.S4 TableMost up-regulated transcripts by ectopic venom allergen-like proteins in Arabidopsis. Differentially expressed genes in transgenic Arabidopsis thaliana overexpressing Hs-VAP2 relative to the corresponding transgenic empty vector control plants.(XLSX)Click here for additional data file.S5 TableArabidopsis thaliana overexpressing Hs-VAP1 and Hs-VAP2 relative to the corresponding transgenic empty vector plants.KEGG pathway enrichment analysis: Statistically enriched pathways among differentially expressed genes in (DOCX)Click here for additional data file.S6 TableNicotiana benthamiana.Plant immune receptors and cognate pathogen elicitors used to induce programmed cell death in leaves of (DOCX)Click here for additional data file.S7 TableOligonucleotide primers used in this study.(DOCX)Click here for additional data file."} +{"text": "MR-guided cardiac electrophysiological (EP) ablations has drawn increasing attention from both the MRI and EP communities, as high-contrast MR images provide images that couple anatomical information with lesion efficacy . CatheteAn optical Fiber Bragg Grating (FBG) sensor was made MR-conditional and installed at the tip of a non-magnetic 8 French EP catheter from St Jude Medical Inc. The FBG sensor was made from an optical fiber, and the sensing signals were transmitted to a measurement setup outside the MRI scanner Figure . The WavFigure Simultaneous force and temperature monitoring based on the proposed FGB-based sensor design provided useful monitoring in MRI-guided EP therapies.NIH U41-RR019703, R43 HL110427-01, AHA 10SDG261039."} +{"text": "The HTLV-1 Tax transactivator initiates transformation in adult T-cell leukemia/lymphoma (ATL), a highly aggressive chemotherapy-resistant malignancy. The arsenic/interferon combination, which triggers degradation of the Tax oncoprotein, selectively induces apoptosis of ATL cell lines and has significant clinical activity in Tax-driven murine ATL or patients. Yet, the role of Tax expression in maintaining the transformed phenotype and of Tax loss in ATL response is disputed and the molecular mechanisms driving degradation remain elusive. Here we demonstrate that ATL-derived or HTLV-1 transformed cells are addicted to continuous Tax expression, suggesting that Tax degradation underlies clinical responses to the arsenic/interferon combination. The latter enforces PML nuclear body (NB) formation and partner protein recruitment. In arsenic/interferon-treated ATL-derived cells, Tax is recruited onto NBs, undergoes PML-dependent hyper-sumoylation by SUMO2/3, but not SUMO1, ubiquitination by RNF4 and proteasome-dependent degradation. Thus, the arsenic/interferon combination clears ATL through degradation of its Tax driver and could have broader therapeutic value by promoting degradation of other pathogenic sumoylated proteins."} +{"text": "Granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) modulate progression of certain solid tumors. The G-CSF- or GM-CSF-secreting cancers, albeit not very common are, however, among the most rapidly advancing ones due to a cytokine-mediated immune suppression and angiogenesis. Similarly, de novo angiogenesis and vasculogenesis may complicate adjuvant use of recombinant G-CSF or GM-CSF thus possibly contributing to a cancer relapse. Rapid diagnostic tools to differentiate G-CSF- or GM-CSF-secreting cancers are not well developed therefore hindering efforts to individualize treatments for these patients. Given an increasing utilization of adjuvant G-/GM-CSF in cancer therapy, we aimed to summarize recent studies exploring their roles in pathophysiology of solid tumors and to provide insights into some complexities of their therapeutic applications. Granulocyte and granulocyte-macrophage colony-stimulating factors regulate maturation of progenitor cells in the bone marrow into differentiated granulocytes, macrophages, and the T cells. In clinical oncology, recombinant G- or GM-CSFs are routinely used to correct neutropenia subsequent to chemotherapy and radiation. However, adjuvant G-/GM-CSF treatments have been suggested to occasionally enable tumor growth. Such adverse treatment outcomes are thought to occur due to certain solid tumors being addicted to G-/GM-CSF-dependent signaling by expressing endogenous cytokines and/or their cognate receptors (G-/GM-CSFR). Clinical case reports reveal that newly diagnosed patients with G-/GM-CSF(R)-positive tumors present with an advanced often metastatic disease suggesting an accelerated progression of such cancers. The putative mechanisms of progression bear similarities to those observed with an adjuvant use of recombinant cytokines, that is, induction of immune tolerance and angiogenesis. It is therefore an imperative to explore roles of G-CSF and GM-CSF in cancer in order to improve treatment outcomes and to better define eligible patients' cohorts. In this review, we aimed to present recent advances in studies addressing putative mechanisms and therapeutic uses of G-CSF and GM-CSF in several cancers of a nonmyeloid origin.\u03b1-subunits and the two nonselective \u03b2c subunits; each \u03b1-/\u03b2c-subunits dimer is thought to bind one ligand molecule Primary and metastatic lung cancers are by far the most frequently occurring type of malignancy driven by the ectopically secreted G-/GM-CSF Various cell types have been proposed as putative sources of G-/GM-CSF in lung cancers. Specifically, tumor-associated endothelial cells may secrete cytokines and thus promote angiogenesis and metastasis via increases in expression of cell adhesion molecules Glioma is the most common type of a primary malignant brain tumor in adults with universally poor prognosis. Gliomas more often than other G-/GM-CSF-secreting cancers also express intratumoral cognate receptors; augmented G-/GM-CSF(R) levels have been found to correlate with higher tumor grade \u03b1-subunits of GM-CSFR in bladder cancers compared to normal tissues A G-CSF was initially purified from the human bladder carcinoma cell line 5637 The elevated GM-CSF plasma levels have been found in certain patients with colorectal cancers thus implying this cytokine contributions to a disease progression \u03b1 augments transcription of a VEGF receptor gene and enhances the antiangiogenic actions of GM-CSF in this model Melanoma is a rapidly progressing incurable skin cancer with a propensity to metastasize early due to immunosuppression and growth induction in part occurring via G-/GM-CSF. Clinical case studies report existence of are albeit severe G-CSF-secreting melanomas A role for G- or GM-CSF in cancers of prostate is less defined. Reports of G-/GM-CSF-secreting prostate tumors in English scientific literature are uncommon thus implying lesser significance of these cytokines for a prostate cancer progression. However, in vitro and animal models of this disease implicate G-/GM-CSF in promoting dissemination and bone metastasis. Namely, costimulation with G-CSF and a stem cell factor enhances cancer stem cell phenotype via upregulation of Oct3/4 transcription factor, NANOGP8 pseudogene and ABCG2 transporter Solid tumors of every origin present with many dissimilarities in their cellular composition and the molecular mechanisms of progression. Tumors addicted to G-/GM-CSF(R) signaling represents a very distinct molecular subset among other malignancies. For example, they utilize signaling pathways of normal hematopoiesis and the cytokine-mediated auto-/paracrine growth augmentation in addition to immune suppression. The aforementioned signaling modalities albeit not common in solid neoplasms contribute to some of the most advanced cases. Table"} +{"text": "However, its use has been limited by reduced blood pool to myocardium contrast for spoiled and balanced steady-state free precession (bSSFP) implementations. T2-preparation techniques are capaAn off-resonance RF pulse was interleaved with whole-heart, respiratory gated 3D radial SPGR sampling [Figure The feasibility of a novel whole-heart functional cardiac acquisition using MT preparation with isotropic spatial resolution in a clinically reasonable scan time is presented. Further studies on optimization of acquisition parameters, including off-resonance frequency, number of projections, and acquired spatial resolution, will improve the applicability of the sequence for clinical situations.NIH grant 2R01HL072260."} +{"text": "Listeria monocytogenes engineered to express the tumor-associated antigen mesothelin. CRS-207 boosts responses against mesothelin and is unique in its capacity to stimulate both innate and adaptive immunity by activating T cells and NK cells. Nivolumab is an antibody against PD-1.A heterologous prime-boost vaccination strategy using GVAX pancreas vaccine and CRS-207 is showing promise in patients with pancreatic adenocarcinoma (PDA) . Furthermore, blockade of the immune checkpoint programmed death-1 (PD-1) is active in some cancers. Combinatorial strategies aimed at priming tumor antigen-specific T cells while simultaneously blocking negative checkpoints may be necessary to improve outcomes in PDA. GVAX is composed of allogeneic pancreatic cancer cells modified to express GM-CSF and induces a broad response against multiple tumor antigens. GVAX is given with low-dose cyclophosphamide (CY) to inhibit regulatory T cells. CRS-207 is live-attenuated Lm- and mesothelin-specific T cell and other immunological responses with OS, progression-free survival and best overall response.This is a Phase II study comparing CY/GVAX and CRS-207 with or without nivolumab in subjects with PDA who failed only one chemotherapy regimen for metastatic disease. Subjects are randomized in a 1:1 ratio to receive either 2 doses of CY/nivolumab/GVAX and 4 doses of nivolumab/CRS-207 (Arm A) or 2 doses of CY/GVAX and 4 doses of CRS-207 (Arm B). The primary objective is to compare OS between Arms A and B. Secondary/exploratory objectives include: assessment of safety and clinical responses (tumor assessments and CA19-9 levels) and correlation of"} +{"text": "In contrast, treatment with VACV-CTLA4 led to activated NK cell accumulation (day +6) followed by a CD8+ T cell infiltrate (day+13) in non-treated tumors. Importantly, CD8+CD44hi T cells isolated from the blood and spleens of the treated mice showed functional specificity to A20 lymphoma cells, but not to MHC-matched tumor cells (CT26) in intra-cellular stains for IFN-\u03b3. Splenocyte-derived A20-specific CD8+CD44hi T cells were induced most efficiently in the Irr-VACV-CTLA4 regimen-treated mice, while blood-derived A20-specific CD8+CD44hi T cells were induced most efficiently in the Irr-VACV regimen-treated mice. Viral plaque assays (VPA) showed lack of live viral particles in both treated and untreated tumors upon sacrificing mice 4 to 10 weeks after treatment initiation. Surprisingly, VPA assays identified live virus in the livers of Irr-VACV-CTLA4 regimen-treated mice, which paralleled a reduced metastatic load.Oncolytic virotherapy is safe and clinically active in solid tumors, however its efficacy in hematologic malignancies as well as in combination with checkpoint inhibitors and radiation is unexplored. To simulate advanced lymphoma, A20 cells were injected subcutaneously on bilateral flanks of BALB/c mice and treatment initiated on day 17 to only the right flank tumor with local irradiation (Irr), intratumoral (i.t.) vaccinia virus (VACV) and i.t. anti-CTLA-4 mAb suggest the effective induction of a potent immune memory response. Effective targeting of distant metastases after intratumoral administration is also an important finding with significant clinical implications. This novel combination immunotherapy with oncolytic viruses and checkpoint inhibitors following local tumor irradiation is now being translated to a Phase I proof-of-concept clinical trial in non-Hodgkin's lymphoma at our institution.Our findings are the first to demonstrate the potential of combination immunotherapy with oncolytic viruses and checkpoint inhibitors in hematologic malignancies. The antitumor activity is attributed to the induction of an effective and specific immune response. This finding is corroborated by the significant infiltration with mature activated NK cells, followed by CD8"} +{"text": "Thus, tumor cell-expressing PD-L1 may present as a crucial mediator of immunosuppression in the tumor microenvironment decreasing cytotoxicity of cetuximab activated PD-1 expressing NK cells. Herein, using The Cancer Genome Atlas (TCGA) data for 500 HNC patients' tumors, we found that PD-1 expression correlates with NK activation markers. Indeed, HNC patients also exhibit higher levels of circulating and tumor infiltrating PD-1+ NK cells, and neoadjuvant cetuximab treatment increased this frequency in vitro and in vivo in a prospective Phase II trial. In addition, anti-PD-1 mAb nivolumab enhanced cetuximab mediated NK cell activation and HNC cell lysis. Therefore, blocking PD-L1/PD-1 axis may be a useful approach to reverse immune evasion of HNC tumors to cetuximab therapy by reversing NK cell dysfunction.Co-inhibitory immune checkpoint receptors have become important targets for cancer immunotherapy. Programmed death 1 (PD-1) has been well-characterized on T cells in many cancer types, including head and neck cancer (HNC), for its ability to mediate activation and eventually T cell exhaustion in the tumor microenvironment. However, PD-1 expression on NK cells, which are crucial innate immune effector cells against cancer, remains largely undefined. In the setting of HNC, NK cells mediate lysis of EGFR-overexpressing tumor targets via cetuximab-mediated antibody dependent cytotoxicity (ADCC). Indeed, cetuximab has shown to be clinically effective but only to a modest extent. Therefore, it is necessary to investigate how cetuximab modulates activation of immune effector cell infiltrates in the tumor microenvironment in order to improve or extend its therapeutic efficacy. We hypothesized that expression of PD-1"} +{"text": "Fatty acids are essential for many aspects of biological activities. They function not only as an energy source but also as signaling molecules, regulating immune responses and other vital cellular functions. However, how to control fatty acid trafficking while sustaining lipid homeostasis inside cells remains largely unknown. As the main cytoplasmic lipid chaperones, fatty acid binding proteins (FABPs) are known to bind a variety of fatty acids and endogenous hydrophobic metabolites, facilitating lipid transportation and coordinating their responses . Thus, FThe FABP family consists of at least nine members of highly homologous proteins, each of which has been named according to the tissue where they were first cloned, such as adipose FABP (A-FABP) and intestinal FABP (I-FABP) . While m+ F4/80+ CD11c+ MHCII+ phenotype, highly express E-FABP and play a pivotal role in E-FABP-mediated protection against mammary tumor growth and metastasis. Furthermore, microarray analysis of genes expressed differentially in wild-type and E-FABP\u2212/\u2212 macrophages demonstrated that E-FABP expression in TAMs strengthens interferon \u03b2 (IFN\u03b2) production and signaling. The E-FABP-regulated IFN\u03b2 responses can further recruit the infiltration of natural killer (NK) cells into the tumor microenvironment to enhance tumor killing activity. Thus, E-FABP is critical in regulating host immune responses to tumor challenges, and host expression of E-FABP may represent a new protective factor towards cancer prevention through enhancing anti-tumor activity of TAMs.In our research focusing on the role of E-FABP in tumor development, we found that mice deficient for E-FABP exhibited increased tumor growth and metastasis in different tumor models as compared to their wild-type counterparts . To dissHowever, everything has two sides. While host expression of E-FABP is able to inhibit tumor growth and metastasis, E-FABP overexpression has been shown to promote inflammatory autoimmune diseases. In a mouse model of human multiple sclerosis, upregulation of E-FABP has been demonstrated to promote the development of experimental autoimmune encephalomyelitis (EAE). On the one hand, E-FABP expression in macrophages and dendritic cells facilitates the production of inflammatory cytokines including IL-6, TNF\u03b1, IL-1\u03b2, etc . On the In exploring the molecular mechanisms of how E-FABP expression in immune cells regulates their differentiation and functions, we observed several interesting phenomena. (1) Macrophages deficient for E-FABP exhibit reduced activation of STAT1 and STAT2 in response to IFN\u03b2 stimulation. (2) E-FABP can modulate gene expression through impacting the activity of nuclear transcriptional factors. For example, E-FABP expression in T cells can bind and restrict PPAR\u03b3 ligands in cytoplasm, thus suppressing PPAR\u03b3 activation. (3) Most importantly, E-FABP-mediated lipid transportation can facilitate immune cells to establish essential lipid platforms (e.g. lipid droplets), which bind specific signaling proteins for the regulation of cytosolic immune responses . All theIn summary, integration of documented research and our studies indicate that E-FABP plays a unique role in regulating immune responses in different diseases. Selective enhancing E-FABP activity in macrophages may represent a novel strategy for tumor prevention and treatment. Meanwhile, it should not be omitted the potential influence of E-FABP overactivity-mediated inflammatory autoimmune diseases."} +{"text": "Postoperative atrial fibrillation (POAF) is a common complication of cardiac surgery and may result in stroke, heart failure and poor prognosis.This study aimed to evaluate a novel index of total atrial conduction time derived from the P-wave onset (lead II) to the peak A'-wave on tissue Doppler imaging (PA-TDI duration). The PA-TDI duration was compared with previously reported predictors of POAF, and the optimal cut-off value of PA-DTI was calculated in patients undergoing mitral valve replacement or repair (MVR) for mitral valve regurgitation (MR).Seventy-two patients undergoing MVR were enrolled. They had transthoracic echocardiography with tissue Doppler imaging preoperatively and were monitored postoperatively with continuous electrocardiographic telemetry for 14 days. Preoperative characteristics, echocardiographic data, operative data and postoperative findings were compared between patients with POAF (44 patients) and without (28 patients).Postoperative cardiac and cerebral events was significantly larger in 44 patients with POAF than in 28 without POAF (14 patients (32%) vs. 2 (7%), p = 0.0190). Multivariate analysis revealed that etiology of degenerative and PA-TDI duration were significant independent predictors of POAF. Receiver-operating-characteristic curve analysis showed the optimal cut-off values of PA-TDI duration was 159.4 ms.ConclusionsThe PA-TDI duration was an independent predictor of POAF after MVR. Patients with PA-TDI duration >159.4 ms should be considered high risk and treated appropriately to improve outcomes."} +{"text": "Malignant pleural effusion (MPE) is a common clinical problem in non-small cell lung carcinoma (NSCLC) patients; however, the underlying mechanisms are still largely unknown. Recent studies indicate that the frequency of the L858R mutant form of the epidermal growth factor receptor (EGFR-L858R) is higher in lung adenocarcinoma with MPE than in surgically resected specimens, suggesting that lung adenocarcinoma cells harboring this mutation tend to invade the adjacent pleural cavity. The purpose of this study was to clarify the relationship between the EGFR-L858R mutation and cancer cell invasion ability and to investigate the molecular mechanisms involved in the formation of MPE. We found that expression of EGFR-L858R in lung cancer cells resulted in up-regulation of the CXCR4 in association with increased cancer cell invasive ability and MPE formation. Ectopic expression of EGFR-L858R in lung cancer cells acted through activation of ERK signaling pathways to induce the expression of CXCR4. We also indicated that Inhibition of CXCR4 with small interfering RNA, neutralizing antibody, or receptor antagonist significantly suppressed the EGFR-L858R\u2013dependent cell invasion. These results suggest that targeting the production of CXCR4 and blocking the CXCL12-CXCR4 pathway might be effective strategies for treating NSCLCs harboring a specific type of EGFR mutation. Lung cancer is the most common cause of cancer death in the world, and patients with this disease have a 5-year survival rate less than 15%13The production of MPE reflects cancer cell invasion into the pleura and accumulation of fluid within the pleural space owing to increased vascular permeability and leakage. MPE is diagnosed by the identification of malignant cells in pleural fluid or upon pleural biopsy668991214151517Although the pathogenesis of MPE is not fully understood, it is generally thought to involve tumor metastasis, angiogenesis, lymphangiogenesis, and tumor-associated inflammation192021112425The purpose of this study was to clarify the relationship between the EGFR-L858R mutation and cancer cell invasion ability and to determine the molecular mechanisms involved in the formation of MPE. Our findings demonstrate that lung adenocarcinomas harboring the EGFR-L858R mutation exhibit increased cancer cell invasive ability and MPE formation through activation of the CXCL12-CXCR4 axis.To assess the role of the EGFR-L858R mutation in cancer cell invasion ability and involvement in the formation of MPE, we used two H1299-derived NSCLC cell lines\u2014H1299-EGFR-WT, overexpressing wild-type EGFR, and H1299-EGFR-L858R, overexpressing EGFR-L858R\u2014as a model system. H1299 cells were selected as permanently transfected cells for wild-type and mutated EGFRs because they express wild-type EGFR and respond well to EGF treatment. Under regular culture condition , all EGFR mutants displayed a higher level of Tyr phosphorylation than did wild-type EGFR in H1299 cellsThe cell proliferation rate were detected by MTT assay. There was no significant difference in the cell proliferation rates between H1299-EGFR-WT and H1299-EGFR-L858R cells . We used5 H1299-EGFR-L858R or control H1299-EGFR-WT cells were injected, 100% of the animal yielded pleural tumors. The size and the number of the pleura tumor has no significantly difference between these two groups. All of the pleural tumors obtained from this study were less than 2 mm. There are no lung nodules and distant metastasis were observed. However, control H1299-EGFR-WT cells failed to promote the development of MPE in 100% of mice tested (5/5) at 14 days after inoculation, whereas H1299-EGFR-L858R cells promoted the development of pleural effusion in 80% (4/5) of mice, with volumes ranging from 100 to 1000\u2009\u03bcl. Collectively, these results indicate that the lung cancer cells harbored the EGFR-L858R mutant promotes the formation of MPE in vivo.To investigate whether the EGFR-L858R mutation plays an important role in the formation of MPE, the animal model of intrapleural injections were performed29Tumor cell migration and metastasis share many similarities with leukocyte trafficking, which is critically regulated by chemokines and their receptors25263134P\u2009<\u20090.01). The expression level of CXCR4 increased at least 3.4-fold in CL1-0-EGFR-L858R cells compared to control CL1-0-EGFR-WT cells and flow cytometric analysis were performed to validate the elevated expression of CXCR4 obtained from GEO public database (GSE11729). As shown in To identify downstream signaling pathways involved in EGFR-L858R\u2013dependent expression of CXCR4, the effective inhibition concentration of pharmacological inhibitors were performed in H1299-EGFR-L858R cells and then stimulated with 40\u2009ng/ml EGF. As showed in To determine if CXCR4 is indeed the major determinant of the EGFR-L858R\u2013dependent increase in invasion potential, we used a CXCR4 siRNA approach. Specific siRNAs targeting CXCR4 effectively knocked down CXCR4 transcript levels and suppressed the invasion ability of H1299-EGFR-WT and H1299-EGFR-L858R cells . The celP\u2009=\u20090.025, Mann-Whitney U Test). The clinical characteristics of the 12 patients, shown in To confirm the association between CXCR4 and the EGFR-L858R mutation, we performed an EGFR mutation analysis of cancer cells in MPEs from 12 lung adenocarcinomas and analyzed the surface expression of CXCR4 by flow cytometry. This analysis included six patients with EGFR-WT and six patients with the EGFR-L858R mutation. As shown in In this study, we show that the EGFR-L858R mutant increases lung adenocarcinoma cell invasion ability and promotes formation of MPE. Specifically, we found that ectopic expression of EGFR-L858R in lung cancer cells acted through activation of ERK signaling pathways to induce the expression of CXCR4. We further found that this increase in CXCR4 expression was central to the effects of EGFR-L858R, showing that siRNA-mediated knockdown of CXCR4 or inhibition of CXCR4 with a neutralizing antibody or antagonist significantly suppressed the invasion ability of H1299-EGFR-L858R cells. Finally, we demonstrated that clinical samples of lung adenocarcinomas harboring the EGFR-L858R mutation exhibited increased surface expression of CXCR4. Collectively, our findings indicate that the EGFR-L858R mutant increases cancer cell invasive ability and MPE formation through ERK-dependent activation of the CXCL12-CXCR4 axis.in vivo pathways37in vitroin vivo exhibit significantly higher levels of CXCL12 protein compared with those observed at the primary tumor site or in plasma, indicating that a chemotactic gradient may be established between the site of the primary tumor and metastatic sites44Over the past decade, accumulating evidence has demonstrated that the chemokine, CXCL12, and it cognate receptor, CXCR4, contribute to metastasis and clinical outcomes in many different types of solid cancers, including lung cancerIn summary, our findings demonstrate that lung adenocarcinomas harboring the EGFR-L858R mutation exhibit increased cancer cell invasive ability and enhanced MPE formation, effects mediated through ERK-dependent activation of CXCL12-CXCR4 signaling pathways. Our results further suggest that targeting the production of CXCR4 and blocking the CXCL12-CXCR4 pathway might be effective strategies for treating NSCLCs harboring specific types of EGFR mutations.The human lung carcinoma cell lines, H1299-EGFR-WT (overexpressing EGFR-WT) and H1299-EGFR-L858R (overexpressing EGFR-L858R), and the expression vectors pcDNA4-EGFR-WT (EGFR-WT in pcDNA4 expression vector) and pcDNA4-EGFR-L858R (EGFR-L858R in pcDNA4 expression vector) kindly provided by Dr. Yi-Rong Chen H1299-EGFR-WT and H1299-EGFR-L858R were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin, 100\u2009ng/ml streptomycin, and 500\u2009\u03bcg/ml of zeocin . The low invasive ability of human lung adenocarcinoma cell lines (CL1-0 cells) were isolated from a 64-year-old male patient with a poorly differentiated adenocarcinoma and selected in our laboratory as previously describedTransfections were performed using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer\u2019s instructions. The CXCR4 siRNA duplexes were purchased from Ambion . Total mRNA was extracted using TRIzol reagent (Invitrogen). cDNA was synthesized from total RNA by reverse transcription using Superscript III reverse transcriptase (Invitrogen), as described by the manufacturer. CXCR4 primers and probes were purchased from Applied Biosystems . EGFR-WT and EGFR-L858R were quantified by real-time RT-PCR (TaqMan) using the primers 5\u2032-CCGCAGCATGTCAAGATCAC-3\u2032 (forward) and 5\u2032-TCCTTCTGCATGGTATTCTTTCTCT-3\u2032 (reverse)After serum-starving for 24\u2009hours, H1299-EGFR-WT and H1299-EGFR-L858R cells were treated with 40\u2009ng/ml EGF for 5\u2009minutes, with or without pretreatment with the pharmacological inhibitors, AG1478 (EGFR inhibitor), AG490 (JAK-STAT3 inhibitor), LY294002 (PI3K-AKT inhibitor), U0126 (ERK inhibitor) or vehicle control (DMSO) for 1\u2009hour. Cells lysates were prepared and analyzed by Western blotting as described previously50. Prior to invasion assay, cells were pretreated for 30\u2009minutes with inhibitors, including the CXCR4 neutralizing antibody (12G5), isotype control antibody, or AMD3100 (Sigma-Aldrich). Cells (2\u2009\u00d7\u2009104) were suspended in 200\u2009\u03bcl of serum-free RPMI-1640 medium placed in the upper chamber; serum-free RPMI-1640 medium containing 30\u2009ng/ml CXCL12 was placed in the lower chamber. Cells were incubated for 18\u2009hours and then removed from the upper surface of the filter by scraping with a cotton swab. The cells that adhered to the bottom of the membrane were stained with Giemsa solution (Sigma-Aldrich).Invasion assays were performed as previously described using 8-\u03bcm-pore-size Costar Transwell chambers and Transwell filters coated with a thin layer of BD Matrigel (BD Biosciences)6 cells) for 15\u2009minutes at room temperature. Cells were then stained with phycoerythrin-conjugated mouse monoclonal anti-human CXCR4 antibody or an isotype-matched control antibody (R&D Systems). After incubation, cells were washed with phosphate-buffered saline (PBS) containing 0.05% bovine serum albumen (BSA) and resuspended in ~300\u2013400\u2009\u03bcl of PBS. Data were collected using a FACSCalibur flow cytometer (BD Biosciences) and analyzed using CellQuest software (BD Biosciences). CXCR4 expression data were presented as fold increase in mean fluorescence intensity (MFI) relative to isotype control staining (MFI ratio).Flow cytometric analyses of surface CXCR4 expression on cultured cancer cells were performed according to the manufacturer\u2019s protocol (R&D Systems). In brief, adherent cells, detached from culture dishes by exposure to 0.5\u2009mM EDTA, were pre-incubated with human IgG of National Taiwan University Hospital. All written informed consent documents regarding the use of these specimens for future molecular studies were completed before thoracentesis. The methods were carried out in accordance with the approved guidelines. Pleural effusions were collected from patients who received thoracentesis in the chest ultrasonography examination room of National Taiwan University Hospital from May to July, 2009. Malignant pleural effusions were confirmed by cytology examination. Some of the samples were previously examined and reported in studies of EGFR mutations. For flow cytometry analyses of CXCR4 surface expression levels, only lung adenocarcinoma patients with EGFR-L858R or EGFR-WT were enrolled. MPE was managed as described previouslyStudent\u2019s t-test was used to compare the means of two groups. Two-sided P-values less than 0.05 were considered significant. All analyses were performed using SPSS software version 15.0 for Windows .How to cite this article: Tsai, M.-F. et al. EGFR-L858R mutant enhances lung adenocarcinoma cell invasive ability and promotes malignant pleural effusion formation through activation of the CXCL12-CXCR4 pathway. Sci. Rep. 5, 13574; doi: 10.1038/srep13574 (2015)."} +{"text": "Here we report the generation of biotinylated, chemically synthesised, modified human CCL19 molecules for optimised cell sorting. Our results demonstrate that these ligands allow the efficient enrichment of mature, antigen-loaded human or murine CCR7+ DC using a rapid and established magnetic bead sorting strategy. CCR7+ murine bone marrow-derived DC (BM-DC) isolated using this GMP-compliant method display significantly improved lymph-node homing ability in vivo over standard (unsorted) BM-DC preparations. Using in vitro models, such as murine OT-I, we also demonstrate the ability of these sorted DC to efficiently generate both effector and memory antigen-specific T cell responses. In conclusion, we show that biotinylated ligands for chemokine receptors constitute a novel, GMP-compliant cell sorting strategy that could enhance the efficacy of existing clinical cell therapy regimens.Cell therapy regimens are frequently compromised by low-efficiency cell homing to therapeutic niches. Improvements in this regard would enhance effectiveness of clinically-applicable cell therapy. The major regulators of tissue-specific cellular migration are chemokines and therefore selection of therapeutic cellular populations for appropriate chemokine receptor expression would enhance tissue-homing competence. We have recently shown that biotinylated ligands for chemokine receptors can be used as efficient cell sorting reagents. These l"} +{"text": "Netta rufina) in southeastern Kazakhstan. Here, we present the complete genome sequence of the virus.An avian paramyxovirus 6 strain was isolated during a wild bird monitoring study in Kazakhstan in 2013. The virus was isolated from a wild duck red-crested pochard ( Avulavirus in the family Paramyxoviridae and include 12 serotypes, APMV-1 to APMV-12 (1Avian paramyxoviruses (APMVs) belong to the genus APMV-12 14. APMV-1During a study in 2013 monitoring wild birds, a hemagglutinating virus was isolated from a cloacal swab sample of a red-crested pochard after inoculation into 10-day-old embryonated chicken eggs. Hemagglutination inhibition tests using reference sera specific to APMV-1 to APMV-9 revealed that the virus belongs to APMV serotype 6.Viral RNA was extracted from infected allantoic fluid using the QIAamp viral RNA minikit (Qiagen). Pan-PMV primers were useFor whole-genome sequencing, viral RNA was used as a template for double-stranded cDNA synthesis (Roche). The Covaris M220 ultrasonicator was used for DNA fragmentation. For library preparation, Illumina adaptors (Bioo Scientific) and SPRIworks fragment library cartridge II (Beckman Coulter) were used, with manual size selection afterward. The quality of the library was checked on a Bioanalyzer 2100 (Agilent Technologies). Quantity was determined via quantitative PCR (qPCR) with the Kapa library quantification kit (Kapa Biosystems). Paired-end sequencing was performed on an Illumina MiSeq instrument using the MiSeq reagent kit version 3. Raw sequence data were analyzed and assembled using the Genome Sequencer software suite .Six out of the 8 available whole-genome sequences of APMV-6 (GenBank) contain a genome length of 16,236 nucleotides: duck/HongKong/18/199/1977, duck/Taiwan/Y1/1998, goose/FarEast/4440/2003, mallard/Belgium/12245/07, mallard/Jilin/127/2011, and mallard/Jilin/190/2011. The remaining two sequences have 16,230 nucleotides . The six-nucleotide difference between the two groups of viruses is due to a deletion in the nontranslating end of the fusion protein gene. The studied virus belongs to the first group, with 16,236 nucleotides, and contains seven genes common to all APMV-6 viruses.Phylogenetic studies of full-length hemagglutinin-neuraminidase (HN) genes and investigations of the antigenic cross-reactivity studies grouped the APMV-6 viruses into two separate clusters , the firKP762799.The complete sequence of APMV-6/red-crested pochard/Balkhash/5842/2013 is available at GenBank under the accession no."} +{"text": "In this article, the data obtained from the uniaxial fully-reversed fatigue experiments conducted on polyether ether ketone (PEEK), a semi-crystalline thermoplastic, are presented. The tests were performed in either strain-controlled or load-controlled mode under various levels of loading. The data are categorized into four subsets according to the type of tests, including (1) strain-controlled fatigue tests with adjusted frequency to obtain the nominal temperature rise of the specimen surface, (2) strain-controlled fatigue tests with various frequencies, (3) load-controlled fatigue tests without step loadings, and (4) load-controlled fatigue tests with step loadings. Accompanied data for each test include the fatigue life, the maximum (peak) and minimum stress\u2013strain responses for each cycle, and the hysteresis stress\u2013strain responses for each collected cycle in a logarithmic increment. A brief description of the experimental method is also given. The test method can be generalized for other semi-crystalline polymers.\u2022The stress\u2013strain responses provided in this paper can be used to obtain the frequency effects on PEEK fatigue behavior.110.7910/DVN/YSFURO.The presented data sets are categorized into four Microsoft Excel workbooks according to the type of tests. The workbook named (1) \u201cNominal Temperature,\u201d contains the strain-controlled fatigue tests with adjusted frequency to achieve nearly fixed strain rates, and thus, similar nominal temperature rise in all fatigue tests, (2) \u201cFrequency Effect Tests,\u201d contains the strain-controlled fatigue tests with various frequencies, (3) \u201cLoad-Controlled Test,\u201d contains load-controlled fatigue tests under constant amplitude loadings, and (4) \u201cLoad-controlled Step Test,\u201d contains load-controlled fatigue tests with step loadings. A summary of each test with corresponding strain/stress amplitudes, test frequency, specimen name, and fatigue life are presented in 2The study was conducted on a neat PEEK polymer All of the uniaxial fully-reversed fatigue tests were conducted under strain-controlled or load-controlled loading condition following the ASTM D7791 standard"} +{"text": "Intrathecal antibody synthesis is a well-documented phenomenon in infectious neurological diseases as well as in demyelinating diseases. Intrathecal antibody synthesis against HTLV-1 has been reported in HAM/TSP, but little is known about the role of B cells and humoral immune responses in the central nervous systems (CNS) of HAM/TSP patients. Here we demonstrate profiles of HTLV-1-specific antibodies in cerebrospinal fluid (CSF) of HAM/TSP patients. Of 36 HAM/TSP patients, antibody responses against Gag and Tax were detected in CSF of all the patients. CSF/Serum antibody ratio was elevated in anti-Gag (mean 1.20) more than in anti-Tax (mean 0.85), but importantly HAM/TSP patients with lesions or atrophy in spinal cord showed higher CSF/Serum anti-Gag antibody ratio. Antibody response against Env was detected in CSF of 94.4% of patients, but CSF/serum anti-Env ratio was significantly lower than those of anti-Gag and anti-Tax (mean 0.18). 19 patients were further studied for oligoclonal band (OCB) specificity to HTLV-1 antigens and all of them were found to have bands specific for at least one antigen studied. Significantly higher proportion of patients had Gag- or Env-specific OCBs than Tax-specific OCBs. Interestingly, CXCL13 (B cell attracting chemokine-1) was increased in CSF of HAM/TSP patients, which was associated with higher HTLV-1-specific antibody responses in CSF and was correlated with decrease of plasma blasts in peripheral blood. These results highlight the importance of the B cell compartment in HAM/TSP where production of HTLV-1-specific antibody may be required to control viral persistence and/or may be associated with HAM/TSP disease progression."} +{"text": "Setaria italica), an elite stress tolerant crop, provided an impetus for the investigation of the NF-Y families in abiotic responses. In the present study, a total of 39 NF-Y genes were identified in foxtail millet. Synteny analyses suggested that foxtail millet NF-Y genes had experienced rapid expansion and strong purifying selection during the process of plant evolution. De novo transcriptome assembly of foxtail millet revealed 11 drought up-regulated NF-Y genes. SiNF-YA1 and SiNF-YB8 were highly activated in leaves and/or roots by drought and salt stresses. Abscisic acid (ABA) and H2O2 played positive roles in the induction of SiNF-YA1 and SiNF-YB8 under stress treatments. Transient luciferase (LUC) expression assays revealed that SiNF-YA1 and SiNF-YB8 could activate the LUC gene driven by the tobacco (Nicotiana tobacam) NtERD10, NtLEA5, NtCAT, NtSOD, or NtPOD promoter under normal or stress conditions. Overexpression of SiNF-YA1 enhanced drought and salt tolerance by activating stress-related genes NtERD10 and NtCAT1 and by maintaining relatively stable relative water content (RWC) and contents of chlorophyll, superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and malondialdehyde (MDA) in transgenic lines under stresses. SiNF-YB8 regulated expression of NtSOD, NtPOD, NtLEA5, and NtERD10 and conferred relatively high RWC and chlorophyll contents and low MDA content, resulting in drought and osmotic tolerance in transgenic lines under stresses. Therefore, SiNF-YA1 and SiNF-YB8 could activate stress-related genes and improve physiological traits, resulting in tolerance to abiotic stresses in plants. All these results will facilitate functional characterization of foxtail millet NF-Ys in future studies.It was reported that Nuclear Factor Y (NF-Y) genes were involved in abiotic stress in plants. Foxtail millet ( Drosophila are lethal , also called heme-activated protein (HAP) or CCAAT binding factor (CBF), is a heterotrimeric transcription factor comprised of three distinct subunits: NF-YA (HAP2 or CBF-B), NF-YB (HAP3 or CBF-A), and NF-YC (HAP5 or CBF-C) by activating ABA-responsive genes in Arabidopsis , an elite stress-tolerant crop, is an important food and fodder grain crop in arid and semi-arid regions of Asia and Africa. However, a collective understanding of NF-Y families in foxtail millet has not been established under abiotic stress conditions. In the present study, we characterized three unique \u201cSi\u201d NF-Y families in foxtail millet, and found that SiNF-YA1 and SiNF-YB8 could enhance stress tolerance in tobacco. This research might serve as an entree to obtain rapid progress in determining the roles of foxtail millet NF-Y genes in abiotic stress responses.Foxtail millet (Arabidopsis and rice. The known NF-Y sequences were retrieved from the Arabidopsis (http://www.arabidopsis.org) and rice (http://rice.plantbiology.msu.edu/) databases. The Hidden Markov Model (HMM) profiles of known NF-Y sequences were also performed to identify the potential foxtail millet NF-Ys . The whole NF-Y protein sequences of foxtail millet were downloaded from the foxtail millet database (http://www.phytozome.org/) (Release 9.0) and plant transcription factor database (http://planttfdb.cbi.pku.edu.cn/). Further, all the sequences identified from BLASTP search and HMM search were queried against Pfam and ProDom (http://prodom.prabi.fr/prodom/current/html/home.php) to confirm their identity as potential NF-Y subunits. The redundant sequences or sequences that do not contain the known core regions were removed manually.Database BLASTP searches were performed to identify foxtail millet NF-Y members using the known NF-Y conserved core regions of http://www.rcsb.org/pdb/) by BLASTP (with the default parameters) to identify the best template having similar sequence and known three-dimensional structure. The data were fed into Phyre2 for predicting the protein structure by homology modeling under the \u201cintensive\u201d mode. The protein structures of NF-Y proteins were modeled at 90% confidence.NF-Y proteins were searched against the Protein Data Bank (PDB) (http://meme.nbcr.net/meme3/meme.html). Discovered MEME motifs (\u22641E-30) were searched in the InterPro database with InterProScan and default program parameters.Multiple sequence alignments were performed using ClustalW with gap open and gap extension penalties of 10 and 0.1, respectively .Phylogenetic analysis was undertaken based on the bootstrap neighbor-joining (NJ) method by MEGA4 with the following parameters: Kimura two-parameter model, pairwise gap deletion and 1000 bootstraps . Tandem duplications were identified manually. Adjacent genes of same sub-group tightly linked within 20 kb of each other and the identity of the genes \u226580% are considered as tandem duplicated genes. The syntenic relationships between foxtail millet and other plant species were then drawn using Circos v0.55 (http://circos.ca/). BLASTP was also performed to ensure unique relationship between the orthologous gene pairs and all hits with E-value \u2264 1E-5 and at least 80% homology were considered significant. The Ka/Ks ratios were estimated for ortholog NF-Y gene pairs through CODEML program in PAML interface tool of PAL2NAL . Ka and Ks were numbers of non-synonymous and synonymous substitutions per site, respectively. Ka/Ks > 1 indicated gene evolution under positive selection, Ka/Ks < 1 indicated purifying (stabilizing) selection and Ka/Ks = 1 suggested a lack of selection or possibly a combination of positive and purifying selection at different points within the gene that canceled each other out.Specific chromosomal positions of the NF-Y genes were plotted according to ascending order of physical position from the short arm telomere to the long arm telomere and finally displayed using MapInspect . The seedlings with the same growth state were used as the control. Construction of subtracted cDNA libraries, sequencing, data analysis of expressed sequence tags (ESTs), differential screening of ESTs by microarray analysis and statistical analysis were performed as described by Puranik for 3 h. Roots, stems and leaves were immediately frozen in liquid nitrogen and stored at \u221280\u00b0C after each treatment. For inhibitor or scavenger treatment, ABA inhibitor fluridone or H2O2 scavenger dimethyl thiourea (DMTU) was used. Foxtail millet seedlings were pretreated with 100 \u03bcM fluridone or 10 mM DMTU for 6 h to stop the production of ABA or H2O2, followed by exposure to dehydration treatment for 2, 6, or 12 h. RNA was extracted using Trizol reagent and the first strand cDNA was synthesized with a PrimeScript 1st Strand cDNA Synthesis kit . SYBR Green and ABI 7300 were used to monitor the kinetics of PCR product formation in qRT-PCR. The amounts of transcript accumulated for SiNF-Y genes normalized to the internal control Actin (AF288226.1) were determined using the 2\u2212\u0394\u0394CT method. For obtaining reproducible results, each experiment was repeated three times.For stress treatments, 21-day-old seedlings were exposed to 10% PEG 6000 (drought stress), 150 mM NaCl , 200 mM mannitol (osmotic stress), and 30 mM HAn expression vector p16318GFP with a green fluorescent protein (GFP) tag was constructed for subcellular localization analysis. The SiNF-Y open reading frame (ORF), lacking a stop codon, was amplified and fused to the N-terminal end of GFP under control of the CaMV 35S promoter. The reconstruction vector was bombarded into onion epidermal cells by a particle gun was cloned into the pBI121 vector driven by the CaMV 35S promoter, and transformed into tobacco (W38 genetic background) using the Agrobacterium-mediated transformation method , chlorophyll content, superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and malondialdehyde (MDA) activity between different chromosomes and the other 4 duplication events within the same chromosome. These results suggested that segmental and tandem duplication events play significant roles in the expansion of SiNF-Y genes.Gene duplication events play crucial roles in the amplification of gene family members in the genome. Arabidopsis, rice and Brachypodium were performed. Among them, 7 pairs existed in both foxtail millet and Arabidopsis genomes were identified to exhibit synteny with their homologs of rice existed in foxtail millet and Brachypodium genomes appear to have undergone strong purifying selection in comparison to foxtail millet-rice (0.1078). These genome synteny data along with Ka/Ks data would assist in understanding the evolution of NF-Y genes in monocot and dicot species.The ratios of non-synonymous (Ka) vs. synonymous (Ks) substitution rate (Ka/Ks) for duplicated gene-pairs as well as between orthologous gene-pairs of SiNF-Ys with those of de novo transcriptome assembly of foxtail millet with drought treatment was performed. Six NF-YAs , three NF-YBs and two NF-YCs (NF-YC3 and NF-YC12) showed up-regulation in expression level under salt and oxidative treatments. By comparison, SiNF-YB8 showed notable changes, mainly in leaves and roots, under drought and osmotic stress treatments. For the two SiNF-YC genes, SiNF-YC1 was up-regulated only in roots under drought treatment. SiNF-YC12 was up-regulated (no more than 4 fold) under salt, mannitol, and H2O2 treatments. Two NF-Y genes, SiNF-YA1 and SiNF-YB8 with notable expression changes, were selected for further study.Accumulating evidence indicated that NF-Ys were involved in responses to various abiotic stresses. The expression profiles of these 11 SiNF-Ys in responses to four abiotic stresses in three tissues were investigated using qRT-PCR. As shown in a heat map Figure , each NFSiNF-YA1 and SiNF-YB8 were selected to perform further investigation due to relatively high up-regulated transcript levels under various stresses, respectively and NaCl at 2 h (4.02 fold) Figure . To expls Figure .SiNF-YB8 was highly induced under PEG at 12 h (8.41 fold) and mannitol treatment at 6 h (9.22 fold) Figure . Similars Figure . These rSiNF-YA1 and SiNF-YB8 were inserted into a subcellular localization vector, respectively. The recombinant vector was transformed into onion epidermal cells and observed by confocal microscopy. The GFP fluorescence of SiNF-YA1 fusion protein suggested a nuclear localization in onion epidermal cells; whereas fluorescence of SiNF-YB8 fusion protein, like the control GFP, was uniformly distributed throughout the cell, including nucleus, cytoplasm and cytomembrane plants was higher than in WT plants , compared with the no-effector control (Luc/Ren 1) assays in tobacco protoplast were used to determine SiNF-YA1 activity against tobacco s Figure . The rel) Figure . Under sd Figure . HoweverNtERD10, NtSOD, NtPOD, and NtLEA5 promoters under normal conditions and the subcellular localization of NF-YB might be influenced by the availability of NF-YC or post-modification. However, NF-YA and NF-YC subunits could be imported into the nucleus without auxiliary assistance. For example, OsHAP2E conferred resistance to pathogens, salinity and drought, and increased photosynthesis and tiller number in rice , suggesting a complex mechanism controlling the functions of NF-YA genes and L1L (AtNF-YB6) that were involved in embryogenesis, seed or silique development stress (Liu and Howell, Previous genomes examination suggested high correlation and high sequence homology within the same NF-Y subunit family resulted in partially redundancy or overlapping functionality, which may be beneficial for protecting the cell from various stress conditions and selecting candidate genes for agriculture production. For NF-YA subunits, SiNF-YA5 was the best orthology match of rice OsHAP2E (namely OsNF-YA8; Figure Figures . These p Figures . SiNF-YB Figures , 4C. OsH Figures . SiNF-YB Figures . SiNF-YBPast studies mainly focused on the functions of NF-Ys in plant development (Zhang et al., 2+ fluxes and sugar sensing occurring both upstream and downstream of the ABA-dependent signaling pathways. The NF-Y complex coordinates oxidative stress response in eukaryotes (Hackenberg et al., 2O2 inhibitor prevented up-regulation of SiNF-YA1 and SiNF-YB8 in dehydration-, NaCl-, and/or mannitol-treated foxtail millet seedlings (Figure SiNF-YA1 and SiNF-YB8 could be part of the ROS-mediation processes. Recently, it was reported that transcripts of rice HAP2E and bermuda grass NF-YC1 in response to drought and salinity stresses might be involved in H2O2 signaling pathway (Th\u00f6n et al., Drought and salt stresses often result from imbalance between ROS producting and ROS scavenging ability that cause damage to various cellular components, such as carbohydrates, lipids, proteins and nucleic acids (Selote and Khanna-Chopra, s Figure . Thus, iNF-YB7 and bermuda grass NF-YC1 transgenic plants (Han et al., PpABI3A along with PpNF-YC1 and PpNF-YB4 synergistically activated PpLEA1 promoter through the ACTT-core ABRE element in Physcomitrella patens (Yotsui et al., NtERD10 and NtLEA5, ABA-dependent LEA genes, were strongly up-regulated in SiNF-YA1 or SiNF-YB8 transgenic tobacco (Figures cis-acting elements were found in promoters of these two tobacco LEA genes (Table SiNF-YA1 and SiNF-YB8 were involved in ABA-dependent stress signal pathway.Many transcription factors responding to stress have been identified, providing an insight that plants develop flexible molecular and cellular mechanisms to tolerate abiotic stress (Xu et al., Figures , and manes Table . This miSiNF-YA1 and SiNF-YB8 up-regulated oxidative stress-responsive genes, such as NtCAT, NtSOD or NtPOD (Figures cis-acting elements were found in promoters of these oxidative stress-responsive genes (Table SiNF-YA1 and SiNF-YB8 could activate expression of oxidative stress-related genes under stress conditions (Figure AtNF-YA5 was likely important for dehydration tolerance via its role in activating oxidative stress-responsive genes (Li et al., SiNF-YA1 or SiNF-YB8 enhanced stress tolerance through enhancing the antioxidant system (Figures In addition, Figures , 9I. CCAes Table . The tras Figure . It was Figures , 9E\u2013G. R Figures . As seve Figures , the antIn conclusion, we proposed foxtail millet NF-YA or NF-YB acted as key component of stress tolerance by being involved in both ABA-dependent and ROS signal pathways.ZX coordinated the project, conceived and designed experiments, and edited the manuscript. ZF conducted the bioinformatic work, generated and analyzed data, and wrote the manuscript. GH performed experiments and analyzed the data. PL, WZ, and MC provided analytical tools. YG and YM contributed with valuable discussions. All authors have read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer Chenghao Li declares that, despite being affiliated with the same institute as the author Wei-Jun Zheng, the review process was carried out objectively."} +{"text": "Modulation of KRAS activity by upstream signals has revealed a promising new approach for pancreatic cancer therapy; however, it is not clear whether microRNA-associated KRAS axis is involved in the carcinogenesis of pancreatic cancer. Here, we identified miR-193b as a tumor-suppressive miRNA in pancreatic ductal adenocarcinoma (PDAC). Expression analyses revealed that miR-193b was downregulated in (10/11) PDAC specimens and cell lines. Moreover, we found that miR-193b functioned as a cell-cycle brake in PDAC cells by inducing G1-phase arrest and reducing the fraction of cells in S phase, thereby leading to dampened cell proliferation. miR-193b also modulated the malignant transformation phenotype of PDAC cells by suppressing anchorage-independent growth. Mechanistically, KRAS was verified as a direct effector of miR-193b, through which the AKT and ERK pathways were modulated and cell growth of PDAC cells was suppressed. Taken together, our findings indicate that miR-193b-mediated deregulation of the KRAS axis is involved in pancreatic carcinogenesis, and suggest that miR-193b could be a potentially effective target for PDAC therapy. MicroRNAs (miRNAs) are a class of very small, non-coding RNAs that are evolutionarily conserved in many organisms. miRNAs suppress the expression of protein-coding genes in metazoans by binding to the 3\u2032 untranslated region (3\u2032-UTR) or even the coding region of their corresponding mRNA \u20133. In thPancreatic cancer is a malignancy with an extremely poor prognosis, with patients exhibiting dismal five-year relative survival rates of 6% . This poIn silico screens using TargetScan (http://www.targetscan.org) have revealed that KRAS is targeted by the miRNA, miR-193b. In addition, Calin et al. found a high correlation between miRNA gene loci and cancer-associated genetic alterations [erations . Notableerations 17]. Mor. MorIn serations \u201320 and merations . Howeverin vitro gain-of-function analysis by transfecting cell lines with miR-193b mimics. miR-193b function in these cells was assessed by examining cell viability, proliferation, apoptosis and colony-formation ability, and the underlying molecular mechanism was probed by testing KRAS as a target of miR-193b.In this study, we found that the expression of miR-193b was downregulated in pancreatic ductal adenocarcinoma (PDAC), the most common type of pancreatic cancer, compared to adjacent benign pancreatic tissue. In order to investigate the role of miR-193b in these disorders, we performed an This study was approved by the Peking Union Medical College Hospital Institutional Review Board. Written informed consent was obtained from all the patients.2 atmosphere.The pancreatic cancer cell lines, MIA PaCa-2, PANC-1, AsPC-1 and BxPC-3, and hTERT-HPNE (Human Pancreatic Nestin Expressing) human pancreatic duct epithelial cells were from American Type Culture Collection (ATCC). The type of hTERT-HPNE cells is \u201cintermediary cells formed during acinar-to-ductal metaplasia\u201d (according to ATCC information). All cell lines were cultured in complete growth medium containing Dulbecco's Modified Eagle's Medium (DMEM) and 10% fetal bovine serum (FBS) at 37\u00b0C in a humidified 5% COCells were transfected with miRIDIAN hsa-miR-193b mimic or Negative Control #2 at a final concentration of 25 nM using the transfection reagent DharmaFECT 4 (Dharmacon), according to the manufacturer\u2019s instructions. Cell density was measured using a Countess Automated Cell Counter (Invitrogen).Pancreatic tissue samples were collected from the Department of Pathology at Peking Union Medical College Hospital. Fresh samples were obtained from patients undergoing pancreatic resection and were immediately snap-frozen and stored in liquid nitrogen. In addition to histological confirmation of PDAC in paraffin-embedded sections immediately after surgery, the cancer status of PDAC and matched adjacent pancreas was reconfirmed in frozen sections of each specimen prior to RNA extraction. Patient characteristics, including sex, age, pathological diagnosis, differentiation and lymph node involvement, are presented in -\u0394\u0394Ct method; U6 snRNA was used as an endogenous control.Total RNA was extracted from frozen tissue samples or cultured cell lines using TRIzol reagent (Life Technologies). RNA concentration was determined on a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific). miRNA was converted to cDNA using a TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems); reverse transcriptase-minus and no-template controls were included. Mature hsa-miR-193b expression levels were assessed by real-time polymerase chain reaction (PCR) using TaqMan MicroRNA assays (Applied Biosystems). Reactions were performed in triplicate on an IQ5 instrument (Bio-Rad) using the 2in situ hybridization was labeled with combined 5\u2032 and 3\u2032 double digoxigenin (DIG). The U6 positive control probe and the scrambled negative control probe were both 5\u2032-DIG labeled. miRNA detection and localization using in situ hybridization were performed according to the manufacturer\u2019s protocols [A miRCURY LNA (locked nucleic acid) miRNA detection probe targeting hsa-miR-193b for rotocols . ParaffiTotal protein was extracted from tissue samples using cold T-PER Tissue Protein Extraction Reagent or from cell lines using RIPA buffer containing Halt Protease Inhibitor Cocktail and Halt Phosphatase Inhibitor Cocktail . Protein concentration in whole-cell extracts was determined with a Pierce BCA Protein Assay Kit (Thermo Scientific), following the manufacturer\u2019s instructions. Proteins in extracts (60 \u03bcg protein) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 12% gels and transferred to a PVDF (polyvinylidene difluoride) membrane (Bio-Rad) for Western blot analysis. Primary antibodies against cyclin D1 , cleaved PARP , cleaved caspase-3 , ERK, p-ERK, Akt and p-Akt were purchased from Cell Signaling Technology. Primary antibodies against K-RAS and \u03b2-actin were from Santa Cruz Biotechnology.MIA PaCa-2 or PANC-1 cells were plated at 3000 cells/well in 96-well plates. One to four days after transfection, cell viability was assessed using a Cell Counting Kit-8 . Cell proliferation was assessed with a BrdU (5-bromo-2\u2032-deoxy-uridine) Labeling and Detection kit I (Roche), using miRNA mimics (GenePharma Co.). After cells were counterstained with 4',6-diamidine-2'-phenylindole dihydrochloride , 6\u20138 representative pictures were taken from each specimen under an EVOS f1 fluorescence microscope (Advanced Microscopy Group). The percentage of BrdU-positive cells was calculated .Pancreatic cells were harvested 24 hours after transfection and subjected to a cell-cycle analysis. Samples were fixed in 75% cold ethanol, digested with 200 \u03bcg/ml ribonuclease A , and stained with 50 \u03bcg/ml propidium iodide , 24. CelEarly-stage apoptosis was detected in MIA PaCa-2 and PANC-1 pancreatic cancer cells by first rinsing cells briefly with 0.125% trypsin and then staining using a FITC Annexin V apoptosis Detection Kit I (BD Biosciences). Stained cells were analyzed on an Accuri C6 Flow Cytometer (BD Biosciences). Colony formation by cells treated with miRNA mimics (GenePharma Co.) was assayed as previously described [T CTA GAA AGG CCA TTT CCT TTT CAC A-3\u20325\u2032-GC and T CTA GAT GCA TGA CAA CAC TGG ATG A-3\u20325\u2032-GC (underlined bases correspond to an XbaI restriction site). Antarctic phosphatase (New England Biolabs Inc.) was used to prevent self-cyclization. A luciferase reporter construct was prepared by digesting the PCR product and inserting it into the pGL3-Control Vector (Promega) at a site immediately downstream of the firefly luciferase gene. Reporter constructs containing mutated KRAS 3\u2032-UTR, synthesized by Sangon Biotech, were also prepared. The miR-193b complementary sequence, prepared by annealing the two primers CTA GAA GCG GGA CTT TGA GGG CCA GTT T-3\u20325\u2032- and CTA GAA ACT GGC CCT CAA AGT CCC GCT T-3\u20325\u2032- (underlined bases correspond to an XbaI restriction site), was used to verify the transfection efficiency.A fragment of wild-type KRAS 3\u2032-UTR was amplified by PCR from PANC-1 genomic DNA using TopTaq DNA Polymerase (QIAGEN) with the primers, 293A cells were co-transfected with luciferase constructs, pRL-TK, and miR-193b mimic or Negative Control #2 using Lipofectamine 2000 (Invitrogen). Luciferase activity was determined 24 hours after transfection using the Dual-Luciferase Reporter Assay System on a Modulus Microplate luminometer (Promega). The pRL-TK vector (Promega), expressing Renilla luciferase, was used to normalize for differences in transfection efficiency between samples.Experiments were repeated at least three times. The results were expressed as means \u00b1 SD. Differences were assessed with a two-tailed Student\u2019s t test or Wilcoxon rank-sum test, and a p-value < 0.05 was considered statistically significant.To explore the expression pattern of miR-193b in PDAC, we determined miR-193b levels in 11 surgically resected PDAC specimens with matched adjacent benign tissues. In addition to primary selection for PDAC based on paraffin-embedded sections, we reconfirmed the PDAC status by histological analysis of frozen sections of each specimen before RNA extraction . Real-tiin situ hybridization of paraffin-embedded PDAC specimens (n = 22). The expression of miR-193b was lower in PDAC samples than matched adjacent tissues oligonucleotide. Transfection efficiency was confirmed by quantitative PCR and luciferase reporter assays , with a consideration of oncogenes crucial in pancreatic carcinogenesis to identify putative target mRNAs. As shown in Fig To investigate the molecular mechanism of miR-193b, we first combined the computational approach, TargetScan is associated with pancreatic intraepithelial neoplasia (PanIN) formation and pancin situ hybridization (n = 22 samples) to visualize the expression pattern of miR-193b in detail. In general, the expression level of miR-193b was high in \u201cnormal\u201d acinar cells (3+), but comparably lower in PDAC cells (1+-2+). To examine the potential involvement of miR-193b in the early stage of PDAC development, we assessed the expression of miR-193b in chronic pancreatitis (CP). In peritumoral CP-like changes, acinar cells were strongly positive for miR-193b, whereas other components showed relatively weaker staining; 20 of 22 samples exhibited downregulated miR-193b in duct-like tissues of the tested bulk specimens from PDAC patients compared to matched adjacent tissues. As tumor tissues comprise a mix of many different cell types, we further used LNA tissues \u2013S3D Fig. tissues Fig.CP is a noted risk factor for PDAC , and ADMwww.atcc.org and www.sanger.ac.uk). Based on these findings, we speculate that the KRAS mutation status may be similar in patient materials and pancreatic cancer cell lines. This would further suggest that the downregulation of miR-193b may be associated with the mutation status of KRAS. But how do oncogenic KRAS mutations affect the function of miR-193b in PDAC development?Many previous studies found that KRAS oncogene is mutated in more than 95% of PDAC tissues. Our present study observed that miR-193b was frequently decreased in PDAC samples. In PDAC cell lines, we found that miR-193b expression is higher in BxPC-3 cells bearing wild-type KRAS compared to the AsPC-1, MIA PaCa-2 and PANC-1 cells, which harbor mutant KRAS , suggests different underlying apoptotic mechanisms in these two cancer cell lines. Our interpretation of these findings is that miR-193b dampens pancreatic cell proliferation, mainly by acting as a brake on the cell cycle.Downstream effectors of KRAS signaling in pancreatic cancer were widely explored. Among them, Ras/Raf/ERK and Ras/PI3K/AKT signals are major effector pathways in PDAC tumorigenesis , especiaIn our work, Cyclin D1 showed decreased level after miR-193b treatment in PDAC cells. Ras-dependent Cyclin D1 expression relies on sustained RAF/MEK/ERK activation, and correspondingly affects G1 to S phase transition . miR-193Collectively, our findings demonstrate that miR-193b is frequently downregulated in PDAC samples and has potential tumor-suppressor activity. Dysregulation of the miR-193b-KRAS axis appears to be involved in pancreatic carcinogenesis, indicating that miR-193b is a potentially new therapeutic target in PDAC.S1 FigThe status of each section was histologically confirmed by senior pathologists before RNA and protein extraction.(TIF)Click here for additional data file.S2 Fig(A) Relative expression of miR-193b in MIA PaCa-2 and PANC-1 cells transfected with 25 nmol/L miR-193b mimic or scrambled oligonucleotide for 48 hours. (B) 293A cells were co-transfected with 50 nmol/L miR-193b mimic and a pGL3 vector containing a complementary miR-193b (c-miR-193b) segment; co-transfected pRL-TK served as a control for transfection efficiency. Upregulation of miR-193b suppressed the luciferase activity of pGL3-c-miR-193b.(TIF)Click here for additional data file.S3 Fig A case with peritumoral CP-like changes: miR-193b staining is lower in duct-like structures compared to acinar cells. A second case with peritumoral CP-like changes: miR-193b staining is lower in the tubular complex compared to the acinar cells. A case of CP without PDAC: miR-193b staining is lower in the duct-like tissues compared to the acinar cells. B, D and F are magnifications of A, C and E, respectively. Horizontal bar represents 500 \u03bcm.(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file."} +{"text": "Spin-echo based cardiac diffusion tensor imaging (DTI) is highly sensitive to myocardial strain . ImagingIt is the objective of the present work to compare second order motion compensated spin-echo DTI of the in-vivo and post-mortem porcine heart on a clinical MR system.2, slice thickness: 6mm, reduced FOV [2, TR/TE: 2R-R/83ms, 10 averages. Fat suppression was established by spectral-spatial excitation. Ten diffusion-encoding directions [2 were applied during early systole. The pig was euthanized by a potassium injection inside the MR scanner and the imaging protocol was repeated. Helix angle maps were calculated upon tensor reconstruction [Second order motion compensated diffusion encoding gradients were incorporated into a cardiac triggered single-shot spin-echo sequence Figure . A pig were acquced FOV : 230\u00d798mrections with a btruction (Figure Example helix angle maps are shown in Figure Good correlation between in-vivo and post-mortem imaging was found proving that bulk motion and strain effects are well suppressed if second order motion compensated diffusion gradients are employed.EU FP7 Marie-Curie fellowship to MG."} +{"text": "En face analysis of the whole aorta revealed that miR-590 significantly decreased aortic atherosclerotic plaque size and lipid content in apoE\u2212/\u2212 mice. Double immunofluorescence staining in cross-sections of the proximal aorta showed that miR-590 agomir reduced CD68 and LPL expression in macrophages in atherosclerotic lesions. MiR-590 agomir down-regulated LPL mRNA and protein expression as analyzed by RT-qPCR and western blotting analyses, respectively. Consistently, miR-590 decreased the expression of CD36 and scavenger receptor A1 (SRA1) mRNA and protein. High-performance liquid chromatography (HPLC)analysis confirmed that treatment with miR-590 agomir reduced lipid levels either in plasma orinabdominal cavity macrophages of apoE\u2212/\u2212 mice. ELISA analysis showed that miR-590 agomir decreased plasma levels of pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-\u03b1), monocyte chemotactic protein-1 (MCP-1), interleukin-1\u03b2 (IL-1\u03b2)and interleukin-6 (IL-6). In contrast, treatment with miR-590 antagomir prevented or reversed these effects. Taken together, these results reveal a novel mechanism of miR-590 effects, and may provide new insights into the development of strategies for attenuating lipid accumulation and pro-inflammatory cytokine secretion.Recent studies have suggested that miR-590 may play critical roles in cardiovascular disease. This study was designed to determine the effects of miR-590 on lipoprotein lipase (LPL) expression and development of atherosclerosis in apolipoprotein E knockout (apoE Cardiovascular disease caused by atherosclerosis is the number one cause of death in Western countries and threatens to become the major cause of morbidity and mortality worldwide. NowadayLipoprotein lipase (LPL) is an important enzyme that hydrolyzes triglyceride (TG) in lipoproteins, especially in TRL such as chylomicrons (CM) and VLDL, to liberate fatty acid (FA). The relationship between LPL and atherosclerosis has been controversial for long time. LPL is generally viewed as an anti-atherogenic enzyme, but none of its atherogenic characters is known to involve macrophage LPL. Macrophage LPL is thought to act as a molecular bridge between proteoglycans and lipoprotein receptors, such as remnant-like particles and LDL receptors, to induce the retention and uptake of atherogenic lipoproteins. In addiThe discovery of microRNAs (miRNAs), small noncoding RNA molecules involved in the posttranscriptional regulation of gene expression, has greatly influenced and furthered our understanding of the pathogenesis of atherosclerosis, 11.Recein vivo. Our results revealed that miR-590 inhibited LPL expression and prevented atherosclerotic lesions in apoE\u2212/\u2212 mice.We previously demonstrated that miR-590 can attenuate lipid accumulation and pro-inflammatory cytokine secretion by targeting LPL gene in human THP-1 macrophages. Thus, i\u2212/\u2212 male mice aged 8-weeks were purchased from Nanjing CAVENS Biological Technology Co, Ltd. ApoE\u2212/\u2212 mice were randomized into four groups: miR-590 agomir group (AG), miR-590 scrambled agomir negative control group (AG-NC), miR-590 antagomir group (AN) and miR-590 scrambled antagomir negative control group (AN-NC).There were 15 mice in each group. All mice were maintained on a 12 h light/dark cycle with unlimited access to food and water. Mice were fed the high fat/high cholesterol Western diet and injected via tail vein with miRNA agomir/antagomir or their respective controls (GuangZhou RiboBio. Co.) at a dose of 80mg /kg wt in 0.2 ml saline once every four weeks. After 8 weeks, these animals were anesthetized (3% isoflurane) andeuthanized by exsanguination. Hearts, aortas, abdominal cavity macrophages and blood samples were collected for the measurements as indicated in other parts. The animal study was performed under the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health and Care and Use guidelines for experimental animals of University of South China. The study protocol was approved by the Animal Ethics Committee of University of South China.ApoEHearts and proximal aortas were removed and fixed with formalin. The heaTissues collected for morphological analysis with aortic roots and ascending aorta were prepared for OCT-embedded cryosections. The sections were fixed in 4% parafor maldehyde for 30 min, washed in PBS, and then incubated in buffered normal goat serum to prevent non-specific binding of antibodies for 1 h at room temperature. The sections were then incubated separately overnight with antibodies against CD68 or LPL , followed by incubation with Alexa Fluor 592-conjugatged goat anti-rabbit IgG for 1 h at 37\u00b0C in a humidified box. Thereafter, the sections were washed in PBS and counter-stained with Hoechst dyetostain DNA and illuminate the nuclei. Photomicrographs were taken at random using an Olympus BX-URA2 camera in 4 sections per mouse sample.Aortic tissues and macrophages in abdominal cavities were lysed for extraction of protein using RIPA buffer containing proteinase inhibitor cocktails (Sigma) as previously described. ProteinAortic tissues and abdominal cavity macrophages were lysed for RNA extraction using the RNeasy Mini Kit (QIAGEN). LPL mRNMice were fasted overnight and then sacrificed. Blood samples were obtained from the retro-orbital plexus. Triglyceride (TG), total cholesterol (TC), and high-density lipoprotein cholesterol (HDL-C) were determined by commercially enzymatic methods , according to the manufacturer\u2019s instructions. The sterol analyses were performed using high-performance liquid chromatography (HPLC) analysis .The quantitation of secreted pro-inflammatory cytokines was performed by enzyme-linked immunosorbent assay (ELISA). Tumor necrosis factor-alpha (TNF-\u03b1), monocyte chemotactic protein-1 (MCP-1), interleukin-1\u03b2 (IL-1\u03b2) and IL-6 in serum were analyzed according to the manufacturer\u2019s instructions. Quantitp values were less than 0.05.All results were expressed as mean \u00b1 standard deviation (SD). Statistically significant differences among groups were analyzed by one-way analysis of variance (ANOVA) or Student\u2019s t-test using SPSS 18.0 software. Statistical significance was indicated when \u2212/\u2212 mice fed high fat diet. In each experimental group, no significant adverse reaction was observed. The atherosclerotic lesion area was examined by an en face analysis of the whole aorta and the cross-sections of the proximal aorta. The en face analysis of the whole aorta showed that atherosclerotic lesions throughout the aorta in apoE\u2212/\u2212 mice were significantly decreased in miR-590 agomir-treated group, but increased in miR-590 antagomir-treated group when compared with their respective negative control mice in peritoneal macrophages in miR-590 agomir-treated group were significantly lower than those in the control group. In comparison with its control group, however, cellular cholesterol levels were increased in response to treatment with miR-590 antagomir . Plasma IL-1\u03b2 levels in apoE\u2212/\u2212 mice as measured by ELISA. Plasma TNF-\u03b1 levels in apoE\u2212/\u2212 mice as measured by ELISA. Plasma MCP-1 levels in apoE\u2212/\u2212 mice as measured by ELISA. Data are shown as the mean\u00b1SD. *: P<0.05 vs. AG-NC. #: P<0.05 vs. AN-NC.Plasma IL-6 levels in apoE(TIF)Click here for additional data file."} +{"text": "Inappropriate activation of epidermal growth factor receptor (EGFR) plays a causal role in many cancers including colon cancer. The activation of EGFR by phosphorylation is balanced by receptor kinase and protein tyrosine phosphatase activities. However, the mechanisms of negative EGFR regulation by tyrosine phosphatases remain largely unexplored. Our previous results indicate that protein tyrosine phosphatase receptor type O (PTPRO) is down-regulated in a subset of colorectal cancer (CRC) patients with a poor prognosis. Here we identified PTPRO as a phosphatase that negatively regulates SRC by directly dephosphorylating Y416 phosphorylation site. SRC activation triggered by PTPRO down-regulation induces phosphorylation of both EGFR at Y845 and the c-CBL ubiquitin ligase at Y731. Increased EGFR phosphorylation at Y845 promotes its receptor activity, whereas enhanced phosphorylation of c-CBL triggers its degradation promoting EGFR stability. Importantly, hyperactivation of SRC/EGFR signaling triggered by loss of PTPRO leads to high resistance of colon cancer to EGFR inhibitors. Our results not only highlight the PTPRO contribution in negative regulation of SRC/EGFR signaling but also suggest that tumors with low PTPRO expression may be therapeutically targetable by anti-SRC therapies. C. elegans and Drosophila, the EGFR pathway controls the development of vulva and several other organs at different stages of embryogenesis, whereas in vertebrates EGFR signaling controls organogenesis of multiple epithelial tissues [EGFR gene as well as overexpression of EGFR and the receptor ligands, are well-characterized. More recent studies also highlight the importance of negative regulation in control of EGFR signaling [Epidermal growth factor receptor (EGFR) plays a crucial role in the regulation of cellular homeostasis in both vertebrates and invertebrates by coordinating cell growth and proliferation. In tissues , 2. EGFR tissues . Multiplignaling . NonetheC. elegans have identified several negative regulators of EGFR including the E3 ubiquitin ligase SLI-1 (c-CBL) and the tyrosine phosphatase SCC-1, a R3 subtype of receptor-type protein tyrosine phosphatases (RPTPs) [Drosophila orthologs of R3 family members, Ptp4E and Ptp10D, have also been shown to negatively regulate EGFR signaling [Genetic screens in (RPTPs) . The Droignaling , 7. LossIn vertebrates RPTPs of the R3 subtype include vascular endothelial\u2013protein tyrosine phosphatase (VE-PTP), density-enriched PTP\u20131 (DEP-1), PTPRO (GLEPP1), and stomach cancer\u2013associated protein tyrosine phosphatase\u20131 (SAP-1). All of these enzymes share a similar structure with a single catalytic domain in the cytoplasmic region and fibronectin type III\u2013like domains in the extracellular region . Recent CRC patients that do not respond to anti-EGFR therapy. The most well-established mechanism of cetuximab resistance in CRC patients is oncogenic KRAS mutations. However, not all patients harboring WT-KRAS benefit from cetuximab treatment. There is accumulating evidence that resistance to anti-EGFR therapy develops due to the loss of negative regulators of EGFR signaling [Anti-EGFR monoclonal antibodies (cetuximab and panitumumab) and small-molecule tyrosine kinase inhibitors (gefitinib and erlotinib) have been recently approved by the Food and Drug Administration (FDA) for the treatment of metastatic colorectal cancer and non-small-cell lung cancer (NSCLC), squamous-cell carcinoma of the head and neck, and pancreatic cancer , 13. Designaling , 13.PTPRO mRNA expression is strongly down-regulated in colon cancer patients with a poor prognosis [To date, only few data have been published about the contribution of PTPRO in colon cancer. Recent gene expression analysis of 688 primary colon tumors revealed that rognosis . In the A recent high-throughput siRNA screen suggested that PTPRO may be implicated in the regulation of EGFR signaling . To elucTo confirm the Antibody Array analysis, we assessed how PTPRO affects the kinetics of EGFR phosphorylation at Y845 upon EGF stimulation. We found that EGFR phosphorylation at Y845 was up-regulated in EGF-dependent manner in HEK293T cells expressing either an empty vector or WT-PTPRO. However, PTPRO overexpression led to more transient and decreased Y845-phosphorylation of EGFR Figure . We alsoWe also found that PTPRO suppression inhibits down-regulation of EGFR expression triggered by EGF stimulation Figure . In addiin vitro substrate trapping assay described in detail in [Drosophila ortholog of PTPRO (Ptp10D) form a complex through their extracellular domains [To test whether EGFR could be a direct substrate of PTPRO, we performed etail in , 17. We in vitro substrate trapping assay revealed a strong interaction between the substrate-trapping PTPRO-DA mutant and the SRC kinase. To confirm these data, we used vanadate competition approach [On the other hand, several reports demonstrated that Y845 is not EGFR autophosphorylation site, instead EGFR phosphorylation at Y845 is regulated by the SRC kinase \u201320. Thisapproach , 17. VanA recent report showed that a truncated form of PTPRO, PTPROt, affects SRC phosphorylation at Y416 in B-cells . Consistin vitro dephosphorylation assay. Purified WT-PTPRO or catalytically inactive PTPRO mutant (PTPRO-DA) were incubated with recombinant active SRC protein for different periods of time. Immunoblotting analysis of Y416-phosphorylated SRC revealed that incubation with WT-PTPRO but not with PTPRO-DA led to a significant decrease of Y416-phosphorylated SRC is observed in tumors with low ) Figure . These dSRC phosphorylation at Y416 plays a crucial role in the upregulation of its enzymatic activity \u201324, suggIn addition to alterations in SRC and EGFR phosphorylation, PTPRO knockdown also affected tyrosine phosphorylation of multiple proteins in a SRC-dependent manner Figure , suggestIn line with the observation that PTPRO suppression led to SRC up-regulation Figure , we founIn addition, SRC has been implicated in phosphorylation of a number of vesicular trafficking proteins such as clathrin, dynamin, caveolin that could also be in involved in modulation of EGFR trafficking \u201329. TherSuppression of the SRC/EGFR pathway by PTPRO suggests its role in negative regulation of MAPK and PI3K/Akt signaling cascades that are two major pathways downstream of EGFR. We assessed the impact of PTPRO overexpression on phosphorylation levels of MAPK and Akt kinases, using the CEER immunoassay platform, a highly sensitive antibody-capture proximity-based immune-microarray . We founWe confirmed the results of the CEER immunoassay by immunoblotting. Analysis of MEK1/2 and ERK1/2 phosphorylation kinetics upon EGF stimulation revealed that PTPRO overexpression dramatically reduced EGF-induced phosphorylation of both ERK1/2 and MEK1/2 Figure . We alsoWe next confirmed that PTPRO affects the MAPK signaling by modulating SRC activity. Indeed, we found that SRC inhibition by AZD0530 treatment completely abolished PTPRO-mediated difference in ERK1/2 phosphorylation Figure . Taken tPTPRO expression in about 15% of all colon cancers. Down-regulation of PTPRO mRNA expression strongly correlates with a poor patient prognosis, highlighting the contribution of PTPRO to colorectal cancer development and progression [PTPRO were dramatically down-regulated in 10 of 14 colon cancer cell lines and PTP activities. Several PTPs have been recently implicated in tumor suppressors by antagonizing the oncogenic effects of RTK signaling through direct RTK dephosphorylation. A high-throughput loss-of-function screen clearly demonstrated that receptor-type PTPs play a crucial role in negative regulation of major RTKs . The resOur study implicates PTPRO in negative regulation of EGFR signaling through direct inactivation of SRC kinase activity Figure . We obseWT-KRAS patients with low PTPRO expression are correlated with progressive disease after cetuximab treatment indicating that this group of patients do not benefit from the therapy. Our results revealed that loss of PTPRO promotes resistance to EGFR inhibitors in colon cancer cells by maintaining activated SRC and EGFR/MAPK pathway. Consistently with these observations, it has been reported that EGFR and SRC kinases cooperate in acquired resistance to cetuximab treatment [We also observed that reatment , 48. Increatment . Furtherreatment . ImportaTaken together, our results further highlight the importance of the negative regulation of SRC/EGFR pathway by tyrosine phosphatases. In addition, our results emphasize that a deeper understanding of the regulation of key kinases by phosphatases is crucial for choosing optimal treatment strategy of colon cancer patients.http://www.broadinstitute.org/rnai/public/resources/protocols). Infected cells were selected by treatment with 3 \u03bcg/ml puromycin (InvivoGen) for 2 days. Wild type (WT) full length PTPRO expression construct was purchased from ORIGENE. GST-tagged catalytic domain of WT-PTPRO (pGEX-5X-1-WT-PTPRO) plasmid was a kind gift of Dr. Hiroyuki Seimiya, The University of Tokyo. To generate a trapping mutant DA-PTPRO, a point mutation resulting in D1102A mutation, was introduced by using QuickChange Site-Directed mutagenesis kit (Stratagene).Human colorectal cell lines were obtained from ATCC; HCT15, HCT116, LOVO, COLO205 were cultured in RPMI 1640 medium (Invitrogen); HCA46, SW480, CACO2, LIM1215, DIFI, DKO4, DLD1, HKE3, HKH2, HEK293T were grown in DMEM medium (Invitrogen) and HT29 were grown in McCoy medium (Lonza). All media were supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. pLKO.1-shPTPRO-1 (TRCN0000002901), pLKO.1-shPTPRO-2 (TRCN0000002903), and pLKO.1-shGFP were purchased from Sigma-Aldrich. Lentiviral infections were carried out as described in TRC protocols containing protease inhibitor and phosphatase inhibitor cocktails (Roche). Proteins were separated by SDS-PAGE, transferred to PVDF membranes, and immunoblotted. Immunoprecipitation was performed as described elsewhere. BrieflyThe following antibodies were used: mouse monoclonal anti-Flag , anti-GAPDH , anti-vinculin , EGFR Antibody FITC , 4G10 Platinum Anti-phosphotyrosine (Millipore); rabbit monoclonal anti-EGFR , anti-p44/42 MAPK (Erk1/2) , anti-phospho-MEK1/2 (S217/221) , anti-SRC , anti-phospho-SRC (Y416) , anti-c-CBL ; rabbit polyclonal anti-phospho-EGFR (Y845) (Invitrogen), anti-PTPRO , anti-phospho-p44/42 MAPK (Erk1/2) (T202/Y204) , anti-phospho-c-CBL (Y731) , anti-phospho-SRC (Y527) .Human EGFR Phosphorylation Antibody Array was purchased from RayBiotech Inc. . Total cell lysates were incubated with membranes overnight. The detection and relative quantification of proteins were performed according to the manufacturer's protocol.Cell lysates were prepared according to the manufacturer's protocol and were sent to Prometheus Laboratories Inc. for further analysis.Cell pre-treated with 1mM pervanadate were lysed on ice in a lysis buffer . 10 mM DTT was then added for 15 min to inactivate iodoacetic acid and orthovanadate. After centrifugation at 14.000g for 15 min, cell lysates were incubated with GST-tagged recombinant PTPRO or GST alone conjugated to GSH sepharose beads overnight at 4\u00b0C. The pulled-downed proteins were resolved on SDS-PAGE gel and detected by immunoblotting.The assay was done as described previously . BrieflyCells were mobilized with enzyme-free cell dissociation buffer (Invitrogen), immunostained with FITC-conjugated anti-EGFR or FITC-conjugated isotype mouse IgG2a control (Santa Cruz) antibodies, and analyzed using FACSCanto flow cytometer (Becton Dickinson).4 cells per well were plated into 6 well plates and allowed to grow for 10 days in the absence and presence of the indicated drugs and then stained with crystal violet. The number of colonies was quantified using ImageJ software.2\u00d710Total RNA was isolated using the RNeasy Mini Kit (Qiagen) according the manufacturer's protocol. 1\u03bcg of RNA was used for reverse transcription with 220 units of Superscript II Reverse Transcriptase (Invitrogen) that was followed by quantitative RT-PCR using a 7500 Real Time PCR System (Applied Biosystems). PTPRO expression was normalized against 3 housekeeping genes used as reference gene. PTPRO probe (Hs00243097_m1) was purchased from Applied Biosystem. Calibrated normalized relative quantities (CNRQ) were calculated with qBasePlus 1.1 software using target-specific PCR efficiencies calculated from the inter-run calibration standard curves (Biogazelle).DSA) designed to work with formalin-fixed paraffin-embedded (FFPE) tissue was used for expression profiling of 65 FFPE CRC stage IV primary tumor samples from patients treated by cetuximab in the KULeuven University Hospital [A CRC specific DNA microarray platform (CRCHospital . 52 WT-KHospital followedFor the experimental data, unpaired student's T test was used. All tests were considered statistically significant for p<0.05."} +{"text": "The presence and extent of hyper-enhancement (HE) on Late Gadolinium Enhancement (LGE) has been associated with adverse events in patients with Hypertrophic Cardiomyopathy (HCM). Signal-threshold techniques are routinely employed for quantification; however, the precision and reproducibility of these versus a gold standard remains uncertain. Full Width Half of Maximum (FWHM) techniques are suggested to provide greater reproducibility than Signal Threshold versus Reference Mean (STRM) techniques, however the precision of these approaches versus manual assignment of optimal signal intensity(SI) thresholds has not been studied. We compare all known semi-automated LGE-quantification techniques for precision and reproducibility among patients with HCM.Seventy-six patients with HCM were studied. Endocardial and epidcardial borders were manually traced. Total HE volume was quantified using 6 semi-automated techniques and compared to expert manual adjustment of the signal intensity threshold. Techniques tested included STRM-based thresholds of > 2, 3, 5 and 6 SD above mean SI of reference myocardium, the FWHM technique, and the Otsu-auto-threshold (OAT) technique. The SI threshold applied for each slice and total HE volume were recorded. Bland-Altman analysis and intra-class correlation coefficients (ICC) were reported for each semi-automated technique versus expert, manually adjusted HE segmentation. Intra- and inter-observer reproducibility assessments were performed.Fifty-two patients showed HE on a total of 201 slices. Total HE volumes by manual segmentation were reproducible . For precision, the STRM > 3SD technique showed the greatest agreement with the manual segmentation while STRM > 6SD, > 5SD and FWHM techniques systematically underestimated total HE volume (Figure FWHM segmentation provides superior reproducibility, however systematically under-estimates total HE volume compared to manual segmentation in patients with HCM. The STRM > 3SD technique provides the greatest precision while retaining acceptable reproducibility and may therefore be the preferred approach for HE quantification in this population.Canada Foundation for Innovation."} +{"text": "Early-onset Alzheimer\u2019s disease (AD) patients present a different clinical profile than late-onset AD patients. This can be partially explained by cortical atrophy, although brain organization might provide more insight. The aim of this study was to examine functional connectivity in early-onset and late-onset AD patients. Resting-state fMRI scans of 20 early-onset (<65 years old), 28 late-onset (\u226565 years old) AD patients and 15 \u201cyoung\u201d (<65 years old) and 31 \u201cold\u201d (\u226565 years old) age-matched controls were available. Resting-state network-masks were used to create subject-specific maps. Group differences were examined using a non-parametric permutation test, accounting for gray-matter. Performance on five cognitive domains were used in a correlation analysis with functional connectivity in AD patients. Functional connectivity was not different in any of the RSNs when comparing the two control groups (young vs. old controls), which implies that there is no general effect of aging on functional connectivity. Functional connectivity in early-onset AD was lower in all networks compared to age-matched controls, where late-onset AD showed lower functional connectivity in the default-mode network. Functional connectivity was lower in early-onset compared to late-onset AD in auditory-, sensory-motor, dorsal-visual systems and the default mode network. Across patients, an association of functional connectivity of the default mode network was found with visuoconstruction. Functional connectivity of the right dorsal visual system was associated with attention across patients. In late-onset AD patients alone, higher functional connectivity of the sensory-motor system was associated with poorer memory performance. Functional brain organization was more widely disrupted in early-onset AD when compared to late-onset AD. This could possibly explain different clinical profiles, although more research into the relationship of functional connectivity and cognitive performance is needed. Alzheimer\u2019s disease (AD) is the most common form of dementia. Age is an important risk factor for developing AD in vivo measures of pathology and brain function in early-onset versus late-onset AD. Hypo-metabolism, measured with Positron Emission Tomography (PET) and fluorine-18 labeled fluorodeoxyglucose ([18F]FDG), was reported to be most pronounced in early-onset AD within parietal brain regions 11C]PIB) PET Only few studies have compared Resting-state functional magnetic resonance imaging (rs-fMRI) can be used to study functional properties of the brain by detecting spontaneous neuronal activity as localized changes in the blood oxyhemoglobin/deoxyhemoglobin ratio 18F]FDG-PET. Functional connectivity in late-onset AD patients was hypothesized to be lower in areas linked to memory function.The aim of this study was to examine functional connectivity in early-onset and late-onset AD patients. To investigate the general effect of aging on functional connectivity, two age-matched control groups were compared. Since very limited data is available describing functional connectivity in early-onset and late-onset AD patients, this was an exploratory study within eight standard RSNs The Ethical Review Board of the VU University Medical Center Amsterdam (VUMC) approved the study. All participants provided written informed consent.11C]PIB PET or Cerebral spinal fluid (CSF) measures were available to confirm AD-pathology. Global cognitive functioning was assessed using the Mini Mental State Examination (MMSE) MRI data of 48 AD patients and 46 healthy controls was available. This was a subset of a larger sample reported earlier Images were acquired on a 1.5 Tesla scanner . A high-resolution T1-weighted magnetization prepared rapid acquisition gradient echo (MPRAGE) image was acquired for every patient. In addition, resting state functional scans consisted of 200 T2*-weighted echo planar imaging (EPI) volumes . Subjects were instructed to lie still with their eyes closed and not to fall asleep during the resting state scan .www.fmrib.ox.ac.uk/fslAll MRI analyses were done using FMRIB\u2019s Software analysis was performed http://fsl.fmrib.ox.ac.uk/fsl/fslwiki/DualRegression; File S1), using standard masks of RSNs Default FSL-settings were used unless otherwise specified. In short, individual rs-fMRI data underwent motion correction, spatial smoothing using a 5 mm full-width-at-half-maximum (FWHM) Gaussian kernel, high-pass temporal filtering (0.01 Hz), removal of non-brain tissue, and registered to standard space (MNI152) via the T1-weighted image; using non-linear registration with a warp resolution of 10 mm. The warp resolution refers to the spacing between control points, and thus the smoothness, of the deformation field. In order to identify individual functional connectivity maps, a dual-regression technique was used To quantitatively evaluate the spatial overlap between atrophy and functional connectivity differences, the Dice Similarity Coefficient (DSC) was calculated. The DSC measures overlap between two segmentations (A and B), and is defined as DSC\u200a=\u200a2(A\u2229B)/(A+B) where \u2229 is the intersection Test scores that were not normally distributed were log-transformed. z-scores were calculated for all test-scores. The inverse (\u22121*zscore) of all tests measuring time were calculated, so that a lower score consistently represented poorer performance. Five cognitive domains were defined based on averaged z-scores; the memory domain included Visual Association Test (VAT) and immediate and delayed recall of the Dutch version of the Rey Auditory Verbal Learning Task (RAVLT). The attention domain included Trail Making Test (TMT) part A and Digit Span forward. The language domain included VAT naming and category fluency. Executive functioning included TMT part B and Digit Span backward. Visuo-constructive functioning consisted only of the Rey Complex Figure Copy test. For a detailed description of neuropsychological tests see Smits et al. Differences between groups were assessed using an Independent Samples T-test, a Mann-Whitney U test or a Chi-square test where appropriate. In order to test the effect of age on functional connectivity in more detail (in a non voxel-wise approach), a linear regression analysis was performed with an interaction term between diagnosis (AD vs. controls) and age (\u201cyoung\u201d <65 years vs. \u201cold\u201d >65 years) on mean functional connectivity (mean z-scores within the RSNs mask), accounting for gender and GM volume. A two-tailed Spearman correlation was used to assess the associations between mean RSNs functional connectivity (z-scores) with neuropsychological test score (z-scores) across AD patients. Second, in order to examine these associations in early-onset and late-onset AD groups separately, a linear regression analysis with an interaction term between age of onset (early-onset AD vs. late-onset AD) and functional connectivity on the relationship with cognitive performance was examined. When the interaction term with age of onset was significant, Spearman correlations were performed for that association within both early-onset and late-onset AD patients separately. A p-value below 0.05 was considered significant. All statistical analyses were performed using SPSS .Based on age at diagnosis, our sample contained 20 early-onset and 28 late-onset AD patients. Data of 15 \u201cyoung\u201d (<65 years old) and 31 \u201cold\u201d (\u226565 years old) age-matched controls were included. As expected, MMSE score and NGMV were found to be significantly lower in AD patients when compared to controls . The voxFunctional connectivity was not different in any of the RSNs when comparing the two control groups (young vs. old controls). This implies that there is no general effect of aging on functional connectivity.Early-onset AD patients showed lower functional connectivity when compared to \u201cyoung\u201d age-matched controls in all 8 RSNs Click here for additional data file."} +{"text": "We recently identified RIG-I-like helicases (RLH) as therapeutic targets of pancreatic cancer for counteracting immunosuppressive mechanisms and apoptosis induction. Here, we investigated immunogenic consequences of RLH-induced tumour cell death.Murine pancreatic cancer cells (Panc02) were treated with RLH ligands to induce apoptosis and were then cocultured with primary dendritic cells (DC). DC maturation marker expression, antigen uptake and antigen cross-presentation were assessed.+ DC effectively engulfed apoptotic tumour material and cross-presented tumour-associated antigen to na\u00efve CD8+ T cells. In comparison, tumour cell death mediated by oxaliplatin, staurosporine or mechanical disruption failed to induce DC activation, antigen uptake or cross-presentation. Moreover, tumour cells treated with sublethal doses of RLH ligands upregulated MHC-I and Fas expression and were sensitised towards CTL- and Fas-mediated killing.RLH ligands induced production of type I IFN, HMGB1 and Hsp70 and translocation of calreticulin to the outer cell membrane of tumour cells. In cocultures, DC upregulated B7 expression, which was mediated by tumour-derived type I IFN, whereas TLR, RAGE or inflammasome signaling was dispensable. CD8aRLH ligands induce a highly immunogenic form of tumour cell death linking innate and adaptive immunity."} +{"text": "Chronic nitroglycerin (GTN) anti-ischemic therapy induces side effects such as nitrate tolerance and endothelial dysfunction. Both phenomena could be based on a desensitization/oxidation of the soluble guanylyl cyclase (sGC). Therefore, the present study aims at investigating the effects of the therapy with the sGC activator BAY 60-2770 and the sGC stimulator BAY 41-8543 on side effects induced by chronic nitroglycerin treatment. Male Wistar rats were treated with nitroglycerin and BAY 60-2 770 (0.5 or 2.5 mg/kg/d) or BAY 41-8543 (1 and 5 mg/kg/d) for 6 days.Therapy with BAY 60-2770 but not with BAY 41-8543 improved nitroglycerin-triggered endothelial dysfunction and nitrate tolerance, corrected the decrease in aortic nitric oxide levels, improved the cGMP dependent activation of protein kinase I in aortic tissue and reduced vascular, cardiac and whole blood oxidative stress . In contrast to BAY 41-8543, the vasodilator potency of BAY 60-2770 was not impaired in isolated aortic ring segments from nitrate tolerant rats. sGC activator therapy improves partially the adverse effects of nitroglycerin therapy whereas sGC stimulation has only minor beneficial effects pointing to a nitroglycerin-dependent sGC oxidation/inactivation mechanism contributing to nitrate tolerance."} +{"text": "To evaluate the feasibility of using coronary magnetic resonance angiography (CMRA) with stress-perfusion and delayed-enhancement MRI as a screening tool for detection of coronary artery disease in asymptomatic patients.Three hundred forty-one self-referred asymptomatic subjects were enrolled in this study. CMRA image quality and factors affecting image quality were evaluated using a 1.5-T scanner with a 32-channel cardiac coil. Coronary artery stenosis, regional wall motion abnormalities, myocardial perfusion abnormalities, and delayed myocardial enhancement were analyzed. The occurrence of new chest pain and cardiac events was assessed with review of medical records and by telephone interviews in 332 subjects (97.3%) during the 29 \u00b1 6 months follow-up period.A total of 3296 (82.4%) of 4000 coronary artery segments exhibited diagnostic image quality on combined whole-heart and volume-targeted MRA. Image quality was affected by heart rates and navigator efficiency in whole-heart CMRA, and heart rates and ages in volume-targeted CMRA. Combined MRI detected significant coronary artery stenosis in eleven (3%) of 341 subjects. Three subjects (0.9%) underwent percutaneous coronary intervention after coronary artery disease was detected on cardiac MRI. There were no cardiac events during the follow-up period in subjects with follow-up.Coronary MRA combined with stress-perfusion and delayed enhancement MRI may help to rule out significant coronary artery disease in asymptomatic individuals.No."} +{"text": "The existing World Health Organization diagnostic algorithms for smear-negative TB perform poorly in HIV-infected individuals. New TB diagnostics such as urine TB lipoarabinomannan (LAM) could improve the accuracy and reduce delays in TB diagnosis in HIV-infected smear-negative presumptive TB. We sought to determine predictors for MTB culture-positivity among these patients.This study was nested into a prospective evaluation of HIV-infected outpatients and inpatients clinically suspected to have TB who were screened by smear-microscopy on two spot sputum samples. Data on socio-demographics, clinical symptoms, antiretroviral therapy, CXR, CD4 count, mycobacterial sputum and blood cultures and TB-LAM were collected. Logistic regression and conditional inference tree analysis were used to determine the most predictive indicators for MTB culture-positivity.3], 96/418 (23%) were sputum and/ or blood culture-positive for Mycobacterium tuberculosis (MTB) complex. Abnormal CXR and positive urine TB-LAM were significantly associated with MTB culture-positivity. Previous TB treatment reduced the likelihood of a positive MTB culture. A conditional inference tree analysis showed that positive urine TB-LAM and abnormal CXR were the most predictive indicators of MTB culture-positivity. A combination of urine TB-LAM test and CXR had sensitivity and specificity of 50% and 86.1% respectively overall, and 70.8% and 84.1% respectively among those with CD4<100 cells/mm3.Of the 418 smear-negative participants [female, 64%; median age (IQR) 32 (28-39) years, median CD4 106 (IQR 22 - 298) cells/mmA positive urine TB-LAM test and an abnormal CXR significantly predict MTB culture-positivity among smear-negative HIV-infected presumptive TB patients while previous TB treatment reduces the likelihood of a positive MTB culture. Validation studies to assess the performance of diagnostic algorithms that include urine TB-LAM in the diagnosis of smear-negative TB in HIV-infected individuals are warranted. Mycobacterium tuberculosis (MTB) culture, which is the gold standard for diagnosis, is not widely available due to technical and biosafety requirements . Majority of the MTB culture-positives were inpatients (80%). A comparison of clinical and laboratory characteristics between MTB culture-positive and negative participants is shown in Of the 418 sputum smear-negative participants, 96/418 (23%) were positive for MTB complex on blood and/or sputum TB cultures; of which, 55/96 were positive on both sputum and blood cultures while 6/96 (6.3%) were positive on blood culture alone. The median CD4 cell count was significantly lower in the MTB culture-positive compared to the culture-negative participants [60 (IQR 14\u2013152) versus 127 (IQR 26\u2013329) cells/mmResults of bivariate analysis are presented in 3. A combination of urine TB-LAM test and CXR had sensitivity and specificity of 50% and 86.1% respectively overall, and 70.8% and 84.1% respectively among those with CD4<100 cells/mm3 (The accuracy values Tables and 4 foells/mm3 . The perDiagnosis of smear-negative TB in HIV-infected individuals is difficult and is often associated with delays. New TB diagnostics techniques such as the urine TB-LAM antigen test have a potential to improve accuracy and to reduce delays in smear-negative TB diagnosis in HIV. We aimed to identify factors that could predict MTB culture-positivity among sputum smear-negative HIV-infected presumptive TB patients in a high prevalence TB/HIV setting and also assessed the accuracy of urine TB-LAM test in smear-negative TB diagnosis. In our cohort, the prevalence of culture-positive TB was high at 23% of the smear-negative HIV-infected presumptive TB patients. The predictors for MTB culture-positivity among smear-negative HIV-infected patients were; positive urine TB-LAM and an abnormal CXR read as highly suggestive of TB by a clinician. Previous TB treatment reduced the likelihood of finding a positive MTB culture. Positive urine TB-LAM test followed by CXR were the most predictive combination and would reduce the total number of CXRs that would have to be performed. CXR facilities are not readily available in many lower level health facilities in RLS and where available, they may not be affordable and maintenance is costly.Similar to earlier reports from other sub-Saharan African studies , 34, 35,3. Our findings are in agreement with earlier reports that urine TB-LAM performs much better among HIV-infected individuals with advanced HIV disease [A positive urine TB-LAM test was another independent predictor for MTB culture-positivity among HIV-infected smear-negative participants in our study, with higher accuracy values seen among participants with CD4<100 cells/mm disease \u201329. This3. Almost two thirds of MTB culture-positive smear negative HIV-infected patients with CD4< 100cells/mm3 in our study were diagnosed by a combination of urine TB-LAM and CXR with a high negative predictive value. Use of this proposed algorithm would reduce on the inaccuracy associated with empirical TB treatment initiation as often used in smear-negative HIV-infected individuals [Our findings suggest that a combination of urine TB-LAM test followed by a CXR significantly improves the accuracy of TB diagnosis in smear-negative HIV-infected patients, especially when CD4 counts are < 100cells/mmividuals . InformaFurther, the application of the urine TB-LAM based algorithm suggested from our findings would be limited to low-income settings where the highly accurate but costly molecular assays are not readily available for use in TB diagnosis. In an earlier report , urine TOur study had some limitations. First, the mycobacterial blood and sputum cultures may have failed to detect some participants with TB and some culture-negative patients could have been misclassified as not TB. This may have underestimated the association between the predicting factors and TB disease in this population as well as the accuracy of the suggested algorithm. Secondly, we did not follow-up the MTB culture-negative participants to obtain information on empirical TB treatment.Despite these limitations, our study has important strengths. In order to accurately classify participants as having TB or not, we performed both solid and liquid MTB cultures in addition to TB blood cultures, making the findings more reliable and accurate. The classification of participants as smear-negative presumptive TB was based on both ZN and FM microscopy, also making our study population definition and findings more accurate and reliable. Further, we report findings from a population that includes both outpatients and inpatients making our findings generalizable and relevant to most HIV/TB prevalent settings.In conclusion, smear-negative culture-positive TB is common in this setting. Positive urine TB-LAM test and abnormal CXR significantly predict MTB culture-positive TB among smear-negative HIV-infected presumptive TB patients while previous TB treatment reduces the likelihood of a positive MTB culture. Positive urine TB-LAM test followed by CXR are the two most predictive factors for MTB culture positivity. Urine TB-LAM test may be applied in the development of diagnostic algorithms for smear-negative TB in HIV. We recommend validation studies to assess the performance of diagnostic algorithms that include urine TB-LAM test in the diagnosis of smear-negative TB in HIV-infected individuals."} +{"text": "Monitoring the genetic diversity of HIV-1 during the early stage of infection offers the best opportunity for understanding viral transmission dynamics and evolution of transmitted/founder viruses. Previous studies of HIV-infected donors in Brazil employing pol region sequencing have shown a predominance of subtype B (~80%) followed by F and C, with rare recombinant viruses. Here, we report application of high-throughput near-full-length (NFLG) and partial HIV-1 proviral genome deep sequencing to characterize HIV in recently infected blood donors at four major blood centers in Brazil.From 2007-2011, 341 HIV+ blood donors from 4 blood centers were recruited to participate in a case control study to identify risk exposure and motivation to donate. Forty-seven were classified as recently infected based on testing by less-sensitive (LS) or \"detuned\" enzyme immunoassay or an LS chemiluminescent immunoassay . Five overlapping amplicons spanning the HIV genome were PCR amplified from peripheral blood mononuclear cells (PBMCs). The amplicons were molecularly bar-coded, pooled, and sequenced by Illumina paired-end protocol.Of the 47 recently infected donor samples studied, 39 (82.9%) NFLGs and 6 (12.7%) partial fragments were de novo assembled into contiguous sequences and successfully subtyped. Subtype B was the only non-recombinant virus characterized in this study and accounted for 60% (27/45) of samples. The remaining 40% (18/45) specimens showed various patterns of subtype discordance in different regions of HIV-1 genomes indicating 2-4 circulating recombinant subtypes derived from clades B, F and C. Over 50% of infection in MG and RJ harbor unique inter-subtype recombinant forms.Our findings revealed a high proportion of HIV-1 recombinants among recently infected blood donors in Brazil which has implications for future diagnosis, therapy and efficient vaccine development."} +{"text": "The patient was a 61-year-old woman with frequent drug-refractory paroxysmal atrial fibrillation (AF). She had history of the systemic hypertension with normal coronary arteriogram. Pre-ablation transesophageal echo showed mild to moderate RV enlargement and medium-sized atrial septal defect (ASD). Multidetector computed tomography (MDCT) showed two left-sided pulmonary veins (PV), three right-sided PVs, and one aberrant large PV draining into mid-LA roof . This ab"} +{"text": "Using different inflammatory mouse models we investigated how RAGE and ICAM-1 are involved in anti-inflammatory functions of protein C . We found that, depending on the stimulus, RAGE and ICAM-1 are cooperatively involved in PC-induced inhibition of leukocyte recruitment in cremaster models of inflammation. During short-term proinflammatory stimulation , ICAM-1 is more important for mediation of anti-inflammatory effects of PC, whereas RAGE plays a major role after longer proinflammatory stimulation (TNF\u03b1). In contrast to WT and \u2212/\u2212Icam-1 mice, PC had no effect on bronchoalveolar neutrophil emigration in \u2212/\u2212RAGE mice during LPS-induced acute lung injury, suggesting that RAGE critically mediates PC effects during acute lung inflammation. In parallel, PC treatment effectively blocked leukocyte recruitment and improved survival of WT mice and Icam-1-deficient mice in LPS-induced endotoxemia, but failed to do so in RAGE-deficient mice. Exploring underlying mechanisms, we found that PC is capable of downregulating intracellular RAGE and extracellular ICAM-1 in endothelial cells. Taken together, our data show that RAGE and ICAM-1 are required for the anti-inflammatory functions of PC.By binding PAR-1), and as a consequence activated protein C (aPC) elicits potent anti-inflammatory and cytoprotective effects besides its common anticoagulatory properties . Due to the antibodies capacity for capturing from plasma, the direct detection of aPC plasma-concentrations is possible [25]. The activity of the captured human PC was determined using a chromogenic substrate [26].Activation of human protein C was analyzed as previously described [24] with some modifications. Briefly, mice were injected with 100 U/kg of human protein C into the tail vein. As positive controls, 50 mU human Click here for additional data file."} +{"text": "Tidal recruitment of nonaerated lung is a main cause of ventilator associated lung injury. CT as the gold standard for quantifying lung collapse (CT-collapse) is associated with certain risks for the patient (e.g. radiation exposure or transportation) and cannot be used for repeated assessments. Electrical impedance tomography (EIT) instead is a bed-side non-invasive radiation-free continuous technique for monitoring of changes in thoracic air content and distribution. EIT may also allow quantification of recruitable lunge collapse (EIT-collapse) .To study correlation and agreement between CT- and EIT-collapse during a decremental PEEP-titration after a lung recruitment maneuver (RM) for further validation of the technique for assessment of EIT-collapse.2/FiO2 remained stable < 200 mmHg. Tidal volume was 6 ml/kg body weight. We performed a RM followed by decremental PEEP-titration (starting from 26cmH2O in steps of 2 cmH2O). We recorded EIT-data and airway pressures simultaneously on each step and obtained end-expiratory CTs. CT-collapse in the entire lung was defined as the lung mass within -200 HU to +100 HU [2O. Recruitable CT-collapse was calculated by multiplying the difference between CT-collapse at a certain PEEP-step and \u201cnon-recruitable collapse\u201d by 100% and then dividing this product by the difference between total lung mass and \u201cnon-recruitable collapse\u201d. EIT-collapse was calculated based on analysis of changes in EIT-pixel compliance [We induced ARDS in anesthetized pigs by pulmonary acid (HCl) instillation until the PaOmpliance . Thus, tmpliance . Bland-AWe analyzed 60 data points from 11 pigs (weight 39 range 37-42) kg). We found a strong within-subject correlation and clinically acceptable agreement between CT- and EIT-collapse . WeOur results support the potential of EIT for non-invasive bedside assessment of recruitable collapse."} +{"text": "Gelatinous Heberden\u2019s nodes (HNs), also termed synovial cysts, are a common form of generalized osteoarthritis (OA). We sought to determine whether HN cases at clinical presentation contained multipotential stromal cells (MSCs) and to explore whether such cells were more closely related to bone marrow (BM) or synovial fluid (SF) MSCs by transcriptional analysis.At clinical presentation, gelatinous material was extracted/extruded from the distal phalangeal joint of OA patients with HNs. From this, plastic adherent cells were culture-expanded for phenotypic and functional characterization and comparison with BM- and SF-MSCs. Mesenchymal related gene expression was studied by using a custom-designed TaqMan Low Density Array to determine transcriptional similarities between different MSC groups and skin fibroblasts.+, CD44+, CD73+, CD81+, and CD90+ and CD14- CD19-, CD31-, CD34-, CD45-, and HLADR-) and exhibited osteogenic, chondrogenic lineage differentiation but weak adipogenesis. Gene cluster analysis showed that HN-MSCs were more closely related to SF- than normal or OA BM-MSCs with significantly higher expression of synovium-related gene markers such as bone morphogenic protein 4 (BMP4), bone morphogenetic protein receptor type 1A (BMPR1A), protein/leucine-rich end leucine-rich repeat protein (PRELP), secreted frizzled-related protein 4 (SFRP4), and tumor necrosis factor alpha-induced protein 6 (TNFAIP6) (P <0.05).In all cases, HN material produced MSC-like colonies. Adherent cultures displayed an MSC phenotype (CD29Gelatinous HNs derived from hand OA at clinical presentation contain a population of MSCs that share transcriptional similarities with SF-derived MSCs. Their aberrant entrapment within the synovial cysts may impact on their normal role in joint homeostasis. Osteoarthritis (OA) represents a common age-related disease characterized by progressive joint destruction, loss of function, and joint failure. Decompensated joint remodeling with florid new bone formation is also a feature of advanced OA, and generalized nodal OA represents a striking example of this bone-forming phenotype. Nodal OA may present acutely with painful joint swelling or gelatinous synovial cysts, known as Heberden\u2019s nodes (HNs), which subsequently are associated with ossification and radiographic features of OA,2. As suSkeletal repair, remodeling, and new bone formation are thought to be linked to multipotential stromal cell (MSC) function, whereby highly proliferative cells from a variety of sources (such as bone marrow (BM), synovium, and adipose tissue) can form mesenchymal lineage tissues. ConsequWe originally identified a population of MSCs in knee synovial fluid (SF) and observed their increase in early knee OA; similarAlthough ossified HNs are common, early manifestations with gelatinous material are infrequent and rarely present to the clinician at a stage when they can be aspirated,2. We unWe identified three female patients (ages 44 to 66 years) with painful distal interphalangeal (DIP) joint HNs. All imaging and samples were collected following informed written consent under ethics approved by the Leeds East NHS Research Ethics Committee. Patients had conventional x-ray of their hands prior to sample collection Figure\u00a0A. HNs we4 cells per cm2, with twice-weekly media changes, and further grown to between p1 and p3.Extruded HN material was diluted (1:4) with non-hematopoetic (NH) expansion medium , centrifuged, and resuspended in NH medium. Samples were plated in triplicate and cultured for 14 days with twice-weekly media changes in NH medium before counting colonies; these cells were considered passage 0 (p0). Thereafter, colonies were trypsinised and further expanded. For expansion, cells were seeded at a density of 10For comparative analysis, MSCs were also expanded from OA knee SF aspirates as described above for HN cells and normal and OA BM. Briefly, normal iliac crest BM (ICBM) (n\u2009=\u200911) was aspirated from orthopedic patients undergoing elective surgery for the removal of metalwork. BM from OA patients\u2014osteoarthritic femoral canal BM (OAFCBM) (n\u2009=\u20093)\u2014was aspirated from the medullary canal at the neck of the femur during hip arthroplasty. Plastic5) were pelleted and cultured in chondrogenic medium for 21 days (reagents and methods as defined in[3 cells per cm2 and cultured for 21 days in either osteogenic or adipogenic differentiation media[Trilineage differentiation assays were performed as previously described. Brieflyfined in). For hion media; cells wFlow cytometry was performed by using an LSRII four-laser flow cytometer with appropriate isotype controls. Passaged MSCs (n\u2009=\u20093 for each group) were stained with combinations of the following antibodies at the dilution recommended by the manufacturers: anti-CD14-allophycocyanin-cyanine (APC-H7), anti-CD19-fluorescein isothiocyanate (FITC), anti-CD34-peridinin chlorophyll protein (PerCP), anti-HLADR-phycoerythrin-cyanine (PE-Cy7), anti-CD73-phycoerythrin (PE), anti-CD90-PE, anti-CD81-FITC, anti-CD44-FITC, and anti-CD29-FITC . Cells were stained with 4\u2032,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich) as a live/dead discriminator immediately prior to acquisition. At leas(-\u0394Ct) method, normalized to the reference gene HPRT1[U) was performed by using SPSS 20 .RNA was isolated from p1- to p3-expanded MSCs from HN, SF, OAFCBM, OAFHBM, ICBM, and primary FBs (n\u2009=\u20093 for each group) by using the Norgen Biotec RNA/DNA/protein kit in accordance with the instructions of the manufacturer. RNA was reverse-transcribed by using the High Capacity cDNA reverse transcription kit for use on a 96-gene custom designed TaqMan Low Density Array . Based on recommendations of the manufacturer, 200 ng cDNA was used per port (two ports per sample). Analysis used the 2ene HPRT1. Hierarcene HPRT1,20. StatSimilar to control SF and ICBM, MSC-likTrilineage differentiation was performed on culture-expanded p2-3) cells , bone morphogenic protein receptor type 1A (BMPR1A), protein/leucine-rich end leucine-rich repeat protein (PRELP), secreted frizzled related protein 4 (SFRP4), and tumor necrosis factor \u03b1-induced protein 6 (TNFAIP6)[Examples of differentially expressed genes between MSCs and FBs are shown in Figure\u00a0TNFAIP6),21-23.ACAN): bone morphogenetic protein 4 (BMP4) and cartilage oligomeric matrix protein (COMP)[Our data also identified increases in the expression of genes associated with chondrogenesis for both HN- and SF-MSCs over BM-derived MSCs Figure\u00a0, includin (COMP),24. TakeAcute HNs are a feature of pre-radiographic hand OA and these gelatinous synovial cysts are thought to be a forerunner of new bone formation-4. The nCEBPA (CCAAT/enhancer-binding protein) and PPRAG. A lack of adiopogenesis has been linked to \u2018in vitro\u2019 aging of MSCs, whereby the ability of cells to differentiate is reduced as the cells approach senescence[Although HNs are a common form of generalized nodal OA, clinical presentation at the acute phase, prior to ossification, is somewhat rare; only three cases have appeared in our clinic in the past five years,3. Consenescence,26. OwinFZD1 (frizzled family receptor 1), IGFBP3 (insulin-like growth factor-binding protein 3), PAPSS2 (3\u2032-phosphoadenosine 5\u2032-phosphosulfate synthase 2), and VEGFC are differentially expressed between MSCs and FBs[GDF5 (growth differentiation factor-5) and HAPLN1 , which are involved in skeletal development, cartilage formation, and maintenance[SFPR4[ACAN, BMP4, PRELP, and TNFIP6 (Figure\u00a0in vivo and to establish their migration patterns following injury or in disease, lineage tracing experiments in animal models are required[in vivo\u2019 functionality in a given environment.Differential transcriptional analysis has previously been shown to reliably distinguish MSCs from FBs and subdivide MSCs from their tissue of origin by identifying commonly expressed genes when cells are grown under the same conditions-30. Herentenance,31. Furtntenance,16. Suchce[SFPR4, and sevrequired. Neverthrequired-30 indicWith both normal human and animal joints having resident SF-MSCs, it is pWe present the first description of MSCs in a small joint at a stage when cartilage damage is minimal (as seen by x-ray; Figure\u00a0ACAN: aggrecan; BM: bone marrow; BMP4: bone morphogenetic protein 4; DIP: distal interphalangeal; FB: fibroblast; FITC: fluorescein isothiocyanate; HN: Heberden\u2019s node; ICBM: iliac crest bone marrow; MSC: multipotential stromal cell; NH: non-hemopotetic; OA: osteoarthritis; OAFCBM: osteoarthritic femoral canal bone marrow; OAFHBM: osteoarthritic femoral head bone marrow; p: passage; PD: population doubling; PDR: population doubling rate; PE: phycoerythrin; PRELP: proline/arginine-rich end leucine-rich repeat protein; qPCR: quantitative polymerase chain reaction; SF: synovial fluid.The authors declare that they have no competing interests.TB participated in the design of the study, drafting of the manuscript, and interpretation of the results; shared responsibility for data acquisition; and helped to perform data analysis. EJ participated in the design of the study, drafting of the manuscript, and interpretation of the results and helped to perform data analysis. DM participated in the design of the study, drafting of the manuscript, and interpretation of the results. SB, SC, and CB shared responsibility for data acquisition. All authors critically evaluated the manuscript and read and approved the final version."} +{"text": "Transgenic mouse model is useful to study pathogenesis of viral genes like tax and HBZ. However, it is impossible to study the host immune responses by these transgenic mice. We used simian T-cell leukemia virus type 1 (STLV-1) infected Japanese monkeys (JMs) to analyze host immune responses after treatment by anti-CCR4 monoclonal antibody (mAb). In the previous study, we reported that anti-CCR4 mAb treatment in STLV-1 infected JMs significantly reduced CCR4 positive cells and STLV-1 proviral load (PVL). In this study, we investigated the long-term effect (48 weeks) on PVL and immune responses to STLV-1 Tax (sTax) and bZIP factor (SBZ) after the anti-CCR4 mAb treatment. STLV-1 PVL in the JMs was kept at the lower level than 0 week even 48 weeks later. To further investigate the anti-CCR4 mAb induced antiviral effect, we analyzed regulatory T (Treg) cells and sTax and SBZ specific T-cell responses. Treg cells, which were CCR4 positive, were rapidly reduced after the treatment. The number of activated Treg cells was severely suppressed. Furthermore, anti-CCR4 mAb treatment of infected CD4 T cells from the JMs enhanced engulfment by phagocytes, which likely enhances antigen-presentation. Treated JMs showed spontaneous activation of cytotoxic T-lymphocytes (CTLs) to sTax and SBZ. However, the immune response to other antigen was not enhanced. In consistent with these data, the enhanced T-cell responses (anti-Tax and HBZ) were also observed in anti-CCR4 mAb treated ATL patients. Taken together, anti-CCR4 mAb induces CTLs to sTax/Tax and HBZ/SBZ by two different mechanisms: reduced Treg cells and enhanced antigen presentation by antibody-dependent cell-mediated phagocytosis. These effects of anti-CCR4 mAb induce prolonged suppression of PVL, which might enable long-term control of ATL and HTLV-1 infected cells."} +{"text": "Bladder cancer is the second most common urological malignancy in the world. In 70% of cases it is initially diagnosed as non-muscle-invasive bladder cancer (NMIBC) and it is amenable to local treatments, with intravesical (IVES) Bacillus-Calmette-Guerin (BCG) immunotherapy being routinely used after transurethral resection of the lesion. However, this treatment is associated with significant side-effects and treatment failures, highlighting the necessity of novel strategies. One potent approach is the suicide-gene mediated therapy/prodrug combination, provided tumor-specificity can be ensured and anti-tumor immune responses induced. Using the mouse syngeneic orthotopic MB49-bladder tumor model, here we show that IVES human papillomavirus non-replicative pseudovirions (PsV) can pseudoinfect tumors with a ten-fold higher efficacy than normal bladders. In addition, PsV carrying the suicide-gene herpes-simplex virus thymidine kinase (PsV-TK) combined to Ganciclovir (GCV) led to immunogenic cell-death of tumor cells in vitro and to MB49-specific CD8 T-cells in vivo. This was associated with reduction in bladder-tumor growth and increased mice survival. Altogether, our data show that IVES PsV-TK/GCV may be a promising alternative or combinatory treatment for NMIBC. Bladder cancer is the fourth and eighth most common malignancy among men and women, respectively ,2. SevenN- and O-sulfation to which HPV PsV bind [For safety reasons, a critical issue is the use of an effective and tumor-specific delivery system, a characteristic that may be provided by human papillomavirus (HPV)-based gene transfer vectors. HPV has a specific tropism for infecting the basal cells of stratified epithelia with binding to heparan sulfate proteoglycans (HSPG) on the basement membrane being an obligatory initial step in HPV infection in vivo . The mecPsV bind are not PsV bind . AccordiPsV bind . Here, wPsV bind . Our datThe cytotoxic effect of pseudovirions encoding for thymidine kinase (PsV-TK) in combination with GCV was first examined in vitro on MB49-cells. PsV-TK or an irrelevant PsV encoding for luciferase (PsV-luc) were added to MB49-cells at a multiplicity of infection (moi) of 10 or 50, followed by addition or not, of GCV 24 h later. Five days later, viable cells were directly quantified using a one-solution colorimetric assay. The data show that only cells infected with PsV-TK and incubated with GCV presented a significantly lower viability, representing less than 10% of control cells when the higher moi of 50 was used .+7AAD\u2212 cells, +aqua\u2212 cells, TCD and its immunogenic potential were further evaluated by flow cytometry. MB49-cells infected with PsV-TK at moi 10 or 50 followed by GCV 24 h later were compared to treatment with GCV alone. Two days later, AnnexinV/7AAD staining was used to determine cells in early apoptosis (AnnexinV6 transducing relative light units transducing relative light units (TRLU) [PsV-luc was used to visualize PsV pseudoinfection of the bladder by in vivo bioluminescence imaging after luciferin injection. IVES administration without prior treatment was ineffective . Howevers (TRLU) ) did not6 transducing relative units of PsV-its (TRU ) and GCVits is derived from a carcinogen induced urothelial carcinoma in male C57Bl/6 mice . Seven t\u00ae Aqueous One Solution Cell Proliferation assay kit according to manufacture instructions. For determination of AnnexinV and CRT expression by flow cytometry, cells were stained with live-dead marker aqua and 7AAD , AnnexinV-PE (from the same apoptosis detection kit), rabbit anti-mouse CRT antibody and anti-rabbit IgG F(ab\u2019)2 APC-Cy7 secondary antibody . Cell acquisition and analysis were performed using Gallios Flow Cytometer and FlowJo software , respectively. HMGB-1 content in the supernatant of the treated cells was determined by an ELISA according to manufacturer instructions . For in vivo PsV/GCV treatment, deeply anesthetized mice, catheterized as described above, received 0.5% Nonoxynol-9 for 5 min and, after a thorough phosphate-buffered saline rinsing of the bladder, were instilled with 106 TRU of PsV. Two days later, mice received daily intraperitoneal (ip) injections of GCV (75 \u00b5g/g of body weight) for 10 days.PsV-TK, PsV-TK-KO and PsV-luc were produced according to Buck et al. using thd-luciferin in the Xenogen imaging system .After IVES instillation of PsV-luc, luciferase expression was monitored by bioluminescence 15 min after ip injection of b restricted Uty246\u2013254 peptide or medium alone (control wells) for 16\u201324 h. Uty-specific responses were defined as the number of IFN-\u03b3 spots/105 cells in the Uty-stimulated wells minus the number of IFN-\u03b3 spots/105 cells in the control wells (<3 spots/well). T cell staining was performed as previously described [246\u2013254 H-2Db-restricted dextramers and APC-labeled CD8\u03b1 . Flow-cytometer cell acquisition and analysis were performed as described above.Single-cell suspensions from the bladders and spleens of sacrificed mice were obtained as previously described ,40. Brieescribed , using Pt-test or log-rank test as indicated in the figure legends.Statistical analyses were performed using Prism 7.00 for Windows . Multiple comparisons were performed using one-way ANOVA and Tukey\u2019s Multiple Comparison Test, Student The bladder is an ideal organ for evaluating novel in situ therapies, because IVES instillation can maximize exposure to the local tumor, while minimizing systemic exposure and limiting toxicity . Bladder"} +{"text": "Regular exercise reduces the risk of cancer and disease recurrence. Yet the mechanisms behind this protection remain to be elucidated. In this study, tumor-bearing mice randomized to voluntary wheel running showed significant exercise related reduction in tumor incidence and growth across several tumor models including transplantable tumors (Lewis lung and B16 melanoma), chemically (diethylnitrosamine (DEN) induced liver cancer, and a model of spontaneous melanoma (Tg(Grm1)EPv transgenic mice). Microarray analysis revealed exercise-induced up-regulation of pathways associated with immune function, prompting further investigations. NK cell infiltration was significantly increased in tumors from exercising mice, and depletion of NK cells by anti-asialo-GM1 administration increased tumor growth and blunted the exercise-dependent tumor suppression. Mechanistic analyses showed that NK cells were engaged through an epinephrine-dependent mobilization, and blockade of this response by \u00df-adrenergic blockade blunted the exercise-dependent tumor inhibition. Moreover, exercise-induced IL-6 facilitated redistribution of NK cells to peripheral tissues and induced a shift towards more cytotoxic NK cells at the tumor site. Together these results link exercise, epinephrine and IL-6 to NK cell mobilization and activation, and ultimately to improved control of tumor growth."} +{"text": "To prospectively assess the safety and technical feasibility of volumetric magnetic resonance-guided high-intensity focused ultrasound (MR-HIFU) ablation with direct skin cooling (DISC) during treatment of uterine fibroids.In this proof-of-concept study, eight patients were selected for clinical MR-HIFU ablation of uterine fibroids with use of an additional DISC device to maintain a constant temperature (T\u224820\u00b0C) at the interface between the HIFU table-top and the patients\u2019 skin Figure . TechnicResults: All MR-HIFU treatments were successfully completed in an outpatient setting. The median NPV-ratio was 0.56 (IQR[0.27-0.72]). Immediately after treatment, two patients experienced coldness related discomfort which resolved the same day. No serious AEs were reported within 30-days\u2019 follow-up. No skin burns, cold injuries or subcutaneous edema were observed in patients treated with the DISC device. Conclusion: This study showed that it is technically feasible and safe to complete a volumetric MR-HIFU ablation with DISC. This technique may further reduce the risk of thermal injury to the abdominal wall during MR-HIFU ablation of uterine fibroids."} +{"text": "MITF is a frequently amplified lineage-specific oncogene in human melanoma, whose role in intrinsic drug resistance has not been systematically investigated. Utilizing chemical inhibitors for major signaling pathways/cellular processes, we witness MITF as an elicitor of intrinsic drug resistance. To search kinase(s) targets able to bypass MITF-conferred drug resistance, we employed a multi-kinase inhibitor-directed chemical proteomics-based differential affinity screen in human melanocytes carrying ectopic MITF overexpression. A subsequent methodical interrogation informed mitotic Ser/Thr kinase Aurora Kinase A (AURKA) as a crucial regulator of melanoma cell proliferation and migration, independent of the underlying molecular alterations, including TP53 functional status and MITF levels. Crucially, assessing the efficacy of investigational AURKA inhibitor MLN8237, we pre-emptively witness the procurement of a molecular program consistent with acquired drug resistance. This involved induction of multiple MAPK (mitogen-activated protein kinase) signaling pathway components and their downstream proliferation effectors (Cyclin D1 and c-JUN) and apoptotic regulators (MITF and Bcl-2). A concomitant AURKA/BRAF and AURKA/MEK targeting overcame MAPK signaling activation-associated resistance signature in BRAF- and NRAS-mutated melanomas, respectively, and elicited heightened anti-proliferative activity and apoptotic cell death. These findings reveal a previously unreported MAPK signaling-mediated mechanism of immediate resistance to AURKA inhibitors. These findings could bear significant implications for the application and the success of anti-AURKA approaches that have already entered phase-II clinical trials for human melanoma. Even so, a previous report showed stromal HGF-mediated resistance to targeted BRAF inhibitors,11 and a recent study reported the role of stroma-mediated immediate resistance to BRAF inhibition,12 the general understanding of the mechanisms of intrinsic resistance has remained quite limited.Detailed molecular investigation of human melanoma has unearthed two key oncogenic driver alterations BRAF(V600E) in ~40% and NRAS (G12D) in ~15% of melanomas.14 MITF has been described as a lineage-specific oncogene in melanoma, which in collaboration with constitutively active mutated BRAF capably transforms melanocytes.14 MITF carries a diverse functionality and has been shown to influence a wide range of cellular phenotypes, including proliferation, apoptosis, migration and differentiation.15 This functional diversity has in-turn been ascribed to different MITF expression levels.15 Although some investigative studies in melanoma cells have also suggested a role for MITF in both intrinsic and acquired resistance to general as well as targeted therapeutics, including MAPK signaling inhibitors,17 a systematic interrogation of this MITF functionality has largely gone unexplored.Microphthalmia-associated transcription factor (MITF) is a basic helix\u2013loop\u2013helix transcription factor that has critical role in melanocytic development and melanomagenesis.In the current study, we systematically investigated the role of MITF in intrinsic drug resistance, followed by development of therapeutic strategies that thwart/bypass the liaison between BRAF(V600E) and MITF.14 (19 introduction of constitutively active BRAF(V600E), while triggering an induction of c-JUN and Cyclin D1 expression, markedly downregulated the expression of endogenous MITF and its target Bcl-2.20 However, ectopic expression of MITF could restore MITF levels and partially rescue its target (Bcl-2) expression activities is essential.To explicitly understand the role of MITF in intrinsic drug resistance, we tested immortalized HMEL cells (Pmel/hTERT/CDK4(R24C)/p53DD), ectopically expressing BRAF(V600E) (referred to as HMEL-B) or BRAF(V600E)+MITF (referred to as HMEL-B/M)14 for thei14 . In linepression . The chohibitors . In conthibitors . With MInaling21 , the heiBRAF and MITF amplification (MITFAmp),14 we developed a multi-step integrative target identification approach\u2014drug-effected integrative identification of target(s) (DEFINIT). This approach, relying strongly on functional assessment, also incorporated the salient feature of \u2018gene expression-disease stage correlation' for therapeutic intervention in a significant proportion of melanomas that harbor dual mut-elation' . With th/M cells . Interes/M cells . These o/M cells . To incr/M cells . This yi/M cells .18 identified AURKA, GSK3A and GSK3B as the shared high-affinity kinases . GSK3A inhibition failed to suppress the colony-forming potential in both HMEL-B/M and HMEL-B cells LC\u2013MS) kinases . As GSK3 kinases , we next-B cells . In cont-B cells . Further-B cells . Corrobo-B cells .25 for AURKA transcript levels. AURKA expression levels showed highly significant increase with melanoma progression from nevi (n=9) to primary (n=31) and from primary to metastatic (n=52) stage 2) stage .To further understand the role of AURKA in melanoma cell biology, we investigated its requirement in cell viability, utilizing a large panel of melanoma cell lines that encompassed the entire gamut of major melanoma-associated molecular alterations . AURKA i26 abrogation of AURKA function triggered a massive accumulation of melanoma cells in G2/M phase and dominant-negative TP53 harboring HMEL-B/M cells excluded the potential requirement of a functional TP53. To conclusively evaluate TP53 requirement in melanoma cells that harbored wt-TP53, we adopted a twofold approach, (1) si-TP53-mediated rescue of AURKA inhibition-associated TP53 and p21Cip1 induction -positive melanomas very frequently develop resistance to targeted BRAF signaling inhibitors, we additionally tested the potential benefit of AURKA-i in melanoma cell lines (451Lu_BR and WM983B_BR) that had developed resistance to BRAF inhibitor PLX-4032. Remarkably, in both the tested resistant cell lines, AURKA inhibition triggered extensive apoptotic cell death tivity27 . Althougll death . Importall death . Lastly,ls in 3D . Further57 cells .TBX2, TRP1 (TYRP1), MLANA and TYR. Providing credence to the transcriptional integrity of upregulated MITF, we observed induction of all the tested MITF transcriptional targets (CDKN1A (p21Cip1) and CDKN1B (p27Kip1)29 and Bcl-2, instead, argued for a MITF-mediated potential resistance program9 29 further program9 . To concactivity . These enduction . Howevernduction , togethema cells excluded21 Additionally, MAPK signaling counterbalances this kinase function-effected MITF degradation through the latter's BRN2-mediated transcription.21 To test whether a MAPK signaling-effected transcription program could potentially explain MITF induction in response to AURKA inhibition, we first analyzed the expression and/or activity changes of key MAPK signaling components in a panel of human melanoma cell lines. AURKA inhibition induced both the expression and activity of ERK -CREB transcriptionally regulates MITF.32 Therefore, our results, in the light of above reports point to CREB as a mediator of MITF upregulation downstream of AURKA inhibition-mediated MAPK signaling induction. Consistently, in contrast to the observations with BRN2, AURKA inhibition induced p(Ser133)-CREB levels in all the tested melanoma cell lines (In response to changes in the cellular cyclic adenosine mono-phosphate (cAMP) levels, followed by protein kinase A (PKA)-mediated phosphorylation, cAMP-response element-binding protein (CREB) has also been shown to induce MITF expression.ll lines .Taken together, these data pre-emptively reveal a potentially significant mechanism of MAPK signaling-mediated early acquired resistance to AURKA inhibition, which involves an upregulation of pERK/ERK and the associated downstream pro-proliferative and anti-apoptotic molecular signature .The robust build-up of MAPK signaling activity and the associated downstream proliferative/pro-survival molecular manifestations alluded Cumulatively, based on these findings, we propose a model detailinMITF14 to identify combinatorial MAPK signaling and AURKA inhibition as more effective therapeutic approach in melanoma. This multi-step target identification is inspired by similar yet distinct target identification approaches.34The target identification approach \u2018DEFINIT', developed and utilized in the current study appreciated the destructive liaison between the oncogenic driver BRAF(V600E) and often concomitantly amplified 35 bears significant implications towards the understanding of molecular mechanisms failing TP53 tumor suppressor activity in this malignancy. Although a recent report suggested the inability of AURKA inhibition to fully switch-on TP53-associated transcriptional function and induce apoptosis,36 our studies with multiple wt-TP53 melanoma cell lines suggest AURKA-i fully capable of inducing TP53, its transcription activity and apoptotic cell death. These data are in conformity with previous reports showing induction of TP53-associated transcriptional activity and apoptotic cell death upon AURKA inhibition.37 Interestingly, one of the wt-TP53 cell lines (Sk-Mel5), also studied by Vilgelm et al.36 showed only subtle increase in TP53 and its target p21Cip1 levels in our hands. However, remaining seven cell lines investigated in the current study showed clear induction in both TP53 and p21Cip1 levels. This underscores the necessity of utilizing multiple cancer cell lines to gain general mechanistic understanding. Furthermore, our data showing AURKA-i-mediated p21Cip1 induction and an anti-proliferative activity even in the mut-TP53 cells, while bearing important mechanistic and therapeutic significance could provide basis for observed TP53 dispensability in AURKA-i-mediated apoptosis. Although p73-mediated p21Cip1 regulation has previously been proposed in mut-TP53 cells,38 additional studies would be required to gain a detailed understanding.Identification of AURKA as a highly overexpressed gene in late-stage melanoma that overcomes the surveillance function of TP53 by orchestrating its proteasome-mediateddegradationAURKA transcript levels, querying the provisional TCGA data consisting of 477 cases of cutaneous melanoma returned only six samples with an amplification of the corresponding (20q) locus. Furthermore, analysis of our own array comparative genome hybridization (aCGH) data (GEO accession no. GSE7606)39 showed merely ~1.6% and ~0.8% of primary and metastatic samples, respectively, with an accompanying copy number gain. Interestingly, a recent report suggested MAPK signaling-mediated transcriptional induction of Aurora kinase family member Aurora B.40 Although this finding together with a highly prevalent melanoma-associated MAPK signaling hyperactivity would argue for a similar mechanism of AURKA regulation, the early procurement of BRAF(V600E) in the evolution of melanoma cells, together with a correspondingly low AURKA levels in melanocytic nevi , by orchestrating the phosphorylation of MITF, promotes its proteasomal degradation.21 On the other hand, MAPK signaling, through the regulation of BRN2 and CREB activity is also required for maintaining MITF levels.32 Our results showing AURKA-i-mediated activation of MAPK signaling, induction of p-CREB levels and the consequential upregulation of MITF expression, suggests the latter signaling mechanism as the predominant player in the setting of AURKA inhibition. AURKA inhibitor MLN8237 has already entered phase III clinical trials for different cancers and phase-II trials in melanoma patients. MAPK cascade induction in human melanoma cells could therefore jeopardize the success of AURKA interference approaches. Therefore, our studies, pre-emptively demonstrating the ability of a concomitant MAPK pathway inhibition to relieve AURKA-i-initiated MAPK signaling-mediated resistance program, recommend a highly promising combination regimen. Although a recent study has also noted the beneficial effects of AURKA and BRAF inhibitor combination,45 the mechanistic rationale for this combination, as elucidated in our studies, had been lacking in this previous work.Detailed reports witnessing the reactivation of PKA-CREB-MITF axis in melanoma patients relapsing from vemurafenib,Notably, whereas BRAF inhibitors have been shown to induce only a change in the activity of the MAPK signaling components, AURKA inhibition triggers both expression and activity changes in MAPK signaling proteins.To conclude, this study, whilst potentially bearing fundamental implications for the application of AURKA inhibitors as melanoma therapeutics, describes a common framework for future target discovery, whereby the functional significance of cooperating oncogenic events is addressed at the outset.14 All utilized small molecule inhibitors are summarized in All melanoma cell lines were culCip1 (H-164), Bcl-2 (100), Raf-1 (C-12), Raf-B (C-19), APAF-1, Phospho-(Ser133)-CREB-1, BRN2 (C20) and GAPDH (FL-335) from Santa Cruz Biotechnology . c-Jun (60A8), Phospho-ERK1/2 (Thr202/Tyr204), ERK1/2 from Cell Signaling Technology . MITF(C5) from Abcam . Cyclin D1 from BD Biosciences . \u03b1-Tubulin antibody from Calbiochem .Phospho-(Ser218/Ser222)-MEK1/2, MEK1 (C-18), p53 (TP53) (DO-1), p2114 were targeted with multi-kinase inhibitors midostaurin and sunitinib, and tested for anchorage-independent growth. In view of the common spectrum of the kinase targets of the inhibitors, the differential effect of the drugs against MITF-expressing HMEL-B/M cells was exploited for further analysis, utilizing drug pull-down-based proteomics.18 Using recently published 1D-LC\u2013MS and iTRAQ labeling combined with gel-free 2D-LC-MS data,18 the kinases with differential affinity for midostaurin were identified. This was followed by functional screening of the identified kinases in soft agar colony formation assay and finally gene expression-disease stage correlation, utilizing previously published gene expression data from melanocytic nevi, primary and metastatic melanoma samples (GEO accession no. GSE8401).25Previously described immortalized human melanocytes (HMEL-B and HMEL-B/M) with isogenic background25 The data have been deposited in the National Center for Biotechnology Information GEO46 and are accessible through GEO Series accession no. GSE8401.Tissue sampling and gene expression profiling were previously performed using Affymetrix U133A microarray platform as described earlier.41 Relative viability was calculated as viability change relative to solvent-treated cells; viability of solvent-treated cells was set to zero. Resistivity index was calculated using the formula ((relative viability of HMEL-B)/(relative viability of HMEL-B/M)); greater the value of resistivity index, higher the resistance of HMEL-B/M cells to the specified treatment in comparison to HMEL-B cells.MTT assay was performed as described previously.TP53-siRNA (sc-416469-NIC-2), MITF-siRNA (110566) and negative control siRNA (AM4636) were performed employing Lipofectamine 2000 transfection reagent (Invitrogen) as per manufacturer's instructions.siRNA transfections using 41 Hypodiploid (necrotic/apoptotic) (Sub-G1 phase), diploid (G1/G0 phase), hyperdiploid (S phase) and tetraploid (G2/M) cell populations were quantified using CellQuest software (BD Biosciences).Cell-Cycle analysis was performed as previously described.41Annexin V/PI-based apoptosis detection and quantification was performed as previously described.47 The cultures were performed either with 40\u2009nM MLN8237 or DMSO control. After 7-day incubation, colonies were stained with AlamarBlue (Invitrogen) according to the manufacturer's instructions. Calorimetric readout was performed using a multi-well plate reader at 570 and 600\u2009nm reference wavelength.Short-term anchorage-independent growth assay was assessed in triplicates in a fluorescence-based 1 week assay as previously described.33 Briefly, 4x104 cells were seeded as above for 4 weeks. The medium was weekly replenished with the drugs being investigated. Images were acquired with Alpha Imager .Long-term anchorage-independent growth assay was carried out in 24-well plate format as previously described with some modifications.33 The cultures contained either 30\u2009nM MLN8237 or DMSO as control. Following incubation for 20\u2009h, cells on the bottom side of the insert membranes were fixed and stained using Kwik-Diff Staining kit according to the manufacturer's instructions. Migrated cells were quantified by counting of six randomly selected microscopic fields.Transwell migration assays were performed as previously described.41\u03b1-Tubulin or GAPDH staining was used as a control for equal sample loading.Western Blotting was performed as previously described.\u03bcg per sample was subjected to reverse transcription, using Superscript II Reverse Transcriptase (Invitrogen). TaqMan gene expression assays for TBX2, TRP1, MLANA, TYR, CDKN1A, CDKN1B, CCND1, MITF and ACTIN-B1 . A StepOne Plus qRT-PCR System was used for amplification and detection. Reverse transcription-negative controls were always included. For relative quantification of gene expression, the 2\u2212\u0394\u0394CT method was used.Total cellular RNA was extracted using TRI Reagent according to the manufacturer's protocol, and 1\u2009http://www.graphpad.com) was used to perform statistical analysis by performing unpaired t-test.Graphpad prism software 5.0 (Graphpad, La Jolla, CA, USA;"} +{"text": "To prospectively evaluate the predictive value of pre-treatment histogram analysis of apparent diffusion coefficients (ADC) at whole body diffusion-weighted imaging (WB-DWI/MRI) for patient outcome in primary ovarian cancers.2), T2-weighted and contrast-enhanced T1-weighted sequences prior to treatment. The primary tumour was delineated using semi-automated software and was analysed by using an ADC histogram approach: mean and median ADC, standard deviation (SD), coefficient of variation , kurtosis and skewness were calculated. Kaplan-Meier with log-rank statistics were used to correlate baseline ADC parameters to disease free survival (DFS). Effects of confounding patients- and tumour-related factors were taken into consideration using Cox proportional hazard model.Institutional review board approval and informed consent were obtained for this prospective study. Forty-four women diagnosed with FIGO stage III or IV ovarian carcinoma underwent 3-Tesla WB-DWI/MRI using 2 b-values . After multivariable analysis, CoV remained an independent prognostic biomarker for DFS (p=0.003) when taking patient\u2019s age, FIGO stage, tumour grade and cancer antigen (CA)-125 level into consideration as clinical prognostic factors.In this pilot study, pre-treatment ADC histogram analysis of primary ovarian cancer using the CoV was an independent predictive marker of DFS suggesting a correlation between tumour heterogeneity and treatment resistance. Further research should elucidate the correlation with overall survival."} +{"text": "Lung transplantation (LTx) is still limited by organ shortage. To expand the donor pool, lung retrieval from non-heart-beating donors (NHBD) was introduced into clinical practice recently. However, primary graft dysfunction with inactivation of endogenous surfactant due to ischemia/reperfusion-injury is a major cause of early mortality. Furthermore, donor-derived human mesenchymal stem cell (hMSC) expansion and fibrotic differentiation in the allograft results in bronchiolitis obliterans syndrome (BOS), a leading cause of post-LTx long-term mortality. Therefore, pretreatment of NHBD with recipient-specific bone-marrow-(BM)-derived hMSC might have the potential to both improve the postischemic allograft function and influence the long-term development of BOS by the numerous paracrine, immunomodulating and tissue-remodeling properties especially on type-II-pneumocytes of hMSC.Asystolic pigs (n = 5/group) were ventilated for 3 h of warm ischemia (groups 2\u20134). 50x106 mesenchymal-stem-cells (MSC) were administered in the pulmonary artery (group 3) or nebulized endobronchially (group 4) before lung preservation. Following left-lung-transplantation, grafts were reperfused, pulmonary-vascular-resistance (PVR), oxygenation and dynamic-lung-compliance (DLC) were monitored and compared to control-lungs (group 2) and sham-controls (group 1). To prove and localize hMSC in the lung, cryosections were counter-stained. Intra-alveolar edema was determined stereologically. Statistics comprised ANOVA with repeated measurements.Oxygenation (p = 0.001) and PVR (p = 0.009) following endovascular application of hMSC were significantly inferior compared to Sham controls, whereas DLC was significantly higher in endobronchially pretreated lungs (p = 0.045) with overall sham-comparable outcome regarding oxygenation and PVR. Stereology revealed low intrapulmonary edema in all groups (p > 0.05). In cryosections of both unreperfused and reperfused grafts, hMSC were localized in vessels of alveolar septa (endovascular application) and alveolar lumen , respectively.Preischemic deposition of hMSC in donor lungs is feasible and effective, and endobronchial application is associated with significantly better DLC as compared to sham controls. In contrast, transvascular hMSC delivery results in inferior oxygenation and PVR. In the long term perspective, due to immunomodulatory, paracrine and tissue-remodeling effects on epithelial and endothelial restitution, an endobronchial NHBD allograft-pretreatment with autologous mesenchymal-stem-cells to attenuate limiting bronchiolitis-obliterans-syndrome in the long-term perspective might be promising in clinical lung transplantation. Subsequent work with chronic experiments is initiated to further elucidate this important field.The online version of this article (doi:10.1186/s13019-014-0151-3) contains supplementary material, which is available to authorized users. Although lung transplantation has been proven to be an effective standard therapy for patients with different end-stage pulmonary diseases, significant scarcity of suitable donor organs still li6 cells per cm2 in T75 cell culture flasks (BD) in specialized hMSC media supplemented with penicillin/streptomycin . Cells were grown at 37\u00b0C and 5% CO2 in a humidified incubator and passaged when confluent. To generate cell numbers sufficient for large animal cell transplantations, 1-5x106 hMSC were seeded into multilayered cell culture vessels and cultured for 14 days. One day before harvesting, hMSC were labeled with fluorescent, paramagnetic microbeads by incubation overnight. For harvesting, cells were washed three times with PBS (Life Technologies) and detached with 0.05% trypsin (Life Technologies). After assessing total cell number using a hemacytometer the cells were labeled with the fluorescent vital stain DiI (Life Technologies) according to the manufacturer\u2019s instructions and then resuspended in PBS at a concentration of 5 million cells per ml. The total number of hMSC for transplantation was 5x107 (2.27-2.5 million hMSC per kilogram of body weight) according to current evidence in the literature . Fu. Fu37]. 6 per animal and and6 perr animal , has stiThe major cause of long-term morbidity and mortality in lung transplant recipients is the bronchiolitis obliterans syndrome (BOS) which is a form of chronic rejection characterized by an irreversible airway obstruction that affects about 50% of recipients surviving 5 and more years ,51]. In. In51]. Recently, a novel form of chronic lung allograft dysfunction (CLAD) was described by Sato et al. ,55]. In. In55]. A specific donor-lung cell therapy with effective pre-ischemic intrapulmonary hMSC transplantation might be a promising therapy to positively influence the onset and/or the extent of both the deleterious bronchiolitis obliterans (BOS) and restrictive allograft syndrome (RAS). Most importantly, such a hMSC-based therapy has a significant safety advantage as no adverse differentiation of transplanted MSC has been observed, as opposed to potential teratoma formation from pluripotent cells and their derivatives ,60]..60].Consequently, according to a well recognized review by Moodley et al. , studies"} +{"text": "Hypertension with left ventricular hypertrophy is a major cause of diastolic heart failure (DHF). Due to its high prevalence and high rate of mortality, DHF represents a major challenge in today's cardiovascular medicine; with limited therapeutic options. Soluble guanylate cyclase (sGC) stimulation is emerging as a promising treatment option in DHF, and is currently under investigation in preclinical and clinical studies. The present study investigates the effect of the sGC stimulator BAY 41-8543 in a transgenic rat model of hypertension-induced heart failure.We used 4 week-old male double transgenic rats expression both human renin and angiotensinogen genes (dTGRs). At 7 weeks of age, dTGRs exhibit striking cardiac hypertrophy with fibrosis and inflammation, ventricular arrhythmias and heart failure, which is accompanied with high mortality. We compared vehicle-treated dTGR to those receiving 3 mg/kg/d BAY 41-8543, and vehicle-treated SD control rats . We performed in vivo echocardiography, hemodynamic monitoring, cardiac electrophysiology studies and blood pressure measurements. Endothelial function was measured in isolated mesenteric arteries. Transcriptional analyses in cardiac tissue were performed using qRT-PCR and gene-microarray. Cardiac tissue was analyzed using histology.Treatment of dTGRs with BAY 41-8543 resulted in 100% survival at week 7, whereas only 24% of vehicle-treated dTGRs survived. Mean arterial pressure in dTGRs was significantly by BAY 41-8543 reduced (197 \u00b1 11 mmHg vehicle vs 133 \u00b1 4 mmHg BAY 41-8543). In addition, BAY 41-8543 significantly decreased in vivo total peripheral resistance and improved endothelium-dependent vasorelaxation of isolated mesenteric arteries. Furthermore BAY 41-8543 prevented fibrosis and inflammation of cardiac tissue. Echocardiography and invasive hemodynamic monitoring revealed BAY 41-8543 significantly increased ejection fraction and cardiac output in dTGR, whereas vehicle-treated had preserved systolic function but reduced diastolic function. In addition, diastolic compliance was significantly enhanced by BAY 41-8543, as shown by myocardial strain analysis and end-diastolic pressure volume relationship (EDPVR); indicative of an improved diastolic function. In vivo programmed electrical stimulation revealed a high ventricular tachycardia induction rate in vehicle-treated dTGRs (46%), which was significantly reduced in BAY 41-8543-treated dTGR (11%). Myocardial gene-microarray analysis showed a reversal of dysregulated genes in dTGR by BAY 41-8543 treatment.Our data demonstrate that BAY 41-8543 improves survival and cardiac performance in a transgenic rat model of hypertension-induced DHF. We postulate that treatment of DHF with sGC stimulators offers a novel therapeutic potential for humans."} +{"text": "We characterized bone marrow stromal cells (BMSC) from patients with pre-fibrotic myeloproliferative neoplasms (MPN). MPN-BMSC showed decreased capacity to stimulate the proliferation of colony-forming units of normal hematopoietic stem and progenitor cells and displayed increased matrix remodelling (in particular fibronectin deposition) compared to control BMSC. This finding was confirmed in pre-fibrotic MPN bone marrow biopsies in a tissue microarray (n\u2009=\u200934), which stained positive for fibronectin in the absence of reticulin as a standard myelofibrosis marker. Fibronectin expression correlated significantly with reduced haemoglobin levels in MPN-patients . Our data show significant cell-intrinsic alterations in MPN-MSC and suggest that Fibronectin expression might be applicable as a biomarker for the identification of early myelofibrotic transformation in reticulin-negative MPN. We applied a three-dimensional collagen culture-system as an patients ,2. The a+-hematopoietic stem and progenitor cells (HSPCs) [To characterize the hematopoiesis-supporting capacity of MSC, secreted cytokines and their biological activity were tested. ET-MSC secreted significantly lower levels of G-CSF and IL-7 compared to controls, indicating a defect in the hematopoiesis-supporting capacity Figure\u00a0c. We the (HSPCs) . Myeloid+ MSC significantly differed in MPN biopsies compared to the control bone marrow in the absence of the hematopoietic clone ,2. MSC iTo validate these findings, we systematically analyzed CD271 and fibronectin in tissue microarrays including ET (n\u2009=\u200913), PV (n\u2009=\u200911), CML (n\u2009=\u200914), PMF (n\u2009=\u200911) and non-MPN controls (n\u2009=\u200917), neoplasms.In conclusion, our data reveal an intrinsic defect of MSC in pre-fibrotic MPN resulting in decreased hematopoiesis-supporting capacities and increased ECM remodelling. Our data suggest that fibronectin up-regulation/distribution detects early myelofibrotic changes in the BM of MPN patients and implies clinical and prognostic application in different myeloproliferative (Ph"} +{"text": "An 80-year-old male presented with an obstructive lower urinary tract and intermittent low back pain lasting approximately 6 months. Digital rectal examination (DRE) revealed asymmetric prostate enlargement. Laboratory examination revealed elevated PSA, CRP, and sedimentation. Multiparametric magnetic resonance imaging (mp-MRI) and lumbar MRI were performed due to suspicion of prostate cancer and bone metastasis. On T2-weighted images (T2-WI), a low-signal intensity lesion with unclear borders was observed in the left anterolateral peripheral zone A. The deIsolated tuberculous prostatitis and concomitant Pott's abscess are extremely rare entity"} +{"text": "Their expression in tumour cells is modulated by a complex interplay of genomic, transcriptomic and post translational factors involving multiple intracellular antigen processing pathways. Ongoing research investigates mechanisms invoked by cancer cells to abrogate MHC-I expression and attenuate anti-tumour CD8+ cytotoxic T cell response. The discovery of MHC-II on tumour cells has been less characterized. However, this finding has triggered further interest in utilising tumour-specific MHC-II to harness sustained anti-tumour immunity through the activation of CD4+ T helper cells. Tumour-specific expression of MHC-I and MHC-II has been associated with improved patient survival in most clinical studies. Thus, their reactivation represents an attractive way to unleash anti-tumour immunity. This review provides a comprehensive overview of physiologically conserved or novel mechanisms utilised by tumour cells to reduce MHC-I or MHC-II expression. It outlines current approaches employed at the preclinical and clinical trial interface towards reversing these processes in order to improve response to immunotherapy and survival outcomes for patients with cancer.Evading immune destruction is one of the hallmarks of cancer. A key mechanism of immune evasion deployed by tumour cells is to reduce neoantigen presentation through down-regulation of the antigen presentation machinery. MHC-I and MHC-II proteins are key components of the antigen presentation machinery responsible for neoantigen presentation to CD8 The advent of immunotherapeutics has revolutionised treatment in cancer. These agents harness our immune system to promote anti-tumour responses and herald the potential for long-term survival in patients with otherwise incurable disease . SpecifiHLA) genes [+ T cells. Immunogenic foreign peptides, such as neoantigens, are recognised by T cell receptors (TCRs) on CD8+ T cells resulting in cell killing.MHC I molecules encoded by human leukocyte antigen (A) genes are presA) genes . They pl+ T cells.The processing of neoantigens is mediated by the ubiquitin-proteasome system . ProteasHLA or B2M loss of heterozygosity (LOH) [HLA or B2M wild-type tumour cells, highlighting the role of non-genetic mechanisms in regulating MHC-I expression [Immune evasion is a hallmark of cancer to reduce visibility of tumour cells to the immune system. Tumour ty (LOH) . Howeverpression ,13\u201315 on chromosome 6 [HLA alleles results in total elimination of MHC-I expression [HLA LOH), reduces MHC-I expression by half. Tumours leverage the genomic instability associated with LOH, whereby a further mutation in the other allele results in complete MHC-I loss, to evade immune recognition [HLA LOH to be 17% [HLA LOH has been shown to be both a negative prognostic and predictive biomarker for ICI therapy [The genes encoding MHC-I are composed of the highly polymorphic class Ia \u2018classical\u2019 human leukocyte antigen genes and melanoma cell lines, and B2M in human melanoma tumours [Somatic mutations in entation . Whole-eentation . HLA mutentation . These f tumours .HLA alleles, B2M and other APM regulatory genes have been described in solid tumours [HLA genes [ERV) genes that trigger cytosolic sensing of dsRNA (double stranded RNA) [ERVs are a relic of ancient infections that comprise 8% of the human genome [Hypermethylation of gene promoters and enhancers of tumours . In brea tumours . This inLA genes , but alsded RNA) . ERVs arn genome . These gn genome ,28. Thisin vitro whole genome CRISPR/Cas9 screen in leukemia cell lines revealed EZH2 (enhancer of zest homolog 2) as a negative regulator of MHC-I, the master regulator of MHC-I transcription NRLC5 (nucleotide-binding domain and leucine-rich repeat caspase recruitment domain-containing 5), and TAP expression [Y641EZH2 mutation, treatment with an EZH2 inhibitor reduced H3K27me3 in the promoter region of NLRC5, resulting in increased NLRC5 and MHC-I expression [in vivo mouse model of head and neck cancer [Histone modifications by trimethylation of lysine residues on histone 3 (H3K27me3) or deacetylation result in gene silencing and have been shown as mechanisms invoked by tumour cells to silence the APM ,32. An ipression . EZH2 capression . EZH2 ink cancer . Histonek cancer , either k cancer .HLA genes [TAP1 and B2M. In vivo deletion of NLRC5 resulted in loss of MHC-I expression in mice, without altering MHC-II expression, demonstrating the critical role of NLRC5 in specifically controlling MHC-I expression. IFN\u03b3 is a key regulator of MHC-I expression through JAK1/2 (janus kinase 1/2)-STAT1 signalling and activation of NLRC5 expression. This transduction pathway also activates IRF1/2 (interferon regulatory factor 1/2) expression that binds the ISRE (interferon-stimulated response element) present in the proximal promoter of HLA genes. Alterations of these key transcription factors through genetic deletions or epigenetic modification results in loss of MHC-I expression. NLRC5 loss has been described in several solid organ cancers [+ T cell mediated cytotoxic responses, thus conferring inferior patient survival [IRF1/2, particularly in melanoma patients with ICI resistance [JAK1 or JAK2 has also been associated with reduced MHC expression and resistance to ICIs [Transcriptomic regulation of MHC-I is tightly controlled to elicit an appropriate immune response. The transcriptional transactivator NLRC5 is a critical regulator of MHC I expression. It forms a scaffold with DNA binding proteins RFX (regulatory factor X), CREB (cAMP responsive element binding protein 1), ATF1 (activating transcription factor 1) and NF-Y (nuclear factor Y) to form the CITA complex (class I transactivator) on the proximal promoter of LA genes . These r cancers ,40. Its survival . Similarsistance . Defects to ICIs ,43. DUX4 to ICIs . DUX4 ov to ICIs .HLA-A and B [Defects in IFN\u03b3 signalling is an important mechanism of primary and acquired resistance to ICIs . Targeti-A and B . NF\u03baB is-A and B ,52. MHC--A and B . Multiplin vitro shRNA screen targeting 526 kinases identified the MAP kinase (MAPK) pathway, including downstream kinases MEK and ERK, as negative regulators of HLA-A expression [Aberrant activation of cell signalling pathways through oncogenic drivers can down-regulate MHC-I. An pression . Tumour pression ,54.The MYC family of proteins regulates transcription of \u223c15% of the human genome . Over-exmir-148a-3p and mir-125a-5p were found in the 3\u2032 untranslated region of HLA-A, -B, -C mRNA and TAP2 mRNA respectively. Overexpression of mir-148a-3p reduced cell surface expression of MHC-I in colorectal and oesophageal cancer [mir-148a-3p restored MHC-I expression and increased T-cell mediated killing in vitro and in vivo [miRNAs are a class of non-coding RNAs that are characterised by their short length (\u223c21\u201325\u2005bp). They can bind to the 3\u2032 untranslated region (UTR) of mRNA and inhibit their translation through mRNA degradation or translational repression. Binding sites for l cancer ,60, whil in vivo .LINK-A was recently found to be a negative regulator of MHC-I and B2M cell surface expression in triple negative breast cancer cells (TNBC), and a negative predictive biomarker in patients treated with ICIs [LINK-A was shown to abrogate phosphorylation of the E3 ubiquitin ligase TRIM71, resulting in increased degradation of the MHC-I peptide loading complex. Other lncRNAs have been associated with positive regulation of MHC-I expression, such as LINC02195 that positively correlated with MHC-I-related protein expression in head and neck squamous cell carcinomas cell lines and patient samples [Like miRNAs, lncRNAs are not translated into proteins. lncRNAs are >200\u2005bp long that predominantly reside in nuclei. They are responsible for diverse processes that result in transcriptional and post-transcriptional regulation of gene expression. The oncogenic lncRNA ith ICIs . LINK-A samples .in vivo models of melanoma and colorectal cancer, resulting in decreased tumour burden [Cancer cells may also evade immune recognition through post translational modification of MHC-I. ER-associated protein degradation (ERAD) constitutes a quality control system to eliminate misfolded or unassembled proteins from the ER . Tumour r burden .MAL2 in patient-derived tumour organoid models resulted in enhanced CD8+ T cell-mediated cytotoxicity, thus making MAL2 a potential therapeutic target.Increased turnover of the antigen loaded MHC-I is another mechanism by which tumour cells evade immune surveillance. Expression of the transmembrane protein MAL2 (myelin and lymphocyte protein 2) is associated with worst prognosis in TNBC cells . Molecul+ T cells. Specifically, high cell surface expression of glycosphingolipids by tumour cells impedes MHC-I and CD8+ T cell interaction [+ T cells. There is considerable interest in inhibiting glycosphingolipids synthesis using clinically approved inhibitors which have demonstrated in vitro efficacy in glioma cell lines [Tumours may also modify their cell membranes to sterically inhibit MHC-I interactions with CD8eraction . Membraneraction . Reducedll lines .in vitro co-culture assay with cytotoxic T cells and enhanced CD8+ T cell infiltration in vivo [Autophagy has been proposed as another mechanism utilised by tumour cells to reduce cell surface expression of MHC-I and avoid immune recognition ,69. Immu in vivo ,69. Thes in vivo .+ T cell responses. However, there is increasing interest in harnessing CD4+ T helper cells to potentiate sustained anti-tumour immunity [+ T cells are activated by MHC-II-bound peptides. MHC-II molecules present exogenously derived peptides and have traditionally been associated with professional antigen presenting cells (APCs) such as dendritic cells, macrophages and B cells [+ T cells [+ T helper cell differentiation is induced by the binding of a na\u00efve CD4+ TCR to an MHC-II peptide complex combined with a second co-stimulatory signal where CD28 on CD4+ T cells binds to CD80/86 found on professional APCs. These T helper cells promote CD8+ T cell mediated responses and immunological memory [+ T cells. Examples of these co-stimulatory molecules include OX40 and CD70, both found in solid cancers [Immuno-oncology research thus far has predominantly focussed on augmenting cytotoxic CD8immunity ,72. CD4+ B cells . While t T cells . CD4+ T l memory ,74. Tumol memory . However cancers ,77.+ T cell-mediated immunosurveillance may be a dominant mechanism for immune control of tumours.The presence of tsMHC-II is associated with increased CD4/CD8 tumour infiltrating lymphocytes, improved survival and responsiveness to ICIs . AnalysiBoth MHC-I and MHC-II genes are highly polymorphic. However, MHC-II can bind a greater diversity of neoantigenic proteins. Their binding pocket can allow peptides of a longer length (>13 amino acid) and accommodates peptide side chains. The regulation of antigen processing and presentation by MHC-II in professional APCs has been reviewed elsewhere . Here weLittle is known on the mechanisms driving the regulation of tsMHC-II. However, some studies are starting to emerge elucidating immune-evasion mechanisms associated with MHC-II complex down-regulation .pI), II (pII), III (pIII), IV (pIV) [pI and pIII. The strongest inducer of CIITA in response to IFN\u03b3 stimulation is pIV [Expression of MHC-II is controlled by the transcriptional master regulator class II transactivator (CIITA) . The CIIIV (pIV) . Constitn is pIV . ModulatCIITA gene, including point mutations and gene fusions have been observed in different types of lymphoid tumours [CIITA or its promoter complex have also been observed in melanoma and microsatellite unstable (MSI-H) colorectal cancer (CRC)[RFX5 gene are also a common event in MSI-H CRC, occurring in approximately a quarter of cases [Genomic alterations in the tumours ,88. Poincer (CRC),90. Framof cases . These aCIITA-promoter sites or directly affecting MHC-II genes. Hypermethylation of CIITA-pIV has been demonstrated in gastric cancer [HLA-DR and HLA-DQ genes and absence of tsMHC-II expression have been associated with inferior survival in patients with oesophageal squamous cell carcinoma [DNA hypermethylation has been described at c cancer . Hypermearcinoma . DNA metarcinoma ,92.HLA-DRA promoter compared with cell lines lacking MHC-II [CIITA or HLA-DRA promoters has been observed in vitro in several solid organ and haematological malignancies, abrogating MHC-II expression [Histone acetylation promotes transcription of MHC-II related genes. B cell lymphoma cells with MHC-II expression were characterised by H3 and H4 acetylation at the g MHC-II . This prg MHC-II . In B ceg MHC-II ,96. Histpression ,97,98. Ppression .Y641EZH2 mutation had low expression of MHC-I and II [CIITA promoter. Importantly this work not only provides a rationale for targeting EZH2 in combination with ICIs, but also identifies EZH2 mutation as a biomarker to stratify patients who may respond to this combination therapy.Histone methylation driven by EZH2 has also been shown to regulate MHC-II expression in DLBCL where tumours with I and II . TreatmeCIITA-pIV promoter [IRF2 loss is described in several cancers and associated with attenuation of MHC-I and MHC-II expression [Loss of interferon signalling can reduce MHC-II transcription. IRF2 has been shown to be a transcriptional activator of the promoter . IRF2 lopression ,101. Actpression . This efCIITA-pIV region, thus preventing transcription. The oncogenes L-MYC and N-MYC have been shown to bind this region in SCLC cell lines, resulting in loss of CIITA transcription [CIITA transcription and is associated with plasma cell differentiation in myeloma [CIITA can also be inhibited by factors that competitively bind to E-box elements in the cription . Over-excription . CIITA ecription ,106. Con myeloma ,108.The MHC-II complex is a heterodimer assembled in the ER with the chaperone protein CD74, also known as the invariant chain, to prevent loading of endogenous peptides. The MHC-II/CD74 complex is transported from the ER and fuses with acidic endosomes where exogenous peptide loading occurs. Cleavage of CD74 leaves the short fragment CLIP (class II-associated invariant peptide) blocking the peptide binding groove of MHC-II . The chaRegulation of the APM in cancer is a critical mechanism that governs the anti-tumour immune response, ultimately determining survival outcomes for patients with cancer. Our review highlights the mechanisms of MHC-I/MHC-II regulation in tumour cells. Considerable studies have been undertaken to elucidate resistance mechanisms contributing to reduced immune visibility, particularly during the current era of immunotherapeutics. Yet more research is required to understand mechanisms of APM down-regulation that underpin resistance to current ICIs and discover novel regulators that may unleash anti-tumour immunity.Despite the promise of long-term survival using immunotherapeutics in patients with otherwise incurable cancer, many do not respond to treatment due to immune evasion by tumour cells. This review outlines the mechanisms that tumour cells utilise to down-regulate neoantigen presentation to avoid immune recognition and highlights current strategies that may reactivate these pathways.Regulation of antigen presentation machinery in tumour cells may occur due to genomic, transcriptomic and post-translational modifications. Whilst most evidence to date focuses on elucidating mechanisms of MHC-I down-regulation, emerging research highlights the ability of tumour cells to express MHC-II and impact adaptive anti-tumour immunity.Ongoing research aims to identify novel mechanisms of neoantigen presentation regulation. Targeting these pathways with novel or repurposed drugs may enable immunotherapy to work for patients with otherwise limited treatment options."} +{"text": "The factors determining whether infection will occur following exposure to SARS-CoV-2 remain elusive. Certain SARS-CoV-2-exposed individuals mount a specific T-cell response but fail to seroconvert, representing a population that may provide further clarity on the nature of infection susceptibility and correlates of protection against SARS-CoV-2. Exposed seronegative individuals have been reported in patients exposed to the blood-borne pathogens Human Immunodeficiency virus and Hepatitis C virus and the sexually transmitted viruses Hepatitis B virus and Herpes Simplex virus. By comparing the quality of seronegative T-cell responses to SARS-CoV-2 with seronegative cellular immunity to these highly divergent viruses, common patterns emerge that offer insights on the role of cellular immunity against infection. For both SARS-CoV-2 and Hepatitis C, T-cell responses in exposed seronegatives are consistently higher than in unexposed individuals, but lower than in infected, seropositive patients. Durability of T-cell responses to Hepatitis C is dependent upon repeated exposure to antigen \u2013 single exposures do not generate long-lived memory T-cells. Finally, exposure to SARS-CoV-2 induces varying degrees of immune activation, suggesting that exposed seronegative individuals represent points on a spectrum rather than a discrete group. Together, these findings paint a complex landscape of the nature of infection but provide clues as to what may be protective early on in SARS-CoV-2 disease course. Further research on this phenomenon, particularly through cohort studies, is warranted. Coronaviridae family, is the causative agent of coronavirus disease 2019 (COVID-19). Canonical immunity to SARS-CoV-2 is well characterised despite its recent emergence: a delay in innate immune activation resulting from viral evasion of interferon (IFN) responses enables infection to occur . This enables the analysis of common and differing patterns of immunity to unrelated viruses through similar modes of exposure.+ and CD4+ T-cell activation in both cohorts compared to unexposed controls, as measured by IFN\u03b3 production following stimulation with Spike (S), Membrane (M), Nucleocapsid (NP), and Envelope peptides. Gallais et\u00a0al. (2021) also identified T-cell responses in close contacts of SARS-CoV-2-infected family members assessed cellular immunity in 90 seropositive individuals and 69 seronegative contacts who had been within 1.5m of a patient for over one hour or in the same household for over 24 hours identified 14 seronegative sexual partners of HCV-infected individuals who generated IFN\u03b3 responses to HCV, although at lower magnitudes than seropositive resolvers identified CD8+ T-cells targeting both structural and non-structural proteins (NSPs) . There wIFI17, an IFN-inducible marker of early infection recruited 26 PCR+ HCWs, 32 seronegative HCWs at high risk of exposure (treated as ESNs here), and 33 community controls . ESNs de\u03b3 to S, M and NP, whilst all generated cellular responses to M and NP by proliferation assay, indicating the potentially higher sensitivity of this assay , where cellular immunity targeting S as well as the rest of the proteome was higher in ESNs compared to controls identified SARS-CoV-2-specific T-cell responses in exposed HCWs: three of 10 generated IFNis assay . T-cell controls .+ responses occurred in four of 10 individuals within eight weeks of injury but were absent after 2.5 years. Heller et\u00a0al. (2013) assayed cellular immunity in HCWs exposed to HCV via needlestick, cut, or mucosal exposure, and identified HCV-specific proliferation and IFN\u03b3 production in 48% (n=30) and 42% (n=26) of individuals, respectively identified T-cell immunity in seronegative HCWs exposed to HCV-contaminated needles . CD4+ rein 48% n= and 42% ectively . The autectively . The dynenv glycoprotein was observed in six of eight ESNs, compared to only one of nine unexposed controls studied eight ESN HCWs following HIV+ needlestick injury. Four to eight weeks after injury, production of interleukin (IL)-2 by T-cells specific for the controls . HoweverViruses that have been well-studied for decades provide valuable information on the roles of exposure frequency and response durability in ESNs. Thurairajah et\u00a0al. (2011) studied seronegative injection drug users exposed to HCV with differing injection behaviours . ContinuAnimal studies also provide insight into the role of antigen exposure for HCV. Shata et\u00a0al. (2003) demonstrated that two chimpanzees exposed at six month intervals to increasing doses of HCV generated transient T-cell responses . 12 montT-cell responses in seronegative household contacts exposed to SARS-CoV-2 suggest that prolonged exposure may not be essential for cellular immunity , 19, 28.Determining which antigens are targeted in SARS-CoV-2 ESNs provides insight into mechanisms of response. T-cells targeting the replication-transcription complex (RTC) of SARS-CoV-2 were described by Swadling et\u00a0al. (2022) in ESNs . The RTC\u03b3-, secreting T-cells in ESNs compared to individuals that later became infected identified higher frequencies of IL-2-, but not IFNinfected . A simil in ESNs . Assays H1-focussed cytokine production has been described in HBV ESNs . A studyTo prevent infection before seroconversion, a rapid cellular response appears critical. Chandran et\u00a0al. (2021) assayed weekly nasopharyngeal swabs and blood samples from HCWs, and demonstrated that SARS-CoV-2 specific T-cell proliferation can occur before PCR positivity . These r+ and CD8+ responses to Middle-East Respiratory Syndrome coronavirus in four highly-exposed seronegative individuals, suggesting that the ESN phenomenon may be common to other human-infective coronaviruses (The RTC is highly conserved between SARS-CoV-2 and HCoVs . Tetrameaviruses .It is unclear whether cross-reactive T-cells contribute to ESN immunity in HCV, HIV, HBV or HSV. Cross-reactivity between HCV and influenza A has been described, with HCV-seronegative individuals generating T-cell responses to a cross-reactive HCV epitope . HoweverKey to understanding correlates of protection against SARS-CoV-2 infection is deciphering the role of cellular versus humoral immunity. Seropositivity may not always be the most appropriate marker if cellular immunity is protective. This is particularly relevant for assessing vaccine-induced protection against disease where neutralizing antibody titres are a common endpoint, and particularly for SARS-CoV-2 where an arms race between booster vaccination and waning antibody titres has begun.+ T-cell responses to SARS-CoV-2 compared to immunocompetent individuals (+ T-cell response was twice as high in infected individuals compared to ESNs. This casts doubt on their role in protection against infection.In a model whereby cellular immunity in ESNs is protective, one would reasonably expect that the magnitude of cellular response in ESNs would be greater than in seropositive individuals, to compensate for the lack of humoral immunity. Cellular immunity is able to clear SARS-CoV-2 infection in isolation; patients with X-linked agammaglobulinemia who cannot produce antibodies eventually clear SARS-CoV-2 infection, and mount higher magnitude CD8ividuals . HoweverIn influenza virus infection, cytotoxic T-cells target conserved non-structural proteins while antibodies target the divergent neuraminidase and hemagglutinin proteins and are thus strain-specific. In 1983, McMichael and colleagues demonstrated that individuals with cross-reactive T-cells targeting influenza A were able to clear infection in the absence of subtype-specific antibody . Later sA model for the dynamics of adaptive immunity in infection versus exposure is shown in Repeated exposure and response durability. Frequent exposure appears critical in the durability of HCV-specific cellular responses. SARS-CoV-2 challenge studies in primates and humans would clarify the role of dose and exposure frequency in ESNs, durability of responses, and the extent to which cellular immune responses correlate with protection against infection.Interaction with innate components. Although not covered here, cross-reactive T-cells likely act in coordination with innate immunity to prevent infection. Natural killer cells have been demonstrated to mediate resistance to HCV infection (IL28B likely contribute to disease susceptibility (nfection \u201361, and tibility . Future Other T-cell subsets. Many of the studies outlined in this review use whole blood samples representing circulating immunity. It is critical for future research to consider mucosal and tissue-resident cells to generate a complete picture wherever possible (possible . This woOur understanding of the ESN phenomenon remains in its infancy yet offers opportunities for development. The remarkable heterogeneity in outcome following SARS-CoV-2 exposure makes understanding infection susceptibility crucial for prevention and treatment. Significant insight can be gained into correlates of protection against SARS-CoV-2 by further investigating this phenomenon and gaining a deeper understanding of the role of cellular immunity in protection against infection.CJ conceived the idea and wrote the manuscript. JR assisted with finalising the manuscript. LT, PG and PK proofread the manuscript. All authors contributed to the article and approved the submitted version."} +{"text": "In addition, significantly prolonged PFS was observed in MED12-Mut patients , After taking into account age, gender, metastasis, treatment and TMB status, the result of multivariable Cox proportional hazards regression showed significantly better PFS . In the MSKCC cohort (n\u2009=\u20091513), overall survival advantage was achieved in MED12-Mut patients , after taking into account same factors in WES cohort, this link still existed , Notably, TMB was also found significantly higher in MED12-Mut patients in both WES and MSKCC cohort. Further tumor-infiltrating lymphocytes and DDR-related gene analysis revealed anti-tumor immunity in MED12-Mut patients. Totally, MED12-Mut successfully predicted better clinical outcomes in ICIs-treated pan-cancer cohort, indicating that MED12-Mut could serve as a potential predictive biomarker for immune checkpoint inhibitors in pan-cancer.Immune checkpoint inhibitors (ICIs) therapy elicits admirable anti-tumor responses across many types of cancer. Growing evidence point to a link to Mediator complex subunit 12 (MED12) and DNA damage repair (DDR) and TGF-\u03b2 signing, while the clinical data on the association of MED12 and ICIs response are lacking. In this study, clinical and whole-exome sequencing (WES) data from published studies were merged as a WES cohort to explore the association between MED12 mutation (MED12-Mut) and ICIs efficiency across cancers. Then, Memorial Sloan Kettering Cancer Center (MSKCC) cohort was used for validating our findings. The Cancer Genome Atlas (TCGA) cohort was used to perform anti-tumor immunity and prognosis analysis. In the WES cohort (The online version contains supplementary material available at 10.1186/s40001-022-00856-z. To the editors,Immune checkpoint inhibitors (ICIs) can elicit impressive improvement of survival in different cancer types. However, few patients can respond well to immunotherapy and reliable biomarkers are urgently needed . Emerginhttp://www.cbioportal.org/) as a WES cohort was surveyed to determine the relationship between MED12-Mut and overall survival (OS). In the MSKCC cohort, MED12-Mut patients achieved significantly longer OS and tumor neoantigen burden (TNB), which are relevant tools for the identification of patients likely to respond to ICIs, were studied first. In the immunotherapy cohort and TCGA cohort, MED12-Wt tumors had a higher TMB than MED12-Wt tumors . B. MSKn et al. ). C. TheAdditional file 2: Fig. S2. The pan-cancer landscape of MED12 mutations across human tumors. The proportion of MED12 mutated tumors identified for each cancer type with alteration frequency in TCGA pan-cancer cohorts.Additional file 3: Fig. S3. Kaplan\u2013Meier curves of OS between the MED12-Mut and wildtype groups in the TCGA cohort.Additional file 4:\u00a0Materials and methodsAdditional file 5: Table S1. Detailed clinical information of the five WES cohorts and the MSKCC cohort."} +{"text": "Multisystem inflammatory syndrome in children (MIS-C) is a severe post-infectious complication occurring weeks after SARS-CoV-2 infection. The exact mechanisms leading to immune dysregulation and organ damage remain incompletely understood. Progress in understanding the immunopathology underlying MIS-C has been halted by limited availability of pre-treatment patient samples and confounding effects of immunomodulatory treatment on previously studied specimens.In this study, we have restricted enrollment to treatment-na\u00efve patients with MIS-C and used a systems biology approach combining CyTOF, single cell transcriptomics, serum cytokine profiling and T cell receptor sequencing to dissect how immune responses in children with MIS-C differ from children with mild SARS-CoV2 infection, adults with severe COVID-19 and healthy individuals. We also integrated single cell transcriptomics datasets from post-treatment MIS-C samples to study how immune responses change along disease course.We identified increased activation markers and antigen presentation across multiple immune cell types in MIS-C patients from both CyTOF and single cell transcriptomics data. Importantly, in PBMCs of MIS-C patients, we identified a distinct subset of proinflammatory monocytes, with increased expression of interferon gamma response genes combined with a signature of enhanced complement expression, antigen processing and presentation, which was not observed in post-treatment MIS-C samples. Interestingly, this monocyte population bears resemblance to a subset of monocytes that emerges after the BNT162b2 mRNA vaccine booster. In addition, in PBMCs of MIS-C patients, we identify increased proportion of proliferating T/NK cells, suggesting distinct T cell expansions in MIS-C. T and NK cells in MIS-C samples also showed increased cell cytotoxicity markers.Taken together, treatment-na\u00efve MIS-C samples display distinct monocyte clusters, activated antigen presentation and complement expression, and increased T and NK cell cytotoxicity, which may account for the clinical presentation of MIS-C.Purvesh Khatri, PhD, Cepheid, Inc.: Advisor/Consultant|Inflammatix, Inc.: Advisor/Consultant|Inflammatix, Inc.: Inflammatix is in negotiations to license the 9-gene signature discussed here.|Inflammatix, Inc.: Stocks/Bonds."} +{"text": "Passive immunization against SARS-CoV-2 limits viral burden and death from COVID-19; however, it poses a theoretical risk of disease exacerbation through antibody-dependent enhancement (ADE). ADE after anti-SARS-CoV2 antibody treatment has not been reported, and therefore the potential risk and promoting factors remain unknown., casirivimab\u00a0and imdevimab were administered as a preemptive approach. The patient developed immune activation and cytokine release syndrome (CRS) occurring within four hours of preemptive anti-SARS-CoV2 antibody (casirivimab/imdevimab) infusion. Immune activation and CRS were evidenced by a rapid increase in serum cytokines , acute respiratory insufficiency, and progressive acute respiratory distress syndrome.A 75-year-old female was admitted to the emergency room with recurrent, unexplained bruises and leukocytopenia, anemia, and thrombocytopenia. Evaluation of a bone marrow biopsy established the diagnosis of an acute promyelocytic leukemia (APL). SARS-CoV-2 RT-PCR testing of nasal and throat swabs on admission was negative. During the routine SARS-CoV-2 testing of inpatients, our patient tested positive for SARS-CoV-2 on day 14 after admission without typical COVID-19 symptoms. Due to disease- and therapy-related immunosuppression and advanced age conferring a high risk of progressing to severe COVID-19The temporal relationship between therapeutic antibody administration and the rapid laboratory, radiological, and clinical deterioration suggests that CRS was an antibody-related adverse event, potentially exacerbated by APL treatment-mediated differentiation of leukemic blasts and promyelocytes. This case highlights the need for careful assessment of life-threatening adverse events after passive SARS-CoV-2 immunization, especially in the clinical context of patients with complex immune and hematological landscapes.The online version contains supplementary material available at 10.1186/s12879-022-07513-0. The profound global health, social, economic disruption from COVID-19 continues , 3. VariA 75-year-old female was admitted to the emergency room with recurrent, unexplained bruises and leukocytopenia, anemia, and thrombocytopenia in mid-2021 , a potentially fatal complication of treatment with differentiation-inducing agents, such as ATRA and ATO, through promoting the differentiation of leukemic cells .\u00a0TherefoAfter ten days of ATRA/ATO, the patient\u2019s CRP increased but she remained subfebrile , but typical COVID-19 symptoms were absent was given for six days to treat a possible concurrent DS. After clinical stabilization and leucocyte decline, ATRA and ATO were reinitiated. Further assessment of the immune cell compartment during this treatment showed decreasing leukocytosis and granulocytosis, and the CD4/CD8 T cell ratio disclosed a relative increase in CD4+ T cells and decrease in CD8+ T cells \u00a0Clinical timeline highlighting the initiated hematological treatments of APL before SARS-CoV-2 detection in nasopharyngeal swabs. (B) Faggot cells with multiple Auer rods (arrow mark) in bone marrow aspirate. Blood differential tests (C), hemoglobin and LDH (D), and vital signs including blood pressure, heart rate, and oxygen saturation (E) from admission to casirivimab/imdevimab infusion. Normal ranges are highlighted in green. Figure S2. Chest X-rays along the clinical course from diagnosis of acute promyelocytic leukemia (APL) to CRS after casirivimab/imdevimab infusion and subsequent period in the ICU. Figure S3. (A)\u00a0Clinical timeline highlighting the initiated treatments in the ICU after casirivimab/imdevimab infusion. (B) Complete blood count after ICU admission. \u00a0(C)\u00a0Flow cytometric analysis of peripheral blood after ICU admission including CD3 T cell characterization. Normal ranges are highlighted in green. Table S1. Microbiological laboratory assessment from diagnosis of acute APL to CRS after casirivimab/imdevimab treatment and during the ICU stay. (A)\u00a0Microbiological culture performed on different patient-derived materials. (B) Microbiological assays for the analysis of certain pathogens. Table S2. Virological laboratory assessment from diagnosis of acute APL to CRS after casirivimab/imdevimab treatment and during the ICU stay. (A) SARS-CoV-2 molecular diagnostics including RT-PCR and virus sequencing at different time points. (B) Serological assessment of the immune status to different viruses. (C) PCR detection of various viruses in blood and bronchoalveolar lavage."} +{"text": "ABCD1 gene and confirmed X-linked adrenoleukodystrophy (X-ALD).A 27-year-old man presented with a two-year history of progressive ataxia. Family history was unremarkable. Examination revealed ataxia and alopecia. Serum cortisol levels were low, suggesting adrenal insufficiency. Brain magnetic resonance imaging (MRI) disclosed cerebellar white matter involvement . Exome sSeveral forms of hereditary ataxias remain undetermined, despite being largely investigated. Whole-exome sequencing is a useful diagnostic approach for undetermined ataxias"} +{"text": "Findings were confirmed, and additional characteristics were explored using public datasets (n\u2009=\u2009555). Drug susceptibilities of tuft cell-like SCLC cell lines were also investigated. By immunohistochemistry, 10\u201320% of SCLC and LCNEC, and approximately 2% of SQCC expressed POU2F3, the master regulator of tuft cells. These tuft cell-like tumors exhibited \u201clineage ambiguity\u201d as they co-expressed NCAM1, a marker for neuroendocrine differentiation, and KRT5, a marker for squamous differentiation. In addition, tuft cell-like tumors co-expressed BCL2 and KIT, and tuft cell-like SCLC and LCNEC, but not SQCC, also highly expressed MYC. Data from public datasets confirmed these features and revealed that tuft cell-like SCLC and LCNEC co-clustered on hierarchical clustering. Furthermore, only tuft cell-like subsets among pulmonary cancers significantly expressed FOXI1, the master regulator of ionocytes, suggesting their bidirectional but immature differentiation status. Clinically, tuft cell-like SCLC and LCNEC had a similar prognosis. Experimentally, tuft cell-like SCLC cell lines were susceptible to PARP and BCL2 co-inhibition, indicating synergistic effects. Taken together, pulmonary tuft cell-like cancers maintain histotype-related clinicopathologic characteristics despite overlapping unique molecular features. From a therapeutic perspective, identification of tuft cell-like LCNECs might be crucial given their close kinship with tuft cell-like SCLC.Tuft cells are chemosensory epithelial cells in the respiratory tract and several other organs. Recent studies revealed tuft cell-like gene expression signatures in some pulmonary adenocarcinomas, squamous cell carcinomas (SQCC), small cell carcinomas (SCLC), and large cell neuroendocrine carcinomas (LCNEC). Identification of their similarities could inform shared druggable vulnerabilities. Clinicopathological features of tuft cell-like (tcl) subsets in various lung cancer histotypes were studied in two independent tumor cohorts using immunohistochemistry ( Tuft cells are epithelial cells with distinct microvilli (tufts) on the apical side. They occur in multiple organs and regulate immune functions, e.g., anti-parasitic immunity \u20134, and tPOU2F3, the tuft cell master regulator ). On the GO analyses pulmonary tuft cell-like cancers across various histotypes have considerably overlapping gene expression profiles, including a hybrid tuft cell/ionocyte-like signature; (ii) tuft cell-like NECs and SQCC nevertheless exhibit distinct histotype-associated clinicopathological features; (iii) in vitro, tuft cell-like SCLCs show higher vulnerability to PARP/BCL2 co-inhibition than to either drug alone.The close kinship among tuft cell-like lung cancers was highlighted by the clustering analysis with combined SCLC and LCNEC datasets, because tuft cell-like SCLC and LCNEC formed a single, small cluster. Also, all the tuft cell-like lung cancer subtypes significantly expressed BCL2 and KIT, and tuft cell-like NECs unlike non-tuft cell-like NECs overexpressed MYC. In addition, tuft cell-like SCLC, LCNEC, and SQCC exhibited \u201clineage ambiguity\u201d, namely with expressions of NCAM1 (NECs\u2009>\u2009SQCC) and KRT5 (SQCC\u2009>\u2009NECs) and infrequent expression of most neuroendocrine markers Fig. . AnotherOn the other hand, we also noticed differences between tuft cell-like NECs and SQCCs, e.g., overexpression of MYC in tuft cell-like NECs but not in tuft cell-like SQCC. Between the two groups, Ki-67 immunohistochemistry was also significantly different. These findings may be related to the poorer prognosis of tuft cell-like NECs compared with SQCCs and underscore that pathological classification remains essential for patient management. Regarding tuft cell-like SCLC and LCNEC, inflammatory signals were enriched in the former and neural differentiation programs in the latter. A more differentiated nature of LCNEC seems consistent with its morphological features, such as the more abundant cytoplasm than SCLC. The inflammatory nature of tuft cell-like SCLC may warrant further studies under (immuno-)therapeutic perspectives .Last, we proposed a therapeutic option for tuft cell-like lung cancers, i.e., co-inhibition of PARP and BCL2. The efficacy of PARP inhibitors for tuft cell-like SCLC was also recently proposed and discussed HPF1 mutations in public datasets [In parallel, functional studies with tuft cell-like SCLC cell lines, and comprehensive genetic and epigenetic profiling of tuft cell-like lung cancers are necessary to provide mechanistic evidence for the unique drug sensitivity of tuft cell-like SCLC; publicly available databases, e.g., SCLC-CellMiner will be datasets , 27 (notThe current study has limitations. The restriction to resection specimens implies a selection bias, as most lung cancers are inoperable but biopsied before neoadjuvant approaches . This biAccumulating evidence suggests that tuft cell-like SCLCs are biologically distinct from the other SCLC subtypes , 54, 55.Supplementary informationRaw data of WB of Fig. 5 and S10"} +{"text": "There is a scarcity of studies comparing percutaneous coronary intervention (PCI) using new-generation drug-eluting stents (DES) with coronary artery bypass grafting (CABG) in patients with multi-vessel coronary artery disease.The CREDO-Kyoto PCI/CABG registry Cohort-3 enrolled 14927 consecutive patients who underwent first coronary revascularization with PCI or isolated CABG between January 2011 and December 2013. The current study population consisted of 2464 patients who underwent multi-vessel coronary revascularization including revascularization of left anterior descending coronary artery (LAD) either with PCI using new-generation DES (N = 1565), or with CABG (N = 899). Patients in the PCI group were older and more often had severe frailty, but had less complex coronary anatomy, and less complete revascularization than those in the CABG group. Cumulative 5-year incidence of a composite of all-cause death, myocardial infarction or stroke was not significantly different between the 2 groups . However, after adjusting confounders, the excess risk of PCI relative to CABG turned to be significant for the composite endpoint . PCI as compared with CABG was associated with comparable adjusted risk for all-cause death , and stroke , but with excess adjusted risk for myocardial infarction , and any coronary revascularization .In this observational study, PCI with new-generation DES as compared with CABG was associated with excess long-term risk for major cardiovascular events in patients who underwent multi-vessel coronary revascularization including LAD. Randomized controlled trials (RCT) in the first-generation drug-eluting stent (DES) era have clearly demonstrated the superiority of coronary artery bypass grafting (CABG) over percutaneous coronary intervention (PCI) in patients with complex multi-vessel coronary artery disease (CAD) , 2. In cThe Coronary Revascularization Demonstrating Outcome Study in Kyoto (CREDO-Kyoto) PCI/CABG registry Cohort-3 is a physician-initiated, non-company sponsored, multi-center registry enrolling consecutive patients who underwent first coronary revascularization with PCI or isolated CABG without combined non-coronary surgery among 22 Japanese centers between January 2011 and December 2013 . The relIn the CREDO-Kyoto PCI/CABG registry Cohort-3, there were 14927 patients who underwent first coronary revascularization with PCI or isolated CABG . In the present study, we excluded those patients who refused study participation (N = 60), those who had left main CAD (N = 1256), or single-vessel disease (N = 5657), those with emergency procedure (N = 2775), cardiogenic shock (N = 11), no LAD target (N = 1464), only LAD target (N = 1033), no stenting (N = 4), bare-metal stents use (N = 127) and first-generation DES use (N = 57). Finally, we identified 2464 patients who received multi-vessel coronary revascularization including revascularization of LAD either by isolated CABG (N = 899) or by PCI with exclusive use of new-generation DES (N = 1565) .The primary outcome measure for the current analysis was a composite of all-cause death, myocardial infarction, or stroke. The secondary outcome measures included all-cause death, cardiovascular death, cardiac death, non-cardiovascular death, non-cardiac death, myocardial infarction, definite stent thrombosis or symptomatic graft occlusion, stroke, hospitalization for heart failure, major bleeding, target-vessel revascularization, ischemia-driven target-vessel revascularization, non-target-vessel revascularization, ischemia-driven non-target-vessel revascularization. any coronary revascularization, ischemia-driven any coronary revascularization, and a composite of all-cause death, myocardial infarction, stroke, or any coronary revascularization. Death was regarded as cardiac in origin unless obvious non-cardiac causes could be identified. Cardiovascular death included cardiac death, and other vascular death related to stroke, renal disease, and vascular disease. Death of unknown cause and any death during the index hospitalization for coronary revascularization were regarded as cardiac death. Myocardial infarction (MI) was categorized into periprocedural and spontaneous MI. We defined \u201cemergency procedure\u201d as those procedures for ACS patients that were performed immediately after clinical presentation. The definition of proximal LAD lesion was the lesion location in segment 6 and/or segment 7 by the American Heart Association (AHA) classification . Severe Clinical, angiographic, and procedural data were collected from hospital charts or hospital databases according to the pre-specified definitions by the experienced clinical research coordinators from an independent clinical research organization . Follow-2 or hemodialysis), and severe frailty, because the number of patients with event was <100 for this outcome measure. Furthermore, we did not perform a multivariable analysis for the outcome measures with <30 patients with event. We performed subgroup analyses for the primary outcome measure stratified by age (> = or < 75 years), sex, diabetes, heart failure, end-stage renal disease , extent of CAD (double-vessel or triple-vessel disease), and complete revascularization (complete or incomplete revascularization). Furthermore, we showed clinical outcomes of two subgroups; patients with two-vessel disease who received coronary revascularization including revascularization of LAD, and patients with multi-vessel coronary revascularization including revascularization of proximal LAD who received PCI with the second-generation DES and 899 patients (36%) who underwent CABG. The patients in the PCI group were older and more often had current heart failure, and severe frailty than those in the CABG group, while the patients in the CABG group had higher prevalence of men, diabetes mellitus, prior heart failure, prior myocardial infarction, peripheral vascular disease, eGFR <30 mL/min/1.73mdialysis . As for dialysis . In termdialysis .Median follow-up duration was 5.8 (interquartile range: 4.7\u20138.2) years, and complete 1-, 3-, 5-year clinical follow-up data were obtained in 97.0%, 91.7%, and 86.3% of patients, respectively without differences between the PCI and the CABG groups .The cumulative 5-year incidence of the primary outcome measure was not significantly different between the PCI and the CABG groups . HoweverIn the subgroup analyses, there was no significant interaction between the subgroup factors and the risks of PCI relative to CABG for the primary outcome measure .In patients with two-vessel disease who underwent multi-vessel revascularization including LAD, the 5-year cumulative incidence of the primary outcome measure was not significantly different between the 2 groups and the risk of PCI relative to CABG remained insignificant for the primary outcome measure after adjusting for the confounders . In patiAfter propensity-score matching in the sensitivity analysis, baseline characteristics of the PCI and the CABG groups were much more comparable than those in the entire population . The resWhile the cumulative 5-year incidence of target-vessel revascularization was not significantly different between patients with complete revascularization (CR) and with incomplete revascularization (ICR) , the cumulative 5-year incidence of non-target-vessel revascularization in patients with CR was significantly higher than that in those with ICR .The main findings in the current study were as follows; (1) PCI with exclusive use of the newer-generation DES was associated with a higher long-term risk for a composite of all-cause death, myocardial infarction, or stroke in patients who underwent multi-vessel coronary revascularization including LAD; (2) PCI as compared with CABG was associated with a comparable long-term risk for all-cause death and stroke, but was associated with a significantly higher long-term risk for myocardial infarction and any coronary revascularization.In the new-generation DES era, there was only one moderate sized RCT comparing PCI with CABG, although RCTs in the first-generation DES era have clearly demonstrated benefit of CABG over PCI in patients with complex multi-vessel CAD in terms of survival, myocardial infarction, and repeat revascularization , 2. The SYNTAX-\u2161 study was initiated in 2014, where clinical outcomes of PCI patients treated with the SYNTAX-\u2161strategy were compared with the historical PCI (SYNTAX-\u2160 PCI) and CABG (SYNTAX-\u2160 CABG) population matched on the basis of SYNTAX score \u2161. The SYNTAX-\u2161strategy included use of a new-generation DES , target lesion selection based on physiological evaluation by instantaneous wave-free ratio (IFR) or fractional flow reserve (FFR) measurement, stent optimization by intravascular ultrasound guidance, and adherence to optimal medical therapy . At 2-yeThe current study has several limitations. First, this is a retrospective observational study. Therefore, selection bias and unmeasured confounding cannot be excluded despite extensive statistical adjustment, because differences in baseline clinical and procedural characteristics between the groups were substantial. Second, the p-value of all-cause death and non-cardiovascular death was not statistically significant but were borderline. Therefore, we could not deny that the mortality risk might be higher in the PCI group than in the CABG group. Third, there was an increase in any coronary revascularization in the PCI group around 1 year, which might be influenced by routine follow-up coronary angiography. We performed a landmark analysis at 1 year and at 2 years and evaluated outcomes with early events excluded . Fourth,In conclusion, in this observational study, PCI with new-generation DES as compared with CABG was associated with excess long-term risk for major cardiovascular events in patients who underwent multi-vessel coronary revascularization including LAD.S1 Appendix(DOCX)Click here for additional data file.S2 Appendix(DOCX)Click here for additional data file.S3 Appendix(DOCX)Click here for additional data file.S1 Method(DOCX)Click here for additional data file.S2 Method(DOCX)Click here for additional data file.S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 FigLandmark analysis at (A)1 year and (B)2 years in any coronary revascularization.(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file."} +{"text": "A 45-year-old male presented with acute-onset left-sided weakness and slurred speech. Non-contrast-enhanced brain magnetic resonance imaging revealed cortical and internal border-zone infarcts compatible with stroke. A survey of ischemic stroke risk factors in young adults excluded coagulopathy, vasculitis, and cardiac disease. Nevertheless, neck-computed tomography angiography revealed a long-segmental narrowing of the right internal carotid artery with wall thickening and a \u201cstring-of-beads\u201d appearance suspicious for fibromuscular dysplasia, which was confirmed on further angiography. His clinical condition stabilized after intensive medical therapy. This case demonstrates cerebrovascular fibromuscular dysplasia as a possible cause of ischemic stroke in young adults."} +{"text": "We retrospectively analyzed clinical, NCS/EMG, and NMRI aspects of five COVID-19 intensive care unit inpatients that received mechanical ventilation. After awakening from sedation, they experienced peripheral neuromyopathic symptoms.Teaching Point: Acquired peripheral nerve injury has been described in COVID-19 infection and knowledge of the clinical, nerve conduction studies/electromyography (NCS/EMG) and neurographic magnetic resonance imaging (NMRI) findings are crucial. Neurological complications have been reported in critically ill COVID-19 patients 13. ClinicFigure 1B and 1C).A 48-year-old woman presented with sudden onset of severe burning pain and progressive weakness in the left shoulder girdle. NCS/EMG suggested acute motor sensory axonal neuropathy (AMSAN) and left brachial neuritis. Left brachial plexus NMRI was consistent with Parsonage-Turner syndrome and Parsonage-Turner syndrome, confirmed by NMRI.Figure 1A).A 78-year-old woman presented with prone positioning for two days. Quadriplegia due to critical illness polyneuropathy and left shoulder pain was present after awakening from sedation. NCS/EMG and NMRI were consistent with Parsonage-Turner syndrome . Mononeuropathy related to COVID-19 was diagnosed.A 35-year-old woman presented with anesthesia and distal left leg muscle strength grade 0. Lumbosacral plexus MRI revealed signs of sciatic neuritis , attributed to ulnar nerve compression lesion.A 48-year-old woman presented with numbness along the fifth and fourth left fingers, weakness and volume loss between first and second metacarpal bones. Elbow MRI was consistent with ulnar neuropathy has expression in the nervous system 1.Post-infectious inflammatory peripheral nerve injury is thought to be immune-mediated and occurs in the setting of several viruses . Periphe1567511Clinical, NCS/EMG, and NMRI are important to assess the potential etiologies and severity of peripheral nerve injury. Early diagnosis may guide treatment decisions, which could improve the clinical outcome."} +{"text": "Teicoplaninis a glycopeptide antibiotic deployed to combat Gram-positivebacterial infection and has recently been associated with developmentof adverse drug reactions, particularly following previous exposureto vancomycin. In this study, we generated teicoplanin-specific monoclonalT-cell populations from healthy volunteers expressing HLA-A*32:01and defined pathways of T-cell activation and HLA allele restriction.Teicoplanin-responsive T-cells were CD8+, HLA class I-restricted,and cross-reacted with the lipoglycopeptide daptomycin in proliferationand cytokine/cytolytic molecule releaseassays. These data show that teicoplanin activates T-cells, whichmay play a role in the pathogenesis of teicoplanin-induced adverseevents, in HLA-A*32:01 positive donors. Despite the incidenceof adverse drug reaction (ADR) associated with teicoplanin being substantiallylower (13.9% vs 21.9%3) compared to vancomycin,the drug still poses a significant risk to patient safety. A recentGWAS has shown an association between vancomycin-induced DRESS andHLA-A*32:01 in European populations.4 Casestudies have reported clinical cross-reactivity and subsequent teicoplanin-inducedDRESS following initial vancomycin hypersensitivity.6 Preliminary in vitro studies using vancomycin-responsiveT-cells generated from HLA-A*32:01 positive healthy donor PBMCs havealready demonstrated low levels of cross-reactivity with teicoplanin.7 Cross-reactivity has been illustrated furtherin patients presenting with suspected vancomycin or teicoplanin-inducedDRESS, with ex vivo data suggesting complex patternsof immunogenicity within the context of HLA class II presentation.8 The aim of the present study was to investigatethe intrinsic immunogenic potential of teicoplanin in terms of evokingT-cell responses in healthy donors (HDs), in addition to further exploringpatterns of cross-reactivity to structurally related glycopeptides.Hypersensitivityto otherwiseefficacious antibiotics is an area of concern to patients, clinicians,and researchers in the field of drug development. Prediction of suchreactions is often difficult due to the elicitation of adverse eventsarising outside of a drug\u2019s known pharmacology. Although rare,reactions of this nature have been associated with activation of theadaptive immune system, with T-cells implicated in the pathogenesisof severe cutaneous adverse reaction, including drug-reaction witheosinophilia and systemic symptoms (DRESS).9 were identified in 3 healthydonors positive for HLA-A*32:01 expression (Teicoplanin-specific T-cell clones (TCCs), generated by serialdilution,pression 1. TCCs gFollowing pretreatment of both APCs and T-cells with anti-HLA blockingantibodies, proliferation of CD8+ TCCs was unaffected after the HLAclass II blockade . However, proliferationwas found to be inhibited in the presence of MHC class I blockingantibodies 2A indicaCytokine and cytolytic molecule secretion of teicoplanin-reactiveTCCs was assessed via ELISpot after a drug rechallenge 3A. Clone10In summary, teicoplanin-responsive T-cells displaying a CD8+phenotypewere generated from 3 drug-na\u00efve healthy donors expressingthe HLA-A*32:01 allele, recently associated with cases of vancomycin-inducedDRESS. Therapeutic concentrations associated with glycopeptide administrationare typically between 10 and 20 \u03bcM, substantially lower thanthe optimal doses used within this study to elicit maximal T-cellresponses for functional analysis. However, we have observed thatglycopeptide-specific TCCs are capable of eliciting proliferativeresponses at lower, more therapeutically relevant doses in line withconcentrations found within the blood plasma of patients. The identificationof TCCs that proliferate and secrete both cytotoxic and DRESS relatedcytokines such as IL-5 suggests T-cell involvement within the pathogenesisof the teicoplanin-induced DRESS syndrome.7 Proliferative T-cell cross-reactivity of teicoplanin-responsiveTCCs generated from healthy volunteers to daptomycin highlights thecomplex patterns of reactivity encountered within clinical settings.The observed in vitro T-cell cross-reactivity maybe explained by structural similarities between both teicoplanin anddaptomycin, specifically the presence of a hydrophobic lipid chain.Conversely, vancomycin\u2019s structure comprises a heptapeptidechain that crucially contains a disaccharide, composed of vancosamineand glucose, instead of the lipid tail found on both teicoplanin anddaptomycin molecules. This potentially explains why some teicoplanin-specificT-cells are able to proliferate in the presence of daptomycin butnot vancomycin. One intriguing avenue to explore the nature of thesecross-reactive responses involves the study of cellular energeticparameters, such as glycolysis, which may provide greater sensitivityfor the determination of T-cell activation thresholds upon antigenpresentation. However, to investigate the specificity of teicoplaninfor HLA-A*32:01, additional cloning experiments focusing on individualsnegative for HLA-A*32:01 expression will need to be conducted. Furthergenetic studies and functional T-cell analysis following HLA-glycopeptidebinding will be required to determine the full pathway of glycopeptidecross-reactivity in addition to the extent of interactions with HLA-A*32:01in order to predict potential susceptibility to severe cross-reactivityand improve patient safety.Mechanistic T-cell assays revealed a processing independentmechanismof activation that hinges on drug presentation via direct interactionwith HLA class I molecules. These data are concordant with previousmechanistic findings relating to T-cell responses to vancomycin forwhich it has been hypothesized glycopeptide compounds possess thecapacity to displace and mimic native HLA peptides."} +{"text": "Carbapenem use correlates with the development of carbapenem-resistant organisms, prompting our antimicrobial stewardship program (ASP) to develop a policy mandating infectious diseases (ID) consultation after 72 hours of meropenem use. Our objective was to evaluate the impact of this policy on meropenem utilization and associated clinical outcomes.Clostridioides difficile (C. difficile) infection incidence. All outcomes were assessed in pre- and post-intervention periods.This quasi-experimental, observational study evaluated the impact of the new ASP policy in adult patients across four campuses. Administered meropenem orders were retrieved retrospectively six months before (11/2020-4/2021) and after (6/2021-11/2021) policy implementation. The primary outcome was meropenem days of therapy per 1000 patient-days (DOTs). Secondary outcomes included DOTs of select broad-spectrum antimicrobials, 30-day all-cause mortality, hospital length of stay (LOS), and Pseudomonas spp. was similar in both groups . ID consultation increased after policy implementation . Meropenem DOTs decreased significantly after intervention . We observed increases in ceftriaxone and cefepime DOTs . An increase in C. difficile incidence was seen. Hospital LOS was similar pre- and post-intervention .There were 1493 and 1404 meropenem orders in the pre- and post-intervention periods, respectively. Pre-intervention group patients had slightly higher modified Charlson Comorbidity Index scores . Pre-intervention group had more patients with penicillin allergies but less patients with sulfa allergies . The most common meropenem indications were bloodstream, respiratory, abdominal, and urinary tract infections. The incidence of extended-spectrum beta-lactamase (ESBL)-producing Enterobacterales and cefepime-resistant The ASP policy mandating ID consultation after 72 hours of meropenem use helped decrease meropenem DOT and encouraged use of antimicrobial agents with narrower spectrum.All Authors: No reported disclosures."} +{"text": "Positron emission tomography (PET) imaging with prostate-specific membrane antigen- (PSMA-) binding tracers has been found incidentally to demonstrate uptake in CNS tumors. Following the encouraging findings of several such case reports, there is a growing interest in the potential application of PSMA-targeted PET imaging for diagnostics, theranostics, and monitoring of CNS tumors. This is a systematic literature review on PSMA-binding tracers in CNS tumors. A PubMed search was conducted, including preclinical and clinical reports. One hundred and twelve records were identified, and after screening, 56 were included in the final report. Tissue studies demonstrated PSMA expression in tumor vascular endothelial cells, without expression in normal brain tissue, though the extent and intensity of staining varied by anti-PSMA antibody and methodology. Most included studies reported on gliomas, which showed strong PSMA ligand uptake and more favorable tumor to background ratios than other PET tracers. There are also case reports demonstrating PSMA ligand uptake in prostate cancer brain metastases, nonprostate cancer brain metastases, and meningiomas. We also review the properties of the various PSMA-binding radiotracers available. Therapeutic and theranostic applications of PSMA-binding tracers have been studied, including labeled alpha- and beta-ray emitting isotopes, as well as PSMA targeting in directing MRI-guided focused ultrasound. There is a potential application for PSMA-targeted PET in neuro-oncology as a combination of diagnostic and therapeutic use, as a theranostic modality for managing CNS tumors. Further research is needed regarding the mechanism(s) of PSMA expression in CNS tumors and its differential performance by tumor type. Standard imaging of central nervous system (CNS) tumors with magnetic resonance imaging (MRI) has demonstrated limitations , and posin vitro and in vivo studies, in order to summarize current knowledge regarding the potential for PSMA-targeted PET in neuro-oncology imaging.PET radioligands targeting PSMA are misleading in their nomenclature as \u201cPSMA tracers,\u201d because they are in reality inhibitors with high affinity for the PSMA-binding motif . Such PShttps://pubmed.ncbi.nlm.nih.gov) in May 2021 using the following search terms: ((brain tumor) OR (spine tumor) OR (CNS tumor) OR (neurooncology) OR (neuro-oncology) OR (glioblastoma) OR (glioma) OR (astrocytoma) OR (oligodendroglioma) OR (meningioma) OR (brain metastasis) OR (spine metastasis) OR (brain metastases) OR (spine metastases) OR (chordoma) OR (craniopharyngioma) OR (gangliocytoma) OR (glomus) OR (pineocytoma) OR (pituitary adenoma) OR (schwannoma) OR (acoustic neuroma) OR (ependymoma) OR (medulloblastoma) OR (hemangioblastoma) OR (rhabdoid)) AND ((positron emission tomography) OR (PET)) AND ((prostate specific membrane antigen) OR (PSMA)). All available years were included.This review utilized a search strategy to identify previous preclinical and clinical research studies of PSMA-targeted PET in neuro-oncologic applications. The search was performed in the PubMed database with oversight by the other authors. Data variables collected included year, study design, study subjects, tumor type, tracer name, and main study results. Data were analyzed and summarized qualitatively.There are 13 published reports of PSMA expression in CNS tumor tissue and preclinical models, including a collective total of 331 patients and 38 animals . The firWernicke et al. (2011) published an immunohistochemistry study of 32 GBMs, using an anti-SMA mAb 3E6 (Dako) stain, quantifying the extent and intensity of vascular endothelial staining . They reThe same group then published in 2014 a PSMA immunohistochemistry study of 14 breast cancer patients with brain metastases . They usThe above data were complemented by several subsequent case reports. Schwenck et al. reported increased PSMA expression in the vascular endothelium of a GBM patient, though not in the normal brain and vasculature 17]. Su. Su17]. The two largest of these studies, by Matsuda et al. and Saffn = 60) . Ar. Ar68Ga- low FDG . Diagnos18F-DCFPyL uptake on PET in metastatic renal cell carcinomas (RCC), one clear cell and one nonclear cell. Rowe et al. reported a clear cell RCC patient who demonstrated intense 18F-DCFPyL uptake in an MRI-diagnosed frontal lobe brain metastasis (SUVmax = 3.9) [18F-DCFPyL SUVmax ranging from 0.5 to 3.4 [Two reports have been published of x = 3.9) . Yin et 5 to 3.4 .68Ga-PSMA uptake in a brain metastasis from melanoma. Hod et al. reported a patient who demonstrated a melanoma metastasis in the occipital lobe on CT and MRI and unexpectedly showed avid 68Ga-PSMA uptake in the lesion [There is only one report of e lesion . Diagnos89Zr-Df-IAB2M uptake in brain metastases from lung cancer. Matsuda et al. report a lung cancer patient with MRI-detected occipital brain metastasis, which demonstrated avid 89Zr-Df-IAB2M uptake [89Zr-Df-IAB2M uptake and PSMA expression.Similarly, there has been only one report of M uptake . There w68Ga-PSMA. Bilgin et al. reported a patient with high 68Ga-PSMA uptake in an orbitofrontal lesion (SUVmax = 3.1) which was subsequently diagnosed as meningioma on MRI [68Ga-PSMA uptake (SUVmax = 1.9), but no FDG avidity [68Ga-PSMA-11 (HBED-CC) uptake than 18F-FDG uptake [68Ga-PSMA uptake, and diagnosis was supported by MRI [68Ga-PSMA uptake in the left frontal lobe, confirmed as a preexisting meningioma [68Ga-PSMA uptake (SUVmax 12.1) in an MRI-detected lesion, suggestive of an intraventricular meningioma of a patient with history of prostate cancer [PSMA targeting is potentially well suited to meningiomas, given that these lesions are generally highly vascularized. There have been eight single case reports published of PSMA-targeted PET imaging in meningioma patients, all of which were incidental findings in patients with prostate cancer . Often ta on MRI . Histopad by MRI . Courtneningioma . Finallye cancer .64Cu-PSMA tracer. Calabria et al. reported an MRI-detected foramen magnum meningioma with SUVmax of 3.8 at 1 hour after tracer administration and 3.9 at four hours postadministration [One report utilized the stration .18F-PSMA-1007 tracer. Haemels et al. reported a patient with moderately avid 18F-PSMA-1007 uptake in the occipital lobe [18F-PSMA-1007 binding, which could be inhibited by 2-PMPA.Similarly, one report utilized the tal lobe . This wan = 4 patients) of 68Ga-PSMA PET imaging in other CNS tumors, including CNS lymphoma, intracranial hemangiopericytoma, and hemangioblastoma. Sasikumar et al. reported two patients with histopathology-confirmed CNS lymphoma [68Ga-PSMA-11 (HBED-CC) uptake than 18F-FDG uptake: in the first patient, 68Ga-PSMA-11 (HBED-CC) uptake showed TBR of 17.0 , and 18F-FDG uptake showed TBR 4.85 ; in the second, 68Ga-PSMA-11 (HBED-CC) uptake showed TBR of 37.2, versus 3.6 for 18F-FDG. Patro et al. reported a patient with an intracranial hemangiopericytoma in the posterior fossa, which demonstrated intense 68Ga-PSMA-11 (HBED-CC) uptake and mild 18F-FDG enhancement [68Ga-PSMA-HBED-CC uptake in the cerebellum on PET/CT, which was confirmed histopathologically as a hemangioblastoma [There are an additional three case reports uptake on initial PET imaging. After four cycles of 177Lu-PSMA-617 therapy totaling 25.5\u2009GBq, the lesions demonstrated reduced size and PSMA expression. This correlated to a decrease in serum PSA from 195\u2009ng/mL to 2.4\u2009ng/mL. The second patient had one brain metastasis that was diagnosed after several hormonal and chemotherapy systemic treatments. This lesion showed 68Ga-PSMA-11 (HBED-CC) uptake on initial PET, and after three cycles of 177Lu-PSMA-617 therapy, the first of which was combined with radiotherapy; it had nearly completely regressed. This was accompanied by a decrease in serum PSA from 112\u2009ng/mL to 3.9\u2009ng/mL. Recently, Kumar et al. reported on the successful application of 177Lu-PSMA-617 therapy in a glioblastoma patient [68Ga-PSMA-11 (HBED-CC) uptake in the MRI-enhancing areas. The patient underwent three cycles of 177Lu-PSMA-617 for a total dose of 3700\u2009MBq, after which MRI and 68Ga-PSMA-11 (HBED-CC) PET demonstrated a reduction in lesion size (from 18\u2009mL to 5.4\u2009mL), which was accompanied by an improvement in ECOG score from 4 to 3. These positive results are tempered by a recent report from Parihar et al., which showed the emergence of new brain metastasis with 68Ga-PSMA uptake, after 177Lu-PSMA and 225Ac-PSMA treatment of prostate cancer [e cancer . The firpatients . The fir patient . The pate cancer .18F-DCFPyL could be administered and targeted to a specific brain focus using MRgFUS for BBB disruption [18F-DCFPyL uptake localized to the foci of BBB disruption and extended minimally beyond the contrast-enhancing MRI lesion. Target uptake plateaued at 80 minutes postadministration, with a maximal TBR of 7. PSMA-binding specificity was confirmed by administering the anti-PSMA ZJ-43 which lowered TBR fourfold. Although this was performed on healthy brains, it demonstrated the possibility of leveraging MRgFUS for opening the BBB to allow hydrophilic 18F-DCFPyL PSMA to enter and reach target tissue within the brain.MRI-guided focused ultrasound (MRgFUS) has been leveraged as a tool for disrupting the blood-brain barrier (BBB) in order to permit the passage of drugs, for a number of CNS indications . In a prsruption . FollowiThe PSMA-binding tracer mechanism of expression is thought to be specific to tumor vasculature, not to invading or proliferating cells. While this may limit its utility as an isolated imaging modality, it may prove to be a useful adjunct to other neuroimaging modalities for CNS tumors. On the other hand, the significant potential advantage of PSMA-targeted PET tracers is in CNS tumor theranostics, which the current literature only begins to describe. There may be the potential for a wide range of future applications, such as small molecule selective PSMA inhibitors and radiosensitizers. Pairing PSMA-targeted diagnostic imaging with PSMA-targeted therapeutics may yield a robust theragnostic option for CNS tumor patients.In summary, this systematic review of the literature on PSMA-targeted tracer use in CNS tumors demonstrates promising results about the potential for this PET tracer for neuro-oncologic imaging. Future efforts should continue to explore the potential for PSMA in neuro-oncologic imaging, specifically focusing on proposed mechanism(s) of PSMA expression in CNS tumors, differential imaging performance by CNS tumor type, direct comparison of PSMA-targeted PET to other neuroimaging modalities for diagnosing and monitoring CNS tumors, and direct comparison of competing PSMA-binding radiotracers. The surreptitious discovery of the utility of PSMA-targeted PET imaging in CNS tumors has invigorated much excitement in the field about its potential clinical application, but more research is needed to better understand this PET tracer before it can be implemented clinically."} +{"text": "Saccharomyces cerevisiaelacking macro-ER-phagy receptors would exhibit enhanced sensitivity to hygromycin B, which reduces translational fidelity and is expected to globally disrupt protein homeostasis, including at the ER. We observed that loss of either of two yeast macro-ER-phagy receptors (Atg39p or Atg40p) compromised cellular resistance to hygromycin B to a similar extent as loss of ER-associated degradation (ERAD) ubiquitin ligases Hrd1p and Doa10p. Our data are consistent with a model whereby macro-ER-phagy and ERAD collaborate to mediate ER protein quality control. Disruptions of macro-ER-phagy have been linked to neuropathy, dementia, and cancer. A dampened capacity to mediate protein quality control may contribute to these conditions.Receptor-mediated autophagic turnover of portions of the endoplasmic reticulum (ER) is mediated by macro-ER-phagy. We hypothesized macro-ER-phagy promotes proteotoxic stress resistance. We predicted Degradation of proteins at the endoplasmic reticulum (ER) is mediated by ER-associated degradation (ERAD) and ER autophagy (ER-phagy). In ERAD, ubiquitin ligases mark individual ER-localized proteins with ubiquitin for proteasomal degradation ). By contrast, ER-phagy involves the lysosomal destruction of larger portions of ER ). ER-phagy contributes to ER homeostasis under a range of conditions, including ER stress and nutrient limitation .Multiple unique forms of ER-phagy exist with distinct regulation and genetic requirements. In micro-ER-phagy, ER segments are directly degraded by the lysosome , independently of characterized core autophagy-related (Atg) proteins and autophagosome formation. Micro-ER-phagy is stimulated by ER stress (elevated abundance of misfolded ER proteins) . By contrast, macro-ER-phagy requires the core autophagic machinery. In macroautophagy, unique receptor proteins interact with organelle-specific cargo at the surface of an organelle . This interaction allows receptor proteins to associate with the phagophore, a double membrane that surrounds the compromised organelle and closes to form an autophagosome . Autophagosomes fuse with lysosomes to form autophagolysosomes, wherein encapsulated contents are degraded . Macro-ER-phagy promotes vacuolar degradation of excess integral membrane proteins and reduces accumulation of aggregation-prone ER proteins .Saccharomyces cerevisiae. Atg39p facilitates turnover of perinuclear ER, while Atg40p promotes cytoplasmic and cortical macro-ER-phagy . The mammalian homolog of Atg40p (FAM134Bp) enables ER-phagy by a similar mechanism .ATG39/ATG40expression and Atg39p/Atg40p-dependent macro-ER-phagy are induced by ER stress, nitrogen starvation, and rapamycin . Whether macro-ER-phagy is required under conditions of global proteotoxic stress has not been determined. We determined whetherATG39andATG40promote resistance to hygromycin B, an aminoglycoside produced byStreptomyces hygroscopicus. Hygromycin B distorts the ribosome aminoacyl site, thereby reducing translational fidelity, and is expected to broadly perturb the cellular proteome . We and others have previously shown several ER and nuclear ubiquitin ligases and associated proteins are required for optimal growth in the presence of hygromycin B . Given that approximately one third of eukaryotic proteins are imported into the ER en route to their subcellular or extracellular destination, we hypothesized that macro-ER-phagy collaborates with ER and nuclear ubiquitin ligases to promote survival during proteotoxic stress.Two proteins, Atg39p and Atg40p, serve as macro-ER-phagy receptors inDOA10andHRD1, and yeast lacking eitherATG39orATG40in the absence or presence of increasing concentrations of hygromycin B . Theatg39\u0394 andatg40\u0394strains were taken from the Yeast Knockout Collection . All strains exhibited similar growth in drug-free media. As previously observed ,hrd1\u0394doa10\u0394 yeast exhibited markedly reduced growth relative to wild type cells with increasing hygromycin B concentrations. Deletion ofATG39orATG40also sensitized yeast to hygromycin B, consistent with our hypothesis that macro-ER-phagy mitigates stress associated with a broadly disrupted proteome. To validate the observation that ER-phagy receptor loss impairs hygromycin B resistance, we evaluated growth of independently generated single and double knockout yeast lackingATG39and/orATG40. Consistent with data in Figure 1A, theseatg39\u0394 andatg40\u0394 yeast exhibited impaired growth in the presence of hygromycin B; simultaneous loss of both genes modestly further enhanced drug sensitivity .To investigate potential general contributions of Atg39p and Atg40p to protein quality control, we cultured wild type yeast, yeast lacking genes encoding both primary ERAD ubiquitin ligases,atg39\u0394atg40\u0394 relative to either single mutant is consistent with Atg39p and Atg40p possessing partially overlapping function, which may be expected given the physical continuity and partially overlapping proteome of the ER and nuclear envelope . Indeed, previous reports indicate contribution of both Atg39p and Atg40p to turnover of the same model macro-ER-phagy substrates .Our results demonstrate that macro-ER-phagy receptors are required for maximal resistance to the aminoglycoside hygromycin B. Modestly enhanced hygromycin B sensitivity ofATG40andSTE24) has been detected in a large-scale interaction study . Future experiments may be conducted to directly assess the overlap between macro-ER-phagy and ERAD in mitigating proteotoxic stress. Macro-ER-phagy defects are associated with a number of ailments in humans and mammalian research models, including neuropathy, dementia, and cancer . The extent to which impaired quality control contributes to the development of these conditions remains to be determined.A role for Atg39p and Atg40p in combatting proteotoxic stress is consistent with quality control functions of macro-ER-phagy receptors suggested by other reports . Additional studies will be required to determine whether (and by what mechanism) Atg39p and Atg40p abundance increases in the face of proteotoxic stress . Further, analyses of macro-ER-phagy-defective mutants cultured in the presence of drugs that perturb proteostasis by different mechanisms may better define the range of stress conditions against which Atg39p and Atg40p protect. Consistent with a role in protein quality control for macro-ER-phagy, a negative genetic interaction between mutations ofYeast growth assays. Yeast growth experiments were performed as described . Four \u03bcl of sixfold serial dilutions were transferred to agar plates containing yeast extract-peptone-dextrose (YPD) medium lacking or possessing hygromycin B (Gibco). Plates were incubated at 30\u00b0C and imaged on the indicated days."} +{"text": "EA \u2248 0.55 eV) observed during DCT measurements after a short trap-filling pulse indicates that current collapse is induced by deep trap states associated with iron (Fe) doping present in the buffer. Interestingly, analogous DCT characterization carried out after a long trap-filling pulse revealed yet another process with time constants of about 1\u20132 s and which was approximately independent of temperature. We reproduced the experimentally observed results with two-dimensional device simulations by modeling the T-independent process as the charging of the interface between the passivation and the AlGaN barrier following electron injection from the gate.Thanks to high-current densities and cutoff frequencies, short-channel length AlGaN/GaN HEMTs are a promising technology solution for implementing RF power amplifiers in 5G front-end modules. These devices, however, might suffer from current collapse due to trapping effects, leading to compressed output power. Here, we investigate the trap dynamic response in 0.15 \u03bcm GaN HEMTs by means of pulsed I-V characterization and drain current transients (DCTs). Pulsed I-V curves reveal an almost absent gate-lag but significant current collapse when pulsing both gate and drain voltages. The thermally activated Arrhenius process (with Short-channel length AlGaN/GaN high-electron mobility transistors (HEMTs) are an interesting technology option for realizing highly efficient power amplifiers in 5G communications systems, due to the high power/frequency handling capability and efficiency they offer . OperatiVGS,B,VDS,B) = V baseline and from = , , and V (VTH is device threshold voltage). Remarkably, the devices-under-test (DUTs) exhibited negligible gate-lag signals peaked the DCT measurements revealed a picture richer in texture compared to the previous case. As shown by Interestingly, when applying a long trap-filling pulse carried out after a short 1 \u03bcs trap-filling pulse revealed that current collapse was mainly due to Fe-doping related traps in the GaN buffer. In addition, DCTs carried out after long 100 s trap-filling pulse disclosed an additional slow"} +{"text": "Staphylococcus aureus (MSSA) infections are an important cause of morbidity and mortality in neonatal intensive care unit (NICU) infants. MSSA colonization is a risk factor for subsequent invasive infection . Active surveillance programs coupled with decolonization measures for MSSA-colonized infants have been associated with decreased invasive MSSA infection rates in the NICU setting.Invasive methicillin-susceptible In June 2020, an active surveillance program was implemented to detect MSSA nasal colonization for infants admitted to the St. Louis Children\u2019s Hospital NICU, a 140-bed quaternary NICU. MSSA-colonized infants were decolonized with a 7 day course of intranasal mupirocin twice daily plus topical chlorhexidine baths on days 1, 4, and 7 . MSSA bloodstream infection (BSI) rates were followed quarterly pre- (September 2018-May 2020) and post- (June 2020-March 2022) implementation of MSSA surveillance and decolonization. Mean MSSA BSI rates pre- and post-implementation were compared via paired T-test. The number of infants treated with mupirocin was calculated pre- and post-implementation.Figure): 0.22 per 1000 patient-days pre-implementation, 0.16 per 1000 patient-days post-implementation (p=0.37). Since initiating MSSA decolonization in June 2020, the number of NICU infants exposed to mupirocin increased nearly fourfold: mean 10 infants treated per month pre-implementation, mean 38 infants treated per month post-implementation.After implementing MSSA surveillance and decolonization there was no significant change in the mean MSSA BSI rate .All Authors: No reported disclosures."} +{"text": "Novel insights into the basic and translational findings on Epstein\u2013Barr virus-related diseases were presented at the \u201c19th International Symposium on Epstein\u2013Barr Virus and Associated Diseases\u201d in Asahikawa, Japan.Novel insights into Epstein\u2013Barr virus (EBV) pathogenicity were presented at the \u201c19th International Symposium on Epstein-Barr Virus and Associated Diseases\u201d in Asahikawa, Japan. In addition, basic and translational findings on EBV-associated tumors, including natural killer (NK)/T-cell lymphoma, gastric cancer, and nasopharyngeal cancer, were presented by an international group of scientists and clinicians. The \u201c19th International Symposium on Epstein-Barr Virus and Associated Diseases\u201d was held from 29\u201330 July 2021, at the Art Hotel in Asahikawa, Japan . It was oriP and single-strand cleavage, which is essential for replication termination and viral episomal maintenance. EBV-associated tumors suppress the expression of lytic and latent EBV antigens to evade immune detection. Rui Guo demonstrated that EBV+ Burkitt cells (BLs) subvert the methionine and folate metabolism pathways to support the EBV genome hypermethylation necessary for viral antigen silencing. Methionine restriction derepresses EBV lytic and latency antigens in EBV-infected BL cells, disrupting type I latency. Chong Wang compared resting B cell and lymphoblastoid cell line (LCL) genome-wide chromatin interaction maps to identify the genome architecture changes arising from EBV infection. The virus partly controls lymphocyte growth by globally reorganizing host genome architecture to facilitate the expression of key oncogenes. Emmanuela N Bonglack (Duke University School of Medicine) showed that the monocarboxylate transporters (MCTs) 1 and 4 responsible for lactate export and cancer cell proliferation are induced by Epstein\u2013Barr nuclear antigen 2 (EBNA2) and latent membrane protein 1 (LMP1), respectively. Dual MTC 1/4 inhibition suppresses the growth of LCLs and sensitizes them to killing by the electron transport chain complex 1 inhibitors phenformin and metformin. Jillian Bristol (University of Wisconsin\u2013Madison) applied bulk/single-cell RNA sequencing (RNA-Seq) and immunoblot analysis to compare cellular and viral gene expression in early-passage LCLs infected with type I or type II EBV. Enhanced lytic viral reactivation in type 2 EBV-infected B cells is associated with B cell receptor (BCR) signaling increased by interferon regulatory factor 4 (IRF4) downregulation. Huanzhou Xu (University of Florida) examined proteins at cellular replication forks, i.e., replisomes, in EBV-transformed B cells using the isolation of proteins on nascent DNA (iPOND) and mass spectrometry. Eight novel proteins are associated with replisomes at cellular replication forks in LCLs and BLs. One zinc finger protein acts directly on fork progression and enhances B-cell proliferation.Session 1 focused on viral latency and pathogenesis. Jayaraju Dheekollu (The Wistar Institute) demonstrated that an amino acid residue in Epstein\u2013Barr nuclear antigen 1 (EBNA1) is required for episomal maintenance in EBV. Tyrosine 518 is indispensable for forming an EBNA1\u2013DNA crosslink with the EBV origin of plasmid replication Session 2 focused on viral replication and reactivation. Eric M Burton (University of Florida) reported that the NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome, a cellular defense system against infectious agents, initiates an EBV lytic cascade in response to danger signals. Entry into the lytic phase allows the virus to escape from damaged cells. Nick Van Sciver (University of Wisconsin\u2013Madison) demonstrated that the Hippo signaling effectors, yes-associated protein 1 (YAP), and tafazzin (TAZ) induce the EBV lytic cycle in epithelial cells. Their known activator lysophosphatidic acid, commonly expressed in saliva, promotes EBV lytic reactivation in epithelial cells via a YAP/TAZ-dependent mechanism. Quincy Rosemarie (University of Wisconsin\u2013Madison) used a library of 293 cells carrying EBV with single-gene knockouts of its lytic replication genes. The reorganization of chromatin in the EBV lytic phase is required for expressing core lytic replication genes but not late lytic genes. Dinesh Verma (University of Utah School of Medicine) revealed that EBV lytic reactivation from latency enhances severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) entry into epithelial cells. The EBV Zta protein, essential for EBV lytic cycle entry, upregulates cellular angiotensin-converting enzyme-2 (ACE2), a SARS-CoV-2 receptor, during EBV lytic replication. Although anti-ACE2 antibodies block the ACE2-dependent entry of SARS-CoV-2 into epithelial cells, EBV infection enhances it. Rodney P Kincaid used fluorescence-activated cell sorting-based genome-wide CRISPR/Cas9 knockout screening and single-cell RNA sequencing. Viral- and host-encoded miRNAs modulate the EBV transcriptional reactivation process at the single-cell level.+) Th1-like state in infected CD4+CCR4+ T cells via T-box transcription factor expression. The cells consequently produce interferon (IFN)-\u03b3 to stimulate astrocytes. Stimulated astrocytes secrete CXCL10, which recruits infected cells via CXCR3, constituting a T-helper type 1-centric positive feedback loop. This loop causes chronic inflammation in the central nervous system in HAM/TSP. Yamano also showed the genomic changes predicting the features that lead to the development of ATLL in patients with HAM/TSP.At the luncheon seminar, Yoshihisa Yamano (St. Marianna University School of Medicine) presented the pathogenesis and genomic changes during leukemic transformation in patients with human T-lymphotropic virus type 1 (HTLV-1)-associated neuroinflammatory diseases. Patients with HTLV-1 can develop aggressive adult T-cell leukemia/lymphoma (ATLL) and/or the debilitating inflammatory disease HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Yamano demonstrated that HTLV-1 induces a C-X-C motif chemokine receptor 3-positive (CXCR3+ tumor-infiltrating T cells. Atsushi Okabe and Harue Mizokami (Chiba University) analyzed the genome-wide chromatin topology of normal and gastric cancer cells obtained by chromosome conformation capture combined with high-throughput sequencing. The EBV genome directly interacts with and remodels the host heterochromatin via epigenetic modifications, facilitating proto-oncogene expression and tumorigenic transformation. This phenomenon, termed \u201cenhancer infestation,\u201d can be observed in EBV+ gastric and nasopharyngeal carcinoma cell lines. Mariko Yasui (The University of Tokyo) analyzed the gene expression of monolayer and sphere-forming cells from EBV+ gastric carcinoma (EBVaGC). The latent membrane protein 2A (LMP2A) contributes to maintaining sphere-forming cancer stem-like cells through the nuclear factor-kappa B (NF-\u03baB) pathway in EBVaGC. Soichiro Fukuda (Yamaguchi University Graduate School of Medicine) proved that MC180295, a novel demethylating compound, could decrease the expression of BRCA1 and cell cycle-related genes and inhibit the proliferation of EBVaGC cell lines by suppressing the DNA and cell cycle. Hisashi Iizasa (Shimane University) reported that Helicobacter pylori increases the levels of low-affinity EBV receptors ephrin type-A receptor 2 (EphA2) and non-muscle myosin IIA (NMHC-IIA) and elevates the efficiency of EBV infection in gastric epithelial cells. This elevation depends on the adhesion of H. pylori to epithelial cells.Session 3 focused on nasopharyngeal cancer (NPC) and gastric cancer. Satoru Kondo (Kanazawa University) showed that Japanese patients with NPC possess type 2 EBV or EBV strain associated with intermediate risk (BALF2 H-H-L), which is different from that in the endemic region of Southern China (BALF2 H-H-H). Xia Yu reported that the diagnostic yield for NPC can be improved by measuring an anti-BNLF2b antibody in a study of more than 30,000 patients with NPC. Anti-BNLF2b antibody has better specificity and sensitivity as a diagnostic marker than EBV-DNA, anti-VCA IgA, or anti-EBNA1 IgA. Qian Zhong (Sun Yat-sen University Cancer Center) performed the single-cell RNA sequencing of NPC biopsy samples. Malignant cells expressing dual immune and epithelial features are associated with poor prognosis. This dual feature positively correlates with the expression of co-inhibitory receptors on CD8In an invited lecture, Kwok-Wai Lo (The Chinese University of Hong Kong) presented the whole-genome profiling of EBV-associated nasopharyngeal carcinoma. It is the most extensive whole-genome catalog of EBV-associated NPC and viral oncogene expression, revealing the mutational pattern of the genome, including a new homologous recombination defect signature prevalent in NPC. Moreover, recurrent structural alterations and homozygous deletions alter NF-\u03baB and transforming growth factor (TGF)-\u03b2 signaling, cell cycle, and impaired immunity. These aberrations and viral gene expression undermine innate and adaptive immunity in 79% and 53% of the cases, respectively, indicating that viral and somatic events subvert antitumor immunity in most patients with NPC. In addition, the catalog showed frequent alterations in TGF-\u03b2 receptor type 2 TGFBR2 and cyclin-dependent kinase inhibitor 2A (CDKN2A), which define a novel viral\u2013somatic interaction promoting persistent EBV infection for NPC pathogenesis.Mei-Ru Chen delivered keynote lecture 1, describing how EBV overcomes cellular barriers and reorganizes cellular organelles for virion replication and assembly. Various EBV lytic proteins regulate and modulate the nuclear envelope structure, such as viral BGLF4 kinase and the nuclear egress complex BFRF1/BFRF2. After nuclear egress, lesser amounts of viral BGLF4 kinase and the single-stranded DNA-binding protein BALF2 are present in the concave nuclear region of TW01-EBV cells, along with tegument proteins. Thus, EBV appears to use multiple types of cellular machineries for maturation, which may disturb cellular homeostasis and lead to EBV pathogenesis.+ T or NK cells in the peripheral blood (EBV-T/NK-LPD). Ayaka Ohashi (St. Marianna University School of Medicine) showed that the plasma level of interleukin (IL)-1\u03b2 in CAEBV could be a biomarker of angiopathy. Peripheral blood monocytes are considered a source of IL-1\u03b2. Yuriko Ishikawa elucidated the role of mucosal-associated invariant T cells in EBV-T/NK-LPD by checking peripheral blood from patients. The activation level of the cells depends on disease severity and IL-18 levels; thus, they may be involved in EBV T/NK-LPD immunopathogenesis. Paul J Collins (University of Birmingham) reported that an increase in myeloid-suppressor cells supports immune evasion, leading to EBV+ T/NK cell proliferation in patients with CAEBV. Conventional steroid therapy for symptom relief might exacerbate the underlying disease. Hydroa vacciniforme-like lymphoproliferative disorder (HV-LPD) is a cutaneous form of CAEBV. Keiji Iwatsuki (Okayama University Graduate School of Medicine) observed atypical \u03b1\u03b2T and \u03b3\u03b4T cells expressing NK cell markers (CD16 and CD56) in HV-LPD. Miki Takahara presented the clinical characteristics of 62 patients with early-stage nasal NK/T-cell lymphoma. The five year disease-specific survival rate of the 18 patients treated with concurrent arterial infusion chemoradiotherapy was 94%. Mayumi Yoshimori (St. Marianna University School of Medicine) demonstrated that EBV+ NK cells produce IFN-\u03b3, enhancing the differentiation and activation of macrophages. The activation leads to lethal complications such as hemophagocytic lymphohistiocytosis. Nenad Sejic reported that B-cell lymphoma 2 (BCL2) homology domain 3 (BH3)-mimetic drugs, small compounds that antagonize anti-apoptotic BCL-2 family proteins, decrease BCL-XL expression, inducing the apoptosis of CAEBV and NK/T-cell lymphoma cell lines. Julien Lupo detected soluble ZEBRA proteins (sZEBRA) with an ELISA assay in plasma samples from more than 300 patients who underwent organ transplantation. The protein levels were higher in patients with PTLD and graft-versus-host disease. Measuring sZEBRA proteins could help identify patients likely to develop severe outcomes during the critical post-transplant period.Session 4 focused on diseases and disorders, including chronic active EBV infection (CAEBV), EBV-associated T/NK cell lymphoproliferative diseases (EBV-T/NK-LPD), nasal NK/T-cell lymphoma, and post-transplant lymphoproliferative disorders (PTLDs). CAEBV is a progressive disease with persistent inflammatory symptoms accompanied by clonally proliferating EBV+ gastric carcinoma.Paul Farrell presented the course of his EBV research history at the Henle Lecture. He first discussed the nature of EBV, which is associated with a wide range of diseases, including infectious mononucleosis, and contributes to several types of cancer, but is also possibly involved in other chronic diseases. A comprehensive understanding of the EBV genome has paved the way for modern EBV research, establishing coherent models for diverse types of EBV infections. The EBV genome variations, type 1 and 2, induce phenotypic changes, and some geographic variations may explain the diverse incidence of several EBV-related diseases. Moreover, some deletions have been observed in the EBV genome infected with T or NK cells and can serve as markers for the disease. In addition, drugs that cause EBV reactivation can be used in patients with EBV+ CAEBV cells. Hence, these pathways may be attractive therapeutic targets for eradicating these cells in CAEBV.Ayako Arai (St. Marianna University School of Medicine) presented recent discoveries on CAEBV in a morning seminar. Ever since CAEBV was added to the World Health Organization classification of tumors of hematopoietic and lymphoid tissues in 2017, numerous reports on the disease have been recorded. Retrospective surveys in Japan have shown that chemotherapies are insufficient to resolve disease activity and eradicate infected cells. Because the only option for curing CAEBV is allogeneic hematopoietic stem cell transplantation, establishing novel treatments is needed. Constitutively activated NF-\u03baB and STAT3 pathways contribute to immortalization and cytokine production in EBV+/CD8+ T cells and the ratio of CD4/CD8 T cells are upregulated in the peripheral blood mononuclear cells (PBMCs) of patients with multiple sclerosis (MS) and correlate with disease severity and the alteration of immune response to EBV. These results suggest that CD4+CD8+ T cells play a pathogenic role in multiple sclerosis. Pascal Polepole (University of Nebraska) found that xenografts of LCL exacerbate experimental autoimmune encephalomyelitis, which mimics a key feature of MS, in C57BL/6 mice, suggesting that EBV-induced B-cell proliferation exacerbates MS. These xenografts have diverse changes in the gut microbiome and modulate the blood\u2013brain barrier. Adityarup Chakravorty (University of Wisconsin\u2013Madison) demonstrated that the EBV oncogene EBNA1 directly suppresses NK cell activation by repressing the NK cell-activating NKG2D ligands ULBP1 and ULBP5 in infected B cells. Because EBNA1 is expressed in all EBV-related tumors, its suppression may inhibit tumor growth. Ashely Campbell (University of Toronto) performed affinity purification coupled with mass spectroscopy, identifying host proteins interacting with EBV protein BGLF2. They interact with the miRNA-induced silencing complex (RISC) and interfere with cellular miRNA function. Moreover, BGLF2 inhibits let-7 miRNA and induces SUMOylation by increasing free SUMO levels. Phillip Ziegler proved that pseudostratified epithelial cells are susceptible to EBV infection using 3D air\u2013liquid interface cultures. Moreover, all four cell types, basal, suprabasal, mucosecretory, and ciliated, can harbor the infection. Single-cell RNA profiling of the cells identified many EBV transcripts associated with latent, abortive, and lytic infections.Session 5 focused on virus\u2013host interactions and immunity. Elliott SoRelle (Duke University) profiled the transcriptomes of five different EBV-infected LCLs with single-cell RNA-Seq analysis, revealing a considerably heterogenic LCL transcriptome profile. These findings indicate that LCL is a continuum of various transcriptional states. Post-infection dynamic studies will provide a complete picture of EBV-induced events. Anna Gil reported that CD4+ tumor cells in a murine xenograft model.Session 6 focused on lymphomagenesis and therapeutics. Ben Gewurz showed that ferrireductase CYB561A3 is critical for the proliferation of Burkitt\u2019s lymphoma, but not LCLs. CYB561A3 knockout Burkitt lymphoma cells show catastrophic lysosomal and mitochondrial damage and impaired mitochondrial respiration. Wei Bu identified a set of human monoclonal antibodies targeting five different antigenic sites on EBV gH/gL. These antibodies prevent EBV infection in both epithelial and B cells. One of the antibodies causes nearly complete protection of humanized mice from viremia after the EBV challenge. Ibukun A Akinyemi (University of Florida) found that the c-Fos/activator protein (AP)-1 inhibitor T-5224 inhibits BamHI Z fragment leftward open reading frame 1 (BZLF1) promoter activity, viral lytic gene expression, and EBV production in Burkitt lymphoma cells. It also retards the immortalization and outgrowth of LCLs, possibly via ZEBRA/phosphoinositide 3-kinase (PI3K) pathway inhibition. Blachy J Davila Saldana (George Washington University) assessed an international cohort of 59 patients with CAEBV outside Asia, showing that hematopoietic stem cell transplantation (HSCT), especially early in the disease course, is the only curative therapy for CAEBV. Sang-Hoon Sin (University of North Carolina at Chapel Hill) generated a transgenic mouse (d.197) carrying the entire 140,000 bp genome of Kaposi\u2019s sarcoma-associated herpesvirus. Within 100 days, 10% of the mice developed angiosarcoma, which mimicked human Kaposi\u2019s sarcoma pathology. Sandhya Sharma (Baylor College of Medicine) successfully induced functional T cells reactive to four EBV type 2 latency proteins from CD45RA-depleted PBMCs. These T cells decrease the metastatic spread of autologous EBVO-GlcNAcylation and the addition of O-linked \u03b2-N-acetyl-d-glucosamine moiety to serine or threonine residues in hematopoiesis. This moiety is conserved throughout eukaryotes and is a fundamental regulator of numerous biological processes. For instance, O-GlcNAcylation has essential and pleiotropic roles in the differentiation, proliferation, and function of various cells, such as T, B, and polyphyletic progenitor cells. Aberrant O-GlcNAcylation promotes hematological malignancies through disordered epigenetics, metabolism, and gene transcription.At the luncheon seminar, Hideaki Nakajima (Yokohama City University Graduate School of Medicine) described the role of Session 7 focused on viral infections and immunity. Takayuki Murata showed that EBV infection induces programmed death-ligand 1 (PD-L1) expression in primary B cells through EBNA2, which binds to the promoter region of the PD-L1 gene. Accordingly, EBV-infected B cells may escape antiviral immunity via the PD-1/PD-L1 pathway. Keiko Nagata (Tottori University) demonstrated that the reactivation of EBV in B cells produces autoreactive antibodies, which may exacerbate Graves\u2019 disease. Danling Dai (Sun Yat-sen University Cancer Center) reported that the EBV transcriptional factor BZLF1 decreases the N6-methyladenosine modification of Kruppel-like factor 4 (KLF4), subsequently increasing its protein level. The KLF4 upregulation promotes EBV infection of nasopharyngeal epithelial cells, resulting in a positive feedback loop between EBV and host molecules. Yoshitaka Sato (Nagoya University Graduate School of Medicine) showed that EBV-infected cells release exosomes containing EBV tegument protein BGLF2. Exosome-encapsulated BGLF2 enhances EBV gene expression, supporting the viral infection. These results suggest that tegument proteins are functional in extra-viral particles, such as exosomes. Xiangwei Kong (Sun Yat-sen University Cancer Center) created a unique model of a varicella-zoster virus (VZV)-based EBV vaccine, displaying the prefusion form of EBV glycoprotein B on its surface. Vaccination against EBV glycoprotein B elicits an EBV neutralization effect in both B and epithelial cells.EBV is associated with 10% of gastric carcinomas (EBVaGCs). Unique clinicopathological features have been indicated in EBVaGC, such as male predominance, predominant location in the proximal stomach, lymphoepithelioma-like histology, and a favorable prognosis. Masashi Fukayama presented his findings on EBVaGC in an invited lecture. In the earliest phase of EBVaGC development, single or a few EBV-infected epithelial cells initiate neoplastic proliferation within the stomach mucosa, potentially via epigenetic regulation such as DNA methylation. EBVaGC cells harbor characteristic genetic alterations, including mutations in AT-rich interaction domain 1A (ARID1A) and PD-L1. ARID1A protein deficiency increases susceptibility to EBV, and PD-L1 overexpression allows the tumor cells to escape the host immune system. Concerning EBV contribution, LMP2A maintains cancer stem-like cells and resistance to apoptosis. Moreover, EBV-derived microRNAs participate in EBVaGC transformation and remodeling of the tumor microenvironment by affecting surrounding stromal cells through exosomes.EBV-associated T/NK-cell malignancies are refractory to conventional chemotherapy and have poor prognoses. Although the transforming capacity of EBV in B cells is well known, the molecular pathogenesis of EBV-caused clonal expansion in T/NK cells is still elusive. As the keynote speaker 2, Hiroshi Kimura (Nagoya University Graduate School of Medicine) described the relationships between EBV and T/NK cells and possible infection of progenitor cells prior to T- or NK-cell differentiation, suggesting a new explanation for how EBV infects T/NK cells. Moreover, EBV gene deletions around the BART microRNA cluster region have been found in CAEBV and EBV-associated T/NK-cell lymphoma, indicating that these deletions are responsible for the neoplastic proliferation of EBV-infected cells.INK4a. Infecting B cells with EBNA3 mutant EBV strain showed that EBNA3C controls p16INK4a. Vishwanath Kumble Bhat (University of Sussex) identified novel variations in EBV EBNA1 sequences from specific geographical regions. Some variations cause amino acid changes in the EBNA1 DNA-binding domain, influencing DNA binding affinity. As EBV manipulates autophagy during the latent and lytic cycles, Maria Pena Francesch (University of Zurich) discovered that the capsid scaffold proteins BVRF2 and BdRF1 interact with the autophagosome marker LC3B. This interaction affects virus production through the virus assembly process.Session 8 focused on viral variation and the environment. Misako Yajima presented an approach to clone and sequence the entire EBV genome obtained from tonsil-derived spontaneous LCLs, uncovering seven distinct EBV strains. Phylogenetic analyses revealed that the subgroup of nasopharyngeal carcinoma-derived EBV strains in endemic areas differs from that in non-endemic areas, such as Japan. Yusuke Okuno found 150 microdeletions in the EBV genome derived from various EBV-related malignancies and diseases. Because most of the deleted lesions are shared between the disorders, specific deletions may contribute to the neoplastic phenotype of infected cells. Xiao Zhang (Sun Yat-sen University Cancer Center) presented the ultrastructure of EBV virion components\u2014the icosahedral capsid, dodecameric portal, and capsid-associated tegument complex\u2014using cryo-electron microscopy. This model helps understand the mechanisms of EBV genome retention and ejection. Yufeng Chen (Karolinska Institute) reported that cigarette smoking is associated with EBV reactivation, as defined by serum IgA antibody levels against EBV capsid antigen and EBNA1. Other environmental factors, such as alcohol and salted fish consumption and various residential and occupational exposures, were not associated with EBV reactivation. Ezgi Akidil succeeded in genome engineering primary resting human B cells using CRISPR-Cas9 technology. In this system, B cells carry a knockout of CDKN2A, which encodes the cell cycle regulator p16In addition to the oral presentations in the main symposium, 112 posters presented online. Ka Wo (The University of Hong Kong) showed that most infectious mono-derived EBV genomes belong to type 1 EBV. A genome-wide association study did not detect any EBV variant that is specific to EBV mono-infection. Yoshitaka Aoki (Kanazawa University) showed the prevalence of EBV in the adenoid and palatine tonsils of adults. Viral DNA is detectable in 74% and 71% of the adenoids and tonsils, respectively. Asuka Nanbo (Nagasaki University) described the mechanism of releasing EBV virions from the host cell into the extracellular milieu. Three Rab GTPases, Rab8a, Rab10, and Rab11a, induce the release of EBV infectious virions via the secretory pathway.Julia Myers (LSUHSC\u2013Shreveport) described a mechanism of viral interference. Because HPV16 E7 facilitates the degradation of retinoblastoma protein (Rb) to promote S-phase progression, Rb knockdown rafts exhibit reduced EBV replication. Xiaomei Deng (Sun Yat-sen University) demonstrated that dengue virus infection reactivates EBV through the PI3K pathway. Likewise, EBV infection promotes dengue virus propagation in EBV-infected cells, suggesting that some viruses can coexist with EBV.p62 is a ubiquitin sensor and signal-transducing adaptor with multiple functions in diverse contexts. Shunbin Ning (East Tennessee State University) showed that p62 participates in LMP1 signal transduction via TRAF6 ubiquitination and activation. Silencing p62 impairs the ability of LMP1 to regulate target gene expression. Ling Wang (East Tennessee State University) reported that p62 depletion causes a substantial activation of DNA repair mechanisms. Eiji Kobayashi (Kanazawa University) described how LMP1 is sorted into exosomes. LMP1 is physically associated with the ubiquitin C-terminal hydrolase L1 (UCH-L1), and the C-terminal farnesylation of UCH-L1 directs LMP1 to exosomes. Inhibiting farnesylation reduces LMP1 amounts in exosome fractions. Thus, restricting the exosome-mediated transfer of prometastatic molecules during cell-to-cell communication is a potential therapeutic target.+ gastric cancer cells. Lok Yiu Avala Ngan (The University of Hong Kong) demonstrated that only early passages of nasopharyngeal cancer cells are activated to undergo lytic infection by hypoxia, suggesting that passage number is critical in research using nasopharyngeal cancer cell lines. Hyemi Kim (Yonsei University College of Medicine) showed that gemcitabine induces lytic activation in EBV-associated cancers. The ZID domain plays a significant role in the lytic cycle entry through p53, which was confirmed in mouse- and patient-derived cancer organoid models. By contrast, Jun Li (Qingdao University) showed that the EBV latent protein LMP1 induces p53 protein expression via the lncRNA H19/miR-675-5p axis. Thus, the function of p53 in the balance between EBV lytic and latent cycles may be context-dependent. The latent EBV protein is considered to drive oncogenesis. However, Hirotomo Dochi (Kanazawa University) demonstrated that ZEBRA, a trigger regulator of the lytic cycle, is expressed in EBV+ nasopharyngeal cancer and is related to a worse prognosis.Blue-leaf A Cordes (University of Wisconsin) showed that hypoxia induces the lytic cycle through BZLF1. Hypoxia-inducing reagents kill EBV+ gastric and nasopharyngeal cancers express PD-L1, a negative immune checkpoint. Yui Hirata-Nozaki found that the c-Met protein contains a helper CD4 epitope that elicits antitumor T cell responses against EBV+ NK/T-cell lymphoma. In addition, HGF-c-Met signaling induces NK/T-cell lymphoma proliferation in an autocrine manner. Sandhya Sharma succeeded in inducing EBV-specific T cells that react to lytic and latent EBV proteins. These T cells produced rapid tumor clearance in a xenograft model.Immunoregulation may be critical to developing curative treatments for virus-related diseases. Shouhei Miyagi (Nagoya University Graduate School of Medicine) reported the inhibition of stimulator of interferon genes (STING), a central adaptor that allows DNA sensors to recognize exogenous cytosolic DNA to activate the type I interferon signaling cascade and reduces the transformation activity of EBV. Sho Sasaki (Yamaguchi University Graduate School of Medicine) and Kina Kase (Kanazawa University) demonstrated in separate studies that EBVDue to the COVID-19 outbreak, this symposium was the first hybrid (online and face-to-face) meeting in the history of EBV meetings. The benefits of online meetings other than infection prevention are the lack of travel expenses, low attendance costs, and saving travel time. The chairs and presenters from the United States, Europe, and Asia successfully presented their results and discussed them online in this symposium. One drawback of the online meeting that we were confronted with during its preparation was organizing it while considering the geographic location of each speaker. Because of this issue, the categorization of each session was challenging, and we regret that the presentation style was not as the presenters wished. This meeting provided an overview of exciting topics in the EBV research field and related diseases. The development of modern technologies, for example, CRISPR-based gene knockout and single-cell analysis, enables us to conduct detailed, in-depth, and accurate research on EBV. Hence, we learned that EBV might play various roles in the tumorigenic transformation of diverse immune and epithelial cells. The geographical disparities in EBV-related diseases have not been fully elucidated; however, they may be explained by the sequence variation from different EBV strains. A greater understanding of EBV would boost the establishment of a novel virus-specific treatment for EBV-related malignancies. Thus, we eagerly look forward to the next EBV symposium, which will be held in Siena, Italy, in 2022."} +{"text": "This cross-sectional quantitative sub-project assessed the association of organizational context (modifiable elements of work environments) with quality indicators (QIs) at the clinical microsystem (care unit) level. We used TREC data collected 09/2019-03/2020. The sample included 285 care units within 91 Western Canadian nursing homes. Outcomes included thirteen practice-sensitive QIs derived from the Minimum Data Set 2.0. Results from random-intercept logistic regression for each dichotomized QI showed that higher unit-aggregated scores on contextual elements as identified by the Alberta Context Tool, specifically care aide participation in decision-making , care aide perceived staffing and time for completing tasks , and care aide rated unit-level leadership , were associated with a better unit-level performance on delirium symptoms, indwelling catheter use, behavioral symptoms, pain, and late-loss physical function. The findings suggest that targeting modifiable contextual elements is an important avenue for quality improvement interventions in nursing homes."} +{"text": "It is not known how ethnic differences in COVID-19 deaths in the Netherlands evolved throughout the pandemic, especially after introduction of ethnicity-oriented COVID-19 prevention measures. We investigated associations between ethnicity and COVID-19 deaths across first wave of the pandemic, inter-wave period, and second wave in the Netherlands.We obtained multiple registry data from Statistics Netherlands spanning from 01 March 2020 to 14 March 2021 comprising of 17.4 million inhabitants. We estimated incidence rate ratios (IRRs) for COVID-19 deaths among ethnic groups using Poisson regression models and adjusted for relevant socio-demographic factors. We used similar models to estimate IRRs for non-COVID-19 deaths among ethnic groups.Ethnic minority populations exhibited higher risk of COVID-19 deaths than the Dutch origin population throughout various study periods. The most elevated risk of COVID-19 deaths was in populations originating from low- and middle-income countries, especially those with Turkish, Moroccan, and Surinamese background. The elevated risk of COVID-19 deaths among ethnic minority groups (as compared to Dutch origin population) was higher in inter-wave period (4 times higher) and second wave (2 times higher) when compared to the first wave (1.5 times as higher). Ethnic differences in COVID-19 deaths were larger compared to non-COVID-19 deaths.Ethnic differences in COVID-19 deaths persisted across first wave, inter-wave period and second wave in the Netherlands despite introduction of ethnicity-oriented prevention measures. Research on explanatory mechanisms and novel prevention measures are needed to address the ongoing differences in COVID-19 deaths across ethnic groups.\u2022\u2002Ethnic differences in COVID-19 deaths persisted in the Netherlands despite introduction of ethnicity-oriented prevention measures.We therefore call for better prevention measures.\u2022\u2002Well known drivers of SARS-CoV-2 infection such as household wealth, did not explain our findings calling for an in-depth understanding of drivers of ethnic differences in COVID-19 deaths."} +{"text": "JCI, Moderbacher et al. characterized SARS-CoV-2\u2013specific CD4+ and CD8+ T cell responses elicited by one or two doses of NVX-CoV2373 in individuals enrolled in a phase I/IIa trial. Substantially increased spike-specific CD4+ and T follicular helper cells were found after the first or second vaccine dose, with some individuals developing a modest spike-specific CD8+ T cell response. Correlation analysis revealed an association between spike-specific CD4+ T cells and neutralizing antibody titers. Notably, preexisting T cell immunity showed negligible effects on NVX-CoV2373\u2013induced T cell responses. These findings indicate that the protein-based vaccine NVX-CoV2373 induces robust T cell immunity capable of recognizing SARS-CoV-2 antigens and supporting humoral immune responses.The SARS-CoV-2 vaccine NVX-CoV2373 is a protein-based vaccine that might circumvent the difficulties in distributing mRNA vaccines to regions with limited access to cold-chain and refrigeration. However, the NVX-CoV2373\u2013induced T cell and antibody responses remain poorly understood. In this issue of the Evaluating the magnitude, longevity, and antigen recognition of T cell and neutralizing antibody responses is the gold standard to understand the quality of the vaccine-elicited immune response. Examining these immunological parameters also helps understand the mechanisms of action and the duration of a vaccine-induced protection, which assists the formulation of future vaccine strategies to combat a pathogen. Vaccine platforms such as mRNA vaccines and a viral vector\u2013based vaccine efficiently induce these functional immune cells upon administration in humans for emergency use in individuals aged 18 and above. It has been shown to be safe and effective in providing protection against severe COVID-19 . Differeadjuvant .+ T cells provide help to antibody and cytotoxic immune responses against pathogens assay with SARS-CoV-2 spike peptide stimulation cells are specialized CD4+ T cells that provide help to the antibody-based immunity in germinal centers T cells after the first and second vaccinations with NVX-CoV2373 compared with baseline CD4+ T cells such as BA.4 and BA.5 show higher neutralization escape than the ancestral strains of SARS-CoV-2 (This clinical study by Moderbacher et al. indicates that protein-based vaccine NVX-CoV2373 induced functional human antibodies as well as CD46 months . It woulRS-CoV-2 , 27. To"} +{"text": "Dear Editor,Patients with cancer, especially with hematological malignancies, were encouraged to be vaccinated early for SARS-CoV-2. Yet, these patients with dysfunctional or depleted B-lymphocytes frequently fail to reach positive SARS-CoV-2 anti-spike IgG titers after vaccination. To prevent severe COVID-19, combined monoclonal neutralizing antibodies were approved as pre-exposition prophylaxis (PreP) for these patients .+/CD20+-positive cells).We report here on three CD20-depleted lymphoma patients, showing negative SARS-CoV-2 anti-spike IgG after two vaccinations. Thus, patients were treated with casirivimab/imdevimab infusions that were recommended to be applied monthly. Due to the development of novel virus variants and its ineffectiveness against BA.1, we stopped the treatment. When the patients were tested for SARS-CoV-2 anti-spike IgG 12\u00a0weeks after the last infusion and four weeks after booster vaccination, we detected extremely high titers of SARS-CoV-2 anti-spike IgG . These IgGs were most likely not induced by active vaccination, but rather by the application of casirivimab/imdevimab. This finding suggests that passive immunization with the monoclonal antibodies casirivimab/imdevimab results in antibody titers that are detectable in the blood much longer than one month, which coincides with the described half-life [The fact that SARS-CoV-2 PrEP causes positive SARS-CoV-2 anti-spike IgG titers over weeks \u2014 especially with high proportion of surrogate neutralization antibodies \u2014 will complicate to define response rates after extra booster vaccinations in patients with dysfunctional B-lymphocytes. We recommend to take this information into consideration, as new combined monoclonal antibodies are currently available for SARS-CoV-2 PrEP that are highly effective against BA.1, for example tixagevimab/cilgavimab has to be administered only once every six months.In summary, (1) testing for SARS-CoV-2 anti-spike IgG titers after extra booster vaccinations is challenging in patients having received monoclonal SARS-CoV2 antibodies and (2) monoclonal antibody treatment has to be taken into account when testing for titers is performed. In case of new vaccines being effective against BA.1 and BA.2, only measurements of live-cell neutralization antibodies would help to discriminate between PrEP and vaccination response rates \u2014 which would not be eligible for standard diagnostics due to the complex analysis."} +{"text": "Tetranychus urticae) is an opportunistic feeder able to utilize an astounding array of plant species is not a typical host. The defense repertoire of Arabidopsis includes amino acid-derived glucosinolates. Once synthesized, glucosinolates, in their glycoside form, are stored in plant cells as innocuous precursors. Upon tissue disruption caused by herbivore attack, for example, these glycosidic precursors are hydrolyzed, releasing bioactive compounds, including isothiocyanates or nitriles to two Arabidopsis genotypes: either the well-defended wild-type Col-0 (Col-0) or the vulnerable cytochrome P450 CYP79B2 CYP79B3 double mutant (cyp79b2 cyp79b3) that lacks the ability to synthesize indole glucosinolates were sequenced in triplicate, enabling the authors to quantify expression level and coding sequence differences. The authors identified 59 transcripts more highly expressed in Col-0-adapted mites than cyp79b2 cyp79b3-adapted and bean-adapted mites, including several involved in detoxification of foreign metabolites (i.e. xenobiotics) (In this issue of biotics) .TuCAS, which encodes \u03b2-cyanoalanine synthase (CAS), stood out to the authors. Baseline TuCAS expression was uniform across the three mite genotypes when fed bean leaves. However, when mites were reared on Arabidopsis leaves (either Col-0 or cyp79b2 cyp79b3), TuCAS expression was induced in Col-0-adapted mites, but not bean-adapted or cyp79b2 cyp79b3-adapted mites. The authors puzzled over why TuCAS would be induced in Arabidopsis-fed spider mites. Due to its ability to detoxify cyanide, TuCAS is required for spider mites to handle consumption of cyanide-releasing cyanogenic glycosides, but these defense compounds are not found in Arabidopsis. What benefit could higher TuCAS expression possibly confer to spider mites feeding on Arabidopsis leaves? Is TuCAS inducibility actually involved in spider mite adaptation to Arabidopsis or is it simply a byproduct of evolution? To answer these questions, the authors needed to understand the role TuCAS plays, if any, in enabling spider mites to feed on Arabidopsis.One of the transcripts enriched in Col-0-adapted mites, TuCAS, the authors employed RNA interference (RNAi) to silence its expression in Col-0-adapted mites. TuCAS-silenced Col-0-adapted mites suffered decreased fecundity compared with untreated Col-0-adapted mites when reared on Col-0 leaves but suffered no ill effects when reared on bean leaves. These results demonstrated that TuCAS enables Col-0-adapted mites to successfully feed on Arabidopsis, though the \u201cwhy\u201d remained elusive. Does the inducible Col-0-adapted TuCAS allele (i.e. gene variant) confers a greater cyanide detoxification capacity? To address this question, they challenged mites with increasing concentrations of potassium cyanide and found that Col-0-adapted mites were, in fact, more cyanide-tolerant than their bean-adapted ancestors.To investigate the function of cyp79b2 cyp79b3, indicating that indole glucosinolates are a source of cyanide in Arabidopsis leaves.Does that mean Arabidopsis releases cyanide upon spider mite herbivory? If so, what is the source? To test these questions, the authors measured leaf cyanide levels directly. They found that cyanide levels were approximately four times greater in untreated leaves from Col-0 than indole glucosinolate-deficient cyp79b2 cyp79b3. Leaves were assayed after 2 h of spider mite feeding, when hydrolysis (i.e. activation) of indole glucosinolates has been triggered but herbivory-induced biosynthesis has not yet occurred indole glucosinolates provide a source of cyanide in the leaves of well-defended Arabidopsis Col-0, (ii) cyanide may be released as a byproduct of indole glucosinolate activation, and (iii) spider mite feeding may favor indole glucosinolate activation-dependent cyanide release. Future experiments are needed to determine the exact conditions under which indole glucosinolate-dependent cyanide release occurs.TuCAS allele, rapidly arose in spider mites during adaptation to well-defended Arabidopsis Col-0. These findings support the hypothesis that changes in gene expression regulation underlie rapid, repeated bouts of adaptation (This study investigated the tempo and mode of evolution by exploring the adaptive changes that occur as spider mites colonize a plant host. While adaptation typically occurs over millions of years, it can also happen in just a few generations, as exemplified by these serial specialists. Dixit and colleagues demonstrated that increased cyanide tolerance, conferred by an inducible aptation .Conflict of interest statement. None declared."} +{"text": "Background: Persistent colonization with resistant gram-negative bacteria (R-GNB) increases risk of clinical infection and intra-facility transmission among nursing home (NH) patients. Limited data exist on the roles of age and function on duration of R-GNB colonization. Methods: Secondary data analysis was performed from a cohort study of patients admitted to 6 Michigan NHs between November 2013 and May 2018. Swabs obtained upon enrollment, day 14, day 30, then monthly until NH discharge from 6 anatomical sites were cultured for GNB. R-GNB were defined as resistant to ciprofloxacin, ceftazidime, or imipenem. Positive R-GNB culture from a single visit followed by negative cultures for the same organism from \u22652 subsequent visits were defined as transient R-GNB colonization. All other patients with positive R-GNB cultures from multiple visits were considered persistently colonized. Demographic data, antibiotic use, device use, and physical self-maintenance scales (PSMSs) were obtained upon enrollment. Characteristics were compared between patients with transient versus persistent R-GNB and uncolonized patients using multinomial logistic regression. Results: We recruited 896 patients and followed them for 2,437 total visits. Of 896 patients, 407 (45.4%) were colonized with \u22651 R-GNB during their stay. Of 171 patients with \u2265 2 follow-up visits after R-GNB detection, 94 (55%) remained persistently colonized with the same R-GNB (Table Escherichia coli (30%) and Proteus mirabilis (22%) were the most frequently identified R-GNB. The most common anatomical colonization sites were perirectal groin , and hands (115 [4.8%] of 2283). Compared to uncolonized patients, patients with persistent and transient R-GNB colonization had lower PSMS (Tables P = .003). Conclusions: R-GNB colonization in vulnerable NH patients is common (407 [45.5%] of 896 and often persistent (94 [55%] of 171 patients with sufficient follow-up to assess persistence). Patients with persistent R-GNB had lower functional status, longer LOS, and higher readmission rates than those without. R-GNB decolonization should be investigated as a strategy to potentially improve outcomes among NH patients.Funding: NoneDisclosures: None"} +{"text": "Home health is the fastest-growing long-term care setting in the U. S.. However, evidence on effective clinician-patient-family communication in home health is lacking. This prospective, two-arm, pre-post randomized controlled trial aimed to assess feasibility, acceptability, and preliminary effectiveness of the COMFORT communication model in home health interprofessional staff (IHHS). IHHS (n = 18) were randomized into two groups: Group 1 (control) (n=10) received seven asynchronous modules, and Group 2 (intervention) (n = 8) received the same modules plus a 2-hour synchronous class with interactive slide presentation and exercises. Measures included completion rates, acceptability ratings, comfort with communication in palliative and end-of-life care (C-COPE), and moral distress in health professionals (MMD-HP). Regardless of group, COMFORT was highly acceptable (>4) to IHHS. COMFORT was positively correlated with improved C-COPE scores (p = 0.037). Moral distress scores did not differ before and after the intervention; however, baseline moral distress scores were found to be higher in IHHS when compared to an academic medical center sample from a previous study. Levels of acceptability of COMFORT were significantly related to clinician levels of considering leaving a job due to moral distress . Findings suggest that COMFORT training increases IHHS comfort with palliative and end-of-life communication, especially among clinicians with histories of considering leaving a job or having left a job due to moral distress."} +{"text": "Rationale: Extracellular vesicles (EVs) play a significant role in cell-cell communication. However, whether and how extracellular vesicles are involved in chronic intermittent hypoxia-induced endothelial dysfunction is unknown.Methods: Comparative transcriptomics analysis and miRNA screening were used to identify the possible pathways or target molecules mediating chronic intermittent hypoxia-induced endothelial function. Serum- or erythrocyte-derived EVs were isolated through ultracentrifugation plus filtration. After in vitro or in vivo treatment with EVs, aortic rings were treated with dihydroethidium staining for superoxidative anion measurement or mounted with wire myography to measure isometric forces. Immunoblotting and qPCR were used for evaluating the molecular mechanism mediating EV miR-144-induced endothelial function under intermittent hypoxia.Results: We revealed a previously undefined importance of circulating extracellular vesicles in regulating endothelial function via delivery of miR-144 to endothelial cells, reducing nuclear factor erythroid 2-related factor 2 expression. Additionally, we identified that erythrocytes were the primary cellular source of miR-144-enriched serum-derived extracellular vesicles and that erythrocyte-derived extracellular vesicles were largely responsible for chronic intermittent hypoxia-impaired endothelial function. Furthermore, silencing of miR-144 by anti-miR-144 confirmed its essential role in endothelial dysfunction elicited by erythrocyte-derived extracellular vesicles from chronic intermittent hypoxia-exposed C57BL/6 mice.Conclusion: The results expand the scope of blood-borne substances involved in vascular homeostasis and suggest that anti-miR-144-loaded extracellular vesicles may represent a promising therapeutic approach against obstructive sleep apnea or chronic intermittent hypoxia-associated endothelial dysfunction. Obstructive sleep apnea (OSA), hallmarked with chronic intermittent hypoxia (CIH) due to recurrent partial or complete pharyngeal collapse during sleep, is a highly prevalent chronic sleep disorder. OSA is identified as an independent risk factor for the development of systemic hypertension. Approximately 50% of OSA patients are hypertensive and an estimated 70-85% patients with resistant hypertensive patients have OSA Oxidative stress leads to endothelial dysfunction by promoting NO uncoupling. Anti-oxidative transcription factor NRF2 preserves endothelial function and prevents Ang II-induced hypertension The secretion of extracellular vesicles (EVs) into the blood or other body fluids is a universal cellular process that occurs in multicellular organisms. EVs, as a vital mediator of intercellular communication, perform multifaceted functions by delivering complex molecules to recipient cells, thus participating in the regulation of multiple physiological and pathological process In this study, we examined the effects of serum-derived EVs as well as erythrocyte-derived EVs from CIH-exposed C57BL/6 mice on endothelial-dependent relaxation and elucidated the underlying mechanisms. This research will help to further understand OSA- or CIH-related endothelial dysfunction and hypertension from a new perspective.The data that support the findings of this study are available from the corresponding author upon reasonable request. See Data Supplement for detailed methods.Experiments were approved by Capital Medical University Animal Experimentation Ethics Committee and in compliance with the National Institutes of Health Guidelines on the Use of Laboratory Animals. C57BL/6 mice were treated under normoxia or chronic intermittent hypoxia as previous report EV was isolated from serum and red blood cells as previous reports Statistical analysis is summarized in the figure legends. In most cases, the results represent the mean \u00b1 standard error of the mean (SEM) of n separate experiments. Concentration-response curves were compared by two-way analysis of variance (two-way ANOVA) followed by Bonferroni post hoc test. Two-tailed Student's t-test was used when two groups were compared. One-way analysis of variance (ANOVA) was performed to determine whether there was a significant difference between more than two datasets, followed by Bonferroni's post hoc test. P < 0.05 indicates statistical difference between groups.https://data.mendeley.com/drafts/zbrmjpdv93) between CIH and normoxia mice, with a concomitant reverse tendency in the Re-Nor post-CIH group. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis demonstrated the cellular processing and functions of differentially expressed genes (Nrf2), to be implicated in all four of the above-mentioned functions staining, which was reversed by acute 30-min exposure to the ROS scavengers, Tempol (100 \u03bcM) or Tiron (1 mM) and DETCA (0.1 mM), as well as by oral treatment with NRF2 agonist, Oltipraz in C57BL/6 mouse thoracic aortas (mean relaxation from 92.1% to 56.4%) and carotid arteries (mean relaxation from 87.3% to 61.8%), and significantly elevated blood pressure, especially systolic blood pressure, but the effects were reversed by re-administering normal oxygen for 15 days after 30-day-CIH treatment Figure A-C. CIH ed genes -3. Baseded genes A-B. Of n p47phox C. Corres) Figure D. Furthe) Figure E.En face staining demonstrated that PKH67-labeled C57BL/6 mouse S-EVs were taken up by endothelial cells in a time-dependent fashion attenuated EDR in mouse aortas, whereas EV-free serum treatment did not produce such an inhibitory effect. Direct exposure to CIH S-EVs for 48 h attenuated EDR in C57BL/6 mouse thoracic aortas and decreased flow-mediated dilatation in C57BL/6 mouse mesenteric arteries . Among them, Nrf2 was the only differentially expressed gene common to both CIH S-EV-treated HUVECs and CIH-treated mouse aortas were not altered in CIH S-EV-treated H5V cells cells after continuous hypoxia, intermittent hypoxia, or DMOG treatment treatment. qPCR results presented that the expression of miR-144 and pri-miR-144 was dominant in erythrocytes rather than in endothelial cells. Under CIH status, the expression of pri-miR-144 and miR-144 was significantly increased in erythrocytes and the tendency was reversed after returning to normoxia for 15 days; whereas, in aortic endothelial cells, only miR-144 was upregulated potentiated superoxide anion production in endothelial cells. However, this effect was largely inhibited in anti-miR-144-loaded CIH E-EV treated groups, as shown by en face DHE staining (Figure in vivo treatment of CIH E-EVs impaired EDR and increased systolic blood pressure in normoxia- and CIH-treated mice. These effects were reduced in groups treated with anti-miR-144-loaded CIH E-EVs (Figure Nrf2 overexpression also effectively blocked CIH E-EV-impaired endothelial function (Figure To further verify the endothelial effect of miR-144 delivery by CIH E-EVs, anti-miR-144 as well as anti-miR-144-loaded CIH E-EVs were utilized to treat aortas d Figure A. Proteid Figure B. Consisg Figure C-D. Means Figure C-E. Simis Figure G-H. Of nn Figure I.This study highlights the role of S-EVs and E-EVs as blood-borne regulators of vascular function, and pinpoints erythrocyte-enriched miR-144 as a critical EV-derived miRNA that participates in CIH-induced endothelial dysfunction via inhibiting NRF2 expression.Nrf2 was the only one commonly implicated in the four vascular-associated responses, namely, contraction and dilation, angiogenesis, ROS production, and inflammation. ROS regulates vascular homeostasis, and ROS overproduction-induced oxidative stress is a primary cause of vascular dysfunction by reducing NO availability OSA is a prevalent disease most frequently associated with secondary hypertension Circulating EVs are constantly in direct contact with and regulate the functions of endothelial cells under pathophysiological conditions Nrf2We previously reported that S-EVs mediate endothelial dysfunction in diabetes through delivery of arginase 1 via HIF-1\u03b1 direct transcriptional regulation. Interestingly, we also demonstrated that both Hif-1\u03b1 overexpression and CIH treatment upregulated GATA1 expression in MEL cells, indicating GATA1 to be a mediator participating in HIF-1\u03b1 indirect regulation of miR-144.The regulation of miR-144 in erythrocytes during hypoxia is not well documented in the literature. Sequencing analysis showed that miR-144 was upregulated after 16 h of hypoxic treatment in HUVECs in vivo mechanism closer to the physiological state should be in future investigations. Due to technical constraints, the CIH paradigm used in the current study does not closely match that observed in clinical OSA status, new specific interfering tools and more rigorous studies are required to further explore the clinical relevance of S-EV or E-EV in the development of OSA-related vasculopathy. In addition, the present study cannot discount other possible signals, such as miR-27a, that are independent of E-EV miR-144/NRF2 might play a role in CIH-induced impairment of endothelial function, which requires future examination.The present study has several limitations. In this study, considering that OSA patients may have complex comorbidities and complex pathophysiological states, which will increase the heterogeneity of EVs and even lead to inconsistent results, we selected serum EVs or erythrocyte-derived EVs obtained under more consistent CIH conditions to treat aortic and endothelial cells to obtain more reproducible results. This result reflects the mechanism of EV-induced vascular impairment in CIH setting, however, this mode of treatment is less clinically relevant than EV of OSA patient origin. Meanwhile, given that many of the miRNAs in mouse EVs are not homologous to human ones, our treatment model may therefore miss other possible functional molecules. We provide clear evidence that miR-144 in erythrocyte-derived EVs is responsible for endothelial dysfunction induced by CIH, the major pathological alteration in OSA. However, it is not known whether the same or different EV miRNAs are involved in the endothelial dysfunction induced by other pathological features of OSA, such as fragmented sleep, sympathetic nerve activation, and increased negative chest pressure. Moreover, we cannot exclude the possible involvement of non-erythroid-derived miR-144, albeit to a lesser degree. Further investigation is warranted to address these problems. Meanwhile, considering that mature erythrocytes would not respond to any stimulus to induce the expression of miRNAs, in the current work, we used MEL to reveal the mechanism by which intermittent hypoxia promoted miR-144 expression, which maybe partially helped to understand the increased levels of miR-144 in mature erythrocytes under CIH status, however, the In summary, we systematically demonstrate that CIH S-EVs and CIH E-EVs can deliver functional miR-144 to endothelial cells, promoting superoxide anion production by reducing NRF2 expression, a critical pathogenic component of endothelial dysfunction and hypertension during CIH. Our study provides experimental evidence for the therapeutic potential of EV-loaded anti-miR-144 or antagomir-144 in treating OSA or CIH-associated vascular complications.Supplementary materials and methods, figures, and tables.Click here for additional data file."} +{"text": "LncRNA RPSAP52 is a newly identified functional molecular in several cancers, but its role in gastric cancer (GC) is currently unclear. This study aimed to investigate the biofunction of lncRNA RPSAP52 in GC. Quantitative polymerase-chain reaction (RT-qPCR) was employed to analyze the gene level of lncRNA RPSAP52 and miR-665. Cell proliferation capacity was evaluated via CCK-8 and colony formation assay. Flow cytometry was applied to detect cell cycle and cell apoptosis. Hematoxylin-eosin staining was conducted for histopathological analysis. Immunochemical staining was carried out to detect expression level of ki-67. Subcellular fractionation was performed to explore the position of lncRNA RPSAP52. The binding relationship among lncRNA RPSAP52, miR-665 and STAT3 was verified via luciferase reporter assay. RNA pull down experiments were used to verify the binding relationship between lncRNA RPSAP52 and miR-665. The STAT3 level was evaluated via Western blot. LncRNA RPSAP52 is significantly elevated in GC cells. Deletion of lncRNA RPSAP52 restrained cell proliferation and induced G0-G1 phase arrest, while expediting apoptosis in GC cells. Tumor growth in vivo was suppressed following lncRNA RPSAP52 depletion. MiR-665 was verified as the target of lncRNA RPSAP52. A ceRNA-sponge mechanism of lncRNA RPSAP52 on miR-665 was identified. Meanwhile, miR-665 functions as STAT3 sponge. MiR-665 overexpression and STAT3 depletion served the same functions as lncRNA RPSAP52 depletion in GC cells. LncRNA RPSAP52 exerted anti-cancer effects via modulating miR-665/STAT3 in GC.Abbreviations: Gastric cancer (GC); Quantitative polymerase-chain reaction (RT-qPCR); Helicobacter pylori (H. pylori); Roswell Park Memorial Institute 1640 (RPMI 1640); fetal bovine serum (FBS); glyceraldheyde 3-phosphate dehydrogenase (GAPDH); propidium iodide (PI); Cell counting kit-8 (CCK-8); radioimmunoprecipitation assay (RIPA); sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE); polyvinylidene fluoride (PVDF); enhanced chemiluminescence (ECL); Statistical Product and Service Solutions (SPSS); standard deviation (SD). The early clinical manifestations of GC are atypical and vague, making it extremely difficult for early diagnosis. The five-year overall survival rate of patients with advanced metastasis GC is poor, whereas, early-stage localized GC patients has a 5-year overall survival rate of more than 50% . LncRNA has been confirmed to work by regulating chromatin organization, gene transcription, gene expression and gene splicing in cancer [15\u201319].LncRNA has been once considered as the transcriptional garbage without biological function. But an accumulating body of research indicates that lncRNA functions as a crucial participant in cell proliferation, invasion, migration, metastasis and so forth in various type of cancer . MiR-665 has been demonstrated to exert anti-proliferation effect in gastric cancer via targeting PPP2R2A [MiRNA-665 has been substantiated as a crucial player in sundry cancers such as hepatocellular cancer, ovarian cancer, colorectal cancer [ PPP2R2A . This co PPP2R2A . ConsistSTAT3 has been confirmed as a well-known oncogene factor. STAT3 is reported to aggravate progress of gastric cancer via promotion of mesothelial\u2013mesenchymal transition . STAT3 iIn this study, we identified a new regulatory axis lncRNA RPSAP52/miR-665/STAT3 in gastric cancer. lncRNA RPSAP52 exerted anti-cancer effects via regulating miR-665/STAT3 in GC. This discovery provides a new idea for the study of the anticancer mechanism of GC, and lays a foundation for the study of the pathological mechanism of GC."} +{"text": "Not all older adults with dementia-related neuropathology in their brains experience cognitive decline or impairment. Instead, some people maintain relatively normal cognitive functioning despite neuropathologic burden, a phenomenon called cognitive resilience. Using a longitudinal, epidemiological, clinical-pathologic cohort study (N=349), the present research investigated associations between well-being and cognitive resilience. Consistent with pre-registered hypotheses, higher eudaimonic well-being and higher hedonic well-being were associated with better-than-expected cognitive functioning and less-than-expected cognitive decline relative to one\u2019s neuropathological burden . The association of eudaimonic well-being in particular was present above and beyond known cognitive resilience factors and dementia risk factors . This research highlights the importance of considering eudaimonic well-being in efforts to prevent dementia."} +{"text": "Cancer patients (CPs) with COVID-19 have an increased risk of adverse outcomes. In addition, CPs seem to have a lower immune response to SARS-CoV-2 vaccination. This study aimed to evaluate SARS-CoV-2 spike antibodies (anti-S Abs) following COVID-19 vaccination in CPs and healthcare workers (HCWs).We conducted a point-seroprevalence study in CPs and HCWs who received a two-dose scheme with either BNT162b2, AZD1222, or Sputnik-V vaccine. We measured anti-S Abs by quantitative immunoassay to assess humoral immune response. Besides, we quantified anti-nucleocapsid antibodies in a subgroup of individuals to determine prior infection. We compared anti-S Abs titers in both groups and stratified by vaccine type, prior infection, and clinical characteristics. We conducted a multivariate logistic regression to determine variables associated with a poor humoral response.Six hundred forty-one individuals were included: 174 (27%) CPs and 467 (73%) HCWs. The median anti-S Abs titter was higher among HCWs compared to CPs . Both CPs and HCWs with prior infection had higher anti-S Abs titter (p< 0.001). Regardless of the time since vaccination, a higher proportion of subjects with titers < 250 U/mL was observed in CPs (p< 0.001) . In the multivariate analysis, older age (p=0.036), AZD1222 (p=0.003), and Sputnik-V (p=0.020) were associated with lower humoral response among the entire cohort.SARS-CoV-2 spike antibody titers among cancer patients and healthcare workers.Global differences in anti-S Abs titers between CPs and HCWs groups (a) and antibody titers in CPs and HCWs groups stratified by type of received vaccine (b). Abbreviations: CP: Cancer patients, HCW: Healthcare workers.SARS-CoV-2 spike antibody titers according to time since vaccination among cancer patients and healthcare workers.Abbreviations: CP: Cancer patients, HCW: Healthcare workers.In this study, both CPs and HCWs showed an adequate response to vaccination; however, CPs had lower anti-S Abs titers and a faster decline over time. Based on our results, new strategies should be assessed to sustain the humoral response to vaccination and thus decrease the COVID-19 burden among the oncologic population.All Authors: No reported disclosures."} +{"text": "Anticoagulant treatment is recommended for at least three months after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-related acute pulmonary embolism (PE), but the persistent pulmonary clot burden after that time is unknown.Lung perfusion was assessed by ventilation-perfusion (V/Q) SPECT/CT in 20 consecutive patients with SARS-CoV-2-associated acute PE after a minimum of three months anticoagulation therapy in a retrospective observational study.Remaining perfusion defects after a median treatment period of six months were observed in only two patients. All patients were on non-vitamin K direct oral anticoagulants (DOACs). No recurrent venous thromboembolism or anticoagulant-related bleeding complications were observed. Among patients with partial clinical recovery, high-risk PE and persistent pulmonary infiltrates were significantly more frequent .Temporary DOAC treatment seems to be safe and efficacious for resolving pulmonary clot burden in SARS-CoV-2-associated acute PE. Partial clinical recovery is more likely caused by prolonged SARS-CoV-2-related parenchymal lung damage rather than by persistent pulmonary perfusion defects. The coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a global infectious disease with an enormous impact on public health. The SARS-CoV-2 infection primarily affects the respiratory system, but it may involve different organ systems and is associated with an increased risk for significant coagulopathy, resulting in high rates of venous thromboembolism (VTE), especially in severely ill patients.There is limited evidence as to how long patients with SARS-CoV-2-related acute pulmonary embolism (PE) should be treated in order to restore pulmonary arterial blood flow. At present, standard VTE management-conform non-COVID recommendations are generally advised, recommending a treatment with anticoagulants for at least three months . HoweverWe therefore evaluated lung perfusion assessed by ventilation-perfusion (V/Q) single photon emission computed tomography/computed tomography (SPECT/CT) after a minimum of three months anticoagulation therapy in patients with SARS-CoV-2-associated acute PE in order to determine the optimal treatment regime.All patients who were diagnosed with SARS-CoV-2-associated acute PE between 1. April 2020 and 31. April 2021 at the University Hospital Augsburg were retrospectively identified. They underwent V/Q SPECT/CT imaging for the re-evaluation of persistent perfusion defects after a minimum period of three months sufficient anticoagulation treatment, unless they had an alternative diagnosis for ongoing anticoagulation therapy . First, ventilation with an 99\u00a0m-technetium- (99\u00a0m-Tc-) labelled aerosol produced by a nebulizer was performed followed by SPECT/CT imaging using a dual-head SPECT/CT gamma camera (GE Optima\u2122 NM/CT 670 or Siemens Symbia T2). Low-dose CT for attenuation correction was acquired in respiratory center position with ventilation SPECT/CT scanning. Iterative and attenuation corrected images were reconstructed using ordered-subset expectation maximization. Subsequently, an activity of about 150 MBq 99\u00a0m-Tc-Pulmocis\u00ae (Curium Pulmocis) was injected intravenously and SPECT scanning was performed again, using the same imaging parameters as described above. Images were reviewed by two board certified nuclear medicine physicians and diagnosis was reached in consensus.All patients gave written informed consent for lung perfusion imaging. The Ethics Committee at the Ludwig-Maximilians-University Munich, Germany exempted this retrospective, non-interventional observational study with a waiver of patient informed consent on May 20, 2021.The statistical analysis was performed using IBM SPSS\u00ae Statistics version 25. Continuous variables were given as mean values with standard deviations. Differences in continuous variables were analysed by Student\u2019s t-test. Categorical variables were specified as amounts and their distribution was further analysed by the chi-square test or by Fisher\u2019s test in case of low frequencies. A p-value of less than 0.05 was considered to indicate statistical significance.Between 1. April 2020 and 31. April 2021, 35 consecutive patients with SARS-CoV-2-associated acute PE were identified. Out of these patients, three patients died during hospitalisation and eight patients had an alternative diagnosis for ongoing anticoagulation therapy. From the 24 eligible patients, three patients were lost for follow-up due to geographical reasons and one patient denied lung perfusion imaging. There were no deaths observed during the follow-up period. Thus, 20 patients could be included in the retrospective study.Patients\u2019 characteristics are summarised in Table\u00a0To the best of our knowledge, the present study is one of the first to evaluate lung perfusion after recovery from SARS-CoV-2-related acute PE. We found persistent pulmonary perfusion defects in 10% of study participants after six months of sufficient oral anticoagulant therapy. However, persistent pulmonary thrombus load does not seem to be associated with an unfavourable clinical short-term outcome.Current recommendations suggest a temporary treatment with anticoagulants for at least three months in VTE patients with COVID-19, as SARS-CoV-2 is considered a relevant, transient provoking factor for VTE , 6. SARSAll patients were set on a DOAC therapy after recovery from COVID-19 in order to prevent VTE recurrence. As DOACs interact with a variety of COVID-19 drugs , DOACs are considered not to be a preferable therapy option during SARS-CoV-2 infections . TherefoIn nearly 40% of our study population, clinical recovery was partial, and persistent pulmonary infiltrates were significantly more frequent in this subgroup. Pulmonary lesions at time of discharge from hospital were found to be quite common even in mild and moderate SARS-CoV-2 infections , but stuIn contrast to one study, which found that chest radiography is a poor predictor of clinical and functional impairment eight weeks after severe COVID-19 pneumonia , other rWe admit that our study is limited by the single-centre design and its very small sample size. Therefore, our data are preliminary and need to be confirmed in a larger study population. However, the demographic profile of our cohort seems to be very representative for a general COVID-19 patient population. Unfortunately, regular lung function tests were not performed during follow-up in our trial. Further limitations are the retrospective study design and the short-term observational period.Despite these limitations, we conclude that a temporary DOAC treatment might be sufficient for resolving pulmonary clot burden after SARS-CoV-2-associated acute PE. In addition, partial clinical recovery seems to be more likely caused by prolonged COVID-19-related parenchymal lung damage than by persistent pulmonary perfusion defects."} +{"text": "Oncoviruses, known as cancer-causing viruses, are typically involved in cancer progression by inhibiting tumor suppressor pathways and uncontrolled cell division. Myeloid cells are the most frequent populations recruited to the tumor microenvironment (TME) and play a critical role in cancer development and metastasis of malignant tumors. Tumor-infiltrating myeloid cells, including tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), tumor-associated dendritic cells (TADCs), and tumor-associated neutrophils (TANs) exert different states from anti-tumorigenic to pro-tumorigenic phenotypes in TME. Although their role in the anti-tumorigenic state is well introduced, their opposing roles, pro-tumorigenic activities, such as anti-inflammatory cytokine and reactive oxygen species (ROS) production, should not be ignored since they result in inflammation, tumor progression, angiogenesis, and evasion. Since the blockade of these cells had promising results against cancer progression, their inhibition might be helpful in various cancer immunotherapies. This review highlights the promoting role of tumor-associated myeloid cells (TAMCs) in the pathophysiology of human virus tumorigenesis. Human oncogenic viruses, known as oncoviruses, potentially contribute to an estimated 12\u201320% of human cancers, accounting for a large fraction of the global cancer burden . It has Myeloid cells exert an immunosuppressive activity to combat the proliferating tumor cells; however, it has been demonstrated that they represent opposing functions from anti-tumorigenic to pro-tumorigenic phenotypes in the TME. Hence, we briefly describe the mechanism of the anti- and pro-tumorigenic function of TAMCs in the immune escape and cancer pathogenesis.+ T-cell responses, however, TAR-DCs exhibited immunosuppressive properties by low expression of costimulatory molecules and high expression of regulatory molecules. Stromal-cell derived factor-1 (SDF-1) which is also known as CXCL12, in the TME of malignant tumors and high expression of CXCL4 ligand results in the accumulation of DCs in TME. Immunoglobulin-like transcript 7 (ILT7) recognizes bone marrow stromal cell antigen 2 (BST2), which is highly expressed on tumor cells, resulting in negative regulation of the interferon responses [Dendritic cells (DCs) are a double-edged sword population in the TME. Plasmacytoid (pDC), conventional (cDC1 or cDC2), and inflammatory DC (moDC) are three phenotypically and functionally distinct subsets of DCs . These iesponses . It has Tumor-associated macrophages (TAMs) are abundant myeloid cells in the TMEwith anti-tumorigenic or strongly pro-tumorigenic phenotypes. Macrophage colony-stimulating factor (M-CSF) is highly expressed in the TME, which recruits the macrophages from the bone marrow or spleen . TAMs ar+ and CD8+ T-cells. Also, it has been suggested that MDSCs suppress NK cells, which may disturb anti-tumor immunity [MDSCs are developmentally immature non-macrophage cells with an immunosuppressive function. These cells potentially prevent the activation of CD4immunity . Therefoimmunity . MDSCs iimmunity . MDSCs simmunity . MDSCs aimmunity .As the first line of immune defense, neutrophils are a substantial population that infiltrates the TME. TANs have a dual function of anti- and pro-tumor activities, modulating anti-tumor immunity . InteresHere we highlight the mechanistic strategies by oncoviral infection in immune disturbances Table .Epstein-Barr virus (EBV), first identified in the tumor cells of Burkitt lymphoma, is now associated with a strikingly diverse variety of lymphoproliferative lesions and malignant lymphomas of B, T, and NK cell origin . Here, wThe latent membrane protein-1 (LMP1) is the primary oncogene of EBV that plays a critical role in the MDSCs proliferation and tumor immunosuppression. A large fraction of MDSCs is found in patients with EBV-associated T/NK cell lymphoproliferative diseases, which may dampen the antiviral T-cell responses . LMP1-me+ tumor cells is dependent on TAMs in the EBV-positive TME [In gastric cancer, the EBV-encoded miR-BART11 targets FOXP1 to enhance the tumor-associated macrophage-induced epithelial-mesenchymal transition . Zhang etive TME . The EBVtive TME .EBV expression has been identified in lesional macrophages of different cancers, ranging from thyroid to uterine carcinoma and some types of lymphoma that possibly elicit EBV lytic infection of macrophages in many tumor-associated macrophages in EBV-related malignancies .In classical Hodgkin\u2019s lymphoma, tumor-infiltrating macrophages are linked to a poor prognosis and the presence of EBV . IncreasThe most frequent kind of liver cancer is hepatocellular carcinoma (HCC). HBV is a chronic infection that affects over 350million individuals worldwide. At least half of all HCC cases globally are caused by chronic hepatitis B virus (HBV) infection . Here, wHCC patients had considerably greater percentages of MDSCs and PMN-MDSCs than chronic hepatitis B patients and healthy controls . Pal et +/CD4+ and Tim3+/CD8+ cells to senescence [Macrophages are monocytic phagocytes with antigen-presentation and cytokine-producing capabilities. The tissue-specific liver macrophages are Kupffer cells dominating other innate immune cells in the organ . From thnescence , 47.The lymphocytes in the human liver tissue are predominantly natural killer cells (NK cells). Patients with persistent HBV and HCV infection have more NK cells in their liver . A liganHCV infection affects more than 270million individuals globally. HCV produces a chronic and lifelong infection in most infected individuals. This persistent inflammation in the liver leads to macronodular cirrhosis in 20% of people who contract it. A 4 to 7% yearly risk of progressing to HCC is associated with these individuals .+Foxp3+ Tregs and inhibited the autologous CD4+ T-cell activation [+ MDSCs, which reduces T-cell responsiveness via the production of ROS [Through the TLR2/PI3K/AKT/STAT3 signaling cascade, HCV induced MDSC-like suppressive monocytes that activated CD4tivation . HCV stin of ROS . IFN-\u03b3 pn of ROS . MDSCs tn of ROS . Hepatitn of ROS .A long non-coding RNA (lncRNA) named HOXA transcript antisense RNA myeloid-specific 1 (HOTAIRM1) targets HOXA1 gene expression to regulate myeloid cell development. HOTAIRM1 enhances MDSCs growth and suppressive activities during HCV infection through the HOXA1-miR124 axis . Exosome+Gr1+ cells with suppressor immune phenotype are induced by vFLIP, which remodels myeloid differentiation and causes their proliferation [The causative agent of Kaposi\u2019s sarcoma (KS) is Kaposi\u2019s sarcoma herpesvirus ). KS is the most prevalent neoplasm among untreated HIV patients, although it may also happen in immunosuppressive conditions after organ transplantation . KSHV vFferation . Based oferation . It has feration . Moreoveferation . Therefo+ effector memory T-cells and controlling MDSCs together allowed protection against cancers caused by the HPV-16 serotype [Cervical cancer is caused by certain HPV (human papillomavirus) genotypes. Other anogenital cancers and a subset of head and neck cancers seem to be caused by the same genotypes. It is necessary to sustain the malignant development of cervical cancer cells by inducing the expression of particular viral oncoproteins, E6 and E7, which specifically inhibit the tumor suppressors p53 and RB . In maliserotype .The oncogenic mechanisms of tumor-promoting myeloid cells in reviewed human oncoviral infections have been depicted in Fig.Due to the immunosuppressive nature of MDSCs and TAMs, these cells have been considered two main potential targets for cancer immunotherapy. Although, different therapeutic approaches to target these immunosuppressive myeloid cells are being investigated. Here, we will provide the strategies for repolarization and revival of tumor-promoting myeloid cells Table .+ TAMs and MDSCs in TME are recruited by the CCL2 chemokine [Blocking the recruitment of MDSCs and TAMs may be a beneficial strategy for reducing tumorigenesis and immunosuppression. CCR2hemokine \u201372. Blochemokine . In breahemokine . Clinicahemokine , 76.The CXCL12/CXCR4 axis governs TAMs\u2019 migration into hypoxic tumor areas through the endothelial barrier . TargetiTAMs are among the most common and important non-neoplastic cell groups in the established TME. The differentiation of macrophages into tumor-suppressive M1 or tumor-promoting M2 types is an important stage in the formation of the TME. Implementing three strategies through this pivotal axis could pave for novel cancer treatment strategies. These strategies could alter M2 TAM survival and apoptotic mechanisms or disrupt their signaling pathways, suppress chemotactic potential toward the tumor, and reprogram M2 TAMs to produce M1 phenotype macrophages .Bisphosphonates elicit myeloid cell cytotoxicity by preferentially targeting phagocytic cells, including TAMs . ZoledroSeveral agents, including growth factors, could modify macrophages\u2019 immune and metabolic responses in their residing microenvironment. This mechanism is reflected in the tricarboxylic acid (TCA) cycle disruption in M1 macrophages with the stimulation of inflammatory mediators resulting in IL-1 and Fatty acid synthesis and switching to pro-inflammatory phenotype , 85\u201388. To induce tumoricidal potential in MDSCs and TAMs, several factors could be used to reprogram their signaling pathways, including colony-stimulating factor 1/colony-stimulating factor 1 receptor (CSF1/CSF1R) blockade, TLR agonists, PI3K inhibitors, CD40 agonists , and ClaTLRs agonists could induce pro-inflammatory and anti-tumor phenotypes in TAMs. Feng et al. developed a glucomannan polysaccharide with acetyl modification to the degree of 1.8 (acGM-1.8), which stimulates TLR2 signaling and promotes macrophages toward becoming anti-tumor . TLR7/8 + T-cell infiltration [CSF1/CSF1R blockade in pancreatic cancer models could enhance immune checkpoint T-cell therapy outcomes while reprogramming TAMs . Moreoveltration . Inapproltration . Pro-tumltration . Indeed,ltration .TAMs are sensitive to profound and abrupt reprogramming in the presence of a CD40 agonist when CSF-1R signaling is inhibited. Despite the short window of macrophage hyperactivation, simultaneous CSF-1R inhibition plus CD40 stimulation is adequate to establish a pro-inflammatory TME that revives an efficient immune response for T-cell immune checkpoint therapy . LikewisA first in vivo evidence revealed that pharmacological suppression of the PI3K p110\u03b4 subunit inhibits the growth of breast cancer by specifically targeting cancer cells and macrophages . Li et a+ T-cell-mediated anti-tumor immune response in breast cancer and reduce metastasis [HDAC inhibition with trichostatin-A increases anti-PD-L1-mediated tumor suppression and potentiates macrophage anti-tumor activity . TMP195,tastasis , 116.The PD-1 and cytotoxic T lymphocyte-associated protein 4 (CTLA-4) immune checkpoints are predominantly produced by effector immune cells, including T and NK cells. Targeting these molecules have exciting therapeutic potential by affecting myeloid biology , 118. BeCD47SIRP\u03b1 axis has been identified as a critical macrophage immune checkpoint. CD47 is a \u201cdon\u2019t eat me\u201d signal that is overexpressed in myeloid malignancies and causes tumors to evade macrophage phagocytosis. CD47 blockade causes leukemic cells to be engulfed and therapeutically eliminated . CD47 blAccording to the clinical and pre-clinical evidence, TAMCs play a dual role in cancer via anti-tumorigenic and pro-tumorigenic effects. TAMCs have pro-tumorigenic and immunosuppressive functions by different mechanisms including TGF-\u03b2 and IL-10 anti-inflammatory cytokine secretion, ROS production, and mediation of angiogenesis through VEGF and HGF production. Hence, TAMCs could be actively involved in cancer progression, and immune escape results in poor prognosis, adverse clinical outcomes, and a low response rate to cancer treatment. Although diverse cancer-related immunotherapies such as ICBs have been investigated, targeting promoting pathways orchestrated by myeloid cells could shed a light on a new therapeutic approach and may improve cancer immunotherapy. Blocking myeloid cells\u2019 recruitment, macrophage population depletion, reprogramming of metabolism, and cellular signaling might be considered helpful strategies for repolarization and revival of tumor-promoting myeloid cells."} +{"text": "Unenhanced influenza vaccines often fail to elicit adequate immune responses in adults \u226565 years due to immunosenescence. Enhanced influenza vaccines, MF59-adjuvanted trivalent inactivated influenza vaccine (aTIV) and high-dose vaccine (HD-TIV), were specifically developed to overcome this problem. The relative vaccine effectiveness (rVE) of aTIV vs HD-TIV and standard, egg-derived quadrivalent influenza vaccines (QIVe) was estimated in 3 retrospective cohort studies during the 2017-2020 US influenza seasons. Influenza-related medical encounters (IRMEs) were identified using diagnostic codes specific to influenza disease (ICD J09*-J11*) from a dataset that integrated electronic primary care medical records with pharmacy and medical claims. rVE was estimated using propensity score methods adjusting for variables including age, sex, race, ethnicity, geographic location, week of vaccination, frailty and health status. Subgroup analyses included specific age groups and those with high-risk medical conditions. aTIV demonstrated a consistent benefit over QIVe and HD-TIV across all three seasons in the overall study populations, and greater rVE versus QIVe was consistently observed over all three seasons in all age subgroups as well . aIIV3 was comparable to HD-TIV and more effective than QIVe in a subgroup with high-risk medical conditions. These findings lend further support to the use of aTIV to prevent influenza-related medical encounters in older individuals."} +{"text": "Previous reports suggest planktonic and under-ice winter microbial communities in Lake Erie are dominated by diatoms. Here, we report the assembled metatranscriptomes of 79 Lake Erie surface water microbial communities spanning both the winter (28 samples) and spring (51 samples) months over spatial, temporal, and climatic gradients in 2019 through 2020. Aulacoseira islandica and Stephanodiscus binderanus . The samples were determined to be DNA-free via the absence of a band in the agarose gel after PCR amplification using 519F/785R 16S rRNA primers (519F-CAG-CMG-CCG-CGG-TAA and 785R-TAC-NVG-GGT-ATC-TAA-TCC) with Escherichia coli K-12 MG1655 DNA as the positive control. The samples were quantified using the Qubit RNA HS assay kit (Invitrogen) and sent to the Department of Energy Joint Genome Institute for rRNA reduction and sequencing using an Illumina NovaSeq S4 2\u2009\u00d7\u2009151-nucleotide indexed run protocol .Opportunistic samples were collected by U.S. Coast Guard Cutter and 2020 . Additiotaxonomy . Water ctaxonomy . RemainiThe reads were filtered using BBDuk v38.92) (2 (9) to Taxonomic annotation of protein coding sequences (CDS) by IMG confirmed diatoms were a transcriptionally active component of the winter microbiome in this Laurentian Great Lake. CDS genes annotated as classes Coscinodiscophyceae (centric) and Bacillariophyceae were highly represented relative to other photosynthetic eukaryotes . ConsideSequences are available through the JGI Genomes Online Database (GOLD) under GOLD Study ID Gs0142002. Assembly and annotation statistics are presented in"} +{"text": "The AP-1 transcription factor, FBJ osteosarcoma oncogene (FOS), is induced in adult muscle satellite cells (SCs) within hours following muscle damage and is required for effective stem cell activation and muscle repair. However, why FOS is rapidly downregulated before SCs enter cell cycle as progenitor cells remains unclear. Further, whether boosting FOS levels in the proliferating progeny of SCs can enhance their myogenic properties needs further evaluation.. We performed various assays to measure cellular proliferation and differentiation, as well as uncover changes in RNA levels and three-dimensional (3D) chromatin interactions.We established an inducible, FOS expression system to evaluate the impact of persistent FOS activity in muscle progenitor cells ex vivoPersistent FOS activity in primary muscle progenitor cells severely antagonizes their ability to differentiate and form myotubes within the first 2 weeks in culture. RNA-seq analysis revealed that ectopic FOS activity in muscle progenitor cells suppressed a global pro-myogenic transcriptional program, while activating a stress-induced, mitogen-activated protein kinase (MAPK) transcriptional signature. Additionally, we observed various FOS-dependent, chromosomal re-organization events in A/B compartments, topologically associated domains (TADs), and genomic loops near FOS-regulated genes.Our results suggest that elevated FOS activity in recently activated muscle progenitor cells perturbs cellular differentiation by altering the 3D chromosome organization near critical pro-myogenic genes. This work highlights the crucial importance of tightly controlling FOS expression in the muscle lineage and suggests that in states of chronic stress or disease, persistent FOS activity in muscle precursor cells may disrupt the muscle-forming process.The online version contains supplementary material available at 10.1186/s13395-022-00303-x. Adult skeletal muscle is one of the few tissues in mammals endowed with a remarkable ability to regenerate after injury. Muscle-specific stem cells, commonly referred to as satellite cells (SCs), are the key cellular source that drives the growth and repair of postnatal skeletal muscle , 2. SCs Recent work has shown that FOS/AP-1 is transiently induced in SCs within hours following muscle trauma \u201310. In aFos can disrupt MyoD and MyoG transcriptional activity from a muscle creatine kinase (MCK) reporter plasmid - log2(100)) - Ct [IP]) \u00d7 100.Primary satellite cells were sorted into 96 well plates and infected with either pSLIK-p value < 0.01 using the DEbrowser package [Total RNA was extracted from GFP or FOS-expressing primary muscle progenitor cells using TRIzol followed by RNeasy Mini Qiagen extraction kit according to the manufacturer\u2019s protocol. RNA-seq libraries were generated using the SMART-Seq ultra low RNA input kit for sequencing coupled with the Nextera XT DNA library kit and then sequenced on the Illumina HiSeq2500 platform. RNA-seq analysis (polyA) was performed by filtering and mapping the reads by Bowtie 2 (mm9) an package . Gene on package . Lower-lHindIII restriction enzyme [HindIII, end-labelled with biotin-14-dCTP, followed by in situ ligation . After DNA extraction, biotin was removed from unligated ends, and the sample was sheared using a Covaris S220 instrument (100\u2013500 bp range) as previously described [In situ Hi-C libraries were generated using the n enzyme , 104. BrR2 > 0.9), indicating the high quality and reproducibility of the datasets. Thus, we pooled all biological replicates for each condition and mapped, filtered, corrected, and binned them as a single Hi-C dataset for all subsequent analyses. Genomic A/B compartments and TADs (insulation method) were defined and visualized using the cworld, HiGlass, and Juicebox toolkits (cworld toolkit codes available at: https://github.com/dekkerlab/cworld-dekker), [hiccups algorithm from the juicer toolkit [The resulting Hi-C sequencing reads were mapped (mm9), filtered, corrected, and binned using the HiC-Pro software v2.8 . There wdekker), \u2013108. Thedekker), , 110. Codekker), . Differe toolkit .Additional file 1: Supplementary Figure S1. Related to Figure Fos and pSLIK-Gfp muscle progenitor cells. n=2295-2816 (PAX7) and n=1432-3779 (MYOG). Mean comparisons using One-way ANOVA with post-hoc Tukey test (B) and Mann Whitney U-test . Supplementary Figure S2. Related to Figure Fos cells were treated with 1 ug/ml DOX for 72-hours and then chased with media devoid of DOX for 3 and 5 days followed by RT-qPCR. Mean relative mRNA expression (+/-SD) and normalized (to Gapdh) for Fos, MyoD, MyoG, Tcap, and Ttn. Data shows that upon removal of DOX, mRNA expression for MyoD, MyoG, Tcap, and Ttn are de-repressed concomitant with FOS returning to basal levels. Mean comparisons were performed with a one-way ANOVA with post hoc Tukey test. N= 5-6 replicate cultures from cells pooled from 4 mice. Supplementary Figure S3. Related to Figure Bmp6, an intergenic region, MyoD, and Ttn in pSLIK-Fos and pSLIK-Gfp cultured muscle progenitor cells. (A) Experimental design: Primary satellite cells were infected with virus expressing pSLIK-Fos or pSLIK-Gfp constructs, selected with 100 \u03bcg/mL of hygromycin for 6 days, and expanded for approximately 3 weeks to generate enough cells for ChIP-qPCR. Once at the desired cell density, pSLIK-Fos and pSLIK-Gfp cells were cultured in GM-supplemented with 1\u03bcg/mL doxycycline (DOX) for 48 hours. Chromatin was IP\u2019d using an H3K27Me3- or immunoglobulin G (IgG)-specific antibodies followed by qPCR. Plot shows mean percent of input (+/- SD) for each amplicon targeting the first 500bps upstream of the TSS for Bmp6 and an intergenic region (left) and MyoD and Ttn (right). Location of primers relative to gene TSS is shown in schematic above each plot. Statistical comparisons were performed with two-way ANOVA with post hoc Holm-Sidak test. N= 8-9 replicate IPs were performed on cells pooled from 4 mice. Supplementary Figure S4. Related to Figure Gfp and pSLIK-Fos datasets showing all the chromosomes and the inter-chromosomal interactions. (B) Scaling plot showing the interaction frequency as a function of genomic distance, demonstrating that pSLIK-Fos and pSLIK-Gfp muscle progenitor cells have similar rates of decay of interaction frequency. (C) Representative higher resolution images of Hi-C datasets of pSLIK-Gfp and pSLIK-Fos muscle progenitor cells. Supplementary Figure S5. Related to Figure Fos and pSLIK-Gfp cells showing the compartment strength, quantified by plotting interaction frequencies in 250kb bins arranged by their values along the first eigenvector (PC1/EV1) to obtain compartmentalization saddle plots. The average interaction frequencies of the observed / expected interactions between pairs of loci (250kb bins) were calculated and arranged by their compartment signal for pSLIK-Fos and pSLIK-Gfp muscle progenitor cells. In these plots the upper left quadrant represents B-B interactions, and the lower right corner represents A-A interactions. (C) Comparison of the intra- and inter-compartmental interaction frequencies indicates that FOS induction results in significantly higher interactions within the A-type and B-type compartments . In contrast, the inter-compartmental (between the A-type and B-type compartments) interactions were significantly decreased in pSLIK-Fos cells when compared to pSLIK-Gfp cells. (D) Gene ontology terms of differentially expressed transcripts whose coding regions have switched from open (A-type) to closed (B-type) compartmentalization. (E). Gene ontology terms of differentially expressed transcripts whose coding regions have switched from closed (B-type) to open (A-type) compartmentalization. Supplementary Figure S6. Related to Figure Fos cells relative to pSLIK-Gfp cells. (B) Violin plot demonstrating similar TAD boundary strengths between pSLIK-Gfp and pSLIK-Fos datasets . Supplementary Figure S7. Related to Figure Gfp and pSLIK-Fos datasets showing a reduction of a loop formation at the Myl1 gene locus, which is associated with a decrease in Myl1 gene expression (pSLIK-Fos vs. pSLIK-Gfp log2FC = -1.7). The z-scores of the looping interaction are depicted on the figure. (B) Overlap in MyoD-specific looping events in [vents in that oveAdditional file 2: Supplementary Table S1. Differentially expressed genes in pSLIK-FOS expressing muscle progenitor cells relative to pSLIK-GFP expressing muscle progenitor cells (Related to Fig. Additional file 3: Supplementary Table S2. Gene ontology terms of up- and down-regulated genes in pSLIK-FOS expressing muscle progenitor cells relative to pSLIK-GFP expressing muscle progenitor cells (Related to Fig."} +{"text": "To explore the expression of circular RNA (circRNA) in gastrointestinal stromal tumors. Gastrointestinal stromal tumors (GIST) are mainly distributed in the stomach and small intestine. Recently, it has been verified that circular RNA (circRNA) has an important function in the regulation of GIST. Nevertheless, detailed investigations of circRNA-miRNA-mRNA regulatory networks in GIST are lacking. To analyze the gastrointestinal stromal tumor circRNA-miRNA-mRNA network, assessing the effect of circle RNA in gastrointestinal stromal tumors. All the differential circRNAs and mRNAs were obtained from Gene Expression Omnibus (GEO) microarray data . Furthermore, a circRNA-miRNA-mRNA network was established. Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment were used to reveal the correlation between the functions of signaling pathways and target genes. The hub genes of protein-protein interaction (PPI) network and cytoHubba were also defined. Quantitative real-time PCR (qRT-PCR) was used to measure the expression levels of hsa-circ-0002917 (circTBC1D4), hsa-miR-590-5p (miR-590-5p), and PLN. PPI network and Cytoscape showed that ATP1A2, PLN, KCNMA1, and SCNN1B were four central DEGs. GO analysis results revealed that DEGs were involved in negative management of myocardial contraction, regulation of myocardial cell contraction, ethanol oxidation, cellular potassium ion homeostasis, and relaxation of cardiac muscle, and KEGG analysis showed that major DEGs were with cGMP-PKG signaling pathway. Moreover, we obtained two pairs of axes, namely, hsa-circ-0039216/hsa-miR-338-3p/ATP1A2 and hsa-circ-0002917/hsa-miR-590-5p/PLN. The target of TBC1D4 is miR-590-5p, and miR-590-5p increased after knocking down TBC1D4. Moreover, PLN was the target of miR-590-5p, and miR-590-5p exerts antitumor effects by reducing PLN. In this study, we constructed a circRNA-miRNA-mRNA management network interrelated with GIST and researched the potential roles of circRNA. Moreover, we discovered a new molecular landmarker for the prediction, diagnosis, and therapy of patients. A gastrointestinal stromal tumor ) is the general common gastrointestinal tumor with an overall incidence of 0.68/100,000 with phencircRNA is an endogenous noncoding RNA with a covalently closed loop structure whose 3\u2032 and 5\u2032 ends are noncollinearly connected by a procedure of \u201creverse splicing\u201d . circRNAIn this research, we found 7 differentially expressed circRNAs (DEcircRNAs) and 12 differentially expressed genes (DEGs) by analyzing two groups of circRNAs and mRNA expression profiles in the GEO dataset. We also performed GO and KEGG analysis on key DEGs to research the role of DEGs. Protein-protein interaction (PPI) network was created, and four key DEGs and one eventful module of the network were defined. The Circular RNA Interactome and TargetScanHuman databases were used to predict miRNAs, and the circRNA-miRNA-mRNA network was succeed in building, which was used to further explore the role of circTBC1D4/hsa-miR-590-5p/PLN axes in stromal tumors.https://www.ncbi.nlm.nih.gov/geo/). Using the keyword \u201cgastrointestinal stromal tumor\u201d to search on the GEO, two circRNA arrays were obtained from GSE131481 and GSE147303 databases; the platforms were GPL22120 and GPL21825. At the same time, two mRNA expression sequences were obtained from GSE131481 and GSE13861 databases; the platforms were GPL22120 and GPL6884. Both circRNA databases included 3 GIST tissues and 3 normal gastric tissues. At the same time, the mRNA databases of GSE131481 included 3 GIST tissues and 3 normal tissues, and GSE13861 included 3 GIST tissues and 19 normal tissues. Because the data in the GEO database publicly available, no ethical approval or informed consent was required for this study.The data applied in the report were retrieved from the GEO database and mRNAs in each dataset I oscillation can be enhanced by exogenous ATP. These results suggest that exogenous ATP regulates pacing cell activity by activating nonselective cationic channels through exogenous Ca(2+) influx and release of the endoplasmic reticulum [Ca(2+)]. Gastrointestinal stromal tumors arise from fusiform mesenchymal cells of Cajal stromal cells (ICC) or stem cell precursors of these cells . Cajal iPhospholamban (PLN), a member of the phospholamban family, regulates calcium reuptake during muscle relaxation. Phosphorylation of phospholamban (PLN) by Ca(2+)/calmodulin-stimulated protein kinase II (CaMKI) accelerates the sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA), which increases the rate of sarcoplasmic Ca(2+) clearance and intracellular calcium release. In most cancer cells, calcium reservoir-operated calcium ion influx mediates the majority of calcium ion influx and may be a factor in regulating intracellular calcium in Cajal and gastrointestinal stromal tumors. Therefore, blocking this mechanism may affect the progress of gastrointestinal stromal tumor . Kurten In recent years, studies found that miR-590-5p played an important role in digestive system diseases. Chen et al. reportedIn conclusion, this study revealed a potential circRNA-miRNA-mRNA network mediated by hsa-circ-0039216/hsa-circ-0002917 in gastrointestinal stromal tumors through total transcriptome analysis, cross-analysis, and correlation analysis. We identified ATP1A2 and PLN as potential inflammatory targets for the therapy of GIST. Our analysis and further experimental verification show that circTBC1D4 promotes the progression of gastrointestinal stromal tumor by regulating miR-590-5p/PLN axis in GIST, providing new insights for GIST. We will conduct further experiments to verify our conclusion."} +{"text": "The original version of this article unfortunately contained a mistake. Author name Alexandra Mavroeidi was incorrectly written as Alexandra Mavroedi. ORCID id for author Alexandra Mavroeidi should be 0000-0001-5213-1596."} +{"text": "Two studies reported the administration of [177Lu]PSMA-radioligands with theragnostic purpose in three patients. Conclusions: The available literature data in this setting are limited and heterogeneous. The employment of PET with PSMA-targeting radiopharmaceuticals in this setting did not affect patient management. Nevertheless, prospective multicentric studies are needed to properly assess its potential role in TC patients.Background: Recently, several studies introduced the potential use of positron emission tomography/computed tomography (PET/CT) with prostate-specific membrane antigen (PSMA)-targeting radiopharmaceuticals in radioiodine-refractory thyroid cancer (TC). Methods: The authors accomplished a comprehensive literature search of original articles concerning the performance of PSMA-targeted PET/CT in TC patients. Original papers exploring this molecular imaging examination in radioiodine-refractory TC patients undergoing restaging of their disease were included. Results: A total of 6 documents concerning the diagnostic performance of PSMA-targeted PET/CT in TC (49 patients) were included in this systematic review. The included articles reported heterogeneous values of PSMA-targeted PET/CT detection rates in TC, ranging from 25% to 100% and overall inferior to [ According to guidelines, its execution is appropriate in TC patients with elevated serum Tg and a negative post-therapeutic whole-body RI scan [18F]F-FDG uptake on PET images is an independent prognostic factor for overall survival in TC patients F-FDG PET/CT [131I]I scintigraphy [18F]F-FDG PET/CT scan, none of the included studies reported whether the exam was performed under TSH stimulation Ga-PSMA-11 I scan as comparative imaging, which has low sensitivity in the setting of RI refractory TC F-FDG PET, despite, in some cases, it could show lesions without significant glucose metabolism Ga-PSMA-11, there are no reports available concerning the differences in the accuracy of the different PSMA radiopharmaceuticals available in this clinical setting.Since the only diagnostic PSMA-targeting radiopharmaceutical employed in all the studies was [177Lu]Lu-PSMA-617 in three RI-refractory TC patients with theragnostic purpose F-FDG PET/CT is the recommended hybrid imaging method of choice in patients with RI [18F]F-FDG PET/CT, PSMA-targeted PET imaging could detect a lower number of lesions, and it was not able to change patient management Lu-PSMA-617 in patients with metastatic castration-resistant prostate cancer [177Lu]Lu-PSMA-617 treatment in three RI-refractory TC patients F-FDG PET/CT in RI-refractory TC was not demonstrated.Overall, a significant advantage in terms of the diagnostic accuracy of PSMA-targeted PET/CT compared to [The potential advantage of the theragnostic value of PSMA-targeted radiopharmaceuticals needs to be further demonstrated (the RI-refractory TC patients performing PSMA radioligand therapy in the literature are scarce).18F]F-FDG PET/CT in RI-refractory TC and studies comparing PSMA-targeted PET findings with immunohistochemical data are warranted.As suggestions for future studies, prospective multicentric studies comparing PSMA-targeted PET/CT with ["} +{"text": "This study triggered a debate on whether and how primary and secondary immune responses occur based on humoral antibody responses to the initial vaccination and revaccination. The anti-SARS-CoV-2 vaccine sheds new light on the interpretation of our previous data. Here, we offer an opinion on the administration of the polyvalent polysaccharide vaccine (PPSV23), which appears to be highly relevant to the primary vaccine against SARS-CoV-2 and its booster dose. Thus, we do not insist this is a secondary immune response but an antibody response, nonetheless, as measured through IgG titers after revaccination. However, we contend that we are not sure if these lower but present IgG levels against pneumococcal antigens are clinically protective or are equally common in all groups because of the phenomenon of \u201chyporesponsiveness\u201d seen after repeated polysaccharide vaccine challenge. We review the literature and propose a new mechanism\u2014caveolae memory extracellular vesicles (CMEVs)\u2014by which polysaccharides mediate prolonged and sustained immune response post-vaccination. We further delineate and explain the data sets to suggest that the dual targets on both Cav-1 and SARS-CoV-2 spike proteins may block the viral entrance and neutralize viral load, which minimizes the immune reaction against viral attacks and inflammatory responses. Thus, while presenting our immunological opinion, we answer queries and responses made by readers to our original statements published in our previous work and propose a hypothesis for all vaccination strategies, i.e., caveolae-mediated extracellular vesicle-mediated vaccine memory. We published a study showing that improvement in response to splenectomy associated defective, in regards to the antibody response to Pneumovax Streptococcus pneumoniae (pneumococcus) in life-threatening proinflammatory response (PR) that triggers severe growth hormone (GH) resistance, suggesting a unique discrete signal transduction switching mechanism through which Cav-1 interfaces with (1177)phospho-SER-endothelial nitric oxide synthase for nitration at the (1007)Y-(1008)Y of JAK2 .E. coli lipopolysaccharide (LPS) in liposomes, which are taken through caveolae-dependent endocytosis onto macrophage plasma membranes, provoking innate immunity pathways in both zebrafish hepatocytes and trout macrophages leads to metabolic deficiencies and mild pulmonary hypertension in mouse models.Macrophages mediate P2X7 receptor-based pro-inflammatory signaling, as modulated by ectonucleotides (CD39) in caveolae and lipid rafts . This LPSOD-bound antibodies against caveolar Plvap lead to CD31 Ab/SOD-related LPS signaling .for pro-Lipopolysaccharides trigger E3 ubiquitin ligase ZNRF1 to act on caveolin-1 ubiquitination and degradation, activating TLR4. TLR4 activation modulates Akt-GSK3\u03b2 through the ZNRF1-CAV1 axis to enhance pro-inflammatory cytokines and inhibit anti-inflammatory cytokine IL-10. Mice with deletion of ZNRF1 in their hematopoietic cells display increased resistance to endotoxic and polymicrobial septic shock due to attenuated inflammation. Our study defines ZNRF1 as a regulator of TLR4-induced inflammatory responses and reveals another mechanism for regulating TLR4 signaling through Cav-1 . We do nResearchers have engineered mammalian extracellular vesicles (EVs) to deliver a host IFITM3 to decrease ZIKV infection . EVs shuThese lines of evidence show that exosomes are associated with a large spectrum of diseases, including cancer, cardiovascular disease, CNS, and viral infection . HoweverIn contrast, immune cells crosstalk innately to adaptive immunity through EVs in the cancer-immunity cycle to modulate cancer progression and metastasis . Neverthhttps://doi.org/10.1016/B978-0-323-42978-8.00008-5, 27 August 2022).Cell membrane-associated receptors, upon agonist\u2013receptor interactions, activate a signaling cascade in physical contact with downstream signaling molecules, depending on the membrane\u2019s lipid composition, which renders the changes in the regulation of lipid bilayer-derived EVs a novel therapeutic strategy . We callCaveolae are clustered with caveolin proteins , high cholesterol and sphingolipid , Src-tyrhttps://doi.org/10.1016/B978-0-323-42978-8.00008-5, 27 August 2022).Ferritin-based nanocarriers of superoxide dismutase (SOD) with uniform size (20 nm diameter) can target caveolar plasmalemmal vesicle-associated protein (Plvap) in endothelial cells to modulate pro-inflammatory cytokines and lipopolysaccharide (LPS) or anti-inflammatory effects . All of Rehmannia glutinosa polysaccharides derived from PEGylation nano-adjuvants stimulate macrophages to secret pro-inflammatory cytokines through macropinocytosis-dependent and caveolae-mediated endocytosis to boost immunity [Along this line, the designer vaccine liposomal LPS-dsRNA activates innate immunity for pro-inflammatory and anti-viral action in zebrafish and trout macrophages . Transcrimmunity . Liposomimmunity . Computaimmunity with enh2 NPs) might act on caveolae in human bone marrow stromal cells to change the molecular profiles based on sensitive single-cell RNA-seq (scRNA-seq) transcriptomes [Similar approaches to the pneumococcal polysaccharide vaccine (PPV) enhance the primary prevention of acute coronary syndrome (ACS) in the elderly in Australia, thereby reducing cardiovascular disease . Recentlriptomes with CRIriptomes . This firiptomes . Omicronriptomes . InvasioSubclonal evolution spatiotemporal tracking system guides diagnostic and treatment. It feels surreal going from caring for the initial onslaught of patients last spring to helping alleviate human suffering through vaccinations. Some readers pointed out that administering a plain Polyvalent polysaccharide vaccine is highly relevant to a messenger RNA vaccine, mRNA-1273, to prevent infection with SARS-CoV-2 after the first vaccination and the These findings suggest that classic vaccines do not block viral entrance but neutralize viral load-derived toxicity. The breathtaking knowledge of COVID-19 vaccines sheds new light on such a grand old field. We suggest a new mechanism by which the dual targets of both Cav-1 and SARS-CoV-2 spike proteins might block the viral entrance and neutralize viral load, minimizing the immune reaction against viral attacks and clearing out the host\u2019s viral survival. Thus, bacteria can activate the host\u2019s immunity to antitumors. For example, Kalaora et al. report tTherapeutic intervention of the tumor microenvironment (TME) with natural products and re-purposed pharmaceuticals illustrates the mechanisms by which exosomes trigger inflammation, leading to immune evasion and tumor progression, and remodeling of the tumor microenvironment, remarkably through EV-mediated signal pathways .Tumor-deStreptococcus pneumoniae (pneumococcus), which appears to be highly relevant to the primary vaccine against SARS-CoV-2 and its booster dose. Conclusion: we indicate that polysaccharides induce memory B cells that are indeed different from the mode of action of protein antigens. Polysaccharide-specific immunoglobulin G (IgG) governs memory B cells that act as T-independent type II immune responses in naive B cells sensitive only to polysaccharides. We offer an opinion regarding caveolae-mediated extracellular vesicle (CMEV) signaling in the administration of the plain Polyvalent polysaccharide vaccine against"} +{"text": "Asparaginyl endopeptidases(AEPs) have recently been widely utilizedfor peptide and protein modification. Labeling is however restrictedto protein termini, severely limiting flexibility and scope in creatingdiverse conjugates as needed for therapeutic and diagnostic applications.Here, we use genetic code expansion to site-specifically modify targetproteins with an isopeptide-linked glycylglycine moiety that servesas an acceptor nucleophile in AEP-mediated transpeptidation with variousprobes containing a tripeptidic recognition motif. Our approach allowssimple and flexible labeling of recombinant proteins at any internalsite and leaves a minimal, entirely peptidic footprint (NGG) in theconjugation product. We show site-specific labeling of diverse targetproteins with various biophysical probes, including dual labelingat an internal site and the N-terminus. Furthermore, we harness AEP-mediatedtranspeptidation for generation of ubiquitin- and ubiquitin-like-modifierconjugates bearing a native isopeptide bond and only one point mutationin the linker region. Developmentof genetic code expansion approaches for site-specific cotranslationalencoding of non-canonical amino acids (ncAAs) bearing bioorthogonalhandles and their reaction with custom-made labels has enabled thegeneration of a diverse range of protein conjugates with an exquisitelevel of control over the labeling site and number of modifications.5 The synthesis and successful incorporation of functionalized ncAAsand probes can however be challenging. Furthermore, most bioorthogonallabeling reactions lead to bulky, hydrophobic, and artificial linkagesbetween the biophysical probe and protein.9Site-specific proteinlabeling with various probes at internalsites and generation of defined protein\u2013protein conjugateshold great promise for studying biological functions of proteins andfor the development of therapeutic and diagnostic bioconjugates.12 In a chemoenzymaticlabeling experiment, the recombinantly expressed target protein isequipped with a peptide recognition motif (4\u201315 amino acids).The respective enzyme (a transferase13 orligase15) binds to this recognition tag and catalyzescovalent attachment of a functionalized substrate to a specific aminoacid within this motif, thereby labeling the protein of interest (POI).The recognition motifs are typically fused to the N- or C-terminusof the POI, allowing installation of modifications close to the targetprotein\u2019s termini. In a few instances, it was also possibleto introduce the recognition sequences into accessible loop regionsof the POI, providing thereby greater flexibility with respect tothe location and number of modifications.16 Still, most of these approaches leave sizable footprints and oftenbulky and artificial linkages in the ligation product.Alternatively, chemoenzymaticlabeling methods have proven to bepowerful tools for attaching probes to specific amino acid side chainswithin recognition sequences under mild conditions.17 sortase,19 and asparaginyl endopeptidases(AEPs)20] have been used successfully forterminal protein labeling and for the generation of protein\u2013proteinconjugates linked via their N- or C-termini. Such enzymes typicallycleave a peptide bond within a recognition motif fused to the POI,forming a labile enzyme-POI intermediate that undergoes specific transpeptidationwith a peptide functionalized with a user-chosen probe.Apartfrom ligases and transferases that covalently attach a functionalizedprobe to an amino acid within the recognition tag, also engineeredproteases and transpeptidases (dubbed OaAEP1 in the following) thatcan be produced recombinantly in E. coli as an ideally suited enzyme for N- and C-terminal labeling of recombinantproteins due to its high catalytic efficiency and minimal recognitionmotif to yield a ligation product. OaAEP1 hasquite stringent sequence requirements for its recognition sequence(P1P1\u2032P2\u2032), with NGL representing an ideal motif, butwas shown to be promiscuous for the incoming nucleophile sequence(P1\u2033P2\u2033). Previous work has found that both GL and GVdipeptides installed at the N-terminus of peptidic probes and proteinsserve as efficient acceptor nucleophiles. Interestingly, the\u2014inthe latter case\u2014resultant NGV ligation product is poorly recognizedby OaAEP1, shifting the reaction equilibrium towardthe product and thereby allowing sequential dual labeling at the N-and C-terminus.23 Furthermore, OaAEP1\u2019s promiscuity for the incoming nucleophilewas harnessed by ligating various primary amines\u2014if presentedat high enough molar excess \u2014toa C-terminal NGL recognition site.24 AEPsthereby represent versatile enzymes for protein labeling, but labelingis so far restricted to protein N- and C-termini.Recentwork has established the engineered AEP onmotif 1a.21,22OOaAEP1-mediated transpeptidationwith genetic code expansion. We show that target proteins carryinga site-specifically introduced isopeptide-linked glycylglycine moiety(GGisoK) serve as acceptor nucleophiles in OaAEP1-mediatedligation with various NGL-bearing probes and proteins, allowing thegeneration of site-specific and user-defined protein conjugates displayinga minimal tripeptidic mark in the ligation product (OaAEP1-mediated transpeptidation forthe generation of defined ubiquitin (Ub)- and Ub-like protein (Ubl)-POIconjugates. Posttranslational modification of target proteins withUb/Ubl presents one of the most common and versatile regulators ineukaryotic biology.26 During ubiquitylation, the C-terminalcarboxylate of Ub is attached to the \u03b5-amino group of a lysinewithin a substrate protein to form an isopeptide bond via a complexmachinery employing E1/E2/E3 enzymes.26 We show that OaAEP1 can be used to covalently attach a Ub/Ubl variantcontaining the NGL recognition motif in its C-terminus to a GGisoK-bearingPOI. OaAEP1-mediated Ub/Ubl conjugates display anative isopeptide bond connecting Ub/Ubl to a specific lysine andbear one point mutation in the linker region. Importantly, we showthat these conjugates are still cleaved by the model deubiquitylaseUSP2,27 and we demonstrate the generalityof our approach by preparing various site-specifically ubiquitylatedsubstrate proteins .On our quest to realizesite-specific protein labeling at internalsites and to generate complex protein\u2013protein conjugates thatare not exclusively linked through their respective N- or C-termini,we here combine product 1b. We moOaAEP1 for nucleophile acceptors (P1\u2033P2\u2033)and we assessed the enzyme\u2019s tolerance and specificity fornucleophiles that can be site-specifically incorporated into proteinsin the form of ncAAs via genetic code expansion. We have recentlyshown that we can site-specifically modify any POI with a GG-dipeptidemoiety attached to the \u03b5-amino group of a lysine residue viagenetic encoding of AzGGisoK and its phosphine-based reduction toGGisoK ,we synthesized GLisoK, GVisoK, and GGisoK and tested their OaAEP1-mediated transpeptidation onto Ub bearing the recognitionsequence NGL at its C-terminus . Unsurprisingly though, thisligation product showed higher stability against enzymatic hydrolysisupon prolonged incubation with OaAEP1 in the absenceof any other acceptor nucleophile, with >85% intact NGG-ligationproductafter 24 h, while NGL- and NGV-bearing ligation products showed \u223c50%hydrolysis under the same conditions (S2). Encouragedby these results, we set out to investigate if GGisoK-bearing proteinscould also function as potential nucleophiles in OaAEP1-mediated transpeptidation. We site-specifically encoded AzGGisoKin response to an introduced amber codon using the previously reported,selective pyrrolysyl-tRNA synthetase-derived AzGGisoKRS/tRNACUA pair.28 The azide moiety in AzGGisoK-modifiedproteins can easily be reduced to its amine analogue via Staudingerreduction using phosphines such as tris(2-carboxyethyl)phosphine or2-(diphenylphosphino)-benzoic acid, generating GGisoK-bearing proteins.Using this approach, we expressed C-terminally H6-tagged Ub, carryingGGisoK at position K48 (Ub-K48GGisoK). In parallel, we synthesizeda desthiobiotin-NGL probe , with the remaining 50% constituting unlabeled Ub-K48GGisoK.Thereby, the released GLH can be quenched by Ni2+ complexation,sequestering the competing nucleophile acceptor . Using dtb-NGLH in the presence of 500 \u03bcMNiSO4, >80% of labeled protein was observed within 1hat 30 \u00b0C. Increasing the OaAEP1 concentrationto 10 \u03bcM and/or using 20-fold excess of the NGLH peptide incombination with 1 mM Ni2+ led to \u223c95% product formationin less than an hour with 50 \u03bcM GGisoK-bearing protein (S3\u2013S5). Importantly, for wild-type Ub(Ub-wt), lacking the GGisoK nucleophile, we could not observe anyligation product formation (S3 and S4). To show thatlabeling progress can indeed be accurately followed by quantifyingMS-peak intensities, we purified dtb-labeled Ub to homogeneity viaStrep-Tag affinity chromatography and determined MS-peak intensitiesof various protein mixtures, confirming a linear correlation betweenprotein concentration and MS-peak intensity . Having established near-quantitative labeling conditions,we next explored the chemical diversity tolerated on NGLH probes forvaried protein labeling. Both alkyne- and fluorophore-NGLH probesallowed efficient labeling of GGisoK-bearing POIs. Furthermore, wesynthesized an NGLH-bearing decapeptide that allowed us to followprotein modification progress via migration differences of unlabeledand labeled protein on SDS-PAGE (S6\u2013S8).We reckoned that the sluggish reaction progressmay stem from thefact that\u2014contrary to traditional labeling approaches\u2014inour approach, the nucleophile-bearing POI (Ub-K48GGisoK) is used asthe substoichiometric component with the recognition motif-containingNGL probe in 10-fold molar excess. As the GL leaving group is releasedfrom the recognition motif over the course of the reaction, this byproductcompetes with the desired GGisoK nucleophile, stagnating in a product-limitingequilibrium. Inspired by previous work,OaAEP1-mediated hydrolysis, wenext explored site-specific sequential protein dual labeling at internalsites and at the N-terminus using differently functionalized NGLH-probes.Therefore, we expressed a Ub-variant bearing GGisoK at position K48and displaying a TEV recognition site at its N-terminus, followedby a GV motif . Additionally, we showed thatthe alkyne moiety could be efficiently used in further Cu(I)-catalyzedalkyne\u2013azide cycloaddition for functionalization with commerciallyavailable azide-bearing fluorophores .As the NGG motif that is installed upon transpeptidationis essentiallyrefractory toward GV motif 3a. In thach step 3b and S932 Guided by the eGFP-nb:eGFP crystal structure(3OGO),33 we selected various surface-exposed positions within eGFP-nbto be replaced by GGisoK. For this, we expressed and purified eGFP-nbvariants bearing GGisoK at individual positions in yields from 1 to 8 mg/L culture (S10). Havingconfirmed via in vitro pull-down assays that all GGisoK-bearing eGFP-nbvariants retained their binding capability toward eGFP , we proceeded with OaAEP1-mediated labeling using a sulforhodamine-NGLH probe . While we observed specific labeling for all sixGGisoK-bearing nanobody variants as judged by in-gel fluorescence,the labeling efficiencies varied greatly among the six variants, presumablydue to disparate steric access of OaAEP1 to the GGisoKacceptor nucleophile. eGFP-nb-R75GGisoK showed >85% labeling within3 h, as assessed by LC\u2013MS, and was purified at preparativescale for further experiments (S11).To demonstrate that GGisoK-directed transpeptidationis suitedfor the preparative generation of defined POI-small-molecule conjugates,we aimed at site-specifically labeling the anti-eGFP nanobody (eGFP-nb)and test its specific binding to eGFP expressed in mammalian cells.34 Cells werefixed and incubated with SuRho-labeled eGFP-nb. Efficient labelingof the cytoskeleton and colocalized green and red fluorescence wasconfirmed by fluorescence microscopy . To show membrane labeling on live cells, we transfectedHEK293T cells with a pDisplay construct where eGFP is fused to thetransmembrane domain of the platelet-derived growth factor receptor(eGFP-PDGFR-TM) to display eGFP on the cell surface.35 Incubation of eGFP-PDGFR-TM-expressing cells with SuRho-labeledeGFP-nb led to specific labeling of cells expressing eGFP at theirplasma membrane .We benchmarked this site-specificallySuRho-labeled eGFP-nb forimaging of eGFP-expressing proteins both in fixed and live mammaliancells. For this, we transfected HEK293T cells with eGFP-LifeAct, afusion between eGFP and a 17-amino-acid-long peptide that binds specificallyto the actin cytoskeleton.croscopy 4e. Imporcroscopy S12. To sOaAEP1 transpeptidation using substrateswith NGL as a recognition motif and GGisoK-bearing proteins as acceptornucleophiles leads to ligation products with an NGG sequence, we hypothesizedthat we could use this approach to build Ub-POI conjugates bearinga native isopeptide bond and harboring only one point mutation inthe Ub C-terminus. Ub is a small, globular, and highly conserved 76amino acid protein.26 Its C-terminus is unstructured,and the last six amino acids have the sequence71-LRLRGG.During ubiquitylation, the carboxylate of G76 is attached to specificlysine residues in a POI (or Ub itself) through a complex enzymaticmachinery, forming an isopeptide bond. We have recently developeda chemoenzymatic approach to build ubiquitylated proteins using thetranspeptidase sortase (sortylation).29 For this,we mutated the Ub C-terminus to contain a sortase (Srt2A)36 recognition motif . Srt2A-mediated transpeptidation between Ub(AT) and a GGisoK-bearingPOI leads to Ub-POI conjugates displaying a native isopeptide bondand two point mutations in the linker region (R72A and R74T). We wereable to show that sortase-generated diUbs largely maintain structuraland functional integrity by retaining their binding affinities tomany Ub-binding domains, a requirement for decoding diverse cellularfunctions. Nevertheless, the two point mutations make sortase-generateddiUbs resistant toward DUBs, indicating that their recognition byDUBs might be impaired. In order to build more native diUbs, we expressedUb bearing an RLNGLH sequence at its C-terminus . K48-diUb(N)could be produced in mg yields, and its identity was confirmed byLC\u2013MS. We next set out to test if the isopeptide bond in K48-diUb(N)would be cleaved by DUBs. We therefore incubated purified K48-diUb-wt,Srt2A-generated diUb [K48-diUb(AT)], and OaAEP1-generateddiUb [K48-diUb(N)] with the catalytic domain of Ub carboxyl terminalhydrolase 2 (USP2CD). Although the Srt2A-generated K48-diUb(AT)is completely resistant to USP2CD cleavage, K48-diUb(N)is as efficiently cleaved to the corresponding monoUbs as observedfor K48-diUb bearing the wt sequence in the linker region (S14).Given that OaAEP1-mediatedubiquitylation,we showed that other natively ubiquitylated substrate proteins canalso be accessed by OaAEP1 and Ub(N). We preparedSUMO2 displaying GGisoK at position K11, as K11 represents a well-knownand the most abundant Ub linkage site on SUMO2.37 Incubation of SUMO2-K11GGisoK with Ub(N) in the presenceof OaAEP1 led to specific formation of the SUMO2-Ubconjugate (tert butyloxycarbonyl-l-lysine (BocK) at position K11 did not generate any SUMO2-Ubconjugate . Among the mostabundant monoubiquitylated proteins are histones H2A and H2B withubiquitylation fulfilling critical roles in regulating transcriptionand other cellular processes.40 Recent reports have shown thatalso histones H3 and H4 are ubiquitylated,41 for example, various large-scale quantitative proteomics studiesidentified several ubiquitylation sites within H3,42 but the functions of these modifications are less understood.To demonstrate that OaAEP1-mediated ubiquitylationis suited to generate defined Ub-H3 conjugates, we prepared H3-constructsbearing amber codons at nine lysine positions and incorporated AzGGKin response to these introduced amber codons . After reduction of the azide group in AzGGK,we purified H3-K23GGisoK, H3-K27GGisoK, and H3-K79GGisoK via affinitychromatography and cation exchange followed by refolding. All threeH3-GGisoK variants were incubated with Ub(N) and OaAEP1, and specific formation of Ub-H3 conjugates was observed within5 min. Ubiquitylation yields ranged between 31 und 35% (S15).To prove the generality of onjugate 5d, whileOaAEP1-mediatedgenerationof Ub conjugates may also be extendable to other Ubls that share Ub\u2019scommon \u00df-grasp fold with a flexible six-residue C-terminal tailand the characteristic GG motif that is attached to a lysine residuein the target protein. We introduced the NGL recognition motif intothe C-terminus of the small-Ub-like-modifier SUMO243 by introducing the point mutation T91N, generating SUMO2(N)with the C-terminal sequence 89-QQNGLH6 .We envisioned that -QQNGLH6 5f. GratiOaAEP1 allows site-specific internal labelingof GGisoK-bearing proteins with a variety of small molecules and biophysicalprobes. OaAEP1-mediated labeling can be conductedat physiological pH in mild and aqueous buffers and leads to near-quantitativeand essentially irreversible formation of the ligation product inthe presence of Ni2+ salts. OaAEP1-mediatedlabeling compares favorably to other chemoenzymatic labeling approachesin terms of size and nature of recognition tag and footprint leftin the ligation product. It requires an NGL recognition motif in thelabeling reagent and the site-specific incorporation of GGisoK intoa target protein, leading to a flexible and minimal tripeptidic NGGmotif in the linker between the label and POI in the conjugation product.This is in contrast to other enzymatic labeling approaches such astransferases and ligases that typically leave a mark of at least 6\u201315 amino acidsin their conjugation products and often result in bulky and artificiallinkages. Importantly, OaAEP1 only requires stablepeptidic probes that can be easily synthesized and/or are often commerciallyavailable and results in entirely peptidic linkers, which may be especiallyadvantageous for generating antibody-drug-like conjugates with reducedimmunogenicity for in vivo administration. As the resulting NGG motifin the ligation product is essentially resistant toward further OaAEP1 hydrolysis, our approach is also suitable for sequentialdual labeling at both an internal site and the N-terminus with diverseprobes. Additionally, only low concentrations of OaAEP1 are required for efficienttranspeptidation, as opposed to, for example, sortase-mediated labeling28 and Ubc9-mediated labeling,16 which mostly rely on >0.2 mol equivalents of their correspondingenzymes for high conversion rates. OaAEP1-mediatedtranspeptidation in combination with site-specific incorporation ofGGisoK via genetic code expansion therefore represents an operationallysimple process for labeling of recombinant proteins at internal sites,leaving a minimal, entirely peptidic scar (NGG) in the ligation product.It is however worth noting that due to the small recognition motifNGL, unwanted cleavage within the target protein may be a side reactionthat can occur during OaAEP1 transpeptidation, especiallyif the POI displays asparagine residues followed by small and hydrophobicamino acids in highly exposed loop regions.We have shown that therecently engineered and chemically improvedAEP OaAEP1-mediated labeling can alsobe harnessed for conjugation of folded proteins to generate novelarchitectures and protein\u2013protein conjugates in a programmablemanner. On our quest to generate and study distinct Ub/Ubl topologies,we were able to show that OaAEP1-mediated transpeptidationallows site-specific monoubiquitylation and monoSUMOylation of targetproteins. We have shown the ubiquitylation of diverse target proteinsincluding the site-specific covalent attachment of Ub to SUMO2 andhistones using OaAEP1. OaAEP1-generatedUb-POI conjugates display a native isopeptide bond connecting Ub andthe POI with only one point mutation in the Ub/Ubl C-terminus. Comparedto our previously developed sortylation approach,29OaAEP1-mediated site-specific conjugation of Ub/Ublto target proteins requires less enzyme and shows faster conversionto product. For sortase-mediated ubiquitylation, a leucine spaceramino acid is often introduced preceding the sortase recognition motif,as this increases accessibility of the sortase to the Ub C-terminusand therefore sortylation yields.28 Interestingly,introduction of such a spacer amino acid was not needed for OaAEP1-mediated ubiquitylation and SUMOylation. Excitingly,this leads to Ub/Ubl-POI conjugates that contain only one single pointmutation in the linker region between Ub/Ubl and the POI. In fact\u2014incontrast to sortase-generated diUbs\u2014OaAEP1-generateddiUbs are cleaved by DUBs to a similar extent as diUbs bearing a wt-linker,confirming their functional and structural resemblance to endogenousUb/Ubl-conjugates. In this sense, OaAEP1-mediatedubiquitylation complements the approach recently reported by Bodeand co-workers that requires the introduction of up to three pointmutations into the POI to make it a substrate for Ubc9-mediated ubiquitylation.46 Ultimately, we envision that OaAEP1-mediated generationof Ub and Ubl conjugates may be combined with sortylation to furtherextend our recently developed approach Ubl-tools 29 as a modularand robust tool for accessing defined Ub/Ubl chains where we can placeDUB-resistant and DUB-susceptible linkages at defined positions.Importantly,"} +{"text": "Leptomeningeal carcinomatosis (LC) is a rare but ominous manifestation of systemic cancer that occurs from the infiltration of cancer cells into the cerebrospinal fluid and leptomeninges. The most common solid tumors exhibiting leptomeningeal spreading include breast, lung, melanoma, gastrointestinal and central nervous system tumors . Within Instituting a standard treatment regimen for LC has been impacted by several factors: heterogenous LC population, rapid progressiveness, low incidence, paucity of adequate randomized clinical trials, and low accrual. The current standard of care for LC management is thus multidisciplinary, including radiotherapy (RT), systemic and intrathecal (ITC) chemotherapies, and surgery \u20138. Reporin vitro and in vivo, thus restricting the spread of HER2+ LC beyond the leptomeningeal surface. Furthermore, cytokine-array-based analysis conducted on conditioned media derived from the various CNS cell types and Lepto cells revealed higher granulocyte-macrophage colony-stimulating factor (GM-CSF) expression in Lepto vs. other CNS cells. Notably, Lepto-bearing mouse brain sections also exhibited elevated levels of the phospho-GM-CSF receptor \u03b1.Our recent comprehensive analysis has defined the signaling events contributing to HER2+ LC development and the pathways that allow its expansion to leptomeninges. To ascertain the role of host glial cells in regulating the growth and development of HER2+ LC, we successfully generated the first-ever expandable primary cell lines from the HER2+ LC patient-derived tumors (\u201cLepto\u201d cells) . Interesin vitro and in vivo.The presence of OPCs significantly reduced the HER2+ LC cell viability, as evident from the enhanced apoptotic signal observed in Lepto cells grown in OPC-conditioned hCSF or hCSF containing anti-GM-CSF antibodies than that in naive Lepto cells. Reduced activation of GM-CSFR \u03b1 and downstream targets STAT5, AKT, and ERK1/2 further corroborated the essential role of OPC in regulating GM-CSF secretion from the HER2+ LC cells. LC/MS-MS-based analysis of secretomes of human astrocytes and OPCs, cultured alone or with Lepto cells, revealed TPP1 as the regulator of GM-CSF; it mediates GM-CSF proteolytic degradation and suppresses signaling, thereby decreasing Lepto cell viability and tumor progression. Remarkably, combinatorial treatment of anti-GM-CSF antibody with a pan-Aurora kinase inhibitor (CCT137690) synergistically halts GM-CSF signaling and HER2+ LC growth Our work has demonstrated neural niche-specific crosstalk between HER2+ LC tumors and OPCs and established the efficacy of intrathecal administration of TPP1 protease, anti-GM-CSF antibodies, and pan-Aurora kinase inhibitors in combating HER2+ LC . This ma"} +{"text": "N-heterocyclic olefins (NHOs)are utilizedas catalysts for the ring-opening polymerization (ROP) of functionalaliphatic carbonates. This emerging class of catalysts provides highreactivity and rapid conversion. Aiming for the polymerization ofmonomers with high side chain functionality, six-membered carbonatesderived from 2,2-bis(hydroxymethyl)propionic acid (bis-MPA) servedas model compounds. Tuning the reactivity of NHO from predominantside chain transesterification at room temperature toward ring-openingat lowered temperatures (\u221240 \u00b0C) enables controlled ROP.These refined conditions give narrowly distributed polymers of thehydrophobic carbonate 5-methyl-5-benzyloxycarbonyl-1,3-dioxan-2-one(MTC-OBn) (\u0110 < 1.30) at (pseudo)first-orderkinetic polymerization progression. End group definition of thesepolymers demonstrated by mass spectrometry underlines the absenceof side reactions. For the active ester monomer 5-methyl-5-pentafluorophenyloxycarbonyl-1,3-dioxane-2-one(MTC-PFP) with elevated side chain reactivity, a cocatalysis systemconsisting of NHO and the Lewis acid magnesium iodide is requiredto retune the reactivity from side chains toward controlled ROP. Excellentdefinition of the products (\u0110 < 1.30) andmass spectrometry data demonstrate the feasibility of this cocatalystapproach, since MTC-PFP has thus far only been polymerized successfullyusing acidic catalysts with moderate control. The broad feasibilityof our findings was further demonstrated by the synthesis of blockcopolymers for bioapplications and their successful nanoparticularassembly. High tolerability of NHO in vitro with concentrations rangingup to 400 \u03bcM further emphasize thesuitability as a catalyst for the synthesis of bioapplicable materials.The polycarbonate block copolymer mPEG44-b-poly(MTC-OBn) enables physical entrapment of hydrophobic dyes insub-20 nm micelles, whereas the active ester block copolymer mPEG44-b-poly(MTC-PFP) is postfunctionalizableby covalent dye attachment. Both block copolymers thereby serve asplatforms for physical or covalent modification of nanocarriers fordrug delivery.Herein, Therefore, controlled polymerization offunctionalized monomers has not been achievable so far.The class of 21 Thus, tuning polymer functionalization provides the ability (a)to anchor or encapsulate therapeutic molecules,23 (b) to enable a (triggered/stimuli-responsive) release,25 and (c) to fine-tune polymer degradation.27 Aliphatic polycarbonates are a promising material class in thisregard due to their functionalizability by side chain introduction,biodegradability, and favorable toxicological profiles.29 Polycarbonate-based block copolymers were self-assembled into nanoparticularcarriers and used for a variety of therapeutic applications.33 An important motif for functional polycarbonates are six-memberedmonomers derived from 2,2-bis(hydroxymethyl)propionic acid (bis-MPA)with a carboxy side chain substituent which, through esterification,can comprise a wide range of different functional groups.34 An important monomer within this group is the hydrophobic 5-methyl-5-benzyloxycarbonyl-1,3-dioxan-2-one(MTC-OBn) that fosters block copolymer self-assembly into micellarnanoparticles and the encapsulation of hydrophobic (aromatic) compounds(supported by \u03c0\u2013\u03c0 stacking).35 Alternatively, the development and polymerization of 5-methyl-5-pentafluorophenyloxycarbonyl-1,3-dioxane-2-one(MTC-PFP), a six-membered carbonate monomer with an active ester sidechain motif, by Hedrick and co-workers enlarged the polycarbonatetoolbox.37 However, previously ROP of MTC-PFP was onlyachieved by using an acidic catalyst which, however, provided polymers of only moderate definition.37 Starting from poly(MTC-PFP) postpolymerization,modification reactions access various materials by amine conjugation.39 Polymeric materials accessed from both monomers were shown to behighly tolerable in vitro33 and even invivo,40 and they can therefore be consideredas biocompatible.Especiallyfor biological applications, functional polymers areneeded to manufacture tailor-made materials.41 Due to its high side chain reactivity,the active ester monomer MTC-PFP was chosen as an appealing, yet challengingmonomer to study the NHO-guided catalyst system. Overall, we intendedto extend NHO-based catalysis to the controlled chain growth polymerizationof functional monomers and to further demonstrate the feasibilityof this approach with respect to accessing micellar nanocarriers,which allow encapsulation or covalent conjugation of molecules fordrug delivery.By selecting the functional carbonate monomersMTC-OBn and MTC-PFPfor our study, we aimed to investigate the influence of ester sidechain motifs on the polymerization process under NHO catalysis. Sinceorganobase ROP catalysts, such as NHOs, often also display pronouncedtransesterification activity, ester side chains are not fully orthogonalfunctionalities and, therefore, pose a major challenge for the controlledROP of carbonates.15 First experiments focused on the polymerization of MTC-OBn and aimedat gaining a deeper insight into general reaction parameters suchas concentration and temperature . Transesterification at the sidechain leads to the release of benzyl alcohol, resulting in polymerchains with benzyl alcohol as initiating end group )which limits achievable high molecular weights and control over theend groups. Further side reactions such as backbiting by transesterificationof hydroxy chain ends at the ester groups of the polymer side chainand at the polycarbonate backbone could be identified as well. Backbitingreactions yield ring-closed polymers that have lost their reactivehydroxy end group and therefore create nonpropagating chains, whichfurther deteriorate the polymerization outcome. Yet, the nonoptimizedpolymerization attempt still confirmed the ability of the NHO to catalyzering-opening of functional six-membered carbonates, however, the observedside reactions limit control and demand further improvements.For the establishment of NHO-catalyzedROP of functional six-memberedcarbonates, the NHO 1,3-dimethyl-2-(1-methylethylidene)imidazolidinewas employed, which was previously identified as a controlled catalystin the ROP of the archetypal carbonate TMC.perature 1A. ReactFigure S3 for NMR spectra). The strong influence of temperature becameevident by the drastically narrowed dispersity (\u0110) from 1.80 (at +25 \u00b0C) to 1.29 (at \u221240 \u00b0C) . Even more clearly, MALDI-ToF spectrometryshows poly(MTC-OBn) with the desired pyrene-butanol initiator groupand hydroxy end groups, proving the end-group defined polymerizationof MTC-OBn using NHO and theabsence of side reactions. By adjusting the reaction parameters, wetuned the catalytic behavior of NHOs and redirected its high activityfrom transesterification at the side chain toward the ring-openingof the cyclic six-membered carbonate (1H NMR spectroscopy) during reaction time,and high conversions of \u223c90% were found after 20 h .To further demonstratethe controlled manner of NHO-catalyzed ROPof MTC-OBn, we investigated the kinetic profile of the reaction bytaking samples from the reaction solution at defined intervals andanalyzing them 1D. Contiter 20 h 1D, top. 19F NMR spectra revealed a minor conversion ofthe active ester side chains, indicated by released pentafluorophenol(PFP), when monomer, catalyst, and initiator were mixed. Due to thehigher side chain reactivity of MTC-PFP compared to MTC-OBn, the samereaction conditions in this case did not result in polymerizationbut in consumption of initiating alcohols through transesterificationreactions. Subsequently, the non-nucleophilic PFP is released whichdoes not initiate polymer chain growth. Although the initial goalof MTC-PFP polymerization could not be achieved, the observed highregiospecificity of the side chain reaction attracted our attention,since it could enable a straightforward synthesis of bis-MPA-basedmonomers with functional side group substituents.Encouraged by these excellent results, weattempted the polymerizationof the reactive ester monomer MTC-PFP 2. Howeve19F NMR spectroscopy,underline the catalytic potential and regiospecificity of NHOs fortransesterification of MTC-PFP (4) product were then identified by 1H NMR spectroscopy and demonstrated the feasibilityof this approach for the straightforward generation of MTC monomers.Therefore, we investigated the feasibility of NHO-catalyzedtransesterificationfor the reaction with tetraethylene glycol monomethyl ether 2A. High MTC-PFP 2B. Respe43 Having established that a direct polymerization of MTC-PFP usingsolely NHO is impossible, we therefore attempted a cocatalyst approachemploying the Lewis acid magnesium iodide (MgI2). Thisspecific metal halide has been found to significantly increase polymerizationcontrol and to moderate NHO reactivity for lactone conversion.43 We thus opted for this particular Lewis acidto prevent transesterification but retune NHO activity toward ROP.Similar to the ROP of MTC-OBn, the cocatalytic approach for MTC-PFPwas attempted at decreased temperature and low monomer molarity (\u0110 \u2248 1.25) of the obtained polymers highlight the controlledmanner of the cocatalyzed polymerization . Detailed mass analysis of the polymersby MALDI-ToF spectrometry confirmed the end group integrity . Successful preparationof homopolymers with targeted DP of 20 and 40 showed the high controlover molar mass and molecular integrity (S11). We have for the first time conclusively shown the ROP of MTC-PFPwith a nonacidic catalyst system . Analysis of theobtained data revealed again a (pseudo)-first-order kinetic . In contrast,when both are applied as a NHO-metal halide cocatalyst system, thereactivity is directed toward the cyclic carbonate functionality,as proposed by the reaction mechanism of In summary,we have shown that NHO itself does not allow ROP ofMTC-PFP but rather catalyzes transesterification at the side chain,which enables the synthesis of new monomers. Also, the Lewis acidmagnesium iodide alone does not trigger any ROP at the chosen reactionconditions . Fortunately, noadverse effects on cell metabolism could be found by MTT -2,5-diphenyltetrazoliumbromide) assay after 24 h .In addition, the cocatalyst\u2019s magnesium salts have alreadybeen applied in several medicinal contexts44 and its abundance in biological environments45 suggests a high tolerability of the Lewis acid magnesiumiodide.Considering an applicationof the obtained materials in a biomedical context, we aimed to explorethe suitability of NHOs and therefore tested the biocompatibilityof the catalyst. Residual amounts of the catalyst might potentiallyremain in the material and would then be applied along with the material.44-OH) to yield polyethylene glycol-polycarbonate block copolymers(44-b-poly(MTC-OBn) block copolymers )were synthesized using the NHO cocatalyst system and gave similarresults as mentioned for MTC-OBn block copolymer (44). The uniqueproperty of the active ester polymer to undergo an additional postpolymerizationmodification was leveraged by covalent attachment of a water-solubleamine-functional fluorescent dye . Subsequently, all remaining active ester sites of the dye-labeledpolymer were converted using benzyl amine to yield an amphiphilicmPEG44-b-poly(MTC-NHBn) block copolymerthat was purified by dialysis to remove PFP and then freeze-driedto gain a red voluminous powder. The conversion of the PFP-ester couldbe monitored by 19F NMR during the aminolysis reaction,while the isolated poly(MTC-NHBn) block copolymer did not provideany fluorine signals anymore demonstrating quantitative modificationof the MTC-PFP block . In principle,the active ester approach therefore allows the conjugation of furthercargos to the polymer chains such as amine-functional therapeuticdrug molecules and is, thus, highly versatile for the generation oftailor-made drug carrying nanoparticles.For accessing nanosized drug delivery systems,amphiphilic blockcopolymers are of high interest due to their ability to self-assembleinto micellar nanoparticles. To demonstrate the general suitabilityof functional materials synthesized by NHO organocatalysis for thesepurposes, we next aimed to prepare amphiphilic polycarbonate-basedblock copolymers. Therefore, we adapted the previous polymerizationparameters and initiated polymerization by polyethylene glycol (mPEGpolymers4A. Usingtroscopy 4B. MTC-P44-b-poly(MTC-OBn)and mPEG44-b-poly(MTC-NHBn) were, respectively,self-assembled to form polymericmicelles by the solvent evaporation method.33 Using this method, polymer chains are first dissolved in acetoneand then added dropwise to water. Upon evaporation of the volatileacetone, polymeric micelles are formed due to the high water solubilityof the mPEG44 block and the insolubility of the benzylester/amide polycarbonate block. Physical interactions of the highlyhydrophobic polycarbonate block stabilize the micelles and can entraphydrophobic cargos. Addition of the hydrophobic dye octadecyl rhodamineB to mPEG44-b-poly(MTC-OBn) in the initialacetone solution thereby provides access to dye containing micelles.For nanoparticle formationblock copolymers of mPEGFigure S23). Further micelle characterization by UV\u2013vis spectroscopyfurther proved either the covalent dye-labeling of mPEG44-b-poly(MTC-NHBn) or the hydrophobic dye encapsulationof mPEG44-b-poly(MTC-OBn), as for bothparticles an additional dye-absorption at long wavelengths was foundby UV\u2013vis spectroscopy , the overall concept provides accessto functional polycarbonate-based materials with promising propertiesfor future drug delivery applications.Both particle solutions were subsequently characterized by DLSanalysis, and successful particle aggregation was confirmed by hydrodynamicdiameters of \u223c15 nm block copolymers and self-assembled dye-containingmicelles by the solvent evaporation method. Using the active estermonomer, mPEG44-b-poly(MTC-PFP) blockcopolymers were yielded by NHO-metal halide cocatalysis. Subsequently,these precursor polymers were covalently equipped with the amine-functionalfluorescent dye tetramethyl rhodamine cadaverine and remaining activeesters were converted with benzyl amine. Dye-labeled mPEG44-b-poly(MTC-NHBn) was then also assembled into polymericmicelles, complementing the fabrication approach of physical dye-loadedmPEG44-b-poly(MTC-OBn) with a covalentattachment approach. Overall, we thereby demonstrated, to the bestof our knowledge, for the first time that NHO catalysis is a feasibleway to polymerize functional monomers and to even generate reactivepostfunctionalizable polymers. The nanoparticular assembly of thesepolymers opens a wide range of possibilities for biomedical applicationsfounding on the rapid and controlled NHO-catalyzed ROP of functionalcyclic carbonates.In conclusion, we herein reported on theNHO-catalyzed polymerizationof functional monomers. After reaction optimization, this organocatalyticapproach allows defined syntheses of ester functional polycarbonatesderived from bis-MPA. For the benzyl ester derivative MTC-OBn, loweringpolymerization temperature was the key to tune NHO\u2019s catalyticactivity from transesterification at the side chain substituent towardROP at the cyclic carbonate motif. Thereby, we accomplished controlledpolymerizations yielding end group defined polymers. However, applicationof these polymerization conditions to a monomer with an activatedester side group (MTC-PFP) resulted exclusively in transesterification.Leveraging the high regiospecificity, we then demonstrated the NHO-catalyzedtransesterification of MTC-PFP as a route to generate ester functionalmonomers. With the aim of directing the reactivity from transesterificationtoward ring-opening polymerization, we implemented a cocatalyst system.By using NHO and the Lewis acid magnesium iodide, we achieved controlledROP of MTC-PFP, which was previously only possible by acid catalysis.The cocatalyst approach not only provides higher control and end groupdefinition but also perspectively allows for the combination of acid-labilemonomers/initiators with the highly versatile MTC-PFP monomer. Movingfurther toward biological application, we confirmed the low toxicityof the NHO organocatalyst in vitro and were thus encouraged to usethe corresponding polymers for the preparation of polymeric micelles.By synthesizing amphiphilic block copolymers grafting onto mPEG"} +{"text": "To overcome technical limitations of detecting low frequencies of antigen-specific-T cells in human patients, we sought to establish a highly sensitive platform for antigen-specific-T cell detection and activation using IBMAM .We first established immortalized B cells in-vitro as antigen-presenting cells. B cells were isolated from CMV-seropositive normal healthy donors (NHDs), stimulated, and transduced with retrovirus containing BCL-6 and BCL-XL. Purified CD4+ T lymphocytes obtained from the NHDs PBMC were co-cultured in-vitro with autologous immortalized B cells transfected with CMVpp65 mRNA fused to MITD (IBMAM) as a model antigen and then measured for activation using IFN-g ELISA. We developed two pathways for IBMAM mediated antigen-specific T cell activation and detection. For the first pathway, if IFN-g is detected, T cells are isolated again from NHDs PBMC and expanded using rapid expansion procedures (REP). After the first REP, antigen-specific T cells are detected using IBMAM, sorted, and a second REP is performed. However, for the second pathway, if IFN-g is not detected, NHDs PBMC is stimulated twice using IBMAM and subsequently antigen-specific T cells are detected using IBMAM again.B cells were successfully immortalized by transfecting at least 5*105 cells with retrovirus (1MOI) and co-cultured with CD40L-IL21 expressing human fibroblasts. We observed a significantly increased activation of antigen-specific CD4+ T cells when using IBMAM in comparison to controls transfected with CMVpp65 mRNA lacking the MITD fusion or a known CMVpp65 peptide pool. For pathway 1 and 2 of our platform, successful detection, activation, and expansion of CMVpp65-specific T cells were observed from low frequency NHDs samples using IBMAM.We have thus established a highly sensitive method of immuno-monitoring that can be used for the detection of antigen-specific T cells in patient samples with low frequencies of antigen-specific-T cells, such as human cancer antigens."} +{"text": "Amp tracer system based on HSV-1 strain H129. The H129Amp tracer system consists of two components: an H129-dTK-T2-pacFlox helper which assists H129Amp tracer\u2019s propagation and transneuronal monosynaptic transmission. The shared viral features of tracer/helper allow for simultaneous single-injection and subsequent high expression efficiency from multiple-copy of expression cassettes in H129Amp tracer. These improvements of H129Amp tracer system shorten experiment duration from 28-day to 5-day for fast-bright monosynaptic tracing. The lack of toxic viral genes in the H129Amp tracer minimizes toxicity in postsynaptic neurons, thus offering the potential for functional anterograde mapping and long-term tracer delivery of genetic payloads. The H129Amp tracer system is a powerful tracing tool for revealing neuronal connectomes.Monosynaptic viral tracers are essential tools for dissecting neuronal connectomes and for targeted delivery of molecular sensors and effectors. Viral toxicity and complex multi-injection protocols are major limiting application barriers. To overcome these barriers, we developed an anterograde monosynaptic H129 Minimizing toxicity of herpes simplex virus 1 (HSV) based tools for anterograde tracing is important for functional studies. Here the authors generate an anterograde monosynaptic tracer system based on HSV amplicon, which shows fast tracing, bright labeling, low toxicity, input-defined postsynaptic neurons\u2019 anterograde monosynaptic tracing feature, and has potential for functional mapping. Although multiple anterograde monosynaptic tracers have been developed based on the herpes simplex virus 1 (HSV-1) strain H129 (H129), functional mapping for output connectome requires further technical development to minimize viral toxicity. Retrograde and anterograde monosynaptic tracing can be achieved by combining two components: (i) a conditionally competent viral tracer (tracer), and (ii) a helper virus (helper). Replication- and/or transmission deficiencies due to the deletion of one or more genes that are required for transneuronal spread to connected neurons of the viral tracer component is a feature that enforces monosynaptic spread. The helper virus component expresses genes that complement the tracer\u2019s deficient gene(s), thus supporting tracer replication and/or transmission6. To maintain the limit of monosynaptic spread, the helper virus must be limited to the initial set of infected cells. To date, all the published monosynaptic tracers use replication-deficient adeno-associated virus (AAV) as the helper virus component9. Due to the slow onset of expression for AAV vectors, the use of AAV necessitates two separate injections, first helper and then tracer, to allow sufficient expression and accumulation of the complementary gene and to support the tracer\u2019s replication and transmission. Subsequently, the tracer is injected into the same location as the previous AAV helper injection 2\u20133 weeks later9. Starting from helper injection to brain collection, the duration of these multi-step experiments typically takes 3\u20134 weeks. One obvious disadvantage of this protocol is that the obligatory sequential injections of the helper and tracer potentially leads to insufficient overlap of their injection locations due to injection variation of the sites of their respective injections, which can lead to variable results between experiments and inefficient tracing due to spatial mismatch of the helper and tracer injection sites.Retrograde monosynaptic tracers derived from rabies virus have been broadly applied for anatomical mapping of the input connectome of neural circuits and functional targeted delivery of genetically encoded sensors and effectors10 which requires no helper and is thus an exception to the above-mentioned scheme for two component helper-dependent tracers11. AAV1 has several disadvantages for neuronal tracing: (i) it fails to perform starter-specific tracing; (ii) it requires high viral dosages which often leads to undesired retrograde labeling and unintended leak to nearby areas4; and (iii) it requires an additional reporter system to amplify the signal to compensate for poor transneuronal transmission efficiency11. Furthermore, AAV1 can only carry small genetic payloads under 5\u2009kb. These combined shortcomings limit the utility of transneuronal AAV112. The second system is a newly developed tracer derived from YFV-17D, a live attenuated yellow fever vaccine virus, and the third is an AAV expressing mCherry fused with the improved wheat germ agglutinin (AAV2-mWGA-mCherry)13. Both YFV-17D derived and AAV2-mWGA-mCherry anterograde tracers can map monosynaptic projectomes in wild-type and Cre mice, and represent promising new systems that merit further investigation. The remaining anterograde tracing systems are all derived from H129, an HSV-1 strain that predominantly spreads anterogradely. The H129-based system has been modified in two ways to include TK deficient tracer versions and gK deficient tracer versions (H129-dgK-G4), representing improved tracing systems14. H129-dTK-tdT and H129-dTK-T2 exhibit limited labeling efficiencies, but H129-dgK-G4 exhibits higher labeling efficiency with improved anterograde specificity14. However, these H129 modified variants all confer strong neuronal toxicity, thus limiting their use for functional connectome mapping for the targeted delivery of sensors and effectors15. These toxicity issues are further complicated by requirements for sequential injection protocols and long experimental durations. These limiting features are shared by most current anterograde and retrograde monosynaptic transneuronal viral tracer systems4. Ongoing efforts focus on circumventing these limitations for the next generations of novel monosynaptic tracer systems.Relative to progress on retrograde neural tracers, the development of useful anterograde tracers is far behind. To date, there are six published anterograde monosynaptic tracer systems that employ to three types of viruses. The first is transneuronal AAV116. The amplicon plasmid contains Ori and pac, two essential cis-elements of HSV-1 genome. Ori plays a critical role in initiating the amplicon plasmid replication and forming the concatameric pseudo-genome, and pac is essential for the pseudo-genome recognition and packaging into the viral capsid18. The HSV-1 amplicon shows promise as a gene transfer vector based on its unique features: (i) broad cell tropism that mimics the infectivity of wild-type HSV-1; (ii) large transgene capacity that supports a maximum of ~150\u2009kb genetic payload20; (iii) high transgene expression efficiency, expressing the target gene(s) simultaneously with the multiunit expression cassettes (multiple-copy of target); and (iv) vastly minimized toxicity as it neither replicates nor produces toxic viral proteins20.The HSV-1 amplicon is a pseudovirus particle that carries a pseudo-genome, a concatemeric form of a multiunit amplicon plasmid. The HSV-1 pseudovirus particle is equipped with the identical capsid, tegument layer, and envelope proteins as the wild-type HSV-1, thus sharing identical host/cell tropism and infection featuresAmp). The H129Amp tracer system is composed of H129Amp tracer and H129-dTK-T2-pacFlox helper. H129-dTK-T2-pacFlox helper has the thymidine kinase gene deleted (dTK), expresses two copies of the fluorescent protein tdTomato (T2), and one copy of pac removed while the remaining one flanked by loxN which can be subsequently excised by Cre-recombinase. The H129Amp tracer contains multiple-copy genetic payloads that express simultaneously, conferring high expression efficiency and high labeling intensity. We generated three different tracers, H129Amp-CTG, H129Amp-DIO-TG, and H129Amp-Flp-DIO-TG to achieve a range of experimental goals. All three tracers utilize the same helper to produce/package the H129 amplicon (\u201ctracer\u201d) to facilitate anterograde spread to postsynaptic neurons. The identical virion structure determines the commonly shared infection features of the tracer/helper and thus requires only a single injection instead of the conventional requirements for sequential injections. As a result, the H129Amp tracer system labels postsynaptic neurons with high labeling intensity within 5 days. Notably, the H129Amp tracer system displays minimized toxicity in the postsynaptic neurons since H129Amp tracers do not express toxic viral proteins. This feature of minimized postsynaptic toxicity offers considerable potential for functional mapping studies using optogenetic effectors combined with electrophysiological assays.In the present study, we developed an anterograde monosynaptic tracer system by using H129 amplicon . H129-dgK tracer pair (helper AAV2/9-DIO-mCh-gK and tracer H129-dgK-G4) were sequentially injected in the 2nd-order neuronal target sites that are innervated by the 1st-order neurons. The newly synthesized H129Amp tracer is transmitted to postsynaptic neurons, making them the input-defined 2nd-order neurons. There, the Cre-recombinase expressed by H129Amp tracer initiates the Cre-dependent AAV helper (AAV2/9-DIO-mCh-gK) expressing gK to support H129-dgK-G4 further anterogradely transmission to the 3rd-order neurons, thus achieving input-defined postsynaptic neurons\u2019 monosynaptic anterograde tracing by labeling the 3rd-order neurons.Input-defined postsynaptic neurons\u2019 monosynaptic anterograde tracing has been successfully acheived by combining H129Amp tracer system represents a significant advance for anterograde monosynaptic tracing, and its applications will contribute to revealing the output connectome by combining anatomical tracing with targeted functional circuit analysis.The H129Ori and pac are two essential cis-elements for replication and packaging of the HSV-1 genome24. We obtained the amplicon plasmid backbone pHSV by cloning Ori and pac from H129 to the pCDNA3.0 plasmid. Subsequently, an expression cassette of Cre-recombinase (Cre), HSV thymidine kinase (TK), and GFP were inserted into the pHSV construct, generating the amplicon plasmid pHSV-Cre-TK-GFP (pHSV-CTG), the basic unit of H129Amp tracer\u2019s pseudo-genome were removed from the bacterial artificial chromosome (BAC) of H129-dTK-T2 (H129-dTK-T2-BAC). The remaining complete pac sequence was flanked by loxN and was reconstituted as the recombinant helper virus H129-dTK-T2-pacFlox (helper) Fig.\u00a0.Fig. 1H1Amp tracer, represented by H129Amp-CTG, was produced in Vero cells as described in the Methods. Briefly, Vero cells were initially transfected with pHSV-CTG, and then infected with the helper, which was separately propagated in Vero cells as described7. TK deficiency severely impairs helper replication in neurons, but not in Vero cells which fully support viral genome replication, viral protein synthesis, as well as production of helper25. In Vero cells, the helper supports the production of high-titer H129Amp tracer, pseudovirus particles, by providing all viral functional and structural proteins necessary for the replication and packaging of pseudo-genome of concatemeric pHSV-CTG18. H129Amp-CTG expresses Cre which efficiently excises the floxed-pac in the H129-dTK-T2-pacFlox genome. Loss of pac disarms encapsidation of H129-dTK-T2-pacFlox genome, thus terminates helper production18 and H129-dTK-T2-pacFlox helper (~5%). These titers of H129Amp tracer, as well as helper \u201ccontamination\u201d, in the raw H129Amp product were determined by plaque-forming assays and judged by their respective fluorescences26. Independently propagated helper was added to the tracer raw product to adjust the tracer and helper to the working titers, which have been carefully optimized for the best tracing efficiency is inevitable and cannot be removed in the raw H129Amp tracer product and the helper co-infect the same neurons when are simultaneously administrated into the brain area of interest. These viral tracer components do not distinguish between outputs of different neuron types in the brain area with the optimized dosage and ratio of the tracer/helper co-infected starter neurons29 was injected into the primary auditory cortex (AC) of wild-type C57BL/6 mice or by helper alone . These singly infected neurons are unable to spread further tracing , lateral amygdaloid nucleus (LA), medial geniculate nucleus (MG), external globus pallidus (GPe) and locus coeruleus (LC) Fig.\u00a0. Due to ons Fig.\u00a0, nor areons Fig.\u00a0 in our eAmp tracer system was further confirmed in Ai14 reporter mice. In these mice, Cre expressed by H129Amp-CTG efficiently drives long-term and robust expression of the Cre-dependent fluorescence reporter. The results show a prolonged tracing observation time window of at least 14 days , no labeled cell bodies in the upstream region are detected, thus indicating the absence of retrograde labeling by the H129Amp tracer system to produce the tracer H129Amp-DIO-TG. The same helper H129-dTK-T2-pacFlox is used in this starter-specific tracer system, but the tracer H129Amp-DIO-TG expresses TK and GFP (without Cre) in a Cre-dependent manner and ratio (1:1), the H129Amp-DIO-TG tracer and H129-dTK-T2-pacFlox helper (H129Amp-DIO-TG tracer system) were co-injected into the AC of Rph3a-Cre transgenic mice . At Day 5, H129Amp-DIO-TG tracer and helper co-labeled neurons (merged as yellow) are observed at the injection site, representing potential starter neurons experimental durations for monosynaptic tracing.The H129Amp tracer system. H129Amp tracer system (H129Amp-CTG tracer and helper) was administrated into the AC in the left hemisphere (L-AC) of wild-type C57BL/6 mice as described above, and AAV2/9-DIO-ChR2-mCh was simultaneously injected into the right hemisphere AC (R-AC) in the same mice are clearly observed in the R-AC R-AC neurons (the H129Amp tracer labeled postsynaptic neurons) maintain apparently normal cell morphology neurons expression cassette to H129Amp-DIO-TG, generated H129Amp-Flp-DIO-TG, which expresses Flp constitutively but expresses TK and GFP restrictively in a Cre-dependent manner was injected into the inferior colliculus (IC) of CaMK2a-Cre transgenic mice that express Cre in glutamatergic neurons. The Flp-dependent reporter AAV2/9-fDIO-ChR2-mCh was simultaneously injected into the subparafascicular thalamic nucleus (SPF), a brain region directly innervated by IC that projects to the lateral superior olive (LSO) .Altogether, these results demonstrate that the H12912. In the present study, we combined H129Amp tracer system with an earlier developed anterograde monosynaptic tracer H129-dgK-G49, and achieved input-defined postsynaptic neurons\u2019 anterograde monosynaptic tracing. H129-dgK-G4 is a GFP-expressing recombinant H129 virus with deletion of the envelope protein glycoprotein K (gK)9. Cre-dependent AAV helper (AAV2/9-DIO-mCh-gK) complementarily restores gK in trans in the presence of Cre-recombinase, thus allowing H129-dgK-G4 tracer to anterogradely transmit to and label postsynaptic neurons from Cre-expressing starter neurons9.Output mapping of the input-defined neurons\u2019 postsynaptic neurons is critical for dissecting neuronal connectomes. While output mapping of the input-defined neurons has been performed only by visualizing the axonal fibers without identifying 3rd-order neurons thus farAmp tracer system (H129Amp-CTG tracer and helper) was administrated in a single injection into the L-AC of wild-type C57BL/6 mice at Day 0. AAV2/9-DIO-mCh-gK and H129-dgK-G4 were injected into R-AC of the same mice by sequential injections at Day 0 and 21, respectively ; (ii) newly produced H129Amp tracer anterogradely transmits through the first synapse to the postsynaptic neurons (2nd-order neurons), including R-AC neurons; (iii) in the R-AC, H129Amp tracer expresses Cre and drives AAV2/9-DIO-mCh-gK to express mCherry and gK, which in turn assists H129-dgK-G4 transmission; and (iv) the newly produced H129-dgK-G4 anterogradely transmits from the R-AC through the second synapse to label neurons with GFP in further downstream regions (3rd-order neurons), shown by labeling in L-AC, R-MG, and R-LA neurons . mCh+ neurons are L-AC-innervating neurons, which were co-infected with AAV2/9-DIO-mCh-gK (Cre-dependant mCherry and gK expressing) and H129Amp tracer (expressing Cre) transneuronally transmitted from L-AC. GFP+ neurons are observed in the R-AC, indicating H129-dgK-G4 infection and labeling. Some neurons are co-labeled with mCherry from AAV2/9-DIO-mCh-gK and GFP from H129-dgK-G4 (merged as yellow). These represent the potential starter neurons (2nd order) of defined inputs from L-RC together with the Flp-dependent H129-dgK tracing set (H129-dgK-G4 and AAV2/9-fDIO-mCh-gK), or Flp-dependent H129Amp tracer system (H129Amp-Flp-TK-GFP and H129-dTK-T2-FRT-pac-FRT) together with the Cre and Flp-dependent H129-dgK tracing set (H129-dgK-G4 and AAV2/9-Con/Fon-mCh-gK) , shorter experimental duration (5 days), and greater potential for functional mapping, as well as input-defined postsynaptic neurons\u2019 anterograde monosynaptic tracing.In the present study, we developed an anterograde monosynaptic tracer system based on HSV amplicon, composed of the H1299. Conventionally, the helper (AAV) and the tracer are administrated by sequential injections. In the H129Amp tracer system, the recombinant self-replication-deficient helper virus not only functions as a helper for the H129Amp tracer production, but also works as a helper to support the H129Amp tracer\u2019s transneuronal transmission. The identical viral particle structures of the H129Amp tracer and helper enable them to co-infect the same neuron, and their shared replication properties results in their synchronized genome replication and protein synthesis following an intracranial single injection. This simplifies experimental operation and reduces potential experimental failures that would occur following lack of precise spatial overlap of helper and tracer injections as can occur by sequential injections required with conventional monosynaptic tracer systems.Monosynaptic viral tracer systems are usually composed of two components: (1) a replication- or transmission-defective viral tracer for transneuronal transmission/labeling, and (2) a non-replicable helper for supporting the defective tracerAmp tracer system achieves anterograde monosynaptic tracing and postsynaptic neuron labeling in as short as 5 days by simultaneous administration and mutual replication. This significant design improvement will accelerate neuroscience research and will streamline neural connectome mapping.Most current monosynaptic tracers, both retrograde and anterograde, use AAVs as helpers. Due to the slow onset of expression for AAV vectors, the use of AAV necessitates two separate injections, first helper and then tracer, to allow sufficient expression and accumulation to support replication and transmission of the tracer. To complementarily express and accumulate a sufficient amount of the required target viral protein, AAV helper has to be injected 2\u20133 weeks prior to the tracer\u2019s administration. Then, an additional 5\u201310 days are required to allow the tracer to replicate and transmit. Taking all of these steps together, conventional monosynaptic tracing usually takes 3\u20134 weeks. In contrast, the H129Amp pseudo-genome is of similar size compared to the wild-type H129 genome (~152\u2009kb) and contains multiunit expression cassettes in a concatemer form34. When the H129Amp tracer infects neurons and releases the pseudo-genome, multiunit expression cassettes transcribe the target genes simultaneously. This leads to rapid accumulation of the target proteins, including the fluorescent protein and directly contributes to strong labeling signal in postsynaptic neurons.The H1294. YFV-17D has a single-stranded positive-sense RNA genome of ~11\u2009kb35, which means that it also has a limited vector capacity. However, the pseudo-genome of H129Amp tracer has a much larger genetic payload capacity of 150\u2009kb, thus enabling delivery of either single large genes or multi-gene clusters20. This payload capacity feature of the HSV amplicon has attracted great interest from many researchers as a promising gene delivery vector. Using the amplicon vector, a multi-gene cluster of 25.6\u2009kb was delivered into the striatum of a rat model of Parkinson\u2019s disease with high-level expression for long-term biochemical and behavioral improvement of Parkinsonian symptoms36. Similarly, the H129Amp tracer created in the present study can also be applied to deliver large genes or other functional elements in addition to marker fluorescent proteins.Most gene delivery vectors and neural circuit tracers have very limited genetic payloads )15. In the H129Amp tracer system, the H129Amp tracer neither replicates nor expresses any toxic viral protein after transmitting to target postsynaptic neurons, thus displaying minimized toxicity. This unique feature of the H129Amp tracer system enables functional assays on the 2nd-order neurons, such as electrophysiological experiments, opto- or chemo-genetic assays, calcium imaging, etc. The potential for functional mapping is another significant improvement for anterograde monosynaptic tracer applications.Conventional H129-derived tracers display strong toxicity in infected neurons and so they cannot be used to perform functional mapping37. TRIO has been performed by the rabies virus tracer (RVdG and AAV-G together with canine adenovirus or retrograde AAV) and transsynaptic AAV2/1 (together with another AAV reporter)40. However, these tracers transmit through a single synapse instead of two sequential synapses. In the present study, input-defined postsynaptic neurons\u2019 anterograde monosynaptic tracing by using the H129Amp tracer system is achieved in combination with another tracer set (H129-dgK-G4 and AAV2/9-DIO-mCh-gK). We are continuing our work on developing the Cre-specific input-defined postsynaptic neurons\u2019 anterograde monosynaptic tracer systems and expect we can make further refinements and performance improvements. Input-defined postsynaptic neurons\u2019 monosynaptic anterograde tracing from the Cre+-neuron could be achieved by combining the H129Amp-Flp-DIO-TG tracer system and Flp-dependent H129-dgK tracer set (AAV2/9-fDIO-mCh-gK and H129-dgK-G4) and FRT-floxed-pac modified helper (H129-dTK-T2-FRT-pac-FRT), together with Cre/ Flp-double-dependent H129-dgK tracer set (AAV2/9-ConFon-mCh-gK and H129-dgK-G4) are required , is required to better understand the neuronal connectome anatomically and functionally. This has been termed as TRIO (trace the relationship between input and output) or cTRIO Amp tracer system still has a few remaining limitations, including the limited observation time window and toxicity in the starter neurons. HSV amplicon expresses the genetic payload rapidly and massively, but the transcription is silenced quickly42. Infection of HSV amplicon may initiate a cascade of immune responses including expression of Type I IFNs, which have been reported to suppress transgene expression at the transcriptional level by activation of STAT142. In addition, the presence of prokaryotic DNA sequences in the pseudo-genome of H129Amp tracer may also lead to transcriptional silencing of the entire vector sequence due to the inactive form of chromatin immediately after infection29. The transgene expression kinetics of HSV amplicon has been shown to peak quickly and then drop by over 3 logs within a week19. We observe fluorescence labeling of the H129Amp tracer system as early as 1 day post-injection at the injection site, but this signal quickly dims below the detection threshold for confocal imaging at 7 days post-injection. Therefore, the time window for observing tracing results is limited between Day 3 to Day 5 following injection. A better reporter may extend the observation time window to 21 days or even longer, such as Ai14 mice or reporter AAV (AAV2/9-DIO-ChR2-mCh and AAV2/9-fDIO-ChR2-mCh). Other improvements could be made by adding insulator and AT-rich sequences or by removing the bacterial sequence in the amplicon plasmid. These modifications could extend the labeling duration19. Second, toxicity does occur in the starter neurons that are co-infected by H129Amp tracer and helper. The tracer and helper act cooperatively to support each other replication and this leads to high levels of viral protein expression and strong toxicity in the starter neurons. This presents a remaining key obstacle that prevents functional assays on starter neurons. Third, the system theoretically may lead to anterograde multi-synaptic transmission. Hypothetically, a small fraction of helper, whose floxed-pac is not excised by Cre, could be packaged and transmitted to the postsynaptic neurons with the tracer. In the 2nd-order neurons, such helper could assist the tracer to transmit to the further downstream neurons. However, we see no evidence for helper transmission to postsynaptic neurons or evidence for tracer transmission to further downstream neurons even in the highly sensitive Ai14 reporter mice. We encourage users to apply proper controls such as tracer/helper titration to exclude the spurious actifactual labeling that might be caused by unintended anterograde multi-synaptic transmission. Further, it is theoretically possible that tracer could be transmitted from a local Cre\u2212 neuron which is infected by helper and recombined tracer (losing Cre-dependency) and transmitted from a nearby Cre+ neuron when H129Amp-DIO-TG tracer system is applied for starter-specific tracing. To determine whether nonspecific transmission occurs, we tested for this possibility in PV-Cre mice that express Cre-recombinase in parvalbumin (PV) interneurons. The H129Amp-DIO-TG tracer system was injected into the AC of PV-Cre mice with optimized tracing titer, and the results were examined at Day 5 .Despite the above-described outstanding advantages, our newly developed H129Amp-based anterograde monosynaptic tracer system for connectome mapping. The unique advantages of our H129Amp anterograde monosynaptic tracer system include simplified operation, fast tracing, bright labeling, large capacity, low toxicity in target neurons, improved potential for functional mapping of target neurons, and input-defined postsynaptic neurons\u2019 anterograde monosynaptic tracing. This H129Amp tracer system will greatly benefit further neuroscience research.In summary, we have developed innovative strategies to create an H129Rph3a coding gene), PV-Cre mice (JAX-017320), and CaMK2a-Cre mice (JAX-005359) were kindly provided by the Laboratory Animal Resource Center at the Chinese Institute for Brain Research , the School of Basic Medicine, Huazhong University of Science and Technology , and the Eye Center of Renmin Hospital, Wuhan University . All mice received food and water ad libitum in a 12\u2009h light/dark cycles and were maintained under conditionals of stable temperature (23\u201325\u2009\u00b0C) and consistent humidity (45\u201355%). All mice used were the C57BL/6 background strains of either sex at 8\u201312 weeks old. All experiments with mice were reviewed and approved by the Institutional Review Board and Institutional Animal Welfare Committee of Wuhan Institute of Virology, Chinese Academy of Sciences (WIVA10201502). All experiments with viruses were reviewed and approved by the Institutional Biosafety Committee, and performed in Biosafety Level 2 (BSL-2) laboratory or Animal Biosafety Level 2 (ABSL-2) facilities following the Institutionally approved standard operating procedures and biosafety guidelines.Wild-type C57BL/6NCr1 mice (C57BL/6) were purchased from Vital River Laboratory Animal Technology Company . Ai14 reporter mice (JAX-007908), Rph3a-Cre mice and 293\u2009T/17 cells were purchased from ATCC, maintained in the laboratory, and tested to be mycoplasma-free. Cells were cultured with DMEM containing 10% fetal bovine serum and penicillin-streptomycin (100 U/ml and 100\u2009\u03bcg/ml) in a humidified incubator with 5% COOri and pac were amplified from the H129 bacterial artificial chromosome (H129-BAC) by PCR7, cloned into pcDNA3.0, and generated the H129 amplicon backbone plasmid pHSV. Then, different expression cassettes, including the recombinases (Cre or Flp), HSV tyrosine kinase (TK) gene, and GFP, were further inserted into pHSV, resulting in a serial of H129 amplicon plasmids. The structure of the amplicon plasmid pHSV-CTG was shown as representative and the other pac was flanked by loxN via galK-based homologous recombination production and monosynaptic tracing.The helper, H129-dTK-T2-ion Fig.\u00a0. FinallyAmp tracer system, represented by H129Amp-CTG, 2\u2009\u00d7\u2009105 Vero cells in a 100\u2009mm tissue culture dish were transfected with 20\u2009\u03bcg of pHSV-CTG. After 24\u2009h, H129-dTK-T2-pacFlox was inoculated to the transfected cells at a multiplicity of infection (MOI) of 1 to rescue the H129Amp-CTG tracers and served as the seed for further propagation. To propagate high-titer H129Amp tracer, the seed was mixed with H129-dTK-T2-pacFlox and then inoculated to Vero cells to reach final MOIs of 0.1 (for H129Amp-CTG) and 1 (for H129-dTK-T2-pacFlox), respectively. The supernatant was harvested at 48 hpi and concentrated as described previously14. Limited by the recombination efficiency of Cre-recombinase, a very small fraction of the replicated helper genome, whose floxed-pac is not excised, packaged and forms the helper \u201ccontamination\u201d (~5%) in the raw tracer product. Then, the titers of the H129Amp-CTG tracer and the H129-dTK-T2-pacFlox helper in the raw tracer product were determined by plaque-forming assay based on GFP and tdTomato, respectively.To produce the H129ers Fig.\u00a0. The suppacFlox helper was added to the titrated raw tracer product to reach the optimized final working titer as specified. This titer-adjusted tracer/helper mixture, designated as the H129Amp tracer system, was aliquoted and stored at \u221280\u2009\u00b0C until administration.Finally, the independently propagated H129-dTK-T2-44. AAV titers were determined according to the copy number of viral genome (vg) by absolute quantitative PCR44. The monosynaptic tracer H129-dgK-G4 was propagated in the Vero-gKmut cell line and was applied for tracing9. After titration using a plaque-forming assay, H129-dgK-G4 was adjusted to 5.0\u2009\u00d7\u2009108 pfu/ml, aliquoted, and stored at \u221280\u2009\u00b0C until use.The recombinant AAVs applied in the present study were created and packaged in 293\u2009T cells following the standard protocol, and stored at \u221280\u2009\u00b0C as aliquots until applicationAmp tracer system and other viral tracers were intracranially administrated into the target brain regions by stereotaxic injection14. The exact coordinates of the injected sites were determined according to the Mouse Brain Atlas (second edition) by the mediolateral (ML), anteroposterior (AP) and dorsoventral (DV) distances to Bregma45. The brains were collected after perfusion with 4% paraformaldehyde at the indicated times. Samples were processed and coronally cryo-sectioned to 40\u2009\u03bcm thick slices using a cryostat Microme . The brain slices were counterstained with DAPI , and then imaged using a Nikon\u2019s A1R MP+ confocal microscope equipped with a fast high-resolution galvanometer scanner14.The H12946. The slices were recovered by incubation in a submersion chamber filled with the prewarmed (35\u2009\u00b0C) artificial cerebrospinal fluid for 30\u2009min, and then gradually cooled to the room temperature. The electrophysiological assay was performed by patch-clamp recordings from AC neurons in slices visualized under a fluorescent infrared-phase-contrast (IR-DIC) Axioskop 2FS upright microscope. The internal solution in the recording electrodes consisted of 140\u2009mM potassium gluconate, 10\u2009mM HEPES, 0.2\u2009mM EGTA, 2\u2009mM NaCl, 2\u2009mM MgATP, and 0.3\u2009mM NaGTP47. Electrophysiological recordings were obtained from Au neurons with Multiclamp 700B amplifiers and pCLAMP 10.3 software. To record light-evoked excitatory postsynaptic currents, 5\u2009ms pulses of 5\u2009mW blue light were delivered through the objective to ChR2-expressing axons originating from the indicated brain region(s).The subjected mouse brains were carefully collected under anesthetization with overdosed pentobarbital , and immediately sectioned to 300\u2009\u03bcm thick coronal slices using a vibrating microtome (Leica VT1200s)Data were collected from at least three independent experiments or animals, each experiment was performed in triplicate, and the results were presented as means\u2009\u00b1\u2009SEM (Standard Error of the Mean) using GraphPad Prism 9.Further information on research design is available in the\u00a0Supplementary InformationPeer Review FileReporting Summary"} +{"text": "Dual-luciferase reporter assay was performed to confirm the binding of tsRNA with its targets. Via using bioinformatics approaches, the hundreds of tsRNAs with available expression abundance were identified in gliomas dataset, most of them derived from D-loop or T-loop fragments of tRNAs. Among tsRNAs derived from tRNA-Cys-GCA, tRFdb-3003a and tRFdb-3003b (tRFdb-3003a/b) were remarkably down-regulated in gliomas. The survival outcome of gliomas patients with low tRFdb-3003a/b expressions was notably worse than that of high-expression patients. In glioma cells, tRFdb-3003a could suppress cells proliferation and colony formation ability. In vivo, tRFdb-3003a suppressed the tumor growth of xenograft gliomas. Enrichment analyses displayed the tRFdb-3003a-related mRNAs were enriched in the specific GO terms, spliceosome and autophagy pathways, and three GSEA molecular signatures. Mechanically, 3'-UTR regions of VAV2 mRNA were predicted to contain the binding positions of tRFdb-3003a/b, tRFdb-3003a and tRFdb-3003b was effective to reduce the relative luciferase activity of cells with VAV2 wild-type reporter. Overexpression of tRFdb-3003a/b could down-regulated the expression levels of VAV2 protein and mRNA in glioma cells. The tRNA-Cys-GCA derived tRFdb-3003a and tRFdb-3003b might act as key player in tumor progressions of gliomas; tRFdb-3003a/b might directly bind to VAV2 and regulate VAV2 expressions in gliomas.The tsRNAs are new types of small noncoding RNAs derived from tRNAs. Gliomas are well-known malignant brain tumors. The study focused on tsRNA characterizations within gliomas. Datasets processing, bioinformatics analyses, and visualizations were performed with the packages of Python and R. Cell proliferations were demonstrated via CCK8 assays and colony formation assays, and The tRNA (transfer RNA) derived small RNAs (tsRNAs) are new types of small noncoding RNAs (sncRNA) that derived from tRNAs, and also known as tRNA-derived fragments (tRFs) . The ideDeep analysis of some sequence data have excavated numerous miRNA isoforms , likewisIn the present study, we aimed at identifying a tsRNA profile within the sncRNA-sequencing data of glioma tissues through the reliable tsRNA mining pipelines, and then reveal the expression patterns and biological roles of tRNA-Cys-GCA derived tsRNAs (tRFdb-3003a and tRFdb-3003b) in gliomas. In addition, a relationship between tRFdb-3003a/b and its target genes is also investigated in order to display the underlying mechanism.http://genome.bioch.virginia.edu/trfdb/) and tRFexplorer (https://trfexplorer.cloud/) databases [http://gtrnadb.ucsc.edu/), the custom annotations of the GRCh37 reference genome containing most known tsRNAs were assembled [The small noncoding RNA sequence data of gliomas on the Cancer Genome Atlas (TCGA), the RNA sequence datasets of LGG (low grade gliomas), and the corresponding patients information were downloaded from GDC data portal . The priatabases , 19. Thessembled .The datasets preprocessing and flow-chart for tsRNA characterizations were displayed in Figure https://cran.r-project.org/web/packages/pheatmap/). Survival curve analyses with Kaplan-Meier methods plus log-rank tests were conducted through the survminer and survival packages within R software [https://ggplot2-book.org). The enrichment analysis was conducted to provide biology views regarding tRFdb-3003a/b-related mRNAs, the MSigDB database (https://www.gsea-msigdb.org/gsea/msigdb/) were applied as potential genes sets for the gene-sets enrichment analysis (GSEA) [https://yulab-smu.github.io/clusterProfiler-book/). The potential binding interactions between tsRNAs and target-genes were predicted through tRFtarget website with RNAhybrid plus IntaRNA tools [The RNA expression data of TCGA were obtained and processed via TCGAbiolinks packages of Bioconductor . Hierarcsoftware . Pearsons (GSEA) . Gene onNA tools . An inteForty glioma tissues were obtained from patients diagnosed with gliomas undergoing surgical resections at the Department of Neurosurgery of Xiangya Hospital of Central South University from July 2015 to December 2018. After excisions, tissues were immediately frozen in liquid nitrogen for subsequent use. Twelve non-tumor brain tissues were obtained from the adult patients with craniocerebral injuries, which required partial resections of brain tissues as decompression treatment to reduce intracranial pressures. This study was approved by the Ethics Committees of Central South University and the patients provided written informed consents .\u03b2-actin) was used as the internal control for mRNA template normalizations, and RNU6 (U6) for tsRNAs template normalizations. Relative quantification of gene expression was calculated by the comparative cycle-threshold methods [Total RNA was extracted from tissues or cultured cells using the TRIzol reagent . One microgram total RNAs of each sample was reversely transcribed into cDNA under standard condition via using PrimeScript RT reagent Kit with gDNA Eraser . Quantitative real-time polymerase chain reaction (qRT-PCR) was performed with the SYBR\u00ae Premix DimerEraser\u2122 on the LightCycler\u00ae 480 system . The Bulge-Loop miRNA qRT-PCR Stater Kit with specific stem-loop RT primers was used to quantify the expression of tsRNAs. ACTB , or inhibitor for tRFdb-3003a (tRF3003a-inhibitor), or negative controls using the Lipofectamine RNAimax reagent . The mimic and inhibitor for tRFdb-3003a/b and negative control were purchased from RiboBio . The micrON\u2122 tRF-3003a agomir and negative control were also purchased from RiboBio . Cell proliferation assays were performed with Cell Counting Kit-8 , the absorbance (A) in each well was measured at 450\u2009nm with a Microplate Reader . Cell proliferation was also assessed by colony formation assay. Visible colonies were manually counted and photographed.Human glioma cell lines T98G and U251 were purchased from the Cell Bank of Chinese Academy of Sciences . The cells were cultured in Dulbecco's Modified Eagle's Medium supplemented with 10% fetal bovine serum at 37\u00b0C in a humidified incubator with 5% COThe mice experiments were performed according to the procedure described previously . BrieflyThe wild-type or mutant sequences of VAV2 3'-UTR (3\u2032-untranslated regions) containing putative tRFdb-3003a and tRFdb-3003b binding sites were subcloned into pmir-RB-Report vector . Glioma cells were cotransfected with pmir-RB-Report vector plus or without tRFdb-3003a/b mimic. The luciferase activity was measured using the Dual-luciferase Reporter Assay System .\u03b2-actin antibody diluted at 1\u2009:\u20091000, Sigma-Aldrich, USA). The \u03b2-actin served as an endogenous protein for normalization. The protein bands were visualized using ChemiDocTM imaging system and Image Lab software .Total protein was extracted from cultured cells using RIPA buffer and the protein concentration was determined using a BCA Protein Assay Kit (Bio-Rad). The specific primary antibody caught our attention. As shown in P < 0.05), although, no significant difference for other tsRNAs was observed between astrocytomas and non-tumors (both P > 0.05). As we known, astrocytomas are the main subtype of gliomas. The clinical tissues detections also displayed the expressions of tRFdb-3003a and tRFdb-3003b was remarkably down-regulated in gliomas compared to non-tumors were down-regulated in astrocytoma specimens compared to non-tumors (n-tumors . These iP = 0.0037) was notably worse than that of the high-expression patients; overall survival of patients with low tRFdb-3003b expression was also worse than that of high-expression patients (P = 0.0037). These data implied that down-regulated tRFdb-3003a and tRFdb-3003b were associated with poor survival outcome of gliomas.Subsequently, we have studied the relationships between tsRNA expressions and clinical survival of glioma patients by Kaplan\u2013Meier curve analyses. Base on median values of tsRNA expressions, primary glioma patients were divided into low-expression group and high-expression group. As shown in \u03c72 test, and was visualized via Hierarchical cluster heatmaps. The expressions of tRFdb-3003a/b was significantly correlated with IDH-mutants status in glioma patients (both P < 0.05), tRFdb-3003a and tRFdb-3003b may tend to raise in the patients with IDH-mutants. Moreover, high tRFdb-3003a/b expression was correlated with 1p19q-codeletion in glioma patients (both P < 0.05). As for transcriptome classifications, tRFdb-3003a and tRFdb-3003b may be more expressed in the proneural subtype of gliomas; although, no significant correlation was found between tRFdb-3003a/b expressions and other pathological characteristics such as sex, grades, MGMT promoter methylation, BRAF, and TERT status . As might be expected, heterozygous mutations in the catalytic arginine residues of isocitrate dehydrogenase gene (IDH1/2) are common in gliomas and acute myeloid leukemia, and contribute to the pathogenesis of several tumors [According to the CNS WHO classification, the primary molecular pathological characteristics of gliomas patient were showed in Table l tumors . These iP < 0.05; In vivo xenograft experiments, the volumes and weights of xenograft tumors in tRF3003a-angmir group were markedly smaller than those in control group of mRNA genes in a manner similar to microRNAs. With bioinformatics methods, the potential target-mRNAs of tRFdb-3003a/b were predicted and picked out. The interactional relationship of tRFdb-3003a/b and the target-mRNAs were showed in V family . The pos-3003a/b . Lucifer/b-mimic . These dThe tRNA-derived small RNAs (tsRNAs) are new types of small noncoding-RNA derived from tRNAs, also known as tRNA-derived fragments . In curretc. Pan's study has identified an inflammatory cytokine-regulated tsRNA tRF-21 was significantly lower in pancreatic ductal adenocarcinoma (PDAC), and patients with low tRF-21 expressions had a poor prognosis [Val, which is down-regulated in breast cancer tissues [Val could suppressed cell viability and colony formations, reduced the migration and invasion of cancer cells, these suggest that 5\u2032-tiRNAVal may serve as a potential antioncogene effect in the development of breast cancers [in vitro; and suppressed tumor growth of xenograft gliomas in vivo. Additionally, the enrichment analyses displayed tRFdb-3003a was associated with some gene ontology terms, including RNA splicing and metabolic process, enzymatic activity, and nuclear speck; tRFdb-3003a-correlated mRNAs might refer to autophagy and spliceosome pathway, along with three GSEA molecular signatures . These data implied that down-regulated tRFdb-3003a could act as anti-oncogene role in glioma progression via specific signal pathways.Recent researches have linked tsRNAs to several human carcinoma diseases, for instance, colon carcinoma , breast rognosis . In PDACrognosis . In brea tissues . Further cancers . In the LATS2 gene, which encoded the component of hippo signaling pathway, and suppressed cells proliferations and promoted cells apoptosis through regulating YAP protein activity in colorectal cancers [VAV2, which was overexpressed in gliomas. Dual-luciferase reporter assays demonstrated that tRFdb-3003a/b could directly bind to the 3\u2032-UTR regions of VAV2 and reduce the relative luciferase activities. Moreover, overexpression of tRFdb-3003a/b could decrease the expressions of VAV2 protein and mRNA within glioma cells. Numerous studies discriminated that VAV2 is a protooncogene of the VAV family, and has been found critical to Rho GTPase activity in tumor development and maintenance [Recent studies have suggested that tsRNAs might bind to Argonaute-1/2 protein and act a key player in genes silencing via targeting the 3\u2032-UTR regions of specific mRNA in the manner similar to microRNAs [ cancers . Bioinfontenance . These fIn conclusion, our studies identified many tsRNAs, especially nine tsRNAs derived from tRNA-Cys-GCA, in the sncRNA-sequence data of glioma tissues. Among them, several tsRNAs were remarkably down-regulated in gliomas, the expressions of tRFdb-3003a and tRFdb-3003b were associated with survival outcome and IDH-mutants status of glioma patients. Overexpression of tRFdb-3003a suppressed cells proliferation and tumor growth of gliomas. Down-regulated tRFdb-3003a might function as anti-oncogene role within glioma progression through specific signaling pathways. Moreover, tRFdb-3003a/b could directly bind to the 3\u2032-UTR regions of VAV2 and regulate VAV2 expression in gliomas. These indicated that tRNA-Cys-GCA derived tsRNAs (tRFdb-3003a and tRFdb-3003b) might regulate tumor- progression of gliomas."} +{"text": "An inflammatory response consists of two consecutive steps: priming and triggering, to prepare and activate inflammatory responses, respectively. The cardinal feature of the triggering step is the activation of intracellular protein complexes called inflammasomes, which provide a platform for the activation of inflammatory signaling pathways. Despite many studies demonstrating the regulatory roles of canonical inflammasomes in inflammatory liver diseases, the roles of newly discovered non-canonical inflammasomes in inflammatory liver diseases are still largely unknown. Recent studies have reported the regulatory roles of the caspase-11 non-canonical inflammasome in inflammatory liver diseases, providing strong evidence that the caspase-11 non-canonical inflammasome may play key roles in the pathogenesis of inflammatory liver diseases. This review comprehensively discusses the emerging roles of the caspase-11 non-canonical inflammasome in the pathogenesis of inflammatory liver diseases, focusing on non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), and inflammatory liver injuries and its underlying mechanisms. This review highlights the current knowledge on the regulatory roles of the caspase-11 non-canonical inflammasome in inflammatory liver diseases, providing new insights into the development of potential therapeutics to prevent and treat inflammatory liver diseases by targeting the caspase-11 non-canonical inflammasome. Although inflammation is a body-protective innate immune response, chronic inflammation is a key determinant of numerous inflammatory diseases and cancers ,2. An infatty liver disease (NAFLD) is a chronic disease caused by excessive fat accumulation and inflammation in the liver. NAFLD can develop into non-alcoholic steatohepatitis (NASH), an aggressive form of fatty liver disease, which is characterized by liver inflammation and may progress to cirrhosis, liver injury, and liver failure [Inflammation also induces liver diseases. Non-alcoholic failure ,13. Many failure ,15,16. Icaspase-11 gene locus, resulting in the expression of truncated and non-functional proteins [caspase-11 gene is not found in humans; instead, caspase-4 and -5 genes have been identified as human homologs of the mouse caspase-11 gene, and studies have demonstrated that the caspase-4 and -5 genes generate caspase-4/5 non-canonical inflammasomes in humans [The caspase-11 non-canonical inflammasome was first discovered in the 129S6 mouse strain, which has a polymorphism in the Canonical inflammasomes are activated in response to their specific ligands ,5. HowevAs described earlier, the direct interaction between LPS and caspase-11 induces the oligomerization of the caspase-11 non-canonical inflammasome. The oligomerized caspase-11 non-canonical inflammasome is subsequently activated by self cleavage at the 285 aspartic acid residue (D285), and this enzymatic activity is mediated by the 254 cysteine residue (C254) of caspase-11 . The two+) efflux induced by GSDMD pore-mediated membrane damage and gate proteins, such as P2X7 channels, pannexin 1 channels, and bacterial pore-forming toxins, play a key role in the activation of the NLRP3 canonical inflammasome [+ efflux through gate proteins and GSDMD pore-mediated membrane damage, leading to the activation of the NLRP3 canonical inflammasome [Although the caspase-11 non-canonical inflammasome induces the secretion of pro-inflammatory cytokines by activating caspase-1, it indirectly activates caspase-1 via functional cooperation with the NLRP3 canonical inflammasome. NLRP3 canonical inflammasome, the most-studied inflammasome, is activated in response to various pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs). Among the PAMPs and DAMPs, the potassium ion - and high-fat-diet (HFD)-fed \u2212/\u2212 mice . Additioese mice . These rese mice . These rNASH is an aggressive form of fatty liver disease characterized by liver inflammation and damage, which may progress to advanced scarring, known as cirrhosis; fibrosis; liver injury and failure; and hepatocellular carcinoma, eventually causing death ,44. NASH\u2212/\u2212Ldlr), which shows hepatic inflammation in Kupffer cells [\u2212/\u2212Ldlr mice transplanted with \u2212/\u2212caspase-11 bone marrow showed less-severe hepatic inflammation and NASH symptoms [\u2212/\u2212Ldlr/\u2212/\u2212caspase-11 mice exerted less cholesterol accumulation and enhanced cholesterol efflux [\u2212/\u2212Ldlr/\u2212/\u2212caspase-11 mice showed decreased autophagy induced upon oxidized low-density lipoprotein (oxLDL) stimulation [\u2212/\u2212caspase-11 mice showed significantly reduced hepatic inflammation, pyroptosis, fibrosis, and injury [\u2212/\u2212caspase-11 mice, and overexpression of caspase-11 exacerbated MCD-induced hepatic steatosis in mice [Many studies have reported the roles of canonical inflammasomes in NASH ,48,49,50er cells . Ldlr\u2212/\u2212symptoms . Cellulal efflux . Moreovemulation . These rmulation ,54. Moremulation . Lebeaupmulation . ER stremulation . These rmulation ,19. Zhu d injury . Additio in mice . These rHepatic inflammation is considered a critical risk factor for liver injury and failure, which triggers various liver diseases associated with poor survival in patients ,59. Ther\u2212/\u2212Hspa12a mice were more susceptible to LPS-induced acute liver inflammation and injury [\u2212/\u2212Hspa12a mice, resulting in the suppression of GSDMD pore formation and GSDMD pore-mediated hepatocyte pyroptosis [Heat shock protein A12A (HSPA12A) is a novel member of the HSP70 family that plays a role in the development of HFD-induced NAFLD and NASH . Liu et d injury . Activatroptosis . These rHepatic ischemia-reperfusion injury (IRI), a major complication of hepatic transplantation, resection, and hemorrhagic shock, often results in systemic hepatic inflammation and liver injury by activating macrophage-induced innate immune responses ,66,67. LSamotolisib is a novel dual inhibitor targeting phosphoinositide 3-kinase (PI3K) and the mammalian target of rapamycin (mTOR), and has undergone several phase-II clinical trials as a potential treatment for different cancers. Zhao et al. screened out samotolisib after a systemic analysis of an FDA-approved compound library and reported the protective effect of samotolisib against LPS-induced hepatic inflammation and acute liver injury in mice. Samotolisib attenuated LPS-induced hepatic inflammation and acute liver injury, and improved survival in mice . A mechaThe regulatory roles of the caspase-11 non-canonical inflammasome in the pathogenesis of NAFLD, NASH, and inflammatory hepatic injury are described in Inflammasomes are inflammatory signalosomes that provide innate immunity against pathogens and cellular dangers, triggering a wide range of human diseases. Several studies have demonstrated that canonical inflammasomes, particularly NLRP3 inflammasomes, are key players in numerous inflammatory diseases ,11, and This review comprehensively summarizes and discusses the current knowledge of the regulatory role of the caspase-11 non-canonical inflammasome in the pathogenesis of inflammatory liver diseases and the underlying molecular mechanism , which mIn conclusion, the caspase-11 non-canonical inflammasome is a key player in the pathogenesis of inflammatory liver diseases. The caspase-11 non-canonical inflammasome induces GSDMD-mediated pyroptosis and the secretion of pro-inflammatory cytokines, and caspase-11 non-canonical inflammasome-induced pyroptosis and pro-inflammatory cytokine secretion are independent of the canonical inflammasomes, which strongly suggests that the caspase-11 non-canonical inflammasome could be an independent therapeutic target of inflammatory liver diseases. Understanding the mechanisms that modulate the activity of the caspase-11 non-canonical inflammasome may contribute to the development of a wide range of therapeutic agents that can selectively target the caspase-11 non-canonical inflammasome in not only inflammatory liver diseases but also various inflammatory diseases caused by the activation of the caspase-11 non-canonical inflammasome."} +{"text": "Polyomavirus-associated nephropathy (BKVAN) is one of the serious causes of allograft dysfunction and allograft loss in kidney transplant (KT) patients. Most of the patients were diagnosed with BKVAN within the first year after transplantation but some more cases of BKVAN occur later. A study focused on the differentiation between early and late-onset BKVAN is needed.We conducted a retrospective cohort study among KT recipients at Ramathibodi Hospital between January 2010 \u2013 December 2020. The incidence of early-onset (diagnosed within 1 year) and late-onset (diagnosed after 1 year) BKVAN, as well as composite kidney allograft outcomes, were compared.From 1032 KT recipients, 645 (62.5%) were screened for BK viral infection. BKVAN was diagnosed in 83 (12.8%) patients. Of those, 46 (55.4%) and 37 (44.6%) were diagnosed with early-onset and late-onset BKVAN, respectively. Composite kidney allograft outcomes of GFR decline\u2265 40% from baseline, graft loss or death were greater in early-onset BKVAN compared to late-onset BKVAN . In multivariate analysis, female gender and living-related kidney transplantation (LRKT) were independently associated with late-onset BKVAN compared to early-onset BKVAN. The more advanced the BKVAN diagnosis, the worse the allograft outcome occurs, even with optimizing immunosuppressive drugs and adjunctive therapy.BKVAN could occur later after one-year post-transplantation, although relatively better composite kidney outcomes were observed compared to early-onset BKVAN. Female individuals who underwent living-related KT should be considered for BK virus preemptive monitoring beyond one year to detect this late-onset BK virus infection.All Authors: No reported disclosures."} +{"text": "Recurrent chronic mucosal inflammation, a characteristic of inflammatory bowel diseases (IBD), perturbs the intestinal epithelial homeostasis resulting in formation of mucosal wounds and, in most severe cases, leads to colitis-associated colon cancer (CAC). The altered structure of epithelial cell-cell adhesions is a hallmark of intestinal inflammation contributing to epithelial injury, repair, and tumorigenesis. P-cadherin is an important adhesion protein, poorly expressed in normal intestinal epithelial cells (IEC) but upregulated in inflamed and injured mucosa. The goal of this study was to investigate the roles of P-cadherin in regulating intestinal inflammation and CAC. P-cadherin expression was markedly induced in the colonic epithelium of human IBD patients and CAC tissues. The roles of P-cadherin were investigated in P-cadherin null mice using dextran sulfate sodium (DSS)-induced colitis and an azoxymethane (AOM)/DSS induced CAC. Although P-cadherin knockout did not affect the severity of acute DSS colitis, P-cadherin null mice exhibited faster recovery after colitis. No significant differences in the number of colonic tumors were observed in P-cadherin null and control mice. Consistently, the CRISPR/Cas9-mediated knockout of P-cadherin in human IEC accelerated epithelial wound healing without affecting cell proliferation. The accelerated migration of P-cadherin depleted IEC was driven by activation of Src kinases, Rac1 GTPase and myosin II motors and was accompanied by transcriptional reprogramming of the cells. Our findings highlight P-cadherin as a negative regulator of IEC motility in vitro and mucosal repair in vivo. In contrast, this protein is dispensable for IEC proliferation and CAC development. Intestinal epithelium is a constantly rejuvenating tissue with the colonic mucosal lining being shed and regenerated every 3\u20135 days. The intestinal rejuvenation involves proliferation of stem cells residing at the base of the crypt with subsequent cell movement to the luminal surface, lined by well-polarized intestinal epithelial cells (IEC) ,2. Such A crucial consequence of the increased phenotypic plasticity of the inflamed intestinal epithelium is profound alterations in intercellular adhesions. The IEC create a tight epithelial barrier by assembling the apical junctional complex, comprising adherens junctions (AJs) and tight junctions (TJs) ,10,11,12A non-canonical cadherin switch has been described in the colonic mucosa of IBD patients. It involves downregulation of E-cadherin level accompanied by a marked expressional upregulation of a homologous adhesion protein, P-cadherin ,21,22. UIn this study, we investigated the roles of P-cadherin in IEC motility, mucosal repair, and colonic tumorigenesis. We found that P-cadherin suppresses collective epithelial cell migration in vitro and mucosal restitution in vivo without having significant effects on IEC proliferation and tumor growth. P-cadherin appears to control multiple molecular pathways responsible for cell migration in IEC monolayers. Among these pathways, activity of the Src family kinases, Rac1 small GTPase and non-muscle myosin II (NM-II) motors are critical for P-cadherin dependent regulation of IEC motility.The following primary polyclonal antibodies (pAb) and monoclonal antibodies (mAb) were used to detect adhesion, cytoskeletal and signaling proteins: anti P-cadherin mAb , and P-cadherin pAb from R & D Systems ; anti P-cadherin mAb , paxillin (Cat# 610568), \u03b21 integrin (Cat# 610467), E-cadherin (Cat# 610181), p120 catenin (Cat# 610133), and \u03b2-catenin (Cat# 610153) mAbs were from BD Biosciences . Anti-E-cadherin (Cat# ab40772) and anti-total actin (Cat# ab1801) rabbit pAbs were from Abcam ; anti-Src (Cat# 2123) and anti-phospho-cofilin S3 (Cat# 3313) rabbit mAbs, as well as anti-phospho-Src (Cat# 2101), total FAK (Cat# 3285), phospho-FAK Y397 (Cat# 8556), phospho-paxillin (Y118) (Cat# 6936), total MLC (Cat# 3672), \u03b24 integrin (Cat# 4707), phospho-MLC S19 (Cat# 3671), di-phospho-MLC T18/S19 (Cat# 3674), and GAPDH (Cat# 2818) pAbs were from Cell Signaling Technology ; anti-cofilin (Cat# SAB2702206); anti-cortactin (Cat# 05-180) mouse mAbs and anti-phospho-cortactin Tyr421 (Cat# AB3852) pAb were from Millipore-Sigma ; anti-\u03b12-integrin (Cat# A7629), \u03b13-integrin (Cat# A17502) and \u03b1V-integrin (Cat# A19071) pAbs were from ABclonal Technology ; anti-NM-IIA (Cat# 909801) and NM-IIB (Cat# 909901) pAbs were from Biolegend ; anti-occludin (Cat# 13409) and \u03b1-catenin (Cat# 12831) pAbs were from ProteinTech ; anti-claudin-7 pAb (Cat# 349100) was from Thermo-Fisher . Horseradish peroxidase (HRP)-conjugated rabbit anti-goat, goat-anti-rabbit and goat anti-mouse secondary antibodies were acquired from Bio-Rad Laboratories . OmniMap anti-Rabbit HRP (Cat# 05269679001) and OmniMap anti-Mouse HRP (Cat# 05269652001) were from Roche . Opal 620 (Cat# FP1495001) and Opal 690 (Cat# FP1497001) fluorophores were from Akoya Biosciences . Both PP2 and EHT 1864 were purchased from Tocris Bioscience . Azoxymethane (AOM) and blebbistatin were obtained from Millipore-Sigma. All other reagents of the highest grade were obtained from either Thermo-Fisher, or Millipore-Sigma. P-cadherin knockout (KO) mice on C57Bl/6J background (JAX stock #003180) were obtw/v) solution of dextran sulfate, sodium salt , in drinking water, ad libitum. Vehicle-treated animals received tap water. Roughly equal numbers of male and female mice were used in each experimental group. At the beginning of colitis experiments, mice weighed 18\u201325 g, with no significant difference between the body masses of mice of different genotypes. Animals were weighed and monitored for symptoms of gastrointestinal disorder daily. The disease activity index was calculated by averaging numerical scores of body weight loss, stool consistency, and intestinal bleeding, as previously described \u2009 < \u20091) before performing differential expression (DE) analysis using R packages edgeR [p-value < 0.05 were considered statistically significant. Volcano plots were created using R package software EnhancedVolcano (https://github.com/kevinblighe/EnhancedVolcano (accessed on 24 March 2022)). Functional analysis of significant DEGs were performed using R package clusterProfiler (v3.14.3) software [https://cytoscape.org/ (accessed on 24 March 2022)). The RNA sequencing data have been deposited in Gene Expression Omnibus. GEO accession number GSE199859. Total RNA was extracted from either control, or two P-cadherin knockout HCA-7 cell lines, using the RNeasy Mini Kit . The RNA quality was assessed using the Agilent bioanalyzer and samples with an RNA quality score of >9.0 were used in RNA-sequencing. The RNA concentrations were determined using Qubit package . Overalles edgeR and Limmes edgeR softwaret-test to compare results obtained with two experimental groups . When data from three experimental groups were compared a one-way ANOVA was used. If the ANOVA test showed significant differences, a Dunnett post-hoc test was used to compare the difference between the control and each P-cadherin-depleted group. p values < 0.05 were considered statistically significant. The statistical analysis was performed using Prism 9.10 software .All data is expressed as means \u00b1 standard error (SE) from three biological replicates. Statistical analysis was performed using a two-tailed unpaired Student As previous studies have documented increased P-cadherin mRNA expression in the intestinal mucosa of IBD patients and in colon cancer tissues ,26,27,28Given the observed upregulation of P-cadherin expression during intestinal inflammation and tumorigenesis, we sought to investigate whether this adhesion protein is involved in the development of experimental colitis and CAC in vivo using P-cadherin knockout mice. Mice with total knockout of P-cadherin are healthy and do not display major physiological abnormalities, except for precocious mammary gland development . We confA classical AOM/DSS model was carried out to investigate the roles of P-cadherin in CAC development ,50. AfteThe observed acceleration of post-colitis recovery of P-cadherin-null mice prompted us to investigate the roles of P-cadherin in modulating IEC migration. We used HCA-7 and SK-CO15 cells, which are well-differentiated human colonic carcinoma cell lines, known to be good models to study intestinal barrier integrity and repair ,62,63,64As healing of intestinal mucosal wounds is known to be driven by two critical mechanisms, epithelial cell migration and proliferation , we sougEpithelial cell migration is a multistep process involving coordinated assembly of cell adhesion to extracellular matrix (ECM) and formation of membrane protrusions at the migrating cell edge ,67. We nNext, we sought to elucidate the signaling mechanisms involved in P-cadherin-dependent regulation of IEC migration. Immunoblotting analysis of total cell lysates obtained from wounded control and P-cadherin knockout HCA-7 cell monolayers was carried out to determine the expression and activation status of most essential regulators of cell-ECM adhesion and cell motility/cytoskeletal remodeling. Surprisingly, diminished adhesiveness of P-cadherin-depleted cells was accompanied by neither downregulated expression of major integrins, nor attenuated phosphorylation of integrin-associated scaffolding and signaling molecules A. OpposiAs the Src family kinases serve as upstream regulators of a number of signaling cascades essential for cell adhesion, migration and cytoskeletal remodeling ,70, we nThe Rac1 small GTPase is a key regulator of cell spreading and migration that could be controlled by Src ,75. NextOur data indicates that in addition to Rac1 activation, loss of P-cadherin also increases activity of RhoA GTPase. This was manifested by the elevated levels of phosphorylated cofilin and di-phosphorylated MLC B, two caOur findings that the loss of P-cadherin triggers multiple functional and signaling changes in migrating IEC monolayers suggest possible alterations of global molecular networks in P-cadherin knockout cells. To investigate if such global molecular alterations are caused by transcriptional reprogramming, we performed whole genome RNA sequencing analysis of control and P-cadherin-depleted HCA-7 cell lines. This analysis showed profound changes in gene expression of P-cadherin depleted cells with a number of significantly upregulated or downregulated genes . A gene P-cadherin is a member of the classical cadherin family with limited tissue-specific expression under homeostatic condition and poorly understood functions in development and diseases. Our study shows marked induction of P-cadherin expression in the colonic epithelium of UC and CAC patients . We demoThe existing literature presents a conflicting view for the roles of P-cadherin in regulating motility of different epithelial and cancer cells, by reporting either promigratory ,43,87,88The results of the present study , togetheOur study revealed several signaling events regulated by P-cadherin in migrating IEC. One important event involves activity of Src kinases. Indeed, loss of P-cadherin resulted in Src activation in HCA-7 and SK-CAn important signaling event controlled by P-cadherin in migrating IEC cells involves regulation of Rho family of small GTPases, such as Rac1 and RhoA. Our data demonstrates that P-cadherin inhibits cell spreading , which iThe observed activation of Src and Rho GTPases appears to be a part of the global rearrangement of molecular/signaling networks caused by P-cadherin depletion in IEC. Indeed, our RNA sequencing analysis revealed an extensive transcriptional reprogramming of P-cadherin-deficient HCA-7 cells . This trneu oncogene [In contrast to the strong evidence implicating P-cadherin in the regulation of IEC matrix adhesion, migration, and mucosal repair, we found little evidence that this cadherin is essential for colon cancer development. Indeed, depletion of P-cadherin affected neither colonic epithelial cell proliferation in vitro nor colooncogene . InteresThe results obtained in our study set the stage for future works aimed at understanding the physiological roles of P-cadherin upregulation in injured, inflamed, or neoplastic colonic mucosa. Such P-cadherin upregulation has pro-adhesive and anti-migratory effects and therefore could impede the excessive and uncoordinated IEC movement. The pathophysiological consequences for induction of such an anti-reparative molecule as P-cadherin remain puzzling. It appears to be detrimental for restoration of acutely injured intestinal mucosa but could be beneficial during multiple rounds of repair in chronically inflamed intestine by preventing excessive IEC loss and orchestrating cell behavior at topographically different regions of the remodeling epithelial layer. Furthermore, attenuated migration of P-cadherin expressing IEC cells is expected to decrease the crypt to villous epithelial cell transit in the inflamed colonic mucosa, which may affect stem cell behavior and differentiation. Finally, induction of P-cadherin expression during colon cancer development could serve as an adaptive antimetastatic response antagonizing either classical EMT or tumor cell invasion/dissemination. Another possible role of P-cadherin upregulation in IEC is alluded to by our RNA sequencing analysis that shows alterations in pathways related to chemokine and cytokine receptor signaling and responses in P-cadherin-depleted IEC cells . This inIn conclusion, our study unravels novel roles for P-cadherin, an important junctional protein upregulated in the colonic mucosa of UC and CAC patients, in regulating migration and signaling in IEC. We discovered that P-cadherin acts as a negative regulator of collective cell migration in vitro and mucosal restitution in vivo. By contrast, we did not find evidence that P-cadherin controls proliferation of human IEC or tumor growth in the mouse model of CAC. The observed anti-migratory effects of P-cadherin involve regulation of cell-ECM adhesion and cell spreading by altering signaling of the Src family kinases, and the Rac1 GTPase and NM-II motors. Additionally, P-cadherin was found to be a modulator of the global transcriptional program in IEC and specifically, molecular pathways related to cell migration, regulation of kinase activity and cytokine/chemokine responses. Therefore, P-cadherin could serve as a potential therapeutic target to accelerate intestinal restitution and increase efficiency of immunotherapeutic interventions in colorectal cancer."} +{"text": "Background: Effective inpatient stewardship initiatives can improve antibiotic prescribing, but impact on outcomes like Clostridioides difficile infections (CDIs) is less apparent. However, the effect of inpatient stewardship efforts may extend to the postdischarge setting. We evaluated whether an intervention targeting inpatient fluoroquinolone (FQ) use in a large healthcare system reduced incidence of postdischarge CDI. Methods: In August 2019, 4 acute-care hospitals in a large healthcare system replaced standalone FQ orders with order sets containing decision support. Order sets redirected prescribers to syndrome order sets that prioritize alternative antibiotics. Monthly patient days (PDs) and antibiotic days of therapy (DOT) administered for FQs and NHSN-defined broad-spectrum hospital-onset (BS-HO) antibiotics were calculated using patient encounter data for the 23 months before and 13 months after the intervention (COVID-19 admissions in the previous 7 months). We evaluated hospital-onset CDI (HO-CDI) per 1,000 PD and 12-week postdischarge (PDC- CDI) per 100 discharges . Interrupted time-series analysis using generalized estimating equation models with negative binomial link function was conducted; a sensitivity analysis with Medicare case-mix index (CMI) adjustment was also performed to control for differences after start of the COVID-19 pandemic. Results: Among 163,117 admissions, there were 683 HO-CDIs and 1,009 PDC-CDIs. Overall, FQ DOT per 1,000 PD decreased by 21% immediately after the intervention and decreased at a consistent rate throughout the entire study period . HO-CDI rates were stable throughout the study period, with a nonsignificant level change decrease of 10% after the intervention. In contrast, there was a reversal in the trend in PDC-CDI rates from a 0.4% per month increase in the preintervention period to a 3% per month decrease in the postintervention period (P < .01). Sensitivity analysis with adjustment for facility-specific CMI produced similar results but with wider confidence intervals, as did an analysis with a distinct COVID-19 time point. Conclusion: Our systemwide intervention using order sets with decision support reduced inpatient FQ use by 21%. The intervention did not significantly reduce HO-CDI but significantly decreased the incidence of CDI within 12 weeks after discharge. Relying on outcome measures limited to inpatient setting may not reflect the full impact of inpatient stewardship efforts and incorporating postdischarge outcomes, such as CDI, should increasingly be considered.Funding: NoDisclosures: None"} +{"text": "The transformation proceeds under mild reaction conditions, has a broad substrate scope, and can be applied to late-stage functionalization of complex small molecules.Sulfoximines are synthetically important scaffolds and serve important roles in drug discovery. Currently, there is no solution to decarboxylative sulfoximination of benzoic acids; although thoroughly investigated, limited substrate scope and harsh reaction conditions still hold back traditional thermal aromatic decarboxylative functionalization. Herein, we realize the first decarboxylative sulfoximination of benzoic acids via photoinduced ligand-to-copper charge transfer. The transformation proceeds at ambient reaction temperature and demonstrates broad substrate scope.We report the first decarboxylative sulfoximination of benzoic acids Promoted to their excited states upon irradiation can result in intramolecular ligand-to-metal charge transfer (LMCT) within the excited state complexes to generate reactive open-shell radical intermediates with reactivity hardly reachable in the ground state.1\u20135 Conventional metal-catalysed or -mediated thermal decarboxylative cross-coupling reactions normally require high reaction temperature and ortho-substituents.6 Radical aromatic decarboxylation proceeds about three orders of magnitude slower than from aliphatic carboxyl radicals,7,8 which generally leads to undesirable side reactions such as hydrogen atom abstraction for benzoyl radicals.7 As a consequence, many aromatic decarboxylative bond forming reactions, including sulfoximination, are still out of reach for conventional reaction chemistry. Here we report the first decarboxylative sulfoximination of benzoic acids enabled by photo-induced ligand to copper charge transfer. Photoactive copper(ii) carboxylates undergo a low-barrier radical CO2 extrusion upon irradiation, with the putative formed aryl radicals subsequently captured by copper complexes to generate CuAr(iii) species for C\u2013N reductive elimination. The synthetic utility of this method was exemplified by late-stage decarboxylative sulfoximination of several complex small-molecule benzoic acids, which are abundantly available from nature.Coordination of substrates including alcohols,ii) complexes have been known as an effective platform for generating reactive radicals for decades. Kochi first studied the addition and C3sp\u2013H abstraction reactivity of chlorine radical generated by the photo-irradiation of CuCl2 in different organic solvents.2 Based on this initial finding, Wan and co-workers developed a vicinal dichlorination of alkenes catalyzed by CuCl2 under air,2 and the Rovis group realized a copper catalyzed olefination of unactivated C3sp\u2013H bonds.2 In addition to chlorine radicals, copper-LMCT is also suitable for N- or C-centered radical generation. In 2018, Rehbein and Reiser found that copper-LMCT was effective for azide radical generation,3 and Wang and Xu suggested the formation of N-centered radical cations via intramolecular LMCT of quinolinyl-8-glycinate ester coordinated alkyl-Cu(iii) adducts.9 For C-centered radicals, Gong and co-workers proposed the generation of alkyl radical intermediates via photolysis of Cu(ii)-alkyl complexes.10 Although copper-LMCT in copper(ii) carboxylate complexes was first described by DeGraff and co-workers during their research on the photolysis of copper(ii)-malonate,4 it was not until 2021 that our group applied this copper-LMCT reactivity to synthetic applications.11 We realized the first aromatic decarboxylative fluorination11 and the decarboxylative hydroxylation11 of benzoic acids. Concurrently, the MacMillan group explored the copper-LMCT reactivity in aromatic decarboxylative borylation12 and halogenation,12 and achieved copper catalysis for transformations with single electron oxidants such as 1-fluoro-2,4,6-trimethylpyridinium tetrafluoroborate (NFTPT). Stoichiometric copper is still required for nucleophiles such as fluoride.11 Nearly at the same time, the Yoon group developed a copper-mediated oxidative decarboxylative functionalization of aliphatic carboxylic acids.4 Most recently, Reiser and co-workers reported a copper-catalysed aliphatic decarboxylative oxygenation methodology, where oxygen was applied as the oxidant.4 While we and others have developed the concept of photo-induced copper-LMCT-enabled aromatic radical decarboxylation to achieve previously unknown reactivity, the coupling counterparts are generally limited to halides, carboxylates or boronate esters but strong coordinating NH-nucleophiles have not been shown to react species, strong coordination of NH-nucleophiles such as NH-sulfoximines will compete with carboxylates and form undesired copper species that can diminish the reaction efficiency. Though thermal aromatic decarboxylative C\u2013N cross couplings under high reaction temperatures have been explored by Jia, Goo\u03b2en, and Xie, electron-deficient ortho-substituted benzoic acids are required for efficient CO2 extrusion halides,21 arylboronic acids,22 aryl siloxanes,23 acyl peroxides,24 arylsulfinates,25 and aryl hydrazides26 but cannot be accessed from benzoic acids (tert-butylpyridine (DTBP) and LiOMe as additives was able to overcome the challenging low reaction efficiency associated with copper-LMCT-enabled aromatic decarboxylative sulfoximination.The study of sulfoximines dates back as far as 1949 when methionine sulfoximine was first synthesized by Bentley and Whitehead.ic acids . Herein,ic acids . We founii) species.16 In the copper-LMCT process, competing coordination of sulfoximines to copper(ii) might hinder the formation of key copper(ii) carboxylate intermediates and, in turn, decrease the reaction efficiency. We hypothesized that initial deprotonation of the benzoic acids to their carboxylate salts might facilitate the generation of photoactive copper(ii) carboxylates, while the careful screening of additives can hinder the formation of undesired sulfoximine-ligated copper(ii) species.Because of the enhanced N\u2013H acidity, NH-sulfoximines can undergo facile deprotonation and readily coordinate with copper(via the decarboxylative sulfoximination of lithium 4-fluorobenzoate (1). A series of reaction condition optimization in MeCN can afford N-arylated sulfoximine 3 in 66% yield, together with side product ester 3a in 8% yield (ii) carboxylates species. The role of LiOMe is not very clear; we propose that the addition of LiOMe might help decrease the concentration of free sulfoximines by forming poorly soluble sulfoximine lithium salts and in turn, accelerate the formation of copper(ii) carboxylate species. Low conversion of starting substrates 1 and 2 was observed when copper sources including Cu(OAc)2 were used instead of Cu(OTf)2 (entry 7). Interestingly, only MeCN as solvent was productive, and DCM only afforded 12% protodecarboxylation side-product fluorobenzene (entry 8). Control experiments confirmed the essential use of 390 nm LEDs irradiation for CO2 extrusion, and no decarboxylation was observed under thermal reaction conditions.As shown in 8% yield , entry 18% yield . We assuN-arylated sulfoximines in moderate to good yields. Owing to the high oxidative potential, radical decarboxylation of electron-deficient benzoic acids is generally problematic,27 however, performed well under our present reaction condition. Ortho-fluoro-substituted benzoic acid (10) gave a moderate yield; yet, benzoic acids with large ortho-substituents failed to afford productive yields, possibly owing to the insufficient generation of copper(ii) carboxylates. Heteroaromatic carboxylic acids such as CF3-substituted isonicotinic acid can also perform efficient decarboxylation to afford the corresponding N-arylated sulfoximine 12. Functional groups including aryl halides , ketone (13), heterocycles , nitriles and sulfonamides were well tolerated. \u03b1-O or \u2013N , benzylic , and tertiary (14) C\u2013H bonds that are sensitive to HAT do not prevent efficient decarboxylative sulfoximination. In addition, strong coordinating or oxidizable functional groups like amines inhibit the transformation. The utility of this decarboxylative sulfoximination was further displayed by the late-stage decarboxylative sulfoximination of several complex small molecules . NH-Sulfoximines with electron-rich and electron-neutral (21\u201324) arenes afforded good yields; however, electron-deficient NH-sulfoximines gave lower yields, possibly due to their weaker N-nucleophilicity. Dialkyl (31) and diaryl (32) NH-sulfoximines furnished their corresponding N-arylated sulfoximines in moderate yields. Benefiting from the mild reaction conditions, N-arylation of enantiopure NH-sulfoximines proceeded in good yields, and no racemization was observed. In most cases, low conversion of the starting benzoate salts accounts for the observed low yield.Subsequently, we next studied the substrate scope of the decarboxylative sulfoximination . Electro2, a strong absorbance (370\u2013470 nm) attributed to the LMCT band of copper(ii) carboxylates was detected carboxylates under the reaction conditions. The coordination of sulfoximines to copper(ii) and the coordination of 2,6-di-tert-butylpyridine (DTBP) to copper(ii) are in agreement with the observation of an absorbance (370\u2013470 nm) of a mixture of sulfoximine 2 and Cu(OTf)2, and a mixture of DTBP and Cu(OTf)2 (ii)-containing mixtures display a broad d\u2013d transitions absorbance at 550\u2212900 nm, which decreased monotonously upon purple LED irradiation, consistent with the reduction of Cu(ii) to Cu(i) (i) was confirmed by the observation of a characteristic purple [CuI(biq)2]+ complex when 2,2\u2032-biquinoline (biq) was added to the irradiated reaction mixture sulfoximine complex, with consumption of the copper(ii) species. This result is consistent with competing coordination of sulfoximines to copper(ii) and may also explain the low reaction reactivity caused by the competing coordination. Based on the above mechanistic investigation, we propose a mechanism as depicted in ii) charge transfer in copper(ii) carboxylates I affords aryl carboxyl radical intermediates II, which then undergo low-barrier radical decarboxylation to afford aryl radicals III. Subsequent copper-assisted aryl radical capture generates arylcopper(iii) intermediates IV that finally undergo C\u2013N reductive elimination to afford N-arylated sulfoximines 2 . All copto Cu(i) . The foroximines . Additioapproach .Copper-LMCT based radical aromatic decarboxylative carbometalation enabled the first decarboxylative sulfoximination of benzoic acids. The broad substrate scope and good functional group tolerance demonstrate the generality of the copper-LMCT concept in aromatic decarboxylative sulfoximination. Conceptually, the success of this transformation demonstrates the expansion of the copper-LMCT concept for aromatic decarboxylative cross-couplings to reactions with strongly coordinating nucleophiles.Procedures and compound characterization are provided in the ESI.\u2020P. X. initiated the project and performed experiments and analyzed the data. W. S. performed and analyzed experiments regarding the mechanism. T. R. directed the project.The authors declare no competing interests.SC-013-D2SC05442F-s001"} +{"text": "A 58-year-old woman presented speech impairment, mental confusion, and left hemiparesis after being found unconscious. Brain magnetic resonance imaging (MRI) showed pyogenic abscess and multiple vascular malformations in the cerebral hemispheres and 2, r"} +{"text": "The Coronavirus Disease 2019 (COVID-19) is well-known for its broad spectrum of immune-related phenotypes similar to those seen in autoimmune or inflammatory diseases. Furthermore, evidence has gradually accumulated that COVID-19 may induce systemic inflammatory manifestations such as multisystem inflammatory syndrome, haemophagocytic syndromes, and systemic vasculitis. Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a small vessel vasculitis characterised by necrotising vasculitis. So far, there have been several case reports regarding AAV occurrence after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, which have indicated a triggering potential of SARS-CoV-2 infection for AAV occurrence. This study investigated the rate of ANCA positivity and its clinical significance in COVID-19 patients.This study included 178 patients infected with SARS-CoV-2 who were enrolled in a cohort of a single center. Myeloperoxidase (MPO)-ANCA and proteinase 3 (PR3)-ANCA from the stored blood sera were measured using the immunoassay kits. Mortality, mechanical ventilator care, and severe infection were assessed as poor outcomes. Severe infection was defined as a medical condition that required a high-flow nasal cannula and/or mechanical ventilator care. The 2022 American College of Rheumatology and the European Alliance of Associations for Rheumatology classification criteria for the three subtypes of AAV were applied only to patients who had MPO-ANCA or PR3-ANCA among the study subjectsThe detection rate of ANCA positivity was 18.5%: MPO-ANCA and PR3-ANCA were found in 22 (12.4%) and 14 (7.9%) patients. Patients with ANCA positivity exhibited a lower cumulative survival rate than those without, but the difference was not statistically significant (P = 0.057). However, neither MPO-ANCA nor PR3-ANCA affected the three poor outcomes. According to the new criteria, 12 (6.7%) and 21 (11.8%) patients were classified as having granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA)Neither ANCA positivity nor ANCA subtype (MPO-ANCA and PR3-ANCA) positivity had a significant influence on poor outcomes of SARS-CoV-2.ANCA: antineutrophil cytoplasmic antibody; MPO: myeloperoxidase; PR3: proteinase 3; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2.SARS-CoV-2 infection may increase the rate of ANCA positivity, which may not affect poor outcomes but contribute to the classification of GPA and MPA despite uncertain clinical significanceAll Authors: No reported disclosures."} +{"text": "Intra-retinal delivery of novel sight-restoring therapies will require the precision of robotic systems accompanied by excellent visualisation of retinal layers. Intra-operative Optical Coherence Tomography (iOCT) provides cross-sectional retinal images in real time but at the cost of image quality that is insufficient for intra-retinal therapy delivery.This paper proposes a super-resolution methodology that improves iOCT image quality leveraging spatiotemporal consistency of incoming iOCT video streams.To overcome the absence of ground truth high-resolution (HR) images, we first generate HR iOCT images by fusing spatially aligned iOCT video frames. Then, we automatically assess the quality of the HR images on key retinal layers using a deep semantic segmentation model. Finally, we use image-to-image translation models (Pix2Pix and CycleGAN) to enhance the quality of LR images via quality transfer from the estimated HR domain.Our proposed methodology generates iOCT images of improved quality according to both full-reference and no-reference metrics. A qualitative study with expert clinicians also confirms the improvement in the delineation of pertinent layers and in the reduction of artefacts. Furthermore, our approach outperforms conventional denoising filters and the learning-based state-of-the-art.The results indicate that the learning-based methods using the estimated, through our pipeline, HR domain can be used to enhance the iOCT image quality. Therefore, the proposed method can computationally augment the capabilities of iOCT imaging helping this modality support the vitreoretinal surgical interventions of the future. Regenerative therapies e.g. , 2) are are 2]) Intra-operative OCT (iOCT), acquired through recently introduced modified biomicroscopy systems such as Zeiss OPMI/Lumera and Leica Proveo/Enfocus, can be delivered in real time but at the expense of image quality with reAn established approach to OCT quality enhancement is denoising. Spatially adaptive wavelets , Wiener Within the deep learning domain, Generative Adversarial Networks can achDespite its superior quality, pre-operative OCT is acquired under different protocols than iOCT, implying a domain gap in addition to deformations that may lead to generated images with artefacts. Therefore, our paper considers iOCT information only. We propose a methodology that uses high-resolution (HR) iOCT images generated offline through registered and fused low-resolution (LR) iOCT video frames (B-scans). Generated HR images are ranked for quality considering metrics that incorporate the quality of segmented retinal layers. High-scoring HR images comprise the target domain for image-to-image translation. Several image quality metrics and a complementary qualitative survey showcase that our super-resolution methodology improves iOCT image quality outperforming filter-based denoising methods and the learning-based state-of-the-art .This section presents the process of creating HR iOCT images, validating their quality and generating SR iOCT images through image-to-image translation.Our data are derived from an internal Moorfields Eye Hospital database of vitreoretinal surgery videos, including intra-operative and pre-operative OCTs. We use a data-complete subset comprising 42 intra-operative retinal surgery videos acquired from 22 subjects. The data contain the surgical microscope view captured by a Zeiss OPMI LUMERA 700 with embedded LR iOCT frames we must verify that the retina is also stationary. Therefore, we manually selected a point at the start of each video sequence that corresponds to a strong feature (e.g. vessel bifurcations), and tracked it using Lucas-Kanade methodIf the aforementioned positions remained constant for more than eight consecutive video frames , the corresponding iOCT B-scans were then rigidly registered to the first B-scan and averaged to generate the corresponding ame Fig. . We applAs the videos depict actual surgical procedures, many incoming LR iOCT images have low signal strength, calculated as signal to noise ratio (SNR) . Thus, tTo assess the quality of the generated images and define which ones should be included in the io (CNR) :1\\documeThe segmentation model utilizes the architecture introduced in and is tGiven the output label maps of the segmentation model, five ROIs are chosen see Fig. : a backgG and a discriminator D in an adversarial manner. Pix2Pix requires supervision in the form of aligned image pairs to update its generator G as it minimizes the L1 loss between images of source (LR) and target (L1 supervised losses for training CycleGAN.To perform super-resolution (SR), we used two state-of-the-art image-to-image translation models: CycleGAN and Pix2The dataset images are estimated by our methodology, full-reference metrics alone are not sufficient in image quality evaluation. Therefore, our analysis uses six different metrics including two full-reference metrics, i.e. Peak signal-to-noise ratio (PSNR) and Structural Similarity Index (SSIM) and four no-reference metrics, i.e. perceptual loss function (t}\u2113feat) , Frechett}\u2113feat) , Global t}\u2113feat) and Natut}\u2113feat) . The mett}\u2113feat) , SR usinmentclasspt{minimat}\u2113feat) (SR-Cyc)mentclass2pt{minimReference metrics were calculated using Network . We alsoAs shown in Table Q1: Can you notice an improvement in the delineation of RPE/Bruchs vs. IS/OS junction in the generated image? (A1: 3.8\u00b10.3)Q2: Can you notice a reduction of artefacts in the generated image?A2: 3.9\u00b10.1)(Q3: Can you notice an improvement in the delineation of the ILM vs. RNFL in the generated image? (A3: 3.7\u00b10.3)Their answers, A1, A2, A3 (mean\u00b1standard deviation), indicate that SR-Cyc images provide improved delineation of RPE vs IS/OS junction (Q1), reduction of artefacts (Q2) and improved delineation of ILM vs RNFL (Q3). Visual results are shown in Fig. To further validate our super-resolution pipeline, we performed qualitative analysis. Our survey included 20 pairs of LR and SR-Cyc images, randomly selected from the test set. We asked 8 retinal doctors/surgeons to evaluate these image pairs by assigning a score between 1 (strongly disagree) and 5 (strongly agree) on the following questions:To demonstrate the denoising effect of our work, as part of the broader aim of image quality enhancement, we compare our optimal (according to the metrics) network (SR-Cyc) with conventional denoising filters. We selected three different state-of-the-art speckle reduction methods for OCT images: Symmetric Nearest Neighbour (SNN) , adaptivAll the filter-based methods demonstrated considerable denoising capabilities, as shown in Fig Quantitative analysis using the aforementioned metrics (see Table This paper addresses the challenge of super-resolution in iOCT images. We overcome the absence of ground truth HR images by a novel pipeline that leverages spatiotemporal consistency of incoming iOCT B-scans to estimate the"} +{"text": "Age-related neurodegenerative disorders, including Alzheimer\u2019s disease and Parkinson\u2019s disease (PD), progressively reduce mobility and quality of life (QoL). Real-world mobility from actigraphy predicts PD disease severity. This pilot analysis assessed utility of actigraphy to screen early physical function-related QoL decline in PD. Method: Mobility was monitored for 4 weeks using wrist-worn ActiGraph recordings in 27 participants with idiopathic PD . Days with >600 mins of wear time during non-sleep times were analyzed (\u00b5 = 29.78 days \u00b1 3.78). Typical activity was quantified as average steps per hour. Participants completed demographic and health assessments. Disease severity and physical function QoL were measured using the clinically-validated Unified PD Rating Scale (MDS-UPDRS) and Short Form-36 (SF-36), respectively. Disease severity, QoL, and typical activity were compared using Spearman correlations.Lower typical activity from actigraphy was associated with more severe motor symptoms and with increased impairment in physical function QoL . Daily activity from actigraphy did not predict symptom severity of non-motor and motor-related complications. Discussion: Pilot results show utility of actigraphic metrics for indexing real-world mobility and QoL declines in neurodegenerative disorders, in line with broader efforts to turn real-world data into actionable evidence for healthcare interventions. Ongoing discovery in larger populations should yield robust, clinically-relevant indices of daily activity in aging and neurodegenerative impairment."} +{"text": "Since the COVID-19 pandemic there is concern for subclinical cardiac pathology in the absence of clinical symptoms in collegiate athletes, we present 4 cases of abnormal left ventricular global longitudinal strain (LVGLS), a \u201cred-flag\u201d for potential COVID-19 myocardial disease, following diagnosis with diverse abnormalities reported via multimodality imaging weeks into recovery. Cardiac imaging studies consisting of transthoracic echocardiography (TTE) and cardiovascular magnetic resonance imaging (CMR) were performed 10 days post-COVID-19 diagnosis and several weeks into recovery. Initial TTE revealed abnormal left ventricular global longitudinal strain (LVGLS), an identified \u201cred-flag\u201d for potential COVID-19 myocardial disease. Further CMR imaging revealed potential recent/prior myocarditis in 1 athlete. Follow-up TTE several weeks later revealed a return to normal LVGLS. Conversely, 2 cases with normal CMR imaging had a LVGLS that remained abnormal >30 days into recovery. These individual cases highlight the substantial differences in echocardiographic and CMR abnormalities between athletes with confirmed COVID-19. With increased concerns for coronavirus disease-19- (COVID-19-) induced cardiac injury in athletes, numerous recommended screening procedures have been proposed; however, the differential pattern of cardiac abnormalities in individual athletes remains unknown [An 18-year-old male collegiate athlete presented with fever, cough, and sinus congestion but denied chest pain and shortness of breath. The test for COVID-19 returned positive. Ten days into recovery, he had normal sinus rhythm, was normotensive, and had a normal 12-lead ECG. High-sensitivity troponin T (hsTnT) was normal (<0.010). Transthoracic echocardiography (TTE) showed mild left ventricular (LV) hypertrophy and cavity dimensions, suggestive of athletic remodeling. Mild dilations of right atria (RA) and left atria (LA) were noted with mild tricuspid valve regurgitation. LV ejection fraction (LVEF) was normal, but global longitudinal strain (GLS) was abnormal (-16%) with normal diastolic function . CardiovA 19-year-old male collegiate athlete tested positive for COVID-19, and 11 days into his recovery, there were no indications of arrhythmias or murmurs with normal hsTnT (<0.010). Contemporaneous TTE revealed a normal camber dimensions, LVEF, and diastolic function, but abnormal GLS (-14.6%) with mitral and tricuspid valve regurgitation was observed. CMR performed 19 days into recovery revealed no signs of myocarditis ). A repeat TTE 35 days into recovery showed a still abnormal but improved GLS of -15.8%, with normal LVEF%.A 20-year-old male collegiate athlete presented with symptoms of a headache, nausea, difficulty breathing, sore throat, and fatigue after testing positive for COVID-19 but denied any symptoms of chest pain or shortness of breath (SOB), and hsTnT was normal (<0.010). TTE was performed 10 days following diagnosis, and athlete reported continuing symptoms of a headache, stuffy nose, and fatigue. He was hypertensive (130/58\u2009mmHg) but had a normal 12-lead ECG and LVEF. TTE showed mild dilation of the RV and RA and abnormal LV GLS of -13%. CMR was performed 22 days into recovery showing no signs of myocarditis ) and normal myocardial perfusion. Follow-up TTE, 31 days into recovery, revealed an abnormal but improved GLS (-14.9%).An 18-year-old male collegiate diagnosed with COVID-19 reported a mild cough during infection but denied chest pain, SOB, or fever. Ten days into recovery, he had a normal ECG and hsTnT (<0.010). TTE revealed an abnormal LVGLS of -16%, but normal LVEF and diastolic function. CMR performed 15 days into recovery showed an area of midmyocardial LGE, indicating potential recent/prior myocarditis, with normal LV wall motion and LVEF . TTE wasEarly reports have indicated that SARS-CoV-2 infection elicits cardiac injury in up to 2 out of 5 hospitalized COVID-19 patients , 6, 9\u201312Unfortunately, to date, most of our knowledge on COVD-19-induced cardiovascular complications is limited to hospitalized patients, with a paucity of information on the use of imaging modalities for diagnosis and follow-up of myocardial involvement in younger nonhospitalized individuals. Recently, Joy et al. evaluated cardiac function in health-care workers (mean age 37 years) 6 months following COVID-19, of which 85% were mildly symptomatic and 15% asymptomatic at the time of diagnosis . Their mAssessment of COVID-19-induced cardiac abnormalities, against the background of athletic remodeling, presents a particularly unique challenge in identifying individuals at risk for pathologic outcomes following infection. In addition, most athletic departments do not have the capabilities or capacity for onsite cardiac evaluation following COVID-19. When follow-up evaluation for cardiac involvement is required, this challenge is further exacerbated when different imaging modalities are utilized. Thus, there is a critical need to improve our understanding on the differential pattern of cardiac abnormalities that may exist in this unique population. Here, we present 4 cases of abnormal LVGLS, an identified \u201cred-flag\u201d for potential COVID-19 myocardial disease [To date, there are a limited number of studies investigating the potential cardiac consequences of COVID-19 in the athletic populations, with fewer extending into recovery . In a stTo date, both direct and indirect effects of the SARS-CoV-2 virus on cardiovascular outcomes have been postulated but remain incompletely understood \u201322, thusThe diverse temporal responses of our cases highlight our limited understanding of the time course of LV function changes as they relate to CMR parameters. Taken altogether, the consequences of COVID-19 infection still remain unclear and future research looking into the long-term effects of this disease is warranted, with clear indication that no screening modality provides a complete picture of potential cardiac abnormalities."} +{"text": "Emergent SARS-CoV-2 variants and waning humoral immunity in vaccinated individuals have resulted in increased infections and hospitalizations. Children are not spared from infection nor complications of COVID-19, and the recent recommendation for boosters in individuals ages 12 years or older calls for broader understanding of the adolescent immune profile after mRNA vaccination. We tested the durability and cross-reactivity of anti-SARS-CoV-2 serologic responses over a six-month time course in vaccinated adolescents against the SARS-CoV-2 D614G (\u201cwild type\u201d) and Omicron antigens. Serum from 77 adolescents showed that anti-Spike antibodies wane significantly over six months. After completion of a two-vaccine series, cross-reactivity against Omicron-specific receptor-binding domain (RBD) was seen. Functional humoral activation against wild type and Omicron SARS-CoV-2 also declines over time in vaccinated adolescent children. Evidence of waning mRNA-induced vaccine immunity underscores vulnerabilities in long-term pediatric protection against SARS-CoV-2 infection, while cross-reactivity highlights the additional benefits of vaccination. Characterization of adolescent immune signatures post-vaccination will inform guidance on vaccine platforms and timelines, and ultimately optimize immunoprotection of children. As we entered the third year of the COVID-19 pandemic, SARS-CoV-2 infection rates surged due to highly infectious viral variants and waning population immunity. Despite full (two-dose) mRNA vaccination [While mortality from COVID-19 is lower in children compared to adults, over 12.7 million children have been diagnosed with COVID-19, leading to roughly 40,000 hospitalizations in the US . AdditioThe emergence of the Omicron variant has called into question the long-term efficacy of current vaccine platforms and dosing regimens in children. Recent data now suggest that BNT16b2 vaccine-induced immune protection declines rapidly in children, especially children aged 5\u201311 years . ChildreHere, we quantified relative antibody responses in adolescent children immediately following the Pfizer-BioNTech mRNA vaccination and six months post-inoculation and analyzed the efficacy of the humoral response against the D614G (\u201cwild type\u201d) SARS-CoV-2 and latest variant of concern (VOC), Omicron.Adolescent children (ages 12\u201317) assented, with parental consent, to participate in the MGH Pediatric COVID-19 Biorepository (MGB IRB #2020P000955) . Young aSerological analyses were performed using an in-house enzyme-linked immunosorbent assay (ELISA) that detects IgG against the D614G (\u201cwild type\u201d) SARS-CoV-2 Spike, the D614G (\u201cwild type\u201d) Receptor-Binding Domain (RBD), or the Omicron SARS-CoV-2 VOC RBD by using the previously described method . BrieflyThe neutralizing activity of vaccine sera against coronaviruses was compared by producing lentiviral particles pseudotyped with different Spike proteins, as previously described . NeutralComplement deposition was performed as previously described . BrieflyTHP-1 cellular phagocytosis assay was performed as previously described . BrieflyNeutrophil phagocytosis assay was performed as previously described . Brieflyt-test for two-way comparisons. Correlations were completed using Pearson correlation. Outliers were removed using a Robust regression and Outlier removal (ROUT) method with a Q value of 0.02%.Analysis was completed by Prism 9.3 using one-way ANOVA for multiple comparisons and Seventy-seven children were enrolled in our study, with an average age of 14 years; sixty-eight children were between the ages of 12\u201315 years; nine were between ages of 16-19 years. Sex was equally distributed, and 19% of the population was Hispanic . Ninety-p < 0.0001, p < 0.0001, p < 0.0001; wild type Spike, wild type RBD, and Omicron RBD, respectively). Interestingly, there was no increase in antibodies against Omicron RBD after the first vaccine dose, but a significant increase in titers was seen following a second vaccine dose. However, there was a subsequent loss of all anti-SARS-CoV-2 antibody responses by six months, as compared to the V2 time point . By six months, antibody responses decreased to levels comparable to titers seen at the V1 time point, following the first vaccine dose. Twenty-four adolescents provided blood samples at all four time points; individual responses align with trends seen in the larger cohort . At peak immunity following the second vaccination, there was a slight correlation in wild type Spike and RBD for both wild type and Omicron , though this correlation plateaued at peak RBD levels. At six-months post mRNA vaccine, despite the fact that wild type RDB titers remained higher than Omicron RBD titers (p < 0.01), there was a strong correlation in declining anti-wild type and Omicron RBD titers with anti-Spike titers . These data underscore the need for long-term vaccine-induced pediatric immunoprotection amidst episodic surges of SARS-CoV-2 infections.As Omicron has become the predominant SARS-CoV-2 variant globally, we assessed the relationship between anti-wild type RBD and anti-Omicron RBD titers to determine if COVID-19 mRNA vaccination displayed cross-coverage and potential protection against the Omicron-specific SARS-CoV-2 RBD. While a single vaccination produces only low titers against Omicron, the second vaccination establishes greater cross-reactivity of RBD responses in many, but not all, adolescents C, and thp = 0.01), although some individuals displayed an increase in neutralization by the six-month time point. No differences in neutralization capacity were seen at the V2 and V6 time points against Omicron . Antibody-dependent cellular (THP-1 monocyte) phagocytosis (ADCP) displayed a significant decrease from the V2 to V6 time point in both wild type (p < 0.0001) and Omicron strains (p < 0.0001). Interestingly, while a pronounced decline in antibody-dependent neutrophil phagocytosis (ADNP) was seen between peak immunity and the six-month post-vaccine time point for the wild type strain (p < 0.0001), neutrophil phagocytosis was sustained, albeit at a lower level, in the Omicron strain showed a sustained response at both the V2 and V6 timepoint in the wild type strain A but shon strain A,B. Immun strain C\u2013F.As the COVID-19 mRNA vaccines represent a new vaccination platform, the longevity of immune responses needs to be characterized across all age ranges, especially in light of emerging variants. Here, we detail the durability of antibody titers and functional capacity of humoral immune responses to the SARS-CoV-2 mRNA vaccine in adolescent children, including responses against the highly infectious and the current predominant variant, Omicron. As expected, and as seen in adult populations, mRNA vaccine-induced immunity in adolescent children wanes significantly over a 6-month time period, with a loss of circulating antibody titers and a reduction in antibody function, including viral neutralization. This finding demonstrates a current vulnerability to infection in adolescent children, many of whom have now received their vaccine series over six months ago.While total anti-SARS-CoV-2 antibody titers and most anti-SARS-CoV-2 antibody functions waned over time in this adolescent cohort, immune complex-induced complement activation against wild type Spike persisted over time. Complement activation augments humoral responses by enhancing antibody neutralization of SARS-CoV-2 and promoting phagocytosis by immune cells . This fiEncouragingly, our data demonstrate that adolescents\u2019 immune responses display some cross-coverage of the VOC, Omicron, with comparable immune responses to those reported in adults . This coOur data support new CDC guidelines that recommend a booster dose five months from the completion of mRNA vaccination series to mitigate waning immunity . AlthougIn conclusion, adolescent children exhibit waning antibody immune responses six months post-mRNA vaccination. mRNA boosters will play a critical role in sustaining durable immune responses in adolescent children, while also reducing pediatric infection, severe illness, and transmission as we traverse the surges of the COVID-19 pandemic."} +{"text": "With the increased implementation of interactive technologies for assessment and rehabilitation, it would be optimal to exhibit the reliability of physical assessment measures via tele-assessment. Aim: To determine the test-retest and intra-rater reliability of physical function outcome measures routinely used in the balance and gait rehabilitation using real-time online tele-assessment.Community-dwelling healthy older adults (N=30) participated in three experimental tele-assessment sessions. During each session, a real-time online tele-assessment was performed on five major domains that evaluate balance and gait function: lower limb strength and endurance (30-second chair stand test), aerobic endurance (2-minute step test), static balance (One-legged stand test), dynamic balance (4-step square test), and gait (Tinetti).Coefficient of determination (R2) was used to determine the test-retest (TRT) and intra-rater (IR) reliability for all outcome variables. Excellent reliability was shown by Tinetti and one-legged stand test . Excellent to good reliability was shown by 4-step square test , 2-min step in place test , and 30-second chair stand test .The reliability of individual outcome measures ranged from good to excellent, suggesting that these outcomes have sufficient sensitivity for detecting change with telerehabilitation."} +{"text": "Indirect immunofluorescence assay (IIFA) based on antineutrophil cytoplasmic antibody (ANCA) testing is a commonly employed test for diagnosing autoimmune vasculitis. Antinuclear antibody (ANA) can give rise to a false interpretation of perinuclear-ANCA (pANCA) in ethanol-fixed granulocyte substrates. Analytical interference could frequently occur in setups where ethanol-fixed substrates are used alone. Here, we intend to investigate this ANA interference in pANCA interpretation. In this retrospective study, we studied anti-MPO-negative but ANA-positive and pANCA (IIFA based) samples. We also correlated immunoblot results (where data were available) and checked the association between grades of blot positivity (an indicator of the concentration of ANA) and frequency of pANCA interpretation. Data were analyzed by appropriate statistical techniques (Chi-square and kappa statistics). About 19.2% of ANA blot (ENA-blot) positive samples displayed a pANCA positive pattern in the ethanol-fixed substrate, while this positivity in ENA-blot negatives was 6.5%. In positive ANA-IIFA samples, about 14.7% yielded pANCA patterns (on ethanol fixed substrates). Out of this, nuclear homogenous pattern yielding samples gave the highest frequency pANCA, that is, in 31.5% followed by speckled (11.1%), DFS (10.3%), and centromere (6.7%).The association of the nuclear homogenous pattern was statistically significant. ANA-positive results may interfere with the interpretation of pANCA as observed in ANA-IIFA and ENA-blot positive samples. ANA-IIFA patterns like nuclear homogenous may strongly associate this pANCA interpretation. This can help laboratories perform ANCA testing more effectively, ruling out ANA interference in ANCA screening. Antineutrophil cytoplasmic antibodies (ANCAs) are crucial in diagnosis and pathogenesis of a group of conditions called ANCA-associated vasculitis , 2. ANCAANCAs are usually detectable in such conditions , 3. In iG, azurocidin, lactoferrin, lysozyme, and bactericidal/permeability-increasing factors can yield pANCA pattern from the scan images of blots. Association was significant between some ANA-IIFA patterns and apparent pANCA positivity, as shown in p=0.0001). Speckled and DFS70 patterns were also linked to a higher proportion of pANCA records (p=0.007) as depicted in records though tIn the current study, about 19.2% of ANA-positive (ENA-blot) subjects (postimmunoblot\u2014see ANCA-associated major antigens (myeloperoxidase and proteinase 3) are localized in cytoplasmic granules of the granulocytes , 8. UponAnti-MPO antibody presence in serum may be due to different types of epitopes present in MPO antigen. It is not possible to have a detection system (ELISA) to cover antibodies against all epitopes\u2014some of these undetectable (by ELISA) may be clinically relevant (vasculitis causing) . Hence, Another area where the current work can be relevant is the testing strategy of a condition like autoimmune hepatitis. A nuclear homogenous ANA and atypical ANCA pattern are the critical laboratory markers for type 1 autoimmune hepatitis . Latter It is pertinent to mention here that in a majority of autoimmune hepatitis related ANA-IIFA-positive cases (2/3rd), the pattern observed is nuclear homogenous (HEp-2 substrate) while rest (1/3rd) is either speckled or nucleolar. The antigens recognized in these cases are single- and double-stranded DNA, nucleolar chromatin, histone, centromere, cyclin A, ribonuclear protein, and unknown (30% cases) .Two limitations of our current study were a small sample size and biased cohort (as ANA-IIFA-positive samples were taken as the starting point in a retrospective manner). We tried to overcome these limitations by taking measures such as long study duration (5\u2009years), performing few tests (immunoblot) on preserved samples, including samples from multiple disciplines/departments covering different age, gender, clinical spectrum etc., and data analysis and data collection by different people.The overall analysis indicates the existence of ANA interference upon ANCA interpretation in our population. In setups performing IIFA-based ANCA tests probably need to adopt an economical workflow to rule out or minimize error due to the issue of ANA interference. Existing clinical/testing guidelines advise IIFA-based ANCA testing low antibody-positive samples or negative antibody samples in the presence of clinical features of AAV , 7. Addi"} +{"text": "MicroRNAs (miRNAs) are a class of small non-coding RNA that can downregulate their targets by selectively binding to the 3\u2032 untranslated region (3\u2032UTR) of most messenger RNAs (mRNAs) in the human genome. MiRNAs can interact with other molecules such as viruses and act as a mediator for viral infection. In this study, we examined whether, and to what extent, the SARS-CoV-2 virus can serve as a \u201csponge\u201d for human miRNAs.We identified multiple potential miRNA/target pairs that may be disrupted during SARS-CoV-2 infection. Using miRNA expression profiles and RNA-seq from published studies, we further identified a highly confident list of 5 miRNA/target pairs that could be disrupted by the virus\u2019s miRNA sponge effect, namely hsa-miR-374a-5p/APOL6, hsa-let-7f-1-3p/EIF4A2, hsa-miR-374a-3p/PARP11, hsa-miR-548d-3p/PSMA2 and hsa-miR-23b-3p/ZNFX1 pairs. Using single-cell RNA-sequencing based data, we identified two important miRNAs, hsa-miR-302c-5p and hsa-miR-16-5p, to be potential virus targeting miRNAs across multiple cell types from bronchoalveolar lavage fluid samples. We further validated some of our findings using miRNA and gene enrichment analyses and the results confirmed with findings from previous studies that some of these identified miRNA/target pairs are involved in ACE2 receptor network, regulating pro-inflammatory cytokines and in immune cell maturation and differentiation.Using publicly available databases and patient-related expression data, we found that acting as a \u201cmiRNA sponge\u201d could be one explanation for SARS-CoV-2-mediated pathophysiological changes. This study provides a novel way of utilizing SARS-CoV-2 related data, with bioinformatics approaches, to help us better understand the etiology of the disease and its differential manifestation across individuals.The online version contains supplementary material available at 10.1186/s12920-022-01243-7. During the past year, the coronavirus disease 2019 (COVID-19) as a major global pandemic has taken the lives of more than 2 million people. The highly transmissible virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is responsible for the world-wide spread of COVID-19. It is a single-strand, positive-sense RNA virus which belongs to betacoronavirus genera together with SARS-CoV and MERS-CoV (Middle East respiratory syndrome coronavirus) , 4, howeMicroRNAs (miRNA) are small (21\u201322\u00a0bp) noncoding RNAs that can selectively repress the expression of target mRNA(s) through binding to targets\u2019 3\u2032 untranslated regions (UTRs) . Human mIn this study, we utilized publicly available databases, including RNA-sequencing (RNA-seq) data and single-cell RNA-sequencing (scRNA-seq) data of COVID-19 patients and healthy controls, and multiple web-services to explore the potential role of SARS-CoV-2 as a miRNA sponge. The process identified multiple candidate miRNA-gene pairs that are likely affected through SARS-CoV-2's miRNA sponge mechanism. Our study can shed new light on the pathogenesis of SARS-CoV-2 infection through the exploration of its potential role as a miRNA sponge.Nine hundred potential viruses targeting miRNAs (VTMs) for the SARS-CoV-2 genome passed the score filter (target score\u2009\u2265\u200950) and 91 miRNAs were categorized into high-confidence VTMs (target score\u2009\u2265\u200990). We identified 24 miRNA families that were over-representated in these VTMs . This highlighted that these mild-to-moderately upregulated genes in our candidate gene list are more likely to be regulated by VTMs. With the presence of SARS-CoV-2, these VTMs that can simultaneously target the virus genome and regulate human genes are likely to have insufficient expression to maintain their normal functionalities. This could result in upregulation of their target genes and would lead to downstream perturbation of normal cellular functions. This miRNA sponge effect is a plausible explanation for the observed phenomenon based on patient RNA-seq data, a high quality in-silico miRNA prediction tool and experimentally validated miRNA-gene interactions in human. The complete list of VTM-gene pairs that are potentially affected by miRNA sponge effect are available in Additional file We further examined if the targets of the high-confidence VTMs are significantly enriched in our candidate gene list (upregulated genes) compared to our background gene list (downregulated genes). Using experimentally validated miRNA-target pairs from miRTarBase, we observed 59 VTMs out of the 373 potential miRNAs that can target genes from candidate gene list and only 28 VTMs out of 330 miRNAs that can target genes from background gene list. Even though the two lists have similar number of targeting miRNAs , our candidate gene list which contained genes that are upregulated in COVID-19 patients showed significantly more binding VTMs . Additionally, these 5 validated genes were also identified in our previous analyses. Based on these multiple lines of evidence, namely gene upregulation, miRNA downregulation, and our bioinformatics investigations, these five VTM-gene pairs are highly likely to be affected by the SARS-CoV-2 virus through miRNA sponge effect.To pinpoint which VTMs are more likely affected by the SARS-CoV-2\u2019s miRNA sponge effect, we obtained 44 significantly upregulated or downregulated miRNAs between SARS-CoV-2 infected and control Calu-3 cells from an independent study . Among tAdditionally, we identified the differentially expressed genes between COVID-19 patients and patients with other upper airway virus infections . Among thttps://github.com/zhangzlab/covid_balf), we annotated all major cell types including epithelial cells, macrophages, neutrophils, myeloid dendritic cells (mDC), plasmacytoid dendritic cells (pDC), mast cells, natural killer (NK) cells, T and B cells. Next, cell-type specific analyses of DEGs were performed bewteen severe COVID-19 patients and moderate COVID-19 patients to examine if miRNA sponge effect could act as an explanation for severe COVID-19 cases. Four of the cell types showed DEGs that passed our fold-change filters, namely B, macrophages, mDCs and T cells samples from bronchoalveolar lavage fluid (BALF) . FollowiIn this study, we suggest that the SARS-CoV-2 virus could act as a miRNA sponge to disrupt the normal miRNA regulatory pathways. Based on multiple lines of evidence, we identified 5 high-confidence VTM-gene pairs that are most likely affected by miRNA sponge effect mediated by the SARS-CoV-2 virus. Some identified pathways such as the cytokine signaling pathway could be one explanation for ARDS and differential disease severity between COVID-19 patients. Additionally, we explored the possibility that miRNAs sponged by the virus could inhibit viral replication at the same time which complicated the functional role of SARS-CoV-2. Through the scRNA-seq analyses, we found out that hsa-miR-302c-5p and hsa-miR-16-5p could potentially affect SARS-CoV-2 infection through modulating ACE2 receptor related network.Since our study and other currently available studies adopted bioinformatics approaches to investigate this issue, one key direction for future studies is to validate our findings including candidate VTM-gene pairs and affected biological pathways using experimental approaches. This can provide invaluable insights into the mechanism of COVID-19 and even other types of viral infections. Additionally, more patient data, especially data with greater heterogeneity, can be helpful in increasing the power to identify key and/or population specific regulatory pathways involved in the sponge mechanism. Further investigations of scRNA-seq data can potentially identify more cell-type specific or housekeeping VTMs. Other bioinformatics studies may use the miRNAs and especially VTMs to design and develop miRNA markers to perform patient risk assessment. In summary, we expect findings reported in our study could provide a valuable starting point for future experimental and functional validations to help us better understand and fight against COVID-19.https://www.ncbi.nlm.nih.gov/nuccore/mn908947.3.A brief summarization of the workflow for this study was shown in Fig.\u00a0http://mirdb.org) to predict potential miRNA target sites in the SARS-CoV-2 genome [http://www.targetscan.org/cgi-bin/targetscan/data_download.vert72.cgi [We used the custom prediction function from miRDB [2 fold change between 0 and 1 (ratio between gene expression in cases over gene expression in controls) [p value of less than 1\u2009\u00d7\u200910\u20136 was used to obtain a high-confidence candidate gene list that was upregulated. Since our focus was mildly-to-moderately upregulated genes, we set a more stringent p value threshold to ensure the quality of the identified DEGs. For our background gene list, we selected genes with fold-change between 0.5 and 1, or equivalently a log2 fold change between -1 and 0, with the same p value cut-off to match their magnitude of change with our candidate gene list. The final transcript names identified from previous step were converted to HUGO Gene Nomenclature Committee (HGNC) gene symbols using R package biomaRt [Differentially expressed genes (DEGs) between COVID-19 patients and no-symptom controls were identified using the gene count data and R package DESeq2 (q2.html) . Based oontrols) . An FDR biomaRt , 22. DEGhttp://mirtarbase.cuhk.edu.cn/php/index.php) [To identify high-confidence miRNA targets for our candiate gene list, we retrieved experimentally verified miRNA-target pairs from miRTarBase (dex.php) , 24 whicTo check if VTMs are enriched in our candidate gene list compared to the background gene list, we performed enrichment anlysis for the number of VTMs in each of these lists. Specifically, using the miRNA-target pairs reported in miRTarBase, the number of VTM-target pairs in our candidate gene list that are reported in miRTarBase (complete VTM-target pairs) and the number of VTM-target pairs in our background gene list that are reported in miRTarBase (background VTM-target pairs) were compared. Chi-squred test statistics was used to claim test significance.. The intersection of these upregulated miRNAs and our candidate VTMs were identified (refined VTM-target pairs).To check which of our candidate VTMs are more likely to be affected by miRNA sponge mechanism, we retrieved a list of downregulated miRNAs in human lung epithial cells between COVID-19 patients and normal controls from a recent study . The inthttps://ccb-compute2.cs.uni-saarland.de/mieaa2/) [p value\u2009<\u20090.05 and a minimum interaction number of 10. Additionally, for our refined VTM-target pairs, we checked if any of these pairs have been previously reported or can be mechanistically associated with pathways related to viral infection through literature search. The VTM-target names and disease terms such as \"COVID-19\", \"SARS-CoV-2\" and \"Viral infection\" were submitted as keywords to google scholar to identify related studies [To better understand the functional impact of our complete VTM-target pairs, we performed miRNA over-representation analysis using the miEAA web-service (mieaa2/) . All VTMmieaa2/) , KEGG pamieaa2/) , target studies .https://github.com/zhangzlab/covid_balf. Cell type annotations were retrieved from the same GitHub page with file name all.cell.annotation.meta.txt. After cell type annotation, DEGs between severe COVID-19 cases and moderate cases for each cell types were identified using the Wilcoxon Rank-Sum test. Again, we limited the expression change of candidate genes to be between 1 and 2 folds, or equivalently a log2 fold change between 0 and 1 (ratio between gene expression in severe cases over gene expression in moderate cases) with Bonferroni corrected p value\u2009<\u20090.05. Potential VTMs associated with these marker genes were identified as previously described.We retrieved single-cell RNA-sequencing (scRNA-seq) data on bronchoalveolar lavage fluid (BALF) samples from a recent study . A totalAdditional file 1. 26 miRNA families enriched in VTMs.Additional file 2. 218 candidate VTM-gene pairs.Additional file 3. 885 genes significantly enriched by targeting VTMs.Additional file 4: miRWalk pathways identified using candidate VTMs.Additional file 5: VTM-gene pairs by cell type identified from single-cell RNA-seq based analyses."} +{"text": "Chemoimmunotherapy combinations have transformed the treatment landscape for patients with triple-negative breast cancer (TNBC). However, the discovery of immune-related biomarkers is needed to optimally identify patients requiring the addition of immune-checkpoint inhibitors (ICIs) to chemotherapy. In this study, we identified immune-related gene signatures via exploratory subgroup discovery algorithm that substantially increase the odds of partial remission for TNBC patients on anti-PD-L1+chemotherapy regimen. We have also uncovered distinct cell populations for TNBC patients with various treatment outcomes. Our framework may result in better risk stratification for TNBC patients that undergo chemoimmunotherapy and lead to overall improvement of their health outcomes in the future.Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with limited therapeutic options. Although immunotherapy has shown potential in TNBC patients, clinical studies have only demonstrated a modest response. Therefore, the exploration of immunotherapy in combination with chemotherapy is warranted. In this project we identified immune-related gene signatures for TNBC patients that may explain differences in patients\u2019 outcomes after anti-PD-L1+chemotherapy treatment. First, we ran the exploratory subgroup discovery algorithm on the TNBC dataset comprised of 422 patients across 24 studies. Secondly, we narrowed down the search to twelve homogenous subgroups based on tumor mutational burden , relapse status (disease-free or recurred), tumor cellularity , menopausal status (pre- or post) and tumor stage . For each subgroup we identified a union of the top 10% of genotypic patterns. Furthermore, we employed a multinomial regression model to predict significant genotypic patterns that would be linked to partial remission after anti-PD-L1+chemotherapy treatment. Finally, we uncovered distinct immune cell populations for TNBC patients with various treatment outcomes. CD4-Tn-LEF1 and CD4-CXCL13 T-cells were linked to partial remission on anti-PD-L1+chemotherapy treatment. Our informatics pipeline may help to select better responders to chemoimmunotherapy, as well as pinpoint the underlying mechanisms of drug resistance in TNBC patients at single-cell resolution. Triple-negative breast cancer (TNBC) occurs in about 10 to 20% of diagnosed breast cancers and defined by the absence or minimal expression of estrogen receptor (ER), progesterone receptor (PR) and epidermal growth factor receptor 2 (HER2) . AlthougTNBC, unlike other breast cancer subtypes, has high tumor mutational burden (TMB), which has been correlated with responsiveness to immune checkpoint inhibitors (ICIs) . Indeed,In this work we determined homogenous TNBC subgroups based on both phenotypic and genotypic parameters using exploratory subgroup mining. We have also identified significant predictors that increase chances of partial remission in TNBC patients on chemoimmunotherapy treatment using multinomial regression model on TNBC scRNA-seq dataset. Lastly, we uncovered distinct immune cell populations for TNBC patients with various treatment outcomes. We interpreted our results using biomedical knowledge, including findings from existing clinical trials, immunohistochemistry experiments and functional characterization of specific genes. The proposed informatics pipeline may assist health care professionals in the selection of chemoimmunotherapy responders, as well as determine the underlying causes of drug resistance in TNBC patients at a single-cell level and resolution. In this study we employed two datasets. Each dataset consisted of multiple phenotypic and genotypic (continuous only) variables. Each categorical variable was labeled based on the National Comprehensive Cancer Network (NCCN) Guidelines in Oncology . For exaThe first TNBC dataset comprised of 422 patients. These patients were selected from 24 breast cancer studies available at the cBioPortal platform . The finPactilaxel) or chemoimmunotherapy treatment (Paclitaxel with Atezolizumab) [The second TNBC dataset consisted of scRNA-seq profiles for 22 TNBC patients that underwent chemotherapy (lizumab) . For thiOur informatics pipeline has three modules: (1) exploratory subgroup discovery, (2) inference module based on multinomial regression model and (3) immune cell populations discovery. Our goal was two-fold: (1) to identify significant genes from exploratory subgroup discovery that increase odds of having partial remission after anti-PD-L1+chemotherapy treatment and (2) to uncover unique immune cell populations for TNBC subgroups with various treatment outcomes.The main goal of exploratory subgroup discovery module was to determine homogenous patient subgroups based on expanatory phenotypic characteristics . The union would represent a set of a collection of genotypic patterns, where each element would not be repetitive. These genotypic patterns were used as covariates for the multinomial regression model in the next section. First, we ran the exploratory subgroup discovery algorithm on the TNBC dataset described in The multinomial regression model on scRNA-seq TNBC dataset was able to identify significant predictors from exploratory subgroup discovery results that increase odds of having partial remission after anti-PD-L1+chemotherapy treatment versus progressive disease after chemotherapy . p = 0.005), and odds ratios for pCR per mut/MB were 2.06 (95% CI 1.33\u20133.20) among all patients, 1.77 (95% CI 1.00\u20133.13) in the Durvalumab arm, and 2.82 (95% CI 1.21\u20136.54) in the chemotherapy arm. Interestingly, the association between pCR and TMB was more pronounced in patients treated with chemotherapy alone. The KEYNOTE-119 trial evaluated metastatic TNBC patients treated with Pembrolizumab monotherapy versus chemotherapy. The positive association was observed between TMB and clinical response to Pembrolizumab but not to chemotherapy . ORR and hazard ratio (HR) for OS also suggested a trend towards increased benefit with Pembrolizumab versus chemotherapy in TNBC patients with high TMB. This clinical trial was constrained by the small sample size and low number of TMB-high cases.Next, we highlight the importance of identified phenotypic features from In terms of relapse status, one study suggested that rapid versus late relapse in TNBC might be characterized by unique clinical and genomic features . Both \u2018rIn connection to menopausal status, TNBC was observed primarily in postmenopausal patients . The oveWe had also discovered novel phenotypic features in TNBC subgroups, such as tumor cellularity and tumor stage. The evaluation of tumor cellularity, defined as the percentage of invasive tumor comprised of tumor cells, may represent an informative histologic measure of the differential response of TNBC to chemoimmunotherapy. To classify the severity of a malignant disease in a particular patient, the tumor staging system is employed during the course of disease. This system is essential in optimizing cancer patients treatment options and their risk stratification. Therefore, these features can be important in the design and analysis of intervention studies, including randomized clinical trials, to better assess their prognostic utility for TNBC patients.This section described immune cell populations that were discovered in scRNA-seq TNBC data based on genotypic patterns from exploratory mining stage. We interpreted our results using biomedical knowledge, including findings from existing clinical trials, immunohistochemistry experiments and functional characterization of specific genes. The summary of our findings is presented in + T-cells (Tprf-MKI67) were exclusively present in TNBC patients achieving progressive disease after chemotherapy. Based on literature findings, the expression of MKI67 gene was significantly correlated with lymph node metastases, tumor invasion and adverse survival outcome in TNBC [p-value = 0.011) [The proliferative MKI67 in TNBC . In addi= 0.011) . Therefo+ T-cells express very high amounts of PD-1 and other co-stimulatory and inhibitory receptors. Therefore, they instrumental to B-cells for efficient antibody responses and their presence in tumor samples is often correlated with a better outcome in patients with solid tumors [+ T-cells, na\u00efve LEF1+ T-cells (Tn-LEF1) were also linked to a favorable response to both anti-PD-L1+chemotherapy and chemotherapy. In a recent study, the magnitude of lymphocytic infiltration was assessed by a four-gene signature\u2014HLF, CXCL13, SULT1E1 and GBP1, which was indicative of favourable outcome in TNBC after neoadjuvant therapy. This signature may help to identify early stage TNBC patients and being a novel prognostic biomarker of this aggressive disease [CD4-Tn-LEF1 and CD4-CXCL13 T-cells were linked to partial remission after anti-PD-L1+chemotherapy treatment. Importantly, these CD4d tumors . Based od tumors ,32. In a disease .+ T-cells (Tact-IFI6) were linked to progressive disease after chemotherapy. The poor metastasis-free survival in breast cancer patients was linked to upregulation of mitochondrial antiapoptotic protein IFI6 that might be involved in regulation of mitochondrial ROS production [The activated IFI6oduction . Therefo+ follicular B-cells (Bfoc-MKI67), NEIL1+ follicular B-cells (Bfoc-NEIL1) and MKI67+ memory B-cells (Bmem-MKI67) were exclusively present in TNBC patients with partial remission after anti-PD-L1+chemotherapy treatment. Based on biomedical literature, follicular B-cells was associated with favorable outcomes for TCGA patients with breast cancer [The MKI67t cancer . The na\u00eft cancer . In regat cancer . Reductit cancer . Hence, + B-cells (pB-IGHG1) were linked to progressive disease after chemotherapy treatment. In TNBC, the expression of IGHG1 indicated the most significant prognostic value compared to trivial clinicopathological parameters [Plasma IGHG1rameters . Intrigurameters . These d+ macrophages (macro-MMP9) were exclusively present in TNBC patients with partial remission after anti-PD-L1+chemotherapy treatment. The literature search revealed that MMPs have a intricate role in cancer progression and may exert both pro- and antitumorigenic activities [The MMP9tivities . Althougtivities , clinicativities . Indeed,tivities . In anottivities . TherefoThe macro-CCL2, macro-CX3CR1, macro-IFI27, macro-IGFBP7, macro-IL1B9, macro-MGP and macro-SLC40A1 cells were exclusively present in TNBC patients achieving progressive disease after chemotherapy. Based on biomedical findings, CCL2 expression in breast carcinomas was highly associated with macrophage infiltration, and its expression was correlated with poor prognosis in breast cancer patients . In anot+ dendritic cells (cDC1-CLEC9A), macro-CFD, macro-FOLR2, macro-MKI67, macro-SPP1, macro-TUBA1B, FCN1+ monocytes (mono-FCN1), mono-S100A89 and mono-SMIM25 cells were linked to progressive disease after chemotherapy treatment. Notably, CFD functioned as an enhancer of tumor proliferation and cancer stem cell properties in breast cancers [+/basal-like subtypes of breast cancer. Therefore, myeloid cell populations expressing CFD, SPP1 and S100A89 might be crucial biomarkers of poor treatment response in TNBC.The CLEC9A cancers . In anot cancers . Interes+ group 2 innate lymphoid cells (ILC2-CNOT2) were exclusively present in TNBC patients achieving partial remission after anti-PD-L1+chemotherapy treatment. Indeed, ILC2s involved in both anti-tumor and pro-tumoral immunity in a variety of human cancers [The CNOT2 cancers . In term cancers . In addi cancers , CXCL1L/ cancers .+ group 1 innate lymphoid cells (ILC1-ZNF683) cells were exclusively present in TNBC patients achieving progressive disease after chemotherapy. The biomedical literature demonstrates that ILC1 cells involved in inhibiting the antitumoral immune response, enabling the differential tumor infiltration of ILC1 cells in patients to improve the levaraging of immunity in cancer therapies [The ZNF683herapies . However+, IL7R+ and VCAM1+ innate lymphoid cells can help determine prognosis for breast cancer patients.ILC3-AREG and ILC3-IL7R cells were linked to partial remission after anti-PD-L1+chemotherapy treatment. It had been shown that ILC3-IL7R could predict a favorable response to both treatment regimens, indicating its potential role in effective antitumor immunity . In cont+ macrophages (macro-MMP9) were exclusively present in TNBC patients with partial remission after anti-PD-L1+chemotherapy treatment, while heterogenous population of macrophages, including macro-CCL2, macro-CX3CR1, macro-IFI27, macro-IGFBP7, macro-IL1B9, macro-MGP and macro-SLC40A1 cells were exclusively present in TNBC patients achieving progressive disease after chemotherapy. Finally, group 3 innate lymphoid cells (ILC3-AREG and ILC3-IL7R) were linked to partial remission after anti-PD-L1+chemotherapy treatment, while ZNF683+ group 1 innate lymphoid cells (ILC1-ZNF683) cells were exclusively present in TNBC patients achieving progressive disease after chemotherapy. Each of these cell populations have distinctive genetic markers that could be useful therapeutic targets for chemoimmunotherapy.The analysis of the TNBC scRNA-seq data revealed distinct immune cell populations that are linked to either partial remission after anti-PD-L1+chemotherapy or progressive disease after chemotherapy only. In terms of T-cells, CD4-Tn-LEF1 and CD4-CXCL13 T-cells were linked to partial remission after anti-PD-L1+chemotherapy treatment, while Tact-IFI6 T-cells were linked to progressive disease after chemotherapy. The na\u00efve B-cells, memory B-cells and follicular B-cells were mainly enriched in tumors responsive to chemoimmunotherapy but not in tumors responsive to chemotherapy treatment. The MMP9The role of T follicular helper and B-cell crosstalk in tumor immunity has been extensively studied over the last decade. Accumulating evidence suggests that tumor infiltrated lymphocyte (TIL) subpopulations constitute of both suppressive (pro-tumor) or effector (anti-tumor) phenotypes whose functions are influenced by the surrounding TME . NaturalDespite of significant survival advantages that could be achieved after treatment with chemoimmunotherapy, most TNBC patients would not benefit. Therefore, more and more attention has been paid to the identification and development of biomarkers for the response of chemoimmunotherapy in recent years. Our informatics pipeline identified novel phenotypic and genotypic predictors in unsupervised manner that indicative of favorable outcome after chemoimmunotherapy. These predictors could be important biomarkers in the design and analysis of intervention studies and ultimately could help to optimize therapy decisions for TNBC patients. In addition, it may help to select better responders to chemoimmunotherapy, as well as pinpoint the underlying mechanisms of drug resistance in TNBC patients at single-cell resolution.To tackle patient heterogeneity, chemoimmunotherapy combinations represent a feasible alternative for TNBC patients. However, matching patient subgroups to effective treatments that increase their chance of survival remains a challenging endeavor. In this work, we augmented our exploratory subgroup discovery algorithm to identify TNBC subpopulations that may benefit from chemoimmunotherapy. Specifically, we identified immune-related gene signatures that increased the likelihood of partial remission after anti-PD-L1+chemotherapy regimen versus progressive disease after chemotherapy in TNBC patients. Our novel informatics pipeline identified immune cell populations that associated with various treatment outcomes in TNBC. We also showed the importance of TMB and menopausal status among the investigated TNBC subgroups. The potential limitations include the usage of two disjoint datasets and the absence of outcome variable for immunotherapy outcomes in TCGA datasets. Further validation of our computational results in wet-lab studies would be a significant step toward improving survival outcomes for TNBC patients."} +{"text": "Chromosomal t translocation is commonly found in adenoid cystic carcinoma (ACC) of the salivary gland. This genetic rearrangement results in the fusion of MYB and NFIB genes. Despite the frequent occurrence of t translocation and MYB-NFIB gene fusion, the nature of chimeric MYB-NFIB proteins and their potential relevance in the development and behavior of ACC remains poorly understood. The lack of validated ACC cell lines, harboring the t translocation with defined MYB-NFIB fusion protein, has restricted fusion-specific functional studies. Hence, we sought to characterize and establish in vitro and in vivo models of a MYB-NFIB fusion protein expressing system in ACC. Defining the nature and functional aspect of MYB-NFIB fusion proteins may not only improve our understanding of the disease but also contribute to the identification of molecular targets that are druggable and can be developed for future therapeutic purpose.Adenoid cystic carcinoma (ACC) is the second most common cancer type arising from the salivary gland. The frequent occurrence of chromosome t translocation leading to the fusion of MYB and NFIB transcription factor genes is considered a genetic hallmark of ACC. This inter-chromosomal rearrangement may encode multiple variants of functional MYB-NFIB fusion in ACC. However, the lack of an ACC model that harbors the t translocation has limited studies on defining the potential function and implication of chimeric MYB-NFIB protein in ACC. This report aims to establish a MYB-NFIB fusion protein expressing system in ACC cells for in vitro and in vivo studies. RNA-seq data from MYB-NFIB translocation positive ACC patients\u2019 tumors and MYB-NFIB fusion transcript in ACC patient-derived xenografts (ACCX) was analyzed to identify MYB breakpoints and their frequency of occurrence. Based on the MYB breakpoint identified, variants of MYB-NFIB fusion expression system were developed in a MYB-NFIB deficient ACC cell lines. Analysis confirmed MYB-NFIB fusion protein expression in ACC cells and ACCXs. Furthermore, recombinant MYB-NFIB fusion displayed sustained protein stability and impacted transcriptional activities of interferon-associated genes set as compared to a wild type MYB. In vivo tumor formation analysis indicated the capacity of MYB-NFIB fusion cells to grow as implanted tumors, although there were no fusion-mediated growth advantages. This expression system may be useful not only in studies to determine the functional aspects of MYB-NFIB fusion but also in evaluating effective drug response in vitro and in vivo settings. Adenoid cystic carcinoma (ACC) is a common malignancy of salivary glands ,2. GenerChromosomal translocations are commonly found in many types of hematologic and solid cancers . They coDespite their frequent occurrence with functional potential that may be crucial in the progression and behavior of ACC, the activities of chimeric MYB-NFIB fusion proteins remain largely unknown. A number of established ACC patient-derived-xenografts (ACCX) are readily available ,19, howeHere, we describe the development of an inducible MYB-NFIB fusion model based on MYB breakpoint sites identified from t translocation positive patient-derived xenograft and patients\u2019 tumors of ACC. This expression system may be useful not only in studies to understand the role of MYB-NFIB fusion but also help in evaluating approaches to test effective drug response in vitro and in vivo models.Patient-derived xenografts of ACC obtained from Dr. C.A. Moskaluk were passaged in mice as previously described , and Dr.ACC patient samples and RNA sequencing (RNA-Seq) have been described in our previous report . In thisTo identify MYB breakpoints from the bulk of RNA-seq data, we combined NeoFuse pipeline (version 1.1.1) and Arriba command-line analysis tools ,25. Briewt) and MYB-NFIB expression in ACCXs, PCR analysis was performed with Taq polymerase using Mex6F (MYB exon 6 forward), and Mex9R (MYB exon 9 reverse) primers, or with Mex6F and Nex11R (NFIB exon 11 reverse) primers set. For positive reference, we used plasmid HA-tagged MYB (MYBwt variant 2) and HA-MYB-NFIB (MYB exon 8-NFIB exon 10 fusion) constructs [Total RNA extracted from ACCXs was used for reverse transcription into cDNA using Superscript First-Strand Synthesis kit . To confirm detection of wild type MYB (MYBnstructs .TM PCR Supermix High-Fidelity (Invitrogen) with MYB-orf-F (located upstream of MYB start codon) and NFIB-orf-R (located downstream of NFIB stop codon) primers set. PCR-amplified bands were gel-extracted, purified, and analyzed by Sanger sequencing . Sequence-verified PCR-product or HA-tagged MYBwt plasmid cDNA were then used as templates for second-round PCR for cloning MYB-NFIB fusion or MYBwt into Gateway pDONR vector using attMYB and attNFIB primers. Following transformation, individual positive pEntry clones (pDONR with inserted PCR fragment) were selected and re-verified by sequencing.For identification of MYB-NFIB transcript, reverse transcribed cDNA from ACCX11, ACCX29 and ACCX20M1 was used. Full-length fusion transcripts were amplified using Platinumwt inserts were recombined into a pLVX-TetOn-hygro-C-3xFLAG expression vector using Gateway cloning system . The various constructs were further verified by sequencing. FLAG-tagged MYBwt or MYB-NFIB constructs along with viral elements were transfected into HEK293T cells to generate lentiviral particles. Virus-transduced ACC stable cell lines were generated after selection in hygromycin (200 \u03bcg/mL). To induce expression of FLAG-tagged MYBwt or MYB-NFIB constructs, stable ACC-01 and HACC-2A cells were cultured in media containing doxycycline (0.5 \u03bcg/mL). All Gateway cloning lentiviral vectors were obtained from Dr. Nevan Krogan\u2019s Lab . Detailed primer information is provided in Next, pEntry constructs with respective MYB-NFIB and MYBwt or MYB-NFIB fusion protein were detected with anti-MYB (N-terminus epitope) D2R4Y, cat #12319 ; anti-MYB (N-teminus epitope) EP769Y, Ab45150 , anti-MYB (C-terminus epitope), D-7, Sc-74512 , or anti-FLAG, M2 . For protein stability assay, time chase experiments were performed in cells expressing MYBwt or MYB-NFIB fusion. Briefly, cells were cultured in doxycycline (dox) containing media for 24 h to induce the expression of MYBwt or MYB-NFIB fusion. Cells were then refreshed with media without dox for up to 6 hr. Alternatively, in cycloheximide (CHX) chase experiment, dox-treated cells were incubated in media containing CHX (100 \u03bcg/mL) to prevent synthesis of new protein. Lysates were then prepared and analyzed by immunoblotting followed by densitometry to determine half-life of MYBwt and MYB-NFIB fusion protein.Protein extracts prepared from fresh or frozen ACCX tissues or cell cultures were resolved in SDS-polyacrylamide gel and analyzed by immunoblotting . MYBwt owt, M8:N10 (MYB-NFIB) and empty vector-transduced ACC-01 cells (control). RNA sample integrity was assessed with an Agilent Bioanalyzer, and corresponding RNA libraries were constructed using TruSeq strand mRNA Library Prep kit (Illumina). The RNA libraries were sequenced using the Illumina HiSeq4000 system with 50 base paired end reads . Sample sequences were aligned to the Ensembl Human GRCh38.78 reference genome using STAR [Total RNA was extracted from four replicates each of doxycycline-induced MYBing STAR after saThe count matrix produced by STAR was used for the RNA-seq analysis. Low abundance genes, defined as having fewer than 10 counts in less than three samples, were excluded. Pairwise differential gene expression analyses of sample types were then performed using the DESeq2 R/Biocon\u2212\u0394Ct or 2\u2212\u0394\u0394Ct equation, respectively.Total RNA purified from dox-induced cell lines or ACCX were reverse transcribed to cDNA with iScript Reverse Transcription mix and amplified with specific primers using iTwt or MYB-NFIB fusion constructs, were implanted subcutaneously into the flanks of NOD.Cg-Prkdcscidtm1WjlIl2rg/SzJ mice . Mice were fed with doxycycline (0.5 mg/mL doxycycline and 5% sucrose) containing water, and tumor growth activity was monitored. After 2.5\u20133 months, mice were sacrificed, and tumors were harvested for further analysis. Mice were housed and cared in animal facilities as approved by the UCSF Animal Care and Use Committee.Parental ACC-01 cells, or cells expressing either FLAG-tagged MYBData presented are representative of replicate experiments wherever indicated. Graphical representations were performed in GraphPad Prism v/9.In a previous study, we found that 44% of tumor samples from ACC patients harbored the MYB-NFIB gene translocation . To idenOf the ten tumors data analyzed, MYB breakpoint occurred at exon 8 for tumors HN332PT, HN348PT, HN325PT and HN312PT, and at exon 15 for tumor samples HN335PT, HN341PT, HN345PT, and HN317PT. A MYB breakpoint at exon 11, and another at exon 16 were detected in tumor samples HN338PT and HN344PT, respectively. Analysis also revealed that all four cases detected with MYB breakpoints at exon 8 contained closely related variants with breakpoints located at nine nucleotides upstream of the C-terminal end of exon 8. Furthermore, tumors HN348PT and HN325PT also showed the presence of an additional variant with a MYB breakpoint occurring at exon 7. These findings indicate that MYB breakpoint occurs predominantly downstream of exon 8. Although not determined in this study, NFIB breakpoint sites linking to MYB detected through Arriba analysis occurred mostly with the last two exons .wt) and MYB-NFIB fusion (C8:N10) constructs were included for reference controls [To examine the expression of MYB-NFIB fusion in ACC patient-derived xenografts (ACCXs), we performed a semi-quantitative RT-PCR analysis of RNA extracted from a panel of nine ACCXs that are known to harbor or lack the MYB-NFIB translocation. Initially, a forward primer Mex6F (MYB exon 6) with either Nex11R (reverse primer from NFIB exon 11) or Mex9R (MYB exon 9) were used for the analysis. Plasmid cDNA of HA-tagged wild type MYB were then cloned into a dox-inducible FLAG-tagged expression system and t translocation, respectively [wt. More importantly, ACC-01 cells are MYB-NFIB fusion negative and with HACC-2A, the fusion can only be verified using nested-PCR techniques, such that on Western blot, we could not identify any appreciable MYB-NFIB fusion protein. Thus, we utilized these two authenticated ACC cell lines for ectopic MYB-NFIB fusions expression studies. Results show that MYBwt and MYB-NFIB fusion proteins were efficiently expressed following dox induction as detected with either anti-FLAG or N-terminus epitope MYB antibody , 1815 genes were found to be significantly differentially expressed between the MYB-NFIB and MYBwt samples. We filtered this set of differentially expressed genes further by applying a fold-change cutoff of two, using MYBwt samples as reference, and retained 215 genes after removing unannotated genes or harbor (ACCX11 and ACCX20M1) the MYB-NFIB fusion. Quantitative PCR results show that a number of the target genes detected appear modest and at a comparable level across ACCX9, ACCX38M1 and ACCX11, while a relatively higher expression pattern of several genes were observed in ACCX20M1 E. Althouwt and parental ACC-01 cells in two mice groups implanted with either 0.5 \u00d7 106 or with 1.0 \u00d7 106 cells. Data showed that in the mice group implanted with 0.5 \u00d7 106 cells, no tumors were formed either with the MYB-NFIB fusion, MYBwt or parental control cells during an 11-week monitoring time period was compared with MYBe period A.6 cells, palpable tumors were formed in parental (3/8), MYB-NFIB fusion (1/6) and MYBwt (1/6) at about 5.2 weeks. And by the end of 8 weeks, tumor formation occurred with parental control (87.5 %), MYBwt (83.3%) and MYB-NFIB fusion (75%) cells. Evaluation of the tumors by Western blotting verified the positive expression of the dox-induced MYB-NFIB fusion and MYBwt proteins translocation, based on which MYB-NFIB expressing system was established for in vitro and in vivo xenograft study model.Our analysis revealed the presence of multiple MYB junctions and indicated that MYB breakpoint location occurs primarily downstream of exon 8. These observations appear consistent with report by others showing the heterogeneity in MYB breakpoints found in ACC. For example, breakpoints detected in ACC from Drs. Caulin and El-Nagar\u2019s group involved MYB exons 8\u201311, 13\u201316 ,37. SimiMYB, a transcription regulator essential in normal tissue homeostasis, developmental process, and as oncogenes in leukemias and other solid cancers, consists of an N-terminal DNA-binding domain (DBD), a centrally located transcriptional activation domain (TAD) and a C-terminus negative regulatory domain (NRD) ,41. EngaThe validated ACC-01 and HACC-2A cells utilized in this work, do not either harbor the t translocation or expreOur study showed that ectopic MYB-NFIB expression upregulated several genes associated in interferon-related signaling pathway. This observation is based on the M8:N10 fusion construct and a single PDX (ACCX20M1) harboring identical fusion variant, and we note that whether such observed transcriptional effects depend on specific fusion variant remains to be ascertained. Nevertheless, it is possible that the substitution of MYB C-terminus with the short NFIB fragment modifies MYB-NFIB function leading to downstream transcriptional activities that are distinct from full-length MYB. Previous work involving domain swapping, N-terminal truncation, and sequential deletion of C-terminal region of MYB has shown to yield unique gene expression profiles ,47,48. Twt and MYB-NFIB cells, suggesting that MYB-NFIB expression by itself does not enhance tumor growth activities. This observation corresponds with our in vitro results showing that MYB-NFIB expression provided no cell growth advantage. Andersson et al. have also reported that MYB variants or MYB-NFIB fusion overexpression is not sufficient for tumor growth and have suggested requiring supplemental signaling mechanisms for tumor formation [We finally evaluated MYB-NFIB expression system and its capacity to promote tumor growth in vivo. When cells were implanted subcutaneously into the flanks of mice, the efficiency of tumor growth formation was comparable between parental, MYBormation . NonetheWe describe here an inducible MYB-NFIB fusion expression system based on MYB breakpoints identified from primary ACC patient and patient-derived xenograft tumors. Currently, the availability of a reliable ACC cell line model that express the various MYB-NFIB fusion for functional evaluation and other critical pre-clinical studies are limited. We believe that the described expression system may be useful not only in studies to further understand the functional aspects of MYB-NFIB fusion but also crucial in determining the impact of MYB-NFIB fusion in drug response studies."} +{"text": "An imbalance in host proteases has been implicated in inflammatory bowel disease (IBD). Recent evidence implicates microbial proteolytic activity (PA) in ulcerative colitis but whether it also plays a role in Crohn\u2019s disease (CD) remains unclear.We therefore investigated the colitogenic potential and underlying pathways of proteolytic CD microbiota.Nod2-/-), and Protease-Activated Receptor 2 (PAR2) cleavage resistant mice (R38E-PAR2) subjected to 2% dextran sodium sulfate in drinking water for 5 days followed by 2 days on water.Adult germ-free (GF) C57BL/6 mice were colonized with CD microbiota selected based on high (CD-HPA) or low fecal proteolytic activity (CD-LPA), and from healthy controls with LPA (HC-LPA), after which total fecal proteolytic, elastolytic and mucolytic activity were analyzed in the mice. Microbial community was assessed by 16S rRNA gene sequencing. Immune function and colonic injury were investigated by inflammatory gene expression (NanoString) and histology. Colitis severity and underlying pathways were investigated in C57BL/6, Nucleotide-binding Oligomerization Domain-2 knock-out were differentially expressed between CD-LPA and CD-HPA. CD-HPA mice had lower alpha diversity, distinct microbial profiles, and higher fecal proteolytic activity compared with CD-LPA. Abundance of several beneficial species was decreased while other taxa were increased in CD-HPA compared to CD-LPA. H. hathewayi as well as the serine protease K04772 were transcriptionally increased in fecal samples from CD-HPA colonized mice. C57BL/6 and Nod2-/- mice, but not R38E-PAR2 mice, colonized with CD-HPA developed earlier and more severe colitis compared with mice colonized with CD-LPA.Colonization with HC-LPA or CD-LPA lowered baseline fecal proteolytic activity compared with GF mice, which was paralleled by lower acute inflammatory cell infiltrate. CD-HPA further increased proteolytic activity compared with GF mice. Fecal supernatants from CD-LPA or HC-LPA colonized mice had lower H. hathewayi correlates with the proinflammatory phenotype through the serine protease K04772 in this model. The results support a role of microbial PA in CD, which could constitute a biomarker for identifying patients who would benefit from anti-proteolytic therapies.CD proteolytic microbiota is proinflammatory through a PAR2 pathway. None Declared"} +{"text": "Coronavirus disease 2019 (COVID-19) is an acute complex systemic disorder caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).While SARS-CoV-2 is known to cause significant pulmonary disease, various extrapulmonary manifestations of COVID-19 have also been reported. Growing evidence suggests that COVID-19 is associated with coagulopathy leading to micro and macrovascular complications. Although in patients with COVID-19, venous thromboembolic events are more frequent, arterial thrombosis also occurs at an increased rate. These often lead to acute life-threatening ischemia, which requires urgent diagnosis and treatment. We present case reports of two patients with an abnormal thrombus formation in the thoracic aorta who recently overcame COVID-19, which led to systemic embolism and splenic infarction. Ambulatory oral factor Xa inhibitor therapy led to aortic thrombosis resolution in both patients. Coronavirus disease 2019 (COVID-19), an acute complex systemic disorder, is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). COVID-19 causes a spectrum of disorders, with various clinical manifestations. Patient condition might vary from asymptomatic or mildly symptomatic respiratory infection to severe interstitial pneumonia progressing to acute respiratory distress syndrome (ARDS), sepsis, and multiorgan failure . In addiA 58-year-old male patient was admitted to our department for left subcostal pain. The patient had a history of symptomatic COVID-19 disease one month prior the hospitalization, leading symptoms were an elevated body temperature of up to 38\u00b0C lasting three days and weakness. The patient had a history of arterial hypertension and was treated with perindopril, amlodipine, and indapamide. Laboratory tests showed an elevated C-reactive protein (CRP) levels, creatinine levels, elevated concentration of D-dimer (3.86\u2009g/mL), and fibrinogen (12.06\u2009g/L). Leukocytosis with neutrophilia was present in the blood count. Other coagulation parameters were normal. Microbiological examinations were negative. A computed tomography pulmonary angiogram (CTPA) was performed without signs of pulmonary embolism. An accessory finding of a mural thrombus on the posterior wall of the descending aorta just behind the subclavian artery was found , togetheA 62-year-old female patient with a history of arterial hypertension, gastroesophageal reflux disease, autoimmune thyroiditis, and recent COVID-19 infection presented with sudden pain and paresthesia in the left lower extremity. Laboratory tests showed slightly elevated CRP (21.6\u2009mg/L) and elevated concentration of D-dimer (1.2\u2009g/mL) and fibrinogen (4.45\u2009g/L). Other coagulation parameters were normal. Antiphospholipid and anticardiolipin antibodies were negative. The patient overcame COVID-19 infection a month before, with bilateral pneumonia requiring hospitalization and oxygen supplementation. CT angiography was performed showing a fluttering thrombus in the descending aorta and a clCOVID-19 may predispose patients to an increased risk of thrombotic complications through various pathophysiological mechanisms . AlthougAnother, yet not answered, question is how to treat COVID-19-related arterial thrombosis and systemic embolism. Therapeutic anticoagulation is being traditionally administrated in acute phase, preferring parenteral heparin or low-molecular-weight heparin (LMWH) administration in COVID-19 patients. Systemic thrombolysis could be an option in severe cases with life-threatening acute ischemia . In our"} +{"text": "Neural stem/progenitor cells derived from olfactory neuroepithelium (hereafter OE-NS/PCs) are emerging as a viable proxy and a valuable tool for translational studies on severe mental illnesses (SMI). In this respect, the use of OE-NS/PCs as a surrogate cellular model of schizophrenia (SZ) has enabled insights into cell signaling and cell cycle dynamics in this disease.We explored whether mitochondrial dysfunction, which has been already associated with SZ, may have a role in the altered proliferation pattern previously observed in OE-NS/PCs of SZ patients.OE-NS/PCs were collected from 20 patients and 20 healthy controls (Hcs) by nasal brushing, cultured in proper medium and expanded. Fresh OE-NS/PCs at passage 3 of both groups underwent BrdU proliferation assays or were frozen for later use. Mitochondrial ATP production was measured in both fresh and thawed OE-NS/PCs by using the ATPlite Luminescence Assay kit.Fresh OE-NS/PCs of patients grew at a higher rate than those of HCs , whereas the proliferation of thawed OE-NS/PCs of both groups exhibited an opposed pattern . Mitochondrial ATP production was significantly lower in OE-NS/PCs of patients than in those of HCs , regardless of freeze-thaw conditions .Mitochondrial ATP production is negatively affected in OE-NS/PCs of SZ patients as compared to those of HCs. This evidence does not differ in fresh OE-NS/PCs and OE-NS/PCs undergoing freeze-thaw cycles, which instead perturb the proliferation pattern of SZ OE-NS/PCs."} +{"text": "AI) and the mean FDG uptake of all lymphoma manifestations (mean-SUVAI). High mean-SUVAI uptake was determined separately for iPET-positive and iPET-negative patients. The endpoint was time-to-progression (TTP). There was a significant interaction of additional rituximab and mean-SUVAI in the iPET-negative group . Patients with high mean-SUVAI had significantly prolonged TTP when treated with 6xR-CHOP\u2009+\u20092\u2009R , whereas max-SUVmanual failed to show an impact of additional rituximab. In the iPET-positive group, patients with high mean-SUVAI had a significantly longer TTP with (R-)CHOP than with the Burkitt protocol . Comprehensive iPET evaluation may provide new prognosticators in aggressive lymphoma. Additional application of rituximab was associated with prolonged TTP in iPET-negative patients with high mean-SUVAI. Comprehensive iPET interpretation could identify high-risk patients who benefit from study-specific interventions.The randomized PETAL trial failed to demonstrate a benefit of interim FDG-PET (iPET)-based treatment intensification over continued standard therapy with CHOP (plus rituximab (R) in CD20-positive lymphomas). We hypothesized that PET analysis of all lymphoma manifestations may identify patients who benefitted from treatment intensification. A previously developed neural network was employed for iPET analysis to identify the highest pathological FDG uptake (max-SUV Fluorodeoxyglucose positron emission tomography/computed tomography (FDG-PET/CT) performed after 1\u20134 cycles of chemotherapy predicts the outcome in aggressive non-Hodgkin lymphoma , 3. The max methods ; Burkitt protocol(1); Supplementary Table\u00a0AI was a statistically significant prognosticator of TTP .The interaction term (treatment regime\u2009\u00d7\u2009mean-SUVAI threshold (SUV 4.78) for patient stratification in the interim PET-positive subgroup was used to group patients into those with high versus low uptake. All patients who were randomly assigned to 8x(R-)CHOP or 2x(R-)CHOP followed by the Burkitt protocol were evaluated in this analysis. In the high mean-SUVAI group, patients treated with the Burkitt protocol showed significantly shorter TTP than patients continuing on (R-)CHOP .The optimal mean-SUVAI parameter identified patients that benefitted from additional application of rituximab as treatment intensification, which could not be achieved using conventional PET metrics.The interim FDG-PET scans of the treatment intensification PETAL trial were re-analyzed in a comprehensive PET analysis to segment all lymphoma manifestations. The following principal findings arise from this analysis: (1) A fully automated analysis of interim FDG-PET/CTs from lymphoma patients is feasible. (2) The biomarkers derived from the comprehensive PET analysis are statistically significant prognosticators of TTP. (3) The mean-SUV(R-)CHOP is the standard first-line treatment for patients with aggressive lymphoma, with cure rates of 60\u201370%. In patients with (multiply) relapsed disease, several treatment options exist, such as high-dose chemotherapy with autologous hematopoietic stem cell transplantation, allogeneic transplantation, CAR-T cell therapy, immunomodulation, and others \u201317. CurrFDG-PET has a long track record of monitoring initial treatment response to systemic anti-cancer therapy , 3, 18. manual, which takes account of a single lymphoma manifestation, no statistically significant interaction of treatment intensification by additional rituximab was found in the present analysis. In contrast, for the mean-SUVAI metric, which averages the FDG uptake of all lymphoma manifestations, a statistically significant interaction with treatment intensification was observed. This indicates that the benefit of treatment intensification through additional rituximab is growing with increasing mean-SUVAI. This was corroborated by looking at patients with high mean-SUVAI who had statistically significantly longer survival when treated with two additional rituximab doses than with 6xR-CHOP alone. Interestingly, patients with high mean-SUVAI had higher baseline SUVmax compared to patients with low mean-SUVAI CHOP versus 2x(R-)CHOP followed by the Burkitt protocol, no statistically significant interaction of a PET parameter and treatment intensification was found. However, patients with high mean-SUVOur study has several limitations. First, it was a retrospective re-analysis of the prospective PETAL trial. The present analysis was not pre-planned, which might cause an observational bias. Additionally, all patients receiving 6xR-CHOP and 6xR-CHOP\u2009+\u20092\u2009R were included, but only a subfraction was truly randomized (178 of 397 patients). However, non-randomized patients receiving 6xR-CHOP or 6xR-CHOP\u2009+\u20092\u2009R were recruited using the same inclusion criteria in the beginning and at the end of the study period, respectively, which should minimize potential biases. Finally, our primary endpoint was TTP which best reflects the impact of therapy on outcome [A comprehensive analysis of interim FDG-PET in patients with aggressive non-Hodgkin lymphoma is feasible. In the PETAL trial, this novel approach identified patients who benefitted from protocol-mandated treatment intensification. This might indicate the superiority of average FDG avidity over conventional metrics restricted to the metabolically most active lesion. Future studies should evaluate the use of automated image analysis for interim PET assessment to identify patients who may benefit from a change in therapy.Supplement"} +{"text": "Tumor cell lysates (TCLs) are a good immunogenic source of tumor-associated antigens. Since whole necrotic TCLs can enhance the maturation and antigen-presenting ability of dendritic cells (DCs), multiple strategies for the exogenous delivery of TCLs have been investigated as novel cancer immunotherapeutic solutions. The TCL-mediated induction of DC maturation and the subsequent immunological response could be improved by utilizing various material-based carriers. Enhanced antitumor immunity and cancer vaccination efficacy could be eventually achieved through the in vivo administration of TCLs. Therefore, (1) important engineering methodologies to prepare antigen-containing TCLs, (2) current therapeutic approaches using TCL-mediated DC activation, and (3) the significant sequential mechanism of DC-based signaling and stimulation in adaptive immunity are summarized in this review. More importantly, the recently reported developments in biomaterial-based exogenous TCL delivery platforms and co-delivery strategies with adjuvants for effective cancer vaccination and antitumor effects are emphasized. Cancer immunotherapy is an emerging antitumor treatment technique, which works via specific antigen-mediated modulation in the patient\u2019s immune system . Convent+ T cells differentiate into various T helper cell subsets, including T helper (Th)1, Th2, Th9, Th17, and T follicular helper cells, in which Th1 cells react with antigen-presenting cells (APCs) and indirectly assist in the differentiation of CD8+ T cells into cytotoxic T lymphocytes (CTLs) by secreting a cytokine, such as interferon (INF)-\u03b3. Additionally, interleukin (IL)-2 secreted from Th1 induces the proliferation of CD8+ T cells . A. A29]. AThe induction of early necrosis using heat shock could be an alternative approach to obtaining TCLs D. A tempFor example, the heat shock treatment of three human melanoma cell lines at 42 \u00b0C for 1 h resulted in an allogeneic TCL mixture (TRIMEL) containing antigen components. The administration of TRIMEL significantly upregulated the release of the pro-inflammatory cytokine IFN-\u03b3 in DCs compared to the application of TCLs without heat shock treatment. Consequently, a previous study reported that TRIMEL showed clinical vaccination effects by developing a delayed type of hypersensitivity response in 64% of patients .The oxidation of source tumor cells prior to the preparation of TCLs could facilitate necrosis and augment the immunogenicity of the antigenic components in TCLs by increasing oxidative stress E. ThrougAs a more stable molecule than HOCl, squaric acid (SqA) has been clinically approved for the treatment of skin papillomas . SqA wasFurthermore, the incorporation of biological substances into source tumor cells could also augment TCL-mediated immune activation. One of these stimulatory substances is known to act as an agonist peptide to activate CD47 in cancer cells. Previous reports demonstrated that CD47 activation using soluble peptides derived from thrombospondin-1(TSP-1) effectively induced cell death in several types of cancer cells F 48,49],4948,49]Phyllanthus amarus induced the reactive oxygen species (ROS)-mediated apoptosis of tumor cells . . 115]. T3) and mesoporous silica NPs (MSNs), have also been used as templates for the encapsulation of proteins and peptide antigens. Lybaert et al. . . 133]. T+ T cells within the spleens and tumors of immunized mice by immune checkpoint blockade was observed. This hydrogel-based combination therapy showed superior immune modulation and anticancer efficacy compared to any single cargo delivery, demonstrating prolonged in vivo antigen-specific T cell immune responses.A similar peptide hydrogel formulation has also been applied for the delivery and in vivo localization of multiple immune stimulants. mPEG-poly (PEA)-based injectable peptide hydrogel could effectively encapsulate (1) melanoma-derived TCLs, (2) GM-CSF, and (3) dual immune checkpoint inhibitors (anti-CTLA-4/PD-1 antibody) during the spontaneous self-assembly of the polypeptide and subsequent gel formation via hydrophobic interactions . Hence, Furthermore, cryogels were al+ T cell response [Some natural compounds possess sufficient adjuvant efficacy to trigger DC activation. Previous studies have used LPS, a membrane component of Gram-negative bacterial cell walls, because of its adjuvant effect on the activation of TLR4 signaling pathways and the CD4response . Hence, response ,138,139.response . Howeverresponse ,142.+ and associated CD8+ T cell-mediated antitumor immunity.Despite LPS-mediated immune activation, a high level of immunosuppressive cytokine secretion (such as IL-10) is usually observed. Therefore, other cellular components in bacterial cells could be used for the upregulated expression of immunoactivators, with reductions in immunosuppressive cytokines to deliver the TCLs . For exaSaccharomyces cerevisiae) are another example of natural compound-based fabrication of a TCL carrier (+ APCs [The \u03b2-glucan particles (GPs) derived from yeast various experimental methods for preparing TCLs as a major immunomodulatory source, (2) TCL-mediated augmentation in DC-T cell interaction, and the subsequently induced activation of T cells, and (3) the recent progress in the biomaterial-based in vivo administration of TCLs. With the aid of co-stimulatory adjuvants, biomaterial-mediated exogenous TCL delivery could be an efficient therapeutic strategy to enhance the stability and sustained release of cargo TCLs, improve the specificity of DC targeting, and activate DCs synergistically. As a result of sufficient DC activation , antigen-specific T cell-mediated tumor suppression and vaccination can be upregulated through the dynamic interplay of immune responses. Therefore, exogenous TCL delivery techniques could be a promising treatment for enhancing the DC-mediated activation of adaptive immune responses, vaccination, and tumor-specific suppression.TCL-mediated cancer immunotherapy has been shown to involve the activation of tumor-specific CD8"} +{"text": "The COVID-19 pandemic has been marked by long-term persistence of SARS-CoV-2 in immunocompromised patients receiving B-cell-depleting therapies, with many individuals experiencing fatal COVID-19.We report an individual treated with rituximab who survived persistent COVID-19 over 9 months. SARS-CoV-2 positive RNA samples were sequenced using targeted amplicon NGS sequencing with backup sequencing on a nanopore platform. The resulting sequences were analyzed for genomic variance over time at the consensus and sub-consensus level.An individual with rheumatoid arthritis (RA) treated with azathioprine and rituximab (last dose in May 2020) was diagnosed with COVID-19 in July 2020 and admitted with pneumonia. After initial incomplete recovery, the patient had persistent infection through March 2021 and received both remdesivir and convalescent plasma (January 2021). The patient received three doses of mRNA vaccine (Pfizer BioNTech) in December 2020, April 2021, and November 2021, but was seronegative for nucleocapsid IgG in both January 2021 and March 2021; positive spike IgG developed by September 2021 (512 AU/ml) and December 2021 (621 AU/ml) . The patient recovered with new oxygen dependence (2-3L) and manages RA off B-cell depletion; they required an extended corticosteroid taper to manage organizing pneumonia and treatment for several opportunistic infections.Viral sequencing over the course of illness indicated a persistent infection with a lineage B.1.585.3 virus that accumulated 14 mutations throughout the infection. Two mutations are associated with therapy resistance and are similar to those found in other immunocompromised individuals with persistent COVID-19. Additional mutations were of unknown consequence.SARS-CoV-2 was able to establish persistent infection and accumulated mutations associated with therapeutic resistance; repeated vaccination was associated with successful resolution following repeated vaccination after stopping rituximab. Cessation of B-cell-depleting therapy was likely the critical factor in the patient\u2019s recovery, but repeated vaccination was associated with a delayed seroconversion in this patient with reversibly immunosuppression.William P. Hanage, PhD, Biobot Analytics Inc: Advisor/Consultant|Merck Vaccines: Advisor/Consultant."} +{"text": "Background: Contact tracing alone is often inadequate to determine the source of healthcare personnel (HCP) COVID-19 when SARS-CoV-2 is widespread in the community. We combined whole-genome sequencing (WGS) with traditional epidemiologic analysis to investigate the frequency with which patients or other HCP with symptomatic COVID-19 acted as the source of HCP infection at a large tertiary-care center early in the pandemic. Methods: Cohort samples were selected from patients and HCP with PCR-positive SARS-CoV-2 infection from a period with complete retention of samples at Rush University Medical Center, a 664-bed hospital in Chicago, Illinois. During this period, testing was limited to symptomatic patients and HCP. Recommended respiratory equipment for HCP evolved under guidance, including a 19-day period when medical face masks were recommended for COVID-19 care except for aerosol-generating procedures. Viral RNA was extracted and sequenced from remnant nasopharyngeal swab samples in M4RT viral transport medium. Genomes with >90% coverage underwent cluster detection using a 2 single-nucleotide variant genetic distance cutoff. Genomic clusters were independently evaluated for valid epidemiologic links by 2 infectious diseases physicians (with a third adjudicator) using metadata extracted from the electronic medical record and according to predetermined criteria (Fig. Conclusions: Using genomic and epidemiologic data, we found that most HCP COVID-19 infections were not healthcare associated. We found weak evidence to support symptomatic patient-to-HCP transmission of SARS-CoV-2 and stronger evidence for HCP-to-HCP transmission. Large genomic clusters without plausible epidemiologic links were identified, reflecting the limited utility of genomic surveillance alone to characterize chains of transmission of SARS-CoV-2 during extensive community spread.Funding: NoneDisclosures: None"} +{"text": "Objectives. This study tested the longitudinal relationship between back pain and mental health and examined the moderating role of subjective social status (SSS). Method. Community-dwelling older men from the MrOS Study provided four study visits of data collected between 2000-2016 . Back pain frequency and severity were assessed at visits 1-4. General mental health was measured at each visit by the 12-item Short Form Survey Mental Component Score . National and community SSS were assessed at visits 1 and 3 with the MacArthur Scale. Growth curve models tested longitudinal within-person change associations after accounting for the repeated measures within each person. Age was used as the primary time variable. Results. At baseline, those with higher back pain-frequency/severity reported lower SF-12 MCS. After accounting for this between-person difference, there were bidirectional within-person associations between back pain frequency/severity and SF-12 MCS. On follow-up visits when back pain frequency/severity increased from baseline, participants reported lower SF-12 MCS (p<.001). On follow-up visits when SF-12 MCS decreased from baseline, participants also reported higher back pain frequency/severity (p<.001). Higher national and community SSS at baseline and having increases or consistently higher SSS over time attenuated the negative relationships between back pain frequency/severity and SF-12 MCS. Results were consistent after controlling for an extensive list of baseline health covariates and pain medications. Discussion. These findings highlight how self-perceived social status may buffer the relationship between greater back pain frequency/severity and lower mental health."} +{"text": "Acinetobacter baumannii (NDM AB). Starting in October 2021, public health shifted from outbreak response to resource-conscious long-term mitigation in affected healthcare facilities, and prevention of spread to interconnected facilities.Since May 2020, the California Department of Public Health and 12 local health jurisdictions (LHJ) have responded to a regional outbreak of highly-resistant New Delhi metallo-\u03b2-lactamase-producing We defined a case as a patient identified with an NDM AB clinical isolate, or NDM positive colonization screening and epidemiologic linkage. In October 2021, we lengthened the screening interval and changed the screening population in outbreak skilled nursing facilities (SNF) and long-term acute care hospitals to only include high-risk patients ; we continued to screen ventilator units in ventilator-equipped SNF, and epidemiologically-linked unit(s) in acute care hospitals. Concurrently, public health initiated a prevention collaborative focused on improving environmental services (EVS) practices in the most affected LHJ, including 3 outbreak and 4 non-outbreak SNF to promote sustainable EVS practice improvement and peer-to-peer learning.Whereas we identified 170 cases during the outbreak response period May 2020 \u2013 September 2021, since October 2021 we identified only 43 cases. Screening test percent positivity decreased from 3.0% (105/3542) through September 2021 to 1.4% (21/1505) since October 2021. During the outbreak response period, we completed 42 infection prevention and control (IPC) onsite assessments that identified poor adherence to core EVS practices; since October 2021, we completed 29 assessments, including 14 for the EVS prevention collaborative.Long-term mitigation of regional, multifacility novel multidrug-resistant organism outbreaks is possible by implementing a coordinated package of interventions including proactive targeted IPC assessment and support at interconnected facilities, and continued routine public health follow-up at outbreak facilities.All Authors: No reported disclosures."} +{"text": "Calorie restriction (CR) slows aging and increases healthy lifespan in model organisms. We tested if CR slowed biological aging in humans using DNA methylation analysis of blood samples from N=197 participants in the Comprehensive Assessment of Long-term Effects ofReducing Intake of Energy (CALERIE\u2122) randomized controlled trial. We quantified CR effects on biological aging by comparing change scores for six epigenetic-clock and Pace-of-Aging measures between n=128 CR-group and n=69 ad-libitum-control-group participants at 12- and24-month follow-ups. CR effects were strongest for DunedinPACE Pace of Aging , followed by DunedinPoAm and the GrimAge epigenetic clock, although effects for these measures were not statistically differentfrom zero (p>0.08). CR effects for other epigenetic clocks were in the opposite direction . CALERIE intervention slowed Pace of Aging but showed minimal effect on epigenetic clocks hypothesized to reflect longer term accumulation of aging burden."} +{"text": "This cohort study compares the risk of infection with SARS-CoV-2 among health care workers by mask preference and level of patient exposure. Although scientific evidence regarding the benefit of respirator vs surgical masks is sparse,3 a previous study has suggested that respirator masks may offer additional protection to HCW with frequent COVID-19-patient exposure.4 In this follow-up study, we analyzed the SARS-CoV-2 risk for HCWs depending on cumulative exposure to patients with COVID-19 and assessed whether this risk can be modulated by the use of respirator compared with surgical masks.Health care workers (HCWs) are at increased risk for acquiring SARS-CoV-2 infection,5 Baseline data (collected in September 2020) included anthropometric characteristics and job descriptions. In weekly follow-up evaluations during 12 months, participants indicated results of symptom-based SARS-CoV-2 nasopharyngeal swabs, exposures, and risk behavior for the increase in SARS-CoV-2 positivity per doubling of contact time were calculated separately for HCWs using respirator masks only and those who used only surgical or both mask types. We used logistic regression to adjust for a priori\u2013defined covariables and included networks as random effects ; 749 participants (26%) were infected with SARS-CoV-2. SARS-CoV-2 positivity was 13% in HCWs without patient exposure. For those exposed to patients, positivity was 21% for HCWs using respirator masks and 35% for those using surgical/mixed masks , showing an increase for surgical/mixed mask users and respirator mask users across categories of patient exposure . VariablIn this study, SARS-CoV-2 positivity in HCWs was associated with cumulative COVID-19 patient exposure. The odds of being SARS-CoV-2\u2013positive were reduced by more than 40% in individuals using respirators irrespective of cumulative exposure, even after adjusting for multiple work- and nonwork-related covariables.4 Self-reporting of preferred mask type and residual confounding are potential study limitations.These data suggest a dose-response association between COVID-19-patient exposure and risk of SARS-CoV-2 infection in HCWs. The presumable protection conferred by respirator use is in line with previous data.6 remains to be seen.Consequent use of respirators and SARS-CoV-2 vaccination might substantially decrease the work-related risk for HCWs exposed to patients with COVID-19. Whether these results are applicable to newer viral variants, which are more contagious and less neutralized by most vaccines,"} +{"text": "Background and Purpose: Myocardial infarction (MI) in diabetic patients results in higher mortality and morbidity. We and others have previously shown that bone marrow-endothelial progenitor cells (EPCs) promote cardiac neovascularization and attenuate ischemic injury. Lately, small extracellular vesicles (EVs) have emerged as major paracrine effectors mediating the benefits of stem cell therapy. Modest clinical outcomes of autologous cell-based therapies suggest diabetes-induced EPC dysfunction and may also reflect their EV derivatives. Moreover, studies suggest that post-translational histone modifications promote diabetes-induced vascular dysfunctions. Therefore, we tested the hypothesis that diabetic EPC-EVs may lose their post-injury cardiac reparative function by modulating histone modification in endothelial cells (ECs).Methods: We collected EVs from the culture medium of EPCs isolated from non-diabetic (db/+) and diabetic (db/db) mice and examined their effects on recipient ECs and cardiomyocytes in vitro, and their reparative function in permanent ligation of left anterior descending (LAD) coronary artery and ischemia/reperfusion (I/R) myocardial ischemic injuries in vivo.Results: Compared to db/+ EPC-EVs, db/db EPC-EVs promoted EC and cardiomyocyte apoptosis and repressed tube-forming capacity of ECs. In vivo, db/db EPC-EVs depressed cardiac function, reduced capillary density, and increased fibrosis compared to db/+ EPC-EV treatments after MI. Moreover, in the I/R MI model, db/+ EPC-EV-mediated acute cardio-protection was lost with db/db EPC-EVs, and db/db EPC-EVs increased immune cell infiltration, infarct area, and plasma cardiac troponin-I. Mechanistically, histone 3 lysine 9 acetylation (H3K9Ac) was significantly decreased in cardiac ECs treated with db/db EPC-EVs compared to db/+ EPC-EVs. The H3K9Ac chromatin immunoprecipitation sequencing (ChIP-Seq) results further revealed that db/db EPC-EVs reduced H3K9Ac level on angiogenic, cell survival, and proliferative genes in cardiac ECs. We found that the histone deacetylase (HDAC) inhibitor, valproic acid (VPA), partly restored diabetic EPC-EV-impaired H3K9Ac levels, tube formation and viability of ECs, and enhanced cell survival and proliferative genes, Pdgfd and Sox12, expression. Moreover, we observed that VPA treatment improved db/db EPC-mediated post-MI cardiac repair and functions.Conclusions: Our findings unravel that diabetes impairs EPC-EV reparative function in the ischemic heart, at least partially, through HDACs-mediated H3K9Ac downregulation leading to transcriptional suppression of angiogenic, proliferative and cell survival genes in recipient cardiac ECs. Thus, HDAC inhibitors may potentially be used to restore the function of diabetic EPC and other stem cells for autologous cell therapy applications. Cardiovascular disease (CVD) causes higher mortality and disability in patients with diabetes and imposes a significant health and economic burden In recent years, emerging consensus suggests that adult stem/progenitor cells benefit myocardial repair and regeneration majorly through paracrine mechanisms. Small extracellular vesicles (EVs), of less than 150 nm in diameter constitute a major functional paracrine entity of stem cells and carry a variety of bioactive molecules cargo such as proteins, small RNAs, and lipids that alter recipient cell behaviorsvice versa, enabling gene repression or activation depending on the accessibility of chromatin Epigenetic dysregulations including DNA methylation and histone modifications result in altered gene expression contributing to diabetic complications This study demonstrates that diabetes deteriorates EPC-EV reparative function in myocardial injuries post permanent LAD ligation (MI) and ischemia-reperfusion (I/R), by decreasing angiogenesis and cardiac functions, increasing maladaptive remodeling, infarct area, cardiomyocyte death and immune cell infiltration. Mechanistically, we show that diabetic EPC-EVs enriched with HDAC1 protein, downregulate the gene activation mark, histone 3 lysine 9 acetylation (H3K9Ac), through increasing histone deacetylase (HDAC) enzymatic activity, which leads to a decrease in a multitude of endothelial functional gene repression in recipient mouse cardiac endothelial cells (MCECs). Furthermore, diabetic EPC-EV-mediated H3K9Ac reduction can be reversed by pretreatment of donor diabetic EPCs with HDAC inhibitor valproic acid (VPA). These findings suggest that diabetes impairs EPC-EV reparative activities in ischemic myocardium. Mechanistically, we report that diabetic EPC-EVs worsen cardiac repair partly through HDAC activity-H3K9Ac-mediated cell survival/proliferative gene inhibition in ECs.All data presented in this study are freely available from the corresponding author upon any reasonable request. The Online Data Supplement provides additional detailed materials and methods.mDock7 +/+ dbLepr/J-homozygous) hereafter db/db and nondiabetic mice (#000642 BKS.Cg-mDock7 +/+ dbLepr/J-heterozygous) hereafter db/+ were purchased from Jackson Research Laboratory for EPC isolation described in our previous paper2 atmosphere. Male mice were used to avoid the reproductive hormone effect in female mice. Human CD34+ Hematopoietic stem cells (HSC) were purchased from AllCells, LLC and were cultured in a human hematopoietic cell culture medium supplemented with 10X CD34+ expansion supplement and 0.25% human albumin . HSCs were cultured in normal glucose media or in high glucose (25 mM) media in 37\u00b0C, 5% CO2 atmosphere.All animal procedures were performed following the approved protocols of the Institutional Animal Care and Use Committee of Temple University. Ten to twelve-week-old male mice (C57BL/6J) were purchased from Jackson Research Laboratory for MI surgery. Eight to ten-week-old diabetic male mice equipped with a sCMOS camera and a 405 nm laser was used. Data acquisition and processing were performed using NTA software version 2.3 build 0025. Background extraction was applied, and automatic settings were employed to determine the minimum expected particle size, minimum track length, and blur settings. Data were obtained at camera level 12 . Three movies of 30 s at 25 frames per second were recorded and assigned a single measurement in triplicates.The ligation of the left anterior descending (LAD) coronary artery was performed as a permeant MI model described previously Transthoracic two-dimensional M-mode echocardiography using the Vevo2100 equipped with 30 MHz transducers was performed before MI (baseline), and 1-, 2-, 3-, and 4- weeks after surgery as described previously Mouse blood was collected from vena cava after 24 hours I/R and immediately centrifuged at 4000 rpm for 10 min at 4\u00b0C. Plasma was then collected, and cTn-I concentrations were measured by kit following the manufacturer's instruction .Mouse heart tissue samples were fixed in 10% formalin for at least 48 hours and embedded in paraffin. Cardiac tissues were cross-sectioned into 4-5 \u03bcm-thick slides. Masson Trichrome staining and TUNEL staining were performed following the manufacturer's instruction and previously described in detail 1X10^4 human or mouse microvascular endothelial cells ) were cultured in EV-depleted culture media while treated with vehicle, 1X 10^6 db/+ EPC-EV or 1X 10^6 diabetic EPC-EV particles for 48 hours. HMVECs or MCECs were then re-plated on Matrigel in a 48-well plate for 16 hours. Images were taken using a phase-contrast microscope to count total branch points.1X10^6 MCECs were treated with 1X 10^9 db/+ EPC-EV, or 1X 10^9 diabetic EPC-EV particles for 24 hours. Cells were then collected, and nuclear proteins were isolated according to the manufacturer's instructions . 10 mg nuclear protein was used to measure HDAC activity according to the manufacturer's instructions .TM Green Master Mix (Applied Biosystems) according to the manufacturer's instructions. Fold changes were normalized to GAPDH with the threshold delta-delta cycle method. Primers are listed in RNA was isolated from cells using miRNeasy Mini kit . NanoDrop-1000 (Thermo Scientific) was used to identify RNA concentration and purity determined by 260A/280A ratio. High-capacity cDNA Reverse Transcription Kit was used to obtain cDNA. RT-PCR was performed on an Applied Biosystems 770 StepOnePlus system using the Fast SYBRMCECs were fixed with 1% formaldehyde for 15 min and quenched with 0.125 M glycine after db/+ or db/db EPC-EV treatment for 24 hours and then processed for ChIP-seq using the antibody against H3K9Ac (Active Motif cat# 39917). Input controls were used in normalizing CHIP-seq signals. ChIP and ChIP analysis was performed by Active Motif, Inc. Detailed methods are provided in Online Data Supplement.All procedures were carried out as reported previously P value of < 0.05 was considered as statistical significance.Statistical analyses were performed using GraphPad Prism 7.0 software . All Data are presented as mean \u00b1 SEM and represent at least 3 independent biological experiments. Unpaired t-test was used to compare 2 sample groups. For comparisons among > 2 groups, one-way ANOVA followed by Tukey multiple comparisons test was performed. For echocardiography parameters with repeated measures over time, two-way ANOVA with Tukey's multiple comparisons test was used. A in vitro. Neovascularization/angiogenesis is crucial for cardiac repair post MI 2O2 stress. We found that db/+ EPC-EVs enhanced MCEC survival but db/db EPC-EVs failed to provide protection from MCEC death or vehicle were injected into three locations in the border zone around the ligation site. At 4 weeks after MI, db/db EPC-EV treated mice exhibited unchanged heart weight to body weight ratio (HW/BW), HW to tibia length ratio (HW/TL) compared to db/+ EPC-EV-treated mice , the portion of differentially upregulated genes (indicated in red color) of db/db EPC-EV treated MCECs is less than db/+ EPC-EV treated MCECs (Figure To elucidate which genes are enriched with H3K9Ac level, we further performed H3K9Ac-ChIP-seq experiments in MCEC treated with db/+ and db/db EPC-EVs. In line with the H3K9Ac protein expression, the ChIP-seq results showed the lowest percentage of H3K9Ac enrichment in the merge peak regions under db/db EPC-EV treatment compared with db/+ EPC-EV treatment, indicating db/db EPC-EV reduced chromatin accessibility for gene transcription Figure A-B. Mores Figure -5. Sox12s Figure . Togethe2O2-induced MCEC death (Figure Recently, it was reported that the pan-HDAC inhibitor, VPA, received FDA approval for the treatment of epilepsy. It was also shown that VPA protected heart function post-AMI h Figure G-H. ThesIt has been demonstrated that specific cargo of EVs, including their RNA, protein, and lipid contents, are reflective of donor cell origin and its physiological state. Db/db-EPC-EVs largely recapitulate the functional deficiency of parent db/db EPCs which we have reported be functionally impaired Lastly, to test the translational aspect of mouse studies, we collected EVs from human CD34+ stem cells stimulated with 25 mM glucose, mimicking the hyperglycemic condition, which is characteristic of diabetic patients. We performed a tube formation assay in HMVECs and found that the tube branch numbers were significantly decreased in cells treated with EVs from hCD34+ cells subjected to 25 mM glucose culture conditions compared with normal hCD34+ EVs and vehicle. In line with our mouse study Figure A-B, thesIn the past decades, numerous preclinical studies reported that autologous BM-EPCs repair ischemic heart by promoting cardiac neovascularization and cardiac function, along with mitigating inflammation, oxidative stress, apoptosis, and maladaptive remodeling in vitro available studies have reported that EVs from diabetic source showed functional deficiencies largely by delivering specific microRNAs to recipient cells As EVs reflect their parental stem cells' cargo and functional properties and since EVs are relatively stable, they appear attractive as cell-free therapeutics and conduits for gene and drug delivery for heart failure in vitro cell culture system and in vivo in two models of myocardial injury. These results establish that healthy non-diabetic EPC-EVs substantially improve physiological and anatomical repair after MI; however diabetic EPC-EVs completely lose their acute cardioprotective and long-term cardiac reparative activities in the ischemic hearts. We also found that diabetic EPC-EVs augmented immune cell infiltration into the injured myocardium, likely due to the systemic and low level of inflammatory environment where EPCs may generate inflamed EVs in diabetic patients Previously, we have reported that EPCs and other stem cells exposed to hyperglycemic culture conditions showed a dramatic alteration in EV RNA contents Epigenetic mechanisms such as DNA and histone modifications leading to altered gene expression are the critical regulatory pathways mediating the initiation, maintenance, and progression of both macro-and micro-vascular complications of diabetes and are also major contributors to epigenetic memory associated with diabetes Preclinical effects of multiple HDAC inhibitors on MI and heart failure have been reported and showed their benefits on reducing the infarct area and improving LV function. Especially, Tian et al. showed that VPA administration protects heart function through Foxm1 pathway post-MI in vivo in MI models. Nonetheless, VPA-treated diabetic EPC-EV did regain their angiogenic and cell protective activities in vitro. In accordance with current notion that EVs secreted from stem cells largely mirror the functional properties of their parent cells Finally, we would like to acknowledge the limitations of our current studies. Although we only examined H3K9Ac modification by diabetic-EPC-EVs, other components of EV cargo may also influence diabetic-EV-mediated recipient cell responses. EVs pack a multitude of bioactive molecules as their cargo including microRNAs and other noncoding RNAs, proteins, kinases, lipids, and metabolites. While establishing a functional role for each of the altered component of diabetic EPC-EVs is not feasible, a comprehensive analysis of diabetic EPC-EV cargo would be needed for identifying the pathways downstream of specifically altered biomolecules. Future studies will attempt at this endeavor. Despite these limitations, data presented in this study is provides a novel level of gene regulatory mechanism induced by diabetic EPC-EVs. Although we tested the efficacy of VPA-pretreatment on enhancing cardiac reparative activity of diabetic-EPCs, we did not test whether EVs from VPA-treated diabetic EPCs regain their cardiac reparative functions In summary, we report that diabetes impairs the therapeutic capability of BM-EPCs as well as of their secreted EVs in two different models of cardiac injury. We demonstrate a new regulatory mechanism of HDAC-mediated epigenetic alteration by diabetic EPC-EVs that mediates functional repression in recipient cells. Inhibiting HDAC enzymes using the small molecule, VPA could rescue H3K9Ac level, which partly reverses cell/EV function. This study provides important implications that EPCs exhibit functionally impaired EVs in diabetes and, at least partly, through HDAC-mediated epigenetic mechanisms.Supplementary materials and methods, figures and tables.Click here for additional data file."} +{"text": "TPR-PDGFRB fusion gene at diagnosis and relapse by using single-cell RNA sequencing (scRNA-seq). Twelve heterogeneous B-cell clusters, four with strong MKI67 expression indicating highly proliferating B cells, were identified. A relapse-enriched B-cell subset associated with poor prognosis was discovered, implicating the transcriptomic evolution during disease progression. Integrative single-cell analysis was performed on Ph-like ALL and Ph+ ALL patients, and revealed Ph-like specific B-cell subpopulations and shared malignant B cells characterized by the ectopic expression of the inhibitory receptor CLEC2D. Collectively, scRNA-seq of Ph-like ALL with a novel TPR-PDGFRB fusion gene provides valuable insights into the underlying heterogeneity associated with disease progression and offers useful information for the development of immunotherapeutic techniques in the future.Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is a refractory and recurrent subtype of B-cell ALL enriched with kinase-activating rearrangements. Incomplete understanding of the heterogeneity within the tumor cells presents a major challenge for the diagnosis and therapy of Ph-like ALL. Here, we exhibited a comprehensive cell atlas of one Ph-like ALL patient with a novel The online version contains supplementary material available at 10.1186/s40164-023-00380-8. To The Editor,+ ALL but lacking the BCR-ABL1 translocation, and 5-year overall survival does not exceed 20% [. It has been reported that drug resistance and subsequent relapse induced by heterogeneous blasts remain the leading causes of death among Ph-like patients [Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is an aggressive neoplasm of B lymphoblasts, with a gene expression profile similar to that of Phpatients . Single-patients , pre-exipatients . HoweverPDGFRB rearrangements account for\u2009~\u20098% of the Ph-like cases [TPR-PDGFRB in-frame fusion gene which consists of exons 1\u201346 of the TPR with exons 11\u201323 of the PDGFRB [Previous study has been reported that patients with ke cases . Our Ph-or (TKI) . Single + ALL patients based on two publicly available datasets [+ ALLs than with other ALL subtypes confirmed the PDGFRB break-apart high and IGLL1 low PreB cells, CD20 low and IGLL1 high PreB cells, IGKC low and IGLL1 high PreB cells, cycling PreB cells (MIK67 high expression), and memory B cells representation was divided on the basis of disease status Fig.\u00a0G, and alD10 Fig.\u00a0G, H, sho+ ALL at the single-cell level [+ T cells) from diagnosis and relapse samples and high cytotoxicity signature scores , is reported to be expressed in T/NK cells, germinal center (GC)-associated B cells, early plasmablasts and GC-derived lymphomas, but not in B-ALL cells [CLEC2D in malignant B-ALL cells , was also comparable with that of T cells and NK cells (clusters 15 and 23) Diagram of the whole treatment process of the Ph-like ALL patient. NR: not remission; PR: partial remission; CR: complete molecular remission; MRD: minimal residual disease. (B) Karyotype analysis showed the normal karyotype at diagnosis for the Ph-like ALL patient. (C) Wright stain of bone marrow aspirate smear from four specimens showed the blast cells clearly. (D) Flow cytometry analysis of immunophenotypic markers for Ph-like ALL patient at diagnosis and three relapse timepoints. Figure.S2 Evaluation of Ph-like expression signature and molecular marker potential for patient with novel TPR-PDGFRB fusion gene. (A) Correlation analysis between the TPR-PDGFRB positive Ph-like patient and different subtypes from TARGET-ALL-P2 cohort. Correlation coefficient was calculated between the TPR-PDGFRB positive Ph-like patient and each of the patients in different subtypes using person method based on the expression level of all overlapped genes. (B) Hierarchical clustering analysis between the Case 13 and other B-ALL patients for three cohorts using the genes defined previously. The red and bold line in each dendrogram for three datasets highlights the location of Case 13. (C) Sanger sequencing of the RT-PCR product validated the TPR-PDGFRB fusion junction. (D) The percentage of blasts evaluated by morphology and immuno-phenotyping at thirteen time points. (E) Amplification plot of qRT-PCR for TPR-PDGFRB fusion transcript and controls. qRT-PCR analysis on the standard reference sample with serial 10-fold gradient dilutions of TPR-PDGFRB fusion transcript copies/\u03bcl and TPR-PDGFRB fusion patient\u2019s specimen at diagnosis. Additional amplifications include a no template control (NTC) and an unrelated control cDNA came from a pediatric B-ALL specimen that does not involve a TPR-PDGFRB fusion. (F) MRD of detected by qRT-PCR for TPR-PDGFRB fusion transcript. (G) MRD of detected by ddPCR for TPR-PDGFRB fusion transcript. Figure.S3 Clusters and transcriptomic feature of different cell types in Ph-like ALL. (A) tSNE visualization of 10,273 individual cells from the patient with Ph-like ALL (TPR-PDGFRB fusion positive) with matched diagnosis and relapse bone marrow samples. (B) Unsupervised t-SNE plot displaying 10,273 cells from one Ph-like ALL patient at diagnosis and relapse, color-coded by 17 clusters. (C) Heatmap of the top-five genes marking 17 clusters. (D) Violin plot of selected genes which are differently expressed in classical monocytes and non-classical monocytes. (E) tSNE projections of selected T cell and NK cell genes. (F) Dot plot showing the average expression levels and cell expression proportions of selected MEP and cell cycle genes in the indicated clusters. (G) Violin plots show the expression of cluster-specific markers for memory B cells. Figure.S4 The functional annotation and clinical outcomes of clusters specific for different disease status in Ph-like ALL. (A) Percentage of cell origin within each clusters (n = 17). (B) Cell types overlaid on the tSNE representation and split by samples, color-coded by cell types. For all non-B cells, the corresponding cluster numbers were labeled using the pink text and cells were indicated by the\u00a0dotted\u00a0oval. (C) Representative GSEA plots of three primary specific clusters comparing with other B cell clusters. The enrichment score (ES) and false discovery rate (FDR) are shown in the graph. (D) Survival analysis of the relapsed B-cell feature (top 10 up-regulated genes) in Figure 2I in the TARGET-ALL-P2 cohort. (E) Expression levels of relapse-specific gene HRK overlaid on the tSNE representation (left panel). Boxplot (right panel) showed that the higher expression of HRK in relapsed patients with CNS-L. Figure.S5 Single-cell transcriptional profiles of Ph-like and Ph+ ALL patients. (A) The tSNE visualization of 25,559 individual cells from one patient with Ph-like ALL (TPR-PDGFRB fusion positive) and two patients with Ph+ ALL with matched bone marrow samples obtained at diagnosis and after relapse. (B) Unsupervised tSNE plot split into Ph-like and Ph+ ALL patients, displaying 25,559 cells color-coded by 23 clusters. (C) Expression levels (x-axis) of cell type-defining genes in each cluster. Violin plots showing the distribution of the normalized expression levels of genes that are color coded on the basis of the cluster, as in (B). (D) Percentage of cell origin within each clusters (n = 23). (E) A volcano plot of the DEGs that were up-regulated (red) or down-regulated (blue) in the Ph-like specific clusters (clusters 1 and 8); the top 10 up-regulated genes are labeled. The p-value is derived using the Wilcoxon rank-sum test. (F) Representative GSEA plots of Ph-like specific clusters comparing with other B cell clusters. The ES and FDR are shown in the graph. (G) Violin plots showing the distribution of the normalized expression levels of cytotoxic genes (left panel), NK cell genes (middle panel) and exhausted genes (right panel) in T cell and NK cell clusters (clusters 15 and 23). (H) Scatter plot for the signature scores about cytotoxicity and NK feature in the T cell clusters. (I) tSNE visualization of cells colored by the expression of cytotoxicity or NK receptor signatures. (J) Violin plot of immune checkpoint genes including CLEC2D and KLRB1."} +{"text": "Hepatocellular carcinoma (HCC) is the fourth leading cause of cancer-related death worldwide. In the recent years nonalcoholic fatty liver disease (NAFLD) is becoming a growing cause of HCCs and the incidence of NAFLD-related HCCs is expected to further dramatically increase by the next decade. Chronic inflammation is regarded as the driving force of NAFLD progression and a key factor in hepatic carcinogenesis. Hepatic inflammation in NAFLD results from the persistent stimulation of innate immunity in response to hepatocellular injury and gut dysbiosis as well as by the activation of adaptive immunity. However, the relative roles of innate and adaptive immunity in the processes leading to HCC are still incompletely characterized. This is due to the complex interplay between different liver cell populations, which is also strongly influenced by gut-derived bacterial products, metabolic/nutritional signals. Furthermore, carcinogenic mechanisms in NAFLD/NASH appear to involve the activation of signals mediated by hypoxia inducible factors. This review discusses recent data regarding the contribution of different inflammatory cells to NAFLD-related HCC and their possible impact on patient response to current treatments. In the last decade nonalcoholic fatty liver disease (NAFLD) has emerged as the most common cause of chronic liver disease worldwide with a global prevalence of about 25%, ranging from 13% in Africa up to 42% in southeast Asia [While simple steatosis has low risk of further liver complications, NASH can progress to fibrosis/cirrhosis and NASH-associated liver fibrosis is the strongest predictor for disease-specific mortality . A growiFrom the histopathologic point of view, NAFLD-related HCCs are often characterized by a specific morphology, known as steatohepatitic HCC, involving the presence of macrovesicular steatosis, cell ballooning along with the presence of Mallory-Denk bodies, inflammation, and variable fibrosis reminiscent of the features of NASH . FurtherSo far, the mechanisms responsible for the onset and evolution of HCC in NAFLD/NASH are still poorly understood. Epidemiological studies have shown that HCC risk is strictly associated with the prevalence of obesity and Type II diabetes ,9. MoreoThe transition from simple steatosis that characterizes NAFLD to NASH is a complex process involving multiple factors including metabolic dysfunctions, lipotoxicity, gut dysbiosis, oxidative stress, and hepatocellular necrosis. All these factors stimulate the induction of chronic inflammation responsible for perpetuating tissue injury, parenchymal cell regeneration, mutagenesis, and HCC progression . The liv\u2212/CD23+ B2-cells, while T-lymphocytes include proinflammatory CD4+ interferon-\u03b3 (IFN-\u03b3)-producing T-helper 1 (Th-1), IL-17 producing T-helper 17 (Th-17) cells and cytotoxic CD8+ T-cells [\u2212/\u2212 mice, lacking mature B-, and T-cells [+ T-cells [+ may provide a stimulus for the proinflammatory activity of macrophages [+ T-cells are cytotoxic against hepatocytes through antigen independent mechanisms [+/FOXp3+ Tregs and exhausted CD8+-T-lymphocytes [Although current view indicates that innate immune mechanisms represent a key element in supporting hepatic inflammation in NASH, increasing evidence points to an additional role of adaptive immunity in NASH progression to fibrosis and HCC . Histolo T-cells ;36. The T-cells as well T-cells . From a rophages ,38, whilchanisms ,38. Howechanisms . Cancer phocytes ,41.+-macrophages accompanies the expansion of CD4+/FOXp3+ Tregs and tumor burden, suggesting a possible contribution of NAMs in promoting an immunosuppressive microenvironment [Inside the liver, the presence of danger signals is sensed through the engagement of pattern recognition receptors (PRRs) expressed on Kupffer cells (KCs) triggering their activation and the ironment which lack conventional NK (cNK) cells without losing the fraction of liver resident NK (LrNK) cells [\u2212/\u2212 mice show an attenuation of the main features of NASH. On the contrary, NKp46-deficient mice lacking both cNK and LrNK cells are not protected from NASH-induced liver injury [Natural killer (NK) cells are a heterogeneous component of innate immunity and in both mice and humans make up a large proportion of hepatic leukocytes ,68. DespK) cells . As compr injury . Altogetr injury . Furtherr injury cells represent a particular subset of T-lymphocytes at the interface between innate and adaptive immunity. NKT cells are characterized by the co-expression of T-cell receptor (TCR) and the NK cell surface receptors (NK1.1 in mice or CD161/CD56 humans) . The majH livers , suggestfat diet . Such anltration .\u2212/CD23+ B2-cells that contribute to hepatic inflammation by producing pro-inflammatory cytokines such as TNF-\u03b1 and IL-6. B2-cell activation is fostered by the up-regulation in the hepatic expression of B-cell activating factor (BAFF) [+ T helper cells [in vitro intrahepatic B-cells directly influence CD4+ T helper (Th) cell functions by promoting Th1 activation [+CD4+ T-cells as compared with control mice [As mentioned above, growing evidence points to the contribution of adaptive immunity in supporting lobular inflammation in NASH. In these settings, B-cell activation appears an early event in NASH ,37, as tr (BAFF) , one of r (BAFF) . Concernr (BAFF) . Furtherer cells . In linetivation . Such anrol mice ,37. Furtrol mice . It is nrol mice . At presrol mice .+ plasma cells associated with NASH-derived HCC have an immunosuppressive phenotype characterized by the expression of PD-L1 and IL-10 and that they inhibit CD8+ T-cell activation [+ T-cell capability to counteract tumour growth [Beside their role in promoting chronic inflammation and fibrogenesis, B-cells also display a pro-carcinogenic action . Indeed,tivation . Along wr growth . Overall+ T-cells in both pediatric and adult NASH patients [+ T-cell depletion ameliorated hepatocellular injury by lowering the expression of IFN-\u03b3 along with classical-activated macrophage markers in OSE-immunized NASH mice [+ T-cells localized in the fibrotic regions and by an increased production of INF-\u03b3 and IL-17A. Of note, in the same mice, CD4+ T-cell depletion decreases NASH-associated immune cell infiltration, fibrosis, and overproduction of inflammatory mediators [+ T-cells that are characterized by the secretion of IL-17A. Although several studies have supported the implication of Th17 cells in NASH pathogenesis, the overall picture remains confused [\u2212/\u2212 mice receiving high-fat diet which are normally protected from NASH [The possible implication of T-cells in NASH has been suggested by the high prevalence of IFN-\u03b3 producing Th1 CD4patients ,89. Morepatients . SimilarASH mice . These rediators . These lconfused . Recentlconfused . These cconfused . Of noteconfused , while trom NASH . Furtherrom NASH .+ T-cells, mounting evidence points out the pathogenetic role of cytotoxic CD8+ T-cells in NASH. These cells are increased in both human and mouse NAFLD/NASH livers [+ T-cells in NASH is further corroborated by the observation that mice with an impaired CD8+ T-cell activation develop less steatosis and fibrosis as compared with control littermates [+ T-cell depletion improves lobular inflammation and fibrosis by lowering the fraction of recruited macrophages and activated HSCs [+ T-cells display an activated phenotype characterized by an increased expression of pro-inflammatory mediators [+ T-cells appears closely related to type I interferon signalling as chimeric mice lacking interferon \u03b1-receptor 1 subunit (INF\u03b1-R1) on CD8+ T-cells show lower hepatic recruitment of CD8+ T-cells. [+ T-cells showing that these cells feature C-X-C motif chemokine receptor 6 (CXCR6), effector molecules and the programmed cell death protein 1 (PD-1), this latter suggesting an activated/exhausted phenotype. Noteworthy, these authors have also reported that CXCR6+/PD1+/CD8+ T cells have an \u2018auto-aggressive\u2019 behaviour and, upon exposure to metabolic stimuli such as acetate and extracellular ATP, kill hepatocytes in an antigen-independent fashion. Overall, these data would suggest a critical role of CD8+ T-cells in perpetuating hepatic injury in NASH leading to tissue scarring. However, it has been recently proposed that tissue-resident memory CD8+ T (CD8+ Trm) cells have a role in controlling liver fibrosis during the resolution of experimental NASH. This effect depends on CD8+ Trm cell cytotoxic activity towards HSCs. Indeed, CD8+ Trm cells attract HSCs in a CCR5-dependent manner and stimulate their death through the Fas/Fas-ligand pathway [+ Trm cells parallels the severity of the disease, suggesting a possible role of these cells in regulating NASH progression [+ T-cells in supporting disease progression. Therefore, further investigations need to define the precise role of distinct CD8+ T cell subsets in the different disease stages.Beside CD4H livers ,100,101 H livers . The pattermates . Similarted HSCs ,101,104.ediators ,105. Not pathway . These ogression . Conside+ T-cells [ex vivo but are instead characterized by the expression of the transcription factor Foxp3 and the proliferation marker Ki67, indicating that HCC development is associated with the expansion of locally proliferating regulatory T-cells (Tregs) [+/Foxp3+ Tregs [+ T-cells that specifically counteract T-cells functions, thus contributing to the loss of cancer immunosurveillance [+ and CD8+ T-cells [+ Tregs and Th17 cells are in contrast with the data showing that selective CD4+ T-cell depletion accelerates HCC growth when NASH is induced in mice with hepatocyte-specific over-expression of Myc [+ T-cells loss results from mitochondrial oxidative stress consequent to disrupted lipid metabolism [+ T-cells observed in many different models of NASH [+ T-cells depletion can favour tumour growth.In parallel with the notions involving T-lymphocytes in the pathogenesis of NASH, increasing evidence point to their possible involvement in the process leading to HCC development. In this setting, multidimensional flow cytometry analysis of human HCC-infiltrating lymphocytes reveals an enrichment of CD4 T-cells which in (Tregs) are fed with a high-fat diet [+ T-cells following the onset of NASH, but prior to HCC development, effectively reduces HCC incidence in mice. Single-cell mapping of CD8+ T-cells has shown that they express activation/exhaustion markers and high levels of the immunomodulating molecule PD-1. Surprisingly, despite the high prevalence of CD8+/PD-1+ T-cells in NASH-driven HCCs these tumours do not respond to anti-PD-1 therapy which instead promotes an earlier HCC onset. A similar behaviour is also evident by inducing HCC on NASH background in PD1-deficient mice [+/PD-1+ T-cells lack immune-surveillance functions and have instead a tissue-damaging action, which is partially counteracted by PD-1 signalling, thus explaining the unfavourable effects of anti-PD-1 agents on tumour development [+/PD-1+ T-cells with a gene expression profile comparable to that observed in rodent NASH are also detectable in human NAFLD/NASH livers suggesting the possibility that liver steatosis/steatohepatitis specifically activates CD8+/PD-1+ T-cells in a manner that favours the disease evolution and limits the response to HCC immunotherapy [+conventional type 1 dendritic cells (cDC1) and CD8+ T cells [Further conflicting data have been obtained in relation to the role of cytotoxic CD8 tumours . Howeverfat diet . These dfat diet . A recenfat diet sheds soent mice . These oent mice and suggelopment is higher as compared with those suffering from viral-related HCC [+/CD8+ T cells positively correlates with serum palmitoleic acid (C16:1n7) to palmitic (C16:0) acid ratio, while in vitro CD8+ T-cells exposure to palmitic acid significantly enhances the fraction of CTLA-4 expressing cells [+ T cells from NASH-HCC also display impairment of multiple metabolic pathways such as glycolysis, fatty oxidation, and mitochondrial respiration. Such metabolic derangements result in altered cell motility that ultimately leads to the loss of anti-tumour capacity [+ T cell functional properties by acting on cellular energy metabolism [The data emerging from recent studies investigating the role of inflammatory and immune cells in the processes leading to NAFLD/NASH evolution to HCC have evidenced that the metabolic derangements associated with the disease evolution not only promotes liver inflammation but can also specifically influence the capacity of the immune system to counteract cancer cell growth. Indeed, NASH livers represent a unique biological microenvironment in which the coexistence of metabolic disorders along with chronic inflammation determine a significant reprogramming of the immune system Figure . In thisated HCC . Notablyng cells . CD8+ T capacity . Of notetabolism ,118.+ and CD8+ T-cells expressing the thymocyte selection associated high mobility group box protein (TOX), a nuclear factor leading to the acquisition of an exhausted phenotype. Notably, TOX expression in effector T-cells promotes the up-regulation of inhibitory receptors such as PD-1 [Among the metabolic imbalances occurring in NASH, aberrant cholesterol metabolism represents a typical hallmark with serious consequences for the disease evolution. In a recent study, Tang et al. have rep as PD-1 . Concomi as PD-1 . MDSCs a+-producing plasma cells express inhibitory molecules such as PDL-1 and secrete IL-10, a potent anti-inflammatory cytokine that strongly inhibits cytotoxic CD8+ T cell function [Finally, chronic tissue injury in NASH is strongly associated with a profound extracellular matrix remodeling resulting in liver fibrosis and cirrhosis which is a fertile ground for cancer development . In thisfunction , thus imfunction Figure . HIFs aret genes . HIFs arnditions . The hetnditions .2 is so far available in human HCC although certain characteristics of these tumours such as hypervascularity, necrotic areas and resistance to therapy may suggest the presence of severe hypoxia in human HCC [in vitro data suggest that the knockdown of HIF-1\u03b1 enhances the expression of HIF-2\u03b1 and vice versa [Beside physiological functions, HIF-controlled target genes are also critical in the process of carcinogenesis in many organs including the liver. Indeed, a growing body of data points to the role of HIFs in regulating HCC angiogenesis, epithelial-to-mesenchymal transition, metastasis, and metabolic reprogramming ,140,141.uman HCC . From thuman HCC ,146. Conuman HCC despite uman HCC . Furtherce versa . However\u2212/\u2212 mice). In these animals, HIF-2\u03b1 depletion halves the number and the size of HCC nodules as compared with wild-type mice [+ macrophages and the up-regulation of pro-inflammatory cytokines [The characterization of HIFs role in NAFLD/NASH has shown that HIF-2\u03b1 regulated genes are involved in fatty acid synthesis/uptake and lipid storage ,154. Hepype mice . Such anype mice . The samype mice . Althougype mice has showytokines . Interesytokines . Howeverytokines . At pres+ T-cells and poor survival. Accordingly, the block of TREM-1 positive TAMs reverse immunosuppression and anti-PDL1 resistance in HCC [+/\u2212 mice, spontaneously develop NASH-related HCC in association with a significant up-regulation of HIF-1\u03b1 in either circulating and liver infiltrating immune cells, but not in hepatocytes. Macrophages from these mice show an increased production of the cytokine CCL5 leading to an increased infiltration of neutrophils and CCL5 neutralization decreases both neutrophil infiltration and HCC development in SART1+/\u2212 [HIF1\u03b1 might have additional roles in modulating TAM function in HCCs as Wu et al. have repe in HCC . Additioe in HCC . Male SASART1+/\u2212 . These rSART1+/\u2212 and thatSART1+/\u2212 . InteresSART1+/\u2212 , while aSART1+/\u2212 . More peSART1+/\u2212 .MDSCs represent another cell population that may be potentially modulated by HIFs in HCC, since MDSCs have been detected mostly in hypoxic regions of human HCC . AccordiIn recent years the contribution of specific immune/inflammatory mechanisms in supporting NAFLD/NASH evolution to HCC has received increasing interest and growing data ha ve shed light on the involvement of different subsets of myeloid and lymphocytic cells. Furthermore, emerging evidence indicates that these cells acquire peculiar phenotypes in the tumour environment that might strongly influence their behaviour during the disease progression and in the carcinogenesis process. The interplay between different liver cell populations is also strongly influenced by gut-derived bacterial products, metabolic/nutritional signals, and hypoxia. These factors are likely capable of modulating in opposite ways cellular responses, possibly explaining why immune/inflammatory cells can sustain HCC development by supporting chronic inflammation and at the same time favour cancer cell immune evasion. Understanding this complexity will represent the challenge for researchers in this field in the next years. The impact of these studies on the diagnosis and the treatment of NASH-related HCC is already appreciable, explaining possible reasons for the poor response to immune checkpoint inhibitors observed in these patients. From this, it is possible to forecast that more detailed information on the actions played by immune/inflammatory cells in NASH-associated carcinogenesis would lead to innovative therapies tailored on this form of HCC."} +{"text": "Defining glioma heterogeneity represents a promising strategy to unravel the mechanisms behind therapy resistance and tumor recurrence. The current review provides a comprehensive overview of experimental and clinical data concerning the visualization and quantification of the tumor microenvironment heterogeneity using molecular imaging, with a special emphasis on positron emission tomography (PET).Glioblastoma is the most common primary brain tumor, highly aggressive by being proliferative, neovascularized and invasive, heavily infiltrated by immunosuppressive glioma-associated myeloid cells (GAMs), including glioma-associated microglia/macrophages (GAMM) and myeloid-derived suppressor cells (MDSCs). Quantifying GAMs by molecular imaging could support patient selection for GAMs-targeting immunotherapy, drug target engagement and further assessment of clinical response. Magnetic resonance imaging (MRI) and amino acid positron emission tomography (PET) are clinically established imaging methods informing on tumor size, localization and secondary phenomena but remain quite limited in defining tumor heterogeneity, a key feature of glioma resistance mechanisms. The combination of different imaging modalities improved the in vivo characterization of the tumor mass by defining functionally distinct tissues probably linked to tumor regression, progression and infiltration. In-depth image validation on tracer specificity, biological function and quantification is critical for clinical decision making. The current review provides a comprehensive overview of the relevant experimental and clinical data concerning the spatiotemporal relationship between tumor cells and GAMs using PET imaging, with a special interest in the combination of amino acid and translocator protein (TSPO) PET imaging to define heterogeneity and as therapy readouts. Glioblastoma (GBM) is a highly proliferative, invasive and neovascularized heterogeneous tumor tissue driven by an immunosuppressive tumor microenvironment (TME). Current therapies for GBM rely on surgical resection, radiotherapy and chemotherapy but patient prognosis remains limited. Therapy response depends on multiple factors, including intra- and inter-tumoral genetic, epigenetic, molecular and metabolic heterogeneity. In addition, radiotherapy and chemotherapy increase the necrotic core with underlying inflammation, vascular changes and blood-brain barrier disruption, leading to enhanced immune cell infiltration and brain edema. Accordingly, subsequent molecular and cellular changes limit the efficiency of the current therapies. In addition, therapy resistance is also established by the synergistic communication between glioma cells and their microenvironment. The secretion of inflammatory mediators and angiogenic factors by the tumor and immune cells promotes a pro-tumorigenic immunosuppressive environment, synergistically promoting tumor cell proliferation and immune cell infiltration within the TME. Furthermore, several immune checkpoint systems implemented by tumor cells also prevent the anti-tumorigenic activity of infiltrating lymphocytes.Therefore, non-invasive imaging techniques, such as magnetic resonance imaging (MRI) and positron emission tomography (PET) could unravel and longitudinally capture the heterogeneity and spatio-temporal dynamics of the glioblastoma microenvironment in vivo, ultimately supporting the development of new immunotherapies targeting the different components of the TME .Single-cell profiling of myeloid cells in glioblastoma across species and disease stages reveals macrophage competition and specialization over time . In non-Like TAMs, myeloid-derived suppressor cells (MDSCs) are a heterogeneous group of cells originating from myeloid precursor cells. MSDCs can be subdivided into polymorphonuclear (PMN)-MDSCs and monocytic (M)-MDSCs. PMN-MDSCs, regarded as pathologically activated neutrophils, are the main population of MDSCs in mice and humans . As for To better understand the role and the dynamics of glioma-associated myeloid cells (GAMs) and other immune players on tumor progression, therapy response and recurrence, the development of new molecular imaging approaches is crucial to longitudinally characterize the immune components of the TME . Moreove2-weighted (T2w), FLAIR and pre- and post-gadolinium enhanced T1-weighted (T1w) imaging. Diagnosis specificity could be improved by diffusion- and perfusion-weighted imaging to distinguish GBM from other tumor types and by nuclear imaging, such as amino acid PET imaging FDOPA dynamics were found to predict the isocitrate dehydrogenase (IDH) mutations and the 1p/19q codeletion , which are determinant in patients\u2019 prognosis FET, [18F]FLT, [11C]MET and [18F]FDOPA were clinically tested in glioma patients [18F]FET is a valuable marker for active glioma volume with significantly higher sensitivity and specificity in the diagnosis of brain tumors over MR, [18F]FDG or [18F]FLT PET imaging [18F]FET or [11C]MET signal exceeded the hyperintense glioma area depicted by T2w- or T1w-Gd-MRI and reported valuable complementary information to conventional MR images [2w and [11C]MET hyperintense signal may delineate therapy-related tissue change, therefore, allowing the distinction between metabolic active tumor tissue and treatment-related changes in patients with gliomas FLT, CMET and FFLT, CME imaging ,20,21, wR images ,23. In p18F]FET to discriminate between glioma recurrence and treatment-induced changes. The results indicated that [18F]FET PET imaging provided a good diagnostic performance (sensitivity: 86.2% (95% CI: 68.3\u201396.1%) and specificity 81.3% (95% CI: 54.4\u201396.1%)) [18F]FET accuracy in diagnosis was reduced in patients with IDH-mutant gliomas. Besides, metabolic changes after bevacizumab treatment could be identified by [18F]FET PET imaging earlier than structural changes detected by MRI, providing an earlier indication of glioma progression FET uptake and volume were significantly reduced after adjuvant temozolomide (TMZ) chemotherapy in an experimental glioma model, in line with the concomitant decrease in gadolinium-enhanced T1w-MR-based tumor volume [18F]FET metabolic tumor volume (MTV) and maximum tumor-to-background ratio (TBRmax) in 41 newly diagnosed glioma patients that underwent resection and two cycles of chemotherapy FET PET imaging in the assessment of the treatment response compared to MRI. Overall, [18F]FET imaging could help patients\u2019 management, including the diagnosis of pseudoprogression after TMZ chemotherapy and assessment of other treatment options.Similarly, PK11195 PET tracer has been investigated but appeared limited by poor pharmacokinetics and pharmacodynamics . The second- and third-generation tracers have shown superior imaging properties, while they are not yet fully characterized [18F]FET PET and MR imaging, (iii) be a suitable biomarker for glioma growth and immune cell infiltration, and (iv) define glioma heterogeneity in combination with other imaging modalities GE-180 tracer indicated that TSPO signal was found in areas beyond contrast-enhanced MR regions, with significant tumor-to-background contrast [18F]GE-180 has been questioned for its ability to cross the intact BBB, [18F]GE-180 uptake seems to be independent of BBB breakdown GE-180, [18F]FET and CE-MRI VOIs in new diagnosed gliomas patients FET PET signal, some patients showed higher [18F]GE-180 signal compared to other patients showing milder tracer uptake, ultimately indicating a higher level of immune infiltration and a potentially worse outcome.The same group recently published a voxel-based analysis of [patients . They co18F]GE-180 (46 \u00b1 27%) and [18F]FET PET (32 \u00b1 18%), as previously reported FET and [123I]CLINDE tracer uptake [18F]FET VOI was larger (79\u201393%) than with [123I]CLINDE SPECT VOI (15\u201330%) while the percentage of overlap between the two VOI tracers was quite limited, reinforcing that the combination of [18F]FET, TSPO and MR imaging allows detecting the tumor mass beyond CE-MRI at baseline scan. Furthermore, follow-up change in gadolinium-CE VOI overlapped to a greater extent with baseline [123I]CLINDE VOI than [18F]FET VOI, indicating that TSPO SPECT could be more representative of progressive tumor areas than [18F]FET [Jensen et al. (2015) reported the temporal change of [r uptake . At the [18F]FET .18F]GE-180 PET uptake was associated with the histological WHO grade, with the highest uptake values observed in WHO grade IV glioblastomas while all TSPO-negative cases were WHO grade II gliomas. Along the same lines, Su et al., indicated that the number of TSPO-positive neoplastic cells increased with glioma grade, with the highest TSPO expression level detected in confirmed glioblastoma patients PK11195 tracer uptake in glioblastoma patients and identified different tracer uptake patterns between low-grade astrocytoma and oligodendroglioma DPA-714 PET imaging as an imaging readout for the degree of immunosuppressive myeloid cell infiltration.On the one hand, Zinnhardt and colleagues supported the use of TSPO PET to determine the degree of immunosuppressive myeloid cell infiltration and, therefore, its use as a prognostic imaging biomarker for mechanisms of resistance in the context of the therapeutic modulation of the immunosuppressive TME 35]. TS. TS35]. 18F]DPA-714 PET signal. Therefore, the authors concluded that TSPO overexpression could be induced by a pro-inflammatory microenvironment.On the other hand, Pannell et al. investigated whether TSPO upregulation in astrocytes and microglia/macrophages was restricted to a specific phenotype . TSPO ovnu/nu mice orthotopically implanted with Gli36dEGFR cells [18F]FET VOI while the exclusive area of [18F]DPA-714 VOI was increased post-treatment. Ex vivo characterizations, in line with the PET imaging data, showed a higher number of TSPO+ Iba-1+ and TSPO+ GFAP+ cells in TMZ-treated mice compared to control mice, indicating GAMs infiltration and increased astrocytic reactivity after treatment. The preclinical data support the use of TSPO PET imaging and the further assessment of exclusive areas of [18F]DPA-714 tracer uptake to detect areas of GAMs infiltration after TMZ.The suitability of combining amino acid with TSPO PET imaging to track therapy response after TMZ chemotherapy has been addressed in NMRIFR cells inhibitor to efficiently deplete GAMs within the TME in an orthotopic syngeneic GL261 mouse model and assessed therapy response using [njection 37]. Th. Th18F]F18F]FET VOI under CSF-1R inhibition and withdrawal. The authors indicated that the TSPO PET volume increase under CSF-1R inhibition-induced GAMs depletion might result from the increase in TSPO+ tumor cell proliferation, as indicated by the simultaneously increased in [18F]FET PET volume.The same tracer dynamics were observed for tumor-based [18F]DPA-714 PET volume. Overall, these preclinical data supported that [18F]DPA-714 PET tracers may highlight an immunosuppressive TME.The authors hypothesized that the increased peripheral immune cell infiltration triggered by GAMs depletion could promote tumor cell proliferation. Interestingly, inhibitor withdrawal may reprogram both GAMs and MDSCs toward an anti-tumorigenic phenotype, in line with the reduction in GE-180 as a PET tracer to visualize and quantify TSPO expression in the brain. Back-translation into a GL261 glioma mouse model supported the use of [18F]GE-180 to longitudinally track TSPO expression in experimental glioma and, therefore, its applicability as a diagnostic tool in patients [18F]GE-180 uptake parameters, including median background activity and TBRmax, are insensitive to polymorphism [18F]GE-180 PET provided a high tumor-to-background ratio (TBR) in untreated and recurrent glioma GE-180 PET tracers remain to be evaluated.Alongside, recent investigations debate the suitability of BR-351 tracer has been reported to efficiently target MMPs, showing a higher affinity for activated MMP-9 and MMP-2 compared to other MMPs DPA-714 (4%) or [18F]BR-351 (11%) VOIs along the tumor rim could be identified. Only a small fraction of the [18F]DPA-714 VOI (14%) and [18F]BR-351 VOI (11%) was not overlapping with [18F]FET VOI. According to the authors, a small part of the [18F]BR-351-derived volume that was not detected by [18F]FET might hint toward regions of glioma invasion. Accordingly, GAMs were found to express MMP-9 and spatially overlapped with [18F]BR-351 PET signal. Overall, multi-tracer and multimodal molecular imaging approaches may allow us to gain important insights into glioma-associated inflammation and differentiate between subpopulations of functionally distinct immune cells.As an example, Zinnhardt et al. investigated the spatiotemporal relationship between TSPO expression and MMPs in a mouse model of human patterns of glioma pathogenesis, hypothesizing that both markers may be found at sites of glioma infiltration . Using aThe combination of different imaging modalities represents a suitable approach to unravel tissue heterogeneity. Given the limitations of MR and amino acid PET imaging, additional TSPO PET imaging seems beneficial to highlight metabolically active regions beyond tumor cells that support therapy resistance. Clinical studies agree on the suitability of quantitative FET and TSPO PET imaging for glioma grading and categorization, which ultimately may help in planning individualized strategies for brain tumor therapy. Additionally, combining amino acid and TSPO imaging represents an interesting way to discriminate glioma cells from glioma-associated myeloid cells. However, it remains unclear if the TSPO signals in glioma-associated myeloid cells could be associated with anti-tumorigenic or immunosuppressive phenotype/functions since their activity is hindered by glioma cells in many studies. Therefore, current research should be supported by in-depth characterization in preclinical glioma models as a back-translation approach or by the combination of different biological markers to characterize the aggressive tumor mass and TSPO."} +{"text": "The genetic underpinning of sexual dimorphism is very poorly understood. The prevalence of many diseases differs between men and women, which could be in part caused by sex-specific genetic effects. Nevertheless, only a few published genome-wide association studies (GWAS) were performed separately in each sex. The reported enrichment of expression quantitative trait loci (eQTLs) among GWAS-associated SNPs suggests a potential role of sex-specific eQTLs in the sex-specific genetic mechanism underlying complex traits.To explore this scenario, we combined sex-specific whole blood RNA-seq eQTL data from 3447 European individuals included in BIOS Consortium and GWAS data from UK Biobank. Next, to test the presence of sex-biased causal effect of gene expression on complex traits, we performed sex-specific transcriptome-wide Mendelian randomization (TWMR) analyses on the two most sexually dimorphic traits, waist-to-hip ratio (WHR) and testosterone levels. Finally, we performed power analysis to calculate the GWAS sample size needed to observe sex-specific trait associations driven by sex-biased eQTLs.cis-eQTLs (FDR 5%). Our phenome-wide association study of the 18 top sex-biased eQTLs on >700 traits unraveled that these eQTLs do not systematically translate into detectable sex-biased trait-associations. In addition, we observed that sex-specific causal effects of gene expression on complex traits are not driven by sex-specific eQTLs. Power analyses using real eQTL- and causal-effect sizes showed that millions of samples would be necessary to observe sex-biased trait associations that are fully driven by sex-biased cis-eQTLs. Compensatory effects may further hamper their detection.Among 9 million SNP-gene pairs showing sex-combined associations, we found 18 genes with significant sex-biased Our results suggest that sex-specific eQTLs in whole blood do not translate to detectable sex-specific trait associations of complex diseases, and vice versa that the observed sex-specific trait associations cannot be explained by sex-specific eQTLs.The online version contains supplementary material available at 10.1186/s13073-022-01088-w. Men and women exhibit sexual dimorphism. Clear examples of sex-biased traits are anthropometric features. However, biological differences between sexes are not limited to physical traits: sex differences are also evident in incidence, prevalence, and severity across diseases. For example, women are much more likely to develop autoimmune , while mDespite the widespread nature of these sexual differences and their noteworthy implications for medical research and treatments, little is known about their underlying biology in complex traits. While the sex chromosomes play key roles in sexual dimorphism, genome-wide association studies (GWAS) have identified dozens of autosomal genetic variants showing sex-specific effects \u201310, suggThe strong enrichment of expression quantitative trait loci (eQTLs) among complex trait-associated loci \u201315 suggePrevious studies that explored this hypothesis revealed that sex-biased eQTLs are associated with traits known to exhibit sex differences, including body mass index, blood pressure, lipid traits, breast cancer, and several autoimmune diseases \u201321. HoweHere we performed a genome-wide analysis of sex-specific whole blood RNA-seq eQTLs from 3447 European individuals included in BIOS Consortium, and sought to replicate in an independent European cohort. To assess the potential contribution of sex-biased eQTLs to sex differences in complex traits, we performed PheWAS in >700 phenotypes from UKBiobank (UKB) and invehttp://www.bbmri.nl/acquisition-use-analyze/bios/) Consortium has been set up in an effort of several Dutch biobanks to create a homogenized dataset with different levels of \u201comics\u201d data layers. Genotyping was performed in each cohort separately, as described before: LifeLines DEEP . Table S8. BIOS results for the sex-biased eQTLs found in Kukurba et al [PMID: 27197214]. Table S9. Replication analyses results - SHIP-Trend cohort. Table S10. PheWAS for top sex-specific eQTLs. Table S11. Independent SNPs associated with WHR. Table S12. Independent SNPs associated with testosterone. Table S13. Sex-stratified TWMR results for testosterone. Table S14. Sex-stratified TWMR results for WHR. Table S15. Sex specific causal genes for WHR and testosterone."} +{"text": "This cohort study examines the association of self-reported postvaccination symptoms with anti\u2013SARS-CoV-2 antibody response among Framingham Heart Study participants contributing to the Collaborative Cohort of Cohorts for COVID-19 Research study. We studied the association of self-reported postvaccination symptoms with anti\u2013SARS-CoV-2 antibody response among Framingham Heart Study (FHS) participants contributing to the Collaborative Cohort of Cohorts for COVID-19 Research (C4R) study.4SARS-CoV-2 messenger RNA (mRNA) vaccines (BNT162b2 [Pfizer-BioNTech] and mRNA-1273 [Moderna]) are associated with local and systemic symptoms; however, whether postvaccination symptoms are associated with vaccine-induced antibody response is unknown. Previous studies5 Associations between postvaccination symptoms and antibody response were assessed by \u03c72 test and multivariable linear regression, with complete case analyses adjusted for batch, time since vaccination, and sociodemographic and clinical characteristics. A 2-sided P\u2009<\u2009.05 was considered statistically significant. Protocols were approved by institutional review boards of participating institutions and the National Heart, Lung, and Blood Institute. Written informed consent was obtained from all participants. This study followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guideline.The FHS is an ongoing, prospective cohort study evaluating cardiovascular disease risk factors. In February 2021, participants were invited to self-administer C4R questions on COVID-19 vaccination (and associated symptoms) and submit a dried blood spot to test for anti\u2013SARS-CoV-2 antibodies . In adjusted models, systemic symptoms were associated with greater antibody response, although associations were attenuated with sequential adjustment for potential confounders completed the C4R questionnaire and blood spot collection and reported 2 doses of BNT162b2 (414 [45%]) or mRNA-1273 (514 [55%]) vaccines (eFigure in the 6 in US health care workers that showed higher postvaccination antibody measurements among those with significant symptoms after an mRNA vaccine. This report identifies age, sex, and Moderna vaccine as factors associated with both vaccine reactogenicity and immunogenicity, consistent with prior observations.6 No association was observed between symptoms after vaccination and race or ethnicity, body mass index, or comorbidities. In this generalizable cohort, nearly all participants exhibited a positive antibody response to complete mRNA vaccine series. Nonetheless, systemic symptoms remained associated with greater antibody response in multivariable-adjusted models, highlighting unexplained interpersonal variability. Further research on biological mechanisms underlying heterogeneity in vaccine response is needed. Limitations of this report include an older, predominantly non-Hispanic White, professional cohort; potential recall bias; and use of MFI, which is not standardized against neutralizing antibody titers. In conclusion, these findings support reframing postvaccination symptoms as signals of vaccine effectiveness and reinforce guidelines for vaccine boosters in older adults.In a sample of twice-vaccinated, older, community-dwelling US adults, self-reported systemic symptoms after SARS-CoV-2 mRNA vaccination were associated with greater antibody response vs local-only or no symptoms. These results agree with a previous report"} +{"text": "Here we describe the emerging roles of ncRNAs in PDAC and highlight how these ncRNAs can be used to detect and control this intractable cancer.An international project on the human genome revealed that various RNAs ) are involved in the pathogenesis of different human diseases, including cancer. Recent studies have highlighted the critical roles of lncRNAs and circRNA in pancreatic ductal adenocarcinoma (PDAC), especially in the epithelial\u2013mesenchymal transition, a phenomenon regulating cancer metastasis. Growing research in this field has indicated that the tertiary structure of lncRNAs supposedly regulates biological function Cancer, a genetic disease, involves multiple mutations in cell growth-promoting and death-inhibiting oncogenes and growth-restricting tumor suppressor genes. These mutations arise from various genetic alterations, including those in both coding and noncoding regions of chromosomes . PositioAlthough ncRNA are generally defined as nonprotein-coding, notably, previous ribosome profiling studies suggest that 40%\u201390% of the annotated lncRNAs undergo translation , indicatSNAI1), SNAI2/SLUG, TWIST family basic helix-loop-helix transcription factor 1, zinc finger E-box-binding homeobox 1 (ZEB1), and ZEB2 -related mechanisms , althougmiR-382 binding to the target enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2), and regulates EMT-related gene expression via epigenetic control gene cluster on chromosome 12 and is coexpressed alongside the HOXC genes -mediated chromatin regulation , which subsequently promotes EMT and the subpopulation of cancer stem-like cell phenotypes by acting as a miR-145 sponge in pancreatic cancer cells, potentially associated with malignant behavior and chemoresistance .ADPGK-AS1 (human chromosome 15q24.1) overexpression promotes PDAC progression via the ceRNA mechanism involving miR-205-5p by activating ZEB1-mediated EMT promotes the invasiveness of PDAC via the miR-665/TGFBR1-TGFBR2/SMAD2/3 pathway overexpression in lung cancer and PDAC suppresses miR-34a and miR-203 expression, thereby upregulating the transcription factors SNAI1 and SNAI2, consequently repressing cadherin 1/E-cadherin expression and type III iodothyronine deiodinase gene (DIO3) locus with MEG3 in EMT regulation is a maternally expressed imprinted gene overexpression in PDAC promotes cancer cell migration, invasion, and EMT via a typical ceRNA mechanism involving the sponging of miR-429 to modulate ZEB1 expression (lncRNA pression .ROR (human chromosome 18q21.31) overexpression in PDAC promotes EMT via the ZEB1 pathway pathway overexpression in PDAC promotes cell metastatic properties by influencing EMT, indicating a poor PDAC prognosis, whereas its silencing elicits apoptosis by impacting the B-cell chronic lymphocytic leukemia/lymphoma 2/caspase-3 pathway is conserved evolutionary from its 3' end overexpression in PDAC facilitates cancer proliferation and metastasis through circ-NEIL3/miR-432-5p/adenosine deaminases acting on the RNA 1 (ADAR1) axis (circ-NEIL3 regulates ADAR1 expression by sponging miR-432-5p to induce RNA editing of glioma-associated oncogene 1 (GLI1), ultimately influencing cell cycle progression and promoting EMT in PDAC cells (via ADAR1 through a negative feedback loop (R1) axis . MechaniAC cells . This prack loop . RNA ediack loop . The ediack loop .circ-0001666 overexpression in PDAC increases transcription factor SOX4 expression, a direct downstream effector of miR-1251, by binding to miR-1251 (circ-0001666 repressed EMT in PDAC cells by upregulating miR-1251 and downregulating SOX4 (circ-0001666 functions via a ceRNA mechanism.miR-1251 . Silenciing SOX4 . This stcirc-0092367 (transcribed from the SNORD116-14 gene [ENSG00000206621]) is significantly downregulated in PDAC and inhibits EMT phenotypes and sensitizes PDAC cells to gemcitabine treatment both in vitro and in vivo via the miR-1206/epithelial splicing regulatory protein 1 (ESRP1) axis axis . ESRP1 rring EMT . circ-00circ-0092314 overexpression in PDAC induces EMT by sponging miR-671, increasing S100P expression overexpression in PDAC activates collagen type XI alpha 1 chain by targeting miR-20a-3p to promote PDAC progression overexpression in PDAC regulates ADP ribosylation factor-like GTPase 4C expression by sponging miR-1306-5p to promote PDAC progression. Circ-UHRF1 expression in PDAC cells was transcriptionally regulated via the interferon regulatory factor 3 . These can be useful for predicting the prognosis of patients with PDAC (myoregulin, an important regulator of skeletal muscle physiology (LINC00961 encodes the small regulatory polypeptide of amino acid response (While clinical approaches indicated improvements in the first-line therapy, the 5-year overall survival only shows an increase from 5% to 10%, and surgical resection is only available for 20% patients with advanced PDAC, indicating that the disease is fatal for humans . This caith PDAC . Neverthith PDAC . Alteredysiology . The stuysiology . For exaresponse . Using tvia binding assays with mutated miRNA binding sites and luciferase translation readout as, similar to mRNAs, a significant portion of the noncoding transcriptome, including lncRNAs and pseudogenes, harbors miRNA-response elements that are targets of ceRNA (Notably, the current information indicates that almost all circRNAs are involved in the ceRNA mechanism, whereas linear lncRNAs are associated with diverse functions and not restricted to the ceRNA mechanism. The lncRNA\u2013miRNA\u2013mRNA ceRNA network has been implicated in various cancers, including lung , tongue of ceRNA . The facof ceRNA ."} +{"text": "Knee osteoarthritis (OA) pain often challenges the physical activity necessary for managing life-threatening chronic diseases. Standard severe knee OA treatment of total knee arthroplasty (TKA) and physical therapy (PT) is effective at improving pain and function but whether such benefits translate to physical activity is unclear. We enrolled 22 participants with severe knee OA scheduled for a TKA and assessed pain , physical function , and physical activity before and 1-month, 3-month, and 6-month after TKA. Using paired-wise ANOVA, pre-to-postTKA pain and function improved but not physical activity. Using regression analyses, outpatient PT sessions during first two months post-TKA were positively related to 3- and 6-month physical activity . Standard TKA and PT for severe OA improved pain and function but not physical activity. However, early post-TKA PT and physical activity relationships are promising, warranting exploration."} +{"text": "Neurophysiological data showed reduced lower-limb maximum voluntary force production and an attenuation of upper-limb and lower-limb skeletal muscle voluntary activation, accompanied by more pronounced upper-limb fatigability in MS-HF versus MS-LF. Results suggest that MS fatigue is characterised by greater cortico-subcortical grey matter atrophy and neural lesions, accompanied by neurophysiological decrements, which include reduced strength and voluntary activation.Fatigue is one of the most debilitating symptoms for people with multiple sclerosis (PwMS). By consolidating a diverse and conflicting evidence-base, this systematic review and meta-analysis aimed to gain new insights into the neurobiology of MS fatigue. MEDLINE, ProQuest, CINAHL, Web of Science databases and grey literature were searched using Medical Subject Headings. Eligible studies compared neuroimaging and neurophysiological data between people experiencing high (MS-HF) versus low (MS-LF) levels of perceived MS fatigue, as defined by validated fatigue questionnaire cut-points. Data were available from 66 studies, with 46 used for meta-analyses. Neuroimaging studies revealed lower volumetric measures in MS-HF versus MS-LF for whole brain (\u00ad22.74\u00a0ml; 95% CI: -37.72 to -7.76\u00a0ml; Prospero registration\u00a0Prospero registration number: CRD42016017934The online version contains supplementary material available at 10.1007/s11065-021-09508-1. Perceived fatigue includes subjective feelings of weariness and an increased subjective perception of effort for everyday tasks and is commonly rated in PwMS with the Fatigue Severity Scale (FSS) and Modified Fatigue Impact Scale (MFIS) experience fatigue symptoms persistently or sporadically neuroimaging and neurophysiological data acquired from senior authors which were not originally presented by fatigue status of PwMS in the published report. By meta-analysing previously reported dichotomised data for MS-HF versus MS-LF, the main aim was to gain an improved insight into structural and neurophysiological correlates of MS fatigue.https://www.crd.york.ac.uk/prospero/display_record.php?RecordID=17934). A systematic literature search of PubMed/MEDLINE, ProQuest, CINAHL and Web of Science from inception until 31 December 2019 was undertaken, blinded to title and authorship, by two reviewers (PE & SG). The search strategy was conducted using Medical Subject Headings (MeSH) and search terms included those related to MS, fatigue, neurophysiology, neuroimaging, MVC, motor nerve stimulation statement , where it was available but not reported as such in the published article, and these authors have been acknowledged. In the case of the same cohort data being reported in\u2009>\u20091 article, only the most recent publication was included. Non-English animal studies, case studies, review articles, randomised controlled trials and other controlled trials, pharmacological trials and studies that only reported other physical/psychological outcomes were excluded.Eligible articles reported data from cross-sectional studies using a validated fatigue scale and defined cut-points for differentiating MS-HF from MS-LF. Adults\u2009>\u200918\u00a0years with definite multiple sclerosis and all types of MS were eligible for inclusion. The included studies must have reported neuroimaging measures or neurophysiological variables for MS-HF and MS-LF. However, 14 authors of 16 peer-reviewed published studies provided original data partitioned by http://www.ncbi.nlm.nih.gov/books/NBK35156/) . Due to variation in clinical or demographic characteristics and fatigue assessments across studies, a random-effects model was applied throughout, and meta-analyses were not guided by the quality assessment data for individual included studies. A p value\u2009<\u20090.05 indicated statistical significance for an overall effect and the magnitude of heterogeneity across studies was tested using the I2 statistic: I2 values\u2009<\u200925%, 25\u201350%, or\u2009>\u200950% indicate low, moderate and high heterogeneity, respectively as follows: (1) Study design; (2) Characteristics of the participants ; (3) Primary outcomes; (4) Secondary outcomes. Means and standard deviations for each variable were extracted for meta-analyses using RevMan 5.0 , diffusion tensor imaging (DTI) and magnetic resonance spectroscopy (MRS). Measures included total normalised brain volume, grey and white matter volumes, T1-weighted hypointense and T2-weighted lesion volumes, white matter microstructural integrity and neuronal/axonal integrity and function (N-acetylaspartate to creatine [NAA/Cr] ratio and choline to creatine [Cho/cr] ratio by MRS). Neurophysiological measures for meta-analyses were obtained using transcranial magnetic stimulation (TMS), electroencephalography (EEG), neuromuscular electrical stimulation (NMES) and electromyography (EMG) and included motor evoked potential (MEP) amplitude, MEP latency, MEP threshold, central motor conduction time, short-interval intracortical inhibition (SICI), and voluntary activation using the twitch-interpolation technique during MVC Merton, . Brain rThe results of meta-analyses are presented as absolute mean differences with 95% confidence intervals (CI) in Table Most of the included studies (70%) were classified as being of \u201cmoderate quality\u201d, 16 24%) studies were rated as \u201clow quality\u201d and four studies (6%) as \u201chigh quality\u201d in MS-HF versus MS-LF, accompanied by a reduction in the volume of grey matter in MS-HF versus MS-LF . There was no significant difference in white matter volume between MS-HF and MS-LF . Larger reductions in mean normalised brain volume, grey and white matter volumes were apparent for MS-LF and MS-HF versus HC and accumbens volumes for MS-HF versus MS-LF. Larger effect-size reductions in thalamus and caudate volumes did not reach statistical significance because of wider confidence intervals and there were high levels of heterogeneity in MS-LF and MS-HF versus HC, and for the accumbens in MS-HF versus HC and for MS-LF and MS-HF versus HC (p\u2009<\u20090.0001). However, there were no differences between MS-HF and MS-LF for T2-weighted lesion volume , white matter microstructural integrity or axonal integrity/function (NAA/Cr or Cho/Cr by MRS). There was an increase in DTI mean diffusivity for MS-HF and MS-LF versus HC, and a reduction in the NAA/Cr ratio in MS-HF versus HC , indicating relative impairment of microstructural and axonal integrity/function and upper-limb MVC force in MS-HF versus MS-LF, with the latter of borderline statistical significance. Reductions in upper-limb MVC force were also apparent in MS-LF and MS-HF versus HC (p\u2009\u2264\u20090.03). Meta-analysis of studies which used the twitch-interpolation technique during a MVC showed reduced voluntary activation in MS-HF versus MS-LF for upper-limb and lower-limb skeletal muscles . Upper-limb muscles included finger and thumb flexors/extensors and lower-limb muscles included the quadriceps and dorsiflexors . A more pronounced level of upper-limb motor fatigability was also observed for MS-HF versus HC skeletal muscle fatigue task (either sustained N\u2009=\u20093] or intermittent N\u2009=\u20092] isometric MVC) revealed greater motor fatigability for MS-HF versus MS-LF , this systematic review and accompanying meta-analyses aimed to provide an improved insight into structural and neurophysiological correlates of MS fatigue. By robustly consolidating an extensive and somewhat conflicting evidence-base, the results suggest that higher levels of MS fatigue are characterised by greater cortico-subcortical atrophy, and with indications of greater neural damage, as evidenced by an increased volume of T1-weighted hypointense lesions in MS-HF versus MS-LF, which consolidates a conflicting body of data on this measure from studies investigating PwMS (irrespective of fatigue status) versus HC in MS-HF versus MS-LF, suggesting MS-HF have a relatively impaired ability to fully activate skeletal muscles in comparison with MS-LF. This may explain the observed reduction in MVC in MS-HF versus MS-LF, as previous studies have reported significant correlations between the decline in MVC and voluntary activation during sustained muscular contractions in PwMS or intracortical facilitation (ICF), despite reports of altered functional connectivity and hyperactivation in fronto-parietal cortical regions, sensorimotor network and subcortical areas important for motor, sensory and cognitive processing in MS-HF compared lower-limb motor fatigability between MS-HF and MS-LF within the same participants. The broad-ranging patient characteristics and lack of participant ethnicity data across different studies may also be considered as a limitation, although confounders such as disease severity, level of disability, sex and age were minimised in the larger data-set meta-analyses. Nevertheless, some of the meta-analyses included a small number of studies and as the overall quality rating of included studies was \u2018moderate\u2019, as such, caution is needed when interpreting these results. In addition, although the Agency for Healthcare Research and Quality , and increased upper-limb motor fatigability. By consolidating an extensive and somewhat conflicting evidence-base, the meta-analysis provides new insights into neurobiological differences that exist between MS-HF and MS-LF.Supplementary file1 (DOCX 13982 KB)Supplementary file2 (DOCX 9677 KB)Supplementary file3 (DOCX 9680 KB)Supplementary file4 (DOCX 15 KB)Supplementary file5 (DOCX 38 KB)Supplementary file6 (DOCX 215 KB)Below is the link to the electronic supplementary material."} +{"text": "We present an updated list of anti-aging/pro-longevity strategies to address limitations of previous food/nutrient combinations. Our list has two unusual features: (i) all interventions should be administered concurrently so no fundamental causes of A-MCI-Alz are without remediation and can inflame and leave smoldering important causal factors, (ii) all interventions should be provided daily or weekly to help avoid re-ignition of key factors driving A-MCI-Alz. These two features can help explain past longevity-trial failures[PMC9310407]: no previous study to our knowledge has included most or all of the following interventions, so untreated causes of A-MCI-Alz likely remained active[PMC9114803]. Our recommended interventions include intermittent-or-continuous calorie restriction or fasting, protein- and/or methionine-restriction, consumption of moderately-strong metal chelators , antioxidant-rich foods[PMC7530501], B vitamins including vitamin B5 (pantothenic acid[PMC9232186, PMC8725342,"} +{"text": "Non-alcoholic fatty liver (NAFL) is the most common chronic liver disease. Activation of mitogen-activated kinases (MAPK) cascade, which leads to c-Jun N-terminal kinase (JNK) activation occurs in the liver in response to the nutritional and metabolic stress. The aberrant activation of MAPKs, especially c-Jun-N-terminal kinases (JNKs), leads to unwanted genetic and epi-genetic modifications in addition to the metabolic stress adaptation in hepatocytes. A mechanism of sustained P-JNK activation was identified in acute and chronic liver diseases, suggesting an important role of aberrant JNK activation in NASH. Therefore, modulation of JNK activation, rather than targeting JNK protein levels, is a plausible therapeutic application for the treatment of chronic liver disease. RO. RO+2 flThe JNK-interacting protein (JIP) family plays an integral role in MAPK cascade activation by creating proximity of upstream kinases and terminal kinases ,70. JIP1de novo lipogenesis. Fat accumulation in hepatocytes causes lipotoxicity and apoptosis allele is associated with an increased risk of diet related hepatocellular carcinoma . The PNPP-JNK activates transcriptional co-repressor NCoR1 and suppresses PPAR\u03b1 activation in NASH, affecting decreased expression of genes involved in fatty acid transport and oxidative degradation in mitochondria and peroxisomes . ConsistNASH is the hepatic steatosis with liver cell inflammation and innate-immune system activation involving Kupffer cells (KC) and production of pro-inflammatory cytokines in the liver. Extracellular vesicles, fatty acids, cholesterol released from steatotic hepatocytes and lipopolysaccharides (LPS) activate KCs and hepatic stellate cells (HSCs) via damage-associated molecular pattern receptors, known as Toll-like receptor 4 (TLR4) and TNF-related apoptosis-inducing ligand death receptor (TRAIL-R2) . Mice wiIn addition, CD62E (E-Selectin) and CD44 which recruit leukocytes into inflammation sites are upregulated in NASH patients . P-selecJNK activation in HSCs in response to TGF-\u03b2 and platelet-derived growth factor (PDGF) activates Smad2/3 leading to \u03b1-smooth muscle actin (\u03b1SMA) expression, migration of resident HSCs and myofibroblasts, and fibrosis in NASH . Notablywww.clinicaltrials.gov, accessed on 18 July 2022). However, the study that aims to reduce level of P-JNK activation via targeting sustained-JNK-activation-loop is very sparse and mentioned here, and prospective targets are discussed.Chronic hepatic inflammation causes fibrosis leading to liver cirrhosis. One-carbon metabolism, mitochondrial stress, ER stress, DNA damage, inflammation, and obesity are involved in carcinogenesis in NAFLD. Patients who develop cirrhosis related to NASH are at risk for hepatocellular carcinoma and/or end-stage liver failure and liver transplantation. Currently there is no drug approved by US Food and Drug Administration (FDA) for the treatment of NASH. Lifestyle alterations remain the only treatment. Steatosis, inflammation, ballooning, and fibrosis were improved in those achieving >5% weight loss, with even greater improvement in patients achieving >10% weight loss. Several molecules are in clinical trials is a scaffolding protein where upstream MAPKs activate JNK. BI-78D3 is able to compete with the D-domain of JIP1 for JNK binding and thus inhibits JNK activation. BI-78D3 effectively prevent CCL4 induced acute liver injury [r injury , yet theASK1 and ASK1 inhibitor: Apoptosis signaling-regulating kinase 1 (ASK1) activity directly involves P-JNK-SAB-ROS activation loop through redox sensitive thioredoxin [oredoxin ,147. ASKoredoxin , but funoredoxin but was oredoxin . SelonseMLKs and MLK inhibitor: The stress kinase mixed lineage kinase is also a MAP3K activating MKK4/7 and then JNK/p38. MLK3-JNK mediates the hepatic extracellular vesicle release [\u2212/\u2212MLK3 mice have reduced CXCL10 levels in their plasma EVs and, hepatoprotection against injury and inflammation. Furthermore, pharmacological MLK3 inhibitor, URMC099, reduces circulating CXCL10 and attenuates murine NASH [ release ,151. Genine NASH ,153. Preine NASH ,155. EffThe understanding the mechanism of kinase activation and signal regulation will generate therapeutic target molecules to develop a safe and effective treatment. However, limitations and caveats do exist because of similarity of the kinase activation and interaction with its substrates through a pattern of amino acid sequence, motif, creating unwanted off target side effects. Finding the targetable proximity molecules in the P-JNK activation loop,"} +{"text": "Remote Infectious Disease (ID) physicians can provide stewardship support through telehealth. Using the RE-AIM framework, we assessed the implementation of telehealth-supported prospective-audit-and-feedback (tele-PAF) across 3 rural Veterans Administration medical centers (VAMC).All 3 invited sites agreed to participate and lacked ID support for stewardship at baseline. During 2021, an ID physician met virtually 3 times/week with the stewardship pharmacist champion at each participating VAMC to review patients on antibiotics in acute-care (mean daily census 3/site) and nursing-homes ; real-time feedback on antibiotic use was given to clinicians. The primary outcome of effectiveness was monthly antibiotic days of therapy (DOT) per 1,000 days-present aggregated across all sites; the secondary outcome was days of antibiotic spectrum coverage (DASC) per 1,000 days-present. An interrupted time-series analysis was performed to asses these outcomes during the 1-year intervention period vs. the 2-year prior baseline. Semi-structured interviews with 20 clinicians and pharmacists were conducted to assess implementation.RE-AIM elements are summarized in Table\u00a01. Tele-PAF reviewed 502 unique patients and made 681 recommendations to 23 clinicians; 77% of recommendations were accepted. The most common recommendations were to stop antibiotics (28%) and change duration (20%). After the start of tele-PAF, antibiotic DOT and DASC immediately decreased in acute-care and NHs . Both metrics began to rise again in acute-care but were stable in NHs . Clinicians generally appreciated feedback, found it compatible with their workflow and responded favorably to collaborative discussions. Barriers included difficulty establishing rapport with some providers.The implementation of tele-PAF was associated with sustained reductions in antibiotic use across 3 NHs but not in the studied small acute-care units. Overall, clinicians perceived the intervention as acceptable and appropriate. Wider implementation of telehealth-supported stewardship activities may achieve reductions in antibiotic use.Daniel J. Livorsi, MD, Merck & Co.: Grant/Research Support."} +{"text": "The plant homeodomain (PHD)-finger gene family that belongs to zinc-finger genes, plays an important role in epigenetics by regulating gene expression in eukaryotes. However, inaccurate annotation of PHD-finger genes hinders further downstream comparative, evolutionary, and functional studies.Arabidopsis thaliana (Arabidopsis), Oryza sativa (rice), Capsicum annuum (pepper), Solanum tuberosum (potato), and Solanum lycopersicum (tomato) to better understand the role of PHD-finger genes in these species. Our investigation identified 875 PHD-finger genes, of which 225 were newly identified, including 57 (54%) novel PHD-finger genes in pepper. The PHD-finger genes of the five plant species have various integrated domains that may be responsible for the diversification of structures and functions of these genes. Evolutionary analyses suggest that PHD-finger genes were expanded recently by lineage-specific duplication, especially in pepper and potato, resulting in diverse repertoires of PHD-finger genes among the species. We validated the expression of six newly identified PHD-finger genes in pepper with qRT-PCR. Transcriptome analyses suggest potential functions of PHD-finger genes in response to various abiotic stresses in pepper.We performed genome-wide re-annotation in Our data, including the updated annotation of PHD-finger genes, provide useful information for further evolutionary and functional analyses to better understand the roles of the PHD-finger gene family in pepper.The online version contains supplementary material available at 10.1186/s12870-022-03580-2. Structural annotation of protein-coding genes is a fundamental process for obtaining essential genetic information for further evolutionary and functional analyses . HoweverArabidopsis [Arabidopsis, the MALE STERILITY1 (MS1) and DUET proteins participate in reproduction by regulating the transcription of genes associated with male gametogenesis and male meiosis, respectively [The plant homeodomain (PHD)-finger proteins are widely distributed in eukaryotes , with mobidopsis , many stbidopsis \u201324. In aectively , 26. PICectively . PKL alsectively , 29. In ectively , 31. HowArabidopsis thaliana (Arabidopsis), Oryza sativa (rice), Capsicum annuum (pepper), Solanum tuberosum (potato), and Solanum lycopersicum (tomato). We identified 875 PHD-finger genes, including 225 genes (26%) that were missed in previous annotations. Domain architecture analysis revealed that integration of diverse domains could contribute to the structural and functional diversification of PHD-finger genes. Based on phylogenetic analysis, PHD-finger genes were classified into 14 subgroups with distinct domain architectures (G1\u2009~\u2009G14). Duplication history analysis revealed that most of the potato and pepper PHD-finger genes were expanded recently via lineage-specific duplication. Microsynteny analysis in the Solanaceae species revealed that most of the G6 genes of potato on chromosome 1 were expanded by recent tandem duplication, resulting in diverse copy number variations in Solanaceae species. We validated the expression of newly identified pepper PHD-finger genes by qRT-PCR. Expression clustering analysis and gene ontology (GO) enrichment testing revealed that pepper PHD-finger genes might be associated with binding or regulation-related functions in response to abiotic stresses. Our study demonstrates a comprehensive evolutionary relationship of the PHD-finger gene family between pepper and the other four plant genomes, thus providing fundamental genomic resources that can be used to accelerate further functional agricultural research.In this study, we conducted re-annotation and comparative analyses of PHD-finger genes in five plant genomes: Arabidopsis, rice, and potato was approximately twice those in pepper and tomato were newly identified. Specifically, 57 (54%) pepper PHD-finger genes were newly annotated, indicating that the re-annotation process could improve previous annotations of PHD-finger genes via new gene identification, especially in the pepper genome Table . Many pr% were neto Table . The lenArabidopsis, rice, and potato, which possess relatively more PHD-finger genes than other species, most of the PHD-finger genes contained specific IDs, such as C1_2 (PF03107) and zf-RING_2 (PF13639). In particular, more than half the rice PHD-finger genes (51%) had zf_RING_2 (PF13639) in chromatin and binding to floral integrators, SUPPRESSOR OF OVEREXPRESSION OF CO1 (SOC1) and FLOWERING LOCUS T (FT) [Functional annotation based on GO analysis was performed to characterize the putative function of PHD-finger genes in the five plant genomes. We determined GO terms for 760 (87%) PHD-finger genes and categorized them based on molecular function, biological process, and cellular component Fig.\u00a0B. The prS T (FT) , 33. OurArabidopsis and rice PHD-finger genes were specifically clustered in G7 and G14, respectively . This suggests that the majority of PHD-finger genes in the same subgroup had expanded after domain integration. We observed specific IDs that consisted mainly of seven subgroups Fig.\u00a0C. The PHKs values between duplicated gene pairs in each subgroup . Our data revealed expression of those genes under abiotic stress treatment after 6 and 12\u00a0h Fig.\u00a0, indicatNext, we then identified differentially expressed genes in pepper, including the newly identified PHD-finger genes, in response to abiotic stresses such as cold 14,698), heat , salt , and mannitol . Our analysis identified 43, 47, 32, and 34 PHD-finger differentially expressed genes (DEGs) in pepper in response to cold, heat, salt, and mannitol treatment, respectively. We conducted expression clustering analysis and grouped these DEGs into four clusters based on their expression pattern under abiotic stress genes under cold stress [We also performed GO enrichment test of clusters that contained an abundant number of PHD-finger genes Fig.\u00a0C. Our anPKL Fig.\u00a0A. A prevongation . This sud stress , 29. TakHigh-quality annotation of protein-coding genes is extremely important and serves as a foundation for comparative analyses of gene families , 3. BecaIn general, evolutionarily conserved domains in protein-coding genes are considered to be significantly related to gene function . When weWe verified via qRT-PCR that newly annotated PHD-finger genes are expressed. Transcriptome analyses and GO enrichment test suggest that many pepper PHD-finger DEGs could participate in binding- or regulation-related functions in response to heat, salt, or mannitol stress.Taken together, we provide: i) updated genomic resources, containing previously omitted PHD-finger genes in five plant genomes including pepper and ii) a more comprehensive understanding of the structure and function of pepper PHD-finger genes.Arabidopsis thaliana [Oryza sativa [Capsicum annuum [Solanum tuberosum [Solanum lycopersicum [http://pfam.xfam.org/).We obtained the genome sequences of thaliana , Oryza sa sativa , Capsicum annuum , Solanumuberosum , and Solpersicum , includipersicum . The dowpersicum and usedWe assigned new gene names for re-annotated PHD-finger genes instead of locus tag names in the published annotations that we used. If PHD-finger genes were already given a gene name, we used the same published name , 44. We http://pfam.xfam.org/). Domains, except for the PHD-finger domain (PF00628), were considered as integrated domains. The bar plots in Fig.\u00a0To identify integrated domains (IDs) of PHD-finger genes, we used TSV files generated by InterProScan 5 accordinhttps://www.biobam.com/omicsbox/). The PHD-finger protein sequences were aligned to the NCBI non-redundant proteins database (nr v5) using BLASTP with an e-value cutoff (<\u200910\u20133). BLAST results were mapped to and annotated with GO terms using default parameters. The GO terms of each PHD-finger protein were classified into three main categories: biological process, molecular function, and cellular component. We selected the GO results at level 2 and visualized them using ggplot2 [To predict the putative function of PHD-finger genes, GO annotation was performed using OmicsBox . Based on the tree, the PHD-finger proteins were clustered and divided into 14 subgroups (G1\u2009~\u2009G14).For phylogenetic analysis, multiple sequence alignment was performed with the re-annotated PHD-finger protein sequences using MAFFT v7.470 . The aliTo estimate the duplication time of PHD-finger genes, we identified recently duplicated PHD-finger gene pairs using DupGen_Finder . The codChromosomal location of PHD-finger genes was obtained using GFF files from the re-annotation results of TGFam-Finder v1.20 and visuMicrosynteny analysis was conducted with genes in G6 located on chromosome 1 of pepper, potato, and tomato. All-by-all comparison for these genes was performed using BLASTP to identhttps://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi). The pepper ubiquitin gene (UBI-3) was used as a reference gene [\u2212\u0394\u0394Ct method [We conducted qRT-PCR to validate the expression of newly identified PHD-finger genes using cDNA isolated from abiotic-stressed pepper leaves . Primersnce gene . We selet method . The bart method in the Rhttps://cran.r-project.org/web/packages/pheatmap/index.html) in the R software. We then identified DEGs with a p-value\u2009<\u20090.05 using Ballgown [2-transformed fold-change values that were calculated from averaged FPKM values.To analyze the expression of pepper PHD-finger genes under abiotic stress, we first downloaded previously reported RNA-Seq data from pepper leaves treated with various stresses . These dBallgown from loghttps://www.biobam.com/omicsbox/). Enrichment test of GO terms in each cluster was performed using Fisher\u2019s exact test .To further investigate the expression pattern of pepper PHD-finger genes, we conducted clustering analysis with the DEGs using Mfuzz in the RAdditional file 1: Supplementary Figures. Figure S1. Chromosomal locations of PHD-finger genes in the five genomes. (A-E) Gene names are listed next to each chromosome bar and written in the same colors of matched subgroups in phylogenetic tree. The PHD-finger genes in (A) Arabidopsis (230), (B) rice (191), (C) pepper (84), (D) potato (192), and (E) tomato (87) are mapped to chromosomes, respectively. Figure S2. Expression profiles of PHD-finger genes under various abiotic stresses. Normalized expression values (log2(FPKM +1)) are shown as a heat map. The colored scale bars in the upper right side of the heat map represents normalized expression values: red indicates high level of expression and green indicates low level of expression. Gene names are matched with subgroup colors in phylogenetic tree.Additional file 2: Supplementary Tables. Table S1. List of the five plant genomic resources. Table S2. Detailed information on the re-annotated PHD-finger genes. Table S3. Description of integrated domain. Table S4. List of primers used in qRT-PCR."} +{"text": "The Coronavirus Disease 2019 (COVID-19) has significantly impacted cancer patients with some reported mortality as high as 25%. The Immunodeficiency Scoring Index (ISI) was developed as a prognostic tool in allogeneic hematopoietic cell transplant recipients with respiratory syncytial virus but also for other respiratory viruses to predict severe infections and mortality. The purpose of our study was to correlate the ISI in HCT recipients with COVID-19 and associated complications such as hospitalization, supplemental oxygen use, and mortality.We performed a cohort study of HCT recipients of all ages with COVID-19 between March 2020 and October 2021. We included only patients who were diagnosed by a PCR-based assay. We excluded patients for whom an ISI score, as previously described, could not be calculated. Outcomes of interest included 60-day mortality, hospital and ICU admission due to COVID-19, and supplemental oxygen requirements. A univariate analysis using Fischer exact testing for nominal variables was performed.Out of the 219 HCT with COVID-19, 101 were excluded due to alternative methods of diagnosis (13), lack of laboratory values needed to calculate an ISI at time of COVID-19 diagnosis (79), or COVID-19 diagnosed prior to transplant (9). Out of the remaining 118 patients, the median age was 60 years (range 6-85), most were male (56%), Caucasian (57%), and had no smoking history (64%). Most patients had an alloHCT (66%) with matched related donor [MRD] (25%), or matched unrelated donor [MUD] (21%) (Table\u00a01). Median time from transplant to COVID-19 was 615 days (range 2-5692), median ISI was 3 (range 0-11), and 92% of patients were unvaccinated prior to COVID-19 (Table\u00a01). On univariate analysis, an ISI of moderate to high (score \u22653) was associated with COVID-19 related hospitalization [p=0.0147] and an ISI \u2265 4 was associated with 60-day all-cause (p=0.045) and COVID-19-related (p-0.019) mortality (Table\u00a02).An ISI of 4 or greater was a prognostic marker for worse outcomes such as COVID-related and all-cause mortality in HCT recipients. Whether an aggressive and prompt management of high-risk patients with COVID-19 may impact these outcomes needs to be determined in future studies.Gabriella Rondon, MD, Omeros: Advisor/Consultant Ella Ariza-Heredia, MD, MERCK: Grant/Research Support Elizabeth Shpall, MD, Adaptimmune: Advisor/Consultant|Affimed: License agreement|Axio: Advisor/Consultant|Bayer Helathcare Pharmaceuticals: Honoraria|Fibroblasts and FibrioBiologics: Advisor/Consultant|Navan: Advisor/Consultant|NY Blood Center: Advisor/Consultant|Takeda: License agreement Roy F. Chemaly, MD/MPH, Karius: Advisor/Consultant|Karius: Grant/Research Support."} +{"text": "In central nervous system, axons fail to regenerate after injury while in peripheral nervous system, axons retain certain regenerative ability. Dorsal root ganglion (DRG) neuron has an ascending central axon branch and a descending peripheral axon branch stemming from one single axon and serves as a suitable model for the comparison of growth competence following central and peripheral axon injuries. Molecular alterations underpin different injury responses of DRG branches have been investigated from many aspects, such as coding gene expression, chromatin accessibility, and histone acetylation. However, changes of circular RNAs are poorly characterized. In the present study, we comprehensively investigate circular RNA expressions in DRGs after rat central and peripheral axon injuries using sequencing analysis and identify a total of 33 differentially expressed circular RNAs after central branch injury as well as 55 differentially expressed circular RNAs after peripheral branch injury. Functional enrichment of host genes of differentially expressed circular RNAs demonstrate the participation of Hippo signaling pathway and Notch signaling pathway after both central and peripheral axon injuries. Circular RNA changes after central axon injury are also linked with apoptosis and cellular junction while changes after peripheral axon injury are associated with metabolism and PTEN-related pathways. Altogether, the present study offers a systematic evaluation of alterations of circular RNAs in rat DRGs following injuries to the central and peripheral axon branches and contributes to the deciphering of essential biological activities and mechanisms behind successful nerve regeneration. Axon injury elicits distal nerve degeneration, impairs neuronal functions, and causes motor incapacity, sensation loss, and neuropathic pain . The recDorsal root ganglion (DRG) neurons are pseudounipolar neurons that have two separate axonal branches stemming from one single axon. The central branch extends into the central nervous system and is generally incapable of regeneration after axon injury while the peripheral branch projects to peripheral target and often obtains a remarkable growth capacity . DespiteRecently, the application of high-throughput RNA sequencing discriminates genetic changes in DRGs after central and peripheral axon injury and reveals the essential roles of reactive oxygen species in axon regeneration by comparing changes between non-regenerative central axon injury and regenerative peripheral axon injury . ATAC anCircular RNAs are endogenous non-coding RNAs with covalently closed loop structures formed by transcript back splicing . AlthougA total of 56 8-week-old male Sprague Dawley rats (\u223c200 g) were obtained from the Animal Center of Nantong University. Rats were randomly divided into central axon branch injury group, central axon branch sham surgery group, peripheral axon branch injury group, and peripheral axon branch sham surgery group, with 14 rats in each group.Animal work was carried out in accordance with the guidelines of Nantong University Institutional Animal Care and Ethical approved by the Administration Committee of Experimental Animals, Nantong University, Jiangsu Province, China.Central and peripheral axon injuries were performed according to a previous publication with modifications . BrieflyCRA006070.Total RNAs was extracted from rat L4-L5 DRGs using TRIzol reagent kit and subjected to RNA quality check using an Agilent Bioanalyzer . mRNA was enriched, fragmented into short fragments, and reverse transcripted to cDNA. cDNA fragments were ligated to Illumina sequencing adapters and sequencing was performed using HiSeq\u2122 4000 platform. RNA libraries were sequenced on the Illumina sequencing platform by Genedenovo Biotechnology Co., Ltd. . Sequencing data were stored in Genome Sequence Archive database with accession number p-value calculation. Circular RNAs with log2 (fold change) > 1 or < \u22121 and p-value < 0.05 were screened and considered as differentially expressed. The functions of host genes of differentially expressed circular RNAs were discovered using kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis to obtain high quality clean reads . Clean ranalysis , reactomanalysis . The sigThe competing endogenous RNA (ceRNA) network was constructed by assembling all co-expression competing circular RNAs, microRNAs, and mRNAs. Expression correlations between circular RNAs and microRNAs as well as microRNAs and mRNAs were analyzed using the Spearman Rank correlation coefficient (SCC) to screen negatively co-expressed circular RNA-microRNA pairs or microRNA-mRNA pairs. Expression correlations between circular RNAs and mRNAs were analyzed using Pearson correlation coefficient (PCC) to screen positively co-expressed circular RNA-mRNA pairs. The interactions between circular RNAs, microRNAs, and mRNAs in the ceRNA network were displayed using Cytoscape software (v3.6.0).\u2013\u0394\u0394Ct method with GAPDH as the reference gene. Primer sequences were as follows (5\u2032\u22123\u2032): novel_circ_000290-F TGACTCCCTCTCTGGTGACA, novel_circ_000290-R GCTTCCTCAACACCATCACC, novel_circ_001817-F AGGCTATTCGCTTAGGATTCCA, novel_circ_001817-R CCAGGTAGTTGTGTCCGATGTA, novel_circ_000991-F CATCCACGTCATGAGGAACAG, novel_circ_000991-R TACAAGACACTCGGTCTACCAA, GAPDH-F ACAGCAACAGGGTGGTGGAC, and GAPDH-R TTTGAGGGTGCAGCGAACTT.Total RNA extracted from rat L4-L5 DRG was reversely transcribed to cDNA using PrimeScript RT Reagent Kit . The characterization of circular RNA was determined using the divergent primers annealing at the distal end. PCR product was subjected to agarose gel electrophoresis and Sanger sequencing . The relative abundance of novel_circ_000290, novel_circ_001817 and novel_circ_000991 was determined using the comparative 2t-test . Significantly difference was set at a p-value < 0.05.Numerical data were expressed as mean with SEM. Comparisons between DR-Exp and DR-sham as well as SN-Exp and SN-sham were generated using unpaired Dorsal root ganglions of rats underwent central axon branch injury, peripheral axon branch injury, or corresponding sham surgery were collected at 24 h after surgery and subjected to sequencing to obtain a global view of circular RNAs in rat DRGs after central and peripheral axon injuries . SequencBioinformatic analysis identified a total of 2,310 circular RNAs in rat DRGs. These circular RNAs showed similar sample expression distributions after central and peripheral axon injuries . The locThe expression levels of circular RNAs after central and peripheral axon injuries were determined to screen differentially expressed circular RNAs. A total of 33 circular RNAs were found to be differentially expressed after central branch injury versus central branch sham surgery, with 9 circular RNAs up-regulated and 24 circular RNAs down-regulated . A largeDetailed information of all differentially expressed circular RNAs, including their chromosome localizations, genomic start/end sites, spliced lengths, annotation types , and host genes, was provided in Among these differentially expressed circular RNAs, circular RNA novel_circ_000991 exhibited relative high abundances in all groups and thus was further examined for its characterization and expression. Consistent with RNA sequencing outcome, Sanger sequencing recognized Mtmr7 (NM_001107312.3) as the host gene of novel_circ_000991 . The cycTo discover biological implications of differentially expressed circular RNAs in DRGs after central axon injury, host genes of these differentially expressed circular RNAs were annotated with KEGG database. Top enrich KEGG pathways were listed in Reactome analysis further demonstrated the considerable participation of Hippo signaling and apoptosis by caspase 3, R-RNO-2028697) in DRGs after central axon injury. The enrichment of reactome pathways WWTR1 (TAZ) binds to ZO-1 (TJP1) (R-RNO-2064417), phosphorylation of Cx43 by c-src (R-RNO-191636), c-src mediated regulation of Cx43 function and closure of gap junctions (R-RNO-191647), and regulation of gap junction activity (R-RNO-191654) implied changes of cellular junction. The identification of significantly involved reactome pathway Caspase-mediated cleavage of TJP1 (R-RNO-351913) further indicated the association of apoptosis and cellular junction regulation. Moreover, the recognition of reactome pathways IL6 binds IL6R-2 (R-RNO-1067640), IL6 binds IL6R (R-RNO-1067667), and IL6:IL6R-2 binds IL6ST-2 (R-RNO-1067676) illuminated the involvement of immune responses in central axon injury-mediated molecular features .Gene ontology term enrichment revealed the involvement of GO biological processes biological regulation, cellular process, regulation of biological process, GO cellular components cell, cell part, and GO molecular function binding .Host genes of differentially expressed circular RNAs in DRGs after peripheral axon injury were subjected to functional enrichment as well. Similar as changes after central axon injury, Hippo signaling pathway and Notch signaling pathway were found to be enriched after peripheral axon injury. A metabolism-related pathway, Inositol phosphate metabolism (ko00562), was also discovered as a commonly involved KEGG pathway in host genes of differentially expressed circular RNAs after both central and peripheral axon injuries. Besides Inositol phosphate metabolism, many other metabolism-related pathways, i.e., Glycosphingolipid biosynthesis-ganglio series (ko00604), Mucin type O-glycan biosynthesis (ko00512), Fatty acid degradation (ko00071), and Fatty acid metabolism (ko01212) were also significantly changed in DRGs after peripheral axon injury .Reactome enrichment analysis showed the essential involvement of PTEN in peripheral axon injury-induced changes in DRGs as many significant reactome pathways were related to PTEN, including Regulation of PTEN gene transcription (R-RNO-8943724), SALL4 recruits NuRD to PTEN gene (R-RNO-8943780), and PTEN Regulation (R-RNO-6807070). Furthermore, TP53-related reactome pathways MTA2-NuRD complex deacetylates TP53 (R-RNO-6805650) and Regulation of TP53 activity through acetylation (R-RNO-6804758) as well as ATF-associated pathway MSK1 activates ATF1 (R-RNO-6804758) were also identified as top enriched reactome pathways .Gene ontology analysis revealed cellular process as the most significantly enriched GO biological process, cell and cell part as the most significantly enriched GO cellular components, and binding as the most significantly enriched GO molecular function .Given the essential participation of PTEN-related pathways in peripheral axon injury-induced changes, circular RNAs sourced from genes involved in PTEN pathways are worthy of note. Novel_circ_002033, a circular RNA cyclizated from Gatad2b, was found to be up-regulated after peripheral axon branch injury versus sham surgery but not significantly altered after central axon branch injury. Therefore, besides the function of the host gene of novel_circ_002033, the regulatory roles of novel_circ_002033 was also explored by generating the ceRNA network of novel_circ_002033. Regulatory network showed that novel_circ_002033 might function as a microRNA sponge and regulate the target genes of miR-6331-z and miR-129-x .Dorsal root ganglion neurons are extensively used to investigate axon regeneration as the central and peripheral axon branches of DRG neurons have different axon regrowth abilities . IdentifPreviously, our laboratory has investigated changes in DRGs at a series of time points, i.e., 0 h, 3 h, 9 h, 24 h, 4, and 7 days after rat sciatic nerve injury and found that many genes exhibited altered expression levels at 24 h post peripheral axon injury . To examFunctional analyses of host genes of circular RNAs provide important biological implications as circular RNAs regulate their host genes . Hippo svia PTEN oxidation and inactivation can be found in the article/The animal study was reviewed and approved by Nantong University Institutional Animal Care and Ethical approved by the Administration Committee of Experimental Animals, Nantong University, Jiangsu Province, China.H-JC, LH, M-RZ, and TZ performed the experiment. L-CX designed the experiment. All authors wrote the manuscript."} +{"text": "Estimates of COVID-19 mRNA vaccine effectiveness (VE) have declined in recent months and 18,027 (8%) occurred duringthe Delta- and Omicron-predominant periods, respectively . A higheAmong 87,904 eligible hospitalizations, 86,327 (98%) and 1,577 (2%) occurred during theDelta- and Omicron-predominant periods, respectively . HospitaIn a multistate analysis of 222,772 ED and UC encounters and 87,904 hospitalizationsamong adults with COVID-19\u2013like illness during August 26, 2021\u2013January5, 2022, estimates of VE against laboratory-confirmed COVID-19 declined during theOmicron-predominant period compared with VE during the Delta-predominant period.During both periods, VE was significantly lower among patients who received theirsecond mRNA COVID-19 vaccine dose \u2265180 days before the medical encounterscompared with those vaccinated more recently. VE increased following a third doseand was highly effective during both the Delta- and Omicron-predominant periods atpreventing COVID-19\u2013associated ED and UC encounters and preventing COVID-19\u2013associated hospitalizations .Estimates of VE for 2 doses of an mRNA vaccine were higher againstCOVID-19\u2013associated hospitalizations than against COVID-19\u2013associatedED or UC encounters, especially during the Omicron period, which is consistent withpossible vaccine attenuation of severity of COVID-19 disease but was not observed inthis network previously inthis observational study might have biased the estimates. Sixth, geneticcharacterization of patients\u2019 viruses was not available, and analysestherefore relied on dates when the Omicron variant became predominant based onsurveillance data. The Omicron period of predominance in this study likely includesmedical encounters associated with the Delta variant. If VE is reduced againstmedical care associated with Omicron variant, this study likely overestimated VE.Finally, although the facilities in this study serve heterogeneous populations in 10states, the findings might not be generalizable to the U.S. population.These findings underscore the importance of receiving a third dose of mRNA COVID-19vaccine to prevent both moderately severe and severe COVID-19, especially while theOmicron variant is the predominant circulating variant and when the effectiveness of2 doses of mRNA vaccines is significantly reduced against this variant. Allunvaccinated persons should get vaccinated as soon as possible. All adults who havereceived mRNA vaccines during their primary COVID-19 vaccination series shouldreceive a third dose when eligible, and eligible persons should stay up to date withCOVID-19 vaccinations.COVID-19 mRNA vaccine effectiveness (VE) in preventing COVID-19 might declinebecause of waning of vaccine-induced immunity or variant immune evasion.VE was significantly higher among patients who received their second mRNACOVID-19 vaccine dose <180 days before medical encounters compared withthose vaccinated \u2265180 days earlier. During both Delta- andOmicron-predominant periods, receipt of a third vaccine dose was highlyeffective at preventing COVID-19\u2013associated emergency department andurgent care encounters and preventingCOVID-19\u2013associated hospitalizations .All unvaccinated persons should start vaccination as soon as possible. Alladults who have received mRNA vaccines during their primary COVID-19vaccination series should receive a third dose when eligible, and eligiblepersons should stay up to date with COVID-19 vaccinations."} +{"text": "S-gene) deletion (\u039469\u201370) in the N-terminal region, which might compensate for immune escape mutations that impair infectivity (S-gene target amplification in certain multitarget reverse transcription\u2013polymerase chain reaction (RT-PCR) assays, a pattern referred to as S-gene target failure (SGTF) (S-gene target presence (SGTP), which alongside genomic sequencing, has facilitated early monitoring of emerging variants. During a period when Omicron BA.5\u2013related sublineages (which exhibit SGTF) predominated, an XBB.1.5 sublineage with SGTP has rapidly expanded in the northeastern United States and other regions.Monitoring emerging SARS-CoV-2 lineages and their epidemiologic characteristics helps to inform public health decisions regarding vaccine policy, the use of therapeutics, and health care capacity. When the SARS-CoV-2 Alpha variant emerged in late 2020, a spike gene were reported to U.S. Department of Health and Human Services (HHS) ProtectAs part of the Increasing Community Access to Testing (ICATT) program,During November 1, 2021\u2013December 24, 2022, national weekly SGTF and SGTP results ranged from 3,104 to 83,805 . Among genomic sequences from ICATT specimens collected through January 2, 2023, 412 (99%) of 415 XBB-related sequences exhibited SGTP; among those collected during December 1, 2022\u2013January 2, 2023, 294 (59%) of 495 SGTP specimens were XBB-related lineages.Trends in SGTP proportions aligned with genomic sequencing results classified by SGTF and SGTP . For theSGTF/SGTP monitoring relies on diagnostic RT-PCR, which is less expensive, permits higher throughput, and faster turnaround of results than sequencing. Using SGTF/SGTP for early studies of emerging variants obviates the need to wait for sequencing results or >50% variant predominance. Limitations are that SGTF/SGTP monitoring is assay-dependent; presumes SARS-CoV-2 lineage classification, requiring further validation by genomic surveillance; cannot discriminate mutations beyond \u039469\u201370 ; and relies on continued changing SGTF/SGTP patterns compared with predominant lineages. SARS-CoV-2 sequencing remains the standard for genomic surveillance because it allows definitive classification of viral lineages and identification of emerging strains for further characterization.When early nowcast estimates of rapidly emerging variants lacked precision and geographic resolution because of lags in genomic sequencing results, SGTF/SGTP estimates were used as complementary data by CDC and the SARS-CoV-2 Interagency Group to support guidance on the use of monoclonal antibody therapies."} +{"text": "Targeted radionuclide therapy (TRT) and immunotherapy are rapidly growing classes of cancer treatments. Basic, translational, and clinical research are now investigating therapeutic combinations of these agents. In comparison to external beam radiation therapy (EBRT), TRT has the unique advantage of treating all disease sites following intravenous injection and selective tumor uptake and retention\u2014a particularly beneficial property in metastatic disease settings. The therapeutic value of combining radiation therapy with immune checkpoint blockade to treat metastases has been demonstrated in preclinical studies, whereas results of clinical studies have been mixed. Several clinical trials combining TRT and immune checkpoint blockade have been initiated based on preclinical studies combining these with EBRT and/or TRT. Despite the interest in translation of TRT and immunotherapy combinations, many questions remain surrounding the mechanisms of interaction and the optimal approach to clinical implementation of these combinations. This review highlights the mechanisms of interaction between anti-tumor immunity and radiation therapy and the status of basic and translational research and clinical trials investigating combinations of TRT and immunotherapies. Metastatic disease carries a poor prognosis for cancer patients, and little progress has been made to change this reality, despite advances in individual applications of immunotherapy, chemotherapy, and radiation therapy. It is estimated that 90% of patients who die from cancer die from metastases . Systemi+ effector T cell activation ICF01012 (a radiolabeled melanin ligand for metastatic melanoma), 223Ra, and 213Bi (an \u03b1-emitter generated from 225Ac/213Bi generator) demonstrated activation of immunogenic cell death via upregulation of calreticulin ([131I]ICF01012 and 223Ra) and HMGB1/heat shock protein 70 (Hsp70) (213Bi) [Many of the key immunogenic effects of EBRT have been evaluated in the context of TRT in preclinical studies with various radiopharmaceuticals. These effects are summarized in (213Bi) ,49,50.+ cytotoxic T lymphocyte (CTL)-mediated killing of cancer cells. Administration of 4 and 10 Gy doses of 223Ra (Xofigo\u00ae) to human breast , prostate , and lung carcinoma showed at least two-fold increases in human leukocyte antigens (HLA)-A, B, and C (HLA-ABC) expression, relative to mock-irradiated controls [153Sm-ethylenediaminetetramethylenephosphonate (Quadramet\u00ae), a radiopharmaceutical complex that binds avidly to hydroxyapatite in bone, the LNCaP prostate cancer cell line showed increased MHC-I expression levels on cells, with no significant change in the percentage of cells positive for MHC-I [223Ra, given at 4- or 10-Gy doses, increased the sensitivity of all previously mentioned cell lines to CTL-mediated lysis targeting the carcinoembryonic antigen , mucin-1 , and brachyury (HLA-A2/A24-restricted) tumor antigens [153Sm-EDTMP showed increased sensitivity to prostate-specific antigen (PSA)-specific CTL-mediated killing , compared with 0 Gy [In cells surviving TRT, several phenotypic changes have been reported. For example, upregulation of major histocompatibility complex-I (MHC-I) expression is important in antigen-specific CD8controls . Anotheror MHC-I . Becauseantigens . Exposurith 0 Gy .90Y-NM600 was shown to activate stimulator of interferon genes (STING) in cells surviving radiation. Administration of 20 \u03bcCi or 100 \u03bcCi 90Y-NM600 in an immunologically cold syngeneic murine model of head and neck cancer, MOC2, resulted in IFN1 activation and induction of immune susceptibility markers ) that were comparable to dose equivalent EBRT treatments in vivo [90Y-NM600 and anti-CTLA4 in B16 melanoma, highlighting the essential role of this pathway in certain combination TRT and immunotherapy regimens [Additionally, in vivo . Additioregimens .213Bi treated MC38 cells increased dendritic cell (DC) maturation and activation in vitro [90Y-NM600 [223Ra, peripheral blood mononuclear cell (PBMC) phenotyping over six months revealed an immunosuppressive phenotype with an increase in both myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs), and an overall decrease in absolute lymphocyte counts. No differences in PBMC populations were noted in subgroup analysis of patients with alkaline phosphatase response [Innate immune populations involved in the initial response to radiation therapy and establishing an adaptive response have been shown to be affected by TRT. Gorin et al. demonstrated that in vitro . Patel e0Y-NM600 . In 30 presponse . Whether90Y-NM600 and anti-CTLA4 therapy increased CD8+ and effector memory CD8+ T cells in B78 melanoma tumors 25 days following 2.5 Gy TRT, but TRT monotherapy did not affect CD8+ populations [+ T cell populations and decreased FOXP3+ cells at day 6 following 90Y-NM600 monotherapy [213Bi-treated MC38 cells protected against future MC38 tumor engraftment. This effect was not observed in immunodeficient athymic (nude) mice, indicating T cell involvement in mediation of the anti-tumor effect [+ T cells in combination 177Lu TRT + anti-PD-L1 treated tumors but not monotherapy at day 4, with loss of the effect at day 14 in MC38 tumors following integrin \u03b1v\u03b23-targeted 177Lu +/\u2212 anti-PD-L1 [v\u03b23 targeted 177Lu, showing a cooperative effect between TRT agents and immune checkpoint blockade [The effect of dose and emission type of TRT on the adaptive immune response over time has not been fully elucidated. Substantial variations have been reported in tumor infiltrating lymphocyte (TIL) populations following TRT. Combination ulations . Howeverotherapy . In the r effect . Chen etti-PD-L1 . Wen et blockade . Despiteblockade .+ colon tumors when treated with a 90Y radiolabeled anti-CEA antibody in combination with a CEA/TRICOM vaccine, as compared to TRT or immunotherapy monotherapy [90Y-veltuzumab and unlabeled veltuzumab initiated one week after TRT [Two of the earliest preclinical examples of the therapeutic interaction between TRT and immunotherapy were reported by Chakraborty et al. in 2008 and Sharotherapy . Sharkeyfter TRT .The most studied form of immunotherapy in combination with TRT are ICIs. A summary of preclinical studies combining TRT and ICIs is presented in 90Y delivered with NM600 and anti-PD-L1 and anti-CTLA-4 in syngeneic murine B78 melanoma models. This 90Y-NM600 + dual ICI resulted in tumor regression and improved survival at approximately 2.5 Gy tumor dose [90Y-NM600 at absorbed doses of 2\u20135 Gy stimulated expansion of T-cell receptor (TCR) clones among tumor infiltrating lymphocytes when combined with anti-CTLA-4 without increasing diversity of the TCR repertoire [90Y-NM600 in combination with dual ICI prolongs survival over each monotherapy and EBRT to the primary tumor in mice bearing two MOC2 head and neck squamous cell carcinoma tumors [90Y-NM600 and ICI [We have observed a cooperative therapeutic interaction of low dose mor dose . Additiopertoire . This enpertoire , in contpertoire . With tha tumors . Finally and ICI . This re177Lu bound to the \u03b1v\u03b23 integrin-targeting peptide Arg-Gly-Asp (177Lu-RGD) improved tumor control and increased overall survival in mice bearing MC38 colon cancer tumors when combined with anti-PD-L1 [+ tumor-infiltrating lymphocytes following combination therapy [177Lu-LLP2A, targeting very late antigen-4 (VLA-4), when combined with anti-CTLA-4, anti-PD-1, or anti-PD-L1 resulted in improved survival of mice bearing B16F10 melanoma tumors. This survival advantage was observed in comparison to dual ICI or TRT monotherapy [131I-ICF01012 (a radiolabeled melanin ligand) + anti-CTLA-4 over ICI therapy alone [Using a different radionuclide and vector, Chen et al. demonstrated that ti-PD-L1 . Of note therapy . In anototherapy . An addipy alone .90Y-labeled granzyme B targeted peptide (GZP) and anti-PD-1 in two murine colon cancer tumor models [131I-anti-PD-L1, Wen et al. observed high tumor to normal tissue ratios of 131I-anti-PD-L1, and a significant survival benefit of combination therapy compared to control groups in MC38 and CT26 murine colon adenocarcinoma [The number of TRT targeting moieties continues to grow, with peptides, small molecules, and antibodies representing the three main classes. Ferreira et al. noted a significant overall survival advantage following treatment with a r models . Employiarcinoma .213Bi in combination with anti-PD-1 led to tumor regression and improved overall survival over ICI monotherapy [177Lu-h8C3 + anti-PD-1, but no benefit to treatment with low or high dose 225Ac-h8C3 +/\u2212 anti-PD-1 [In addition to these reports, a growing number of studies , a beta emitter that binds osteoblastic bone lesions, and PSA/TRICOM vaccine, a therapeutic mCRPC vaccine (NCT00450619) [As is evident from 0450619) , 18 pati0450619) .\u00ae), an autologous cellular therapy designed to stimulate an effector T cell response against tumor cells [223Ra, or other hormonal agents, leading to its 2010 FDA approval [223Ra in a Phase II clinical trial with 32 patients, where patients receiving combination therapy had longer overall and progression-free survival and were more likely to demonstrate a >50% PSA decline; however had decreased prostatic acid phosphatase (PAP)-specific peripheral T cell proliferation compared to patients receiving sipuleucel-T alone [Another Phase II mCRPC clinical trial (NCT02463799) incorporated the immunotherapy Sipuleucel-T , three combining 223Ra with ICIs , and one pairing PSMA-targeting 225Ac-J591 with pembrolizumab (NCT04946370). In addition to assessing the safety of these therapies, the collective results of these trials will begin to inform how high LET, alpha-emitting 223Ra and 225Ac differ from low LET, beta-emitting 177Lu in terms of enhancing response to ICIs. Two case reports described patients with neuroendocrine tumors [213Bi-DOTATOC and 225Ac-PSMA-617, respectively, following disease progression or prohibitive hematologic toxicity on corresponding beta-emitter therapy. Alpha-emitting TRT resulted in neuroendocrine tumor responses with lower hematologic toxicity than beta-emitting TRT for a neuroendocrine tumor patient with disseminated bone marrow disease, an advantage gained from the short tissue range of alpha particles [Six of the ongoing combination TRT-ICI trials are targeting patients with mCRPC\u2014two combining e tumors and mCRPe tumors who resparticles .\u00ae monotherapy [223RaCl2 combination therapy have not demonstrated a therapeutic benefit. In an ongoing Phase II study (NCT03093428) randomizing 42 patients with mCRPC to receive 223RaCl2 +/\u2212 pembrolizumab, the combination therapy group did not have improved overall survival nor progression free survival compared to 223RaCl2 monotherapy [223RaCl2 in men with mCRPC having progressed on androgen pathway inhibitor treatment, combination therapy had greater toxicity than either monotherapy, without clinical benefit. Out of 44 patients enrolled, 23 (52.3%) experienced a grade 3/4 adverse event [177Lu-PSMA-617 and pembrolizumab in mCRPC with encouraging interim results [Despite positive Phase III results of Xofigootherapy , early iotherapy . In a phse event . The PRI results . Out of results .68Ga-DOTATATE on PET/CT of the seven patients evaluated [Phase I results from a trial studying combination nivolumab (anti-PD-1) and Lutathera for extensive-stage small cell lung cancer (NCT03325816) reported a tolerable toxicity profile. Of seven patients with CT-measurable disease, one patient with extensive-stage disease had a partial response, and two with pulmonary atypical carcinoid disease had stable disease for six months. Of note, the patient with extensive-stage disease with a partial response showed the highest tumor uptake of valuated .177Lu-DOTATATE/DOTATOC) and ICIs in ICI-refractory metastatic Merkel cell carcinoma (mMCC), a skin cancer with occasional responses to PRRT, have been described in recent case reports [177Lu-DOTATOC. This response was maintained for five months (until time of manuscript submission) [177Lu-DOTATATE in combination with pembrolizumab. Both patients had PET/CT demonstrated responses at all disease sites for 3.6 and 4.8 months, respectively [177Lu-DOTATATE in combination with pembrolizumab in patients with MCC (NCT05583708). Additionally, survival data from the GoTHAM trial (NCT04261855) combining 177Lu-DOTATATE and avelumab for treatment of mMCC will provide further insight into treatment efficacy when available. Notably, despite promising preclinical results from dual immune checkpoint blockade therapies in combination with TRT, there is yet to be a randomized controlled trial investigating this combination. Collectively, these preliminary results will lay the foundation for future Phase III trials of combination TRT-immunotherapy.Impressive responses to combination peptide receptor radionuclide therapy (PRRT; reports ,85. In oectively . These eAs more developments are made in the field of combination TRT-immunotherapy, several critical differences between TRT and conventional EBRT must be understood to ensure effective and timely implementation of these exciting novel therapeutic options for metastatic disease. A detailed understanding of TRT dosimetry, toxicity, and immunomodulatory effects, as well as tumor immune escape pathways will be essential to enable effective translation of these treatments. Synchronously with mechanistic studies, determination and standardization of multidisciplinary approaches to implement these therapies will be vital to effective application of TRT-immunotherapy combinations . PotentiCombinations of TRT and immunotherapy have the potential to change patient outcomes in oncology clinics. The therapeutic effectiveness of engaging a patient\u2019s own immune system to eradicate tumor cells at all disease sites has already proven immense. Preclinical and clinical studies are starting to build on the momentum of promising TRT agents. Yet, the radiobiologic and immunologic effects of TRT are not well understood. Therefore, detailed dosimetry studies are required to enable understanding of the effects of absorbed dose, dose rate, dose range, LET, and tissue and cellular distribution of absorbed dose and how these properties affect tumor cells, normal cells, and the TME. It is important for these studies to be performed with different radionuclides and combination therapies in order to define the immuno-radiobiological interface.Well-designed preclinical veterinary trials with larger subjects, such as companion canines, will aid in clinical translation of TRT-immunotherapy combinations. Ultimately, well designed clinical trials in cancer patients will be essential to widespread TRT-immunotherapy implementation. Development of new training pathways to establish a workforce with TRT-specific expertise, and collaboration between multiple disciplines of medicine and science will be essential to the effective design, conduct, and outcome of these clinical trials. The interface between TRT and immunotherapies proffers abundant potential as a novel approach to treating metastatic cancer, and this is motivating a growing field of scientific and clinical investigation."} +{"text": "Background: We evaluated the impact of a comprehensive SARS-CoV-2 (COVID-19) infection prevention (IP) bundle on rates of non\u2013COVID-19 healthcare-acquired respiratory viral infection (HA-RVI). Methods: We performed a retrospective analysis of prospectively collected respiratory viral data using an infection prevention database from April 2017 to January 2021. We defined HA-RVI as identification of a respiratory virus via nasal or nasopharyngeal swabs collected on or after hospital day 7 for COVID-19 and non\u2013COVID-19 RVI. We compared incident rate ratios (IRRs) of HA-RVI for each of the 3 years (April 2017 to March 2020) prior to and 10 months (April 2020 to January 2021) following full implementation of a comprehensive COVID-19 IP bundle at Duke University Health System. The COVID-19 IP bundle consists of the following elements: universal masking; eye protection; employee, patient, and visitor symptom screening; contact tracing; admission and preprocedure testing; visitor restrictions; discouraging presenteeism; population density control and/or physical distancing; and ongoing attention to basic horizontal IP strategies including hand hygiene, PPE compliance, and environmental cleaning. Results: During the study period, we identified 715 HA-RVIs over 1,899,700 inpatient days, for an overall incidence rate of 0.38 HA-RVI per 1,000 inpatient days. The HA-RVI IRR was significantly higher during each of the 3 years prior to implementing the COVID-19 IP bundle (Table Conclusions: Implementation of a comprehensive COVID-19 IP bundle likely contributed to a reduction in HA-RVI for hospitalized patients in our healthcare system. Augmenting traditional IP interventions in place during the annual respiratory virus season may be a future strategy to reduce rates of HA-RVI for inpatients.-2 COVID-9 infecti-2 COVID-9 infecti"} +{"text": "Canine mammary tumor (CMT) is prevalent in female dogs. As tumor recurrence and metastasis occur in malignant CMT (MMT) dogs after surgery, serum prognostic biomarkers are needed to facilitate prediction of disease outcomes. We have identified CMT-associated autoantibodies, namely TYMS-AAb, IGFBP5-AAb, HAPLN1-AAb, and AGR2-AAb, which are present in serum of CMT dogs. In this study, we demonstrated that the serum AAb levels decreased at 3- and 12-months post-surgery in 11 MMT dogs. We also evaluated the correlation between the presurgical AAb level and overall survival (OS) of 90 CMT dogs after surgery. Data reveal that higher levels of IGFBP5-AAb and TYMS-AAb are significantly correlated with worse OS. On the contrary, a lower level of AGR2-AAb is correlated with poorer OS. Notably, MMT dogs presenting higher levels of TYMS-AAb and IGFBP5-AAb plus a lower level of AGR2-AAb have worst OS. This is the first study revealing an association between serum AAb levels and clinical outcomes of CMT and provides a starting point to verify the validity of utilizing this AAb panel in prediction of tumor relapse and therapy responses.p < 0.05). We evaluated the correlation between the presurgical AAb level and overall survival (OS) of 90 CMT dogs after surgery. Kaplan-Meier survival analysis reveals that IGFBP5-AAbHIgh and TYMS-AAbHigh are significantly correlated with worse OS , while AGR2-AAbLow is correlated with somewhat poorer OS (p = 0.086). Areas under a time-dependent receiver operating characteristic curve (AUC) of IGFBP5-AAb and TYMS-AAb in predicting OS of MMT dogs are 0.611 and 0.616, respectively. Notably, MMT dogs presenting TYMS-AAbHigh/IGFBP5-AAbHigh/AGR2-AAbLow have worst OS (p = 0.0004). This study reveals an association between the serum AAb level and CMT prognosis.Canine mammary tumor (CMT) is the most prevalent neoplasm in female dogs. Tumor recurrence and metastasis occur in malignant CMT (MMT) dogs after surgery. Identification of serum prognostic biomarkers holds the potential to facilitate prediction of disease outcomes. We have identified CMT-associated autoantibodies against thymidylate synthetase (TYMS), insulin-like growth factor-binding protein 5 (IGFBP5), hyaluronan and proteoglycan link protein 1 (HAPLN1), and anterior gradient 2 (AGR2), i.e., TYMS-AAb, IGFBP5-AAb, HAPLN1-AAb, and AGR2-AAb, respectively, by conducting serological enzyme-linked immunosorbent assays (ELISA). Herein we assessed serum AAb levels in 11 MMT dogs before and after surgery, demonstrating that IGFBP5-AAb and HAPLN1-AAb significantly decrease at 3- and 12-months post-surgery ( Canine mammary tumor (CMT) is the most prevalent neoplasm in female dogs, and approximately 50% of CMTs are malignant . A prospProteins overexpressed or aberrantly localized in tumor cells likely act as autologous immunogenic antigens, so-called tumor-associated antigens (TAAs). Humoral immune responses toward TAAs result in production of tumor-associated autoantibodies (AAbs) which are less subject to proteolysis and highly stable in serum compared to soluble TAAs. As a result, tumor-associated AAbs can persist at lasting levels in serum even when corresponding TAAs are no longer detectable, and have been proposed as serum biomarkers for predicting cancer outcomes and patients\u2019 survival in various types of human cancer ,13. DespWe previously identified thymidylate synthetase (TYMS), insulin-like growth factor-binding protein 5 (IGFBP5), hyaluronan, and proteoglycan link protein 1 (HAPLN1), and anterior gradient 2 (AGR2) as CMT-associated proteins overexpressed in malignant CMT (MMT) tissues compared with normal counterparts . TYMS isWe have verified TYMS, IGFBP5, and HAPLN1 as CMT-associated antigens and the presence of corresponding autoantibodies, namely TYMS-AAb, IGFBP5-AAb, and HAPLN1-AAb, respectively, in serum of CMT patients. These AAbs show validity in distinguishing early-stage MMT from benign CMT (BMT) or healthy dogs . WhetherAGR2 acts as a protein disulfide isomerase (PDI) and mainly resides at the endoplasmic reticulum (ER) to modulate ER homeostasis and the quality control of proteins ,27. On tIn the present study, we first assessed serum AGR2-AAb in CMT patients and then evaluated the correlation between serum levels of CMT-associated AAbs and CMT outcomes. Furthermore, we monitored the changes in the serum levels of tailored panels of AAbs in CMT patients before and after tumor resection by mastectomy.g, 4 \u00b0C for 15 min. The resulting supernatants (sera) were collected and supplemented with a protease inhibitor cocktail . Sera were distributed into aliquots of 50 \u03bcL and stored at \u221280 \u00b0C until utilization.Serum samples were collected at Veterinary Medical Teaching Hospital (VMTH), National Chung Hsing University (NCHU), Taichung, Taiwan, from 2017 to 2021. All experimental procedures and sample collection were approved by Institutional Animal Care and Use Committee (IACUC) of NCHU (Code: 109-002). Written informed consent was provided by each dog owner for the experimental procedures. Blood samples were collected from 27 healthy female dogs and 90 female CMT dogs before partial or complete mastectomy, including 20 dogs with benign CMT (BMT) and 70 dogs with malignant CMT (MMT) which were classified and staged according to the modified WHO TNM classification for CMT .An in-house ELISA was established for serum AAb detection . The recNinety CMT dogs were periodically followed up through telephone interviews every 6 months. Overall survival time (OST) was calculated as the survival period from surgery to death or to the last of follow-up visit . The patients who died within one week after surgery or died from an unrelated cause were excluded from this study. No patient was performed with euthanasia because of CMT.U test. Difference of serum AAb levels before and after surgery was analyzed by paired Wilcoxon matched-pairs signed-rank test and Wilcoxon signed-rank test. For overall survival (OS) analysis, the median serum levels (OD) of TYMS-AAb (0.370), IGFBP5-AAb (0.313), HAPLN1-AAb (0.344), and AGR2-AAb (0.274) were respectively set as the cutoff to dichotomize the analyzed cases into high (the AAb level > median) and low (the AAb level \u2264 median) groups. OS analysis was conducted using the Kaplan-Meier method, and the statistical significance was evaluated using the log-rank test. All statistical analyses were performed by using the GraphPad Prism V8.4 software . All p values were two-tailed; a p < 0.05 was considered statistically significant. The performance of the serum AAb level in OS prediction was estimated by using the \u201csurvival ROC\u201d package with a time-dependent ROC curve in RStudio V1.1463 software as previously described [Comparison of presurgical serum AAb levels between two categories was conducted using the Mann\u2013Whitney escribed .To verify the correlation between CMT progression and serum levels of TYMS-AAb, IGFBP5-AAb, HAPLN1-AAb, and AGR2-AAb, we conducted an in-house ELISA established previously to assesp < 0.001 and p < 0.05, respectively) or with those in the BMT group . Moreover, HAPLN1-AAb levels in the metastasized MMT dogs were significantly higher than those in the healthy dogs compared with those in the BMT group and remained significantly low compared with the pre-surgery levels .To evaluate the responses of CMT-associated AAbs after tumor resection, serum samples of 11 MMT dogs were collected before surgery and at 3 and/or 12 months post-surgery, respectively. Signalments of these 11 MMT cases are listed in To better monitor the changes in serum AAb levels in individual patients after surgery, a postsurgical-to-presurgical ratio of the serum AAb was calculated by dividing the postsurgical AAb levels by the matched presurgical levels. As shown in We further investigated the prognostic correlation between the presurgical serum AAb level and CMT patient\u2019s outcomes in terms of overall survival time (OST). The median and the range of OST of 90 CMT dogs or those of the subgroups were summarized in High (n = 49) and TYMS-AAbLow (n = 41); IGFBP5-AAbHigh (n = 48) and IGFBP5-AAbLow (n = 42); AGR2-AAbHigh (n = 46) and AGR2-AAbLow (n = 44); HAPLN1-AAbHigh (n = 49) and HAPLN1-AAbLow (n = 41). Kaplan-Meier survival analysis revealed that higher serum IGFBP5-AAb and TYMS-AAb levels were significantly correlated with inferior OS to estimate the prognostic potential of the serum AAb level in OS prediction. As listed in Furthermore, for predicting OS of non-metastasized MMT dogs (stage I/II/III), utilizing TYMS-AAb and utilizing IGFBP5-AAb achieved best AUC . Notably, the AUC of utilizing HAPLN1-AAb in predicting OS of non-metastasized MMT dogs also reached 0.614. Collectively, the data indicate that TYMS-AAb and IGFBP5-AAb are potentially useful in OS prediction for MMT dogs, particularly for non-metastasized patients.High) and/or high levels of IGFBP5-AAb (IGFBP5-AAbHigh), plus a low level of AGR2-AAb (AGR2-AAbLow). Data revealed that CMT dogs exhibiting TYMS-AAbHigh plus AGR2-AAbLow had worse OS when compared with the others. Notably, CMT dogs exhibiting TYMS-AAbHigh, IGFBP5-AAbHigh plus AGR2-AAbLow have worst OS (p < 0.0001). Moreover, a combination of TYMS-AAbHigh, IGFBP5-AAbHigh and AGR2-AAbLow also stratifies MMT dogs having poorest OS (p = 0.0004). These data reveal a correlation of serum levels of TYMS-AAb, IGFBP5-AAb, and AGR2-AAb with CMT outcomes, suggesting a possibility of utilizing this AAb panel for prognostic prediction of CMT. As there is a limited number of prognostic AAb identified for CMT, it is critically needed to expand the panel of CMT-associated AAbs possessing prognostic potential.Due to tumor heterogeneity and variable profiles of tumor-associated AAbs among patients, it could be less effective to use a single AAb for cancer prognostication ,34. DeteIn summary, the present study is the first to demonstrate an association of circulating TYMS-AAb, IGFBP5-AAb, and AGR2-AAb with CMT prognosis. This study provides a starting point to further verify the validity of this tailored AAb panel in prediction of tumor relapse and therapy responses of CMT in the future."} +{"text": "The design and fabrication ofsynthetic self-assembled systemsthat can mimic some biological features require exquisitely sophisticatedcomponents that make use of supramolecular interactions to attainenhanced structural and functional complexity. In nature, nucleobaseinteractions play a key role in biological functions in living organisms,including transcription and translation processes. Inspired by nature,scientists are progressively exploring nucleobase synthons to createa diverse range of functional systems with a plethora of nanostructuresby virtue of molecular-recognition-directed assembly and flexibleprogrammability of the base-pairing interactions. To that end, nucleobase-functionalizedmolecules and macromolecules are attracting great attention becauseof their versatile structures with smart and adaptive material propertiessuch as stimuli responsiveness, interaction with external agents,and ability to repair structural defects. In this regard, a rangeof nucleobase-interaction-mediated hierarchical self-assembled systemshave been developed to obtain biomimetic materials with unique properties.For example, a new \u201cgrafting to\u201d strategy utilizingcomplementary nucleobase interactions has been demonstrated to temporarilycontrol the functional group display on micellar surfaces. In a differentapproach, complementary nucleobase interactions have been exploredto enable morphological transitions in functionalized diblock copolymerassembly. It has been demonstrated that complementary nucleobase interactionscan drive the morphological transformation to produce highly anisotropicnanoparticles by controlling the assembly processes at multiple lengthscales. Furthermore, nucleobase-functionalized bottle brush polymershave been employed to generate stimuli-responsive hierarchical assembly.Finally, such interactions have been exploited to induce biomimeticsegregation in polymer self-assembly, which has been employed as atemplate to synthesize polymers with narrow polydispersity. It isevident from these examples that the optimal design of molecular buildingblocks and precise positioning of the nucleobase functionality areessential for fabrication of complex supramolecular assemblies. Whilea considerable amount of research remains to be explored, our studieshave demonstrated the potential of nucleobase-interaction-mediatedsupramolecular assembly to be a promising field of research enablingthe development of biomimetic materials.This Account summarizesrecent examples that employ nucleobaseinteractions to generate functional biomaterials by judicious designof the building blocks. We begin by discussing the molecular recognitionproperties of different nucleobases, followed by different strategiesto employ nucleobase interactions in polymeric systems in order toachieve self-assembled nanomaterials with versatile properties. Moreover,some of their prospective biological/material applications such asenhanced drug encapsulation, superior adhesion, and fast self-healingproperties facilitated by complementary nucleobase interactions areemphasized. Finally, we identify issues and challenges that are facedby this class of materials and propose future directions for the explorationof functional materials with the aim of promoting the developmentof nucleobase-functionalized systems to design the next generationof biomaterials. VarlasS.; HuaZ.; JonesJ. R.; ThomasM.; FosterJ. C.; O\u2019ReillyR. K.Complementary Nucleobase InteractionsDrive the Hierarchical Self-Assembly of Core\u2013Shell BottlebrushBlock Copolymers toward Cylindrical Supramolecules. Macromolecules2020, 53, 9747\u20139757.1This article reports thermally inducedsupramolecular assembly of bottlebrush polymers facilitated by complementaryH-bonding between nucleobase pairs.HuaZ.; JonesJ.R.; ThomasM.; ArnoM. C.; SouslovA.; WilksT. R.; O\u2019ReillyR. K.Anisotropic polymer nanoparticleswith controlled dimensions from the morphological transformation ofisotropic seeds. Nat. Commun.2019, 10, 540631776334.2This communicationreveals a method to produce highly anisotropic nanoparticles withcontrolled dimensions by means of a morphological transformation processdriven by complementary nucleobase interactions.HuaZ.; KeoghR.; LiZ.; WilksT. R.; ChenG.; O\u2019ReillyR. K.Reversibly Manipulating the SurfaceChemistry of Polymeric Nanostructures via a \u201cGrafting To\u201dApproach Mediated by Nucleobase Interactions. Macromolecules2017, 50, 3662\u2013367028529382.3This article describes a supramolecular \u201cgraftingto\u201d method to fabricate highly functionalized mixed polymericnanostructures exploiting multiple H-bonding interactions betweenthymine- and adenine-containing complementary polymers.HuaZ.; Pitto-BarryA.; KangY.; KirbyN.; WilksT. R.; O\u2019ReillyR. K.Micellar nanoparticles withtunable morphologies through interactions between nucleobase containingsynthetic polymers in aqueous solution. Polym.Chem.2016, 7, 4254\u20134262.4This article highlights the role of complementary nucleobaseinteractions to induce nanostructure reorganization to achieve differentnanostructures of variable shape and size.5 nucleobase interactions have been widely explored.The development of highly organized biomimetic materials through theprogrammed assembly of bioinspired molecular building blocks has beena rapidly expanding field in the areas of functional materials, biomedicine,and bioengineering processes.7 The rich chemistry betweennucleobase pairs with unique features such as reactivity, responsiveness,chirality transfer, and biocompatibility as well as their inexpensivecommercial availability has motivated scientists to use nucleobasefunctionality in molecular and macromolecular assembly.12 Exploiting nucleobases to generate functional materials can offerflexibility because of the different binding properties of differentnucleobases. For example, the A:T base pair interacts via two-pointH-bonding with a binding constant (Ka)of \u223c102 (in chloroform), whereas the G:C pair involvesthree-point H-bonding with a 2 orders of magnitude increase in bindingconstant (Ka \u223c 104 inchloroform) and thymine (T) and between guanine (G) and cytosine (C),oroform) 1a.13 Int18 Therefore, the possibilitiesare endless for creating elegant nanostructures by designing suitablebuilding blocks with different topologies and vinylbenzyl derivatives of thenucleobases adenine and thymine (VBA/VBT). The supramolecular coassemblyof VBA-co-DMA and VBT-co-DMA polymers(PVBA/PVBT) of the same molecular weight by postpolymerization modification led to an alternate copolymer because of the strongintermolecular H-bonding between the adenine and thymine monomers,whereas in dimethylformamide (DMF) statistical copolymers were formedbecause of the absence of nucleobase interactions. Interestingly,self-assembly of the block copolymers prepared in DMF and CHCl3 led to distinctly different morphologies depending on thepolymerization conditions, further highlighting the role of nucleobaseinteractions in tuning hierarchical assembly (PDLLA) polymers containingperipheral A and T units (PDLLA-A and PDLLA-T) and PDLLA without thenucleobase units as a control system. Association of the nucleobase-functionalizedstar-shaped polymers in CHCl3 resulted in higher viscositycompared with the coassembly of the corresponding non-nucleobase-functionalizedprecursors, confirming the formation of a supramolecular structure.Job\u2019s plots and Benesi\u2013Hildebrand analysis suggestedthe formation of a 1:1 complex with strong intermolecular H-bonding.To investigate further the effect of molecular weight on complementaryrecognition, a series of PDLLA-A and PDLLA-T polymers were preparedby variation of the chain length, and it was found that the molecularrecognition property of the A:T unit decreased with increasing molecularweight.Apart from linear polymerchains, nucleobase-containing star-shapedpolymers have also been explored to investigate the effect of complementarynucleobase interactions. Long and co-workers29 explored the assembly of amphiphilic nucleobase-containingpolymers in aqueous media. Block copolymers (PEG-b-A/PEG-b-T) -b-poly amphiphilic block copolymers where the A and T units werepresent in the hydrophobic block methyl ether methacrylate and hydrophobic n-butyl methacrylate connecting an A or T unit either at the hydrophilicend (PCL) and thymine-modified PEG and prepared nanoparticles in situby comixing the polymers. Moreover, they highlighted the advantageof complementary base pairing to achieve polymer micelles with a verynarrow size distribution compared with conventional amphiphilic assemblyof diblock polymers. By the same principle, water-soluble luminescentnanoparticles were prepared by coassembly of thymine-functionalized\u03c0-conjugated fluorescent polymers with adenine-containing PEG.33 In this regard, we have reported a unique wayto introduce fluorescence in a novel class of nucleobase-containingdiblock copolymers containing an adenine or thymine unit as the hydrophobicblock and polymorpholine as hydrophilic segment (PS-b-PVBT) as a function of the DMF:H2O ratio aswell as the molecular weight of the PS-b-PVBT block to tune the sizeand morphology of the polymer nanostructures blocksand either A- or T-conjugated polypropylacrylamide blocks that utilizes A:T H-bonding interactions to drive the insertion ofthe complementary polymer into an isotropic polymer nanoparticle followedby elongation to produce wormlike nanostructures. The dimensions ofthe worms were dependent on the amount of complementary polymer added.It is worth noting that the size of the worm and the amount of complementarypolymer added displayed a linear relationship, which allowed us toget a desired particle of a specific length on demand.We have further demonstrated controlled growthof polymer nanoparticlesthat exploits base-pair interactions to produce anisotropic structureswith a high aspect ratio.1 Thymine-and adenine-containing bottlebrush polymers (TBB and ABB) were foundto form anisotropic nanoparticles individually in aqueous solutionwith corona-forming poly(4-acryloylmorpholine) and core-forming poly(thymineacrylamide)(TBB polymer) or poly(adenineacrylamide) (ABB polymer) blocks blocks 5. Mixing37 A series of amphiphilic block copolymers of similar molecularweight were prepared using different nucleobase (A/T/G/C)-functionalizedstyrene or methacrylate and N-isopropylacrylamide(NIPAM) monomers having flexible (methacrylate) or rigid (polystyrene)backbones. Notably, spherical nanoparticles were obtained for individualpolymer assembly, whereas very different nanostructures were observedfor 1:1 coassembly of complementary polymers, such as spindlelikestructures or short rodlike morphology depending on the base pair(A:T or G:C) and the rigidity of the polymer backbone.Along with A:T interactions, G:C-interaction-inducedmorphologicalevolution has been explored by Thang and colleagues.3 The thymine-containingdiblock copolymer PNAM-b-PTAm was used to preparecore\u2013shell micelles with a T-containing core and poly(4-acryloylmorpholine)corona. We successfully obtained stimuli-responsive micelles withdifferent sizes by adding a series of complementary diblock copolymers(PNIPAM-b-PAAm) containing thermoresponsive (PNIPAM)blocks with various chain lengths. Next, we targeted achieving temperaturecontrol with functional group display of polymer nanoparticles. Weused the carbohydrate d-mannose, which is known to bind withlectin protein concanavalin A (Con A), as a functional group. A mannoseunit was conjugated to PNAM-b-PTAm micelles witha mannose-functionalized corona. Addition of the complementary polymerchain PNIPAM-b-PAAm to these micelles afforded mixed-coronamicelles in which the mannose groups were buried inside the PNIPAMcorona and showed no interaction with Con A at 25 \u00b0C, whereasabove the LCST the PNIPAM chains were collapsed, revealing the mannoseligands bound with Con A proteins.Along with inducingmorphological changes, nucleobase interactionshave been utilized for surface functionalization to impart desiredproperties to polymer nanoparticles. During the past decade, we havedemonstrated a versatile \u201cgrafting to\u201d strategy to preparefunctionalized mixed-corona micelles by exploiting A:T H-bonding betweencomplementary diblock copolymers 6.3 The t38This base-pair-interaction-mediated \u201cgrafting to\u201dapproach was further explored with cellulose-grafted complementary-nucleobase-functionalizedbottlebrush polymers to fabricate a series of polymer nanostructureswith similar shapes but varying surface charge as well, to achievethermally induced reversible morphological transformation from a sphereto a worm.41Templated synthesis is afundamental biological process in nature,where the information stored in DNA strands in terms of sequence andspatial arrangement of the nucleic acids/nucleobases can be transferredprecisely to its daughter strands. To mimic this phenomenon, nucleobase-containingpolymers have been implemented to prepare precision polymers suchas sequence-controlled polymers or polymers with similar degree ofpolymerization (DP) and dispersity as their parent polymers.39 demonstrated thetemplatedsynthesis of nucleobase-functionalized polymers by alignment of theadenine monomers on a thymine-containing block copolymer templateby virtue of A:T intermolecular H-bonding interactions followed bySonogashira polymerization. The daughter polymer was found to copythe chain length of the parent polymer and possess narrow polydispersity,whereas similar polymers prepared by a non-templated method generatedshort oligomers with higher polydispersity. The success of this nucleobase-templatedpolymerization stems from excellent programmability due to the highlydirectional complementary nucleobase interaction.Lo and Sleiman40 For this goal, thelow-molecular-weight block copolymer poly(styrene-b-vinylbenzylthymine (PSt-b-PVBT) was synthesizedto obtain core\u2013shell micelles with thymine units at the coreof the micelles that acted as a template to polymerize adenine-containingvinyl monomers. Herein, the micro confined zone in the micellar coreenabled segregation of the propagating radical chains, leading toa complementary daughter polymer with high molecular weight (Mw up to \u223c400\u202f000 g mol\u20131) and low polydispersity (\u22641.08). Afterward, such template-directedradical polymerizations were explored by Garcia et al.41 using uridine-derived polymer templates immobilizedon a solid support to selectively achieve an adenine-containing daughterpolymer within a mixture of different nucleobase-containing monomers.We have combinedself-assembly-mediated segregation of nucleobaseunits and a templating approach to synthesize polymers via free radicalpolymerization in order to obtain unprecedented control over the molecularweight of the daughter polymer 7.40 For 48 For example, Zhu and co-workers42 exploitedadenine-terminated PCL (PCL-A) and uracil-terminated PEG (PEG-U) tofabricate amphiphilic supramolecular block copolymers that subsequentlyformed micelles. The drug release profile of the encapsulated doxorubicin(DOX) revealed enhanced DOX delivery at mildly acidic pH comparedwith that at physiological pH due to disruption of the H-bonding betweenthe A:U pair. In a related study, Huang and co-workers43 reported dual pH-responsive supramolecular micellesusing nucleobase-conjugated dextran polymers assembled via A:T interactionsthat disassembled at lower pH because of disruption of the complementaryH-bonding as well as hydrolysis of the ketal group of the dextranbackbone. Recently, Chen and co-workers44 reported the formation of bionic nanocapsules designed by cross-linkingof thymine- and adenine-containing polymer precursors and demonstratedthe role of nucleobase interactions in preventing premature drug leakagein their in vivo circulation in addition to the controlled diffusionof the encapsulated drug.Formation of nucleobase-mediated cross-linked polymernetworkshas been proven to be a promising approach for drug delivery applicationsbecause it has several advantages, such as increased micellar stability,enhanced cellular uptake, increased drug loading capacity, reduceddye leakage, and stimuli-responsive controlled release properties.46 For instance, an adenine-terminatedstar-shaped PCL polymer and monofunctionalized uracil-connected PEGchain (U-PEG) were utilized by Zhu and co-workers to form a supramolecularamphiphilic hyperbranched copolymer that assembled into pH-responsivemicelles.45 Subsequently, they furtherexplored the assembly of A-functionalized brush copolymers with U-PEGthat coassembled via A:U interactions to form polymer nanoparticleswith pH- and salt-responsive properties in order to utilize this asa drug delivery vehicle.46Nucleobase-functionalized non-linearpolymers have also been exploredas drug delivery vehicles.49 A similar concept wasalso explored by Lee and co-workers50 usinguracil-functionalized PPG and an adenine-connected model drug. Inthat study, the formation of supramolecular micelles by U:A interactionsfollowed by irradiation-mediated photo-cross-linking of the U unitsresulted in long-term structural stability of the micelles in serumsolution.Another interesting strategy hasbeen explored in the developmentof drug delivery systems by the use of a nucleobase-conjugated drugalong with a complementary-nucleobase-conjugated polymer chain. Forinstance, Cheng and co-workers recently reported a coassembled systemusing uracil end-capped poly(propylene glycol) (U-PPG-U) and adenine-modifiedrhodamine 6G (A-R6G) featuring an extremely high A-R6G loading andexcellent structural stability in biological media along with pH-triggeredrelease of A-R6G.51 Acentral thymine oligomer (T10) connected to a hydrophobic poly(lactic-co-glycolic acid) (PLGA) block and a hydrophilic PEG blockassembled into core\u2013shell micelles where the middle T10 blockwas utilized to bind complementary DNA strands with high loading capacity.They further used this polymer to encapsulate a prodrug-activatingenzyme, cytosine deaminase (CodA), connected with a complementaryDNA strand by intermolecular H-bonding along with DOX via hydrophobicinteractions and demonstrated the advantage of dual encapsulationon anticancer activity (52Cha and co-workers synthesized a unique designed polymer(PEG-T10-PLGA)to use as a gene delivery vehicle.activity 8.5254 using the complementary and reversible intermolecularH-bonding ability of the nucleobase functionality.Self-healing materialsare ubiquitous in nature and play a criticalrole in repairing damage and replacing degenerated parts in livingorganisms. For example, DNA repairs itself by radical scission ofits strand, inducing a DNA repair cascade. This has been employedas a source of inspiration for the development of self-healing polymericmaterials53 developed a seriesof self-healing nucleobase-functionalized hyaluronicacid-based hydrogels derived from G:C intermolecular interactions.The G:C-interaction-mediated hydrogels showed better mechanical propertiesand higher healing efficiencies than the corresponding single-component(G or C) hydrogels, confirming the significance of the complementaryH-bonding interaction. In a different design, Jiang and co-workers54 produced a type of self-healing polymer hydrogelwith improved elasticity, tensile strength, and recoverable mechanicalproperty by conjugating nucleobase (A and T) precursors to the knownelastomer bis(3-aminopropyl)-terminated polydimethylsiloxane.In this context,Gu and co-workers58 Long and co-workers55 prepared nucleobase-containing statistical copolymersby polymerizing A/T-functionalized acrylate monomers. Blending ofA- and T-containing polymers led to supramolecularly cross-linkedmaterials with tunable adhesive and cohesive strength depending onthe A:T interaction. Yu and co-workers56 developed a new class of adhesive hydrogels prepared by introducingnucleobase functionality into the biodegradable polyphosphoesterbackbone. This nucleobase-tackified polyphosphoester adhesive gelshowed excellent adhesive performance, controllable biodegradation,and outstanding biocompatibility due to multiple H-bonding interactions.Liu and co-workers57 developed a seriesof nucleobase (A and T)-functionalized homopolymers and statisticalcopolymers and demonstrated that compared with the individual homopolymers,a comixture of the complementary homopolymers exerted superior outstandingmechanical properties and higher adhesive propensity due to intra-and intermolecular H-bonding interactions. We have fabricated hydrophobicnucleobase-containing adhesives by synthesizing a random copolymerusing hydrophobic long aliphatic chain-containing monomers as wellas both A- and T-containing monomers (58 This elegantlydesigned supramolecularly assembled polymer network produced the strongestunderwater adhesives reported to date, outperforming most previouslyreported underwater hydrogel-based adhesives.Adhesion is another common property of living organisms, such asmussels clinging on rocks and geckos walking on vertical walls. Asvarious non-covalent interactions are responsible for such adhesiveproperties, researchers have been interested in exploring adhesivematerials based on nucleobase-pair interactions.monomers 9.58 This30 to vesicles,22 one-dimensional fibers,38 helices, and even more complexthree-dimensional37 structures, along with controlof the functional design. These nucleobase-functionalized moleculesand macromolecules have shown promising results in their prospectiveapplications such as drug48 and gene52 delivery, templated polymerization,41 and superior adhesive58 and self-healing54 properties.The association constant of nucleobase interactions can be tuned from102 to 106 M\u20131 as a functionof several parameters such as the nature of the H-bonding unit,37 solvent polarity,27 temperature,26 pH,46 and number, sequence, and position of the nucleobases31 present in the system. Intermolecular interactionsin these novel systems can be tuned upon requirement to create programmableassemblies featuring adaptive properties. Furthermore, with excellentperformance in terms of responsiveness, tunable mechanical properties,biocompatibility, and biodegradability, these supramolecular materialscertainly are expected to have many possibilities for the design andpreparation of next-generation biomimetic materials.As illustrated in this Account,nucleobase-functionalized molecularand macromolecular systems have proven to be a promising area of researchbridging the gap between materials chemistry and biology. Base-pairinteractions have been explored to drive self-assembly to fabricatea wide variety of structures ranging from spherical micellesNotwithstandingthe aforementioned advantages, there are certainchallenges that still need to be addressed in order to expand theirimpact and utility in biomaterials research. One such constraint isthe often poor solubility of the nucleobases in many organic solvents,making it sometimes difficult to prepare new functional moleculesor monomers. Second, resolving the poor solubility of nucleobase-containingmolecules and macromolecules in aqueous solutions, which preventsscientists from exploring these nucleic acid analogues under physiologicalconditions, should be a next step forward to biomimicking. Explorationof new monomers connected to a suitable solubilizing group as wellas synthesis of monomers with non-natural nucleobases or other complementaryH-bonding systems such as diaminotriazine or ureidopyrimidinone canprovide a feasible route to address this.Built on the knowledgefrom the previous discussions, it is evidentthat investigations of nucleobase-containing substances have mainlybeen limited to A:T interactions and not much explored with G:C pairs.This could be due to undesirable interactions between G and C nucleobasemonomers, which interact via a stronger three-point H-bonding, leadingto monomer aggregation and poor polymerization control with largepolydispersity and incomplete polymerization of the monomer. Thisissue could be overcome by protection of the nucleobases during polymerizationfollowed by deprotection or by the use of postpolymerization functionalizationtechniques in order to fully explore supramolecular systems with G:Ccomplementary pairs. Moreover, judicious incorporation of both A:Tand G:C pairs in synthetic building blocks could open an excitingpath for future research.Looking forward, nucleobase-functionalizedmolecules and macromoleculesare an intriguing platform to prepare complex nanostructures. However,it is worth emphasizing that we are still far from devising highlyprogrammable multifunctional complex nanostructures like those presentin nature. For instance, accurate positioning of \u03b1-helixes and\u03b2-sheets followed by macromolecular folding in a specific wayto generate specific tertiary and quaternary structures in proteinsis responsible for the transfer of molecular information necessaryto play a large range of functions in our body. To harness such informationtransfer with wholly synthetic systems, one needs to judiciously designthe constituent building blocks for logical reprogramming of hierarchicalmolecular organization. On that account, more research must be conductedto rationally derive engineered nanostructures by combining the molecularrecognition properties of nucleobases with other directional interactionsalong with responsive functional groups in order to introduce multipleswitchable properties. Additionally, more sophisticated macromolecularstructures such as sequence-controlled polymers can be used, whichcan effectively control the chain folding or single-chain manipulationbecause of the high structural precision of the position of a specificnucleobase functionality. New approaches and strategies are requiredto produce sequence-controlled polymers, as to date genetically encodedsequence-defined polymers are limited only to polymerase- and ribosome-mediatedsynthesis, which imposes constraints on structural diversity. Moreimportantly, these sequence-defined polymers need to be explored toconstruct more complex supramolecular nanostructures with high programmabilityand adaptable properties, which could prove to be a major breakthroughin supramolecular biomaterials research."} +{"text": "Correction: BMC Geriatrics 22, 318 (2022)https://doi.org/10.1186/s12877-022-03014-6After publication of this article , the autThe last paragraph of the Results section should read: \u201cAssociations between early prescription of anti-osteoporosis agents and long-term medical costs were analyzed and presented in Table 3. A total of 233 patients had their anti-osteoporosis agent prescribed within one month post discharge, and 345 patients had not. Both the number of patients returned for overall medical services and the average expenditure (per returned patient or per patient in treatment group) for overall medical services were significantly lower in patient on anti-osteoporosis agent , whilst average expenditure (per returned patient or per patient in treatment group) for outpatient clinic service was significantly higher in patient group on anti-osteoporosis agent (p\u2009<\u20090.001 and p\u2009<\u20090.001 respectively), as compared to patient group not on anti-osteoporosis agent.\u201dThe table legend should read: \u201cTable 3. Relation of early anti-osteoporosis agent prescription with 1-year medical visits and expenditure.\u201dThe original article has been"} +{"text": "This systematically excludes adolescents and young adults who have been perceived to have worse outcomes. We have previously reported long-term survival of our adolescent and young adult cohort that were largely comparable to childhood medulloblastoma. We now report on molecularly characterized WNT-subgroup patients treated between 2004\u20132020 with risk-stratified multi-modality therapy to identify differences between childhood (<15 years) versus adolescent and young adults (>15 years). Despite modest differences in disease status at presentation and treatment modality, there were no significant differences in patterns of failure or survival between childhood versus adolescent and young adult WNT-pathway medulloblastoma. Two de-intensification trials in low-risk WNT-pathway medulloblastoma \u2013 first testing omission of upfront craniospinal irradiation and second a primary chemotherapy approach after surgery \u2013 had to be terminated prematurely due to unacceptably high relapse rates suggesting that craniospinal irradiation remains an integral component of treatment. The presence of TP53 mutations and OTX2 gains have recently been reported as independent negative prognostic factors in a multi-institutional cohort of WNT-pathway medulloblastoma raising questions on eligibility of such patients for de-escalation trials. The definition of low-risk WNT-pathway medulloblastoma may need to be refined in light of recent clinical data and newer biological information.Novel biological insights have established that medulloblastoma is a heterogenous disease comprising four broad molecular subgroups - WNT, SHH, Group 3, and Group 4 respectively, resulting in the incorporation of molecular/genetic information in 5th edition of WHO classification and contemporary risk-stratification. Concerns regarding therapy-related late toxicity in long-term survivors have led to systematic attempts at treatment de-intensification in good-risk medulloblastoma. Given the excellent survival (>90%) of WNT-pathway medulloblastoma, prospective clinical trials have focused on optimization of therapy to balance survival versus quality of survival. The currently accepted definition of low-risk WNT-pathway medulloblastoma includes children <16 years of age with residual tumour <1.5 cm The cur disease \u20136. Conve therapy .Novel biological insights have vastly improved our fundamental understanding of pediatric brain tumors with potential to transform therapy , 3. It i2 and no evidence of metastases. This systematically excludes adolescents and young adults (AYAs) who have been perceived to have worse outcomes compared to their childhood counterparts. An analysis of the Surveillance, Epidemiology, and End Results (SEER) database [n = 616) and adult MB (n = 349). We have also previously reported long-term survival outcomes of our AYA-MB cohort [p-value <0.05 was considered as statistically significant. All analysis was done on SPSS version 24.0 and RStudio version 4.03.Contemporary aggressive multi-modality treatment provides excellent long-term survival particularly in WNT-MB with 5-year survival exceeding 90% , 13. Howdatabase from 199B cohort that werp = 0.80, p = 0.30, During this time-period, a total of 67 patients - 44 children (<15 years) and 23 AYAs (\u226515 years) were diagnosed with WNT-MB at our institute. Patient, disease, treatment characteristics and patterns of failure are summarized in n = 93). Fifteen patients with relapse were identified, 12 in metastatic compartment including one with extra-neuraxial metastases and 3 in the surgical cavity. Lower cumulative dose of cyclophosphamide/ifosfamide (<12 mg/m2) during maintenance chemotherapy (p = 0.032) and male gender (p = 0.033) were associated with significantly increased risk of relapse. Age at diagnosis, extent of resection, metastases, CSI dose, and additional molecular/genetic alterations did not influence the risk of relapse. More recently, 5-year PFS and OS of 100% has been reported in WNT-MB in conventionally classified average-risk (n = 46) as well as high-risk disease (n = 7) from a prospective cohort study of risk-adapted therapy (SJMB-03) [n = 64) confirming excellent outcomes regardless of risk-stratification and age. In accordance with above retrospective and prospective clinical data, we believe that age alone should not preclude WNT-MB patients from participation in prospective clinical trials testing treatment de-intensification.Our results are in accordance with previously published reports. Nobre et al. reportedSJMB-03) . AnotherSJMB-03) that incAttempts at treatment de-escalation in non-metastatic childhood MB are not new and have been attempted systematically since the last 40 years. The first successful de-intensification was the reduction from full-dose CSI (36 Gy) to reduced-dose CSI 23.4 Gy) with addition of adjuvant systemic chemotherapy in children with average-risk MB .4 Gy wit or reducTP53 mutations or OTX2 gains emerged as independent poor prognostic markers [Various molecular markers and associated signalling pathways involved in metastases from MB have been described . However markers raising markers has idenConcerns regarding therapy-related late toxicity have prompted systematic attempts at treatment de-intensification in good-risk MB over the last four decades. However, results of prior studies should be used to inform and guide controlled de-intensification of therapy even in low-risk and favourable biology disease. The definition of low-risk WNT-MB may need to be further refined in light of recent clinical data and newer biological information."} +{"text": "In the current extended clinical case, we sought to delineate cardiac uptake of 68Ga-FAPI-04 in a patient after MI with clinical indication for the evidence of fibroblast activation. Carcinoma patients without cardiac disease underwent 68Ga-FAPI-04-PET/CT as control. The patient with one-vessel disease underwent dynamic 68Ga-FAPI-04-cardiac-PET/CMR for 60 minutes. Correlation of cardiac 68Ga-FAPI-04 uptake with clinical findings, ECG, echocardiography, coronary-arteriography and enhanced cardiac-MRI with T1 MOLLI and ECV mapping were performed. No uptake was found in normal myocardium and in mature scar. A focal intense 68Ga-FAPI-04 uptake with continuous wash-out in the infarct territory of coronary occlusion correlating with T1 and ECV mapping was observed. The uptake of 68Ga-FAPI-04 extends beyond the actual infarcted area and overestimates the infarct size as confirmed by follow-up CMR.Our previous study has demonstrated the feasibility of noninvasive imaging of fibroblast activation protein (FAP)-expression after myocardial infarction (MI) in MI-territory in a rat model with Following myocardial infarction (MI), an orchestrated inflammatory response and reparative pathways are initiated, aiming to produce a robust and collagen-rich scar.68Gallium-labeled FAP-inhibitor (FAPI) compound 04 (68Ga-FAPI-04) was initially introduced for PET imaging of cancer-associated fibroblasts.7The serine protease fibroblast activation protein (FAP) is a membrane-anchored enzyme which is specifically expressed in fibroblasts activated to differentiate to (proto-)myofibroblasts, but not in dormant fibroblasts or mature fibrocytes.568Ga-FAPI-04-PET investigations in patients after MI have not been reported yet. For this case presentation, we retrospectively evaluated fibroblast activation in a patient after MI und carcinoma patients without cardiac disease.Further retrospective evaluations of cardiac FAPI distribution in a heterogeneous patient population with metastasized cancer associated with preexisting coronary artery disease, cardiovascular risk factors or metabolic disease were reported.68Ga-FAPI-04-PET/CT was the possible compassionate use of 177Lu-FAPI-radiotherapy68Ga-FAPI-04-PET/MR was the compassionate use for chimeric antigen receptor T-cell-therapyAll examinations had clinical indications and complied to the conditions of the updated Declaration of Helsinki and the German Pharmaceutical Law . In the controls, the indications for 68Ga-FAPI-04 of similar intensity as blood pool activity indicating no specific uptake are summarized in Table Patient population of control group is summarized in Table e Figure . The ave\u22121. The patient complained about persistent dyspnea and fatigue. To investigate the cardiac fibroblast activation, a dynamic PET/MR imaging was performed 6 days after STEMI immediately after intravenous injection of 165 MBq 68Ga-FAPI-04.A 33-year-old male was referred to intensive cardiac care unit due to acute STEMI followed by ventricular fibrillation. Return of spontaneous circulation after defibrillation. The present clinical findings at admission are summarized in Table 68Ga-FAPI-04-PET of this patient showed focal intense uptake in the anterior and anterior-septal wall, which correlated well with the sub-occluded LAD territory revealed in cine sequences a myocardial thickening of the anterior septum (14 mm end-diastolic) and thinning of the inferior apex with hypokinesia. The left ventricular function was moderately reduced (46%). In T2 weighted imaging, the anterior wall showed increased signal, suggesting myocardial edema. In dynamic myocardial perfusion imaging moderate hyperemia of the anterior wall could be observed. Early gadolinium enhancement sequences demonstrated a transmural enhancement of the anterior wall and adjacent septal segments, while late gadolinium enhancement revealed a sub-endocardial enhancement anterior-septal, but also a sub-endocardial enhancement in inferior apex. In T1 Modified Look-Locker Inversion Recovery (MOLLI) Figure A the ant68Ga-FAPI-04 scan. The left ventricular function recovered to almost normal with LVEF 60%. No hypokinetic ventricular wall motion was detected, except a discrete hypokinesia in the apex. Apart from the anterior-septal sub-endocardial scar was similar to normal myocardium of the control group, indicating the absence of active fibroblasts in healthy myocardium68Ga-FAPI-04 .68Ga-FAPI-04 uptake do not represent solely myocardial scaring process. But it also indicates more likely overlapping enhanced myocardial fibroblast migration in an inflammatory, proliferative process, such as in viable but ischemic jeopardized border zone or hibernating myocardium. The presence of active fibroblasts in the cardiac interstitium of the hibernating myocardium is reported to be an important indicator in determining recovery of function after revascularization.A major determinant of post-MI remodeling severity is the infarct size. In the present study, we observed that the extent of 68Ga-FAPI-04 uptake with the alteration in T1 and ECV mapping registered in our patient is remarkable (Figures 68Ga-FAPI-04-PET, however, may have a potential to specifically visualize fibrotic process already at its onset. It could therefore provide unique opportunities to study cardiac remodeling at molecular level over time and to monitor therapeutic interventions that aim to prevent a progressive decline of ventricular function.68Ga-FAPI-04-PET/CMR may boost the diagnostic potential of 68Ga-FAPI-04-PET by additional information achieved from CMR in identification of early manifestation of remodeling amenable to preventive intervention. To confirm our preliminary results and to further investigate the comprehensive pathophysiology, further studies with a larger population are encouraged.Contrast-enhanced CMR offers high spatial resolution and can identify acute myocardial infarction and myocardial scar. Figures , 3. Howe68Ga-FAPI-04-PET. Noninvasive assessment of activated fibroblasts may provide unique opportunities to identify early manifestation of cardiac remodeling amenable to preventive intervention.As shown in this case of a patient after STEMI, the enhanced FAP activation in acutely injured myocardium was identified und visualized with"} +{"text": "Although picornaviruses are conventionally considered \u2018nonenveloped\u2019, members of multiple picornaviral genera are released nonlytically from infected cells in extracellular vesicles. The mechanisms underlying this process are poorly understood. Here, we describe interactions of the hepatitis A virus (HAV) capsid with components of host endosomal sorting complexes required for transport (ESCRT) that play an essential role in release. We show release of quasi-enveloped virus (eHAV) in exosome-like vesicles requires a conserved export signal located within the 8 kDa C-terminal VP1 pX extension that functions in a manner analogous to late domains of canonical enveloped viruses. Fusing pX to a self-assembling engineered protein nanocage (EPN-pX) resulted in its ESCRT-dependent release in extracellular vesicles. Mutational analysis identified a 24 amino acid peptide sequence located within the center of pX that was both necessary and sufficient for nanocage release. Deleting a YxxL motif within this sequence ablated eHAV release, resulting in virus accumulating intracellularly. The pX export signal is conserved in non-human hepatoviruses from a wide range of mammalian species, and functional in pX sequences from bat hepatoviruses when fused to the nanocage protein, suggesting these viruses are released as quasi-enveloped virions. Quantitative proteomics identified multiple ESCRT-related proteins associating with EPN-pX, including ALG2-interacting protein X (ALIX), and its paralog, tyrosine-protein phosphatase non-receptor type 23 (HD-PTP), a second Bro1 domain protein linked to sorting of ubiquitylated cargo into multivesicular endosomes. RNAi-mediated depletion of either Bro1 domain protein impeded eHAV release. Super-resolution fluorescence microscopy demonstrated colocalization of viral capsids with endogenous ALIX and HD-PTP. Co-immunoprecipitation assays using biotin-tagged peptides and recombinant proteins revealed pX interacts directly through the export signal with N-terminal Bro1 domains of both HD-PTP and ALIX. Our study identifies an exceptionally potent viral export signal mediating extracellular release of virus-sized protein assemblies and shows release requires non-redundant activities of both HD-PTP and ALIX. Mechanisms underlying nonlytic release of canonical nonenveloped viruses from infected cells are poorly understood. We show here that release of hepatitis A virus from cells in exosome-like vesicles requires nonredundant activities of two distinct Bro1-domain proteins associated with host cell machinery for endosomal sorting (ESCRT), HD-PTP and ALIX. We demonstrate both Bro1 domain proteins are recruited to the viral capsid by the pX segment of the 1D capsid protein, and that they act in a non-redundant manner to mediate virus release. Fusing pX to a self-assembling nanocage protein resulted in ESCRT-dependent release mediated by a short pX peptide sequence conserved in hepatoviruses from bats to humans. Mutations within the pX sequence ablate release and result in noncytolytic virus accumulating intracellularly. Our study identifies an exceptionally potent viral export signal mediating extracellular release of virus-sized protein assemblies and shows nonlytic release of quasi-enveloped virus is an ancient evolutionary trait of hepatoviruses. Picornaviridae, a large and diverse family of positive-strand RNA viruses . Alt. Alt1]. ng HIV-1 ,60,61. Ung HIV-1 ,62,63.Picornaviridae xLR[L/M]xxGxxRxxxA\u2019, as well as a second conserved motif (\u2018[L/V]ESxVD\u2019) within the pentamer assembly domain . The ExpD sequence logo was generated from the 22 sequences above it with WebLogo version 2.8.2 (https://weblogo.berkeley.edu). Two bat Hepatovirus G viruses shown at the bottom have a large deletion in the pX region. *Bat virus pX sequences shown to mediate EPN secretion , then manually adjusted. See The alignment was constructed from 24 near genome-length hepatovirus sequences available in GenBank, recovered from 18 different mammalian species. Residues that are relatively well conserved are shown in red font, those less conserved in blue, followed by very poorly conserved residues in grey font. Residues that are absolutely conserved are underlined. Predicted VP1 and protein 2B sequences flanking pX are shaded in grey; the polyprotein cleavage sites have not been determined experimentally other than for (PDF)Click here for additional data file.S4 FigHep\u201d = Hepatovirus species). GenBank accession numbers and genotype of Hepatovirus A viruses are indicated. Arrows denote pX sequences studied as EPN fusions ((A) Phylogenetic tree showing relatedness of amino acid sequences of pX in 9 recognized hepatovirus species infecting 18 different mammalian species Imm(PDF)Click here for additional data file.S5 Fig(A) pX sequences in expression vectors used for LFQ analysis of pX-interacting proteins. EPN-PAD contains only the pentamer assembly domain (PAD) of pX. (B) Anti-Myc immunoblot showing triplicate protein precipitates subjected to proteomics analysis from 293T cells expressing the EPN-pX and EPN-PAD constructs. (C) Mean LFQ intensities of pX peptides in both protein precipitates . Intensities of peptides identified in the EPN-PAD sample (common to both EPN-pX and EPN-PAD) are shown in solid black bars; intensities of peptides unique to EPN-pX are shown in red. (D) Percentage of the pX sequence covered by peptides found in the EPN-PAD and EPN-pX samples. (E) Spectral counts of selected proteins identified in the proteomics samples. C-term pX = pX sequence uniquely present in EPN-pX and absent in EPN-PAD. (F) Peptide coverage of selected proteins identified in the proteomics samples. (G) Cellular component of all 349 proteins enriched over 2-fold in EPN-pX compared to EPN-PAD precipitates (FDR<0.05). Significance for protein clustering with each component is plotted on the right axis (hatched bars).(PDF)Click here for additional data file.S6 Fig(A) Co-immunoprecipitation of HA-ALIX and EPN-pX or EPN-pX mutants containing single amino acid substitutions of conserved ExpD residues co-expressed in 293T cells. Lysates of 293T cells transfected with DNAs expressing the indicated nanocage proteins and HA-tagged ALIX were precipitated with anti-Myc antibody, then immunoblotted with anti-HA antibody. (B) Quantitative comparison of the efficiency of co-immunoprecipitation of EPN constructs with ALIX shown in panel A versus the efficiency of EPN release when fused to pX with mutations in the ExpD domain . Data ar(PDF)Click here for additional data file.S7 Fig(A) Low magnification view and (B) super-resolution fluorescent microscopy images of a cell infected with 18f virus, demonstrating close proximity of viral antigens recognized by polyclonal human anti-HAV , endogenous ALIX (red), and endogenous HD-PTP (magenta). High magnification single- and dual channel images of the region bounded by the dashed yellow box are shown below in three dimensions below.(PDF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Tablehttps://scicrunch.org/resources.RRID: Research Resource Identification Portal, (PDF)Click here for additional data file."} +{"text": "Sus scrofa domesticus) [Pigs (esticus) ,2;Ovis aries) [Sheep (s aries) ,4;Capra aegagrus hircus) [Goats ( hircus) ,6;Bos taurus taurus) [Cattle ( taurus) ,8,9;Equus ferus caballus) [Horses ;Equus asinus \u00d7 Equus ferus caballus) [Mules ;Camelus dromedarius) [Dromedary camels (edarius) ;Camelus bactrianus ferus) [Bactrian camels (s ferus) ;Bubalus bubalis) [Water buffalos ;Oryctolagus cuniculus) [Rabbits (niculus) ;Felis silvestris catus) [Domestic cats (s catus) ;Canis lupus familiaris) [Domestic dogs (iliaris) ;Mus musculus musculus) [Mice (usculus) ;Rattus norvegicus domestica) [Rats (mestica) ;Mustela putorius furo) [Ferrets (us furo) ;Ovis aries/ammon musimon) [Mouflon (musimon) ;Bos gaurus) [Gaur ( gaurus) ;Cervus elaphus) [Red deer (elaphus) ;Capra pyrenaica pyrenaica) [Pyrenean ibex ;Felis silvestris lybica) [African wild cat ( lybica) ;Felis margarita margarita) [Sand cat (rgarita) ;Canis lupus lupus) [Gray wolf (s lupus) ;Canis latrans) [Coyote (latrans) ;Macaca fascicularis) [Cynomolgus monkey/macaque (cularis) .Thus far, nearly 25 mammalian species have been cloned by intra- or interspecies somatic cell nuclear transfer (SCNT). Among them, non-transgenic and transgenic representatives of such domesticated and wild-living animals that have been propagated and/or multiplied by intraspecific or interspecific SCNT-based cloning are:Despite the above-indicated abundant variety of SCNT-derived mammalian species, the effectiveness of SCNT-based cloning remains immensely or considerably low and oscillates between 0.1% and 5%, while estimating the outcomes of offspring born in relation to the total numbers of nuclear-transferred oocytes ,31. For It is beyond any doubt that the relatively or extremely low efficiency of mammalian SCNT-mediated cloning, including both its intra- and interspecies model, can only be improved by comprehensively recognizing molecular and epigenetic determinants and mechanisms affecting the developmental competences of SCNT-derived embryos . A wide In summary, this Special Issue will publish research articles and comprehensive review papers aimed at highlighting the state of the art and mechanistic insights into precisely identifying and unravelling a wide array of genomic, epigenomic, transcriptomic and proteomic factors which cumulatively determine the molecular parameters which are of paramount importance for the quality of nuclear donor cells, nuclear recipient oocytes and SCNT-derived embryos. Thoroughly deciphering the multifaceted nature of all the aforementioned factors and insightful interpretation of the biological crosstalk between them can finally bias the augmentation of the overall efficiency of SCNT-based cloning. This is a preponderant condition indispensable for the practical implementation of SCNT-mediated ARTs to various research fields and interdisciplinary studies at the interface of experimental and applied embryology, biotechnology, transgenics, biomedicine, biopharmacology, the creation of animal models for etiopathogenesis and physiopathology of human diseases and the genetic rescue and/or resurrection of endangered/extinct mammalian species."} +{"text": "Dear Editors,Since the beginning of the COVID-19 pandemic, messenger RNA vaccination has been highly effective for preventing SARS-CoV-2 infections . Little In a prospective cohort study, we assessed SARS-CoV-2-specific immune response in 14 ICU patients (7 vaccinated and 7 non-vaccinated) between January and February 2022. Vaccinated patients received at least two doses of BNT162b2 vaccine with a delay ranging from two to six months. Of them, two had pre-existing immunosuppression (one kidney transplant and one mixed connective tissue disease). Demographic and clinical characteristics of the study population are provided in Additional file We evaluated humoral response by measuring anti-Spike IgG titers. Cellular response was assessed by T-cell proliferation assay and whole blood Interferon-Gamma Release Assay (IGRA) after stimulation with SARS-Cov-2 proteins .Upon ICU admission, vaccinated patients presented high anti-Spike IgG titers that were significantly higher than non-vaccinated patients Fig.\u00a0A. In conAlthough obtained in a relatively small cohort of patients, these results suggest that cellular response is also an important determinant of COVID-19 evolution in vaccinated patients. They raise concern about using humoral response as a sole metric of protective immunity following vaccination for SARS-CoV-2 especially in high-risk patients. Specific tools measuring cellular response, usable in a standardized routine practice, could guide the administration of COVID-19 vaccine booster in patients who did not mount any cellular response. This may help preventing evolution towards the most severe forms of the disease. Considering that an uncoordinated T cell and antibody responses have been associated with disease progression , the undAdditional file 1. Complementary methods and description of clinical data."} +{"text": "Behavioral-variant frontotemporal dementia (bvFTD) is characterized by impairment in socioemotional functioning. Spouses caring for individuals with bvFTD often experience profound health/well-being declines, compared to Alzheimer\u2019s disease (AD) caregivers and non-caregiving older adults. We hypothesized that disrupted positive emotional connections between spousal caregivers and individuals with bvFTD contribute to caregivers\u2019 lower emotional well-being. 23 bvFTD-caregiver, 23 AD-caregiver, and 17 control dyads had a 10-minute conflict conversation in the laboratory. Positive emotional connections were measured as the covariation of partners\u2019 positive emotional behaviors during the conversation. Caregiver emotional well-being was assessed via questionnaire (SF-36). We found that bvFTD caregivers had lower emotional well-being than AD caregivers and controls , c=-.70, p<.01. Importantly, this effect was fully mediated by bvFTD caregivers' lower positive emotional connections, c\u2019=-.38, n.s. We speculate that lower positive emotional connections can cause social isolation and contribute to bvFTD caregivers\u2019 health/well-being declines."} +{"text": "DMD variants is sometimes rather difficult, mainly due to complex structural variants (SVs) and deep intronic splice-altering variants. We performed genomic long-read whole DMD gene sequencing in a boy with asymptomatic hyper-creatine kinase-emia who remained genetically undiagnosed after standard genetic testing, dystrophin protein and DMD mRNA studies, and genomic short-read whole DMD gene sequencing. We successfully identified a novel pathogenic SV in DMD intron 1 via long-read sequencing. The deep intronic SV consists of a long interspersed nuclear element-1 (LINE-1) insertion/non-tandem duplication rearrangement causing partial exonization of the LINE-1, establishing a genetic diagnosis of Becker muscular dystrophy. Our study expands the genetic spectrum of dystrophinopathies and highlights the significant role of disease-causing LINE-1 insertions in monogenic diseases.The precise identification of pathogenic DMD variants and characterized by primary involvement in skeletal and/or cardiac muscle fibers, are among the most common inherited muscular dystrophies and mainly affect male patients and small pathogenic DMD variants, which can occur in coding and/or non-coding regions and short-read sequencing of 79 exons and adjacent intronic regions of DMD insertion/non-tandem duplication rearrangement, causing partial exonization of the LINE-1 in addition to the normal splicing of DMD, which ultimately established a genetic diagnosis of BMD in the patient.Herein, we performed standard genetic testing in a boy with a highly suspected BMD based on his clinical and pathological characteristics, which failed to detect pathogenic variant(s) in him. In order to discover potential deep intronic splice-altering variants in The patient enrolled in the study is a 3.5-year-old boy with an asymptomatic hyper-creatine kinase-emia phenotype. He presented to Peking University First Hospital Neuromuscular Center at the age of 3.5 years because of an incidental finding of hyper-creatine kinase-emia. His serum creatine kinase was markedly elevated in every test . No one else in his family had hyper-creatine kinase-emia. He had no delayed motor milestones and no exercise-induced muscle pain or cramps. Physical examination confirmed that he had mild calf hypertrophy and no muscle weakness, which was further validated by his muscle MRI examinations, showing no muscle fatty infiltration of the pelvis and thigh muscles. His muscle biopsy revealed myopathic changes, including several clusters of necrotic and regenerating muscle fibers; a few hypertrophic, atrophic, and hypercontracted muscle fibers; and a small number of internal nuclei . The immDMD (copy number variations) and a short-read sequencing panel for inherited neuromuscular disorders using the reverse transcription-polymerase chain reaction (RT-PCR) approach . The Human BLAT Search tool was adopted to search genomic sequences (GRCh37/hg19) that were homologous to the aberrant 111-bp sequence insertion. The homology search confirmed that the aberrant insertion consisted of a 15-bp sequence originating from DMD intron 1 (NM_004006.2:c.31+1941_31+1955), a single guanine nucleotide, and a 95-bp sequence homologous to a deep intronic region in KCBN2 intron 2 . The 95-bp sequence was part of an L1HS/LINE-1 element annotated by the RepeatMasker Web Server with default conditions (http://www.repeatmasker.org/cgi-bin/WEBRepeatMasker), that is, the partial exonization of LINE-1. According to the Human Genome Variation Society nomenclature flanked by a non-tandem duplication of 17 bp sequence originating from DMD intron 1 (chrX:33227444-33227460), which was finally described as follows causing the aberrant splicing event. As no potential splice-altering variants were detected through short-read whole DMD gene sequencing, genomic long-read whole DMD gene sequencing was performed in the patient, which detected a novel pathogenic SV in the deep intronic region of DMD intron 1. The novel deep intronic SV is a \u223c6-kb LINE-1 insertion/non-tandem duplication rearrangement identified through long-read sequencing, which further indicates that short reads are not enough to cover variants involving repetitive elements or large-scale SVs (DMD variants involving repetitive elements, such as the novel genomic SV causing partial exonization of the LINE-1 identified in our patient, might be a significant cause of genetically unsolved dystrophinopathies. This study highlights the importance of long-read sequencing in the detection and construction of pathogenic DMD variants involving repetitive elements. Precise detection and construction of deep intronic splice-altering variants in DMD have potential implications for the genetic therapy targeting normal dystrophin expression.In addition to exonic opathies . The precale SVs . The patDMD SV identified in our patient, can cause inherited diseases through various pathogenic mechanisms, including de novo LINE-1 insertions, LINE-1 insertion-mediated deletions, and genomic rearrangements (DMD variants involving LINE-1s. One of the eight patients is caused by a LINE-1 insertion in the 5\u2032 untranslated region of DMD, causing instability of the mature DMD mRNA (DMD causing multiple exons-skipping (DMD variants and highlighting the significant role of disease-causing LINE-1 insertions in monogenic diseases.Long interspersed nuclear elements, typically spanning about 6\u00a0kb in length, belong to the retrotransposons, which cover about 17% of the human genome . LINE-1sngements . To our DMD mRNA , one is skipping , two areskipping , and fouskipping . Our stu"} +{"text": "The mucus network provides innate immune defense to protect our gastrointestinal tract from pathogens, and promote homeostasis with our resident microbiota. This network is constituted by the mucin MUC2 (Muc2 in mouse), which is ~80% complex O-linked glycans by weight. Sialic acid (Sia) is a key capping monosaccharide on complex O-glycans which has recently been linked to preserving mucus integrity. Sia can undergo enzymatic modifications including the addition of O-acetyl groups. The 9-O-acetyltransferase CasD1 is responsible for the 9-OAc Sia variants. Functionally, the OAc-modification is known to inhibit microbial sialidase activities which may preserve Sia\u2019s protective roles on mucins. However, the extent of these OAc modifications in human and murine Mucin-2, and how they influence mucus function is unclear.Purpose: To determine whether and how Sia O-acetylation on colonic mucus regulates mucus integrity, host-microbe interactions, and colitis susceptibility.Casd1flox/flox;VillinCre or IEC Casd1-/- mice) and analyzed their mucins. Sialidase activities were quantified in the supernatants of colon fecal materials from WT and IEC Casd1-/- mutants mice using a fluorogenic substrate 4-MU-NeuNAc. Colitis susceptibility was monitored using 1.5% w/v Dextran Sodium Sulfate (DSS).We used viral-derived probes that target specific OAc-Sia analogues on mucus on sections from human feces and mouse feces and colon tissues to visualize their spatial arrangement and microbial interaction in situ. For glycomics, OAc-Sia analogues were quantitated on purified human MUC2 and mouse Muc2 by HPLC-MS after derivatization with 4,5-dimethyl-1,2-diaminobenzamine (DMBA). O-glycans were released via non-reductive ammonia-catalyzed \u03b2-elimination and analyzed by mass spectrometry. For in vivo work, we generated intestinal epithelial cell-specific Casd1 KO mice (Casd1-/- mice were viable and healthy with knockdown confirmed by 9-OAc staining, western blot of protein lysates and mucins, and sialylomics. The mucus encapsulation appeared overall intact regardless of OAc status. However, IEC Casd1-/- mice showed heightened susceptibility to 1.5% DSS colitis, linked to thinning of the mucus in IEC Casd1-/- vs WT littermates after challenge. Consistent with the role of OAc Sia in sialidase inhibition, loss of OAc Sia was associated with increased sialidase activities as assessed by heightened 4 MU signal in fecal supernatants in WT vs littermate IEC Casd1-/- mice. O-glycomics also showed reduction in the number of sialylated O-glycan structures upon loss of 9-OAc Sia.We found Sias on both human MUC2 and murine Muc2 were heavily O-acetylated, with ~75% and ~45% of Sias having 9-OAc-based modification in humans and mice respectively, and were distributed throughout the niche and barrier layers of mucus in situ. IEC Sia O-acetylation appears important in maintaining key aspects of Sia-dependent mucus function and protecting from inflammatory insult.Please acknowledge all funding agencies by checking the applicable boxes below: CCCNone Declared"} +{"text": "Teaching Point: In patients with familial multiple cavernous malformation syndrome with acute focal neurological deficit, a symptomatic spinal cavernous malformation must be included in the differential diagnosis. A 29-year-old male patient with a known history of familial multiple cavernous malformation syndrome (FCMS) presented with new-onset pyramidal signs during routing follow-up. Initial brain computed tomography (CT) showed multiple hyperdense cerebral lesions corresponding to known cerebral cavernous malformations (CCM), without apparent acute hemorrhage. Because of persisting symptoms, magnetic resonance imaging (MRI) of the thoracic spine was performed 10 days later. T2-weighted images (T2-WI) showed a intramedullary round hyperintense focus with a hypo-intense rim at the level of the 4th thoracic vertebral body (Th4) . Typical (type 2) lesions have a mixed T2-signal centrally with a hypo-intense hemosiderin rim. Bi-directional T2- and T1-hypo-intense extension in the spinal cord representing intramedullary hemorrhage is often seen in SCM. Unlike in CCM, popcorn-like appearance is uncommon. Appearance on T1-WI is variable [Most patients are treated conservatively but surgical resection may be necessary in cases with severe or recurrent symptoms. Patients with FCMS may benefit from follow-up using brain and spinal MRI although no widely used guidelines exist."} +{"text": "Nausea is a discomforting sensation of gut malaise that remains a major clinical challenge. Several visceral poisons induce nausea through the area postrema, a sensory circumventricular organ that detects blood-borne factors. Here, we use genetic approaches based on an area postrema cell atlas to reveal inhibitory neurons that counteract nausea-associated poison responses. The gut hormone glucose insulinotropic peptide (GIP) activates area postrema inhibitory neurons that project locally and elicit inhibitory currents in nausea-promoting excitatory neurons through \u03b3-aminobutyric acid (GABA) receptors. Moreover, GIP blocks behavioral responses to poisons in wild-type mice, with protection eliminated by targeted area postrema neuron ablation. These findings provide insights into the basic organization of nausea-associated brainstem circuits and reveal that area postrema inhibitory neurons are an effective pharmacological target for nausea intervention. Nausea is an unpleasant sensation of visceral malaise that remains poorly understood at a molecular and cellular level. Zhang et al. uncover brainstem inhibitory neurons that suppress nausea-related behaviors through selective blockade of nausea-promoting, GDF15 responsive-excitatory neurons. Nausea-suppressing neurons can be pharmacologically engaged by the gut hormone glucose insulinotropic peptide (GIP). Nausea is one of the most encountered symptoms in healthcare, with current anti-nausea medications displaying variable clinical success . New strSingle-cell cDNA sequencing approaches recently provided a cell atlas of the area postrema, revealing four excitatory and three inhibitory neuron types . One excGad2-ires-Cre mice, and genetic tracing previously revealed their projections to be local and largely confined to the area postrema, with minor projections observed in the adjacent nucleus of the solitary tract (NTS) and not in other brain regions that receive excitatory area postrema inputs (The functions of area postrema inhibitory neurons are unclear. All three area postrema inhibitory neuron types (and none of the excitatory neurons) are marked in a inputs . Based oGad2-ires-Cre, Rosa26-lsl-L10GFP mice were injected in the area postrema with an AAV containing a Cre-dependent ChR2-mCherry allele (AAV-Flex-ChR2-mCherry); post hoc histological analysis confirmed that viral infection was largely restricted to the area postrema in area postrema excitatory neurons -assisted circuit mapping (CRACM) to investigate the connectivity patterns of area postrema inhibitory neurons. postrema and 1B. postrema . Post-syuculline . Light-euculline and S1B,uculline and S1D.Gad2-ires-Cre mice in the area postrema with or without adeno-associated viruses (AAVs) containing Cre-dependent genes encoding designer G\u03b1s-coupled receptors (AAV-Flex-GsDREADD-mCherry) activated by the synthetic agonist clozapine-N-oxide (CNO) (s-coupled receptors were used based on commonly expressed area postrema receptors (see below). Chemogenetic activation of area postrema inhibitory neurons was validated by measuring CNO-induced Fos expression in mCherry-labeled neurons (We activated area postrema inhibitory neurons using chemogenetic approaches and observed the consequences on mouse behavior. We injected de (CNO) ; G\u03b1s-cou neurons . Proper neurons , and aniWe used an established behavioral paradigm to measure conditioned flavor avoidance . BrieflyWe focused on one subpopulation of area postrema inhibitory neurons that express the receptor (GIPR) for glucose insulinotropic peptide (GIP). GIP is a gut-derived hormone and incretin released upon nutrient intake that rapidly promotes insulin release . Small mGipr is expressed in a subset of area postrema inhibitory neurons using the genetically encoded fluorescent cAMP sensor cADDis , Slc6a2-p2a-Cre; Rosa26-lsl-tdTomato (cluster 2), Agtr1a-t2a-Cre; Rosa26-lsl-tdTomato (cluster 3), Gfral-p2a-Cre; Rosa26-lsl-tdTomato (cluster 4), and Glp1r-ires-Cre; Rosa26-lsl-tdTomato (clusters 2\u20134) mice. We obtained area postrema tissue slices from mice of each genotype and examined GIP-evoked responses by whole-cell, patch-clamp electrophysiology in the presence of ionotropic glutamate receptor antagonists to block excitatory transmission .We asked whether area postrema cluster 6 neurons evoked inhibitory postsynaptic currents in particular classes of area postrema excitatory neurons. Various excitatory neuron types were marked in smission . GIP indGFRAL neurons mediate nausea-associated responses to several visceral threats , so we rGipr-Cre; Rosa26-lsl-DTR mice (Gipr-ABLATEAP mice) or of Cre-negative Rosa26-lsl-DTR mice (non-ABLATE mice) as a control. Gipr-ABLATEAP mice displayed near-complete loss of cluster 6 neurons, but not other area postrema neuron types, and some loss of GIPR neurons in the NTS but not loss of GIPR neurons in other brain regions such as the hypothalamus (Gipr-ABLATEAP mice and non-ABLATE mice were examined using the conditioned-flavor-avoidance paradigm. We observed that GIP protected against GDF15-conditioned flavor avoidance in non-ABLATE mice but no longer suppressed GDF15 responses in Gipr-ABLA-TEAP mice (We asked whether area postrema cluster 6 neurons were required for the suppression of nausea-related behavior by GIP. We used a genetic approach involving diphtheria toxin (DT) to ablate Cre-expressing GIPR neurons in the area postrema. Mouse cells are DT resistant but can be made susceptible by Cre-guided expression of the DT receptor (DTR) . DT was thalamus and 4C. EAP mice . Thus, GNausea evoked by visceral poisons can be counterproductive, causing patients to forego life-saving medications for cancer, diabetes, and other diseases. Studies here establish area postrema inhibitory neurons as a target for suppressing behavioral responses to at least some nausea-inducing toxins . GIP is GIP directly stimulates cluster 6 neurons in slice preparations , and perStephen_liberles@hms.harvard.edu).Further information and requests for resources and reagents should be directed and will be fulfilled by the Lead Contact, Stephen Liberles .All primary images and data have been deposited at Mendeley Data , and all protocols were approved by the institutional animal care and use committee (IACUC) at Harvard Medical School. Slc6a2-p2a-Cre , Rosa26-lsl-tdTomato (007908), Gad2-ires-Cre (010802), Agtr1a-t2a-Cre (031487), and Rosa26-lsl-DTR (007900) mice were purchased (Jackson Laboratory). Both male and female mice between 8\u201324 weeks old were used for all other studies, and no differences based on sex were observed.All animal husbandry and procedures were performed in compliance with institutional animal care and use committee guidelines. All animal husbandry and procedures followed the ethical guidelines outlined in the NIH Guide for the Care and Use of Laboratory Animals ]. Then, slices were maintained and recorded at room temperature in oxygenated artificial cerebral spinal fluid (ACSF) solution . Inhibitory synaptic currents were recorded using a cesium methanesulfonate-based intracellular solution with high chloride, which contained (in mM): 120 CsCl, 15 CeMeSO3, 8 NaCl, 0.5 EGTA, 10 HEPES, pH 7.3, 290 mOsm. Photostimulation-evoked IPSCs were recorded in the whole-cell, voltage-clamp mode, with membrane potential clamped at \u221260 mV. To photo-stimulate channelrhodopsin-positive cells, an LED light source was used and controlled by the pClamp 10.2 software (Axon Instruments), with a photostimulation involving five 50 ms blue light laser pulses administered 1 s apart. In some experiments, 10 \u03bcM bicuculline methiodide (Tocris 2503) and 100 nM tetrodotoxin (Tocris 1078) were applied during recordings. GIP responses were measured by whole-cell recordings in current-clamp mode and zero holding current. Intracellular solution contained (in mM) 130 K-Gluconate, 15 KCl, 4 NaCl, 0.5 CaCl2, 10 HEPES, 1 EGTA, pH 7.2, 290 mOsm. As indicated, some experiments involved administration of cyanquixaline , D-AP5 , and bicuculline methiodide (10 \u03bcM). Recordings were obtained using borosilicate glass microelectrodes (2\u20135 M\u03a9) and Molecular Device 700B amplifier with data filtered at 1 kHz.Brain tissue was dissected from mice (6\u201310 weeks of age) following anesthesia (isoflurane) and decapitation, and brain slices (200 \u03bcm) were cut with a Leica VT1000S vibratome in slicing solution (4\u00b0C) . The area postrema was visualized by microscopy and harvested based on anatomical landmarks. Harvested tissues were digested with a papain dissociation system , washed , and gently triturated with three pipettes of decreasing diameters. Isolated single cells were centrifuged and resuspended in culture medium . Cells were plated on laminin-coated coverslips , and transfected with a viral vector encoding Green Up cADDis cAMP sensor (Montana Molecular U0200G). Real-time cAMP transients were imaged using a Leica SP5 II confocal microscope with cells under continuous gravity-based perfusion of Ringer\u2019s solution alone or containing 100 nM [D-Ala2]-GIP or 25 \u03bcM forskolin (Tocris 1099). Responses were processed with Matlab (MathWorks), and for display, smoothened with the smoothdata function, as a moving average with an automated window length. The baseline activity for each neuron was defined as the average green fluorescence intensity over a five-minute period preceding stimulus delivery, and cells were excluded if they failed to display responses to positive controls.Neuronal responses were measured in acutely dissociated area postrema neurons. Brains were acutely harvested from 13 vg/mL, 35 nL, 2 nL/second) or DT . Animals recovered for 3\u20134 weeks for behavioral analysis. Injected viruses included AAV-Flex-ChR2-mCherry -mCherry, prep #20297-AAV9), AAV-Flex-G\u03b1s-DREADD -mCherry, plasmid 50458, custom virus (Mice were anaesthetized (avertin) and placed on a stereotaxic frame (David Kopf Instruments) with heads facing down ~45\u00b0. The fourth ventricle and area postrema were surgically exposed after removal of the meninges, and a Nanoject III Injector (Drummond) was positioned directly into the area postrema for injections of virus following the HCR-FISH 3.0 protocol . Gipr probe was designed to be associated with the B3 initiator sequence and detected by hairpins labeled with Alexa Fluor 647 (Lot# PRL060). Fluorescent images were analyzed with a Leica SP5 II confocal microscope.Hybridization chain reaction (HCR) RNA protocol , 2020. GImmunohistochemistry and reporter fluorescence analysis was performed on cryosections (40 \u03bcm) of tissues fixed by intracardial perfusion with cold fixative and cryopreserved . Slide-mounted sections were blocked , incubated with primary antibody , washed , incubated with fluorophore-conjugated secondary antibodies . Antibody solutions were Rabbit-anti-RFP , Chick-anti-GFP , Goat-anti-HB-EGF . Fluorescent images were analyzed with a Leica SP5 II confocal microscope and processed with FIJI .ad libitum water access in the test arena. In the first three days (habituation days), both water bottles contained unflavored water. On day four (conditioning day), both water bottles were filled with either grape-flavored or cherry-flavored water (grape or cherry Kool-Aid) sweetened with 0.2% saccharin, each on half of the trials. Immediately after test arena occupancy on the conditioning day, mice were injected with saline alone or containing either CNO , GDF15 , [D-Ala2]-GIP (374 \u03bcg/kg), or LiCl (168 mg/kg). On day five (recovery day), both water bottles contained unflavored water. On day six (testing day 1), one water bottle contained cherry-flavored water, while the other contained grape-flavored water on a randomized arena side, and consumption from each water bottle scored using an automated lickometer based on a published design (Conditioned flavor avoidance assays involved a seven-day protocol with daily 30-minute introductions to a test arena containing two water bottles soon after dark onset. Mice had no access to water in the home arena but given d design . On day 2]-GIP (374 \u03bcg/kg) 2.5 hours before tissue harvest. For Fos studies involved IP injection of CNO or [D-AlaAll data points are derived from different mice except for some data points related to expression quantification and S2B F/F exceeded three standard deviations above the baseline mean. Statistical significance was measured on Prism 9 software (Graphpad) using a Mann-Whitney test (In neuronal imaging experiments, cells were counted as responsive if stimulus-evoked \u0394ney test and 4D aney test .1"} +{"text": "The present report evaluated the Enterobacterales (493 from blood samples) non-duplicate clinically significant isolates, were collected from 2019-2020. In vitro activity of Ceftazidime-avibactam and its comparators were assessed against Enterobacterales blood isolates.A total of 2126 Enterobacterales. with highest susceptibility to Ceftazidime-avibactam. Overall highest susceptibility (97%) was noted for Tigecycline for Enterobacterales(n=476).The susceptibility to colistin was lower(83%) in Enterobacterales (n=409). Among 168 CR-Enterobacterales (CRE), Ceftazidime avibactam showed 29% susceptibility (n=49) while susceptibility to Tigecycline was 96%(n=161) followed by Colistin at 84%(n=141). Molecular analysis for CRE isolates showed 55 % (n=93) to be OXA-48 like producers (with or without NDM) ,68%(n=114)to be NDM producers (with or without OXA- 48) and 30%(n=51) to be co producers of OXA-48 like & NDM.Overall susceptibility to the \u03b2 -lactam/\u03b2 -lactamase inhibitor (BL-BLI) was 60 -76% for In vitro activity of commonly used antibiotics against Enterobacterales, blood isolates (2019-2020)Enterobacterales. Tigecycline followed by Colistin showed the highest susceptibilities. Tigecycline attains very low plasma concentrations and is not licensed for use in blood stream infections, while the use of colistin may be limited by the lack of CLSI susceptibility breakpoints and safety concerns.Caz-Avi showed overall good susceptibility against the blood isolates tested hence it may be a valuable therapeutic option for treating BSI caused by MBL-negative Abhisek Routray, PhD, Pfizer India Limited: Advisor/Consultant Akshata Mane, MD, Pfizer India Limited: Advisor/Consultant|Pfizer India Limited: Stocks/Bonds."} +{"text": "The endoplasmic reticulum (ER) fills the cell with a continuous network of sealed membrane tubules and sheets. The ER is subdivided into microdomains mediating one-third of total protein biosynthesis, oxidative protein folding, secretion, protein quality control, calcium signaling, marcoautophagy/autophagy, stress sensing, and apoptosis. Defects in ER-calcium homeostasis underlie several diseases. Damage to the ER by misfolded membrane proteins is suppressed by specific HSPA/Hsp70 and DNAJ/Hsp40 chaperone pairs that select intermediates for ubiquitination and ER-associated degradation (ERAD) via the proteasome. The ER-transmembrane Hsp40 chaperone DNAJB12 and HSPA/Hsp70 also target toxic intermediates of misfolded membrane proteins for ER-associated autophagy (ERAA). DNAJB12-HSPA/Hsp70 maintain membrane protein degradation intermediates in detergent-soluble and degradation-competent states. DNAJB12-HSPA/Hsp70 also interact with the autophagy initiation kinase ULK1 on ER tubules containing ERAD-resistant misfolded membrane proteins (ERAD-RMPs). Omegasomes are ER microdomains where the autophagosome precursor or phagophore (PG) forms. ER tubules loaded with ERAD-RMPs enter omegasomes where they are converted into ER-connected PG (ER-PG). The Atg8 (autophagy related 8)-family member GABARAP (GABA type A receptor-associated protein) facilitates transfer of ERAD-RMPs from ER-PGs to autolysosomes (AL) that dock transiently with omegasomes. This article describes a model for DNAJB12-HSPA/Hsp70 action during the conformation-dependent triage in the ER of misfolded membrane proteins for folding versus proteasomal or AL degradation. P23H, misfold with degradation intermediates stabilized via a disulfide. RHOP23H is therefore difficult to extract from the ER and is ERAD-resistant [P23H cause retinal degeneration and blindness, possibly through disruption of ER-membrane integrity. ERAA is proposed to suppress the accumulation of toxic and ERAD-resistant RHOP23H , ATP binding cassette transporters (ABC-transporters), and P-type ATPases occurs in the ER. Folding inefficiencies in wild-type and disease-related mutant membrane proteins challenge the ER with the accumulation of potentially toxic misfolded intermediates. Retinitis pigmentosa is largely due to missense mutations in RHO (rhodopsin), a GPCR, that cause its misfolding, retention in the ER, and premature degradation. RHO contains a conserved disulfide whose formation is required for binding of the light sensing cofactor 9-cis-Retinal. Misfolded RHO intermediates lacking a disulfide are clients of ERAD. RHO mutants that cause retinitis pigmentosa, such as RHOesistant . Dominan RHOP23H .P23H, are not cleared from the ER and remain associated with DNAJB12-HSPA/Hsp70. This leads to the association of DNAJB12-HSPA/Hsp70 with autophagy initiation machinery and entry of ERAD-RMPs into ERAA.ERAA is a pathway for the degradation of misfolded membrane protein intermediates in energized cells. ERAA occurs in parallel to ERAD, and the triage of misfolded membrane proteins between these pathways is mediated by hybrid protein quality control complexes containing DNAJB12-HSPA/Hsp70 and ERAD or autophagy initiation machinery. DNAJB12-HSPA/Hsp70 complexes help fold and assemble subunits of ion channels. DNAJB12-HSPA/Hsp70 also targets misfolded membrane proteins for ubiquitination through interaction with ER-associated ubiquitin ligases. Kinetically-trapped DNAJB12-HSPA/Hsp70 clients that contain tertiary structure, but fail to complete folding, such as RHOP23H intermediates into ERAA. The mechanism for DNAJB12 action in ERAA requires its interaction with HSPA/Hsp70 and involves at least two functions. First, DNAJB12-HSPA/Hsp70 maintain nascent membrane proteins in a detergent-soluble conformation. The downstream fate of individual DNAJB12-HSPA/Hsp70 clients being determined by their innate folding kinetics on a case-by-case basis. Second, RHOP23H stimulates the association of DNAJB12-HSPA/Hsp70 with the ULK1 complex. DNAJB12-HSPA/Hsp70 may target ULK1-RB1CC1/FIP200 (RB1 inducible coiled-coil 1) to ER tubules containing RHOP23H to facilitate their conversion into ER-PGs -decorated omegasomal rings. RHOP23H-containing ER tubules located within omegasomes are decorated with Atg8-family proteins, which is a feature of ER-PGs. RHOP23H is concentrated from the ER tubular network into ER-PGs in a selective process that excludes forms of RHOP23H that are mutated to block disulfide formation. The transmembrane ER-chaperone CANX as well as DNAJB12, which both bind RHOP23H in the ER, are excluded from ER-PGs. The exclusion of DNAJB12 and CANX from ER-PGs is consistent with ERAA being a selective process.In support of this model, spots of RB1CC1, the targeting subunit of the ULK1 complex, are painted on ER tubules containing RHOP23H reveals them to dynamically interact with WIPI-decorated omegasomal surfaces. Notably, ALs marked with LAMP1 appear to dynamically dock with omegasomes and could therefore contact ER-PGs containing RHOP23H. AL association with omegasomes lasts on average for 22s and varies from short (10 s) to longer (70 s) associations. During apparent docking it appears that RHOP23H is transferred from ER-PGs to ALs because after departure ALs contain new ~100-nm patches of RHOP23H. AL association with omegasomes is suggested to stimulate the autophagic transfer of RHOP23H from ER-PGs to ALs.Live-cell imaging of ER-PGs containing RHOP23H from ER-PGs to ALs. Knockdown of all three GABARAP subfamily members blocks the accumulation of RHOP23H in ALs. Yet, knockdown of all three LC3 subfamily members does not significantly hinder autophagic degradation of RHOP23H. Interestingly, GABARAP knockdown does not hinder the entry of RHOP23H into ER-PGs, nor the association of ALs with omegasomes. Instead, loss of GABARAP hinders the transfer of RHOP23H from ER-PGs to ALs.A knockdown screen for Atg8-family protein requirements in ERAA revealed a role for GABARAP in the transfer of RHOP23H from the ER. The GABARAPs interact with ULK1 with higher affinity than the LC3s, are implicated in focal activation of ULK1, and facilitate autophagosome fusion with ALs. These data provide a road map to explore the unknown mechanism for GABARAP action in RHOP23H transfer from ER-PGs to omegasome-associated ALs.LC3 family members appear sufficient to support early steps in ERAA, and GABARAPs play a specific role in autophagic exit of RHOP23H does not aggregate in the ER-membrane due to the action of DNAJB12-HSPA/Hsp70. The individual knockdown of six different reticulophagy receptors and the simultaneous knockdown of all six does not block autophagic degradation of RHOP23H. In ERAA, DNAJB12-HSPA/Hsp70 can play a role analogous to reticulophagy receptors. DNAJB12-HSPA/Hsp70 can also function with a yet to be identified reticulophagy receptor that facilitates ERAA. Proteomic studies are being conducted to identify new components of the machinery that triages ERAD-RMPs for ERAA instead of ERAD.A family of reticulophagy receptors that mediate the selective lysosomal degradation of ER that is damaged by stress and protein aggregates help maintain ER-homeostasis. RHO"} +{"text": "NN)(BINAP)BF4 were synthesized bearing \u03c0-extended diimine ligands. Their behavior in several photocatalytic processes were evaluated and revealed acceptable levels of activity in an SET process, but negligible activity in PCET or ET processes. Suitable activity in ET processes could be restored through modification of the ligand. The BINAP-derived complexes were then evaluated for activity against triple-negative breast cancer cell lines. Controls indicated that copper complexes, and not their ligands, were responsible for activity. Encouraging activity was displayed by a homoleptic complex Cu(dppz)2BF4.A series of copper-based photocatalysts of the type Cu( Copper-based complexes have demonstrated their potential across photocatalysis . As an aNN)(PP)X) bearing the wide-bite-angle bisphosphine DPEPhos possessed unusually long excited-state lifetimes . Gi. GiBINAPifetimes . For exaBINAP-containing complexes against triple-negative breast cancer cell lines (MBA-MB-231) (dpq)(BINAP)BF4 and Cu(dppz)(BINAP)BF4 complexes and both displayed approximately 25\u201335% viability. Controls performed from the dpq, dppz, and BINAP ligands (entries 10\u201312) demonstrated that biological activity was originating from the metal complexes themselves. While a homoleptic complex Cu(BINAP)2BF4 was poorly active, the homoleptic complexes derived from the diimines showed low cell viabilities, with Cu(dppz)2BF4 being the most active of all complexes tested. Finally, given that the BINAP used in the above photocatalysis and biological evaluations was racemic, we prepared and evaluated the enantiomer variants of the dpq- and dppz-containing complexes. Interestingly, for the dpq complexes, the (S)-BINAP-containing complex Cu(dpq)((S)-BINAP)BF4 was approximately twice as active as the analogous (R)-BINAP complex. The copper complexes of dppz bearing either (S)- or (R)-BINAP did not show any difference in activity. The complex [Cu(MeCN)4]BF4 (10 \u00b5M) had negligible effects on cell viability (>75%), indicating that the complexes, rather than free copper, were responsible for biological activity.Given the recent interest in copper-containing complexes for medicinal chemistry ,35,36, i-MB-231) . The viaNN)(BINAP)BF4 were synthesized bearing \u03c0-extended diimine ligands. Their behavior in several photocatalytic processes was evaluated and revealed the following:BINAP with \u03c0-extended diimine ligands without ortho-substitution did not show significant different photophysical properties when compared to analogous complexes with the exception of the excited state lifetime, which decreased by approximately an order of magnitude.Copper-based complexes derived from BINAP-containing complexes were active in the visible-light Appel-type process, with the Cu(dpq)(BINAP)BF4 complex having slightly better activity than analogous complexes derived from phen of dmp ligands.The new BINAP-derived complexes did not afford complexes active for a PCET process.The new BINAP or the dpq, dppz, and ddppz diimines through judicious choice of the accompanying ligand. For example, Cu(dmp)(BINAP)BF4 and Cu(dppz)(XantPhos)BF4 afforded quantitative yields of the product.In an energy transfer process, high yields of the desired product could be obtained with either dppz)2BF4.In addition to the photocatalysis, the copper complexes were evaluated for the first time in a medicinal chemistry context against triple-negative breast cancer cell lines. Controls indicated that copper complexes, and not their ligands, were responsible for activity. Encouraging activity was displayed by a homoleptic complex Cu(In summary, a series of copper-based photocatalysts of the type Cu("} +{"text": "Efforts to protect assisted living residents from COVID-19 by limiting contact warrant attention. Assisted living was developed as a social model where care is provided in a home-like environment. Given the social dimensions of assisted living, we sought to better understand the effects of COVID-19-based restrictions in assisted living. We surveyed (online) assisted living community (ALC) administrators (N=130) between October 2020 and March 2021 as part of a larger project on COVID-19 in Florida. We then interviewed a subset of participants (N=26). Administrators of chain-affiliated ALCs (compared to non-chain) were 2.7 times more likely to report resident-contact limitations had disrupted care (p=0.02). Larger ALCs (25 or more beds) were marginally more likely to report care disruptions (p<0.10). Three main themes emerged from our qualitative interviews \u2013 varying interpretation of COVID-19 guidelines, effect of precautions on residents, assisted living as a home. Policy implications of these findings will be discussed."} +{"text": "Determining if a patient with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains infectious can be challenging in patients with severe disease or those profoundly immunosuppressed. We use an assay that detects minus-strand RNA as a surrogate for actively replicating SARS-CoV-2. Here, we describe a cohort of patients who had strand-assay reversion: a detectable minus strand-specific assay after having had an undetectable value which may signify relapse of infection or reinfection.We used a 2-step rRT-PCR specific to the minus strand of the SARS-CoV-2 envelope gene. The strand-specific assay is used to evaluate for infectivity in asymptomatic patients with a positive screening admission or pre-procedural test or in cases when ongoing infection was suspected . We collected minus strand-specific assays performed at Stanford Healthcare during August 2020\u2013April 2022. We describe basic demographics and clinical characteristics for patients who reverted from undetectable to detectable using the minus strand-specific assay.A total of 2,505 strand-specific tests were collected from August 2020\u2013April 2022 from 2,064 patients. A total of 292 (14%) had two or more strand-specific tests. Of them, 19 (7%) had an undetected minus strand-specific assay followed by a subsequent detectable value. Of them, seven (37%) had a minus strand-specific CT value of < 33. Most were male (n=4), median age was 54 (range: 8\u201362). All were profoundly immunosuppressed: Four had hematologic malignancies and three were post transplantation . Median time from onset of symptoms or first positive test to reversion was 41 days (range:27\u2013159). Median time from undetectable to detectable minus strand specific test was 26 days (range:4\u201334). All cases were considered relapses or ongoing infection rather than a new infection.Among patients with SARS-CoV-2 infection and consecutive strand-specific testing, a small proportion reverted from negative to positive. Most were profoundly immunosuppressed. The strand-specific assay can be an important tool in the evaluation of suspected cases of relapse or reinfection.Aruna Subramanian, MD, Gilead Sciences: Grant/Research Support|Regeneron, Inc: Grant/Research Support."} +{"text": "A general linear mixed model was used, and included fixed effects for cohort , time , and the cohort-time interaction term. P-values were adjusted for multiple hypothesis testing using Tukey\u2019s method. We observed that POIT responders had reduced TNFa producing myeloid dendritic cells (mDCs) compared to non-responders. Additionally, non-responders had increased OX40L expressing mDCs at 18-weeks compared to responders. In conclusion, our findings suggest that a reduced pro-inflammatory phenotype in DCs could potentially serve as a predictor of early outcome and success of POIT desensitization.Dendritic cells are important mediators in the early presentation of antigen and regulation of the differentiation of T cells. Peanut oral immunotherapy (POIT) results in desensitization in most peanut allergic individuals (responders), but not in others due to allergic reactions (non-responders). Delineation of early immunologic changes contributing to desensitization would help clarify the POIT mechanism of action. We analyzed dendritic cells in 15 pediatric subjects (5\u201312 years) undergoing a phase 1 single-center POIT study. We examined dendritic cells at baseline, 6-, 12-, 18- and 24-weeks after initiation of POIT and responders of therapy were compared to non-responders and healthy controls. The distribution frequency of myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) from peripheral blood samples were measured Peanut oral immunotherapy (POIT) has been shown to help circumvent the morbidity of accidental food exposures . The mecStudies suggest that immunotherapy induces antigen-specific T-regulatory cells (Tregs), which attenuate allergen-driven Th2 cytokine responses . AntigenThe open label study was approved by the Baylor College of Medicine Institutional Review Board (H-26819). All patients and parents provided written and informed assent/consent. This trial was registered under ClinicalTrials.org (NCT 02203799). Fifteen subjects between the ages of 5\u201312 years with IgE-mediated peanut allergy were recruited for a 3-year high-dose open label POIT pilot study. Patient baseline demographic and clinical characteristics are summarized in Patients were required to have a peanut specific IgE value >7kU/L and positive peanut specific skin prick test (SPT) . All participants underwent a double-blind placebo-controlled food challenge (DBPCFC) to peanut prior to enrollment. Once enrolled, each patient underwent peanut oral immunotherapy (POIT) build-up every 2 weeks, with a final goal maintenance dose of 3900mg of peanut protein (approx. 20 peanuts), which was achieved within 1 year of starting therapy. Patients underwent DBPCFC up to 6000mg peanut protein within 1 month of achieving the maintenance dose. \u201cResponders\u201d successfully achieved the maintenance dose and had no adverse events requiring epinephrine during POIT build-up. \u201cNon-responders\u201d had >/ = 1 adverse events requiring epinephrine, had more than 1 dose drop in the dose level during build-up, and were unable to achieve the maintenance dose. The maintenance dose for each non-responder was maintained below 3900mg and adjusted to prevent any allergic reactions. Blood samples were collected at baseline (prior to each DBPCFC) and then every 6 weeks thereafter, until the maintenance dose was achieved.Patients were excluded from study enrollment if they met any of the following exclusion criteria: History of severe anaphylaxis to peanut (Grade 3 or higher per WAO guidelines); participation in another study using an investigational new drug; participation in any interventional study for the treatment of food allergy in the past 12 months or while participating in this study; poor control of persistent atopic dermatitis; a diagnosis of asthma; inability to discontinue antihistamines for skin testing and oral food challenges; pregnant females; chronic medical conditions requiring frequent use of oral steroids, chronic psychiatric illness or history of substance abuse; active eosinophilic esophagitis requiring therapy 12 months prior to study enrollment; subjects with known wheat or oat allergy; subjects on the build-up phase of environmental allergen immunotherapy injection; and/or subject living more than 175 miles away from Texas Children\u2019s Hospital.Blood was collected from each subject at baseline and every 6-weeks during their POIT build-up phase. Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples by density gradient centrifugation over Ficoll-Paque, and cryopreserved in 10% dimethyl sulfoxide in fetal bovine serum and stored in liquid nitrogen.Escherichia coli 0111:B4 (Sigma) or crude peanut extract (CPE) (200ug/mL) for 24 hours at 37C\u00b0, 5% CO2. LPS is a negative stimulator of type 2 allergic inflammation, therefore we used LPS as a control to assess perturbations in the non-type 2 allergic response between peanut allergic and non-peanut allergic subjects. CPE is a stimulant promoting Type 2 allergic inflammation in peanut allergic subjects, thus it would not promote a type 2 response in non-peanut allergic subjects. CPE was used to assess the peanut antigen effect on DCs in the peanut allergic subjects undergoing POIT treatment, since CPE clinically promotes Type 2 inflammation in peanut allergic subjects. To capture intracellular cytokines, brefeldin A and monensin (BD Biosciences 0.7uL/mL) were added to each culture at the beginning of incubation.Cryopreserved PBMCs were thawed and then cultured in media and incubated in 96-well plates with media alone, lipopolysaccharide (LPS) (1ng/mL) from Cell cultures were collected after 24 hours of incubation. Cells were stained with a viability dye/Ghost V510 and incubated at room temperature for 15 minutes protected from light. Cell were stained with cell surface antibody for 30 minutes at room temperature and protected from light, followed by washing with wash buffer (PBS+0.25% BSA) twice. Surface marker antibody cocktail was made using 2.5uL of each antibody for every 1e6 cells: anti-CD123 , anti-HLA-DR , anti-CD11c , anti-CD14 , anti-CD252 (OX40L) , anti-CD19 , anti-CD56 and anti-CD3 . Cells were incubated with surface cocktail for 30 minutes protected from light at room temperature. For staining intracellular cytokines, cells were washed twice with fixation/permeabilization buffer (BD Biosciences). Permeabilized cells were stained with an intracellular marker antibody cocktail of the following antibodies (5uL of each antibody for every 1e6 cells): anti-IL4 , anti-IL6 , anti-IL-10 , and anti-TNFa . Cells were incubated with intracellular cocktail for 30 minutes protected from light at room temperature. Cells were washed once with permeabilization buffer and once with wash buffer. In order to standardize samples across time, all cryopreserved POIT samples were run in separate batches of 3 analyzing all timepoints using the same techniques.\u00ae, San Jose, CA), and analyses were completed using FlowJo Version 10.5.0 software (FlowJo\u00ae LLC). All samples were run on an 18-parameter flow cytometer , at the 1-year time point compared with baseline using a paired t-test. A difference in proportions of 0.60 units (.2 at baseline and .8 at 1 year) would be clinically important. Assuming a common SD = 0.6 at each time point, correlation between repeated measures is 0.50 and alpha = 0.05, a sample size of 10 subjects would be required to detect this difference with 80% power. Therefore, assuming 50% attrition, we enrolled a total of 15 subjects. A general linear mixed model was used, and included fixed effects for cohort , time , and the cohort-time interaction term. The matrix of correlated residuals assumed a first-order auto-regressive structure. The model was used to estimate mean frequency (95% CI) and compare all possible pairwise comparisons. P-values were adjusted for multiple hypothesis testing using Tukey\u2019s method. A separate model was fit for each cell type-stimulant combination. Statistical significance was assessed at the 0.05 level.Tumor necrosis factor alpha (TNFa), a pleotropic cytokine, has both pro-inflammatory and anti-inflammatory effects . The bioOX40L (CD252) is a surface glycoprotein found on professional antigen-presenting cells (APCs), such as mDCs. It is a member of the TNFR/TNF superfamily that is important in directing T-helper type 2 (Th2) cell polarization . To inveNo significant differences were observed between baseline healthy controls compared to the responders and non-responders for TNFa and OX40L. Additionally, no significant differences were seen in the production of IL-4, IL-6 or IL-10 from mDCs \u2013S6 Figs,Suppression of Th2-driven allergic inflammation and type-I hypersensitivity has been associated with successful desensitization to allergens. Specifically this has been observed by an increase in IL-10 producing CD4+ T-cells and reduced Th2 cytokines during POIT . LimitedThe overall increased frequency of TNFa-producing mDCs in non-responders may contribute to a pro-inflammatory state that allows for persistent sensitization and allergy. Increased TNFa production has been associated with either promotion of inflammation through the activation of TNFR1 or maintenance of immune homeostasis and tolerance through activation of TNFR2 , 6. In oSignaling through OX40L can mediate Th2 differentiation and break T-cell tolerance by binding to OX40 on the surface of T-cells . In thisPOIT responders and non-responders were expected to differ in their DC response to CPE, and that DC responses to LPS would be similar in these two cohorts. However, our findings demonstrated DC differences occurring in certain DC populations following LPS stimulation as well as some populations following CPE stimulation between POIT responders and non-responders. Dendritic cells from POIT non-responders demonstrated that their universal ability to respond to different stimuli was hindered broadly and this was not limited to just peanut antigen stimulation, which was the driver of their peanut allergy. Using LPS as a control allowed us to assess perturbations in the non-type 2 allergic response between peanut allergic and non-peanut allergic subjects. If CPE was the only stimulant assessed, then it still would not be understood if the DC effects seen were just peanut specific or due to a broader context. By testing LPS and CPE, we are able to determine a broader inflammatory signature of dendritic cells in the context of non-specific antigens and assess the susceptibility and persistence for Type 2 inflammation.POIT may promote a shift away from Th2 or Type 2 inflammation and allow for development of adaptive Tregs thus inducing desensitization . Our finS1 FigChanges in frequency of IL-4 expressing mDCs following LPS stimulation between responders (diamond) and non-responders (square) of peanut oral immunotherapy during the first 24-weeks of therapy. Healthy controls (circle) were not treated and only assessed at baseline.(TIF)Click here for additional data file.S2 FigChanges in frequency of IL-4 expressing mDCs following CPE stimulation between responders (diamond) and non-responders (square) of peanut oral immunotherapy during the first 24-weeks of therapy. Healthy controls (circle) were not treated and only assessed at baseline.(TIF)Click here for additional data file.S3 FigChanges in frequency of IL-6 expressing mDCs following LPS stimulation between responders (diamond) and non-responders (square) of peanut oral immunotherapy during the first 24-weeks of therapy. Healthy controls (circle) were not treated and only assessed at baseline.(TIF)Click here for additional data file.S4 FigChanges in frequency of IL-6 expressing mDCs following CPE stimulation between responders (diamond) and non-responders (square) of peanut oral immunotherapy during the first 24-weeks of therapy. Healthy controls (circle) were not treated and only assessed at baseline.(TIF)Click here for additional data file.S5 FigChanges in frequency of IL-10 expressing mDCs following LPS stimulation between responders (diamond) and non-responders (square) of peanut oral immunotherapy during the first 24-weeks of therapy. Healthy controls (circle) were not treated and only assessed at baseline.(TIF)Click here for additional data file.S6 FigChanges in frequency of IL-10 expressing mDCs following CPE stimulation between responders (diamond) and non-responders (square) of peanut oral immunotherapy during the first 24-weeks of therapy. Healthy controls (circle) were not treated and only assessed at baseline.(TIF)Click here for additional data file.S7 FigChanges in frequency of IL-4 expressing pDCs following LPS stimulation between responders (diamond) and non-responders (square) of peanut oral immunotherapy during the first 24-weeks of therapy. Healthy controls (circle) were not treated and only assessed at baseline.(TIF)Click here for additional data file.S8 FigChanges in frequency of IL-4 expressing pDCs following CPE stimulation between responders (diamond) and non-responders (square) of peanut oral immunotherapy during the first 24-weeks of therapy. Healthy controls (circle) were not treated and only assessed at baseline.(TIF)Click here for additional data file.S9 FigChanges in frequency of I6-4 expressing pDCs following LPS stimulation between responders (diamond) and non-responders (square) of peanut oral immunotherapy during the first 24-weeks of therapy. Healthy controls (circle) were not treated and only assessed at baseline.(TIF)Click here for additional data file.S10 FigChanges in frequency of IL-6 expressing pDCs following CPE stimulation between responders (diamond) and non-responders (square) of peanut oral immunotherapy during the first 24-weeks of therapy. Healthy controls (circle) were not treated and only assessed at baseline.(TIF)Click here for additional data file.S11 FigChanges in frequency of IL-10 expressing pDCs following LPS stimulation between responders (diamond) and non-responders (square) of peanut oral immunotherapy during the first 24-weeks of therapy. Healthy controls (circle) were not treated and only assessed at baseline.(TIF)Click here for additional data file.S12 FigChanges in frequency of IL-10 expressing pDCs following CPE stimulation between responders (diamond) and non-responders (square) of peanut oral immunotherapy during the first 24-weeks of therapy. Healthy controls (circle) were not treated and only assessed at baseline.(TIF)Click here for additional data file.S13 FigChanges in frequency of TNFa expressing pDCs following LPS stimulation between responders (diamond) and non-responders (square) of peanut oral immunotherapy during the first 24-weeks of therapy. Healthy controls (circle) were not treated and only assessed at baseline.(TIF)Click here for additional data file.S14 FigChanges in frequency of TNFa expressing pDCs following CPE stimulation between responders (diamond) and non-responders (square) of peanut oral immunotherapy during the first 24-weeks of therapy. Healthy controls (circle) were not treated and only assessed at baseline.(TIF)Click here for additional data file.S15 FigChanges in frequency of total mDCs following LPS stimulation between responders (diamond) and non-responders (square) of peanut oral immunotherapy during the first 24-weeks of therapy. Healthy controls (circle) were not treated and only assessed at baseline.(TIF)Click here for additional data file.S16 FigChanges in frequency of total mDCs following CPE stimulation between responders (diamond) and non-responders (square) of peanut oral immunotherapy during the first 24-weeks of therapy. Healthy controls (circle) were not treated and only assessed at baseline.(TIF)Click here for additional data file.S17 FigChanges in frequency of total pDCs following LPS stimulation between responders (diamond) and non-responders (square) of peanut oral immunotherapy during the first 24-weeks of therapy. Healthy controls (circle) were not treated and only assessed at baseline.(TIF)Click here for additional data file.S18 FigChanges in frequency of total pDCs following CPE stimulation between responders (diamond) and non-responders (square) of peanut oral immunotherapy during the first 24-weeks of therapy. Healthy controls (circle) were not treated and only assessed at baseline.(TIF)Click here for additional data file.S19 Fig(A) Mean percent frequency of TNFa-production mDCs following LPS stimulation; (B) Mean percent frequency of TNFa-production mDCs following CPE stimulation.(PNG)Click here for additional data file.S20 Fig(A) Mean percent frequency (95% CI) of OX40L mDCs following LPS stimulation, (B) Mean percent frequency (95% CI) of OX40L mDCs following CPE stimulation.(PNG)Click here for additional data file.S21 FigChanges in frequency of IL-4 expressing mDCs following LPS stimulation between responders (diamond) and non-responders (square) of peanut oral immunotherapy during the first 24-weeks of therapy. Healthy controls (circle) were not treated and only assessed at baseline.(PNG)Click here for additional data file.S22 FigChanges in frequency of IL-4 expressing mDCs following CPE stimulation between responders (diamond) and non-responders (square) of peanut oral immunotherapy during the first 24-weeks of therapy. Healthy controls (circle) were not treated and only assessed at baseline.(PNG)Click here for additional data file.S23 FigChanges in frequency of IL-6 expressing mDCs following LPS stimulation between responders (diamond) and non-responders (square) of peanut oral immunotherapy during the first 24-weeks of therapy. Healthy controls (circle) were not treated and only assessed at baseline.(PNG)Click here for additional data file.S24 FigChanges in frequency of IL-6 expressing mDCs following CPE stimulation between responders (diamond) and non-responders (square) of peanut oral immunotherapy during the first 24-weeks of therapy. Healthy controls (circle) were not treated and only assessed at baseline.(PNG)Click here for additional data file.S25 FigChanges in frequency of IL-10 expressing mDCs following LPS stimulation between responders (diamond) and non-responders (square) of peanut oral immunotherapy during the first 24-weeks of therapy. Healthy controls (circle) were not treated and only assessed at baseline.(PNG)Click here for additional data file.S26 FigChanges in frequency of IL-10 expressing mDCs following CPE stimulation between responders (diamond) and non-responders (square) of peanut oral immunotherapy during the first 24-weeks of therapy. Healthy controls (circle) were not treated and only assessed at baseline.(PNG)Click here for additional data file.S27 FigChanges in frequency of IL-4 expressing pDCs following LPS stimulation between responders (diamond) and non-responders (square) of peanut oral immunotherapy during the first 24-weeks of therapy. Healthy controls (circle) were not treated and only assessed at baseline.(PNG)Click here for additional data file.S28 FigChanges in frequency of IL-4 expressing pDCs following CPE stimulation between responders (diamond) and non-responders (square) of peanut oral immunotherapy during the first 24-weeks of therapy. Healthy controls (circle) were not treated and only assessed at baseline.(PNG)Click here for additional data file.S29 FigChanges in frequency of I6-4 expressing pDCs following LPS stimulation between responders (diamond) and non-responders (square) of peanut oral immunotherapy during the first 24-weeks of therapy. Healthy controls (circle) were not treated and only assessed at baseline.(PNG)Click here for additional data file.S30 FigChanges in frequency of IL-6 expressing pDCs following CPE stimulation between responders (diamond) and non-responders (square) of peanut oral immunotherapy during the first 24-weeks of therapy. Healthy controls (circle) were not treated and only assessed at baseline.(PNG)Click here for additional data file.S31 FigChanges in frequency of IL-10 expressing pDCs following LPS stimulation between responders (diamond) and non-responders (square) of peanut oral immunotherapy during the first 24-weeks of therapy. Healthy controls (circle) were not treated and only assessed at baseline.(PNG)Click here for additional data file.S32 FigChanges in frequency of IL-10 expressing pDCs following CPE stimulation between responders (diamond) and non-responders (square) of peanut oral immunotherapy during the first 24-weeks of therapy. Healthy controls (circle) were not treated and only assessed at baseline.(PNG)Click here for additional data file.S33 FigChanges in frequency of TNFa expressing pDCs following LPS stimulation between responders (diamond) and non-responders (square) of peanut oral immunotherapy during the first 24-weeks of therapy. Healthy controls (circle) were not treated and only assessed at baseline.(PNG)Click here for additional data file.S34 FigChanges in frequency of TNFa expressing pDCs following CPE stimulation between responders (diamond) and non-responders (square) of peanut oral immunotherapy during the first 24-weeks of therapy. Healthy controls (circle) were not treated and only assessed at baseline.(PNG)Click here for additional data file.S35 FigChanges in frequency of total mDCs following LPS stimulation between responders (diamond) and non-responders (square) of peanut oral immunotherapy during the first 24-weeks of therapy. Healthy controls (circle) were not treated and only assessed at baseline.(PNG)Click here for additional data file.S36 FigChanges in frequency of total mDCs following CPE stimulation between responders (diamond) and non-responders (square) of peanut oral immunotherapy during the first 24-weeks of therapy. Healthy controls (circle) were not treated and only assessed at baseline.(PNG)Click here for additional data file.S37 FigChanges in frequency of total pDCs following LPS stimulation between responders (diamond) and non-responders (square) of peanut oral immunotherapy during the first 24-weeks of therapy. Healthy controls (circle) were not treated and only assessed at baseline.(PNG)Click here for additional data file.S38 FigChanges in frequency of total pDCs following CPE stimulation between responders (diamond) and non-responders (square) of peanut oral immunotherapy during the first 24-weeks of therapy. Healthy controls (circle) were not treated and only assessed at baseline.(PNG)Click here for additional data file.S1 File(CSV)Click here for additional data file.S2 File(RTF)Click here for additional data file."} +{"text": "The second best conduit for coronary artery bypass grafting is uncertain. Theobjective of this study is to determine the second best conduit according tograft patency results from randomized controlled trials using a networkmeta-analysis.A systematic literature search was conducted for randomized controlled trialscomparing the angiographic patency rate of the no-touch saphenous vein(NT-SV), the radial artery (RA), the right internal thoracic artery (RITA),and the gastroepiploic artery (GEA) in reference to the conventionallyharvested saphenous vein (CON-SV). The primary outcome was graft occlusion,and the secondary outcome was all-cause mortality.A total of 859 studies were retrieved, of which 18 were included. A total of6,543 patients and 8,272 grafts were analyzed. The weighted meanangiographic follow-up time was 3.5 years. Compared with CON-SV, RAand NT-SV demonstrated lower graft occlusion.NT-SV and RA were ranked as the best conduits . There was nosignificant difference in late mortality between different conduittypes.RA and NT-SV are associated with significantly lower graft occlusion ratesand are comparably ranked as the best conduit for patency. A comprehensive networkmeta-analysis (NMA) of graft patency in randomized controlled trials (RCTs) waspreviously completed by our group4][. In the 2005 trial by Gaudino etal.15][. Tian et al.3][. Deb et al.13][. The NT-SV received aClass IIa recommendation in the 2018 European Revascularizationguidelines23][ and was a Best Practice in the 2021 American College ofCardiology/American Heart Association revascularization guidelines22][.Several large RCTs support the long-term patency of RA over CON-SV22][. These results are further substantiated in theRADIAL 10-year extension study\u2019s post-hoc analysis for survival24][. The factorial trial by Angelini et al.involving CON-SV vs. NT-SV and low- vs.high-pressure graft dilation reported that low-pressure distention of CON-SV canachieve wall thickening comparable to NT-SV8][.Limitations of this meta-analysis included a small sample size causing certainpairwise analyses to be underpowered, varying quality of the RCTs included, andno data collected on renal disease, secondary prevention, and antispasmodictherapy, which are additional factors that influence graft patency. It isworthwhile to note that the included studies involving NT-SV grafts used pedicleharvest technique withIn this NMA of 18 angiographic RCTs, the current randomized evidence showssignificantly better patency rates for RA and NT-SV compared with CON-SV, while allconduits were associated with similar rates of late mortality compared with CON-SV.NT-SV and RA were identified as the second best conduits using data from this NMA ofangiographic trials."} +{"text": "A heavy smoker 49-year-old man, American Society of Anestesiologists (ASA) physical status III, with positive history of gradually worsening dyspnea was proposed for Suspension Microlaryngoscopy surgery. Airway evaluation showed a grade-III Mallampati score and no apparent or palpable cervical mass. After preoxygenation and induction, orotracheal intubation was performed with C-MAC D blade Videolaryngoscope\u00ae, and founded an omega-shaped epiglottis (OSE) , with voOSE is a variant configuration of epiglottis in which the lateral folds are curled inwards."} +{"text": "Residential care communities (RCC) and adult day services centers (ADSC) were greatly impacted by COVID-19. As state-regulated home- and community-based care settings (HCBSs), they experienced changing regulations, outbreaks among care recipients and staff, and new challenges to safely provide services. Using a nationally representative sample of over 11,600 RCCs and census of nearly 5,500 ADSCs from the 2020 National Post-acute and Long-term Care Study, this study examined the prevalence and regional variation (p<.05) of practices to mitigate COVID-19. Preliminary data show that most RCCs (87%) and ADSCs (72%) screened for symptoms. Most RCCs limited communal activities (83%) and ADSCs reduced hours or temporarily closed (73%). More RCCs used video telemedicine than audio-only (40% vs 34%) to assess, diagnose, or treat users. However, more ADSCs used audio-only than video telemedicine (25% vs 17%). Over 80% of all providers always or sometimes imposed in-person restrictions on family, visitors, volunteers, and non-essential services providers. More ADSCs in the Midwest (82%) and South (80%) screened symptoms than Northeast (68%) and more ADSCs in the Midwest (85%) limited hours/temporarily closed than Northeast (72%). More RCCs in the Midwest (43%) and South (41%) used video telemedicine than Northeast (36%); however, a lower percentage of ADSCs in the Midwest (9%) and South (12%) used video telemedicine than Northeast (25%). Results show a variety of experiences in the first year of the COVID-19 pandemic among these two HCBSs. Practices to mitigate COVID-19 while continuing to provide needed services were common, with some differences across settings and US regions."} +{"text": "Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induces immune-mediated type 1 interferon (IFN-1) production, the pathophysiology of which involves sterile alpha motif and histidine-aspartate domain-containing protein 1 (SAMHD1) tetramerization and the cytosolic DNA sensor cyclic-GMP-AMP synthase (cGAS)\u2013stimulator of interferon genes (STING) signaling pathway. As a result, type I interferonopathies are exacerbated. Aspirin inhibits cGAS-mediated signaling through cGAS acetylation. Acetylation contributes to cGAS activity control and activates IFN-1 production and nuclear factor-\u03baB (NF-\u03baB) signaling via STING. Aspirin and dapsone inhibit the activation of both IFN-1 and NF-\u03baB by targeting cGAS. We define these as anticatalytic mechanisms. It is necessary to alleviate the pathologic course and take the lag time of the odds of achieving viral clearance by day 7 to coordinate innate or adaptive immune cell reactions. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induces immune-mediated inflammasome diseases . The innOur current perception of coronavirus disease-2019 (COVID-19) immunopathology does not fully elucidate how the innate and adaptive host immune systems appear to miscommunicate to worsen the viral impact and patient prognosis. The bronchoalveolar immune response has a unique local profile in COVID-19 that strongly differs from the immune profile in peripheral blood: T cell activation in blood, but not in bronchoalveolar lavage fluid (BALF), was found to be higher in fatal COVID-19 cases, and increased levels of inflammatory mediators were more pronounced in BALF than in plasma . ChronicThe pathophysiologic mechanisms driving multiorgan damage include direct viral infection, maladaptive functions of the renin\u2013angiotensin\u2013aldosterone system (RAAS), impacts on endothelial cell damage and the generation of a thrombotic milieu, dysregulation of the immune response, and cytokine release syndrome. Nonetheless, many of the morphological data collected are nonspecific, variable, and possibly associated with other coexisting factors, such as the mechanisms contributing to endothelial\u2013epithelial barrier breakdown during the monocyte and neutrophil invasion process . The extInnate immune cells, including macrophages, monocytes, dendritic cells, neutrophils, and innate lymphoid cells (ILCs) such as natural killer (NK) cells, recognize pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) to induce inflammatory signaling pathways and immune responses. They are armed with an arsenal of five pattern recognition receptors (PRRs) that include Toll-like receptors (TLRs), retinoic-acid-inducible gene I (RIG-I)-like receptors (RLRs), nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs), C-type lectin receptors, and absent in melanoma 2 (AIM2)-like receptors . SeveralThe sterile alpha motif (SAM) is a protein interaction domain involved in developmental regulation. SAM is an evolutionarily conserved protein-binding domain that regulates numerous developmental processes among diverse eukaryotes . SAM andSAMHD1 can restrict retroviruses . Mutations in SAMHD1 are linked to the pathogenesis of chronic lymphocytic leukemia and Aicardi\u2013Gouti\u00e8res syndrome (AGS). SAMHD1 holds a target motif for cyclin-dependent kinase 1 (CDK1), whose activity is needed for SAMHD1 phosphorylation. SAMHD1 is phosphorylated at residue threonine 592 (T592) in cycling cells, and phosphorylated SAMHD1 on T592 cannot block retroviral infection. Phosphorylation modulates SAMHD1 to block retroviral infection without affecting its ability to decrease cellular dNTP levels . A phospApoenzymes are catalytically inactive, whereas holoenzymes are catalytically active. Inactive apo-SAMHD1 interconverts between monomers and dimers. The binding of deoxyguanosine triphosphates (dGTP) to four allosteric sites promotes tetramerization. It induces a conformational change in the substrate-binding pocket to yield the catalytically active tetramer . The binThe SAMHD1 tetramer structure could provide a mechanistic understanding of its rapid function in SARS-CoV-2. SAMHD1 negatively regulates the interferon (IFN)-1 signaling pathway: the elevated innate immune response and IFN activation upon genetic loss of SAMHD1 effectively suppress SARS-CoV-2 replication . The spoIfnb and Ifna4) [cGAS catalyzes the production of cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) upon sensing cytosolic DNA, which activates STING\u2013tank-binding kinase 1 (TBK1)\u2013interferon regulatory factor 3 (IRF3) signaling. cGAS is present in the nucleus, and nuclear cGAS is sequestered at chromatin in an inactive state. It recruits protein arginine methyltransferase 5 (Prmt5), facilitating IRF3 access upon viral infection. Nuclear-localized cGAS, in innate immunity, interacts with Prmt5 to catalyze the symmetric dimethylation of histone H3 arginine 2 at IRF3-responsive genes (d Ifna4) . Active d Ifna4) . cGAS\u2013STd Ifna4) . A SARS-d Ifna4) interaction but not IRF3-type I IFN induction . SARS-CoProfiling skin manifestations, a STING-dependent type I IFN signature is mediated by macrophages adjacent to areas of endothelial cell damage, and cGAS\u2013STING activity was detected in lungs from COVID-19 patients with prominent tissue destruction, which was associated with IFN-1 responses. A lung-on-chip model with SARS-CoV-2 infection activates cGAS\u2013STING signaling in endothelial cells through mitochondrial DNA release. A STING inhibitor shows anti-inflammatory potential by alleviating detrimental immune responses . Among pSARS-CoV-2 may move from the periphery into the CNS through neurons or the vagus nerve from the lungs or gut ,34. In aDifferent cell types, including dopaminergic neurons, cortical neurons, brain microvascular endothelial cells, and choroidal epithelial cells, are susceptible to SARS-CoV-2, but there are differences in permissiveness. SARS-CoV-2 is neurovirulent, not restricted to patients with mild or severe diseases, and can cause the development of neurological complications. We found activated microglia in the olfactory bulb, midbrain , hindbrain, the dorsal motor nucleus of the vagus nerve, and pre-B\u00f6tzinger complex on the medulla . ClinicaIn many cases, there is no direct virus attack on vulnerable structures. Instead, the neurological disease manifestations may be due to an immune reaction against the virus, and pre-existing neurological disease may become clinically evident or worsen with COVID-19, which explains why various nervous system manifestations react favorably to immune suppression or immune modulation ,42. The Early in the COVID-19 pandemic, there was concern that non-steroidal anti-inflammatory drugs (NSAIDs) could be related to adverse cardiovascular and pulmonary outcomes. Some suggested potential harm with NSAID prescription for COVID-19 patients . HoweverAspirin inhibited cGAS-mediated interferon production and alleviated DNA-induced autoimmunity in AGS mouse models and patient cells. However, another specific COX inhibitor, diclofenac, did not affect cGAS activation . cGAS acAspirin strongly inhibited cGAS-mediated TBK1\u2013IRF3 signaling. However, aspirin does not affect STING activator cGAMP- or cyclic di-guanosine monophosphate (c-di-GMP)-induced downstream TBK1\u2013IRF3 signaling and does not inhibit STING-associated vasculopathy with onset in infancy (SAVI)-associated STING mutant-mediated IRF3 activation. Aspirin inhibits the DNA\u2013cGAS\u2013STING\u2013TBK1\u2013IRF3 axis by inhibiting cGAS . With an\u00ae, Tamiflu\u00ae), was shown to inhibit NF-\u03baB activation and alleviate influenza symptoms in hospitalized patients [Patients progress rapidly to acute respiratory distress syndrome (ARDS), and in the underlying form, septic shock, irreversible metabolic acidosis, blood coagulation dysfunction, and hemostatic and thrombotic anomalies are the leading causes of death due to COVID-19. Platelets play a crucial role in developing severe disease cases. They are the enucleated cells responsible for thrombus formation and the hyper-reactivity induced by proinflammatory microenvironments in the more aggressive course of COVID-19 . Dissemipatients .There are many aspirin contraindications to consider, such as in DIC and other bleeding disorder settings. Aspirin use can result in uncontrolled hemorrhage and poses a risk of Reye\u2019s syndrome in children. However, we found no evidence of a harmful effect of NSAIDs on COVID-19-related deaths. The risks of COVID-19 do not need to influence decisions about the therapeutic use of NSAIDs . In thisDapsone is a small molecule with anti-inflammatory, immunosuppressive, antibacterial, and antibiotic properties. Dapsone passes through the blood\u2013brain barrier (BBB) ,70. DDS 2 (fraction of inspired oxygen) and no further progression of hypoxia. The results were statistically significant according to the chi-square test and Fisher\u2019s exact test for the criteria for the effectiveness of dapsone [Kanwar et al. administered standard treatments plus dapsone for COVID-19 ARDS patients in the intensive care unit (ICU) from 21 December 2020 to 2021. The treatments were administered to patients with decreased FIO dapsone ,75.Statistics 1: Chi-square test for ARDS onset and mortalityp value was 0.016 (significant at p < 0.05). The group with ARDS onset treated with dapsone was more likely to survive than those with ARDS onset not treated with dapsone.A chi-square test was performed to analyze the mortality relationship of ARDS onset with dapsone and without dapsone. The chi-square statistic was 5.81, and the Statistics 2: Chi-square statistics and Fisher\u2019s exact test2 was significant in the entire dapsone-administered (+) and nonprescribed (\u2013) groups, which applied to only the ARDS-onset stage . This analysis showed a lower risk of death or discharge to long-term acute care hospitals and a higher likelihood of discharge home with dapsone compared with those receiving the usual standard of care without dapsone [In the complete overall analysis, the median age was 64 years, 58% were men, and 42% were women. The most common risk factors for deteriorating hypoxia and death were CRP data over 150 (in 45%) and oxygen requiring >10 L/min on admission (37%). By specific measures such as initial CRP and oxygen, the patients in the dapsone group were not less sick than those in the control group. Of 30 patients, 16 with dapsone were discharged home, requiring less than 8 L oxygen/min\u2014mostly not more than 5 L oxygen/min; only 6 of 30 patients without dapsone were discharged home fluvoxamine, could prevent clinical deterioration in early stage COVID-19 outpatients ,93. HoweThe genome-wide DNA methylation profiles of severe COVID-19 cases revealed increased methylation of IFN-related genes, while inflammatory genes were hypomethylated . An ISG Clinical trials of COVID-19 vaccines found high vaccine efficacy, and observational studies evaluated vaccine effectiveness in several countries ,127. MulWhile an immune response is necessary for clearing SARS-CoV-2 infection and establishing immunological memory to combat reinfection, hyperinflammatory responses have also been shown to underlie the pathology of exacerbated COVID-19. COVID-19 therapeutic interventions could have regulated and anticatalyzed immune responses to SARS-CoV-2 in multiple ways. Anticatalytic medicines with acetylating capacity can treat COVID-19."} +{"text": "Remodeling of tumors into inflamed ones by mJX-594 led to a response to combined anti-PD-1 treatment, but not to mJX-594 or anti-PD-1 monotherapy. mJX-594 treatment increased T cell factor 1-positive stem-like T cells among cancer-specific CD8+ T cells, and anti-PD-1 combination treatment further increased proliferation of these cells, which was important for therapeutic efficacy. The presence of functional cancer-specific CD8+ T cells in the spleen and bone marrow for an extended period, which proliferated upon encountering cancer antigen-loaded splenic dendritic cells, further indicated that long-term durable anticancer immunity was elicited by oncolytic VACV.Oncolytic virotherapy has garnered attention as an antigen-agnostic therapeutic cancer vaccine that induces cancer-specific T cell responses without additional antigen loading. As anticancer immune responses are compromised by a lack of antigenicity and chronic immunosuppressive microenvironments, an effective immuno-oncology modality that converts cold tumors into hot tumors is crucial. To evaluate the immune-activating characteristics of oncolytic vaccinia virus , diverse murine syngeneic cancer models with different tissue types and immune microenvironments were used. Intratumorally administered mJX-594, a murine variant of JX-594, potently increased CD8 The efficacy of oncolytic virotherapy for cancer was originally thought to be dependent on selective lysis of infected cancer cells; therefore, efficient cancer targeting and extensive intratumoral virus replication and propagation were considered limiting factors for cancer treatment . However+ T cells ..+ T cell+ T cells was compromised by the expression of several negative immune checkpoint molecules on their surfaces, which led to their exhaustion [+ PD-1+, which are referred to as either stem-like or progenitor-exhausted T cells [+ T cells in the tumor tissue and secondary lymphoid tissue, such as the spleen and bone marrow. Moreover, the quantity of these stem-like T cells correlated with the therapeutic efficacy of mJX-594 and was further increased by (and correlated with) combination therapy involving anti-PD-1 potently increased CD8"} +{"text": "Several surveys report that post-COVID-19 patients (pts) could be at risk of persistent emotional distress, fatigue and impaired neurocognitive function (NCF).The aim was to assess emotional distress, fatigue and NCF in order to provide adequate care.Patients with persistent physical or mental symptoms, at least 8 weeks post-COVID-19, were eligible for this ongoing prospective longitudinal single center trial. Data on depression, anxiety, cognition, post-traumatic stress symptoms (PTSS) and fatigue were collected using 4 validated questionnaires at study entry (T0) and at 6 months (T1).Ninety-three pts were recruited between November 2020-March 2021. Test results from 64 eligible pts were analyzed at T0; 63 pts (98%) were treated in outpatient settings. Median age was 47 years [range 27-75]). Median time since COVID-19 was 29 weeks [range 8-53]. Twenty-two pts (34%) had a history of psychiatric disorders. According to the Hospital Anxiety Depression Scale (HADS), 44 pts (73%) reported anxiety symptoms and 26 pts (41%) reported depressive symptoms; 48 pts (69%) reported cognitive complaints according to the Cognitive Failure Questionnaire and 29 pts (45%) suffered from PTSS, according to the Post-Traumatic Stress Disorder Checklist-Civilian Version (PCL-C). Fifty-five pts (86%) had an elevated score on the Fatigue Severity Scale, indicating severe fatigue. Twenty-seven pts (42%) were still on sick leaf. Diminished social support and psychiatric history were predictive factors for neurocognitive dysfunction and PTSS.A majority of patients who recovered physically from COVID-19, are at risk for suffering from persistent anxiety, PTSS and neurocognitive dysfunction.No significant relationships."} +{"text": "Dear Editor,The coronavirus disease 2019 (COVID-19), which was declared to be a pandemic by the World Health Organization (WHO) on March 11, 2020, is an important issue worldwide.We conducted a systematic search for Peruvian scientific articles on COVID-19 in the PubMed/MEDLINE and SciELO databases, and directly in the RPMESP archives up to May 21, 2020. The search strategy for PubMed/MEDLINE was (COVID-19 [MeSH] OR COVID-19 diagnostic testing [MeSH] OR COVID-19 drug treatment [MeSH] OR COVID-19 serotherapy [MeSH] OR COVID-19 vaccine [MeSH] OR severe acute respiratory syndrome coronavirus 2 [MeSH] OR COVID-19 [Title/Abstract] OR SARS-CoV-2 [Title/Abstract] OR coronavirus disease 2019 [Title/Abstract] OR Wuhan coronavirus [Title/Abstract]) AND (Peru [MeSH] OR Peru [Title/Abstract] OR Peruvian [Title/Abstract] OR Andean [Title/Abstract]). For SciELO, it was (COVID-19) OR (infections coronavirus) OR (coronavirus disease 2019) OR (SARS-CoV-2). Articles that did not provide scientific knowledge were excluded.We included 24 articles . Eleven The topics addressed mostly were related to epidemiology, with emphasis on the probability of effectiveness or failure regarding pandemic control measures. The technological contribution of telemedicine and tele-education was also addressed. In addition, research on mental health in the era of COVID-19 pointed out the possibility of undesired outcomes such as development of psychotic symptoms due to inadequate management of anxiety caused by COVID-19.We found that around 10% of the articles were published in Q1 journals. Scientific publication in higher-quartile journals increases the likelihood of citation because of higher impact factors and h-indexes, which are important indicators of scientific-academic publication.In Peru, UNMSM and UPCH provide the highest scientific production within medicine.In conclusion, Peruvian scientific production on COVID-19 has emphasized epidemiological research, and has mainly been produced within institutions located in Lima. Therefore, there is a need to decentralize collaborative networks and promote clinical studies in order to generate more evidence with which to combat the pandemic."} +{"text": "The COVID-19 pandemic and ensuing public health emergency has emphasized the need to study SARS-CoV-2 pathogenesis. The human microbiome has been shown to regulate the host immune system and may influence host susceptibility to viral infection, as well as disease severity. Several studies have assessed whether compositional alterations in the nasopharyngeal microbiota are associated with SARS-CoV-2 infection. However, the results of these studies were varied, and many did not account for disease severity. This study aims to examine whether compositional differences in the nasopharyngeal microbiota are associated with SARS-CoV-2 infection status and disease severity.We performed Nanopore full-length 16S rRNA sequencing on 194 nasopharyngeal swab specimens from hospitalized and community-dwelling SARS-CoV-2-infected and uninfected individuals. Sequence data analysis was performed using the BugSeq 16S analysis pipeline.We found significant beta (PERMANOVA p < 0.05), but not alpha diversity differences in the nasopharyngeal microbiota among our study groups. We identified several differentially abundant taxa associated with SARS-CoV-2 infection status and disease severity using ALDEx2. Finally, we observed a trend towards higher abundance of Enterobacteriaceae in specimens from hospitalized SARS-CoV-2-infected patients.This study identified several alterations in the nasopharyngeal microbiome associated with SARS-CoV-2 infection status and disease severity. Understanding the role of the microbiome in infection susceptibility and severity may open new avenues of research for disease prevention and treatment. COVID-19 is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2); a positive-sense RNA virus of the Coronaviridae family . Primers and probes were designed to target the N501Y and E484K mutations; and PCR conditions are provided in S1 Table in Routine diagnostic testing was performed using either a Roche MagNA Pure extraction system in combination with a lab developed (LDT) real time PCR (RT-PCR) assay detecting the E-gene and RdRp gene targets, or the Panther Fusion SARS-CoV-2 assay , detecting two targets in ORF1ab. The LDT RT-PCR assay was developed at the British Columbia Center for Disease Control (BCCDC) and is based on the World Health Organization\u2019s guidelines for COVID-19 RT-PCR diagnostic screening . RT-PCR Total nucleic acids were extracted from NPS specimens using the MagNA Pure 24 Total NA Isolation Kit on the MagNA Pure 24 extraction system . The Pathogen 1000 protocol was used with a 500\u03bcL sample and 50\u03bcL elution volume. Total nucleic acid extracts were quantified with a Qubit 4 Fluorometer (Thermo Fisher Scientific) using the high-sensitivity dsDNA assay kit.We utilized the Oxford Nanopore Technologies (ONT) MinION sequencing platform for this study, which enables real-time, long read sequencing of biological samples. This approach may confer more accurate taxonomic resolution than short-read 16S rRNA sequencing approaches , and hasdevice cuda:0 flag to enable GPU basecalling. Basecalled fastq files were analyzed using the BugSeq 16S sequencing analysis pipeline [vegan R package [randomForest R package [Raw sequence data were basecalled using Guppy with default parameters and the\u2013pipeline . Downstrpipeline . Relativ package . Multiva package was used package . Our modThis study included de-identified samples collected from VGH and has obtained ethics board approval from the research ethics board at the University of British Columbia (H20-02152). All raw sequence data for this study is available on NCBI SRA under the accession number PRJNA868394.t values , low complexity (fastp complexity < 30%), and reads shorter than 1000bp or longer than 1850bp were discarded . Samplesversus SARS-CoV-2-uninfected community-dwelling samples. This trend was not observed in hospitalized SARS-CoV-2-infected versus uninfected samples.We did not observe any significant differences in alpha diversity among our study groups , althougadonis within the vegan R package. We observed significant clustering at all three taxonomic ranks , suggesting that there are differences in microbial community composition among our study groups, although, there was a large degree of overlap in the 95% confidence ellipses.Bray-Curtis dissimilarity ordination was performed to assess differences in beta diversity among our study groups using PCoA at the species-, genus-, and family-level taxonomic ranks . StatistStaphylococcus was the most abundant genus on average for both SARS-CoV-2-uninfected study groups, whereas Acinetobacter was the most abundant genus in the hospitalized infected group and Moraxella was the most abundant genus in the community-dwelling SARS-CoV-2-infected group . An \u201cuncultured\u201d genus assigned to the family Neisseriaceae was also found to be differentially abundant. All the differentially abundant taxa were enriched in community-dwelling SARS-CoV-2 infected individuals. Our Random Forest predictive models for the binary outcome variable (infected vs non-infected) and our four study groups produced out-of-bag error estimates of 44.9% and 66.5% respectively.ADLEx2 was used to assess which of the taxa were significantly differentially abundant. The species Streptococcus spp. and Corynebacterium spp. among samples from infected individuals collected during the first three waves of the pandemic in British Columbia . We did not observe significant differences in alpha , or beta diversity (adonis p > 0.05) associated with viral load or by SARS-CoV-2 variant infection status.Significant beta (PERMANOVA p < 0.05), but not alpha diversity differences in the nasopharyngeal microbiome composition were observed when stratified by sample collection date . ALDEx2 In this study, we used full-length 16S rRNA sequencing data to examine differences in the nasopharyngeal microbiota associated with SARS-CoV-2 infection and COVID-19 severity. We found significant beta , but notStaphylococcus spp. among our SARS-CoV-2-uninfected groups . We identified a high proportion of reads mapping to the genus Alishewanella, an environmental microbe Click here for additional data file.S2 Fig(PNG)Click here for additional data file.S3 Fig(PNG)Click here for additional data file.S1 File(DOCX)Click here for additional data file."} +{"text": "Little is known about the relationship between family caregivers and persons living with dementia communication at micro-level . Micro-level communication was assessed second-by-second using 75 in-home video recordings from 19 caregiver-care recipient dyads. Each care recipient (N = 38) and caregiver (N = 42) behavior unit was paired and compared using Spearman\u2019s partial correlation. Two-hundred twenty care recipient-caregiver behavior unit pairs were correlated . Ninety-one caregiver facilitative-care recipient engaging behavior unit pairs were positively correlated . Eighteen caregiver disabling-care recipient challenging behavior unit pairs were positively correlated . Individualized caregiver education for matching and adjusting communication is needed to facilitate meaningful interaction."} +{"text": "Background: SARS-CoV-2 N95 mask contamination in healthcare providers (HCPs) treating patients with COVID-19 is poorly understood. Method: We performed a prospective observational study of HCP N95 respirator SARS-CoV-2 contamination during aerosol-generating procedures (AGPs) on SARS-CoV-2\u2013positive patients housed in a COVID-19\u2013specific unit at an academic medical center. Medical masks were used as surrogates for N95 respirators to avoid waste and were worn on top of HCP N95 respirators during study AGPs. Study masks were provided to HCPs while donning PPE and were retrieved during doffing. Additionally, during doffing, face shields were swabbed with Floq swabs premoistened with viral transport media (VTM) prior to disinfection. Medical masks were cut into 9 position-based pieces, placed in VTM, vortexed, and centrifuged (Fig. Results: We enrolled 31 HCPs between September and December 2021. HCP and patient characteristics are presented in Table Conclusions: Overall, mask contamination in HCPs treating patients with COVID-19 undergoing AGPs was not detectable while wearing face shields, despite patient contact and performing AGP.Funding: NoneDisclosures: None"} +{"text": "KTM2A, previously known as MLL) gene rearrangement KMT2A-r, which is associated with neprilysin (CD10)-negative immature B-cell precursor phenotype and a very poor prognosis [KTM2A-r based on transcription factors (iroquois-class homeodomain protein IRX and homeobox protein HOXA), fusion partners, and corresponding stages of B-lymphopoietic and hemato-endothelial development has been suggested for genomic-driven diagnostics and potential therapeutic strategies in infant ALL [DUX4)-rearranged (DUX4-r), tyrosine-protein kinase ABL (ABL)-class fusions, and myocyte-specific enhancer factor 2 D (MEF2D)-rearranged (MED2D-r) [DUX4-r [Acute lymphoblastic leukemia (ALL) is the most common childhood cancer, in which nearly 5% of the cases are diagnosed before the first year of age . Around fant ALL . Interesfant ALL . Nearly MED2D-r) ,7,8. A s [DUX4-r . Last yeilysin CD-negative [DUX4-r .ALL treatment typically involves several phases: induction , prophylaxis , consolidation ) and maintenance . Targeted therapy protocols including the use of tyrosine kinase inhibitors to Philadelphia chromosome-positive ALL patients and of immunotherapeutic agents such as antibody and chimeric antigen receptor (CAR) T-cell therapies are now being examined to be part of the upfront setting ,13.Acute myeloid leukemia (AML) is an aggressive and heterogeneous hematological cancer. Although most patients with newly diagnosed AML achieve complete remission (CR) after intensive induction and consolidation therapy, more than half of them relapse within the next three years ,15. BecaFLT3 mutations), combined epigenetic therapy with the BCL-2 inhibitor venetoclax, immunotherapy or restricted approaches for AML with myeloid-related changes (AML-MRC), or therapy-related AML (CPX-351) cases [Besides intensive induction and consolidation therapy, AML treatment has improved in recent years, and several new therapeutic options have been approved. Most of them include mutation-specific approaches cases .The genetic and clinical heterogeneity of ALL and AML cases represent a challenge even for the new therapies. Moreover, relapse and treatment resistance seriously hinder ALL and AML treatment. To understand the biology underlying those differences, omic sciences have increasingly been utilized to produce useful knowledge.https://proteomics.se/forall accessed on 8 January 2023) identified the diacylglycerol-analog bryostatin-1 as a therapeutic candidate in the myocyte enhancer factor 2D (MEF2D)-heterogeneous nuclear ribonucleoprotein U-like protein 1 (HNRNPUL1) fusion high-risk subtype, for which this drug activates pro-apoptotic mitogen-activated protein kinase (ERK) signaling associated with the molecular mediators of pre-B-cell negative selection [Multi-omic analyses of 49 childhood ALL cell lines using proteomics, transcriptomics, and pharmacoproteomic characterization , at IDH1/2 mutations displayed a high expression of the 2-oxoglutarate\u2013dependent histone demethylases KDM4A/B/C, despite no changes in messenger RNA levels for these genes, and samples with FLT3-tyrosine kinase domain (TKD) mutationsassociated with the activation of the SRC-family tyrosine kinases FGR and HCK) [To uncover the molecular changes allowing AML cells to escape treatment, two proteomic studies with liquid chromatography\u2013mass spectrometry (LC\u2013MS) and serial time-point samples during the disease progression of patients have shown that the proteomic profile at relapse is enriched for mitochondrial ribosomal proteins and subunits of the respiratory chain complex, indicative of reprogrammed energy metabolism from diagnosis to relapse ,24. Usinand HCK) . Using sand HCK) .Altogether, these studies show us the importance and strength of single- and multi-omic approaches to decode the complex and heterogenous molecular basis of acute leukemias and to improve the current treatment and survival of ALL and AML patients.https://github.com/j-rehn/RaScALL accessed on 10 January 2023) [In the past few decades, single-omic strategies have been used for research leading to ALL- and AML-related biomarker discovery. Genomic approaches have described several causative variants that are now part of international diagnosis guidelines. However, the integration of genome sequencing into a healthcare setting still lags behind. Nevertheless, examples of genomic incorporation, such as the reported initiative across multiple clinical entities of rare diseases and the ry 2023) .Numerous single-omic studies with patient cohorts have revealed a high number of leukemia biomarkers ,30,31. RMulti-omic approaches that integrate the data from distinct levels of the cellular organization, i.e., from genes to metabolites, have recently been reported, which offer new perspectives for innovation in ALL and AML diagnostics and therapeutics. Multi-omic strategies bring together researchers with different expertise, including data managers and bioinformaticians, to provide protein and metabolite targets of specific genomic backgrounds. The incorporation of more clinicians in research teams appears to be more necessary than ever to transfer clinical innovation into practice."} +{"text": "BACKGROUND: Novel chimeric antigen receptor T-cells (CAR-T) target the B-cell maturation antigen (BCMA) expressed on multiple myeloma cells. Assays monitoring CAR-T cell expansion and treatment response are being implemented in clinical routine. METHODS: Plasma levels of soluble BCMA (sBCMA) and anti-BCMA CAR-T cell copy numbers were monitored in the blood, following CAR-T cell infusion in patients with relapsed multiple myeloma. sBCMA peptide concentration was determined in the plasma, applying a human BCMA/TNFRS17 ELISA. ddPCR was performed using probes targeting the intracellular signaling domains 4-1BB und CD3zeta of the anti-BCMA CAR-T construct. RESULTS: We report responses in the first five patients who received anti-BCMA CAR- T cell therapy at our center. Four patients achieved a complete remission (CR) in the bone marrow one month after CAR-T infusion, with three patients achieving stringent CR, determined by flow cytometry techniques. Anti-BCMA CAR-T cells were detectable in the peripheral blood for up to 300 days, with copy numbers peaking 7 to 14 days post-infusion. sBCMA plasma levels started declining one to ten days post infusion, reaching minimal levels 30 to 60 days post infusion, before rebounding to normal levels. CONCLUSIONS: Our data confirm a favorable response to treatment in four of the first five patients receiving anti-BCMA CAR-T at our hospital. Anti-BCMA CAR-T cell expansion seems to peak in the peripheral blood in a similar pattern compared to the CAR-T cell products already approved for lymphoma treatment. sBCMA plasma level may be a valid biomarker in assessing response to BCMA-targeting therapies in myeloma patients. Multiple myeloma (MM) is a neoplasm of clonal plasma cells. MM is considered treatable, but incurable. Remission may be induced by treatment options involving steroids, chemotherapy, antibodies, targeted compounds, and autologous stem cell transplantation (ASCT). Advancements in treatments, including the introduction of immunomodulatory drugs (IMID), proteasome inhibitors (PI), and monoclonal antibodies have prolonged survival to a five-year survival rate of about 50% ,2,3,4. NThe B-cell maturation antigen is a cell surface receptor of the TNF receptor superfamily preferentially expressed in malignant and normal plasma cells and mature B lymphocytes, specifically binding to TNFSF13B/TALL-1/BAFF (B-cell activating factor) and to various TRAF family members, and, thus, transducing signals for cell survival and proliferation ,6. ProteIdecaptagene-vicleucel (bb2121) is an anti-BCMA chimeric antigen receptor T-cell therapy for the treatment of multiple myeloma, approved by the FDA in 2021 for the treatment of adults with relapsed or refractory multiple myeloma, who have received at least three lines of anti-CD38/PI/IMID treatment. In clinical studies the median progression-free survival was 11.8 months in phase I and 8.8 The assays monitoring CAR-T expansion and assessing response to this novel therapeutic option await implementation into clinical routine. Here, we evaluate the first five patients with relapsed MM treated with anti-BCMA CAR-T cell therapy at the Insel-spital Bern, Switzerland.In the work described here, we aimed to monitor response in anti-BCMA CAR T-cell recipients, by establishing a panel of laboratory assessments. We evaluated whether introducing routine laboratory parameters might facilitate timely identification of response and trigger therapeutic interventions. We established a digital droplet PCR (ddPCR) assay for CAR-T\u2212specific T-cell receptor (TCR) measurement from peripheral blood (PB) and introduced sBCMA assessments at consecutive time points before and after CAR-T cell infusion.The first five patients with relapsed/refractory multiple myeloma (RR/MM) receiving anti-BCMA CAR-T therapy at Bern University Hospital, Switzerland, were included in this study. They received their individual anti-BCMA CAR-T cell infusion between May and September 2021 after several lines of prior therapy. All patients gave written informed consent, and the study was approved by the local ethics committee of Bern, Switzerland (No. 2018-00628).2 + cyclophosphamide 300 mg/m2.Lymphocyte collections with the Spectra Optia (Terumo BCT) device were performed using the continuous mononuclear cell collection (CMNC) procedure. Peripheral CD3+ cell counts of blood and lymphocyte products were analyzed by multi-parameter flow cytometry (BD FACSCanto II). Five patients received idecabtagene vicleucel . Ide-cel was manufactured following leukapheresis and then infused at dose levels aiming at 4.5E + 08 CAR+ T cells after 2 days interval, following lympho-depletion with 3 days of fludarabine 30 mg/mThe criteria for assessing response were according to the International Myeloma Working Group ,17. NegaIn analogy to our previous study on CD19-CAR-T cell specific digital PCR , we desiThe Human BCMA/TNFRSF17 ELISA Kit is a solid-phase sandwich enzyme-linked immunosorbent assay (ELISA) designed to detect and quantify the level of human BCMA in cell culture supernatants, plasma, and serum. ELISA assay was done according to the manufacturer\u2019s instructions using BCMA protein standard. sBCMA levels were represented as the mean of triplicate samples for each specimen.We evaluated the first five myeloma patients treated with bb2121 CAR-T cell therapy at a single academic center . After CFive patients with relapsed/refractory multiple myeloma were included in this study . They rePatient 1 (C-80), first diagnosed with multiple myeloma, stage III (R-ISS) received three lines of prior therapy and autologous stem cell transplant (ASCT). After the third relapse, anti-BCMA CAR-T treatment resulted in stringent complete remission four weeks after CAR-T cell infusion. CAR-T copy number in the peripheral blood peaked on day 7 at high levels (>3E + 05 copies/\u03bcg gDNA), followed by decline to minimal levels after six months, then remained at low levels (100 copies/\u03bcg gDNA) until end of observation. sBCMA plasma levels, initially at 115 ng/mL, started dropping immediately after infusion, and reached minimal levels (5 ng/mL) eleven weeks after infusion. sBCMA plasma levels started to rise to normal levels 4 months after infusion, and 10 months after infusion the response status was still sCR.Patient 2 (C-85), first diagnosed with multiple myeloma, stage II (R-ISS), had received two lines of prior therapy and ASCT. After second relapse, anti-BCMA CAR-T treatment resulted in stringent complete remission four weeks after CAR-T cell infusion. CAR-T copy number in the peripheral blood peaked on day 7 at intermediate levels (2E + 05 copies/\u03bcg gDNA) and subsided below the detection limit after five months. sBCMA plasma levels, initially at 100 ng/mL, started dropping immediately after CAR-T cell infusion, and reached minimal levels (10 ng/mL) four weeks after infusion, persisting below normal levels for nine months when the response status was still sCR.Patient 3 (C-86), first diagnosed with multiple myeloma, stage II (R-ISS) received five lines of prior therapy and ASCT. After second relapse, anti-BCMA CAR-T treatment resulted in complete remission four weeks after infusion. CAR-T copy number in the peripheral blood peaked on day 13 at high levels (3.2E + 05 copies/\u03bcg gDNA) and was still at 128 copies/\u03bcg gDNA after six months. sBCMA plasma levels, initially at 120 ng/mL, started dropping 10 days after infusion, and reached minimal levels (10 ng/mL) two months after CAR-T cell infusion. sBCMA plasma levels started to rise above normal levels (rebound) three months after infusion. Eight months after CAR-T infusion sBCMA plasma concentration had reached pretreatment levels, while the response status was still CR.Patient 4 (C-89) had received two lines of prior therapy, but was not eligible for ASCT. After second relapse anti-BCMA CAR-T, therapy resulted in complete remission four weeks after infusion. CAR-T copy number in the peripheral blood peaked on day 9 at high levels (6E + 05 copies/\u03bcg gDNA) and subsided below the detection limit after five months. sBCMA plasma levels, initially at 280 ng/mL, started dropping immediately after infusion and reached minimal levels two months after CAR-T cell infusion. Six months after CAR-T infusion, the response determination indicated a relapse.Patient 5 (C-91), first diagnosed with multiple myeloma, stage II (R-ISS), had received two lines of prior therapy and ASCT. Anti-BCMA CAR-T treatment resulted in stable disease (SD) four weeks after infusion. CAR-T copy number in the peripheral blood peaked on day 14 at low levels (1.3E + 04 copies/\u03bcg gDNA) and was at 100 copies/\u03bcg gDNA after three months when disease was progressive (PD). sBCMA plasma levels, initially at 200 ng/mL, started dropping immediately after infusion; however, they soon reached a plateau at 120 ng/mL. The disease progressed, and the patient died 100 days after CAR-T infusion.We determined the dynamics of CAR-T cell presence in the peripheral blood by evaluating the copy number, as well as persistence after infusion, similarly to the analysis done in the phase I and phase II studies ,14. ExpaProteolytic shedding of the BCMA receptor reduces its cell-surface expression and ligand-mediated survival of B cell subsets. This shedding is mediated by protease \u03b3-secretase, and is partially dependent on ligand binding and receptor interactions. sBCMA plasma levels are elevated in MM sera (100\u2013300 ng/mL) compared to healthy donors 10\u201380 ng/mL), with no difference between newly diagnosed patients and those with relapsed disease 0 ng/mL, ,10. A reFour of the first five patients receiving anti-BCMA CAR-T cells at the University Hospital, Bern, demonstrated favorable response to anti-BCMA CAR-T treatment. Complete remission (CR) in the bone marrow was prevalent in four patients (80%) one month post CAR-T infusion, with stringent CR (sCR) present in three patients (60%), as determined by flow cytometry techniques. This is in accordance with results from a phase II study , where, Responses to anti-BCMA CAR-T treatment in this small cohort were found to associate with peak expansion by qPCR (>2E + 05 copies/\u03bcg DNA for CR vs. 1E + 04 copies/\u03bcg for PR/SD), similarly to data reported by Cohen et al., 2019 , who desWith respect to sBCMA levels, a clinical reference value has not been defined. However, sBCMA peptide concentrations are elevated in MM plasma (100\u20131000 ng/mL) compared to healthy donors (10\u201380 ng/mL) ,22,23. IsBCMA levels above normal levels after the decline of CAR-T cells may indicate the impending end of remission. sBCMA plasma concentrations of 100 ng/mL can represent normal levels in one patient, and indicate recurrence of myeloma in another patient. sBCMA plasma levels may be easily determined together with standard remission controls on a monthly basis and can serve, not only as response monitoring tool, but also as an early predictor of response.A remarkable decrease in sBCMA level was observed in the four patients with good responses to bb2121 therapy. With this limited number of patients, definite conclusions on the utility of sBCMA as a response monitoring marker are not possible. However, the study may present a proof of principle, suggesting that sBCMA may represent a promising biomarker of response to BCMA-targeted immunotherapy. sBCMA concentrations were found to decline more significantly after CAR-T cell infusions in responders (CR/sCR) than in non-responders (SD). sBCMA was detectable in all five patients at the end of the study, suggesting that BCMA antigen loss is not a prevalent mechanism of escape from ide-cel therapy. sBCMA concentrations remained at low levels in long-term responders, confirming sBCMA plasma levels as a useful adjunctive biomarker for assessing myeloma disease response and progression. Larger studies are required to evaluate the prognostic significance of sBCMA plasma levels for their potential as biomarker of response and predictor of response to BCMA-targeting therapies in relapsed multiple myeloma patients.Identification of the observed CAR-T copy number kinetics suggests investigation of whether the pattern \u2018rapid rise to high peak levels, followed by decline, with persistence at low levels\u2019 may be associated with superior efficacy of CAR-T treatment. Identification of the observed sBCMA plasma level dynamics suggests further investigation of whether the pattern \u2018rapid decline to minimal levels < 10 ng/mL\u2019, as well as \u2018duration at minimal levels\u2019, may be associated with superior efficacy of CAR-T treatment. These questions can be addressed once the therapy is approved and more MM patients can be admitted to BCMA CAR-T therapy."} +{"text": "Autoimmune diseases result from a breach of immunological self-tolerance and tissue damage by autoreactive T lymphocytes. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection is characterized by an inflammatory dysregulation that has been associated with the development of autoimmune processes .Molecular mimicry has been suggested as a potential mechanism for these associations as well as \u2018bystander activation\u2019 where the infection may lead to activation of antigen presenting cells that may activate autoreactive T-cells, with the production of pro-inflammatory mediators and tissue damage .There is a potential antigenic cross-reactivity between SARS-CoV-2 and human tissue possibly linked to an increase in autoimmune diseases. A recent study showed that antibodies against the spike protein S1 of SARS-CoV-2 had high affinity against some human tissue proteins such as transglutaminase 2 and 3, or myelin basic protein, among others , COST ACTION CA17112-Prospective European Drug-Induced Liver Injury Network and IMI2- Translational Safety Biomarker Pipeline (TransBioLine Horizonte 2020). CM21/00074 (Rio Hortega contract: J.M.P.B.).None declared.goac014_Supplementary_DataClick here for additional data file."} +{"text": "Patients\u2019 attitudes and subjective experience are crucial in the management of severe mental illness, but their practical value is overlooked.To identify predictors of future adherence to LAI antipsychotic maintenance treatment of schizophrenia among socio-demographic, clinical, and psychometric characteristics \u2013 including Drug Attitude Inventory-10 (DAI-10) and Subjective Well-being under Neuroleptics short form (SWN-K) scores.Retrospective baseline data from 53 clinically stable outpatients with schizophrenia switched from oral to LAI therapy were collected. Patients continuing treatment at the time of analysis (n=29) were compared to those who had discontinued it (n=24). Selected variables were further evaluated in survival analyses.Between-group differences are presented in Table 1 .Cox regression analysis included instruction, employment, hospitalisations, PANSS subscales and DAI-10 scores: a protective role against treatment discontinuation was outlined only for employment and higher DAI-10 scores . DAI-10 scores delineated distinct adherence trajectories .Baseline DAI-10 scores may identify patients at risk of dropout after switching to LAI.No significant relationships."} +{"text": "S-glutathionylation, induced by oxidative stress, and its deletion causes fatty liver in mice. Although Glrx regulates various pathways, including metabolism and apoptosis, the impact of Glrx on liver fibrosis has not been studied. Therefore, we evaluated the role of Glrx in liver fibrosis induced by aging or by a high-fat, high-fructose diet. We found that: (1) upregulation of Glrx expression level inhibits age-induced hepatic apoptosis and liver fibrosis. In vitro studies indicate that Glrx regulates Fas-induced apoptosis in hepatocytes; (2) diet-induced NASH leads to reduced expression of Glrx and higher levels of S-glutathionylated proteins in the liver. In the NASH model, hepatocyte-specific adeno-associated virus-mediated Glrx overexpression (AAV-Hep-Glrx) suppresses fibrosis and apoptosis and improves liver function; (3) AAV-Hep-Glrx significantly inhibits transcription of Zbtb16 and negatively regulates immune pathways in the NASH liver. In conclusion, the upregulation of Glrx is a potential therapeutic for the reversal of NASH progression by attenuating inflammatory and fibrotic processes.Liver fibrosis is a sign of non-alcoholic fatty liver disease progression towards steatohepatitis (NASH) and cirrhosis and is accelerated by aging. Glutaredoxin-1 (Glrx) controls redox signaling by reversing protein Liver fibrosis is a sign of non-alcoholic fatty liver disease (NAFLD) progression towards steatohepatitis and cirrhosis. The higher level of fibrosis in non-alcoholic steatohepatitis (NASH) is associated with a higher risk of developing liver cirrhosis, hepatocellular carcinoma ,2, and cAging is also a risk factor for fibrosis progression in chronic liver diseases . Older pS-glutathionylation). Glrx is often categorized as an antioxidant, but its deletion does not necessarily increase sensitivity to oxidative stress [We previously showed that mice with deletion of the glutaredoxin-1 (Glrx) gene develop NAFLD with regular chow and are more susceptible to diet-induced inflammation . Glrx, oe stress ,24. Glrxe stress ,25 and pe stress . In addie stress . Howeverper se induced mild fibrosis and increased glutathionylated protein levels in the liver of mice fed a regular chow diet. As a proof of concept, we administrated hepatocyte-specific adeno-associated virus (AAV-Hep)-mediated Glrx and showed decreased apoptosis and collagen accumulation in the liver of aged mice. Furthermore, we tested the effects of AAV-Hep-Glrx on high-fat, high-fructose diet-induced NASH in mice and demonstrated that AAV-Hep-Glrx attenuated diet-induced fibrosis and improved hepatic function. Gene expression profiling of the liver indicated that AAV-Hep-Glrx suppressed leukocyte-related inflammatory pathways. In particular, the expression of Zbtb16 (zinc finger and BTB domain-containing 16) was significantly decreased by Glrx administration, suggesting that Zbtb16 may be a key downstream effector controlling liver fibrosis. Interestingly, the diet change to regular chow indicates that AAV-Hep-Glrx has an anti-fibrotic effect independent of steatosis.In this study, we examine the extent to which Glrx can control age-induced liver fibrosis. We found that biological aging In summary, hepatocyte-targeted upregulation of Glrx inhibits liver fibrosis in chow-fed aged mice and in diet-induced NASH, and AAV-Hep-Glrx is a potential therapeutic for the reversal of NASH progression by attenuating inflammatory and fibrotic processes.The following antibodies were purchased; anti-glutaredoxin-1 (Glrx) , anti-cleaved caspase 3 , anti-\u03b1-smooth muscle actin (\u03b1SMA) , anti-glutathione (Pr-SSG) , anti-4-hydroxynonenal (HNE) (Abcam), anti-goat IgG-HRP , anti-rabbit IgG-HRP, and anti-mouse IgG-HRP . All cell culture reagents were purchased from Gibco , Lonza , and Atlanta Biologicals . Chemicals for liver perfusion and AAV production were purchased from Sigma and Fisher Scientific unless specified otherwise.Glrx KO mice were originally generated by Dr. Y.S. Ho , backcroLiver samples were fixed in 10% formalin overnight and replaced into 10% and 20% sucrose before embedding in OCT compound. Frozen OCT-embedded samples were cut to 10 \u00b5m sections with a Leica cryostat. The specimens on a slide were washed in PBS for 10 min and incubated in Picro Sirius Red solution (Sirius Red F3B in a saturated solution of picric acid) for 1 h. After dehydration by sequential ethanol and xylene immersions, a cover glass was placed using Permount mounting medium. Images were taken by Olympus microscope using a polarized filter . Collagen type I signal appears bright orange in the dark background under polarized light. For quantification of the collagen area, we measured 6\u20137 images of each liver section. The images were split into 3 colors with ImageJ. Red signals were converted to black and white images. The collagen area (%) was quantified by the percent of the white area in the entire field by ImageJ. Each dot in the graph indicates the average of 6\u20137 images from one mouse. H&E staining of formalin-fixed Chow/NASH-diet liver was conducted with H&E staining kit following the protocol provided by the manufacturer.Tissue samples were powdered in liquid nitrogen. RNA was isolated from tissue powders or cells using TRIzol reagent following the manufacturer\u2019s protocol. Reverse transcription was performed with RNA to generate cDNA using iScript cDNA synthesis kit . Quantitative PCR was performed with TaqMan assays or validated SYBR Green assays (BioRad) using a CFX Real-Time System (BioRad). The following TaqMan primers were used: human GLRX (Hs00829752_g1) (for detecting the human GLRX gene in Glrx Tg mice), Acta2 (Mm01546133_m1), Tnf (Mm00443258_m1), and 18S (Mm03928990_g1). Primers for SYBR Green assays are listed in Preparation of the solution for hepatocyte isolation was performed according to Mederacke et al. . Mice weS-glutathionylated protein (Pr-SSG), which requires non-reducing conditions). In addition, 10 mM N-ethylmaleimide (NEM) was added to alkylate free protein thiol to detect Pr-SSG. Samples were loaded on a NuPAGE 4\u201312% Bis-Tris gel (Invitrogen) using NuPAGE MES SDS running buffer (Invitrogen) and transferred to a PVDF membrane using a Trans-BlotTM Turbo Transfer System (Bio-Rad). The membrane was blocked with 3% non-fat dry milk in TBS-T for 1 h and incubated with primary antibodies overnight at 4 \u00b0C in 3% BSA. After washing with TBS-T, membranes were incubated for 1 h with the corresponding HRP-conjugated antibodies: anti-goat IgG-HRP (1:5000), Digital anti-rabbit IgG-HRP (1:3000), or Digital anti-mouse IgG-HRP (1:3000). The chemiluminescent signal was detected using the Hi/Lo Digital-ECLTM Western Blot Detection Kit and the KwikQuant Imager. The signals were quantified by ImageJ software and normalized by Ponceau S staining intensity.Tissue powder or cultured cells were homogenized in lysis buffer containing 50 mM Tris pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% NP-40, and Protease Inhibitor Cocktail . After centrifugation , the supernatant was mixed with LDS sample buffer (Invitrogen); 2-mercaptoethanol was added to 2.5% final concentration (except for the detection of 12 vg) was administrated per mouse by intraperitoneal injection. The promoter of TBG induces a hepatocyte-specific gene expression [The AAV plasmid for hepatocyte-specific overexpression pAAV.TBG.PI.Null.bGH was purcpression ,34. ThusHepatic triglyceride and cholesterol levels were measured by the Infinity triglycerides and total cholesterol reagent kit (Thermo Fisher). Serum alanine aminotransferase (ALT) level and aspartate aminotransferase (AST) level were measured by Catalyst Dx Chemistry Analyzer (IDEXX) in the Boston University Analytical Instrumentation Core.n = 5 or 6 each), pooled into 3 samples per group. The samples were analyzed after confirming the quality of the RNA by 2100 Bioanalyzer . Raw CEL files were normalized to produce gene-level expression values with the Robust Multiarray Average (RMA) [bioconductor.org) and Entrez Gene-specific R packages from the Molecular and Behavioral Neuroscience Institute (Brainarray) at the University of Michigan [t-test implemented in the limma Bioconductor R package . Correction for multiple hypothesis testing was accomplished using the Benjamini\u2013Hochberg false discovery rate (FDR). Human homologs of mouse genes were identified using the NCBI HomoloGene resource . All microarray analyses were performed using the R environment for statistical computing . Gene Set Enrichment Analysis (GSEA) [t statistic, and any mouse genes with multiple human homologs (or vice versa) were removed prior to ranking thus that the ranked list represents only those human genes that match exactly one mouse gene. This ranked list was then used to perform pre-ranked GSEA analyses (default parameters with random seed 1234) using the Entrez Gene versions of the H , C2 CP , C3 (transcription factor and microRNA motif), and C5 gene sets obtained from the Molecular Signatures Database (MSigDB), .We examined the transcriptome of the livers of mice NASH model after AAV-Glrx or AAV-control injection by Affymetrix Mouse Gene 2.0 ST GeneChip microarray (Thermo Fisher) at the Microarray and Sequencing Resource at Boston University School of Medicine. RNA was isolated from mouse liver (ge (RMA) using thich.edu) . Differegdb.org) was usedFor standard statistical analysis, we used GraphPad Prism 9.0.t-test was used for normally distributed data. For the analysis of more than two groups, one-way or two-way ANOVA was used, followed by Bonferroni post-hoc tests. p < 0.05 values were considered significant.Student\u2019s unpaired n = 7 each, p < 0.05) . The quantitative assessment of collagen-stained areas indicated an increase in collagen deposition in the livers of 12- and 18-month-old mice compared to 4-month-old mice A,B. In a < 0.05) F,G. In a < 0.05) H. Our da+/\u2212 (Het), and Glrx\u2212/\u2212 (KO) male mice. Protein levels of \u03b1SMA (Acta2) were increased in a dose-dependent manner by Glrx deficiency with a trend of increased cleaved caspase-3 into aged WT mice to evaluate whether Glrx overexpression in hepatocytes protects against apoptosis and fibrosis in the aged liver. Three months after AAV administration, we confirmed that AAV-Hep-Glrx injection-induced expression of HA/FLAG-tagged Glrx and attenuated expression of cleaved caspase-3 in the liver D,E. In aTo directly evaluate the anti-apoptotic role of Glrx in hepatocytes, we used primary hepatocytes treated with an agonistic antibody to FAS receptor (anti-CD95). Anti-CD95 antibody induces apoptosis through the activation of FAS signaling pathway. To see the effect of Glrx overexpression in hepatocytes, we injected AAV-Hep-Glrx or AAV-Hep-control vectors into WT mice, and isolated primary hepatocytes at 2 months post-injection. Then, we induced apoptosis in cultured cells by stimulation with an anti-CD95 . CD95-induced expression of cleaved caspase-3 was attenuated in Glrx-overexpressing hepatocytes compared with the control AAV vector A. ConverWe then examined the effect of Glrx overexpression on liver fibrosis in a diet-induced NASH model. Wild-type male mice (10-week-old) that are fed a high-fat high fructose diet (NASH diet) become obese and develop fatty liver with fibrosis and higher serum levels of AST and ALT at 15\u201316 weeks (about four months) . H&E staTo determine whether increased expression of Glrx could attenuate these effects, we injected AAV-Hep-Glrx or AAV-Hep-control vectors after 20 weeks of the NASH diet, when fibrosis had already appeared and continued the diet for another 12 weeks A. AAV-HeClinically, dietary therapy to reduce fat and calorie intake is a primary intervention for NAFLD/NASH. To model this dietary modification, we changed the NASH diet to regular chow for a month for a cohort of mice and found that the liver size and bodyweight of these animals returned close to those of animals fed only regular chow . Furtherp = 3.3 \u00d7 10\u22126, FDR q = 0.09), although the expression of several inflammatory markers also trended downward in AAV-Hep-Glrx-treated animals (q < 0.25) by AAV-Hep-Glrx compared to AAV-Hep-control . Quantit= 0.060) A. We the-control , includi-control B. These In this study, we have shown that Glrx deficiency elevates markers of fibrosis and apoptosis both in vivo and in vitro, whereas augmenting Glrx expression protects hepatocytes from cell death and decreases collagen levels in the aged liver and diet-induced NASH liver. Age-induced fibrosis is mild and may not affect liver function per se but promotes fibrosis and NASH progression in the context of a high-fat diet ,19. We, S-glutathionylation. Accordingly, our data show that Glrx regulates Fas-induced apoptosis in hepatocytes and may, therefore, suppress fibrotic processes in the liver through anti-apoptotic regulation.Glrx inhibits Fas signaling, protects lung epithelial cells from apoptosis, and attenuates experimental lung fibrosis , and canHigh-fat-diet-induced lipotoxicity causes hepatocyte damage and cell death, which in turn activates immune cells and stellate cells, leading to fibrosis and to the progression of NAFLD and NASH . Apoptot+/\u2212 mice [Intriguingly, AAV-Hep-Glrx treatment also significantly downregulated transcription of the gene Zbtb16 (zinc finger and BTB domain-containing 16), which encodes the transcription factor PLZF (promyelocytic leukemia zinc finger) that controls lymphocyte differentiation and meta+/\u2212 mice , suggest+/\u2212 mice ,49, and +/\u2212 mice . TherefoS-glutathionylation in adipocytes [Hepatocytes produce innate immunity proteins and pro-inflammatory cytokines under stress conditions. One of the liver-enriched transcription factors that may secrete immune mediators in an acute response is CCAAT/enhancer-binding protein (C/EBP) \u03b2 ,52. C/EBipocytes and may,ipocytes , but theWe have demonstrated that the upregulation of Glrx can reverse age-induced apoptosis and fibrosis in the liver and ameliorate fibrosis in diet-induced NASH. AAV-mediated hepatocyte-specific Glrx expression in vivo decreased collagen accumulation, improved hepatic function, and suppressed immune cell activation and inflammation in NASH livers. The data indicate crosstalk between hepatocytes and other cells to control fibrotic processes in the liver, showing that Glrx has the potential to be a powerful anti-fibrotic therapeutic molecule."} +{"text": "Correction: Cellular and Molecular Life Sciences (2022) 80:6 10.1007/s00018-022-04634-2In the published article reference 41 was missed in the third paragraph, under discussion section in the page 15, and the below mentioned reference has been incorrectly processed, and the error in the below references has been now updated.Previously, we have reported that aberrant activation of the MET receptor modulates the cellular response to IR by rewiring key DNA damage response (DDR)-related phosphorylations in some tumor cell lines featuring MET activation [41]. Assuming that a MET\u2013DDR interface underlies MET dependency, here we monitored 116 DDR- and RTK signalling-associated phosphosites in a panel of MET-positive, MET-responsive as well as non-responsive tumor models following targeted MET inhibition. This analysis revealed 14 METi-modulated phosphorylation events that were present solely in MET-addicted models, thus representing \u2018MET-asa-driver\u2019 footprints.The original article has been updated."} +{"text": "Somatostatin-expressing interneurons (SOM-INs) are a major subpopulation of GABAergic cells in CA1 hippocampus that receive excitation from pyramidal cells (PCs) and provide feedback control of synaptic inputs onto PC dendrites. Excitatory synapses from PCs onto SOM-INs (PC-SOM synapses) exhibit long-term potentiation (LTP) mediated by type 1a metabotropic glutamate receptors (mGluR1a). LTP at PC-SOM synapses translates in lasting regulation of metaplasticity of entorhinal and CA3 synaptic inputs on PCs and contributes to hippocampus-dependent learning. A persistent form of PC-SOM synapse LTP lasting hours is prevented by blockers of transcription and translation, and a more transient form of PC-SOM synapse LTP lasting tens of minutes requires mTORC1-signaling, suggesting an involvement of protein synthesis. However, the role of protein synthesis in these forms of plasticity has not been directly demonstrated. Here we use the SUrface SEnsing of Translation (SUnSET) assay of protein synthesis to directly show that the induction protocols for both forms of LTP at PC-SOM synapses stimulate protein synthesis in SOM-INs. Moreover, protein synthesis stimulated by persistent LTP induction was prevented in mice with a SOM-IN conditional knock-out of Raptor, an essential component of mTORC1, indicating a critical role of mTORC1 in the control of translation in PC-SOM synapse plasticity. Moreover, protein synthesis induced by both forms of LTP may share common mechanisms as transient LTP induction occluded further stimulation of protein synthesis by persistent LTP induction. Our findings highlight a crucial role of protein synthesis and its control by mTORC1 in SOM-INs that is important for hippocampus-dependent memory function.The online version contains supplementary material available at 10.1186/s13041-022-00967-y. S51A) in SOM-INs upregulates their general mRNA translation, gates CA1 network plasticity and increases long-term contextual fear memory [Somatostatin-expressing interneurons in CA1 hippocampus (SOM-INs) are a major subpopulation of local inhibitory interneurons . SOM-INsr memory . Thus, pr memory ,\u00a08,\u00a09. HHere, we investigate the involvement of protein synthesis in plasticity at PC-SOM synapses using the SUrface SEnsing of Translation (SUnSET) assay in SOM-ISstires\u2212Cre;Rosa26lsl\u2212EYFP mice (SOM-EYFP-WT mice) [First, we established the specificity of puromycin labeling in the SUnSET assay ,\u00a013 of ESstires\u2212Cre;Rosa26lsl\u2212EYFP;Rptorfl/fl\u00a0knock-out mice; SOM-EYFP-Raptor-KO mice) [Synaptic mechanisms that induce chemical late LTP in CA1 pyramidal cells are diffopto), an effective protocol to induce transient LTP at PC-SOM synapses [opto) was then given as previously [opto relative to unstimulated slices from hChR2-expressing mice , puromycin immunofluorescence was increased in SOM-INs after DHPG treatment of Schaffer collateral synapses in PCs mediated by mGluR5 and protein synthesis . Our obsAdditional file 1. Materials and methods.Additional file 2: Figure S1. Specificity of puromycin labeling in SUnSET assay. (a) Diagram of puromycin entering the ribosomal A-site, arrest of protein synthesis and release of premature peptides, later detected using a puromycin specific antibody. (b) Representative images of EYFP and puromycin immunofluorescence showing specificity of puromycin antibody in absence (upper panels) or presence (middle panels) of puromycin, and no puromycin labeling without puromycin antibody (bottom panels). Hippocampal slices were exposed with or without puromycin to Sham-treatment of late LTP protocol. Arrows indicate cells with colocalization of EYFP and puromycin fluorescence signal. Scale bar, 100 \u00b5m. (c) Summary bar graph of puromycin colocalization in EYFP cells expressed as percentage of total EYFP cells . Figure S2. Intact SOM-IN basal protein synthesis in mice with conditional Rptor knock-out, and unchanged pyramidal cell layer puromycin immunofluorescence after chemical persistent LTP induction. (a) Representative images and summary bar graph of puromycin immunofluorescence in SOM-INs of SOM-EYFP WT and SOM-EYFP-Raptor KO mice, showing no difference of puromycin fluorescence in SOM-INs (sham-treatment) of control and knockout mice. Arrows indicate cells with colocalization of EYFP with puromycin immunofluorescence. Summary bar graph . (b-c) Representative images and summary bar graphs of puromycin immunofluorescence in the CA1 pyramidal layer of SOM-EYFP WT (b) and SOM-EYFP-Raptor KO (c), showing no difference in puromycin fluorescence following chemical persistent LTP induction. Summary bar graph . Scale bars, 50 \u00b5m. Student\u2019s t-tests, ns not significant"} +{"text": "We report a case of methotrexate (MTX)-induced stroke-like encephalopathy in an 18-year-old woman, with acute lymphoblastic leukemia, who developed a sudden neurological deficit mimicking a cerebrovascular event. Bain MRI showed hyperintensities on diffusion-weighted-imaging (DWI) with matching apparent diffusion coefficient hypointensities, which also represent the commonest MRI findings in acute cerebral infarction. DWI changes spared the cerebral cortex and did not respect vascular territories, supporting a non-vascular mechanism. MRI plays a crucial role in the diagnostic work-up and is essential to avoid unnecessary intervention such as thrombolytic therapy.Teaching Point: Methotrexate-induced stroke like neurotoxicity should be considered in patients treated with methotrexate and presenting with a stroke-like clinical picture and radiological findings consistent with acute cerebral infarction. Methotrexate (MTX) is a folic-acid antagonist commonly used to treat a range of autoimmune diseases and malignancies. Rarely, MTX neurotoxicity may present as stroke-like onset, causing diagnostic dilemma. We herein present a novel observation of MTX-induced stroke-like encephalopathy in a young patient with acute lymphoblastic leukemia (ALL) and who developed a sudden neurological deficit mimicking a cerebrovascular event.ClinicalTrials.gov Identifier: NCT02617004). Morphological remission was achieved after induction therapy. In December 2019, three days after completing the fifth consolidation cycle, consisting of vincristine , 6-mercaptopurine (60 mg/m2), intravenous MTX (1g/m2), triple intrathecal therapy , she presented abrupt onset of aphasia and right facial weakness at 11:30 a.m. She was admitted to the Emergency Department at 12:40 p.m. Initial National Institutes of Health Stroke Scale (NIHSS) score was 8. Blood sugar was normal; vital signs were unremarkable. Due to the sudden onset of symptoms, stroke was first considered. An urgent brain magnetic resonance imaging (MRI), performed within 2h of symptom onset, showed bilateral diffusion-weighted imaging (DWI) hyperintensities involving the subcortical white matter a direct toxicity of homocysteine on the vascular endothelium, (ii) an increased excitatory effects on N-methyl-D-aspartate receptors by homocysteine-derived metabolites, (iii) alterations of adenosine metabolism, (iiii) a chronic folate depletion in brain tissue, and (iiiii) a direct neuronal damage by MTX . In most1Our observation underlines the importance of considering MTX-induced stroke like neurotoxicity in patients treated with MTX and presenting with a stroke-like clinical picture. Emergency MRI with DWI is the mainstay of imaging to discriminate this stroke-mimicking condition from a real stroke."} +{"text": "Homalodisca vitripennis ) . MAGs representing the obligate symbionts Candidatus Sulcia muelleri and Candidatus Baumannia cicadellinicola and the facultative symbiont Wolbachia were obtained from imidacloprid-resistant and imidacloprid-susceptible sharpshooters.The role of microbes in insecticide resistance is an emerging question. Here, we describe six metagenome-assembled genomes (MAGs) associated with the glassy-winged sharpshooter ( Homalodisca vitripennis) is an invasive xylem-feeding leafhopper that is native to the southeastern United States and Mexico and was recently introduced into California with Minimap2, given that these obligate symbionts have reduced genomes (https://doi.org/10.5281/zenodo.6493649) (Sequence reads for imidacloprid-susceptible (A6) and imidacloprid-resistant (C9) e (PGAP) . Related6493649) ."} +{"text": "In China, the duck industry has been severely impacted by the newly emerging duck Tembusu virus (DTMUV). For DTMUV to successfully infect host cells, it employs several strategies that subvert the host's innate immune response. It has been found that several viral proteins encoded by DTMUV have strategically targeted the crucial molecules of the RIG-I-like Receptor (RLR) signaling pathway to antagonize host antiviral responses. However, it is not well known how the host proteins manipulated by DTMUV contribute to innate immune evasion. The present study reports that duck TRIM35 (duTRIM35) antagonizes DTMUV-induced innate immune responses by targeting duck RIG-I (duRIG-I) in duck embryo fibroblasts. A significant increase in duTRIM35 expression occurred during DTMUV infection. DuTRIM35 overexpression suppressed DTMUV-triggered expression of interferon beta (IFN-\u03b2) and interferon-stimulated genes (ISGs), promoting viral replication, whereas knockdown of duTRIM35 augments the innate immune response, reducing viral replication. Furthermore, duTRIM35 significantly impaired the IFN-\u03b2 expression mediated by duRIG-I but not by other RLR signaling molecules. Mechanistically, duTRIM35 interfered with duRIG-I-duTRIM25 interaction and impeded duTRIM25-mediated duRIG-I ubiquitination by interacting with both duRIG-I and duTRIM25. Our findings indicate that duTRIM35 expression induced by DTMUV infection interfered with the duRIG-I-mediated antiviral response, illustrating a novel strategy in which DTMUV can evade the host's innate immunity.IMPORTANCE Duck Tembusu virus (DTMUV), an emerging flavivirus pathogen causing a substantial drop in egg production and severe neurological disorders in duck populations, has led to massive economic losses in the global duck industry. DTMUV has employed various strategies to subvert the host's innate immune response to establish a productive infection in host cells. In this study, we report that duck TRIM35 (duTRIM35) expression was upregulated upon DTMUV infection in vitro and in vivo, and its expression antagonized DTMUV-induced innate immune responses by targeting duck RIG-I (duRIG-I) in duck embryo fibroblasts. Further studies suggest that duTRIM35 interfered with duRIG-I-duTRIM25 interaction and impeded duTRIM25-mediated duRIG-I ubiquitination by interacting with both duRIG-I and duTRIM25. Together, these results revealed that duTRIM35 expression induced by DTMUV infection downregulated duRIG-I-mediated host antiviral response, which elucidated a novel strategy of DTMUV for innate immune evasion. Flavivirus in the family Flaviviridae (\u20136\u2013\u2013Tembusu virus (TMUV), an emerging iviridae , causes iviridae \u20134. This viridae \u2013, 6. The ridae \u20136\u2013. Accumulridae \u20136\u2013\u201311.The host's innate immune response to pathogen-associated molecular patterns (PAMPs) is the first line of defense against invading pathogens , 13. Dur16\u2013Although RIG-I is a crucial receptor to trigger innate immune responses, different flaviviruses have developed various mechanisms to attenuate its activity to evade host immune responses. For instance, Zika virus (ZIKV) NS5 protein hampers the K63-linked polyubiquitination of RIG-I by interacting with RIG-I, thus dampening RIG-I-mediated signaling . PreviouThe tripartite motif-containing (TRIM) proteins are found in all vertebrates including avian, most of which contain a conserved RING domain plus one or two B-boxes and an N-terminal coiled-coil (CC) domain . By theiin vivo was performed by quantitative real-time PCR (qRT-PCR) to determine the transcription levels of duTRIM35 mRNA in different tissues from healthy or DTMUV-infected ducks. As illustrated in in vitro and in vivo, DTMUV infection significantly induces the expression of duTRIM35.To assess the role of duTRIM35 in modulating DTMUV-induced innate immunity, we analyzed its expression in duck embryo fibroblasts (DEFs) at different time points following infection with DTMUV. Significant increases in protein abundance and mRNA expression levels of duTRIM35 were observed during DTMUV infection and B. FWe first confirmed the expression of the duTRIM35 eukaryotic expression plasmid in DEFs by Western blotting . To deteTo determine whether duTRIM35 promotes DTMUV replication by modulating the host's innate immunity, the mRNA expression levels of IFN-\u03b2, viperin, and PKR mRNA in the duTRIM35-expressing DEFs were detected using qRT-PCR. We observed that overexpression of duTRIM35 downregulated the DTMUV-induced mRNA expression of IFN-\u03b2, viperin, and PKR . In contTo further determine the target of duTRIM35 in the IFN production signaling, plasmids encoding essential molecules in RLR signaling and duTRIM35, along with the IFN-\u03b2 luciferase reporter plasmid, were cotransfected into DEFs. As illustrated in To identify which domain of duRIG-I interacts with duTRIM35, we cotransfected the plasmids encoding the Flag-tagged duRIG-I deletion mutants and HA-tagged duTRIM35 in HEK-293T cells. Through Co-IP assays, we found that duTRIM35 was coprecipitated with full-length duRIG-I but not with its deletion mutants , suggestSince the K63-linked ubiquitination of RIG-I is essential for triggering the antiviral responses , we inveIt has been demonstrated that duck TRIM25 contributes to duRIG-I ubiquitination and enhancement of interferon production , which pTo investigate whether duTRIM35 binding to duTRIM25 and duRIG-I impairs duRIG-I-mediated antiviral signaling, we evaluated the effects of duTRIM35 on duRIG-I-duTRIM25 interaction. For this purpose, we cotransfected the plasmids expressing Myc-tagged duRIG-I and HA-tagged duTRIM25 together with the duTRIM35 expression plasmid. The Co-IP experiments showed that the association between duRIG-I and duTRIM25 was remarkably decreased in the presence of duTRIM35 , suggestin vitro and in vivo inhibit MDA5/RIG-I-mediated IFN-\u03b2 expression via blocking TBK1 phosphorylation, whereas NS4A and NS5 efficiently interfere with RIG-I-induced IRF3 activation , 36. Lik in vivo , and its in vivo . Secondl in vivo . Thirdly in vivo and 5. L in vivo and 7.TRIM35 was originally discovered as a tumor suppressor with the potential to reduce tumor cell proliferation, clonogenicity, and tumorigenicity , 39. SevRIG-I is an important member of the RLRs family that recognizes viral RNAs and recruits MAVS to activate downstream kinases TBK1 and IKK\u03b5, resulting in IRF3 translocation and type I IFN production. Several cellular factors have been reported to downregulate RIG-I activity and suppress RLR-mediated antiviral signaling . For exaAccumulating research has found that RIG-I activity could be regulated by ubiquitination modification. By catalyzing the process of K63-linked polyubiquitination, E3 ligases, such as TRIM25, Riplet, TRIM4, and MEX3C, positively regulate the activity of RIG-I \u201347. In c\u201348\u2013In conclusion, we present a schematic model that illustrates how DTMUV inhibits RIG-I signalosome activation to suppress type I interferon production by duTRIM35 . During 2 incubator. HeLa and HEK-293T cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS. DTMUV strain MC (GenBank accession number KX452096) was isolated by our laboratory as previously described (Duck embryo fibroblasts (DEFs) were cultured in minimum essential medium (MEM) containing 10% fetal bovine serum (FBS) at 37\u00b0C in a 5% COescribed . Variousescribed . Tissue escribed . Mouse aDuTRIM35 and duTRIM25 were amplified from the cDNA of DEFs and subcloned into the pCAGGS expression vector in-frame with an HA or Flag tag at the N terminus. The plasmids encoding duRIG-I, duRIG-I-truncated mutants, duMAVS, duTBK1, duIKK\u03b5, and duIRF7 were constructed as previously described . All plaTC) method with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal reference. Quantitative PCR (qPCR) primers employed in this study are listed in Table S1.Total RNA was extracted from DEFs and selected tissues of ducklings using TRIzol reagent according to the manufacturer's instructions. RNA was reverse-transcribed to cDNA using the Transcriptor First Strand cDNA synthesis kit . FastStart Universal SYBR green master mix was used in a ViiA 7 system for quantitative real-time PCR (qRT-PCR). The relative expression levels were determined using the comparative threshold cycle following the manufacturer's instructions. Representative data were presented as the relative firefly luciferase activities with normalization to the Renilla luciferase activities from three independent assays.In a 48-well plate, ~80% confluence DEFs were cotransfected using Lipofectamine 2000 (Invitrogen) with a luciferase reporter plasmid and an internal control pRL-TK, along with appropriate expression plasmids. After transfection, the cells were inoculated with DTMUV for 24 h. Subsequently, the cells were collected, and firefly and After seeding HeLa cells on coverslips in 24-well plates, the cells were transfected with the expression plasmids. The transfected cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked with 5% bovine serum albumin (BSA). Subsequently, the cells were incubated separately with a mouse anti-HA antibody (1:200) or a rabbit anti-Flag antibody (1:50) and then stained with secondary antibodies Alexa Fluor 488-conjugated donkey anti-mouse IgG and Alexa Fluor 594-conjugated donkey anti-rabbit IgG (Invitrogen), followed by treatment with 4\u2032,6-diamidino-2-phenylindole (DAPI) (Invitrogen). Fluorescence was visualized by a Zeiss LSM 880 confocal microscope.Protein samples from the indicated cells were prepared using radioimmunoprecipitation assay (RIPA) buffer (Beyotime) supplemented with 1\u2009mM phenylmethanesulfonyl fluoride (PMSF). The lysates were separated by 12% SDS-PAGE, electroblotted onto polyvinylidene difluoride (PVDF) membranes (Millipore), and then blocked with Tris-buffered saline-Tween (TBST) containing 10% skim milk. The appropriate antibodies were used to probe specific protein bands. An enhanced chemiluminescence (ECL) system (Bio-Rad) was used to detect antibody-antigen complexes.HEK-293T cells were cultured in 10-cm dishes and cotransfected with the protein expression plasmids. After transfection, cells were collected with Co-IP buffer (Beyotime) supplemented with EDTA-free protease inhibitor cocktail (Roche). Cell lysate (0.4\u2009mL) was incubated for each immunoprecipitation with anti-Flag or anti-Myc monoclonal antibodies overnight at 4\u00b0C. After further incubation with 40\u2009\u03bcL protein A/G plus agarose (Santa Cruz Biotechnology) for 4 h, the beads were collected by centrifugation and washed 4 times with cold IP buffer. Finally, the precipitates were analyzed by standard immunoblot procedures with the appropriate antibody.DEFs were cotransfected with Myc-duRIG-I, Flag-duTRIM35, or an empty vector in the presence or absence of HA-Ub or its mutations. After transfection, cells were harvested with Co-IP buffer (Beyotime), and cell lysates were immunoprecipitated with anti-Myc MAb, followed by incubating with protein A/G plus-agarose (Santa Cruz Biotechnology). After washing with lysis buffer, the immunoprecipitates were analyzed by standard immunoblot procedures with the appropriate antibodies.P value was determined using an unpaired two-tailed Student's t test. A P value\u2009of <0.05 was regarded as statistically significant, and a P value\u2009of <0.01 was regarded as highly significant.The data were statistically evaluated using GraphPad Prism software . The"} +{"text": "Positron emission tomography/computed tomography (PET/CT) with prostate-specific membrane antigen (PSMA)-targeting radiopharmaceuticals is a relatively novel technique currently employed in the management of prostate cancer patients. These radiopharmaceuticals target PSMA, also known as carboxypeptidase type II, which is mainly expressed by prostate cancer cells. Nevertheless, its expression has been observed in the neovasculature of various kinds of cancer, including clear cell renal cell carcinoma (ccRCC). Based on this biological mechanism, several authors postulated a potential role for this diagnostic method in ccRCC patients in different clinical settings. This systematic review provides an overview of the possible applications of PET/CT with PSMA-targeting radiopharmaceuticals in ccRCC.Background: Recent articles proposed the employment of positron emission tomography/computed tomography (PET/CT) with prostate-specific membrane antigen (PSMA)-targeting radiopharmaceuticals in clear cell renal cell carcinoma (ccRCC). Methods: The authors performed a comprehensive literature search of studies on the performance of PET/CT with PSMA-targeting radiopharmaceuticals in ccRCC. Original articles concerning this imaging examination were included in newly diagnosed ccRCC patients and ccRCC patients with disease recurrence. Results: A total of sixteen papers concerning the diagnostic performance of PSMA-targeted PET/CT in ccRCC (331 patients) were included in this systematic review. The included articles demonstrated an excellent detection rate of PSMA-targeting PET/CT in ccRCC. Conclusions: PSMA-targeted PET/CT seems promising in detecting ccRCC lesions as well as in discriminating the presence of aggressive phenotypes. Prospective multicentric studies are warranted to strengthen the role of PSMA-targeting PET/CT in ccRCC. Kidney cancer is a common kind of tumor with an estimated incidence of approximately 400,000 new diagnoses every year worldwide ,2. RenalDue to the tumor cells\u2019 overproduction of platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF), ccRCC is considered a highly vascularized tumor .To date, most ccRCC diagnoses result from incidental findings as a consequence of the widespread use of noninvasive radiological techniques, including ultrasonography (US), magnetic resonance imaging (MRI), or computed tomography (CT), performed for another reason . Moreove18F]FDG) positron emission tomography (PET)/CT [18F]FDG PET/CT is not currently recommended as an imaging method in ccRCC by practice guidelines [18F]FDG and its metabolites, seeing as how their physiological renal excretion hinders the characterization of primary lesions, making the differentiation from the physiologic background challenging [18F]FDG PET/CT is not routinely employed to evaluate ccRCC because of its relatively low [18F]FDG uptake; the underlying mechanism for this phenomenon is partially unclear yet. Nevertheless, a recent study showed that [18F]FDG uptake reflected FBP1 expression levels in patients with ccRCC since it was higher in ccRCCs with low fructose 1,6-bisphosphatase 1 (FBP1) expression compared to patients with high FBP1 expression FDidelines ,18,19 anllenging . Furtherpression .18F]FDG uptake, exploring different biological processes than glucose metabolism. Furthermore, since a combination of immunotherapy and antineoangiogenetic therapy are the standard of care in metastatic ccRCC FDG PET/CT (Comparator); the predetermined outcomes were the evaluation of PSMA-targeting radiopharmaceuticals uptake in ccRCC and PSMA-targeted PET detection rate in ccRCC.In agreement with the Population, Intervention, Comparator, Outcomes (PICO) framework, two reviewers (G.T. and A.R.) accomplished a literature search, establishing criteria for the eligibility of the studies found in the literature search: patients with ccRCC diagnosis (Population) submitted to PET with PSMA-targeting radiopharmaceuticals (Intervention) compared or not to traditional imaging or FDG PET/CT [18F]FDG PET/CT and diagnostic CT Ga-PSMA-11 [18F]F-DCFPyL was injected (mean injected activity: 333 MBq) [18F]F-PSMA-1007 (activity range: 217\u2013268 MBq); in two papers, an alternate between [68Ga]Ga-PSMA-11 and [18F]F-PSMA-1007 was reported based on radiopharmaceuticals availability [68Ga]Ga-PSMA-11 and [18F]F-DCFPyL were used FDG PET/CT FDG PET imaging relies on the increased glucose demand by rapidly dividing tumor cells. Membrane glucose transporters, mainly GLUT-1, actively transport [18F]FDG into the cell, where it is converted by hexokinase into [18F]FDG-6-phosphate, which is trapped in the cytoplasm and accumulates. Tumor cells usually show increased [18F]FDG uptake due to the overexpression of GLUT-1 transporter, increased intracellular hexokinase, and low FBP1 [18F]-FDG presents a variable uptake in ccRCC lesions; from this biological mechanism, PET/CT with [18F] FDG is not currently recommended in ccRCC management. Among the included studies, two compared the performance of [18F]FDG and PSMA-targeted PET/CT and both reported superior performance of the latter Zr for the imaging of ccRCC [89Zr]Zr-girentuximab were found in the literature search.Carbonic anhydrase IX (CAIX), a cell membrane antigen, has been recently used as a target for molecular imaging in ccRCC patients , since iof ccRCC ,55. They68Ga]Ga-PSMA-11, [18F]F-DCFPyL, and [18F]F-PSMA-1007 [18F]-PSMA-targeting radiopharmaceuticals counts on several advantages such as reduced costs through large-scale production, higher-quality images through lower positron energy and a longer half-life, none of the included studies compared the diagnostic performances between different PSMA-targeting radiopharmaceuticals.Since the introduction of carboxypeptidase type II as a possible target for molecular imaging and theragnostics, an increasing number of PSMA-targeting radiopharmaceuticals has been developed, including Lu-PSMA-617 and [225Ac]Ac-PSMA-617, and their outstanding results in metastatic castration-resistant prostate cancer patients FDG PET/CT was superior in detecting gastrointestinal and pancreatic tumors [Consistently with the studies included in this systematic review, Yin et al. reported a poor accuracy of PSMA-targeted PET/CT in patients with metastatic non-ccRCC, inferior to conventional imaging with CT and/or MRI . These dc tumors .None of the studies included in this systematic review addressed the cost-effectiveness of PET with PSMA-targeting radiopharmaceuticals in managing ccRCC patients if it was actually introduced in clinical practice as a standard of care. Since, to date, this examination is employed in a research setting, cost-effectiveness studies are needed to strengthen and refine the role of this imaging technique in ccRCC.Two previously published reviews explored the potential role of PSMA-targeted PET/CT in RCC patients ,62. The Concerning the limitations and biases of this systematic review, a limited number of prospective studies was available and most of them enrolled a restricted number of patients. Furthermore, we clearly demonstrated a significant heterogeneity among the included studies about the study methodology, patient characteristics, index test characteristics, and reference standard or comparison used. Prospective multicentric studies are needed to strengthen the role of PSMA-targeting PET/CT in ccRCC.The qualitative data provided by this systematic review enhanced the promising role of PSMA-targeted PET/CT in patients with ccRCC. Nevertheless, more studies are warranted to overpower these findings and deepen how PET imaging with PSMA-targeting radiopharmaceuticals could integrate with conventional imaging in different clinical settings."} +{"text": "Innovations in CRISPR/Cas allow the use of online tools for single guide RNA design and multiplexing, cloning , robust CRISPR/Cas constructs, efficient transformation protocols such as Agrobacterium, and DNA-free protoplast method for Cas9-gRNAs ribonucleoproteins (RNPs) complex, Cas9 variants like PAM-free Cas12a, and Cas9-NG/XNG-Cas9, homologous recombination (HR)-based gene knock-in (HKI) by geminivirus replicon, and base/prime editing (Target-AID technology). This mini-review highlights the current research advances in CRISPR/Cas for fast and efficient breeding of tomato.The narrow genetic base of tomato poses serious challenges in breeding. Hence, with the advent of clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein9 (CRISPR/Cas9) genome editing, fast and efficient breeding has become possible in tomato breeding. Many traits have been edited and functionally characterized using CRISPR/Cas9 in tomato such as plant architecture and flower characters , fruit ripening, quality and nutrition , disease resistance , abiotic stress tolerance , C-N metabolism, and herbicide resistance. CRISPR/Cas9 has been proven in introgression of Solanum lycopersicum L.) is one of the most economically important vegetable crops, which is consumed as fresh and processed products. Tomato is considered a functional and protective food because of its health-beneficial compounds such as vitamins A and C, minerals, and antioxidants mainly lycopene and beta-carotene and CRISPR-associated 9 protein (CRISPR/Cas9) genome editing tool. CRISPR/Cas9 is a powerful and precise genome editing technology that allows trait-specific targeted mutants and functional characterization of genes. CRISPR/Cas9 has been used to edit various traits in tomato . RecentlCRISPR/Cas9 protein functions on the principles of a bacterial or archaeal adaptive immunity system that confronts the invading viruses or phages. CRISPR/Cas has been categorized into several types and sub-types such as the class 1 includes numerous proteins to form complexes, whereas the class 2 includes only one protein. Among them, Cas9 is the most widely deployed machinery in crop improvement. CRISPR/Cas9 uses specific, designed nucleases to cause double-stranded break (DSB) in DNA (dsDNA). Further, the DSB is repaired by the mechanisms called non-homologous end joining (NHEJ) or homology-directed repair (HDR)/homologus recombination (HR). The NHEJ mechanism is error-prone, allows random small insertions or deletions and substitutions, and probably causes gene knock-out (KO) mutations. The HDR mechanism mostly generates point mutations or deletions caused by gene knock-in (KI), but this method has a very low success rate so far. CRISPR/Cas9 makes breeding much easier by producing gene knock-out mutants for desired traits. Gene knock-in is possible through HDR by providing template DNA with overlapping flanking regions. Nevertheless, CRISPR/Cas9 has many limitations, such as the availability of NGG protospacer adjacent motif (PAM) motifs in the genome sequence. Hence, emphasis has been driven to diversify Cas9 proteins and search for other Cas9 proteins in different bacteria, which have different PAM sequences.https://crispr.dbcls.jp), CRISPR-P (http://cbi.hzau.edu.cn/cgi-bin/CRISPR), CRISPR-PLANT (http://omap.org/crispr/index.html), CRISPR-GE (http://skl.scau.edu.cn/), Breaking-Cas (http://bioinfogp.cnb.csic.es/tools/breakingcas/), CRISPOR.org (http://crispor.org), CRISPR-BETs . Cas9 requires the PAM sequence at the target site. The approximately 20 nucleotide-long gRNA spacer sequence could be readily programmed to target DNA sites with PAM using the online tools available. The freely available online tools for sgRNA design and quality check are CRISPRdirect (SPR-BETs and so oSPR-BETs to assemSPR-BETs protocolThe Golden Gate cloning strategy is a very fast and flexible assembly for CRISPR/Cas construct designing and applAgrobacterium-mediated transformation is the most commonly used method in CRISPR/Cas9 research in plants including tomato (via mutagenesis in the cis-regulatory regions of the CLV3 (Clavata3) gene in tomato complexes. Therefore, preassembled Cas9-gRNA RNPs have been directly delivered into the plant cells via protoplast-mediated transformation by polyethylene glycol (PEG) fusion or biolistic methods. To remove CRISPR/Cas background, the Cas9-gRNA RNPs system is completely DNA-free and devoid of genetic segregation (i) avoiding progeny screening by selfing or backcrossing; ii) no off-target effects; iii) better control over CRISPR/Cas endonuclease; iv) direct and easy mutagenesis process after transfection without any lagging phase; and v) direct delivery of Cas9-sgRNA assembly or preassembled isolated RNPs into the plant cells. However, RNP\u2019s system has problems like delivery through the plant cells and regeneration from cell-wall-free cells. As a result, protoplast culture protocol is dependent on genotype, species, tissue-specific, and cell wall responses. Thus, minimizing the proportion of off-targets and checking the possibilities of transgene integration is important for enhancing precise Cas9 endonuclease activity.regation . There acis-elements for random promoter engineering (via selfing or backcrossing is possible for diploid tomato. The availability of PAM sequences (2-5 bp) is highly lacking in the genome, so it is difficult to edit target genes. Hence, diversity in protospacer adjacent motif (PAM) sequence specificities has become a key requirement of CRISPR/Cas9.Cas9 can be used to edit DNA highly efficiently to target gene knock-outs within coding sequences or random changes in non-coding sequences like ineering . FurtherLachnospiraceae bacteria is a new category of the CRISPR system, which is quite analogous to Cas9 and enables editing of AT-rich genomic regions like the 5\u2019 and 3\u2019 UTR and promoter domains. Cas9 endonuclease recognizes G-rich PAM sequences and produces a blunt end cut, whereas Cas12a has a shorter crRNA by 60 nucleotides and no spacer is required, tracrRNA is not required to generate mature crRNA, and recognizes T-rich PAM sequences, producing cohesive ends requires PAM sequences (5\u2019-TTTN-3\u2019). This is quite useful for the tomato genome, as it has AT-rich sequences and uses Cas12a nuclease. Hence, new variants of Cas9 with diverse sequence specificities of PAM would be greatly useful for the restoration of genetic diversity. The Cas9 variant, a PAM-free Cas12a nuclease, is smaller in size and highly thermostable than Cas9 . Cas12a ive ends . Thus, nPAM-free Cas9 has advantages like: i) it can target any sequence; ii) selection of the target site is simple and flexible with more on-target and less off-target; iii) it allows easy placement of base-editing over specific nucleotides; and iv) it is beneficial for multiplexing because only one nuclease targets most genes. Nevertheless, a major limitation of PAM-free nuclease is that the gRNA that is expressed from the DNA constructs would self-target the parent DNA immediately, ultimately leading to unsuccessful results. Another drawback is that a nuclease without a specific PAM would search every sequence in the genome and would take much longer time to find the target sequence, hence increasing the chance of off-targets. Importantly, Cas12a has much scope for PAM-free nuclease activity in plants. Therefore, the development of a nuclease repertoire could be a novel way by which any gene sequence can be targeted by the CRISPR/Cas system.Streptococcus pyogenes Cas9 (dSpCas9) and demonstrated in tomato . On the contrary, DSB repair by HDR requires donor template DNA with homologous flanking sequences that allow gene KI mechanism. NHEJ is the most commonly deployed to develop KO mutants, whereas HDR or KI is promising but has a low success rate in eukaryotic cells. Therefore, the KI mechanism requires more research to handle the technical difficulties in placing the donor templates in the vicinity of the DSB in the recipient plant cells.Agrobacterium-mediated delivery mechanism is most commonly applied in plants, but it is less efficient for HDR-mediated gene editing. Therefore, to address these issues virus-induced genome editing (VIGE) involves plant virus-derived vectors for fast and efficient delivery of sgRNAs (via the HDR mechanism provides an innovative breeding method in tomato.f sgRNAs and has f sgRNAs . HR-basePetromyzon marinus cytidine deaminase1 (PmCDA1) fused with nuclease-deficient Cas9 (dCas9) or nCas9 (nickase Cas9 has single-strand DNA cleavage activity). Target-AID using dCas9 results in highly efficient and accurate substitution of C to T, whereas the use of nCas9 causes base substitution and insertion-deletion in the target sites at high efficiency to introduce point mutations (C to T or A to G) without any off-target , or A/T to G/C , but no CRISPR system has been reported until now for transversion base changes in the plant . The ABEf-target . Third, f-target . The HKIf-target . Thus, aCRISPR/Cas9 genome editing has revolutionized crop breeding, including tomatoes. CRISPR/Cas9 is the most commonly used genome editing system for precise, efficient, easy, versatile, cost-effective, and targeted gene editing at the desired genomic regions. After the first report in 2013, this technology has been used extensively to edit tomato genotypes. The successful examples of the application of CRISPR/Cas9 genome editing in tomatoes are summarized in SP (Self pruning) gene, a 3-fold increase in fruit size by the gene FAS (fasciated), an oval shape the ovate (O) gene, a 10-fold increase in fruit number by the gene MULT (multiflora), an increased fruit weight by the gene FW2.2 (fruit weight), and a very high lycopene content that has been improved by 500% (CYC-B/CycB). Moreover, many other traits have also been edited such as male sterility for hybrid seed production via co-knockouts of the Ms1035 , GSTAA (Glutathione S-Transferase) genes (J2 (Jointless-2) gene for mechanical harvesting genes , J2 gene encoding the MADS-box transcription factor in fruit ripening is well known, as evidenced by various researchers (SGR1 (Stay-Green1), LCY-E (Lycopene e-cyclase), Blc (Beta-lycopene someri), and LCY-B1/LCY-B2 (Lycopene b-cyclase 1/2) have been illustrated in the carotenoid metabolic pathways by using CRISPR/Cas9 gene knockout mechanism. Lycopene content in the mutants was increased by about 5.1-fold along with other advantages such as high efficiency, rare off-target mutations, and stable heredity (CYC-B/CycB (Lycopene beta someri) gene-mediated Cas9 editing increased the lycopene content in tomato (AGL6 (Agamous-like 6) gene resulted in parthenocarpic fruit development under heat stress conditions in tomato (GAD2/3 (Glutamate Decarboxylase 2/3) genes genes . High toTy-5) and powdery mildew diseases were developed via Cas9 editing of the Pelo and Mlo1 genes (via CRISPR/Cas9 editing of DMR6-1 (Downy mildew resistance 6-1) for P. syringae, P. capsici, and Xanthomonas spp. (Phytophthora infestans) resistant mutants were also developed by CRISPR/Cas9 editing of the MYBS2 (MYB transcription factor S2) gene (Tomato is affected seriously by various biotic (disease and insect-pest) and abiotic stresses. To overcome these problems, in the recent years many traits have been edited by CRISPR/Cas9 tools Table\u00a01.o1 genes . Furthernas spp. . They shcid (SA) . Late blS2) gene .CPK28 gene targeting APX2 (Ascorbate peroxidase 2) improved thermotolerance in tomato (GRXS14/15/16/17 (CGFS-type glutaredoxin 14/15/16/17) genes showed increased tolerance to multiple abiotic stresses such as heat, chilling, drought, heavy metal toxicity, and nutrient deficiency , RAD51/54 (DNA repair and recombination protein 51/54) (PR-1 (Pathogenesis-related protein 1) , and PR-otein 1) . Taken tvia the induction of double nickage mutants or truncation of the gRNA. It is also important to reduce the problems caused by the low frequency of HDR in plants. Third, multiplex editing is an efficient strategy for the simultaneous dissection of multiple genes, proteins, and metabolites at specific precision. Fourth, problems are associated with the selection of a large number of bases, which is possible with HKI. Fifth, dealing with in-planta transformation issues in Agrobacterium-mediated with diverse PAM sequence specificities and base or prime editing. Second, off-target mutants are reduced by increasing the specificity of Cas9 cleavage mediated or nanopmediated transforvia base editing, prime editing, and HKI. Allele introgression via targeted mutagenesis, as well as base editing and HKI in tomato, has become easier and less time-consuming. The discovery of programmable Cas9 variant and Cas12a nucleases has widened more scope without any off-targets. The use of a multiplexing approach with a robust CRISPR/Cas9 array having single or multiple sgRNAs or RNPs targeting conserved sequences of target genes and protoplast-mediated transformation could be deployed for precision editing. Homozygous edited lines can be obtained through haploid-inducer-based genome editing. Cas9 components can be removed via genetic segregation, and transgene-free genome-edited mutants can be obtained to meet regulatory requirements. More importantly, public awareness is necessary for the popularity of genome-edited plants, which do not contain any foreign genes. Hence, the regulatory process needs to be harmonized in public for genome-edited mutants. There is no concern about biosafety regulation in genome-edited plants because they are free of foreign genes, particularly site-directed nucleases (SDN1 and SDN2), and the vector backbone is removed through genetic segregation via backcrossing. The United States Department of Agriculture provides an exemption for these genome-edited crops, e.g., mushroom and corn; on the contrary, the Court of Justice of the European Union considered genome-edited plants under the same transgenic category. Further, the USA has released six virus-resistant genome-edited tomatoes. Genome-edited plants could be accepted as transgene-free (non-GM) by the public and regulatory bodies, which would open more avenues for the application of CRISPR/Cas in tomatoes. Taken together, there is a tremendous potential to deploy CRISPR/Cas9 for trait modifications in tomato.Innovations in Cas9 offer precise editing of targeted genes and even simultaneous editing of multiple genes by multiplexing to accelerate breeding at reduced costs. Diversity in CRISPR/Cas toolbox is required to mediate the catalytic activities of CRISPR/Cas along with environmentally friendly protocols for efficient delivery of Cas9. New CRISPR/Cas tools have created more efficient and precise editing JT: conceptualized and wrote the manuscript; AS and TB critically edited the manuscript. All authors contributed to the article and approved the submitted version."} +{"text": "N-glycans on LEs can be phosphorylated in the Golgi apparatus through two-step enzyme reactions. The consequent M6P moiety is recognized by M6P receptors (MPRs) on the trans-Golgi network membrane and delivered through the endo-lysosomal pathway. On the other hand, secreted LEs containing M6P glycans can be recaptured by MPRs on the plasma membrane and targeted to the lysosome. Enzyme replacement therapy (ERT) for lysosomal storage diseases exploits this M6P-MPR-dependent endocytosis to deliver recombinant enzymes to lysosomes. This review discusses various engineering and application technologies using M6P\u2019s lysosomal targeting. Glyco-engineering for increasing M6P contents developed \u2018Bio-better\u2019 ERT enzymes with enhanced therapeutic efficacy. M6P-decorated peptides, proteins, liposomes, and nanoparticles have been developed for drug delivery and subcellular imaging. A recently developed lysosome-targeting chimera uses an M6P-based bifunctional binder to degrade specific extracellular and membrane proteins. The success and efficiency of M6P-based lysosomal targeting will boost further technological developments with new applications in the biomedical field.A lysosome, an acidic membrane-bound organelle, contains hydrolytic enzymes to digest macromolecules for recycling. Many lysosomal enzymes (LEs) traffic to the lysosome through the mannose-6-phosphate (M6P)-dependent pathway. Some mannose residues of high-mannose type Secreted LEs can then be recaptured by MPRs located on the plasma membrane and delivered to lysosomes through MPR-mediated endocytosis produced in the ER are trimmed to Man5GlcNAc2 glycans in the cis-Golgi for further processing into complex glycans. On the other hand, some high-mannose glycans (Man6-9GlcNAc2) on LEs are recognized in a conformation-dependent manner by GlcNAc-1-phosphotransferase (GlcNAc-PT) structure in the TGNs (Bao et cNAc-PT) . GlcNAc-After M6P glycan formation, LEs are recognized by MPRs at the TGN membrane. MPRs are classified as cation-independent (CI)-MPR or cation-dependent (CD)-MPR depending on the requirement of divalent cations for M6P binding Kornfeld . CI-MPR,Some fractions of LEs that escape binding to MPRs in the TGN are secreted to the extracellular spaces. These LEs can be recaptured by a small population (\u223c10%) of CI-MPR existing at the plasma membrane through clathrin-mediated endocytosis Braulke; Oh 2015Although more than 50 LSDs have been discovered, only 11 LSDs have been treated with 23 FDA-approved therapeutics, including 15 ERTs, as of May 2019 mouse experiments, possibly because of a higher M6P glycan level deficiency, which results in the accumulation of globotriaosylceramide (Gb3) and lysoGb3 in the lysosome. Two approved recombinant human GLAs (rhGLAs), agalsidase alfa and agalsidase beta, are produced from human fibroblasts and Chinese hamster ovary (CHO) cells, respectively deficiency, accumulates glycogen in the lysosome, damaging muscle and nerve cells throughout the body. Recombinant human GAA (rhGAA), an algucosidase alfa, was approved by two brand names, Myozyme and Lumizyme, owing to the scale-up problem mice because the unreacted high-mannose type N-glycans provoked clearance by binding mannose-binding lectins and mannose receptors was synthesized and conjugated to rhGAA to generate neo-rhGAA . Avalglucosidase alfa-ngpt containing a 15-fold increased M6P content improved cellular uptake and enhanced glycogen clearance in target tissues.Sanofi Genzyme researchers developed a series of chemical conjugation strategies to increase the M6P content. First, they modified M6P glycans isolated from rhGLA and conjugated them to periodate-oxidized sialic acids of rhGAA , resembling \u2018GlcNAc-1-phosphate-6O-mannose\u2019 in mammals, can be converted to M6P (phosphate-6O-mannose) by removing the outer mannose residue , which showed greatly enhanced efficacy in Pompe mice to develop an efficient enzymatic process for M6P glycan generation technologies have attracted attention since proteolysis-targeting chimera (PROTAC) showed great promise by degrading \u2018undruggable\u2019 target proteins (Verma et al. LYTAC consists of a target protein binder and glycopeptide containing CI-MPR ligands (Banik et al. M6P glycan\u2019s crucial role in lysosomal targeting has been elucidated in studies on lysosome dysfunction. Therefore, many engineering and application technologies have initially focused on ERT to develop \u2018Bio-better\u2019 therapeutics for LSDs. The success of M6P-based lysosomal targeting in ERT has boosted the development of other technologies. M6P-decorated proteins, liposomes, and nanoparticles have been developed to deliver drugs selectively to tumors that overexpress CI-MPRs. In particular, the molecules that destroy the membrane integrity of the lysosome showed synergistic anti-cancer effects because they could induce LMP with cathepsin leakage and subsequent apoptosis. The recent development of LYTAC has opened up promising possibilities for generating next-generation therapeutics. Existing TPD technologies, including PROTACs, are limited in that their targets are restricted to cytosolic proteins. LYTAC has been shown to degrade extracellular and membrane proteins, which is also different from target-binding therapeutics, including monoclonal antibodies. The LYTAC technology can be employed to upgrade and repurpose antibodies that have not obtained FDA approval. The strategy using M6P-based lysosomal targeting will provide new applications, especially in the biomedical field."} +{"text": "Group Name: CDC Prevention Epicenters Program Background: Reverse-transcriptase polymerase chain reaction (RT-PCR) tests are the reference standard for diagnosing SARS-CoV-2 infection, but false positives can occur and viral RNA may persist for weeks-to-months following recovery. Isolating such patients increases pressure on limited hospital resources and may impede care. Therefore, we quantified the percentage of patients who tested positive by RT-PCR yet were unlikely to be infectious and could be released from isolation. Methods: We prospectively identified all adults hospitalized at Brigham and Women\u2019s Hospital who tested positive for SARS-CoV-2 by RT-PCR (primarily Hologic Panther Fusion or Cepheid Xpert platforms) between December 24, 2020, and January 24, 2021. Each case was assessed by infection control staff for possible discontinuation of isolation using an algorithm that incorporated the patient\u2019s prior history of COVID-19, current symptoms, RT-PCR cycle threshold (Ct) values, repeat RT-PCR testing at least 24 hours later, and SARS-CoV-2 serologies tested positive for SARS-CoV-2 by RT-PCR during the study period. Of these, 201 (81.7%) were deemed new diagnoses of active disease on the basis of low Ct values and/or progressive symptoms. Moreover, 44 patients (17.9%) were deemed noninfectious: 35 (14.2%) had prior known resolved infections (n = 21) or unknown prior infection but positive serology (n = 14), high Ct values on initial testing, and negative or stably high Ct values on repeat testing. Also, 5 (2.0%) had recent infection but >10 days had passed since symptom onset and they were clinically improving. In addition, 4 (1.6%) results were deemed false positives based on lack of symptoms and at least 1 negative repeat RT-PCR test (Figure Conclusions: During the winter COVID-19 second surge in Massachusetts, nearly 1 in 5 hospitalized patients who tested positive for SARS-CoV-2 by RT-PCR were deemed noninfectious and eligible for discontinuation of precautions. Most of these cases were consistent with residual RNA from prior known or undiagnosed infections. Active assessments of SARS-CoV-2 RT-PCR tests by infection control practitioners using clinical data, Ct values, repeat tests, and serologies can safely validate the release many patients from isolation and thereby conserve resources and facilitate patient care.Funding: NoDisclosures: None"} +{"text": "Vipera Aspis Francisciredi) (PanelA), complaining of vomiting, and mouth\u2019s dryness. The first electocardiogram (ECG) was unremarkable. However, during observation patient developed five episodes of ventricular fibrillation (VT), treated with Direct-Current Defibrillation (DC) shock and Advanced Cardiovascular Life Support (ACLS). Repeated ECG demonstrated anterolateral ST-elevation myocardial infarction (MI) (Panel B). In 2018, for an acute coronary syndrome, he underwent everolimus-eluting-stent (EES) implantation on the left anterior descending artery (LAD) and first diagonal (D1) branch with a T-modified-stent-technique, and he was discharged with ticagrelor 180 mg/day for 1 year.A 60-year-old male was referred to the emergency department after a European snake bite (Panel D) which was satisfactorily treated with mechanical thrombectomy and kissing balloon inflations with non-compliant balloons (Panel A). Intracoronary imaging was not performed to assess the potential mechanisms underlying ST due to refractory VT episodes. The patient was treated with bolus and infusion and integrilin. Ticagrelor 90 mg/b.i.d. followed by 60 mg/b.i.d. after 1 year plus aspirin was planned. He was treated with anti-snake venom (ANTYTOKSYNA JADU ZMIJ) and other supportive measures. The subsequent course was uneventful. The patient developed massive oedema of the limb point, eyelid ptosis associated with weakness of the musculature associated with chewing, with progressive improvement.Coronary angiograms revealed a very late EES thrombosis in mid-LAD-D1 shows a dissected European viper, Vipera Aspis Francisciredi, killed by the patient after being bitten; (Panel B) repeated ECG after the admission demonstrates ST-elevation anterolateral myocardial infarction (STEMI); (Panel C) coronary angiography performed during STEMI illustrates the angiographic appearance of a thrombus (arrow) causing very late stent thrombosis on drug-eluting-stent on the left anterior descending artery (LAD) and first diagonal branch (D1) LAD-D1 bifurcation; (Panel D) satisfactory final results after primary percutaneous coronary intervention (PCI) and thrombus aspiration of the infarct-related artery;("} +{"text": "Background: Diagnostic tests for COVID-19 are in high demand. Serologic assays are of interest as diagnostic adjuncts to SARS-CoV-2 quantitative polymerase chain reaction (PCR); however, many of the commercially available assays have limited validation data and clinical utility is unknown. We describe the utilization of SARS-CoV-2 IgG enzyme-linked immunosorbent assay (ELISA) for healthcare workers with acute respiratory tract infection (ARTI) who underwent SARS-CoV-2 PCR testing. Methods: The MetroHealth System is the largest public hospital system in Ohio, employing ~8,000 staff. COVID-19 detection began in early March 2020. EDI novel coronavirus COVID-19 IgG ELISA (KT-1032) targeting antibody response to viral nucleocapsid was obtained for diagnostic and seroprevalence analyses. Manufacturer reports of sensitivity and specificity of the assay are 100% and 99%, respectively. A 2-part test strategy for employees with symptoms of ARTI was implemented. Qualifying symptoms for SARS-CoV-2 PCR testing included fever and either cough or shortness of breath. Additional symptoms were included to reflect expanding knowledge of COVID-19. Employees who underwent SARS-CoV-2 PCR testing (Luminex ARIES) were offered serologic testing on day 14 following PCR result. Education accompanied the offer for serologic testing as well as the receipt of test result to aide interpretation. Results: From April 16, 2020, through July 6, 2020, 588 employees underwent PCR testing. Overall, 70 cases of COVID-19 were detected. Of the 197 employees who opted for serologic testing, IgG positivity was 12.6%. The mean time to IgG collection following PCR result was 30 days . Using PCR results obtained in the clinical setting of ARTI as the diagnostic gold standard, IgG was 84.6% sensitive and 98.2% specific , a positive SARS-CoV-2 IgG has a low positive predictive value, which may falsely imply immunity and may negatively affect infection prevention practices.Funding: NoDisclosures: None"} +{"text": "Introduction: Cutaneous melanoma remains a leading cancer with sobering post-metastasis mortality rates. To date, the ligand-receptor interactome of melanomas remains weakly studied despite applicability to anti-cancer drug discovery. Here we leverage established crosstalk methodologies to characterize important ligand-receptor pairs in primary and metastatic cutaneous melanoma. Methods: Bulk transcriptomic data, representing 470 cutaneous melanoma samples, was retrieved from the Broad Genome Data Analysis Center Firehose portal. Tumor and stroma compartments were computationally derived as a function of tumor purity estimates. Identification of preferential ligand-receptor interactions was achieved by relative crosstalk scoring of 1380 previously established pairs. Results: Metastatic cutaneous melanoma uniquely enriched PTH2-PTH1R for tumor-to-stroma signaling. The Human R-spondin ligand family was involved in 4 of the 15 top-scoring stroma-to-tumor interactions. Receptor ACVR2B was involved in 3 of the 15 top-scoring tumor-to-tumor interactions. Conclusions: Numerous gene-level differences in ligand-receptor crosstalk between primary and metastatic cutaneous melanomas. Further investigation of notable pairings is warranted. Cutaneous melanoma (CM) remains one of the most aggressive and deadliest forms of skin cancer, with epidemiological studies depicting the last few decades witnessing dramatic increases in incidences worldwide, with over 200,000 new cases diagnosed each year ,2. HowevBulk RNA sequencing (RNA-seq) provides extensive utilities for the classification of cancer, identification of biomarkers, disease diagnosis, and the optimization of treatments . In the The cellular composition of tumors consists of highly heterogenous tumor cells due to somatic genetic alterations and cells within the tumor microenvironment that results from the infiltration of the stroma and other immune cell types . The spahttps://gdac.broadinstitute.org/, Accessed on 4 October 2022) (N = 470). These data are represented by The Cancer Genome Atlas (TCGA) effort.Expression data. Bulk transcriptome data for primary and metastatic cutaneous melanomas were retrieved from the Broad GDAC Firehose portal compartmental gene expression was expressed as previously described . For a giTumor and stroma compartment expression levels, defined in the above equation, were obtained via negative least-squares regression, with the assumption that these average compartment expression levels are constant across tumor samples. 95% confidence intervals for tumor and stroma point estimates were derived via bootstrapping.Ligand-receptor interaction scoring. Compartmental expression values were annotated using a combined set of 1380 ligand-receptor pairs ,17. ApplIf the following conditions are assumed: (1) the inferred mRNA expression values are reasonable proxies for ligand and receptor concentrations, (2) ligand-receptor kinetics are constant across all samples, and (3) the law of mass action assumptions are met, then the following relative crosstalk (RC) score equation can be applied. Here, the numerator represents the ligand-receptor complex of interest, and the denominator represents all possible ligand-receptor complexes . Example given below for tumor-stroma ligand-receptor interaction scoring.Cancelling out the dissociation constant, the relative crosstalk score for a specified tumor ligand and stromal receptor pair, can be modeled by the following final equation:Software. All analyses were conducted with Python (v3.10.7) and R (v.4.2.2). Initial data wrangling and figure creation was carried out with R (v.4.2.2).The highest-scoring ligand-receptor interactions in primary and metastatic lesions were evaluated for each signaling direction . In tumoLigand-receptor pairs with a preference for stroma-to-tumor signaling include BMP10-ACVR2B, FGF16-FGFR4, FGF5-FGFR4, INSL3-RXFP2, RLN3-RXFP2, RSPO1-LRP6, RSPO3-LGR4, and RSPO3-LRP6 . These sAmong the highest-scoring tumor-to-tumor interactions, EFNA2-EPHA6 was decisive in both primary and metastatic lesions . HoweverChemokine-receptor and interleukin-receptor pairs scored highly among stroma-to-stroma signaling . These iFGF ligand-receptor interactions were implicated across multiple signaling directions in cutaneous melanoma. Previously, FGF signaling dysregulation has been identified across many types of cancer, although the underlying mechanism is not well understood . HoweverIn our study, RSPO1 and RSPO3 were identified as important stroma-to-tumor ligands, each interacting with both the LGR4 and LRP6 receptors in cutaneous melanoma. The role of RSPO proteins in cancer has been largely derived from studies in the intestinal tract . HoweverIn our study, EFNA2-EPHA interactions scored highly among the tumor-to-tumor and tumor-to-stroma signaling directions. While dysregulation of EFN signaling has been shown to increase susceptibility to skin carcinogenesis , EFN-EPHIn the tumor-to-tumor signaling direction, activin receptor type-2B (ACVR2B) was implicated in three of the fifteen top-scoring interactions with corresponding ligands of bone morphogenetic protein 5 (BMP5), growth differentiation factor 11 (GDF11), and inhibin beta C chain (INHBC). In the stroma-to-tumor signaling pathway, BMP5-ACVR2B signaling was highlighted as well. All three ligands belong to the transforming growth factor-beta (TGF-\u03b2) superfamily, in which increased expression has been associated with cancer cachexia . BarretoOur results further demonstrated stroma-to-stroma signaling as predominantly characterized by interleukin (IL) and chemokine signaling. This ligand-receptor signaling architecture has practical applications in melanoma treatment. Cytokines, including the interleukin IL-2 and IL-21 ligands identified in cutaneous melanoma, regulate innate and adaptive immunity . High-doA primary limitation of the present analysis is the use of average gene expression for each compartment and treatment as constant across CM samples. It is well understood that tumor and stromal cell populations are not evenly distributed in space, thereby rendering the CM microenvironment highly variable from sample to sample. Additional limitations include the lack of both a replicative dataset and association with clinical parameters . Still, this semi-supervised approach valuably identified numerous molecular pairs eligible for deeper investigation, potentially proposing novel therapeutic targets with specificity for the melanoma disease course.Cutaneous melanoma continues to rank among the deadliest cancers, yet efforts to analyze key ligand-receptor interactions remain paltry at the time of writing. In the present study, we reported a first-use case of crosstalk scoring for transcriptome data derived from primary and metastatic CM samples. Notable findings include the following: enrichment for tumor ligand PTH2 interactions with stromal receptor PTH1R in metastatic lesions; enrichment for human R-spondin family proteins in stroma-to-tumor signaling; enrichment for tumor ligand EFNA2 interactions with stromal receptor EPHA6; enrichment for chemokine and interleukin stromal receptors interacting with stromal ligands. These data offer novel protein targets of interest and support genomic change in the post-metastasis CM microenvironment. Future interactome efforts should aim to build onto this work with greater power by incorporating microarray and single cell expression datasets."} +{"text": "To differentiate IDH-mutant grade 4 astrocytomas from IDH-wild-type glioblastomas, two MRI sequences (post-contrast T1 and T2-FLAIR) were acquired from 57 patients. The images were resliced, resampled, and realigned. In the next step, tumors were segmented semi-automatically into subregions including whole tumor, edema region, core tumor, enhancing region, and necrotic region. A total of 105 radiomic features were extracted from each subregion. The data were divided randomly into training and testing sets. A deep learning-based data augmentation method (CTGAN) was implemented to synthesize 200 datasets. A total of 18 classifiers were used to distinguish two genotypes of grade 4 astrocytomas. The best discriminatory power was obtained from core tumor regions overlaid on post-contrast T1 using the K-best feature selection algorithm and a Gaussian na\u00efve Bayes classifier.n = 23) and IDH-wild-type GBMs (n = 34) underwent anatomical imaging on a 3T MR system with standard parameters. Post-contrast T1-weighted and T2-FLAIR images were co-registered. A semi-automatic segmentation approach was used to generate regions of interest (ROIs) from different tissue components of neoplasms. A total of 1050 radiomic features were extracted from each image. The data were split randomly into training and testing sets. A deep learning-based data augmentation method (CTGAN) was implemented to synthesize 200 datasets from the training sets. A total of 18 classifiers were used to distinguish two genotypes of grade 4 astrocytomas. From generated data using 80% training set, the best discriminatory power was obtained from core tumor regions overlaid on post-contrast T1 using the K-best feature selection algorithm and a Gaussian na\u00efve Bayes classifier . Similarly, high diagnostic performances were obtained from original and generated data using 50% and 30% training sets. Our findings suggest that conventional MR imaging-based radiomic features combined with machine/deep learning methods may be valuable in discriminating IDH-mutant grade 4 astrocytomas from IDH-wild-type GBMs.This study aimed to investigate the potential of quantitative radiomic data extracted from conventional MR images in discriminating IDH-mutant grade 4 astrocytomas from IDH-wild-type glioblastomas (GBMs). A cohort of 57 treatment-na\u00efve patients with IDH-mutant grade 4 astrocytomas ( Glioblastomas (GBMs) are devastating and universally fatal brain cancers in adults despite advancements in diagnostic and therapeutic strategies . ApproxiTherefore, non-invasive identification of IDH-mutant gliomas is vital for making informed decisions on therapeutic intervention and prognosticating these patients. IDH mutations confer the neomorphic activity of an enzyme leading to the conversion of alpha-ketoglutarate (\u03b1-KG) to 2-hydroxyglutarate (2HG) . Prior sn = 31) ; in-plane resolution = 1 \u00d7 1 mmThe overview of the image processing and radiomics pipeline, which includes image registration, tissue segmentation, feature extraction, feature selection, and radiomics model building, is shown in From each segmented ROI, 105 original radiomic features from categories were extracted using the PyRadiomics package in python . These oIt is important to eliminate irrelevant or redundant variables that may cause data overfitting and may bias the performance of the prediction model. Multiple feature selection algorithms, including recursive feature elimination (RFE), minimum redundancy, maximum relevance (mRmR), and K-best were employed to select image features. Patients were divided into 2 mutually exclusive training and testing sets using the random shuffling method. Ten percent of the training set was split off to serve as the validation set. All data were normalized by MinMax normalization. The mRmR feature selection technique was used to select 15 features.The current study implemented a deep learning method based on generative adversarial networks (GAN) for data augmentation . CTGAN iTo develop a prediction model for distinguishing IDH-mutant grade 4 astrocytomas from IDH-wild-type GBMs, a total of 18 single and ensembled machine learning classifiers [Bernoulli na\u00efve Bayes (BNB), multilayer perceptron (MLP), support vector classifier (SVC), Gaussian na\u00efve Bayes (GNB), quadratic discriminant analysis (QDA), bagging classifier, linear discriminant analysis (LDA), logistic regression (RG), ridge, ada boost (AD), hist gradient boosting (HGB), K-neighbors (KN) (K = 5), random forest (RF), gradient boosting (GB), extra trees (ET), decision tree (DT), nearest centroid (NC), and passive aggressive were used to train the classifiers, and an internal validation was performed from the testing cohort . Receiver operative characteristic (ROC) curve analyses were performed to evaluate the diagnostic potentials of prediction models in distinguishing 2 groups (IDH-mutant grade 4 astrocytomas and IDH-wild-type GBMs). Area under the ROC curve (AUC), area under the precision-recall curve (PR_AUC), accuracy (ACC), sensitivity, specificity, and negative and positive predictive values were determined for each prediction model as performance metrics.n = 57) were used in discriminating IDH-mutant grade 4 astrocytomas from IDH-wild-type GBMs, the best discriminatory performance was obtained from solid/contrast enhancing, and core tumor (solid + necrotic region) overlaid on post-contrast T1-weighted images using various combinations of feature selection algorithms and machine learning classifiers. The predictive power, accuracy, sensitivity, specificity, and PR_AUC of the best 10 methods in distinguishing two genotypes of grade 4 astrocytomas are summarized in When original MRI data , predictive accuracy (ACC), sensitivity (SEN), and specificity (SPE) for discriminating IDH-mutant grade 4 astrocytomas from IDH-wild-type GBMs utilizing a variety of feature selections , and machine learning algorithms applied to distinct subregions of neoplasms, are shown in n = 18) to build a reliable prediction model in distinguishing IDH-mutant grade 4 astrocytomas and IDH-wild-type GBMs. In the testing cohort, our best prediction model consisted of a central necrotic region from post-contrast, T1-weighted images when a combination of the K-best feature selection algorithm and Gaussian na\u00efve Bayes classifier were used together. This prediction model achieved a high diagnostic performance in discriminating two genotypes of grade 4 astrocytomas.In this study, we investigated the clinical utility of a conventional neuroimaging-based radiomics approach with deep learning in determining the IDH status of grade 4 astrocytomas. A total of 1050 radiomic features were extracted from different tumor habitats , encompassing post-contrast T1-weighted and T2-FLAIR images. Our work is an extension of previous studies as we used a GAN-based algorithm to increase our sample size and used a large number of machine learning classifiers ,8. MoreoMechanistically, wild-type IDH normally catalyzes the reversible, NADP+-dependent oxidative decarboxylation of isocitrate to alpha-ketoglutarate (\u03b1-KG) in the TCA cycle. However, IDH mutations confer a neomorphic enzyme activity converting \u03b1-KG to 2HG. Therefore, the oncometabolite 2HG has been proposed as a putative biomarker for IDH-specific genetic profiles for gliomas. A few studies have employed modified spectroscopic sequences and post-processing tools for detecting spectral resonances of 2HG from IDH-mutant gliomas ,46,47,48Radiomics is a quantitative analytical method of medical images that provides information that is generally difficult to perceive by visual inspection. Compared with conventional analytical approaches, radiomic analysis can provide a more efficient and unbiased quantification of imaging information. Readily interpretable and quantitative features, such as intensity distributions, spatial relationships, textural heterogeneity, and shape descriptors are extracted from a pre-defined ROI encompassing both solid and peritumoral regions of neoplasms in a typical fashion . The traIDH mutation occurs only in 10% of grade 4 astrocytomas, so we could only include data from 23 IDH-mutant cases in the present study. Due to this small sample size and imbalance in data distribution, our data was prone to overfitting. Furthermore, in situations with an insufficient number of training datasets, the model is often overtrained. Consequently, the model performs well during the training stage but comparatively poorly during the subsequent testing stage. To address this challenge of small sample size, we leveraged the use of a well-established GAN method for synthesizing high-quality images and, in turn, raised the total sample size from 57 to 200. GAN is a deep learning architecture in which two neural networks compete against each other in a zero-sum game framework . A GAN mIn a previous study , IDH mutIn conclusion, a prediction model based on conventional MRI-extracted radiomic features achieved promising diagnostic power in distinguishing IDH-mutant grade 4 astrocytomas from IDH-wild-type GBMs."} +{"text": "Scientific Reports 10.1038/s41598-022-11492-2, published online 07 May 2022Correction to: The original version of this Article contained an error in the Methods section. The IRB number was incorrect.\u201cThis study was approved by the institutional review board (IRB) of Seoul National University Hospital (IRB No. 2001-139-1096).\u201dnow reads:\u201cThis study was approved by the institutional review board (IRB) of Seoul National University Hospital (IRB No. 1912-099-1089).\u201dThe original Article has been corrected."} +{"text": "Edis (Ect4-derived immune suppressor) plays an essential role in neuronal development in Drosophila. We show that depletion of Edis in vivo causes defects in axonal projection patterns of mushroom body (MB) neurons in the brain, as well as impaired locomotor activity and shortened lifespan of adult flies. In addition, we find that the castor gene, which encodes a transcription factor involved in neurodevelopment, is upregulated in Edis knockdown neurons. Notably, castor overexpression phenocopies Edis knockdown, and reducing castor levels suppresses the neurodevelopmental phenotypes in Edis-depleted neurons. Furthermore, chromatin immunoprecipitation analysis reveals that the transcription factor Relish, which plays a key role in regulating innate immunity signaling, occupies a pair of sites at the castor promoter, and that both sites are required for optimal castor gene activation by either immune challenge or Edis depletion. Lastly, Relish mutation and/or depletion can rescue both the castor gene hyperactivation phenotype and neuronal defects in Edis knockdown animals. We conclude that the circular RNA Edis acts through Relish and castor to regulate neuronal development.Circular RNAs (circRNAs) are a new group of noncoding/regulatory RNAs that are particularly abundant in the nervous system, however, their physiological functions are underexplored. Here we report that the brain-enriched circular RNA Edis (Ect4-derived immune suppressor) plays an essential role in neuronal development in the fruitfly Drosophila melanogaster, as its depletion causes defects in the development of neurons in a brain structure called mushroom body (MB), as well as impaired locomotor activity and shortened lifespan of adult flies. In addition, we show that the castor gene, which encodes a protein involved in neurodevelopment, is upregulated in Edis knockdown neurons. Notably, flies with increased levels of castor or reduced levels of Edis display similar phenotypes, and reducing castor levels rescues the developmental defects in Edis-depleted neurons. Furthermore, we find that the immune regulator Relish occupies a pair of sites at the castor gene locus, and that both sites are required for optimal castor gene activation by either immune challenge or Edis depletion. Lastly, Relish mutation and/or depletion can rescue both the castor gene hyperactivation phenotype and neuronal defects in Edis knockdown animals. We conclude that the circular RNA Edis acts through Relish and castor to regulate neuronal development.Circular RNAs (circRNAs) are a new group of noncoding/regulatory RNAs that are particularly abundant in the nervous system, although their physiological functions are underexplored. Here we report that the brain-enriched circular RNA Circular RNAs (circRNAs) are the latest addition to the noncoding and regulatory RNA collection. They are characterized as covalently closed RNA loops generated by \u201chead-to-tail\u201d back-splicing events ,2. With CDR1as binds to microRNA-7 (miR-7) and regulates miR-7 biogenesis, thereby impacting brain development ) were obtained. Gene with poor read counts in all samples were excluded from further analysis. The differences in gene expression between knockdown and GFP control conditions were assessed using BioConductor edgeR package [RNA-Seq datasets were generated from RNA samples extracted from (v2.0.4) with the package . The resS1 FigA-F\u2019) Fluorescence in situ hybridization (FISH) was employed to visualize Edis (red) in adult fly brains of control (Elav>shgfp) and Edis knockdown (Elav>shEdis) animals. Nuclei were marked by DAPI (blue). A and B, and D and E are split channels of C and F, respectively. A\u2019-C\u2019 and D\u2019-F\u2019 show high magnification images of boxed regions in A-C and D-F, respectively. The scale bars indicate 50 \u03bcm in A-F, and 10 \u03bcm in A\u2019-F\u2019. (G) UAS-laccase 2-Edis or UAS-laccase 2 vector (control) flies were crossed to Elav-Gal4 flies and MB morphology was revealed by anti-FasII antibody staining. (H) MB morphology phenotypes of indicated genotypes were quantified. Chi-squared test was employed in statistical analysis. Sample numbers in each genotype are shown. (I) Levels of Edis were measured by real time PCR (n = 3).((TIF)Click here for additional data file.S2 FigDptA, Dro, Def, Drs) in Elav>shEdis brain tissues and control samples were measured by quantitative PCR (n = 3).Levels mRNA encoding various AMPs ((TIF)Click here for additional data file.S3 FigA-B) Overexpression of castor in neurons led to MB morphology defects. UAS-castor-3XHA or UAS-mCD8GFP (control) flies were crossed to ok107-Gal4 flies. Brains of progeny of indicated genotypes were stained by anti-FasII antibody. The scale bar indicates 50 \u03bcm. (B) MB morphology phenotypes of indicated genotypes were quantified. Chi-squared test was employed in statistical analysis. Sample numbers and percentages of samples showing normal MB morphology in each genotype are shown. (C-D) The short lifespan phenotype and mobility defects elicited by Edis depletion can be rescued by reducing castor expression. Various combinations of UAS-shgfp, UAS-shEdis, UAS-shmcherry, and UAS-dscastor transgenes were crossed with Elav-Gal4 flies. Lifespan (C) and locomotor activity (D) of flies with indicated genotypes are shown.((TIF)Click here for additional data file.S4 FigA) RNA levels of genes encoding a series of temporal transcription factors in fly brain samples were measured by real-time PCR and normalized to control samples. grh was among the upregulated genes in Edis-depleted fly brain (n = 6). (B) Overexpression of grh in the MB neurons led to MB morphology defects. UAS-grh or UAS-mCD8GFP (control) flies were crossed to ok107-Gal4 flies. Brains of progeny of indicated genotypes were stained by anti-FasII antibody. MB morphology phenotypes of indicated genotypes were quantified. Chi-squared test was employed in statistical analysis. Sample numbers of each genotype are shown. (C) Various combinations of UAS-shgfp, UAS-shmcherry, UAS-shEdis and UAS-shgrh transgenes were crossed with Elav-Gal4 flies. Levels of Edis and grh transcripts in flies of the indicated genotypes were measured by quantitative PCR (n = 3). (D) Levels of Edis, Relish, and grh transcripts were measured in control Elav>shgfp or Elav>shEdis animals in wildtype, Relish heterozygous (RelE20/+ or RelE38/+) or homozygous (RelE20/E38) mutant background (n = 3). Levels of grh mRNA in Elav>shEdis flies are significantly lower in Relish homozygous (RelE20/E38) compared with Relish heterozygous or wildtype background.((TIF)Click here for additional data file.S5 FigA) Integrative genomics viewer (IGV) plot of Rel-N-GFP ChIP sequence result showing the Relish peaks at the promoter region of castor. (B-C) RelN overexpression in MB neurons led to MB morphology defects. UAS-Flag-RelN or UAS-mCD8GFP (control) flies were crossed to Elav-Gal4 flies. Brains of progeny of indicated genotypes were stained by anti-FasII antibody (B). The scale bar indicates 50 \u03bcm. MB morphology phenotypes of indicated genotypes were quantified in C. Chi-squared test was employed in statistical analysis. Sample numbers and percentages of samples showing normal MB morphology in each genotype are shown.((TIF)Click here for additional data file.S1 TableElav>shEdis or Elav>shGFP (control) fly heads. Differential expressed genes (DEGs) in knockdown vs Control were defined using cut-off criteria of absolute fold change of > = 2.0 and p value of = < 0.05.RNA-seq analysis was performed using RNA samples extracted from (XLSX)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.S2 Data(RAR)Click here for additional data file.S1 Information(DOCX)Click here for additional data file."} +{"text": "Vitis spp.) in Japan were determined.Two complete and three partial genome sequences of grapevine red globe virus (GRGV) from grapevines ( Maculavirus (family Tymoviridae) and has a genome consisting of approximately 6,850 nucleotides (nt) of single-stranded positive-sense RNA. GRGV has been reported from at least 14 countries and reported to cause no obvious symptoms on grapevines (Vitis spp.) (1Grapevine red globe virus (GRGV) is a tentative member of the genus s spp.) 15. FourteTo verify whether commercial grapevine nursery stocks in Japan were infected with GRGV, total RNA was extracted from petioles of 32 stocks as described by Iandolino et al. using 3-GRGV genomes were amplified by RT-PCR, using the primer pairs described in https://www.ncbi.nlm.nih.gov/orffinder/). ORF1 encoded a putative 220-kDa replicase polyprotein, and ORF2 encoded a putative 24.9-kDa coat protein (CP). The partial sequences of JP-YS2, JP-Ch, and JP-PN were also predicted to contain the full ORF2. A phylogenetic analysis of the CP showed that the Japanese viruses formed a monophyletic group with all known GRGVs tail. Their structures were identical, and two putative GRGV open reading frames (ORFs) were predicted by NCBI ORF Finder , LC704875 (JP-PN), LC704876 (JP-YS1), LC704877 (JP-YS2), and LC704878 (JP-YS3).The viral genome sequences have been deposited in the DDBJ under accession numbers"} +{"text": "Severe COVID-19 survivors experience long-term neuropsychiatric morbidity, particularly those who developed delirium, with a negative impact on health-related quality of life (HRQoL).To identify the cases of delirium in severe COVID-19 patients and to describe its association with post-hospital discharge HRQoL.In the context of the longitudinal MAPA project, we included adult patients (\u2265 18 years old) admitted with COVID-19 to the Intensive Care Medicine Department (ICMD) of a Portuguese University Hospital (October 2020-April 2021). Exclusion criteria were: ICMD length of stay \u226424h, terminal illness, major auditory loss, or inability to communicate at the time of assessment. Delirium during ICMD stay was ascertained based on patients\u2019 clinical records. HRQoL was evaluated using the 5-Level EQ-5D questionnaire (EQ-5D-5L), at a scheduled telephone follow-up appointment on average 1-2 months after hospital discharge.Overall, 124 patients were included with a median age of 62 (range: 24-86) years, being mostly male (65%). About 19% had delirium, 42% were deeply sedated and 43% required invasive mechanical ventilation. Most survivors reported problems on the EQ-5D-5L domains: usual activities (85%), mobility (73%) and anxiety/depression (65%). Patients with delirium reported more pain/discomfort and considerably anxiety/depression .These findings pointed that COVID-19 patients who experienced delirium reported worse HRQoL, regarding pain/discomfort and anxiety/depression. This study highlights the importance of not only prevention but also early screening of delirium during hospital stay, as well as the crucial role of the timely interventions at discharge, in order to minimize delirium long-term impacts.No significant relationships."} +{"text": "Various genetic polymorphisms have been associated with attention-deficit/hyperactivity disorder (ADHD), and some of these have also been implicated in individual differences in affective processing. Yet, no studies to date have examined the complex interrelations across these genetic polymorphisms, ADHD, and affective processing. Several variables have been shown to affect whether a given genetic variant confers risk. Our aim was to examine whether relevant genetic variants differentially confer risk for negative affectivity (NA) and/or emotion dysregulation (ED), depending on ADHD status.n = 297 adolescents with (n = 83) and without (DSM-5) ADHD. ADHD- and affective processing-related dopaminergic and serotonergic polymorphisms were genotyped , dopamine receptor DRD4 exon-3 48 bp VNTR, and serotonin transporter linked polymorphic region 5-HTTLPR including the rs25531). Affectivity and ED were measured via parent- and/or self-report.Participants were r to z-transformed) values between with and without ADHD groups. There were no correlations that were significant \u2013 but several differed \u2013 across groups. In youth without ADHD, carrying the DRD2 rs1800497 T-allele was negatively associated both with negative affectivity (pcorr=.033) and with self-rated ED (pcorr=0.039). In youth with ADHD, carrying the DRD4 VNTR 7-repeat allele was positively associated with self-rated ED (pcorr=0.008), and carrying the L'L\u2019 relative to the low-expression S\u2019 serotonergic allele was also positively associated with parent-rated ED (pcorr=.042)We first calculated bivariate correlations between polymorphisms, affectivity, and ED then compared the obtained suggest these results may be robust."} +{"text": "Hand hygiene (HH) is one of the most important measures to prevent healthcare-associated infections (HAIs). Several indicators have been proposed to measure HH practices, including alcohol-based handrub consumption (AHC). The objective of this study was to evaluate whether AHC is associated with HAI transmission.All 25 public hospitals/trusts of Piedmont, in Northern Italy, are required to provide data each year concerning HAIs and infection prevention and control (IPC) activities, as part of a mandatory regional indicator system. Data on AHC and HAIs were extracted from reports provided concerning the years 2017-2019. The mean annual AHC for each facility was expressed as liters per patient-day. The rate of hospital-wide Methicillin-resistant Staphylococcus aureus (MRSA) and carbapenem-resistant Enterobacteriaceae (CRE) infections was calculated per 1000 patient-days (pd). Mean ventilation-associated infections (VAP) for 1000 days of ventilation (vd) and mean catheter-related bloodstream infections (CR-BSI) for 1000 days of catheterization (cd) were calculated for intensive care units (ICUs). Spearman's correlational analysis was conducted between AHC and HAIs: hospital-level, ICU-level.Hospital-level: mean AHC was 15.4 liters/pd; mean HAI rate was 39.6/1000 pd (MRSA) and 18.5/1000 pd (CRE). No significant correlation was found. ICU-level: mean AHC was 39.6 liters/pd; mean HAI rate was 1.7/1000 cd (CR-BSI) and 8.2/1000 vd (VAP). A moderate correlation was found between AHC and CR-BSI rate . Concerning AHC and VAP, higher AHC was reported from facilities with lower VAP rates, however no significant correlation was found .Results of this study support the validity of AHC as an indicator of IPC practices in ICUs. The hospital-level analysis could have been affected by important differences in AHC among ward types.Results of this study support the association between higher AHC and reduced HAI transmission in intensive care units.Stratifying data by ward type could provide further insight in the association between AHC and HAI rates."} +{"text": "The current literature demonstrated the superior diagnostic accuracy of this imaging modality, especially for lesions difficult to be detected or characterized on conventional imaging protocols, such as skull base or transosseous meningiomas. [68Ga]Ga-DOTA-SSTR PET tracers also seem to provide superior volume contouring for radiotherapy planning and may also be used to evaluate the tumor\u2019s overexpression of somatostatin receptors for devising patient-tailored peptide receptor radionuclide therapy. In this review, we comprehensively analyzed the current literature discussing the implementation of [68Ga]Ga-DOTA-SSTR PET imaging in brain tumors, further presenting ongoing clinical trials and suggesting potential future applications.[68Ga]Ga-DOTA-SSTR PET tracers has garnered interest in neuro-oncology, to increase accuracy in diagnostic, radiation planning, and neurotheranostics protocols. We systematically reviewed the literature on the current uses of [68Ga]Ga-DOTA-SSTR PET in brain tumors. Methods: PubMed, Scopus, Web of Science, and Cochrane were searched in accordance with the PRISMA guidelines to include published studies and ongoing trials utilizing [68Ga]Ga-DOTA-SSTR PET in patients with brain tumors. Results: We included 63 published studies comprising 1030 patients with 1277 lesions, and 4 ongoing trials. [68Ga]Ga-DOTA-SSTR PET was mostly used for diagnostic purposes (62.5%), followed by treatment planning (32.7%), and neurotheranostics (4.8%). Most lesions were meningiomas (93.6%), followed by pituitary adenomas (2.8%), and the DOTATOC tracer (53.2%) was used more frequently than DOTATATE (39.1%) and DOTANOC (5.7%), except for diagnostic purposes (DOTATATE 51.1%). [68Ga]Ga-DOTA-SSTR PET studies were mostly required to confirm the diagnosis of meningiomas (owing to their high SSTR2 expression and tracer uptake) or evaluate their extent of bone invasion, and improve volume contouring for better radiotherapy planning. Some studies reported the uncommon occurrence of SSTR2-positive brain pathology challenging the diagnostic accuracy of [68Ga]Ga-DOTA-SSTR PET for meningiomas. Pre-treatment assessment of tracer uptake rates has been used to confirm patient eligibility (high somatostatin receptor-2 expression) for peptide receptor radionuclide therapy (PRRT) for recurrent meningiomas and pituitary carcinomas. Conclusion: [68Ga]Ga-DOTA-SSTR PET studies may revolutionize the routine neuro-oncology practice, especially in meningiomas, by improving diagnostic accuracy, delineation of radiotherapy targets, and patient eligibility for radionuclide therapies.Background: The development of [ Articles were excluded if they: (1) were literature reviews, cadaver studies, animal studies, or study protocols, (2) described different uses of [68Ga]Ga-DOTA-SSTR PET imaging not for brain tumors, (3) reported the use of different molecular nuclear medicine imaging techniques. [68Ga]Ga-DOTA-SSTR PET imaging defined the use of [68Ga]Ga-DOTA-chelator PET studies specifically targeting tissues and/or lesions expressing somatostatin receptors (SSTR).A priori inclusion and exclusion criteria were defined. Articles written in English were included if they described the use of Ga-DOTA-SSTR PET studies, sample size, number of lesions, pathology and location, tracer and administered dose, SUV max, clinico-radiological findings.Two independent reviewers (G.W. and C.O.) extracted data, which were then confirmed by an additional reviewer (P.P.). Missing data were not reported by the authors. Extracted data included: authors, year, the reason for the use of [68Ga]Ga-DOTA-SSTR PET studies in patients with brain tumors and their impact on clinical practice. For each article, the level of evidence was appraised using the 2011 Oxford Centre For Evidence-Based Medicine guidelines Ga-DOTA-SSTR PET in patients with brain tumors. A total of 1030 patients with 1277 lesions were included. [68Ga]Ga-DOTA-SSTR PET studies were mostly implemented for diagnostic purposes in 644 patients (62.5%) with 867 lesions (67.9%), followed by radiotherapy/surgery planning in 337 patients (32.7%) with 356 lesions (27.9%), and by assessment of somatostatin receptor-2 (SSTR2) expression for peptide receptor radionuclide therapy (PRRT) in 49 patients (4.8%) with 54 lesions (4.2%). Meningioma comprised the most common pathology in all groups, followed by pituitary adenomas . DOTATOC was the most frequent tracer used overall , but DOTATATE was used more in patients undergoing diagnostic [68Ga]Ga-DOTA-SSTR PET studies . Purandare et al. [e et al. to deline et al. lacked a68Ga]Ga-DOTA-SSTR PET for diagnostic purposes in 644 patients with 867 lesions. Most lesions were meningiomas (93%), followed by pituitary adenomas (4.2%). Less common SSTR2-positive brain tumors were also described, including: brain metastases from breast cancer (6 patients) [68Ga]Ga-DOTA-SSTR PET studies were implemented to: (1) evaluate intraosseous or infracranial extension [68Ga]Ga-DOTA-SSTR PET studies were utilized to confirm biologically active post-surgical residual tumor tissue in concomitance with FDG PET/CT studies Ga-DOTA-SSTR PET for planning radiotherapy protocols and/or surgical resection in 337 patients with 356 lesions. All lesions were meningiomas (99.7%), except for one pituitary carcinoma reported by d\u2019Amico et al. [68Ga]Ga-DOTA-SSTR PET studies were mostly fused with CT/MRI studies and implemented to improve volume contouring for radiation planning, such as stereotactic radiotherapy (SRT) [68Ga]Ga-DOTA-SSTR PET proved to significantly improve target definition in meningiomas of the skull base, parafalcine, intraosseous, or affecting the optic nerve sheath, also favoring reduced radiation doses to adjacent organs at risk. Guinto-Nishimura et al. [68Ga]Ga-DOTA-SSTR studies to their neuronavigation system for intraoperative PET-guided meningioma resection. D\u2019Amico et al. Ga-DOTA-SSTR PET for planning neurotheranostics therapy in 49 patients with 54 lesions. Most lesions were meningiomas (74.1%) followed by high-grade gliomas (22.2%) and pituitary carcinomas (3.7%). DOTATOC was used in 23 patients (46.9%), DOTATATE in 6 patients (12.2%), and unclear use of DOTATOC or DOTATATE was described in 20 patients (40.8%). 68Ga-DOTATATE PET studies were mostly utilized for assessing SSTR2 expression and eligibility for [177Lu]Lu-DOTATATE therapy in both recurrent meningiomas [68Ga]Ga-DOTATOC uptake by targeted tumors and estimate [90Y]Y-DOTATOC uptake, planning to develop a specific probe used for intraoperative radiotracer-guided tumor resection. Verburg et al. [68Ga]Ga-DOTATATE in meningiomas and compared tracer uptake rates to those following venous [68Ga]Ga-DOTATATE infusion, with the goal of better evaluating eligibility for [177Lu]Lu-DOTATATE therapy in patients with insufficient tracer uptake after venous [68Ga]Ga-DOTATATE infusion.ingiomas and pituingiomas ,68. Collingiomas devised g et al. evaluate68Ga]Ga-DOTATATE PET/MRI in meningiomas and other SSTR2-positive brain tumors compared to MRI alone, and secondarily correlate rates of tracer uptake to the expressions of SSTR2, Ki67, progesterone receptor, and EGFR. The interventional trial led by Johnson [68Ga]Ga-DOTATATE PET/CT in residual meningiomas and measuring their metabolic response to radiotherapy. The interventional trial led by Merrell [68Ga]Ga-DOTATATE PET/MRI to assess meningioma eligibility for [177Lu]Lu-DOTATATE therapy and simultaneously assess rates of progression-free survival, overall survival, and adverse events. Filipsson Nystr\u00f6m [68Ga]Ga-DOTATOC uptake between tumors and normal pituitary tissue, and secondarily to correlate tracer uptake rates to SSTR2 expression, to report adverse events, and to detect post-surgery tumor recurrences.Four clinical trials are currently ongoing\u2014three interventional ,82,83 an Nystr\u00f6m is curre68Ga]Ga-DOTA-SSTR PET studies in neuro-oncology, which have proved to be effective and safe for diagnostic and treatment planning purposes. However, the high variability in applications, SSTR2-positive diseases, and findings may pose some challenges in defining the pros and cons of their implementation in routine practice. In this review, we aimed to provide a comprehensive summary of the current literature reporting the use of [68Ga]Ga-DOTA-SSTR PET for brain tumors, hoping to assist all physicians involved in the multidisciplinary management of neuro-oncology patients.A growing body of literature is currently focusing on analyzing the role of [18F]F-FDG and amino acid tracers are mostly used in patients with gliomas, brain metastases, and primary central nervous system lymphomas (PCNSL). They show variable sensitivity and specificity in differentiating tumor tissue from the normal brain tissue, distinguishing post-treatment changes from tumor recurrences, and, more recently, predicting molecular patterns and patient prognosis when implemented for radiomics analyses [68Ga]Ga-labeled DOTA tracers targeting SSTR, primarily developed for neuroendocrine tumors, have been largely used to allow highly selected meningioma uptake, low healthy brain tissue uptake, and, thus, higher specificity in the tumor-to-background contrast with excellent tumor visualization Ga-DOTA-SSTR PET the preferred modality in SSTR-positive tumors, including meningiomas and pituitary neoplasms appears insufficient to universally prove their findings, which would require further external validation with larger studies, their threshold has largely been used in the literature to differentiate residual or recurrent meningiomas from post-treatment changes, such as radiation necrosis, scarring, and pseudoprogression [68Ga]Ga-DOTA-SSTR PET has also been studied to quantify the extent of meningioma resection in comparison to postoperative MRI and intraoperative Simpson grading, showing lower rates of false-negative and superior detection of tumor remnants [68Ga]Ga-DOTATOC PET and biopsy confirmation of peri-cavitary \u201careas of doubt\u201d, has been preliminarily proposed to evaluate the completeness of meningioma resection.Rachinger et al. defined gression ,53. Simiremnants . From th68Ga]Ga-DOTA-SSTR PET studies has also been investigated in pituitary adenomas and carcinomas, in view of their SSTR overexpression. As the normal pituitary gland is characterized by physiological tracer uptake, Zhao et al. [18F-FDG and [68Ga]Ga-DOTA-SSTR PET studies may assist in postoperatively differentiate residual adenomas from the remaining functioning pituitary tissue . In contrast, pituitary carcinomas present higher tracer uptake compared to the normal pituitary gland, with previous studies reporting high diagnostic accuracy of [68Ga]Ga-DOTA-SSTR PET in confirming the diagnosis and enabling the detection of concurrent systemic metastases Ga-DOTA-SSTR PET for treatment planning has been first described by Milker-Zabel et al. [68Ga]Ga-DOTA-SSTR PET detection of additional information on meningiomas not detected at MRI [68Ga]Ga-DOTA-SSTR PET for tumor volume contouring has been validated for FSRT [68Ga]Ga-DOTA-SSTR PET for radiosurgery planning in pituitary carcinomas infiltrating the cavernous sinus, as it allows precise tumor contouring of residual post-surgery volumes.Accurate tumor volume contouring and target definition are of vital importance for optimizing the planning of surgical resection and radiotherapy protocols. This is especially true in recurrent tumors, such as malignant meningiomas or pituitary carcinomas, characterized by aggressive invasive growth patterns, which often require high doses of radiation, and by post-treatment changes, which may pose some challenges in the delineation of target volumes using morphological imaging studies ,97,98,99l et al. , who impd at MRI ,69 and sd at MRI ,29. The for FSRT ,66, radifor FSRT ,18, and for FSRT ,69 plannfor FSRT confirme68Ga]Ga-DOTA-SSTR PET for navigation-guided meningioma resection has been less investigated. To date, only Guinto-Nishimura et al. [68Ga]Ga-DOTATOC PET-guided resection of one primary intraosseous meningioma. In this technical note, the authors noted their ability to achieve gross total tumor removal by including in the resection peripheral bone areas showing high tracer uptake, which appeared macroscopically intact and extended beyond the tumor margins identified on the MRI. The postoperative pathology report confirmed the presence of tumor cells in the PET-positive peripheral bone and their absence in the specimen\u2019s surgically resected margins. In addition, the fusion of PET/CT with MRI images, coupled with their integration with the navigation system, allowed for high accuracy for intraoperatively visualizing radiological anatomical structures and tumor margins, without altering the surgeon\u2019s performance, compared to routine navigation-guided surgical protocols. Hence, [68Ga]Ga-DOTA-SSTR PET-guided meningioma resection may be feasible and effective in challenging cases with great intraosseous and/or skull base extension, but further surgical studies should be conducted to analyze the surgical performances from multiple centers and operators.In contrast to the well-described PET-guided glioma surgery , the usea et al. reported68Ga]Ga-DOTA-based theranostic agents in neuro-oncology, the only validated theranostic pair is composed of [68Ga]Ga-DOTATATE (diagnostic) and [177Lu]Lu-DOTATATE (therapeutic), clinically used for meningiomas [68Ga]Ga-DOTATATE) predicts longer and more favorable treatment responses [68Ga]Ga-DOTATOC and [90Y]Y-DOTATOC theranostic pair for radioguided high-grade glioma and meningioma resection, their findings still need to be externally validated before being implemented in clinical practice.Precision medicine approaches are constantly expanding in neuro-oncology to devise patient-tailored treatments directed against individual molecular and genetic profiles, responsible for the high intra-tumor and between-tumor heterogeneity, so as to selectively target cancer cells while minimizing damage to the healthy brain tissue . The devingiomas or pituiingiomas ,68. Receesponses ,41,68. Vesponses also demesponses devised 68Ga]Ga-DOTATOC and [68Ga]Ga-DOTATATE in patients with meningiomas [68Ga]Ga-DOTA-SSTR diagnostic accuracy: (1) compared to MRI for meningiomas [68Ga]Ga-DOTATATE and [177Lu]Lu-DOTATATE) for meningiomas in terms of progression-free survival, overall survival, and adverse events. While the trial conducted by Filipsson Nystr\u00f6m [68Ga]Ga-DOTA-SSTR PET studies for different populations. Future studies should also evaluate the role of intraoperative [68Ga]Ga-DOTA-SSTR PET-guided resection of skull base and transosseous meningiomas in terms of surgical feasibility, additional operating time, the extent of tumor resection, and its impact on postoperative patient performance status.Four ongoing clinical trials are currently evaluating Ga-DOTA-SSTR in clinical settings and the reduced availability of studies currently published, we have also included many case reports. These case reports may limit any statistical evaluation of the accuracy, sensitivity, and specificity of this technique as of the current day, but offer valuable information on the several potential uses of [68Ga]Ga-DOTA-SSTR in neuro-oncology. The high costs and recent development of [68Ga]Ga-DOTA-SSTR tracers may have prevented the implementation of such a technique worldwide, limiting published studies and our findings only to experiences from a few selected institutions. Due to a lack of granular data, we could neither comprehensively assess differences in SUVmax rates among different tumors nor the impact of [68Ga]Ga-DOTA-SSTR PET studies on post-treatment patient outcomes. Future studies should better analyze the diagnostic accuracy of [68Ga]Ga-DOTA-SSTR PET imaging compared to other imaging modalities for each type of brain tumor and how these studies impact the management of affected patients.Our review has some limitations. All included studies were retrospective case reports and case series likely exposed to selection bias. Owing to the recent introduction of [68Ga]Ga-DOTA-SSTR PET tracers in brain tumors has provided a valuable diagnostic adjunct primarily in the management of patients with meningiomas and pituitary adenomas/carcinomas. In particular, current routine applications of [68Ga]Ga-DOTA-SSTR PET imaging are shown to correlate with improved diagnostic accuracy, delineation of radiotherapy targets, and patient eligibility for radionuclide therapies. Ongoing trials are set to better define the diagnostic performance of these approaches, and future studies should evaluate the impact of [68Ga]Ga-DOTA-SSTR PET studies for imaging-guided surgical tumor resections.The recent development of ["} +{"text": "Aging is significantly associated with cognitive decline. A growing number of US adults ages \u2265 65 years have neurocognitive impairment resulting in compromised immediate and/or long-term health outcomes. Interventions to mitigate cognitive decline and promote healthy aging are needed. Research in intermittent fasting (IF) suggests positive health outcomes related to improvements in circadian rhythm and metabolism, which influence cognition. IF regimens may therefore result in improved neurocognitive health. We conducted an IF single-group, pre/post pilot study to explore changes in neurocognitive. Older adults with self-reported memory decline engaged in an 8-week, remotely-delivered, prolonged nightly fasting (PNF) intervention . Our primary exploratory outcome was 8-week change in neurocognitive function assessed via composite score of the Memory and Attention Phone Screener (MAPS). Trends in outcome change were assessed with paired t-tests. Participants were mean age 69.7 years, non-Hispanic White, predominantly female (94%), married (50%), and employed (65%). Completion defined as percentage of participants that completed the intervention from those that started the intervention; completion rate was 90%. Paired t-test indicated a significant increase in scores on a neurocognitive screen (MAPS) pre/post-intervention (p=0.02) with a medium effect size (Cohen\u2019s d=0.58). Findings suggest that PNF, a type of IF regimen targeted to align food intake with circadian rhythms, may significantly improve neurocognitive function among older adults with self-reported memory decline. These promising pilot results suggest a need for fully-powered, randomized controlled trials to test the efficacy of this non-pharmacological, low cost-to-burden ratio intervention."} +{"text": "Nature Communications 10.1038/s41467-022-28674-1, published online 25 February 2022.Correction to: The original version of this article contained errors in the Methods section, where the incorrect suppliers for two antibodies were listed. The sentence \u2018HRP-conjugated goat anti-human Fc antibody (Novus)\u2019 should read \u2018HRP-conjugated goat anti-human Fc antibody (Bethyl Laboratories)\u2019, and the sentence \u2018HRP-conjugated goat anti-mouse Fc antibody (Novus)\u2019 should read \u2018HRP-conjugated goat anti-mouse Fc antibody \u2019. These errors have now been corrected in both the PDF and HTML versions of the Article."} +{"text": "There is an urgent need to identify effective strategies to prevent leukemic transformation and induce sustained deep remissions in adult high-risk myelodysplastic syndrome (MDS) patients. This article discusses the clinical impact potential of bispecific antibodies (BiAB) capable of redirecting host T-cell cytotoxicity in an MHC-independent manner to malignant clones as well as immunosuppressive myeloid-derived suppressor cells (MDSC) as a new class of anti-MDS drug candidates. T-cell engaging BiAB targeting the CD123 antigen may help delay disease progression in high-risk adult MDS and potentially reduce the risk of transformation to secondary AML. Adult myelodysplastic syndrome (MDS), a heterogeneous group of clonal malignant hematologic disorders with an incidence rate of 4.5 per 100,000 persons per year, is characterized by ineffective hematopoiesis, abnormal differentiation of progenitor cells in the myeloid, erythroid, and megakaryocytic compartments, emergence of dysplastic myeloid cells, and an enhanced risk of transformation to acute myeloid leukemia (AML) . There i+CD123+ immature myeloid cells within the bone marrow mononuclear cell fraction contribute to the immunosuppressive tumor microenvironment (TME) by inhibiting both memory and cytotoxic effector T-cell populations as well as natural killer (NK) cells, thereby promoting the immune evasion of MDS clones (The immunosuppressive bone marrow microenvironment (BMME) in adult MDS has been implicated in clonal evolution and disease progression . ExpandeS clones as a treatment-emergent and potentially life-threatening complication . CRS was+ cells to create \u201ccytolytic synapses\u201d as a short bridge between MDS cells and CTLs, triggering CTL activation and destruction of targeted MDS cells within close vicinity of target CD123DS cells . APVO436DS cells , and T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) antigens as well as functional cytotoxic T-cell (CTL) deficiency . Further+ MDS clones as well as leukemic stem cells. Notably, Venetoclax has recently been shown to augment T-cell effector function by increasing the production of reactive oxygen species and Azacitidine has been shown to enhance sensitivity of AML cells to cytotoxic T-cells by activating the STING pathway . Louka ess CD123 . TherefoRecombinant T-cell engaging humanized bispecific antibodies can be designed to redirect host T-cell cytotoxicity in an MHC-independent manner and in a target-dependent manner, selectively, to CD123-expressing blast cells in patients with hematologic malignancies.+ MDSC (+CD34+ blast cells.They have clinical impact potential in high-risk MDS as it may both prevent disease progression/immune evasion by reducing the numbers of CD123+ MDSC and dela"} +{"text": "This study aimed to explore the impact of short-term exercise of varying intensity on traditional cardiometabolic disease (CMD) risk factors.One hundred-and-nine children (11.07 \u00b1 0.81 y) were conveniently assigned to 5-weeks of either: moderate intensity continuous training set at 65% - 70% of maximum heart rate (MHR); high intensity interval training ; combined training ; or no training . A two-way analysis of variance (group x time) was used to evaluate the effects of training on all traditional CMD risk parameters. Effect sizes (ES) were calculated to assess the magnitude of difference.MICT, HIIT and HIIT + MICT significantly improved resting heart rate , fasting glucose , peak oxygen consumption and c-reactive protein , respectively. The HIIT + MICT group significantly reducedwaist circumference and waist-to-hip ratio compared with the MICT and HIIT groups, respectively.The findings from this study indicate that short-term HIIT and MICT interventions are both effective for improving cardiometabolic health in children. HIIT + MICT may provide superior reductions in central obesity indicators."} +{"text": "As the population recovers from the coronavirus disease 2019 (COVID-19) pandemic, a subset of individuals is emerging as post-coronavirus disease (post-COVID) patients who experience multifactorial long-term symptoms several weeks after the initial recovery from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. The aim of this systematic review is to present the latest scientific reports that evaluate changes in glucose levels, blood pressure readings and lipid profiles after recovery from COVID-19 to verify the hypothesis that new-onset diabetes mellitus, arterial hypertension and dyslipidaemia are a possible sequela of a COVID-19 infection. The open access databases PubMed and Google Scholar were searched. Articles investigating patients with residual clinical signs and biochemical alteration indicating diabetes, hypertension and dyslipidaemia at least a month after recovering from COVID-19 were included. It has been shown that a select number of patients were diagnosed with new-onset diabetes, arterial hypertension and dyslipidaemia after COVID-19 infection. Alterations in glucose levels, blood pressure and lipid profiles months after initial infection shows the importance of considering diabetes mellitus, arterial hypertension and dyslipidaemia as part of the multifactorial diagnostic criteria post-COVID to better provide evidence-based clinical care. Despite most individuals infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) recovering within weeks of infection, a subset of individuals experiences post-coronavirus disease (post-COVID) conditions. Post-COVID conditions, also known as long-coronavirus disease (long-COVID), is a multisystem disease which causes patients to experience a cluster of debilitating symptoms four or more weeks after being infected with SARS-CoV-2 . The incEmerging evidence shows that new-onset diabetes mellitus, hypertension and dyslipidaemia are detected during the acute phase of a COVID-19 infection, but limited reviews have been conducted concerning similar outcomes occurring concurrently with post-COVID ,7,8,9. AStudies included in this review were searched for using PubMed (MEDLINE) and Google Scholar. Predefined search terms included multiple combinations of the following: (post-acute sequelae of SARS-CoV-2 OR post-COVID condition OR post-COVID-19) AND AND (new-onset).Included articles were original English language articles published from 1 December 2019 to 30 January 2022 investigating patients with residual clinical signs and biochemical alterations at least a month after recovering from COVID-19; specifically, articles investigated fasting glucose and C-peptide levels, blood pressure and lipid profiles. The eligible articles focused on patients over eighteen years of age. Confirmed infections were defined as positive real-time reverse-transcriptase polymerase chain reaction (RT-PCR) results from a nasopharyngeal and/or throat swab. Only studies reaching the statistical significance have been incorporated.Excluded articles were published before 1 December 2019, non-English language, non-original studies. Articles were ineligible if sample population did not meet long-COVID definition of new or persistent symptoms four weeks after initial infection.http://www.bristol.ac.uk/media-library/sites/social-community-medicine/robis/ROBIS%201.2%20Clean.pdf (accessed on 30 January 2022)). Characteristics of the studies that were mainly assessed in bias analysis: specifications of the studies met the eligibility criteria, relevance of studies in context of review aim. See The screening was mechanically performed by checking the publication date, record of patients testing positive for COVID-19 from a nasopharyngeal swab using the RT-PCR test during acute phase, persistent clinical alteration at least a month after recovery and biochemical investigations comparing baseline status to follow-up. The databases searched a total of 1027 records. After reading the abstracts, 916 studies were removed. The browsing led to the elimination of 96 papers, leaving 10 ,16,17,18This review was conducted following Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelines. Evaluation of the quality of the included studies was based on selecting only published peer-reviewed studies, excluding preprint manuscripts and case reports.The outcome measure was the alteration from baseline and prevalence of diabetes, arterial hypertension, or dyslipidaemia several months after infection with COVID-19.p = 0.001) were significantly raised in patients who recovered from COVID-19, with 127 diagnoses per 1000 patient years [Despite apparent clinical recovery at discharge from initial infection with COVID-19, many patients have residual medical problems and persistent hyperglycaemia when evaluated after several months . A longint years .p = 0.027) [= 0.027) .p = 0.001) three months post-recovery from SARS-CoV-2 infection than in control peers with no history of COVID-19 [COVID-19 .This systematic review compared studies that evaluated changes in glucose levels, blood pressure readings and lipid profiles after recovery from COVID-19 to verify the hypothesis that new-onset diabetes mellitus, arterial hypertension and dyslipidaemia are a possible sequela of a COVID-19 infection. Compiled studies investigating glucometabolic abnormalities showed that a select number of recovered COVID-19 patients were diagnosed with diabetes and had continuous hyperglycaemia several months after infection ,11,12,13Multiple hypotheses have been proposed to explain the association between new-onset diabetes and COVID-19 infection. A possible mechanism for altered glucometabolic control is SARS-CoV-2 damaging the pancreas. The angiotensin-converting enzyme 2 (ACE2) plays a crucial role in glucose homeostasis and insulin secretion by regulating beta-cell physiology. Within the pancreas, ACE2 is expressed within the pancreas\u2019 endocrine islet cells, including beta cells ,21. ACE2Hypertension is a major risk factor for stroke, coronary artery disease (CAD), renal disease, heart failure and peripheral vascular disease; therefore, early diagnosis and timely intervention are crucial in preventing these complications. Possible mechanisms explaining the development of new-onset hypertension due to SARS-CoV-2 infection include renin-angiotensin-aldosterone system (RAAS) dysregulation. It is hypothesised that SARS-CoV-2 binds to the ACE2 receptor via its spike (S) protein provoking transient ACE2 down-regulation consequently deregulating RAAS signalling . ACE2 faThe measurement of plasma lipids and lipoproteins is critical in cardiovascular disease (CVD) risk management. Multiple possible mechanisms may explain why patients experience new-onset dyslipidaemia after COVID-19 infection. For one, SARS-CoV-2 is an enveloped virus meaning a lipid bilayer surrounds it; therefore, lipid metabolism plays a crucial role in the viral life cycle . The virPersistent pathophysiological and clinical alterations following COVID-19 infection call attention to the importance of mitigation strategies in COVID-19 prevention. A longitudinal observational study amongst vaccinated and unvaccinated SARS-CoV-2 infected healthcare workers not requiring hospitalisation in Italy showed lower prevalence of long-COVID amongst vaccinated . Though This systematic review compared studies that evaluated changes in glucose levels, blood pressure readings and lipid profiles several weeks after recovery from COVID-19 to verify the hypothesis that new-onset diabetes mellitus, arterial hypertension and dyslipidaemia may be a sequela of a COVID-19 infection. The review had a couple of strengths, such as geographical diversity and the statistical validity of selected studies that compared clinical presentation at acute versus post-COVID period. The review also had some limitations, such as the selected studies did not distinguish between disease characterisation, such as diabetes type 1 or type 2. Another limitation of this review was the scarcity of studies investigating clinical presentation and biomarkers of post-COVID condition at the point in time the systemic review was conducted.It can be concluded from this review that people older than eighteen years of age are at an increased risk of developing new-onset incidents of diabetes, hypertension, or dyslipidaemia several months after COVID-19 infection. In addition to other post-COVID symptoms, patients presented with altered glucose levels, blood pressure and lipid profiles several months after recovery from COVID-19 infection. Therefore, it is imperative that hyperglycaemia and insulin resistance, increased systolic blood pressure and altered lipid profiles be considered a part of the multifaceted post-COVID. Evidence-based clinical guidelines for the diagnosis and management of post-COVID syndrome that consider alteration in glucose levels, blood pressure and lipid profiles need to be established to provide patients with integrated clinical care, follow-up and monitoring after COVID-19 recovery. Further investigation of the multifactorial long term medical consequences after recovery from COVID-19 need to be conducted."} +{"text": "An X-ray co-crystal structure of the compound bound to PLpro validated our design strategy and established the molecular basis for covalent inhibition and selectivity against structurally similar human DUBs. These findings present an opportunity for further development of covalent PLpro inhibitors.Direct-acting antivirals are needed to combat coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). The papain-like protease (PLpro) domain of Nsp3 from SARS-CoV-2 is essential for viral replication. In addition, PLpro dysregulates the host immune response by cleaving ubiquitin and interferon-stimulated gene 15 protein (ISG15) from host proteins. As a result, PLpro is a promising target for inhibition by small-molecule therapeutics. Here we have designed a series of covalent inhibitors by introducing a peptidomimetic linker and reactive electrophile onto analogs of the noncovalent PLpro inhibitor GRL0617. The most potent compound inhibited PLpro with"} +{"text": "This study aims to examine the effects of low-quality employment trajectories on severe common mental disorders (CMD) according to Swedish and foreign background.This is a longitudinal study based on Swedish population registries . Low- and high-quality employment trajectories observed across five years (2005-2009) are the exposure with severe CMD as outcome (2010-2017). Adjusted hazard ratios (HR) were calculated using Cox regression stratified according to background (first-generation (i) EU migrants, (ii) non-EU migrants, (iii) second-generation migrants, (iv) Swedish-born with Swedish background) and sex. The reference group were Swedish-born with Swedish background in a Constant high-quality employment trajectory.Second-generation migrants had an increased risk of CMD compared to Swedish-born with Swedish background when following low-quality employment trajectories . Female migrant workers, especially first-generation from non-Western countries in low-quality employment trajectories , had a higher risk of CMD compared to female Swedish-born with Swedish background. The confidence interval for CMD risk showed little differences between migrant groups (1st and 2nd generation) compared to the reference group.Low-quality employment trajectories appear to be determinants of risk for CMD, having a differential impact according to background of origin and sex. We observe a higher risk for severe CMD across migrant groups, especially second-generation migrants, compared to Swedish-born with Swedish background. Further qualitative research is recommended to understand the mechanism behind the differential mental health impact of low-quality employment trajectories according to foreign background.\u2022\u2002First and second-generation migrants in low quality employment have higher risk of severe common mental disorders compared to Swedish born with Swedish background workers in low quality employment.\u2022\u2002Policies targeting working conditions in low-quality employment and promoting workers mental well-being are essential to reduce this higher risk for developing CMD, especially for migrant populations."} +{"text": "We report the near-complete genome sequence of an avian orthoavulavirus 13 (AOAV-13) strain isolated from a wild goose fecal sample collected in South Korea in early 2020. The AOAV-13 sequence had a unique 3\u2032 trailer region, including an 84-nucleotide\u2009(nt) deletion and a 24-nt insertion, compared to the most closely related Chinese genome sequence from 2015. Avulavirinae of the family Paramyxoviridae. The subfamily Avulavirinae is composed of three genera, namely, Metaavulavirus (avian metaavulavirus [AMAV]), Orthoavulavirus (avian orthoavulavirus [AOAV]), and Paraavulavirus (avian paraavulavirus [APAV]) (Avian paramyxoviruses (APMVs) have been reported all around the world, infecting poultry as well as diverse species of wild birds. APMVs belong to the recently assigned subfamily [APAV]) . Avulavi, and L) . In thisMN150295.1) with Geneious Prime v2021.2.2 using default parameters. A total of 544,667 reads were mapped to produce a near-complete genome of AOAV-13. Putative insertions and deletions identified in the initial assembly were verified with Sanger sequencing with primers designed to amplify the nucleotide position 14508 to 16032 region. The genome positions were calculated based on the AOAV-13/wild goose/China/Hubei/V93-1/2015 (China/V93-1) genome (GenBank accession no. MN150295).A hemagglutinating agent was isolated from allantoic fluid harvested from 9- to 11-day-old embryonated chicken eggs inoculated with a fecal sample from a wild migratory bird that had been collected during avian influenza virus surveillance from October 2019 to April 2020. The host species of the isolated virus was determined from wild bird feces by the mitochondrial DNA barcoding PCR technique by using the primers AvesF (GCATGAGCAGGAATAGTTGG) and AvesR (AAGATGTAGACTTCTGGGTG), as described previously (112V-R-E-N-R-L117) as found in other AOAV-13 strains. The monobasic amino acid at the C terminus of the cleavage site and leucine at residue 117 suggest low pathogenicity in domestic poultry (\u2013n = 55) as observed in most genomes of the subfamily Paramyxovirinae databases under the accession no."} +{"text": "The unmet clinical need for effective treatments in ovarian cancer has yet to be addressed using monoclonal antibodies (mAbs), which have largely failed to overcome tumour-associated immunosuppression, restrict cancer growth, and significantly improve survival. In recent years, experimental mAb design has moved away from solely targeting ovarian tumours and instead sought to modulate the wider tumour microenvironment (TME). Tumour-associated macrophages (TAMs) may represent an attractive therapeutic target for mAbs in ovarian cancer due to their high abundance and close proximity to tumour cells and their active involvement in facilitating several pro-tumoural processes. Moreover, the expression of several antibody crystallisable fragment (Fc) receptors and broad phenotypic plasticity of TAMs provide opportunities to modulate TAM polarisation using mAbs to promote anti-tumoural phenotypes. In this review, we discuss the role of TAMs in ovarian cancer TME and the emerging strategies to target the contributions of these cells in tumour progression through the rationale design of mAbs. The unmet clinical need for effective treatments in ovarian cancer has yet to be addressed using monoclonal antibodies (mAbs). In this review we discuss the role of tumour-associated macrophages (TAMs) in ovarian cancer and consider how TAMs can be modulated by novel mAb therapies to provide unique opportunities for clinical efficacy. Ovarian cancer has the highest mortality rate among gynaecological malignancies . This poOver the past 30 years, mAb therapies have become widely used in cancer treatment, offering significant advantages relative to conventional chemotherapy and radiotherapy, including high specificity and affinity for a single epitope target, which limits off-target effects . TherapeDespite significant successes in other tumour types, mAb therapies in ovarian cancer frequently report disappointing clinical trial results . In receMacrophages are highly abundant mononuclear phagocytic cells present in almost every human tissue .Macrophages are both important for inflammatory reactions and homeostatic functions. Monocyte-derived macrophages (MDMs) rapidly increase in number during inflammatory events such as infection, to aid the restoration of homeostasis through the promotion of pathogen clearance and subsequently tissue repair . MoreoveTAMs frequently constitute a highly abundant population within TMEs, typified in ovarian cancer, where they can account for over 50% of all cells in peritoneal tumours and ascites . InitialIn ovarian cancer, total TAM density exhibits no prognostic significance , 45. HowIn vivo, especially in TMEs, the binary M1/M2 model has proven oversimplified, with a spectrum model possibly offering a more accurate representation of macrophage polarisation . In. In77]. A key requirement for the development of both the primary and metastatic tumours is the establishment of access to the circulatory system via neo-angiogenesis. TAMs potently promote neo-angiogenesis, displaying enrichment at sites with high angiogenic requirements, including hypoxic tumour nests, nascent peritoneal tumours, and perivascular regions , 78, 79 The IgG1 mAb Bevacizumab blockades VEGF-A/VEGFR-mediated neo-angiogenesis through binding to VEGF-A 87]. Be. Be87]. TAMs may play a significant role in this resistance. In a murine model of Bevacizumab-resistant ovarian cancer, tumours exhibited restored response when treated with a TAM-depleting anti-CSF-1 mAb . SpecifiThe potential utility of combination therapy for targeting neo-angiogenesis has been indicated clinically. Firstly, in a Phase III trial, peptide-Fc fusion protein Trebananib, which targets Ang2, as well as additional Tie2 ligand, Ang1, was found to enhance PFS in ovarian cancer patients 23]. Se. Se23]. Due to the redundancy of neo-angiogenesis pathways and the centrality of TAMs in driving them, a more durable therapeutic strategy may be to specifically target pro-angiogenic TAM subsets via mAbs and 3. TInstead of inhibiting the pro-tumoural activity of TAMs, specific mAbs have sought to promote the anti-tumour function of TAMs.One such approach is through an antibody\u2019s simultaneous engagement of tumour-associated antigens (TAAs) on tumour cells and antibody Fc receptors on TAMs, to trigger tumouricidal TAM effector function . This caAlthough TAA-targeting IgG mAbs have displayed efficacy in solid tumours, clinical results in ovarian cancer have been disappointing . This diOne emerging strategy to enhance the efficacy of TAA-targeting IgG mAbs is through mAb-mediated blockade of \u2018don\u2019t eat me\u2019 signals expressed on tumour cells, to enhance the contribution of TAM effector functions to their mechanism of action. For example, tumour cell CD47 and CD24 engage cognate receptors on TAMs, signal regulatory protein-\u03b1 (SIRP\u03b1) and sialic acid-binding Ig-like lectin 10 (Siglec-10), respectively, to trigger signalling cascades which inhibit macrophage phagocytosis , 103 Fi. CD47 exin vitro, as well as inhibition of tumour growth in murine ovarian cancer models ..26].Moreover, in view of the enhanced pre-clinical efficacy of TAA-targeting mAbs when in combination with anti-CD47 mAbs, several trials are currently underway using this regimen in non-ovarian solid tumours. For example, a phase II study in colorectal cancer patients of anti-CD47 IgG4 mAb Hu5F9-G4 in combination with Cetuximab, reported stable disease in 45% of patients, as well as increased macrophage tumour infiltration . It remahighIL-12low secretome upregulation and downregulation of M2-associated CSF1R. However, a Phase I/II trial of ABBV-428, a bispecific mAb targeting CD40 (as an agonist) and tumour cell marker mesothelin, was not able to replicate these promising anti-tumoural effects in ovarian cancer patients . De. De57]. Clinically, a Phase I trial using anti-CD40 IgG2 mAb CDX-1140 with anti-PD-1 IgG4 mAb Pembrolizumab (Keytruda) is currently recruiting ovarian cancer patients (NCT03329950). This will provide the first insight into whether anti-CD40 mediated TAM repolarisation can skew the ovarian TME immune landscape towards ICB efficacy .Ka for IgG of 108\u2013109 M\u22121, the high-affinity IgE receptor, Fc \u03b5 receptor I (Fc\u03b5RI), exhibits a substantially higher affinity for IgE (Ka = 1010\u20131011 M\u22121) [Currently, all licensed full-length mAbs are of the IgG isotype . IgE ant011 M\u22121) , 123. Th011 M\u22121) , 124. Mo011 M\u22121) . Further011 M\u22121) , 127. Ac011 M\u22121) . ConsequAmong several developed IgE mAbs targeting TAAs, MOv18 IgE, represents the first-in-class to enter clinical testing, in an ongoing phase I trial involving predominantly ovarian cancer patients (NCT02546921). MOv18 IgE is targeted against TAA, folate receptor \u03b1 (FR\u03b1), which is overexpressed in 82% of serous epithelial ovarian cancers and inversely correlated with patient OS and disease-free interval (DFI) , 130.In pre-clinical models, MOv18 IgE exerted anti-tumoural effects via Fc engagement of monocytes and macrophages in a two-armed mechanism, comprising tumour killing and crucially, pro-inflammatory polarisation . In an oIn vitro interrogation of tumour cell killing determined that MOv18 IgE predominantly induces cancer cell ADCC by monocytes, as well as ADCP, when monocytes are IL-4-stimulated to induce expression of low-affinity IgE receptor, CD23 [or, CD23 . In compin vitro and in vivo studies have found that cross-linking of TAA-bound IgE initiates a TNF-\u03b1/CCL2-mediated monocyte and macrophage pro-inflammatory recruitment feedback loop, to potentiate tumour killing and drive pro-inflammatory polarisation [in vitro cross-linking of the TAA-specific IgE mAb SF-25 [Consistently, risation . Explorarisation . ConsistAb SF-25 .in vivo anti-tumour activity, may permit sustained efficacy, which has hitherto eluded TAA-targeting IgG mAbs in ovarian cancer. Recent interim results from the Phase I clinical trial of MOv18 IgE have demonstrated the therapy to be well tolerated and, although preliminary, anti-tumour activity was displayed by one patient [Collectively, TAA-targeting IgE mAbs may drive a unique monocyte and macrophage polarisation signature, which combined with an alternative tumour killing mechanism and superior patient .High frequency, broad Fc receptor expression, extensive potential pro-tumoural activities and strong phenotypic plasticity, render TAMs and their associated functions as attractive mAb targets to meet the unmet clinical need in ovarian cancer . AntibodConsequently, instead of viewing TAMs as a deleterious population for removal, recent experimental mAb strategies have frequently sought to exploit their extensive plasticity and immunoregulatory function via phenotypic repolarisation, to mediate a TAM-driven shift in the TME towards anti-tumour immunostimulation and 3. TOverall, mAb-mediated TAM repolarisation may facilitate a therapeutic window in which the ovarian TME is biased towards anti-tumour activity. Clinical exploitation of this opportunity therefore may best be achieved through combination with established cytotoxic therapies. For example, potential synergistic anti-tumour activity between TAM repolarisation and both tumour-targeting chemotherapy and adaptive immunity-targeting ICB have begun to emerge.Further investigation of these mAb candidates and possible combination regimens is warranted in larger clinical studies, to determine whether therapeutic targeting of TAMs may represent a viable pathway for efficacy, which has generally eluded mAbs in ovarian cancer."} +{"text": "Science1 showing that immune imprinting patterns might differentially influence immune responses against variants of concern (VOCs) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). They also demonstrated that infection with B.1.1.529 (Omicron variant) of SARS-CoV-2 confers only partial and transient protection against itself, resulting in frequent reinfections with Omicron in short time intervals.Recently, Reynold et al. published a study in 3 it is still unclear how distinctive histories of infections differentially shape immune responses against Omicron. Furthermore, it is unknown whether Omicron infection can act as a natural booster of immunity against SARS-CoV-2 VOCs. To address these questions, Reynold et al. analyzed adaptive immune responses in BNT162b2 mRNA-vaccinated London healthcare workers (HCWs) with different histories of SARS-CoV-2 infections , P.1 (\u03b3 variant), and Omicron variant than infection-na\u00efve HCWs. Interestingly, HCWs who had been infected with both Wuhan Hu-1 and B.1.617.2 (\u03b4 variant) showed enhanced neutralizing antibody (nAb) responses against Wuhan Hu-1 and \u03b4 variants. However, serum nAb responses against the Omicron variant were markedly diminished, when compared to the other VOCs, regardless of the history of SARS-CoV-2 infections. Moreover, MBC frequencies against the Omicron variant 2\u20133 weeks after the third vaccination and 20\u201321 weeks after the second vaccination were significantly lower than those against the ancestral Wuhan Hu-1 and \u03b4 variants, regardless of the infection history.Reynold et al. first demonstrated that antibody responses and memory B cell (MBC) counts were lower in HCWs who had received three doses of mRNA vaccine when they were infected with Omicron than those infected with Wuhan Hu-1 or other VOCs. These results showed that previous infection(s) and heterologous antigen exposure could alter and define immune responses to future infection(s) with VOCs, which agrees with the results from previous studies.In addition, responses of T cells against Omicron were significantly weaker than those against Wuhan Hu-1 and \u03b4 variants. Based on these findings, patterns of T-cell responses against a mapped epitope pool (MEP) derived from Omicron spike subunits S1 and S2, and a matched pool from Wuhan Hu-1 were compared to explore how Omicron spike mutations influence T-cell recognition. It is worth noting that T-cell responses against the Omicron MEP were lower than those against the matched Wuhan Hu-1 pool, regardless of the infection history, suggesting that the cross-recognition by T cells of the Omicron spike epitopes was reduced in triple-vaccinated HCWs.Omicron spike mutations led to a loss of HLA-DR4-restricted T-cell epitopes at four specific sites and a gain of nascent HLA-DR4 T-cell epitopes at four additional sites. To analyze the gain and loss of T-cell epitopes associated with Omicron spike mutations, the ancestral Wuhan Hu-1 or Omicron sequence-specific peptide pool was used to immunize HLA-DRB*04:01 transgenic mice. Intriguingly, priming T-cell responses with one pool led to reduced responses to the other, suggesting a causative mechanism underlying \u201chybrid immune damping\u201d, wherein protection against future infection might be altered by incidental immune imprinting patterns.Reynold et al. further analyzed B-cell immunity following Omicron infection and demonstrated that triple-vaccinated HCWs who were infected with Omicron as the first infection showed increased MBC frequency and antibody responses against all VOCs, including Omicron. However, cross-reactive antibody responses in triple-vaccinated HCWs who were infected initially with the ancestral Wuhan Hu-1 and later with Omicron were not significantly induced, compared to those in uninfected HCWs. Moreover, infection-na\u00efve HCWs showed only marginal antibody responses against Omicron.Reynold et al. next determined T-cell responses against Omicron infection in triple-vaccinated HCWs. Surprisingly, Omicron infection failed to induce significant T-cell responses against itself, regardless of the infection history. The observation that T-cell responses against Omicron were impaired in triple-vaccinated HCWs who were infected initially with the ancestral Wuhan Hu-1 and later with Omicron is consistent with reduced antibody responses against Omicron, suggesting that Omicron infection is not a powerful natural booster against itself, especially for HCWs who were infected during the Wuhan Hu-1 wave. On the other hand, infection with Omicron was found to enhance cross-reactive T-cell immune recognition against spike MEPs of the Wuhan Hu-1 and \u03b4 variant.Finally, by analyzing B- and T-cell responses in the longitudinal HCW cohort, Reynold et al. demonstrated that hybrid immunity (single vaccination plus prior infection) could lead to a significant increase in antibody responses against Omicron. Besides, for HCWs who experienced infection with Wuhan Hu-1 or \u03b1 variant, antibody responses against Omicron were significantly increased 2\u20133 weeks after the second vaccination and then declined after 20\u201321 weeks. Importantly, triple-vaccinated HCWs who experienced the ancestral Wuhan Hu-1 infection showed no increase in antibody-binding responses against Omicron. In short, immune imprinting mediated by Wuhan Hu-1 significantly diminished the antibody responses against Omicron in HCWs who were infected with Omicron.In summary, this study demonstrated that various infection and vaccination histories, so-called immune imprinting, might alter adaptive immune responses against the Omicron variant. Accordingly, immune responses against Omicron were not effectively induced by previous infections with Wuhan Hu-1 or other SARS-CoV-2 VOCs in triple-vaccinated HCWs. In addition, Omicron infection failed to serve as a natural immune booster against itself. On the other hand, antibody responses against Omicron were induced in several cohorts of HCWs with specific histories of infection and vaccination. These findings may provide novel insights into the development of effective anti-Omicron strategies."} +{"text": "A total of 675 LN-stations were sampled in 180 patients. The logistic and RF models identified SUVmax, the short-axis LN-diameter and the echelon of the considered LN among the most important parameters for EBUS-positivity. Adjusting the sensitivity of machine-learning classifiers to that of the expert-rater of 94.5%, MLP (P\u2009=\u20090.0061) and RF models (P\u2009=\u20090.038) showed lower misclassification rates (MCR) than the standard-report, weighting false positives and false negatives equally. Increasing the sensitivity of classifiers from 94.5 to 99.3% resulted in increase of MCR from 13.3/14.5 to 29.8/34.2% for MLP/RF, respectively. PET/CT-based machine-learning classifiers can achieve a high sensitivity (94.5%) to detect EBUS-positive LNs at a low misclassification rate. As the specificity decreases rapidly above that level, a combined test of a PET/CT-based MLP/RF classifier and EBUS-TBNA is recommended for radiation target volume definition.Accurate determination of lymph-node (LN) metastases is a prerequisite for high precision radiotherapy. The primary aim is to characterise the performance of PET/CT-based machine-learning classifiers to predict LN-involvement by endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) in stage-III NSCLC. Prediction models for LN-positivity based on [ This may allow and establish further treatment pathways with combined chemoradiation3.For patients with newly diagnosed locally advanced non-small cell lung cancer (NSCLC) finding the right treatment choice is often a challenge. In this context, radiotherapy represents one of the principal treatments. Improvements in systemic therapy have enabled more patients to live longer, even with metastatic disease4, and 11.4% and 6.5% in the large randomised PACIFIC trial with or without durvalumab consolidation5. Detection of involved lymph nodes (LNs) with high sensitivity and inclusion into the radiation target volume is an important determinant for a high loco-regional tumour control after definitive radiochemotherapy.Regional recurrences at five years after definitive radiochemotherapy for stage-III NSCLC were observed at cumulative incidence rates of 38.2% in the RTOG 0617 trial18F]fluoro-D-glucose positron emission tomography/computed tomography (2-[18F]FDG-PET/CT) is the standard diagnostic procedure for definition of the radiation therapy target volume7. Positive LNs are identified and delineated by visual inspection9. Machine-learning classifiers are not readily characterised to support this routine. Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is recommended for mediastinal nodal staging in patients suspected of mediastinal LN involvement by PET/CT10. Although the use of EBUS-TBNA increased over time11, its additional value for target volume definition has not been analysed in prospective studies so far.2-deoxy-2-FDG-PET/CT imaging for primary staging obtained at the same time point before treatment. Patients were excluded if they had severe systemic disease or previous tumour disease.This work develops on our previous publications18F]FDG-PET/CT imaging was performed on the Biograph_mCT PET/CT scanner after intravenous injection of 250\u2013400\u00a0MBq 2-[18F]FDG ligand-complex. One expert radiologist and one nuclear medicine physician independently rated the PET/CT images and, in case of discrepancies, approved the standard report by a consensus reading. Orthogonal short and long-axis LN-diameters were also measured on CT. LNs with diffuse mediastinal infiltration were classified by an additional binary classification variable and assigned to the largest size group with a diameter of\u2009\u2265\u20096 cm14.Contrast-enhanced FDG-PET/CT maximum standardised uptake value (SUVmax) were carried out in all LN-stations sampled with EBUS-TBNA.Obtained tissue was placed in formalin-solution to allow the preparation of a cell block for histologic evaluation and immunocytochemical analysis. Measurements of the FDG-PET/CT features and EBUS-TBNA prove to be valuable instruments for determining the regional pattern of nodal spread and radiation treatment planning in lung cancer.PET/CT based machine-learning classifiers demonstrate the potential to reduce the MCR compared to the standard report. Because misclassification increases substantially at higher sensitivities, a combined test of a PET/CT-based machine-learning classifier with a systematic EBUS-TBNA of PET-negative LN-stations is recommended. The classifiers can support the specialist to increase sensitivity. This dual test performed better than machine-learning classifiers alone. Combined, classifiers based on [Supplementary Legends.Supplementary Figure 1."} +{"text": "Anxiety and depression often co-occur and may be risk factors for neurodegenerative diseases such as Alzheimer\u2019s disease and related dementias. We examined associations between symptoms of anxiety and cognitive change and impairment in middle-aged and older Hispanics/Latinos. Method: 6,162 participants aged \u226550 were enrolled in the Study of Latinos-Investigations of Neurocognitive Aging study (SOL-INCA), an ancillary to the Hispanic Community Health Study/Study of Latinos (HCHS/SOL). Exposures included the Spielberger State-Trait Anxiety Inventory 10-item (STAI-10) and the Center for Epidemiology Scale for Depression 10-item (CESD-10) at HCHS/SOL Visit 1 (2008\u20132011). Outcomes include cognitive tests at Visit 1 and 2 about 7 years later in SOL-INCA and mild cognitive impairment diagnosis . Separate models were created to examine associations between cognition and anxiety adjusting for demographic variables, antianxiety medication, and depressive symptoms.After adjusting for demographic variables and medication use, STAI-10 scores were associated with Visit 2 cognitive function (p < 0.05) and MCI (p < 0.01), but not with changes in cognitive function over time. After adjusting for depressive symptoms, most effects of anxiety were attenuated, with exception of B-SEVLT-Recall (p < 0.05). Discussion: Anxiety symptoms were associated with lower memory function 7 years later among diverse older Hispanics/Latinos. Anxiety was associated with 7-year cognitive change and MCI, but those associations were explained by depressive symptoms and other covariables. Future research should examine cognition in relation to anxiety and depression since the two commonly co-occur."} +{"text": "Depression is the most common comorbidity of migraine. The brain of migraineurs with depression shows differences compared to migraine only or depression only patients. The comorbidity may affect specific regions such as the periaqueductal gray matter (PAG) which is important in negative emotion regulation and pain modulatory system.We hypothesized that the alterations in PAG functional connectivity (FC) may play a role in migraineurs vulnerability for depression.A resting-state fMRI was conducted with 34 episodic migraine without aura patients and 41 control subjects. All participants were medication free and they did not have any psychiatric or chronic disorders. Depressive symptoms were measured with Zung Self-Rating Depression Scale. To investigate the relationship between depressive symptoms and PAG functional connectivity, Zung scores were used as covariates in each groups\u2019 PAG-FC analysis using the Statistical Parametric Mapping (SPM12) toolbox in MATLAB environment.FWE<0.05) in migraine group. However, there was no significant correlation between Zung scores and PAG-FC in healthy control group.There were no significant difference between migraine and control group in Zung scores (p=0.394). Negative correlation was found between Zung scores and PAG-FC with thalamus, fusiform gyrus, middle occipital gyrus and calcarine ; 2020-4.1.1.-TKP2020; ERA PerMed (2019-2.1.7-ERA-NET-2020-00005); \u00daNKP-21-4-I-SE-15 (DB)."} +{"text": "Background: Metastatic prostate cancer (PCa) predicts a poor prognosis and lower likelihood of survival. Osteoblasts (OBs) are known to be responsible for the synthesis and mineralization of bone, although it is unclear as to whether PCa in the prostate gland cooperates with OBs in bone to promote PCa malignant transformation. We aimed to elucidate how primary PCa cells cooperate with distal OBs and contribute to the vicious cycle that leads to metastatic PCa.Methods: N-cadherin, E-cadherin, and Twist protein expression were measured by Western blot. Twist translocation into the nucleus was detected by the immunofluorescence (IF) assay. Enzyme-linked immunosorbent assay (ELISA) detected protein levels in human serum samples. Levels of candidate protein expression were examined by the human cytokine array. Prostate tumor growth and metastasis were analyzed by orthotopic and metastatic prostate cancer models, respectively. Immunohistochemistry (IHC) staining was used to observe ADAM metallopeptidase domain 9 (ADAM9) and WNT1 inducible signaling pathway protein 1 (WISP-1) expression in tissue.Results: Our in vitro and in vivo analyses have now discovered that primary PCa expressing ADAM9 protein enables the transformation of OBs into PCa-associated osteoblasts (PCa-OBs), inducing WISP-1 secretion from PCa-OBs in the bone microenvironment. The upregulation of WISP-1 in bone provided feedback to primary PCa and promoted PCa cell aggressiveness via epithelial-mesenchymal transition (EMT) activity. Elevated levels of WISP-1 expression were detected in the serum of patients with PCa. ADAM9 levels were overexpressed in tumor tissue from PCa patients; ADAM9 blockade interrupted OB-induced release of WISP-1 and also suppressed primary tumor growth and distal metastasis in orthotopic PCa mouse models.Conclusion: Our study suggests that the ADAM9/WISP-1 axis assists with metastatic PCa progression. Thus, targeting the ADAM9/WISP-1 axis may help to prevent the malignant phenotypes of PCa cells. Prostate cancer (PCa) commonly affects the urinary tract of men, whose susceptibility for this cancer increases with age Secretion of androgen by testicular Leydig cells plays a pivotal role in prostate epithelial cell growth and differentiation in vivoOsteoblasts (OBs) play a critical role in the repair and regeneration of the bone matrix In this study, our data indicate that primary PCa cells may distally influence OBs in bone, transforming OBs into PCa-OBs, with subsequent increases in WISP-1 expression in the bone microenvironment. Positive feedback from PCa-OBs expressing WISP-1 elevates the invasiveness of primary PCa cells via EMT functioning. WISP-1 enhances EMT activity in PCa cells by suppressing the epithelial marker E-cadherin and inducing the mesenchymal markers N-cadherin and Twist. Cytokine array analysis revealed that soluble ADAM9 (ADAM9-S) is a main mediator secreted from primary PCa cells, capable of regulating PCa-OB formation and WISP-1 expression. Higher ADAM9 levels were detected in clinical PCa tumor tissue compared with normal tissue specimens, while ADAM9 blockade suppressed PCa tumor growth and reduced the formation of distant metastasis in orthotopic PCa mouse models. Our results not only elucidate the mechanism underlying cooperation between primary PCa tumor cells and distal OBs, but also indicate that targeting the ADAM9/WISP-1 axis may interrupt the progression to metastatic disease.2 atmosphere.The human osteoblastic-like cell line MG-63 was grown in DMEM medium. The human prostate epithelial cell line (PZ-HPV-7) was grown in keratinocyte serum-free medium supplemented with bovine pituitary extract. All PCa cell lines were grown in RPMI-1640 medium. All cell types were cultivated at 37\u00b0C in a 5% CO6/10 mL each) in a 10 cm dish with fetal bovine serum (FBS) for 24 h, then the medium was replaced with serum-free medium. After 48 h, CMs were collected as osteoblast CM (OBCM), PZ-HPV-7 CM, LNCaP CM, PC-3 CM, and DU145 CM.MG-63 cells, human prostate epithelial cells (PZ-HPV-7), and PCa cell lines were seeded (at a rate of 2x106) were seeded in a 10 cm dish for 24 h, then incubated with serum-free medium for another 24 h. The cells were treated with 30% PZ-HPV-7 (PZ) CM, PC-3 CM, or DU145 CM for 24 h, then incubated in serum-free medium for 48 h. CMs were collected as PZ-OBCM, PC-3-OBCM, and DU145-OBCM. Long-term OBCM (OBCM-L) was also collected without PCa CM pretreatment , Oncomine and UALCAN tools.Cell migratory and invasive activity was evaluated through Transwell inserts in 24-well dishes , as according to our previous research A Magic RT Master Mix cDNA synthesis kit was used to convert cDNA from RNA fragment, according to the manufacturer's recommendations. Sequence-specific primers and Taqman one-step PCR Master Mix were used to perform qRT-PCR analysis, as according to our previous study A human cytokine array containing a total of 32 different cytokine antibodies spotted in duplicate on two membranes was used to analyze protein expression in CM, according to the manufacturer's recommendations. Levels of 32 proteins were detected through enhanced chemiluminescence and quantified by UN-SCAN-IT gel 6.1 software.The ADAM9 ELISA kit assayed ADAM9 protein concentrations in CM, according to the manufacturer's instructions.5 PC-3 cells suspended in 50 \u03bcL Matrigel via a 22-gauge needle. Tumor development and distal metastasis was monitored using the IVIS Spectrum scanner . After 2 weeks, the mice in the orthotopic model (n=7) were humanely sacrificed in a CO2 chamber. The tumors and tibias were collected before immunostaining. Mice in the metastatic model (n=9) were humanely sacrificed after 5 weeks and distant organs, including the liver and lungs, were harvested. Hematoxylin and eosin (H&E) staining was used to investigate distal metastasis in liver and lung specimens.Six\u2010week\u2010old male nude mice, bought from BioLASCO Taiwan Co., Ltd. , were injected in the anterior prostate with 5 \u00d7 10All procedures in the animal studies were approved by the Institutional Animal Care and Use Committee and performed according to the Guidelines of Animal Experimentation issued by Shin Kong Wu Ho-Su Memorial Hospital . Serum samples were collected from 11 patients undergoing surgical resection in China Medical University Hospital, under approval granted by the Institutional Review Board of China Medical University Hospital . Written informed consent was obtained from each study participant before enrollment.t-test. One-way ANOVA followed by Bonferroni's post hoc comparison tests compared the means of more than two groups. The results are reported using means \u00b1 standard deviations. Statistical significance was indicated if P < 0.05.A statistical comparison between two samples was performed using the Student's In order to determine whether primary PCa cells cooperate with OBs to promote the malignant transformation of PCa, samples of PCa CM, OBCM, PZ-OBCM and PCa-OBCM were collected for Transwell assay analyses of cancer cell motility and invasion Fig. A. Our stA cytokine array that examined levels of candidate protein secretion from PCa-OBs showed higher WISP-1 expression in PCa-OBCM compared with OBCM Fig. A&B. TreaTo examine WISP-1 protumor functions, recombinant human WISP-1 (rhWISP-1) protein was used to measure EMT function in PCa cells. We observed that rhWISP-1 treatment elevated N-cadherin and Twist expression and reduced E-cadherin expression in PCa cell lines DU145 and PC-3 and cathepsin L (cathepsin L-S) levels were elevated in DU145 CM compared with PZ CM and promote tumor development IMGC936 (an anti-ADAM9 antibody drug conjugate developed by MacroGenics) is undergoing Phase 1 investigations involving patients with TNBC, nonsquamous non-small cell lung cancer, colorectal cancer, gastroesophageal cancer, or pancreatic cancer (NCT04622774). Identifying novel ADAM9 inhibitors from cell-based assays may encourage the design of more selective compounds that help to block ADAM9-induced promotion of cancer + cancer stem-like cells can switch to EMT cells via TGF\u03b21-CD44 signaling and drive PCa metastasis The initiation of metastasis requires tumor cells to exhibit an invasive ability, which is enabled by the development of the EMT process in cancer cells in vivoSince PCa-derived factors are favorable for OB differentiation and mineralization in the bone microenvironment, PCa bone metastases are mainly characterized as osteoblastic (forming new bone), according to radiographic and/or pathologic appearances of the lesions In conclusion, our study demonstrated that the ADAM9/WISP-1 axis triggers primary prostate tumor growth and distant metastasis by cooperating with OBs in bone. Interfering with ADAM9 expression in primary PCa interrupts the formation of the vicious cycle between primary PCa cells and OBs, impairing PCa-OB formation, tumor growth and metastatic spread Fig. .Supplementary materials and methods, figures.Click here for additional data file."} +{"text": "The diversification of B-cell receptor (BCR), as well as its secreted product, antibody, is a hallmark of adaptive immunity, which has more specific roles in fighting against pathogens. The antibody diversification is from recombination-activating gene (RAG)-initiated V(D)J recombination, activation-induced cytidine deaminase (AID)-initiated class switch recombination (CSR), and V(D)J exon somatic hypermutation (SHM). The proper repair of RAG- and AID-initiated DNA lesions and double-strand breaks (DSBs) is required for promoting antibody diversification, suppressing genomic instability, and oncogenic translocations. DNA damage response (DDR) factors and DSB end-joining factors are recruited to the RAG- and AID-initiated DNA lesions and DSBs to coordinately resolve them for generating productive recombination products during antibody diversification. Recently, cohesin-mediated loop extrusion is proposed to be the underlying mechanism of V(D)J recombination and CSR, which plays essential roles in promoting the orientation-biased deletional end-joining . Here, we will discuss the mechanism of DNA damage repair in antibody diversification. H (variable), D (diversity), and JH (joining) gene segments in different combinations (L and JL segments (The B-cell receptor (BCR) and antibody comprise two pairs of immunoglobulin heavy (IgH) and light (IgL) chains . The N-tinations . Also, Isegments . In a gisegments and V(D)segments to furthsegments .Hs in the 2.4\u00a0Mb distal region, a 100\u00a0Kb intervening region, and a 60\u00a0Kb region with multiple Ds followed by 4 JHs J exons J rby 4 JHs . RAG is ncer iE\u03bc . RAG binncer iE\u03bc that flasegments . The twosegments (Zhao etsegments . V(D)J r)J exons (Alt et )J exons .Hs) that lie 100\u2013200\u00a0kb downstream, to change the antibody isotype with different pathogen-elimination functions (H has an inducible (I) promoter exon, long (1\u201312\u00a0kb) repetitive switch (S) region, and several CH exons (After V(D)J recombination is completed, immature B cells migrate to some peripheral lymphoid organs such as the spleen and further develop to become mature B cells . Withoutunctions , where they are further matured by introducing somatic hypermutation (SHM) into the V(D)J exons Pilzeck. In respRAG endonuclease is a Y-shaped heterotetramer, which contains two units of RAG1 catalytic enzymes and two units of RAG2 regulatory co-factors . Both RARAG not only binds the bona fide RSSs flanked by the V, D, and J gene segments for physiological V(D)J recombination but also can capture and cut cryptic targets besides RSSs at a low frequency , which mH recombination and VH to DJH recombination. The plasmid-based studies indicate that the RSS sequence, not RAG scanning, determines the utilization of D-RSSs (v-Abl progenitor (pro)-B-cell lines supports that RSS orientation, not the RSS sequence, plays a key role in deletional D to JH recombination, indicating that RAG scanning promotes the utilization of the downstream D-RSSs during physiological D to JH recombination J recombination. RAG activity mainly focuses on the targets within the dynamic chromatin impediments including the CTCF-bound chromatin, highly transcribed chromatin, RAG-bound chromatin, and even catalytic-dead Cas9-bound chromatin (D to J(3\u2032CBEs) . VH to Dream VHs . The dep-B cells . Moreovestal VHs , which ihromatin . The afoIntrinsic and extrinsic stress-induced DSBs are the most harmful DNA lesions to genome integrity, which trigger DNA damage response (DDR) by recruiting DDR factors to the DSBs for repairing. ATM and its downstream phosphorylated targets are the key DDR factors, which play crucial roles in repairing general DSBs and maintaining genome stability .RAG-initiated DSBs also recruit DDR factors during V(D)J recombination. ATM and ATM-phosphorylated p53 are recruited to the RAG-initiated DSBs to surveil the intermediates in V(D)J recombination, protecting against the potentially aberrant oncogenic translocations . Also, cIntrinsic and extrinsic stress-induced DSBs are mainly repaired by homologous recombination (HR) and C-NHEJ. HR mainly functions in the late S and G2 phases, which uses sister chromatids as templates for error-free DNA repair. C-NHEJ repairs almost all DSBs outside of S and G2 phases and is the major DSB repair pathway in both dividing and non-dividing cells .RAG-initiated DSBs are exclusively repaired by C-NHEJ, resulting from the synapsis of breaks held by the RAG post-cleavage complex (PCC) Teng an. RAG2 trIn addition to the conserved core C-NHEJ factors, there are several other C-NHEJ factors including DNA-PKcs, Artemis, XLF, and PAXX. DNA-PKcs is recruited to the RAG-initiated coding ends and phosAID is essential for both CSR and SHM . As a paBER and MMR are two complex DNA repair processes which can function as error-free repair and error-prone repair Hwang e. BER repDDR factors can also be recruited to the AID-initiated DNA lesions, and these DDR factors are required for CSR as the deficiency of the individual ATM, H2AX, or 53BP1 decreases the CSR frequency . 53BP1 aThe core C-NHEJ factor deficiency completely abolishes V(D)J recombination and blocks lymphocyte development, while core C-NHEJ factor-deficiency only decreases but not abrogates CSR, suggesting other less efficient end-joining pathways can join the AID-initiated breaks when C-NHEJ is absent during CSR . This levia an unprecedented mechanism during CSR.C-NHEJ is the major DSB end-joining pathway during CSR . The defA-EJ is activated when C-NHEJ or DDR factors are absent during CSR . The defAID-initiated CSR occurs within the \u223c200\u00a0kb constant region of the IgH locus in mature B cells. Chromatin loop extrusion is proposed to be the underlying mechanism of CSR, which promotes the formation of the CSR center, transcriptional activation of acceptor S regions, synapsis of donor S\u03bc and an activated acceptor S region, and deletional joining of AID-initiated DSBs during CSR Zhang X. In addiIn resting B cells, cohesin is loaded onto either the active iE\u03bc-S\u03bc region or the downstream 3\u2032RR enhancer region to initiate loop extrusion. Cohesin-mediated loop extrusion brings these two active regions, namely, iE\u03bc-S\u03bc and 3\u2032RR into proximity to form a basal loop, in which the iE\u03bc-S\u03bc and 3\u2032RR serve as dynamic loop anchors. This basal loop is termed as a dynamic CSR center . When B AID can target different locations of the synapsed donor S\u03bc and acceptor S region at different times within the CSR center. Once AID initiates a DSB within an S region, the DSB ends will be pulled toward the opposite direction by loop extrusion and stalled by the associated cohesin rings. The two pairs of ends held by cohesin rings will be joined deletionally to generate the productive CSR products . The disvia the ATR/Chk1 axis is downregulated by the transcription factor Bcl-6 in GC B cells, suggesting that negative regulation of the ATR/Chk1 axis is required for efficient SHM in vivo J exons get mutated . Unlike in vivo . However in vivo . So, theBCRs and antibodies play vital roles in protecting against antigens. The diversification of BCRs and antibodies from RAG-initiated V(D)J recombination, AID-initiated CSR, and V(D)J exon SHM is crucial for efficient elimination of antigens. However, the mechanisms of these complicated antibody diversification processes are still not well understood. The immunoglobulin genes must be tightly regulated to generate the large amounts of highly efficient antibodies, meanwhile, suppress the generation of undesired translocations or mutations. So, there are still many puzzling questions: how do B cells minimize the off-target effects of RAG and AID during antibody diversification and what are the mechanisms of their specificities? How DNA repair factors/pathways are differentially regulated for the general DNA damage and immunoglobulin gene recombination? Whether cohesin-mediated loop extrusion plays more roles in antibody diversification? Answers to these questions provide not only insights into the understanding of antibody diversification during B-cell development but also the basis for understanding the immune-related diseases. Moreover, the mechanism of antibody diversification has a wide range of applications for drug development of related diseases such as COVID-19 and HIV."} +{"text": "STRN-ALK fusion is a rare ALK rearrangement identified in non-small cell lung cancer (NSCLC) patients. Here, we reported a case of lung adenocarcinomas with brain metastasis, harboring STRN-ALK fusion, responded well to ensartinib. This case report could provide more information for the therapeutic strategy selecting of NSCLC patients harboring STRN-ALK fusion. EML4-ALK fusion is the most common ALK rearrangement detected in non-small cell lung cancer (NSCLC) patients. Effectiveness of ALK tyrosine kinase inhibitors (TKIs) in NSCLC patients with EML4-ALK fusion could be easily obtained from randomized controlled trials. However, response of NSCLC with non-classic ALK fusions to ALK-TKIs has rarely been reported. Ensartinib is a second-generation small molecule inhibitor that inhibits ALK tyrosine kinase activity. eXalt3 trial demonstrated ensartinib had superior efficacy as compared with crizotinib for the treatment of ALK-positive NSCLC patients . In addiA-67-year old male patient presented with chief complaint of cough for 1 month in June 2019. He had a history of smoking for 40 years and no alcohol consumption. Computed tomography (CT) demonstrated: (1) a nodule of 20 \u00d7 19 mm in his upper lobe of the left lung; (2) nodular thickening of both side of the pleura; (3) mediastinal lymph nodes enlargement; (4) left pleural effusion . No bonehttp://bigd.big.ac.cn/gsub/ accessed on 1 August 2022) with accession number HRA002593.According to NSCLC guidelines, molecular testing was preformed using patient\u2019s tissue sample. DNA next generation sequencing (NGS), including 825 cancer-related genes panel, revealed an STRN:exon3-ALK:exon20 fusion with variant allele frequency (VAF) 24% A, an ALKCrizotinib was administrated as first-line treatment of this patient, with a dose of 250 mg twice daily, according to previous case reports at that time. The patient responded well two months post-treatment . And meaSimultaneously, DNA-NGS assay of patient\u2019s cerebrospinal fluid biopsy was performed, but no resistance mutations was identified. Ensartinib was administrated as second-line treatment, with a dose of 225 mg once daily. However, dose reduction was conducted because of persistent drug-induced fever , to 100 mg once daily. Two months after switch to ensartinib, MRI showed his brain metastasis disappeared, and serum CEA level reduced to 9.60 ng/mL . This reSTRN-ALK fusion is a rare type of ALK rearrangement, especially in NSCLC. Treatment design for NSCLC patients with rare ALK fusion usually depended on previous case reports. In recent years, several cases of NSCLC with STRN-ALK fusion have been reported . From thEnsartinib is a second-generation ALK-TKI. In the phase I/II eXalt2 trial, ensartinib demonstrated to be associated with high systemic as well as central nervous system response rates in patients harboring ALK fusion who resistant to prior crizotinib treatment . In addiIn conclusion, this case provided a valuable clinical evidence of NSCLC patient with brain metastasis harboring STRN-ALK fusion treated effectively with ensartinib."} +{"text": "Major advances in cancer treatment have emerged with the introduction of immunotherapies using blocking antibodies that target T-cell inhibitory receptors, such as programmed death-1 (PD-1) and cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4), known as immune checkpoints. However, most cancer patients do not respond to immune checkpoint blockade (ICB) therapies, suggesting the development of resistance mechanisms associated with either an insufficient number of preexisting tumor-specific T-cell precursors and/or inappropriate T-cell reactivation. To broaden clinical benefit, anti-PD-1/PD-1 ligand (PD-L1) neutralizing antibodies have been combined with therapeutic cancer vaccines based on non-mutant and/or mutant tumor antigens, to stimulate and expand tumor-specific T lymphocytes. Although these combination treatments achieve the expected goal in some patients, relapse linked to alterations in antigen presentation machinery (APM) of cancer cells often occurs leading to tumor escape from CD8 T-cell immunity. Remarkably, an alternative antigenic peptide repertoire, referred to as T-cell epitopes associated with impaired peptide processing (TEIPP), arises on these malignant cells with altered APM. TEIPP are derived from ubiquitous non-mutant self-proteins and represent a unique resource to target immune-edited tumors that have acquired resistance to cytotoxic T lymphocytes (CTLs) related to defects in transporter associated with antigen processing (TAP) and possibly also to ICB. The present review discusses tumor-associated antigens (TAAs) and mutant neoantigens and their use as targets in peptide- and RNA-based therapeutic cancer vaccines. Finally, this paper highlights TEIPP as a promising immunogenic non-mutant neoantigen candidates for active cancer immunotherapy and combination with TAA and mutant neoantigens. Combining these polyepitope cancer vaccines with ICB would broaden T-cell specificity and reinvigorate exhausted antitumor CTL, resulting in the eradication of all types of neoplastic cells, including immune-escaped subtypes. These mutant neoantigens have opened up new perspectives in active immunotherapy to a wide range of cancer types without the need for isolating tumor-reactive CTL clones and establishing autologous cancer cell lines [Current cancer immunotherapies are designed to boost spontaneous antitumor T-cell response either via 1) administration of blocking monoclonal antibodies targeting T-cell inhibitory receptors, such as cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) and programmed death-1 (PD-1) ; 2) adopnt cells , 4; or 3nt cells \u20137. In thnt cells . More rell lines , 9.2m) complexes. A CTL response to tumor cells was demonstrated by isolating CD8+ T cells from patients with cancers such as melanoma and lung carcinoma, capable of mediating specific cytotoxic activity against autologous tumor cells [+ T lymphocytes in spontaneously regressing melanomas further strengthened the concept of tumor-specific CTL immunity [+ T cells and CD8+CD103+ resident memory T cells correlated with better survival for treatment-naive cancer patients and, to some extent, improved response to immune checkpoint blockade (ICB) [It is generally agreed that CTL is major effector of adaptive T-cell immunity and an ideal weapon to specifically combat cancers. They are capable of destroying transformed cells upon recognition, via the TCR, of specific epitopes presented on the target surface by major histocompatibility complex class I (MHC-I)-beta-2-microglobulin \u201314. Respde (ICB) \u201317. Thesde (ICB) . The prede (ICB) , 20.The identification in the early 1990s of TAA recognized by T lymphocytes represents a paramount advance in our knowledge of cancer immune surveillance and the antitumor T-cell response. It also opened up new perspectives in the field of immunooncology and cancer immunotherapy. The first human tumor antigen recognized by autologous CTL, named melanoma-associated antigen-1 (MAGE-1), was identified using a genetic method . SubsequTAA was used for the development of therapeutic cancer vaccines aimed at priming and/or strengthening a preexisting antitumor CTL response . Unlike + T-cells, long peptides (25\u201350 amino acids) must be processed by APCs to trigger a specific T-cell response [+ and CD4+ T-cell immunity, leading to a stronger antitumor response [+ T-cells and helps to generate potent CTL and broaden the CD8+ T-cell repertoire. Furthermore, the development of messenger RNA (mRNA)-based vaccines offer promising opportunities in cancer therapeutics. In this regard, technological innovations have made mRNA an attractive tool candidate with rapid, inexpensive, and large-scale production compared to other vaccines [in vivo half-life can be regulated by various delivery methods and modifications [in vitro transcription [Saccharomyces cerevisiae showed strong activation of immune cells that inhibited tumor growth in various syngeneic mouse models [The efficacy of peptide-based vaccines is dependent on the vaccination route, the quality of the adjuvant used, and the length of the synthetic peptides. While short synthetic peptides (8\u201311 amino acids) bind directly to human leukocyte antigen class I (HLA-I) molecules to prime antigen-specific CD8response , 29. In response , 30\u201332. vaccines . Althougvaccines . Indeed,ications . To thiscription , 37. Thecription . Anothercription . Particucription \u201342. Recee models .First-generation peptide vaccines with non-mutant TAA, such as MAGE-A3, NY-ESO-1, tyrosinase, TRP-2, or MART-1 delivered with an adjuvant or pulsed on autologous or allogeneic DC, resulted in clinical responses in only a limited number of cancer patients , 44\u201346. + T-cell response, 6/6 of neoantigen-pulsed DC-based vaccines induced strong T-cell response [WES combined with RNAseq and T-cell epitope prediction programs permitted the identification of cancer-specific antigens generated by somatic mutations in individual patient tumors . These nresponse .In humans, mutant neoantigens were used for the design of personalized immunotherapy aimed at specifically targeting tumors of individual patients . It shouin vivo transfer of mutation-specific T cells induced by RNA mutanome vaccine resulted in beneficial antitumor effects [2m-deficient malignant cells as an acquired resistance mechanism, was observed in responding melanoma patients [2m, is frequently used by tumors to evade CTL recognition and elimination. Among additional mechanisms used by malignant cells to escape specific CD8 T lymphocytes, alterations in TAP play an important role by inducing a severe decrease in the expression of MHC-I/\u03b22m-peptide complexes on the surface of cancer cells enabling escape from TCR-mediated cytotoxicity [Unfortunately, most personalized peptide vaccines do not improve the survival of patients with advanced NSCLC . The lim effects . In hepa effects . Howeverpatients . Indeed,toxicity \u201394. Constoxicity , 20.2m complexes and are presented on the membrane of APCs for CD8 T-cell recognition. However, under continuous CTL pressure, cancer cells frequently downregulate TAP1 and/or TAP2 to evade T-cell destruction [low cancer cells to TCR-mediated killing. Remarkably, an alternative antigenic peptide repertoire, referred to as TEIPP, arises on these MHC-Ilow neoplastic cells [16\u201325) [16\u201325 epitope was recognized on the HLA-A2low NSCLC cell line by an autologous CTL clone isolated from patient tumor-infiltrating lymphocytes. It is derived from the C-terminal region of the leader sequence of the ppCT precursor protein and is processed independently of TAP, by signal peptidase and signal peptide peptidase [16\u201325, represent attractive candidates for therapeutic vaccines targeting immune-escape tumors. Indeed, tumors often evade CD8 T-cell immunity by downregulating TAP1 and/or TAP2 peptide transporters. In this context, TEIPP constitutes a category of immunogenic non-mutant neoantigens that emerge during tumor immune evasion, and thus correspond to promising candidates for cancer immunotherapy [Most antigenic peptides recognized by CTL originate from the degradation of intracellular proteins in proteasomes, and the transport of the generated peptides from the cytosol into the endoplasmic reticulum by TAP complexes. Peptides of nine to ten amino acids then bind to MHC-I-\u03b2truction , 96. Indic cells . These eic cells . In this [16\u201325) , 98. Theeptidase , 98. Thieptidase . These notherapy , 100.16\u201325 TEIPP, delivered with poly(I:C) adjuvant. This active immunotherapy was able to induce efficient antitumor T-cell responses against APM-impaired tumors transplanted into HLA-A*0201/HLA-DR3-transgenic (HHD-DR3) mice and NOD-scid-Il2r\u03b3null (NSG) mice adoptively transferred with human HLA-A2+ peripheral blood mononuclear cells (PBMCs). This resulted in the control of tumor growth and regression of established tumors expressing low levels of HLA-A2/ppCT complexes [The development of innovative immunotherapies based on tumor non-mutant neoantigens that are selectively presented by malignant cells carrying APM defects and that are competent in inducing a specific CTL response able to destroy such transformed cells represents both a major challenge and a new hope in cancer treatment. In this context, we recently provided proof of concept of a ppCT peptide vaccine based on a cocktail of five immunogenic peptides, including the ppCTomplexes . Along tomplexes . These romplexes . Therefoomplexes , 100.Recent technological advances in identifying mutation-derived tumor antigens have enabled the development of patient-specific therapeutic vaccines that target individual cancer mutations. The association of mutant neoantigens with shared TAA and non-mutant neoepitopes would allow targeting tumor heterogeneity to eliminate all types of malignant cells, including those with APM defects. These polyepitope cancer vaccines combined with ICB will broaden T-cell specificities and reinvigorate exhausted antitumor CTL. However, a difficulty remains in the selection of the type of antigens to include in the vaccine. The conformation of the antigen and the targeted HLA could promote distinct responses between individual patients , 103. Th"} +{"text": "Despite literature\u2019s proofs about their safety, concerns arose regarding adverse events due to Covid-19 vaccines, including the possible impact on fertility, accentuated by misinformation and anti-vaccine campaigns. The aim of this study was to evaluate the Covid-19 vaccines\u2019 impact on male and female fertility.2 statistics was used to assess statistical heterogeneity.PubMed, Scopus, Web of Science, Cochrane and Embase databases were searched for eligible studies until March 7th, 2022. Primary studies investigating the Covid-19 vaccines impact on male and female fertility, were included. Studies\u2019 quality was assessed by the Newcastle-Ottawa and the Before and After Quality Assessment scales for cohort and pre-post studies, respectively. Random-effect meta-analyses were performed for parameters considered in \u2265 2 studies, calculating means, p-values and 95% Confidence Intervals (CIs). IOut of 1406 studies screened, 20 studies were included in the systematic review. These studies, conducted in Israel (35%), USA (30%), Russia (25%), China (5%) and Italy (5%), were of poor (15%), moderate (75%) and good (10%) quality. Meta-analyses among five studies considering several vaccines were performed for pre- and post-vaccination sperm progressive motility and concentration . Subgroup meta-analyses based on the type of vaccine showed no significant difference: between vaccinated with mRNA vaccines and non-vaccinated regarding biochemical pregnancy rates; pre- and post-vaccination with Gam-COVID-Vac regarding testosterone, FSH and LH levels; pre- and post-vaccination with BNT162b2 vaccines regarding sperm volumes.There is no scientific proof of any association between Covid-19 vaccines and infertility in men or women. Misinformation and doubts about vaccines should be properly addressed.\u2022\u2002The doubts regarding Covid-19 vaccines\u2019 impact on both male and female fertility resulted to be unfounded. Covid-19 vaccines remain the most important weapon to fight the pandemic.\u2022\u2002It is important to keep providing to public opinion and health care providers evidence-based scientific information, in order to effectively combat misinformation and anti-vaccines campaigns."} +{"text": "Clinical rebound of COVID-19 after nirmatrelvir/ritonavir treatment has been reported. We performed clinical, virologic, and immune measurements in seven patients with symptomatic rebound, six after nirmatrelvir/ritonavir treatment and one without previous treatment. There was no evidence of severe disease or impaired antibody and T-cell responses in people with rebound symptoms. To address these questions, we performed detailed virologic and immunologic evaluations of seven patients with rebound COVID-19.Nirmatrelvir-ritonavir (NMV-r) has been granted an Emergency Use Authorization for treatment of early mild-moderate COVID-19 after demonstrating an 89% relative risk reduction of hospitalization or death in unvaccinated patients at high risk for severe disease, with an associated decrease in nasopharyngeal viral load at day-five.NCT04401436. Soluble biomarkers were measured using enzyme-linked immunosorbent assay (ELISA) or electrochemiluminescence assays. Viral isolation on Vero E6 cells and targeted viral sequencing were performed on SARS-CoV-2 positive nasal swab samples, as previously described.6 Serum SARS-CoV-2 nucleocapsid protein was measured using a single-molecule immunobead assay.7 ELISA was performed for SARS-CoV-2 anti-spike (anti-S) and anti-receptor binding domain (anti-RBD) IgG, IgA, and IgM and for IgG and IgM anti-nucleocapsid antibodies using a previously validated technique8. The GenScript cPass assay, a surrogate viral neutralization test (sVNT)9, was done to detect neutralizing antibodies against wild-type and Omicron spike protein. T-cell stimulation assays using peptide pools corresponding to SARS-CoV-2 spike, nucleocapsid, and membrane proteins were performed.Detailed methods are provided in Seven patients with rebound COVID-19 symptoms were seen, two with samples also collected during their acute presentation. Five COVID-19 (Omicron)-infected patients were included as controls. Based on time from symptom onset to sample collection, patients were subdivided into an acute and late group that were compared to the rebound patients who received NMV-r (n=6) . All par10 such as IL-6 or IL-8, were downtrending at rebound, whereas markers of T-cell activation like interferon-gamma and soluble CD25 were stable or increasing (NMV-r was started 1\u20134 days after initial symptom onset. All rebound patients experienced significant symptomatic improvement prior to worsening. Median time to symptom recurrence was 12.5-days after initial symptom onset and 6.5-days after completing NMV-r. On rebound, five patients reported milder, one worse and one similar symptom severity from initial illness . No addicreasing .9 assays showed high levels of wild-type spike neutralizing antibodies in all patients with lower percent binding inhibition to Omicron-spike protein, especially in the acute group (Anti-S and anti-RBD IgG antibodies were at high levels in both groups \u2013G, consite group \u2013J. More te group .+ T-cells were seen in those with rebound or late presentation (+ T-cell responses against nucleocapsid and membrane proteins were also higher in the late and rebound cases (+ T-cells (CD4 and CD8 T-cell counts increased in rebound and late cases \u2013B. Robusentation \u2013E. CD4+ nd cases \u2013E. Two pnd cases . They bond cases \u2013G at reb T-cells \u2013I.2 Currently, NMV-r is widely used in breakthrough infections by the Omicron variant, which may impact the incidence of clinical rebound. Larger studies are required to determine the incidence and risk factors for rebound COVID-19 in those treated versus not treated with NMV-r.Nirmatrelvir/ritonavir has been a long-awaited addition in the COVID-19 therapeutic armamentarium as an outpatient oral medication that can improve disease prognosis in high-risk patients. Cases of clinical rebound after NMV-r reported recently have raised concerns about clinical deterioration and interference of early antiviral administration with adaptive immune responses. The licensing trial did not identify significant differences in viral rebound incidence among NMV-r versus placebo in unvaccinated patients during the delta variant wave.11 Additionally, we detected rising T-cells and robust SARS-CoV-2 specific T-cell responses at rebound, that were greater than those in early acute COVID-19. Two patients with longitudinal sampling demonstrated an increase in both antibody and cellular immune responses during rebound compared to their acute presentation. These findings refute the hypothesis that impaired adaptive immune responses contribute to symptomatic rebound.In our case series, which includes one patient with rebound without prior antiviral therapy, no patients developed severe disease or required additional therapy. High levels of SARS-CoV-2 anti-S IgG antibodies were detected in all patients consistent with prior vaccination. Anti-nucleocapsid IgG and Omicron-specific neutralizing antibodies were higher in patients with rebound consistent with development of humoral immunity, that usually occurs 2\u20133-weeks post-infection.12 SARS-CoV-2 was isolated from culture in one of seven patients with rebound and in three of four tested controls with acute infection probably signifying higher transmission potential in early disease. In a recent report, virus growth was observed in samples from three of seven rebound patients after NMV-r12, however, different methodologies may explain this disparity. Interestingly, our rebound patient with culturable virus had underlying immune suppression raising the question of longer treatment for immunocompromised persons.Resistance mutations were not identified at COVID-19 rebound, consistent with prior reports.13 Symptoms may be more clinically evident after use of potent antiviral treatment with quick clinical improvement and re-appearance of antigen at the time of a maturing immune response.Overall, our findings of lower levels of serum nucleocapsid antigen and downtrending innate immune markers with an emerging adaptive immune response in rebound COVID-19, do not support uncontrolled viral replication driving inflammation with significant risk for impending disease progression. Increases in total and virus-specific T-lymphocytes, biomarkers of T-cell activation, and antibodies suggest that the rebound symptoms may in fact be partially driven by the emerging immune response against residual viral antigens possibly shed from dying infected cells due to cytotoxicity and tissue repair throughout the respiratory tract.In conclusion, this case series provides important insights into the pathophysiology of rebound COVID-19. None of these patients developed severe disease, and adaptive immunity against SARS-CoV-2 appeared intact. Our findings support the need for isolation and the consideration for prolonged or additional therapies for immunocompromised patients who cannot rely on adaptive immune responses. Evaluation of larger cohorts is required to further assess the incidence, clinical and, importantly, epidemiologic implications of rebound COVID-19.Supplement 1"} +{"text": "Metastatic disease is the leading cause of morbidity and mortality in prostate cancer. Current therapeutic strategies mostly target cancer cells with important systemic effects injuring normal cells and healthy tissues. In prostate cancer patients with less than five metastases , this could be circumvented by implementing metastases-directed therapies , potentially avoiding systemic toxicity. In this setting, more accurate disease staging will likely result in more patients receiving the appropriate treatment, with expected better oncological results. On this basis, we verified the impact of two different radiotracers for Positron Emission Tomography/Computed Tomography imaging used as the guide for metastases-directed therapy on the oncological outcome in a retrospective sample of prostate cancer patients having less than five distant metastases. Obtained data showed that using next-generation imaging with [68Ga]Ga-prostate-specific membrane antigen-11 Positron Emission Tomography/Computed Tomography might favourably impact the oncological outcome of oligometastatic prostate cancer patients treated with metastases-directed therapy.p < 0.05). Coherently, the radiotracer used for PET-guided MDT resulted in predictive PFS at the univariate analysis, as well as the castration-resistant status at the time of MDT and the PSA nadir after MDT. However, in the multivariate analysis, castration resistance and PSA nadir after MDT remained the sole independent predictors of PFS. In conclusion, in the present proof-of-concept study, [68Ga]Ga-PSMA-11 provided higher PFS rates than [18F]F-Fluorocholine imaging in oligometastatic PCa patients receiving PET-guided MDT. Although preliminary, this finding suggests that enlarging the \u201ctip of the iceberg\u201d, by detecting a major proportion of the submerged disease thanks to next-generation imaging may favourably impact the oncological outcome of oligometastatic PCa treated with MDT.The superior diagnostic accuracy of [68Ga]Ga-prostate-specific membrane antigen-11 (PSMA) ([68Ga]Ga-PSMA-11) compared to [18F]F-Fluorocholine Positron Emission Tomography/Computed Tomography (PET/CT) in Prostate Cancer (PCa) is established. However, it is currently unclear if the added diagnostic accuracy actually translates into improved clinical outcomes in oligometastatic PCa patients treated with [68Ga]Ga-PSMA-11 PET-guided metastasis-directed therapy (MDT). The present study aimed to assess the impact of these two imaging techniques on Progression-Free Survival (PFS) in a real-world sample of oligometastatic PCa patients submitted to PET-guided MDT. Thirty-seven oligometastatic PCa patients treated with PET-guided MDT were retrospectively enrolled. MDT was guided by [18F]F-Fluorocholine PET/CT in eleven patients and by [68Ga]Ga-PSMA-11 PET/CT in twenty-six. Progression was defined as biochemical recurrence (BR), radiological progression at subsequent PET/CT imaging, clinical progression, androgen deprivation therapy initiation, or death. Clinical and imaging parameters were assessed as predictors of PFS. [18F]F-Fluorocholine PET-guided MDT was associated with significantly lower PFS compared to the [68Ga]Ga-PSMA-11 group (median PFS, mPFS 15.47 months, 95% CI: 4.13\u201338.00 vs. 40.93 months, 95% CI: 40.93\u201340.93, respectively; The primary management of metastatic prostate cancer (PCa) is androgen deprivation therapy (ADT). However, even though many patients can undergo ADT for years before progression or failure, it rarely eradicates metastatic disease permanently . This isThe role of MDT has been widely explored in oligometastatic PCa. Patients treated with MDT presented a significantly prolonged time to initiation of ADT ,8,9. MorIn recent years, Positron Emission Tomography/Computed Tomography (PET/CT) has dramatically improved the detection of PCa recurrences compared to conventional imaging . It is pSimilarly, the hypothetical advantage of displaying cell membrane phospholipid synthesis as an index of cell growth (with radio-labelled choline) or targeting the expression of Prostate-Specific Membrane Antigen (PSMA) on PCa tumour cells through PET/CT still needs to be verified in this clinical setting. For several years, [18F]F-Fluorocholine and [11C]C-choline PET/CT have been recommended by international guidelines as the gold-standard approach for PCa restaging in the presence of biochemical relapse . RecentlOn these bases, the present study aimed to preliminarily compare the impact of these two tracers on the oncological outcome in a retrospective real-world sample of oligometastatic PCa patients subjected to PET-guided MDT.We retrospectively enrolled consecutive oligometastatic PCa patients submitted to PET-guided MDT between June 2017 and December 2021 at our institution. Patients were informed that their clinical and imaging data could have been used for retrospective research purposes and gave their written consent for usage and publication in an anonymized form. The study was conducted according to the guidelines of the Declaration of Helsinki. The local ethical committee approved the study .Inclusion criteria were: (i) histologically confirmed diagnosis of PCa; (ii) pelvic or extra-regional nodal relapse (M1a), bone metastases (M1b) detected by either choline or PSMA PET/CT imaging; (iii) the presence of no more than five lesions at PET/CT imaging preceding MDT administration (oligometastatic PCa); (iv) upfront MDT delivered through SBRT +/\u2212 systemic therapy. The sole exclusion criterion was the unavailability of the subsequent clinical follow-up. This process led to the identification of 37 oligometastatic PCa patients (mean age 73.7 \u00b1 7.6 years old).The following demographic and clinical variables were collected: Gleason score, International Society of Urological Pathology (ISUP) grade at diagnosis, primary radical treatment, salvage radiation therapy, the time interval between diagnosis and oligometastatic state, age at MDT, number of CRPC patients at MDT, the addition of systemic treatment to MDT, number and sites of metastases treated with MDT, MDT total dose and biologically effective dose, PSA nadir after MDT, time to PSA nadir, and the number of patients which experienced progression at follow-up.[18F]F-Fluorocholine and [68Ga]Ga-PSMA-11 PET/CT were performed according to current guidelines ,16.Integrated PET/CT scanners using either Hirez-Biograph 16 or Biograph mCT Flow were used to perform a whole-body (skull vertex to the upper thighs) PET acquisition in the three-dimensional mode . A low-dose CT was performed for attenuation correction and anatomical allocation. No diagnostic contrast-enhanced CT scans were performed. Reconstruction was performed with an ordered subset expectation maximization algorithm with four iterations per eight subsets. Images were corrected for random coincidences and scatter.Images were analyzed using Syngo.via software by two experienced nuclear medicine physicians in consensus. For each lesion identified on transaxial images, a volume of interest (VOI) was drawn with an isocounter threshold based on 40% of the SUVmax. Maximum standardized uptake values (SUVmax), metabolic tumor volume , and total lesion glycolysis were collected.As established by our institutional protocol, patients were managed according to current International Guidelines and goodFollowing MDT, patients entered a short-term clinical follow-up according to our institutional protocol, including clinical evaluation and PSA blood test every 3 to 4 months. 18F]F-Fluorocholine or [68Ga]Ga-PSMA-11 PET/CT restaging was performed using the corresponding MDT-guiding radiotracer in case of detectable PSA or biochemical progression post-MDT, defined according to PC Working Group 3 [FF-FluoroThe primary endpoint of the study was Progression-Free Survival (PFS) in oligometastatic PCa patients submitted to 68Ga]Ga-PSMA-11 versus [18F]F-Fluorocholine guided MDT. Progression was defined as a composite endpoint as previously described GaGa-PSMA. Brieflyt-test, while categorical variables were tested with the chi-square test. Cox regression analysis was performed assuming PFS as the dependent variable. Univariate analyses were performed to correlate PFS with the available clinical and imaging parameters. All variables with a p < 0.100 entered the multivariate analysis. Hazard ratios (HR) for Cox regression models were reported with a 95% confidence interval (CI) and p-value. The Kaplan-Meier method (log-rank test) was also applied once continuous variables were dichotomized. Statistical significance was set at p < 0.050. Data analysis was conducted by using SPSS\u00ae software version 26 .All patients\u2019 variables were assessed with the Kolmogorov-Smirnov to evaluate data distribution. Continuous variables were compared with the The clinical characteristics of the entire sample are summarized in p = ns for all) (p = ns for both), and PSA serum levels were not significantly different between the two subgroups of patients . PET/CT imaging identified 48 lesions, including 37 lymph node metastases (77%) and 11 bone lesions (23%), while no visceral metastases were detected. Most recruited patients had one metastatic site (100% for patients receiving [18F]F-Fluorocholine-guided MDT), and the percentage of nodal and bone metastases were balanced between the two study groups (p = ns for both). Notably, the concurrent administration of medical therapy in addition to MDT was equally balanced between the two subgroups .The two subgroups were balanced concerning baseline bioptical ISUP grade, primary treatment, previous salvage radiotherapy, and time from PCa diagnosis to oligometastatic disease onset . At the y groups . No othey groups . In partp = ns). However, the PSA level at the nadir was significantly lower in patients submitted to [68Ga]Ga-PSMA-11 PET/CT guided MDT (p = 0.018). After MDT, patients were clinically and biochemically followed up for a median interval of 20.48 months (range 5.37\u201360.13), and the median PFS (mPFS) was 40.93 months (95% CI 15.93\u201340.93) .p = 0.001). Among relapsed patients, 14 (93.3%) were restaged to PET/CT imaging , and 1 died . In all cases, PET/CT restaging was performed using the same tracer previously driving MDT. In 5 cases, the persistence of the oligometastatic status was shown by the restaging PET/CT (2/5 in the [18F]F-Fluorocholine subgroup and 3/5 in the [68Ga]Ga-PSMA-11 subgroup), thus guiding a second-line of MDT. Second-line MDT was performed in the same sites of initial treatment in 3 cases (2/3 in the [18F]F-Fluorocholine subgroup and 1/3 in the [68Ga]Ga-PSMA-11 subgroup).Of the 15 (40.5%) patients presenting biochemical relapse after MDT, the number of progressions was higher in the [18F]F-Fluorocholine compared to the [68Ga]Ga-PSMA-11 subgroup , PSA nadir after MDT and by the use of [18F]F-Fluorocholine PET/CT imaging as a guide for MDT . Kaplan-Meier analysis was performed to explore better the impact of the two PET/CT radiotracers on PFS (p = 0.047). CRPC status and PSA nadir after MDT turned out to be independent predictors of PFS at the multivariate analysis III Group was published, with a strong recommendation for the use of next-generation imaging in patients with biochemically recurrent prostate cancer . ShortlyThe small number of patients in our study is the major limitation of this work. Moreover, among 37 patients recruited, patients who submitted to 68Ga]Ga-PSMA-11 PET/CT-guided MDT were more numerous with respect to the [18F]F-Fluorocholine subgroup . Thus, further studies are needed to confirm our findings in larger and more homogeneous samples of oligometastatic PCa patients. Second, the real-world design of this study led to the inclusion of both hormone-sensitive and CRPC patients. Again, the limited number of patients did not allow for performing dedicated subanalyses comparing these two subgroups. Finally, it is known that ADT can negatively affect the quality of life in PCa patients 8GaGa-PSM. On thisIn conclusion, the present data suggest that enlarging the \u201ctip of the iceberg\u201d by detecting a major proportion of the submerged disease thanks to next-generation imaging may favourably impact the oncological outcome of oligometastatic PCa patients treated with MDT. Further, prospective studies with randomized designs are still needed to support the clinical adoption of [68Ga]Ga-PSMA-11 PET/CT-guided MDT."} +{"text": "Human T-cell leukemia virus type 1 (HTLV-1) is a human delta retrovirus that causes adult T-cell leukemia/lymphoma (ATL) in 3\u20135% of the infected population after decades of clinical latency. HTLV-1 Tax is a potent activator of IKK/NF-\u03baB and a clastogen. While NF-\u03baB activities are associated with cell survival and proliferation, constitutive NF-\u03baB activation (NF-\u03baB hyperactivation) by Tax leads to senescence and oncogenesis. Until recently, the mechanisms underlying the DNA damage and senescence induced by Tax and NF-\u03baB were unknown. Current data indicate that NF-\u03baB hyperactivation by Tax causes the accumulation of a nucleic acid structure known as an R-loop. R-loop excision by the transcription-coupled nucleotide excision repair (TC-NER) endonucleases, Xeroderma pigmentosum F (XPF), and XPG, in turn, promotes DNA double-strand breaks (DSBs). NF-\u03baB blockade prevents Tax-induced R-loop accumulation, DNA damage, and senescence. In the same vein, the silencing of XPF and XPG mitigates Tax senescence, while deficiency in either or both frequently occurs in ATL of all types. ATL cells maintain constitutively active NF-\u03baB, accumulate R-loops, and resist Tax-induced senescence. These results suggest that ATL cells must have acquired adaptive changes to prevent senescence and benefit from the survival and proliferation advantages conferred by Tax and NF-\u03baB. In this review, the roles of R-loops in Tax- and NF-\u03baB-induced DNA DSBs, senescence, and ATL development, and the epigenetic and genetic alterations that arise in ATL to reduce R-loop-associated DNA damage and avert senescence will be discussed. Human T-cell leukemia virus type-1 (HTLV-1) is a complex human delta retrovirus that causes an aggressive CD4+ T-cell malignancy called Adult T-cell Leukemia/Lymphoma (ATL) . HTLV-1 De novo infection within infected individuals occurs continually [HTLV-1 is transmitted by cell-to-cell contact between infected T-lymphocytes and uninfected target cells. Human-to-human transmission occurs via the transfer of virus-infected cells through breastfeeding, transfusion of cell-containing blood or blood products, organ transplantation, and sexual intercourse. In infected individuals, HTLV-1 proviral DNAs integrate into host chromosomes. Latently infected cells are maintained by mitotic expansion. tinually , likely env and pX regions [The HTLV-1 proviral DNA is approximately 9 kb in size and is flanked by 5\u2032- and 3\u2032- long terminal repeats (LTR). The 5\u2032 side of the proviral DNA harbors retroviral genes that encode structural and enzymatic proteins . In contrast, the 3\u2032 pX region contains several overlapping open reading frames (ORFs) that encode regulatory proteins [ regions .Tax and HBZ are regulatory proteins that play indispensable roles in the HTLV-1 viral life cycle and ATL development. Tax is a weak or conditional DNA binding protein. It drives LTR-mediated viral mRNA transcription by forming protein complexes with the basic domain-leucine zipper transcription factors, CREB and ATF-1, to assemble on three composite enhancer DNA elements in the U3 region of the LTR. LTR-bound Tax, in turn, recruits transcriptional co-activators including CBP/p300 to promote robust viral trans-activation . Tax als6 bases of ATL DNA and 59.5 structural variations per ATL genome [ATL exhibits extensive genomic instability and chromosomal abnormalities compared to other lymphoid malignancies. The whole-genome and exome sequencing of 426 ATL patients had previously identified, on average, 7.9 point mutations/10L genome . The genL genome . In Tax-L genome .HBZ antagonizes many activities of Tax, including LTR and NF-\u03baB activation . It is ccis, due to RNA polymerase stalling caused by excess transcriptional activation/derepression or at sites of DNA damage) or post-transcriptionally .An R-loop is a three-stranded nucleic acid structure consisting of an RNA-DNA hybrid and a displaced single-stranded DNA loop . R-loopsV12 upregulates genome-wide transcription and R-loop formation in immortalized human fibroblasts [Co-transcriptionally formed R-loops have recently gained much attention as they regulate gene expression and predispose the genome to DNA damage. The non-template single-stranded DNA of the R-loop is vulnerable to DNA altering agents like activation-induced cytidine deaminase or DNA endonuclease and causes lesions or nicks. R-loops can also interfere with DNA replication to cause replication fork collapse and DNA DSBs . Activatroblasts . More reroblasts , a lysine 63 (K63) ubiquitin E3 ligase critical for signaling DNA DSB repair, and another ubiquitin ligase, linear (M1) ubiquitin assembly complex (LUBAC), to promote the assembly of K63-M1 hybrid polyubiquitin chains in the cytosol ,32. ThisIt should be pointed out that the impact of RNF8 dysregulation by Tax extends beyond the cytosolic activation of the kinase cascades mentioned above. Aberrant RNF8 activation by Tax in the nucleus leads to the assembly of K63-linked polyubiquitin chains that sequester DNA damage response (DDR) factors, including RNF8, BRCA1, DNA-PK, MDC1, etc., into Tax-containing pseudo-DNA damage foci known asNF-\u03baB impacts immune and inflammatory responses broadly and potently. As such, cellular pathways that lead to NF-\u03baB activation are stringently regulated by negative feedback mechanisms to ensure NF-\u03baB is active only in a short duration . As NF-\u03baCip1/Waf1 (p21) and p27Kip1 (p27) [As HTLV-1 causes ATL in infected individuals and transforms T cells in culture, it was initially thought that HTLV-1 infection induces T cells to proliferate . And sinp1 (p27) ,51,52. Np1 (p27) .Of note, the transcriptional activity of NF-\u03baB is critical for Tax-induced senescence . RNA silTax-induced senescence occurs after cellular passage through an aberrant cell cycle during which the S and G2 phases stall and mitosis is disrupted or impaired ,58. As dTranscription-coupled nucleotide excision repair (TC-NER) pathway is conserved and ubiquitous in organisms ranging from unicellular bacteria and yeast to mammals. It consists of a multiprotein repair system capable of recognizing and processing DNA structural distortions and lesions induced by UV irradiation, reactive oxygen and nitrogen species, and mutagens that introduce bulky chemical adducts in DNA. TC-NER pathway repairs DNA damage by a \u201ccut and patch\u201d mechanism. Initiation of repair occurs upon the physical blockage of RNA polymerase II (RNAPII) at the sites of DNA lesions/distortions during transcription. RNAPII stalling, in turn, gives rise to an R-loop formed by the nascent RNA transcript, the stalled RNAPII, the DNA template containing the lesion, and the single-stranded complementary DNA strand that loops out see . TC-NER When R-loops accumulate due to Tax-induced NF-\u03baB hyperactivation, they are thought to be excised much like those that form co-transcriptionally at UV-induced DNA lesions, leading to single-stranded DNA gaps. DNA replication or additional DNA incision then gives rise to DNA DSBs. Indeed, NF-\u03baB blockade prevents Tax-induced R-loop accumulation, DNA damage, and senescence ,53. The Excess R-loop accumulation threatens genomic integrity and contributes to oncogenic, neurodegenerative, and inflammatory disorders ,63,64,65The survival and proliferative advantages conferred by NF-\u03baB for ATL cells outweigh the disadvantages associated with R-loops. NF-\u03baB is constitutively active in ATL cells, and, as expected, R-loop levels therein are significantly elevated (2\u20133 fold of non-ATL control) . These dDo ATL cells mitigate the risk of excess R-loops by preventing their formation or resolving them via the cellular factors described above? Transcriptional repression of RNF8, the K63 ubiquitin E3 ligase hijacked by Tax for canonical NF-\u03baB activation, is common in ATL cells of all types . Many ATOther changes in ATL cells likely prevent the senescence response mediated by p21 and p27. In this vein, it is interesting to note that Kaposi\u2019s sarcoma-associated herpesvirus (KSHV) viral cyclin (vCyclin) forms a vCyclin-CDK6 complex that resists p21 and p27 inhibition and targets p27 for degradation to drive cell proliferation ,67. IndeR-loop accumulation is often a result of aberrant mRNA transcription, processing, or export. ,68. Excede novo HTLV-1 infections continued to occur. And clones that appeared soon after the initial infections persisted and, on rare occasions, underwent significant expansion [HTLV-1 infection in cell culture causes most cells to become senescent or senescent-like due apparently to the cytopathic effect of Tax-related DNA damage. Only a minor fraction of infected cells that do not express Tax or express it at low levels manage to survive and persist . The fatxpansion .Recent evidence demonstrates that latent HTLV-1 provirus reactivates sporadically and expresses Tax in a short, intense burst of ~19 h to induce anti-apoptotic factors that enhance cell survival. Tax inhibits DNA damage response ,34, inacRepeated cycles of viral reactivation likely also select for subclones harboring genetic or epigenetic alterations that mitigate R-loop-associated DNA DSBs and prevent senescence induction by Tax and NF-\u03baB during reactivation, setting the stage for the acquisition of gain-of-function mutations that promote Tax-independent NF-\u03baB activation. Identifying and targeting these alterations can reinstate R-loop-induced DNA damage and senescence (or apoptosis) in NF-\u03baB-addicted ATL cells."} +{"text": "Dear Editor,1 Single-cell assay for transposase-accessible chromatin using sequencing (scATAC-seq) on the next-generation sequencing (NGS) platform is a well-established method to detect open chromatin regions within an individual cell.2 However, there remain challenges in detection of large-scale structural variations and haplotype phasing from scATAC-seq data, which can be well resolved by third-generation sequencing (TGS) platform-based single-molecule long-read sequencing. To integrate advantages of long-read sequencing into scATAC-seq, we developed single-cell assay for transposase\u2010accessible chromatin on Nanopore sequencing platform (scNanoATAC-seq), a plate-based scATAC-seq method suitable for TGS platform were comparable between long-read and short-read scATAC-seq to profile distributions of peaks called from NGS-based scATAC-seq data and TGS-based scNanoATAC-seq data of GM12878 cells. Among scNanoATAC-seq peaks, we found that the proportions of CTCF-only elements and distal enhancer-like signatures (dELS) increased, while the proportions of promoter-like signatures (PLS) and proximal enhancer-like signatures (pELS) were relatively lower than NGS-based scATAC-seq as well as in human peripheral blood mononuclear cells (PBMCs). The medians of read length of our scNanoATAC-seq libraries ranged from 4000\u2009bp to 4900\u2009bp. We evaluated the quality of the data produced by our method using transcriptional start site (TSS) enrichment, fragment numbers and fractions of read ends in peaks (FRIP) and two mouse cell lines (mESC and MEF). In two technical replicates, 2.2% (5 out of 223) and 1.6% (4 out of 248) of single cells were identified as doublets of total peaks were allele-specific in GM12878. Among them, 22.4% (86 out of 384) of ASPs contained heterozygous SNPs within the peaks, and were verified by short-read bulk ATAC-seq. 90.9% and 88.1% of maternal and paternal peaks got validated, respectively in individual cells using scNanoATAC-seq data. Distinct CNV patterns were observed among different cell lines. eHAP1 is a fully haploid cell line without any large-scale CNVsSOX4 gene locus to demonstrate this strategy within an individual cell simultaneously. Meanwhile, we revealed ASPs even without heterozygous SNPs inside a peak, which is not feasible for NGS platform-based short-read scATAC-seq. We provided the direct evidence of co-accessibility between neighboring peaks from scNanoATAC-seq, where the chromatin accessibility of two sites in the same single cell and in fact on the same allele was detected simultaneously by a long read. The cost of scNanoATAC-seq library is less than 2.5 dollars per cell under current sequencing depth. As the cost of ONT is still higher than that of NGS platform at the same sequencing depth, there is a trade-off between more fragments to enrich stronger epigenetic signals and longer reads to detect long read-specific features such as SVs.Supplementary information, Figures and DataSupplementary information, Table S1Supplementary information, Table S2Supplementary information, Table S3"} +{"text": "Acute liver failure (ALF) and acute-on-chronic liver failure (ACLF), respectively, occur in patients with normal liver and patients with chronic liver diseases, including cirrhosis . In geneIn the COVID-19 era, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is also an important acute insult in ACLF patients . To someIn patients with COVID-19, drug-induced liver injury (DILI) has often been observed . In totaAlthough a recent study reportedAIH was occasionally observed after COVID-19 vaccination 14,15].,15.14,15The outbreak of acute severe hepatitis of unknown origin in children has recently been reported . Some caIn certain cases, bacterial infection is also related to the development of ACLF. Takaya et al. reported that endotoxin level was a predictive factor independently associated with ACLF development . They alA meta-analysis of published studies on patients following liver resection for hepatocellular carcinoma (HCC) demonstrated that albumin-bilirubin (ALBI) grades 2 and 3 showed increased rates of post-hepatectomy liver failure compared with patients with grade 1 ALBI 1 . ALBI grNovel strategies to treat patients with ACLF have also been under development ,25. We a"} +{"text": "Short sentences improve readability. Short sentences also promote social justice through accessibility and inclusiveness. Despite this, much remains unknown about sentence length perception\u2014an important factor in producing readable writing. Accordingly, we conducted a psychophysical study using procedures from Signal Detection Theory to examine sentence length perception in naive adults. Participants viewed real-world full-page text samples and judged whether a bolded target sentence contained more or fewer than 17 words. The experiment yielded four findings. First, na\u00efve adults perceived sentence length in real-world text samples quickly (median = 300\u2013400 ms) and precisely (median = ~90% correct). Second, flipping real-world text samples upside-down generated no reaction-time cost and nearly no loss in the precision of sentence length perception. This differs from the large inversion effects that characterize other highly practiced, real-world perceptual tasks involving canonically oriented stimuli, most notably face perception and reading. Third, participants significantly underestimated the length of mirror-reversed sentences\u2014but not upside-down, nor standard sentences. This finding parallels participants\u2019 familiarity with commonly occurring left-justified right-ragged text, and suggests a novel demonstration of left-lateralized anchoring in scene syntax. Fourth, error patterns demonstrated that participants achieved their high speed, high precision sentence-length judgments by heuristically counting text lines, not by explicitly counting words. This suggests practical advice for writing instructors to offer students. When copy editing, students can quickly and precisely identify their long sentences via a line-counting heuristic, e.g., \u201ca 17-word sentence spans about 1.5 text lines\u201d. Students can subsequently improve a long sentence\u2019s readability and inclusiveness by omitting needless words. Omit needless words. That self-exemplifying advice from a writing style guide helps geth grade level. Doing so fosters a demographically fair distribution of research costs and research benefits. This embraces the justice principle described in ethics documents such as the Belmont Report . On November 4, 2021, we collected data with the written informed consent of each participant. To promote reproducibility, the Open Science Framework [https://osf.io/89myj/] contains the complete data set and all software needed to replicate the experiment and the statistical analyses. In the Results, we distinguish pre-registered from exploratory analyses .To avoid the Gaussian-distribution assumption required by parametric tests, we assessed statistical significance non-parametrically. Specifically, we used a Monte Carlo bootstrapping procedure to evaluate median differences among conditions at the 0.05 alpha level. The bootstrapping procedure involved computing a simulated median difference after randomly shuffling the empirically observed data between the experimental conditions under comparison. Repeating this 10,000 times generated a distribution of simulated differences. Statistical significance occurred when the empirically observed median difference exceeded the 95th percentile of the simulated distribution. Larger median differences reflect larger effect sizes. This procedure parallels that used by and the The statistical analyses included data from participants who satisfied each of two criteria. First, as noted above, the participant had to demonstrate criterion accuracy after at least 20 practice trials . Second, on the subsequent 140 trials for analysis the participants had to achieve at least 62.86% correct .Each of the 88 participants who met criterion accuracy on practice trials also met criterion accuracy on trials for analysis. We included the data from each of those 88 participants. Nominally, this would suggest a 100% inclusion rate. However, we have no information regarding how many online participants may have started practice trials but subsequently withdrew or failed to reach criterion accuracy.Our pre-registered data analysis plan required describing the data with psychometric functions. We used the psychometric functions in precision with which participants judged sentence length. Higher d-Prime values reflect greater precision. Visually inspecting each sample reveals a slight inversion effect, i.e., slightly lower precision for flipped text (yellow boxes) than for standard text (green boxes). To evaluate this inversion effect statistically, we ran the pre-registered Monte Carlo simulations on the main effect of text-orientation: flipped versus standard text. The preregistered simulations indicated that the inversion effect reached statistical significance in Sample 2 (p = 0.021) but not in Sample 1 (p = 0.1261). An exploratory simulation combined the data from the two samples (n = 88) and revealed a statistically significant (p = 0.0067) but small inversion effect. Specifically, relative to standard text, flipped text impaired precision by 0.0976 d-Prime units. For context, this effect size corresponds to non-biased responding at 90.7% correct for standard text compared to 89.0% correct for flipped text. The main effect of flip-type (mirror-reversed versus upside-down text) was non-significant.The boxplots in The boxplots in bias with which participants judged sentence length. Within each plot the gray horizontal line at 1 marks neutral responding, i.e., using the \u201cMore-than-17-word\u201d and \u201cFewer-than-17-word\u201d response options equally often. A bias toward underestimating sentence length corresponds to criterion (Beta) values greater than 1. A bias toward overestimating sentence length corresponds to criterion (Beta) values less than 1.The boxplots in and standard sentences (green boxes). By contrast, participants randomly assigned to our upside-down groups tended to neutrally judge the length of upside-down sentences (yellow boxes) and standard sentences (green boxes). Stated differently, the main effect of flip-type (mirror-reversed versus upside-down) mattered more than did the main effect of text-orientation (flipped versus standard).Surprisingly, visually inspecting Monte Carlo simulations support these visually evident patterns. First, our pre-registered Monte Carlo simulations showed a non-significant main effect of text-orientation (flipped versus standard) within each sample. This effect remained non-significant even after increasing the statistical power by combining the samples in exploratory simulations. Second, exploratory simulations on the combined samples showed that our mirror-reversed groups underestimated sentence length significantly more than did our upside-down groups (p = 0.0023).underestimation bias by altering the miss and false alarm rates relative to those of the upside-down groups\u2019 unbiased responses. An example entails increasing the miss rate from 9.6% to 21.6% and reducing the false alarm rate from 9.6% to 4.1%. Indeed, these miss and false alarm rates generate the empirically observed median criterion (Beta) and median d-Prime values from the mirror-reversed and upside-down groups. Misses reflect sentence length underestimates; false alarms reflect sentence length overestimates.Regarding effect size, the mirror-reversed groups\u2019 median Beta value (1.351) exceeded that of upside-down groups by 35.1%. Equivalently, one can model the mirror-reversed groups\u2019 Our preregistered methods operationally defined lapses as incorrect responses on the two longest and two shortest sentence lengths. These relatively extreme sentence lengths correspond to more than three times the subsequently observed median JND threshold in each condition.Further evidence for the specificity of this under-estimation effect comes from contrasting In summary, the Lapse analyses demonstrate that participants significantly underestimated the length of mirror-reversed\u2014but not upside-down, nor standard\u2014sentences. In the Discussion we address how the specificity of this inversion effect relates to scene syntax \u201380, 85.overestimates) on the 3-line-16-word mischievous sentence than on the 2-line-16-word sentences.Recall that during our study\u2019s demonstration and practice phases, we primed participants with a sentence-length heuristic: 17-word sentences typically span ~1.5 text lines. Per our pre-registered hypotheses and research design, we probed participants\u2019 use of this heuristic via our \u201cmischievous sentence\u201d. The mischievous sentence contained only 16 words, yet appeared in three consecutive lines of text. Specifically, it began near an edge of its first line, spanned its second line, then ended near the opposite edge of its third line. This differed from the other 16-word sentences, which each spanned no more than two text lines. If used, our heuristic would generate more errors (sentence length worse (higher) than predicted by mere random responding (upper gray line at error rate = 0.68). This significant mischievous sentence effect replicated across all eight experimental conditions: flipped (yellow bars) and standard (green bars) text in each of the four groups. By contrast, the other 16-word sentences typically generated error rates lower than expected by chance (lower gray line at error rate = 0.32). The one exception (sentence 3) generated worse-than-chance (higher) error rates in one experimental condition, and chance-level error rates in the remaining seven experimental conditions. Overall, the specificity in Lastly, our pre-registered data analyses for the mischievous sentence required conducting Monte Carlo simulations to test the sentence-by-text-orientation interaction effects. Each of those simulations showed non-significant interactions. Likewise, exploratory simulations on mischievous sentence trials showed non-significant interactions between flip-type (mirror-reversed versus upside-down) and text-orientation (standard versus flipped). To summarize, the findings from our mischievous sentence manipulation suggest that, regardless of flip-type and text-orientation, participants judged sentence length by heuristically counting text lines.Short sentences play a critical role in readability . Short sFirst, na\u00efve participants precisely and quickly perceived sentence length in real-world text samples. Regarding precision, participants achieved ~90% correct responding on median, with median sentence-length Weber fractions ranging between 8.98% and 10.65%. Regarding speed, median reaction times ranged between 300 and 400 milliseconds. Moreover, 88 of 88 naive participants met the inclusion criteria. Taken together, these findings demonstrate the ease with which our naive adult participants perceived the length of target sentences in real-world English text samples.Second, flipping the text generated no reaction-time cost and nearly no loss in the precision of sentence length perception. The text-orientation effect size corresponded to non-biased 90.7% correct responding for standard text compared to non-biased 89.0% correct responding for flipped text. This robustness to global text orientation variability contrasts sharply with the large inversion effects previously reported for diverse stimuli and tasks. These include the perception of faces \u201366, bodyThird, our three-line 16-word \u201cmischievous sentence\u201d consistently generated more errors\u2014specifically, sentence length overestimates\u2014than did any of our two-line 16-word sentences. Also, unlike any of our two-line 16-word sentences, our three-line 16-word \u201cmischievous sentence\u201d consistently generated more errors (sentence-length overestimates) than predicted by mere random responding. The reproducibility and specificity of this finding suggests that participants took advantage of the heuristic that 17-word sentences typically span ~1.5 text lines. This in turn implies that the participants\u2019 high speed, high precision, and largely orientationally invariant sentence-length judgments reflect subitizing text lines \u201333, not Fourth, participants significantly underestimated the length of mirror-reversed sentences\u2014but not upside-down, nor standard sentences. Evidence for this came from our lapse analysis. Here, participants exhibited a significant bias toward classifying 23- and 24-word sentences as having fewer than 17 words, but only for mirror-reversed text. The specificity in underestimating mirror-reversed sentence length partially matches predictions from our scene syntax hypothesis. In preregistration, we predicted that participants would underestimate flipped-sentence length because mirror-reversing the text or flipping it upside-down repositions words from high-probability to low-probability locations. The data support the predicted underestimation-bias for mirror-reversed text only.Given that mirror-reversed text and upside-down text each occur rarely in real world settings, why would significant sentence-length-underestimates occur only for mirror-reversed text? One possible explanation comes from research demonstrating that spatial anchors influence visual search , 101. AnWhile left-laterally anchored scene syntax would account for the significant sentence-length underestimates observed here, we emphasize that our pre-registered hypotheses did not include that explanation. In fact, left-lateral anchoring occurred to us only after the data showed significantly greater sentence-length underestimates for mirror-reversed text than for standard and upside-down text. The post hoc nature of this explanation warrants future attempts to replicate the significant sentence-length underestimation bias observed here for mirror-reversed text.Other future studies might provide new insights about sentence length perception by building on the present experiment\u2019s task and stimuli. Our stimuli comprised real-world text examples containing a bolded target sentence among non-bolded distractor sentences. Our two-step task required (1) searching for the bolded target sentence and then (2) judging its length relative to a reference length. However, real-world text pages often contain no bolded sentences, and their absence would complicate the visual search component of the task. This suggests a future conventional visual search experiment comprising non-bolded short distractor sentences and, on half the trials, a non-bolded target sentence of reference length. Participants would report \u201ctarget-absent\u201d or \u201ctarget-present\u201d on each trial. Here, sentence length\u2014rather than bold font\u2014would distinguish targets from distractors, paralleling real-world text conditions. A finding that performance on this visual search task benefits from a line-counting heuristic\u2014as our results suggest\u2014could help writers produce more readable writing.Short sentences improve readability . ReadabiS1 Fig(JPG)Click here for additional data file.S2 Fig(JPG)Click here for additional data file.S3 Fig(JPG)Click here for additional data file.S4 Fig(JPG)Click here for additional data file.S5 Fig(JPG)Click here for additional data file.S6 Fig(JPG)Click here for additional data file."} +{"text": "Ongoing outbreaks of Middle East respiratory syndrome coronavirus (MERS-CoV) continue posing a global health threat. Vaccination of livestock reservoir species is a recommended strategy to prevent spread of MERS-CoV among animals and potential spillover to humans. Using a direct-contact llama challenge model that mimics naturally occurring viral transmission, we tested the efficacy of a multimeric receptor binding domain (RBD) particle-display based vaccine candidate. While MERS-CoV was transmitted to na\u00efve animals exposed to virus-inoculated llamas, immunization induced robust virus-neutralizing antibody responses and prevented transmission in 1/3 vaccinated, in-contact animals. Our exploratory study supports further improvement of the RBD-based vaccine to prevent zoonotic spillover of MERS-CoV.The online version contains supplementary material available at 10.1186/s42522-022-00068-9. MERS-CoV is associated with severe pneumonia and lethal disease in humans with high case-fatality rates in the Middle East . The virDromedaries are the main reservoir, although other camelid species such as llamas and alpacas are also susceptible to MERS-CoV \u201310. CameTo date, commercial vaccines and therapeutics against MERS-CoV are lacking, and the World Health Organization has advised animal vaccination as a strategy to control the spread of MERS-CoV to animals and humans . DiffereHere, we used the same direct-contact model to assess the efficacy of a virus-like particle vaccine to block MERS-CoV transmission in llamas. The vaccine was composed of self-assembling multimeric protein scaffold particles (MPSP) expressing the receptor-binding domain (RBD) of the MERS-CoV S protein . The MPSn\u2009=\u20093) and adjuvant-control administrated animals (n\u2009=\u20092) were put in direct contact with na\u00efve llamas (n\u2009=\u20092) infected with MERS-CoV throughout the study were then in direct contact with na\u00efve (grey) and vaccinated (red) animals.Additional file 2: Fig. 2. Temperature and rhinorrhoea after MERS-CoV exposure to llamas. MERS-CoV experimentally inoculated llamas (black) were, two days later, put in contact with na\u00efve (grey) and vaccinated (red). (a) Rectal temperature was measured daily after MERS-CoV. Each line/sign represents an individual animal. One na\u00efve (b) and one vaccinated, contact animal (c) showed moderate mucus excretion at 5-9 and 8-19 days post-inoculation procedure, respectively.Additional file 3. Materials and methods."} +{"text": "Several studies have examined the effects of repetitive transcranial magnetic stimulation (rTMS) on associative memory (AM) but findings were inconsistent. Here, we aimed to test whether twice-daily rTMS could significantly improve AM.In this single-blind, sham-controlled experiment, 40 participants were randomized to receive twice-daily sham or real rTMS sessions for five consecutive days . The stimulation target in left inferior parietal lobule (IPL) exhibiting peak functional connectivity to the left hippocampus was individually defined for each participant. Participants completed both a picture-cued word association task and Stroop test at baseline and 1 day after the final real or sham rTMS session. Effects of twice-daily rTMS on AM and Stroop test performance were compared using two-way repeated measures analysis of variance with main factors Group and Time (baseline vs. post-rTMS).post hoc analysis after multiple comparison correction. Further, AM improvement in the twice-daily real group was not superior to a previously reported once-daily rTMS group receiving 8,000 pulses. Then, we combined the twice- and once-daily real groups, and found a significant Group \u00d7 Time interaction effect. Post hoc analysis indicated that the AM score was significantly enhanced in the real group after multiple comparisons correction.There was a significant Group \u00d7 Time interaction effect. AM score was significantly enhanced in the twice-daily real group after rTMS, but this difference could not survive the Our prospective experiment did not show significant rTMS effect on AM, but this effect may become significant if more participants could be recruited as revealed by our retrospective analysis. Transcranial magnetic stimulation (TMS) is a non-invasive technique for modulating brain network connectivity with demonstrated therapeutic efficacy against neurological and neuropsychiatric illnesses . Wang etHowever, the physiological response to non-invasive brain stimulation is known to be highly variable among individuals , and sevIncreasing the stimulation dose is one potential method to achieve a more robust effect on AM . A recenIn the present study, we aimed to investigated whether a higher rTMS dose could produce significantly greater AM improvement, beyond the sham rTMS. To this end, we modified the paradigm of our previous study in two pForty healthy subjects with no history of rTMS, transcranial electric stimulation, neuropsychological disorders, or psychoactive drug use were recruited for this study. All participants met the safety criteria for MRI and rTMS and provThis was a randomized, single-blind, sham-controlled, parallel design study consisting of two arms, real 20-Hz rTMS over the IPL and sham rTMS (control). Forty subjects were included based on our previous work using single daily rTMS sessions. Subjects were assigned to the real or sham group according to random number selection while ensuring 20 per group. All subjects were unaware of the stimulation protocols until the end of the study (single-blind).Each participant received twice-daily rTMS sessions over five consecutive days, for a total of ten sessions and 16,000 pulses. A face-cued word recall task was using to test AM and the Stroop test to assess non-associative memory cognitive processing. Tasks were performed both 1 day prior to the first real or sham stimulation session (baseline assessment) and 1 day after the final session (post-rTMS assessment). Structural MRI and rs-fMRI were conducted on each subject prior to baseline testing to identify the IPL target site . After e2; 256 \u00d7 256 matrix; section thickness, 1 mm, without intersection gap; voxel size, 1 \u00d7 1 \u00d7 1 mm3; 188 sections). Following structural MRI scanning, functional images (217 volumes) were acquired using a single-shot gradient-recalled echo planar imaging sequence . Images of 46 transverse sections were acquired parallel to the anteroposterior commissure line. Foam fillers and earplugs were used to minimize head motion and scanner noise during image acquisition. All participants were asked to keep their eyes close and rest without falling asleep during scanning. Scanning was performed prior to AM and Stroop testing to exclude potential carry-over effects.Magnetic resonance images (MRI) were collected at the University of Science and Technology of China using a 3.0 T scanner . Functional and structural images were acquired using the same parameters as in our previous studies ,b, 2021.Cortical stimulation locations over the IPL were identified from individual resting-state functional connectivity maps with the left hippocampus as the seed region . The hip2Preprocessing consisted of seven steps: (1) deleting the first five functional volumes; (2) slice timing correction and realignment; (3) co-registration of structural and functional images; (4) normalization of functional images by the matrix computed in structural segmentation and normalization; (5) smoothing of functional images using a 4-mm full-width at half-maximum isotropic Gaussian kernel; (6) temporal band-pass filtering (0.01\u20130.1 Hz); (7) regressing out 27 nuisance signals . All processing steps were performed using Statistical Parametric Mapping (SPM) 12 and in-house software TMStarget.2 stimulator through a 70-mm air-cooled figure-of-eight coil under the guidance of a frameless stereotactic optical tracking neuronavigation system . Individ Canada) . For theFor the sham stimulation condition, participants received the same rTMS protocol using a sham coil with the same appearance as the real coil to avoid participants identifying rTMS group allocation. This sham coil generated only sound and sensations on the scalp similar to the real coil but no current .3 Subjects were instructed to pay attention and try to remember the face-word associations. After the learning phase, subjects were given a rest of approximately 1 min, followed by face ire-presentation in a different and random order. Subjects were instructed to recall the word that accompanied each face during the learning phase, and each word response was scored as correct or incorrect (with no errors relating to pronunciation). Participants received no prompts or feedback on the correctness of their answers. The number of correctly recalled face\u2013word pairs was recorded as the AM score. To account for inter-subject differences in baseline AM performance, the individual improvement in AM following rTMS was expressed as a percentage change relative to baseline [AM score percentage change = (post correct \u2013 baseline correct)/baseline correct \u00d7 100%] adapted to local Chinese was also conducted . The test-test and categorical baseline variables by 2\u03c7 test. Differences in AM and Stroop test performance were compared by two-way repeated measures analysis of variance (RT-ANOVA) with main factors Group and Time (baseline vs. post-rTMS), followed by post hoc Sidak\u2019s multiple comparison tests. AM score percentage change was compared between groups using the independent samples t-test. Outliers were identified by non-linear regression using GraphPad Prism and removed from subsequent analysis.Continuous baseline variables were compared between groups by independent samples Twice-daily dataset including Twice-daily(R) and Twice-daily(S) groups). The second analysis tested if the higher rTMS dose produced more prominent effects on AM by comparing the Twice-daily(R) group to the Once-daily(R) group. This Once-daily(R) group included 16 subjects from Twice-daily(R) and Once-daily(R) groups to produce a Combined(R) group and investigated the effects on AM compared to the Twice-daily(S) group.Three separate analyses were performed using two-way ANOVA. The primary analysis included only the current data from participants receiving sham rTMS and participants receiving real rTMS (termed the Twice-daily dataset) but one participant randomized to the sham group [Twice-daily(S)] dropped out for personal reasons. Thus, data from 39 subjects were included in the primary analyzes. Twice-daily(R) and Twice-daily(S) groups did not differ significantly in age, gender ratio, RMT, test delay , baseline AM score, or baseline Stroop test score (p = 0.12) between the Twice-daily(R) and Twice-daily(S) groups. In general, both sham and real rTMS were well tolerated, with only slight discomfort reported by some participants. But this effect disappeared after the stimulation ended. The average (\u00b1 SEM) MNI coordinate of IPL stimulation was x = \u221245.4 (0.92), y = \u221272.0 (0.83), z = 33.1 (1.01) .Twice-daily(R) and Twice-daily(S) groups are presented in Twice-daily(R) vs. Twice-daily(S)] \u00d7 Time (baseline vs. post-rTMS) interaction , but no main effect of Time or Group . Post hoc analyses using Sidak\u2019s multiple comparison test indicated that AM score was not increased significantly after real stimulation and Twice-daily(S) groups .The AM scores for the Twice-daily(R) vs. Twice-daily(S)] and Time, and no significant Group [Twice-daily(R) vs. Twice-daily(S)] \u00d7 Time (baseline vs. post-rTMS) interaction and Once-daily(R) groups, while test delay was slightly but significantly lower in the Twice-daily(R) group group .Twice-daily(R) and Once-daily(R)] \u00d7 Time (baseline and post-rTMS) interaction (Twice-daily(R) and Once-daily(R) rTMS. There was a significant main effect of Time but not Group . Average percentage change in AM score did not differ significantly between Twice-daily(R) and Once-daily(R) groups suggesti = 0.80] .Once-daily(R) and Twice-daily(R) groups are presented in Twice-daily(R) vs. Once-daily(R)] \u00d7 Time (baseline vs. post-rTMS). See details in the The response times and error rates for the Stroop tests from Combined(R) and Twice-daily(S) groups (There were no significant differences in any of the aforementioned baseline characteristics between ) groups .Combined(R) and Twice-daily(S) groups are presented in Combined(R) vs. Twice-daily(S)] \u00d7 Time (baseline vs. post-rTMS) interaction effect on AM change but no main effect of Time or Group . Further, post hoc analyses using Sidak\u2019s multiple comparison tests indicated that real stimulation significantly enhanced AM score compared to baseline (Combined(R) group than the Twice-daily(S) group (Associative memory scores for the = 0.003) while sh = 0.37) . In addi = 0.09] .Combined(R) and Twice-daily(S) groups are presented in Combined(R) vs. Twice-daily(S)] \u00d7 Time (baseline vs. post-rTMS) interaction. See details in the The Stroop test response times and error rates for post hoc analysis in real group survived the multiple comparisons correction.In the present study, we found that the twice-daily rTMS enhanced AM significantly better than the placebo effect. However, the improvement in the twice-daily real stimulation group was not significant after multiple comparisons correction. Furthermore, the twice-daily protocol was not superior to the once-daily sessions previously reported by post hoc multiple comparison tests for the twice-daily real stimulation group. As small sample size reduces statistical power (Twice-daily dataset and Once-daily dataset from our previous experiment (N = 36), and post hoc multiple comparison test analyses showed significant AM improvement. An insufficient sample size could contribute to variability and affect the reliability of non-invasive brain stimulation studies . Future large-sample prospective studies are warranted to document the reliability and duration of this AM-enhancing effect. Second, the RMT was measured only once rather than prior to each session, and a previous study reported that RMT varied significantly across days among subjects receiving rTMS .Possibly due to the small sample size, our prospective experiment did not find significant rTMS effect on memory. But this effect may become significant if more participants could be recruited as revealed by our retrospective analysis.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Anhui Medical University. The patients/participants provided their written informed consent to participate in this study.QH, G-JJ, and KW: conceptualization. QH, YZ, QL, XG, RD, YW, QZ, TZ, LZ, and JS: data acquisition. QH and G-JJ: methodology and formal analysis. QH: writing \u2013 original draft. G-JJ and KW: writing \u2013 review and editing and funding acquisition. All authors had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis."} +{"text": "Dear Editor,3. Although heterologous booster with mRNA vaccine (BNT162b2) has been shown to boost significantly higher neutralization against BA.2 than homologous booster, neutralization against the latest omicron sublineages is lacking4. Till now, there is no studies comparing the boosting effects against the latest omicron sublineages by multi-platform COVID-19 vaccines comprehensively. More importantly, the long-term durability against omicron sublineages remains to be explored. Here, we report firstly the vaccination-induced cross-neutralization data against omicron sublineages, including BA.2.75 and BF.7, in head-to-head comparison of COVID-19 vaccines from four platforms within a 3-month follow-up period.As of July 2022, over 89% of Chinese population have finished COVID-19 vaccine primary immunization, mostly with two-dose inactivated vaccines. A booster dose has been deployed to combat waning immunity over time and immune escape due to evolving virus variants. However, previous studies revealed that homologous booster with CoronaVac/BBIBP-CorV or heterologous booster with ZF2001 (recombinant RBD subunit protein vaccine) in participants primed with two-dose CoronaVac or BBIBP-CorV induced limited cross-neutralization against the more recently prevalent omicron sublineages BA.4/BA.55, including RQ30136, ChAdTS-S8, ZR202-CoV9, and CoronaVac. RQ3013 is a pseudouridine-modified mRNA vaccine encoding a near full-length Spike protein of alpha strain (B.1.1.7) with additional mutations from beta variant (B.1.351). ZR202-CoV is a recombinant protein vaccine based on a prefusionstabilized Spike ectodomain trimer of wild-type SARS-CoV-2 with two mutation sites, including a \u201cGGSG\u201d substitution at the furin cleavage site (residues 682\u2013685) and proline substitutions at residues 986 and 987. ChAd-TS-S is a chimpanzee adenovirus serotype 68 (AdC68) vector-based vaccine encoding the full-length Spike protein of wild-type SARS-CoV-2. CoronaVac is an inactivated vaccine of the whole wild-type SARS-CoV-2 (CN02 strain) (see Supplementary Methods). In this RCT, a total of 234 participants aged 18\u201359 years, who had received a prime two-dose CoronaVac (3\u20135 weeks apart) vaccination 100\u2013270 days before, were enrolled and randomized to receive one of the four COVID-19 vaccines or placebo as a booster dose. Their median age was 28 years with interquartile range from 24 to 34 years, and 126/234 (53.85%) were females. All their demographic characteristics and baseline immunogenicity measurements distributed comparably across five groups5. This trial has been performed in accordance with the Declaration of Helsinki and approved by the Committee on Human Subject Research and Ethics of The Affiliated Hospital of Yunnan University (ID: 2022026). Signed informed consent has been obtained from all participants. The results revealed varying levels of magnitude and breadth in neutralizing antibodies against live SARS-CoV-2 across four vaccines, with the mRNA vaccine RQ3013 demonstrating the highest levels of neutralizing antibodies5. In this work, we further tracked the variants of concerns (VOCs) of SARS-CoV-2 and tested neutralizing antibodies comprehensively against 8 omicron sublineages measured by the pseudovirus test10 (see Supplementary Methods) (ChiCTR.org.cn Identifier: ChiCTR2200057758) to evaluate immunogenicity in head-to-head design of four COVID-19 vaccines in China representing four major platformsds) Fig. .Fig. 1Ma11. The durable humoral response of adenovirus-vectored vaccine ChAdTS-S could potentially attribute to longer half-life of immunogen-expressing adenovirus vector than mRNA, which was also observed in the Ad26.COV2.S vaccine12. Additionally, the broad host cell tropism of the vector AdC68 and the expression of membrane-anchored rather than soluble Spike protein by ChAdTS-S might also contribute to the relative stronger durability8. Notably, despite of slower waning immunity for the adenovirus-vectored vaccine, our head-to-head comparison revealed much higher absolute neutralizing titers for ZR202-CoV and RQ3013 at 3 months after vaccination. Thus, ZR202-CoV and RQ3013 demonstrated superior protective capacity over time than ChAdTS-S and CoronaVac.Firstly, we analyzed time-series anti-RBD specific IgG data (see Supplementary methods) before and after the booster dose to explore the dynamics of antibody responses to the booster dose Fig. . The basFor each omicron sublineage, we observed a wide range of boosting effects across vaccine types by a third dose Fig. . Althoug5, was also essential for vaccine efficacy, especially for preventing disease severity13.In this study, we mainly focused on the broadness of neutralizing antibody elicited by vaccine and several limitations should be mentioned. First, different assay systems exhibit different levels of neutralizing antibodies, and antibody titers measured by different assays are not directly comparable. However, the neutralizing antibody titers measured by our pseudovirus neutralization assay correlated well with those measured by live virus assay to boost immunity against omicron sublineages following an initial course of CoronaVac/CoronaVac. Specifically, a booster dose with the mRNA RQ3013 elicited the strongest immune responses , while the recombinant protein vaccine ZR202-CoV with CpG as adjuvant optimized the durability (Supplementary Table Supplementary Figures and Tables"} +{"text": "Correction to: BMC Public Health 22, 47 (2022)https://doi.org/10.1186/s12889-021-12481-2The PRISMA Flow diagram contained incorrect values298 (Full-text articles assessed for eligibility) should be 28953 should be 5039 (Studies reflecting only work environment outcomes) should be 36The following record-values were incorrect:The original publication of this article containeThe original article has been updated to correct these errors. This does not impact the conclusions of the article."} +{"text": "There is a lack of data on Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) vaccination safety in children and young people (CYP) with rheumatic and musculoskeletal diseases (RMDs) as they were excluded from initial vaccine trials. Vaccination guidance is based on data from adults with or CYP without RMDs.Our objective was to describe the safety of SARS-COV-2 vaccination in adolescents with inflammatory RMDs and adults with JIA. We described patient characteristics, flares, and adverse events in adolescent cases under 18 with inflammatory RMDs and adult cases aged 18 or above with JIA submitted to the European Alliance of Associations for Rheumatology (EULAR) COVAX registry.Thirty-six adolescent cases were reported from 4 countries, mostly female (58%) with JIA and a median age of 15 . Most were in remission (64%) or had minimal (22%) disease activity at the time of vaccination. Over half of the adolescent group (56%) reported early reactogenic-like AEs. One mild polyarthralgia flare and one serious AE of special interest were reported. No CYP reported SARS-CoV-2 infection post-vaccination. No cases of paediatric inflammatory multi-system syndrome or myocarditis adverse events were reported.Seventy-four adult JIA cases were reported from 11 countries; 73% were female with a median age of 26 . Eight-five percent had ns-JIA and 15% had s-JIA. Almost two thirds (62%) reported early reactogenic-like AEs and two flares were reported . No serious AEs of special interest were reported among adults with JIA. Three 20-30 year old females were diagnosed with SARS-CoV-2 post-vaccination; all fully recovered.In this observational registry dataset, SARS-CoV-2 vaccines appeared safe in adolescents with RMDs and adults with JIA, with a low frequency of disease flares, serious AEs, and SARS-CoV-2 re-infection seen in both populations."} +{"text": "Depressive disorders (Dd) in childhood have a prevalence about 1-2%. Sometimes depression may be underdiagnosed with the risk of complications: comorbidity, chronicity or development of psychiatric diseases in adulthood. Although children often do not show a clear sad mood, they usually presents irritability as a cardinal symptom. Other common symptoms in children\u00b4s depression are lack of attention, difficult of concentration and impulsivity. These symptoms actually could define as well an Attention Deficit and Hyperactivity Disorder (ADHD), highly prevalent in school-aged children (5-7%).-To deep into diagnosis and evolution of depressive disorder in primary school-aged children (7-12 years-old). -To contrast clinical evidence about specific aged-symptoms observed in the boy and follow-up until remission.-Case study. Graphic description of diagnosis path and treatment in a 8-years-old boy suffers from depression. -Clinical case attended in Mental Health Unit, ambulatory consultation (outpatient). -Diagnosis tools: Clinical examination, family interview, evaluation tests and school psychopedagogical assessment.-Treatment methods: psychotherapy, psychopharmacology and theater. -Specific depressive symptoms depends on childhood stages (*chart by ages). -Pharmacological treatment used: psychostimulants, benzodiazepines and antidepressants. -Efficacy of monotherapy with Fluoxetine 20mg/day 6-months. -Importance of individual psychotherapy and group activities 12-months. -Episode resolution and functional recovery 15-months.Variability of symptoms in children\u00b4s depression can be confused with other psychiatric disorders like decreased school performance (ADHD), that may make diagnosis difficult. Sometimes, both disorders coexist, especially when the mood disorder is secondary to academic problems caused by ADHD. Early diagnosis and continued follow-up in specialized units is necessary to avoid progression and complications of Dd."} +{"text": "Handwriting disorder is commonly observed in Developmental Coordination Disorder (DCD) (87-88%) and is often noted in children with high Intellectual Quotient (HIQ).Two mainly pure DCD subtypes: ideomotor-DCD (IM), visuospatial/or visuoconstructional-DCD (VSC) and a mixed subtype (MX) were identified in the literature but nothing is known regarding IQ and dysgraphia.To refine the specific clinical features of dysgraphia related to DCD subtypes regarding IQ levels.Neurovisual, neuropsychological, neuropsychomotor functions, and handwriting performances of 38 children diagnosed with DCD (DSM-5 criteria) were collected. Two matched groups were analyzed according to their IQ: 19 (TC) typical children (IQ=90-110) and 19 HIQ children (IQ> 120).IQ scores were not significantly associated with dysgraphia. There isa significant difference between TC vs HIQ with a lower rate of IM-DCD respectively 11% vs 5% (p=.035) and 68% vs 37% for VSC-DCD (p=.03) but 21% vs 58% in MX-DCD (p=.41). Dysgraphia was significantly more present in TC group with MX-DCD and in HIQ with VSC-DCD. A negative correlation between Kho\u2019s\u2019 cubes test failure (p=.006), visual-spatial memory (p=.05) and VSC-DCD was noted in HIQ group. The deficit of visual spatial memory was significantly related to dysgraphia in HIQ children (p=.01) associated to visual gnosis impairment (p=.03).Dysgraphia was significantly found with VSC-DCD subgroup in FIQ>120 with specific features of visual perception disorders suggesting more involvement of the right cortex. These results suggest that VSC-DCD in HIQ could be a neurovisual impairment rather than a pure VSC-DCD."} +{"text": "Following publication of the original article , the autThe incorrect co-first authors:\u2020Co-first author: Jayshil J. Patel and James ZeltenThe correct co-first authors are:\u2020Co-first author: Luis Ortiz\u2011Reyes and Jayshil J. PatelThe co-first authors have been updated above and the original article has been"} +{"text": "N/AOBJECTIVES/GOALS: Vitamin D [25(OH)D], known to have anti-inflammatory and anti-fibrotic effects in other tissues, may also impact adipose tissue. We designed parallel studies in humans and rodents to define the effects of vitamin D on adipose tissue inflammation and fibrosis, and on systemic insulin resistance. METHODS/STUDY POPULATION: We performed a randomized, double-blinded placebo-controlled trial to examine the effects of repleting vitamin D at to two levels (to >30 ng/ml and to > 50 ng/ml) in 25(OH)D-deficient (<20 ng/ml), insulin resistant, overweight-to-obese humans (n = 19). A comprehensive study of whole-body insulin action was undertaken with euglycemic stepped hyperinsulinemic clamp studies, both before (1st visit) and after administration of vitamin D or placebo (2nd visit and 3rd visit). Adipose tissue fibrosis and inflammation were quantified by \u2018real-time\u2019 rt-PCR and immunofluorescence. To determine whether vitamin D\u2019s effects are mediated through adipocytes, we performed hyperinsulinemic clamp studies and adipose tissue analysis in an adipocyte-specific vitamin D receptor knockout (VDR KO) mouse model. RESULTS/ANTICIPATED RESULTS: 25(OH)D repletion (to >30 ng/ml) was associated with reductions in adipose tissue expression of inflammatory and pro-fibrotic factors, decreased collagen VI immunofluorescence (p = 0.02) and improved hepatic insulin sensitivity in humans, with suppression of endogenous glucose production (EGP) . Compared to wild type (WT), VDR KO mice exhibited increased adipose tissue expression of several pro-inflammatory and pro-fibrotic genes , in concert with hepatic insulin resistance . DISCUSSION/SIGNIFICANCE OF IMPACT: Collectively, these complementary human and rodent studies establish a beneficial role of vitamin D to improve hepatic insulin resistance, likely by restraining adipose tissue inflammation and fibrosis. Thus, normalizing 25(OH)D levels could have metabolic benefits in targeted individuals. CONFLICT OF INTEREST DESCRIPTION:"} +{"text": "As a common endocrinopathy of reproductive-aged women, polycystic ovary syndrome (PCOS) is characterized by hyperandrogenism, oligo-anovulation and polycystic ovarian morphology. It is linked with insulin resistance through preferential abdominal fat accumulation that is worsened by obesity. Over the past two millennia, menstrual irregularity, male-type habitus and sub-infertility have been described in women and confirm that these clinical features of PCOS were common in antiquity. Recent findings in normal-weight hyperandrogenic PCOS women show that exaggerated lipid accumulation by subcutaneous (SC) abdominal stem cells during development to adipocytes in vitro occurs in combination with reduced insulin sensitivity and preferential accumulation of highly-lipolytic intra-abdominal fat in vivo. This PCOS phenotype may be an evolutionary metabolic adaptation to balance energy storage with glucose availability and fatty acid oxidation for optimal energy use during reproduction. This review integrates fundamental endocrine-metabolic changes in healthy, normal-weight PCOS women with similar PCOS-like traits present in animal models in which tissue differentiation is completed during fetal life as in humans to support the evolutionary concept that PCOS has common ancestral and developmental origins. Polycystic ovary syndrome (PCOS) is characterized by ovarian hyperandrogenism from altered hypothalamic-pituitary-ovarian function in combination with hyperinsulinemia from insulin resistance. With a prevalence of 6\u201320% in the general population, depending upon the definition of PCOS , its cliAs a heritable syndrome with a polygenic origin, large genome-wide association studies (GWAS) have identified several PCOS susceptible loci in candidate genes involving insulin action, androgen biosynthesis and gonadal function . These PThis review examines how the endocrine-metabolic characteristics of women with PCOS originally favored survival of humans in ancient times of food deprivation, but now predispose to endocrine-reproductive dysfunction in today\u2019s obesogenic environment. It integrates clinical characteristics of PCOS women with similar PCOS-like traits present in prenatally testosterone-treated monkeys and sheep to provide evidence of developmental programming in PCOS, given that tissue differentiation in these species, as in humans, occurs during fetal life.Several variables affect the endocrine-metabolic characteristics of women with PCOS. As one such variable, different PCOS phenotypes by Rotterdam criteria vary in their degree of reproductive and metabolic dysfunction . Women wIn addition, obesity coexists with abnormal insulin action in most women with PCOS \u201311. AlthIn the context of metabolic function, women with NIH-defined PCOS have two distinct PCOS subtypes with different genetic heterogeneity. A reproductive endocrine subtype (23% of cases) is characterized by higher luteinizing hormone (LH) and sex hormone binding globulin (SHBG) levels with relatively low BMI and insulin levels, while a metabolic subtype (37% of cases) is characterized by higher BMI, glucose and insulin levels, with lower SHBG and LH levels. These PCOS subtypes may differ in their developmental origins , with thMost women with PCOS have some degree of insulin resistance due to perturbed insulin receptor/post receptor signaling, altered adipokine secretion and abnormal steroid metabolism in combi, defined by the product of fasting circulating free fatty acid (FFA) and insulin levels [In women with NIH-defined PCOS, preferential abdominal fat accumulation , 27 promn levels , 24, 26.Intra-abdominal adipose in humans is highly lipolytic and resists androgen inhibition of catecholamine-induced lipolysis (lipid breakdown) despite expressing androgen receptors . InsteadSubcutaneous (SC) abdominal adipose normally protects against insulin resistance through a balance between lipogenesis (lipid formation) and lipolysis (lipid breakdown) in mature adipocytes combined with new adipocyte formation , whereby adipose stem cells (ASCs) initially undergo commitment to preadipocytes and then differentiate into newly-formed adipocytes \u201337. Subc2-adrenergic receptor and hormone-sensitive lipase (HSL) protein expression [2-adrenergic receptor, HSL and protein kinase A regulatory-II\u03b2 component (PKA-RegII\u03b2) [Within SC adipose, androgen normally inhibits early-stage adipogenesis, diminishes insulin-stimulated glucose uptake and impairs catecholamine-stimulated lipolysis through reduced \u03b2pression , 35, 40.pression and cate-RegII\u03b2) , 43. As -RegII\u03b2) , yet inc-RegII\u03b2) , likely -RegII\u03b2) .Consequently, adipose-IR is often increased in healthy normal-weight women with NIH-defined PCOS compared to age- and BMI-matched normal women . Moreove\u03b3 (PPAR\u03b3) and CCAAT enhancer binding protein a (CEBPa) during adipocyte maturation in vitro accompanies altered dynamic chromatin remodeling, with enrichment of binding motifs for transcription factors of the activator protein-1 (AP-1) subfamily that govern adipocyte differentiation [The dynamic process of adipocyte development begins in early life . Subcutantiation . These fntiation . SimilarZFP423 upregulation due to epigenetic changes in its promoter region [Linked with this exaggerated ZFP423-induced ASC commitment to preadipocytes in vitro is a greater formation of small SC abdominal adipocytes in normal-weight PCOS women , 26, 50.r region . An incrr region . It alsor region , perhapsr region , 58.PPAR\u03b3 and CEBPa in some PCOS SC abdominal stem cells accompanies upregulation of AKR1C3 during adipocyte maturation in vitro. These findings correspond with an inverse relationship of serum total T/A4 ratio with serum TG level in normal-weight PCOS women [Finally, overexpression of OS women , and sugOS women , 60. AKROS women . These dOS women , 50.Several endocrine-metabolic characteristics of PCOS women worsen with increased adiposity. Overweight/obese compared to normal-weight women with PCOS exhibit greater preferential abdominal fat accumulation, hyperandrogenism and insulin resistance accompanLipotoxicity refers to the ectopic lipid accumulation in non-adipose tissue where it induces oxidative/endoplasmic reticulum stress tightly linked with insulin resistance and inflammation . OverweiThrough an evolutionary perspective, the high worldwide prevalence of PCOS in today\u2019s environment and its negative impact on reproduction should have disappeared over millennia unless a beneficial effect favored both survival and reproduction . There iDENND1A involved in regulating androgen production, have been identified in ~\u200950% of families with PCOS [DENND1A, DENND1A.v2, is over-expressed in some women with PCOS [Indeed, commonality among >\u200920 PCOS candidate genes in women with differing PCOS phenotypes, including those diagnosed by Rotterdam or NIH criteria, or by self-report, strongly suggests shared molecular and developmental origins despite heterogeneity of PCOS phenotypic expression . Moreoveith PCOS . In thisith PCOS , while eith PCOS .A separate whole-genome sequencing study found in ~\u20093% of families with PCOS, rare gene variants in anti-mullerian hormone (AMH) and its type 2 receptor (AMHR2) . Both geTHADA) and insulin receptor (INSR), have been associated with metabolic syndrome and impaired glucose regulation in PCOS and type 2 diabetes [In addition, some PCOS candidate genes, such as thyroid adenoma associated , induces reproductive and metabolic PCOS-like phenotypes resembling those of women with PCOS . Such PCAs precocial species, in which tissue differentiation is completed during fetal life as in humans , prenataMid-gestational prenatal T-treatment in rhesus monkeys and sheep also programs adipose dysfunction with insulin resistance in adult offspring , 103. AdNaturally-occurring female hyperandrogenism also occurs in some adult female macaques accompanied by PCOS-like endocrine-metabolic traits , 107. A Potentially programmed in part during gestation, an altered metabolic phenotype in women with PCOS could predispose them to excess weight gain in today\u2019s obesogenic environment, emphasizing the need for appropriate clinical strategies to improve their health, reduce their risks of developing maternal-fetal complications and optimize the long-term health of their offspring . AlthougPolycystic ovary syndrome has persisted from antiquity to become the most common reproductive-metabolic disorder of reproductive-aged women. Its ancestral traits once favored abdominal fat deposition and increased energy availability through hyperandrogenism and insulin resistance, respectively, for reproduction during food deprivation. These same traits in today\u2019s environment, however, underlie different PCOS phenotypes with variable risks for subfertility and metabolic dysfunction that are worsened by obesity. Recent studies of healthy normal-weight women with NIH-defined PCOS show enhanced AKR1C3 activity in SC abdominal adipose favoring lipid storage in combination with preferential intra-abdominal fat deposition accompanying hyperandrogenemia and low-normal insulin sensitivity. Potentially programmed as an ancestral trait by genetic inheritance and epigenetic events during early life, such a metabolic adaptation in these normal-weight PCOS women provides a balance between enhanced SC adipose TG storage and increased circulating glucose and FFA availability as energy substrate for crucial target tissues, including brain and muscle Fig.\u00a0. It also"} +{"text": "Patients undergoing transcatheter aortic valve replacement (TAVR) usually have multiple comorbidities, such as severely impaired left ventricular function (LVF) and heavily calcified coronary lesions. When they undergo pre-TAVR high-risk percutaneous coronary interventions (HR-PCIs) for severely calcified left main (LM) lesions, potential life-threatening intra-procedural complications associated with the different techniques available to treat calcified lesions can arise. In this setting, mechanical circulatory support proves its usefulness. However, the choice of device can be troublesome.We report two clinical scenarios of intravascular lithotripsy (IVL) for the treatment of heavily calcified LM coronary lesions, wherein peripheral veno-arterial extracorporeal membrane oxygenation (VA-ECMO), alone or combined with an intra-aortic balloon pump (IABP), were used as an upfront strategy to support the procedure. The use of these techniques was particularly effective during multi-vessel HR-PCIs and TAVR, and no complications occurred, which suggested their safety.These cases provide multiple insights into the strategy of using IVL + VA-ECMO, alone or with IABP, to treat heavily calcified LM coronary lesions in patients with severely compromised LVF undergoing TAVR. IVL safely and effectively overcame shortcomings related to other plaque ablation techniques, and VA-ECMO proved to be effective when facing the combination of high-risk coronary and valve interventions. Learning pointsIntravascular lithotripsy proved to be safe and useful for overcoming shortcomings related to other aggressive plaque debulking techniques used in the treatment of heavily calcified coronary lesions.Veno-arterial extracorporeal membrane oxygenation, alone or combined with intra-aortic balloon pump, could be used to avoid ventricular decompensation in the setting of a complex pre-transcatheter aortic valve replacement high-risk percutaneous coronary intervention.,Transcatheter aortic valve replacement (TAVR) has emerged as a therapeutic option for severe aortic stenosis (AS) in high-, intermediate-, and low-risk patients.Recently, intravascular lithotripsy (IVL) has proven to be safe and effective while minimizing plaque disruption and distal-embolization risks.A 78-year-old male ex-smoker was admitted after experiencing cardiac arrest and was resuscitated for sustained ventricular tachycardia with two electric shocks. Upon arrival at the emergency department, physical examination showed cold sweat, weak pulse, and a harsh aortic systolic murmur.1 reduction of 40%. An electrocardiogram (ECG) showed anterior\u2013lateral T-wave inversion. In the hospital, the two-dimensional echocardiogram (2D-echo) showed a severely calcified aortic valve and a left ventricular ejection fraction (LVEF) of 23% (Video 1). The right ventricle appeared severely compromised, with pulmonary artery systolic pressure of 68 mmHg. Coronary angiography (CAG) revealed extensive calcification of both coronary arteries with multiple critical stenoses of the LM stem (Video 2), the left anterior descending artery (LAD) and the dominant right coronary artery with FEVAfter ketamine sedation of the patient, we used two pre-closure devices on the left common femoral artery, inserted the cannulae percutaneously with ultrasound (US) guidance and activated VA-ECMO. Unfractionated heparin was used, with activated clotting time ranging from 180 to 250 s.Video 2). Finally, after suboptimal pre-dilation, IVL successfully pre-treated the RCA stenosis, allowing the implantation and optimization of a 3.5/28-mm DES of the focal LM stem stenosis was ineffective as balloon waist persisted. Therefore, intravascular ultrasound (IVUS) was performed, and multiple \u2018short-shock\u2019 pulses of IVL were applied with a 4.0/12-mm balloon inflated up to 4 ATM .Video 1), and the mean aortic gradient was 38 mmHg. On Day 9, low-dose dobutamine-stress echocardiography (DSE) was performed to confirm true low-flow/low-gradient severe AS before proceeding with TAVR. On 10 \u00b5g/kg/min low-dose DSE, the mean aortic gradient increased to 45 mmHg, and AVA was 0.78 cm2, confirming a true severe low-flow/low-gradient AS. Two days later, a CoreValve Evolut-R 29 bioprosthesis was implanted through right femoral access without any MCS (Video 3). Medical therapy at discharge was aspirin + ticagrelor, amiodarone, diuretics, and calcium antagonists. At 1-month follow-up, the patient was asymptomatic for dyspnoea and angina. At the 4-month 2D-echo evaluation, LVEF was 45%, and pulmonary artery systolic pressure of 50 mmHg.The patient was weaned from VA-ECMO in the catheterization laboratory. On Day 7, the in-hospital 2D-echo showed that LVEF had risen to 41% (1 = 63%). In-hospital 2D-echo showed LVEF of 26% and severe AS . CAG revealed severe and heavily calcified LM stenosis of the bifurcation or VA-ECMO increase risks. In our patients, MSCT imaging showed that peripheral vessel size could allow large cannulae. Furthermore, VA-ECMO can be inserted using US guidance only, unlike other MCS devices, which require fluoroscopy. Finally, as closure devices see more widespread use, most VA-ECMO procedures could be fully percutaneous, and haemostasis could be immediately achieved.A growing number of single-centre experiences support the role of VA-ECMO used electively during complex HR-PCI or TAVR.Impella demonstrated its effectiveness in this settingFinally, when a large ischaemic burden and a long-lasting, complex PCI are expected, VA-ECMO combined with IABP can overcome some of the side effects of VA-ECMO and limitations of the Impella heart pump.In the presented cases, IVL proved to be effective in managing heavily calcified LM coronary lesions, especially in the presence of an impaired LVF. In this setting, VA-ECMO was a valid MCS device to support high-risk multi-vessel PCIs and TAVR. Randomized studies are needed to evaluate the effect of VA-ECMO or VA-ECMO + IABP on the short-term outcomes of these high-risk patients.Dr Alfredo Marchese is the Chief of the Interventional Cardiology at Ospedale Santa Maria, in Bari, Italy. He has a prominent role in the executive board of the Italian Society of Interventional Cardiology. He has a particular expertise in the treatment of the left main coronary artery and of bifurcatiotion lesions. His research interests include optimizing antiplatelet therapy during elective CHIP-PCI and minimizing coronary access failure during TAVR procedures.European Heart Journal - Case Reports online.Slide sets: A fully edited slide set detailing this case and suitable for local presentation is available online as Consent: The authors confirm that written consent for submission and publication of this case report including images and associated text has been obtained from the patient in line with Committee on Publication Ethics (COPE) guidance.Conflictof interest: None declared.Funding: This work was supported by \u2018Societ\u00e0 Italiana di Cardiologia Interventistica-Gruppo Italiano di Studi Emodinamici\u2019 (SICI-GISE).ytab498_Supplementary_DataClick here for additional data file."} +{"text": "The novel coronavirus disease 2019 (COVID-19) pandemic remains a global challenge. Accurate COVID-19 prognosis remains an important aspect of clinical management. While many prognostic systems have been proposed, most are derived from analyses of individual symptoms or biomarkers. Here, we take a machine learning approach to first identify discrete clusters of early stage-symptoms which may delineate groups with distinct symptom phenotypes. We then sought to identify whether these groups correlate with subsequent disease severity.The Epidemiology, Immunology, and Clinical Characteristics of Emerging Infectious Diseases with Pandemic Potential (EPICC) study is a longitudinal cohort study with data and biospecimens collected from nine military treatment facilities over 1 year of follow-up. Demographic and clinical characteristics were measured with interviews and electronic medical record review. Early symptoms by organ-domain were measured by FLU-PRO-plus surveys collected for 14 days post-enrollment, with surveys completed a median 14.5 days post-symptom onset. Using these FLU-PRO-plus responses, we applied principal component analysis followed by unsupervised machine learning algorithm k-means to identify groups with distinct clusters of symptoms. We then fit multivariate logistic regression models to determine how these early-symptom clusters correlated with hospitalization risk after controlling for age, sex, race, and obesity.P < 0.001] of hospitalization which remained significant after controlling for other factors .Using SARS-CoV-2 positive participants (n = 1137) from the EPICC cohort , we transformed reported symptoms into domains and identified three groups of participants with distinct clusters of symptoms. Logistic regression demonstrated that cluster-2 was associated with an approximately three-fold increased odds [3.01 (95% CI: 2-4.52); (A) Baseline characteristics of SARS-CoV-2 positive participants. (B) Heatmap comparing FLU-PRO response in each participant. (C) Principal component analysis followed by k-means clustering identified three groups of participants. (D) Crude and adjusted association of identified cluster with hospitalization.Our findings have identified three distinct groups with early-symptom phenotypes. With further validation of the clusters\u2019 significance, this tool could be used to improve COVID-19 prognosis in a precision medicine framework and may assist in patient triaging and clinical decision-making.David A. Lindholm, MD, American Board of Internal Medicine Involved: Self): Member of Auxiliary R&D Infectious Disease Item-Writer Task Force. No financial support received. No exam questions will be disclosed ., Other Financial or Material Support Ryan C. Maves, MD, EMD Serono (Advisor or Review Panel member)Heron Therapeutics (Advisor or Review Panel member) Simon Pollett, MBBS, Astra Zeneca )"} +{"text": "At least 2/3 of people with mild to moderate COVID-19 infection will experience long-haul COVID symptoms that persist for weeks or months, however, risk factors that modify the likelihood that one develops these symptoms are unknown. Patients referred to the Post-COVID Recovery Program at Rutgers in New Brunswick (n= 108) through primary care referral or self-submitted online request and experiencing a wide variety of Post-Acute COVID-19 Syndrome (PACS) symptoms were stratified by those without self-reported cognitive complaints (n=54), those with self-reported cognitive complaints who scored well on cognitive testing (n=29), and those with self-reported cognitive complaints who scored poorly on cognitive testing (n=25). Comparisons between groups were made using ANOVAs and Chi Squared: for COVID-19 disease severity, COVID-19 disease treatment, comorbid COVID-19 symptoms during infection, comorbid PACS symptoms post-infection, pre-existing health conditions, levels of depression and anxiety, level of fatigue, and social determinants of health . Preliminary analyses indicated that whereas people without complaints were normally distributed according to age (p>0.200 for Kolmogorov\u2013Smirnov test), people with complaints and deficits were skewed towards the older age group (p<0.001 for K-S test) suggesting age to be a risk factor for cognitive impairment in PACS. Participants that reported cognitive complaints also reported increased symptoms of depression, anxiety, and fatigue, compared to participants without cognitive complaints. These data provide insight into associations between PACS symptoms and risk factors relevant in understanding this novel disease."} +{"text": "In our data, all extranodal lesions located in subcutaneous-tissue, lung and liver were detected on PET, while some of those located in other tissues were not detected on PET. We also evaluated the predictive role of PET in these patients, and found that increased [18F]FDG-uptake in extranodal lesions was associated with worse disease progression.MALT lymphoma represents a relatively rare lymphoma subtype that arises in different extranodal anatomic locations. In the current paper we summarize our experience in assessing disease extent and metabolic characteristics of MALT lymphomas with [18F-fluorodeoxyglucose ([18F]FDG) positron emission tomography\u2014computed tomography (PET-CT) in assessing mucosa-associated lymphoid tissue (MALT) lymphoma is debatable. We retrospectively explored the role of [18F]FDG PET-CT in staging and predicting progression-free-survival (PFS) of patients with newly-diagnosed MALT lymphoma. Sixty-six studies were included. The maximum standardized uptake value (SUVmax), metabolic tumor volume (MTV) and total lesion glycolysis (TLG) were documented in the \u201chottest\u201d extranodal and nodal lesions. Extranodal lesions and accompanying nodal disease were detected on PET in 38/66 (57.6%) and 13/66 (19.7%) studies, respectively. Detection rate of extranodal lesions differed significantly between those located in tissues with high/heterogeneous vs low/homogenous physiologic [18F]FDG-uptake . Nodal lesions had significantly lower SUVmax, MTV and TLG compared with extrandodal lesions in the same patients. Detection and [18F]FDG-avidity of extranodal lesions were higher in patients with advanced, bulky disease and concomitant marrow/nodal involvement. Increased SUVmax of extranodal lesions predicted shorter PFS . Higher SUVmax and TLG showed trends towards shorter PFS in patients with localized disease. In conclusion, detection rate of extranodal MALT lymphoma lesions located in tissues with low/homogeneous physiologic [18F]FDG-uptake is excellent on [18F]FDG PET-CT. When detected, SUVmax of extranodal lesions may predict PFS.The role of Mucosa-associated lymphoid tissue (MALT) lymphoma, also known as extranodal marginal zone lymphoma (MZL), involves the mucosa-associated lymphoid tissue and can potentially develop at any mucosal site, most frequently in the stomach, but also in the orbit, lung and other tissues ,2.MALT lymphomas usually follow an indolent clinical course and patients diagnosed with MALT lymphoma usually have a favorable prognosis. However, due to the large variability in clinical presentation, management of this heterogeneous group of patients is often patient-tailored ,4. At st18F-fluorodeoxyglucose ([18F]FDG) positron emission tomography\u2014computed tomography (PET-CT) is the preferred imaging modality for staging most lymphoma subtypes FDG PET-CT in patients with MALT lymphoma. A wide range of reported detection rates and [18F]FDG-avidity have led to a continuous debate as to whether MALT lymphoma should be categorized as [18F]FDG-avid [18F]FDG PET-CT in patients with MALT lymphoma FDG PET-CT in MALT lymphoma continues to be a subject of debate, in the current study we aimed to summarize our experience in this topic, and particularly use a retrospective cohort of patients to describe and explore the role of semiquantitative PET parameters in staging studies of patients with MALT lymphoma.As the role of [18F]FDG PET-CT in the nuclear medicine department at Tel-Aviv Sourasky Medical Center for primary staging.We retrospectively screened the medical records of all patients that met the following criteria: (i) age \u2265 18 years (ii) diagnosed with histologically proven MALT lymphoma between April 2010 and August 2021 (diagnosis confirmed by hematopathology expert) (iii) clinically evaluated and treated at the hematology institute at Tel-Aviv Soursaky Medical Center (iv) underwent a whole-body FDG PET-CT studies were performed on PET-CT scanners , according to our standard protocol, with the administration of a diluted oral contrast agent and injection of 3.7 MBq/kg [18F]FDG approximately 60 min prior to the study.The FDG-avidity and staging, laboratory and pathological parameters were assessed. On a survival analysis, the endpoint evaluated was progression-free-survival (PFS), defined as the time from the staging [18F]FDG PET-CT study to disease progression that necessitated a change in the primary clinical strategy, or to death from any cause.Associations between FDG-uptake [18F]FDG-avid nodal disease identified on PET imaging, and nine patients had non-[18F]FDG-avid lymphadenopathy identified on the CT part of the scan, suggesting involvement by lymphoma . Rates of [18F]FDG-avid nodal lymphoma were not significantly different among patients with MALT lymphoma of different extranodal locations study patients based on imaging. Thirteen patients had [ocations . The nodocations . Among p lesions .18F]FDG-avidity (SUVmax < median SUVmax) and 19 patients whose extranodal lesions showed high [18F]FDG-avidity on PET. p = 0.02), rates of bulky disease (p < 0.01), BM involvement (p = 0.02) and coexisting [18F]FDG-positive nodal involvement (p = 0.02).We analyzed the difference between three groups of the total cohort: 28 patients whose extranodal lesions were not detected on PET, 19 patients whose extranodal lesions showed low [18F]FDG PET-CT. At the time of the analysis, the study patients had a median follow-up of 31.2 (IQR 14.4\u201474.9) months, and their median PFS was 38.0 (IQR 12.4-110.1) months from their staging FDG-uptake can make image interpretation difficult. In our data, more than two-thirds of the studied extranodal lesions were located in tissues that have high/heterogenous physiologic [18F]FDG-uptake, e.g., along the digestive tract and in proximity to extraocular muscles. In most of such cases we found that the interpreting physician could not confidently identify the extranodal lesion on PET. Such extranodal lesions are not necessarily non-[18F]FDG-avid, but their [18F]FDG-avidity might not be high enough to overcome the background physiologic [18F]FDG-uptake. In contrast, our study highlight the excellent detection (100%) found among extranodal MALT lymphoma lesions located in tissues that have low/homogenous physiologic [18F]FDG-uptake, such as subcutaneous-tissue, lungs, and liver. In such cases, [18F]FDG PET-CT could be the modality of choice for primary anatomical staging.[18F]FDG-avid nodal disease was detected in one fifth of the study patients. The detection rate of nodal disease on PET, as well as the SUVmax, MTV and TLG calculated in these lesions did not differ significantly in patients with extranodal lesions located in different sites. However, these PET-derived parameters were all significantly lower in nodal lesions compared with matched extranodal lesions. These observations, which were not described in previous studies, possibly reflect that in patients with concomitant nodal and extranodal involvement, extranodal sites may possess different biological characteristics than those nodal sites do.[18F]FDG-avidity of extranodal lesions and staging variables, including lymphoma\u2019s stage, rates of bulky disease, BM involvement, and [18F]FDG-avid nodal involvement, associations that were reported in previous studies [18F]FDG-avidity and mitotic index FDG PET-CT findings did not correlate with PFS FDG PET-CT in patients with MALT lymphoma.The present study suffers several limitations. Since the incidence of MALT lymphoma is low and the number of included patients was hence relatively small, we had to include all patients in the PFS analysis, despite their heterogeneity, in order to gain the statistical power to find significant predictors. Therefore, and in light of contradicting results on such a debated topic, larger studies or subgroup-specific studies are warranted to better define the prognostic role of [18F]FDG-uptake appears to be low on [18F]FDG PET-CT. However, detection rate of extranodal MALT lymphoma lesions located in other tissues is excellent, and [18F]FDG PET-CT should be recommended for staging such cases. Our results indicate that PET-based parameters measured in detected extranodal MALT lymphoma lesions may be associated with PFS, although verification of this observation in larger studies is still required.Detection rate of extranodal MALT lymphoma lesions located in tissues with high/heterogeneous physiologic ["} +{"text": "ABSTRACT IMPACT: Demonstrate the role of astrocyte released MMPs in response to pathogenic HIV protein Tat. OBJECTIVES/GOALS: In the presence of the pathogenic HIV protein Tat, astrocytes have been demonstrated to adopt an inflammatory phenotype as well as release extracellular matrix degrading enzymes, MMPs. Our work aims to identify whether MMPs alter perineuronal net integrity and working memory in a mouse model of Tat-induced neuroinflammation. METHODS/STUDY POPULATION: Stereotaxic Injection: C57BL6/J mice were injected bilaterally with HIV-1 IIIB Tat 5ug in 5uL or Vehicle , into the hippocampus . All outcome measurements were performed 14-days post injection. Behavior: T-maze was used to assess working memory following Tat exposure. qRT-PCR: TaqMan probes were used according to manufacturer on extracted whole hippocampus mRNA. IF: GFAP and CD68 immunofluorescence was used to determine inflammation post injection. Inhibitory interneurons and peri-neuronal nets (WFA positive) were quantified. WB: Synaptosomes from whole hippocampi (Syn-PER) were isolated and synaptic excitatory markers were quantified . RESULTS/ANTICIPATED RESULTS: Tat exposure resulted in impairments in working memory as measured by T-maze alternations and an increase in hippocampal mRNA expression of MMP-13 and IL-1\u03b2, indicative of neuroinflammation. We also noted an increase in GFAP+ injection site width 14 days post-Tat injection, suggesting robust gliosis. While there were no changes in the excitatory pre and post synaptic markers we found a significant decrease in the percent of PV+ interneurons with peri-neuronal nets (PNNs) following Tat exposure. Taken together, this preliminary data supports a role for inflammation and PNN integrity in Tat-induced alterations in working memory. DISCUSSION/SIGNIFICANCE OF FINDINGS: Our findings suggest that Tat contributes to cognitive impairment and that astrogliosis with elevated MMP-13 facilitates the degradation of peri-neuronal nets (PNNs) within the hippocampus. Since PNN degradation can alter neuronal circuitry future studies will focus on Tat-induced changes in hippocampal signaling."} +{"text": "Iatrogenic immunodeficiency-associated lymphoproliferative disorders (IA-LPD) may arise in patients treated with immunosuppressive drugs for autoimmune disease or other conditions. Polymorphic EBV-positive B-lymphoproliferations often have features mimicking Hodgkin lymphoma and typically a self-limited, indolent course. We present an unusual case with isolated, intracerebral manifestation of polymorphic B-LPD with features of classic Hodgkin-lymphoma in an immunosuppressed patient treated with methotrexate and infliximab, including clinical-radiological features and a detailed description of morphological findings, together with a literature review on reported cases\u00a0 of\u00a0primary CNS manifestation of cHL and IA-LPD with Hodgkin-like morphology. The patient achieved complete remission following neurosurgery with gross total tumor resection and drug withdrawal without any additional treatment. Post-operative staging revealed no evidence for focal relapse or systemic disease during the 18 months follow-up period. Among the previously reported 24 cases of primary, isolated Hodgkin lymphoma in the central nervous system, three similar cases of iatrogenic, IA-LPDs were identified and are discussed here. Polymorphic B-LPD are destructive lesions with a range of morphologic features and disease manifestations. It is clinically important to recognize the spectrum of proliferations with features of classic Hodgkin lymphoma in immunodeficiency, iatrogenic settings, because they are likely to impact the choice of treatment strategies.The online version contains supplementary material available at 10.1007/s12308-021-00478-0. Iatrogenic, immunodeficiency-associated lymphoproliferative disorders (IA-LPDs) are defined as lymphoid proliferations or lymphomas, arising in patients after long-term treatment with immunosuppressive drugs for an underlying autoimmune disease or other disease in the non-transplant setting . MethotrWe present a case of iatrogenic, immunodeficiency-associated, EBV-positive polymorphic LPD with morphology mimicking Hodgkin lymphoma in the CNS after long-term treatment with both methotrexate and infliximab (TNF inhibitor). The patient had no evidence of systemic disease and achieved complete remission after neurosurgery and drug withdrawal without any additional treatment. Among the previously reported 24 cases of primary, isolated CNS manifestation of HL, three additional cases of iatrogenic, immunodeficiency-associated EBV-positive LPD were identified and are discussed here.A 75-year-old woman presented to the emergency department with acute onset of hemiparesis, impaired consciousness, and facial droop. Her past medical history included a diagnosis of neurosarcoidosis and long-term immunosuppressive treatment with methotrexate and infliximab (TNF inhibitor) for at least 6 years. A magnetic resonance imaging scan (MRI) of the brain revealed a solitary, well-circumscribed, contrast-enhancing, left-sided supratentorial lesion measuring approximately 2 cm with significant perifocal edema and mass effect Fig. The patThe tissue was fixed in 4% buffered paraformaldehyde solution and embedded in paraffin. Tissue sections were stained with hematoxylin-eosin and Giemsa. Immunohistochemical stainings included CD3 (2GV6), CD20 (clone L26), CD15 (MMA), CD30 (Ber-H2), CD45 (RP2-18), CD68 (PG-M1), BOB-1 SP-92), OCT-2 (MRQ-2), CD79a (SP-18), CD23 (SP-23), MUM-1 (EP190), PAX5 (SP34), p53 (DO-7), Ki67 (30-9), CD138 (clone B-A38) , kappa/lambda , and LMP-1 using an automated immunostaining system . In situ hybridization for Epstein-Barr virus RNA (EBER transcripts) was performed on formalin fixed paraffin sections (FFPE) using the EBER-1 probe . PCR-analysis for immunoglobulin (IG) gene rearrangements was performed using genomic DNA isolated from FFPE tissue blocks (QIAGEN Tissue kit) and BIOMED-2 primers according to the BIOMED-2 PCR based protocol [2, OCT-2 Routine H&E\u00a0staining demonstrated a well-circumscribed, intracerebral lesion consisting of a polymorphous infiltrate with a mixture of small lymphocytes, numerous polyclonal plasma cells, eosinophils, histiocytes and scattered large, atypical mono- and multinucleated, Hodgkin- and Reed-Sternberg-like cells Fig. . The larAmong the 24 published reports on primary, isolated CNS manifestation of cHL between 1980 and 2020 \u201333, threAmong the other reported cases in the literature diagnosed as isolated cHL in the CNS (summarized in Suppl. Table 1), one patient had an underlying autoimmune hyperthyroidism (Graves\u2019 disease) treated with radioactive iodine , and anoWe describe an unusual case of immunodeficiency-associated polymorphic B-cell LPD with cells mimicking Hodgkin cells which presented as a solitary lesion in the brain without evidence of systemic disease. The patient had a history of neurosarcoidosis and been treated with Methotrexate and Infliximab. Three similar cases with isolated CNS manifestation have been reported in immunocompromised patients, all with an indolent clinical course. In contrast to our case, these patients received combined radio-chemotherapy or radiotherapy only. Importantly, no evidence for systemic disease was detected during clinical follow-up. The described lesions had morphological features of classic Hodgkin lymphoma, e.g., presence of Hodgkin/Reed-Sternberg cells in an appropriate reactive/inflammatory background. Acknowledging the diverse overlapping histological patterns of cHL and EBV+ B-LPD with cells that mimic Hodgkin cells in the setting of iatrogenic immunodeficiency-associated LPD, a combination of clinical, morphological, and immunophenotypic findings is needed for reaching the correct diagnosis. For example, the lack of CD15-expression and variable expression of B-cell markers in CD30-positive HRS-like cells have been considered a feature against the diagnosis of cHL . An addiIatrogenic immunodeficiency-associated LPD constitute a spectrum of disease manifestations, including EBV+ B-LPD with Hodgkin-like cells and cases that fulfil the criteria of classic Hodgkin lymphoma. The primary, isolated CNS manifestation of cHL is exceedingly rare \u201339. CorrSupplementary file1 (DOCX 27 KB)Below is the link to the electronic supplementary material."} +{"text": "Correction to: Basic Clin Androl 31, 14 (2021)https://doi.org/10.1186/s12610-021-00132-wFollowing publication of the original article , an erroThe incorrect article title read: Revised manuscript R2, clean version are serum levels of 25-hydroxy vitamin D reduced following orchiectomy in testicular cancer patients?The correct article title is: Are serum levels of 25-hydroxy vitamin D reduced following orchiectomy in testicular cancer patients?The article title has been updated above and in the original article. The publisher apologises to the authors and readers for the inconvenience caused by this mistake."} +{"text": "To determine the prevalence of anxiety and depression amongst participants with severe or complex obesity randomised and selected for bariatric surgery in a large multi-centre trial.To describe the change in prevalence of anxiety and depression amongst participants who had undergone bariatric surgery, within 6 months of randomisation and at 12 months post-randomisation.The By-Band-Sleeve (BBS) study is a multi-site randomised controlled trial evaluating the surgical management of severe or complex obesity and is the largest trial of its kind. Participants completed the Hospital Anxiety and Depression Scale (HADS) on study enrolment (pre-randomisation) and at 12 months post-randomisation. In this sub-study, we describe provisional data concerning the baseline prevalence of anxiety and depression along with change in median HADS symptom score amongst those who actually underwent bariatric surgery.758 participants met the criteria for study inclusion with 716 (94.46%) and 712 (93.93%) individuals fully completing questionnaires for HADS-A and HADS-D. At pre-randomisation, the prevalence of possible (HADS A/D = 8-10) and probable (HADS A/D >11) anxiety or depression was 46.19% (n 330/716) and 48.17% (n 48.17%) respectively. Paired and complete HADS-A and HADS-D questionnaires were available for 70.25% (n 503/716) and 69.94% (n 498/712) participants. There was a highly statistically significant decrease in median HADS-A and HADS-D scores at 12 months post-randomisation (Wilcoxon signed-rank test p < 0.001). This was coupled with a statistically significant reduction in the proportion of cases with possible and probable anxiety and also depression at 12 months post-randomisation.Our results characterise the high rate of psychological comorbidity amongst patients with severe or complex obesity selected for bariatric surgery. Whilst bariatric surgery remains the most clinically effective treatment for severe obesity, its effects on long-term post-operative mental health outcomes are less clear. These findings contribute to the growing body of evidence calling for increased pre/post-operative mental health surveillance and integrated care for this cohort of patients."} +{"text": "We investigated nasopharyngeal microbial community structure in COVID-19-positive and -negative patients. High-throughput 16S ribosomal RNA gene amplicon sequencing revealed significant microbial community structure differences between COVID-19-positive and -negative patients. This proof-of-concept study demonstrates that: (1) nasopharyngeal microbiome communities can be assessed using collection samples already collected for SARS-CoV-2 testing and (2) SARS-CoV-2 infection is associated with altered dysbiotic microbial profiles which could be a biomarker for disease progression and prognosis in SARS-CoV-2.The online version contains supplementary material available at 10.1186/s12575-021-00148-6. COVID-19 is primarily transmitted through the respiratory tract via aerosolized droplets containing viral particles . Due to i.e., PCR reagent blanks; n\u2009=\u20095) were amplified and sequenced with samples. Sequencing was performed using an Illumina MiniSeq with a mid-output kit and paired-end 153 base reads.Isolation of viral nucleic acids and bacterial DNA from nasal swab VTM (200ul) samples were performed using the NucleoMag Pathogen manufacturer\u2019s protocol . Microbiome characterization was performed using a PCR-next-generation sequencing (NGS) approach with a two-stage PCR protocol, as described previously . The V4 Raw sequences obtained from the sequencer were merged using the PEAR (Paired-End read merger) algorithm (v0.9.11) . Merged p\u2009<\u20090.05. Permutation Multivariate Analysis of Variance (PERMANOVA) was used to assess global differences in microbial communities between groups [Analyses of alpha- and beta-diversity were used to compare nasopharyngeal microbial communities in COVID-19-positive and -negative patients; all analyses were performed on feature (ASV) counts. Alpha-diversity metrics were calculated on rarefied data . Differences in alpha-diversity indices and bacterial ratios were assessed for significance using unpaired t-test or Mann\u2013Whitney test, based on the outcome of Shapiro-Wilks normality test. Significance levels were set at n groups . SignifiRandom forest models were used to predict featured taxa of importance using the R version of the Boruta algorithm . Analysiq\u2009=\u20090.016) in nasopharyngeal microbial community structure were observed between positive and negative patients in beta diversity analyses conducted on microbial features Proteobacteria-to-Actinobacteria ratio relative to COVID-19-negative patients less abundant in COVID-19-positive relative to -negative patients, with nine other genera trending (p \u02c2 0.05) towards significance Fig.\u00a0. Microbints Fig.\u00a0. ANCOM iCorynebacterium (Actinobacteria) [Streptococcus (Firmicutes), previously shown to be affected by influenza virus infection [This proof-of-concept study provides evidence that current biorepositories with nasopharyngeal VTM can be utilized to assess microbiome communities. Importantly, this study is the first to show that COVID-19-positive patients have a dysbiotic nasopharyngeal microbial community characterized by loss of putative nasal commensal bacteria and an increase in putative pro-inflammatory bacteria. Specifically, this study revealed a characteristic nasal microbiome in COVID-19-positive individuals with (1) a trend toward reduced microbial alpha diversity, (2) an alteration in the relative abundance of multiple taxa within the phylum Proteobacteria and a significantly increased ratio of Proteobacteria:Actinobacteria, and (3) decreased relative abundance of nasal commensal organisms such as acteria) and Strenfection . These rMostafa et al. [Propionibacteriaceae and lower Corynebacterium accolens in COVID-19-positive relative to -negative individuals. In comparison, VTM data in this study indicated a similarly reduced (though not significant) microbial diversity, a significant difference between microbial communities, and a markedly decreased abundance of genera Corynebacterium in COVID-19-positive relative to -negative patients. Due to mismatches between the 16S rRNA gene sequence of bacteria from the genus Propionibacterium and the V4 primers employed in this study , Propioquencing . DiffereLimitations include the relatively small sample size that may have constrained our ability to identify additional significant difference between groups. We acknowledge a lack of inclusion of in-depth clinical medical information, as only basic demographic data was collected from patients at the COVID-19 rapid COVID-19 testing sites from where these samples were obtained. Additionally, internal household spousal or live-in close members as COVID-19 negative controls would be an ideal comparison cohort to help exclude environmental effects. We suggest that future research studies should attempt to include these data.In conclusion, this proof-of concept study demonstrates the feasibility of using VTM from nasopharyngeal swab collections to study nasopharyngeal microbiota in COVID-19. As hospitals have collected and stored COVID-19 testing swabs and VTM over the entirety of the pandemic, this opens a huge potential dataset for interrogating the nasal microbiome. Secondly, this proof-of-concept study provides preliminary data suggesting the presence of a dysbiotic and pro-inflammatory nasopharyngeal microbiota in COVID-19-positive patients. Our study provides a strong scientific rationale for future studies to investigate the relationship between nasal microbiome and SARS-CoV-2 infection and COVID-19 severity, and also the relationship to the long-lasting effects of COVID-19.Additional file 1: Supplementary Table 1. Demographics and clinical characteristics of COVID-19-positive individuals. Supplementary Table 2. Alpha-diversity values in nasopharyngeal samples. Supplementary Table 3. Global community analysis of nasopharyngeal microbial community structure in COVID-19-positive and -negative patients, as assessed by Permutational Multivariate Analyses of Variance (PERMANOVA). Supplementary Table4. Relative abundances of bacterial taxa in COVID-19-positive and -negative nasopharyngeal samples. Supplementary Table 5. Differential relative abundance of nasopharyngeal bacterial phyla in COVID-19-positive and -negative samples identified by DESeq2 analysis. Supplementary Table 6. Genus taxonomic level differential abundance DeSeq2 analysis between COVID-19-positive and -negative patient\u2019s nasopharyngeal samples.Additional file 2: Supplementary Figure1. Microbial features driving differentiation of COVID-19-positive and -negative patients. A predictive model based on the genus-level relative abundance was generated using Boruta. Green boxes are bacterial genera that are strongly associated with differentiating the groups using the Boruta feature selection algorithm. Blue boxes are the shadow genera introduced into Random Forest classifier to act as benchmarks. Red boxes are bacterial genera that were not associated with differentiating the groups."} +{"text": "Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has led to more than 150 million infections and about 3.1 million deaths up to date. Currently, drugs screened are urgently aiming to block the infection of SARS-CoV-2. Here, we explored the interaction networks of kinase and COVID-19 crosstalk, and identified phosphoinositide 3-kinase (PI3K)/AKT pathway as the most important kinase signal pathway involving COVID-19. Further, we found a PI3K/AKT signal pathway inhibitor capivasertib restricted the entry of SARS-CoV-2 into cells under non-cytotoxic concentrations. Lastly, the signal axis PI3K/AKT/FYVE finger-containing phosphoinositide kinase (PIKfyve)/PtdInsP2 was revealed to play a key role during the cellular entry of viruses including SARS-CoV-2, possibly providing potential antiviral targets. Altogether, our study suggests that the PI3K/AKT kinase inhibitor drugs may be a promising anti-SARS-CoV-2 strategy for clinical application, especially for managing cancer patients with COVID-19 in the pandemic era. Although the detailed pathology and molecular mechanisms of COVID-19 are not completely clear yet, the virology investigation provided some knowledge and potential treatment avenues. The virus which causes COVID-19, named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), first enters into host cells by binding to the cellular receptor angiotensin-converting enzyme 2 (ACE2) via receptor-binding domain (RBD) interacting with the C-terminal domain of SARS-CoV-2 spike protein (S) Pandemic coronavirus disease-2019 (COVID-19) has caused more than 150 million infections and about 3.1 million deaths up to date Upon SARS-CoV-2 virions attachment to the host cells, viral spike protein shows a conformation change which mediates the viral envelope fusion with the host cell membrane via an endosomal pathway Given the similarity of signaling of cancer and COVID-19, a recent study revealed a large scale kinases mediated global phosphorylation of both viral and host cell proteins in a cell line of monkey kidney Vero E6 cells by whole proteomics analysis upon SARS-CoV-2 infection 2.Vero cells were purchased from the China Center for Type Culture Collection (CCTCC) and were cultured in DMEM supplemented with 10% FBS (Gibco-Invitrogen) and 1% penicillin/streptomycin at 37 \u00b0C in an incubator filled with 5% COhttps://www.ncbi.nlm.nih.gov), GenCLiP3 GeneCards, using key words above and \u201cHomo sapiens\u201d for human species plus the input of targets from recent published pool http://www.bioinformatics.com.cn). For capivasertib (AZD5363) based drug targets-COVID-19 PPI, the targets were identified based on the drug bank by dissociation constant (Kd) of 100 nM to 10 \u00b5M range of dose (https://lincs.hms.harvard.edu/db/sm/10510-101-1/), and PPIs were established after screening of crosstalk intersection targets by venny 2.1.0 tool with STRING database and through Cytoscape 3.7.2 tool as mentioned above.The drug targets of COVID-19 and kinases related targets were collected by searching databases of NCBI glycoprotein deficient VSV exogenously expressing firefly luciferase (VSV-dG-Luc) and SARS-CoV-2 pseudotyped virus whose S protein was packaged using the VSV-dG-Luc were kindly offered by Professor Huan Yan . VSV-dG-Luc and SARS-CoV-2 S pseudovirion were used to transduce Vero cells at a multiplicity of infection (MOI) of 1 seeded into 96-well plates. After overnight incubation with the different concentrations of a phosphoinositide 3-kinase (PI3K)/AKT inhibitor capivasertib (Beyotime Biotechnology), the firefly luciferase activity was measured by Dual Luciferase Reporter Assay Kit . Each concentration of capivasertib had 3-5 duplicates. The firefly luciferase activity in the control group without capivasertib was normalized to 1. Then, the relative luciferase activity in the group with capivasertib was calculated and drawed. The experiment was repeated at least three times.The database re-analysis based on the transcripts per kilobase million (TPM) was performed for patient overall survival according to instructions as described in the website https://www.ncbi.nlm.nih.gov), GenCLiP3 (http://ci.smu.edu.cn/genclip3/GeneAssociation.php), GeneCards (https://www.genecards.org/), databases, and Venny 2.1.0 (https://bioinfogp.cnb.csic.es/tools/venny/) tool for mapping the common targets. We obtained the predicted encoding intersection targets suggesting the interaction proteins involved in kinase mediated COVID-19 infection, progression, and host cell reaction (Figure Firstly, we searched a variety of databases for \u201cCOVID-19\u201d and \u201ckinase\u201d targets from NCBI (in vitro. In fact, luciferase activity could reflect viral infection rate changes from two steps of viral entry and viral genome replication/expression. In order to exclude the role of capivasertib during the step of viral genome replication/expression, we tested the effect of capivasertib on VSV-dG-Luc in Vero cells where SARS-CoV-2 spike was overexpressed or not. The data indicated that the treatment of capivasertib had no effect on the luciferase activity of VSV-dG-Luc infecting Vero cells with SARS-CoV-2 spike overexpression or not, suggesting that capivasertib was not related to the genome replication and expression of VSV-dG-Luc (Figure Next, we aimed to explore the influence of AKT kinases signaling on COVID-19 by testing the efficacy of the predicted drug in a SARS-CoV-2 model system. Capivasertib is a potent pan-AKT kinase inhibitor drug that inhibits AKT1, AKT2 and AKT3. Capivasertib had significant antitumor activities, and was being used as an oral small-molecule AKT inhibitor for drug-resistant breast cancer in clinical trials c Figure . ClearlyFinally, we searched database of gene expression profiling of the targets and listed the poor outcome of overall survival which correlated to the gene overexpression encoding for AKT kinases. The significant cancer types are urothelial bladder carcinoma, kidney chromophobe carcinoma, prostate adenocarcinoma, uterine corpus endometrial carcinoma for AKT Figure . Thus, oHere, we reported a whole kinase-protein-protein interaction network, which identified a top ranked AKT kinase-mediated pathway and the related clinical anti-cancer drugs for possibly treating COVID-19 patients by targeting AKT. The clinical applicable drug capivasertib has a great potential for clinical trial of anti-SARS-CoV-2.It was very interesting to investigate the detailed relationship between PI3K/AKT signal pathway and viral infection. The previous many studies reported that the activation of the PI3K/AKT signaling pathway facilitated viral replication PI3K/AKT signaling plays a key role in cell proliferation, survival, growth, migration, invasion, and can inhibit apoptosis and promote angiogenesis. Abnormal PI3K/AKT signaling pathway can cause diseases, such as cancer. Cancer has always been one of the major health problems. With the continuous progress of life science, targeted therapy has become the latest popular method for malignant tumor. Among them, inhibitors of the PI3K/AKT pathway play an essential role in cancer treatment. PI3K and AKT are potential tumor drug targets, and their anti-tumor therapies show attractive prospects. Capivasertib is a potent pan-AKT kinase inhibitor drug that inhibits AKT1, AKT2 and AKT3. It had significant antitumor activity and was an attractive lead of antitumor drugs. Capivasertib is being used as an oral small-molecule AKT inhibitor for drug-resistant breast cancer in clinical trials Our study showed that a kinase inhibitor capivasertib, also an AKT-targeted anti-cancer drug, could prevent the entry of SARS-CoV-2 to cells. There are so many antitumoral drugs targeting PI3K/AKT signal pathway in clinical trials, which would possibly be valuable drug resources to combat the current COVID-19 pandemic and be worth managing the critical urological cancer patients with COVID-19 in the pandemic era."} +{"text": "Here, we propose Fourier ring correlation-based quality estimation (FRC-QE) as a new metric for automated image quality estimation in 3D fluorescence microscopy acquisitions of cleared organoids that yields comparable measurements across experimental replicates, clearing protocols and works for different microscopy modalities.https://github.com/PreibischLab/FRC-QE.FRC-QE is written in ImgLib2/Java and provided as an easy-to-use and macro-scriptable plugin for Fiji. Code, documentation, sample images and further information can be found under Bioinformatics online. Three-dimensional (3D) organoids are a powerful tool for studying cellular processes in tissue-like structures, enabling in vitro experiments in an organ-specific context . HoweverFourier ring correlation (FRC) relies on two independent realizations of the same signal to measure image resolution in frequency space and was used for both electron and supeTo validate FRC-QE, we generated human induced pluripotent stem-cell (hiPSC)-derived cerebral organoids of a defined size (\u223c600\u2009\u00b5m) that were stained with the nuclear dye Draq5 and subsequently chemically fixed. We chose three straight-forward to implement clearing methods . ClearedWe first compared FRC-QE to the NR-IQA method DCT Shannon entropy, which was previously used in the context of automated microscopy .We further validated FRC-QE for different microscopy modalities comparing Fructose-Glycerol-cleared organoids to uncleared organoids using a spinning-disk confocal microscope, where FRC-QE recapitulated differences in image quality across image stacks and protocols .Next, we used FRC-QE to identify the clearing protocol yielding the best image quality. Across all protocols nuclear structures can be easily visually identified at the surface of organoids. However, image quality differs at the center, with ClearT2 and ScaleA2 protocols resulting in blurred objects compared to the Fructose-Glycerol protocol , indicatWe additionally compared FRC-QE to the established machine learning based NR-IQA algorithm BRISQUE . HoweverWe introduce FRC-QE, implemented in ImgLib2 (btab160_Supplementary_DataClick here for additional data file."} +{"text": "Although dozens of terminal selectors have been described thus far for individual neuron types of the nematode C. elegans , the identification of their target genes has primarily relied on candidate approaches and availability of markers for neuronal terminal identity. Hence, unbiased methods are needed to identify the full spectrum of terminal selector target genes in individual neuron types. This study focuses on the phylogenetically conserved terminal selector UNC-3/Ebf (member of the Collier/Olf/Ebf family), which controls cholinergic motor neuron (MN) identity in the ventral nerve cord of the nematode C. elegans. To identify novel UNC-3 target genes, we took advantage of the genome-wide binding map of UNC-3 from our previous Chromatin Immunoprecipitation followed by Sequencing (ChIP-Seq) analysis . We generated transgenic reporter lines for ten putative terminal identity genes , whose expression patterns were largely unknown in C. elegans. Six of these reporter lines showed expression in ventral nerve cord MNs , whereas the remaining four showed expression in head and tail neurons, as well as some non-neuronal cells. Importantly, the number of ventral nerve cord MNs showing expression of the nep-21, D2007.2, and dmsr-2 reporters was significantly reduced in unc-3 null mutant animals, thereby expanding the repertoire of known UNC-3 target genes in these cells. Altogether, this study demonstrates that transgenic reporter analysis guided by ChIP-Seq results is a relatively efficient approach for the identification and validation of transcription factor target genes.Terminal selector-type transcription factors are key regulators of neuronal identity and function . Mechanistically, terminal selectors are thought to act directly through binding at the Through a candidate approach, more than 50 terminal identity genes have been identified genetically as downstream targets of UNC-3. These genes encode proteins essential for the proper functions of cholinergic MNs and include acetylcholine biosynthesis components, ion channels, neurotransmitter receptors, and neuropeptides . To test whether UNC-3 directly controls the expression of these genes, we recently performed Chromatin Immunoprecipitation followed by Sequencing (ChIP-Seq) analysis for UNC-3. Indeed, UNC-3 directly binds to the cis-regulatory region of these ~50 known target genes via a consensus DNA binding site, termed COE motif . This suggests that activating terminal identity genes through physical binding to their loci is a key strategy employed by UNC-3 to regulate cholinergic MN identity.The role of UNC-3 in establishing and maintaining the cell identity of cholinergic motor neurons (MNs) has been investigated in several studies . We focused on testing 10 putative terminal identity genes that showed strong UNC-3 binding signal in their cis-regulatory region . To test the hypothesis that UNC-3 binding is required for gene expression, we first generated transgenic reporter lines for these 10 genes by amplifying the genomic region bound by UNC-3 and fusing it with a fluorescent reporter tagRFP. We found 6 reporter lines with strong expression in ventral nerve cord MNs. Intriguingly, all these six genes have been independently validated by the CeNGEN consortium ; RNA-Sequencing analysis shows high expression levels of nep-21, D2007.2, dmsr-2, ncs-2, npr-29, and drn-1 in ventral cord MNs. To assess whether their expression in MNs is dependent on UNC-3, we crossed these reporter lines with unc-3(n3435) null mutants .Here, we sought to expand the list of UNC-3 targets by taking advantage of the genome-wide binding map of UNC-3 from our previous ChIP-Seq analysis , (b) dmsr-2 is predicted to encode a protein that enables G protein-coupled peptide receptor activity , and (c) D2007.2 is an uncharacterized gene encoding a protein with an Immunoglobulin-like domain . The number of cholinergic MNs showing expression of nep-21::RFP, dmsr-2::RFP and D2007.2::RFP is significantly decreased in unc-3(n3435) animals when compared to wild-type animals animals -C, sugge animals -F. This do not show expression in ventral nerve cord MNs, despite UNC-3 binding on their cis-regulatory regions . Future studies are needed to determine whether pxd-1, cal-2, lgc-4, and ldb-1 are expressed in any of these unc-3-expressing neurons. Lastly, our observations suggest that transgenic reporter analysis guided by ChIP-Seq can be used for the generation of tissue and neuron-specific reporter lines.We also found that RFP reporter lines for the remaining four genes regions . Since t regions . UNC-3 iC. elegans, whose expression patterns were largely unknown. These tagRFP reporter lines will be useful tools for researchers who intend to investigate further the functions of these genes in the C. elegans nervous system. Lastly, our reporter analysis of ChIP-Seq-defined enhancer regions identified three novel target genes of the terminal selector UNC-3 in cholinergic MNs, expanding the repertoire of its target genes.Altogether, we generated transgenic reporter lines for ten putative terminal identity genes in Generation of transgenic animals carrying transcriptional fusion reporterscis-regulatory analyses and validation of newly identified UNC-3 target genes were made with PCR fusion . Genomic regions were amplified and fused to the coding sequence of tagrfp followed by the unc-54 3\u2019 UTR. PCR fusion DNA fragments were injected into young adult pha-1(e2123) hermaphrodites at 50 ng/\u00b5l together with pha-1 (pBX plasmid) as co-injection marker (50 ng/\u00b5l).Reporter gene fusions for"} +{"text": "The brain tumor immune microenvironment (TIME) continuously evolves during glioma progression, but a comprehensive characterization of the glioma-centric immune cell repertoire beyond a priori cell states is uncharted. In this study, we performed single-cell RNA-sequencing (scRNA-seq) and single cell- Assay for Transposase-Accessible Chromatin using sequencing (sc-ATAC-seq) on ~100,000 tumor-associated immune cells from seventeen isocitrate dehydrogenase (IDH) mutation classified primary and recurrent human gliomas and non-glioma brains (NGBs). Our analyses revealed sixty-two transcriptionally distinct myeloid and lymphoid cell states within and across glioma subtypes and we noted microglial attrition with increasing disease severity concomitant with invading monocyte-derived cells and lymphocytes. Specifically, certain microglial and monocyte-derived subpopulations were associated with antigen presentation gene modules, akin to cross-presenting dendritic cells (DCs). We identified cytotoxic T cells with poly-functional cytolytic states mostly in recurrent IDH-wt gliomas. Furthermore, ligand-receptor interactome analyses showed a preponderance of antigen presentation and phagocytosis over the checkpoint axis in IDH-wt compared to IDH-mut gliomas.+ exhausted T cells from IDH-wt showed strong enhancer accessibility on Cytotoxic T-lymphocyte-associated protein 4, Layilin and Hepatitis A Virus Cellular Receptor 2 but no enrichment on PDCD1 (gene encoding Programmed cell death protein 1) was seen. In summary, our study provides unprecedented granular detail of transcriptionally defined glioma- specific immune contexture that can be exploited for immunotherapy applications.Additionally, our sc-ATAC-seq analyses revealed differences in regulatory networks in NGBs, IDH-mut and IDH-wt glioma associated immune cells. In particular, we noted abundant usage of inflammatory transcription factors (TFs) as exemplified by Nuclear factor kappa B and Activator Protein-1 TF family in IDH-wt microglia when compared with microglia from IDH-mut and NGBs. Unique features such as amplification of 11- Zinc Finger Protein accessibility were restricted to monocyte derived cells and were not observed in microglia. Finally, sc-ATAC-seq profiles of CD8This study in K.B. laboratory was supported by the generous philanthropic contributions to The University of Texas (UT) MD Anderson Cancer Center (MDACC) Moon Shots Program\u2122, Marnie Rose Foundation, NIH grants: R21 CA222992 and R01CA225963. This study was partly supported by the UT MDACC start-up research fund to L.W. and CPRIT Single Core grant RP180684 to N. E. N."} +{"text": "A repeated contrast-enhanced MRI showed new, intense leptomeningeal enhancement of the cerebellar hemispheres, highly suspicious for leptomeningeal metastases. Leptomeningeal biopsy confirmed the diagnosis of leptomeningeal spread of the known meningioma, with rhabdoid transformation (WHO grade 3). Diffuse cerebellar cortical hypermetabolism on 18F-FDG-PET/CT is a rare phenomenon reported in a few cases with paraneoplastic cerebellar degeneration [18F-FDG-PET/CT can be an early imaging marker for leptomeningeal metastases in solid tumors, appearing even before contrast enhancement on MR imaging. Recognizing this phenomenon may prevent delayed diagnosis and treatment.A 20-year-old woman was referred to our center with a 3-month history of progressive nausea, headache, and meningismus. She had surgery followed by proton-therapy three years earlier for a right-sided parasagittal atypical meningioma. Extensive blood and CSF examinations were done, but cultures remained negative and no infectious agents or malignant cells were identified. An"} +{"text": "Strategies to address the problem of poorly water-soluble drugs encompass the conversion of a crystalline drug into an amorphous form to promote its apparent solubility and dissolution. Co-amorphous systems (CAMs) incorporate low molecular mass molecules (co-formers), which are mixed with the drug to form one single phase ,2. The al-aspartate (ASP); l-tryptophan (TRY); l-arginine (ARG); l-proline (PRO); citric acid (CIT); tartaric acid (TAR); oxalic acid (OXA); saccharine (SAC); potassium acessulfame (ACE); cyclamic acid (CYC)] in 1:1 molar ratios were submitted to ball milling (BM), solvent evaporation (SE) and quench cooling (QC). CAMs were evaluated for the OLZ solubility increase, co-formability and storage stability over time, by differential scanning calorimetry (DSC), X-ray powder diffraction (XRPD) and Fourier-transform infra-red spectroscopy (FTIR).Mixtures (2\u2009g) of olanzapine (OLZ) and each co-former [Discussion and conclusions: The study has shown the possibility of converting a crystalline drug into an amorphous entity, particularly when in presence of co-formers which stabilise the amorphous structures formed. In fact, with sulphonic acids, both SE and BM, achieved complete amorphization and successfully stabilised the CAMs obtained. Due to the noteworthy increase in solubility, resulting from co-amorphization, this technique is considered to be adequate to process active compounds with poor water solubility, such as OLZ.The BM technique presented the most promising results followed by QC and SE , since m"} +{"text": "Staphylococcus aureus (MRSA) acquisition at UVA Hospital (P=0.016). This \u201cnatural experiment\u201d allowed us to analyze 3 key mechanisms by which the pandemic may have influenced nosocomial transmission: 1) enhanced infection control measures , 2) patient-level risk factors, and 3) networks of healthcare personnel (HCP)-mediated contacts.The COVID-19 pandemic was associated with a significant (28%) reduction of methicillin-resistant Figure 1. Monthly MRSA Acquisition Rates Pre- and Post-COVD-19Hospital MRSA acquisition was defined as a new clinical or surveillance positive in patients with prior unknown or negative MRSA status occurring >72h after admission. 10 month time periods pre- (5/6/2019 to 2/23/2020) and post-COVID-19 (5/4/2020 to 2/28/2021) were chosen to mitigate the effects of seasonality. A 6-week wash-in period was utilized coinciding with the onset of several major hospital-wide infection control measures . Box and whisker plots depict quartile ranges, median (dotted line), and mean values. Mean MRSA acquisition rates pre- significantly declined post-COVD-19 . Independent-samples t tests were used (2-tailed) for statistical comparisons except for variables without a normal distribution (Shorr Scores), for which a Mann-Whitney U test was used.Census-adjusted hospital-acquired MRSA acquisition events were analyzed over 10 months pre- (5/6/2019 to 2/23/2020) and post-COVD-19 (5/4/2020 to 2/28/2021), with a 6-week wash-in period coinciding with hospital-wide intensification of infection control measures . HCP hand hygiene compliance rates were examined to reflect adherence to infection control practices. To examine impacts of non-infection control measures on MRSA transmission, we analyzed pre/post-COVD-19 differences in individual risk profiles for MRSA acquisition as well as a broad suite of properties of the hospital social network using person-location and person-person interactions inferred from the electronic medical record. Figure 2. Social Network ConstructionWe constructed a contact network of hospitalized patients and staff at University of Virginia Hospital to analyze the properties of both person-location and person-person networks and their changes pre- and post-COVID-19. Colocation data -patient interactions recorded in the electronic health record, e.g., medication administration) were used to construct contact networks, with nodes representing patients and HCP, and edges representing contacts. The above schematic shows how the temporal networks are inferred. In the figure, circles represent patients and the small filled squares represent HCP, while the larger rectangles represent patient rooms. The first room is a shared room with two patients. At each time step, co-location is inferred from the EMR data, which specifies interactions between HCP and patients. This can be represented as the temporal network (t) at the bottom.Hand hygiene compliance significantly improved post-COVD-19, in parallel with other infection control measures. Patient Shorr Scores were statistically similar pre-/post-COVD-19. Analysis of various network properties demonstrated no trends to suggest a reduced outbreak threshold post-COVD-19.Figure 3. Hand Hygiene Compliance RatesAnalysis of hospital-wide hand hygiene auditing data ) demonstrated a statistically significant (6%) improvement in average monthly hand hygiene compliance .Figure 4. Individual MRSA Risk FactorsWe calculated the Shorr Score :2205-10) for patients using data from the electronic health record to test the hypothesis that individual risk factors in aggregate did not change significantly in the post-COVD-19 period to explain changes in MRSA acquisition. Values for this score ranged from 0 to 10 with the following criteria: recent hospitalization (4), nursing home residence (3), hemodialysis (2), ICU admission (1). Pictured are frequency distributions of Shorr scores in the pre-COVID-19 and post-COVID-19 periods. The Mann-Whitney effect size (E), 0.53 (P=0.51), indicated that pre- and post-COVD-19 distributions were very similar.Figure 5. Social Network Properties and Analysis We analyzed three major types of network properties for this analysis: (1) Node properties of the pre- and post-COVID-19 networks consisted of all the edges in the pre- and post-COVID-19 periods, respectively. We considered a number of standard properties used in social network analysis to quantify opportunities for patient-patient transmission: degree centrality (links held by each node), betweenness centrality (times each node acts as the shortest \u2018bridge\u2019 between two other nodes), closeness centrality (how close each node is to other nodes in network), Eigenvector centrality (node\u2019s relative influence on the network), and clustering coefficient (degree to which nodes cluster together) in the first five panels ; . Each panel shows the frequency distributions of these properties. These properties generally did not have a normal distribution and therefore we used a Mann Whitney U test on random subsets of nodes in these networks to compare pre- and post-COVID properties. The mean effect size (E) and P-values are shown for each metric in parenthesis. We concluded that all of these pre- versus post-COVID-19 network properties were statistically similar. (2) Properties of the ego networks (networks induced by each node and its \u2018one-hop\u2019 neighbors). We considered density and degree centrality (number of links held by each node) of ego networks (middle right and bottom left panels). The mean effect size and p-values using the Mann Whitney test are shown in parenthesis; there were no statistically significant differences in these properties in the pre- and post-COVID networks. (3) Aggregate properties of the weekly networks, consisting of all the interactions within a week. We considered modularity and density of the weekly networks (bottom middle and bottom right panels). The modularity in the post-COVID weekly networks was slightly lower , while density was slightly higher, the differences of which were statistically significant; a caveat is that these are relatively small datasets (about 40 weeks). These differences both increase the risk of transmission in the post-COVID networks. In summary, the post-COVID networks either have similar properties as the pre-COVID networks, or had changes which are unlikely to have played a role in reducing MRSA transmission.A significant reduction in post-COVD-19 MRSA transmission may have been an unintended positive effect of enhanced infection control measures, particularly hand hygiene and increased mask use. A modest (11.6%) post-COVD-19 reduction in surveillance testing may have also played a role. Despite pandemic-related cohorting and census fluctuations, most network properties were not significantly different post-COVID-19, except for aggregate density and modularity which varied in a direction that instead favored transmission; therefore, HCP-based networks did not play a significant role in reducing MRSA transmission. Multivariate modeling to isolate relative contributions of these factors is underway.Figure 6. Surveillance Testing and Clinical CulturingPost-COVD-19, there was a modest (11.6%) but statistically significant reduction in surveillance PCR testing . There was not a statistically significant difference in rates of clinical cultures sent .All Authors: No reported disclosures"} +{"text": "COVID 19 is associated with a hypercoagulable state with cytokine storm syndrome and thrombocytopenia leading to complications across various systems. COVID-19 infection, its treatment, resultant immunosuppression, and pre-existing comorbidities have made patients vulnerable to secondary infectionsWe systematically reviewed COVID-19 cases between Jan to May 2021 for pulmonary and extrapulmonary complications. Patients with recent COVID-19 vaccination and neurological symptoms were also included.Figure 1. \u201cBlack turbinate\u201d sign of mucormycosisContrast enhanced coronal T1 FS images of paranasal sinuses shows necrotic non-enhancing right superior and middle turbinates (*) with extension into the right orbital fat.FIGURE 2 - A composite image of Coronal CT of upper abdomen in arterial phase and lung bases in lung window showing wedge showing right renal infarcts (line arrow) due to inferior polar artery thrombosis and ground glass opacities (solid arrow) in lung bases.Neurological complications: Neurological complications include ischemic and haemorrhagic strokes. Other complications are encephalopathy, encephalitis, Guillain-Barr\u00e9 syndrome, acute hemorrhagic necrotizing encephalopathy. Demyelination and radiculopathies are seen as post vaccination complications. Mucormycosis: Unprecedented high rate of invasive fungal sinusitis in association with COVID -19 is reported from the Indian subcontinent. This has a propensity for intra orbital and intracranial extension. COVID -19 associated coagulopathy: COVID -19 is a pro-inflammatory hypercoagulable state. Pulmonary thromboembolism, deep venous thrombosis and catheter related thrombosis are well documented. Cardiac complications: Cardiac manifestations include Myocardial Injury with non-obstructed coronary arteries (MINOCA), myocarditis, myocardial ischemia, cardiomyopathy. Pulmonary complications and sequelae of COVID -19: Progression of lung injury to ARDS during the initial phase and fibrosis of parenchyma in the recovery phase. Spontaneous pneumomediastinum, pneumatoceles and pneumothorax and secondary infections are identified in our study. COVID- 19 associated gastrointestinal complications: Patients evaluated for renal colic, pancreatitis, cholecystitis showed, ground glass opacities or subpleural bands in typical Covid-19 distribution. COVID-19 may lead of acute kidney and bowel injury due to arterial thrombosis. COVID - 19 associated myonecrosis: Ischemia of the small caliber vessels may result in myonecrosis.FIGURE 3 - Coronal STIR image shows thickened and hyperintense trunks and divisions of the right brachial plexus suggestive of plexopathy in a COVID -19 patient with H/O recent COVID-19 vaccination.Figure 4. Axial CT chest section in lung window showing pneumothorax (white arrow) and pneumatocele ( grey arrow) with peripheral ground glass opacities and consolidations in both lungs.Awareness of these unusual manifestations will facilitate an early diagnosis, improve management and help reduce morbidity and mortalityAll Authors: No reported disclosures"} +{"text": "Successful aging depends on avoiding disease and disability, maintaining high physical and cognitive function, and psychological adaptation. Research examining the relationship of pet ownership (PO) or human-animal interaction (HAI) to human health supports contributions to these successful ag-ing-related outcomes at some point in the life-cycle, mostly in populations with diseases or disabili-ties. We examine the contributions of PO to maintaining physical capacity among generally healthy community-dwelling older participants in the Baltimore Longitudinal Study of Aging (BLSA). Partici-pants\u2019 completed a standardized physi-cal function test battery (among other measures) every 1-4 years and a ten-year PO history. Linear mixed, or generalized linear mixed, models with time varying PO were used to examine change in successful aging-related outcomes over up to 13 years according to PO. Physi-cal function declined across all domains examined, but was observed to be less severe with PO in overall physical performance (p<0.001), rapid gait speed (p=0.041), 400-meter walk time (p<0.001), and reported physical wellbeing (p=0.032). No differences were observed for grip strength (p=0.56), usual gait speed (p=0.07), and leisure time physical activity (p=0.26) after con-trolling for age. This study provides the first longitudinal evidence that PO may promote successful aging among community-dwelling healthy older adults by moderating age-related declines in physical functional status in late-life."} +{"text": "We found that the differences between ROS levels generated from TFN and tobacco-derived nicotine-containing vape products vary by flavor. TFN tobacco flavored and fruit flavored products are more toxic in terms of ROS generation than menthol/ice and drink/beverage flavored products using TFN. Our study provides further insight into understanding how flavoring agents used in vape products impact ROS generation from e-cigarettes differently in TFN e-cigarettes than e-cigarettes using tobacco-derived nicotine.Electronic nicotine delivery systems (ENDS) containing synthetic nicotine have yet to be classified as tobacco products; consequently, there is ambiguity over whether Food and Drug Administration (FDA) regulatory authority can be extended to include tobacco-free nicotine (TFN) e-cigarettes. In recent years, a more significant number of e-cigarette companies have been manufacturing TFN-containing e-cigarettes and e-liquids to circumvent FDA regulations. While studies have shown that aerosols generated from tobacco-derived nicotine-containing e-cigarettes contain significant reactive oxygen species (ROS) levels, no comparison studies have been conducted using TFN e-cigarettes. This study uses a single puff aerosol generator to aerosolize TFN and tobacco-derived nicotine-containing vape products and subsequently involves semi-quantifying the ROS generated by these vape products in H Based on data from the 2021 National Youth Tobacco Survey (NYTS), a report published in the Morbidity and Mortality Weekly Report estimated 11.3% 1.72 million) of high school students and an estimated 2.8% of middle school students currently use e-cigarettes ,5,6. Add2 millionThe 2021 NYTS found that out of all youth e-cigarette users surveyed, 85% used flavored e-cigarettes . RegardiThree different TFN vape-bars and three different TFN e-liquids were analyzed in this study . In addi2O2 equivalents. Aerosols from each individual TFN vape-product used in the study were generated using a Buxco Individual Cigarette Puff Generator , St. Paul, MN, USA) (Cat#601-2055-001) (2DCF-DA) (Cat#287810), phosphate (PO4) buffer, and horseradish peroxidase . Each TFN e-liquid was aerosolized using a new, empty refillable JUUL Pod (Model: WO1 JUUL Pods) inserted into a JUUL device (Model: Rechargeable JUUL Device w/USB charger). Subsequently, this JUUL device was inserted into the Individual Cigarette Puff Generator.ROS levels within aerosols generated from all twelve vape-products were quantified via spectrofluorometry and in H055-001) . Upon in055-001) . One puf2O). The tubing was also rinsed with 70% ethanol and ddH2O prior to generating puffs from a different e-cigarette model.Each vape-bar and JUUL Pod containing TFN e-liquid had undergone three separate puffing regimens to prepare three individual samples of 10 mL dye solution exposed to e-cigarette aerosols. For our negative control, filtered air was passed through fluorogenic dye using the previously mentioned puffing regimen and inserting a filter into the Individual Puff Generator instead of an e-cigarette. For our positive control, the smoke generated from a conventional cigarette (Model Reference: 3R4F) was exposed to fluorogenic dye under the previously mentioned puffing regimen. To avoid cross-contamination, once a specific e-cigarette had undergone a single puffing regimen, the tubing connecting the Puff Generator to the 50 mL conical tube containing dye was rinsed with 70% ethanol and then double-distilled water (Cat#H323-500) and ddH2O. After aerosolizing each vape product and exposing its generated aerosols to three separate 10 mL samples of fluorogenic dye, each resulting fluorogenic dye sample and standard was placed in a 37 \u00b0C degree water bath for fifteen minutes. After placing each sample and standard into the water bath, the resulting solutions were analyzed via fluorescence spectroscopy (Ex = 475 nm and Em = 535 nm). Readings were taken on a spectrofluorometer (Model: FM109535) in fluorescence intensity units (FIU) and measured as H2O2 equivalents.Subsequently, 0 \u03bcM, 10 \u03bcM, 15 \u03bcM, 20 \u03bcM, 30 \u03bcM, 40 \u03bcM, and 50 \u03bcM Hp values < 0.05.One-way ANOVA and Tukey\u2019s post-hoc test for multiple pairwise comparisons via GraphPad Prism Software version 8.1.1 was used to conduct statistical analyses of significance. Samples were run in triplicates. The results are shown as mean \u00b1 SEM with triplicate analyses. Data were considered to be statistically significant for For the blueberry-raspberry-flavored vape-products analyzed, the level of ROS generated from the Hyppe: Blue Raz (5.0% tobacco-derived nicotine) bar (4.92\u20136.61 \u03bcM) did not significantly differ from that generated from the GLAS Basix Blue Razz (5.0% synthetic nicotine) e-liquid (4.97\u20137.44 \u03bcM) a. Among Regarding minty/iced (cooled) flavored vape products, there appear to be significant differences in ROS levels generated between TFN vape products and their corresponding flavor-specific tobacco-derived nicotine counterparts . The levWhen comparing tobacco-flavored vape products, the level of ROS generated from the aerosolized Salty Man: Creamy Tobacco (5.0% synthetic nicotine) e-liquid (2.32\u20133.96 \u03bcM) did not significantly differ from that generated from the JUUL: Virginia Tobacco (5.0% tobacco-derived nicotine) bar (1.26\u20135.14 \u03bcM) a. HoweveOur data suggest that the type of nicotine salt used in e-liquids and vape-bars, tobacco-derived or synthetic, plays a role in modulating ROS generation upon component e-liquid aerosolization. To further explain, significant differences in ROS generation were observed between TFN and tobacco-derived nicotine-containing vape-products containing drink and minty/iced flavoring. However, non-significant differences in ROS generation were observed between TFN and tobacco-derived nicotine-containing vape-products with fruity and tobacco flavoring. Our data suggest that flavoring agents used in e-cigarettes containing synthetic nicotine play a role in modulating ROS levels within generated aerosols. Our data also indicate that flavoring agents used in e-liquids affect acellular ROS generation from synthetic-nicotine-containing e-cigarettes and tobacco-derived nicotine-containing e-cigarettes of comparable flavors differently.Similarly, the results of our study seem to concur with our previous study, the data of which suggested that flavoring agents used in tobacco-derived nicotine-containing vape-bars play a role in modulating ROS generation upon component e-liquid aerosolization . RegardiInterestingly, we noticed that amongst the minty/cooled flavored vape-products analyzed (Spearmint and Banana Ice), the level of ROS generated by the synthetic-nicotine vape-product was significantly less than that generated by its flavor specific tobacco-derived nicotine-containing counterpart. Additionally, amongst the drink/beverage-flavored vape-products analyzed, the synthetic nicotine-containing vape product generated significantly less ROS than its tobacco-derived nicotine-containing counterpart. Synthetic nicotine lacks the impurities contained within tobacco-derived nicotine ,17. VapeRegarding the limitations of this study, due to there being very few companies that manufacture both TFN and tobacco-derived nicotine-containing vape-products, we could not control for the e-cigarette brand in our pairwise comparisons between TFN products and their flavor specific tobacco-derived nicotine-containing counterparts, as well as differences between enantiomers or stereoisomers (R-nicotine vs. S-nicotine) of nicotine in both the products. Many vendors which utilize synthetic nicotine in their vape products either never sold e-cigarettes using tobacco-derived nicotine or stopped selling them entirely due to the cost-burden associated with submitting PMTAs and lack of public interests, and confirming the validity of synthetic vs. natural nicotine. One study has shown that even amongst e-cigarettes of the same flavor, ROS levels within generated aerosols vary by brand . Future Our data suggest that TFN tobacco flavors and fruit flavors are more toxic in terms of ROS generation than menthol/ice and drink/beverage flavored products using TFN. In other words, beverage flavor and minty/iced (cool) flavored TFN products generate significantly less ROS than their corresponding flavor-specific tobacco-derived nicotine-containing counterparts. Our study provides insight into how interactions between flavoring agents and salt-nicotine used in e-cigarettes impact ROS levels generated by TFN e-cigarettes differently than e-cigarettes using tobacco-derived nicotine."} +{"text": "We provide evidence of concurrent and close sequential infections between SARS-CoV-2 and select arboviruses\u2014namely, chikungunya virus (CHIKV); dengue viruses 1, 2, and 3 (DENV1\u20133), and Zika virus (ZIKV)\u2014in patients in Guerrero, southwest Mexico, in 2020\u20132021. The study population consisted of 176 febrile patients with laboratory evidence of SARS-CoV-2 infection. Sera from all patients were serologically and antigenically tested for seven arboviruses known to occur in Guerrero. Eighteen patients contained CHIKV IgM, six of whom also contained CHIKV RNA. Another 16 patients contained flavivirus antigen. The flaviviruses responsible for the infections were identified by plaque reduction neutralization test as DENV1 (two patients), DENV2 (five patients), DENV3 (three patients), ZIKV (three patients), and an undetermined flavivirus (three patients). In summary, we identified patients in Guerrero, Mexico, with concurrent or recent sequential infections between SARS-CoV-2 and select arboviruses, exemplifying the importance of performing differential diagnosis in regions where these viruses cocirculate. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent of COVID-19, which is characterized by various clinical manifestations, including acute undifferentiated febrile illness, in humans.The study population consisted of 176 patients from Guerrero who had laboratory confirmed acute SARS-CoV-2 infections, probable acute or recent SARS-CoV-2 infections, or probable past SARS-CoV-2 infections. The patients presented with acute undifferentiated febrile illness in June 2020 to March 2021 at three participating sites in Guerrero: the Hospital General Adolfo Prieto in Taxco de Alarc\u00f3n (HGAPTA), Laboratorio de An\u00e1lisis Cl\u00ednicos Avellaneda in Chilpancingo (LACAC), and Labymedic Laboratorios in Acapulco (LLA). As noted earlier, all patients presented with unspecified febrile illness, but the medical personnel at the participating performance sites were not willing to provide any other clinical information because of the time needed to compile these data. Nasopharyngeal swabs were collected from select patients and serum samples were collected from all patients. If a swab was collected, the patient was tested for SARS-CoV-2 RNA by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) using the DeCoV19 Kit Triplex kit . If a swab was not collected, the patient was serologically assayed for SARS-CoV-2 using the Panbio COVID-19 IgG/IgM rapid test .- Serologic tests were performed at the participating sites and qRT-PCRs were performed at the Laboratorio MicroTec, a reference laboratory in Mexico City. Swabs were not taken from patients who considered the qRT-PCR testing to be cost-prohibitive. Patients with positive qRT-PCR results had confirmed acute SARS-CoV-2 infections. Patients that contained SARS-CoV-2 IgM, either in the presence or absence of SARS-CoV-2 IgG, were considered to have probable acute or recent SARS-CoV-2 infections. Patients with SARS-CoV-2 IgG in the absence of IgM had probable past SARS-CoV-2 infections.\u2013An aliquot of each serum was transported to Iowa State University and serologically and antigenically tested for seven arboviruses known to occur in Guerrero .Taq polymerase (ThermoFisher) in accordance to the manufacturer\u2019s instructions. Primers specific to a 445-nt region of the CHIKV E1 gene were used . RT-PCR products were purified using the purelink gel extraction kit (ThermoFisher) and sequenced using a 3730\u2009\u00d7\u20091 DNA sequencer . All sera with CHIKV IgM were also assayed by plaque reduction neutralization test (PRNT) using an isolate of CHIKV (strain CH-R-1950) originally recovered from a patient in Tamaulipas, Mexico, in 2015.90).All sera with CHIKV IgM were further assayed by RT-PCR. Complementary DNAs were generated using Superscript III reverse transcriptase , and PCRs were performed using high-fidelity 90 antibody titer to the respective virus needed to be at least 4-fold greater than that to the other flaviviruses tested.To identify patients with acute flavivirus infections, sera were assayed using the Human Dengue Virus NS1 Antigen ELISA Kit . The ELISA is not DENV-specific because the flavivirus nonstructural protein 1 contains group-reactive epitopes.The ages of the patients in the study population ranged from 4 to 89 years, with mean of 45.7 years. There were 87 females and 89 males. Half of the patients presented at the LLA (88 patients) and the remainder at the HGAPTA and LACAC . Twenty (11.4%) patients had laboratory-confirmed acute SARS-CoV-2 infections, 96 (54.5%) patients had probable acute or recent SARS-CoV-2 infections and 60 (34.1%) patients had probable past SARS-CoV-2 infections.90 titers ranging from 20 to 1280. Six of these patients also contained CHIKV RNA (Genbank Accession Nos. OL440054-OL440059). The number of CHIKV RNA-positive patients is likely an underestimate because the sera were transported to Iowa State University on ice packs instead of dry ice, which is not sold in Guerrero. Of the 18 CHIKV IgM-positive patients, two patients had confirmed acute SARS-CoV-2 infections, seven had probable acute or recent SARS-CoV-2 infections, and nine had probable past SARS-CoV-2 infections. The two CHIKV IgM-positive patients with confirmed acute SARS-CoV-2 infections were negative for CHIKV RNA. One patient was a 24-year-old man who developed symptoms in May 2020. The other patient was a 62-year-old man with illness onset in July 2020.Eighteen patients with evidence of SARS-CoV-2 infection contained CHIKV IgM in their sera Table . These pSera from 16 patients contained flavivirus antigen Table . Of thes\u2013Our data indicate that a subset of patients had concurrent or close sequential infections between SARS-CoV-2 and various arboviruses\u2014namely CHIKV, DENV1, DENV2, DENV3, and ZIKV. Other studies have reported patients with concurrent SARS-CoV-2 and DENV infections.,In conclusion, we report apparent concurrent and close sequential infections between SARS-CoV-2 and select arboviruses in Guerrero, Mexico. SARS-CoV-2 and the arboviruses under investigation produce overlapping clinical manifestations , complicating the diagnosis of coinfections."} +{"text": "Nilaparvata lugens can develop into either long-winged or short-winged adults depending on environmental stimuli received during larval stages. The transcription factor NlFoxO serves as a key regulator determining alternative wing morphs in BPH, but the underlying molecular mechanism is largely unknown. Here, we investigated the transcriptomic profile of forewing and hindwing buds across the 5th-instar stage, the wing-morph decision stage. Our results indicated that NlFoxO modulated the developmental plasticity of wing buds mainly by regulating the expression of cell proliferation-associated genes.The brown planthopper (BPH) Nilaparvata lugens, can develop into either short-winged (SW) or long-winged (LW) adults according to environmental conditions, and has long served as a model organism for exploring the mechanisms of wing polyphenism in insects. The transcription factor NlFoxO acts as a master regulator that directs the development of either SW or LW morphs, but the underlying molecular mechanism is largely unknown. Here, we microinjected SW-destined morphs with double stranded-RNA (dsRNA) targeting NlFoxO (dsNlFoxO) to change them into LW-winged morphs. In parallel, SW-destined morphs microinjected with dsRNA targeting the gene encoding green fluorescence protein (dsGfp) served as a negative control. The forewing and hindwing buds of 5th-instar nymphs collected at 24, 36, and 48 h after eclosion (hAE) were used for RNA sequencing. We obtained a minimum of 43.4 million clean reads from forewing and hindwing buds at a single developmental time. Differentially expressed genes (DEGs) were significantly enriched in various Gene Ontology (GO) terms, including cellular process, binding, and cell part. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment pathway analysis showed that up-regulated genes in dsNlFoxO-treated forewing and hindwing buds were largely associated with the cell cycle and DNA replication. Furthermore, most up-regulated genes displayed higher expression at 24-, and 36-hAE relative to 48 hAE, indicating that wing cells in LW-destined wings might actively proliferate during the first 36 h in 5th-instar nymphs. Our findings indicated that LW development in BPH was likely dependent on the duration of cell proliferation in the 5th-instar stage, which sheds light on the molecular basis of wing polymorphism in insects.The brown planthopper (BPH), Nilaparvata lugens (Hemiptera: Delphacidae), is the most destructive rice pest in Asia [N. lugens FoxO homolog NlFoxO relied on the insulin/insulin-like signaling (IIS) activity to direct wing buds developing into SW or LW morphs during the wing-morph decisive stage (the 5th-instar stage). High IIS activity inhibited NlFoxO activity, leading to LW morphs, and vice versa [NlFoxO influences alternative wing morphs remains largely unknown.The brown planthopper (BPH), in Asia . BPH fee in Asia ,3. BPH i in Asia . As a hece versa ,7. HowevFoxO is associated with increased cancer in mammals [Caenorhabditis elegans, the FoxO homolog daf-16 was initially isolated as a dauer defective mutant [daf-16 can extend lifespan via dauer formation [Drosophila melanogaster, fly FoxO homolog dFoxO mutants were viable and of normal size, but lost protection against oxidative stress [dFoxO caused a decrease in cell number and cell size [FoxO transcription factors belong to the large Forkhead family of proteins, which are characterized by a conserved DNA-binding domain termed the \u201cforkhead box\u201d . FoxO pr mammals ,10. In te mutant , and oveormation . In addiormation ,16,17,18ormation . FoxO trormation ,21. In te stress . In contell size ,23,24, lAphis gossypii), genes associated with flight-reproduction trade-offs were differentially expressed in winged versus wingless morphs through RNA-seq analysis [Toxoptera citricida), RNA-seq identified both lipid and glycogen metabolism-associated genes that were differentially expressed between winged and wingless adults, indicating that these genes might contribute to energy metabolism during aphid wing development [The emergence of next-generation sequencing technology has profoundly improved our understanding of the molecular basis of wing polymorphism in insects. Our early RNA sequencing (RNA-seq) study identified hundreds of genes including those correlated to respiration and energy metabolism were up-regulated in LW versus SW BPH adults, indicating LW BPH adults might require more energy than SW for flight . In the analysis . In the elopment .NlFoxO (dsNlFoxO) to change them into LW-winged morphs. In parallel, SW-destined morphs microinjected with dsRNA targeting the gene encoding green fluorescence protein (dsGfp) served as a negative control. Forwing and hindwing buds were dissected from dsNlFoxO- and dsGfp-treated nymphs at the wing-morph decisive stage (the 5th-instar stage), and were then used for RNA-sequencing. Comparative transcriptomic analysis indicated that NlFoxO-regulated alternative wing morphs were likely through modulating genes involved in cell proliferation. These results advanced our understanding of the molecular basis of wing dimorphism in insects.In the present study, we microinjected SW-destined morphs with double stranded-RNA (dsRNA) targeting N. lugens strain (SW > 95%) was initially collected from a rice field in Hangzhou, China, in 2009 [The SW in 2009 . InsectsNlFoxO to generate LW-destined morphs. For a negative control, 4th-instar nymphs were microinjected with dsGfp, which produced SW-destined adults as the SW N. lugens strain. For transcriptome sequencing, forewing and hindwing buds were dissected from 5th-instar nymphs at 24, 36, and 48 h after ecdysis (hAE). Three independent biological replicates were performed at each designed time. Total RNA was isolated from wing buds using TRIzol , and the RNA concentration and quality were determined using a NanoDrop ND-2000 spectrophotometer . RNA samples (>2 \u03bcg) were subsequently used for cDNA library construction using a NEBNext Ultra RNA Library Prep Kit for Illumina according to manufacturer\u2019s recommendations, and index codes were added to each sequence. Library fragments of 250\u2013300 bp in length were preferentially purified using an AMPure XP system , and library quality was assessed using the Agilent Bioanalyzer 2100 system. Clustering of the index-coded samples was performed on a cBot Cluster Generation System using a HiSeq PE Cluster Kit v4-cBot-HS according to the manufacturer\u2019s instructions. The cDNA libraries were sequenced on an Illumina Hiseq platform and 150-bp paired-end reads were generated.4th-instar nymphs were collected for microinjection with dsNlFoxO versus dsGfp was analyzed using DEseq2 [p-value in multiple tests, and transcripts with fold change \u22652 and FDR < 0.05 were subjected to differentially expressed gene (DEG) analysis [Clean reads were derived from raw reads by removing adapter reads, low-quality reads, and reads containing poly-N and ambiguous bases using the trimmomatic . Clean rg DEseq2 . False danalysis ,33.https://www.omicshare.com/tools/) on 15 February 2021. GO terms with corrected p-value <0.05 were defined as significantly enriched GO terms [p-value < 0.05 [Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were analyzed using an online platform , 2533 (1496 up-regulated and 1037 down-regulated), and 2279 (1381 up-regulated and 898 down-regulated) DEGs were identified in dsNlFoxO-treated 5th-instar nymphs at 24, 36, and 48 hAE, respectively , 2902 (1473 up-regulated and 1429 down-regulated), and 1813 (732 up-regulated and 1081 down-regulated) DEGs in forewing buds of dsectively B.NlFoxO, DEGs were used for GO term and KEGG pathway enrichment analyses. For forewing buds at 24 hAE, 2128 DEGs were enriched in 54 GO terms associated with 24 biological process (BP) categories (GO:0008150), 16 cellular component (CC) categories (GO:0005575), and 14 molecular function (MF) categories (GO:0003674) with FDR < 0.05 as the criterion were categorized into 24 BP, 14 MF and 16 CC GO terms based on FDR < 0.05 . As showFor hindwing buds at 36 hAE, 2533 (1496 up-regulated and 1037 down-regulated) DEGs were annotated using GO classifications, including 25 BP, 13 MF, and 16 CC terms, among which cellular process (807 up-regulated and 412 down-regulated), binding (753 up-regulated and 327 down-regulated), and cell part (691 up-regulated and 300 down-regulated) were the top-ranked terms B. In linFor forewing buds at 48 hAE, the 1813 (732 up-regulated and 1081 down-regulated) DEGs were mapped to 52 GO terms A. AnalogFor hindwing buds at 48 hAE, greater percentages of the 2279 DEGs (1381 up-regulated and 898 down-regulated) were mapped to the single-organism process, binding, and cell part terms B. KEGG aNlFoxO treatment; p-value <0.05 as a cutoff criterion. A total of 39 cell-cycle- and 25 DNA-replication-associated genes were significantly regulated by dsNlFoxO versus dsGfp in forewing and hindwing buds [Drosophila caused by ectopic expression of dFoxO. In the present, our results show that knockdown of NlFoxO significantly enhanced the expression of cyclin D2 ,41. ThisGadd45) ,23,24 anGadd45) ,23 in DrGfp) to LW (dsNlFoxO) in 5th-instar nymphs, the wing-morph decisive stage. Comparative transcriptome analysis revealed that a large percentage of genes (10\u201319%) were significantly differentially expressed in both forewing and hindwing buds. GO enrichment analysis showed that DEGs were mainly related to cellular process, binding, and cell part categories. The up-regulated genes were significantly enriched in DNA replication (ko03030), and cell cycle (ko04110 and ko04111) pathways in KEGG analysis. Thus, the duration of cell proliferation might contribute significantly to long wing development modulated by NlFoxO.This study investigated the transcriptional profile of wing-morph transition from SW (ds"} +{"text": "Walking and talking on the phone are common high-cognitive-load-situations , requiring extra attentional allocation and increasing perceived stress. We explored whether two load types, 1) single-task (ST) walking or talking on a phone and 2) HCLS walking while talking on a phone, influenced walking and/or cognitive performance among young , middle-aged , and older adults while controlling for perceived stress. Participants completed 3-minute trials of single-task walking (ST-W), single-task phone conversations with common and uncommon topics , and walking while talking on a phone (HCLS-C and HCLS-U). Walking speed was analyzed with 3 x 3(Age) ANCOVA. HCLS resulted in slower walking speed (p<.001). Older adults exhibited slower speed across conditions compared to young (p=.015). Cognitive complexity ) on the Linguistic Inquiry and Word Count (LIWC) were analyzed with 2(Cvs.U) x 2(STvs.HCLS) x 3(Age) ANCOVAs. Older age was associated with less cognitive complexity; positive tone (p=.014) and SIXLTR (p=.016), respectively in conversations. Uncommon topics reduced positive tone (p=.022) and SIXLTR (p=.003). Effects of HCLS on tone (p=.040) and SIXLTR (p=.005) varied with age. HCLS with different conversation topics resulted in reduced walking and cognitive complexity while controlling for perceived stress. The analysis of cognitive complexity using common/uncommon conversation topics is a novel method to assess the impact of HCLS. This research will disrupt the transformation of aging leading to a better understanding of attentional allocation and its effects on function."} +{"text": "SARS-CoV-2 infection can present with a broad clinical differential that includes many other respiratory viruses; therefore, accurate tests are crucial to distinguish true COVID-19 cases from pathogens that do not require urgent public health interventions. Co-circulation of other respiratory viruses is largely unknown during the COVID-19 pandemic but would inform strategies to rapidly and accurately test patients with respiratory symptoms.Respiratory Pathogen Panel for 17 other pathogens, this study examines the prevalence of 18 potentially co-circulating pathogens and their relative rates in prior years versus since COVID-19 emerged, including four endemic coronaviruses.This study retrospectively examined 298,415 respiratory specimens collected from symptomatic patients for SARS-CoV-2 testing in the three months since COVID-19 was initially documented in the province of Alberta, Canada . By focusing on 52,285 specimens that were also tested with the Luminex p\u2009<\u20090.0001), suggesting very low rates of SARS-CoV-2 co-infection. Furthermore, the overall co-infection rate was significantly lower among specimens with SARS-CoV-2 detected (p\u2009<\u20090.0001). Finally, less than 0.005% of all specimens tested positive for both SARS-CoV-2 and any of the four endemic coronaviruses tested, strongly suggesting neither co-infection nor cross-reactivity between these coronaviruses.SARS-CoV-2 was identified in 2.2% of all specimens. Parallel broad multiplex testing detected additional pathogens in only 3.4% of these SARS-CoV-2-positive specimens: significantly less than in SARS-CoV-2-negative specimens (Broad respiratory pathogen testing rarely detected additional pathogens in SARS-CoV-2-positive specimens. While helpful to understand co-circulation of respiratory viruses causing similar symptoms as COVID-19, ultimately these broad tests were resource-intensive and inflexible in a time when clinical laboratories face unprecedented demand for respiratory virus testing, with further increases expected during influenza season. A transition from broad, multiplex tests toward streamlined diagnostic algorithms targeting respiratory pathogens of public health concern could simultaneously reduce the overall burden on clinical laboratories while prioritizing testing of pathogens of public health importance. This is particularly valuable with ongoing strains on testing resources, exacerbated during influenza seasons. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused significant disease COVID-19) and deaths worldwide [9 and deaSince SARS-CoV-2 emerged, there has been little research on the concurrent circulation of these other respiratory viruses, which had been the subject of broad surveillance in the years prior : only a Furthermore, co-circulation of SARS-CoV-2 and endemic coronaviruses (eCoVs) may pose a particular diagnostic challenge due to potential cross-reactivity of SARS-CoV-2 with pre-existing assays targeting eCoVs and vice versa. While laboratories must perform in-lab validations of the specificity of their assays, in situ studies are an essential complement, showing real-world data that may indicate necessary and opportune changes in diagnostic approaches.Accordingly, we sought to conduct a retrospective analysis of all specimens submitted to the provincial public health laboratory in Alberta, Canada for SARS-CoV-2 simultaneously tested for 17 additional respiratory pathogens\u2014including influenza viruses and endemic coronaviruses\u2014to inform our diagnostic algorithms during the COVID-19 pandemic and assess for potential co-infection or cross-reactivity of SARS-CoV-2 and eCoVs in a clinical setting.Mycoplasma pneumoniae.The NxTAG Respiratory Pathogen Panel was used to test respiratory specimens from symptomatic patients admitted to hospital, emergency departments, in long-term care, or amid a suspected respiratory outbreak in the community. Following collection, respiratory specimens were transported to the provincial Public Health Laboratories (ProvLab) for testing. The RPP was validated to detect nucleic acids from 17 respiratory pathogens: influenza viruses A-B, parainfluenza viruses 1\u20134, respiratory syncytial viruses A-B, rhinovirus/enterovirus, adenovirus, human metapneumovirus, four eCoVs , and SARS-CoV-2 testing was performed at ProvLab using multiple assays with different analytical sensitivities (Sn): singleplex and multiplex laboratory-developed tests using the TaqMan Fast Virus One-Step real-time reverse transcription (RT)-PCR Master Mix , or theSARS-CoV-2 and RPP tests performed between January 1\u2013June 6, 2020, and RPPs between January 1\u2013June 6 in 2018/2019 were compiled. Only tests from validated, respiratory tract specimen types and from patients with a primary address in Alberta were assessed. Analyses of coronavirus test positivity in 2020 focused on March 7\u2013May 28, wherein RPP testing criteria were consistent with those used in prior years, except for providing RPP tests to symptomatic patients in long-term care in 2020.Differences between categorical variables were compared using Fisher\u2019s exact test or Chi-square analysis. Continuous variables were compared by Kruskal\u2013Wallis test or Student\u2019s t-test.n\u2009=\u200952,285/53,661; Fig.\u00a0On March 5, 2020, Alberta reported its first identified case of COVID-19n\u2009=\u20092; Table To assess co-circulation of (or cross-reactivity with) other respiratory pathogens causing COVID-19-like symptoms in a clinical setting, we further examined the 1,141 specimens from which SARS-CoV-2 was detected. Of these, 96.6% were negative for all 17 RPP pathogens (Table p\u2009<\u20090.0001) and the predominant eCoV detected in Alberta in 2020 was NL63, accounting for 100.0% and 60.5% of the eCoV-positive specimens among SARS-CoV-2-positive and -negative specimens, respectively (n\u2009=\u20092/2 and 565/933). In total, 401 specimens had more than one respiratory pathogen detected in this study, including 39 (3.4%) of the SARS-CoV-2-positive specimens and 362 (0.7%) of the SARS-CoV-2-negative specimens and positivity decreased more sharply over time than in prior years , the patient age increased significantly from previous years even when these patients were excluded years. More flexible test panel designs and algorithms could better capture local epidemiology and changing needs, whether that is to identify circulating pathogens in routine \u2018surveillance mode\u2019 or with panels focused on public health, infection prevention and control, and novel pathogens in \u2018pandemic mode.\u2019 Ultimately, more flexible panel designs may support clinical and operational effectiveness in diagnostic laboratories, both during \u2018surveillance mode\u2019 , and critically in \u2018pandemic mode\u2019 (including when novel pathogens like SARS-CoV-2 emerge). Indeed, flexible syndromic panels that identify pathogens of public health and infection control concern would be more easily adaptable to novel pathogens and could play an important role in the current pandemic response and future pandemic preparedness.During a pandemic, clinical laboratories require greater test capacity to support public health efforts to limit further spread , 30. WitThis study highlights the importance of ongoing diagnostic stewardship to best align laboratory resources with public health efforts. Consequently, as of May 29, 2020, our laboratory pandemic response shifted from routine \u2018surveillance mode\u2019 to a prioritized \u2018pandemic mode\u2019 by no longer routinely performing the RPP multiplex test with every SARS-CoV-2 test. This further demonstrates how clinical laboratories have adapted throughout this pandemic, as laboratory-driven alternatives continue to mitigate the myriad of ongoing challenges such as test supply shortages , 31, 32.By maintaining broad respiratory pathogen testing from previous years through the emergence of SARS-CoV-2, this study reveals concurrent respiratory virus circulation during the COVID-19 pandemic. Due to the massive surge in test volumes, this became the largest single-year eCoV study and the largest eCoV study during the COVID-19 pandemic. There was no evidence of assay cross-reactivity in a clinical setting between SARS-CoV-2 and the 17 respiratory pathogen targets, and less than 0.01% of specimens tested positive for both SARS-CoV-2 and any eCoV. In fact, specimens containing SARS-CoV-2 had a significantly lower viral co-infections rate. Overall, broad panels had limited clinical or surveillance value and high cost in this pandemic, and more flexible panel designs may better support clinical and operational effectiveness in diagnostic laboratories.As a consequence of this data, our provincial laboratory eliminated reflexive multiplex testing in a shift from routine \u2018surveillance mode\u2019 to a prioritized \u2018pandemic mode.\u2019 Focused diagnostic stewardship can help mitigate ongoing challenges to better address future case surges\u2014including future waves of COVID-19\u2014without compromising public health benefits of ongoing testing."} +{"text": "Several diseases are linked to increased risk of Coronavirus disease 19 (COVID-19). Our aim was to investigate whether depressive and anxiety symptoms predict subsequent risk of COVID-19, as has been shown for other respiratory infections.We based our analysis on UK Biobank participants providing prospective data to estimate temporal association between depressive and anxiety symptoms and COVID-19. We estimated whether the magnitude of these symptoms predicts subsequent diagnosis of COVID-19 in this sample. Further, we evaluated whether depressive and anxiety symptoms predicted (i) being tested for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and (ii) COVID-19 in those tested.N = 135 102 participants, depressive symptoms (odds ratio (OR) = 1.052; 95% confidence interval (CI) 1.017\u20131.086; absolute case risk: (moderately) severe depression: 493 per 100 000 v. minimal depression: 231 per 100 000) but not anxiety predicted COVID-19. While depressive symptoms but not anxiety predicted (i) being tested for SARS-CoV-2 , (ii) neither predicted COVID-19 in those tested . Results remained stable after adjusting for sociodemographic characteristics, multimorbidity and behavioural factors.Based on data from Depressive symptoms were associated with a higher risk of COVID-19 diagnosis, irrespective of multimorbidities. Potential underlying mechanisms to be elucidated include risk behaviour, symptom perception, healthcare use, testing likelihood, viral exposure, immune function and disease progress. Our findings highlight the relevance of mental processes in the context of COVID-19. We performed all calculations at sciCORE . Estimates remained stable when adjusting for potential sociodemographic confounders , as well as when adjusting for individual physical diseases and behavioural factors .Depressive symptoms but not anxiety symptoms predicted COVID-19 . Estimates remained stable when adjusting for potential sociodemographic confounders , as well as when additionally adjusting for individual physical diseases and behavioural factors .Depressive symptoms but not anxiety symptoms predicted being tested for SARS-CoV-2 as well as in the adjusted models .Neither depressive symptoms nor anxiety symptoms predicted COVID-19 in those tested for SARS-CoV-2 in the crude models .Depressive symptoms have a dose\u2013response effect on COVID-19 depressive symptoms but not anxiety are linked to an increased likelihood of being tested for SARS-CoV-2 and (b) depressive symptoms are associated with an increased risk of a diagnosis of COVID-19, irrespective of potential confounders. While depressive symptoms but not anxiety were linked to an increased likelihood of being tested for SARS-CoV-2, there was no such association with a COVID-19 diagnosis in those tested. This stresses the need for a better understanding of potential underlying mechanisms, including risk behaviour, viral exposure, immune function, disease progress, symptom perception, health care use and testing likelihood. Our findings highlight the relevance of mental processes in the context of COVID-19."} +{"text": "ABSTRACT IMPACT: Our work unveils a novel mechanism of ischemia repurfusion injury driven by pre-existing autoimmunity following lung transplant and a potential therapeutic strategy for blocking complement-dependent injury thereby reducing risk of lung transplant rejection. OBJECTIVES/GOALS: Our goal was to determine if pre-existing autoimmune autoantibodies, such as those resulting from cigarette smoke (CS), contribute to graft rejection in lung transplantation (LTx) and if autoreactive-mediated graft injury is complement-dependent. METHODS/STUDY POPULATION: For in vivo experiments, we utilized our emphysema mouse model. Briefly, eight-week-old C57BL/6J mice are exposed to 3R4F reference cigarette smoke 5 hours per day, 5 days a week for 6 months. Upon completion, cigarette smoked (CS) mice and control (NS) mice received syngeneic orthotopic left-lung transplant from age-matched C57BL/6J donors. To determine if pre-existing autoreactivity mediated graft injury was complement-dependent we treated CS-LTx mice with a novel, bifunctional complement inhibitor. Autoantibody levels were measured by ELISA and lung injury was assessed by blinded histopathological analyses. Complement inhibition was verified by immunofluorescence. RESULTS/ANTICIPATED RESULTS: We found that CS-exposure leads to production of autoreactive antibodies towards extracellular matrix (ECM) components and contributes to graft injury. Interestingly, LTx into CS exposed mice further increased de-novo ECM autoantibody development. Lastly, treatment with our novel, bifunctional complement inhibitor blocked autoantibody spreading and significantly reduced graft rejection. DISCUSSION/SIGNIFICANCE OF FINDINGS: These data demonstrate that smoking induces pre-LTx autoreactivity to ECM proteins that promotes graft injury following LTx. Furthermore, complement inhibition reduces autoantibody production and protects the graft from injury."} +{"text": "Scientific Reports 10.1038/s41598-020-69318-y, published online 09 September 2020Correction to: The original version of the Article contained an error in the Author Information section.\u201cThese authors contributed equally: Dong Won Paik, Jun Sang Park, Dong Hui Lim and Tae-Young Chung.\u201dnow reads:\u201cThese authors contributed equally: Dong Won Paik and Jun Sang Park.These authors jointly supervised this work: Dong Hui Lim and Tae-Young Chung.\u201dThe original Article has been corrected."} +{"text": "ABSTRACT IMPACT: The proposed research study will provide critical pilot data on the effect of using the prebiotic (HAMS-AB) on the gut microbiome profile, Beta-cell function and immune markers in humans with T1D. OBJECTIVES/GOALS: The overall objective of this study is to assess how the prebiotic high amylose maize starch that has been acetylated and butyrylated (HAMS-AB) impacts the gut microbiome profile, short chain fatty acid (SCFA) production, glycemia, Beta-cell function/health and immune responses in newly diagnosed youth with type 1 diabetes (T1D). METHODS/STUDY POPULATION: We are performing a pilot randomized cross-over trial. We plan to recruit 12 newly-diagnosed T1D youth with residual Beta-cell function between 12-16 years of age. We will profile the gut microbiome using metagenomics, measure stool SCFA levels using mass spectrometry, assess glycemia using continuous glucose monitoring, assess insulin production using mixed meal tolerance testing, assess Beta-cell stress using proinsulin/C-peptide levels, and test immune responses by examining cytokine levels and frequency, phenotype and function of T cell markers in peripheral blood. RESULTS/ANTICIPATED RESULTS: Thus far, we have enrolled 3 participants, 1 has completed the study. Baseline assessments indicate that we have technical feasibility of performing the above studies and measurements. Recruitment and enrollment are ongoing. We hypothesize that the use of HAMS-AB in newly diagnosed youth with T1D will (i) improve the gut microbiome profile, (ii) increase SCFA production, (iii) improve overall glycemia and Beta-cell function and (iv) modulate the immune system and mitigate autoimmunity. DISCUSSION/SIGNIFICANCE OF FINDINGS: Given the failure to develop a cure for T1D despite multiple completed intervention studies and the unknown long-term effects of immune-modulatory therapy on those at risk for or those diagnosed with T1D, prebiotics such as HAMS-AB may offer a simple, safe, yet inexpensive and tolerated dietary alternative approach to mitigating disease."} +{"text": "Two coding-complete sequences of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were obtained from samples from two patients in Arkansas, in the southeastern corner of the United States. The viral genome was obtained using the ARTIC Network protocol and Oxford Nanopore Technologies sequencing. Two coding-complete sequences of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were obtained from samples from two patients in Arkansas, in the southeastern corner of the United States. The viral genome was obtained using the ARTIC Network protocol and Oxford Nanopore Technologies sequencing. Betacoronavirus, in the family Coronaviridae. In this work, we used Oxford Nanopore Technologies (ONT) MinION sequencing technology, which provided a consensus viral genome from SARS-CoV-2-positive samples within 1 day. Importantly, the device can be easily used in environments with very limited resources, such as in rural areas without access to traditional laboratory facilities.As the novel coronavirus disease 2019 (COVID-19) outbreak continues to worsen around the world, the daily death toll in the United States is currently averaging more than 1,000 deaths per day. Rapid sharing of genome sequences in conjunction with other epidemiological data can facilitate early decision-making in an attempt to control the local transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), an RNA virus that belongs to the genus https://www.protocols.io/view/ncov-2019-sequencing-protocol-v3-locost-bh42j8ye). The PCR amplification process was slightly modified from the ARTIC Network protocol by changing the annealing and extension temperature from 65\u00b0C to 63\u00b0C. The libraries were prepared using a ligation-based sequencing kit , loaded onto a MinION flow cell (ONT), and sequenced with the MinION Mk1B device (ONT). Base calling of the resulting FAST5 files was performed in real time using Guppy (v3.4.5) (https://github.com/artic-network/rampart) was used to monitor sequencing in real time. The minimum coverage we used for each region on the genome was 300\u00d7. For quality control and filtering of reads (fragments of 400 to 700\u2009bp), the guppyplex script of the ARTIC Network bioinformatics protocol (https://artic.network/ncov-2019/ncov2019-bioinformatics-sop.html) was used, followed by a reference assembly using the MinION script with madeka polishing against the sequence of the Wuhan-Hu-1 isolate (GenBank accession number MN908947.3). The quality metrics for the reference-based assemblies are shown in A set of two residual, deidentified nasopharyngeal samples (USA/AR-UAMS001/2020 and USA/AR-UAMS002/2020) that tested positive for SARS-CoV-2 by quantitative reverse transcription-PCR (qRT-PCR) were obtained from patients at the University of Arkansas for Medical Sciences (UAMS) hospital. Total RNA was extracted by the QIAamp viral RNA minikit according to the manufacturer\u2019s instructions. Samples were reverse transcribed as described in the PCR tiling of COVID-19 virus protocol (vPTC_9096_v109_revF_06Feb2020) published by the ARTIC Network (http://nextstrain.org/ncov) were used for phylogenetic analysis. The phylogenetic analysis was performed following the standard protocol for analysis of SARS-CoV-2 genomes provided by Nextstrain (rg/ncov) .MT766907 and SRA accession number SRR12277392 for USA/AR-UAMS001/2020 and GenBank accession number MT766908 and SRA accession number SRR12277391 for USA/AR-UAMS002/2020) and in the Cancer Imaging Archive (TCIA) (www.gisaid.org).The coding-complete sequences of the two isolates were deposited in GenBank (GenBank accession number"} +{"text": "GRM2, Tent5b, Fos, Sstr2, and Gadd45b) in a Cre-specific manner. To illustrate the versatility of this tool, we created a parallel CRISPRi construct that successfully inhibited expression from a luciferase reporter in HEK293T cells only in the presence of Cre. These results provide a robust framework for Cre-dependent CRISPR-dCas9 approaches across different model systems, and enable cell-specific targeting when combined with common Cre driver lines or Cre delivery via viral vectors.Site-specific genetic and epigenetic targeting of distinct cell populations is a central goal in molecular neuroscience and is crucial to understand the gene regulatory mechanisms that underlie complex phenotypes and behaviors. While recent technological advances have enabled unprecedented control over gene expression, many of these approaches are focused on selected model organisms and/or require labor-intensive customization for different applications. The simplicity and modularity of clustered regularly interspaced short palindromic repeats (CRISPR)-based systems have transformed genome editing and expanded the gene regulatory toolbox. However, there are few available tools for cell-selective CRISPR regulation in neurons. We designed, validated, and optimized CRISPR activation (CRISPRa) and CRISPR interference (CRISPRi) systems for Cre recombinase-dependent gene regulation. Unexpectedly, CRISPRa systems based on a traditional double-floxed inverted open reading frame (DIO) strategy exhibited leaky target gene induction even without Cre. Therefore, we developed an intron-containing Cre-dependent CRISPRa system (SVI-DIO-dCas9-VPR) that alleviated leaky gene induction and outperformed the traditional DIO system at endogenous genes in HEK293T cells and rat primary neuron cultures. Using gene-specific CRISPR sgRNAs, we demonstrate that SVI-DIO-dCas9-VPR can activate numerous rat or human genes ( This manuscript reports a novel set of clustered regularly interspaced short palindromic repeats (CRISPR) tools for Cre-dependent transcriptional targeting in neurons and non-neuronal dividing cells. Our results demonstrate that these tools perform well at multiple gene targets and can be used for both transcriptional activation or repression. Compared with traditional Cre-dependent overexpression or knock-out models, Cre-dependent CRISPR activation (CRISPRa) and CRISPR interference (CRISPRi) provide several advantages, including targeting of one or multiple endogenous genomic loci, titration of effect size, and bidirectional regulation using the same CRISPR sgRNA. Further, SVI-DIO-dCas9 tools can be used in existing transgenic animal models or wild-type models via viral delivery. This advance enables applications in less common animal models and can be used to target endogenous genomic loci for fine-tuned transcriptional activation or repression.Genetic and epigenetic targeting are fundamental strategies to study gene regulation and function, and also provide novel therapeutic avenues for genetic diseases. In recent years, clustered regularly interspaced short palindromic repeats (CRISPR) systems have revolutionized the field as tools for site-specific DNA and RNA editing . In thesDNA recombinases are commonly used to enable inversion, deletion, or integration of transgenes in a cell-specific manner. The Cre/Lox system is a commonly used recombination approach in which the Cre recombinase recognizes specific 34-bp palindromic Lox sites within a DNA sequence . Cre-medWhile CRISPR/dCas9 approaches have enabled targeted induction of transcriptional or epigenetic states at selected genes, inducible cell type-specific CRISPR tools based on these platforms remain limited . For exa2O. Dissected tissues were incubated with papain for 25\u2009min at 37\u00b0C. After rinsing in HBSS, a single-cell suspension of the tissue was re-suspended in Neurobasal media (Invitrogen) by trituration through a series of large to small fire-polished Pasteur pipets. Primary neuronal cells were passed through a 100 \u03bcm cell strainer, spun and re-suspended in fresh media. Cells were then counted and plated to a density of 125,000 cells per well on 24-well culture plate with or without glass coverslips . Cells were grown in Neurobasal media plus B-27 and L-glutamine supplement for 11 days in vitro (DIV) in a humidified CO2 (5%) incubator at 37\u00b0C.Primary rat neuronal cultures were generated from embryonic day 18 rat striatal tissue as described previously . Briefly9 GC/ml, with a target multiplicity of infection (MOIs) of at least 1000. After an 8- to 16-h incubation period, virus-containing media was replaced with conditioned media to minimize toxicity. A regular half-media change followed on DIV8. On DIV11, transduced cells were imaged and virus expression was verified before RNA extraction. EGFP and mCherry expression was also used to visualize successful transduction using a Nikon TiS inverted epifluorescence microscope.For virus experiments, cells were transduced with lentiviruses on DIV4 or DIV5. All viruses had a minimum titer of 1\u2009\u00d7\u200910Total RNA was extracted with DNase treatment , and reverse-transcribed . cDNA was subject to qPCR for genes of interest, as described previously . A list http://www.rgenome.net/cas-offinder/). sgRNAs were designed to target GRM2, Tent5b, Sstr2, Gadd45b, and Fos, respectively. A list of the target sequences is provided in To achieve transcriptional activation or inactivation, lentivirus-compatible plasmids were engineered to express dCas9 fused to either VPR or KRAB-MeCP2, based on existing published plasmids [Addgene plasmid #114196 ; AddgeneViruses were produced in a sterile environment subject to BSL-2 safety by transfecting HEK293T cells with specified CRISPR-dCas9 plasmids, the psPAX2 packaging plasmid, and the pCMV-VSV-G envelope plasmid (Addgene plasmids #12260 and #8454) with FuGene HD (Promega) for 40\u201348 h as previously described . VirusesCVCL_0063) and cultured in standard HEK media: DMEM supplemented with 10% bovine serum and 1 U penicillin-streptomycin (Invitrogen 15140122). Cells were maintained in T75 or T225 tissue flasks. At each passage, cells were trypsinized for 1\u20133\u2009min at room temperature. For transfection experiment cells were plated in 24-well plates and transfected with FuGene HD (Promega).HEK293T cells were obtained from American type Culture Collection . The DIO-dCas9-VPR construct was tested with or without transfection of a Cre-2A-mCherry plasmid that was driven under the human synapsin (hSYN) promoter promoter resulted in strong upregulation of Tent5b mRNA with both the constitutive and Cre-dependent constructs. However, in neurons the DIO system also exhibited leaky expression, indicated by an 8-fold increase of Tent5b mRNA in the absence of Cre. Tent5b is a highly inducible gene which could be more susceptible to baseline/leaky induction compared with other genes. Therefore, we tested the DIO-dCas9-VPR system at two other genes including a gene encoding the neuropeptide receptor Sstr2 and DNA-damage inducible gene Gadd45b (Tent5b (with induction rates of 8- to 19-fold compared with lacZ controls), we still observed non-specific gene induction in the absence of Cre (2- to 5-fold induction without Cre). While we chose efficient gRNAs validated in prior studies into the dCas9 sequence to create a constitutively active, intron-containing construct (SVI-dCas9-VPR) in which the dCas9 cassette is divided into two segments by the SV40 intron A. We creUsing the more efficient SVI-dCas9-VPR 2.0, we next generated a split-dCas9 DIO cassette by inverting and flanking the first segment of dCas9 with LoxP and Lox2272 sites to prevent leaky transgene expression E. With oWe next transfected HEK293T cells with each of these three constructs to compare recombination, expression, and splicing efficiency of the SVI-DIO-dCas9-VPR construct. The SVI-DIO-dCas9-VPR groups also received a Cre-EGFP plasmid that was driven under the hSYN promoter to initiate recombination. PCR amplification of cDNA generated from the transfected HEK293T experiments with intron-spanning primers revealed strong signals for the short, spliced PCR product for all three groups G. This ilacZ control). Both the intron-containing SVI-dCas9-VPR and constitutive dCas9-VPR caused strong induction of GRM2 compared with their respective lacZ controls , but not in the absence of Cre , but only in the presence of Cre recombinase and striatal neurons (Tent5b), without undesired gene induction in the absence of Cre recombinase promoter to specifically target and knock-out the Th gene in dopaminergic neurons. While this approach allowed for cell type-specific expression of sgRNA constructs, a concern with similar systems is the untargeted overexpression of Cas9 or dCas9 fusion proteins, which could potentially cause unintended and non-specific effects on gene expression.One of the first cell type-specific CRISPR systems used in neurons was based on Cre-dependent expression of CRISPR sgRNAs. B\u00e4ck et al., developed a Lox-stop-Lox based sgRNA construct and used it for CRISPR-Cas9 mediated gene knock-out . This stOur system extends this previous work in two ways. First, the SVI-DIO-dCas9 approach alleviates some of these concerns, as the dCas9 construct itself is Cre dependent, and therefore, functional fusion proteins are not expressed without Cre recombinase. Second, our SVI-DIO-dCas9 system is compatible with traditional validated sgRNA constructs, which can be multiplexed for effect size titration at a single gene . AdditioPrior work has incorporated temporal specificity into Cre/Lox systems, either via use of optically or chemically inducible proteins. For example, selective estrogen receptor modulator (SERM) inducible systems have been generated by fusion of the ligand binding domain of the ER to Cre (Cre-ER). In this approach, a mutated version of the mouse ER that binds tamoxifen but not estrogen was used to create a Tamoxifen-inducible Cre system . ActivitFos or Arc transgenic animals were generated to express GFP or channelrhodopsin (ChR2) under an immediate early gene promoter such as s or Arc . Using aFosb gene in the ventral hippocampus and injection of a monosynaptic rabies virus expressing a sgRNA targeting the us vHPC; . While tWhile the use of our SVI-DIO-dCas9 system in Cre driver animal models is an expected application, this approach does not require transgenic organisms. In addition to sgRNA and SVI-DIO-dCas9-VPR or SVI-dCas9-KRAB-MeCP2 delivery, a separate construct expressing Cre can be delivered as necessary to target brain tissues. Additionally, this intron-containing dCas9 provides a basic framework that can be customized and combined with a number of effector proteins to introduce a variety of genetic or epigenetic modifications. The system can easily be customized for expression in various cell types and brain regions and adjusted for inducible efforts and enhanced temporal specificity."} +{"text": "We further evaluated the effect and explored the molecular mechanism of a PARP inhibitor (PARPi) in CAR-T cell immunotherapy by administering the PARPi to mouse xenografts model derived from human RCC cells. Treatment with the PARPi promoted CAR-T cell infiltration by stimulating a chemokine milieu that promoted CAR-T cell recruitment and the modulation of immunosuppression in the TME. Moreover, our data demonstrate that PARPi modulates the TME by activating the cGAS-STING pathway, thereby altering the balance of immunostimulatory signaling and enabling low-dose CAR-T cell treatment to induce effective tumor regression. These data demonstrate the application of CD70 CAR-T cell therapeutic strategies for RCC and the cross-talk between targeting DNA damage responses and antitumor CAR-T cell therapy. These findings provide insight into the mechanisms of PARPis in CAR-T cell therapy for RCC and suggest a promising adjuvant therapeutic strategy for CAR-T cell therapy in solid tumors.Chimeric antigen receptor T-cell (CAR-T) therapy has shown tremendous success in eradicating hematologic malignancies. However, this success has not yet been extrapolated to solid tumors due to the limited infiltration and persistence of CAR-T cells in the tumor microenvironment (TME). In this study, we screened a novel anti-CD70 scFv and generated CD70 CAR-T cells that showed effective antitumor functions against CD70The online version contains supplementary material available at 10.1186/s13045-021-01168-1. This sAdditional file 1: Supplementary Figures.Additional file 2: Supplementary materials and methods."} +{"text": "Aging augments postischemic apoptosis via incomplete mechanisms. Our previous animal study suggests that in addition to proapoptotic effects, lncRNAs also exert antiapoptotic effects in cardiomyocytes. However, whether this unexpected phenomenon exists in humans is unknown. In the present study, we investigated the relationship between aging and apoptosis regulation in human blood samples and confirmed their role by utilizing the cardiomyocyte lines (AC16 cells). Human blood samples were collected from 20 pairs of older adult and young volunteers. Age-different apoptotic regulatory lncRNAs and miRNAs were identified by microarray and bioinformatics analysis. The results indicated that lncRNA (NONHSAT069381 and NONHSAT140844) and miRNA (hsa-miR-124-5p and hsa-miR-6507-5p) were increased in aging human blood, confirmed by both bioinformatics analysis and polymerase chain reaction (PCR). Overexpression of NONHSAT069381 in AC16 cells increased caspase-3 levels and increased cardiomyocyte apoptotic cell death (determined by TUNEL staining and caspase activity assays) after hypoxia/reoxygenation (H/R), while overexpression of NONHSAT140844 increased X-chromosome-linked inhibitor of apoptosis protein (XIAP) content and decreased the myocardial apoptotic cell death. Furthermore, luciferase reporter assay revealed that hsa-miR-124-5p might be a mediator between NONHSAT069381 and mCASP3 and hsa-miR-6507-5p might be a mediator between NONHSAT140844 and mXIAP. Overexpression of hsa-miR-124-5p decreased caspase-3 levels and overexpression of hsa-miR-6507-5p decreased XIAP content in AC16 cells. We have found evidence that lncRNAs are important regulatory molecules in aging-mediated effects upon apoptosis. More interestingly, besides apoptosis-promoting effects, aging also inhibits myocardial apoptosis after H/R. This phenomenon also exists in the human cardiomyocyte line. Age is one of the most important risk factors for a wide range of diseases. Unfortunately, the overall increasing age of the world's population is accompanied by the rise in diseases including cardiovascular and neurodegenerative diseases and cancer and immune disorders . Given tAging is an independent risk factor for ischemia-related heart failure, which promotes the continuous decline of cardiac function after ischemia. Apoptosis plays a vital role in heart failure development. In 2012, through a combination of small-scale clinical trials and animal experiments, we demonstrated aging may promote cardiac apoptosis, a significant cause of ischemia-induced heart failure . StudiesOur previous study indicated that aging augmented in vivo reactive oxygen species (ROS) and reactive nitrogen species (RNS) levels after ischemia/reperfusion (I/R) . Liu et Long noncoding RNAs (lncRNAs) range from 200\u2009bp to several kilobases in length and do not encode any protein . Due to With mouse data, it can only be postulated that lncRNAs regulate apoptosis regulatory effects in vivo. However, the precise roles that lncRNAs in aging-related cardiomyocyte apoptosis play in humans remain uncertain. In our current study, we aim to (1) examine the effects of aging-related lncRNA on apoptosis in human cardiomyocyte lines, (2) analyze whether lncRNA has a bidirectional regulatory effect on cardiomyocyte apoptosis, and (3) explore the exact mechanism related to lncRNA affecting cardiomyocyte apoptosis.We enrolled 40 healthy men (20 older adults and 20 young). All members of the older adult group met the following inclusion criteria: (1) above 65 years of age; (2) absence of any significant clinical symptoms ; (3) absence of diagnosed chronic diseases ; (4) absence of any previous surgeries ; (5) absence of any significant family medical history; (6) absence of tobacco and alcohol use; (7) blood pressure (BP) \u2264 150/90\u2009mmHg at the time of enrollment; (8) absence of chronic medication use ; and (9) absence of autoimmune diseases . Inclusion criteria for the group of young men were the same as the older adult group, except that these men were all younger than 35 years old. Exclusion criteria for any patient in this study included major infection within the last 2 weeks.All human sample harvests were carried out in accordance with the Declaration of Helsinki. The study protocol was approved by the institutional ethics committee in Beijing An Zhen Hospital, Capital Medical University. After full disclosure of the study's purpose, nature, and inherent risk of participation, all subjects gave written informed consent prior to study inclusion.For Affymetrix microarray profiling, total RNA was isolated from 40 blood samples (20 older adult and young pairs) by TRIzol reagent , digested by DNase treatment, and purified with a RNeasy Mini Kit , per manufacturer's protocol. The amount and quality of RNA were determined by a UV-Vis Spectrophotometer at 260\u2009nm absorbance. The lncRNA and miRNA expression profiling was measured by GeneChip Human Clariom\u2122 D Assay , containing 134,700 gene-level probe sets. The microarray analysis was performed by Affymetrix Expression Console Software (version 4.0). Raw data (CEL files) were normalized at the transcript level using a robust multiarray average method (RMA workflow). Median summarizations of transcript expressions were calculated. Gene-level data were then filtered to include only those probe sets present in the \u201ccore\u201d metaprobe list representing RefSeq genes.t-test was used to identify differentially expressed genes for the older adult and young groups. This model has more power than standard tests to detect large changes in expression, without increasing the rate of false positives. After using significant and false discovery rate (FDR) analyses, we selected the differentially expressed genes according to predefined P value thresholds (0.05). The results of differentially expressed genes were subjected to unsupervised hierarchical clustering (Cluster 3.0) and TreeView analysis .The random variance model (RVM) A competing endogenous RNA (ceRNA) network was constructed to discover the ceRNA mechanism based on the differentially expressed lncRNAs and miRNAs. RNA transcripts would combine with miRNAs by miRNA response element (MRE), so we could identify the competition relationship between RNA transcripts in the process of combining MRE by predicting MRE and computing free energy. First, in miRNA-mRNA and miRNA-lncRNA, the target relationships were predicted by target prediction database. Pearson's correlation coefficient (PCC) between matched lncRNA-mRNA was computed based on their expression data. Then, the PCC between miRNA-mRNA and miRNA-lncRNA was computed. For a given lncRNA-mRNA pair, both mRNA and lncRNA were targeted by a common miRNA and coexpressed negatively with this miRNA. Finally, this miRNA-mRNA-lncRNA was identified as competing triplets.\u03bcL) contained SYBR Green Real-Time PCR Master Mix (TIAN-GEN), 0.5\u2009\u03bcL primers, and 0.5\u2009\u03bcL of template cDNA. Cycling conditions comprised of an initial, single cycle of 2 minutes at 94\u00b0C, followed by 40 cycles of 15 seconds at 94\u00b0C, 20 seconds at 63\u00b0C, and 30 seconds at 68\u00b0C. PCR amplifications were performed in three duplicates for each sample. Gene expression levels were quantified relative to the expression of 18S using an optimized comparative Ct (DDCt) value method.Real-time RT-PCR verified the differential expression of 12 genes detected by the lncRNA/miRNA Expression Microarray. It also assessed the lncRNA, miRNA, mCASP3, and mXIAP levels in AC16 cells. NCode\u2122 miRNA RT-qPCR System from Invitrogen is an example of a nondirect RT approach, requiring poly(A)-tailing of miRNAs prior to the RT reaction. NCode\u2122 miRNA First-Strand cDNA Synthesis Kits were used for polyadenylation and cDNA synthesis of miRNA. Then, amplify the miRNA of interest using qPCR primers and DNA polymerase. Detection is through the binding of the SYBR\u00ae Green dye to the amplified product. The PrimeScript\u2122 RT Reagent Kit with gDNA Eraser and gene-specific primers or random primers were used to generate cDNA of lncRNAs and mRNAs. Each real-time RT-PCR reaction expression were purchased from Oligobiobio Co., Ltd. . To construct lentivirus-mediated overexpression of lncRNA/miRNA, the full-length coding sequences of human lncRNA/miRNA C-terminally tagged with GFP or GFP alone (control) were cloned into the lentivirus vector. Empty vector lentivirus served as negative control (NC) (termed overexpression/knockdown control in this study). To generate lentivirus-mediated overexpression vector, the expression sequence targeting human lncRNA/miRNA was cloned into the lentivirus overexpression vector (termed overexpression in this study). Similarly, to produce lentivirus-mediated silencing system targeting human lncRNA/miRNA, a small interfering (5\u2032-gtcacagtccaacactgaggg-3\u2032) sequence was cloned into the lentivirus knockdown vector.2 at 37\u00b0C. In this study, overexpression and knockdown and their control lentiviruses were used to infect AC16 cells. Fluorescence microscopy monitored infection efficiency after 48 hours postlentivirus infection.Human adult ventricular cardiomyocyte cell line AC16 was obtained from the American Type Culture Collection . Cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 1% penicillin/streptomycin in a humidified incubator with 5% COAC16 cells were homogenized in an ice-cold lysis buffer. After homogenization, the lysates were centrifuged. The supernatant was saved and separated by electrophoresis on SDS-PAGE and transferred onto polyvinylidene difluoride-plus membranes. After blocking buffer, the immunoblots were probed with anti-total caspase 3 (lot: GR3356520-3), anti-XIAP (lot: GR3298310-4), and anti-actin (lot: GR333517-1) antibodies overnight at 4\u00b0C, followed by incubation with fluorescent-conjugated secondary antibodies at room temperature for 1 hour.2 and 5% CO2) for 6 hours and then normoxia for 2 hours to simulate H/R.Seventy-two hours after AC16 cell transfection, all groups were treated with hypoxia . Total nuclei were stained by DAPI . Apoptotic index was determined in a blinded manner.After 2 hours of reoxygenation, AC16 cells were homogenized in an ice-cold lysis buffer for 30 seconds (PRO 200 homogenizer). Homogenates were centrifuged for 5 minutes at 10,000g at 4\u00b0C. Supernatants were collected, and protein concentrations were measured using the BCA method . For each well in a 96-well plate, the supernatant containing 200\u2009mg of protein was loaded and incubated with 25\u2009mg Ac-DEVD-pNA, Ac-IETC-pNA, or Ac-LEHD-pNA at 37\u00b0C for 1.5 hours. pNA was cleaved from DEVD , IETD , or LEHD . Free pNA was quantified using a SpectraMax Plus microplate spectrophotometer at 405\u2009nm. Changes in caspase activity in I/R tissue samples were calculated and expressed as nmole pNA/mg/h .The amplified human 3\u2032-untranslated repeat segments of genes containing the predicted hsa-miR-124-5p/hsa-miR-6507-5p binding site were inserted into psiCHECK-2 vectors containing Renilla luciferase (Promega) to generate the wild plasmid or mutant plasmid construct. For luciferase assays, 239T cells were seeded in 96-well plates and transfected with pLuc-3\u2032-untranslated repeat, 10\u2009ng Renilla, and the mimic/NC miRNA using Lipofectamine 2000 reagent . Cells were collected and analyzed using the Dual-Luciferase Reporter Assay System (Promega) after 48 hours. The luciferase activity values were normalized relative to the Renilla luciferase internal control. Successful transfection of all cell treatment groups was confirmed prior to biological testing. Each experiment was repeated three times in duplicate.t-test for comparison between 2 groups and ANOVA followed by Bonferroni multiple comparison test for comparison among \u22653 groups. Probabilities of.05 or less were considered statistically significant.The data was analyzed with Prism 5.0 . All values in the text and figures are presented as mean \u00b1 SD. Statistical differences were determined by Student's 40 blood samples were initially taken. Two blood samples (one old adult and one young) were excluded because of sample contamination. 38 blood samples were used in the final results. In general, 19 pairs of older adult and young people were healthy. Age and BMI were the major differences between them .lncRNA/miRNA microarray was conducted in nineteen pairs of human blood samples, with a total of 75,434 lncRNAs and 4012 miRNAs detected Figures . Among tThe results of network analysis suggested that miRNAs may have synergistic effects on relative lncRNAs and mRNAs. We got all lncRNAs and miRNAs, which have significant relationship with aging and apoptotic factors. Within these RNAs, we selected 8 pairs of lncRNAs and miRNAs (a lncRNA and a relative miRNA as a pair) at random. The 8 pairs of RNAs met the following inclusion criteria: (1) aging resulted in increasing >1.2 or (2) aging resulted in decreasing <0.8. To confirm the microarray results, the 8 pairs were subjected to real-time RT-PCR. The results of real-time RT-PCR indicated that there are no significant aging-related differences in the 2 pairs. The expression levels of the other 6 pairs of genes were consistent with the microarray results . FurtherTo obtain direct evidence to support the relationship between these molecules, cultured AC16 cells were divided into four groups: (1) overexpression control (NONHSAT069381-overexpression control lentivirus), (2) overexpression (NONHSAT069381-overexpression lentivirus), (3) knockdown control (lncRNA NONHSAT069381 knockdown control lentivirus), and (4) knockdown (lncRNA NONHSAT069381 knockdown lentivirus). Transfection efficacy is reported in Cultured AC16 cells were divided into four groups: (1) overexpression control (NONHSAT140844-overexpression control lentivirus), (2) overexpression (NONHSAT140844-overexpression lentivirus), (3) knockdown control (lncRNA NONHSAT140844 knockdown control lentivirus), and (4) knockdown (lncRNA NONHSAT140844 knockdown lentivirus). Transfection efficacy is reported in lncRNA (NONHSAT069381 and NONHSAT140844) expression was upregulated or downregulated by lentiviral vectors. The effects of gene manipulation upon AC16 cell apoptosis were assessed using TUNEL staining and caspase activity measurement.Compared with the control, a significant increment of total TUNEL-positive nuclei was observed in the NONHSAT069381 overexpression group Figures and NONHThe results of the NONHSAT140844 group are the opposite. Compared to the control, the total TUNEL-positive nucleus of the NONHSAT140844 overexpression group was significantly reduced Figures and downhttp://www.targetscan.org/vert_71/) and Miranda (http://www.microrna.org/microrna/home.do). We then conducted a luciferase reporter assay to validate the binding of miRNA , lncRNA , and mRNA . hsa-miR-124-5p transfection significantly inhibited NONHSAT069381 and CASP3 luciferase activity overexpression control (hsa-miR-124-5p-overexpression control lentivirus), (2) overexpression (hsa-miR-124-5p-overexpression lentivirus), (3) knockdown control (hsa-miR-124-5p knockdown control lentivirus), and (4) knockdown (hsa-miR-124-5p knockdown lentivirus). Firstly, no significant efficacy difference existed among the four lentivirus-treated groups, but RT-PCR results indicated that hsa-miR-124-5p levels increased significantly in AC16 cells subjected to overexpression-encoding lentivirus treatment . ConversFurthermore, cultured AC16 cells were divided into four groups: (1) overexpression control (hsa-miR-6507-5p-overexpression control lentivirus), (2) overexpression (hsa-miR-6507-5p-overexpression lentivirus), (3) knockdown control (hsa-miR-6507-5p knockdown control lentivirus), and (4) knockdown (hsa-miR-6507-5p knockdown lentivirus). The results of the hsa-miR-6507-5p group indicated that increased levels of hsa-miR-6507-5p led to a significantly decreased XIAP activity, and decreased levels of hsa-miR-6507-5p led to a significant increment in XIAP activity Figures . SimilarThe increased mortality rate in the geriatric population related to cardiovascular disease suggests that cardiac aging itself may be a major risk factor for cardiovascular pathologies such as ischemic heart disease. However, the potential mechanisms were unclear. Apoptosis is a well-established cell death process after I/R injury. Previously, we demonstrated that cardiomyocyte apoptosis increases after ischemia-reperfusion and apoptosis contributes to heart failure , 7.Our present study showed that lncRNAs (NONHSAT069381 and NONHSAT140844) were increased in aging human blood, confirmed by both bioinformatics analysis and PCR. However, the precise roles that lncRNAs in aging-related cardiomyocyte apoptosis play in humans remain uncertain. Therefore, we further verified their role by utilizing cardiomyocyte lines (AC16 cells) and found that lncRNAs act as ceRNAs in the effects of aging-related cardiomyocyte apoptosis.There have been some novel findings in our present study. First, our results indicated that lncRNAs played vital roles in hypoxia-induced myocardial apoptosis in the human cardiomyocyte line. Second, we found that in addition to proapoptotic effects, lncRNAs also decreased the occurrence of myocardial apoptosis via antiapoptotic factors. We identified NONHSAT069381 and NONHSAT140844 as lncRNAs regulating apoptosis. Overexpression of NONHSAT069381 or knockdown of NONHSAT140844 augmented the myocardial apoptotic ratio after H/R, whereas overexpression of NONHSAT140844 or knockdown of NONHSAT069381 decreased the apoptotic ratio of cardiomyocytes. Lastly, we proved that lncRNA NONHSAT069381 played a ceRNA role in regulating mCASP3 expression by binding to hsa-miR-124-5p, while lncRNA NONHSAT140844 played a ceRNA role in regulating XIAP expression by binding to hsa-miR-6507-5p.The Encyclopedia of DNA Elements (ENCODE) Project Consortium indicated that more than 28,000 lncRNAs were transcribed in the whole human genome . The funCaspases are the key effector molecules of apoptosis. Sequential activation of caspases plays a central role in the execution phase of cell apoptosis . PreviouAdditionally, increasing experimental evidence supports that lncRNA functions as competitive endogenous RNA (ceRNA), which compete for miRNA to upregulate the expression of a target gene . The ceRAlmost all age-related apoptosis studies, including our previous study, demonstrated aging augmented postischemia apoptosis. However, our previous data suggests that aging may also reduce apoptosis through lncRNA, an unknown pathway in mouse cardiomyocytes. The present study confirmed that in addition to apoptosis-promoting effects, aging also inhibits cardiomyocyte apoptosis after H/R. It is the first report to our knowledge of an age-mediated antiapoptotic cardiac effect in the human cardiomyocyte line.In summary, we have provided evidence that lncRNA may both promote and inhibit cardiomyocyte apoptosis after hypoxia or ischemia. NONHSAT069381/hsa-miR-124-5p/CASP3 and NONHSAT140844/hsa-miR-6507-5p/XIAP may be important regulatory axes in aging-mediated effects upon apoptosis. In addition, the apoptosis regulatory effects of aging are complex. The high burden of comorbidities is also an important factor in the clinical setting.This study is aimed at elucidating the changes of lncRNA and miRNA in cardiomyocytes after aging and their potential regulatory functions in apoptosis. Through the above experiments, we can clarify the relationship between human aging and myocardial apoptosis. However, it is difficult to obtain human myocardium of different ages. Therefore, we innovatively employed a \u201cbridge\u201d study model to find aging-related lncRNA and miRNA in human blood and then verify the apoptosis-regulating function of aging-related lncRNA and miRNA in human cardiomyocyte cell lines. In addition, BMI was different between the older adult group and the young group. However, the size of difference seems clinically irrelevant. Although the above methods cannot fully confirm the effect of human aging on cardiomyocyte apoptosis, it indirectly reflects the regulatory effect of human myocardial aging on apoptosis."} +{"text": "We sequenced \u224850% of coronavirus disease cases imported to Hong Kong during March\u2013July 2021 and identified 70 cases caused by Delta variants of severe acute respiratory syndrome coronavirus 2. The genomic diversity detected in Hong Kong was similar to global diversity, suggesting travel hubs can play a substantial role in surveillance. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lineage B.1.617 (2 test). This observation aligns with previous findings that the Delta variant virus can induce more severe clinical symptoms (Hong Kong adopted an elimination strategy to control coronavirus disease (COVID-19). A previous study reported the use of stringent measures to detect and prevent SARS-CoV-2 importation by COVID-19\u2013positive travelers and idenAll Kappa variant cases were imported from India, where the Kappa variant predominantly circulated . Delta vhttps://doi.org/10.1101/2021.06.19.21259169), a global-level surveillance network using airports in different geographic locations might enable more feasible and cost-effective worldwide genomic surveillance. SARS-CoV-2 sequence information thus obtained, combined with relevant metadata, could strengthen current surveillance systems designed for other travel-related sources of illness and death (https://www.gisaid.org), but specific electronic tools and pipelines would need to be developed to enable timely, robust analyses. Although genomic sequencing has been used extensively to track SARS-CoV-2 transmission in specific geographic locations . There were 2 independent local Delta variant cases in which the infection was acquired at the Hong Kong airport Figure 1https://www.gisaid.org; https://github.com/Leo-Poon-Lab/HK-Delta-variants).Virus sequences reported in this study are available from GISAID (Additional information about monitoring incoming travelers arriving at Hong Kong for genomic surveillance of SARS-CoV-2. GISAID information for study of monitoring incoming travelers arriving at Hong Kong for genomic surveillance of SARS-CoV-2."} +{"text": "Clostridiodes difficile colitis during autologous stem cell transplantation (https://www-ncbi-nlm-nih-gov.proxy.libraries.uc.edu/pubmed/29594489). To biological validate these findings, we sought to evaluate the development of chemotherapy-associated Clostridiodes difficile infections by assessing the effect of C.difficile toxin B (TcdB) and of using melphalan in beta-catenin protein expression in Caco2 cells. METHODS/STUDY POPULATION: To determine the effect of melphalan and/or C.difficile toxin B on expression of Beta-catenin from human gut epithelial cells:Adenocarcinoma cells (Caco-2) cells were seeded and allowed to grow into monolayersMonolayers were treated with PBS, TcdB, melphalan and/or TcdB + melphalan for 24 hours and then washed with PBSImmunofluorescence was measured on the monolayers to visualize three markers -DAPI-Nuclear Stain (blue),Actin-ccytoskeletal stain (red), B-Catenin (green)Analysis of images with ImageJ (NIH). Statistical analysis of the effect of TcdB and/or melphalan on \u03b2-catenin protein levels was determined by One-way ANOVACells stained with a primary anti-\u03b2 catenin antibody and an Alexa-488 secondary antibody were evaluated by flow cytometry to quantify the effect of melphalan and/or C. difficile toxin B on Caco2 cells. RESULTS/ANTICIPATED RESULTS: Immunofluorescent intensity was higher in the control (PSS exposed) cells when compared to melphalan, TcdB and mephalan+TcdB exposed cells DISCUSSION/SIGNIFICANCE OF IMPACT: A significant difference was seen in \u03b2 catenin expression in Caco-2 monolayers exposed to TcdB and/or melphalan. These data support the a role of \u03b2-catenin in the pathophysiology of CDI during chemotherapy and support GWAS findings reporting a difference in CDI susceptibility based on \u03b2-catenin genotype.OBJECTIVES/GOALS: We previously reported that genetic polymorphisms in the beta-catenin gene (CTNNB) are associated with the development of"} +{"text": "Additionally, sponge-derived compounds such as dihydrogracilin A and avarol showed immunomodulatory activity that can target the cytokines storm. Here, we reviewed the potential use of sponge-derived compounds as promising therapeutics against SARS-CoV-2. Despite the reported antiviral activity of isolated marine metabolites, structural modifications showed the importance in targeting and efficacy. On that basis, we are proposing a novel structure with bifunctional scaffolds and dual pharmacophores that can be superiorly employed in SARS-CoV-2 infection.The current pandemic caused by SARS-CoV2 and named COVID-19 urgent the need for novel lead antiviral drugs. Recently, United States Food and Drug Administration (FDA) approved the use of remdesivir as anti-SARS-CoV-2. Remdesivir is a natural product-inspired nucleoside analogue with significant broad-spectrum antiviral activity. Nucleosides analogues from marine sponge including spongouridine and spongothymidine have been used as lead for the evolutionary synthesis of various antiviral drugs such as vidarabine and cytarabine. Furthermore, the marine sponge is a rich source of compounds with unique activities. Marine sponge produces classes of compounds that can inhibit the viral cysteine protease (M The current outbreak caused by the novel coronavirus (SARS-CoV-2) and designated COVID-19 by the World Health Organization (WHO), spread aggressively worldwide . As of tRemdesivir is a prodrug that is once entered the cell converted to a triphosphate nucleoside analogue with significant inhibition activity against viral RNA-dependent RNA polymerase (RdRp) . RemdesiCryptotethya sponge, were investigated for antiviral activity and serine (TMPRSS2) proteases.Structure-based modelling indicated that both pseudotheonamide and aeruginosin may also show potent inhibitory activity against SARS-CoV-2 Mpro . HoweverThe aforementioned data can be of great benefit to fight against SARS-CoV-2 once the biological activity of the compounds is validated.\u0251, G-CSF, IP-10, MCP-1, and MIP and exhaustion of T cells. Therefore, strategies to boost the immune system at the earlier stage (mild condition) and those to modulate or suppress the cytokine storm at a later stage (severe condition) are required to manage SARS-CoV-2 infection . Octa-peactivity . Contign of IL-6 . Puupehe T-cells .Agelas oroides, showed inhibitory activity to IL-2 production (Disidea sp. (Eryloside E, isolated from Erylus goffrilleri sponge, demonstrated specific immunosuppressive activity at IC50 1.3\u2002\u03bcg/ml . Pateamioduction . Severalidea sp. de Almeiidea sp. .Ianthella quadrangulata sponge produces the polyketide iso-iantheran A, which is capable to activate the P2Y11 receptor Dendrilla membranosa sponge (a sponge .The data described here indicated that several metabolites derived from marine sponge showed promising immunomodulatory activity. Some of these compounds shared structural similarity including the terpenoid and/ or the sugar moieties .pro, respectively, while guanine derivatives can inhibit the human TMPRSS2 (The aforementioned classes of sponge-derived compounds provided an insight into pharmacophores with shared structures that can be employed in the development of novel scaffold with potent antiviral activity and improved efficacy against SARS-CoV-2. A promising strategy as indicated in TMPRSS2 . Further"} +{"text": "Metabolic regulation is a necessary component of all stress response pathways, because all different mechanisms of stress-adaptation place high-energy demands on the cell. Mechanisms that integrate diverse stress response pathways with their metabolic components are therefore of great interest, but few are known. We show that stress granule (SG) formation, a common adaptive response to a variety of stresses, is reciprocally regulated by the pathways inducing lipid droplet accumulation. Inability to upregulate lipid droplets reduces stress granule formation. Stress granule formation in turn drives lipid droplet clustering and fatty acid accumulation. Our findings reveal a novel connection between stress response pathways and new modifiers of stress granule formation. Lipid droplets (LD) are ubiquitous lipid storage organelles that are involved in regulating energy homeostasis and membrane synthesis. Their importance to the cell has been thought to derive from the need to sequester excess fatty acids, as an energy reserve and to prevent lipotoxicity -mediated transcription, which leads, among other things, to LD biogenesis , SG formation in many cases maybe unrelated to a specific effect of the inhibitor, as we demonstrated for Fasnall , 1% penicillin/streptomycin at 37\u00b0C/5% CO2. Cells modified via CRISPR/Cas9 were maintained as above with addition of puromycin during selection of clonal populations.HEK293T and U2OS (WT and G3BP1/2 KO) cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, at 37\u00b0C/5% COFatty acids were added 30 min prior to quantification , polyclonal anti-TIA1 produced in rabbit (Sigma-Aldrich SAB4301803), anti-GAPDH , anti-PPARA , anti-PPARG , anti-PPARD , antiphospho-4EBP , and anti-4EBP .Secondary antibodies for immunofluorescence: anti-rabbit IgG Cy3-conjugated (Sigma-Aldrich C2306), anti-mouse IgG Cy3 conjugated (Sigma-Aldrich C2181), and anti-rabbit IgG Cy5 conjugated (Invitrogen A10523).10505, Cayman Chemical).BODIPY\u2122 558/568 C12 -4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid, Thermo Fischer Scientific), Hoechst (Sigma), sodium arsenite , cycloheximide (Sigma), BODIPY\u2122 493/503 , Rosiglitazone (Sigma), Clofibrate (Sigma), GW501516 (Sigma), 2-bromopalmitic acid (Sigma), streptavidin-HRP (Thermo Scientific), fatty acid-free BSA (PAN Biotech), DMEM (PAN Biotech), FBS (PAN Biotech), PBS (PAN Biotech), methanol (Roth), chlorophorm (Sigma), aprotinin (Roth), leupeptin (Roth), phenylmethylsulfonyl fluoride , Fasnall (Sigma), and kinase screening library (http://www.rgenome.net/cas-offinder/) . Movies for kymographs were acquired in resonant-scanning mode. Image processing was performed using NIS-Elements software.For live cell imaging we used four-well microscope glass-bottom plates (IBIDI) or Cellview cell culture dish (Greiner Bio One). Plates were coated with Concanavalin A (Sigma) for live cell imaging of yeast. Confocal images and movies were acquired using a dual point-scanning Nikon A1R-si microscope equipped with a PInano Piezo stage (MCL), temperature and COP-values were calculated by two-tailed Student t-test or one-way ANOVA for samples with N > 10 following normal distribution. Normal distribution of the data was verified using Shapiro-Wilk test, and the equality of variances was verified by Levene's test. Mann-Whitney or Kruskal-Wallis tests were used for experiments with < 5 samples or when samples did not follow a normal distribution. The sample sizes were not predetermined. Scatter plots were generated using Matplotlib (Hunter, Three or more independent experiments were performed to obtain the data. Hunter, .The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.All aspects of the work comprising the manuscript were carried out jointly by DK and TA.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Half of all urinary tract infections (UTI) are probably unnecessary. We conducted a cluster-randomized trial in which a toolkit to enhance the diagnosis and treatment of UTIs was introduced in study NHs via usual implementation versus an enhanced implementation approach based on external facilitation and peer comparison reporting.Thirty Wisconsin NHs were randomized to each treatment arm in a 1.5:1 ratio. NHs used an online portal to report urine culture and antibiotic treatment data over a 6-month pre-intervention period (Jan-June 2019), a pre-COVID 8-month post intervention period (July 2019-Feb 2020) and an 8-month post-COVID intervention period (Mar-Oct 2020). Study outcomes included urine culture (UC), antibiotic start (AS), and antibiotic days of therapy (DOT) rates per 1,000 resident days. A generalized estimating equation model for panel data was used to assess differences in study outcomes between treatment arms before and after onset of the COVID-19 pandemic. STATA 16.1 was used for all analyses.A total of 802 UCs , 724 AS , and 6,454 DOT (3553 pre-COVID and 2901 post-COVID) were reported over the 16-month intervention period. No significant differences in the study outcomes were observed during the pre-COVID intervention period, however, UC rates in NHs assigned to the usual care arm of the study increased while those in the enhanced arm declined following onset of COVID-19 . AS and DOT rates followed a similar pattern although the differences between the study arms were not statistically significant.Figure 1. Post Implementation PeriodsOur findings suggest that NHs assigned to usual implementation regressed in their diagnosis and treatment of UTIs during the COVID-19 pandemic while those receiving external facilitation and peer comparison reports were more resilient to the effects of COVID-19.All Authors: No reported disclosures"} +{"text": "ABSTRACT IMPACT: The key to advancing precision medicine is to deepen our understanding of drug modes-of-action (MOA). This project aims to develop a novel method for predicting MOA of potential drug compounds, providing an experimental and computational platform for more efficient drug discovery. OBJECTIVES/GOALS: To develop (1) a targeted CRISPR-Cas9 chemical-genetic screen approach, and (2) a computational method to predict drug mode-of-action from chemical-genetic interaction profiles. METHODS/STUDY POPULATION: Screening drugs against a gene deletion library can identify knockouts that modulate drug sensitivity. These chemical-genetic interaction (CGI) screens can be performed in human cell lines using a pooled lentiviral CRISPR-Cas9 approach to assess drug sensitivity/resistance of single-gene knockouts across the human genome. A targeted, rather than genome-wide, library can enable scaling these screens across many drugs.CGI profiles can be derived from phenotypic screen readouts. These profiles are analogous to genetic interaction (GI) profiles, which represent sensitivity/resistance of gene knockouts to a second gene knockout rather than a drug. To computationally predict a drug\u2019s genetic target, we leverage the property that a drug\u2019s CGI profile will be similar to its target\u2019s GI profile. RESULTS/ANTICIPATED RESULTS: Five proof-of-principle screens will be conducted with compounds that have existing genome-wide profiles and well-characterized MOA. I will generate CGI profiles for these five compounds and identify genes that are drug-sensitizers or drug-suppressors. I will then evaluate whether targeted library screens can recapitulate the CGIs found in genome-wide screens. Finally, I will develop a computational tool to integrate these CGI profiles with GI profiles (derived from another project) to predict gene-level and bioprocess-level drug targets. These predictions (from both targeted and genome-wide profiles) will be benchmarked against a drug-target and drug-bioprocess standard. DISCUSSION/SIGNIFICANCE OF FINDINGS: This work will develop a scalable, targeted chemical-genetic screen approach to discovering how putative therapeutics work. The targeted screen workflow provides a method for higher-throughput drug screening. The computational pipeline provides a powerful tool for exploring the MOA of uncharacterized drugs or repurposing FDA-approved drugs."} +{"text": "The current randomized controlled trial (RCT) will be conducted to assess the effect of green tea intake on disease symptoms and laboratory parameters including C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), and complete blood count (CBC) in patients with mild-to-moderate Covid-19 infection.Randomized, double-blinded, parallel (1:1 ratio) clinical trial exploratory studyWe will recruit patients with COVID-19 infection admitted to Yasuj Shahid Jalil Hospital in Yasuj City, Kohgiluyeh and Boyer-Ahmad Province, Iran.Patients aged \u226518 yearsCOVID-19 diagnosis according to real-time polymerase chain reaction (RT-PCR)Inclusion CriteriaPregnancy or lactationDisseminated intravascular coagulation or any other types of coagulopathySevere congestive kidney failureHaving a history of participating in a clinical trial during the last 30 daysExclusion CriteriaParticipants\u2019 inclusion criteria are as follows:Intervention: Two capsules containing 450 mg green tea extract along with routine treatment for COVID-19 patients in the intervention group. Two capsules containing placebo plus routine treatment for patients with COVID-19 infection. Capsules will be taken twice a day, after lunch and dinner, for 14 days.Changes in disease symptoms and laboratory parameters including CRP, ESR, and CBC after 14 days of the intervention compared to control group.Randomization.com)Eligible patients will be randomly assigned into the intervention or control group in a 1:1 ratio. Randomization will be performed based on 8 permuted blocks with block sizes of 10, and patients in the intervention and control groups will be matched according to sex and age categories. Randomization will be done using computer-generated random numbers retrospectively registered on June 4, 2021IRCT20150711023153N3 (The full protocol is attached as an additional file, accessible from the Trials website (Additional file The online version contains supplementary material available at 10.1186/s13063-021-05462-8. Additional file 1. Full study protocol."} +{"text": "OBJECTIVES/GOALS: Patient beliefs and goals can facilitate discussion of recovery expectations, patient-provider collaboration and maximization of goal achievement. In this study, we sought to address an evidence gap and examine the association of preoperative self-assessment of goals with preoperative and 6-week knee function and gait speed among total knee arthroplasty (TKA) patients. METHODS/STUDY POPULATION: We conducted a secondary analysis of data from the VERITAS randomized, controlled trial conducted from 11/2016-03/2018 that included adults age \u2265 18 years with scheduled and completed unilateral TKA followed by post-surgical physical therapy. Patients rated their ability to perform various activities of daily living goals scaled from 0 (unable to perform) to 10 (full performance). Patients were categorized by pre-surgical (baseline) goal rating: low = 0-2, intermediate = 3-4, and high = 5-10. Outcomes including gait speed and the KOOS were assessed within 10 days prior to surgery and 6-weeks post-surgery. Descriptive statistics and outcomes were compared for patients by preoperative goal rating using Chi-square or Fisher\u2019s exact tests and ANOVA or Kruskal-Wallis tests as appropriate. RESULTS/ANTICIPATED RESULTS: Of 288 patients , 102 had a low goal rating (GR), 86 intermediate, and 99 high. Patients with low GR preoperatively generally had lower baseline mean scores than intermediate and high GR patients, respectively, on the KOOS and lower gait speed (m/s) compared to intermediate and high GR patients at baseline . The low, intermediate, and high GR groups, respectively, showed no difference across mean KOOS scores or gait speed (m/s) at 6 weeks postoperative. DISCUSSION/SIGNIFICANCE OF IMPACT: In this study, adults who perceived greater difficulty with a pre-selected activity goal, exhibited lower function prior to TKA but showed no differences in function 6-weeks after surgery. Follow-up studies will describe the association between goal-setting preoperatively and patient goal attainment and satisfaction following surgery."} +{"text": "Current guidelines recommend empiric antibiotics be used only for severe cases of coronavirus disease 2019 (COVID-19) or in cases where there is high clinical suspicion for bacterial co-infection. Level of adherence to guideline-recommended prescribing is unknown and high rates of antimicrobial prescribing may lead to increased development of resistance.We reviewed antimicrobial prescribing patterns for patients with COVID-19 managed at The Alfred Hospital in Melbourne, Australia in 2020. Adherence to World Health Organization (WHO) guideline-based prescribing was assessed by manual review of case notes. Monthly hospital-wide antibacterial consumption April-Dec 2020 (post-pandemic period) was compared to Jan 2019-Mar 2020 (pre-pandemic period), measured as days of therapy (DOT) per 1000 patient-days. Rates of multi-drug resistant organisms (MRO) were compared between months in 2019 and 2020 after pandemic onset (April 2020) and expressed as isolates per 1000 patient-days.P=0.0065). Antimicrobial use patterns varied, with significant decreases in commonly used antibiotics such as ceftriaxone, piperacillin-tazobactam, azithromycin and ciprofloxacin but no change in vancomycin or meropenem . There was a mean decrease of 0.77 MRO isolates/1000 patient-days (P=0.026) when each month in 2020 was compared with the corresponding month in 2019 .147 patients were managed for COVID-19 in 2020 at our centre. 101 patients required hospital admission and 58 (39%) were classified as either severe or critical in severity. 80 (54%) patients received empiric antimicrobial treatment, including 78/101 (77%) of hospital inpatients and 24/26 (92%) of ICU-admitted patients. 59 (73%) of antimicrobial prescriptions were adherent to WHO guidelines. Monthly antibacterial consumption was significantly lower post-pandemic than in the pre-pandemic period (mean 853 vs 902 DOT/1000 patient-days, Antibacterial consumption in 2019 and 2020 by month, expressed as days of therapy/1000 patient-days.Rates of isolated multi-drug resistant organisms in 2019 and 2020 by month, expressed as isolates/1000 patient-days.A high proportion of admitted patients with COVID-19 received empiric antibiotics. In spite of this, we observed a significant reduction in total antimicrobial consumption and reduced rates of MRO isolation in the post-pandemic period.All Authors: No reported disclosures"} +{"text": "Bone Marrow Transplantation (2021) 56:1550\u20131557; 10.1038/s41409-020-01200-x, published online 29 January 2021Correction to: Due to a processing error, the shared first authorship between Sebastian Schober and Uwe Thiel nor the shared last authorship between Stefan Burdach and Peter Lang was not taken.The original article has been corrected."} +{"text": "ABSTRACT IMPACT: Patients living in overcrowded zip codes were at increased risk of contracting severe COVID-19 after controlling for confounding disease and socioeconomic factors OBJECTIVES/GOALS: This study sought to examine whether residences in over-crowded zip codes with higher reported over-crowding represented an independent risk factor for severe COVID-19 infection, defined by presentation to an emergency department. METHODS/STUDY POPULATION: In this zip code tabulated area (ZCTA)-level analysis, we used NYC Department of Health disease surveillance data in March 2020 merged with data from the CDC and ACS to model suspected COVID-19 case rates by zip code over-crowdedness . We defined suspected COVID-19 cases as emergency department reported cases of pneumonia and influenza-like illness. Our final model employed a multivariate Poisson regression models with controls for known COVID-19 clinical and related socioeconomic risk factors after accounting for multicollinearity. RESULTS/ANTICIPATED RESULTS: Our analysis examined 39,923 suspected COVID-19 cases across 173 ZCTAs in NYC between March 1 and March 30 2020. We found that, after adjusted analysis, for every quartile increase in defined over-crowdedness, case rates increased by 32.8% . DISCUSSION/SIGNIFICANCE OF FINDINGS: Over-crowdedness by zip code may be an independent risk factor for severe COVID-19. Social distancing measures such as school closures that increase house-bound populations may inadvertently worsen the risk of COVID-19 contraction in this setting."} +{"text": "There is an increasing interest in identifying aging-related factors which may be permissive of Alzheimer\u2019s Disease (AD) emergence. We previously used machine learning to derive an index of neuroanatomic risk of dementia called AD pattern similarity (AD-PS) score using MRIs obtained in the Atherosclerosis Risk in Communities (ARIC) study. Here, we investigate the potential of the AD-PS scores as a brain-focused measure of biologic age. Among 1970 ARIC participants with MRI collected at ARIC Visit 5, we related AD-PS scores to three measures of aging: mortality (n=356) over 8 years of follow-up; an a priori panel of 32 proteins related to aging (N=1647); and a deficit accumulation index (DAI) based on 38 health-related measures. We found lower AD-PS scores associated with significantly lower mortality after adjusting for age, race, smoking and hypertension. Among the 32 proteins, nine were significantly associated to AD-PS scores (p < 0.05) with 4 remaining significant adjusting for multiple comparisons chain). Finally, in a linear regression model after adjusting for age, race, sex, hypertension and smoking, AD-PS scores were associated with the DAI (p < 0.001). The consistent patterns of associations suggest that a data-driven measure of AD neuroanatomic risk may be capturing aspects of biologic age in older adults."} +{"text": "Bcr-Abl oncogene that results from a reciprocal translocation between human chromosome 9 and 22, is associated with several hematological malignancies. However, the role of lncRNAs in Bcr-Abl-induced leukemia remains largely unexplored.Dysregulation of long noncoding RNAs (lncRNAs) has been linked to various human cancers. LncRNA cDNA microarray was employed to identify key lncRNAs involved in Bcr-Abl-mediated cellular transformation. Abl-transformed cell survival and xenografted tumor growth in mice were evaluated to dissect the role of imatinib-upregulated lncRNA 1 (IUR1) in Abl-induced tumorigenesis. Primary bone marrow transformation and in vivo leukemia transplant using lncRNA-IUR1 knockout (KO) mice were further conducted to address the functional relevance of lncRNA-IUR1 in Abl-mediated leukemia. Transcriptome RNA-seq and Western blotting were performed to determine the mechanisms by which lncRNA-IUR1 regulates Bcr-Abl-induced tumorigenesis.We identified lncRNA-IUR1 as a critical negative regulator of Bcr-Abl-induced tumorigenesis. LncRNA-IUR1 expressed in a very low level in Bcr-Abl-positive cells from chronic myeloid leukemia patients. Interestingly, it was significantly induced in Abl-positive leukemic cells treated by imatinib. Depletion of lncRNA-IUR1 promoted survival of Abl-transformed human leukemic cells in experiments in vitro and xenografted tumor growth in mice, whereas ectopic expression of lncRNA-IUR1 sensitized the cells to apoptosis and suppressed tumor growth. In concert, silencing murine lncRNA-IUR1 in Abl-transformed cells accelerated cell survival and the development of leukemia in mice. Furthermore, lncRNA-IUR1 deficient mice were generated, and we observed that knockout of murine lncRNA-IUR1 facilitated Bcr-Abl-mediated primary bone marrow transformation. Moreover, animal leukemia model revealed that lncRNA-IUR1 deficiency promoted Abl-transformed cell survival and development of leukemia in mice. Mechanistically, we demonstrated that lncRNA-IUR1 suppressed Bcr-Abl-induced tumorigenesis through negatively regulating STAT5-mediated GATA3 expression.These findings unveil an inhibitory role of lncRNA-IUR1 in Abl-mediated cellular transformation, and provide new insights into molecular mechanisms underlying Abl-induced leukemogenesis.The online version contains supplementary material available at 10.1186/s12943-021-01478-5. Bcr-Abl oncogene that results from the reciprocal translocation between human chromosome 9 and 22, and occurs in more than 90% of CML cases [Bcr-Abl oncogene is also involved in tumorigenesis of other leukemia such as acute lymphoid leukemia (ALL) [v-Abl is the oncogene of Abelson murine leukemia virus, which contributes to the malignant transformation of mouse pre-B cells and lymphoid tumorigenesis in mice [Chronic myeloid leukemia (CML) is a hematological malignancy mainly caused by the ML cases . Bcr-Abl in mice . Owing t in mice \u20136. AlthoThe majority of human transcripts lack protein-coding capacity, which are defined as noncoding RNAs (ncRNAs) . Long noOur group has previously identified several functional lncRNAs involved in Bcr-Abl-induced oncogenic transformation. For example, we identified lncRNA beta globin locus 3 (BGL3) as a critical tumor suppressor in Bcr-Abl-mediated tumorigenesis. LncRNA-BGL3 can act as a competitive endogenous RNA (ceRNA) to positively regulate the expression of phosphatase and tensin homolog (PTEN), thereby promoting Abl-transformed leukemic cell apoptosis and suppressing xenografted tumor growth in vivo . In addiIn the current study, we identified a novel imatinib-upregulated lncRNA, designated lncRNA-IUR1 that critically regulated Abl-mediated cellular transformation. The expression of lncRNA-IUR1 was highly induced by imatinib treatment in Abl-transformed leukemic cells. Loss of lncRNA-IUR1 promoted leukemic cell survival and tumor growth in mice. Knockout of lncRNA-IUR1 in mice facilitated Abl-mediated transformation of primary bone marrow cells and the progression of Abl-mediated leukemia in mice. Furthermore, we revealed that lncRNA-IUR1 suppressed Abl-induced tumorigenesis through regulation of STAT5-mediated GATA3 expression. These findings highlight the functional involvement and physiological significance of lncRNAs in Abl-mediated oncogenic transformation, and provide new insights into complicated mechanisms underlying hematopoietic malignancies.The mouse experimental design and protocol used in this study were approved by the Regulation of the Institute of Microbiology, Chinese Academy of Sciences of Research Ethics Committee (Permit Number: SQIMCAS2018043). All mouse experiments were performed in accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals approved by the State Council of People\u2019s Republic of China. All participants signed informed consent prior to using the peripheral blood cells for scientific research.The lncRNA cDNA microarray was from Agilent . Total RNAs from three independent groups of K562 cells treated with imatinib or control cells were prepared using Trizol reagent . Sample labeling, hybridization and data analysis were performed as previously described . The micTotal RNAs isolated from three independent groups of K562 cells expressing shRNA targeting lncRNA-IUR1 or control shRNA, were used for RNA-seq. RNA libraries were prepared for sequencing using standard Illumina protocols. Illumina Casava software was employed for basecalling during the data procession. Sequenced reads were trimmed for adaptor sequence, masked for low-complexity or low-quality sequence, and then mapped to hg38 whole genome. Reads Per Kilo bases per Million reads (RPKM) were calculated and analyzed using samtools v0.1.19. RNA-seq data have been deposited on GEO public database (Accession number GSE181535).K562 and HEK293T cell lines were purchased from ATCC (American Type Culture Collection) . The v-Abl-transformed mouse cell lines NS2 and W44 were generated as described previously\u00a0[\u2212\u20091 of polybrene and a spin infection was performed by centrifugation at 2200\u2009rpm for 2\u2009h.DNA transfection was performed using VigoFect (Vigorous Biotechnology). Lentiviruses expressing shRNAs were packaged in HEK293T cells in which shRNA constructs, and lentiviral packaging plasmids were co-transfected. Lentiviral vector pNL/EGFP/CMV/WPRE\u0394U3 was utilized for overexpression experiments. LncRNA-IUR1 or lncRNA-mIUR1 cDNA was cloned into the lentiviral vector using Nhe1 and Xho1 sites. The lncRNA-IUR1 or lncRNA-mIUR1 cDNA construct along with lentiviral packaging vectors were transfected into HEK293T cells. For the generation of stable lncRNA-IUR1 knockdown or overexpressing cells, the virus-containing supernatant was collected 48\u2009h after transfection and passed through a 0.45\u2009\u03bcm filter to eliminate cells. Cells in 6-well tissue culture plates were infected with the virus in medium containing 8\u2009\u03bcg\u2009mlhuman lncRNA-IUR1 shRNA: 5\u2032-GCTCTTCAGTTCTGACCTTCC-3\u2032,mouse lncRNA-IUR1 shRNA: 5\u2032-GTGCATACAGTAGGCCTAGCA-3\u2032,mouse GATA3 shRNA: 5\u2032-GCAATGCCTGCGGACTCTACC-3\u2032.The following shRNAs were used in this study:\u2212\u0394\u0394Ct method was used to calculate the relative RNA levels against GAPDH or \u03b2-Actin. The peripheral blood cells were treated with red blood cell lysis buffer to remove the red blood cells, and then subjected to RNA isolation followed by RT-PCR.RNA was isolated with TRIzol RNA Isolation Reagents (Thermo Fisher). Reverse transcription was performed with PrimeScript\u2122 RT Reagent Kit (Takara), and quantitative real-time PCR was performed with Power SYBR Green Master Mix (Thermo Fisher). For quantification, the 25\u2032 and 3\u2032 RACE experiments were performed using the SMARTer RACE cDNA amplification Kit according to the manufacturer\u2019s instruction.Cell apoptosis assay was performed as previously described . BrieflyCell cytotoxicity assay was performed using the CCK-8 kit (Beyotime) according to the manufacturer\u2019s instruction. For colony formation assay, K562 cells were washed with PBS and plated in methylcellulose (STEMCELL). Colonies were counted 7-10\u2009days later.For immunostaining experiment, fresh tissues were embedded in optimal cutting temperature compound, sectioned onto slides and fixed with 4% paraformaldehyde for 15\u2009min as previously described . TissuesNude mice injection was performed as previously described . In brie7) were injected into sub-lethally irradiated recipients (C57BL/6\u2009N mice) through tail vein.Leukemia transplantation was performed as previously described . GFP-posMurine lncRNA-IUR1 knockout mice were generated by CRISPR/Cas9-based genome editing system as previously described . BrieflyPrimary bone marrow transformation was carried out as previously described . Transfot test, and p value <\u20090.05 was considered to be significant. Statistical significance is represented in figures by: *P\u2009<\u20090.05; **P\u2009<\u20090.01; ***P\u2009<\u20090.001.Data are presented as mean\u2009\u00b1\u2009SEM of at least three independent experiments. Statistical analyses were performed in Microsoft Excel 2010 with the Student\u2019s two-tailed To identify key long noncoding RNAs (lncRNAs) involved in Bcr-Abl-mediated tumorigenesis, an lncRNA microarray was employed to analyze the expression of lncRNAs in Bcr-Abl\u2013positive K562 cells treated with or without the Abl kinase inhibitor imatinib. Numerous lncRNAs were found to display differential expression in response to imatinib treatment Fig.\u00a0. Notablypurinergic receptor P2Y2 (P2ry2) gene and the FCH and double SH3 domains 2 (Fchsd2) gene on human chromosome 11 mice Fig.\u00a0. The defNext, we established the leukemia model using lncRNA-mIUR1 KO mice, and evaluated the effect of lncRNA-mIUR1 knockout on Abl-induced leukemia in mice. Sub-lethally irradiated WT or lncRNA-mIUR1 KO mice were injected with GFP-positive NS2 cells, and the development of Abl-mediated leukemia was examined to analyze the differential expression of genes in control and lncRNA-IUR1 knockdown K562 cells Fig.\u00a0. Of partTo test this possibility, we first determined whether the expression of GATA3 was regulated by lncRNA-IUR1 in Abl-transformed leukemic cells. Indeed, the expression of GATA3 vastly increased in lncRNA-IUR1 knockdown K562 cells, whereas lncRNA-IUR1 overexpression led to a significant decrease of GATA3 levels in the cells Fig. . DisruptSTAT5 has been reported as a transcription factor of GATA3 , which pNext, we asked whether lncRNA-IUR1 suppresses Abl-induced tumorigenesis through regulating GATA3 expression. To test this, we first evaluated the role of GATA3 in Abl-mediated transformation. Control and GATA3 knockdown NS2 cells stably expressing control or GATA3 shRNA were generated, treated with imatinib, and subjected to cell survival analysis Fig.\u00a0. We obseThen, we further investigated the involvement of GATA3 in regulation of Abl-induced tumorigenesis by lncRNA-IUR1. We generated K562 cells stably overexpressing empty vector (EV), lncRNA-IUR1, or combination of lncRNA-IUR1 and GATA3. These cells were treated with imatinib, and subjected to cell survival analysis Fig. . As showAbl oncogenes is associated with tumorigenesis of several types of leukemia including CML. It is well known that Abl-induced tumorigenesis is a complicated process involving dysregulation of multiple signal pathways that regulate cell survival and proliferation. Although considerable progress has been made to address the molecular mechanisms accounting for Abl-mediated oncogenic transformation, the roles of lncRNAs in Abl-induced leukemia remain largely unexplored and attract increasing attention. In the present study, we identified an lncRNA IUR1 as a critical negative regulator of Abl-induced tumor formation. LncRNA-IUR1 is expressed at a very low level in Bcr-Abl-positive cells from chronic myeloid leukemia patients. Importantly, it was significantly induced in Abl-positive leukemic cells treated by imatinib. Depletion of lncRNA-IUR1 promoted Abl-positive leukemic cell survival and tumor growth in the xenograft mouse model, while enhanced expression of lncRNA-IUR1 sensitized cell to apoptosis and suppressed xenografted tumor growth in mice. Furthermore, we identified the mouse homologous lncRNA-IUR1. Knockout of lncRNA-mIUR1 in mice facilitated Abl-mediated transformation of primary bone marrow cells, Abl-transformed cell survival, and the development of leukemia in mice. These findings suggest that low expression of lncRNA-IUR1 is required for efficient tumorigenesis induced by Abl oncogenes. However, how Bcr-Abl downregulates the expression of lncRNA-IUR1 and evades from the inhibitory effect of lncRNA-IUR1 remain to be further defined.Cellular transformation mediated by Imatinib can competitively bind to the adenosine triphosphate (ATP) binding pocket of Bcr-Abl, thereby effectively inhibiting its tyrosine kinase activity . ImatiniThe STAT5 signaling is constitutively activated by Bcr-Abl in CML, leading to uncontrolled cell survival and proliferation, which is indispensable for the initial and maintenance of Bcr-Abl\u2013positive leukemia , 47. OurIn this study, lncRNA-IUR1 knockout mouse model was generated and employed to evaluate the functional relevance of lncRNA-IUR1 in Abl-induced leukemia under a more sophisticated and physiological circumstance. Loss of murine lncRNA-IUR1 promoted Abl-mediated transformation of primary bone marrow cells, leukemic cell survival, and the development of Abl-mediated leukemia in mice, supporting that lncRNA-IUR1 acted as a key negative regulator of Abl-induced tumorigenesis. Longitudinal analysis in a large cohort of CML patients is necessary to address the association between CML patient survival and the expression of lncRNA-IUR1, and the relationship of lncRNA-IUR1 with imatinib resistance in CML patients, and further to evaluate the translational potential of lncRNA-IUR1 for diagnosis and treatment of Bcr-Abl\u2013positive leukemia. These deserve further studies in the future.In summary, this study identified lncRNA-IUR1 as a critical negative regulator of Bcr-Abl-induced tumorigenesis. Deficiency of lncRNA-IUR1 promoted Abl-transformed cell survival and development of leukemia in mice. Mechanistically, lncRNA-IUR1 suppressed Bcr-Abl-induced tumorigenesis through negatively regulating STAT5-mediated GATA3 expression. These findings reveal the vital involvement and physiological significance of lncRNAs in Abl-mediated oncogenic transformation, and provide new insights into molecular mechanisms underlying Abl-induced leukemogenesis.ESM 1."} +{"text": "To the Editor:Clinical and experimental evidence suggests that acute lymphoblastic leukemia (ALL) is highly resistant to natural killer (NK) cell-mediated killing, even in the allogeneic setting , 2. The n\u2009=\u200917) HLA-E levels were restored to normal ranges and stayed within this range during the observation time , restored HLA-E levels after the induction phase dropped significantly indicating relapse stably maintained HLA-E expression levels comparable to initial values . Among these, six patients with late relapse (>6 months after HSCT) managed to reach reconstituted HLA-E levels initially after HSCT but eventually experienced HLA-E downregulation at the time of relapse . Initial values were within the range of the respective stem cell donors (data not shown). Again, patients who achieved long-term remission following HSCT and by flow-cytometry [\u22123 (PCR) and >0.01% (flow-cytometry), marking the onset of molecular relapse. Once having achieved a further remission, similar impacts on cell surface expression could be observed: following 2nd HSCT, HLA-E levels dropped significantly accompanied by increasing MRD loads. These observations appeared to be independent of the conditioning regimen, donor type, graft-versus-host disease (GvHD) prophylaxis, and presence of GvHD. Furthermore, the correlation was also independent of the HCMV status of the patients ts Table\u00a0.Fig. 2LeIn the next step, we wanted to address the question of whether HLA-E downregulation on peripheral leukemic blasts reflects the degree of expression on immature precursor B cells found in BM samples or is a leukemia-specific mechanism, possibly in order to escape immune surveillance. For this purpose, we assigned each ALL to a specific stage of B cell development according to the expression of stage-specific markers and dire+ NK cells mAb increased the cytolytic activity of healthy NK cells significantly, making leukemic blasts susceptible to recognition by NKG2Alls Fig.\u00a0. The datlls Fig.\u00a0, documenPresently, qPCR from bone marrow biopsies represents the most validated and standardized method for MRD detection, which significantly correlates with clinical outcomes in leukemia . The pre+ NK cells. The functional data, demonstrating restored killing of ALL blasts by specific blocking of HLA-E/NKG2A interaction, suggest that HLA-E-mediated resistance to host immune surveillance is only given within a small corridor enabling leukemic escape from both T cell and NK-mediated control. Moreover, they challenge the prevailing view that B-ALL is generally resistant to NK cell surveillance. In fact, cellular therapy combining allogeneic NK cells with antibody-mediated blocking of the HLA-E/NKG2A inhibitory axis, as provided by the NKG2A-specific therapeutic reagent Monalizumab, could open novel avenues for treatment of B-ALL [Mechanistically, the study suggests strong selective pressure for expression of low but still tolerogenic HLA-E levels on ALL blasts, in turn indicating tight surveillance of ALL by NKG2Aof B-ALL , 12.SUPPLEMENTAL MATERIAL"} +{"text": "The diagnosis of dengue disease, caused by the dengue virus (DENV) (a flavivirus), often requires serologic testing during acute and early convalescent phases of the disease. Some symptoms of DENV infection, such as nonspecific fever, are similar to those caused by infection with SARS-CoV-2, the virus that causes COVID-19. In studies with few COVID-19 cases, positive DENV immunoglobulin M (IgM) results were reported with various serologic tests, indicating possible cross-reactivity in these tests for DENV and SARS-CoV-2 infections transmitted by Laboratory diagnosis of dengue disease focuses on the detection of viral RNA by real-time reverse transcription\u2013polymerase chain reaction (RT-PCR) or nonstructural protein 1 (NS1) antigen tests in blood specimens. These tests identify a large percentage of cases during the first few days of illness (Recent reports indicated possible cross-reactivity in serologic (IgM) tests for DENV in specimens from confirmed COVID-19 cases and SARS-CoV-2. A previous study reported a similar test specificity of the SARS-CoV-2 pan-Ig Spike Protein ELISA assay (99%) for pathogens unrelated to those evaluated in this study immunoglobulin M results were reported with various serologic tests, indicating possible cross-reactivity in serologic tests for DENV and SARS-CoV-2 infections. In a large cohort of febrile patients in Puerto Rico (where DENV is endemic) with recently confirmed SARS-CoV-2, DENV, or Zika virus infections, the specificity of DENV and SARS-CoV-2 enzyme-linked immunosorbent assays was \u226598%.These findings indicate that diagnoses of dengue or Zika virus diseases with the serological assays described in this report are not affected by COVID-19, nor do dengue or Zika virus diseases interfere with the diagnosis of COVID-19."} +{"text": "Scientific Reports 10.1038/s41598-021-86991-9, published online 13 April 2021Correction to: In the original version of this Article the Acknowledgments section was incomplete.\u201cWe are very grateful to The Atat\u00fcrk University, Scientific Research Projects Foundation for generous financial support (Project Number FBA-2019-7219).\u201dnow reads:\u201cWe are very grateful to The Atat\u00fcrk University, Scientific Research Projects Foundation (Project Number FBA-2019-7219) and Humintech GmbH for generous support.\u201dThe original Article has been corrected."} +{"text": "There is an increasing interest in identifying aging-related factors which may be permissive of Alzheimer\u2019s Disease (AD) emergence. We previously used machine learning to derive an index of neuroanatomic risk of dementia called AD pattern similarity (AD-PS) score using MRIs obtained in the Atherosclerosis Risk in Communities (ARIC) study. Here, we investigate the potential of the AD-PS scores as a brain-focused measure of biologic age. Among 1970 ARIC participants with MRI collected at ARIC Visit 5, we related AD-PS scores to three measures of aging: mortality (n=356) over 8 years of follow-up; an a priori panel of 32 proteins related to aging (N=1647); and a deficit accumulation index (DAI) based on 38 health-related measures. We found lower AD-PS scores associated with significantly lower mortality after adjusting for age, race, smoking and hypertension. Among the 32 proteins, nine were significantly associated to AD-PS scores (p < 0.05) with 4 remaining significant adjusting for multiple comparisons chain). Finally, in a linear regression model after adjusting for age, race, sex, hypertension and smoking, AD-PS scores were associated with the DAI (p < 0.001). The consistent patterns of associations suggest that a data-driven measure of AD neuroanatomic risk may be capturing aspects of biologic age in older adults."} +{"text": "Gallium 68-tetraazacyclododecane-tetraacetic acid-octreotate ([68Ga]Ga-DOTA-TATE) is a selective somatostatin analogue ligand, which shows increased affinity for somatostatin receptor subtype (SSTR) 2 and has been used routinely for imaging neuroendocrine tumors with PET/CT. We investigated the utility of [68Ga]Ga-DOTA-TATE positron emission tomography/computed tomography (PET/CT) in patients with suspected pituitary pathology. We reviewed imaging for twenty consecutive patients with suspected pituitary pathology who were referred for [68Ga]Ga-DOTA-TATE PET/CT.Nine patients presented with recurrent Cushing\u2019s syndrome following surgical resection of pituitary adenomas due to recurrent Cushing\u2019s disease (seven patients) and ectopic ACTH secreting tumor (2 patients). All seven patients with recurrent Cushing\u2019s disease showed positive pituitary [68Ga]Ga-DOTA-TATE uptake while both cases of ectopic hormonal secretion had absented pituitary uptake. In 1 of these 2 patients, [68Ga]Ga-DOTA-TATE was able to localize the source of ectopic ACTH tumor.Six patients presented de novo with Cushing\u2019s due to ectopic ACTH secretion; [68Ga]Ga-DOTA-TATE PET/CT was able to localize ectopic tumors in six of eight patients There was high uptake [68Ga]Ga-DOTA-TATE in 3 cases of recurrent central hyperthyroidism (SUVmax 6.6\u201314.3) and 2 cases of prolactinoma (SUVmax 5.5 and 11.3).Absent [68Ga]Ga-DOTA-TATE activity in the pituitary fossa is useful in excluding pituitary disease in recurrent Cushing\u2019s. Recurrent pituitary thyrotropinomas and prolactinomas showed moderate to high pituitary activity. In addition, in Cushing\u2019s syndrome, [68Ga]Ga-DOTA-TATE is useful for detection of ectopic sources of ACTH production, especially where anatomic imaging is negative. Neuroendocrine tumors (NET) cover a heterogeneous group of tumors, which originate from endocrine glands or other endocrine organs like thyroid, pancreas, respiratory, and gastrointestinal tissue.As most NETs express somatostatin receptors, they can be adequately targeted and visualized with somatostatin receptor radio-labeled analogs in vivo is based on the increased affinity of [68Ga]Ga-DOTA-TATE labeled somatostatin receptor ligands relative to The aim of our study was to evaluate the utility of [68Ga]Ga-DOTA-TATE PET/CT imaging scan in patients with suspected pituitary pathology. Patients were divided into two broad groups: those with ACTH dependent Cushing\u2019s syndrome and those with recurrent prolactinomas and thyrotropinomas.Cushing\u2019s syndrome is a hormonal imbalance due to abnormally increased levels of cortisol hormone in blood. Cushing\u2019s syndrome is divided into 2 types: ACTH-dependent and ACTH-independent forms. In ACTH-dependent type, there is over-synthesis of ACTH from pituitary adenoma, called Cushing\u2019s disease (CD), or ectopic secretion of ACTH from peripheral tumors ) who underwent [68Ga]Ga-DOTA-TATE PET/CT for evaluation of pituitary pathology.Suspected recurrent Cushing\u2019s disease following previous surgical resectionACTH dependent Cushing syndrome secondary to suspected ectopic ACTH productionRecurrent central hyperthyroidismRecurrent prolactinomaThe indication for [68Ga]Ga-DOTA-TATE PET/CT were as follows Table :SuspectImages were acquired 45\u201360 min after injection of 120\u2013200 MBq of [68Ga]Ga-DOTA-TATE. Imaging was performed using a dedicated GE Discovery STE camera combining a PET unit and a 16-slice CT unit; whole-body examinations (brain to mid-thigh) were performed with the patient supine. The CT exposure factors for all examinations were 120 kVp and 80 mA in 0.8 s. Maintaining patient position, we performed a whole-body PET emission scan covering an area identical to that covered by CT. PET scans were acquired at a rate of 4 min per bed position, and PET images were reconstructed using CT for attenuation correction. The [68Ga]Ga-DOTA-TATE PET acquisitions were performed in 3 dimensions with a 5-slice overlap between consecutive bed positions. Ga-68-DOTATATE PET images were reconstructed using an ordered-subsets expectation maximization algorithm with 3 iterations and 25 subsets. The CT data for [68Ga]Ga-DOTA-TATE were reconstructed to axial slices 3.75 mm thick with a soft-tissue reconstruction algorithm and 2.5 mm thick with a lung reconstruction algorithm.The documented clinical reports were used to determine results of [68Ga]Ga-DOTA-TATE PET/CT scans. The presence or absence of uptake at the suspected lesion level allowed to classify the 20 patients in \u201cpositive\u201d and \u201cnegative\u201d respectively. In addition, scans were retrospectively reviewed to document standardized uptake value (SUVmax) in all lesions.Histological confirmation of tumor type was available for all patients except for one case where ectopic ACTH source for Cushing syndrome was unknown.All patients had informed consent, and institutional board ethics approval was received for this retrospective study.Tumor overview, histology assessment, and [68Ga]Ga-DOTA-TATE uptake are summarized in Table Fifteen patients had Cushing\u2019s syndrome. Of these 15, nine presented with recurrent Cushing\u2019s following surgical treatment for Cushing\u2019s disease. Six out of 15 patients presented de novo with ectopic ACTH-dependent Cushing\u2019s syndrome. In 7/9 patients with recurrent Cushing\u2019s syndrome, there was recurrent pituitary disease. In 2/9 patients, recurrent Cushing\u2019s syndrome was due to ectopic ACTH producing tumor.The source of ectopic ACTH was due to bronchial carcinoid (3 patients), pancreatic NETs (2 patients), and mid gut NET (1 patient). Of 3 bronchial carcinoid tumors, 2 were typical carcinoid (0.8 and 1.7 cm) and 1 was atypical carcinoid (1.5 cm). In one patient, ectopic source of ACTH production was unknown.In all seven patients with recurrent Cushing\u2019s secondary to recurrent Cushing\u2019s disease, there was positive uptake of [68Ga]Ga-DOTA-TATE within pituitary . In both cases of recurrent Cushing\u2019s due to ectopic ACTH production, there was absent uptake of [68Ga]Ga-DOTA-TATE in the pituitary. Pituitary uptake in those with recurrent pituitary adenomas was less intense than pituitary uptake seen in patients presenting de novo with ectopic Cushing\u2019s .[68Ga]Ga-DOTA-TATE was able to depict source of ectopic ACTH production in six of eight patients . [68Ga]Ga-DOTA-TATE showed positive but low uptake as well absent pituitary [68Ga]Ga-DOTA-TATE activity Fig. . In anotIn one case with unknown primary site on conventional CT/MRI imaging Ga-[68Ga]Ga-DOTA-TATE showed site of primary tumor .Three patients presented with recurrent central hyperthyroidism due to thyroid stimulating hormone (TSH) secreting adenoma following previous surgical resection, with increased TSH and free-thyroid hormone levels, and residual pituitary macro adenomas on MRI . All patients with recurrent thyrotropinomas showed high tracer uptake Fig. within pOur study suggests that, in selected indications, [68Ga]Ga-DOTA-TATE has a useful role in evaluating patients with suspected pituitary pathology.[68Ga]Ga-DOTA-TATE activity within the pituitary fossa is a marker for functioning pituitary tissue, a property which can help assess patients with recurrent Cushing\u2019s syndrome following resection of corticotrophin secreting pituitary tumors. Positive pituitary uptake indicates the presence of functioning pituitary tissue; in all seven patients with recurrent Cushing\u2019s disease, there was positive uptake within pituitary although this was less than normal pituitary activity seen in those with Cushing\u2019s due to ectopic ACTH secretion. Our findings are in keeping with Zhao et al. who showed that [68Ga]Ga-DOTA-TATE had higher uptake in normal remaining pituitary tissue than in recurrent or residual pituitary adenomas for evaluating patients with ectopic Cushing syndrome with majority of published studies . The results are in line with the literature data, although absolute values of SUV are generally lower in our case (Kakade et al. Thyrotropinomas are a rare cause of hyperthyroidism in clinical practice often diagnosed as macro adenomas due to delayed diagnosis. Suppression of TSH secretion is mediated via both SSTR 2 and SSTR 5 subtypes (Shimon et al. [68Ga]Ga-DOTA-TATE, with integrated PET/CT, is a useful diagnostic modality for the evaluation of patients with suspected pituitary pathology. Recurrent Cushing\u2019s disease is associated with positive pituitary uptake of [68Ga]Ga-DOTA-TATE. Although in these cases it would not be possible to distinguish pathological from physiological uptake, positive [68Ga]Ga-DOTA-TATE is useful as it indicates the presence of functioning pituitary tissue. Absence of pituitary uptake in patients with recurrent Cushing\u2019s suggests source of ACTH is ectopic. Moderate to high pituitary tracer uptake of [68Ga]Ga-DOTA-TATE was seen in patients with recurrent thyrotropinomas and prolactinomas indicating [68Ga]Ga-DOTA-TATE may be useful for detection of disease post-surgery.[68Ga]Ga-DOTA-TATE may be helpful in detecting source of ectopic lesion in Cushing\u2019s syndrome particularly in those where CT imaging is negative. Finally, locally aggressive or metastatic pituitary tumors may show [68Ga]Ga-DOTA-TATE uptake and therefore indicate potential for treatment with radio-labeled somatostatin receptor analogues such as[177Lu]Lu DOTA-TATE."} +{"text": "Porcine reproductive and respiratory syndrome virus (PRRSV) causes PRRS and is known to effectively suppress host innate immunity. The current strategies for controlling PRRSV are limited and complete understanding of anti-PRRSV innate immunity is needed. Here, we utilized nine porcine innate immune signaling adaptors which represent all currently known innate immune receptor signaling pathways for screening of anti-PRRSV activity. The analysis of PRRSV N gene transcription and protein expression both suggested that the multiple ectopic adaptors exhibited varying degrees of anti-PRRSV activities, with TRIF and MAVS most effective. To better quantify the PRRSV replication, the GFP signal of PRRSV from reverse genetics were measured by flow cytometry and similarly varying anti-PRRSV activities by different signaling adaptors were observed. Based on the screening data, and considering the importance of viral nucleic acid in innate immune response, endogenous TRIF, MAVS and STING were selected for further examination of anti-PRRSV activity. Agonist stimulation assay showed that MAVS and STING signaling possessed significant anti-PRRSV activities, whereas siRNA knockdown assay showed that TRIF, MAVS and STING are all involved in anti-PRRSV activity, with TLR3-TRIF displaying discrepancy in anti-PRRSV infection. Nevertheless, our work suggests that multiple pattern recognition receptor (PRR) signaling pathways are involved in anti-PRRSV innate immunity, which may have implications for the development of future antiviral strategies. Arteriviridae with a genome of positive-sense single-stranded RNA. PRRSV has a genome approximately 15.4 kb in length and is divided into two species (PRRSV1 and PRRSV2) based on the genomic sequence identity. The genome contains at least 11 open reading frames (ORFs), encoding 16 nonstructural proteins (nsp1-12) and 8 structural proteins [Porcine reproductive and respiratory syndrome (PRRS) is one of the most widespread swine diseases in the world , charactproteins . The ORFThe innate immune system is the first line of defense against infections. Pathogen-associated molecular patterns (PAMPs) are recognized by host germline-encoded pattern recognition receptors, including Toll-like receptors (TLRs), RIG-I-like receptors (RLRs), NOD-like receptors (NLRs), C-type lectin receptors (CLRs), and cytosolic DNA receptors (CDRs) ,5,6. TLRType I IFNs (IFN\u03b1/\u03b2) are the most potent component of innate immunity fighting against invading viruses, including PRRSV . HoweverRegardless of the IFN antagonism by PRRSV, some host factors have been identified as having antiviral functions against PRRSV, including interferon-induced protein with tetratricopeptide repeats 3 (IFIT3) , guanyla2 in a humidified incubator. The PRRSV strain used in this study were highly pathogenic PRRSV XJ17-5 , which we isolated from Xinjiang Province, China [PRRSV-permissive Marc-145 and HEK293T cells were cultured in Dulbecco\u2019s Modified Eagle\u2019s Medium supplemented with 10% fetal bovine serum (FBS) and 100 U/mL of penicillin plus 100 \u03bcg/mL streptomycin. Porcine alveolar macrophages were cultured in RPMI containing 10% FBS with penicillin/streptomycin. All cells were maintained at 37 \u00b0C with 5% COe, China . The virBglII/EcoRI), TRIF (EcoRI/BamHI), MAVS (BglII/KpnI), RIPK2 (BglII/EcoRI), CARD9 (BglII/KpnI), BCL10 (BglII/EcoRI), MALT1 (XhoI/KpnI) and STING (BglII/EcoRI) DNA fragments were cloned into pmCherry-C1 and the expressed proteins carried an N-terminal red fluorescent protein (mCherry). The BglII/KpnI digested ASC DNA fragment was cloned into pmCherry-N1, and the expressed ASC was fused with a C-terminal mCherry.The nine porcine adaptors ,28 we prNcoI and XhoI restriction sites of the pET-28a expression vector, creating the construct pET-28a-ORF7. The protein was expressed in recombinant bacteria DE3/BL21 after 3 h induction with 1 mM isopropyl-b-D-thiogalactopyranoside (IPTG) at 30 \u00b0C. The bacterial pellet was then resuspended in PBS and subjected to sonication and centrifugation. The soluble protein in the supernatant were mixed with loading buffer and run on SDS-PAGE. The gel containing the target protein was excised and processed according to the instructions of the Micro Protein PAGE Recovery Kit . The purified protein was obtained and evaluated for purity and amount by SDS-PAGE. Mice were immunized with 20 \u03bcg purified N protein plus immune adjuvant (1:1) three times, and the polyclonal serum antibody was isolated from eyeball blood. The experiment was carried out in strict accordance with the institutional animal care and use protocol of Yangzhou University.The coding regions of the PRRSV N gene were amplified by PCR from PRRSV cDNA using the primers listed in PacI, AflII, AscI and NotI). The rXJ17-5-EGFP clone was constructed by inserting the EGFP coding sequence between the non-structural and structural genes as previously described [AscI-KpnI-EGFP-BclI-BglII fragment (1.1kb) synthesized was ligated into the AscI and BglII sites of pACYC177-XJ17-5-F3. The F3-EGFP fragment was cut from the positive and sequence confirmed pACYC177-XJ17-5-F3-EGFP by AscI and NotI, and ligated into the same sites of pACYC177-XJ17-5-F1-F2 to generate full-length pACYC177-XJ17-5-EGFP (rXJ17-5-EGFP). The obtained full-length rXJ17-5-EGFP clone was transfected into BHK-21 cells using Lipofectamine 3000 reagent and cell culture supernatants obtained at 48 h post transfection were serially passaged on Marc-145 cells to rescue PRRSV rXJ175-EGFP. The real-time infection of rXJ17-5-EGFP virus was visualized by fluorescence microscopy.To generate an infectious clone of PRRSV carrying the EGFP sequence, the full-genome cDNA clone of the HP-PRRSV XJ17-5 isolate was firstly constructed by the similar strategy as we previously described . Brieflyescribed . The AscAll siRNAs targeting monkey TRIF (GenBank accession: NM_001130428.1), MAVS (GenBank accession: KC415016.1) and STING (GenBank accession: MF622060.1) were designed and synthesized by Invitrogen and the siRNA sequences are listed in \u00ae 1st Strand cDNA Synthesis Kit according to the manufacturer\u2019s instructions, quantitative PCR (qPCR) reaction (20 \u03bcL) was carried out on a StepOne Plus real-time PCR system using ChamQ Universal SYBR qPCR Master Mix (Vazyme) and regular qPCR amplification program. All the gene primers used for qPCR amplification are listed in \u2212\u0394\u0394Ct method.Infected Marc-145 cells were washed three times with PBS, and the total RNAs were extracted using TRIzol reagent . The extracted RNA was reverse transcribed into cDNA with HiScriptCells were collected and lysed in radio-immunoprecipitation assay (RIPA) buffer , and the cellular proteins were separated by 15% SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes. After being blocked with 5% skim milk for 1 h at room temperature, the membranes were incubated with either mouse anti-PRRSV N protein (N) antiserum (1:1000) or other primary antibodies. Membranes were washed three times with TBST, followed by incubation with HRP-conjugated goat anti-mouse or rabbit IgG as the secondary antibody. The protein signal was visualized by Western blot imaging system using an ECL chemiluminescent detection system (Tanon) according to the manufacturer\u2019s instructions.Marc-145 cells were transfected using Lipofectamine 3000 with various adaptor plasmids or stimulated with agonists, and then infected with PRRSV XJ17-5-EGFP. At different time points post infection, cells were collected and analyzed by FACSVerse flow cytometer (BD Biosciences) for EGFP and mCherry expressions. All samples were gated based on forward scatter (FSC) and side scatter (SSC) to gate out cellular debris or dead cells. The levels of EGFP and mCherry were detected using the wavelength pairs 488/525 nm and 510/600 nm, respectively. The final analysis and graphical output were performed using FlowJo software .Marc-145 cells grown in 96-well plates were infected with ten-fold serial dilutions of PRRSV samples. After 1 h incubation at 37 \u00b0C, the supernatants were replaced with fresh DMEM containing 2% FBS. Five days post infection, the cytopathic effect (CPE) characterized by clumping and shrinkage of cells was obviously visible in Marc-145 cells and the viral titers, expressed as 50% tissue culture infective dose (TCID50), were calculated according to the method of Reed\u2013Muench.+ standard deviations (SD) (n = 3). Statistical significance between multiple groups was determined by performing ANOVA analysis with GraphPad Prism 6.0 software. The p value of <0.05 was considered significant.The data are presented as the means We previously cloned and characterized the nine porcine innate immune signaling adaptors which represent all currently known innate immune signaling pathways ,28. HereIn order to better evaluate the antiviral effects of innate immune adaptor proteins, we obtained the GFP-labeled PRRSV virus rescued by reverse genetics and its replication can be measured by flow cytometry A. To ensBased on the screening results and considering the importance of the nucleic acid-sensing signal in the antiviral activity, The nucleic acid signaling related adaptors TRIF, MAVS and STING were chosen and their endogenous signaling were investigated for antiviral roles in PRRSV infection. Several agonists were used to trigger the endogenous innate signaling, including the poly(I:C)-HMW for TLR3-TRIF, poly(I:C)-LMW for RIG-I/MDA5-MAVS, 2\u20323\u2032-cGAMP for STING, and poly(dA:dT) for cGAS-STING. The poly(I:C)-HMW as the agonist for TLR3 when added to cells is able to specifically activate the downstream adaptor TRIF signaling, however, compared with non-stimulated control cells, there was no significant changes of PRRSV infection in poly(I:C)-HMW treated Marc-145 cells as shown by N gene transcription left, A and N pTo further explore the roles of endogenous TRIF, MAVS and STING in the PRRSV replication, the siRNAs were used to knockdown the TRIF, MAVS and STING in Marc-145 cells before PRRSV infection. Three siRNA duplexes were designed for TRIF, MAVS and STING, and knockdown efficiency was examined by RT-qPCR A\u2013D. The Due to the discrepancy of endogenous TRIF in the inhibition of PRRSV replication, we wondered whether there is a defect of poly(I:C)-HMW-TLR3 signaling in Marc-145 cells. The poly(I:C)-HMW was added to the Marc-145 cells to trigger TLR3-TRIF signaling and the downstream gene transcriptions were examined by RT-qPCR. In parallel, the poly(I:C)-LMW was transfected into Marc-145 cells to trigger RLR (RIG-I/MDA5)-MAVS signaling and the downstream gene transcriptions were examined. Indeed, no obvious downstream gene transcriptions of IFN\u03b1, IFN\u03b2, ISG15, IFIT1, IL-8 and TNF\u03b1 were observed in poly(I:C)-HMW-treated Marc-145 cells C,D. The PRRS is an economically important viral disease of swine all over the world; at present, because of the high genomic diversity, antigenic heterogeneity and antibody-dependent enhancement of PRRSV infection, vaccination strategies are obviously ineffective for controlling PRRSV infection . The comTLR signaling is broadly divided into MyD88 and TRIF dependent signaling pathways. All TLRs except TLR3 utilize MyD88 as the signaling adaptor to relay NF-\u03baB signaling, whereas TLR3 and TLR4 utilize TRIF as signaling adaptors and TRIF is the only signaling adaptor for TLR3 ,33. In tRLRs play a critical role in sensing RNA infections by recognition of the cytosolic double stranded RNA from virus infections. Similarly, our results demonstrate that MAVS, which mediates RLR signaling, plays a critical role against PRRSV infection: Both expression of MAVS and poly(I:C)-LMW stimulation exhibited effective anti-PRRSV activity, whereas knockdown of MAVS enhanced PRRSV replication. In fact, the important role of MAVS in PRRSV infection has been previously shown by several studies: The production of zinc finger anti-PRRSV protein, ZAP, was dependent on MAVS ; IFI16 eNLR signaling adaptor RIPK2 is involved in the activation of NF-\u03baB and MAPK , and anoIn conclusion, our data show for the first time that the nine innate signaling adaptor proteins could inhibit PRRSV infection in varying degrees, which is helpful to understand the host antiviral response against PRRSV infection and design anti-PRRSV therapeutics."} +{"text": "Gliomas present a formidable challenge for translational progress. Heterogeneity within and between tumors may demand empirically individualized and adaptive paradigms requiring rapid mechanistic feedback. We asked if tumor-associated metabolic biomarkers from glioma extracellular fluid could impart mechanistic \u201cintelligence\u201d reflecting intra- and inter-tumoral heterogeneity.Five live human gliomas , were evaluated in situ with high molecular weight (100kDA) intraoperative microdialysis using 3 disparately placed catheters. Isotonic 3% dextran perfusate was collected in 20 min (40mL) aliquots. CSF samples (n=21) were additionally evaluated from these and other patients with diverse brain tumors. The IDH-mutant glioma-associated oncometabolite D2-hydroxyglutarate (D2-HG) was quantified with targeted Liquid Chromotography-Mass Spectrometry (LC-MS). Over 200 metabolites were further evaluated via untargeted LC-MS using the Metabolon platform. Correlation, clustering, ROC and enrichment analyses were employed to identify correlations within and between patient samples.CSF samples from patients with IDH-mutant gliomas contained over twenty-fold higher levels of D2-HG compared to those from IDH-wild type tumors . Microdialysate from IDH-mutant gliomas contained 10-953mM D2-HG, 9-63x higher than paired CSF samples. Interestingly, IDH status failed to predict the global metabolic signature of microdialysate. Microdialysate samples clustered into 2 major metabolic phenotype clusters with IDH-WT and IDH-mutant gliomas in each cluster. A superimposed metabolic signature distinguishing enhancing from non-enhancing tumor, was conserved in both patient clusters. Amino acid and carnitine metabolism predominated in microdialysate signatures. TCA cycle and Warburg-associated metabolites were differentially enriched in CSF samples after prior therapy independent of tumor burden.Intra-operative micro-dialysis may complement currently available \u201cintelligence\u201d regarding the phenotype, burden, and metabolism of live human gliomas and is feasible within standard-of-care surgical procedures. Future work will evaluate utility for pharmacodynamic feedback following novel early phase candidate therapies."} +{"text": "Multiple Myeloma (MM) is a clonal plasma cell malignancy characterized by low blood counts and increased risk of infection, and primarily afflicts older adults. Although MM is incurable, advances in treatment, including autologous stem cell transplant (ASCT) has improved the lifespan of patients. MM patients commonly use over-the-counter complementary and alternative medicines (CAM) alongside conventional cancer therapies which, often without recognition by health care practitioners, may impact their treatment. Using data from an 18-month ethnographic study, we applied conversation analysis to examine 1180 minutes of audio-recordings to describe how patients and nurses interacted about CAM during ASCT education visits. Patients (n=12) had a median age of 62 years (IQR= 54-73), were mostly white , male , and had a moderate score on the FACT-G7 of 15 (IQR= 10-20). All patients had a caregiver present during their visit. Nurses (n=3) were aged 39 (IQR= 29-49) all with at least five years providing care to patients with blood cancers. Results suggested that nurses rarely provided direct feedback about CAM modalities, instead providing brief responses, and moving on to other topics. Excerpts were categorized into three groups, (1) demonstration of implicit epistemic authority, (2) demonstration of deferred epistemic authority in patient-initiated conversations, and (3) demonstration of deferred epistemic authority in nurse-initiated conversations. Understanding how conversations surrounding CAM are navigated can provide insights into patient-communication in general, and methods for improving ASCT education."} +{"text": "Porcine reproductive and respiratory syndrome virus (PRRSV) modulates host innate immunity which plays a key role against PRRSV infection. As a RNA virus, PRRSV is mainly sensed by innate immune RNA receptors, whereas the role of innate immune DNA sensors in the PRRSV infection has not been elucidated. Here, we investigated the roles of DNA sensing cGAS-STING pathway in both PRRSV infected Marc-145 cells and porcine macrophages. The results show that in Marc-145 cells, the stable expression of STING with or without stimulations exhibited anti-PRRSV activity, and STING knockout heightened PRRSV infection. In CD163-3D4/21 porcine macrophages, either expression of STING or stimulation of cGAS-STING signaling obviously suppressed PRRSV infection, whereas in STING knockdown macrophages, the PRRSV infection was upregulated. Our results clearly demonstrate that the host cGAS-STING signal exerts an important antiviral role in PRRSV infection. It is covirales , has a govirales . The ORFovirales . The innate immune system acts as the first line of defense against pathogen infection. Pathogen-associated molecular patterns (PAMPs) during infection are recognized by host germline-encoded pattern recognition receptors (PRRs), including Toll-like receptors (TLRs), RIG-I-like receptors (RLRs), NOD-like receptors (NLRs), C-type lectin receptors (CLRs), and cytosolic DNA receptors (CDRs) ,7,8. ThePRRSV has coevolved with host in an intricate and delicate way. On the one hand, PRRSV has evolved a number of strategies to modulate host immune responses, especially antiviral type I IFNs in swine . SpecifiThe cGAS-STING has been defined as the most critical members of CDRs, which plays a key role in sensing DNA virus infection . The DNA2 in a humidified incubator. The PRRSV strain used in this study was highly pathogenic (HP) PRRSV XJ17-5 (GenBank ID: MK759853.1), which was isolated from Xinjiang Province, China, being the representative strain of HP-PRRSV2 predominant in Chinese swine herds [\u00ae LR ClonaseTM II Enzyme mix were from ThermoFisher Scientific polydA:dT and 2\u20323\u2032-cGAMP were bought from InvivoGen .PRRSV-permissive Marc-145 cells were cultured in Dulbecco\u2019s Modified Eagle\u2019s Medium supplemented with 10% fetal bovine serum (FBS) and 100 U/mL of penicillin plus 100 \u03bcg/mL streptomycin. Porcine alveolar macrophages were cultured in RPMI containing 10% FBS with penicillin/streptomycin. All cells were maintained at 37 \u00b0C with 5% COMarc-145 cells were transfected with the plasmid pEGFP-pSTING or vector control pEGFP-C1 using the Lipofectamine 3000 reagent (ThermoFisher Scientific) according to the manufacturer\u2019s protocol. GFP-STING and vector GFP expressing cells were both selected with 800 \u03bcg/mL of G418 diluted in DMEM containing 10% FBS. The medium containing G418 was replenished every 3\u20134 days during selection. After selection, individual cell clones were isolated by a limited dilution method using 96-well cell culture plates, and the cell clones expressing the GFP-STING and vector GFP were chosen and designated as Marc-145-GFP-STING and control Marc-145-GFP, respectively.PacI, AflII, AscI and NotI). The dsRed was PCR amplified from pDsRed-C1 using the primers shown in KpnI and BclI sites of F3 segment of pACYC177-XJ17-5-F3-EGFP prepared in our separate study, where the dsRed gene coding is to replace EGFP coding located between the non-structural and structural genes. Then, the generated new F3 segment was cut off by AscI and NotI from recombinant pACYC177-CMV-XJ17-5-F3, and ligated into the same sites of pACYC177-CMV XJ17-5-F1-F2 to obtain the full length of pACYC177-CMV-XJ17-5 (rXJ17-5-dsRed) [To generate an infectious clone carrying the dsRed encoding sequence, a full-genome cDNA clone of the HP-PRRSV XJ17-5 isolate was firstly constructed as we previously described . Briefly5-dsRed) ,30. The www.benchling.com, accessed on 9 September 2021). Two gRNA 1 and gRNA 2 were chosen according to the predicted scores, and the encoded DNA sequences are shown in BbsI digested vector pSpCas9(BB)-2A-GFP . Subsequently, gRNA expressing pX458 was transfected into Marc-145 cells using Lipofectamine 3000 reagent and the GFP-positive cells were sorted out from transfected cells using a FACS Aria SORP cell sorter (Becton Dickinson). The individual Marc-145 cell clones obtained by limited dilution method from the sorted GFP expressing cells were screened by PCR using the designed primers shown in \u2212/\u2212 and one STING+/\u2212 Marc-145 cell clones were obtained (The CRISPR gRNAs targeting monkey STING (GenBank accession: NC_041759.1) were designed using the web tool from Benchling . The sequence confirmed pCD163-EGFP were transferred from pENTR4 vector to lentiviral vector pLenti CMV Puro DEST (Addgene) by LR recombination. The CD163-EGFP expressing lentiviruses were generated by co-transfecting pCD163-EGFP lentiviral vector with package plasmids psPAX2 and pMD2.G into 293T cells using Lipofectamine 2000 (ThermoFisher Scientific). The supernatant containing pCD163-EGFP expressing lentiviruses were used to infect the porcine alveolar macrophages , and the infected cells were selected with 2 \u03bcg/mL puromycin, by replacing with new medium every 3\u20134 days. About 2 weeks later, individual cell clones were screened for GFP expression and supporting PRRSV replication. The desired cell clone was used for subsequent PRRSV infection experiments.The coding region of the pCD163-EGFP fusion protein was PCR amplified from pEGFP-N1-pCD163 preserved in our lab, using the primer pairs: Forward-All siRNAs targeting porcine STING (mRNA accession number: NM_001142838.1) were designed and synthesized by Invitrogen (ThermoFisher Scientific) and the siRNA sequences are listed in \u00ae 1st Strand cDNA Synthesis Kit according to the manufacturer\u2019s instructions. Quantitative PCR (qPCR) reaction (20 \u03bcL) was performed on a StepOne Plus real-time PCR system using ChamQ Universal SYBR qPCR Master Mix and regular qPCR amplification program. The gene primers used for qPCR amplification are listed in \u2212\u0394\u0394Ct method.PRRSV infected Marc-145 or CD163-PAMs were washed three times with PBS, and the total RNAs were extracted using TRIzol reagent . The extracted RNA was reverse transcribed into cDNA with HiScriptCells were collected and lysed in radio-immunoprecipitation assay (RIPA) buffer . The cleared cell lysates were separated by 15% SDS-PAGE and proteins were transferred onto polyvinylidene difluoride (PVDF) membranes. After being blocked with 5% skim milk for 1 h at room temperature, the membrane was incubated with either mouse anti-PRRSV N protein (Npro) antiserum (1:1000) or other primary antibodies. Next, the membrane was incubated with HRP-conjugated goat anti-mouse or rabbit IgG after TBST washing. The protein signal was visualized by Western blot imaging system using an ECL chemiluminescent detection system according to the manufacturer\u2019s instructions.Marc-145 or CD163-PAMs were transfected or stimulated with agonists, and then infected with PRRSV XJ17-5-dsRed. At different time points post infection, cells were collected and analyzed by FACSVerse flow cytometer for GFP and dsRed expressions. All samples were gated based on forward scatter (FSC) and side scatter (SSC) to gate out cellular debris or dead cells. The levels of GFP and dsRed were detected using the wavelength pairs 488/525 nm and 510/600 nm, respectively. The final analysis and graphical output were performed using FlowJo software .Marc-145 cells grown in 96-well plates were infected with ten-fold serial dilutions of PRRSV samples. After 1 h incubation at 37 \u00b0C, the supernatants were replaced with fresh DMEM containing 2% FBS. Five days post infection, the cytopathic effect (CPE) characterized by clumping and shrinkage of cells was obviously visible in Marc-145 cells and the viral titers, expressed as 50% tissue culture infective dose (TCID50), were calculated according to the method of Reed-Muench .n = 3). Statistical significance between groups was determined by performing a Student t test with GraphPad Prism 6.0 software. The p value of <0.05 was considered statistically significant.The data are presented as the means \u00b1 standard deviations control stable Marc-145 cells, both of which have GFP signal maintained after G418 selection A. To quaThe rPRRSV-dsRed infection of GFP-STING and GFP control stable Marc-145 cells were quantified by flow cytometry, and the results show that either dsRed signal or dsRed/GFP double signals were decreased in GFP-STING cells relative to GFP vector control cells, in particular at 48 h post infection (hpi), indicating the antiviral role of STING A. SimilaFurther, the stable Marc-145 cells were stimulated with poly (dA:dT) and 2\u20323\u2032-cGAMP to activate cGAS-STING and STING, respectively, before PRRSV infection. Both agonists inhibited the PRRSV replication as evidenced by Western-blotting E and TCIPreviously, we were able to observe the upregulated PRRSV replication in STING siRNA treated Marc-145 cells in another study. Here, we further made the homologous STING knockout Marc-145 cell clones by CRISPR-Cas9 method. Four homologous STING knockout cell clones were obtained and two cell clones (2-7-1 and 1-4-2) were selected for subsequent experiments . CompareTo investigate the anti-PRRSV role of STING in porcine macrophages, we developed CD163-PAMs (3D4/21), which stably express GFP tagged porcine CD163 and successfully support PRRSV replication. First, the CD163-PAMs were transfected with porcine STING and infected with PRRSV. The results from the experiment show that ectopic STING suppressed the N gene transcription in RT-qPCR A, N protSecond, the CD163-PAMs were stimulated with poly (dA:dT) and 2\u20323\u2032-cGAMP to activate cGAS-STING and STING, respectively, before PRRSV infection. The results show that both agonists suppressed the PRRSV infection, at 48 and 72 hpi, as evidenced by N gene transcription in RT-qPCR A, N protThird, the CD163-PAMs were treated with STING siRNA to knockdown STING for subsequent PRRSV infection. To this end, three pairs of STING siRNAs were tested for the knockdown efficiency by RT-qPCR, and the most effective siRNA 529 was selected for the next experiments D. In STIThe complex interaction between PRRSV and host immune response remains to be fully understood . SpecifiFlaviviridae and Coronaviridae have been shown to be restricted by a cGAS and/or STING signaling, and on the other side, the RNA viral proteins can function as antagonists of cGAS-STING pathway, which has been summarized in review [Caliciviridae [Paramyxovirdae [Togaviridae [Arteriviridae to the list.Although the role of cGAS-STING in sensing of DNA viruses is clearly elucidated, many RNA viruses of families n review . More reiviridae , nipah vxovirdae , chikungaviridae . Here, wPRRSV replicates within the cytoplasm using its own RNA-dependent RNA polymerase (nsp9) and its replication cycle does not rely on a DNA intermediate step . While cSince PRRSV infection is sensed and controlled by multiple innate immune signaling pathways, it would raise several questions. First, how much is the relative contribution of these individual signaling pathways in sensing and controlling PRRSV replication during infection? Currently, this is still unknown even though our studies suggested RLR-MAVS is likely the major sensor and antiviral pathway. To address the question, the individual receptors and/or signaling adaptors need to be knockout from cells and then PRRSV replication be examined and compared with each other. Second, what are the interrelationships between these different anti-PRRSV innate immune signaling pathways? Are they separate, cooperative or antagonizing? This information can be obtained by investigation of the PRRSV infection of various knockout cells and the anti-PRRSV activity by different signaling agonists. Such information will be critical for guidance and the design of anti-PRRSV combo therapeutics.PRRSV functional pathogenesis may vary depending on the PRRSV genotypes. In China, the epidemiological studies including ours showed that highly pathogenic (HP)-PRRSV2 (lineage 8) has been the predominant genotype, whereas the NADC30-like PRRSV2 (lineage 1) isolates increasingly appear in recent years, and QYYZ-like PRRSV2 (lineage 3), CH-1a-like PRRSV2 (lineage 5) and PRRSV1 isolates also coexist in Chinese swine herds . HP-PRRS"} +{"text": "B-cell lymphoma-extra large (Bcl-xL) is an anti-apoptotic protein that regulates energy metabolism in neurons. In this study, we found that primary hippocampal neurons transduced with Bcl-xL shRNA or treated with a pharmacological inhibitor of Bxl-xL had a decrease in the population of motile mitochondria. Primary hippocampal neurons lacking Bcl-xL failed to retain ATP at their neurites, which hindered the formation of complex neurite arbors, and ultimately had enhanced vulnerability to excitotoxic challenge.B-cell lymphoma-extra large (Bcl-xL) is a mitochondrial protein known to inhibit mitochondria-dependent intrinsic apoptotic pathways. An increasing number of studies have demonstrated that Bcl-xL is critical in regulating neuronal energy metabolism and has a protective role in pathologies associated with an energy deficit. However, it is less known how Bcl-xL regulates physiological processes of the brain. In this study, we hypothesize that Bcl-xL is required for neurite branching and maturation during neuronal development by improving local energy metabolism. We found that the absence of Bcl-xL in rat primary hippocampal neurons resulted in mitochondrial dysfunction. Specifically, the ATP/ADP ratio was significantly decreased in the neurites of Bcl-xL depleted neurons. We further found that neurons transduced with Bcl-xL shRNA or neurons treated with ABT-263, a pharmacological inhibitor of Bcl-xL, showed impaired mitochondrial motility. Neurons lacking Bcl-xL had significantly decreased anterograde and retrograde movement of mitochondria and an increased stationary mitochondrial population when Bcl-xL was depleted by either means. These mitochondrial defects, including loss of ATP, impaired normal neurite development. Neurons lacking Bcl-xL showed significantly decreased neurite arborization, growth and complexity. Bcl-xL depleted neurons also showed impaired synapse formation. These neurons showed increased intracellular calcium concentration and were more susceptible to excitotoxic challenge. Bcl-xL may support positioning of mitochondria at metabolically demanding regions of neurites like branching points. Our findings suggest a role for Bcl-xL in physiological regulation of neuronal growth and development. B-cell lymphoma-extra large (Bcl-xL) is an anti-apoptotic member of the Bcl2 protein family . Bcl-xL Mitochondria are dynamic organelles that change location, population, and morphology, and alterations of mitochondrial dynamics contribute to the loss of neuronal function ,17,18,19In this study, we investigated whether Bcl-xL is required during the development of primary hippocampal neurons. Neurons lacking Bcl-xL demonstrated impaired mitochondrial movement in both the anterograde and retrograde directions and were associated with local energy depletion at neurites. Failure of ATP retention at neurites impaired neurite arborization and increased susceptibility to excitotoxic challenges. We suggest that Bcl-xL is an important contributor to the physiologic development of neurite complexity by supporting neurite energy retention.6 cells/35 mm plate) were seeded on poly-l-lysin coated plates and grown in a neurobasal medium supplemented with B-27, glutamine, and antibiotics for 21 days in vitro (DIV). shRNA lentiviral particle transduction: Primary hippocampal neurons were transduced with control shRNA lentiviral particles or Bcl-xL shRNA lentiviral particles ; copGFP control lentiviral particles or Bcl-xL shRNA-GFP lentiviral particles (Santa Cruz Biotechnology) at DIV 7. Glutamate treatment: Glutamate was freshly prepared in sterile PBS (Thermo Fisher Scientific) and added to the cell culture medium . The vehicle control group for the glutamate experiment was treated with isovolumetric sterile PBS. ABT-263 treatment: ABT-263 was prepared in dimethyl sulfoxide (DMSO) and added to the cell culture medium . The vehicle control group for ABT-263 was treated with isovolumetric DMSO. The protocol was approved by the Institutional Animal Care Committee (IACUC) of the University of Alabama, Tuscaloosa, AL, USA.Primary rat hippocampal neurons were prepared from rat feti as described previously ,32,33,34TM mitochondria-RFP and image sequences were generated via fluorescent imaging with images taken every 60 s over a 60 min period. Image sequences were then used to generate kymographs using the KymoAnalyzer 1.01 software for ImageJ [Mitochondria were labeled with CellLightr ImageJ . To genePrimary hippocampal neurons transduced with GFP control lentiviral particles or Bcl-xL shRNA-GFP lentiviral particles (Santa Cruz Biotechnology) were imaged with a Zeiss Axio Vert.A1 microscope . Sholl analysis was used to quantify neurite branches as previously described ,37. In b488nm/F405nm. Pseudo-color ratiometric images were made using NIS-Elements 5.11 with the Ratio View Live Ratio Graphing Module (Nikon) [Primary hippocampal neurons were transfected at DIV7 with GW1-PercevalHR, a sensor developed in Gary Yellen\u2019s laboratory ,39. NeurMitochondrial membrane potential (\u0394\u03c8) was measured using the fluorescent lipophilic cationic dye tetramethylrhodamine methyl ester (TMRM) , which accumulates within mitochondria in a potential-dependent manner ,42. PrimNeurons were scraped and lysed in 1X cell signaling buffer and protein concentration was determined using BCA protein reagents . Samples (50\u2013100 \u00b5g of protein/lane) were separated on a 4\u201312% SDS-polyacrylamide gel and probed with anti-Bcl-xL antibody and anti-beta actin . Scanned images were analyzed using ImageJ software . Viable or dead cells were stained with Calcein-AM or PI as previously described . After tPrimary hippocampal neurons were treated with 2.5 \u00b5M Fluo-4 (Invitrogen) solution prepared in a light-protected vessel, then incubated for 30 min at 37 \u00b0C as per the manufacturer\u2019s protocol . MicrogrPrimary hippocampal neurons fixed in 10% buffered formalin were blocked in 10% goat serum for 1 h, then incubated with anti-Bassoon , anti-synaptophysin , anti-MAP2 antibodies overnight at 4 \u00b0C. Cells were washed and incubated with Alexa-fluor 488 antibody or Alexa-568 antibody for 1 h at room temperature and mounted on glass slides. Images were taken with a Zeiss Axiovert A1 microscope and processed using AxioVision 4.9.p < 0.05 was considered statistically significant. p values are provided in figure legends.Data are reported as the mean \u00b1 SEM of at least three independent cultures with multiple independent experimental designs, such as independent neuronal isolation, independent performance dates, and independent plates within a culture using separately prepared reagents. All quantitative graphs were made from at least three independent neuronal isolations. 1Fo ATP synthase [Bcl-xL is reported to improve neuronal energy metabolism by enhancing mitochondrial ATP production via direct interaction with the Fsynthase ,13. Howesynthase , thus nesynthase . The proTo determine whether Bcl-xL is required to localize mitochondria at neurites, we monitored mitochondrial motility using neurons with or without expressing Bcl-xL. Primary hippocampal neurons were transduced with control shRNA or Bcl-xL shRNA followed by mitochondrial labeling using mito-RFP BacMam2.0. Image sequences visualizing mitochondrial movement were converted to kymographs A, and moBcl-xL depletion affected the amount of mitochondrial movement. We, therefore, tested if Bcl-xL depletion affected the density of mitochondria in the neurite. Primary hippocampal neurons expressing Bcl-xL also displayed higher densities of moving mitochondria and number of motile mitochondria per micrometer of neurite than Bcl-xL depleted neurons H\u2013J. The Furthermore, we applied a pharmacological approach to target Bcl-xL Primary hippocampal neurons were treated with or without 1 \u00b5M ABT-263, an inhibitor of Bcl-xL. As previously described, kymographs were generated A and theTMRM fluorescence was also analyzed in ABT-263-treated neurons to measure mitochondrial inner membrane potential (\u0394\u03c8) H\u2013J. ABT-We have previously shown that Bcl-xL is necessary for neurite outgrowth during the development of primary hippocampal neurons . To testWe then further tested to see if Bcl-xL depletion impacted synapse formation. Immunocytochemistry was performed on primary hippocampal neurons C,D to viAlteration of neurite morphology does not cause the immediate death of developing neurons; however, it does increase the vulnerability of neurons during maturation . To deteNeurons were then labeled with calcein-AM, to indicate live, healthy cells, and Hoechst, to show all cells present. Consistent with our previous report , treatmeBcl-xL is traditionally known as a blocker of apoptotic death via its ability to bind pro-apoptotic proteins. In this study, we found an additional function of Bcl-xL under physiological conditions. Depletion of Bcl-xL significantly decreased the population of motile mitochondria in primary hippocampal neurons, and this may hinder the delivery of mitochondria, impairing local energy metabolism at neurites. Indeed, we found that neurons transduced with Bcl-xL shRNA failed to maintain mitochondrial inner membrane potential and depleted ATP at their neurites, arresting arborization of neurites compared to neurons expressing Bcl-xL. Although Bcl-xL depletion did not immediately change neuronal viability, failure to achieve neurite complexity eventually increased the vulnerability of neurons against excitotoxic challenge.1Fo ATP synthase [1Fo ATP synthase enhances the efficiency of ATP production by blocking a leak channel activity. Although there are several candidates to form mitochondrial permeability transition pore (mPTP), the c-subunit of the F1Fo ATP synthase, a ring-shaped subunit found in the Fo complex, exhibits non-selective voltage-gated and calcium-activated channel activities resembling mPTP [1Fo ATP synthase showed mPTP-like activities, whereas treatment with oligomycin A closed the channel [1Fo ATP synthase in neurons lacking Bcl-xL may lead to failure of intracellular transport. Additionally, we found that primary hippocampal neurons expressing Bcl-xL have approximately 22% motile and 78% stationary mitochondrial populations, whereas neurons lacking Bcl-xL maintained only 7% motile mitochondria (1Fo ATP synthase.Bcl-xL regulates the efficiency of ATP production in mitochondria via direct interaction with the Fsynthase ,13. In psynthase . Interacing mPTP ,47,48. A channel ,50,51, ichondria F,G. Conschondria A,B. SincPreviously, we reported that depletion of Bcl-xL does not directly cause neuronal death under physiological conditions, but neurons lacking Bcl-xL show significantly altered neurite morphology leading to death during hypoxic insult . SimilarIn this study, we showed that full-length Bcl-xL is required for neurite development. Bcl-xL supports local energy retention at neurites and enhances both the efficiency of ATP production and the recruitment of mitochondria to intracellular locations. We suggest a physiological function of Bcl-xL during neuronal development and under neurotoxic challenge."} +{"text": "Here we report the case of concomitant favorable-risk prostate cancer and Hodgkin Lymphoma in a 38-year old male. 68Ga-Prostate Specific Membrane Antigen-11 Positron Emission Tomography/Computed Tomography (68Ga-PSMA-11 PET/CT) was performed for staging purposes, showing the focal PSMA prostatic uptake as well as the presence of enlarged low-PSMA expressing mediastinal lymphadenopathies, thus raising the suspicion of another malignancy. A subsequent 18F-Fluorodeoxyglucose (18F-FDG) PET/CT demonstrated a high FDG-avidity by mediastinal lymphadenopathies as opposed to the low prostate cancer FDG uptake. Of note, both tumor entities were clearly detected by the two scans. However, different ranges in terms of Maximum Standardized Uptake Value (SUVmax) uptake allowed the discrimination between the two tumor entities. At the subsequent mediastinal lymph nodal biopsy, the coexistence of Hodgkin lymphoma was documented. The present case suggests that even if specific for prostate cancer, 68Ga-PSMA-11 PET/CT may raise the suspicion of other concurrent malignancies thanks to its non-receptor bounding mechanism. Further, it shows that in certain cases, the combination of 18F-FDG and 68Ga-PSMA PET/CT imaging may non-invasively guide the clinical management, optimizing the diagnostic process and the subsequent therapeutic interventions. A 38-year-old man was referred to our Institution in May 2020 for the diagnostic workup for weight loss, asthenia, and Prostate-Specific Antigen (PSA) elevation (10.49 ng/mL). A right lobe prostatic adenocarcinoma (Gleason score 3 + 3) was diagnosed after pelvic Magnetic Resonance Imaging (MRI) and fusion biopsy. Given the favorable risk, the active surveillance strategy was started [The present case represents an excellent demonstration of lesion characterization using molecular imaging, as the two different PET-tracers provided complementary information able to guide the diagnostic workup leading to a prognosis-priority treatment plan. PET/CT imaging with PSMA-labelled radiotracers is influencing more and more the clinical practice in the initial staging of prostate cancer, given its superior sensitivity, specificity and diagnostic accuracy compared to the standard of care (bone scan and CT) and compared to other PET tracers ,5,6. HowOnly a few cases available in the literature previously described a combined 68Ga-PSMA-11 and 18F-FDG PET/CT approach in patients with concurrent prostate cancer and follicular lymphoma ,14,15,16Even if specific for prostate cancer, 68Ga-PSMA-11 PET/CT may raise the suspicion of other con-current malignancies thanks to its non-receptor bounding mechanism. In certain cases, the combination of 18F-FDG and 68Ga-PSMA-11 PET/CT imaging may non-invasively guide the clinical management, optimizing the diagnostic process and the subsequent therapeutic interventions."} +{"text": "Pleurotus sajor-caju GAPDH) in response to abiotic stress, we generated transgenic rice plants with single-copy/intergenic/homozygous overexpression PsGAPDH (PsGAPDH-OX) and investigated their responses to salinity stress. Seedling growth and germination rates of PsGAPDH-OX were significantly increased under salt stress conditions compared to those of the wild type. To elucidate the role of PsGAPDH-OX in salt stress tolerance of rice, an Illumina HiSeq 2000 platform was used to analyze transcriptome profiles of leaves under salt stress. Analysis results of sequencing data showed that 1124 transcripts were differentially expressed. Using the list of differentially expressed genes (DEGs), functional enrichment analyses of DEGs such as Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were performed. KEGG pathway enrichment analysis revealed that unigenes exhibiting differential expression were involved in starch and sucrose metabolism. Interestingly, trehalose-6-phosphate synthase (TPS) genes, of which expression was enhanced by abiotic stress, showed a significant difference in PsGAPDH-OX. Findings of this study suggest that PsGAPDH plays a role in the adaptation of rice plants to salt stress.In plants, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a main enzyme in the glycolytic pathway. It plays an essential role in glycerolipid metabolism and response to various stresses. To examine the function of PsGAPDH ( Oryza sativa) is a major staple food crop in the world. It is also a model plant for the genomics research of monocotyledons . We. WePsGAPce lines C. SinglePsGAPDH expression, the distribution of PsGAPDH in maize and Arabidopsis protoplasts was examined. PsGAPDH\u2013GFP proteins were transiently expressed in protoplasts isolated from leaves using a polyethylene glycol (PEG)-mediated transfection system [GAPDH catalyzes the conversion of glyceraldehyde-3-phosphate to 1,3-bisphospho glycerate. Both cytosolic (GAPCs) and plastidial (GAPCps) GAPDH activities have been described in plants. To determine the subcellular localization of n system . A fusiop < 0.05) on MS media supplemented with 100 mM or 200 mM NaCl of NaCl. Their germination rates and seedling growths were monitored. There was no significant difference between the wild-type and PsGAPDH-OX cultured in MS medium without NaCl . However mM NaCl . These rBased on quantified gene expression, relationships among samples and stages were investigated using the MDS method. Multi-dimensional plot indicated that samples could be distinguished by transgenic rice . After cp-value < 0.05 (The top 10 down-regulated DEGs and top 10 up-regulated DEGs matched with Oryzabase gene symbols and Oryzabase gene name synonym(s) with an adjusted e < 0.05 . Typicale < 0.05 . The heae < 0.05 . A totaltrehalose-6-phosphate synthase (TPS) gene for transferring Gluc-6P to T6P was down-regulated, whereas the trehalose-6-phosphate phosphatase (TPP) gene for transferring T6P to trehalose was up-regulated genes, OsTPS1 (Os11t0156600) and OsTPs5 (Os02t0790500), and four for trehalose-6-phosphate phosphatase (TPP) genes, OsTPP6 (Os08t0409100), OsTPP3 (Os09t0332100), OsTPP4 (Os02t0753000), and OsTPP9 (Os03t0386500). Expression levels of OsTPS1 and OsTPS5 genes were decreased in PsGAPDH-OX plants compared to those in the wild-type. Furthermore, the transcript levels of the TPP genes were significantly induced and trehalose-6-phosphate phosphatase (TPP). Among various osmolytes, trehalose (\u03b1-D-glucopyranosyl-\u03b1-D-glucopyranoside), a non-reducing disaccharide, is a principal compatible solute and a potential signal metabolite in plants. In plants, there is only one pathway for trehalose biosynthesis. It consists of a two-step process involving TPS and TPP that synthesize and subsequently dephosphorylate trehalose-6-phosphate (T6P) as an intermediate. Previous studies have shown that genetic modification of the TPS/TPP gene can increase stress resistance in different species [OsTPP1 can improve the resistance to salt and cold. It can also trigger the expression of a series of abiotic response genes, thus increasing the stress tolerance of rice [TPS/TPP genes are directly involved in stress tolerance by improving trehalose contents in several plants [Our RNA sequencing analysis data indicated that overexpression of species ,43. Hete species . Overexp of rice . Previoul plants .PsGAPDH gene might participate in salt tolerance through coordination with these responsive genes. Hence, we speculate that PsGAPDH is possibly involved in various cellular processes in addition to the metabolism of starch and sucrose. Taken together, our findings suggest that PsGAPDH plays an important role in the metabolism of starch and sucrose during the adaptation of plants to salt stress.Our results suggest that the"} +{"text": "Neurospora crassa, expression from an unpaired gene is suppressed bya mechanism known as meiotic silencing by unpaired DNA (MSUD). MSUD utilizes common RNAinterference (RNAi) factors to silence target mRNAs. Here, we report thatNeurospora CAR-1 and CGH-1, homologs of two Caenorhabditiselegans RNA granule components, are involved in MSUD. These fungal proteins arefound in the perinuclear region and P-bodies, much like their worm counterparts. Theyinteract with components of the meiotic silencing complex (MSC), including the SMS-2Argonaute. This is the first time MSUD has been linked to RNA granule proteins.In Neurospora crassa grows as an interconnected network of tubular cells(hyphae). Because the cross walls between individual cells are usually incomplete, virusesand transposons can potentially infiltrate the entire fungal colony. To defend against theseinvasive elements, Neurospora maintains several genome surveillance systems are cytoplasmic granulesassociated with the regulation of RNA translation, storage, and degradation . Althougor decay . In addior decay . In thishttp://www.fgsc.net/Neurospora/NeurosporaProtocolGuide.htm). Genotypes ofNeurospora strains used are provided in Standard fungal protocols were followed throughout this study and sexual (SRR957218) RNA-seq datasets were obtainedfrom the European Bioinformatics Institute (EBI)\u2019s European Nucleotide Archive (ENA) strains were grown for six days in 24-wellmicroplates (Corning #3524) at room temperature for use as designated females. Conidia from each male strain were suspended in sterile water and adjusted to aconcentration of 1000 counts/\u00b5l. For fertilization, 50\u2009\u00b5l conidial suspension wasinoculated on the female strain within a well. Ascospores were collectedfrom the lids 21 days post-fertilization and counted on a hemocytometer.cgh-1-null crosses, sexual development is too impaired for proficientspore shooting. Accordingly, spores from these crosses were extracted out of the fruitingbodies for progeny phenotyping.Most assessments of MSUD proficiency were performed according to the method of Green fluorescent protein (GFP) and mCherry tagging vectors were constructed usingdouble-joint polymerase chain reaction are brought together by the association oftwo interacting proteins (Bimolecular fluorescence complementation (BiFC) is an Light microscopy images of protoperithecia , perithecia (fruitingbodies), and asci (spore sacs) were captured according to the methods of P-values werecalculated using the two-tailed Student\u2019s t-test.Images of asci in the single-nucleus (diploid) stage were analyzed with Fijiv2.0.0-rc-69-1.52p and tagged them with GFP. As seen in CAR-1 (Sm-domain protein) and CGH-1 (RNA helicase) family proteins are conserved P-bodycomponents . InCaenNeurospora CAR-1 and CGH-1 colocalize .CAR-1 and CGH-1 family proteins are known to have direct or indirect interaction witheach other . Becauselocalize , we testlocalize . It is pcar-1 andcgh-1, we examined their transcript levels through RNA-seq datasets.While presumed MSUD-exclusive genes have relatively low vegetative expression , car-1 and cgh-1 areabundantly expressed in both developmental stages (To determine the expression profiles of l stages . These rcar-1 or cgh-1 homolog in yeast(SCD6/DHH1) is not lethal to the fungus also leads to severe mating defects . The latter localization brings to mindthe case in Using BiFC, we have shown that CGH-1 has interaction with the SMS-2 Argonaute. This issimilar to the situation in human cells, in which a CGH-1 homolog (RCK) interacts withArgonaute proteins . Future work on these and other RNA granule components will shed lighton how they regulate gene expression and diverse processes.Although many components of the meiotic silencing machinery have been revealed, the finalfates of MSUD-targeted mRNAs remain unclear. Our current model proposes that the SMS-2Argonaute uses siRNAs to guide the slicing of complementary mRNAs (presumably in theperinuclear region). It is conceivable that targeted mRNA slicing is not 100% efficient andthat unsliced (and possibly sliced) mRNAs are associated with certain RNA granule proteinsfor degradation, repression, and/or storage. CAR-1 and CGH-1 family proteins are presumed tobe translation repressors and/or decapping activators (https://doi.org/10.25387/g3.14575689.Strains are available upon request. Supplementary material is available at figshare:"} +{"text": "In order to mitigate the spread of SARS-CoV-2 and the COVID-19 pandemic, public health officials have recommended self-isolation, self-quarantine of exposed household contacts (HHC), and mask use to limit viral spread within households and communities. While household transmission of SARS-CoV-2 is common, risk factors for HHC transmission are poorly understood.In this prospective cohort study, we enrolled 37 households with at least one reverse transcription polymerase chain reaction-confirmed (RT-PCR) COVID-19 index case from March 2020 - March 2021, in order to calculate secondary attack rates (SAR) and define risk factors for secondary infections. Participants were tested daily for SARS-CoV-2 via RT-PCR, using self-collected lower nasal samples. Households were followed until all members tested negative for seven consecutive days. We collected demographics, medical conditions, relationship to index case, and socioeconomic indicators. Subgroup data analysis was conducted and stratified by positivity status.Of 99 enrolled participants, 37 were index cases and 62 were household contacts (HHC), of whom 25 HHC were infected (40.3%). Secondary attack rate (SAR) was highest among adults caring for a parent and parents of index cases . Households whose income came from service work had greater risk of transmission compared to households whose primary income was technology . Pediatric contacts were at lower risk of infection when compared to adult contacts .This study suggests that household transmission represents a key source of community-based infection of SARS-CoV-2. Allocating resources for education/training regarding prevention among infected individuals and their close contacts will be critical for control of future outbreaks of SARS-CoV-2.All Authors: No reported disclosures"} +{"text": "PRODH/POX (proline dehydrogenase/proline oxidase) is a mitochondrial enzyme that catalyzes proline degradation generating reactive oxygen species (ROS). Estrogens limit proline availability for PRODH/POX by stimulating collagen biosynthesis. It has been considered that estrogens determine efficiency of troglitazone (TGZ)-induced PRODH/POX-dependent apoptosis in breast cancer cells. The studies were performed in wild-type and PRODH/POX-silenced estrogen-dependent MCF-7 cells and estrogen-independent MDA-MB-231 cells. DNA and collagen biosynthesis were determined by radiometric method, ROS production was measured by fluorescence assay, protein expression was determined by Western blot and proline concentration by LC/MS analysis. We found that: i/TGZ-induced apoptosis in MDA-MB-231 occurs only in the absence of estradiol or ER\u03b2, ii/the process is mediated by PRODH/POX, iii/and is facilitated by proline availability for PRODH/POX by TGZ-dependent inhibition of collagen biosynthesis (proline utilizing process). The data suggest that combined TGZ and anti-estrogen treatment could be considered in experimental therapy of ER negative breast cancers.The impact of estradiol on troglitazone (TGZ)-induced proline dehydrogenase/proline oxidase (PRODH/POX)-dependent apoptosis was studied in wild-type and PRODH/POX-silenced estrogen receptor (ER) dependent MCF-7 cells and ER-independent MDA-MB-231 cells. DNA and collagen biosynthesis were determined by radiometric method, prolidase activity evaluated by colorimetric method, ROS production was measured by fluorescence assay. Protein expression was determined by Western blot and proline concentration by LC/MS analysis. PRODH/POX degrades proline yielding reactive oxygen species (ROS). Estrogens stimulate collagen biosynthesis utilizing free proline and limiting its availability for PRODH/POX-dependent apoptosis. TGZ cytotoxicity was highly pronounced in wild-type MDA-MB-231 cells cultured in medium without estradiol or in the cells cultured in medium with estradiol but deprived of ER\u03b2 (by ICI-dependent degradation), while in PRODH/POX-silenced cells the process was not affected. The TGZ cytotoxicity was accompanied by increase in PRODH/POX expression, ROS production, expression of cleaved caspase-3, caspase-9 and PARP, inhibition of collagen biosynthesis, prolidase activity and decrease in intracellular proline concentration. The phenomena were not observed in PRODH/POX-silenced cells. The data suggest that TGZ-induced apoptosis in MDA-MB-231 cells cultured in medium without estradiol or deprived of ER\u03b2 is mediated by PRODH/POX and the process is facilitated by proline availability for PRODH/POX by TGZ-dependent inhibition of collagen biosynthesis. It suggests that combined TGZ and antiestrogen treatment could be considered in experimental therapy of estrogen receptor negative breast cancers. Troglitazone is a member of thiazolidinediones (TZD), the new class of antidiabetic drugs ,2 and agPPAR-\u03b3 is expressed in different types of cancer cells, including breast cancer cells. Activation of the receptor was found to attenuate cell growth and induce cell death . AnotherWe have come up with the assumption that the mechanism of PPAR-\u03b3 dependent apoptosis could involve PRODH/POX, a mitochondrial membrane associated enzyme that catalyzes proline degradation into \u03941-pyrroline-5-carboxylic acid (P5C). During this reaction, electrons are transferred via flavin adenine dinucleotide to cytochrome c in the respiratory chain producing ATP. However, when electrons are transferred directly to oxygen then the reactive oxygen species (ROS) are formed inducing apoptosis or autophagy ,8,9. AltEstrogen receptor (ER) activation is considered an important factor in breast cancer progression . In factPRODH/POX is also up-regulated by AMP-activated protein kinase (AMPK) . This spAs TGZ induces PRODH/POX , and estThe link between estrogens, collagen biosynthesis and degradation, PRODH/POX, PPAR-\u03b3 and apoptosis/survival led us to evaluate the impact of estrogen receptor activation on the above processes and PRODH/POX-dependent apoptosis in MCF-7 and MDA-MB-231 breast cancer cells as is outlined on the 2. At about 80% of confluency the cells were treated for 24 h with estradiol , troglitazone or both compounds in DMEM without phenol red supplemented with 10% CPSR1.MCF-7 and MDA-MB-231 cells were obtained from ATCC . PRODH/POX-silenced MCF-7 and MDA-MB-231 cells were obtained as we descripted previously . MCF-7 c3H]-thymidine incorporation into DNA as described previously [3H]-thymidine. Incorporation of the tracer into DNA was measured by Liquid Scintillation Analyzer Tri-Carb 2810 TR and calculated using Quanto Smart TM software.DNA biosynthesis was evaluated by [methyl-eviously . The cel3H]-proline (5 \u03bcCi/mL) incorporation into proteins susceptible to bacterial collagenase. The cells were cultured in the presence of tracer in medium with or without estradiol (E) and troglitazone (TGZ) for 24 h. Incorporation of tracer into collagen was measured in accordance to the method of Peterkofsky et al. [Collagen biosynthesis was evaluated by 5[y et al. . IncorpoThe activity of prolidase was determined according to the method of Myara et al. . The celProtein analysis was performed by Western blot as previously described ,29. CellProline concentration was measured according to the method of Klupczynska et al. . Briefly\u00ae M200 PRO . The results were presented as a percent of the control value.Intracellular reactive oxygen species accumulation was measured using DCFH-DA as a fluorescent probe. Briefly, cells were pre-incubated with DCFH-DA (20 \u00b5M) in culture medium for 30 min, washed twice with PBS and treated with increasing concentrations of for 24 h with estradiol , troglitazone or both compounds in DMEM without phenol red. The fluorescent intensity was measured at excitation/emission wavelength of 488/535 nm using TECAN Infinitep < 0.01 level and are denoted by an asterisk (*).In the experiments presented in ER-positive, MCF-7 cells (expressing \u03b1 and \u03b2 estrogen receptor), wild-type (wt of MCF-7 cells) and PRODH/POX-silenced (shPRODH/POX MCF-7 cells), and ER-negative, MDA-MB 231 cells (expressing only \u03b2 estrogen receptor), wild-type (wt of MDA-MB-231 cells) and PRODH/POX-silenced (shPRODH/POX MDA-MB-231 cells), were used to study the effect of estrogen receptor activation on the PRODH/POX-dependent apoptosis and related processes. Preparation of PRODH/POX-silenced MCF-7 and MDA-MB-231 cells were described previously ,29. The TGZ inhibits DNA biosynthesis in both MCF-7 A,B and MTGZ induces PRODH/POX expression in both wild type breast cancer cells independently of the presence or absence of estradiol A,B. In PTGZ did not significantly affect ROS production in both lines of MCF-7 cells cultured in the presence or absence of estradiol C,D and iTGZ induced expression of cleaved caspase-3, caspase-9 and PARP in both cell lines (MCF-7 and MDA-MB-231) cultured in the presence or absence of estradiol A,B. HoweIt suggests that TGZ-dependent apoptosis in breast cancer cells is highly pronounced in ER\u03b2 expressing MDA-MB-231 cells cultured in estradiol free medium.PRODH/POX-induced apoptosis (through ROS generation) in breast cancer cells is dependent on proline availability. Intracellular free proline content is regulated mainly by collagen biosynthesis (proline utilizing process) and prolidase activity (proline releasing enzyme). In wild type and PRODH/POX-silenced MCF-7 cells cultured in medium containing estradiol, TGZ induced dose-dependent increase in proline concentration A and inhIn both wild type and PRODH/POX-silenced MDA-MB-231 cells cultured in medium containing estradiol, TGZ contributed to decrease in proline concentration A, collagThis study investigated the role of estrogen receptor activation on troglitazone (TGZ)-induced PRODH/POX -dependent apoptosis in four models of breast cancer cells. Although cell line models have some limitations , they are a powerful tool which offer several advantages. Certainly, the cell models allow strict control of the conditions of the experiment in order to establish the critical factor affecting the studied processes. They are especially helpful in case of limited availability of clinical samples or in vivo models . Therefore, results on cell models allow to predict the consequences of pharmacotherapeutic manipulation in human. Different treatment regimens and combinations of therapies have been tested using cell lines which have yielded interesting and potentially promising results that currently have an application value ,36. In tThe data presented in this report suggest that TGZ-induced PRODH/POX-dependent apoptosis is conditioned by the status of the ERs in breast cancer cells. In MDA-MB-231 cells cultured in medium without estradiol or deprived of ER\u03b2, TGZ induces PRODH/POX-dependent apoptosis and the process is facilitated by proline availability for PRODH/POX by TGZ-dependent inhibition of collagen biosynthesis. The underlying mechanism involves activation of PRODH/POX that under availability of proline (substrate for PRODH/POX) and dependently on the ER status (absence of estradiol or ER\u03b2) induces apoptosis in ER negative breast cancer cells. The role of PRODH/POX in this process was confirmed by experiments showing that in PRODH/POX silenced cells apoptosis does not occur. The role of proline availability for PRODH/POX-dependent functions was well established . AlthougCollagen biosynthesis is the most effective process in utilization of intracellular proline, substrate for PRODH/POX. Previously we found that PPAR-\u03b3 ligands evoke collagen biosynthesis inhibiting activity . It was However, in contrast to these results, it has been suggested that activation of ER\u03b2 (expressed in ER negative MDA-MB-231 cells) contributes to pro-apoptotic phenotype of breast cancer cells, while activation of ER\u03b1 (expressed in ER positive MCF-7 cells) induces anti-apoptotic effects . It is pIt cannot be excluded that pro-apoptotic phenotype of TGZ-treated breast cancer cells, cultured in the absence of estradiol is due to other mechanisms. Some studies documented that there is a cross talk between ERs and PPAR-\u03b2 , or ERs The absence of estradiol or ER\u03b2 in the cell culture is critical requirement for TGZ-induced PRODH/POX-dependent apoptosis in MDA-MB-231 cells. Probably, in vivo such a situation never happens, as estradiol in tissues is ubiquitous. This finding may be of importance in the experimental therapy of ER negative breast cancers by combined use of TGZ and anti-estrogens. In fact, it has been well established that estrogen receptor status determine efficacy of breast cancer therapy and its determination has predictive and prognostic value, particularly in triple-negative breast cancer . HoweverDownregulation of AMPK, metabolic regulator involved in control of cell growth and survival has been established as a major contributor to carcinogenesis in many types of human cancer . We haveAnother interesting factor in studies on PRODH/POX dependent apoptosis is p53 protein. This transcription factor is the best characterized apoptosis inducing factor and also the most potent factor regulating expression of PRODH/POX. The presence of response element for p53 protein in the promoter sequence of the gene coding PRODH/POX has been demonstrated. It shows direct participation of p53 in the transcription of PRODH/POX . HoweverPresently, the selective ER modulator, tamoxifen, is the only endocrine agent with approval for prevention and treatment of ER positive breast cancer . In viewThe data suggest that TGZ-induced apoptosis in MDA-MB-231 cells cultured in medium without estradiol or deprived of ER\u03b2 is mediated by PRODH/POX and the process is facilitated by proline availability for PRODH/POX by TGZ-dependent inhibition of collagen biosynthesis . It sugg"} +{"text": "Recent declines in heart failure (HF) prevalence and increases in mortality among older adults in the US suggest the need for research to investigate the relative contribution of the epidemiological determinants of these two processes to their historical and current trends. Study data were derived from a 5% sample of Medicare beneficiaries, 1991-2017. Partitioning analysis was used to decompose age-adjusted prevalence and incidence-based mortality (IBM) into their constituent components. HF prevalence trend decomposition demonstrated three phases: (a) Decelerated Increasing Prevalence (1994-2006) mainly driven by decreasing incidence, overpowering increasing survival, (b) Accelerated Declining Prevalence (2007-2014) and (c) Decelerated Declining Prevalence (2015-2017), mainly driven by declining incidence, overpowering declining survival. For HF IBM four phases were identified: (a) Decelerated Increasing Mortality (1994-2001) with declining incidence and increasing survival driving deceleration, (b) Accelerated Declining Mortality (2002-2012), (c) Decelerated Declining Mortality (2013-2016), mainly driven by declining incidence, overpowering declining survival, and (d) Accelerated Increasing Mortality (2017) mainly driven by declining survival, overpowering declining incidence. Study findings suggest that the recent decade-long decline in HF prevalence and 15-year decline in HF mortality mainly reflected decreasing incidence, while the most recent increase in mortality was due to declining survival, which may be associated with the Hospital Readmission Reduction Program. If current trends of incidence and survival persist, HF prevalence and mortality are forecasted to grow, suggesting that actions to reduce HF risk factors and improve treatment and management of HF after diagnosis are warranted."} +{"text": "PFWER\u2009=\u20090.033), seeminglydriven by association with the Fantasy subfacet. Trait Extraversion was significantlynegatively associated with functional connectivity between the visual and dorsal attentionnetworks and positively associated with functional connectivity between the frontoparietaland language networks. Our findings provide evidence that resting-state DMN is associatedwith trait Openness and gives insight into personality neuroscience.Evaluating associations between the five-factor personality domains and resting-statefunctional connectivity networks highlights distributedneurobiological systems linked to behaviorally relevant phenotypes. Establishing theseassociations can highlight a potential underlying role for these neural pathways inrelated clinical illness and treatment response. Here, we examined associations betweenwithin- and between-network resting-state functional connectivity with functional magneticresonance imaging and the five-factor personality domains: Openness to experience(Openness), Extraversion, Neuroticism, Agreeableness and Conscientiousness. We includeddata from 470 resting-state scan sessions and personality assessments in 295 healthyparticipants. Within- and between-network functional connectivity from 32 a priori definedregions was computed across seven resting-state networks. The association betweenfunctional connectivity and personality traits was assessed using generalized leastsquares. Within-network DMN functional connectivity was significantly negativelyassociated with trait Openness (regression coefficient\u2009=\u2009\u22120.0010; [95% confidenceinterval]\u2009=\u2009; The five-factor model of personality is a widely recognized model for personality, asmeasured by the NEO Personality Inventory (NEO PI-R), consisting of trait Openness toExperience (Openness), Extraversion, Neuroticism, Agreeableness and Conscientiousness . OpennesResting-state functional magnetic resonance imaging (rs-fMRI) is a non-invasive brainimaging tool that can estimate so-called \u2018resting-state networks\u2019 (RSNs) and provides aframework for delineating neural pathways underlying individual variability in personalitytraits . RestingMultiple studies have examined associations between resting-state functional connectivityand core personality traits applying different analysis methodologies , which iA comprehensive neuroimaging study in 884 healthy participants from the Human ConnectomeProject reported that Openness alone and a combination personality trait derived fromOpenness, Neuroticism and Extraversion were best predicted by resting-state data database 01-2006-20\u2009+\u2009appendix (KF)23830, VEK H-15003302, VEK (KF)01280377, VEK H-1-2010-085,VEK H-15017713, VEK H-3-2013-100, VEK H-2-2010-108, VEK (KF)01-2006-20, VEK H-6-2014-057,VEK H-16026898, VEK H-15004506. Some of the functional connectivity data presented herewere included in a previous study (https://www.hogrefe-online.com).Personality factor and facet scores were determined by summing the scores from relevantitems, loading on to respective factors and facets. Internal consistency, as measured byCronbach\u2019s alpha, for each personality trait was as follows: Neuroticism\u2009=\u20090.90,Extraversion\u2009=\u20090.88, Openness\u2009=\u20090.88, Agreeableness\u2009=\u20090.88 and Conscientiousness\u2009=\u20090.90.For participants with multiple completed NEO PI-R assessments (n\u2009=\u2009148),we paired each rs-fMRI scan session with the personality assessment nearest in time to thecorresponding rs-fMRI scan date.All participants completed a Danish version of either the NEO Personality InventoryRevised (PI-R) or theuMRI scan sessions were completed on one of five 3 Tesla (3T) MRI scanners: 3T Trio, 3TmMR, 3T Prisma and two 3T Verio MRI scanners. Resting-state fMRI scans were 10\u2009minutes inlength. Detailed scanner information and scanner-specific sequence parameters can be foundin https://www.fil.ion.ucl.ac.uk/spm/, Wellcome Department of CognitiveNeurology, London, UK) and MATLAB R2019b . Functional images wereslice-timing corrected, field map distortion corrected and realigned to the first volume.The high-resolution, T1-weighted structural image was co-registered to the functionalimages; segmented into gray matter, white matter and cerebrospinal fluid; and normalizedinto Montreal Neurological Institute (MNI) space. Functional images were normalized intoMNI space using the estimated deformations and smoothedusing an 8-mm full-width half-maximum Gaussian kernel to limit spatial variance introducedby the normalization. Additional denoising of functional images was performed in \u2018Conn\u2019 between denoised regional time series for all pairs of regions defined a priori bythe \u2018networks\u2019 atlas in \u2018Conn\u2019. This atlas defines eight RSNs from 32 discrete brainregions: DMN, sensorimotor network (SMN), visual network (VN), dorsal-attention network(DAN), salience network (SN), frontoparietal network (FPN), language network (LN) andcerebellar network (CN) (r-to-z transformation(i.e. 0.5\u00a0\u00d7\u00a0[ln(1\u2009+\u2009r)\u00a0\u2212\u00a0ln(1\u00a0\u2212\u00a0r)], where r is the correlationcoefficient and \u2018ln\u2019 is the natural logarithm). Within- and between-network functionalconnectivity estimates were calculated as the mean of allr-to-z values for a specific within- orbetween-network . Thisresulted in eight within-network and 28 between-network estimates per rs-fMRI scansession.For each rs-fMRI scan session, functional connectivity was estimated as the Fisher\u2019sork (CN) . Pearsonn and percentage, as appropriate. We assessed theassociation between the big five personality factors and the mean within- andbetween-network functional connectivity of DMN, SMN, VN, SN, DAN, FPN, LN and CN bygeneralized least-squares regression, using \u2018corSymm\u2019 as a covariance structure to accountfor the correlation between repeated measurements over the same subject and was included as a covariate with sex, age andMRI scanner. Residuals from generalized least-squares regression models were plotted in QQplots and inspected visually for normal distribution assumption. Statistical significanceestimates for the associations between a personalityfactor and all within- and between-network functional connectivity estimates (i.e. 36tests) were adjusted for multiple comparisons using Dunnett\u2019s procedure(PFWER), which controls the family-wise error rate(\u03b1\u2009=\u20090.05) , or \u2009=\u20090.05) .SubfaceP-value\u2009<\u20090.05. All reported analyses were performed in thestatistical software package R or by SPSS Statistics .All statistical analyses were two-tailed and the level of statistical significance wasset to We included 295 healthy participants . Post hoc analyses betweenOpenness facets and DMN functional connectivity identified Fantasy as the only facetshowing a statistically significantly negative correlation ]\u2009=\u2009, PFWER\u2009=\u20090.031) and FPN\u2013LN between-network functionalconnectivity was statistically significantly positively associated with Extraversion.Post hoc facet-level analysis of Extraversion indicated that Warmth wassignificantly negatively associated with VN\u2013DAN . Between-network functional connectivity of VN\u2013DAN was statistically significantlynegatively associated with Extraversion .Beyond effects reported above, no other associations between personality factors andwithin- or between-network resting-state functional connectivity were statisticallysignificant , which may affectconnectivity variance. Simon and colleagues excluded negative correlation values whencomputing DMN functional connectivity, stemming from uncertainty about how to interpretnegative correlations (ell time andinfoell time , butareThe negative association between DMN functional connectivity and Openness is convergentwith serotonin psychedelic studies reporting decreased resting-state functional connectivitywith DMN regions (Intriguingly, recent studies suggest a link between Openness and psychoticism, acharacteristic of psychotic-like symptoms but without severe schizophrenic illness (We observed that Extraversion was statistically significantly associated with twobetween-network estimates, namely a negative association with VN-DAN and a positiveassociation with FPN-LN functional connectivity. We are not aware of a previous studydirectly evaluating the between-network effect that we observed but our finding suggestsbetween-network functional connectivity should be considered in future personalityneuroimaging studies. People with high Extraversion scores tend to be outgoing (Post hoc analyses of personality facets indicated facets of Openness andExtraversion were associated with resting-state functional connectivity. The Openness facetFantasy, which describes persons with an imaginative mind (ive mind was negaive mind , was negive mind , was posetal. was able topredict four out of five personality factors using relevance vector machine learning (etal. was only able to significantly predict Openness(We observed only three statistically significant associations between resting-statefunctional connectivity and any of the five-factor personality traits examined; we did notobserve a significant association for three of the personality traits, i.e. Agreeableness,Conscientiousness and Neuroticism. Previous studies have reported a similar scale ofassociations (Certain limitations should be taken into consideration when interpreting our reportedresults. Although we considered a large dataset of 470 rs-fMRI scans from 295 uniqueindividuals, data acquisition was carried out across five different 3T MRI scanners, addingheterogeneity to our dataset. We attempted to model this variability by including MRIscanner as a covariate in all regression models. If anything, we expect scannerheterogeneity would decrease our ability to detect statistically significant associationswith Openness. The participants in our sample have significantly higher Openness scorescompared to a reported Danish norm sample (In conclusion, we observed a negative association between DMN functional connectivity andOpenness and its facet Fantasy. Extraversion was significantly negatively associated withVN\u2013DAN between-network functional connectivity and significantly positively associated withFPN\u2013LN between-network functional connectivity. The Extraversion facet Warmth was negativelyassociated with VN\u2013DAN functional connectivity, whereas the facet Positive Emotions waspositively associated with FPN\u2013LN functional connectivity. Openness was not significantlyassociated with any other network functional connectivity estimate, nor were any otherfive-factor personality measures. These findings reinforce the relevance of DMN functionalconnectivity as a neurobiological correlate of a core personality trait.nsab048_SuppClick here for additional data file."} +{"text": "It is of interest to document the moelcular docking analysis of SARS-CoV-2 linked RNA dependent RNA polymerase (RdRp) with compounds from Plectranthus amboinicus. Hence, we report the binding features of rutin, Luteolin, Salvianolic acid A, Rosmarinic acidand p-Coumaric acid with the target protein SARS-CoV-2 linked RNA dependent RNA polymerase (RdRp) for further consideration. The new strain of coronavirus SARS-CoV-2 (Severe Acute Respiratory Syndrome) is the infectious disease COVID-19 . RdRp isThe three-dimensional structure of the protein RdRp of SARS-CoV-2 (PDB ID: 6M71) was downloaded from the Protein Data Bank (www.rcsb.org/pdb). This structure is solved with 2.9Thirty compounds from the Plectranthus amboinicus plant were gleaned from literature. The compound structures were downloaded in .sdf format from the database of PubChem compounds (www.pubchem.ncbi.nlm.nih.gov/). All the compounds were translated to .Pdb formatby using the online smiles converter. The energy of all ligands was minimized and translated to the PDBQT file format.The grid box around the binding pocket is positioned using a standard protocol . PyRx haThe Lipinski filters (http:/www.scfbio-iitd.res.in / software / drugdesign / lipinski.jsp) were used to measure the drug likeness of the compounds from the docking calculation. Four of the five parameters defined for drug likeness are molecular mass, cLogP,hydrogen donor and acceptor and molar refractive index .It is of interest to document the moelcular docking analysis of SARS-CoV-2 linked RNA dependent RNA polymerase (RdRp) with compounds (Table 1 - see PDF) from Plectranthus amboinicus. Hence, we report the binding features of rutin, Luteolin, Salvianolic acid A,Rosmarinic acid and p-Coumaric acid with the target protein SARS-CoV-2 linked RNA dependent RNA polymerase (RdRp) for further consideration (Table 2 - see PDf). The interactions between the targeted protein and the ligands were analysed using the Pymol MolecularVisualization Tools as shown in We report the binding features of rutin, Luteolin, Salvianolic acid A, Rosmarinic acid and p-Coumaric acid with the target protein SARS-CoV-2 linked RNA dependent RNA polymerase (RdRp) for further consideration."} +{"text": "Approaches based on expression signatures of prostate cancer (PCa) have been proposed to predict patient outcomes and response to treatments. The transcription factor NF-Y participates to the progression from benign epithelium to both localized and metastatic PCa and is associated with aggressive transcriptional profile. The gene encoding for NF-YA, the DNA-binding subunit of NF-Y, produces two alternatively spliced transcripts, NF-YAs and NF-YAl. Bioinformatic analyses pointed at NF-YA splicing as a key transcriptional signature to discriminate between different tumor molecular subtypes. In this study, we aimed to determine the pathophysiological role of NF-YA splice variants in PCa and their association with aggressive subtypes.Data on the expression of NF-YA isoforms were extracted from the TCGA (The Cancer Genome Atlas) database of tumor prostate tissues and validated in prostate cell lines. Lentiviral transduction and CRISPR-Cas9 technology allowed the modulation of the expression of NF-YA splice variants in PCa cells. We characterized 3D cell cultures through in vitro assays and RNA-seq profilings. We used the rank-rank hypergeometric overlap approach to identify concordant/discordant gene expression signatures of NF-YAs/NF-YAl-overexpressing cells and human PCa patients. We performed in vivo studies in SHO-SCID mice to determine pathological and molecular phenotypes of NF-YAs/NF-YAl xenograft tumors.NF-YA depletion affects the tumorigenic potential of PCa cells in vitro and in vivo. Elevated NF-YAs levels are associated to aggressive PCa specimens, defined by Gleason Score and TNM classification. NF-YAl overexpression increases cell motility, while NF-YAs enhances cell proliferation in PCa 3D spheroids and xenograft tumors. The transcriptome of NF-YAs-spheroids has an extensive overlap with localized and metastatic human PCa signatures. According to PCa PAM50 classification, NF-YAs transcript levels are higher in LumB, characterized by poor prognosis compared to LumA and basal subtypes. A significant decrease in NF-YAs/NF-YAl ratio distinguishes PCa circulating tumor cells from cancer cells in metastatic sites, consistently with pro-migratory function of NF-YAl. Stratification of patients based on NF-YAs expression is predictive of clinical outcome.Altogether, our results indicate that the modulation of NF-YA isoforms affects prostate pathophysiological processes and contributes to cancer-relevant phenotype, in vitro and in vivo. Evaluation of NF-YA splicing may represent a new molecular strategy for risk assessment of PCa patients.The online version contains supplementary material available at 10.1186/s13046-021-02166-4. Prostate cancer (PCa) is characterized by high clinical heterogeneity and multiple levels of molecular signatures . DespiteThe binding site for the heterotrimeric transcription factor (TF) NF-Y has been identified as biologically relevant within the molecular network of genes up-regulated during PCa progression, from benign epithelium to localized and hormone-refractory metastatic PCa . These dhigh subtypes being characterized by cell-cycle transcriptional signature and NF-YAlhigh tumors by a pro-migration transcriptional profile . N. N23]. NThe importance of NF-YA in cancer-associated cellular processes has been highlighted in various cellular contexts and NF-YA knock-down triggers cell cycle arrest and apoptotic cell death , 35\u201337. . Scramble and shNF-YA-infected PC3 cells were sub-cutaneously (s.c.) injected into immune-compromised mice and xenograft tumor growth was monitored for 5 consecutive weeks. NF-YA loss severely impaired tumor incidence and growth of engrafted tumors were grown in scaffold-based or -free conditions, depending on the ability of PCa cell lines to spontaneously form spheroids in anchorage-independent circumstances. NF-YAs overexpression increased spheroids size of PC3 Fig. D, F.We assessed cell proliferation and viability in MTSs through Ki-67 and Calcein-AM/Propidium Iodide (live/dead) stainings: all MTSs showed an external proliferating zone and an internal necrotic area Fig. F, hintinInterestingly, morphological analysis of Calcein-AM/Propidium Iodide-stained MTSs by confocal microscopy Fig. H, I showTaken together, these findings demonstrate that NF-YAs enhances cell proliferation and triggers unique cell-ECM interactions in three-dimensional culture conditions recapitulating complex interactions.NF-YA gene to definitely ascribe phenotypic alterations to isoform-specific activities. We designed a sgRNA to guide Streptococcus pyogenes Cas9 (SpCas9) to a 5\u2032-TGG-3\u2032 PAM sequence present on the reverse complementary strand of human NF-YA exon 2 cannot survive, differently from the cells expressing NF-YAs-only. We thus selected two NF-YAs-only clones for further analyses Fig. A, we knoses Fig.\u00a0B. Additises Fig.\u00a0C.Fig. 4EThese results suggest a unique role for NF-YAs in PCa cell viability.To shed light on the transcriptional effects induced by NF-YA isoforms, leading to different tumor phenotypes, we performed RNA-seq profilings of transduced cells cultured as MTSs. Compared to Empty-cells, 872 and 620 differentially expressed genes (DEGs) were retrieved in NF-YAl cells, and 962 and 686 genes were found in NF-YAs cells as up- and down-regulated, respectively. The analysis of Gene Ontology enrichment (GO<\u2009500 and |Log2FC|\u2009>\u20091) Fig.\u00a0 showed tThe ECM signature clearly hints at a role for NF-YAs in modulating the interaction between epithelial cells and the environment, which is consistent with the budding ring-shaped structures previously described Fig. H, I but These results suggest that NF-YAs expression characterizes localized tumors, while both NF-YAs and NF-YAl could be involved in the metastatization process.We then used NF-YA transduced cells for mouse xenograft studies to investigate whether the above described properties were obvious also in vivo. Equal number of exponentially growing PC3 cells were injected s.c. into SCID Hairless mice and tumor volumes were determined from prostatectomy has been recommended . StratifThese results further corroborate that high level of NF-YAs transcript is a significant independent prognostic factor associated with poor clinical outcome in PCa patients.PCa disease is characterized by extreme clinical heterogeneity and unpredicted therapeutic response of patients. The transcriptional profile of PCa is different from the majority of tumors, being an indolent and slow-proliferating cancer in which cell growth and cell cycle gene categories are not retrieved among up-regulated terms. Despite this, large-scale analyses identified NF-Y among TFs involved in molecular networks inducing the progression from benign epithelium to both localized and hormone-refractory metastatic PCa . MoreoveData analysis from TCGA database highlights the increase in total NF-YA gene transcription in PCa compared to healthy tissues, in particular in high GS samples. This increase is associated to the up-regulation of the NF-YAs isoform, while NF-YAl transcript decreases. Of particular relevance is the demonstration that changes in the expression of NF-YA isoforms are associated to key clinical and molecular features of aggressive PCa. The ratio between NF-YAs and NF-YAl increases in higher GS adenocarcinomas and in luminal B/PCS1 tumor subtype, which includes tumors with the poorest outcome with GS\u2009\u2265\u20098 but also with GS\u2009\u2264\u20097 associated to metastatic progression . WesterMTSs, models for self-assembled cell aggregates, came in support to better elucidate whether NF-YAs overexpression can sustain tumor aggressiveness, as suggested by TCGA transcriptional data from PCa patients. A significant increase in BrdU+ cells demonstrates the enhanced proliferative ability conferred by NF-YAs to tumor cells compared to both Empty and NF-YAl cells, accompanied by reduced cell death determined by Annexin V staining. Surprisingly, no categories associated to cell proliferation or cell cycle progression are observed in highly proliferative NF-YAs tumors, although these terms are usually described as up-regulated by NF-YA overexpression \u201317, 20. Taking into consideration that high NF-YAs/NF-YAl ratio characterizes both primary tumors and metastatic ones, we hypothesized that high NF-YAs is a condition necessary to allow tumor cell expansion locally as well as in distant metastasis, while NF-YAl could be required for short-time specific invasive behaviors, such as migrating abilities. Indeed, the metastatic process requires three consecutive steps: firstly, neoplastic cells break down the basement membrane of tumor blood vessels, allowing stroma invasion and intravasation. Afterwards, the cells have to survive through the circulation and they finally extravasate in a distant organ, where they adapt and start to proliferate . The proPFI and regression analyses highlight the clinical significance of our studies. In particular, we propose the analysis of NF-YAs levels as a new molecular strategy for risk assessment in PCa patients. High NF-YAs expression is associated with PCa outcome, independently of other clinical variables. Optimal management of PCa is majorly associated to the diagnostic process rather than the selection of appropriate active treatment. While Gleason score, TNM staging and pre-treatment PSA values provide key prognostic information on the expected behaviour of the tumor, they don\u2019t always allow to discriminate biologically aggressive tumors (for a review see ).In this scenario, NF-YAs can represent a new biomarker for stratification of PCa patients Fig.\u00a0. FurtherRegulation of the splicing process of the NF-YA subunit of the transcription factor NF-Y is highly relevant for understanding PCa hallmarks. Unbalanced NF-YAs/NF-YAl ratio is associated not only to localized adenocarcinoma, but also to metastatic PCa that eventually develops resistance to therapies. Evaluation of the expression of NF-YA isoforms may be adopted as a new strategy for risk stratification of PCa patients and can be useful for defining biological properties of PCa Fig. .Additional file 1:\u00a0Suppl. Figure S1.\u00a0Effects of NF-YA inactivation by RNAi in vitro and in vivo. (A) Cell proliferation curve measured by MTT assay of PC3 cells infected with scramble (shCTR) and NF-YA-targeting shRNA (shNF-YA). Data represent mean \u00b1 SEM . (B) Images of xenograft tumors dissected from SCID Hairless Outbred (SHO\u00ae) mice after 5 weeks from s.c. injection of shCTR and shNF-YA cells (7 mice per group).Additional file 2: Suppl. Figure S2.\u00a0Effects of the overexpression of NF-YA isoforms in PCa cell lines. (A) Validation of the overexpression of NF-YAl and NF-YAs by western blot of whole cell extracts from LNCaP stable cell lines. Tubulin was used as loading control. (B) Colony number of Empty, NF-YAl and NF-YAs LNCaP cells cultured in anchorage-dependent growth condition. Data represent mean \u00b1 SEM . (C) Colony number of Empty, NF-YAl and NF-YAs LNCaP cells cultured in anchorage-independent growth condition. Data represent mean \u00b1 SEM . (D) Time course analysis of cellular growth of LNCaP cultured as MTSs, calculated as projected area fold change relative to day 2, arbitrarily set at 1. Data represent mean \u00b1 SEM . (E) Western blot analysis of NF-YA expression in Empty, NF-YAl and NF-YAs DU145 stable cell lines. Tubulin was used as loading control. (F) Time course analysis of cellular growth of DU145 cultured as MTSs, calculated as projected area fold change relative to day 4, arbitrarily set at 1. Data represent mean \u00b1 SEM . (G) Western blot of total extracts from PC3 MTSs with the indicated antibodies. Tubulin has been used as loading control.Additional file 3: Suppl. Figure S3. CRISPR/Cas9-mediated knock out of endogenous hNF-YA. (A) Schematic representation of CRISPR/Cas9 strategy to knock down human NF-YA. The picture illustrates the sgRNA targeting endogenous hNF-YA (blue line) and the PAM sequence specific for the human gene (red line). Sequence alignment of the reverse complementary strand of exon 2 of human and mouse NF-YA gene is shown. (B) Indel spectrum determined by TIDE analysis on human NF-YA gene in a representative experiment of bulk CRISPR-treated PC3 cells overexpressing murine NF-YAl (left panel) or NF-YAs (right panel). Editing frequencies are shown (p < 0.05). (C) Indel spectrum determined by TIDE analysis on murine NF-YA in CRISPR-treated and GFP-sorted PC3 cells overexpressing murine NF-YAl (left panel) or NF-YAs (right panel). (D) Indel spectrum determined by TIDE analysis on human NF-YA gene in PC3 NF-YAs clone #11 (left panel) or #23 (right panel). Frequencies of editing are reported (p < 0.05) and show biallelic editing.Additional file 4: Suppl. Figure S4.\u00a0Gene signature of MTSs overexpressing NF-YAs or NF-YAl. Top 25 enriched GO terms of down regulated genes in NF-YAl (left panel) and NF-YAs (right panel) overexpressing PC3 MTSs vs Empty control ones. The size of each circle represents the number of genes included in each GO term and the color of the circle indicates the adjusted p value.Additional file 5: Suppl. Figure S5. Unique gene signature of MTSs overexpressing NF-YAs or NF-YAl. (A) Top 25 enriched GO terms of up and down regulated genes in NF-YAl overexpressing cells vs Empty cells by setting |Log2FC| >1 and discarding genes with |Log2FC| >0.5 in NF-YAs vs Empty. (B) Top 25 enriched GO terms of up and down regulated genes in NF-YAs overexpressing cells vs Empty cells by setting |Log2FC|>1 and discarding genes with |Log2FC| >0.5 in NF-YAl vs Empty. The size of each circle represents the number of genes enriched in each GO term. Color bar indicates adjusted p-value for the labeled GO term.Additional file 6: Suppl. Figure S6. Effect of NF-YA overexpression on tumor growth in vivo and analysis of NF-YA isoforms in PCa metastatic sites. (A) Volumes (mm3) of Empty, NF-YAs and NF-YAl xenograft tumors at the indicated time points. Data represent mean \u00b1 SEM . (B) Images of xenograft tumors dissected from SCID Hairless Outbred (SHO\u00ae) mice after 5 weeks from s.c. injection. (C) Analysis of NF-YAs and NF-YAl transcripts in metastatic sites from PCa samples (GEO147250) and TCGA dataset (Prostate primary).Additional file 7: Table S1. Oligonucleotides used in the experiments."} +{"text": "Despite considerable progress, it remains unclear why some patients admitted for COVID-19 develop adverse outcomes while others recover spontaneously. Clues may lie with the predisposition to hypoxemia or unexpected absence of dyspnea (\u2018silent hypoxemia\u2019) in some patients who later develop respiratory failure. Using a recently-validated breath-holding technique, we sought to test the hypothesis that gas exchange and ventilatory control deficits observed at admission are associated with subsequent adverse COVID-19 outcomes .N\u2009=\u200950) performed breath-holds to obtain measurements reflecting the predisposition to oxygen desaturation (mean desaturation after 20-s) and reduced chemosensitivity to hypoxic-hypercapnia . Associations with the primary composite outcome were modeled adjusting for baseline oxygen saturation, obesity, sex, age, and prior cardiovascular disease. Healthy controls (N\u2009=\u200923) provided a normative comparison.Patients with COVID-19 (N\u2009=\u200911/50) was associated with breath-holding measures at admission ; specifically, greater mean desaturation and greater maximal breath-holding duration . COVID-19 patients who did not develop the adverse composite outcome had similar mean desaturation to healthy controls.The adverse composite outcome (observed in Breath-holding offers a novel method to identify patients with high risk of respiratory failure in COVID-19. Greater breath-hold induced desaturation (gas exchange deficit) and greater breath-holding tolerance (ventilatory control deficit) may be independent harbingers of progression to severe disease.The online version contains supplementary material available at 10.1186/s13054-021-03630-5. The breath-holding model\u2014but not the reference model\u2014passed cross validation analysis .The parsimonious model Table describiial Fig.\u00a0D-left: mv. Borg\u2009=\u20090) was also associated with reduced risk of the primary outcome in COVID-19 in the current sample . Inclusion of dyspnea in the above models did not meaningfully alter the findings reported above we performed additional analysis of dyspnea at admission: In fully adjusted analysis, dyspnea are associated with deficits in gas exchange and ventilatory control revealed using a validated breath-holding technique. Specifically, we demonstrated that with increasing predisposition to oxygen desaturation during breath-holding there is higher risk of progression to severe disease, independent of baseline oxygenation, and other key covariates. Greater maximal breath-hold duration\u2014adjusted for hypoxemia (i.e. baseline saturation)\u2014was also an independent risk factor among patients with COVID-19. Our finding that blunted ventilatory control \u201328 is a 2, breath-holding desaturation is an independent risk factor for adverse outcomes in COVID-19. Indeed, breath-holding is expected to provide unique information on gas exchange deficits on the basis that V/Q heterogeneity (lower V/Q regions readily desaturate during apnea) [2 per unit time) [2. Of note, baseline SpO2 can be insensitive to reduced PaO2 when on the plateau of the SpO2/PaO2 curve. Our findings also withstood adjustment for obesity and pre-existing cardiovascular disease as confounders, suggesting that these particular non-COVID sources of variability do not explain away the associations observed.Early signs of gas exchange deficits in patients who later develop severe COVID-19\u2014such as regional ventilation/perfusion (V/Q) heterogeneity and reduced lung gas volumes\u2014have been inferred from chest imaging , 14, 15;g apnea) and reduit time) , 18 infl2-adjusted [n.b. impaired chemosensitivity may mitigate dyspnea [v. healthy controls; thus, such individuals may escape adverse outcomes of COVID-19 partly through a more robust ventilatory control defense against hypoxia/hypercapnia. Overall, our data do not support the concept that robust chemoreflexes exacerbate lung injury via greater chemoreflex-related transpulmonary pressures (P-SILI) [The finding that longer maximal breath-hold duration (SpOadjusted ) confersadjusted \u201328 may p dyspnea \u201340). Not dyspnea or centr dyspnea . Regardl(P-SILI) , 24. Ins(P-SILI) , 34, 35.2, and early analysis suggests\u00a0that the approach has potential predictive value. Exploratory inclusion of existing biomarkers C-reactive problem and d-dimer, and adjustments for haemoglobin levels did not change our findings. Our translational work therefore demonstrates the feasibility of using physiological testing to estimate the risk of adverse COVID-19 outcomes days in advance of patient deterioration, enabling prioritization of limited resources to the high risk patients who need them most and longer maximal breath-holds (interpreted as lower chemosensitivity) are independent risk factors. Simplified physiological measures of gas exchange and neurophysiological deficits in COVID-19 may hold utility for future translational use in early triage to scarce health care resources or early administration of medical interventions. Our study also raises the possibility that blunted ventilatory control is a therapeutic target for preventing severe disease in COVID-19, a concept that will require interventional studies to assess.Additional file 1: Supplemental text (methods and results) and tables.Additional file 2: Tool to assess probability of the primary adverse outcome ."} +{"text": "COVID-19 patients may experience \u201ccytokine storm\u201d when human immune system produces excessive cytokines/chemokines. However, it remains unclear whether early responses of inflammatory cytokines would lead to high or low titers of anti-SARS-CoV-2 antibodies.This retrospective study enrolled a cohort of 272 hospitalized patients with laboratory-confirmed SARS-CoV-2. Laboratory assessments of serum cytokines , anti-SARS-CoV-2 IgG/IgM antibodies, and peripheral blood biomarkers were conducted during hospitalization.At hospital admission, 36.4% patients were severely ill, 51.5% patients were\u2009\u2265\u200965\u2009years, and 60.3% patients had comorbidities. Higher levels of IL-2R and IL-6 were observed in older patients (\u226565\u2009years). Significant differences of IL-2R (week 2 to week \u22655 from symptom onset), IL-6 (week 1 to week \u22655), IL-8 (week 2 to week \u22655), and IL-10 (week 1 to week 3) were observed between moderately-ill and severely ill patients. Anti-SARS-CoV-2 IgG titers were significantly higher in severely ill patients than in moderately ill patients, but such difference was not observed for IgM. High titers of early-stage IL-6, IL-8, and TNF-\u03b1 (\u22642\u2009weeks after symptom onset) were positively correlated with high titers of late-stage IgG (\u22655\u2009weeks after symptom onset). Deaths were mostly observed in severely ill older patients (45.9%). Survival analyses revealed risk factors of patient age, baseline COVID-19 severity, and baseline IL-6 that affected survival time, especially in severely ill older patients.Early responses of elevated cytokines such as IL-6 reflect the active immune responses, leading to high titers of IgG antibodies against COVID-19.The online version contains supplementary material available at 10.1186/s12979-022-00271-2. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the aetiological agent of coronavirus disease 2019 (COVID-19), causing severe pneumonia, multiorgan dysfunction, or even death . AlthougPrevious studies suggest that serum levels of cytokines and chemokines are likely associated with the disease severity and clinical outcomes of COVID-19 \u201319. ManyAmong the long list of cytokines/chemokines, IL-6 has been recognized as a key COVID-19-associated cytokine. First, IL-6 may serve as an early biomarker to monitor inflammatory and immune responses in COVID-19 patients. Early diagnosis of serum IL-6 can be achieved within the first 10\u2009days after symptom onset; other cytokines are often detected at the later stage . The earDuring the innate immune response to SARS-CoV-2 infection, inflammatory cytokines are produced at the early stage . Later, We performed a retrospective study to collect cytokine and antibody records of hospitalized COVID-19 patients during the early outbreak of the pandemic (January to March 2020) when approved vaccines and cytokine-targeted drugs were not available or administered. We analyzed a cohort of 272 COVID-19 patients who were hospitalized from 2020/02/01 to 2020/03/31 in the Sino-French New-City Tongji Hospital in Wuhan, China. At hospital admission, all patients were confirmed to have SARS-CoV-2 infection based on real-time RT\u2013PCR tests of nasopharyngeal swab specimens. During their hospitalization, all patients received neither vaccines nor cytokine-targeted drugs, according to the COVID-19 guidelines before 2020 April. Archived records during the inpatient hospital stay were analyzed in this retrospective study; thereby, written informed consent was waived. This study was conducted under the Helsinki Declaration and was approved by the Ethics Committees of The Second Xiangya Hospital .2/FiO2 \u2264300\u2009mmHg; (iv) chest imaging tests that exhibit >\u200950% progression of lung lesions within 48\u2009h; and (v) shock or organ failure that requires intensive care unit, or respiratory failure that requires mechanical ventilation. Moderately ill cases were defined based on mild/moderate symptoms of fever, respiratory difficulties, and/or radiological findings of pneumonia.Based on the New Coronavirus Diagnosis and Treatment Guidelines in China, a severely ill case was identified if any of the following five conditions were fulfilled at baseline or during hospitalization: (i) the breathing rate of \u226530 breaths per minute; (ii) oxygen saturation of \u226493% at rest; (iii) PaOElectronic medical records of COVID-19 patients were retrieved to collect datasets of epidemiological and demographic information, laboratory biomarkers, onset symptoms, disease severity and clinical comorbidities at hospital admission. All medical records were retrieved based on a standard collection form from the Sino-French New-City Tongji Hospital. Two authors cross-checked the medical records and communicated with doctors for data accuracy.Real-time RT\u2013PCR tests were performed to detect SARS-CoV-2 nucleic acid; the detailed protocols were described previously . During We analyzed either the proportion of categorical variables or the median/interquartile range (IQR) of continuous variables. Chi-square tests were applied to compare proportions of categorical variables, and Fisher\u2019s exact tests were applied within the context of limited data. Logarithmic transformation of continuous variables with all-positive data was conducted, and normality tests of continuous variables on the original scale and the log-transformed scale were performed using Shapiro-Wilk tests. We employed two-tailed t-tests for continuous variables following normal distributions, Wilcoxon rank-sum tests for non-normal continuous variables in paired groups, and Kruskal-Wallis tests for non-normal continuous variables in unpaired groups. To analyze correlations between biomarkers, Pearson\u2019s and Spearman\u2019s correlation coefficients were measured for normal and non-normal continuous variables, respectively. Hierarchical clustering analysis was applied to show clustering of correlating biomarkers. Cox proportional hazards models were utilized in survival analyses to assess hazard ratios (HR) indicating the effects of risk factors on survival outcomes: either death for non-survivors or hospital discharge for survivors. Baseline values of all factors were initiated in univariate survival analysis, and significant variables were subsequently used as inputs in stepwise multivariate survival analyses. Kaplan-Meier curves were built to show survival time and log-rank tests were used to compare survival distributions between different groups. The pairwise deletion approach was applied to handle missing data. All statistical analyses were descriptive, and no random sampling was conducted. For all statistical tests, two-sided tests at the level of <\u20090.05 were considered significant. Above statistical analyses were performed using MATLAB R2016.A cohort of 272 hospitalized patients with laboratory-confirmed COVID-19 were enrolled in this retrospective study. At hospital admission, 99 (36.4%) patients were severely ill, 139 (51.1%) were males, and 164 (60.3%) had comorbidities (Table\u00a0N\u2009=\u2009132) and age\u2009\u2265\u200965\u2009years (N\u2009=\u2009140). The proportion of males was higher in patients aged \u226565\u2009years than in those aged <\u200965\u2009years . Compared with patients aged <\u200965\u2009years, older patients had more severe cases and comorbidities . In patients aged \u226565\u2009years, highly prevalent comorbidities included hypertension (55%), diabetes mellitus (31.4%), and cardiovascular disease . In contrast, patient age correlated negatively with the decreasing levels of biomarkers, including albumin, lymphocytes, eosinophils, and platelets .Baseline levels of blood biomarkers were associated with patient age in subsets of moderately ill patients and severely ill patients. As shown in Fig.\u00a0To monitor disease progression, 1769 blood samples were collected from 272 COVID-19 patients to measure the serum levels of cytokines and blood biomarkers during the hospitalization. Blood samples were collected at the time of physician requests . Because patient age plus disease severity may play a role in biomarker dynamics, our subsequent analyses focused on the weekly dynamics of the above biomarkers in four subgroups: (i) moderately ill patients aged <\u200965\u2009years, (ii) severely ill patients aged <\u200965\u2009years, (iii) moderately ill patients aged \u226565\u2009years, and (iv) severely ill patients aged \u226565\u2009years., Fig.\u00a0, Fig. , Fig. Serum levels of IL-2R, IL-6, IL-8, and IL-10 were higher in severely-ill patients than in moderately ill patients. Significant differences in IL-2R was identified between white blood cells and neutrophils to predict late-stage IgG responses (the variable Y) based on early-stage IL-6 levels (the variable X). This significant correlation was independently observed in the subsets of moderately-ill patients and severely-ill patients . Due to a steady decrease in IgM titers from \u22655\u2009weeks, significant correlations of IgG titers with early-stage biomarkers were not observed.We next asked whether late-stage IgG responses (from week 5 to 10) are associated with early-stage biomarker responses (\u2264 2\u2009weeks) in COVID-19 patients. To this end, we analyzed a subcohort of 47 hospitalized patients whose blood samples were tested within two weeks after symptom onset and whose late-stage IgG titers were also tested from weeks 5 to 10. Among all available biomarkers, early responses of six biomarkers correlated significantly with high titers of anti-SARS-CoV-2 IgG antibodies .In our cohort of 272 hospitalized patients, 42 15.4%) died, and the median length of hospital stay was 21\u2009days , baseline severity , baseline D-dimer , and baseline IL-6 .Using 62 baseline parameters as predictors, Cox proportional hazards models were applied to analyze risk factors potentially associated with the clinical outcome of either death for non-survivors or hospital discharge for survivors , baseline severity , baseline D-dimer , and baseline IL-6 . Of note, the area under the curve for three factors reached 0.923 and chemokines , thereafter driving induction of SARS-CoV-2-specific B cell and T cell responses . SubsequIn agreement with previous studies \u201338, our Responses to anti-SARS-CoV-2 antibodies are associated with many baseline factors such as COVID-19 severity, patient age, and baseline symptoms \u201353. Our Age-related features of COVID-19 cytokine profiles and immune responses have been observed by our study and in other studies \u201356. We dN\u2009<\u2009100 in total) have been reported after December 2020 because of the strict adherence to a zero-COVID-19 policy in China. Second, our study reported the interplay between early-stage cytokines and late-stage IgG due to active immune responses, but any causal effect remains unclear and future studies need to address the exact molecular mechanisms. Third, blood samples in our retrospective study were not collected prior to SARS-CoV-2 infection due to the sudden outbreak in early 2020. Future studies should address the baseline impact of inflamm-ageing in older patients with COVID-19. Fourth, more than 10 cytokines/chemokines are possibly involved in the disease progression of COVID-19, but our study was limited to those biomarkers that had been tested in our hospital laboratory. It is worth examining a full panel of cytokines/chemokines in future studies. Whether cytokine antagonists reduce the risk of cytokine storms and improve the clinical outcomes of different variants should also be addressed.There are several limitations to this study. First, this retrospective study analyzed a cohort of COVID-19 patients hospitalized during the early pandemic to avoid the potential impact of different vaccines, cytokine antagonists, and SARS-CoV-2 variants. Although our study sought to analyze immune responses of different variants , >\u200990% oBased on a cohort of hospitalized COVID-19 patients with detailed records of cytokines, antibodies, and blood biomarkers, this retrospective study supports the hypothesis that early responses of elevated cytokines such as IL-6 may reflect active responses of the humoral immune system Fig. S. Active Additional file 1: Table S1. Summary of laboratory-confirmed biomarkers in our study. Fig. S1. Dynamics of albumin (A), D-dimer (B), lymphocytes (C), C-reactive protein (D), neutrophils (E), procalcitonin (F) in COVID-19 patients. Four groups were visualized in light blue (moderately-ill patients aged <\u200965\u2009years), blue (severely-ill patients aged <\u200965\u2009years), light red (moderately-ill patients aged \u226565\u2009years), and red (severely-ill patients aged \u226565\u2009years). Fig. S2. Dynamics of creatine (A), creatine kinase (B), prothrombin time (C), total cholesterol (D), urea nitrogen (E), total bilirubin (F), troponin I (G), N-terminal brain natriuretic peptide (H) in COVID-19 patients. Four groups were visualized in light blue (moderately-ill patients aged <\u200965\u2009years), blue (severely-ill patients aged <\u200965\u2009years), light red (moderately-ill patients aged \u226565\u2009years), and red (severely-ill patients aged \u226565\u2009years). Fig. S3. Dynamics of monocytes (A), platelets (B), eosinophils (C), white blood cells (D), hemoglobin (E), and fibrinogen (F) in COVID-19 patients. Four patient groups were visualized in light blue (moderately-ill patients aged <\u200965\u2009years), blue (severely-ill patients aged <\u200965\u2009years), light red (moderately-ill patients aged \u226565\u2009years), and red (severely-ill patients aged \u226565\u2009years). Fig. S4. Dynamics of aspartate aminotransferase (A), alanine aminotransferase (B), lactate dehydrogenase (C), and erythrocyte sedimentation rate (D) in COVID-19 patients. Four patient groups were visualized in light blue (moderately-ill patients aged <\u200965\u2009years), blue (severely-ill patients aged <\u200965\u2009years), light red (moderately-ill patients aged \u226565\u2009years), and red (severely-ill patients aged \u226565\u2009years). Fig. S5. Our hypothesis of the cytokine - antibody associations during the disease progression of COVID-19. This figure is adapted from previous publications. Briefly, the disease progression of SARS-CoV-2 can be intuitively divided into the early stage (nearly 2\u2009weeks after symptom onset) and the late stage (>\u20092\u2009weeks after symptom onset). During the early stage, SARS-CoV-2 infects airway epithelial cells with the surface receptors such as ACE2 and TMPRSS2. The active replication and release of viral particles cause host cells to trigger the generation of pro-inflammatory cytokines , chemokines , and interferons (type I/III interferons). This attracts monocytes, macrophages, and T cells to the infected cells and establish a pro-inflammatory feedback loop. The innate immunity responses to SARS-CoV-2 by activating many signaling pathways during the early stage of infection, while the adaptive immune responses take over during the late stage with production of antibodies such as anti-SARS-CoV-2 IgG. The defective immune response causes the overproduction of cytokines, resulting in cytokine storm that causes fatal symptoms such as acute respiratory distress syndrome (ARDS), severe pneumonia, multiorgan failure, and coagulation damage. The red lines indicate findings of our study that early responses of cytokines are associated with IgG responses at the late stage, and baseline cytokines and other factors such as older age can be early predictors of death outcome."} +{"text": "Radioligand theranostics (RT) in oncology use cancer-type specific biomarkers and molecular imaging (MI), including positron emission tomography (PET), single-photon emission computed tomography (SPECT) and planar scintigraphy, for patient diagnosis, therapy, and personalized management. While the definition of theranostics was initially restricted to a single compound allowing visualization and therapy simultaneously, the concept has been widened with the development of theranostic pairs and the combination of nuclear medicine with different types of cancer therapies. Here, we review the clinical applications of different theranostic radiopharmaceuticals in managing different tumor types that support the combination of innovative oncological therapies such as gene and cell-based therapies with RT. The name \u201ctheranostics\u201d reflects the combination of a therapeutic marker with a diagnostic tool to enable therapy and visualization simultaneously or sequentially. Theranostics in nuclear medicine, or radiopharmaceutical theranostics, is currently one of the leading medical fields promoting the development of theranostics, with the potential to make an important contribution to cancer therapy. Radioligand theranostics (RT) rely on the combination of disease-related biomarkers with the delivery of a radioactive compound that can be visualized by molecular imaging (MI), including planar scintigraphy, positron emission tomography-computed tomography (PET-CT), positron emission tomography-magnetic resonance imaging (PET-MRI) and single-photon emission computed tomography (SPECT).99mTc, T1/2 = 6.0 h), iodine-123 and positron emitters such as carbon-11 , fluorine-18 and gallium-68 are commonly used for diagnostic purposed using SPECT and PET imaging respectively, while \u03b1- and \u03b2- emitters are mostly, but not exclusively, used for radionuclide therapy NaI) is trapped by thyroid follicular cells, establishing the basis of radioiodine therapy (RAI) metaiodobenzylguanidine ([131I]MIBG) allows visualization and concomitant therapy of neuroendocrine tumors that express the norepinephrine transporter (NET) [131I]MIBG radiotherapy is recommended in case of relapsed/refractory neuroblastoma (stage III or IV), inoperable metastatic phaeochromocytoma, paraganglioma, carcinoid tumors or recurrent medullary thyroid carcinoma MIBG is trapped by active endocytosis or by passive diffusion in NET-expressing cells of the sympathetic nervous system and either remains in the cytoplasm or is stored in neurosecretory granules [123I]/[131I]MIBG imaging prior to radiotherapy allows detection of MIBG-avid tissue and prediction of the beneficial effect of [131I]MIBG therapy. Overall, the response rate to [131I]MIBG therapy in pheochromocytomas and paragangliomas increases with dose: the response rate increases with a higher radiation dose than 18.5 MBq or with repeated exposure, leading to a progression-free survival of up to 85 months [The agent [er (NET) . [131I]Marcinoma . NET is granules ,23. Diag5 months ,25,26.123I]MIBG offers better image quality with planar/SPECT imaging [123I]MIBG positive metastases have a better prognosis and longer survival after therapy, while [123I]MIBG negativity indicates a worse outcome [123I]MIBG scintigraphy showed a sensitivity of 88% and a specificity of 83% in a prospective trial to test diagnostic performance in neuroblastoma [123I]MIBG imaging in the diagnosis of phaeochromocytoma and paraganglioma [18F-metafluorobenzylguanidine ([18F]MFBG), which shows greater contrast and detects additional lesions that are not observed with [123I]MIBG imaging, is currently been evaluated in a prospective study (NCT02348749) [18F]MFBG PET imaging in patients with neural crest and neuroendocrine tumors in comparison to [123I]MIBG imaging (NCT04258592).In-pentetreotide. Over the past decade, a plethora of diagnostic agents has been investigated for PET imaging. The increased interest in imaging SSTRs was triggered by the establishment of [68Ga]Ga-DOTATATE, [68Ga]Ga-DOTATOC and [68Ga]Ga-DOTANOC tracer imaging, which showed high sensitivity (82\u201397%) and specificity (80\u201392%) for detecting neuroendocrine tumors [111In]In-pentetreotide SPECT tracer [68Ga]Ga-DOTATATE shows higher selectivity for SSTR2, [68Ga]Ga-DOTATOC binds SSTR2 and SSTR5 while [68Ga]Ga-DOTANOC has a wider binding profile, including SSTR2, SSTR3 and SSTR5 Lu-DOTATATE (Lutathera\u00ae). Lutathera\u00ae was approved by the EMA in 2017 and the FDA in 2018 for the treatment of SSTR-positive gastroenteropancreatic (GEP) neuroendocrine tumors [111In]In-pentetreotide or 68Ga-labeled somatostatin receptor tracer uptake in the tumor tissue [The most promising and currently used theranostic paradigm in neuroendocrine tumors employs [e tumors . Inclusir tissue .177Lu]Lu-DOTATATE to treat midgut NETs (NCT01578239) [177Lu]Lu-DOTATATE therapy combined with standard of care treatment (7.4 GBq every 8 weeks + 30 mg octreotide LAR) compared to high-dosage octreotide (60 mg every 4 weeks) resulted in markedly longer progression-free survival , higher overall survival and higher response rate (18 vs. 4% for the octreotide arm) in NET patients. In both arms, assessment of long-term safety issues revealed 1.8% myelodysplastic syndrome in [177Lu]Lu-DOTATATE arm, and no significant effect in nephrotoxicity (5.4 vs. 3.6%) s. 3.6%) ,42.225Ac]Ac-DOTATATE; [225Ac]Ac-DOTATOC or [212Pb]Pb-DOTAMTATE (Alphamedix) are currently under clinical development in patients with \u03b2-radiation refractory metastatic NET to enhance the anti-tumor effect or overcome therapy resistance. They have already brought clinical benefits and long-time survival to patients with metastatic NETs [225Ac]Ac-DOTATATE showed partial remission [225Ac]Ac-DOTATOC \u03b1- PRRT, including reduced tumor volume and fewer lesions MIP-1072 and [123I]MIP-1095 were the first to show clinical potential for imaging prostate cancer, and the replacement of 123I by 131I led to the first theranostics application, showing positive tumor response to therapy in 70% of the patients after a single dose [68Ga]Ga-PSMA-11 represented a step forward in diagnosing recurrent PCa [68Ga]Ga-PSMA-617 and [68Ga]Ga-PSMA-I&T, which were converted into 177Lu- or 90Y-labeled agents for therapeutic purposes [68Ga]Ga-PSMA-617 PET tracer shows the highest uptake in the kidneys and salivary glands and accumulates in the metastatic lesions at 2\u20133 h post-injection. Toxicity in salivary glands and kidneys is dose-limiting, inducing xerostomia in patients, but theranostics support the safety strategy as it can identify organs at risk.Many small-molecule PSMA-targeting radiopharmaceuticals are available. Lu-PSMA-617, binds to PSMA with high affinity in castration-resistant metastatic PCa and low toxicity profile. It reduced pain and gave a high positive response rate (more than 50%) in those patients who progressed after standard treatment [177Lu]Lu-PSMA-617 has promising anti-tumor activity in men with resistant and metastatic PCa. Although planar scintigraphy with [177Lu]Lu-PSMA-617 allowed accurate follow-up, [68Ga]Ga-PSMA-11 PET imaging is preferred for patient selection and final response assessment together with serum prostate-specific antigen (PSA) level as a well-established surrogate marker of therapy efficiency Lu-PSMA-617 PRRT, a considerable number of patients were not responsive to \u03b2-emitters PRRT and showed adverse treatment effects such as xerostomia or hematologic toxicity. Currently [225Ac]Ac-PSMA-617 is tested in clinical trials and showed already durable complete responses in metastatic prostate cancer in heavily pretreated patients but also chemotherapy-na\u00efve patients [225Ac]Ac-PSMA-617 PRRT as a salvage last-line therapy on 38 eligible heavily pretreated advanced PCa patients [177Lu]Lu-PSMA-617 [225Ac]Ac-PSMA-617 PRRT with no relevant treatment adverse effects, overall supporting that [225Ac]Ac-PSMA-617 could represent a new salvage therapy in advanced PCa [225Ac]Ac-PSMA-617 as a first-line therapy in chemotherapy-naive patients with advanced prostate cancer, in which good anti-tumor response was observed in 16/17 patients following two or three cycles of [225Ac]Ac-PSMA-617, with 11/17 patients showing complete resolution of all metastatic lesions Zr-trastuzumab was used for imaging approaches and 227Th-labeled HER2 (NCT04147819) antibodies are currently developed for therapeutic purposes [89Zr-trastuzumab PET imaging clinical trial in patients with metastatic BCa [89Zr]Zr-trastuzumab to visualize HER2-positive metastases from HER2-negative primary cancer and lesions that do not respond to treatment [- emitting radionuclides for theranostic purposes [177Lu]Lu-DOTA-trastuzumab uptake in primary and metastatic BCa lesion and no uptake in HER2-negative sites, attesting on its specificity GMIB-anti-HER2-VHH1 is the lead candidate in the therapeutic development [131I]GMIB-anti-HER2-VHH1 was well-tolerated, safe, and showed tracer uptake in HER2-positive metastatic lesions [68Ga]Ga-HER2-sdAb ([68Ga]Ga-NOTA-HER2) was successfully conducted in clinical trials and a Phase II is currently ongoing Ga-Pentixafor and [177Lu]Lu/[90Y]Y-Pentixather showed clinical benefit in combination with chemotherapy and autologous stem cell transfer in advanced multiple myeloma. Further clinical trials in lymphoma and multiple myeloma are ongoing FHGB or [124I]FIAU for visualization which has been used extensively as a reporter system in conjunction with radiotracers such as FLT supported the identification of viable target tissue, which may benefit from HSV-1-tk gene therapy. Additionally, the same study localized the transduced tissue dose of HSV-1 amplicon vector-mediated therapeutic gene expression using in vivo [18F]FHBG PET imaging [18F]FHBG PET imaging. There have been many trials of mesenchymal stem cells (MSCs) as cell-based drug delivery systems in which tumors homing and assessment of their therapeutic effects were monitored by transducing the cells with HSV-1-tk to allow their visualization using PET [+ engineered T lymphocytes expressing both HSV1-tk and interleukin-13 (IL-13) zetakine chimeric antigen receptor (CAR) could be tracked longitudinally by [18F]FHBG imaging in patients with high-grade glioma [18F]-FHBG was high due to a low background signal. Taken together, these studies indicated that the development of brain cancer cell-based therapies and immunotherapies could benefit greatly from a constitutive-promoter-driven reporter gene system to enable longitudinal monitoring of specific cell populations.In experimental glioblastoma gene therapy, MI using FIAU as the reporter probe Ga-DOTATATE- and [68Ga]Ga-DOTATOC PET imaging [90Y]Y-DOTATOC in hSSTR2-transduced xenografts Lu-DOTATATE in GEP-NET (NCT02936323) and SSTR2-positive breast cancer (NCT04529044). However, the combination of PET/SPECT imaging with SSTR2-based gene therapy has not yet been reported in humans.Recent clinical trials reporting on hSSTR2-positive tumors (i) assessed the diagnostic agents in different types of cancer that partly express SSTR2 (NCT04298541), (ii) optimized imaging protocols, and (iii) assessed the potential of [68Ga]Ga-DOTATOC [90Y]Y-DOTATOC and showed delayed tumor growth, supporting further research as a therapeutic approach in cancer In-pentetreotide [68Ga]Ga-DOTATOC PET imaging tetrafluoroborate ([18F]BF4\u2212 or [18F]TFB) for PET imaging [99mTc]pertechnetate ([99mTc]TcO4\u2212) for SPECT imaging of hNIS expression in myeloma patients treated with Measles virus-NIS (MV-NIS) and endometrial cancer patients treated with vesicular stomatitis virus co-expressing interferon and hNIS (VSV-hINF-NIS) (NCT03456908) , multiple myeloma combined with cyclophosphamide , and res3456908) B. Result18F]MFBG PET imaging probe was the most sensitive system, which was capable of detecting 35\u201340 \u00d7 103 T-cells at the site of T-cell injection in a preclinical model FLT was used to assess the effects of gene therapy on tumor cell proliferation by performing PET imaging before and after gene therapy in glioblastoma [18F]FHBG PET, was correlated with the therapeutic effect on cell proliferation, as assessed by [18F]FLT PET imaging. Other imaging probes such as [18F]FET , [18F]FDG (glucose consumption) and [18F]FDOPA (amine precursor) can be used to characterize the metabolic state of the cancer cells in conjunction with vascular MR imaging [18F]DPA-714 and [18F]BR-351 inform on the therapeutic effect of the gene therapy on tumor-associated inflammation FHBG tracer represents one of the most important clinical applications of the reporter system in humans. However, the use of gene and cell-based imaging strategies in theranostic approaches is still rare, partly due to the complex nature of clinical gene- and cell-based therapy protocols, the limitations in radiotracer production and the quantification of the imaging signal. However, it already provides a more comprehensive picture of underlying tumor biology, extending the range of theranostic applications, guiding personalized management, and supporting treatment refinement.The development of, and progress in, the field of theranostics demonstrate the benefits of combining nuclear medicine with different types of cancer therapies, including gene and cell-based therapies. With the increasing understanding of the inter- and intra-individual heterogeneity of tumors, theranostics turns out to be a smart therapeutic approach for personalized patient management. Successful clinical approaches such as LUTHATERA and PSMA-617 pave the way for further development in the field of theranostics. Expanding the field of theranostics using \u03b1-emitting radionuclides allowed to overcome some of the limitations of \u03b2- emitters with similar or enhanced anti-tumor activity. Additionally, there are now seven well-defined human reporter genes, including hNIS, hNET and hSSTR2 with complementary radiolabeled marker substrates available for clinical application; hNIS being the best characterized for theranostic applications and hNET being the most sensitive system to detect transduced cells. In this field, the use of HSV-1-tk in CAR-T cells together with the well-established ["} +{"text": "Opioid binding assays unequivocally demonstrated that only hybrids SBL-OPNK-5, SBL-OPNK-7 and SBL-OPNK-9, bearing the KGOP01 scaffold, conserved nanomolar range \u03bc-opioid receptor (MOR) affinity, and slightly reduced affinity for the \u03b4-opioid receptor (DOR). Moreover, NK binding experiments proved that compounds SBL-OPNK-5, SBL-OPNK-7, and SBL-OPNK-9 exhibited (sub)nanomolar binding affinity for NK2 and NK3, opening promising opportunities for the design of next-generation opioid hybrids. Opioid agonists are well-established analgesics, widely prescribed for acute but also chronic pain. However, their efficiency comes with the price of drastically impacting side effects that are inherently linked to their prolonged use. To answer these liabilities, designed multiple ligands (DMLs) offer a promising strategy by co-targeting opioid and non-opioid signaling pathways involved in nociception. Despite being intimately linked to the Substance P (SP)/neurokinin 1 (NK1) system, which is broadly examined for pain treatment, the neurokinin receptors NK2 and NK3 have so far been neglected in such DMLs. Herein, a series of newly designed opioid agonist-NK2 or -NK3 antagonists is reported. A selection of reported peptidic, pseudo-peptidic, and non-peptide neurokinin NK2 and NK3 ligands were covalently linked to the peptidic \u03bc-opioid selective pharmacophore Dmt-DALDA (H-Dmt- From the ancient use of the plant alkaloid morphine up until the discovery of modern medicine technologies, opioid receptor-targeting analgesics still occupy a prominent place in the management of acute or chronic pain. Through a competition with endogenous neuropeptides , these small molecule drugs work by primarily activating the \u03bc-opioid receptor (MOR) , and to Defined as a single chemical entity bearing two pharmacophores with affinity and selectivity toward two (or more) distinct molecular targets, designed multiple ligands (DMLs) aim at improved pharmacokinetic/pharmacodynamic and safety profiles compared to combination therapies. Treatment with a unique molecule drastically simplifies therapeutic procedures and allows for the overall doses to be lowered. Such multitarget-directed ligands are thus anticipated to balance therapeutic efficacy and bioavailability, while minimizing minor to severe and harmful side effects . More spVia the pioneering discovery of Substance P (SP), a whole family of endogenous neuropeptides was unveiled and rapidly considered as an attractive target for brain-related disorders. The neurokinin ligands NKA and NKB, in particular, and their preferred transmembrane receptors, neurokinin-2 (NK2) and neurokinin-3 (NK3), respectively, both belonging to the GPCR family, are essentially present in the central nervous system (CNS) and the periphery, where they exert neuromodulatory activities in a wide range of patho- and physiological processes ,13. TheiSBL-OPNK-1, SBL-OPNK-2, and SBL-OPNK-3 (A2 = 8.08 \u00b1 0.1 in RPA\u2013endothelium-deprived rabbit pulmonary artery against neurokinin A as an agonist), hence offering an adequate peptide-based candidate for this study -labeled, commercially available ligands from a commercially available cell membrane preparation from Chinese hamster ovary (CHO) cells expressing human recombinant receptors of interest (Perkin Elmer).Binding affinities for opioid receptors were determined by displacing [3H]DAMGO for MOR, [3H]deltorphin-2 for DOR, and [3H]nor-binaltorphimine ([3H]nor-BNI) for KOR. The following radioligands were used: -labelled NKA and [3H]-labeled SR 142801 and Hill coefficients (nH) were determined by non-linear regression analysis of the competition curves generated with mean replicate values using Hill equation curve fitting:Y = specific binding; A = left asymptote of the curve; D = right asymptote of the curve; C = compound concentration; C50 = IC50; and nH = slope factor. This analysis was performed using software developed at Cerep (Hill software) and validated by comparison with data generated by the commercial software SigmaPlot\u00ae 4.0 for Windows\u00ae .The Ki) were calculated using the Cheng Prusoff equationL = concentration of radioligand in the assay; and DK = affinity of the radioligand for the receptor. A Scatchard plot is used to determine the DK.The inhibition constants ("} +{"text": "To the Editor:Adult T cell leukemia\u2013lymphoma (ATL) is a peripheral T cell lymphoid malignancy caused by human T cell leukemia virus type 1 (HTLV-1). The clinical subtypes of ATL are closely associated with its prognosis, which is extremely poor in aggressive subtypes compared to indolent subtypes (favorable chronic and smoldering) [Human leukocyte antigen (HLA) plays an important role in T cell-mediated elimination of cancer cells. Downregulation of HLA occurs in various cancers and has been linked to poor prognosis . Structuin phase high-resolution typing, which includes nucleotide differences in both the coding and non-coding regions of HLA genes [Conventional polymerase chain reaction (PCR)-based HLA typing mainly focuses on the polymorphic exons encoding the antigen recognition domains. Therefore, the genetic variations in the non-coding regions or in the exons outside of the polymorphic exons have largely remained ignored. In addition, methods for precisely deciphering HLA alleles are limited due to chromosomal phase ambiguity. To overcome a line of difficulties, we successfully developed the super high-resolution single-molecule sequence-based typing (SS-SBT) method , which cLA genes . Here, wWe evaluated 25 patients diagnosed as ATL between 2012 and 2018. Their characteristics are summarized in Supplementary Tables\u00a0Through magnetic cell sorting, more than 97% pure CADM1-positive cells (ATL cells) and more than 90% CADM1-negative cells (non-ATL cells) were isolated in CADM1-positive and -negative fractions, respectively of the HLA class I genes.To detect somatic mutations in HLA genes, HLA allele sequences were determined in ATL cells. Mutational events were observed in the ATL cells but not in the non-ATL cells. We found a total of 18 somatic mutations in ATL cells from 8 patients, including 13 single nucleotide variants (SNVs) and five insertions/deletions (indels) ratio of HLA class I (relative MFI ratio of ATL cells to non-ATL cells) in each patient. Representative plots of a patient without HLA-LOH/NSVs (ATL06) and patients with HLA-LOH/NSVs (ATL20 and ATL27) are shown in Supplementary Fig. n\u2009=\u20099) showed a trend for worse survival compared with patients without LOH/NSVs (n\u2009=\u200911) , albeit without statistical significance , 8. HoweWe also found that HLA-LOH and/or NSVs frequently occurred in both alleles of the same HLA loci, suggesting that both HLA class I alleles would lack the antigen-presenting function to CTLs in patients with HLA-LOH/NSVs. Because most patients with aggressive ATL cannot be cured with conventional chemotherapies , allo-SCHLA-A or HLA-B, whereas there were only two mutations within the HLA-C gene, which is in agreement with the results of a previous report [The present study demonstrated that most NSVs were located in s report . All HLAs report . NK cellAlthough we could not draw any definitive conclusions due to the small number of patients included, a trend of low survival rate in patients with HLA-LOH/NSVs was observed compared with those without HLA-LOH/NSVs. Further work is required to evaluate the impact of the HLA gene alterations occurring in ATL cells on the clinical outcomes.In conclusion, SS-SBT\u2013based HLA gene analyses at full-length level revealed that HLA-LOH/NSVs frequently and predominantly occur in HLA class I loci in ATL cells from patients with acute-type ATL. Because allo-HSCT is an essential therapeutic option for aggressive ATL, comprehensive knowledge of HLA gene abnormalities would substantially help to optimize therapeutic modalities.Supplementary FiguresSupplementary TablesSupplementary methods"} +{"text": "COVID-19 is a multi-system infection with emerging evidence-based antiviral and anti-inflammatory therapies to improve disease prognosis. However, a subset of patients with COVID-19 signs and symptoms have repeatedly negative RT-PCR tests, leading to treatment hesitancy. We used comparative serology early in the COVID-19 pandemic when background seroprevalence was low to estimate the likelihood of COVID-19 infection among RT-PCR negative patients with clinical signs and/or symptoms compatible with COVID-19.Between April and October 2020, we conducted serologic testing of patients with (i) signs and symptoms of COVID-19 who were repeatedly negative by RT-PCR , (ii) signs and symptoms of COVID-19 but with a potential alternative diagnosis , (iii) no signs and symptoms of COVID-19 , (iv) RT-PCR confirmed COVID-19 patients (N\u2009=\u200940), and (v) pre-pandemic samples (N\u2009=\u200955).Probables had similar seropositivity and levels of IgG and IgM antibodies as propensity-score matched RT-PCR confirmed COVID-19 patients , but multi-fold higher seropositivity rates than Suspects and matched Non-suspects . However, Probables were half as likely to receive COVID-19 treatment than the RT-PCR confirmed COVID-19 patients with similar disease severity.Findings from this study indicate a high likelihood of acute COVID-19 among RT-PCR negative with typical signs/symptoms, but a common omission of COVID-19 therapies among these patients. Clinically diagnosed COVID-19, independent of RT-PCR positivity, thus has a potential vital role in guiding treatment decisions.The online version contains supplementary material available at 10.1186/s12879-022-07095-x. COVID-19 (\u2018COVID\u2019), a multi-system infection caused by the novel coronavirus SARS-CoV-2, can manifest along a clinical spectrum from asymptomatic and mild upper respiratory infections to severe pneumonia with respiratory and multi-organ failure . DespiteReverse transcriptase polymerase chain reaction (RT-PCR) is currently the primary test for diagnosis of acute COVID. The FDA issued emergency use authorizations to commercial RT-PCR tests based on test performance with known positive material from a patient or contrived upper respiratory specimens . Use of This study was approved by Rutgers University Institutional Review Board (Pro2020000861) and conducted at University Hospital (UH) in Newark, NJ, which had adopted early guidelines to screen all patients by SARS-CoV-2 RT-PCR. UH patients were screened 1\u20132 times per week by an Infectious Disease physician between April and October 2020 using the electronic medical records (EMR) for one of four cohorts: (1) PCR-confirmed COVID-19 (\u2018PCR-confirmed\u2019), (2) COVID Probable (\u2018Probables\u2019), (3) COVID Suspects (\u2018Suspects\u2019), and (4) COVID Non-suspects (\u2018Non-suspects\u2019) . SpecifiFor the study, blood was collected in accordance to routine UH hematology lab procedure that detTo validate our findings on a separate serologic assay with additional estimation of neutralizing antibody titers, we evaluated all cohorts using the SARS-CoV-2 surrogate neutralization test kit which measures percentage of inhibition of binding of RBD to recombinant human ACE2, as per manufacturer\u2019s instructions . InhibitPrior to data analysis, a pre-determined target sample size for Probable and Non-suspects was estimated by Fisher\u2019s Exact Test for difference of Proportion, using SAS 9.4 . Sample size calculations were driven by the following assumptions: (a) Seropositivity rate among Non-suspects of 15% was assumed to be similar to the background seroprevalence at the time; (b) seropostivity rate among Probables would always be higher than among Non-suspects; (c) seropositivity rate among Probables was estimated to be\u2009>\u200955% ; (d) power\u2009\u2265\u200980%; (e) alpha of 0.05; and (f) ratio of Non-suspects to Probables would be 2:1 given a larger recruitment pool of Non-suspects. With these assumptions as inputs we computed the required sample size for a One-sided Fisher\u2019s Exact Conditional Test for Two Proportions. Per our calculation a total of 48 study patients (32 Non-suspects: 16 Probables) would be required to detect a 40% excess seropositivity in the Probables. In our study we oversampled and enrolled 43 Non-suspects and 20 Probables with an actual power of 89%.All categorical data were described using numbers and percentage, and comparisons were made between groups using Fisher\u2019s exact test. All continuous data were described as median [25th\u201375th percentile] and comparisons between groups were made using non-parametric two-sample Wilcoxon Signed Rank test. Statistical analyses were primarily conducted using R and Graph-Pad Prism version 9. Propensity score matching of PCR-confirmed to Probables was performed with PSMATCH procedure using SAS 9.4.We identified 314 PCR-confirmed, 58 Probables, 46 Suspects, and 75 Non-suspects who met clinical criteria, of whom leftover blood was available for 198 (63.1%), 20 (34.5%), 15 (32.6%), and 43 (57.3%), respectively , although their median percent inhibition were similar Fig.\u00a0. ProbablAlthough the numbers were small, the 15 Probables with neutralizing antibodies were more likely to have a longer median duration of symptoms, more severe disease, and receive COVID-directed treatment compared to the Probables without neutralizing antibodies were low but woulThe study had several limitations. The primary limitation is that the Probables in this study with available blood underestimated the total number of Probables during this period given the lack of a comprehensive method to screen for all individuals at UH with clinical signs and symptoms of COVID-19. The small sample size based on statistical estimates may not capture important variability across populations and centers of COVID Probables and Suspects. While expansion to larger, multicenter studies could increase confidence in the generalizability of the findings, the ability to reproduce this study now is diminished by high background seroprevalence rates in the present post-vaccination era. Secondly, the study was also not designed nor powered to describe the epidemiology or outcomes of RT-PCR negative COVID. Although we were not able to evaluate the prevalence of RT-PCR negative COVID-19 suspects in our patient population, their considerable prevalence has been suggested previously . ThirdlyThis study identified a cohort of RT-PCR negative patients with clinical signs/symptoms of acute COVID with serologic evidence of SARS-CoV-2 infection that was multifold higher than Non-suspects and comparable to that of RT-PCR confirmed COVID. Despite similar (matched) disease severity, these RT-PCR negative patients were approximately half as likely to receive treatment as the RT-PCR confirmed COVID patients. Among the ongoing threat of highly-infectious variants and emergence of new evidence-based COVID therapies, these findings suggests a critical role for clinically-diagnosed COVID, whereby a negative RT-PCR test should not preclude evidence-based COVID treatment in the presence of clinical suspicion and no potential alternative diagnosis.Additional file 1. Study Dataset.Additional file 2. Additional Methods\u00a0and Results. Table S1. Clinical characteristics of PCR-confirmed or Probable cases with high neutralizing antibodies. Table S2. Clinical characteristics of Probables positive or negative of neutralizing antibodies. Table S3. Clinical characteristics of seropositive vs seronegative Probables for IgG or IgM."} +{"text": "Human endogenous retroviruses (HERVs) are \u2018fossil viruses\u2019 that resulted from stable integrations of exogenous retroviruses throughout evolution. HERVs are defective and do not produce infectious viral particles. However, some HERVs retain a limited coding capacity and produce retroviral transcripts and proteins, which function in human developmental process and various pathologies, including many cancers and neurological diseases. Recently, it has been reported that HERVs are differently expressed in COVID-19 disease caused by infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this review, we discuss the molecular structure and function of HERV ENV proteins, particularly syncytins, and their conventional roles in human development and diseases, and potential involvement in COVID-19 regarding the newly reported mental symptoms. We also address COVID-19 vaccine-related infertility concerns arising from the similarity of syncytin with the spike protein of SARS-CoV-2, which have been proved invalid. SU and TM contain functional domains for the protein\u2019s physiological activities: receptor-binding domain (RBD), fusion peptide (FP), the fusion core N- and C-terminal heptad repeats , immunosuppressive domain (ISD), transmembrane domain (TD), and intracytoplasmic tail (CYT) . Syncytin-1-expressing astrocytes are cytotoxic to oligodendrocytes, releasing redox reactants. In a mouse MS model, syncytin-1 mediates neuroinflammation and cell death of oligodendrocytes, which are prevented by antioxidant ferulic acid.Syncytin-1 mRNA and protein levels are higher in the brain tissue from multiple sclerosis (MS) patients than in that from normal individuals. Syncytin-1 is specifically expressed in astrocytes, glial cells, and activated macrophages of MS lesions , a transporter for the essential omega-3 fatty acid and bipolar disorder (BD) and COVID-19, a viral infectious pandemic disease whose patients commonly suffer from psychological distress. Especially, the expression level of syncytin-1, the product of HERV-W1, is often changed in these illnesses and associated with those of inflammatory markers.Retroviral RNA of HERV-W family is detected in the cell-free cerebrospinal fluids (CSFs) of 10 of 35 individuals with recent-onset SZ or schizoaffective disorder but not in the CSFs of 22 individuals with noninflammatory neurological diseases or from 30 individuals with no neurological or psychiatric diseases via directly regulating its expression or downregulating disrupted-in-schizophrenia 1 (DISC1), a susceptibility factor for SZ, which is a scaffold protein interacting with various proteins to regulate synaptic processes and dopamine signaling , PTSD (28.6%), somatization (41.8%), obsessive-compulsive (19.8%), depression (11.5%), anxiety (28%), phobic-anxiety (24.2%) and psychoticism (17.6%) , HERV-K (HML-3) and HERV-K (HML-1) is significantly upregulated in bronchoalveolar lavage fluid (BALF) of COVID-19 patients compared to that in BALF of healthy individuals but not in the peripheral blood monocytes (PBMCs) and SARS-CoV-2 spike protein (1273aa) indicates no homology between these two proteins; They are similar in only one 5-amino acid stretches, VVLQN for syncytin-1 and VVNQN for the spike protein, only two 2-amino acids identities (VV and QN). Considering the lack of homology between these two proteins, it is very unlikely that any antibodies raised against SARS-CoV-2 spike protein cross-react with endogenous human syncytin-1 (Kloc et al. Expectedly, a cohort study conducted in 3,958 pregnant women shows no risk of increased miscarriage in women vaccinated in early pregnancy, compared to the general female population (Shimabukuro et al. HERVs have been participating in human evolution. Human development requires HERV ENV proteins to build a highly elaborate placenta. When dysregulated, those proteins unbalance cellular activities to aggravate human diseases such as cancers and mental illnesses. Recent studies indicate that the essential HERV ENV proteins may be involved in developing the psychotic and cognitive symptoms of COVID-19 disease. Based on clear scientific evidence, the speculation regarding COVID-19 vaccinations causing infertility due to a misunderstanding of viral protein structure has been proved wrong. HERV activities in COVID-19 disease strongly suggest that the ancient relics in the human genome play a significant role in human evolution even in the unprecedented pandemic of COVID-19."} +{"text": "Three differentiation pathways of highly proliferative inducible costimulatory molecule (ICOS)+- and less proliferative ICOS\u2212-CD45RA+CD31+-recent-thymic-emigrant (RTE)-Tregs/Tresps via CD45RA\u2212CD31+-memory-Tregs/Tresps (CD31+-memory-Tregs/Tresps), their direct proliferation via CD45RA+CD31\u2212-mature na\u00efve (MN)-Tregs/Tresps, and the production and differentiation of resting MN-Tregs/Tresp into CD45RA\u2212CD31\u2212-memory-Tregs/Tresps (CD31\u2212-memory-Tregs/Tresps) were examined in 115 healthy controls, 96 SLE remission patients, and 20 active disease patients using six color flow cytometric analysis. In healthy controls an appropriate sequence of these pathways ensured regular age-dependent differentiation. In SLE patients, an age-independently exaggerated differentiation was observed for all Treg/Tresp subsets, where the increased conversion of resting MN-Tregs/Tresps particularly guaranteed the significantly increased ratios of ICOS+-Tregs/ICOS+-Tresps and ICOS\u2212-Tregs/ICOS\u2212-Tresps during remission. Changes in the differentiation of resting ICOS+-MN-Tresps and ICOS\u2212-MN-Tregs from conversion to proliferation caused a significant shift in the ratio of ICOS+-Tregs/ICOS+-Tresps in favor of ICOS+-Tresps and a further increase in the ratio of ICOS\u2212-Tregs/ICOS\u2212-Tresps with active disease. The differentiation of ICOS+-RTE-Tregs/Tresps seems to be crucial for keeping patients in remission, where their limited production of proliferating resting MN-Tregs may be responsible for the occurrence of active disease flares.Dysregulations in the differentiation of CD4 Historically, the Th1/Th2 balance is reckoned as decisively important [Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by periods of active disease and remission. The pathophysiology includes strong hyperactivity of B- and T-cells, resulting in autoantibody production against nuclear self-antigens and tissue injury in multiple organs, with lupus nephritis being the most common cause of morbidity and mortality ,2,3,4,5.mportant . Other smportant ,8,9 and mportant ,11,12. Important .+-T-cell signaling in active disease patients [+-T-helper cells in SLE patients, but indicated a shift in the composition of the total CD4+-T-helper cell pool in favor of Tregs. This shift was significantly more pronounced in patients with active disease compared to those in remission [Initially, a general imbalance between Tregs and Tresps was presumed to cause disturbed CD4patients . In contemission . Other semission ,17,18. Semission . Meanwhiemission . However+CD31+-recent thymic emigrant (RTE)-Tregs/Tresps may be disturbed under special pathological conditions such as renal insufficiency, forcing the already distributed RTE-Tregs/Tresp to differentiate more intensely into CD45RA\u2212CD31\u2212-memory Tregs/Tresps (CD31\u2212-memory-Tregs/Tresps). Thus, the reinforcement of individual pathways via CD45RA\u2212CD31+-memory-Tregs/Tresps (CD31+-memory-Tregs/Tresps) or resting CD45RA+CD31\u2212-mature na\u00efve (MN)-Tregs/Tresps influenced both functional properties of Tregs/Tresps, as well as quantitative changes concerning their ratio within total CD4+-T cells [+-T cell system we investigated which differentiation pathways of Tregs or Tresps are used during normal aging in healthy controls compared to SLE patients in remission or in patients with active disease. Moreover, we examined which pathways are age-independently strengthened and therefore probably become exhausted during active disease, as well as which ones are successfully suppressed by immunosuppressive therapy. Here, we distinguished between inducible costimulatory molecule (ICOS)+- and ICOS\u2212-Tregs/Tresps, since our previous studies already showed that an accumulation of na\u00efve cells occurred within total ICOS+-Tregs, indicating that immune senescence of these cells may play a central role in the development of active disease [+-Tregs and, presumably, ICOS+-Tresps with increased proliferation and survival abilities and therefore identifies both ICOS+-Tregs/Tresps as outstanding T-helper cell populations that may potentially affect the regulation of immune responses during autoimmune diseases [We recently demonstrated that the thymic release of CD45RA-T cells . Since S disease . Therebydiseases ,22. Morediseases ,24,25,26+-MN Tregs converted while resting ICOS+-MN Tresps proliferated into CD31\u2212-memory-Tregs/Tresps, causing a shift in the ratio of ICOS+-Tregs/ICOS+-Tresps in favor of ICOS+-Tresps. In contrast, converting resting ICOS\u2212-MN Tregs changed to proliferation while resting ICOS\u2212-MN-Tresps still converted, causing a shift in the ratio of ICOS\u2212-Tregs/ICOS\u2212-Tresps in favor of ICOS\u2212-Tregs.In this present study, we show that different pathways ensure adequate differentiation of these Treg/Tresp subsets with age. In SLE patients with active diseases, the individual pathways exhaust independent of age, while others still proceed and allow age-dependent differentiation. An increased differentiation via resting MN-Tregs/Tresps was ascertained for all Treg/Tresp subsets in active disease. Thus, resting ICOS+- and ICOS\u2212-Tregs/Tresps between healthy volunteers (Group 1) and SLE patients in remission (Group 2) or SLE patients with a flare of the disease (Group 3), we determined the composition of the total CD4+-ICOS+- and ICOS\u2212-Treg/Tresp pools with RTE-, MN-, CD31+- and CD31\u2212-memory-cells in both healthy and affected individuals of different ages and second via their direct proliferation into CD31\u2212-memory-Tregs/Tresps (pathway 2). The additional differentiation of resting MN-Tregs/Tresps into CD31\u2212-memory-Tregs/Tresps (pathway 3) was examined by correlating the percentage of resting MN-Tregs/Tresps within total CD31\u2212-Tregs/Tresps with their own Ki67 expression, as well as with that of CD31\u2212-memory-Tregs7Tresps. Our investigations revealed that in healthy controls a regular combination of these three pathways ensured a stable CD4+-Treg/Tresp cell homeostasis with an increasing ratio of ICOS+-Tregs/ICOS+-Tresps and a consistent ratio of ICOS\u2212-Tregs/ICOS\u2212-Tresps with age. Presumably, the increasing ICOS+-Treg/ICOS+-Tresp ratio contributes to the maintenance of self-tolerance with age. Recently, we showed in different pathologic conditions, such as preterm labor, renal insufficiency, and patients on renal replacement therapy, the described ratios to have a decisive role in maintaining proper Treg- and Tresp-cell functioning [In this study, we examined the differentiation of highly proliferative ICOSctioning ,41.+-Tregs/ICOS+-Tresps ratio, which simultaneously increases in an age-dependent manner. Obviously, this was achieved by an age-independent increase in the conversion of resting ICOS+-MN-Tregs/Tresps, where the differentiation of resting ICOS+-MN Tresps changed from proliferation in healthy controls to conversion in SLE remission patients. In addition, we observed an age-dependent proliferation of ICOS+-RTE-Tregs into ICOS+-CD31\u2212-memory Treg, which could not be assured for ICOS+-RTE-Tresps. An age-independently increased conversion of resting MN-T-cells into CD31\u2212-memory-T-cells was also observed for both ICOS\u2212-Tregs and ICOS\u2212-Tresps, significantly increasing the ICOS\u2212-Tregs/Tresps ratio. In SLE remission patients, the immunosuppressive therapy suppressed exaggerated differentiation of all these T-cell subsets substantially. However, it became apparent that despite a highly sophisticated immunosuppression, an increased additional consumption of resting na\u00efve MN-Tregs/Tresps of these T-cell subsets was detectable. This phenomenon has been known for almost 20 years . Yet, un+-Tregs/ICOS+-Tresps broke down and was significantly decreased, while that of ICOS\u2212-Tregs/ICOS\u2212-Tresps continued to rise significantly, compared to that of healthy controls. In this regard, our data showed an exhaustion of RTE-Treg/Tresp differentiation in all Treg/Tresp T-cell subsets, with an age-independently increased differentiation of resting MN-Tregs/Tresps into CD31\u2212-memory Tregs/Tresps appearing to be most decisively involved in the transition from remission to active disease. Here, the drop in the ratio of ICOS+-Tregs/ICOS+-Tresps seemed to be because differentiation of resting ICOS+-MN Tresps switched from conversion back to proliferation upon breakthrough of the immunosuppressive therapy, increasing the ICOS+-Tresp pool. In these patients, age-dependent differentiation of ICOS+-RTE-Tregs/Tresps via resting MN-Tregs/Tresps was no longer possible, but was replaced by pathways 1 and 2 for ICOS+-RTE-Tresps, but only in a limited way for ICOS+-Tregs. In contrast, the age-independent increase in the ratio of ICOS\u2212-Tregs/ICOS\u2212-Tresps was found to be since the differentiation of ICOS\u2212-resting MN-Tregs changed from conversion to proliferation upon breakthrough of the immunosuppressive therapy, increasing the ICOS\u2212-Treg pool. In these patients, age-dependent differentiation of ICOS\u2212-RTE-Tregs/Tresps via resting MN-Tregs/Tresps was maintained, demonstrating that the differentiation of resting ICOS\u2212-MN-Tregs/Tresps represents the final pathway by which CD31\u2212-memory Tregs/Tresps are produced. However, although this final proliferation of the resting ICOS\u2212-MN Tregs can significantly increase the total Treg pool, it may have a weakening effect on the functionality of the total Treg cell pool [\u2212-CD31\u2212-memory-Tregs with strong proliferative capacity and high apoptosis sensitivity may arise and thus can hardly be re-stimulated. On the other hand, the inability of resting ICOS\u2212-MN-Tresps to proliferate into ICOS\u2212-CD31\u2212-memory Tresps, resulting in ICOS\u2212-CD31\u2212-memory Tresps with lower proliferative capacity and apoptosis sensitivity may be responsible for the increased resistance of these cells for Treg suppression [+- and ICOS\u2212-Tregs/Tresps are differently affected by these mechanisms, the immune homeostasis may be characteristically changed, as shown for SLE patients in remission or active disease. With active disease, the age-independently increased ratio of ICOSell pool , as highpression ,21. Cons+- or ICOS\u2212-Treg/Tresp subsets are specifically involved in the pathogenesis of SLE, further investigations for the identification of characteristic markers on both ICOS+- and ICOS\u2212-Tregs/Tresps may be necessary. It is known that ICOS is predominantly expressed on both follicular and regulatory follicular T helper cells (Tfh and Tfr cells) which regulate antagonistically the quantity and quality of humoral immunity [+-Tregs/Tresps were not further characterized by Tfr or Tfh specific markers, such as BCl6, CXCR5 or PD-1, our data also suggest that the dual role of ICOS in both Treg and Tresp expansion, may contribute decisively to sensitive changes in their ratio in active disease patients. Thereby, our data reveal that excessive differentiation of thymus-derived ICOS+- or ICOS\u2212-RTE-Tregs/Tresps and of the resulting mature na\u00efve Tregs/Tresps affects significantly the ICOS+-Treg/Tresp or ICOS\u2212-Treg/Tresp ratios. Differential expression of ICOS within the thymus was already shown for Tregs [+ or ICOS\u2212-Tregs may be expanded in active SLE in order to compensate for autoreactive effector responses, which were shown for FoxP3+Helios+ Tregs and more recently for a special CD25low+FoxP3+ Helios+-Treg subset [However, regarding the question of which specific ICOSimmunity ,42. Actiimmunity . Howeverimmunity ,45. Althor Tregs . Therefog subset ,47,48. +- or ICOS\u2212-Tresp subsets would be suitable to deliver meaningful results in such ratios in combination with ICOS+- or ICOS\u2212-Tregs. Beside germinal center Tfh cells, various circulating Tfh-like populations and peripheral helper T (Tph) cells, expressing ICOS and producing IL-21, are now described. These cell populations resemble phenotypically Tfh cells but provoke autoantibody production outside of germinal centers [\u2212-Tresps which represent the largest subset within CD4+-T-helper cells, we observed a strong decline of these cells within total CD4+-T-helper cells in association with disease activity. These findings are to some extent consistent with previous studies describing lymphopenia as a common clinical feature and diagnostic criterion for SLE patients [+- and CD8+-T-cells, or Tresps and Tregs. It should be noted that the Treg fraction of the diminished CD4+-T-helper cell pool is increased in SLE patients, whereas the Tresp fraction is decreased. Therefore, these findings presumably rather apply for Tregs than for Tresps. Our findings, showing proliferation of ICOS\u2212-resting MN-Tregs but conversion of ICOS\u2212-resting MN-Tresps rather suggest the formation of apoptosis-sensitive ICOS\u2212-Tregs with diminished functionality, but apoptosis-resistant ICOS\u2212-Tresps with reduced sensitivity for Treg suppression. In summary, our data show strong exhaustion of individual differentiation pathways of RTE-Tregs/Tresps in SLE patients with active disease, where final differentiation via proliferation or conversion of resting ICOS\u2212-MN-Tregs/Tresps may provoke mainly functional changes of the arising ICOS\u2212-memory Tregs/Tresps. In contrast, similar mechanisms may cause quantitative changes of ICOS+-Tregs/Tresps causing disease flares in SLE remission patients. Thereby, the generally restricted differentiation capacity of ICOS+-RTE-Tregs via all three pathways was found to represent a reliable marker for the discrimination of SLE patients in remission or active disease. Hence, immunosuppressive medication targeting ICOS+-Tresp differentiation in SLE patients while enabling Treg differentiation could be a goal of further investigations. Our calculations and the resulting interpretations may have limitations such as unequal numbers of subjects in the groups and that our strategy may be further confirmed. Meanwhile, further studies suggest accelerated immune senescence, not only for CD4+-T-cells, but also for other immune cells, such as CD8+-T-cells and B-cells, as well as for cells of the innate immune system, proposing similarities in immune dysfunctions between SLE patients and elderly people with advanced age [Nevertheless, it remains elusive which ICOS centers . Regardipatients . The undpatients , where rpatients . Howevernced age . Moreoven = 96) or suffering from active disease . Of all SLE patients, 89 (77%) showed kidney involvement. Blood samples were collected during routine visits or during a hospital stay at the Department of Nephrology, University of Heidelberg. Peripheral blood samples were collected from 115 healthy controls (Group 1) and 116 SLE patients. All SLE patients fulfilled the 1983 revised and 1997 updated criteria of the American College of Rheumatology (ACR) for SLE ,55 and w+-T-cells were isolated by immune affinity chromatography with the FABian system , according to the manufacturer\u2019s instructions. This cell isolation process is performed in the FABian column prefilled with a Strep-Tactin-Fab-anti-CD4 coated agarose matrix. The process starts with sucking up whole blood samples. CD4+-T-cells bind to the matrix, while non-target cells are washed away. Adding D-biotin to the matrix causes a dissociation of Fab and target cells from the beads so the CD4+-T-cells can be recovered. Subsequently the isolated CD4+-T-cells are analyzed using six-color flow cytometry.Peripheral venous blood samples (9 mL) were collected from all participants into EDTA-containing tubes. CD46 CD4+-T-cells were surface stained with 5 \u00b5L peridinin-chlorophyll-protein-Cy5-5 (PerCpCy5.5)-conjugated anti-CD127 , 20 \u00b5L phycoerythrin (PE)-conjugated anti-ICOS , 5\u00b5L allophycocyanin-H7 (APC-H7)-conjugated anti-CD45RA (BD Biosciences), and 5 \u00b5L phycoerythrin-cyanine 7 (PE-Cy7)-conjugated anti-CD31 (eBioscience) mouse monoclonal antibodies. Intracellular staining for the detection of FoxP3 was performed using a fluoresceinisothiocyanat (FITC)-conjugated anti-human FoxP3 staining set according to the manufacturer\u2019s instructions. Detection of Ki67+ cells within the different Treg/Tresp subsets was performed by incubating the fixed cells with 2 \u00b5L Alexa-flour 647-conjugated anti-Ki67-conjugated mouse monoclonal antibodies . Negative control samples were incubated with isotype-matched antibodies. FSC-H versus FSC-A and FSC versus SSC gating was used for doublet and debris discrimination for healthy controls, SLE remission patients, and patients with active disease using separate models. The same approach was used for evaluating the changes with age in the composition of the na\u00efve ICOS+- or ICOS\u2212-Treg/Tresp cell pools with RTE- and MN-Tregs/Tresps, as well as the changes with age in the composition of the ICOS+-or ICOS\u2212CD31\u2212-memory Treg/Tresp cell pools. In addition, we calculated the Pearson correlation coefficients (r) between age and the change of variables. A P-value < 0.05 was considered significant.Linear regression was used to evaluate the influence of age on the composition of total ICOS+- or ICOS\u2212-RTE-Tregs/Tresps via CD31+-memory-Tregs/Tresps or proliferation via MN-Tregs/Tresps, an analogous procedure was chosen by correlating the changes in the percentages of ICOS+- or ICOS\u2212-RTE-Tregs/Tresp within their na\u00efve CD45RA+-Tregs/Tresps with the Ki67 expression of their respective RTE-, MN, CD31+-memory, and C31\u2212-memory Tregs/Tresps. Similarly, in order to examine the differentiation of resting ICOS+- or ICOS\u2212-MN-Tregs/Tresps into ICOS+- or ICOS\u2212CD31\u2212-memory Tregs/Tresps, the changes in the percentages of MN-Tregs/Tresps within CD31\u2212-Tregs/Tresps were correlated with the Ki67 expression of their respective MN- and CD31\u2212-memory-Tregs/Tresps. A P-value of < 0.05 was considered significant. Age-independent differences concerning the above listed Treg/Tresp subsets between healthy volunteers, active SLE patients, and inactive SLE patients, were examined using multiple regression analysis adjusted for the age variable (centered on the mean), wherein an interaction term of the age and the patient group was included. Software package BiAS for Windows (version 10.06) was used for all tests.In order to discover the differentiation pathway of ICOS"} +{"text": "In January\u2013March 2020, the Centers for Disease Control and Prevention (CDC) issued multiple warnings regarding COVID-19 travel-associated risks. We sought to describe US travelers seeking pretravel consultation regarding international travel at US Global TravEpiNet (GTEN) sites before and after the initial COVID-19 travel warnings.We prospectively collected data at 22 GTEN sites pre-COVID-19 (January\u2013December 2019) and 18 GTEN sites during the COVID-19 pandemic (April 2020\u2013March 2021). We excluded travelers evaluated during January\u2013March 2020, when CDC travel guidance was evolving rapidly. Travelers used standardized questionnaires to self-report data regarding demographics and travel-related characteristics. Providers confirmed these data and documented their recommendations during pretravel consultation, which could be performed virtually. We conducted descriptive analyses of differences in demographics, travel-related characteristics, vaccinations, and medications . Compared with 16,903 pre-COVID-19 consultations, only 1,564 consultations occurred during the COVID-19 pandemic, a 90% reduction (Table). During COVID-19, a greater proportion of travelers were children aged 1\u20135 years, visiting friends and relatives (VFR), with itineraries \u2265 30 days, and going to Africa; a smaller proportion of travelers were aged > 55 years, or traveling to Southeast Asia or the Western Pacific. During COVID-19, fewer vaccine-eligible travelers received vaccines at the pretravel consultation except for yellow fever, and a greater proportion were referred to another provider for vaccination . Table. Demographics and travel-related characteristics of international travelers seeking pretravel consultation at Global TravEpiNet sites before and during the COVID-19 pandemicTable continued. Demographics and travel-related characteristics of international travelers seeking pretravel consultation at Global TravEpiNet sites before and during the COVID-19 pandemicFigure. Vaccinations and reasons for nonvaccination among vaccine-eligible international travelers at pretravel consultations at Global TravEpiNet (GTEN) sites before and during the COVID-19 pandemic.Among vaccine-eligible travelers, we summarized those who were vaccinated at the visit (blue) and not vaccinated (orange). We then categorized reasons for nonvaccination into: provider decision (solid), referral to another provider (dots), traveler refusal (striped), or other (hatched). COVID-19 vaccination was not available at GTEN sites during the analysis period; although COVID-19 vaccinations outside of GTEN sites might have affected vaccination recommendations, they were unlikely to have had a large effect given their limited availability in January-March 2021.Compared with pre-COVID-19, US travelers seeking pretravel consultations at GTEN sites during the pandemic might be at higher risk for travel-related infections given VFR status, traveling for \u2265 30 days, and going to Africa. Fewer vaccine-eligible travelers were vaccinated at pretravel consultations, which could reflect more virtual pretravel consultations. Counseling and vaccination for international travelers continue to be priorities during the COVID-19 pandemic.All Authors: No reported disclosures"} +{"text": "As a result of FDA approval of CAR-T cell treatments in the last few years, this immunotherapy has provided further direction to precision medicine through its combination with other therapeutic approaches. In the past year, several review articles have been published focusing on advances in this fast-developing field, especially with respect to efforts to overcome hurdles associated with applying CAR-T cells in solid tumors. This review paper focuses on combining CAR-T cell therapy with small molecule drugs, up-to-date progress in CAR-T cell therapy research, and advances in combined CAR-T immunotherapy with other treatments targeting solid tumors.Chimeric antigen receptors (CAR) T cells are T cells engineered to express membrane receptors with high specificity to recognize specific target antigens presented by cancer cells and are co-stimulated with intracellular signals to increase the T cell response. CAR-T cell therapy is emerging as a novel therapeutic approach to improve T cell specificity that will lead to advances in precision medicine. CAR-T cells have had impressive outcomes in hematological malignancies. However, there continue to be significant limitations of these therapeutic responses in targeting solid malignancies such as heterogeneous antigens in solid tumors, tumor immunosuppressive microenvironment, risk of on-target/off-tumor, infiltrating CAR-T cells, immunosuppressive checkpoint molecules, and cytokines. This review paper summarizes recent approaches and innovations through combination therapies of CAR-T cells and other immunotherapy or small molecule drugs to counter the above disadvantages to potentiate the activity of CAR-T cells. CAR T-cell therapy entails the engineered modification of autologous T cells to robust T cells that can initiate anti-tumor reactivity of the target tumor cells. These therapeutics have produced meaningful clinical outcomes in the treatment of hematologic cancers, but have not produced comparable responses in solid malignancies. Notably, CAR-T therapy with the most investigated target thus far, CD19, is a promising therapy for hematologic cancer and has dramatically improved the treatment of lymphoid malignancies, predominantly diffuse large B-cell lymphoma (DLBCL) and acute lymphoblastic leukemia (ALL). Besides CD19, other common targets include B cell maturation antigen (BCMA), CD20, mesothelin (MSL), and PD-1/PD-L1 . The CARPreviously, TCR-like antibody CAR-T cells with scFv recognizing the MHC/peptide complex have improved T cell activation and proliferation by targeting a variety of intracellular antigens of solid tumors . SubsequNanobody enhanced immunoactivity resulting from the presence of an antigen-binding domain on CAR-T cells through its high affinity and stability has been applied in different generations of CARs. Nanobody second-generation CAR-T cells exhibited over 50% positive expression on the cell surface, thus secreting IL-2 and IFN-\u03b3 and boosting the cytotoxicity in VEGFR2 expressing cells . AdditioBivalent nanobodies in bi-specific CAR-T cells significantly reduced tumor escape by simultaneously recognizing two targets, for example, CD20 plus either HER2, PD-L1, or EIIIB, on the cells with a resultant loss of target expression ,14. MostOver the past seven years, extensive research on the inhibition of immune checkpoints targeting PD-1 and PD-L1 has resulted in the rapid expansion of the class of checkpoint inhibitor drugs. More than 2000 clinical trials have been studied that focused on over 30 anti-PD-1 or anti-PD-L1 antibodies . Among tInhibitors specific to immune response checkpoint ligands on tumor cells or receptors on T cells are designed to block the immune response to the tumor. CAR-T cell therapy incorporated with PD-1 blockade has demonstrated a synergistic effect in the treatment of hematological malignancies: for example, PD-1 blockade coupled with CD19 CAR-T cell therapy. Masked CAR is another strategy to overcome cytotoxicity in normal cells while showing tumor-killing activity on EFGR-positive human tumors. Researchers have designed EGFR-targeting CARs that lead to inactive PD-1 in the non-cancerous environment but are selectively activated through proteolytic cleavage by protease present in the tumor environment ,21. In cHowever, CAR-T cell combination therapy with inhibitory checkpoint PD-1 blockade could present potential side effects, especially cytokine release syndrome (CRS). Checkpoint inhibition has notably shown one of the most significant adverse effects in CAR-T immunotherapies . The mosThe encouraging synergistic therapeutic effect of checkpoint inhibitors and CAR-T cells gives rise to the development of armored CAR-T cells that secrete immune checkpoint inhibitors to overcome immunosuppression within solid tumors, which possess physical and immune obstacles that limit CAR-T effectiveness . Novel aTumor cells suppress the immune response at tumor sites through a strategy involving increased TGF-\u03b2-mediated repression of T-cell proliferation coupled with a Treg-mediated hostile microenvironment . CAR-T cCytokines are released as part of the immune response and play a role in the immunosuppressive environment at tumor sites that promotes tumor progression. Certain cytokines upregulate the expression of PD-1 and PD-L1 to inhibit T cell activation and proliferation, leading to immunosuppression and negative feedback inhibition. Investigators modified a third-generation CAR to express PD-L1 and CD28 fusion receptors aimed at suppressing the inhibition with CD28. They fused the extracellular domain of PD-1 with the intracellular domain of CD28. Upon binding its extracellular ligand PD-L1, the fusion receptor transmitted an activation signal instead of an inhibitory signal with the CD28 cytoplasmic domain, hence a \u201cswitch\u201d. These switch receptors improved cytokine release, proliferation, and cytotoxicity of CAR-T cells . FurtherTargeted therapies such as monoclonal antibodies and small-molecule inhibitors have improved CAR-T cell infiltration and specificity. Monoclonal antibodies target specific antigens found on the cell surface, whereas small-molecule drugs can translocate through the plasma membrane to interfere with the enzymatic activity and signaling pathways associated with tumor growth, survival, angiogenesis, and metastasis . MonocloThe majority of small molecules target signal transduction mechanisms of proliferation and survival in many tumors; for example, tyrosine or serine/threonine kinases and the mitogen-activated protein kinase (MAPK) signaling cascade including RAS, RAF, MEK, and ERK. Kinase inhibitors can target several growth factor receptors with aberrant activation or deregulation in tumors such as EGFR, VEGFR, IGFR, and FGFR. VEGF signaling induces the suppression of CD8-positive T cell proliferation and reduces their cytotoxic activity . MoreoveGiordano-Attianese et al. designed CAR-T cells that can be switched off through its chemically disruptable heterodimer by administering a small-molecule drug. These switchable CAR-T cells provide a controllable means to improve the safety of CAR-T cell therapy and reduce the risk of toxicity while mounting similar anti-tumor killing activity as second-generation CAR-T cells . EquallyCollectively, these results suggest that a systematic and integrated understanding of the molecular consequences of small molecules on CAR-T cells, tumor cells, and the microenvironment has been implemented for the optimal design of combined therapies to limit the risk of toxicities.These studies collectively suggest that novel CAR-T cell modifications and combinations are being rapidly introduced with the goal of enhancing the efficacy of CAR-T therapy or reducing unfavorable responses. These new CAR-T therapies can be partitioned into several major groups .Multi-target CAR-T cells address tumor antigen escape through multiple antigen-targeting CARs on a single CAR-T cell to overcome antigen loss or faded expression of a single antigen. Each unispecific CAR has a different individual binding domain. Multi-target CAR-T cells exert efficiency in the cytolysis of tumor tissues due to the high density of CAR expression on the surface of T cells. An example is multi-target CAR-T cells simultaneously expressing a receptor for the activation of T cells via CD3z and the antigen-MHC complex and another receptor such as CD28 and/or CD137 for a co-stimulatory signaling process . Given hAdditionally, multi-specific CAR-T cells can be more potent and efficient than unispecific CAR-T cells in improving the durability of CAR response . A biCARAnother multi-target CAR-T cell approach is Fc-gamma chimeric receptor T cell-based immunotherapy ,73. ThisThus, in theory, this approach would enable improved ADCC-associated T-cell cytotoxicity toward any cancer cell expressing tumor-associated antigens (TAAs) that are targetable by therapeutic mAbs. This concept of potential universality of TAA targeting is further illustrated by a study of mogamulizumab (Mog), a humanized mAb that targets a different TAA, namely CC chemokine receptor 4 (CCR4). Mog exhibits ADCC through CD16-expressing effector cells, and a CD16 retroviral vector construct expressing 158V/V-CD3\u03b6, (cCD16\u2013CD3\u03b6) was infused in combination with Mog into NOD/scid/\u03b3cnull (NOG) female mice along with autologous cCD16\u03b6-T cells from a patient with aggressive adult T-cell leukemia cells . This thIn a recent comparison of CD16-CAR-T and CD32-CAR-T cells in KRAS-mutated colorectal carcinoma, even though CD32 was able to effectively bind soluble mAbs and provide a cytotoxic signal, CD32-CAR-T cells did not produce pro-inflammatory cytokines when co-cultured with KRAS-mutated HCT116 colorectal cancer cells, and they had no effect on HCT116 cell viability. However, CD16158V-CAR-T cells combined with cetuximab decreased HCT116 cell viability and SCID mice injected subcutaneously with KRAS-mutated HCT116 cells, followed in one hour by injection of CD16158V-CAR-T cells plus cetuximab near the HCT116 injection site, showed decreased tumor growth and increased disease-free survival . This saTandem CAR-T (TanCAR) cells express one CAR containing two binding domains linked in tandem, sharing a common intracellular domain. Similar to dual-signaling CARs, TanCAR-T cells expressing two scFv domains on one CAR exert an effect similar to that of dual-signaling CAR-T cells . TanCAR Despite promising results in clinical trials, lack of control over-engineered cells once infused into the patient can result in severe adverse events requiring control strategies to improve safety. Excessive cytotoxicity activity and poor control of CAR-T cells have been challenges limiting the application of CAR-T therapies ,85. In aB cells targeting CD19, CD22, and HER2 showed promising results of switchable CAR-T cell potency, demonstrating that clinicians could control the anti-tumor reactivity switch molecule ,89,90. ITAAs express with low density in normal cells and T cells targeting TAAs possibly induce on-target off-tissue toxicity. EGFR is overexpressed in tumor cells and has been the target of therapies against various types of cancer. Nevertheless, on-target off-tissue toxicity occurs when normal cells express EGFR at lower levels. Liu et al. generated T cells modified with EGFR-specific CARs to reduce the \u201con-target off-tissue\u201d toxicity . The novInhibitory CARs (iCARs) recognize specific antigens expressed on normal or non-cancer cells to direct the CAR-mediated attack away from healthy cells. Thus, induction of the negative signaling keeps CAR-T cells from attacking normal cells. For example, Fedorov et al. evaluated PSMA iCARs, PSMA-targeted CAR-T cells expressing PD-1 and CTLA-4 for negative signaling when encountering PSMA in normal cells . With adApplication of suicide switches by inserting suicide genes into CAR-T cells that migrate to tumors and initiate the process of cell suicide can limit the CAR-T cell toxicity . The eliPooled CAR-T cell therapy involves the infusion of multiple single-targeting CAR-T cells to acquire lower tumor relapse. Each CAR-T cell targets a different cognate antigen. Pooled CAR-T cells secrete a higher level of cytokines and anti-tumor activity than individual CAR-T cells. However, it is of concern that one CAR-T cell may expand over the other as anti-CD19 CAR-T cells are known to increase predominantly over anti-CD20 CAR-T cells, eventually resulting in a net decline of anti-CD20 CAR-T cells. Dual-targeted CAR-T cell therapy is preferred over single-targeted treatment because it prevents CD19-negative recurrence after CAR-T cell treatment. More studies are needed on CD19/CD20, CD19/CD22, or CD33/CD123 dual-targeted CAR-T cells for enhanced CAR-T cell function ,107. A pCancer cells typically outgrow the vascular system around tumor tissue and require neovascularization for further growth. Angiogenesis and metastasis depend on the expression of vascular endothelial growth factors (VEGF) and their receptors (VEGFRs) in the tumor. Therefore, the unique vascular network around the tissue is a target for CAR-T therapy ,109. CARTargeting other factors in the tumor microenvironment is another therapeutic strategy for CAR-T cell therapy. One example is CAR-T cells constructed against the fibroblast activation protein (FAP) to target cancer-associated fibroblasts (CAPs), which secrete growth factors to the extracellular matrix (ECM) to support tumor growth . FAP-speHeparan sulfate proteoglycans (HSPGs) prevent CAR-T cell transport through the ECM in the stroma-rich tumor. Decreased heparanase (HPSE) mRNA expression reduces the anti-tumor function of CAR-T cells in solid tumors because HPSE aids in the penetration of HSPGs, which in turn increases the abundance of the ECM. ThereforThe infiltration of CAR-T cells to the tumor site is significantly reduced when the chemokine receptor expressed by T cells is not compatible with chemokines secreted by tumor cells, as noted in the case of CXCL-1, CXCL-5, and CXCL-12. CAR-T cells designed to increase the expression of those chemokine receptors that recognize chemokine secreted by tumors are known to improve the migration of CAR-T cells to a target site. Kershaw et al. developed CAR-T cells expressing chemokine receptor CXCR2 that binds to the ligand CXCL1 on tumor cells. They found that CXCR2 CAR-T cells effectively migrate toward melanoma . In anotIn the last decade, CAR-T immunotherapy has become an exciting approach with great potential in precision medicine. Novel strategies have been developed to enhance the tumor-killing activity, endurance, infiltration to solid tumor tissues, and regulation of the immune microenvironment. Combined therapies are being developed to improve immune response, minimize \u201con-target off-tumor\u201d effects, and transform immunologically \u201ccold\u201d to \u201chot\u201d tumors. More studies are focused on identifying ideal target selection and targeting multiple tumor-associated antigens. Equally important, development in optimal treatment timing, persistence of CAR-T cells, and expansion of the CAR-T cells within the tumor immunosuppressive microenvironment have been further addressed. The configuration of these therapies will result in more effective treatment and improved response in tumors."} +{"text": "Recent emergence of SARS-CoV-2 and associated COVID-19 pandemic have posed a great challenge for the scientific community. In this study, we performed bioinformatic analyses on SARS-CoV-2 protein sequences, trying to unravel potential molecular similarities between this newly emerged pathogen with non-coronavirus ssRNA viruses. Comparing the proteins of SARS-CoV-2 with non-coronavirus positive and negative strand ssRNA viruses revealed multiple sequence similarities between SARS-CoV-2 and non-coronaviruses, including similarities between RNA-dependent RNA-polymerases and helicases (two highly-conserved proteins). We also observed similarities between SARS-CoV-2 surface (i.e. spike) protein with paramyxovirus fusion proteins. This similarity was restricted to a segment of spike protein S2 subunit which is involved in cell fusion. We next analyzed spike proteins from SARS-CoV-2 \u201cvariants of concern\u201d (VOCs) and \u201cvariants of interests\u201d (VOIs) and found that some of these variants show considerably higher spike-fusion similarity with paramyxoviruses. The \u2018spike-fusion\u2019 similarity was also observed for some pathogenic coronaviruses other than SARS-CoV-2. Epitope analysis using experimentally verified data deposited in Immune Epitope Database (IEDB) revealed that several B cell epitopes as well as T cell and MHC binding epitopes map within the spike-fusion similarity region. These data indicate that there might be a degree of convergent evolution between SARS-CoV-2 and paramyxovirus surface proteins which could be of pathogenic and immunological importance. Current COVID-19 pandemic which is caused by a newly emerged betacoronavirus has led to immense health and socio-economic problems around the world. First human coronaviruses were discovered in 1960s, but human coronavirus infections have likely existed for centuries . In the SARS-CoV-2 nucleic acid and protein sequences did become available shortly after the start of COVID-19 pandemic and biological aspects of these genes/proteins including their interactions with immune system have been under intense investigation. In this study, we performed a comprehensive bioinformatic analysis on SARS-CoV-2 protein sequences to determine whether there might be any biologically interesting similarities between SARS-CoV-2 proteins with non-coronavirus ssRNA viruses. We performed protein similarity searches for individual SARS-CoV-2 proteins against available protein sequences from different ssRNA virus families. This revealed some interesting similarities between SARS-CoV-2 spike protein and paramyxovirus surface proteins as well as SARS-CoV-2 RdRp (RNA-dependent RNA polymerase) and helicase proteins with Togaviruses and Caliciviruses. Considering the importance of spike proteins in viral infectivity and immune response, we then performed more analyses on other coronavirus spike proteins as well as analyses to determine potential immunological significance of these similarities.https://www.ncbi.nlm.nih.gov/protein). Redundant sequences as well as polyprotein sequences (i.e. ORF1ab and ORF1a) were removed. RefSeq accession numbers and their official gene names are shown in S1 Table in https://viralzone.expasy.org).SARS-CoV-2 Reference Protein sequences were obtained from NCBI Protein database was used to extract the list of viruses which are pathogenic for humans .Virus-Host database (Peptide sequences from SARS-CoV-2 spike protein which could act as B cell or T cell epitopes or MHC binders were determined using immune epitope database (IEDB). Epitope search was limited to experimentally verified epitopes. Linear epitopes were considered that had exact matches with substrings of SARS-CoV-2 spike protein sequence. Pairwise BLASTP search was used to find the distribution of these epitopes on spike protein.Phylogenetic analysis was performed using RNA-dependent RNA polymerase (RdRP) protein sequences from different ssRNA viruses. RdRP sequences from representative positive strand and negative strand ssRNA viruses were obtained from NCBI Protein Database. Multiple sequence alignment (MSA) and phylogenetic analysis were performed using MEGA X and PhyML . MUSCLE SARS-CoV-2 is a member of betacoronaviruses, which belong to Nidovirales order of positive sense ssRNA viruses . To inveWe next compared SARS-CoV-2 proteins with negative strand ssRNA viruses, with Paramyxoviridae, Orthomyxoviridae, Rhabdoviridae and Filoviridae, analyzed individually. Similarity searches against Paramyxoviridae showed similarities between SARS-CoV-2 spike protein and fusion proteins of various paramyxoviruses . SequencResults of all similarity searches for positive and negative sense ssRNA viruses are illustrated in overlay plots . Furthergenome.jp/virushostdb/) and extracted their alignment score- E values from Delta-BLAST search. As shown in The region of SARS-CoV-2 spike protein which shows similarity with paramyxovirus fusion proteins in displayed in Since its discovery in late 2019, SARS-CoV-2 has been undergoing genetic changes that have influenced its transmissibility and pathogenesis. Based on criteria including their virulence, transmissibility and susceptibility to antiviral drugs, SARS-CoV-2 variants have been broadly categorized to \u2018variants of concern\u2019 (VOC) and \u2018variants of interest\u2019 (VOI) . Varioushttps://www.genome.jp/virushostdb) and obtained their spike protein sequences from NCBI protein database is widely used for performing phylogenetic studies on RNA viruses. Representative pathogenic viruses were selected from each group demonstrated a higher score-log E value compared to other human coronaviruses. The degree of similarity differed between SARS-CoV-2 genetic variants and included several B cell, T cell and MHC binding epitopes.Paramyxoviruses include several major human and animal pathogens, with well-recognized pathophysiology and immune behavior \u201330. HeniEvolution from a common ancestor is usually the underlying reason for the existence of similarities between nucleic acid and/or protein sequences. Indeed, the concept of \u201csequence homology\u201d entails \u201cshared ancestry\u201d and sequence similarities between taxa which do not share a recent common ancestor are referred to as \u2018homoplasy\u2019 , 32. PhyUnraveling different aspects of the evolution of microbial pathogens is of paramount importance in predicting, understanding and controlling infectious disease epidemics. Ordinary bioinformatic analyses on nucleic acid and/or proteins might give us some hints about the relatedness or dissimilarities between pathogenic microorganisms, but grasping the complexity of this dynamism would likely require integrated analyses of host(s)-pathogen(s) co-evolution.S1 File(DOCX)Click here for additional data file."} +{"text": "EGFR amplification and CDKN2A/B homozygous deletion. We also investigated the immune cluster-specific distribution of four malignant cellular states in the IDH-wildtype cohort. We identified two major immune-based subgroups of IDH-mutant gliomas, which largely aligned with 1p/19q co-deletion status. Non-codeleted gliomas showed distinct proportions of a key genomic aberration (CDKN2A/B loss) among immune cell-based groups. We also observed significant positive correlations between monocyte proportion and expression of PD-L1 and PD-L2 . Overall, the findings highlight specific roles of the TME in biology and classification of CNS tumors, where specific immune cell admixtures correlate with tumor types and genomic alterations.It is recognized that the tumor microenvironment (TME) plays a critical role in the biology of cancer. To better understand the role of immune cell components in CNS tumors, we applied a deconvolution approach to bulk DNA methylation array data in a large set of newly profiled samples (n\u2009=\u2009741) as well as samples from external data sources (n\u2009=\u20093311) of methylation-defined glial and glioneuronal tumors. Using the cell-type proportion data as input, we used dimensionality reduction to visualize sample-wise patterns that emerge from the cell type proportion estimations. In IDH-wildtype glioblastomas , we identified distinct tumor clusters based on immune cell proportion and demonstrated an association with oncogenic alterations such as The online version contains supplementary material available at 10.1186/s40478-021-01249-9. NF1 and RB1 [EGFR-amplified and PTEN-deleted) [Glial and glioneuronal tumors represent a wide range of tumor types with distinct biology and clinical outcomes and are currently assigned WHO grades 1\u20134 based on histopathologic and molecular features . The tum and RB1 . Conversdeleted) . IDH-mutdeleted) . Finallydeleted) , consistdeleted) , 78.PDGFRA amplification and RTKII group showed combined chromosome 7 gain/chromosome 10 loss (+\u20097/\u221210), CDKN2A loss and amplification of epidermal growth factor receptor (EGFR) [Aberrant DNA methylation is recognized as a key process for tumor development . Distincr (EGFR) , 81, 85.r (EGFR) . Recentlr (EGFR) .Deconvolution of bulk tumors has typically involved in situ-based techniques, such as immunohistochemistry (IHC), as well as fluorescence-activated cell sorting (FACS), and more recently single-cell RNA sequencing (RNA-seq). Recent in silico techniques have allowed deconvolution on a much larger scale by utilizing high-throughput assays such as gene expression and DNA methylation microarrays , 83, 84.http://bioconductor.org/packages/conumee/) as implemented in the classifier package. Batch effects were examined using variables of formalin-fixed, paraffin-embedded specimens (FFPE) versus frozen specimens. We also compared the 2 array types (450\u00a0k vs. EPIC). We did not observe major batch effects with respect to material type and array type.Samples (n\u2009=\u2009741) were profiled as part of clinical methylation testing and analyzed as previously described by Capper et al. , and appWe collated published literature to collect additional data for glial/glioneuronal tumor subtypes. The following publicly-available datasets were included for downstream analyses: GSE104293 , GSE1286\u03b2-value difference of 0.2 and false discovery rate of 0.01.\u00a0We selected 1,290 probes from the generated signature matrix differentiating 13 cell types: B-cells, cancer, CD4T, CD8T, endothelial, eosinophil, glia, microglia, monocyte, neuron, neutrophil, NK cells and Treg. To validate the matrix represents a homogeneous signature for each putative cell type, we performed dimensionality reduction (t-SNE) of pure reference cell types and heatmap with unsupervised clustering of selected probes and all cancer cell lines used to create the signature matrix. We generated scatter plots between cancer fraction and two purity measures (ESTIMATE purity and ABSOLUTE purity) [To construct the reference immune cell signature matrix, we collected methylation profile of pure non-neoplastic cell types. Raw data files (idat) were obtained from publicly-available sources, including B-cells , 72, 77, purity) to demon purity) . To vali purity) . We obse purity) .We classified all samples N\u2009=\u20094052) into three representative cohorts: IDH-wt (N\u2009=\u20092072), IDH-mut (N\u2009=\u20091178) and LIGGNT (N\u2009=\u2009802). Our main objective was to investigate an immune cell fraction-based clustering pattern within these three major cohorts. We first investigated the clustering distribution by both including and excluding the inferred cancer proportion in each dataset as described by Capper et \u00a0al. [Somatic copy number variants were computed from raw signal intensities (IDAT) using the Conumee R package . This me et \u00a0al. and we oTo investigate a possible association of monocyte proportion with gene expression and promoter methylation of the immune checkpoint ligands PD-L1 and PD-L2, we used a subset of the data (n\u2009=\u2009594) which contained matched gene expression profiles (RNA-seq). RNA-seq trimmed mean M-values (TMM) was performed using the calcNormFactors function in the edgeR package . Using tsurvminer and survival packages in R [Survival data was collected from original source of data (if available) as described in data collection sub-section of material and methods. To examine the association of immune cell proportion with overall survival, we performed Kaplan\u2013Meier survival analyses with the log-rank test using the ges in R . SamplesR\u2009=\u20090.59, p value\u2009<\u20092.2e\u221216) and ABSOLUTE purity . This set consisted of seven methylation-defined glioma types: GBM-G34, GBM-MES, GBM-MID, GBM-MYCN, GBM-RTKI, GBM-RTKII and GBM-RTKIII that were defined using DKFZ DNA methylation classifier calibrated scores (version 11b4) . From thWe first examined clustering of IDH-wt GBM based on the calculated immune cell proportions. K-means consensus clustering was performed to identify an optimal number of clusters across all IDH-wt tumor samples N\u2009=\u20092,072). The optimal number of clusters was determined by NbClust R\u00a0package by providing 30 indices for determining the number of clusters . Consens,072. TheImmune cell proportions showed a distinct relationship with GBM methylation classes. As shown in Fig.\u00a0conumee R package to analyze frequent genomic aberrations (amplifications/deletions) in GBM [EGFR amplification and was approximately twice as frequent as tumors in clusters 3 and 4 based on genomic aberrations.Next, we investigated select copy number alterations in IDH-wt gliomas with respect to the defined immune clusters Table. . We used) in GBM . Clusterd 4 Fig.\u00a0e. Tumorsups Fig.\u00a0f. Interep\u2009<\u20090.01 and p\u2009=\u20090.05, respectively) have been identified based on single-cell RNA sequencing data . Here, wThe IDH-mutant cohort (N\u2009=\u20091178) was next examined and comprised the methylation classes O-IDH (oligodendroglioma), A-IDH (astrocytoma) and A-IDH-HG (high-grade astrocytoma). The presence of an IDH mutation was inferred from the methylation data. Surprisingly, visualization (UMAP) revealed apparent discrimination of these three methylation classes according to relative immune cell proportion Fig.\u00a0a. K-meanWe next focused on oligodendroglioma samples in isolation N\u2009=\u2009444), where consensus clustering on immune cell proportion indicated four major clusters , showing a higher proportion in cluster 2 consistent with the higher overall tumor grade in this cluster , we identified five optimal immune-based clusters Fig.\u00a0a, b. Sigter Fig.\u00a0e. Similater Fig.\u00a0: Fig.\u00a08.Immune-based clustering of low-to-intermediate grade glioneuronal tumors (LIGGNT) (N\u2009=\u2009802) consisted of 11 DNA methylation-based types: anaplastic pilocytic astrocytoma/high grade astrocytoma with piloid features (ANA-PA), desmoplastic infantile ganglioglioma and astrocytoma (DIG-DIA), dysembryoplastic neuroepithelial tumor (DNT), ganglioglioma (GG), low-grade glioma with MYB/MYBL1 alteration (MYB), midline pilocytic astrocytoma (PA-MID), pilocytic astrocytoma, posterior fossa (PA-PF), supratentorial pilocytic astrocytoma/ganglioglioma (PA/GG-ST), rosette-forming glioneuronal tumor (RGNT), subependymal giant cell astrocytoma (SEGA), and pleomorphic xanthoastrocytoma (PXA) Fig.\u00a0a. K-meanp\u2009<\u20092.2e\u221216 and 0.68; p\u2009<\u20092.2e\u221216, respectively). Consistently, we found high negative correlations between monocytes and promoter methylation of PD-L1 in contributing to these malignant cell states, suggesting cross-talk between tumor cell states and the immune microenvironment.Studies of cell states in glioblastoma have previously demonstrated an association with chromosomal alterations . We therA recent study showed that IDH wild-type tumors associated with high degree of immune cell infiltration, and were suggested as immune-hot phenotype, whereas IDH mut-tumors with 1p/19q codeletion cohort showed low degree of immune cell infiltration as immune-cold phenotype . Our ImmRecently, it has been shown that CDKN2A homozygous deletion was associated with poorer outcome among IDH-mutant gliomas lacking 1p/19q codeletion (IDH-mutant astrocytoma) as well as among anaplastic oligodendrogliomas, IDH-mutant-1p/19q codeleted . We alsoImmune clustering of low-to-intermediate grade glioneuronal tumors (LIGGNT) showed some promising outputs regarding tumor subtype specific overall infiltration of immune cells. We observed that several tumor subtypes, including MYB, DNT, GG and RGNT showed lower infiltration of immune cells as compared to other subtypes and these observations are in line with a recent study .Given that cells of the monocyte lineage are one of the most dominant and key modulator of tumor microenvironment in glio-neuronal tumors, we examined the association of monocytes with programmed cell death ligands such as PD-L1 and PD-L2 gene expression, which plays crucial role in immunotherapy. It has been shown in literature that GBM EVs (extracellular vesicles) induce immunosuppressive monocytes, including myeloid-derived suppressor cells (MDSCs) and nonclassical monocytes (NCMs) and glioIn conclusion, our analysis relies on a large sample set of glial/glioneuronal tumors to demonstrate relationships of tumor immune microenvironmental factors with tumor type and key genomic aberrations. We also highlight the prominent role of monocytic lineage cells in these tumors, including associations with expression of immune checkpoint ligand PD-L1 and PD-L2 in these tumors. Results of this investigation provides insights for future investigation into glioma biology and immunotherapeutic approaches in gliomas.Additional file 1: To validate our MethylCIBERSORT output we selected a data subset (N=394) with available gene expression data. CIBERSORTx method was used with LM6 signature matrix to get fraction of six major cell types. We found significant positive correlations between MethylCIBERSORT fraction and CIBERSORTx derived fraction of six major cell types.Additional file 2: Assessment of clustering pattern in all three major cohorts IDH-wt (N=2072), IDH-mutant (N=1178) and Low-to-intermediate grade glioneuronal tumors (LIGGNT) (N=802) by including and excluding cancer part and scaling non-cancer part from 0 to 1. (a) UMAP clustering for IDH-wt cohort with all cell type (including cancer). (b) UMAP clustering for IDH-wt cohort with all normal cell type scaled 0 to 1 (non-cancer part). (c) UMAP clustering for IDH-wt cohort with all immune cells type scaled 0 to 1 (non-cancer part). (d) UMAP clustering for IDH-mutant cohort with all cell type(including cancer). (e) UMAP clustering for IDH-mutant cohort with all normal cell type scaled 0 to 1 (non-cancer part). (f) UMAP clustering for IDH-mutant cohort with all immune cells type scaled 0 to 1 (non-cancer part). (g) UMAP clustering for LIGGNT cohort with all cell type (including cancer). (h) UMAP clustering for LIGGNT cohort with all normal cell type scaled from 0 to 1 (non-cancer part). (i) UMAP clustering for LIGGNT cohort with all immune cells type scaled 0 to 1 (non-cancer part).Additional file 3: Cluster selection process was based on the majority rule, which is available in the NbClust package. We compared three different methods; kmeans, ward.D and ward.D2. (a) For each method we selected optimum number of clusters (given in brackets) proposed by maximum number of indices out of 30. We compared output of each method and found similar results. Finally, we selected kmeans clustering as a uniform approach to select optimum number of cluster (proposed by maximum number of indices out of 30) in each cohort. (b) In IDH wild type 11 indices proposed five cluster. (c) In IDH mutant type 8 indices proposed two cluster. (d) In O-IDH cohort, 6 indices proposed two and four clusters respectively. In this case we selected four cluster as ward.D2 method also suggested four clusters in O-IDH cohort. (e) In A-IDH/A-IDH-HG 8 indices proposed five clusters. (f) In LIGGNT 10 indices proposed two clusters.Additional file 4: (a) Tumor subtype specific bar plot distribution of mean Immune cell proportion . (b) Overall cell fractions of each cell type . (c) Cancer proportion shown by boxplots for each tumor subtype indicated significant differences. T-test and Wilcoxon test were used to calculate statistical significance.Additional file 5: Sankey diagram-based associations between immune cells and tumor subtype in IDH-wt cohort (N=2072). Sankey plot showing proportions shared between each immune clusters and tumor subtype.Additional file 6: Analysis of the IDH-wt glioblastoma cohort (N=100) shows cluster specific distribution of AC-like, OPC-like, MES-like and NPC-like cellular states, respectively. Tumor cell states were derived from single cell data of IDH-wild type GBMs (PMID: 31327527). A signature matrix was derived and applied to TCGA samples for which gene expression data were available. Cell state estimations for each sample were performed using CIBERSORTx. T-test and Wilcoxon test were used to calculate statistical significance.Additional file 7: Kaplan-Meier plot of (a) GBM-MES for Monocyte proportion high and low groups . (b) GBM-RTK-I dataset with high and low proportion of monocytes . (c) Kaplan-Meier plot for Endothelial cell high and low groups in GBM-MES .Additional file 8: To investigate homozygous and heterozygous deletion of CDKN2AB as a separate event we selected a range of log2ratio and annotated samples as an event of homozygous deletion, heterozygous deletion, and no deletion. Significance was calculated by applying Fisher's exact test; p-value <."} +{"text": "Chloropsis cochinchinensis sensu lato), which have evolved from ancestral quasi-ordered channel-type nanostructures. Self-assembled avian photonic crystals may serve as inspiration for multifunctional applications, as they suggest efficient, alternative routes to single gyroid synthesis at optical length scales, which has been experimentally elusive.Vivid, saturated structural colors are conspicuous and important features of many animals. A rich diversity of three-dimensional periodic photonic nanostructures is found in the chitinaceous exoskeletons of invertebrates. Three-dimensional photonic nanostructures have been described in bird feathers, but they are typically quasi-ordered. Here, we report bicontinuous single gyroid \u03b2-keratin and air photonic crystal networks in the feather barbs of blue-winged leafbirds ( Many animals produce vivid, saturated structural colors via constructive light interference from diverse integumentary nanostructures with mesoscopic (\u223c100 nm to 350 nm) long-range order or short-range translational order . StructuChloropsis cochinchinensis s. l., Chloropseidae), revealed by synchrotron small-angle X-ray scattering (SAXS) and scanning electron microscopy (SEM). We compare these ordered morphologies to homologous channel-type nanostructures with short-range order and intermediate structures from other closely related Chloropsis species, and their sister group, the fairy bluebirds , 8.I4132) space group is a measure of crystallite domain size, and thus the extent of long-range translational order (Chloropsis species (green line) exhibits a continuum of intermediate states (green triangles), from ancestral channel-type nanostructures of Irena (blue dashed line) to the derived single gyroids of C. cochinchinensis (\u0394q) relative to their dominant length scales (qpk), Q = qpk/\u0394q \u2248 4, suggesting that scale-invariant or size-independent processes G. Intereipennis) exhibit rocesses underlieChloropsis and Irena are homologs that first evolved in the most recent common ancestor of these two monophyletic sister genera . In bothittidae) result iittidae) .Single gyroids that can exhibit large complete bandgaps have long been a target for photonic and photovoltaic engineering (reviewed in ref. Quasi-ordered channel-type photonic nanostructures of avian feather barbs are understood to self-assemble within medullary cells likely via self-arrested phase separation of polymerizing \u03b2-keratin from the cytoplasm, in the absence of any cytoskeletal prepatterns or membraneous precursor templates (reviewed in ref. Dataset S1. SAXS assays, SEM, spectrophotometry, and photonic bandgap modeling of biophotonic nanostructures have been described previously (R. Further details are provided in SI Appendix, SI Text.Plumage sampling information is provided in eviously , 7, 9. ASupplementary FileSupplementary File"} +{"text": "Our study demonstrates the utility of the CRISPR/Cas13d nuclease system to target acute and latent HIV infection and provides an alternative treatment modality against HIV.Recently discovered Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas13 proteins are programmable RNA-guided ribonucleases that target single-stranded RNA (ssRNA). CRISPR/Cas13-mediated RNA targeting has emerged as a powerful tool for detecting and eliminating RNA viruses. Here, we demonstrate the effectiveness of CRISPR/Cas13d to inhibit HIV-1 replication. We designed guide RNAs (gRNAs) targeting highly conserved regions of HIV-1. RfxCas13d (CasRx) in combination with HIV-specific gRNAs efficiently inhibited HIV-1 replication in cell line models. Furthermore, simultaneous targeting of four distinct, non-overlapping sites in the HIV-1 transcript resulted in robust inhibition of HIV-1 replication. We also show the effective HIV-1 inhibition in primary CD4 The development of combination therapy with multiple highly active antiretroviral drugs (HAART) has led to significantly more diverse treatment options and better quality of life for patients by mitigating against HIV-associated physical deterioration [+ T-cell count [Globally, 37.6 million people are infected with HIV. It is estimated that there are 5000 new infections every day represents one possible approach to achieve a functional cure for HIV. Small RNAs (si/shRNA) that use cellular RNAi machinery mediate HIV RNA decay efficiently. However, RNA structural constraints, HIV sequence diversity ,11,12, vWe used the recently discovered Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR/RfxCas13d (CasRx) proteins to meet our above hypothesized criteria. It is a programmable RNA-guided ribonuclease targeting single-stranded RNA (ssRNA) to target HIV-1 transcripts and genome. It has been reported that this ribonuclease is highly efficacious at degrading RNA in mammalian cells, with reduced dependency on RNA structure and significantly lower off-target effects than current siRNA/shRNA technologies ,24,25,26The CasRx, , dCasRx plasmids, gRNA expression vectors , Vesicular Stomatitis Virus glycoprotein (VSV-G) envelope expression vector , lentiviral packaging plasmid , iRFP670 fluorescent reporter vector and pKLV2-U6gRNA5(BbsI)-PGKpuro2ABFP-W (Addgene #67974) were obtained from the Addgene repository .\u00ae High-Fidelity DNA Polymerase and primers encoding NcoI and XmaI digestion sites . The purified products were ligated using T4 DNA ligase . The ligated product was transformed into Escherichia coli (E. Coli) Stbl competent cells . The clones were picked and screened using colony PCR (Construction of pBR43IeG-nef+-iRFP670 HIV vector: The open reading frame (ORF) sequence of iRFP670 amplified from piRFP670-N1 vector by polymerase chain reaction (PCR) using Q5on sites . The PCRlony PCR . The pos\u00ae High-Fidelity DNA polymerase to encode MluI and BamHI digestion sites as described in E. coli Stbl competent cells , colonies were screened by PCR, positive clones were grown, and the vector sequence was further confirmed by Sanger sequencing as described above. Sanger Sequencing primers are described in Construction of pKLV2-U6-CasRx-(pre-gRNA)-PGKpuro2ABFP vector: The pKLV2-U6-CasRx-(pre-gRNA)-PGKpuro2ABFP vector was constructed using restriction enzyme digestion. The pre-gRNA cassette was PCR amplified using Q5TM cells cells, followed by visualization of eGFP by flow cytometry and confirmation of CasRx expression by qPCR by restriction enzyme cloning. The PCR product was digested with KpnI and XbaI and ligated into pVaxI vector. The expression of CasRx-eGFP was evaluated by transient transfection in HEK293T derived Lenti-XE. coli Stbl for in vivo cloning as previously described [Construction of CasRx-NES vector: Sequence from PXR001 vector was PCR amplified with specific oligonucleotide to exclude NLS sequence and add nuclear export signal of mitogen-activated protein kinase (MAPK). The rest of the backbone sequence of PXR001 was amplified in a separate PCR. Two DNA fragments with overlapping ends were prepared by consecutive PCR reactions with Q5 DNA polymerase. These fragments were then directly transformed into escribed . Plasmidhttp://sivirus.rnai.jp/HIV/; accessed on 2 September 2021). These gRNAs were then confirmed for >70% genetic conservation with all HIV-1 variants within the Los Alamos National Laboratory HIV database . The blast-like alignment tool was used to analyze gRNA sequences for potential off-target binding in the human genes. We confirmed that all gRNAs showed at least three or more mismatches and sequenced with U6 promoter primer listed in Single gRNA design and cloning: The single gRNAs were designed to target HIV-1 transcript at highly conserved and siRNA targetable sites using the siVirus software , along with direct repeat sequences, were collinearly arranged. The entire sequence was commercially synthesized and cloned into pKLV-U6-CasRx-(pre-gRNA)-PGKpuro2ABFP vector using the golden gate assembly with some modifications. The modified 10 \u00b5L golden gate reaction contained 50 ng of synthesized polygRNA fragment, 25 ng of the pKLV-U6-CasRx-(pre-gRNA)-PGKpuro2ABFP, 0.5 \u00b5L BbsI (10 U/\u00b5L), 0.5 \u00b5L T4 DNA Ligase (400 U/ \u00b5L), 1 \u00b5L of 10\u00d7 T4 DNA Ligase Buffer, and distilled water.TM cells and TZM-bl cells [\u00ae, Manassas, VA, USA) and HIV latency model J1.1 cells [+T cells were isolated from peripheral blood lymphocytes and activated with ImmunocultTM human CD3/CD28 T cell activator , in the RPMI-1640 media supplemented with 2mM L-Glutamine, 10% bovine calf serum, and 1\u00d7 antibiotic-antimycotic and human recombinant IL-2 . Activated CD4+ T cells were transfected with pVax-CasRx and gRNA plasmids by nucleofection using Lonza 4d nucleofector. Eighteen hours after nucleofection, the cells were infected with HIV-iRFP virus particles. Virus replication was measured by flow cytometry after 48 h of infection.HEK293T derived Lenti-XProgram) were culTM cells using Transit-X2 to make XR-4 tropic or VSV-G pseudotyped HIV-iRFP virus, respectively. Antiviral supernatant was harvested 48 and 72 h following transfection, centrifuged at 500g for 10 min at 4 \u00b0C to remove cellular debris, and concentrated 10-fold using Lenti-X concentrator . CasRx-GFP lentiviral particles were produced by co-transfecting plasmids encoding CasRx-GFP with VSV-G envelope and packaging into Lenti-XTM cells. Culture supernatants were harvested 48 h post-transfection, clarified cell debris by centrifugation at 500\u00d7 g for 10 min at 4 \u00b0C, and concentrated 10-fold using Lenti-X concentrator . To obtain cells with stable CasRx-GFP expression, Lenti-XTM cells were transduced by spinoculation at 800 g for 4 h with CasRx-GFP lentivirus in growth media containing 8 \u00b5g/mL polybrene. Forty-eight hours after transduction, the top 5% of cells expressing the highest levels of CasRx-GFP were sorted using BD FACS ARIA II . The CasRx-GFP expressing cells were expanded and screened for CasRx expression using qPCR (primers listed in The pBR43IeG-nef+-iRFP670 vector was transfected either by itself or with VSV-G envelop plasmid (PMD2.G) into Lenti-XTM cells (LRx) were plated at ~80,000 cells per well in a 96 well flat bottom plate and incubated overnight to achieve ~90% confluency before transfection. Two hundred nanograms (ng) of gRNA plasmids and 100 ng of pBR43IeG-iRFP670-nef+ plasmids were co-transfected per well. Each gRNA was transfected in three wells. Forty-eight hours after transfection, the cells were detached and made into a single cell suspension using Versene. HIV replication was measured using a BD Accuri C6 Flow Cytometer as percent HIV-iRFP670 expressing cells in HIV-guide RNA vs. the non-targeting control (NT) guide RNA transfected cells.CasRx-gRNA transfection and measurement of HIV replication by flow cytometry Twenty-four hours before transfection, stable CasRx-GFP expressing Lenti-XJ1.1 cells were transfected with pVax-CasRx-GFP and gRNAs using Lonza 4d nucleofector. Eight hours post-nucleofection, the cells were stimulated with phorbol 12-myristate 13 acetate (PMA) and ionomycin , and cell supernatants were collected after 24 h.TM reagent as previously described [HIV production in the cell supernatants was measured using TZM-bl reporter cell line. Supernatants were added to the TZM-bl reporter cells, and the cells were incubated for 48 h. Cells were lysed, and luciferase activity was measured using Bright-Gloescribed .http://siVirus.RNAi.jp/HIV/; accessed on 2 September 2021) [We employed the siVirus online tool (er 2021) and the TM cell line with the NLS-CasRx-GFP lentiviral particles and isolated cells expressing the highest NLS-CasRx-GFP using a fluorescence-activated cell sorter and a fluorescently labeled HIV-1 molecular clone. This allowed us to visualize the effect on HIV replication using flow cytometry. We used CasRx-GFP lentiviral construct encoding RfxCas13d fused to nuclear localization signal (NLS) at the N- and C-terminal and an enhanced green fluorescent protein (eGFP) . A self-er FACS; . We confer FACS; . We refeWe designed a molecular clone of HIV-1 expressing a fluorescent tag to allow easy detection and quantification of virus infection, replication, spread, and inhibition by the CRISPR/CasRx system. We used dpBR43IeG-nef+ clone # 11349 (NIH AIDS reagent program) ,34, a fuE. coli \u03b2-galactosidase under the control of an HIV-1 long terminal repeat (LTR) [We co-transfected the plasmids encoding NT or HIV-gRNAs, and HIV-iRFP vector in LRx cells, and 48 h (h) post-transfection, measured viral replication by flow cytometry as percent iRFP expressing cells and mean fluorescent intensity (MFI) of iRFP670 expression . We alsoat (LTR) , permittTM cell line was transduced with the CasRx-GFP-polyNT or CasRx-GFP-polyHIV lentiviral particles, and single cells expressing the highest GFP were isolated using FACS. The cells with stable expression of CasRx-polyNT (LRx-polyNT) or CasRx-polyHIV (LRx-polyHIV) were transfected with HIV-iRFP. The cells expressing CasRx-polyHIV showed a 99% reduction in HIV replication, as indicated by both the percent iRFP expressing cells . Inhibition of virus infection in the polyHIV vs. polyNT transfected cells was assessed by flow cytometry 48 h post-infection. Cells transfected with nuclear (NLS) and cytoplasmic (NES) CasRx significantly reduced HIV replication in polyHIV gRNA transfected cells compared to polyNT transfected cells. However, the nuclear localization of CasRx was more effective in inhibiting HIV-1 replication than the cytoplasmic CasRx with the MAPK nuclear export signal (NES) such that CasRx-NES protein is localized to the cytoplasm. We transfected Lenti-Xic CasRx .+ T cells, we cloned CasRx in a mammalian expression vector, pVax1 (pVax-CasRx). We transfected activated CD4+ T cells with plasmids encoding pVax-CasRx and gRNAs (polyHIV or polyNT) using nucleofection. The cells were infected with the X4-tropic HIV-iRFP virus particles 18 h post-transfection. HIV-iRFP expression was measured by flow cytometry 48 h after infection. The cells expressing pVax-CasRx and polyHIV significantly reduced HIV-iRFP expression compared to pVax-CasRx and polyNT . We co-tIn this study, we present data demonstrating the CRISPR/CasRx nuclease system as an alternative treatment option against HIV. A major problem in developing effective countermeasures against HIV is viral diversity due to the error-prone nature of reverse transcriptase . To overpol gene. HIV pol and gag are the two most conserved regions of the group M HIV clades [pol gene in patients often result in reduced viral replicative fitness and pathogenicity [Wessels et al. also developed an algorithm to reliably predict predict functional efficacy of gRNAs based on specific sequence and structural features, and partition gRNAs into four quartiles based on the efficacy score. The gRNAs in the top two predicted efficacy quartiles (Q4 and Q3) showed high knockdown efficiency. Using their freely available algorithm , we calcV clades . Mutatiogenicity ,40. To mThe cellular location of CasRx nuclease is critical for efficient degradation of target RNA. It has been reported that cytoplasmic expression of CasRx is more effective against viruses that complete their life cycle in the cytoplasm. In our study, the nuclear localization of CasRx was found to be more effective against HIV than its cytoplasmic expression. Konerman et al. showed that nuclear localization signal increased CasRx activity in mammalian cells . The abi+T cells [+T cells as well as lymphocytic cell lines. CasRx and gRNA plasmid nucleofection inhibited HIV replication in primary CD4+ T cells and suppressed the expression of viral RNA from activated latent HIV-1 provirus. These results indicate that the CRISPR/CasRx system works in primary cells and can be effective in vivo. We are now developing methods to deliver our antiviral approach in animal models efficiently.The main target of HIV-1 infection is the CD4+T cells . We testIn vitro, RNAi against the virally transcribed RNA is highly effective in the short term at inhibiting HIV-1 replication ,44,45,46Type VI CRISPR nucleases have recently been discovered as site-specific RNA-guided, RNA-targeting effectors ,25,26,49CRISPR/Cas9, a popular gene-editing tool, has been recently shown to treat HIV-1 effectively in vitro ,66,67,68CRISPR/Cas13d is encoded in the bacterial genome, and its optimized version (CasRx) can function in human cells to decay or edit cellular RNA without using endogenous components . Cas13d/CRISPR/CasRx nuclease system is a promising alternative tool against acute and chronic viral infections. We have demonstrated the effective use of this technology against HIV-1. Simultaneous delivery of a single vector with four gRNAs targeting non-overlapping conserved regions of HIV-1 is a robust approach to inhibit HIV replication. This approach will impede the generation of escape variants which is a significant problem in the field. This RNA-targeting strategy is easily programmable to target any host factor or RNA virus. We anticipate that it will be highly efficient against any RNA virus, especially highly mutable and infectious viruses. Our promising in vitro results warrant further optimization of in vivo delivery and assessment of efficacy, immunogenicity, and safety of this promising therapeutic modality against HIV."} +{"text": "Infectious complications in cancer patients (pts) who have received T-cell therapies are similar to those in autologous hematopoietic stem cell transplant (HCT) recipients, who - because they lose prior acquired immunity after undergoing conditioning regimens and transplantation- may be at an increased risk for vaccine-preventable infections. We sought to determine seroprotection rates against pneumococcus and tetanus-diphtheria before and after cellular therapies.In this ongoing prospective observational cohort study, we enrolled pts with any type of cancer who received cellular therapy with chimeric antigen receptor modified T cell (CAR-T), natural killer CAR-T, or T-cell receptor- directed immunotherapies at MD Anderson Cancer Center from January 2020 through May 2021. We performed antibody assays for diphtheria, tetanus, and pneumococcus before, at 1 month, and between 3-6 months after T-cell therapy for each pt regardless of vaccination history. Of 38 pts enrolled, 27 (71%) were men and 25 (66%) had non-Hodgkin lymphoma (Table 1); 38 (100%) and 17 (45%) had a history of previous diphtheria-tetanus-acellular pertussis (Tdap) and pneumococcal vaccination, respectively (Table 2). Tetanus serologies were positive for all pts tested before, at 1 month and 3-6 months after T cell therapy . Diphtheria serologies were positive for most pts tested before, at 1 month and 3-6 months after therapy Ansun Biopharma Chimerix Clinigen (Consultant)Genentech Janssen Karius (Grant/Research Support)Merck Molecular Partners Novartis (Grant/Research Support)Oxford Immunotec Partner Therapeutics (Consultant)Pulmotec Shire/Takeda Viracor (Grant/Research Support)Xenex (Grant/Research Support) Fareed Khawaja, MBBS, Eurofins Viracor (Research Grant or Support) Ella Ariza Heredia, MD, Merck (Grant/Research Support)"} +{"text": "OBJECTIVES/GOALS: Specific aims are to: Develop a new measure of cancer-related self-efficacy in partners (BCSES-P) and obtain feedback on the items (Quantitative) Evaluate the psychometric properties of the BCSES-P including: dimensionality, factor analysis, and construct validity assessing the relationships posited METHODS/STUDY POPULATION: 2-Phase Approach: 1) Item development and 1) Item testing Phase 1 Stage 1: Literature review to identify additional covariates Stage 2: Focus groups and individual interviews to determine partners\u2019 needs Sample size: 20 partners Design: cross-sectional, qualitative interviews Stage 3: Develop candidate items Stage 4: Cognitive interviews Stage 5: Finalize items with research team Phase 2 Preliminary psychometric testing Dimensionality Internal consistency reliability Construct validity Sample size: 150 partners Design: cross-sectional, online survey RESULTS/ANTICIPATED RESULTS: The BCSES-P will be unidimensional as assessed by exploratory factor analysis. The BCSES-P will demonstrate an internal consistency coefficient of 0.70 or above. Construct validity of the BCSES-P will be demonstrated by support of the following theoretical relationships: Cancer-related self-efficacy will be positively related to marital satisfaction and sexual functioning and the distal outcome, overall QoL. Cancer-related self-efficacy will be negatively related to fatigue , fear of recurrence, depression, and anxiety . DISCUSSION/SIGNIFICANCE OF IMPACT: Findings will guide intervention development to improve partners\u2019 quality of life The BCSES-P will be the first scale to measure partners\u2019 cancer-related self-efficacy. This study will highlight a holistic approach to studying the long-term effects of breast cancer on partners."} +{"text": "Fusobacterium nucleatum and Salmonella), and functions as a key bridge molecule linking oncogenic infection to colonic inflammatory and malignant processes. Accumulating studies verified the overexpression of TLR4 in colitis and CAC, and the over-expressed TLR4 might promote colitis-associated tumorigenesis via facilitating cell proliferation, protecting malignant cells against apoptosis, accelerating invasion and metastasis, as well as contributing to the creation of tumor-favouring cellular microenvironment. In recent years, considerable attention has been focused on the regulation of TLR4 signaling in the context of colitis-associated tumorigenesis. MicroRNA (miR)-155 and TLR4 exhibited a similar dynamic expression change during CAC development and shared similar CAC-promoting properties. The available data demonstrated an interplay between TLR4 and miR-155 in the context of different disorders or cell lines. miR-155 could augment TLR4 signaling through targeting negative regulators SOCS1 and SHIP1; and TLR4 activation would induce miR-155 expression via transcriptional and post-transcriptional mechanisms. This possible TLR4-miR-155 positive feedback loop might result in the synergistic accelerating effect of TLR4 and miR-155 on CAC development.Ulcerative colitis (UC) has closely been associated with an increased risk of colorectal\u00a0cancer. However, the exact mechanisms underlying colitis-associated cancer (CAC) development remain unclear. As a classic pattern-recognition receptor, Toll like receptor (TLR)4 is a canonical receptor for lipopolysaccharide of Gram-negative bacteria (including two CAC-associated pathogens Video abstractThe online version contains supplementary material available at 10.1186/s12964-021-00771-6. Fusobacterium nucleatum (a Gram-negative anaerobic bacterium) and Salmonella (a Gram-negative facultative anaerobic bacterium) might be the potential pathogens that play key roles in the progression of CAC .In summary, both clinical and experimental evidences showed a gradual increase of TLR4 expression during different stages of CAC development. As a classic PRR, TLR4 might act as a key bridge molecule between the infection of oncogenic pathogens (such as"} +{"text": "Neurodegenerative disorders are characterized by insidious progression with poorly-delineated long latent period. Antecedent clinical insult could rarely unmask latent neurodegenerative disorders. Here, we report an autopsy-proven case of corticobasal degeneration which was preceded by a lacunar infarction.A 58-year-old man presented with acute ataxia associated with a lacunar infarction in the right paramedian pons. His ataxia persisted with additional progressive gait difficulty and left arm clumsiness. Six months later, a follow-up neurological examination showed asymmetrical bradykinesia, apraxia, dystonic posturing, postural instability, and mild ataxia of the left limbs. Cognitive examination revealed frontal executive dysfunction and visuospatial difficulties. Dopamine transporter imaging scan demonstrated bilateral reduced uptakes in mid-to-posterior putamen, more prominent on the right side. Levodopa-unresponsive parkinsonism, asymmetric limb dystonia, and ideomotor apraxia became more conspicuous, while limb ataxia gradually vanished. The patient became unable to walk without assistance after 1\u2009year, and died 4\u2009years after the symptom onset. Autopsy findings showed frontoparietal cortical atrophy, ballooned neurons, and phosphorylated tau-positive astrocytic plaques and neuropil threads with gliosis and neuronal loss, confirming the corticobasal degeneration.The case illustrates that precedent clinical events such as stroke might tip a patient with subclinical CBS into overt clinical manifestations.The online version contains supplementary material available at 10.1186/s12883-021-02178-9. Corticobasal degeneration (CBD) is a pathologic entity characterized by widespread tau-immunoreactive depositions in neurons and glia with a specific topographical distribution . The cli18F-N-3-fluoropropyl-2\u03b2-carboxymethoxy-3\u03b2-(4-iodophenyl)-nortropane showed decreased dopamine transporter bindings in both mid-to-posterior putamen, more apparent on the right side , some clinical events such as subdural hematoma or the use of dopamine receptor blocking agents could uncover latent PD . In CBD,"} +{"text": "An 85-year-old woman with chronic obstructive pulmonary disease and a smoking history of 40 packs per year was admitted to the emergency room with a 9-day history of dyspnea, fever, cough, and diffuse myalgia. She tested positive for coronavirus disease 2019 (Covid-19). A chest computed tomography (CT) examination performed eight days after symptom onset demonstrated bilateral ground-glass opacities associated with a pattern suggestive of honeycombing cysts (fibrosis), predominantly in the upper lobes A-C. A ch,Recent studies have documented the development of fibrosis, both histologically and radiologically, during the evolution of Covid-19 pneumonia"} +{"text": "Distinguishing between tumor and treatment effect in post-treatment glioma, although crucial for clinical management, is difficult because contrast-enhancing regions are mixtures of recurrent tumor and reactive tissue, and definitive histopathological criteria do not exist. This study disentangles the marked intra-tumoral heterogeneity in the treatment-recurrent setting by developing an unsupervised framework to algorithmically categorize intraoperative MRI-localized biopsies into three clinically-relevant tissue clusters based on joint analysis of RNA sequencing and histopathological data.A retrospective cohort of 84 MRI-localized biopsies from 37 patients with post-treatment recurrent glioblastoma underwent mRNA extraction and quantification via PLATEseq protocol. For 48 of 84 biopsies, a neighboring piece of tissue underwent quantitative histopathology based on labeling index (LI) for SOX2, CD68, NeuN, Ki67, and H&E. Correlation between LIs and gene expression for these 48 samples was performed. Genes significantly correlated (p<0.05) with \u22651 marker were used for hierarchical clustering of correlation matrix, identifying three mutually-exclusive tissue-specific gene sets. These sets were then used to perform ssGSEA to categorize each of 84 biopsies into one of three tissue types.Correlation analysis identified 7779 genes significantly correlated with \u22651 histopathological marker. Clustering revealed three gene sets associated with specific markers: SetA-3688 genes associated with SOX2/Ki67/H&E; SetB-2418 genes associated with CD68; SetC-1673 genes associated with NeuN. ssGSEA using these sets categorized each biopsy into one of three tissue types: 27 biopsies enriched in SetA, 28 in SetB, and 29 in SetC.Using MRI-localized biopsies with both RNAseq and histopathological data, this algorithmic approach allows development of three orthogonal tissue-specific gene sets that can be applied to characterize the heterogeneity in post-treatment recurrent glioma: SetA: genes correlated with SOX2/Ki67/H&E, representing recurrent tumor; SetB: genes correlated with CD68 (microglia) representing reactive tissue consistent with treatment effect; SetC: genes correlated with NeuN (neurons), representing infiltrated brain."} +{"text": "ABSTRACT IMPACT: My work is on the development of a novel tumor immunotherapy to treat various types of cancer OBJECTIVES/GOALS: As iNKT cells can have direct and indirect killing effects on tumor cells, we propose a novel strategy for activating iNKT cells, via a PLGA nanoparticle delivery platform, to promote anti-tumor immune responses. METHODS/STUDY POPULATION: Poly-lactic-co-glycolic acid (PLGA) nanoparticles can be reproducibly loaded with an iNKT cell glycolipid agonist, alpha-galactosylceramide , and a tumor associated antigen, ovalbumin (OVA). We then test our nanoP prophylactically and therapeutically against a murine model of melanoma, B16F10-OVA. RESULTS/ANTICIPATED RESULTS: These dual-loaded PLGA nanoparticles rapidly activate iNKT cells in vivo to produce IFNgamma. Furthermore, in an in vivo model of melanoma, using B16F10-OVA cells, both prophylactic and therapeutic administration of nanoparticles containing \u03b1GalCer and OVA led to decreased tumor cell growth and increased survival. We also show our nanoparticle therapy has synergistic potential with clinically used immune checkpoint blockade (ICB) therapies, anti-PD-1 and anti-CTLA-4, indicated by the significance increase in survival and lower tumor growth rate of ICB +nanoP treated mice compared to either ICB or nanoP alone. DISCUSSION/SIGNIFICANCE OF FINDINGS: This novel delivery system provides a platform with tremendous potential to harness iNKT cells for cancer immunotherapy purposes against many cancer types."} +{"text": "MprF regulates surface charge by modulating the content and translocation of the positively charged cell membrane phospholipid, lysyl-phosphatidylglycerol (LPG). The precise cell membrane adaptations accompanying such \u03b2-lactam-induced mprF perturbations are unknown. This study examined key cell membrane metrics relevant to antimicrobial resistance among three daptomycin-resistant MRSA clinical strains, which became daptomycin-susceptible following prolonged exposure to cloxacillin (\u2018daptomycin-resensitized\u2019). The causal role of such secondary mprF mutations in mediating daptomycin resensitization was confirmed through allelic exchange strategies. The daptomycin-resensitized strains derived either post-cloxacillin passage or via allelic exchange (vs. their respective daptomycin-resistant strains) showed the following cell membrane changes: (i) enhanced BODIPY-DAP binding; (ii) significant reductions in LPG content, accompanied by significant increases in phosphatidylglycerol content (p < 0.05); (iii) no significant changes in positive cell surface charge; (iv) decreased cell membrane fluidity (p < 0.05); (v) enhanced carotenoid content (p < 0.05); and (vi) lower branched chain fatty acid profiles (antiso- vs. iso-), resulting in increases in saturated fatty acid composition (p < 0.05). Overall, the cell membrane characteristics of the daptomycin-resensitized strains resembled those of parental daptomycin-susceptible strains. Daptomycin resensitization with selected \u03b2-lactams results in both definable genetic changes and a number of key cell membrane phenotype modifications, which likely facilitate daptomycin activity.The reversal of daptomycin resistance in MRSA to a daptomycin-susceptible phenotype following prolonged passage in selected \u03b2-lactams occurs coincident with the accumulation of multiple point mutations in the S. aureus is a leading cause of bacteremia and other endovascular infections including endocarditis, vascular catheter sepsis, and intracardiac device infections -LPG), was visualized by ninhydrin staining. The identity of each of the major TLC spots was made in relation to known positive control PLs. Data were presented as the mean (\u00b1SD) percentages of the three major PLs .Fluorescamine labeling was performed, using the same 2D TLC plates ,34,35,36BODIPY-DAP fluorescence microscopy. DAP binding was performed using confocal microscopy with BODIPY-labeled DAP. Cells were incubated with BODIPY-labeled DAP as previously described . Th. ThResencroscopy .CM phospholipid (PL) content. As expected, the DAP-R variants showed CM PL profiles featuring increased total LPG content vs. the respective DAP-S parental strainconsistent with other previously described DAP-R strains , a more negative surface charge was observed as compared to respective DAP-R strains (p < 0.05) and similar to the DAP-S parental strains.Fatty acid content. We observed considerable shifts in the patterns of saturated fatty acids (SFAs) as well as iso- and anteiso-branched chain fatty acid (BCFA) profiles among the DAP-resensitized strains in comparison to both the parental DAP-S and their respective DAP-R mutant strains (p < 0.01). The DAP-resensitized strains and their DAP-S parental strains had similar levels of anteiso-BCFAs. In addition, no consistent pattern of iso-BCFAs differences was observed among the strain-sets to resist the microbicidal action of DAP and other cationic peptides [p < 0.05). These data in line with the re-establishing of a more rigid, parental-level CM.peptides ,34,35. Tpeptides . This isCM order (fluidity/rigidity). There appears to be an optimal degree of CM order for the interaction of most CM-targeting cationic peptides, including calcium-complexed DAP [exed DAP ,35,36. Aexed DAP , the thrmprF [mprF mutations in either their translocase or synthase domains [mprF mutations in our LOX-post passage variants correlated with a significant drop in DAP MICs, which is often below that of the DAP-S parental strains [mprF are often associated with distinct compensatory changes in PL composition, surface charge, and CM biophysics . Understanding the CM-associated adaptations in DAP-R mutants when exposed to selected \u03b2-lactams has important therapeutic implications, as infections with such strains are now being more frequently treated with DAP-\u03b2-lactam combinations [Our recent studies suggest that \u03b2-lactams can synergize with DAP against DAP-R MRSA through the inhibition of PBP-1 without necessarily enhancing DAP binding . FurthermprF ,29,30. O domains . The acc strains . The emeinations ,36,38,42mprF mutations can yield a decrease-of-function paradigm, featuring both reversal of DAP MICs, as well as a shift in PL profiles, paralleling DAP-S parental phenotypes. As noted above, prolonged LOX passage induces the accumulation of secondary mutations in mprF; however, the causal nature of this event for daptomycin resensitization was unclear, since other genetic mutations were also observed, most notably in div1b and rpoC. In this present study, we confirmed that these secondary mprF mutations are sufficient to restore parental level daptomycin MICs, as well as induce prototypical modifications in CM phenotypes. Studies are in progress to understand the mechanism(s) by which \u03b2-lactams can trigger the accumulation of secondary mprF mutations.Several interesting themes emerged from our study. First, MprF mutations among DAP-R clinical strains have been noted in a variety of \u201chot spot\u201d loci within this protein, most frequently (>50%) in its central bifunctional domain ,30. Of iSecond, DAP-R MRSA strains can modulate their surface charge toward a more relatively positive charge phenotype, potentially creating a \u201ccharge-repulsive\u201d surface milieu against cationic molecules such as calcium-complexed DAP ,27,28. HThird, all three DAP-resensitized strains demonstrated substantially decreased CM fluidity as compared to their respective DAP-R strains and similar to their respective DAP-S parental strains. There are optimal biophysical metrics within the CM microenvironment that appear to maximize interactions of cationic peptides with the CM of MRSA ,21,22,24S. aureus strains [Finally, the above differences in CM order within the strain-sets were well correlated with changes in both carotenoid content and the patterns of anteiso-BCFAs; thus, changes in CM carotenoid content can lead to a buildup of C30 precursor species that shuttle into the menaquinone-fatty acid oxidation pathways . In addi strains .mprF mutation [mprF and its associated CM changes play a critical role in the DAP-resensitization phenomenon.We recognize that the current investigation has several key limitations: (i) only three DAP S/DAP-R/DAP-resensitized strain-sets were studied; (ii) only a relatively focused cadre of phenotypic characteristics were interrogated in comparing the strain-sets; (iii) only a single \u03b2 lactam antibiotic was used for prolonged passage, leaving unresolved whether other PBP-specific or PBP-promiscuous \u03b2-lactams can elicit the same adaptations; (iv) the linkage of our CM perturbations with specific metabolomics modifications was not explored ; and (vimutation ; thus, i"} +{"text": "We have recently established mosaic analysis by dual recombinase-mediated cassette exchange (MADR), which permits stable labeling of mutant cells expressing transgenic elements from precisely defined chromosomal loci. MADR provides a toolkit of elements for combinatorial labeling, inducible/reversible transgene manipulation, VCre recombinase expression, and genetic manipulation of human cells. Further, we have demonstrated the versatility of MADR by creating glioma models with mixed, reporter-identified zygosity or with \u201cpersonalized\u201d driver mutations from pediatric glioma. For example, introducing H3f3a (aka H3.3) mutation variants with MADR regulates the spatiotemporal profile of glioma, and single-cell RNA and ATAC sequencing analysis demonstrates a recapitulation of developmental hierarchy seen in K27M mutant human glioma. Moreover, we have generated novel models of H3.3 WT glioma, H3.3 G34R glioma, and supratentorial ependymoma using patient-derived oncofusion transgenes. These models display a high degree of phenotypic fidelity and we now compare these models on a single-cell level with our previous models, mouse single-cell RNA glioma datasets from other studies, and human tumor cell transcriptomes. Our multi-omics approach includes integration of ChIP-seq/Cut&Tag datasets, single-cell ATAC, and single-cell Cut&Tag datasets.Moreover, we have engineered a novel methodology for inducible gain- and loss-of function perturbation studies in vivo. Using ETS transcription factors as a proof-of-principle, we overlay these genetic perturbations on the glioma atlas to examine the gene networks altered by precise molecular manipulations.We hope that these combined approaches will enable researchers to discover disease mechanisms with increased resolution and test therapeutics in credentialed pre-clinical disease models."} +{"text": "Human immunodeficiency virus (HIV) is a global health concern with major risks for opportunistic infections and predisposition to malignancies including Kaposi sarcoma associated with Human Herpes Virus-8 (HHV-8) and non-Hodgkin lymphoma (NHL) commonly associated with Epstein Barr Virus (EBV). Although the exact mechanisms of predisposition to certain malignancies are unclear, HIV (+) cancer patients typically have poorer prognosis.We included all five HIV positive NHL patients receiving antiretroviral therapy (ART) and chemotherapy in our clinic and aim to determine their follow-up outcomes associated with ART.The use of ART in conjunction with chemotherapy regimens lead to better therapeutic outcome in our cases with no mortality over three years of follow-up despite high rates of poor prognostic factors and studies demonstrating 1-year survival rates of approximately 30% in HIV-associated lymphoma. No significant adverse effect has been recorded.We recommend use of ART along with chemotherapy regimens in HIV positive lymphoma patients for better treatment response. Possible hypothesis include increased apoptotic rates in lymphocytes, defects at natural killer cells, hypergammaglobulinemia and over-proliferation of T-cells at lymph nodes . In addition to being a predisposing factor for malignancy, HIV-infection is a poor prognostic factor after malignant transformation in terms of mortality and morbidity (Ji and Lu 2017). HIV infection is related to complex karyotypes and leukemic presentation both of which are independent poor prognostic factors, a retrospective study conducted with forty-nine HIV positive Burkitt lymphoma patients . However, HIV viral load was not associated with mortality in a study conducted with eighty-six participants in Sub-Saharan Africa . 25-40% of HIV-infected population has shown to develop malignancy in their life-time while Kaposi sarcoma, cervical cancer and non-Hodgkin lymphoma (NHL) are the most common HIV-associated malignancies. 20% of HIV-infected patients develop NHL during life-time, while 2-3% of those are diagnosed with NHL at the time of HIV diagnosis . Most predominant subtype of NHL is diffuse large B-cell lymphoma (DLBCL) composing 75% of the cases followed by Burkitt lymphoma (Rabkin and Yellin 1994). NHL incidence was 25-100 fold higher in HIV-infected population before the use of ART, while malignancies remain the most common cause of mortality in developed countries despite the use of ART . In this study we report five cases of HIV-infected patients developing lymphoma at our clinic over the last 3 years treatment, who received ART in addition to chemotherapy regimens.Human immunodeficiency virus (HIV) is a predisposing factor for certain malignancies including lymphoma. Despite high incidence of concomitant infections with Human Herpes Virus-8 (HHV-8) and Epstein Barr Virus (EBV), primary mechanism underlying malignant transformation is thought to be HIV-related immunosuppression and splenic infiltrative lesion at 31\u00d732 mm size. Excisional lymph node biopsy specimen was suggestive of DLBCL. The clinical stage was III and the patient was started on combination chemotherapy with rituximab, cyclophosphamide, doxorubicin, vincristine, prednisolone (R-CHOP) and involved field radiotherapy . Bone marrow biopsy was not suggestive of lymphoma. The disease recurred after 3 years in which the patient presented with progressive cervical lymphadenomegaly. Meanwhile, the patient was positive for anti-HIV test. Anti-viral therapy and simultaneous chemotherapy with rituximab and bendamustine regimen due to the comorbidities of patient were initiated . Due to A 30-year-old male patient presented to our clinic with the diagnosis of high grade diffuse large B-cell lymphoma/Burkitt lymphoma after excisional biopsy of cervical lymphadenomegaly. He was found to be HIV positive during initial laboratory work-up. Bone marrow biopsy showed no lymphoma involvement. Positron emission tomography (PET)-CT scan revealed left cervical, bilateral axillary, lymph nodes located inferior to left parathyroid gland and bilateral nasopharyngeal involvement. The clinical stage was II and the patient was started on combination chemotherapy with R-CHOP simultaneously with the anti-viral therapy . Follow-A 63-year-old male patient presented to internal medicine policlinic with a year history of fatigue, fever and dyspnea on exertion. HIV RNA test previously performed in another center was positive. The patient had been followed up and anti-viral therapy was initiated . PET scaA 43-year-old HIV (+) male patient presented with nasal congestion and fever to ear, nose and throat (ENT) specialist. Biopsy obtained from non-regressing lesion at left maxillary sinus revealed DLBCL while laboratory work-up showed positivity for EBV at that time. Past medical history of the patient was significant for eczema with exanthematous skin lesions and VDRL positivity treated with penicillin. Further evaluation demonstrated CD4 cell count of 21 cells/\u03bcL. Anti-viral therapy and 6 cycles of R-CHOP regimen were initiated. Following 6 cycles of chemotherapy complete metabolic response was achieved. Later on, patient presented to our clinic with nasal congestion during which biopsy obtained from right maxillary sinus revealed 10\u00d77\u00d72 mm soft irregular mass containing malignant kappa light chain plasma cell infiltration with CD38 positivity and lambda negativity considered as plasmacytoma. Bone marrow biopsy was obtained by the differential diagnosis of multiple myeloma demonstrated normocellular bone marrow without any atypia. Patient received radiotherapy for plasmacytoma. Follow-up of the patient has been uneventful since then.A 33-year-old HIV (+) male patient with undetectable HIV RNA over 3 months was admitted to our clinic with bulky neck mass. MRI revealed an irregular 63\u00d747\u00d780 mm mass located posterior to sternocleidomastoid muscle after which PET scan demonstrated hypermetabolic profile of the cervical mass in addition to suspicious hypermetabolic lesions at spleen and adrenal gland. Patient described the absence of B-symptoms. Past medical history was significant for tonsillectomy and surgically-resected anal condyloma while family history was significant for hepatocellular carcinoma in father. Biopsy revealed germinal center DLBCL with positivity for Bcl-6 and c-myc, however, FISH analysis ruled out c-myc and Bcl-6 positivity. Patient received 2 cycles of R-CHOP regimen which was followed by 2 cycles of dose-adjusted R-EPOCH due to lack of metabolic response. Patient is still on the regimen.et al., 2018). In our case series; 3 of 5 patients had at least 2 poor prognostic factors, while all patients received ART . Therefore, our study is significant by demonstrating beneficial effects of HIV-related lymphoma cases in Turkey that received ART in addition to chemotherapy regimens.HIV infection is a strong predisposing factor for malignant transformation, therefore, high risk patients presenting with advanced staged NHL or fast disease progression should be checked for concomitant HIV infection. Poor prognostic factors include low CD4 cell count (< 100 cells/ml), high LDH values, IV drug abuse, advanced stage and patients over age 35 . Combination of rituximab and DA-EPOCH therapy should be considered in cases with poor prognostic factors, though, results of clinical trials are inconclusive .Two commonly preferred chemotherapy regimens for HIV-related NHL cases are R-CHOP and dose-adjusted etoposide-vincristine-doxorubicin-oral prednisolone-dose-escalated cyclophosphamide (DA-EPOCH), while choice should be individualized. DA-EPOCH is recommended in CD20 (+) patients with plasmablastic histology or high growth fraction disease (Ki67>80%) unless CD4 cell count is below 50 cells/ml while none were present in our study population . No significant adverse effects of ART have been detected in our cases.et al., 2016). Moreover, most HIV-associated lymphoma cases reported in maxillary sinus have been reported as plasmablastic lymphoma in contrast to our case presenting with DLBCL.Another feature of HIV-associated lymphoma is the atypical presentation sites. Similarly, one of our cases presented with maxillary sinus involvement, while only limited number of cases with maxillary sinus lymphoma were present . We assume that therapeutic outcomes of HIV (+) patients presenting with NHL might improve in the upcoming years with the improvements in ART options. Thus, we strongly suggest utilization of ART combined with chemotherapy in HIV-positive patients presenting with lymphoma to improve therapeutic outcomes.Despite studies indicating 1-year survival rate of 30% in HIV-associated lymphoma, individualized treatment modalities involving multidisciplinary approach have led to uneventful follow-up of all cases with HIV-associated lymphoma in our clinic for the last 3 years (Yancheva HIV-Human immunodeficiency virusART-Antiretroviral therapyHHV-8-Human Herpes Virus-8EBV-Epstein Barr VirusNHL-Non-Hodgkin lymphomaDLBCL- Diffuse large B-cell lymphomaCD-Cluster of differentiationR-CHOP Rituximab-cyclophosphamide-doxorubicin-vincristine-prednisoloneGDP-Gemcitabine-dexamethasone-cisplatinR-DHAP-Rituximab-cisplatin-cytosine arabinoside-dexamethasoneDA-EPOCH-Dose-adjusted etoposide-vincristine-doxorubicin-oral prednisolone-dose-escalated cyclophosphamidePET-Positron emission tomographyFDG-FluorodeoxyglucoseCT-Computed tomographyMRI-Magnetic resonance imagingSUVmax-Maximal measuring standardized uptake valueFISH-Fluorescence in situ hybridizationVDRL-Venereal Disease Research LaboratoryECOG- The Eastern Cooperative Oncology GroupIPI- The International Prognostic IndexBcl- B-cell lymphomaMyc-MyelocytomaMum1- Multiple myeloma oncogene 1."} +{"text": "Salivary gland tumors are rare neoplasms which vary in terms of origin and malignant potential. 2-[18F]-fluoro-2-deoxy-d-glucose (FDG)-positron emission tomography (PET) has limited ability to differentiate between different types of salivary gland tumors because both Warthin\u2019s tumors and pleomorphic adenomas usually show increased FDG uptake, with no statistically significant difference in standardized uptake value (SUV) compared with malignant salivary gland tumors. Here, we discuss 4\u2032-[methyl-11C]-thiothymidine (4DST) PET, which provides cell proliferation imaging capable of demonstrating intense uptake in parotid carcinoma and Warthin\u2019s tumor, but no uptake in parotid pleomorphic adenoma. This is the first report of the potential of proliferation PET/ computed tomography (CT) imaging for characterizing salivary gland tumors based on the molecular pathogenesis of the tumor. PreviouFDG-PET/CT generally shows intense uptake in Warthin\u2019s tumors, mimicking malignancy. The numerous mitochondria, immunoglobulin A, and lymphoid stroma in the epithelial cells of Warthin\u2019s tumors cause increased glucose metabolism that leads to increased FDG uptake in the tumors .max 1.9), whereas there was positive FDG uptake (SUVmax 3.2) in Warthin\u2019s tumor, in which no Ki-67-positive cells were found [18F]-fluoro-3\u2032-deoxy-3\u2032-L-fluorothymidine (FLT) uptake, representing cell proliferation related to the salvage pathway of DNA synthesis had faint uptake (SUVF-fluoro-re found . The prePleomorphic adenoma (PA) is one of the most common benign neoplasms of the salivary glands and is characterized as an encapsulating tumor with epithelial and myoepithelial components in addition to mesenchymal and stromal components. High GLUT-1 immunoexpression (>25% immunostained cells) has been reported in 57% of PA , and theIn conclusion, we report the potential of proliferation PET/CT imaging for characterizing salivary gland tumors based on the molecular pathogenesis of the tumor. Further study is required to clarify the utility of 4DST-PET/CT for the assessment of salivary gland tumors."} +{"text": "MicroRNAs (miRNAs) classified as non-coding RNAs regulate various metabolic systems and viral life cycles. To date, numerous DNA viruses, many of which are members of the herpesvirus family, and a relatively small number of RNA viruses, including retroviruses, have been reported to encode and express miRNAs in infected cells. A few retroviruses have been shown to express miRNAs, and foamy viruses (FVs) were initially predicted by computational analyses to possess miRNA-coding regions. Subsequent studies on simian and bovine FVs confirmed the presence of functional and biologically active miRNA expression cassettes. We herein identified feline FV-derived miRNAs using a small RNA deep sequencing ana\u00adlysis. We confirmed their repressive functions on gene expression by dual-luciferase reporter assays. We found that the seed sequences of the miRNAs identified in the present study were conserved among all previously reported FFV isolates. These results suggest that FFV-derived miRNAs play a pivotal role in FFV infection. Spumaretrovirinae of the family Retroviridae, do not cause tumors or other diseases, in contrast to other retroviruses in the subfamily Orthoretrovirinae (Foamy viruses (FVs), which belong to the subfamily MicroRNAs (miRNAs) are defined as non-coding RNAs. They bind to the 3\u2032 untranslated regions (UTRs) of target mRNAs complementarily and induce their cleavage or repress their translation . After bChlorocebus aethiops) (Macaca fuscata) cells, infected with FFV isolate 159 and identified 8 mature miRNAs. Dual-luciferase reporter assays revealed that these FV-derived miRNAs exhibited repressive activities on the target reporter genes.\u20131), and streptomycin at 37\u00b0C in a humidified atmosphere of 5% CO2 in the air. Cells were purchased from ATCC and routinely monitored for mycoplasma contamination using the Plasmo Test\u2122 (InvivoGen). The GFP-based FFV-infection indicator cell line, FFG, previously reported (CRFK cells (ATCC CCL-94) (g) for 5\u200d \u200dmin to remove debris and then filtered through 450-nm disc filters (PALL). L-1-Tosylamide-2-phenylethyl chloromethyl ketone-treated trypsin (Sigma-Aldrich) was added to the samples at a final concentration of 0.1\u200d \u200d\u03bcg mL\u20131, and samples were incubated at 37\u00b0C for 15\u200d \u200dmin. The mixture was inoculated into CRFK cells grown in 25-cm2 flasks in serum-free DMEM (Sigma-Aldrich) supplemented with antibiotics. After 16 h, inocula were replaced with DMEM supplemented with 2% heat-inactivated FCS and antibiotics (see above). Cultures were incubated at 37\u00b0C in a humidified atmosphere with 5% CO2 in the air and observed daily for cytopathic effects (CPEs) by light microscopy to confirm virus isolation (\u20131 of polybrene (hexadimethrine bromide) (Sigma-Aldrich) for viral adsorption. After 2 days, cells were passaged (10-fold dilution) and GFP fluorescence in inoculated cells was observed after 2 days. GFP fluorescence was observed in FFG cells inoculated with the supernatant of FFV-infected cells showing CPE, but not in FFG cells inoculated with the supernatant of na\u00efve CRFK (data not shown). We excluded contamination by other viral agents, including feline morbillivirus and exogenous feline leukemia viruses, by reverse transcription-PCR and PCR, respectively (data not shown) . We desit shown) .Genomic DNA was extracted from CRFK/FFV(PI) cells using the DNeasy Blood & Tissue kit (Qiagen). The viral genome was sequenced using PCR-based Sanger sequencing by a commercial DNA sequencing company (FASMAC). The sequence of this FFV has been deposited in GenBank as FFV isolate 159 (GenBank accession number: MW389244). The phylogenetic relationship was inferred by the maximum likelihood method using RAxML v7.2.8 . To infeHindIII/BamHI to become pUC18/FFV-mir-F1 and pUC18/FFV-mir-F3, respectively (Genomic DNAs were extracted from uninfected CRFK cells and CRFK/FFV(PI) cells using the QIAamp DNA Blood Mini kit (Qiagen). Genomic DNAs were used as templates to construct miRNA expression plasmids. To clone miRNA expression sites, primers were designed for sequences tens to hundreds of bases upstream/downstream of miRNAs, including the predicted regulatory sequences of miRNAs (NheI/HindIII sites of pGL3-basic (Promega) to become pGL3-hCMV (XbaI site of pGL3-hCMV using NEBuilder HiFi DNA Assembly (New England Biolab) for the construction of pGL3-hCMV/ FFV-miR-F1-3p or pGL3-hCMV/ FFV-miR-F3-3p enhancer/promoter was cloned into the Small RNA sequencing and transcriptome analyses were performed on CRFK and CRFK/FFV(PI) cells. Total RNAs were extracted from cells using RNAzol (Promega), followed by a DNase I (Roche) treatment without a heat inactivation step, and re-extracted using RNAzol. Re-extracted RNAs were further processed by a commercial next-generation sequencing company (Novogene). Sequencing libraries were constructed from total RNAs using the NEBNext Multiplex Small RNA Library Prep Set for Illumina (New England Biolab), which generates strand-specific small RNA libraries. Size selection (18 to 40 nt) of the cDNA libraries was performed using native polyacrylamide gel (12%)-electrophoresis to isolate a fraction containing miRNA-derived cDNAs. Single-end 50-bp sequencing was then performed on an Illumina Hiseq 2500 platform. Sequencing adapter sequences were trimmed from the pre-processed reads using Trimmomatic (ver. 0.38) followed by Fastp (ver. 0.19.4) and convt-test.Dual-luciferase reporter assays were conducted to verify that miRNA expression plasmids produce the intended miRNAs. CRFK cells grown in collagen-coated 24-well plates (Iwaki) were co-transfected with 40\u200d \u200dng of each firefly luciferase reporter plasmid, 4\u200d \u200dng of pRL-TK (Renilla luciferase reporter plasmid) (Promega), and 456\u200d \u200dng of each expression plasmid, as indicated in Illumina Hiseq sequencing data and nucleotide sequences identified in the present study were deposited to the database of the DNA Data Bank of Japan (DDBJ) with the accession number DRA011435. The nucleotide sequence data of FFV isolate 159 obtained in the present study has been deposited in Genbank with the accession number MW389244.We initially attempted to isolate viral agents from cat urine samples using CRFK cells. We successfully isolated feline morbilliviruses and reported them elsewhere A and B. To identify miRNAs in FFV-infected cells, we performed small RNA sequencing analyses of CRFK cells persistently infected with FFV isolate 159 (CRFK/FFV[PI]). We mapped small RNA reads to FFV isolate 159 and identified 8 mature miRNAs mapping to the U3 region of LTR . To obseFelis catus), 2 FFVpco isolates derived from pumas (Puma concolor), 2 FFVpbe isolates derived from leopard cats , available in the NCBI database, and the isolate 159 analyzed in the present study were idnt study . These rThe expression of FFV-miR-F1-3p and FFV-miR-F3-3p was high among the identified miRNAs. We then attempted to conduct functional analyses of these miRNAs. A dual-luciferase reporter assay was performed on CRFK cells to examine whether these miRNAs repress gene expression using pUC18/FFV-mir-F1 and pUC18/FFV-mir-F3, respectively A. To enset al. examined the relationship between FFV infection and renal disease in 125 Australian pet cats with and without chronic kidney disease and found no apparent association Identification of Feline Foamy Virus-derived MicroRNAs. https://doi.org/10.1264/jsme2.ME21055Supplementary Material"} +{"text": "The coagulation study and auto-immune markers were normal. Skin biopsy showed mild perivascular dermatitis (A 73-year-old woman with pulmonary hypertension and high-risk myelodysplastic syndrome (MDS) developed asymptomatic erythematous-violaceous macules on her thorax . Maculesrmatitis and negaMDS is frequently associated with autoimmune disorders, which may worsen survival rates ,2. In th"} +{"text": "Primary care providers (PCP) - internists, family practitioners, nurse practitioners, physician assistants - play an integral role in the care of older adults, although many receive limited geriatrics education. We sought to examine what questions community-based PCPs had about geriatrics and clinical care of older adults. As part of large clinical continuing medical education (CME) conferences across 12 states , PCPs attended a live in-person 60-minute geriatrics-focused lecture and entered questions into a mobile application. Questions were then qualitatively analyzed using constant-comparison and tie-break methodology. At all sites, 103 questions were asked with 158 upticks (PCPs could check off that they had similar question) with a range of 3-18 questions per lecture. PCPs asked questions on the following common themes: 1.) Medication-related , 2.) Dementia 3.) Medicare Coding 4.) Falls 5.) Weight loss, and 6.) Insomnia. There were a number of questions referencing incorrect practices . In conclusion, community-based PCPs nationally experience gaps in geriatric knowledge and several utilize practices that could jeopardize older adult health. While attending CME-based lectures is one means of overcoming these gaps, some PCPs may not find time or realize geriatrics as an educational need. PCPs need to be better supported with opportunities to ask geriatric care-related questions in order to improve the care of older adults."} +{"text": "EGFR-mutant non\u2013small cell lung cancer is limited by the development of drug resistance. One mechanism of EGFR inhibitor resistance occurs through amplification of the human growth factor receptor (MET) proto-oncogene, which bypasses EGFR to reactivate downstream signaling. Tumors exhibiting concurrent EGFR mutation and MET amplification are historically thought to be codependent on the activation of both oncogenes. Hence, patients whose tumors harbor both alterations are commonly treated with a combination of EGFR and MET tyrosine kinase inhibitors (TKIs). Here, we identify and characterize six patient-derived models of EGFR-mutant, MET-amplified lung cancer that have switched oncogene dependence to rely exclusively on MET activation for survival. We demonstrate in this MET-driven subset of EGFR TKI-refractory cancers that canonical EGFR downstream signaling was governed by MET, even in the presence of sustained mutant EGFR expression and activation. In these models, combined EGFR and MET inhibition did not result in greater efficacy in vitro or in vivo compared to single-agent MET inhibition. We further identified a reduced EGFR:MET mRNA expression stoichiometry as associated with MET oncogene dependence and single-agent MET TKI sensitivity. Tumors from 10 of 11 EGFR inhibitor\u2013resistant EGFR-mutant, MET-amplified patients also exhibited a reduced EGFR:MET mRNA ratio. Our findings reveal that a subset of EGFR-mutant, MET-amplified lung cancers develop dependence on MET activation alone, suggesting that such patients could be treated with a single-agent MET TKI rather than the current standard-of-care EGFR and MET inhibitor combination regimens.The clinical efficacy of epidermal growth factor receptor (EGFR)\u2013targeted therapy in EGFR mutations, most commonly short in-frame deletions of exon 19 (del19) or missense mutations resulting in the amino acid substitution L858R. These activating mutations promote constitutive, ligand-independent receptor activation is a cell surface receptor tyrosine kinase that promotes cell proliferation and survival through activation of downstream signaling cascades , 2. A sutivation , 4 and ttivation .EGFR-mutant tumors initially respond to EGFR TKIs, the clinical success of these inhibitors is limited by the development of acquired drug resistance is a conserved mechanism of resistance to EGFR inhibitors, observed in 5 to 20% of patients who develop clinical resistance to treatment with EGFR TKIs .EGFR-mutant, EGFR inhibitor\u2013resistant patient-derived models, we identified several models harboring MET amplification that exhibited an unexpected sensitivity to single-agent MET inhibition. Here, we provide a mechanistic basis for these observations and propose a potential strategy to identify patients with EGFR-mutant cancer who may respond to single-agent MET TKI therapy.In our ongoing effort to develop EGFR-activating mutations , no evidence of EGFR secondary mutations, and genomic MET copy number gain in all three models sequencing (MET amplification by fluorescence in situ hybridization (FISH) analysis (We established three patient-derived xenograft (PDX) models of lung adenocarcinoma from the drug refractory tumors of patients who developed resistance to single-agent EGFR TKI therapy. One model, DFCI81, was established from the tumor-derived cell line of a patient who developed acquired resistance after an initial long-term response to treatment with EGFR-targeted therapy, whereas the other two models, DFCI161 and DFCI307, exhibited de novo resistance to EGFR TKI . Targetee models 14). Al. AlEGFR-quencing , and exhanalysis 20, 21), 21EGFR-Treatment of all three PDX models showed sensitivity to single-agent MET inhibition and resistance to single-agent EGFR inhibition in vivo. MET inhibition led to sustained tumor regression, and Western blot analysis revealed that MET inhibition alone induced up-regulation of the proapoptotic Bcl2-like protein 11 (BIM) in the tumor xenografts . We also50) values in the nanomolar range (EGFR wild-type EBC-1 line. This sensitivity contrasted with the EGFR/MET codependence observed in the HCC827GR6 cells and the single-agent gefitinib sensitivity of the EGFR-dependent controls PC9 and HCC827 (P < 0.05) increased by single-agent crizotinib treatment in DFCI81 and DFCI161 cells, compared to EGFR-dependent and EGFR/MET-codependent controls . DF. DFEGFR-ll lines and C.P < 0.0035 and P < 0.009, respectively) to diminish the prevalence of ERBB3-p85 complexes , EGFR/MET-codependent cells (HCC827GR6), and MET-dependent cells (EBC-1 and H1993) and DFCI282 (EGFR L858R/T790M) . We noteR/T790M) \u201337 than nografts .EGFR-mutant patients likely to benefit from single-agent MET inhibition. For instance, the EGFR-dependent H1975 cells (EGFR L858R/T790M) exhibited lower EGFR expression, confounding our observation analysis, the signal area of MET compared to mutant EGFR counts per cell was higher in DFCI81 and DFCI161 cells higher EGFR:MET ratios by BaseScope analysis compared to both EGFR-mutant MET-dependent DFCI81 and DFCI161 cells and EGFR wild-type MET-dependent EBC-1 and H1993 cells (EGFR:MET transcript ratios of our confirmed MET-dependent DFCI307 PDX model to the EGFR wild-type control xenograft DFCI315 (HER2 exon20 V777_G778insGSP) tumor specimens, we evaluated 93 cells . We next8insGSP) 37) and andEGFR:nografts .EGFR:MET transcript ratios against results obtained by an RT droplet digital PCR (RT-ddPCR) assay. Using our existing ddPCR assay platform (EGFR L858R and EGFR del19 (both ELREA and LREAT), as well as wild-type MET . Ad. AdEGFR: in vivo . RetrospDX model .EGFR-mutant, MET-amplified tumor of an osimertinib-resistant patient lower EGFR L858R:MET signal ratio compared to the EGFR-dependent DFCI282 PDX model evaluated by BaseScope was derived from the tumor of a treatment-na\u00efve patient. Although we observed a range of mutant EGFR:MET expression ratios among the patient tumors, the majority (10 of 11) revealed low mutant EGFR:MET transcript ratios, within the same range as observed in our patient-derived models or a red fluorescent protein (RFP)\u2013encoding control vector. Although overexpressing mutant EGFR in a doxycycline-inducible manner in the DFCI81 and DFCI161 models had no effect on the single-agent gefitinib resistance of the cells inhibitors in ALK-rearranged NSCLC, cetuximab in colorectal cancers, and antiangiogenic therapies in glioblastoma to EGFR inhibitors, without prior in vitro propagation. In addition to PDXs, our models spanned a spectrum of modalities including patient-derived cell line and organoid models and a PDX-derived organoid model (DFCI81). Hence, our observations of MET dependence in EGFR-mutant lung cancers are unlikely to be an experimental artifact. In contrast, the HCC827GR6 cell line was selected in vitro to be EGFR inhibitor resistant. Whether this difference alone accounts for the distinct drug sensitivity profiles remains to be determined. It is likely that both MET dependency and EGFR/MET codependency exist in the clinic. Recent reports have described single-agent MET inhibitor sensitivity in EGFR-mutant patients, suggesting that a switch to MET dependence can occur clinically mice were implanted with expanded primary cells or tumors, as previously described (6 cells per mouse in 30% Matrigel (Corning) were implanted subcutaneously in mice. All PDX tumors and DFCI81 cells were implanted in 8- to 10-week-old female NSG mice purchased from the Jackson Laboratories . After implantation, tumor establishment and growth were monitored by caliper measurements twice per week. Sample sizes of tumor-implanted mice were calculated to minimize the number of animals used, based on predetermined tumor take rates, variations among PDX growth kinetics, and number of treatment arms for each study. When patient- and cell line\u2013derived PDX tumors reached 100 to 200 mm3 (with sizes varying based on PDX model and tumor growth kinetics), mice were randomized by tumor volume into various treatment groups. Mice harboring PDX tumors for DFCI81, DFCI161, and DFCI202 were treated with crizotinib hydrochloride (100 mg/kg) and with erlotinib hydrochloride (30 to 50 mg/kg) . Mice harboring patient-derived DFCI307 and MR007 xenograft tumors were treated with 12.5 and 15 mg/kg, respectively, of savolitinib and with 25 and 10 mg/kg, respectively, of osimertinib, provided by AstraZeneca. Vehicle control mice were treated orally with either 0.5% hydroxypropyl methylcellulose (DFCI161 and DFCI307) or 6% Captisol (DFCI81 and DFCI202) in autoclaved water, depending on the drug formulations for each study. Mice were dosed once daily by oral gavage, and tumor measurements were taken using calipers twice a week, as previously described \u2013accredited vivarium and in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. For sequencing and pharmacodynamic studies, mice with 200 to 400 mm3 of tumor volumes were dosed for 3 days with kinase inhibitors or vehicle control, and tumors were harvested and snap-frozen in liquid nitrogen for analysis. Tumor tissues used for protein and RNA extraction were first homogenized using FastPrep-24 in corresponding lysis buffers per manual instructions.All in vivo studies completed at Dana-Farber Cancer Institute were conducted in strict accordance with Dana-Farber Animal Care and Use Committee guidelines. Briefly, NSG or Supp_Data_S1Supplement"} +{"text": "SARS-CoV-2 might spread through the nervous system, reaching respiratory centers in the brainstem. Because we recently reported neurophysiological brainstem reflex abnormalities in COVID-19 patients, we here neuropathologically assessed structural brainstem damage in two COVID-19 patients.corpora amylacea (CA) numbers /mm2 were histopathologically assessed. Other features studied were the immunohistochemical expression of the SARS-CoV-2 nucleoprotein (NP) and the Iba-1 antigen for glial activation.We assessed neuropathological features in two patients who died of COVID-19 and in two COVID-19 negative patients as controls. Neuronal damage and Autopsies showed normal gross brainstem anatomy. Histopathological examination demonstrated increased neuronal and CA damage in Covid-19 patients\u2019 medulla oblongata. Immunohistochemistry disclosed SARS-CoV-2 NP in brainstem neurons and glial cells, and in cranial nerves. Glial elements also exhibited a widespread increase in Iba-1 expression. Sars-Co-V2 was immunohistochemically detected in the vagus nerve fibers.Neuropathologic evidence showing SARS-CoV-2 in the brainstem and medullary damage in the area of respiratory centers strongly suggests that the pathophysiology of COVID-19-related respiratory failure includes a neurogenic component. Sars-Co-V2 detection in the vagus nerve, argues for viral trafficking between brainstem and lung.The online version contains supplementary material available at 10.1007/s00415-021-10604-8. Dear Sirs,Increasing evidence over time has associated SARS-CoV-2 infection with widely ranging neurological complications , 15, 18.To see whether the neurological and neurophysiological findings we previously described depend on structural damage , we neurNeuropathological data were acquired from four autopsies done\u2009<\u20093 h after death: two were from patients who died of COVID-19 and two from COVID-19 negative subjects who died of non-neurological conditions . None of these 4 patients suffered from known underlying neurological diseases or had a history of neurological disorders. The brainstem was formalin-fixed for 48 h as previously described . The fixmedulla oblongata (MO). A further tissue damage marker assessed was the corpora amylacea (CA) number per mm2, at the pontine and medullary level. CA are predominantly glial and extracellular glycoprotein inclusions, suggesting astroglial activation [To evaluate neuronal damage we calculated morphologically damaged cell percentages at trigeminal nuclei level in the pons and tivation . SARS-Comedulla than in the pons . The number of damaged neurons in the MO was higher in COVID-19 positive than in COVID-19 negative patients . The number of CA per mm2, was higher in the medulla than in the pons and in COVID-19 patients than in controls agrees with recent data [Our findings show SARS-CoV-2-related brainstem involvement. In our cases Below is the link to the electronic supplementary material."} +{"text": "Adults with cognitive limitations and diabetes may be less able to adhere to treatment recommendations. Our aims were to: 1) estimate and compare the prevalence of cognitive limitations and diabetes among foreign-born non-Hispanic whites, blacks, Hispanics, Asians, and Arab Americans to US-born non-Hispanic whites; and 2) examine associations after controlling for covariates. We linked 2002-2016 National Health Interview Survey and 2003-2017 Medical Expenditure Panel Survey data . The prevalence of cognitive limitations was highest among foreign-born non-Hispanic whites (9.71%) and Arab Americans (9.40%) and lowest among foreign-born blacks (5.19%). Foreign-born non-Hispanic whites had higher odds of cognitive limitations than their US-born counterparts. Foreign-born Hispanics with diabetes had greater odds of cognitive limitations compared to US-born non-Hispanic whites. Additional findings will be discussed focused on stressors that may contribute to cognition disparities using the immigrant health paradox framework."} +{"text": "Tumor-infiltrating myeloid cells are a prominent pro-tumorigenic immune cell population that limit host anti-tumor immunity and present a significant obstacle for many cancer immunotherapies. Targeting the mechanisms regulating myeloid cell function within the tumor microenvironment may overcome immunotherapy resistance in some cancers. Recent discoveries in the emerging field of immunometabolism reveal that the metabolic profiles of intratumoral myeloid cells are rewired to adapt to the nutrition-limited tumor microenvironment, and this shapes their pro-tumor phenotypes. Interestingly, metabolic modulation can shift these myeloid cells toward the immune-stimulating anti-tumor phenotype. In this review, we will highlight the roles of specific metabolic pathways in the activation and function of myeloid cells, and discuss the therapeutic value of metabolically reprogramming myeloid cells to augment and improve outcomes with cancer immunotherapy. The tumor microenvironment (TME) is often infiltrated by a large number of myeloid cells, which represent a prominent immune component in tumors and play a critical role in modulating anti-tumor immunity . The tumvia de novo fatty acids synthesis, which leads to the accumulation of cancer-associated fibroblasts and adipocytes , macrophages, and neutrophils . During via CCAAT/enhancer-binding protein beta and induces the production of GM-CSF and granulocyte CSF (G-CSF), which promotes emergency myelopoiesis in bone marrow pathway induces the expression of glycolysis-related enzymes including HK1, hexokinase-2 (HK2), phosphofructokinase-1 (PFK1), pyruvate kinase M2 (PKM2), and lactate dehydrogenase (LDH), which are vital for M-MDSC lineage commitment . Inhibitl-norleucine (DON) on MDSCs (d-aspartate (NMDA) glutamate transport receptor control the immunosuppressive function of human immature myeloid cells, while glutamine-derived \u03b1-ketoglutarate (\u03b1KG) is crucial for their expansion . It plays an important role in MDSC maturation and immunosuppressive function. A recent report studied the role of a small molecule glutamine antagonist, 6-diazo-5-oxo-on MDSCs . DON limon MDSCs . Supportxpansion . Blockinxpansion . More inl-arginine by MDSCs acts as a major immunosuppressive pathway that obstructs T cell activation and function. Elevated expression of ARG1 is a hallmark of MDSCs, which generates urea and l-ornithine from l-arginine , fatty acids are oxidized to produce acetyl CoA which enters the oxidative phosphorylation (OXPHOS) and TCA cycles for energy generation. Several studies discovered that tumor-infiltrating MDSCs frequently utilize the fatty acids oxidation process for energy production . Compare+ cells exhibit the enriched gene signature and immunomodulatory activity of suppressive PMN-MDSCs receptor lectin-type oxidized LDL receptor 1 (LOX-1) is a specific marker for human intratumoral PMN-MDSC . LOX-1+ MN-MDSCs . As a keMN-MDSCs . ActivatMN-MDSCs . On the MN-MDSCs . This LXMN-MDSCs . Another of TGF\u03b2 . It also of TGF\u03b2 . AltogetTAMs are one of the most abundant TIMs, and have been extensively described in various solid tumors. Recent studies revealed that TAMs can replenish from either circulating monocytes of hematopoietic origin or tissue-resident macrophages of embryonic origin. In a simplified view, TAMs are comprised of two phenotypically and functionally distinct subsets. Anti-tumorigenic TAMs are similar to M1-like macrophages stimulated by toll-like receptor (TLR) ligands and interferon-\u03b3 (IFN-\u03b3), which produce ROS and NO and secrete pro-inflammatory cytokines , as well as IL-12 and IL-23, and chemokines such as C-X-C motif ligand 9 (CXCL9) and CXCL10. Their primary functions are activating anti-tumor immune response and direct phagocytosis of cancer cells. In contrast, pro-tumorigenic TAMs closely resemble IL-4- and IL-13-induced anti-inflammatory M2-like macrophages. Pro-tumorigenic TAMs produce an array of inhibitory molecules such as ARG1, IL-10, and IDO that exert immunosuppression functions, and angiogenesis factors, such as VEGF and TGF-\u03b2 that promote tumor progression. These TAM subsets co-exist in the same tumor and play various roles at different stages of oncogenesis. M1 macrophage-mediated chronic inflammation promotes the growth of epithelial cells that can spontaneously acquire cancer-associated mutations and enhance transformation during the cancer-initiating stage. In contrast, the late-stage tumor microenvironment fosters anti-inflammatory TAMs. The majority of TAMs from late-stage tumors behave in an M2-like manner to suppress anti-tumor immunity and have been shown to contribute to therapeutic resistance .Macrophage subsets acquire distinct metabolic programs that support their different energy demands and regulate the expression of pro- or anti-inflammatory genes. For instance, TLR and IFN-\u03b3 induce M1 macrophages to engage in aerobic glycolysis and exit the TCA cycle, which leads to the accumulation of an intermediate metabolite, succinate. Succinate can stabilize HIF-1\u03b1, which activates glycolytic gene transcription and reinforces the glycolytic metabolism in M1 macrophages. In M2-like macrophage, IL-4 induces expression of PPAR\u03b3, which transcriptionally upregulates genes associated with FAO and mitochondrial biogenesis. Thus, M2 macrophages exhibit more metabolic flexibility since they can rely on OXPHOS with intact TCA using glutamine and fatty acids. However, these oversimplified models cannot fully recapture the highly dynamic and heterogeneous TAMs, which exhibit a complex spectrum of metabolic and functional profiles across different types of tumors or different malignant lesions from the same patient.Unlike the traditional M2-like macrophages, TAMs depend on glycolysis for metabolism. TAMs derived from circulating monocytes extravasate from the oxygen-rich bloodstream into the oxygen-depleted TME in response to chemokines and pro-inflammatory signals. During this transition, the gradual decrease in oxygen availability induces HIF-1\u03b1 expression, which transcriptionally induces glycolysis pathway genes such as pyruvate dehydrogenase kinase 1 (PDK1), phosphoglycerate kinase 1 (PGK1), glucose transporter 1 (GLUT1), glucokinase (GCK), and PKM2. Consistently, the per-cell glucose uptake ability of TAMs is significantly higher than tumor cells and other immune cells . Comprehin vitro tumor-conditioned macrophages, two studies demonstrated that TAMs with enriched glycolytic signatures support vascular network formation and extravasation of tumor cells induces an epigenetic landscape favoring the differentiation of anti-inflammatory macrophages. \u03b1KG can serve as a vital co-factor for Jumonji domain-containing 3 (Jmjd3)-mediated demethylation of histone H3 at lysine 27 (H3K27). Increased trimethylation of histone H3 at lysine 27 (H3K27me3) is a repressive epigenetic mark dramatically reduces the expression of M2 marker genes. Meanwhile, \u03b1KG also promotes the prolyl hydroxylase (PHD)-mediated post-translational modification of inhibitor of nuclear factor kappa-B kinase subunit beta (IKK\u03b2), which disrupts the NF-\u03baB pathway to limit M1 polarization therapy in EG7 lymphoma and 3LL lung carcinoma in mice signaling, and reduces T cell metabolic flexibility. TAM-mediated arginine depletion forces T cells to switch their primary energy source to glucose, the majority of which is consumed by cancer cells and TAMs. Genetic ablation of in mice . Unfortu in mice . Enhance in mice . In earl in mice . Support in mice . However in mice . In well in mice . The arg reduced . This loteinases . In additeinases . Finallyteinases . Despiteteinases .in vivo and oxidized lipoproteins, and many studies have confirmed that TAMs exhibit increased triglyceride uptake through CD36 and enhanced FAO. Since the TME is enriched with fatty acids, fatty acid metabolism likely plays a pivotal role in TAM polarization. The anti-tumor activity of TAMs is associated with the epidermal fatty acids-binding protein (E-FABP), a lipid chaperone that induces lipid droplet formation and IFN-\u03b2 production. As a result, E-FABP-expressing macrophages promote recruitment of anti-tumor immune cells, including CD8 T cells and NK cells to inhibit tumor initiation . Howeverin vivo . OverallFurthermore, lipid content has a differential impact on the function of TAMs. Macrophages enriched with polyunsaturated fatty acids (PUFA) and linoleic acid (18:3) exhibit cytotoxic activity against P815 tumor cells, while this effect is absent in macrophages enriched with saturated stearic acid (18:0) . In PDACIn the TME, DCs are the primary antigen-presenting cell and specializes in priming different types of effector T cells; they are one of the most important predictors of response to immunotherapy. TADCs capture and process tumor antigens, upregulate the expression of MHC-peptide complexes and costimulatory molecules, and secrete cytokines. DC-mediated antigen presentation is essential for a productive immune response against cancer cells. Upon activation, DCs enter glycolytic flux within minutes to support the high biosynthetic requirements associated with antigen presentation. Blocking this early activation of glycolysis can dramatically dampen multiple functions of DCs, such as antigen presentation and expression of cytokine stimulatory molecules. Since glucose deprivation is predominant in tumors, these tumor-infiltrating DCs may exhibit bioenergetic defects. However, the detailed mechanism and impact of nutrient limitation in TADC metabolism and function remain largely unexplored. Due to the highly glycolytic nature of tumor cells, intratumoral lactate accumulation can activate G-protein-coupled receptor (GPR81) in DC . As a rede novo lipogenesis, fatty acids synthase (FASN), is overexpressed and produces an excessive amount of fatty acids, which can be ingested by DC via scavenging receptors (macrophage scavenger receptor 1 and CD204) and stored inside DC as lipid droplets and enha (FATP2) . Overall (FATP2) .Metabolic regulation is increasingly recognized as the major factor determining pro or anti-tumorigenic function of TIMs. Therefore, the metabolic reprogramming of TIMs may unleash anti-cancer immunity and augment available therapeutic modalities. Such approaches are supported by many immunometabolism studies mentioned and have gained substantial interest. Although there are limited strategies to specifically target TIM metabolism, numerous clinical trials have assessed the feasibility of generic metabolic modulators to combat cancer .d-glucose (2-DG) can competitively suppress glycolysis, which can abolish TIM-mediated immunosuppression. This approach is being tested in phase I clinical trials alone or in combination with docetaxel against advanced solid tumors , and an additional phase I trial is assessing the synergic effect between ADI-PEG 20 and pembrolizumab (NCT03254732). Targeting amino acid metabolism with an IDO inhibitor showed no clinical promise in a phase III trial of metastatic melanoma patients . FinallyTargeting lipid metabolism may also switch TIMs toward an anti-tumorigenic phenotype. Metformin is used to treat hypoglycemia, but can also promote FAO to reduce the proportion of M2-like TAMs and increase M1-like TAMs. Several clinical trials are examining the benefit of metformin alone or in combination with carboplatin/paclitaxel to treat prostate, breast, ovarian and NSCLC . Blocking the TCA cycle by reducing the intermediate metabolite \u03b1-KG is another approach to interrupt fatty acid metabolism. \u03b1-KG inhibitors such as enasidenib (AG-221), ivosidenib (AG-120), AGI-5198, and AG-881 are being evaluated in various hematological malignancies and solid tumors (NCT02074839). More recently, aspirin and celecoxib, which block the polyunsaturated fatty acid pathway mediator COX-2, are being tested in advanced-stage colorectal cancers (NCT03638297). However, designing studies that target fatty acid metabolism must consider the caveat that anti-tumor T cells utilize similar metabolic pathways for survival and function. Several elegant studies have reported that mitochondrial function is vital for exhausted CD8 T cells to sustain their effector activity such as producing IFN\u03b3 and granzyme B . TherefoRecent immunometabolism research provides appealing evidence to suggest that metabolic regulation plays an important role in the differentiation and function of TIMs. This bioenergetic profile can be influenced by tumor cells in various ways. First, tumor cells alter nutrient availability, such as glucose deprivation and lipid enrichment, which induces metabolic reprogramming in myeloid cells. Secondly, tumor-derived factor, metabolic byproduct and waste such as hypoxia and lactate, can modulate signaling pathways to induce a metabolic shift in TIMs. This metabolic rewiring can subvert the anti-tumor function of TIM and promote the pro-tumorigenic phenotype. These discoveries provide a unique opportunity to selectively modulate myeloid cell metabolism to fight cancer.Although metabolic targeting holds great promise as an anti-cancer therapeutic approach, several challenges need to be addressed. One is an improved understanding of the underlying mechanisms of metabolic regulation. Many elegant studies clearly show that the metabolic switch between the different phenotypes of myeloid cells both satisfies the biogenetic need and impacts epigenetic regulation of gene expression . It is w"} +{"text": "KATP) variants predict the risk of increased Apo B concentration (\u2265 80 mg/dL) and related ASCVD remain less clear. We recruited 522 subjects with elevated Apo B concentration (\u2265 80 mg/dL) and 522 counterpart subjects (< 80 mg/dL) from South China to assess the associations of KATP variants with the risks of increased Apo B serum concentration (\u2265 80 mg/dL), carotid artery stenosis (CAS) \u2265 50% and new-onset ischemic stroke (IS). Our results showed that only KATP SNP rs11046182 (GG genotype) was associated with increased risk of Apo B \u2265 80 mg/dL and CAS \u2265 50% . After median 50.6-months follow-up, subjects carrying GG genotype of rs11046182 were associated with higher risk of new-onset IS . Further, the exosome-derived microRNAs (exo-miRs) expression profile was identified by next-generation sequencing. 41 exo-miRs were significantly differentially expressed under cross-talk status between high Apo B level (\u2265 80 mg/dL) and KATP rs11046182. Our study demonstrated that KATP variant rs11046182 was associated with higher risks of elevated serum Apo B levels and its related ASCVD, and the possible mechanism was related to specific exo-miRs expression profile of KATP rs11046182.Serum concentration of apolipoprotein B (Apo B) is causally associated with arteriosclerosis cardiovascular disease (ASCVD) risk. Whether ATP-sensitive potassium channels ( Apolipoprotein B (Apo B) is the major protein constituent of low-density lipoprotein cholesterol (LDL-C). Increased serum level of LDL-C is recognized to be an independent risk factor for atherosclerotic-related events , such asKATP) plays as essential well-fidelity metabolic sensors, and also as an important end effector of ischemic preservation, indicating that KATP couples metabolic abnormalities to protection against ischemic-related injury. This also emphasizes KATP as novel targets for prevention and treatment of ASCVD. The structure of KATP is large heteromultimeric protein complex, consisted of four inwardly-rectifying potassium channel subunits and four sulfonylurea receptor subunits . The Kir6.x pore-forming subunits are encoding respectively by KCNJ8 (Kir6.1) and KCNJ11 (Kir6.2) while SURx regulatory subunits are respectively by ABCC8 (SUR1) and ABCC9 (SUR2) . The subunit constitution of KATP possibly remodel with different physiological and pathological circumstances, involving in substitute splicing of these coding genes as mentioned above, which can result in different subunits being functional in different status. The KATP has extremely high genetic diversity. KATP mutations were not only correlated with serum lipid disorder \u20136 and AS(HDL-C)] , 8 but aeiotropy .KATP variants) to phenotype (elevated Apo B serum levels) remain elusive. In present study we investigate possible associations of KATP variants with the risks of increased Apo B serum levels (\u2265 80 mg/dL) and ASCVD in South China, and identify the plasma expression profile of exo-miRs among subjects under specific genotype (KATP variants)-phenotype (Apo B \u2265 80 mg/dL) correlations.The occurrence and development of elevated Apo B serum concentration and its related ASCVD arises from complex interaction between genetic and environment factors. The exosome-derived microRNAs (exo-miRs) are one of the main classes of non-coding RNAs, and play a critical role as bridge that links genetic and environment factors. Exosomes are important extracellular vesicles with lipid bilayer membrane, and carry cell-specific medium for mediating intercellular communication, especially microRNAs (miRs). The miRs are a class of small (about 22-25 nucleotides long) and endogenous single-stranded RNAs, with an established function of regulating genes at transcriptional and post-transcriptional steps. The exo-miRs take part in almost every physiological or pathological processes ranging from elevated Apo B level to ASCVD. However, the circulating expression profile of exo-miRs and its effect in the process from genotype (P<0.001), as shown in P>0.05), as shown in Participants with or without higher Apo B serum concentration (\u2265 80 mg/dL) showed significant differences on serum concentration of TRIG, TC and LDL-C , as shown in Only KATP rs11046182 was also correlated with elevated CAS \u2265 50% risk while rs78148713 and rs147265929 were not , as shown in KATP rs145456027 will not be estimated due to possible bias.KATP rs11046182 were correlated with elevated new-onset IS risk via a median follow-up of 50.6 months, as shown in Subjects carrying GG genotype of P>0.05) between the two genotypes (AA+GA vs. GG) of KATP rs11046182 among subjects with increased Apo B serum concentration (\u2265 80 mg/dL).As shown in P < 0.05 as threshold cutoff to exclude the low expression of exo-miRs, 41 exo-miRs were then found to be obviously DE between the two genotypes (AA+GA vs. GG) of rs11046182, as shown in e.g., 193a-5p and 193b-5p), miR-320 , and miR-378 . In addition, only 6 of the 41 DE exo-miRs were found to be obviously DE between the two genotypes (AA+GA vs. GG) of rs11046182 in participants with decreased serum Apo B levels (< 80 mg/dL). 2 exo-miRs (miR-31-5p and miR-497-5p) were up-regulated and 4 exo-miRs were down-regulated in subjects carrying GG genotype of rs11046182 compared to those with AA+GA genotype, as shown in KATP rs11046182.A total of 615 exo-miRs were detected by implementing strict data quality control. Using reads per million (RPM) values < 10, and e.g., protein, ion, DNA, enzyme and ATP) and activity regulation .Three terms of GO enriched categories analysis was carried out for those candidate target genes (CTGs) regulated by the top 10 DE exo-miRs, as shown in e.g., endocytosis, signaling pathways of PI3K-Akt, MAPK and Ras, and protein processing in endoplasmic reticulum, etc), metabolism-related diseases , and organismal systems , as shown in KEGG analyses of top 10 DE exo-miRs showed that the top 30 enrichment pathways were mainly associated with metabolic pathways, environmental information/genetic information processing subjects with GG genotype of rs11046182, as shown in KATP SNPs with elevated Apo B serum concentration Apo AI, KATP variants . Interestingly, in this study there was no significant difference on body mass index (BMI), SBP, P2hBS, hypersensitive C-reactive protein (HsCRP) in subjects with or without higher Apo B serum levels (\u2265 80 mg/dL) (2), SBP (> 130 mmHg), P2hBS (> 7.8 mmol/L), HsCRP (> 3 mg/L) were higher than normal especially in higher Apo B level group, which was also complicated with higher serum levels of TRIG, TC and LDL-C. On the other hand, subjects with GG genotype of KATP rs11046182 were indeed related to higher serum levels of P2hBS and HsCRP besides high Apo B serum levels (KATP rs11046182 sustain a status of metabolic disorders and inflammation. Insulin resistance (IR) acts a major role in the pathogenesis of this process. Indeed, a previous study reported that KATP rs5219 was correlated with IR [KATP variants (rs1799854 and rs1799859), the effect was not existed in Caribbeans (rs5219) [To the best of our knowledge, this is the first comprehensive study to examine the possible associations of 80 mg/dL ) and ASC0 mg/dL) , but itsm levels . It is s with IR . The locKATP rs61688134 was associated with AMI among Italians [KATP rs1799854 was with stroke in diabetic Polish [KATP rs11046182 may be an optimal marker of elevated risk of Apo B related ASCVD.Cholesterol-rich, Apo B-containing lipoproteins are now widely accepted as the most important causal agents of ASCVD . CAS no Italians while KAKATP rs11046182) to phenotype (Apo B \u2265 80 mg/dL) is still largely unclear. Our results firstly characterized the circulating expression profile of exo-miRs run through the whole pathological processes from the accumulation of cardiovascular risk factors to the occurrence of atherosclerotic-related events and even death. In addition, there were another four exo-miRs as follows: miR-1291, miR-4488, miR-4726-5p, and miR-7704. However, the relationships of the 4 exo-miRs with cardiovascular disease are still unknown, needing further research.The interplay between genetic and environment factors causes the development of dyslipidemia and related ASCVD. The circulating exo-miRs, as bridge that links genetic and environment factors, play an essential effect in physiological or pathological processes from elevated Apo B level to ASCVD. However, the expression profile of exo-miRs in biological process from genotype , iiR-17-3p , miR-210iR-17-3p and miR-iR-17-3p were sigiR-17-5p , miR-210iR-17-5p , miR-218iR-17-5p , miR-422iR-17-5p , miR-497iR-17-5p , miR-442iR-17-5p , miR-208iR-17-5p , miR-320iR-17-5p and miR-iR-17-5p reportediR-17-5p who repoiR-17-5p was relaiR-17-5p . Under te.g., glucose/lipid/protein metabolism, etc) under disease status [To further evaluate the roles of exo-miRs under interaction between genetic and environment factors, GO and KEGG analysis for the top 10 DE exo-miRs related 1156 CTGs were carried out among elevated Apo B \u2265 80 mg/dL subjects with GG genotype of rs11046182. GO analyses showed tand T2D) . These rKATP mutations with the risk of increased Apo B concentration (\u2265 80 mg/dL) and ASCVD in South China, and characterize the circulating expression profile of exo-miRs under interplay status between genetic (KATP variants) and environmental (elevated Apo B serum levels) factors, intimating that the possible epigenetic modification effect of exo-miRs in development of dyslipidemia and its related atherosclerotic vascular events. The major disadvantages of the study were as follows: Firstly, due to the sample size (N=1044), large-scale subgroup analysis based on Apo B serum level (< 80 mg/dL vs. \u2265 80 mg/dL) and genotypes (GG vs. AA + GA) of KATP rs11046182 will help to further verify the hypothesis that the occurrence and development of increased Apo B serum concentration and its related ASCVD arises from complex interaction between genetic and environment factors. Secondly, Bonferroni correction was executed to correct significance thresholds, but false-positive results may still occur. Thirdly, a rudimentary bioinformatics analysis was only executed, and the non-specific effect and miss-distance effect may exist, owing to lack of verification at the cellular and molecular levels. Therefore, the results of the study should be interpreted carefully.The major advantage of the research was firstly evaluate the associations of KATP rs11046182 was correlated with increased risks of elevated serum Apo B concentration (\u2265 80 mg/dL) and ASCVD, suggesting that this variant is a prospective clinical translational target for precision prevention and early-detection strategies for those disorders, and needs further verification by prospective studies with large sample sizes in different ethnic populations. The potential molecular regulatory may be involved in these significantly DE exo-miRs and metabolic related pathways under those cross-talk status, warrant further research.2) of estimated glomerular filtration rate (eGFR), (4) or (and) any other medical disorders or drugs that could result in kinds of dyslipidemia as mentioned above. Participant\u2019s medical records were assembled via interviewing patient himself and physicians as well as reviewing of medical records. Standard analytical methods were performed to assess blood biochemical indexes on admission to the study. Bilateral carotid ultrasound was executed on enrollment to the study referring to relevant recommendations [The ethics approval (K-2017-043-02) of this study was granted from Guangzhou First People\u2019s Hospital, South China University of Technology. The present study was conducted in consistent with the Helsinki Declaration and the ethics guidelines of the institutional. A total of 522 participants with increased Apo B serum levels (\u2265 80 mg/dL) and 522 counterpart subjects (< 80 mg/dL) were enrolled to the research from South China. All participants with blood lipid disorder were newly identified referring to guidelines as follondations .KATP single nucleotide polymorphisms (SNPs) were genotyped with the MassARRAY (Sequenom) system as previously described methods [KATP variants based on the KATP gene sequence information in GenBank . The SNPs determination accuracy was 100% for each variant of KATP.Four methods . Primer 1521T>G) . The spePrimary follow-up end-point was new-onset IS. All stroke subjects were survivors of IS, and determined by magnetic resonance image and/or computed tomography scanning of the brain referring to relevant guidelines . Subjecthttp://www.mirbase.org).Another total of 10 participants from South China with only elevated serum Apo B concentration (\u2265 80 mg/dL) were newly enrolled to the study . The Cox proportional hazards regression model was carried out to access the crude hazard ratios (HRs) for event free analysis of new-onset IS, adjusted HRs and their 95% confidence intervals (CIs) with corrections for potential covariates. A P value < 0.05 is statistically significant. All probabilities are two-tailed.The SPSS software was used for statistical analyses. The Hardy-Weinberg equilibrium was evaluated for control subjects as shown in P value less than 0.05. Gene Ontology (GO) category and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment were analyzed with the Fisher\u2019s exact test and \u03c72 test, and followed by false discovery rate (FDR) correction. A corrected P value < 0.05 was performed to choose significant GO categories and KEGG pathways.The differentially expressed (DE) exo-miRs in increased Apo B levels (\u2265 80 mg/dL) subjects with different genotypes (GG vs. AA+GA) of rs11046182 were analyzed with edgeR software referring to the criteria of |log2 (Fold Change)| no less than 1 and Supplementary MaterialSupplementary FiguresSupplementary Tables"} +{"text": "Adipocytes in the breast tumour microenvironment promotes acquired treatment resistance. We used an in vitro adipocyte-conditioned media approach to investigate the direct paracrine effects of adipocyte secretory factors on MDA-MB-231 breast cancer cells treated with doxorubicin to clarify the underlying treatment resistance mechanisms. Cell-viability assays, and Western blots were performed to determine alterations in apoptotic, proliferation and lipid metabolism protein markers. Free fatty acids (FFA) and inflammatory markers in the collected treatment-conditioned media were also quantified. Adipocyte secretory factors increased the cell-viability of doxorubicin-treated cells (p < 0.0001), which did not correspond to apoptosis or proliferation pathways. Adipocyte secretory factors increased the protein expression of hormone-sensitive lipase (p < 0.05) in doxorubicin-treated cells. Adipocyte secretory factors increased the utilization of leptin (p < 0.05) and MCP-1 (p < 0.01) proteins and possibly inhibited release of linoleic acid by doxorubicin-treated cells (treatment-conditioned media FFA profiles). Adipocyte secretory factors induced doxorubicin treatment resistance, by increasing the utilization of inflammatory mediators and inhibiting the release of FFA by doxorubicin-treated cells. This further promotes inflammation and lipid metabolic reprogramming (lipid storage) in the tumour microenvironment, which breast cancer cells use to evade the toxic effects induced by doxorubicin and confers to acquired treatment resistance. Globally, breast cancer is the most prevalent type of cancer diagnosed in women ,2, of whvia increased secretion of chemokine ligand-5 and matrix metallopeptidase-9, which promotes metastasis in MDA-MB-231 cells ) thiophene . These regions were scraped off the plates into glass tubes with screw caps followed by FA methyl esters (FAMEs) production by adding 2 ml methanol: sulphuric acid . The individual FAMEs were quantified using heptadecanoic acid as an internal standard and the results are expressed as total \u00b5g FAMEs/ml treatment conditioned medium was used for all statistical analysis. Normality was assessed using the Shapiro\u2013Wilks test, and results were reported as means \u00b1 SEM. A one- or two-way ANOVA was used to describe differences between three or more groups, followed by the Fisher\u2019s LSD vs control, p <\u00a00.0001), whereas-viability was reduced in cells cultured in adipocyte-conditioned media containing Doxorubicin compared to those cultured in CM only . FollowiTo evaluate whether adipocyte-conditioned media could alter doxorubicin-induced apoptosis, we assessed the protein expression of poly (ADP-ribose) polymerase (PARP) activation (116 kDa) and cleavage (89 kDa), total Caspase-3 (35 kDa) and cleaved caspase 3 (15 kDa).vs Dox+CM, p <\u00a00.05; Following 48 hours of doxorubicin treatment, cleaved-PARP protein expression was increased in CM treated cells (CM To ascertain if the activation of cellular proliferation pathways could explain the Doxorubicin treatment resistance as observed with our cell-viability results (increased cell-viability in Dox+CM), protein expression of various cellular proliferation pathways, i.e. PI3K/Akt, ERK and nuclear factor kappa B-p65 (NF\u0138B-p65) was assessed.Phosphorylated PI3K (Tyr 467 and Tyr 199) protein expression was increased in CM-treated cells compared to both the control (p\u00a0<\u00a00.05) and Dox+CM-treated cells (p\u00a0<\u00a00.05) . PhosphoNo significant differences were observed for Phosphorylated ERK1/2 and NF\u0138BConflicting results are reported on the role of adipocyte-derived factors in cancer invasion and metastasis, which are both associated with acquired treatment resistance ,50. Thisde novo synthesis of FA evident by increased expression of various enzymes regulating this pathway, i.e. Acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS) [de novo FA synthesis (FAS and SCD-1) and lipolysis (ATGL and HSL) with or without doxorubicin treatment.Breast cancer cells disrupt normal lipid metabolism by increasing se (FAS) and stease (FAS) . Additiose (FAS) . Therefovs Dox, p <\u00a00.05; No differences were observed for FAS and SCD-1 between any of the experimental treatment groups and b. CTo determine if secretory factors in the adipocyte-conditioned media can stimulate the utilization or release of FFA by MDA-MB-231 TNBC cells treated with/without doxorubicin, we assessed the FFA composition of collected treatment-conditioned media in all the experimental treatment groups, to assess any alterations within the media FFA profile.The total \u00b5g FFA per 1\u00a0ml adipocyte-conditioned media and the treatment-conditioned media are illustrated in Supplementary First, we observed that the percentage of total saturated FA (\u03a3 SFA) was lower in the treatment-conditioned media of the Dox and Dox+CM experimental groups (after 48 hours) when compared to the adipocyte-conditioned media (unexposed to cells and treatments), suggesting the utilization/internalization of these FFA by the MDA-MB-231 cells and oleic acid in both Dox and Dox+CM treatment groups, indicating higher cVA and OA content compared to the control group and Dox+CM (p\u00a0<\u00a00.01) treatment-conditioned media showed higher \u03a3 MUFA content compared to treatment-conditioned media from the control group, whereas Dox+CM showed higher \u03a3 MUFA content compared to the treatment-conditioned media from the CM group and the collected treatment-conditioned media of all experimental groups (after 48 hours).Various adipokines were identified in the adipocyte-conditioned media ranging from highest (adiponectin) to lowest (IL-1\u03b2) concentration , efflux (increased) and alterations in drug transporter proteins ,63, incrIncreased leptin, IL-8, IL-6, and IL-1\u03b2 concentrations is reported in MDA-MB-231 and MDA-MB-468 cells after co-cultivation with adipocytes or adipocyte-conditioned medium favouring tumour cell survival and progression ,67. ThesAdipocytes also induce signalling pathways in breast cancer cells that facilitate proliferation, invasion and metastasis, which is associated with acquired treatment resistance ,20,50,75Adipocytes induce morphological changes (loss of stellate and gain of round/mass colony phenotype), and changes the MET protein profile (increased E-cadherin and Claudin-7 expression) in mesenchymal MDA-MB-231 cells , 76. Adiin vitro model.Reports on how adipocyte-derived factors induce EMT and MET remain conflicting ,77,78. Ovia an additional signalling pathway, such as STAT-3 [via other mechanisms of action, such as lipid accumulation resulting from increased uptake/utilization of adipocyte-derived FFA, which lead to the uncoupling of FA oxidation favouring invasion due to EMT, but not the activation of proliferation pathways [Similar to the findings reported by others ,79, we ds STAT-3 ,50,78. Apathways .de novo synthesis of FA and the uptake of adipocyte-derived exogenous FFA to sustain its high-energy demand via \u03b2-oxidation [Lipid metabolic reprogramming is evident in breast cancer cells showing a strong dependence on both xidation ,36,42. Axidation and are xidation , all of xidation . Here, Hxidation ,25,34.Evidence also showed that chemotherapeutic agents 5-Fluorouracil and Irinotecan decreased the expression of lipogenesis (ACC and FAS) and lipolysis (HSL) and also decreased the SFAs (PA) and MUFAs (PTA) content, thereby inducing cancer cell invasion and metastasis by increasing lipid accumulation . DoxorubWe further propose that adipocyte-derived factors may induce MDA-MB-231 TNBC cells to redirect and incorporate n-6 PUFAs into lipid droplets (PUFA-TAGs) rather than membrane structures in order to limit doxorubicin-induced toxicity , by inhiLinoleic acid is an essential FA , which iin vitro adipocyte-conditioned media model. The increased cell viability may in part be due to adipocyte secretory factors inducing increased HSL protein expression in doxorubicin-treated MDA-MB-231 TNBC cells, including the increased utilization of inflammatory mediators (leptin and MCP-1) and possibly the inhibition of FFA release .To summarize, we provide evidence that adipocyte-derived factors induced doxorubicin treatment resistance in an Lastly, the present study was limited by the following. The adipocyte-conditioned media did not induce apoptosis or a proliferative state and could therefore not account for the increased cell-viability observed in doxorubicin-treated MDA-MB-231 cells. Considering this, it should be highlighted that the apoptosis (PARP and Caspase-3) and proliferation signalling pathways assessed , could have been activated at earlier time points which we did not assess; this could probably also explain the increased cell-viability observed at 48\u00a0hours. We therefore propose that apoptosis and proliferation signalling pathways analysis should be assessed at these earlier time points. In addition, the FFA profile of both the normal growth media and adipocyte-conditioned media unexposed to cells/treatments was not determined, which for future investigations should be done to serve as additional internal controls. For future investigations, we also propose to assess the phospholipid-FA and FFA profiles of the MDA-MB-231 cells themselves, which will be more informative in terms of intracellular FA uptake, utilization and release.via induction apoptosis or cellular proliferation. Here we identified other adipocyte-derived mechanisms underlying treatment resistance, whereby adipocyte secretory factors induced a partial MET phenotype and induced hormone-sensitive lipase protein expression in doxorubicin-treated MDA-MB-231 cells in a paracrine manner. Adipocyte-conditioned media also increased the utilization of inflammatory mediators (leptin and MCP-1) and possibly inhibited the release of FFA (decreased linoleic acid) by doxorubicin-treated MDA-MB-231 cells. This confers to acquired treatment resistance by promoting survival mechanisms including inflammation and metabolic reprogramming (lipid storage) in order to evade the toxic effects induced by doxorubicin (We highlight the complex role that adipocyte-derived factors play in doxorubicin treatment resistance in TNBC cells within tumour microenvironment. Adipocyte secretory factors induced doxorubicin treatment resistance (increased cell-viability), but not orubicin .Figure Click here for additional data file."} +{"text": "OBJECTIVES/GOALS: High blood-pressure (BP) is a common adverse effect of erythropoietin (EPO) therapy among patients with chronic kidney disease on hemodialysis, and even among otherwise healthy individuals who receive EPO. In human genetics, EPO is associated with not only red blood cell traits, but hypertension (HTN) as well. Currently, there is no vascular gene expression data available in the setting of EPO-induced HTN that may explain precise role of key cellular players in its hypertensive etiology. Our aim is to characterize vascular transcriptome to identify key cellular players in EPO-induced HTN. METHODS/STUDY POPULATION: 10-12 week C57BL/6 male and female mice were randomly divided into two groups 1. Vehicle , 2. EPO, (N = 4). VEH and EPO were intraperitoneal administered for 20 days. Blood-pressure was measured non-invasively via tail-cuff plethysmography. We characterized in-vivo transcriptome response of mouse descending aorta to EPO-HTN and vehicle control group by high-throughput RNA-sequencing. RESULTS/ANTICIPATED RESULTS: Systolic blood pressure (SBP) was significantly higher in EPO treatment, compared to vehicle . Comparison of in-vivo transcriptional differences between vehicle and EPO-treated reveal statistically significant changes in cellular pathways consistent with hypertension such as upregulation of RAS signaling pathway and oxidative stress. In-vitro mouse aortic smooth muscle cells, EPO markedly increased phosphorylated-ERK activity, suggesting increased RAS activity. DISCUSSION/SIGNIFICANCE OF IMPACT: This study highlights the importance of previously unknown vascular key players and advances our understanding of the transcriptional events associated with EPO-induced hypertension."} +{"text": "Financial exploitation (FE) perpetrators are usually seen in a position of trust, such as family members or friends, whereas perpetrators of scam tend to be unknown individuals. Few empirical studies have examined victim risk factors, and this study aimed to systematically compare risk factors of FE versus scam. One-hundred-and-ninety-five adults (ages 18-89) were recruited to complete a 60-minute survey and interview at Purdue University in Indiana (n1=97) and Scripps College in California (n2=98). Risk factors assessed included cognitive tasks , socio-emotional questionnaires , financial measures , physical health and demographics . Additionally, participants reported experiences of FE and scam, including (1) the 11-item short-form Older Adult Financial Exploitation Measure, (2) seven questions on scam from the Health and Retirement Study, and (3) likelihood to contact a scammer after reviewing lottery scam materials. The three dependent variables were log-transformed before OLS regression models were built. Each dependent variable was associated with different risk factors. Lower standard of living (p=.02) and ostracism (p<.05) independently predicted FE. Lower physical health was the strongest predictor of scam, with lower level of financial well-being (p=.02) serving as an independent predictor. For lottery scams contact likelihood, ostracism and being male were the strongest predictors. Since risk factors differed between FE and scam, prevention and intervention programs should target the unique profiles of risk factors for each."} +{"text": "NC), the SARS-CoV-2 anti-spike protein IgM assay (IgMSP), and the SARS-CoV-2 anti-spike protein IgG II assay (IgGSP). CD4+ T-cell activity was assessed as a marker of immune competence using the ImmuKnow\u00ae assay. Results: About 25% (18/73) of SARS-CoV-2 uninfected-LT patients generated a positive spike-IgG response following 2D of vaccines, with 36% (9/25) in the Moderna cohort and only 19% (9/48) in the Pfizer cohort. 2D in LT patients elicited a significantly lesser median IgGSP response compared to non-transplanted, uninfected na\u00efve subjects . In LT patients, the Moderna-evoked seropositivity trend was higher than Pfizer. Conclusion: 2D COVID-19 vaccination elicits a dampened serological response in LT patients. Whether assessing other arms of host immunity combined with a higher vaccine dose can better capture and elicit improved immunogenicity in this immunocompromised population warrants investigation.Background: Lung-transplant (LT) recipients are at high risk for COVID-19 due to immunosuppression and respiratory tropism of SARS-CoV-2. The information on the effect of COVID-19 mRNA vaccines to elicit immunogenic responses after a two-dose (2D) regimen in LT recipients is sparse. Thus, we assessed the effect of Pfizer-BioNTech and Moderna mRNA vaccines\u2019 2D regimen on anti-spike responses in immunocompromised LT recipients. Methods: We utilized serum samples from LT recipients vaccinated for SARS-CoV-2 with 2D of either the Pfizer-BioNTech or Moderna vaccines and 2D-vaccinated na\u00efve (non-transplanted and non-exposed to COVID-19) group. Antibody responses were assessed using the FDA-approved SARS-CoV-2 anti-nucleocapsid protein IgG assay (IgG Lung-transplant (LT) recipients are at high risk for severe COVID-19 due to immunosuppression (IS) and respiratory tropism for SARS-CoV-2. Although SARS-CoV-2 vaccine-related immunogenicity in solid organ transplant (SOT) has emerged . ImportaWe and others have demonstrated that the serological assessment of IgG and IgM against SARS-CoV-2 nucleocapsid (NC) and spike (SP) proteins is reliable in differentiating humoral responses to infection versus vaccination ,4,5,6. ANC, SP-specific-IgG (IgGSP), and IgM (IgMSP) analysis.Given this knowledge of conservative threshold and the need to understand the impact of vaccine regimens on humoral immunity to optimize the COVID-19 immunization in this immunocompromised cohort, we sought to address the effect of Pfizer-BioNTech and Moderna mRNA vaccines\u2019 two-dose (2D) regimen on humoral responses in immunocompromised LT recipients, using a combination of an orthogonal approach comprising of IgGLT recipients and na\u00efve (non-transplanted and non-exposed to COVID-19) group vaccinated for SARS-CoV-2 with 2D of either the Pfizer-BioNTech or Moderna vaccines between 15 December 2020 and 19 March 2021 were included in this study. The University of Texas Southwestern Medical Center\u2019s institutional review board waiver of consent approval was obtained for the use of excess, remnant samples available from these patients in our clinical diagnostic laboratory.NC), the SARS-CoV-2 anti-spike protein IgM assay (IgMSP), or the SARS-CoV-2 anti-spike protein IgG II assay (IgGSP), as previously described. Two Index values of \u22651.4 (IgGNC), \u22651.0 (IgMSP), and \u226550 AU/mL (IgGSP) were interpreted as positive per the manufacturer\u2019s recommended threshold. IgGNC positivity informs natural SARS-CoV-2 infection, while IgGSP/IgMSP positivity strongly correlates with the emergence of natural or vaccine-driven neutralizing immunity [\u00ae assay . Results were interpreted as either low, moderately, or strongly correlating with manufacturer-established Adenosine tri phosphate (ATP) ranges of \u2264225, 226\u2013524, and \u2265525 ng/mL, respectively.Antibody responses were semi-quantitatively assessed using serum samples analyzed on the Alinity i platform using the FDA-approved SARS-CoV-2 anti-nucleocapsid protein IgG assay displayed positive IgGSP levels. In contrast, at a median time of 19 days after 2D of Moderna-mRNA vaccine, 36% (9/25) had IgGSP-positivity (Only 25% 18/73) of LT recipients demonstrated IgGsitivity . In addi/73 of LTSP responses in 44% (4/9) of the patients having moderate, and 50% (1/2) of the patients having strong Cylex assay values. In contrast, the Pfizer vaccine elicited a positive IgGSP response only in 18% (3/17) of patients with a moderate Cylex response and none (0/6) in patients with strong Cylex ImmuKnow levels stimulation. The released ATP was then processed through a luminometer to calculate the numeric result, and based on these numeric ATP values, the CD4 T-cell response was stratified as low (ATP < 225 ng/mL), moderate (ATP 226\u2013524 ng/mL), and strong (ATP > 525 ng/mL) . The Cylw levels . For eitSP levels when compared to the non-transplant, na\u00efve (never SARS-CoV-2 infected) participants .Comparison of SARS-CoV-2 specific antibody responses following a 2D regimen of mRNA vaccine clearly illustrated that the immunosuppressed LT recipients had significantly decreased median IgGImmunization against SARS-CoV-2 has proven effective, with the potential to restrain viral propagation and prevent severe illnesses in the general population. However, studies evaluating the effectiveness of the COVID-19 vaccine in immunocompromised subgroups, including LT recipients, are a work in progress. In this study, we estimated the extent to which the Pfizer and Moderna COVID-19 vaccines following the 2D regimen evoked SARS-CoV-2-specific antibody responses in a cohort almost twice the size of previous studies with LT patients . Our datSP responses. Additionally, when compared with the na\u00efve vaccinated (immunocompetent) group, the vaccinated LT patients without prior infection that mounted an antibody response appeared to generate a response not comparable to that of a neutralizing antibody titer, as derived from the application of the conservative threshold of >4160 AU/mL [SP serology data with the conservative neutralizing threshold value may imply an inadequate protective response. While this correlation approach may serve as a proxy for neutralization and has been previously used to obtain a simple immunological meaning [Importantly, our data indicated that receiving two doses of vaccine does not mean assured protection against a COVID-19 infection for the majority of LT patients, as the majority exhibited either nil or negligible IgG60 AU/mL . The obs meaning , its cli meaning . How and meaning . HoweverNotably, the current study does not include the assessment of immunogenicity that may result from the administration of FDA-authorized adenovirus (Adv)-based replication-incompetent vector vaccines due to a lack of Adv vaccines-immunized subjects at the time of communication. However, early reports from other laboratories indicated that both the immunocompetent controls and the immunocompromised transplant patients immunized with Adv-based vaccines showed an impaired humoral immune response . ContrasLimitations include the small sample size, lack of demographic data in the non-transplant group, absence of serial measurements after vaccination, and shorter time for follow-up. Despite these limitations, we demonstrated that a two-dose COVID-19 mRNA vaccine elicits a serological response, though not in a majority of LT patients. Further studies are needed to understand the efficacy and longevity of vaccine-derived COVID-19 immunity in this vulnerable population. Since most lung transplant recipients are maintained at a higher level of immunosuppression compared to other solid organ transplant recipients, larger studies with a longer duration of follow-up are needed to confirm our preliminary findings of antibody response after completing the SARS-CoV-2 mRNA vaccination.In sum, the current standard two-dose mRNA vaccine regimen resulted in a restrained SARS-CoV-2-specific antibody response in LT recipients. Further, a relatively higher anti-spike response was observed in Moderna compared to Pfizer-vaccinated LT patients, implying the potential benefit of a higher antigenic dose (as in the case of Moderna) with possible room for an additional vaccine booster in these selected candidates. Importantly, while arguing in favor of vaccinating these immune-suppressed LT cohorts, our data also cautions that, due to immune paresis, it is imperative that these candidates continue to strictly follow the hand hygiene protocols, masking, and physical distancing regardless of immunization."} +{"text": "Inhabitants of the Greater Mekong Subregion in Cambodia are exposed to pathogens that might influence serologic cross-reactivity with severe acute respiratory syndrome coronavirus 2. A prepandemic serosurvey of 528 malaria-infected persons demonstrated higher-than-expected positivity of nonneutralizing IgG to spike and receptor-binding domain antigens. These findings could affect interpretation of large-scale serosurveys. Plasmodium infections (https://www.medrxiv.org/content/10.1101/2021.05.10.21256855v1), or previously uncharacterized betacoronaviruses in wildlife populations in the rural GMS (Serosurveys for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the Greater Mekong Subregion (GMS) of Cambodia have been limited to those screening healthcare workers in 2 urban hospital-based settings for IgG reactive to SARS-CoV-2 spike and receptor-binding domain (RBD) proteins by using ELISA possess structural proteins capable of infecting humans, we selected highly specific ELISAs for the SARS-CoV-2 structural proteins varying https://www.genscript.com). Of the 24 persons who were seronegative by in-house assay and 11 who were seropositive by in-house assay, 18 tested negative and 9 tested positive by the commercial test, yielding an overall concordance of 77.1% between assays , which is highly immunogenic and an indicator of parasite exposure, and P. falciparum Pfs25 protein (Pfs25), which is poorly immunogenic and expressed only during the mosquito stages of parasite development , but we cannot discard the hypothesis that nonneutralizing SARS-CoV-2\u2013reactive antibodies in prepandemic serum samples might be linked to the ability of betacoronaviruses to evade immune recognition because of their complex surfaces of the samples with the highest reactivity to SARS-CoV-2 total IgG and performed neutralization assays Figure. We found in a widely used, highly specific, and validated ELISA that \u22484%\u201314% of prepandemic serum samples from malaria-infected persons in Cambodia were positive for nonneutralizing antibodies to SARS-CoV-2 spike and RBD antigens by using various standardized optical density cutoff values (Additional information about SARS-CoV-2 cross-reactivity in prepandemic serum from rural malaria-infected persons, Cambodia"} +{"text": "HLTF gene in colorectal cancer (CRC) cells occurs more frequently in men than women. Progressive epigenetic silencing of HLTF in tumor cells is accompanied by negligible expression in the tumor microenvironment (TME). Cell line-derived xenografts (CDX) were established in control (Hltf+/+) and Hltf-deleted male Rag2-/-IL2rg-/- mice by direct orthotopic cell microinjection (OCMI) of HLTF+/+HCT116 Red-FLuc cells into the submucosa of the cecum. Combinatorial induction of IL6 and S100A8/A9 in the Hltf-deleted TME with ICAM-1 and IL8 in the primary tumor activated a positive feedback loop. The proinflammatory niche produced a major shift in CDX metastasis to peritoneal dissemination compared to controls. Inducible nitric oxide (iNOS) gene expression and transactivation of the iNOS-S100A8/A9 signaling complex in Hltf-deleted TME reprogrammed the human S-nitroso-proteome. POTEE, TRIM52 and UN45B were S-nitrosylated on the conserved I/L-X-C-X2-D/E motif indicative of iNOS-S100A8/A9-mediated S-nitrosylation. 2D-DIGE and protein identification by MALDI-TOF/TOF mass spectrometry authenticated S-nitrosylation of 53 individual cysteines in half-site motifs (I/L-X-C or C-X-X-D/E) in CDX tumors. POTEE in CDX tumors is both a general S-nitrosylation target and an iNOS-S100A8/A9 site-specific (Cys638) target in the Hltf-deleted TME. REL is an example of convergence of transcriptomic-S-nitroso-proteomic signaling. The gene is transcriptionally activated in CDX tumors with an Hltf-deleted TME, and REL-SNO (Cys143) was found in primary CDX tumors and all metastatic sites. Primary CDX tumors from Hltf-deleted TME shared 60% of their S-nitroso-proteome with all metastatic sites. Forty percent of SNO-proteins from primary CDX tumors were variably expressed at metastatic sites. Global S-nitrosylation of proteins in pathways related to cytoskeleton and motility was strongly implicated in the metastatic dissemination of CDX tumors. Hltf-deletion from the TME played a major role in the pathogenesis of inflammation and linked protein S-nitrosylation in primary CDX tumors with spatiotemporal continuity in metastatic progression when the tumor cells expressed HLTF.Methylation of the Metastasis is responsible for nearly 90% of deaths from cancer . This esHLTF has been implicated in the malignant transformation of adenomas to carcinomas [HLTF gene is silenced by hypermethylation in 43% of colon cancers [HLTF DNA in serum is significantly correlated with tumor size, more aggressive tumors, advanced stage (III or IV) metastatic disease, including micro-metastasis, and shorter survival [HLTF expression from tumor cells correlates with tumor staging [HLTF was not detected in any of the fibroblast populations despite measured changes in mRNA levels between normal and tumor cells. Epigenetic silencing of HLTF in tumor cells has long been the experimental focus to the exclusion of HLTF in the TME [HLTF protein\u2014the multidomain mammalian ortholog of yeast Rad5\u2014has a DNA-binding domain, a HIRAN domain, and a C3HC4-type RING-finger embedded between SNF2 helicase motifs . HLTF cDrcinomas because cancers . Methylasurvival \u201318. Prog staging . There a staging . Three f the TME .2-D/E motif [CRC progression is accompanied by overexpression of nitric oxide (NO)\u2014a diffusible/pleiotropic regulator of many normal cellular processes\u2014implicated in the activation of oncogenic signaling pathways , 23. NO /E motif . However/E motif : free su/E motif in the bHltf-deleted and control male mice by direct OCMI of HLTF+/+HCT116 Red-FLuc cells [HLTF+/+HCT116 cells in Hltf-deleted mice shifted their metastatic direction. Species-specific RNAseq analysis of primary CDX tumors arising from the same passage of HLTF+/+HCT116 Red-FLuc cells in the TME of Hltf-deleted and control mice revealed striking coordination of an inflammation pathway. Transactivation of iNOS expression and the iNOS-S100A8/A9 signaling axis in Hltf-deleted TME drove the comparison of S-nitroso-proteome of primary tumor with metastatic tumors in Hltf-deleted TMEs to reveal remarkable spatiotemporal continuity in S-nitrosylation signaling.To investigate the functional importance of negligible HLTF expression in fibroblasts of the TME, we developed an HCT116 cell line-derived xenograft model of metastatic CRC. Primary tumor xenografts were established in uc cells . The HCTuc cells , displayuc cells , and recuc cells . HoweverHLTF+/+ human HCT116 Red-FLuc cells were used to establish an orthotopic xenograft model in Hltf+/+ (control mice) and Hltf-deleted mice a timeline of 35 days was established during which primary tumor size and metastasis was assessed weekly by BLI. At necropsy, primary tumors were \u2264 10 mm, i.e. below the maximum allowable tumor size of 20 mm tumor development/metastasis did not interfere with daily activities . Additionally, none of the mice experienced bowel obstruction or changes in stooling or stool consistency. There was no evidence of blood associated with stooling. There was no evidence of anemia, i.e. loss of pink condition of mouse footpads. There was no evidence of compromised behavior, i.e. mice did not become lethargic and there was no incidence of piloerection, poor grooming or inability to thermoregulate. The mice experienced no sustained weight loss. Comparison survival curves for Hltf-deleted mice (n = 20) and control mice (n = 12) mice with the logrank (Mantel-Cox) test , and the Gehan-Breslow-Wilcoxon test , indicated Hltf-deletion had no significant effect on the mortality of the mice during the 35-day post-surgery timeline indicated Hltf-deleted mice at any time during the treatment protocol were 5-times more likely to die than control mice. This approach determined an earlier humane endpoint of 28 days for Hltf-deleted mice in future experiments. The mice were treated alike and maintained under identical conditions.There is a negative correlation between HLTF expression in tumor cells and survival in mice . Therefotimeline . HoweverThere is a negative correlation between HLTF expression and the progression of CRC in humans \u201316. PrevHltf-deleted (n-20) and control (n = 12) mice. However, a shift in the pattern of metastases in control male mice by direct OCMI between the mucosa and the muscularis layers of the cecal wall . Care watastases to regioeted TME . PC is aHomo sapiens [Hltf in RNA-seq samples from control mouse TME. A 4:1 ratio of full-length 4956 nucleotide (nt) message isoform (NM_09210) to a long non-coding (Lnc) 3643 nt transcript variant 4 was identified. This implicates Lnc-Hltf-4 in CRC progression.Alternative splicing increases the complexity of HLTF gene expression in human and mouse cells. Because of its putative role in tumor development, alternative splicing in cells of the tumor and TME were evaluated. Two transcript variants encode the same HLTF protein in sapiens . Cuff.di sapiens , 35\u201337, Cuff.diff FPKM tracking files were analyzed with iPathwayGuide (Advaita Bioinformatics)\u2013using the q value of 0.05 for statistical significance and a log fold change of expression with an absolute value of at least 0.06\u201330 differentially expressed (DE) genes were identified out of a total of 15,857 genes with measured expression in the mouse TME . SimilarHltf-deletion from the TME had little or no effect on the oxygenated/metabolic state of CDX tumors. In fact, cytokine-receptor interaction (KEGG: 04060) was the top biological pathway in CDX tumors (p = 0.002) and mouse TME (p = 2.546e-5) with Bonferroni corrected p-values. Increased mRNA (IL8) and intercellular adhesion molecule-1 (ICAM-1) was identified in CDX tumors with an Hltf-deleted TME. Transactivation of interleukin 6 (IL6) and all components of iNOS-S100A8/A9 signaling in the Hltf-deleted TME (HP) and SERPINA3, and decreased hepatocyte growth factor (HGF) in CDX tumors in heart . Howeversed mRNA and protsed mRNA expressieted TME coincideX tumors . CompartHltf-deleted TME promoted metastasis in the CDX model via S-nitrosylation. A snapshot of the entire CDX tumor S-nitroso-proteome in the presence/absence of Hltf in the TME was obtained when iodoTMT enriched S-nitrosylated human proteins were interrogated by nanoLC-MS/MS. NanoLC-MS/MS-based protein identification provided a comprehensive collection of 136 SNO-proteins in CDX tumors with an Hltf-deleted TME compared to 178 SNO-proteins in CDX tumors with control TME increased from 11 protein candidates to 18 protein candidates in CDX tumors with the Hltf-deleted TME. The emPAI value (0.28) for POTEE, the primate-specific POTE ankyrin domain family member E that promotes CRC growth [Hltf-deleted and control TME.We tested the hypothesis that iNOS derived nitric oxide (NO) production in the trol TME . ProteinC growth , indicatHltf-deleted and control mice were covalently tagged with either Cy3 (Hltf-deleted) or Cy2 (control) for 2D-DIGE of three members (E/F/I) of the primate-specific POTE gene family comprised of 14 members and subdivided into four groups. Group 3 POTE-actin genes (POTEs E/F/I/J/KP) encode proteins that are actin chimeras with a full-sized long inverted repeat, seven ankyrin repeats and a C-terminal coiled-coil domain. The consensus iNOS-S100A8/A9 target sequence (aa 636\u2013641) immediately precedes the start of the coiled-coil domain (aa 642\u2013698). POTEE-SNO in CDX tumors is both a general S-nitrosylation target and an iNOS-S100A8/A9 site-specific target in the Hltf-deleted TME. When additional very weak spots (30\u201335) were added to the analysis, authentic S-nitrosylation of Cys238 in the consensus sequence motif (I/L-X-C-X-X-D/E) was identified in the tripartite motif (TRIM) family member TRIM52. Dysregulation of TRIM family members characterized by a tripartite motif\u2013RING domain, one or two B-box domains, and a coiled-coil domain\u2013has been implicated in CRC cell proliferation [284). There was no evidence of iNOS-S100A8/A9 site-specific S-nitrosylation (Cys117). 2D-DIGE and protein identification by MALDI-TOF/TOF led to the authentication of S-nitrosylation of 53 individual cysteines in half-site motifs of either I/L-X-C or C-X-X-D/E in proteins from CDX tumors from both Hltf-deleted and control TME now includes SNO-myosins and provides continuity between the primary CDX tumor and all distant locations. Functional enrichment assessment with STRING included actin filament-based process, movement, and binding in conjunction with muscle filament sliding and contraction in primary CDX tumors from an Hltf-deleted TME, and the encoded protein is an S-nitrosylation target (Cys143) in all metastatic sites. However, expression is increased (14-fold) in tissue from the inguinal region compared with the primary CDX tumor. Vinculin\u2014a general adhesion protein\u2014dramatically distinguished the metastatic peritoneal carcinomatosis (39-fold) and the lymph nodes extending into the scrotum (168-fold) from the other metastatic sites. However, vinculin appears to be a co-immunoprecipitate because we were unable to authenticate an S-nitrosylated cysteine residue.As shown in traction . These f myosins has threThe CDX model\u2014male HCT-116 cells in male mice\u2014is predicated on the fact that male gender is a risk factor in metastatic CRC . GeneralHltf+/+ TME of a CDX tumor model. It remains to be determined if silencing Lnc-Hltf-4 expression in the Hltf-deleted TME actively promoted metastasis.RNAseq and transcriptomic analyses have identified LncRNAs , 46 thatHltf-deleted TME. Changes in CDX gene expression further promoted metastasis as ICAM-1 increases cancer cell invasion/intravasation into the microvasculature [In this study, the proinflammatory mediator S100A8/A9 in combination with species-specific expression of proinflammatory cytokines IL6 (mouse) and IL8 (human) established a pro-metastatic primary tumor niche in the culature and mediculature HP promoculature and SERPculature . Decreasculature . Combinaculature . These fculature , 56.Hltf-deleted TME. INOS contributed to the metastatic phenotype via S-nitrosylation of cytoskeletal target proteins . One of the newest components of the TME under investigation is the mechanical microenvironment [Increased HP known to preserve vascular NO signaling occurredironment with an ironment especialironment where S-ironment . Most inironment \u2014metastatironment , 62 and ironment \u2014considerironment , 65\u2014has ironment .2-D/E motif indicative of iNOS-S100A8/A9-mediated S-nitrosylation. Moreover, it appears there is iNOS-S100A8/A9 half-site selective S-nitrosylation. Jai et al. [in vitro mutagenesis experiments in which the target cysteine was changed, or knockdown experiments (siRNA) in which individual components of the heteroduplex (S100A8/A9) were eliminated. However, in this in vivo situation, in the presence of both S100A8 and S100A9 proteins, half-site S-nitrosylation was authenticated. Global S-nitrosylation has a role in the regulation of gene transcription [Hltf+/+TME. Collectively, these findings support ongoing efforts to find selective iNOS inhibitors as chemo-preventive agents against CRC [Functional protein posttranslational modifications include phosphorylation, ubiquitination and S-nitrosylation. Despite the ongoing search for S-nitrosylation motifs , iNOS-S1i et al. authenticription . Zinc-ficription . HLTF isinst CRC .Hltf-deletion from the TME promotes inflammation in the shared TME-primary tumor niche. Induction of iNOS in the TME produced general and iNOS-S100A8/A9 site-specific S-nitrosylation of previously unidentified human tumor proteins. We establish continuity between global S-nitrosylation of proteins in pathways related to cytoskeleton and motility under physiological conditions in the metastatic dissemination of CDX tumors in an Hltf-deleted TME. We provide the first evidence of cross-talk between increased gene transcription and S-nitrosylation of the encoded protein. This new role for Hltf-deletion in NO-mediated protein S-nitrosylation to promote metastasis extends our understanding of Hltf as a tumor suppressor.In this study, we show for the first time that \u2122 reagents: S-nitrosylation Western blot kit (90105), HENS buffer (90106), mouse anti-TMTantibody (90075), immobilized anti-TMT resin (90076), TMT elution buffer (90104) and, iodoTMTsixplex\u2122 label reagent set (90101). BioRad was the source of 7.5% Mini-PROTEAN TGX precast protein gels (4561024), Clarity Western ECL-substrate (170\u20135060), Precision Protein StrepTactin-HRP conjugate (1610381) and Kaleidoscope SDS-PAGE Standards (1610324). PerkinElmer was the source of Bioware\u00ae Brite Cell Line HCT116 Red-FLuc (BW124318). All protocols are accessible in protocols.io (dx.doi.org/10.17504/protocols.io.bs5xng7n).Abcam was the source of the following antibodies: mouse monoclonal (ab22506) to S100A9 + Calprotectin (S100A8/A9 complex), mouse monoclonal (ab18672) to IL8, rabbit polyclonal anti-iNOS (ab15323), rabbit monoclonal anti-ICAM-1 (ab109361), goat anti-rabbit IgG H&L , goat anti-mouse ; and mouse on mouse polymer IHC kit (ab 127055). For immunoprecipitation and Western blotting, Abcam was the source of rabbit polyclonal anti-HLTF (ab17984) to human HLTF aa 600\u2013700 that reacts with mouse and human. For immunohistochemistry, Sigma was the source of rabbit polyclonal anti-HLTF (HPA015284) to human HLTF aa 164\u2013300 that reacts with mouse and human. Biotinylated goat-anti-mouse (BA-9200) and goat anti-rabbit (BA-1000) IgG antibodies and the ABC-enzyme complex were purchased from Vector Laboratories, Inc. . Harris Modified Hematoxylin (HHS16) was purchased from MilliporeSigma . XenoLight D-luciferin potassium salt (122799) was purchased from PerkinElmer . ThermoScientific was the source of the following Pierce\u00ae Brite Cell Line HCT116 Red-FLuc is a cell line derived from the parental cell line from adult male colorectal carcinoma by stable transduction with red-shifted lentivirus containing firefly luciferase from Luciola Italica (Red-FLuc) under the control of human ubiquitin C promoter. HCT116 Red-FLuc cells were confirmed to be pathogen free by the IMPACT Profile I (PCR) at the University of Missouri Research Animal Diagnostic and Investigative Laboratory. HCT116 Red-FLuc cells have a mutation in codon 13 of the ras proto-oncogene, and express transforming growth factor beta 1 (TGF\u03b21) and tumor protein 53 (TP53), HCT116 Red-FLuc cells grown in McCoy\u2019s 5a Modified Medium supplemented with 10% fetal bovine serum and puromycin (2\u03bcg/mL) have an average doubling time of 16 hours. For each experiment, cell stocks in liquid nitrogen at passage 2 were thawed using T25 flasks, expanded in T150 flasks for 2 days, passaged overnight and harvested at 70\u201375% confluency. This protocol provided a unified framework for comparative gene expression analysis.BiowareHltf-deleted mice were developed in collaboration with genOway as previously described [Hltf-deleted mice present a neonatal lethal phenotype. However, when the Hltf-deletion line was bred (IACUC# 02007) into the recombinase activating gene 2 (Rag2)/common gamma (IL2rg) double knockout background [Global escribed , and breescribed . Global ckground , i.e. miad libitum and bedding was changed 2\u20133 times/week. Routine testing of sentinel mice ensured the colony was disease free. All studies and the anticipated mortality were conducted in accord with the NIH Guidelines for the Care and Use of Laboratory Animals, as reviewed and approved by the Animal Care and Use Committee at Texas Tech University Health Sciences Center and HLTF+/+ (n = 12) male Rag2-/-IL2rg-/- mice received direct orthotopic cell microinjections (OCMI) of HLTF+/+HCT116 Red-Fluc cells (2x106 cells/10 \u03bcl) between the mucosa and the muscularis layers of the cecal wall. Hereafter the mice were designated Hltf-deleted and control. All surgery was performed with isoflurane (Isothesia) and the SomnoSuite\u00ae Low-Flow anesthesia system (Kent Scientific) with far infrared warming pads during surgery and recovery. Additional efforts to minimize suffering included an IP injection of Buprenorphine prior to surgery to manage incisional pain followed by a second dose 4\u20138 hours later. The cecum was exteriorized via a small midline laparotomy on the vertical linea alba to eliminate bleeding. Non-invasive bioluminescence imaging (BLI) with an IVIS Spectrum In Vivo Imaging System was used to validate the quality and accuracy of the injection, and to track and quantify tumor growth and metastasis. Histopathology at necropsy confirmed placement of the inoculum. Mouse behavior and well-being were monitored daily. Tumor growth/metastasis was monitored weekly with BLI.The orthotopic Necropsy was performed at 35 days post CDX establishment. Mice under continuous isothesia were imaged and killed immediately (< 15 seconds) by cervical dislocation. Primary tumor xenografts and metastatic tumors were quickly removed, rinsed in physiological saline and either flash frozen for biochemical evaluation or fixed in formalin and processed for either routine histopathology or immunohistochemistry.Tissue blocks were serially sectioned (3\u20134 \u03bcm). Two sections were placed on each slide and deparaffinized prior to staining. Beginning with the first slide, sections on every fifth slide were stained with hematoxylin and eosin (H&E) for evaluation by light microscopy. Sections on alternate slides were processed for immunohistochemistry with heat-induced epitope retrieval. Two tissue sections per slide facilitated the use of one section for positive immunostaining, and the serial section for negative (minus primary antibody) control staining. All primary antibodies (1:50) were paired with an appropriate HRP-conjugated secondary antibody (1:200) depending upon the species in which the primary antibody was generated. Nuclei were counterstained (blue) with hematoxylin.Immunoprecipitation and Western analysis were performed as previously reported , 35. BriHltf-deleted and control male mice = 6 total samples) were flash frozen and sent to Otogenetics Corp. for RNA-seq assays as previously described [q-value of 0.05 for statistical significance and a log-fold change (logFC) of expression with an absolute value \u2264 0.6. Cuffdiff generated q-values are adjusted p-values that consider the false discovery rate (FDR). The q-value is an essential statistic when measuring thousands of gene expression levels from a relatively small sample set because it has a greater ability (power) to identify significant changes in gene expression. All RNA-seq data in this publication are accessible through NCBI\u2019s Gene Expression Omnibus (GEO) Series accession number GSE161961 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE161961).Primary tumor xenografts database , gene ontologies from the Gene Ontology Consortium database (2019-Apr26), miRNAs from the miRBase and TARGETSCAN databases, network of regulatory relations from BioGRID: Biological General Repository for Interaction Datasets v3.5.171. March 25th, 2019, and diseases from the KEGG database .Hltf-deleted (n = 2 mice) and control (n = 2 mice) male mice were homogenized/sonicated (Polytron) in 4 volumes HENS buffer . Protein concentrations were determined (OD280) with NanoDrop One (ThermoFisher), and adjusted to a final concentration of 2 \u03bcg/\u03bcl with HENS buffer. Two hundred microgram samples were incubated for 30 minutes at room temperature with 20 mM methyl methanethiosulfonate (MMTS) in dimethylformamide (DMF) to block free cysteine thiols. Proteins were precipitated with 6 volumes pre-chilled (-20\u00b0C) acetone for a minimum of 60 minutes to remove MMTS, pelleted by centrifugation for 10 minutes at 4\u00b0C, and dried for 10 minutes. Following sample resuspension in HENS buffer (100 \u03bcl), S-nitrosylated cysteines were selectively reduced with ascorbate (protected from light) and irreversibly labeled with iodoTMTzero reagent for 2 hours at room temperature. Proteins were precipitated with 6 volumes pre-chilled (-20\u00b0C) acetone for a minimum of 60 minutes, pelleted by centrifugation for 10 minutes at 4\u00b0C, and dried for 10 minutes. Samples were resuspended in HENS buffer (100 \u03bcl), added to anti-TMT resin and incubated overnight with end-over-end mixing at 4\u00b0C. Resin was washed 3X with 1XTBS, 3X with water, and eluted with 4 volumes TMT elution buffer. Eluates were frozen and lyophilized to dryness. In experiment 1, global S-nitrosylation of CDX tumors from TMT-tagged Hltf-deleted and control samples was analyzed by NanoLC-MS/MS with a data base search for human genes in SwissProt using MASCOT. Exponentially Modified Protein Abundance Index (emPAI defined as 10PAI-1) values, where PAI (Protein Abundance Index) is the ratio of observed to observable peptides, is approximately proportional to the logarithm of protein concentration and indicates absolute protein abundance.Step-wise tumor S-nitroso-proteomic analysis leading to iNOS-S100A8/A9 site-specific analysis was performed with iodoTMT-switch labeling , affinitHltf-deleted and control TMT-tagged samples were resolved by two-dimensional gel electrophoresis (2DE) with mouse anti-TMT as the primary antibody and Cy5-labeled donkey anti-mouse IgG (1:2000) as the second antibody. In experiment 3, TMT-labeled proteins were co-labeled with different color fluorescent dyes, i.e. Cy3 for Hltf-deleted and Cy2 for control for two-dimensional difference gel electrophoresis (2D-DIGE), which detected as little as 1.0 fmol of protein in each sample. Spots were excised and proteins identified by matrix-assisted laser-desorption ionization time-of-flight (MALDI-TOF/TOF). Database search was for human proteins in SWISSProt using MASCOT.In experiment 2, Hltf-deleted mice were blocked with MMTS, selectively reduced with ascorbate (protected from light), and individually tagged with iodoTMTsixplex isobaric reagent such that a unique mass reporter (126\u2013131 Da) in the low-mass region of the MS/MS spectrum was generated for samples from six locations. Equal amounts of six different samples (1 mg each) were combined into a single sample for enrichment with anti-TMT resin and NanoLC-MS/MS analysis. Relative expression for each protein fragment, i.e. the average ratio(s) for the protein, together with the number of peptide ratios that contributed (N) and the geometric standard deviation (SDgeo) were calculated.In experiment 4, unmodified cysteines in proteins (1 mg/ml) from primary tumor xenografts (n = 4 mice) and metastatic tumors (n = 2 mice) from Protein ANalysis THrough Evolutionary Relationships (PANTHER) database [In all experiments, peptides labeled with iodoTMT were quantified. Off-target (non-cysteine) labeling was <5%, and authentic cysteine nitrosylation as well as authentic iNOS/S100A8/A9 consensus sequences were condatabase for funcdatabase with EMPS1 Checklist(PDF)Click here for additional data file.S1 TableCuffdiff, part of Cufflinks, uses RPKM values to calculate changes in gene expression. Cuffdiff data were input into iPathwayGuide. Met-analysis calculated long fold change (logFC) and an adjusted p-value (adjpv) for each comparison. The minus sign in the column for logFC shows 12 genes were downregulated and 18 genes were upregulated.(XLSX)Click here for additional data file.S2 TableCuffdiff, part of Cufflinks, uses RPKM values to calculate changes in gene expression. Cuffdiff data were input into iPathwayGuide. Met-analysis calculated long fold change (logFC) and an adjusted p-value (adjpv) for each comparison. The minus sign in the column for logFC shows 136 genes were downregulated and 15 genes were upregulated.(CSV)Click here for additional data file.S1 FileControl and test files each contain three sections: section 1 is the legend (how to read the data sheets); section 2 is the protein peptide summary with peptide sequence, and section 3 is the list of significant hits (95% confidence). The summary file contains a comparative list of proteins with emPAI values for control or test or both.(ZIP)Click here for additional data file.S2 FileWestern blot analysis of test and control samples, 2D-DIGE analysis, and spot identification. Spots were excised and proteins identified by matrix-assisted laser-desorption ionization time-of-flight (MALDI-TOF/TOF).(ZIP)Click here for additional data file.S3 FileAnalysis was conducted in two phases in which spots with a unique or strong signal were analyzed first followed by analysis of spots with a very weak signal Two files each have a section on how to read MS report, a protein peptide summary and a protein ID summary. Only high confidence matches are reported.(ZIP)Click here for additional data file.S4 FileHltf-deleted TME.There is a legend showing how to read the file, a peptide list and a protein list. The protein list contains ratio data show the relationship of protein expression in the secondary metastatic sites to the primary CDX tumor in the (ZIP)Click here for additional data file."} +{"text": "ABSTRACT IMPACT: We describe a novel methodology that combines CRISPR/Cas9-induced double stranded DNA breaks with homology dependent repair from an adeno associated virus (AAV) encoded template to generate single-allele edited isogenic cell line models of cancer-associated mutations with high efficiency. OBJECTIVES/GOALS: Conventional approaches to creating isogenic knock-ins, to model disease-associated mutations, are limited by poor efficiency and loss of the mutant allele on extended culture. We present an optimized editing approach combining CRISPR/Cas9 with Adeno-associated virus, using mutant SF3B1 as a prototype. METHODS/STUDY POPULATION: Left and right homology arms for SF3B1 were PCR amplified and cloned into pAAV-SEPT-Acceptor plasmid . The disease-associated K700E mutation was introduced by site-directed mutagenesis. Single guide RNA (sgRNA) complexed with recombinant Cas9 along with the AAV donor were delivered into K562 cells, G418 resistant clones selected, and screened for integration by PCR. Confirmed clones were then transduced with a doxycycline-inducible Cre-recombinase containing lentiviral vector. Inducible expression of Cre-recombinase and expression of the mutant allele were confirmed by Western blot and Sanger sequencing respectively. RESULTS/ANTICIPATED RESULTS: Targeted-integration efficiencies among the Neo-resistant clones, generated by AAV-alone and AAV+CRISPR/Cas9, were 16% and 94%, respectively. Single cell cloning after Cre-mediated excision of loxp was unsuccessful presumably due to toxicity of the K700E mutation. To overcome this limitation, clones were transduced with doxycycline-inducible Cre-recombinase lentiviral vector. Doxycycline induction of Cre-recombinase resulted in reliable excision of the loxp cassette and expression of mutant allele at about 50% variable allele frequency (as determined by Sanger sequencing). The approach was validated in additional cell lines and for introduction of N-terminal FLAG tag for SF3B1. DISCUSSION/SIGNIFICANCE OF FINDINGS: Combining AAV and CRISPR/Cas9 can generate scalable single-dominant allele mutants with high precision and efficiency compared to AAV or CRISPR alone. Together with inducible Cre-recombinase, our approach can generate isogenic models where the mutation confers a growth disadvantage."} +{"text": "To demonstrate our transvaginal high uterosacral ligament (HUL) hysteropexy technique as an alternative mesh-free uterine-preserving pelvic organ prolapse (POP) repair approach and present our institutional outcomes. Concurrent hysterectomy with POP repair is controversial as uterine-preserving techniques may beneficially allow fertility, body image and sexual function preservation , 2.This video illustrates a step-by-step sequence of our HUL hysteropexy technique in a symptomatic Stage III POP patient. Retrospective single-institution, single-surgeon analysis of patients treated by either HUL hysteropexy or hysterectomy with HUL suspension for symptomatic prolapse was performed with minimum 2 years of follow-up. Patient demographics, operative characteristics, pre and post-operative POP-Q evaluation, American Urological Association Symptom scores (AUASS) and post-operative Pelvic Floor Distress Inventory (PFDI-20) were compared.Surgery time was 3 hours 24 minutes. No immediate/early complications were noted, with successful repair on follow-up. Outcomes of 18 patients were assessed . The onlWe present our HUL hysteropexy technique. Although limited by sample size and retrospective design, resulted in significantly reduced blood loss and operative time with comparable post-operative 2 year outcomes to non-uterine-preserving techniques. In our opinion, HUL hysteropexy is a safe, durable POP management option for women without significant endometrial pathology risk factors."} +{"text": "Pneumocystis jirovecii pneumonia (PCP).Lymphopenia, corticosteroids and immunomodulatory therapeutics frequently used in COVID-19 patients with acute respiratory distress syndrome (C-ARDS) may be contributing factors to opportunistic infection such as We conducted a retrospective study to compare the prevalence of PCP between patients with C-ARDS and those with non-SARS-CoV-2 viral ARDS (NC-ARDS).Pneumocystis jirovecii DNA by real-time polymerase chain reaction (qPCR) .Two NC-ARDS patients fulfilled proven PCP diagnostic criteria, with a positive direct examination, a single \u00df-D-glucan\u2009>\u200980\u00a0pg/mL Table , and recThree other NC-ARDS patients were classified as colonized, while no patient fulfilled possible PCP diagnostic criteria. Time between ICU admission and positive sample for PCP Table was shorPneumocystis colonisation in COVID-19 patients. In our cohort, qPCR was positive in 13% of NC-ARDS. This result is in accordance with a previous study showing 7% of positive qPCR in ICU-admitted influenza patients [In this study of patients with viral ARDS, we found a low risk for possible or proven PCP. Our findings are in accordance with two smaller studies in France , 5 retripatients .The strengths of our study are the analysis of a large ARDS cohort with fungal analyses. Our study also has limitations: monocentric design, NC-ARDS patients more frequently immunocompromised, and a long cohort period."} +{"text": "Rationale: T cell therapeutic strategy using CD19-targeting chimeric antigen receptor (CAR) is a revolutionary, novel, and successful treatment for B-cell malignancies. However, the dependency on T-cell mediated cytotoxicity restricts CAR-T therapy as a patient-specific individualized therapy with severe side effects, such as cytokine release syndrome (CRS). Whether a non-T-cell based universal cellular therapy can substitute CAR-T therapy is largely unknown.Methods: Various artificial antigen-recognizing cells were prepared to determine whether non-T-cell-derived CD19-scFv bearing effector cells could cause target cell death. A universal strategy for CRS-free cellular therapeutics was proposed, utilizing artificial antigen-recognizing cells (AARC), which can be manufactured universally and routinely as \u201coff-the-shelf\u201d mesenchymal stromal cells (MSCs) or other types of non-autologous cells expressing anergic CARs.Results: We demonstrated that T-lymphocytic and non-lymphocytic cells could cause CD19 internalization and subsequent depletion when armed with a CD19-recognizing moiety. This CD19 antigen depletion could efficiently induce T-cell independent apoptosis in target cancer cells whose survival depends on CD19 expression, suggesting that CD19 antigen depletion constitutes a crucial tumor destroying mechanism for CD19-CAR-T, especially for its long-term efficacy.Conclusion: Our results uncovered an unrecognized CAR-T cytotoxicity and antigen loss mechanism and provided new insights into a shift from unique patient-specific autologous therapeutics to universal and standardized allogeneic treatment. Autologous CAR-T cells constitute a promising novel therapeutic approach for the treatment of hematological malignancies. Although this individualized therapy-based approach has resulted in outstanding clinical results to date, it has several intrinsic disadvantages, such as the risk of manufacturing failure in certain patients, the risk of delay in treatment due to time-costing manufacturing procedures, as well as financial burdens. Therefore, developing the next-generation allogeneic cellular therapeutic strategy to address these issues is an active area of research Cytotoxic T lymphocytes (CTL) mediate their anti-tumor effects predominantly through the granule exocytosis axis, the death ligands axis, and the release of cytokines in vivo activation and expansion of CAR-T-cell products, which inevitably cause the well-characterized side effect of cytokine release syndrome (CRS). Therefore, the development of T-cell independent universal cellular therapy strategies may provide an alternative option for \u201coff-the-shelf\u201d and standardized treatments and reduce the CRS risk. Here, we have demonstrated that CAR-T cells can destroy target cells through a T-cell independent mechanism. Based on this finding, we propose the concept of a universal cellular therapy strategy, which could be used in conjunction with current CAR-T therapeutics.Successful CAR-T therapeutics depends heavily upon the SEM, REH, RAJI, Jurkat, and K562 cell lines were obtained from DSMZ. KOPN8, KOBP26, and NALM6 cell lines were obtained from ATCC. Mesenchymal stem cells (MSCs) were obtained from Shanghai Nerostem Tech. CD3+ T cells were isolated using EasySep Human T Cell Isolation Kit (STEMCELL Technologies) and then cultured in CTS T Cell Expansion medium (Thermo) containing 10% fetal bovine serum and 100 IU/ml human IL-2 (PeproTech). The CellTiter 96 MTS assay (Promega) was used to determine cell viability and proliferation.Fragments encoding CD19-, CD22-, and CD133-specific competent CARs and anergic CARs (scFvs) that lack co-stimulatory and \u03b6-chain signaling domains were inserted into the lentiviral vector pCDH-T2A-copGFP (System Biosciences). The CD19-mRuby2 fusion was generated by fusing the mRuby2 sequence at the C terminus of CD19 and cloned into the pCDH lentiviral vector. Target sequences (CTTCAACGTCTCTCAACAGAT #1 and CCGAGTTCTATGAGAACGACT#2) against human CD19 and a control scrambled sequence (CTCAATCAACAGATCTCGTCT) were inserted into the pLKO.1 vector (Sigma).The CellTrace Far Red Proliferation Kit, the CellTrace CFSE Cell Proliferation Kit and the CellTrace Violet Proliferation Kit (Invitrogen) were used for cell labeling. The human CD19-APC and CD69-APC antibodies were obtained from BD Biosciences and the human CD133-PE antibody was purchased from Miltenyi Biotec. A human CD22 antibody was obtained from Biolegend . Apoptosis was measured using the Annexin V Apoptosis Detection Kit (BD Bioscience). Flow cytometry was performed on LSRFortessa or FACSAria sorter (BD Biosciences). Data were analyzed by the FlowJo software.Bortezomib (Velcade), Sc-79, CsA, and dynago-4a were obtained from Selleck Chemicals. Bafilomycin A1 (Baf-A1), DC661, MK-2206, and M\u03b2CD were acquired from MedChemExpress.Human CD19 and Akt antibodies were obtained from ABclonal Technology. Antibodies against CD133, p44/42 MAPK (Erk1/2), phosphor-p44/42 MAPK (p-Erk1/2), and phosphor-Akt (p-Akt) were purchased from Cell Signaling Biotechnology. MYC antibody was obtained from Santa Cruz Technology. Mouse anti-GAPDH antibody was obtained from Sigma Aldrich, and immunoblot signals were acquired by the Amersham Imager 600 .SEM cells labeled with Cell Trace Far Red and CD19 CAR-Jurkat T cells expressing copGFP were co-cultured at the ratio of 1:1 for 1 h. Cells were re-suspended in 4% PFA for 30 min, and images were acquired on the Amnis Imagestream Mk II Imagine flow cytometer (Luminex).REH cells expressing CD19-mRuby2 fusion labeled with Cell Trace Violet and CD19 CAR-Jurkat T cells expressing copGFP were seeded in cell culture imaging dishes. Protease inhibitor cocktail was added to prevent CD19 antigen degradation. Images were acquired on the GE Delta Vision OMX SR imaging system, and ImageJ software was used to generate the figures.GAPDH. Primers used for qRT-PCR assays are listed below:qRT-PCR was performed using 7500 Real-Time PCR Systems (Applied Biosystems). The data represent relative mRNA levels normalized to CD19-Forward: CCCAAGGGGCCTAAGTCATTG,CD19-Reverse: AACAGACCCGTCTCCATTACC;GAPDH-Forward: GGCACAGTCAAGGCTGAGAATG,GAPDH-Reverse: ATGGTGGTGAAGACGCCAGTA.SEM cells simultaneously expressing GFP and luciferase have been described previously t-test was used to analyze the differences between the groups.All statistical analyses were performed using the GraphPad Prism 6 software. The Student's in vitro capability of CTLs or CAR-T cells is usually tested using a short time-specific cell lysis assay. Whether these effector cells have any chronic effects on target cells when co-cultured for an extended time has received scant attention to date.Since cytotoxic CTL- or CAR-T cells mediated target cell destruction is an acute process, the In a co-culture system, B cell acute lymphoblastic leukemia (B-ALL) target cells caused quick and robust activation of CD19-CAR-Jurkat T cells that was substantially reduced after 24 h as demonstrated by attenuation in ERK phosphorylation to understand whether T cell activation was required for CAR T cell-mediated target cell death and tested whether target cells could still be destroyed by these anergic CAR-T cells. As expected, while CsA continuously blocked the activation of CAR T cells, as evidenced by diminished CD69 expression Figure F, a largWe examined whether target cells could be killed by non-signaling CD19-CAR-Jurkat (hereafter referred to as CD19-scFv-Jurkat) cells or T cell activation was necessary for target cell death B. As eviCollectively, these results suggested that CAR-T cells, especially CD19-CAR-T cells, could achieve target cell death through two distinct pathways, one involving the classical CTL pathway, causing rapid death and was dependent on T-cell activation and the other with slower kinetics and was T-cell activation independent.Since the activation of T cells is not crucial for target cell death, we reasoned that the target cell death effect could alternatively be achieved by other non-lymphocyte-derived antigen-recognizing cells. To this end, we determined whether non-T-cell-derived CD19-scFv bearing effector cells could cause target cell death B. IntereWe tested whether the targeted killing capabilities of scFv-bearing K562 cells recognizing antigens other than CD19 was a general phenomenon. Among the scFv-bearing K562 cells, CD22 targeting K562 cells could cause efficient target cell death in CD22-positive KOPN8 cells Figure D-F. ThesWe next addressed the question of how AARCs can kill or inhibit target cancer cells. Since target cells can no longer activate effector CAR-T cells in the late period of co- culture, we suspected that the death or inhibition of target cells might be related to the antigen loss on target cells.Previous studies have found that CD19-CAR-T cells provoke reversible CD19 antigen loss through trogocytosis, an active process in which the target antigen is transferred to T cells We next examined whether the phenomenon of antigen depletion could extend to other cell types and antigens. Even though CD19-scFv-K562 cells could not kill RAJI cells, they could induce effective CD19 antigen depletion in RAJI cells B. FurtheCD19 mRNA expression (Figure Although CD19 was depleted upon exposure to CD19-targeting effector cells, there was little variation in n Figure A. Moreovn Figure B, indican Figure B, even tWe addressed the mechanism underlying CD19 depletion by first tracking the behavior of CD19 proteins in the co-culture system of REH and CD19-CAR-Jurkat cells. To this end, CD19-mRuby2 fusion proteins were ectopically expressed in REH cells A, and thThe CD19 depletion after co-culturing suggested that endocytosis of CD19 was ultimately terminated. One of the main degradation pathways for endocytosed proteins is lysosomal degradation As a cell surface signaling protein, CD19 is required for several processes involved in B-cell development and function, and is, therefore, essential for the survival of malignant B cells, and serves as a crucial therapeutic target for B-cell malignancies The PI3K/AKT pathway is known to be instrumental in BCR signaling in B cells. High expression of CD19 on the malignant B cell surface may activate AKT and up-regulate MYC to support the survival of these cells We further confirmed that MYC downregulation was essential for B-ALL target cell death by examining whether the reestablishment of MYC protein levels could rescue the target cell death. As expected, overexpression of MYC or rescuing the AKT/MYC axis using the AKT activator SC-79 in REH cells could mitigate the target cell death by CD19-scFv-K562 Figure E-G. Thesin vivo efficacy of CD19-AARC cells in a previously established B-ALL xenograft mouse model in vitro and in vivo efficacies of CD19-scFv presenting MSCs. Similar to other CD19-AARC cells, CD19-scFv-MSCs caused CD19 depletion and apoptosis efficiently in the co-cultured B-ALL target cells (Figure in vitro and in vivo efficacies of CD19-scFv-MSCs, suggesting the ability of CD19-AARC cells to serve as a novel cellular therapeutic approach for B-ALL leukemia treatment.We next determined the s Figure A-C. Subss Figure D. Leukemin vitro efficacy of CAR-T cells. However, the chronic effect of CAR-T cells has seldom been addressed. Here we have shown that the cytotoxic effect of CAR-T cells exerts both acute and chronic effects (Figure Target cell death by CTLs is thought to be a rapid process. Therefore, a 4-hour cytotoxicity assay is routinely used to test the s Figure . The forThe chronic effect of CAR-T cells is largely dependent on antigen selection. In this context, our study provides valuable insights into antigen selection and CAR design. The first choice is that the antigen won't be internalized and depleted when recognized by the corresponding CAR. In this way, the escape of the antigen can be avoided and the target cells can be continuously recognized and destroyed. Alternatively, if the antigen is critical for the target cell survival, it can be internalized and depleted when recognized by effector cells. Thus, dysfunction of the antigen can lead to cell destruction.Here we showed CD19 is a superior target because CD19-recognizing effector cells can maintain the target cells being deprived of cell-surface CD19, and therefore effectively cause chronic cell death in the target cells whose survival is dependent on CD19 signaling. This can partly explain why CD19-CAR-T remains the most successful CAR-T therapeutic. However, various antigens may have specific characteristics in different cancer cells, at least partly rationalizing the distinct response of cancers to different CAR-Ts or even the same CAR-T.Consistent with previous reports The AARC could maintain the antigen depletion status of target cells through a transient interaction, which can endow them with cell death capability as a serial killer. Although antibody-based drugs, such as CD19 antibodies, can also cause antigen depletion The AARC can serve as the \u201coff-the-shelf\u201d ready-to-use therapeutic agent for large-scale clinical applications. The AARC treatment has a lower financial burden, no risk of manufacturing failure in certain patients or delay in treatment due to lengthy manufacturing procedures. Moreover, the non-lymphocytes based cell therapy is independent of T-cell activation, thus will greatly reduce the risk of CRS. Therefore, these universal cells are paving the way for a new generation of the allogeneic cellular therapeutic strategy being delivered to multiple recipients without re-editing of T cells.For those who are not suitable to be treated with CD19 CAR-T, AARC treatment might provide an alternative option. In our study, MSCs were chosen as an AARC source because of the low immunogenicity, widely demonstrated clinical safety, good transplant potential, and tumor-homing features Supplementary figures.Click here for additional data file."} +{"text": "To comparatively analyse the aberrant affinity maturation of the antinuclear and rheumatoid factor (RF) B cell repertoires in blood and tissues of patients with Sj\u00f6gren\u2019s syndrome (SjS) using an integrated omics workflow.Peptide sequencing of anti-Ro60, anti-Ro52, anti-La and RF was combined with B cell repertoire analysis at the DNA, RNA and single cell level in blood B cell subsets, affected salivary gland and extranodal marginal zone lymphomas of mucosa-associated lymphoid tissue (MALT) of patients with SjS.Affected tissues contained anti-Ro60, anti-Ro52, anti-La and RF clones as a small part of a polyclonal infiltrate. Anti-Ro60, anti-La and anti-Ro52 clones outnumbered RF clones. MALT lymphoma tissues contained monoclonal RF expansions. Autoreactive clones were not selected from a restricted repertoire in a circulating B cell subset. The antinuclear antibody (ANA) repertoires displayed similar antigen-dependent and immunoglobulin (Ig) G1-directed affinity maturation. RF clones displayed antigen-dependent, IgM-directed and more B cell receptor integrity-dependent affinity maturation. This coincided with extensive intra-clonal diversification in RF-derived lymphomas. Regeneration of clinical disease manifestations after rituximab coincided with large RF clones, which not necessarily belonged to the lymphoma clone, that displayed continuous affinity maturation and intra-clonal diversification.The ANA and RF repertoires in patients with SjS display tissue-restricted, antigen-dependent and divergent affinity maturation. Affinity maturation of RF clones deviates further during RF clone derived lymphomagenesis and during regeneration of the autoreactive repertoire after temporary disruption by rituximab. These data give insight into the molecular mechanisms of autoreactive inflammation in SjS, assist MALT lymphoma diagnosis and allow tracking its response to rituximab. Sj\u00f6gren\u2019s syndrome features activated B cells in affected tissues, aberrancies in circulating B cell populations and autoantibodies, including anti-Ro60/SSA, antiRo52/SSA, anti-La/SSB and rheumatoid factors (RFs).RF and antinuclear antibody (ANA) clones are enriched in affected tissues where they occur as a small part of a polyclonal repertoire. RF and ANA clones affinity maturate in divergent fashion, which increases during secondary RF lymphomagenesis and after temporary disruption by rituximab (RTX).Analysis of RF clones in affected tissues may assist identification of mucosa-associated lymphoid tissue lymphomas and tracking of their response to RTX.Sj\u00f6gren\u2019s syndrome (SjS) is a systemic autoimmune disease, principally affecting exocrine glands. It features activated B cells in affected tissues, aberrancies in circulating B cell populations and circulating autoantibodies (AutoAbs), including antinuclear antibodies (ANAs) anti-Ro60/SSA, anti-Ro52/SSA, anti-La/SSB and rheumatoid factors (RFs).The generation of the ANA-specific and RF-specific B cell repertoires and how they breach self-tolerance checkpoints has not been precisely determined. In experimental animal models, autoreactive B cells affinity maturate in antigen-dependent fashion in germinal centres in lymphoid tissues or in extrafollicular sites such as the splenic marginal zone.Evidence suggests that the same mechanisms operative in SjS might also contribute to generation of SjS-associated RF-derived MALT lymphomas. Lymphomagenesis of RF clones results from gradual accumulation of lymphoma driver mutations.Herein, we combined mass spectrometry (MS) approach for serum antibody sequencing with methods to analyse the B cell repertoire at the RNA, DNA and single cell level to investigate the selection and affinity maturation of the autoreactive B cell repertoire in blood and tissues of patients with SjS.The autoreactive B cell repertoire was analysed in six patients with SjS, fulfilling the 2016 ACR-EULAR classification criteria, in comparison to 4 age-matched and 3 elderly healthy controls . Among p10.1136/annrheumdis-2021-221604.supp1Supplementary data10.1136/annrheumdis-2021-221604.supp12Supplementary dataIn six patients and four age-matched and three elderly healthy controls, the B cell repertoire was determined by heavy chain Ig mRNA analysis using next-generation sequencing in blood and tissue samples. In blood samples, a comparison was made between whole blood samples and sorted B cell subsets. This was done to account for the bias toward abundant plasmablast/-cell reads in the blood samples. Sorting of B cells from tissues for clonality is challenging and cannot be performed on stored tissue samples. Therefore, in affected patient tissues, we made a comparison between Ig-RNAseq and NGS at the DNA level for Ig heavy and light chain (Ig-DNAseq) that we validated for clonality testing.10.1136/annrheumdis-2021-221604.supp2Supplementary dataFirst, we analysed the anti-Ro/anti-La and RF repertoire in blood and affected tissues from six patients with SjS. Ig-RNAseq showed that a higher fraction of Ig reads from affected tissues compared with blood mapped to MS proteomic sequences of AutoAbs p<0.01; .To gain more insight in the B cell repertoire in blood of patients with SjS with anti-Ro/anti-La, we compared these (n=26) with patients with other anti-Ro/anti-La positive autoimmune disease (n=10) and healthy controls n=24). Most Ig sequences detected in blood were low abundant clones, but a number of sequences was highly expressed , switched memory (SM) and double-negative (DN) B cells in contrast to na\u00efve B cells .Tissues contained a similar number of HESs compared with circulating memory B cell subsets. The SHM load was similar between circulating memory B cell subsets and affected tissues . The num10.1136/annrheumdis-2021-221604.supp4Supplementary dataWe analysed the relationship between HESs and AutoAbs in blood. A small proportion of the B cell repertoire in whole blood and sorted B cell subsets of patients with SjS mapped to amino acid sequences of AutoAbs . RFs werTo gain more insight in the autoreactive repertoire in affected tissues, we comparatively analysed clones with immunohistochemistry (IHC), Ig-RNAseq and Ig-DNAseq using a protocol that we developed and validated for detection of B cell clonality by NGS in stored tissues.Morphology/IHC for CD20, CD79a, kappa and lambda showed a variable infiltration by B cell follicles (CD20+) and plasma cells (CD79a+/CD20\u2212) in affected SG tissues . The MAL10.1136/annrheumdis-2021-221604.supp3Supplementary dataBoth Ig-RNAseq and Ig-DNAseq showed the presence of a polyclonal repertoire with a variable extent of HESs in all tissues. ANA and RF clones constituted a small fraction of clones . MALT lyWe analysed the regeneration of the autoreactive repertoire in affected tissues after temporary perturbation with RTX monotherapy in two patients with MALT lymphoma see . Ig-RNAsSimilarly, the ANA clones persisted or disappeared after RTX and new clones appeared . SimilarWe next analysed gene segment usage of ANA and RF clones in pooled samples. The number of anti-Ro60, anti-La and anti-Ro52 clones was higher than the number of RF clones (p<0.05).Analysis of IGHV gene use revealed that RF clones used a limited number of IGHV gene segments with predominant use of IGHV1-69, combined with IGHJ4 and an IGKV3-20 light chain . IGHV1-610.1136/annrheumdis-2021-221604.supp5Supplementary data10.1136/annrheumdis-2021-221604.supp7Supplementary data10.1136/annrheumdis-2021-221604.supp8Supplementary dataEarlier studies suggest that ANA may be derived from polyreactive B cell precursors that share sequence motifs.10.1136/annrheumdis-2021-221604.supp6Supplementary dataAnalysis of affinity maturation showed that the SHM load of RF clones was relatively low compared with ANA clones . The SHMBefore RTX intra-clonal diversification was similar between ANA and RF clones . RF MALTWe analysed the antigen dependence of affinity maturation in ANA and RF clones using the BASEline tool see . AnalysiIn an earlier study, selection of recombinantly expressed Igs from SjS tissues was enhanced by N-glycosylation sites in the B cell receptor variable region FR1, resulting in activation by C-type lectins.Finally, we analysed the potential utility of Ig-seq to assist lymphoma diagnosis in patients with SjS. For this, Ig clonality assessment was performed in affected tissues of two additional patients with SjS (B012 and B013), who had been referred from other hospitals because of challenging lymphoma diagnostics levels, which may facilitate clonal expansion of new and persisting clones.Finally, from a clinical perspective, we found that RTX did not succeed in abrogating lymphomatous B cell clones. Possibly, other B cell depletive treatments may have superior efficacy. Besides this, it can be challenging to discriminate MALT lymphoma from SjS-associated inflammation, determine the best treatment regimen and assess response to treatment. A diagnosis is made by assessing the combination of clinical presentation, histology, phenotype and sometimes clonality analysis and/or genetic studies. Also in the study patients, the diagnosis of MALT lymphoma had been challenging. Ig-seq retrospectively could have assisted in establishing a diagnosis earlier and more precisely. Future prospective investigations should investigate in more patients in more detail the added value of Ig-seq for diagnostic problems in patients with SjS with one or more mass lesions and for detection of small (pre-)lymphomatous clones in major SGs.To summarise, we used for the first time an integrated omics workflow to analyse the generation of the autoreactive repertoire in circulating B cell populations and affected tissues of patients with SjS, and demonstrated tissue restricted, aberrant affinity maturation of RF clones compared with ANA clones in inflamed tissues. These data give insight into the molecular mechanisms of autoreactive inflammation and MALT lymphoma, and help to analyse the clinical response to RTX treatment in individual patients.10.1136/annrheumdis-2021-221604.supp9Supplementary data10.1136/annrheumdis-2021-221604.supp10Supplementary data10.1136/annrheumdis-2021-221604.supp11Supplementary data"} +{"text": "Human-to-feline and airborne transmission among cats of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) has been described, though documented feline-to-human transmission has not been reported. In October 2020, all 3 Malayan tigers at a Tennessee AZA accredited zoo were diagnosed with symptomatic SARS-CoV-2 infection. We investigated to determine source and prevent further transmission.Tiger nasal swab specimens were tested at the National Veterinary Services Laboratories (NVSL). An environmental assessment at the zoo was completed. We interviewed 18 staff who interacted with the tigers during the 2 weeks before animal symptom onset. Confirmed human cases were defined as persons testing positive for SARS-CoV-2 by RT-PCR during September 28\u2013October 29, with tiger interaction during their 14-day incubation period. Interviewed staff had repeat SARS-CoV-2 RT-PCR and serum IgG testing on October 29. Tigers and staff testing positive had specimens sent to CDC for genomic sequencing. Tiger sequences were compared phylogenetically with 30 geographically associated human cases collected within 2 weeks of the outbreak and > 200 background sequences from TN.NVSL confirmed SARS-CoV-2 infection in all 3 tigers. Environmental assessment identified fencing between humans and animals allowing airflow and an open outdoor exhibit observation point above the habitat. Confirmed cases were identified in a tiger keeper and veterinary assistant; both developed symptoms after exposure to symptomatic tigers and one sample was genotyped. Staff did not report known contact with ill visitors. All staff were negative for SARS-CoV-2 IgG. The tigers and most temporally and geographically associated cases had genetic sequences in clade 20G and B.1.2. Tiger sequences were 3-6 single nucleotide polymorphisms different from the positive tiger keeper .Figure. Whole-genome phylogenetic analysis.Whole-genome phylogenetic analysis from a portion of clade 20G showing divergence estimates from SARS-CoV-2 Wuhan-Hu-1 reference genome with sequences from humans living in Tennessee and Malayan tigers sampled during the outbreak investigation in October 2020. Sequence analysis showed 3-6 single nucleotide polymorphisms (SNPs) differences between one human tiger keeper and all three tiger sequences. Differences are indicated by one-step edges (lines) between colored dots . Numbers indicate unique sequences. Note not all analyzed sequences are shown in this figure.Using a One Health approach, we concluded the index tiger was likely infected via transmission from an ill visitor at an exhibit observation point or unidentified asymptomatic staff. Infection spread to the other 2 tigers and tiger-to-human transmission to 2 staff is possible thereafter. The zoo was advised on infection control practices for humans and animals, and no additional cases were identified.William Schaffner, MD, VBI Vaccines (Consultant)"} +{"text": "Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) has been associated with a significantly increased risk of venous and arterial thromboembolism, particularly in severely sick patients. Recently, cerebral venous sinus thrombosis (CVST) cases have been reported in the context of coronavirus disease-2019 (COVID-19). These cases either had an active COVID infection with a positive reverse transcription\u2010polymerase chain reaction (RT\u2010PCR) or were symptomatic during the presentation. We present here a 41-year-old male with CVST who had negative RT-PCR and positive immunoglobulin G (IgG) COVID-19 antibodies. He was neither diagnosed nor had a flu-like illness before admission. This case highlights that CVST can be a late sequela of previously undiagnosed asymptomatic COVID-19 infection. Coronavirus disease-2019 (COVID-19) has been associated with a heightened risk of arterial and venous thromboembolic complications including stroke, myocardial infarction, acute limb ischemia, and venous thromboembolism. The proposed mechanisms include direct viral endothelial invasion followed by activation of the coagulation cascade, cytokine storm-related prothrombotic state, and/or increased platelet activity. Various virus infections have been associated with coagulation disorders including herpes simplex, human immunodeficiency virus (HIV), cytomegalovirus (CMV), severe acute respiratory syndrome (SARS), and Middle East respiratory syndrome (MERS) [A 41-year-old right-handed male with no significant past medical history presented with sudden onset of severe frontal headache started on the day of admission. He denied any aggravating or relieving factors. It was associated with progressively worsening difficulty in speaking started 1-2 hours after the beginning of the headache. He denied vision loss, focal weakness, sensory loss, vomiting, abnormal body movements, involuntary loss of urine or stool, and loss of consciousness. The patient was afebrile at admission and remained afebrile throughout hospitalization. Other vital signs at admission were - blood pressure 141/76, heart rate 77, respiratory rate 18, and oxygen saturation 97% on room air. Physical examination was remarkable for significant expressive aphasia without other neurological deficits. Laboratory data showed elevated\u00a0erythrocyte sedimentation rate (ESR) and c-reactive protein (CRP) on admission. SARS-CoV-2 reverse transcription\u2010polymerase chain reaction (RT\u2010PCR) was negative, immunoglobulin G (IgG) antibodies were positive. Complete blood count (CBC), comprehensive metabolic panel (CMP), and lipid panel were within normal limits. Imaging of the head was consistent with an acute dural sinus thrombosis of the superior sagittal sinus extending into the left transverse and sigmoid sinus Figures -1B. AlsoCVST is a rare neurovascular emergency caused by several systemic or local risk factors. These include hematologic prothrombotic states, infections, malignancies, pregnancy, hormonal contraceptives, dehydration, head injury, and connective tissue diseases. Prothrombotic state induced by hyperactivation of cytokines in COVID-19 has been associated with arterial and venous thrombosis, especially in severely sick patients ,4. The tOur case reveals that a hypercoagulable state can be seen as a late manifestation even if the patient is immune after an asymptomatic COVID-19 infection. All patients with CVST should be tested for COVID-19 during the current pandemic, regardless of symptoms. Due to potential association, COVID-19 should be considered as one of the differentials for CVST."} +{"text": "GBM is a highly metabolic cancer phenotype that confers sustained growth and evasion of cell death mechanism via mitochondrial dysregulation. Efforts to re-engage mitochondrial metabolism via anti-cancer therapeutics has not been successful. BPM 31510 is a CoQ10-lipid conjugate nanodispersion for delivery of CoQ10 preferentially to mitochondria of human cells. BPM has demonstrated anti-cancer effects across multiple cancers, without adversely affecting normal tissue. The anti-cancer mechanism of CoQ10 was elucidated by Interrogative Biology, a data-driven approach to understand disease biology, identify targets and biomarkers of disease. Specifically, oncogenic and corresponding non-disease normal cell-based models were subjected to cancer specific perturbations . Comprehensive multi-omic and functional endpoints data were profiled. A Bayesian artificial intelligence analytics was used to generate network models in a data driven manner to identify BPM 31510 mechanism . BPM 31510 re-capitulated its anti-cancer effect in GBM models, including LN-229 xenograft and C6 glioma allograft, both as monotherapy and in combination with temozolomide (TMZ)/radiation. The platform generated network maps from longitudinal pharmacodynamic samples (20 samples/28 days) collected from GBM patient refractory to TMZ/radiation/bevacizumab identified alterations in intermediary metabolism as drivers of Progression Free Survival (PFS) and Overall Survival (OS) in response to BPM 31510 treatment. The platform supports the ongoing Phase 2 trial of adjuvant BPM 31510 plus TMZ/radiation in newly diagnosed GBM patients and potential accelerated approval."} +{"text": "The imaging method of choice for diagnosis or exclusion of CVST is magnetic resonance imaging (MRI) combined with contrast-enhanced venous MR angiography (MRA). On T2*-weighted or susceptibility weighted MR sequences, the thrombus causes susceptibility artefacts (blooming), that allow for the detection even of isolated cortical vein thromboses. The diagnosis of TTS-CVST can usually be made reliably in synopsis with the clinical and laboratory findings. A close collaboration between neurologists and neuroradiologists is mandatory. TTS-CVST requires specific regimens of anticoagulation and immunomodulation therapy if thrombocytopenia and/or pathogenic antibodies to PF4/polyanion complexes are present. In this review article, the diagnostic and therapeutic steps in cases of suspected TTS associated CSVT are presented.Cerebral venous and sinus thrombosis (CVST) after adenovirus-vectored COVID-19 ChAdOx1 nCov-19 (Oxford\u2013AstraZeneca) and Ad26.COV2.S (Janssen/Johnson & Johnson) is a rare complication, occurring mainly in individuals under 60 years of age and more frequently in women. It manifests 4\u201324 days after vaccination. In most cases, antibodies against platelet factor-4/polyanion complexes play a pathogenic role, leading to thrombosis with thrombocytopenia syndrome (TTS) and sometimes a severe clinical or even fatal course. The leading symptom is headache, which usually increases in intensity over a few days. Seizures, visual disturbances, focal neurological symptoms, and signs of increased intracranial pressure are also possible. These symptoms may be combined with clinical signs of disseminated intravascular coagulation such as petechiae or gastrointestinal bleeding. If TTS-CVST is suspected, checking The online version contains supplementary material available at 10.1007/s00234-022-02914-z. Reporting of several fatal cases with cerebral venous sinus thrombosis (CVST) and other thromboses at various sites in combination with a thrombocytopenia 4 to 28 days after vaccination with the SARS-CoV-2 vaccines ChAdOx1 nCov-19 (Oxford\u2013AstraZeneca) and Ad26.COV2.S (Janssen/Johnson & Johnson) led to restriction of vaccination in several countries. This condition has been introduced as vaccine-induced thrombotic thrombocytopenia (VITT) or vaccine-induced postthrombotic immune thrombocytopenia (VIPIT) . ClinicaHeadache is the leading symptom of CVST as well as of CVST-TTS. Therefore, an increasing requirement of MRI examinations of the cranium (cMRI) has been recognized. For avoidance of under- or overdiagnosis, a coordination of the diagnostic procedure between neurologists and neuroradiologists is required. In this review, we aim to summarize the current available literature on the diagnostic management of CVST-TTS. Furthermore, we propose an interdisciplinary agreed diagnostic and therapeutic approach, if vaccine-induced CVST with or without TTS is suspected with reference to the recommendations of the German Society for Thrombosis and Haemostasis Research (GTH) .Thrombotic thrombocytopenia after vaccination has been described occasionally for vaccination against influenza, rabies, and H1N1 . HoweverThe number of reported cases in Germany associated with dangerous CVST-TTS after ChAdOx1 nCov-19 vaccination was 45 by May 2021 . ConsideCVST-TTS occurred usually within 4\u201324 days after vaccination with ChAdOx1 nCov-19 (Oxford\u2013AstraZeneca) . RecentlSubacute or acute, mostly holocephalic headacheEpileptic seizuresPersonality changes, deliriumVisual disturbancesCentral paresis and/or other focal neurological symptomsQuantitative and/or qualitative disturbance of consciousnessThe neuropsychiatric symptoms of CVST-TTS are similar as compared to spontaneous aseptic CVST without TTS:Cutaneous hematomasPetechiaePersistent secondary bleeding at cutaneous puncture sitesGastrointestinal bleedingA recent multicentre cohort study analyzed characteristics of CVST-TTS in comparison with CVST without TTS. Frequency of symptoms seemed to be comparable, the possible impairment of consciousness seemed be more severe in CVST-TTS . Since vd-dimer levels is detectable in venous thrombosis of any location. A normal d-dimer value, however, does not exclude a CVST, especially in patients with (1) isolated headache or (2) duration of symptoms for longer than 1 week [Heterozygous or homozygous factor V Leiden mutation (10\u201325% of cases)Heterozygous or homozygous prothrombin mutation G20210ACongenital antithrombin deficiencyCongenital protein C or protein S deficiencyPersistently increased factor VIIIAntiphospholipid antibodiesHyperhomocysteinemiaVery rarely dysfibrinogenemiaUsually, an increase in plasma n 1 week . Generaln 1 week :Heterozd-dimer (or fibrin degradation products), and decreased fibrinogen plasma level.A severe, rapidly developing DIC is confirmed by evidence of thrombocytopenia, prolonged partial thromboplastin time (PTT) and prolonged prothrombin time (PT), increased levels of plasma Blood count (platelet count!)d-dimers!)Coagulation parameters The laboratory work-up is initially directed by the grading of the clinical suspicion of CVST-TTS: more moderate suspicion or high level of suspicion Fig. . The lab\u2192 At presence of thrombocytopenia: order supplementary test for EDTA-associated pseudothrombopenia and request a manual blood count (with reference to platelet activation). Go on with cranial imaging as specified in the chapters below.d-dimer level is normal, CVST-TTS is unlikely: still, standard MRI with negative MRA or CT with contrast-enhanced venography should be considered. If plasma d-dimer level is increased, MRI with contrast-enhanced venography or CT with contrast-enhanced venography is obligatory.\u2192 At absence of thrombocytopenia: if also plasma Screening ELISA for HIT-2 Levels of immunoglobulins Search for hereditary or immunogenic thrombophilia (see earlier chapter)The laboratory work-up at high suspicion of CVST-TTS or imaging-proven CVST should in addition include the following:\u2192 If HIT-2 screening assay is positive: specialized laboratory workup with heparin-induced platelet aggregation (HIPA) assay and\u2014if HIPA is negative\u2014the modified HIPA assay to detect elevated serum IgG antibodies against PF4-polyanion complexes ; only a positive HIPA or PIPA assay proves vaccine-induced thrombosis [\u2192 If HIT-2 screening assay is positive, CVST is proven on cranial imaging decide urgently on the administration of intravenous Immunoglobulins (Cave: IgA deficiency has to be excluded prior to this therapy! [d-dimers\u2014rather unreliable). The choice of method\u2014MRI or CT unenhanced or with a contrast medium\u2014is based on the in-house imaging protocol, local conditions, the patient\u2019s condition, any potential contraindications, and the available neuroradiological expertise. If only unenhanced imaging is performed , MRI is the method of choice. An alternative is contrast-enhanced CT. CT and MRI should each be performed with venous angiography ; native MRA is sufficient in cases of moderate suspicion of CVST (see above) along with normal platelet count and plasma d-dimer level. To avoid radiation exposure, MRI should be preferred in younger patients and during pregnancy. In pregnancy, unenhanced MRA can avoid the administration of contrast medium [Radiologic imaging in suspected CVST is well established . The prot medium . In addiThe in-house examination protocol on the Siemens Avanto (1.5 Tesla) as well as the Siemens Vida (3.0 T) with corresponding imaging findings may be found in the Supplementary files and Ad26.COV2.S (Janssen/Johnson & Johnson). One course of a patient with CVST-TTS and intracranial bleeding was fatal.The following three case studies show that after vaccination with ChAdOx1 nCov-19 (Oxford\u2013AstraZeneca) or with Ad26.COV2.S (Janssen/Johnson & Johnson) vaccine with headache, sinus or bridging vein thrombosis should be considered as a possible complication.Bridging vein thrombosis after ChAdOx1 nCov-19 (Oxford\u2013AstraZeneca) vaccination 12 days ago.A 25-year-old female patient was vaccinated with ChAdOx1 nCov-19 (Oxford\u2013AstraZeneca) vaccine. From the 12th day after vaccination, she experienced persistent headache and a feeling of pressure bifrontally\u2014pain scale 6\u20137 out of 10. This increased especially when turning her gaze. In addition, there was a photo- and phonophobia with staggering dizziness, especially when standing up.The typical morphologic correlate of a thrombosis of bridging veins is the \u201cblooming sign\u201d in T2* see Fig. . PC-MRA Bridging vein thrombosis, SAB and thrombosis of the sigmoid sinus after ChAdOx1 nCov-19 (Oxford\u2013AstraZeneca) vaccination 11 days ago.d-dimers and a thrombocytopenia. Figure A 31-year-old man after ChAdOx1 nCov-19-vaccination (11 days ago) showed flu-like symptoms for 1\u20132 days with improvement. Now cephalgia was present for about 4 days. SARS CoV2-PCR-test was negative. At the time of clinical presentation, he had a holocephalic headache with a range of 8 of 10 in subjective pain scale. Decreasing pain symptoms undergoing intravenous paracetamol application to 6 of 10. The clinical laboratory values show elevated Atypical bleeding after Ad26.COV2.S (Janssen/Johnson & Johnson) vaccination 12 days ago.A 29-year-old patient had a Sars-CoV2-vaccination using \u201cone-shot\u201d of Ad26.COV2.S (Janssen/Johnson & Johnson). At the time of his clinical presentation, he was somnolent, disoriented, had headache since 3 o\u2019clock in the night, and recurrent nausea and vomiting. In neurological examination, he had no paresis. The laboratory findings showed evidence of vaccine-induced immune thrombotic thrombocytopenia.The immediately performed CT and MRI scans revealed atypical hemorrhages of the right hemisphere with thrombosis of the left bridging veins as well as the transverse and sigmoid sinus Fig. .Fig. 4AHemicraniectomy and hematoma evacuation right hemispheric followed. Indication for anticoagulation with argatroban, a drug used to inhibit blood clotting by direct inhibition of thrombin, was given.If vaccine-induced CVST with or without TTS is suspected, or if petechial skin hemorrhage is present, there is indication of emergency hospital admission. In the case of a clinically unclear assessment in outpatient setting, the blood count should be checked urgently. In the case of thrombocytopenia, the immediate hospital admission is indicated .Because of the potentially foudroyant course, the patient with proven vaccine-induced CVST-TTS should be treated during the first few days on a Stroke unit or, if the course is severe, in the (neuro-) intensive care unit with regular neuromonitoring. The general measures follow the guidelines for therapy for any CVST . The speIn Germany, cases of vaccine-induced CVST-TTS have to be reported to the Paul-Ehrlich-Institute (PEI), and as well the pharmaceutical manufacturer should be informed. In addition, there are scientific case collections at national and at European level. For example, all neurological clinics in Germany were asked by the German Society for Neurology (DGN) to include cases of CVST-TTS, intracerebral hemorrhage, or temporal cerebral ischemia related to a COVID-19 vaccination to a national vaccination survey; by mid-April 2021, data of more than 60 patients were evaluated and published promptly . Additiod-dimers level, platelet count, and HIT2 screening assay is mandatory. The imaging method of choice for confirming or excluding CVST is MRI with venous MRA. On T2 *w/SWI sequences, the thrombus causes susceptibility artefacts, in association with pronounced signal cancelation (blooming). MRI/MRA as well as CT/CTA can usually reliably confirm the diagnosis of CVST in synopsis with clinical and laboratory findings. Vaccine-induced CVST-TTS requires specific anticoagulation and immunomodulation therapies, especially in the case of thrombocytopenia and/or the detection of pathogenic antibodies against PF4/polyanion complexes. A close interaction between neurologists, neuroradiologists, and hemostaseology specialists is mandatory.The main symptom of vaccine-induced CVST-TTS is headache beginning 4\u201324 days after vaccination. First ever seizures, visual disturbances, focal neurological symptoms, and signs of increased intracranial pressure may also occur. If CVST-TTS is suspected, the urgent control of plasma Supplementary Figure S1Summary of pathognomonic signs of sinus and cerebral venous thrombosis on CT and MRI. A, hyperdense internal cerebral veins on CT scan; B, atypical bleeding on CT scan; C, sulcal SAB, hyperintense in FLAIR, which are hypointense in T2*-weighted images (D); E, blooming of the bridging veins in T2*; \u201ccord sign\u201d in CE-images ; H, missing venous and sinus contrast in CE-MRI. (PNG 837 kb)High resolution image (TIF 260 kb)Supplementary Table S1DWI diffusion weighted imaging , FLAIR fluid attenuated inversion recovery, FOV field of view, CM contrast media, MIP maximum intensity projection, MPR multiplanar reconstruction, CE-MRA contrast enhanced MR-angiography, PC phase contrast (phase-contrast-angiography), TR repetition time, TE echo time (DOCX 14 kb)Proposed protocol for MRI in case of suspicion of vaccine-induced CVST . Supplementary Table S2SWI susceptibility weighted imaging, FLAIR fluid attenuated inversion recovery, FOV field of view, CM contrast media, MIP maximum intensity projection, MPR multiplanar reconstruction, MRA MR-angiography, TWIST time-resolved angiography with interleaved stochastic trajectories, PC phase contrast (phase-contrast-angiography), TR repetition time, TE echo time (DOCX 14 kb)Proposed protocol for MRI in case of suspicion of vaccine-induced CVST . Supplementary Table S3CM contrast media, PCA phase contrast angiography, SWI susceptibility-weighted imaging, TOF time-of-flight angiography (DOCX 12 kb)Signal of thrombosis in MRI depending on thrombus age (modified ). CM con"} +{"text": "Teaching Point: Contrast-enhanced FLAIR images have unsurpassed value for the radiological depiction of hypertensive papilledema. FLAIR acquisition should therefore be performed after intravenous contrast, especially in the of work-up of intracranial hypertension and/or tumor. Figure 1A) and post-contrast T1-WI images, but far less than on fat-suppressed post-contrast FLAIR (fluid-attenuated inversion recovery) .A healthy 31-year-old woman presented with a three-month history of increasing holocranial headaches, weight loss, and nausea. Initial contrast-enhanced (CE) computed tomography (CT) demonstrated a large right-sided temporal malignant tumor surrended by edema. Magnetic resonance (MR) work-up supported the hypothesis of a high-grade glioma (not shown). Edema of the optic disc matching the clinical signs of intracranial hypertension (ICHT) was additionally highlighted on MR. The feature had been suspected on CT (not shown), becoming more obvious on T2-weighted (WI) (Ophthalmologic examination revealed a bilateral grade IV papilledema and a left lateral homonymous hemianopia. Histopathologic examination of resected specimen revealed a WHO grade II astrocytoma with IDH1 mutation.The exquisite depiction of the papilledema on post-contrast FLAIR views was hypothesized to be synergistically due to both the high protein content and the leakage of contrast agent molecules within the fluid filling the protruding disks. The combined paramagnetic effect of proteins and contrast agent results in a strong signal intensity on FLAIR images, contrasting with nulled signal intensity of the adjacent vitreous fluid. Because of this, contrast-enhanced FLAIR images surpassed all other imaging techniques for the radiological depiction of hypertensive papilledema . FLAIR a"} +{"text": "Xuanwei area, in southwestern China, harbors the highest female lung cancer rate in the country (Supplementary Table 1) . The genThe patients recruited for this study were 117 non-smoker females with untreated primary lung adenocarcinoma (LUAD) from the Xuanwei area, who were receiving surgical treatment at Yunnan Cancer Hospital (Supplementary Table 2). Samples were taken for whole-exome sequencing (WES) , and 33 normal and 115 tumor samples were sequenced with mRNA-Seq technology. Datasets of 168 TCGA-LUAD female smokers (TLSF) and 102 TCGA-LUAD female non-smokers (TLNF) were adopted from The Cancer Genome Atlas (TCGA) program for genomic comparison between lung cancer associated with cigarettes and that associated with smoky coal . RNA-seq dataset for mouse_cigarette model analyses were downloaded from GEO accession: GSE76205 [https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE76205]. RNA-seq and somatic mutation datasets of TCGA-LUAD and TCGA-LUSC were downloaded from XENA (https://xena.ucsc.edu/). All other relevant data are available within the article, Supplementary data or available from the authors upon request.Clinical data (deidentified) are listed in Supplementary Table 2. Raw sequencing data including WES and RNA-seq datasets from human samples have been deposited in the Genome Sequence Archive under the accession code HRA000124. The RNA-seq dataset from rat model samples has been deposited in the Gene Expression Omnibus (GEO, nwab152_Supplemental_FilesClick here for additional data file."} +{"text": "Dysregulated formation of neutrophil extracellular traps (NETs) is observed in acute viral infections. Moreover, NETs contribute to the pathogenesis of acute viral infections, including those caused by the dengue virus (DV) and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Furthermore, excessive NET formation (NETosis) is associated with disease severity in patients suffering from SARS-CoV-2-induced multiple organ injuries. Dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) and other members of C-type lectin family have been reported to interact with viral glycans to facilitate virus spreading and exacerbates inflammatory reactions. Moreover, spleen tyrosine kinase (Syk)-coupled C-type lectin member 5A (CLEC5A) has been shown as the pattern recognition receptor for members of flaviviruses, and is responsible for DV-induced cytokine storm and Japanese encephalomyelitis virus (JEV)-induced neuronal inflammation. Moreover, DV activates platelets via CLEC2 to release extracellular vesicles (EVs), including microvesicles (MVs) and exosomes (EXOs). The DV-activated EXOs (DV-EXOs) and MVs (DV-MVs) stimulate CLEC5A and Toll-like receptor 2 (TLR2), respectively, to enhance NET formation and inflammatory reactions. Thus, EVs from virus-activated platelets (PLT-EVs) are potent endogenous danger signals, and blockade of C-type lectins is a promising strategy to attenuate virus-induced NETosis and intravascular coagulopathy. Listeria (spleen tyrosine kinase (Syk)-coupled C-type lectin member 5A, CLEC5A) . Ev. Ev58]. It is interesting to note that EV serum level increased in COVID-19 patients, and correlates with clinical symptoms and lethality , 110. ItIt is also interesting to note that severe pulmonary inflammation in COVID-19 patients is associated with thrombotic complications, such as microangiopathy and pulmonary embolism , 115. AsEVs have been implicated in regulation of infectious and autoimmune diseases, but the underlying molecular mechanisms are still unclear. We have demonstrated that platelets secrete EVs after incubation with DV, LPS, and thrombin. Interestingly, all the"} +{"text": "To limit the spread of severe acute respiratory syndrome coronavirus 2, the government of China has been monitoring infected travelers and minimizing cold-chain contamination. However, other factors might contribute to recurring outbreaks. We analyze the role of undocumented migrants as potential transmitters of severe acute respiratory syndrome coronavirus 2 in China. As a result, the focus of epidemic control and prevention work has shifted from local to imported cases of COVID-19. Although viral spread has been contained by mandates minimizing travel and cold-chain contamination (China\u2019s efforts to suppress coronavirus disease (COVID-19), the illness caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), rely on rigorous quarantine measures. These measures contributed to a decline in COVID-19 cases; no new locally acquired cases were reported in China on March 18, 2020 (mination .http://www.yn.gov.cn/ztgg/yqfk/zcfk/202004/t20200401_201604.html), which outlined strict measures to prevent COVID-19 importation from land and water ports. This notice discouraged citizens of adjacent countries from entering Yunnan Province; if entry was required, then those citizens should enter Yunnan Province via 1 of 19 official land ports under accession nos. EPI_ISL_632934 and EPI_ISL_632935. Sequence alignment analyses (To evaluate potential variations in SARS-CoV-2 sequences for the 2 cases, we conducted whole-genome sequencing (analyses Figure 2Although the SARS-CoV-2\u2013infected migrants did not cause a COVID-19 outbreak, the event illustrates a transmission pathway distinct from air travel and cold-chain food transmission (Additional information on undocumented migrants reintroducing COVID-19."} +{"text": "This new method, featuring a low catalyst loading, fast reaction rate, and solvent-free conditions, provided facile access to a diversity of carbamate-protected Mannich bases. A mechanistic investigation indicated that the three-component reaction proceeds via sequential aldol condensation and aza-Michael addition, but not the Mannich-type pathway.Hafnium(IV) triflate (Hf(OTf) The replacement of amine with much less reactive carbamate radically changes the reaction pathway to sequential aldol condensation and aza-Michael addition.In summary, we developed a Hf(OTf)"} +{"text": "Dear Sirs,Since the end of 2019, the coronavirus disease 2019 (COVID-19) pandemic induced by an infection with severe acute respiratory coronavirus 2 (SARS-CoV-2) has led to millions of deaths worldwide. The unprecedented fast development of a vaccination against SARS-CoV-2 led to the approval of several vaccinations by the authorities since December 2020. Rare severe side effects are sometimes not observed during the pivotal trials but get noticed during daily clinical practice such as thrombotic thrombocytopenia after ChAdOx1 nCov-19 vaccination [A 28-year-old healthy female received the first dosage of mRNA vaccination against SARS-CoV-2 (Moderna). Five days later, she complained about muscle pain of her thigh muscles, radiating to the lower legs, accompanied by an asymmetrical weakness of the lower limbs. Seven days following vaccination, a blood test revealed marked elevation of creatine kinase and transaminases. At first presentation, she had a mild predominantly left-sided weakness of hip flexor and knee extension (MRC 4-/5 vs. MRC 4/5) with marked subcutaneous leg edema Fig.\u00a0a, the crCOVID-19 mRNA vaccine-associated side effects include pain, redness and pain at the injection site, fatigue, headache, myalgia or arthralgia , 3. One In summary, we present a new and so far unknown complication of mRNA vaccination against SARS-CoV-2. Clinicians should be vigilant especially in patients developing myalgia with paresis following COVID-19 vaccination to detect rhabdomyolysis and start treatment without delay."} +{"text": "Additionally, panobinostat co-treatment or pretreatment synergized with NK cells to mediate tumor cell cytolysis. Mechanistically, panobinostat treatment increased the expression of cell adhesion and tight junction-related genes, promoted conjugation formation between NK and tumor cells, and modulates NK cell-activating receptors and ligands on tumor cells, contributing to the increased tumor cytolysis. Finally, panobinostat therapy led to better tumor control and synergized with anti-PD-L1 therapy. Our data highlights the anti-tumor potential of HDAC inhibition through tumor-intrinsic toxicity and enhancement of NK \u2013based immunotherapy.Histone deacetylases (HDAC) are frequently overexpressed in tumors, and their inhibition has shown promising anti-tumor effects. However, the synergistic effects of HDAC inhibition with immune cell therapy have not been fully explored. Natural killer (NK) cells are cytotoxic lymphocytes for anti-tumor immune surveillance, with immunotherapy potential. We showed that a pan-HDAC inhibitor, panobinostat, alone demonstrated anti-tumor and anti-proliferative activities on all tested tumors Natural killer (NK) cells are cytotoxic components of the innate immune system and are the first line of host anti-tumor immune surveillance . Aside fin vivo inhibition of HDAC activity does not impair NK cell functions (Histone deacetylases (HDACs) belong to a family of 18 proteins (HDAC1\u201311 and SIRT1\u20137) known to deacetylate histones and non-histone proteins . Over-exunctions ; thus, tHere, we assessed the cytotoxicity of panobinostat against tumor cell lines both alone and in combination with NK cells. We demonstrated the anti-tumor activities of panobinostat on human skin carcinoma (A375), cervical carcinoma (HeLa), hepatocellular carcinoma (HepG2), and hepatocarcinoma (Huh7) cell-lines with its ability to synergize with and further enhance NK cell cytolysis. Therefore, our results support the immunomodulatory efficacy of panobinostat in the treatment of tumors.NK (YTS) and RMA-S (MHC class I-deficient variant of RBL-5) cell lines were preserved in-house. A375, HeLa, HepG2, Huh7, B16F10, and CT26 cell lines were all obtained from ATCC; HPDE, LO-2, HK-2, and MCF10A cell lines were used as control normal cells. The YTS and RMA-S cell lines were grown in RPMI-1640 (Corning) medium, HPDE was grown in DMEM/F12 + endothelial growth factor (EGF) while A375, HeLa, HepG2, Huh7, LO-2, HK-2, MCF10A, and CT26 cell lines were cultured in DMEM medium. All media were supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/ml of penicillin, and 100 \u00b5g/ml of streptomycin , 2mM L-glutamine as described and wereTo obtain mouse primary NK cells, mice splenocytes were sorted. For the primary human NK cells: fresh blood was obtained from healthy donors with informed consent in compliance with the approval from the ethics committee and Institutional Review Board of Shenzhen Institutes of Advanced Technology. Peripheral blood mononuclear cells (PBMC) were isolated using Ficoll-Hypaque density gradient centrifugation. For NK cell activation and expansion, the PBMC was cultured for 21 days using the TBD\u2122NK2013-S Cell Activation and Expansion Kit according to the manufacturer instructions.Panobinostat (Cat. No.: HY-10224) and EGTA (Cat. No: HY-D0861) were obtained from MedChemExpress , DMSO , InVivoMAb anti-mouse NK1.1 (clone PK136. BioXCell BE0036-5), InVivoMAb Mouse IgG2a, \u03ba . InVivoMab anti-mouse PD-L1 .Flow cytometry antibodies: all antibodies used were obtained from BioLegend unless otherwise indicated. Anti-human antibodies used were: PE Fas-L (306406), PE-NKG2D (320806), PC5.5-CD11C (301624), APC-CD28 (983406), APC-CD56 (362504), PE-CD69 (310906), APC-CD80 (305219), APC-CD86 (374207), PE-CD107A (328607), PE-CD112 (337410), APC-CD226 (338311), FITC-CD3 (317305), PC5.5-CD19 (302229), PE-CD16 (302007), PE-NKp46 (331907), PE-NKp44 (325107), PE-NKp30 (325207), PE-NKG2A (375103).Anti-mouse antibodies: FITC-CD3\u03f5 (145-2C11), BV-510\u00a0-CD45 (103138), PE-CD45 (103106), APC-NK-1.1 (Clone PK136) (108710), BV-510 -NK-1.1 (Clone PK136) (108737), PE-CD69 (104507), PE-CD107A (121612), PE-NKG2D (115606), APC-CD226 (128809), PE- Ly-49C/F/I/H (108207), APC-NKG2A (142807), PE- PD-1H (159604). Isotype control antibodies: APC-Rat IgG1 (401904), PE-Rat IgG1 (401906), BV-Rat IgG1 (401911).Cell apoptosis was evaluated using Pacific Blue\u2122 Annexin V Apoptosis - 7-aminoactinomycin D (7-AAD) Detection Kit . The assay was performed as recommended by the manufacturer. Cells were analyzed by flow cytometry, and the gating was done to show viable cells, apoptotic cells and excluded cellular debris.4 cell/50ul (per well) were seeded into the 96X E-Plates and monitored with impedance-based xCELLigence System until a log growth forming a monolayer is obtained (approximately 18-24 hours). Increasing concentration of panobinostat or DMSO control was then added into each well to make 200ul complete media. The cell index (CI) was measured continuously following \u226570 hours of incubation. CI was normalized at the end of the experiment, and it represents several cellular indicators such as cell-cell attachment, cell number, or cell size, and increased CI indicates cell growth.The xCELLigence RTCA SP System (ACEA Biosciences Inc.) was used to assess the real-time analysis of the cellular response of A375, HeLa, HepG2, and Huh7 cell lines \u201327 to pain vitro cytolytic effect of panobinostat and NK cell on the tumor cells, target cells were assessed under different conditions: (i) without panobinostat and NK cells, (ii) panobinostat only, (iii) NK cell only (iv) panobinostat and NK cells (co-treatment). For this assay, NK cell was directly added into the panobinostat treated tumor cells.NK cell cytotoxicity against the target tumor cells was performed using the xCELLigence real-time cell analyzer (RTCA) as described previously . To evalTo show NK cytolysis of panobinostat and vehicle-only treated tumor cells, the target cells were pretreated with 50nM of panobinostat or vehicle for 12-18 hours; the treatment media was gently removed and replaced with complete media. Cultured human primary NK cells, NK (YTS) or primary mouse NK cells were directly added to the pretreated target cells at different effector: target ratios (E: T). To rule out the possibility of lymphocytes contributing to impedance in the cytolytic assay, sorted B cells were added to the target cells; NK cells only were also seeded to wells without target cells. Cell index for individual wells was continuously measured every 15 minutes for the specified number of hours after effector cell addition. The Cell Index (CI) was normalized using the RTCA software Pro (version 2.3.0) to remove any well-well variations, and percentage cytolysis was calculated at the end of the experiment as:Each cytolysis was performed in three independent experiments at four or more replicates at various effector-to-target (E: T) ratios.To investigate the role of panobinostat-induced cell adhesion and tight junction-related gene expression in tumor cells relative to their enhanced NK cytolysis, 0.5mM EGTA (ethylene glycol tetraacetic acid) was added to block calcium-dependent adherence mediated cytolysis.EGTA was not toxic to both target and effector cells and was used in NK cytotoxicity experiments to determine the function of adherence-conjugate mediated increased tumor cell death.A375 cells were treated with 50nM of panobinostat or DMSO for 24 hours, the cells were wash and suspended in Trizol. Cells were then sent to GENEWIZ, Inc. for RNA isolation, library construction, and RNA sequencing. Gene set enrichment analysis based on KEGG pathways was based on significantly differentially expressed genes.Total RNA was extracted with TRIzol (Invitrogen), and 1\u03bcg RNA was reverse-transcribed using PrimeScript\u2122 RT Master Mix . Quantitative Real-Time PCR (qPCR) was performed using SYBR Green qPCR Premix Ex Taq II (Tli RNaseH Plus) in CFX96 Touch\u2122 Real-Time PCR Detection System (Bio-Rad) using gene-specific primers related to the top differentially expressed Adhesion molecules and Tight junction related gene sets from the GSEA (0 C for the indicated time at a 1:2 effector-target ratio. This was immediately followed by fixing the cells using True-Nuclear\u2122 Transcription Factor Buffer (BioLegend). Samples were analyzed by CytoFLEX LX Flow Cytometer (Beckman Coulter) and conjugates formed are indicated by double-positive signals for CFSE and CD56-APC.Conjugation formation between NK (YTS) cell, A375, and Hela cells was performed as described . Target 0C, washed again, and then directly analyzed by FACS. To analyze splenic NK cells, splenocytes were prepared from spleens obtained from panobinostat treated and control tumor-bearing mice. Splenocytes were washed and resuspended in cell staining buffer. The cells were labeled with the specific fluorophore-conjugated anti-mouse antibodies. Data was acquired using CytoFLEX LX Flow Cytometer (Beckman Coulter) and analyzed using FlowJo software (version 10).To assess the cellular effect of panobinostat on modulating the surface expression of receptors and ligands on primary human NK cells, NK(YTS) and tumor cells, this was done as earlier described . BrieflySix to eight-week-old BALB/c and C57BL/6 mice were used in this study. Animal handling and the experimental procedures followed the guidelines and approved protocols of the Shenzhen Institute of Advanced Technology (SIAT), Chinese Academy of Sciences (CAS).5 cells/mice), and B16F10 (2 X 105 cells/mice), RMA-S (4 X 105 cells/mice) cells respectively. Therapy commenced two days post-tumor challenge, and the mice were randomly divided into two groups (6 mice per group). Mice were administered with panobinostat (10 mg/kg) three times a week or PBS (untreated group) intraperitoneally (i.p). Tumor size and body weight were recorded every two days, and all mice were sacrificed when tumor volume reached 1500 mm3.BALB/c and C57BL/6 mice were subcutaneously injected with CT26 or Isotype control one day before tumor challenge, followed by panobinostat therapy post-tumor challenge. For the combination therapy, mice received 200 \u03bcg/ml anti-PD-L1 (clone 10F.9G2) intraperitoneally at days 3, 6, and 9. Other groups received panobinostat only, panobinostat and PD-L1, and PBS.All experiments were performed at least three independent times. Data were analyzed with GraphPad Prism9 software.In order to investigate the tumor \u2013suppressive effects of HDACi panobinostat (PANO) on tumor cells, we exposed the tumor cell lines to increasing concentrations of panobinostat. Nanomolecular concentration of panobinostat was sufficient to initiate apoptosis in A375 cells cells, four normal human cell lines, and human primary NK cells viability. NK(YTS), HPDE, LO-2, HK-2, MCF10a, and human primary NK cells were exposed to increasing doses of panobinostat for 24 hours, and their cell viability was assessed. As shown in The data from the x-CELLigence system showed that panobinostat significantly inhibited proliferation in all the treated tumor cells in a dose-dependent manner in comparison with the control without panobinostat and NK cells, (ii) panobinostat only, (iii) NK cell only, and (iv) panobinostat and NK cells. We observed that tumor cells co-treated with panobinostat and NK cell showed a synergistic increased cytolytic effect compared to panobinostat or NK cell treatment only or vehicle pretreatment of tumors prior to NK(YTS) cytolysis. As shown in We further evaluated the cytolysis of panobinostat treated tumor cells against primary mouse NK cells (M-P) and human NK cells (Q-T). The data showed that panobinostat pretreated\u00a0B16F10 and CT26 mouse tumor cells \u2013NKp46, NKp44, NKp30 and NKG2A by FACS revealed no observable differences\u00a0between the treated and control groups albeit with slight reduction in CD16 expression of panobinostat group cells with panobinostat or vehicle control, followed by the assessment of its surface receptors by flow cytometry, revealed no significant difference between panobinostat treatment relative to the vehicle-treated control , and CD112 (another activating ligand for NK cell) were assessed on all the four target cells. Our data showed a significantly higher surface expression of these ligands by panobinostat treatment relative to the control was added to block the calcium-dependent adherence and conjugation formation between NK and tumor cells. As shown in in vivo. Our findings showed significant tumor growth suppression in panobinostat-treated tumor-bearing mice relative to their untreated control and inhibitory surface receptors on the splenic NK cells showed that panobinostat promoted the expression of activating receptors with significantly reduced expression of the inhibitory receptors or a combination of anti-PD-L1 and panobinostat to CT26 tumor-bearing BALB/c mice. We observe a significant tumor growth suppression in the combination therapy relative to the control and the monotherapies of panobinostat and anti-PD-L1 on the surface of panobinostat-treated primary human NK cells showed no significant observable difference in comparison to the control. In the same vein, FACS analysis of the activating surface receptors on panobinostat-treated NK(YTS) cells revealed no significant difference in expression compared to the vehicle-treated control. Although there appears to be relatively low surface expression of other activating receptors, we found an enhanced expression of CD56 (cell adhesion surface receptors) that play an important role in cell-cell contact for panobinostat-treated NK(YTS) cells relative to control. Our data is consistent with earlier reports showing the importance of CD56 \u2013neural cell adhesion molecule (NCAM) . CD56 on panobinostat-treated target tumor cells revealed an enhanced expression of these ligands relative to their controls. These findings provide a further mechanistic explanation for the increased NK cell cytolysis of panobinostat-treated tumor cells, as well as support for Panobinostat\u2019s immunomodulatory roles. Collectively, our findings revealed that panobinostat could modulate the expression pattern of surface activating receptors on NK cells and their ligands on tumor cells, leading to enhanced tumor cell immunogenicity to NK cell cytotoxicity. These findings are consistent with our previous research on a small molecule\u2019s ability to modulate the surface expression of activating receptors on NK cells and its ligands on tumor cells .2+) abrogated the enhanced cytolysis observed for panobinostat pretreated tumor cells such as NK cells for total tumor remission are required.https://www.ncbi.nlm.nih.gov/sra/), accession number(s) SRX11149113 - SRX11149117 and NCBI BioProject, accession number PRJNA737652.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: NCBI Sequence Read Archive (SRA) database . The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by Shenzhen Institute of Advanced Technology (SIAT), Chinese Academy of Sciences (CAS).LA and JB designed the experiments, analyzed the data, and wrote the manuscript. LA performed the experiments. JB performed transcriptome analysis. XL contributed new reagents/analytic tools. XL, AA, FA, HW, DY, and LC validated the investigation and reviewed the final draft. XW supervised the project and oversaw the writing process. All authors contributed to the article and approved the submitted version.This work was funded by the National Key R&D Program of China (2019YFA0906100 and 2020YFA0710802), the Natural Science Foundation of China (82071768), Key-Area Research and Development Program of Guangdong Province (2019B020201014), the Natural Science Foundation of Guangdong Province (2019A1515011412), Shenzhen Basic Science Research Project (JCYJ20170818164619194), Shenzhen Basic Science Research Project (JCYJ20170413153158716), Special funds for major science and technology of Guangdong province (2019B020201014), and Nanshan pilot team project (LHTD20160004), 2018 Science and Technology Talents Boost-boosting Program of Shenzhen University General Hospital (SUGH2018QD029).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."} +{"text": "NAMPT is the rate-limiting enzyme in the salvage metabolic pathway leading to NAD+ generation. Tumor cells which are deficient in de novo pathway enzyme NAPRT1 are addicted to NAMPT. In clinical trials, treatment with NAMPT inhibitors resulted in dose-limiting toxicities. In order to dissect the mechanism of toxicity, mice were treated with KPT-9274 and resulting toxicities were characterized histopathologically and biochemically. KPT-9274 treatment caused gender-dependent stomach and kidney injuries and anemia. Female mice treated with KPT-9274 had EPO deficiency and associated impaired erythropoiesis. KPT-9274 treatment suppressed SIRT3 expression and concomitantly upregulated acetyl-manganese superoxide dismutase (MnSOD) in IMCD3 cells, providing a mechanistic basis for observed kidney toxicity. Importantly, niacin supplementation mitigated KPT-9274-caused kidney injury and EPO deficiency without affecting its efficacy. Altogether, our study delineated the mechanism of KPT-9274-mediated toxicity and sheds light onto developing strategies to improve the tolerability of this important anti-AML inhibitor.KPT-9274 is a phase 1 first-in-class dual PAK4/NAMPT inhibitor for solid tumor and non-Hodgkin\u2019s lymphoma. It demonstrates pre-clinical efficacy toward a broad spectrum of acute myeloid leukemia (AML) subtypes by inhibiting NAMPT-dependent NADThe online version contains supplementary material available at 10.1186/s13045-021-01107-0. To the Editor,+ biosynthetic enzyme, has been demonstrated in several cancers. Several NAMPT inhibitors have entered phase I trials to date [The therapeutic potential of targeting NAMPT, an NADPT-9274) , 6 recapPT-9274) , the preTumor-free mice were treated with a therapeutically effective dose of KPT-9274 or the vehicle once daily. Mild cell death/loss of gastric epithelial cells was increased and concomitantly orthochromatic erythroblasts (EryC) was markedly reduced, suggesting inhibition of erythroblast differentiation and production of mature erythroblast subsets.Anemia and kidney injury have been linked to erythropoietin (EPO) production deficiency. Female KPT-9274-treated mice had consistently lower levels of EPO Fig.\u00a0G. Concom+-dependent lysine deacetylase that participates in mitochondrial respiration. SIRT3 is also implicated in renal function through the regulation of reactive oxygen species (ROS). In IMCD3 cells treated with KPT-9274, we observed a dose-dependent decrease in SIRT3 expression and a concomitant rise in acetyl-manganese superoxide dismutase is a NADase Fig.\u00a0J and ROSase Fig.\u00a0K. Additiase Fig.\u00a0L. These + production, has been shown to circumvent the toxicity seen with NAMPT inhibitors. Tumor-specific promoter hypermethylation and loss of NAPRT1 protein expression have also been observed in subtypes of lung, pancreatic, and ovarian cancers [+ production. We showed that co-administration of 30\u00a0mg/kg of niacin orally decreased the magnitude of renal lesions without affecting serum levels of creatinine and BUN in KPT-9274-treated NSG mice (Fig.\u00a0The activation of the NAPRT1-depended salvage pathway through the supplementation of niacin as an al cancers , making In conclusion, our study delineated the mechanism of KPT-9274-mediated toxicity and sheds light onto developing strategies to improve the tolerability of this important anti-AML inhibitor. Our data also reaffirm that stratifying patients by NAMPT and NAPRT1 levels may determine the patient population that will benefit the most from NAMPT inhibition.Additional file 1. Supplementary Table I\u00a0and Supplementary Figures.Additional file 2.\u00a0Supplementary Methods."} +{"text": "Despite remarkable progress in the outcome of childhood acute myeloid leukemia (AML), risk of relapse and refractory diseases remains high. Treatment of the chemo-refractory disease is restricted by dose-limiting therapy-related toxicities which necessitate alternative tolerable efficient therapeutic modalities. By disrupting its immune environment, leukemic blasts are known to gain the ability to evade immune surveillance and promote disease progression; therefore, many efforts have been made to redirect the immune system against malignant blasts. Deeper knowledge about immunologic alterations has paved the way to the discovery and development of novel targeted therapeutic concepts, which specifically override the immune evasion mechanisms to eradicate leukemic blasts. Herein, we review innovative immunotherapeutic strategies and their mechanisms of action in pediatric AML.Acute myeloid leukemia is a life-threatening malignant disorder arising in a complex and dysregulated microenvironment that, in part, promotes the leukemogenesis. Treatment of relapsed and refractory AML, despite the current overall success rates in management of pediatric AML, remains a challenge with limited options considering the heavy but unsuccessful pretreatments in these patients. For relapsed/refractory (R/R) patients, hematopoietic stem cell transplantation (HSCT) following ablative chemotherapy presents the only opportunity to cure AML. Even though in some cases immune-mediated graft-versus-leukemia (GvL) effect has been proven to efficiently eradicate leukemic blasts, the immune- and chemotherapy-related toxicities and adverse effects considerably restrict the feasibility and therapeutic power. Thus, immunotherapy presents a potent tool against acute leukemia but needs to be engineered to function more specifically and with decreased toxicity. To identify innovative immunotherapeutic approaches, sound knowledge concerning immune-evasive strategies of AML blasts and the clinical impact of an immune-privileged microenvironment is indispensable. Based on our knowledge to date, several promising immunotherapies are under clinical evaluation and further innovative approaches are on their way. In this review, we first focus on immunological dysregulations contributing to leukemogenesis and progression in AML. Second, we highlight the most promising therapeutic targets for redirecting the leukemic immunosuppressive microenvironment into a highly immunogenic environment again capable of anti-leukemic immune surveillance. Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy that originates from transformed myeloid precursor cells arising from a hijacked bone marrow microenvironment (BMM). Leukemogenesis is characterized by uncontrolled clonal proliferation of malignant leukemic cells (blasts) that have lost the ability of proper differentiation at various stages of maturation. Our knowledge today suggests that leukemic blasts transduce the surrounding BMM into a leukemia-supportive niche and vice-versa, pointing at a bidirectional crosstalk between leukemic blasts and BMM reciprocally supporting further disease progression . In adulConsidering its heterogeneous characteristics, treatment of pediatric AML is adapted to different risk groups, stratified based on different genetic, cytogenetic, and clinical properties. Primarily though, treatment in all groups consists of intensive chemotherapeutic regimens with severe systemic side effects, emphasizing the urgent need for more tolerable, less toxic, and highly efficient treatments. Stepping towards this goal, numerous research works have uncovered substantial mechanisms underlying leukemogenesis and provided pivotal knowledge regarding the biology of AML, paving the way for identification of promising novel therapeutic approaches . HoweverIn this review, we outline immunosuppressive strategies and the pathophysiological background of disrupted leukemic blasts and microenvironment. In a second step, existing and promising potential immuno-therapeutic approaches are highlighted.In addition to oncogenic alterations in hematopoietic cells and BMM, immunological dysregulations contributes to leukemogenesis as well. During the leukemic transition, leukemic stem cells undergo immunoediting, a process that comprises the acquisition of multiple strategies to successfully evade immune surveillance. Consequently, the selected leukemic population is characterized by different immune-evasive mechanisms .One known mechanism of immune tolerance in AML was initially observed in patients with relapsed AML following mismatched HSCT, i.e., partially human-leukocyte-antigen (HLA)-incompatible HSCT, which contributes to downregulation of the mismatched HLA class I and II in AML blasts. Upon pressure from the transplanted immune system, this strategy confers \u2018survival advantage\u2019 to outgrowing immune-resistant mutant AML clones, characterized by genomic loss of the mismatched histocompatibility determinants. Leukemic blasts evade alloreactive donor T-cell-recognition and killing through this genomic loss of mismatched HLA haplotype which hampers the GvL effect following allogenic HSCT; thus paving the way for leukemia relapse ,18,19,20The non-classical HLA class I molecule HLA-G physiologically suppresses the immune system by direct inhibition of dendritic cells (via the inhibitory receptors immunoglobulin-like transcript (ILT)-2 and ILT-4), T-cells (via ILT-2), natural killer (NK)-cells (via ILT-2 and the killer-immunoglobulin-like-receptor (KIR)-2DL4), and monocytes (via ILT-2) . HLA-G hTo evade immune surveillance, leukemic blasts (relapse/refractory rather than newly diagnosed patients) express programmed-cell-death ligand-1 (PD-L1), a checkpoint marker that compromises cytotoxic T cells expressing PD-1 . BlockadIn the leukemic microenvironment, T-cells are proposed to reveal altered functional and phenotypic profiles, thereby contributing to immune-suppressive surroundings ,36,37. CApart from Treg-cell-mediated T-cell suppression, leukemic cells have also been proposed to orchestrate arginase and STAT-3 pathways to reduce T-cell proliferation, which was restored after selective inhibition, confirming the immune-suppressive impact of these mediators ,38. InhiIn conclusion, as suggested by numerous research works ,50,51,52Multiple studies provide evidence of deregulated anti-leukemic NK-mediated cytotoxicity in AML ,57,58,59As the immunological microenvironment is hijacked by leukemic blasts leading to these malignant cells evading the immune surveillance, multiple novel therapeutic approaches target immune-evasive strategies to restore anti-leukemic immune activity .In AML, expression of checkpoint molecules, such as PD-1 and CTLA-4 by immune cells or PD-L1 and Tim-3 by leukemic blasts has been reported as a sufficient strategy to escape immune surveillance. Clinical trials evaluating immune checkpoint-inhibitor (CPI) targeting PD-1 and CTLA-4 (Ipilimumab) achieved encouraging success in solid tumors . FeasibiTreatment of recurrent hematologic malignancies after allogenic HSCT with the monoclonal CTLA-4 inhibitor Ipilimumab achieved objective clinical response results in less than 10% of cases; however, Ipilimumab treatment did not lead to GvHD whilst tolerable immune-mediated adverse events occurred in 4 of 29 patients (14%) with a positive treatment response . In a coTo overcome this hurdle, which could be a major cause for modest response rates of CPI monotherapy, combinatory treatment approaches are currently under investigation, striving for better patient outcomes. Studies combining CPIs with chemotherapy in AML are currently under investigation . Preliminary results from a phase II trial assessing additional administration of Nivolumab three weeks after standard chemotherapy (anthracycline and cytarabine) in adult patients, including 42 newly diagnosed AML and 2 high-risk MDS demonstrate efficacy and feasibility, of note 6 out of 44 patients had grade 3/4 immune-related toxicities . MoreoveEffective CPI therapy requires proper effector lymphocyte reactivity, which is reduced in the immunosuppressive leukemic BMM, thus presumably hampering therapeutic effectiveness. Recognition by T-cells could be further reduced due to a relatively lower tumor mutation burden in AML, which has been determined as predictive response marker for CPI therapy . ConsideSince different studies revealed distinct co-expression patterns of immune checkpoints with a prognostic impact ,34,92, dDespite the paucity of truly tumor-specific antigens, leukemia-associated surface molecules preferentially expressed by AML-blasts were developed as potential therapeutic targets.CD33 and CD123 are partly expressed on healthy HSCs as well as most AML blasts, including LSCs ,95,96,97Gemtuzumab ozogamicin (GO), a CD33-directed ADC loaded with cytotoxic calicheamicin, showed feasibility and efficacy in early clinical studies conducted on a compassionate-use basis in pediatric patients with relapsed/refractory AML ,99. GO iWhilst CD33 and CD123 are also expressed on subsets of HSCs ,103, CLLPreliminary results of a phase I study evaluating a dual affinity re-targeting anti-CD123-CD3 antibody in refractory/relapsed AML and MDS reported T-cell-mediated efficacy and acceptable toxicity with cytokine release (grades 1 and 2) as a frequent but manageable adverse event . InteresThe Fc-engineered CD123 antibody with increased CD16-dependent NK cell affinity (CSL362) efficiently achieved NK cell-mediated cytotoxicity of AML blasts and LSCs in preclinical investigations ,111. HowFor treating solid cancers, tetra-specific antibodies were developed, consisting of CD16 crosslinked to IL-15 as an NK cell activating moiety fused to single-chain variable fragments (scFvs) binding cancer-associated antigens . RegardiInitially, cellular therapeutic approaches were developed to target leukemia inspired by powerful T-cell-mediated anti-leukemic immunity in the setting of allogenic HSCT . The infIn a preclinical setting, anti-leukemic activity has been proven for genetically modified chimeric-antigen-receptor (CAR)-expressing T-cells directed against CD123 or CD33 on leukemic cells ,123. TraHowever, increased T-cell-mediated toxicity, especially regarding elevated cytokine release and GvHD should be considered when using CAR T-cells . ConversSo far, different NK-based immunotherapeutic strategies have been developed. Cytokine-stimulated and lentiviral-transduced CAR-engineered killer cells targeting CD123 exhibited safety and anti-leukemic cytotoxicity against leukemic blasts in preclinical investigations . Adoptiv6/kg, transfusion of a lower dose of 12.5 \u00d7 106/kg could not achieve beneficial results under otherwise comparable conditions in children with AML in first complete remission [6 to 5 \u00d7 106 /kg was found to be clinically efficient and feasible [Transfusion of highly purified, allogenic haploidentical KIR-HLA mismatched NK cells following low-dose immunosuppression (with cyclophosphamide and fludarabine) and administration of IL-2 yielded beneficial response rates at low toxicity without GvHD and has been proposed as an innovative alternative consolidation therapy for pediatric and adult AML patients ,132. Comemission ,133. Thefeasible . Therefofeasible .Since different pro-inflammatory cytokines, such as IL-12, -15, -18, and -21 were shown to reinforce leukemia-directed immunogenicity ,137, cytTo maximize anti-leukemic immunogenicity, ex vivo stimulation using a mix of IL-12, IL-15, and IL-18 provokes differentiation into memory-like NK cells, which exhibit enhanced leukemia-directed reactivity compared to non-activated/native NK cells ,138. TheDifferent strategies have been developed to bypass this obstacle. To promote effectiveness of allogenic NK cell-based therapy, administration of IL-2 diphtheria toxin fusion protein, that depletes Treg cells bearing IL2R\u03b1 (CD25), restored anti-leukemic NK cell immunogenicity when administered prior to NK-infusion and IL-2-administration . In phasThe IL-15/IL-15Ra super-agonist (N-803) is supposed to mimic the immunostimulatory effect exerted by antigen-presenting cells that trans-present IL-15 linked to IL-15Ra to T-cytotoxic- and NK cells through a shared IL-2/IL-15-alfa/beta receptor . AdminisEven though class one interferons are proposed to exercise anti-leukemic and pro-immunogenic functions on malignancies, utility as a potent therapeutic opportunity was modest when investigated as sole treatment ,148.Considering the complexity of the leukemic BMM, various further immunosuppressive factors also restrict anti-leukemic immunogenicity. These limitations could be offset using drugs that alleviate leukemia-driven lymphocyte suppression. In this regard, additional suppression of inhibitory mediators TGF-\u03b2 or IDO 2,3 may present promising approaches to overcoming leukemic restrictions and increasing the success of cell-based therapy in AML.Inhibition of IDO 2,3 was found to promote cellular T- and NK cell-mediated immunoregulation and to inhibit Treg-cell conversion ,75. AntaSince research explored the immuno-suppressive role of TGF-\u03b2 in different cancers, antagonizing TGF-\u03b2 reinforces the lymphocyte-mediated immune response in fighting malignant disease, as has been shown for non-hematologic malignancies . In thisIncreased levels of cell-bound and soluble HLA-G are overexpressed in distinct AML subtypes and presumably present a highly efficient approach to restoring immune response as HLA-G regulates NK, T-, and dendritic cells. In addition, HLA-E also represses NK cell functionality binding NKG2A, thus presenting another potential target. To date, preclinical and clinical data focusing on the feasibility of stated approaches are limited or even do not exist.Vaccination has attracted rising attention as a potential therapeutic tool to facilitate anti-leukemic immunological surveillance. To provoke leukemia-directed immune response, two main vaccine-based strategies are focused on (i) leukemia-associated antigens and (ii) antigen-presenting dendritic cells, either stimulated by AML cells, used as fusion-product or genetically modified.Wilms tumor gene (WT-1) peptide-based vaccines were generally well-received and achieved specific cytotoxic T-cell reactivity resulting in reduced leukemic burden with markedly decreased MRD markers in the majority (60%) of treated AML patients after consolidation therapy .Intracutaneous administration of the WT-1 vaccine is suggested as a safe and efficient promising second-line maintenance therapy strengthening GvL effect post HSCT when given at the lowest point of leukemic load . This coIn phase I/II trials (NCT00665002 and NCT01266083) administration of the multivalent WT-1 vaccine, galinpepimut-S, triggered a specific immune response related to improved survival without causing relevant toxicity in treated AML patients after achieving first complete remission . These eDendritic cells (DCs) are considered as the most powerful antigen presenting cells and are therefore ideally suited as cellular adjuvants for therapeutic vaccination. Results from a phase II trial (NCT00965224) suggest WT1-mRNA loaded DCs as an effective strategy to induce antigen-specific T-cell response and subsequently prevent or delay relapse after standard chemotherapy , Table . VaccinaAn injectable cryogel vaccination which delivers the immunostimulatory CpG-oligodeoxynucleotide and GM-CSF together with leukemia-related antigens induced a sound anti-leukemic immunity including DC activation which prevented treated AML-bearing mice from leukemic engraftment and eradicated established leukemia when administered together with chemotherapy . InteresFacing limitations in clinical attempts of new immune-therapeutic strategies and of the intricate immunological leukemic microenvironment, a combination of cellular- and non-cellular-based immunotherapeutic strategies may be strong enough to efficiently overcome leukemic immune-evasion.Oncolytic virus (OV) has been determined as another appealing therapeutic approach to directly kill malignant cells and boost anti-tumorigenic immunity in various malignant diseases ,159.Due to a natural tumor-specific tropism, OVs selectively contaminate, proliferate in, and destroy tumor cells without harming healthy cells. Simultaneous release of tumor-specific damage-associated molecular patterns (DAMP) including highly immunogenic tumor-derived peptides alert the immune system and cause anti-tumor immunity re-modifying BMM into a highly inflammatory site . TherefoSeveral in vitro and in vivo studies evaluated the use of (modified) OVs alone or in combination with other drugs in hematologic malignancies including AML. Measles vaccine virus (MeV) activated IFN signaling and directly lysed AML cells expressing the measle entry receptor CD46, a complement regulatory cofactor. Combination of MeV with leukemia site-specific generation of the cytotoxic active drug 5\u00b4flour uracil using modified MeV equipped with super cytosine deaminase (MeV-SCD) additionally intensified this anti-leukemic effect .The engineered adenoviral vector, zA4, coated by TNF-apoptosis-inducing related-ligand (TRAIL) efficiently evokes cytotoxicity and significantly inhibits leukemia-cell proliferation, and additive ginsenoside (Rh2) enforces anti-tumorgenicity by inducing the expression of TRAIL-related receptors on leukemic blasts .UV-light-inactivated herpes simplex virus-1 (UV-HSV-1) exerts cytolysis of AML cells via activation of leukemia-directed immune response and, synergistically with IL-15 and IL-2, induces cytolytic activity of stimulated NK cells, suggesting UV-HSV-1 as a therapeutic supplement to reinforce adoptive cell therapy .Despite the fact that AML cells are resistant to the direct lytic effect of the coxsackievirus strain CVA21, this OV exerts a powerful innate and adaptive immune response, killing leukemic blasts . This coTo further augment treatment efficacy, genetically modified OVs are in development. Transduced OVs, expressing distinct immunogenic TAA, have been assessed to strengthen desirable anti-tumoral immunity ,168. OthThe variety of modified OVs leads to the hypothetical concept to utilize OV as a supplier of leukemia-specific antigens, potentially addressing the challenging lack of unique targetable antigens in AML. For solid tumors, the combination of CD19 CAR-T cells with engineered OVs delivering CD19 as an amenable target to tumor cells turned out to be safe and effective in preclinical proof of concept trial .Together, OVs appear as an exciting novel immunotherapeutic strategy, that needs to be followed-up in further investigations. Clinical trials in AML are pending to determine the most potent viral strain and combinatorial immunotherapeutic setting for the efficient killing of AML blasts in pre-treated patients.In AML, immunotherapeutic approaches have recently emerged as a promising step towards the long-term goal of an improved, more specific anti-leukemic treatment. Findings of disrupted immunoregulation in AML, that contributes to leukemogenesis and promotes disease progression led to innovative therapeutic attempts to reconstitute anti-leukemic immunosurveillance. Promising results of preclinical and clinical trials point at significant therapeutic values of oncolytic viruses, vaccines, and cytokines as well as cellular- and antibody-based immunological approaches. Combining these different immunotherapies could increase treatment efficacy and mark future directions towards a new generation of successful, targeted therapy in AML. However, clinical success may be limited by possible adverse toxic side-effects. To enhance safety and therapeutic effectiveness, a deeper understanding of immune dysregulation in AML is urgently needed. This may further enrich our repertoire of potent immunological drugs to eliminate leukemic blasts in AML."} +{"text": "Neurological long-term sequelae are increasingly considered an important challenge in the recent COVID-19 pandemic. However, most evidence for neurological symptoms after SARS-CoV-2 infection and central nervous system invasion of the virus stems from individuals severely affected in the acute phase of the disease. Here, we report long-lasting cognitive impairment along with persistent cerebrospinal fluid anti-SARS-CoV-2 antibodies in a female patient with unremarkable standard examination 6 months after mild COVID-19, supporting the implementation of neuropsychological testing and specific cerebrospinal fluid investigation also in patients with a relatively mild acute disease phase.The online version contains supplementary material available at 10.1186/s42466-021-00135-y. There is mounting evidence for neurological long-term sequelae after COVID-19 . In the n\u2009=\u20092), we detected elevated levels of SARS-CoV-2- specific IgG S1 antibodies and a trend towards elevated IgG nucleocapsid antibodies in the CSF (Table n\u2009=\u20093) (Table We report a 57-year-old female with mild but disabling neuropsychological symptoms 6 months after mild COVID-19. During the acute infection, the patient self-referred to a hospital after a positive result of SARS-CoV-2 PCR-testing, which was performed in an outpatient setting due to fever, headache, ubiquitous limb pain, and diarrhea. Upon admittance, vital parameters including peripheral oxygen levels were normal. Laboratory routine examinations were unremarkable despite slightly increased levels of C-reactive protein. Neither intensive care, nor ventilation were required during the hospitalization, and she was discharged home after 5 days. Two weeks after the acute illness, she experienced impaired concentration and action planning. As this condition persisted for several months, preventing her from resuming her professional activity as a nurse and limiting herself in the organization of her activities of daily living, she was referred to our university hospital by a local neurologist. A detailed neurological examination did not detect any abnormalities. In particular, no apraxia or aphasia, and no hypokinetic-rigid signs were found. Apart from a depressive episode in the past, her medical history was unremarkable. A brain MRI examination, standardized testing of the olfactory function \u2013 BSIT, In conclusion, we demonstrate long-lasting neuropsychologic sequelae along with persistent SARS-CoV-2-specific CSF antibodies several months after a relatively mild acute disease course and encourage the investigation of CSF SARS-CoV-2-specific antibodies and neuropsychological testing to detect possible \u2018Long COVID\u2019. Given the mounting evidence for neurological sequelae including cognitive impairment after acute SARS-CoV-2 infection , the incAdditional file 1."} +{"text": "The recent COronaVIrus Disease (COVID)-19 pandemic has placed an unprecedented burden on the drug development opportunity to prevent the onset of multi-organ failure.Emerging experimental reports have highlighted the beneficial effects of mesenchymal stem cell (MSC) administration against COVID-19. MSCs and their derived exosomes may attenuate SARS-CoV-2-induced inflammatory response through managing the immune cell function and cytokine expression. Although these are promising results, the exposure of MSCs to chemical compounds with pharmacological activities may further improve their homing, survival, and paracrine machinery.Nicorandil (N-[2-hydroxyethyl]-nicotinamide nitrate), an established adenosine triphosphate-sensitive potassium channel opener, is recently hypothesized to modulate inflammation as well as cell injury and death in COVID-19-affected lungs through inhibiting reactive oxygen species levels and apoptosis. Since it also exerts protective effects against hypoxia-induced MSC apoptosis, we assumed that transplanted MSCs combined to long-term nicorandil administration may survive longer in a severely inflamed microenvironment and have more beneficial effects in the treatment of SARS-CoV-2 infection than MSCs alone. The recent COronaVIrus Disease (COVID)-19 pandemic has placed an unprecedented burden on the opportunity for improving therapy development to prevent the onset of multi-organ failure in SARS-CoV-2-infected patients.Initial experimental reports revealed beneficial effects of mesenchymal stem cell (MSC) administration against COVID-19 . However+ channel opener with anti-inflammatory and anti-oxidant properties, is conventionally used to treat patients with ischemic heart diseases [Nicorandil (N-[2-hydroxyethyl]-nicotinamide nitrate), a nitric oxide (NO) donor and ATP-sensitive Kdiseases . Recentldiseases . This sudiseases . Moreovediseases .Although mechanisms underlying COVID-19 are still under investigation, regulatory effects of the abovementioned cellular signaling may impact on the pathogenesis, severity, and long-term sequelae of severe SARS-CoV-2-induced pneumonia .In light of its pleiotropic protective effects , 6, 7, wInterestingly, nicorandil also promotes MSC survival under conditions mimicking the ischemic microenvironment, such as hypoxia and oxidative stress, through the activation of the PI3K/Akt signaling pathway and the reduction of reactive oxygen species production . MoreoveLastly, we cannot exclude that paracrine factors may play a key role in mediating synergistic effects of combined MSC-nicorandil therapy. In particular, exosomes, the smallest extracellular vesicles that serve as mediators for cell-to-cell communication, recapitulate the efficacy of MSCs in treating critical illness . The fir"} +{"text": "COVID-19 vaccines are of great importance in reducing SARS-CoV-2 infection and severe cases. Patients with autoimmune inflammatory rheumatic diseases (AIIRDs) have been strongly recommended to be vaccinated according to the novel guidance because they are more vulnerable to SARS-CoV-2 infection.We conducted a real-world survey to evaluate the safety profiles and disease flare in patients with AIIRDs who received any dose of inactivated COVID-19 vaccines in China. From 1 Aug 2021 to 15 Oct 2021, eligible participants completed a predefined 25-question-based questionnaire by invitation on social media or visiting the outpatient department. There was no restriction on the time interval from vaccination to completing the survey.10.1136/annrheumdis-2021-221736.supp1Supplementary dataIn total, 1507 adults patients with AIIRDs who received inactivated COVID-19 vaccine participated in this study participants experienced adverse events (AEs) after vaccination . Local A10.1136/annrheumdis-2021-221736.supp2Supplementary dataFlare of existing AIIRDs was reported by 158 (10.5%) participants, with requirement of treatment escalation in 53 (3.5%) patients. Joint pain and swelling were the most common manifestations of disease flare, followed by skin rash , morning stiffness and febrile recurrence . Interestingly, the frequencies of AE and flare of AIIRDs were generally lower in inflammatory arthritis patients (RA or PsA/PsO) than those in patients with systemic AIIRDs . MultivaOur data confirmed the safety profiles, and for the first time demonstrated the disease flare after inactivated COVID-19 vaccination in patients with AIIRDs. Overall, 29.9% of participants experienced AEs after vaccination and no fatal AEs occurred, indicating the well tolerability of inactivated COVID-19 vaccines in AIIRDs population. Importantly, our results aligned with a large real-world study supported by European League against Rheumatism(EULAR) COVID-19 database (83% mRNA vaccines), whose vaccine-related AEs were observed in 31% of patients."} +{"text": "The elderly and chronically ill are among groups at the highest risk for morbidity and mortality to several infections, including SARs-CoV-2. Cellular senescence contributes to inflammation, multiple chronic diseases, and age-related dysfunction, but effects on responses to viral infection are unclear. Old mice acutely infected with pathogens that included a SARS-CoV-2-related mouse \u03b2-coronavirus experienced increased senescence and inflammation with nearly 100% mortality. Targeting SCs using senolytic drugs before or after pathogen exposure significantly reduced mortality, cellular senescence, and inflammatory markers and increased anti-viral antibodies. Thus, reducing the SC burden in diseased or aged individuals should enhance resilience and reduce mortality following viral infection, including SARS-CoV-2."} +{"text": "Following cisplatin-based concurrent chemo-radiotherapy (CCRT), the post-treatment PET/CT illustrated the absence of an abnormal metabolic accumulation over the suspicious region as observed in post-treatment CT. A further trans-ostia re-biopsy confirmed complete tumor remission. This case demonstrates the remarkable ability of 18F-FDG PET/CT to differentiate between a persistent malignancy and post-treatment changes. Furthermore, a definite CCRT might provide comparable outcomes to traditional surgery.Non-keratinizing squamous cell carcinoma (NKSCC) of the lacrimal apparatus is extremely rare. It is usually very aggressive in destroying local tissue and has a grave prognosis for relentless recurrence and distant failures. Though the current evidence cannot make confident recommendations regarding the best management, curative surgical excision with adjuvant radiotherapy remains the most commonly used strategy. Here, we report a 71-year-old woman presented with progressive right medial canthal swellings for six months. A transnasal endoscopic biopsy revealed NKSCC of the lacrimal sac. She then underwent a combination of magnetic resonance images (MRI) and 2-deoxy-2-(18F)fluoro-D-glucose positron emission tomography/computed tomography ("} +{"text": "In patients mounting an immune response to the vaccine, a common SYE(N)E TCR motif known to bind CMVpp65 was detected. CMV-peptide-vaccination-responder patients had TCR features distinct from those of non-responders. In a non-responder patient, a monoclonal inflammatory T-cell response was detected upon CMV reactivation. The identification of vaccine-induced CMV-reactive TCRs motifs might facilitate the development of cellular therapies for patients wait-listed for kidney transplantation.After solid-organ transplantation, reactivation of the cytomegalovirus (CMV) is often observed in seronegative patients and associated with a high risk of disease and mortality. CMV-specific T cells can prevent CMV reactivation. In a phase 1 trial, CMV-seronegative patients with end-stage renal disease listed for kidney transplantation were subjected to CMV phosphoprotein 65 (CMVpp65) peptide vaccination and further investigated for T-cell responses. To this end, CMV-specific CD8 The most vulnerable time period is early after transplantation when patients are under high immunosuppression , where uClassical prophylaxis or preemptive therapy using ganciclovir or foscavir is effective, with nephron- and myelotoxicity limiting the use of these drugs .In addition to drug treatment, vaccination offers an intriguing option in development for patients at risk : severalRecently, we published the results of a first phase I trial for a CMVpp65-derived vaccine in HLA-A*02-positive CMV-seronegative end-stage renal disease patients on the kidney transplant waiting list . The stuHowever, further investigation of both the affected patient cohort and of possible response factors is highly warranted. + T cells by bulk TCR sequencing as well as combined single-cell RNA and TCR sequencing.For this reason, we aimed at a deeper understanding of T-cell-receptor (TCR) repertoire dynamics and specificities and, in addition, report here on the characterization of CMV-specific CD8To understand the clonal evolution of T-cell clonotypes following repetitive CMVpp65 peptide vaccinations and to identify TCR sequences dominating peptide-induced peripheral T-cell responses, we performed deep repertoire TCRA and TCRB sequencing. In both responder and non-responder patients, repetitive vaccinations were not associated with consistent longitudinal alterations in the most-abundant clonotypes in the peripheral blood . One resvdjdb.cdr3.net (accessed on 15 August 2019) was found in responders, making it a predictor of response [high and MKI67high) (vdjdb.cdr3.net (accessed on 1 November 2021)).Five months after transplantation, non-responder patient #002 experienced CMV reactivation. To obtain a more detailed immunological understanding of this reactivation, we performed combined single-cell (sc) RNA (scRNA) and scTCR sequencing of CMV-reactive PBMC-derived T cells. Upon reactivation, the patient mounted a monoclonal (80%) CMV-specific T-cell response with a unique TCR beta chain pairing with two different alpha chains . FurtherZMBhigh) ,16 and pI67high) . Using tIn contrast, the scTCR-sequencing of tetramer-sorted CMV-reactive T cells from vaccine-responding patient #003 revealed that the second-most-abundant post-enrichment clonotype was found at all the available post-vaccine time points, and its abundance peaked, in congruence with the peripheral total T-cell response, at time point 3 . Of noteCMV reactivation after solid-organ transplantation constitutes a serious clinical problem . It is kWithin our phase 1 trial, ten patients received four subcutaneous vaccinations as per protocol. The vaccine was well tolerated, and 5/10 patients mounted a T-cell response with an emulsified CMVpp65 nonamer peptide . Further+ T cells by bulk TCR sequencing as well as combined single-cell RNA and TCR sequencing.For this reason, we analyzed the T-cell response to CMV-specific peptide vaccination in exemplary patients in more detail for a deeper understanding of T-cell receptor (TCR) repertoire dynamics and specificities and the characterization of CMV-specific CD8vdjdb.cdr3.net). As patient #002 is also HLA-A*31 positive and HLA-A*03 and HLA-A*31 fall into the same HLA supertype family, it remains unknown if this observation is causally related to both the previously reported ineffective T-cell response (tetramer staining) but, at the same time, weak T-Track\u2122 [Previous reports had demonstrated that immunodominant CMV antigens can induce highly diverse changes in T-cell-receptor (TCR) repertoires, which, nevertheless, contain convergently selected motifs and/or public clones indicative of patient response and HLA type ,22,23. IT-Track\u2122 assay reT-Track\u2122 .In general, transplantation-associated immunosuppression interferes with anti-CMV immune responses . Specifi\u00ae) and combined with a local application of the Toll-like receptor (TLR)-7 agonist imiquimod (Aldara\u00ae 5% cream) [Vaccination strategies have been highly efficacious for several decades in controlling infectious diseases, highlighted more recently by the COVID-19 pandemic. Vaccines that include whole organisms or large proteins appear to have some adverse side effects attributable to the inclusion of an unnecessary antigenic load . In prin% cream) . Both ad% cream) . It remaIn summary, 50.0% of the vaccinated patients mounted a peripheral immune response during prophylactic CMV-specific peptide vaccination prior to kidney transplantation. Although only a small number of patients were enrolled in this phase 1 clinical trial, CMV-associated TCR motifs could be identified. Further follow-up studies with an increased patient number and multi-center assessment are necessary to confirm the clinical as well as exploratory translational results. Future vaccination strategies might also incorporate MHC class II antigens or other immunotherapeutic strategies such as mRNA vaccines or TCR-transgenic cellular therapies.Detailed information for both the CMVpp65 peptide vaccine and the investigated patients has been described earlier in the published clinical results of the phase I peptide vaccination trial .+ T cells in tetramer-based flow cytometry nor significant (>10/200.000) interferon gamma (IFN-\u03b3) spot-forming cells (SFC). In this phase I study, 10 CMV-seronegative end-stage renal disease patients waiting for kidney transplantation were vaccinated four times biweekly. All the enrolled patients were CMV IgM/IgG negative prior to the CMVpp65 peptide vaccination. At baseline, the participants showed neither pre-existing CMV-specific CD8+ T cells, were observed within the core study of 56 days lasting until 14 days after the last vaccination [In 5 of 10 patients , any immune responses, as evidenced by the detection of an increase in IFN-\u03b3 production in the T-Track\u2122 assay and/or an increase in CMV-specific CD8cination . PatientDetails on the manufacturing process and administration of the vaccine as well as all the clinical results and side effects are provided in the previous publication .+ T cells was determined as described earlier [The frequency of CMV-specific CD8 earlier . The acq6 viable peripheral blood mononuclear cells (PBMCs) per time point for each patient were frozen in DMSO/freezing medium. Following thawing, RNA extraction was performed and the RNA was concentrated using a SpeedVac\u2122 . Where sufficient RNA was available, duplicate random amplification of cDNA ends (RACE) reactions targeting the alpha- and beta-chains of the TCR (TCRA and TCRB) were set up using 250 ng of RNA as the template. Preliminary repertoire analysis was conducted with VDJ tools [A total of 5 \u00d7 10DJ tools , and theDJ tools . The TCRDJ tools ,18 and DDJ tools .For one responder (patient #003) and one non-responder (patient #008), single-cell RNA-seq and single-cell TCR-seq libraries were prepared using the Single Cell Immune Profiling Solution Kit , according to the manufacturer\u2019s instructions. For the gene-expression library construction, amplified cDNA was fragmented and end-repaired, double-sided size-selected with SPRIselect beads (Beckman Coulter), PCR-amplified with sample-indexing primers and double-sided size-selected with SPRIselect beads . For TCR library construction, TCR transcripts were enriched from 2 \u03bcL of amplified cDNA by PCR. Following TCR enrichment, the PCR product was fragmented and end-repaired, size-selected with SPRIselect beads , PCR-amplified with sample-indexing primers and size-selected with SPRIselect beads . The single-cell RNA libraries were sequenced on an Illumina HiSeq 4000\u2122 . The single-cell TCR libraries were sequenced on an Illumina NextSeq 550\u2122 paired-end 150 mid-output flow cell to a minimum sequencing depth of 5000 reads per cell.The sequencing data were aligned using cellranger (v6.1.2) . The count matrix was then imported into R for further downstream analysis. The data were normalized and transformed using the Seurat package sctransform and subsequently clustered via the Louvain algorithm using Seurat. A UMAP and heatmap were also plotted using Seurat (v.4.0.4).\u00ae CMV assays were used for the assessment of CMVpp65 antigen-specific IFN-\u03b3 release as previously reported [Commercially available IFN-\u03b3 ELISpot T-Trackreported .The exploratory results are presented in a descriptive manner, with numbers and percentages."} +{"text": "In this review, I discuss our recent finding that lysophospholipid metabolism is essential for the maintenance of chronic myelogenous leukemia (CML) stem cells. Lysophospholipids have only one fatty acid chain and so are more hydrophilic than phospholipids, allowing them to act as lipid second messengers. We demonstrated that the stem cell quiescence and TKI resistance displayed by CML stem cells in vivo are sustained by the Gdpd3 enzyme involved in lysophospholipid metabolism. At the mechanistic level, Gdpd3 function allows lysophospholipid metabolism to suppress the AKT/mTORC1-mediated cell growth pathway while activating the stemness factors FOXO and \u03b2-catenin. Our results thus link lysophospholipid metabolism to CML stemness, and may thereby open up new therapeutic avenues to overcome CML relapse post-TKI therapy.It is well known that mature chronic myelogenous leukemia (CML) cells proliferate in response to oncogenic BCR\u2013ABL1-dependent signaling, but how CML stem cells are able to survive in an oncogene-independent manner and cause disease relapse has long been elusive. Here, I put into the context of the broader literature our recent finding that lysophospholipid metabolism is essential for the maintenance of CML stem cells. I describe the fundamentals of lysophospholipid metabolism and discuss how one of its key enzymes, Glycerophosphodiester Phosphodiesterase Domain Containing 3 (Gdpd3), is responsible for maintaining the unique characteristics of CML stem cells. I also explore how this knowledge may be exploited to devise novel therapies for CML patients. BCR\u2013ABL1 fusion oncogene forms and is activated in hematopoietic stem cells (HSCs) [BCR\u2013ABL1 can be blocked by tyrosine kinase inhibitors (TKIs), which has dramatically improved the prognosis of many CML patients [Chronic myeloid leukemia (CML) arises when the s (HSCs) . CML stepatients . The firpatients ,4,5. HowIt turns out that the CML stem cells that generate most mature CML cells are responsible for disease relapse post-TKI therapy ,7,8. WhiTo date, it is reportedly known that several signaling pathways maintain the self-renewal capacity in CML stem cells, such as JAK/STAT, Hedgehog, Wnt/\u03b2-catenin, PI3K\u2013Akt and TGF-\u03b2-FOXO signaling within the bone marrow microenvironmental niche ,7,8. PelThe lipid bilayer in the plasma membrane of most cells consists of the familiar glycerophospholipids (commonly referred to as \u201cphospholipids\u201d) that contain two fatty acid ester chains and one polar group . In contGdpd3 gene more highly than normal LT-HSCs [Gdpd3-deficient mouse strain using genome-editing methodologies. Then, we established a CML-like disease model by retroviral BCR\u2013ABL1 transduction into HSCs isolated from WT and Gdpd3-deficent mice, followed by bone marrow transplantation (BMT) into recipient mice. We isolated CML stem cells from these CML-affected mice and evaluated their self-renewal capacity in serial BMT experiments in vivo. After a first-round of BMT, mice that were transplanted with CML stem cells from Gdpd3-deficient mice (Gdpd3-deficient CML stem cells) and developed CML disease succumbed more rapidly than recipients transplanted with CML stem cells from WT mice (WT-CML stem cells). We then purified CML stem cells from these first-round CML-affected mice and transplanted them into a second set of recipients. To our surprise, Gdpd3-deficient CML stem cells showed a significant decrease in their ability to induce CML, whereas WT-CML stem cells maintained this capacity in a second-round of BMT. These results demonstrate that Gdpd3 is critical for the long-term maintenance of CML stem cells in vivo.To investigate gene expression changes specific to CML stem cells, we performed a comparative RNA-Seq analysis of murine normal HSCs and CML stem cells. We found that the most primitive long-term (LT) CML stem cells expressed the LT-HSCs . While lGdpd3-deficient and WT-CML stem cells in CML-affected mice after a first-round of BMT. Importantly, the frequency of S-phase cells was strikingly increased among Gdpd3-deficient CML stem cells compared to WT-CML stem cells, indicating that Gdpd3 loss activates the cell division of quiescent CML stem cells. Because sustained stem cell quiescence is the key to TKI resistance, we then investigated whether loss of Gdpd3 would affect TKI resistance in vivo. Strikingly, recipient mice that harbored Gdpd3-deficient CML stem cells and were treated with the TKI dasatinib showed a decrease in disease relapse even after a first-round of BMT. Thus, Gdpd3-mediated lysophospholipid metabolism in CML stem cells is critical for the maintenance of their quiescence and thus TKI resistance in vivo.Given that stem cell quiescence is vital for the maintenance of CML stem cells, we used in vivo BrdU incorporation assays to evaluate the cell cycle distribution of Atx gene encoding the lysophospholipase D enzyme Autotaxin promoted tumor cell metastasis in a mouse model of breast cancer [Gdpd3-deficient CML-affected mice. We observed that the mutant CML stem cells showed decreased LPAs compared to WT-CML stem cells [Researchers have used numerous animal models and human patient samples to explore the roles of lysophospholipids in human diseases. For example, enforced transgenic expression of the t cancer ,23. In at cancer . Severalt cancer ,26. Howeem cells . These rGdpd3-deficient CML cells compared to WT-CML cells [Lipid mediators and the signaling pathways they trigger play important roles in immune responses, inflammation and carcinogenesis ,28,29. AML cells . While iGdpd3-deficient LT-CML stem cells. Levels of phospho-Akt and phospho-S6 were increased in Gdpd3-deficient LT-CML stem cells compared to WT-LT-CML stem cells, indicating that Gdpd3 loss activates the Akt\u2013mTORC1 signaling pathway in CML stem cells. Consistent with this finding, Foxo3a was exported to cytoplasm and inactivated in Gdpd3-deficient LT-CML stem cells, in contrast to its nuclear (activated) localization in WT-LT-CML stem cells [We have sought to define the underlying molecular mechanisms by which lysophospholipid metabolism affects stem cell quiescence and CML stemness. Because Gdpd3 loss activated the cell division of CML stem cells but attenuated their self-renewal capacity, we examined the phosphorylation of Akt and S6 ribosomal protein in WT- and em cells . Thus, GGdpd3-deficient LT-CML stem cells. We observed that expression levels of several genes encoding prototypical seven-transmembrane G-protein-coupled receptors (GPCRs) were attenuated in Gdpd3-deficient CML stem cells -containing GPCR, were decreased in Gdpd3-deficient LT-CML stem cells compared to WT-LT-CML stem cells. Lgr4/GPR48 is known to be involved in the canonical Wnt/\u03b2-catenin signaling pathway [Gdpd3-deficient CML mouse model or an Lgr4/Gpr48-hypomorphic mutant CML mouse model [Lgr4/Gpr48-hypomorphic mutant LT-CML stem cells, but not in Gdpd3-deficient LT-CML stem cells (where Foxo3a was inactivated in the cytoplasm). These results suggest that Gdpd3-mediated lysophospholipid metabolism may maintain the self-renewal capacity of CML stem cells by activating the stemness factors Foxo3a and \u03b2-catenin [To understand gene expression patterns related to lysophospholipid metabolism in CML stem cells, we conducted comparative RNA-Seq analysis of WT- and em cells . Among t pathway ,31,32, a pathway . \u03b2-caten pathway ,34. Thusse model . We alsose model . To our -catenin ,15.Lpar4/Gpr23 gene were decreased in Gdpd3-deficient LT-CML stem cells compared to WT-LT-CML stem cells. Igarashi et al. previously showed that bone marrow stromal cells isolated from Lpar4-deficient mice have a deficit in hematopoiesis-supporting capacity compared to those from WT mice [How exactly does Gdpd3-mediated lysophospholipid metabolism suppress the Akt\u2013mTORC1 pathway and thus activate Foxo3a, leading to the quiescence essential for self-renewal capacity? . Taniguc WT mice . It is tarachidonate 5-oxygenase (Alox5) or Alox15 display decreased self-renewal capacity in vivo [Lpar3-deficient female mice show decreased Cox2 mRNA and defects in embryo implantation, a phenotype similar to that of Cox2-deficient mice lacking the Cox2 enzyme essential for generating prostaglandins [Lpar3-deficient female mice with PGE2 or cPGI partially rescued these defects [Gdpd3-deficient CML stem cells showed decreased PGE2 levels, and in vitro treatment of Lgr4-hypomorphic mutant CML stem cells with PGE2 restored the interaction between active Foxo3a and \u03b2-catenin [Although it has yet to be clarified how lysophospholipid metabolism is involved in the biosynthesis of downstream lipid mediators, several previous reports have established that certain lipid mediators and their downstream signaling targets are required for CML stemness. For example: (1) Murine CML stem cells isolated from mice lacking either in vivo ,39. (2) in vivo . (3) Tre in vivo . (4) Lpaglandins ,42. Trea defects . (5) In -catenin . Collect13C-stable isotopic metabolite tracing experiments. The results of these studies will shed much-needed additional light on how lysophospholipid metabolism is linked to vital lipid mediators in CML stem cells.Our knowledge is still limited as to precisely how lysophospholipids are involved in the production of the lipid mediators needed to maintain CML stem cells. It is possible that lysophospholipids are direct sources of such lipid mediators, and/or that lysophospholipids regulate the expression of genes critical for lipid mediator biosynthesis. To distinguish between these possibilities, we should determine the substrates and products of the metabolic reaction governed by Gdpd3 by using GPCR mRNAs. We observed that Gdpd3-deficient LT-CML stem cells showed decreased expression of GPCR genes, including Lgr4/GPR48, compared to WT-LT-CML stem cells [Lgr4/GPR48-hypomorphic mutant mice had a defect in active Foxo3a/\u03b2-catenin interaction that could be rescued in vitro by PGE2. However, this treatment could not restore Foxo3a/\u03b2-catenin interaction lost due to lack of Gdpd3. Notably, Beulac et al. reported that Foxo3 induces Gdpd3 expression in a mouse model of noise-induced hearing loss [Another unanswered question in the field is how lysophospholipid metabolism regulates the transcription of em cells . Interesem cells ,31,32. Rem cells , paralleem cells . We alsoing loss . These dIn this review, I have attempted to put into a broader biological context our recent findings on the role of lysophospholipid metabolism in general, and Gdpd3 in particular, in CML stem cells in vivo. Importantly, healthy Gdpd3-deficient mice show no obvious defects ,45, sugg"} +{"text": "Physical activity especially at moderate-to-vigorous intensity may preserve brain structure in old age. However, current findings are cross-sectional and rely on absolute intensity. This study aimed to examine whether relative or absolute vigorous-intensity physical activity (VPA) predicts brain microstructural changes. We analyzed 260 initially cognitively normal and well-functioning participants(age=70.5yrs) who had VPA data via ActiHeart and longitudinal brain microstructure by DTI(follow-up=3.7yrs). Associations of VPA with microstructural changes were examined using linear mixed-effects models, adjusted for demographics. Each SD higher relative VPA defined by heart rate reserve (i.e. 21 min/day) was significantly associated with less decline in memory-related microstructural integrity, including mean diffusivity of entorhinal cortex and parahippocampal gyrus and fractional anisotropy of uncinate fasciculus and cingulum-hippocampal part, and not executive/motor-related microstructure. Absolute VPA was not associated with microstructural markers. Among well-functioning older adults, participating in VPA defined by heart rate reserve may predict less brain microstructural decline in memory-related areas."} +{"text": "Monoclonal antibodies and antibody cocktails are a promising therapeutic and prophylaxis for coronavirus disease 2019 (COVID-19). However, ongoing evolution of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) can render monoclonal antibodies ineffective. Here, we completely map all of the mutations to the SARS-CoV-2 spike receptor-binding domain (RBD) that escape binding by a leading monoclonal antibody, LY-CoV555, and its cocktail combination with LY-CoV016. Individual mutations that escape binding by each antibody are combined in the circulating B.1.351 and P.1 SARS-CoV-2 lineages . In addition, the L452R mutation in the B.1.429 lineage escapes LY-CoV555. Furthermore, we identify single amino acid changes that escape the combined LY-CoV555+LY-CoV016 cocktail. We suggest that future efforts diversify the epitopes targeted by antibodies and antibody cocktails to make them more resilient to the antigenic evolution of SARS-CoV-2. Map of all SARS-CoV-2 mutations that escape LY-CoV555 (bamlanivimab) antibody bindingLY-CoV555 is sensitive to mutations E484K and L452R in variants of concernSingle mutations and circulating combinations escape the LY-CoV555/LY-CoV016 cocktailTherapeutic antibodies that target subdominant epitopes are needed Starr et\u00a0al. report a complete map of all of the SARS-CoV-2 mutations that escape binding by the LY-CoV555 (bamlanivimab) antibody and its cocktail with LY-CoV016. These maps highlight high-frequency mutations that may affect LY-CoV555 efficacy and enable immediate interpretation of the effects of future viral variation on this antibody. This question has taken on growing importance with the recent emergence of SARS-CoV-2 lineages containing mutations in the spike receptor-binding domain (RBD),17,,To enable more comprehensive and prospective assessment of the effects of viral mutations, we recently developed a method to completely map how all single amino acid mutations in the SARS-CoV-2 RBD affect antibody binding.Here, we prospectively map how all mutations to the RBD affect binding by LY-CoV555 alone and in a cocktail with LY-CoV016. . The maps show that LY-CoV555 is escaped by mutations at a focused set of sites, with site E484 standing out as a hotspot of escape . Note that some of the other smaller cocktail escape mutations in the cocktail maps may reflect a higher potency of LY-CoV555 in the 1:1 cocktail rather than representing mutations that truly escape binding by both antibodies. Mutations at position Q493 are notably well tolerated with respect to ACE2 binding and RBD expression . LY-CoV016 and LY-CoV555 bind opposite sides of the receptor-binding ridge, a structurally,To gain insight into the structural basis for the escape mutations, we projected our escape maps onto crystal structures of\u00a0the antibodies bound to the RBDWe generated complete maps of mutations that escape a leading antibody and antibody cocktail being used to combat COVID-19. Our maps highlight the need to consider circulating SARS-CoV-2 diversity in regions where these antibodies are deployed, as several viral lineages already have mutations that escape binding from LY-CoV555 and its cocktail with LY-CoV016. The maps we report will continue to enable the immediate assessment of the effects of newly observed mutants on these antibodies and their cocktail, although it will of course remain necessary to validate key findings with additional virological experiments.,,More broadly, our maps suggest that it may be advisable to more systematically consider possible escape mutations when devising antibodies for clinical use against SARS-CoV-2. It is now clear that human coronaviruses undergo antigenic evolution in response to immune pressure,in\u00a0vitro neutralization or therapeutic protection. We note that our antibody binding-escape maps generated in prior studies have proven highly concordant with the effects of mutations on the antibody neutralization of mutant spike-pseudotyped viral particles.,,,Our approach maps escape from antibody binding, but we do not directly measure the effects of mutations on jbloom@fredhutch.org)Further information and requests for resources should be directed to and will be fulfilled by the Lead Contact, Jesse Bloom on a BD FACSAria II. FACS selection gates were drahttps://jbloomlab.github.io/dms_variants/, version 0.8.2) to process Illumina sequences into counts of each barcoded RBD variant using the barcode/RBD look-up table from Starr et\u00a0al.https://github.com/jbloomlab/SARS-CoV-2-RBD_MAP_LY-CoV555/blob/main/results/summary/summary.md.Escape fractions were computed as described in Starr et\u00a0al.https://github.com/jbloomlab/SARS-CoV-2-RBD_MAP_LY-CoV555/blob/main/data/gisaid_hcov-19_acknowledgement_table_2021_03_15.pdf).All spike sequences present on GISAIDhttps://jbloomlab.github.io/dmslogo/). Interactive visualizations of the escape maps and their projection onto the ACE2-bound (PDB: 6M0Jhttps://jbloomlab.github.io/SARS-CoV-2-RBD_MAP_LY-CoV555/ were created using dms-view (https://dms-view.github.io/docs/).7KMG7C01.Static logoplots were created using dmslogo (Experiments were conducted in full biological duplicate, using independently generated and assayed mutant libraries. Values used throughout the text are the average of these duplicate measurements, as described in"} +{"text": "Primary or secondary central nervous system (CNS) lymphoma is frequently associated with a poor prognosis. CAR T-cells are being established as a relevant treatment approach in hematological B-cell malignancies. Unfortunately, most clinical studies on chimeric antigen-receptor (CAR) T-cells have excluded patients with CNS involvement but several clinical trials on CAR T-cell therapy in CNS lymphoma patients are currently ongoing. Preclinical and preliminary clinical data suggest an overall acceptable safety profile and considerable anti-tumor effects might be extrapolated for CAR T-cell therapy in CNS lymphoma.Primary CNS lymphomas (PCNSL) represent a group of extranodal non-Hodgkin lymphomas and secondary CNS lymphomas refer to secondary involvement of the neuroaxis by systemic disease. CNS lymphomas are associated with limited prognosis even after aggressive multimodal therapy. Chimeric antigen receptor (CAR) T-cells have proven as a promising therapeutic avenue in hematological B-cell malignancies including diffuse large B-cell lymphoma, B-cell acute lymphoblastic leukemia, and mantle-cell lymphoma. CARs endow an autologous T-cell population with MHC-unrestricted effectivity against tumor target antigens such as the pan B-cell marker CD19. In PCNSL, compelling and long-lasting anti-tumor effects of such therapy have been shown in murine immunocompromised models. In clinical studies on CAR T-cells for CNS lymphoma, only limited data are available and often include both patients with PCNSL but also patients with secondary CNS lymphoma. Several clinical trials on CAR T-cell therapy for primary and secondary CNS lymphoma are currently ongoing. Extrapolated from the available preliminary data, an overall acceptable safety profile with considerable anti-tumor effects might be expected. Whether these beneficial anti-tumor effects are as long-lasting as in animal models is currently in doubt; and the immunosuppressive tumor microenvironment of the brain may be among the most pivotal factors limiting efficacy of CAR T-cell therapy in CNS lymphoma. Based on an increasing understanding of CAR T-cell interactions with the tumor cells as well as the cerebral tissue, modifications of CAR design or the combination of CAR T-cell therapy with other therapeutic approaches may aid to release the full therapeutic efficiency of CAR T-cells. CAR T-cells may therefore emerge as a novel treatment strategy in primary and secondary CNS lymphoma. Primary central nervous system lymphoma (PCNSL) represent a rare group of extranodal B-cell non-Hodgkin lymphomas arising from the brain parenchyma, spinal cord, eyes, or meninges without systemic, extra-axial involvement . Such tuAdoptive immunotherapy with chimeric antigen receptor (CAR) T-cells has emerged as an efficient therapy for relapsed or refractory hematological malignancies . FollowiAnti-tumor effects of CD19-directed CAR T-cells against PCNSL have not only been demonstrated in vitro, but also in murine in vivo models ,17. MulaPCNSL regression after local but not intravenous administration of CAR T-cells was recently corroborated in another immunoincompetent mouse model (lacking function T- and B-cells) of PCNSL . Wang etThese findings made from preclinical studies appear therefore promising in controlling PCNSL; however, they have not yet been validated in immunocompetent animal models. Given that only a limited number of preclinical studies on CAR T-cells and PCNSL is available, a high level of suspicion is therefore required when interpreting these results; however, some anti-tumor effects against PCNSL and CNS lymphoma in general might be assumed.Patients with active involvement of the brain were excluded from almost all clinical trials on CAR T-cells, mainly due to dreaded more severe neurotoxic side effects. These trials have resulted in US Food and Drug Administration (FDA) approval of CD19-directed CAR T-cells for patients with systemic but not CNS disease ,12. So fIn 2017, a first case report on CAR T-cell efficacy in secondary CNS lymphoma was published . Abramso8 to 6.0 \u00d7 108 CAR T-cells). Only mild neurotoxic or systemic side effects were encountered, and none of these patients experienced CAR T-cell-mediated toxicities necessitating therapy with the anti-interleukin 6-receptor antagonist tocilizumab or steroids. Response assessment on day 28 after CAR T-cell infusion showed complete response in two patients, partial response in two more patients, and disease progression in four patients . Further follow-up on day 90 revealed ongoing disease control in three of the four patients who initially responded to CAR T-cells, and long-term follow up on day 180 was available in one of those patients showing complete response.Another CD19-directed CAR T-cell product, tisagenlecleucel (formerly known as CTL019), has been approved in 2017 for large B-cell lymphoma patients with systemic but also secondary (not primary) CNS involvement. Based on the granted FDA approval, Frigault et al. treated and reported on a retrospective cohort of eight patients with secondary CNS involvement of the brain, spine, and leptomeninges [8 to 6 \u00d7 108 CAR T-cells) following lymphodepletion, whereas when no life-threatening toxicities occurred, tocilizumab was provided for moderate cytokine release syndrome in two patients and steroids for neurotoxicity in three patients. Four patients had disease responses to CAR T-cells with one patient showing complete response and three patients showing partial response. On a cautionary note, follow-up time was only in the range of several weeks and it is unclear whether this response was durable.These results suggesting considerable anti-tumor effects in the treatment of CNS disease were recently corroborated by preliminary data from an ongoing prospective trial of CD19-directed CAR T-cells for B-cell non-Hodgkin lymphoma NCT02153580) [53580 [216 to 7.1 \u00d7 106/kg body weight) and one infusion of CD22-directed CAR T-cells (3.1 \u00d7 106 to 7.0 \u00d7 106/kg). CD22 is another pan B-cell marker which offers an additional target in the case of CD19 antigen loss [Data from longer follow-up intervals after treatment of CNS disease were reported from Li et al. (ChiCTR-OPN-16008526) . One patgen loss . In thisBased on the encouraging results of above-mentioned studies, different clinical phase I and phase II trials are currently testing safety and efficiency of CD19 CAR T-cells in primary and secondary CNS lymphoma patients .The above-mentioned studies suggest an acceptable safety profile of CAR T-cells for CNS lymphoma. Furthermore, considerable anti-tumor effects have been reported. Whether these anti-tumor effects are as long-lasting and profound as it has been described for extracranial disease might be in doubt. A number of CNS-specific aspects may hamper clinical success of such therapies.Primary brain tumors including PCNSL represent complex compositions of neoplastic and non-neoplastic cells which individually contribute to tumor formation . Tumor-anu/nu mice (lacking T- but not B-cell function). However, the murine CD19 on B-cells from Foxn1nu/nu mice differs substantially from human CD19 which the CAR T-cells were directed against in the study by Mulazzani et al. It might therefore be indeed speculated that these models may underestimate CAR T-cell effectivity.In addition to metabolic barriers for tumor infiltration by CAR T-cells, physical barriers including the blood\u2013brain barrier may limit treatment success. Under physiologic conditions, the brain is virtually free of leucocytes, and their influx is tightly regulated. CAR T-cells have been shown to migrate across the blood\u2013brain barrier and can be found in brain and CSF ,32. In tClinical studies for glioblastoma show that local CAR T-cell delivery (delivery into a resection cavity or administration into the CSF) is feasible and effective ,18,35. SAnother factor contributing to recurrence after CAR T-cell therapy may be the loss or downregulation of the tumoral target antigens. Of patients with B-cell leukemia, 7\u201325% experience CD19-negative relapse ,36. The CAR T-cell therapy directed against CD19, but also other antigens might be accompanied by unique toxicities including cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), on-target\u2013off-tumor toxicities, and prolonged cytopenia ,40.CRS represents the most commonly encountered adverse effect and is characterized by a systemic increase of pro-inflammatory cytokines translating into sepsis-like symptoms . A high ICANS is the second most commonly observed toxicity following CAR T-cell therapy. Clinical presentation varies and includes moderate symptoms such as headaches, fatigue, or aphasia , but alsHematological toxicities are among the most common, yet underreported adverse effects of CAR T-cell therapy . As all Whereas patients with glioblastoma and primary brain tumors other than PCNSL often experience lymphopenia before, during, and after CAR T-cell therapy as stated above, patients with CNS lymphoma are at a particularly high risk due to aggressive myeloablative first-line therapies which also include the use of stem cell transplantation ,57,58. IMoreover, autoimmune disorders requiring immunosuppressive medication are overrepresented among patients with CNS lymphoma . The patGiven the substantial adverse effects of CAR T-cells, anti-tumor effects will need to be carefully weighed against such side effects. Improvement of CAR T-cell efficacy might therefore be critical to consider such a therapy in patients in which other therapeutic approaches might still be available.Recent CAR T-cell constructs have been evolving with novel design approaches emerging to optimize clinical efficacy and safety. The basic structure of every CAR consists of an extracellular ligand recognition domain, typically a single-chain variable fragment, providing tumor antigen specificity, a transmembrane domain, and an intracellular T-cell-activating domain that includes a CD3 zeta chain. Such \u2018first-generation CARs\u2019 showed limited efficacy in early clinical trials due to their insufficient signaling capability and low persistence ,66. To eIn an effort to mitigate the effects of the immunosuppressive lymphoma microenvironment, featuring TAM/M activation and immunosuppressive cytokines, CARs expressing an additional transgenic inducible-cytokine have been designed . Such \u2018fTo circumvent tumor recurrence due to loss of the target antigen, CAR T-cell products able to recognize multiple tumor antigens have been designed. As such, Tu et al. described a single PCNSL patient who was treated simultaneously with CD19- and CD70-directed CAR T-cells, and durable complete remission at 17 months follow-up was reported . MoreoveApart from improving CAR constructs, there is a growing interest in developing alternative cell lines endowing antigen-specificity such as CAR NK-cells and CAR-macrophages, both showing properties that could enhance anti-tumor activity B,C. In aGiven the peculiar role of metabolic and physical barriers for CAR T-cell efficiency in brain tumors, as well as other solid tumors outside the CNS, the combination of CAR T-cells with other therapies to ameliorate such effects might be helpful in increasing therapeutic effects. CNS lymphoma frequently shows PD-L1 expression, and PD-1-positive T-cells in the tumor microenvironment showed increased exhaustion markers compared to PD-1-negative T-cells . Based oColony-stimulating factor 1 receptor (CSF-1R) controls the formation, differentiation, and function of M2 macrophages and strategies to limit myeloid recruitment or reprogram the myeloid populations by CSF-1R-targeting have been proven beneficial ,83. In pSimilar to CAR T-cell delivery to CNS tumor sites, the ideal mode of administration of these additional combinatorial therapies is still an area of uncertainty. Different mechanisms such as direct injection via viral vectors or polymer systems using reservoir systems but also infusion via convection-enhanced delivery are currently being investigated ,88.In conclusion, CAR T-cells appear as a promising therapeutic approach in CNS lymphoma. Preclinical data seem promising, but there is a lack of in vivo studies in immunocompetent animals. Moreover, adverse effects of CAR T-cell therapy are not adequately depictured in animal models and need peculiar attention when translation this therapy into clinical studies . Based o"} +{"text": "TP53, etc.), only a small proportion of these molecular alterations are therapeutically actionable. In an attempt to address this therapeutic impasse, our group has proposed an innovative extreme outlier model to identify novel cooperative molecular vulnerabilities in high-risk GI cancers which dictate prognosis, correlate with distinct patterns of metastasis, and define therapeutic sensitivity or resistance. Our model also proposes comprehensive investigation of their downstream transcriptomic, immunomic, metabolic, or upstream epigenomic cellular consequences to reveal novel therapeutic targets in previously \u201cundruggable\u201d tumors with high-risk genomic features. Leveraging this methodology, our and others\u2019 data reveal that the genomic cooperativity between Ras and p53 alterations is not only prognostically relevant in GI malignancy, but may also represent the incipient molecular events that initiate and sustain innate immunoregulatory signaling networks within the GI tumor microenvironment, driving T-cell exclusion and therapeutic resistance in these cancers. As such, deciphering the unique transcriptional programs encoded by Ras-p53 cooperativity that promote innate immune trafficking and chronic inflammatory tumor-stromal-immune crosstalk may uncover immunologic vulnerabilities that could be exploited to develop novel therapeutic strategies for these difficult-to-treat malignancies.Despite increasingly thorough mechanistic understanding of the dominant genetic drivers of gastrointestinal (GI) tumorigenesis (e.g., Ras/Raf, TP53 mutations resulting in p53 inactivation, loss of SMAD4, etc., only a small proportion of these molecular alterations are therapeutically actionable , Ras-TP53 cooperativity was associated with earlier local and distant recurrence in CRLM patients undergoing complete resection followed by adjuvant systemic and liver-directed chemotherapy. Not only is Ras-p53 genomic cooperativity oncologically significant, but also has broad clinical relevance by virtue of its frequent occurrence in colorectal cancers, with studies reporting concurrent Ras-p53 mutations occurring in nearly one-third of patients with colorectal liver metastasis [http://www.cbioportal.org) corroborate these statistics [Leveraging this methodology, our group is actively pursuing the genomic cooperativity between Ras and p53 alterations in GI malignancy. In a recent manuscript in \u201cClinical Cancer Research\u201d, we utilized an extremes-of-survivorship approach in patients with resected colorectal cancer liver metastasis (CRLM) and demonstrated that concurrent mutations in both atistics , with Rain vivo [CREB1 upregulate the pro-metastatic transcription factor FOXA1, while promoting Wnt/\u03b2-catenin signaling, together driving metastasis [Data from The Cancer Genome Atlas (TCGA) also demonstrate that the RTK-Ras and p53 oncogenic pathways are co-altered in a substantial portion of patients with non-colorectal gastrointestinal cancers, particularly pancreatic ductal adenocarcinoma (PDAC) in which more than half of all patients demonstrate Ras-p53 cooperative alterations . These din vivo , 15. Rectastasis . Finallytastasis . Interestastasis , 19. TheR175H) or activated H-Ras (RasG12V) were profiled by microarray analysis to delineate a set of gene transcripts\u2014encompassing a broad range of functional annotations such as signal transduction, metabolism, cell adhesion, etc.\u2014that are synergistically regulated by Ras-p53 cooperative signaling [While the prognostic relevance of Ras-p53 genomic cooperativity and its mechanistic underpinnings are undoubtedly compelling, neither mutant Ras nor p53 are yet considered therapeutically actionable. As mentioned previously, we believe that deciphering how these cooperative driver mutations orchestrate tumor-promoting and immunosuppressive tumor-stromal-immune interactions in the tumor microenvironment (TME) to promote treatment resistance may reveal novel therapeutic opportunities for these aggressive cancers. As such, our group is particularly interested in understanding and targeting novel immune repercussions of Ras-p53 cooperativity in GI cancers. While the independent roles of both oncogenic Ras activation and p53 loss in establishing pro-inflammatory signaling and the recruitment and activation of immunosuppressive cells is well established \u201322, how ignaling . Furtherin vitro transformation model using immortalized human fibroblasts transduced with RasG12V and p53R175H constructs to identify an inflammation-associated gene signature synergistically induced by cooperative Ras-p53 alterations [CXCL1, CXCL2, CXCL3, and CXCL6) and well as the pro-inflammatory ligands and p53 versus tumors with Ras alterations alone. Ras-p53 cooperative COAD/READ tumors exhibited increases in immunosuppressive innate immune populations such as tumor-associated neutrophils, tumor-associated macrophages (TAMs), monocytes, inducible T-regulatory type 1 (Tr1) cells, and \u03b3\u03b4 T-cells . To expl T-cells and 2B. c cancer . These fTaken together, prior and emerging data indicate that Ras-p53 genomic cooperativity is an ideal model to investigate mechanisms of innate immune regulation in gastrointestinal cancers. Innate immune populations such as neutrophils, neutrophilic myeloid-derived suppressor cells (MDSCs), and TAMs are not only critically important for the initiation and progression of solid organ cancers , but havhttp://www.cbioportal.org [n = 935) at MSKCC that had undergone sequencing using the MSK-IMPACT platform.Previously published genomic data , 8 was crtal.org , 33 and KRAS, NRAS, HRAS) and TP53, and the frequency of co-altered Ras-TP53 mutations across selected GI cancers was tabulated. TCGA samples in the COAD and READ datasets were dichotomized into Ras-p53 cooperative and Ras-alone altered, and resulting fragments per kilobase of transcript per million (FPKM) reads from these two groups were entered into a publicly available single cell immune deconvolution pipeline ImmuneCellAI (available at: http://bioinfo.life.hust.edu.cn/ImmuCellAI#!/) [The TCGA pan-cancer database was queried for all hotspot mutations in Ras family members (llAI#!/) . ImmuneC"} +{"text": "Tumor-associated macrophages (TAMs) promote key processes in the modulation of tumor microenvironment (TME). However, the clinical significance of heterogeneous subpopulations of TAMs in hepatocellular carcinoma (HCC) remains unknown.high (Siglec-10hi) TAMs and explore their impact on the TME of HCC. The effect of Siglec-10 blockade was evaluated in vitro based on fresh human tumor tissues.HCC tissues from Zhongshan Hospital and data from The Cancer Genome Atlas were obtained and analyzed. Immunohistochemistry and flow cytometry were performed to detect the characteristics of sialic acid-binding immunoglobulin-like lectin 10+ cell enrichment was associated with unfavorable prognosis in patients with HCC. Notably, multiple anti-inflammatory cytokines and inhibitory receptors were enriched in Siglec-10hi TAMs. RNA sequencing data also revealed that numerous M2-like signaling pathways were significantly upregulated in Siglec-10hi TAMs. High infiltration of Siglec-10hi TAMs was associated with impaired CD8+ T cell function in HCC. Of note, blocking Siglec-10 with the competitive binding antibody Siglec-10 Fc led to decreased expression of immunosuppressive molecules and increased the cytotoxic effects of CD8+ T cells against HCC cells. Moreover, blocking Siglec-10 promoted the anti-tumor efficacy of the programmed cell death protein 1 (PD-1) inhibitor pembrolizumab.Our data revealed that Siglec-10 was abundant in a large proportion of HCC specimens and prominently distributed on macrophages. Kaplan\u2013Meier curves and Cox regression analysis showed that intratumoral Siglec-10hi TAMs are associated with immune suppression in the TME, and indicate poor prognosis in patients with HCC. Targeting Siglec-10hi TAMs may serve as a promising immunotherapy approach for HCC.Siglec-10The online version contains supplementary material available at 10.1186/s40164-021-00230-5. Hepatocellular carcinoma (HCC), the most common primary liver cancer, is one of the top causes of cancer-related death worldwide , 2. The Immunotherapy targeting the tumor microenvironment (TME) has revolutionized tumor treatment. The results of the CheckMate 040 and KEYNOTE-224 studies were extraordinary milestones in the battle against HCC. However, the response rate to monoclonal anti-programmed cell death protein 1 (PD-1) antibody has been disappointing due to the high heterogeneity of immune evasion mechanisms . Thus, iTumor-associated macrophages (TAMs) are the most abundant immune cells within the TME, and promote vital processes in tumor progression . Increashi macrophages may promote immune evasion in HCC.Sialic acid-binding immunoglobulin-like lectin 10 (Siglec-10) is widely expressed on subsets of human leukocytes, and plays an important part in distinguishing between self and non-self in the immune system . As an ihi TAM infiltration was positively associated with CD8+ T cell inactivation. Notably, inhibition of Siglec-10 restrained the secretion of anti-inflammatory cytokines from macrophages and suppressed the expression of inhibitory receptors on CD8+ T cells, resulting in restored cytotoxic activities against tumor cells. Our study identifies targeting Siglec-10hi TAMs as a promising therapeutic option for HCC.In this study, we found that Siglec-10, which was associated with poor overall survival (OS) and recurrence-free survival (RFS) in patients with HCC, was predominately expressed on macrophages. Furthermore, we demonstrated that intratumoral Siglec-10We retrospectively evaluated 246 patients with HCC who underwent hepatectomy from 2008 to 2010. The exclusion criteria were: pathological diagnosis other than HCC or combined with other pathological types, distant metastatic disease, or unavailability of tumor tissues or follow-up information. None of the patients enrolled in this study had received neoadjuvant therapy before surgery. Two patients with mixed HCC/intrahepatic cholangiocarcinoma (ICC) and five patients with unavailable follow-up data were excluded. Four specimens were lost on tissue microarray (TMA) when immunohistochemistry (IHC) was performed. Finally, 235 eligible HCC patients were randomly divided into discovery set (n\u2009=\u2009115) and validation set (n\u2009=\u2009120). Patients\u2019 clinical characteristics are shown in Additional file The clinical tumor stage was determined according to the American Joint Committee on Cancer/International Union Against Cancer (AJCC/UICC) Tumor-Node-Metastasis (TNM) staging system 8th edition. All follow-up data were collected from the date of surgery to December 2018. OS was defined as the time period between surgery and death or last follow-up. RFS was defined as the time period between surgery and the first documented recurrence or death, whichever occurred first. This study was approved by the Clinical Research Ethics Committee of Zhongshan Hospital, Fudan University . An informed consent form was signed by every patient.IHC and immunofluorescence (IF) staining were performed on TMAs constructed with formalin-fixed and paraffin-embedded HCC specimens. IHC staining was performed according to a previously described protocol . In brieSeventy-seven fresh human specimens were collected from HCC patients, who underwent liver resection at Zhongshan Hospital. Single-cell suspension was prepared as previously described . After rhi TAMs (CD45+ CD14+ Siglec-10hi) and Siglec-10lo TAMs (CD45+ CD14+ Siglec-10lo) were freshly isolated from three HCC specimens using the MoFlo XDP Cell Sorter and directly lysed in lysis buffer. The cDNA libraries were constructed using the TruSeq Stranded mRNA LT Sample Prep Kit according to the manufacturer\u2019s instructions. Then the libraries were sequenced on the Illumina HiSeq X Ten platform. About 6G raw reads for each sample of fastq format were processed using Trimmomatic [Siglec-10mmomatic and low-hi TAMs and Siglec-10lo TAMs were performed respectively using R software based on the hypergeometric distribution. Gene set enrichment analysis (GSEA) was performed by the Molecular Signature Database for gene functional annotation.The clean reads were aligned to the human genome (GRCh38) using HISAT2 . The FPKhttps://www.cbioportal.org on July 15, 2020. Six cases were excluded due to lack of RNA-seq data. Finally, data from 366 patients were enrolled in this study. The median value of Siglec-10 expression was set as the cut-off value.The Cancer Genome Atlas Liver Hepatocellular Carcinoma (TCGA-LIHC) mRNA and clinical data, including RNA sequencing (RNA-seq) and clinicopathological data for 372 tumors, were downloaded from In vitro intervention studies were performed according to previously described methods . DissociThe antibodies used in this experiment were IgG1 isotype control , recombinant human Siglec-10 Fc chimera , and pembrolizumab . After overnight culture in RPMI 1640 medium containing 10% fetal bovine serum and corresponding antibodies, cells were subjected to flow cytometry analysis to examine the apoptosis of tumor cells by using the FITC Annexin-V Apoptosis Detection Kit I (BD Biosciences) or corresponding fluorescence-activated cell sorting antibodies.t-test or one-way analysis of variance was used for continuous variables. Spearman\u2019s correlation was employed to evaluate the correlation between different variables. Kaplan\u2013Meier method was used to determine OS and RFS. Cox proportional hazards regression model was used for multivariate analysis with hazard ratios (HRs) and 95% confidence intervals (CIs). To obtain the best prognostic efficacy, X-Tile Software was used as previously described [hi TAM infiltration(Siglec-10hi CD68+ cells / CD68+ cells)were defined as cut-off value. Calculations were done with SPSS 22.0 . Graphical illustrations were carried out with GraphPad Prism v7 . A two-tailed P value less than 0.05 indicated statistical significance.Results are expressed as the mean\u2009\u00b1\u2009standard deviation. Categorical variables were reported as numbers and percentages, and were analyzed by the Pearson\u2019s chi-squared test or Fisher\u2019s exact test. Student\u2019s escribed . The cut+ cells in HCC, Kaplan\u2013Meier curves were used to compare the prognosis of patients stratified by intratumoral Siglec-10+ cell infiltration. Patients with low Siglec-10+ cell infiltration had better OS , T cell immunoglobulin and mucin domain 3 (TIM3), T cell immunoreceptor with IG and ITIM domains (TIGIT), cytotoxic T-lymphocyte-associated protein 4 (CTLA4), V-set immunoregulatory receptor (VSIR), CD274, and lymphocyte-activation gene 3 (LAG3) were defined according to median cut-off value. HCC with high Siglec-10hi TAM infiltration exhibited higher levels of CD4+ FoxP3+ regulatory T cells compared to tumors with low Siglec-10hi TAM infiltration were investigated. HCC specimens with high Siglec-10hi TAM infiltration exhibited lower proportions of granzyme B+ (GZMB+), IFN-\u03b3+, IL-2+ and perforin-1+ (PRF-1+) CD8+ CTLs and M1 macrophage marker compared with Siglec-10lo TAMs , compared to Siglec-10lo TAMs production, and T helper type 1 (Th1) and Th2 cell differentiation, whereas intratumoral Siglec-10hi TAMs showed marked upregulation of genes involved in metabolic pathways cell mediated cytotoxicity, immunological synapse, and immunoglobulin receptor binding significantly increased after blocking Siglec-10, whereas the levels of anti-inflammatory molecules were markedly decreased compared with the isotype group and M2 (CD206) markers. Compared to Siglec-10lo TAMs, Siglec-10hi TAMs showed higher levels of immune stimulatory cytokines , and higher levels of immune inhibitory molecules . Interestingly, the expression of immune inhibitory molecules was significantly suppressed after blocking Siglec-10, whereas the levels of TNF-\u03b1 and IL-12 were further increased. It is known that despite functional impairment, exhausted T cells with overexpression of inhibitory receptors retain a crucial level of control over tumor growth [hi TAMs, retain the anti-tumor effect in some degree. Indeed, our findings indicate that Siglec-10 expression may facilitate the secretion of macrophage-derived anti-inflammatory cytokines in HCC. However, once released from inhibition, Siglec-10hi TAMs may be transformed into an active role in anti-tumor immunity. In summary, Siglec-10 represents a novel immune inhibitor in HCC. Targeting Siglec-10 may serve as a promising approach to restore anti-tumor immunity.In our study, we also found that intratumoral Siglec-10r growth \u201355. Targhi NK cells, Siglec-10hi dendritic cells, or other cells expressing Siglec-10 in the TME. Second, the study lacked in vivo experiments to further characterize the role of Siglec-10hi TAMs in HCC. Based on the current findings, we will further explore the mechanism underlying the results that blocking Siglec-10 promotes tumor cell death.This study had several limitations. First, Siglec-10 is widely expressed on various leucocytes and predominantly distributed on macrophages in HCC tissues. In this study, we did not investigate the effects of Siglec-10hi TAMs may play a key role in tumor immune evasion via expression of immune inhibitory molecules and inactivation of CD8+ CTLs. Blocking Siglec-10 led to markedly declined secretion of anti-inflammatory cytokines and increased the cytotoxic effects of CD8+ T cells against tumor cells. These findings highlight the importance of Siglec-10hi TAMs in immune modulation of the TME and provide a novel promising immunotherapy approach for HCC.As immune suppressors, Siglec-10Additional file 1: Figure S1. Flow chart of patient selection. Figure S2. Gating strategy for selection of Siglec-10hi macrophages. Figure S3. Representative images of CD8+ T cells by flow cytometry analysis. Table S1. Characteristics of HCC patients and relationship with intratumoral Siglec-10+ cell infiltration. Table S2. Immunohistochemistry, immunofluorescence and flow cytometry antibodies."} +{"text": "Individuals with chronic lymphocytic leukemia (CLL) have significant immune disfunction, often further disrupted by treatment. While currently available COVID-19 vaccinations are highly effective in immunocompetent individuals, they are often poorly immunogenic in CLL patients. It is important to understand the role a heterologous boost would have in patients who did not respond to the initial two-dose mRNA vaccine series. SARS-CoV-2 specific immune responses, including antibodies and memory B-cells, CD4 and CD8 T-cells were assessed prior to vaccination, as well as postinitial vaccination series and post-third dose in two subjects. One subject seroconverted, had RBD-specific memory B-cells and spike-specific CD4 T-cells while the other did not. Both subjects had a spike-specific CD8 T-cell response after the original mRNA vaccination series that was further boosted after the third dose or remained stable. The results of this study, however small, are especially promising to CLL individuals who did not seroconvert following the initial mRNA vaccination series. Chronic lymphocytic leukemia (CLL) is characterized by the monoclonal proliferation of dysfunctional B-cells, leading to a broad range of immune defects. CLL patients face significant risk of morbidity and mortality from infections , includiAlthough current COVID-19 vaccines elicit robust immunity in immunocompetent hosts , the antGiven decreased vaccine efficacy in CLL, an additional dose of vaccine may be beneficial in CLL patients, especially given the rise of variants of concern (VoCs). Initial data from solid organ transplant recipients on immunosuppression as well as individuals with solid tumors on active therapy showed a role for additional vaccination , 9. ThisHere we describe two CLL patients who \u201cself-referred\u201d to outside pharmacies for an additional vaccination with the J&J COVID-19 vaccine following 2 doses of the BNT162b2 vaccine (Pfizer-BioNTech). Both patients had previously been enrolled as study subjects in an IRB-approved observational study (OHSU IRB# 21230) to investigate immune response following COVID-19 vaccination. The additional J&J dose was subsequently self-reported to the study team. On initial enrollment, demographics, CLL disease characteristics, and treatment details were collected , and bas\u03b3 and TNF\u03b1 secretion following spike protein-derived peptide stimulation. Briefly, PBMCs were stimulated with 2 peptide pools of overlapping (10AA) 17mers representing the SARS-CoV-2 spike protein (BEI Resources). Following stimulation, the cells were stained as previously described [SARS-CoV-2 spike receptor binding domain (RBD)-specific antibody levels were tested by ELISA and endpoint titers were calculated as previously described . In addiescribed , 14. DatNeither subject had prevaccination B-cell responses as measured by RBD-specific antibodies or MBCs. Neither had a virus-specific CD8+ response at baseline. While Subject 2 had spike peptide-reactive CD4+ T-cells at baseline, these cells were unresponsive and did not expand following vaccination. In contrast, CD8+ responses were observed after mRNA vaccination in both subjects . It has Approximately four weeks after initial vaccination, neither subject had detectable RBD-specific SARS-CoV-2 antibodies or MBCs. Both had measurable vaccine-induced CD8+ T-cell responses following mRNA vaccination, although CD4+ responses did not appear to increase above baseline .6 B-cells, and 166 spike-specific CD4+ T-cells/106, and a spike-specific CD8+ T-cell response that remained stable and did not boost appreciably following the 3rd vaccination , the most notable difference between the subjects' baseline characteristics is that Active treatment with Bruton's tyrosine kinase (BTK) inhibitors like ibrutinib may have a profound impact on B-cell survival, differentiation, and production of antibodies as the absence of intact BTK-dependent B-cell receptor mediated signaling prevents B-cells from differentiating into mature peripheral B-cells. Immune responses following vaccination or natural infection are limited in these patients . Recall rd vaccination against COVID-19 with the heterotypic vaccine Ad26COV2.s results in an immune response that was not observed following the recommended 2-dose mRNA vaccination series. This is especially promising news to subjects who are treatment na\u00efve, not currently in active treatment, or who may consider vaccination before beginning active treatment.The results of this study, however small, provide initial evidence that a 3"} +{"text": "The Prospective Assessment of SARS-CoV-2 Seroconversion (PASS) study is following over 200 healthcare workers who have received the Pfizer-BioNTech BNT162b2 COVID-19 mRNA vaccine. A major aim of the study is to determine whether baseline antibody titers against the seasonal human coronaviruses are associated with altered levels of vaccine-induced antibody responses to SARS-CoV-2.Serial serum samples obtained pre-vaccination and 1 month after the second dose were tested for IgG antibodies against the full pre-fusion spike protein and the receptor binding domain (RBD) of SARS-CoV-2, as well as the full pre-fusion spike proteins of OC43, HKU1, 229E, and NL63. Antibodies were measured using highly sensitive and specific multiplex assays based on Luminex-xMAP technology. nd vaccine dose. Preliminary analysis demonstrates no association between baseline antibody titers against spike protein of OC43 and antibody titers against SARS-CoV-2 spike protein or RBD one month after vaccination. Future analyses will evaluate whether there is an association with baseline seasonal coronavirus antibody titers and either SARS-CoV-2 neutralization titers or anti-SARS-CoV-2 spike protein titers at 6 months after vaccination. Preliminary analyses of the first 103 subjects in whom we have 1 month post-vaccination serum demonstrate development of high IgG geometric mean titers (GMT) to both the full spike protein and the RBD of SARS-CoV-2 after the 2These preliminary results suggest that baseline antibody responses to seasonal coronaviruses neither boost nor impede SARS-CoV-2 vaccine-induced antibody responses. Longitudinal sampling will enable assessment of vaccine durability and determination of whether baseline seasonal coronavirus antibody levels are associated with altered duration of detectable COVID-19 vaccine-induced antibody responses.Simon Pollett, MBBS, Astra Zeneca ) David Tribble, M.D., DrPH, Astra Zeneca )"} +{"text": "Risk-adapted multiagent chemotherapy has led to a remarkable improvement in the life expectancy of patients with acute lymphoblastic leukemia (ALL). Nevertheless, in high-risk subgroups such as BCR-ABL+ ALL, relapse rates remain high without allogeneic hematopoietic stem cell transplantation, and the adverse effects of chemotherapy may cause acute and chronic cardiac complications or dysfunction. Here, we demonstrated that chemotherapy-free targeted therapies designed to optimize apoptosis induction in BCR-ABL+ ALL may circumvent cardiac on-target side effects and may even activate cardiac recovery.Targeted therapies are currently considered the best cost\u2013benefit anti-cancer treatment. In hematological malignancies, however, relapse rates and non-hematopoietic side effects including cardiotoxicity remain high. Here, we describe significant heart damage due to advanced acute lymphoblastic leukemia (ALL) with t encoding the bcr-abl oncogene (BCR-ABL+ ALL) in murine xenotransplantation models. Echocardiography reveals severe cardiac dysfunction with impaired left ventricular function and reduced heart and cardiomyocyte dimensions associated with increased apoptosis. This cardiac damage is fully reversible, but cardiac recovery depends on the therapy used to induce ALL remission. Chemotherapy-free combination therapy with dasatinib (DAS), venetoclax (VEN) , respectively), and dexamethasone (DEX) can fully revert cardiac defects, whereas the depletion of otherwise identical ALL in a genetic model using herpes simplex virus type 1 thymidine kinase (HSV-TK) cannot. Mechanistically, dexamethasone induces a pro-apoptotic BCL2-interacting mediator of cell death (BIM) expression and apoptosis in ALL cells but enhances pro-survival B-cell lymphoma extra-large (BCLXL) expression in cardiomyocytes and clinical recovery with the reversion of cardiac atrophy. These data demonstrate that therapies designed to optimize apoptosis induction in ALL may circumvent cardiac on-target side effects and may even activate cardiac recovery. In the future, combining the careful clinical monitoring of cardiotoxicity in leukemic patients with the further characterization of organ-specific side effects and signaling pathways activated by malignancy and/or anti-tumor therapies seems reasonable. Acute lymphoblastic leukemia (ALL) is a heterogeneous cancer disease of lymphoid blood cells, characterized by the accumulation of large numbers of immature lymphocytes, often harboring multiple genetic aberrations. The Philadelphia translocation t9;22)q34;q11) defines a very high-risk subtype of B cell precursor (BCP) ALL . As . As 9]. This cardiac phenotype included a significantly reduced heart size and substantially impaired left ventricular (LV) function due to reduced cardiac output (LV CO) and stroke volume (ESV) . Post-moLC3B cross-sectional area (CSA) e,f assocLC3B and BNIPLC3B [18,27,2LC3B j,k. In lLC3B , Table 2We next used a genetic model to analyze the leukemia dependency of this cardiac phenotype. NSG mice were injected with BV173 cells stably expressing both luciferase and HSV-TK for chemotherapy-free selective ablation of transduced leukemic cells upon treatment with GCV . Upon adHowever, no significant improvement in ALL-induced cardiac failure was detected three weeks after remission induction with no recovery of cardiac dimensions (decline in diastolic LV area (LVAd)) and cardiac dysfunction (impaired LV CO and ESV) at the end of GCV therapy .The cardiotoxicity of GCV therapy could be excluded by the treatment of healthy mice with GCV for four weeks with no detectable effects on cardiac function and output . These dAs we have shown previously, the chemotherapy-free combination therapy consisting of DAS/VEN/DEX induces long-term leukemia-free survival after initial treatment blocks of several weeks .12 \u00b1 0.7 \u00d7 1012 p/s) a using e012 p/s) b. In con012 p/s) . In addi012 p/s) d,e and n012 p/s) f, with n8 \u00b1 3.2 \u00d7 108 p/s) and cardiac and morphometric analyses were performed in a follow-up cohort of long-term survivors after 38 weeks. No signs of cardiac dysfunction or alterations in cardiac and CM dimensions ,10.In the BCR-ABL+ ALL cell line, BV173, DEX and DAS enhance pro-apoptotic BIM expression and, at the same time, reduce anti-apoptotic BCLXL and MCL1 protein levels . As a coSurprisingly, and in contrast to its effect on ALL cells, the DAS/VEN/DEX triple-therapy does not enhance the low level of apoptosis in the heart but rather induces cardiac recovery from ALL-induced damage c\u2013f. BIM BNIP3 and its binding partner BCLXL in cell cultures of primary adult mouse CM , the induction of BCLXL in CM by DEX has cytoprotective effects ,43. FurtIn addition, treatment with DAS/VEN/DEX normalized BNIP3 expression and the LC3 B II/I ratio in cardiac tissue, indicating cardiac recovery.The combination therapy of DAS, DEX and VEN in NSG mice was started at a stage of advanced leukemia. Since ALL patients in the clinics would be treated at a much earlier timepoint, we used a more clinically relevant BV173 model by starting anti-leukemic combination therapy at a much earlier timepoint (one week after cell injection). In this follow-up cohort of long-term survivors (after 38 weeks), no signs of cardiac dysfunction or alterations in cardiac and CM dimensions were found, without any differences from age-matched healthy controls. Therefore, and in contrast to GCV-treatment, cardiac outcome depends on the kind of anti-leukemic therapy in this model.Although cell-, tissue-, organ- and species-specific effects, and their underlying molecular mechanisms, remain to be determined in detail, these data demonstrate that murine xenograft leukemia models can be used to simultaneously optimize anti-leukemic therapies and analyze disease- and/or therapy-dependent cardiotoxicity. The cardiac comorbidity of current anti-tumor therapies may have a tremendous impact on the long-term survival and morbidity of tumor patients, as was most prominently described for anthracycline-induced cardiomyopathy . TransieIn conclusion, our data provide evidence that BCR-ABL+ ALL induces severe and persisting cardiac damage with cardiomyocyte atrophy and functional impairment. The DAS/VEN/DEX triple-therapy not only eradicated the BCR-ABL+ ALL by the highly efficient induction of apoptosis in leukemic cells but also prevented cardiac damage and improved cardiac recovery. Leukemic animal models could be useful tools to optimize anti-leukemic therapy and monitor the safety of combination therapies to avoid unexpected adverse cardiac events from anti-cancer therapies."} +{"text": "Using this model, we demonstrated that loss of Smyd1b, a lysine methyltransferase, disrupted F-actin filament organization in cardiomyocytes of zebrafish embryos. Our studies, however, also demonstrated that strong Lifeact-GFP expression in cardiomyocytes was detrimental to actin filament organization in cardiomyocytes that led to pericardial edema and early embryonic lethality of zebrafish embryos. Collectively, these data suggest that although Lifeact-GFP is a good probe for visualizing F-actin dynamics, transgenic models need to be carefully evaluated to avoid artifacts induced by Lifeact-GFP overexpression.Lifeact-GFP is a frequently used molecular probe to study F-actin structure and dynamic assembly in living cells. In this study, we generated transgenic zebrafish models expressing Lifeact-GFP specifically in cardiac muscles to investigate the effect of Lifeact-GFP on heart development and its application to study cardiomyopathy. The data showed that transgenic zebrafish with low to moderate levels of Lifeact-GFP expression could be used as a good model to study contractile dynamics of actin filaments in cardiac muscles Actin is one of the most abundant proteins in eukaryotic cells and exists as either a free monomer called G-actin (globular) or a linear polymer microfilament called F-actin (filamentous). The F-actin is an important component of the cytoskeleton in eukaryotic cells and thin filaments in myofibrils of muscle cells. F-actin participates in many important cellular processes, including cell division, intracellular cargo transport, cell migration, cell morphogenesis, and muscle contraction . A largeVisualization of actin filament structures and dynamic assembly in living cells is critical for the study of various cellular processes of cell division, migration, polarization, and muscle contraction. Several different techniques and approaches are currently applied to analyze and visualize cellular actin structures and their dynamic assembly and disassembly in various cell and model systems . Phalloiin vitro or in vivo. Thus, Lifeact-GFP surpasses other reagents like fluorescent phalloidin, actin-GFP, and anti-actin antibodies. It enables live imaging of the actin cytoskeleton and therefore the study of many fundamental biological processes in vivo.To avoid the problem of steric clashes with actin from using large and bulky Actin-GFP chimeras, small fluorophore labeled peptides, such as Lifeact-GFP, were developed. Lifeact-GFP is the most popular and widely used actin probe for visualizing F-Actin structures in various cell types and model organisms. Lifeact-GFP is generated by fusing a short 17-amino-acid peptide (Lifeact) from yeast Abp140 with GFP . The speArabidopsis before fixation in 4% paraformaldehyde (PFA) for whole mount observation and immunostaining.smyd1bsa15678 mutant was obtained from ZIRC (Tg(myl7:Lifeact-GFP) and Tg(acta1b:Lifeact-GFP) constructs used to generate the transgenic lines and transient expression assay were gifts from Dr. Didier Stainier , Tg(acta1b:Lifeact-GFP) and pTol2-\u03b1-actin-EGFP were dissolved in sterile water to a final concentration of 50 or 100 ng/\u03bcl. For transient expression analysis, approximately 1\u20132 nl of DNA construct (50\u2013100 pg) was injected into each embryo at one- or two-cell stage. For the generation of transgenic zebrafish lines, the DNA constructs were mixed with Tol2 transposase mRNA (50 ng/\u03bcl). Approximately 1\u20132 nl of mixed DNA construct/Tol2 transposase mRNA was injected into each embryo at one- or two-cell stage. Transgenic zebrafish founders (P1) were screened by examining GFP expression in their F1 embryos at 24 hpf under a fluorescence microscope .DNA constructs of Immunostaining of zebrafish embryonic hearts was carried out at 3 and 14 days-post-fertilization (dpf) as described previously . In brieHaeIII, and then circularized by ligation. The ligated circular DNA was purified and used for two rounds of nested PCR to isolate the sequence at the 5\u2032 junction of the transgene. The first round of PCR was carried out using Tol2-5\u2032/f1 (5\u2032-AAGTACTTTTTACTCCTTACAA-3\u2032) and Tol2-5\u2032/r1 5\u2032-TGATTTTTAATTGTACTCAAGT-3\u2032) primers. The second round was performed using Tol2-5\u2032/f2 (5\u2032- TTACAGTCAAAAAGTACTTA-3\u2032) and Tol2-5\u2032/r2 (5\u2032-CAAGTAAAGTAAAAATCCC-3\u2032) primers. The PCR products were purified and cloned into pGEM-T easy for sequencing.Inverse PCR was performed as described with somThe standard control-MO and the Lifeact-GFP-MO (GATCAAATCTGCGACACCCATCCCC) targeted to the ATG start site of Lifeact were purchased from Gene Tools . The morpholino antisense oligos were dissolved in 1 \u00d7 Danieau buffer to a finTM SYBRTM Green Master Mix . The GFP and elongation factor 1\u03b1 (EF1\u03b1) specific primers are: GFP-qpcr-F (5-CAAGCAGAAGAACGGCATCAAGGTG-3), GFP-qpcr-R (5-G GACTGGGTGCTCAGGTAGTGGTTG-3), EF1\u03b1-P3 (5\u2032 \u2013 CTT CAACGCTCAGGTCATCAT \u2013 3\u2032) and EF1\u03b1-P4 (5\u2032 \u2013 ACAGC AAAGCGACCAAGAGGA \u2013 3\u2032). The PCR was performed with the following parameters including 2 min initial activation at 50\u00b0C and followed by 40 cycles of PCR reaction. Each cycle includes 15 s denaturation at 95\u00b0C, 10 s annealing at 64\u00b0C, and 20 s elongation at 72\u00b0C. For each qPCR experiment, samples were run with three technical replicates. The relative amount of a given mRNA was calculated by using the formulae 2\u2013\u0394\u0394Ct with EF1\u03b1 as reference.Total RNA was isolated from transgenic larvae at 5 days post-fertilization (dpf) using Trizol . Three biological replicates were carried out for each line. The RNA samples were treated with RNase-free DNase . For each sample, 0.5 \u03bcg of RNA was used for reverse transcription using Maxima First Strand cDNA Synthesis Kit . qPCR was performed on the Applied Biosystems QuantStudio 3 using the PowerUpGFP fluorescent images were collected under a fluorescent microscopy (Leica Microsystems) with the same parameters. For each transgenic line, three embryos were randomly chosen for imaging at 24 h post-fertilization (hpf). ImageJ was used to manually measure GFP fluorescent intensity for each heart tube.t test. P value < 0.05 was set as the threshold for statistical significance.Data are presented as the mean \u00b1 sd. Statistical differences were analyzed by using a student\u2019s SMYD1 are associated with congenital and hypertrophic cardiomyopathies in human transgene (mb21Tg(myl7:Lifeact-GFP) zebrafish embryos transgenic line that showed normal development was selected to cross with smyd1bsa15678/+ mutant to generate smyd1bsa15678/+; mb21Tg(myl7:Lifeact-GFP) heterozygous transgenic mutants. The smyd1bsa15678/+; mb21Tg(myl7:Lifeact-GFP) heterozygous transgenic mutants were subsequently in-crossed to generate smyd1bsa15678; mb21Tg(myl7:Lifeact-GFP) homozygous transgenic mutant embryos (smyd1bsa15678 mutant transgenic embryos (mb21Tg(myl7:Lifeact-GFP) transgenic embryos mutant transgenic fish embryos (mb21Tg(myl7:Lifeact-GFP) transgenic zebrafish are a useful model to analyze F-actin myofibril defects in cardiomyopathy.The embryos . Lifeact embryos . However embryos , Lifeact embryos . Lifeact embryos . The homTg(myl7:Lifeact-GFP) transgenic embryos from six out of the eight transgenic founders showed normal development (mb22Tg(myl7:Lifeact-GFP) and mb23Tg(myl7:Lifeact-GFP), gave F1 transgenic embryos with pericardial edema and died at early larval stages (n = 23) from the mb22Tg(myl7:Lifeact-GFP) founder showed severe edema phenotype and were 100% embryonic lethal around 6 dpf. The non-transgenic sibling from the same mb22Tg(myl7:Lifeact-GFP) transgenic founder were normal. In contrast, transgenic embryos from the mb23Tg(myl7:Lifeact-GFP) founder showed normal cardiac morphology at early stage. However, they developed an edema phenotype around 14 dpf (mb23Tg(myl7:Lifeact-GFP) transgenic larvae died around 25\u201330 dpf. Together, these data suggest that expression of Lifeact-GFP might have an adverse effect on heart development.elopment . They wel stages . All trad 14 dpf . Visibled 14 dpf . All Tg(mb22Tg(myl7:Lifeact-GFP) and mb23Tg(myl7:Lifeact-GFP) transgenic embryos that showed edema phenotypes (mb21Tg(myl7:Lifeact-GFP) line that showed no edema phenotype (mb21Tg(myl7:Lifeact-GFP) transgenic embryos with no edema phenotype had a lower Lifeact-GFP fluorescent intensity in the heart (mb22Tg(myl7:Lifeact-GFP) transgenic embryos with a large pericardial edema showed a strong Lifeact-GFP expression (mb22Tg(myl7:Lifeact-GFP) transgenic embryos that displayed the edema phenotype at a later stage showed moderate levels of Lifeact-GFP fluorescent intensity (mb22Tg(myl7:Lifeact-GFP) transgenic line had higher levels of Lifeact-GFP expression, we compared the GFP fluorescence intensity and quantified the levels of GFP mRNA expression in cardiac muscles of three transgenic lines that showed the sever, moderate and no heart defects and mb22Tg(myl7:Lifeact-GFP) transgenic embryos by direct observation of Lifeact-GFP localization and phalloidin staining. F-actin thin filaments were clearly detected in cardiomyocytes of mb21Tg(myl7:Lifeact-GFP) transgenic embryos that showed no edema phenotype (mb22Tg(myl7:Lifeact-GFP) transgenic embryos displayed a significant disruption of F-actin thin filaments in sarcomeres of cardiomyocytes (mb22Tg(myl7:Lifeact-GFP) transgenic embryos by Lifeact-GFP (mb21Tg(myl7:Lifeact-GFP) and mb22Tg(myl7:Lifeact-GFP) transgenic embryos. The data showed that the number of sarcomeres were dramatically reduced in cardiomyocytes of the mb22Tg(myl7:Lifeact-GFP) transgenic larvae compared with the mb21Tg(myl7:Lifeact-GFP) transgenic line (mb22Tg(myl7:Lifeact-GFP) disrupted F-actin filament organization in cardiomyocytes of zebrafish embryos.To further characterize the effect of Lifeact-GFP overexpression on cardiac muscle differentiation, we compared F-actin thin filament organization in cardiomyocytes of henotype . Staininhenotype . In contmyocytes . Very feeact-GFP or phalleact-GFP . To bettnic line . Togethemb21Tg(myl7:Lifeact-GFP) transgenic line displayed striated Z-line structures (mb22Tg(myl7:Lifeact-GFP) transgenic embryos with the edema phenotype (mb22Tg(myl7:Lifeact-GFP) embryos exhibited irregular F-actin structure and poor Z-line organization. Lifeact-GFP and \u03b1-actinin staining appeared as dispersed punctate within the cardiomyocytes transgenes into zebrafish genome could disrupt expression and function of genes at the integration sites (mb22Tg(myl7:Lifeact-GFP) and mb23Tg(myl7:Lifeact-GFP) transgenic lines that displayed the edema phenotype, we mapped the transgene integration sites by inverse PCR. The data revealed that in the mb22Tg(myl7:Lifeact-GFP) transgenic line, the transgene was inserted into the third intron of the cadherin2 gene (mb23Tg(myl7:Lifeact-GFP) transgenic line, the transgene was integrated into the last exon of traf4a and mb23Tg(myl7:Lifeact-GFP) hemizygous transgenic lines were unlikely caused by the transgene integration in these genes.Integration of on sites . To assein2 gene , which ein2 gene . In the f traf4a , which eormality , indicatmb22Tg(myl7:Lifeact-GFP) transgenic zebrafish embryos was indeed caused by Lifeact-GFP expression, we decided to knock down Lifeact-GFP expression in the transgenic embryos and assess the effect on heart development. A Lifeact-GFP specific antisense morpholino (Lifeact-GFP-MO) was designed that targets the translational start site of Lifeact-GFP (mb22Tg(myl7:Lifeact-GFP) transgenic fish. Compared with the control MO (Con-MO) injected embryos that showed normal Lifeact-GFP expression and a strong edema phenotype transgenic embryos injected with Con-MO and Lifeact-GFP-MO. Compared with the Con-MO injected embryos, the number of sarcomeres was dramatically increased in cardiomyocytes of Lifeact-GFP-MO injected embryos transgenic zebrafish models to study cardiomyopathy in smyd1b mutants and to investigate the effect of Lifeact-GFP overexpression on heart development. We showed that Lifeact-GFP transgenic zebrafish is a useful model for studying contractile dynamics of F-actin filaments in cardiomyocytes in vivo. Using this model, we found that loss of smyd1b resulted in poor actin filament organization in cardiac myocytes. Moreover, our studies also demonstrated that strong Lifeact-GFP expression was detrimental to F-actin filament organization in cardiomyocytes, causing pericardial edema and early embryonic lethality of the transgenic embryos. Collectively, these data indicate that although Lifeact-GFP is a powerful probe for studying actin dynamic in cardiomyocytes in vivo, Lifeact-GFP could alter actin filament arrangement and dynamics in cardiomyocytes. Lifeact-GFP mediated artifacts are concentration-dependent and thus the use of Lifeact requires a closer examination and optimization to minimize the Lifeact-GFP induced adverse effects.In this study, we generated smyd1b is required for actin filament assembly in cardiac myocytes of zebrafish embryos. This finding is consistent with data from previous studies using phalloidin and immunostaining that showed that Smyd1 is required for sarcomere assembly in skeletal and cardiac muscles transgenic zebrafish could be a useful model for future studies of cardiomyopathy from Smyd1 deficiency.Smyd1 function in cardiac muscles is conserved during evolution from fish to human. Recent studies demonstrated that genetic mutations in human myopathy . An infant stage . A peripnt stage . The sarnt stage . In micer system . In zebrtraction . Tg(myl7Tg(myl7:Lifeact-GFP) transgene (Tg(myl7:Lifeact-GFP) transgenic founders could produce healthy and viable transgenic offspring. All these six transgenic lines had lower levels of Lifeact-GFP expression. The other two lines with higher levels of Lifeact-GFP expression developed edema and were early embryonic lethal. The varied levels of gene expression were likely due to the positional effect of transgene integration. We have mapped the integration sites in mb22Tg(myl7:Lifeact-GFP) and mb23Tg(myl7:Lifeact-GFP) to the cadherin2 and traf4a genes, respectively. We do not think the heart phenotype in these lines was caused by disruption of these host genes at the integration sites because the heterozygous mutant of cadherin2 or traf4a in zebrafish showed no edema phenotypes (mb22Tg(myl7:Lifeact-GFP) could be rescued by knockdown of Lifeact-GFP expression.Data from this study showed that overexpression of Lifeact-GFP disrupted actin filament organization in cardiac myocytes, leading to pericardial edema and early larval lethality. This adverse effect was not observed in a previous study using the same ransgene . Reischaenotypes , and morDrosophila, and plant cells transgenic zebrafish is a useful model to study contractile dynamics of F-actin filaments in cardiomyocytes in vivo and to investigate cardiomyopathy from defective thin filament organization. Data from this study also demonstrated that strong Lifeact-GFP expression in cardiomyocytes could alter actin filament arrangement and dynamics in cardiomyocytes causing heart development defect and embryonic lethality. The Lifeact-GFP induced artifacts are concentration-dependent and thus the use of Lifeact requires careful evaluation of the transgenic model to minimize the adverse effects.In summary, our studies show that The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by the Institutional Animal Care and Use Committee of University of Maryland Baltimore.RX and SD conceived and designed the research project and analyzed the data. RX performed the experiments, data collection, and wrote the first draft of the manuscript. SD revised the manuscript. Both authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."} +{"text": "To conduct an individual patient data meta-analysis of randomised controlled trials (RCTs) of manualised psychological treatments for obsessive-compulsive disorder (OCD), and examine the differential efficacy of psychological treatments by treatment type and format.Previous meta-analyses conclude that efficacious psychological treatments for OCD exist. However, determining the efficacy of psychological treatments requires multiple forms of assessment across a range of indexes, yet most previous meta-analyses in OCD are based solely on effect sizes.We evaluated treatment efficacy across 24 RCTs by conducting clinical significance analyses (using standardised Jacobson methodology) and standardised mean difference within-group effect-size analyses. Outcomes were Yale-Brown Obsessive Compulsive Scale (Y-BOCS) scores, evaluated at post-treatment and follow-up (3-6 months post-treatment).Post-treatment, there was a large significant within-group effect size for treated patients (g = 1.28) and a small significant effect size for controls (g = 0.30). At follow-up, large within-group effect sizes were found for both treated patients (g = 1.45) and controls (g = 0.90). Clinical significance analyses indicated that treated patients were significantly more likely than controls to recover following an intervention, but recovery rates were low; post-intervention, only 32% of treated patients and 3% of controls recovered; rising to 38% and 21% respectively at follow-up. Regardless of allocation, only approximately 20% of patients were asymptomatic at follow-up. Across the different analysis methods, individual cognitive therapy (CT) was the most effective intervention, followed by group CT plus exposure and response prevention. Self-help interventions were generally less effective.Reliance on aggregated within-group effect sizes may lead to overestimation of the efficacy of psychological treatments for OCD. More research is needed to determine the most effective treatment type and format for patients with OCD."} +{"text": "Hematologic malignancy patients have high rates of antibiotic exposure, and increasing resistance is a major concern, particularly with extended-spectrum beta lactamases (ESBL) in Enterobacterales blood stream infections (BSIs). Identifying risk factors for ESBL-producing Enterobacterales (ESBL-E) BSIs may facilitate faster appropriate antibiotic use and decrease mortality.Escherichia coli or Klebsiella spp. bacteremia admitted to Carolinas Medical Center in Charlotte, NC from January 2010 through September 2020. The primary objective was to compare 30-day mortality rates for patients with ESBL-E BSIs to those with non-ESBL-E BSIs. Fisher\u2019s exact or Mann-Whitney U tests were used for primary and secondary clinical outcomes as appropriate. Risk factors associated with 30-day mortality and ESBL production were assessed as secondary objectives using logistic regression models.This was a retrospective study of patients with hematologic malignancies and A total of 28 patients with ESBL-E BSIs and 60 patients with non-ESBL-E BSIs were included. The 30-day mortality rate with ESBL-E BSIs was 25% compared to 15% with non-ESBL-E BSIs (P = .373). In-hospital mortality, 30-day infection recurrence, intensive care unit (ICU) admission, and length of stay after culture were not significantly different. However, time to optimal therapy was longer in the ESBL-E group . Multivariate logistic regression analysis showed an association of 30-day mortality with ICU admission and longer time to optimal therapy . Prior ESBL-positive culture was associated with ESBL-E BSI in the univariate logistic regression . Additionally, prolonged neutropenia and prior intravenous antibiotic use were associated with ESBL-E BSI in the multivariate analysis. Significantly longer time to optimal therapy was seen in ESBL-E BSIs and was associated with mortality in patients with hematologic malignancies. The identified ESBL risk factors create an opportunity to decrease delay in optimal therapy through risk stratification during initial antibiotic selection.Ekaterina Kachur, PharmD, BCOP, Bristol Myers Squibb (Advisor or Review Panel member)Genentech (Employee)Glaxosmithkline (Advisor or Review Panel member)Kyowa Kirin (Advisor or Review Panel member)"} +{"text": "Phylogenomic studies over the past two decades have consolidated the major branches of the arthropod tree of life. However, especially within the Chelicerata , interrelationships of the constituent taxa remain controversial. While sea spiders (Pycnogonida) are firmly established as sister group of all other extant representatives (Euchelicerata), euchelicerate phylogeny itself is still contested. One key issue concerns the marine horseshoe crabs (Xiphosura), which recent studies recover either as sister group of terrestrial Arachnida or nested within the latter, with significant impact on postulated terrestrialization scenarios and long-standing paradigms of ancestral chelicerate traits. In potential support of a nested placement, previous neuroanatomical studies highlighted similarities in the visual pathway of xiphosurans and some arachnopulmonates . However, contradictory descriptions of the pycnogonid visual system hamper outgroup comparison and thus character polarization.To advance the understanding of the pycnogonid brain and its sense organs with the aim of elucidating chelicerate visual system evolution, a wide range of families were studied using a combination of micro-computed X-ray tomography, histology, dye tracing, and immunolabeling of tubulin, the neuropil marker synapsin, and several neuroactive substances . Contrary to previous descriptions, the visual system displays a serial layout with only one first-order visual neuropil connected to a bilayered arcuate body by catecholaminergic interneurons. Fluorescent dye tracing reveals a previously reported second visual neuropil as the target of axons from the lateral sense organ instead of the eyes.Ground pattern reconstruction reveals remarkable neuroanatomical stasis in the pycnogonid visual system since the Ordovician or even earlier. Its conserved layout exhibits similarities to the median eye pathway in euchelicerates, especially in xiphosurans, with which pycnogonids share two median eye pairs that differentiate consecutively during development and target one visual neuropil upstream of the arcuate body. Given multiple losses of median and/or lateral eyes in chelicerates, and the tightly linked reduction of visual processing centers, interconnections between median and lateral visual neuropils in xiphosurans and arachnopulmonates are critically discussed, representing a plausible ancestral condition of taxa that have retained both eye types.The online version contains supplementary material available at 10.1186/s12915-021-01212-z. In the last two decades, significant advances of phylogenetic analyses leveraging steadily increasing amounts of transcriptomic and genomic data sets have led to the stabilization of the major branches in the arthropod tree of life , 2. HoweNeuroanatomical studies on Arthropoda have a long history , 14. Alrvs. vs. )globulin .The neuropeptide proctolin (PROC) is expressed in neurons of the central nervous system in different arthropod groups, including chelicerates , 138. InDiploptera punctata-allatostatin I (APSGAQRLYGFGL amide) coupled to bovine thyroglobulin with glutaraldehyde [The polyclonal rabbit antiserum against allatostatin (AST) used in this study was raised against A-type aldehyde . Allatosaldehyde , 140.Endeis spinosa. PFA-fixed samples were used, having been previously shown to be suitable for lipophilic dye tracing in arthropod brains [The lipophilic fluorescent dye DiI was used to trace afferents from the eyes\u2019 R-cells and the sensory cells in the LO of d brains , 142. SpThe nucleic acid marker Hoechst 33342 was applied after all other labeling procedures. Incubation lasted minimally 1\u2009h at RT and was occasionally extended overnight at 4\u2009\u00b0C. After rinsing in PBS, samples were transferred into non-hardening Vectashield\u00ae Mounting Medium and placed on microscopic slides. To avoid squeezing of whole-mount brains, small pieces of Surgident periphery wax were placed under the cover slip corners and gradually compressed under a stereomicroscope. The flexible spacers enabled fine adjustment of the orientation of the whole-mount brains via horizontal movement of the cover slip.Histological sections were documented with a Nikon Eclipse 90i epifluorescence microscope, equipped with a Nikon DS Fi3 camera. XY-tiling and combination into images with extended depth of field was managed in the accompanying NIS Elements AR software .Confocal laser scanning microscopy (CLSM) was performed with a Leica DMI 6000 CS microscope coupled to a Leica TCS SP5 II scan unit (RRID:SCR_018714). Laser lines were chosen according to the excitation spectra of the fluorochromes applied . The Z-increment between optical planes ranged between 0.50 and 1.50\u2009\u03bcm, depending on objective used and required resolution.Analysis and visualization of the CLSM and \u03bcCT image stacks was performed with the software packages Imaris and Amira . Software tools were applied as previously described . Figures were assembled with Adobe Illustrator .Neuroanatomical terminology follows for the most part Richter and colleagues . SpeciesAdditional file 1: Table S1: List of species studied, collection sites and methods applied.Additional file 2: Figure S1: Selected details of the sense organs and protocerebral structures in E. spinosa. Extended optical sections of immunolabeled samples (MIP). A: Tubulin (TUB) labeling, para-sagittal section through the ocular tubercle. A subepidermal network of neurite bundles (arrows point to selected examples) spreads in the ocular tubercle and dorsal cephalon, being connected to the lateral thickening via a postero-lateral nerve (black arrowhead). R-cell axon bundles (white arrowheads) and the optic nerve (star) have been severed during vibratome sectioning. The asterisks mark muscle attachment sites. B: Tyrosine hydroxylase and histamine , para-sagittal section through anterior portion of the brain. TH-ir somata of type 1 interneurons (arrowheads) are found adjacent to the visual neuropil. Note indications for TH-ir neurites in the optic nerve (arrows). C,D: Orcokinin (ORCO), mid-sagittal and oblique horizontal brain sections . C: Orcokinin-ir neurons (arrowheads) send projections through the antero-median tract (arrows) to the lower layer of the arcuate body. Note orcokinin labeling of the antero-median neuropil. D: \u201cNaked\u201d orcokinin-ir axonal projections run through the antero-median tract and form dense synaptic varicosities upon entry (arrow) into the lower arcuate body layer. Note scattered synaptic varicosities (arrowhead) also in the upper arcuate body layer. E: Serotonin (5HT), oblique horizontal brain section. Serotonin-ir axons from the antero-median tract project into the lower arcuate body layer (arrow) and form dense synaptic varicosities. Note also a loose network of synaptic varicosities in the dorso-posterior lobe. Abbreviations: AB \u2013 arcuate body; ABL \u2013 lower arcuate body layer; ABU \u2013 upper arcuate body layer; AE \u2013 anterior eye; AMN \u2013 antero-median neuropil; AMT \u2013 antero-median tract; DPL \u2013 dorso-posterior lobe; LO \u2013 lateral sense organ; PE \u2013 posterior eye; RTA \u2013 R-cell axons; SVD \u2013 sub-visual domain, VN \u2013 visual neuropil.Additional file 3: Movie S1: Serotonin and tyrosine hydroxylase expression in the anterior protocerebral region of Endeis spinosa. Tyrosine hydroxylase (magenta) and serotonin (green) immunolabeling with nuclear counterstain (gray), 3D volume of anterior protocerebral region (blend mode). The brain is shown in anterior view (in terms of neuraxis) and rotates counterclockwise to show the spatial arrangements of the visual neuropil, arcuate body (upper and lower layer), dorso-posterior lobe and antero-median tract. Note that the arcuate body is antero-dorsally covered by the soma cortex (see start of the movie). Abbreviations used in the movie: ABL \u2013 lower arcuate body layer; ABU \u2013 upper arcuate body layer; AMT \u2013 antero-median tract; DPL \u2013 dorso-posterior lobe; VN \u2013 visual neuropil.Additional file 4: Movie S2: Serotonin expression in the anterior protocerebral region of Endeis spinosa. Serotonin immunolabeling, 3D volume of anterior protocerebral region (blend mode). The brain is shown in anterior view (in terms of neuraxis) and rotates clockwise to show the spatial arrangement of the lower arcuate body layer, antero-median tract and the group of antero-dorsal neurons that project past the antero-median tract into the central brain neuropil. Note the ventral neurons with projections that loop into the antero-median tract and contribute to the dense synaptic varicosities in the lower arcuate body layer. Abbreviations used in the movie: AB \u2013 arcuate body; ABL \u2013 lower arcuate body layer; AMT \u2013 antero-median tract; DPL \u2013 dorso-posterior lobe.Additional file 5: Movie S3: Tyrosine hydroxylase expression in the anterior protocerebral region of Endeis spinosa. Tyrosine hydroxylase immunolabeling, 3D volume of anterior protocerebral region (blend mode). The brain is shown in anterior view (in terms of neuraxis) and rotates clockwise to show the spatial arrangement of the TH-ir projections and synaptic varicosities in the visual neuropil and upper arcuate body. The somata of anterior type 1 and antero-ventral type 2 neurons are highlighted as the brain rotates. Note also medial projections from the sub-visual domain into the central brain neuropil. Abbreviations used in the movie: ABU \u2013 upper arcuate body layer; VN \u2013 visual neuropil.Additional file 6: Figure S2: Tyrosine hydroxylase (TH) expression in the anterior protocerebrum of various pycnogonid families. Horizontal sections of immunolabeled samples (MIP). The left image column shows extended sections that include the visual neuropil, sub-visual domain and arcuate body. In the central image column, major parts of the apical visual neuropil have been removed by a clipping plane. The right image column depicts the apical visual neuropil only. White arrows mark selected type 2 neurons, white arrowheads indicate type 1 neurons. In some samples , some neurites in the optic nerve are visible (black arrowheads). Black arrows indicate weakly or strongly labeled projections from the sub-visual domain into the underlying central brain neuropil. Light blue arrowheads point to somata of dorsal neurons extending projections into more posterior brain regions (not shown), sometimes looping anteriorly around the narrow median portion of the arcuate body . A:Pycnogonum litorale (Pycnogonidae). Note weak TH signal in the visual neuropil and the presumptive type 1 neurons. Small white arrows highlight small TH-ir somata in the lateral thickening. B:Anoplodactylus australis (Phoxichilidiidae). C:Endeis spinosa (Endeidae). D:Pallenopsis sp. . Note weak TH signal in the visual neuropil and the presumptive type 1 neurons. E:Austropallene cornigera . F:Nymphon tenuipes (Nymphonidae). Abbreviations: AB \u2013 arcuate body; SVD \u2013 sub-visual domain, VN \u2013 visual neuropil.Additional file 7: Figure S3: Protocerebral sense organs and their brain connections in Phoxichilidium femoratum (Phoxichilidiidae). Extended optical sections and 3D-curved optical sections (B-D) of immunolabeled samples (MIP). Yellow arrowheads point to the optic nerve branch extending to the antero-median neuropil. A: Tubulin and synapsin with nuclear counterstain and autofluorescence , para-sagittal section. The tubulin-labeled lateral sense organ and its nerve (arrow) to the lateral thickening as well as the proximal portion of the nerve (black arrowhead) connecting to the subepidermal neurite network of the ocular tubercle have been segmented and highlighted in different colors . B,C: Tubulin (green) and synapsin (magenta), detail of the optic nerve as it enters the brain, para-sagittal section. In the optic nerve, the neurites from the lateral sense organ and the axons targeting the visual neuropil remain separated as they enter the soma cortex. D: Tubulin and orcokinin with nuclear counterstain and autofluorescence (gray), para-sagittal section. A subset of orcokinin-ir lateral sense organ cells (white arrows) appears to project orcokinin-ir projections (white arrowheads) through the lateral sense organ nerve toward the brain. Note absence of orcokinin labeling in the visual neuropil. E: Tubulin (magenta) and serotonin with nuclear counterstain , cross section through anterior brain region. Note additional branch from the optic nerve that passes the antero-median neuropil (red arrowhead). The antero-median tract shows strong serotonin signal and also the antero-median neuropil is weakly labeled. Abbreviations: AE \u2013 anterior eye; AEL \u2013 anterior eye lens; AMN \u2013 antero-median neuropil; AMT \u2013 antero-median tract; BRN \u2013 brain neuropil; LO \u2013 lateral sense organ; LONV \u2013 lateral sense organ nerve; LT \u2013 lateral thickening; PE \u2013 posterior eye; PEL \u2013 posterior eye lens; RTA \u2013 R-cell axons; VN \u2013 visual neuropil.Additional file 8: Figure S4: The eyes and lateral sense organ in all pycnogonid families. Volume renderings of the ocular tubercle, \u03bcCT scans in lateral view. If the eye lenses or lateral sense organs where not clearly discernible by external inspection alone, their presence was additionally confirmed by the study of the internal anatomy in the scans. A:Austrodecus glaciale (Austrodecidae). B:Rhynchothorax australis (Rhynchothoracidae). C:Pycnogonum litorale (Pycnogonidae). Note absence of the lateral sense organ. D:Colossendeis angusta (Colossendeidae). Note absence of the eyes. E:Endeis spinosa (Endeidae). F:Phoxichilidium femoratum (Phoxichilidiidae). G:Pallenopsis cf. aulaeturcarum . H:Achelia echinata (Ammotheidae). I:Ammothea longipes (Ammotheidae). J:Tanystylum orbiculare (Ammotheidae). K:Ascorhynchus ramipes (Ascorhynchidae). Note upward shift of the posterior eye and the unusual position of the lateral sense organ below the latter. L:Nymphon gracile (Nymphonidae). M:Callipallene tiberii . N:Pallenella sp. . O:Parapallene avida . P:Stylopallene cheilorhynchus . Note external subdivision of the eye lenses. Abbreviations: AEL \u2013 anterior eye lens; LO \u2013 lateral sense organ; PEL \u2013 posterior eye lens.Additional file 9: Figure S5: Histamine expression in eyes and first-order visual neuropil of Ammotheidae and Phoxichilidiidae. Extended optical sections of immunolabeled samples (MIP). A,C,E: Tubulin and histamine with nuclear counterstain and autofluorescence , horizontal sections through the ocular tubercle. Note absence of histamine labeling in the lateral sense organ. B,D,F: Histamine (green) with nuclear counterstain , horizontal sections through the anterior protocerebral region. Note absence of histamine labeling in the median area housing the antero-median neuropil. A,B:Achelia echinata (Ammotheidae). C,D:Tanystylum orbiculare (Ammotheidae). E,F:Phoxichilidium femoratum (Phoxichilidiidae). Abbreviations: AE \u2013 anterior eye; LO \u2013 lateral sense organ; LONV \u2013 lateral sense organ nerve; LT \u2013 lateral thickening; PE \u2013 posterior eye; RTA \u2013 R-cell axons; VN \u2013 visual neuropil.Additional file 10: Figure S6: The first-order visual neuropil in various pycnogonid families. Tubulin and synapsin immunolabeling with nuclear counterstain , optical cross sections through the anterior protocerebral region. A:Austrodecus glaciale (Austrodecidae). B:Rhynchothorax australis (Rhynchothoracidae). C:Pycnogonum litorale (Pycnogonidae). D:Endeis spinosa (Endeidae). E:Phoxichilidium femoratum (Phoxichilidiidae). F:Pallenopsis sp. . G:Achelia echinata (Ammotheidae). H:Callipallene brevirostris . I:Nymphon gracile (Nymphonidae). Abbreviations: BRN \u2013 brain neuropil; VN \u2013 visual neuropil.Additional file 11: Figure S7: Selected details of protocerebral structures in Ammotheidae and Nymphonidae. Optical sections of immunolabeled samples (MIP). A:Achelia echinata, tyrosine hydroxylase with nuclear counterstain , cross section through anterior protocerebral region. TH-ir somata of type 1 interneurons (arrowheads) are located next to the TH-ir visual neuropil. Note also TH labeling in the sub-visual domain underlying the visual neuropil. B:Nymphon cf. multituberculatum, TH and serotonin , extended horizontal section through anterior protocerebral region. Note TH and serotonin co-labeling in the visual neuropil. C:Nymphon gracile, serotonin, horizontal section through the antero-median tract and arcuate body. Serotonin-ir axons of ventral neurons project through the antero-median tract, bifurcate upon entry into the lower arcuate body layer (arrows) and form dense synaptic varicosities in its lateral arms. Note fine (and in part columnar) collaterals extending into the upper arcuate body layer (arrowheads). Abbreviations: ABL \u2013 lower arcuate body layer; ABU \u2013 upper arcuate body layer; AMT \u2013 antero-median tract; BRN \u2013 brain neuropil; SVD \u2013 sub-visual domain; VN \u2013 visual neuropil.Additional file 12: Figure S8: The antero-median neuropil and antero-median tract in various pycnogonid families. A-C: Tubulin and synapsin immunolabeling with nuclear counterstain , optical cross sections through the anterior protocerebral region. Yellow arrowheads indicate the branch of the optic nerve looping toward the antero-median neuropil. A:Achelia echinata (Ammotheidae). B:Pallenopsis sp. . C:Nymphon gracile (Nymphonidae). Note that the antero-median tract is not readily discernible in cross section, due to its rather diffuse, neuropil-rich nature in Nymphonidae immunolabeling, extended optical sections through the anterior protocerebral region (MIP). White arrows point to orcokinin-ir somata of anterior neurons that contribute neurites to the antero-median neuropil. Yellow arrowheads mark orcokinin-ir projections in the optic nerve branch extending to the antero-median neuropil. D:Tanystylum orbiculare (Ammotheidae). Note absence of distinct labeling in the antero-median tract. E:Phoxichilidium femoratum (Phoxichilidiidae). Note relatively weak labeling in the antero-median neuropil. Black arrowheads mark orcokinin-ir somata in the lateral thickening. F:Callipallene brevirostris . Note distinct orcokinin-ir projections (yellow arrowheads) from the optic nerve to the antero-median neuropil and strong signal in the antero-median neuropil as well as the antero-median tract. Abbreviations: AMN \u2013 antero-median neuropil; AMT \u2013 antero-median tract; BRN \u2013 brain neuropil; LT \u2013 lateral thickening.Additional file 13: Figure S9: Serotonin expression in antero-median tract and arcuate body of various pycnogonid families. Serotonin (5HT) immunolabeling, extended horizontal sections through the brain (MIP). Arrowheads point to selected somata of ventral neurons that send projections along the antero-median tract into the arcuate body. A:Anoplodactylus pygmaeus (Phoxichilidiidae). B:Phoxichilidium femoratum (Phoxichilidiidae). C:Pallenopsis sp. . Note relatively diffuse, neuropil-rich nature of the antero-median tract. D:Tanystylum orbiculare (Ammotheidae). E:Achelia echinata (Ammotheidae). F:Ammothella biunguiculata (Ammotheidae). Note relatively diffuse, neuropil-rich nature of the antero-median tract. G:Ascorhynchus auchenicus (Ascorhynchidae). H:Callipallene brevirostris . I:Austrodecus glaciale (Austrodecidae). Due to long-term PFA storage of the available material, the signal is extremely weak but still points to the presence of the serotonin-ir antero-median tract and lower arcuate body layer. Abbreviations: AB \u2013 arcuate body; AMT \u2013 antero-median tract; DPL \u2013 dorso-posterior lobe.Additional file 14: Table S2: Overview of protocerebral sense organs and brain structures identified across pycnogonid families, with details on immunoreactivity for neuroactive substances."} +{"text": "Scientific Reports 10.1038/s41598-022-07211-6, published online 28 March 2022Correction to: The Acknowledgements section in the original version of this Article was incomplete.\u201cWe acknowledge the access to data from PCAWG Consortium which provided SVs data. We thank the patients and their families for their participation in the ICGC and TCGA projects. Among others, this study has been supported by projects: SAF2017-89450-R (TransTumVar) and PID2020-119797RB-100 (BenchSV) from Science and Innovation Spanish Ministry. It has also been supported by the Spanish Government (contract PID2019-107255GB), Generalitat de Catalunya (contract 2014-SGR-1051) and Universitat Polit\u00e8cnica de Catalunya (45-FPIUPC2018).\u201dnow reads:\u201cWe acknowledge the access to data from PCAWG Consortium which provided SVs data. We thank the patients and their families for their participation in the ICGC and TCGA projects. Among others, this study has been supported by projects: BenchSV (PID2020-119797RB-I00/AEI/10.13039/501100011033) from Science and Innovation Spanish Ministry and SAF2017-89450-R (TransTumVar). It has also been supported by the Spanish Government (PID2019-107255GB-C21/AEI/10.13039/501100011033), Generalitat de Catalunya (contract 2014-SGR-1051) and Universitat Polit\u00e8cnica de Catalunya (45-FPIUPC2018).\u201dThe original Article has been corrected."} +{"text": "Follow-up of low-risk monoclonal gammopathy of undetermined significance (MGUS) is debated as multiple myeloma (MM) progression risk is low. Worse MM outcome was reported for patients followed for low-risk MGUS, possibly due to less optimal follow-up. However, it is unknown whether progressing low-risk MGUS is associated with aggressive tumor behavior. Understanding these patterns is crucial for MGUS management. Here, we investigated whether progression from low-risk MGUS is associated with worse MM outcome in patients who had no MGUS follow-up before myeloma diagnosis. We retrospectively determined the MGUS status in repeated pre-diagnostic blood samples prospectively collected from 42 myeloma patients in median 11.6\u00a0years (first sample) and 3.3\u00a0years (repeated sample) before myeloma diagnosis. At first pre-diagnostic blood draw, 12 had low-risk and 30 had MGUS of other risk. MM bone disease was more common in patients with low-risk MGUS at first blood draw . Median survival since myeloma diagnosis was worse in low-risk than other MGUS at first blood draw . Modest progression was observed between first and repeated blood draw for the majority of low-risk MGUS as 67% remained as low- or low-intermediate-risk MGUS at repeated blood draw. Our study, albeit limited by its small size, indicates that progression from low-risk MGUS is associated with worse MM outcome regardless of MGUS follow-up. Although further investigation is needed, progressing low-risk MGUS could belong to a group of aggressive tumors with progression that is difficult to predict.The online version contains supplementary material available at 10.1186/s40164-022-00259-0. Multiple myeloma (MM) is preceded by monoclonal gammopathy of undetermined significance (MGUS) , 2. GuidThe Ume\u00e5 University review board approved this study using samples from the Northern Sweden Health and Disease Study, a large prospective cohort. Linkage to the Swedish Cancer Registry facilitated identification of myeloma patients with a first and repeated pre-diagnostic blood sample before myeloma diagnosis. We could study natural progression patterns in relation to MM outcome because 42 had detectable MGUS (protein and immunofixation electrophoresis and free light-chain assays) in both pre-diagnostic samples without MGUS follow-up before myeloma diagnosis. Kaplan\u2013Meier plots and multivariable Cox regression were used to study overall survival (Table 58 (TableWe compared MM progression trajectories in patients who had low-risk vs. other MGUS (restricted to IgG isotype for better comparison) at first pre-diagnostic blood draw. At repeated pre-diagnostic blood draw, progression to smoldering multiple myeloma (M spike\u2009\u2265\u200930\u00a0g/L) was observed in 8% 1 of 12) in low-risk vs. 20% (3 of 15) in other MGUS (P\u2009=\u20090.605). More patients with low-risk MGUS at first pre-diagnostic blood draw had lower MGUS risk (low- or low-intermediate-risk) at repeated blood draw compared to other MGUS . This was pronounced excluding four patients who did not progress to symptomatic MM . These observations could indicate a more rapid progression process in low-risk MGUS closer to MM initiation. Investigating this, we plotted M spikes in both groups. M spike trajectories were visually largely similar in both groups with some patients experiencing rapid clonal evolution as indicated by fast increasing paraprotein levels at first blood draw."} +{"text": "Accumulating evidence pinpoints sex differences in stroke incidence, etiology and outcome. Therefore, more understanding of the sex-specific mechanisms that lead to ischemic stroke and aggravation of secondary damage after stroke is needed. Our current mechanistic understanding of cerebral ischemia states that endothelial quiescence in neurovascular units (NVUs) is a major physiological parameter affecting the cellular response to neuron, astrocyte and vascular smooth muscle cell (VSMC) injury. Although a hallmark of the response to injury in these cells is transcriptional activation, noncoding RNAs such as microRNAs exhibit cell-type and context dependent regulation of gene expression at the post-transcriptional level. This review assesses whether sex-specific microRNA expression (either derived from X-chromosome loci following incomplete X-chromosome inactivation or regulated by estrogen in their biogenesis) in these cells controls NVU quiescence, and as such, could differentiate stroke pathophysiology in women compared to men. Their adverse expression was found to decrease tight junction affinity in endothelial cells and activate VSMC proliferation, while their regulation of paracrine astrocyte signaling was shown to neutralize sex-specific apoptotic pathways in neurons. As such, these microRNAs have cell type-specific functions in astrocytes and vascular cells which act on one another, thereby affecting the cell viability of neurons. Furthermore, these microRNAs display actual and potential clinical implications as diagnostic and prognostic biomarkers in ischemic stroke and in predicting therapeutic response to antiplatelet therapy. In conclusion, this review improves the current mechanistic understanding of the molecular mechanisms leading to ischemic stroke in women and highlights the clinical promise of sex-specific microRNAs as novel diagnostic biomarkers for (silent) ischemic stroke. Accumulating evidence suggests that there are sex differences in stroke, in terms of incidence and clinical presentation, etiology, and outcome. Although young women (25\u201344 year age groups) have a higher stroke incidence than young men , this trend is reversed in older age groups, with a higher acute stroke incidence ratio in men . MechaniA deeper mechanistic understanding of ischemic brain injury is becoming available with the concept of dysfunctional cell communication in neurovascular units (NVUs). This functional unit consists of neurons, astrocytes, endothelial cells (ECs), pericytes and vascular smooth muscle cells (VSMCs), and controls, among other processes, neurovascular coupling, thereby regulating cerebral blood flow (CBF) A 13]. N. N13]. NIncreasing evidence suggests that the cellular response to ischemic injury is largely regulated at the post-transcriptional level, involving microRNAs which reThis narrative review describes the sex-specific expression of microRNAs in NVUs and their actual and potential clinical application. We first provide an overview of the mechanisms of sex-specific microRNA expression. Second, we demonstrate that these microRNAs function as a post-transcriptional regulatory network of a sex-specific cellular response to injury in cerebral ECs, VSMCs and neurons. This either promotes or prevents dysfunctional cell\u2013cell communication in NVUs leading to ischemic stroke. Lastly, we describe how these microRNAs could function as a diagnostic and prognostic biomarker in stroke, and aid in predicting therapeutic response to antiplatelet therapy.X-chromosome gene dosage disequilibrium is prevented by the neutralization of one X-chromosome by random X-chromosome inactivation (XCI) during embryonic development . XCI is Collectively, these studies indicate that the incomplete silencing of X-chromosomes in NVU cells can promote elevated X-linked microRNA expression in NVUs C, therebn = 30). These miR-30b levels increased 2-fold in cultured glioblastoma cells exposed to estrogen and were present in higher levels in female mouse brain compared to male mouse brain [Experimental studies in noncerebral cells have demonstrated that estrogen receptor (ER) binding to estrogen response elements (EREs) in gene promotors, drives microRNA expression thereby regulating multiple downstream target genes . Furtherse brain . Howeverse brain . In humase brain . This cose brain . Whetherse brain .Taken together, these studies on estrogen-regulated miR biogenesis were predominantly carried out in MC7- and glioblastoma cell cultures, and thus, provide no definite answers to the question of whether estrogen regulates each step in microRNA biogenesis B. HoweveA striking pathophysiological feature of ischemic stroke is BBB disruption . The higFollowing local ischemia, altered cell-specific metabolomic adaptations perturb BBB function and integrity and immu+ T cell activation and as such the acute inflammatory response in women [n\u2009 =\u2009 24, 8\u201310 weeks old), a phenotype induced by elevated miR-106a expression [Various in vitro cell models have addressed the role of microRNA expression in BBB integrity and function, for instance by investigating TNF-\u03b1 stimulation of cultured murine brain vascular endothelial (bEnd3) cells. In these cells, this experimental setting significantly increased the expression of the X-linked miR-501-3p . This miin women . More suin women . Also, mpression .+ T cells which partly controls the immune response at the post-transcriptional level.In contrast to these detrimental effects on BBB integrity via increased expression of X-chromosome located microRNAs, their increase may also exhibit NVU protective effects, as was observed for the X-linked miR-98. The expression of this miR is reduced in OGD cultured primary brain microvascular endothelial cells (BMVECs) and in mice following transient MCAO which was found to disrupt BBB integrity. By restoring its expression in experimental studies, EC barrier function improved, BBB breakdown was rescued and diminished infiltration of brain leukocyte influx and neuroinflammation in mice was seen . InteresCollectively, these studies suggest that differential expression of X-linked microRNAs alters endothelial tight junction expression and thus BBB integrity in response to ischemic injury in stroke-like conditions. The fact that both the X-linked miR-424 and miR-501 regulate the expression of ZO-1 protein, and thereby, EC tight junction function, pinpoints the synergistic function of microRNAs (regulating the same gene) in BBB integrity. As such, this simultaneous repression of ZO-1 protein directly influences the output of functionally related biological pathways (BBB integrity) and thusVSMCs play a key role in vessel wall remodeling in response to cerebral ischemic injury and neurodegenerative disease. Following ischemia and EC activation, VSMCs switch from a contractile phenotype, that normally coordinates vascular vasodilation and constriction, to a synthetic phenotype that proliferates and migrates , and insn = 110) compared to control patients (n = 84) [n = 103) compared to healthy controls (n = 77), inversely related to carotid intima-media thickness (CIMT) and displayed a high sensitivity and specificity for atherosclerosis (AUC 0.897) [A striking example of X-linked microRNA expression in VSMC activation is miR-362-3p of which plasma levels were significantly decreased in atherosclerotic patients (ontrols) . Subsequontrols) . This suC 0.897) . However\u2212/\u2212 C56BL/6J mice (n = 5) fed a high-fat diet. In these experimental settings, the X-linked miR-188 decreased in VSMCs from mice fed a high-fat diet compared to controls. As a consequence, the expression of its gene target, fibroblast growth factor 1 (Fgf1) was elevated [n = 62) compared to healthy controls (n = 60) [In cerebral VSMC calcification, another pathological event in atherosclerosis development, several X-linked microRNAs are involved, while the subsequent differentiation of these cells into osteoblast- and chondrocyte-like cells following microRNA expression changes can occur as well. Studies in VSMCs from aged rats have demonstrated that aged VSMCs have decreased miR-542-3p expression compared to VSMCs from younger rats . Howeverelevated . Functioelevated . Further(n = 60) . This pa(n = 60) . Moreove(n = 60) . The res(n = 60) .In summary, these studies demonstrate that following carotid artery atherosclerosis, the expression of particular X-linked microRNAs is decreased and promotes VSMC proliferation. Although gene expression results in these studies were not stratified for women and men, these findings could provide a novel molecular mechanism for sex-specific atherosclerotic phenotypes given that extracranial atherosclerosis is less often present in women compared to men .2+ influx promotes nitric oxide synthase (nNOS) and calpain I release, thereby increasing cellular reactive oxygen species (ROS) [Cellular hypoxia and the loss of glucose and ATP supply following stroke, promote Na+/K+ pump failure, depolarization of neurons and glutamate release. Following an ensuing activation of glutamate receptors like the N-methyl-D-aspartate (NMDA) receptors, cellular excitation due to increased Caes (ROS) . This in2+ influx, cellular oxidative stress and apoptosis). Particular the X-linked miR-223 was found to be elevated in neurons from rats at different time intervals after ischemia (10-fold in cortex neurons after 48 h and 20\u201330 fold in the striatum 24 h) while its target gene Nckx2, a member 2 of the K+-dependent Na+/Ca2+ exchanger family, was decreased [2+ ions in neurons, X-linked microRNAs also control neuronal apoptosis by regulating cellular oxidative stress and mitochondrial respiration. One study into neuronal mitochondrial hemodynamics after stroke demonstrated that neurons isolated from a rat cerebral ischemia/reperfusion (CI/R) injury model display increased expression of the cerebral NADPH oxidase 2 (Nox2), which promotes ROS production in neurons. This phenomenon coincided with less expression of miR-652, suggesting that this microRNA protects against ischemic events, a conclusion emphasized by systemic injection of miR-652 agomirs, which improved cellular apoptosis and infarction in rats [Interestingly microRNAs derived from the X-chromosome were found to play a significant role in regulating multiple sets of functionally related genes that modulate the neuronal effects induced by cellular hypoxia and ischemia reduced cell viability, leading to apoptosis of neurons (as determined by MTT assays and TUNEL staining of cells) [In vitro stroke models of the aforementioned mechanisms of neuronal cellular stress (like cell culture of neurons in OGD conditions) have demonstrated marked upregulation of the X-linked miRs involved in ischemic cell death (caspase) pathways. A clear example is the elevated expression of the X-linked miR-188-5p in OGD-cultured neurons, the expression of which coincided with lower cell proliferation over time and higher expression of apoptosis markers caspase-3 and caspase-8 . OGD simf cells) . More nef cells) . This prf cells) .In conclusion, these studies indicate that the neuronal response to OGD regulates cellular ion balance and proliferation (miR-223), and activates caspase-mediated cell-death pathways (regulated by miR-188-5p) and apoptosis of neurons (miR-502 and miR-764). Because the majority of these studies were performed in mice and rats, additional studies are needed to investigate whether these miRs have a conserved role in NVU homeostasis in humans as well, as was demonstrated for miR-188-5p, of which the altered expression in humans contributes to Alzheimer disease (AD) pathogenesis . CellulaIn contrast to the aforementioned detrimental effects of X-chromosome located microRNAs, some X-linked microRNAs prevent cellular apoptosis of neurons across distinct neuronal cellular pathways, such as cellular excitation and mitochondrial respiration. Overstimulation of the glutamate receptor (glutamate excitotoxicity) is such a mechanism in neuronal cell death during stroke, because it results in abnormally high intracellular calcium concentrations. In neurons, N-methyl-D-aspartate (NMDA) receptors regulate this calcium influx, which requires membrane depolarization induced by sodium influx through 2-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPAR) receptors. MiR-223 prevents NMDA-induced calcium influx, thereby protecting neurons against NMDA induced excitotoxicity and cellular apoptosis . The genMore beneficial effects on neuronal apoptosis were seen in the MCAO model in C57/BL6 mice, which had elevated levels of the X-linked miR-424 at 1 and 4 h in the peri-infarct cortex but decreased levels after 24 h . This paCollectively, these studies illustrate that miR-98, -223 and -424 are involved in the regulation of cellular excitation and oxidative stress in neurons following cellular ischemia. These autocrine functions may counteract the detrimental caspase-mediated cell-death pathways as described for miR-188, -223, -502 and -764 were involved. The fact that miR-223 has both roles underscores the likelihood that most miRs function pleiotropically, depending on cellular contexts and functional networks .p < 0.05) and subsequent experiments determined that this microRNA therefore exerts neuroprotective functions, because expressed infarct volumes were higher in female rats following an antagomiR-363 injection. Interestingly, the intravenous infusion of this microRNA preserved microvascular density of female mice compared to controls and reduced the expression of caspase-3, an executioner caspase protein involved in apoptotic pathways [2O2- induced cell death via the increased expression of miR-223. Mechanistically, miR-223 lowered the level of another microRNA namely miR-27b, thereby suppressing the expression of IKK\u03b1, an upstream kinase for NF-kB. Experimentally induced overexpression of miR-223 in C6 astrocytes and primary cultured astrocytes lowered miR-27b levels by suppressing the expression of IKK\u03b1, suggesting that miR-223 increased Sdf-1 expression by downregulating miR-27b [Astrocyte end feet cover cerebral capillaries in order to increase BBB integrity . Moreovepathways . Anotherpathways . This supathways . This el miR-27b .In conclusion, these astrocyte-specific X-linked microRNAs exert neuroprotective functions. Given that the inhibition of miR-363 in female mice increased infarct volumes after MCAO, this suggests that sex-specific microRNA expression in astrocytes determines cerebral cellular fate in women with stroke. The fact that the coordination of caspase mediated cell death of neurons starts via X-chromosome-located miRs could increase the likelihood that this regulatory control will develop differently between men and women.Brain astrocytes express the estrogen receptor-\u03b1 (ER-\u03b1) and -\u03b2 (ER-\u03b2), thereby enabling estrogen to influence astrocyte to neuron signaling . One exap-value 0.028) and in cerebral spinal fluid [antagomir alone provided a significant reduction of infarct volume, i.e., 24%, in female mice only [Also, within the CNS microRNAs are influenced by estrogen levels. For instance in human studies, miR-126 correlated with both estradiol levels in serum . Howeverice only . Interesice only . Next toice only . Interesice only . Estrogeice only .Collectively, estrogen-regulated microRNAs in astrocytes counteract sex-specific apoptotic pathways in neurons, thereby regulating infarct volume. In this way, estrogen renders women less susceptible to ischemic cell death pathways. This pinpoints the consequences of alterations in sex hormone levels which could affect stroke incidence and outcome in young women and postmenopausal women. The study by Leppert et al. demonstrClinical studies have demonstrated that sex-specific microRNAs could provide a valuable biomarker potential for the diagnosis and progression of ischemic stroke. Particularly the X-chromosome origin of plasma microRNAs may result in a sex-specific diagnostic biomarker for stroke in women. Although many studies identified the involvement of these circulating X-linked microRNAs in ischemic stroke, a stratification of circulating microRNA levels for women is oftentimes not implemented within these studies in contrast to studies into sex-specific microRNAs in female-specific cardiovascular disease pathophysiology . Therefop = 0.039). Its levels were significantly increased in patients with moderate-severe stroke (n = 65) compared to control patients, but not in minor stroke (n = 67) compared to control patients [n = 22), elevated plasma levels of the X-linked miR-505 differentiated stroke patients from controls (n = 22) and its levels normalized following stroke treatment [n = 46) [n = 167) compared to healthy controls (n = 157) which demonstrated that stroke risk increased by 10-fold following the decrease of this microRNA [One of the sex-specific circulating microRNAs involved in stroke pathophysiology is the X-linked miR-503. MiR-503 levels were positively associated with acute ischemic stroke diagnosis . Subsequ[n = 46) . These r[n = 46) . SignifimicroRNA .n = 79) compared to healthy controls (n = 75). These levels displayed a negative correlation with NIHSS scores and infarct volume but a positive correlation to plasma IGF-1 [n = 22), the X-linked miR-224-3p and miR-532-5p decreased following ischemic stroke [n = 45), plasma levels of the X-linked miR-424 were decreased and displayed a negative correlation with inflammatory cytokines like IL-6, IL-4, and TNF-\u03b1 serum levels compared to controls (n = 45) [n = 40) within 6 h of symptom onset, elevated miR-424 in lymphocytes and neutrophils associated with ischemic stroke. The neutrophil-specific expression levels in this study displayed a negative correlation with infarct volume, while lymphocyte and neutrophil miR-424 levels were both inversely correlated with plasma TNF-\u03b1, IL-10, or IGF-1 levels [Elevated X-linked miR-223 levels were also seen in ischemic stroke patients (ma IGF-1 . Becausema IGF-1 their sec stroke . Gene onc stroke . In a si(n = 45) . Similar1 levels . This su1 levels . Interes1 levels while a 1 levels . This st1 levels .In both the acute and chronic phase of stroke, antiplatelet therapy (aspirin and clopidogrel) is currently the first-line treatment to prevent recurrent stroke , albeit It has become clear that post-transcriptional networks in NVUs drive the cellular transcriptome and its response to injury . As such"} +{"text": "The models converged on stable fits that accurately reproduced observations from meta-analyses of Streptococcus pneumoniae datasets. The estimates of invasiveness, the progression rate from carriage to invasive disease, in cases per carrier per year correlated strongly with the dimensionless values from meta-analysis of odds ratios when sample sizes were large. At smaller sample sizes, the Bayesian models produced more informative estimates. This identified historically rare but high-risk S. pneumoniae serotypes that could be problematic following vaccine-associated disruption of the bacterial population. The package allows for hypothesis testing through model comparisons with Bayes factors. Application to datasets in which strain and serotype information were available for S. pneumoniae found significant evidence for within-strain and within-serotype variation in invasiveness. The heterogeneous geographical distribution of these genotypes is therefore likely to contribute to differences in the impact of vaccination in between locations. Hence genomic surveillance of opportunistic pathogens is crucial for quantifying the effectiveness of public health interventions, and enabling ongoing meta-analyses that can identify new, highly invasive variants.The disease burden attributable to opportunistic pathogens depends on their prevalence in asymptomatic colonisation and the rate at which they progress to cause symptomatic disease. Increases in infections caused by commensals can result from the emergence of \u201chyperinvasive\u201d strains. Such pathogens can be identified through quantifying progression rates using matched samples of typed microbes from disease cases and healthy carriers. This study describes Bayesian models for analysing such datasets, implemented in an RStan package ( Opportunistic pathogens are microbes that are commonly carried by healthy hosts, but can occasionally cause severe disease. The progression rate quantifies the risk of such a pathogen transitioning from a harmless commensal to causing a symptomatic infection. The incidence of infections caused by opportunistic pathogens can rise with the emergence of \u201chyperinvasive\u201d strains, which have high progression rates. Therefore methods for calculating progression rates of different pathogen strains using surveillance data are crucial for rapidly identifying emerging infectious disease threats. Existing methods typically measure progression rates relative to the overall mix of microbes in the population, but these populations can vary substantially between locations and times, making the outputs challenging to combine across studies. This work presents a new method for estimating progression rates from surveillance data that generates values useful for modelling pathogen populations, even from relatively small sample sizes. Neisseria meningitidis . Th. Thk stareliable . Hence mreliable . The Bayreliable .This study-adjusted type-specific negative binomial model was therefore applied to the full dataset. Both the MCMC traces and erations . This enerations , includi\u22122 IPD cases per carrier per year, with the minimum point estimates below 10\u22124 IPD cases per carrier per year. However, the incidence of IPD in the Navajo population is high, with excellent disease surveillance ,749V-3 [9V-3 ,37 identWhether these models are useful long-term, or will ultimately be superseded by alternatives based on genome-wide association studies, will depend on the extent to which progression rates are affected by interactions between genetic loci . It may Even using more conventional approaches to estimating invasiveness, whole genome sequencing clearly represents an important tool in future case-carrier studies of opportunistic pathogen invasiveness. In addition to assigning isolates to strains, genomic data is a reliable and cost-effective means of classifying microbes according to their capsular structures ,97. FurtSome of these problems with detecting colonisation can be addressed using new techniques with improved sensitivity for detecting multiple serotypes in carriage . HoweverS1 Text(DOCX)Click here for additional data file.S2 Text(DOCX)Click here for additional data file.S1 Fig(A) Stacked bar plot showing the distribution of carriage and disease isolates between studies. (B) Stacked bar plot showing the distribution of carriage and disease isolates between serotypes.(PNG)Click here for additional data file.S2 FigEach panel shows the fit of eight different models, indicated by colour, to data simulated from a single model structure. Points represent the median estimate, and the error bars show the 95% credible intervals. The models from which data were simulated were (A) null Poisson, (B) null negative binomial, (C) type-specific Poisson, (D) type-specific negative binomial, (E) study-adjusted Poisson, (F) study-adjusted negative binomial, (G) study-adjusted type-specific Poisson, and (H) study-adjusted type-specific negative binomial. For panels (A), (B), (E) and (F), all types had the same progression rate, and therefore the dashed horizontal line represents the single true value used in the simulations. For panels (B), (C), (G) and (H), each of the 20 types had a different progression rate, which determines the horizontal position of the point on the graph. In these four plots, the dashed line indicates the line of identity.(PNG)Click here for additional data file.S3 FigThe horizontal position of the point corresponds to the model that was both used to generate the data, and to then infer the parameter value from these data. The vertical position of the point indicates the median estimate of the parameter, and the error bars represent the 95% credibility interval. The horizontal dashed line indicates the true value of the parameter used to simulate data, \u03d5 = 0.1.(PNG)Click here for additional data file.S4 Fig(A) Stacked bar plot showing the distribution of carriage and disease isolates between studies. (B) Stacked bar plot showing the distribution of carriage and disease isolates between serotypes.(PNG)Click here for additional data file.S5 FigThe horizontal axis shows the generation of the MCMC, with values for the two chains shown by orange and purple lines. Each panel corresponds to a different model: (A) null Poisson model; (B) null negative binomial model; (C) type-specific Poisson model; (D) type-specific negative binomial model; (E) study-adjusted Poisson model; (F) study-adjusted negative binomial model; (G) study-adjusted type-specific Poisson model; (H) study-adjusted type-specific negative binomial model.(PNG)Click here for additional data file.S6 FigEach panel corresponds to a different model: (A) null Poisson model; (B) null negative binomial model; (C) type-specific Poisson model; (D) type-specific negative binomial model; (E) study-adjusted Poisson model; (F) study-adjusted negative binomial model; (G) study-adjusted type-specific Poisson model; (H) study-adjusted type-specific negative binomial model.(PNG)Click here for additional data file.S7 FigBlue crosses represent the individual observations.(PNG)Click here for additional data file.S8 FigThe points represent the median estimates from the MCMCs, and the error bars show the 95% credibility intervals.(PNG)Click here for additional data file.S9 Fig(A) Histogram showing the distribution of (PNG)Click here for additional data file.S10 FigThe reference study, for which the value was fixed at one, was the Navajo post-PCV7 dataset, which had the greatest sample size in this meta-analysis .(PNG)Click here for additional data file.S11 Fig(A) Histogram showing the distribution of (PNG)Click here for additional data file.S12 FigThe reference study, for which the value was fixed at one, was the Navajo post-PCV7 dataset, which had the greatest sample size in this meta-analysis .(PNG)Click here for additional data file.S13 FigThe carriage duration estimates were derived from a multi-variate lasso regression that included both serotype and antibiotic resistance phenotypes. Values were available for 14 serotypes, all relative to the carriage duration of serotype 6A/C, which was assigned a value of zero days.(PNG)Click here for additional data file.S14 Fig(A) Stacked bar plot showing the distribution of carriage and disease isolates between studies. (B) Stacked bar plot showing the distribution of carriage and disease isolates between serotypes.(PNG)Click here for additional data file.S15 Fig(A) Stacked bar plot showing the distribution of carriage and disease isolates between studies. (B) Stacked bar plot showing the distribution of carriage and disease isolates between serotypes.(PNG)Click here for additional data file.S16 FigThe horizontal axis shows the generation of the MCMC, with values for the two chains shown by orange and purple lines. Each panel corresponds to a different model: (A) null Poisson model; (B) null negative binomial model; (C) type-specific Poisson model; (D) type-specific negative binomial model; (E) study-adjusted Poisson model; (F) study-adjusted negative binomial model; (G) study-adjusted type-specific Poisson model; (H) study-adjusted type-specific negative binomial model.(PNG)Click here for additional data file.S17 FigEach panel corresponds to a different model: (A) null Poisson model; (B) null negative binomial model; (C) type-specific Poisson model; (D) type-specific negative binomial model; (E) study-adjusted Poisson model; (F) study-adjusted negative binomial model; (G) study-adjusted type-specific Poisson model; (H) study-adjusted type-specific negative binomial model.(PNG)Click here for additional data file.S18 Figci,j). The plots in the right column display data on disease in adults . The points are coloured by the study to which they correspond, and represent the observed value on the horizontal axis, and the median predicted value on the vertical axis. The error bars show the 95% credibility intervals. The red dashed line shows the line of identity, corresponding to a perfect match between prediction and observation. Each row corresponds to a different model: (A) null Poisson model; (B) null negative binomial model; (C) type-specific Poisson model; (D) type-specific negative binomial model; (E) study-adjusted Poisson model; (F) study-adjusted negative binomial model; (G) study-adjusted type-specific Poisson model; (H) study-adjusted type-specific negative binomial model.The plots in the left column display data on carriage in children Click here for additional data file.S19 Fig(A) Histogram showing the distribution of (PNG)Click here for additional data file.S20 FigThe reference study, for which the value was fixed at one, was the Navajo post-PCV7 dataset, which had the greatest sample size in this meta-analysis .(PNG)Click here for additional data file.S21 Fig(A) Stacked bar plot showing the distribution of carriage and disease isolates between studies. (B) Stacked bar plot showing the distribution of carriage and disease isolates between serotypes.(PNG)Click here for additional data file.S22 Fig(A) Stacked bar plot showing the distribution of carriage and disease isolates between studies. (B) Stacked bar plot showing the distribution of carriage and disease isolates between serotypes.(PNG)Click here for additional data file.S23 FigEach panel corresponds to a model with a different method of associating isolates with an invasiveness estimate: (A) serotype-determined, Poisson-distributed invasiveness; (B) serotype-determined, negative binomially-distributed invasiveness; (C) strain-determined, Poisson-distributed invasiveness; (D) strain-determined, negative binomially-distributed invasiveness; (E) serotype-determined, strain-modified Poisson-distributed invasiveness; (F) serotype-determined, strain-modified negative binomially-distributed invasiveness; (G) strain-determined, serotype-modified Poisson-distributed invasiveness; (H) strain-determined, serotype-modified negative binomially-distributed invasiveness; (I) strain- and serotype-determined Poisson-distributed invasiveness; (J) strain- and serotype-determined negative binomially-distributed invasiveness.(PNG)Click here for additional data file.S24 FigEach panel corresponds to a different model: (A) serotype-determined, Poisson-distributed invasiveness; (B) serotype-determined, negative binomially-distributed invasiveness; (C) strain-determined, Poisson-distributed invasiveness; (D) strain-determined, negative binomially-distributed invasiveness; (E) serotype-determined, strain-modified Poisson-distributed invasiveness; (F) serotype-determined, strain-modified negative binomially-distributed invasiveness; (G) strain-determined, serotype-modified Poisson-distributed invasiveness; (H) strain-determined, serotype-modified negative binomially-distributed invasiveness; (I) strain- and serotype-determined Poisson-distributed invasiveness; (J) strain- and serotype-determined negative binomially-distributed invasiveness.(PNG)Click here for additional data file.S25 Figci,j,k), and the right column shows the correspondence for disease isolates . Each row corresponds to a different model: (A) serotype-determined, Poisson-distributed invasiveness; (B) serotype-determined, negative binomially-distributed invasiveness; (C) strain-determined, Poisson-distributed invasiveness; (D) strain-determined, negative binomially-distributed invasiveness; (E) serotype-determined, strain-modified Poisson-distributed invasiveness; (F) serotype-determined, strain-modified negative binomially-distributed invasiveness; (G) strain-determined, serotype-modified Poisson-distributed invasiveness; (H) strain-determined, serotype-modified negative binomially-distributed invasiveness; (I) strain- and serotype-determined Poisson-distributed invasiveness; (J) strain- and serotype-determined negative binomially-distributed invasiveness.The points are coloured by the study to which they correspond, and represent the observed value on the horizontal axis, and the median predicted values on the vertical axis. The error bars show the 95% credibility intervals. The red dashed line shows the line of identity, corresponding to a perfect match between prediction and observation. The left column shows the correspondence for carriage data Click here for additional data file.S26 FigThe points represent the median estimates from the MCMCs, and the error bars show the 95% credibility interval.(PNG)Click here for additional data file.S27 FigBlue crosses represent the individual observations.(PNG)Click here for additional data file.S28 FigThe reference study, for which the value was fixed at one, was the South Africa post-PCV7 dataset, which had the greatest sample size in this meta-analysis, and was associated with a known carriage sample size .(PNG)Click here for additional data file.S29 Fig(A) Histogram showing the distribution of (PNG)Click here for additional data file.S30 FigThe shape of each point represents the number of isolates of each combination observed across carriage and disease samples. The serotype-strain combinations with the highest combined invasiveness that are not targeted by current PCV designs are labelled.(PNG)Click here for additional data file.S31 Fig(PNG)Click here for additional data file.S32 Fig(A) Histogram showing the distribution of (PNG)Click here for additional data file.S33 FigPoints represent the median estimates, and are coloured by the vaccine formulations in which the corresponding serotype is present. The error bars represent 95% credibility intervals. The shape of the point represents the sample size on which the estimate is based. (A) Estimates of the invasiveness associated with serotypes, arranged by the strains in which they are found. Only strains expressing multiple serotypes are displayed. (B) Estimates of the coefficient by which strains modify the invasiveness of their expressed serotype, arranged by the serotypes with which they are associated. Only serotypes associated with multiple strains are displayed.(PNG)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 TableThese values were generated using the logarithm of the likelihoods calculated within the models. Models are ranked by their expected log pointwise predictive density (ELPD), calculated from the individual pointwise log predictive densities across all observed data points. The ELPD difference column shows the difference between the ELPD of a model and that of the most likely model . The ELPD difference standard error is calculated from the distribution of individual pointwise log predictive densities from the same comparison.(DOCX)Click here for additional data file.S3 TableThese values were generated using the logarithm of the likelihoods calculated for the observations of isolates from disease only, as these were more constrained than the modelling of isolate counts from carriage. The table is displayed as described for Table S2.(DOCX)Click here for additional data file.S4 TableEach row corresponds to data simulated from the specified model. All eight models were fitted to each set of simulated data. The table shows the models adjudged to be the first and second best-fitting to each dataset, using bridge sampling. The final column shows the logarithm of the Bayes factor by which the best-fitting model was favoured over the second best-fitting model.(DOCX)Click here for additional data file.S5 TableModels are ranked by their logarithmic marginal likelihoods. The logarithmic Bayes factors were calculated for each model relative to that which was found to be the most likely given the data; hence the value is zero for the first row.(DOCX)Click here for additional data file.S6 TableThe table is displayed as described for Table S5.(DOCX)Click here for additional data file.S7 TableThe disease isolates from Portugal came from a mixture of infants and adults, but are tabulated based on them primarily arising from the latter age category.(DOCX)Click here for additional data file.S8 TableThese values were generated using the logarithm of the likelihoods calculated for the observations of isolates from carriage and disease. The table is displayed as described for Table S2.(DOCX)Click here for additional data file.S9 TableThese values were generated using the logarithm of the likelihoods calculated for the observations of isolates from disease only. The table is displayed as described for Table S2.(DOCX)Click here for additional data file.S10 TableThe table is displayed as described for Table S5.(DOCX)Click here for additional data file.S11 TableIn this analysis, the carriage sample size for three studies was increased 100-fold, to test the sensitivity of the model comparisons to the uncertainty in this parameter (Text S2). The table is displayed as described for Table S5.(DOCX)Click here for additional data file.S12 TableThe exclusion of strain and serotype combinations represented by fewer than ten isolates, summed across carriage and disease, meant that some within-serotype differences between strains were not tested in this analysis.(DOCX)Click here for additional data file.S1 DatasetThe median estimates, and lower and upper bounds of the 95% credibility interval, are listed for each serotype.(XLSX)Click here for additional data file.S2 DatasetThe median estimates, and lower and upper bounds of the 95% credibility interval, are listed for each serotype.(XLSX)Click here for additional data file.S3 DatasetEach row corresponds to a serotype-strain combination. Both the invasiveness associated with the serotype, and the invasiveness coefficient associated with the strain, are listed, along with the corresponding lower and upper bounds of their 95% credibility intervals.(XLSX)Click here for additional data file."} +{"text": "Staphylococcus aureus bloodstream infections (BSIs) in patients with febrile neutropenia (FN) is associated with a mortality rate of up to 49%. For documented infections in patients with FN, guidelines recommend narrowing therapy once susceptibilities result and fever has resolved. Although anti-staphylococcal beta-lactams are the mainstay of treatment for Methicillin-Susceptible and Penicillin-Susceptible Staphylococcus aureus (MSSA and PSSA) BSIs, some clinicians opt to continue broad antibiotics against Pseudomonas during FN. Studies evaluating treatment modalities and outcomes of MSSA and PSSA BSI in patients with FN are lacking.We conducted a retrospective cohort study of adult patients with MSSA or PSSA BSI who received antibiotics for the treatment of FN (absolute neutrophil count < 500 cells/L and temperature > 100.4F) at Brigham and Women\u2019s Hospital and Dana-Farber Cancer Institute from 1/2010 to 4/2021. Patients who received < 72-h of antibiotics were excluded. The primary outcome was composite clinical failure . Other outcomes included inpatient mortality, 60-day readmission, 60-day infection outcomes, incidence of acute kidney injury and hepatotoxicity. Data was analyzed using Chi-Square test or Fisher\u2019s Exact test.Among 108 patients who met our criteria, 58% were male, median age was 57 years , 94% had a hematologic malignancy, 4% had a solid tumor, and 2% had both. A total of 41 (38%) received combination therapy with broad spectrum and anti-staphylococcal beta-lactam, 48 (44%) received broad spectrum beta-lactam followed by anti-staphylococcal beta-lactam after neutrophil recovery, and 19 (18%) were narrowed to an anti-staphylococcal beta-lactam prior to resolution of neutropenia. Clinical failure was similar across all treatment arms (Table).Table. OutcomesDe-escalation to an anti-staphylococcal beta-lactam prior to neutrophil recovery in FN patients with MSSA or PSSA BSIs did not result in significantly higher clinical failures. Further prospective studies are needed to support antimicrobial stewardship initiatives in oncology patients.All Authors: No reported disclosures"} +{"text": "Caenorhabditiselegans UNC-62 homothorax/Meis/TALE homeodomain protein functions sequentially to regulate general identity of the AWC olfactory neuron pair and the stochastic choice of asymmetric AWC subtypes during embryogenesis. Here we analyze the expression pattern of unc-62 during AWC development using an integrated unc-62::GFP fosmid rescuing transgene. UNC-62::GFP was not detected in AWC neurons in early or late embryos. These results are consistent with previous single-cell RNA sequencing data and also suggest an undetectable level of unc-62 expression and/or low stability of UNC-62 protein in AWC neurons during embryogenesis.The An integrated unc-62::GFP fosmid transgene, in which all UNC-62 protein isoforms are tagged with GFP at the C- terminus (unc-62(lf) mutant phenotypes of AWC general identity, determined by odr-1p::DsRed expression, and AWC asymmetry, determined by str-2p::GFP expression . These results suggest that UNC-62::GFP fusion protein expressed from the unc-62::GFP fosmid transgene is functional for AWC development. It has been shown that this integrated unc-62::GFP fosmid transgene is expressed in sensory neurons, touch neurons, interneurons, ventral nerve cord motor neurons, and head motor neurons, but it is not expressed in AWC in late-stage larvae or young-stage adult worms using the multicolor transgene NeuroPAL .The UNC-62 homeodomain protein regulates AWC general identity and subsequently plays a cell autonomous role, determined by mosaic analysis, in AWC asymmetry during embryogenesis , rescuedet al., 1983; Chuang and Bargmann, 2005). To determine whether unc-62 is expressed in AWC neurons at the embryonic stages of AWC development, the expression pattern of the integrated unc-62::GFP fosmid transgene . Together, these results suggest that unc-62 may be expressed at an undetectable level and/or UNC-62 protein may have a very short half-life in embryonic AWC neurons.The AWC neurons are born near the end of gastrulation; AWC asymmetry is established around the 1.5-fold and 3-fold embryonic stage , was ana embryos -D. Consi"} +{"text": "Background: Patients developing acute kidney injury (AKI) during critical illness or major surgery are at risk for renal sequelae such as costly and invasive acute renal replacement therapy (RRT) and chronic dialysis (CD). Rates of renal injury may be reduced with use of chloride-restrictive intravenous (IV) resuscitation fluids instead of chloride-liberal fluids.Objectives: To compare the cost-effectiveness of chloride-restrictive versus chloride-liberal crystalloid fluids used during fluid resuscitation or for the maintenance of hydration among patients hospitalized in the US for critical illnesses or major surgery.Methods: Clinical outcomes and costs for a simulated patient cohort (starting age 60 years) receiving either chloride-restrictive or chloride-liberal crystalloids were estimated using a decision tree for the first 90-day period after IV fluid initiation followed by a Markov model over the remainder of the cohort lifespan. Outcomes modeled in the decision tree were AKI development, recovery from AKI, progression to acute RRT, progression to CD, and death. Health states included in the Markov model were dialysis free without prior AKI, dialysis-free following AKI, CD, and death. Estimates of clinical parameters were taken from a recent meta-analysis, other published studies, and the US Renal Data System. Direct healthcare costs (in 2015 USD) were included for IV fluids, RRT, and CD. US-normalized health-state utilities were used to calculate quality-adjusted life years (QALYs).Results: In the cohort of 100 patients, AKI was predicted to develop in the first 90 days in 36 patients receiving chloride-liberal crystalloids versus 22 receiving chloride-restrictive crystalloids. Higher costs of chloride-restrictive crystalloids were offset by savings from avoided renal adverse events. Chloride-liberal crystalloids were dominant over chloride-restrictive crystalloids, gaining 93.5 life-years and 81.4 QALYs while saving $298 576 over the cohort lifespan. One-way sensitivity analyses indicated results were most sensitive to the relative risk for AKI development and relatively insensitive to fluid cost. In probabilistic sensitivity analyses with 1000 iterations, chloride-restrictive crystalloids were dominant in 94.7% of iterations, with incremental cost-effectiveness ratios below $50 000/QALY in 99.6%.Conclusions: This analysis predicts improved patient survival and fewer renal complications with chloriderestrictive IV fluids, yielding net savings versus chloride-liberal fluids. Results require confirmation in adequately powered head-to-head randomized trials."} +{"text": "Antigen tests for SARS-CoV-2 offer advantages over nucleic acid amplification tests , including lower cost and rapid return of results, but show reduced sensitivity. Public health organizations recommend different strategies for utilizing NAATs and antigen tests. We sought to create a framework for the quantitative comparison of these recommended strategies based on their expected performance.We utilized a decision analysis approach to simulate the expected outcomes of six testing algorithms analogous to strategies recommended by public health organizations. Each algorithm was simulated 50,000 times in a population of 100,000 persons seeking testing. Primary outcomes were number of missed cases, number of false-positive diagnoses, and total test volumes. Outcome medians and 95% uncertainty ranges (URs) were reported.Algorithms that use NAATs to confirm all negative antigen results minimized missed cases but required high NAAT capacity: 92,200 tests at 10% prevalence. Selective use of NAATs to confirm antigen results when discordant with symptom status resulted in the most efficient use of NAATs, with 25 NAATs (95% UR: 13-57) needed to detect one additional case compared to exclusive use of antigen tests.No single SARS-CoV-2 testing algorithm is likely to be optimal across settings with different levels of prevalence and for all programmatic priorities. This analysis provides a framework for selecting setting-specific strategies to achieve acceptable balances and trade-offs between programmatic priorities and resource constraints.The online version contains supplementary material available at 10.1186/s12889-021-12489-8. The COVID-19 pandemic, caused by the SARS-CoV-2 virus, continues to cause significant morbidity, mortality, and economic hardship worldwide. Diagnostic testing is a cornerstone of COVID-19 response strategies in the U.S. and globally . HoweverDespite the advantages of lower costs and faster turnaround time, antigen tests are generally less sensitive than NAATs for diagnosis of COVID-19, particularly for persons without COVID-19 symptoms . In manyWe evaluated outcomes of a modeled population of 100,000 persons seeking community-based SARS-CoV-2 testing in settings of 5%, 10%, 15%, and 20% prevalence of SARS-CoV-2 infection. Prevalence levels can vary substantially over time and geographically and thes(A) NAAT Only \u2013 each person is tested for SARS-CoV-2 infection by a NAAT. (B) Antigen (Ag) Only \u2013 each person is tested using a single antigen test, the result of which is used as a definitive diagnosis. This algorithm represents settings with access to point-of-care antigen tests, but no access to NAAT. (C) NAAT Confirmation for Symptomatic Antigen-Negative (Sx/Ag-neg) and Asymptomatic Antigen-Positive (Asx/Ag-pos) Results \u2013 each person receives an antigen test and NAAT is used to confirm diagnoses in persons for whom antigen results do not match binary symptom status . (D) NAAT Confirmation of Negative Antigen Results (Ag-neg) \u2013 each person receives an antigen test and NAAT is used to confirm negative antigen test results. (E) Repeat Antigen Confirmation of (Ag-neg) \u2013 each person receives an antigen test and, for those with initial negative results, a repeat antigen test is used to confirm negative diagnoses. (F) NAAT for Asymptomatic Persons (Asx) & Symptomatic Persons with Positive Antigen Results (Sx/Ag-pos) \u2013 asymptomatic persons receive a NAAT; symptomatic persons receive an antigen test followed by a NAAT for those with positive antigen results.We evaluated six diagnostic algorithms which were adapted from current recommendations for SARS-CoV-2 antigen testing in various settings. These algorithms are illustrated in Fig.\u00a0Parameters from empirical studies used for model simulations are summarized in Table\u00a0https://github.com/CDCgov/SARS-CoV-2-NAAT-and-Antigen-Testing-Algorithms).Parameters were sampled from triangular distributions , false positive diagnoses , and numbers of antigen tests and NAATs performed per 100,000 persons evaluated. Secondary outcomes (including person-time of lost productivity) and sensitivity analyses are available in the(A) NAAT Only algorithm] the incremental number of missed cases and saved NAATs [how many fewer NAATs were needed]under each algorithm. These incremental measures, calculated as a quotient representing the number of NAATs saved for each additional missed case compared to the (A) NAAT Only algorithm, provide an indication of the number of NAATs saved under different algorithms and the consequent trade-off of additional missed cases.To characterize the potential consequences of adopting different testing algorithms in settings of varying NAAT capacity, we calculated and 2280 missed cases [(E) Repeat Ag for Ag-neg 95% UR: 1507-3067], respectively. Algorithms in which NAATs were performed prior to all definitive negative diagnoses [(A) NAAT Only and (D) NAAT Confirmation of Ag-neg], resulted in zero missed cases (due to assumed 100% sensitivity of NAATs). The remaining algorithms in which some but not all negative antigen results are confirmed by NAAT [(C) NAAT Confirmation for Sx/Ag-pos & Asx/Ag-neg and (F) NAAT Confirmation for Asx & Sx/Ag-pos], resulted in intermediate numbers of missed cases. At 10% prevalence, these algorithms result in 1409 missed cases (95% UR: 815-2100) and 1389 missed cases (95% UR: 622-2280), respectively.Primary outcomes for each algorithm are presented in Fig. (B) Ag Only, (D) NAAT Confirmation for Ag-neg, and (E) Repeat Ag for Ag-neg. The first two of these algorithms resulted in identical numbers of false positive diagnoses as both consider initial positive antigen results as definitive, while (E) Repeat Ag for Ag-neg resulted in higher numbers due to false positive diagnoses following the repeat antigen test. Algorithms where NAATs were performed prior to all definitive positive diagnoses [(A) NAAT Only and (F) NAAT Confirmation for Asx & Sx/Ag-pos], resulted in zero false positive diagnoses (assumed 100% specificity of NAATs). The remaining algorithm [(C) NAATConfirmation for Sx/Ag-pos & Asx/Ag-neg], where some but not all positive antigen results are confirmed by NAAT, resulted in low numbers of false positive diagnoses .False positive diagnoses were greatest in algorithms in which positive antigen results were not confirmed by NAATs\u2014(A) NAAT Onlyand (B) Ag Only algorithms, at 100,000 NAAT or antigen tests, respectively. Antigen testing also remained constant at 100,000 testsfor (C) NAAT Confirmation for Sx/Ag-neg & Asx/Ag-posand (D)NAAT Confirmation for Ag-neg algorithms. Antigen testing volume was highest for the (E) Repeat Ag for Ag-neg algorithm and varied depending on the number of initial negative antigen results and total volume ranged from a median of 185,100 tests at 20% prevalence to a median of 195,700 tests at 5% prevalence. Among algorithms using antigen testing, antigen testing volume was lowest for (F) NAATConfirmation for Asx & Sx/Ag-pos and varied depending on the prevalence of symptoms among persons evaluated, ranging from a median of 35,500 tests at 5% prevalence to a median of 40,700 tests at 20%prevalence . Among algorithms using NAATs, NAAT testing volume was lowest for (C) NAAT Confirmation for Sx/Ag-neg & Asx/Ag-pos: at 10% prevalence, a median of 34,100 NAATs were used . NAAT testing volume was higher for the (F) NAAT Confirmation for Asx & Sx/Ag-pos and the (D)NAAT Confirmation for Ag-neg: at 10% prevalence, a median of 68,300 NAATs and 92,200 NAATs were used, respectively.Total testing volume remained constant for (A) NAAT Onlyalgorithm are depicted in Figure (A) NAAT Only)at a level of 10% prevalence. The quotient of these measures is defined as the ratio of NAATs saved per additional missed case in Fig. (D) NAAT Confirmation for Ag-negalgorithm had a ratio of positive infinity,resulting from zero additional missed cases . The (C) NAAT Confirmation for Sx/Ag-neg & Asx/Ag-posalgorithm had the most favorable ratio among remaining algorithms: at 10% prevalence, a median of 46 NAATs were saved per additional missed case (95% UR: 29-83) compared to (A) NAAT Only.Incremental outcomes of simulations under algorithms compared to corresponding simulations under the (B) Ag Onlyalgorithm are depicted in Fig. (E) Repeat Ag for Ag-neg algorithm (which uses zero NAATs) had a ratio of zero additional NAATs needed per additional case. Among the remaining algorithms, the (C) NAAT Confirmation for Sx/Ag-neg & Asx/Ag-posalgorithm had the most favorable ratio: at 10% prevalence, a median of 25 NAATs were needed to detect each additional case (95% UR: 13-57) compared to (B) Ag Only. For both incremental outcomes, the order of algorithm favorability remained constant across prevalence levels; however, the absolute differences between algorithms shrank as prevalence increased. A summary and synthesis of algorithms to achieve a key programmatic priority, balancing missed cases and NAAT volume, is presented in Table Incremental outcomes compared to the 95% UR: 1-57 compa(A) NAAT Only and (D) NAAT Confirmation for Ag-neg algorithms maximize the use of NAATs to confirm negative antigen results and yielded the smallest numbers of missed cases. However, use of confirmatory NAATs for negative antigen results also incurred a need for high NAAT capacity. A strategy that selectively confirms negative antigen results with NAAT was found to be the most efficient use of limited NAATs [(C) NAAT Confirmation for Sx/Ag-neg & Asx/Ag-pos]. When uninfected people are erroneously diagnosed with SARS-CoV-2 infection , consequent isolation orders and case investigations result in lost productivity, unnecessary use of limited public health resources, and, when resulting in co-isolation with true cases, puts them at risk for ongoing exposure. Therefore, algorithms which maximize NAATs to confirm positive antigen results yielded the smallest numbers of false-positive diagnoses [(A) NAAT Only and (F) NAAT for Asx & Sx/Ag-pos]. NAATs are often more costly to perform than antigen tests and may require logistical arrangements for timely off-site transport and testing. Strategies which minimize the use of NAATs [(B) Ag Only and (E) Repeat Ag for Ag-neg] offer benefits for resource-limited testing programs. Each of these algorithms may be advisable depending on the programmatic goals and resource limitations of community-based SARS-CoV-2 testing programsIn this analysis, we utilized a decision analysis approach to provide a quantitative comparison of different strategies for the use of antigen tests and NAATs in SARS-CoV-2 testing programs. The six algorithms evaluated reflect differing priorities testing in populations based on resources, SARS-CoV-2 prevalence, and tolerance for missed cases and false positives. Multiple reports have found that antigen tests are less sensitive than NAATs \u201318 and w(A) NAAT Only, (C) NAAT Confirmation for Sx/Ag-neg & Asx/Ag-pos, and (D) NAAT Confirmation for Ag-neg are most preferable; selecting between these algorithms depends on tolerance for missed cases and available NAAT capacity. For programs intended to minimize NAAT volume, algorithms (B) Ag Only, (C) NAAT Confirmation for Sx/Ag-neg & Asx/Ag-pos, and (E) Repeat Ag for Ag-neg are most preferable; selecting between these algorithms depends on tolerance for missed cases and available NAAT and antigen test capacity. Predictive values NAAT Only, an idealized baseline]. Each strategy recommended is articulated with important nuances; algorithms analyzed here are intended to be analogous to, but not exact reproductions of these strategies. Guidance from WHO and ECDC distinguishes strategies for antigen testing in communities with low and high prevalence of SARS-CoV-2 infection. In high prevalence settings, WHO recommends considering repeat antigen testing for those with negative results [(E) Repeat Ag for Ag-neg; ECDC indicates that negative tests should be confirmed with RT-PCR [(D) NAAT Confirmation for Ag-neg. In low prevalence settings following negative antigen results, WHO recommends clinical evaluation for suspect cases in lieu of confirmatory NAATs [(B) Ag Only; ECDC does not recommend antigen testing for asymptomatic persons and recommends confirmatory RT-PCR for symptomatic persons with positive antigen results [(F) NAAT for Asx & Sx/Ag-pos. CDC interim guidance recommends a unified strategy for testing across settings analogous to (C) NAAT Confirmation for Sx/Ag-neg & Asx/Ag-pos [Each algorithm evaluated in this analysis is rooted in strategies currently recommended by public health organizations [except for in test performance, such as patient age or sex. . This aThe results of our analysis are dependent on the accuracy and generalizability of the input parameter estimates used. Several reports have described the performance characteristics of several antigen tests, with comparable results across reports \u201318. ProgOur results provide the first quantitative comparison of the expected performance of different strategies for community-based SARS-CoV-2 testing programs recommended by public health organizations. None of the algorithms evaluated in this analysis is likely to be optimal in all settings and for all programmatic priorities, and this analysis provides a framework for selecting setting-specific strategies to achieve an acceptable balance and trade-offs between programmatic priorities and constraints. As global responses to the COVID-19 pandemic continue to evolve and adapt, our results contribute to the body of evidence informing SARS-CoV-2 testing strategies.Additional file 1."} +{"text": "ABSTRACT IMPACT: Understanding the women community leaders\u2019 sense of relational and financial empowerment in the social entrepreneurship context will be key to developing a sustainable pathway to scale-up community-based HPV self-sampling programs in low resource settings. OBJECTIVES/GOALS: The Hope Project, a social entrepreneurship (SE) near Lima, Peru, trains women leaders (Hope Ladies) to promote human papillomavirus (HPV) self-sampling in their communities. This study aims to evaluate the Hope Ladies\u2019 own relational/financial empowerment after participating in the program. METHODS/STUDY POPULATION: The Hope Ladies participated in semi-structured in-depth interviews (n= 9) and 8-question 5-point Likert-scale survey (n=16) that evaluated their relational/financial empowerment after participating in the social entrepreneurship. The interview and the survey questions were developed using validated empowerment frameworks, indicators, and theory, respectively: 1) Kabeer\u2019s conceptual framework, 2) International Center for Research on Women (ICRW), and 3) Relational Leadership Theory (RLT). Direct content analysis was used to deductively evaluate the interviews with predetermined codes and categories of empowerment. Descriptive statistics were used to analyze the survey results. RESULTS/ANTICIPATED RESULTS: All reported experiencing empowerment in the SE. Interviews: The codes were mapped onto 3 categories/9 sub-categories: 1) voicing confidence ; 2) social resources ; 3) financial gains . Survey: 75% indicated an increase in social contacts, confidence in discussing reproductive topics (75%), comfort with medical facilities (44%), ability to help the community (62.5%), and ability to make household purchasing decisions (36%) since joining the program. DISCUSSION/SIGNIFICANCE OF FINDINGS: The Hope Ladies\u2019 experience in this SE demonstrated the complex relationship between various domains of empowerment . More studies are needed to elucidate the relationship between empowerment and worker retention/performance to inform scale-up of HPV self-sampling SE\u2019s."} +{"text": "Correction to: Trials 22, 780 (2021)https://doi.org/10.1186/s13063-021-05755-yFollowing the publication of the original article , we were\u201cFor example, Brookes and Butler 2021 inaccurately claim that \u2018the Danish mask study showed the overall effects from mask wearing and social distancing were modest.\u201dOriginal:\u201cFor example, Oraby et al., 2021 inaccurately claim that \u2018the Danish mask study showed the overall effects from mask wearing and social distancing were modest.\u201d [citation: 10.1038/s41598-021-82873-2]Correction:The original article has been corrected."} +{"text": "However, its role in regulation of biliary cholesterol secretion and gallstone formation remains unknown. Hence, mice with conventional knockout (KO), hepatocyte-specific knockout (\u0394Hepa) / knockdown (KD) or gain expression of miRNA-223 were included in the study and were subjected to lithogenic diet (LD) for various weeks. The gall bladders and liver tissues were harvested for cholesterol crystal imaging, gallstone mass measurement and molecular analysis. Levels of cholesterol, bile salt, phospholipids, and triglyceride were determined in serum, liver tissues, and bile by enzyme color reactive assays. A 3' UTR reporter gene assay was used to verify the direct target genes for miRNA-223. LD-induced gallstone formation was remarkably accelerated in miRNA-223 KO, \u0394Hepa, and KD mice with concurrent enhancement in total cholesterol levels in liver tissues and bile. Key biliary cholesterol transporters ABCG5 and ABCG8 were identified as direct targets of miRNA-223. Reversely, AAV-mediated hepatocyte-specific miRNA-223 overexpression prevented gallstone progression with reduced targets expression. Therefore, the present study demonstrates a novel role of miRNA-223 in the gallstone formation by targeting ABCG5 and ABCG8 and elevating miRNA-223 would be a potentially novel approach to overcome the sternness of cholesterol gallstone disease. Abcg5 and/or Abcg8 KO mice ABCG5/8 loci mutations are closely associated with gallstone diseases Abcg5/8 KO mice, LD-induced gallstone formation was greatly attenuated Scarb1 in mice, has also been identified for efficient conduction of biliary cholesterol secretion in physical condition Abcg5 KO mice Scarb1 KO or Scarb1/Abcg 5 double KO mice exhibited reduced biliary cholesterol secretion Gallstone is a disease of the digestive system with high incidence and approximately 10-20% prevalence in developed countries phox pathway and protects the liver from alcoholic injury et al.i.e. attenuating hepatocyte cholesterol uptake from blood by targeting SR-BI expression, suppressing cholesterols synthesis via targeting HMGCS1 and SC4MOL, and promoting cholesterol efflux by indirectly upregulating basolateral ABCA1 expression in hepatocytes. Given that, we further wonder whether or not miRNA-223 also influences biliary cholesterol secretion pathway as well as consequential gallstone formation.The miRNAs play an important role in modulating pathophysiological processes in a fine-tuning manner by suppressing their target mRNA translation. In the liver, miRNAs are reported to actively contribute to metabolic homeostasis of glucose The purpose of the present study is to determine the significance of hepatocyte miRNA-223 in gallstone pathogenesis with a special focus on its role in regulating the expression of key transporters in the biliary cholesterol secretion pathway.Tg and Lyz2-CreTg mouse lines provided by Biomodel Organism Science & Technology Development Co., Ltd.miRNA-223 KO and conditional KO mice (cKO) were generated in C57BL/6J line by Shanghai Biomodel Organism Science & Technology Development Co., Ltd. Conventional, hepatocyte-specific, and myeloid-specific miRNA-223 KO mice were achieved by crossbreeding miRNA-223 cKO with Alb-Cre11 virus genome) or AAV8-TBG-GFP was injected intravenously via tail vein into miRNA-223 cKO mice. For hepatocyte-specific overexpression of mouse miRNA-223 precursor sequence, wild-type (WT) mice were received a one-time injection of AAV8-U6-miRNA-223 precursor/CMV-GFP or AAV8-CMV-GFP (each 1\u00d71011 virus genome) via the tail vein.Adeno-Associated Viruses (AAVs) used in this study were purchased from Hanbio Biotechnology Co., Ltd. For hepatocyte-specific knockdown of miRNA-223 expression, AAV8-TBG-Flag-Cre-T2A-GFP or chow (LAD0011) purchased from TROPHIC Animal Feed High Tech Co. Ltd, China for the indicated time periods.The procedure was as described in previous publication Animals were maintained in Dalian Medical University Laboratory Animal Center under the specific pathogen-free condition, and all animal study procedures were approved (#AEE17036) by the Ethics Committee for Biology and Medical Science of Dalian Medical University.2 and 95% air humidified incubator at 37 \u00b0C. miRNA-223 mimics and negative control were transfected into primary hepatocytes via Lipofectamine RNAi MAX Transfection Reagent (Thermo Fisher Scientific) according to the manufacturer's instructions.Mouse primary hepatocytes were isolated by gradient density centrifugation of 30% Percoll solution after liver perfusion using Collagenase IV (Sigma). Primary hepatocytes were cultured in William's E Medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum, 1% ITS (Sigma), 2 mM L-glutamine, and 100 nM dexamethasone. HEK293T cells were maintained in Dulbecco's Modified Eagle's Medium supplemented with 10% fetal bovine serum plus penicillin (100 mg/mL) and streptomycin (100 mg/mL). Cells were kept in a 5% COin vivo digestion, hepatic cell suspension or purified hepatocytes were incubated with indicted primary antibodies at room temperature for 20 min in the dark and different cell populations were sorted and further analyzed by MOFLO ASTRIOSEQ, BECKMAN COULTER. The liver cell mixtures was firstly scattered by FSC/SSC, and signal cells population was gated by FSC-A/FSC-H, living cells population was further gated by DAPI negative selection, and the CD11b+/Gr1+ positive cell percentages were determined from those \u201cliving cell\u201d population.After Collagenase IV Abcg5 and Abcg8 were obtained by RT-PCR from liver tissue cDNA and subcloned into the pMIR-Reporter plasmid. The mutants for miRNA-223 binding sequences in 3' UTRs of Abcg5 and Abcg8 were obtained by using the Fast Mutagenesis System (TRANSGEN BIOTECH). The primer sequences for cloning and mutation are all shown in The partial 3' UTR regions of mouse Total cholesterol , high-density lipoprotein cholesterol , low-density lipoprotein cholesterol , total bile acid , and triglycerides concentrations were analyzed using Kits according to the manufacturer's instructions. Phospholipid (PL) concentrations were quantified using Wako Kits according to the manufacturer's instructions. The activities were measured using the Multi-Mode Microplate Reader (BioTek Synergy NEO). Bile cholesterol saturation index was calculated as described in the previous report The contents of gallbladders were placed onto glass slides or centrifugal tubes after cholecystectomy, followed by measuring the weights of gallstone after removal of bile and dry by air. Bile cholesterol crystal was evaluated under polarized-light microscopy (Olympus BX63) and the crystal type was classified according to previous study After scarification, animal livers were embedded in paraffin or optimal cutting temperature compound (OCT). Paraffin-embedded tissue sections were stained with hematoxylin and eosin (H&E) for morphological studies. OCT-embedded tissue cryosections were incubated with rhodamine-phalloidin (Sigma) and DAPI for fluorescence analysis.18S was used as a standard reference. For genotyping, the genomic DNA from indicated tissues or primary cells were prepared by TIANamp Genomic DNA Kit (TIANGEN) and further identified by PCR using specific primers. The primer sequences used in this study are presented in Total RNA was extracted from mouse liver tissues using the TRIzol (Invitrogen) according to the manufacturer's instructions and cDNAs were reverse-transcribed using the FastKing RT Kit (TIANGEN). The cDNA samples were amplified by Real-time quantitative PCR (RT-qPCR) using SYBR Green qPCR Master Mix (Bimake) and Mouse tissues or primary hepatocytes homogenates were subjected to SDS-PAGE, transferred to nitrocellulose (NC) membranes, and incubated with specific primary antibody. After washing, the membranes were incubated with anti-mouse Dylight 680 or anti-rabbit Dylight 800 secondary antibody (Abbkine). The membranes were then scanned using the Odyssey CLx Imaging System (LICOR), and the images were generated employing the Image Studio software.5/well) were cultured in 24-well plates one day before transfection, and then co-transfected with 60 ng 3\u2032-UTR luciferase reporter plasmids, 20 ng \u03b2-galactosidase plasmids, 20 nM miRNA-223 mimics or the negative controls by using Lipofectamine RNAi MAX Transfection Reagent (Thermo Fisher Scientific) according to the manufacturer's instructions. Cells were harvested 24 h post-transfection and further subjected to luciferase and \u03b2-galactosidase activitydetection by using the D-Luciferin (BD Biosciences) and o-nitrophenyl-\u03b2-d-galactoside (ONPG), respectively. The firefly luciferase and \u03b2-galactosidase activities were measured using the Multi-Mode Microplate Reader (BioTek Synergy NEO). The results were normalized as the ratio of firefly luciferase activities to \u03b2-galactosidase activities.HEK293T cells for 5 weeks. Along with LD-induced gallstone generation Figure A miRNA-2Tg mice content with no change in levels of total bile acid (TBA) Figure G, resultTg mice by crossbreeding the cKO mice with Alb-CreTg mice B. IntereKDHep, KDHepa) was performed by using Flag-tagged Cre recombinase under control of TBG promoter. Three weeks after AAV-Cre infection, cKO mice was feed with LD for another 5 weeks which has been seen in \u0394Hepa mice -mediated hepatocytes-specific knockdown (KD) of miRNA-223 expression of murine Hmgcs, Acat2 and Sc4mcl), uptake (Scarb1) and efflux (Abca1) Hmgcs and Acat2 were only detected in \u0394Hepa livers, however the Hmgcs protein expression was not changed as well as reduced gallstone mass or AAV8-CMV-GFP (GFP) towards WT mice was conducted in the third week during an eight-weeks LD feeding frame Figure A. Additis Figure C & D.Taken together, we proposed a novel mechanistic role of miRNA-223 in regulating LD-induced cholesterol gallstone development Figure : miRNA-2The purpose of this study was to determine the importance of hepatocytes miRNA-223 in regulating cholesterol biliary secretion and gallstone formation by using a series of genetically modified mouse models. The major finding is that miRNA-223 plays a pivotal role in regulating hepatobiliary cholesterol secretion and cholesterol gallstone formation by targeting key cholesterol transporters.in vitro studies, in which cholesterol deprivation would time-dependently suppress miRNA-223 expresison in cultured human hepatocytes We found that the LD with 1.25% cholesterol readily increases miRNA-223 expression in mouse livers. However, the increased miRNA-223 expression in liver tissue would be mainly attributed to infiltrated myeloid cells or Lyz-2 positive nonparenchymal cells because there was a 78% reduction of miRNA-223 expression in livers from myeloid specific miRNA-223 KO mice. Due to relative lower content of miRNA-223 in hepatocytes, we suggested liver miRNA-223 alternation would not reflect the issues occurred in hepatocytes. Therefore, we prepared primary hepatocytes and determined miRNA-223 expression was significantly increased in freshly isolated primary hepatocytes from LD treated animals. The data were consistent with previous Hep) in miRNA-223 cKO mice. By phenotypic analysis, LD feeding promoted gallstone development in KO, \u0394Hep and KDHep mice but not in \u0394Mye mice, fully supporting the importance of hepatocytes miRNA-223 in regulating hepatic/biliary cholesterol homeostasis. Besides, we noticed miRNA-223 expression levels were correspondingly decreased or increased in primary hepatocytes isolated from \u0394Hep, KDHep or OE mice, however unchanged miRNA-223 levels were observed in liver tissues from those mice mice model and obtained conventional (KO), hepatocytes-specific (\u0394Hepa), and myeloid-specific KO (\u0394Mye) mice by crossbreeding with EIIA-Cre, Alb-Cre, and Lyz-Cre transgenic mice. We also performed AAV-TBG-Cre-mediated hepatocytes-specific knockdown were all shown to be less correlation with miRNA-223, suggesting other targets or mechanism might contribute to the phenotype. Inconsistantly, neither miRNA-223 KO nor \u0394Hepa and KD would support the links between miRNA-223 deficency with hypercholesterolemia. Of the note, upregulation of SR-BI and down regulation of ABCA1 in human hepatocyte cell line was addressed to be involved in miRNA-223 mediated hepatocyte cholesterol uptake and efflux in vivo data from miRNA-223 \u0394Hepa and OE mice proved the both were all negatively regulated by miRNA-223, herein balancing cholesterol exchanges between liver and blood. Our current study focused on in vivo functional identidication by using hepatocyte specific loss or gain expression of miRNA-223 to dessect its role in regulating liver cholesterol transportation in mice. The differences of our fingdings with previous study might be reasoned by different experimental settings such as genetic modified animals, animal modles or species etc. Although inconsistence exisited, our data and previous study all strongly support the important role of hepatocytes miRNA-223 in regulating cholesterols balance. Based on consisitant higher cholesterol contents in liver and less influenced serum cholesterol levels in miRNA-223 KO, \u0394Hepa and KD livers, we wonder how to process the excess cholesterol in miRNA-223 deficent livers. Our data further reported hepatocyte specific loss of miRNA-223 expression greatly promote cholesterol gallstone formation, revealing a completely new function of miRNA-223 in hepatic cholesterol homeostasis.Although the lower expression level, hepatocyte expressing miRNA-223 was previously reported to play important roles in hepatic biological synthesis, cholesterol uptake, and efflux by targeting a complex gene set including Our first novel finding is that miRNA-223 in hepatocytes plays a pivotal role in controlling biliary cholesterol secretion by direct targeting the canalicular cholesterol transporters ABCG5 and ABCG8. ABCG5/8 has been found to contribute ~75% biliary cholesterol secretion, and its genetic mutation, or dysregulation would strongly disturbs cholesterol secretion and cholelithiasis progression. Our data solidly demonstrate that in LD feeding condition distrupting miRNA-223 expression in hepatocytes would significantly increases ABCG5 and ABCG8 expression and consequently elevates cholesterol levels in both liver secreted and bladder storaged bile. Meanwhile, we also detected increased SR-BI protein expression, which is believed to be a ABCG5/8 independent cholesterol transporter localized in canalicular membranes and conducting biliary cholesterol secretion in physical condtion Abcg5 and Abcg8 mRNA, suggesting a possible directly regulating manner. Therefore, hypothsis was experimental confirmed by classical 3'UTR reporter assays.Reversely, AAV-mediated hepatocytes-specific miRNA-223 OE in WT mice reduces expression of ABCG5, ABCG8 and decreases cholesterol content in the bile. In current study, we demonstrated a negative regulation manner between miRNA-223 and those cholesterol transporters in mouse hepatocyte. Although not noted in current available miRNA targets prediction databases, imperfect binding sequences of miRNA-223 were found in 3'UTR regions of To our knowledge, conventional miRNA-223 KO mice were wildly used in various research fields, however, the tissue or cell type specific miRNA-223 KO animal is emergently required for precisely verifying its functional importance and molecular mechanism in interested cell types or tissues. Therein, we generated miRNA-223 KO, hepatocyte specific miRNA-223 KO and KD mice, demonstrating importance of hepatocyte expressing miRNA-223 in regulating gallstone development via targeting cholesterol transporters. Meanwhile, we also suggested certain retrospective studies should be considerd by using cell specific miRNA-223 KO mice, which whould be able to enrich, verify or correct our current knowledges and understandings towards miRNA-223 biological functions.Our second important finding is the novel role of miRNA-223 in cholesterol gallstone formation. Gallstone crystallization occurs in supersaturated bile where BA and PL cannot dissolve excessive cholesterol. Supersaturated bile could be attributed by 1) hyper-cholesterol secretion; 2) reduced biliary BA or PL secretion with unaffected cholesterol secretion; 3) increased cholesterol and reduced BA or PL secretion More interestingly, insufficient biliary PL levels were also determined in miRNA-223 deficient mice that would further decline cholesterol solubility, which also caused our interest. ABCG4 is known as a main PL transporter faciliateing hepatic PL secretion towards bile and its mRNA expression level was slightly increased in miRNA-223 deficent livers, it would be explained by a negative feedback way in which insufficient PL storage in bile requires more ABCG4 to increase biliary PL transportation. Similary, increased BA transporter ABCG11 in miRNA-223 deficent livers might attempt to balance the oversaturated cholesterol status in bile. It is a complexed gene regulating network for PL and BA metabolism in hepatocytes and more detailed works needed in the future studies to determine the potent role of miRNA-223 in regulating hepatic PL and BA homeostasis.Besides, recent research also indicates that gallbladder motility insufficiency is greatly affected by the loss of interstitial Cajal-like cells and telocytes that further contributes to the cholelithiasis Lastly, this study provides the evidence for the miRNA-223 to be an effective therapeutic target in cholesterol gallstone disease. To evaluate the therapeutic efficacy by manipulating miRNA-223 expression, we conducted AAV-mediated miRNA-223 overexpression in WT mouse liver. The results demonstrated the strategy would efficiently decrease bile cholesterol content and reduced gallstone formation, supporting a novel therapeutic strategy for cholesterol gallstone disease by targeting miRNA-223.In summary, the present study uncovers an important role of miRNA-223 in regulating hepatobiliary cholesterol secretion and cholesterol gallstone formation by targeting key cholesterol transporters ABCG5 and ABCG8 in cholesterol biliary secretion pathway. Proof-of-concept study using AAV-mediated miRNA-223 OE in WT mouse liver shows promising therapeutic efficacy in treating LD-induced cholesterol gallstone. Hence, miRNA-223 has been identified as a novel potential therapeutic target in cholesterol gallstone disease.Supplementary figures and tables.Click here for additional data file."} +{"text": "Scientific Reportshttps://doi.org/10.1038/s41598-021-97566-z, published online 13 September 2021Correction to: The original version of this Article contained an error in the Funding section.\u201cThis research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.\u201dnow reads:\u201cThis work was partially funded by the Ruth L. Kirschstein National Research Service Award (NRSA) Individual Fellowship F30CA250254 (AL).\u201dThe original Article has been corrected."} +{"text": "The recent emergence of the SARS-CoV-2 Omicron variant of concern (VOC) containing a heavily mutated spike protein capable of escaping preexisting immunity, identifies a continued need for interventional measures. Molnupiravir (MK-4482), an orally administered nucleoside analog, has demonstrated efficacy against earlier SARS-CoV-2 lineages and was recently approved for SARS-CoV-2 infections in high-risk adults. Here we assessed the efficacy of MK-4482 against the earlier Alpha, Beta and Delta VOCs and Omicron in the Syrian hamster COVID-19 model. Omicron replication and associated lung disease in vehicle treated hamsters was reduced compared to the earlier VOCs. MK-4482 treatment inhibited virus replication in the lungs of Alpha, Beta and Delta VOC infected hamsters. Importantly, MK-4482 profoundly inhibited virus replication in the upper and lower respiratory tract of hamsters infected with the Omicron VOC. Consistent with its mutagenic mechanism, MK-4482 treatment had a more pronounced inhibitory effect on infectious virus titers compared to viral RNA genome load. Histopathologic analysis showed that MK-4482 treatment caused a concomitant reduction in the level of lung disease and viral antigen load in infected hamsters across all VOCs examined. Together, our data indicate the potential of MK-4482 as an effective antiviral against known SARS-CoV-2 VOCs, especially Omicron, and likely future SARS-CoV-2 variants. MK-4482 inhibits replication of multiple SARS-CoV-2 variants of concern, including Omicron, in the Syrian hamster COVID-19 model Now in its third year, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) coronavirus disease 2019 (COVID-19) pandemic has become characterized by the serial emergence of variants of concern (VOCs) that rapidly and globally replace earlier, previously predominant strains. In November 2021, Omicron (B.1.1.529) emerged to rapidly replace Delta (B.1.617.2), the predominant VOC at the time , 4. The in vitro compared to therapeutic in vivo effect. In the present study we investigate the ability of MK-4482 to inhibit several SARS-CoV-2 VOCs in the Syrian hamster COVID-19 model.Molnupiravir MK-4482) is an orally available antiviral nucleoside analogue that targets SARS-CoV-2 polymerase fidelity rather than the spike protein . It has 2 is an o3 TCID50 as previously established for Omicron (Hamsters in vehicle and MK-4482 treatment groups remained largely asymptomatic with noticeable but not significant weight loss (3\u20134%) in vehicle treated animals over the 4-day study period . Quantitn animal . MK-4482n animal . sgE loa Omicron . MK-4482 Omicron . SimilarCID50/g) . InfectiCID50/g) .Histopathologic analysis of lung tissues revealed minimal to moderate broncho-interstitial pneumonia for vehicle treated Alpha, Beta and Delta VOC infected hamsters. Omicron infected animals showed only mild lesions reflecting reduced virus replication of this VOC in the lower respiratory tract Fig. 2)Fig. 2). 4 TCID50) to account for the decreased replication of the Omicron VOC in lung tissue , a difference that was not statistically significant (10 TCID50/mL (10 following MK-4482 treatment (50) consistent with lower replication of Omicron in this tissue in general (10 (10 (10 higher in trachea (9 to 11 log10 copies/g) compared to lung tissue of vehicle treated hamsters. sgE loads were less than a log10 lower in trachea tissue from MK-4482 treated animals or VOCs circulating throughout the world . As eachImportant for transmission, a key driver of any epidemic and pandemic, is the impact of an antiviral intervention on virus shedding. In contrast to SARS-CoV-2 replication in the lower respiratory tract, MK-4482 treatment only slightly decreased replication and shedding from the upper respiratory tract for the Alpha, Beta and Delta VOCs. Interestingly, MK-4482 treatment significantly reduced replication and shedding of Omicron, indicating potent efficacy against the VOC driving the current SARS-CoV-2 pandemic. This is a somewhat surprising but rather encouraging result with the potential of positively influencing the course of the current pandemic wave.The antiviral activity of MK-4482 is not affected by mutations in the spike protein and remains active against all variants of SARS-CoV-2. Thus, MK-4482 remains a potent alternative to monoclonal antibody treatment which is rather vulnerable to mutations in the spike protein and performed in high biocontainment at Rocky Mountain Laboratories (RML), NIAID, NIH. IBC-approved Standard Operating Protocols were followed for sample removal from biocontainment. Institutional Animal Care and Use Committee approved all animal work which was performed by certified staff in an Association for Assessment and Accreditation of Laboratory Animal Care International accredited facility. The institution\u2019s guidelines for animal use, the guidelines and basic principles in the NIH Guide for the Care and Use of Laboratory Animals, the Animal Welfare Act, United States Department of Agriculture and the United States Public Health Service Policy on Humane Care and Use of Laboratory Animals were followed. Syrian hamsters were group housed in HEPA-filtered cage systems enriched with nesting material and were provided with commercial chow and water 3 or 104 TCID50 (high dose) of SARS-CoV-2 (25 \u03bcL/nare). Animal weights were collected once daily, and animals were monitored twice daily for disease signs and progression. All procedures were performed on anesthetized animals. Oral swabs were collected on days 2 and 4 post-infection. Animals were euthanized on day 4 post-infection and trachea and lung tissues were collected at necropsy for analysis.Male and female 8\u201310-week-old hamsters were divided into vehicle or treatment groups prior to infection and treatments with MK-4482 (MedChemExpress). MK-4482 was dissolved in DMSO and then resuspended in sterile saline for delivery at 250mg/kg. Hamsters were treated 12 hours following infection, and treatment was continued every 12 hours until the completion of the study 84 hours post-infection (day 4). Vehicle control animals received the same dosing schedule and volume as VOC infection groups. All groups were infected intranasally with 10The SARS-CoV-2_2hCOV_19_England_204820464_2020 isolate was provided by BEI Resources. The stock was sequence confirmed and had amino acid changes at ORF1AB (D3725G: 13%) and ORF1AB (L3826F: 18%) when aligned to the GISAID sequence (GISAID # EPI_ISL_683466). SARS-CoV-2 isolate nCoV-hCoV-19/USA/MD-HP01542/2021 was provided by Andy Pekosz (Johns Hopkins). The virus stock was sequence confirmed and found to have amino acid changes at NSP5 (P252L: 17%) and NSP6 (L257F: 57%) when aligned to the GISAID sequence (GISAID # EPI_ISL_890360). SARS-CoV-2 variant hCoV-19/USA/KY-CDC-2\u20134242084/2021 was obtained with contributions from B. Zhou, N. Thornburg and S. Tong . SARS-CoV-2 variant hCoV-19/USA/GA-EHC-2811C/2021 was obtained from Mehul Suthar, Emory University. All viral stocks were sequenced via Illumina-based deep sequencing to confirm identity and exclude any contaminants. Virus propagation was performed in DMEM (Sigma) supplemented with 2% fetal bovine serum (Gibco), 1 mM L-glutamine (Gibco), 50 U/ml penicillin and 50 \u03bcg/ml streptomycin (Gibco). Vero E6 cells, kindly provided by R. Baric, University of North Carolina, were maintained in DMEM (Sigma) supplemented with 10% fetal calf serum, 1 mM L-glutamine, 50 U/mL penicillin and 50 \u03bcg/mL streptomycin.qPCR was performed on RNA extracted from swabs or tissues (30 mg or less) using QiaAmp Viral RNA kit. A one-step real-time RT-PCR assay was used to amplify a portion of the E gene to detect subgenomic RNA . Dilutio50 was calculated via the Reed-Muench formula.Virus end-point titrations were performed in Vero E6 cells. Briefly, tissue was homogenized in 1ml DMEM using a TissueLyzer (Qiagen) and clarified by low-speed centrifugation. Cells were inoculated with 10-fold serial dilutions of homogenized lung samples or oral swabs in 100 \u03bcl DMEM (Sigma-Aldrich) supplemented with 2% fetal bovine serum, 1 mM L-glutamine, 50 U/ml penicillin and 50 \u03bcg/ml streptomycin. Cells were incubated for six days and then scored for cytopathogenic effects (CPE) and TCIDTissues were embedded in Pureaffin paraffin polymer and sectioned at 5 \u03bcm for hematoxylin and eosin (H&E) staining. For immunohistochemistry (IHC), tissues were processed using the Discovery Ultra automated stainer with a ChromoMap DAB kit (Roche Tissue Diagnostics cat#760\u2013159). Specific immunoreactivity was detected using the GenScript U864YFA140\u20134/CB2093 NP-1 SARS-CoV-2-specific antiserum at a 1:1000 dilution. The secondary antibody was an anti-rabbit IgG polymer (cat# MP-6401) from Vector Laboratories ImPress VR. As all animals in this study were inoculated with SARS-CoV-2, uninfected tissues from previous age-matched porcine studies were used for control tissues.Statistical analysis was performed in Prism 9. The difference in weight, viral load and infectious titers between study arms was assessed by ordinary one-way ANOVA.1"} +{"text": "Multiple imputation is a welhttps://www.universiteitleiden.nl/en/staffmembers/joost-van-ginkel#tab-1. A manual of the subroutine is also included in this zip file. The syntax files can be applied using SPSS 22.0 and later versions for Windows. SPSS 21.0 has the option to install SPSS Python Essentials during setup. For SPSS versions 18.0, 19.0, and 20.0, SPSS Python Essentials can be downloaded from the IBM SPSS website.The SPSS syntax file \u201cGPA.sps\u201d contains the subroutine that was partly written in SPSS for Windows using the MATRIX command and partClick here for additional data file.Supplemental material, sj-pdf-3-apm-10.1177_0146621621990757 for SPSS Syntax for Combining Results of Principal Component Analysis of Multiply Imputed Data Sets using Generalized Procrustes Analysis by Bart van Wingerde and Joost van Ginkel in Applied Psychological MeasurementClick here for additional data file.Supplemental material, sj-sav-1-apm-10.1177_0146621621990757 for SPSS Syntax for Combining Results of Principal Component Analysis of Multiply Imputed Data Sets using Generalized Procrustes Analysis by Bart van Wingerde and Joost van Ginkel in Applied Psychological MeasurementClick here for additional data file.Supplemental material, sj-sav-4-apm-10.1177_0146621621990757 for SPSS Syntax for Combining Results of Principal Component Analysis of Multiply Imputed Data Sets using Generalized Procrustes Analysis by Bart van Wingerde and Joost van Ginkel in Applied Psychological MeasurementClick here for additional data file.Supplemental material, sj-sps-2-apm-10.1177_0146621621990757 for SPSS Syntax for Combining Results of Principal Component Analysis of Multiply Imputed Data Sets using Generalized Procrustes Analysis by Bart van Wingerde and Joost van Ginkel in Applied Psychological MeasurementClick here for additional data file.Supplemental material, sj-sps-5-apm-10.1177_0146621621990757 for SPSS Syntax for Combining Results of Principal Component Analysis of Multiply Imputed Data Sets using Generalized Procrustes Analysis by Bart van Wingerde and Joost van Ginkel in Applied Psychological Measurement"} +{"text": "Peng et al.), multifunctional magnetic nanobubbles , folic acid functionalized gelatin\u2013AuNPs composite scaffolds , Zinc oxide nanocrystals , and Near-Infrared responsive Phase-shifting nanoparticles are presented through original research works. These novel nanoparticles with tailor-made properties offer a universal approach for anti-cancer therapy as their responsiveness depends on the general physiological properties commonly found in all tumors.The Research topic entitled \u201cStimuli-Responsive Nanoparticles for Anti-Cancer Therapy\u201d addresses the current advances in the stimuli-responsive nanoparticles for anti-cancer. This issue comprises nine selected peer-reviewed manuscripts discussing the latest updates on various stimuli-responsive nanoparticles used in oncology. Different types of nanoparticles, including activated polymeric delivery systems . In this study, amino-propyl functionalized ZnO nanocrystals (ZnO NCs) combined with ultrasound shock waves (SW) were used to treat cancer cells. The ZnO NCs demonstrated synergism in combination with SW stimulus. In another study by Jin et al., multifunctional magnetic nanobubbles (MF-MNBs) comprising of poly - polyethylene glycol\u2013folate (PLGA-PEG-FA) polymer-based nanobubbles were evaluated as tumor-targeted ultrasound (US)/magnetic resonance (MR) imaging and focused ultrasound (FUS)-triggered drug delivery system. The MF-MNB exhibited ligand-receptor mediated tumor accumulation and focused ultrasound FUS-triggered drug delivery for efficient cancer treatment. Chen et al. have demonstrated photothermal ablation using near-infra-red irradiation as an external stimulus for killing cancer cells. This study synthesized folic acid (FA)-functionalized composite scaffold by hybridizing FA-conjugated gelatin and FA-modified AuNPs and using ice particulates as porogen material. In vitro and In vivo studies demonstrated that FA-functionalized gelatin\u2013AuNPs composite scaffolds could elicit local photothermal ablation of breast cancer cells. Next, near-infrared responsive phase-shifted nanoparticles (NRPNs) have been designed by Xu et al. for magnetically targeted MR/US imaging and photothermal therapy of tumors. The near-infrared responsive phase-shifted nanoparticles (NRPNs) comprise PLGA nanoparticles encapsulated with indocyanine green (ICG), magnetic Fe3O4 nanoparticles, and perfluoro pentane (PFP). Upon irradiating with a NIR laser, the NRPNs, a phase-shifted expansion effect due to the quick conversion from light to heat by ICG and Fe3O4, can be used for ultrasound (US) imaging. In another study, Peng et al. have reported on an activated nanoparticle system comprising of poly -containing iron oxide nanoparticles (IOs) for biological imaging, using fucoidan/hyaluronic acid (FU/HA) to achieve targeting activity and applying polyethylene glycol-modified gelatin (PG)-carrying a phytochemical, epigallocatechin gallate (EGCG) to eradicate prostate tumors. This study demonstrated that the combination of therapeutic and molecular imaging could effectively target prostate cancer cells.The first example of external stimuli-responsive nanoparticles is presented by , pathological pH-responsive polymeric nano biosensors , tumor microenvironment responsive nanoparticles , and plant virus nanoparticles . As a starting example, Kumar et al. have summarized the recent developments in the design, preparation, and characterization of pH-responsive nanobiosensors and their ability to behave as efficient in vivo nano theranostics agents in acidic cancer environments. Thomas et al. have summarized the different types of internal stimuli-responsive nanoparticle drug delivery systems, Mohapatra et al., have outlined the role of different metallic nanotherapeutics in anti-cancer therapy, as well as their combinational effects with multiple stimuli for enhanced anti-cancer treatment. Finally, Hefferon et al. explore plant viruses as epitope-carrying nanoparticles and novel tools in cancer immunotherapy.Besides the original research articles, this research topic also has a series of review articles that summarized the recent advances in anti-cancer therapy using external and internal stimuli-responsive metallic nanoparticles (To summarize, we hope that this research topic will provide insights into the recent trends in nanomedicine, especially in oncology, using stimuli-responsive nanoparticles, providing insights into the development of targeted nanomedicine. The editors hope that the Research Topic \u201cStimuli-Responsive Nanoparticles for Anti-Cancer Therapy\u201d will contribute to the progress of research and development activities in the field of nanomedicine, inspiring and offering a universal approach for anti-cancer therapy by taking advantage of the physiological properties commonly found in all tumors."} +{"text": "O-GlcNAcylation, is one well-defined form of PTM that is catalyzed by a single pair of enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). Previous studies have discovered critical roles of protein O-GlcNAcylation in many fundamental biological activities via modifying numerous nuclear and cytoplasmic proteins. A common mechanism by which O-GlcNAc affects protein function is through the cross-regulation between protein O-GlcNAcylation and phosphorylation. This is of particular importance to innate immune cell functions due to the essential role of protein phosphorylation in regulating many aspects of innate immune signaling. Indeed, as an integral component of cellular metabolic network, profound alteration in protein O-GlcNAcylation has been documented following the activation of innate immune cells. Accumulating evidence suggests that O-GlcNAcylation of proteins involved in the NF-\u03baB pathway and other inflammation-associated signaling pathways plays an essential role in regulating the functionality of innate immune cells. Here, we summarize recent studies focusing on the role of protein O-GlcNAcylation in regulating the NF-\u03baB pathway, other innate immune signaling responses and its disease relevance.Metabolite-mediated protein posttranslational modifications (PTM) represent highly evolutionarily conserved mechanisms by which metabolic networks participate in fine-tuning diverse cellular biological activities. Modification of proteins with the metabolite UDP-N-acetylglucosamine (UDP-GlcNAc), known as protein O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) catalyzes the modification of nuclear and cytoplasmic proteins with UDP-GlcNAc on their serine or threonine residue, known as protein O-GlcNAcylation (O-GlcNAcase (OGA) catalyzes the hydrolysis of this sugar modification. Thus, as a dynamic and reversible modification, protein O-GlcNAcylation is tightly controlled by this single pair of enzymes OGT and OGA or genetic strategy resulted in increased expression of NF-\u03baB target genes, which was accompanied with enhanced RelA O-GlcNAcylation (O-GlcNAcylation sites on RelA (Oga gene-deletion (+/\u2212Oga) mouse model demonstrated that enhanced O-GlcNAcylation of RelA on T322 and T352 led to an increased binding of RelA to its target promoter regions, resulting in the hyperactivation of NF-\u03baB signaling and increased cytokine production. This in vitro immune phenotype was recapitulated by hyperinflammatory response and exacerbated inflammation-driving tumor growth in the dextran sodium sulfate (DSS)-induced colitis and azoxymethane (AOM)/DSS-induced colitis-associated cancer (CAC) animal models , a necessary modification for its transcriptional activity -mediated OGT gene-knockdown caused a reduced RelA phosphorylation, nuclear translocation, NF-\u03baB transcriptional activity, as well as target gene expression in human pancreatic ductal adenocarcinoma cells as an important modification to promote its DNA-binding capacity and transcriptional activity was mutated to alanine (S393ATab1) . No obviO-GlcNAcylation has been shown to exaggerate inflammatory response by counteracting anti-inflammatory signaling such as STAT3 signaling in innate immune cells. One study discovered that OGT-mediated O-GlcNAcylation of STAT3 at T717 negatively regulates phosphorylation of STAT3 at tyrosine-705 (Y705) . STAT3 i5 (Y705) \u201336. It w in vivo . O-GlcNAcylation . Based oO-GlcNAcylation exerts both positive and negative impact on immune signaling at diverse molecular levels. Indeed, several recent studies discovered an anti-inflammatory role of OGT-mediated protein O-GlcNAcylation, which is opposite to the observations showing increased NF-\u03baB activation and inflammatory response induced by protein O-GlcNAcylation. One study defined a transcriptional repression protein complex containing OGT, the transcriptional corepressor mammalian Sin3A (mSin3A), and histone deacetylase 1 (HDAC1) (Nos2 gene expression (via a nutrient-sensing mechanism (Ogt gene-deletion mouse strain and observed an inhibitory effect of OGT on innate immune activation through O-GlcNAcylation of RIPK3 (receptor-interacting serine/threonine kinase 3). As one of seven members of the RIP serine/threonine kinase family, RIPK3 forms a complex with RIPK1 and plays an essential role in inflammatory cytokine production -induced obesity model, Yang et al. observed an elevated inflammatory cytokine production in Ogt-deficient macrophages, which subsequently exacerbates HFD-induced metabolic dysfunctions in liver and muscle antagonized its phosphorylation and mTORC1 signaling, thus downregulating macrophage inflammation. Collectively, genetic evidence with Ogt knockout suggests that OGT-mediated protein O-GlcNAcylation negatively regulates myeloid cell immune activation and inflammatory response. Consistent with this concept, several studies observed that administration of glucosamine or OGA inhibitor thiamet G (TMG) reduced the levels of inflammatory cytokines such as IL-6 and TNF-a and improved organ function in multiple inflammation-associated animal models such as sepsis . It was pression . A folloechanism . To examoduction \u201343 and toduction . It was roptosis . This alfibrosis . Higher tability . Thus, Od muscle . Mechanis sepsis , trauma-s sepsis and stros sepsis .Oga knockout , it willO-GlcNAcylation in promoting antiviral immune responses against both RNA and DNA viruses. One study observed that infection of macrophages with an RNA virus, vesicular stomatitis virus (VSV), caused an elevated HBP activity and protein O-GlcNAcylation. Deletion of OGT in macrophages impaired activation of antiviral immune signaling and reduced inflammatory cytokine expression , a critical adaptor protein for downstream of RLR activation, was a required step for K63-linked MAVS ubiquitination and subsequent activation of antiviral immune signaling. Moreover, myeloid cell-specific deletion of OGT caused an enhanced susceptibility to VSV and IAV challenge in vivo, highlighting the importance of OGT-mediated protein O-GlcNAcylation in promoting host defense against RNA viruses. Furthermore, Wang et al. reported that deletion of OGT in myeloid cells attenuated IAV-induced cytokine storm and identified O-GlcNAcylation of interferon regulatory factor-5 (IRF5) at S430 as an important mechanism promoting the activation of antiviral immune response (O-GlcNAcylation on the infectivity of hepatitis C virus (HCV), another RNA virus, in human hepatocytes played a critical role in promoting antiviral effects , 62. HBVus (HBV) . Wang etproteins . The criproteins , suggest effects . Wang etimmunity Figure\u00a01O-GlcNAcylation provides antiviral benefit against both RNA and DNA viruses, a possibility is raised that protein O-GlcNAcylation may function through some unified cellular mechanism(s) to restrict virus replication. It has been well recognized that profound metabolic changes occur during viral infection, leading to an increased nutrient availability and permissive intracellular environment that are beneficial for viruses to accomplish their life cycles. For example, viral infection reprograms lipid metabolism in host cells towards an enhanced activity of the fatty acid biosynthesis pathway, causing increased accumulation of neutral lipid species in lipid droplets (LDs) as an important mechanism affecting the transcription of lipid metabolic enzymes \u201368. Thists (LDs) . As a rets (LDs) , 70\u201372, ts (LDs) . It has tabolism , 75. One enzymes . Deletio+ T cell-mediated immune response is another key host immune strategy to eliminate virus in a more specific and efficient manner. Of the two lymphocytes that carry out adaptive immunity, T cells function as a versatile \u201cplayer\u201d in multiple aspects, including pathogens killing, immunoregulation, and homeostasis. Inevitably, such intensive immune activities are associated with increased glucose consumption and protein modification. UDP-GlcNAc has been reported to participate in\u00a0T cell activation, self-renewal and immunosuppression through\u00a0OGT-mediated protein O-GlcNAcylation elicits the initiate signal for T cell activation. Following the activation of TCR, multiple transcription factors, including NF-\u03baB and nuclear factor of activated T cells (NFAT), are activated to regulate the expression of activation-associated genes. Golks et al. have demonstrated that NFAT and NF-\u03baB were modified by O-GlcNAc during T cell activation grants R01GM120496 and R01GM135234 (HW).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."} +{"text": "Herein, we aimed to analyze the outcomes of the methicillin sensitive (MS) versus methicillin resistant (MR) culture-proven Staphylococcus spp. nosocomial meningitis (S-NM) in our setting.Staphylococcus spp. (c) Presence of at least two of three clinical/laboratory criteria as meningitis findings: (i) Body temperature >38oC; (ii) CSF finding; >250 leucocytes/mm3; (iii) at least one of the following clinical findings, ie. impairment of consciousness, neck stiffness, nausea/vomiting. Identification of the infecting bacteria and determination of antimicrobial susceptibility were performed using the VITEK 2 automated system and conventional methods. Resistance to methicillin was tested by E-test (bioM\u00e9rieux). Antibacterial susceptibility tests were evaluated according to Clinical Laboratory Standards Institute (CLSI) criteria until 2014 and EUCAST between 2015 and 2021. Chi-square and Student T tests were used for statistical comparison.We extracted data and outcomes for all adult patients (age >18 years) consulted by the Infectious Diseases Consultants and diagnosed NM between January 2006 and 2021 and fulfilled the following study inclusion criteria: (a) Age \u226518-year-old; (b) CSF culture is positive for Acinetobacter baumannii and Pseudomonas aeruginosa after 12 and 30 days end of treatment. A total of 9 patients in MSS-NM, 41 patients in MRS-NM group fulfilled the study inclusion criteria. Age, gender, and CSF findings (except CSF glucose was significantly lower in MSS-NM) were similar in both groups (Table 1). Besides, EOT clinical success and overall success rates were similar (Table 1). Relapse and reinfection rates during post-treatment one month period were 0%-0% and 0%-6.6% in MSS/MRS-NM, respectively. In MRS-NM group reinfection pathogens were Characteristics of NMOverall success in MSS-NM was acceptable while it was non-significantly lower in MRS-NM. The medical community should seek better infection control measures from NM. All Authors: No reported disclosures"} +{"text": "Pseudomonas aeruginosa (PA), multidrug-resistant (MDR) metallo-beta-lactamase (MBL)-negative-PA, MBL-positive-PA, carbapenem-susceptible Acinetobacter baumannii (AB), and carbapenem-resistant AB. (3) Results: Piperacillin\u2013tazobactam or fourth-generation cephalosporins represent the first therapeutic choice in IVACs caused by multi-susceptible PA. A carbapenem-sparing approach favouring the administration of novel beta-lactam/beta-lactamase inhibitors should be pursued in the management of MDR-MBL-negative PA infections. Cefiderocol should be used as first-line therapy for the management of IVACs caused by MBL-producing-PA or carbapenem-resistant AB. Fosfomycin-based combination therapy, as well as inhaled colistin, could be considered as a reasonable alternative for the management of IVACs due to MDR-PA and carbapenem-resistant AB. (4) Conclusions: The implementation of algorithms focused on prompt revision of antibiotic regimens guided by results of conventional and rapid diagnostic methodologies, appropriate place in therapy of novel beta-lactams, implementation of strategies for sparing the broadest-spectrum antibiotics, and pharmacokinetic/pharmacodynamic optimization of antibiotic dosing regimens is strongly suggested.(1) Background: To develop evidence-based algorithms for targeted antibiotic therapy of infection-related ventilator-associated complications (IVACs) caused by non-fermenting Gram-negative pathogens. (2) Methods: A multidisciplinary team of four experts had several rounds of assessments for developing algorithms devoted to targeted antimicrobial therapy of IVACs caused by two non-fermenting Gram-negative pathogens. A literature search was performed on PubMed-MEDLINE (until September 2021) to provide evidence for supporting therapeutic choices. Quality and strength of evidence was established according to a hierarchical scale of the study design. Six different algorithms with associated recommendations in terms of therapeutic choice and dosing optimization were suggested according to the susceptibility pattern of two non-fermenting Gram-negative pathogens: multi-susceptible Pseudomonas aeruginosa and Acinetobacter baumannii) are responsible for a remarkable amount of IVACs in critically ill patients, second only to Staphylococcus aureus in terms of prevalence or FiO2 followed by a rise in PEEP of 3 cm H2O or a rise in FiO2 of 0.2 sustained for 48 h) coupled with the occurrence of body temperature <36 \u00b0C or >38 \u00b0C and the start of at least one antibiotic agent continued for over 96 h. VAP was considered a subgroup of IVACs, consisting in the presence of at least 25 neutrophils/field coupled with positive semi-quantitative/quantitative culture for pathogenic organisms at bronchoalveolar lavage [Pseudomonas aeruginosa, MDR metallo-beta-lactamase-negative Pseudomonas aeruginosa, MDR metallo-beta-lactamase-positive Pseudomonas aeruginosa, carbapenem-susceptible Acinetobacter baumannii, and carbapenem-resistant Acinetobacter baumannii). MDR Pseudomonas aeruginosa isolates were defined according to the classification proposed by Magiorakos et al. [A multidisciplinary task force, composed by one intensive care physician (B.V.), one infectious disease consultant (P.V.), one clinical microbiologist (G.M.R.), and one MD clinical pharmacologist (F.P.) met virtually on several occasions with the intent of developing algorithms for targeted antimicrobial therapy of IVACs caused by r lavage . The defs et al. . A hieraA researcher (M.G.) retrieved the scientific evidence needed for supporting the specific choices included in the algorithms by means of a PubMed-MEDLINE literature search (until October 2021). Key terms were selected antibiotics, HAP, VAP, IVACs, and bacterial pathogens with genotype of resistance and/or antibiotic susceptibility pattern. Quality of evidence was established according to a hierarchical scale of the study design, as reported in the evidence pyramid : randomiIn an era characterized by widespread diffusion of MDR Gram-negative pathogens and continuous increase in antibiotic resistance, the implementation of a multidisciplinary taskforce focusing on targeted therapy in critically ill patients has become a real need. Our approach is focused on prompt revision of inappropriate/unnecessary antibiotic therapy, implementation of \u201ccarbapenem-sparing\u201d strategies, and PK/PD optimization of antibiotic exposure hopefully guided by real-time TDM whenever feasible. Rational use of broad-spectrum antibiotics, especially carbapenems, could represent a powerful strategy for tackling resistance spread in the ICU setting . We beli"} +{"text": "A 78-year-old male patient with symptomatic aortic valve stenosis and history of surgical therapy of a preductal aortic coarctation by an aorto-aortic bypass was admitted to our Heart Center through the aortic coarctation . A Medtronic EvolutPro\u00ae 29\u00a0mm TAVR-prosthesis was implanted without any problems after pre-dilatation by balloon-valvuloplasty (peak-to-peak gradient\u2009>\u2009100\u00a0mmHg) with a good result of excellent hemodynamics and mild paravalvular leakage.Our case demonstrates that advanced guidance by fusion imaging can facilitate safe TAVR-procedures even in uncommon and complicated anatomies.Fusion imaging: fusion of real-time fluoroscopy and planning CT-scan: the TAVR-prosthesis is advanced over the stiff wire through the native kinked and stenotic aortic arch . For details, see text. (MP4 12627 kb)Below is the link to the electronic supplementary material."} +{"text": "Scientific Reportshttps://doi.org/10.1038/s41598-021-90923-y, published online 31 May 2021Correction to: The original version of this Article contained an error in the list numerals in the IGTD algorithm section, where,(xxii) (xxiii)(xxiv)now reads:\u201cOtherwise, the algorithm does the following:\u201cOtherwise, the algorithm does the following:(v)(vi)(vii)The original Article has been corrected."} +{"text": "Dostarlimab (500 mg intravenously every 3 weeks) was then introduced. The subsequent evaluation after three perfusions demonstrated a complete metabolic response on 18F-FDG PET/CT according to immunotherapy-modified PET response criteria in solid tumors (imPERCIST) criteria, then confirmed by MRI according to immune response evaluation criteria in solid tumors (iRECIST). This clinical description suggests that 18F-FDG PET/CT might take place among available tools for guiding the preoperative management for recurrent endometrial cancer patients receiving dostarlimab immunotherapy that should be further explored through clinical trials.Dostarlimab is an immune checkpoint inhibitor (ICI) targeting the Programmed-Death-1 (PD-1) co-receptor, recently approved by the European Medicines Agency (EMA) and the Food and Drug Administration (FDA) as a novel therapy for recurrent or advanced endometrial cancer. We report the case of a 64-year-old woman, experiencing vaginal recurrence with microsatellite instability high/hypermutated of a FIGO stage IA grade 2 endometrial endometrioid adenocarcinoma. She received preoperative chemotherapy with four cycles of carboplatin plus paclitaxel, with stable disease on pelvic magnetic resonance imaging (MRI) and fluorine-18 fluorodeoxyglucose positron emission tomography ( This rare description highlights the utility of 18F-FDG PET/CT for guiding the preoperative management for recurrent endometrial cancer patients using PET-based immune-related response criteria [In this case , 18F-FDGcriteria .The very recent literature reports that dostarlimab monotherapy TSR-042) is associated with significant antitumor activity and promising response rates for relapsed endometrial cancer patients ,4, espec2 is asso18F-FDG PET/CT might take a place among available tools for a reliable strategy to manage preoperative therapy and should be further explored through clinical trials evaluating the clinical benefit with dostarlimab.Given this update on the evolving role of immunotherapy as treatment of patients with recurrent endometrial cancer ,8, we st"} +{"text": "ABSTRACT IMPACT: Limited research has been conducted on the survival of men with castration-resistance prostate cancer (CRPC) with a pre-existing history of cardiovascular disease, receiving oral androgen signaling inhibitors. This study highlights all-cause and prostate cancer-specific mortality for elderly patients with CRPC with pre-existing history of cardiovascular disease. OBJECTIVES/GOALS: Inadequate knowledge is known about the survival of men with castration-resistance prostate cancer (CRPC) with pre-existing history of cardiovascular disease (CVD), receiving oral androgen signaling inhibitors (OASI). We compared all-cause and prostate cancer-specific mortality for elderly patients with CRPC with pre-existing history of CVD. METHODS/STUDY POPULATION: An active comparator, new user design, was used to identify 2,608 men older than age 65 years with CRPC using the Surveillance, Epidemiology, and End Results (SEER)-Medicare linked database from 2011 to 2015. Patients were grouped into two analytical cohorts by CVD history. Within each analytical cohort patients were divided into two arms based on their new-user status (OASI vs. chemotherapy). All demographics and clinical characteristics were adjusted by inverse probability treatment weights (IPTWs). Unadjusted and IPTW-adjusted time-dependent Cox models, and Fine and Gray\u2019s models were conducted to evaluate associations between OASI and all-cause and prostate cancer-specific mortality. RESULTS/ANTICIPATED RESULTS: Nearly 64.5% of patients had pre-existing CVD. We observed a lower all-cause mortality in the pre-existing CVD cohort compared to the no pre-existing CVD cohort . Similarly, the prostate cancer specific-mortality was showed to be lower in the pre-existing CVD cohort compared to the no pre-existing CVD cohort when comparing OASI versus chemotherapy by the IPTW-adjusted time-dependent Fine and Gray\u2019s models . DISCUSSION/SIGNIFICANCE OF FINDINGS: OASI showed a significant protective effect against all-cause and prostate cancer-specific mortality compared with chemotherapy; however, were less protective among patients without pre-existing CVD. Further studies are needed to investigate OASI in patients with and without pre-existing CVD."} +{"text": "Staph aureus (anti-MRSA) and Pseudomonas aeruginosa (anti-PAER) for patients hospitalized for pneumonia. To explore this variation further, we conducted (1) quantitative analyses of facility-level versus provider-level variation, and (2) qualitative interviews with emergency department providers. We previously found widespread variation in the empiric use of antibiotics against methicillin-resistant For each hospitalization, we predicted the probability of anti-MRSA and anti-PAER use by fitting machine learning models from 75 patient variables. We estimated the predicted risk of anti-MRSA/anti-PAER and facility features among patients hospitalized at upper versus lower 10% facilities after controlling for patient characteristics. We plotted density curves with the variance attributed to facility and provider alone and together. We then interviewed 16 emergency department (ED) providers at 8 VA facilities using a cognitive task analysis. Figure 1). Providers reported social influences from the opinions of other providers during decision-making and a high trust in guidelines and order sets. Consideration of pathogens was not mentioned by any providers at high-prescribing facilities.Among 215,803 hospitalizations at 128 VA facilities 1/1/2006-12/31/2016, 31% received empiric anti-MRSA and 29% received empiric anti-PAER antibiotics. Hospitalizations at upper-decile facilities had a 50% and 45% adjusted probability of receiving anti-MRSA and anti-PAER antibiotics, compared to 15% and 20% in the lower-decile facilities. Facility features most predictive of anti-MRSA or anti-PAER use after adjusting for patient characteristics were complexity level (33% and 30% in high versus 15% and 20% in low complexity facilities). Variation in empiric anti-MRSA and anti-PAER use was almost completely at the facility level (Variation in empiric use of anti-MRSA and anti-PAER antibiotics in pneumonia clustered nearly completely at the facility level. ED providers report social influences during decision-making and a high trust in guidelines and order sets. Guidelines, order sets, and facility-level clinical champions that promote consideration of pathogens could be important strategies for de-adoption. All Authors: No reported disclosures"} +{"text": "BRAF alterations are recognized as a significant driver of disease in pediatric low-grade glioma (pLGG) but the implications of BRAF alterations on the natural history and response to treatment are unclear in adult glioma. We characterized the molecular and clinical features of a multi-institutional cohort of adults with BRAF-mutated gliomas. We identified patients with glioma containing BRAF alterations on sequencing in multi-institutional cohorts . BRAF alterations were grouped into previously defined classes: I , II (RAS-independent/dimerization-dependent), III (RAS-dependent/dimerization-dependent) in addition to BRAF copy number gains, fusions, and other. We interrogated 289 BRAF-altered gliomas , and observed histopathologic and molecular differences between BRAF-altered gliomas in adults versus pediatric patients. Amongst adults, the most common BRAF alterations were Class I followed by copy number gains, with glioblastoma (GBM) the most prevalent histology. In comparison, pediatric gliomas in our cohort frequently harbored Class I mutations followed by BRAF fusions, with primarily pilocytic astrocytoma and pLGG histologies. Principal component analysis and correlation analysis revealed molecular features associated with gliomas of different BRAF alterations and histologies, including mutation of NF1, a negative regulator of RAS, which was significantly associated with class II/III BRAF alterations (64.3%) and not observed in BRAFV600E-mutated gliomas (p<0.0001). Demographic and molecular features were evaluated for correlates for adult glioma risk stratification. Comparative survival analysis showed no significant difference between adult GBM harboring Class I compared to other BRAF alterations, whereas young adult age (18-35yrs) was associated with improved outcomes (p<0.05). Among 86 GBM patients with detailed clinicopathologic data, 7 received RAF-targeted therapy, with variable clinical response. This cohort of BRAF-altered adult gliomas demonstrates a broad range of molecular alterations with implications for treatment sensitivity and patient risk stratification."} +{"text": "Drosophila melanogaster through genetic manipulations of brummer (bmm), which encodes a triglyceride lipase orthologous to mammalian Adipose Triglyceride Lipase. RNAi-mediated knock-down of bmm in all tissues or metabolic specific tissues results in reduced locomotor activity, altered sleep patterns and reduced lifespan. Metabolomic analysis on flies in which bmm is downregulated reveals a marked reduction in medium chain fatty acids, long chain saturated fatty acids and long chain monounsaturated and polyunsaturated fatty acids, and an increase in diacylglycerol levels. Elevated carbohydrate metabolites and tricarboxylic acid intermediates indicate that impairment of fatty acid mobilization as an energy source may result in upregulation of compensatory carbohydrate catabolism. bmm downregulation also results in elevated levels of serotonin and dopamine neurotransmitters, possibly accounting for the impairment of locomotor activity and sleep patterns. Physiological phenotypes and metabolomic changes upon reduction of bmm expression show extensive sexual dimorphism. Altered metabolic states in the Drosophila model are relevant for understanding human metabolic disorders, since pathways of intermediary metabolism are conserved across phyla.Disruption of lipolysis has widespread effects on intermediary metabolism and organismal phenotypes. Defects in lipolysis can be modeled in Metabolic syndrome and metabolic diseases impact a large proportion of the world population: a global analysis of 195 countries showed that 604 million adults and 108 million children had obesity in 2015 . ObesityDrosophila provides a powerful model system for comprehensive analyses of physiological, behavioral and metabolic consequences of disruption of intermediary metabolism through selective tissue-specific disruption of target genes under controlled dietary conditions [Drosophila are further facilitated through the public availability of a wide array of genetic resources that can facilitate in-depth systems genetic studies of metabolic regulation [Drosophila has organ systems analogous to those of mammals that control the uptake, storage and metabolism of nutrients [nditions \u20139. Comprgulation \u201315. Drosutrients . Digestiutrients . Followiutrients .brummer (bmm) [Lipids in the fat body are accumulated in lipid droplets. Their release is controlled by adipokinetic hormone (Akh), the functional counterpart of glucagon in humans, via a G protein-coupled Akh receptor (Akh-R) pathway ,20. Akh-er (bmm) ,22.bmm is the ortholog of the human PNPLA2 gene that encodes Adipose Triglyceride Lipase (ATGL), the major mammalian TAG lipase. ATGL hydrolyzes TAGs at the sn-1 and sn-2 positions to release fatty acids for catabolism. Homozygous bmm null alleles exhibit embryonic lethality, but some \u201cescapers\u201d reach the adult stage and present excessive fat storage [bmm ortholog PNPLA2 have neutral lipid storage disease with myopathy (NLSDM), characterized by abnormal accumulation of fat in different tissues [bmm is sexually dimorphic and under neural control [Drosophila , but lit storage . Humans tissues . Regulat control . Defectsbmm results in impaired locomotion and altered sleep patterns. Metabolomic analysis shows that these effects are accompanied by shifts in intermediary metabolism and changes in levels of neurotransmitters in the brain.Here, we show that RNAi-mediated suppression of bmm. We employed in all experiments only one copy of the Gal4 driver and UAS-bmm-RNAi to generate hypomorphic effects. First, we analyzed bmm transcript levels with ubiquitous expression of bmm-RNAi in adult flies using RT-qPCR. We used two different UAS-bmm-RNAi constructs, bmm-RNAiV37877 and bmm-RNAiV37880. For both Ubi > bmm-RNAi flies, levels of bmm transcripts were significantly lower compared to their controls for both sexes, but the effect of Ubi > bmm-RNAiV37877 was more pronounced than the effect of Ubi > bmm-RNAiV37880. The reduction of bmm transcripts was greater in males than in females for both genotypes system. The 24h locomotor activity was significantly lower in Ubi > bmm-RNAiV37877 males than control males than Ubi > bmm-RNAiV37880 females and males . However, there were quantitative changes in metabolites common to both genotypes: 124 in females and 118 in males than Ubi > bmm-RNAiV37880 (bmm transcripts (RT-qPCR), and greater effects on lifespan, locomotor activity and sleep parameters.To gain insights in the mechanisms by which suppression of identity . Principidentity . Therefo control . The metin males . The pron males) . These dbmm-RNAi lines for differentially abundant metabolites of single comparisons than Ubi > bmm-RNAiV37880 females . Conversely, Ubi > bmm-RNAiV37877 males had fewer changes in metabolite levels than Ubi > bmm-RNAiV37880 males .We analyzed 298 metabolites from 49 lipid metabolism sub-pathways in flies in which controls . Ubi > bUbi > bmm-RNAiV37880 males and in two lipid metabolites of Ubi > bmm-RNAiV37880 females and in Ubi > bmm-RNAiV37880 females . However, more carbohydrate metabolites showed altered abundances in Ubi > bmm-RNAiV37877 males than in Ubi > bmm-RNAiV37880 males . We observed more changes in metabolites associated with energy metabolism in Ubi > bmm-RNAiV37877 females than any other genotypes (bmm knockdown strains could compensate for impaired lipid mobilization.Impairment of lipid mobilization for energy production resulted in marked changes in the intermediates of the glycolysis pathway and the tricarboxylic acid (TCA) cycle in both strains . We eval strains . The samenotypes . Our datUbi > bmm-RNAiV37877 females had altered quantities of 65 metabolites (58\u2191/7\u2193), while only 39 metabolites changed (27\u2191/12\u2193) in Ubi > bmm-RNAiV37880 females. Ubi > bmm-RNAiV37877 males showed changes in 64 metabolites (54\u2191/10\u2193), while Ubi > bmm-RNAiV37880 males only showed alterations in 32 compounds (18\u2191/14\u2193).The shifts in energy metabolism described above are accompanied by changes in intermediaries of amino acid and peptide metabolism pathways . We evalUbi > bmm-RNAiV37877 flies showed more changes in levels of nucleotide intermediaries than Ubi > bmm-RNAiV37880 flies. In Ubi > bmm-RNAiV37877 females 65 metabolites underwent changes in abundance (58\u2191/7\u2193), whereas only 39 metabolites changed (27\u2191/12\u2193) in Ubi > bmm-RNAiV37880 females. In Ubi > bmm-RNAiV37877 males 64 metabolites showed altered levels of abundance (54\u2191/10\u2193) and levels of 32 metabolites changed in Ubi > bmm-RNAiV37880 males (18\u2191/14\u2193). Thus, dysregulation of lipid metabolism has widespread consequences in the metabolome.We also identified 76 metabolites from 9 nucleotide metabolism sub-pathways . Ubi > bbmm suppression on locomotion, sleep and lifespan, we explored changes in neurotransmitter levels resulting from bmm knockdown. We observed significant alterations, dependent on RNAi genotype and sex, of different neurotransmitters in bmm knockdown flies N6-carboxymethyllysine, which serves as a biomarker of high blood glucose levels in humans; this might suggest a diabetes-like phenotype. High levels of AGEs together with high-fat diet produces liver dysfunction in mice [Ubi > bmm-RNAiV37877 flies is not clear, since glucose did not increase significantly in these flies, nor did they exhibit an increase in trehalose production by the fat body. These results provide a framework for future studies on compensatory metabolic mechanisms through manipulation of carbohydrate metabolism in the bmm mutant. in mice , and liv in mice . Whetherbmm expression results in elevated levels of the neurotransmitters serotonin and dopamine, which may account for impairment of locomotor activity and altered sleep patterns [1A receptors in Drosophila are required for insulin-producing cells to regulate lipid content [bmm-sensitive phenotypes in flies requires assessment in future experiments.Inhibition of patterns \u201344. Sero content . Interes content . Also, i content . Causal Drosophila model are relevant to advancing our understanding of human metabolic disorders.Impairment of fatty acid mobilization results in a shift in intermediary metabolism toward utilization of carbohydrates as an energy source. In addition, amino acid, nucleotide, and neurotransmitter metabolic pathways are affected by disruption of lipid catabolism. This altered metabolic state gives rise to changes in morphological and physiological organismal phenotypes. Because pathways of intermediary metabolism are conserved across phyla, studies on the w1118; UAS-bmm-RNAi lines (#37877 and #37880) and their GD control from the Vienna Drosophila Resource Center [Gal4 driver lines were obtained from the Bloomington Drosophila Stock Center: w*; Ubi-Gal4/CyO (#32551) for ubiquitous expression, w1118; Lsp2-Gal4 (#6357) for specific expression in fat body, and w*; Dsat1-Gal4 (#65405) and w*; OK72-Gal4 (#6486) for expression in oenocytes. Flies were reared on molasses-cornmeal medium (Nutri-Fly\u00ae) with propionic acid and Tegosept added as fungicides. They were maintained at 25\u00b0C and a 12h:12h light-dark schedule (lights on at 6:00 am). Matings were performed with 6 pairs per vial and flies were maintained at a controlled population density. UAS-bmm-RNAi flies and control GD flies were crossed with Ubi-Gal4/CyO. The progeny with one copy of Ubi-Gal4 and UAS-bmm-RNAi (Ubi > bmm-RNAiV37877 and Ubi > bmm-RNAiV37880) or Ubi-Gal4 and GD control background (Ubi > +), were subjected to RT-qPCR, lifespan assay, locomotor activity/sleep assay and metabolomic analysis.We obtained two e Center . All GalUbi-Gal4 and UAS-bmm-RNAi (Ubi > bmm-RNAiV37877 and Ubi > bmm-RNAiV37880) or the GD control (Ubi > +), were anesthetized with CO2 and frozen on dry ice. All flies were collected at the same time of day to avoid effects of circadian rhythms and stored at -80\u00b0C. Total RNA was extracted using the RNeasy Plus Mini Kit and single strand cDNA was generated with High-Capacity cDNA Reverse Transcription . We designed primers to generate a PCR fragment of 116 bp for bmm and 102 bp for Gapdh1 . Three technical replicates were used for each sample, and qPCR was performed in a QuantStudio 3 instrument (Applied Biosystems).Three biological replicates of 3 to 5-day old virgin females and males, with one copy of r Gapdh1 . RelativUbi-Gal4 and UAS-bmm-RNAi (Ubi > bmm-RNAiV37877 and Ubi > bmm-RNAiV37880) or the GD control (Ubi > +) for lifespan assays. We set up 50 vials of molasses-cornmeal media with 3 males or 3 females for each genotype, transferred them to fresh food every 2\u20133 days, and scored for survival daily. Flies that escaped during transfers or were stuck in the food were discarded from the analysis , the fat body driver or oenocyte drivers . Offspring with one copy of driver-Gal4 and UAS-bmm-RNAi or the GD control were subjected to activity monitoring using Drosophila Activity Monitors , which record movement by counting interruptions of an infrared beam. Tubes were plugged with 2% agar and 5% sucrose for normal feeding assays and 1% agar for starvation assays [n assays .Two-day old males and females of the three genotypes were collected simultaneously and unmated flies were used for the assay. Two full 32-tube replicates were analyzed for activity behavior for each sex and genotype . The incSix replicates of ~150 6-day old females and males for each genotype (36 samples) were collected at the same time of day on dry ice and stored at -80\u00b0C. Unmated flies were used for metabolomic profiling by Metabolon, Inc., NC. Samples were prepared using the automated MicroLab STAR system from Hamilton Company, adding several recovery standards for quality control prior to the extraction process. Proteins were precipitated with methanol under vigorous shaking (Glen Mills GenoGrinder 2000) for 2 min., followed by centrifugation . The extract was divided into five fractions: two for analysis by two separate RP/UPLC-MS/MS methods with positive ion mode ESI, one for analysis by RP/UPLC-MS/MS with negative ion mode ESI, one for analysis by HILIC/UPLC-MS/MS with negative ion mode ESI, and one sample was reserved for backup. Organic solvent was removed placing samples on a TurboVap (Zymark) and the sample extracts were stored overnight under nitrogen before preparation for analysis. Metabolites were extracted from the flies and loaded in an equivalent manner across the analytical platforms and the values for each metabolite were normalized based on Bradford protein concentrations.Metabolon\u2019s hardware and software were used for raw data extraction, peak-identification, and quality control processing. Compounds were identified by comparison to library entries of purified standards (or recorded as unknown entities) and peaks were quantified using area under the curve. Data normalization was performed to correct variation from instrument inter-day tuning differences. Each compound was corrected in run day blocks by registering the medians to equal one (1.00) and normalizing each data point proportionately. The detailed procedure for metabolomic profiling from Metabolon Inc. has been described previously .https://www.metaboanalyst.ca). Mapped metabolites in the Human Metabolome Database (HMDB) were used for over-representation analysis of lipid structure comparing to the super-class dataset.Metabolic networks are rendered using a private custom software application made by Metabolon that is based on the Cytoscape application. This software uses the Cytoscape JS toolkit and the D3 JavaScript library ,52 and iUbi > bmm-RNAi females and males were analyzed separately with one-way ANOVA. ANOVA contrasts between experimental groups and controls were performed using two approaches: single comparisons of each strain with the corresponding control or comparisons of both strains with the proper control (V37877-F + V37880-F and V37877-M + V37880-M). Mean, p-values and q-values, and the magnitude of changes are in Two-way ANOVA tests were performed for RT-qPCR and locomotor activity/sleep, followed by Tukey\u2019s multiple testing correction (p<0.05) to assess statistically significant results Tables. S1 FigUAS-bmm-RNAi flies were mated with Lsp2-Gal4 (a-c), Dsat1-Gal4 (d-f) and OK72-Gal4 (g-i), and F1-flies were used for the assay in normal feeding. Average of whole-day locomotor activity. Average of locomotor activity during the daytime and nighttime. Average activity profiles in Zeitgeber time. Averages were calculated from 7 days of behavior assay. For this and Lsp2 > + F (n = 60), Lsp2 > + M (n = 63), Lsp2 > bmm-RNAiV37877 F (n = 55), Lsp2 > bmm-RNAiV37877 M (n = 57), Lsp2 > bmm-RNAiV37880 F (n = 57), Lsp2 > bmm-RNAiV37880 M (n = 61), Dsat1 > + F (n = 62), Dsat1 > + M (n = 63), Dsat1 > bmm-RNAiV37877 F (n = 63), Dsat1 > bmm-RNAiV37877 M (n = 63), Dsat1 > bmm-RNAiV37880 F (n = 63), Dsat1 > bmm-RNAiV37880 M (n = 63), OK72 > + F (n = 58), OK72 > + M (n = 54), OK72 > bmm-RNAiV37877 F (n = 61), OK72 > bmm-RNAiV37877 M (n = 60), OK72 > bmm-RNAiV37880 F (n = 63) and OK72 > bmm-RNAiV37880 M (n = 61). Asterisks indicate significant differences at p < 0.05 compared with the appropriate female or male control, following Tukey\u2019s correction for multiple tests. Error bars are SEM. ANOVA tests are reported in GD control and (JPG)Click here for additional data file.S2 FigUAS-bmm-RNAi flies were mated with Lsp2-Gal4 (a-c), Dsat1-Gal4 (d-f) and OK72-Gal4 (g-i), and F1-flies were used for the assay in normal feeding. Average number of sleep bouts. Average length of sleep bout. Average sleep during the daytime and the nighttime. Sleep was calculated using the standard definition of continuous period of inactivity lasting at least 5 minutes. Averages were calculated from 7 days of behavior assay and recalculated to periods of 30 minutes of sleep. Asterisks indicate significant differences at p < 0.05 compared with the appropriate female or male control, following Tukey\u2019s correction for multiple tests. Error bars are SEM. ANOVA tests are reported in GD control and (JPG)Click here for additional data file.S3 FigUbi > bmm-RNAiV37877 females compared with control females. (b) Metabolites that significantly changed in Ubi > bmm-RNAiV37880 females compared with control females. (c) Metabolites that significantly changed in Ubi > bmm-RNAiV37877 males compared with control males. (d) Metabolites that significantly changed in Ubi > bmm-RNAiV37880 males compared with control males. Yellow nodes represent the sub-pathways analyzed Metabolites that significantly changed in yzed see . Red and(JPG)Click here for additional data file.S4 FigUbi > bmm-RNAiV37877 females compared with control females. (b) Metabolites that significantly changed in Ubi > bmm-RNAiV37880 females compared with control females. (c) Metabolites that significantly changed in Ubi > bmm-RNAiV37877 males compared with control males. (d) Metabolites that significantly changed in Ubi > bmm-RNAiV37880 males compared with control males. Yellow nodes represent the sub-pathways analyzed Metabolites that significantly changed in yzed see . Red and(JPG)Click here for additional data file.S5 FigUbi > bmm-RNAiV37877 females compared with control females. (b) Metabolites that significantly changed in Ubi > bmm-RNAiV37880 females compared with control females. (c) Metabolites that significantly changed in Ubi > bmm-RNAiV37877 males compared with control males. (d) Metabolites that significantly changed in Ubi > bmm-RNAiV37880 males compared with control males. Yellow nodes represent the sub-pathways analyzed Metabolites that significantly changed in yzed see . Red and(JPG)Click here for additional data file.S6 FigUbi > bmm-RNAiV37877 females compared with control females. (b) Metabolites that significantly changed in Ubi > bmm-RNAiV37880 females compared with control females. (c) Metabolites that significantly changed in Ubi > bmm-RNAiV37877 males compared with control males. (d) Metabolites that significantly changed in Ubi > bmm-RNAiV37880 males compared with control males. Yellow nodes represent the sub-pathways analyzed Metabolites that significantly changed in yzed see . Red and(JPG)Click here for additional data file.S7 FigUbi > bmm-RNAiV37877 females compared with control females. (b) Metabolites that significantly changed in Ubi > bmm-RNAiV37880 females compared with control females. (c) Metabolites that significantly changed in Ubi > bmm-RNAiV37877 males compared with control males. (d) Metabolites that significantly changed in Ubi > bmm-RNAiV37880 males compared with control males. Yellow nodes represent the sub-pathways analyzed Metabolites that significantly changed in yzed see . Red and(JPG)Click here for additional data file.S8 FigUbi > bmm-RNAiV37877 females compared with control females. (b) Metabolites that significantly changed in Ubi > bmm-RNAiV37880 females compared with control females. (c) Metabolites that significantly changed in Ubi > bmm-RNAiV37877 males compared with control males. (d) Metabolites that significantly changed in Ubi > bmm-RNAiV37880 males compared with control males. Yellow nodes represent the sub-pathways analyzed Metabolites that significantly changed in yzed see . Red and(JPG)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.S5 Table(PDF)Click here for additional data file.S1 Dataset(XLSX)Click here for additional data file.S2 Dataset(XLSX)Click here for additional data file.S3 Dataset(XLSX)Click here for additional data file.S4 Dataset(XLSX)Click here for additional data file.S5 Dataset(XLSX)Click here for additional data file.S6 Dataset(XLSX)Click here for additional data file.S7 Dataset(XLSX)Click here for additional data file.S8 Dataset(XLSX)Click here for additional data file.S9 Dataset(XLSX)Click here for additional data file.S10 Dataset(XLSX)Click here for additional data file."} +{"text": "Single-cell DNA template strand sequencing (Strand-seq) enables chromosome length haplotype phasing, construction of phased assemblies, mapping sister-chromatid exchange events and structural variant discovery. The initial quality control of potentially thousands of single-cell libraries is still done manually by domain experts. ASHLEYS automates this tedious task, delivers near-expert performance and labels even large datasets in seconds.github.com/friendsofstrandseq/ashleys-qc, MIT license.Bioinformatics online. The Strand-seq protocol affords unique insights in diverse applications, e.g. locating sister chromatid exchange events . ASHLEYS is based on established machine learning technology and ships with ready-to-use classification models trained on a large cohort of Strand-seq libraries. ASHLEYS pretrained classifiers have been vetted on independent test data to ensure stable generalization performance on new Strand-seq data with similar feature characteristics. Next, we describe ASHLEYS\u2019 feature model and summarize the performance of the default classifier recommended for QC of new Strand-seq libraries.Strand-seq is a single-cell short-read sequencing technique that assays sister chromatid inheritance patterns at the level of individual chromosomes and machine learning (n\u2009=\u20092304) generated as part of the Human Genome Structural Variation Consortium (HGSVC) (n\u2009=\u2009456) labeled by the same domain expert (ASHLEYS pretrained classifiers were tuned on a large dataset ( (HGSVC) . Model (HGSVC) . Model gn expert , suggestIn conclusion, ASHLEYS\u2019 high performance and the resulting gains in efficiency facilitate scaling Strand-seq to even larger cohorts without burdening domain experts with an overwhelming amount of repetitive QC. This raises promising expectations for addressing further challenges such as extensive aneuploidy in cancer.btab221_Supplementary_DataClick here for additional data file."} +{"text": "Limited options currently exist for treatment of patients diagnosed with symptomatic coronavirus 2019 (COVID-19). Monoclonal antibody therapy (MAT) has been investigated as a therapeutic option for symptomatic COVID-19 patients in the outpatient setting at high-risk for progression to severe disease based on emergency use authorization (EUA) criteria. No published studies have compared outcomes for patients treated with different MAT for COVID-19.This was a single-center, retrospective cohort study at The Ohio State University Wexner Medical Center to compare COVID-19-related emergency room (ER) visits, admissions, and mortality at 30 days after MAT infusion for adult patients with symptomatic SARS-CoV-2 between November 16, 2020 and February 2, 2021 who received bamlanivimab versus those who received casirivimab-imdevimab. Statistical analysis used logistic regression analysis to determine the odds ratio (OR) to evaluate the relationship between patient characteristics, MAT, and outcomes.2 were associated with increased risk for COVID-19 related mortality at 30 days.The cohort included 943 patients with SARS-CoV-2 who received MAT, including 658 patients who received bamlanivimab and 285 who received casirivimab-imdevimab. Outcome results between patients who received bamlanivimab and casirivimab-imdevimab showed no statistically significant difference seen in the number of COVID-19 related ER visits , hospital admissions , or mortality . Multivariate analysis showed no statistically significant difference in outcomes between the groups when accounting for potential confounders. As reflected in the Table, chronic lymphocytic leukemia (CLL), gender, and asthma were associated with increased COVID-19 related ER visit within 30 days of infusion and age, chronic obstructive pulmonary disease, CLL, and lupus were associated with increased risk for COVID-19 related admission within 30 days of infusion. Age and obesity with body mass index greater than 35 mg/kgCOVID-19 related outcomes were similar when comparing patients with COVID-19 treated with bamlanivimab versus those treated with casirivimab-imdevimab.Mohammad Mahdee Sobhanie, M.D., Regeneron (Scientific Research Study Investigator)Regeneron Carlos Malvestutto, M.D., Lilly (Scientific Research Study Investigator)Regeneron Inc. (Scientific Research Study Investigator)ViiV Healthcare (Advisor or Review Panel member)"} +{"text": "N-isopropylacrylamide) (PNIPAm), design of alternative homo- and copolymer gels with controllable swelling properties has recently become a hot topic. This study focuses on equilibrium swelling of five potential candidates to replace PNIPAm in biomedical and biotechnological applications: poly(N-vinylcaprolactam), poly(vinyl methyl ether), poly hydrogels require these materials to be biocompatible, non-cytotoxic, and non-immunogenic. Due to serious concerns regarding potential toxicity of poly( T. TR gels of the low critical solution temperature (LCST) type gels form to a special group of stimuli-sensitive hydrogels whose equilibrium water uptake is strongly affected by temperature bic gels ) swell nove VPTT . Equilibove VPTT as smartove VPTT ,5, (ii) ove VPTT , and (iiove VPTT .N-isopropylacrylamide) (PNIPAm) is the most extensively studied member of the family of temperature-sensitive polymers. PNIPAm gels exhibit abrupt volume phase transitions at temperature close to Q at Poly(ove VPTT . As VPTTer gels) ,10 and (er gels) .N-isopropylacrylamide (NIPAm), N-isopropylmethacrylamide (NIPMAm), N-ethylacrylamide (NEAm), N-methyl acrylamide (NMAm), N-cyclopropylacrylamide (NCPAm) (VA-086) as an initiator . The exa et al. ). Figuren et al. ). In Fign et al. ).T, The equilibrium degree of swelling of a microgel nd PEGMA coincideN-vinylcaprolactam-co-2-methoxyethyl acrylate) P(VCL-MEA) microgels of aqueous solutions on monomers . FigureEquation with \u03c7\u02dc=Poly(vinyl methyl ether) (PVME) is a thermo-responsive polymer that undergoes volume phase transition close to the physiological temperature. Hydrogels prepared by cross-linking of commercially available poly chains (Gantrez AN-139) with poly(ethylene glycol) demonstrate good biocompatibility . Among bEquilibrium swelling diagrams on PVME gels are reported in a et al. ).VME gels A\u2013C, whilsite gel D. These PVME gels are characterized by rather large (exceeding 2) values of the equilibrium degree of swelling above VPTT: unlike PNIPAm gels that expel water molecules in the collapsed state (Equation ), PVME gT>Tc . This isPoly mixture of water and isobutanol by using ethylene glycol dimethacrylate as a cross-linker and Equilibrium swelling diagrams on four PDMAEMA homopolymer gels are presented in h et al. ).v/v) mixture of water and ethanol by using BIS .Q in the collapsed state .Two characteristic features of PDMAEMA gels: (i) very low values of their elastic moduli in the swollen state v/v) mixture of water and ethanol by using BIS . As AAmEquation with \u03c7\u02dc=Poly(2-oxazoline)s (POx) form a large family of thermo-responsive polymers that have attracted substantial attention in the past decade ,80,81,82A characteristic feature of POx gels is that these materials are temperature-sensitive, but do not reveal the volume phase transition . To examine this property, three sets of experimental data on homopolymer networks are analyzed .a et al. ).In t et al. ).Experimental data on poly(2-isopropenyl-2-oxazoline) (PIPOx) gel are depicted in a et al. ).T are reported in Although POx homopolymer gels do not exhibit volume phase transition, copolymerization of POx with hydrophobic monomers results in formation of networks where aggregation of segments takes place. In traditional TR gels copolymerized with hydrophobic monomers, VPTT decreases monotonically with molar fraction of comonomers hobicity . Unlike To confirm this assertion, three sets of experimental data are approximated on poly(2-ethyl-2-oxazoline-co-2-hydroxyethyl methacrylate) (PEtOx-HEMA), poly(2-ethyl-2-oxazo line-co-2-hydroxypropyl acrylate) (PEtOx-HPA), and poly(2-ethyl-2-oxazoline-co-methyl meth acrylate) (PEtOx-MMA) gels . Copolyma et al. ).Q is strongly influenced by hydrophobicity of comonomers and their molar fraction both below and above the VPTT point. The effect of comonomers is accounted for by three parameters: the ultimate value of the FH parameter Poly(2-(2-methoxyethoxy) ethyl methacrylate) mixture of water and ethanol by using EGDMA (molar fraction with respect to monomers 0.02) as a cross-linker, and KPS as an initiator .Q is calculated from the equationIn z et al. ). Given i et al. ).In a et al. ).T under consideration. To confirm this conclusion, the experimental dependencies All swelling diagrams in To demonstrate that POEGMA gels with small number of EG units (low hydrophilicity) do not show VPT, we analyze observations on a series of microgels prepared by polymerization of oligomers with various chain lengths. Observations in equalibrium water uptake tests on microgels prepared by polymerization of di(ethylene glycol) methyl ether methacrylate (molar mass 188 g/mol), tri(ethylene glycol) methyl ether methacrylate (molar mass 232 g/mol), and oligo(ethylene glycol) methyl ether methacrylate (molar mass 300 g/mol) are depicted in Experimental data in Equation below VPWe proceed with matching observations on POEGMA macro- and microgels reported in r et al. ).v/v) mixture of water and ethanol under ambient conditions . Figurec et al. ).To analyze equilibrium swelling of P. ExperiT is correctly described by Equation (Equation in the eThe other set of observations is presented in -Garrido ).The dimensionless elastic modulus ymer gel A. AfterwT in To confirm that P.T . The comonomers . ChangesEquation with A cT is correctly described by Equation methyl ether acrylate) P(MEA-OEGA) microgels with two molar fractions monomers . The gelu et al. ). FigureEquation in the eThis study deals with the analysis of equilibrium swelling of thermo-responsive homo- and copolymer gels to be employed in biomedical applications. Due to the concern regarding cytotoxicity of substituted acrylamide gels, we focus on the response of five groups of biocompatible hydrogels with poly(vinylcaprolactam), poly(vinyl methyl ether), poly(dimethylaminoethyl methacrylate), poly(oxazoline)s, and poly(methoxyethoxy ethyl methacrylate) as temperature-sensitive monomers. The aim is to compare their equilibrium water uptake curves with those on substituted acrylamide gels and to assess how molar fraction of comonomers affects VPTT of these gels.For this purpose, a unified model is developed for equilibrium swelling of TR gels. The model is grounded on the conventional scenario, according to which the Flory\u2013Huggins parameter The entire set of biocompatible thermo-responsive gels under consideration can be split into three groups depending on the strength of hydrophobic interactions between segments.TR gels with strong hydrophobic interactions (PNIPAm and PVCL) demonstrate abrupt volume phase transitions. Their homo- and copolymer gels expel practically all water molecules when they are in the collapsed state .TR gels with intermediate hydrophobic interactions (PMVE and PDMAEMA) show sharp volume phase transitions from the swollen state into a sponge-like state with degrees of swelling above VPTT ranging from 2 to 10 . This feThe volume phase transition temperature of copolymer gels prepared by copolymerization of TR monomers with strong and intermediate hydrophobic interactions and temperature-insensitive monomers is adequately predicted by Equation , see FigTR gels with weak hydrophobic interactions demonstrate sharp volume phase transitions. TR gels manufactured by copolymerization of MEO"} +{"text": "Repeat-associated non-AUG (RAN) translation was discovered in 2011 in spinocerebellar ataxia type 8 (SCA8) and myotonic dystrophy type 1 (DM1). This non-canonical form of translation occurs in all reading frames from both coding and non-coding regions of sense and antisense transcripts carrying expansions of trinucleotide to hexanucleotide repeat sequences. RAN translation has since been reported in 7 of the 53 known microsatellite expansion disorders which mainly present with neurodegenerative features. RAN translation leads to the biosynthesis of low-complexity polymeric repeat proteins with aggregating and cytotoxic properties. However, the molecular mechanisms and protein factors involved in assembling functional ribosomes in absence of canonical AUG start codons remain poorly characterised while secondary repeat RNA structures play key roles in initiating RAN translation. Here, we briefly review the repeat expansion disorders, their complex pathogenesis and the mechanisms of physiological translation initiation together with the known factors involved in RAN translation. Finally, we discuss research challenges surrounding the understanding of pathogenesis and future directions that may provide opportunities for the development of novel therapeutic approaches for this group of incurable neurodegenerative diseases. HTT) cause Huntington's disease (HD) [FMR1) gene in Fragile X-associated syndromes [C9ORF72) in the most common genetic forms of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) [Microsatellite expansions have been characterised in a large number of incurable neurodegenerative diseases subdivided into polyglutamine (polyQ) and non-polyglutamine (non-polyQ) disorders . Autosomase (HD) ,3 while ase (HD) ,5. Non-pyndromes ; thousanyndromes ,8 and tryndromes ; GAA repyndromes ; thousania (FTD) ,12. AltoThe transcription of repeat expansions located in coding and non-coding regions of genes generates pathological transcripts with polymorphic RNA-repeat sequences. The microsatellite loci are moreover bi-directionally transcribed in HD, DM, C9ORF72-ALS/FTD and in some SCAs and Fragile X-associated syndromes leading to expression of both sense and antisense repeat transcripts. These are thought to cause neuronal injury through complex intertwined mechanisms involving: (i) translation of proteins with expanded stretches of glutamine in poly-Q disorders; (ii) protein gain-of-functions caused by repeat-associated non-AUG (RAN) translation of toxic repeat proteins; (iii) RNA toxic gain-of-functions through the sequestration of RNA-binding proteins within RNA foci and onto repeat transcripts; (iv) protein loss-of-functions via haploinsufficiency .ATXN8OS (ATXN8 Opposite Strand) gene [ATXN8 were reported and shown to result in the expression and accumulation of a polyQ protein that forms neuronal inclusions [Polymorphic CAG repeat expansions in HD and poly-Q SCAs encode long stretches of poly-glutamine and the translation of proteins with polyQ domains. These promote misfolding/ aggregation, inhibit interactions with physiological binding protein partners and generate abnormal interactions with other proteins, mediating thus both toxic protein loss- and gain-of-functions . The nonnd) gene . Later, clusions .FMR1 cause Fragile X syndrome (FXS) [FMR2 gene; [CYSTB gene; [C9ORF72 lead to decreased expression levels of C9ORF72 mRNAs, encoding a protein involved in autophagy regulation [in vitro and in vivo models and post-mortem brains [Loss-of-function of the genes harbouring the repeat expansions can directly contribute to the pathophysiology of the microsatellite repeats. Over 200 CGG repeats in the 5\u2032-UTR of me (FXS) , the mosR2 gene; ) and myoTB gene; ). Loss-oTB gene; . Hexanucgulation , vesiclegulation ,27 and igulation ,29 in sem brains ,30\u201333. HC9ORF72-repeat transcripts retaining pathological expansions in intron-1 [RNA-mediated cellular toxicity results in either protein gain- and loss- of-functions via sequestration of RNA-processing proteins on repeat transcripts which may either be co-transcriptionally processed or aggregated into RNA foci. Protein loss-of-functions have been implicated in a wide range of expansion disorders via RNA-repeat sequestration of mRNA-binding proteins which may loose their normal cellular functions including: muscleblind-like splicing regulator (MBLN) and CUG-binding protein and ETR3-like factor (CELF) families of proteins in myotonic dystrophy ; MBLN anintron-1 .ATXN8 CAG-repeat transcripts were also characterised in SCA8 mice and human brain tissue [ATXN8OS CUG-repeat transcripts in transfected cells. Since this discovery, RAN translation of non-coding transcript regions was highlighted to occur from CGG repeats in FXTAS which produce toxic poly-glycine FMRpolyG and poly-alanine FMRpolyA proteins [HTT open reading frame leading overall to both canonical translation of the polyQ-expanded HTT mutant protein and to four RAN-translated sense and antisense homo-polymeric repeat proteins in HD [In 2011, Laura Ranum's group demonstrated that CAG-repeat transcripts lacking canonical AUG start codons are remarkably translated into homo-polymeric proteins in all frames by repeat-associated non-AUG (RAN) translation . RAN-tran tissue . Interesproteins and fromproteins . Moreoveysteine) . To dateDrosophila, mice, patient-derived neurons and other cell models [The recent discovery of RAN translation challenged the initial hypothesis that non-coding repeat expansion disorders are primarily caused by RNA foci and protein loss-of functions due to sequestration of RNA-binding proteins since polymeric repeat proteins exhibit aggregating properties and high levels of cytotoxicity in multiple cell and animal models of repeat expansion disorders. A range of polypeptides are produced through these mechanisms from the homo-polymeric proteins derived from trinucleotide repeat expansions through to dipeptide repeat proteins found in C9ORF72-ALS/FTD and SCA36 to more complex polypeptide repeat proteins expressed in transfected reporter cell models in SCA31 and DM2 . Repeat l models while pol models . Mechanil models , transcrl models , broad dl models , alteredl models and nucll models ,66,67, il models , mitochol models ,69 and gl models ,70,71 tol models .Translation involves three distinct mechanisms in Eukaryotes: (i) canonical AUG-driven cap-dependent initiation of translation for the vast majority of mRNAs; (ii) IRES-mediated cap-independent translation and (iii) canonical translation using alternative near-cognate codons.Met to form the 48S pre-initiation complex, which scans along the mRNA 5\u2032-UTR with the RNA-helicase eIF4A and its cofactors eIF4B and eIF4H unwinding any secondary structures until the AUG codon is reached. Further initiation proteins facilitate the joining of the 60S subunit to produce the initiating 80S complex [Translation initiation of canonical mRNAs is a complex process which requires many eukaryotic initiation factors (eIFs) and is one of the key rate-limiting steps for the regulation of gene expression . Transla complex . Regulat complex . A schemAlternative initiation mechanisms using internal ribosome entry site (IRES) elements are utilised by many viral and a growing number of cellular mRNAs . IRES elMet and methionine as the initiating amino acid [Leu at a CUG codon in the case of the major histocompatibility complex class I molecules [Met [Met or a GTP-independent manner through Leu-tRNALeu [Finally, near-cognate start codons , which differ from the AUG start codon by one nucleotide, initiate translation in mammalian cells at a much lower efficiency, using the non-AUG initiator tRNAiino acid or an elolecules ,82. Nearles [Met , when fi-tRNALeu of 55 nucleotides which is located in intron 1 and flanked by an AUG start codon and 2 downstream stop codons (UGA and UAA) Figure . Howevergh eIF2A (Figure in vitro in 13 reporter repeat expansion cell models and in patient bio-samples from seven diseases, including SCA8, DM1 [The pathogenesis driven by nucleic acid repeat expansions and RAN-translated products is complex and still poorly understood. Multiple mechanisms of neuronal injury involve toxic RNA gain-of-functions, haploinsufficiency as well as protein gain-of-functions via canonical translation of proteins with extended polyQ domains and/or RAN translation of toxic repeat polypeptides which have been characterised CA8, DM1 ,106, C9OCA8, DM1 ,120 and CA8, DM1 . HoweverCA8, DM1 ,41 howevCA8, DM1 . SimilarCA8, DM1 ,54,121. CA8, DM1 .If expression of polymeric repeat proteins can kill cells and animal models, it is difficult to evaluate which levels are RAN-translated in patients and which expression levels trigger toxicity depending on the nature of the repeat expansion, disease and cell type. Understanding the molecular mechanisms of RAN translation will be key to improving our understanding of pathogenesis. Additional mechanisms such as ribosome frameshifting events also need to be explored. For example, frameshifting was suggested to occur during translation of CAG-repeat expanded transcripts in the \u22121 frame in SCA3 as well Mechanisms of RAN translation and RAN-translated proteins/peptides still remain poorly characterised despite discovery in DM1/SCA8 patient samples in 2011, and later in C9ORF72-ALS and HD in 2013 and 2015, respectively.RAN-translation occurs in absence of the canonical methionine start codon, in all frames, and from coding and non-coding regions of transcripts encoding proteins of various functions. So far, it is known to involve RNA secondary structures formed by repeat sequences and general translation initiation factors, which exhibit/stimulate RNA-helicase activities, or play a role in the fidelity of start codon recognition.In the future, it will be fundamental to fully identify the RAN-translation machinery components and mechanisms in the context of the sequences flanking each microsatellite repeat expansions, as well as examine further the pathological contribution of RAN-translated products for the future development of much-needed disease treatments."} +{"text": "Given the limited collaborative international studies that evaluated COVID-19 in patients with cancer in comparison to patients without cancer, we aimed to determine the independent risk factors associated with increased 30-day mortality and the impact of novel treatment modalities in a large group of cancer and non-cancer patients with COVID-19 from multiple countries.We retrospectively collected de-identified data on cancer and non-cancer patients diagnosed with COVID-19 between January and November 2020, at 16 centers in Asia, Australia, Europe, North America, and South America. A logistic regression model was used to identify independent predictors of all-cause mortality within 30 days after COVID-19 diagnosis.p=0.04).Of the total 4015 COVID-19 confirmed patients entered, we analyzed 3966 patients, 1115 cancer and 2851 non-cancer patients. Cancer patients were older than non-cancer patients ; more likely to be pancytopenic , had pulmonary disorders, hypertension, diabetes mellitus. In addition, they were more likely to present with higher inflammatory biomarkers , but were less likely to present with clinical symptoms. By multivariable logistic regression analysis, cancer was an independent risk factor for 30-day mortality . Older age (\u226565 years) was the strongest predictor of 30-day mortality in all patients . Remdesivir was the only therapeutic agent independently associated with decreased 30-day mortality . Among patients on low-flow oxygen at admission, patients who received remdesivir had a lower 30-day mortality rate than those who were on high flow oxygen . Patients transfused with convalescent plasma within 1 day of diagnosis had a lower 30-day mortality rate than those transfused later Ansun Biopharma Chimerix Clinigen (Consultant)Genentech Janssen Karius (Grant/Research Support)Merck Molecular Partners Novartis (Grant/Research Support)Oxford Immunotec Partner Therapeutics (Consultant)Pulmotec Shire/Takeda Viracor (Grant/Research Support)Xenex (Grant/Research Support) Fareed Khawaja, MBBS, Eurofins Viracor (Research Grant or Support) Monica Slavin, MBBS,MD, F2G (Advisor or Review Panel member)Merck (Advisor or Review Panel member)Pfizer (Advisor or Review Panel member) Dimitrios P. Kontoyiannis, MD, Astellas (Consultant)Cidara Therapeutics (Advisor or Review Panel member)Gilead Sciences"} +{"text": "Increasingly, it has become apparent that mitochondria serve as reservoirs for distinct cues that trigger immune responses and that alterations in mitochondrial morphology may also tip infection outcomes. Furthermore, new data are foreshadowing pivotal roles for classic, homeostatic facets of this organelle as host-virus interfaces, namely, the tricarboxylic acid (TCA) cycle and electron transport chain (ETC) complexes like respiratory supercomplexes. Underscoring the importance of \u201chousekeeping\u201d mitochondrial activities in viral infection is the growing list of viral-encoded inhibitors including mimics derived from cellular genes that antagonize these functions. For example, virologs for ETC factors and several enzymes from the TCA cycle have been recently identified in DNA virus genomes and serve to pinpoint new vulnerabilities during infection. Here, we highlight recent advances for known antiviral functions associated with mitochondria as well as where the next battlegrounds may be based on viral effectors. Collectively, new methodology and mechanistic insights over the coming years will strengthen our understanding of how an ancient molecular truce continues to defend cells against viruses.Mitochondria are dynamic organelles vital for energy production with now appreciated roles in immune defense. During microbial infection, mitochondria serve as signaling hubs to induce immune responses to counteract invading pathogens like viruses. Mitochondrial functions are central to a variety of antiviral responses including apoptosis and type I interferon signaling (IFN-I). While apoptosis and IFN-I mediated by Mito(OXPHOS) .mitochondrial antiviral-signaling (MAVS) protein (\u2013pattern-recognition receptors (PRRs), like retinoic acid-inducible gene I (RIG-I) , that deCell death is an evolutionarily conserved means to restrict viral replication and protect the host organism \u201313. Deat\u201314\u2013cysteine-aspartic proteases initially synthesized as inactive procaspases family of proteins. Internal stress cues can stimulate pro-apoptotic BCL-2 proteins, such as BCL-2-interacting mediator of cell death (BIM) (xtra-large (BCL-xL) and myeloid cell leukemia-1 (MCL-1) can inhibit the progression of apoptosis by counteracting the activated proapoptotic proteins (BCL-2-associated X protein (BAX) and BCL-2 antagonist/killer (BAK). This leads to oligomerization of BAK and BAX on the outer mitochondrial membrane (OMM), which drives the formation of macropores in a process known as mitochondrial outer membrane permeabilization (MOMP) (cytochrome c (CytC). Binding of CytC to the scaffold protein apoptotic protease activating factor-1 (Apaf-1) in the cytoplasm promotes the formation of a supramolecular complex known as the apoptosome , 16. Antproteins . Apoptosn (MOMP) 14, 15), 15B-celoptosome 14, 18)B-cell lyoptosome 14, 22)B-cell lyA valuable signature illuminating essential host activities in immune defense, like apoptosis, is the identification of viral-encoded antagonists of specific cellular functions. Chiefly, the premise is based on the limited coding capacity of viruses. Namely, viruses are restricted to only encoding and maintaining what may be deemed required. Indeed, many viruses encode factors that counteract apoptosis through diverse strategies, one of which is mimicry . Viral mvaccinia virus (VACV) F1L, a BCL2 homolog, which interacts with BAK to inhibit BAK/BAX activation are encoded in the genomes of poxviruses, herpesviruses, iridoviruses, and asfarviruses , 24. vBCtivation .gasdermin (GSDM) family proteins (NOD-like receptors (NLRs) (absent in melanoma 2 (AIM2) (pathogen associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs). This sensing results in the formation of a large, multi-protein complex termed the inflammasome. The inflammasome consists of the PRR, an adaptor protein named apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and (pro)caspase-1 (interleukin-1 beta (IL-1\u03b2) and interleukin-18 (IL-18) as well as gasdermin D (GSDMD). Proteolytic processing of GSDMD licenses cell lysis mediated by this factor , 33 and 2 (AIM2) that detaspase-1 . Here, centerovirus 71 (EV71) degrades GSDMD during infection in 293T cells activates the NOD-, LRP, and pyrin domain-containing 3 (NLRP3) inflammasome (2+ influx via its viroporin 2B protein (herpes simplex virus (HSV-1) ICP27 counteracts this pathway . Sp. Spenter pathway . Caspase (mtROS) . In cont (mtROS) notwiths (mtROS) \u201346. Neveinner mitochondrial membrane (IMM). PB1-F2 induces apoptosis complex through interactions with voltage dependent anion channel (VDAC) and adenine nucleotide translocator 3 (ANT3). This pro-apoptotic function is hypothesized to be proviral by killing off immune cells accessory factors, which are micropeptides (<100 amino acids), encoded by divergent viruses circuit. The host MISTR factors are differentially regulated by stress signals including cytokines and hypoxia , which is induced by immune signals, attenuates chemical- and virus-induced apoptosis (supercomplexes (SC) . One posxes (SC) , a procexes (SC) .hepatitis C virus (HCV) NS3/4A, a serine protease, localizes to mitochondria, where it cleaves MAVS and thus blocks IFN-I induction due to volatile alterations of mitochondrial morphology. MAVS is the prototype for antiviral signaling at the mitochondrial surface. Given MAVS is instrumental for IFN-I signaling during RNA virus infection, several virus-encoded proteins that directly antagonize MAVS functions have been characterized. For example, nduction 63). Co. Cohepatnduction .In addition to HCV, other viruses encode mitochondrial proteins that can degrade MAVS. For instance, the rotavirus RNA capping enzyme, VP3, localizes to mitochondria, where it induces phosphorylation and subsequent proteasomal degradation of MAVS 65). Wh. Wh65). Along with signal transduction on the surface, release of molecules from within mitochondria results in gene expression changes. Prominent, emerging agonists are mtDNA and mtdsRNA. When these nucleic acids are leaked from mitochondria, they are sensed as DAMPs by proteins that stimulate antiviral gene expression programs. Documented means for the release of mtDNA involve the mPTP , 69, VDA71\u201374\u201377\u2013Tfam (+/-) in MEFs results in IFN-I induction that is dependent on cGAS/STING (dideoxycytidine (ddC), a nucleoside analog and an antiviral drug that reduces mtDNA copy number and nucleoid size. The aforementioned defines packaging of mtDNA as a regulator of its release (absent in melanoma-like receptors (ALRs), as well as membrane-bound PRRs such as toll-like receptors (TLRs) , 92. Hers (TLRs) \u201396 and A (TLRs) \u2013, 97 infl (TLRs) \u2013 via recr (TLRs) \u2013 99)..Tfam (+/mitogen activated protein kinase (MAPK) pathways induces \u039dF-\u03ba\u0392 signaling and \u039dF-\u03ba\u0392 target genes (cytosine-phosphate-guanine (CpG) dinucleotides, which is common to bacteria and DNA viruses (dengue virus (DENV), which has an RNA genome, also activates TLR9 signaling in human dendritic cells (DCs) due to mtDNA release during the infection and exo/endonuclease G (EXOG) (Epstein-Barr virus (EBV) DNA-binding protein, Zta, co-opts the mitochondrial single-stranded binding (mtSSB) protein, through direct binding, and relocalizes it to the nucleus, where the genome of this virus resides . Another resides . This inmelanoma differentiation-associated protein 5 (MDA5) detection of cytostolic mtdsRNA, which accumulates during MOMP in a BAX/BAK-dependent manner, leads to IFN-I expression through MAVS signaling (polynucleotide phosphorylase (PNPase) is dysfunctional. Forthcoming studies will surely shed light on the role and regulation of this DAMP in host defense.Along with mtDNA, mtdsRNA released into the cytosol is sensed by PRRs. Indeed, ignaling 106). N. NmelanoE. coliRas-like 1 (ERAL1) during RNA virus infection by BAX/BAK enhances MAVS signaling in vitro and in vivo during RNA virus infection . T. TE. colnfection . ERAL1 pnfection , 110. Comitofusin1 (MFN1), MFN2, and optic atrophy 1 (OPA1) (dynamin related protein 1 (DRP1) . In cont1 (DRP1) . Fission1 (DRP1) . MitochoParkinson\u2019s disease (PD) \u2013123. In sirtuin 3 (SIRT3) promotes mitochondrial fusion, which suppresses human cytomegalovirus (HCMV) viral production . D. Dsirtuioduction . Interacoduction . A subseoduction .hepatitis Bvirus (HBV) genome or the HBV master regulator, HBx, also triggers fission by inducing S616 phosphorylation of DRP1 and thus mitochondrial translocation . M. Mhepatilocation . This cllocation .2+ signaling, mitochondrial division, and metabolism (mitochondria-associated membrane (MAM), are critical in induction of MAVS signaling , triggers mitophagy by phosphorylation of Unc-51 Like Autophagy Activating Kinase 1 (ULK1) . Consisttivation .matrix protein (M) of human parainfluenza virus type 3 (HPIV3) binds a receptor for mitophagy initiation, Tu translation elongation factor mitochondrial (TUFM), to induce Parkin-independent mitophagy in HeLa cells. By an unclear mechanism, M-induced mitophagy leads to inhibition of IFN-I response . A. Amatrixresponse and funcresponse , 139.Metabolism has exploded as an important regulator of immunity, especially functions characteristic of immune cells discussed below, an area termed immunometabolism . Cells llactate dehydrogenase A (LDHA). Consistently, chemical inhibition and genetic deletion of LDHA enhance restriction of multiple RNA viruses both in vitro and in vivo . L. L147). More defined are major changes in metabolism occurring during viral infection . Upregulimmune-responsive gene 1 (IRG1) conversion of cis-aconitate to itaconate induces expression of anti-inflammatory genes by nuclear factor erythroid 2-related factor 2 (Nrf2) to counter pro-inflammatory responses (Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in Vero cells are infected with VACV . During VACV infection, VGF induces epidermal growth factor receptor (EGFR) signaling to activate the noncanonical signal transducer and activator of transcription 3 (STAT3) pathway (latency-associated nuclear antigen (LANA) encoded by Kaposi's sarcoma-associated herpesvirus (KSHV) . Lysine s (KSHV) , 161.nucleo-cytoplasmic large DNA viruses (NCLDV), which include giant viruses, have independently acquired not only specific TCA enzymes but also different combinations of TCA enzymes (citrate synthase (CS), aconitate (ACON), isocitrate dehydrogenase (ICD), isocitrate lyase (ICL), succinate dehydrogenase subunits A, B, and C , fumarate hydratase (FH), and malate dehydrogenase (MDH). Phycodnaviridae encodes SDB and SDC. Iridoviridae only encodes ICL. These data indicate extensive, yet unexplored biology for the TCA cycle during viral infection.Strikingly, diverse enzymes 53). No. Nonuclefatty acid synthase (FASN) from the cytosol to sites of viral replication . Specifirus (MV) . Anothermic acid . Furthermic acid 165). I. Imeasle2 to reduce O2 to water. As electrons are transferred between the ETC complexes, CI, III, and IV pump protons from the mitochondrial matrix into the intermembrane space (IMS). This process generates an electrochemical gradient that fuels the catalysis of ATP from ADP and phosphate by ATP synthase (Complex V) \u2013171. Theplex V) \u2013, 173. Asbone marrow-derived macrophages (BMDMs) from Parkin-deficient mice exhibit higher clearance of viral replication via the mtROS-NLRP3 axis appear important for IMM bending and cristae formation is a host negative regulator of SC formation . Recognition of the bacteria through TLR- and NLRP3-dependent pathways decreased levels of CI and CI-containing SCs. Functionally, reduced activity of CI was accompanied by increased activity of CII , more than 1,000 nuclear-encoded proteins have evidence for mitochondrial localization under homeostatic conditions , 67. We 195\u2013in vivo models in mice and novel exotic animals (Cell culture studies undoubtedly have been powerful in defining many aspects of known biology, yet the impact of different nutrients in cell culture media on mitochondrial functions during virus infection in cultured cells is largely unexplored. However, it has been established that replacing glucose with galactose in media serves to reprogram uninfected cultured cells from glycolysis to oxidative phosphorylation , 202. Th206\u2013 animals .Over the coming years, we suspect that many textbook activities associated with mitochondria will be revisited and perhaps even reimagined through studies of host\u2013virus interfaces. The fact that mitochondria are such a hot spot during pathogen infection is not altogether surprising when considering that this entire organelle is a shell of a former organism. Although now extinct, the ongoing battle between extant pathogens and hosts at this site may shed light on pivotal adaptations that occurred for modern complex cells to co-opt an energy-rich prokaryote."} +{"text": "To the EditorInterventions to address viral persistence of COVID-19 in immunosuppressed individuals are urgently needed for both patient and societal benefit , 3. ThesA 37-year-old Caucasian male with Wiskott-Aldrich syndrome (WAS) (IVS6 + 5G>A) developed anosmia and ageusia, followed by cough, dyspnoea, and fatigue (henceforth day 0). Community nasopharyngeal reverse-transcription PCR (RT-PCR) testing 1 day later was positive for SARS-CoV-2. Contact tracing suggested workplace exposure. Past medical history included childhood splenectomy for thrombocytopenia, eczema, recurrent infections, early bronchiectasis, asthma, and persistent molluscum contagiosum. Following diagnosis of specific antibody deficiency aged 24 years . A contemporaneous rise in RT-PCR cycle threshold (Ct) for detection by 6 cycles was noted across both viral nucleocapsid (N)-gene and ORF1a-gene targets. Given a difference of 1\u2009Ct unit is approximately equivalent to a factor of 2 in the number of viral particles per sample, this represents a 64-fold decrease in viral genetic material recovered by nasopharyngeal swabbing 2 weeks following initial vaccination. SARS-CoV-2 clearance followed 72 days following first therapeutic vaccination dose .Given persistent RT-PCR positivity, ongoing fluctuating symptoms and impact on both work and mental health of prolonged self-isolation, we adopted an individualized therapeutic approach. A growing range of therapeutic strategies have been suggested to counter SARS-CoV-2 infection in immunocompromised individuals including antibody therapies , hyperimmune IgG, and monoclonal antibodies) and antivirals. Rapid symptomatic and virological response have been reported following convalescent plasma therapy (CPT) in B cell-deficient individuals with protracted SARS-CoV-2, including where antiviral therapy with remdesivir has failed . HoweverThis case highlights persistent SARS-CoV-2 infection as an important outcome in immunodeficient individuals, beyond commonly used metrics of mortality or critical illness. Persistent infection is often associated with a symptomatic burden to the individual, further amplified here by the psychological and economic impacts of prolonged isolation. Diagnosis may be reliant on a high index of clinical suspicion in order to minimize the risk of cross-infection, particularly in the hospital setting. To our knowledge, this is the first description of therapeutic mRNA vaccination in the context of persistent SARS-CoV-2 infection. Importantly, vaccination proved well-tolerated and successfully elicited humoral and cellular responses which had not been induced after 120 days of PCR-confirmed SARS-CoV-2 infection. The timing of viral clearance following induction of host immunity strongly supports a causal role for vaccination; however, we cannot exclude that viral clearance may have occurred independently. Further studies are required to assess the reproducibility and generalizability of our findings, particularly given the likely heterogeneity of vaccine-induced immune response across the spectrum of inborn errors of immunity. In conclusion, we highlight the potential for therapeutic vaccination in persistent SARS-CoV-2 infection where sufficient immunological function remains to produce relevant humoral and T cell responses.Supplementary file1 (DOCX 185 KB)Below is the link to the electronic supplementary material."} +{"text": "This study examined elder mistreatment victims\u2019 experiences at the beginning of the COVID-19 pandemic. San Francisco Adult Protective Services (APS) caseworkers conducted phone interviews to inquire about clients\u2019 awareness of COVID-19 and unmet needs. Nine-hundred-and-thirty-four (71%) of 1,313 APS\u2019 past clients or their collaterals were interviewed, with 741 (79%) responding positively to COVID-19-awareness questions, and 697 (75%) having no unmet needs. Binary logistic regression with Firth adjusted maximum likelihood estimation method revealed that older persons (p < .05), self-neglectors (p < .05), and victims of neglect (p < .05) were less aware of COVID-19. Unmet needs varied by mistreatment type. Victims of isolation were more likely to have medical needs (p < .05), while victims of emotional abuse were more likely to report loneliness (p < .001). Collaboration between service providers is key in assisting victims experiencing unmet needs to live safely in a public health crisis."} +{"text": "Non-ideal donor pursuit rate was defined as the proportion of non-ideal donors at each OPO from whom consent for lung donation was requested with lower numbers indicating increased risk aversion. We estimated the correlation between non-ideal and overall donor pursuit using a Spearman correlation coefficient. Adjusted non-ideal donor pursuit rates were estimated using multivariable logistic regression. RESULTS/ANTICIPATED RESULTS: Overall, 18,333 deceased donors were included and classified as ideal or non-ideal. Among 58 OPOs, rates of non-ideal donor pursuit ranged from 0.24-1.00 Figure). Of 5 non-ideal characteristics, DCD and IRD status were associated with the most and least risk aversion, respectively. Non-ideal donor pursuit was strongly correlated with overall donor pursuit (r = 0.99). On adjusted analysis, older age , smoking history , low P/F ratio , and DCD status were all independently associated with significant risk aversion, corresponding to decreased rates of donor pursuit. DISCUSSION/SIGNIFICANCE OF IMPACT: OPOs differ in their levels of risk aversion in LTx and risk aversion is not uniform across selected categories of non-ideal lung donor. Consideration of new OPO performance metrics that encourage the pursuit of non-ideal lung donors is warranted.OBJECTIVES/GOALS: Lung transplant (LTx) candidates benefit from use of non-ideal donor organs. Each organ procurement organization (OPO) defines \u201cacceptable\u201d donor organs introducing unmeasured variation in donor pursuit. We characterized non-ideal donor pursuit among OPOs to identify drivers of risk aversion in LTx. METHODS/STUDY POPULATION: We queried the UNOS registry for adult donors who donated \u22651 organ for transplantation from 12/2007-12/2018. Non-ideal donors were those with any of age>50, smoking history \u226520 pack-years, PaO"} +{"text": "Multisystem Inflammatory Syndrome in Children (MIS-C) is a rare, life-threatening, hyperinflammatory condition presumed to follow SARS-CoV-2 infection. Whether MIS-C can also follow SARS-CoV-2 vaccination is not clear, making MIS-C an adverse event of special interest following immunization. Monitoring for post-vaccine MIS-C is complicated by the clinical overlap of MIS-C with numerous other inflammatory conditions including Kawasaki Disease, toxic shock syndrome, and viral myocarditis. A case definition for MIS-C was recently created with the Brighton Collaboration (BC). We aimed to determine the performance of the BC MIS-C case definition among a large, single-center MIS-C cohort.Retrospective review was performed for the first 100 MIS-C cases at our institution (May 2020-February 2021). All cases met the Centers for Disease Control and Prevention (CDC) case definition. Data on age, presentation, laboratory results and cardiac studies were collected and used to determine cases that fulfilled the BC case definition for MIS-C (see figure).Case Definition: Definite CaseOf 100 children (age < 21 years) diagnosed with MIS-C using the CDC case definition, 93 patients also fulfilled the BC definition. All 100 patients had elevated laboratory markers of inflammation and positive SARS-CoV-2 antibodies. However, 1 patient was excluded for significant respiratory symptoms (pulmonary hemorrhage), 5 were excluded due to only 1 clinical feature, and an additional patient was excluded for having none of the measures of disease activity. Among the 93 patients fulfilling the revised case definition, 88 (95%) met criteria for a definite case. Five of the 93 patients (5%) were considered probable cases, 1 reported only 1 day of fever and 4 had only 1 measure of disease activity.The original case definitions for MIS-C were created rapidly following the first emerging reports of this hyperinflammatory state. Knowledge of the varied clinical presentations of this disorder has grown substantially. Modification of the case definition to include features truly representative of MIS-C will allow for more precise diagnosis in the face of conditions which mimic MIS-C, and for accurate and reliable monitoring for adverse events following immunization.Flor M. Munoz, MD, Biocryst (Scientific Research Study Investigator)Gilead (Scientific Research Study Investigator)Meissa Moderna Pfizer Virometix"} +{"text": "After decades-long investigations of their biological functions, including potential as drug targets, only very recently has atomic-resolution information of NOX enzymes been made available. In this graphical review, we summarize the present structural biology understanding of the NOX enzymes afforded by X-ray crystallography and cryo-electron microscopy. Combined molecular-level insights predominantly informed by DUOX1 full-length Cryo-EM structures suggest a general structural basis for the control of their catalytic activity by intracellular domain-domain stabilization.The NADPH Oxidases (NOX) catalyze the deliberate production of reactive oxygen species (ROS) and are established regulators of redox-dependent processes across diverse biological settings. Proper management of their activity is controlled through a conserved electron transfer (ET) cascade from cytosolic NADPH substrate through the plasma membrane to extracellular O With re density ,22. Furtnumbers) , these c4csNOX5 TM domain was solved with X-ray crystallography defining the fold of the transmembrane helices and the Fe heme cofactors [2 [All NOX enzymes comprise a six-pass transmembrane \u0251-helix domain that complex two b-type hemes responsible for transferring electrons from NADPH through the cell membrane ,22. The ofactors , and pleckstrin homology-like domain (PHLD). Purification of mDUOX1-DUOXA1 complexes yields either a 1:1 heterodimer \u201d."} +{"text": "Acute kidney injury (AKI) subtypes combining kidney functional parameters and injury biomarkers may have prognostic value. We aimed to determine whether neutrophil gelatinase-associated lipocalin (NGAL)/hepcidin-25 ratio (urinary concentrations of NGAL divided by that of hepcidin-25) defined subtypes are of prognostic relevance in cardiac surgery patients.We studied 198 higher-risk cardiac surgery patients. We allocated patients to four groups: Kidney Disease Improving Global Outcomes (KDIGO)-AKI-negative and NGAL/hepcidin-25 ratio-negative (no AKI), KDIGO AKI-negative and NGAL/hepcidin-25 ratio-positive , KDIGO AKI-positive and NGAL/hepcidin-25 ratio-negative , KDIGO AKI-positive and NGAL/hepcidin-25 ratio-positive (combined AKI). Outcomes included in-hospital mortality (primary) and long-term mortality (secondary).We identified 127 (61.6%) patients with no AKI, 13 (6.6%) with subclinical, 40 (20.2%) with clinical and 18 (9.1%) with combined AKI. Subclinical AKI patients had a 23-fold greater in-hospital mortality than no AKI patients. For combined AKI vs. no AKI or clinical AKI, findings were stronger (odds ratios (ORs): 126 and 39, respectively). After adjusting for EuroScore, volume of intraoperative packed red blood cells, and aortic cross-clamp time, subclinical and combined AKI remained associated with greater in-hospital mortality than no AKI and clinical AKI . Cox proportional hazard models found a significant association of biomarker-informed AKI subtypes with long-term survival compared with no AKI .In the presence or absence of KDIGO clinical criteria for AKI, the urinary NGAL/hepcidin-25-ratio appears to detect prognostically relevant AKI subtypes.https://clinicaltrials.gov/ct2/show/NCT00672334.NCT00672334, clinicaltrials.gov, date of registration: 6th May 2008, Definition of AKI subtypes: subclinical AKI (KDIGO negative AND Ratio-positive), clinical AKI (KDIGO positive AND Ratio-negative) and combined AKI (KDIGO positive AND Ratio-positive) with urinary NGAL/hepcidin-25 ratio-positive cut-off at 85% specificity for in-hospital death. AKI, acute kidney injury. AUC, area under the curve. NGAL, neutrophil gelatinase-associated lipocalin. KDIGO, Kidney Disease Improving Global Outcomes Initiative AKI definition.The online version contains supplementary material available at 10.1007/s40620-021-01063-5. Acute kidney injury (AKI) is an independent risk factor for morbidity and mortality after cardiac surgery . AlthougThe use of cardiopulmonary bypass is associated with inflammation, hemolysis and the release of catalytic iron. Catalytic iron leads to the formation of injurious free radicals . NGAL anAccordingly, we aimed to test the hypothesis of whether an increased NGAL/hepcidin-25-ratio (urinary concentrations of NGAL divided by that of hepcidin-25) at ICU admission can be used to define AKI subtypes in cardiac surgery patients that carry specific and different associations with subsequent in-hospital and long-term mortality.The current study is a prospective observational study using samples from a previously approved, randomized, multicenter study , the BICFull study details have been previously described . In brieThe primary outcome measure was defined as in-hospital mortality. Secondary outcome was long-term mortality. We collected preoperative, peri-operative and postoperative data from medical records and calculated the EuroScore .Urine samples were obtained at ICU admission as previously described . NGAL coKDIGO criteria-based AKI status [during the first seven postoperative days (present/absent) and their NGAL/hepcidin-25 ratio status at ICU admission . Acute kidney injury was defined according to the criteria of the KDIGO Initiative [KDIGO-based AKI-negative and NGAL/hepcidin-25 ratio-negative (no AKI)KDIGO-based AKI-negative and NGAL/hepcidin-25 ratio-positive KDIGO-based AKI-positive and NGAL/hepcidin-25 ratio-negative KDIGO-based AKI-positive and NGAL/hepcidin-25 ratio-positive (combined AKI)Patients were grouped according to their I status during tFollow up of patients was done in July 2015. Patient\u2019s vital status was obtained at least 5 years after discharge and cross-referenced when possible. We made telephone calls and sought contact by mail at the patients\u2019 homes and through their physicians, and reviewed hospital and physicians\u2019 records. Survival was recorded, if confirmed by the patient or their contact person.t-test, Mann\u2013Whitney U test, Kruskal\u2013Wallis test, \u03c72 test, or Fisher\u2019s exact test were used where appropriate. The relationship of AKI subtypes with primary or secondary endpoint was calculated after adjustment for reference model in a logistic regression, and adjusted AUC values and odds ratios (ORs) with 95% confidence intervals were provided. The association of AKI subtypes with long-term patient survival was assessed using Cox proportional-hazard regression analysis adjusting for reference model. Kaplan\u2013Meier curves were plotted. Differences of curves were evaluated using the log-rank test. Information regarding missing data is provided in the table footnotes. Alpha was set at 0.05 and all tests were 2-tailed. SPSS, version 26.0 was used for statistical analysis.Study size was predetermined by the size of a previous RCT . MaximumTwo hundred patients underwent cardiac surgery at the German Heart Center among 350 patients enrolled in a previous multicenter randomized controlled trial . NGAL and hepcidin-25 concentrations were available for 198 patients. Patient flow through the study is shown in Supplemental Fig. p\u2009=\u20090.027; adjusted OR 3.737, 95% CI 1.746\u20137.998, p\u2009=\u20090.001), even after adjusting for EuroScore, volume of intraoperative packed red blood cells, and aortic cross-clamp time . The adjusted AUC of the reference model increased from 0.811 to 0.929 or 0.961 after including subclinical or combined AKI, respectively .Overall, 127 (61.6%) patients had no AKI, 13 (6.6%) had subclinical AKI, 40 (20.2%) had clinical AKI and 18 (9.1%) had combined AKI. Patients with subclinical AKI (NGAL/hepcidin-25 ratio\u2009\u2265\u20090.5 but no KDIGO-AKI) had a 23-fold increased risk of in-hospital mortality compared with patients without AKI, almost seven times greater than clinical AKI .Long-term follow-up showed separation of survival according to AKI subtypes Fig. . Subclinp\u2009<\u20090.001) and pooled subclinical and clinical AKI with long-term survival compared with no AKI as reference.After adjustment for EuroScore, volume of intraoperative packed red blood cells and aortic cross-clamp time, Cox proportional hazard regression analyses found a significant association of combined AKI had a much higher risk of in-hospital mortality than either no AKI or clinical AKI. Its long-term mortality was also higher than in patients with no AKI. The highest mortality rates were observed in patients with combined AKI (9.1% of patients) compared with no AKI or with other forms of AKI. The study findings remained essentially unchanged after adjustment for clinical risk score or important intraoperative covariates.\u2018subclinical AKI\u2019. Such subclinical AKI was associated with increased length of hospital stay and mortality [\u2018hemodynamic AKI\u2019 to describe a serum creatinine increase in the absence of increased kidney biomarker concentration, as may occur in cardiorenal syndrome or with renin\u2013angiotensin\u2013aldosterone system inhibition [A recent systematic review described a state of increased kidney biomarker concentration (NGAL) in the absence of KDIGO creatinine-based criteria for AKI as ortality . Howeverhibition . Recentlhibition \u20139. For ehibition . HoweverThe outcome pattern for AKI subtypes observed in our study was similar to that of AKI subtype definition using other urinary biomarkers , 9. SubcOur findings imply that the urinary NGAL/hepcidin-25 ratio at the end of surgery, alone or combined with creatinine- or urine output-based criteria, identifies a subpopulation of patients at high mortality risk. Such identification may provide an opportunity for differential therapeutic options , 25, andOur study has several strengths. We investigated typical patients at risk of adverse kidney outcome in a relatively homogeneous and well-defined patient cohort after cardiac surgery. These are the patients for whom allocation to distinct AKI subtypes may be crucial. Study results remained stable after adjustment for established risk scores and other important intraoperative covariates. We acknowledge some limitations. The number of events was limited thereby affecting generalizability of the study results. Hepcidin-25 measurement on a clinical laboratory platform is not yet available, thus large scale application of our research findings to practice is not yet possible. We were not able to provide direct measurement of glomerular filtration rate or histopathological findings of AKI subtypes to better delineate their pathophysiological meaning.In conclusion, the urinary NGAL/hepcidin-25-ratio appears to detect prognostically relevant AKI subtypes including subclinical and combined AKI. Cautiously considering the limited number of events in this study, subclinical AKI appears to detect patients with a type of otherwise undetected clinically highly relevant form of AKI. If confirmed in larger prospective studies, the urinary NGAL/hepcidin-25 ratio might enable the early identification of high-risk patients for future interventional studies.Supplementary file1 (DOCX 97 KB)Below is the link to the electronic supplementary material."} +{"text": "Coronavirus disease 2019 (COVID-19) has had a devastating impact on older adult nursing home residents (NHR). NHRs comprise greater than one-third of COVID-19 U.S. deaths, emphasizing the importance of engaging in end-of-life discussions. At South Texas Veterans Health Care System (STVHCS), we implemented early documentation of patient\u2019s Life-Sustaining Treatment (LST) or end-of-life goals-of-care preferences prior COVID-19 infection. We now aim to examine the association between early LST documentation (prior to COVID-19 diagnosis) and hospital admissions for COVID-19 by conducting a retrospective cohort study of Veteran NHRs at STVHCS from March 2020-January 2021. Inclusion criteria were NHRs with COVID-19 diagnosis, LST documentation, and clear timing of whether the LST documentation occurred before or after COVID-19 diagnosis. Logistic regression was used to determine the likelihood of hospitalization by whether LST was documented before or after COVID-19 diagnosis. 208 NHRs were diagnosed with COVID-19 and 160 (76.9%) had LST documentation. Of these, 148 were included in the analysis: 84 (56.8%) had a completed LST note prior to diagnosis and 64 (43.2%) after diagnosis. The hospitalization rate was 46% for those with LST prior to diagnosis compared to 78% in those with LST after diagnosis , showing that early LST documentation was associated with 76% lower likelihood of hospitalization. Early interventions for LST documentation can reduce hospitalization in high-risk populations. These findings may have implications for reducing unnecessary hospitalizations, diminishing healthcare costs, and resolving ethical dilemmas related to potential resource allocation during a pandemic."} +{"text": "Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies decay but persist 6 months postvaccination; lower levels of neutralizing titers persist against Delta than wild-type virus. Of 227 vaccinated healthcare workers tested, only 2 experienced outpatient symptomatic breakthrough infections, despite 59/227 exhibiting serologic evidence of SARS-CoV-2 infection, defined as presence of nucleocapsid protein antibodies. We report bAb and nAb levels as well as clinically overt and asymptomatic breakthrough infections that occurred among US healthcare workers in the Prospective Assessment of SARS-CoV-2 Seroconversion (PASS) study in compliance with all applicable federal regulations governing the protection of human participants. Written consent was obtained from all study participants.For the PASS study, we enrolled and followed generally healthy, adult healthcare workers (HCWs) at Walter Reed National Military Medical Center who were seronegative for IgG to SARS-CoV-2 spike glycoprotein (spike) and had no history of COVID-19, as previously described , we interpolated IgG levels against an internal standard curve calibrated to the Human SARS-CoV-2 Serology Standard Figure 1>15 days per month, and had similar rates of direct interaction with COVID-19 positive patients (monthly average of 47% for vaccinated and 45% for unvaccinated participants).Excluding persons infected before January 31, 2021, the study followed 227 participants fully vaccinated with BNT162b2 vaccine and 17 unvaccinated participants. Participants were generally healthy, had a mean age of 41.7 (range 20\u201369) years, and were predominantly women . VaccinaWe observed seroconversion in all participants 1 month after the second vaccine dose , panel AIn addition to spike IgG bAbs, we also monitored for seroconversion of IgG bAbs to NP. Of vaccinated participants, 26.0% (59/227) had NP seroconversion during March\u2013August 2021 . Only 2 In this prospective cohort study of generally healthy, adult HCWs, we found that SARS-CoV-2 spike IgG bAbs and nAbs induced by BNT162b2 mRNA COVID-19 vaccination wane but remained detectable through 6 months after vaccination, corroborating results of another study enabled detection of asymptomatic and pauci-symptomatic SARS-CoV-2 exposures. Although our study was powered to show clear differences in antibody titers over time, limitations include the moderate size of the cohort and the small number of unvaccinated participants. Further, seasonal human coronavirus (HCoV) infections may drive cross-reactive IgG responses against SARS-CoV-2 NP. We mitigated the likelihood of HCoV-driven false-positives by using convalescent serum samples from persons with PCR-confirmed HCoV infections to establish the threshold for SARS-CoV-2 NP IgG positivity, which had a specificity of 94% in our multiplex assay . In a separate study, NP seroconversion reportedly occurred in only 71% of PCR-confirmed vaccine-breakthrough infections of healthy adults 6 months after vaccination with BNT162b2. Neutralizing activity against Delta virus was lower,; only 47% (23/49) of participants maintained nAb titers above the lowest dilution at 6 months postvaccination. The decrease in nAb does not necessarily mean that persons have lost protection against severe COVID-19, however, given that nAb titers required for protection remain unknown and virus neutralization is only 1 function of antibodies. In addition, memory B cells and T cells have been detected 8\u201312 months after SARS-CoV-2 infection, demonstrating that adaptive immune memory can be long-lasting (Additional information about durability of antibody response and frequency of SARS-CoV-2 infection 6 months after COVID-19 vaccination in healthcare workers."} +{"text": "Moreover, optical coherence tomography (OCT) revealed a disrupted fibrous cap with a residual lipid-rich plaque (LRP). The minimum lumen area (MLA) was 4.4 mm2 , a minimum fibrous cap thickness increase, disrupted fibrous cap disappearance, and ruptured plaque healing with an expanding MLA (10 mm2) for 99% stenosis of the proximal right coronary artery. He underwent a successful PCI with drug-eluting stent implantation under the guidance of near-infrared spectroscopy intravascular ultrasound (NIRS-IVUS). However, non-culprit ruptured plaques were identified distal to the culprit lesion Fig.\u00a0A. The mamm2 Fig.\u00a0A. An aggPrevious intravascular imaging studies reported on the presence of plaque ruptures in both culprit and non-culprit lesions in patients with acute coronary syndrome (ACS). , 2 Non-cIn our patient, NIRS-IVUS and OCT revealed morphological details and drastic changes of the ruptured non-culprit plaque with a residual LRP. A combination of an aggressive lipid-lowering therapy, consisting of a strong statin and a PCSK9 inhibitor, might have healed and stabilized the non-culprit vulnerable ruptured plaques, without significant stenosis.These imaging findings support the possibility of administering lipid-lowering therapy for the healing and stabilization of non-culprit ruptured plaques and provide historical evidence for its clinical benefits."} +{"text": "Antibiotic prescribing for pyogenic liver abscess(es) (PLA) is highly variable with literature primarily aimed at assessing surgical intervention with a scarcity of data for antibiotic selection and duration of therapy. Given the lack of data, there is no clear consensus for treatment options or length of treatment. Our Antimicrobial Support Network (ASN) in collaboration with the hepatopancreatobiliary (HPB) team created a treatment and management algorithm to guide duration of therapy and antibiotic selection. C. difficile and multi-drug resistant organism (MDRO) colonization at 90 days from diagnosis, and hospital length of stay (LOS). Additional a priori subgroup analyses of duration of therapy, treatment failure, all-cause and abscess-related readmissions were also conducted based on surgical intervention. A retrospective, quasi-experimental cohort study was performed at Carolinas Medical Center in hospitalized patients with PLA with an HPB and/or infectious diseases consult. The primary outcome was antipseudomonal beta-lactam days of therapy (DOT) per 1000 patient days (PD) in the pre-versus post-intervention group. Secondary outcomes included rates of treatment failure at 90 days, 90-day all-cause and abscess-related hospital readmission, C. difficile or MDRO rates.A total of 93 patients were included, 49 patients in the pre-intervention group and 44 patients in the post-intervention group. Baseline characteristics were similar between the groups. The majority of liver abscesses were unilocular and monomicrobial. Anti-pseudomonal beta-lactam DOT per 1000 PD decreased by 13.8% (507.4 versus 437.5 DOT/1000 PD). Treatment failure occurred in 30.6% of pre-intervention patients and 18.2% of post-intervention patients (p = 0.165). Patients in the post-intervention group were discharged a median of 2.4 days sooner than the pre-intervention period . No significant differences resulted in 90-day readmission rates or 90-day Table 1. Primary Outcome for Patients with Pyogenic Liver Abscesses Treated Pre- and Post-Antibiotic Stewardship AlgorithmTable 2. Secondary Outcomes for Patients with Pyogenic Liver Abscesses Treated Pre- and Post-Antibiotic Stewardship AlgorithmThe implementation of a PLA treatment and management algorithm led to a decrease in anti-pseudomonal beta-lactams without impacting clinical outcomes and a trend towards decreased LOS. All Authors: No reported disclosures"} +{"text": "Background: Alcoholic liver disease (ALD) caused by chronic ethanol overconsumption is a common type of liver disease with a severe mortality burden throughout the world. The pathogenesis of ALD is complex, and no effective clinical treatment for the disease has advanced so far. Prolonged alcohol abstinence is the most effective therapy to attenuate the clinical course of ALD and even reverse liver damage. However, the molecular mechanisms involved in alcohol abstinence-improved recovery from alcoholic fatty liver remain unclear. This study aims to systematically evaluate the beneficial effect of alcohol abstinence on pathological changes in ALD.Methods: Using the Lieber-DeCarli mouse model of ALD, we analysed whether 1-week alcohol withdrawal reversed alcohol-induced detrimental alterations, including oxidative stress, liver injury, lipids metabolism, and hepatic inflammation, by detecting biomarkers and potential targets.Results: Alcohol withdrawal ameliorated alcohol-induced hepatic steatosis by improving liver lipid metabolism reprogramming via upregulating phosphorylated 5\u2032-AMP -activated protein kinase (p-AMPK), peroxisome proliferator-activated receptor-\u03b1 (PPAR-\u03b1), and carnitine palmitoyltransferase-1 (CPT-1), and downregulating fatty acid synthase (FAS) and diacylglycerol acyltransferase-2 (DGAT-2). The activities of antioxidant enzymes, including superoxide dismutase (SOD) and glutathione peroxidase (GSH-px), were significantly enhanced by alcohol withdrawal. Importantly, the abstinence recovered alcohol-fed induced liver injury, as evidenced by the improvements in haematoxylin and eosin (H&E) staining, plasma alanine aminotransferase (ALT) levels, and liver weight/body weight ratio. Alcohol-stimulated toll-like receptor 4/mitogen-activated protein kinases (TLR4/MAPKs) were significantly reversed by alcohol withdrawal, which might mechanistically contribute to the amelioration of liver injury. Accordingly, the hepatic inflammatory factor represented by tumour necrosis factor-alpha (TNF-\u03b1) was improved by alcohol abstinence.Conclusion: In summary, we reported that alcohol withdrawal effectively restored hepatic lipid metabolism and reversed liver injury and inflammation by improving metabolism reprogramming. These findings enhanced our understanding of the biological mechanisms involved in the beneficial role of alcohol abstinence as an effective treatment for ALD. Alcoholic liver disease (ALD), one of the main causes of chronic liver disease, ranges from alcoholic fatty liver (AFL) to alcohol hepatitis, alcoholic hepatic fibrosis, alcoholic cirrhosis, and even hepatocellular carcinoma . ALD is de-novo biosynthesis of free fatty acids (FFAs) caused by alcohol consumption play an important role in the development of AFL . All killings were performed under avertin anaesthesia, and efforts were made to minimise animal suffering.n = 8 per group): pair-fed (PF) group, alcohol-fed (AF) group, abstinence control (PF-PF) group, and alcohol abstinence (AF-PF) group. Mice in the PF or AF group were fed with isocaloric control liquid diet or ethanol-containing Lieber-DeCarli diet for 4\u00a0weeks, respectively weighing 18.51 \u00b1 1.21\u00a0g were group-housed in cages in a temperature-controlled vivarium (22 \u00b1 2\u00b0C) and maintained on a 12-h light/dark cycle. After 2\u00a0weeks of adaption, the 8-weeks-old mice were randomly divided into four groups ( States) . PF-PF aPlasma alanine aminotransferase (ALT), triglyceride (TG), glycerol, and FFAs levels were determined by using commercial kits from Nanjing Jiancheng Bioengineering Institute according to the manufacturer\u2019s instructions.Liver malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) were measured with commercial kits based on enzymatic methods .Histological examination was performed as previously described . In brieProtein isolation from liver tissue and Western blot analysis were performed as previously described . The folTotal RNA extraction, reverse transcription, and real-time polymerase chain reaction were performed as previously described . Brieflyp < 0.05.Statistical analysis was performed using one-way analysis of variance (ANOVA) and followed by post-hoc test with Fisher\u2019s least significant difference (LSD). Data were presented as means \u00b1 SD. Differences between each group were considered to be statistically significant at A schematic diagram of the research design was shown in We subsequently detected lipids levels in animal blood. Our data showed that chronic alcohol exposure significantly elevated plasma TG and FFAs levels . The glyOxidative stress caused by alcohol metabolism is an important pathological mechanism of ALD. Therefore, we measured the levels of oxidative stress products and the activities of antioxidant enzymes in the liver. The results showed that 1\u00a0week alcohol withdrawal was not sufficient to reverse alcohol-induced excessive formation of MDA in the liver . Howeverde novo synthesis of fatty acids, such as mature-SREBP-1c and phosphorylated-ACC were activated by chronic alcohol intake promotes fatty acid biosynthesis by upregulating the expression of lipogenesis-related genes, including ACC, FAS and DGAT-2 (via decreasing the de novo lipogenesis.In the initial stage of ALD, the imbalance between FA synthesis and metabolism contributed to hepatic steatosis . SREBP-1d DGAT-2 . The expd DGAT-2 . ConsistLipotoxicity is a vital pathological factor in ALD. Previous studies showed that TLR4/MAPKs pathway was mechanistically involved in lipotoxicity-induced hepatotoxicity . TLR4 acvia upregulating Bax and down-regulating Bcl-2, leading to mitochondrial permeabilization increase, release of cytochrome c and activation of caspases 3 (Mitochondrial dysfunction has been detected in both experimental animal and patients with ALD . Chronicspases 3 . In thisTaken together, alcohol withdrawal effectively reversed chronic alcohol intake-caused liver metabolic reprogramming, and in hence improved hepatic steatosis, liver injury, and inflammation. Our findings provide a theoretical basis for the understanding of alcohol withdrawal on ALD recovery."} +{"text": "OBJECTIVES/GOALS: Variants in voltage-gated sodium channels (VGSC) are a common cause of severe early onset epilepsy. Changes in CSF neurotransmitters (NT) were identified in 2 cases of VGSC-related epilepsy. Here we investigate NT changes in patients and a novel mouse model of VGSC-related epilepsy. METHODS/STUDY POPULATION: We conducted a single site IRB approved retrospective chart review of patients with VGSC-related epilepsy who underwent CSF NT testing for diagnostic purposes. In parallel, we examined NT levels from the brains of wild-type (WT) and a novel VGSC-related epilepsy mouse model after obtaining IACUC approval. We rapidly isolated forebrain, cortex, striatum, and brainstem from 5-6 animals per sex and genotype. A combination of HPLC with electrochemical detection and mass spectrometry were used to quantify NT levels from brain samples. RESULTS/ANTICIPATED RESULTS: We identified 10 patients with VGSC-related epilepsy who received CSF NT testing. Two of these patients had abnormal NT results including changes to dopamine (DA) or serotonin (5-HT) metabolites. We analyzed NT levels from four brain regions from male and female WT and VGSC-related epilepsy mice. We anticipate that most of the NT levels will be similar to WT, however subtle changes in the DA or 5-HT metabolites may be seen in VGSC-related epilepsy. DISCUSSION/SIGNIFICANCE OF IMPACT: Patients with VGSC-related epilepsy often have autism spectrum disorder, sleep, and movement disorders. Understanding the role of aberrant NT levels in VGSC-related epilepsy may provide additional therapeutic targets that address common neuropsychological comorbidities as well as seizures."} +{"text": "P\u2009<\u20090.05 to P\u2009<\u20090.0001) suppressed MERS-CoV replication in human lung adenocarcinoma and human small airway epithelial (HSAEC) cells. Both MERS-CoV and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection increased the total and phosphorylated forms of hnRNP C to activate the downstream CRK-mTOR pathway. Treatment of MERS-CoV- (IC50: 0.618 \u00b5M) or SARS-CoV-2-infected (IC50: 1.233 \u00b5M) Calu-3 cells with the mTOR inhibitor OSI-027 resulted in significantly reduced viral loads. Collectively, our study identified hnRNP C as a key regulator of MERS-CoV-perturbed circRNAs and their cognate mRNAs, and the potential of targeting hnRNP C-related signalling pathways as an anticoronaviral strategy.Host circular RNAs (circRNAs) play critical roles in the pathogenesis of viral infections. However, how viruses modulate the biogenesis of host proviral circRNAs to facilitate their replication remains unclear. We have recently shown that Middle East respiratory syndrome coronavirus (MERS-CoV) infection increases co-expression of circRNAs and their cognate messenger RNAs (mRNAs), possibly by hijacking specific host RNA binding proteins (RBPs). In this study, we systemically analysed the interactions between the representative circRNA\u2013mRNA pairs upregulated upon MERS-CoV infection and host RBPs. Our analysis identified heterogeneous nuclear ribonucleoprotein C (hnRNP C) as a key host factor that governed the expression of numerous MERS-CoV-perturbed circRNAs, including hsa_circ_0002846, hsa_circ_0002061, and hsa_circ_0004445. RNA immunoprecipitation assay showed that hnRNP C could bind physically to these circRNAs. Specific knockdown of hnRNP C by small interfering RNA significantly ( Eukaryotic splicing machineries tightly modulate the outputs of pre-messenger RNAs (mRNAs) in mammalian cells by initiating canonical splicing to produce linear mRNAs or back-splicing to produce circular RNAs (circRNAs) , 2. HumaThe roles of host circRNAs in viral infections have been increasingly recognized in recent years , 11. HowMERS-CoV (strain HCoV-EMC/2012) was kindly provided by Ron Fouchier . Clinical isolates of SARS-CoV (GZ50) and SARS-CoV-2 HKU-001a were available at the Department of Microbiology at The University of Hong Kong as we previously described , 14. CalThe in-house guinea pig anti-MERS-CoV nucleocapsid protein (NP) serum and negative control guinea pig serum were used for MERS-CoV NP detection in flow cytometry experiments as we previously described . Mouse aP values (cPs) were calculated based on the expression level of these DE circRNAs and mRNAs at three time points. CircRNAs and their cognate mRNAs that stably expressed [average expression >10 transcripts per million (TPM)/fragments per kb for a million reads (FPKM)], both significantly upregulated and positively co-expressed were filtered out as potential MERS-CoV pathogenic circRNA-mRNA pairs for further study. To predict the splicing factors regulating their expression, correlation analysis between DE splicing factors and DE circRNAs or mRNAs identified above was performed simultaneously by the same method. At the same time, RBPmap (http://rbpmap.technion.ac.il/) database was used to predict the potential binding motifs between circRNAs and splicing factors to furtherly filter the dominant functional splicing factors.Firstly, differential expression (DE) analysis was performed to identify DE splicing factors, DE circRNAs and their cognately expressed mRNAs according to our previous methods . Then, Phttps://circinteractome.nia.nih.gov/) (Supplementary Table 1). Fold changes of circRNA and host gene expression were calculated through the comparative CT (2\u2212 \u0394\u0394CT) method and normalized with the expression of housekeeping gene GAPDH. For viral load quantification, RT-qPCR assays were performed for MERS-CoV, SARS-CoV, and SARS-CoV-2 as we described previously were identified . Then, we synthesized and transfected them into Calu-3 cells (P\u2009<\u20090.05 to P\u2009<\u20090.001). The inhibition of viral load was over 1 log in supernatant samples collected at 24 hpi (P\u2009<\u20090.001) by the area-under-the-curve calculation. hnRNPs could directly interact with viral RNAs, structural and non-structural proteins [in vitro binding between human hnRNP A1 and SARS-CoV NP [Recent studies have uncovered the role of circRNAs as host pathogenic factors utilized by multiple viruses , 11, 37.-3 cells A and fou-3 cells B. Among -3 cells B. Knockdt 24 hpi C. Moreovt 24 hpi D. MERS-Cproteins , 42. PreS-CoV NP , as wellS-CoV NP . In the P\u2009<\u20090.05) and extracellularly (P\u2009<\u20090.001) relative to scrambled siRNA-transfected cells during SARS-CoV-2 infection. This inhibition might have resulted from the similar alterations of MERS-CoV-perturbed proviral circRNAs and cognate mRNAs, as we observed diminished hsa_circ_0004445 and its cognate gene CRK after hnRNP C knockdown during SARS-CoV-2 infection, similarly to MERS-CoV infection (P\u2009<\u20090.05) but did not significantly suppress the expression of hsa_circ_0004445 during SARS-CoV infection and SARS-CoV-2 by about 28.6% (P\u2009<\u20090.01). We observed that CRK depletion negatively regulated the phosphorylation of mTOR during MERS-CoV and SARS-CoV-2 infections and Src homology 3 (SH3) domains upon different stimuli . Our prertmannin , 58. Corrtmannin , exhibitin vivo evaluation of the effect of hnRNP C inhibition or inhibitors targeting the CRK-mTOR signaling pathway should be considered for MERS-CoV and other coronavirus infections.Collectively, our study identified a novel role of the RBP hnRNP C in the regulation of MERS-CoV-perturbed circRNAs. The intervention of the downstream signalling pathway of hnRNP C by the use of the mTOR inhibitor OSI-027 led to significantly reduced coronavirus replication. Further Click here for additional data file."} +{"text": "Subjective feelings of energy and tiredness may reflect different neural processes. Functional connectivity (FC) was measured in 272 HealthABC participants via resting state functional MRI in striatal-associative, striatal-limbic and striatal-sensorimotor networks. Subjective energy level (scored 1-10) and tiredness (tired/not-tired) during the prior month were collected via self-report from year 2 to year 10 . Participants who never reported being tired during follow-up (N=119) had significantly lower FC in the striatal-limbic network . Participants with stable energy level over time had significantly higher FC in the striatal-associative network . Associations were similar when adjusted for brain atrophy, demographics, and education. Although based on subjective measures, the distinct spatial patterns of these associations support our hypothesis that neural basis of energy and fatigue may differ."} +{"text": "O-methylation of small molecules is a common modification widely present in most organisms. Type III polyketides undergo O-methylation at hydroxyl end to play a wide spectrum of roles in bacteria, plants, algae, and fungi. Mycobacterium marinum harbours a distinctive genomic cluster with a type III pks gene and genes for several polyketide modifiers including a methyltransferase gene, mmar_2193. This study reports functional analyses of MMAR_2193 and reveals multi-methylating potential of the protein. Comparative sequence analyses revealed conservation of catalytically important motifs in MMAR_2193 protein. Homology-based structure-function and molecular docking studies suggested type III polyketide cores as possible substrates for MMAR_2193 catalysis. In vitro enzymatic characterization revealed the capability of MMAR_2193 protein to utilize diverse polyphenolic substrates to methylate several hydroxyl positions on a single substrate molecule. High-resolution mass spectrometric analyses identified multi-methylations of type III polyketides in cell-free reconstitution assays. Notably, our metabolomics analyses identified some of these methylated molecules in biofilms of wild type Mycobacterium marinum. This study characterizes a novel mycobacterial O-methyltransferase protein with multi-methylating enzymatic ability that could be exploited to generate a palette of structurally distinct bioactive molecules. L-methionine (SAM) to a nucleophile containing carbon (C), sulfur (S), nitrogen (N) and oxygen (O) - at m/z 122.9031 and m/z 136.9603 from resorcinol primed reaction. Reaction with phloroglucinol led to product ions with [M-H]- at m/z 138.9629, m/z 152.9535 and m/z 166.9535 in UFLC peaks shown in Fig 3F. Olivetol primed reactions formed product ions with [M-H]- at m/z 191.1410 and m/z 207.0473 in profile observed in Fig 3G.Our Fig 4A\u20134G, confirmed these product ions as mono-methylated resorcinol ; di-methylated resorcinol ; mono-methylated phloroglucinol ; di-methylated phloroglucinol ; tri-methylated phloroglucinol ; mono-methylated olivetol and di-methylated olivetol . Table 2 in S1 Text provides details of mass spectrometric characterization of the methylated molecules. Our functional characterization of MMAR_2193 as summarized in Fig 4H provided evidence for the O-methylation potential of the mycobacterial protein. Notably, MMAR_2193 displayed a potential to perform multiple O-methylations on a single substrate molecule.Tandem MS/MS analyses as shown in \u03b1-alkyl pyrones that are many times co-produced with alkyl-resorcinols or acyl-phloroglucinols. Our biochemical studies with resorcinol, phloroglucinol and olivetol provided evidence for the O-methylation potential of MMAR_2193. We further set out to examine MMAR_2193 mediated possible methylation of \u03b1-alkyl pyrones. MtbPKS18, a type III PKS from Mycobacterium tuberculosis has been previously characterized to catalyze biosynthesis of long-chain triketide- and tetraketide-\u03b1-alkyl pyrones in cell-free assays - at m/z 335.1987 and m/z 377.2351. Tandem MS/MS of these precursor ions confirmed the identity of these molecules as methylated \u03b1-alkyl pyrones, 16A\u2019 and 16B\u2019, respectively . Our studies displayed the ability of MMAR_2193 to catalyze O-methylations on \u03b1-alkyl pyrone polyketides.Type III polyketide synthases catalyze formation of products with diverse scaffolds. The most commonly biosynthesized products include triketide-and tetraketide-e assays . In a siO-methylated polyketides in wild-type Mmar biofilms. Biofilm pellicles were developed for Mmar cells and extracted for HRMS metabolomics analyses. A multiple-reaction-monitoring (MRM) based metabolomics approach identified two ions of [M-H]- at m/z 279.2257 and m/z 335.1987. Tandem MS/MS confirmed identities of O-methylated triketide-\u03b1-lauroylpyrone (12A\u2019) and triketide-\u03b1-palmitoylpyrone (16A\u2019) corresponding to the two identified ions, respectively . Our results revealed presence of O-methylated polyketides in wild-type Mmar biofilms.Mmar harbours several genomic clusters with polyketide biosynthetic genes including the MMAR_2193 cluster that is exclusively identified in pathogenic species. Biofilms have lately been associated with several pathogenic diseases \u201335 and ammar_2193, a probable methyltransferase was identified to be clustered with a type III pks and other modifying genes in pathogenic genomes. Our homology-based sequence/structure analyses predicted a SAM-dependent catalysis for MMAR_2193 protein. Structural modeling and docking studies predicted polyketide cores as probable substrates for methylation.Pathogenic mycobacterial genomes reveal several genomic clusters dedicated to virulent lipid biosynthesis. Polyketide synthases (PKSs) work in conjunction with fatty acid synthases to biosynthesize these molecules. Post-synthesis modification of polyketide cores is crucial for biological activity of these metabolites. Mycobacterial genomes reveal a plethora of genes homologous to methyltransferases that are important polyketide modifiers. In a distinct organization, in silico analyses. High-resolution mass spectrometry confirmed methylated polyketide products from MMAR_2193 catalyzed reactions. It was interesting to note that this mycobacterial protein utilized SAM as a donor to biosynthesize variably methylated products. Notably, MMAR_2193 exhibited the potential to methylate several hydroxyl positions on a single polyphenolic substrate molecule generating a palette of variably methylated products. Polyphenolic compounds in differing methylated states could play diverse physiological roles. MMAR_2193 utilized phloroglucinol to produce methylated products, including tri-methylated phloroglucinol or tri-methoxy benzene (TMB). TMB is the key volatile molecule that imparts typical floral scent to the Chinese rose, Rosa chinensis. However, the plant requires two separate classes of O-methyltransferases to achieve complete methylation of phloroglucinol precursor and production of TMB [O-methytransferases involved in secondary metabolism generally display strict substrate specificity and through methylation direct small molecules into various metabolic pathways [O-methytransferase proteins have been shown to change substrate specificity though with limited region-selectivity [O-methytransferase from Mycobacterium tuberculosis was reported to display promiscuous substrate specificity and relaxed region-selectivity [Interestingly, our docking studies proposed a multi-methylating potential of MMAR_2193 protein. Our biochemical studies using polyketide core molecules as substrates corroborated the n of TMB , 37. Plapathways \u201341. Atteectivity \u201344. Receectivity . The proO-methyltransferases occur in several secondary metabolite generating genomic clusters. A sequencial assay of MMAR_2193 with reaction products of MtbPKS18 generated methylated \u03b1-pyrones in vitro. Based on the genomic placement of mmar_2193 in a type III pks gene cluster and further the capability of the protein to methylate polyketide cores and products, it is tempting to speculate that MMAR_2193 could play crucial roles in modifying type III polyketides in M. marinum. Interestingly, O-methylated triketide \u03b1-pyrones could be identified in M. marinum biofilms, suggesting roles of these molecules in mycobacterial physiology. This study provides functional analyses of an unusual O-methyltransferase from M. marinum. The distinctive ability of MMAR_2193 to perform multi-methylations on varied substrates could be utilized to generate a palette of novel methylated bioactive metabolites.O-methyltransferase, mmar_2193 belongs to a part of type III pks cluster found exclusively in pathogenic bacterial strains. Efficient O-methylation of hydroxyl groups seems to be essential to produce varied methylated type III PKS products. Our study reveals multiple O-methylating potential of MMAR_2193 to methylate all hydroxyl positions on a given substrate. O-methylation is reported to determine bacterial pathogenicity and survival in adverse conditions. The presence of O-methylated products in biofilm culture of wildtype M. marinum suggest the importance of O-methylation. Further, the enzyme can be utilized for generation of novel methylated scaffolds.S1 Text(DOCX)Click here for additional data file.S1 Raw images(PDF)Click here for additional data file."} +{"text": "V600 mutation\u2013targeting inhibitors leads to disease relapse in a majority of melanoma patients. In many instances, this resistance is promoted by upregulation of mitochondrial oxidative phosphorylation (OxPhos) in melanoma cells. We recently showed that a novel electron transport chain (ETC) complex I inhibitor, IACS-010759 (IACS), abolished OxPhos and significantly inhibited tumor growth of high-OxPhos, BRAF inhibitor (BRAFi)\u2013resistant human melanomas. However, the inhibition was not uniform across different high OxPhos melanomas, and combination with BRAFi did not improve efficacy.Primary and posttreatment resistance to BRAFin vivo molecular basis of sensitization to the combination treatment.We performed a high-throughput unbiased combinatorial drug screen of clinically relevant small molecules to identify the most potent combination agent with IACS for inhibiting the growth of high-OxPhos, BRAFi-resistant melanomas. We performed bioenergetics and carbon-13 metabolite tracing to delineate the metabolic basis of sensitization of melanomas to the combination treatment. We performed xenograft tumor growth studies and Reverse-Phase Protein Array (RPPA)\u2013based functional proteomics analysis of tumors from mice fed with regular or high-fat diet to evaluate in vitro cell growth and induce tumor regression or stasis of some BRAFi-resistant melanomas. Bioenergetics analysis revealed a dependence on fatty acid metabolism in melanomas that responded to the combination treatment. RPPA analysis and carbon-13 tracing analysis in these melanoma cells showed that IACS treatment decreased metabolic fuel utilization for fatty acid metabolism, but increased substrate availability for activation of the mevalonate pathway by HMGCR, creating a dependence on this pathway. Functional proteomic analysis showed that IACS treatment inhibited MAPK but activated AKT pathway. Combination treatment with STN counteracted AKT activation.A combinatorial drug screen and subsequent validation studies identified Atorvastatin (STN), a hydroxymethylglutaryl-coenzyme A reductase inhibitor (HMGCRi), as the most potent treatment combination with IACS to inhibit STN and other clinically approved HMGCRi could be promising combinatorial agents for improving the efficacy of ETC inhibitors like IACS in BRAFi-resistant melanomas.The online version contains supplementary material available at 10.1186/s40170-022-00281-0. Many cancers including melanomas acquire unique metabolic dependencies over their lifetimes \u20133. TheseV600-mutant melanoma tumors, but weakly inhibits the growth of others with a similar metabolic phenotype, signifying the importance of specific dependencies for efficacy -glucose or [U-13C]-glutamine and treated with the indicated inhibitors for 12 h. [U-13C]-glucose and [U-13C]-glutamine tracing analyses were performed at the MDACC Metabolomics Core Facility as described before -GLC (glucose) (H) or [U-13C]-GLN (glutamine) (I) following treatment with IACS (I) and STN (S) or their combination (I+S) for 12 h. Figure S3. (A) Heatmap showing unsupervised clustering analysis of significantly (p<0.005) altered proteins in UCSD354L tumors harvested from mice fed with regular (Reg) or high-fat keto diet and analyzed by RPPA. The heatmap shows Pearson correlation of significantly (p<0.005) different proteins in tumors from mice fed with high fat keto (Hi-fat) versus regular (Reg) diet. Intensity ranges of lowest (blue) and highest (red) protein levels are indicated at the bottom of the heatmap. (B) Sub-cutaneous tumor growth of parental A375 cells in mice fed with Regular diet or high-fat keto diet. (C and D) Percent changes in body weights of A375R1 tumor bearing mice fed with regular (Reg) diet (C) or high-fat keto diet (Hi-fat) (D), and treated with Vehicle, 5mg/kg IACS, 1mg/kg STN or their combinations over the number of days shown. Mice weight data is from eight mice/group, error bars, standard error of mean (SEM).Additional file 3: Table S1. Inhibitors (probes) in the combinatorial drug screen, their molecular targets and cellular pathways targeted. Table S2. IC50 values of single agent inhibitors (probes) and their combinations with IACS-010759 (IACS), derived from growth inhibition curve analysis using GraphPad Prism."} +{"text": "The expression of sialosyl-Tn (STn) antigen was evaluated by immunohistochemistry in primary gastric cancers. Twenty-one of 31 (68%) gastric cancers expressed STn, regardless of tumour location, stage or histological type. Eighty-one per cent of patients with STn-positive tumours died of their disease or had recurrent cancer, compared with 20% of patients with STn-negative tumours (P < 0.002). STn may be a useful prognostic marker in patients with gastric cancer."} +{"text": "Tissue specimens from 150 patients with localised prostatic carcinomas and 116 patients with prostatic carcinomas with distant metastases were analysed for histological grade (WHO and Gleason) and immunoreactivity for prostate acid phosphatase (PAP), prostate-specific antigen (PSA), neurone-specific enolase (NSE), p53 protein, c-erbB-2 protein, cytokeratins (AE1/AE3) and vimentin. After stratification for the presence or absence of distant metastases, multivariate regression analysis revealed that WHO grading was the most powerful independent prognosticator, followed by age and prostate acid phosphatase expression. There was a trend towards reduced survival with decreasing prostate-specific antigen reactivity. The Gleason system showed poor prognostic ability. The analysis predicted reduced survival in the presence of extensive neurone-specific enolase reactivity, mostly because of one case of small-cell carcinoma."} +{"text": "Surgical biopsy specimens of 179 breast carcinoma were studied by steroid-binding and immunohistochemical assays or oestrogen and progesterone receptors in order to explore reasons for discordant results between the two assay types. Receptor statuses in 18% of ER assays and 30% of PR assays were in disagreement. Immunohistochemistry-positive steroid-binding-negative status predominated among the discordant ER assays, while the discordant PR assays displayed the opposite situation. In discordant assays receptor concentration was significantly more often close to the cut-off (10-50 fmol mg-1) than in the concordant ones. Low binding affinity (high Kd) was also significantly associated with disagreeing assay results. These observations clearly indicate that immunohistochemical ER and PR assays measure high-affinity binding components (i.e. type I receptors) in steroid-binding assays. ER but not PR assays in premenopausal women disagreed more often than those in post-menopausal women. Such factors as histological type, specimen size in steroid-binding assay, grade of malignancy and tumour necrosis were statistically unrelated to agreement or disagreement of receptor assays."} +{"text": "We examined bone marrow aspirates from 100 metastasis-free primary breast cancer patients. In 38/100 patients (38%), tumour cells were detected in the marrow using an immunocytochemical technique with a cocktail of two monoclonal antibodies: anti-EMA and anti-cytokeratin. Median follow-up was 34 months: 15/38 (39%) tumour cell-positive patients have since relapsed, but only 9/62 (15%) tumour cell-negative patients. The median interval between tumour cell detection and relapse was 11.4 months. No statistically significant correlation existed between tumour cell presence and 'established' prognostic factors. However, relapse-free survival was significantly shorter in tumour cell-positive patients. Multivariate analysis showed tumour cell presence as a strong, significant prognostic factor for relapse-free as well as overall survival. We conclude that screening for tumour cells in bone marrow of primary breast cancer patients identifies high-risk patients for early relapse. In particular, patients with node-negative tumours who have tumour cells in their bone marrow may require subsequent systemic therapy."} +{"text": "The regulation of extracellular proteolytic activity via the plasminogen activation system is complex, involving numerous activators, inhibitors, and receptors. Previous studies on monocytic and colon cell lines suggest that plasmin pre-treatment can increase plasminogen binding, allowing the active enzyme to generate binding sites for its precursor. Other studies have shown the importance of pre-formed receptors such as annexin II heterotetramer. However, few studies have used techniques that exclusively characterise cell-surface events and these mechanisms have not been investigated at the breast cancer cell surface.O-tetradecanoylphorbol-13-acetate) stimulation, allowing flexible and transient modulation of cell-surface uPA. Similar experiments were also performed using MDA-MB-231 cells, which overexpress uPAR/uPA endogenously. Using techniques that preserve cell integrity, we characterise the role of uPA as both a plasminogen receptor and activator and quantify the relative contribution of pre-formed and cryptic plasminogen receptors to plasminogen binding.We have studied plasminogen binding to MCF-7 in which urokinase plasminogen activator receptor (uPAR) levels were upregulated by PMA (12-Cell-surface plasminogen binding was significantly enhanced in the presence of elevated levels of uPA in an activity-dependent manner and was greatly attenuated in the presence of the plasmin inhibitor aprotinin. Pre-formed receptors were also found to contribute to increased plasminogen binding after PMA stimulation and to co-localise with uPA/uPAR and plasminogen. Nevertheless, a relatively modest increase in plasminogen-binding capacity coupled with an increase in uPA led to a dramatic increase in the proteolytic capacity of these cells.We show that the majority of lysine-dependent plasminogen binding to breast cancer cells is ultimately regulated by plasmin activity and is dependent on the presence of significant levels of active uPA. The existence of a proteolytic positive feedback loop in plasminogen activation has profound implications for the ability of breast cancer cells expressing high amounts of uPA to accumulate a large proteolytic capacity at the cell surface, thereby conferring invasive potential. The uPA A-chain contains a growth factor-like domain (amino acids 1 to 48) and a kringle domain (amino acids 50 to 131), whereas the B-chain contains the protease domain [The components of the plasminogen activation system (PAS) are important determinants of metastatic capacity, participating in both proteolytic and non-proteolytic pathways during cancer progression ,2. Plasmide bond . Howevere domain . The recKd) values ranging from 0.1 to 2 \u03bcM. Receptor candidates can be grouped into three classes: (a) those that possess a pre-existing C-terminal lysine residue (pre-formed), (b) those that are cleaved to expose a lysine residue (cryptic), and (c) those that bind plg through a lysine-independent mechanism [4 to 107 binding sites per cell) and relatively low affinity (Kd 0.1 to 2 \u03bcM) of cell-surface plg binding suggest the presence of multiple plg receptors that are responsible for the total plg-binding capacity of a cell [Receptor-mediated cell-surface localisation of the various components of the PAS is critical for the spatial and temporal regulation of proteolysis. Proteins, gangliosides, and free fatty acids are among the mediators that regulate cell-surface plg binding ,10. Seveechanism -19. In aechanism . The lysf a cell -23. Togein vitro and these cells are capable of binding larger amounts of Glu-plg at the cell surface than non-metastatic breast cancer cells [Mounting evidence implicates uPA as a key regulator of breast cancer metastasis and demonstrates a role for uPA in a number of processes that facilitate tumour progression, including extracellular matrix degradation, cell proliferation, migration, and adhesion ,25. Furter cells . Moreoveer cells . This hi135 residue) has been shown to mediate the activation of plg by uPA and the reciprocal activation of pro-uPA by pln [in situ on breast cancer cells. Using techniques that preserve cell integrity and exclude the contribution of intracellular plg-binding moieties [A direct, non-active-site interaction between the uPA A-chain and plg has been shown to exist at the cell surface of the monocytic cell line, U937 . In addiA by pln . Thus, tmoieties , we char158) and mouse anti-cytokeratin 8/18 monoclonal antibody (mAb) (CBL195) were purchased from Chemicon International . Bovine aprotinin, rabbit anti-actin C-11 polyclonal antibody (pAb) (A2066), rabbit immunoglobulin G (IgG) (I5006), goat IgG (I9140), fluorescein isothiocyanate (FITC)-labelled goat anti-mouse Fab-specific pAb (F4018), FITC-labelled rabbit anti-goat pAb (F7367), FITC-labelled goat anti-rabbit pAb (F6005), and cyanine (Cy) 3-labelled goat anti-mouse pAb (C2181) were purchased from Sigma-Aldrich Pty. Ltd. . Spectrozyme PL, mouse anti-uPAR mAb (no. 3936), mouse anti-uPA A-chain mAb (no. 3921), and mouse anti-uPA B-chain mAb (no. 394) were purchased from American Diagnostica Inc. . Goat anti-S100A10 (p11) mAb (AF1698) was purchased from R&D Systems, Inc. . Human \u03b12-antiplasmin was purchased from Molecular Innovations, Inc. . Goat anti-annexin II (C-16) pAb (sc-1924) was purchased from Santa Cruz Biotechnology, Inc. . Irrelevant isotype-matched control antibodies mouse anti-sheep lymphocyte antigen IgG1 mAb (SBU-T6) and IgG2\u03b1 mAb (SBU-LCA) were obtained from the Centre for Animal Biotechnology .Active human urine-derived uPA and MDA-MB-231 cell lines were maintained in RPMI-1640 medium supplemented with 5% heat-inactivated foetal calf serum (FCS) at 37\u00b0C in a humidified incubator with 5% COHuman Glu-plg was affinity-purified from plasma using Lysine Sepharose\u2122 4B as described . Glu-plgPurified human uPA (0.5 mg/ml) was inactivated by incubation overnight with alpha-toluenesulfonyl fluoride (PMSF) (1 mM) in phosphate-buffered saline (PBS) (pH 7.4) at 4\u00b0C . PMSF inactivation of uPA was maintained for the duration of the assays (data not shown). Pln activity assays confirmed that residual excess PMSF in the preparation was negligible after overnight incubation and dilution for use in the assays (data not shown).O-tetradecanoylphorbol-13-acetate) without change of media at 37\u00b0C for a further 16 hours as previously described [MCF-7 cells were cultured for 32 hours in 5% FCS/RPMI-1640 and then incubated with PMA to distinguish between viable and non-viable cells as previously described . Only ce2-antiplasmin to inhibit pln activity in solution and to ensure that only cell-surface pln activity was assayed. Pln activity was measured at 405 nm at 37\u00b0C using a Spectramax 250 plate reader with Softmax Pro version 4.0 software .MCF-7 cells were grown, PMA-treated, and pre-incubated with uPA or PMSF-uPA (50 nM) for 10 minutes at room temperature as per plg binding assays in modified (no phenol red) binding buffer (MBB) (pH 7.4). Cells were then washed twice and resuspended in MBB containing 0.5 \u03bcM Glu-plg in the presence and absence of 1.6 TIU aprotinin (to measure pln-dependent effects). Cells were incubated for 10 minutes at room temperature before addition of 0.4 \u03bcM of the colorimetric pln substrate Spectrozyme PL. Plg activation assays were performed in the presence of 0.25 \u03bcM \u03b1Cell-surface uPAR, uPA, cytokeratin 8/18, actin, annexin II, and S100A10 (p11) expression was analysed by indirect immunofluorescence staining as previously described . Only ce5 cells per millilitre) were cultured onto sterile glass coverslips in 24-well tissue culture plates in 1 ml of 5% FCS/RPMI-1640 for 32 hours and PMA treated as above. Coverslips were washed twice with ice-cold MBB and fixed by incubation with freshly prepared, ice-cold 3.75% paraformaldehyde in PBS (pH 7.4) for 15 minutes on ice. Coverslips were washed twice and blocked for 30 minutes with MBB on ice. Samples were incubated for 10 minutes at room temperature with MBB containing 50 nM uPA. Cells were washed and incubated for 45 minutes with Cy5-labelled Glu-plg (0.5 \u03bcM) in the presence and absence of 5 mM tranexamic acid in MBB on ice in the absence of light. Cells were washed and incubated for 30 minutes with 10 to 20 \u03bcg/ml mouse anti-uPAR mAb (no. 3936), mouse anti-uPA mAb (no. 394), or goat anti-annexin II (C-16) pAb (sc-1924) in MBB on ice in the absence of light. After two washes, samples were incubated for 30 minutes either with 1:50 FITC- or Cy3-labelled goat anti-mouse pAb or with FITC-labelled rabbit anti-goat pAb in MBB on ice in the absence of light and washed three times with ice-cold MBB. Samples were mounted and examined as previously described [For confocal microscopy, MCF7 cells . Where variances differed significantly, data were log-transformed prior to analysis by t test.Graphs shown represent mean \u00b1 standard error (performed in duplicate) and are representative of experiments performed on multiple days. The means of the data were compared statistically using Student's 149-Lys158) of the uPA A-chain has been shown to exist on U937, a monocytic cell line [The presence of a non-active site, direct binding determinant for plg in the C-terminal region , bindinell line , which dHigh levels of uPA on breast cancer cell lines are linked to increased plg binding ,28. We a158 residue contribution to plg binding and thus this model is highly useful for investigating the role of uPA activity in cell-surface plg binding and activation.Dose-dependent cell-surface binding of exogenous uPA was observed on PMA-stimulated MCF-7 cells. Saturation of enhanced uPAR was observed at 50 nM uPA Figure , represeTo investigate the spatial relationship between cell-surface-bound plg and putative plg receptors, we performed co-localisation experiments using immunofluorescence staining and confocal microscopy. Results for both control and PMA-stimulated MCF-7 cells pre-incubated with exogenous uPA are shown Figure . In the In MCF-7 cells, annexin II displays punctuate cell-surface and diffuse intracellular staining. After PMA stimulation, there was both a subtle increase and a distinct redistribution of cell-surface annexin II into clustered surface puncta Figure . A simil2-antiplasmin. Hence, without uPA catalytic activity at the cell surface, increased plg binding alone is not sufficient for increased plg activation. Nevertheless, increased lysine-dependent plg binding has been clearly linked to increased plg activation in the presence of uPA activity [Several studies in other cell types have shown that limited proteolysis of the cell surface by pln can reveal cryptic plg-binding sites ,22,40. Tactivity .de novo expression of plg-binding moieties after PMA stimulation. Furthermore, amongst the PMA-stimulated cells, the additional and significant increase in plg binding observed after exogenous active uPA treatment was entirely pln-dependent given that there was no corresponding increase in residual plg binding in the presence of aprotinin and pln-independent binding . Plg binding on control (unstimulated) cells, regardless of uPA treatment, was entirely pln-dependent as very little residual plg binding was observed in the presence of aprotinin Figure . In contn Figure . A similA number of clinical and functional studies have shown that uPA-mediated plg activation is integral to the processes of cancer cell invasion and metastasis ,2,6. We Progression to the metastatic phenotype is clearly associated with deregulation of the uPA system ,24,41,42Pro-uPA, though termed an 'inactive' precursor, has been shown to possess some minimal intrinsic plg activating capability . In a prde novo synthesis) has been demonstrated on neutrophils and monocytic and colon cancer cell lines by pretreatment of cells with pln [in vitro. Moreover, we have previously shown that non-viable cell populations bind approximately 100 times more plg, masking changes in cell-surface binding on viable cells [125I-plg (which cannot distinguish between intracellular and cell-surface binding) to measure cell-surface plg binding is unsuitable because considerable artifactual contributions of intracellular plg binding cannot be excluded. In contrast, the use of dual-colour flow cytometry is far superior because it allows clear distinction of cell-surface from intracellular plg binding by exclusion of non-viable cells. Furthermore, the use of aprotinin in this study to quantify the contribution of pln activity to plg binding removes the inherent complicating effect of pln treatment on cell viability. These modifications therefore represent significant improvements to previous works and allow careful characterisation of cell-surface events.The ability of pln to generate its own plg-binding sites in a positive feedback loop , cytokeratin 8, and \u03b1-enolase, have been shown to both bind and enhance the activation of plg to pln on a number of cell types and asso monomer , again sifically .Surface plasmon resonance studies have demonstrated that only AIIt or isolated p11 subunit (S100A10) is able to bind tPA, plg, and pln with moderate affinity, whereas annexin II subunit is able to bind only pln ,61. P11 The invasive capacity of breast cancer cells may be linked to the presence of significant levels of active uPA, which in concert with even a moderate amount of bound plg can generate large amounts of pln at the cell surface through a positive feedback mechanism. Mounting evidence points to uPA as an integral facilitator of breast cancer progression. Recent studies have indicated that uPA deficiency in a breast cancer mouse model significantly reduces lung and lymph node metastases , and recThis study characterises plg binding and activation at the breast cancer cell surface using techniques that exclude artifacts derived from intracellular proteins, contributions from non-viable cells, and the C-terminal lysine from the uPA A-chain. We show for the first time that several mechanisms regulate breast cancer cell plg binding, including direct binding of plg to uPA/pro-uPA, expression of various plg binding proteins, and pln-mediated generation of plg-binding sites through limited proteolysis of cell-surface proteins. Critically, cells with increased uPA and a moderate increase in plg binding displayed dramatic increases in pln activity. This effect was not observed in MCF-7 cells with moderate plg binding alone but was observed in MDA-MB-231 cells, which have naturally high levels of uPA, pln generation, and invasive capacity. Our data thus support the model that increased uPA expression at the cell surface substantially increases pln generation, and therefore invasive potential, via a positive feedback mechanism. These results also strengthen the evidence implicating uPA as a strong therapeutic target for the treatment of metastatic breast cancer.Kd = dissociation constant; mAb = monoclonal antibody; MBB = modified (no phenol red) binding buffer; MFI = mean fluorescence intensity; pAb = polyclonal antibody; PAI-1 = plasminogen activator inhibitor type-1; PAI-2 = plasminogen activator inhibitor type-2; PAS = plasminogen activation system; PBS = phosphate-buffered saline; PI = propidium iodide; plg = plasminogen; pln = plasmin; PMA = 12-O-tetradecanoylphorbol-13-acetate; PMSF = alpha-toluenesulfonyl fluoride; PMSFuPA = alpha-toluenesulfonyl fluorideinactivated urokinase plasminogen activator; TIU = trypsin inhibitor units; tPA = tissue-type plasminogen activator; uPA = urokinase plasminogen activator; uPAR = urokinase plasminogen activator receptor.AIIt = annexin II heterotetramer; Cy = cyanine; FCS = foetal calf serum; FITC = fluorescein isothiocyanate; Glu = glutamic acid; IgG = immunoglobulin G; The authors declare that they have no competing interests.GES contributed to the study concept and design, data acquisition, data analysis/interpretation, and manuscript drafting and editing. DNS contributed to data analysis/interpretation and manuscript drafting and editing. MR contributed to the study concept and design, data analysis/interpretation, and manuscript drafting and editing. All authors read and approved the final manuscript."} +{"text": "The knowledge of the three-dimensional structure of globular proteins is fundamental for a detailed investigation of their functional properties. Experimental methods are too slow for structure investigation on a large scale, while computational prediction methods offer alternatives that are continuously being improved. The international Comparative Assessment of Structure Prediction (CASP), an \"a posteriori\" evaluation of the quality of theoretical models when the experimental structure becomes available, demonstrates that predictions can be successful as well as unsuccessful, and this suggests the necessity for evaluations able to discard \"a priori\" the wrong models.We analyzed different structural properties of globular proteins for experimentally solved proteins belonging to the four different structural classes: \"mainly alpha\", \"mainly beta\", \"alpha/beta\" and \"alpha+beta\". The properties were found to be linearly correlated to protein molecular weight, but with some differences among the four classes. These results were applied to develop an evaluation test of theoretical models based on the expected globular properties of proteins. To verify the success of our test, we applied it to several protein models submitted to the sixth edition of CASP. The best theoretical models, as judged by CASP assessors, were in agreement with the expected properties, while most of the low-quality models had not passed our evaluations.This study supports the need for careful checks to avoid the diffusion of incorrect structural models. Our test allows the evaluation of models in the absence of experimental reference structures, thereby preventing the diffusion of incorrect structural models and the formulation of incorrect functional hypotheses. It can be used to check the globularity of predicted models, and to supplement other methods already used to evaluate their quality. Globular proteins are critical players in the cell whose function is dictated by their characteristic structure. Because the number of proteins with known sequence far exceeds the number with known structure, the ability of computational methods to predict the structure from sequence is considered extremely valuable to investigate their functional properties . Proper In 1951 Pauling was the first to consider the importance of the intra-molecular H-bonds in protein structures, emphasizing their role in stabilizing the alpha-helices and beta-strands ,5. Many Therefore, we first studied and analyzed a valuable set of experimental protein structures belonging to the four known structural classes in terms of H-bonds, voids, solvent-accessible surface area, and water molecules in a layer of 5 \u00c5ngstroms. This analysis allowed us to deduce structural parameters useful in determining the protein globularity and to define operative criteria to evaluate models, particularly those predicted by \"ab-initio\" methods. These structural parameters were combined together as an index of globularity by which we tested thirteen sets of models submitted to CASP6 protein structure prediction experiment in the New Fold (NF) and difficult Fold Recognition Analogous (FR/A) categories.Protein structures, solved by NMR or X-Ray crystallography with resolution of 2.5 \u00c5 or better, were extracted from PDBselect . These pThe H-bonds in these protein structures were evaluated using the Hbplus package and classified as main-chain donor to main-chain acceptor (MM), main-chain donor to side-chain acceptor (MS), side-chain donor to main-chain acceptor (SM) and side-chain donor to side-chain acceptor (SS). The total number of H-bonds ranged between 14 and 377 in \"mainly-alpha\", 9 and 145 in \"mainly-beta\", 15 and 351 in \"alpha+beta\" and 41 and 400 in \"alpha/beta\" proteins. The correlation coefficients between the total number of H-bonds or MM-, MS-, SM- and SS-type H-bonds are reported in Table For each selected structure the total accessible surface area was evaluated by summing the related polar and non-polar components. The total area accessible to solvent increased in a linear way with the protein molecular weights Figures , 2, 3 wiA layer of 5 \u00c5 around each protein was considered, and the number of water molecules and of H-bonds between the water molecules and the protein residues was evaluated. The number of water molecules increased linearly with the molecular weight of the proteins in all four sets with correlation coefficients higher than 0.9 Table , 2, 3.The AVP program was run on the four protein sets using two different probes: the first to identify the holes in the interior of a protein (a zero-sized probe) and the second to delimit the solvent accessible regions on the surface (probe with a radius of 1.4). The total void volume is obtained summing the total buried and surface void volumes. This increased linearly with the protein size with correlation coefficients higher than 0.9 . The list of the thirteen target models selected is reported in Table In this work four sets of protein structures were selected from the PDB, classified as \"mainly-alpha\", \"mainly-beta\", \"alpha+beta\" and \"alpha/beta\" , with its polar and non-polar components, was evaluated for all proteins of the four structural classes. The correlation coefficients between the non-polar components and the molecular weights were found to be higher than those obtained for the polar components in \"mainly-alpha\", \"mainly-beta\" and \"alpha/beta\" proteins, but not in \"alpha+beta\" proteins. In fact, in these structures the non-polar component was slightly higher than the polar one, as indicated by the mean ratios between non-polar accessibility values and the relative total accessibility Table .Moreover, as for the 5 \u00c5 layer around each protein, we verified that the number of water molecules increased linearly with the molecular weight of the proteins in all four sets Figures , 2, 3. T3 in \"mainly-alpha\" and \"mainly-beta\" proteins, and >3000 \u00c5 3 in those \"alpha/beta\" and \"alpha+beta\", in which more buried voids are also present , shown in Additional File a priori because they do not have the structural properties expected in globular proteins.To test the applicability and usefulness of these criteria, thirteen targets of CASP experiment were selected. Only the full-atom structures were chosen for each testing set ,17. Our An interesting aspect concerns the subdivision of prediction methods present in CASP6 as \"human\" and \"server\" predictors . The resThe threshold value has been applied in our analysis as a cutoff which creates two subsets of models, the one below the cutoff should include models with globularity features in agreement with those expected in crystallographic structures, while the subset of models above the cutoff should include models with poor globularity features. To validate this, we evaluated the average model quality for the two subsets by using root-mean square deviations (RMSD) and Global Distance Test Total Score (GDT_TS) reported in CASP6 tables, as well as MaxSub score , by consFor each target and for the whole set, the average values of RMSD and GDT_TS were evaluated for the two subsets of models , RMSD and GDT_TS reported in CASP6 tables, as well as MaxSub score. In addition, we applied to the models other quality assessment programs, i.e. ProsaII Z-score , ModchecThe correlation coefficients between our globularity score, as well as all quality assessment programs listed above, and the correct quality measures were evaluated for the whole set , only beta- strands (\"mainly-beta\") and alpha-helices and beta- strands on the basis of the clearly defined classification . The \"alHydrogen atoms were added to the protein structures with the \"Modify/Add Hydrogens tool\" in InsightII package . The Hbplus package was used to evaluate the putative formation of H-bonds. It identifies H-bonds within a distance of 2.5 \u00c5ngstroms and a minimum angle of 90\u00b0 . SolventA layer of water molecules around every protein was added using the tool \"Assembly\u2192Soak\u2192Layer\" in InsightII. The H-bonds between the residue atoms in protein and the water molecules was evaluated by Hbplus program.Further Perl scripts were written to apply the AVP, Hbplus and NACCESS programs to the selected protein dataset.y values to the x values (i.e. molecular weights). For each of four properties the Root Mean Squared Error (RMSE) was calculated, that is the average distance of all the points from the fitted line, measured along a vertical lineThe total accessible surface area (ASA), the number of MM-type H-bonds, voids and water molecules versus the molecular weights of selected proteins in each structural class fit to linear regressions described by four equations relating the NC is the total number of structures in each class, yi is the value predicted by linear equation in each of the four cases and y'i is the corresponding calculated value.where EiC).A score value was calculated for all the proteins, belonging to one class, by summing the ratios of the differences between the calculated and predicted values for each of the four properties versus the related errors and difficult Fold Recognition Analogous (FR/A) categories ,17. To aNext, our globularity scores were compared with some model evaluation parameters, i. e. gross violations of distance constraints (err), root-mean square deviations (RMSD) and Global Distance Test_Total Score (GDT_TS) reported in CASP6 tables.Finally, all models were analyzed also by MaxSub program , PROSA IA web site has been devoted to the additional materials we produced during this work and to add in the time more evaluations. The site is freely accessible .AMF and GC conceived of the study and participated in its design and coordination. SC carried out the computational experiments and analysis of the results. All authors read and approved the final manuscript.Figure1S. Parameters plotted against values of molecular weights obtained for each protein, belonging to \"mainly-beta\" class. (A) MM-type H-bonds (B) total accessibility (C) void number (D) water molecules.Click here for fileFigure2S. Some parameters plotted against values of molecular weights obtained for each protein, belonging to \"alpha/beta\" class. (A) MM-type H-bonds (B) total accessibility (C) void number (D) water molecules.Click here for fileFigure3S. Some parameters plotted against values of molecular weights obtained for each protein, belonging to \"alpha+beta\" class. (A) MM-type H-bonds (B) total accessibility (C) void number (D) water molecules.Click here for fileTable1S. Theoretical parameters obtained analysing protein structures belonging to four structural classes.Click here for fileTable2S. We have reported for each target and for the whole model set the cutoff value of globularity score, and the average values of RMSD and GDT_TS for the models above and below the cutoff. In parenthesis are reported the standard deviations.Click here for fileFigure4S. Model evaluation parameters plotted against values of globularity score obtained for T0198 target (A) gross violations of distance constraints (err) (B) root-mean square deviations (RMSD) (C) Global Distance Test_Total Score (GDT_TS) (D) MaxSub score (E) PROSA II Z-score (F) Victor/FRST function (G) Modcheck score (H) Anolea Z-score.Click here for fileFigure5S. Model evaluation parameters plotted against values of globularity score obtained for T0201 target (A) gross violations of distance constraints (err) (B) root-mean square deviations (RMSD) (C) Global Distance Test_Total Score (GDT_TS) (D) MaxSub score (E) PROSA II Z-score (F) Victor/FRST function (G) Modcheck score (H) Anolea Z-score.Click here for fileFigure6S. Model evaluation parameters plotted against values of globularity score obtained for T0212 target (A) gross violations of distance constraints (err) (B) root-mean square deviations (RMSD) (C) Global Distance Test_Total Score (GDT_TS) (D) MaxSub score (E) PROSA II Z-score (F) Victor/FRST function (G) Modcheck score (H) Anolea Z-score.Click here for fileFigure7S. Model evaluation parameters plotted against values of globularity score obtained for T0238 target (A) gross violations of distance constraints (err) (B) root-mean square deviations (RMSD) (C) Global Distance Test_Total Score (GDT_TS) (D) MaxSub score (E) PROSA II Z-score (F) Victor/FRST function (G) Modcheck score (H) Anolea Z-score.Click here for fileFigure8S. Model evaluation parameters plotted against values of globularity score obtained for T0242 target (A) gross violations of distance constraints (err) (B) root-mean square deviations (RMSD) (C) Global Distance Test_Total Score (GDT_TS) (D) MaxSub score (E) PROSA II Z-score (F) Victor/FRST function (G) Modcheck score (H) Anolea Z-score.Click here for fileFigure9S. Model evaluation parameters plotted against values of globularity score obtained for T0248 target (A) gross violations of distance constraints (err) (B) root-mean square deviations (RMSD) (C) Global Distance Test_Total Score (GDT_TS) (D) MaxSub score (E) PROSA II Z-score (F) Victor/FRST function (G) Modcheck score (H) Anolea Z-score.Click here for fileFigure10S. Model evaluation parameters plotted against values of globularity score obtained for T0216_1 target (A) gross violations of distance constraints (err) (B) root-mean square deviations (RMSD) (C) Global Distance Test_Total Score (GDT_TS) (D) MaxSub score (E) PROSA II Z-score (F) Victor/FRST function (G) Modcheck score (H) Anolea Z-score.Click here for fileFigure11S. Model evaluation parameters plotted against values of globularity score obtained for T0216_2 target (A) gross violations of distance constraints (err) (B) root-mean square deviations (RMSD) (C) Global Distance Test_Total Score (GDT_TS) (D) MaxSub score (E) PROSA II Z-score (F) Victor/FRST function (G) Modcheck score (H) Anolea Z-score.Click here for fileFigure12S. Model evaluation parameters plotted against values of globularity score obtained for T0239 target (A) gross violations of distance constraints (err) (B) root-mean square deviations (RMSD) (C) Global Distance Test_Total Score (GDT_TS) (D) MaxSub score (E) PROSA II Z-score (F) Victor/FRST function (G) Modcheck score (H) Anolea Z-score.Click here for fileFigure13S. Model evaluation parameters plotted against values of globularity score obtained for T0199_3 target (A) gross violations of distance constraints (err) (B) root-mean square deviations (RMSD) (C) Global Distance Test_Total Score (GDT_TS) (D) MaxSub score (E) PROSA II Z-score (F) Victor/FRST function (G) Modcheck score (H) Anolea Z-score.Click here for fileFigure14S. Model evaluation parameters plotted against values of globularity score obtained for T0209_1 target (A) gross violations of distance constraints (err) (B) root-mean square deviations (RMSD) (C) Global Distance Test_Total Score (GDT_TS) (D) MaxSub score (E) PROSA II Z-score (F) Victor/FRST function (G) Modcheck score (H) Anolea Z-score.Click here for fileFigure15S. Model evaluation parameters plotted against values of globularity score obtained for T0209_2 target (A) gross violations of distance constraints (err) (B) root-mean square deviations (RMSD) (C) Global Distance Test_Total Score (GDT_TS) (D) MaxSub score (E) PROSA II Z-score (F) Victor/FRST function (G) Modcheck score (H) Anolea Z-score.Click here for fileFigure16S. Model evaluation parameters plotted against values of globularity score obtained for T0273 target (A) gross violations of distance constraints (err) (B) root-mean square deviations (RMSD) (C) Global Distance Test_Total Score (GDT_TS) (D) MaxSub score (E) PROSA II Z-score (F) Victor/FRST function (G) Modcheck score (H) Anolea Z-score.Click here for fileFigure17S. Model evaluation parameters plotted against values of globularity score obtained for all models (A) gross violations of distance constraints (err) (B) root-mean square deviations (RMSD) (C) Global Distance Test_Total Score (GDT_TS) (D) MaxSub score (E) PROSA II Z-score (F) Victor/FRST function (G) Modcheck score (H) Anolea Z-score.Click here for fileTable3S. Correlation coefficients between all methods, as well as for our four individual features , and three correct quality measures evaluated for each of 13 target.Click here for fileTable4S. A. For each target, columns report the number of models analyzed and the ratios of models for which the voids, MM-type H-bonds, water molecules, the total accessibility and the score values resulted within expected ranges for globular proteins and calculated as reported in Methods resulted in the expected ranges for globular proteins and calculated as reported in Methods (see Additional File Click here for file"} +{"text": "Cre recombinase. As recombinant plasmids tend to be more stable in RecA-deficient strains, we aimed to construct a recA- bacterial strain for generation of minicircle vector DNA with less chance of unwanted deletions.Minicircle DNA is the non-replicating product of intramolecular site-specific recombination within a bacterial minicircle producer plasmid. Minicircle DNA can be engineered to contain predominantly human sequences which have a low content of CpG dinucleotides and thus reduced immunotoxicity for humans, whilst the immunogenic bacterial origin and antibiotic resistance marker gene sequences are entirely removed by site-specific recombination. This property makes minicircle DNA an excellent vector for non-viral gene therapy. Large-scale production of minicircle DNA requires a bacterial strain expressing tightly controlled site-specific recombinase, such as Escherichia coli HB101Cre with a chromosomally located Cre recombinase gene under the tight control of the araC regulon. The Cre gene expression cassette was inserted into the chromosomal lacZ gene by creating transient homologous recombination proficiency in the recA- strain HB101 using plasmid-born recET genes and homology-mediated chromosomal \"pop-in, pop-out\" of the plasmid pBAD75Cre containing the Cre gene and a temperature sensitive replication origin. Favourably for the Cre gene placement, at the \"pop-out\" step, the observed frequency of RecET-led recombination between the proximal regions of homology was 10 times higher than between the distal regions. Using the minicircle producing plasmid pFIXluc containing mutant loxP66 and loxP71 sites, we isolated pure minicircle DNA from the obtained recA- producer strain HB101Cre. The minicircle DNA preparation consisted of monomeric and, unexpectedly, also multimeric minicircle DNA forms, all containing the hybrid loxP66/71 site 5'-TACCGTTCGT ATAATGTATG CTATACGAAC GGTA-3', which was previously shown to be an inefficient partner in Cre-mediated recombination.We describe here the construction of the RecA-deficient minicircle DNA producer araC controlled Cre gene into the lacZ gene on the chromosome of E. coli recA- strain HB101. The resultant recA- minicircle DNA producer strain HB101Cre was used to obtain pure minicircle DNA, consisting of monomeric and multimeric minicircle forms. The obtained recA- minicircle DNA producer strain is expected to decrease the risk of undesired deletions within minicircle producer plasmids and, therefore, to improve production of the therapeutic minicircle vectors.Using transient RecET-driven recombination we inserted a single copy of the Thus, for reliable identification of the LacZ+ phenotype of the bacterial colonies, the sparsely seeded LB-agar indicator plates supplemented with X-gal and IPTG were incubated overnight at 37\u00b0C and then for additional 2\u20133 days at room temperature for the blue colour of the colonies to mature.The bacteria were cultured as described before . A lacY Cre gene containing plasmid pBAD75Cre were described previously . (B) A part of the electropherogram showing the sequence 5'-ACC CTG TTA CGT ATA GCC GAA ATT GCC AGG ATC AGG-3' of the Cre gene (obtained using primer CRF), which encodes a stretch of amino acids TLLRIAEIARIR including R173 (shown in bold) from the Cre recombinase catalytic site [(C) A part of the electropherogram showing the same sequence as in (B), read from the opposite strand . (D) A part of the electropherogram showing the sequence 5'-TCT GGA CAC AGT GCC CGT GTC GGA GCC GCG CGA GAT-3' of the Cre gene (obtained using primer CRF), which encodes a stretch of amino acids SGHSARVGAARD including H289 and R292 (shown in bold) from the Cre recombinase catalytic site [(E) A part of the electropherogram showing the sequence 5'-TGG ACC AAT GTA AAT ATT GTC ATG AAC TAT ATC CGT AAC-3' of the Cre gene (obtained using primer CRF), which encodes a stretch of amino acids WTNVNIVMNYIRN including W315 and Y324 (shown in bold) from the Cre recombinase catalytic site [tic site . (C) A ptic site . (E) A ptic site .Click here for fileSequencing of the minicircle DNA. Minicircle mFIXluc was isolated from the recA- E. coli strain HB101Cre and recA+ E. coli strain MM219Cre, both harbouring the minicircle producer plasmid pFIXluc before L-arabinose induction of Cre recombinase expression. (A) A diagram showing minicircle DNA sequencing strategy. Primers SEQ-CMV and SEQ-D were used to sequence opposite strands of the hybrid loxP66/71 site and the flanking regions. (B) A part of the electropherogram showing the sequence of loxP66/71 (5'-TACCGTTCGT ATAATGTATG CTATACGAAC GGTA-3') of the minicircle mFIXluc extracted from E. coli HB101Cre (SEQ-CMV primed extension). (C) A part of the electropherogram showing the sequence of loxP71/66 (5'-TACC GTTCGTATAG CATACATTAT ACGAACGGTA-3') of the minicircle mFIXluc extracted from E. coli HB101Cre (SEQ-D primed extension). (D) A part of the electropherogram showing the sequence of loxP66/71 site of the minicircle mFIXluc extracted from E. coli MM219Cre (SEQ-CMV primed extension). (E) A part of the electropherogram showing the sequence of loxP71/66 site of the minicircle mFIXluc extracted from E. coli MM219Cre (SEQ-D primed extension).Click here for file"} +{"text": "Deregulated expression of myc proto-oncogenes is implicated in several human neoplasias. We analysed the expression of c-myc, N-myc, L-myc, max and RB1 mRNAs in a panel of human gliomas and glioma cell lines and compared the findings with normal neural cells. The max and RB1 genes were included in the study because their protein products can interact with the Myc proteins, being thus putative modulators of Myc activity. Several gliomas contained c/L-myc mRNAs at levels higher than those in fetal brain, L-myc predominantly in grade II/III and c-myc in grade III gliomas. High-level N-myc expression was detected. In one small-cell glioblastoma and lower levels in five other gliomas. In contrast, glioma cell lines totally lacked N/L-myc expression. The in situ hybridisations revealed mutually exclusive topographic distribution of myc and glial fibrillary acidic protein (GFAP) mRNAs, and a lack of correlation between myc expression and proliferative activity, max and RB1 mRNAs were detected in most tumours and cell lines. The glioma cells displayed interesting alternative splicing patterns of max mRNAs encoding Max proteins which either suppress (Max) or augment (delta Max) the transforming activity of Myc. We conclude that (1) glioma cells in vivo may coexpress several myc genes, thus resembling fetal neural cells; but (2) cultured glioma cells expression only c-myc; (3) myc, max and RB1 are regulated independently in glioma cells; and (4) alternative processing of max mRNA in some glioma cells results in delta Max encoding mRNAs not seen in normal fetal brain."} +{"text": "Expression of alpha-1-antitrypsin (AAT) in tumour cells of 102 surgically resected lung adenocarcinomas was examined by immunohistochemical method using anti-AAT antiserum. While only 13 cases (13%) were negative for AAT expression, 89 cases (87%) contained AAT at varying degrees. The degree of AAT-positive tumour cells was significantly higher in advanced cases than in early cases. Clinical follow-up study of the patients, particularly in stage I, showed that strongly AAT-positive cases have poor prognosis than weak-to-moderately AAT-positive or AAT-negative cases. Thus, AAT expression status in tumour cells of lung adenocarcinoma may be a biological marker of prognostic significance in regard to tumour growth."} +{"text": "A receptor antagonist or chloride channel blocker, suggesting that postsynaptic GABAA receptors in VB neurons were involved in the shunting inhibition. GABAA receptor-mediated inhibitory postsynaptic currents (IPSCs) were evoked in VB neurons by electrical stimulation of the reticular thalamic nucleus. Propofol markedly increased amplitude, decay time, and charge transfer of GABAA IPSCs. The results demonstrated that shunting inhibition of thalamic somatosensory relay neurons by propofol at clinically relevant concentrations is primarily mediated through the potentiation of the GABAA receptor chloride channel-mediated conductance, and such inhibition may contribute to the impaired thalamic responses to sensory stimuli seen during propofol-induced anesthesia.Propofol is a widely used intravenous general anesthetic. Propofol-induced unconsciousness in humans is associated with inhibition of thalamic activity evoked by somatosensory stimuli. However, the cellular mechanisms underlying the effects of propofol in thalamic circuits are largely unknown. We investigated the influence of propofol on synaptic responsiveness of thalamocortical relay neurons in the ventrobasal complex (VB) to excitatory input in mouse brain slices, using both current- and voltage-clamp recording techniques. Excitatory responses including EPSP temporal summation and action potential firing were evoked in VB neurons by electrical stimulation of corticothalamic fibers or pharmacological activation of glutamate receptors. Propofol (0.6 \u2013 3 \u03bcM) suppressed temporal summation and spike firing in a concentration-dependent manner. The thalamocortical suppression was accompanied by a marked decrease in both EPSP amplitude and input resistance, indicating that a shunting mechanism was involved. The propofol-mediated thalamocortical suppression could be blocked by a GABA General anesthesia consists of five distinct components: analgesia, amnesia, unconsciousness, immobility, and blunted autonomic responsiveness ,2. WhileExcitatory input regulates the functional state of thalamic neurons, and such input is provided by both ascending activating systems in the brain stem and hypothalamus and the descending pathway . CorticoA receptors amino-2-hydroxypropyl] (phenylmethyl) phosphinic acid (CGP55845), 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX), D-2-amino-5-phosphopentanoic acid (D-AP5), (\u00b1)1-aminocyclopentane-trans-1, 3-dicarboxylic acid - 1] \u00d7 100. Temporal summation was defined as % increase in depolarization occurring at the soma during a train [t-test or one-way ANOVA. Data were expressed as means \u00b1 SE.Membrane voltages and currents were analyzed using both Clampfit 9.0 and MiniAnalysis 6 . To analyze temporal summation containing five responses, the peak of the first and fifth responses were measured from baseline and expressed as \u0394V a train . StatistThe author(s) declare that they have no competing interests.(SWY) \u2013 study design, data collection and analysis, manuscript preparation. (PAG) \u2013 study design, manuscript preparation."} +{"text": "The pharmacokinetics of Zn-phthalocyanine (Zn-Pc) in mice bearing a transplanted MS-2 fibrosarcoma has been studied using dipalmitoyl-phosphatidylcholine (DPPC) liposomes and low density lipoproteins (LDL) as drug delivery systems. LDL induce a higher Zn-Pc uptake by the tumour and improve the selectivity of tumour targeting as compared to DPPC liposomes. Experimental photodynamic therapy (PDT) of the MS-2 fibrosarcoma has been performed using liposome-delivered Zn-Pc and the efficiency of tumour necrosis has been measured following four different irradiation protocols. We found that Zn-Pc doses as low as 0.07-0.35 mg kg-1 are sufficient for inducing an efficient tumour response that is linearly dependent on the injected dose. The amount of Zn-Pc in the tumour decreases very slowly as a function of time, hence PDT gives satisfactory results even if performed at relatively long time intervals after administration."} +{"text": "Successful generation of adherent lymphokine-activated killer (A-LAK) cells, highly-enriched in CD3-CD56+ antitumour effector cells, from the peripheral blood of ten patients with acute myelogenous leukaemia (AML) is described. The AML patients were either untreated or in remission. In vitro proliferation of A-LAK cells in patients with AML was generally poor, unless the cells were cocultured with irradiated concanavalin A (ConA)--prestimulated allogeneic PBL or selected lymphoblastoid cell lines (LCL) as feeder cells. Using this method, the median fold proliferation was 290 for A-LAK cells cultured with ConA-activated feeders and 291 for those grown with LCL, both significantly higher (both P less than 0.001) than the median of 2-fold expansion observed in cultures without feeders. A-LAK cultures generated in the presence of feeders consistently showed good enrichment (up to 90%) in CD3-CD56+ NK cells. Although NK activity was not significantly increased on a per cell basis in A-LAK cells grown with feeder cells, total lytic activities against both NK-sensitive target, K562, and NK-resistant target, Daudi, were significantly greater (P less than 0.02 for ConA-PBL feeders and P less than 0.005 for LCL feeders) as compared to those in paired cultures without feeders. In the presence of irradiated allogeneic feeder cells, 7/10 AML patients generated A-LAK cultures characterised by good proliferation and increased purity as well as cytotoxic activity."} +{"text": "One hundred and nine samples comprising carcinomas, adenomas, dysplastic, inflamed and normal mucosa from patients with sporadic colon cancer and ulcerative colitis (UC) were analysed for c-Ki-ras mutations. DNA was extracted from archival paraffin-embedded material, amplified using the polymerase chain reaction (PCR) and the PCR products analysed using restriction enzyme digestion. Forty-two per cent (14/33) of the sporadic carcinoma controls contained Ki-ras codon 12 mutations in contrast to 24% (8/33) of ulcerative colitis carcinomas. A significantly higher c-Ki-ras mutation rate was observed in rectal carcinomas (72%) in comparison to colonic carcinomas (28%) in control patients (P less than 0.04), while the opposite was observed in UC patients. The difference between the incidence of c-Ki-ras mutations in rectal carcinomas in UC (9%) and in sporadic rectal carcinomas (72%) was also significant (P less than 0.01). This lower prevalence rate and different site distribution of c-Ki-ras mutations in UC carcinomas compared to sporadic carcinomas suggests that specific genetic differences may underlie the causation of carcinomas arising in these situations."} +{"text": "Mice bearing progressively growing syngeneic methylcholanthrene-induced sarcomas are immunologically hyporeactive. However, both basal (steady-state) and bacterial lipopolysaccharide (LPS)-induced synthesis of mRNA for interleukin-1 (IL-1) in peritoneal exudate cells (PEC) or spleen cells were comparable in control and tumour-bearing animals. Furthermore, the production of IL-1 by PEC stimulated with LPS in the presence of indomethacin was same in control and tumour-bearing mice. The results thus demonstrate that LPS-stimulated cells from animals bearing progressively growing syngeneic sarcomas synthesise the same quantities of mRNA for IL-1 and produce comparable amounts of IL-1 as do cells from normal animals, in spite of the profound immunological hyporeactivity of the former."} +{"text": "SDZ 280-446 is a semi-synthetic derivative of a natural cyclic peptolide. Its ability to sensitise in vitro tumour cells whose resistance is due to P-glycoprotein-mediated anticancer-drug efflux was shown using four different pairs of parental drug-sensitive (Par-) and multidrug-resistant (MDR-) cell lines, from three different species representing four different cell lineages , and using four different drug classes . By measuring its capacity to restore normal drug sensitivity of MDR-cells in culture in vitro, it appeared that SDZ 280-446 belongs to the same class of very potent chemosensitisers as the cyclosporin derivative SDZ PSC 833: both are about one order of magnitude more active than cyclosporin A (CsA), which is itself about one order of magnitude more active than other known chemosensitisers . Low concentrations of SDZ 280-446 could also restore cellular daunomycin retention in MDR-P388 cells to the levels found in the Par-P388 cells. SDZ 280-446 was also effective as a chemosensitiser when given orally in vivo. In a syngeneic mouse model, combined therapy with vinca alkaloids given i.p. and SDZ 280-446 given per os for 5 consecutive days significantly prolonged the survival of MDR-P388 tumour-bearing mice, when compared with mice receiving vinca alkaloids alone. Another protocol, using three cycles of i.p. doxorubicin at 4 day intervals, could also not increase MDR-P388 tumour-bearing mouse survival unless the mice received SDZ 280-446 orally 4 h before each doxorubicin injection. Though only very few combined therapy treatment protocols have been tested so far, clear increases in survival time of MDR-tumour-bearing mice were regularly obtained, leaving hope for major improvement of the therapy using other dosing schedules."} +{"text": "Drosophila CTCF has only been characterised recently. To date only one endogenous binding location for CTCF has been identified in the Drosophila genome, the Fab-8 insulator in the Abdominal-B locus in the Bithorax complex (BX-C). We carried out chromatin immunopurification coupled with genomic microarray analysis to identify CTCF binding sites within representative regions of the Drosophila genome, including the 3-Mb Adh region, the BX-C, and the Antennapedia complex. Location of in vivo CTCF binding within these regions enabled us to construct a robust CTCF binding-site consensus sequence. CTCF binding sites identified in the BX-C map precisely to the known insulator elements Mcp, Fab-6, and Fab-8. Other CTCF binding sites correlate with boundaries of regulatory domains allowing us to locate three additional presumptive insulator elements; \u201cFab-2,\u201d \u201cFab-3,\u201d and \u201cFab-4.\u201d With the exception of Fab-7, our data indicate that CTCF is directly associated with all known or predicted insulators in the BX-C, suggesting that the functioning of these insulators involves a common CTCF-dependent mechanism. Comparison of the locations of the CTCF sites with characterised Polycomb target sites and histone modification provides support for the domain model of BX-C regulation.Insulator or enhancer-blocking elements are proposed to play an important role in the regulation of transcription by preventing inappropriate enhancer/promoter interaction. The zinc-finger protein CTCF is well studied in vertebrates as an enhancer blocking factor, but Drosophila genome. In particular, we have focussed on the Hox gene cluster in the Bithorax complex; a region whose regulation has been extensively characterised. Previous investigations have identified independent regulatory domains that control the expression of Bithorax complex genes in different segments of the fly, however the molecular nature of the domain boundaries is unclear. Our major result is that we find CTCF binding sites precisely located at the boundaries of these regulatory domains, giving a common molecular basis for these boundaries. This provides a clear example of the link between the positioning of insulators and the organisation of gene regulation in the Drosophila genome.There is still much to learn about the organisation of regulatory elements that control where, when, and how much individual genes in the genome are transcribed. Several types of regulatory element have been identified; some, such as enhancers, act over large genomic distances. This creates a problem: how do such long-range elements only regulate their appropriate target genes? Insulator elements have been proposed to act as barriers within the genome, confining the effects of long-range regulatory elements. Here we have mapped the locations of one insulator-binding protein, CTCF, in several regions of the Insulator elements are DNA sequences that regulate interactions between promoters and enhancers. By preventing inappropriate enhancer/promoter communication, insulators are believed to play a key role in the genomic organisation of transcriptional regulation. Their mode of action is still unclear but may involve the formation of chromatin loops that partition the genome into separate regulatory domains \u20135.Drosophila CTCF has been identified , abdominal-A [abd-A], and Abdominal-B [Abd-B]) into distinct enhancer domains ) or normal rabbit serum (control IP). The immunopurified DNA preparations were labelled with either Cy3 or Cy5 and hybridised to a 1-kb tiling-path genomic microarray covering the 3-Mb Adh region together with other selected genomic regions including the BX-C, the Antennapedia complex (ANT-C), and the achaete-scute region. As a positive control, the immunopurification reactions were assessed using specific PCR primers to amplify a 378-bp fragment from the Fab-8 region, containing characterised CTCF binding sites ) in TAE-buffer. Electrophoresis was performed at 4 \u00b0C with a field strength of 12 V/cm for 3 h.Radiolabelled DNA probes (150\u2013250 bp) were generated by PCR with eviously . The binDataset S1t-value, and p-value derived by CyberT from the ChIP-array data.Table shows the Array Spot ID, chromosome coordinates, Fragment ID, the values for the four biological replicate ratios, number of observations, Mn, standard deviation, (483 KB TDS)Click here for additional data file.Dataset S2t-value, and p-value for the selected fragments with Mn > 0.45 and p < 0.05.Table shows Fragment ID, start coordinate, stop coordinate, CyberT Mn, (13 KB PDF)Click here for additional data file.Figure S1Drosophila S2 cells as in Fab-8 is the positive control, and the negative controls were pre-immune serum or a nonbinding sequence (Fab-8 5\u2032-control). Fab-7 shows significant enrichment, although less enrichment than Fab-8.ChIP was performed with chromatin from (54 KB PDF)Click here for additional data file.Table S1(29 KB DOC)Click here for additional data file.Table S2(12 KB PDF)Click here for additional data file.http://www.ncbi.nlm.nih.gov/geo) accession number for the genomic tiling array is GEO Platform GPL5028 XC003 and for the ChIP data is series GSE7351.The Gene Expression Omnibus (GEO) (http://www.ncbi.nlm.nih.gov) accession numbers of the genes discussed in this paper are: CTCF human, 10664 and Ctcf mouse, 13018.The Entrez Gene (http://flybase.bio.indiana.edu) accession numbers of the genes and gene products discussed in this paper are: abdominal-A (abd-A), FBgn0000014; Abdominal-B (Abd-B), FBgn0000015; achaete (ac), FBgn0000022; Alcohol dehydrogenase (Adh), FBgn0000055; Antennapedia (Antp), FBgn0000095; BEAF32, FBgn0015602; cactus (cact), FBgn0000250; CTCF, FBgn0035769; Cyclin E (CycE), FBgn0010382; Enhancer of zeste (E(z)), FBgn0000629; kuzbanian (kuz), FBgn0015954; lethal of scute (l(1)sc), FBgn0002561; outspread (osp), FBgn0003016; Polycomb (Pc), FBgn0003042; Posterior sex combs (Psc), FBgn0005624; scute (sc), FBgn0004170; smell-impaired 35A (smi35A), FBgn0016930; suppressor of Hairy wing (su(Hw)), FBgn0003567; Su(var)3\u20139, FBgn0003600; Ultrabithorax (Ubx), FBgn0003944; and Zw5, FBgn0000520.The Flybase ("} +{"text": "Cathespin D (Cath D) is a proteolytic enzyme secreted by human breast cancer cells with a growth promoting activity in vitro. In the present study, we measured Cath D and Epidermal Growth Factor/alpha Transforming Growth Factor concentrations in the breast cyst fluid (BCF) of 43 patients with gross cystic disease of the breast. Both Cath D and EGF/alpha-TGF levels were higher in BCF of apocrine than flattened cysts . Premenopausal patients showed higher concentrations of Cath D (P less than 0.05) and EGF/alpha-TGF (P less than 0.05) than postmenopausal patients. A positive correlation was obtained between intracystic concentrations of Cath D and EGF/alpha-TGF (P less than 0.00001). The higher levels of Cath-D and EGF/alpha-TGF found in apocrine cysts could provide an explanation for the increased risk of subsequent breast cancer in women with this type of cyst."} +{"text": "O6-methylguanine-DNA methyltransferase (MGMT) renders glioma cells resistant to the treatment, indicating that identification of mechanisms underlying the gene regulation of MGMT is highly required. Although glioma-derived cell lines have been widely employed to understand such mechanisms, those models harbor numerous unidentified genetic lesions specific for individual cell lines, which complicates the study of specific molecules and pathways.A novel alkylating agent, temozolomide, has proven efficacious in the treatment of malignant gliomas. However, expression of MGMT genes, which is being used in the clinic to nominate patients for temozolomide treatment. Furthermore, we discovered that Valproic acid, one of histone deacetylase inhibitors, suppressed growth of the transformed astrocyte cells without increasing MGMT protein, suggesting that such epigenetic compounds may be used to some types of gliomas in combination with alkylating agents.We established glioma models by transforming normal human astrocyte cells via retroviral-mediated gene transfer of defined genetic elements and found that MGMT was downregulated in the transformed cells. Interestingly, inhibitors of DNA methylation and histone deacetylation failed to increase MGMT protein levels in the transformed astrocyte cells as well as cultured glioblastoma cell lines, whereas the treatment partially restored mRNA levels. These observations suggest that downregulation of MGMT may depend largely on cellular factors other than promoter-hypermethylation of Normal human astrocyte cells allow us to generate experimental models of human gliomas by direct manipulation with defined genetic elements, in contrast to tumor-derived cell lines which harbor numerous unknown genetic abnormalities. Thus, we propose that the study using the transformed astrocyte cells would be useful for identifying the mechanisms underlying MGMT regulation in tumor and for the development of rational drug combination in glioma therapies. O6-methylguanine-DNA methyltransferase (MGMT) provides resistance to treatment with temozolomide, unless expression is lost by promoter methylation or there is direct inhibition of MGMT activity . Myc-His-tagged active form of mouse AKT1 cDNA, which has N-terminal myristoylation, was isolated from the pUSEamp-myr-AKT plasmid and subcloned into pCX4bleo retroviral vector [GenBank: AB086388]. Full-length cDNAs for human MGMT and p53 were generated by PCR and subcloned into pCX4bleo and pCX4gfp [GenBank: AB296083] retroviral vectors, respectively. Primer sequences used in this experiment included 5'-ATG GAC AAG GAT TGT GAA-3' and 5'-TCA GTT TCG GCC AGC AGG-3' for human MGMT and 5'-CTG AAT TCA TGG AGG AGC CGC AGT CAG-3' and 5'-CCG AAT TCA GTC TGA GTC AGG CCC TTC-3' for human p53. Other retroviral vectors and the procedure of retroviral-mediated gene transfer were described previously [EcoVR was first introduced into NHA cells by using amphotropic virus, in order to make human cells susceptible to the subsequent infection with ecotropic viral vectors. Infected cell populations were selected in blasticidin S (20 \u03bcg/ml), G418 (1000 \u03bcg/ml), puromycin (500 ng/ml), or zeocine (500 \u03bcg/ml) for two weeks. In all cases, cultures arose from polyclonal expansion of infected cells.A cDNA fragment encoding murine ecotropic retrovirus receptor using the primers described above. PCR primers for Glyceraldehydes-3-phosphate dehydrogenase (GAPDH) were described previously [Total RNA was isolated with the TRI Reagent (Sigma) and reverse transcribed into cDNA using the oligo-dT primer and the Superscript II (Invitrogen). The levels of eviously .nu/nu) were purchased from Clea Japan and all animal procedures were carried out according to the protocol approved by the institutional Animal Care and Use Committee at Hokkaido University Graduate School of Medicine.Soft-agar colony formation assay and xenoFormalin-fixed paraffin-embedded tissues were sectioned and stained with haematoxylin and eosin (H&E) using standard protocols. Immunohistochemistry was performed using anti-Ki-67 and anti-p53 monoclonal antibodies.KIP1 monoclonal antibodies ; anti-dimethylated Histone-H3 (Me-H3) and anti-acetylated Histone-H3 (Ac-H3) polyclonal antibodies (Upstate); anti-MGMT (MT3.1) and anti-ACTIN monoclonal antibodies ; an anti-p21WAF1 monoclonal antibody ; an anti-hTERT (L20) polyclonal antibody .Protein determination, SDS-PAGE and immunoblotting were carried out as described previously , and rea6-methylguanine-DNA methyltransferaseMGMT: OGBM: glioblastoma multiformeNHA: normal human astrocytehTERT (T): human telomerase catalytic subunitH-RasV12 (R): activated H-RasmyrAKT (A): myristoylated form (active form) of AKTSV40ER (S): simian virus 40 early regionHDAC: histone deacetylaseVPA: Valproic acid5-aza-dC: 5-aza-2'-deoxycytosineRT-PCR: reverse transcriptase polymerase chain reactionGAPDH: Glyceraldehydes-3-phosphate dehydrogenaseThe author(s) declare that they have no competing interests.KS designed the research, carried out all experiments except for immunohistochemistry, and drafted the manuscript. TA participated in the design of the study and contributed to new reagents. EA carried out H&E staining and immunohistochemistry. KT participated in epigenetic studies. SK collected and analyzed clinical samples. ST carried out histological analyses, conceived of the study, participated in its design and coordination, and helped to draft the manuscript. All authors read and approved the final manuscript.Histopathological analysis of human brain tumors. Formalin-fixed paraffin-embedded tissue sections were stained with H&E. Low- (\u00d7100) and high- (\u00d7400) magnification images are shown . Tissue sections were also processed for immunohistochemistry using Ki-67 and p53 antibodies. Staining intensities were summarized in Table Click here for file"} +{"text": "Anti-tumour effector cells were generated through 4 days culture of normal C57BL/6 splenocytes in a medium containing concanavalin A supernatant and then fractionated with Dolichos biflorus lectin (DBA) into DBA+ (agglutinable with DBA) and DBA- (non-agglutinable with DBA) cells. The DBA- cells, infused intravenously into mice together with B16 melanoma cells, or adoptively transferred into mice 3 days after the injection of B16 cells, caused a marked decrease in the number of lung nodules, while the DBA+ cells exerted no effect. On the other hand, the DBA+ cells exhibited higher cytolytic activity in vitro than the DBA- cells in short-term 51Cr-release assays. Then, we analysed the mechanism of the strong anti-tumour activity of DBA- cells in vivo. We found that DBA- cells showed higher response to recombinant interleukin-2 (rIL-2) than DBA+ cells and proliferated very well with a small amount of IL-2. In addition, DBA- cells adhered more strongly to lung endothelial cells than DBA+ cells in response to rIL-1 or rTNF. Furthermore, DBA- cells produced larger amounts of macrophage activating factor (MAF) including IFN-gamma when cultured with B16 melanoma. Taken together, our results show that DBA- cells are effective in reducing experimental pulmonary metastases not only by the direct lytic activity but also by the indirect killing activity through the activated macrophage."} +{"text": "Human rhinoviruses (HRV), the most frequent cause of respiratory infections, include 99 different serotypes segregating into two species, A and B. Rhinoviruses share extensive genomic sequence similarity with enteroviruses and both are part of the picornavirus family. Nevertheless they differ significantly at the phenotypic level. The lack of HRV full-length genome sequences and the absence of analysis comparing picornaviruses at the whole genome level limit our knowledge of the genomic features supporting these differences.cis-acting replication element (cre) in HRV-B that is not present in HRV-A, and that had been previously characterized only in HEV. In contrast to HEV viruses, HRV-A and HRV-B share also markedly lower GC content along the whole genome length.Here we report complete genome sequences of 12 HRV-A and HRV-B serotypes, more than doubling the current number of available HRV sequences. The whole-genome maximum-likelihood phylogenetic analysis suggests that HRV-B and human enteroviruses (HEV) diverged from the last common ancestor after their separation from HRV-A. On the other hand, compared to HEV, HRV-B are more related to HRV-A in the capsid and 3B-C regions. We also identified the presence of a 2C Our findings provide basis to speculate about both the biological similarities and the differences of these three groups of viruses. Human rhinovirus (HRV) is the most frequent cause of infection across all age groups of the population . ReplicaPicornaviridae family and are closely related to HEVs, another genus of the same family. The genome organization of Picornaviridae is conserved among the family with a long 5'-untranslated region (UTR), a single open reading frame (ORF) encoding a polyprotein, a short 3'UTR, and a poly(A) tail . They vary in sequence length from 7124 nucleotides (HRV-12) to 7219 nucleotides (HRV-17) which is similar to the length range of previously sequenced genomes (from 7102 to 7208 nucleotides). The average size ofHRVs type A (7131 nt) is smaller than the average size of HRVs type B (7215 nt), whereas the average size of 14 HEVs analysed in this study is 7417.The 12 newly sequencedHRV genomes have been deposited in GenBank , 2 [GenBank: X02316], 14 [GenBank:X01087], 16 [GenBank: L24917], 39 [GenBank:AY751783], 89 [GenBank:M16248], 85 and 9 whose sequences were directly downloaded from the Picornaviridae sequence database [AY064708] analyzed in this study, were obtained from GenBand at the NCBI. The 14 HEV sequences include the two members of the HEV-D subspecies: EV-68 [GenBank: EF107098] and EV-70 [GenBank:DQ201177], the three members of the poliovirus subspecies: PV-1 [GenBank:V01148], PV-2 [GenBank:X00595] and PV-3 [GenBank: X00925] as well as three representatives of the HEV-A, B and C subspecies randomly chosen: Coxsackie (CV)-A2 [GenBank:AY421760], CV-A6 [GenBank:AY421764]and CV-A14 for HEV-A [GenBank:AY421769]; Echovirus (E)-1 [GenBank:AF029859], E-6 [GenBank:AY302558] and CV-B2 [GenBank:AF081485]for HEV-B; and CV-A1 [GenBank:AF499635], CV-A17 [GenBank:AF499639] and CV-A18 [GenBank:AF499640] for HEV-C. A list of all viruses with their corresponding GenBank accession numbers can be found in the additional file The full-length genome sequences of the 8 additional HRV serotypes (HRV-1B [GenBank:database ), as welComplete genome sequences were determined for each of the 12 above-mentioned strains. Reverse transcription was performed with random hexamers on TRIzol- extracted (Invitrogen) RNA . OverlapOpen reading frames (ORFs) were extracted from the whole-genome nucleotide sequences of each virus species using the getorf programme from the EMBOSS package , using aThe maximum-likelihood phylogenetic analyses were performed using PhyML with estAll-against-all protein product identity scores were produced using the Belvu programme , and refA polyprotein multiple alignment was constructed (as described above) with 14 HEV sequences, 13 HRV-A sequences, 7 HRV-B sequences, and 1 SV-2 sequence. This alignment was subjected to bootscanning (as described in ) with a We extracted sub-alignments for HEV, HRV-A, and HRV-B from the above-described nucleotide-level, whole-genome alignment of 14 HEV, 13 HRV-A, 7 HRV-B sequences. This allows direct comparison of the GC content at the orthologous positions using a sliding window of 600 nt along the alignment, computing GC percentage over all sequences within the window, and with a step of 10 nt. The resulting set of three measures of local GC percentage content, one each for HEV, HRV-A, and HRV-B were plotted.The complete genome alignment of all 34 genomes spanning 7852 positions (5'UTR+ORF+3'UTR) was scanned for thermodynamically stable and structurally conserved RNA structures using RNAz The struHRV: Human RhinovirusHEV: Human EnterovirusCV: Coxsackie VirusE: EchovirusPV: PoliovirusEV-68: HEV-68SV: Simian Picornaviruscre: cis-acting replication elementUTR: untranslated regionUMP: Uridine monophosphateIRES: internal ribosomal entry siteORF: open reading frameML: maximum-likelihoodcre sequencing and revised the manuscript. TJ conducted all the sequences, phylogenetic analyses and calculation of GC content and participated in the writing of the manuscript. DG analyzed the RNA secondary structure, identified the cre elements and participated in the writing of the manuscript. LP participated to the analysis and the writing of the manuscript. EZ supervised and designed all the bioinformatics work and corrected the manuscript. LK designed the original project, supervised the complete work and corrected the manuscript. All authors read and approved the final manuscript.CT designed the original project, conducted and supervised the experiments and drafted the manuscript. SVB conducted most of the experiments, SC conducted the Whole-protein maximum likelihood phylogenetic trees for the 11 individual picornavirus proteins. Each individual protein tree was performed as the whole polyprotein phylogenetic tree 5'cloverleaf consensus structure for HRV-A, HRV-B and HEV identified by comparative sequence analysis. B) IRES consensus structure for HRV-A, HRV-B and HEV identified by comparative sequence analysis. See legend to Figure Click here for file3'UTR structure conservation. 3'UTR consensus structure for HRV-A, HRV-B and HEV identified by comparative sequence analysis. See legend to Figure Click here for fileConserved stem-loop structure in the ORF of HRV-A. Conserved secondary structure located close to the 3'UTR of HRV-A and corresponding structures in HRV-B and HEV located in the same alignment region. See legend to Figure Click here for filecre conservation among HRV-A and HRV-B serotypesInternal . A) Internal 2A cre conservation among HRV-A serotypes. B) Internal VP1 cre conservation among HRV-B serotypesClick here for fileVirus accession number. List of all the accession numbers for the viruses used in the analyses.Click here for filePrimer list. Degenerate and specific primers used to amplify and sequence the new rhinovirus genomes.Click here for file"} +{"text": "The mechanism by which tumour necrosis factor (TNF)-alpha increases the susceptibility of U87-MG human glioblastoma cells to lysis by natural killer (NK) cells was studied. Treatment with TNF-alpha (100 units ml-1) for 48 h enhanced the susceptibility of tumour cells to lysis by NK cells. Increased susceptibility to lysis was associated with enhanced expression of intercellular adhesion molecule 1 (ICAM-1) and HLA class I antigen. Antisense ICAM-1 oligonucleotide inhibited lysis by NK cells of TNF-alpha-treated tumour cells. In contrast, acid treatment following TNF-alpha treatment increased lysis by NK cells. These findings indicate that TNF-alpha treatment of glioblastoma cells increased their susceptibility to lysis by NK cells, since ICAM-1 up-regulation would have more profound effects on NK susceptibility than would HLA class I antigen up-regulation."} +{"text": "A lipopolysaccharide (BP-LPS) isolated from killed Bordetella pertussis (Tohama strain) was determined to have low toxicity based on the mortality and decrease in body weight of BP-LPS-injected mice. BP-LPS, administered intradermally or intraperitoneally, clearly inhibited the growth of an MM46 murine mammary carcinoma. When compared with a toxic Escherichia coli-derived LPS, BP-LPS displayed excellent anti-tumour activity against MH134 hepatoma and Meth A fibrosarcoma. As part of a combined chemotherapy/immunotherapy regimen, BP-LPS also seemed to prolong the lifespan of mice inoculated with Lewis lung carcinoma. BP-LPS thus appears to have valuable characteristics as an anti-tumour agent."} +{"text": "Metastatic variant sublines of the murine large-cell lymphoma cell line RAW117 were tested for their growth and migration properties in vitro in medium conditioned by soluble factors released from syngeneic mouse liver-, lung-, and brain-derived microvessel endothelial cells. Medium conditioned with hepatic sinusoidal endothelial cells stimulated the growth of highly liver-colonising (RAW117-H10) and highly liver- and lung-colonising (RAW117-L17) sublines at higher rates than the poorly metastatic parental line (RAW117-P) (H10 greater than L17 greater than P). Medium conditioned with lung microvessel endothelial cells selectively stimulated the growth of the lung-colonising RAW117-L17 subline. Medium conditioned with brain microvessel endothelial cells showed no growth selectivity, and equivalently stimulated the growth of various RAW117 cell sublines. Medium conditioned with hepatic sinusoidal endothelial cells preferentially promoted the migration of the liver-colonising H10 and L17 sublines, and medium conditioned with lung endothelial cells differentially stimulated the migration of the lung-colonising L17 subline; whereas medium conditioned with brain endothelial cells only slightly stimulated the migration of L17, but not H10 or P cells. Fractionation of medium conditioned with hepatic sinusoidal endothelial cells by DEAE Sephacel anion exchange chromatography revealed that the growth-stimulating activities were clearly separable from migration-stimulating activities. The growth- and migration-stimulating activities released from organ microvessel endothelial cells may be important in determining the ability of RAW117 cells to selectively form metastatic colonies in particular organs."} +{"text": "Resistance to multiple antitumour drugs, mostly antibiotics or alkaloids, has been associated with a cellular plasma membrane P-glycoprotein (Pgp), causing energy-dependent transport of drugs out of cells. However, in many common chemotherapy resistant human cancers there is no overexpression of Pgp, which could explain drug resistance. In order to characterise early steps in multidrug resistance we have derived a series of P-glycoprotein-positive (Pgp/+) and P-glycoprotein-negative (Pgp/-) multidrug resistant cell lines, from a human non-small cell lung cancer cell line, SW-1573, by stepwise selection with increasing concentrations of doxorubicin. These cells were exposed to doxorubicin and its fluorescence in nucleus (N) and cytoplasm (C) was quantified with laserscan microscopy and image analysis. The fluorescence N/C ratio in parent cells was 3.8 and decreased both in Pgp/+ and Pgp/- cells with increasing selection pressure to 1.2-2.6 for cells with a resistance factor of 7-17. N/C ratios could be restored partly with verapamil only in Pgp/+ cells. N/C ratio measurements may define a general Pgp-independent type of defense of mammalian cells against certain anticancer agents which may precede Pgp expression in early doxorubicin resistance."} +{"text": "A human breast epithelial cell line (Hu-MI), established by microinjecting SV40 DNA into human milk epithelial cells, exhibits the phenotype of luminal epithelial cells and is neither clonogenic nor tumorigenic. From this cell line we have selected two sublines, HuMI-T and HuMI-TTul, reflecting different stages of spontaneous transformation. HuMI-T cells grow anchorage-independently, but do not induce tumours in nude mice. HuMI-TTul cells are clonogenic as well as tumorigenic. Cells from both lines exhibit polymorphic structural and numerical chromosome aberrations. Immortalisation of normal luminal epithelial cells from human mammary gland with SV40 DNA alone may thus cause random genetic changes eventually resulting in tumorigenic cell lines. Since Hu-MI, HuMI-T and HuMI-TTul represent some of the consecutive stages taking place during cellular transformation, they are particularly suited as a novel in vitro model system to study progression of human breast cancer."} +{"text": "We have examined the expression of receptors for epidermal growth factor (EGFR) by the ZR-75-1 human breast cancer cell line and tamoxifen resistant and oestrogen independent/tamoxifen sensitive (ZR-PR-LT) variants. The parent line expressed a single class of high affinity binding sites . ZR-75-9al 8 microM cells, routinely maintained in medium containing 8 microM tamoxifen, were negative for oestrogen receptor (ER) and progesterone receptor (PGR) and expressed a markedly increased number of EGFR . Receptor affinity was unchanged. Time dependent reversal of the tamoxifen resistant phenotype was accompanied by a return to ER and PGR positivity and a fall in EGFR numbers to parent cell levels. In contrast ZR-PR-LT cells had a greatly reduced EGFR content (803 +/- 161 receptors/cell) accompanying elevated PGR numbers. Pre-treatment of these cells with suramin or mild acid stripping failed to expose receptors which may have been occupied by endogenously produced ligand. Increased proliferation of ZR-75-1 cells treated with EGFR (0.01-10 ng ml-1) was only observed in serum-free medium lacking insulin and oestradiol. Under these conditions untreated cells failed to proliferate. Both variant lines continued to proliferate in serum free medium in the absence or presence of insulin and oestradiol but failed to respond to exogenous EGF."} +{"text": "The cell surface receptor for antigen in mature B (BCR) and T lymphocytes (TCR) is central to the adaptative immune response. Structurally, these receptors entails the association between clonotypic antigen binding chains (TCR\u03b1 and TCR\u03b2 for the TCR), coupled to signaling chains . Emergence of many non-Hodgkin B cell lymphomas subtypes is commonly associated with antigenic BCR activation, activating mutations in BCR signaling chains and downstream adapters/effectors. Likewise, progression of certain T cell lymphomas is associated with gain-of function alterations in TCR signaling components. For example, Sezary syndrome cutaneous T cell lymphoma shows upregulation of the TCR LAT adaptor in most cases and frequent activating mutations in the adaptor CARD11 and in phospholipase C\u03b31 [In vitro treatment with anti-CD3\u03b5 specifically induced TCR signaling followed by apoptosis in 24/24 TCR-positive diagnostic T-ALL cases while sparing TCR-negative cases. Most importantly, in vivo expansion of 6/6 TCR-positive xenografts belonging to different T-ALL molecular subtypes was severely impaired by anti-CD3\u03b5 OKT3 mAb treatment, an anti-leukemic effect that translated into improved survival. OKT3 anti-leukemic effects can result from induction of a cell-intrinsic cell death program and/or antibody-dependent cell cytotoxicity (ADCC)-type responses. The fact that LAT expression knockdown in T-ALL strongly impaired the anti-leukemic response to OKT3 shows that, at least in NSG mice, OKT3-induced TCR signaling rather than ADCC-type responses is responsible for the anti-CD3 anti-leukemic effects [in vitro [in vivo (our unpubl. obs.) and found it to resemble that of thymocyte negative selection, but to be distinct from that resulting from inactivation of T-ALL oncogenes [T cell acute lymphoblastic leukemia (T-ALL) originates from the transformation of T cell progenitors, resulting in the accumulation of lymphoblasts arrested at specific stages of differentiation. T-ALL are classified into molecular subtypes characterized by abnormal expression of specific transcription factors , involved in differentiation blockade. A number of additional genetic alterations are found across these subtypes, including activating mutations in NOTCH1 or the JAK/STAT pathway and inactivating mutations in several tumor suppressor genes . TCR signcogenes . CharactThe selection of acquired mutations during T-ALL progression is associated with clonal evolution, resulting in coexistence at diagnosis of related clones endowed with distinct leukemogenic potential. Whether anti-CD3 treatment can impair the leukemia initiating (stem) potential of TCR-positive T-ALL remains to be investigated.OKT3 has been used in the clinics since 1986 to treat allograft rejection. Its side effects in humans include strong immunogenicity and a cytokine-release (flu-like) syndrome. Since then, several humanized anti-CD3\u03b5 mAb further mutated in their Fc domain to impair FcR recognition have been developed ["} +{"text": "Chiung-Ying Liao's named appeared incorrectly as Chun-Ying Liao.In the article, \u201cRole of breast magnetic resonance imaging in predicting malignant invasion of the nipple-areolar complex: Potential predictors and reliability between inter-observers\u201d,"} +{"text": "We found coordinate regulation of precursor and mature miRNAs under hypoxia suggesting their regulation mainly at transcriptional level. Hypoxia response elements were located upstream of 97% of upregulated hypoxia regulated miRNAs (HRMs) suggesting hypoxia-inducible-factor (HIF) driven transcription. HIF binding to the candidate cis-elements of specific miRNAs under hypoxia was confirmed by Chromatin immunoprecipitation coupled with qPCR. Role analysis of a subset of upregulated HRMs identified linkage to reported inhibition of differentiation while a downregulated subset of HRMs had a putative role in the promotion of differentiation. MiRNA-target prediction correlation with published hypoxic hESC and hMSC gene expression profiles revealed HRM target genes enriched in the cytokine:cytokine receptor, HIF signalling and pathways in cancer. Overall, our study reveals, novel and distinct hypoxia-driven miRNA signatures in hESCs and hMSCs with the potential for application in optimised culture and differentiation models for both therapeutic application and improved understanding of stem cell biology.MicroRNAs are reported to have a crucial role in the regulation of self-renewal and differentiation of stem cells. Hypoxia has been identified as a key biophysical element of the stem cell culture milieu however, the link between hypoxia and miRNA expression in stem cells remains poorly understood. We therefore explored miRNA expression in hypoxic human embryonic and mesenchymal stem cells (hESCs and hMSCs). A total of 50 and 76 miRNAs were differentially regulated by hypoxia (2% O In vitro experimentation has established that hypoxic culture of hESC correlates closely with increased clonogenicity, reduced spontaneous differentiation, increased genetic stability, and transcriptional homogeneity alongside improved epigenetic profiles The Low Cell ChIP Kit protocol was followed with some minor modifications. Briefly, cells were cross-linked using 1% formaldehyde in PBS with the reaction stopped after 10 min using 100 \u03bcl of 125 mM glycine. The cells were then washed with PBS followed by suspension in ChIP buffer. The chromatin was next fragmented using Diagenode Bioruptor plus. Fragmented chromatin was immunoprecipitated using HIF-1 antibody (M/s Santa Cruz) and IgG antibody as per Low Cell ChIP kit guidelines. After overnight incubation, the beads were washed and immunoprecipitated and input DNAs were proceeded for DNA isolation using IPure kit following the manufacturer\u2019s instructions. qPCR reactions were performed using 3\u03bcl of DNA in a CFX96Probable targets for differentially regulated miRNAs were identified with the DIANA-MicroT-CDS prediction program . DIANA-m\u2122 real time system (Bio-RAD) using cDNA specific forward primer and a universal reverse primer as listed in Candidate miRNAs were reverse transcribed to cDNA using specific stem-loop RT primers . Quantit2) or hypoxia (2% O2) followed by RNA extraction and determination of miRNA expression using Affymetrix GeneChip\u00ae miRNA Arrays.Previous studies, including ours, have described hypoxia-induced differential gene expression in hESCs and hMSCs , 23, 8, Comparison of normalized normoxic and hypoxic hESCs miRNA profiles identified differential expression of 50 miRNAs ; 31 up-regulated and 19 down-regulated in response to hypoxia . UpregulTo explore uniformity of hypoxic miRNA expression modulation in stem cell populations we next explored hMSC isolated from bone marrow. We identified 76 differentially expressed miRNAs in hMSC (35 up-regulated and 41 down-regulated) in response to hypoxia . Up-reguTaken together the hypoxic miRNA profiles of hESCs and hMSCs were largely distinct with only miR-25-3p (upregulated), mir-1275 and miR-23a-5p (both downregulated) overlapping between the two stem cell populations. Further evidence of distinct behaviour was noted in relation to miR-138-5p which was downregulated and upregulated by hypoxia in hESC and hMSC, respectively.We next sought to determine co-regulation of miRNA clusters by hypoxia. To determine the overall trend of differential expression within the miRNA cluster, we considered all miRNAs showing >\u00b11.5 fold change. We identified 3 miRNA clusters which contained 19 up-regulated miRNAs while a further 6 miRNA clusters contained 15 down-regulated miRNAs in hESCs . The miRFurther evidence of the distinct hESC and hMSC HRM profiles emerged via miRNA clusters- miR-379/656, mir-532/502, miR-17/92 and its paralog miR-106b/25 being downregulated by hypoxia in hESCs while conversely up-regulated in hMSCs. For instance miR-17/92 cluster members were upregulated in hMSC (miR-106b-5p and miR-25-3p) but downregulated (miR-93-3p and miR-25-5p) in hESCs . This suMany hypoxia regulated transcripts contain consensus HIF responsive elements (HRE) in their promoter regions which promote HIF1/HIF2 binding and induction of transcription . The 5 kHIF binding to candidate HREs was examined by CHIP-qPCR in hESCs and hMSCs under normoxia and hypoxia. In hypoxia treated hMSCs, direct HIF binding to the cis-elements was observed for 5/7 miRNAs tested and hMSC upregulated HRMs were predicted to target HIF pathway inhibitors; HIF1A inhibitor (HIF1AN) and HIF3A, respectively, creating a prospective HIF pathway positive feedback loop. In contrast, downregulated HRMs in hESCs (miR-92a-1-5p and miR-92a-2-5p) were predicted to target HIF1A directly creating a negative feedback loop to suppress HIF signal transduction. Overall, this suggests that specific HRMs may operate as a check and balance system to regulate HIF levels during exposure to hypoxia .Utilising previously generated transcript array data sets for both hESC and hMSC in normoxia/hypoxia we next sought to determine miRNA:gene correlations by identifying the inverse correlation of expression in miRNA:target pairs , 8. TargFinally we explored pathways enriched by the differentially expressed miRNA target genes in both hESCs and hMSCs. This identified strong associations with the HIF-1 signalling pathway (p = 2.40E-11) for hESC, pathways in cancer (p\u22640.000975) for hESC and hMSC, and cytokine-cytokine receptor interaction pathway (p = 0.001933) for hMSCs Tables. In the 15 years since the first demonstration of hypoxic regulation of miRNA expression in a range of cancer cell lines, the hypoxia: miRNA correlation has become widely accepted. Several HRMs have now been shown to be critical regulators of hypoxic signal transduction by regulating cellular properties such as apoptosis, proliferation, metabolism, angiogenesis, DNA repair and stemness \u201315. WhilA number of miRNA species have been identified in previous studies as having roles in determining hESC biology. These include miR-145 (pluripotency), -148b, -146a (differentiation), -302 , -195 (apoptosis), -372 (cell division) and miR-9 (migration) \u201331. CoupSimilarly, miRNAs have been shown to play important roles in the regulation of hMSC biology. MiRNAs such as miR-335, -21, -146a-5p, -377, -494, -141, -10a, -138-5p, -140, -17-5p, -143/145, -302 and miR-210 have been identified as pivotal players in governing specific aspects of hMSC biology related to proliferation, migration, differentiation, angiogenesis, aging, and apoptosis , 16. A nThe largely distinct HRM profiles observed between hESC and hMSC illustrate the likely existence of divergent hypoxia signalling pathways in these two cell types. This concept is underlined by a comparison of the hypoxia induced transcriptomes in hESC and hMSC which bear little resemblance to each other. Intriguingly we noted that the canonical upregulated HRM, miR-210 though induced in hMSC, in agreement with other studies , 56, wasWe also noticed that hypoxia based regulation of most of these HRMs is mainly at transcriptional level. The appearance of HIF1A binding site consensus sequences in the majority of HRM promoter sequences coupled to predicted targets including HIF or HIF regulating genes was suggestive of the existence of a feedback-loop in HIF signalling. The cell type specific upregulated miRs were predicted, or previously experimentally validated, to target HIF pathways inhibitors-HIF1AN and HIF3A while downregulated miRNAs were predicted to target HIF1A , 58. TheTwo further pathways enriched in gene ontology analyses of the HRM target genes were cytokine: cytokine receptor (hMSCs) and pathways in cancer (hESCs and hMSCs). In confirmation of our findings previous reports have described oxygen-dependent expression of cytokines and linked them with proliferation and differentiation of hMSCs , 66. A rOverall, our findings add richly to the growing body of data surrounding hypoxic stem cell biology for both hESCs and hMSCs. In consideration of the strong influence of hypoxia on stem cell self-renewal and differentiation the regulatory and functional characterization of hypoxia regulated miRNAs or genes may provide strategies to design novel cellular therapies for regenerative medicine.S1 Fig(a) up-regulated and (b) down-regulated HRMs in SHEF2. QRT-PCR data showing levels of Drosha and DICER in hESCs grown in normoxia and hypoxia (c). Graphical data points in a, b and c represent mean \u00b1 S.D. of a minimum of three independent experiments. .qRT-PCR data of (TIF)Click here for additional data file.S2 Fig(a) up-regulated and (b) down-regulated HRMs in a different human bone-marrow derived MSCs. QRT-PCR data showing levels of Drosha and DICER in hMSCs grown in normoxia and hypoxia (c). Graphical data points in a, b and c represent mean \u00b1 S.D. of a minimum of three independent experiments. .Quantitative RT-PCR data of (TIF)Click here for additional data file.S3 FigGraphical data points in a-d represent mean \u00b1 S.D. of a minimum of three independent experiments. .(TIF)Click here for additional data file.S4 FigA figure showing miRNA:target gene interaction network for HIF-1 signalling pathway in hESCs drawn using Cytoscape software. The green color refers up-regulation while the red color refers down-regulation.(TIF)Click here for additional data file.S5 Fig(a) and hMSCs (b) drawn using Cytoscape software. The green color refers up-regulation while the red color refers down-regulation.A figure showing miRNA: target gene interaction network for pathways in cancer of hESCs (TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file."} +{"text": "The importance of long non-coding RNAs (lncRNAs) in the pathogenesis of various malignancies has been uncovered over the last few years. Their dysregulation often contributes to or is a result of tumour progression. In prostate cancer, the most common malignancy in men, lncRNAs can promote castration resistance, cell proliferation, invasion, and metastatic spread. Expression patterns of lncRNAs often change during tumour progression; their expression levels may constantly rise , or steadily decrease . In prostate cancer, lncRNAs likewise have diagnostic , prognostic , and predictive functions. Considering their dynamic role in prostate cancer, lncRNAs may also serve as therapeutic targets, helping to prevent development of castration resistance, maintain stable disease, and prohibit metastatic spread. ERG) and ETS translocation variant 4 (ETV4) with the regulatory sequence of androgen-regulated genes such as transmembrane protease, serine 2 (TMPRSS2) brings transcription factors under androgen control . C. C61]. CGAS5 gene on 1q25, a PCA-associated locus . A. A71]. ADuring ADT, expression levels of CTBP1-AS constantly increase. Especially in androgen-deprived conditions, the lncRNA promotes cell cycle progression and tumour cell proliferation . During The HOX transcript antisense RNA (HOTAIR) lncRNA is usually repressed by the AR. Consequently, HOTAIR is significantly overexpressed in mCRPC as compared with early-stage PCA . By bindOverexpression of HOTAIR enhances proliferation and invasion of castration-resistant cells. Additionally, levels of HOTAIR constantly increase in LNCaP cell lines upon treatment with enzalutamide . This coAs its name implies, the metastasis-associated lung adenocarcinoma transcript-1 (MALAT-1) was first identified as a prognostic factor in bronchial carcinoma . In the Clinically, MALAT-1 correlates with advanced tumour stage, elevated PSA levels and resistance to ADT . MoreoveIn mCRPC, signalling through the oestrogen receptor \u03b1 (ER\u03b1) constitutes an effective mechanism to bypass the androgen\u2013AR axis . Whilst NEAT1 is recruited to the promoter regions of specific PCA genes. It epigenetically induces an environment in favour of active transcription by binding to histone H3 . MoreoveNotably, NEAT1 levels increase upon long-term treatment with either tamoxifen, bicalutamide or enzalutamide . AccordiFusions of ETS family transcription factors with regulatory sequences of androgen-regulated genes are found in over 50% of PCA patients . By actiThe lncRNA prostate cancer-associated transcript 5 (PCAT5) is particularly overexpressed in ERG-positive mCRPC compared to healthy prostate tissue C 82]. K. K82]. KAs with PCAT5, the lncRNA Second chromosome locus associated with prostate-1 (SChLAP1) is associated with ETS-positive PCA types and is overexpressed in about one-quarter of all PCA. Elevated SChLAP1-levels are even more frequent in mCRPC . It promSChLAP1 levels do not only predict early biochemical recurrence in localised PCA , but alsThe lncRNA Suppressor of cytokine signalling 2-antisense transcript 1 (LncRNA SOCS2-AS1) is located at the opposite strand of the protein coding gene region for SOCS2 . ThroughTNFSF10), a gene downregulated most by SOCS2-AS1, belongs to pro-apoptotic protein ligands of the tumour necrosis factor superfamily [Likewise, in the hormone-sensitive cell line LNCaP and castration-resistant cell line LTDA, the knockdown of SOCS2-AS1 and SOCS2 diminishes cell proliferation . Converserfamily .Particularly in castration-resistant cell lines, knockdown of SOCS2-AS1 markedly induces expression of TNFSF10 and other apoptosis-related genes . ConsequDue to the prolonged and varied disease process, treatment of prostate cancer has to be planned individually for each patient. On the grounds of thorough basic medical research performed over the last few years, molecular mechanisms underlying the pathogenesis of prostate cancer have been gradually uncovered. The introduction of novel anti-androgens into clinical practice has markedly improved life expectancy of patients resistant to conventional anti-hormonal therapy. Treatment may be adapted upon detection of specific biomarkers such as the AR-V7 splice variant in mCRPC. LncRNAs are involved in all these stages of tumour progression. They may preserve androgen-related pathways upon androgen deprivation, promote the progress towards castration-resistant states or maintain cellular proliferation and invasion independent from androgens. Some lncRNAs are already\u2014or may be in the future\u2014used as diagnostic biomarkers. Distinct lncRNA expression patterns can be prognostic or predictive. As therapeutic targets, lncRNAs could likewise enhance efficacy of anti-tumour agents and aid deceleration of prostate cancer progression.The expression of lncRNAs can be regulated by using the RNA-interference (RNAi) technology. In this method, short double-stranded RNAs induce a RISC (RNA Induced Silencing Complex) -mediated degradation of their target lncRNA . Therefo"} +{"text": "Negative symptoms are a core feature of schizophrenia and are a major determinant of functional impairment. Few studies have been conducted to examine patterns of longitudinal course of negative symptoms in the early stage of illness. Differential relationships of negative symptom trajectories with long-term clinical and functional outcomes remain to be clarified. This study aimed to investigate patterns of negative symptom trajectories over 3 years, utilizing latent class growth analysis (LCGA), in patients presenting with first-episode non-affective psychosis. Predictive capacity of symptom trajectories on 13-year functional and negative symptom outcomes was also examined.One hundred thirty-six Chinese patients aged 18\u201355 years presenting with DSM-IV first-episode schizophrenia, schizoaffective disorder, schizophreniform disorder, brief psychotic disorder or delusional disorder were assessed at clinical stabilization for first psychotic episode (baseline), 1, 2, 3 and 13 years of follow-up. Assessments encompassing premorbid adjustment, baseline symptom and cognitive profiles and functional levels were conducted. Negative symptoms were measured by High Royds Evaluation of Negativity (HEN) Scale. Individual class membership of negative symptoms derived from LCGA was based on HEN ratings at baseline, 1, 2 and 3-year follow-up.Three distinct negative symptom trajectories were identified including low-stable , moderate-stable and high-increasing trajectories. Multinomial regression analysis revealed that poorer premorbid adjustment, lower baseline cognitive composite scores and more severe baseline depression predicted high-increasing trajectory membership . At 13 years, 88 patients (64.7%) completed follow-up assessment, with attrition analysis indicating lack of significant differences in demographic, premorbid and baseline characteristics between completers and non-completers. Analysis of covariance followed by post-hoc comparison analyses found that high-increasing trajectory was significantly associated with poorer global functional outcome and higher negative symptom levels at 13-year follow-up.Our results indicate that 11% of first-episode non-affective psychosis patients displayed persistently high levels of negative symptoms with gradual symptom worsening over 3-year follow-up. This trajectory membership was predictive of poorer negative symptom and functional outcomes 13 years after presentation. High-increasing negative symptom trajectory identified in the initial 3 years of treatment for first-episode psychosis may represent a subgroup of patients having markedly elevated risk of developing deficit syndrome in the later course of illness."} +{"text": "The upregulated expression of heparin binding EGF-like growth factor (HB-EGF) in the vessel and circulation is associated with risk of cardiovascular disease. In this study, we tested the effects of HB-EGF targeting using HB-EGF-specific antisense oligonucleotide (ASO) on the development of aortic aneurysm in a mouse aneurysm model.Low-density lipoprotein receptor (LDLR) deficient mice were injected with control and HB-EGF ASOs for 10 weeks. To induce aneurysm, the mice were fed a high fat diet at 5 week point of ASO administration and infused with angiotensin II for the last 4 weeks of ASO administration. We confirmed that the HB-EGF ASO administration significantly downregulated HB-EGF expression in multiple tissues including the liver. Importantly, the HB-EGF ASO administration significantly suppressed development of aortic aneurysms including thoracic and abdominal types. Interestingly, the HB-EGF ASO administration induced a remarkable anti-hyperlipidemic effect by suppressing very low density lipoprotein (VLDL) level in the blood. Mechanistically, the HB-EGF targeting suppressed hepatic VLDL secretion rate without changing heparin-releasable plasma triglyceride (TG) hydrolytic activity or fecal neutral cholesterol excretion rate.This result suggested that the HB-EGF targeting induced protection against aneurysm development through anti-hyperlipidemic effects. Suppression of hepatic VLDL production process appears to be a key mechanism for the anti-hyperlipidemic effects by the HB-EGF targeting. Heparin binding EGF-like growth factor (HB-EGF), which is a member of epidermal growth factor (EGF) family member and a ligand for EGF-receptor (EGFR) , is invoHyperlipidemia is a key risk factor for the development of vascular diseases including aneurysm and atherosclerosis , 15. ForInfusion of angiotensin II (AngII) into hyperlipidemic mouse models have been widely used as aneurysm models for the last decade , 20. AngIn this study, we targeted HB-EGF gene transcription using HB-EGF-specific antisense oligonucleotide (ASO) administration to determine the targeting effects on aortic aneurysm development. In summary, we observed that the HB-EGF ASO administration induced an efficient protection against aneurysm developments in ascending and abdominal aorta. The HB-EGF targeting induced a remarkable anti-hyperlipidemic effect by suppressing hepatic VLDL secretion, which appears to be a key mechanism for the protection.GGCCAATACGCCGTCA -3\u20325\u2032- ) and HB-EGF ASO (597622: TACATTATAGTCTTGG -3\u20325 \u2032-) were synthesized and purified by Ionis Pharmaceuticals as previously described for the last 5 weeks of the study. At the 6 week point, osmotic mini-pumps were filled with AngII and implanted subcutaneously. (B) Weekly body weight changes of the disease model mice. Starting points for HFD feeding and AngII infusion are marked with arrows. Values are mean plus standard deviation (SD). (C) Representative examples of aortic arch intimal images for the control and HB-EGF ASO groups. The \u2018a\u2019 indicates location of aortic dissection; and \u2018b\u2019 indicates lesion area covered with plaque accumulation in subendothelial space. The intimal perimeter of the ascending aorta was traced in the right panel image. Scale bars inserted have units of mm.((TIF)Click here for additional data file.S3 FigA-B) Images of aortas for control and HB-EGF ASO treatment groups. * indicates AAA located at the suprarenal area of the abdominal aorta. (C) At termination, the maximal diameter of the suprarenal abdominal aorta was measured. (D) En face measurement of aortic arch intimal atherosclerotic lesion area as a percent of total aortic arch lumen area.Refer to (TIF)Click here for additional data file.S4 FigA) At the termination step, liver, aorta, heart, and kidney tissues were isolated for the measurement of HB-EGF expression levels by qRT-PCR analyses. (B) After removing adventitia from the aortic structure, the diameters of aortic arch and suprarenal area were measured. (C) Alignment of images of intact aortas linked with heart and kidney tissues. (D) The size of heart and kidney was measured for long and short dimensions of tissues.Male LDLR deficient mice were injected weekly intraperitoneally with either control or HB-EGF ASOs (40 mg/kg/wk) for 6 weeks (N = 5 per group). The mice were fed normal standard diet. There was no treatment of AngII in the mice (as non-disease control mice). ((TIF)Click here for additional data file.S5 FigA) The representative images of heart sections (B) Morphology of the heart muscle cells (x 200) Systolic blood pressure and heart rate as measured by tail-cuff method as described in the Procedure section. At the termination step, plasma samples were collected by heart puncture. The levels of total cholesterol and TG in the plasmas were quantified.Refer to (TIF)Click here for additional data file.S6 FigA, B) At the termination step, the plasma samples of each animal were collected by heart puncture bleeding. The levels of plasma total cholesterol and TG were quantified.C57BL/6 mice were injected weekly intraperitoneally with either control or HB-EGF ASOs (40 mg/kg/wk) for 6 weeks (N = 5 per group). The mice were fed normal standard diet. There was no treatment of AngII (as non-disease wild type control mice) ((TIF)Click here for additional data file.S7 FigA) Heparin-releasable plasma TG hydrolytic activities were measured in C57BL/6 mice, which is genetic background of LDLR KO mice, after 3 weeks of control or HB-EGF ASO administrations (50 mg/kg/wk) (N = 5). Downregulation of hepatic HB-EGF expression levels by the HB-EGF ASO administration was separately confirmed by qRT-PCR. (B) Heparin-releasable plasma TG hydrolytic activities were measured in C57BL/6 mice after one time tail-vein injection of either saline or recombinant HB-EGF at 2 hours before heparin injection. (N = 5) (C). The HB-EGF ASO administration for 6 weeks (40 and 20 mg/kg/wk for 4 and 2 weeks consequently) in LDLR deficient mice under normal diet did not change fecal neutral sterol excretion rate. (N = 5) Refer to Supplemental Procedure-Extended for the procedure details. n.s. = not significant.((TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file."} +{"text": "N-acetyl-p-aminophenol (APAP)]-induced acute liver injury (ALI) not only remains a persistent clinical challenge but likewise stands out as well-characterized paradigmatic model of drug-induced liver damage. APAP intoxication associates with robust hepatic necroinflammation the role of which remains elusive with pathogenic but also pro-regenerative/-resolving functions being ascribed to leukocyte activation. Here, we shine a light on and put forward a unique role of the interleukin (IL)-1 family member IL-18 in experimental APAP-induced ALI. Indeed, amelioration of disease as previously observed in IL-18-deficient mice was further substantiated herein by application of the IL-18 opponent IL-18-binding protein (IL-18BPd:Fc) to wild-type mice. Data altogether emphasize crucial pathological action of this cytokine in APAP toxicity. Adding recombinant IL-22 to IL-18BPd:Fc further enhanced protection from liver injury. In contrast to IL-18, the role of prototypic pro-inflammatory IL-1 and tumor necrosis factor-\u03b1 is controversially discussed with lack of effects or even protective action being repeatedly reported. A prominent detrimental function for IL-18 in APAP-induced ALI as proposed herein should relate to its pivotal role for hepatic expression of interferon-\u03b3 and Fas ligand, both of which aggravate APAP toxicity. As IL-18 serum levels increase in patients after APAP overdosing, targeting IL-18 may evolve as novel therapeutic option in those hard-to-treat patients where standard therapy with N-acetylcysteine is unsuccessful. Being a paradigmatic experimental model of ALI, current knowledge on ill-fated properties of IL-18 in APAP intoxication likewise emphasizes the potential of this cytokine to serve as therapeutic target in other entities of inflammatory liver diseases.Acetaminophen [paracetamol, N-acetyl-p-aminophenol (APAP)] is regarded a major cause of acute liver failure provoking roughly 50,000 emergency room admissions, 2,500 hospitalizations, and 500 fatalities per year in the United States. The global burden on health-care systems that connects to APAP is based on a narrow therapeutic margin and supported by its broad over-the-counter availability. In fact, adverse consequences of APAP (self-)pharmacotherapy fuel a sustained discussion on safety issues and regulations regarding this fairly weak but frequently used analgesic drug , an APAP metabolite generated by hepatic Cyp2e1 and Cyp1a2. Under the influence of NAPQI hepatocytes endure oxidative stress, malfunction of mitochondrial respiration, a drop in ATP, and predominantly necrotic cell death. Here, standard therapy with N-acetylcysteine interferes by providing NAPQI detoxifying glutathione (GSH) and by counteracting APAP-associated oxidative stress. Aforementioned noxious chain of events is amplified by cell intrinsic processes, among others activation of c-Jun N-terminal kinase (Murine models of APAP-induced acute liver injury (ALI) are well established and adequately resemble key features of human intoxication . A crucil kinase \u20137. Sincel kinase , an addil kinase , 10 and l kinase were repl kinase , 13. Intl kinase , 14, 15.l kinase or supprl kinase , 18.IL22BP-deficient mice suggest a pathogenic role for endogenous IL-22 particularly during early intoxication in adult onset still\u2019s disease actually revealed an acceptable safety profile of this agent\u2014besides specific therapeutic efficacy . MoreoveAll animal experiments using C57Bl/6 mice were carried out in accordance with the recommendations of the Animal Protection Agency of the Federal State of Hessen . The protocol was approved by the Regierungspr\u00e4sidium Darmstadt (Germany).MB: performed all experiments, analyzed the data, and contributed to manuscript writing and editing. JP: analyzed the data and contributed to manuscript editing. HM: analyzed the data, designed the study, wrote the paper, and performed manuscript editing.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "AT7519 was able to override this phenotype to induce apoptosis in ARDS neutrophils with reduced expression of the pro-survival protein Mcl-1. We demonstrate the first pharmacological compound to induce neutrophil apoptosis in sepsis-related ARDS, highlighting cyclin-dependent kinase inhibitors as potential novel therapeutic agents.Acute respiratory distress syndrome (ARDS) is a neutrophil-dominant disorder with no effective pharmacological therapies. While the cyclin-dependent kinase inhibitor AT7519 induces neutrophil apoptosis to promote inflammation resolution in preclinical models of lung inflammation, its potential efficacy in ARDS has not been examined. Untreated peripheral blood sepsis-related ARDS neutrophils demonstrated prolonged survival after 20\u2005hours Acute respiratory distress syndrome (ARDS) is a neutrophil-dominant disease with significant morbidity and mortality but no effective pharmacological therapies exist. Pulmonary neutrophil accumulation results in release of chemokines, proteases and reactive oxygen species perpetuating inflammation and tissue injury. Augmenting neutrophil apoptosis in order to accelerate the resolution of inflammation has been proposed as a treatment strategy.Neutrophils are short-lived granulocytes that undergo energy-dependent and caspase-dependent programmed cell death (apoptosis) within hours. Apoptosis, in concert with clearance by phagocytes, results in a pro-resolution phenotype restoring tissue homeostasis. Neutrophil survival is governed by both pro-apoptotic and anti-apoptotic factors in the intracellular and extracellular environment. Granulocyte macrophage-colony stimulating factor (GM-CSF), tumour necrosis factor (TNF), hypoxia and bacterial endotoxins increase lifespan partly through upregulation of intracellular proteins including the Bcl-2 family member Mcl-1. Spontaneous apoptosis is therefore delayed in various diseases including ARDS, cystic fibrosis and sepsis and is thought to contribute to disease pathogenesis.in vitro by phosphoinositide 3-kinase (PI3K) and cyclin-dependent kinase (CDK) inhibitors, polyphenolic flavones and lipid mediators.in vitro including GM-CSF and bacterial endotoxins. The CDKi AT7519 potently inhibits CDK9 and, at higher concentrations, other CDKs including CDK2 and CDK5 but not non-CDK kinasesApoptosis in neutrophils isolated from healthy human volunteers has been achieved Venous blood was collected from mechanically ventilated patients with ARDS (defined according to Berlin criteria)10.1136/thoraxjnl-2016-209229.supp1supplementary tableDemographic and clinical data for ARDS patients and control subjects6\u2005cells/mL; 5% autologous or fetal calf serum) in the presence or absence of AT7519 . Neutrophil apoptosis was examined by flow cytometry (Annexin-V (Roche) and propidium iodide (Sigma)) and confirmed by cytocentrifuge and Diff-Quik staining (Gamidor).Neutrophils, isolated by dextran sedimentation and Percoll gradient, were cultured in Iscove's modified Dulbecco's medium (Gibco) and statistical analyses with Prismv7 (GraphPad); significance was accepted at p<0.05.Unstimulated peripheral blood neutrophils from patients with ARDS had greater survival compared with age-matched and sex-matched healthy controls following 13 and 20\u2005hours of culture A. This eAT7519 induced neutrophil apoptosis in healthy volunteer neutrophils within 6\u2005hours. In ARDS neutrophils, AT7519 induced apoptosis but only after 13\u2005hours of culture. By 20\u2005hours apoptosis was at a level equivalent to AT7519-treated healthy control cells, thus completely overriding the pro-survival phenotype A\u2013C. NecrWithin the highly complex proinflammatory milieu, increased neutrophil survival provides the opportunity for continued release of toxic mediators to exacerbate tissue injury and potentiate inflammation. Indeed, delayed neutrophil apoptosis correlates with disease severity in sepsis and associated lung injury,Recent elegant phenotyping of peripheral blood and alveolar neutrophils in ARDS has been described.Taken together, this suggests that intrinsic, PI3K-independent factors act to delay neutrophil apoptosis in ARDS and supports the conclusion that Mcl-1 targeted therapies may be beneficial in human disease. CDKi-augmented neutrophil apoptosis may therefore enhance resolution of lung inflammation and serve as a potential therapeutic strategy in ARDS."} +{"text": "Tz) and targeted to PSMA expressing cells using trans-cyclooctene (TCO)-functionalized anti-PSMA antibodies (TCO-anti-PSMA). The extent of MB binding to PSMA positive cells for two different targeting strategies was determined using an in vitro flow chamber. The initial approach involved pretargeting, where TCO-anti-PSMA was first incubated with PSMA expressing cells and followed by MBTz, which subsequently showed a 2.8 fold increase in the number of bound MBs compared to experiments performed in the absence of TCO-anti-PSMA. Using direct targeting, where TCO-anti-PSMA was linked to MBTz prior to initiation of the assay, a 5-fold increase in binding compared to controls was observed. The direct targeting approach was subsequently evaluated in vivo using a human xenograft tumor model and two different PSMA-targeting antibodies. The US signal enhancements observed were 1.6- and 5.9-fold greater than that for non-targeted MBs. The lead construct was also evaluated in a head-to-head study using mice bearing both PSMA positive or negative tumors in separate limbs. The human PSMA expressing tumors exhibited a 2-fold higher US signal compared to those tumors deficient in human PSMA. The results demonstrate both the feasibility of preparing PSMA-targeted MBs and the benefits of using bioorthogonal chemistry to create targeted US probes.Prostate specific membrane antigen (PSMA) targeted microbubbles (MBs) were developed using bioorthogonal chemistry. Streptavidin-labeled MBs were treated with a biotinylated tetrazine (MB Prostate cancer (PCa) is the second leading cause of cancer-related deaths in men . It is eOne approach to improving PCa detection is to employ imaging agents that target prostate specific membrane antigen (PSMA). PSMA is a transmembrane glycoprotein that is expressed at low levels in normal prostate, liver, kidney and brain tissue, but is expressed at much higher levels in PCa tumors \u201317. HighA microbubble based (MB) contrast agent that targets PSMA would provide the opportunity to use US imaging to detect and characterize primary and recurrent PCa . PSMA is+) cells in vitro, however the evaluation of this agent under dynamic flow conditions or in preclinical animal models has not yet been reported. Wang and coworkers prepared nano-scale US contrast bubbles (NBs) coated with streptavidin and loaded with a biotinylated derivative of an anti-PSMA antibody . Th. Thtransding MBs . The fireted MBs , prior t8 MBs/vial) from a MicroMarkerTM, Target-Ready Contrast Agent Kit . Streptavidin coated magnetic beads and MACSiMAGTM separator magnet were used during the purification of MBs. Conjugated antibodies were analyzed by mass spectrometry on a MALDI Bruker Ultraflextreme Spectrometer. MB size and concentration were determined using Z2 Coulter counter . The syringe pump used in the flow chamber assay was a PhD 2000 . Western blot images were generated using a STORM 840 imaging system Microbubbles (MBs) were obtained (8.4 \u00d7 102CO3 (aq). (E)-Cyclooct-4-enyl-2,5-dioxopyrrolidin-1-yl carbonate (TCO-NHS) was then added in 9 \u03bcL of DMSO. The solution was left on a shaker overnight at 4\u00b0C. The desired product (TCO-J591) was isolated from excess TCO using an Amicon Ultra-0.5 Centrifugal filter (30 kDa) and washed with PBS three times. Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) analysis of the antibody, before and after conjugation to TCO, showed an average of 1.2 TCO groups per antibody using a MALDI Bruker Ultraflextreme Spectrometer that binds the extracellular domain of human PSMA , was protrometer . TCO wasN-benzyl)-6-(5-((4S)-2-oxohexahydro-1H-thienoimidazol-4-yl)pentanamido)hexanamide (biotin-Tz) was synthesized as previously described imidazol-4-yl)-pentanamido)- hexanamide -cyclooct-4-enyl-2,5-dioxopyrrolidin-1-yl carbonate (TCO-NHS) at pH 9\u20139.5 and 4\u00b0C overnight. The number of TCO moieties per antibody was 1.2 as determined by MALDI-TOF MS , they have low adhesive properties in culture, and therefore were not suitable for use in the flow chamber system. Instead, more adherent PC-3 cells that were PSMA+ (human PSMA gene-transfected) or PSMA- (non-transfected) were used. Western blot analysis of the two PC-3 cell lines showed PSMA protein expression in the PSMA+ PC-3 cells at levels comparable to LNCaP cells, and no expression by PSMA- PC-3 cells . Both the MBTz (pretargeting) and the MBTz-TCO-J591 conjugates (direct targeting) were added at a standard flow rate. After increasing the flow rates to remove any non-covalently bound MBs, bright-field microscopy images were collected and images were analyzed using FIJI software following a literature procedure [Both the pretargeting and direct targeting approaches were evaluated using the rocedure .Tz to PSMA+ PC-3 cells (Tz by pretargeting was still 2.8 times higher than controls in which non-conjugated MBs (no Tz) or PSMA- PC-3 cells were used . Qualitatively, the parametric US images showed higher signal enhancement when using the MBTz-TCO-J591 direct targeting construct, as compared to non-targeted MBTz that did not reach significance. The apparent difference may indicate greater benefit for targeted US imaging in xenograft models through the use of antibodies that bind to both mouse and human PSMA. Irrespective of the differences in signal enhancement, both direct targeting constructs demonstrated enhanced accumulation of PSMA-targeted MBs in the tumor compared to the control non-targeted MBTz. Furthermore, these results demonstrate the feasibility and ease of using bioorthogonal chemistry for screening different targeting vectors.For all studies, imaging was performed using the same plane of view as that for MBs containing no targeting agent mice that had both PSMA+ and PSMA\u2212PC-3 tumors in each animal. This model was selected as it has been used to evaluate the ability of other molecular imaging probes [+ and PSMA\u2212tumors. In this study, MBTz-TCO-ARP was injected and allowed to circulate for 4 min. Using the disruption replenishment sequence, images were collected in a random order between the PSMA+ and PSMA\u2212tumors. The parametric US images showed qualitatively higher signal enhancement in PSMA+ PC-3 tumors compared to PSMA\u2212tumors in PSMA+ tumors compared to that in PSMA\u2212tumors (Tz-TCO-ARP in PSMA-expressing tumors was shown to be 1.97 fold higher (\u00b1 0.55) than that in PSMA-deficient tumors. These results indicate that the presence of PSMA promoted enhanced binding of the targeted MBs.The signal enhancement attributed to binding of MB, n = 7) . OverallIt is worth noting that these results show a larger signal enhancement for targeted MBs compared to the previous reports using PSMA-targeted nanobubbles ,36. For Tz that when combined with a humanized anti-PSMA antibody (e.g. J591) will create PSMA-targeted MBs suitable for translation to clinical trials. This work will leverage the high yield of the Tz-TCO reaction and allow for production of targeted MBs with minimal modification to both the contrast agent and targeting moiety. While US imaging has a number of attractive features, the issues of user dependent variability and potential cost of the agent will need to be considered in the future when comparing the reported approach with other diagnostic methods. In the interim, the targeted MBs described here can be produced via straightforward chemistry and a commercially available MB kit. These PSMA-targeted MBs can be used for preclinical US imaging studies in animal models of PSMA-expressing cancers.The experiments demonstrated the effectiveness of TCO-Tz bioorthogonal chemistry for constructing US MBs that can bind to PSMA expressing tumors. Studies are ongoing to create a human compatible MBS1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file.S3 File(PDF)Click here for additional data file.S4 File(PDF)Click here for additional data file.S5 File(PDF)Click here for additional data file."} +{"text": "Olea europaea L.) pollen during in vitro germination and assessment of the S-nitroso- and nitro-proteomes by computational predictive methods\u201d doi:10.1016/j.niox.2017.06.005 S-nitrosylation and Tyr-nitration sites in proteins derived from a de novo assembled and annotated pollen transcriptome from olive tree (Olea europaea L.) were obtained after using well-established predictive tools in silico. Predictions were performed using both default and highly restrictive thresholds. Numerous gene products identified with these characteristics are listed here. An experimental validation of the data, consisting in nano-LC-MS (Liquid Chromatography-Mass Spectrometry) determination of olive pollen proteins after immunoprecipitation with antibodies to anti-S-nitrosoCys and anti-3-NT (NitroTyrosine) allowed identification of numerous proteins subjected to these two post-translational modifications, which are listed here together with information regarding their cross-presence among the predictions.The data presented here are related to the research article entitled \u201cGeneration of nitric oxide by olive ( Specifications TableValue of the data\u2022Obtained data can be compared with dataset obtained from other sources or predictions, enabling for example comparisons between vegetative/reproductive tissues, diploid/haploid material or signaling capabilities throughout differential metabolic pathways.\u2022in vivo, determining under what conditions they can be modified, the effects of such modifications, reversibility of the modifications (S-nitrosylation), and many other aspects.nano-LC-MS determinations allowed building a list of structural, enzymatic, regulatory etc. proteins that should be further assessed regarding whether these gene products are really modified by NO \u2022in silico tools are a good start point for such improvements.Information listed here can be used to improve performance of the predictive tools used here and to create new programs: algorithms are usually modified after feedback from experimental studies. Proteins listed here after MS identification and non-predicted by the 1S-nitrosoproteome of olive pollen is provided through 3 different tables ortholog, FLN status and FLN definition and TAIR database ortholog.Predicted t tables , which iS-nitrosylation position/s, FLN ortholog, FLN status and FLN definition and TAIR database ortholog.S-nitrosoproteome of olive pollen is provided in the form of a table http://sno.biocuckoo.org) http://csb.cse.yzu.edu.tw/SNOSite/) http://reprolive.eez.csic.es) http://yno2.biocuckoo.org) For 2.2S-nitrosoCys polyclonal (Sigma-Aldrich) antibodies conjugated to protein A agarose beads (Sigma-Aldrich), which were then collected by centrifugation and washed. Samples were subjected to SDS-PAGE gels. Bands of interest were excised after colloidal Coomassie staining of gel, and subjected to nano LC-MS analysis using an NanoAcquity nano-HPLC (Waters), equipped with a Waters BEH C18 nano-column (LC step). Mass spectrometry was performed in a Synapt G2Si ESI Q-Mobility-TOF spectrometer (Waters) equipped with an ion mobility chamber (T-Wave-IMS). Database searching was performed using MASCOT 2.2.07 against an ad hoc in house made database corresponding to Olea europaea L Proteins from 'Picual' mature pollen homogenates were subjected to immunoprecipitation with either an anti-3-NT polyclonal (Millipore) or anti-European Regional Development Fund (ERDF) co-funded projects BFU2011-22779, BFU2016-77243-P, RTC-2015-4181-2 and RTC-2016-4824-2 (Spanish MINECO), P2011-CVI-7487 and 201540E065 (CSIC).This work is part of Ph.D. thesis by Mar\u00eda Jos\u00e9 Jim\u00e9nez-Quesada, and was supported by"} +{"text": "Nicotiana benthamiana transient overexpression systems offer unique advantages for rapid and scalable biopharmaceuticals production, including high scalability and eukaryotic post-translational modifications such as N-glycosylation. High-mannose-type glycans (HMGs) of glycoprotein antigens have been implicated in the effectiveness of some subunit vaccines. In particular, Man9GlcNAc2 (Man9) has high binding affinity to mannose-specific C-type lectin receptors such as the mannose receptor and dendritic cell-specific intracellular adhesion molecule 3-grabbing non-integrin (DC-SIGN). Here, we investigated the effect of kifunensine, an \u03b1-mannosidase I inhibitor, supplemented in a hydroponic culture of N. benthamiana for the production of Man9-rich HMG glycoproteins, using N-glycosylated cholera toxin B subunit (gCTB) and human immunodeficiency virus gp120 that are tagged with a H/KDEL endoplasmic reticulum retention signal as model vaccine antigens. Biochemical analysis using anti-fucose and anti-xylose antibodies as well as Endo H and PNGase F digestion showed that kifunensine treatment effectively reduced plant-specific glycoforms while increasing HMGs in the N-glycan compositions of gCTB. Detailed glycan profiling revealed that plant-produced gp120 had a glycan profile bearing mostly HMGs regardless of kifunensine treatment. However, the gp120 produced under kifunensine-treatment conditions showed Man9 being the most prominent glycoform (64.5%), while the protein produced without kifunensine had a substantially lower Man9 composition (20.3%). Our results open up possibilities for efficient production of highly mannosylated recombinant vaccine antigens in plants. Nicotiana benthamiana expression systems using viral and non-viral vectors have become viable platforms for the production of recombinant proteins .Over the past decade, 9GlcNAc2, a high-mannose-type glycan (HMG) with 9 mannosyl residues (Man9) has a higher binding affinity to DC-SIGN than other HMGs with fewer mannoses (Man5-8) and complex-type N-glycans -type and Golgi \u03b1-mannosidase-I proteins are responsible for the initial step of mannose trimming bound to cell-surface DC-SIGN in addition to GM1-ganglioside receptors, indicating that the glycosylated vaccine antigen may elicit additional immunomodulatory activity via several C-type lectin receptors . See Supplemental Methods for vector construction, expression, and purification of gp120. The purified protein was analyzed via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) whereas its concentration was measured by the bicinchoninic acid (BCA) protein assay using HEK293 cell-produced gp120DU156 as a reference control.Proteins were transiently overexpressed using the magnICON\u00ae deconstructed tobamovirus vector treatments with each group containing 3 plants, viz., control receiving no kif at all (0 kif), plants receiving kif only once (1 kif), twice (2 kif) and/or thrice (3 kif) during post inoculation growth . Plant tissues were lysed by grinding the tissue using liquid nitrogen and with a mortar and pestle. The samples were prepared by QIAShredder (Qiagen) and RNAqueous Phenol-free total RNA Isolation Kits , following manufacturer's protocol. After total RNA isolation, TURBO DNA free kit (Thermo Fisher Scientific) was used to eliminate genomic DNA. For reverse transcription, first strand cDNA (2 \u03bcg) was synthesized using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). RT-qPCR was performed on an StepOnePlus\u2122 Real-Time PCR System (Thermo Fisher Scientific) with SYBR Green PCR master mix (Thermo Fisher Scientific). The primers for BiP, PDI, and bZIP60 and RT-qPCR conditions were followed as described previously digestions were performed as described previously and peptide-GM1-ganglioside-capture enzyme-linked immunosorbent assay (GM1-ELISA) was used for the detection and quantification of gCTB using a commercial CTB , as described previously was subjected to Endo H and PNGase F digestions. Endo H is a glycosidase which cleaves within the chitobiose core of high mannose and some hybrid oligosaccharides from N-linked glycoproteins. PNGase F, on the other hand, cleaves mammalian N-glycans (including HMGs) between the innermost N-acetylglucosamine (GlcNAc) and Asn residues but fails to cleave those containing \u03b1-linked fucose, which are mostly found in plant and some insect glycoproteins at 5 days post vector inoculation -based ion-exchange chromatography. The lectins of G. nivalis bind to D-mannose and have been used for the purification of HIV gp120 (Man5\u22129GlcNAc2) Figure . A previ) Figure . However) Figure . This mi) Figure . Neverth) Figure .N. benthamiana with kifunensine allows us to obtain Man9-rich HMG-displaying recombinant glycoproteins upon transient overexpression. Our findings warrant further studies evaluating the effectiveness of kifunensine treatment for other glycoproteins, particularly those without a H/KDEL tag, optimization of hydroponic culture conditions, and feasibility of this approach for large-scale production. With additional investigations for glycosylation and bioprocess optimizations, our strategy discussed here opens up new possibilities of producing mannosylated recombinant vaccine antigens that can be efficiently targeted to C-type lectin receptors. Identification and characterization of N. benthamiana mannosidase(s) targeted by kifunensine may aid in understanding the glycosylation regulation in plants and developing glyco-engineered host plants for vaccine production.To conclude, we have shown that the hydroponic treatment of NM: Conceived of and designed the study; SR and YO: Performed experiments and contributed equally to the work; HK: Performed glycan analysis; SR, YO, KH, KF, and NM: Analyzed data; SR, YO, and NM: Wrote the manuscript. All authors reviewed the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "P. berghei-infected male Sprague-Dawley rats. Oral glucose test (OGT) responses to OA-pectin patch and CHQ-OA combination matrix patch were monitored in non-infected and infected rats. To evaluate the short-term effects of treatment, percentage parasitaemia, blood glucose, glycogen and plasma insulin were monitored in separate groups of animals treated with either OA-patch monotherapy or CHQ-OA combination pectin patch over a 21-days period. Animals treated with drug-free pectin and CHQ acted as untreated and treated positive controls, respectively. Infected control rats exhibited significantly increased parasitaemia which was accompanied by hypoglycaemia. Both OA monotherapy and CHQ-OA combination therapy reduced and cleared the malaria parasites within a period of 4 and 3 days, respectively. Compared to respective controls groups, OGT responses of animals treated with OA monotherapy or CHQ-OA combination therapy exhibited lower blood glucose levels at all time points. A once-off transdermal application of OA-patch or CHQ-OA combination patch significantly improved blood glucose concentrations inducing any changes in insulin concentration. Transdermal OA used as a monotherapy or in combination with CHQ is able to clear and reduce the malaria parasites within a shorter period of time without eliciting any adverse effects on glucose homeostasis of P. berghei-infected rats.The present study investigated the effects of transdermally delivered oleanolic acid (OA) monotherapy and in combination with chloroquine (CHQ) on malaria parasites and glucose homeostasis of Plasmodium falciparum. This decrease in blood glucose has been attributed to increased utilisation of the host\u2019s glucose stores by the malaria parasites commonly known as clove has been widely used in folkloric medicine for the treatment of a variety of ailments. Reports indicate that S. aromaticum-derived oleanolic acid (OA) and maslinic acid (MA) possesses anti-inflammatory, antibacterial, antioxidant, and hepatoprotective properties [Myrtaceae] (cloves) flower buds using a standard protocol that has been validated in our laboratory with slight modifications [S. aromaticum flower buds (500 g) were sequentially extracted twice at 24-hour intervals at room temperature with 1 L dichloromethane (DCM), and ethyl acetate (720 mL) on each occasion. Removal of the solvent from the extract under reduced pressure at 55\u00b11\u00b0C using a rotary evaporator yielded dichloromethane solubles and ethyl acetate solubles . The EAS containing mixtures of oleanolic/ursolic acid was purified by silica gel 60 column chromatography with a hexane: ethyl acetate 9:1, 8:2 solvent system increasing polarity. OA was yielded. The structure of OA was confirmed by spectroscopic analysis using 1D and 2D, 1H and 13C Nuclear Magnetic Resonance (NMR) techniques.OA was isolated from ications \u201326. Brieet al., (2003) with slight modifications [2 solution was added to allow for cross-linking and formation of the matrix patch. The patches were then stored in a refrigerator at 4\u00b0C until use.Amidated pectin hydrogel OA and CHQ-OA matrix patches were prepared using a well-established protocol by Musabayane ications . BrieflyP. berghei-infected control animals were closely monitored, for ethical reasons the infected control animals were sacrificed on day 12 of the 21 days study to alleviating any pain and distress. The animals were sacrificed by exposing to halothane (100 mg/kg) for 3 minutes via a gas anaesthetic chamber. All animal experimentation was reviewed and approved by the Animal Ethics Committee of the University of KwaZulu-Natal .Male Sprague-Dawley rats (90-120g body weight) bred and maintained at the Biomedical Research Unit, University of KwaZulu-Natal were used. The animals had free access to standard rat chow and water, with a 12-hour light/12 h-hour dark cycle. The animals were monitored twice daily at 8h00 and 17h00. Humane endpoints were also monitored daily and the animals were assessed on the following: alertness upon handling, mobility, type of breathing change in body weight, haematocrit, % baseline weight change, food and water consumption. Animals that stopped eating, drinking water or lost more than 20% of baseline weight at any time point were euthanised by exposing to halothane (100 mg/kg) for 3 minutes via a gas anaesthetic chamber. Untreated P. berghei parasitized red blood cells (105) [Malaria was induced in male Sprague-Dawley rats with a single intraperitoneal injection of ls (105) . ControlRats were shaved on the dorsal region of the neck using the Oster Golden A5 heavy duty single-speed animal clipper 24 hours prior to the application of hydrogel matrix patches. The dermal patches were secured in place with adhesive hydrofilm and rat jackets which were adjusted according to the size of the animal.P. berghei-infected rats as previously described by Musabayane et al [OGT responses following transdermal application of either OA-pectin patches (34 mg/kg) or CHQ-OA (56 /34 mg/kg) pectin patches and oral OA (160 mg/kg) were evaluated in separate groups of non-infected and ne et al . Brieflyrd day during the pre-treatment and post-treatment periods. However, following the application of dermal patches, parameters were monitored on selected days (9 and 12). Blood glucose concentrations were measured using blood glucose testing strips, .To evaluate the short-term effects of transdermally delivered OA (34 mg/kg) and a CHQ-OA (56/34 mg/kg) combination, patches were topically applied onto the shaved skin area on the back of the neck. The patches were only applied once at 9h00 on day 7 of the treatment period. Oral CHQ (30 mg/kg) and OA (160 mg/kg) were administered twice daily for 5 consecutive days. Animals treated with drug-free pectin and CHQ acted as negative and positive controls, respectively. Blood glucose, body weights, amounts of water and food consumed were measured in control and treated animals at 09h00 every 3P. berghei-infected animals throughout the 21-day experimental period. The blood smears were collected 24 hours after treatment at 9h00. Parasitaemia was scored on Giemsa-stained tail-blood films under a microscope with a x50\u2013x100 oil immersion objective .Daily malaria parasite density was monitored by microscopic counting of Giemsa-stained thin blood smears of On selected days , separate groups of animals were sacrificed by exposing to halothane (100 mg/kg) for 3 minutes via a gas anaesthetic chamber. Blood was collected by cardiac puncture into individual pre-cooled heparinized tubes. Separated plasma was analysed for insulin concentrations. Thereafter, the liver and muscle tissues were removed snap frozen in liquid nitrogen and stored in a BioUltra freezer at -80\u00b0C until use. The liver and muscle were analysed for glycogen content.Plasma insulin concentrations were determined in blood samples of separate groups of non- infected and infected animals following treatment with various patch formulations. The assays were performed on ultrasensitive rat insulin ELISA kit . The lower limit of detection was 0.020 \u03bcg/L. The intra- and interassay analytical coefficients of variation ranged from 2.0 to 2.9% and from 3.5 to 4.8% respectively.et al [Glycogen concentrations were determined as previously described by Khathi et al . Liver aThe effects of dermal-hydrogel matrix patch applications on the skin were evaluated using histological analysis. Following collection, the skin samples were fixed in formalin solution (10%). This was followed by rehydration in decreasing grades of ethanol and embedding in paraffin wax. The samples were then sectioned using a microm rotary microtome . Subsequently, the sections were stained with haematoxylin and eosin (H and E) followed by dehydration in increasing grades of ethanol and cleared in xylene. The sections were viewed and captured using Leica light microscope .All data were expressed as means \u00b1 standard error of means (S.E.M.). Statistical comparison of the differences between the control means and experimental groups was performed with GraphPadInStat Software , using one-way analysis of variance (ANOVA), followed by Tukey-Kramer multiple comparison test. A value of p\u02c20.05 was considered significant.The isolated OA was identified by 1H NMR and 13C NMR (1D and 2D). This data compared with those reported in the literature . The purThe infected untreated control showed a continuous increase in percentage parasitaemia reaching 61.2 \u00b1 2.3% by day 12 post infection. These control animals were sacrificed on day 12 due to ethical reasons. A once-off topical application of OA and CHQ-OA dermal patches significantly decreased percentage parasitaemia to undetectable levels by day 12 and 11 respectively. Oral administration of either OA or CHQ twice daily, reduced and cleared all residual parasites from the systemic circulation by day 14 .P. berghei-infected animals reaching hypoglycaemic values by the end of the experimental period.Following a glucose load, there was a significant increase in blood glucose concentrations of non-infected (NIC) and infected animals (IC) . A once-Infected control (IC) animals exhibited reduced blood glucose concentrations in comparison to the non-infected control (NIC) . TopicalInfected control (IC) animals showed significantly reduced hepatic and muscle glycogen content in comparison to the non-infected control animals . TransdeP. berghei-infected or transdermally (TD OA) had no effects on terminal plasma insulin concentrations of non-infected and P. binfected animals.infected .P. berghei- infected (NIC) or transdermally (TD OA) had no effect on plasma insulin concentrations throughout the experimental period. There were no significant changes in plasma insulin concentrations following a once-off transdermal application of the CHQ-OA combination (TD CHQ-OA) patch . Oral aded (NIC) .P. berghei-infected control (IC) and oral CHQ (O CHQ) treated animals showed a significant (p<0.05) reduction in food and water intake. This reduction in food and water intake was associated with a reduction in body weight. There were no changes in food and water intake in animals treated with either transdermal OA (TD OA) or CHQ-OA combination patch (TD CHQ-OA). However, a significant reduction in body weight during the treatment period was observed in these animals. During the post-treatment, period transdermally treated animals gained weight, reaching values comparable to the non-infected control (Food and water intake of non-infected control (NIC) animals remained unchanged throughout the study, whilst the animals progressively gained weight . When co control .P. berghei-infected rats treated with a once-off topical application of the pectin OA matrix patch (TD OA) and oral CHQ (O CHQ). P.berghei-infected control (IC) and photomicrograph (6C) shows P.berghei-infected oral CHQ treated group. Photomicrograph (6D) represents intact secretory ducts (ISD), uninjured stratum basale (USB) and intact sebaceous glands (ISG) of the P. berghei infected treated with once-off topical application of the OA pectin patch. Compared to the non-infected control animals for 10 days induced liver injury in mice, which indicates the toxic effects of this triterpene when using high doses [Plasmodium falciparum in vivo remain to be investigated.The results presented in this study show that a once-off transdermal application of OA-alone or in combination with CHQ clears the malaria parasites in d manner \u201330. Simiin vitro \u201332. Beroin vitro . Howeveratocytes . Interesthe drug \u201334. As aculation . This fugh doses . OA dosegh doses . The dosThe acute blood glucose lowering effect of the OA-pectin patch demonstrates the ability of this novel formulation to deliver OA at a sustained controlled manner. Indeed, transdermal delivery systems have been shown to offer controlled release of drugs over longer periods of time \u201338. The Plasmodium falciparum malaria, particularly in children and pregnant women. The ability of OA treatment to increase blood glucose concentrations in infected rats demonstrates the beneficial effects of using OA as an antiplasmodial agent. The utilisation of the host\u2019s glucose stores by the malaria parasites has been reported to cause of hypoglycaemia in malaria infection [Hypoglycaemia is an important complication of severe nfection .We belienfection . The hepnfection \u201343. FurtGlycogen concentrations of animals treated with transdermal OA were comparable to those of the non-infected control animals. This data further demonstrates the ability of OA to improve glucose homeostasis without having any adverse effects in rats. The data also shows that OA-associated increase in blood glucose levels is mainly mediated through the removal of the malaria parasites from the systemic circulation. Hepatic and muscle glycogen contents were increased following treatment with oral CHQ. The increase in glycogen concentration might attribute to increased plasma insulin following treatment with CHQ. We speculate that this increase in insulin concentrations promotes glucose uptake in the liver.Transdermal delivery offers a convenient and non-invasive alternative route of drug delivery to oral and subcutaneous injections. The skin plays a major role in the delivery of drugs via the transdermal route. Accordingly, we evaluated the effects of transdermal patch application on the skin morphology. Transdermal OA and CHQ-OA formulations did no elicit any adverse effects on the skin morphology. We have previously reported on the ability of pectin-insulin to deliver therapeutic doses of insulin without any adverse effects on the underlying tissues of the skin . The preP. berghei-infected animals. The antimalarial formulations presented in the current study not only provide novel alternatives treatments for malaria, but also introduce an enhanced route of administration for CHQ which might improve patient compliance. Additionally, the transdermal route also offers a convenient dosing schedule which only requires a once-off application of the patch as opposed to multiple doses that are associated with oral administration and painful intravenous injections of CHQ. OA and OA-CHQ patches provide a novel, pain-free and convenient formulations which may be may be used for the treatment of malaria.In summary, the data presented in this study shows for the first time that a once-off transdermal administration OA alone or in combination with CHQ is able to clear the malaria parasites in S1 TableIC- Infected control; O CHQ- Orally administered chloroquine; O OA- Orally administered oleanolic acid; TD OA- Transdermally administered oleanolic acid; TD CHQ-OA- Transdermally administered chloroquine-oleanolic acid combination.(DOCX)Click here for additional data file.S2 TableIC- Infected control; O CHQ- Orally administered chloroquine; O OA- Orally administered oleanolic acid; TD OA- Transdermally administered oleanolic acid; TD CHQ-OA- Transdermally administered chloroquine-oleanolic acid combination.(DOCX)Click here for additional data file.S3 TableIC- Infected control; O CHQ- Orally administered chloroquine; O OA- Orally administered oleanolic acid; TD OA- Transdermally administered oleanolic acid; TD CHQ-OA- Transdermally administered chloroquine-oleanolic acid combination.(DOCX)Click here for additional data file.S4 TableIC- Infected control; O CHQ- Orally administered chloroquine; O OA- Orally administered oleanolic acid; TD OA- Transdermally administered oleanolic acid; TD CHQ-OA- Transdermally administered chloroquine-oleanolic acid combination.(DOCX)Click here for additional data file.S5 TableIC- Infected control; O CHQ- Orally administered chloroquine; O OA- Orally administered oleanolic acid; TD OA- Transdermally administered oleanolic acid; TD CHQ-OA- Transdermally administered chloroquine-oleanolic acid combination.(DOCX)Click here for additional data file.S6 TableIC- Infected control; O CHQ- Orally administered chloroquine; O OA- Orally administered oleanolic acid; TD OA- Transdermally administered oleanolic acid; TD CHQ-OA- Transdermally administered chloroquine-oleanolic acid combination.(DOCX)Click here for additional data file."} +{"text": "Of 130 carbapenem-resistant isolates tested, 45 were Carba NP\u2013positive; 43 harbored blaNDM, and 2 harbored blaVIM. Multidrug-resistant microbial pathogen surveillance and antimicrobial drug stewardship are needed to prevent further spread of New Delhi metallo-\u03b2-lactamase variants.During 2013\u20132016, we isolated Enterobacteriaceae can efficiently hydrolyze carbapenems and most \u03b2-lactam drugs. Since the identification of New Delhi metallo-\u03b2-lactamase-1 (NDM-1) in 2008 (blaIMP-harboring Enterobacteriaceae were reported in 2014 (Escherichia coli (sequence type [ST] 131) isolate harboring blaNDM-1 was reported in 2014 (Klebsiella pneumoniae (ST626 and ST903) isolates harboring blaNDM-1 and blaNDM-7 genes were reported in 2016 on 1,516 gram-positive and gram-negative isolates from patients admitted to various wards in the V. Luna Medical Center, a tertiary-care military hospital in Manila, the Philippines, during August 2013\u2013April 2016. To better assess the distribution of carbapenem resistance and the underlying molecular mechanisms of resistance, we selected gram-negative isolates with imipenem or meropenem (or both) MICs of blaNDM; 25 (58%) of the blaNDM-carrying isolates were identified as K. pneumoniae of the 8 isolates were positive for blaNDM and identified as K. pneumoniae had positive Carba NP test results and 43 (33%) harbored eumoniae Table. Noeumoniae Table.blaNDM-positive bacterial isolates in several genera of Enterobacteriaceae and nonfermentative bacteria in the Philippines. This finding is particularly significant because NDM-like enzymes have a broad range of activity against most \u03b2-lactam antimicrobial drugs and are often associated with serious clinical infections (blaNDM, which suggests the possibility of nosocomial transmission and local circulation. We report the identification of blaNDM, blaKPC, blaVIM, blaIMP-1, and blaOXA-48 and did not investigate clonality; thus, further investigation into other carbapenemase genes should be conducted. In addition, further experiments should be performed to characterize the plasmids carrying the carbapenemase genes. Strengthening of multidrug-resistant microbial pathogen surveillance and antimicrobial drug stewardship is urgently needed to better characterize drug-resistance patterns and improve early detection and containment strategies in developing countries.We conducted multiplex real-time PCR testing only for Molecular resistance mechanisms of carbapenem-resistant clinical and environmental isolates from a tertiary-care military hospital, Manila, Philippines, August 2013\u2013April 2016."} +{"text": "Immunoglobulin G (IgG) against the NR1-subunit of the N-methyl-D-aspartate (NMDAR) receptor mediates NMDAR-antibody encephalitis (NMDAR-Ab-E). This multi-stage illness presents with an acute severe psychiatric syndrome, alongside other neurological features, similar to human and animal NMDAR antagonist models. The disease is associated with an ovarian teratoma in around 20% of cases. The cellular immunity underlying this disease is not well understood. While antibody-modifying immunotherapies often promote disease resolution, the illness can be refractory to these treatments correlating with sub-optimal outcomes.NR1-IgG can be detected several years after clinical resolution, which may be via ongoing germinal centre reactions or the establishment of antibody-secreting cells as long-lived plasma cells in bone marrow niches. These two divergent models implicate use of differing immunotherapies to target these cells. Here we investigate the contribution of ongoing germinal centre reactions to disease progression, potentially informing disease mechanisms and guide targeted immunotherapy.We hypothesised that recurrent antigen-driven germinal centre reactions would be associated with active generation of NR1-specific IgM and IgG and NR1-specific circulating B cells. We validated a NR1-IgM cell based assay establishing specificity cut-offs by screening healthy and disease control cohorts alongside a previously collected NMDAR-Ab-E cohort (n=46). Following this we went on to explore the temporal evolution of NR1-IgG and NR1-IgM titres in a prospective cohort (n=12).To investigate the lymphocyte characteristics, we stimulated ovarian teratoma lymphocytes and peripheral blood mononuclear cells (PBMCs) from multiple time points under varying cytokine conditions to understand whether these circulating cells showed capacity for NR1-IgG and IgM generation.We found a 43% prevalence rate of NR1-IgM in the historic cohort. We then confirmed that NR1-IgM binding was specific by its selective depletion after anti-IgM precipitation but not with protein G. In the prospective cohort, we noted often high titres of IgM (up to 1:500) most commonly early in the disease but persisting for around 2 years. NR1-IgM levels varied in titre alongside NR1-IgG spikes. Consistently, culture experiments of patient lymphocytes (PBMCs and tumour-derived) produced varying degrees of NR1-IgM and NR1-IgG under conditions associated with B cell proliferation. The NR1-IgG levels correlated with serum NR1-titres suggesting these circulating B cells made a proportional contribution to serum levels.Ongoing germinal centre reactions likely contribute much of the circulating NR1-specific B cell population in NMDAR-Ab-E. Autoimmunisation at these centres represents an as yet unexplored therapeutic target in this and potentially other autoimmune encephalopathies. Regional specificity of these reactions including lymph nodes draining sources of NR1-antigen require further direct evaluation."} +{"text": "Drosophila leg morphogenesis occurs under the control of a relatively well-known genetic cascade, which mobilizes both cell signaling pathways and tissue-specific transcription factors. However, their cross-regulatory interactions, deployed to refine leg patterning, remain poorly characterized at the gene expression level. Within the genetically interacting landscape that governs limb development, the bric-\u00e0-brac2 (bab2) gene is required for distal leg segmentation. We have previously shown that the Distal-less (Dll) homeodomain and Rotund (Rn) zinc-finger activating transcription factors control limb-specific bab2 expression by binding directly a single critical leg/antennal enhancer (LAE) within the bric-\u00e0-brac locus. By genetic and molecular analyses, we show here that the EGFR-responsive C15 homeodomain and the Notch-regulated Bowl zinc-finger transcription factors also interact directly with the LAE enhancer as a repressive duo. The appendage patterning gene bab2 is the first identified direct target of the Bowl repressor, an Odd-skipped/Osr family member. Moreover, we show that C15 acts on LAE activity independently of its regular partner, the Aristaless homeoprotein. Instead, we find that C15 interacts physically with the Dll activator through contacts between their homeodomain and binds competitively with Dll to adjacent cognate sites on LAE, adding potential new layers of regulation by C15. Lastly, we show that C15 and Bowl activities regulate also rn expression. Our findings shed light on how the concerted action of two transcriptional repressors, in response to cell signaling inputs, shapes and refines gene expression along the limb proximo-distal axis in a timely manner. Drosophila leg provides a good paradigm to dissect the molecular mechanisms underlying gene regulation. Here, we used the bric-a-brac2 (bab2) gene as a model to study the integrated regulation of patterning genes implicated in tarsal segmentation. bab2 expression in the leg primordium is dynamic and complex, going from initial broad distal expression to precisely positioned tarsal rings. By genetic and molecular analyses, we show here that the cell signaling-responding TFs C15 and Bowl interact directly with the limb-specific bab2 enhancer as a repressive duo. Moreover, C15 acts independently of its partner Aristaless through physical interaction with the Dll activator. We propose that Dll induces early circular bab2 expression pattern, then EGFR signaling-induced C15 in the distalmost cells competes with Dll for LAE binding and resolves bab2 pattern as a ring. Taken together our data shed light on how the concerted action of a quartet of transcription factors reshapes gene expression during limb proximo-distal axis development.Limb morphogenesis is controlled by a well-known genetic cascade, mobilizing both cell signaling and tissue-specific transcription factors (TFs). However, how their concerted action refines gene expression remains to be deciphered. It is thus crucial to understand how cell signaling inputs are integrated by transcriptional \u201cenhancers\u201d. The HoweDrosophila leg provides a paradigm with which to tackle the issue of the molecular mechanisms underlying proximo-distal limb development and Bowl [5\u2019-(C/A)C(A/G)G(T/A)AG(C/T)] ZF domains by a bacterial one-hybrid approach . Two seqequences . In addiomestica . Moreove species .in vitro, we therefore expressed a 163 amino-acid long segment of the Bowl protein encompassing its zinc-finger domain (termed BowlZF) as a glutathione S-transferase (GST) fusion protein in E. coli. Purified GST-BowlZF was examined for specific DNA binding in an electrophoretic gel mobility shift assay (EMSA) (ZF BS) previously shown to interact with a recombinant GST-OddZF fusion protein . Howeverin vitro. Given that C15 and Al interact physically through contacts between their homeodomain cells (Novagen), from which soluble forms were purified on glutathione-Sepharose beads and then isolated through elution with free glutathione. Purified proteins were used in EMSA, as described in cells, from which soluble forms were purified by glutathione-Sepharose affinity. For control EMSA and pull-down experiments, transformation with empty pGex4T was also performed, to produce and then purify soluble non-fused GST.The Open Reading Frame (ORF) encompassing the C15 HD-encoding sequence (codons 192 to 292) was amplified by PCR, using as a template a full length (FL) cDNA insert (IP08859), obtained from the A insert , and insribed in . Given tin vitro translated Al HD (see below). Protein-DNA complexes were separated by electrophoresis on 6% native polyacrylamide gels and revealed by PhosphoImager detection. DNA Probes were obtained from coupled in vitro transcription/translation from DNA matrices generated by PCR, using 5\u2019 oligonucleotides comprising each a T7 RNA polymerase promoter and a translation initiating ATG within an optimal Kozak context (sequences of oligonucleotides are available upon request). Radiolabeled C15 and Dll HDs were produced from full-length cDNA clones (described above). In vitro translated Al HD (amino acids 86\u2013156) was produced from a full-length cDNA clone (RE68460), obtained from the Drosophila Genomic Resource Center (DGRC).Pull down was down as previously described , in duplbric-\u00e0-brac locus sequences were recovered from dipteran genomic sequences and aligned as previously described , indicating that Bowl-insensitive LAEm3-GFP is still repressed in the developing pretarsus, including in a row of Bowl-expressing cells [white bracket in panel (D\u201d)].(A-B) Late L3 leg discs expressing either the (TIF)Click here for additional data file.S2 FigC. capitata, G. morsitans and M. domestica LAE-like sequences were identified though BLAST analyses using the Trace archive nucleotide blast server at NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and were aligned with the D. melanogaster LAE sequence using MAFFT . Homology shading was performed using BoxShade (http://www.ch.embnet.org/software/BOX_form.html). Bowl BS positions are indicated by blue dashed boxes. The near-palindromic site within CR2 is indicated by an orange dashed box.(A-B) CR2 (A) and 3\u2019-neighboring region of CR2 (B) sequences of 27 dipterans are aligned. Drosophilidae, (TIF)Click here for additional data file.S3 FigC152 homozygous mutant (B) expressing LAE-RFP. Merged RFP fluorescence (red) and Bowl immunostaining (magenta) are shown, as well as the latter in isolation, in (A\u2019) and (B\u2019). It is noteworthy that bowl expression at the ts5/pretarsal (pt) boundary [yellow arrows in (A) and in (A\u2019)] is no longer detected in the C15 loss-of-function developing distal leg, while remaining unaffected at the tibial (ti)/ts1 boundary [white arrows in (B) and (B\u2019)].(A-B) Late L3 late discs from wild-type (A) or (TIF)Click here for additional data file.S4 FigHD and unfused GST as a negative control. A non-specific retarded complex is indicated by asterisks. See in vivo. GFP (green) and RFP (red) fluorescence are shown for late L3 leg discs expressing LAEwt-GFP together with wild-type (upper panels) or mutated (lower panels) LAE-RFP constructs. The F2LAES3+4+6m construct mutated for both C15 binding sites within CR1 does not expressed RFP within the leg discs, suggesting that the S3-4 and S6 motifs encompass activating sequences.(A) C15 homeodomain binds distinct A/T-rich motifs within the LAE CR1 sequence. EMSA experiment with retarded complexes obtained from purified GST-C15(TIF)Click here for additional data file.S5 FigLAE-GFP (A-C) or LAE\u2013RFP (D-E) and harboring either rotund null clones (detected by the loss of RFP) (A-C) or FO clones overexpressing the Rn protein (D-E). Merged RFP fluorescence (red), GFP fluorescence (green) and Dll , Bowl or C15 (C) immunostaining (magenta) are shown, as well as the latter and GFP or RFP markers in isolation, in (A\u2019-E\u2019) and (A\u201d-E\u201d), respectively. Mitotic clones are circled with white dashed lines. In striking contrast to LAE-RFP , Dll, bowl and C15 expression remained unchanged in rn null clones. Similarly, misexpressed Rn TF never detectably affected Dll, bowl nor C15 (see LAE-RFP was slightly up-regulated in ts3-5 FO clones (white arrow).Mosaic late L3 leg discs expressing C15 see expressi(TIF)Click here for additional data file.S6 FigLAE-RFP and harboring FO clones ectopically-expressing C15 (A), Bowl (B-C) or Dll (D-E). Merged markers and Bowl, C15 or Rn immunostaining (magenta) are shown, as well as LAE-RFP fluorescence in isolation. FO mitotic clones are circled with white dashed lines and some are indicated with white or yellow arrows. Lateral confocal views are shown in (A) and (B) with distalmost pretarsal cells on the left and right side, respectively. Note that ectopically-expressed C15 protein down-regulated bowl expression at the pt-ts5 boundary, while the converse was not observed (yellow vs white arrows). As expected LAE-RFP was autonomously extinguished in Lines-depleted tarsal FO cells (C\u201d), but C15 protein was never detected there (C\u2019) (yellow arrows), thus discarding the possibility that C15 misexpression contributes to LAE-RFP repression by ectopically-stabilized nuclear Bowl. Lastly, whereas rn remained unaffected in all Dll-misexpressing FO clones , a faint cell-autonomous LAE-RFP up-regulation was observed in some proximalmost clones , indicating that misexpressed Dll is sufficient to activate the LAE in proximal cells, as shown in C15-expressing distal cells Click here for additional data file.S7 FigLAE-RFP or LAE-GFP (C) and harboring loss- or gain-of-function clones for bowl or C15 (C-D). Merged RFP fluorescence (red), GFP fluorescence (green) and Dll immunostaining (magenta) are shown, as well as the Dll and RFP or GFP markers in isolation, in (A\u2019-D\u2019) and (A\u201d-D\u201d), respectively. Mitotic clones are circled with white dashed lines. In striking contrast to bab2 reporters, Dll expression was never detectably affected in any mosaic discs (white arrows). As expected, LAE reporter activity was down-regulated in FO clones misexpressing Bowl and C15 proteins. LAE-RFP up-regulation was observed in all bowl mutant cells, provided that they are located within the Dll-expressing cells .Mosaic late L3 leg discs expressing (TIF)Click here for additional data file."} +{"text": "A sialic acid-targeted near-infrared profluorophore with pH-responsive fluorescence and photothermal properties was developed for fluorescence-guided staging and photothermal therapy of viable tumors exposed during surgery. in vivo tumor targeting and a profluorophore which undergoes lysosomal acidity-triggered fluorogenic isomerization. SA-pNIR displays a number of advantageous biomedical properties in mice, e.g. high tumor-to-normal tissue signal contrast, long-term retention in tumors and low systemic toxicity. In addition, SA-pNIR effectively converts NIR light into cytotoxic heat in cells, suggesting tumor-activatable photothermal therapy. With high performance tumor illumination and lysosome-activatable photothermal properties, SA-pNIR is a promising agent for detection and photothermal ablation of surgically exposed tumors.Agents enabling tumor staging are valuable for cancer surgery. Herein, a targetable sialic acid-armed near-infrared profluorophore (SA-pNIR) is reported for fluorescence guided tumor detection. SA-pNIR consists of a sialic acid entity effective for The utility of fluorescence guided surgery is demonstrated by recent clinical treatment of ovarian cancer with the aid of fluorescein-conjugated folate.2 Recently, optical agents that could direct surgeons to tumor foci evasive to visual inspection have been intensively explored.3With the increasing morbidity and mortality imposed by cancers, approaches that could improve the outcome of existing treatment modalities are of significance.4 Rhodamine derivatives with intramolecular spirorings are poised for proton-triggered fluorogenic opening of the intramolecular rings within acidic lysosomes, enabling high performance tumor imaging in mice models.5 Relative to rhodamines, near-infrared (NIR) dyes are advantageous for in vivo imaging as biological tissues display the least optical absorption and autofluorescence in the NIR window (650\u2013900 nm).6 Nanomaterials that could convert NIR irradiation into cytotoxic heat are being actively explored for photothermal cancer therapy.7 Given concerns regarding the biosafety of nanomaterials, small molecular NIR dyes could be biocompatible. For instance, indocyanine green (ICG) has been approved for clinical applications.8 Analogous to acid-responsive rhodamines, probes displaying lysosomal acidity-activatable NIR fluorescence and photothermal properties have been largely unexplored for intraoperative tumor therapy.High tumor-to-background signal contrast is paramount for tumor imaging. As such, optical probes that could be activated to a \u201csignal-on\u201d state within tumors while remaining silent in off-target settings are critical to achieve low background signals.in vivo tumor imaging, dyes are often deliberately armed with tumor-homing entities, such as monoclonal antibodies, folates, aptamers, etc.9 Sialic acids (SA) are anionic monosaccharides commonly located at termini of cell surface glycans,10 and hypersialylation of cell surface constituents has been identified in a broad spectrum of cancers,11 suggesting enhanced metabolic demand for SA by these tumors cells. Fluorescein isothiocyanate-labelled sialic acid (SA-FITC) was recently employed for high performance tumor detection in mice, showing effective uptake of SA monosaccharide by metabolically active tumor cells.12 Albeit selectively accumulated in tumors, SA-FITC suffers from \u201calways-on\u201d green fluorescence which has limited tissue penetration, and quick in vivo clearance which might lead to fast attenuation of tumor-associated signals during surgery.12 Herein, we report a sialylated pH-activatable NIR profluorophore (SA-pNIR) for targeted tumor imaging and photothermal therapy in mice with dramatically improved pharmacokinetics critical for clinical translation. SA-pNIR consists of a sialic acid entity for effective in vivo tumor uptake and an activatable NIR profluorophore which becomes photothermal and fluorescent within acidic lysosomes.To achieve the high tumor-to-background signal contrast required for 4 We recently reported the use of rhodamines with intramolecular spirorings for in vivo tumor detection via lysosomal acidity-triggered fluorogenic opening of the rings to give rhodamines.5 Relative to red-emissive rhodamines, NIR dyes are advantageous for bioimaging due to the enhanced tissue penetration of NIR fluorescence and the minimal light absorption and autofluorescence of biological tissues in the NIR region.6 Hence, we set out to develop a NIR probe, akin to rhodamine\u2013lactams, for tumor imaging via lysosomal acidity-mediated fluorescence activation within tumors -2-(2-(9-(2-Carboxyphenyl)-6-(diethyl-amino)-2,3-dihydro-1H-xanthen-4-yl)vinyl)-1,3,3-trimethyl-3H-indol-1-ium perchlorate), a NIR dye reported by Lin et al.,13 was first amidated with ethylenediamine and then conjugated with 9-amino-9-deoxy-5-N-acetylneuraminic acid to afford the desired SA-pNIR in 32% overall yield supplemented with SA-pNIR and then stained with LysoTracker Green DND-26, referred to herein as Lysotracker green. Confocal microscopy images show that NIR fluorescence is clearly observed within all the three cell lines tested and colocalizes with Lysotracker green specific for acidic lysosomes , which is a potent inhibitor of V-ATPase and effectively alkalinizes lysosomes in BFA-treated cells.via tail vein. The mice were imaged for whole body NIR fluorescence over the course of 144 h after injection. No NIR signal was observed in mice 30 min after administration was prepared and then administered into tumor-bearing ICR mice via tail vein at the C-9 position of sialic acid.Hypersialylation of cell surface glycoconjugates is a hallmark of a broad spectrum of cancersin vivo administration. We first examined the effects of SA-pNIR on the survival of HeLa cells by trypan blue exclusion test. No obvious detrimental effects on cell viability were observed on cells treated with SA-pNIR for 24 h at doses up to 100 \u03bcg mL\u20131 for clinical applications. Inspired by the emerging use of NIR dyes in photothermal therapy,20 we proceeded to examine the capability of SA-pNIR as a pH-responsive photothermal agent. SA-pNIR was spiked into buffers of pH 7.5 and 4.5. The solutions were exposed to 660 nm laser illumination at a power density of 0.5 W cm\u20132 and the temperature of the solutions was monitored over the irradiation time. Reagents that could convert optical energy into cytotoxic heat are attractive tools for light-mediated photothermal tumor therapy.SA-pNIR was then evaluated for its photothermal effects on host cells. HeLa, U87-MG and Raw 264.7 cells pre-loaded with SA-pNIR or SA were either irradiated with an NIR laser, or not subjected to irradiation. The viability of these cell populations was assayed using 3--2,5-diphenyltetrazolium (MTT). Cells treated with SA-pNIR and light illumination display further decreased viability relative to that of cells treated with NIR laser irradiation or SA-pNIR alone , demonstComplete manual cytoreduction of small-sized or embedded tumor foci is often challenging during surgery. Photothermal tumor ablation during surgery is applicable due to surgical exposure of cancerous tissues that are otherwise inaccessible to exogenous laser irradiation. Theranostics allowing intraoperative tumor staging and simultaneous photothermal tumor therapy are of clinical significance to complement surgical dissection. The lysosomal acidity-triggered photothermal effect of SA-pNIR on targeted cells supports the potential utility of SA-pNIR as a theranostic probe for dual imaging and photothermal killing of tumor foci in intraoperative settings.in vivo clearance, SA-pNIR displays signal activation in viable tumor cells, high tumor-to-normal tissue signal contrasts, and long-term retention in tumors, rendering optical imaging over an adequate duration which is critical for practical surgical intervention. In addition, SA-pNIR effectively converts NIR irradiation into heat in acidic lysosomes and leads to obvious cell death upon NIR irradiation, suggesting its utility for photothermal ablation of surgically exposed tumor foci that are otherwise inaccessible to exogenous light. With superior in vivo pharmacokinetics, high performance tumor illumination, and acid-responsive photothermal properties, SA-pNIR is a promising small molecular theranostic for fluorescence guided tumor detection and possibly photothermal tumor therapy in intraoperative settings.SA-pNIR, a sialylated lysosome-activatable NIR dye, has been developed for intraoperative tumor therapy. The sialic acid entity enables effective tumor targeting in mice and the NIR profluorophore undergoes lysosomal pH-triggered isomerization to give an NIR signal. In contrast with SA-FITC which is compromised by \u201calways-on\u201d green fluorescence and quick"} +{"text": "Period1 (Per1) transcription in mammalian clocks. Here, we demonstrate a light-independent role of Drosophila CRTC in sustaining circadian behaviors. Genomic deletion of the crtc locus causes long but poor locomotor rhythms in constant darkness. Overexpression or RNA interference-mediated depletion of CRTC in circadian pacemaker neurons similarly impairs the free-running behavioral rhythms, implying that Drosophila clocks are sensitive to the dosage of CRTC. The crtc null mutation delays the overall phase of circadian gene expression yet it remarkably dampens light-independent oscillations of TIMELESS (TIM) proteins in the clock neurons. In fact, CRTC overexpression enhances CLOCK/CYCLE (CLK/CYC)-activated transcription from tim but not per promoter in clock-less S2 cells whereas CRTC depletion suppresses it. Consistently, TIM overexpression partially but significantly rescues the behavioral rhythms in crtc mutants. Taken together, our data suggest that CRTC is a novel co-activator for the CLK/CYC-activated tim transcription to coordinate molecular rhythms with circadian behaviors over a 24-hour time-scale. We thus propose that CRTC-dependent clock mechanisms have co-evolved with selective clock genes among different species.Light is one of the strongest environmental time cues for entraining endogenous circadian rhythms. Emerging evidence indicates that CREB-regulated transcription co-activator 1 (CRTC1) is a key player in this pathway, stimulating light-induced Most living organisms have evolved endogenous time-keeping mechanisms known as circadian clocks to anticipate and adapt to daily changes in the environment. External time cues, such as cycles of light, temperature or food availability, entrain the circadian oscillators to sustain 24-hour rhythms. Timing information is subsequently translated into other physiological pathways of the organism, such as sleep, metabolism, immune responses and so forth12Drosophila, CLK-CYC, a heterodimeric transcription factor composed of CLOCK and CYCLE, binds E-box sequences and activates the transcription of period (per) and timeless (tim) genes from the late afternoon to midnight. PER-TIM complexes accumulate in the cytoplasm at early night and are then translocated to the nucleus, where they inhibit the transcriptional activity of CLK-CYC after midnight. In the morning, light-dependent TIM degradation and cumulative PER phosphorylation destabilize the PER-TIM complex. The consequent de-repression of CLK-CYC activity leads to a new daily cycle5678101112131516At the molecular level, a transcriptional feedback network of circadian transcription factors that regulates daily rhythmic gene expression constitutes a basic framework for cell-autonomous molecular clocksPer expression via CREB (cAMP response element binding protein)-dependent transcriptional activation, playing important roles in the photic entrainment of mammalian clocks1819Sik1 transcription, and then elevated SIK1 feeds back to phosphorylate CRTC proteins, blocking their nuclear entry2223Light stimulates mammalian Drosophila clocks and demonstrate that Drosophila CRTC activates tim transcription to align circadian gene expression on a 24-hour time-scale and drive robust free-running rhythms in circadian behaviors. Light-independent effects of Drosophila crtc are evident in the molecular rhythms of both central pacemaker neurons and peripheral clock tissues, implicating an ancestral origin of CRTC-dependent clocks. Given distinct clock functions of CRTC homologs, we suggest a model on how CRTC-dependent clock mechanisms have co-evolved with selective clock targets among different species.Here, we identify an unexpected role of CRTC in Drosophila, we first examined how CRTC loss-of-function affects circadian behaviors. We tested a crtc25-3 allele lacking the entire crtc locus as a result of imprecise excision of a transposable element insert transgene decreased endogenous levels of CRTC proteins in fly heads by 30~60% when overexpressed by the pan-neuronal Elav-Gal4 driver or by the clock cell-specific tim-Gal4 driver . When CRTC depletion was more restricted to large and small ventral lateral neurons (LNv) expressing the circadian neuropeptide PDF (pigment-dispersing factor) (referred to as PDF neurons hereafter)3031Pdf\u2009>\u2009DCR2, crtc RNAi). Consistent with the RNAi phenotype, PDF neuron-specific overexpression of Drosophila SIK2, a negative regulator of CRTC33There are ~150 circadian pacemaker neurons that exhibit daily circadian gene expression in the adult fly brain . Based o2627284 driver . As expecrtc mutant flies and compared their circadian behaviors to those in transgenic controls. Indeed, PDF neuron-specific CRTC overexpression restored 24-hour periodicity in circadian behaviors of crtc mutants whereas it partially but significantly rescued the rhythmicity phenotype -containing food but not on food containing ethanol (vehicle control), to drive transgenic expression in PDF neuronscrtc RNAi flies crtc mutants displayed a ~4-hour delay in the increasing phase of circadian mRNA expression in LD cycles while crtc effects on the declining phase were relatively weak may arise from cell-type specific context of CRTC-dependent transcription.Molecular rhythms in fly head extracts largely reflect the circadian pace of peripheral clock tissues nt flies , top. PE L phase but the DD cycle as compaproteins . In addicrtc mutants, we hypothesized that CRTC might facilitate a rate-limiting circadian transcription that coordinates molecular rhythms with 24-hour periodicity. However, the interlocked nature and feedback regulation of circadian transcription factors as well as the possibility of genetic compensation in mutant animals make it difficult to discriminate direct CRTC targets from indirect crtc effects on circadian transcription. To determine if the overall phase-delay in crtc mutants results from the attenuated transcription of general clocks or a specific CRTC target, we tested crtc effects on individual clock reporter genes in clock-less Drosophila S2 cells. Of note, S2 cells endogenously express the cyc gene, so CLK overexpression alone is sufficient to support the transcriptional induction of CLK-CYC target reporter genes, such as per-luciferase (luc) or tim-lucOn the basis of gene expression analyses in tim-luc, but not per-luc, in a dose-dependent manner synergistically activated the expression of cre-luc, a reporter gene whose transcription is driven by tandem repeats of cAMP-responsive element (CRE), thus faithfully responding to CREB-dependent transcription in S2 cells since lack of CRTC failed to potently induce tim transcription within a short window of its increasing phase. We thus reasoned that if tim transcription is rate-limiting in crtc mutants, TIM overexpression would rescue their circadian behaviors. To test this hypothesis, we overexpressed TIM or other clock proteins in PDF neurons of crtc mutants and examined their free-running locomotor behaviors in DD cycles. Indeed, TIM overexpression partially, but significantly, rescued long but weak behavioral rhythms in crtc mutants, whereas PDP1 or CRY (CRYPTOCHROME) overexpression did not show any comparable rescue particularly on the rhythmicity phenotype 181921Drosophila CRTC that serves to coordinate circadian gene expression with 24-hour locomotor rhythms even in the absence of light. CRTC may regulate several clock-relevant genes, including those clock output genes that might be involved in the rhythmic arborizations and PDF cycling of the circadian pacemaker neurons. However, we identified tim transcription as one of the primary targets of Drosophila CRTC to sustain circadian rhythms in the free-running conditions, thus defining its light-independent clock function.CREB-dependent transcription has long been implicated in different aspects of circadian gene expression. In mammalian clocks, light exposure triggers intracellular signaling pathways that activate CREB-dependent Drosophila CBP associates with CLK, titrating its transcriptional activityDrosophila CLK-CYC heterodimer, to stimulate their transcriptional activity51tim transcription is that Drosophila CRTC may analogously target the CLK-CYC heterodimer to stimulate CLK-CYC\u2013dependent tim transcription. However, this model does not explain tim-specific crtc effects among other CLK-CYC\u2013induced clock genes. Moreover, CRTC associates with the bZIP domain in CREB protein, whereas CBP/p300 binds CREB through the phosphorylated KID domain53Drosophila S2 cells to activate CRE-dependent transcription47S2 cells . Thus, itim transcription indirectly, then why do crtc effects require CLK? A recent study suggested that mammalian CLOCK-BMAL1 may regulate the rhythmic access of other DNA-binding transcription factors to their target promoters in the context of chromatin, acting as a pioneer-like transcription factorDrosophila CLK-CYC and mammalian CLOCK-BMAL1, the presence of CLK-CYC in the tim promoter might allow the recruitment of additional transcription factors and their co-activators including CRTC for maximal tim transcription . Interestingly, chromatin immunoprecipitation with V5-tagged CLK protein revealed that CLK-CYC heterodimers associate with both tim and Sik2 gene promoters in fly headsSik2 promoter is phase-delayed by ~4.5\u2009hours compared with that to the tim promoter. These modes of transcriptional regulation may gate crtc effects on tim transcription in a clock-dependent manner, particularly in the increasing phase of tim transcription.If CRTC augments CLK-CYC\u2013dependent cription , middle.Drosophila and mammals195757585960dCREB2 and per at the transcription level has been reported to sustain free-running circadian rhythms in DrosophilaBmal1 expressionTranscription from CREB-responsive reporter genes shows daily oscillations, both in Drosophila, light is accessible directly to circadian pacemaker neurons in the adult fly brain. Therefore, TIM degradation by the blue-light photoreceptor CRY63Drosophila clocks although the photic induction of CLK/CYC-dependent tim transcription has been reported specifically at lower temperaturesDrosophila CRTC retained a constitutive co-activator function from the ancestral TTFL to support CLK/CYC-activated tim transcription and sustain free-running circadian behaviors. In homeothermic mammals, light input to the SCN is indirectly mediated by neurotransmitter release from presynaptic termini of the retinohypothalamic tract (RHT)21per took over a role in the light-entrainment pathway by retaining CREB-CRTC1\u2013dependent transcriptional regulation from the primitive TTFL. Consequently, mammalian clocks have lost a homolog of the Drosophila-like cry gene family, but instead evolved CRY homologs of the vertebrate-like cry gene family with transcriptional repressor activities in CLOCK-BMAL1\u2013dependent transcriptionOn the basis of these observations, we propose a model for the evolution of CRTC-dependent clocks to explain the distinctive circadian roles of CRTC homologs . CRTC isDrosophila253469Regulation of metabolism and stress responses by neuronal CREB-CRTC-SIK pathways has been well documented in crtc25-3 and UAS-CRTC (gifts from M. Montminy), as well as UAS-SIK (a gift from J. Chung), Pdf-GeneSwitch-Gal4 (a gift from M. Ceriani), Pdf-Gal4, and tim-Gal4 have been described previously25323438Df(3L)ED4710, Df(3L)BSC415, UAS-crtc RNAi (stock number 28886), and UAS-DCR2 were obtained from the Bloomington Drosophila Stock Center.All flies were reared on standard cornmeal-yeast-agar medium at 25\u2009\u00b0C under 12\u2009hour:12\u2009hour LD cycles. Drosophila Activity Monitor (DAM) system (TriKinetics). Behavioral data were collected from the first to the 6th DD cycle and analyzed to determine the free-running period length and power of rhythmicity in individual flies using the ClockLab analysis software (Actimetrics). The confidence interval of the chi-squared periodogram was set to 0.05 with the testing range of period lengths from 15-hour to 35-hour. The power of rhythmicity (P-S) was calculated by subtracting a Significance value from a Power value (the observed rhythmicity measurement at the given period length). All the genotypes were tested in multiple behavioral runs. Per each genotype, circadian periods were averaged from rhythmic flies only whereas the power of rhythmicity values were averaged either from all the flies tested or from rhythmic flies as shown in Individual male flies were placed into glass vials containing 5% sucrose and 2% agar, entrained by three LD cycles at 25\u2009\u00b0C and then transferred to constant darkness (DD). Locomotor activities were recorded using the Fifty fly heads were homogenized in lysis buffer consisting of 25\u2009mM Tris-Cl pH 7.5, 300\u2009mM NaCl, 10% glycerol, 1\u2009mM PMSF, 1\u2009mM DTT, 0.5% NP-40, and protease/phosphatase inhibitor cocktail. Soluble extracts were clarified by centrifugation, resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to nitrocellulose membranes (Amersham) for immunoblotting. Membranes were probed with rabbit anti-CRTC (a gift from J. Chung), guinea pig anti-TIMMale flies were entrained in three LD cycles and then transferred to constant darkness. Brains were dissected at six different time-points in LD cycles or from the first to the second DD cycle. Whole-mount immunostaining was performed as described previously72rp49 (control), 5\u2032-GAA GAA GCG CAC CAA GCA CT-3\u2032 (forward) and 5\u2032-TTG AAT CCG GTG GGC AGC AT-3\u2032 (reverse); tim, 5\u2032-GAC TTG CCA AAT CCC TCA TC-3\u2032 (forward) and 5\u2032-GAA GCA CTG CAA CTC GAT CA-3\u2032 (reverse); per, 5\u2032-GAC CGA ATC CCT GCT CAA TA-3\u2032 (forward) and 5\u2032-GTG TCA TTG GCG GAC TTC TT-3\u2032 (reverse); Clk, 5\u2032-TAC TGC GTG AGG ATA TCG-3\u2032 (forward) and 5\u2032-GTT GTT GTT CTG GTT GC-3\u2032 (reverse); Pdp1e, 5\u2032-GTC GCC CTC CTC CTT GTA TT-3\u2032 (forward) and 5\u2032-CCA ATG AGC ATC ACA ACC AT-3\u2032 (reverse); cry, 5\u2032-CCA CCG CTG ACC TAC CAA A-3\u2032 (forward) and 5\u2032-GGT GGA AGC CCA ATA ATT TGC-3\u2032 (reverse); and pdfr, 5\u2032-CAG CTC GTT AGC ATT GTC CA-3\u2032 (forward) and 5\u2032-ACG TTT AAG TTG GCC ACA GG-3\u2032 (reverse).Flies were entrained in three LD cycles and then transferred to constant darkness. For transcription analyses of clock genes, flies were harvested at six different time-points in LD cycles or during the transition from the first to the second DD cycle. Total RNA was extracted from 50 fly heads using TRIzol reagent (Ambion), according to the manufacturer\u2019s instructions. Purified RNA was digested with RQ1 DNase to remove genomic DNAs, extracted with a mixture of phenol:chloroform:isoamyl alcohol (25:24:1), and then reverse-transcribed with M-MLV reverse transcriptase using oligo dT primers, according to the manufacturer\u2019s instructions (Promega). Quantitative polymerase chain reaction (PCR) was performed on a CFX96 system (Bio-Rad) using TOPreal qPCR 2X PreMIX (Enzynomics) and the following specific primer pairs: Drosophila S2 cells were cultured in Shields and Sang M3 insect medium (Sigma) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin (Invitrogen). Transient transfection was performed using Effectene according to the manufacturer\u2019s instructions (Qiagen). crtc cDNA (a gift from J. Chung) was subcloned into a modified pAc vector for expression of HA-tagged CRTC. pAc-CLK-V5, pAc-PKA-V5, per-luc, tim-luc, Clk-luc, cre-luc and renilla-luc vectors have been described previously1044in vitro-transcribed using a MEGAscript RNAi kit (Ambion). S2 cells were treated with dsRNA against EGFP (control) or CRTC for 2 days before transfection. Luciferase assays were performed 44\u2009hours after transfection using the Dual-Luciferase Reporter Assay System according to the manufacturer\u2019s instructions (Promega). Luminescence was measured using a TriStar2 plate reader (Berthold Technologies).How to cite this article: Kim, M. et al. CRTC Potentiates Light-independent timeless Transcription to Sustain Circadian Rhythms in Drosophila. Sci. Rep.6, 32113; doi: 10.1038/srep32113 (2016)."} +{"text": "E. coli-derived L-asparaginase might be a novel means for the therapy of one of the most recalcitrant neoplasms and for which no efficient treatment currently exists - glioblastoma (WHO grade IV). Our results suggest that certain glioma cell cultures are particularly susceptible to inhibition of proliferation by L-asparaginase, while others display a more resistant phenotype. In sensitive cells, L-asparaginase induces apoptosis with dissipation of mitochondrial membrane potential and activation of effector caspases. L-asparaginase-mediated apoptosis was accompanied by modulation of pro- and anti-apoptotic Bcl-2 family members, including Noxa, Mcl-1 and the deubiquitinase Usp9X. Given the impact of L-asparaginase on these molecules, we found that L-asparaginase potently overcomes resistance to both intrinsic apoptosis induced by the Bcl-2/Bcl-xL inhibitor, ABT263, and extrinsic apoptosis mediated by TRAIL even in glioma cells that are resistant towards L-asparaginase single treatment. RNA interference studies showed that Usp9X, Mcl-1, Noxa and Bax/Bak are involved in ABT263/L-asparaginase-mediated cell death. In vivo, combined treatment with ABT263 and L-asparaginase led to an enhanced reduction of tumor growth when compared to each reagent alone without induction of toxicity. These observations suggest that L-asparaginase might be useful for the treatment of malignant glial neoplasms.Cancer cells display a variety of global metabolic changes, which aside from the glycolytic pathway largely involve amino acid metabolism. To ensure aggressive growth, tumor cells highly depend on amino acids, most notably due to their pivotal need of protein synthesis. In this study, we assessed the overall hypothesis that depletion of asparagine by Amino acid metabolism might represent an \u201cAchilles heel\u201d in cancer since a number of tumors acquire an altered dependency on some of these metabolic pathways -3. For iWith regards to solid malignancies, L-asparaginase is not commonly employed due to the fact that most solid tumors display relatively high levels of ASNS and are therefore primarily resistant , 10. NevE. coli-derived L-asparaginase. Our results suggest that certain glioma cells are particularly sensitive to L-asparaginase. Moreover, we demonstrate that L-asparaginase treatment overcomes resistance to intrinsic and extrinsic apoptosis and therefore may be applicable in the context of combination therapies. Finally, our results suggest that the combination therapy of a BH3-mimetic along with L-asparaginase exerts anti-glioma activity in vivo.In order to establish a potential new therapeutic strategy for patients with glioblastoma, we analyzed different glioma cell cultures, including established, stem cell-like and patient-derived xenograft (PDX) cells, for their susceptibility to in vitro. Established glioblastoma cells , glioma stem-like cells and glioblastoma cells derived from a PDX-model (GBM12) or a murine transgenic model were treated for 72h with increasing concentrations of L-asparaginase prior to performing MTT- or CellTiter-Glo\u00ae-assays. As shown in Figure We first assessed whether treatment with L-asparaginase Figure and 1B hTo assess the mechanism of the anti-proliferative effect of L-asparaginase, we treated SF188 and U251 glioblastoma cells with L-asparaginase prior to staining with annexin V and propidium iodide. In both cell lines, treatment with L-asparaginase lead to an enhanced fraction of annexin V-positive cells (apoptotic cells) in a dose-dependent manner Figure . ConsistSince L-asparaginase induced apoptosis in glioblastoma cells, we next examined whether this observation is at least in part due to activation of intrinsic apoptosis. We therefore performed staining for JC-1 in SF188 glioblastoma cells treated with increasing concentrations of L-asparaginase. As shown in Figure Our observations so far pointed towards an apoptotic response that is at least in part mitochondrially-driven. Therefore, we focused next on effects of L-asparaginase on the expression of anti-apoptotic Bcl-2 family members. Treatment with L-asparaginase resulted in a marked down-regulation of Mcl-1 in T98G, U251 and LN229 glioblastoma cells Figure . ExpressTo further assess by which mechanism Mcl-1 is down-regulated, we performed real-time PCR analysis in T98G glioblastoma cells. Treatment with L-asparaginase did not result in reduced Mcl-1 mRNA expression after 6h, but even in a markedly enhanced expression after 24h indicating a post-transcriptional mechanism Figure .TP53-mutated, it appears likely that L-asparaginase-mediated up-regulation of Noxa is independent of p53 signaling.We next assessed by which mechanism Noxa is up-regulated. Real-time PCR analysis in T98G glioblastoma cells showed that treatment with L-asparaginase resulted in increased Noxa mRNA expression after 6h which was even more pronounced after 24h suggesting at least in part a transcriptional mechanism Figure . Since NEnhanced expression or function of Mcl-1 has been shown to represent a major mediator of resistance towards ABT compounds such as ABT263. Given that treatment with L-asparaginase resulted in either down-regulation of Mcl-1 or up-regulation of its pro-apoptotic counterpart, Noxa, we next assessed whether this molecular observation would sensitize for treatment with ABT263. As shown in Figure Since we observed that treatment with L-asparaginase sensitizes for mitochondrially driven apoptosis, we next assessed whether L-asparaginase also sensitizes for apoptosis mediated through death-promoting ligands. We therefore treated SF188 and LN229 glioblastoma cells with L-asparaginase, TNF\u03b1-related apoptosis-inducing ligand (TRAIL) or the combination of both as indicated. Combined treatment leads to a pronounced synergistic anti-proliferative effect in both cell lines tested Figure . To asseTo examine whether the pro-apoptotic synergism of the combination treatment is at least in part due to an enhanced activation of the mitochondrial pathway we performed JC-1-staining. As shown in Figure Given that L-asparaginase modulated Mcl-1 levels in various glioblastoma cells tested, we examined whether knock-down of Mcl-1 would be sufficient to enhance apoptosis induced by ABT263. SiRNA-mediated knock-down of Mcl-1 enhanced apoptosis induced by ABT263 in T98G glioblastoma cells and thereby phenocopied the sensitizing effect of L-asparaginase on ABT263 Figure . Since wSince treatment with L-asparaginase leads to a marked down-regulation of Usp9X, we next assessed whether knock-down of Usp9X would mirror the pro-apoptotic synergism of L-asparaginase and ABT263 or TRAIL. We therefore performed knock-down experiments with Usp9X-siRNA. As shown in Figure Our data showed that L-asparaginase treatment causes a decrease in the mitochondrial membrane potential. We therefore next assessed whether knock-down of the pro-apoptotic multi-domain effector proteins Bax and Bak would decrease the apoptotic response towards combined treatment with ABT263 and L-asparaginase. As shown in Figure Based on our observation, that treatment with L-asparaginase lead to an up-regulation of Noxa protein levels under certain conditions, we next examined whether Noxa is involved in the pro-apoptotic effect of the combination therapy. As shown in Figure in vivo. Therefore, MGPP-3 glioblastoma cells, that were derived from a transgenic proneural mouse model, were implanted subcutaneously into SCID SHO mice. After tumor formation, the mice were randomized and treatment with ABT263 (25 mg/kg), L-asparaginase (1500 IU/kg), both agents or solvent was started. Combined treatment with ABT263 and L-asparaginase resulted in statistically significant smaller tumors when compared to single-agent or vehicle treatments and stem cell-like glioma cells. Our findings showed that SF188 pediatric glioblastoma cells display a remarkable sensitivity to L-asparaginase in contrast to LN229 GBM cells. These observations are in agreement with earlier reports, showing that LN229 is resistant to glutamine withdrawal-induced cell death, while in contrast SF188 is highly susceptible. In sensitive cells, L-asparaginase-induced apoptosis was accompanied by dissipation of the mitochondrial membrane potential and activation of effector caspases. These findings are in agreement with previous reports in solid and non-solid malignancies -2, 5-diphenyltetrazolium bromide (MTT) assays were performed as previously described for adherent cells , 52. AntFor annexin V/propidium iodide (PI) staining the FITC Annexin V Apoptosis Detection Kit I was used according to the manufacturer's instructions. Staining for PI was performed as previously described . The datRT-PCR was performed as described before using the following primers: Usp9X forward: GTG TCA GTT CGT CTT GCT CAG C; Usp9X reverse: GCT GTA ACG ACC CAC ATC CTG A; Mcl-1 forward: CCA AGA AAG CTG CAT CGA ACC AT; Mcl-1 reverse: CAG CAC ATT CCT GAT GCC ACC T; GAPDH forward: GTC TCC TCT GAC TTC AAC AGC G and GAPDH reverse: ACC ACC CTG TTG CTG TAG CCA A.Specific protein expression in cell lines was determined by Western blot analysis as described before using th\u00aeUsp9X siRNA I #6308 was purchased from CST. Non-targeting siRNA-pool , BAX and Mcl-1 were purchased from Thermo Fisher Scientific. PMAIP1 siRNA and BAK siRNA were purchased from Ambion. Transfections were performed as previously described [\u00ae2000 and the respective siRNA (12-well condition) in DMEM without FBS and antibiotics. After 6h, FBS was added to a total concentration of 1.5%.SignalSilenceescribed , 55. Bri6MGPP-3 proneural glioblastoma cells suspended 1:1 in Matrigel\u00aeMatrix were implanted subcutaneously into the flanks of 6-8 week-old SCID SHO mice as previously described [1 \u00d7 10Subcutaneous tumors and samples from organs were extracted from SCID SHO mice and fixed for at least 24h in 10% PBS-buffered formalin . Then tit-test using Prism version 5.04 . A p \u2264 0.05 was considered statistically significant. The CompuSyn software was used for the drug combination analysis including the calculation of the combination index (CI) and isobologram as described before [50) is normalized to 1, plotted on x- or y-axis and connected by a line which represents the ED50 isobologram. Data points of drug combinations plotted below the connecting line represent a synergistic interaction, data points located on the line represent an additive interaction and data points located above the connecting line represent an antagonistic interaction.Statistical significance was assessed by Student's d before . A CI <"} +{"text": "ZNF521 mRNA overexpression and MLL-rearranged acute myeloid leukemia (AML), leaving open the question on the role of ZNF521 in this subtype of leukemia. In this study, we sought to analyze the effect of ZNF521 depletion on MLL-rearranged AML cell lines and MLL-AF9 xenograft primary cells. Knockdown of ZNF521 with short-hairpin RNA (shRNA) led to decreased leukemia proliferation, reduced colony formation and caused cell cycle arrest in MLL-rearranged AML cell lines. Importantly, we showed that loss of ZNF521 substantially caused differentiation of both MLL-rearranged cell lines and primary cells. Moreover, gene profile analysis in ZNF521-silenced THP-1 cells revealed a loss of MLL-AF9-directed leukemic signature and an increase of the differentiation program. Finally, we determined that both MLL-AF9 and MLL-ENL fusion proteins directly interacted with ZNF521 promoter activating its transcription. In conclusion, our findings identify ZNF521 as a critical effector of MLL fusion proteins in blocking myeloid differentiation and highlight ZNF521 as a potential therapeutic target for this subtype of leukemia.Zinc finger protein 521 (ZNF521) is a multiple zinc finger transcription factor and a strong candidate as regulator of hematopoietic stem cell homeostasis. Recently, independent gene expression profile studies have evidenced a positive correlation between MLL encodes an H3K4 methyltransferase that forms multiprotein chromatin-modifying complexes required in controlling transcriptional program necessary for the development and maintenance of hematopoiesis [MLL count more than 60 different fusion partners, which have been identified in AML, acute lymphoid leukemia, and biphenotypic or chemotherapy-related leukemias [MLL translocations contribute to leukemogenesis subverting self-renewal program and block of hematopoietic differentiation [MLL targets genes, such as HOXA9 and MEIS1, it is well established the crucial role played in MLL-induced leukemia [Acute myeloid leukemias (AMLs) are heterogeneous tumor blood diseases characterized by deregulated cell proliferation, survival and differentiation of hematopoietic/stem progenitor cells . One of eukemias \u20137. MLL tntiation , 8. Tranntiation , 9\u201310. Aleukemia , 12, howEBF1) [Zinc finger protein 521 (ZNF521) is a C2H2-type zinc finger transcription factor containing an amino-terminal motif that binds to the nucleosome remodeling and histone deacetylase (NuRD) complex, which is associated with transcriptional repression and conserved among other zinc finger proteins, including Friend of GATA (FOG1 and FOG2), BCL11A, and SALL family members . InitialEBF1) .ZNF521 mRNA has been observed in medulloblastoma, lymphoblastic lymphoma and acute leukemia [E2A-HLF and E2A-PBX1 involving fusion genes in B-lineage acute lymphoblastic leukemia (B-ALL) have demonstrated that enhanced expression of Zfp521, the murine counterpart of human ZNF521, cooperates to leukemia transformation, and an upregulation of ZNF521 was found in human B-ALL samples bearing E2A-HLF or E2A-PBX1 fusion oncogenes. Therefore, an altered expression of ZNF521 may be an important cofactor contributing to hematopoietic cell transformation. Recently, high expression of ZNF521 has been observed in pediatric AML, particularly in those cases carrying MLL gene rearrangements [MLL-rearranged AML is currently unknown. In this study, we examined the role of ZNF521 in MLL-rearranged AML cell lines and primary ex vivo MLL-AF9-expressing cells and showed that depletion of ZNF521 impaired AML progression by inducing myeloid differentiation.Deregulated expression of leukemia \u201319. Recengements , 21; howMLL-fusion genes, we performed quantitative real-time PCR (qRT-PCR) in an independent cohort of 50 pediatric AML patients . In addition, we analyzed the expression of ZNF521 in 6 MLL-rearranged and 6 non-MLL-rearranged human leukemic cell lines. Similarly, leukemic cell lines with MLL rearrangements, with the exception of those carrying MLL-AF4 fusion transcripts, showed significantly higher ZNF521 mRNA levels compared to cell lines with other abnormalities , ML-2 (expressing MLL-AF6) and OCI-AML4 (expressing MLL-ENL). To suppress ZNF521, we used GFP-tagged lentiviral vectors expressing anti-ZNF521 shRNAs (ZNF004 and ZNF710) or a non-targeting shRNA sequence (shScram). After assessing transduction efficiency by flow cytometry (range 30\u201380%) in three out of four cell lines expressing anti-ZNF521 shRNAs. This was most likely due to S phase reduction (from 29% to 65%) rather than G2/M alterations and p27 (CDKN1B) cell cycle inhibitors [ZNF521 knockdown cells, suggesting a prolonged G1/S transition as the main reason for the aforementioned cell cycle arrest and with Securinine, two differentiation agents administered to THP-1 and NOMO-1 AML cells, respectively , 58 were upregulated while 100 were downregulated [ZNF521 expression negatively modulates genes involved in myeloid differentiation, and is required to maintain expression programs associated with MLL-induced transformation.To investigate the gene expression pattern in MLL-AF9 AML cells expressing high levels of d Figure . Gene Seed genes \u201333 and iusashi2) was subdivided in 4 fragments and inserted into a pGL4-basic reporter plasmid spanning the ZNF521P3 fragment and in two non-MLL-associated fusion genes such as AML1-ETO and PML-RARA\u03b1. We observed that both AML1-ETO and PML-RARA\u03b1 yielded only a minimal luciferase activity compared with MLL-ENL that showed even a higher promoter binding affinity than MLL-AF9 (> 2.5 fold) and an anti-MLL C-terminal (MLLC) antibodies were used for this experiment. ChIP assays showed that MLLN bound specifically to the ZNF521 promoter region in NOMO-1 but not in HL60 Figure . Besidesn Figure . Consists Figure . In ordeMLL translocations have a significantly upregulation of ZNF521 expression independently of the fusion partner involved in the translocation with MLL. The overexpression of ZNF521 is a robust transcriptional feature of MLL-rearranged AML, consistent across independent adult and pediatric microarray datasets [MLL-rearranged AML, and understand if it might deserve attention as potential therapeutic target.We present data showing that pediatric AML patients carrying datasets \u201321. FromZNF521 depletion was to enhance myeloid differentiation of leukemia cells as evidenced by changes in cell morphology, immunophenotype and increase of a myeloid-specific gene expression in MLL-rearranged cell lines and primary cells. The requirement of ZNF521 in the maintenance of an undifferentiated status associated with MLL-rearranged AML was also supported by the fact that ZNF521 expression drastically decreased upon treatment with specific differentiation-induced agents, such as ATRA. The observed growth defect, cell cycle arrest and reduced colony formation upon ZNF521 depletion were secondary to cells entering into a differentiation program. Thus, MLL fusion proteins might promote leukemogenesis not only by HOXA9 and MEIS1 upregulation, but also by keeping the ZNF521 overexpressed, which in turn contributes to a block of differentiation or to the maintenance of an undifferentiated state of leukemia cells.A major hallmark of leukemia and a consequence of MLL fusion proteins expression is a block in hematopoietic differentiation . On thisZNF521 enhanced erythroid differentiation and increased B-lineage maturation in cell lines and primary hematopoietic progenitor cell, respectively [ZNF521 showed enrichment of hematopoietic stem cells (HSCs)- and ESC-associated downregulated genes and sets of genes associated with differentiation program [ZNF521 depletion, which in part resemble what has been previously observed in MLL-rearranged cells after loss of HOXA9, gave a further support that ZNF521 plays a critical role in MLL-fusion-mediated leukemia. Interestingly, the expression of either HOXA9, a canonical downstream target for MLL-rearranged leukemia [ZNF521 have been shown to be restricted in CD34+ progenitor cells [Consistent with this finding, others have reported that loss of ectively , 16. Morectively \u201339. Cons program , 26. Bas program , 30, 40.leukemia , 41 or Zor cells , 30.ZNF521 does not affect HOXA9 expression, implying that both are MLL-dependent but might act in a non-mutually exclusive and additive manner.Nevertheless, in our gene-expression analysis, loss of ZNF521 is a direct target of both MLL-AF9 and MLL-ENL fusion proteins. We defined a genomic region of 555 bp in 5\u2032 ZNF521 promoter that is thought to be crucial for ZNF521 activation by MLL fusion proteins. This finding is consistent with prior observations that showed how the modulation of MLL-AF9 levels resulted in concordant changes in ZNF521 expression in different human in vitro models [Zfp521 gene in an MLL-AF9 mouse leukemia model. Of note, this is also observed for other well-known targets of MLL fusion proteins such as EVI1 and PLZF [Zfp521 is primarily expressed in the HSC fraction and significantly reduced in granulocyte-monocyte-progenitor cells (GMPs) (http://servers.binf.ku.dk/bloodspot/?gene=ZFP521&dataset=nl_mouse_data), in which the analysis has been done. Future ChIP-seq experiments on human transformed HSC will likely shed further light on ZNF521-MLL-AF9 target gene specificity.Supporting the idea that ZNF521 is particularly required for MLL-mediated leukemia, our data of luciferase reporter and ChIP assays revealed that o models , 43. Suro models did not and PLZF , 46 AbouIn summary, this study unravels the anti-differentiation function of ZNF521 in MLL-rearranged cells and showed the mechanism by which ZNF521 participates in MLL-fusion mediated transformation. This data also indicate that ZNF521 is highly expressed in the majority of MLL-rearranged AML pediatric patients, and thus ZNF521 could be a potential molecular target for this subtype of aggressive leukemia.All of the pediatric AML patient samples were obtained at the time of diagnosis from the University-Hospital of Padua and stratified according to the AIEOP AML 2002/01 protocol AML 2002/01 . PatientZNF521, PU.1, CEBPa, HOXA9 and MEIS1 were measured by quantitative RT-PCR (qRT-PCR) with SYBR green on an AB 7900HT real time system (Applied Biosystem) using the comparative Ct method and the GAPDH gene expression as internal control [Total RNA was extracted with Trizol reagent (Invitrogen) and reverse transcribed into cDNA using the Superscript III First-Strand Synthesis System (Life Technology). The mRNA levels of control . The priZNF521 and a control scrambled shRNA (shScram) were used is described in Renilla luciferase reporter vector (Promega) as internal control for normalization of transfection efficiency, for a total of 2 \u03bcg of combined plasmids per well. The cells were then harvested at 48 hours after transfection using a Dual-Luciferase reporter assay system (Promega) and the Victor3 TM 1420 Multilabel Counter (PerkinElmer). Data are presented as the mean ratio for triplicate experiments.pMSCV-neo-Flag-MLL-AF9, MSCV-PML-RARA-IRES-GFP, MSCV-AML1-ETO-GFP, pCMVMLL-3xFlag and pCMVMLL-ENL-3xFlag have been previously described , 50\u201351. 6 cells) were transfected with 10 \u03bcg of Flag-MLL-AF9 or Flag-MLL-ENL expression plasmids. 48 hours post transfection, were cross-linked with 1% formaldehyde (Sigma) for 15 minute at room temperature. Subsequently, the lysed cells were isolated and sonicated on ice to shear DNA into fragments of 200 bp to 1 kb. Then, the chromatin complexes were incubated into pre-treated Stripwells (Sigma) with anti-Flag M2 monoclonal antibody (Sigma), or normal mouse IgG (Sigma) as indicated. The input DNA was isolated from sonicated lysates before immunoprecipitation as a positive control. Purified DNA was then resuspended in TE buffer for PCR. ChIP assay from 2 \u00d7 106 of MLL-AF9-expressing NOMO-1 cells or HL60 cells was performed as above reported using a N-terminal MLL monoclonal antibody (Santa Cruz Biotechnology) or a C-terminal MLL polyclonal antibody (Sigma) or a mouse IgG (Sigma) as indicated. Purified ChIP DNA was amplified by regular PCR. Primers amplifying the ZNF521 promoter region and the HOXA9 promoter region used for the ChIP PCR are listed in ChIP assay was performed using the Imprint Chromatin Immunoprecipitation kit (Sigma), according to the manufacturer's protocol with minor modifications. Briefly, 293T cells (3.5 \u00d7 10http://www.r-project.org/) with BioConductor package (www.bioconductor.org). Shrinkage t test was used to identify differentially expressed genes between shScram and shRNA ZNF521 THP-1 cells selected with a local FDR < 0.05 (FDR). Hierarchical clustering analyses were performed using Euclidian distance and Ward's methods. Gene set enrichment analysis (GSEA) was performed using GSEA version 2.0 software with genes ranked by difference of class and statistical significance by 1000 gene set permutations. Gene set permutation was used to enable direct comparisons between shScram and shRNA ZNF521 results (< 7 replicates). Median of probes was used to collapse multiple probe sets to a single value per gene for each sample. Gene sets with a FDR < 0.05 were declared to be statistically significant. The microarray gene expression data have been submitted in NCBI's Gene Expression Omnibus under accession GSE79110Total RNA from sorted THP-1 cells transduced with shRNAs was isolated using Trizol as above reported and processed for microarray analysis using the Affymetrix GeneChip 3\u2032IVT express Kit (Affimetrix) after RNA quality control using Agilent 2100 Bioanalyzer (Agilent). Gene expression profile was performed using a Human Genome U133 2.0 Plus chip (Affymetrix), as previously described . The datWestern blot and immunofluorescence were performed using standard procedures. The antibody against ZNF521 was from Novus Biologicals (72009). Gamma-Tubulin (T6557), Actin (A5316), Flag M2 (F3165) antibodies were from Sigma Aldrich. For immunofluorescence analysis, antibody against CDKN1A/p21 (2947S) was from Cell Signaling and antibody against CDKN1B/p27 (610241) was from BD Biosciences. Detail method for the immunofluorescence is described in Cell viability, clonogenicity, cell cycle, apoptosis, expression of CD11b and CD14, morphological analysis and cell differentiation induction were performed as detailed in MLL-AF9-expressing cells were obtained from BM samples of diagnosed AML pediatric patients stored in the BioBank of the laboratory of Pediatric Hematology of the University Hospital of Padua, according to the guidelines of the local ethics committee. Initial AML xenografts were established by tail vein injection with 8 \u00d7 106 primary cells suspended in 300 \u03bcl of PBS in 6- to 8-week-old NSG mice, which were purchased from Charles River . All animal experiments were performed in accordance with institutional guidelines and established protocols [ex vivo experiments, two independent biological replicates were performed.Primary rotocols . Engraftt test, Mann\u2013Whitney U-test or kruskal-Wallis one-way analysis of variance followed Dunn's test as appropriate . Results were considered significant at P < 0.05.Data are presented as mean \u00b1 SD. Each experiment was performed at least 3 times, except where stated otherwise. The differences were examined using 2-tailed"} +{"text": "Glutamatergic dysfunction, deregulated mitochondrial metabolism and alterations of membrane phospholipids are considered as core pathology of psychosis, and have been studied in schizophrenic illness using magnetic resonance spectroscopy (MRS). Combining 1H- and 31P-MRS, this study investigates these aspects in Ultra-high risk (UHR-T) patients right after transition to psychosis (T0) and after a two years interval (T1) in a naturalistic longitudinal design, including treatment as usual by cognitive-behavioral therapy (CBT) and pharmacotherapy with second generation antipsychotics.We applied 3 T chemical shift imaging and hippocampal single-voxel 1H-MRS in 29 neuroleptic-na\u00efve UHR-T patients and 27 healthy controls matched for age and gender. Glutamate (Glu) and N-acetyl-aspartate (NAA) reflect neuronal functioning, phosphocreatine (PCr), adenosine triphosphate (ATP) and NAA indicate mitochondrial function and energy metabolism, and phosphomono- and diester indicate the balance of phospholipid synthesis (PME) and -breakdown (PDE). Psychopathology was assessed using the CAARMS, BPRS-E and SCL-90-R. Generalized linear mixed models were used to examine case-control differences in metabolite changes over time, and associations with clinical improvement.At T0, cross-sectional analysis revealed decreased NAA, Glu and PME levels in the left dorsolateral prefrontal cortex (DLPFC) and thalamus of UHR-T patients as well as higher PCr and lower PDE levels in the right hippocampus. (ii) Follow-up analysis (T1) showed in patients a significant increase of Glu in the bilateral DLPFC and the right thalamus, while a decrease of PCr was observed in the right hippocampus.The observed metabolite pattern at T0 likely reflects a hypofunction of glutamatergic neurons and a disturbance of membrane phospholipid turnover in fronto-thalamo-hippocampal networks during the first acute onset phase of psychotic illness. The pattern of changes at T1 is suggestive for an improvement of neuronal functioning in these networks that is caused by therapy, and presumably underlies the observed clinical improvement in terms of negative symptoms and cognitive impairment."} +{"text": "The repertoire of auxin-related cis-elements and their involvement in different modes of auxin response are not yet known. Here we analyze the enrichment of nucleotide hexamers in upstream regions of auxin-responsive genes associated with auxin up- or down-regulation, with early or late response, ARF-binding domains, and with different chromatin states. Intriguingly, hexamers potentially bound by basic helix\u2013loop\u2013helix (bHLH) and basic leucine zipper (bZIP) factors as well as a family of A/T-rich hexamers are more highly enriched in auxin-responsive regions than canonical TGTC-containing AuxREs. We classify and annotate the whole spectrum of enriched hexamers and discuss their patterns of enrichment related to different modes of auxin response.The phytohormone auxin regulates virtually every developmental process in land plants. This regulation is mediated via de-repression of DNA-binding auxin response factors (ARFs). ARFs bind TGTC-containing auxin response Whereast manner .cis-element may compete , C-box (GACGTC), and G-box (CACGTG) sequences are bound more preferentially motif CACATG for each hexamer, we computed the permutation P-value by P=(m+1)/(M+1), where m is a number of recorded P-values not greater than the meta P-value.In the third step, we determined the statistical significance of selected hexamers by a permutation test. In each permutation, we mixed promoters between genes so that each promoter was used exactly once . Then weP-value <0.005.We considered the association between the presence of the hexamer and the auxin responsiveness as significant with the permutation Bonferroni-corrected cis-regulatory elements using TOMTOM . We considered the matches with an E-value <0.05 as significant. For hexamers without significant matches, we additionally screened the literature.We compared the detected hexamers with known P-values of enrichment with Bonferroni multiple testing correction. We considered over-representation as significant at a family-wise error rate (FWER) below 0.05.To study if the obtained hexamers are enriched within ARF-binding regions, we used available whole-genome data on ARF6 ChIP-Seq the number of positions which this hexamer occupies across all upstream regions of 21 098 genes relative to the total number of all possible positions; and (ii) the similar one across all promoter segments associated with a specific chromatin state.Thirdly, we tested if the hexamers were enriched in the upstream regions located within specific chromatin states of auxin-responsive genes (positive set) relative to the same promoter segments of non-regulated genes (control set). For each hexamer, we compared two proportions via one-tailed Fisher\u2019s exact test: (i) the number of positions this hexamer occupies in the promoter segments of a specific chromatin state within a positive gene set relative to the cumulative number of all possible positions in these segments; and (ii) the same proportion for the control gene set.P-values of enrichment with Bonferroni multiple testing correction, considering the association as significant at FWER <0.05.In each step, we adjusted To expand our knowledge on the scope of transcriptional regulation in auxin response, we aimed to detect putative AuxREs from meta-analysis of auxin-responsive transcriptome data sets without prior assumptions on the transcription factors binding these.A. thaliana , which were substantially enriched in upstream regions of auxin-responsive genes . We consWe found the canonical AuxRE core TGTCTC and its analog TGTCCC enriched in upstream regions of auxin-up-regulated genes, thus confirming previous findings , were alcis-elements with more than five A/Ts in a row to be the most abundant and the most significant in our search , 3. Two-Analyzing the A/T-rich hexamers against the data on Arabidopsis transcription factor-binding sites generated by Among non-A/T-rich hexamers, TOMTOM found significant matches with MYB-binding sites for GATAAG and AGGGTT, a FUS3-binding site for CATGCA, TCP-binding sites for TGGGCC and GTCCCC, and a number of ACGT-containing sequences, which resemble the binding sites for several transcription factors families . A closeWhat could over-representation of these hexamers in auxin-responsive regulatory regions mean? The identified hexamers could be the core sequences for transcription factor-binding sites mediating primary or secondary response. They could be the coupling hexamers for TGTC-containing AuxRE, constituting with it the composite element and bound by ARF partner transcription factors hexamers is significantly enriched within ARF2-binding regions , which pAvailable whole-genome maps . Upstream regions of both auxin up- and down-regulated genes appeared to be enriched with the chromatin in state 4 (FWER <0.001). Up-regulated genes additionally possess significantly higher portions of chromatin state 1 (FWER <0.001) and state 3 (FWER <0.05) in the upstream regions comparing non-responsive with auxin-responsive genes. Down-regulated genes were additionally enriched with chromatin states 2 (FWER <0.01) and 5 (FWER <0.001).Analyzing the distribution of putative AuxREs over difWe next tested if putative AuxREs are specMany A/T-rich putative AuxREs are specifically over-represented in chromatin states 2 and 5 of both auxin-activated and repressed genes, with the repressed genes showing a greater variety . Most ofNegatively and positively auxin-responsive genes differed by non-A/T-rich hexamers associated with specific chromatin states . All chrInterestingly TGTC-containing cores were not enriched in any chromatin state. Recently we showed that ethylene-responsive genes possess an EIN3-binding site within specific chromatin state 4 found by the TOMTOM tool of putative AuxREs with the transcription factor-binding sites identified by TOMTOM (Fig. S2. Distribution of A/T-rich putative AuxREs along the upstream regions of auxin-responsive genes.Supplementary Figures_S1_S2Click here for additional data file.Supplementary Tables_S1_S4Click here for additional data file."} +{"text": "Several G-protein-coupled receptors (GPCRs) have been shown to be important signaling mediators between neurons and glia. In our previous screening for identification of nerve injury-associated GPCRs, G-protein-coupled receptor 84 (GPR84) mRNA showed the highest up-regulation by microglia after nerve injury. GPR84 is a pro-inflammatory receptor of macrophages in a neuropathic pain mouse model, yet its function in resident microglia in the central nervous system is poorly understood.We used endogenous, natural, and surrogate agonists for GPR84 and examined their effect on mouse primary cultured microglia in vitro.GPR84 gene.6-n-Octylaminouracil (6-OAU), embelin, and capric acid rapidly induced membrane ruffling and motility in cultured microglia obtained from C57BL/6 mice, although these agonists failed to promote microglial pro-inflammatory cytokine expression. Concomitantly, 6-OAU suppressed forskolin-induced increase of cAMP in cultured microglia. Pertussis toxin, an inhibitor of Gi-coupled signaling, completely suppressed 6-OAU-induced microglial membrane ruffling and motility. In contrast, no 6-OAU-induced microglial membrane ruffling and motility was observed in microglia from DBA/2 mice, a mouse strain that does not express functional GPR84 protein due to endogenous nonsense mutation of the GPR84 mediated signaling causes microglial motility and membrane ruffling but does not promote pro-inflammatory responses. As GPR84 is a known receptor for medium-chain fatty acids, those released from damaged brain cells may be involved in the enhancement of microglial motility through GPR84 after neuronal injury. G-protein-coupled receptors (GPCRs) form the largest superfamily of membrane proteins, and several GPCRs are important signal mediators between neurons and glia, protecting neurons from pathological stressors that cause disease , 2. To iGPR84 gene has also been discovered in some classical inbred mouse strains. This deletion generates a premature stop codon, resulting in a truncated protein that lacks transmembrane domains 4\u20137 GTP\u03b3S binding assay and identified a highly effective agonist compound, 6-OAU, which activated human GPR84 in the presence of Gqi5 chimeric G proteins. Furthermore, EGFP-labeled human GPR84 internalization was observed in a 6-OAU-dependent manner. Altogether, these findings suggest that 6-OAU activates GPR84 [Potent surrogate agonists are a useful tool for characterizing functionally unknown or less known GPCRs. Suzuki et al. screenedes GPR84 . GPR84 ies GPR84 , 17, 18.es GPR84 . Collectes GPR84 , 33, sugSeveral fatty acids regulate microglial morphology and activity \u201321. GiveThis study demonstrates the functional significance of GPR84 agonists in microglia in vitro. Our findings show that GPR84 agonist-mediated signaling affects morphology and motility of microglia, suggesting that microglial GPR84 could be a therapeutic target in microglia-associated diseases such as multiple sclerosis and Alzheimer\u2019s disease [The present study demonstrates that GPR84 does not modulate pro-inflammatory responses in primary cultured microglia. However, endogenous and natural ligands and a surrogate agonist for GPR84 rapidly induce microglial ruffling and motility. 6-OAU induced activation of microglia was suppressed by PTX, which inhibits the Gi/o pathway located downstream of GPR84. Concomitantly, 6-OAU failed to induce membrane ruffling and motility in primary cultured microglia from DBA/2 mice lacking functional GPR84. Since GPR84 is known as a receptor for medium-chain fatty acids such as capric acid, fatty acids released from damaged brain cells may act on microglial GPR84 to increase cellular motility."} +{"text": "Concordance indices (Ct) from time-dependent receiver-operating characteristics (TDROC) were compared between patients with post-IMRT undetectable and those with detectable titers. After a median follow-up duration of 3.4 years (range 1.4-4.6 years), patients with post-IMRT 8th week and 6th month undetectable plasma EBV DNA titers enjoyed longer 3-year survival endpoints than those who had detectable titers at the same time points. Post-IMRT 8th week, and more significantly, post-IMRT 6th month undetectable plasma EBV DNA were the only significant prognostic factors of 3-year survival endpoints. Ct values for all 3-year survival endpoints for both post-IMRT 8th week and 6th month undetectable plasma EBV DNA were significantly higher in those with stage IVA\u2013IVB diseases compared to stage I-III counterparts. Early post-IMRT undetectable plasma EBV DNA titers were prognostic of 3-year survival endpoints in patients with non-metastatic NPC. Intensified treatment should be further explored for patients with persistently detectable titers after IMRT.Plasma Epstein-Barr virus (EBV) DNA titers have been used to monitor treatment response and provide prognostic information on survival for nasopharyngeal carcinoma (NPC). However, the long-term prognostic role of pretreatment and posttreatment titers after radical contemporaneous radiation therapy remains uncertain. We recruited 260 evaluable patients with non-metastatic NPC treated with radical intensity-modulated radiation therapy (IMRT) with or without adjunct chemotherapy. Plasma EBV DNA titers at baseline and then 8 weeks and 6 months after IMRT were measured. Cox regression models were employed to identify interaction between post-IMRT 8 The most common histological types are undifferentiated non-keratinizing carcinoma (WHO Type III) and differentiated non-keratinizing carcinoma (WHO Type II). Radiation therapy alone is offered for stage I and II disease while concurrent chemoradiation is indicated for locally advanced stage III to IVB disease fluorodeoxyglucose (planning . Two indescribed 12]. Al. Al18F-Fescribed 38]. Br. Br18F-Fing IMRT . Patientth week and 6th month undetectable plasma EBV DNA titers as covariates. Time-dependent receiver-operating characteristics (TDROC) analyses were performed to obtain time-dependent concordance indices (Ct) and area under the curve (AUC) for advanced stage IVA to IVB disease versus stage I to III disease [P < 0.05 (two-sided). The database-lock date for analysis was December 30, 2015.The prespecified survival endpoints including local failure-free survival (LFFS), regional failure-free survival (RFFS), distant metastasis-free survival (DMFS), progression-free survival (PFS), cancer-specific survival (CSS) and overall survival (OS) were defined and evaluated by log rank tests . Univari disease . Nonpara disease . The othst American Society of Clinical Oncology (ASCO) Annual Meeting (Abstract 6007), Chicago, IL, May 29-June 2, 2015. Clinical trial information: NCT02476669 .This study was presented in part as an oral abstract at 51"} +{"text": "Activation of indirect pathway medium spiny neurons (MSNs) via promotion of cAMP production is the principal mechanism of action (MOA) of current antipsychotics with dopamine D2 receptor antagonism. Phosphodiesterase 10A (PDE10A) inhibitors activate both direct and indirect pathway MSNs by increasing both cAMP and cGMP levels by inhibiting their degradation, which might be expected to promote activation of intracellular signaling similar to that of D2 antagonists in the indirect pathway MSNs. Thus, the activation of the indirect MSN pathway through the distinct MOA of these compounds raises the possibility of augmented pharmacologic effects with combined treatment.In this study, we compared gene-regulation patterns in the indirect pathway MSNs induced by the PDE10A inhibitors T-773 and T-609, and the D2 antagonist haloperidol, using a cell-type-specific comprehensive gene expression analysis in Drd2-bacTRAP (translating ribosome affinity purification) mice. The pharmacologic effects of combined treatment with another PDE10A inhibitor, TAK-063, and clinically used antipsychotics, haloperidol (HAL) and olanzapine (OLA), were evaluated in multiple rodent models.Male ICR mice, Drd2-bacTRAP mice, and Sprague-Dawley rats were used. The indirect pathway MSN-specific gene expression changes by T-773, T-609, and HAL were investigated using RNA sequencing of striatal samples of Drd2-bacTRAP mice. The activation of MSNs in rats was evaluated by measuring glutamate receptor subunit 1 phosphorylation (pGluR1) levels. An in vitro electrophysiological study on the corticostrial pathway in rats was conducted in a slice preparation. The activation of each MSN pathway was assessed by inducing genes as pathway-specific markers: enkephalin for the indirect pathway and substance P for the direct pathway. Suppression of MK-801- or methamphetamine (METH)-induced hyperactivity was assessed by measuring locomotor activity for 2 hours after administration of these stimulants to rats. Improvement of prepulse inhibition (PPI) was investigated in a MK-801-induced PPI deficit mouse model.Translational profiling in Drd2-bacTRAP mice treated with the PDE10A selective inhibitors, T-773 and T-609, and with HAL suggested regulatory of a largely overlapping signaling pathway by these compounds in the indirect pathway MSNs: 87% of the genes regulated by HAL were also regulated by both T-773 and T-609. Combined treatment with TAK-063 and either HAL or OLA produced an augmented effect on pGluR1 in the rat striatum. An electrophysiological study in rat brain slices indicated that TAK-063 enhanced synaptic responses to a similar extent in both direct and indirect pathway MSNs. Additional evaluation using MSN pathway-specific markers revealed that coadministration of TAK-063 with HAL or OLA additively activated the indirect pathway, but not the direct pathway. Combined treatment with TAK-063 (0.1 mg/kg p.o.) and either HAL (0.3 mg/kg p.o.) or OLA (3 mg/kg p.o.) at subeffective doses produced augmented effects on METH- or MK-801\u2013induced hyperactivity in rats and MK-801\u2013induced PPI deficits in mice. TAK-063 at 0.1 mg/kg did not affect plasma prolactin levels and cataleptic response induced by HAL or OLA in rats.PDE10A inhibitors and HAL showed similar patterns of gene regulation in indirect pathway MSNs in mice. Combined treatment with TAK-063 and either HAL or OLA at subeffective doses produced significant antipsychotic-like effects but no augmentation of the plasma prolactin level and cataleptic response. Although further preclinical and clinical studies will be needed, TAK-063 may provide a novel mechanism as a PDE10A inhibitor for use as combination therapy in schizophrenia."} +{"text": "Purpose. To monitor the inflammatory response (IR) following traumatic brain injury (TBI) before and after the rehabilitation of the blood-brain barrier (BBB) in rabbits using USPIO- and Gd-enhanced MRI. Materials and Methods. Twenty white big-eared rabbits with mild TBI (mTBI) were randomly and equally divided into four groups. Rabbits were sacrificed for the brain specimens immediately after the last MRI-monitoring. Sequences were tse-T1WI, tse-T2WI, Gd-T1WI, and USPIO-T1WI. Dynamical MRI presentations were evaluated and compared with pathological findings for each group. Results. Twenty-four hours after injury, all rabbits displayed high signal foci on T2WI, while only 55% lesions could be found on Gd-T1WI and none on USPIO-T1WI. The lesions were enhanced on Gd-T1WI in 100% subjects after 48\u2009h and the enhancement sizes augmented to the largest after 72\u2009h. At the time point of 72\u2009h after TBI, 90% lesions were enhanced by USPIO. Five days after injury, 19 lesions showed decreased Gd-enhancement and one disappeared; however, USPIO-enhancement became larger than before. Pathological findings showed microglias slightly appeared in dense leukocytes at 48\u2009h, but became the dominant inflammatory cells after five days. Conclusions. Dynamic IR following injury could be monitored by combination of Gd- and USPIO-MRI in mTBI rabbits. Traumatic brain injury (TBI) is thought to be triggered by a single event; however, patients usually show a highly variable outcome even from the same initiator event . Patient\u03b1 either in serum or cerebrospinal fluid (R)-PK11195-PET, [(18)F] fluoroethyl-DAA 1106-PET, and [(18)F] fluorodeoxyglucose- PET [USPIO enabled more sensitive assessment of leukocyte (mainly macrophage) infiltration by the MR technologies independent from a disturbance of the BBB . Combinaose- PET , which mIn this study, IR following TBI before and after the rehabilitation of the blood-brain barrier (BBB) in rabbits was accessed via both USPIO-enhanced and Gd-enhanced MRI. The edema areas of the lesions sharply reached peak 72\u2009h after injury and became alleviated five days after TBI. Peripheral blood leucocytes were the major contributor of the edema before the BBB rehabilitation, which was indirectly shown by Gd-enhanced T1WI. USPIO-enhancement turned into predominance regardless of the BBB rehabilitation. These findings supported that IR following injury was dynamically evolved in a distinct mode before and after the BBB rehabilitation, which could be monitored by the combination of Gd-MRI and USPIO-MRI."} +{"text": "Overexpression of miR-17-92 blocked the induction of apoptosis upon oncogene inactivation in the MYC and RAS-driven but not in the BCR-ABL-driven ALL leukemia. Hence, our results provide novel insight into the mechanism of apoptosis upon oncogene inactivation and suggest that induction of BIM-mediated apoptosis may be an important therapeutic approach for ALL.Oncogene inactivation in both clinical targeted therapies and conditional transgenic mouse cancer models can induce significant tumor regression associated with the robust induction of apoptosis. Here we report that in MYC-, RAS-, and BCR-ABL-induced acute lymphoblastic leukemia (ALL), apoptosis upon oncogene inactivation is mediated by the same pro-apoptotic protein, BIM. The induction of BIMin the MYC- and RAS-driven leukemia is mediated by the downregulation of Although cancers evolve through a multistage process with accumulation of both genetic and epigenetic changes, many cancers are dependent on specific driver oncogenes for maintenance of the malignancy . This phBim deficient mice often develop autoimmune diseases, and lymphocytes from these mice are refractory to apoptotic stimuli. More recently, BIM, together with other BCL-2 family proteins, have been implicated in the mechanism of apoptosis and therapeutic sensitivity of BCR-ABL positive cells treated with imatinib, lung adenocarcinoma cells treated with EGFR inhibitors, and breast cancer cells treated with HER2 inhibitors [The inactivation of driver oncogenes in mouse cancer models and human targeted therapy often leads to tumor regression associated with the induction of apoptosis \u20135. The mhibitors \u201318.MYC, RAS, and BCR-ABL, are frequently involved in the pathogenesis of human acute lymphoblastic leukemia (ALL) [E\u03bc) is used to regulate the expression of Tet transactivator (tTA) in lymphocytes. By crossing of E\u03bc-tTA mice with mice carrying different oncogenes controlled by a tetracycline-responsive element (TRE), we are able to regulate oncogene expression in lymphocytes . We and ia (ALL) , 4. The s Figure . E\u03bc-tTA/cell ALL , 4, 20. cell ALL , 4. HereE\u03bc-tTA/TRE-MYC leukemia cells underwent significant cell death as shown by 7-AAD staining [miR-17-92 expression blocked the induction of BIM upon MYC inactivation . miR-17-tivation . The apoS Figure \u20134E. Thess Figure .To explain how BIM is induced in the BCR-ABL model, we tested other potential mechanisms, such as transcriptional stimulation , 26 and miR-17-92, regulates BIM induction and apoptosis in MYC- and RAS- but not in BCR-ABL-driven ALLs. The regulation of BIM-mediated apoptosis in BCR-ABL-driven ALL appears to be post-transcriptional and related to JNK phosphorylation.We used three conditional transgenic mouse models of ALL driven by different oncogenes to demonstrate that BIM activation is the convergent mechanism of apoptosis associated with oncogene addiction. Interestingly, the mechanism of BIM activation was different depending on the specific driver oncogene. The microRNA cluster, via inhibition of the MEK/ERK pathway and/or the PI3K/mTOR pathway [miR-17-92 and/or JNK phosphorylation in regulating BIM-mediated apoptosis upon oncogene inactivation particularly in the leukemia contexts. Thus, our work provides novel insight into the mechanism of apoptosis associated with oncogene addiction.Multiple prior studies have correlated BIM activation with clinical response to targeted therapies. Lung adenocarcinoma cells treated with gefitinib, breast cancer cells treated with lapatinib, and melanoma cells treated with B-RAF inhibitors all exhibit BIM activation. In these solid tumors, BIM is often activated pathway , 31\u201333. The activity of BIM seems to be particularly critical in regulating the balance between pro-apoptotic and pro-survival signals. Even modest changes in BIM activity can significantly influence tumorigenesis and therapeutic responses of cancer. For examples, the loss of one allele of BIM is sufficient to substantially accelerate lymphomagenesis , 35. EpiE\u03bc-tTA/TRE-MYC, E\u03bc-tTA/TRE-KRAS, and E\u03bc-tTA/TRE-BCR/ABL mice were cultured in RPMI-1640 media (Gibco) supplemented with 10% fetal bovine serum under standard culture condition. Inactivation of the Tet-regulated oncogenes was achieved with doxycycline treatment (20ng/ml) in the culture media.The murine ALL leukemia cell lines derived from Western blots were performed as described before . The folmiR-30-based shRNAs with puromycin selection marker (V2LMM_220682 from Open Biosystems). Retrovirus was prepared using the phoenix retrovirus packaging system. The leukemia cells were spin infected and selected with puromycin (2\u03bcg/ml).The knockdown of BIM expression was accomplished using LMP The tumors cells were labeled with MSCV-luciferase and transplanted subcutaneously into FVB/N mice. When tumors reach approximately 1.0cm in diameter, mice were imaged and then treated with doxycycline (20\u03bcg/ml). For imaging procedure, the mice were injected intraperitoneally with 100\u03bcl of d-luciferin (33mg/ml) about ten minutes before detection using the IVIS 200 cooled CCD camera system (Xenogen). Mice were imaged daily for 18 days following doxycycline treatment. Quantification of bioluminescence signals was done using Living Imaging software 4.3.1 (Perkin Elmer). All animal experiments were approved by Stanford's Administrative Panel on Laboratory Animal Care (APLAC) and were performed in accordance with institutional and national guidelines.Bim mRNA expression was quantified in triplicates using SYBR based quantitative PCR and normalized to Ubc mRNA level. Primers used are: Bim-F 5\u2032-CACCTGCTGTGTGCTTCCTA-3\u2032 and Bim-R 5\u2032-TTCAGTGAGCCATCTTGACG-3; Ubc-F 5\u2032-AGCCCAGTGTTACCACCAAG-3\u2032 and Ubc-R 5\u2032-ACCCAAGAACAAGCACAAGG-3\u2032. Total microRNA was extracted with miRNeasy microRNA extraction kit (Qiagen). The microRNAs were first reverse-transcribed and then quantified with TaqMan microRNA assay kits (Applied Biosystems) following manufacturer's protocols. Each sample was run in triplicate and normalized to U6 snRNA.Propidium iodide staining was used for the study of cell cycle distribution and apoptosis. Briefly, leukemia cells were resuspended in PBS and fixed with ice-cold ethanol. Cells were treated with RNase and propidium iodide and analyzed on a FACScan flow cytometer (Becton Dickinson). For live/dead staining, cells were stained with 7-AAD. FACS data was analyzed with FlowJo software (Tree Star)."} +{"text": "ERG intragenic deletions. ERG-related patients have remarkably favorable outcome despite a high incidence of inauspicious IKZF1 aberrations.ERG-related leukemia is a B cell precursor acute lymphoblastic leukemia (BCP ALL) subtype characterized by aberrant expression of DUX4 and ERG transcription factors, and highly recurrent We describe clinical and genomic features of the ERG-related cases in an unselected cohort of B-other BCP ALL pediatric patients enrolled in the AIEOP ALL 2000 therapeutic protocol. We report a small noncoding RNA signature specific of ERG-related group, with up-regulation of miR-125b-2 cluster on chromosome 21 and several snoRNAs in the Prader-Willi locus at 15q11.2, including the orphan SNORD116 cluster. Childhood B cell precursor acute lymphoblastic leukemia (BCP ALL) is an heterogeneous disease characterized by recurrent genetic aberrations and frequent mutations of genes involved in hematopoietic development, patients that lack common aberrations are defined \u201cB-others\u201d .IGH-DUX4 rearrangements, and rare ERG-DUX4 rearrangements, as universal feature and founder event in the ERG-related leukemia subtype was used to detect ERG proteins, anti-\u03b2-actin-HRP was used as loading control.Event Free Survival (EFS) and overall survival were estimated according to Kaplan-Meier, with Greenwood standard error and with the log-rank test for comparison; Cumulative Relapse Incidence (CRI) was estimated adjusting for competing risks of other events and compared with the Grey test. To assess associations between patients\u2019 features, the Chi-Square test was applied. GraphPad Prism software and SAS 9.2 were used for analyses ."} +{"text": "Unlike for other retroviruses, only a few host cell factors that aid the replication of foamy viruses (FVs) via interaction with viral structural components are known. Using a yeast-two-hybrid (Y2H) screen with prototype FV (PFV) Gag protein as bait we identified human polo-like kinase 2 (hPLK2), a member of cell cycle regulatory kinases, as a new interactor of PFV capsids. Further Y2H studies confirmed interaction of PFV Gag with several PLKs of both human and rat origin. A consensus Ser-Thr/Ser-Pro (S-T/S-P) motif in Gag, which is conserved among primate FVs and phosphorylated in PFV virions, was essential for recognition by PLKs. In the case of rat PLK2, functional kinase and polo-box domains were required for interaction with PFV Gag. Fluorescently-tagged PFV Gag, through its chromatin tethering function, selectively relocalized ectopically expressed eGFP-tagged PLK proteins to mitotic chromosomes in a Gag STP motif-dependent manner, confirming a specific and dominant nature of the Gag-PLK interaction in mammalian cells. The functional relevance of the Gag-PLK interaction was examined in the context of replication-competent FVs and single-round PFV vectors. Although STP motif mutated viruses displayed wild type (wt) particle release, RNA packaging and intra-particle reverse transcription, their replication capacity was decreased 3-fold in single-cycle infections, and up to 20-fold in spreading infections over an extended time period. Strikingly similar defects were observed when cells infected with single-round wt Gag PFV vectors were treated with a pan PLK inhibitor. Analysis of entry kinetics of the mutant viruses indicated a post-fusion defect resulting in delayed and reduced integration, which was accompanied with an enhanced preference to integrate into heterochromatin. We conclude that interaction between PFV Gag and cellular PLK proteins is important for early replication steps of PFV within host cells. Orthoretrovirinae subfamily from the genus lentiviruses, numerous important virus-host interactions have been described. In contrast, only a few cellular proteins are known to influence the replication of foamy viruses , an intriguing type of complex retrovirus of the Spumaretrovirinae subfamily that combines features of both retroviruses and hepadnaviruses in its replication strategy. Given the increasing interest in FVs as gene transfer tools and their unique status within the retrovirus family, this discrepancy urged the identification of novel host cell interaction partners of FV structural components. This study focused on prototype FV (PFV), the best-characterized member of FVs, and its capsid protein, Gag, as the central player of viral replication. Members of the mitosis-regulatory, polo-like kinase (PLK) family were identified as novel Gag binding partners. The Gag interaction with PLK1 facilitated efficient PFV genome integration into host chromatin, ensuring successful replication and viral spread in infected target cell cultures. Collectively, our results elucidate the first link between cell cycle regulatory networks and the mitosis-dependent PFV integration process.Viruses are masters at exploiting host cell machineries for their replication. For human immunodeficiency virus type 1 (HIV-1), the best-studied representative of the As obligatory intracellular parasites, retroviruses depend on host cell machineries for successful replication. Over the years, many cellular proteins counteracting or aiding retroviral replication have been identified and indicated truncations) were tested for interaction with human [hPLK], mouse [mPLK] and rat PLK proteins [rPLK] or, where indicated, respective PBDs. PFV Gag was provided fused to the N-terminus [Gag-DB] or C-terminus [DB-Gag] of the GAL4 DB in combination with Tsg101- or PLK proteins fused to the C-terminus [AD-Prey] of the GAL4 AD. Presence and absence of interaction between each two partners is marked by either \u201c+\u201d or \u201c-\u201c, respectively; nd: not determined. Data of n = 2\u20136 independent experiments are summarized. (A+B) Results of PFV Gag C-terminal truncation mutant interaction with human and mouse PLK proteins. (C) Minimal Gag interaction domains for binding to PLK2 protein variants. iKD: inactive kinase domain; caKD: constitutively active kinase domain; iPBD: inactive polo-box domain.(TIFF)Click here for additional data file.S2 Fig960-T961-P962 and S1057-T1058-P1059 motifs highlighted. Solid vertical arrow: primary Pol processing site; dashed vertical lines: Pol subdomain boundaries. (B) Different variants of the PFV Pol protein were tested for interaction with human [hPLK] or, where indicated, respective PBDs. PFV Pol-iPR or IN was provided fused to the N-terminus (Pol-iPR-DB) or C-terminus (DB-Pol-iPR) of the GAL4 DB in combination with PLK proteins, Pol-iPR or IN fused to the N-terminus (Prey-AD) or C-terminus (AD-Prey) of the GAL4 AD. Presence and absence of interaction between each two partners is marked by either \u201c+\u201d or \u201c-\u201c, respectively. Data of n = 2\u20135 independent experiments are summarized. (C) PFV virions were produced by transient transfection of 293T cells with the four-component PFV vector system containing combinations of Gag and Pol variants as indicated. Titers of harvested viruses were determined by flow cytometry analysis of infected HT1080 target cells three days post-infection. The mean values and standard deviation for each supernatant were calculated from samples of cells infected with serial virus dilutions as described in Material and Methods. The values obtained using wt PFV Gag and Pol expression plasmids were arbitrarily set to 100%. Relative means and standard deviations normalized for Gag content (except uninfected) from independent experiments (n = 3) are shown. Differences between means of wt Gag and wt Pol containing virus and the individual mutants were analyzed by Welch\u2019s t test . Absolute titers of wt supernatants ranged between 1.2 x 106 and 1.6 x 106 eGFP ffu/ml.(A) Schematic representation of full-length PFV Pol with protease (PR), reverse transcriptase (RT), RNase H (RH), integrase (IN) enzymatic domains and C-terminal S(TIFF)Click here for additional data file.S3 Fig293T cells were transfected with eGFP-PLK-expressing constructs alone (left panels) or a combination of eGFP or eGFP-PLK and Gag-mCherry encoding expression constructs, as indicated above each panel of images. Forty-eight hours post-transfection, protein localization patterns were examined in fixed cells by confocal laser scanning microscopy (CLSM). Channels of the individual fluorescence micrographs are indicated on top, and the PLK variant used is indicated on the left. Data are representative of n = 2\u20135 independent experiments. (A) Localization patterns of eGFP-tagged PLK proteins (detected in eGFP-PLK channel) in mitotic and interphase cells transfected with the corresponding constructs. (B) Localization patterns of eGFP-tagged PLK and wt mCherry-tagged Gag proteins detected in corresponding channels in mitotic and interphase cells. (C) Localization of eGFP-tagged PLK and T225A Gag-mCherry in mitotic and interphase cells. (D) Localization patterns of wt mCherry-tagged Gag and various eGFP-tagged rPLK proteins detected in corresponding channels in mitotic cells. Scale bar: 10 \u03bcm. iKD: inactive kinase domain; caKD: constitutively active kinase domain; iPBD: inactive polo-box domain.(PDF)Click here for additional data file.S4 Fig(A) Coomassie staining of concentrated and purified, cell-free cell culture supernatants harvested from transfected 293T cells and separated by SDS-PAGE. Boxes with white dashed lines indicate gel regions at around 65\u201375 kDa corresponding to PFV Gag in supernatant lysates of cells transfected with PFV 4-component vector (wt) or respective mock transfected (mock) cells that were excised for proteolytic digest and mass spectrometric analysis. No PFV Gag derived peptides were detectable in mock supernatant lysates. \u00d8: empty lane; mwm: molecular weight standard . (B) Extracted ion chromatogram for precursor ions with m/z 989.469 and 1016.125 corresponding to triply charged un- and mono-phosphorylated tryptic peptide aa 222 to 250 ATSTPGNIPWSLGDDNPPSSSFPGPSQPR of particle-associated Gag protein. Arrows indicate peaks corresponding to non-phosphorylated peptide and phosphorylated peptide pool. (C) High resolution fragmentation spectrum of singly phosphorylated peptide aa 222\u2013250. Unique peaks corresponding to phosphorylated N-terminal 8-mer fragment are marked with a red box. Signals assigned to b(0)3 and b(4)-98 ions\u2014water loss of non-phosphorylated aa 3-mer fragment 222-ATS-224 and characteristic phosphogroup loss of mono-phosphorylated aa 4-mer fragment 222-ATSTp-225 \u2013suggest that the P-group could be located at T225.(TIF)Click here for additional data file.S5 Fig6 and 1.2 x 107 eGFP ffu/ml. (B) Replication-competent PFV virions were produced by transient transfection of proviral expression vectors, containing either the wt Gag or one of the denoted iSTP Gag variants into 293T cells. Viruses were harvested two days post-transfection and Gag content normalized amounts of viral supernatants were used to infect MRC5 fibroblasts using a 1:100 dilution. Cell-free supernatants of infected MRC5 cultures were harvested at the time points indicated. Virus titers were determined by titration on HT1080 PLNE target cells and flow cytometry analysis one day post-infection. Virus titers of one representative experiment out of two are shown.(A) Replication-deficient PFV virions were produced by transient transfection of 293T cells with the four-component PFV vector system, containing either the wt Gag or T225A iSTP Gag variant. Titers of harvested viruses were determined by flow cytometry analysis of infected HT1080, human MRC-5 fibroblasts or immortalized primary mouse C57BL/6 embryonic fibroblasts (MEF) three days post-infection. The mean values and standard deviation for each supernatant were calculated from samples of cells infected with serial virus dilutions as described in Material and Methods. The values obtained using wt PFV Gag expression plasmids were arbitrarily set to 100%. Relative means and standard deviations not normalized for Gag content from independent experiments (n = 4\u20139) are shown. Differences between means of wt virus and the individual mutants were analyzed by Welch\u2019s t test . Absolute titers of wt supernatants ranged between 1.2 x 10(TIF)Click here for additional data file.S6 FigReplication-deficient PFV virions were produced by transient transfection of 293T cells with the four-component PFV vector system, containing either the wt Gag (wt) or one of the denoted iSTP- and pmSTP (T225E) Gag variants. As controls, viruses containing wt Gag in combination with Pol with inactive reverse transcriptase (iRT) or inactive integrase (iIN) domain or particles produced in the absence of PFV Env expression construct (\u0394Env) were produced. The mock control (mock) included cells transfected with pUC19 alone. (A) Representative Western blot analysis of viral particles (virus) purified from 293T cell culture supernatant by ultracentrifugation through 20% sucrose and 293T cell lysates (cell). PFV proteins were detected using polyclonal antibodies specific for PFV Gag (\u03b1-Gag) or PFV Env LP (\u03b1-LP), a mixture of hybridoma supernatants specific for PFV Pol PR/RT and IN (\u03b1-PR/RT + \u03b1-IN), or a commercial monoclonal antibody specific for GAPDH (\u03b1-GAPDH). Serial dilutions of the wt samples were quantified to determine their relative protein contents compared to other samples. The identity of the individual proteins detected is indicated on the right. (B) Viral particle release was determined by quantitative Western blot analysis of viral particle lysates. Mean values and standard deviations (n = 2) are shown as relative values compared to the wild type control and normalized for cellular expression levels. (C) Quantification of PFV vgRNA in virus producing cells (vgRNA cell) and released particles (vgRNA virus) and particle-associated vgDNA (vgDNA virus). Mean values and standard deviation (n = 2) are shown as relative values compared to the wt control. Cellular values were normalized to GAPDH levels, viral particle values were normalized for Gag content.(TIF)Click here for additional data file.S7 FigP values of resulting pairwise comparisons are shown. RefSeq gene and LAD P values were determined using Fisher\u2019s Exact Test, whereas gene density P values were calculated by Wilcoxon Rank Sum Test. WT-1, S224A-1, and T225A-1 infections were performed on the same day, whereas S224A-2 and T225A-2 infections were performed side-by-side on a separate day. WT-2 and WT-3 infections were performed on separate independent days. P values > 0.05 are highlighted in bold, italic type.(TIF)Click here for additional data file.S1 Tablea FAM: 6-carboxyfluorescein; HEX: hexachloro-fluorescein; BHQ1: Black Hole Quencher 1; BHQ2: Black Hole Quencher 2.PFV: prototype foamy virus; LTR: long terminal repeat; EGFP: enhanced green fluorescent protein; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; fwd: forward; rev: reverse. (PDF)Click here for additional data file."} +{"text": "TDP-43 proteinopathy is a prominent pathological feature that occurs in a number of human diseases including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and inclusion body myositis (IBM). Our recent finding that TDP-43 represses nonconserved cryptic exons led us to ask whether cell type-specific cryptic exons could exist to impact unique molecular pathways in brain or muscle.In the present work, we investigated TDP-43\u2019s function in various mouse tissues to model disease pathogenesis. We generated mice to conditionally delete TDP-43 in excitatory neurons or skeletal myocytes and identified the cell type-specific cryptic exons associated with TDP-43 loss of function.Comparative analysis of nonconserved cryptic exons in various mouse cell types revealed that only some cryptic exons were common amongst stem cells, neurons, and myocytes; the majority of these nonconserved cryptic exons were cell type-specific.Our results suggest that in human disease, TDP-43 loss of function may impair cell type-specific pathways.The online version of this article (doi:10.1186/s13024-016-0144-x) contains supplementary material, which is available to authorized users. TARDBP) [Recent genetic evidence has established the linkage between the neurological disorders amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) \u20135. The kTARDBP) . Since tTARDBP) \u201312. Of pTARDBP) , 14. To in vitro inducible stem cell model of TDP-43 deletion. However, we have yet to establish the cell type-specific cryptic exons that arise in vivo. Here, we generated conditional Tdp-43 knockout mice to specifically delete Tdp-43 in excitatory neurons and skeletal myocytes. We found that Tdp-43 cryptic exons are highly variable between cell types and that many distinct pathways are altered\u2014novel findings that have mechanistic and therapeutic implications for human diseases exhibiting TDP-43 proteinopathy.We have recently found that TDP-43 plays a major role in repressing nonconserved cryptic exons . These cTardbp knockout mice (TardbpF/+) with CamKIIa-Cre transgenic mice to obtain a cohort of CamKIIa-Cre;TardbpF/+ mice which were subsequently crossbred to TardbpF/+ mice to generate the final cohort: CamKIIa-Cre;Tardbp+/+, CamKIIa-Cre;TardbpF/+ and CamKIIa-Cre;TardbpF/F mice. A similar strategy was applied when crossbreeding the MLC-Cre driver line to TardbpF/+ mice. All mouse experiments were approved by the Johns Hopkins University Animal Care and Use Committee.We crossbred our conditional CamKIIa-Cre line, wildtype and floxed mice were anaesthetized and perfused with 4% paraformaldehyde. Brains were embedded into paraffin, cut into 10\u00a0\u03bcm sections and stained according to standard protocols. For the MLC-Cre line, wildtype and floxed mice were anaesthetized and sacrificed by decapitation. Muscle tissue was then rapidly dissected and flash frozen in liquid nitrogen cooled isopentane. Frozen cryosections were cut at 10\u00a0\u03bcm thickness and stained according to standard protocols. Immunoreactivity was visualized using the Vectastain ABC Kit and diaminobenzidine peroxidase substrate (Vector Laboratories). Images were obtained using Olyumpus BX53 microscope.For the CamKIIa-Cre line, wildtype and floxed mice were anaesthetized and sacrificed by decapitation. Brain tissue was then rapidly dissected and manually homogenized in RIPA buffer (Sigma) containing an EDTA-free protease inhibitor cocktail (Thermo Scientific). For the MLC-Cre line, wildtype and floxed mice were also anaesthetized and sacrificed by decapitation. Muscle tissue was snap frozen in isopentane cooled with liquid nitrogen, manually ground into a powder, and then homogenized in RIPA buffer with protease inhibitor cocktail. Protein concentration was determined using the BCA assay (Pierce). Proteins were resolved using the NuPAGE 4-12% Bis-Tris Gel (Novex) with NuPAGE MES SDS Running Buffer (Novex), and transferred to PVDF membrane (Millipore) with NuPAGE Transfer Buffer (Invitrogen).For the The following antibodies were used for protein blots, immunofluorescence, and immunohistochemical analyses: rabbit anti-TDP-43 (Proteintech 10782-2-AP and 12892-1-AP), anti-NeuN monoclonal antibody (Chemicon), anti-GAPDH monoclonal antibody (Sigma), Alexa Fluor 488-conjugated Donkey anti-Guinea Pig IgG (H\u2009+\u2009L) antibody (Jackson ImmunoResearch), Alexa Fluor 594- and 647-conjugated Donkey anti-goat and anti-rabbit IgG (H\u2009+\u2009L) antibodies (Life Tech.).CamKIIa-Cre;TardbpF/F and littermate control mice using TRIzol (Life Tech.) and RNeasy Mini kits (Qiagen). Total RNA from 2\u00a0month old male MLC-Cre;TardbpF/F and littermate control mice was also extracted in a similar manner. For the CamKIIa-Cre line, 3 control brains and 3 knockout brains were analyzed and all mice were female. For the MLC-Cre line, 2 control quadriceps and 2 knockout quadriceps were analyzed and all mice were male. 100-bp paired end RNA-seq libraries were generated using Illumina Tru-seq kits and then sequenced on an Illumina HiSeq 2000. For RT-PCR analysis, total RNA was isolated using RNeasy Mini Kit (Qiagen). cDNA was synthetized using RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific) with random primers. RNA-seq analysis was performed using HISAT [Total RNA was extracted from hippocampi of 3\u00a0month old female ng HISAT and Cuffng HISAT softwareng HISAT . Crypticng HISAT . To idenng HISAT .Tardbp knockout alleles [CaMKII\u03b1-Cre [MLC-Cre [CaMKII\u03b1) drives expression primarily in the excitatory neurons of the cortex and hippocampus whereas the promoter of the myosin light chain 1/3 locus (MLC) drives expression in type II fast-twitch skeletal muscle fibers. Efficient deletion of Tdp-43 can be detected by immunoblot in brain .To identify the cryptic exons of mouse neurons, RNA-sequencing (RNA-seq) analysis was performed using RNA extracted from hippocampi of 3\u00a0month old deletion , we alsoice Fig.\u00a0. Neuron-ice Fig.\u00a0 and coulice Fig.\u00a0-e.Fig. MLC-Cre;TardbpF/F knockout mice and controls. Indeed, numerous muscle-specific cryptic exons could be identified . For example, the cryptic exon in Ube2d1 is highly incorporated in stem cells, moderately incorporated in myocytes, and absent in neurons do not induce nonsense mediated decay, but still have an impact on protein structure. These cryptic exons do not contain any stop codons and have sequence lengths that are multiples of three, thereby preventing detrimental frameshifts will be difficult to envision due to the sizeable number of nonconserved cryptic exons per cell. Furthermore, because nonconserved cryptic exons are different between mouse and human, testing splicing modulators for human cryptic exons in animal models is essentially impossible. However, the general splicing repression function of TDP-43 is conserved. Thus, it may be possible to use mouse models of TDP-43 deletion to specifically test therapeutic strategies that rescue TDP-43 mechanism of action rather than directly targeting individual cryptic exons. One strategy would employ gene therapy to introduce designer splicing factors\u2014chimeric proteins that would couple the UG binding domain of TDP-43 with non-aggregating splicing repressor domains \u2014into neuIf neuron loss or skeletal muscle degeneration can be attenuated, such a therapeutic strategy could be rapidly translated into the clinic. Moreover, the observation that cryptic exons can occasionally introduce inframe insertions into mRNA suggests that certain human TDP-43 cryptic exons could represent biomarkers for human disease. We envision the development of specific antibodies to detect neoantigens introduced by human inframe cryptic exons in CSF or blood from patients, serving as either diagnostic biomarkers or tools to monitor the efficacy of treatments in future clinical trials.This study demonstrates that Tdp-43 represses a unique set of cryptic exons, depending on cellular context. Thus, the pathways impacted by Tdp-43 loss-of-function and cryptic exon incorporation are likely distinct for each cell type. These results have important implications for human disease, given that Tdp-43 proteinopathy can manifest in various tissues."} +{"text": "Hepatocellular carcinoma (HCC) is a major cause of cancer-related death worldwide. As vectors for intercellular information exchange, the potential role of extracellular vesicles (EVs) in HCC formation, progression and therapy has been widely investigated. In this review, we explore the current status of the researches in this field. Altogether there is undeniable evidence that EVs play a crucial role in HCC development, metastasis. Moreover, EVs have shown great potential as drug delivery systems (DDSs) for the treatment of HCC. Exosomal miRNAs derived from HCC cells can enhance transformed cell growth in recipient cells by modulating the expression of transforming growth factor-\u03b2 activated kinase-1(TAK1) and downstream signaling molecules. Furthermore, vacuolar protein sortin 4 homolog A(VPS4A) and insulin-like growth factor(IGF)-1 regulate exosome-mediated miRNAs transfer. Immune cells- derived EVs containing integrin \u03b1M\u03b22 or CD147 may facilitate HCC metastasis. In addition, EVs-mediated shuttle of long non-coding RNAs (lncRNAs), specifically linc- VLDLR and linc-ROR promote chemoresistance of malignant cells. Heat shock proteins (HSPs)-harboring exosomes derived from HCC tumor cells increase the antitumor effect of natural killer (NK) cells, thus enhancing HCC immunotherapy. Indeed, inhibition of HCC tumor growth has been associated with tumor cell-derived exosomes (TEX)-pulsed dentritic cells (DCs). Exosomes are also essential in liver metastasis during colorectal carcinoma (CRC) and pancreatic ductal adenocarcinomas (PDAC). Therefore, as nucleic acid and drug delivery vehicles, EVs show a tremendous potential for effective treatment against HCC. Hepatocellular carcinoma (HCC) is the sixth most common incident cancer worldwide and the third leading cause of cancer death annually , 2. In CAlthough EVs - including exosomes and microvesicles (MVs) - were previously considered as cellular debris, they are currently well-recognized vectors for intercellular exchange of information . EVs medin vitro.HCC tumor cell-derived EVs have been reported to potentially contribute to local spread, intrahepatic metastases and multifocal growth of HCC . EVs-medAccumulating evidence indicate that EVs are involved in tumor progression, metastasis, and treatment failure, thus showing great potential as DDSs for the treatment of HCC. In the present article, current studies investigating the mechanism on the contribution of EVs to HCC development, progression, metastasis and treatment are reviewed.To rapidly and efficiently extract exosomes secreted by tumor cells, different methods were explored -34. Amonvia a ceramide dependent manner [The RNA expression profile of exosomes derived from human HCC cell lines Hep3B and PLC/PRF/5 was investigated . Interest manner , 35. Intt manner .HCC-derived exosomes mediated miRNA transfer is an important mechanism of environmental modulation of HCC growth and progress . While bet al. reported that enhanced expression of miR-429 in liver tissue - caused by hypomethylation may be used as a prognosis factor in HCC patients [+ -tumor initiating cells (T-ICs) could lead to shedding of miR-429-harboring exosomes, thus facilitating tumor formation. These exosomes could shuttle and relocate miR-429 into their surrounded target cells, targeting Rb binding protein 4 (RBBP4) expression and further promoting the transcriptional activity of E2F1, finally upregulating the expression of POU class 5 homeobox 1(POU5F1) [via the tricarboxylic acid cycle in recipient cells. Similarly, Takayuki and collaborators demonstrated that the most highly expressed lncRNA in HCC cell-derived EVs was TUC339. Suppression of TUC339 with short interfering RNA (siRNA) significantly reduced HCC cell proliferation and adhesion. Therefore, EVs-mediated transfer of lncRNA-TUC339 is a unique signaling mechanism to promote HCC growth and metastasis [Some exosomal miRNAs and long non-coding RNAs (lncRNAs) are involved in HCC progression and treatment failure. For the first time, Li patients . The enr(POU5F1) in recip(POU5F1) . The lev(POU5F1) potentia(POU5F1) . High exin vitro, Vps4A was identified as a HCC suppressor [via selectively packaging oncogenic miR-27b-3p and miR-92a-3p into exosomes and accumulating tumor-suppressive miR-193a-3p, miR-320a, and miR-132-3p in HCC cells. Moreover, they demonstrated that Vps4A decreased the recipient HCC cell response to exosomes via selective uptake of exosomal tumor-suppressive miR-122-5p, miR-33a-5p, miR-34a-5p, miR-193a-3p, miR-16-5p, and miR-29b-3p.Based on the evidence that vacuolar protein sorting 4 homolog A (Vps4A) is frequently down-regulated in human HCC tissue and that Vps4A represses the colony formation, migration, growth and invasion of HCC cells ppressor . Vps4A uppressor . In thisHowever, insulin-like growth factor-1 (IGF-1) is considered as a HCC promoter since it can override homeostasis and lead to tumor progression during the initial steps of HCC development . Expressin vitro and metastasis in vivo. Indeed, MPs mediate the acquisition of a metastatic phenotype by HCC cells via the effective relay of integrin \u03b1M\u03b22 (CD11b/CD18) from stimulated innate immune cells. These findings reveal that HCC tumor cells may usurp the phenotype of innate immune cells through MPs in order to metastasize. In patients with HCC, stromal and vascular invasions contribute to tumor progression. Kornek and colleagues indicated that MPs containing CD147 can be released by human T cells, stimulating the expression of matrix metalloproteinases (MMP) in fibroblasts and thus facilitating tumor invasion and metastasis [Immune cells such as immature myeloid cells, macrophages, and mast cells are considered the roots of metastasis of tumor cells , 48. In tastasis .in vitro study [The uptake of exosomes from invasive HCC cell lines can trigger the activation of phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) and mitogen-activated protein kinase (MAPK) signaling pathways, which resulting in increased secretion of active MMP, enhanced migratory and invasive abilities of non-motile immortalized hepatocytes . This maro study demonstrro study . Progresro study Table 1)in vitro HCC is a chemorefractory cancer and highly resistant to chemotherapy, limiting the effectiveness of anticancer agents . Thus th+ cells and colony growth partly due to the selective enrichment and high expression of lincRNA-ROR (linc-ROR) within exosomes, overall inducing increased resistance of HCC cells to chemotherapy [EV-mediated transfer of linc-VLDLR is involved in HCC tumor cell response to chemotherapy by modulating cell-cell communication in the tumor microenvironment . The essotherapy . ConsideHowever, MVs secreted by tumor-associated macrophages (TAMs) mediate effective HCC chemotherapy. In fact, propofol inhibits HCC cell invasiveness, viability and proliferation . Macrophin vitro [HCC is notoriously difficult to treat due to the unique immune tolerogenicity nature of the liver , howeverin vitro . HCC-resin vitro . Thus, Hin vitro . More imin vitro . Additioin vitro , 65.The chief cause of death in cancer patients is tumor metastasis and the in vitro results in enhanced ability of HepG2 cell migration via activation of MAPK and extracellular signal regulated kinases (ERK)1/2 in recipient cells [Several groups have demonstrated the pivotal role of exosomes in metastatic progression of colorectal carcinoma (CRC) . The incnt cells . Besidesnt cells and miR-nt cells ) might mnt cells indicateet al. clarified that PDACs-derived exosomes prime the liver for metastasis via pre-metastatic niche formation in the liver, in which highly expressed migration inhibitory factor (MIF) in PDAC-derived exosomes is very likely to be involved [The liver is also the most common organ in metastases of pancreatic ductal adenocarcinomas (PDACs) . Costa-Sinvolved , 73 , and proteins involved in the proteasome complex [O. viverrini EV uptake process by cholangiocytes have shown great potential in the treatment of infection-derived cholangiocarcinoma.A recent study found that EVs secreted by the liver fluke giocytes . Authors complex . Vaccinein vitro and in vivo, by exosomal delivery of selective proteins, mRNAs and miRNAs into HCC cells. In stem cell-based therapies, adipose- and bone marrow-derived MSCs are commonly used.Mesenchymal stem cells (MSCs) are multipotent cells with intrinsic properties of migration and tumor tropism. In recent years, a considerable number of studies have investigated the biological effect of MSCs on HCC tumor growth and progression -77. Mostet al. conducted the first animal study using adipose-derived mesenchymal stem cells (AMSC)-derived exosomes for the treatment of HCC. The authors evaluated the effects of AMSCs-derived exosomes on N1S1 rat HCC cells inoculation-induced ectopic hepatoma in vivo [+ NK T-cells and circulating protective NK T-suppressing HCC growth. Considering that several miRNAs are associated with potential antitumor activity, AMSC exosomes were used for these miRNAs delivery [in vitro and in vivo [Sheung-Fat in vivo . Interes in vivo . In theidelivery , 83. Sindelivery , miR-122 in vivo , indicatin vitro. Moreover, in vivo intra-tumor administration of BMSCs-derived MVs in tumors remarkably inhibited tumor growth [22 cells in vitro, as indicated by cell cycle arrest at G0/G1phase and significantly decreased PCNA protein expression in these cells. Previous studies [in vitro and suppress growth of HCC xenografts in SCID mice by delivering antitumor miRNAs , that down-regulate MDR1, MIF, ras-related protein 14(RAB14) and E2F-2 [Similarly, MVs derived from bone marrow MSCs (BMSCs) inhibit cell cycle progression and induce apoptosis in HepG2 cells r growth . The posr growth reported studies -88 foundnd E2F-2 .Altogether, these studies suggest that MVs derived from stem cells may inhibit HCC tumor growth and stimulate apoptosis in a variety of ways, including delivery of selected miRNAs, modulation of NK T-cell responses or antigen-specific T cellular immune responses. Regardless of the mechanisms, the complex antitumor capacity of MVs derived from MSCs or MSCs pulsed with homologous/autologous TEX in HCC treatment needs further investigation.in vivo [via AMSC exosomes with use of chemotherapeutic agents results in enhancement of cell apoptosis and cell cycle arrest at G0/G1, what represents a promising strategy for HCC chemotherapy. In addition, exosomes from AMSCs have also been tested as an effective vehicle to package and deliver therapeutic siRNA [Contrary to lincROR mediating TGF \u03b2-dependent chemoresistance, miR-122 promotes chemosensitivity of HCC cells. MiR-122 is a liver-specific anti-proliferative miRNA that can be transferred via exosomes between human hepatoma cells. The loss or down-regulation of miR-122 has been associated with HCC development and progression and is closely related to poor prognosis and metastasis of HCC , 90. Morin vivo . MSC is in vivo . AMSC-dein vivo . Deliveric siRNA and actiic siRNA . ImproveSimilarly, the ability of intestinal epithelial cells Caco-2-derived MVs has been investigated as a vehicle for transfer of miR-168a to HCC cells. Caco-2 transfected by miR-168a expression plasmid secrete MVs containing these plant miRNAs. Thus, co-culture of these MVs with HepG2 might trigger transfer of specific miR-168a to human hepatocytes resulting in a 100-fold increase of the miR-168a levels and significant decreased expression of the miR-168a target protein .HCC is highly resistant to chemotherapy. Sorafenib, 5-fluorouracil and doxorubicin are currently being used for systemic or locoregional therapies against HCC but exhibit limited efficacy. Therefore, the discovery of new therapeutic targets and the development of novel clinical approaches to enhance HCC chemosensitivity are urgently needed. Therefore, the use of exosomes/EVs as nucleic acid and drug delivery vehicles has gained considerable interest due to their excellent biodistribution and biocompatibility.Altogether, these data suggest that EVs play multiple roles in mediating progression, metastasis and thus can be used as a potential therapy for the treatment of HCC. Further investigations are needed to shed more light on the role of EVs in HCC development and its application in the clinic."} +{"text": "This interaction was mediated by the PDZ2 domain of NHERF1 and the carboxyl terminal PDZ binding motif of GPER. NHERF1 was demonstrated to facilitate GPER expression at post-transcriptional level and improve GPER protein stability by inhibiting the receptor degradation via ubiquitin-proteasome pathway in a GPER/NHERF1 interaction-dependent manner. In addition, GPER protein levels are positively associated with NHERF1 protein levels in a panel of estrogen receptor (ER)-positive breast cancer cells. Furthermore, analysis of clinical IBC data from The Cancer Genome Atlas (TCGA) showed no significant difference in GPER mRNA levels between ER-positive IBC and normal breast tissues. However, gene set enrichment analysis (GSEA) showed that GPER signaling is ultra-activated in ER-positive IBC when compared with normal and its activation is positively associated with NHERF1 mRNA levels. Taken together, our findings identify NHERF1 as a new binding partner for GPER and its overexpression promotes protein stability and activation of GPER in ER-positive IBC. Our data indicate that regulation of GPER stability by NHERF1 may contribute to GPER-mediated carcinogenesis in ER-positive IBC.G protein-coupled estrogen receptor (GPER) plays an important role in mediating the effects of estradiol. High levels of GPER have been implicated to associate with the malignant progress of invasive breast cancer (IBC). However, the mechanisms by which GPER protein levels were regulated remain unclear. In this study, PDZ protein Na GPERetc. . Estrogeetc. , calciumetc. , 7, PI3Ketc. and EGFRetc. . GPER haetc. .In vivo study from transgenic mice tumor model showed that deletion of GPER reduced the size of mammary tumors and lung metastasis, indicating that GPER is critical in breast tumor growth and distant metastasis [Recently, high protein levels of GPER have been reported to positively correlate with increased tumor size, distant metastasis and poor prognosis of breast cancer , 12. In tastasis . The dettastasis \u201316. Meantastasis . However+/H+ exchanger regulatory factor (NHERF1) is a PDZ protein with well reported roles in the regulation of stability of its binding partners [The stability of some GPCRs could be regulated by binding with PDZ domain containing proteins. Our previous study showed that interaction with postsynaptic density-95 protein (PSD-95) enhanced Mas protein level by increasing its stability . Anotherpartners , 25. NHEpartners \u201328. Our partners . InteresIn this study, we found that NHERF1 interacted with GPER via the PDZ2 domain of NHERF1 and the C-terminal of GPER. Further, NHERF1 enhanced the stability of GPER through inhibition of GPER ubiquitination. By analyzing clinical breast cancer data from TCGA, we also found a positive correlation between NHERF1 expression and GPER pathway activation in ER-positive breast cancer. These findings provide a new insight into the regulatory mechanism of the GPER protein by NHERF1 in breast cancer cells.To test the possibility of GPER and NHERF1 interaction, we first performed GST pull-down analysis. GST-NHERF1 fusion protein was used to pull down the lysates of HEK-293 cells stably transfected with Flag-GPER (HEK-GPER). As shown in Figure To verify this interaction in cells, HEK-293 cells were transfected with GFP-NHERF1 in the presence or absence of Flag-GPER. Immunoprecipitation (IP) of Flag-GPER was followed by Western blotting and a strong NHERF1 signal was found in Flag-GPER-IP complex Figure . These r375 residue of GPER, which is the main determinant for its binding to PSD-95 [NHERF1 possesses two non-identical tandem PDZ domains and a carboxyl-terminal (CT) ezrin-binding domain . To ideno PSD-95 , was mutTo further confirm GPER/NHERF1 interaction in intact cells, MCF-7 cells were double stained using anti-GPER and anti-NHERF1 antibodies. Our data showed strong co-localization of GPER with NHERF1 intracellularly by forming multiple spots in cytoplasm, especially areas surrounding the nucleus in MCF-7 cells Figure . To examWe next explored the potential functional significance of the GPER/NHERF1 interaction. In the immunoprecipitation studies, we noted that the expression level of GPER-wt was significantly higher than that of the GPER-V375A mutant when the same amount of plasmid was co-transfected with GFP-NHERF1. The expression of Flag-GPER-wt and Flag-GPER-V375A were adjusted to similar levels through transfection with different amounts of the respective constructs, which would allow comparison of NHERF1 association with the wild-type receptor or the mutant one Figure . In furtgper transcriptional regulation and the GPER/NHERF1 interaction is required for enhanced GPER stability by NHERF1.We next explored whether NHERF1 increases GPER level by regulating GPER mRNA expression. Our results showed that although GPER protein levels varied after NHERF1 knock-down or overexpression, the GPER mRNA levels were unchanged in both cases Figure . These dIntracellular protein degradation occurs through the lysosomal and/or the ubiquitin-proteasome degradation pathway. To explore which pathway is involved in GPER degradation, Western blotting analysis of HEK-GPER cell lysates was performed following treatment with either the lysosome inhibitor chloroquine or the proteasome inhibitor MG132. As shown in Figure To further verify that the regulation of NHERF1 on GPER stability is associated with the ubiquitin-proteasome pathway, we assessed the effects of NHERF1 on GPER protein ubiquitination in HEK-293 cells. As shown in Figure Based on the findings that GPER-NHERF1 interaction enhanced the stability of GPER protein, we further defined whether these findings have clinical relevance. Considering the findings by other group that GPER expression levels were different between ER-positive and ER-negative breast cancer , we firsGPER plays an essential role in the rapid responses of estrogen and high level of GPER has been reported to associate with cancer development, especially in IBC . The exp375 to Ala375 (V375A) in the C-terminal of GPER resulted in abolishment of this association . Lipofectamine 2000 Reagent was used for cell transfection. HEK-293 cells that stably express Flag-GPER or Flag-GPER-V375A were selected with the growth medium containing 1 mg/mL G418 and then maintained in growth medium containing 500 \u03bcg/mL G418 .Human embryonic kidney (HEK)-293 cells and breast carcinoma MCF-7 cells were grown in Dulbecco's modified eagle media (DMEM) supplemented with 10% fetal bovine serum (FBS). Human breast cancer cell lines T47D, ZR-75-1 and SK-BR-3 were maintained in RPMI-1640 with a supplement of 10% fetal bovine serum (FBS). BT474 cells were grown in RPMI-1640 supplemented with 10% FBS and 0.01 mg/ml insulin. All cells were cultured at 37\u00b0C in a humidified incubator with 5% COThe monoclonal rabbit anti-GPER (sc-48525-R) and anti-ubiquitin (#3933S) antibodies were purchased from Santa Cruz Biotechnology and Cell Signaling Technology respectively. The monoclonal mouse anti-NHERF1 IgG2b (MA1-19292) was purchased from Thermo Fisher , the monoclonal mouse anti-NHERF1 IgG (#611161) was purchased from BD . The polyclonal rabbit anti-HA (#561) and anti-proteasome 20s \u03b1/\u03b2 (ab22673) antibodies were purchased from MBL and Abcam respectively. Monoclonal mouse anti-Flag (F3165) antibody was purchased from Sigma-Aldrich .The constructs encoding Flag-GPER (EX-M0792-M12) were purchased from Gene Copoeia . The V375A mutation of Flag-GPER, the wild type HA-GPER and HA-GPER-V375A were amplified by PCR using indicated primers shown in Table Whole cell lysates or immunoprecipitated samples were resolved in 10% SDS-PAGE gels and transferred to PVDF membrane . After being blocked with 5% non-fat dried milk for 1 hour at room temperature, membranes were incubated in primary antibody overnight at 4\u00b0C. Horse radish peroxidase (HRP)-conjugated or infrared fluorescent dyes (IRDye)-conjugated secondary antibodies were used to detect the immunoreactivity by enhanced chemiluminescence (ECL) detection reagents and Odyssey infrared imaging system respectively.GST pull-down assay was performed as previously described . BrieflyCo-immunoprecipitation was performed as described previously . Flag-GPTotal RNA of HEK-293 that transfected with indicated Flag-GPER (HEK-GPER) was isolated using Trizol reagent according to the manufacturer's instruction. The GPER and NHERF1 mRNA levels were determined using an RT-PCR kit and primers listed in Table Immunofluorescence was performed as described previously . Cells oHEK-GPER or HEK-GPER-V375A cells were transiently transfected with GFP or GFP-NHERF1, scrambled sequence or siNHERF1 was added and treated for 4 hours. Then cell lysates were precipitated using anti-Flag affinity gel, followed by Western blotting analysis with anti-ubiquitin antibody.http://cancergenome.nih.gov/) was downloaded from Sage Synapse (www.synapse.org/).Gene expression profile of invasive breast carcinoma (unc.edu BRCA IlluminaHiSeq RNASeqV2.geneExp) from The Cancer Genome Atlas (TCGA) database (http://www.broad.mit.edu/gsea/) [< 0.05.The association between gene expression and biological processes was analyzed using Gene Set Enrichment Analysis . GSEA cap < 0.05.Statistical analyses were performed using the software GraphPad Prism 5. Results are expressed as mean \u00b1 SEM. One-way analysis of variance followed by Tukey's multiple comparison test was used to determine statistical significances. Statistical significance was accepted for"} +{"text": "To the Editor: Risk factors for primary acquisition of Middle Eastrespiratory syndrome (MERS) coronavirus (CoV) include recent direct contact withdromedary camels 1a gene reported direct camel contact , and 4 (24%) reported indirect camelcontact during the exposureperiod . ThreecViruses from 13 of the 27 case-patients were sequenced, and all belonged to the MERS-CoVrecombinant subclade NRC-2015 . Full geA novel nucleotide substitution was identified in the MERS-CoV sequence from 1case-patient atpositSince the emergence of MERS-CoV in 2012, virus acquisition has been associated withdirect exposure to camels (Characteristics of 27 MERS cases-patients, Saudi Arabia,January\u2013February 2016Phylogeny of MERS-CoV genome sequences, Saudi Arabia, January\u2013February2016."} +{"text": "Mitral valve abnormalities are an important cause of patient morbidity in hypertrophic cardiomyopathy (HCM) contributing to left ventricular outflow tract obstruction (LVOTO). Anterior mitral valve leaflet (AMVL) elongation predisposes to LVOTO. CMR detects this in overt HCM and in sarcomere gene mutation carriers without left ventricular hypertrophy (G+LVH-). However, the geometry of the mitral valve, the subvalvular apparatus and the LVOT is more complex. The purpose of this study was analyse the whole aorto-mitral area and track its dynamic motion to explore mechanisms involved in LVOTO-predisposition in subclinical HCM.1 - hinge point of the posterior MVL; ROI2 - intertrigonal mitral annulus; ROI3 - tip of the AMVL; ROI4 - anterior aortic annulus was performed on 35 G+LVH- patients without left atrial (LA) enlargement , 31 patients with a clinical diagnosis of HCM with preserved ejection fraction , and 53 matched healthy volunteers . Direct assessment of the aortomitral apparatus was performed on the 3-chamber view acquired using a breath-held steady-state free precession sequence. Cines were interrogated frame-by-frame, semi-automatically using an in-house script developed for MATLAB\u00ae. The motion tracking software interactively assigned, tracked and graphed, the dynamic excursion of 4 pre-defined aortomitral regions of interest (ROI) throughout one cardiac cycle: ROI1 (P = 0.005), ROI2 (P = 0.01) and ROI4 , ROI2 (P = 0.007), ROI3 (P < 0.0001) and ROI4 (P < 0.0001).Qualitative examination of normalized two-dimensional displacement-versus-time plots in G+LVH- patients revealed subtle systolic anterior motion (SAM) of the intertrigonal mitral annulus and reduced longitudinal excursion compared to controls. Statistically significant differences were present for ROIThe AMVL is longer in preclinical HCM, but in addition, there are more widespread abnormalities of the aorto-mitral apparatus motion, including SAM of the mitral annulus before the development of LVH or LA enlargement. These data have the potential to improve our understanding of early phenotype development and LVOTO-predisposition in HCM."} +{"text": "Tumor cells acquire metastasis-associated (MA) phenotypes following genetic alterations in them which cause deregulation of different signaling pathways. Earlier, we reported that an upregulation of the Wnt-beta-catenin pathway (WP) is one of the genetic salient features of triple-negative breast cancer (TNBC), and WP signaling is associated with metastasis in TNBC. Using cBioPortal, here we found that collective % of alteration(s) in WP genes, CTNNB1, APC and DVL1 among breast-invasive-carcinomas was 21% as compared to 56% in PAM50 Basal. To understand the functional relevance of WP in the biology of heterogeneous/metastasizing TNBC cells, we undertook this comprehensive study using 15 cell lines in which we examined the role of WP in the context of integrin-dependent MA-phenotypes. Directional movement of tumor cells was observed by confocal immunofluorescence microscopy and quantitative confocal-video-microscopy while matrigel-invasion was studied by MMP7-specific casein-zymography. WntC59, XAV939, sulindac sulfide and beta-catenin siRNA (1) inhibited fibronectin-directed migration, (2) decreased podia-parameters and motility-descriptors, (3) altered filamentous-actin, (4) decreased matrigel-invasion and (5) inhibited cell proliferation as well as 3D clonogenic growth. Sulindac sulfide and beta-catenin siRNA decreased beta-catenin/active-beta-catenin and MMP7. LWnt3ACM-stimulated proliferation, clonogenicity, fibronection-directed migration and matrigel-invasion were perturbed by WP-modulators, sulindac sulfide and GDC-0941. We studied a direct involvement of WP in metastasis by stimulating brain-metastasis-specific MDA-MB231BR cells to demonstrate that LWnt3ACM-stimulated proliferation, clonogenicity and migration were blocked following sulindac sulfide, GDC-0941 and beta-catenin knockdown. We present the first evidence showing a direct functional relationship between WP activation and integrin-dependent MA-phenotypes. By proving the functional relationship between WP activation and MA-phenotypes, our data mechanistically explains (1) why different components of WP are upregulated in TNBC, (2) how WP activation is associated with metastasis and (3) how integrin-dependent MA-phenotypes can be regulated by mitigating the WP. The triple negative (TN) subtype of breast cancers (BC) represents 15-20% of breast tumors (BT) which are more commonly diagnosed in younger African American women with the prevalence of BRCA1/2 mutations . TNBC suThe Wnt-beta-catenin pathway (WP) is a ligand-driven signaling pathway which regulates several cellular phenotypes in development and disease , 17. Actet al., regarding an association of WP signaling in poor-prognosis TNBC patients with metastasis [26 [26In viTNBC cells were plated on fibronectin and treated with sulindac sulfide under conditions similar to that of the migration experiments. Cells were processed for Phalloidin-555 staining for filamentous actin. Nuclei were counterstained with DAPI. Cells were photographed using a Zeiss LSM 510 Meta confocal microscope with a 63 x or 40 x Plan-Apochromat oil objective as mentioned earlier . LamelliDigitized bright-field time-lapse real-time images of the movement of MDA-MB231 cells (placed in live-cell imaging chambers) into the scratched area (for migration assay) were acquired with a Perkin Elmer Ultraview ERS disk-spinning confocal system, mounted on a Zeiss Axiovert 200M inverted microscope. To account for the axial focal changes of cells as they move, optical Z-sections were collected at 0.95 \u03bcm interval spacing & M2.Image analysis was carried out to measure the motility from the time-lapse images of the effect of sulindac sulfide on the fibronectin-directed migration of the TNBC cells in the scratch-wound assay on fibronectin. Nuclear tracking paths of migrating cells were shown as track overlays. The trajectory of the movement of cells is characterized by two quantitative motility descriptors . The position of the nucleus is taken as the point of \u201corigin\u201d. Time-lapse images are acquired with a Perkin Elmer Ultraview ERS confocal system. Bright-field images are acquired with a Hamamatsu Orca-ER camera (10x objective) at 10 minutes intervals.Enzymatic activity of the secreted MMP7 from the conditioned media was determined by zymography using Bio-Rad precast casein gels as described earlier .The cell survival assay was performed using cell Titer-GLO luminescent cell viability assay kit as per manufacturer's direction (Promega). The real-time proliferation of GFP positive MDA-MB231BR cells was performed using time-lapse imaging without labels . Using live cells we generated long-term growth and growth inhibition curves and monitored morphology. The time course of the percentage of the confluence of LWnt3ACM stimulated GFP positive MDA-MB231BR cells (Mean Vs time) was represented by four days following two doses of sulindac sulfide (25 \u03bcM and 50 \u03bcM).ex vivo. We tested the involvement of WP in 3D ON-TOP cultures of the laminin-rich extracellular matrix as many of the crucial micro-environmental cues are restored using this form of culture as previously described [Appropriate three-dimensional (3D) culture provides a more physiologically relevant approach to the analysis of gene function and cell phenotype escribed .Wnt3A gene encodes a secreted glycoprotein. The cells are engineered to secrete biologically active Wnt3A protein. We used these cells as our source for production of Wnt3A conditioned medium (Wnt3ACM) (as described by the ATCC). L Cell (ATCC\u00ae CRL2648\u2122) was used as the parental line for the LWnt3A cell line.LWnt3A conditioned medium was obtained from the LWnt-3A (ATCC\u00ae CRL-2647\u2122) cells. These L-M(TK-) cells were transfected with a Wnt-3A expression vector and stable clones were selected in medium containing G418. The The transient transfection of beta-catenin siRNA was performed as reported before . GFP-posEach experiment was independently repeated 4-5 times. The Student t-test was used to evaluate differences observed between treated groups and vehicle treated controls for other experiments. Data presented in the graphs represent the Mean \u00b1 S.D. of results. The minimum level of statistical significance was set at p<0.05. Inter-group comparison was made with a paired two-sample (Student t-test). Phenotypic experiments are performed in quadruplets."} +{"text": "The acquired resistance, however, will also occur and limit their response. Understanding the mechanisms of resistance to irreversible EGFR-TKIs plays an important role in the choice of subsequent treatment. In this review, we show the currently known mechanisms of resistance which can be summarized as EGFR dependent and independent mechanisms and potential therapeutic strategies to irreversible EGFR-TKIs.Epidermal growth factor receptor (EGFR) T790M mutation is the most frequent mechanism which accounts for about 60% of acquired resistance to first-generation EGFR tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC) patients harboring EGFR activating mutations. Irreversible EGFR-TKIs which include the second-generation and third-generation EGFR-TKIs are developed to overcome T790M mediated resistance. The second-generation EGFR-TKIs inhibit the wide type (WT) EGFR combined with dose-limiting toxicity which limits its application in clinics, while the development of third-generation EGFR-TKIs brings inspiring efficacy either The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) significantly improve the outcomes as an initial treatment in non-small cell lung cancer (NSCLC) patients with activating EGFR mutations compared with standard platinum-doublet chemotherapy. However, acquired resistance will inevitably occur after initial treatment of EGFR-TKIs. It is reported that EGFR T790M mutation is the most frequent mechanism of acquired resistance, which accounts for about 60% and may be more common in patients with mutant EGFR L858R than EGFR 19del . The poiSecond-generation EGFR-TKIs is a quinazoline-based irreversible pan- human epidermal growth factor receptor (HER/ERBB) inhibitor. It has electrophilic functionality, covalently binding to cysteine residue of EGFR (afatinib to Cysteine-773 and dacomitinib to Cysteine-797) , which ain vitro or in vivo. The acquired resistance, however, will still emerge and limit their response. Therefore, understanding the mechanisms of acquired resistance to irreversible EGFR-TKIs plays an important role in guiding subsequent treatment. We review the currently known mechanisms of resistance and potential therapeutic strategies to irreversible EGFR-TKIs.The third-generation EGFR-TKIs, AZD9291, CO-1686, WZ4002, EGF816, and ASP8273 are irreversible small-molecule inhibitors being tested in clinical trials. Like other irreversible EGFR-TKIs, they irreversibly react with the cysteine-797 residue in ATP bonding pocket. But they are more specific in targeting EGFR T790M and EGFR activating mutation than WT EGFR, because the anilinopyrimidine scaffold can be better adapted to the conformation after T790M mutation and the binding force to T790M mutant is 100\u2013200 times than WT EGFR. Thus they have greater clinical efficacy than second-generation EGFR-TKIs to inhibit EGFR T790M mutation , 9. The EGFR-dependent resistant mechanism is usually caused by secondary mutation in ATP-binding pocket of EGFR. The mutation can change the conformation of EGFR and thus the EGFR-TKI can no longer bind to EGFR. In EGFR-dependent resistant cells, the activated EGFR signaling is still playing the major role in promoting survival through its downstream signaling . Therefore, EGFR-targeted therapies should be potential approaches to this type of resistance. Figure , Table 1In vitro, the second-generation EGFR-TKIs can effectively inhibit EGFR T790M, thus researchers speculated whether these irreversible EGFR-TKIs could be superior to first-generation EGFR-TKIs in preventing or delaying the emergence of the resistance as a first-line therapy. In resistant cell lines derived from PC-9 cells (EGFR exon 19del) at clinically relevant concentration of neratinib (HKI-272), an EGFR/pan-HER inhibitor, EGFR T790M gatekeeper mutation was first revealed as an acquired resistance mechanism of NSCLC to second-generation EGFR-TKIs [GFR-TKIs . High doGFR-TKIs . DacomitGFR-TKIs , 12, 13.GFR-TKIs , 15. AndGFR-TKIs , 17. Thep = 0.017) [p < 0.0001) [T790M mutation was confirmed to be the resistance mechanism of second-generation EGFR-TKIs in tissue specimen. It is still the most important mechanism accounting for 30\u201350% of the resistance both in reversible EGFR-TKIs naive patients and treated patients according to the rencent studies. , 19. Ta But the = 0.017) . And the 0.0001) . These dPatients with acquired resistance of T790M mutation to first/second-generation EGFR-TKIs exhibited good efficacy to third-generation TKIs in clinical trials , 22. TheAccording to a recent study, atypical mutations occur in 14% of NSCLC patients haboring EGFR-TKIs sensitizing mutation, the majority of which have nothing to do with drug sensitivity . The atyCys-797 site in ATP binding pocket of EGFR is the one where irreversible EGFR-TKIs covalently bound to. Thus the point mutation of Cys797Ser located in exon 20 of EGFR results in acquired resistance to third-generation EGFR-TKIs. In cell lines and xenograft model with EGFR activating mutation (exon 19 deletion or L858R mutation) alone or EGFR activating mutation/T790M, the emerging of C797S caused drug resistant to AZD9291, CO-1686 and WZ4002 \u201334. C797The emerging data shows the allocation pattern of T790M and C797S mutation may help the choice of treatment after acquire resistance based on C797S. In resistant cell lines, the double mutants (EGFR activating mutation/ C797S) were still sensitive to quinazoline-based EGFR-TKIs such as gefitinib and afatinib, while the cell lines harboring triple mutation (EGFR activating mutation/ T790M/ C797S) were resistant to all quinazoline-based EGFR inhibitors . The resEGFR L718Q and L844V are another two drug resistance mutations which disrupt the covalent bond of inhibitor onto C797 , 38. UnlIn CO-1686 resistant PC-9 cell lines, Nukaga et al. found EGFR WT allele was amplified. When expressed the WT EGFR in H1975 cell lines, they became resistant to CO-1686 or AZD9291 through activation of WT EGFR and downstream pathways induced by the ligand of EGFR . The comWhen EGFR independent resistance occurs, EGFR phosphorylation still can be shut down by inhibitors, but the resistant cells acquire an alternative way for survival and proliferation. EGFR-independent mechanisms of irreversible EGFR-TKIs mainly include the activation of other parallel signaling, the aberrant downstream pathways, and phenotypic transformation. The combined therapy targeting these resistance mechanisms with EGFR-TKIs may be potential treatment strategies. Figure The activation of alternative receptor tyrosine kinases (RTKs) signaling reactivates mitogen-activated protein kinases (MAPK) and phosphatidylinositol-3-kinase (PI3K)/ protein kinase B (AKT) downstream pathways regardless of the inhibited EGFR. Consequently, the dependence of these resistant cells on EGFR for survival and proliferation is weakened or disappeared. The interactive cross-talk between EGFR and other RTKs such as c-Met, HER2, and IGF1R may be an underlying event mediating EGFR-TKIs resistance based on this kinase switch , 51.Human mesenchymal-epithelial transition factor (c-Met) belongs to RTK superfamily whose ligand is hepatocyte growth factor (HGF) . C-Met aMet signaling provides an alternative route bypassing EGFR which can limit the activity of irreversible EGFR-TKIs in EGFR-mutant NSCLC. In-vitro studies showed that c-Met inhibitor (SU11274 or crizotinib) could improve the sensitivity of H1975 (EGFR L858R/ T790M) and HCC827ER (EGFR exon 19 del/c-Met amplification) cell lines to irreversible EGFR-TKIs (afatinib and WZ4002) even when HGF was present , 60. WheHGF, the ligand of c-Met, can also induce resistance to irreversible EGFR-TKIs. When HCC827 and PC-9 (EGFR exon 19 deletions) or H1975 (EGFR L858R/T790M) cells were co-cultured with MRC-5 cells (human embryonic lung-derived fibroblast cell line that secreted HGF), the cell lines became resistant to irreversible EGFR-TKIs mediated by high levels of HGF in a paracrine manner , 63. In Met kinase inhibitor plus EGFR-TKIs is a potential therapy to overcome TKIs resistance induced by the abnormity of Met signaling. In preclinical studies, SGX-523 (a selective c-Met inhibitor) , 68, criInsulin-like growth factor (IGF) signaling consists of its ligands , receptors and IGF binding proteins (IGFBPs) . IGF1 revivo study. But only the combination therapy of IGF1R and EGFR inhibition is demonstrated to be effective in preclinical studies, which perhaps because of the disruption of the existing crosstalk between EGFR and IGF1R or other RTKs [IGF inhibitors, such as BMS536924, recombinant IGFBP3 , BI83684her RTKs . An ongoHER2 is a member of ERBB family which lack its specific ligand so that it only can form heterodimers with other ERBB receptors . EGFR isin vitro and vivo studies [The second-generation EGFR-TKIs such as afatinib, dacomitinib, and neratinib were proved to be potent inhibitors in blocking HER2 signaling studies , 91, 92. studies . It was studies . But in studies . Besides studies , 96. But studies . Further studies . HER2 amGrowth arrest specific 6 (Gas6)/ anexelekto (AXL) activation is a kinase switch resistant mechanism of EGFR-TKIs. This resistance is often caused by increased expression of Gas6/AXL at RNA or protein level, while lack the genetic alteration, such as AXL mutation or AXL amplification , 48, 99.in vitro. In afatinib resistant PC-9 cells and AZD9291 resistant HCC4006 cells, FGFR1 was activated by its ligand FGF2 through autocrine fasion and the resistant cell lines were sensitive to FGFR inhibitors, such as PD173074, axitinib and BGJ398 [Fibroblast growth factor receptor 1 (FGFR1) activation mediated acquired resistance to afatinib and AZD9291 d BGJ398 , 100. Ind BGJ398 , 49, 101In an in-vitro study, activation of Interleukin 6 receptor (IL-6R)/ Janus kinase 1 (JAK1)/ Signal transducer and activator of transcription 3 (STAT3) mediated de novo resistance to afatinib and dacomitinib through IL-6 autocrine or paracrine loop in T790M mutant cell lines. Increased sensitivity to afatinib was observed when blocking IL-6R/JAK1/STAT3 signaling .The phosphorylation of Src family kinases (SFK) and focal adhesion kinase (FAK) were increased after AZD9291 treatment and SFK inhibitor and FAK inhibitor (PF573228) could improve the proliferation inhibition of AZD9291. Besides, amplification of YES1, a SFK family member, contributed to acquired resistance to AZD9291 in T790M mutant cell lines . RecentlIn vitro, Amato et al. found that erlotinib resistant PC-9 and HCC827 cell lines depended on EPH receptor A2 (EPHA2) signaling for survival. The overexpression of EPHA2, a kind of transmembrane glycoprotein with RTK activity, mediated acquired resistance to erlotinib and EPHA2 inhibitor could inhibit cell viability in erlotinib resistant cell lines. They also found that blocking EPHA2 signaling by ALW-II-41-27 could inhibit the survival of AZD9291 resistant cells, indicating that EPHA2 played a role in AZD9291 resistance [sistance .MAPK and PI3K/AKT are two important downstream pathways of EGFR. MAPK pathway is mainly correlated with cell proliferation in tumor, while the AKT signaling is mainly involved in anti-apoptotic regulation . The altRAS-RAF-MEK1/2-ERK1/2 signaling is one of the most characteristic pathways in MAPK pathways . RAS amplification and the decreased of negative regulators in MAPK signaling such as dual specific phosphatase 6 (DUSP6) led to WZ4002 resistance , 126. CRThe combination of irreversible EGFR-TKIs with MEK inhibitor (selumetinib or trametinib) could overcome or delay the emergence of acquired resistance based on the activation of MAPK according to the in-vitro study . An in-vPI3K, AKT and PTEN are the important components in PI3K-AKT pathway. The activated PI3K can catalyse phosphorylation of PIP2 into PIP3, while phosphatase and tensin homologue deleted on chromosome ten (PTEN) plays a negative role in this process . PIP3 biPIK3CA encode p110\u03b1 subunit which has catalytic activity in PI3K, thus the alteration of PIK3CA including mutation, amplification and over-expression would lead to activation of PI3K-AKT pathway , 132. Whin vitro study. The decreased PTEN expression was detected in 1 out of 10 tumor samples from NSCLC patients with progression disease after gefitinib treatment [PTEN deletion and decrreatment . While Preatment .There are three AKT isoforms which express in the majority of tissues\u2014AKT1, AKT2, AKT3. A recent preclinical study showed that the expression of AKT3 and AKT2 were up-regulated in CO-1686 resistant H1975 cell lines compared with parental cell lines, while no increase in AKT1 [EMT is a biological process that cells with epithelial phenotype are converted into the mesenchymal phenotype and acquire the ability of migration . The morAs an acquired resistance mechanism of EGFR-TKIs, EMT was firstly revealed in a study from an analysis of tumor biopsies of 37 NSCLC patients who developed resistance to EGFR-TKIs. It was an independent mechanism having no overlap with others. In order to figure out how EMT occurs and leads to drug resistance, some vitro studies are carried out. Transforming Growth Factor (TGF)-\u03b2 and other contextual signals such as Wnt and Notch signal which epithelial cells receive from their microenvironment may induce and maintain this phenotype change , 148. ThEMT has been proved to be a mechanism of acquired resistant to irreversible EGFR-TKIs. In the afatinib-resistant/afatinib-crizotinib-resistant HCC827 cell lines (AR/ACR) and afatinib-resistant HCC4006 cell lines, EMT was observed. In addition to the downregulation of E-cadherin and upregulation of vimentin, micro RNA-200c, a negative regulator of zinc finger E-Box binding homeobox 1 (ZEB1), was downregulated and modified in these resistant cell lines . The majThe EMT may not be a pan-resistance character; instead, these resistant cells exhibit sensitivity to other agents. Researches showed that the cells with mesenchyma phenotype were resistant to common cytotoxic chemotherapies, such as docetaxel , pemetreThe small cell lung cancer (SCLC) transformation is observed in ~14% of the re-biopsy tumors from patients after resistance to first-generation EGFR-TKIs , 154. ThIn a cohort of T790M mutant patients who had developed resistance to CO-1686, about 17% (2/12) patients underwent SCLC transformation. Both of them retained EGFR activating mutation and lost RB . SCLC trT790M loss may be a common acquired resistant mechanism of third-generation TKIs, rencent studies showed that about half of T790M-mutant patients developed resistance to third-generation TKIs due to T790M loss , 49, 101Bcl-2-like protein 11 (BIM) is a family member of Bcl-2 protein which promotes apoptosis. A 2903-bp intronic deletion of BIM gene lead to the splice of exon 3 and exon 4, thus BIM isoforms without pro-apoptotic effect are expressed. It was reported BIM deletion polymorphism was detected in 12% of East Asians and associated with primary resistance to first-generation EGFR-TKIs. A recent study revealed that BIM deletion polymorphism could also cause apoptosis resistance to afatinib and AZD9291 in EGFR-mutant cell lines, the combined treatment with histone deacetylase 3 (HDAC3) inhibitor could cope with this resistance by disrupt alternative mRNA splicing of exon 3 to exon 4 .The decrease of EGFR expression and lossAlthough great effort has made to discover the resistant mechanism of TKIs, there are still patients with unknown resistant mechanism. Evidence showed that 18\u201330% of the patients who progressed on first-generation EGFR-TKIs lacked clear resistant mechanism , 161. Anp = 0.27) and OS of placebo group was longer than gefitinib group though the data were immature, which suggested the standard second-line treatment was chemotherapy after progressed on first-line EGFR-TKIs. Both of the two studies do not consider the resistant mechanisms of patients and thus may give some implications for treatment of patients with unknown resistant mechanisms. The complicated clinical failure mode, however, should be taken into consideration for further investigations. And in fact, the previous treatment, performance status, willingness for treatment of patients, etc. may be more complicated after irreversible TKIs progression, which are also of importance in therapeutic decision-making.The ideal situation is that we choose the appropriate target-treatment based on acquired resistant mechanisms, but in most practical cases, clinical failure mode are the main basis of treatment, especially for those without clear resistant mechanisms , 166. ThNew strategies of EGFR-TKIs in combination with other novel agents regardless of resistant mechanism are currently ongoing to delay or decrease the appearance of resistances. Table .The irreversible EGFR-TKIs have their own characteristics on inhibition of EGFR signaling when compared with first-generation EGFR-TKIs. The second-generation EGFR-TKIs have broader inhibitory profiles, while the third-generation EGFR-TKIs are more specific to T790M mutation, so the resistant mechanisms have their own features. Some of new resistance mechanisms are introduced, such as C797S/G mutation, T790M loss. And some of mechanisms are absent when compared to reversible EGFR-TKIs, for example HER2 amplification was not reported as resistance mechanism of second-generation EGFR-TKIs. But in short, figuring out whether EGFR-dependent resistance or independent resistance can help the clinician to determine the next therapy. Besides, available data also suggest that > 13% of T790M mutant patients have multiple resistance mechanisms after progression on AZD9291 and CO-1686, so combination and multitargeted therapeutics may be promising strategies to overcome acquired resistance. For the moment, the majority of studies about resistance mechanisms of irreversible EGFR-TKIs are in-vitro, therefore large studies analyzing the biopsy samples of patients are expected."} +{"text": "Flow-Independent Dark-blood DeLayed Enhancement technique (FIDDLE) that allows visualization of tissue contrast-enhancement while suppressing blood-pool signal. We validate FIDDLE in an animal model of myocardial infarction (MI) and demonstrate feasibility in patients.A fundamental component of the CMR exam is contrast enhanced imaging, which is crucial for delineating diseased from normal tissue. Unfortunately, diseased tissue adjacent to vasculature often remains hidden since there is poor contrast between hyperenhanced tissue and bright blood-pool. Conventional black-blood double-IR methods are not a solution; these were not designed to function after contrast administration since they rely on the long native T1 of blood (~2s at 3T) and adequate blood flow within this time period. We introduce a novel Z < tissue MZ. CMR was performed acutely or chronically at 3T. FIDDLE and delayed-enhancement CMR (DE-CMR) were acquired using matched settings ~15 minutes after contrast (0.2 mmol/kg). We enrolled patients with enyzmatically confirmed MI and identifiable infarct-related-artery by X-ray angiography, as well as controls with Framingham Risk Score = 0. The patient CMR protocol was similar to that in canines. FIDDLE & DE-CMR analysis were performed separately and masked to identity and pathology (canines) or angiography results (patients).A canine model with variable coronary occlusion times was employed to create a range of MI size/transmurality. Following CMR, hearts were stained with TTC to provide a histopathology reference standard. The main components of FIDDLE are (1) a prep pulse that differentially saturates tissue compared with blood (eg. MT-prep); (2) phase-sensitive IR; and (3) inversion time selection under condition: blood MIn all canines (n = 22) and patients , black-blood images were successfully acquired using FIDDLE Fig . Slow-flWe demonstrate that FIDDLE is more sensitive and accurate than standard DE-CMR for the diagnosis of MI. Although validation and feasibility is demonstrated for diagnosis of MI, the technique is easily transferable beyond cardiac imaging and additional applications are expected in other settings where there is need to distinguish abnormal tissue enhancement from blood-pool."} +{"text": "Delivery systems for nanoparticle PrEP facilitate administration of nano-encapsulated ARVs to high-risk tissues. In this mini-review, we summarize the comparative nanoparticle and drug solution studies and the potential of two delivery methods: thermosensitive gels and polymeric nanoparticle films for direct prophylactic applications.HIV continues to be one of the greatest challenges facing the global health community. More than 36 million people currently live with HIV and, in 2015 2.1 million new infections were reported globally. Pre-Exposure Prophylaxis (PrEP) prevents HIV infection by inhibiting viral entry, replication, or integration at the primary site of pathogenic contraction. Failures of large antiretroviral drug (ARV) PrEP clinical trials indicate the current insufficiencies of PrEP for women in high-risk areas, such as sub-Saharan Africa. A combination of social, adherence, and drug barriers create these insufficiencies and limit the efficacy of ARV. Nanotechnology offers the promise of extended drug release and enhances bioavailability of ARVs when encapsulated in polymeric nano-particles. Nanoparticle encapsulation has been evaluated As a result, between 2000 \u2013 2015, HIV infection rates fell 35%, and Acquired Immune Deficiency Syndrome (AIDS)-related deaths fell 27%. Despite these recent successes, HIV infection and AIDS continue to be a challenging healthcare problem in the 21st century. According to a report by the American Foundation for AIDS Research, over 36 million people worldwide continue to live with HIV-1 and 2.1 million new HIV infections were reported in 2015,2. Of those newly infected individuals, 47% were women and 8% were children less than 15 years old. Young people between the ages of 15 and 24 accounted for 35% of all new adult infections, with infection rates of young women in this age group accounting for 20% of the global sum of HIV infections,2 In sub-Saharan Africa, 15 \u2013 24 year old females are eight times more likely to be infected with HIV than their male counterparts. Greater than 80% of HIV infections are contracted through sexual transmission and 86% of female transmission has been attributed to heterosexual intercourse,4. Factors such as mode of viral transmission, female physiology, and social, economic, and legal disadvantages contribute to increased rates of HIV infection in women. HIV/AIDS remains the leading cause of death for pre-menopausal women worldwide. Given international efforts to reduce the annual global HIV infection rates by 90% by 2030, highly efficacious therapeutic and preventative HIV therapeutic options must be available to at-risk populations, particularly women.Over the last fifteen years, international initiatives have designed more potent, new antiretroviral drugs(ARV) to reduce HIV infection and HIV-related deaths. ART has been shown to significantly decrease the incidence of HIV transmission among serodiscordant couples when therapies are followed consistently by patient populations. However, current oral therapies for (PrEP) are costly and are often in limited supply in developing countries and/or to at-risk populations. Pre-Exposure Prophylaxis (PrEP) holds the promise of eliminating new infections and thereby the associated risks of HIV infection. Consequently, there is a need for the development of innovative, cost-effective, and highly efficacious PrEP. Nanotechnology has garnered considerable interest in the field of HIV PrEP because of its potential to extend the release, target and increase cellular uptake, and improve the chemical, enzymatic and metabolic stability of therapeutic drugs,10. Various types of nanocarriers such as dendrimers, liposomes, polymeric nanoparticles and nanosuspensions are being evaluated for PrEP. Vaccines are another promising area of innovative PrEP research and development but are beyond the focus of this review. This mini-review article presents the history of and the latest development in those nanofabrications showing promise for female PrEP with specific emphasis on nanoparticle fabrications involving antiretroviral drugs (ARVs).Post-infection HIV treatment using daily, highly active oral delivery of combination antiretroviral drug (ARV) therapies has significantly reduced HIV infection rates when such oral therapies are readily accessible and are reviewed elsewhere\u201314. As concerns over drug-resistance waned, and awareness that women need access to cost-effective HIV prevention strategies increased, investigators focused on ARV-mediated prevention. ARVs acting before integration of viral genomic material into the host cells became the strongest candidates for preventative treatments,11. Initially, two classes of reverse transcriptase inhibitors, nucleoside reverse transcriptase inhibitors (NRTIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs), were the focus of PrEP efforts/strategies. Two nucleoside reverse transcriptase inhibitors, tenofovir disoproxil fumarate (TDF) and emtricitabine (FTC) were delivered orally for PrEP. Daily oral regimen of TDF demonstrated a 48.9% reduction in HIV infection among injection-drug users. Daily oral regimen of TDF/FTC (Truvada) showed similar reduction in the incidence of HIV infection (44%) in men who have sex with other men,21. The high efficacy of Truvada (44 \u2013 75%) seen in clinical trials led to Truvada\u2019s approval for PrEP in both men and women by the US Food and Drug Administration in 2012,20,22\u201324. Importantly, these clinical trials established a correlation between plasma drug levels and prophylactic capacity of ARV PrEP. Seroconversions are frequently associated with low plasma drug concentrations in treatment groups suggesting that maintenance of plasma drug levels is important for PrEP efficacy. Low plasma drug levels associated with a lack of adherence and observed in the FEM-PrEP and VOICE clinical trials where adherence was below 40% resulted in reduced efficacy of TDF PrEP.For a decade, it has been recognized that new infections of HIV must be reduced and that effective PrEP will be required to reach the worldwide goals for reducing the number of HIV infected individuals. The objective is to design PrEP that will block HIV at the mucosal membrane without causing tissue irritation or carrying the risk of developing resistance to ARVs. In order to eliminate the possibility of resistance, initial PrEP design involved the use of vaginal microbicides such as detergents, polyanionic inhibitors, or pH buffers that did not contain ARVs. Macromolecular entry inhibitors were largely unable to block HIV infection\u201318. The focus of the well-known VOICE trial was to assess the effectiveness of daily treatment with vaginal TDF gel and oral TDF and oral TDF/FTC (Truvada) in preventing sexually transmitted HIV-1 infection in women. Results from VOICE reinforce the importance of adherence to PrEP regimens. No significant difference was seen between drug and placebo treatments either vaginally or orally. Drug levels detected in the blood were low or absent for the majority of participants unless they were older than 25, married, or their sexual partner was older than 28. CAPRISA 004 clinical trial investigated the efficacy of 1% TDF gel for PrEP revealing similar adherence-mediated effects. In this case, drug delivery specified for pre- and postcoital gel applications found the gel to be 39% effective in preventing HIV infection and concluded an overall efficacy of 54% in cohort with greater than 80% adherence. A subsequent study carried out over 2.5 years in 9 locations across South Africa demonstrated that adherence of high-risk female populations (> 70%) exhibited enhanced PrEP for HIV, but only 20% of the overall sample size was in this cohort. More recently, three double-blind placebo controlled randomized trials demonstrate that daily oral TDF-based PrEP is quite successful given adherence and detectable TDF-blood levels. These three studies found that daily oral PrEP reduced the risk of HIV infection in women. The Partners PrEP study included 1785 Kenyan and Ugandan women with HIV-infected partners. PrEP efficacy was 66% and 71%. In the TDF2 study conducted in Botswana among heterosexual men and women, efficacy was 49% with a small sample size of 557 women. A tenofovir study in Bangkok (BTS) showed that PrEP reduced the risk of HIV infection in women by 79%. All five studies demonstrate that acquisition of HIV occurs during periods of low or no adherence to PrEP.TDF was also formulated into a vaginal gel and evaluated for pharmacokinetic, safety, and antiviral efficacyTaken together, these studies demonstrate that participant adherence directly influences PrEP efficacy. Surmounting the adherence barrier necessitates the development of cost-effective, easily used, and long-lasting PrEP fabrications.,30. Nanoparticle sustained drug delivery is likely to reduce the required frequency of drug application for proper efficacy. Reducing dosing complexities and frequency are likely to increase treatment adherence and effectiveness while decreasing cost and high dosage toxicity. Current developments of ARV-encapsulated NP treatments for PrEP typically utilized poly(lactic-co-glycolic acid) (PLGA) based prophylactic modalities. Other polymers such as Cellulose Acetate Phalate (CAP) and Polycaprolactone (PCL) are being explored. Specific polymers, such as CAP, have anti-microbicidal function and may serve not only as a nanoparticle polymer but also enhance PrEP efficacy.Polymeric nanoparticles for ARV drug delivery can encapsulate various drug formulations for selective and enhanced drug delivery. Polymeric NP pharmacokinetic and material development are reviewed elsewhere,3. Cellular in vitro assays examined the efficacy of ARV in solution verses encapsulation of ARV in PLGA-NPs. In many studies, encapsulation of ARV in PLGA-NP has shown increased prophylactic efficacy of PLGA-ARV-NP as compared to ARV in solution alone. Mandal et al., 2016 encapsulated FTC in PLGA-NPs via a water-in-oil-in-water emulsion method assays demonstrated that PLGA-FTC-NPs significantly reduced FTC IC50 levels against HIV as compared with FTC solution,32. PLGA-FTC-NPs protected PBMCs for up to 21 days post-HIV exposure.PLGA is a common nanoparticle polymer. Studies suggest that PLGA-NPs undergo endosomal uptake allowing for delivery of encapsulated drug directly to cellular cytoplasm and thus enhancing ARV drug uptake into the celln method . In vitret al., 2013 examined the encapsulation of low-solubility ARV drugs. Efavirenz (EFV), a non-nucleoside reverse transcriptase inhibitor (NNRTI), and saquinavir (SQV), a protease inhibitor, were encapsulated in PLGA-NP. PLGA-EFV-NP were loaded using single solvent emulsion evaporation fabrication,34. PLGA-SQV-NP were formulated by nanoprecipitation. Individually, PLGA-EFV-NP and PLGA-SQV-NP showed increased efficacy over their respective free-drug solutions in TZM-bl indicator cell assays following 24hr pretreatment. PLGA-EFV-NP and PLGA-SQV-NP showed significantly reduced ARV IC50 levels against HIV as compared to drug in solution and RAL/ETR-NP (IC50: 0.40nM) paired treatments compared to the ETR solution (ETR-Sol) combined with MVC-Sol (IC50:3.02nM) and RAL-Sol (IC50: 4.21nM). ETR-NP combinations were also three times more efficacious in blocking cell-cell HIV transmission. Drug synergy was only observed when ETR was paired with RAL or MVC and encapsulated into a polymeric nano-formulation. Interestingly, triple NP treatments did not show any increased potency over the double drug combinations (IC50:0.40nM). However, triple combination NP treatments were associated with higher intracellular concentrations than free-drug triple combination as measured by LC-MS/MS. Triple combination NP treatments were also protective against RT-SHIV challenges in macaque cervico-vaginal explants tissue. Differential encapsulation efficiencies for RAL were observed in fabrication of combination PLGA-EFV-RAL NPs. Oil-in-water emulsion with PLGA: Pluronic 127 at 1:2 w/w ratio resulted in EFV entrapment efficiency of 55.8% and RAL at 98.2% , the NNRTI etravirine (ETR), and the ISTI raltegravir (RAL) into PLGA-NPs were developed . Drugs w . Single-at 98.2% [38]. Desl assays [38].. Encapsulation efficiency of EFV, LPV/r were 81.0, 79.8, and 79.5%, respectively . EFV and LPV/r delivered by PLGA-NPs remained in different cellular compartments for as long as seven days in a HIV-1NL4-3 challenged human T cell line as determined by sub cellular fractionation and HPLC analysis. Only ritonavir solution was found at detectable levels in cells at seven days while combination NPs showed measurable drug levels in membrane, nuclear, and cytoskeletal fractions indicating sustained release of drug through NP delivery,42.Multiple drug encapsulations of efavirenz (EFV) boosted by two protease inhibitors lopinavir/ritonavir (LPV/r) in PLGA-NPs using an emulsion-solvent-evaporation method with a high-pressure homogenization component to increase encapsulation efficiency were designedectively . \u201345. Addition of Poly-Ethylene Oxide (PEO), Sodium Lauryl Sulfate (SLS), and Cetyl-Trimethyl Ammonium Bromide (CTAB) were surface modifications compared in PCL-NP fabrications. Each fabrication encapsulated dapivirine (DAP), a NNRTI, using a modified solvent displacement method,45 that yielded higher DAP association efficiencies demonstrated lower EC50 values. However, CTAB surface coatings were found to be 4-fold more cytotoxic than the free dapivirine solution. PEO/SLS-PCL demonstrated significantly less cytotoxicity than free drug. Anti-HIV efficacy of these NPs using PBMC assays challenged with HIV after 2 hrs NP treatment resulted in EC50 values in the nano-molar range, 3\u20137 fold lower than free drug,48. PEO-PCL exhibited the lowest cytotoxic concentration (CC50) in PMBCs at 105 nM, half that of SLS-PCL and approximately 200-fold less than CTAB. These NPs inhibited HIV infection of monocyte-derived dendritic cells (Mo-DC) and CD4+ T cell co-culture for 14 days,50. Single drug applications to Mo-DC showed EC50 for NP-encapsulated DAP at 7\u201312 fold lower than free DAP. Cytotoxicity of NP treatments to Mo-DCs mirrored PBMC assays with CTAB-coated NP (PCL-CTAB) having the highest cytotoxicity , 20-fold less than PCL-SLS , and 40-fold less than PCL-PEO . PEO-PCL-DAP NPs were chosen for in vivo pharmacokinetic analysis due to their enhanced inhibition of infection and comparably low cytotoxicity profile. Application of PEO-PCL-DAP NP or free DAP solution intravaginally to female mice showed enhanced retention of NPs in vaginal fluid. PEO-PCL-DAP NP retained drug levels above the previously established drug level for DAP for 24hrs while free DAP solution maintained threshold levels for 4 hrs. These results indicate the extended protective capacity of DAP NPs in vivo,52.Polymer alternatives to PLGA such as poly(\u03b5-caprolactone) (PCL) surface coated NPs were shown to enhance encapsulated drug bioavailability and intracellular retentionciencies than pre,54. CAP also may destroy viral particles by stripping envelope glycol-proteins and causing HIV\u201355. CAP is pH a sensitive polymer that depolymerizes at pH higher than 6.2. Since vaginal mucosal pH is lower than 6.2, CAP-NPs are likely to remain stable in the acidic pH environment. CAP-EFV-NPs were formulated by nano-precipitation method and yielded an EFV entrapment efficiency of 98.1% \u00b1 1.2% (. Short term (4 hr) and long term (3 day) PrEP of CAP-EFV-NPs against HIV-1NL4-3 challenge were assessed in vitro using TZM-bl assays. CAP-EFV-NPs significantly reduced HIV infection at concentrations below 50 ng/mL compared to EFV drug solution. At 3 days the EFV solution had significantly lower antiretroviral activity compared to CAP-EFV-NPs treatment at equivalent concentrations (5 ng/mL). CAP-EFV-NPs reduced the cytotoxicity of EFV on HeLa cells with significantly higher cell viability at 48 h and 96 h. CAP may be another cost-effective polymer option for NP synthesis and PrEP.Another nanoparticle polymer under investigation for PrEP is Cellulose Acetate Phthalate (CAP). CAP is unique to other functionally inert polymers because CAP has anti-microbicial properties that inhibit HIV-1 entry. CAP has been shown to bind to gp 120 and to gp 41 on HIV and to form six-helix bundles with R4 and R5 tropic viruses% \u00b1 1.2% [57]. Sho,59. Mechanisms for gelation have been explored. Osmolarity is an important consideration for gels as failures in large clinical trials including CAPRISA-004 have been attributed to hyperosmolar gels causing inflammation and increased susceptibility to HIV-1 infection. Combinations of pluronic polymers (F127/F68) are used to tailor the rhelogical properties with citrate-buffered NP solutions, DMSO, and N-Methyl pyrrolidone through thermosensitive gels into HeLa cells. Fluorescence of PLGA-Rhod6G-NP was observed in HeLa cells after 30 minutes of incubation showing rapid release and uptake of NPs into cells. Rhodamine 6G fluorescence was maintained for up to seven days in vitro. PLGA-Rhod6G-NP delivered to the vaginal tissues of humanized mice showed uniform distribution in vaginal tissues. PLGA-Rhod6G-NP was specifically localized in the vaginal epithelium for up to 24 hours. As proof-of-concept experiments, CAP-EFV-NP were incorporated into TMS and examined efficacy. HIV-1NL4-3 antiviral efficacy was measured in vitro using TZM-bl assays following CAP-EFV-NP-TMS, CAP-NP-TMS, and EVF-TMS pre-treatment. TZMbl cells were challenged with HIV-1 four hours post-treatment and CAP-EVF-NP-TMS showed higher efficacy with 90% antiviral activity at 500pg/mL of EFV. These studies indicated enhanced efficacy of CAP-ARV-NP-TMS and expanded the study of TMS delivery to ARVs more likely to be used in human clinical studies.For ease of topical application to reproductive tissues some NP fabrications have utilized thermosensitive (TMS) gels. Topical gels coupled with polymeric NP encapsulated ARVs may offer direct application to principal sites of HIV exposure prior to sexual intercourse, ensure uniform drug application, and control drug kinetics for elongated release. Thermosensitivity modulates rheological properties by increasing viscosity as a function of increasing body temperature upon application to facilitate delivery and enhance vaginal retentionrolidone . RecommeIn vivo efficacy of PLGA-ARV-NP-TMS has been recently demonstrated in humanized mice. PLGA-Rilpivirine (RPV) \u2013NPs were formulated by encapsulation using ion-solvent-evaporation technique for incorporation into TMS gel. Kovarova et al., 2015 achieved 98% RPV association efficiency and embedded their NPs in 20:1 Pluronic F127:F68 ratio TMS gel. Humanized BLT mice treated with PLGA-RPV-NP-TMS (17.5\u03bcg RPV) were completely protected when challenged with high dose HIV-1RHPA 1.5 hrs post-application. Only half of these mice were protected from HIV challenge 24 h after application of PLGA-RPV\u2013NPs-TMS were individually applied intra-vaginally to Hu-BLT mice. Following TDF-NP-TMS treatment mice were challenged with 2 transmission/founder HIV-1 strains at three time points. The four hour (n = 4) and 24 hour (n = 6) challenge groups showed 100% protection against HIV-1 challenge as determined by plasma viral load (pVL). All mice challenged at seven days showed HIV-infection at 14 days post-inoculation, signifying TDF-NP-TMS gel capacity for intermediate protective capacity, but, currently, not for longer time durations (>24hrs).\u2013NPs-TMS as deterof 52.9% [67], sig. Clinical trials of film-encapsulated dapivirine indicated the efficacy of these treatments in maintaining plasma drug levels comparable to that of gel fabrications. Using solvent casting with glycerin as a plasticizer, prepared films of PLGA/stearylamine (SA)-Tenofovir-NPs were investigated for efficacy. PLGA/SA-Tenofovir-NPs were produced by double emulsion/solvent evaporation and demonstrated much higher NP-drug association efficiency (PLGA/SA: 53.5 \u00b1 4.9%) than pure PLGA-based NPs (18.5 \u00b1 2.5%). NP-embedded films were thicker and weaker than pure films, potentially complicating fabrication, handling, and applications, but they maintained minimum pharmaceutical thresholds. Like gels, the physiochemical properties of films must conform to physiologic osmolarity and pH levels to ensure safe vaginal applications,71. PLGA/SA-Tenofovir-NP-film formulations were within physiologic thresholds. Tenofovir release was sustained further in Tenofovir-NP-film fabrications compared to Tenofovir-NP and Tenofovir-film fabrications . Non-gel EFV-NP treatments released at a much faster rate indicating the potential of the film-matrix to extend NP drug release in SVF. PLGA-EFV-NP/TFV-films examined in vivo using female CD-1 mice showed enhanced retention of TFV for two hours but overall rapid decreases in drug concentration. Similar decreases have been observed with intra-vaginal tablet-tenofovir drug formulations in macaques and rabbits,76. EFV concentrations were also sustained at early time points (30 min) using NP-EFV/TFV-solution films compared to EFV solution/TFV solution-film formulations, indicating the ability of NPs in films to elongate drug release of both EFV and TFV.Machado was high . . Films were optimized for physiologic physiochemical properties and drug loaded to 1.5% wt/wt (drug/film),78. In vitro drug release was measured in continuous flow in-line Franz cells,79 and showed significantly elongated release of IQP-0528-NP from films (24hr: 51.65% \u00b1 7.22% release) compared to free IQP-0528 films (1hr: 100% release). However, in vivo pharmacokinetic analysis on pigtailed macaques found that median drug levels at 24hrs were higher in the free-IQP-0528 films as opposed to the IQP-0528-NP-films in the distal and proximal vaginal fluid indicating uniform coverage and enhanced retention of drug in the vaginal environment.Film fabrication differing in PVA: HPMC polymer excipient ratio has been designed. Polymer film embedded with IQP-0528, an NNRTI with entry inhibiting capabilities, was encapsulated in PLGA-NPs (PLGA/Eudragit S100-IQP-0582-NPs) by double emulsionNP-films [72]. AllIn vitro models with PLGA/SA-TFV NPs and PLGA-EFV-NPs both showed elongated release compared to free-drug film fabrications (2 film articles). However, in vivo pharmacokinetic studies using PLGA-IQP-0582-NPs exhibited drug clearance rates similar to that of the IQP-0582 molecule in solution. Currently, there are no studies directly comparing gels and films as delivery systems for ARV-NPs.Films have shown mixed results as a NP delivery modality. . Prophylaxis that enhances current nanoparticle technology to deliver higher and sustained concentrations of ARV drugs is likely to provide enhanced efficacy. Future studies will show the viability of nanoparticle fabrications for PrEP.Recent clinical studies have shown that PrEP can be highly efficacious given patient adherence. Widespread use of PrEP must be cost-effective and stable. New highly efficacious PrEP that can be delivered to at risk populations must be developed. Nanoparticle fabrications of ARVs delivered in thermosensitive gels or polymeric films may provide a means for low cost, highly effective PrEP and is an important goal of current PrEP research. There is an increased need for studies investigating new prophylaxis for women"} +{"text": "NP model for triple-negative breast cancer allows the analysis of parameters influencing immunotherapeutic approaches. Except for WAP-TNP tumors expressing the immune-dominant LCMV NP-epitope within SV40 T-antigen (T-AgNP) which is not expressed by T-Ag of WAP-T tumors, the tumors are extremely similar. Comparative anti-PD1/PD-L1 immunotherapy of WAP-T and WAP-TNP mice supported the hypothesis that the immunogenicity of tumor antigen T-cell epitopes strongly influences the success of immune checkpoint blockade therapy, with highly immunogenic T-cell epitopes favoring rapid CTL exhaustion. Here we analyzed the immune response in NP8 mice during early times of tumor development. LCMV infection of lactating NP8 mice induced lifelong tumor protection by memory CTLs. Immunization with LCMV after involution and appearance of T-AgNP expressing parity-induced tumor progenitor cells could not cure the mice, as memory CTLs became exhausted. However, immunization significantly prolonged the time of tumor outgrowth. Elimination of exhausted CTLs and of immunosuppressive cells by sub-lethal \u03b3-irradiation, followed by adoptive transfer of NP-epitope specific CTLs into NP8 tumor mice with early lesions, completely prevented tumor outgrowth, when lymphocytes obtained after injection of weakly immunogenic NP8 tumor-derived cells into BALB/c mice were transferred. Transfer of lymphocytes obtained after infection of BALB/c mice with highly immunogenic LCMV into such mice delayed tumor outgrowth for a significant period, but could not prevent it. We conclude that eliminating exhausted CTLs and immune-suppressive cells followed by transfer or generation of low-avidity tumor antigen-specific CTLs might be a promising approach for curative tumor immunotherapy.The SV40 transgenic BALB/c mouse based WAP-T/WAP-T Upon transgene induction after parturition SV40 early proteins are expressed, with T-antigen (T-Ag) being the major tumor antigen. In WAP-TNP mice, the SV40 transgene additionally encodes a highly immunogenic T-cell epitope, the NP118-126-epitope within the nucleoprotein (NP) of lymphocytic choriomeningitis virus (LCMV). While SV40 T-Ag expressed in WAP-T tumor mice is only weakly immunogenic in the BALB/c mouse background, the chimeric T-Ag/NP protein (T-AgNP) in WAP-TNP tumor mice is highly immunogenic. Except for this immunological difference, WAP-T and WAP-TNP tumors are histologically and molecularly extremely similar.One way of systematically addressing at least some parameters influencing immunotherapy is the use of suitable animal systems. For this purpose, our laboratory has developed the cross-species validated transgenic BALB/c mouse based WAP-T models for triple negative breast cancer. For immunological studies, we use two different lines of tumor mice NP-epitope of LCMV within SV40 T-Ag (T-AgNP in NP8) or not (T-Ag in T1). Tumor development and tumor characteristics are similar for T1 and NP8 mice [Transgene induction, tumor growth, as well as histological and molecular tumor characteristics have been summarized recently in Bruns et al., 2015 . In brieNP8 mice .NP tumors, this difference could only be ascribed to the presence or absence of the NP-epitope in WAP-TNP and WAP-T tumors, respectively. Indeed, we were able to show that the NP-epitope elicited a fast and strong epitope-specific CTL response, but at the same time also promoted rapid CD8+ T-cell exhaustion. On the other hand, the relatively good efficacy of the anti-PD1/PD-L1 treatment in WAP-T tumor mice supported the idea that tumors expressing weak tumor antigen T-cell epitopes respond much better to immune checkpoint blockade therapy because re-establishing an exhausted status of CTLs against these epitopes will take much longer [Immunization of NP8 tumor mice with LCMV led to transient tumor regression, due to the presence of exhausted, programmed death-1 protein (PD1)-expressing NP-epitope specific CTLs. CTL activity could be largely restored by treatment of the mice with anti-PD1 antibodies . Followih longer .We envisioned that the idea of \u201cweak beats strong\u201d regarding immunogenicity of T-cell epitopes in cancer immunotherapy could beNP is induced during late pregnancy and reaches its peak during lactation on day 7 pp. We previously showed that LCMV infection or adoptive transfer of NP epitope-specific CTLs eliminates T-AgNP expressing cells in lactating mammary glands of NP8 mice [NP expressing cells in lactating mammary glands, but from parity-induced tumor progenitor cells arising after involution [NP positive ducts, which, however, failed to form hyperplastic lesions . Therefore, we considered to improve our therapy scheme by eliminating exhausted CTLs and immune-suppressive cells before therapy. Sub-lethal \u03b3-irradiation with 4 Gray (Gy) was chosen as the most efficient treatment option, as it eliminates, amongst other immune cells, all T cells, including (exhausted) CD8+ T-cells and Treg, as well as Breg cells. Alternatively, we also applied PD-L1 treatment. For immunization, we opted for adoptive transfer of NP-epitope specific CTLs from immunized BALB/c mice out of two reasons: First, we previously found that CTLs derived from LCMV infected BALB/c mice had been very effective in clearing lactating mammary glands of NP8 mice from T-AgNP expressing cells [The insufficient therapeutic efficiency of LCMV immunization of NP8 mice with early stage tumors is due to exhaustion of NP-epitope specific CD8latory T and B [1latory T cells for tumor free survival until sacrifice Figure , expressNP expression during lactation (day 7 pp), were completely protected from tumor outgrowth by NP-specific memory T-cells that had developed after LCMV infection. This finding can be explained by assuming that the non-replicating, T-AgNP expressing mammary epithelial cells did not significantly influence the immune response to LCMV, and that at this stage the immune response of infected NP8 mice reflected that of lytic infection of BALB/c mice, which protects mice life-long against re-infection by LCMV [In this study we analyzed the effects of LCMV NP-epitope specific immunotherapy of NP8 mice at early stages of tumor development, as we hoped to achieve better responses than in tumor mice harboring large tumors. Indeed, induced NP8 mice, LCMV treated at the peak of T-Ag by LCMV . ImmunizNP expressing cell clusters, local hyperplastic lesions and very few MIN can be detected in involuted mammary glands. However, in contrast to non-replicating T-AgNP expressing epithelial cells in lactating mammary glands, T-AgNP expressing cells in involuted mammary glands represent replicating parity-induced tumor progenitor cells arising after involution [Surprisingly, the immune status of induced NP8 mice changed dramatically already very early after weaning. As can be deduced from the data listed in volution . These cThe most important finding of this study, however, is that at least for early stage malignancies in NP8 tumor development curative immunotherapy was nevertheless possible: removal of immune cells, specifically exhausted T-cells and immune-suppressive cells, by sub-lethal \u03b3-irradiation, followed by adoptive transfer of CTLs obtained from BALB/c mice transplanted with H8N8 cells completely prevented tumor outgrowth up to 300 days pw . Clearly, a major prerequisite for this positive result was the removal of exhausted immune as well as of immune-suppressive cells, but as important was the choice of CTLs for adoptive transfer. This is demonstrated by comparing the transfer of H8N8-specific CTLs with that of LCMV-specific CTLs, respectively, into NP8 tumor mice with or without prior irradiation of NP8 mice: While transfer of H8N8-specific CTLs into non-irradiated NP8 tumor mice did not significantly prolong the time for tumor outgrowth, transfer of LCMV-specific CTLs was very effective in prolonging this time. On the other hand, prior \u03b3-irradiation did not significantly boost the effect of the transferred LCMV-specific CTLs. We interpret this result as further support of the pattern outlined in our previous study: weak immunogenic stimulation will result in a weak immune response, but will not induce CTL exhaustion, or at least delay it. Strong immunogenic stimulation will induce a strong immune response, but also induce rapid CTL exhaustion. This interpretation is in line with previous experimental data showing that upon contact with their targets high avidity CTLs become more easily eliminated or exhausted than low avidity CTLs , 14.NP, which is also the major immunogenic T-cell epitope in LCMV infected BALB/c mice, the question arises why CTLs generated by injection of H8N8 cells into BALB/c mice elicit a rather weak immune response (i.e. are of low avidity) compared to high avidity CTLs derived from LCMV infected BALB/c mice. In addition to the different routes of immunization , the simplest explanation is the difference in amount of NP-epitope presented by H8N8 cells compared to NP-epitope presentation during lytic infection. This interpretation corresponds well to our observation that H8N8 cells, despite presenting the NP-epitope, are able to transiently grow in BALB/c mice in a dose dependent manner and WAP-T (T1) lines containing either the BALB/c mouse specific CTL NP-epitope of LCMV within the SV40 T-Ag (T-AgNP in NP8) or not (T-Ag in T1), respectively [Inbred BALB/c mice were crossed with BALB/c-based transgenic mice and female transgenic offspring mice were selected for use in the experiments presented here. We used the transgenic WAP-Tectively , 3. TumoIf not stated otherwise, at least three mice per group were used in each experiment. Statistic analyses were performed with the Single-Sample Confidence Interval Calculator and the One-way ANOVA Calculator programs. Mice were kept in S1 animal facilities, held under specific pathogen-free conditions and handled according to German regulations for animal experimentations. All protocols had been approved by the Hamburg administration .5 PFU of LCMV intravenously (iv) or intraperitoneally (ip).The triple plaque-purified WE strain of LCMV was prop2.Cultures of tumor-derived G-2 or H8N8 cancer cells served in particular for the precise calculations of tumor growth during therapeutic treatments. The procedures for their isolation and their growth characteristics were described elsewhere , 33, 34.For anti-PD-L1/PD1 treatment experiments we used goat polyclonal B7-H1/PD-L1 or PD1 antibodies (R&D Systems). The dose-dependency for clearance of the exhausted immune status using the antibodies in NP8 and T1 tumor mice was evaluated in previous analyses . An iv d2 PFU of LCMV iv or were alternatively treated with 105 H8N8 tumor cells ip. Prior to the transfer of immune cells from donor (BALB/c) mice, the acceptor (NP8 tumor) mice were \u03b3-irradiated sub-lethally with a radiation dose of 4 Gy by the Cs-137 source of a LISA I apparatus (Conservatome) to remove exhausted CTLs for about two to three weeks . A similar extensive discharge of CTLs was obtained in mice, when they were treated with 400 \u03bcg of monoclonal anti-CD8 antibodies (mAbs) iv [Adoptive transfer experiments were performed as already described . BrieflymAbs) iv , 36. In 2 to 1\u00d7105 tumor cells were harvested from cultures and re-suspended in 50 \u03bcl of a 1:1 mixture of serum-free DMEM and BD Matrigel Matrix high concentration, growth factor reduced (BD Bioscience). Between 10 and 20 weeks old non-induced (virgin) female mice were anaesthetized by ip injection of ketamine/xylazine. After an incision of about 5 mm into the skin the cell suspensions were injected into the left or right abdominal mammary gland (MG #3 or MG #6); carprofen (50 mg/ml) was applied as analgesic; the skin was closed by interrupted sutures after implantations.For transplantation experiments 1\u00d7102O2 solution in phosphate buffered saline. Na\u00efve rabbit serum served as control. Sections were slightly counterstained with hemalum. All photographs were taken by the Zeiss Axioplan2 imaging microscopic equipment with the camera ProgRes C12plus of Jenoptic using the Software ProgRes CapturePro 2.9.0.1.gy.Histopathology and analysis of transgene expression were essentially as already described . In brieT-Ag protein and mRNA contents were measured by enzyme-linked immuno-sorbent assay (ELISA) and quantitative real-time polymerase chain reaction (qRT-PCR), respectively, as already described . In shor"} +{"text": "Emerging evidence indicates that microRNAs, a class of small and well-conserved noncoding RNAs, participate in many physiological and pathological processes. RNase III endonuclease DICER is one of the key enzymes for microRNA biogenesis. Here, we found that DICER was downregulated in tumor samples of colorectal cancer (CRC) patients at both mRNA and protein levels. Importantly, intestinal epithelial cell (IEC)-specific deletion of Dicer mice got more tumors after azoxymethane and dextran sulfate sodium (DSS) administration. Interestingly, IEC-specific deletion of Dicer led to severe chronic inflammation and epithelium layer remodeling in mice with or without DSS administration. Microarray analysis of 3 paired Dicer deletion CRC cell lines showed that miR-324-5p was one of the most significantly decreased miRNAs. In the intestinal epithelium of IEC-specific deletion of Dicer mice, miR-324-5p was also found to be markedly reduced. Mechanistically, miR-324-5p directly bound to the 3\u2032untranslated regions (3\u2032UTRs) of HMG-box containing 3 (HMGXB3) and WAS protein family member 2 (WASF-2), two key proteins participated in cell motility and cytoskeleton remodeling, to suppress their expressions. Intraperitoneal injection of miR-324-5p AgomiR (an agonist of miR-324-5p) curtailed chronic inflammation and cytoskeleton remodeling of colorectal epithelium and restored intestinal barrier function in IEC-specific deletion of Dicer mice induced by DSS. Therefore, our study reveals a key role of a DICER/miR-324-5p/HMGXB3/WASF-2 axis in tumorigenesis of CRC by regulation of cytoskeleton remodeling and maintaining integrity of intestinal barriers. RNase III endonuclease DICER, a key player in the biogenesis of microRNAs (miRNAs), has been widely studied in many physiological and pathological programs including cancer. Reduced expression of DICER is associated with poor prognosis in many types of cancers including lung cancer , breastMiRNAs are a class of single-stranded sequences (\u223c22nt) that can either mediate mRNA degradation or prevent mRNA translation , and haPersistent chronic inflammation is a general inducer in colorectal tumorigenesis, as patients with Crohn\u2019s disease and ulcerative colitis have an increased risk of suffering from colitis-associated colorectal cancer , 15 . InHerein, we found DICER is a tumor suppressor in CRC using clinical samples from CRC patients and an azoxymethane (AOM) plus DSS induced mouse CRC model. Importantly, DICER deletion led to cytoskeleton remodeling and disruption of intestinal barrier by downregulation of miR-324-5p, which targets HMGXB3 and WASF-2. Based on these findings, we propose that DICER/miR-324-5p/ HMGXB3/WASF-2 axis plays a dominant role in cytoskeleton remodeling, intestinal barrier integrity maintaining and CRC tumorigenesis.To understand the clinical significance of DICER in CRC, DICER expression levels were evaluated in clinical samples of CRC patients. In 78% (53/68) of the clinical CRC samples, DICER mRNA levels were reduced in the tumour tissues compared with their counterpart adjacent tissues . Using ploxp/loxp&VillinCre mice) could not survive beyond one month (loxp/+&VillinCre mice) were used for the remainder of the experiments. Examination of intestinal gross morphology showed that there were more and larger colorectal tumors developed in the Dicerloxp/+&VillinCre mice than the wild type mice survived for no more than one month, severe intestinal epithelial injury and inflammatory response in both small intestine were exploited to identify the key miRNAs affected by DICER deletion using Human MiRNA Microarray analysis. Expressed data were normalized using the Median normalization (GEO accession number: GSE93177). Finally, hierarchical clustering was performed to determine distinguishable miRNA expression profiles among the samples. Compared with wild-type cells, hsa-miR-324-5p was one of the top two declined microRNAs after DICER deletion and has-miR-324-5p levels were decreased significantly in three DICER deletion cells . RT-PCR experiments validated decreased has-miR-324-5p expressions in all the three DICER deletion cell lines , mmu-miR-324-5p mRNA levels were also significantly decreased in the intestine epithelial layer , has been widely studied in many processes including mammalian embryogenesis , 26 , DNloxp/+&VillinCre mice). Although Dicer has been suggested as a tumour suppressor for a long time [Dicer1 in intestinal epithelial cells promotes tumorigenesis in AOM and DSS induced mouse CRC model [Here, we found Dicer as a tumour suppressor in CRC using both clinical samples of CRC patients and in vivo mouse CRC model with IECs specific deletion of Dicer. We found that Dicer was downregulated in tumour samples of CRC patients at both mRNA and protein levels. Importantly, in AOM and DSS induced mouse CRC model, more tumours were developed in mice with IECs specific deletion of Dicer . Therefore, cytoskeleton remodeling of intestinal epithelial cells will be the key step for gut inflammatory response to any stress or inflammation. Interestingly, a recent study showed that Dicer is involved in formation and maintenance of cell-cell junctions of mouse seminiferous epithelium . Our stMore importantly, we found a key mechanism that Dicer suppresses cytoskeleton remodeling of colorectal epithelium by miR-324-5p mediated suppression of HMGXB3 and WASF-2. As one of most abundant miRNAs, miR-324-5p is significantly regulated by Dicer and has a key role in maintaining intestinal epithelial integrity and intestinal homeostasis. We found that HMGXB3 and WASF-2 are two direct targets involved in cytoskeleton remodeling of colorectal epithelium for miR-324-5p. HMGXB3 (HMG-box containing 3), one of the non-canonical high mobility group genes, belonging to the High Mobility Group superfamily, participates in a range of cellular process including cell migration and proliferation , 36. WASIn summary, we found DICER as a tumour suppressor in CRC using clinical samples and an AOM and DSS induced mouse model with Dicer deletion in IECs. Importantly, we suggest a new DICER/miR-324-5p/HMGXB3/WASF-2 axis plays a dominant role in cytoskeleton remodeling, intestinal barrier integrity maintaining and tumorigenesis.Cre mice) and mice carrying the floxed allele of Dicer (Dicerloxp/loxp mice) were purchased from the Jackson Laboratory. VillinCre and Dicerloxp/loxp mice were crossed to obtain intestinal-specific Dicer knockout mice (Dicerloxp/loxp&VillinCre mice). The mice used in the present study were maintained in the barrier facilities at the Laboratory Animal Center in Soochow University (China). All the experiments were performed according to the protocols approved by the Committee on Animal Research of Soochow University.Villin promoter-driven Cre recombinase transgenic mice . HCT116, SW480, RKO and DLD1 were cultured in an RPMI-1640 medium (HyClone) with 10% fetal bovine serum (FBS). Caco-2 were maintained in Dulbecco\u2019s modified Eagle\u2019s medium (DMEM) with 20% FBS. Dicer-knockout cell lines of were kind gifts of Professor Bert Vogelstein from the Sidney Kimmel Comprehensive Cancer and Howard Hughes Medical Institute. For in vivo studies, the mmu-miR-324-5p agomir (modified miR-324-5p mimic) and the negative control were from RiboBio . MiR-324-5p mimic and inhibitor were purchased from RiboBio . Rhodamine Phalloidin was purchased from Cytoskeleton, Inc.The luciferase reporter gene vectors including HMGXB3 and WASF-2 3\u2032 untranslated region (3\u2032UTR) sequences of the human miR-324-5p binding sites were constructed. Primers of HMGXB3 and WASF-2 3\u2032 UTR including miR-324-5p binding sites were amplified respectively. The PCR products were digested and transferred into pMIR-REPORT vector to construct the recombinant pMIR-promoter-HMGXB3 3\u2032UTR-wild type and pMIR- promoter-WASF-2 3\u2032UTR-wild type respectively. siRNAs for human DICER, #1: GGAAGAGGCUGACUAUGAA, and #2: UGCUUGAAGCAGCUCUGGA. Transient transfections were performed with Lipofectamine 2000 (Invitrogen) following the manufacture\u2019s protocol. Cells were lysed 24-48h after transfection and fluorescence intensity was assayed using a luciferase reporter assay system (Promega). Each transfection also included \u03b2-gal for normal control. Experiments were performed in triplicate wells and repeated at least three times.Whole cell protein was extracted using RIPA buffer supplemented with a protease inhibitor tablet (Roche). Western blot analyses were performed with anti-DICER1 (Santa Cruz), anti-HMGXB3 , anti-WASF2 (Santa Cruz), anti-a-tubulin (Invitrogen) antibodies.Prepared frozen section or the transfected cells for the following staining. Slides or cells were then fixed, permeabilized at room temperature and then incubated with 100nM rhodamine phalloidin in the dark for 30 min. Counterstain the nuclei for 30s with DAPI and seal the slides in anti-fade mounting media. Store the slides in the dark at 4\u00b0C before observation.t test. P-value < 0.05 was considered significant and marked with *. Each experiment was performed in at least three independent experiments. Additional methods employed in this study are described in Supplementary materials and methods (see The results are presented as the mean\u00b1 standard deviation. Statistical significance was determined by Student two-tailed hods see ."} +{"text": "We aimed to determine the inter-study reproducibility of left ventricular (LV) mechanical dyssynchrony measures based on standard cardiovascular magnetic resonance (CMR) cine images.Steady state free precession (SSFP) LV short-axis stacks as well as 2-, 3- and 4-chamber views were acquired on the same day at 9:00 (Exam A), 9:30 (Exam B) and 14:00 (Exam C) in 16 healthy volunteers. Circumferential strain systolic dyssynchrony indexes (SDI), area SDI as well as circumferential and radial uniformity ratio estimates were derived from CMR myocardial feature tracking (CMR-FT) based on the tracking of 3 short-axis planes . Furthermore, 4D LV-Analysis based on short-axis stacks and all three longitudinal planes was performed to quantify 4D volume SDI. Exam A and B were compared to assess the inter-study reproducibility. Morning and afternoon scans were compared to study possible diurnal variation.AVG) showed the lowest inter-study variation (CoV 6.4%) with an ICC of 0.80. Dyssynchrony indexes were not measurably affected by diurnal variation between morning and afternoon scans.CMR-FT derived CURE and RURE as well as 4D LV-Analysis derived volume SDI showed good inter-study reproducibility . Conversely, circumferential strain and area SDI showed higher variability between the repeated measurements . Averaged CURE and RURE indexes (CURE:RUREAVG and 4D volume SDI showed good inter-study reproducibility. Their clinical value should next be explored in patient groups who potentially benefit from cardiac resynchronization therapy.Derivation of LV mechanical dyssynchrony measures from standard cine images is feasible using CMR-FT and 4D LV-Analysis tools. CURE:RURE"} +{"text": "N-acetylglucosamine (UDP-GlcNAc). UDP-GlcNAC serves as a donor substrate during O-GlcNAcylation (O-linked \u03b2-N-acetylglucosamine or O-GlcNAc) [O-GlcNAcylated, competing with phosphorylation. O-GlcNAcylation is catalyzed by one unique enzyme called O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT). O-GlcNAcylation is cleaved and removed by another one enzyme called N-acetyl-\u03b2-D-glucosaminidase (OGA) [Glucose is partly metabolized through the glucose sensing hexosamine biosynthetic pathway (HBP) leading to the formation of an end product called acetylated amino sugar nucleotide uridine 5\u2032-diphospho--GlcNAc) . Serine se (OGA) . The exise (OGA) .O-GlcNAcylation levels may occur in human breast cancer and hepatocellular carcinoma (HCC) tissues [O-GlcNAcylation at Thr-58 stabilizes c-MYC, promoting tumorigenesis [O-GlcNAcylation levels are very dynamic and cycles rapidly, fluctuating in response to glucose concentrations influencing cell signaling pathways . O-GlcNA tissues . The oncigenesis .Unconventional prefoldin RPB5 interactor (URI) binds and modulates OGT activity in response to glucose concentrations. In presence of glucose, URI, OGT and protein phosphatase 1 gamma (PP1\u03b3) form a heterotrimeric complex. Glucose deprivation induces anaplerotic reactions, increasing ATP/cAMP levels, thereby activating PKA which in turn, phosphorylates URI at Ser-371. Phosphorylated URI frees PP1\u03b3 from the heterotrimeric complex and, URI becomes a potent inhibitor of OGT . AbnormaURI-regulated OGT is reported to confer c-MYC-dependent survival functions in response to glucose fluctuations . In the These findings delineate an adaptive mechanism for cells to cope with metabolic stress in order to have an opportunity to survive. Prolonged exposure of cells to inadequate and low glucose concentrations induces cell survival functions allowing cells to cope with glucose restrictions or to fix a perturbed cellular homeostasis. Apoptosis of pancreatic \u03b2 cells is critical in the development of T2D. Low glucose levels in \u03b2 cells may thus favor OGT inhibition by URI protecting cells from death maintaining a certain cellular homeostasis. Reducing OGT activity and O-GlcNAcylation levels may be beneficial for diabetic patients, in agreement with the fact that OGT overexpression in mice causes insulin resistance and T2D . URI losOGT inhibition by URI is also an adaptive molecular mechanism enabling cancer cell survival and tumor development during glucose restrictions. Nutrients such as oxygen and glucose are delivered to various tissues via an efficient vasculature network. However, during tumor development with increasing tumor burden, an early avascular stage or collapsed vasculature inside the tumor often affect the delivery and diffusion of nutrients leading to a state of nutrient restrictions. Poor nutrient supply is demonstrated by several mathematical models to impact tumor growth, confirming experimental studies in which hypoxia supports tumor aggressiveness . Not onl"} +{"text": "PDCD1 -CD274 axis have shown unprecedented clinical benefits in the treatment of refractory neoplasms . Recently, the U.S. Food and Drug Administration (FDA) approved the anti-PDCD1 (PD-1) antibody pembrolizumab for treating solid tumors with high-level microsatellite instability (MSI) or mismatch repair deficiency. This approval attracted great attention as the first of an agent based on a tumor biomarker rather than the primary cancer site. High-level MSI is commonly present in colorectal carcinomas, and abundant neoantigens due to frequent frameshift mutations have been proposed as a potential explanation of the survival benefits seen with the immune checkpoint blockade in this tumor subtype . Taking the inter-tumor heterogeneity into account, large cohort studies suggest that survival benefits from postdiagnosis aspirin use are pronounced for colorectal cancer with PTGS2 overexpression or activating PIK3CA mutations [PTGS2 and prostaglandin E2 (PGE2) synthesis that are enhanced by activated PI3K signaling. Beyond these potential anti-tumor effects associated with inhibition of oncogenic signaling pathways, accumulating evidence points to immune-enhancing effects of aspirin on adaptive and innate immune response, including T cell-mediated anti-tumor immunity [Aspirin is a common nonsteroidal anti-inflammatory drug (NSAID) that inhibits utations , 5 [DomiCD274 (PD-L1) expression than in cancer with higher-level CD274 expression . This differential survival association was consistently observed regardless of levels of tumor-infiltrating lymphocytes defined by histopathologic examination. These findings indicate that activation of the CD274-PDCD1 immune checkpoint pathway may confer resistance to aspirin therapy, and that this could be overcome with the immune checkpoint blockade. This study was the first population-based study to suggest an interaction between immune checkpoint status and PGE2 inhibition via aspirin in regulating the progression of human colorectal cancer.Recently, we reported a U.S. population-based study which suggested a stronger survival association of postdiagnosis aspirin use in colorectal cancer with lower-level D274 PD-L expressiantibody . In the antibody , PTGS (cCD274-low vs. CD274-high). Furthermore, the aspirin studies utilized the methodology of pharmacoepidemiology, clearly illustrating the successful integration of this traditional discipline with MPE (termed \u201cpharmaco-MPE\u201d) [These aspirin studies were based on the paradigm of molecular pathological epidemiology (MPE) for survival analyses , 3. In cCD274-low colorectal cancer) and in those receiving immune checkpoint inhibitors. Given the importance of better understanding of the tumor-immune microenvironment, consideration of immune parameters in MPE research would provide ample opportunities for development of novel immunomodulatory strategies of cancer prevention and treatment (which can be termed \u201cimmuno-MPE\u201d) [In summary, our study underscono-MPE\u201d) ."} +{"text": "Nature Communications8:122 doi: 10.1038/s41467-017-00172-9; Article published online: 25 Jul 2017The original version of this Article contained errors in the Abstract. \u2018Chro also prevents NSCs from ire-entering quiescence at later stages. NSC-specific in vivo profiling has dentified many downstream targets of Chro, including a temporal transcription factor Grainy head (Grh) and a neural stem cell quiescence-inducing factor Prospero (Pros)\u2019 now reads \u2018Chro also prevents NSCs from re-entering quiescence at later stages. NSC-specific in vivo profiling has identified many downstream targets of Chro, including a temporal transcription factor Grainy head (Grh) and a neural stem cell quiescence-inducing factor Prospero (Pros).\u2019 These errors have now been corrected in both the PDF and HTML versions of this Article."} +{"text": "Although most triple-negative breast cancer (TNBC) patients initially respond to chemotherapy, residual tumor cells frequently persist and drive recurrent tumor growth. Previous studies from our laboratory and others' indicate that TNBC is heterogeneous, being composed of chemo-sensitive and chemo-resistant tumor cell subpopulations. In the current work, we studied the invasive behaviors of chemo-resistant TNBC, and sought to identify markers of invasion in chemo-residual TNBC.in vitro was studied using transwell invasion assays and an experimental metastasis model. mRNA expression levels of neural cadherin (N-cadherin), an adhesion molecule that promotes invasion, was assessed by PCR. Expression of N-cadherin and its precursor form (pro-N-cadherin) was assessed by immunoblotting and flow cytometry. Pro-N-cadherin immunohistochemistry was performed on tumors obtained from patients pre- and post- neoadjuvant chemotherapy treatment.The invasive behavior of TNBC tumor cells surviving short-term chemotherapy treatment TNBC cells surviving short-term chemotherapy treatment exhibited increased invasive behavior and capacity to colonize metastatic sites compared to untreated tumor cells. The invasive behavior of chemo-resistant cells was associated with their increased cell surface expression of precursor N-cadherin (pro-N-cadherin). An antibody specific for the precursor domain of N-cadherin inhibited invasion of chemo-resistant TNBC cells. To begin to validate our findings in humans, we showed that the percent cell surface pro-N-cadherin (+) tumor cells increased in patients post- chemotherapy treatment.TNBC cells surviving short-term chemotherapy treatment are more invasive than bulk tumor cells. Cell surface pro-N-cadherin expression is associated with the invasive and chemo-resistant behaviors of this tumor cell subset. Our findings indicate the importance of future studies determining the value of cell surface pro-N-cadherin as: 1) a biomarker for TNBC recurrence and 2) a therapeutic target for eliminating chemo-residual disease. Most triple-negative breast cancers respond initially to chemotherapy. However, residual tumor cells frequently persist. These residual tumor cells are thought to be responsible for recurrent tumor growth , which frequently occurs within 3 years of treatment , accountIt is now well-appreciated that tumors are heterogeneous, being composed of chemotherapy-sensitive and chemotherapy-resistant tumor cell subpopulations , 3. BecaPreviously we described a method for studying TN breast cancer cell subpopulations enriched by short-term chemotherapy treatment . In thisWe have developed a short-term chemotherapy treatment model that enriches for a chemo-resistant subset of TN breast tumor cells Figure . In thisBased on their increased invasive phenotype, we next sought to determine if these chemo-resistant tumor cells, when injected into the tail vein of immunocompromised mice, exhibited increased ability to colonize the lung compared to untreated tumor cells. First, luciferase-expressing SUM159 TN tumor cells were subjected to short-term docetaxel treatment as in Figure 3, at which point they were measured every 2-3 days until volumes reached 2000 mm3. As shown in Previous studies indicate that long-term chemotherapy selection models drive the growth of cancer stem-like cells -8. We thLong-term chemotherapy selection models drive an epithelial-mesenchymal transition in estrogen receptor-positive breast tumors, characterized by reduced epithelial adhesion marker (E-cadherin) and acquired mesenchymal adhesion marker (N-cadherin) expression. By contrast, triple-negative breast cancers are typically mesenchymal in nature, expressing significant N-cadherin prior to chemotherapy treatment. We performed real-time PCR to determine relative levels of N-cadherin in parental (untreated) and chemo-resistant SUM159 cells from our short term chemotherapy treatment model. As shown in Figure In untransformed cells, only the mature form of N-cadherin, and not the precursor protein, is transported to the cell surface . By contPro-N-cadherin is expressed on the surface of melanoma and glioma cells, and contributes to their invasive behavior . We hypovs pro-N-cadherin-negative sorted populations from untreated SUM159 tumor cells. As shown in Figure Based on the knowledge that the cell surface pro-N-cadherin- positive population was enriched by chemotherapy treatment of SUM159 cells Figure , we nextn = 6 cases) pre- and post- neoadjuvant chemotherapy treatment. Pro-N-cadherin expression levels were assessed in these tumor tissues by immunohistochemistry. Notably, we observed nuclear/peri-nuclear pro-N-cadherin in both pre- and post-chemotherapy cases underscore the clinical significance of our results.We next sought to validate our findings in chemo-residual tumor cells from TN breast cancer patients. Matched tumor biopsies were obtained from TN breast cancer patients have been achieved in patients . Cells were lysed on ice for 20 minutes then centrifuged at 3500 rpm for 5 minutes at 4\u00b0C. Supernatant containing cytosolic proteins was removed and stored at -80\u00b0C. Nuclear proteins were extracted from the pellet on ice for 15 min using nuclear extraction buffer . Extracts were centrifuged at 4\u00b0C at 14000 rpm for 10 min. Supernatant containing nuclear proteins was removed and stored at -80\u00b0C. Protein concentrations were determined using BCA Protein Assay Kit (Pierce).Cells were harvested using 2mM EDTA / HBSS and washed 2X with HBSS (Gibco). Cytosolic extracts were prepared using lysis buffer and MCF7 breast tumor cells (Pro-N-cadherin(-)] were prepared as positive and negative controls respectively for Pro-N-cadherin reactivity.Two pathologists (blinded to patient samples) assigned scores for percent tumor cells positive for cell surface (membrane) pro-N-cadherin staining, as well as intensity of staining .2 overnight. The next day, culture medium was removed and treatments were added [no treatment, Docetaxel was added. Every three days, culture medium was changed and plates were examined for colony formation. To stain colonies, plates were fixed in 10% acetone /10%methanol solution for 10 min. Colonies were stained with 2% crystal violet for 30 minutes. The plates are then washed with water and allowed to air dry. Plates were imaged on GelCount and colonies were counted with Image J.Pro-N-cadherin-positive and Pro-N-cadherin-negative populations were sorted from untreated SUM159 cells, seeded into 6 well plates (4 wells per condition) at varying cell densities, and incubated at 37\u00b0C, 5% CO"} +{"text": "Six hours after intraperitoneal SAA injection both groups of transgenic mice demonstrated markedly higher (~2-5-fold) expression levels of inflammatory mediators in the liver and kidney compared to wild type mice. Histological examinations of hepatic and renal tissue from SAA-treated mice revealed moderate level of damage in the liver of both transgenic but not in the wild type mice. Activities of plasma transaminases, biomarkers of liver injury, were also moderately higher in hSR-B transgenic mice when compared to wild type mice. Our findings identify hSR-BII as a functional SAA receptor that mediates SAA uptake and contributes to its pro-inflammatory signaling via the MAPKs-mediated signaling pathways.Serum amyloid A (SAA) is an acute phase protein with cytokine-like and chemotactic properties, that is markedly up-regulated during various inflammatory conditions. Several receptors, including FPRL-1, TLR2, TLR4, RAGE, class B scavenger receptors, SR-BI and CD36, have been identified as SAA receptors. This study provides new evidence that SR-BII, splice variant of SR-BI, could function as an SAA receptor mediating its uptake and pro-inflammatory signaling. The uptake of Alexa Fluor488 SAA was markedly (~3 fold) increased in hSR-BII-expressing HeLa cells when compared with mock-transfected cells. The levels of SAA-induced interleukin-8 secretion by hSR-BII-expressing HEK293 cells were also significantly (~3\u20133.5 fold) higher than those detected in control cells. Moderately enhanced levels of phosphorylation of all three mitogen-activated protein kinases, ERK1/2, and p38 and JNK, were observed in hSR-BII-expressing cells following SAA stimulation when compared with control wild type cells. Transgenic mice with pLiv-11-directed liver/kidney overexpression of hSR-BI or hSR-BII were used to assess the Serum amyloid A (SAA) is a 12-14-kDa highly conserved acute phase apolipoprotein that is predominantly secreted by hepatocytes. Normally present in plasma in only trace amounts, SAA is a major acute phase reactant, whose plasma levels may increase up to 1000-fold . While tIn addition to its well-established acute response to inflammatory stimuli, SAA elevation can also be observed in multiple chronic inflammatory conditions, such as secondary amyloidosis , atherosMultiple studies suggest that SAA may have profound effects on innate immunity as a result of its chemotactic and cytokine-inducing activities. A-SAA induces the secretion of pro-inflammatory cytokines tumor necrosis factor-\u03b1 (TNF-\u03b1), interleukin-1\u03b2 (IL-1\u03b2), and interleukin-8 (IL-8) , and actThe diverse effects suggest that SAA may interact with more than one receptor and activate multiple signaling pathways. Earlier studies revealed several proteins that are capable of binding and/or mediating various SAA functions. FPRL1 (formyl peptide receptor like-1) protein was shown to mediate SAA\u2013induced chemotactic migration of leukocytes as well SR-BI, its splice variant SR-BII, and CD36 are members of the scavenger receptor family class B, that have high structural homology and all localize in plasma membrane caveolae-like domains which facilitate lipid exchange and cell signaling . These rOur previous studies demonstrated SAA binding to and signaling through the CLA-1 , human orthologue of rodent SR-BI , and CD3Despite serum amyloid A proteins being well-recognized markers of sepsis, and multiple reports demonstrated SAA presence at various inflammation sites \u201311, its in vitro and in vivo gain-of-function models\u2014human cell lines overexpressing human SR-BI and SR-BII and transgenic mice with pLiv-11-directed liver/kidney overexpression of these two receptors [in vitro, as well as in inflammation and tissue damage in vivo.Our most recent studies that used transgenic mice overexpressing human SR-BI and SR-BII revealed that hSR-BII, and to a lesser extent hSR-BI, have a major contribution to the LPS-induced pro-inflammatory response and organ injury in a model of non-lethal endotoxemia . Consideeceptors . This apvia hSR-BII might also involve MAPKs-mediated pathways. Additionally, the results of our in vivo experiments indicate that both hSR-BI and hSR-BII contribute to SAA-mediated organ injury and local tissue inflammation.Findings of this study demonstrate that hSR-BII is a functional SAA receptor that mediates its uptake and contributes to SAA-induced pro-inflammatory signaling. Our data suggests that similar to previously reported hSR-BI- and CD36-dependent signaling of SAA, its signaling Recombinant synthetic human apo-SAA was purchased from PeproTech. The lipid content of the recombinant apo-SAA was analyzed by the phospholipid B enzymatic method , and the cholesterol content was determined by an enzymatic cholesterol method on a Cobas Fara II analyzer (Roche Applied Science). These assays indicated that the SAA preparation contained only small amounts of phospholipids (<5 ng/\u03bcg) and cholesterol (<2 ng/\u03bcg) and hence was considered a lipid-poor form of SAA throughout this study. The synthetic amphipathic peptides were synthesized by a solid-phase procedure as previously reported . All reaThe liver-specific expression vector pLiv-11, which contains the human apoE promoter was usedMice were kept at the NIH animal facility under specific pathogen free conditions. All animal studies were approved by the Animal Care and Use Committee (ACUC) of the NHLBI under protocols H-0050R2 and H-0100R2 or NIDDK ACUC under protocol K100-KDB-15. The mice were monitored immediately after intervention, then after one and three hours to ensure that mice were not ill. Criteria for premature euthanasia were based on a points system of clinical scoring, where animals with a score exceeding 5 would be euthanized immediately. Points were scored as follows: depressed respiratory rate (2), apneustic respiration (5), spontaneous activity without stimulus (0), activity in response to tactile stimuli (1) delayed activity in response to tactile stimuli (2) unresponsive to tactile stimuli (5) piloerection (1) and lack of eye grooming (1). All mice had a score of 0 throughout the experiment.In vivo studies were performed as follows: 11\u201312 week old male wild-type (WT), hSR-BI tgn or hSR-BII tgn mice were injected intraperitoneally (IP) with SAA (2 mg/kg) or PBS . None of the mice had a score above zero. Six hours after SAA/PBS injection, mice were anesthetized by ketamine/xylazine/acepromazine , then blood and organs were collected, and mice were euthanized by exsanguination.Wild-type HeLa cells were transfected with human SR-BI and SR-BII expressing pcDNA 3.1 plasmids by using the lipofectamine reagent and further selected in the presence of 800 \u03bcg/ml G418. Human embryonic kidney cells were also stably transfected to express hSR-BI and hSR-BII, respectively) as described previously ,41.Human apolipoprotein E-free high density lipoproteins (HDL) were isolated from the plasma of healthy donors as previously reported . HDL, L3All incubations were performed in Dulbecco's modified Eagle's medium containing 0.1% bovine serum albumin at 37\u00b0C. Uptake experiments with HeLa cells were performed using Alexa 488\u2013labeled ligands at concentrations between 2.5 and 40 \u03bcg/ml, in triplicate, in a 96-well plate. After 2 hours of incubation the cells were rinsed 3 times with ice-cold PBS and read in a fluorescence plate reader . Competition experiments were performed using 5 \u03bcg/ml of Alexa 488-SAA and unlabeled ligands ranging in concentration from 0 to 125 \u03bcg/ml. Following 2-hour incubation and washing with ice-cold PBS, cell-associated fluorescence was analyzed by a fluorescence plate reader.For RNA isolation, tissue samples preserved in RNAlater stabilization solution, were homogenized in TRIzol Reagent . RNA was isolated with the PureLink RNAMini Kit after DNase treatment. RNA (2 \u03bcg) was reverse-transcribed using a TaqMan Reverse Transcriptase Reagent Kit. Real-time qPCR assays were performed with a StepOne Real-TimePCR System (Applied Biosystems), using 40 ng of cDNA per reaction. A list of TaqMan Gene Expression assays used in the study is shown in Relative levels of gene expression were measured by the comparative CT (\u0394\u0394CT) method with mouse \u03b2-actin or GAPDH genes as reference genes. All gene expression results were analyzed using the 2-\u2206\u2206CT formula and presented as normalized fold changes, compared to WT control (without LPS treatment).The IL-8 secretion by HEK293 cells was analyzed in culture supernatants after a 20h incubation period in serum-free medium with or without BSA (2 mg/ml), utilizing an ELISA kit for human IL-8. Plasma levels of cytokines, corticosterone, cortisol, and nitrate (NOx) were quantified with corresponding ELISA or colorimetric kits. All samples and standards were measured in duplicate.202/Tyr204) antibody, anti-ERK1/2 antibody, anti-phospho-SAPK/JNK (Thr183/Tyr185) antibody, anti-SAPK/JNK antibody, anti-phospho-p38 MAPK (Thr180/Tyr182) antibody and anti-p38 MAPK antibody . The immunoreactive bands were detected using an alkaline phosphatase-conjugated secondary antibody and chromogenic substrate for alkaline phosphatase (Invitrogen).Wild type and hSR-BII-overexpressing HEK293 cells were grown in 6-well culture plates to confluence. Before the MAPKs activation assay, the cells were incubated for 6 hrs in serum-free DMEM. The cells were stimulated for varying periods of time with SAA (0.5ug/ml) at 37\u00b0C. After stimulation, the culture medium was immediately aspirated; the cells were placed on ice and washed three times with ice-cold PBS. Afterwards, the cells were scrapped into 100 \u03bcl of lysis buffer Triton X-100, 150uM NaCl and 1% (v/v) protease/phosphatase inhibitor cocktail (Thermo Fisher Scientific). After a 10-min incubation on ice the samples were centrifuged at 12,000 g for 10 min at 4\u00b0C. The cell extracts were collected and mixed with the 2\u00d7 SDS sample buffer. The samples were separated on SDS-PAGE in 10% Tris-glycine pre-cast gels (Thermo Fisher Scientific) and then transferred to nitrocellulose membranes. After the transfer, the membranes were blocked with Tris-buffered saline containing 0.1% Tween 20 and 1% (w/v) nonfat dry milk and then probed with either one of three anti-phospho-MAPK antibodies or corresponding antibodies that recognize both active and inactive forms of each subfamily of MAP kinases, according to the manufacturer's protocols. The MAPK antibodies used in this study included anti-phospho-ERK1/2 (ThrAspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities in plasma were determined using corresponding colorimetric assay kits supplied by Sigma-Aldrich. For histological analyses formalin-fixed, paraffin-embedded 4 \u03bcm thin liver and kidney sections were stained with periodic acid-Schiff reagent (PAS) . Kidney histological changes were assessed in a blind manner in 10 different randomly selected 400X fields per animal from the cortex and 10 fields from the outer stripe of the outer medulla (OSOM). Kidney tubular damage was defined as tubular epithelial swelling, loss of brush border, vacuolar degeneration, necrotic tubules, cast formation, and desquamation. Liver damage was semi-quantitatively scored as previously described : the amoFor immunofluorescent staining, specimens of liver tissue were embedded in OCT compound and frozen in a dry ice-acetone bath. The blocks were cut into 10 \u03bcM sections using a Leica CM 1900 cryostat and placed onto microscope slides. Sections were fixed with 3.7% formaldehyde for 10 min, washed 3 x 5 min with 0.5% Saponin in PBS and were blocked with 5% Goat Serum-0.05% Saponin-1% BSA-PBS for 1 h. Next, sections were incubated overnight at 4\u00b0C with rabbit antibodies against CD11b followed by a 1h incubation with secondary antibodies conjugated with AlexaFluor 488 (Thermo Fisher Scientific). After two washes with PBS, sections were counterstained for nuclei with Hoechst 33342 , mounted using Vectashield antifade reagent , and visualized using Zeiss LSM 710 confocal microscope.Differences between the groups were examined for statistical significance by one-way analysis of variance (ANOVA). Alternatively, a two-tailed Student\u2019s t-test was used. All data are expressed as mean values \u00b1 standard deviation (SD) with a p value of < 0.05 considered as significant.To test functional activity of hSR-BII as a potential SAA receptor, we measured cellular uptake of Fluor 488-labelled SAA using hSR-BII-expressing HeLa cells. Compared with mock-transfected cells, expression of hSR-BII markedly (~3-fold) increased the uptake of fluorescently labeled SAA . Two othSAA is a potent pro-inflammatory mediator capable to induce secretion of pro-inflammatory cytokines in cultured human phagocytic cells such as neutrophils ,26 and TTo test if hSR-BII is a potential SAA receptor we performed competition experiments using other well-known ligands of SR-Bs. As seen in Our previous study demonstrated that the hSR-BI-dependent pro-inflammatory response in HeLa cells induced by SAA involves activation of 2 mitogen-activated protein kinases\u2014ERK1/2 and p38 . To invein vivo, control WT, transgenic hSR-BI and hSR-BII mice were injected with PBS or SAA and inflammatory responses were assessed by measuring pro-inflammatory cytokines and nitric oxide (NO) serum levels 6 hours following injection. In order to avoid non-physiological effects of lipid-poor recombinant SAA reported by Christenson et al [To investigate the influence of each splice variant\u2019s (hSR-BI and hSR-BII), on SAA-induced pro-inflammatory activity on et al , our stuon et al , can stivs. WT mice, versus hSR-BI mice.To investigate if any inflammation associated changes have occurred locally, hepatic and renal tissues of SAA- and PBS-treated mice were assessed for the gene expression of several pro-inflammatory mediators by quantitative PCR six hours after SAA or PBS injection. Despite the relatively low increase of systemic plasma cytokines to acute SAA injection, we detected a very strong increase of all tested pro-inflammatory markers in both organs. Hepatic expression of all cytokines and aspartate aminotransferase (AST), were measured 6 hours after SAA injection. We observed a moderate, but statistically significant, increase of AST activity in plasma of hSR-BI (~by 45%) and hSR-BII (~by 40%) transgenic mice, while no changes in AST activity were found in plasma of wild type mice . Of all Histological changes were examined in the livers and kidneys of mice 6 hours following PBS or SAA injection via the optical microscopy. As shown in Serum amyloid A is an endogenous damage-associated molecular pattern (DAMP) molecule which, when recognized by the host, initiates tissue-controlled immune responses . SAA levin vitro, and SAA-induced cytokine production in human microvascular endothelial cells can be down-regulated by SR-BI antagonists, ApoA-I mimetic peptides, and a specific anti\u2013SR-B1 antibody. Hong et al [To date several receptors, including FPRL-1 , TLR2 2,51 and Tng et al demonstrSR-BII, a splice variant of SR-BI, another member of SR-B family, is known mainly as a lipoprotein receptor involved with cholesterol transport, whereas its role as a receptor for pathogens, including various bacterial products, and danger-associated ligands, such as SAA, has not received proper attention until recently. In our opinion, SR-BII physiological importance was underestimated by the researches in particular because most studies failed to detect its expression when commercial LIMP2 sequence-based antibody was mistakenly used instead of anti-SR-BII antibody . Our recin vivo.Our earlier studies demonstrated that all three SR-B family members, SR-BI, SR-BII and CD36, are able to recognize and mediate pro-inflammatory signaling of bacterial products, such as LPS and GroEL, suggesting their important roles in innate immunity and host defense . In thisThe results of this study provide new evidence that hSR-BII may function as a SAA receptor, involved in its uptake and pro-inflammatory signaling. Our data demonstrate that both hSR-BI- and hSR-BII-expressing HeLa cells have significantly increased Alexa Fluor 488-SAA uptake when compared to mock-transfected control cells. The specificity of SAA uptake by hSR-BII was further confirmed in competition experiments, where non-labeled SAA as well as two other SR-B ligands, HDL and L37pA, were shown to efficiently block Alexa Fluor 488 SAA uptake in both hSR-BI and hSR-BII- expressing HeLa cells, whereas L37pA-3D peptide had no inhibitory effect. In addition to the enhanced uptake of SAA via the hSR-BII in HeLa cells we also found significant (~3\u20133.5-fold) increase in SAA-induced pro-inflammatory cytokine IL-8 secretion in hSR-BII-expressing HEK293 cells.MAPK signaling pathways have been implicated in SAA-induced pro-inflammatory cytokine/chemokine production in several cell types including neutrophils , monocytin vivo, we compared several pro-inflammatory markers levels both in plasma and gene tissue expression in liver and kidney of SR-B transgenic and wild-type mice 6 hours following SAA administration. SAA treatment caused modest responses for all measured plasma pro-inflammatory markers that were not statistically different between the groups of animals. Although the SAA dose used in this study exceeded the normal range of plasma SAA levels, it was considerably lower than those observed at pathological inflammatory conditions. SAA-treated mice were exposed to only one injection of SAA while in patients with chronic inflammatory conditions are continuously exposed to SAA for a much longer period of time. Additionally, the pro-inflammatory potential of SAA could be reduced due to its association with plasma lipoproteins. Therefore, we did not expect to see any significant changes in systemic inflammatory markers in our experimental setting. Despite the relatively low systemic pro-inflammatory response, all groups of SAA-treated mice demonstrated significant release of plasma corticosterone, reaching levels that are typically found during more severe inflammation.Utilizing hSR-BI- and hSR-BII-transgenic mice as gain-of-function models allowed us to access the potential in vivo contribution of both receptors to SAA-induced acute inflammation. The dose of SAA used in this study (2mg/kg) corresponded to SAA levels found during some mild chronic inflammatory conditions, with estimated plasma SAA to HDL ratio 1:20 (w/w). To assess the potential role of hSR-BI and hSR-BII as mediators of SAA-induced inflammatory response in vitro and in vivo studies demonstrated important role played by SAA in chemotaxis of human monocytes and polymorphonuclear leukocytes [We also assessed the effects of acute SAA injection on tissue expression of several genes, related to inflammation and cell adhesion, in hSR-B transgenic and wild type mice. We found that gene expression of all inflammation-associated mediators measured in liver and kidney was markedly up-regulated in all SAA-treated groups of mice. However, both hepatic and renal expression of almost all pro-inflammatory markers was significantly higher in SAA-treated hSR-BI and hSR-BII transgenic mice than in WT mice. Importantly, the strongest increases were found for both pro-inflammatory chemokines, CXCL1 and CCL2. Earlier ukocytes . ApparenIt has been suggested that at local sites of inflammation, upon release of proteolytic enzymes by activated monocytes or leukoRecently our group described the roles of SR-BI and SR-BII as important receptors for lipopolysaccharide (LPS): hSR-BI and hSR-BII transgenic mice subjected to IP LPS injection exhibited increased systemic inflammation, increased hepatic and renal expression of inflammation\u2013related genes, and more importantly, more liver and kidney histological lesions than LPS-treated WT mice . While tin vivo studies revealing higher SAA-induced pro-inflammatory responses along with moderate liver damage in hSR-BI- and hSR-BII-transgenic mice further highlight the important role of the class B scavenger receptor family as mediators of PAMP- and DAMP-induced inflammation and support SR-BI/BII\u2019s potential contribution to the host immune response.In conclusion, we found that human SR-BII, a splice variant of hSR-BI, is a functional receptor of SAA, capable of mediating its uptake and pro-inflammatory signaling. The S1 FigFrozen liver sections from PBS-treated (panels 1\u20133) and SAA-treated (panels 4\u20136) mice were stained using an anti-CD11b antibody, followed by the Alexa 488 Fluor-conjugated secondary antibody (green), according to the protocol described in Material and Methods. Hoechst 33342 nucleic counterstain appears blue. Scale bars, 50 \u03bcM.(TIF)Click here for additional data file."} +{"text": "Bombyx mori (B. mori), Fem piRNA originates from the W chromosome and is responsible for femaleness. The Fem piRNA-PIWI complex targets and cleaves mRNAs transcribed from the Masc gene. Masc encodes a novel CCCH type zinc-finger protein and is required for male-specific splicing of B. mori doublesex (Bmdsx) transcripts. In the present study, several silkworm strains carrying a transgene, which encodes a Fem piRNA-resistant Masc mRNA (Masc-R), were generated. Forced expression of the Masc-R transgene caused female-specific lethality during the larval stages. One of the Masc-R strains weakly expressed Masc-R in various tissues. Females heterozygous for the transgene expressed male-specific isoform of the Bombyx homolog of insulin-like growth factor II mRNA-binding protein (ImpM) and Bmdsx. All examined females showed a lower inducibility of vitellogenin synthesis and exhibited abnormalities in the ovaries. Testis-like tissues were observed in abnormal ovaries and, notably, the tissues contained considerable numbers of sperm bundles. Homozygous expression of the transgene resulted in formation of the male-specific abdominal segment in adult females and caused partial male differentiation in female genitalia. These results strongly suggest that Masc is an important regulatory gene of maleness in B. mori.In Bombyx mori, a W-chromosome-linked gene Feminizer (Fem) determines femaleness. Fem transcript yields a piRNA (Fem piRNA) and Fem- piRNA-PIWI complex targets and cleaves mRNAs transcribed from the Masculinizer (Masc). Masc is required for male-specific expression of Bmdsx, which is an important regulatory gene for sexual differentiation, and therefore, Masc is considered to be essential for maleness. However, there has been no direct evidence that Masc indeed causes maleness in sexually dimorphic structures. To clarify this point, we established silkworm strains carrying a transgene that expresses Fem-piRNA-resistant Masc gene (Masc-R). Transgenic expression of the Masc-R induced male mode of expressions of downstream sex-determining genes in females. Notably, ovaries in these females exhibited testis-like structures that contained sperm bundles. Homozygous expression of the Masc-R caused formation of the male-specific abdominal segment in adult females and induced partial male differentiation in female genitalia. Thus, Masc can induce maleness at the morphological level and is sufficient for spermatogenesis. This is the first report to our knowledge on a gene that can masculinize a wide variety of sexual characteristics in lepidopteran insects.In the silkworm, Daphnia magna, a shortening of the photoperiod, lack of food, and increase in population density leads to the production of males that are genetically identical to females vector , was produced by the following procedure. sc-R-His using KO] vector using InBamHI or BglII. Digested genomic DNAs were then separated on a 1.0% agarose gel and subsequently transferred to a Hybond-N+ membrane . Southern blot analysis was performed using probes labeled with Amersham AlkPhos Direct Labeling Reagents, and DNA bands were visualized using Amersham CDP-Star Detection Reagent following the manufacturer\u2019s guidelines . SDS-PAGE analysis of hemolymph was performed according to the method of Mine et al. . Bri. Bri55].S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S1 FigMasc-R transgenes. R/+ and G/+ animals carried either BmA3-GAL4 or UAS-Masc-R, respectively. +/+ animals had no transgenes.R/+ (DsRed-positive); G/+ (EGFP-positive) animals possessed both BmA3-GAL4 and UAS-(TIF)Click here for additional data file.S2 FigBmdsx were analyzed by RT-PCR. Amplified products were separated by 1% agarose gel electrophoresis. The panel indicates the female- and male-specific splice variants of Bmdsx . cDNAs prepared from larvae at L1D1. (B) Quantification of Masc mRNA at L1D1 using qRT-PCR. EF2 was served as an internal standard. Error bar: SD. R/G animals possessed both BmA3-GAL4 and UAS-Masc-R. G/+ animals carried UAS-Masc-R transgene.(A) Expression patterns of (TIF)Click here for additional data file.S3 FigMasc-R expression. The second panel from the top indicates the female- and male-specific splice variants of Bmdsx . The third panel shows ImpM expression. The bottom panel shows amplification of the GAPDH transcript, which served as a positive control for RNA extraction and RT-PCR. Masc-R/+, Sumi13-3 females heterozygous for the UAS-Masc-R transgene; +/+, Sumi13-3 sister females, which did not have the UAS-Masc-R transgene. cDNAs prepared from fat bodies within 3 hours after puation (A) and ovaries at the third instar larval stage (B) were subjected to the RT-PCR analyses.Amplified products were separated by 1% agarose gel electrophoresis. The top panel indicates the (TIF)Click here for additional data file.S4 FigMasc-R transgene. The lower panel (TG+) shows the PCR amplifications using Sumi13-3F and ks129, which can amplify the DNA fragment between the transgene and its flanking genomic region. Only TG- DNA fragment was amplified from the Sumi13-3 females, which did not have the UAS-Masc-R transgene (+/+), while both TG- and TG+ DNA fragments were amplified from the Sumi13-3 females heterozygous for the UAS-Masc-R transgene (Masc-R/+). The same PCR reactions detected only a TG+ DNA fragment from females numbered 1 through 17. These individuals served as females homozygous for UAS-Masc-R transgene (Masc-R/Masc-R) in the present study.(A) Schematic diagram of the primer locations used in the PCR-based genotyping. Red arrows indicate primers. (B) PCR products were separated by 1% agarose gel electrophoresis. The upper panel (TG-) indicates amplified products with Sumi13-3F and Sumi13-3R primers that specifically annealed to the region flanking the insertion site of the UAS-(TIF)Click here for additional data file.S5 FigEF2 was used as an internal standard. Error bar: SD.qRT-PCR was performed to quantify mRNA levels of Bmgn015522, Bmgn012518, and Bmgn012517 genes at L1D1. (TIF)Click here for additional data file.S6 FigBmdsx were injected into eggs, and hatched larvae were subjected to the phenotypic analysis. (A) Target site of TALENs within female specific exon of Bmdsx. Splicing patterns of Bmdsx gene from male and female are shown. Exons are described by box, and female specific exons are in black. TAL effector-binding sequences within the female-specific exon 3 are shown in blue. (B) Variation of sequences around the TALEN target site from two G0 female lines (No. 5 and 6). Molecular sexing of G0 animals was determined by PCR using W chromosome RAPD markers, Musashi. The native sequence is shown at the top of the alignment (Bmdsx). Deleted nucleotides are shown as dashed line in red. 6\u20135 contained unknown inserted sequences (red) and lacked approximately 1 kbp of sequence downstream of the target site. The ovaries of the day-5 fifth instar larvae of normal females (C) and G0 females with mutations in BmdsxF (D). (E) The abnormal globular tissue observed in the ovary of the day-12 G0 female pupa.Knockout silkworms were generated using transcription activator-like effector nuclease (TALENs), as described previously , 61. mRN(TIF)Click here for additional data file."} +{"text": "While trans-eQTL scans suffer from high testing dimensionality, recent evidence indicates most trans-eQTL associations are mediated by cis-regulated genes, such as transcription factors. Leveraging a data-driven gene co-expression network, we conducted a comprehensive cis-mediator analysis using RNA-Seq data from 471 normal prostate tissue samples to identify downstream regulatory associations of previously identified prostate cancer risk variants. We discovered multiple trans-eQTL associations that were significantly mediated by cis-regulated transcripts, four of which involved risk locus 17q12, proximal transcription factor HNF1B, and target trans-genes with known HNF response elements . We additionally identified evidence of cis-acting down-regulation of MSMB via rs10993994 corresponding to reduced co-expression of NDRG1. The majority of these cis-mediator relationships demonstrated trans-eQTL replicability in 87 prostate tissue samples from the Gene-Tissue Expression Project. These findings provide further biological context to known risk loci and outline new hypotheses for investigation into the etiology of prostate cancer.Large-scale genome-wide association studies have identified multiple single-nucleotide polymorphisms associated with risk of prostate cancer. Many of these genetic variants are presumed to be regulatory in nature; however, follow-up expression quantitative trait loci (eQTL) association studies have to-date been restricted largely to Prostate cancer (PRCA) is one of the most heritable cancers, with latest estimates of the genetic contribution to total risk near 58% . To datecis-acting regulation (cis-eQTLs). Thus, tested associations are limited to genes near the variants of interest. However, a growing number of studies have identified trans-eQTLs are also likely to play a major role in disease etiology [trans-associations of trait-associated loci. For example, Yao et al. [cis- and trans-eQTLs among SNPs associated with cardiometabolic traits relevant to cardiovascular disease. In PRCA, Chen et al. [trans-associations of reported risk loci in a relatively small set of tumor-adjacent stromal tissue samples. Other approaches, including adaptive false discovery rate estimation [trans-eQTL \u201chotspots\u201d, whereby genetic loci are associated in trans with expression levels of multiple transcripts.Due to the high testing-dimensionality presented by evaluating transcriptome-wide associations, most eQTL studies of trait-associated genetic variation focus on etiology \u201321. As to et al. , 23 haven et al. applied timation and crostimation , have fotrans associations are likely mediated by the products of cis-regulated transcripts [trans-eQTL associations under such a model is leveraging patterns of gene co-expression with cis-regulated genes. Gene co-expression analysis is a powerful data-driven approach for uncovering relevant regulatory networks in high-dimensional expression data. Recent improvements in the construction of sparse undirected graphs using regularized Gaussian graphical models have enabled sparse network inference on large gene expression datasets [trans-acting dysregulation of protein-coding gene expression by PRCA risk loci using cis-mediator analysis on a large prostate tissue eQTL dataset. We first substantially reduce the search space of trans-eQTL associations by constructing an undirected co-expression network of genes exhibiting at least modest eQTL associations with PRCA risk loci. We then identify cis-eQTL genes as potential mediators of trans-eQTL associations. We then apply a network-driven strategy to determine if neighboring genes in the expression graph exhibit trans-eQTL associations that are mediated by cis-regulatory effects using causal inference analyses. Finally, we interpret putative regulatory targets of dysregulated cis-eQTL genes in the context of PRCA susceptibility.Multiple studies have indicated nscripts , 27, sucdatasets . In thiscis-eQTL target genes, we identified 86 significant cis-genes associated with 72 unique PRCA risk loci variants and SEMA6A (mediation P = 3.0E-04). As HNF1B encodes the transcription factor HNF-1B, these results highlight multiple putative targets of trans-acting dysregulation via PRCA risk SNP rs11263762.A total of 3763 expressed transcripts met our transcriptome-wide eQTL screening criteria for inclusion in the co-expression network inference (FDR < 0.2). The estimated undirected graph for this gene subset consisted of 36,728 connections involving 3757 unique transcripts. Of the 3130 candidate variants . A totalM for significant cis-mediator relationships indicated incomplete attenuation of the trans-eQTL by the mediating cis-genes, ranging from 0.50 to 0.83 .Quantification of eQTL effect mediation by to 0.83 . Howevertrans-eQTL associations that corresponded to the cis-mediator relationships presented in Table HNF1B, we allowed any of 19 genotyped positions in LD with rs11263762 to be considered. Of the seven trans-associations reported, four corresponded to trans-eQTLs with p-values < 0.05 , with GTEx effect estimate directionality consistent with all seven discovery findings. Co-expression scatterplots for each of the four cis-mediator results with GTEx-replicated trans-eQTLs are presented in Figure The latest release of Gene Tissue Expression (GTEx) project currentlHNF1B have indicated potentially multiple variants independently contributing to disease susceptibility for prostate [cis-eQTL association may not completely capture downstream regulatory effects on potential HNF-1B targets. We subsequently relaxed the constraint of investigating trans-associations with peak HNF1B cis-SNP rs11263762 to all 33 potential PRCA risk cis-variants within the LD block.Fine-mapping studies of the PRCA risk locus near prostate and endoprostate . Thus, tvariants for the 20 SNPs . The sigFLRT3 and SLC14A1) that exhibited reduced expression in accordance with HNF1B over-expression in PC3. In our analyses, none of these genes exhibited compelling evidence of trans-eQTL effects with HNF1B cis-eQTLs consistent with HNF-1B regulation in normal prostate epithelium did account for the largest mediation effects across all significant cis-mediator trios and exhibited the strongest eQTL association with rs11263762 , only four additional trios were detected , a transcription factor that plays a critical regulatory role in nephron and pancreas development [HNF1B locus [HNF1B in benign prostate tissue. These results have led to differing perspectives on the role of HNF-1B in progression of PRCA. For example, Griziano et al. [HNF1B expression levels with the rs4430796-A allele across multiple ethnicities, and HNF1B knockdown in the LNCaP PRCA cell line resulted in reduced colony formation, proliferation, and viability. In contrast, Ross-Adams et al. [HNF1B expression in tumor samples, with additional evidence indicating these variants are associated with reduced promoter methylation. In our analyses, risk-associated alleles exhibited patterns of upregulatory effects on HNF1B expression, suggesting oncogenic properties of HNF1B in the development of PRCA. Similar results were observed in the fine-mapping analysis of the HNF1B locus by Painter et al. [2 with rs11263762 = 0.61; HNF1B cis-eQTL P = 4.8E-12) to be associated with reduced HNF1B promoter activity. Recent analysis of HNF1B in breast cancer has also indicated HNF1B overexpression induces transformation and epithelial-to-mesenchymal transition in the NMuMG epithelial cell-line [HNF1B in cancers of epithelial origin.elopment . HNF-1B elopment . Althougelopment , and may1B locus , althougo et al. identifis et al. found nor et al. in endomHNF1B corresponded to four out of seven of the reported cis-mediator associations we identified, indicating potential dysregulation of multiple HNF-1B transcription factor targets by PRCA susceptibility variants near HNF1B. SRC encodes the proto-oncogene c-Src, a member of the Src family kinases [SRC expression has also been shown to be directly regulated by HNF-1A via an alternative tissue-specific HNF-1 promoter in multiple cell-types [MIA2 encodes the melanoma-inhibitory activity 2 (MIA2) protein, which belongs to the MIA gene family, and is similarly transcriptionally regulated by HNF-1A [MIA2 exhibits protumoral properties in oral squamous cell carcinoma, demonstrating increases in invasion, survival, and angiogenesis [MIA2 has also been implicated in pancreatic cancer [KIF12 has been shown to be directly regulated by HNF-1B in kidneys [KIF12 has been implicated as a disease severity modifier of renal cystic disease via HNF-1B-induced transcription [ kinases , and Src kinases , 46. SRCll-types . MIA2 eny HNF-1A . Althougy HNF-1A , MIA2 exogenesis . HNF-1A c cancer . Additio kidneys . While Kcription , its potMSMB encodes the prostate secretory protein 94 (PSP94), which is predominantly expressed in prostate. Reduced or lost MSMB expression is commonly observed in PRCA tumors [MSMB in prostate epithelial cells also promotes anchorage-independent growth [trans-assocition of NDRG1 expression with PRCA risk SNP rs10993994 to be mediated by MSMB expression, with the two genes exhibiting positively correlated co-expression patterns. Although the potential biological mechanisms linking MSMB expression to dysregulation of NDRG1 are not immediately clear, it is hypothesized that PSP94 peptides may activate signal transduction pathways relevant to apoptosis via cell surface receptors [NDRG1 encodes the N-myc downstream regulated gene 1 (NDRG1) protein, and NDRG1 gene expression is repressed by N-myc and c-myc [NDRG1 has been shown to suppress cell growth and proliferation [MSMB may confer protective effects against PRCA via signaling cascades that upregulate expression of NDRG1.A tumors , 55 and A tumors , 57, altA tumors . Supprest growth . Multiplt growth \u201362 have t growth , 64. Cont growth . In our eceptors . NDRG1 end c-myc . The NDRferation . NDRG1 hferation . Specififeration , and immferation . Thus, Mcis-associated genes. The relatively small sample size in comparison to the large gene count may also have limited our ability to estimate the graphical network structure, and smaller partial correlations may have gone undetected. Second, other types of RNA beyond protein-coding transcripts are also known to possess regulatory effects, including long non-coding RNAs and miRNAs. For example, HNF-1B was recently identified to regulate the miR-200 cluster in renal cells [There are a number of limitations to our study that warrant mention. First, our network inference is based solely on mRNA expression, and only captures a fraction of the biomolecular intermediaries of causal pathways impacted by dysregulation of the al cells . IntegraMSMB and HNF1B. Our work provides the foundation for novel hypotheses for further investigation into the functional genetics of PRCA susceptibility and tumor progression.By integrating gene co-expression patterns and causal mediation analyses in the evaluation of transcriptional dysregulation by PRCA risk loci, we identified multiple plausible downstream effects mediated by PRCA risk genes All analyses were conducted on a normal prostate tissue eQTL dataset comprised of 471 samples that passed strict quality control criteria (dbGap accession phs000985.v1.p1), previously detailed elsewhere , 73. BriDNA was extracted using the Puregene tissue extraction protocol per the manufacturer\u2019s recommendations and DNA quality was assessed by examining 260/280 ratio and DNA yield. Samples were genotyped using Illumina Infinium 2.5M bead arrays based on the manufacturer\u2019s protocol . Standard quality control analyses were performed to identify poor-quality samples or SNPs. Untyped SNPs as well as missing genotypes for typed SNPs were imputed using SHAPEIT and IMPURNA was extracted using the QIAGEN miRNeasy Mini Kit and the QIAcube instrument in accordance with the manufacturer\u2019s instructions, and RNA quality was assessed by evaluating the RNA integrity number (>7) and the 260/280 ratio. RNA libraries were prepared using the TruSeq RNA Sample Prep Kit v2 according to the manufacturer\u2019s instructions. Paired-end sequencing was performed on an Illumina HiSeq 2000 using TruSeq SBS sequencing kit version 3 and HCS v2.0.12 data collection software. A minimum of 50 million total reads per sample was required for analysis; samples with insufficient reads were re-sequenced and resultant BAM files were merged.RNA-seq data were analyzed with the use of the MAP-R-Seq pipeline . Paired-To remove potential biases such as GC content and differences in sequencing depth, gene read counts were normalized using conditional quantile normalization . To accor2 > 0.3) within 200kb and with linkage disequilibrium (LD) r2>0.5 eligible for eQTL analysis as candidate risk variants, resulting in a total of 8073 variants of interest. All eQTL association analyses were conducted using the MatrixEQTL R package [cis if the eQTL SNP was within 1 Mb of the transcript. To avoid spurious associations due to long-range LD patterns, we declared all transcripts at least 10 Mb from a cis-gene to be eligible as cis-mediated trans-genes.For the 202 previously reported PRCA risk SNPs , we declhuge R package [To identify a large subset of potentially relevant PRCA susceptibility genes for network inference, we conducted an initial transcriptome-wide eQTL screening for expressed transcripts with all tag PRCA risk SNPs under liberal selection criteria. The rationale for this strategy was the notion that risk SNP associations would propagate through relevant co-expression networks. The smallest eQTL association p-value per transcript across all tested SNPs was Bonferroni-adjusted for the number of original risk loci , and all transcripts corresponding to a FDR < 0.20 were selected for network analysis. This permissive significance threshold accommodated efficient dimensionality reduction while limiting the potential exclusion of false negative results. We estimated an undirected graph using the Meinshausen-Buhlmann method as part of the package . Defaultcis-mediator causal relationship is comprised of the eQTL variant, denoted L, the cis-regulated transcript (or cis-gene), denoted C, and the trans-regulated transcript (or trans-gene), denoted T. Thus, the eQTL variant, cis-gene, and trans-gene comprise a candidate cis-mediator trio , where the causal relationship can be characterized as L \u2192 C \u2192 T with arrows indicating causal direction were considered to be significant. All mRNAs connected to significant cis-genes in the expression network and in trans with the corresponding peak cis-eQTL variant were declared to be eligible cis-mediator trios trios to investigate whether trans-gene associations with cis-SNPs were mediated by the corresponding cis-gene expression. We employed model-based causal inference analysis using the cit R package [cit correspond to a conservative omnibus intersection-union test for four constituent association relationships that comprise the causal mediation relationship L \u2192 C \u2192 T, returning the maximum p-value across these tests. We additionally quantified mediation by calculating the proportion of the trans-eQTL effect estimate \u03b2^T attenuated by the additional adjustment of the corresponding cis-gene expression values as a covariate. For cis-gene adjusted trans-eQTL effect \u03b1-level of 0.05 were reported as significant cis-mediated trans-eQTLs.Causal inference was conducted for all eligible ( package , 83. The"} +{"text": "Besides, Mel-18 was lower-expressed in ovary metastatic lesions compared with that in primary lesions of gastric cancer, and Mel-18 overexpression inhibited the migration ability of gastric cancer cells. Interestingly, overexpression of Mel-18 resulted in down-regulation of miR-21 in gastric cancer cells and the expression of Mel-18 was negatively correlated with the expression of miR-21 in gastric cancer tissues. Furthermore, miR-21 overexpression partially restored sphere-forming ability, migration potential and chemo-resistance in Mel-18 overexpressing gastric cancer cells. These results suggests Mel-18 negatively regulates stem cell-like properties through downregulation of miR-21 in gastric cancer cells.Mel-18, a polycomb group protein, has been reported to act as a tumor suppressor and be down-regulated in several human cancers including gastric cancer. It was also found that Mel-18 negatively regulates self-renewal of hematopoietic stem cells and breast cancer stem cells (CSCs). This study aimed to clarify its role in gastric CSCs and explore the mechanisms. We found that low-expression of Mel-18 was correlated with poor prognosis and negatively correlated with overexpression of stem cell markers Oct4, Sox2, and Gli1 in 101 gastric cancer tissues. Mel-18 was down-regulated in cultured spheroid cells, which possess CSCs, and overexpression of Mel-18 inhibits cells sphere-forming ability and tumor growth Cancer stem cells(CSCs) refer to a small subset of cancer cells within tumors, which have the ability of self-renewal and generating diverse tumor cells , 2, and Mel-18 is a member of polycomb group (PcG) proteins, which are epigenetic chromatin modifiers. Mel-18 is similar in structure but opposite in some function to another PcG member Bmi-1 , which iThe ability of self-renewal is one key property of CSC, and a recognized experimental verification method is spheroid colony formation assay, in which cancer cells are cultured without serum, but with growth factors, such as epidermal growth factor (EGF) and recombinant basic fibroblast growth factor (bFGF) . BesidesTo explore the role of Mel-18 in gastric CSCs, we firstly detected the expressions of Mel-18 and stem cell markers or related proteins CD44, CD133, Oct4, Sox2, and Gli1 in samplOur former research has revealed that serum-free culture microsphere formation is available for isolating stem cell-like cells in gastric cancer. Spheroid cells overexpressed stem cell markers including Bmi-1, Oct-4, Nanog, \u00df-catenin, and Sox2, and acquire higher tumorigenicity, higher metastatic potential and higher chemo-resistance, suggesting micro-sphere enrich CSCs or stem cell-like cells . To explin vivo is also considered as self-renewal properties of CSCs, so we tested whether Mel-18 overexpression inhibited tumor growth in vivo. The control and Mel-18 overexpressing gastric cancer cells SGC7901 were injected subcutaneously in one rear flank of severe combined immunodeficient (SCID) mice and tumor growth was examined. Mice injected with Mel-18 overexpressing cells formed smaller tumors compared to those injected with control cells within 30 days supplemented plus2ml of DMEM/F12 medium (Invitrogen) with 10mM HEPES, human recombinant epidermal growth factor (EGF) (Invitrogen) at the concentration of 20 ng/ml, and human recombinant basic fibroblast growth factor (bFGF) (Invitrogen) at the concentration of 10 ng/ml. After 3~4 weeks, each well was examined using light microscope and spheroid colonies in 5 random fields were counted.Gastric cancer cell line SGC-7901 was cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) and antibiotics. Spheroid Colony Formation Assay was carried out as described previously . Cells whttp://www.dojindo.com) following the manufacturer's instructions, and the optical absorbance at wavelength 450 nm was measured for the supernatant of each well using the plate reader Multiskan EX .Cells were inoculated into 96-well plates (5000 cells per well) in triplicate supplied with RPMI-1640 medium containing 10% FBS, along with different concentrations chemotherapy reagent epirubicin (EPI) or irinotecan IRI and no drug as control. The number of viable cells was evaluated after 2 days cultivation using the Cell Counting Kit-8 (CCK8) .Stable cell lines expressing Mel-18 was generated by transfection of Mel-18 overexpressing plasmid and selected by puromycine as described previously . LentiviTotal RNA of cultured cell lines and tissue samples were extracted using TRIzol reagent (Invitrogen). For Mel-18, CD133, Oct4, Gli1, Sox2, TIMP3, VEGF and PTEN mRNA, GAPDH acted as an internal control. As to miR-21, total RNA was poly(A) tailed using poly(A) polymerase and then reverse-transcribed into first-strand cDNA using miRcute miRNA cDNA kit (Tiangen).5S acted as an internal control and the SYBR Green-based real-time PCR was conducted using 7900HT fast real-time PCR System (Applied Biosystems).The primer of related gene for qRT-PCR as follows:SGC-7901 cells transfected with either Mel-18-overexpressing plasmid or the mock plasmid were injected subcutaneously into the flanks of SCID mice. Tumor sizes were detected terminally by vernier caliper. After 4 weeks, mice were sacrificed by cervical dislocation, and tumors were removed and imaged. All experiments involving animal abided by protocols approved by the Shanghai Medical Experimental Animal Care Commission.Mel-18 was detected in cell lysate and tumor tissues with western blot (WB) and immunohisto-chemistry (IHC), respectively, as standard procedures . For incAll data were shown as mean \u00b1 SEM, the Student t test was used for statistical analysis unless otherwise noted, with P < 0.05 considered significant. In IHC assays of GC samples, Spearman's Rank correlation assay was used to determine the correlation between Mel-18 and stem cell markers expression. In QRT-PCR analysis of fresh tissues, the correlation between Mel-18 and miR-21 expression levels was analyzed by the Pearson coefficient test."} +{"text": "The 40-Hz auditory steady-state response (ASSR) probing gamma-band oscillations may reflect N-methyl-D-aspartate receptor (NMDAR) dysfunction in patients with schizophrenia (SZ). Diminished gamma oscillations are reported in SZ, although increased spontaneous gamma oscillations are also reported. We investigated the 40-Hz ASSR and its association with brain volumes and clinical symptoms of SZ.The 40-Hz ASSR was measured using electroencephalography in 33 patients with SZ and 30 healthy controls (HCs). Four gamma oscillation components , and inter-trial phase coherence (ITC)) were assessed. Brain volumes were assessed using high-resolution magnetic resonance imaging and voxel-based morphometry.Patients with SZ had larger evoked and total powers and higher ITC than HCs. In HCs, evoked power showed significant positive correlations with bilateral superior temporal gyrus (STG) volume. In SZ, the effect of positive symptoms on the path from evoked power to left STG volume was significantly moderated. In SZ with elevated positive symptoms, large evoked power predicted small left STG volume, whereas large evoked power predicted large left STG volume in those with low positive symptoms. Increased baseline power was associated with a smaller left middle frontal gyrus (MFG) volume in SZ, whereas increased ITC correlated with larger MFG volume in HCs.Our results support the NMDAR hypofunction model of SZ, and suggest significant involvement of the STG and MFG in gamma oscillations."} +{"text": "Lung cancer is one of the deadliest malignancies. The immune checkpoint-blockade (ICB) tumor therapy has led to striking improvement of long-term survival for some lung cancer patients. However, the response rate of immunotherapy is still low for lung cancer. Studying the tumor microenvironment (TME) should shed light on improvement of immunotherapy of lung cancer. Interleukin-33 (IL-33), an \u201calarmin\u201d cytokine, has been implicated in tumor associated immune responses and inflammatory diseases of the lung. The role of IL-33 in lung cancer progression, however, remains elusive. This study is designed to characterize IL-33 expression in lung tumor tissues and establish the clinical significance of IL-33 in non-small cell lung cancer lung cancer (NSCLC).Tumor tissue specimens from patients suffering from NSCLC were analyzed for expression of IL-33 protein by immunohistochemistry and expression of IL-33 and ST2 mRNA by RT-quantitative PCR (RT-QPCR). The expression data were analyzed for their association with clinical and pathological parameters of NSCLC. In addition, the association between expression levels of IL-33 mRNA and patient survival was determined using 5 independent expression profiling datasets of human lung cancer.The expression levels of IL-33 and ST2 were significantly down-regulated in both adenocarcinoma and squamous cell carcinoma of the lung when compared to adjacent normal lung tissues. In addition, the level of IL-33 protein was inversely correlated with tumor grade and size. Moreover, analysis of TCGA and GEO lung cancer expression datasets revealed that higher expression levels of IL-33 mRNA were correlated with longer overall survival of patients suffering from adenocarcinoma of the lung. These data indicate that the expression levels of IL-33 are inversely associated with lung cancer progression, consistent with the hypothesis that IL-33 is involved in immune surveillance of NSCLC. Cancer progression is inhibited by tumor immune surveillance, because cancer cells express unique tumor antigens, which trigger T cell-mediated antitumor immune responses \u20135. In or+ T cells, Th1 cells, NK cells, and \u03b3\u03b4T cells .\u201338.-/- Aruitment .+ T cells, NK cells, NKT cells and DC. Whereas low levels of IL-33, combined with a dampened type 1 lymphocyte-mediated antitumor immune response, promotes tumor progression through Treg, MDSC, M2 and mast cells. Thus, IL-33 can promote type 1 immunity against cancer in the setting where a cancer therapy induces tumor cell death and release of large amounts of IL-33. This might explain why lung cancer patients with higher expression levels of IL-33 survive longer. Nevertheless, the exact role of IL-33 in lung cancer development needs to be established using appropriate mouse models of lung cancer.In summary, the effect of IL-33 on tumorigenesis is dependent on its predominant target cell types. High levels of IL-33 in the tumor tissue can drive anti-tumor responses via CD8S1 FigData are collected from GEO database. Accession numbers are GSE37745 GSE42127 GSE50081 (D)GSE68465 (E)GSE30219. Log-rank test was performed.(PPTX)Click here for additional data file.S2 FigData are collected from TCGA. Log-rank test was performed.(PPTX)Click here for additional data file."} +{"text": "Mycoplasma bovis Ningxia-1 strain was tested by Pacific Biosciences (PacBio) single-molecule real-time (SMRT) sequencing technology. The strain was isolated from a lesioned calf lung in 2013 in Pengyang, Ningxia, China. The single circular chromosome of 1,033,629 bp shows differences between complete Mycoplasma bovis genome in insertion-like sequences (ISs), integrative conjugative elements (ICEs), lipoproteins (LPs), variable surface lipoproteins (VSPs), pathogenicity islands (PAIs), etc.A genome sequence of the Mycoplasma bovis is the main cause of bovine respiratory disease syndrome. At present, the pathogen is prevalent worldwide, which causes huge economic losses to national cattle industries technology were identified, totaling 845,928\u00a0bp , and occupied 81.84% of the whole genome, with an average length of 1121 bp and a mean GC content of 29.77%. A total of 577 coding sequence (CDS) genes could be classified into clusters of orthologous groups (COG) families, which have 19 functional categories. Seventy-four pseudogenes were predicted by GeneMarkS+ . A cluster of 8 variable surface protein (VSP)-related ORFs were found in the genome. Sixty-six lipoproteins (LPs) revealed signatures denoting distinct mutation-based mechanisms of phase variation were without an IS element but still denoted a pathogenicity island. These characteristics may contribute to the emergence of bacterial pathogens with new virulence properties (We found 60 insertion-like sequence (IS) elements that comprised three distinct categories (ariation with a lCP023663.This whole-genome sequence assembly has been deposited at GenBank under the accession no."} +{"text": "Here, we developed a new technology for obtaining neural stem cells (NSCs) from iPSCs through chimera formation, in an in vivo environment. iPSCs contributed to the neural lineage in the chimera, which could be efficiently purified and directly cultured as NSCs in vitro. The iPSC-derived, in vivo-differentiated NSCs expressed NSC markers, and their gene-expression pattern more closely resembled that of fetal brain-derived NSCs than in vitro-differentiated NSCs. This system could be applied for differentiating pluripotent stem cells into specialized cell types whose differentiation protocols are not well established.Like embryonic stem cells, induced pluripotent stem cells (iPSCs) can differentiate into all three germ layers in an Therefore, developing proper protocols for differentiating pluripotent stem cells into specific cell types is a critical step for studying developmental biology and advancing applications to the clinical stage. For these purposes, long-term expandable somatic cell types have been derived from pluripotent stem cells, including embryonic stem cell (ESC)- or induced pluripotent stem cell (iPSC)-derived neural stem cells (NSCs) > 2). In iPS-NSCs, 323 genes were up-regulated compared to fetal brain-derived NSCs . In totaNext, we performed Gene Ontology (GO) analysis of the differentially expressed genes identified in iPS-NSCs and iPS-cNSCs. Tables Fin vivo differentiated NSCs (iPS-cNSCs) from iPSCs by chimera formation, which were similar to fetal brain-derived NSCs in terms of morphology and gene-expression patterns. These results indicated that iPSCs contributed to the neural lineage in chimeras (in vivo environment), which could be purified and then directly cultured as NSCs in vitro. NSCs established from iPSCs through in vitro and in vivo differentiation systems are very similar to fetal brain-derived NSCs, but NSCs differentiated from an in vivo system were slightly more similar to fetal brain-derived NSCs than in vitro-differentiated NSCs. Araki et al. (2013) used in vivo-differentiated somatic cells from iPSCs for transplantations. They transplanted skin cells, a type of in vivo-differentiated somatic cells, from the tails of chimeric mice and showed that the transplanted cells were sustained over 10 months, which exceeded the period in which in vitro-differentiated somatic cells were sustained post-transplantation [in vivo differentiation system. Specific cell types could be isolated from pluripotent stem cells through teratoma formation, which was based on the ideas of teratoma contained diverse cell types [in vitro differentiation. These results suggested that the differentiation environment influences gene expression in differentiated cells and could be a crucial factor determining the characteristics of the differentiated cells from iPSCs.Here, we established antation . There ii et al. 13 used ill types . We alsoll types . Chimerain vitro, which could be maintained in culture, while retaining the ability to differentiate into neuronal or glial cells [in vitro differentiation of retrovirus-derived iPSCs into NSCs was slower (4\u20137 weeks) than that of ESCs (1\u20132 weeks) [in vivo differentiation following chimera formation could overcome the differentiation delay problem of retrovirus-derived iPSCs. The iPSCs developed using retroviral transgene expression efficiently differentiated into NSCs through chimera formation without a delay of NSC formation in brain tissue. Furthermore, we could not see re-reprogramming in the iPS-cNSCs, which was observed in NSCs differentiated in vitro [Differentiating pluripotent stem cells into specialized cell types is potentially useful, not only for clinical transplantation applications, but also as a research tool for studying the basic mechanisms of diseases. Recently, pluripotent stem cells such as ESCs and iPSCs were reported to be differentiated into NSCs al cells , 14. Howal cells . Transgeal cells . The in in vitro [Oct4 expression was detected in Nestin-positive neural progenitor cells and in TuJ1- and TH-positive neurons differentiated from human iPSCs induced by a lentivirus [in vivo-differentiated NSCs did not contain undifferentiated cells Gene expression levels of the NSC marker Nestin and Sox2 in brain-derived NSCs, iPS-NSCs, and iPS-cNSCs. Data are presented as mean\u00b1SD of triplicates (n = 3).(TIF)Click here for additional data file.S2 Fig(A) Neurosphere formation and establishment of iPS-cNSC-Single cell-line (iPS-cNSC-S) from single cell. (B) iPS-cNSCs expressed NSC markers, such as NESTIN and SOX2, as determined by immunocytochemistry.(TIF)Click here for additional data file.S3 Fig(A) iPS-cNSC-S and NED-NSCs could differentiate into glial (GFAP+) or neural (MAP2+) lineage. (B) Quantification of the lineage-specific differentiation efficiency by NED-NSCs and iPS-cNSC-S. Error bars indicate the standard error of the mean.(TIF)Click here for additional data file.S4 Fig(A) Expression levels of exogenous 4 Factor in iPSCs (Negative control), iPS-NSCs , iPS-cN(TIF)Click here for additional data file.S5 Fig(A) iPS-cNSC-S and iPS-NSCs were negative for Oct4-GFP transgene expression.(TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file."} +{"text": "Right hind limbs were immobilized by an external fixation procedure for 13 days. Muscle mass of the plantaris muscle in the immobilized groups was reduced by 16% in comparison with the sedentary control group. High-intensity running and immobilization increased both mRNA and protein levels of matrix metalloproteinase type 2 (MMP-2) in plantaris. Running and immobilization decreased the percentages of transverse sectional area of fast-twitch glycolytic (FG) type IIb fibers, running increased relative cross-sectional area of fast-twitch oxidative glycolytic (FOG) type IIa muscle fibers, whereas immobilization increased relative cross-sectional area of slow-twitch oxidative (SO) muscle fibers (type I). Our results suggest that both high-intensity running and immobilization are enough to induce overwhelming changes in plantaris.The effect of 2-week, high-intensity running and a 2-week immobilization on muscle fiber type composition of the plantaris muscle from 18 female, 6-month-old Wistar rats was bio- and histochemically investigated. The high-intensity treadmill running began with 20 min (32 m/min, 0% gradient, 75% VO"} +{"text": "The correct citation is: Okano J, Okuda N (2018) Effects of resource-dependent cannibalism on population size distribution and individual life history in a case-bearing caddisfly. PLoS ONE 13(2): e0191925."} +{"text": "In vitro release profiles show a significant controlled release effort that the release concentration of PTX from micelles was significantly increased with the exposure of NIR light. In vitro and in vivo antitumor studies demonstrate that, compared with chemo or PTT treatment alone, the combined treatment with the local exposure of NIR light exhibited significantly enhanced anti-tumor efficiency. These findings indicate that this system exhibited great potential in tumor-targeting imaging and synchronous chemo- and photo-thermal therapy.A combination of chemo- and photo-thermal therapy (PTT) has provided a promising efficient approach for cancer therapy. To achieve the superior synergistic chemotherapeutic effect with PTT, the development of a simple theranostic nanoplatform that can provide both cancer imaging and a spatial-temporal synchronism of both therapeutic approaches are highly desired. Our previous study has demonstrated that near-infrared (NIR) light-triggered biodegradable chitosan-based amphiphilic block copolymer micelles (SNSC) containing light-sensitive 2-nitrobenzyl alcohol and NIR dye cypate on the hydrophobic block could be used for fast light-triggered drug release. In this study, we conjugated the SNSC micelles with tumor targeting ligand c(RGDyK) and also encapsulated antitumor drug Paclitaxel (PTX). The results show that c(RGDyK)-modified micelles could enhance the targeting and residence time in tumor site, as well as be capable performing high temperature response for PTT on cancer cells and two-photon photolysis for fast release of anticancer drugs under NIR irradiation. Multifunctional nanomaterials, where therapeutic and imaging features are integrated within a single nanoplatform, has offered a promising opportunity for concurrent diagnosis and treatment of cancer tumors \u20133. Up toIncreasing interest has been showed on developing nanomedicine for cancer therapy by designing nanomicelles capable of carrying therapeutic agents to the target tissues/organs while possessing triggered drug release property. Stimuli-responsive nanocarriers which could control micelle dissociation and triggered drug release in response to the pathological environment-specific stimuli and/or externally applied signals provide advantages of regulating the therapy process both spatially and temporally \u201322. AmonDespite intensive research efforts on developing light-sensitive nanocarriers for cancer theranostics, the use of such drug-loaded micelles with active-tumor-targeting capability, as well as PTT-chemotherapy combination for tumor treatment has not been fully explored yet. Hence, in this study, to get the highest drug concentration to the tumor tissue as possible, we developed a multifunctional micellar drug delivery platform with the capability of active tumor targeting, NIR light-triggered anticancer drug release for two-photon stimulated combination of PTT and chemotherapy, as well as NIR imaging. The nanoplatform was designed for co-loading cypate as an imaging agent as well as a photo-thermal agent, and paclitaxel (PTX) as a chemotherapeutic drug into the inner core of the SNSC micelles, with cyclic RGD as an active targeting ligand conjugated to the surface of the micelles to increase the selectivity for tumor cells and enhance intracellular drug delivery, while reducing systemic toxicity and adverse side effects compared to untargeted micelles and systemic chemotherapy.c(RGDyK)-SUC = 704.3). Furthermore, by comparing the FTIR spectra of SNSC and c(RGDyK)-SNSC (\u22121 peak is attributed to stretching vibration of secondary amides and free hydroxyl groups of c(RGDyK). The increasing of carboxyl groups of the compound made the peak 1675.9 cm\u22121 strengthen and the intensified in-plane bending vibration peak of 1527.6 cm\u22121 was shown in As described in the method section, the cyclic RGD was reacted with succinyl anhydride to form amide bond. The carboxyl group was activated by EDC and NHS system and then reacted with the amino group of NIR triggered chitosan micelles to synthesize tumor-targeted c(RGDyK)-modified SNSC micelles Figure . The masyK)-SNSC , the shaAs described, cypate (Ex/Em: 780/808 nm) and PTX were encapsulated into the hydrophobic core of the micelles to form nanostructure c(RGDyK)-SNSC- cypate-PTX. When the molar ratio of PTX and drug-loading system was 1:4, the micelles exerted the best drug loading property with highest drug loading content (DLC) and encapsulation efficiency (EE) Table . Thus, tThe distinct absorption and fluorescence spectra of different components in the nanocarrier further confirmed the conjugation of c(RGDyk) and encapsulation of cypate and PTX onto the light-sensitive nanocarrier. The adsorption spectra Figure illustraOur previous work have demonstrated that under 765 nm light irradiation, the emission light (808 nm) of cypate could be re-absorbed by the 2-nitrobenzyl group in the core of SNSC micelles, accelerating the dissociation of the hydrophobic core to lead the collapse of the micelles. In this study, to further investigate the effect of cypate on fast photocleavage of nanomicelles, we measured the temperature changes of c(RGDyK)-SNSC-cypate-PTX under NIR light irradiation Figure . The temThe photostability of c(RGDyK)-SNSC-cypate- PTX was further evaluated and compared to cypate. Both c(RGDyK)-SNSC-cypate- PTX and cypate solution were continuously irradiated by a semiconductor laser at 765 nm (light intensity = 400 mW) and their photoluminescence emission intensities were monitored over a period of 60 min by a S2000 eight-gap optical fiber spectrometer. As shown in Figure v\u03b23-positive (MDA-MB-231) and negative (MCF-7) cell lines were used [v\u03b23, MCF-7 cells have a very weak affinity to both SNSC-cypate and c(RGDyK)-SNSC-cypate -SNSC-cypate has potential to increase delivered anticancer drug amount to the tumor sites.The specificity of c(RGDyK)-SNSC-cypate uptake via integrin \u03b1in vitro antitumor efficacy of c(RGDyK)-SNSC- cypate-PTX, the cumulative release profile of PTX from c(RGDyK)-SNSC-PTX and c(RGDyK)-SNSC-cypate-PTX micelles in the presence or absence of NIR illumination was studied -SNSC-cypate-PTX, cell viability assays were carried out in cancer cells (MDA-MB-231 and MCF-7). As anticipated, the higher the drug loading concentrations were, the stronger the growth inhibition effect exerted Figure and 4C. in vitro drug release. The results also inferred that there was no significant difference of inhibitory effects on integrin-negative cells between SNSC-cypate-PTX and c(RGDyK)-SNSC-cypate- PTX regardless of NIR irradiation, further confirmed the cell uptake of c(RGDyK)-SNSC-cypate-PTX was mediated by integrin receptor.Similarly, the samples inhibitory effect on MCF-7 cells was enhanced by NIR irradiation Figure . CompareTo further confirm the efficacy of c(RGDyK) targeted NIR illuminated PTX-loaded SNSC micelles in tumor cells, the cell uptake and cell morphology were displayed after incubated in MDA-MB-231 cells for 6 h Figure , with cevia both EPR effect and integrin-receptor.The temperature measurement was also performed to confirm the PTT mechanisms was activated under exposure of c(RGDyK)-SNSC-cypate-PTX-treated cancer cells to 765 nm laser light of 800 mW power density. After exposure, the MDA-MB-231 cells incubated with c(RGDyK)-SNSC-cypate-PTX exhibit rapid heating with a starting temperature of 35\u00b0C. The maximum temperature reached as high as 55\u00b0C -SNSC-cypate was assessed in mice implanted with \u03b1v\u03b23-positive MDA-MB-231. As shown in Figure in vivo blocking experiment was conducted and displayed in Figure v\u03b23-negative MCF-7 bearing mice treated with SNSC-cypate or c(RGDyK)-SNSC-cypate -SNSC-cypate-PTX were exposed for 20 min to a 765 nm laser at a power density of 800 mW. SNSC-cypate-PTX and PTX MDA-MB-231 tumors were as control. The anti-tumor effects of various treatments were then quantified based on tumor volume, animal weight change, and survival rate , with deacetylation degrees of 90% and an average molecular weight (MW) of 50 kDa. N,N'-dicyclohexylcarbodiimide (DCC), N-hydroxysuccinimide (NHS), 2-nitrobenzyl alcohol, pyridine were all purchased from Sigma-Aldrich . Indocyanine Green (ICG) derivative cypate (MW 689) was prepared in our laboratory. Cyclic RGD (c(RGDyK)) was purchased from Shanghai Jier Biochemistry Co. Methyl thiazolyltetrazolium (MTT), RPMI 1640 medium and fetal bovine serum (FBS), penicillin, streptomycin, trypsin and ethylenediamine tetra-acetic acid (EDTA) were purchased from Invitrogen-Life Technologies . All other analytical reagent grade chemical reagents used in the study were commercially acquired from Shanghai Chemical Reagent Company .Human breast cancer cell lines (MCF-7), human glioma cell lines (U87MG) and human breast cancer cell lines (MDA-MB-231) were all purchased from American Type Culture Collection . Kunming normal mice and Athymic nude mice were purchased from SLAC Laboratory Animal Co. Ltd. .P = 300 W for 50 times and then centrifuged. The supernatant c(RGDyK)- N-succinyl-N\u2032-4-(2-nitrobenzyloxy)-succinyl chitosan (c(RGDyK)-SNSC) micelles were kept at room temperature for further research.The c(RGDyK)-modified photoactive amphiphilic diblock copolymer was synthesized as depicted in Figure Our previous study has shown that the dissociation of the NIR fluorescent dye cypate-encapsulated micelles was accelerated under NIR (765 nm) illumination. The NIR emission triggered the photo-cleavage reaction, resulting in accelerated dissociation of SNSC micelles and concomitant release of co-loaded hydrophobic species. Here, we encapsulated cypate and antitumor drug PTX to the NIR light-triggered c(RGDyK)-SNSC nanocarrier. Specifically, 1 mg cypate dissolved in 100 \u03bcL DMSO was added dropwise into 2 mL c(RGDyK)-SNSC or SNSC micelles. Likewise, we added 2 mg PTX of 100 \u03bcL DMSO into micelles. The solution was stirred continuously for 10 min and sonicated at 200 W for 30 times. Then the solution was purified by dialysis in a dialysis tube (MWCO 10000) against distilled water and centrifuged to remove free PTX. The entrapment efficiency and loading content were calculated according to the following Equation: Entrapment efficiency = \u00d7 100%; Loading content = (mass of drug loaded in micelles/mass of drug loaded micelles) \u00d7 100%.The Fourier Transform Infrared (FTIR) spectra of c(RGDyK)-SNSC micelles was recorded by FTIR spectrometer . The average diameters and distribution of c(RGDyK)-SNSC, c(RGDyK)-SNSC-cypate and c(RGDyK)-SNSC- cypate-PTX were measured by a Mastersizer 2000 Laser Particle Size Analyzer with a helium\u2013neon laser as the light source at the scattering angle of 90\u00b0. The available detecting size interval is 2 nm\u20133 \u03bcm.The UV-Vis spectra were acquired by 754-PC UV-Vis spectrophotometer . S2000 eight-channel optical fiber spectrographotometer , and a NL-FC-2.0-763 semiconductor laser light were utilized for fluorescence spectra detection. All optical measurements were performed at room temperature.A continuous NL-FC-2.0-763 semiconductor laser light was employed. To evaluate the lased induced temperature increase, 500 \u03bcL of c(RGDyK)-SNSC-cypate-PTX aqueous solution was irradiated under NIR light for 20 min, while the temperature was monitored with a temperature sensor at designated time intervals. In addition, 500 \u03bcL of water or cypate were irradiated at the same laser settings for temperature recording.For the study of photostability, c(RGDyK)-SNSC-cypate-PTX or cypate aqueous solution were continuously irradiated by a semiconductor laser at 765 nm (light intensity = 400 mW) and their photoluminescence emission intensities were monitored over a period of 60 min by a S2000 eight-gap optical fiber spectrometer.\u03c5\u03b23 receptor specificity of c(RGDyK)-SNSC-cypate, integrin \u03b1\u03c5\u03b23 highly expressed tumor cells MDA-MB-231 was used. On the other hand, MCF-7 with low integrin \u03b1\u03c5\u03b23 expression was served as negative control. Briefly, MDA-MB-231 or MCF-7 cells (3 \u00d7 105 cells/mL) were seeded at the confocal petri dish and incubated at 37\u00b0C for 24 h. 200 \u03bcL of SNSC-cypate or c(RGDyK)-SNSC-cypate solution (1 mmol/L) were added into cell culture media. After cultured for 8 h, the adherent cells were washed with PBS thrice and the cells' nucleus were stained with Hochest (12 \u03bcg/mL) for 0.5 h and then imaged using laser confocal microscopy.To evaluate the integrin \u03b1\u03c5\u03b23 receptor mediation, in vitro receptor blocking experiment with cyclic RGD (c(RGDyK)) was conducted on MDA-MB-231 cells. After cultured the MDA-MB-231 cells at 37\u00b0C for 24 hours, c(RGDyK) (50 mmol/L) was preliminarily added to the cells for 30-minute incubation. Subsequently, c(RGDyK)-SNSC-cypate was added to the dishes and cultured for another 8 h. After washing with PBS, the cells were imaged using laser confocal microscopy.To confirm the integrin \u03b1in vitro were tested by a dialysis method. SNSC-cypate-PTX or c(RGDyK)-SNSC- cypate-PTX micelles (2 mL) were dialyzed in dialysis tubes against phosphate buffered saline (PBS) at 37.5\u00b0C and 30 rpm with or without the exposure of NIR light . The released drug in the incubation buffer was collected and the aliquots taken from the dialysate were replaced with fresh PBS (containing 0.1% tween 80) at pre-determined time intervals to keep the volume constant during the assay. Remove water by a freeze-drying method and re-dissolve the drug by methanol for High Performance Liquid Chromatography (HPLC) analysis.The release profiles of paclitaxel (PTX) from c(RGDyK)-SNSC-cypate micelles in vitro drug efficacy of c(RGDyK)-SNSC-PTX, MTT assay was conducted following the standard protocol reported previously [3 cell) with 10% FCS (serum) was added into each well in a 96-well plate and incubated for 24 h in humidified atmosphere containing 5% CO2 at 37.0\u00b0C. The culture medium in each well was replaced by 200 mL of RPMI 1640 containing c(RGDyK)-SNSC-cypate-PTX with particular concentrations . The mixture was then exposed to the laser diode and the temperature changes were measured by fiber optic temperature probe. After light irradiation, the cells were incubated for another 48 h. Finally, the medium was replaced by 180 mL of fresh RPMI 1640 and 20 mL of MTT solution (5 mg/mL). After incubating for another 4 h, the medium containing MTT was removed from each well and 200 mL of DMSO was added and shaken at room temperature. The optical density (OD) was measured at 570 nm with a Microplate Reader . The viable rate could be calculated by the following equation: viable rate = (ODtreated/ODcontrol) \u00d7 100%, where ODtreated was obtained in the presence of c(RGDyK)-SNSC-cypate-PTX and ODcontrol was obtained in the absence of c(RGDyK)-SNSC-cypate- PTX. Furthermore, the following control groups were employed in the study to evaluate the efficacy of combinatorial of PTT and chemotherapy: untreated cells, cells treated with PTX, SNSC-cypate-PTX, c(RGDyK)-SNSC-cypate-PTX under dark conditions, cells treated with SNSC-PTX, SNSC-cypate-PTX and c(RGDyK)-SNSC-PTX exposed to the laser diode . In addition, the morphological changes of cells treated with c(RGDyK)-SNSC-cypate-PTX under irradiation were imaged using laser confocal microscopy.To assess the eviously . The MDAin vivo study, tumor cell MDA-MB-231 or MCF-7 suspension was injected into the mice subcutaneously after the mice were anesthetized by isoflurane. When the volume of the tumor reaches to 100 ~ 200 mm3, they were used for in vivo targeting experiments.Thymus defect nude mice were used in this investigation. For vivo fluorescence images of animal were obtained at designated time intervals (1\u201396 h).The tumor bearing mice were divided into 2 groups (5 mice per group), which was intravenously injected with either SNSC-cypate-PTX solution or c(RGDyK)-SNSC-cypate-PTX solution (0.2 mL) via the tail vein. To capture the real time dynamics and biodistributions of the c(RGDyK)-SNSC-cypate-PTX in the tumor bearing mice, a custom-constructed NIR imaging system was used. The 3, drug or saline was given every other day. During this period, we recorded the weight and volume of the tumor.The tumor bearing mice were divided into 6 groups (8 mice per group), shown in Table 2, where a represents the length of the tumor and b represents the width of the tumor. The relative tumor volume (RTV) is found by the equation: RTV = Vt/V0, where Vt represents the tumor volume before administration and V0 represents the recorded tumor volume. Relative tumor inhibitory rate (T/C) is the assessment of antitumor activity of human xenograft tumor model and is calculated by the equation T/C(%)=TRTV/CRTV\u00d7100%, where TRTV is obtained by RTV of control group and CRTV of treatment group.All of the groups were intravenously injected with samples via the tail vein, respectively. The E-H group were irradiated by NIR laser at 6 h post-injection. Tumor volume (TV) is calculated by the equation: TV = 1/2*a*bn = 8). The sliced organs were stained with H&E and examined under a microscope.To confirm the combinatorial PTT and chemotherapy efficacy of c(RGDyK)- SNSC-cypate-PTX for tumor therapy, histology analysis of tumor tissues was performed after treatment. Tumor tissues were separated and fixed with 10% neutral buffered formalin and embedded in paraffin (t-test with statistical significance assigned for P value of <0.05.Data was expressed as mean \u00b1 standard deviation. Statistical analysis was performed by using students' in vitro drug release of the micelles. Experimental results show that, under exposure of NIR light, the release rate of PTX increased significantly from micelle and exerted significant controlled release effect. In vitro and in vivo anti-tumor studies revealed that the micelles exhibit an excellent PTT and synergistic chemotherapy of cancer via NIR light-triggered release of PTX from micelles, eventually resulting in decreased cancer recurrence rates. Our results support that this NIR light-triggered targeted therapeutic system has the advantages of precisely locating on tumor sites, sensitive imaging for diagnosis, controlled drug release and accurate therapy. This novel system has huge potential in the new era of combination therapies for tumor treatment.In this manuscript, we developed NIR light-triggered micelles that could simultaneously deliver anticancer drug PTX and NIR dye cypate to tumor sites for combined chemo-photothermal therapy. Cypate as a NIR imaging agent not only enhanced the dissociation of the micelles under irradiation, but also revealed high temperature response for PTT. Conjugation of cyclic peptide RGD to the micelle surface enhanced the uptake of the micelles to integrin receptor-positive breast cancer cell line MDA-MB-231, and the results further confirmed that active targeting ability was mediated by integrin receptor. To investigate the anti-tumor effect, we first investigate the"} +{"text": "The aim of this study was to investigate the antitumor roles of miR-217 in PDAC cells and to identify miR-217-mediated molecular pathways involved in PDAC aggressiveness. The expression levels of miR-217 were significantly reduced in PDAC clinical specimens. Ectopic expression of miR-217 significantly suppressed cancer cell migration and invasion. Transcription of actin-binding protein Anillin (coded by ANLN) was detected by our in silico and gene expression analyses. Moreover, luciferase reporter assays showed that ANLN was a direct target of miR-217 in PDAC cells. Overexpression of ANLN was detected in PDAC clinical specimens by real-time PCR methods and immunohistochemistry. Interestingly, Kaplan\u2013Meier survival curves showed that high expression of ANLN predicted shorter survival in patients with PDAC by TCGA database analysis. Silencing ANLN expression markedly inhibited cancer cell migration and invasion capabilities of PDAC cell lines. We further investigated ANLN-mediated downstream pathways in PDAC cells. \u201cFocal adhesion\u201d and \u201cRegulation of actin binding protein\u201d were identified as ANLN-modulated downstream pathways in PDAC cells. Identification of antitumor miR-217/ANLN-mediated PDAC pathways will provide new insights into the potential mechanisms underlying the aggressive course of PDAC.Analysis of our microRNA (miRNA) expression signature of pancreatic ductal adenocarcinoma (PDAC) revealed that Due to the aggressive nature of pancreatic ductal adenocarcinoma (PDAC), it is one of the most lethal malignancies in the world , 2. RecemicroRNA (miRNA) belongs to a family of small non-coding RNAs that fine-tune the expression of protein coding/noncoding RNAs by repressing translation or cleaving RNA transcripts in a sequence-dependent manner . A uniqumiR-375, and the observation that it regulated oncogenic zinc finger protein 36 ring finger protein-like 2 (ZFP36L2) in PDAC cells . Expression levels of miR-217 in PDAC cells were significantly elevated by 5-aza-dc treatment in cancer tissues by GEO database analyses . We identified 167 genes that were putative targets of miR-217 and were upregulated in PDAC specimens. Finally, we found that 19 genes had conserved sequences that were putatively targeted by miR-217 (Table To gain further insight into the molecular mechanisms and pathways regulated by tumor-suppressive http://www.oncolnc.org/). Kaplan-Meier survival curves showed that high expression of 6 genes was associated with poor prognosis in PDAC by TCGA database searching , whereas transfection with the deletion vector (binding site had been removed) blocked the decrease in luminescence Figure .NLN in PDAC cells, we carried out loss-of-function studies using si-ANLN transfectants. First, we evaluated the knockdown efficiency of si-ANLN transfection in PDAC cell lines. In the present study, we used two types of si-ANLN (si-ANLN-1 and si-ANLN-2). Based on qRT-PCR assessment and Western blot analyses, both siRNAs effectively downregulated ANLN expression in both cell lines and showed strong nuclear immunoreactivity Figure .ANLN, genome-wide gene expression and in silico analyses were performed in a PDAC cell line (PANC-1) transfected with si-ANLN. Our selection strategy is shown in Figure 2 ratio < - 1.0) in si-ANLN-transfected PANC-1 cells. We also assessed the downregulated genes using KEGG pathways and the GENECODIS program. With that approach, we identified 8 pathways and 15 genes that were significantly enriched had a significantly poorer overall survival for patients with PDAC . Expression of these clustered miRNAs were significantly downregulated in PDAC cells (data not shown). Past studies demonstrated that miR-217 were downregulated in esophageal adenocarcinoma cells and leukemia cells [in silico database search to identify antitumor miR-217 targets in PDAC cells. Nineteen genes were identified as putative miR-217 targets. Among them, we selected the actin-binding protein gene ANLN because our past studies showed that several actin-binding protein genes were overexpressed in cancer tissues and were deeply involved in promoting human cancer cell migration and invasion [miR-1,miR-133a, miR-145 and miR-218) [ANLN was directly regulated by antitumor miR-217 and knockdown of ANLN significantly inhibited cancer cell aggressiveness in PDAC cells.In this study, we confirmed the downregulation and antitumor function of ia cells , 28. Morinvasion \u201332. IntemiR-218) . Our preANLN gene was cloned as a human homologue of the Anln gene in Drosophila melanogaster that encodes a 1,125\u2013amino acid actin-binding protein. ANLN has a unique multi-domain structure: an actin-binding and a myosin-binding domain at the N-terminus and a pleckstrin homology domain at the C-terminus [The human terminus . ANLN isterminus . A smallterminus .ANLN was reported in several cancers and elevated expression appears to be involved in the metastatic potential of human cancers [ANLN was confirmed in PDAC cells, and Kaplan\u2013Meier survival curves showed that high expression of ANLN predicted shorter survival in patients with PDAC by TCGA dataset analysis. Moreover, our data showed that reduced expression of ANLN in PDAC cells suppressed cancer cell migration and invasion. Past studies showed that knockdown of ANLN expression in breast cancer cells arrested the cell cycle in S/G2 or G2/M transition [ANLN has multiple functions, (2) its expression affects several oncogenic pathways and (3) overexpression of ANLN enhances cancer cell aggressiveness.Overexpressed cancers \u201325. In n cancers . Likewis cancers . Our preansition . These fANLN in PDAC cells, we applied a genome-wide gene expression analysis using si-ANLN transfectants. Our data showed that several pathways were downstream from ANLN, such as the \u201cfocal adhesion\u201d, \u201cpathways in cancer\u201d, \u201cECM-receptor interaction\u201d and \u201cregulation of the actin cytoskeleton\u201d. Finally, we identified 15 genes that were upregulated in PDAC specimens and involved in those pathways. Among the 15 genes, high expression of ITGA3, GNA15, PLAU, FZD2, MSM and SERPINE1 predicted shorter survival in patients with PDAC as determined by TCGA database analysis. Our previous study showed that overexpression of ITGA3 enhanced cancer cell migration and invasion and directly regulated antitumor expression of miR-223 in prostate cancer [To investigate the molecular pathways regulated by e cancer .PLAU and PLAUR has been linked to tumor progression, metastasis, and shortened survival in cancer [miR-217/ANLN-mediated genes are deeply involved in PDAC pathogenesis. However, the detailed molecular mechanism how downstream genes of ANLN are regulated is still unclear. Future analysis is needed. Exploration of novel antitumor miR-217-mediated pathways may lead to the development of new treatment protocols for this disease.Integrins play an important role in cell adhesion by linking the cytoskeleton of cells to components in the extracellular matrix (ECM) by integrin-mediated signaling pathways . Severaln cancer \u201349. ThesmiR-217 was detected in PDAC clinical specimens. miR-217 acts as an antitumor miRNA through its targeting of ANLN expression in PDAC cells. Expression of ANLN enhanced cancer cell aggressiveness and its high expression predicts poorer survival of PDAC patients. Elucidation of miR-217/ANLN-mediated molecular networks may lead to a better understanding of PDAC pathogenesis and the development of new treatment protocols.In conclusion, downregulation of Clinical tissues specimens (n = 27) were collected from patients with PDAC who underwent curative surgical resection at Kagoshima University Hospital between 1997 and 2015. Normal pancreatic tissue specimens (n = 14) were obtained from noncancerous tumor-adjacent tissue. Each surgical specimen was histologically classified according to the TNM classification system . All patTotal RNA, including miRNA, was isolated using ISOGEN according to the manufacturer's protocol.miR-217 , ANLN , human GUSB and RNU48 were used as internal controls. Expression fold-changes were determined using the \u0394\u0394Ct method.Quantification of miRNA was performed using qRT-PCR as previously described , 51, 52.TM miRNA precursors for miR-217 (product ID: PM 12774), negative control miRNA (product ID: AM 17111), two ANLN siRNAs (product IDs: HSS122893 and HSS122895) and negative control siRNA (product ID: D-001810-10) were purchased from Thermo Fisher Scientific. The transfection efficiencies of miRNA in PANC-1 and SW 1990 cells were calculated as described in previous studies [PDAC cell lines were transfected with a miRNA mimic for gain-of-function experiments and siRNA for loss-of-function experiments. Pre-miR studies , 52.XTT assays were used to assess cell proliferation . Cell migration assays were performed with BD Falcon Cell Culture Inserts . Modified Boyden chambers containing Transwell-precoated Matrigel membrane filter inserts were used to quantitate cellular invasion. The protcols of these assays were described as previously , 51, 52.Protein lysates were collected 96 h after transfection and 20 \u03bcg of protein were separated using gel electrophoresis on e-PAGEL 5-20% gels before transfer to polyvinylidene fluoride membranes. Solutions of mouse anti-ANLN antibodies were diluted 1:750 for immunoblotting. Anti-GAPDH antibodies at a 1:1000 dilution were used as an internal loading control. A detailed description of the Western blotting procedure was published elsewhere , 51, 52.ANLN was evaluated in cancer-rich fields using high-power microscopy (400\u00d7).Tissue sections were incubated overnight at room temperature with anti-ANLN antibodies diluted 1:200 . Overnight, antibodies were visualized using an avidin-biotin complex (ABC) detection kit and a diaminobenzidine substrate system according to the manufacturer's protocol. The expression of miR-217 target genes, we used in silico analyses conducted as described previously [http://www.targetscan.org/vert_71) database and TCGA . Figure To identify eviously , 52. TheANLN in PDAC was assessed by RNA sequencing in TCGA OncoLnc (http://www.oncolnc.org/) [https://gdc-portal.nci.nih.gov/projects/TCGA-PAAD). We selected high and low ANLN expression groups defined by the median value, and data were analyzed by Kaplan-Meier survival curves and log-rank statistics.The clinical significance of nc.org/) , 54. We ANLN 3\u2032-untranslated region (UTR) was inserted between the XhoI\u2013PmeI restriction sites in the 3\u2032-UTR of the hRluc gene in the psiCHECK-2 vector . Alternatively, we used sequences that were missing the miR-217 target sites (position 132 - 139 or position 660 - 666). The synthesized DNA was cloned into the psiCHECK-2 vector. PANC-1 and SW1990 cells were transfected with 20 ng of the vector, 20 nM microRNAs, and 1 \u03bcL Lipofectamine 2000 in 100 \u03bcL Opti-MEM (Invitrogen). The procedure of dual-luciferase reporter assay was described previously [The partial wild-type sequence of the eviously , 52.ANLN. Genes downregulated by ANLN were categorized by KEGG pathways using the GENECODIS program.We used genome-wide gene expression analysis in a PDAC cell line (PANC-1) transfected with si-miR-217 and ANLN was evaluated using Spearman's rank test. The relationships among more than 3 variables and numerical values were analyzed using the Bonferroni-adjusted Mann-Whitney U-test. Overall survival (OS) after surgery was gauged using Kaplan\u2013Meier curves. Patients were divided into two groups based on ANLN expression, and differences in survival were estimated using the log-rank test. We used Expert StatView software for these analyses [The relationships between 2 groups and expression values obtained by RT-qPCR were analyzed using the Mann-Whitney U-test. The correlation between expression of analyses , 13, 17."} +{"text": "Epidermal growth factor receptor (EGFR) plays a pivotal role in the pathophysiology of esophageal squamous cell carcinoma (ESCC). However, the clinical effects of EGFR inhibitors on ESCC are controversial. This study sought to identify the factors determining the therapeutic efficacy of EGFR inhibitors in ESCC cells.Immortalized-human esophageal epithelial cells (EPC2-hTERT), transformed-human esophageal epithelial cells (T-Epi and T-Mes), and ESCC cells were treated with the EGFR inhibitors erlotinib or cetuximab. Inhibitory effects on cell growth were assessed by cell counting or cell-cycle analysis. The expression levels of genes and proteins such as involucrin and cytokeratin13 (a squamous differentiation marker), E-cadherin, and vimentin were evaluated by real-time polymerase chain reaction or western blotting. To examine whether mesenchymal phenotype influenced the effects of EGFR inhibitors, we treated T-Epi cells with TGF-\u03b21 to establish a mesenchymal phenotype . We then compared the effects of EGFR inhibitors on parental T-Epi cells and mesenchymal T-Epi cells. TE-8 - or TE-11R -derived xenograft tumors in mice were treated with cetuximab, and the antitumor effects of EGFR inhibitors were evaluated.Cells were classified as epithelial-like or mesenchymal-like phenotypes, determined by the expression levels of E-cadherin and vimentin. Both erlotinib and cetuximab reduced cell growth and the ratio of cells in cell-cycle S phase in epithelial-like but not mesenchymal-like cells. Additionally, EGFR inhibitors induced squamous cell differentiation (defined as increased expression of involucrin and cytokeratin13) in epithelial-like but not mesenchymal-like cells. We found that EGFR inhibitors did not suppress the phosphorylation of EGFR in mesenchymal-like cells, while EGFR dephosphorylation was observed after treatment with EGFR inhibitors in epithelial-like cells. Furthermore, mesenchymal T-Epi cells showed resistance to EGFR inhibitors by circumventing the dephosphorylation of EGFR signaling. Cetuximab consistently showed antitumor effects, and increased involucrin expression in TE-11R -derived xenograft tumors but not TE-8 -derived xenograft tumors.The factor determining the therapeutic effects of EGFR inhibitors in ESCC cells is the phenotype representing the epithelial-like or mesenchymal-like cells. Mesenchymal-like ESCC cells are resistant to EGFR inhibitors because EGFR signaling is not blocked. EGFR inhibitors show antitumor effects on epithelial-like ESCC cells accompanied by promotion of squamous cell differentiation.The online version of this article (doi:10.1186/s13046-017-0572-7) contains supplementary material, which is available to authorized users. Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive cancers with a poor prognosis despite recent advances in therapeutics . Among tTyrosine kinase inhibitors and monoclonal antibodies that inhibit EGFR signaling have been developed as EGFR inhibitors and haveRecently, EGFR signaling blockade has been shown to promote squamous cell differentiation in skin keratinocytes as well as cutaneous SCCs . The conIn this study, we treated various human esophageal epithelial cells including immortalized- or transformed-esophageal epithelial cells and human ESCC cells with EGFR inhibitors and identified factors that could explain the inhibitory effects of EGFR signaling on these cells.R175H (T-Epi) or cetuximab [100\u00a0\u03bcg/mL]) for 72\u00a0h. No inhibition of cell growth was observed in FEF3 cells treated with erlotinib or cetuximab. (C) Phosphorylated- and total-EGFR protein level in FEF3 cells treated with human recombinant EGF (rEGF) for 24\u00a0h determined by western blotting. Untreated EPC2-hTERT cells were used as a positive control. rEGF did not activate EGFR signaling in FEF3 cells. (D) Involucrin protein production levels in FEF3 cells treated with or without erlotinib or cetuximab for 72\u00a0h determined by western blotting. Untreated EPC2-hTERT cells were used as positive controls. Treatment with EGFR inhibitors did not increase involucrin protein production levels in FEF3 cells. (E) Phosphorylated- and total-EGFR protein levels in FEF3 cells treated with or without erlotinib or cetuximab for 72\u00a0h determined by western blotting. Untreated EPC2-hTERT cells were used as a positive control. Neither erlotinib nor cetuximab suppressed the phosphorylation of EGFR signaling in FEF3 cells . (JPEG 381 kb)Effects of EGFR inhibitors on esophageal fibroblast cells. (A) E-cadherin and vimentin protein production levels in FEF3 cells determined by western blotting. EPC2-hTERT cells were used as a control. (B) Cell growth of FEF3 cells treated with or without erlotinib or cetuximab. Results are represented as means\u00a0\u00b1\u00a0SD (bars). (n.s., not significant, vs vehicle control;"} +{"text": "Colorectal cancer (CRC) tends to occur at older age; however, CRC incidence rates have been rising sharply among young age groups. The increasing prevalence of obesity is recognized as a major risk, yet the mechanistic underpinnings remain poorly understood. Using a diet-induced obesity mouse model, we identified obesity-associated molecular changes in the colonic epithelium of young and aged mice, and we further investigated whether the changes were reversed after weight loss. Transcriptome analysis indicated that obesity-related colonic cellular metabolic switch favoring long-chain fatty acid oxidation happened in young mice, while obesity-associated downregulation of negative feedback regulators of pro-proliferative signaling pathways occurred in older mice. Strikingly, colonic DNA methylome was pre-programmed by obesity at young age, priming for a tumor-prone gene signature after aging. Furthermore, obesity-related changes were substantially preserved after short-term weight loss, but they were largely reversed after long-term weight loss. We provided mechanistic insights into increased CRC risk in obesity. Li et al. find that obesity-induced DNA methylation changes reprogram the colonic transcriptome, leading to a metabolic switch favoring long-chain fatty acid oxidation in young mice and a more tumor-prone gene signature after aging. Obesity-related changes are substantially preserved after short-term weight loss, but they are largely reversed after long-term weight loss. Colorectal cancer (CRC) is the third most common cancer worldwide . CRC tenEpigenetic mechanisms are fundamental to phenotypic changes induced by environmental and lifestyle factors. Regarding epigenetic mechanisms underlying the link between obesity and CRC, DNA methylation is a prime candidate. First, aberrant DNA methylation is observed in virtually all CRCs ; second,To mimic human obesity, we employed a diet-induced obesity mouse model, which exhibits metabolic dysfunctions and incrComparing young obese mice with age-matched control mice, 287 differentially expressed genes (DEGs) were identified using DESeq2 . Gene exAcss1, Acss2, and Acsm3) and Acyl-CoA dehydrogenase (Acadsb) that are specifically responsible for short- and medium-chain fatty acid activation and dehydrogenation during fatty acid oxidation (Slc27a2 and Acaa1b (Slc2a4 (glucose transporter), Pfkm (phosphofructokinase), and Pdp1 (PDH phosphatase) . Collectphatase) , as normphatase) . Moreovephatase) .Slc7a9, an amino acid transporter defined using mouse Encyclopedia of DNA Elements (ENCODE) data from over 100 mouse cell types and tissues ; 4,123 regions gained DNA methylation (hyper-DMRs) and 4,076 regions lost DNA methylation (hypo-DMRs) in obese mice compared with control mice . Althoug tissues . BETA inTo infer the biological impacts of obesity-associated DNA methylation changes, we performed IPA of DMR target genes. They were predominantly involved in cancer (267 of 321) and gastrointestinal disease (229 of 321), and they were significantly enriched with lipid and carbohydrate metabolism genes . CorrelaTo test whether obesity-related DNA methylation changes prime for future gene expression changes at a later stage of life, we ran BETA with obesity-related DMRs from young mice and differential gene expression data from aged mice. We reasoned that, if that is true, those DMRs should also show significant association with obesity-related DEGs from aged mice. In fact, those DMRs exhibited even more significant associations with DEGs from aged mice, especially with downregulated genes, than with DEGs from young mice .Next, we explored the mechanistic explanation for that. Since a substantial number of DMRs overlapped with regulatory regions and S2D Errfi1, Spry1, Rasa4, Dusp1, and Dusp5) (Cish) (Mark4) , nuclearTnfaip3) , transfod Spsb1) , JAK/STA) (Cish) , and mTO (Mark4) . These sMap4k2, Map4k5, and Mapk12) (Chin et Mapk12) . SAPK pa Mapk12) , the att Mapk12) , some of Mapk12) , includi Mapk12) ; the maj Mapk12) , presuma Mapk12) , we inve Mapk12) . A subst Mapk12) . CollectNdufa3, Ndufa4, Ndufa8, and Ndufa12) and a subunit of mitochondrial ATP synthase (Atp5g1) were also upregulated with persistent changes after weight loss were shown were also significantly dysregulated in formerly obese mice in the same direction as in obese mice . These cConsidering that those formerly obese mice just returned to normal weight after 5 weeks of diet-switching, to investigate whether obesity-related dysregulation of gene expression can be completely reversed after long-term weight loss, we performed RNA-seq in the colonic epithelium of mice with diet-switching for 28 weeks. Body weights of these mice returned to normal after 5 weeks of diet-switching, and they stayed at the same level as control mice since then . Using hCollectively, obesity-related changes in DNA methylation and gene expression were substantially preserved after short-term weight loss, but gene expression changes mostly went back to normal after long-term weight loss.The genes altered in obese mice at both life stages are involved in fatty acid metabolism . The besWe characterized metabolic features of CRCs based on their changes in fatty acid metabolism and glycolysis. Single-sample gene set enrichment analysis (ssGSEA) was usedCellular metabolism was once thought to be a mere consequence of cellular state; it is now recognized as a key player in cell fate determination . ColonicThe epigenetic machinery is highly responsive to metabolic cues, because it relies on intermediate metabolites as substrates or cofactors . ObesityA striking feature of colonic gene expression changes, in aged obese mice compared with age-matched controls, is the downregulation of integral components of SAPK pathways and the downregulation of negative feedback regulators of pro-survival and pro-proliferative signaling pathways, including the EGFR/RTK-RAS-ERK/MAPK cascade, NF-\u03baB signaling, TGF-\u03b2 signaling, JAK/STAT signaling, and mTORC1 . These cRemarkably, obesity-related changes in DNA methylation and gene expression were substantially preserved after short-term weight loss, but gene expression changes were largely reversed after long-term weight loss and S6, In summary, we profiled colonic DNA methylome, transcriptome, and metabolome to identify obesity-related molecular pathophysiological changes in the colon. Those changes were not due to alterations of cell composition in the colonic epithelium, since no significant expression changes of cell-type-specific marker genes were observed in obese mice . We provAlthough colon tumors occur less frequently than small intestine tumors in mice unlike humans , we usedSix-week-old male C57BL/6J mice were fed either a low-fat diet or a high-fat diet . The source of fat in the diets is lard. Specifically, five mice were put on each dietary regimen as follows: (1) low-fat diet for 20 (or 43) weeks; (2) high-fat diet for 20 (or 43) weeks; and (3) high-fat diet for an initial 15 weeks and then switched to a low-fat diet for another 5 (or 28) weeks. The mice were housed in a specific pathogen-free facility. Body weight was measured weekly after diet-switching. At the end of the study, the mice were humanely euthanized and the colons were collected for further experiments. All animal experiments were approved by the NIEHS Animal Care and Use Committee, and they were performed according to NIH guidelines for care and use of laboratory animals.The colonic epithelium was isolated as previously described . GenomicmRNA sequencing libraries were prepared using the TruSeq Stranded mRNA Sample Prep Kit (Illumina) and sequenced on HiSeq 2000 (paired-end 50 bp). Sequencing reads were mapped against mm10 reference genome using TopHat . Mapped Genomic DNA was sonicated to an average size of 200 bp using a Covaris S220 instrument. DNA fragments were end-repaired, adenylated, and ligated to Illumina-compatible adaptors using BIOO NEXTflex Bisulfite-Seq Kit. Bisulfite conversion was performed using EZ DNA Methylation-Lightning Kit (Zymo Research). PCR was then carried out to enrich bisulfite-converted and adaptor-ligated fragments. The libraries were sequenced on Nextseq 500 (paired-end 75 bp). According to the coverage recommendations for WGBS , we sequSequencing reads were mapped to mm10 reference genome via Bismark with BowRao Scott Likelihood Ratio Test , 1987 waRNA-seq libraries were prepared using TruSeq Stranded Total RNA Library Prep Kit with Ribo-Zero H/M/R Gold (Illumina) and sequenced on Illumina NextSeq 500 (paired-end 76 bp). Read pairs were filtered by a mean base quality score >20, followed by adaptor-trimming with CutAdapt, and then they were mapped to mm10 reference genome with spliced transcripts alignment to a reference (STAR) . Mapped Data analysis was performed using Partek and GraphPad Prism. The heat-maps were prepared using Partek. Overlap significance was calculated using hypergeometric test. Body weights and gene expression data were represented as mean \u00b1 SEM.12345678"} +{"text": "The tumor-associated Thomsen-Friedenreich glycoantigen (TF-Ag) plays an important role in hematogenous metastasis of multiple cancers. The LTQ Orbitrap LC-MS/MS mass spectrometry analysis of cell surface TF-Ag proteome of metastatic prostate cancer cells reveals that several cell surface glycoproteins expressing this carbohydrate antigen in prostate cancer are either known as stem cell markers or control important cancer stem-like cell functions. This outcome points to a potential link between TF-Ag expression and prostate cancer stem-like phenotype. Indeed, selecting prostate cancer cells for TF-Ag expression resulted in the enrichment of cells with stem-like properties such as enhanced clonogenic survival and growth, prostasphere formation under non-differentiating and differentiating conditions, and elevated expression of stem cell markers such as CD44 and CD133. Further, the analysis of the recent literature demonstrates that TF-Ag is a common denominator for multiple prostate cancer stem-like cell populations identified to date and otherwise characterized by distinct molecular signatures. The current paradigm suggests that dissemination of tumor cells with stem-like properties to bone marrow that occurred before surgery and/or radiation therapy is largely responsible for disease recurrence years after radical treatment causing a massive clinical problem in prostate cancer. Thus, developing means for destroying disseminated prostate cancer stem-like cells is an important goal of modern cancer research. The results presented in this study suggest that multiple subpopulation of putative prostate cancer stem-like cells characterized by distinct molecular signatures can be attacked using a single target commonly expressed on these cells, the TF-Ag. Thomsen-Friedenreich antigen is a core 1 glycan structure Gal\u03b21\u20133GalNAc\u03b11- O-linked to serine or threonine, which is commonly present in a cryptic form on many normal cell types, but is unmasked and reactive on the vast majority about 90%) of all human cancers including colon, breast, bladder, prostate, liver, ovary and stomach , 3, pros0% of allTo date, in various types of cancer TF antigen has been shown to be expressed on several glycoproteins. Specifically, the mucin MUC1 has been shown to express TF-Ag in breast and coloIndeed, selecting TF-Ag expressing PC-3 prostate cancer cells yielded a subpopulation of cells with enhanced stem cell properties such as clonogenic survival, clonogenic growth, prostasphere formation under both non-differentiating and differentiating conditions, and enhanced expression of stem cell markers compared with their TF-Ag negative counterparts. Taken together, these results strongly suggest that TF antigen expression in prostate cancer cells is associated with the stem cell like phenotype. It appears that TF antigen could be a common denominator for multiple populations of prostate cancer stem like cells identified to date and otherwise characterized by distinct cell surface signatures . ConsequIn the present work, we employed a method combining biotinylation of cell surface proteins followed by a streptavidin agarose pull-down, lectin affinity chromatography, and LC-MS/MS mass spectrometry analysis to identify cell surface TF-Ag glycoprotein carriers expressed on PC-3 and DU-145 human prostate carcinoma cell lines.The cell surface proteins were first labeled by Sulfo-NHS-SS-Biotin, followed by the affinity purification using streptavidin-coated agarose beads. Next, upon reduction with DTT, disulfide bonds were broken to release biotinylated proteins from the beads , which wThe enriched TF-Ag carrying glycoproteins were digested and analyzed by LC-MS/MS using LTQ Orbitrap mass spectrometry. Figure In three independent experiments, the LTQ Orbitrap analysis resulted in the identification of 45, 152, and 97 proteins from biotinylated PNA-lectin tandem affinity purification fractions from DU-145 cells. Similarly, 123, 39, 102, and 102 proteins were identified in four independent experiments with PC-3 cells. However, due to the limitations of immunoprecipitation technique and high sensitivity of the LTQ Orbitrap mass spectrometry, not all of the proteins identified in these experiments are cell surface TF-Ag carriers. A significant number of them could be either non-specifically absorbed high abundance intracellular proteins or co-precipitated proteins interacting with the TF-Ag expressing glycopeptides. Thus, our next task was to screen out such proteins and select only cell surface glycoproteins. To achieve this, a consensus transmembrane prediction strategy combining the prediction results from several different transmembrane prediction software tools was used.A consensus prediction strategy employing several transmembrane prediction tools has been proven to improve overall accuracy compared to any of the individual methods. . Here, tAlthough TMHMM has the highest accuracy to predict transmembrane proteins among these three prediction software tools, the consensus strategy can provide a better result than any single prediction method alone. For example, isoform 1 of plectin was predicted as a non-transmembrane segment containing by TMHMM, but as having two transmembrane segments by MPEx and three transmembrane segments by TopPred. Thus, based on the consensus prediction strategy, plectin should be considered as protein with transmembrane segments, which is consistent with the published literature . All posOne remarkable outcome of this analysis was that a significant fraction of glycoproteins identified as cell surface TF-antigen carriers in PC-3 and DU-145 cells Figure are assoTo validate the mass spectrometry identified TF-Ag expressing cell surface proteins, eight of the identified glycoproteins were individually immunoprecipitated, resolved by SDS PAGE, and probed with anti-TF-Ag monoclonal antibody JAA-F11 using WeThe fact that the majority of prostate cancer cell surface TF-Ag-expressing glycoproteins are known as stem cell markers prompted us to ask a question whether selecting TF-Ag-positive prostate cancer cells will enrich for the cells with stem-like properties. To interrogate this question, we have used magnetic activated cell sorting (MACS) with biotinylated anti-TF-Ag antibody JAA-F11 to isolate TF-Ag-positive (TF+) and TF-Ag-negative (TF-) prostate cancer cells. Of note, in multiple isolation experiments, the average yield of TF+ cells was 3.93% \u00b1 0.901% for PC-3 and 0.36% \u00b1 0.21% for DU-145 cells (mean \u00b1 standard error of mean).One of the fundamental unique properties attributed to stem cells, including prostate cancer stem-like cells, is self-renewal. There are several commonly accepted tools for analyzing putative stem-like cells self-renewal capacity including clonogenic assay, which is based on seeding a small number of cells and monitoring colony formation over a defined time period. Remarkably, in contrast to TF- cells, the TF+ population exhibited clonogenic growth patterns even when plated at the regular densities and culturing conditions Figure and 3B. Another approach to analyzing self-renewal capacity of cancer stem-like cells is the sphere formation assay. The sphere formation assay is based on low adherence cultures in defined serum-free medium (non-differentiating conditions), or serum-supplemented medium (differentiating conditions) to produce multicellular spheroid aggregates called tumorspheres (prostaspheres in prostate cancer), which are enriched for cells with stem-like phenotypes.In our experiments, under non-differentiating conditions (serum free cultures in Matrigel) TF+ PC-3 cells formed greater than 2-fold more prostaspheres of a much bigger size than TF- cells did Figure . Under dAs TF+ prostate cancer cells demonstrated much higher ability for clonogenic survival and growth, as well as the propensity to form prostaspheres under both differentiating and non-differentiating conditions compared to TF- cells, our next question was whether TF-Ag selection leads to the enrichment of cells with elevated expression of stem cell markers such as CD44 and CD133. The comparison of CD44 and CD133 expression in TF+ and TF- cells by Western blot analysis revealed that TF+ cells express significantly higher levels of both CD44 and CD133 Figure . FurtherThe results of LTQ Orbitrap LC-MS/MS mass spectrometry analysis of prostate cancer cell surface TF-Ag proteome presented in this study demonstrate that many of TF-Ag expressing cell surface glycoproteins in prostate cancer cells are in fact either well known stem cell markers or the proteins controlling important cancer stem-like cell functions . This outcome strongly suggests that there is a link between TF-Ag expression and prostate cancer stem-like phenotype. Indeed, selecting prostate cancer cells for TF-Ag expression resulted in the enrichment of cells with stem-like properties characterizing self-renewal capacity such as enhanced clonogenic survival and growth Figure and prosFinding efficient approaches to targeting prostate cancer stem-like cells is an important challenge of modern cancer research. In the recent review, Yu et al. indicate that \u201cEach year, ~40,000 men who \u2018should\u2019 have been cured of their PCa [prostate cancer] by surgery or radiation therapy present with incurable metastatic disease that will manifest itself as metastatic lesions in the bone, usually years after primary treatment\u201d . The bes2 humidified atmosphere using RPMI-1640 medium supplemented with L-glutamine, 10% FBS, sodium pyruvate, and non-essential amino acids.Human prostate carcinoma cell lines PC-3 and DU-145 were from ATCC . The identity of cell lines was confirmed by IDEXX BioResearch using short tandem repeat (STR) profile following the completion of the experiments. Both cell lines were routinely maintained on plastic in 5% COFor each experiment, cell surface proteins in four T75 flasks of 70\u201380% confluent live cultures of either PC-3 or DU-145 cells were biotinylated and purified using Pierce Cell Surface Protein Isolation Kit according to the manufacturer instructions. Next, TF-Ag expressing glycoproteins were enriched using TF-Ag specific PNA (peanut agglutinin) lectin affinity chromatography and further purified using C18 zip-tips. Tryptic peptides were loaded onto a C8 trap column and analyzed on a system with Proxeon Easy nLC system attached to the LTQ Orbitrap mass spectrometer using CID and ETD fragmentation. All MS/MS spectra were searched against the IPI database (IPI_human_20090713.fasta) using SEQUEST\u2122 algorithm on the Sorcerer 2 integrated data appliances (IDA) server with default peak list extraction parameters. Post-search analysis was performed using the Scaffold, implementing PeptideProphet and ProteinProphet algorithms.http://www.cbs.dtu.dk/services/TMHMM-2.0/), MPEx 3.2 and TopPred 0.01 were used to identify the transmembrane proteins with biotinylated anti-TF-Ag antibody JAA-F11 was usedCD44 positive prostate cancer cells were isolated by MACS using CD44 MicroBeads and probed for TF-Ag expression using anti-TF-Ag antibody JAA-F11 and goat anti-mouse Alexa Fluor 594 conjugated antibody . For Western blot analysis, see complete list of antibodies used in"} +{"text": "Previous studies using zymosan-triggered inflammation and ALI model revealed that zymosan promotes inducible NO synthase (iNOS) expression in neutrophils, and that isoflurane inhibits zymosan-induced oxidative stress and iNOS biosynthesis. However, the underlying mechanisms remain largely unknown. We found here that in zymosan-primed neutrophils, iNOS is transcriptionally activated by NF-\u03baB, whose nuclear translocation is triggered by excessive reactive oxygen species (ROS) and consequently activated p38 MAPK. ROS production is attributed to zymosan-initiated Toll-like receptor 2 (TLR2) signaling, in which the adaptor MyD88 recruits and activates c-Src, and c-Src activates NADPH oxidase to generate ROS. Subanesthetic isoflurane counteracts the aforementioned zymosan-induced signaling by targeting N-methyl-D-aspartic acid (NMDA) glutamate receptor and thereby suppressing calcium influx and c-Src activation. Whereas iNOS accelerates NO/ONOO\u2212 production in neutrophils which eventually promote protein leak from pulmonary microvascular endothelial cells (PMVEC), isoflurane reduced NO/ONOO\u2212 release from zymosan-treated neutrophils, and thus relieves trans- PMVEC protein leak. This study provides novel insights into the roles of neutrophils and the underlying mechanisms in zymosan-induced ALI, and has implications for the therapeutic potential of subanesthetic isoflurane in attenuating inflammatory responses causing lung endothelial cell damage.Neutrophil release of NO/ONOO A 96-well microplate reader was used to measure the absorbance at 540 nm. Data were analyzed using Softmax Pro software. Sodium nitrite was dissolved in double-distilled water for use as standards.Production of nitrite .Quantitative data were expressed as means \u00b1 SEM. Groups were compared using Student's two-tailed unpaired"} +{"text": "PET/CT revealed extensive 18F-FDG avid lymphadenopathy as well as innumerable intensely 18F-FDG avid lung, liver and splenic nodules, highly concerning for malignancy. A PET-guided bone marrow biopsy of the posterior superior iliac spine revealed several non-necrotizing, well-formed granulomas, consistent with sarcoidosis. The patient was managed conservatively and remained clinically well over the subsequent 9 years of follow-up.A 60-year-old female with no significant medical history presented with hematuria. A computed tomography (CT) scan revealed extensive lymphadenopathy with hypodensities in the liver and spleen, and she was referred for an"} +{"text": "Caenorhabditis elegans , in germlines. These phasiRNAs, known as one of PIWI-interacting RNAs (piRNAs), mainly repress transposable elements. Similarly, reproduction-specific phasiRNAs have been identified in the family Poaceae, although DICER LIKE (DCL) protein-dependent phasiRNA biogenesis in rice is distinct from piRNA biogenesis in animals. In plants, phasiRNA biogenesis is initiated when 22-nt microRNAs (miRNAs) cleave single-stranded target RNAs. Subsequently, RNA-dependent RNA polymerase (RDR) forms dsRNAs from the cleaved RNAs, and dsRNAs are further processed by DCLs into 21 to 24-nt phasiRNAs. Finally, the phasiRNAs are loaded to ARGONAUTE (AGO) proteins to induce RNA-silencing. There are diverse types of phasiRNA precursors and the miRNAs that trigger the biogenesis. Their expression patterns also differ among plant species, suggesting that species-specific combinations of these triggers dictate the spatio-temporal pattern of phasiRNA biogenesis during development, or in response to environmental stimuli.It has been almost 30\u00a0years since RNA interference (RNAi) was shown to silence genes via double-stranded RNAs (dsRNAs) in Caenorhabditis elegans, double-stranded RNAs (dsRNAs) induce RNA silencing, called RNA interference (RNAi) bound to AGO4 cause RNA-directed DNA methylation (RdDM) and TGS. TGS, mediated by rasiRNAs via DNA methylation, suppresses many transposable elements (TEs) and repeat regions to prevent their transposition and transmission to the next generation , which are one type of phased small interfering RNAs (phasiRNAs) in plants.RNA silencing is classified into two types: transcriptional gene silencing (TGS) and post-transcriptional gene silencing (PTGS). In Arabidopsis thaliana, play a role in leaf phase transition from juvenile to adult phases of vegetative stages family, pentatricopeptide repeat (PPR) genes, and MYB transcription factors that include complement sequences recognized by AGO1/7-tasiRNAs complexes identified as precursor RNAs are single-stranded. They are transcribed with poly(A) tails without introns during early reproductive stages prior to meiosis in rice is a rice AGO protein that functions in the development of pre-meiotic germ cells and the progression of meiosis in both male and female organs. MEL1 binds dominantly to 21-nt phasiRNAs bearing a 5\u2032-terminal cytosine, although AGOs interacting with 24-nt phasiRNAs remain unknown encodes the 1.2-kbp non-coding RNA that regulates PSMS. The abundant LDMAR transcript induces male sterility during long days, and is processed into small RNAs of pms3 mutants, although the male sterility phenotype in pms3 is distinct from that of mel1 in both male and female tissues. is a phenomenon where particular rice strains show male sterility under long-day conditions, but are fertile under short-day conditions. Long-day-specific male-fertility-associated RNA , related to rice MEL1, is believed to function in female gametogenesis, since a semi-dominant ago5-4 mutant is defective in the initiation of mega-gametogenesis degradome, a modified RACE that detects cleaved transcripts, and a transcriptome analysis of mel1 and dcl4 mutants, have failed so far to identify any cleaved target transcripts with complementary sequences of these phasiRNAs, indicating that MEL1-phasiRNAs may not trans-regulate targets modifications are depleted in early meiosis I and AGO3 in Medicago truncatula (Fabaceae), the 22-nt miR1507, miR2109, and miR2118 act as triggers to initiate generation of 21- and 22-nt phasiRNAs from mRNAs that encode nucleotide binding and leucine-rich repeat (NB-LRR) pathogen-defense genes and these 22-nt miRNAs are conserved in other legumes and nonlegumes, members of the family Solanaceae . The functions of phasiRNAs derived from the large gene families of MYB and PPR remain unknown. In contrast, NB-LRR gene families are regulated by both triggered-miRNAs and phasiRNAs generated from NB-LRRs, and the phasiRNAs also regulates in trans at other NB-LRR loci in legumes. Thus, NB-LRR-phasiRNA regulation shows a self-reinforcing network involving cis- or trans- regulation , over 2000 phasiRNA-produced loci (PHAS loci) have been identified, including ~800 loci derived from NB-LRRs and ~600 from non-coding RNAs, recognized by 41 species of miRNAs. Interestingly, miR418/miR2118 super-families in gymnosperms target both NB-LRR RNAs and reproductive non-coding RNAs, resulting in production of phasiRNAs derived from both NB-LRR RNAs and reproductive non-coding RNAs and 21-nt phasiRNAs accumulate in pollen sacs, and miR2118 localizes in epidermis of premeiotic anthers in maize and African rice. In contrast, lincRNAs (24-nt PHAS), miR2275, and 24-nt phasiRNAs are expressed in tapetum and meiocytes in meiotic anthers How are numerous lincRNAs regulated during specific developmental stages and pathogenesis? (2) What is the molecular mechanism of lincRNA cleavage by 22-nt miRNAs? (3) What is the primary role of phasiRNAs in plants? Answering these questions will require experiments exploiting technologies such as genomics/epigenetics using non-coding RNA mutants, cell sorting, biochemistry, and structural analysis. Such studies are expected to help us to understand the significance of non-coding regions in both plants and animals."} +{"text": "TP63 or TNFRSF10B. The promoter activity of LTR12 is largely confined to the testes, silenced in testicular carcinoma, but reactivated in testicular cancer cells by broad-range histone deacetylase (HDAC) inhibitors. Here we show that inhibition of HDAC1-3 is sufficient for LTR12 activation. Importantly, HDAC inhibitors induce LTR12 activity not only in testicular cancer cells, but also in cells derived from many additional tumor species. Finally, we characterize the transcription factor NF-Y as a mediator of LTR12 promoter activity and HDAC inhibitor-induced apoptosis, in the context of widespread genomic binding of NF-Y to specific LTR12 sequences. Thus, HDAC inhibitor-driven LTR12 activation represents a generally applicable means to induce proapoptotic genes in human cancer cells.A considerable proportion of the human genome consists of transposable elements, including the long terminal repeats (LTRs) of endogenous retroviruses. During evolution, such LTRs were occasionally inserted upstream of protein-coding genes, contributing to their regulation. We previously identified the LTR12 from endogenous retrovirus 9 (ERV9) as a regulator of proapoptotic genes such as In-e LTR12s revealede LTR12s . NF-Y bie LTR12s . In line . The inactive promoter region of the myoglobin gene MB was analyzed as a negative control. CCNB1/Cyclin B1, which is strongly bound by NF-Y [To determine whether NF-Y was also present on LTR12 in a testicular context, and whether its binding pattern was subject to regulation by HDAC inhibitors, we assessed NF-Y chromatin binding in testicular cancer cells. Coordinates of NF-Y peaks within LTR12 sequences were first retrieved from the genome-wide analyses in HeLa, GM12878 and K562 cells . Of note by NF-Y , was detFinally, we determined how NF-Y affects the promoter activity of LTR12 in the presence or absence of HDAC inhibitors. To this end, we removed the subunits alpha , H1299 (adenocarcinoma of the lung), HeLa and U2OS (osteosarcoma) cells were maintained in Dulbecco's Modified Eagle medium supplemented with 10% FBS. Ovcar-3 (ovarian carcinoma), K562 (chronic myelogenous leukemia) and HuT-78 (cutaneous T-cell lymphoma) cells were maintained in RPMI 1640 medium with 10% FBS. The HDAC inhibitors Trichostatin A (Sigma-Aldrich), SAHA, Mocetinostat, Entinostat, PCI-34051, Droxinostat and Tubastatin A hydrochloride were dissolved in DMSO and added as indicated. Corresponding amounts of DMSO alone were added to controls.\u00ae (Invitrogen), followed by reverse transcription with Moloney Murine Leukemia Virus reverse transcriptase (New England Biolabs) and a mixture of oligo(dT) and random nonamer primers. A SYBR Green master mix including Taq polymerase (Primetech) was used for real-time PCR. The primer sequences are shown in RPLP0or to RSP20 as reference genes as indicated and calculated using the 2-\u0394\u0394Ct method.Total RNA was isolated using TRIzol6 cells was incubated with 2 \u03bcg anti-NF-YB antibody or corresponding amounts of anti-IgG antibody and 30 \u03bcl protein A/G plus agarose beads (Santa Cruz). After multiple washing steps and purification, the ChIP samples were analyzed by qPCR. The primer sequences were designed according to the target sequences, and the following data sets were used. GSM935429, GSM935433, GSM935408, GSM935508, GSM935506 and GSM935507.Based on the NF-YA and NF-YB binding information provided by Fleming et al. and bed-et al. . The infScrambled control (shSC), NF-YA (shNF-YA) and NF-YB (shNF-YB) shRNAs were cloned into the pLKO.1 vector (Sigma Aldrich). Viral supernatants expressing sh-scramble (control vector), sh-NF-YA and sh-NF-YB were prepared by transfecting HEK293T packaging cells. Briefly, shRNA plasmids and second generation packaging plasmids (VSVG and pCMV-dR8.74) were transfected into HEK293T cells. Lentivirus-containing supernatants were collected 48 h after transfection, filtered and frozen until use.Hela cells were transduced with sh-SC or sh-NF-YA or sh-NF-YB, treated with DMSO or TSA 54 hours after transduction, and collected at 18 hrs after treatment. Knockdown and treatment efficiency were assayed by PCR on cDNAs and by Western blot analysis on whole cell protein extracts using anti-NF-YA (Santa Cruz), anti NF-YB (GeneSpin), anti H3K9Ac (Abcam) and anti-Vinculin (Sigma) antibodies. Total RNA was prepared by Trizol extraction and reverse transcribed using the Iscript cDNA Synthesis kit (BIORAD 170-8890).After SDS-polyacrylamide gel electrophoresis and transfer on nitrocellulose, blots were incubated overnight with antibodies to PARP or Caspase 3 , each diluted 1:1000 in TBST with 4% BSA, followed by incubation with secondary antibodies coupled to peroxidase (1:10000) and chemiluminescent detection."} +{"text": "Drosophila melanogaster testis for progress through spermatogenesis. Phenotypic analysis of candidate genes pinpointed the stage of germline development disrupted. Bioinformatic analysis revealed that particular gene classes were associated with specific developmental transitions. Requirement for genes associated with endocytosis, cell polarity, and microtubule-based transport corresponded with the development of spermatogonia, spermatocytes, and spermatids, respectively. Overall, we identify mechanisms that act specifically in the somatic cells of the testis to regulate spermatogenesis.Spermatogenesis is a dynamic developmental process requiring precisely timed transitions between discrete stages. Specifically, the germline undergoes three transitions: from mitotic spermatogonia to spermatocytes, from meiotic spermatocytes to spermatids, and from morphogenetic spermatids to spermatozoa. The somatic cells of the testis provide essential support to the germline throughout spermatogenesis, but their precise role during these developmental transitions has not been comprehensively explored. Here, we describe the identification and characterization of genes that are required in the somatic cells of the Drosophila melanogaster testis. Using fertility assays we identified 281 genes that are required in somatic cyst cells for fertility. To better understand the role of these genes in regulating spermatogenesis we classified the resulting phenotypes by the stage of germline development disrupted. This revealed distinct sets of genes required to support specific stages of spermatogenesis. Our study characterizes the somatic specific defects resulting from disruption of candidate genes and provides insight into their function in the testes. Overall, our findings reveal the mechanisms controlling Drosophila melanogaster spermatogenesis and provide a resource for studying soma-germline interactions more broadly.Sexual reproduction in animals requires the production of male and female gametes, spermatozoa and ova, respectively. Gametes are derived from specialized cells known as the germline through a process called gametogenesis. Gametogenesis typically takes place in a gonad and requires the germ cells to be surrounded by specialized somatic cells that support germline development. While many prior studies have identified germline specific genes required for gametogenesis few have systematically identified genes required in the somatic cells for gametogenesis. To this end we performed an RNAi screen where we disrupted the function of genes specifically in the somatic cyst cells of the Spermatogenesis is a highly choreographed developmental process that involves dramatic changes in cell morphology and the integration of multiple regulatory cues. Orchestrating such a process would be challenging even if spermatogenesis involved only one cell type, but it in fact includes two very different types of cells, somatic cells (soma) and germ cells (germline). Close association between the soma and the germline is a conserved feature of spermatogenesis in animal testes . ImportaSpermatogenesis can be divided into three distinct developmental stages: the spermatogonial-stage, characterized by mitotic germ cells; the spermatocyte-stage, characterized by meiotic germ cells; and the spermatid-stage, characterized by the morphogenetic changes that form spermatozoa . The speDrosophila melanogaster testis provides a powerful model to study the conserved process of spermatogenesis as it allows convenient imaging of the transitions between different stages of germline development , raised at 20\u00b0C and shifted to 29\u00b0C for 3 days during pupation to kill males. Individual male fertility assays were done in triplicate and listed as sterile if no larva/pupae were present 14 days post-mating. If the majority of fertility assays yielded sterile males, more males were tested and additional RNAi lines targeting the gene were screened.Female flies were collected from bam-GFP. Spermatocytes were identified by expression of Vasa, dispersed DAPI staining, and their large size; meiotic spermatocytes identified by expression of both Vasa and Boule. Early spermatids identified by expression of Boule, polarization of their nuclei, and their elongated shape; individualizing spermatids identified by expression of DonJuan::GFP. Spermatozoa identified by expression of DonJuan::GFP and their accumulation in the seminal vesicle as individual cells. Phenotypes were assigned based on the stage of germline development that was disrupted in the majority of samples. Germline disruption was defined as over-proliferation, disintegration, death, or other disorders the germ cells including the failure of spermatozoa to reach the seminal vesicle.Spermatogonia were identified by expression of Vasa, dense DAPI staining nuclei, and their small size; differentiating spermatogonia were identified by expression of both Vasa and Protein-protein interaction network built ustj-Gal4 (P-element insertion {GawB}NP1624) obtained from the Bloomington Drosophila Stock Center [BDRC]). Additional lines from the BDSC included the Virginator G,hs-hid), UAS-Dcr2 (UAS-Dicer2), UAS-mGFP (UAS-mCD8::GFP), UAS-mRFP (UAS-mCD8::Tomato), UAS-RFP.nls (UAS-RedStinger), Baz::GFP (bazCC01941), Dlg1::GFP (dlg1YC0005), DonJuan::GFP (dj-GFP.S), and hh-LacZ (hhP30). Vasa::GFP (vas.EGFP.HA) provided by the Kyoto Drosophila Genetic Resource Center (DGRC). upd-LacZ (upd1-PD) courtesy of David Bilder, University of California Berkeley, USA. bam-GFP (bam-GFP-799/+133) courtesy of Christian B\u00f6kel, Center for Regenerative Therapies Dresden, Germany. UAS-RNAi lines obtained from the Transgenic RNAi Project (TRiP), the Vienna Drosophila RNAi Centre (VDRC), and the National Institute of Genetics (NIG). RNAi lines used in Figs CG10483.RNAi (TRiP.HMS01023), UAS-ance.RNAi (TRiP.HMS03009), UAS-AP-2\u03bc.RNAi (TRiP.JF02875), UAS-syx7.RNAi (TRiP.JF02436), UAS-msn.RNAi (TRiP.HMJ02084), UAS-pyr.RNAi (VDRC.36523), UAS-rab5.RNAi , UAS-rab7.RNAi (VDRC.40338), UAS-baz.RNAi (VDRC.2914), UAS-par-6.RNAi (VDRC.108560), UAS-aPKC.RNAi (VDRC.2907), UAS-dlg1.RNAi (TRiP.JF01365), UAS-scrib.RNAi (TRiP.JF03229), UAS-l(2)gl.RNAi (TRiP.HMS01522), UAS-\u03b23tub.RNAi (VDRC.34607), UAS-glued.RNAi (TRiP.JF02803), UAS-Dhc64C.RNAi (TRiP.JF03177). See Samples fixed using 4% PFA in PBS for 15 minutes. Antibodies incubated in PBS supplemented with 0.2% BSA and 0.3% Triton-X at 4\u00b0 with the exception of rabbit-anti-STAT incubated at 20\u00b0. Primary antibodies included rat-anti-Filamin.C\u2019terminus , rabbit-anti-Boule , guinea pig-anti-Tj , rabbit-anti-Zfh1 , guinea pig-anti-Zfh1 , rabbit-anti-STAT , rabbit-anti-pMad , rabbit-anti-Baz , rabbit-anti-\u03b23tub , chicken-anti-GFP , goat-anti-Vasa , rat-anti-dsRed , chicken-anti-LacZ , mouse-anti-\u03b1Spectrin , mouse-anti-Eya , mouse-anti-FasIII , mouse-anti-Patched , mouse-anti-Dlg1 , mouse-anti-Cora , rat-anti-DEcad . Secondary antibodies included CF405S, Alexafluor-488, Cy3, and Cy5 conjugates.\u03b11tub-Gal80ts;tj-Gal4,\u03b11tub-Gal80ts/UAS-rab5.RNAi flies raised at 18\u00b0C and split into two cohorts 1\u20133 Days Post Eclosion (DPE) and raised at 18\u00b0C or 29\u00b0C for 14 days. Cyst cell clones made using c587-Gal4,UAS-RFP.nls,UAS-mGFP,hs-Flp;\u03b11tub-Gal80,Frt40A/Frt40A;UAS-rab5.RNAi flies and the \u2018Coloc 2\u2019 plugin were utilized for Pearson co-localization analysis. Terminal epithelium and seminal vesicle images Click here for additional data file.S2 Fig(A-B) Germline development defect resulting from cyst cell specific knockdown of Rab5. Germ cells labelled by Vasa, spectrosomes and fusomes labelled by \u03b1-Spectrin, and CySCs labelled by Zfh1. Single channel showing \u03b1-Spectrin. (A) GSCs in the apical tip of the testis contain small dot shaped spectrosomes. Differentiating spermatogonia remain connected by thin, branching fusomes (Arrowhead) while spermatocytes are connected by large, branching fusomes (Arrow). (B) Knockdown of Rab5 in cyst cells leads to overgrowth of germ cells connected by thin, branching fusomes similar to those found in spermatogonia (Arrowhead). Scale bars are 50\u03bcm.(TIF)Click here for additional data file.S3 Fig(A-C) Changes in BMP signalling after Rab5 knockdown in cyst cells. CySCs labelled by Zfh1, germ cells labelled by Vasa, BMP signalling detected by phosphorylated-Mad (pMad). Males aged 14 days post eclosion (DPE). Single channel showing pMad. (A) In control testes pMad is detectable in GSCs indicating active BMP signalling (Arrowhead). (B) Knockdown of Rab5 in cyst cells leads to increased levels of pMad in the germ cells near an enlarged stem cell niche (Arrowhead). (C) Increased levels of pMad are also found in the germ cell tumour-like growths that develop after knockdown of Rab5 in cyst cells. (D) Hh signalling is detected in the cyst cell tumour-like growths that develop outside of the stem cell niche after knockdown of Rab5. CySCs labelled by Zfh1, hub cells labelled by the Hh ligand reporter hh-LacZ, Hh signalling detected by Patched accumulation in CySCs. Males aged 14 DPE. (D\u2019) Single channel showing Patched. (E) JAK-STAT signalling is detected in the cyst cell tumour-like growths that develop outside of the stem cell niche after knockdown of Rab5. CySCs labelled by Zfh1, hub cells labelled by the JAK-STAT ligand reporter upd-LacZ, JAK-STAT signalling detected by STAT expression in CySCs. Males aged 14 DPE. (E\u2019) Single channel showing STAT. Scale bars are 20\u03bcm.(TIF)Click here for additional data file.S4 Fig(A) A single cyst cell clone at the early spermatocyte-stage labelled by membrane bound GFP (mGFP). The Par polarity module protein Baz and the Scribble polarity module protein Dlg1 both localize to the membrane at the junction between the two encapsulating cyst cells (Arrowheads). The labelled cyst cell clone has a thin membranous extension that spreads along the sheath of the testis. (A\u2019) Single channel showing Baz. (A\u201d) Single channel showing Dlg1. (B) Individual spermatocyst extracted from the testis and labelled for the Par polarity module protein Baz::GFP and the Scribble polarity module protein Dlg1. Both proteins extend around the entire circumference of the junctional belt connecting the two encapsulating cyst cells. A section of this image appears in (B\u2019) Single channel showing Baz::GFP. (B\u201d) Single channel showing Dlg1. Scale bars are 50\u03bcm.(TIF)Click here for additional data file.S1 TableDrosophila melanogaster testis for fertility. List includes gene names and identification, a summary of their Gene Ontology annotations, the phenotype when knocked-down in somatic cyst cells using RNAi, and mouse homologs with stage specific expression in mouse Sertoli cells (Asterisk) [List of genes required in the somatic cyst cells of the sterisk) .(PDF)Click here for additional data file.S2 Table(RNAi) Is a list of all UAS-RNAi lines that were expressed in the cyst cells of the testis using tj-Gal4 in our screen as well as the raw data from the male fertility assays that were carried out subsequently. (Genes) Is a list of all genes targeted by RNAi knockdown in the cyst cells and a summary of the results of male fertility assays. Additional data includes gene classification, knockdown phenotype, and a comparison to prior gene annotations including the Gene Ontology (GO) term \u2018spermatogenesis\u2019, male sterile alleles listed in Flybase, phenotypes identified by other genetic screens in the somatic cells of the fly the gonad, and mouse homologs expressed in a stage specific manner in Sertoli cells. (Sterile genes) Is a list of candidate genes required in the somatic cyst cells of the testis for fertility Is a list of enriched GO terms associated with candidate genes in each phenotypic class characterized using the DAVID algorithm [(Screen comparison) Is a list comparing the results of our screen to prior screens in the somatic cells of the fly gonad including screens using tj-Gal4>UAS-RNAi in male fly cyst cells [tj-Gal4>UAS-RNAi in female fly follicle cells [This Excel file contains a number of individual sheets as follows: see also . Click here for additional data file."} +{"text": "Both preclinical and clinical investigations suggest that Notch signalling is critical for the development of many cancers and for their response to chemotherapy. We previously showed that Notch inhibition abrogates stromal-induced chemoresistance in lymphoid neoplasms. However, the role of Notch in acute myeloid leukemia (AML) and its contribution to the crosstalk between leukemia cells and bone marrow stromal cells remain controversial. Thus, we evaluated the role of the Notch pathway in the proliferation, survival and chemoresistance of AML cells in co-culture with bone marrow mesenchymal stromal cells expanded from both healthy donors (hBM-MSCs) and AML patients (hBM-MSCs*). As compared to hBM-MSCs, hBM-MSCs* showed higher level of Notch1, Jagged1 as well as the main Notch target gene HES1. Notably, hBM-MSCs* induced expression and activation of Notch signalling in AML cells, supporting AML proliferation and being more efficientin inducing AML chemoresistance than hBM-MSCs*. Pharmacological inhibition of Notch using combinations of Notch receptor-blocking antibodies or gamma-secretase inhibitors (GSIs), in presence of chemotherapeutic agents, significant lowered the supportive effect of hBM-MSCs and hBM-MSCs* towards AML cells, by activating apoptotic cascade and reducing protein level of STAT3, AKT and NF-\u03baB.These results suggest that Notch signalling inhibition, by overcoming the stromal-mediated promotion of chemoresistance,may represent a potential therapeutic targetnot only for lymphoid neoplasms, but also for AML. HES-1). Depending on the cell system, Notch signalling may act as tumor suppressor or oncogene -2,5-diphenyltetrazolium bromide metabolic activity assay. Apoptosis was evaluated by AnnexinV-FITC assay (MiltenyiBiotec). To study cell proliferation, AML cells were labelled with carboxyfluoresceinsuccinimidylester (CFSE) (Life Technologies) (5mM). For primary leukemia cells proliferation assay, complete RPMI medium was supplemented with IL-3, IL-6 and SCF, as previously described (15). After 4 days, AML cells were harvested, stained with anti-CD45 PerCP-Vio700 antibody and analyzed by FACS CantoII (BD Biosciences). MTT, AnnexinV-FITC and CFSE assay are described in detailed in BM-MSCs were expanded and characterized as previously described , 5. Co-cTHP1 cells were transfected with reporter plasmids encoding for an inducible RBP-Jk-responsive GFP reporter (Qiagen) using MACSfectin transfection reagent (MiltenyiBiotec). GFP signal were quantitatively measured by flow cytometry. Notch activity was determined by normalizing the activity of RBP-Jk-GFP to that of CMV-GFP plasmid.The pLKO.1-puro-CMV-TurboGFP lentivirus plasmid (SHC003) was obtained from Sigma. For gene silencing, shRNA plasmids targeted to RBP-jk, TRCN0000016207 (shRBP-jk), Scramble shRNA SHC016 were purchased from Sigma, as well asLentiviral Packaging Mix (SHP001). Plasmids were co-transfected into 293T cells usingMACSfectin transfection reagent (MiltenyiBiotec), at a ratio of 1:1(plasmid of shRNA:Packaging Mix). Media were replaced with DMEM + 10% FBS at 16 hours after transfection; viruses were collected at 48 and 72 hours after transfection and filtered through a 0.45 \u03bcm filter. HL-60 cells and THP1 cells were transduced by the lentivirus in the presence of the polycation Polybrene, and the stably transduced cells were then selected by puromycin (1\u03bcg/ml) for 4 weeks.hBM-MSCs or hBM-MSCs* were seeded in 24 wells plates. Once reached the confluence, cells were induced to differentiate by replacing growth medium by adipogenic or osteogenic differentiation media. Adipogenic differentiation medium consisted in DMEM supplemented with 18% FBS, 10mg/ml IBMX (Sigma-Aldrich), 100U/ml Insulin (Lilly) and 1mM Dexamethasone (Sigma-Aldrich). For Osteogenic differentiation, cells were incubated with StemMACSOsteo Diff Medium (MiltenyiBiotec). Differentiation media were changed each 3 days and cells were allowed to differentiate for 3 weeks; then cells were characterized with Oil Red O staining and Alizarin red for adipogenic andosteogenic differentiation, respectively.3VO4. Proteins were quantified using BCA protein assay kit and separated on a 10% or 12% polyacrylamide gel. Subsequently, proteins were transferred onto nitrocellulose membrane , labelled with appropriate antibody and acquired by LAS4000 instrument. Densitometric analyses were performed on scanned immunoblot images using the ImageJ analysis tool . GAPDH density was used to normalize density of each protein.Cells were lysed with an appropriate amount of RIPA buffer supplemented with complete Protease Inhibitor (Roche) and 1 mM Nat-test was used to compare 2 groups and one-way ANOVA followed by the Tukey's range test were applied to compare multiple groups. Mann-Whittney test was used for non-coupled, non-parametric comparison.Statistical analysis was performed using GraphPad Prism software . Data were expressed as mean \u00b1 standard error means (SEM). Student"} +{"text": "Our findings demonstrate a miR-532/miR-3064-mediated mechanism responsible for hTERT upregulation in OC cells, and reveal a possibility of targeting miR-532/miR-3064 for future treatment of OC.Human telomerase reverse transcriptase (hTERT) plays a crucial role in ovarian cancer (OC) progression. However, the mechanisms underlying hTERT upregulation in OC, and the specific microRNAs (miRNAs) involved in the regulation of hTERT in OC cells, remains unclear. We performed a bioinformatics search to identify potential miRNAs that bind to the 3'-untranslated region (3'-UTR) region of the hTERT mRNA. We examined the expression levels of miR-532/miR-3064 in OC tissues and normal ovarian tissues, and analyzed the correlation between miRNA expression and OC patient outcomes. The impacts of miR-532/miR-3064 on hTERT expression were evaluated by western blot analysis and hTERT 3'-UTR reporter assays. We investigated the effects of miR-532/miR-3064 on proliferation and invasion in OC cells. We found that miR-532 and miR-3064 are down-regulated in OC specimens. We observed a significant association between reduced miR-532/miR-3064 expression and poorer survival of patients with OC. We confirmed that in OC cells, these two miRNAs downregulate hTERT levels by directly targeting its 3'-UTR region, and inhibited proliferation, EMT and invasion of OC cells. In addition, the overexpression of the hTERT cDNA lacking the 3'-UTR partially restored miR-532/miR-3064-inhibited OC cell proliferation and invasion. The silencing of hTERT by siRNA oligonucleotides abolished these malignant features, and phenocopied the effects of miR-532/miR-3064 overexpression. Furthermore, overexpression of miR-532/miR-3064 inhibits the growth of OC cells Ovarian cancer (OC) is a highly aggressive disease and represents the most lethal gynecologic tumor in the world , 2. The Small regulatory RNAs such as microRNAs (miRNAs), have been linked to cancer development, and are reported to suppress hTERT expression in various cancer cells \u201315. It iThis study shows that miR-532 and miR-3064 that were down-regulated in OC tissues, can suppress the proliferation, EMT and invasion of OC cells by directly repressing hTERT expression.We obtained 60 specimens of serous OCs form the Department of Obstetrics and Gynecology, the First People's Hospital of Shangqiu . A total of 20 normal ovarian tissues were collected as controls from patients following surgery for uterine fibroids. All subjects provided written consent. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of the First People's Hospital of Shangqiu (reference number 2010\u2013104). All tissue samples were immediately snap-frozen in liquid nitrogen. Total RNA was isolated using TRIzol reagents .Human OC cell lines (SKOV-3 and ES-2) were purchased from the Cell Bank of the Chinese Academy of Science . Normal ovarian epithelial cells (NOEC) were obtained from Pricells Biotechnology & Medicine . All cell lines used in this study were maintained in DMEM/F12 supplemented with 10% fetal bovine serum . All miRNA mimics (30 nM), miRNA inhibitors (30 nM) and siRNAs (5 nM) were obtained from GenePharma , and were transiently transfected into OC cells using Lipofectamine 2000 following the manufacturer\u2019s protocol. Transfection of hTERT cDNA plasmids were also performed using Lipofectamine 2000 .5\u2032-CGGAAGAGTGTCTGGAGCAA-3\u2032, hTERT (reverse): 5\u2032-GGATGAAGCGGAGTCTGGA-3\u2032, glyceraldehyde 3-phosphate dehydrogenase : 5\u2032-CAATGACCCCTTCATTGACC-3\u2032 and GAPDH (reverse): 5\u2032-GACAAGCTTCCCGTTCTCAG-3\u2032. For the measurement of mRNA, real-time PCRs were performed using the Takara SYBR Premix Ex Taq II in a 7500 Real-Time PCR System (Applied Biosystems). The NCode miRNA qRT-PCR analysis was used for detecting mature miRNA levels according to the manufacturer\u2019s instructions. Forward primers are the exact sequences of the mature miR-532 and miR-3064. The housekeeping gene GADPH and small noncoding RNA U6 were used as internal controls for mRNA and miRNA quantification, respectively. All results were represented as the fold change relative to respective controls.The total RNA was isolated using TRIzol reagents (Invitrogen), and was reverse-transcribed into cDNA using the PrimeScript RT reagent kit according to the manufacturer\u2019s instructions. The resulting cDNAs were PCR amplified using the following gene-specific primers : hTERT , 10 ng of Renilla report plasmid , together with and 30 nM miRNA mimics or miRNA inhibitors with Lipofectamine 2000 . Then cells were lysed 24 hours later and assayed for luciferase activity using the Dual Luciferase Assay Kit . Predicted miR-532- or miR-3064-binding sites were mutated by RiboBio Co., Ltd. using the QuickChange II XL Site-Directed Mutagenesis Kit . The relative luciferase activity was calculated as a ratio of firefly luciferase activity divided by Renilla luciferase activity.3 per well in 96-well plates for 24 hours, and transfected as indicated. After 72 hours, 10 \u03bcl of Cell counting kit-8 solution was added to each well and incubated at 37\u00b0C for 2 hours; followed by the measurement of absorbance using an automated micro-plate reader (Corona Electric) at 450 nm.OC cells were seeded at a density of 5 \u00d7 104) were plated into upper chamber of Boyden chambers coated with Matrigel as described previously [OC cells for overexpression of miR-532/miR-3064 and the negative control lentiviral vector were purchased from Applied Biological Materials Inc. , and were prepared in accordance with standard protocols. ES-2 cells were infected with lentivirus and selected using 1 \u03bcg/ml puromycin for 4 weeks, to establish stable miR-532, miR-3064 or negative control (Neg) transfectants.6 ES-2 cells were subcutaneously injected in 0.1 ml of PBS into nude mice (n = 8 per group). After implantation for 7 days, tumor volume measurement began and was performed every 4 days, using the following formula: volume = length (mm) x width2 (mm2)/2. All the mice were sacrificed on the 26th day post-injection and the xenografts tissues were collected for immunohistochemical staining analysis. The xenografts tissues were formalin-fixed/paraffin-embedded and cut into 4 \u03bcm slides. The primary antibody used was anti\u2011Ki-67 . Staining was performed using the IHC detection kit .The animal protocol was approved by the First People's Hospital of Shangqiu\u2019s Institutional Animal Care and Use Committee and Ethics committee. BALB/c nude mice (five weeks old) were purchased from Beijing HFK Bioscience and maintained under pathogen-free conditions. For the subcutaneous tumor growth assay, 1 \u00d7 10t-test or Fisher's exact test was used for statistical analysis. Kaplan-Meier survival curves were plotted and the log-rank test was performed. Data are shown as mean \u00b1 SD of at least three independent experiments performed in triplicate. P < 0.05 were considered statistically significant.Statistical differences were analyzed using SPSS 17.0. 2-tailed Student\u2019s Using target prediction programs (TargetScan and DIANA-MicroT-CDS), we found that hTERT contains seed sequences for two putative miRNAs (miR-532 and miR-3064) in its 3'-UTR. Detailed information about these two miRNAs and their binding site sequences in the hTERT 3'-UTR are summarized in We screened OC patient clinical tissues (n = 60) and normal ovarian epithelial tissues (n = 20) for the endogenous expression of miR-532 and miR-3064 using qPCR. We found that the expression of miR-532/miR-3064 was significantly down-regulated in OC tissues compared with normal specimens . We furtTo delineate the clinical significance of miR-532 or miR-3064, we determined the correlations between the levels of miR-532/miR-3064 and clinicopathological factors. 60 OC patients were divided into two groups with higher (n = 30) or lower (n = 30) expression of miR-532 or miR-3064, using the median expression values of miR-532 or miR-3064 in OC samples. More importantly, lower levels of miR-532/miR-3064 were significantly associated with advanced tumor stage, higher tumor grade and higher incidence of lymph node metastasis . FurtherTo ascertain whether hTERT is directly targeted by miR-532/miR-3064, we performed gain-of-function experiments using ES-2 cells that express relatively low levels of miR-532/miR-3064. Our luciferase assays suggested that, the overexpression of either miR-532 or miR-3064 mimic markedly suppressed the luciferase activity of the wild-type (WT) version of hTERT 3'-UTR in ES-2 cells . ConversFurthermore, the western blotting analysis demonstrated that the transfection with miR-532/miR-3064 mimics reduced hTERT expression , while tin vitro cell invasion assay, and found that the ectopic expression of miR-532/miR-3064 strongly suppresses the proliferative and invasive capacity of OC cells , and has a key role in enhancing OC cell proliferation and invasion. Re-expression of miR-532 or miR-3064 might be a possible strategy for the treatment of OC.S1 Table(DOCX)Click here for additional data file."} +{"text": "Acer tegmentosum extract, has been shown to protect against liver damage caused by alcohol consumption. Therefore, in this study, we aimed to determine whether HIMH0021 could regulate alcoholic fatty liver and liver injury in mice. Oral administration of 10 days of Lieber-DeCarli ethanol plus a single binge of 30% ethanol (chronic-plus-binge model) induced steatosis and liver injury and inflammation in mice, which appears similar to the condition observed in human patients with alcohol-related diseases. HIMH0021, which was isolated from the active methanol extract of A. tegmentosum, inhibited alcohol-induced steatosis and attenuated the serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) during hepatocellular alcohol metabolism, both of which promote lipogenesis as well as liver inflammation. Treatment with HIMH0021 conferred protection against lipogenesis and liver injury, inhibited the expression of cytochrome P4502E1, and increased serum adiponectin levels in the mice subjected to chronic-plus-binge feeding. Furthermore, in hepatocytes, HIMH0021 activated fatty acid oxidation by activating pAMPK, which comprises pACC and CPT1a. These findings suggested that HIMH0021 could be used to target a TNF\u03b1-related pathway for treating patients with alcoholic hepatitis.Chronic alcohol consumption causes alcohol-induced lipogenesis and promotes hepatic injury by preventing the oxidation of hepatocellular fatty acids through the suppression of the activation of AMP-activated protein kinase (AMPK). HIMH0021, an active flavonoid compound, which is a component of the Alcohol-related diseases can be divided into two main categories: alcohol dependence and alcohol abuse \u20132. AlcohAlcohol exposure can also induce fatty liver by increasing the NADH/NAD+ ratio, increasing sterol regulatory element-binding protein-1 (SREBP-1) activity, decreasing peroxisome proliferator-activated receptor-\u03b1 (PPAR-\u03b1) activity, and increasing complement C3 hepatic levels. Alcohol exposure also increases NADH production in the process of metabolism by cytosolic alcohol dehydrogenase (ADH) and mitochondrial aldehyde dehydrogenase (ALDH2). Several studies have also shown that NADH is implicated in the disruption of many alcohol-related reactions in the cytoplasm and mitochondria, which leads to suppression of energy supply and fatty acid oxidation, which in turn results in alcoholic fatty liver \u20139.Long-term alcohol exposure can increase the levels of sterol regulatory element-binding protein-1 (SREBP-1), a master transcription factor that regulates lipogenic enzyme expression, by decreasing the activities of AMP-activated protein kinase and sirtuin-1 ,11. UpreAMPK is known to activate the phosphorylation of the target enzymes involved in lipid metabolism in many tissues, thus promoting fatty acid oxidation by inactivation of acetyl-CoA carboxylase (ACC) and activation of malonyl Co-A decarboxylase (MCD). ACC, a rate limiting enzyme for fatty acid biosynthesis, catalyzes the conversion of acetyl-CoA to malonyl-CoA, which is a potent inhibitor of carnitine palmitoyltransferase-1 (CPT-1). CPT-1 plays an important role in the transportation of fatty acids, which are metabolized via the mitochondrial \u03b2-oxidation pathway. MCD degrades malonyl-CoA . SubsequAcer tegmentosum extract has been shown to protect against liver injury caused by alcohol consumption -, positive mode m/z 435 [M+H] + and 457 [M+Na] +.(TIF)Click here for additional data file.S1 Table(d in ppm).(TIF)Click here for additional data file."} +{"text": "BRCA1-mutation carriers at high risk of developing breast cancer. Beyond its well-recognized bone-targeted activity impeding osteoclastogenesis, denosumab has been proposed to interfere with the cross-talk between RANKL-producing sensor cells and cancer-initiating RANK+ responder cells that reside within premalignant tissues of BRCA1-mutation carriers. We herein tested the alternative but not mutually exclusive hypothesis that BRCA1 deficiency might cell-autonomously activate RANKL expression to generate cellular states with cancer stem cell (CSC)-like properties. Using isogenic pairs of normal-like human breast epithelial cells in which the inactivation of a single BRCA1 allele results in genomic instability, we assessed the impact of BRCA1 haploinsufficiency on the expression status of RANK and RANKL. RANK expression remained unaltered but RANKL was dramatically up-regulated in mut/+BRCA1 haploinsufficient cells relative to isogenic +/+BRCA1 parental cells. Neutralizing RANKL with denosumab significantly abrogated the ability of BRCA1 haploinsufficient cells to survive and proliferate as floating microtumors or \u201cmammospheres\u201d under non-adherent/non-differentiating conditions, an accepted surrogate of the relative proportion and survival of CSCs. Intriguingly, CSC-like states driven by epithelial-to-mesenchymal transition or HER2 overexpression traits responded to some extent to denosumab. We propose that breast epithelium-specific mono-allelic inactivation of BRCA1 might suffice to cell-autonomously generate RANKL-addicted, denosumab-responsive CSC-like states. The convergent addiction to a hyperactive RANKL/RANK axis of CSC-like states from genetically diverse breast cancer subtypes might inaugurate a new era of cancer prevention and treatment based on denosumab as a CSC-targeted agent.Denosumab, a monoclonal antibody to the receptor activator of nuclear factor-\u03baB ligand (RANKL), might be a novel preventative therapy for BRCA1/2 mutations, a group of woman predisposed to high lifetime risks of breast and ovarian cancer [BRCA1/2-related carcinomas in clinical trials, the repurposing of a bone-targeted agent such as denosumab for oncology indications might inaugurate a new era of molecularly targeted pharmacological cancer prevention therapies and perhaps cancer treatments using RANKL inhibitors for millions of people worldwide [Two recent studies have provided strong evidence that denosumab, a fully humanized monoclonal antibody that binds and inactivates the receptor activator of nuclear factor-\u03baB ligand (RANKL), might be a novel preventative therapy for carriers of n cancer \u20136. If prorldwide .BRCA1 deficiency [BRCA1 mutations revealed that highly proliferative, genomically unstable RANK+ cells were the key target cancer-driven population in this high-risk group [BRCA1-deficient mouse models using the RANKL inhibitor OPG-Fc or a RANKL-specific monoclonal antibody significantly delayed tumor onset, confirming the therapeutic value of targeting the RANKL/RANK pathway to prevent breast oncogenesis in carriers of BRCA1 mutations [BRCA-D\u201d pilot window study, which aims to evaluate the biological effects of denosumab on normal breast tissue from carriers of BRCA1 and BRCA2 mutations and high-risk, non-BRCA carriers [BRCA1 mutations.The study by Penninger and colleagues demonstrated that genetic inactivation of RANKL in mammary epithelium was sufficient to significantly delay tumor onset, reduce incidence, and attenuate tumor progression in multiple models of mammary carcinogenesis . Moreovesk group . Pharmacutations . Importacarriers , revealeBRCA1/2 mutations, such as tamoxifen treatment, prophylactic mastectomy and salpingo-oophorectomy [+ luminal \u201cresponder\u201d progenitors residing within premalignant tissues of carriers of BRCA1 mutations [While the aforementioned landmark studies provide genetic and pharmacological models supporting RANKL-targeted approaches as novel preventative strategies for delaying and possibly eliminating the need for existing risk-reducing approaches in carriers of orectomy , 10, theorectomy \u201316, it wBRCA1 deficiency might cell-autonomously activate RANKL expression to generate RANKL-addicted CSC-like cellular states, we employed isogenic pairs of nontumorigenic, normal-like human breast epithelial cells in which a knock-in of the 185delAG mutation in a single BRCA1 allele results in genomic instability and accurately mimics the cell-autonomous consequences of one-hit BRCA1 inactivation occurring in the breast epithelium of carriers of BRCA1 mutations [BRCA1 haploinsufficient cells, we took advantage of the functional ability of breast cancer cell lines to display a subpopulation of cells with CSC-like properties defined experimentally by their ability to to self-renew and form anchorage-independent multicellular microtumors or \u201cmammospheres\u201d in non-adherent, non-differentiating conditions in vitro at low frequency [BRCA1 haploinsufficient cells but also in genetically diverse breast cancer subtypes in which CSC-like states are known to be driven by molecular traits such as epithelial-to-mesenchymal transition (EMT) or HER2-oncogene overexpression (22\u201330). We now report the ability of denosumab to efficiently target tumorsphere-initiating, RANKL-addicted CSC-like cells in cancer-prone BRCA1mut/+ breast epithelial cell populations, and also in the presence of key amplifiers of breast cancer CSC-like cellular states including EMT phenomena and HER2 activation.To evaluate whether utations \u201319. To erequency , 21. Themut/+BRCA1 carriers, we used a well-defined experimental system of spontaneously immortal and genetically stable, non-tumorigenic MCF10A breast epithelial cells. A common BRCA1 pathogenic mutation, 185delAG, a 2-bp deletion in the coding region close to the N-terminus is introduced into BRCA1 by gene targeting, leading to haploinsufficiency (185delAG/+BRCA1) and genomic instability [To investigate the functional importance of the RANKL/RANK signaling pathway in phenotypically relevant models of early tumorigenesis in tability .BRCA1 deficiency might suffice to cell-autonomously alter the RANKL/RANK axis in breast epithelial cells, we first assessed the impact of BRCA1 haploinsufficiency on the expression status of RANK and RANKL molecules in MCF10A cells by RT-PCR. As shown in Figure mut/+BRCA1 cells relative to isogenic +/+BRCA1 parental counterparts. The expression status of RANK, however, remained unaltered in BRCA1 haploinsufficient cells their MSFE .When cell monolayers were pre-treated or not with denosumab for 3 days, trypsinized and re-plated for mammosphere assays in the absence of denosumab, no differences were observed in the mammosphere-forming ability of E Figure . These rBRCA1/2 mutations, and likely in women with non-BRCA1/2 mutations at very high risk of developing breast/ovarian cancer. Using a monoallelic BRCA1 germline mutation model that accurately mimics the molecular events of rapid and tissue-specific predisposition of breast cancer development associated with BRCA1 haploinsufficiency, we demonstrate the ability of denosumab to robustly impair tumorsphere forming ability, a functional marker that correlates with CSC numbers in cancer cell lines and with progenitor activity in nontransformed mammary epithelial cells of mut/+BRCA1 breast epithelial populations. Given the robust tumorsphere-lowering/CSC-targeting output of denosumab in cancer-prone mut/+BRCA1 breast epithelial cells and the unresponsiveness to denosumab of CSC-driven tumorsphere formation in +/+BRCA1 isogenic cells, and considering also the absence of any confounding effect from different genetic backgrounds in the distinctive and specific anti-CSC activity of denosumab against mut/+BRCA1 cell populations, these findings provide unbiased evidence to suggest that BRCA1 haploinsufficiency is sufficient to generate CSC-like states that functionally depend on the occurrence of hyperactive autocrine/paracrine RANKL/RANK signaling. Indeed, it is noteworthy that not all the cells within mut/+BRCA1 populations are necessarily addicted to the same extent to autocrine/paracrine RANKL/RANK signaling, which appears to necessarily and exclusively operate in those cell states with tumor-initiating capacity since denosumab treatment does not affect cell viability or proliferation of 2D mut/+BRCA1 cultures but efficiently reduces the mammosphere forming capability of CSC-like cellular states, including those that might pre-exist in 2D monolayers.Based on the recent landmark studies by the Penninger & Lindeman groups , 2, clinversus dispensability of the autocrine/paracrine RANKL/RANK pathway in CSC-like versus non-CSC cellular states, respectively. In other words, the capacity of denosumab to operate as a bona fide CSC-targeting agent might depend on the convergent ability of unrelated CSC drivers to activate and maintain hyperactive RANKL/RANK signaling pathway to which the CSC-like cellular states become addicted. Supporting this notion of denosumab-responsive RANKL/RANK-driven stemness, denosumab might also target de novo generation of cancer stemness via induction of the EMT program or HER2-oncogene overexpression (Box 1).The different degrees of local production of RANKL, its specific reception by RANK, and/or the potency of RANKL/RANK signaling might dictate the degree of indispensability Box 1BRCA1 haploinsufficiency cell-autonomously activates RANKL expression and generates denosumab-responsive CSC-like cellular states, we preliminary evaluated whether RANKL/RANK signaling could also favor the maintenance of CSC populations in genetically diverse subtypes of breast carcinoma cells.Having confirmed that Denosumab and EMT-driven CSC-like cellular states. By activating NF-\u03baB, the RANKL/RANK pathway has been shown to promote cell migration, invasion, and metastasis via the induction of EMT in cancer cells [er cells \u201337. Accoer cells , supporter cells \u201341. To eer cells , 23.shECad cells is vastly superior to that of HMLERshCntrl cells, which mostly failed to form bona fide mammospheres (data not shown). Of note, the EMT-promoted spheroid formation capacity of HMLER cells was apparently reduced in the presence of denosumab within breast cancer cell populations, we employed V12H-RAS-transformed derivatives of immortalized mammary epithelial cells driven to undergo EMT by E-cadherin knockdown to assay the ability of denosumab to selectively reduce the EMT-driven enrichment of CSC-like cells \u201326. We fin situ (DCIS) [MCF10DCIS.com cells as an in vitro model representative of clinical comedo, basal-like DCIS that behaves as a precursor to invasive basal-like, triple-negative breast cancer (TNBC) [MCF10DCIS.com cells cultured as spheroids secrete vast amounts of the oncogenic protein osteopontin [mut/+BRCA1 and EMT-like HMLERshECad cell models, denosumab slightly but significantly reduced by the capacity for MCF10DCIS.com cells to form mammospheres \u201345, we ur (TNBC) . It is neopontin , a CD44 eopontin \u201349. AlthThird, to evaluate the generality of the above-mentioned findings, we assessed whether claudin-low breast subtypes displaying increased activation of the EMT program might also contain CSC-like cells responsive to denosumab. Mammosphere formation ability of MDA-MB-231 and SUM-159 cells, two highly aggressive models for claudin-low TNBC breast cancer , 30, wasDenosumab and HER2-driven CSC-like cellular states. We examined whether denosumab treatment might prevent the well-recognized ability of HER2 signaling, even in the absence of HER2 gene amplification, to expand CSC-like breast cancer populations [neo isogenic parental cells might suggest that hyperactive RANKL/RANK signaling not only suffices to induce EMT-like phenomena \u201337, but currence , 59\u201361. currence . Indeed,ce [HER2 might bece [HER2 , 57. Yetce [HER2 , it shouBox 2shECad cells to survive and proliferate as floating spherical colonies under non-adherent/non-differentiating conditions in the presence of graded concentrations of zoledronic acid and denosumab in cancer patients might go beyond prevention of skeletal complications \u201371. In eBRCA1-associated breast carcinomas appears to be driven by specific molecular and cellular alterations triggered by inherited mutations in BRCA1 in breast epithelial differentiation before development of cancer. Here, we established that BRCA1 haploinsufficiency cell-autonomously elicits a vicious cycle involving RANKL/RANK signaling favoring the pool of breast epithelial cells with CSC-like properties [BRCA1 mutation carriers [BRCA1 patients. The observations by the Penninger & Lindeman groups [BRCA1 haploinsufficiency, EMT phenomena and HER2 activation, might inaugurate a new era of cancer prevention and treatment based on the previously unrecognized CSC-targeted capabilities of bone-targeted agents such as the bisphosphonate zoledronic acid (Box 2) and the anti-RANKL monoclonal antibody denosumab.The propensity for tumor formation in operties . By bindcarriers , prelimicarriers , 66 and carriers should bn groups togetherex vivo experiments using freshly isolated CSC-enriched cell populations from specific BRCA1 KO animals and primary breast tumor samples of distinct molecular subtypes should definitely clarify the role of the anti-RANKL antibody denosumab as a potential breast CSC-specific inhibitor.We should acknowledge that our mammospheres studies have used too few cell lines to reliably interpret data in the context of breast cancer-initiating cells derived from molecularly heterogeneous primary human tumors. Additional \u00ae) and zoledronic acid (Zometa\u00ae) were kindly provided by the Hospital Universitari de Girona Dr. Josep Trueta Pharmacy .Denosumab (ProliaBRCA1 (185delAG/+) MCF10A cells with a heterozygous knock-in of a 2-bp deletion of BRCA1 resulting in a premature termination codon at position 39 and MCF10A isogenic parental cells were obtained from Horizon Discovery Ltd., Cambridge, UK . Cells were routinely grown in DMEM/F-12 including 2.5 mmol/L L-glutamine, and 15 mmol/L HEPES, supplemented with 5% (v/v) horse serum (HS), 10 \u03bcg/mL insulin, 20 ng/mL hEGF, 0.5 \u03bcg/mL hydrocortisone and 0.1 \u03bcg/ mL cholera toxin.Human v/v) heat-inactivated fetal bovine serum (FBS), insulin, hydrocortisone, and Clonetics\u2122 MEGM\u2122 .HMLER cells expressing a control shRNA (shCntrl) or an shRNA targeting E-cadherin (shEcad) were generated as described , 23 and MCF10DCIS.com and SUM-159PT cells were purchased from Asterand .DCIS.com cells were cultured in DMEM/F12 with L-glutamine supplemented with 5% HS and penicillin/streptomycin. SUM159PT cells were cultured in Ham's F12 with 5% FBS, 5 \u03bcg/mL insulin, and 1 \u03bcg/mL hydrocortisone.MDA-MB-231 cells were purchased from American Type Culture Collection and grown in Improved MEM supplemented with 5% FBS and 2 mmol/L L-glutamine.neo cells were kindly provided by Mien-Chie Hung . Breast cancer cell lines were routinely grown in improved MEM containing 5% (v/v) FBS and 2 mmol/L L-glutamine.HER2-overexpressing MCF-7/HER2 (clone 18) cells and their matched isogenic control (empty vector-transfected) MCF-7/2. Cells were screened periodically for Mycoplasma contamination.All cells were maintained at 37\u00b0C in a humidified atmosphere of 95% air/5% COTotal RNA was extracted from cell cultures using the Qiagen RNeasy Kit and QIAshredder columns according to the manufacturer's instructions. One microgram of total RNA was reverse-transcribed to cDNA using the Reaction Ready\u2122 First Strand cDNA Synthesis Kit . PCR arrays were processed according to the SABiosciences RT-PCR manual and analyzed using an Applied Biosystems 7500 Fast Real-Time PCR System with an automated baseline and threshold cycle detection. The data were interpreted using the web-based PCR array analysis tool from SABiosciences.Single cell suspensions of cell lines were plated in 6-well tissue culture plates previously coated with poly-2-hydroxyethyl-methacrylate to prevent cell attachment, at a density of 1000 cells/mL in serum-free DMEM/F-12 supplemented with 1% L-glutamine, 1% penicillin/streptomycin, 2% B27 , 20 ng/mL EGF (Sigma) and 20 ng/mL FGFb (Invitrogen). The medium was made semi-solid by the addition of 0.5% methylcellulose to prevent cell aggregation. Mammosphere-forming efficiency (MSFE) was calculated as the number of sphere-like structures (diameter >50 \u03bcm) formed after 7 days in the absence or presence of denosumab or zoledronic acid, divided by the original number of cells seeded and expressed as a percentage (mean \u00b1 SD).570 of the treated sample/OD570 of the untreated sample)\u00d7100.Cell viability was determined using the standard colorimetric MTT reduction assay. For each treatment, cell viability was evaluated as a percentage using the following equation: . All statistical tests were two-sided.All observations were confirmed by at least three independent experiments. Data are presented as mean \u00b1 SD. Two-group comparisons were performed using Student's"} +{"text": "BM742401 is a tumor suppressor lncRNA downregulated in gastric cancer. As the promoter region and the entire transcript are embedded in a CpG island, we postulated that BM742401 is a tumor suppressor lncRNA inactivated by DNA methylation in chronic lymphocytic leukemia (CLL). The promoter of BM742401 was unmethylated in normal controls including three each of normal bone marrow, peripheral blood buffy coats, and CD19-sorted peripheral B-cells, but methylated in four (57.1%) CLL cell lines. Methylation of BM742401 correlated inversely with expression. In the completely methylated WAC3CD5+ CLL cells, 5-Aza-2\u2032-deoxycytidine treatment led to promoter demethylation and re-expression of BM742401 transcript. Functionally, stable overexpression of BM742401 resulted in inhibition of cellular proliferation and enhanced apoptosis through caspase-9-dependent intrinsic but not caspase-8-dependent extrinsic apoptosis pathway, suggesting a tumor suppressor role of BM742401 in CLL. In primary CLL samples, methylation of BM742401 was detected in 43/98 (43.9%) of patients. Moreover, among CLL patients with standard-risk cytogenetic aberrations, methylation of BM742401 correlated with advanced Rai stage (\u2265 stage 2)(P = 0.002). Furthermore, BM742401 methylation was associated with miR-129-2 methylation (P = 0.05). BM742401 is a tumor suppressor lncRNA frequently methylated in CLL. The mechanism of BM742401 as a tumor suppressor warrants further studies. Bisulfite treatment of DNA was performed to convert unmethylated cytosine to uracil (but unaffecting methylated cytosine) using the EpiTect Bisulfite Kit kit . Each bisulfite-treated sample was amplified using primer sets specific to methylated DNA and unmethylated DNA respectively. Details of primers and conditions for M-MSP and U-MSP of BM742401 were given in Table DNA was extracted from 98 diagnostic marrow samples, seven CLL cell lines and nine healthy normal controls was compared to those with standard-risk karyotypes . Association between methylation of BM742401 and miR-129-2 was studied by chi-square test. All P values were two-sided.In 98 primary CLL samples, the correlation between"} +{"text": "Recently, BZA was also shown to exhibit potent anti-cancer effects that were both estrogen receptor (ER)-dependent and ER-independent. Our results suggest that BZA inhibits IL-6 signaling by disrupting IL-6R/gp130 protein-protein interactions. BZA treatment of CAL27-IL-6 (IL-6 overexpressing cells) or UM-SCC-74A significantly inhibited cell proliferation, migration and colony formation ability in a dose-dependent manner. In addition, BZA significantly decreased IL-6-mediated tumorsphere formation by markedly reducing nanog expression. BZA treatment also markedly reduced chemo and radioresistance in head and neck cancer cells by downregulating ERCC-1, XRCC-1 and survivin expression. In a SCID mouse xenograft model, BZA significantly enhanced the anti-tumor effects of cisplatin and radiation treatment with no added systemic toxicity. Furthermore, combination treatments significantly decreased tumor metastasis, pSTAT3 expression and nanog expression, in vivo. Taken together, our results suggest that targeting IL-6 signaling with bazedoxifene could be an effective treatment strategy for the treatment of HNSCC patients.Recent studies have shown that IL-6 signaling plays an important role in the aggressive and metastatic phenotype of head and neck squamous cell carcinoma (HNSCC). Therefore, we hypothesized that targeting of IL-6 signaling in HNSCC could enhance the therapeutic efficacy of standard chemoradiation treatment. We used both After 27 days, primary tumors and lungs were carefully removed. Primary tumors were analyzed by immunohistochemistry for pSTAT3 and naong expression whereas lungs were examined for metastatic disease.6-8 week old SCID mice (NCI) were used in all the eriments . Tumor cThe xenograft tumor tissues and lungs were fixed in 4% paraformaldehyde overnight and paraffin embedded. Tissue sections were deparaffinized and pretreated with antigen retrieval buffer . For nanData from all the experiments are expressed as mean \u00b1 SEM from a minimum of 3 independent experiments. The data was statistically analyzed by two-way analysis of variance (ANOVA) or Student's t test and a p value of <0.05 was considered significant."} +{"text": "This report describes a patient with a third recurrence in lymph nodes. The recurrence was treated with 177Lu-PSMA radioligand therapy instead of chemotherapy with docetaxel. The effect was in part evaluated relative to that of two established salvage treatments. Prior salvage radiotherapy and abiraterone of the first and second recurrence in lymph nodes had given only a partial reduction of PSA. Nevertheless within five months of follow-up, 177Lu-PSMA radioligand therapy of the third recurrence in lymph nodes reduced PSA for a period to unmeasurable levels. 177Lu-PSMA radioligand therapy gave only mild adverse effects. In conclusion, for a patient with lymph node metastatic prostate cancer, 177Lu-PSMA-617 radioligand therapy had an attractive therapeutic profile. A follow-up study of similar patients is being planned.Prostate specific membrane antigen (PSMA) is expressed in unfavorable prostate cancer. PSMA is basis for new diagnostics and theranostics. PET/CT using PSMA is more sensitive than choline PET/CT. Up to a third of patients with prostate cancer (PC) who undergo radical prostatectomy (RP) later develop a continuous rise of prostate specific antigen (PSA). A low but rising PSA level has been denoted biochemical recurrence (BCR). In this phase of recurrence, conventional imaging do not detect the sites of recurrence. Patients with BCR may be treated with salvage external beam radiotherapy (SRT) with or without androgen deprivation therapy (ADT) , 2. Five68Ga-PSMA HBED-CC PET/CT detected cancer lesions for most of these patients [177Lu-PSMA-617 radioligand therapy (RLT) was effective for patients with PSMA-positive mCRPC [68Ga-PSMA HBED-CC PET/CT and 177Lu-PSMA-617 RLT [177Lu-PSMA-617 RLT is mainly used for patients with end-stage mCRPC who had failed with established systemic treatments.Most poor-risk PC express prostate membrane specific antigen (PSMA). Correspondingly, patients . 177Lu-Pve mCRPC . Guideli-617 RLT , 6. Howe177Lu-PSMA-617 RLT.We report a patient who had three episodes of recurrence with lymph node metastatic prostate cancer. The first two episodes had been treated with SRT and abiraterone. The third episode was treated with 18F-fluorobutane-1-carboxylic acid (18F-FACBC) PET/CT showed one lesion in a pelvic lymph node. He was given SRT with a boost for the lesion. The boost was given with 2 Gy per fraction and a conventional fractionation scheme to a cumulative dose of 78 Gy. A follow-up PET/CT showed that the patient had achieved a partial remission [11C-choline PET/CT was carried out. The new PET/CT showed he had developed a new site in a retroperitoneal lymph node. Therefore his treatment was changed to abiraterone 150 mg a day and prednisone 5 mg twice daily. Three months later, the patient also underwent a second course of SRT with a cumulative dose of 60 Gy. The second SRT was targeting the new site. Again, the combined treatment transitorily reduced PSA. The new nadir PSA was 0.3 ng/ml.A 50-year-old patient was diagnosed with PC in May 2007. At diagnosis, he underwent RP and limited bilateral pelvic lymphadenectomy ). He started androgen deprivation therapy (ADT) shortly after RP. Two years later, he had continuously rising PSA levels in both cycles [177Lu-PSMA-617 RLT caused acute nausea. But with use of anti-emetic drugs, the patient avoided nausea in connection with the second cycle. Otherwise the 177Lu-PSMA-617 RLT did not cause acute adverse effects.In 2016, the patient had a third episode of continuously rising PSA. A restaging s Figure . At that68Ga-PSMA HBED-CC PET/CT was carried out in November 2016 and February 2017 .A new study of patients with lymph node metastatic castration-resistant PC treated with 177Lu-PSMA RLT for lymph node metastatic castration-resistant PC.In conclusion, our study is a good example of clinical efficacy with"} +{"text": "Emerging evidence shows this decline mainly results from defects in negative selection, but there is insufficient evidence regarding whether tTreg cell generation is also impaired. We mechanistically studied tTreg cell generation in the atrophied thymus by utilizing both postnatal TEC-defective and naturally aged mouse models. We found that the capacity of tTreg cell generation was not impaired compared to CD4+ thymic conventional T cells, suggesting thymic atrophy positively influences tTreg cell generation. This is potentially attributed to decreased T cell receptor (TCR) signaling strength due to inefficiency in promiscuous expression of self-antigens or presenting a neo-self-antigen by medullary TECs, displaying decreased negative selection-related marker genes (Nur77 and CD5high) in CD4 single positive (SP) thymocytes. Our results provide evidence that the atrophied thymus attempts to balance the defective negative selection by enhancing tTreg cell generation to maintain central T-cell tolerance in the elderly. Once the balance is broken, age-related diseases could take place.Postnatal thymic epithelial cell (TEC) homeostatic defect- or natural aging-induced thymic atrophy results in a decline in central T-cell tolerance establishment, which is constituted by thymocyte negative selection and cluster of differentiation (CD) 4 Chronic inflammation in the elderly is partially attributed to atrophy of the thymus\u2014an organ that regulates the immune system\u2014and in particular the ability of organisms to recognize their own cells\u2014a phenomenon known as central tolerance. Immune central tolerance is established by two processes: first, immune cells that react strongly to self are eliminated in a process called negative selection, and second, thymic regulatory T (tTreg) cells are generated to suppress self-reactive immune reactions. The former has already been reported to be defective in the aged thymus, but whether the generation of new tTreg cells is also impaired has remained unclear. Here, we analyzed the effect of aging on tTreg cell generation and found that the atrophied thymus is still able to make new tTreg cells; indeed, we show that tTreg cell generation capacity is enhanced when compared with other na\u00efve T cells from the same thymus. We conclude that the balance of defective negative selection with enhanced tTreg cell generation may be necessary to avoid autoimmune diseases during aging. Central T-cell tolerance, which refers to the mechanism by which newly developing T cells are rendered nonreactive to self , plays aFOXN1 (Forkhead box protein N1) gene 5high) were decreased in certain cell subsets of the atrophied thymus compared to those in the young thymus. Additionally, this declined TCR signaling strength is due to an inefficient presentation of self-pMHC on mTECs, which was demonstrated in a neo/mock self-antigen rat insulin promoter (RIP)-driven membrane-bound ovalbumin (mOVA) transgenic (Tg) mouse model. Together, our results provide evidence that the atrophied thymus attempts to balance the defective negative selection by relatively enhancing tTreg cell generation to maintain central T-cell tolerance in the elderly.In this study, we explored how tTreg cell generation in the atrophied thymus is affected and by what mechanism in mouse models. We demonstrated that although thymic atrophy perturbs negative selection, resulting in an inability to sufficiently deplete self-reactive T clones, it was compensated for by relative enhancement of tTreg cell generation, as shown by an increased ratio of percentage of tTreg cells to percentage of tTcon cells and unreduced absolute tTreg cell numbers despite a dramatically decreased whole thymic cellularity. This relative enhancement was attributed to the new generation/differentiation of tTreg cells, rather than any changes in apoptotic or anti-apoptotic features, which was demonstrated by enhanced phosphoralated-Zap70 (p-Zap70) and unchanged apoptotic (Annexin Vloxp-flanked FoxN1 gene (FoxN1fx/fx) [T) mediation through either a tamoxifen (TM) induction or a CreERT autoleakage with age. We named this model the FC (FoxN1fx/fx/CreERT) mouse and FF (FoxN1fx/fx without CreERT for controls) mouse. One-month-old FC thymi without any TM treatment exhibit the same thymocyte profiles as those in wild-type (WT) young mice. However, when young FC mice are treated with TM for 3 consecutive days (TM x3), or FC mice are housed for more than 6 months without any TM treatment but with an age-related CreERT autoleakage, their thymi exhibit thymic atrophy analogous to aged (approximately 18-month-old) WT thymi (details in rag-gfp reporter gene was utilized in this model to mark newly generated thymocytes [+) CD4 single positive (SP) (CD4SP) thymocytes, including tTreg cells and tTcon cells in CD4SP population, in the acute atrophied thymus (TM x3) CD4SP population (gate shown in -neg in the thymus was decreased with age (-neg populations (+ population (To determine whether the capacity of newly generated tTreg cells is impaired in aged thymus and/or iN1fx/fx) can be dymocytes , which w (TM x3) . We foun (TM x3) image wiecreased , implyinver time ). The raBim (a pro-apoptotic gene) expression [FoxN1fx/fx-deleted mice have a relative young periphery since only the thymus undergoes an accelerated aging. Therefore, the young FC mouse does not have increased/accumulated pTreg cells. In order to account for the peripheral interference of new tTreg cell generation in the naturally age-related atrophied thymus, we investigated the same parameters in WT (containing Rag-GFP reporter) mouse thymi mouse, pTreg cells are increased due to tpression , 20. Howse thymi . As we eSP cells showed ted cells . These rSPCD25-neg thymocytes) from a pool of young OT-II+ TCR Tg mice [Rag\u2212/\u2212 background, details in FoxN1fx/fxCreERT with a TM x3 induction) thymi containing RIP-mOVA Tg TECs [+ TCR, these grafted OT-II+ (bearing V\u03b12 and V\u03b25 TCR subunits which recognize mOVA antigen) CD4SP clones from young mice, which do not have any aging-related imprints or characteristics, undergo either negative selection-driven apoptosis or tTreg cell development-related differentiation, dependent on TCR signaling strength produced by the interaction [We believe that the atrophied thymic microenvironment promotes the activity of new tTreg cell generation. To exclude any intrinsic alterations from tTreg cells in the atrophied thymus, such as tTreg cells acquiring any refractory behavior for anti-apoptosis or loss of suppressive function, we designed an adaptive transfer experiment based on a published protocol . We intric) mice to differentiate into tTreg cells in an increased proportion compared to tTcon cells (We first verified that the atrophied thymic microenvironment promoted the grafted pre-tTreg cells (CD4on cells and thaton cells compared+ TCR-Tg pre-tTreg cells (+CD4SPCD25+) from young WT, middle-aged WT, and middle-aged FC mice and CD4SPCD5low populations were decreased in mTECs of the FoxN1-conditional knockout (cKO) atrophied thymus [TCR signaling strength is produced through an interaction between the self-pMHC presented on mTECs and the self-recognizing TCR expressed on self-T clones. Self-peptide presentation in mTECs is also termed \u201cpromiscuous expression\u201d of peripheral tissue-specific antigens (TSAs), which distinguishes the TEC-autonomous gene expression. In addition, expression of most TSAs on mTECs is in TECs and the d thymus , this suire gene , and thever time , see S1 reduced , which vd thymus .To further identify the underlying mechanism by which thymic atrophy relatively enhances tTreg cell generation, we also considered that tTreg cell generation-required cytokines, such as interleukin-2 (IL-2) and TranUsing a combination of naturally aged and multiple genetically engineered mouse models and investigating signaling molecules in either the thymocyte or mTEC thymic cellular compartments, we have demonstrated that the aged, atrophied thymic microenvironment does not impair, but may relatively promote, tTreg cell generation. This evidence has revealed how central tolerance is established in the age-related atrophied thymic microenvironment, which we determined to be a heterogeneous effect of defective negative selection accompanied by relatively enhanced tTreg cell generation. This is probably a potential attempt by the atrophied thymus to desperately balance the defective negative selection by enhancing tTreg cell generation to maintain central T-cell tolerance. As we know, under normal conditions, two arms\u2014namely, negative selection and tTreg cell generation\u2014work in tandem for central T-cell tolerance establishment. However, in the age-related atrophied thymus, as one arm of tolerance induction (negative selection) fails, we assert that the second arm (tTreg cell generation) attempts to compensate, resulting in relative tTreg cell enhancement.Aire gene expression [Aire gene in positive selection of Treg cells [Aire regulates Aire-dependent self-peptide expression on mTECs. Moreover, total [Aire deficient models. Therefore, the interaction of self-pMHC and self-reactive TCR produces weak signals in the age-related atrophied thymus, which results in a similar outcome as illustrated by the second model: ineffective mTECs [This mechanism can be explained by changes in TCR signaling strength in the thymus, which acts as a gate-keeper to determine thymocyte fate and reentered (Rag-GFP-neg) portions only in the same subset, rather than reflecting the changes in an integrated total CD4SP population. Therefore, it does not exhibit integrated changes in newly generated and reentered Treg cells compared to their counterpart Tcon cells and within the entire CD4SP cell population. Regarding inhibition of the de novo tTreg cell generation by reentered pTreg cells in the thymus, these cells may be importantly involved in a potential negative feedback loop of regulation [Bim gene-resulted accumulation [Bim gene [Our results presented here are inconsistent with a published report in which the phenotype of \u201csubstantially decreased new tTreg cells and augmented recirculating pTreg cells among thymic Treg cells with age\u201d was observed . These pgulation . Since pmulation , it is aBim gene ) or old Development of tTreg cells in the thymus is primarily dependent on thymic microenvironments. We found that the atrophied thymic microenvironment favors tTreg cell development because they demonstrated more activity compared to their counterparts in the normal thymus Figs and comp+) Treg cells in the spleens of aged mice were increased, while Rag-GFP+ CD4SP Tcon cells in the spleens of aged mice were decreased approved by the Institutional Animal Care and Use Committee (IACUC) of the University of North Texas Health Science Center, in accordance with guidelines on animal welfare of the National Institutes of Health. All efforts were taken to minimize mouse usage to maximize necessary results; provide the best veterinary care; and minimize discomfort, distress, and surgery with anesthetic procedures and euthanasia.FoxN1fx/fx [T (ubiquitous promoter-driven Cre-recombinase and estrogen-receptor fusion protein) Tg mice, then with rag-gfp reporter mice. The FoxN1fx/fx cKO was through either a TM-induced acute deletion or CreERT autoleakage-mediated time-course deletion [T age-related autoleakage are the same as in the naturally aged WT (\u226518-month-old) thymus (details in + TCR Tg mice (#004194) were purchased from Jackson Lab. OT-II+ TCR Tg mice were cross-bred with Rag\u2212/\u2212 mice (Jackson Lab #002216). These mice were termed OT-II+/Rag \u2212/\u2212 mice and their crossbreeding schemes have been described in our previous publication . BrieflyxN1fx/fx mice werdeletion . Both demice see . Aged WTSP and CD8+CD4+ DP OT-II+ TCR-Tg thymocytes were depleted through an anti-CD8 cytotoxic antibody (clone HO-2.2) and Low-Toxic-M complement. Then, the live thymocytes were sorted by flow cytometry for pre-tTreg cells (CD4SP and CD25-neg thymocytes). The sorted pre-tTreg cells were i.t. injected into the thymus (details in our previous publication [6 cells per recipient mouse), respectively. Five days after the pre-tTreg cell injection, the thymocytes with V\u03b12+V\u03b25+ TCR chain types (mostly OT-II+ TCR-Tg thymocytes) in recipient FF- or FC-mOVA thymi were collected for analysis of newly generated tTreg cells (CD4SPCD25+FoxP3+) versus tTcon cells (CD4SPCD25-negFoxP3-neg). Meanwhile, recipient mice were intraperitoneally (i.p.) injected with TM (1 mg/10 g body weight/day) for 3 consecutive days (TM x3): one day before, one day along with i.t. injection, and one day after to induce thymic atrophy in FC-mOVA mice (detailed procedure is outlined in CD8lication ) of youn+ TCR-Tg mice under the kidney capsule of Nur77 and CD5 in CD4SP cells carrying V\u03b12+V\u03b25+ TCR chain type for detecting cells bearing OT-II+ TCR-Tg T cells.The surgical operation of the kidney capsule transplantation was performed as previously described . BrieflyFor thymocyte staining, single-thymocyte suspensions were prepared from the thymi of mice using a 70-\u03bcm cell strainer. Samples were then stained with specific fluorochrome-conjugated antibodies of cell surface CD markers, indicated in each figure legend, and then fixed and permeabilized with fixation/permeabilization buffer , per company\u2019s instruction, followed by intracellular staining for PE-anti-FoxP3 (eBioscience Cat# 12-5773-82) or FITC-anti-FoxP3 (eBioscience Cat# 11-5773-82), and/or PE-anti-p-Zap70 , PE-anti-Nur77 , and AlexaFluor488-anti-GFP . For TEC staining, the thymus was cut into pieces, then was digested with Collagenase-V/DNase-I, as per previously published methods , then st2) for 15 min at room temperature in the dark. The cells were then washed with PBS and fixed and permeabilized with fixation/permeabilization buffer, followed by eBioScience intracellular staining protocol for FoxP3 and GFP. Positive control cells were aliquoted from the thymocytes and made by incubating at 55\u00b0C for 20 min to induce cell death before staining.Thymocytes were harvested from young (2 months old) and aged (16 months old) Rag-GFP reporter mice, and stained with surface CD markers as described above. Then, the cells were washed with Annexin-V binding buffer and incubated in APC-Annexin-V at a 1:20 dilution with Annexin-V binding buffer and were treated with irradiation of 2,000 Rads. T effector (Teff) cells were prepared from young WT spleen through direct flow cytometric sorting of CD4+CD25-neg spleen T cells; details were outlined in our and others\u2019 previous publications [SPCD25+Rag-GFP+) were sorted from 3 groups of thymocytes: young (approximately 6\u20138 weeks old) Rag-GFP WT; middle-aged (approximately 12 months old) Rag-GFP WT; and middle-aged (\u2265 8 months old) FC-Rag-GFP mice , Teff cells (5 \u00d7 104/well), and tTreg cells (2.5 \u00d7 104/well) supplied with CD3\u03b5 (1 \u03bcg/ml) and CD28 (2 \u03bcg/ml) antibodies were set in 96-well U-bottom plates for 72 hours. Proliferation of cells was determined using CellTiter 96 Aqueous One Solution Reagent following the company\u2019s protocol: adding 20 \u03bcl of solution per well for additional 2-hour culture, and absorbance was measured at 490 nm using an ELISA 96-well plate reader (BioTek ELx800).Antigen presenting cells (APCs) were prepared from young WT mouse bone marrow-derived dendritic cells to cDNA with the SuperScriptIII cDNA kit (Invitrogen/ThermoFisher Scientific). Real-time RT-PCR was performed with TaqMan reagents and primers [t test was used, assuming equal variance. Differences were considered statistically significant at values of * p < 0.05; ** p < 0.01; and *** p < 0.001. Curves in Figs For evaluation of group differences, the unpaired two-tailed Student S1 FigA) Flow cytometric profile of CD4 versus CD8 from the 5 groups of various mice. (B) Total thymocyte number in four thymocyte subsets of 5 mouse groups. (C) Proportions of thymocytes in four thymocyte subsets of 5 mouse groups. Mouse numbers in each group are 5\u201315 animals. The thymus of FoxN1fx/fx-CreERT (FC)-Young (1 month old) was not treated with tamoxifen (TM), showed the same profile as WT-young; while treated with TM 3 times (TM x3), showed the same profile as WT-aged (18 Months old) thymus. FC-6-month-old thymus without treatment with TM (with CreERT-mediated auto-leaky deletion of FoxN1fx/fx), also showed the same profile as WT-aged (18 Months old) thymus. NS = not significant between groups; SDs = significant differences (either p < 0.05 or p < 0.01) between any of these groups . The results suggest that WT-young and FC-young (without TM treatment) have the same profile, while WT-aged (18 Months old), FC-young (TM x3), and FC-6 Months old (without TM treatment) possess the same profile. Underlying data used in the generation of this figure can be found in T, ubiquitous promoter-driven Cre-recombinase and estrogen-receptor fusion protein; FC, FoxN1fx/fx/CreERT; FF, FoxN1fx/fx without CreERT for controls; Foxn1, Forkhead box protein N1; FoxN1fx/fx, loxp-flanked FoxN1 gene; mOVA-Tg, membrane-bound ovalbumin transgenic mouse; TM, tamoxifen; WT, wild-type.((TIF)Click here for additional data file.S2 FigA and B) The curves are nonlinear one-phase decay; the results demonstrated that absolute cell numbers of tTreg cells were not reduced with age, while the numbers of tTcon cells were dramatically reduced with age. (C) Summarized results of absolute cell numbers of tTcon and tTreg at the ages of 4 weeks and 55 weeks from the FoxN1fx/fx-CreERT (autoleakage-induced deletion with time) and naturally aged thymus, respectively, from which the tTreg cells were not different between the two age groups, while tTcon cells were significantly reduced in the aged group compared to young groups. SD = Standard Deviation; NS = Not Significant. Underlying data used in the generation of panels A and B can be found in T, ubiquitous promoter-driven Cre-recombinase and estrogen-receptor fusion protein; FoxN1fx/fx, loxp-flanked FoxN1 gene; Tcon, conventional T cell; tTcon, thymic conventional T cell; tTreg, thymic regulatory T cell.((TIF)Click here for additional data file.S3 FigA) Comparison of V\u03b12 and V\u03b25 double TCR positive CD4SP thymocytes in WT, OT-II TCR-Tg only, Rag\u2212/\u2212 only, and OT-II TCR-Tg with Rag\u2212/\u2212 background mice; (B) Comparison of V\u03b12 and V\u03b25 double TCR positive CD3+CD4+ splenic cells in four genotypic mice. The results indicated that we successfully generated OT-II TCR-Tg with Rag\u2212/\u2212 background mice, in which V\u03b12V\u03b25 TCR+ CD4SP population is dramatically increased, while CD8SP and B cells are dramatically decreased. CD, cluster of differentiation; OT-II+ TCR Tg, MHC class-II restricted ovalbumin-specific TCR transgenic; RAG, Recombination activating gene; TCR, T cell receptor; Tg, transgenic; WT, wild-type.((TIF)Click here for additional data file.S4 Fig+) tTreg (CD4+CD25+) cells from young WT, middle-aged WT, and middle-aged FC sorted from young WT splenic cells. Cells were cultured for 3 days, then CellTiter 96 AQ reagent (Promega) was added for 2\u20134 hours of culture, then reaction was analyzed by Absorber Reader at 490 nm. (A) Results of tTreg cell sorting purity test; (B) Summarized results of tTreg cell suppressive capacity from the tTreg cell sorting of three times with at least four animals in each group. The results suggest that there is no difference of tTreg cell suppressive capacity among young, middle-aged, and middle-aged FC mice. Underlying data used in the generation of this figure (panel B) can be found in T, ubiquitous promoter-driven Cre-recombinase and estrogen-receptor fusion protein; FC, FoxN1fx/fx/CreERT; FoxN1, Forkhead box protein N1; FoxN1fx/fx or FoxN1-floxed, loxp-flanked FoxN1 gene; GFP, green fluorescent protein; Teff, T effector cell; tTreg, thymic regulatory T cell; WT, wild-type.Sorted newly generated (Rag-GFP(TIF)Click here for additional data file.S5 FigTop) The mRNAs from the thymi of WT young and naturally aged mice; (Bottom) The mRNAs from thymi of FF and FC mice (both groups treated with TM x3). A TaqMan-based real-time RT-PCR was conducted with TaqMan primers and probes to TGF\u03b2, NF-\u03baB (p105/p50), and IL-2, along with house-keeping genes, GAPDH and 18sRNA, for normalization. A Student t-test was used to determine statistical significance between groups. Data are expressed as mean \u00b1 SEM. Each symbol represents an animal. Underlying data used in the generation of this figure can be found in T, ubiquitous promoter-driven Cre-recombinase and estrogen-receptor fusion protein; FC, FoxN1fx/fx/CreERT; FoxN1, Forkhead box protein N1; FoxN1fx/fx, loxp-flanked FoxN1 gene; IL-2, interleukin-2; NF-\u03baB, Nuclear Factor kappa Beta; RT, reverse transcribed; PCR, polymerase chain reaction; tTreg, thymic regulatory T cell.((TIF)Click here for additional data file.S6 FigA) Flow cytometric gate strategy shows gates of splenic Treg cells and Tcon cells from isotype control sample (mixture of young and old spleen cells stained with isotype control antibody for FoxP3); Young and old Rag-GFP reporter mice. (B) A summary of percentages of splenic Tcon cells and Treg cells in young and old mice, showing decreased RTE Tcon cells and increased RTE Treg cells in the old spleens. Underlying data used in the generation of this figure can be found in ((TIF)Click here for additional data file.S1 Data(XLSX)Click here for additional data file."} +{"text": "We report a case of 68-years-old gentleman who developed a delayed local recurrence, 30 years following curative radiation treatment for muscle-invasive bladder cancer. This case emphasizes the importance of lifelong post-treatment surveillance for bladder cancer."} +{"text": "Here, we identify Brain Tumor (Brat), a tripartite motif protein, as a new regulator of midline crossing in the Drosophila CNS. Genetic analysis indicates that Brat acts independently of the Netrin/Fra pathway. In addition, we show that through its B-Box domains, Brat acts cell autonomously to regulate the expression and localization of Adenomatous polyposis coli-2 (Apc2), a key component of the Wnt canonical signaling pathway, to promote axon growth across the midline. Genetic evidence indicates that the role of Brat and Apc2 to promote axon growth across the midline is independent of Wnt and Beta-catenin-mediated transcriptional regulation. Instead, we propose that Brat promotes midline crossing through directing the localization or stability of Apc2 at the plus ends of microtubules in navigating commissural axons. These findings define a new mechanism in the coordination of axon growth and guidance at the midline.Commissural axons must cross the midline to establish reciprocal connections between the two sides of the body. This process is highly conserved between invertebrates and vertebrates and depends on guidance cues and their receptors to instruct axon trajectories. The DCC family receptor Frazzled (Fra) signals chemoattraction and promotes midline crossing in response to its ligand Netrin. However, in Netrin or The establishment of neuronal connections that cross the midline of the animal is essential to generate neural circuits that coordinate the left and right sides of the body. Axons that cross the midline to form these connections are called commissural axons and the molecules and mechanisms that control midline axon crossing are remarkably conserved across animal evolution. In this study we have used a genetic screen in the fruit fly in an attempt to uncover additional players in this key developmental process, and have identified a novel role for the Brain Tumor (Brat) protein in promoting commissural axon growth across the midline. Unlike its previous described functions, in the context of midline axon guidance Brat cooperates with the microtubule stabilizing protein Apc2 to coordinate axon growth and guidance. Molecular and genetic analyses point to the conserved B box motifs of the Brat protein as key in promoting the association of Apc2 with the plus ends of microtubules. Brat is highly conserved and future studies will determine whether homologous genes play analogous roles in mammalian neural development. Drosophila, are a highly conserved chemoattractive guidance pathway [netrinAB double mutants or fra mutants in Drosophila. This indicates that additional pathways must promote midline crossing [Organisms with bilateral symmetry coordinate the left and right sides of their body by establishing reciprocal connections in the central nervous system. During development, commissural axons navigate across the midline to form contralateral connections by responding to attractant and repellant cues expressed at the midline and in other cells . To alte pathway \u20135. Loss pathway \u201310. Despcrossing ,12. Indecrossing , Sema2a/crossing , and VEGcrossing . In addicrossing \u201320. DespDrosophila embryo resulting in an easily quantifiable defect in midline crossing [C. elegans to humans. Brat contains two B-box domains (BB), a Coiled-coil domain (CC) in the N-terminus, and a NHL domain in the C-terminus [hunchback mRNA in the posterior part of the embryo during early development [To identify additional pathways implicated in the midline crossing process, we performed a genetic screen using a sensitized genetic background. In this background a dominant negative Fra receptor (Fra\u0394C- missing its entire cytoplasmic domain) is expressed in a subset of commissural neurons in the crossing . From thterminus . Identifterminus , Brat haterminus ,25, and terminus . Moreoveelopment , and forelopment . In addielopment . This prelopment . The desDrosophila sensory neuron dendrites, Apc2 interacts with EB1 (for End Binding) to control microtubule polarity [Drosophila Apc2 [Interestingly, in addition to its role in the destruction complex, Apc2 is a microtubule plus-end binding protein (+TIP) ,32. In Dpolarity . In growila Apc2 , regulatila Apc2 ,35. In tbrat mutants, results in enhanced commissural guidance defects in the Fra\u0394C sensitized background. Moreover, Apc2 expression and localization are altered in brat mutant embryos suggesting that Brat function in this context is critically dependent on Apc2. These data suggest a model where Brat promotes the elongation of the axon before crossing by maintaining Apc2 at the microtubule plus-ends.In this study, we report that Brain Tumor maintains Apc2 at the plus-ends of microtubules to promote axon elongation and midline crossing. Brat acts independently of the Fra/Netrin pathway and independently of its common partners Pumilio, Nanos and d4EHP, which are required for the inhibition of mRNA translation. In addition, we show that this process requires the B-Box domains of Brain Tumor. Reducing the function of Apc2 in fra mutants, EW axons fail to cross the midline in 36% of embryonic segments, while the axons of EG neurons are unaffected [wild type embryos expressing Fra\u0394C specifically in eagle neurons missing its cytoplasmic domain that functions as a dominant negative . By targaffected is expressed selectively in eagle neurons antibody, in fra mutants, brat mutants and brat, fra double mutants. In wild type embryos, HRP staining reveals that thick anterior and posterior commissures form in each segment and GFP staining reveals that EW and EG axons cross the midline and Pumilio (Pum) to repress the translation of target mRNAs ,37,38. Tguidance . In addi protein . Express defects . Importa defects .UASBratG774D or UASBratR837D variants can also fully rescue the mushroom body axon maintenance defects observed in brat mutants [brat mutants, we observe no difference in Src64B levels in CNS axons in brat mutant embryos , which is known to play a role in regulating asymmetric protein segregation, and the pair of B-box domains (BB1 and BB2), which have been implicated in the control of intermediate neural progenitor (INP) cell identity . We usedckground , deletiockground , althougckground .Apc2, a component of the destruction complex, either with specific point mutations or with a deletion of the Apc2 locus, significantly enhances the EW crossing defects in the Fra\u0394C sensitized background . The following stocks were from Bloominton: Df(3R)Exel6198, Df(2L)Exel8040, arm8, UAS-arm-GFP, UAS-Apc2-GFP, UAS-arm.S10, UAS-TCF and UAS-\u0394TCF. The stock Src64B-GFP was from the Kyoto Stock Center. The following stocks were a gift from C-Y Lee: brat11, UAS-brat-Myc, UAS-brat\u0394NHL-Myc, UAS-brat\u0394CC-Myc, UAS-brat\u0394BB-Myc, UAS-brat\u0394BB1-Myc, and UAS-brat\u0394BB2-Myc. The following stocks were a gift from F Besse: UAS-brat-HA, UAS-bratGD-HA, and UAS-bratRD-HA. The following stocks were a gift from M. Peifer: Apc2g10. The following transgenes were used UAS-Fra\u0394C-HA, UAS-A5CD8-GFP. The UAS-EB1-RFP stock was a gift from Yuanquan Song. GAL4 drivers used were elav-GAL4 and eg-GAL4. All crosses were carried out at 25\u00b0C. Embryos were genotyped using balancer chromosomes carrying lacZ markers or by the presence of epitope-tagged transgenes. See The following Dechorionated, formaldehyde-fixed, methanol devitellinized embryos were fluorescently stained as previously described . The folFluorescent mRNA in situ hybridization was performed as described, with digoxigenin labeled probes . BrieflyUAS-Apc2GFP or brat(-); UAS-Apc2GFP, +/+; src64bGFP or brat(-); scr64bGFP) were imaged using identical settings. Max projections were generated using ImageJ. After subtracting the staining background, the average pixel intensity was measured on twelve to sixteen clusters of EW neurons or across five regions within longitudinal axons for each embryo. The values from the five to ten embryos for each phenotype were averaged.Phenotypes were analyzed and images were acquired using a spinning disk confocal system (Perkin Elmer) built on a Nikon Ti-U inverted microscope using a Nikon OFN25 60x 40x or 10x objective with a Hamamatsu C10600-10B CCD camera and Yokogawa CSU-10 scanner head with Volocity imaging software. Images were processed using ImageJ and Adobe Illustrator software. For fluorescence quantification of GFP antibody staining in embryos, ten embryos per genotype failed to reach the midline. Embryos were scored blind to genotype when possible.For statistical analysis, comparisons were made between genotypes using the Student\u2019s t-test, ANOVA or Chi-squared test. For multiple comparisons, significance was assessed by using a Bonferroni correction.S1 Figbrat mRNA, Anti-GFP labels cell bodies and axons of the eagle neurons (EG and EW). Scale bar represents 10\u03bcm , 5\u03bcm (F\u2019). (A-E) In whole mount embryos, brat mRNA (in green) is detected in the ventral nerve cord and the brain during all the stages of development (Stages 13 to 17), when axons grow and cross the midline. (F-J\u2019) Dissected embryos reveal that brat mRNA (in green) is expressed in Eagle neurons (magenta) during stage 13 to 17.(A-J\u2019) Stage 13\u201317 embryos of the indicated genotypes carrying eg-GAL4 and UAS-tauMycGFP transgenes, stained with anti-DIG (green) (A-J\u2019) and anti-GFP (magenta). Anti-DIG reveals (TIF)Click here for additional data file.S2 Fig(A-B\u2019) Stage 15\u201316 embryos of the indicated genotype carrying the elav-GAL4 transgene, stained with anti-FasII (green) (A-B) and anti-Myc (red) (A\u2019-B\u2019) antibodies. Anti-FasII labels the ipsilateral axons, anti-Myc reveals the UAS-Brat transgene expression. Scale bar represents 10\u03bcm (A) and 5\u03bcm (A\u2019). (C-D) Stage 15\u201316 embryos of the indicated genotype carrying eg-GAL4 and UAS-Fra\u0394C transgenes, stained with anti-GFP antibodies. Anti-GFP labels cell bodies and axons of the eagle neurons (EG and EW). Scale bar represents 10\u03bcm (C). (A) In wild-type embryos Fas2 positive ipsilateral axons turn before reaching the midline to grow longitudinally in all segments. (B) Expressing UAS-Brat in all neurons does not induce any ectopic crossing of ipsilateral axons. (C) EW axons fail to cross in 27% of segments when UAS-Fra\u0394C is selectively expressed in eagle neurons. (D) In the Fra\u0394C background the expression of UAS-Brat in eagle neurons reduces the EW crossing defects to 17%. (E) Quantification of EW midline crossing defects in the genotypes shown in (C-D). Data are presented as mean \u00b1 SEM. 20 embryos were scored for each genotype. Significance was assessed using the Student\u2019s t-test (p<0.05).(TIF)Click here for additional data file.S3 FigGDHA (B), UAS-BratRDHA (C), UAS-Bratmyc (D), UAS-BratNHLMyc (E), UAS-BratCCMyc (F), UAS-BratBBMyc (G), UAS-BratBB1Myc (H) or UAS-BratBB2Myc (I) transgenes, stained with anti-HA (A-C) or anti-Myc (D-I) (green) and anti-HRP (blue (A-C) or magenta (D-I)) antibodies. Anti-HA and Anti-Myc labels cell bodies and axons of the eagle neurons (EG and EW), Anti-HRP reveals all of the CNS axons. Scale bar represents 10\u03bcm (A and D). (A-I) When driven by the eg-GAL4 transgene, the three UAS-Brat tagged HA and the six UAS-Brat tagged Myc transgenes are expressed at similar levels in the cell bodies and axons during the studied development stages.(A-I) Stage 15\u201316 embryos of the indicated genotype carrying eg-GAL4 and UAS-BratHA (A), UAS-Brat(TIF)Click here for additional data file.S4 Figwild type embryos, the average of the GFP signal intensity, reflecting Src64b expression, corresponds to 73%. (D-F) In brat mutant embryos, the GFP signal remains the same intensity compare to wild type embryos (70%). (G) Quantification of the GFP staining signal intensity shown in (A-F). Data are presented as mean \u00b1 SEM. 10 embryos were scored for each genotype. Significance was assessed using the Student\u2019s t-test .(A-F\u2019) Stage 14\u201317 embryos of the indicated genotype, stained with anti-GFP (green) and anti-HRP (magenta) antibodies. Anti-GFP labels the fusion protein Src-GFP, Anti-HRP reveals all of the CNS axons. Scale bar represents 10\u03bcm (A). (A-F) Src-GFP is expressed in all neurons from stage 13 to 17. (A-C) In (TIF)Click here for additional data file.S5 FigApc2 homozygous mutant embryos, show a similar HA signal intensity in cell bodies (73%), the absence of Apc2 does not perturb the Brat transgene expression. (B) Quantification of the HA staining signal intensity shown in (A-B\u2019). Data are presented as mean \u00b1 SEM. 3 embryos were scored for each genotype. Significance was assessed using the Student\u2019s t-test (A-B\u2019) Stage 15\u201316 embryos of the indicated genotype carrying eg-GAL4 and UAS-bratHA transgenes, stained with anti-HA antibodies. Anti-HA labels cell bodies of the eagle neurons (EG and EW). (A) and (A\u2019) In control embryos the average of the HA signal intensity reflecting the Brat transgene expression, corresponds to 71% in cell bodies. (B) and (B\u2019) (TIF)Click here for additional data file.S1 TableRelated to Figs (PDF)Click here for additional data file."} +{"text": "Intracranial injection of RCAS-PTN did not induce glioma formation when administrated alone, but significantly enhanced RCAS-platelet derived growth factor (PDGF)B-induced gliomagenesis. PTN co-treatment augmented PDGFB-induced Akt activation in neural progenitor cells in vitro, and enhanced neural sphere size associated with increased proliferation. Our data indicates that PTN expression is associated with chromosome 7 gain, and that PTN enhances PDGFB-induced gliomagenesis by stimulating proliferation of neural progenitor cells.Pleiotrophin (PTN) augments tumor growth by increasing proliferation of tumor cells and promoting vascular abnormalization, but its role in early gliomagenesis has not been evaluated. Through analysis of publically available datasets, we demonstrate that increased PTN mRNA expression is associated with amplification of chromosome 7, identified as one of the earliest steps in glioblastoma development. To elucidate the role of PTN in tumor initiation we employed the RCAS/ The vast majority of gliomas are adult grade II-IV oligodendrogliomas or astrocytomas that grow invasively into the cortex . Glioblaagenesis PDGFA amPTN), encoding a heparin-binding cytokine. PTN binds to and inactivates receptor-type protein tyrosine phosphatase receptor \u03b6 (PTPR\u03b6), leading to increased phosphorylation of its substrates [One of the genes located on chromosome 7 is pleiotrophin , or DF1 RCAS-PDGFB in combination with DF1 RCAS-PTN (RCAS-PDGFB+RCAS-PTN). Tumor incidence, as determined by the presence of Ki-67+ cells, was strikingly increased in mice injected with RCAS-PDGFB+RCAS-PTN (66.7%) as compared to mice injected with RCAS-PDGFB+RCAS-ev (38.7%) Figure .PDGFB cDNA was detected in all gliomas, and hPTN cDNA was present in all mice where RCAS-PDGFB and RCAS-PTN were combined . We did G/tv-a wt mice [+, and apoptotic cells were present . Data was cross-referenced to previously reported subtype classification [To determine the level of PTN expression in different glioblastoma subtypes, patient information and mRNA expression data from glioblastoma samples were collected as described (The Cancer Genome Atlas Research Network 2008). Processed datasets were obtained from the public access data portal (http://gliovis.bioinfo.cnio.es/). Gene expression data was correlated to gene copy numbers determined by Genomic Identification of Significant Targets in Cancer (GISTC) [The correlation between Chromosome 7 gain and PTN mRNA expression was analyzed using the GlioVis database ( (GISTC) .Co-expression analysis was performed at cBioPortal database using dataset of Glioblastoma . 41 genes were identified with Pearson's correlation coefficient more than 0.61.\u00ae 2000 transfection agent .hPTN cDNA was cloned into RCAS-Y to generG/tv-a wt [G/tv-a;Arf-/- [G/tv-a mice, DNA was extracted from paraffin embedded tissue. PCR-detection of gene was done on with specific primers , 10ng/ml PDGFB or the combination of PTN and PDGFB. Cells were harvested 4 hours after treatment.Neural progenitor cells were isolated from eviously . Cells wNeural progenitor cells were seeded at 250 cells/ml in neural stem cell medium in ultra-low attachment 6-well plate . 25ng/ml PTN, 10ng/ml PDGF or the combination of PTN and PDGF were added every second day. On day 6, microscopic images of all spheres were taken using a phase contrast microscope to allow size quantification. Then the spheres were fixed with 4% PFA on ice for 10 min, and embedded in OCT medium (Tissue-Tek Sakura) for frozen section. Immunostaining was performed on 7 \u03bcm sections. Slides were blocked with 3% bovine serum albumin (Roche Diagnostics) in phosphate-bufferd saline (PBS) and incubated with primary antibody diluted Data was analyzed using GraphPad Prism 5.0. The Mann-Whitney test was used for comparison between two groups, and ANOVA with Newman-Keuls test was used for multiple groups\u2019 comparison. Error bars indicate standard deviation from the mean (s.d). Statistical tests were two-sided, and p-values smaller than 0.05 were considered statistically significant."} +{"text": "Drosophila platonic (plt) males court females, but fail to copulate. Here we show that plt is an allele of scribbler (sbb), a BMP signalling component. sbb knockdown in larvae leads to the loss of approximately eight serotonergic neurons, which express the sex-determinant protein Doublesex (Dsx). Genetic deprivation of serotonin (5-HT) from dsx-expressing neurons results in copulation defects. Thus, sbb+ is developmentally required for the survival of a specific subset of dsx-expressing neurons, which support the normal execution of copulation in adults by providing 5-HT. Our study highlights the conserved involvement of serotonergic neurons in the control of copulatory mechanisms and the key role of BMP signalling in the formation of a sex-specific circuitry. Drosophila platonic (plt) mutant males court with females but fail to copulate. Here, the authors find plt is an allele of scribbler and may disrupt courtship behaviour via developmental disruption of a subgroup of serotonergic Doublesex+ neurons in the abdominal ganglion. This approach led to the identification of pC2l, a dsx-expressing (dsx[+]) neural cluster in the brain, which plays a role in triggering attempted copulationdsx[+] neurons in the abdominal ganglion with roles in regulating the copulation durationserotonin receptor 1A (5-HT1A) knockdown in IPCs reduces the latency to abdominal curling and increases the incidence of attempted copulation5-HT1A knockdown in IPCs also shortens the latency to courtship initiationplatonic (plt), whose males fail to copulate despite vigorous courtship towards a female in the abdominal ganglion. The plt mutation impairs the BMP signalling required for the survival of eight serotonergic interneurons in the abdominal ganglion, and thereby induces copulation defects in mutant males via a 5-HT deficiency. Our results highlight the conserved roles of 5-HT in the regulation of copulation and of BMP signalling in the development of cellular substrates for sex-specific behaviours.When successful, male courtship culminates in copulation, yet the neural mechanism underlying copulation has attracted limited attention in biologyplt homozygotes are semilethal, and exhibit a visible phenotype in the wing vein: the posterior-most wing vein fails to reach the wing margin BSC334 and Df(2R)ED3683, when placed in trans to plt, uncovered the no-copulation phenotype as well as the short-wing vein phenotype in theseplt hemizogotes, whereas four other deficiencies, (Df(2R)BSC335, Df(2R)BSC399, Df(2R)BSC483 and Df(2R)Exel7153), similarly tested failed to uncover either phenotype (scribbler (sbb). Indeed, genetic complementation tests where plt was combined with different sbb mutations demonstrated that plt is allelic to sbb: these heteroallelic mutants manifested both the copulation defect and wing vein defect expression as detected by PCR with reverse transcription and doublesex (dsx)1314sbb knockdown with UAS-sbbRNAi as driven by fru-GAL4 (fruGAL4 or fruNP21) or dsx-GAL4 (dsxGAL4). In contrast to the partial suppression of copulation success with fru-GAL4 . This result indicated that sbbRNAi expression via dsx-GAL4 completely blocked copulation irrespective of whether repo-GAL80 was present or not , while others express either dsx or fru , and the rest express neither of these genes1617dsx-positive cells requires sbb+ for normal copulation, we restricted the action of dsx-GAL4 by expressing GAL80 in a subset of dsx-positive cells using three different intersection strategies. In the first strategy, we used a conditional GAL80 transgene, tubP>GAL80>. Here the two > indicate the FRT sequences, that is, thetarget of the flippase FLP; the FLP binds to the FRTs flanking GAL80, thereby excising GAL80 and allowing GAL4 to act. We combined tub>GAL80> with fruFLP, which expresses FLP from the endogenous fru gene promoter, and in this way activated GAL4 only within dsx[+]/fru[+] cells or mechanosensory cells (ppk-GAL4), as these peripheral neurons may guide a male fly to potential copulation targetssbb knockdown was targeted to all the central nervous system (CNS) dsx cells except those in the head, the copulation defect was still produced was not amenable to testing due to the lack of tools for its specific manipulation. These results collectively indicate that the dsx-expressing neurons in the non-thoracic ventral nerve cord (VNC), dsx-positive TN2 and dsx-positive SN remain as candidate cells. Peripheral mechano- and chemo-sensory neurons were judged not to make a large contribution to the no-copulation phenotype induced by sbb knockdown (GAL4s representing six neurotransmitter species were tested in conjunction with dsxFLP and tubP>GAL80>: acetylcholine (Cha-GAL4), octopamine (Tdc2-GAL4), dopamine (TH-GAL4), 5-HT (Trh-GAL4),L-glutamate (Vglut0k371-GAL4 and Vglut-GAL4 on X) and GABA . The most striking inhibition of copulation was observed when sbbRNAi was expressed under the control of Trh-GAL4 or GAD-GAL4, although Cha-GAL4, TH-GAL4 and Vglut-GAL4 also yielded significant reductions in copulation success when used as drivers for sbbRNAi expression . It isnoteworthy that only a total of eight dsx-expressing cells were labelled by the intersection of Trh-GAL4 and dsxFLP also inhibited copulation (n=24) nor did a solitary male display motions associated with copulation. This might mean that 5-HT secreted by these neurons gates execution of copulation when the released dose exceeds a threshold, but elicits no further effect at higher doses beyond the threshold. To determine whether 5-HT is necessary for executing copulation at the adult stage or for normal development of neural components, we examined the effect of supplementing foods with the 5-HT precursor 5-hydroxytriptophan (5-HTP) after adult emergenceTrh knockdown in dsx-expressing cells were fed with 5-HTP only at the adult stage, the emerged adult males exhibited higher copulation success , a gene encoding atranscription factor also acting downstream of Dpp; Omb transcriptionally represses the Dpp receptor gene thickvein (tkv), and failure in this repression results in tkv upregulation, which ultimately causes cell deathtkv expression in the larval wing discdsx neurons at a defined time window before the imaginal metamorphosis for proper differentiation of these neurons. The BMP family proteins to which Dpp belongs are conserved across phyla, thereby playing central roles in a variety of morphogenetic events, including sexual character development. For example, the formation of papillary processes in the anal fin, a masculine sexual characteristic in Medaka fish, is induced by androgen via the enhancement of BMP7 expressionOverall, our results indicate that sbb+, 5-HT produced by Trh-positive dsx neurons in the VNC is required for the control of copulation in the adult stage. The exact role of serotonergic dsx neurons in the VNC in the control of copulatory mechanisms remains to be clarified. Higher-resolution motion recordings of a fly attempting copulation might help in detecting subtle yet critical changes in the specific movements of abdominal structures in sbb mutant males.In contrast to the developmental role of dsx single-positive neurons called pC2l fruGAL4, fruFLP, UAS>stop>mCD8::GFP, UAS>stop>TNT, UAS>stop>TNTin and UAS>stop>dTrpA1 were the kind gift from Barry J. Dickson (HHMI-Janelia Farm Research Campus)30dsxGAL4, dsxFLP and elav-GAL80 were the kind gifts from Stephen F. Goodwin (University of Oxford)17repo-GAL80 was the kind gift from Leslie B. Vosshall (Rockefeller University)fruLexA was the kind gift from Bruce S. Baker (HHMI-Janelia Farm Research Campus)Otd-FLP was the kind gift from David J. Anderson tsh-GAL80 was the kind gift from Julie Simpson (HHMI-Janelia Farm Research Campus). w;; poxn-GAL4-14-1-7, UAS-mCD8::GFP was a gift from Ken-ichi Kimura (Hokkaido University of Education). w, ppk-GAL4 was the kind gift from Kazuo Emoto (University of Tokyo). w;; GAD-GAL4 on 3rd was the kind gift from Yuh Nung Jan w1118; Df(2R)ED3610/CyO (#9066), w1118; Df(2R)BSC334/CyO (#24358), w1118; Df(2R)ED3683/SM6a (#8918), w1118; Df(2R)BSC335/CyO (#24359), w1118; Df(2R)Exel7153/CyO (#7893), w1118; Df(2R)BSC399/CyO(#24423), w1118; Df(2R)BSC483/CyO (#24987), w; Tdc2-GAL4 (#9313), w;; TH-GAL4 (#8848), Vglut-GAL4 on X (#24635), w1118; VglutOK371-GAL4 (II) (#26160), w; Cha-GAL4, UAS-GFP (#6793), w; Trh-GAL4 (#38388), w; GAD-GAL4 on 2nd (#51630), w; UAS-Dcr2 (#24650), w;;UAS-Dcr2 (#24651), tubP>GAL80>; Bl/CyO; TM2/TM6 (#38879), w; tubP>GAL80>/CyO; TM2/TM6 (#38880), w; Sp/CyO; tubP>GAL80>/TM6B (#38881), w; tubP>stop>GAL80>; MKRS/TM6 (#38878), w; wgSp-1/CyO; tubP>stop>GAL80 (#39213), w; UAS>stop>mCD8::GFP; UAS>stop>mCD8::GFP(#30032), w;; LexAop-GAL80 (#32213) and UAS-Trh-RNAi (#25842). The w1118;UAS-sbbRNAi (#41845) strain was obtained from the Vienna Drosophila Resource Center.Flies were reared on cornmeal-yeast medium under a 12:12 light:dark cycle at 25\u2009\u00b0C, except for those carrying a transgene for RNA interference (RNAi), which were raised at 29\u2009\u00b0C. For the heat-shock induction of nded RNA , larvae Trh-RNAi and dsx-GAL4 were collected upon emergence, and kept singly with food containing 100\u2009mM 5-HTPpost hoc comparisons for the courtship index, or by the \u03c72-test for the mating success.The virgin males and females were collected at eclosion. Males were placed singly in food vials, while 10 females were placed together in single food vials. They were kept at 25\u2009\u00b0C until being subjected to behavioural assays. For the 5-HTP treatment , virgin The CNS was dissected from flies in PBS, and fixed in 4% paraformaldehyde in PBS for 60\u2009min. Immunostaining was carried out as described previouslyThe authors declare that the data supporting the findings of this study are available within the article and its files, oHow to cite this article: Yilmazer, Y. B. et al. Serotonergic neuronal death and concomitant serotonin deficiency curb copulation ability of Drosophila platonic mutants. Nat. Commun.7, 13792 doi: 10.1038/ncomms13792 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures 1-12 and Supplementary Note 1."} +{"text": "The introduction of ABL Tyrosine Kinase Inhibitors (TKIs) has significantly improved the outcome of Chronic Myeloid Leukemia (CML) patients that, in large part, achieve satisfactory hematological, cytogenetic and molecular remissions. However, approximately 15\u201320% fail to obtain optimal responses according to the current European Leukemia Network recommendation because of drug intolerance or resistance.Moreover, a plethora of evidence suggests that Leukemic Stem Cells (LSCs) show BCR-ABL1-independent survival. Hence, they are unresponsive to TKIs, leading to disease relapse if pharmacological treatment is discontinued.All together, these biological events generate a subpopulation of CML patients in need of alternative therapeutic strategies to overcome TKI resistance or to eradicate LSCs in order to allow cure of the disease.non ABL-directed inhibitors\u201d targeting signaling pathways downstream of the BCR-ABL1 oncoprotein and describe immunological approaches activating specific T cell responses against CML cells.In this review we update the role of \u201c BCR-ABL1 fusion oncogene encoding for a multi-domain BCR-ABL1 oncoprotein [Chronic Myeloid Leukemia (CML) is a myeloproliferative disorder characterized by neoplastic transformation of the Hematopoietic Stem Cell (HSC) which displays a cytogenetic marker derived from a reciprocal t9;22 translocation . The ensoprotein . BCR-ABLoprotein \u20137.In 2001 the introduction of Imatinib Mesylate (IM), a semi-specific BCR-ABL1 tyrosine kinase inhibitor, improved the outcome of CML patients in chronic phase, generating unprecedented rates of hematologic, cytogenetic and molecular response \u201310. IndeSeveral biological mechanisms responsible for IM failure have been described including BCR-ABL1-dependent and \u2013independent mechanisms.i) mutations in the ABL kinase domain which prevent TKI binding [ii) amplification of the BCR-ABL1 oncogene [iii) high expression levels of the BCR-ABL1 mRNA [The former include: binding ; ii) amponcogene , 17; iiiBL1 mRNA .i) up-regulation of drug efflux pumps [ii) down regulation of drug influx transporters [iii) Lyn overexpression (Src-family kinase protein) [iv) other BCR-ABL1-independent mechanisms [The latter comprise: ux pumps ; ii) dowsporters ; iii) Lyprotein) and iv) chanisms .To overcome IM-resistance, more potent second-generation and third-generation (3G i.e. Ponatinib - PON) TKIs have been developed and approved for the treatment of the disease \u201326.However, while 2G and 3G TKIs present higher BCR-ABL1 inhibitory activity if compared to IM, they have failed to generate meaningful survival advantages for CML patients \u201330. MoreIn this review, we provide an update on the current knowledge of non ABL-directed inhibitors and immunological-targeting approaches as treatment strategies for CML patients achieving unsatisfactory responses to TKIs. In detail, we will focus on findings generated in primary CML cells, CML murine models and clinical trials.Farnesyl Transferase Inhibitors (FT-Is) inhibit farnesyl transferase activity preventing isoprenoid-group transfer on different protein targets , 35. IsoIn CML, constitutive RAS activation is promoted by BCR-ABL1 interaction with Grb2 (Growth factor receptor bound protein), SOS (Son Of Sevenless) and Gab2 (Grb2-associated binder 2) and plays a critical role in leukemogenesis Fig.\u00a01a1a. Tipifn\u2009=\u20091 T315I, n\u2009=\u20092\u00a0M244V, n\u2009=\u20091 E255K) [Clinical data obtained from twenty-two CML patients with chronic or advanced disease that had failed Interferon-alpha\u00a0(INF\u03b1) treatment demonstrated that Tipifarnib, as a single agent, induced complete or partial hematological responses and transient minor cytogenetic responses with a median duration of only 9\u00a0weeks . In Phas1 E255K) .A pilot study investigated Lonafarnib efficacy in CML patients resistant or intolerant to IM. Only two of thirteen enrolled subjects showed hematological responses . HoweverIn summary, these data demonstrate that FT-I monotherapy showed little benefit for CML patients. However, their combination with IM may prove useful for CML subjects unresponsive to IM.mTOR Inhibitors (mTOR-Is) target the mammalian Target of Rapamycin (mTOR) , a serinRapamycin induces mTOR dephosphorylation resulting in reduced CML cell viability and incrEverolimus blocks mTOR constitutive activation, reducing CML proliferation and increasing IM sensitivity , 55. IntEverolimus therapeutic efficacy in CML patients,\u00a0both alone and in combination with IM, is being evaluated in different clinical trials (NCT00081874), (NCT00093639).BEZ235\u00a0is a dual PI3K-mTOR inhibitor tested on BCR-ABL1-positive cell lines. Published data demonstrated that the combination of BEZ235 and NIL induces apoptosis, inhibits tumor growth in CML xenograft models and impairs NIL resistance , 58. A PTo date, Temsirolimus is being investigated in a clinical trial in combination with IM (NCT00101088).Even if the mTOR-Is have been thoroughly investigated in primary CML cells and in CML murine models recipient showing the ability to kill LSCs, to date, no data on\u00a0CML patients are available, hence their therapeutic efficacy remains to be established.Smo Antagonists (Smo-As) inhibit Smoothend (Smo), a putative seven-transmembrane domain receptor which is a component of the Hedgeohg (Hh) pathway involved in a\u00a0broad number of\u00a0cellular mechanisms such as stem cell renewal, cell proliferation and survival Fig. . BindingCML patients showed higher Hh expression compared to healthy donors and IM treatment did not reduce these mRNA levels, suggesting that Hh over-expression was not dependent on BCR-ABL1 kinase activity , 61.Smo knock-out mice, compromised both leukemic stem cell renewal and propagation [Dierks et al. reported that Smo up-regulation improves expansion of BCR-ABL1-positive LSCs . Moreovepagation . Hence, Smo-As have been investigated in ex-vivo studies as well as in several clinical trials.LDE225 significantly reduced colony forming ability and re-plating efficiency of CML CD34-positive cells and\u00a0also decreases their Long Term Culture - Initiating Cell (LTC-IC) frequency. Furthermore, the combination of LDE225 with NIL reduced the engraftment of CML CD45-positive cells in NSG (NOD scid gamma) mice. . At the Two clinical trials have evaluated the efficacy of BMS833923 in CML. In the first study (NCT01218477) CML and Ph\u2009+\u2009Acute Lymphoblastic Leukemia (ALL) patients resistant to IM or NIL were exposed to the combination of BMS833923 and\u00a0DAS. Only 1 of 27 patients in chronic phase attained a complete cytogenetic response while no patients with Ph\u2009+\u2009ALL or advanced CML displayed any clinical benefit . In the BCL2 (B-Cell Lymphoma 2)\u00a0and/or ABCA2 (ATP-Binding Cassette sub-family A member 2) oncogenes [In preclinical studies, PF-04449913 impaired the multi drug resistance (MDR) mechanism in LSCs by down-regulating the ncogenes . Furtherncogenes . A Phasencogenes . HoweverIn conclusion, data obtained by ex-vivo studies or in mouse models suggest that inhibition of the Hh pathway interferes with both self-renewal and propagation of pluripotent BCR-ABL1-positive hematopoietic cells. Unfortunately, the unsatisfactory results obtained in CML patients currently preclude any significant role for these drugs in CML treatment.JAK2 inhibitors (JAK2-Is) suppress JAK2 catalytic activity that modulates STATs transcription factors regulating the expression of genes involved in cell proliferation, differentiation and apoptosis Fig. . PublishJAK2 inhibitors (JAK2-Is) have also been combined with IM, NIL and DAS killing CML cells and restoring TKI-sensitivity in resistant CML cell lines \u201373.BCR-ABL1 mRNA levels [Using a combination of Ruxolitinib with NIL, Gallipoli and colleagues observed an increased apoptotic rate in CML cell lines and a reduction of the leukemic engraftment in CML murine models . These dA levels . SeveralBMS-911543 displays cytotoxic effects in CML cell lines when administrated in combination with TKIs. Specifically, the exposure of BCR-ABL1-positive CD34 cells to BMS-911543 and DAS, eliminates TKI-insensitive leukemic stem cells, suggesting that the dual targeting strategy involving inhibition of both BCR-ABL1 and JAK2 may reduce the risk of developing TKI resistance in CML patients .In conclusion, JAK2-Is combined with TKIs may represent a useful therapeutic approach for patients with advanced or resistant CML and may also contribute to the eradication of LSCs.Heat shock protein 90 (Hsp90) is a member of the Hsp family that encompass several ATP-dependent molecular chaperones constitutively expressed or induced by stress conditions such as hypoxia or toxin exposure (proteotoxic stress). They act preserving the correct folding of their client proteins and blocking their proteosomal degradation. Hsp90 shows high intratumoral expression and represents a poor prognostic indicator in cancer patients. Hsp90 inhibitors (Hsp90-Is) represent compounds of great interest as potential anti-leukemic agents \u201379.Since, high Hsp90\u00a0expression inhibits BCR-ABL1 degradation, Hsp90-Is reduce BCR-ABL1 half-life Fig. limitingIn preclinical experiments, 17-allylamino-17-demethoxygeldanamycin (17-AAG) showed low efficacy when used as monotherapy but increased apoptotic rates when administrated in combination with Histone Deacetylase Inhibitors (HDAC-Is) or IM , 81. TwoUsing in-vitro CML experimental models, Ying et al., compared the anticancer properties of STA-9090 and 17-AAG. STA-9090 was more potent than 17-AAG in reducing the proliferation of CML cells, suggesting that it may be a useful agent for CML patients . Both PhBIIB021 reduces BCR-ABL1 protein expression thereby inducing significant growth inhibition in CML cell lines both sensitive and resistant to TKIs. In addition, BIIB021 also triggers autophagy by repressing the AKT-mTOR pathway and thus reactivating autophagy-inducer Ulk1 (unc-51 like autophagy activating kinase 1) .Novobiocin is a potent inhibitor of CML cell proliferation, with weak effects on CD34-positive cells derived from healthy donors. Furthermore, co-treatment of\u00a0Novobiocin with\u00a0IM reduced the proliferation of TKI-resistant cells, suggesting that this combination may be useful to overcome the mechanisms leading to IM failure .In summary, Hsp90-Is generated promising results against primary and immortalized CML cells and in CML mouse models. However, the lack of data in CML patients requires further studies to asses the effectiveness of Hsp90-Is for CML treatment.Histone Deacetilase Inhibitors (HDAC-Is) are small-molecules that block HDAC enzymes involved in epigenetic modifications that regulate histone acetylation state. In general, while histone acetylation carried by Histone Acetyl Transferases (HATs) determines a chromatin permissive state that favors gene expression, histone deacetylation performed by HDACs, overturn this biological event inducing gene repression [pression or TKIs kills primary CML cells, Baf3 cells\u00a0expressing different BCR-ABL1 mutants and also shows antileukemic properties in CML mouse models. , 89. TheLBH589 is an HDAC-I with potent antiproliferative activity in several cancer cell lines . LBH589 These results suggest that HDAC-Is have questionable efficacy as single agents while they may be promising therapeutic agents when administrated in combination with additional anti-cancer drugs in patients failing TKIs.Sirtuin Inhibitors (Sirt-Is) are a broad range of pharmaceutical agents inhibiting class III HDAC enzymes called Sirtuins (SIRTs) Fig. . These ptivities . Among ativities . SIRT1 icipients , 96. SirIs a small-molecule that inhibits SIRT1 and SIRT2 resulting in p53 acetylation and activation\u00a0. The comUnlike TV-6, sirtinol is a SIRT1 specific inhibitor with anti-cancer properties in different tumors . Wang etIn conclusion, the ability of Sirt-Is to maintain genomic stability and to reduce the LSCs pool, makes these compounds promising tools for CML treatment.Studies of gene and protein expression have shown that alternative splicing of multiple BCL2 family members facilitate the expansion of quiescent CML stem cells , 100 andAs BCL2 inhibitors (BCL2-Is) overturn these biological effects Fig. , they haSabutoclax, a pan-BCL2 inhibitor, sensitizes LSCs in the bone marrow niche to TKIs. A recent study has shown that exposure of CML CD34-positive cells to Sabutoclax increases DAS efficacy reducing engraftment of LSCs in mice .Preclinical evidence suggests that the pan-BCL2 inhibitor Obatoclax reduces colony formation in Ph\u2009+\u2009CD34-positive progenitors . A PhaseUnlike Sabutoclax and Obatoclax, Venetoclax displays BCL2-selective antagonism with modest activity against CML progenitors when used as single agent. However, Ko and colleagues have recently shown that Venetoclax enhances IM cytotoxicity on CML progenitors .In conclusion, although BCL2 inhibition may become a useful strategy in the future, the lack of clinical data in CML patients currently excludes this class of drugs from CML therapy.Aurora kinase inhibitors (AURK-Is) suppress the serine-threonine kinase activity of the AURK family that regulates cell division \u2013107 Fig. Three iMK-0457 is active against immortalized CML cell lines and has also shown the ability to revert advanced CML patients expressing the T315I mutant to the chronic phase of the disease , 109. ThUnlike MK-0457, PHA-739358 is a dual inhibitor of AURK and ABL , which showed promising activity both in leukemia and solid tumors. In detail, Danusertib exerts growth inhibition in immortalized BCR-ABL1-positive cells and in CML CD34-positive progenitors derived from patients sensitive or resistant to TKIs , 112. InAKI603 is an aurora kinase A inhibitor that exerts its antiproliferative activity by arresting CML cells sensitive or resistant to IM in the G2/M phase of the cell cycle. . AKI603 Like AKI603, MLN8237 is an Aurora Kinase A inhibitor but it induces CML cell death by decreasing expression of Apollon, a protein that modulates cell division and apoptosis. In-vitro CML experimental models showed that MLN8273 induces apoptosis in cells expressing both wt and mutant BCR-ABL1. Moreover, MLN8273 improves NIL activity increasing CML CD34-positive cell death and reducing tumor growth in recipient mice .This multitarget kinase inhibitor, shows activity against CML cell lines and is able to reduce the engraftment of primary BCR-ABL1-positive cells . A PhaseAll together, these data indicate a likely role for AURK-Is as a useful therapeutic resource for patients with advanced CML resistant to TKIs.Omacetaxine binds the ribosome aminoacyl-tRNA acceptor site, thereby inhibiting the synthesis of different oncoproteins including BCR-ABL1 Fig.\u00a02)2). ExperCortes and colleagues used Omacetaxine in CML patients resistant or intolerant to TKIs and obtained meaningful hematological and cytogenetic remissions , 121. FuFollowing these clinical data, the FDA approved Omacetaxine for the treatment of CML patients that do not benefit from TKIs with specific attention to patients carrying the T315I substitution.Clinical studies and results from non ABL-directed Inhibitors are summarized in Table The immune response against cancer is impaired by an immune escape of the tumor cells . Over thUsually BCR-ABL1 immunogenic peptides are formed by an amino acid sequence of the e13a2 or e14a2 break-point region . DiffereThe EPIC study accrued nineteen patients that were vaccinated using e14a2 peptides. Thirteen patients, in cytogenetic remission after IM, showed late T cell immune response to BCR-ABL1 peptides and achieved a 1-log decrease in BCR-ABL1 transcripts .Nitin and colleagues investigated the efficacy of a mixture of immune-peptides in ten CML patients expressing e13a2 or co-expressing e13a2/e14a2 BCR-ABL1 isoforms. Three patients achieved a 1-log reduction in BCR-ABL1 mRNA levels and 3 additional patients developed a major molecular response. However, these responses have not been stable over time, suggesting that this therapeutic approach may only transiently improve molecular response in CML patients .In a Phase 2 trial (NCT00267085), patients previously exposed to IM and showing complete cytogenetic remission but not a major molecular response were subjected to vaccination using the CMLVAXB2 or CMLVAXB3 peptides against the e13a2 and e14a2 BCR-ABL1 isoforms, respectively. Three patients out of ten achieved a 1-log reduction in BCR-ABL1 mRNA levels.BCR-ABL1 mRNA levels reduction in patients previously exposed to IM and/or IFN [An interim analysis of a Phase II Multicenter GIMEMA CML Working Party trial reported that CML patients with minimal residual disease during IM treatment obtained a reduction of their disease burden after being exposed to the peptide vaccine CMLVAX100 . Furtherd/or IFN .BCR-ABL1 transcripts in both peripheral blood and bone marrow [The same group also described a patient that received a vaccine based on the e13a2 BCR-ABL1 isoform (CMLb2a2\u201325mer), achieving undetectable e marrow .In summary, the vaccines against BCR-ABL1 break-points have shown the ability to reduce residual disease in TKI-treated patients achieving cytogenetic remission. Several clinical trials are being this therapeutic approach (NCT00428077), (NCT00466726), (NCT00004052).i. the immunopeptide against the Wilms tumor oncogene (WT1), frequently overexpressed in CML patients. When this immunopeptide associated with IM, it may induce deep molecular response [ii. K562/GM-CSF (GVAX), a cell-based vaccine derived from K562 cells genetically modified to produce granulocyte-macrophage colony-stimulating factor (GM-CSF) and a number of LAAs which recruit dendritic cells and activate T cell-mediated CML-specific immune responses. GAVX has been shown to reduce BCR-ABL1 transcript levels in CML patients [Leukemia Associated Antigens (LAAs) are overexpressed in multiple leukemias including CML. Different LAAs have been identified as potential targets for vaccine synthesis and CML therapy , 130. Amresponse . Currentpatients .Overall, the data generated in CML preclinical models and clinical report indicate a promising role for immune-dependent therapies for CML treatment.DCs are antigen-presenting cells that induce humoral and cellular immune responses. In CML, progenitor cells drive the formation of both leukemic clones and DCs. Since 98% of them express the BCR-ABL1 oncoprotein, these cells represent a potential target for immunological therapy . PreviouIn conclusion, DCs-based vaccines appear unlikely to be of any meaningful value for CML treatment in the foreseeable future.Cancer immunotherapy based on immune-checkpoint blockade (ICB) employs monoclonal antibodies against negative immune-regulator checkpoints such as cytotoxic T-lymphocyte antigen 4 (CTLA-4), programmed death 1 (PD-1) and its ligands .CML-specific cytotoxic T Lymphocytes (CTLs) show high PD-1 levels, whereas CML cells express PD-L1. In murine CML models, abrogation of PD-1 expression increases overall survival , 138 sugRecently, Schutz demonstrated a correlation between CTLA-4-ligand CD86 expression and risk of disease relapse after TKI discontinuation. Indeed of 122 patients that had ceased TKIs, those expressing lower CD86 levels showed a 70% relapse-free survival suggesting that CD86 expression may be an early indicator of poor treatment-free remission probability .A clinical trial (NCT01822509) is presently evaluating the efficacy of the combination ipilimumab (anti-CTLA-4) plus nivolumab (anti-PD-1) in patients with hematologic malignancies, including CML, relapsed after allogeneic hematopoietic cell transplantation.Clinical studies and results from immune strategies are summarized in Table TKIs that interfere with BCR-ABL1 signaling currently represent the first line and second line treatment of choice for most CML patients .However, BCR-ABL1-dependent or \u2013independent resistance as well as BCR-ABL1-independent LSCs survival, partially undermine TKIs efficacy. Hence, a subgroup of CML patients is clearly in need of alternative therapeutic approaches. In this review we focused our attention on a range of pharmacological agents -non ABL-directed inhibitors- against different targets involved in BCR-ABL1-dependent leukemic transformation.BCR-ABL1 mRNA levels or induce cytogenetic remissions. However, with the exception of Omacetaxine, none of the above indicated compounds have received approval for CML treatment. Furthermore, while there are several ongoing clinical trials evaluating the association of Ruxolitinib with NIL, at the current time it appears unlikely that other promising agents will undergo clinical development for the treatment of the disease.We summarized data showing that FT-Is in combination with TKIs, Omacetaxine, AURK-Is and JAK2-Is have demonstrated efficacy in CML patients. We have also outlined clinical data demonstrating that vaccination against WT1 antigen, in combination with IM may represent a potential strategy to reduce i) a WNT (homologus wingless)-targeting drug to modulate stem cell survival , ii) HDM2 inhibition to increase p53 half-life , iii) a CXCR4 (CXC-chemokine receptor 4) antagonist as a hematopoiesis regulator , iv) an ABL allosteric modulator .The unsatisfactory results obtained with most of the non ABL-direct inhibitors has fostered additional research in the field that is currently investigating alternative strategies including: In summary, non ABL-directed inhibitors have often showed ability to overcome TKI resistance in primary CML cells or to eradicate the LSCs in mouse models. However, they displayed questionable efficacy in CML patients. Likewise, immunological approaches may be useful to improve molecular response, but this effect is often transient.Finally, while the use of ICB may represent promising approaches to eradicate LSCs and predict molecular relapse of the disease after TKI discontinuation, these immune-based strategies seem far from achieving clinical relevance for CML therapy."} +{"text": "Myocardial oedema is typically imaged using a pre-contrast T2-weighted short tau inversion recovery (T2w-STIR) sequence on cardiovascular magnetic resonance (CMR) imaging. However, this sequence is prone to motion and rhythm artefact, signal dropout, blood-pool artefact, surface coil signal inhomogeneity and potentially prohibitive long breath-hold duration. This susceptibility to artefacts limits utility of T2w-STIR in large clinical trials where attainment of diagnostic quality oedema imaging in the majority is necessary to determine myocardial salvage: a measure of reperfusion success and a strong predictor of adverse remodeling and prognosis post ST-segment elevation myocardial infarction (STEMI). We compare AAR quantified on T2w-STIR imaging with novel T1-mapping on 3.0T CMR post STEMI.Fifty-five patients underwent CMR 1-5 days following presentation with STEMI. AAR was quantified using semi-automatic thresholding on T2w-STIR images and resulting parametric colour maps from T1 Modified Look Locker Inversion Recovery (MOLLI) sequences performed with the patient breathing freely and with motion correction algorithm applied (MOCO-T1). Paired t-tests were used to compare AAR derived using the two sequences. Pearson's correlation coefficient was used to assess correlation. Inter-sequence agreement was assessed using the Bland-Altman method, coefficient of variation (CoV) and two-way mixed-effect intra-class correlation coefficient (ICC) for absolute agreement.See Table MOCO-T1-mapping is more robust than T2w-STIR for identification of reversible myocardial injury and prediction of functional recovery following STEMI. Furthermore, MOCO-T1 imaging may allow AAR to be accurately determined without long breath holds, often required for the acquisition of oedema imaging, in acutely unwell patients. This requires validation in larger studies and may have implications for sample size calculations in trials using CMR surrogate markers of myocardial injury."} +{"text": "Some authors have evaluated the possibility of using post-surgical Tg (ps-Tg) values in deciding for or against TRA. The aim of our study was to verify the diagnostic accuracy of 131I-pT-WBS and SPECT/CT imaging (post-therapeutic imaging) compared to serum Tg levels in detecting metastases in early stage of DTC patients.Differentiated thyroid cancer (DTC) work-up is based on (near)total-thyroidectomy plus thyroid remnant ablation (TRA) with 131-radioiodine in many patients, and long-life follow-up. Post-therapeutic imaging revealed metastases in 82 out of 570 (14.4%) patients. Metastases were successively confirmed by other diagnostic tools or by histology (sensitivity and PPV = 100%). Seventy-three out of 82 patients (90.2%) showed ps-Tg levels \u22641 ng/ml. In fifty-four per cent of patients, serum Tg levels at TRA remained \u22641 ng/ml.131I-pT-WBS and SPECT/CT) is an accurate method of detecting metastases, also in patients with stimulated serum Tg values \u22641 ng/mlIn conclusion, ps-Tg levels cannot be used in deciding for or against TRA. In early stage of DTC, post-therapeutic imaging , referred to our Nuclear Medicine Units in the last five years to perform TRA after (near)-total-thyroidectomy.All patients underwent TRA 3-4 months after thyroid surgery either in euthyroid or in hypothyroid state. Serum Tg values evaluated in post-surgical period and at TRA were matched with post-therapeutic imaging results. Thyroid cancers occur in 2-5% of thyroid nodules, with an incidence of 3.8% of all new cancer diagnosis in 2014 plus radioiodine (RAI) thyroid remnant ablation (TRA) in most patients, and long-life follow-up.According to the latest American Thyroid Association (ATA) guidelines, the role of TRA has been revised. TRA is clearly indicated in high risk patients while it is not indicated in low risk patients and discouraged in many intermediate risk cases.131I-post therapy whole body scan (pT-WBS), with or without SPECT/CT imaging (post-therapeutic imaging) and serum thyroglobulin (Tg) measurements, are used in identifying metastatic patients. In addition, in the recent past, some authors have evaluated the possibility of using post-surgical Tg (ps-Tg) values (both in L-T4 therapy and/or after TSH-stimulation) in deciding for or against TRA and/or histology [16 out of 82 patients (19.5%)].In all patients, metastases were successively confirmed (sensitivity and PPV = 100% for both) by targeted morphological and/or morpho-metabolic studies or in hypothyroid state .Patients underwent TRA 3-4 months after thyroid surgery either in euthyroid state [by intra-muscular administration of recombinant human TSH (rhTSH) . Colour-Doppler study was also performed, if necessary. nUS evaluation included the thyroid bed and both central and lateral lymph-nodes stations. Patients with suspicious lymph-nodes underwent fine needle aspiration cytology (FNAC) and Tg measurement in the aspirate fluid. If the lymph-node was positive for malignancy, the patient underwent selective or radical lymphoadenectomy. These patients were not included in the present study. However, in this study, patients without lymph-nodes suspicious for malignancy were assessed.Measurement of serum TSH , Thyroglobulin (Tg) , and anti-Thyroglobulin Antibodies (Tg-Ab) was obtained. Tg assay had a functional cut-off of 0.6 ng/ml and a sensitivity cut-off of 0.15 ng/mL.131I tracer activity (1.8 MBq) administration, as elsewhere described [RAIU was evaluated 24 hours after Na-escribed , 16.Radioiodine activities administrated for TRA ranged from 1110 to 9250 MBq (median: 3700 MBq). Higher radioiodine activities were administered to patients with higher risk factors .pT-WBS was obtained using a double-headed gamma camera equipped with high-energy low-resolution parallel-hole collimators (HEHRPAR). In order to obtain a better target/background ratio, patients were required to drink at least 1.5 litres of water and take laxatives drugs some days before the study. The pT-WBS was integrated by single photon emission tomography-computed tomography (SPECT-CT) on request of attending nuclear medicine physician. When SPECT/CT images were obtained, nuclear medicine physicians verified if the abnormal radioiodine uptake corresponded to a defined morphological lesion .During retrospective analysis of the data, all post-therapeutic images were independently re-evaluated by two nuclear medicine physicians with more than twenty years of expertise, blinded to serum Tg levels. If they disagreed, they discussed and reached a consensus in all cases. Radioiodine uptake in the thyroid bed was considered as thyroid remnant while the radioiodine uptake located outside the thyroid bed and/or corresponding to lymph-node was considered as lymph-node metastasis.In patients with neck lymph-node metastases discovered by post-therapeutic imaging, a targeted nUS was also performed.18F-FDG-PET/CT studies were required in selected cases on indication of the attending physician.Contrast-enhanced CT and/or magnetic resonance (MR) and/or 99mTc-MIBI scan of the neck-thoracic regions was also obtained, as previously described [In 60 out of 82 metastatic patients (73.2%), a escribed .In patients with lateral neck lymph-node metastases, an FNAC for cytological examination and Tg measurement in the aspirate fluid was also performed.Patients with loco-regional and/or distant metastases at post-therapeutic imaging underwent surgery (whenever possible) and/or RIT with high radioiodine activity (5550-7400 MBq) 6-8 months after TRA.Patients without loco-regional and/or distant metastases at post-therapeutic imaging were revaluated 6\u201312 months after TRA to verify the effectiveness of treatment by Tg measurements and nUS. In patients with large thyroid remnant and/or undetectable serum Tg values at TRA, a diagnostic (185 MBq) radioiodine whole body scintigraphy was also obtained after rhTSH administration (standard protocol).Kolmogorov Smirnov test; consequently, the non-parametric approach has been used.Numerical data are expressed as median and categorical variables as number and percentage. Examined variables did not present normal distribution as verified by Mann Whitney test was applied in order to evaluate the existence of statistically significant differences between patients with and without metastases, regarding age, RAIU, TSH and lymph-nodes size; the same test was used to assess differences between patients treated in euthyroid or in hypothyroid state.Chi Square test was applied in order to assess differences in gender distribution in patients with and without metastases.Receiver Operating Characteristic (ROC) curve was realized, to verify the possible optimal thyroglobulin cut-off value in differentiating between non-metastatic and metastatic patients, and the area under the curve (AUC) was calculated. Sensitivity, specificity, negative predictive value (NPV) and positive predictive value (PPV) of thyroglobulin were evaluated.Statistical analyses were performed using SPSS 17.0 for Window package.P < 0.050 two sided was considered to be statistically significant."} +{"text": "Glioblastoma multiforme (GBM) is the most common primary malignant brain tumor in adults. Patients with GBM have poor outcomes, even with the current gold-standard first-line treatment: maximal safe resection combined with radiotherapy and temozolomide chemotherapy. Accumulating evidence suggests that advances in antigen-specific cancer vaccines and immune checkpoint blockade in other advanced tumors may provide an appealing promise for immunotherapy in glioma. The future of therapy for GBM will likely incorporate a combinatorial, personalized approach, including current conventional treatments, active immunotherapeutics, plus agents targeting immunosuppressive checkpoints. Glioblastoma multiforme (GBM) is the most common primary malignant brain tumor in adults, accounting for approximately 60\u201370% of gliomas and 15% h1 immune response through tumor vaccines, nonspecific immune stimulants, or cellular vaccines, and passive immunotherapy, to induce an antitumor effect by transferring effector immune cells into patients. In 2010, the first antigen-specific vaccine for castration-resistant prostate cancer, sipuleucel-T, was approved by the FDA. In 2011, the first checkpoint inhibitor for advanced melanoma, ipilimumab, was also approved. Since then, immunotherapy has proven effective in the treatment of melanoma, Hodgkin's lymphoma, renal cell carcinoma, and non-small-cell lung cancer (NSCLC) in which conventional therapies have gained limited success [Immunotherapy, harnessing the power of the host's immune system by inducing, enhancing, or suppressing immune responses to reject cancer cells, is rapidly becoming a pillar of anticancer therapy. Immunotherapeutic approaches can be classified as active immunotherapy aimed at promoting a T success \u20139 has been traditionally viewed as an immune-privileged site, secondary to the blood-brain barrier (BBB) that prevents free diffusion of cells and molecules and lack of a conventional lymphatic drainage system \u201313. Para\u03b2, and STAT3 [\u03b2/Smads signaling can restore immune surveillance in glioma models [\u03b2 (PDGF-\u03b2). Second, another immunosuppressive pathway mediated by interactions between programmed death 1 (PD-1) and programmed death-ligand 1 (PD-L1) contributes to the inhibition of T-cell activation and proliferation. Examination of 135 GBM specimens demonstrated that PD-L1 was positively expressed in 88% newly diagnosed GBM patients and 72% recurrent GBM patients [In GBM, a high level of vascular endothelial growth factor (VEGF) expression and pathologically structured microvessels can introduce increased permeability of BBB, enhancing the interaction between tumor cells and the immune system. GBM cells express high levels of MHC and Fas which play a role in the adaptive immune response. However, GBM has been traditionally considered an immunosuppressive tumor, effective in evading the immune response through a variety of mechanisms . First, nd STAT3 \u201324. IDO nd STAT3 . IDO1 fund STAT3 . Inhibita models which copatients . Althougpatients and 25\u20133patients ). Moreovpatients , 32. A tpatients , 34. TheNCT01480479) and relapsed (NCT01498328) GBM. Unfortunately, since EGFRvIII is only present in 20\u201330% of newly diagnosed GBM [NCT02193347) has shown greater efficacy [Recent expansion in our knowledge of immune-mediated mechanisms has led to the rapid development of immune-targeted therapeutic strategies . Among aosed GBM , the ideefficacy ; the mutefficacy .NCT01795313). On the other hand, peptide elution from GBM cells was demonstrated capable of identifying 10 novel GBM-associated antigens, brevican, chitinase 3-like 2, Chondroitin sulphate proteoglycan, fatty acid-binding protein 7, insulin-like growth factor 2 messenger RNA-binding protein 3, neuroligin 4, X-linked, neuronal cell adhesion molecule, protein tyrosine phosphatase, receptor-type, Z polypeptide, tenascin C, were overexpressed in 80\u2013100% of GBM patients, making a peptide vaccine possible [\u221702+ glioblastoma, of which over 3000 were restricted by HLA-A\u221702. They prioritized investigation of these 10 glioblastoma-associated antigens, to which GBM patients showed no T-cell tolerance. Moreover, researchers demonstrated that these 10 peptides were highly immunogenic not only in healthy individuals but also in GBM patients, 9 of which were being developed in a multipeptide therapeutic vaccine designated IMA950. Moreover, peptide elution from GBM cells identified 10 novel GBM-associated antigens which are overexpressed in 80\u2013100% of GBM patients, making a peptide vaccine a potential reality [NCT01403285, NCT01920191, NCT01222221), using CD8+ T-cell epitopes with different adjuvants. Other trials aiming at eliciting both CD4 and CD8 T-cell responses use whole proteins as immunogen to construct the TAA vaccines .Considering that heterogeneity of TSAs in the patient population as a potentially limiting factor in treatment efficacy, tumor-associated antigens (TAAs), which are not tumor exclusive but are relatively overexpressed compared to normal tissues, may be a more viable target in tumor vaccines. Clinical trials in GBM patients, using peptide-pulsed dendritic cells or peptides alone in adjuvant, demonstrated that TAA-based vaccine could elicit T-cell responses without collateral autoimmunity, showing benefit in some patients \u201350. Earlpossible . In this reality . Three tVaccines that target single antigens are restricted to the relatively small subset of patients with tumors that express those TSAs and TAAs. Moreover, the heterogeneity of tumor cells in expressing such antigens may also potentially limit the utility and efficacy of these single-antigen vaccines. Accordingly, alternative vaccine approaches have been created to target a broad range of antigens. Among these, heat-shock protein (HSP) peptide complexes (HSPPC-96) have generated particular interest. HSPPC-96 is a primary resident chaperone of the endoplasmic reticulum and binds various client proteins that are involved in the antigen-presenting pathway . When coNCT00045968). A preclinical study demonstrated that modulation of CMV-specific DCs with tetanus/diphtheria (Td) preconditioning could increase DC migration to vaccine site-draining lymph nodes (VDLNs) [The concept of vaccine immunotherapy involves priming antigen-presenting cells (APCs) with tumor-derived antigens in order to accelerate the eradication of tumor cells Figure . Of the (VDLNs) . This DC (VDLNs) .\u03b12) are considered GSC-associated, has shown promising results in phase II trial for newly diagnosed GBM patients [Another approach uses an immunotherapeutic strategy to target glioma stem cells (GSCs). With their more active DNA repair mechanisms and highly expressed multidrug resistance genes, GSCs may play a role in mediating the resistance of GBM to radiotherapy and chemotherapy and contribute to local immunosuppression in the GBM microenvironment \u201366. Sevepatients . Anotherpatients . Accordipatients .Several studies have demonstrated that GSC-antigens-loaded DC vaccines could induce immune-reactivity and a survival benefit in rodent orthotopic GBM models , 70. CliIt has been recognized that coinhibitory receptors on T-cells play an essential role in attenuating the strength and duration of T-cell-mediated immune responses. These inhibitory receptors are referred to as immune checkpoint molecules responsible for maintaining self-tolerance and preventing autoimmune reactions , 73. To NCT02337491, NCT02336165). The most promising results have been achieved in a randomized control trial with combinatorial CTLA-4/PD-(L)1 blockade for advanced melanoma, in which combination of CTLA-4 and PD-1 blockade demonstrated an improved objective response rate (ORR) of 58%, compared to monotherapy of anti-CTLA-4 (19%) and monotherapy of anti-PD-1 (44%) [NCT02017717). Another two phase I/II trials will analyze the effectiveness of combinatorial pembrolizumab with bevacizumab (NCT02337491) and combinatorial pembrolizumab with MRI-guided laser ablation (NCT02311582) in recurrent GBM patients. In addition, MEDI4736, a humanized PD-Ll mAb, is currently being tested in clinical trials for GBM patients combined with radiotherapy and bevacizumab (NCT02336165).Conversely, efforts aimed at inhibiting the PD-1/PDL1 pathway have shown more promising results. In a preclinical study using the GL261 glioma mouse model, combination of anti-PD-1 antibodies and radiotherapy doubled median survival and elicited long-term survival in 15\u201340% of mice compared with either treatment alone . Clinica-1 (44%) . A rando+ and CD8+ T-cells into normal tissues in company with elevated levels of proinflammatory cytokines [NCT00729664). These agents have shown the capability of inducing durable tumor regression with less grade 3 or 4 adverse events compared with CTLA-4 mAb and PD-1 mAb [However, relatively high frequency of immune-related adverse effects, such as endocrinological, hepatic, gastrointestinal, and dermatological toxicities, have limited enthusiasm for immune checkpoint blockade as a immunotherapeutic strategy against cancer . These aytokines . RecentlPD-1 mAb . Overall adoptive T-cell therapies provide an alternative strategy: in vitro amplification of tumor-specific autologous T-cells followed by venous infusion into the same individual. Adoptive T-cell therapy has evolved during the past two decades in concert with the development of genetic engineering, resulting in the generation of high avidity tumor-specific T-cells. Tumor-reactive T-cells are often achieved by transducing the patient's autologous T-cells with vectors encoding T-cell receptors (TCR) or chimeric antibody receptors (CAR) [NCT00693095). The next step of adoptive T-cell therapy for GBM patients will likely involve transducing autologous T-cells with CAR. CAR, which consist of the antigen-binding region of a monoclonal antibody fused with a T-cell cytoplasmic signaling domain, acts independently of MHC I expression on tumors [NCT02209376, NCT01454596), HER2 (NCT01109095), and IL-13R\u03b12 (NCT02208362) are underway, and therapeutic benefits without unacceptable toxicity are anticipated.While previously described therapeutic strategies endeavored to induce endogenous T-cell responses,rs (CAR) . Althougrs (CAR) , 86, Herrs (CAR) , 88, Ephrs (CAR) , and EGFrs (CAR) , 91) havrs (CAR) . A clinirs (CAR) . Anothern tumors . ClinicaCurrent open clinical trials of immunotherapy predominantly focusing on DC vaccines and antibodies targeting immunosuppressive checkpoints have achieved promising immune activity and clinical responses (see Tables"} +{"text": "The development of site-specific modification of alkyne-functionalized proteins using dimethylarylsilanes and substoichiometric or low-loading of Ru(ii) catalysts is reported. Furthermore, the resultant gem-vinylsilane can undergo further targeted chemical modifications, highlighting its potential for single-site, dual-modification applications. ii) system that enables the first example of alkyne hydrosilylation between dimethylarylsilanes and O-propargyl-functionalized proteins using a substoichiometric amount or low-loading of Ru(ii) catalyst to achieve the first C\u2013Si bond formation on full-length substrates. The reaction proceeds under physiological conditions at a rate comparable to other widely used bioorthogonal reactions. Moreover, the resultant gem-disubstituted vinylsilane linkage can be further elaborated through thiol\u2013ene coupling or fluoride-induced protodesilylation, demonstrating its utility in further rounds of targeted modifications.Transition metal catalysis has emerged as a powerful strategy to expand synthetic flexibility of protein modification. Herein, we report a cationic Ru( Previou3]PF6 (1) and dimethylaryl hydrosilane derivatives, this methodology enables the efficient labeling of multiple protein targets modified both stochastically and site-specifically with an alkyne-containing moiety g moiety . In addi35 aqueous alkyne hydrosilylation is largely underdeveloped. Inspired by the development of a cationic ruthenium catalyst [Cp*Ru(MeCN)3]PF61 by Trost and Ball,37 we examined the catalyst's ability to catalyze hydrosilylation under biocompatible, aqueous conditions. We started our investigation by reacting 3,6,9,12-tetraoxapentadec-14-yne 2 as a model alkyne and a variety of water-soluble hydrosilanes with 1 (5 mol%). Despite previously reported reactivities of trialkoxy and trialkyl silanes, no vinylsilane products were observed under the reaction conditions tested cross-metathesis and strain-promoted alkyne\u2013azide cycloadditions. Furthermore, 4 was found to be stable in buffered conditions at neutral pH, with a half-life (t1/2) > 1 week in the hope to increase selectivity for hydrosilylation over silanol formation. However, none of the tested analogues gave better selectivity or reaction rates (6 and 7 showed incomplete conversion despite prolonged reaction times (One of the side reactions of aqueous hydrosilylation is the hydrolysis of hydrosilane to form silanol (Si\u2013OH). In an effort to reduce silanol formation, we installed substituents adjacent to the Si\u2013H bond with varying degree of steric hindrance at pH 7.4 gave the corresponding vinylsilane in 99% isolated yield as an additive/ligand helped to stabilize the Ru(ii) complex from rapid exchange processes with, for example, histidine39 and aspartic acid40 residues in plasma protein and restored the activity of 1, with the corresponding vinylsilane product isolated in a good yield , which is one of the most important metabolites of l-DOPA. We first investigated whether nearby chalcogens on the terminal alkyne group could increase the rate of hydrosilylation. With no nearby coordinating groups, the reaction with alkyne 9 proceeded slowly, requiring a long reaction time to reach 68% yield aqueous hydrosilylation protocol to modify more complex biomolecules and irradiated at 365 nm to give doubly-modified derivative 34 in excellent isolated yield (81%). Furthermore, the gem-disubstituted vinylsilane linkage can be cleaved by treatment with TBAF to give the corresponding O-allyl 35, demonstrating the potential for chemical Si-modifications installed via hydrosilylation to be selectively removed.Most site-selective dual-labeling efforts require the incorporation of two unique bioorthogonal functional groupsctions57 . To illuvia hydrosilylation on different protein systems. First, lysine residues on lysozyme (Lyz) were non-selectively modified with 36 to give O-propargyl modified Lyz (OP-Lyz) , dose- and time-dependent labeling was observed, even at very low catalyst loading (2 mol%) . When trlabeling . SimilarO-propargyl groups site-specifically into a super-folder GFP (sfGFP) protein at position 150 (sfGFP150TAGHis6) were introduced into E. coli. Addition of 37 (5 mM) led to the amino acid dependent synthesis of full-length sfGFP-37150 in good yield (15 mg L\u20131 of culture). A similar approach was used to obtain sfGFP-38150 as a negative control for labeling experiments in PBS (pH 7.4) at 37 \u00b0C. A fluorescent band was detected after 24 h of incubation with limited background fluorescence observed. This result is particularly noteworthy because no fluorescence was observed when sfGFP-38150 was reacted under the same conditions, highlighting the bioorthogonality and specificity of this reaction towards O-propargyl groups was converted to OP-C2Am via the dehydroalanine-tagged protein intermediate in >95% conversion (1 successfully mediated hydrosilylation of OP-C2Am with 8 at 37 \u00b0C for 1 h to afford VS-C2Am as detected by LC-MS (ii) complex 1 mediated aqueous hydrosilylation offers a milder alternative and these results highlight its potential for site-specific chemical protein modification using either substoichiometric or low-catalyst loading systems.As an alternative to recombinant techniques, we also site-specifically incorporated the alkyne handle through chemical modifications at cysteine. Using the methodology developed by Davis and co-workers,nversion . Gratifyby LC-MS . Compareh\u03bd irradiation yielded Cys-Lyz, as detected by LC-MS -catalyzed aqueous hydrosilylation.Having succinctly demonstrated the ability to carry out alkyne hydrosilylation on numerous protein systems, efforts were then directed towards ascertaining whether it was possible to modify the protein-incorporated vinylsilane through our earlier described radical thiol\u2013ene and protodesilylation reactions. Vinylsilane-modified lysozyme (VS-Lyz) was chosen for initial studies. We found that treatment of VS-Lyz with a protected cysteine amino acid and DMPA under O-propargyl groups and dimethylaryl hydrosilanes (HSiMe2Ar) are effective coupling partners for Ru(ii) complex 1 catalyzed aqueous hydrosilylation, where alkyne-labeled small-molecules and peptides are site-specifically modified in good to excellent yields. Furthermore, hydrosilylation is shown to have orthogonal reactivity to the widely used bioorthogonal hydrazine condensation reaction, giving rise to potential biomolecule dual-labeling applications. Furthermore, O-propargyl tagged proteins successfully undergo site-specific hydrosilylation in the presence of substoichiometric or low loading of 1 to achieve the first C\u2013Si bond on protein substrates. Finally, the resultant gem-disubstituted vinylsilane linkage serves as a reactive chemical handle for thiol\u2013ene coupling and protodesilylation, highlighting the potential for single-site dual-modification and the selective removal of vinylsilane modifications. Hence, we believe this work greatly expands the reaction conditions and substrate complexity of hydrosilylation and complements the growing interest in metal-mediated protein modification strategies.In conclusion, we have demonstrated that"} +{"text": "MRI-guidance of cardiovascular catheterization offers improved soft-tissue contrast and reduced ionizing radiation exposure. The application of MRI-guidance to complex catheterization procedures has been limited by the unavailability of guidewires that are safe and visible under MRI. Here, we use RF-efficient spiral imaging for MR-guided cardiovascular catheterization, with real-time off-resonance reconstruction for improved visualization of off-the-shelf nitinol guidewires.MRI-guided left and right heart catheterizations were performed on a swine using a commercial nitinol guidewire and balloon-tipped catheter with spiral imaging . To enhance guidewire visualization, we exploited the off-resonance signal near the guidewire. Using a custom reconstruction framework affixed to the guidewire tip measured temperature during 2 minutes of continuous scanning with the spiral sequence and our standard real-time imaging sequence .The spiral sequence generated 6 frames/s. Guidewire-enhanced images offered improved delineation of the guidewire shaft, compared to standard signal void visualization Figure , and a uSubstantial heating (\u0394T = 80.5\u00b0C) was observed using our standard real-time Cartesian bSSFP sequence. Heating was reduced to below allowable limits using spiral gradient echo imaging (\u0394T = 1.63\u00b0C) Figure .Figure 2This visualization method is particularly flexible because it uses a targeted reconstruction of standard anatomical imaging data. This method may enable safe MRI-guided cardiovascular catheterizations using commercially available nitinol guidewires."} +{"text": "The effects of increased Ca2+ on BDNF up-regulation and neurite outgrowth remain unclear. We showed here that ES increased phosphorylation of the cAMP-response element binding protein (CREB). Blockade of Ca2+ suppressed CREB phosphorylation and neurite outgrowth. Down-regulation of phosphorylated (p)-CREB reduced BDNF transcription and neurite outgrowth triggered by ES. Furthermore, blockade of calmodulin-dependent protein kinase II (CaMKII) using the inhibitors KN93 or KN62 reduced p-CREB, and specific knockdown of the CaMKII\u03b1 or CaMKII\u03b2 subunit was sufficient to suppress p-CREB. Recombinant BDNF or hyperforin reversed the effects of Ca2+ blockade and CaMKII knockdown. Taken together, these data establish a potential signaling pathway of Ca2+-CaMKII-CREB in neuronal activation. To our knowledge, this is the first report of the mechanisms of Ca2+-dependent BDNF transcription and neurite outgrowth triggered by ES. These findings might help further investigation of complex molecular signaling networks in ES-triggered nerve regeneration in vivo.Electrical stimulation (ES)-triggered up-regulation of brain-derived neurotrophic factor (BDNF) and neurite outgrowth in cultured rat postnatal dorsal root ganglion neurons (DRGNs) is calcium (Ca The initial rise in [Ca2+]i activates downstream CaMKII, which subsequently triggers CREB phosphorylation, thereby promoting BDNF gene expression and neurite outgrowth (schematic shown in via a CREB-independent signaling pathway. Future studies will investigate the role of CaMKK in ES-induced neurite outgrowth in neuronal cells.The current findings together with a previous report support"} +{"text": "In addition to its well-established neurotrophic action, brain-derived neurotrophic factor (BDNF) also possesses other neuroprotective effects including anti-apoptosis, anti-oxidation, and suppression of autophagy. We have shown before that BDNF triggers multiple mechanisms to confer neuronal resistance against 3-nitropropionic acid (3-NP)-induced mitochondrial dysfunction in primary rat cortical cultures. The beneficial effects of BDNF involve the induction of anti-oxidative thioredoxin with the resultant expression of anti-apoptotic B-cell lymphoma 2 (Bcl-2) as well as erythropoietin (EPO)-dependent stimulation of sonic hedgehog (SHH). We further revealed that BDNF may bring the expression of sulfiredoxin, an ATP-dependent antioxidant enzyme, to offset mitochondrial inhibition in cortical neurons. Recently, we provided insights into another novel anti-oxidative mechanism of BDNF, which involves the augmentation of sestrin2 expression to endow neuronal resistance against oxidative stress induced by 3-NP; BDNF induction of sestrin2 entails the activation of a pathway involving nitric oxide (NO), cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG), and nuclear factor-\u03baB (NF-\u03baB). Apart from anti-apoptosis and anti-oxidation, we demonstrated in our most recent study that BDNF may activate the mammalian target of rapamycin (mTOR) with resultant activation of transcription factor c-Jun, thereby stimulating the expression of p62/sequestosome-1 to suppress heightened autophagy as a result of 3-NP exposure. Together, our results provide in-depth insight into multi-faceted protective mechanisms of BDNF against mitochondrial dysfunction commonly associated with the pathogenesis of many chronic neurodegenerative disorders. Delineation of the protective signaling pathways elicited by BDNF would endow a rationale to develop novel therapeutic regimens to halt or prevent the progression of neurodegeneration. Neurotrophic factors are critical ligands for neuronal cells to proliferate, differentiate, and grow during developmental stages and play important roles to maintain survival, network connectivity, and neuronal plasticity in adult brains. Among the neurotrophic factors expressed in the brain, the neurotrophin family, which includes neurotrophin 3 (NT-3), neurotrophin-4 (NT-4), nerve growth factor (NGF), and brain-derived neurotrophic factor (BDNF), have been extensively investigated. BDNF and its well-known transmembrane receptor, tropomyosin-related kinase B (TrkB), have gained much attention in neuropsychiatric disorders ,2. BDNF Increasing evidence suggests that synaptic dysfunction is a crucial pathophysiological feature of neurodegenerative disorders . SynaptiIt is well appreciated that low extent of reactive oxygen species (ROS) and reactive nitrogen species (RNS) are critical for maintenance of neuronal function . On the Alteration of the activities and levels of BDNF have been shown in several neurodegenerative disorders. Among them, with gain- and loss-of-function experiments, a possible mechanistic link between BDNF with HD has been established . BDNF le3-Nitropropionic acid (3-NP) is a well-known toxin of Indigofera that irreversibly inhibits succinate dehydrogenase activity in the complex II of the mitochondrial electron transport chain . 3-NP maN-methyl-d-aspartate receptor (NMDAR) triggered excitotoxicity, promoting dendritic regeneration, and possesses anti-apoptotic effects via B-cell lymphoma 2 (Bcl-2) protein and/or by post-translational modifications of apoptosis-related proteins such as Bad and Bim [BDNF has been recognized as an important growth factor that exerts a beneficial effect on neuronal function under various stressful conditions . BDNF bi and Bim . Impaire and Bim .Apart from its well-known neurotrophic actions and anti-apoptotic effects, anti-oxidative effects of BDNF may also contribute considerably to its protective characteristics in various experimental paradigms mimicking neurodegenerative conditions, which have gained less attention. BDNF increases the expression levels of superoxide dismutases (SODs) and glutathione reductase in cultured hippocampal neurons of rats . BDNF mal-nitroarginine methylester (l-NAME). These effects may occur partially through the activation of the signaling pathways involving NOS/NO, thioredoxin, cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG), and Bcl-2 [In the past decade, we have been working in this field in an attempt to reveal the neuroprotective mechanisms of BDNF ,30,31,32nd Bcl-2 . BDNF ennd Bcl-2 .Erythropoietin (EPO) is a 34-kDa cytokine and is vital for the survival and differentiation of red blood cells during erythropoiesis . EPO posIt was reported that BDNF could augment the antioxidant enzymes activities such as glutathione peroxidase, glutathione reductase, and superoxide dismutases (SODs) in cultured cortical neurons and servHi95) that may be elicited by prolonged hypoxia [+), a mitochondrial complex I inhibitor, in neuroblastoma SH-SY5Y cells [Sestrin2 was first recognized as one of the hypoxia-inducible genes, hence named hypoxia-inducible gene 95 , which selectively blocks degradation of cGMP, improved neurologic scores and reduced loss of striatal lesion volumes that were produced by the systemic infusion of 3-NP in rats and, importantly, the striatal expression of BDNF was significantly increased in the sildenafil-treated rats . This inted mice . Memantited mice . Cannabited mice . In addited mice . Transplted mice . OverallMultiple and perhaps reciprocally regulated signaling cascades exist to mediate BDNF-dependent neuroprotective effects that together convey neuronal protection against 3-NP-induced mitochondrial dysfunction and the resultant neurotoxicity . These p"} +{"text": "Haploidentical and human leukocyte antigen (HLA)-identical sibling hematopoietic stem transplantation are two main ways used in allogeneic hematopoietic stem cell transplantation . In recent years, remarkable progress has been made in haploidentical allo-HSCT (HID-SCT), and some institutions found HID-SCT had similar outcomes as HLA-identical sibling allo-HSCT (ISD-SCT). To clarify if HID-SCT has equal effects to ISD-SCT in hematologic malignancies, we performed this meta-analysis.Relevant articles published prior to February 2017 were searched on PubMed. Two reviewers assessed the quality of the included studies and extracted data independently. Odds ratio (OR) and 95% confidence intervals (CIs) were calculated for statistical analysis.P < 0.00001). The risk of acute graft-versus-host disease (GVHD) is significantly higher after HID-SCT versus ISD-SCT , but the relapse rate is lower in HID-SCT group . The incidence rates of overall survival (OS) and disease-free-survival/leukemia-free survival/relapse-free survival (DFS/LFS/RFS) after ISD-SCT are all significantly superior to HID-SCT . There is no significant difference in transplantation related mortality (TRM) rate after HID-SCT and ISD-SCT.Seven studies including 1919 patients were included. The rate of platelet engraftment is significantly lower after HID-SCT versus ISD-SCT while there is no difference in neutrophil engraftment . Currently, HID-SCT with GVHD prophylaxis (CsA + MTX + MMF + ATG) may not replace ISD-SCT when HLA-identical sibling donor available. Allogeneic hematopoietic stem cell transplantation with human leukocyte antigen (HLA)-identical sibling or unrelated donor is the main way for treatment for high-risk hematological malignancies. For patients without a suitable donor, especially those in urgent need of transplantation, haploidentical allo-HSCT (HID-SCT) is an option . HID-SCTTwo reviews independently identified relevant studies by searching PubMed. Search terms included \u201chaploidentical stem cell transplantation\u201d, \u201chaploidentical\u201d and \u201cidentical\u201d. All studies published prior to February 2017 were eligible. The title and abstracts of all potentially relevant publications were reviewed. Studies that met the inclusion criteria were selected for the analysis. The reference lists from the selected articles were then hand-searched to identify further relevant trials.This meta-analysis included hematologic malignancies who received HSCT (HID-SCT or ISD-SCT). T cell replete HID-SCT for hematologic malignancies using GVHD prophylaxis (CsA + MMF + MTX + ATG) was included. Studies with data concerning grades 3\u20134 acute GVHD were included if no suitable data of grades 2\u20134 acute GVHD were available. The inclusion criteria were as follows: randomized/nonrandomized studies that compared the outcomes of HID-SCT versus ISD-SCT; the key outcomes including engraftment, OS, DFS/LFS/RFS, acute/chronic-GVHD, relapse and NRM/TRM; myeloablative conditioning regimens and HID group GVHD prophylaxis (CsA + MMF + MTX + ATG).We excluded ongoing studies or studies with data inaccessible. If the same authors had more than one publication based on same population, only the most recent or most complete report was included.Two reviewers independently assessed the quality of the included studies using Newcastle-Ottawa scale . StudiesI2 metric . We performed meta-analysis using a fixed or random effect model (Mantel\u2013Haenszel method for dichotomous data). A funnel plot was applied to detect the presence of publication bias.The extracted information was analyzed on the Cochrane statistical program Review Manager 5.3. For the key outcomes, odds ratio (OR) and 95% confidence intervals (CI) were calculated for each trial. Dichotomous outcomes were determined by the number of participants with events and the total number of participants in HID-SCT and ISD-SCT. Heterogeneity was checked by Q-test and defined as P < 0.1. Heterogeneity was quantified using the Fig 1). Among these, 46 publications were excluded for review, 120 studies were excluded for not fulfilling the inclusion criteria and 16 were excluded for meeting the exclusion criteria. Additionally, 2 articles were included through searching the reference lists. Finally, 7 studies including 1919 patients were included [Table 1). Specifically, 936 patients were treated with HID-SCT and 983 patients received ISD-SCT. Both non-randomized and non-blinded comparative studies were included. All data from the comparative studies of HID-SCT versus ISD-SCT were clinical trials. The key outcomes were neutrophil and platelet engraftment, OS, DFS/LFS/RFS, acute/chronic-GVHD, relapse and NRM/TRM, myeloablative conditioning regimen and HID group with GVHD prophylaxis (CsA + MMF + MTX + ATG) were also included.A total of 187 potentially relevant publications were retrieved from our initial search , acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), chronic myelogenous leukemia (CML), and lymphoma. Subgroups were divided by the follow up time \u2264 3 year and \u2265 4 year. The outcomes of each study are shown in Table 3.The characteristics of all included studies are shown in P = 0.66, I2 = 0). The platelet engraftment was significantly faster following ISD-SCT than HID-SCT .Five studies reported neutrophil and platelet engraftment rates, including 705 HID-SCT-treated patients and 672 ISD-SCT-treated patients. For neutrophil engraftment, a fixed effect model was used. There were no significances differences in the incidence rates of neutrophil engraftment between the two groups . No significant difference was found in the incidence rates of chronic GVHD between HID-SCT and ISD-SCT .There is a significant difference between HID-SCT and ISD-SCT regarding the incidence rates of acute GVHD, but not chronic GVHD. We extracted data regarding acute GVHD from seven studies including 1919 patients. The fixed effect model was used, results showed the risk of acute GVHD after HID-SCT was significantly higher than ISD-SCT . Four studies reported the NRM rates and three reported TRM rates, respectively. The risk of TRM was not significantly different between HID-SCT-treated and ISD-SCT-treated patients . Significant difference in TRM was found, but it was unreliable because of unacceptable heterogeneity .Seven studies regarding relapse involving 1919 patients were analyzed with the fixed effect model. The risk of relapse after HID-SCT was significantly lower than ISD-SCT . Significant differences in OS and longer life expectancy were found between ISD-SCT-treated and HID-SCT-treated group .All seven studies reported DFS/LFS/RFS and OS. Fixed effect model showed the heterogeneity of outcomes was acceptable. The rates of DFS/LFS/RFS after HID-SCT were significantly lower compared with ISD-SCT (Table 4). For chronic GVHD, publication bias might cause the heterogenity.As mentioned above, we found significant heterogeneity in chronic GVHD and NRM rates. Thus, subgroup analysis by follow-up time was performed to further investigate NRM. It was found the cumulative incidence rates of NRM for HID-SCT and ISD-SCT were similar at \u2264 3 year, but not at \u2265 4year Click here for additional data file."} +{"text": "Immunohistochemical staining revealed weak PSMA positivity of the bone lesion supporting the hypothesis that neovasculature might explain positive PSMA-PET/CT findings in Paget disease.A 67-year-old man diagnosed with Gleason score 4 + 5 = 9 clinically localized prostate cancer with The s disease . In view68Ga-labeled prostate-specific membrane antigen-targeted ligand positron emission tomography/computed tomography (PSMA-PET/CT) showed moderate PSMA positivity of this lesion . A bone scan revealed increased pelvic tracer uptake that was considered suspicious for Paget disease . 68Ga-las lesion . Since Ps lesion . After rPaget disease is a common disorder of the skeleton characterized by hypertrophic and abnormally structured remodeling of bone , 10. ManEndothelial expression of PSMA in neovasculature known to occur in Paget disease has been postulated as the mechanism causing the PSMA-PET/CT positivity of this condition \u20138. In th"} +{"text": "We investigated the potential protective effects of montelukast (MLK) on cecal ligation and puncture (CLP)\u2013induced tissue injury in vital organs \u2014 liver, heart, kidneys, and especially lungs \u2014 through inhibition of the proinflammatory cytokine response and the generation of reactive oxygen species (ROS) in rats. The rat groups were (1) a 10-mg/kg MLK-treated CLP group; (2) a 20-mg/kg MLK-treated CLP group; (3) a 20-mg/kg MLK-treated, sham-operated group; (4) a CLP control group; and (5) a sham-operated control group. MLK treatment significantly decreased proinflammatory cytokine levels following CLP. The lipid peroxide level increased in the lung, heart, liver, and kidney tissues after CLP-induced sepsis, and myeloperoxidase activity increased in the lung, heart, and liver tissues. MLK attenuated this elevation in all tissues except the kidney, dose dependently. The glutathione levels and superoxide dismutase activity were significantly increased in the lung, liver, and kidney tissues after MLK treatment. MLK treatment after CLP also potentially reduced mortality. The lung and kidney tissues were the most protected by MLK under sepsis conditions. We can suggest that MLK reverses the systemic inflammatory reaction to polymicrobial sepsis and thereby reduces multiple organ failure."} +{"text": "Tritium aestivum L.) production is essential for global food security. Infection of barley yellow dwarf virus-GAV (BYDV-GAV) results in wheat showing leaf yellowing and plant dwarfism symptom. To explore the molecular and ultrastructural mechanisms underlying yellow dwarf symptom formation in BYDV-GAV-infected wheat, we investigated the chloroplast ultrastructure via transmission electron microscopy (TEM), examined the contents of the virus, H2O2, and chlorophyll in Zhong8601, and studied the comparative transcriptome through microarray analyses in the susceptible wheat line Zhong8601 after virus infection. TEM images indicated that chloroplasts in BYDV-GAV-infected Zhong8601 leaf cells were fragmentized. Where thylakoids were not well developed, starch granules and plastoglobules were rare. Compared with mock-inoculated Zhong8601, chlorophyll content was markedly reduced, but the virus and H2O2 contents were significantly higher in BYDV-GAV-infected Zhong8601. The transcriptomic analyses revealed that chlorophyll biosynthesis and chloroplast related transcripts, encoding chlorophyll a/b binding protein, glucose-6-phosphate/phosphate translocator 2, and glutamyl-tRNA reductase 1, were down-regulated in BYDV-GAV-infected Zhong8601. Some phytohormone signaling-related transcripts, including abscisic acid (ABA) signaling factors and nine ethylene response factors, were up-regulated. Additionally, reactive oxygen species (ROS)-related genes were transcriptionally regulated in BYDV-GAV infected Zhong8601, including three up-regulated transcripts encoding germin-like proteins (promoting ROS accumulation) and four down-regulated transcripts encoding peroxides (scavenging ROS). These results clearly suggest that the yellow dwarf symptom formation is mainly attributed to reduced chlorophyll content and fragmentized chloroplasts caused by down-regulation of the chlorophyll and chloroplast biosynthesis related genes, ROS excessive accumulation, and precisely transcriptional regulation of the above-mentioned ABA and ethylene signaling- and ROS-related genes in susceptible wheat infected by BYDV-GAV.Wheat ( Luteovirus or Polerovrus or unassigned to a genus ..\u00ae wheat http://harvest.ucr.edu/, Affymetrix Wheat 1 Chip version 1.58, e value \u2264 10\u221210) and based on the best BlastX search, the corresponding protein sequence were analyzed in non-redundant protein sequence database at the National Center for Biotechnology Information . Gene Ontology (GO) terms [http://www.uniprot.org/) with protein IDs from HarvEST. All differentially expressed transcripts were classified into functional categories according to Gene Ontology analysis using AgriGO (http://bioinfo.cau.edu.cn/agriGO) combined with UniProt.Putative functions were assigned to the differentially expressed transcripts by HarvEST (O) terms were obtactin gene was used as the internal control to normalize all data. The 2Ct\u2212\u25b3\u25b3 method [RT-qPCR technique was used to verify the expression of eight differentially expressed transcripts identified by microarray analysis. The sequences of primers specific to the tested genes are listed in t method was usedCab, GPT2, and GluTR1)\u2014and ROS excessive accumulation are mainly responsible for the formation of yellowing leaves. The dwarfism formation in the infected wheat plants is mainly attributed to the elevated transcription of ET signaling and ABA signaling\u2013related genes (PLD1 and CBL9) and the precisely transcriptional regulation of JA-GA signaling related genes. Based on the above results, we proposed a model to explain major mechanisms underlying the yellow dwarf symptom formation induced by BYDV-GAV infection in susceptible wheat (After BYDV-GAV infection in the susceptible wheat, the lower chlorophyll content, fragmentized chloroplast, poorly-developed thylakoids, rare starch granules and plastoglobules\u2014which were mainly caused by down-regulation of chlorophyll and chloroplast biosynthesis related genes (i.e., le wheat . This re"} +{"text": "Pseudomonas fluorescens strain EK007-RG4, which was isolated from the phylloplane of a pear tree. P.\u00a0fluorescens EK007-RG4 displays strong antagonism against Erwinia amylovora, the causal agent for fire blight disease, in addition to several other pathogenic and non-pathogenic bacteria.Here, we report the first draft whole-genome sequence of Pseudomonas fluorescens is a Gram negative, rod-shaped bacterium that is widely distributed in various environments . The RAST and SEED (Genomic DNA was extracted using the DNA blood and tissue kit from Qiagen. The whole-genome sequencing library was prepared using the Nextera XT DNA library preparation kit (Illumina) and quantified by a fragment analyzer . Sequencing was completed with 2 \u00d7 250-bp paired-end reads using the Illumina MiSeq platform (Illumina). Standard protocols were used for all of the above kits, as provided by the manufacturers. The reads were cleaned and trimmed using CLC Genomics Workbench version 7 (CLC bio). Next, quality-filtered reads were assembled into contigs -Leu-Asp-Thr-Ile-Leu-Ser-Leu-Ser-Ile using antiSMASH (Genome comparison showed closest similarity between EK007-RG4 and the biocontrol agents ntiSMASH . The potMRST00000000. The version described in this paper is the first version, MRST01000000.This whole-genome shotgun project has been deposited in DDBJ/ENA/GenBank under the accession number"} +{"text": "Ultraviolet (UV) light is a leading cause of diseases, such as skin cancers and cataracts. A main process mediating UV-induced pathogenesis is the production of reactive oxygen species (ROS). Excessive ROS levels induce the formation of DNA adducts and result in stalled DNA replication forks. In addition, ROS promotes phosphorylation of tyrosine kinase-coupled hormone receptors and alters downstream energy metabolism. With respect to the risk of UV-induced photocarcinogenesis and photodamage, the antitumoral and antioxidant functions of natural compounds become important for reducing UV-induced adverse effects. One important question in the field is what determines the differential sensitivity of various types of cells to UV light and how exogenous molecules, such as phytochemicals, protect normal cells from UV-inflicted damage while potentiating tumor cell death, presumably via interaction with intracellular target molecules and signaling pathways. Several endogenous molecules have emerged as possible players mediating UV-triggered DNA damage responses. Specifically, UV activates the PIKK (phosphatidylinositol 3-kinase-related kinase) family members, which include DNA-PKcs, ATM (ataxia telangiectasia mutated) and mTOR , whose signaling can be affected by energy metabolism; however, it remains unclear to what extent the activation of hormone receptors regulates PIKKs and whether this crosstalk occurs in all types of cells in response to UV. This review focuses on proteomic descriptions of the relationships between cellular photosensitivity and the phenotypic expression of the insulin/insulin-like growth receptor. It covers the cAMP-dependent pathways, which have recently been shown to regulate the DNA repair machinery through interactions with the PIKK family members. Finally, this review provides a strategic illustration of how UV-induced mitogenic activity is modulated by the insulin sensitizer, ursolic acid (UA), which results in the metabolic adaptation of normal cells against UV-induced ROS, and the metabolic switch of tumor cells subject to UV-induced damage. The multifaceted natural compound, UA, specifically inhibits photo-oxidative DNA damage in retinal pigment epithelial cells while enhancing that in skin melanoma. Considering the UA-mediated differential effects on cell bioenergetics, this article reviews the disparities in glucose metabolism between tumor and normal cells, along with (peroxisome proliferator-activated receptor-\u03b3 coactivator 1\u03b1)-dependent mitochondrial metabolism and redox (reduction-oxidation) control to demonstrate UA-induced synthetic lethality in tumor cells. Adjacent pyrimidines are subject to dimerization upon UVB irradiation, and the resultant cyclobutane pyrimidine dimers (CPDs) may result in cytosine-thymine base transitions in case of insufficient DNA repair . . versusigf-1 transactivation can be inhibited by UV-mediated down-regulation of PPAR\u03b1, leading to reduced mitochondrial oxidation ,12418,12 i.e., by energy deprivation. Under UA-mediated energy deprivation, UVR-attenuated cAMP can be further restricted to mediate p53 proteosomal degradation via AKT-dependent p53-MDM2 interaction, leading to apoptosis of p53-reactive tumor cells and a utations . In the utations . AdditioAs many malignant cancer cell lines encompass over-expressed 3-phosphoinositide-dependent kinase 1 or a loss of PTEN function, the anabolism activator, insulin or its mimetics, usually exacerbate AKT-mediated PGC-1\u03b1 suppression and reduce mitochondrial metabolism and antioxidant defenses ,146. TheUA modulates cellular sensitivity to UV by agonizing insulin-/(IGF-1)-mediated anabolism and up-regulating (PGC-1\u03b1)-mediated catabolism. Insulin-/(IGF-1)-mediated anabolism reduces intracellular cAMP, which in contrast is increased by (PGC-1\u03b1)-mediated catabolism. Differential expression of insulin/IGF-1 receptors in different cell lines, therefore, controls the intracellular cAMP pool and affects cellular responses to UV . Through"} +{"text": "X/perovskite /PC61BM/BCP/Ag configuration were measured with Keithley 2400 source meter unit under 100\u00a0mW/cm2 (AM 1.5 G). The data collected in this article compares the hysteresis of PSCs with different solvents and is directly related to our research article \u201cHigh-Performance Planar Perovskite Solar Cells: Influence of Solvent upon Performance\u201d fabricated using dimethylformamide (DMF), gamma-butyrolactone (GBL), methyl-2-pyrrolidinone (NMP), dimethylsulfoxide (DMSO), DMF-DMSO, GBL-DMSO and NMP-DMSO as perovskite precursor solutions according to different scan directions, sweep times, and current stability. The hysteresis analyses of the planar PSCs prepared with a glass-ITO /NiO Specifications TableValue of the data\u2022The data article presents the variations of hysteresis curves in PSCs with DMF, GBL, NMP, DMSO, DMF-DMSO, GBL-DMSO, and NMP-DMSO.\u2022Different sweep directions, different scan times, and current stability characteristics of PSCs with different solvents would be useful for insight of hysteresis behavior.\u2022These data can provide better understanding for research into the influence of solvent in planar PSCs.1We investigated the hysteresis of PSCs fabricated using different solvents according to different scan directions, sweep times, and current stability 22 illumination at AM (air mass) 1.5 G condition with a Keithley 2400 instrument calibrated with a Si solar cell . For accurate comparisons, the collected J-V curve was chosen as close to statistical analysis of each PSC Seven different perovskite films were prepared for planar PSCs"} +{"text": "Linc-ROR knockdown in ovarian cancer cell lines inhibited the epithelial-to-mesenchymal transition (EMT) program, which led to ovarian cancer cell metastasis through the repression of canonical Wnt/\u03b2-catenin signaling. Together, our results indicated that linc-ROR induces EMT in ovarian cancer cells and may be an important molecule in the invasion and metastasis of ovarian cancer.Long intergenic non-protein coding RNA, regulator of reprogramming (linc-ROR) is an intergenic long non-coding RNA (lncRNA) previously shown to contribute to tumorigenesis in several malignancies. However, little is known about whether linc-ROR has a role in ovarian cancer progression. In this study, we found that linc-ROR expression was increased in high-grade ovarian serous cancer tissues compared with normal ovarian tissues or normal fallopian tube tissues. Furthermore, the level of linc-ROR expression was associated with ovarian cancer International Federation of Gynecology and Obstetrics stage and lymph node metastasis. Linc-ROR promoted ovarian cancer cell proliferation both Ovarian cancer has the highest mortality rate among all gynecological malignancies. Compared with early ovarian cancer, patients who are diagnosed with advanced ovarian cancer are more likely to have invasion and peritoneal metastasis to adjacent organs , 2. HighPrevious studies have indicated that the epithelial-to-mesenchymal transition (EMT) is closely related to the invasion and metastasis of ovarian cancer . The EMTLong non-coding RNAs (lncRNAs) are a new group of RNAs that were recently discovered by the rapid development of sequencing techniques . LncRNAsin vitro and in vivo, and its roles in cell invasion and metastasis. We demonstrate that the linc-ROR-induced changes in EMT in ovarian cancer cell lines are the result of alterations in the canonical Wnt/\u03b2-catenin signaling pathway. This study explored a new regulatory mechanism for linc-ROR in ovarian cancer invasion and metastasis, and provides new evidence for the therapeutic and prognostic value of linc-ROR in ovarian cancer.In this study, we assessed linc-ROR expression in high-grade ovarian serous cancer tissues, normal ovarian tissues, and normal fallopian tube tissues, and further analyzed the relationship between the level of linc-ROR expression and ovarian cancer International Federation of Gynecology and Obstetrics (FIGO) stage and lymph node metastasis. We then investigated the functions of linc-ROR in ovarian cancer cell proliferation both P<0.01). Table P<0.01), while patients with lymph node metastases had higher linc-ROR expression than those without (P<0.01). These results suggest that up-regulated linc-ROR expression may be associated with ovarian carcinogenesis and tumor progression.Linc-ROR mRNA expression was assessed by quantitative real-time (qRT)-PCR and shown to be significantly higher in the 39 high-grade ovarian serous cancer tissue samples than in either the 20 normal ovarian tissue samples or the 20 normal fallopian tube tissue samples Figure , P<0.01.P<0.05); linc-ROR expression was significantly higher after transfecting the linc-ROR expression plasmid than in cells transfected with empty vector . siRNA-1 had the best inhibitory effect on linc-ROR expression, so we used it for subsequent experiments.To further investigate the effects of linc-ROR on the biological functions of ovarian cancer cells, we exogenously decreased linc-ROR expression by transfecting small interfering (si)-linc-ROR, and increased linc-ROR expression by transfecting the pIRES2-EGFP-linc-ROR vector. The levels of linc-ROR expression after transfection were confirmed by qRT-PCR. After siRNA transfection, linc-ROR expression was significantly lower in the si-linc-ROR groups than in the si-NC group Figure , P<0.05;r Figure , P<0.01.in vitro.We then detected the proliferation rates of experimentally manipulated SKOV3 cells using the CCK-8 assay. Compared with the si-NC group, si-linc-ROR-transfected cells showed significantly reduced proliferation rates Figure . ConversWe next investigated the biological effects of linc-ROR on the migration and invasion of ovarian cancer cells. Wound healing assays showed that linc-ROR knockdown reduced SKOV3 cell migration Figure , while lTo investigate the oncogenic role of linc-ROR in ovarian cancer EMT, we detected EMT marker expression via western blotting. Levels of the epithelial marker E-cadherin were reduced in SKOV3 cells exogenously overexpressing linc-ROR compared with cells transfected with empty vector. Accordingly, levels of the mesenchymal marker vimentin and the EMT-associated proteins \u03b2-catenin and c-myc were increased in linc-ROR-overexpressing SKOV3 cells compared with cells carrying empty vector Figure . Conversin vivo, we performed assays in immunodeficient mice. SKOV3-ROR-LV, SKOV3-ROR-p, and control SKOV3 cells were inoculated to establish a subcutaneous xenograft model using nude mice. Compared with SKOV3-ROR-LV and control SKOV3 cells, mice inoculated with SKOV3-ROR-p cells formed larger xenograft tumors have increased tumoral linc-ROR expression; furthermore, linc-ROR expression was higher in ovarian cancer tissues from patients with than without lymph node metastases. These results suggest that linc-ROR may be associated with the development and metastasis of ovarian cancer. To investigate the oncogenic role of linc-ROR in ovarian cancer, we exogenously silenced or overexpressed linc-ROR in ovarian cancer cells using siRNA, or lentivirus and expression plasmids. Ectopically expressed linc-ROR promoted proliferation MKK4) and promotes Twist expression, which then inhibits E-cadherin expression and induces EMT in ovarian cancer cells; in their experiments the invasive and metastatic abilities of ovarian cancer cells were accordingly enhanced [EMT is one of the most important steps of tumor metastasis and plays a central role in tumor progression. During this process, cells lose epithelial marker expression and instead express mesenchymal markers, which induces the loss of cell\u2013cell and cell\u2013matrix adhesion, and increases migratory, invasive, and stem cell properties . EMT is enhanced . In our A variety of signaling pathways, including transforming growth factor-\u03b2, mitogen-activated protein kinase, Notch, and Wnt, can trigger EMT. The canonical Wnt/\u03b2-catenin pathway has been proven to induce EMT in ovarian cancer, breast cancer, and colon cancer cells . When thIn conclusion, this study demonstrated that linc-ROR is up-regulated in high-grade ovarian serous cancer tissues, where it promotes proliferation, invasion, and metastasis. Furthermore, we showed that linc-ROR acts as a potential oncogene in ovarian cancer by initiating an EMT program, and that the Wnt/\u03b2-catenin pathway may also be involved in this process. These results suggest that linc-ROR is a potential biomarker and therapeutic target for ovarian cancer.This investigation has been conducted in accordance with the ethical standards and according to the Declaration of Helsinki and according to national and international guidelines and has been approved by the authors\u2019 institutional review board. All experimental procedures were approved by the Institutional Review Board of the Ethics Board of the Affiliated Hospital of Qingdao University, and all subjects signed written informed consent forms.Ovarian cancer tissue specimens were obtained from patients who had undergone surgical treatment for ovarian cancer at the Affiliated Hospital of Qingdao University between March 2015 and March 2016. Normal ovarian tissue and normal fallopian tube tissue specimens came from patients who had undergone surgical treatment for hysteromyoma at the Affiliated Hospital of Qingdao University in the same timeframe. All cases were confirmed by postoperative pathological diagnosis. Patients who had received neoadjuvant chemotherapy or radiation therapy before surgery were excluded from this study. We collected 39 high-grade ovarian serous cancer tissue samples , 20 normal ovarian tissue samples, and 20 normal fallopian tube tissue samples. Linc-ROR mRNA expression was evaluated by qRT-PCR in all samples, which were stored at \u221280\u00b0C immediately after the operation.2.The human epithelial ovarian cancer cell lines SKOV3 and A2780 were purchased from the Chinese Academy of Science . Cells were cultured in RPMI-1640 medium or DMEM (Hyclone), both supplemented with 10% fetal bovine serum , 100 U/mL penicillin, and 100 mg/mL streptomycin in a humidified incubator at 37\u00b0C with 5% COGAPDH) genes were amplified in separate wells and run in triplicate. Statistical analyses of the results were performed using the 2\u2212\u0394\u0394CT relative quantification method.Total RNA was extracted from tissue samples and cells on ice using TRIzol reagent and reverse-transcribed into cDNA using the TRUEscript 1st Strand cDNA Synthesis Kit following the manufacturer\u2019s protocol; reactions were incubated for 30 min at 42\u00b0C, 5 min at 85\u00b0C, and then stored at \u221220\u00b0C. qRT-PCR reactions were performed using 2\u00d7SYBR Green qPCR Mix (Aidlab) on an ABI 7900HT sequence detection machine ; reactions were incubated at 95\u00b0C for 3 min, followed by 40 cycles of 95\u00b0C for 15 s and 60\u00b0C for 40 s. GAPDH was used as an internal control. The primer sequences were as follows: linc-ROR forward: 5'-GAAGGTTCAACATGGAAACTGG-3', and reverse: 5'-TGAGACCTGCTGATCCCATTC-3'; GAPDH forward: 5'-CTCAGACACCATGGGGAAGGTGA-3', and reverse: 5'-ATGATCTTGAGGCTGTTGTCATA-3'. Both target (linc-ROR) and reference . Ovarian cancer cells cultured in 6-well plates were transfected with the plRES2-EGFP-linc-ROR or empty vector using Lipofectamine 2000 (Invitrogen) according to the manufacturer\u2019s instructions. Linc-ROR knockdown was performed using siRNA purchased from RiboBio Co. . Ovarian cancer cells cultured in 6-well plates were transfected with si-linc-ROR or siRNA-negative control (siNC) using riboFECT\u2122 CP (RiboBio Co.) according to the manufacturer\u2019s instructions. Cells were harvested 48 h after transfection for qRT-PCR to detect transfection efficiencies.Cell proliferation was monitored using the Cell Counting Kit-8 . After 48-h transfection with the linc-ROR expression plasmid or si-linc-ROR, ovarian cancer cells were seeded into 96-well plates (3000 cells/well), and cell proliferation was documented every 24 h for 6 d following the manufacturer\u2019s instructions. All experiments were performed in quadruplicate. The number of viable cells was assessed by measuring absorbance at 450 nm using an enzyme-linked immunometric meter (Thermo Fisher Scientific).5 cells/well). When the cells reached approximately 90% confluence, cell layers were wounded using a sterile 200-\u03bcL pipette tip and washed with phosphate-buffered saline to remove cell debris. The cells were then cultured in medium with 1% FBS. Scrape lines were photographed with a light microscope at the indicated time points. Each experiment was performed in triplicate.After 48-h transfection with the linc-ROR expression plasmid or si-linc-ROR, ovarian cancer cells were seeded into 6-well plates transwell inserts . After 48-h transfection with the linc-ROR expression plasmid or si-linc-ROR, 5\u00d710Cells were washed twice with PBS and lysed in RIPA buffer supplemented with protease inhibitor cocktail . Protein concentrations were measured using the BCA Protein Assay Kit (Beyotime). Protein lysates were subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membranes and incubated with primary antibodies for 24 h at 4\u00b0C. Membranes were then washed and incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature, and immunoreactive bands were visualized with ECL chromogenic substrate . GAPDH was used as an internal control. Primary antibodies were as follows: E-cadherin , vimentin , \u03b2-catenin , c-myc , and GAPDH .6) were subcutaneously injected into the armpit of nude mice. The mice were killed 40 days after inoculation, and xenograft tumors were surgically isolated. Tumors were photographed and weighed later, collected to stain with hematoxylin and eosin for histological analysis and the expression of linc-ROR was assessed by qRT-PCR. All animal experiments were approved by the Animal Care Committee Affiliated Hospital of Qingdao University.Female BALB/c nude mice (4-weeks-old) were purchased from Slac Laboratory Animal Company . Lentivirus LV-LINC-ROR-RNAi was transfected into SKOV3 cells to stably decrease the linc-ROR expression level, and plasmid plRES2-EGFP-linc-ROR was used to stably increase linc-ROR expression in SKOV3 cells. SKOV3-ROR-LV cells (transfected with lentivirus LV-LINC-ROR-RNAi), SKOV3-ROR-p cells (transfected with plasmid plRES2-EGFP-linc-ROR), and control SKOV3 cells and GraphPad Prism 5.0 were used for statistical analysis and the graphical presentation of data. Data are presented as means\u00b1SD. The statistical significance of the results was analyzed by fold change and the Student\u2019s"} +{"text": "Blood. 2015;126(26):2821-31). However, the underlying mechanisms of c-MET activation within PEL cells remain largely unknown. To solve this puzzle, here we have utilized the next generation sequencing (NGS) based bioinformatics approach to investigate the genomic landscape of the c-MET gene and we found that there's no single nucleotide variations (SNVs) occurred in the c-MET genomic regions in a cohort of PEL samples. Consistently, Sanger sequencing analysis of frequently mutated exons such as exon 10, 14 and 19 shows no mutation of these c-MET exons in PEL cell-lines, which further supports the notion that mutations are not the major mechanism responsible for c-MET activation in PEL. Further, we found that a transmembrane receptor protein, Plexin-B1, is expressed in PEL cell-lines, which is required for c-MET-mediated PEL cell survival via direct protein interaction.The oncogenic Kaposi's sarcoma\u2013associated herpesvirus (KSHV) is a principal causative agent of primary effusion lymphoma (PEL), which is mostly seen in immunosuppressed patients. PEL is a rapidly progressing malignancy with a median survival time of approximately 6 months even under the conventional chemotherapy. We recently report that the hepatocyte growth factor (HGF)/c-MET pathway is highly activated in PEL cells and represents a promising therapeutic target ( The DNA tumor virus, Kaposi's sarcoma-associated herpesvirus (KSHV) can cause several human cancers especially in the immunocompromised populations . One sucMet proto-oncogene, and the hepatocyte growth factor (HGF) is the only known c-MET ligand [One of receptor tyrosine kinases (RTKs), c-MET, is encoded by the T ligand , 7. SincT ligand . MultiplT ligand . PreviouT ligand \u201315. CandT ligand , 16; 2) T ligand ; 3) Y100T ligand .We recently showed an abnormal activation of the HGF/c-MET signaling pathway within KSHV+ PEL cell-lines . One of The human c-MET gene consists of 21 exons and encodes a protein of 1408 amino acids (aa). Structurally, the c-MET protein contains both extracellular and intracellular domains. Its extracellular domain can be further divided into the semaphoring domain, the plexin-semaphorin-integrin domain, and four immunoglobulin-plexin-transcription domains. The intracellular domain of c-MET carries its tyrosine kinase activity having the same length of exon 10, 14 and 19 as that from A549, one of NSCLC cell-lines without c-MET missense mutations passages.The PEL cell-line BCBL-1 (KSHV+/EBV-) was cultured as described previously . The othRaw whole exome sequencing reads from five kidney/renal carcinoma samples and five lung adenocarcinoma samples generated through the National Institutes of Health, The Cancer Genome Atlas (TCGA) project were obtained from the National Cancer Institute Genomic Data Commons. Raw sequencing reads from 16 PEL cell-lines were obtained from the National Center for Biotechnology Information short reads archive (accession no. SRP032975). Raw sequence data were first aligned to the human reference genome hg19 with the Burrows-Wheeler Aligner. Next, the resulting alignment files were analyzed using a detection pipeline consisting of Samtools mpileup and VarScan to identify candidate SNVs. Lastly, the identified SNVs were annotated using the ANNOVAR software and the c-MET specific SNVs were then plotted as a heatmap based on the number of reads covering that specific SNV on a log10 scale.Genomic DNA from PEL cells was extracted using the Wizard Genomic DNA Purification Kit (Promega) according to the manufacturer's instructions. Primers used for amplification of specific exons of c-MET gene are listed in Table Plexin-B1 ON-TARGET plus SMART pool siRNA, or negative control siRNA (n-siRNA) (Dharmacon), were delivered using the DharmaFECT transfection reagent according to the manufacturer's instructions.For RNA interference, For cell apoptosis assays, the fluorescein isothiocyanate (FITC)\u2013Annexin V and propidium iodide (PI) Apoptosis Detection kit I (BD Pharmingen) was used as previously described . For celTotal cell lysates (20\u03bcg) were resolved by 10% SDS\u2013PAGE, transferred to nitrocellulose membranes, and immunoblotted with antibodies for cleaved caspase 3/9, cleaved PARP, phosphor (p)-c-MET/total (t)-c-MET, p-Chk1/2, Cyclin A2, Cyclin B1 , Plexin-B1 (Santa Cruz), and \u03b2-Actin (Sigma) for loading controls. Immunoreactive bands were identified using an enhanced chemiluminescence reaction (Perkin-Elmer), and visualized by autoradiography. Immunoprecipitation assays were performed using Catch and Release Immunoprecipitation Kit (Milipore) according to the manufacturer's instructions.Significance for differences between experimental and control groups was determined using the two-tailed Student's t-test (Excel 8.0), and p values <0.05 were considered significant."} +{"text": "Inhibition of DHDPS is supposed to a promising therapeutic target due to its specific role insporulation, cross-linking of the peptidiglycan polymers and biosynthesis of amino acids. In this work, a known inhibitor-based similaritysearch was carried out against a natural products database towards identification of more potent phyto-inhibitors.Molecular interaction studies were accomplished using three different tools to understand and establish the participation of active siteresidues as the key players in stabilizing the binding mode of ligands and target protein. The best phyto-compound deduced on the basisof binding affinity was further used as a template to make similarity scan across the PubChem Compound database (score > = 80 %) to getmore divesred leads. In this search 5098 hits were obtained that further reduced to 262 after drug-likeness filtration. These phytochemicallikecompounds were docked at the active site of DHDPS.Then, those hits selected from docking analysis that showing stronger bindingand forming maximum H-bonds with the active site residues . Finally, we predicted onephytochemical compound (SN00003544), two PubChem-compounds akin to phytochemical moleculeshowing better interactions in comaprison of known inhibitors of target protein.These findings might be further useful to gain thestructural insight into the designing of novel leads against DapA family. Mycobacterium tuberculosis (Mtb), a brutal killer of the humanpopulation by spreading most infectious disease, tuberculosis (TB)has been avowed a big threat to public health across the globe .The GloThe 4-hydroxy-tetrahydrodipicolinate synthase P9WP25) is a keyenzyme of Lysine/DHDPS biosynthetic pathway of Mtbresponsible for synthesis of D, L diaminopimelic acid (meso-DHDPS) and lysine ,4.Apart is a keyDHDPS is an important enzyme of lysine biosynthesis pathwaythat catalyses the condensation of aspartate-\u03b2-semialdehyde andpyruvate to 4-hydroxy-tetrahydrodipicolinate (HTPA).Dihydropicolinate (DHDP) is released from active site (Lys-171) with the elimination of water molecule [Numerous inhibitors against Mtb-DHDPS have been identified sofor, but quest to find the best is still unexplored. Towards thisdirection a comparison between experimentally known andpredicted inhibitor was made by Garg et al., 2010 throughmolecular dynamics simulation study. They proposed thatPUB475318 is bestowed better inhibition potential as compared tothe previously reported inhibitors of Mtb-DHDPS. Keeping thesefacts on consideration we have used it as a template for search andidentification of novel phyto-ligands and diversified PubChemcompounds instead of considering experimentally knowninhibitors as template.In the proposed study three different computational tools , and AutoDock Tools),7 was usThe crystal structure (3D) of Mtb-DHDPS (PDB ID: 1XXX) wasextracted from RCSB Protein Data Bank. The coordinates of thechloride ion, magnesium ion, 2, 3-dihydroxy-1, 4-dithiobutane(DDT), and water molecules were removed to prepare the proteinfor molecular docking. The protein was energetically minimizedusing the CHARMm force field.The structure of PUB475318, a newly predicted inhibitor of DHDPS[Lipinski rule of five (RO5) was employed to predict the druglikenessof ligands. RO5 includes molecular mass ,high lipophilicity (Log p < = 5) H-bond donors (< = 5), H-bondacceptors (< = 10) and molar refractivity 40-130). These filtrationsensure drug-likeness for molecules obeying two or more features ofRO5 0. These .BioPredicta tool of VlifeMDS package , MVD, Piecewise Linear PairwisePotential (PLP) and Grid algorithms energy minimization by usingMMFF force fields. The Dock scoring function was used to assessthe binding efficacies of ligands. This scoring function take intoaccount the terms for van der Walls interaction, hydrophobiceffects, hydrogen bonding and deformation penalty. BioPredictatool uses following fitness function for searching rigid dockingspace.E = InterEq; E = InterEvdW + InterEq; E = EEPIC; Where, InterEq =intermolecular electrostatic energy of complex; InterEvdW =intermolecular vdW energy of complex; EEPIC = electrostaticpotential for intermolecular complexAll other required parameters were set as default during theprocess of molecular interactions.It integrates highly efficient PLP and MolDock scoring function formolecular docking. Docking parameters and other requiredparameters were set to default values . MolDockPolar H-atoms, Kollman united atom and atom type parameterswere added and further, non-polar H-atoms were merged duringgeneration of the protein pdbqt file. During preparation of ligandpdbqt file, polar H-atoms added, non-polar H-atoms merged,number of torsions, and rotatable bonds were defined. Cubicvolume of 40 \u00d7 40 \u00d7 40 \u212b 3 with 0.408 \u212b grid points spacing and X:3.163, Y: 39.286, Z: 70.258 centre coordinates was set to cover theentire active site and accommodate ligand to move freely.Lamarckian genetic algorithm was employed for the receptor-fixedligand-flexible docking calculations. The conformer having lowestfree energy of binding (\u0394G) was considered for further analysis ,11.Two approaches were implemented to search and find out thepotent leads against Mtb-DHDPS. Virtual screening of phytocompoundsfrom the natural products database of the UEFS(http://www.uefs.br) was performed as first approach usingrecently predicted inhibitor, PUB475318 as template [Among all phyto-compounds docked with the Mtb-DHDPS,SN00003544 was found to bind with the best efficacy in the Nterminal(\u03b2/\u03b1)8-barrel domain of Mtb-DHDPS comprises of 1-233residues .To search and identify a diverse classes of ligands having antitubercularpotential, 3-D similary search (similarity score > = 80 %)against the PubChem compound database was carried out usingbest phyto-ligand (SN00003544) as template. Initially SN00003544-akin 5098 compounds were retrieved. These molecules weresubjected to RO5 filtration before going for docking studies. The268 molecules out of 5098 were succeeded the RO5 filtration.Molecular docking studies of these compounds were performed forthe best binding orientation prediction into the active site of Mtb-DHDPS using the same docking procedure and parameters asmentioned earlier for phyto-compounds. Out of 268 only 50 compounds exhibited plausible binding along with the formation ofH-bond with the active site residue Lys171 of Mtb-DHDPS. Further,out of 50 only 10 ligands were observed consistent as bestowed byall three adopted computational tools . SimilarA comparison of top 10 PubChem hits were made with the fiveexperimentally known inhibitors for example piperidine-2,6-dicarboxylic acid (CID557515), dimethylpiperidine-2,6-dicarboxylate (CID12265924), pyridine-2,6-dicarbxylic acid(CID10367), 1,4-dihydro-4-oxopyridine-2,6-dicarboxylic acid(CID11390199), and dimethyl-1,4-dihydro-4-oxopyridine-2,6-dicarboxylate (CID68297515), and a novel predicted inhibitorPUB475318 of Mtb-DHDPS in order to screen the best phyto-leadlikechemical agents. Only 4 out of 10 hits exhibited strongerbinding affinity in comparison of 5 experimentally knowninhibitors. Furthermore, out of four only two compounds(CID54025334 and CID41032023) were depicted as strongerinhibitors in comparison of PUB475318 as shown by scoringfunctions of adopted docking tools . DockingThe active site residues Thr54, Thr55, Arg148, Lys171 of N-terminaldomain and Gly256 of C-terminal domain were stabilized themolecular interaction of first potent PubChem ligand(CID41032023) and protein (Mtb-DHDPS) through hydrogen bondformation. Apart from H-bonding, Arg148 is also engaged in saltbridge formation and enhancing the stability of complex .FurtherLikewise, higher binding affinity of second potent PubChemcompound (CID54025334) towards Mtb-DHDPS was attributed bythe five hydrogen bonding , two hydrophobic interactions , and onesalt bridge formation (Lys171) , Table 5Validation of docking procedure adopted in the study wasaccomplished by superimposing all the ligands showing strongerbinding activity into the active site of Mtb-DHDPS ,19,20. In the present study, we employed two virtual screeningapproaches towards the identification and elucidation of noveldrug leads against one of the oldest malady of humankind. In thefirst approach we screened out a potent natural compound(SN00003544) from the UEFS (http://www.uefs.br) database thatbestowed strong binding affinity with Mtb-DHDPS as shown byfive hydrogen bonding and one slat bridge formation (Arg148). In the second approach twocompounds akin to phyto-leadwith extremely different scaffold from template molecule wereidentified. These two compounds demonstrated better bindingmode into the active site of Mtb-DHDPS and establishing strongbonded and non-bonded molecular interactions as compared byknown inhibitors. In hydrogen bonding distance between donorand acceptor atoms (<4.1 \u212b), and angle between donor, acceptorand hydrogen atoms (>100\u00b0) were found in significant range.Similarly in salt bridges, distance between centers of charge (<5.5 \u212b)and in hydrophobic interactions, distance between interactionscarbon atoms (<4.0 \u212b) were found significant . Since aAuthors would like to declare no conflict of interest."} +{"text": "Atherosclerosis is a chronic inflammatory disease with complex pathological processes. MicroRNA-33 (miR-33), a novel non-coding RNA that coexpresses with sterol regulatory element-binding proteins (SREBPs), affects macrophage actions to prevent atherosclerosis. Fibroblast growth factor 21 (FGF21) is an important regulator of lipid metabolism, especially for macrophage-related cholesterol export, but the mechanism is not fully studied. Interestingly, FGF21 has been evidenced to prevent atherosclerosis via inhibiting SREBP-2 expression. Therefore, we speculate that FGF21 may be a potential regulator for miR-33 with an aim of insight into novel anti-atherosclerotic mechanisms and research fields. Atherosclerosis is a chronic inflammatory disorder characterized by the deposition of excess lipids in the arterial intima. Reverse cholesterol transport (RCT) could counteract the pathogenic events by promoting cholesterol efflux to high-density lipoprotein (HDL) from the artery wall, which involves in a series of factors including macrophage or non-macrophage related cholesterol efflux, cell membrane bound transporters, plasma lipid acceptors, plasma proteins and enzymes, and hepatic cellular receptors . MacrophInflammation response is of central importance for the initiation and progression of atherosclerosis. Macrophage counts in symptomatic carotid plaques are significantly increased and considered to discriminate between symptomatic and asymptomatic patients . MoreoveMicroRNAs (miRNAs) are small, non-coding RNAs that regulate gene expression and control a wide range of biological functions by base pair with specific mRNAs. Recent reports have identified specific miRNAs as major regulators of lipid homeostasis and anti-atherosclerosis; and the best-characteristic one is miRNA-33 (miR-33) . AlthougLdlr-/- mice model, inhibition of miR-33 could delay progression of atherosclerosis by inhibiting monocyte recruitment and changing macrophage-induced inflammation in atherosclerotic plaques, which was independent of the effect of ABCA1 induced cholesterol export. Thus, inhibiting miR-33 could promote macrophage conversion from pro-inflammatory M1 to anti-inflammatory M2 phenotype to prevent atherosclerosis and stabilize plaque.Well-documented evidences of miR-33-induced anti-atherosclerotic mechanisms are focused on regulating macrophage actions. MiR-33 was reported to inhibit the expression of ABCA1 and ABCG1 in macrophages, thereby attenuating cholesterol efflux to apoA-I and nascent HDL . While iP\u2009<\u20090.05) and triglyceride (P\u2009<\u20090.01). Thus, serum FGF21 elevation under atherosclerosis conditions may be a regulatory compensation mechanism and indicate a promising therapeutic target.Fibroblast growth factor 21 (FGF21), a member of the fibroblast growth factor (FGF) family, has been described as an important regulator of glucose and lipid metabolism \u201316. ReceP\u2009<\u20090.05); and this process was related to upregulating the expression of ABCA1 and ABCG1 in THP-1 macrophage-derived foam cells. Similarly, Lin et al. [Mechanically, FGF21 was also received wide attention in the effect of macrophage-derived cholesterol efflux. Shang et al. found thn et al. reportedSREBP-2 and the intron 17 of SREBP-1 genes [SREBPs gene [Sterol regulatory element\u2013binding proteins (SREBPs) are a family of membrane-bound transcription factors that mainly regulate lipid homeostasis . SREBPs -1 genes . And miRBPs gene . Hence, SREBP-2 gene. Lin et al. [apoE-/- mice and exacerbation of atherosclerosis followed by significantly worsened lipid profile and inflammatory cytokines. In mechanism, they found that SREBP-2 was FGF21 targeted gene to regulate cholesterol efflux. Therefore, we speculate that FGF21 could inhibit SREBPs expression as well as the expression of miR-33.More interestingly, recent findings indicated that FGF21 regulate cholesterol efflux by targeting n et al. investigTogether, we hypothesis that FGF21 potentially inhibits miR-33 expression, and thereby enhancing macrophage related cholesterol efflux and increasing anti-inflammatory macrophages to prevent atherosclerosis (Fig."} +{"text": "H\u00b0) and entropy (\u0394S\u00b0), showed that the adsorption of Pb(II) onto nanofibres was spontaneous and exothermic. The novel nanofibres exhibited higher potential removal of Pb(II) ions than plain PVA nanofibres. Ubiquitous cations adsorption test was also investigated and studied.Plain polyvinyl alcohol (PVA) nanofibres and novel polyvinyl alcohol benzene tetracarboxylate nanofibres incorporated with strontium, lanthanum and antimony ((PVA/Sr-TBC), (PVA/La-TBC) and (PVA/Sb-TBC)), respectively, where TBC is benzene 1,2,4,5-tetracarboxylate adsorbents, were fabricated by electrospinning. The as-prepared electrospun nanofibres were characterized by scanning electron microscope (SEM), Fourier transform infrared (FTIR) and thermogravimetric analysis (TGA). Only plain PVA nanofibres followed the Freundlich isotherm with a correlation coefficient of 0.9814, while novel nanofibres followed the Langmuir isotherm with correlation coefficients of 0.9999, 0.9994 and 0.9947, respectively. The sorption process of all nanofibres followed a pseudo second-order kinetic model. Adsorption capacity of novel nanofibres was twofold and more compared to that of plain PVA nanofibres. The thermodynamic studies: apparent enthalpy (\u0394The online version of this article (doi:10.1186/s11671-016-1631-2) contains supplementary material, which is available to authorized users. The chemisorption predominated adsorbents (PVA/La-TBC and PVA/Sr-TBC nanofibres) removed the Pb(II) ions much better than the physisorption dominant adsorbents (plain PVA and PVA/Sb-TBC nanofibres). PVA/Sb-TBC, PVA/Sr-TBC and PVA/La-TBC nanofibre sorption data fitted best with the Langmuir isotherm, indicating the homogeneous nature of the monolayer sorption of Pb(II) on the modified nanofibres. Sorption of Pb(II) onto the nanofibres was rapid and spontaneous. This research proposes a convenient approach for the application of modified nanofibres in the field of practical water treatment."} +{"text": "Drosophila repressor complex contains the fly CSL orthologue Suppressor of Hairless [Su(H)] and Hairless (H). The Su(H)-H crystal structure revealed a large conformational change within Su(H) upon H binding, precluding interactions with NICD. Based on the structure, several sites in Su(H) and H were determined to specifically engage in complex formation. In particular, three mutations in Su(H) were identified that affect interactions with the repressor H but not the activator NICD. To analyse the effects these mutants have on normal fly development, we introduced these mutations into the native Su(H) locus by genome engineering. We show that the three H-binding deficient Su(H) alleles behave similarly. As these mutants lack the ability to form the repressor complex, Notch signalling activity is strongly increased in homozygotes, comparable to a complete loss of H activity. Unexpectedly, we find that the abundance of the three mutant Su(H) protein variants is altered, as is that of wild type Su(H) protein in the absence of H protein. In the presence of NICD, however, Su(H) mutant protein persists. Apparently, Su(H) protein levels depend on the interactions with H as well as with NICD. Based on these results, we propose that in vivo levels of Su(H) protein are stabilised by interactions with transcription-regulator complexes.Cell fate choices during metazoan development are driven by the highly conserved Notch signalling pathway. Notch receptor activation results in release of the Notch intracellular domain (NICD) that acts as transcriptional co-activator of the DNA-binding protein CSL. In the absence of signal, a repressor complex consisting of CSL bound to co-repressors silences Notch target genes. The Drosophila, repression of Notch target genes involves the CSL homologue Suppressor of Hairless [Su(H)] and the Notch antagonist Hairless (H). H binding to Su(H) excludes simultaneous NICD binding. Based on structural information, amino acids important for Su(H)-H interactions were mutated, generating Su(H) molecules that still bind NICD but no longer H, thereby preventing repressor but not activator complex formation. Three such mutations were introduced into the native Su(H) locus to analyse their consequences on fly development in vivo. All three alleles are homozygous lethal, demonstrating the essential role of Su(H) as repressor during fly development. Moreover, all three H-binding deficient Su(H) alleles show marked Notch gain of function. Unexpectedly, protein abundance of mutant Su(H) variants is reduced due to the loss of interactions with H. Moreover, we find that Su(H)-NICD interaction increased mutant Su(H) protein levels. Taken together, we propose that Su(H) protein is stabilised in vivo by interactions with transcription-regulator complexes.Notch signalling activity plays a major role in determining cell fates. Notch signals are transduced into gene expression changes by the transcription factor CSL and the activated Notch receptor intracellular domain (NICD). CSL can also function as a transcriptional repressor, depending on its bound cofactors. In Cell-to-cell communication is essential to development in higher animals and relies in part on the Notch signalling cascade which specifies the lineage of cells in a multitude of tissues ,2. As a Notch Intracellular Domain), and assembly of a transcriptional activator complex with the DNA-binding protein CSL and the co-activator Mastermind (Mam) and lin-12 and Glp-1 phenotype [Lag1]). In the absence of a Notch signal, a repressor complex consisting of CSL and co-repressors silences transcription from Notch target genes. Most mammalian co-repressors compete with NICD for binding to a hydrophobic pocket within the beta-trefoil domain (BTD) of CSL . Th. ThSu(H)LLL-mCh and Su(H)LLF-mCh proteins appeared specifically enriched in these cells (If we hypothesize that Su(H) is protected from degradation when nuclear and assembled within the repressor complex, it might likewise be protected within the activator complex when bound to NICD. Notch signalling is specifically activated along the dorso-ventral boundary of the wing imaginal disc ,35. Indese cells .To further substantiate this idea, we turned to RNAi analyses. Knock down of H protein levels along the antero-posterior border of wing imaginal discs resulted in a strong decrease of Su(H) protein levels\u2013yet, a weak signal was still present specifically along the dorso-ventral boundary . As predSu(H)LL, Su(H)LLF, or Su(H)LLL, however, where Su(H) protein levels are low, the signal enhancement was clearly seen at places of the strongest Notch activation, i.e. exactly along the border of Cut induction (In order to test the influence of NICD on Su(H) protein accumulation, we specifically induced the pathway in an ectopic location by misexpressing the ligand Serrate (Ser) along the antero-posterior boundary in the wing imaginal disc. In this setting, the Notch signalling cascade is induced specifically within the ventral domain of the wing disc \u201338. Morenduction . Next wenduction . These dSu(H) alleles, Su(H)LL, Su(H)LLF, and Su(H)LLL, were generated by genome engineering. These mutations were based on our previous findings that alanine substitutions at these positions specifically affected repressor but not activator complex formation = Crew}DH1 (BL1092) as described before , established by homologous recombination. (B) The white+ marker and flanking sequences were floxed out by Cre-mediated recombination, resulting in Su(H)attP. This founder line served as origin for the integration of any Su(H)* DNA construct cloned into pGE-attBGMR, as outlined for the genomic rescue construct below. (C) The genomic Su(H)gwt rescue construct was inserted as a proof of principle, giving Su(H)gwt [w+]. (D) The final control line Su(H)gwt was established by deleting the white+ marker. The same procedure was applied for any other construct as well. Primer pair used for RT-PCR in (E) is not to scale. (E) Splicing of Su(H) mRNA occurs normally in the rescue strain Su(H)gwt compared to the wild type strain Oregon R. RT-PCR was performed on cDNA from the respective strains, using primer pair shown in (D), to obtain the expected 238 bp fragment (arrowhead). Unlike the control (c), the probe (p) contained reverse transcriptase. Size standard (M) was a 100 bp ladder; some bands are labelled for clarity (* unspecific products). (F) Quantification of Western blots (n = 5) was performed with Image J gel analysis program. Beta-tubulin signals served as internal standard; mutants were compared to Su(H)gwt control. Error bars represent standard deviation. Significance was tested by ANOVA two-tailed Tukey-Kramer approach for multiple comparisons .(TIF)Click here for additional data file.S2 Fig(A) Structure of the Su(H) primary transcript. Dashed lines indicate positions of deletion and reintegration into the founder line Su(H)attP. Relevant restriction sites used for cloning are indicated. (B) Scheme of the control and mutant Su(H) constructs used for the generation of the respective Su(H) alleles. Note the absence of intron 3 in Su(H)LL and Su(H)LLF, and of both introns 2 and 3 in Su(H)LLL. (C) Viability of the new Su(H) alleles balanced over CyO was assessed by a cross to y1w67c23 control in comparison to Su(H)\u039447 and Su(H)SF8 alleles. The expected 1:1 ratio of mutant and balancer chromosome CyO was observed, yet Su(H)LLL has a slightly lowered viability. (D) The new Su(H) alleles were crossed with Su(H)attP/CyO-GFP and the number of hemizygous pupae counted relative to the heterozygotes recognized by green fluorencence. Su(H)\u039447 and Su(H)SF8 served for comparison. (E) The homozygous control lines Su(H)gwt, Su(H)gwt\u0394i3, or Su(H)gwt\u0394i2i3 are like wild type, exemplified by a normal Wg staining. All three are able to complement the Su(H)attP allele: they are viable and have normal looking wing discs in hemizygosis. Likewise, Su(H)gwt-mCh appears wild type, whereas Su(H)LLL-mCh and Su(H)LLF-mCh display hypertrophied wing discs similar to the untagged strains (compare with (F) Survival rates of the hemizygous controls Su(H)gwt, Su(H)gwt\u0394i3, or Su(H)gwt\u0394i2i3 over Su(H)attP was determined relative to wild type stock Oregon R (OreR). Inset numbers in (C), (D), (F) represents animals analysed.(TIF)Click here for additional data file.S3 Fig(A) Wg is expressed along the dorso-ventral boundary of the disc. Control clones of Su(H)gwt do not disturb the wild type pattern. (B) Wg protein expression is lost from Su(H)attP homozygous cells (arrow) due to the absence of Su(H) protein. (C-E) A slight broadening of Wg expression is seen in cell clones homozygous mutant for any of the H-binding deficient Su(H) alleles, Su(H)LL(C), Su(H)LLF(D), or Su(H)LLL(E). (F-G) A similar broadening is observed in cells lacking H activity (arrow), as in HattP(F) or HLD (G). Size bar represents 25\u03bcm in all panels.Expression of Wg (red) was analysed in cell clones homozygous mutant for the allele indicated. Mutant clones are within the central wing blade; they are marked by the absence of GFP (green) and are outlined. Examples of altered Wg expression are highlighted by arrows. (TIF)Click here for additional data file.S4 Fig(A) In contrast to Su(H)gwt control clones, (B)Su(H)attP null mutant cells show little Su(H) protein signals. (C-E) Note the reduction of Su(H) protein levels in H-binding deficient Su(H) mutant cells, and alike in cells lacking H activity. . Size bar represents 50\u03bcm in all panels.Clones were induced in wing imaginal discs; magnification of the central wing blade is shown. Genotypes are indicated on the left. Cells were stained with a polyclonal rat anti-Su(H) antiserum (red) , anti-GF(TIF)Click here for additional data file.S5 Fig Salivary glands from homozygous third instar larvae of the given genotype were doubly stained for Pzg as nuclear marker and for Su(H) and mCherry, respectively. Drastic reduction of nuclear Su(H) protein expression is apparent in the mutants. (C) Quantification of staining intensity was performed on stacks of pictures cutting through the entire gland using Image J. Intensity of all the nuclei from one gland was compared with the total cytoplasmic signal from that gland (n = 3\u20135). Loss of nuclear staining and no concurrent cytoplasmic enrichment is observed. (D) Signal intensity of Su(H) and Pzg staining of individual nuclei were recorded using Image J. A four- to five-fold drop in Su(H) expression is seen in the H-binding deficient Su(H) as well as in the H alleles indicated. Differences between mutants and control are highly significant by ANOVA two-tailed Tukey-Kramer approach for multiple comparisons . Size bar represents 100\u03bcm in all panels.(TIF)Click here for additional data file.S6 Fig(A)Su(H) mRNA expression was quantified by qPCR relative to cyp33 and Tbp as reference genes. Efficiencies for Su(H) (0.97), for cyp33 (0.94) and for Tbp (0.92) were taken into account for the relative quantification ; homozygous wild type cells show high GFP levels, mutant cells lack GFP. The clones are outlined for clarity. Size bar represents 50\u03bcm in all panels.((TIF)Click here for additional data file."} +{"text": "MicroRNAs (miRNAs) play important roles in cancer formation and progression by suppressing the production of key functional proteins at the post-transcriptional level in a sequence-specific manner. While differential expression of miRNAs is widely observed in cancers including prostate cancer (PCa), how these miRNAs are transcriptionally regulated is largely unknown. MiRNA-221 and miRNA-222 (miR-221/-222) are well-established oncogenes and overexpressed in breast, liver, pancreas, and lung cancer, but their expression and biological functions in PCa remain controversial. Both up and down regulation have been observed in patient samples. Specifically, studies have demonstrated miR-221/-222 function as oncogenes, and promote PCa cell proliferation and the development of castration-resistant prostate cancer (CRPC). However, the expression level of miR-221/-222 is downregulated in several miRNA expression profiling studies. In this study, we demonstrate miR-221/-222 are androgen receptor (AR)-repressed genes and reside in a long primary transcript (pri-miRNA). Derepression of miR-221/-222 after androgen deprivation therapy (ADT) may enhance PCa cell proliferation potential through promoting G1/S phase transition. This function is likely transient but important in the development of CRPC. Downregulation of miR-221/-222 subsequently occurs once AR activity is restored through AR overexpression in CRPC. Our findings shed light on the complexity of transcriptional regulation of miRNAs in PCa and suggest context-dependent targeting of oncogenic miRNAs. MicroRNAs (miRNAs) are small non-coding RNAs about 22 nucleotides in length that regulate the expression of target mRNA post-transcriptionally and influence a multitude of cellular processes during development and disease. Dysregulation of miRNAs has been widely observed in numerous cancers and different stages of cancer. These miRNAs function as oncogenes or tumor suppressors based on their inhibition of tumor-suppressive and oncogenic target mRNAs, respectively. Prostate cancer (PCa) is the most frequently diagnosed cancer in American men. While miRNA profiling shows that many miRNAs are differentially expressed in PCa tissues versus the corresponding normal tissues, only a small number of them have been experimentally determined to be involved in the development and progression of PCa.MiRNA-221 and miRNA-222 (miR-221/-222) are two highly homologous miRNAs, which are clustered on the short arm of chromosome X. They are overexpressed in the majority of epithelial cancers, including breast, liver, pancreas, and lung cancer . It is bIn general, a miRNA is processed from a primary transcript (referred to as pri-miRNA) that is transcribed by RNA polymerase II (Pol II). The pri-miRNA can extend hundreds of kilobases in length and include more than one precursor miRNA hairpin (pre-miRNA). Studies have shown that approximately 50% of miRNAs are intragenic and mostly located within introns of protein-coding genes , 24. WhiHere, we present integrated genomic data at the miR-221/-222 locus. We define and characterize a pri-miRNA for miR-221/-222 in CRPC cells. We investigate whether and how miR-221/-222 are regulated by AR, which may explain the disparity of miR-221/-222 expression level in different PCa tumors. Because miR-221/-222 target key cell cycle genes and increase cellular proliferation potential in cancer cells, a complete understanding of their regulation during PCa progression may have clinical applications in the future.Human prostate cancer LNCaP and C4-2B cells were described previously . All celAnti-AR (1:200 dilution), anti-FOXA1 (1:1000 dilution), and anti\u2014\u03b2-tubulin (1:2000 dilution) were used as primary antibodies in Western blot. Experiments were performed as previously described .C4-2B cells were co-transfected with miR-221 and miR-222 precursors or inhibitors (Ambion) at a final concentration of 20 nM each using Lipofectamine RNAiMAX Transfection Reagent (Life Technologies) according to the manufacturer\u2019s instruction. Cell number was counted 3 days after transfection. Cell cycle analysis was performed in parallel using Propidium Iodide Flow Cytometry Kit (Abcam). Cell cycle distribution of 10,000 gated cells is presented.\u2122 and Hy5\u2122 as labeling dyes with dye swap were conducted for comparison of differential expression between LNCaP and C4-2B cells. In addition, miR-221/-222 were quantified by RT-qPCR using TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems) and FastStart Universal Probe Master (Roche) according to the manufacturer\u2019s instruction. U6 snRNA was used for normalization. All primer sets for TaqMan MicroRNA Assays were purchased from Applied Biosystems. Values are means \u00b1 standard deviations (SD) of triplicate wells.LNCaP and C4-2B cells were grown in RPMI 1640 supplemented with 10% FBS. Total RNA was extracted using TRIzol Reagent (Thermo Fisher Scientific) and submitted to Exiqon for miRNA expression profiling using miRNA microarrays. Dual-color experiments using Hy35\u2019-GTCATAATGGCAGAGTCCTCAT-3\u2019; reverse, 5\u2019-TACATGGCAGAAGAGCAGAAG-3\u2019; Site B-forward, 5\u2019-CAGAAGTTCATGGATGGGAGAG-3\u2019; reverse, 5\u2019-TGCTTTGTACTCTTCGGGATTAG-3\u2019. Primers for AR, PSA, and GAPDH mRNA were previously described [After treatments as indicated, total RNA from cells or tissues was extracted using TRIzol Reagent. Complementary DNA (cDNA) was prepared using the iScript cDNA Synthesis Kit (Bio-Rad), and qPCR was conducted using SYBR Green PCR Master Mix (Applied Biosystems). Triplicate PCR reactions were conducted. The primers are: Site A-forward, escribed , 28.For AR and FOXA1 knockdown, cells were seeded in 6-well plate and transfected with gene-specific siRNA at a final concentration of 20 nM using Lipofectamine RNAiMAX Transfection Reagent according to the manufacturer\u2019s instruction. SMARTpool siRNAs against AR, FOXA1, and non-specific (NS) control were purchased from Thermo Fisher Scientific. Cells were collected for miR-221/-222 RT-qPCR analysis 3 days after transfection.Cells were transfected with miRNA luciferase reporters, pMiR-221-Luc or pMiR-222-Luc (Signosis). These two vectors are firefly luciferase-based reporter constructs, which have a unique miRNA target site at 3\u2019UTR perfectly complementary to miR-221 and miR-222 respectively. pRL-TK Renilla luciferase reporter (Promega) was co-transfected as an internal control. Luciferase activity was measured 24 hours after transfection using Dual-Luciferase Reporter Assay System (Promega). The results are represented as Firefly/Renilla ratio.http://epigenomics.wustl.edu/liData/C42B_riboM_1.bw.ChIP-seq data at the miR-221/-222 locus are from our previously generated datasets . These d5\u2019-TCTTTGCAATCTGAACACAGCA-3\u2019; reverse, 5\u2019-TGCCCGACTTCTAAGCATTAGC-3\u2019; miR-221/-222 TSS-forward, 5\u2019-CTCCATTAAACCCTTGTCCAAAC-3\u2019; reverse, 5\u2019-GGAATGGGTTTGCTGAACTTAC-3\u2019. Primers for the PSA enhancer and promoter were described previously [ChIP experiments were performed as described previously . FAIRE eeviously .LuCaP 35 and LuCaP 35CR xenografts were established from metastatic human prostate tumors in lymph nodes . LuCaP 3MiR-221/-222 and AR expression data from 111 PCa patient samples were obtained from microRNA microarray (GSE21036) and whole exon microarray (GSE21034) datasets respectively. Patients were ranked according to AR expression level and grouped into high-AR and low-AR . Differential expression of miR-221/-222 between two groups was analyzed by non-parametric Mann-Whitney test. Boxplot shows the mean \u00b1 95% confidence interval (CI).First, we conducted miRNA expression profiling in androgen-dependent LNCaP and LNCaP-derived C4-2B cells using miRNA microarray analysis . C4-2B iWhile it has been reported that miR-221/-222 are regulated by NF \u03baB in PCa , androgeNext, we sought to determine whether and how androgen inhibits expression of miR-221/-222 through the AR at this locus. We studied this in C4-2B cells because a greater inhibitory effect was observed. It is known that miR-221/-222 are transcribed in a single primary transcript . ComputaThe mechanism underlying AR-mediated transcriptional activation has been well studied, whereas AR-mediated transcriptional repression is poorly understood. FOXA1 functions as a pioneer factor, which facilitates AR recruitment to AR binding sites. Like the PSA enhancer, we detected strong pre-existing FOXA1 occupancy, which coincides with the AR binding site at the miR-221/-222 locus in the absence of androgen . FOXA1 bAR binding sites can be epigenetically marked by specific histone modifications and fluctuation of these modifications has been linked to transcriptional regulation. To understand chromatin modifications associated with AR-mediated activation versus repression, we examined several specific histone modification marks at the AR binding site of the miR-221/-222 locus versus that of the PSA locus . ChIP-qPWhile miR-221/-222 are considered as oncogenes in CRPC, it is not clear why their expression level is downregulated in CRPC patients in several studies , 16, 20.The molecular mechanisms by which PCa cells progress from androgen-dependent to castration-resistant status have been extensively studied. Much of the previous work has been focused on how AR activity is restored through AR overexpression, amplification, mutations and AR variants, which leads to reactivation of some AR-stimulated genes including PSA. Nevertheless, emerging evidence has suggested that one of the molecular mechanisms for PCa cells to survive ADT is derepression of AR-repressed genes that contribute to androgen synthesis, DNA synthesis, and cellular proliferation . While AOur studies revealed that AR-mediated repression is associated with suppression of histone H3 acetylation and pol II binding at this locus. Repressive effects of AR on miR-221/-222 expression restrains their oncogenic potential. ADT may unlock miR-221/-222, which in turn promote G1/S transition through downregulation of cell cycle genes, such as p27Kip1. Recent studies further suggested HECTD2 and RAB1A as miR-221/-222 targets . Downregin vitro [in vitro [in vivo approaches and confirmed that overexpression of miR-221 in LNCaP cells confers a high growth advantage and inhibition of miR-221/-222 in PC3 cells reduces tumor growth in mice [Several studies showed downregulation of miR-221/-222 in metastatic PCa and CRPC specimens, suggesting a tumor suppressor role for miR-221/-222 , 13, 16.in vitro . In contin vitro . Mercate in mice . These oMiR-221/-222 are well-established oncogenes in all epithelial cancers except prostate cancer (PCa). In the present study, we show miR-221/-222 are AR-repressed genes and their expression and oncogenic function are associated with AR status in PCa cells. The findings provide an explanation of why miR-221/-222 act as oncogenes in the development of CRPC, but their overexpression is not observed in CRPC tumors. Our findings shed light on the complexity of transcriptional regulation of miR-221/-222 in PCa and suggest context-dependent targeting of oncogenic miR-221/-222.S1 Table(XLSX)Click here for additional data file."} +{"text": "Rice transcription factor OsNF-YB1 is specifically expressed in the aleurone layer of developing endosperm and forms protein complexes consisting of OsNF-YB1, OsNF-YC and ERF to regulate grain filling and endosperm development. OsNF-YB1 expression by RNAi significantly retarded grain filling, leading to small grains with chalky endosperm as well as altered starch quality. Whereas OsNF-YB1 shows subcellular localization in both the cytosol and the nucleus in roots, it was specifically targeted to the nucleus of aleurone layer cells, which was facilitated by interacting with OsNF-YC proteins preferentially expressed in the aleurone layer. RNA sequencing analysis revealed that genes related to membrane transport and ATP biosynthesis were enriched in the down-regulated category in OsNF-YB1 RNAi plants, which is consistent with the crucial role of OsNF-YB1 in rice grain filling and endosperm development. Identification of the genome-wide targets of OsNF-YB1 by ChIP sequencing showed that OsNF-YB1 directly regulates genes involved in the transport of nutrients such as sugar and amino acids. Interestingly, different from the binding sites reported for other NF-Y complexes, the GCC box, the binding motif of ERF transcription factors, was enriched in the binding peaks of OsNF-YB1. Indeed, further analyses confirmed the interaction of OsERF#115 with OsNF-YB1, and OsERF#115 directly binds to the GCC box. It is proposed that OsNF-YB1 specifically regulate the transcription of downstream genes during rice endosperm development by forming protein complexes consisting of OsNF-YB1, OsNF-YC and ERF, providing informative insights into the molecular functional mechanisms of the NF-Y factor.Grain yield and quality of rice mainly depend on grain filling and endosperm development. Here we report that a rice NUCLEAR FACTOR Y (NF-Y) transcription factor, OsNF-YB1, is specifically expressed in the aleurone layer of developing endosperm and regulates grain filling and endosperm development. Knockdown of The formation of the embryo and endosperm are key processes in seed development. Endosperm development plays an important and active role in regulating embryo and seed development and the cereal endosperm is important biologically as well as nutritionally and economically as a major source of human food. Elucidation of the molecular mechanisms regulating endosperm development will help to improve grain yield and quality.Rice endosperm consists of starchy endosperm and the aleurone layer. During grain filling, nutrients are transported into the developing caryopsis through the dorsal vascular bundle, then into the nucellar projection and finally to the endosperm. As there are no plasmodesmatal connections between the maternal and filial tissues , the aleGRAIN INCOMPLETE FILLING 1 (GIF1) is expressed in the ovular vascular trace and encodes a cell-wall invertase required for carbon partitioning and grain filling , also known as CCAAT-binding factor (CBF) and heme-associated protein (HAP), binds to the CCAAT box. NF-Y is composed of NF-YA, NF-YB, and NF-YC subunits in most species, and is evolutionarily conserved in all eukaryotes. Extensive studies of the structure and function of the NF-Y complex in mammals showed that NF-YB and NF-YC form a tight dimer via histone-fold domains, and that this interacts with DNA non-specifically, while NF-YA is responsible for sequence-specific contact with the CCAAT box to the Studies have shown that NF-Y is required for the early embryonic development of mouse, which is consistent with its role in cell-cycle regulation in proliferating cells , droughtOther transcription factors can interact with NF-Y subunits to regulate the expression of downstream target genes. NF-Y proteins physically interact with bZIP, MADS, and the conserved CCT domain proteins to regulate various processes was used for rice transformation. Transgenic rice plants were grown in a phytotron with a 12 h light (28 oC)\u201312 h dark (22 oC) cycle. To measure grain-related traits, plants were grown in an experimental field under natural conditions.Japonica rice (OsNF-YB1 (LOC_Os02g49410) RNAi plants, a 311-bp fragment specific to the OsNF-YB1 coding region (15\u2013325 nt) was amplified by PCR and inserted in the sense orientation into the HindIII/PstI sites of the pSK-Int vector. The same fragment was subsequently cloned and inserted in the antisense orientation into the KpnI/BamHI sites of pSK-Int carrying the sense fragment. Finally, the fragment in pSK-Int containing the 478-bp intron flanked with two 311-bp opposite fragments was digested with HindIII and cloned into pUN1301, resulting in the RNAi construct OsNF-YB1. The resultant construct was transformed into ZH11 by Agrobacterium-mediated transformation and homozygous T2 lines were analyzed. Primers for creating the RNAi construct are listed in the Supplementary Table S1 at JXB online.To generate OsNF-YB1-overexpressing plants, an approximately 7.3-kb DNA fragment containing the whole OsNF-YB1 gene and ~5.6-kb upstream of the translation initiation codon was digested with XbaI/SacI from bacterial artificial chromosome clone OSJNBa0096K12 (Arizona Genomics Institute) and subcloned into the XbaI/SacI sites of the pCAMBIA1300 vector (CAMBIA) for transformation. Homozygous T2 rice lines were analyzed.To generate the Actin (LOC_Os03g50885) gene was used as an internal standard to normalize the expression of tested genes. Relevant primer sequences are listed in Supplementary Table S1.Total RNA was extracted using TRIzol reagent (Life Technologies) and reverse transcribed into cDNA according to the manufacturer\u2019s instructions . Quantitative RT-PCR (qRT-PCR) was performed by using the SYBR Green Realtime PCR Master Mix (Toyobo). Rice OsNF-YB1 (upstream of ATG) was amplified by PCR and subcloned into XbaI/BamHI sites of pCAMBIA1300+pBI101. The resultant construct was transformed into ZH11 and independent lines of positive T2 transgenic progeny were used to detect GUS activity. Photography was performed using a Nikon microscope (SMZ1500) with a digital camera. Primer sequences are listed in Supplementary Table S1.The ~3.4-kb putative promoter region of OsNF-YB1 was amplified by PCR (primer sequences are listed in Supplementary Table S1), subcloned into pGEM-T easy vector (Promega), and used as a template to generate digoxigenin-labeled sense and antisense probes (Roche). Caryopses of ZH11 were fixed by 4% paraformaldehyde in 0.1 M sodium phosphate buffer, dehydrated through a graded ethanol series, replaced with xylene, embedded in paraffin (Sigma-Aldrich), and sectioned at 8 mm. In situ hybridization was performed according to the previous description , and the chain length distributions of amylopectin were analyzed as described previously . Rapid VOsNF-YB1 coding region was amplified by PCR and subcloned into pGBKT7 bait plasmid. Coding regions of OsNF-YC8 (LOC_Os01g01290), OsNF-YC9 (LOC_Os01g24460), OsNF-YC10 (LOC_Os01g39850), OsNF-YC11 (LOC_Os05g23910), OsNF-YC12 (LOC_Os10g11580), OsERF#074 (LOC_Os05g41780), OsERF#114 (LOC_Os06g42910), OsERF#115 (LOC_Os08g41030), and OsERF#072 (LOC_Os09g26420) were amplified and cloned into pGADT7 prey plasmid. Yeast strain AH109 was co-transformed with specific bait and prey constructs through a lithium acetate-mediated method. Interactions were tested using SD/\u2013Leu/\u2013Trp/\u2013His/\u2013Ade medium. Primer sequences are listed in Supplementary Table S1.Whole Os07g19070 promoter containing the intact GCC box was repeated three times and inserted into pHIS2.1 (Clontech). OsERF#115 or NF-YB1 was fused to GAL4 transcriptional activation domain (AD). Yeast strain AH109 was co-transformed with the indicated constructs through a lithium acetate-mediated method and a one-hybrid assay was performed following the manufacturer\u2019s manual (Clontech).A twenty base pair DNA fragment of OsERF#115 were amplified by PCR, subcloned into pGBKT7 and fused with the BD domain. The resultant construct was transformed into AH109 and transcriptional activity was examined by observing the yeast cell growth on SD/\u2013Trp/\u2013His/\u2013Ade medium.Coding regions of OsERF#115 was amplified by PCR, subcloned into pBridge and fused with the BD domain. The OsNF-YB1 coding region was then subcloned into the pBridge-OsERF#115 construct. Coding regions of OsNF-YC11 or OsNF-YC12 were subcloned into modified pGADT7 without GAL4-activation domain. Yeast strain AH109 was co-transformed with the indicated constructs and transformed yeast cells were selected on SD/\u2013Leu/\u2013Trp medium. Transcriptional activity was examined by observing the cell growth on SD/\u2013Leu/\u2013Trp/\u2013His/\u2013Met medium.The coding region of Supplementary Table S1.Sequences of primers used are listed in OsNF-YB1 were amplified by PCR and subcloned into pA7 vector. The resultant construct was introduced into onion epidermal cells (pA7 vector as control) and green fluorescence was observed with a confocal laser scanning microscope (Olympus FV1000).Coding regions of Nicotiana benthamiana (tobacco) leaf epidermal cells, a DNA fragment in pA7 containing OsNF-YB1-GFP was digested with HindIII/SacI and cloned into PHB vector (OsNF-YC2 (LOC_Os03g14669), OsNF-YC8, OsNF-YC9, OsNF-YC10, OsNF-YC11 and OsNF-YC12 were amplified by PCR and subcloned into a modified pCambia1300 containing mCherry reporter. Constructs were co-transformed with the P19 silencing suppressor into tobacco leaves through Agrobacterium infiltration and examined with a confocal laser scanning microscope (Olympus FV1000) after infiltration for 2 days.For transient expression of fusion protein in pUbi:OsNF-YB1-GFP, a DNA fragment in pA7 containing OsNF-YB1-GFP was digested with XhoI/EcoRI and cloned into pUN1301. The resultant construct was transformed into ZH11 and homozygous T2 lines were analyzed.To construct Supplementary Table S1.All primer sequences are listed in the The starchy endosperm and embryo of caryopses were removed, and aleurone layer cells were isolated from the remaining caryopsis coats as described previously . Inner lOsNF-YB1-GFP, OsNF-YC11-mCherry, or OsNF-YC12-mCherry were firstly co-transformed with the P19 silencing suppressor into tobacco leaves. After 2 days, the infiltrated leaves were ground in liquid nitrogen, lysed with the extraction buffer , and centrifuged at ~10 000 g for 20 min. Supernatant was incubated with anti-GFP antibody and coupled to Dynabeads Protein G (Life Technologies) for 2 h. Beads were washed three times with wash buffer . The bound proteins were eluted with 2\u00d7 SDS sample buffer, and subjected to immunoblot analysis using monoclonal anti-GFP antibody and anti-mCherry antibody .For co-immunoprecipitation (Co-IP) analysis, constructs One hundred caryopses at 8 days after flowering (DAF) of 20 independent plants were used to isolate the aleurone layer cells as described above. Throughout the process, cordycepin was added to a final concentration of 1 mM to inhibit transcription. Total RNA was extracted using Trizol, and Illumina sequencing libraries were constructed according to the manufacturer\u2019s instructions. The libraries were sequenced with Hiseq2500. Reads were aligned to the rice genome (MSU7.0) using Tophat. Differentially expressed genes were indicated as false discovery rate (FDR)<0.05 with Cuffdiff.http://www.geneontology.org/), Rice Genome Annotation (http://rice.plantbiology.msu.edu/), and Arabidopsis annotation (ftp://ftp.arabidopsis.org/home/tair/Ontologies/Gene_Ontology/) was used. The hypergeometric test was used with subsequent Benjamini and Hochberg FDR corrections. GO annotation terms were considered significant if the corrected P-value was <0.05 and if there were at least five genes associated with the same annotation.Gene ontology (GO) enrichment was performed using BiNGO. GO information from the gene ontology website (pUbi:OsNF-YB1-GFP (lines 16 and 22) were used for ChIP assay. For each biological repeat, 1 g of caryopsis coats at 8\u201312 DAF was harvested. Tissue fixation, nuclei extraction, and chromatin immunoprecipitation using anti-GFP antibody were performed as described previously and weaker but broader regions with default settings. Overlapping peaks (having a stronger but narrower peak in at least one replicate) between two biological replicates were used for further analysis. Target genes were defined when overlapping peaks appeared on their genic or within 3-kb upstream regions. The summits of overlapping peaks were used to define the location types in the genome as described previously (http://repeatmasker.org), and then used for motif search.The short reads given were aligned to the rice genome (MSU7.0) by Bowtie2 with default parameters. MACS2 .NF-Y families, OsNF-YB1, is positively correlated with the endosperm-preferentially expressed starch synthesis genes by co-expression analysis . Results showed that OsNF-YB1 was specifically expressed in the developing caryopses and reached a maximum at 8 days after fertilization . Further promoter-\u03b2-glucuronidase (GUS) fusion analysis revealed that OsNF-YB1 was expressed in the outer surface of the endosperm, possibly the aleurone layer of developing caryopses , which is similar to a report by A member of the analysis . To studOsNF-YB1, RNA in situ hybridization analysis was performed using caryopses at different developmental stages. Results showed that OsNF-YB1 was expressed in the dorsal aleurone layer cells at 4 DAF, immediately after the completion of cellularization. Along with endosperm differentiation, OsNF-YB1 was expressed at the dorsal and lateral aleurone layer cells, and in the entire aleurone layer at 10 DAF construct containing a non-conserved region of OsNF-YB1 was generated and transformed into rice . Analysis of the T0 transgenic lines revealed they were independent lines by genomic Southern blot . Further analysis confirmed the significantly suppressed OsNF-YB1 expression in the T2 generation of transgenic lines, while the expression levels of OsNF-YB6 and OsNF-YB8, which present nucleotide sequences homologous with OsNF-YB1, were not affected .To study the physiological role of OsNF-YB1 RNAi plants, including height, flowering timing, panicle number per plant, and grain number per panicle , while the brown grains were smaller, resulting in a reduced 1000-brown-grain weight . Further comparison revealed the decreased grain-filling rate of OsNF-YB1 RNAi plants during grain development and degree of endosperm chalkiness (DEC) . The Rapid Visco Analyzer (RVA) profile provides a comprehensive evaluation of starch quality and further analyses showed that OsNF-YB1 RNAi grains had a different RVA profile , indicating the obviously altered physicochemical characteristics of starch under suppressed OsNF-YB1.Interestingly, compared with those of ZH11, ss (DEC) . The app reduced , and anaOsNF-YB1 RNAi seeds presented a normal aleurone layer composed of multilayers at the dorsal side adjacent to the major vascular bundle and one or two layers at the ventral and lateral sides and analysis of seven selected independent OsNF-YB1-overexpressing lines showed that compared with ZH11, the PGWC and DEC of five OsNF-YB1-overexpressing lines were reduced cells showed that the green fluorescent protein (GFP)-tagged OsNF-YB1 protein was located in both the cytosol and the nucleus , which was the same as with OsNF-YB1 in rice root cells expressing pUbi:OsNF-YB1-GFP are preferentially expressed in endosperm , and thus the possible interactions of them with OsNF-YB1 was examined by yeast two-hybrid analysis. Results showed that all these five NF-YCs interact with OsNF-YB1 in yeast cells . A further transient expression assay using tobacco epidermal cells showed that OsNF-YC11-mCherry and OsNF-YC12-mCherry co-expression mediated the OsNF-YB1-GFP nuclear localization , while OsNF-YB1-GFP was observed in both the cytosol and nucleus when co-expressing with mCherry, OsNF-YC8-mCherry, OsNF-YC9-mCherry, OsNF-YC10-mCherry, or OsNF-YC2-mCherry .Studies in fungi and mammals showed that the nuclear import of NF-YB and NF-YC requires heterodimer formation . Further transient expression assay using rice protoplast cells showed that OsNF-YC11 or OsNF-YC12 mediated the OsNF-YB1 nuclear localization in rice cells using aleurone layer cells of ZH11 and OsNF-YB1 RNAi plants. Aleurone layer cells were isolated enzymatically from the caryopses at 8 DAF at 8\u201312 DAF were collected from two independent transgenic lines expressing pUbi:OsNF-YB1-GFP, and then used to carry out the ChIP assays with an anti-GFP antibody . ChIP-seq data were analyzed with the statistical software MACS2 (Target genes of OsNF-YB1 were firstly identified using ChIP sequencing (ChIP-seq). Based on the specific expression of re MACS2 .The 933 binding peaks linked to 743 neighbor genes and are considered as the OsNF-YB1 target genes. Functional analysis by GO annotation showed that many terms related to nutrients transport were significantly enriched . ChIP-qPInterestingly, a search for motifs using DREME led to tOsERF#115 had the highest expression in aleurone layer (see Supplementary Table S3), and it was selected for further analysis. In addition we selected and analyzed at same time OsERF#072 and OsERF#074, which are relatively highly expressed in aleurone layer, OsERF#114, which is preferentially expressed in developing endosperm but lowly expressed in aleurone layer , which indicates a weak interaction of OsNF-YB1 with OsERF#115 in vivo, identification of the GCC box by CHIP-seq analysis suggests the possible in vivo interaction between OsNF-YB1 and ERF proteins.Yeast two-hybrid assays showed that OsNF-YB1 could interact with OsERF#115, but not other ERF members . In addiConsidering that ERF transcription factor binds to the GCC box and OsERF#115 interacts with OsNF-YB1, we further performed yeast one-hybrid analysis and results confirmed that OsERF#115 binds to the GCC box, while OsNF-YB1 does not . This inFurther transcriptional activity assay using yeast cells showed that OsEFF#115 did not possess transactivational activity , and theOur studies have identified a possible novel non-canonical NF-Y trimeric complex consisting NF-YB, NF-YC and ERF protein that forms in rice aleurone layer cells and plays important roles in regulating endosperm development and hence grain filling. OsNF-YB1 is imported into the nucleus through heterodimerization with OsNF-YC11 or OsNF-YC12, which are preferentially expressed in aleurone layer cells. By forming the transcriptional complexes with transcription factor OsERF#115, OsNF-YB\u2013NF-YC\u2013ERF regulates the expression of target genes containing the GCC box and hence grain filling and endosperm development. Enrichment of transport-related genes reveals the crucial role of the OsNF-Y factor and the aleurone layer in regulating nutrient transport into endosperm, and further in rice, filling and seed size/quality .Studies of the crystal structure of NF-Y trimer bound to DNA showed that while the NF-YB\u2013NF-YC dimer interacts with DNA non-specifically, NF-YA provides sequence-specific contact to the CCAAT box , suggesting that NF-Y complexes may play important roles in endosperm development of various plant species.Our results demonstrate the importance of Arabidopsis AP2 has a significant effect on early endosperm development show a dual cytosolic\u2013nuclear localization in tobacco cells and are accumulated in the nucleus after coexpression, suggesting that the nuclear import of NF-YB and NF-YC requires dimer formation, which is consistent with the observations in mammals .Figure S5. A dual cytosolic\u2013nuclear localization of OsNF-YB1 in onion and tobacco epidermal cells.Figure S6. Expression pattern and subcellular localization of OsNF-YCs.Figure S7. Overview of the ChIP assays.Table S1. List of the primers used in this study.Table S2. List of the examined genes identified by RNA-seq or Chip-seq analysis.Table S3. Expression of OsERF genes in rice aleurones."} +{"text": "These data support the idea that HCV assembly occurs in concert with budding at the ER membrane. Furthermore, capsid-containing particles did not accumulate in the absence of SP-catalyzed cleavage, suggesting the quality of newly formed viral particles is controlled before secretion.In hepatitis C virus (HCV) polyprotein sequence, core protein terminates with E1 envelope signal peptide. Cleavage by signal peptidase (SP) separates E1 from the complete form of core protein, anchored in the endoplasmic reticulum (ER) membrane by the signal peptide. Subsequent cleavage of the signal peptide by signal-peptide peptidase (SPP) releases the mature form of core protein, which preferentially relocates to lipid droplets. Both of these cleavages are required for the HCV infectious cycle, supporting the idea that HCV assembly begins at the surface of lipid droplets, yet SPP-catalyzed cleavage is dispensable for initiation of budding in the ER. Here we have addressed at what step(s) of the HCV infectious cycle SP-catalyzed cleavage at the core-E1 junction is required. Taking advantage of the sole system that has allowed visualization of HCV budding events in the ER lumen of mammalian cells, we showed that, unexpectedly, mutations abolishing this cleavage did not prevent but instead tended to promote the initiation of viral budding. Moreover, even though no viral particles were released from Huh-7 cells transfected with a full-length HCV genome bearing these mutations, intracellular viral particles containing core protein protected by a membrane envelope were formed. These were visualized by electron microscopy as capsid-containing particles with a diameter of about 70 nm and 40 nm before and after delipidation, respectively, comparable to intracellular wild-type particle precursors except that they were non-infectious. Thus, our results show that SP-catalyzed cleavage is dispensable for HCV budding Hepacivirus belonging to the Flaviviridae family. HCV is an enveloped virus with a single-strand positive RNA genome. This genome encodes a single polyprotein precursor that undergoes a series of proteolytic cleavages to generate functional viral proteins . HCV core protein is the most N-terminal component of the viral polyprotein, and terminates with E1 signal peptide . Cell lysates were subjected to western blot analysis with mAb against HCV E2 glycoprotein (anti-E2) or HCV core protein (anti-core). Positions on blots of protein molecular mass standards are indicated (in kDa). The same membrane reprobed with the different antibodies is shown.BHK-21 cells were electroporated with the recombinant RNAs SFV-(PPTX)Click here for additional data file.S3 FiglacZ (LacZ), SFV-HCV1b (WT), SFV-HCV1b/Sp1mt (Sp1mt), or SFV-HCV1b/Sp2mt (Sp2mt). Transfected cells were fixed, permeabilized, and subjected to double-label immunofluorescence staining for confocal microscopy detection of HCV core protein (red) and marker (green), BODIPY 493/503 (lipid) or calnexin (ER).BHK-21 cells were electroporated with the recombinant RNAs SFV-(PPTX)Click here for additional data file.S4 FiglacZ RNA (LacZ). Cells were processed for (A) conventional EM, or (B) immunogold labeling with mAbs against HCV core protein (+ anti-core) or HCV E2 envelope glycoprotein (+ anti-E2).Representative electron micrographs of ultra-thin sections of BHK-21 cells electroporated with the recombinant SFV-(PPTX)Click here for additional data file."} +{"text": "O-MTase activity of CoVs for attenuation. With clear understanding of the IFN/IFIT (IFN-induced proteins with tetratricopeptide repeats)-based mechanism, NSP16 mutants provide a suitable target for a live attenuated vaccine platform, as well as therapeutic development for both current and future emergent CoV strains. Importantly, other approaches targeting other conserved pan-CoV functions have not yet proven effective against MERS-CoV, illustrating the broad applicability of targeting viral 2\u2032O-MTase function across CoVs.Coronavirus (CoV) emergence in both humans and livestock represents a significant threat to global public health, as evidenced by the sudden emergence of severe acute respiratory syndrome CoV (SARS-CoV), MERS-CoV, porcine epidemic diarrhea virus, and swine delta CoV in the 21st century. These studies describe an approach that effectively targets the highly conserved 2\u2032 O-methyltransferase (2\u2032O-MTase) that encodes critical functions in immune modulation and infection. Using reverse genetics, we disrupted a key motif in the conserved KDKE motif of Middle East respiratory syndrome CoV (MERS-CoV) NSP16 (D130A) and evaluated the effect on viral infection and pathogenesis. While the absence of 2\u2032O-MTase activity had only a marginal impact on propagation and replication in Vero cells, dNSP16 mutant MERS-CoV demonstrated significant attenuation relative to the control both in primary human airway cell cultures and in vivo. Further examination indicated that dNSP16 mutant MERS-CoV had a type I interferon (IFN)-based attenuation and was partially restored in the absence of molecules of IFN-induced proteins with tetratricopeptide repeats. Importantly, the robust attenuation permitted the use of dNSP16 mutant MERS-CoV as a live attenuated vaccine platform protecting from a challenge with a mouse-adapted MERS-CoV strain. These studies demonstrate the importance of the conserved 2\u2032O-MTase activity for CoV pathogenesis and highlight NSP16 as a conserved universal target for rapid live attenuated vaccine design in an expanding CoV outbreak setting.Coronaviruses (CoVs) encode a mixture of highly conserved and novel genes, as well as genetic elements necessary for infection and pathogenesis, raising the possibility of common targets for attenuation and therapeutic design. In this study, we focused on highly conserved nonstructural protein 16 (NSP16), a viral 2\u2032IMPORTANCE Coronavirus (CoV) emergence in both humans and livestock represents a significant threat to global public health, as evidenced by the sudden emergence of severe acute respiratory syndrome CoV (SARS-CoV), MERS-CoV, porcine epidemic diarrhea virus, and swine delta CoV in the 21st century. These studies describe an approach that effectively targets the highly conserved 2\u2032O-MTase activity of CoVs for attenuation. With clear understanding of the IFN/IFIT (IFN-induced proteins with tetratricopeptide repeats)-based mechanism, NSP16 mutants provide a suitable target for a live attenuated vaccine platform, as well as therapeutic development for both current and future emergent CoV strains. Importantly, other approaches targeting other conserved pan-CoV functions have not yet proven effective against MERS-CoV, illustrating the broad applicability of targeting viral 2\u2032O-MTase function across CoVs. The emergence of Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012 represents the second severe CoV to strike the human population since the beginning of the 21st century . ImportaO-methyltransferase (2\u2032O-MTase), CoV NSP16 has been implicated in the capping of viral RNA and prevention of its recognition by the intracellular sensor MDA5 and antiviral effectors, including members of the IFIT (interferon [IFN]-induced proteins with tetratricopeptide repeats) family (in vitro and in vivo attenuation of both mouse hepatitis virus (MHV) and SARS-CoV family . GeneratSARS-CoV , 10. Thein vivo studies in a MERS-CoV mouse model demonstrated robust attenuation of dNSP16 mutant growth and pathogenesis. Notably, attenuation was both IFN and IFIT1 dependent, providing a clear mechanism for attenuation. Importantly, the dNSP16 mutant also provided robust protection against a lethal MERS-CoV challenge and maintained attenuation in the mouse-adapted backbone. Together, the results illustrate the broad conservation and necessity of NSP16 in CoV pathogenesis and highlight the targeting of this protein as a rapid-response platform for future emergent CoV strains.Using reverse genetics to target residues in the highly conserved active site, we evaluated MERS-CoV infection outcomes in the context of inactive NSP16 (dNSP16). Consistent with previous studies of SARS-CoV , the dNSO-MTase activity , similar to observations with NSP16 mutant SARS-CoV compared with wild-type (WT) SARS-CoV and only modest differences in host induction (zero genes with a logSARS-CoV . HoweverSARS-CoV . Over thin vivo infection by using an adenovirus BALB/c mouse transduction model (Dpp4 at positions 288 and 330 (288-330+/+) conferring efficient WT MERS-CoV infection and growth in mice but no clinical disease CRISPR mice with the WT virus (IFIT1 and IFIT2 gene expression in this attenuation phenotype in previously constructed stable short hairpin RNA (shRNA) knockdown cell lines . Ext. Extin vreatment . In addiDpp4 288-330+/+ mutant mice were vaccinated with dNSP16 mutant MERS-CoV and subsequently challenged with a mouse-adapted MERS-CoV strain . Fo. FoDpp4 V strain . In addiV strain and C. IV strain . TogetheDespite conferring protection in the WT MERS-CoV backbone, it was unclear if the NSP16 mutant would be sufficiently attenuated in a virulent MERS-CoV backbone. To address this question, we inserted the dNSP16 mutation (D130A) into the mouse-adapted MERS-CoV backbone . Followiin vitro and in vivo. Similar to MHV and SARS-CoV at each time point ; fragment 2, EMC:EmuC(+) (NNNNNNGCTCTTCCGCGATGTATGATCCTACTACTAAG) and EMC:E#6(\u2212) (CAACCTCAATACAAGCAGAC)]. The two resulting products were digested with SapI (in boldface) and ligated overnight with T4 DNA ligase. This product was then digested with PpuMI and NsiI and used to replace the region of the EMC E plasmid (puc57) that had been similarly digested. Thereafter, plasmids containing WT and mutant MERS-CoV genome fragments were amplified, excised, ligated, and purified. In vitro transcription reactions were then performed to synthesize full-length genomic RNA, which was transfected into Vero E6 cells. The medium from transfected cells was harvested and used as seed stock for subsequent experiments. Viral mutants were confirmed by sequence analysis prior to use. Synthetic construction of NSP16 mutants was approved by the University of North Carolina Institutional Biosafety Committee.Both WT and mutant viruses were derived from either MERS-CoV EMC or a corresponding mouse-adapted (MA1) infectious clone as previously described . For NSP2 change of >1.5-fold and a false-discovery rate-adjusted P\u00a0value of <0.05 for a given time point.RNA isolation and microarray processing, quality control, and normalization from Calu-3 2B4 cells were carried out as previously described . Differehttps://david.ncifcrf.gov/) was used to acquire functional enrichment results for the genes in each cluster. David output was manually summarized for each cluster. Plots were generated with R.Genes identified as differentially expressed were used to generate clustered expression heat maps. Hierarchical clustering (using Euclidean distance and complete linkage clustering) was used to cluster gene expression according to behavior across experimental conditions. The David online resource , National Institutes of Health. The Institutional Animal Care and Use Committee (IACUC) of the University of North Carolina at Chapel Hill approved the animal study protocols used in this study (IACUC protocols 15-009 and 13-072).+/+ C57BL/6 mice were anesthetized with ketamine and xylazine (in accordance with UNC IACUC guidelines) and intranasally inoculated with a 50-\u00b5l volume containing 106\u00a0PFU of WT MERS-CoV, dNSP16 mutant MERS-CoV, mouse-adapted variants, or PBS (mock inoculation) as indicated in the figure legends. Infected animals were monitored for weight loss, morbidity, and clinical signs of disease, and lung virus titers were determined as described previously or CRISPR-Cas9-targeted 288-330eviously . In vivoescribed . For vacGSE65574.Raw microarray data for these studies were deposited in publicly available databases at the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus and are"} +{"text": "Bordetella pertussis CyaA-hemolysin (CyaA-Hly) domain was previously demonstrated to be an important determinant for hemolysis against target erythrocytes and ion-channel formation in planar lipid bilayers (PLBs). Here, net-charge variations in the pore-lining helix of thirteen related RTX cytolysins including CyaA-Hly were revealed by amino acid sequence alignments, reflecting their different degrees of hemolytic activity. To analyze possible functional effects of net-charge alterations on hemolytic activity and channel formation of CyaA-Hly, specific mutations were made at Gln574 or Glu581 in its pore-lining \u03b13 of which both residues are highly conserved Lys in the three highly active RTX cytolysins . All six constructed CyaA-Hly mutants that were over-expressed in E. coli as 126 kDa His-tagged soluble proteins were successfully purified via immobilized Ni2+-affinity chromatography. Both positive-charge substitutions and negative-charge elimination (E581Q) appeared to increase the kinetics of toxin-induced hemolysis while the substitution with a negatively-charged side-chain (Q574E) completely abolished its hemolytic activity. When incorporated into PLBs under symmetrical conditions , all five mutant toxins with the increased hemolytic activity produced clearly-resolved single channels with higher open probability and longer lifetime than the wild-type toxin, albeit with a half decrease in their maximum conductance. Molecular dynamics simulations for 50 ns of a trimeric CyaA-Hly pore model comprising three \u03b12-loop-\u03b13 transmembrane hairpins revealed a significant role of the positive charge at both target positions in the structural stability and enlarged diameter of the simulated pore. Altogether, our present data have disclosed functional contributions of positively-charged side-chains substituted at positions Gln574 and Glu581 in the pore-lining \u03b13 to the enhanced hemolytic activity and ion-channel opening of CyaA-Hly that actually mimics the highly-active RTX (repeat-in-toxin) cytolysins.The Bordetella pertussis is a causative agent of human whooping cough which has now re-emerged globally as a consequence of pathogen adaptation to vaccination and/or waning protection from acellular pertussis (aP) vaccines \u00d7 100. An equal amount of erythrocytes lysed with 0.1% Triton X-100 was defined as 100% hemolysis. All samples were tested in triplicate for three independent experiments as previously described [The purified toxin (~10 \u03bcg/mL) was assayed with sheep erythrocyte suspension on a 250-\u03bcm aperture in a 1 mL-Delrin cup as previously described [2) were added into both cis and trans compartments containing recording buffer . Single-channel currents were recorded with Geneclamp-500 amplifier and signals (filtered at 10 kHz) were digitized with PCI-6221 analog-to-digital converter using LabVIEW 7.1 software at a 50 kHz sampling frequency. Channel conductances and lifetimes were determined from the observed current steps (~100 open channels) at 100 mV applied voltage.PLBs were formed by painting method 20 mg/mL of 1,2-diphytanoyl-escribed . Toxin ssn-glycero-3-phosphocholine) bilayer using CHARMM GUI server [+ ions and 27 Cl\u2212 ions (at 150 mM KCl) was energetically minimized, equilibrated, and finally ran for 50 ns at temperature 300 K and pressure 1 atm using the NAMD program [\u03b1 atom of residue T time-steps, respectively.A trimeric pore model of CyaA-Hly was constructed by docking three copies of the \u03b12-loop-\u03b13 hairpin using ZDOCK server as previNY, USA) and thenNY, USA) . The fin program and CHAR program on a com program . The dyn"} +{"text": "Open Biology would like to thank all reviewers who have worked on articles published in the journal in 2016. We appreciate that most reviewers value recognition of their efforts by the journal and through services such as Publons, which is partnered with Open Biology.The Editors of Lun ATLHachani ADiwan ABlanchard AQuintana AHergovich AMiserez AMaloyan ACarmena AMusacchio APreiss Avon Philipsborn ACLambeir A-MBoudaoud AQueliconi BBCoutard BLarsen CNorden CPrahalathan CFaherty CSCooper CDOhttp://orcid.org/0000-0002-9197-8041Scheffers D-JHeymann DYoung RCaspari TTromer Ehttp://orcid.org/0000-0003-3540-7727Lou EHuber EMZietkiewicz ECernilogar FMUhlmann FMacintyre GPolese Gvan Wezel GPLillywhite HBhttp://orcid.org/0000-0002-6410-4832Huang H-HDjagaeva IDavie JRTagne J-BChen JYang JZhang Jhttp://orcid.org//0000-0001-8683-509XNilvebrant JAtkins JFHiggins JTatzelt JJimenez JIRood JIWelburn JMotohashi Khttp://orcid.org/0000-0002-8414-2836Hermo LKoziol MJPeres MAGiansanti MGhttp://orcid.org/0000-0002-6753-7262Peifer MWaldor MYun MHShao MLaurent MRSendtner MH\u00e4rk\u00f6nen PDiederichs SKaufman Jhttp://orcid.org/0000-0002-7216-8422Gaur RDas RMStrugnell RAHaltiwanger RSKhochbin SWu SYeh S-DCragg SMAra\u00fajo SJRocha SScholpp SSweet SMEvans SEMitchell TJTanaka TBollag WBYang Xhttp://orcid.org/0000-0002-2537-5752Yoshida KTo provide an opportunity for recognition, we list the names of all reviewers (who opted for inclusion). This article is made permanent and citeable by its digital object identifier (DOI). We encourage you to quote your contribution in your applications for tenure and grants or other forms of research assessment. Where you have provided it, we have included your ORCID so your contribution can be unambiguously assigned to you."} +{"text": "Direct immuno-fluorescence test (IFAT) and multiplex real-time RT-PCR have been central to RSV diagnosis in Kilifi, Kenya. Recently, these two methods showed discrepancies with an increasing number of PCR undetectable RSV-B viruses.Establish if mismatches in the primer and probe binding sites could have reduced real-time RT-PCR sensitivity.Nucleoprotein (N) and glycoprotein (G) genes were sequenced for real-time RT-PCR positive and negative samples. Primer and probe binding regions in N gene were checked for mismatches and phylogenetic analyses done to determine molecular epidemiology of these viruses. New primers and probe were designed and tested on the previously real-time RT-PCR negative samples.N gene sequences revealed 3 different mismatches in the probe target site of PCR negative, IFAT positive viruses. The primers target sites had no mismatches. Phylogenetic analysis of N and G genes showed that real-time RT-PCR positive and negative samples fell into distinct clades. Newly designed primers-probe pair improved detection and recovered previous PCR undetectable viruses.An emerging RSV-B variant is undetectable by a quite widely used real-time RT-PCR assay due to polymorphisms that influence probe hybridization affecting PCR accuracy. The Scientific and Ethical Review Unit, KEMRI, Kenya and the Coventry Research Ethics Committee, UK approved the study protocols for the surveillance and informed consent was sought accordingly. All samples (n\u00a0=\u00a01251) were tested by IFAT and total RNA, for the IFAT positive samples , was extracted by RNeasy 96 QIAcube HT kit (Qiagen) using 140\u00a0\u03bcl of the swab sample, followed by one-step multiplex rRT-PCR . IFAT is used as the gold standard assay for RSV detection. The RSV-B specific primers and probe (Old N assay) used are listed in Fast-track Diagnostics\u00ae (FTD) FLU/HRSV rRT-PCR commercial kit and published primers and probe geneious v.8.1.8 KX775772-KX775940).G and N genes were amplified by one-step RT-PCR (Qiagen) and sequenced (ABI 3700) for all samples that were IFAT positive. G gene amplification PCR is described in 3TC) value >35.0). RT-PCR products were obtained for N and G genes as revealed by gel electrophoresis, despite the lack of detection by real-time RT-PCR. G gene RT-PCR was successful for 46 of the discordant 58 samples: 45 were RSV-B genotype BA, 1 was RSV group A (Fifty-eight samples (22.5%) positive for RSV by the IFAT method were negative by rRT-PCR , 14 (C/A) and 17 (A/G) at the probe target site were unique to the rRT-PCR negative viruses . MajoritTC value of 27.1 (range 19.8\u201334.6). The new primers and probe were also specific to RSV-B in presence of other respiratory viruses and have been shown to detect RSV-B in both singleplex and multiplex reactions.We designed new primers and probe upstream of the old target site within N gene and test4Sensitive detection of RSV is important for molecular epidemiology analyses and for tracking virus transmission. Our RSV-B rRT-PCR primers and probe was adopted from Gunson et al. Newly designed N gene rRT-PCR primers and probe effectively detected the novel RSV-B variant in addition to other circulating RSV-B variants without decreasing assay specificity. Furthermore, re-testing using a commercial and previously published RSV rRT-PCR assays showed positive results; however, these alternative assays do not distinguish RSV-A and RSV-B and they target the matrix gene.Based on phylogenetic analyses, rRT-PCR negative viruses genetically clustered separately from the detectable RSV-B viruses and the clustering does not appear to be random. Our results extend earlier reports and observations Wellcome Trust (grant ref: 102975).This work was funded by the The Scientific and Ethical Review Unit, KEMRI, Kenya and the Coventry Research Ethics Committee, UK approved the study protocols for the surveillance and informed consent was sought accordingly.None declared."} +{"text": "Scientific Reports7: Article number: 4071010.1038/srep40710; published online: 01182017; updated: 04262017Salmonella isolates\u2019, where incorrect primers were quoted.This Article contains errors in the Materials and Methods section under subheading \u2018Prevalence investigation of P1-like bacteriophage in blaCTX-M-27 gene was detected using the following primers: IS-fw (AGAATCATCGC CGAAGGGCTGTAACTGGTTTT) and IS-rev (GCGAACATCATCCGTTGCACT CTCTTTGT)\u201d.\u201cThe presence of the insertion sequence on the other nontypeable plasmids carrying should read:blaCTX-M-27 gene was detected using the following primers: IR-fw-(GTTGCTGGCTGACGCCTATGAAG) and IR-rev-(ATGTTTGCCATTTCATAGGGGAG)\u201d.\u201cThe presence of the insertion sequence on the other nontypeable plasmids carrying"} +{"text": "Preoperative chemoradiotherapy (pre-CRT) has been represented as the standard treatment for locally advanced rectal cancer (LARC), but large variations of tumor radiation response to CRT have been reported in the clinic. To explore the function of microRNAs as potential therapeutic predictors of pre-CRT pathological response in LARC, we analyzed global miRNA expression in CRT-sensitive and CRT-resistant groups before treatment. MiR-345 was significantly elevated in the CRT-resistant group. Therefore, miR-345 was selected as a candidate for further analysis. We assessed the correlation between the miRNA signatures and the chemoradiotherapeutic response in 20 randomly selected LARC tissue samples and 87 serum samples (Training set) by qRT-PCR. Further, we validated the results in 42 randomly selected LARC serum samples . High miR-345 expression was significantly correlated with unfavorable pre-CRT pathological response in tissue and serum. Moreover, low miR-345 levels predicted superior 3-year local recurrence free survival (LRFS). Taken together, circulating serum miR-345 correlates with unfavorable pre-CRT response and poor locoregional control in LARC. It might be a promising biomarker to facilitate patient stratification for personalized treatment. Preoperative chemoradiotherapy (pre-CRT) followed by total mesorectal excision (TME) has been recommended as the standard care for patients with locally advanced rectal cancer (LARC). Patients who undergo pre-CRT achieve higher rates of R0 resection, anal sphincter preservation and lower local recurrence rates , 2. NeveMicroRNAs (miRNAs) are small, highly conserved, non-coding sequences 18\u201325 nucleotides in length and that act as post-transcriptional regulators in tumorigenesis and development . MiRNAs To address this issue, we performed a global miRNA analysis in CRT-sensitive and CRT-resistant patients. MiR-345 was significantly elevated in CRT-resistance patients. As a potential candidate biomarker, subsequently, we investigated and validated whether miR-345 in serum correlated with unfavorable pathological response and poor prognosis in LARC before CRT initiation.A total of 149 LARC patients were enrolled in this study. All patients were treated with concurrent CRT with doses of 50-50.4 Gy. Most patients were male (70%) and moderate differentiation (48%). The clinical stages were relatively balanced (44% stage II and 56% stage III). The clinical characteristics of the tissue validation set, serum training set, and serum validation set were summarized in Table Pathological response was evaluated according to Mandard criteria . TRG 1 sP<0.05) and CRT-resistant group (TRG 4) Table . Of thes5) Table . NotablyGiven the low incidence of TRG 1 in the entire cohort, in all three tissue validation sets, the serum training and validation sets, the radiosensitive group was defined as TRG 1/2 and the radioresistant group was defined as TRG 3/4.P=0.046).To validate miRNA array data, we examined miR-345 expression using qRT-PCR in 20 randomly selected LARC tissues . MiR-345 expression was consistent with miRNA array data. As shown in Figure KRAS mutant population in CRC [P=0.002). Receiver operating characteristic (ROC) analysis of miR-345 expression yielded an area under the curve (AUC) value of 0.69 to distinguish CRT-sensitivity from resistance . ROC analysis of miR-345 expression yielded an AUC value=0.75 . As expected, miR-345 expression was significantly down-regulated in the CRT-sensitive group as compared to the CRT-resistant group (P=0.002), but was not associated with 3-year DFS. This result indicated that circulating serum miR-345 correlates with unfavorable pathological response to pre-CRT in LARC.To further evaluate whether serum miR-345 levels can serve as a predictor of patient outcome, we performed Kaplan-Meier survival analysis. As shown in Figure In this study, we preformed miRNA array to screen chemoradiosensity-related miRNAs from LARC tissue specimens. First, we found that the circulating serum miR-345 predicted the pathological response to pre-CRT. The low miR-345 expression in serum appeared to correlate with favorable local control in LARC. Serum miRNA signature could be obtained before initiating CRT, thus miR-345was probably used as a noninvasive predictive biomarker for prescribing a personalized treatment strategy in LARC.Previous studies have revealed that patients with LARC who underwent pathological down-staging or pCR after pre-CRT achieved more favorable outcomes \u201321. RegaTo accurately select radiosensitive patients, conventional examinations such as magnetic resonance imaging (MRI) or positron emission tomography/computed tomography were used to differentiate good radiotherapy responders from the poor responders \u201324. HoweMiRNAs function as negative regulators of their target genes , 29. CurMiR-345 was first reported to be highly expressed in malignant mesothelioma tissues . SubsequRecent studies have showed that serum miR-345 served as a strong adverse prognostic factor in metastatic CRC after adjusting for sex, age, KRAS, PI3KCA and performance status. Patients with high miR-345 expression were 1.75 and 1.63 times more likely to develop mortality and progression risk, and high expression was associated with poor response to chemotherapy and targeted therapy (cetuximab combined with irinotecan) , which wBased on our microarray analysis, miR-345 expression was significantly elevated in the CRT-resistant group, and had been identified as a circulating biomarker in our pre-experiment and by other researchers . As our Two recent studies confirmed that post-CRT diffusion-weighted (DW) MRI volumetry and volume reduction (\u0394 volume) after pre-CRT provided high and accurate diagnostic performance in assessing the good radiation response of pCR , 39. We Previous studies showed that P21, BCL2-associated athanogene 3 (BAG3) and BCL2 were confirmed to be the targets of miR-345. Shiu et al. indicated that miR-345 could directly down-regulate the crucial tumor suppressor P21 to facilitate the hepatocarcinogenesis under the chronic HCV infection . BAG3, aThis study included 149 patients with previously untreated and histologically confirmed rectal adenocarcinoma from 2006 to 2015 at the Chinese Academy of Medical Sciences Cancer Hospital. All patients underwent standard pre-CRT plus TME. Surgery was scheduled 6\u20138 weeks after pre-CRT, and adjuvant chemotherapy was administered according to postoperative pathology diagnosis. This study was designed as an initial screening phase and a subsequent validation phase. For screening, we characterized the miRNA expression profiles of three neoadjuvant CRT-sensitive and three neoadjuvant CRT-resistant LARC samples using miRNA array. Pathological response was evaluated according to TRG as described by Mandard . ConsideThis study protocol has been reviewed and approved by the Chinese Academy of Medical Sciences Cancer Hospital ethics committee. All participants provided written informed consent.The pre-therapeutic biopsies from patients with LARC were stored in liquid nitrogen, and then subjected to miRNA array analysis at CapitalBio company using Affymetrix GeneChip miRNA 4.0 Arrays containing 761 miRNAs.Caenorhabditis elegans miR-39 was added to the serum prior to RNA extraction as the internal control. U6 small nuclear RNA was used as the internal control for the tissue samples. All reactions were run in triplicate, and miRNA expression was quantified using the comparative threshold cycle (2\u2212\u0394Ct) method [Tissue and serum samples were collected from patients before pre-CRT. Tissue RNA was isolated using TRIzol\u00ae reagent . MiRNA extraction from 200 \u03bcl serum was performed with miRNeasy RNA isolation Kits according to the manufacturer's instructions . For qRT-PCR, total 1 \u03bcg RNA was reverse-transcribed with Bulge-Loop miRNA-specific reverse transcription primers using a miScript II RT Kit . The miR-345 looped RT primer sequence was 5\u2032-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGGAGCCCTG-3\u2032; the miR-345 PCR primer sequence was 5\u2032-ACACTCCATCTGGGGCTGACTCCTAGTCCA-3\u2032. The qRT-PCR was performed using a miScript SYBR Green PCR kit . Synthetic spiked-in ) method .Statistical analysis was performed using SPSS version 19.0 . Normal distribution of data was verified using the Kolmogorov-Smirnov test. The Mann-Whitney U test was used to analyze the different miRNA expression between the CRT-sensitive and CRT-resistant groups in the tissue validation set, serum training set and validation set. ROC curves were generated to evaluate the diagnostic performance in differentiating the CRT-sensitive from CRT-resistant samples. DFS was measured by the date of initial treatment to the date of disease recurrence. LRFS was evaluated from the date of surgery until the date of local or regional lymph node recurrence (or last follow-up). Survival was calculated using the Kaplan-Meier method, and compared using the log-rank test. P<0.05 was considered significant."} +{"text": "We present three patients with bronchial carcinoids, in which a more probed study emphasized the presence of three multiple endocrine neoplasia (MEN). Assessment included a total-body computerized tomography, a total-body single-photon emission computerized tomography by In-DTPA-D-Phe octreotide, and genetic map. Two patients presented an atypical MEN 1 and one patient showed an atypical MEN 1 with a familial medullary thyroid carcinoma. All patients were operated upon: two are still alive and one died 50 months after the first intervention. Precocious diagnosis of MEN permits a good long-term outcome."} +{"text": "CSF abnormalities and a neuroinflammatory pathophysiology have been discussed for affective and non-affective psychosis for more than 30-years. Recent studies pointed towards a specific phenotype of autoimmune-antibody mediated psychosis, but evidence is still sparse. Especially CSF data investigating autoimmune antibodies in large-scale CSF cohorts of affective and non-affective psychoses are lacking.We analyzed a retrospective naturalistic cohort of 592 patients with A) schizophrenia-spectrum disorders (N = 330) or B) depressive disorders (N = 262) who underwent a lumbar puncture as part of the clinical routine in the Department of Psychiatry and Psychotherapy at the Ludwig-Maximilians University Munich between July 2012 and May 2017. We used a predefined systematic algorithm for the database search in the clinical documentation system and data was extracted by TO and AG. The study was approved by the local ethics committee.We identified 592 patients with standard CSF parameters. Schizophrenia spectrum patients did not differ from depressive patients with regard to the white blood cell count (cells/\u00b5l) (p = 0.774) or albumin quotient (p = 0.663). The general prevalence of oligoclonal bands did not differ between groups . However, schizophrenia patients showed higher frequencies for intrathecal oligoclonal bands compared to depressive patients . 124 schizophrenia-spectrum patients (54 first-episode patients) received CSF analyses for neural antibodies. None of the patients showed positive CSF results in any of the tested autoimmune-encephalitis panel (NMDA(N=119), AMPA-1(N=114), AMPA-2(N=114), CASPR(N=111), LGI-1(N=110) and GABA-B(N=112)-Antibodies) in CSF. The results for the intracellular onconeuronal and synaptic antibodies were also negative (Amphyphysin(N=93), Yo(N=58), Hu(N=94), Ri(N=94), CV2(N=93), Ma1(N=93) and Ma2(N=93)-Antibodies). Three of these patients with negative CSF titers did have low-titer neuronal antibodies in serum: CASPR-2-AB: 1:10, CASPR-2-AB: 1:50, Yo-AB: low band-intensity. 36 depression patients were also tested for autoimmune antibodies and again no positive reports could be identified in CSF.This is the first analyses of autoimmune antibodies in first-episode and recurrent schizophrenia and depressive mood disorder showing no positive CSF titers. However, schizophrenia patients have a higher prevalence of intrathecal oligoclonal bands compared to affective patients pointing towards more immunological disturbances in this population. The here presented analyses are exploratory and need to undergo confirmatory analyses and quality control."} +{"text": "Aegilops tauschii sometimes show abnormal growth phenotypes, and the growth abnormalities inhibit generation of wheat synthetic hexaploids. In type II necrosis, one of the growth abnormalities, necrotic cell death accompanied by marked growth repression occurs only under low temperature conditions. At normal temperature, the type II necrosis lines show grass-clump dwarfism with no necrotic symptoms, excess tillers, severe dwarfism and delayed flowering. Here, we report comparative expression analyses to elucidate the molecular mechanisms of the temperature-dependent phenotypic plasticity in the triploid wheat hybrids. We compared gene and small RNA expression profiles in crown tissues to characterize the temperature-dependent phenotypic plasticity. No up-regulation of defense-related genes was observed under the normal temperature, and down-regulation of wheat APETALA1-like MADS-box genes, considered to act as flowering promoters, was found in the grass-clump dwarf lines. Some microRNAs, including miR156, were up-regulated, whereas the levels of transcripts of the miR156 target genes SPLs, known to inhibit tiller and branch number, were reduced in crown tissues of the grass-clump dwarf lines at the normal temperature. Unusual expression of the miR156/SPLs module could explain the grass-clump dwarf phenotype. Dramatic alteration of gene expression profiles, including miRNA levels, in crown tissues is associated with the temperature-dependent phenotypic plasticity in type II necrosis/grass-clump dwarf wheat hybrids.Triploid wheat hybrids between tetraploid wheat and Arabidopsis and other plant species of 5%) probes were extracted and compared. Based on the GO term enrichment analysis , the differentially encountered probes were input for biological processes (level 3).BLASTx searches conditions for 8 weeks using Sepasol-RNA I Super G solution . For each sample, crown tissues of at least two independent plants were bulked with no biological replications. Small RNA libraries were prepared using a TruSeq Small RNA Library Preparation Kit and RNA 3\u2019 adapter and RNA 5\u2019 adapter were respectively added using truncated T4 RNA ligase 2 and T4 RNA ligase 1 (New England BioLabs). cDNA was synthetized using the 3\u2019 adapter-recognizing primer, and after PCR, products of around 150 bp were selected. Single-end sequencing was performed with a TruSeq SBS Kit v3-HS (Illumina) on a HiSeq2000 platform (Illumina) according to the manufacturer\u2019s instructions. Files containing raw sequence data were deposited in the sequence read archive of DDBJ (accession number DRA004554).http://www.repeatmasker.org).From the raw sequence reads, stop oligonucleotides, binding to the 3' adapter to prevent further ligation, were removed and then adapter sequences were trimmed . The trihttp://sourceforge.net/projects/mireap) with the following parameters: minimal miRNA length of 18 bp and maximal of 30 bp, minimal reference miRNA length of 18 bp and maximal of 26 bp, maximal space between miRNA and miRNA* of 400 bp, maximal bulge of miRNA and miRNA* of 3 bp, flank sequence length of miRNA precursor of 20 bp and maximal free energy allowed for a miRNA precursor of -20. The obtained miRNAs were BLASTn searched against the miRBase 19.0 plant miRNA database [The aligned data were used to predict novel miRNAs with mireap version 0.2 version 12 and Triticum aestivum cDNA, Ensembl Plants, release-31. Targets with maximum expectation values > 2 were discarded. Unannotated genes were BLASTx searched against the NCBI non-redundant protein database .For miRNA target prediction, psRNATarget (2017 Update) were use20 primer. The accumulation of each gene transcript was detected by qRT-PCR using a LightCycler 480 Real-Time PCR System with the gene-specific primer sets listed in Actin gene was used as an internal control. The rate of amplification was monitored using THUNDERBIRD SYBR qPCR mix according to the manufacturer\u2019s protocol. The relative expression level was calculated as 2-\u0394\u0394Ct of three technical replicates [Actin.For qRT-PCR, each plant was grown at 23\u00b0C for 3 weeks and total RNA was extracted from the crown tissues using Sepasol-RNA I . Crown tissues of at least two independent plants were bulked for each synthetic hexaploid line. First-strand cDNA was synthesized from DNase I-treated RNA samples using ReverTra Ace Reverse Transcriptase and an oligo(dT)plicates , where \u03942 individuals of the Ldn/KU-2075//Ldn/KU-2025 population. Bulked RNA samples of each Net2 genotype were extracted from the crown tissues of five F2 individuals. The accumulation of each miRNA was detected by qRT-PCR using the first-strand cDNA samples synthesized by a Mir-X miRNA First-Strand Synthesis Kit . qRT-PCR was conducted using SYBR Advantage premix (BD Biosciences Clontech) with a miRNA-specific primer and mRQ3\u2019 adapter primer. The miRNA-specific primers for qRT-PCR are listed in -\u0394\u0394Ct of three technical replicates.For validation of the first screening of miRNA assocaited with grass-clump dwarfism via small RNA sequencing, we conducted qRT-PCR of miRNAs. Total RNA was extracted from crown tissues of the synthetic hexaploid lines and FSQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) and MADS-box genes were based on recent reports [http://rice.tigr.org/), Arabidopsis , barley and wheat , and the deduced amino acid sequences were predicted by ORF Finder (http://www.ncbi.nlm.nih.gov/projects/gorf/gorf.html). Conserved domains of the amino acids were confirmed using InterProScan (http://www.ebi.ac.uk/Tools/pfa/iprscan/). Multiple sequence alignments of the amino acid sequences were carried out using the ClustalW computer program of MEGA ver. 5.05 software [The cDNA sequences of reports \u201352, and software , and a pSPL sequences, gene-specific primers were designed for determining the 5\u2019 ends of the wheat SPL cDNAs, and rapid amplification of cDNA ends (RACE)-PCR was conducted using a BD SMART RACE cDNA Amplification Kit (BD Biosciences Clontech). Each amplified PCR product was cloned into vector pMD20-T using Mighty TA-cloning Reagent Set for PrimeSTAR (Takara Bio). The nucleotide sequences of the 5\u2019-RACE PCR products were determined using a Big Dye terminator cycle sequencing kit and an Applied Biosystems 3730xl DNA Analyzer. The nucleotide sequences were analyzed using GENETYX-MAC version 12.00 software .Based on the wheat R = 0.651, P<0.001) was detected (To comprehensively compare gene expression profiles between WT (Ldn/KU-2059) and two type II necrosis lines (Ldn/KU-2025 and crt/KU-2025) under normal temperature, we analyzed their transcriptomes using a wheat-specific 38k oligo DNA microarray . After hdetected .P<0.001) between crown tissues of the Ldn/KU-2025 and crt/KU-2025 lines of differences in signal intensity of the 37,826 probes under normal temperature relative to that under LT were observed between the WT synthetic line Ldn/KU-2059 and type II necrosis line Ldn/KU-2025 (P<0.001) correlated with those under normal temperature. These observations indicated that the similar set of genes was transcriptionally altered by LT between WT and type II necrosis. On the other hand, differences in the probe signal intensity between the two growth conditions were negatively (P<0.001) correlated with those between Ldn/KU-2059 and Ldn/KU-2025. Thus, changes of gene expression levels via the growth temperatures were inconsistent with those via development of the growth abnormalities, and expression of some genes was specifically changed in the type II necrosis line in response to the growth temperatures.Correlation of the logus study and comp/KU-2025 . Out of /KU-2025 . Five /FRUITFULL (FUL), controls the vernalization requirement and acts as a flowering promoter [Vrn-A1-derived probe (wheat0130contig14238) showed 0.064 of the signal ratio relative to Ldn/KU-2059 in the microarray analysis. The signal ratios of two wheat Vrn-1 homologs, TaAP1-2 (rwhfl48m16) and TaAP1-3 (wheat0130contig14728), were respectively 0.011 and 0.408 in the crown tissues of Ldn/KU-2025. Their rice orthologs OsMADS18 and OsMADS15 play important roles in the promotion of flowering [AP1/FUL-like MADS-box genes. Transcript accumulation of the MADS-box genes acting as flowering promoters was markedly decreased in the crown tissues of Ldn/KU-2025 and crt/KU-2025 compared with Ldn/KU-2059 showed similar ratios of expression under LT to normal temperature for Ldn/KU-2025 and Ldn/KU-2059 and a KU-2075/KU-2025 heterozygote at the Net2 chromosomal region. These were predicted to have differentially expressed miRNAs in the crown tissues between Ldn/KU-2059 and Ldn/KU-2025 . Two samples of the heterozygous bulks more abundantly accumulated the four miRNAs than samples of the KU-2075-allele homozygous bulks were down-regulated in crown tissues of crt/KU-2025 under the normal temperature condition (SPL (TaSPL) cDNAs, and the putative amino acid sequences of TaSPLs were compared with those of Arabidopsis and rice SPLs mRNA, 5\u2019-RACE PCR with a TaSPL-specific primer was performed using the crown tissue-derived RNA of Ldn/KU-2059. One of six 5\u2019-ends of the TaSPL mRNA was found in the target region of tae-miR156 molecules.bidopsis ,50,63. Ion wheat ,65. Our ondition . We survice SPLs . Based or TaSPLs . Under n/KU-2159 . Under LAe. tauschii, and 22 of our 122 Ae. tauschii accessions display the type II necrosis phenotype in ABD triploids in the winter season [AP1/FUL-type MADS-box genes, the orthologs of which act as flowering promoters in rice [AP1/FUL-type MADS-box genes could explain at least partly two of the characteristics of the temperature-dependent phenotypic plasticity in the type II necrosis lines.Type II necrosis is one of the major hybrid incompatibilities between tetraploid wheat and r season . As prevr season , necrotir season ,18. Ther in rice ,59, was SPLs, direct targets of miR156 [TaSPL mRNA [SPLs and in morphological changes including dwarfism, increased tiller number and late flowering in maize, switchgrass and rice [TaSPLs could be direct targets of miR156 in the crown tissues of wheat. Thus, the excess tiller numbers and dwarfism could be caused by the increased miR156 expression and repressed TaSPL expression in the grass-clump dwarf lines. Moreover, the miR156/SPLs module also controls flowering time in Arabidopsis. miR156 molecules decrease the expression of miR172 through the cleavage of SPL transcripts, and miR172 directly down-regulates APETALA2-like genes TOE1 and TOE2 to promote the transition from vegetative to reproductive phase [Arabidopsis SPLs directly activate flowering promoting the MADS-box genes FUL and SOC1, and miR156 molecules negatively regulate the MADS-box genes through the cleavage of SPL transcripts [AP1/FUL-type MADS-box genes could be due to the increased miR156 expression.Information on molecular mechanisms underlying the other two characteristics of the grass-clump dwarf phenotype was obtained from deep-sequencing analysis of small RNAs. Accumulation of miR156 molecules was altered in response to the growth temperature in the crown tissues of wheat synthetic hexaploids . LT treaf miR156 ,50,66, wSPL mRNA ,65. In aand rice ,66,67, wand rice . The preve phase . Therefonscripts . TherefoSPLs module could at least partly explain the three characteristics of dwarfism, excess tillers and late flowering under normal temperature.Evolution of allopolyploids is accompanied by changes in genome organization and gene expression patterns . As receSPLs module is at least partly associated with the grass-clump dwarf phenotype in the type II necrosis lines of synthetic hexaploid wheat. Namely, the Net1-Net2 interaction regulates the temperature-dependent phenotypic plasticity through the miR156/SPLs module in the wheat crown tissues. With Net1-Net2 interaction, accumulation of some wheat miRNAs including miR156 showed unexpected alterations compared with the WT lines in the wheat ABD hybrids and synthetic hexaploids Click here for additional data file.S2 FigP<0.001). The regression lines are also shown.Scatter plots of differential signal intensities in crown tissues of the grass-clump dwarf lines at the normal temperature are represented for the six indicated categories of probes. The correlations were significant (***(PDF)Click here for additional data file.S3 FigSPL genes contain the miR156-target site, and their transcripts are directly cleaved by miR156 in Arabidopsis and rice.Recent reports have shown ,63 that (PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file."} +{"text": "PRTN3 gene transcription. MicroRNA-941 (miR-941) has been shown to target KDM6B mRNA and inhibit JMJD3 production. We therefore investigated whether polymorphonuclear granulocytes (PMNs) from patients suffering from granulomatosis with polyangiitis (GPA) have lower expression of miR-941 than healthy control donors as a biological cause for higher JMJD3 levels. We found no significant difference in the degree of maturation of PMNs from GPA patients (n = 8) and healthy controls (n = 11) as determined from cell surface expression of the neutrophil maturation marker CD16 and gene expression profile of FCGR3B. The expression of PRTN3 and KDM6B mRNAs and miR-941 was not significantly different in GPA patients and healthy controls. Transfection of pre-miR-941 into the neutrophil promyelocyte cell line PLB-985 cells did not result in reduction of the KDM6B mRNA level as shown previously in a hepatocellular carcinoma cell line. The amount of PR3 in PMNs from GPA patients and healthy controls was comparable. In conclusion, we found that PRTN3 mRNA, KDM6B mRNA, and miR-941 expression levels in PMNs do not differ between GPA patients and healthy controls, and that miR-941 does not uniformly regulate KDM6B mRNA levels by inducing degradation of the transcript. Thus, decreased miR-941 expression in PMNs cannot be part of the pathogenesis of GPA.Jumonji Domain-Containing Protein 3 (JMJD3)/lysine demethylase 6B (KDM6B) is an epigenetic modulator that removes repressive histone marks on genes. Expression of KDM6B mRNA is elevated in leukocytes from patients with ANCA-associated vasculitis (AAV) and has been suggested to be the reason for higher proteinase 3 (PR3) mRNA expression in these cells due to derepression of Granulomatosis with polyangiitis (GPA), formerly known as Wegener\u2019s granulomatosis, is an anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). GPA is a granulomatous inflammation involving the respiratory tract and a necrotizing vasculitis that affects small- to medium-sized blood vessels. Necrotizing glomerulonephritis is common in GPA . The majMicroRNAs (miRNAs) are small (~22 nt), non-coding RNAs that play a major role in many cellular processes such as differentiation and proliferation ,6. miRNA3 on the PRTN3 gene by JMJD3 was the reason for higher PRTN3 mRNA expression [It has been shown that there is a higher expression of the transcripts for PR3 and the epigenetic regulator JMJD3 in total leukocytes from AAV patients compared to healthy controls and it was hypothesized that removal of the inhibitory epigenetic mark H3K27mepression . Concomipression . Based opression .Bone marrow aspirates and peripheral blood samples from patients and healthy controls (HC) were obtained after informed and written consent according to permissions H-1-2011-65 and H-2-2009-103 in compliance with the Helsinki Declaration and guidelines from the local ethics committee of the Capital Region of Denmark.GPA patients (n = 8) referred to the Department of Rheumatology, Rigshospitalet, University of Copenhagen, were included in the study based on clinical presentation and recognizable active disease confirmed by Birmingham Vasculitis Activity Score (BVAS). Healthy control donors (n = 11) were staff members.\u2122 (Axis-Shield). Monocytes were purified from the top layer by immunomagnetic cell sorting (MACS\u2122 (Miltenyi)), using murine anti-CD14 antibodies (eBioscience 14-0149-82) and bead-labeled rat-anti-mouse antibodies (MACS 130-047-101). Residual erythrocytes in the granulocyte cell pellet (after density centrifugation) were destroyed by hypotonic lysis. Eosinophils were removed by MACS using anti-CD49d antibodies and bead-labeled rat-anti-mouse antibodies (MACS 130-047-101). Purity of the isolated neutrophils and their stage of maturation was evaluated by inspection of May-Gr\u00fcnwald-Giemsa (MGG) stained cytospins and expression of maturation markers by flow cytometric analysis and quantitative real-time PCR (qRT-PCR).Granulocytes were isolated from peripheral blood as described in . BrieflyExpression of PRTN3 mRNA, KDM6B mRNA, and miR-941 was examined in neutrophil precursors from human bone marrow aspirates isolated by density centrifugation on a Percoll gradient followed by immunomagnetic depletion of non-neutrophil cells as described in . The thrFlow cytometry was carried out on a FACS-Calibur (BD Biosciences) followed by data analysis on Cell Quest (BD Biosciences) and FlowJo software. Cells were stained with a FITC-labeled antibody against CD16 (555407) using mouse-anti-human IgG1\u043a (555749) as isotype control (both BD Biosciences).6 PLB-985 cells was performed according to the manufacturers recommendations (AMAXA) using program T-019 with 30 pmole pre-miR-941 (PM 12273), pre-miR negative control #1 (AM17110), siRNA against KDM6B (Silencer Select P/N 4392420), or siRNA negative control #1 (Silencer Select P/N 4390843), respectively\u2014all from Applied Biosystems. Transfections were done in triplicate. Cells were analyzed 24 hours post-transfection.PLB-985 cells (DSMZ ACC-139) were grown in RPMI 1640 (Gibco BRL) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco BRL) and 1% 100 U/ml penicillin and 100 \u03bcg/ml streptomycin (P/S) (Gibco BRL). Electroporation of 2 x 10\u00ae Reagent (Invitrogen) according to the manufacturer\u2019s instructions. qRT-PCR was performed on an MX 3000P real-time PCR system (Stratagene) as described earlier [RNA was extracted using TRIzol earlier with Taq5 cells analyzed on a 4\u201312% NuPage Bis-Tris gradient gel (Invitrogen). Antibodies: Rabbit anti-human proteinase 3 succeeded by HRP-conjugated goat-anti-rabbit . The membrane was stripped and reprobed with goat-anti-human-\u03b2-actin succeeded by HRP-conjugated rabbit-anti-goat . The membrane was developed by chemiluminiscence using SuperSignal West Pico (Pierce) and analyzed on a Bio-Rad Chemidoc (Bio-Rad). Quantification of band intensities was performed with ImageLab software (Bio-Rad).Western blotting was performed as described in . PurifieGene expression profiles for PR-3-ANCA samples (datasetMean fluorescence intensity (MFI) data were analyzed by Mann-Whitney U-test. For qRT-PCR data, unpaired Student\u00b4s t-test was applied on \u0394Ct values as they meet the requirements for parametric analysis. The relative expression is shown in graphs together with medians and interquartile ranges. PRTN3 intensity on Western blot is analyzed by Mann-Whitney U-test. Significance level was set to 0.05, and statistical analysis was performed using GraphPad Prism 5.0 (GraphPad Software).In a previous study, we examined the miRNA expression profile during normal granulopoiesis and found by miRNA microarray analysis that miR-941 was expressed at low levels in the most immature precursors myeloblasts/promyelocytes (MB&PM), reaching a high expression level in the myelocytes/metamyelocytes (MC&MM), i.e. at the stage where cell proliferation ceases and terminal differentiation commences, followed by an intermediate expression level in band cells/segmented cells (BC&SC) and PMNs Fig 1)..Fig 1). www.targetscan.org), the epigenetic regulator JMJD3/KDM6B is predicted as a miR-941 target. In a recent publication, it was shown that the mean expression level of KDM6B mRNA is almost two-fold higher in total leukocytes from AAV patients compared to healthy controls, whilst there is an almost twentyfold increase in mean PRTN3 mRNA expression level in total leukocytes from AAV patients compared to healthy controls (HCs) [PRTN3 promoter through demethylation of the repressive epigenetic mark histone 3 lysine 27 (H3K27), thereby causing higher PR3 expression in mature neutrophils of AAV than in healthy individuals. Consequently, more PR3 may be expressed on the PMN membrane and trigger development of AAV [Using the online miRNA-target prediction software TargetScan , late promyelocytes (late_PM), myelocytes (MY), metamyelocytes (MM), band cells (BC), PMNs, and monocytes (Mono). Expression data are from the HemaExplorer database demonstr(TIF)Click here for additional data file.S2 FigGenes found to be differentially expressed in specific populations of cells and expression levels in these. Cell populations represented are: MY: myelocytes, MM: metamyelocytes, BC: band cells, PMN: polymorphonuclear granulocytes, Mono: monocytes, PR3: PR3-positive GPA leukocytes.(TIF)Click here for additional data file.S3 Fig(A) five healthy controls (HC) and (B) two patients with granulomatosis with polyangiitis (GPA). PRTN3 expression is shown relative to the expression in TL, which is given the value 1. Error bars are mean \u00b1 SD.Relative expression of PRTN3 mRNA in total leukocytes (TL), monocytes (MC), and PMNs (PMN) from peripheral blood of (TIF)Click here for additional data file."} +{"text": "Electrocardiogram (ECG)-gated breath-hold cardiac cine magnetic resonance imaging (MRI) is generally accepted as the gold standard for left-ventricular (LV) volume assessment. However, it may fail in patients with arrhythmia, impaired breath-hold capacity, and poor ECG gating. Recently, sparse real-time (RT) cine using a prototype sequence with sparse sampling and iterative reconstruction has been proposed to accelerate cine MRI . The purpose of this study was to evaluate the diagnostic quality and accuracy of sparse free-breathing (FB) RT cine MRI for the quantification of LV function compared with standard multi-breath-hold cine MRI.3). The image quality, ejection fraction (EF), end-diastolic volume (EDV), end-systolic volume (ESV), stroke volume (SV), and LV mass for sparse FB RT cine and standard cine were compared.50 patients underwent both standard segmented cine MRI (Acc. factor 3) and sparse FB RT cine with a prototype sequence using sparse sampling and iterative reconstruction (acc. factor 12.8) on a clinical 3T MRI scanner . The cine images were obtained in a stack of 8 short-axis slices spanning the entire LV from base to apex ; EDV ; ESV ; SV ; LV mass . The intra-observer and inter-observer agreement for all parameters was good.Sparse FB RT cine MRI evaluates LV function with good accuracy compared with conventional multi-breath-hold cine MRI. For patients with impaired breath-hold capacity, FB RT cine MRI may be clinically useful for quantitative assessment of LV function."} +{"text": "MicroRNAs (miRNAs) are small noncoding RNAs that modulate gene expression at the post-transcriptional level. Different types of cells express unique sets of miRNAs that can be exploited as potential molecular markers to identify specific cell types. Among the variety of miRNA detection methods, a fluorescence-based imaging system that utilises a fluorescent-reporter gene regulated by a target miRNA offers a major advantage for long-term tracking of the miRNA in living cells. In this study, we developed a novel fluorescence-based miRNA-monitoring system using a non-integrating cytoplasmic RNA vector based on a replication-defective and persistent Sendai virus (SeVdp). Because SeVdp vectors robustly and stably express transgenes, this system enabled sensitive monitoring of miRNAs by fluorescence microscopy. By applying this system for cellular reprogramming, we found that miR-124, but not miR-9, was significantly upregulated during direct neuronal conversion. Additionally, we were able to isolate integration-free human induced pluripotent stem cells by long-term tracking of let-7 expression. Notably, this system was easily expandable to allow detection of multiple miRNAs separately and simultaneously. Our findings provide insight into a powerful tool for evaluating miRNA expression during the cellular reprogramming process and for isolating reprogrammed cells potentially useful for medical applications. Many miRNAs are evolutionarily conserved among different organisms and play critical roles in controlling various biological processes, including metabolism, proliferation, and differentiation3. Comprehensive expression analyses show that different types of cells express unique sets of miRNAs5. Importantly, some miRNAs are highly abundant in specific cell types and in many cases are directly involved in determining cell identity5.MicroRNAs (miRNAs) are a class of small noncoding RNAs that act as post-transcriptional regulators of gene expression8. Stem cells have the ability to self-renew and differentiate into various types of committed tissue-specific cells. Thus far, numerous protocols for the in vitro differentiation of stem cells have been explored to develop potential cell sources for clinical applications, including transplantation therapy9. Recently, cellular reprogramming, through which forced expression of defined factors induces cell-fate conversion, has been extensively studied to obtain human induced pluripotent stem cells (hiPSCs) or desired tissue-specific cells11. However, currently available protocols often produce cell mixtures exhibiting various stages of differentiation. Therefore, specific molecular markers are commonly used to isolate target cells from highly heterogeneous cell populations. Although cell-specific proteins are widely used in this context, recent studies demonstrated that miRNAs can also serve as unambiguous molecular markers12. miR-302a, miR-122, and miR-208a can be used to specifically identify embryonic stem cells (ESCs), hepatocytes, and cardiomyocytes, respectively13, and, interestingly, miR-375 can be used as a marker for isolation of insulin-producing cells lacking available specific surface markers8.Because of their unique expression profiles, miRNAs have been suggested to serve as potential molecular markers in stem cell research14, and in situ hybridisation and molecular beacons can visualise miRNA expression in cultured cells or in vivo14. However, the transient nature of these approaches is unlikely to be suitable for tracking miRNAs over time during differentiation or cellular reprogramming processes. By contrast, a fluorescence-based imaging system containing a fluorescent reporter gene harbouring multiple binding sites for an miRNA of interest in its 3\u2032 untranslated region (UTR) offers a major advantage for tracking of miRNAs in living cells14. Because binding of miRNA to its target sequences results in inhibition of reporter synthesis, miRNA expression can be readily evaluated based on the decrease in fluorescence intensity. Furthermore, systems based on chromosomal-integrative vectors can stably express reporter genes, thereby allowing long-term monitoring of miRNAs16. However, the activities of cellular and viral promoters, which are commonly used to drive reporter gene expression, vary considerably depending on cell type, genomic context, and epigenetic control20. Such characteristics might impede sensitive and stable monitoring of miRNAs during cell differentiation and reprogramming processes. Additionally, although these systems have been successfully applied for isolation of hiPSCs after somatic cell reprogramming22, chromosomal integration of reporter genes is a critical disadvantage for the safe use of hiPSC-derived cells for clinical applications.With the identification of miRNAs as novel molecular markers, a sophisticated system for detecting intracellular miRNAs is in high demand. To examine miRNA expression, northern blots, microarrays, and reverse-transcription PCR (RT-PCR) are broadly exploited as standard techniques23. In contrast to typical cellular and viral promoters, transgene expression mediated by SeVdp vectors depends entirely upon the activity of a Sendai virus (SeV) RNA-dependent RNA polymerase (RdRp)24. Therefore, this vector confers robust and stable expression of transgenes in various types of mammalian cells26. Importantly, SeVdp vectors also enable prolonged transgene expression without chromosomal integration because RdRp persistently replicates the SeVdp RNA genome in the cytoplasm of infected cells25. Additionally, if needed, the SeVdp genome can be completely erased from infected cells by inhibiting RdRp function, resulting in the ability to obtain transgene-free cells23. These unique properties make the SeVdp vector a versatile gene delivery tool for various applications27.To overcome these limitations, we designed a novel fluorescence-based miRNA-monitoring system using a replication-defective and persistent Sendai virus (SeVdp) vector. SeVdp vectors accommodate multiple transgenes into a single vector backbone and simultaneously deliver these genes into target cellsIn this study, we demonstrated that a novel SeV-based fluorescence-imaging system, termed SeVdp-miR-Sensor, could be used to reliably evaluate miRNA expression in human stem cells and somatic cells. Based on its stable reporter gene expression, SeVdp-miR-Sensor enabled sensitive monitoring of miR-124 and let-7 during direct neuronal conversion and hiPSC generation, respectively. We were able to isolate reprogrammed hiPSCs by tracking let-7 expression, and the subsequent erasure of the SeVdp genome facilitated generation of transgene-free hiPSCs. Furthermore, we showed that SeVdp-miR-Sensor can be easily expanded to detect two distinct miRNAs separately and simultaneously. Our findings offer insight into a powerful tool for evaluating miRNA expression over time during cellular reprogramming and for isolating transgene-free reprogrammed cells.in vitro differentiation of hiPSCs. To this end, we prepared a vector encoding Kusabira-Orange (KO) and blasticidin S deaminase (Bsr), termed SeVdp(Bsr/KO) genome. We detected KO expression at 54 days post-infection without significant loss of fluorescence intensity infection did not affect the morphological features or proliferative capacity of hiPSCs during long-term culture were then cultured on a non-adherent plate to induce spontaneous differentiation. Notably, we observed the maintenance of robust KO expression in embryoid bodies (EBs) and further differentiated cells , enabling KR to be used as an internal reference to ensure reliable interpretation of EGFP levels in infected cells, whereas Hygr enabled selection of infected cells.To design SeVdp-miR-Sensor, we prepared an SeVdp vector containing a gene encoding enhanced green fluorescent protein (EGFP) and four copies of the complementary sequence of a target miRNA at the 3\u2032 UTR of GFP Fig.\u00a0. Bindingels Fig.\u00a0. The vecTo investigate the potency of SeVdp-miR-Sensor, we constructed vectors containing target sequences for let-7a (SeVdp-let-7aT), miR-302a (SeVdp-302aT), miR-9 (SeVdp-9T), or miR-124 (SeVdp-124T), as well as a vector containing complementary sequences for a portion of the firefly luciferase gene (SeVdp-FlucT) as a control. Initially, we infected hiPSCs with each sensor vector and examined EGFP expression by fluorescence microscopy. We observed decreases in the EGFP signal in SeVdp-302aT-infected hiPSCs, whereas that of SeVdp-let-7aT- and SeVdp-9T-infected hiPSCs was comparable to that of SeVdp-FlucT-infected hiPSCs Fig.\u00a0. Additio23. Therefore, we expected that EGFP expression would be reliably normalised along with KR expression. Infected cells exhibiting robust KR intensity were gated, and EGFP levels in those cells were analysed. As shown in Fig.\u00a0To quantitatively compare EGFP levels between cell types, fluorescent protein expression was measured by flow cytometry. We previously demonstrated that SeVdp vectors could express multiple transgenes at a pre-fixed balance when the transgenes were incorporated onto the same vector backbone28. Furthermore, the combination of NeuroD1 and these three factors facilitates neuronal conversion of human fibroblasts29.We then examined whether SeVdp-miR-Sensor could allow the monitoring of miRNA expression during direct neuronal conversion of mouse embryonic fibroblasts (MEFs). Previous studies demonstrated that ectopic expression of Ascl1, Brn2, and Myt1L efficiently reprogrammed MEFs into induced neuronal cellsASCL1, BRN2, MYT1L, and NEUROD1 [SeVdp(ABMN)] on the vector backbone and SeVdp-miR-Sensor, including SeVdp-FlucT, SeVdp-302aT, SeVdp-9T, and SeVdp-124T, and fluorescent protein expression was examined at 8 days post-infection. We observed a significant reduction in EGFP signal in SeVdp-124T-infected MEFs, whereas SeVdp-302aT- and SeVdp-9T-infected MEFs exhibited EGFP signals similar to those observed in SeVdp-FlucT-infected MEFs Fig.\u00a0. Time-laGFP gene connected with the target sequences of let-7a at the GFP 3\u2032 UTR was exploited to isolate hiPSCs following somatic cell reprogramming22. Because let-7 family members are highly expressed in differentiated cells, but not in hiPSCs30, only reprogrammed hiPSCs harbouring the vector exhibit GFP fluorescence. Although this system definitively distinguished hiPSCs from partially reprogrammed cells through examination of GFP expression, chromosomal integration of the reporter gene by the lentiviral vector is unlikely to be suitable for preparing cells derived from the hiPSCs for future medical applications.Ectopic expression of Oct4, Sox2, Klf4, and c-Myc can reprogram human somatic cells into hiPSCs; however, this reprogramming process is relatively inefficient. Previously, a lentiviral vector containing a KLF4, OCT4, SOX2, and c-MYC [SeVdp(KOSM)] efficiently reprogrammed human somatic cells into hiPSCs32. To examine whether SeVdp-miR-Sensor could monitor let-7 expression during hiPSC generation, NHDFs were co-infected with SeVdp(KOSM) and SeVdp-let-7aT, and the cells were cultured on feeder cells. Time-lapse imaging revealed that the EGFP signal of SeVdp-let-7aT-infected NHDFs was detectable at ~10 days post-infection, and that the intensity continued to increase gradually variant lacking the KR reporter from the SeVdp-let-7aT vector colonies after infection of NHDFs with SeVdp(KOSM) and SeVdp-let-7aT and transfected these cells with siRNA against the SeV polymerase P gene (siP234) to remove the SeVdp genomes from the colonies. Following repeated treatment with siP234, we confirmed that expression of SeV nucleocapsid protein (NP) mRNA was not detected by qRT-PCR, indicating that the SeVdp genomes were effectively erased from the hiPSC clones against the SeV polymerase gene facilitated erasure of the SeVdp genome from iPSC colonies, resulting in generation of transgene-free iPSCstro Fig.\u00a0. These dKR or EGFP created two different sensor vectors, SeVdp-302aT/let-7aT and SeVdp-302aT/17T . Subsequent incorporation of four copies of the target sequence for miR-302a, let-7a, or miR-17 into the 3\u2032 UTR of 17T Fig.\u00a0, with Sehed Fig.\u00a0. By conthed Fig.\u00a0. Additiohed Fig.\u00a0, whereashed Fig.\u00a0. We alsohed Fig.\u00a0. To quanhed Fig.\u00a0. These dhed Fig.\u00a0. These rpiggyBac-transposon37. These vectors require cellular and viral promoters to drive the expression of reporter genes. Elongation factor-1\u03b1 (EF1\u03b1) and \u03b2-actin promoters exhibit potent transcriptional activities in mouse and human ESCs38, and phosphoglycerate kinase and ubiquitin C (UbC) promoters direct robust expression in murine neocortical cultures39. However, the transcriptional activities of these promoters would likely be altered based on cell type and culture conditions. The activities of the EF1\u03b1 and UbC promoters gradually decreased during the long-term culture of hESCs19, with the EF1\u03b1 promoter losing most of its activity during the neuronal differentiation of mESCs17. These characteristics represent clear disadvantages for stable transgene expression during cell conversion processes. In contrast to typical expression systems, SeVdp employs RdRp and cis-acting elements incorporated into the SeVdp genome to synthesise mRNAs40. Given the availability of host-independent transcriptional machinery, SeVdp vectors are able to confer stable transgene expression in a broad range of mammalian cells and even during hiPSC differentiation in combination with miR-124 facilitates the reprogramming of human fibroblasts into neurons45, suggesting a positive role for miR-9 in direct neuronal conversion. Although the exact effect of miR-9 expression during neuronal conversion remains elusive, its expression might be affected by induction or culture conditions . Alternatively, miR-9 might be preferentially expressed in specific neuronal subtypes. It will be interesting to investigate the roles of miR-9 in direct neuronal-conversion processes in future work.Following discovery of the reprogramming of somatic cells into iPSCs, several lines of evidence demonstrated that ectopic expression of lineage-related factors could directly reprogram fibroblasts into tissue-specific cellsEFs Fig.\u00a0. AdditioEFs Fig.\u00a0. miR-124ion Fig.\u00a0, even th12. Notably, stepwise expression of these marker genes during hiPSC generation facilitates identification of reprogramming states. Although AP and SSEA4 are detected during the early phase of reprogramming, TRA-1-60 and NANOG expression appears during relatively later phases33. Here, we observed that significant reductions in let-7 expression occurred later as compared with TRA-1-60 expression during NHDF reprogramming and following infection with SeVdp(KOSM) , SSEA4, TRA-1-60, and NANOG have been commonly used as molecular markers to identify hiPSC coloniesSM) Fig.\u00a0. This reies Fig.\u00a0. These f46. Although this system allowed long-term monitoring of miR-302a-5p expression during hiPSC differentiation, episomal vectors may be rapidly cleared from highly proliferating cells46. By contrast, the replication of the SeVdp genome is remarkably stable, even in hiPSCs , pEGFP-1 , phdKeima-Red-S1 , pE2-Crimson Vector , and pCX4-bsr51 as templates, respectively. cDNA encoding Hygr was synthesised by GeneScript . These cDNAs were used to construct SeVdp(Bsr/KO), SeVdp(Hygr/EGFP), SeVdp(KR/Hygr/EGFP), and SeVdp(Crimson/KR/Hygr/EGFP). To construct SeVdp-302aT, SeVdp-let-7aT, SeVdp-9T, and SeVdp-124T, four copies of the miRNA target sequence were inserted into the 3\u2032 UTR of the EGFP gene in the SeVdp(KR/Hygr/EGFP) vector. To construct SeVdp-let-7aT(KR\u2212), four copies of the let-7a target sequence were inserted into the 3\u2032 UTR of the EGFP gene in the SeVdp(Hygr/EGFP) vector. To construct SeVdp-302aT/let-7aT and SeVdp-302aT/17T, four copies of the miRNA target sequence were inserted into the 3\u2032 UTRs of the KR and EGFP genes in the SeVdp(Crimson/KR/Hygr/EGFP) vector. To construct SeVdp-FlucT and SeVdp-FlucTx2, four copies of the complementary sequence for a portion of the firefly luciferase gene52 were inserted into the 3\u2032 UTR(s) of the KR and/or EGFP genes in the SeVdp(KR/Hygr/EGFP) and SeVdp(Crimson/KR/Hygr/EGFP) vectors, respectively. To construct SeVdp-scrT, four copies of the scrambled sequence were inserted into the 3\u2032 UTR of the EGFP gene in the SeVdp(KR/Hygr/EGFP) vector. The scrambled sequence was designed using siRNA Wizard v3.1 software . The DNA sequences that included miRNA target sequences are listed in Supplementary Table\u00a026.SeVdp genomic cDNAs were constructed as described previously26 were cultured in mTeSR1 on a plate coated with iMatrix-511 .NHDFs and MEFs were cultured in Dulbecco\u2019s modified Eagle\u2019s medium supplemented with 10% fetal bovine serum and penicillin-streptomycin . WJSCs were cultured in H-GRO medium (DV biologics). H9-NSCs were cultured in StemPro NSC SFM (Thermo Fisher Scientific) on a plate coated with CTS CELLstart substrate (Thermo Fisher Scientific). hiPSCsBsr/KO) at a multiplicity of infection (MOI) of four for 2\u2009h at room temperature, followed by incubation for 1\u2009h at 32\u2009\u00b0C. The viral medium was replaced with mTeSR1, and 10\u2009\u00b5g/mL blasticidin S (Bs) was added at 4 days post-infection. The cells were continuously cultured in the presence of Bs.hiPSCs were infected with SeVdp using customised filters. Fluorescence was analysed with iVision-Mac software . Co-imaging of fluorescence for EGFP, AlexaFluor488 (Thermo Fisher Scientific), AlexaFluor555 (Thermo Fisher Scientific), and 4\u2032,6-diamidino-2-phenylindole (DAPI) was performed using Axio Observer or BIOREVO BZ-9000 with a BZ-II analyzer . Time-lapse fluorescence microscopy was performed using a DMi8 , and data were analysed with LAS X software (Leica). Flow cytometry was performed using a Gallios flow cytometer , and the mean fluorescence intensity (MFI) was calculated using Kaluza software (Beckman Coulter).For direct neuronal conversion, MEFs were seeded onto an iMatrix-511-coated plate and infected with SeVdp(ABMN) at an MOI of 18. The cells were then co-infected with SeVdp-miR-Sensor at an MOI of two for evaluation of miRNA expression. Viral medium was replaced with neuronal culture medium . The medium was changed every 2 to 3 days.23 using Lipofectamine RNAi MAX reagent (Thermo Fisher Scientific) at 27 days post-infection. The transfection was performed three additional times every 2 to 4 days. siP234 was synthesised by GeneDesign To generate hiPSCs, NHDFs were co-infected with the SeVdp(KOSM) vector and SeVdp-miR-Sensor (SeVdp-FlucT or SeVdp-let-7aT) at an MOI of four each. The infected cells were then seeded onto mitomycin C-treated MEFs and cultured in Primate ES medium (ReproCELL) with 5 ng/mL basic fibroblast growth factor (Wako). For the feeder-free culture, NHDFs were co-infected with the SeVdp(KOSM) vector and SeVdp-let-7aT or SeVdp-let-7aT(KR\u2212) at an MOI of four each. The infected cells were seeded onto an iMatrix-511-coated plate and cultured in Stem Fit AK02N . To remove the SeVdp genome from reprogrammed colonies, cells were transfected with 40\u2009nM siP23454. NP and GAPDH mRNA levels were determined by quantitative real-time PCR (up to 45 cycles) using SsoAdvanced Universal SYBR Green Supermix and the following primers: NP , and GAPDH .Total RNA was extracted using the ISOGEN reagent . For miRNA expression analysis, cDNAs were synthesised using the TaqMan miRNA reverse transcription kit , and miRNA levels were determined using the TaqMan miRNA assays (Applied Biosystems). Levels of RNU48 (human) or snoRNA202 (mouse) were used to normalise data. For mRNA expression analysis, total RNA was treated with DNase I (Nippon Gene) to digest residual DNA, and cDNAs were synthesised using the SuperScript III first-strand synthesis system (Thermo Fisher Scientific). PCR was performed using GoTaq Green master mix with primer sets described previouslyCells were fixed with 3.7% formaldehyde in phosphate-buffered saline (PBS). After permeablisation with 0.1% to 0.2% Triton X-100/PBS, cells were incubated with a primary antibody, followed by staining with a secondary antibody conjugated with AlexaFluor488 (1:500) or AlexaFluor555 (1:500). The following primary antibodies were used in this study: anti-SSEA4 , anti-OCT4 , anti-NANOG , anti-TRA-1-60 , anti-\u03b2-III Tubulin , anti-SOX17 , anti-smooth muscle actin , anti-Desmin , anti-MAP2 , and anti-Synapsin I . Nuclei were counterstained with DAPI using VECTASHIELD mounting medium with DAPI .55, and fluorescence was immediately monitored by time-lapse fluorescence microscopy.Cells were labelled with 10\u2009\u00b5g/mL Fluo-4AM (Thermo Fisher Scientific) in DMEM/Ham\u2019s F12 medium (minus phenol red) with GlutaMAX (Thermo Fisher Scientific) for 20\u2009min at 37\u2009\u00b0C. The labelling medium was replaced with Ringer\u2019s solutionhiPSCs were treated with TrypLE Express (Thermo Fisher Scientific) and transferred onto Nunclon Sphera Microplates (Thermo Fisher Scientific) in Primate ES cell culture medium supplemented with 10\u2009\u00b5M Y27632 (Wako). Cells were cultured for 3 to 5 days to allow EB formation. EBs were attached to a gelatin-coated plate and cultured in DMEM supplemented with 10% FBS for an additional 10 to 12 days.Supplementary InformationSupplementary Video S1Supplementary Video S2aSupplementary Video S2bSupplementary Video S2cSupplementary Video S2d"} +{"text": "Twelve new platinum(II) complexes, analogs of cisplatin, containing a 2-aminoethanethiol N-substituted by several benzyl groups have been prepared and characterized in good yields. The ligands were obtained by reaction between 2-aminoethanethiol hydrochloride and different benzyl halides"} +{"text": "Abnormalities in left ventricular (LV) strain as detected during pharmacological or physiological exercise stress have been demonstrated to be earlier and more sensitive markers of contractile dysfunction than global ejection fraction. Recent developments allow for in-scanner exercise using MR-compatible ergometers. Therefore, the objective of this study was to analyze LV strain with cardiovascular magnetic resonance myocardial feature tracking (CMR-FT) in volunteers during exercise using an in-scanner ergometer.15 healthy volunteers were enrolled for supine cycle ergometry on the CMR scanner table using a MR-compatible ergometer . Imaging was performed at 3T (Siemens Skyra). Long-axis 2- and 4-chamber steady state free precession (SSFP) cine images as well as short-axis stacks were acquired at rest and after 3 minutes of cycling at 50W and 100W during a 30 sec break to minimize motion artifacts. CMR-FT was performed in 2- and 4-chamber views to quantify left ventricular global longitudinal strain (EII), and in all short-axis slices to quantify global radial strain (Err) and global circumferential strain (Ecc). Furthermore, LV volumes and ejection fraction (EF) were analyzed from the whole short-axis stack .Results are displayed in table CMR-FT derived quantification of LV strain is feasible during dynamic exercise using a supine MR-compatible ergometer. It provides a potential technique for assessing radial, circumferential and longitudinal LV strain in different patient groups during physiological exercise."} +{"text": "Drosophila embryo. Two signaling pathways act sequentially to orchestrate this dynamic morphogenetic process. First, c-Jun N-terminal kinase (JNK) signaling activity in the dorsal-most leading edge (LE) cells of the epidermis induces expression of decapentaplegic (dpp). Second, Dpp, a secreted TGF-\u03b2 homolog, triggers cell shape changes in the adjacent, ventrally located lateral epidermis, that guide the morphogenetic movements and cell migration mandatory for DC. Here we uncover a cell non-autonomous requirement for the Epidermal growth factor receptor (Egfr) pathway in the lateral epidermis for sustained dpp expression in the LE. Specifically, we demonstrate that Egfr pathway activity in the lateral epidermis prevents expression of the gene scarface (scaf), encoding a secreted antagonist of JNK signaling. In embryos with compromised Egfr signaling, upregulated Scaf causes reduction of JNK activity in LE cells, thereby impeding completion of DC. Our results identify a new developmental role for Egfr signaling in regulating epithelial plasticity via crosstalk with the JNK pathway.Dorsal closure (DC) is a developmental process in which two contralateral epithelial sheets migrate to seal a large hole in the dorsal ectoderm of the Drosophila embryogenesis has provided many fundamental insights into conserved mechanisms controlling epithelial dynamics and cell migration, and therefore serves as a model for studying tissue morphogenesis and wound repair. DC is known to require the coordinated action of two signaling pathways: (i) the c-Jun N-terminal kinase (JNK) pathway, which is activated in the dorsal-most leading edge (LE) cells of the migrating epithelia; and (ii) the Decapentaplegic (Dpp) pathway, induced by the JNK pathway, which signals to neighboring lateral epidermis cells to drive the cellular changes required for DC. Here we uncover a new tier of regulation essential for DC, mediated by the Epidermal growth factor receptor (Egfr) pathway. Specifically, we demonstrate that Egfr signalling in the lateral epidermis suppresses the expression of the gene scarface, which encodes a secreted JNK antagonist. Through this regulatory switch the Egfr pathway facilitates JNK signalling in LE cells, ensuring full induction and activity of Dpp signaling that is pivotal for orchestrating the synchronized morphogenetic movements characteristic of DC. Our study thus identifies a novel mechanism of signal integration between the Egfr and JNK pathways, linking Egfr signalling to the core regulatory network controlling DC.The developmental process of dorsal closure (DC) in Drosophila melanogaster. In this developmental setting, two contralateral epithelial sheets from opposing sides of the embryo migrate and converge at the dorsal midline above the amnioserosa (AS), an extraembryonic epithelium tissue, thereby generating a continuous epidermis that seals a large dorsal hole (decapentaplegic (dpp), encoding the TGF-\u03b2/BMP family member Dpp . Two. TwoDrosmber Dpp \u20136. Secrember Dpp ,8. Accormber Dpp \u201311. Thusdpp and other JNK pathway target genes requires input by two negative feedback regulators, which are expressed in response to JNK signaling in LE cells .,27.26,27e extent . InsteadCollectively, our findings identify a requirement for functional Egfr signaling in the process of DC, which is already apparent at the level of the cell shape changes that normally occur during early DC.dpp in LE cells of rhomboid mutants, as well as in embryos expressing EgfrDN in the ectoderm, finding that it is reduced in both genotypes -Gal4 -Gal4 . Reciprom of Ras ; S4 Fig.dpp (Mad) .,14.scaf guration . By contLE cells .scaf expression, and provide a plausible explanation for the positive, non-autonomous effect of the Egfr pathway on JNK signaling in the LE. According to this scenario, in wild-type embryos Egfr signaling inhibits scaf expression in lateral epidermis cells, thereby blocking its secretion and antagonistic effect on JNK signaling in neighboring LE cells. Under Egfr loss-of-function conditions, scaf is up-regulated in lateral epidermis cells, leading to reduced JNK activity in LE cells. Accordingly, scaf expression in the LE itself is reduced relative to normal embryos [scaf expression indirectly by inducing an intermediary repressor of this gene. To begin testing this idea, we expressed a non-phosphorylatable derivative of Yan, YanAct, that is insensitive to attenuation by Egfr-mediated signaling [pnr>YanAct embryos display dorsal open phenotypes . Therefoignaling and, henenotypes , loss ofstaining . Remarka embryos , consistscaf is under complex transcriptional control, involving several activators as well as at least two repressors: Engrailed (En) and Abdominal-A (Abd-A) [en, scaf is derepressed only in LE cells, perhaps due to the combined repressor activity of Abd-A. Nevertheless, when they expressed a form of En that was converted into an activator, scaf was ectopically expressed in the lateral epidermis [scaf. Indeed, we find that En is dominantly repressed in the lateral epidermis of embryos expressing pnr>YanACT, specifically in the pnr domain . They shpidermis . This rar domain ; cf. 8E.in . In general, mutant chromosomes were maintained over wg-LacZ- or dfd-YFP-marked balancer chromosomes, allowing the unambiguous identification of embryos of the correct genotype. yellow white flies served as wild-type controls.Flies were cultured and crossed on standard yeast-cornmeal-molasses-malt extract-agar medium at 25\u00b0C. The following mutant stocks and Gal4 drivers were used: , EgfrEB , scaf\u03941.UAS-Scaf , rhomboiwhite; pnr-Gal4, puc-lacZ/UAS-GFP (control); 2) white; UAS-EgfrDN; pnr-Gal4, puc-lacZ; 3) and UAS-RasDN; pnr-Gal4, puc-lacZ.To quantify the proportion of embryos compromised in Egfr signaling that fail to complete closure at st16, the following genotypes were used: 1) Unhatched larvae (>24 hours old) were dechorionated in bleach, devitellinised in 1:1 methanol/heptane, rehydrated in PBS/methanol and mounted in 1:1 Hoyer\u2019s medium/double-distilled water and cleared overnight at 70\u00b0C.dpp and scaf were visualized by whole-mount in situ hybridization using digoxigenin-UTP labeled antisense RNA probes and anti-digoxigenin antibodies conjugated to alkaline phosphatase (Roche).Embryos were dechorionated in bleach and fixed in 8% formaldehyde/PBS/heptane for 20 minutes. Expression patterns of Fluorescent immunodetection of dpErk, in freshly fixed embryos , was attained using rabbit \u03b1dpErk . Other aLight microscope images were acquired using a Zeiss Axioplan2 microscope and confocal images were taken using a Zeiss LSM710 confocal microscope. Images were processed using Adobe Photoshop software, and the ZEN 2012 blue edition was used to measure LE cell length in embryos compromised in Egfr signaling.S1 Figpnr-Gal4>GFP, puc-lacZ enhancer-trap embryo, stained for LacZ and for GFP . (C) Merge. Note that ectodermal expression of GFP, driven by pnr-Gal4, is restricted to the lateral epidermis and LE cells.(A-C) A (TIF)Click here for additional data file.S2 Figpuc-lacZ enhancer-trap line embryos, in which pnr-Gal4 drives the expression either of GFP (control), RasDN or EgfrDN, stained for LacZ to demarcate LE cells. The numbers of st16 embryos, displaying a dorsal-open hole and therefore incomplete closure , or those that have completed closure , were scored. (C) Percentage of st16 embryos, expressing GFP, RasDN or EgfrDN via pnr-Gal4, that have completed closure (blue) or not (red). n = number of embryos from each definitive genotype that were scored.(A-B) Confocal images of st16 (TIF)Click here for additional data file.S3 Figbsk, spi and rhomboid mutant embryos, as well as in embryos expressing pnr>EgfrDN or pnr>RasDN. The data represent the mean \u00b1 SD derived from 8\u201310 different embryos. *** P<0.0001 compared to wild-type embryos (Mann-Whitney U-test). n = number of LE cells from each definitive genotype that were scored.Quantification of LE cell length in wild-type or in (TIF)Click here for additional data file.S4 Figdpp (blue). Small insets show the full embryos. (A) Wild-type embryo showing the normal dpp pattern. Levels of dpp are reduced in a rhomboid mutant (B) as well as in embryo expressing pnr>EgfrDN (C). Conversely, the dpp domain expands ventrally in embryo expressing pnr>RasV12 (D).(A-D) High magnification (x40) lateral views of embryos hybridized using a digoxigenin-labeled RNA probe for (TIF)Click here for additional data file.S5 Figprd>EgfrDN showing an open dorsal phenotype (white arrowhead). (B) St13 embryo expressing prd>EgfrDN hybridized using a digoxigenin-labeled RNA probe for dpp (blue). Loss of dpp expression (black arrowheads) in both stripe and inter-stripe regions of prd>EgfrDN embryos indicates that the resulting ectopic Scaf acts on LE cells non- autonomously (see below). White asterisks and arrowheads mark JNK-independent dpp expression in the visceral mesoderm and lateral ectoderm, respectively.(A) Cuticle preparation of embryo expressing (TIF)Click here for additional data file.S6 Figpuc-lacZ, pnr-Gal4 embryo expressing GFP (A), or puc-lacZ, pnr-Gal4 embryos expressing EgfrDN (B) and RasDN (C), stained for pMad (red) and LacZ (blue). Note that LE cells, distinguishable by LacZ staining, co-stain for pMad, whereas pMad staining is markedly reduced in the lateral epidermis.Control (TIF)Click here for additional data file.S7 FigEgfrSH2 allele, maintained at permissive (18\u00b0C) (A) or restrictive (29\u00b0C) (B) temperatures. The embryo in (B) was shifted from the permissive to the restrictive temperature at the onset of dorsal closure (st12). Note the dorsal open phenotype (arrowhead). Wild-type embryos subjected to the same regime hatched normally. The embryo in (A) has mild segmental defects. The domain of pMad staining (red) decreases in EgfrSH2 embryo shifted to 29\u00b0C at st12 (D) but not in embryo of the same genotype raised at 18\u00b0C (C). Embryos stained for DE-cadherin (green) to outline cell membranes. Corresponding primed panels (E\u2019 and F\u2019) show magnified views of the regions marked with arrowheads. Note the occurrence of cell elongation defects in F\u2019. Cuticle preparations of embryos carrying the temperature sensitive (TIF)Click here for additional data file.S8 Figpnr>GFP, stained for (A) GFP and (B) pMad. (C) Merge. (D-F) Embryo co-expressing pnr>RasV12;GFP, stained for (D) GFP and (E) pMad. (F) Merge. Note the strong pMad staining in pnr>RasV12;GFP embryo.(A-F) Lateral views of st13 embryos stained for GFP (green) and pMad (red). (A-C) Control embryo, expressing (TIF)Click here for additional data file.S9 Figbsk mutant embryo (B) or upon pnr>HepAct expression (C), compared to control (A). The signal in the AS is an artifact caused by auto-florescence.(A-C) Embryos stained for dpErk (red). No significant change in the dpErk pattern is observed in (TIF)Click here for additional data file."} +{"text": "Brentuximab vedotin (BV) is an antibody-drug conjugate composed of a CD30-directed monoclonal antibody and monomethyl auristatin E . BV mono"} +{"text": "Maternal embryonic leucine zipper kinase (MELK) is known to modulate intracellular signaling and control cellular processes. However, the role of MELK in oncogenesis is not well defined. In this study, using two microarray datasets of neuroblastoma (NB) patients, we identified that MELK expression is significantly correlated to poor overall survival, unfavorable prognosis, and high-risk status. We found that MELK is a direct transcription target of MYCN and MYC in NB, and MYCN increases MELK expression via direct promoter binding. Interestingly, knockdown of MELK expression significantly reduced the phosphorylation of target protein Retinoblastoma (pRb) and inhibited NB cell growth. Furthermore, pharmacological inhibition of MELK activity by small-molecule inhibitor OTSSP167 significantly inhibited cell proliferation, anchorage-independent colony formation, blocked cell cycle progression, and induced apoptosis in different NB cell lines including a drug-resistant cell line. Additionally, OTSSP167 suppressed NB tumor growth in an orthotopic xenograft mouse model. Overall, our data suggest that MELK is a novel therapeutic target for NB and its inhibitor OTSSP167 is a promising drug for further clinical development. MYCN amplification is a strong characteristic of high-risk NB patients and serves as a genetic marker of disease [Neuroblastoma (NB) is the most common extracranial neoplasm in children and contributes to about 15% of all pediatric cancer-related deaths (1). Despite major advances in therapies over the past decade, the overall outcome for high-risk NB patients is still unacceptable [ disease , 3. FindMELK expression predicts a poor prognosis of many cancer types, including but not limited to breast cancer [Protein kinases play an essential role in the regulation of cell survival and proliferation . Differet cancer , astrocyt cancer and gliot cancer . MELK hat cancer . Furthert cancer , 16. Thet cancer .MELK gene promoter and promotes transcription. Additionally, the MYCN or MYC knockdown led to decreased MELK mRNA and protein levels in NB cells, whereas MYCN or MYC overexpression led to increased mRNA and protein levels of MELK in NB cells. Furthermore, MELK small molecule inhibitor OTSSP167 inhibited NB growth both in vitro and in vivo by inducing apoptosis. This is the first report that shows the oncogenic role and regulation of MELK in NB. Novel MELK inhibitor OTSSP167 is shown to reduce growth of various cancer types [ClinicalTrails.gov # NCT01910545) and is fast approaching for further clinical development. Our pre-clinical data suggest that MELK is an attractive therapeutic target in MYCN- and MYC-driven cancers, such as NB.The molecular mechanism regulating MELK overexpression in cancer cells and the role of MELK in NB tumorigenesis remains ambiguous. In this study, we analyzed a large cohort of NB patients and identified that higher MELK expression is correlated with poor overall survival, prognosis, and overall outcome. Moreover, MELK expression is higher in tumors from high-risk NB patients. Using ChIP-qPCR assays, we showed that MYCN directly binds at the E-box binding motifs present at the er types . The oraMELK correlates with NB outcomes, we analyzed large clinical cohorts of NB patients using the R2-data analysis platform. Kaplan-Meier analyses of datasets revealed that low MELK transcript levels strongly correlated with better overall and event-free survival for the Tumor Neuroblastoma SEGC dataset (n = 498) (p < 9.7E-27) and Kocak dataset (n = 649) (p < 1.3e-12) in MYCN-amplified NB tumors compared to those in MYCN-nonamplified ones Figure . Interess Figure . There wt Figure . In addiy Figure . These fMYCN in two MYCN-amplified NB cell lines, NGP and IMR-32, as well as knockdown of MYC in two MYCN-non-amplified NB cell lines, SK-N-AS and SH-SY5Y. Results showed that stable knockdown of MYCN or MYC led to both reduced MELK mRNA and protein in NB cell lines tested . As expected, the stable knockdown of MELK reduced MELK protein levels with both of the sh-RNA knockdown constructs used to less sensitive in a drug-resiistant NB cell line LA-N-6 (334.8 nM) Figure . Along w) Figure . In addi) Figure . Further) Figure . This soe arrest . To furtin vivo effects of OTSSP167, we used an orthotopic NB xenograft mouse model. This mouse model recapitulate the highly aggressive and invasive growth pattern of human NB [in vivo NB tumor growth in comparison to controls or DMSO (vehicle control) daily for 48 hours. Subsequently, tumors were harvested and analyzed. We found that MELK and p-Rb levels were significantly inhibited and PARP cleavage levels were increased in tumors from the OTPSS167 treated mice in comparison to tumors from vehicle treated mice . Although related off-target effects are yet to be elucidated, OTSSP167 is still capable in directly reducing MELK levels and inducing apoptosis in NB cell lines tested.MELK inhibitor OTSSP167 with a potential off-target effect should be considered. Studies have reported that MELK-knockout cells remain sensitive to OTSSP167 since OTSSP167 blocks cell proliferation through an off-target mechanism . In addiAnother confounding issue is to determine which signaling pathways regulate MELK kinase activity. MELK kinase mediates a large variety of cell characteristics including but not limited to cell cycle control, cell proliferation, apoptosis, cell migration, oncogenesis, cancer resistance, and recurrence . FurtherIn summary, our data showed that MELK is a transcriptional target of MYCN/MYC and plays an oncogenic role in NB. Inhibition of MELK, inhibit NB growth by blocking the cell cycle progression due to inhibition of Rb functions. The small molecule MELK inhibitor, OTSSP167 potently inhibit NB tumor growth, and reprents a promising therapeutic strategy for high-risk NB.http://hgserver1.amc.nl/cgibin/r2/main.cgi). This platform also support the multi-parametric analysis of NB patient outcome with gene expression.The Kocak neuroblastoma patient dataset (N = 649) and tumor neuroblastoma dataset SEGC (N = 498) that includes microarray profiles of unique primary tumors are publically available in the R2: Genomic Analysis and Visualization Platform database solution and were analyzed by flow cytometry (FCS Express 4 Software). The data showed the distribution of the three cell cycle phases .Cell proliferation was measured using Cell Counting Kit-8 (Dojindo Laboratories) in accordance to the manufacturer's instructions. NB cells were seeded at 5000 cells/well in a 96-well microtiter plate and after 24 hours of incubation, were treated with increasing concentrations of OTSSP167. Cellular proliferation was measured 72 hours post-incubation by adding CCK-8 reagent and was monitored for optical density at 450 nm using a microplate reader. Each experiment was performed in replicates of six and background reading of the media was subtracted from each well for result standardization. Soft agar colony formation assays were performed using standard conditions as described previously [MYC, MYCN and MELK knockdown cell lines. The following were shRNA sequences used: MYCN-sh-1: 5\u2019- AATTCTTACACTGCCTGTATA-3\u2019, MYCN-sh-2: 5\u2019-AATCTCTGTTATGTACTGTAC-3\u2019. MYC-sh-1: AATGTCCTGAGCAATCACCTA, MYC-sh-2: AATGTCCTGAGCAATCACCTA. MELK-sh-1: 5\u2019-AAGTTCATTGGAACTACCAAC-3\u2019, MELK-sh-2: 5\u2019-AATTGATGGATTCTTCCATCC-3\u2019. The lenti-viral expression vectors encoding human MYCN and MYC open reading frame (ORF) were used to generate MYC and MYCN overexpression NB cell lines. These vectors were used to generate virus and transduce NB cell lines as described previously [Trc2 lentiviral shRNA vectors (Sigma-Aldrich Inc.) were used to generate eviously .MYCN: Forward 5\u2019-AGAGGACACCCTGAGCGATTC-3\u2019, Reverse 5\u2019-CATAGTTGTGCTGCTGGTGGA-3\u2019; MYC: Forward 5\u2019-CTCCATGAGGAGACACCGCCCA-3\u2019, Reverse 5\u2019-AAGGTGATCCAGACTCTGACCT-3\u2019; MELK: Forward 5\u2019-ATAGCTACCATCTCTCCAGTA-3\u2019 and Reverse 5\u2019-CTTGCAAGAGGACTATGAAAG-3\u2019, respectively.Gene transcription levels were measured using the qRT-PCR method as described previously . Total Rwww.SimGene.com). ChIP-MELK primers were Forward- 5\u2019-GAGAACTGTGACTGCCAGAGG-3\u2019 and Reverse- 5\u2019-TGTGGAGCCGTGAAAGGGAT-3\u2019. ChIP-negative control primers were Forward- 5\u2019- ATGGTTGCCACTGGGGATCT -3\u2019 and Reverse- 5\u2019- TGCCAAAGCCTAGGGGAAGA -3\u2019.ChIP-qPCR assays were performed using the ChIP-IT Express Kit (Active Motif) in accordance to the manufacturer's instructions as described previously . The ant6 NGP NB cells were surgically implanted in the sub-renal capsule through a transverse incision over the left flank. Tumor growth was monitored twice a week with bioluminescent imaging . Mice bearing similar sized tumors were randomly divided into groups and were treated with either DMSO or OTSSP167 for 14 days. After treatment, tumors were harvested. Tumors were either weighted and photographed for analysis or lysed for immunoblotting.Four to six week-old female inbred athymic immunodeficient Nude mice (Nu/Nu) were purchased from Taconic Biosciences and used for all xenograft studies. Mice were implanted using a previously described orthotopic xenograft model , 34. AllP < 0.05 was considered to be statistically significant. The IC50 value was calculated with Graphpad Prism 5 software . Survival analyses were performed using Kaplan-Meier method and two-sided log-rank tests.All values were presented as the mean \u00b1 standard deviation (SD). A two-tailed Student's t-test and ANVOA were used to determine the statistical significance among drug treatment groups. Each assay was repeated at least twice, and representative results were presented."} +{"text": "KitW-sh mice to generate URO-OVA/KitW-sh mice that retained urothelial OVA expression but lacked endogenous mast cells. We compared URO-OVA mice with URO-OVA/KitW-sh mice with and without mast cell reconstitution in response to cystitis induction. URO-OVA mice developed profound bladder inflammation with increased mast cell counts and LUTD, including increased total number of voids, decreased mean volume voided per micturition, and decreased maximum volume voided per micturition, after cystitis induction. In contrast, similarly cystitis-induced URO-OVA/KitW-sh mice developed reduced bladder inflammation with no mast cells and LUTD detected. However, after mast cell reconstitution URO-OVA/KitW-sh mice restored the ability to develop bladder inflammation and LUTD following cystitis induction. We further treated URO-OVA mice with cromolyn, a mast cell membrane stabilizer, and found that cromolyn treatment reversed bladder inflammation and LUTD in the animal model. Our results provide direct evidence for the role of mast cells in cystitis-associated LUTD, supporting the use of mast cell inhibitors for treatment of certain forms of IC/BPS.Bladder inflammation frequently causes cystitis pain and lower urinary tract dysfunction (LUTD) such as urinary frequency and urgency. Although mast cells have been identified to play a critical role in bladder inflammation and pain, the role of mast cells in cystitis-associated LUTD has not been demonstrated. Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic and debilitating inflammatory condition of the urinary bladder characterized by the hallmark symptoms of pelvic pain and LUTD. In this study we investigated the role of mast cells in LUTD using a transgenic autoimmune cystitis model (URO-OVA) that reproduces many clinical correlates of IC/BPS. URO-OVA mice express the membrane form of the model antigen ovalbumin (OVA) as a self-antigen on the urothelium and develop bladder inflammation upon introduction of OVA-specific T cells. To investigate the role of mast cells, we crossed URO-OVA mice with mast cell-deficient Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic inflammatory condition of the urinary bladder characterized by the hallmark symptoms of pelvic pain and lower urinary tract dysfunction (LUTD) such as urinary frequency and urgency . IC/BPS The etiology of IC/BPS remains elusive and may involve multiple causes. Although autoimmunity is debated as a potential cause of IC/BPS, evidence suggests that it may play an important role in the pathophysiology of the disease. It has been reported that IC/BPS patients develop antinuclear and anti-urothelium autoantibodies , overexpAnimal models with bladder autoimmune inflammation have been actively used in IC/BPS research for over two decades ,19\u201324. E2 asphyxiation, followed by physical confirmation of euthanasia.All animal experiments were approved by University of Iowa Animal Care and Use Committee (Permit Number: 1308153) and performed according to the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Animals were euthanized via COKitW-sh mice (KitW-sh) were obtained from Jackson Laboratories . URO-OVA/KitW-sh mice were generated through crossbreeding of the two strains and selected as described previously [KitW-sh mice (homozygous) retained urothelial OVA expression but lacked endogenous mast cells. OT-I mice that express CD8+ T cell receptor (V\u03b12V\u03b25) specific for the H2-Kb/OVA257\u2013264 epitope were used to provide OVA-specific CD8+ T cells for cystitis induction [URO-OVA mice were previously developed in our laboratory . Mast ceeviously ,28. URO-nduction . All micKitW-sh mice (6 weeks old) were intravenously (i.v.) injected with 1 X 106 mast cells and used 9 weeks after mast cell reconstitution for cystitis induction.Bone marrow cells were prepared from the femurs of C57BL/6 mice (8 weeks old) and cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, penicillin (100 units/ml), streptomycin (100 \u03bcg/ml), \u03b2-mercaptoethanol (50 \u03bcM), and mouse recombinant IL-3 for 5 weeks . The purKitW-sh mice were injected i.v. with OT-I splenocytes (5 X 106 cells per mouse) that had been pre-activated with OVA257\u2013264 peptide in culture for 3 days as described previously [Age- and sex-matched URO-OVA and URO-OVA/URO-OVA mice (8 weeks old) were injected intraperitoneally (i.p.) with cromolyn sodium , a mast cell membrane stabilizer , at 10 mBladders were collected and processed for formalin fixation, paraffin embedment, section preparation, hematoxylin and eosin (H&E) staining, and photography as described previously . BladderAs described previously , mice weBladder single-cell suspensions were prepared, stained with FITC-CD8 and/or PE-V\u03b12 antibodies , and analyzed by flow cytometry as described previously .5\u2019-TGAACGCTACACACTGCATCT-3\u2019 and 5\u2019-GACTCCTTTTCCGCTTCCTGA-3\u2019; 459 bp), IL-6 , tumor necrosis factor (TNF)-\u03b1 , NGF , tachykinin-1 (SP precursor) , and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) . Based on our pre-established PCR kinetics, GAPDH was amplified for 25 cycles, IFN-\u03b3, TNF-\u03b1, NGF and SP precursor were amplified for 36 cycles, and IL-6 was amplified for 40 cycles. The PCR products were run on 1% agarose gels, stained with ethidium bromide, and imaged by Gel Doc EZ Imager .Bladder total RNAs were extracted using Qiagen RNeasy Mini Kit and cDNAs were synthesized using Invitrogen SuperScript III Reverse Transcriptase and Oligo dT as described previously . PCR amp+V\u03b12+ cells in the bladder and voiding habit changes after cystitis induction. Data was compared using Student\u2019s t-test (two groups) or ANOVA followed by LSD post hoc tests (multiple groups). A value of p<0.05 was considered statistically significant.Results were analyzed using GraphPad Prism (Version 6.0g) and presented as mean \u00b1 s.d. for mast cells and CD8KitW-sh mice developed reduced bladder inflammation , together with URO-OVA mice, in micturition cages for a 24-hour recording of voiding habits at day 6 after cystitis induction. Baseline voiding habits were recorded for comparison. As observed previously [p = 0.0053), decreased maximum volume voided per micturition (p = 0.012), and increased total number of voids (p = 0.0002). The number of voids in the dark period was increased (p = 0.0107). Unlike URO-OVA mice, URO-OVA/KitW-sh mice developed no significant changes in voiding habits after cystitis induction . These observations indicate a critical role of mast cells in cystitis-associated LUTD in the URO-OVA model.To identify whether mast cells play a role in cystitis-associated LUTD, we placed URO-OVA/eviously , URO-OVAnduction . Howeverf voids) . Excessi 0.0638) . Fig 2 r 0.0638) , URO-OVA. There were also significant differences between cromolyn- and saline-treated groups in voiding habits . The total voided volumes in 24 hours were similar between normal, cystitis-induced plus saline-treated, and cystitis-induced plus cromolyn-treated groups . These observations indicate that blocking mast cell activation reverses bladder inflammation and voiding dysfunction in the URO-OVA model.To conform the role of mast cells in bladder inflammation and associated LUTD, we treated URO-OVA mice with cromolyn or control saline during the development of bladder inflammation in the animal model . Comparenduction . The blanduction . In paranduction . In contMast cells are considered to play an important role in IC/BPS, as many patients exhibit increased mast cell counts in the bladder and elevated levels of mast cell mediators in the urine ,3. To suKitW-sh mice to generate mast cell-deficient URO-OVA/KitW-sh mice. We observed that URO-OVA/KitW-sh mice developed reduced bladder inflammation with no detectable LUTD after cystitis induction. However, reconstitution of mast cells in URO-OVA/KitW-sh mice restored their ability to develop bladder inflammation and LUTD in response to cystitis induction. In addition, treatment of URO-OVA mice with systemic cromolyn, a mast cell membrane stabilizer [To facilitate investigation of the role of mast cells, we crossed URO-OVA mice with mast cell-deficient abilizer , reverseAlthough multiple treatment modalities are currently available for IC/BPS patients, they are largely empirical and often dissatisfactory and vary in efficacy. Therefore, effort to develop new therapies for IC/BPS is greatly needed. Since mast cells are considered to play a critical role in the pathophysiology of IC/BPS, pharmacotherapy with drugs specific for mast cell inhibition may benefit IC/BPS patients. In addition to traditionally used antihistamine drugs , inhibitWe previously demonstrated that the URO-OVA model was responsive to intravesical treatment with anti-inflammatory agents such as dimethyl sulfoxide and RDP5The present study demonstrates the role of mast cells in LUTD at an acute cystitis setting. We will extend to identify the role of mast cells at a chronic cystitis setting to more closely mimic human IC/BPS. Our future studies will also include to identify which mast cell mediator(s) play a predominant role in cystitis-associated LUTD in the URO-OVA model and use this model to develop new therapies for LUTD and pain seen in IC/BPS patients.Our study provides direct evidence for the role of mast cells in cystitis-associated LUTD, supporting the use of mast cell inhibitors for treatment of certain forms of IC/BPS.S1 FigMagnifications: X200 and X1000.(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 FigKitW-sh mice but not in URO-OVA/KitW-sh mice at day 7 after cystitis induction. Mast cells are indicated by red arrows. MC, mast cells. Magnification: X400.Mast cells were detected in both URO-OVA and mast cell-reconstituted URO-OVA/(TIF)Click here for additional data file."} +{"text": "The PET/CT showed unilateral diffuse skeletal muscle 18F-FDG uptake as well as bilateral salivary gland uptake artifacts, suggestive of non-compliance with patient preparation instructions. The PET/CT nurse noted that during the 18F-FDG uptake phase, the patient appeared intoxicated, and she found two beer cans hidden in the waste disposal beside his chair just prior to imaging. The patient only admitted to eating a cookie approximately 30 minutes after the injection of 18F-FDG PET/CT and denied consuming alcohol during the uptake phase. We present the imaging findings of non-compliance with patient instructions during the uptake phase of 18F-FDG.A 49-year-old male patient with a prior history of poor compliance with medical appointments was referred for an"} +{"text": "O-methacryloyl-d-galactopyranose)-b-poly(6-cholesteryloxyhexyl methacrylate) , with cholesterol/galactose grafts were prepared through a sequential reversible addition-fragmentation chain transfer (RAFT) polymerization and deprotection process. The glycopolymers could self-assemble into aggregates with various morphologies depending on cholesterol/galactose-containing block weight ratios, as determined by transmission electronic microscopy (TEM) and dynamic laser light scattering (DLS). In addition, the lectin recognition and bovine serum albumin (BSA) adsorption of the PMAgala-b-PMAChol aggregates were evaluated. The SK-Hep-1 tumor cell inhibition properties of the PMAgala-b-PMAChol/doxorubicin (DOX) complex aggregates were further examined in vitro. Results indicate that the PMAgala-b-PMAChol aggregates with various morphologies showed different interaction/recognition features with RCA120 and BSA. Spherical aggregates (d \u2248 92 nm) possessed the highest RCA120 recognition ability and lowest BSA protein adsorption. In addition, the DOX-loaded spherical complex aggregates exhibited a better tumor cell inhibition property than those of nanofibrous complex aggregates. The morphology-variable aggregates derived from the amphiphilic glycopolymers may serve as multifunctional biomaterials with biomolecular recognition and drug delivery features.In this study, a series of diblock glycopolymers, poly(6- In fact18-b-PMAChol amphiphilic glycopolymers with galactose and cholesterol grafts were designed and prepared. These glycopolymers could self-assemble into morphology-variable nanoscale aggregates from spherical nanoparticles to nanofibers. These glycol-containing amphiphilic aggregates showed different lectin RCA120 recognition and BSA adsorption behaviors, largely relying on their molecular structure/aggregate morphology. MTT assay of the PMAgala18-b-PMAChol aggregates (up to 500 \u03bcg/mL) indicated their low cytotoxicity toward SK-Hep-1 cells, enabling them to serve as promising drug carriers in vivo. Furthermore, the PMAgala18-b-PMAChol/DOX complex aggregates were prepared, and the DOX-loading and cell proliferation inhibition properties of the complex aggregates were also found to be molecular structure/morphology-dependent. The spherical complex aggregates gave rise to higher tumor cell inhibition efficiency than those of the nanofibrous and nanospindles counterparts under the same DOX dosages. The study may provide a potential molecular structure/morphology-control approach towards the design and preparation of efficient amphiphilic glycopolymers for protein recognition/adsorption and drug delivery.In summary, a new series of diblock PMAgala"} +{"text": "VHL) tumor suppressor gene is inactivated in the vast majority of human clear cell renal carcinomas. The pathogenesis of VHL loss is currently best understood to occur through stabilization of the hypoxia-inducible factors, activation of hypoxia-induced signaling pathways, and transcriptional reprogramming towards a pro-angiogenic and pro-growth state. However, hypoxia also drives other pro-tumorigenic processes, including the development of genomic instability via down-regulation of DNA repair gene expression. Here, we find that DNA repair genes involved in double-strand break repair by homologous recombination (HR) and in mismatch repair, which are down-regulated by hypoxic stress, are decreased in VHL-deficient renal cancer cells relative to wild type VHL-complemented cells. Functionally, this gene repression is associated with impaired DNA double-strand break repair in VHL-deficient cells, as determined by the persistence of ionizing radiation-induced DNA double-strand breaks and reduced repair activity in a homology-dependent plasmid reactivation assay. Furthermore, VHL deficiency conferred increased sensitivity to PARP inhibitors, analogous to the synthetic lethality observed between hypoxia and these agents. Finally, we discovered a correlation between VHL inactivation and reduced HR gene expression in a large panel of human renal carcinoma samples. Together, our data elucidate a novel connection between VHL-deficient renal carcinoma and hypoxia-induced down-regulation of DNA repair, and identify potential opportunities for targeting DNA repair defects in human renal cell carcinoma.The von Hippel-Lindau ( VHL) gene is mutated, deleted, or silenced in 60\u201380% of human clear cell renal cell carcinomas (ccRCC), the most common type of kidney cancer [VHL into VHL-deficient renal carcinoma cells suppresses tumor formation, establishing its role as a tumor suppressor [VHL protein (pVHL) is as the substrate-recognition subunit of an E3 ubiquitin ligase that targets hypoxia-inducible factor (HIF) \u03b1-subunits for degradation under normoxic conditions [VHL-deficient RCC cell lines [The von Hippel-Lindau (y cancer . Reintroppressor . The besnditions . HIF standitions \u20137). In tll lines .VHL deficiency, hypoxia is an important physiological component of solid tumors and contributes to tumor progression and metastasis . Hypiewed in . Hypoxiatability \u201321.BRCA1 and MLH1 gene promoters [Although hypoxia induces genomic instability, it does not directly generate DNA damage. Instead, hypoxic stress facilitates the down-regulation of cellular DNA repair pathways through transcriptional, translational, and epigenetic mechanisms (reviewed in \u201324). DNAromoters , 35. Nucromoters , 37. In romoters , 38\u201340. romoters , 42.VHL mutations and physiologic hypoxia, we hypothesized that VHL-deficient RCC may have reduced DNA repair capacity that could be exploited for therapeutic gain. Interestingly, several possible connections between pVHL and DNA repair have been reported. First, pVHL has been shown to positively regulate p53 by inhibiting Mdm2-mediated p53 ubiquitination [VHL-deficient cells have been shown to have reduced nucleotide excision repair (NER) capacity [VHL clearly plays a role in maintaining genomic stability, but whether the mechanisms induced by hypoxia leading to coordinated down-regulation of DNA repair pathways occur in VHL-deficient cells has not yet been investigated.Given the similar downstream effects of tination , 44. Lostination . Recentltination . Finallycapacity . Thus, VVHL mutations, through induction of hypoxia-like signaling pathways, may lead to down-regulation of DNA repair pathways and sensitivity to DNA damage. We have found that VHL-deficient human renal carcinoma cells have reduced protein and mRNA expression of key HR and MMR genes down-regulated by hypoxia, including BRCA1, RAD51, FANCD2, and MLH1. Using siRNA depletion, we have demonstrated that this reduced gene expression is directly linked to loss of pVHL. We have further established that this decrease in HR gene expression is associated with reduced repair of DNA double-strand breaks by HR and consequent sensitivity to PARP inhibitors in VHL-deficient renal carcinoma cells. Finally, by analyzing mRNA expression in human renal carcinoma samples in The Cancer Genome Atlas (TCGA), we have identified a correlation between VHL deficiency in RCC and reduced expression of HR and MMR genes, supporting the significance of our findings in human RCC.In this study, we have investigated the possibility that VHL-deficient human ccRCC cell lines, 786-OVHL\u2013/\u2013 and RCC4VHL\u2013/\u2013, and their wild-type VHL-complemented matched pairs, 786-O+VHLWT and RCC4+VHLWT. We confirmed that these cells overexpress the HIF \u03b1-subunits (HIF-2\u03b1 in 786-OVHL\u2013/\u2013 cells and both HIF-1\u03b1 and HIF-2\u03b1 in RCC4 VHL\u2013/\u2013 cells) and that VHL complementation blocks HIF overexpression as previously described for 24 and 48 h using the neutral comet assay. The 786-O and RCC4 matched pair cell lines were treated with 10 Gy IR and assayed 24 h post-irradiation. In both cell lines, we observed a significantly higher median comet tail moment in the irradiated VHL-deficient cells compared to the irradiated WTVHL-complemented cells, indicative of more DNA double-strand breaks persisting 24 h post-irradiation and less efficient repair in the VHL-deficient cells Kidney Renal Clear Cell Carcinoma database to analyze DNA repair gene expression in VHL-deficient and WTVHL human renal tumor samples. VHL deficiency in renal carcinomas can occur via inactivating mutations or allelic loss. We therefore sorted the tumor samples into groups with and without VHL point mutations and groups with copy number alterations of 0/1, -1, or -2. In addition, we determined the putative VHL genotype of each tumor sample based on the combination of point mutations and copy number alterations and sorted the samples into homozygous WT (+/+), heterozygous (+/\u2013), and homozygous mutant (\u2013/\u2013) groups capacity using a luciferase reporter reactivation assay in VHL-deficient renal cells [VHL-deficiency-induced DNA repair repression.As with hypoxia, we find that A damage , 64. pVHA damage . Metcalfear foci . In thatal cells . Hypoxiaal cells , furtherBRCA1 or BRCA2 mutations [VHL-deficient renal carcinoma shares some features with BRCAness tumors. However, renal cancer is known to be highly resistant to radiotherapy and cytotoxic chemotherapy, and advanced ccRCC, found in about a third of patients at diagnosis, has an extremely poor 5-year survival rate of about only 10% [The term \u201cBRCAness\u201d has been coined to describe tumors with a defect in DNA double-strand break repair by homologous recombination in the absence of utations , 67. BRCutations . Our finonly 10% . Hence, VHL-deficient cells could contribute to this finding and provide a rationale for treatment with drugs displaying hallmark sensitivity in BRCAness tumors. Given the crucial need for better treatment options in RCC, we believe that our findings support the investigation of PARP inhibitors in VHL-deficient human renal cell carcinoma.Current first- and second-line treatment of ccRCC consists of targeted therapy with VEGF receptor tyrosine kinase inhibitors and immunotherapy with IFN-\u03b1 . DespiteVHL loss in renal cell carcinoma. These findings have important implications for renal carcinoma, but may also be applicable to other cancer types. Synergism between hypoxia-induced repression of HR and PARP inhibitors has been demonstrated in vitro [In conclusion, our findings elucidate a novel connection between hypoxia-induced repression of DNA repair and in vitro , 42, 73,in vitro \u201376. ThisWT) or pRC vector alone (786-OVHL\u2013/\u2013) were generously provided by Dr. W.G. Kaelin and were authenticated by short tandem repeat profiling at the Yale DNA Analysis Facility and comparison to the published profile. RCC4 cell lines stably expressing pcDNA3-VHL (RCC4+VHLWT) or pcDNA3 vector alone (RCC4VHL\u2212/\u2212) were obtained through Sigma-Aldrich from the European Collection of Authenticated Cell Cultures. Cells were grown in high-glucose DMEM supplemented with 10% FBS (Invitrogen). Selection of cells containing the pVHL or empty vector constructs was maintained with 500 \u03bcg/mL G418 (American Bio). Hypoxic conditions were established as previously described [786-O cell lines stably expressing pRC-HA-VHL (786-O+VHLescribed .Olaparib (Sigma) in DMSO at 50 mM and BMN-673 in DMSO at 10 mM were diluted in DMSO to make 1000X solutions and added directly to cell culture media, with 0.1% DMSO used as a control. 5-Bromo-2\u2032-deoxyuridine (BrdU) (Sigma) at 1 mM in DMSO was diluted directly in cell culture media for a final concentration of 10 \u03bcM.4P2O7, 10 mM NaF) supplemented with Protease Inhibitor Cocktail (Roche) on ice for 20 min. Cellular debris was cleared by centrifugation and lysate protein concentration was quantified using the DC Protein Assay (Bio-Rad). Equal amounts of protein were subjected to SDS-PAGE in Mini-PROTEAN TGX gradient gels (Bio-Rad) and then transferred to nitrocellulose membrane. The following primary antibodies were used for western blot analysis: mouse monoclonal anti-FANCD2 , mouse monoclonal anti-BRCA1 , mouse monoclonal anti-RAD51 , mouse monoclonal anti-MLH1 , mouse monoclonal anti-Vinculin , mouse monoclonal anti-VHL , rabbit polyclonal anti-VHL , mouse monoclonal anti-HIF-1\u03b1 , and rabbit monoclonal anti-HIF-2\u03b1 . Band intensities were quantified using ImageJ software and normalized to Vinculin expression.Frozen cell pellets were lysed in AZ lysis buffer . A StepOnePlus Real-Time PCR System (Thermo Fisher Scientific) was used to measure fluorescence intensity in real-time and to calculate cycle thresholds. Ct values were normalized to 18S rRNA and relative expression was calculated using the \u2013\u0394\u0394Ct method.Total RNA was prepared using the RNeasy Mini Kit (Qiagen). The optional on-column DNase digestion was performed with the RNase-Free DNase Set (Qiagen) to eliminate genomic DNA. Complementary DNA (cDNA) was synthesized using 750 ng RNA in the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). The resulting cDNA was diluted 1:5 and used in triplicate PCRs containing TaqMan Gene Expression Assay premixed primers and probes for 5 cells/well, allowed to adhere overnight and then transfected with 50 nM SMARTpool ON-TARGETplus VHL siRNA (Dharmacon) or ON-TARGETplus GAPD Control Pool (Dharmacon) using DharmaFECT 1 Transfection Reagent (Dharmacon). Culture media was replaced 8 h post-transfection and cells were assayed by western blotting 48 h or 72 h post-transfection.Cells were plated at 10Cells were plated, allowed to adhere overnight, and treated with 10 Gy IR. Cells were harvested 24 h post-irradiation and resuspended in LM Agarose (Trevigen). Neutral single-cell gel electrophoresis was conducted using the CometAssay Electrophoresis System (Trevigen) according to the manufacturer's protocol. Data were collected with an EVOS FL microscope (Advance Microscopy Group) and analyzed with CometScore software (TriTek Corporation). Statistical analyses were performed with the Kruskal-Wallis one-way ANOVA followed by Dunn's Multiple Comparison Test using GraphPad Prism Version 7.0a for MAC OS X (GraphPad Software).For IR clonogenic assays, cells were treated with varying doses of IR and immediately reseeded in triplicate at 100, 300, and 1000 cells/well in 6-well plates. For Olaparib and BMN-673 clonogenic assays, cells were seeded in triplicate at 100\u2013500 cells/well in 6-well plates and allowed to adhere overnight. Cells were then treated continuously with varying concentrations of Olaparib or BMN-673. In all assays, cells were cultured for 7\u201310 days until colonies formed, replacing culture media every 3 days. Cells were permeabilized with 0.9% saline solution and stained with crystal violet in 80% methanol. Colonies with >50 cells were counted manually.The assay was performed as previously described using an HR luciferase reporter plasmid: a modified gWIZ.Luciferase vector (Gelantis) with an inactivating I-SceI recognition site 56 amino acids into the firefly luciferase gene and a promoter-less copy of the firefly luciferase open reading frame 700 base pairs downstream as donor template for HR . BrieflyVHL mutation and copy number alteration data, and VHL, FANCD2, BRCA1, RAD51, MLH1, VEGFA and CA9 mRNA expression Z-scores (RNA Seq V2 RSEM) for 446 complete tumor samples in the TCGA Provisional Kidney Renal Clear Cell Carcinoma dataset generated by the TCGA Research Network (http://cancergenome.nih.gov) were downloaded via cBioPortal [VHL mutation status, VHL copy number, or putative VHL genotype and mRNA expression of each gene was compared between groups.ioPortal , 78. On ioPortal . The rem"} +{"text": "Hypofunction of the N-methyl-D-aspartate receptor (NMDA-R) has been implicated as a possible mechanism underlying cognitive deficits and aberrant neuronal dynamics in schizophrenia (ScZ).In a single-blind cross-over design, 14 participants received either a subanaesthetic dose of S-ketamine (0.006 mg/Kg) or saline while Magnetoencephalographic (MEG) data were recorded during a visual task. In addition, MEG-data were obtained in a sample of unmedicated first-episode psychosis (FEP) patients (n = 10) and in patients with chronic ScZ (n = 16). MEG-data were analyzed at source-level in the 1\u201390 Hz frequency range in occipital and thalamic regions of interest (ROIs). In addition, directed functional connectivity analysis was performed using Granger Causality (GC). Psychopathology was assessed by the Positive and Negative Syndrome Scale (PANSS).Behavioral impairments were accompanied by increased amplitude and frequency of gamma-power (63\u201380 Hz) in occipital regions during Ketamine administration, while low-frequency (~5\u201330 Hz) activity was upregulated. Moreover, Ketamine disrupted feedforward (FF) and feedback (FB) signaling at high and low frequencies leading to hypoconnectivity in thalamo-cortical (TC) networks. In contrast, FEP and chronic ScZ patients showed a different pattern of MEG-activity, characterized by decreased task-induced high-gamma band oscillations and increased FF/FB-mediated GC-connectivity.The effects of Ketamine on high-frequency oscillations and their connectivity profile are not consistent with observations in FEP and chronic ScZ-patients. Accordingly, the current data have implications for theories of cognitive dysfunctions and circuit impairments in the disorder, suggesting that the effects of acute NMDA-R hypofunction are not consistent with impairments in visuo-perceptual oscillations in ScZ-patients."} +{"text": "Telomerase (TERT) is a ribonucleoprotein enzyme that preserves the molecular organization at the ends of eukaryotic chromosomes. Since TERT deregulation is a common step in leukaemia, treatments targeting telomerase might be useful for the therapy of hematologic malignancies. Despite a large spectrum of potential drugs, their bench-to-bedside translation is quite limited, with only a therapeutic vaccine in the clinic and a telomerase inhibitor at late stage of preclinical validation. We recently demonstrated that the adoptive transfer of T cell transduced with an HLA-A2-restricted T-cell receptor (TCR), which recognize human TERT with high avidity, controls human B-cell chronic lymphocytic leukaemia (B-CLL) progression without severe side-effects in humanized mice. In the present report, we show the ability of our approach to limit the progression of more aggressive leukemic pathologies, such as acute myeloid leukaemia (AML) and B-cell acute lymphoblastic leukaemia (B-ALL). Together, our findings demonstrate that TERT-based adoptive cell therapy is a concrete platform of T cell-mediated immunotherapy for leukaemia treatment. On the contrary, preclinical experiments using a selective hCD123 CAR able to eliminate human AML cells caused complete eradication of normal bone marrow (BM) cells in mice engrafted with human CD34+ stem cells [Acute myeloid leukaemia (AML) and B-cell acute lymphoblastic leukaemia (B-ALL) are still incurable diseases for a number of patients and a leading cause of death in children and adults, respectively \u20133. Curre*0201 BiTEs or*0201 \u201327. BiTE+T cells were detected, with a higher frequency, in the blood of patients affected by chronic lymphocytic leukemia (B-CLL), as well as breast, lung and colorectal cancers, compared to healthy donors (HD) [865-873-specific, TCR-engineered T-cells both to efficiently recognize different solid human tumour cells and restrict human B-CLL tumour progression in vivo without inducing dramatic toxicity. In fact, the hTERT-specific ACT did not induce myeloid precursor depletion in tumour-bearing humanized mice, affecting only BM resident mature granulocytes and preserving the ability of hCD34+ cells to generate mature leukocytes [Human Telomerase (TERT) has been identified as a common hallmark of cancer, since it plays a critical role in aberrant cell proliferation and immortalization in the majority of tumours . Variablors (HD) \u201342. Howeors (HD) . To overukocytes . We desc865-873-specific, TCR-engineered T-cells transferred to human tumour-bearing immunocompromised (NOG) mice. In particular, we emphasized the ability of TERT-based ACT to promote a strong control of tumor growth in several cancer settings, while ignoring hematopoietic progenitors and activated T lymphocytes [865-873-specific, TCR-engineered T-cells specifically induced the contraction of the mature granulocytic cell subset resident in the BM of human immune reconstituted (HIR) mice [865-873-specific TCR-engineered T-cells on AML cells. We initially verified TERT expression and activity in both PBMCs isolated from HLA-A2+ AML patients or HD and THP1 human myeloid leukaemia-immortalized cells -\u03b3 in response to patients\u2019 samples, at similar levels to those reached using THP1 cells as target mice . Startins Figure . All leus Figure . Indeed,e Figure . These d865-873-specific TCR-engineered T-cells on controlling AML progression, we subcutaneously (s.c.) challenged immunodeficient NOG mice with THP1 cells. Our immunotherapeutic treatment based on anti-TERT CTLs infusion significantly controlled tumour growth inducing a survival benefit on treated mice compared to mice treated with hHCV1406-1415-specific TCR-engineered T-cells injection delivery, to mimic the disseminated disease in patients. THP1-Luc cells were recognized in vitro by the hTERT865-873-specific TCR-engineered T-lymphocytes at levels comparable with wild type (WT) THP1 cells (data not shown). Immunocompromised NOG mice were injected i.v. with 3\u00d7105 THP1-Luc cells and treated with three weekly infusions of either hTERT865-873-specific TCR-engineered or hHCV1406-1415-specific TCR-engineered T-cells seven days after tumour challenge. Tumour progression was evaluated through bioluminescence imaging. Figure 6 p/s/cm2/sr). Lymphopoietic organs (spleen and BM) isolated from control HCV-based ACT treated mice presented a more severe leukemic cell infiltration compared to TERT-based ACT treated mice were characterized by higher levels of telomerase expression and activity, when compared to B cells isolated from HD PBMCs . We engrafted immunocompromised NOG mice with 1.5\u00d7106 hCD19+ cells and then assessed the leukemic progression by quantifying the frequency of circulating hCD19+ lymphocytes. Mice were treated with three consecutive ACT, starting one week after tumour challenge. The TERT-based immunotherapeutic approach significantly controlled leukemic expansion compared to the control therapy in TERT-treated mice compared to control mice, therefore suggesting an effective control of leukemic spread hTERT865-873-specific TCR-engineered T-cells show specificity in recognizing both primary and immortalized AML and ALL cells but not target cells pulsed with unrelated antigen, normal PBMCs or B-cells; d) TERT-based ACT controls in vivo the systemic dissemination of AML and ALL cells; e) TERT-based ACT significantly prolongs the survival of ALL affected hosts. All these findings depict TERT-targeting immunotherapy as a promising strategy for treating leukemic patients.The results of our study demonstrated that: a) AML and ALL cells not only overexpress TERT protein but also show an higher TERT activity compared to normal cells; b) AML and ALL cells process TERT protein, generate the immunodominant hTERTvia TERT overexpression [865-873-specific TCR-engineered T-cells ACT to control different human solid tumour growth [Although the outstanding clinical success achieved by CAR-mediated immunotherapy in contrasting B-ALL progression has comprehensibly attracted the interest of scientific community for the development and improvement of this type of engineered T-cell therapy, the idea of redirecting T-lymphocytes with high affinity TCR selective for universal tumour-associated antigens, such as TERT, may open new avenues on the clinical procedure. Indeed, TERT is shared and actively expressed in about 85% of human tumours with various histological type . Transfopression . These fpression . We addir growth . Finallyr growth , as wellr growth , 53 or dr growth , such asr growth , within in vivo persistence of hTERT865-873-specific TCR-engineered T-cells rather than their exhaustion. In fact, before their in vivo administration, TCR-transduced T-cells had an effector/memory phenotype (CD45RA\u2212CD62L\u2212CCR7\u2212 cells) characterized by the low-expression of exhaustion markers (LAG3/PD-1/Tim-3). Other immune-evasion mechanisms, such as antigen loss or antigen presentation deficiency, seem not to play a critical role in our experimental setting since residual leukemia cells in TERT-treated mice maintained telomerase expression and HLA-A2 molecules (data not shown). As recently reported, the identification and characterization of T memory stem cells (TSCM) generated an increased interest for their therapeutic use for cancer treatment. TSCM cells, that are the earliest developmental stage of memory T cells, exhibit stem cell-like properties and display a gene profile between na\u00efve and central memory T cells [SCM cells show in preclinical models an intense self-renewing capacity following antigen loss, a long lifespan since they are refractory to apoptosis and a robust proliferative potential [SCM cells in human showed an extended longevity persisting in the host for up to 12 years post infusion [SCM cells in vivo persistence not required cytokine supply [SCM cells might completely eliminate all the adverse effects produced by the administration of high dose of IL-2 [865-873-specific TCR-engineered TSCM-cells, taking advantage of recent protocols for TSCM cells in vitro isolation and expansion, to provide their increased benefit on tumor control [in vivo analysis of anti-leukemic cell-based therapy in the presence of a human immune system [The limited therapeutic impact of our immunotherapy is probably due to the low T cells . Moreoveotential but overinfusion . TSCM cee supply ; therefo of IL-2 . We plan control , 60, in control , 62. A pe system .+ T cells that caused a transient B-cell lymphopenia. This toxicity might be related to the schedule and type of immunotherapeutic approach. In fact, the side effects were observed when repeated infusions of TERT-specific CTLs were associated with the injection of an adenovirus encoding mouse TERT protein and the administration of high dose of IL-2, whereas DNA vaccination did not induce any detectable adverse event [in vitro activation of hTERT865-873-specific TCR-engineered T-cells in contact with vital tissue slides of specimens with different histology. Moreover, to limit an uncontrolled activation of our transduced T cell that may produce severe immunopathologies, we plan to incorporate a suicide gene in the retroviral vector encoding TERT-specific TCR sequences. Indeed the use of an inducible caspase 9 (iCas9) \u201csafety switch\u201d has already produced excellent results on controlling graft-versus-host disease (GVHD) development in patients of haploidentical stem-cell transplants [The most serious concern about the impact of TERT-based ACT in clinic is related to its safety. In fact, even if tumour tissues showed a higher TERT expression and telomere length compared to normal tissues, it is impossible to predict whether and to what extent normal cells would be recognized by TERT-specific T cells. Prudence is needed when designing a selective TERT-targeting immunotherapy, as pointed out by our previous data about the treatment of prostate tumor-bearing mice with three cycles of ACT with mouse TERT-specific CD8se event , 64. HowCollectively, our data proved and reinforced the potential clinical translation of TERT-based immunotherapy on treating different leukemic pathologies.PrkdcscidIl2rgtm1Sug/JicTac) were purchased from Taconic. All animal experiments were approved by Verona University Ethical Committee (http://www.medicina.univr.it/fol/main?ent=bibliocr&id=85) and authorized by Ministerial Decree (16/2014-B). B-ALL animal experiments were in accordance with the Amsterdam Protocol on animal protection and welfare and conducted according to the guidelines of Federation of European Laboratory Animal Science Associations (FELASA).NOG mice (NOD.Cg-2 atmosphere.PBMCs isolated from AML patients and HDs and BM (BM) cells from B-ALL were collected at the Hematology Unit, Azienda Ospedaliera Universitaria Integrata (AOUI) in Verona . All participating people provided written informed consent for the collection and use of their samples for research purposes, in compliance with the Declaration of Helsinki. The study was approved by the local Ethics Committee . THP1 acute monocytic leukemia cell line were obtained from American Type Culture Collection (ATCC), and were maintained in RPMI 1640 medium (Lonza). ALL-CM acute lymphocytic leukemia cell line were provided by Dr. A. Bondanza under restricted condition with Leiden University Medical Center and cult865-873 -specific TCR sequences were isolated as previously described in [1406-1415 (KLVALGINAV) were used. hTERT865-873 - and hHCV1406-1415- specific T cells were obtained by transduction of OKT-3-activated PBMCs with the viral supernatant of hTERT865-873/PG13 or HCV1406-1415/PG13 cell lines in the presence of hIL-15 and rIL-2 . Selected T cells were then expanded in AIM-V medium (Gibco) supplemented with 5% human serum (Gibco) with OKT-3 , recombinant IL-2 and human IL-15. The percentage of CD4+ and CD8+ T cells in the culture was always tested before in vitro or in vivo studies. In general, CD8+ T lymphocytes represent about 70-80% of the total T cells and numbers for in vivo treatments were adjusted in order to inject 2.5\u00d7106 CD8+ T cells.hTERTribed in . As cont\u00ae XL telomerase detection kit; Millipore), following manufacturer's instructions.Telomerase expression was assessed by western blotting as previously described . TelomerSingle-cell suspensions were labelled with either fluorochrome-conjugated Ab anti-mouse CD45.1 (A20) or Ab anti-human CD3 (OKT-3), CD19 (HIB19), CD45 (2D1), HLA-A2 (BB7.2), purchased from either Biolegend or eBiosciences and the relative isotype controls purchased from the same companies. B-ALL samples were acquired with a FACSCanto II (BD) and analyzed with FlowJo software (Treestar Inc.).+ cells.Crio-preserved BM aspirates from ALL patients were thawed and the total cells were sorted with hCD19-Microbeads (Miltenyi) according with the standard procedures. Purified cells were analyzed by flow citometry and they reached the ~95% of CD195 engineered CTLs were co-cultured at 3:1 ratio in presence of with human malignant cells or control cell targets (PBMCs or purified hCD19+ cells isolated from HDs). Negative and positive controls were represented by HD PBMCs pulsed with either hHCV1406-1415 or hTERT865-873 peptides, respectively. After 24 hours (h) of co-culture, the supernatants were harvested to measure the released IFN-\u03b3. Moreover, functional response was also evaluated by flow cytometric cytotoxic activity assay as previously described [IFN-\u03b3 secretion was quantified by a sandwich enzyme-linked immunosorbent assay according to manufacturer's instructions. Briefly, 3\u00d710escribed . In briein vivo testing of the therapeutic activity of hTERT865-873-specific T cells, 5\u00d7106 THP-1 or 5\u00d7106 ALL-CM cells were inoculated s.c. in the left flank of NOG mice. Tumour volume was calculated according to the following equation: V (mm3) = (d2 \u00d7 D)/2, where d (mm) and D (mm) are the smallest and largest perpendicular tumour diameters, respectively, as assessed by caliper measurement. Treatments started when tumour volume reached 100 mm3. hTERT865-873- or control HCV1406-1415-specific T cells were injected i.v. in the tail vein once a week, for 2 consecutive weeks. Every adoptive transfer was followed by the administration of recombinant human IL-2 intraperitoneally every 12h, for a total of 6 doses.For 6) or with hCD19+ cells isolated from a HLA-A2+ B-ALL patient (1.5\u00d7106). Mice were then treated with three, weekly ACT of hTERT865-873-specific or control HCV1406-1415-specific T-cells starting one week after tumour challenge, followed by IL-2 administration, as previously indicated. Leukemic spread was monitored as percentage of circulating hCD19+ cells in peripheral blood and we set up a survival threshold of hCD19+ circulating leukemic cells in the amount of either 60% in the case of ALL-CM tumour setting or 80% in the case of patient derived ALL tumour setting.Eight-week-old NOG mice were intravenously (i.v.) injected with ALL-CM cells . THP1-Luc cells were sorted to obtain a 95% of EGFP+ cell population that was used for the study. A total of 3 infusions of 2.5\u00d7106 hTERT865-873- or hHCV1406-1415-specific CTLs were retro-orbitally injected every 7 days, starting 1 week after tumour injection, followed by IL-2 administration, as previously indicated. Leukaemia burden was monitored by BLI (photons/second/cm2/sr) using the IVIS Spectrum Imaging System (PerkinElmer). Images were acquired prior to treatment and then weekly, 7 days after the last treatment. Animals were anesthetized with isoflurane/oxygen. For each animal, dorsal and ventral images were obtained pre and 10 minutes after i.p. administration with 150 mg/kg of D-luciferin (PerkinElmer) in PBS in order to discriminate specific light signal from basal emission. Five mice were imaged simultaneously with the subsequent parameters: exposure time = 5 minutes, field of view = 19\u00d719 cm, binning B = 8 and f/stop = 1. Images were quantified tracing the region of interest (ROI) on the entire animal body. Living Image Software 4.4 (PerkinElmer) was used to acquire and quantify the bioluminescence.NOG mice (8-10 weeks of age) were firstly injected i.v. with 3\u00d710Spleen and BM samples were fixed in 10% neutral buffered formalin; after fixation, BM samples were decalcified with 10% EDTA (pH 7.4) for 3 weeks and embedded into paraffin; 5 \u03bcm slides were cut and stained with Hematoxylin (BioOptica) and Eosin (BioOptica) for histological examination. For immunohistochemistry, sections were deparaffinized, serially rehydrated and after the appropriate antigen retrieval procedure, incubated with the following primary antibodies: monoclonal Mouse Anti-Human CD45 or monoclonal Mouse Anti-Human CD20 , followed by the secondary antibody Dako EnVision System-HRP Labelled Polymer Anti-mouse . After chromogen incubation, slides were counterstained in Hematoxylin and images were acquired by Leica DMRD optical microscope (Leica). The absence of cross-reactions between human and mouse antigens was verified testing anti-human antibodies on a normal mouse spleen. The percentage of positive cells was evaluated on the digital images of 5-6 samples per group (6-10 \u00d7 200 microscopic fields per sample) by 2 pathologists, independently and in a blind fashion.P values less than 0.05 were considered statistically significant.Data were indicated as the mean \u00b1 SD. Student's t test was used to determine statistically significant differences between two treatment groups, while ANOVA test was used in case of multiple comparisons. Growth curves were analyzed with Repeated Measures (RM) ANOVA. Survival analysis was performed using the Kaplan-Meier survival analysis (Log-Rank) method."} +{"text": "Since this up-regulation has not been detected in T cells in GCs and in Concanavalin A-stimulated T cells in vitro, selective function of GANP molecule on B cell proliferation and differentiation might exist.Adaptive immunity is dependent on proliferation of antigen-driven B cells for clonal expansion in germinal centers (GCs) against T cell-dependent antigens (TD-Ag), accompanied with somatic hypermutation of variable-region gene and class switching of B cell antigen receptors. To study molecular mechanisms for B cell differentiation in GCs, we have identified and studied a 210 kDa GANP protein expressed in GC-B cells. GANP has domains for MCM3-binding and RNA-primase activities and is selectively up-regulated in centrocytes surrounded with follicular dendritic cells (FDCs) upon immunization with TD-Ag"} +{"text": "Characterization of genetic diversity among locally prevalent parasite strains and the ensuing strain-specific immunity will be particularly important in a specific geographical setting for development of vaccine constructs and for planning future vaccination strategies.Plasmodium vivax, 42 kDa fragment of the Merozoite Surface Protein -1 (MSP-142), Apical Membrane Antigen-1 domain II (AMA-1 DII) and the Duffy Binding Protein region II (PvDBPII), in patients infected with P. vivax malaria from two endemic areas, Anuradhapura and Kataragama, in Sri Lanka, where unstable malaria prevails with low transmission, and from a malaria non-endemic area, Colombo, where the patients contracted the infections while visiting areas endemic to vivax malaria. Based on nucleotide sequences, extensive allelic polymorphism was evident in the N-terminal region of MSP-142(Pvmsp-133), Pvama- 1dII and PvdbpII, due to recombination, mutations, natural selection and genetic differentiation. Nucleotide sequences of 42Pvmsp-1(N=95), Pvama-1dII (64) and PvdbpII (100), generated 27, 21 and 33 amino acid (a.a) haplotypes, respectively. Clinical isolates with amino acid haplotypes available for all three antigens (N=24) revealed uniquely different haplotype combinations in each isolate. Conversely, PvMSP-119, domain II loop of PvAMA-1 and the six a.a. in the critical binding domain of PvDBPII responsible for direct binding with the Duffy antigen on human RBC, showed 100% conservation of the amino acid sequences.The present study characterized the prevailing genetic diversity of three major asexual stage putative vaccine antigens of 42, PvAMA-1DII and PvDBPII were aligned with the homologous total (IgM + IgG) antibody responses assayed by ELISA against the recombinant protein of each antigen, and the seven (P01-P07) PvAMA-1DII peptides. Though Anti-PvMSP-119 and anti-P04 antibody prevalence precludes strain-specific immune responses against the highly conserved PvMSP-119 and the P04 peptide on domain II loop of PvAMA-1, a protective immune response was clearly elicited to these two regions by the marked isotype switch from IgM to IgG with increasing exposure in areas of endemicity. Further, the presence of strain transcending (cross reactive) PvDBPII specific functionally active binding inhibitory antibodies were demonstrated among locally exposed P. vivax patients. Thus this study may support the inclusion of PvMSP-119, P04 region of PvAMA-1 domain II and region II of PvDBP as veritable vaccine components for a multi component \"cocktail\" asexual stage vaccine against P. vivax malaria in Sri Lanka.In order to evaluate whether sequence differences among each antigen was indicative of strain-specific immunity, amino acid sequences of PvMSP-1"} +{"text": "Camptothecins are DNA topoisomerase I-directed anti-tumour drugs with a novel mechanism of action. Topotecan (TPT), a hydrophilic derivative of camptothecin, is currently undergoing phase II clinical trials in small-cell lung cancer (SCLC). Human SCLC OC-NYH cells were made more than 6-fold resistant to topotecan by stepwise drug exposure and resistance was stable for 70 passages without drug. NYH/TPT cells had half the topoisomerase I level and activity of wild-type cells. However, no difference in camptothecin or topotecan inhibition of topoisomerase I-mediated DNA relaxation was found, indicating that the enzyme itself was unchanged in the resistant cell. In NYH/TPT cells, topoisomerase II alpha and beta levels were increased approximately 2-fold. Accordingly, the topoisomerase II-directed drug etoposide (VP-16) induced an increased number of DNA single-strand breaks in NYH/TPT cells. However, sensitivity to different topoisomerase II-targeting agents in NYH/TPT cells varied from increased to decreased, indicating a role for as yet unidentified factors acting on the pathway to cell death after topoisomerase II-induced DNA damage has occurred. Of 20 anti-cancer agents tested, only hydroxyurea showed marked collateral hypersensitivity in NYH/TPT cells."} +{"text": "Nitric oxide (NO), an endogenous free radical, has been implicated in a wide range of biological functions. NO is generated enzymatically from the terminal guanidinonitrogen of L-arginine by nitric oxide synthase (NOS). Despite intensive investigations, the role of NO--either as the primary product of the L-arginine/NOS pathway or provided from the NO donor sodium nitroprusside (SNP)--in carcinogenesis and tumour cell growth remains unclear and controversial. The objective of this study was to examine the growth effects of NO on a ductal pancreatic adenocarcinoma in the rat and on a human pancreatic tumour cell line (HA-hpc2). In vivo, both SNP and endogenous induction of NO by endotoxins [lipopolysaccharide (LPS)] plus L-arginine significantly reduced the tumour growth. To investigate the mechanisms of NO anti-tumour growth action, the effects of either the SNP or L-arginine/NOS pathway were analysed on the HA-hpc2 cell line. Nitrite/nitrate production, NOS activity and iNOS expression [assessed by reverse transcription-polymerase chain reaction (RT-PCR)] were tested and related to growth (assessed by [3H]thymidine incorporation assay) and apoptosis . SNP exerted a dual effect on tumour cells: stimulation of the proliferation up to 1 mM and inhibition at higher concentrations. These effects were related to NO production. Both proliferative and cytostatic responses were inhibited by NO scavenger 2-phenyl-4,4,5,5-tetramethyl-hemidazoline-1-oxyl3-oxide (carboxy-PTIO). The marked apoptotic DNA fragmentation induced by SNP was also abolished by PTIO association. Unlike macrophages, the human pancreatic tumour cells did not seem to express intrinsically the L-arginine/NOS pathway. Macrophages were activated by HA-hpc2 cells as well as by LPS plus cytokines [interleukin (IL)-1beta plus tumour necrosis factor (TNF)-alpha and interferon (IFN)-gamma]. In HA-hpc2/macrophage co-cultures, NOS activity and inducible NOS (iNOS) transcription were stimulated, whereas an antiproliferative response was observed. These effects were related to both macrophage amount and NO production. Addition of LPS plus cytokines to co-cultures doubled iNOS activity, nitrite/nitrate production and tumoricidal effect. These data suggest the involvement of NO in pancreatic tumour growth and support the fact that generation of high levels of NO with potential production of endogenous reactive nitrogen intermediates may contribute to induction of apoptosis and tumour growth inhibition."} +{"text": "To the Editor: Nontyphoidal salmonellosis (NTS) is a major food-borne illness worldwide. Extended-spectrum cephalosporins (ESCs) are currently preferred drugs for treatment of children with NTS. However, resistance to ESCs has emerged worldwide and has become a serious public health problem. This resistance is caused by production of various class A extended-spectrum \u03b2-lactamases (ESBLs) and class C cephalosporinases in Salmonella enterica , the Moroccan National Institute of Hygiene reported only 210 human non-Typhi isolates and 999 animal non-Gallinarum isolates during 1999\u20132003. Antimicrobial drug resistance data are extremely rare. We report the presence of nontyphoidal Salmonella isolates resistant to ESCs during an outbreak of food poisoning and in food products in Morocco.According to the World Health Organization Global Salm Surv database standard pulsed-field gel electrophoresis (PFGE) of blaTEM, blaSHV, and blaCTX-M group genes, as described , ceftriaxone (MIC 64 mg/L), ceftazidime (MIC 128 mg/L), streptomycin, sulfonamides, chloramphenicol, and tetracycline. We identified the blaCMY-2 gene carried by a 210-kb nonconjugative plasmid of replicon IncA/C. These CMY-2\u2013producing isolates are also known as Salmonella Newport multidrug-resistant (MDR)\u2013AmpC. XbaI-PFGE showed a profile similar to the New8a profile described in 2003 in France during a small outbreak linked to consumption of imported horse meat (ESC-resistant Salmonella Newport MDR-AmpC strain in Africa. Salmonella Newport MDR-AmpC isolates were reported in 1998 in the United States (Although CMY-2 was originally identified in a serotype Senftenberg isolate from a child in Algeria ("} +{"text": "Activation of cell surface receptors transduces extracellular signals into cellular responses such as proliferation, differentiation and survival. However, as important as the activation of these receptors is their appropriate spatial and temporal down-regulation for normal development and tissue homeostasis. The Cbl family of E3-ubiquitin ligases plays a major role for the ligand-dependent inactivation of receptor tyrosine kinases (RTKs), most notably the Epidermal Growth Factor Receptor (EGFR) through ubiquitin-mediated endocytosis and lysosomal degradation.Drosophila cbl (D-cbl) during eye development. D-cbl mutants display overgrowth, inhibition of apoptosis, differentiation defects and increased ommatidial spacing. Using genetic interaction and molecular markers, we show that most of these phenotypes are caused by increased activity of the Drosophila EGFR. Our genetic data also indicate a critical role of ubiquitination for D-cbl function, consistent with biochemical models.Here, we report the mutant phenotypes of cbl genes.These data may provide a mechanistic model for the understanding of the oncogenic activity of mammalian Normal cellular function and tissue homeostasis is dependent on the precise regulation of several signal transduction pathways that control cell proliferation, cell differentiation and cell survival. Each cell integrates an array of extracellular signals into appropriate cellular responses. Deregulation of these processes causes developmental abnormalities and human diseases including cancer. However, we still lack a clear understanding of how these processes are integrated in the context of a developing organism.Drosophila compound eye has long been a model system to study how extra-cellular signaling generates precise cellular differentiation patterns , the epidermal growth factor receptor (EGFR) and Sevenless (Sev), contribute to retinal development EGFR (DNEGFR), or shifting a temperature-sensitive EGFR allele to the non-permissive temperature leads to an impairment of differentiation argos, gap1 and sprouty, three negative regulators of EGFR, contain extra R and cone cells surrounded by more secondary and tertiary pigment cells in the lattice Drosophila eye development, due to its negative regulation of hid, a cell death-inducing gene sev is required only for R7 differentiation The specification of cell fate in the developing C.elegans in which Sli-1, the Cbl ortholog, attenuates the activity of Let-23, the EGFR equivalent, in vulval development. c-cbl, cbl-b and cbl-3 have no obvious developmental phenotypes except in the immune system suggesting that they are functionally redundant Drosophila has only one cbl gene, referred to as D-cblD-cbl mutants may reveal more information about its oncogenic role. For example, an isoform of D-cbl, which mimicked the oncogenic viral cbl (v-cbl), demonstrated that v-cbl acts in a dominant negative manner C.elegans and mammalian cell culture, D-Cbl has been shown to function as a negative regulator of EGFR during dorsoventral patterning in oogenesis and guided migration of border cells D-cbl for eye development in Drosophila has not been reported.As important as the activation of cell surface receptors is their inactivation for appropriate control of cell number and differentiation. The proto-oncogene Casitas B-lineage lymphoma (Cbl) was first identified as a retroviral transforming gene product that induces pre-B cell lymphomas and myeloid leukemia D-cbl encodes two alternatively spliced isoforms, D-cblSHORT (D-cblS) and D-cblLONG (D-cblL), both of which contain the TBK and the RING E3 ubiquitin ligase domains, while D-CblL also has proline-rich (SH3 binding) and UBA domains similar to c-Cbl and Cbl-b Mechanistically, Cbl binds tyrosine-phosphorylated EGFR through its tyrosine kinase binding (TKB) domain D-cbl mutant phenotype for eye development. D-cbl mutants display overgrowth and lack of developmental apoptosis. Mutant ommatidia contain increased numbers of photoreceptors (mostly R7), cone and pigment cells. Genetic interaction tests indicate that D-cbl regulates the EGFR pathway during eye development consistent with its proposed role as negative regulator of EGFR. Our genetic data indicate a critical role of ubiquitination for D-cbl function, in accord with biochemical models. In summary, these data provide a genetic model for the understanding of the oncogenic activity of mammalian cbl genes.Here, we present the characterization of the D-cbl alleles as recessive suppressors of the small eye phenotype caused by expression of the pro-apoptotic gene hid under control of the eye-specific GMR enhancer as a marker of RTK activity is increased in D-cbl clones in third instar larval eye discs , the increased p-Tyr labeling is mainly caused by increased EGFR activity.To further confirm this notion we tested three molecular markers in loss-of-function and gain-of-function analyses of ye discs Fig. 3A.. In D-K26cbl mutant clones, developmental cell death is significantly blocked (D-cbl mutants as suppressors of GMR-hid (In the D-cbl mutant clones display differentiation defects. The eye disc is fully differentiated by 42 hrs APF D-cbl mutant ommatidium contains on average 11.45\u00b11.03 (n\u200a=\u200a35) R cells compared to eight in wild-type ommatidia and Seven-up did not reveal significant differences between D-cbl and wild-type tissue , a second RTK, is involved in eye development. D-cbl clones result from hyper-activity of Sev, then these cells should require sev for their specification. However, genetic removal of sev using the null allele d2sevK26D-cbl mosaic background , containing more than four R7 cells per ommatidium , further supporting an essential role of ubiquitylation for D-cbl function.In contrast to G domain Fig. 1K.D-cbl clones. The fact that D-cbl clones contain up to four additional R7 cells is likely due to the fact that R7 and the four cone cells are developmentally equivalent. These five cells express sev and all have the capacity to become R7 if Sev or downstream components are activated D-cbl clones likely represent transformed cone cells.It is unclear why only the number of R7 cells is affected whereas the remaining R cells are normal in number although R1\u2013R6 also require the EGFR for specification. However, it suggests that the sequence of events during R cell specification is normal in D-cbl clones. In contrast, we even observe an over-recruitment of cone cells. Interestingly, the cone cell over-recruitment in D-cbl mutants does not occur during pupal stages as suggested for gap1D-cbl clones follows the same rules of reiterative use of the EGFR as compared to wild-type.However, this transformation does not mean that the cone cells are lost in D-cbl mutant phenotypes are similar to the ones described for argos, gap1 and sprouty, we noticed at least two phenotypes which appear to be specific for D-cbl. First, D-cbl mutant heads and imaginal discs are overgrown results in an eye ablation phenotype (GMR-hid ey-FLP (GheF) method GMR-hid eye ablation phenotype. This method induces homozygous mutant clones in the eye by ey-FLP/FRT-mediated mitotic recombination in otherwise heterozygous background GheF screening, ey-FLP; FRT80 males were incubated with 25 mM EMS in 5% sucrose solution for 24 hours. After recovery for 3 hours, they were mated to GheF; +]FRT80 P FRT80B , UASp-P35DNUAS-EGFRN17sev-rasactsev-yand2sevD-cbl mutant clones, K26 FRT80BD-cbl and D-7 FRT80Bcbl flies were crossed to ey-FLP; P[ubi-GFP] FRT80B. Clones are marked by loss of GFP. DNGMR-EGFR is DNGMR-Gal4 UAS-EGFR.Fly crosses were conducted using standard procedures at 25\u00b0C. Pupal developmental ages are expressed as hours after puparium formation (APF) with white pre-pupae defined as 0 hour APF. The following stocks were used: Eye imaginal discs from the indicated larval or pupal stages were dissected and immunohistochemical labeling was performed as described"} +{"text": "The present report describes the non-haematological toxicity and the influence of growth factor administration on haematological toxicity and haematopoietic recovery observed after high-dose carboplatin (1200 mg m(-2)), etoposide (900 mg m(-2)) and melphalan (100 mg m(-2)) (CEM) followed by peripheral blood progenitor cell transplantation (PBPCT) in 40 patients with high-risk cancer during their first-line treatment. PBPCs were collected during the previous outpatient induction chemotherapy programme by leukaphereses. CEM administration with PBPCT was associated with low non-haematological toxicity and the only significant toxicity consisted of a reversible grade III/IV increase in liver enzymes in 32% of the patients. Haematopoietic recovery was very fast in all patients and the administration of granulocyte colony-stimulating factor (G-CSF) plus erythropoietin (EPO) or granulocyte-macrophage colony-stimulating factor (GM-CSF) plus EPO after PBPCT significantly reduced haematological toxicity, abrogated antibiotic administration during neutropenia and significantly reduced hospital stay and patient's hospital charge compared with patients treated with PBPCT only. None of the patients died early of CEM plus PBPCT-related complications. Low non-haematological toxicity and accelerated haematopoietic recovery renders CEM with PBPC/growth factor support an acceptable therapeutic approach in an adjuvant or neoadjuvant setting."} +{"text": "The ability of tumour necrosis factor alpha , a potent endogenous inflammatory agent, to promote malignant transformation of Syrian hamster embryo cells (SHE) initiated by a 0.5-Gy dose of alpha-particles was investigated. Opsonized zymosan particles, which were phagocytosed by a human macrophage-like cell line, triggered TNF-alpha production from U937 cells. This cell supernatant could significantly increase the transformation frequency (TF) of primary SHE cells previously irradiated by a 0.5-Gy dose of alpha-particles. The TF decreased significantly if monoclonal antibody against TNF-alpha was added to the supernatant. Similarly, recombinant human TNF-alpha increased the TF of alpha-irradiated primary SHE cells to an even greater extent. Addition of TNF-alpha to subcultures of irradiated SHE cells permitted the continuous propagation of these primary cells. In contrast, both TNF-alpha-treated control and alpha-irradiated cells without subsequent TNF-alpha treatment senesced after 7-15 passages. Irradiated SHE cells treated continuously with TNF-alpha could be subcultured over 40 passages and produced fibrosarcomas upon inoculation into nude mice. Our results provide the first evidence that TNF-alpha released by activated macrophages may contribute to the process of malignant transformation initiated by low-dose alpha-particles."} +{"text": "Imatinib mesylate, a selective inhibitor of Abl tyrosine kinase, is efficacious in treating chronic myeloid leukaemia (CML) and Ph+ acute lymphoblastic leukaemia (ALL). However, most advanced-phase CML and Ph+ ALL patients relapse on Imatinib therapy. Several mechanisms of refractoriness have been reported, including the activation of the Src-family kinases (SFK). Here, we investigated the biological effect of the new specific dual Src/Abl kinase inhibitor AZD0530 on Ph+ leukaemic cells.Bcr-Abl infected Ba/F3 cells, p185Bcr-Abl mutant infected Ba/F3 cells, SupB15 (Ph+ ALL) and Imatinib resistant SupB15 (RTSupB15) (Ph+ ALL) cells. Cells were exposed to AZD0530 and Imatinib. Cell proliferation, apoptosis, survival and signalling pathways were assessed by dye exclusion, flow cytometry and Western blotting respectively.Cell lines used included BV173 (CML in myeloid blast crisis), SEM t, Ba/F3 (IL-3 dependent murine pro B), p185Bcr-Abl was overcome by AZD0530. Combination of AZD0530 and Imatinib showed an additive inhibitory effect on the proliferation of CML BV173 cells but not on Ph+ ALL SupB15 cells. An ongoing transphosphorylation was demonstrated between SFKs and Bcr-Abl. AZD0530 significantly down-regulated the activation of survival signalling pathways in Ph+ cells, resistant or sensitive to Imatinib, with the exception of the RTSupB15.AZD0530 specifically inhibited the growth of, and induced apoptosis in CML and Ph+ ALL cells in a dose dependent manner, but showed only marginal effects on Ph- ALL cells. Resistance to Imatinib due to the mutation Y253F in p185Our results indicate that AZD0530 targets both Src and Bcr-Abl kinase activity and reduces the leukaemic maintenance by Bcr-Abl. The cytogenetic hallmark of chronic myeloid leukaemia (CML) and a subset of acute lymphoblastic leukaemia (ALL) is the Philadelphia (Ph) chromosome. It is a shortened chromosome 22, generated by a reciprocal translocation between chromosome 9 and 22 tq34;q11) [1 [1].The most exciting breakthrough in the treatment of Ph+ leukaemias has been the development of Imatinib as an orally bioavailable therapeutic agent . AlthougRecent reports have demonstrated a requirement for Src kinase activity in Bcr-Abl transformation and oncogenic signal transduction . Bcr-AblIn Imatinib resistant patients, a non-Bcr-Abl dependent up-regulation in SFK expression has been observed . Up-regu2-terminal portion of Abl bears 42% identity to the SFK and shares a similar domain organisation [The NHnisation . Src inhnisation . Moreovenisation .Based on this rationale, we investigated the effects of a new dual Src/Abl kinase inhibitor, AZD0530 with the aim of inhibiting both Src and Bcr-Abl kinases irrespective of their conformations to explore the possibility of overcoming resistance to Imatinib with the use of AZD0530.Stratagene's QuickChange site-directed mutagenesis Kit protocol. For the generation of mutated plasmid DNA the following primers were used (mutated base pairs are underlined): Mut255_Fwd: 5'-G GGG CCA GTA CGGG GAA ATG TAC GAG GGC GTG-3', and Mut255_rev: 5'-CAC GCC CTC GTA CAC TTT CCC GTA CTG GC-3' (pEp185Bcr-AblMutE255K); Mut315_Fwd: 5'-GTT CTA TAT CAT CAT AGA GTT CAT GAC CTA C-3' and Mut315_rev: 5'-GGT CAT GAA CTC TAT GAT GAT ATA GAA CGG-3' (pEp185Bcr-AblMutT315I); and Mut253_Fwd: 5'-GGG CGG GGG CCA GTT TGG GGA GGT GTA CGA GGG C-3'and Mut253_rev: 5'-CCT CGT ACA CCT CCC CAA ACT GGC CCC CGC CCA GC-3' (pEp185Bcr-AblMutY253F). Mutated plasmid DNA was sequenced using the primer Bcr-Abl 2436: 5'-CTT GAT GGA GAA CTT GTT GTA GGC-3'. All PCR-products were controlled for the presence of mutations by sequencing. The resulting cDNAs were cloned into the pENTR1A vector for further recombination into the PINCO vector as described in Beissert et al. 2008 [Invitrogen, Karslruhe, Germany).Bcr-Abl cDNAs harbouring E255K, T315I, and Y253F mutations were obtained by site-directed mutagenesis using a modification of al. 2008 using th2 in humidified atmosphere. Human leukaemic cell lines, BV173, SEM, SupB15, and murine Ba/F3 were obtained from the German Collection of Microorganisms and Cell Cultures . The ecotropic packaging cells Phoenix were obtained from Harald von Melchner at the Medical School of Johann Wolfgang Goethe, Frankfurt. Ba/F3 were cultured in RPMI 1640 (Invitrogen) supplemented with 10% fetal calf serum (FCS) (Invitrogen), 10ng/ml murine IL-3 , 1% Glutamine and 1% Penicillin/Streptomycin (Invitrogen). BV173, Ba/F3PINCOp185Bcr-Abl, Ba/F3PINCOp185Bcr-AblMutE255K, Ba/F3PINCOp185Bcr-AblMut T315I, Ba/F3PINCOp185Bcr-AblMutY253F were maintained in the same medium but without IL-3. SEM cells were cultured in Iscove's MDM supplemented with 10% FCS, 1% Glutamine and 1% Penicillin/Streptomycin. WTSupB15 were maintained in RPMI 1640 supplemented with 15% FCS, 1% Glutamine and 1% Penicillin/Streptomycin. RTSupB15 were cultured in supplemented RPMI 1640 medium with the addition of 1 \u03bcM Imatinib.Cells were cultured at 37\u00b0C in 5% CO5cells/ml in medium without IL-3.Ecotropic Phoenix packaging cells were transiently transfected with the indicated retroviral vectors as described before . TransfeViability of cells was detected by the trypan blue dye exclusion and apoptosis was determined by 7-AAD staining as described previously .Cell Signaling Technologies, Frankfurt, Germany). Monoclonal mouse anti-c-Abl (clone 24-11)-IgG1-antibody (\u03b1-Abl), polyclonal rabbit anti-c-Abl (clone K-12)-IgG-antibody (\u03b1-Abl), monoclonal mouse anti-phospho-Erk (clone E-4)-IgG2a-antibody (\u03b1-p-Erk), polyclonal goat anti-p-Hck (Tyr 411)-IgG-antibody (\u03b1-p-Hck), polyclonal goat anti-Hck (clone N-30)-antibody (\u03b1-Hck), and polyclonal goat anti-Hck (clone M-28)-IgG-antibody (\u03b1-Hck) were purchased from Santa Cruz . Monoclonal mouse anti-Hck (clone 18)-IgG1-antibody (\u03b1-Hck) and monoclonal mouse anti-Stat5 (clone 89)-IgG2b-antibody (\u03b1-Stat5) were from Becton Dickinson .Western blot analysis was performed accordingly to widely used protocols. The following primary antibodies were used: Rabbit polyclonal antibodies included anti-phospho-c-Abl (Tyr245)-antibody (\u03b1-p-c-Abl), anti-phospho-Akt (Tyr326)-antibody (\u03b1-p-Akt), anti-Akt-antibody (\u03b1-Akt), anti-p44/p42 MAP Kinase-antibody (\u03b1-p44/42), anti-Hck-antibody (\u03b1-Hck), anti-phospho-Lyn (Tyr507)-antibody (\u03b1-p-Lyn), anti-Lyn-antibody (\u03b1-Lyn), anti-PARP-antibody (\u03b1-PARP), anti-phospho-Src family (Tyr416)-antibody (\u03b1-p-Src), Polyclonal rabbit anti-Stat5-antibody (\u03b1-Stat5). Monoclonal rabbit anti-Src (clone 36D10)-IgG-antibody (\u03b1-Src) and monoclonal mouse anti-phospho-Stat5 (Tyr694) (clone 14H2) IgG1-antibody (\u03b1-p-Stat5) .Secondary polyclonal goat-anti-rabbit-IgG-HRP conjugate-antibody, polyclonal goat-anti-mouse-IgG-HRP conjugate-antibody, and polyclonal mouse-anti-goat-IgG-HRP conjugate-antibody were purchased from t test; p values < 0.05 were considered to be significant.Data were compared by a two-tailed Student To determine the effect and specificity of AZD0530 on Src and Bcr-Abl mediated growth inhibition of Ph+ cells, the CML blast cell line BV173, was treated with various concentrations of AZD0530, and cell proliferation was measured by trypan blue exclusion of viable cells. As control for specificity the Ph- ALL cell line SEM, was used. Figure These data show that AZD0530 is able to specifically block growth of Ph+ patient derived cells.Clinical relapse and resistance to Imatinib has been shown to develop rapidly in the advanced phases of CML and Ph+ ALL patients mainly due to Bcr-Abl-dependent mechanisms such as amplification or mutations in the Abl portion of the Bcr-Abl gene. To examine the role of other potential mechanisms of Imatinib-resistance, the Imatinib-resistant Ph+ ALL cells RTSupB15 were established by long term culture of WTSupB15 cells with increasing amounts of Imatinib . The RTSIn summary, these data show that AZD0530 is unable to overcome non-mutational Imatinib resistance.Bcr-Abl showed a concentration-dependent growth inhibition.Recent data have demonstrated that Bcr-Abl-dependent cell lines are sensitive to growth arrest induced by dual Src/Abl kinase as well as Src selective kinase inhibitors such as Dasatinib, PP2 and A-419259 [Bcr-Abl cells treated in a similar manner Ba/F3 cells expressing these mutants were treated with the dual Src/Abl kinase inhibitor AZD0530, and proliferation was assessed comparing them with the Ba/F3 infected p185r Figure . Here weTaken together these results indicate that AZD0530 is able to overcome resistance of Bcr-Abl Mut Y253F and E255K but not of T315I.To answer the question whether the dose-dependent inhibition of Ph+ cell proliferation by AZD0530 was associated with the induction of apoptosis, both BV173 (Ph+) and SEM (Ph-) cell lines were treated in parallel with increasing concentrations of AZD0530 and Imatinib for three days and apoptosis was measured by 7-AAD staining. At the protein level, poly(ADP-ribose)polymerase (PARP) cleavage was used as a sign of apoptosis, and was examined in whole cell lysates by immunoblotting. As shown in Figure These results confirmed that the inhibition of SFKs and Bcr-Abl by both compounds was associated with the induction of apoptosis and was Bcr-Abl dependent.50 of 0.5 \u03bcM) correlated well with PARP cleavage (bottom panel), with a strong effect of 2 \u03bcM ADZ0530. This is contrary to the RTSupB15 cells with not more than 20% of cells undergoing apoptosis at the highest concentration (5 \u03bcM AZD0530) used, as compared to the control cells. This could be confirmed by a lack of PARP cleavage (bottom panel).To further investigate the influence of AZD0530, apoptosis measurement and PARP cleavage were assessed in the RTSupB15 cells and the results were compared to that of the parental WTSupB15 cell line Figure . In the To determine the effects of AZD0530 and Imatinib on SFK activity in the CML cell line BV173 cells were exposed to the same inhibitor concentrations used in the proliferation and apoptosis assays Figure . Whole cIn contrast SFK and Hck inhibition in SEM cells was observed in the presence of AZD0530 but not Imatinib, and neither led to growth inhibition nor apoptosis induction.In Ph+ leukaemic cells, it is not clear if the interaction between Bcr-Abl and SFKs is solely due to activation of Src kinases by Bcr-Abl, activation of Bcr-Abl by SFKs or, if there exists a transphosphorylation mechanism between both tyrosine kinases.To investigate the influence of Abl kinase inhibition on SFK activation we exposed BV173 cells to Imatinib. Figure in vitro [It has already been shown that Hck, Lyn, Fyn and Fgr each bind the kinase domain, at the C-terminal tail, and SH3/SH2 region of Bcr-Abl . It is kin vitro . These fActivation of the Raf/Mek/Erk, PI3K/Akt and Stat pathways synergize to promote Ph+ leukaemic cell growth. To investigate if inactivation of Src kinases and Bcr-Abl by AZD0530 could interfere with the leukaemic survival signalling pathways, lysates from BV173 cells treated with AZD0530 or Imatinib were probed with antibodies (activated and whole protein) against Stat5, Erk, and Akt kinases. Results were compared to Ph- SEM cells Figure . In the in vitro inhibitory profiles against Imatinib resistance due to Bcr-Abl mutations and resistance independent of Bcr-Abl [6 cells/ml) compared to the single agents alone (0.1 \u03bcM Imatinib (1.23 \u00d7 106 cells/ml) and 1 \u03bcM AZD0530 (0.8 \u00d7 106 cells/ml)))Figure . No majo))Figure and RTSu))Figure between Recent reports have demonstrated a requirement for Src kinase activity in Bcr-Abl transformation and oncogenic signal transduction .Here, we present AZD0530, a new, orally administered, potent and highly selective dual Src/Abl kinase inhibitor, whose effects have been investigated in solid tumours . In thisA significant induction of apoptosis in the Ph+ ALL cell line WTSupB15 occurred at lower concentrations (0.5 \u03bcM) when compared to the CML cell line, BV173 (2 \u03bcM). It could be concluded that the SFK inhibited by AZD0530, contribute to a greater extent to proliferation and survival in Ph+ ALL cells than the CML cells. Neither proliferation nor apoptosis induction was influenced by AZD0530 in the Imatinib resistant RTSupB15 cells (Ph+ ALL). This confirmed that resistance to Imatinib in these cells is not Src dependent.Analysis of the mechanisms of action of AZD0530 showed that higher concentrations of AZD0530 and Imatinib were needed to inhibit the activation of Bcr-Abl, when compared to the concentrations needed to inhibit activated SFK in BV173 cells. Growth and survival of the Ph- ALL cell line SEM was not influenced by both compounds, though AZD0530 inhibited the activation of SFKs in these cells. This also confirmed that Src kinases influence the survival of Bcr-Abl positive cells.In this study, it could not be clearly defined if the inhibitory effects of AZD0530 were a result of its direct effect on Src kinases, Bcr-Abl or on both kinases. Since Src kinases are inhibited by Imatinib in BV173 cells but not in Ph- SEM cells, it could be hypothesized that SFKs are transphosphorylated and acting downstream of Bcr-Abl in the CML blast cell line BV173. In contrast to the BV173 cells, in the SupB15 cells, Bcr-Abl was described as acting downstream of SFKs. In SupB15 cells, AZD0530 down-regulated the activated forms of both SFK and Bcr-Abl, in contrast to Imatinib, which inhibited only the activity of Bcr-Abl, and not SFK. This can be explained by the fact that inhibition of Bcr-Abl alone (by Imatinib) has no effect on SFK. Hence Bcr-Abl can only be activated by SFK in this cell line and not vice versa.In BV173 cells, both AZD0530 and Imatinib blocked Erk, Akt and Stat5 activation at concentrations that inhibited SFKs but did not affect Bcr-Abl tyrosine phosphorylation. This was an indication that SFKs coupled Bcr-Abl to its survival and signalling molecules, enhancing disease progression. This was in contrast to SEM cells in which AZD0530 inhibited the activation of SFKs but not the activation of Erk, Akt and Stat5. This confirmed that the activity of these substrate proteins was Src independent in Ph- cells.Treatment of the BV173 cells but not the WTSupB15 cells with a combination of AZD0530 and Imatinib yielded an additive antiproliferative effect, when compared to the single agents alone. Taken together these data suggest an additional effect of AZD0530 on Imatinib in BV173 cells that seems to be cell type specific.In advanced Ph+ leukaemia (Ph+ ALL or CML blast crisis) clinical trials with Imatinib have shown that Bcr-Abl inhibition is not sufficient to prevent disease progression or to restrict expansion of resistant cells. This has necessitated the development of treatment regimens, where single compounds target both Bcr-Abl and its substrate proteins as seen in the case of AZD0530.in vitro data strongly suggest that inhibition of SFK augments growth inhibition achieved by Bcr-Abl kinase inhibitors. Two of the most frequent domain mutations, which show resistance to Imatinib (MutY253F and E255K), responded to treatment with AZD0530.In recent investigations we have been able to show that in primary patient cells resistant to Imatinib, Src kinase inhibition is more effective in the presence of AZD0530 when compared to the Imatinib treated cells. In summary, our The authors declare that they have no competing interests.PMG \u2013 carried out most of the experiments and drafted the manuscript. AR \u2013 participated in the design of the study and contributed to write the paper. KS \u2013 performed experiments and statistical analysis. BB \u2013 performed experiments and provided experimental tools. MR \u2013 conceived of the study, participated in its design and contributed to write the paper. OGO \u2013 conceived of the study and participated in its design and coordination. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/9/53/prepubAZD0530 overcomes Imatinib resistance and induces growth arrest in Imatinib resistance Ba/F3MutT253F cells. Ba/F3MutT253F cells were grown in the presence of AZD0530 (left) and Imatinib (right) for three days. Proliferation was assessed by trypan blue exclusion of viable cells, and the percentage of apoptotic cells was measured by staining with 7-AAD. Results are the mean of 3 independent experiments carried out in duplicates +/- S.D.Click here for fileAZD0530 inhibits Bcr-Abl activation and its downstream signalling pathways in Imatinib resistance Ba/F3MutT253F cells. The Imatinib resistant cell line Ba/F3MutT253F was treated with AZD0530 and Imatinib for three days. Whole cell lysates were blotted for the indicated antibodies. Tubulin was probed and used as control for equal protein loading. One representative experiment out of 3 is given.Click here for file"} +{"text": "Human glioma usually shows intrinsic multidrug resistance because of the blood-brain barrier (BBB), in which membrane-associated P-glycoprotein (P-gp), encoded by the human multidrug resistance gene MDR1, plays a role. We studied drug sensitivity to vincristine (VCR), doxorubicin (DOX) and nimustine (ACNU) in both intracerebrally and subcutaneously xenotransplanted human glioma. We examined the levels of MDR1 and murine mdr3 gene expression in the xenografts by reverse transcriptase polymerase chain reaction and the localization of P-gp by immunohistochemistry. Six of seven subcutaneously transplanted xenografts (scX) were sensitive to the above three drugs. In contrast, all three intracerebrally transplanted human glioma xenografts (icX) were resistant to P-gp-mediated drugs VCR and DOX, but were sensitive to the non-P-gp-mediated drug ACNU. Neither icX nor scX showed any MDR1 expression. Intracerebrally transplanted human glioma xenografts showed an increased level of murine mdr3 gene expression, whereas scX showed only faint expression. The localization of P-gp was limited to the stromal vessels in icX by immunohistochemistry, whereas scX expressed no P-gp. Our findings suggest that the P-gp expressed on the stromal vessels in icX is a major contributing factor to multidrug resistance in human glioma in vivo."} +{"text": "HIV-infected individuals harbour a latent reservoir, which is not accessible to current treatments and constitutes a major obstacle for virus eradication. Post-integration latency could result from transcriptional inhibition of the provirus. Efficient transcription of the viral genome requires recruitment of the positive transcription elongation factor b (P-TEFb) to the long terminal repeat by the viral protein, Tat. To better understand the regulation of viral transcription, we aimed at characterizing nuclear Tat-associated protein complexes.HeLa-S3 cells were stably transduced with C-terminally TAP-tagged Tat. Tat-associated complexes were purified from Dignam nuclear extracts by tandem affinity chromatography and interactors identified by tandem mass spectrometry. Biochemical and functional analysis using the siRNA approach were applied to understand the role of the newly identified Tat cofactors.Tat forms two distinct and stable complexes. Tatcom1 consists of core active P-TEFb, MLL-fusion partners involved in leukemia and PAF1 complex. Importantly, Tatcom1 formation relies on P-TEFb, while optimal CDK9 CTD-kinase activity is AF9 dependent. MLL-fusion partners and PAF1 associate with elongating PTEFb as part of the regular cellular physiology. Tat increases the assembly or stability of this complex. Tatcom2 is composed of CDK9, Cyclin T1 and 7SK snRNP lacking HEXIM. Tat remodels 7SK snRNP by interacting directly with 7SKRNA, leading to the formation of a stress-resistant 7SK snRNP particle (Figure We identified new factors required for Tat transactivation and important for P-TEFb function. Given the involvement of P-TEFb in HIV-1 transcriptional latency, they may represent new potential targets against HIV."} +{"text": "Nitric oxide (NO) is an important molecule in regulating tumour blood flow and stimulating tumour angiogenesis. Inhibition of NO synthase by L-NAME might induce an anti-tumour effect by limiting nutrients and oxygen to reach tumour tissue or affecting vascular growth. The anti-tumour effect of L-NAME after systemic administration was studied in a renal subcapsular CC531 adenocarcinoma model in rats. Moreover, regional administration of L-NAME, in combination with TNF and melphalan, was studied in an isolated limb perfusion (ILP) model using BN175 soft-tissue sarcomas. Systemic treatment with L-NAME inhibited growth of adenocarcinoma significantly but was accompanied by impaired renal function. In ILP, reduced tumour growth was observed when L-NAME was used alone. In combination with TNF or melphalan, L-NAME increased response rates significantly compared to perfusions without L-NAME (0\u201364% and 0\u201363% respectively). An additional anti-tumour effect was demonstrated when L-NAME was added to the synergistic combination of melphalan and TNF (responses increased from 70 to 100%). Inhibition of NO synthase reduces tumour growth both after systemic and regional (ILP) treatment. A synergistic anti-tumour effect of L-NAME is observed in combination with melphalan and/or TNF using ILP. These results indicate a possible role of L-NAME for the treatment of solid tumours in a systemic or regional setting. \u00a9 2000 Cancer Research Campaign"} +{"text": "A brain tumour-associated marker, urokinase (UK), was investigated using rabbit anti-UK polyclonal and murine anti-UK monoclonal antibodies, which were prepared by immunization with low molecular weight UK (LMW-UK) and high molecular weight urokinase (HMW-UK) synthetic peptide respectively. The polyclonal antibody cross-reacted with both LMW-UK and HMW-UK, whereas the murine MAbs were specific for HMW-UK. These immunological probes were used to study urokinase in glioma extracts, tissues, sera and cell lines that had been prepared from primary cultures of freshly dissected gliomas. Radioimmunoassays showed that glioma extracts had much higher level (5- to 44-fold) of UK than normal human brain extracts. This result was confirmed by immunoblotting of electrophoresis gels of glioma and human brain extracts. Immunohistochemical study using anti-UK MAb demonstrated much higher levels of UK in glioma tissue than normal brain tissue. Immunohistochemical study using anti-UK MAbs localized UK on the cell surface of glioma cells. Anti-UK MAbs inhibited the proliferation of AA cell lines and GB cell lines (50% to > 90%) and exerted minor effects (< or = 20%) on normal human liver, intestine and lymphocyte cell lines. Taken together, these results suggest that anti-UK MAbs may have therapeutic potential for human gliomas and cancer metastasis."} +{"text": "Multiple cultures from GM-CSF-/-and wild type mice were established and compared for cell production. GM-CSF-/- LTCdeveloped more slowly, but by 16 weeks produced cells resembling DC in numbers comparableto wild type cultures. LTC maintained distinct populations of small and large cells, the latterresembling DC. Cells collected from GM-CSF-/- LTC were capable antigen presentingcells (APC) for T cell stimulation and morphologically resembled DC. Large cells expressedthe CD11b, CD11c, CD86, 33D1 and Dec-205 markers of DC. Addition of GM-CSF toGM-CSF-/- LTC increased the proportion of large, mature DC present in culture. Stromal cellsfrom GM-CSF-/- LTC could support the differentiation of DC from early progenitors maintainedin LTC without addition of GM-CSF. However, GM-CSF is not a critical factor in the in vitro generation of DC from progenitors. It can, however, substitute for stromal cells inincreasing the survival of mature DC.The production of dendritic cells (DC) from haemopoietic progenitors maintained in longterm stroma-dependent cultures (LTC) of spleen or bone marrow (BM) occurs independentlyof added granulocyte/macrophage colony stimulating factor (GM-CSF). The possibility thatcultures depend on endogenous GM-CSF produced in low levels was tested by attempting togenerate LTC from spleen and BM of GM-CSF"} +{"text": "In teleosts, the Major Histocompatibility Complex (MHC) class I and class II molecules reside on different linkage groups as opposed to tetrapods and shark, where the class I and class II genes reside in one genomic region. Several teleost MHC class I regions have been sequenced and show varying number of class I genes. Salmonids have one major expressed MHC class I locus (UBA) in addition to varying numbers of non-classical genes. Two other more distant lineages are also identifyed denoted L and ZE. For class II, only one major expressed class II alpha (DAA) and beta (DAB) gene has been identified in salmonids so far.Sasa-DBA) and beta (Sasa-DBB) genes in addition to NRGN, TIPRL, TBCEL and TECTA. The region was not linked to the classical class II genes and had some synteny to genomic regions from other teleosts. Two additional divergent and expressed class II sequences denoted DCA and DDA were also identified in both salmon and trout. Expression patterns and lack of polymorphism make these genes non-classical class II analogues. Sasa-DBB, Sasa-DCA and Sasa-DDA had highest expression levels in liver, hindgut and spleen respectively, suggestive of distinctive functions in these tissues. Phylogenetic studies revealed more yet undescribed divergent expressed MHC class II molecules also in other teleosts.We sequenced a genomic region of 211 kb encompassing divergent MHC class II alpha (We have characterised one genomic region containing expressed non-classical MHC class II genes in addition to four other genes not involved in immune function. Salmonids contain at least two expressed MHC class II beta genes and four expressed MHC class II alpha genes with properties suggestive of new functions for MHC class II in vertebrates. Collectively, our data suggest that the class II is worthy of more elaborate studies also in other teleost species. UBA with additional non-classical MHC class I genes in two duplicated MHC class I regions was performed by primer walking. Rainbow trout sequences denoted Onmy-DBB as locus definition name was available in Genbank, but sequence identity in the beta2 domain suggested this may be an Onmy-DAB allele rather than a new locus and four divergent MHC class II alpha sequences , which all share most of the characteristics typical for vertebrate MHC class II molecules.In summary, Atlantic salmon harbours at least two divergent MHC class II beta sequences , while no substitutions were found in the Sasa-DBA sequences. As the included sequences were derived from both Norwegian as well as Canadian waters, one may assume these genes have little or no polymorphism analogous to the nonclassical MHC class II genes identified in higher vertebrates. As for Sasa-DCA and Sasa-DDA, we have not performed any extensive polymorphic studies, but based on available EST information these loci contain little or no polymorphismTo evaluate the polymorphic content of the BAC genes Sasa-DAB expression was detected in spleen, followed by gills, hindgut and head kidney. Lower Sasa-DAB levels were observed in heart, liver and foregut with muscle showing the lowest expression levels. This Sasa-DAB expression pattern fits with the observations done by Koppang et al. descend from a liver cDNA library, confirming the expression of this gene in liver. Both macrophage and interhepatocytic cell populations have been found in liver of Atlantic salmon .The 630N19 BAC clone positive for DAA was subjected to nucleotide sequencing using a shotgun strategy. BAC DNA was isolated and purified by an alkaline lysis procedure using Nucleobond columns (BD Biosciences ClonTech). 15 microgram (or more) of isolated DNA was nebulized (Invitrogen) (20PSI/15s), trimmed and end filled with Mung Bean Nuclease (NEB), T4 DNA polymerase (NEB) and Klenow Fragment (NEB). The blunt-ended DNA was size fractioned by electrophoresis and the fragments corresponding to 1\u20134 kb were excised and gel purified using GeneClean III (Qbiogene). Fragments were ligated into a pUC19/s method . More th Pregap4 ,48. The Pregap4 ,50, asse Pregap4 and then Pregap4 . Remaini Pregap4 within CDIGIT and GENSPhylogenetic trees were created using neighbour-joining method in MEGA3 . Consensmicro mRNA Purification Kit . 1 \u03bcl of mRNA sample was used for quantification with Nanodrop spectrometer . All samples were DNase treated using Turbo DNA-free\u2122 . Gene specific PCR primers (Table T) were set manually and CT values > 37 were rejected. Relative expression of mRNA in relation to the housekeeping gene elongation factor 1\u03b1 (EF1A) was calculated using the \u0394\u0394CT method , Sasa-DBB [GenBank: DY726096], Onmy-DAB [GenBank: AAA79133], Onmy-DBB [GenBank: AAD53026], Orla-DAB [GenBank: BAA94279], Orla-DBB [GenBank: BAA94280], Xima-DAB [GenBank: AAC05652], Gaac-DAB [GenBank: AAU01918], Gaac-DBB [GenBank: AAU01920], Gaac-EST [Genbank: DN681207], Dila-DAB [GenBank: ABH09450], Fuhe-EST1 [GenBank: CN976662], Fuhe-EST2 [GenBank: CN984097], Taru-EST [GenBank: CA846190], Icpu-DAB [GenBank: AAB67871], Cyca-DAB [GenBank: CAA64709], Dare-DAB [GenBank: NP_571551], Dare-DCB [GenBank: CAD56804], Dare-DDB [GenBank: AAA87893], Dare-EST [Genbank: CK126567], Pipr-EST1 [GenBank: DT084791], Pipr-EST2 [GenBank: DT351641], Pipr-EST3 [GenBank: DT139435], Pipr-EST4 [GenBank: DT355684], Ximu-DXB [GenBank: AAS55041], Gici-DAB [GenBank: AAF82681] and HLA_DRB1 [GenBank: AAA59781]. Dots indicate identities, dashes indicate gaps or missing sequence information and + indicate peptide binding sites based on HLA_DRB1. Individual domains and regions are defined based on mammalian class II sequences.Click here for fileAmino acid sequence alignment of teleost MHC class II alpha sequences. Sequence references are as follows: Sasa-DAA [GenBank: AAL40122], Sasa-DBA [GenBank: EG757342], Sasa-DCA [GenBank: DW549478], Sasa-DDA [GenBank: DW557800], Onmy-DAA [GenBank: CAB96451], Onmy-DBA [GenBank: CX137594], Onmy-DCA [GenBank: CR376525], Onmy-DDA [GenBank: BX085673], Gaac-DAA [GenBank: AAU01917], Gaac-DBA [GenBank: AAU01919], Gaac-EST [GenBank: DN737221], Spau-DAA [GenBank: AAY42849], Orla-EST1 [GenBank: DC261023], Orla-EST2 [Genbank: BJ884671], Fuhe-EST [Genbank: CV816904], Dila-DAA [Genbank: ABH09446], Orni-DAA [Genbank: AAF66843], Cyca-DXA [GenBank: CAA64707], Pipr-EST1 [Genbank: DT253073], Pipr-EST2 [Genbank: DT092734], Pipr-EST3 [Genbank: DT311896], Dare-DAA [Genbank: NP_571565], Dare-EST1 [Genbank: CK018982], Dare-EST2 [Genbank: CO928661], Icpu-DAA [GenBank: AAD39865], Gici-DAA [GenBank: AAA49310], HLA-DRA [Genbank: NP_061984]. Dots indicate identities, dashes indicate gaps or missing sequence information and + indicate peptide binding sites based on HLA_DRA. Individual domains and regions are defined based on mammalian class II sequences.Click here for file"} +{"text": "Regressor serum from MSV-M-infected mice markedly reduced MSV-M oncogenesis when administered i.p. (0-1 ml/mouse) as much as 30 days before i.m. MSV-M infection in adult BALB/c mice. The regressor serum activity appeared to be directly dependent on the amount of IgG, as shown by: (1) inactivity of sera which have low virus-neutralizing antibody content; (2) high effectiveness only of the IgG serum fraction; (3) inactivity of regressor serum incubated with anti-mouse gamma-globulin serum. The regressor serum activity was specific and could not be ascribed to interferon or interferon-inducing factors, antigen-antibody complexes or free antigen. The activity was not suppressed by sublethal irradiation (380 rad) of recipient mice. These results suggest that the activity of regressor serum administered before MSV-M infection is mediated through sensitization of host cells which are not radiosensitive."} +{"text": "The reverse transcriptase polymerase chain reaction (RT-PCR) is a sensitive technique that can detect prostate-specific messenger RNA in circulating blood. Many authors have studied the potential of RT-PCR as a staging technique in prostate cancer (PC). Clinical sensitivity and in some cases specificity has been disappointing. Few authors have been able to correlate RT-PCR result with patient stage. We have compared the results of using two different RT-PCR protocols with different sensitivities on blood samples from prostate cancer patients. An 80-amplification-cycle nested primer RT-PCR assay had a detection limit of 10 prostate cells and a 50-cycle RT-PCR could detect 20 cells in 5 ml blood. The 80-cycle assay detected prostate mRNA in four of 10 female samples, whereas the 50-cycle assay detected it in none. There was little difference in the assays\u2019 ability to detect prostate mRNA in advanced PC patients. The 50-cycle assay could differentiate between hormone-escaped, stable hormone-treated and untreated localized PC patients, whereas the 80-cycle assay could not. Each blood sample must be assayed several times with RT-PCR to avoid false-negative results and, if this is done, assay specificity can be increased with little effect on clinical sensitivity. \u00a9 2000 Cancer Research Campaign"} +{"text": "Withdrawal of L-arginine from culture medium resulted as potent as the higher inhibition obtained when blocking nitric oxide synthase with L-arginine analogues. Furthermore, intermedial concentrations of Larginine and exogenous nitric oxide donors were found for achieving optimal IL2-induced proliferation of CTLL-2. These findings prompted us to suggest that intra- and/or inter-cellular nitric oxide signalling may contribute to the modulation of the IL2 mitogenic effect upon cytotoxic T lymphocytes.The role of the L-arginine\u2013nitric oxide metabolic pathway was explored for interleukin-2-induced proliferation in the cytotoxic T lymphocyte clone CTLL-2. Specific inhibition of nitric oxide synthase significantly diminished, in a concentration-dependent manner,"} +{"text": "This paper reviews the synthesis, characterization, biodegradation and usage of bioresorbable polymers based on polydepsipeptides. The ring-opening polymerization of morpholine-2,5-dione derivatives using organic Sn and enzyme lipase is discussed. The dependence of the macroscopic properties of the block copolymers on their structure is also presented. Bioresorbable polymers based on polydepsipeptides could be used as biomaterials in drug controlled release, tissue engineering scaffolding and shape-memory materials. Thesel-lactic acid) (PLLA), poly (PDLLA) are synthesized by ring-opening polymerization of the corresponding six-membered cyclic diesters, i.e., glycolide, l-lactide and d,l-lactide. PGA and PLLA are semicrystalline polymers, whereas PDLLA is amorphous. PGA has a glass transition temperature (Tg) close to body temperature (Tg = 36 \u00b0C), whereas PLLA and PDLLA have Tg values above body temperature , Cyclo-[DLLA-Tyr(Bzl)] was performed as shown in The synthesis of morpholine-2,5-diones with pendant functional groups, i.e. Cyclo[DLLA-Asp(OBzl)], Cyclo[DLLA-Glu(OBzl)], Cyclo[DLLA-Lys(Z)], Cyclo[DLLA-Cys-[hydroxypropionyl]-N\u03b5-(benzyloxylcarbonyl)-(S)-lysine. Attempts to synthesize Cyclo[DLLA-Asp(OBzl)] and Cyclo[DLLA-Glu(OBzl)] using similar methods failed. Most probably transesterification reactions at the protective benzyl ester groups of \u03b2-benzyl-N-[2-hydroxy-propionyl]-(S)-aspartate and \u03b3-benzyl-N-[2-hydroxypropionyl]-(S)-glutamate occur during the cyclization procedure. Cyclo[DLLA-Asp(OBzl)], Cyclo[DLLA-Glu(OBzl)] and Cyclo[DLLA-Tyr(Bzl)] were synthesized according to Cyclo[DLLA-Lys(Z)] was prepared in 25% yield by intramolecular cyclization of pNBzl)] was prepared by treatment of N-[2-bromopropionyl]-S-p-nitrobenzyl-(R)-cysteine with TEA in DMF for 17 h at room temperature. When the reaction was carried out at 100 \u00b0C for 3 h, partial deprotection of the thiol group resulted in the formation of several side products. Yao synthesized Cyclo[DLLA-Lys(Z)] via intramolecular cyclization of N\u03b1-[-bromoacetyl]-N\u03b5-(benzyloxylcarbonyl)-(S)-lysine with TEA in DMF for 24 h at 60\u00b0C with 66% yield . The DSDOP initiated polymerizations gave slightly higher molecular weights than the polymers obtained using Sn(Oct)2, but the molecular weights were rather low in all cases and was not proportional to the M/I-ratio.Kricheldorf p-dioxanone and unsubstituted or alkyl-substituted morpholine-2,5-diones , poly(LLA-Lys) and poly[LLA-(Glc-Lys)], were obtained by ring-opening polymerization of LLA with cyclodepsipeptides consisting of Glc and Asp or Lys [et al. reported the preparation of biodegradable microspheres from poly[LLA-(Glc-Asp)] and poly[LLA-(Glc-Lys)] with low contents of depsipeptide units by the oil-in-water/solvent evaporation method . Wh. WhS)-is4.S)Lac-Val) and poly in vitro at 70 \u00b0C -6-methylmorpholine-2,5-dione with DLLA [Feijen d groups . The pen residue 83]. Po. Poet alith DLLA . The carr-LA], poly[(Glc-Lys)-r-LA], and poly[(Glc-Cys)-r-LA], had amphiphilic structures, i.e. hydrophilic side-chain groups and hydrophobic LA main chains s having cationic pendant groups (PGK(+)-b-PDLLA) and having anionic pendant groups (PGD(\u2212)-b-PDLLA) acted as good biodegradable surfactants to stabilize the corresponding primary water inner phases having proteins in PLGA-based MSs has higher hydrophilicity than PLGA, because amide groups in poly(LLA-BMD) chain improved hydrophilicity of the material and resulted in the increase of water absorption. Poly(LLA-BMD) almost degraded completely in PBS at 37\u00b0C in 90 days. The chemical structure of poly(LLA-BMD) was not observed any change during degradation [Poly-CL] > poly[(dl-DMO-DLLA)-CL] > poly[(d-DMO-DLLA)-CL], i.e. this enzyme has the highest substrate specificity for naturally occurring l-alanine. The enzymatic degradability rate of poly(l-DMO-DLLA) is greater than that of poly(l-DMO-LLA) copolymers with an identical DMO/LA ratio. This is probably due to the greater permeability of water into amorphous poly(l-DMO-DLLA) copolymers than the crystalline poly(l-DMO-LLA).Shirahama investigated the enzymatic degradation of optically active polydepsipeptides based on -alanine . Among tet al. studied the enzymatic degradation of polydesipeptides with functionalized side-chain groups using trypsin, V8 protease and papain as enzymes [Ouchi enzymes . The pol2PO4/Na2HPO4 buffer (pH = 7.0) at 37\u00b0C using papain as an enzyme [in vitro. The degradation rate of poly[LA-(Glc-Cys)] with papain was faster than that of nonenzymatic degradation. The susceptibility of enzyme to amino acid units was reflected on the specific cleavage of ester bonds of (Glc-Cys) units of the main chain in poly[LA-(Glc-Cys)].The degradation behavior of poly[LA-(Glc-Cys)] with 3.0 mol% of Cys was investigated in KHn enzyme . The maiet al. synthesized thermosensitive biodegeradable polymers based on polydepsipeptide. The thermosensitive polydepsipeptide poly[Glc-Asn(N-isopropyl)] could be degraded in vitro at room temperature by cleavage of the ester bonds in the main chain [N-isopropyl)] decreased to 25% of that of the initial value after 7 d. As expected, the poly[Glc-Asn(N-isopropyl)] degraded to a monomer level through cleavage of the ester bonds in the main chain in water. The degradation products of poly[Glc-Asn(N-isopropyl)] after 7 d of degradation did not show any cytotoxic activity against L929 fibroblast cells. The polymer and its degradation products are non-toxic and biocompatible. The cloud point at 29\u00b0C (between room and body temperature) of this thermosensitive polydepsipeptide make it attractive for implants and other biomedical applications.Ohya in chain . The molet al. synthesized multiblock copolymers based on oligodepsipeptides with shape-memory properties \u2013100S)-seIn vitro degradation experiments revealed that the block copolymers based on depsipeptides, DLLA and PEO8000 lost their weight faster than the block copolymers based on depsipeptides, LLA and PEO. The weight loss rates depended on the weight fraction of PEO in the block copolymers and the starting molecular weight. The molecular weight decreased quickly, while the molecular weight distribution increased up to a polydispersity from 1.63 to 4.29 with increasing degradation time.ABA block copolymers were synthesized via ring-opening polymerization of morpholine-2,5-diones and DLLA or LLA in the presence of PEO8000 as an initiator and stannous octoate as a catalyst at 140 \u00b0C for 9 h and 13. et al. synthesized amphiphilic AB-type diblock copolymers composed of hydrophobic polylactide segment and hydrophilic polydepsipeptide segment with amino groups or carboxyl groups [block-poly(l-lactide) and poly[Glc-Asp(OBzl)]-block-poly(l-lactide). Subsequent deprotection of Z and OBzl groups obtained the amphiphiles poly(Glc-Lys)-block-poly(l-lactide) and poly(Glc-Asp)-block-poly(l-lactide), respectively. These amphiphilic AB-type diblock copolymers of PLA and polydepsipeptides with amino or carboxyl groups formed core\u2013shell polymeric micelles (50\u2013100 nm in diameter) with chemically modifiable surfaces in aqueous solutions.Ouchi l groups . The mor5.l-lactide and 6,6\u2019-dimethylmorpholine-2,5-dione containing ester and amido functional groups in the backbone had lower glass transition temperatures and crystallinity compared to homopolymer PLLA. The adhesion of D1 mouse stem cells on copolymer films with low amido functional groups content was attenuated compared to pure PLLA -PEG- poly[LLA-co-(Glc-Leu)] were used to develop a biodegradable anti-adhesive membrane. The poly[LLA-co-(Glc-Leu)] was used as the hydrophobic segment A, and the PEG with Mn 10,000 and Mn 20,500 as the hydrophilic segment B. The degradation rate of the triblock copolymer films varied with changed in the molecular architecture, specifically, the molecular weight of the hydrophilic segment and the depsipeptide unit content in the A segment were more prominent [in vivo without inflammation.Amphiphilic ABA-type triblock copolymers of poly[LLA-rominent . The filco-LA] and poly[(Glc-Lys)-co-LA] film surfaces with respect to cell attachment and growth of mouse fibroblast L929 cells were investigated. The films with reactive surfaces had higher cell attachment ability than PLLA film [in vivo. These copolymers are expected to be applicable as degradable and chemically modifiable temporary scaffolds for tissue engineering in various situations.The effects of charged groups on poly[(Glc-Asp)-LLA film . During co-LA] and poly[(Glc-Lys)-co-LA] materials by freeze-drying method were used as biodegradable carriers for drug delivery systems [in vivo degradation of these copolymers was strongly influenced by Ala units during subcutaneous implantation in the backs of male rats. Poly(Ala-Ala-Glu(OEt)-Lac) showed the highest degradability, in which 100% degradation was observed in 24 weeks implantation.Sequential polydepsipeptides poly[(Ala) systems . The in 5.The polydepsipeptides with reactive carboxyl or amino groups as functional groups are suitable for covalently binding bioactive molecules to control cell interaction, such as immobilization of RGD peptides to enhance the cell attachment ability. Moreover, the microspheres prepared from copolymers and block copolymers containing functional (hydrophilic and ionic) groups exhibit more efficient entrapment of ionic substances by electrostatic interaction than PLLA microspheres. The microspheres showed controlled release of drugs and growth factors from the biodegradable matrix to promote rapid growth of cells and the regeneration of tissue. The degradation of polydepsipeptides is expected to reduce the local concentration of acid formed upon degradation as compared to polylactides which in certain cases might be advantageous for other biodegradable polyesters. Bioresorbable polymers based on polydepsipeptides could be used as biomaterials in drug controlled release, tissue engineering scaffolding, and shape-memory materials."} +{"text": "Recombinant human interferon alpha inhibits growth of a human colon cancer cell line, Colo 205. To explore the mechanisms of IFN induced growth inhibition, quiescent Colo 205 cells were stimulated to proliferate in serum-free media by defined growth factors. Addition of insulin, transferrin and selenium (ITS) stimulated DNA synthesis, as measured by 3H-thymidine incorporation, in a dose-dependent manner. IFN-alpha (at concentrations greater than 100 U ml-1) inhibited ITS stimulated DNA synthesis by 63%. Inhibition of cell cycle traverse was confirmed by flow cytometric analysis. Although IFN inhibited growth of ITS-treated cells, steady state levels of c-myc mRNA remained above levels observed in unstimulated cells. IFN inhibited DNA synthesis only when added prior to mitogen stimulation. IFN, added 6 h after exposure of quiescent cells to ITS, failed to inhibit cell growth. Addition of increasing concentrations of ITS failed to overcome the IFN-induced growth inhibition. These results suggest IFN may inhibit cell growth in part by antagonizing the action of growth factors."} +{"text": "Objective: The impact of anaerobic growth conditions on the Staphylococcus aureus toxic shock syndrometoxin-1 (TSST-1) production was studied. Methods: Ten strains of S. aureus derived from patients with toxic shock syndrome (TSS), 10isolates of S. aureus, and documented TSST-1-producing strains recovered from patients with eitherstaphylococcal septicemia or staphylococcal nongenital abscesses were grown under aerobic andanaerobic conditions. The bacterial growth was measured using optical density (OD) determinationsat 520 nm. The toxin production was assayed using the TS-RPLA latex agglutination test. Results: Both TSS and non-TSS strains of S. aureus grown under aerobic and anaerobic conditionsexhibited comparable OD patterns of growth, and the levels of toxin production remained constantduring the logarithmic phase. Toxin titers developed during the logarithmic growth phase andpeaked after 24 h of incubation. When stationary-phase isolates grown initially under aerobicconditions were subjected to strict anaerobic conditions, subsequent toxin titers, compared withisolates grown in the continued presence of oxygen, were depressed 2-fold, peaking at a later time. Conclusions: TSST-1 production is diminished under continued anaerobic conditions."} +{"text": "The human pancreatic tumour cell line PSN1/ADR, stepwise selected in 17-510 nM doxorubicin, displayed a multidrug resistance not conferred by P-glycoprotein (P-gp). Resistance to 17-51 nM doxorubicin was accompanied by overexpression of the vesicular marker lung resistance-related protein (LRP). Further selection in 170 nM doxorubicin led to the activation of multidrug resistance-associated protein (MRP) and to the development of drug accumulation/retention defects sensitive to verapamil. In addition, these defects were reversible by the vesicular traffic inhibitors brefeldin A, fluoroaluminate and nocodazole. In contrast, in human ovarian H134AD cells that are resistant to 1700 nM doxorubicin and used as P-gp-positive controls, the drug efflux was inhibited only by verapamil. The tyrosine kinase inhibitor genistein was a potent blocker of doxorubicin efflux in the PSN1/ADR cells but showed no activity in the H134 AD cells. The doxorubicin cytotoxicity in the PSN1/ADR cells was enhanced both by verapamil and brefeldin A, whereas in the parental PSN1 cells they demonstrated the opposite effects, being respectively sensitising and protecting. The P-gp-negative PSN1/ADR cells adapted to 510 nM doxorubicin retained brefeldin A-sensitive doxorubicin accumulation defects while MRP declined. The persistence of brefeldin A-responsive phenotype on the background of variable MRP expression suggests this agent as a useful functional probe for non-P-gp-mediated resistance to plasma-achievable doxorubicin concentrations."} +{"text": "The H&N cancer patients' SF-36 scores did not differ significantly from those of an age- and sex-matched sample (n = 871) from the Swedish normative population, except on the role-physical functioning scale. On the other hand, treatment-related side-effects and disease-specific problems measured by the H&N cancer module were, with few exceptions, significantly worse than norm values. Gender comparisons revealed that female H&N cancer patients generally scored better than the norms on both the SF-36 and the EORTC QLQ-C30, while the male patients scored significantly worse on most SF-36 scales. Patients \u226565 years more often scored worse than the norm than did patients <65. Clinically relevant differences were found on the majority of SF-36 scales in comparison of tumour sites, however, comparisons of patients with small (stage I+II) versus advanced (stage III+IV) tumours revealed few differences. Three years after diagnosis H&N cancer patients still suffer significant functional limitations/problems related to their disease and its treatment but these problems do not generally affect their overall HRQL. Tumour stage no longer differentiates HRQL at 3 years, however, factors related to the patients' age, gender and location of the tumour appear to have bearing on their reported health status. \u00a9 2001 Cancer Research Campaign http://www.bjcancer.comTo examine the health-related quality of life (HRQL) in long-term head and neck (H&N) cancer survivors compared with general population norms. HRQL was assessed with three standardized questionnaires: the SF-36 Health Survey (Short Form 36) and the EORTC QLQ-C30 and QLQ-H&N35 . Altogether 135 H&N cancer patients of 151 survivors (89% acceptance) from a longitudinal HRQL study ("} +{"text": "Under appropriate organ-culture conditions, ES celsdifferentiate into lymphoidlike cells at a stage equivalent to lymphoid cells found in fetalliver. These hematopoietic precursors are located in cup-shaped structures found in someembryoid bodies; we called such embryoid bodies \u201cES fetuses.\u201d In this study, we havefollowed the maturation of hematopoietic cells after implantation of ES fetuses into nudemice for 3 weeks. ES-cell-derived lymphoid cells-pre-B cells, mature B cells, and mature Tcells were found in all lymphoid organs. Interestingly, there was also an increase of T cellsof host origin. Because native nude mouse lack thymus, these T cells might be educated bythymuslike epithelium generated from ES fetuses. Practical applications of this combined"} +{"text": "Human adenoviruses (HAdV) are causing a broad spectrum of diseases. One of the most severe forms of adenovirus infection is a disseminated disease resulting in significant morbidity and mortality. Several reports in recent years have identified HAdV-31 from species A (HAdV-A31) as a cause of disseminated disease in children following haematopoetic stem cell transplantation (hSCT) and liver transplantation. We sequenced and analyzed the complete genome of the HAdV-A31 prototype strain to uncover unique sequence motifs associated with its high virulence. Moreover, we sequenced coding regions known to be essential for tropism and virulence of HAdV-A31 clinical isolates from patients with disseminated disease.The genome size of HAdV-A31 is 33763 base pairs (bp) in length with a GC content of 46.36%. Nucleotide alignment to the closely related HAdV-A12 revealed an overall homology of 84.2%. The genome organization into early, intermediate and late regions is similar to HAdV-A12. Sequence analysis of the prototype strain showed unique sequence features such as an immunoglobulin-like domain in the species A specific gene product E3 CR1 beta and a potentially integrin binding RGD motif in the C-terminal region of the protein IX. These features were conserved in all analyzed clinical isolates. Overall, amino acid sequences of clinical isolates were highly conserved compared to the prototype (99.2 to 100%), but a synonymous/non synonymous ratio (S/N) of 2.36 in E3 CR1 beta suggested positive selection.Unique sequence features of HAdV-A31 may enhance its ability to escape the host's immune surveillance and may facilitate a promiscuous tropism for various tissues. Moderate evolution of clinical isolates did not indicate the emergence of new HAdV-A31 subtypes in the recent years. Adenoviridae are non-enveloped, double-stranded DNA viruses with an icosahedral capsid . Human AHuman adenoviruses have long been recognized as pathogens causing a broad spectrum of different diseases depending on the type-related organotropism and virulence. For example, infections of the upper respiratory tract are caused by HAdV-C1, -C2, -C5, -B3, and -B7 , the morMoreover, immunocompromised patients can develop a sepsis-like, disseminated adenovirus syndrome that is associated with high levels of immunosuppression as a crucial risk factor . A wide One of the essential sequence features for HAdV types causing dissemination may be the viral RGD (integrin binding) motif of the penton base protein, because HAdV-F types lacking the RGD motif have never caused a disseminated infection (with exception of a single case report) in spite of their high prevalence -18. In ade novo HAdV-A31 infections . Therefore, this region was re-sequenced a second time and additionally sequenced for all seven clinical isolates; our previous result was confirmed. Most likely, the databank sequence relates to a subtype of HAdV-A31 or is mislabelled.The predicted homologue of protein V has a molecular weight of 39.8 K and is 347 amino acids in length. A 20.4 K gene product is homologue to the pVII, and a predicted product of 7.9 K represents the pX protein of HAdV-A31. All three proteins are described as being core proteins and are associated with the virus DNA . The seqThe L3 transcription unit encodes three proteins; we could identify ORF for pVI, the hexon protein (II) and the virus encoded protease. The predicted pVI protein of HAdV-A31 is 260 amino acids in length and has a molecular weight of 28.5 K. It revealed two nuclear localization signals (KRPRP at amino acid 136-140 and KRRR at amino acid 255-258) close to its C-terminus. Corresponding motifs of HAdV-C2 play an important role in directing cytoplasmatic proteins to the nucleolus and thus might be functionally active as nuclear localization signals , HAdV-2 [GenBank: NC_001405], HAdV-3 [GenBank: DQ086466], HAdV-4 [GenBank: AY599837], HAdV-5 [GenBank: AC_000008], HAdV-7 [GenBank: AC_000018], HAdV-8 [GenBank: AB448768], HAdV-9 [GenBank: AJ854486], HAdV-11 [GenBank: AC_000015], HAdV-12 [GenBank: AC_000005], HAdV-16 [GenBank: AY601636], HAdV-17 [GenBank: AC_000006], HAdV-19 [GenBank: AB448771], HAdV-21 [GenBank: AY601633], HAdV-22 [GenBank: FJ404771], HAdV-26 [GenBank: EF153474], HAdV-34 [GenBank: AY737797], HAdV-35 [GenBank: AY128640], HAdV-37 [GenBank: DQ900900], HAdV-40 [GenBank: L19443], HAdV-41 [GenBank: DQ315364], HAdV-46 [GenBank: AY875648], HAdV-48 [GenBank: EF153473], HAdV-49 [GenBank: DQ393829], HAdV-50 [GenBank: AY737798], HAdV-52 [GenBank: DQ923122].HAdV-1 [GenBank: DQ149611, pX is GenBank: U14653.The previously sequenced HAdV-31 hexon protein's accession number is GenBank: SH carried out the laboratory work, molecular genetic studies, genome annotation, bioinformatic analysis and drafted the manuscript. IM participated in the design of the study and performed the phylogenetic analysis. SD participated in sequencing and analysis of the fiber gene regions. FR participated in sequencing and bioinformatic analysis. AH conceived the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript."} +{"text": "A 62 old-year lady with previous long-standing history of non ischemic dilated cardiomyopathy with an LVEF between 20 to 27%, and complete LBBB underwent a cardiac resynchronization therapy-pacemaker (CRT-P) implantation in another institution. The patient behaved as a non-responder since implant despite AV interval optimization therapy. Chest X-ray (AP and lateral views,"} +{"text": "Using a doxorubicin-resistant subline (K562/ADM) of human leukaemia K562 cells , the effect of immunoliposomes that targeted a cellular transferrin receptor (TFR) was examined by neutralization of doxorubicin (DOX) resistance. OKT9-CIL, prepared by conjugation of DOX-encapsulated liposome with an anti-TFR monoclonal antibody, OKT9 , showed similar binding to both K562 and K562/ADM. Although an 80-fold higher sensitivity to free DOX on cell growth inhibition in K562 than in K562/ADM was found, the difference was clearly diminished after OKT9-CIL treatment through the increased sensitivity of K562/ADM. The cellular DOX level 30 min after the exposure of free DOX was 45-fold lower in K562/ADM than in K562, whereas nearly equivalent DOX levels were detected in K562 and K562/ADM after OKT9-CIL treatment. In addition, DOX in K562/ADM in the free DOX treatment was efficiently excreted by 54% within 120 min of incubation, whereas almost all DOX supplied by OKT9-CIL remained uncleared. Fluorescence microscopic observation showed that OKT9-CIL was internalized into juxtanuclear vesicles in K562/ADM cells. These results suggest that OKT9-CIL has a potency to accumulate DOX, resulting in augmentation of DOX cytotoxicity in DOX-resistant tumour cells."} +{"text": "Glioblastoma multiforme (GBM) is an invariably fatal central nervous system tumor despite treatment with surgery, radiation, and chemotherapy. Further insights into the molecular and cellular mechanisms that drive GBM formation are required to improve patient outcome. MicroRNAs are emerging as important regulators of cellular differentiation and proliferation, and have been implicated in the etiology of a variety of cancers, yet the role of microRNAs in GBM remains poorly understood. In this study, we investigated the role of microRNAs in regulating the differentiation and proliferation of neural stem cells and glioblastoma-multiforme tumor cells.We used quantitative RT-PCR to assess microRNA expression in high-grade astrocytomas and adult mouse neural stem cells. To assess the function of candidate microRNAs in high-grade astrocytomas, we transfected miR mimics to cultured-mouse neural stem cells, -mouse oligodendroglioma-derived stem cells, -human glioblastoma multiforme-derived stem cells and -glioblastoma multiforme cell lines. Cellular differentiation was assessed by immunostaining, and cellular proliferation was determined using fluorescence-activated cell sorting.erbB tumors and cluster of differentiation 133+ human glioblastoma multiforme-derived stem cells (SF6969). Transfection of microRNA-124 or microRNA-137 also induced G1 cell cycle arrest in U251 and SF6969 glioblastoma multiforme cells, which was associated with decreased expression of cyclin-dependent kinase 6 and phosphorylated retinoblastoma (pSer 807/811) proteins.Our studies revealed that expression levels of microRNA-124 and microRNA-137 were significantly decreased in anaplastic astrocytomas and glioblastoma multiforme relative to non-neoplastic brain tissue (P < 0.01), and were increased 8- to 20-fold during differentiation of cultured mouse neural stem cells following growth factor withdrawal. Expression of microRNA-137 was increased 3- to 12-fold in glioblastoma multiforme cell lines U87 and U251 following inhibition of DNA methylation with 5-aza-2'-deoxycytidine (5-aza-dC). Transfection of microRNA-124 or microRNA-137 induced morphological changes and marker expressions consistent with neuronal differentiation in mouse neural stem cells, mouse oligodendroglioma-derived stem cells derived from S100\u03b2-v-microRNA-124 and microRNA-137 induce differentiation of adult mouse neural stem cells, mouse oligodendroglioma-derived stem cells and human glioblastoma multiforme-derived stem cells and induce glioblastoma multiforme cell cycle arrest. These results suggest that targeted delivery of microRNA-124 and/or microRNA-137 to glioblastoma multiforme tumor cells may be therapeutically efficacious for the treatment of this disease. MicroRNAs (miRNAs) are a class of small non-coding RNAs that regulate diverse cellular processes through RNA interference-based mechanisms. miRNAs are transcribed as primary RNA transcripts (pri-miRNAs), processed in the nucleus to smaller precursor hairpin structures (pre-miRNAs), and then exported to the cytoplasm where they are processed further by the Dicer nuclease to become mature, functional miRNAs approximately 21 nucleotides in length. Mature miRNAs, the endogenous equivalent of short interfering RNAs (siRNAs), are then incorporated into the RNA-induced silencing complex, which facilitates their interaction with, and inhibition of, target messenger RNAs (mRNAs) by translational repression or message cleavage cells and mouse tumors. For example, expression of miR-124 and miR-9 increases during differentiation of mouse ES cell-derived neural progenitors, and experimental manipulation of miR-124 and miR-9 expression affects neural lineage differentiation in the ES cell-derived cultures . These rThe discovery of a rare, highly tumorigenic, self-renewing sub-population of glioblastoma multiforme (GBM) cells that express the cell surface marker cluster of differentiation (CD) 133 see ,15), the, the15])Fresh frozen primary human tissues were acquired from the Brain Tumor Research Center tissue core at the University of California San Francisco (UCSF) in accordance with the Committee on Human Research approved procedures. All samples were thoroughly reviewed by a neuropathologist (S Vandenberg), and anaplastic astrocytomas (AAs) and GBM tumors were confirmed to contain at least 90% tumor. Non-neoplastic brain tissues were derived from the temporal lobes of epileptic patient surgeries and comprised primarily cortex with mild to moderate reactive astrocytosis and neurons. For further details about the samples see Additional file \u00ae miRNA assays human panel-early access kit . Expression of the six high-grade astrocytomas (HGA)-miRNAs during NSC differentiation was quantitated using individual TaqMan\u00ae MicroRNA Assays. The comparative Ct (\u0394\u0394Ct) method was used to determine the expression fold change.Total RNA was extracted using the miR-Vana RNA isolation system . Expression of 192 human miRNAs was quantitated in human tissues using TaqMant-statistics were obtained as described elsewhere [Statistical analysis of miRNA expression in primary tissues was performed on log2 transformed fold change data using freely available R language. The limma package in Bioconductor was used to compare the three types of primary tissues . Moderated lsewhere and P-va5 cells per well of a six-well plate, incubated for 24 hours in Dulbecco's Modified Eagle's Medium (DMEM) high glucose 10% serum, and then supplemented with fresh media containing 5-aza-dC for 72 hours or trichostatin A (TSA) for 12 hours. For the combination study, 1 or 5 \u03bcM 5-aza-dC was present for 72 hours and TSA was added for the last 12 hours. The media containing drugs were changed every 24 hours.U87 and U251 glioma cell lines were seeded at 1 \u00d7 10miRIDIAN miRNA mimic negative control (cel-miR-67) and miRIDIAN miRNA mimics were purchased from Dharmacon and validated using the pMIR-REPORT miRNA Expression Reporter Vector System . For results, see Additional file HindIII and SpeI restriction sites of pMIR-REPORT: CDK6-UTR-WT fw 5'-AGCTTGATCACAGAAATATTGCTAGCTGATACATATTATTGCATTTCATAAAACTA CDK6-UTR-WT rv 5'-CTAGTAGTTTTATGAAATGCAATAATATGTATCAGCTAGCAATATTTCTGTGATCA CDK6-UTR-MUT fw 5'-AGCTTGATCACAGAAATTAACGAAGCTGATACATATTATTGCATTTCATAAAACTA CDK6-UTR-MUT rv 5'-CTAGTAGTTTTATGAAATGCAATAATATGTATCAGCTTCGTTAATTTCTGTGATCA Cells were transfected with (1) miR-137 or cel-miR-67-negative-control mimics (50 nM), (2) pMIR-REPORT vectors containing WT or MUT miR-137 binding sites (400 ng) and (3) pRL-SV40 (Promega) expressing Renilla luciferase (400 ng) for normalization. Cells were grown in high-glucose DMEM supplemented with 10% fetal bovine serum, and luciferase measurements were performed 48 hours post-transfection using the Dual-Luciferase Reporter Assay System (Promega).Cyclin-dependent kinase 6 (CDK6)-3'UTR reporter assays were performed in U251 cells. pMIR-REPORT vectors harboring CDK6-3'UTR sequences with wild type (WT) miR-137 binding sites or mutated (MUT) miR-137 binding sites were generated by cloning the following oligonucleotides into the Adult mouse subventricular zone (SVZ)-NSC cultures were derived and grown as described previously with a fFor transfection of miR-124/137 into SVZ-NSCs, 50,000 cells were plated into eight-well culture slides pretreated with 0.1 mg/ml poly-D-lysine (Sigma) and 10 \u03bcg/ml laminin (Invitrogen) 24 hours prior to transfection. A total of 100 nM miRIDIAN miRNA mimics (50 nM each for miR-124 and miR-137 co-transfections) were complexed with LipofectAMINE 2000 (Invitrogen) and added directly to cells growing in proliferating medium. Transfection and proliferating medium was removed 12 to 24 hours post-transfection and cells were induced to differentiate as described above.erbB transgene under control of the S100\u03b2 promoter [Adult tumor stem cells were derived from a low-grade oligodendroglioma of a 120-day-old FVB/N transgenic mouse expressing the v-For miRNA transfections, 25,000 cells were plated into eight-well pre-coated culture dishes (Nunc), 24 hours prior to transfection. Transfections and differentiation procedures were performed as described for SVZ-NSC cultures.Human GBM tissue was acquired by surgical removal after informed consent at UCSF and washed with Hank's buffered saline solution without magnesium and calcium. Tumors were then enzymatically dissociated with papain (Worthington) for 30 minutes at 37\u00b0C. After centrifugation and one wash with phosphate buffered saline, pH 7.4, cells were transferred to NBE media consisting of neurobasal media without retinoic acid (Invitrogen), N2 and B27 supplements , 20 ng/ml human recombinant bFGF (Peprotech) and 20 ng/ml human recombinant EGF (Sigma-Aldrich). Cells were plated in ultra-low adherent plates (Corning). Medium was changed every 3 to 5 days.Cells cultured in suspension were dissociated using accutase (Innovative Cell Technologies) for 30 minutes at 37\u00b0C. After one wash in RinseMACS buffer (Miltenyi Biotech), cells were incubated with magnetic beads conjugated with an antibody against the CD133/1 epitope. The cells were incubated with beads for 30 minutes at 4\u00b0C. Thereafter, cells were washed with 20\u00d7 RinseMACS buffer, centrifuged and added on to large cell columns connected to a pre-separation filter. Fluorescence-activated cell sorter analyses confirmed a pure CD133- fraction and a highly enriched CD133+ fraction.For transfections, both CD133+ and CD133- cells were plated in 24-well plates coated with polyornithine and laminin. Cells were transfected with miR-124 and/or miR-137 (100 nM) or a negative control oligonucleotide for 4 hours using lipofectamine. Cells were then washed and cultured for 10 days in NBE media without growth factors.Stem cell cultures were fixed, washed and preblocked prior to incubation with primary antibodies ab, 1:500, Sigma). Cells were then stained with Alexa488- or Alexa594-conjugated secondary antibodies and the nuclei counterstained with Hoechst 33258 (Molecular Probes) or DAPI (Sigma).Cell cycle analyses were conducted using the fluorescein isothiocyanate BrdU Flow Kit following manufacturer's recommendations .Immunoblotting was performed using standard protocols with antibodies CDK6 , Phospho-Rb , and \u03b2-actin .For more detailed information regarding experimental methods, see Additional file To identify deregulated miRNAs that have not been previously implicated in GBM cells ,22, we uWe next performed statistical analyses of our miRNA expression data to identify novel miRNAs of interest in HGAs (GBM and AA). For a summary of these analyses see Additional file We observed that the majority of the HGA-miRNAs show expression changes during, or have been implicated in, differentiation of various cell lineages: miR-7 during photoreceptor differentiation ; miR-124Our differentiation studies in mNSCs suggested that growth factor signaling, which is recurrently activated in HGAs, suppresses expression of miR-124 and miR-137. It has also been shown that miR-124 expression is epigenetically suppressed in a number of tumor types including colorectal and breast cancers . Furtherin vitro.To test whether up-regulation of miR-124 and miR-137 promote differentiation of adult mNSCs, we transfected proliferating mNSCs with double-stranded RNA oligonucleotides corresponding to the mature sequences of each miRNA. In each experiment, at least 80% to 90% transfection efficiencies were achieved. NSCs were maintained in proliferation medium during transfection in which cells generally have a spindle, non-neuronal morphology, with high expression of GFAP, a stem cell and astrocyte marker, but low expression of the neuronal marker Tuj1 Figure . Growth erbB transgenic mouse oligodendrogliomas [erbB tumor stem cells relative to mNSCs and expanded as tumor spheres in nonadherent plates. Cells were sorted using magnetic beads conjugated to an antibody against CD133, a putative marker of GBM stem cells ,15. BothTo further investigate the role of miR-137 in neuronal differentiation of GBM cells, we assessed expression of an additional neuronal marker, MAP2, following overexpression of miR-137. Unsorted SF6969 GBM cells were transfected with miR-137 and cultured for 10 days in NBE media without growth factors. In addition to the expected increase of cells positive for Tuj-1 after 10 days, we also observed an evident increase in MAP2-postive cells following transfection of miR-137 Figure . Again, Since exit from the cell cycle is required for induction of differentiation, we tested whether miR-124 and miR-137 inhibit proliferation of GBM cells. Relative to control oligonucleotides, transfection of miR-124 or miR-137 resulted in a marked reduction in the number of cells in the S-phase of the cell cycle and a marked increase in the number of cells in G0/G1 in U251 GBM cells Figure and earlTo ascertain the molecular mechanisms by which miR-124 and miR-137 induce G0/G1 cell cycle arrest in GBM cells, we assessed expression of CDK6, a regulator of the cell cycle and differentiation (reviewed in ), followTo validate that the 3' UTR of CDK6 is a direct target of miR-137, we used a luciferase reporter system in which the predicted miR-137 binding site of CDK6 was cloned downstream of luciferase. A control reporter vector was also developed in which the seed region of the miR-137 binding site was mutated Figure . Co-tranTo identify miRNAs that are recurrently deregulated in high-grade gliomas, we used quantitative RT-PCR to profile expression of 192 miRNAs in human non-neoplastic brain tissues, AAs and GBMs. From our analyses, we identified a number of miRNAs that have been described previously in GBM tumors such as miR-10b (see ) and theAnother notable discrepancy is that of miR-221 expression, which was not overexpressed in any of the tumors tested in our study, but was shown to be overexpressed in five out of nine of GBMs studied by Ciafre et al. . The mosOur expression analyses revealed a total of 35 miRNAs that were differentially expressed between high-grade gliomas and non-neoplastic brain tissue , which Our results reveal two potential mechanisms by which miR-124 and miR-137 may be suppressed in stem cells and/or tumor cells. The first mechanism is growth factor signaling: removal of EGF, and FGF from the culture media resulted in robust increases in miR-124 and miR-137 expression in adult NSCs. Given that activation of EGF and FGF The second mechanism by which miR-124 and miR-137 expression may be suppressed in GBM stem cells is via epigenetic modification of their transcriptional regulatory sequences. Indeed, epigenetic modification of specific miRNAs in other tumor types has been reported recently. For example, miR-127, which is down-regulated in prostate, colon and bladder tumors relative to matched normal tissues, is up-regulated in cell lines derived from these tumor types following inhibition of DNA demethylation and histone deacetylase . Of partPrevious studies have demonstrated that miR-124 is up-regulated during development of the rodent nervous system ,42, and The ability of miR-124 to induce robust stem cell differentiation appears to be dependent on cell type, developmental timing and other, as yet unidentified, factors. For example, in mouse neuroblastoma cell lines CAD and Neuro2a, ectopic up-regulation of miR-124 alone is sufficient to induce neuron-like differentiation, whereas in mouse embryonic carcinoma cells (P19), miR-124 enhances neuronal differentiation only in the presence of retinoic acid, an established inducer of P19 neuronal differentiation . InvestiRecent studies have begun to shed light on the molecular mechanisms by which miR-124 regulates differentiation and proliferation. For example, miR-124 directly targets PTBP1 (PTB/hnRNP I) mRNA, a global repressor of alternative pre-mRNA splicing in non-neuronal cells, resulting in the transition from non-neuronal- to neuronal-specific alternative splicing patterns . miR-124The ability of miR-124 and miR-137 to induce potent antiproliferative and prodifferentiation effects in CD133+ and CD133- human GBM cells suggests their potential value for treatment of this disease. RNAi-based therapeutics holds great promise for the development of entirely novel therapeutic strategies for disease treatment , and earWe have investigated the role of miRNAs in adult human HGAs and hGSCs and in adult mNSCs and mOSCs. Our studies showed, for the first time to the best of the authors' knowledge, that miR-124 and miR-137: (1) are expressed at significantly lower levels in GBM tumors relative to non-neoplastic brain tissue; (2) are up-regulated during neuronal differentiation of adult mNSCs induced by growth factor withdrawal; (3) promote neuronal-like differentiation of growth-factor-deprived mNSCs, mOSCs and hGSCs; (4) promote G0/G1 cell cycle arrest in GBM cells and growth-factor-deprived hGSCs; (5) inhibit expression of CDK6 mRNA, CDK6 protein and phosphorylated RB in GBM cells. These results suggest that targeted delivery of miR-124 and/or miR-137 to GBM tumor cells may be therapeutically valuable for GBM disease treatment.AA: anaplastic astrocytomas; bFGF: basic fibroblast growth factor; CD: cluster of differentiation; CDK6: cyclin-dependent kinase 6; DMEM: Dulbecco's Modified Eagle's Medium; EDTA: ethylene diamine tetraacetic acid; EGF: epidermal growth factor; ES: embryonic stem; FCS: fetal calf serum; FGF: fibroblast growth factor; GBM: glioblastoma multiforme; GFAP: glial fibrillary acidic protein; HGA: high grade astrocytoma; hGSC: human GBM-derived stem cell; MAP2: microtubule-associated protein 2; miRNA: microRNA; mNSC: mouse neural stem cells; mOSC: mouse oligodendroglioma tumor stem cells; mRNA: messenger RNA; MUT: mutated; NCS: neural stem cells; PDGF: platelet-derived growth factor; RB: retinoblastoma; RT-PCR: reverse transcriptase polymerase chain reaction; siRNA: short interfering RNA; SVZ: subventricular zone; TSA: trichostatin A; TSC: tumor stem cell; UCSF: University of California San Francisco; WHO: World Health Organization; WT: wild type.David Ginzinger, a collaborator and co-author of the manuscript, was an employee of Applied Biosystems at the time the experiments were conducted. The miRNA TaqMan assays were provided by David Ginzinger.JS performed the miRNA expression studies, proliferation assays in GBM cells, CDK6 and Rb Western blotting, luciferase reporter assays, developed the miRNA transfection protocols and helped to draft the manuscript. DAL designed, implemented, interpreted and described experiments involving SVZ-NSCs. CP designed, implemented, interpreted and described experiments involving mOSCs. AIP designed, implemented, interpreted and described experiments involving hGSCs. AKM designed, implemented, interpreted and described demethylation and deacetylation experiments. MY assisted with design, implementation and interpretation of miRNA expression studies. SRV provided a histopathological review of human tissues and critical revision of the manuscript. DGG provided design and support for the miRNA expression analyses. CDJ provided overall conception and design support and critical revision of the manuscript. JFC provided design and analyses support for the demethylation and deacetylation experiments and critical revision of the manuscript. GB provided design and analyses support for the differentiation studies of mOSCs and critical revision of the manuscript. WAW provided design and analyses support for the differentiation studies of hGSCs and critical revision of the manuscript. AA\u2013B provided design and analyses support for the miRNA expression and differentiation studies of SVZ-NSCs and critical revision of the manuscript. JGH conceived the study, participated in its design and coordination and drafted the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:Characteristics of the patient tissues used in this study.Click here for fileValidation of miR-124 expression and function in glioblastoma multiforme cells.Click here for fileSupplementary methods. This pdf file provides further details related to the methods used in this study.Click here for fileLog2 fold change of microRNAs in non-tumor brain samples (glioses), anaplastic astrocytomas and glioblastoma multiforme tumors.Click here for fileValidation of let-7a and miR-16 as appropriate control miRNAs in primary tumor samples and neural stem cells.Click here for fileStatistical comparisons of miRNA expression in glioblastoma multiforme and anaplastic astrocytoma tumors versus non-tumor brain tissue (gliosis).Click here for fileAssessment of miR-124a and miR-137 expression in mouse oligodendroglial stem cells.Click here for filemiR expression in glioblastoma multiforme cell lines following treatment with DNA demethylating and deacetylating agentsClick here for file"} +{"text": "Accordingly,both subcapsulary and outer cortex increase in size, whereas the deep cortex and principallythe thymic medulla almost disappear in DCx embryos. In contrast, other T-cell subsets ofDCx embryos, largely CDgglowCD4- cells and TcR \u03b3\u03b4-expressing cells do not undergosignificant variations throughout thymic ontogeny.We have evaluated the immunohistological and cytofluorometric changes that occur in thethymus of chicken embryos partially decapitated at 33-38 hr of incubation (DCx embryos)in an attempt to analyze possible neuroendocrinological influences on T-cell differentiationand, indirectly, the ontogeny of the so-called neuroendocrine-immune network. Thethymus of DCx embryos shows important variations that profoundly and selectively affectdifferent T-cell subsets, but not the nonlymphoid cell components of thymic stroma. Thesemodifications include the accumulation of cell precursors, mainly DN (CD4"} +{"text": "It is unknown whether breast cancer (BC) characteristics among young women treated with radiotherapy (RT) for Hodgkin's lymphoma (HL) differ from sporadic BC.Using population-based data, we calculated BC risk following HL according to clinicopathologic features.P=0.002) than that of ER-positive/PR-positive BC.Compared with BC in the general population, risks of oestrogen receptor (ER)-positive/progesterone receptor (PR)-positive and ER-negative/PR-negative BC in young, irradiated HL survivors were increased five-fold =3.81\u20136.35) and nine-fold (95% CI=6.93\u201312.25), respectively. Among 15-year survivors, relative risk of ER-negative/PR-negative BC exceeded by two-fold (Radiotherapy may disproportionately contribute to the development of BC with adverse prognostic features among young HL survivors. Young women treated with radiotherapy (RT) for Hodgkin's lymphoma (HL) have an elevated risk of developing breast cancer (BC) compared with the general population Programme in the United States during 1973\u20132000 and followed through 2005 . The SEEStandardised incidence ratios (SIRs) were calculated as the ratio of observed (Obs)-to-expected number of second or higher order invasive BC using methods previously described . Age-, r*Stat Incidence Rate module. BC age-specific IRs were calculated among HL survivors diagnosed with HL before 35 years of age and treated with RT by dividing the number of BC cases diagnosed during 1990\u20132005 at specified attained ages by the HL population at-risk. There were no cases of BC diagnosed before 15 years of age or at the age of 65 years and older among HL survivors. All IRs were expressed per 100 person-years and plotted on a log-linear scale as previously described for first primary female BC in the general population diagnosed during 1990\u20132005 were calculated using the SEERPRR>0.5). Ductal adenocarcinoma and upper outer quadrant tumours were diagnosed most frequently. Relative risks did not differ significantly according to histology or laterality.Overall, 2645 young female 5-year survivors of HL diagnosed between 1973\u20132000 and treated with RT as part of initial therapy had a six-fold risk of developing invasive BC compared with that expected in the general population . StandarPTrend=0.7), regional (PTrend=0.6), distant (PTrend=1.0) or unknown (PTrend=0.9) stages according to calendar year groups. Among LN-negative BC diagnosed during 1990\u20132005, we observed somewhat lower risks for larger (>2\u2009cm) than smaller (\u2a7d2\u2009cm) tumours; however, this difference was not statistically significant in multivariate RR analyses (PRR=0.12).In comparison with that expected for primary BC in the general population, there were no significant differences in risks for developing localised, regional or distant stage BC following RT for HL. Multivariate analyses comparing SIRs (while adjusting for age at HL diagnosis and time since HL diagnosis) did not demonstrate statistically significant differences in risk of BC for regional stage (RR=0.87) or distant stage (RR=0.88) disease compared with localised stage BC. We also considered the possibility that screening for subsequent radiation-related BC could increase the likelihood of detecting localised BC in the more recent calendar years, and therefore assessed differences in RT-related BC risk by stage over time . There was no significant trend observed for localised (PRR=0.008). The risk of developing high-grade BC was modestly elevated, but did not differ significantly from risk of low-grade tumours .Irradiated HL survivors had a greater than nine-fold risk of developing ER-negative/PR-negative BC during 1990\u20132005 as compared with a nearly five-fold risk of ER-positive/PR-positive tumours. In age- and time-adjusted multivariate analyses, the RR of ER-negative/PR-negative disease was 66% higher than ER-positive/PR-positive BC (PRR=0.002). Fifteen-year survivors also had a 52% significantly greater risk of high-grade compared with low-grade tumours .To assess for a possible radiation effect, we further evaluated hormone receptor status and grade according to time since HL diagnosis. The SIR of ER-negative/PR-negative BC and high-grade BC showed greater increases with time since HL diagnosis than did ER-positive/PR-positive or low-grade BC, respectively . Among 1Age-specific BC IRs among HL survivors and primary BC IRs in the general SEER population are depicted in This is the first study to demonstrate that young women treated with RT for HL have an increased risk of developing ER-positive/PR-positive and ER-negative/PR-negative BC compared with expected values in an age-matched series of BC in the general population. Overall, the RR of ER-negative/PR-negative BC was 66% higher than ER-positive/PR-positive BC among 5-year HL survivors, and nearly two-fold higher among 15-year survivors. In addition, 15-year survivors had a significant 50% greater risk of developing high-grade than low-grade BC compared with primary BC. Although longer follow-up is needed, temporal patterns suggest that radiation may contribute disproportionately to the development of BC with more adverse prognostic features than would be expected in the general population.In contrast to our findings, after accounting for patient age, hospital-based case\u2013control studies of BC among HL survivors have not found a significant variation in hormone-receptor status when compared with primary BC controls (Among HL survivors, higher SIRs for ER-negative/PR-negative BC than ER-positive/PR-positive BC may reflect lower baseline IRs for ER-negative/PR-negative BC in the general population. However, in our study, the incidence patterns by BC subtype among individuals with HL were distinct from those of primary BC. Whereas incidence of hormone receptor-positive BC in the general population exceeds that of ER-negative/PR-negative BC with advancing age, IR patterns by subtype among irradiated HL survivors revealed no such divergence. It is also important to consider that young women treated for HL may experience premature ovarian failure related to HL therapy ; therefoSimilar to others studies (Other studies of HL survivors (The strengths of our study include the large number of second BCs occurring among nearly 2700 irradiated HL survivors diagnosed over a 30-year period. A unique feature is the population-based study design, which compared BC following HL to that expected in the general population using age-matched, subtype-specific BC IRs. Despite decentralised hormone receptor testing in various laboratories, SEER data have been shown to be reasonably reliable for ER-positive/PR-positive and ER-negative/PR-negative BC subtypes (Our population-based study suggests that long-term survivors of HL treated with RT before 35 years of age have a significantly higher risk of developing ER-negative/PR-negative than ER-positive/PR-positive BC, particularly among 15-year survivors. Fifteen-year HL survivors also had a significantly higher risk of developing high-grade than low-grade tumours. Although confirmatory studies are needed, the temporal patterns we observed suggest that radiation may contribute disproportionately to the development of BC with adverse prognostic features."} +{"text": "Inhibition of protein kinase C (PKC) is discussed as a new approach for overcoming multidrug resistance (MDR) in cancer chemotherapy. For evaluation of this concept we applied the bisindolylmaleimide GF 109203X, which shows a highly selective inhibition of PKC isozymes alpha, beta 1, beta 2, gamma, delta and epsilon in vitro. The efficacy of this compound in modulation of MDR was examined using several P-glycoprotein (P-gp)-overexpressing cell lines including a MDR1-transfected HeLa clone, and was compared with the activities of dexniguldipine-HCI (DNIG) and dexverapamil-HC1 (DVER), both of which essentially act via binding to P-gp. As PKC alpha has been suggested to play a major role in P-gp-mediated MDR, cell lines exhibiting different expression levels of this PKC isozyme were chosen. On crude PKC preparations or in a cellular assay using a cfos(-711)CAT-transfected NIH 3T3 clone, the inhibitory qualities of the bisindolylmaleimide at submicromolar concentrations were demonstrated. At up 1 microM final concentrations of the PKC inhibitor GF 109203X, a concentration at which many PKC isozymes should be blocked substantially, no cytotoxic or MDR-reversing effects whatsoever were seen, as monitored by 72 h tetrazolium-based colorimetric MTT assays or a 90 min rhodamine 123 accumulation assay. Moreover, depletion of PKC alpha by phorbol ester in HeLa-MDR1 transfectants had no influence on rhodamine 123 accumulation after 24 or 48 h. MDR reversal activity of GF 109203X was seen at higher final drug concentrations, however. Remarkably, [3H]vinblastine-sulphate binding competition experiments using P-gp-containing crude membrane preparations demonstrated similar dose dependencies as found for MDR reversion by the three modulators, i.e. decreasing efficacy in the series dexniguldipine-HCl > dexverapamil-HCl > GF 109203X. Similar interaction with the P-gp in the micromolar concentration range was revealed by competition of GF 109203X with photoincorporation of [3H]azidopine into P-gp-containing crude membrane preparations. No significant effect of the PKC inhibitor on MDR1 expression was seen, which was examined by cDNA-PCR. Thus, the bisindolylmaleimide GF 109203X probably influences MDR mostly via direct binding to P-gp. Our work identifies the bisindolylmaleimide GF 109203X as a new type of drug interacting with P-gp directly, but does not support the concept of a major contribution of PKC to a P-gp-associated MDR, at least using the particular cellular model systems and the selective, albeit general, PKC inhibitor GF 109203X."} +{"text": "The glycolipid content of human non-seminomatous germ cell tumour cell lines correlates with their differentiation lineage. To analyse whether this reflects the situation in primary tumours, we studied five embryonal carcinomas, five yolk sac tumours and nine (mixed) non-seminomas, using thin-layer chromatography and carbohydrate immunostaining. We also analysed the glycolipid content of 19 seminomas to reveal their relationship with non-seminomas. Lactosylceramide (CDH) was detected in all embryonal carcinomas, but in fewer than half of the seminomas. Seminomas and embryonal carcinomas contained globoseries glycolipids, including globotriosylceramide (Gb3), globoside (Gb4), galactosy globoside (Gb5) and sialy1 galactosyl globoside (GL7). The lacto-series glycolipid Le(x) was found in all embryonal carcinomas, but only in one seminoma. Gangliosides GD3 and GT3 were detected in many seminomas, but rarely in embryonal carcinomas. Yolk sac tumours displayed a heterogeneous glycolipid profile. Compared with seminomas and pure embryonal carcinomas, differentiated non-seminomas had reduced levels of globo-series glycolipids, especially Gb3 and Gb5, whereas CDH, Le(x), GD3 and GT3 were found in the majority of cases. Thus, the glycolipid content of non-seminoma cell lines reflects the situation in primary tumours. Globo-series glycolipids are similarly expressed in seminomas and embryonal carcinomas. The expression of Gb3 and Gb5 is reduced in non-seminomas upon differentiation. Le(x) expression in non-seminomas, including embryonal carcinomas, allows discrimination from seminomas. Expression of gangliosides in seminomas might indicate their maturation from ganglioside-negative precursor cells. Reprogramming of these precursors would result in the formation of Le(x)-expressing embryonal carcinomas."} +{"text": "Classically protein kinase A (PKA) and transcription factor activator protein 1 (AP-1) mediate the cyclic AMP (cAMP) induced-corticotrophin releasing hormone (CRH) expression in the placenta. However enteric Gram (-) bacterial cell wall component lipopolysaccharide (LPS) may also induce-CRH expression via Toll like receptor (TLR)4 and its adaptor molecule Myd88. Here we investigated the role of MyD88, TRIF and IRAK2 on cAMP-induced CRH promoter activation in JEG3 cells in the absence of LPS/TLR4 stimulation.JEG3 cells were transfected with CRH-luciferase and Beta-galactosidase expression vectors and either empty or dominant-negative (DN)-MyD88, DN-TRIF or DN-IRAK2 vectors using Fugene6 (Roche). cAMP-induced CRH promoter activation was examined by using a luminometer and luciferase assay. Calorimetric Beta-galactosidase assays were performed to correct for transfection efficiency. Luciferase expression vectors of cAMP-downstream molecules, CRE and AP-1, were used to further examine the signaling cascades.cAMP stimulation induced AP-1 and CRE promoter expression and led to dose-dependent CRH promoter activation in JEG3 cells. Inhibition of MyD88 signaling blocked cAMP-induced CRE and CRH promoter activation. Inhibition of TRIF signaling blocked cAMP-induced CRH but not CRE expression, while inhibition of IRAK2 did not have an effect on cAMP-induced CRH expression.MyD88 and TRIF exert direct regulatory effect on cAMP-induced CRH promoter activation in JEG3 cells in the absence of infection. MyD88 most likely interacts with molecules upstream of IRAK2 to regulate cAMP-induced CRH expression. In the placenta cAMP induces CRH expression via PKA, cAMP response element (CRE) and transcription factor activating protein (AP)-1 signaling . Our dacAMP regulates gene transcription via PKA. In the basal state, PKA resides in the cytoplasm as an inactive heterotetramer of paired regulatory (R) and catalytic (C) subunits. cAMP liberates the C subunits, which passively diffuse into the nucleus and phosphorylate CREB . CREB me2) rich regions of the plasma membrane through its amino-terminal phosphatidylinositol 4,5-bisphosphate (PIP2)-binding domain [cAMP is known to inhibit immune activation in macrophages since 1970s . Scaffolg domain . MyD88 mHere we demonstrate that inhibition of MyD88 and TRIF signaling block cAMP-induced CRH promoter activation in the JEG3 cells in the absence of infection. These data add to our previous findings on infection induced placental CRH expression and sugg2): phosphatidylinositol-4,5-bisphosphate; (PIP2): phosphatidylinositol 4,5-bisphosphate; (AKAPs): A-kinase anchoring proteins; (PKA): protein kinase A; (CRE): cAMP-response element.(TLR): Toll like receptor; (AP-1): activator protein 1; (TIR): Toll/Interleukin-1 receptor; (TRIF): TIR-domain-containing adapter-inducing interferon-\u03b2; (TIRAP): TIR-domain-containing adaptor molecule; (MyD88): myeloid differentiation primary response; (CRH): corticotrophin releasing hormone; (AC): Adenylate cyclases; (cAMP): cyclic adenosine monophosphate; PThe authors declare that they have no competing interests.AU conducted the transfection, luciferase and galactosidase experiments; CFS provided funds, helped with data analyses and manuscript preparation; CTB conducted the statistical analyses; AFG helped with the study design, data analyses, and manuscript preparation, he developed and provided help with the CRE and AP-1 vectors; HA helped with data analyses and manuscript preparation; RN developed the CRH-luciferase vectors, he helped with the study design, data analyses, manuscript preparation; HK and NK conducted the transfection, luciferase and galactosidase experiments; OE initiated and designed the project, analyzed the data, wrote the manuscript. All authors have read and approved the final manuscript."} +{"text": "Despite being a mainstay for treating superficial bladder carcinoma and a promising agent for interstitial cystitis, the precise mechanism of Bacillus Calmette-Guerin (BCG) remains poorly understood. It is particularly unclear whether BCG is capable of altering gene expression beyond its well-recognized pro-inflammatory effects and how this relates to its therapeutic efficacy. The objective of this study was to determine differentially expressed genes in the mouse bladder following repeated intravesical BCG therapy.Mice were transurethrally instilled with BCG or pyrogen-free on days 1, 7, 14, and 21. Seven days after the last instillation, urothelia along with the submucosa was removed and amplified ds-DNA was prepared from control- and BCG-treated bladder mucosa and used to generate suppression subtractive hybridization (SSH). Plasmids from control- and BCG-specific differentially expressed clones and confirmed by Virtual Northern were then purified and the inserts were sequenced and annotated. Finally, chromatin immune precipitation combined with real-time polymerase chain reaction assay (ChIP/Q-PCR) was used to validate SSH-selected transcripts.Repeated intravesical BCG treatment induced an up regulation of genes associated with antigen presentation and representatives of two IFN\u03b3-induced small GTPase families: the GBPs and the p47GTPases . Genes expressed in saline-treated bladders but down-regulated by BCG included: the single-spanning uroplakins (UPK3a and UPK2), SPRR2G, GSTM5, and RSP 19.Mycobacterium. It will be of tremendous future interest to determine whether these immune response cascades play a role in the anti-cancer effects exerted by BCG.Here we introduced a hypothesis-generator approach to determine key genes involved in the urothelium/sumbmucosa responses to BCG therapy. Urinary bladder responds to repeated BCG treatment by up-regulating not only antigen presentation-related genes, but also GBP and p47 small GTPases, both potentially serving to mount a resistance to the replication of the Intravesical Bacillus Calmette-Guerin (BCG) has been presented as a promising option for treatment of interstitial cystitis . It is eIt is not clear, however, how BCG alters the course of cystitis or cancer progression. One theory is that intravesical BCG corrects an aberrant immune imbalance in the bladder, leading to long-term symptomatic improvement .1 response). This cell activation leads to phase 3, which consists of amplification of cytotoxic-populations: CD8 T cells, gamma-delta lymphocytes, macrophages, and natural killer (NK) cells. All these cells also release cytokines which then regulate the immune response to BCG , is a robust and expertly curated database containing up-to-date information on over 20,000 mammalian genes and proteins, 1.4 million biological interactions, and one hundred canonical pathways incorporating over 6,000 discreet gene concepts. This information is integrated with other relevant databases such as EntrezGene and Gene Ontology . IPA comce alone . The scoce alone . The sigordingly .\u00ae-Aventis-Pasteur; total dose of 1.35 mg) or pyrogen-free saline on days 1, 7, 14, and 21, as described above. Mice were euthanized with pentobarbital 24 hours after a single instillation (BCG acute) or 7 days after 4 weekly instillations (Control and BCG repeated). A total of 60 mice were used (20 mice per group). Bladders were removed rapidly, frozen, and were shipped to Genpathway ) and is expressed by all nucleated cells. Class I molecules play a central role in the immune system mainly by presenting peptides to class I-restricted CD8+ T cells. Class I-binding peptides are typically derived from the endoplasmic reticulum lumen but also include 'cross-presented' exogenous antigens . The claOur results also show, for the first time, that BCG treatment selectively alters the expression of some of the uroplakins. Uroplakins are a group of integral membrane proteins found exclusively in differentiated mammalian urothelial cells -66. TherWe used the subtraction technique in an attempt to find alterations in the host urothelium and chronically infiltrating host immune cells by subtracting out the control urothelium messages. Thus, we expected to find changes related to (chronic) host immunity, host-pathogen relationships and alterations in the mucosa. Our results indicate that BCG induces both host adaptive immune response (HLA) and host mediated pathogen destruction (GTPases) with a down-regulation of uroplakins. The GTPases appear to be dedicated to the BCG response, since with the exception of GBP2, their resting levels in normal urothelium are negligible Figure . Most ofThe author(s) declare that they have no competing interests.in silico analysis, and drafted the manuscript.MRS participated in its design, carried out the animal experiments, removed the tissues, extracted the RNA, performed suppression subtractive hybridizations, and performed sequence alignments; HLH participated in the design of the study regarding SSHs and target validation; CS treated the animals with BCG; CAD was responsible for the animal husbandry; MLL consulted RS regarding HLA, NADPH, and helped drafting the manuscript; MAI participated in the experimental design and helped drafting this manuscript; MAO consulted RS regarding clinical implications of BCG treatment, data interpretation, and helped drafting the manuscript; W-RW consulted RS regarding uroplakins, data interpretation, and helped drafting the manuscript. RS conceived of the study, developed annotation and The pre-publication history for this paper can be accessed here:Agarose gel electrophoresis of RNA samples. C = saline-treated and T=BCG-treated.Click here for fileRsa I digestion. Lane 1-SMART-amplified C cDNA (driver); Lane 2-SMART-amplified T cDNA (tester); Lane 3-Rsa I digested C cDNA; and Lane 4-Rsa I digested T cDNA M = 1 kb DNA size markers.Agarose gel electrophoresis of ds cDNA synthesis and Click here for fileTable 1 Oligonucleotides usedClick here for fileAgarose gel electrophoresis of primary and secondary PCR products. Lane M = 1 kb DNA size markers, Lane 1 = primary PCR of C subtracted cDNA; Lane 2 = primary PCR of T subtracted cDNA; Lane 3 = secondary PCR of C subtracted cDNA; Lane 4 = secondary PCR of T subtracted cDNA; Lane 5 = unsubtracted C cDNA; and Lane 6 = unsubtracted T cDNA.Click here for fileTable 2. QOCR PrimersClick here for fileDifferential screening of plate C-1 from C (driver) subtracted library was subjected to differential screening using driver-specific (A) and tester-specific (B) subtracted probes.Click here for fileDifferential screening of plate C-2 from C (driver) subtracted library was subjected to differential screening using driver-specific (A) and tester-specific (B) subtracted probes.Click here for fileDifferential screening of plate T-1 from T (tester) subtracted library was subjected to differential screening using driver-specific (A) and tester-specific (B) subtracted probes.Click here for fileDifferential screening of plate T-2 from T (tester) subtracted library was subjected to differential screening using driver-specific (A) and tester-specific (B) subtracted probes.Click here for fileDifferential screening of plate T-3 from T (tester) subtracted library was subjected to differential screening using driver-specific (A) and tester-specific (B) subtracted probes.Click here for fileDifferential screening of plate T-4 from T (tester) subtracted library was subjected to differential screening using driver-specific (A) and tester-specific (B) subtracted probes.Click here for fileDifferential screening of plate T-5 from T (tester) subtracted library was subjected to differential screening using driver-specific (A) and tester-specific (B) subtracted probes.Click here for fileVirtual Northern blot analysis of differential clones obtained from control bladder (C) subtracted library. A = Plate C-1 and B = Plate C-2Click here for fileVirtual Northern blot analysis of differential clones obtained from BCG-treated bladder (T) subtracted library. A = Plate T-1; B = Plate T-2; C = Plate T-3; D = Plate T-4; and E = Plate T-5.Click here for fileTable 3. Genes up-regulated by BCGClick here for fileTable 4. Genes down-regulated by BCG.Click here for fileTable 5. Ingenuity Summary BCG.Click here for fileTable 6. Ingenuity Summary Control.Click here for file"} +{"text": "Microcystis proliferates in a wide range of freshwater ecosystems and is exposed to changing environmental factors during its life cycle. Microcystis blooms are often toxic, potentially fatal to animals and humans, and may cause environmental problems. There has been little investigation of the genomics of these cyanobacteria.The colonial cyanobacterium Microcystis aeruginosa PCC 7806 has revealed the high plasticity of its genome: 11.7% DNA repeats containing more than 1,000 bases, 6.8% putative transposases and 21 putative restriction enzymes. Compared to the genomes of other cyanobacterial lineages, strain PCC 7806 contains a large number of atypical genes that may have been acquired by lateral transfers. Metabolic pathways, such as fermentation and a methionine salvage pathway, have been identified, as have genes for programmed cell death that may be related to the rapid disappearance of Microcystis blooms in nature. Analysis of the PCC 7806 genome also reveals striking novel biosynthetic features that might help to elucidate the ecological impact of secondary metabolites and lead to the discovery of novel metabolites for new biotechnological applications. M. aeruginosa and other large cyanobacterial genomes exhibit a rapid loss of synteny in contrast to other microbial genomes.Deciphering the 5,172,804 bp sequence of Microcystis aeruginosa PCC 7806 appears to have adopted an evolutionary strategy relying on unusual genome plasticity to adapt to eutrophic freshwater ecosystems, a property shared by another strain of M. aeruginosa (NIES-843). Comparisons of the genomes of PCC 7806 and other cyanobacterial strains indicate that a similar strategy may have also been used by the marine strain Crocosphaera watsonii WH8501 to adapt to other ecological niches, such as oligotrophic open oceans. As archstralia) .Microcystis are distributed worldwide, and are involved in numerous proliferation events in stratified lakes [Microcystis cells are organized in large colonies of various sizes and shapes, which were used to define various morphospecies. Five of these have recently been reunified as a single species, Microcystis aeruginosa [Freshwater cyanobacteria of the genus ed lakes . In theiruginosa . The detM. aeruginosa is characterized by an annual life cycle comprising a spring and summer pelagic phase, and an overwintering benthic phase [M. aeruginosa colonies migrate daily in the water column [The ecology of ic phase . During r column and may Microcystis cells to synthesize toxins, in particular variants of microcystin [In the last decade, cyanobacterial blooms have been involved in numerous cases of animal and humarocystin . Many otrocystin . Other pMicrocystis aeruginosa, we deciphered the genome sequence of the toxic strain PCC 7806. The results presented here associate descriptive genomics and comparisons with the genomes of other cyanobacteria isolated from freshwater and marine ecosystems to highlight the ecophysiological peculiarities of this strain, and put its particularly high genome plasticity into a cyanobacterial context.To gain further insight into the ecophysiology of AM778843\u2013AM778958). The genome sequence of M. aeruginosa PCC 7806 (Mic-PCC7806), represented by these contigs, consists of 5,172,804 bases, with an average G+C content of 42%. These values are consistent with those previously determined using thermally denatured DNA [The 12\u00d7 shotgun sequencing project produced 90,000 sequence reads, and their assembly resulted in more than 500 contigs. After the first steps of a long finishing process performed using CAAT-Box and Consured DNA . The conAll the genomes used for the comparative studies described below are listed in the Methods section.Microcystis form a well-supported group (BV of 853\u2030) with Synechocystis sp. (Syn-PCC6803), Crocosphaera watsonii (Cwa-WH8501) and Cyanothece sp. (Cth-CCY0110 and Cth-ATCC51142). Within this group, Microcystis is most closely related to Syn-PCC6803 (BV of 990\u2030).A concatenated dataset of large and small subunit rRNA sequences 23S and 16S rRNA) was used to construct a phylogenetic tree including Mic-PCC7806 and 37 other cyanobacterial strains [Microcystis strains independently. Although the two genomes contain the same proportions of large DNA repeats , their distribution and size partly differ since Mic-PCC7806 contains 48 repeats longer than 3,000 bases for only 11 in Mic-NIES843. The comparison of the location of similar genes in the largest contig of the Mic-PCC7806 assembly (contig328) and in the Mic-NIES843 genome shows numerous genomic rearrangements . AlthougThe 5292 CDSs of the Mic-PCC7806 genome were also compared to the proteomes of 44 strains representing the diversity of the cyanobacterial lineages . The distribution of the best High Scoring Pairs (HSPs) found using Blastall software indicates a high similarity between the proteome of Mic-PCC7806 and a group of three strains Cth-ATCC51142, Cth-CCY0110 and Cwa-WH8501 specific to the Mic-PCC7806 genome and not found in the 15 selected genomes; 438 (8.3%) of them have no homolog in the uniprot database;- The \"core40\" group comprised 652 proteins (12.3%) sharing significant Blastp scores with at least one CDS in each of the 15 other genomes tested;- The last group, designated \"other40\", consisted of 3,876 CDSs (73%) sharing significant Blastp scores with CDSs in only some of the other 15 genomes tested.The small percentage of CDSs in the core40 group reflects the wide diversity of the cyanobacterial genomes analyzed. In the other40 group, the distribution of the Mic-PCC7806 CDSs among the tested genomes matches their phylogenetic distances based on 23S-16S rDNA sequences. For example, in this group, 10% of the CDSs were present in all the genomes, apart from that of Gvi-PCC7421, which is the most distant phylogenetically . The genome of Cwa-WH8501 also contains numerous putative transposases. One third of them are associated to IS5, but none to IS30; the DNA repeated sequences are therefore different in each genome, and cannot account for the close phylogenetic relationship between these two strains.The Mic-PCC7806 genome includes a very large number of DNA sequences containing more than 1000 bases that are repeated at least twice in the genome with more than 90% identity. A comparative analysis of all the cyanobacterial genome sequences available in databases showed that Mic-PCC7806, Mic-NIES843 and Cwa-WH8501 are particularly rich in such DNA repeats. Indeed, they account for 11.7%, 11.7% and 19.8% of the total DNA length, respectively Figure . The cum PCC7806 , but a lMicrocystis strains and two close relatives, Cwa-WH8501 and Syn-PCC6803 (68% mean CDS similarity). Since the same observation was made for all the cyanobacterial genomes tested, we compared the dynamics of these genomes using a large set of other bacterial genomes chosen on the basis of their sizes and phylogenetic distances. To this end, a synteny score was calculated for a number of genome pairs (see Methods), and then compared to their evolutionary distance based on the 23S-16S rDNA tree. This analysis showed that the synteny scores for cyanobacterial genomes were significantly lower than those obtained for pairs of non-cyanobacterial genomes with similar genome lengths and 23S-16S phylogenetic distances than that of Gvi-PCC7421 (4.6 Mb), but harbors longer intergenic sequences. Although the role of long intergenic sequences in most cyanobacterial genomes remains unclear, we can surmise that they might be involved in the modulation of gene expression, which would allow cells to acclimate to rapid environmental changes.Microcystis genomes contain a higher proportion of genes recently acquired by lateral transfers than the other genomes studied.In order to explore the plasticity of the Mic-PCC7806 genome further, the number of CDSs with an atypical dinucleotide composition was determined using a one-order Markov chain-based methodology . This meMicrocystis aeruginosa strains might thus constitute a rich source of novel restriction enzymes potentially useful in biotechnology. According to Zhao et al. [Anabaena, Spirulina and Nostoc strains) contain more restriction and modification genes than unicellular cyanobacteria . Based on COG annotations, at least as many restriction-modification genes were found in Mic-PCC7806, Mic-NIES843 and Cwa-WH8501 as in filamentous cyanobacteria. Thus, rather than corresponding to a difference between filamentous and unicellular cyanobacteria, the restriction-modification gene content of Microcystis aeruginosa may reflect the potential exposure of the cells to high concentrations of foreign DNA due to the presence of numerous other bacterial cells or viruses associated with Microcystis colonies [Crocosphaera remains an open question.Blast searches for restriction enzymes and examination of genes surrounding DNA methylases, identified 21 potential restriction enzymes . The dnd gene products incorporate sulfur into the DNA backbone as a sequence-selective, stereospecific phosphorothioate modification [et al. [dndB homologs is not clear in the genomes of cyanobacteria, the rest of the cluster was found in several of them including Mic-PCC7806 coding for a protein similar to Hik33 that perceives osmotic stress and cold stress in Syn-PCC6803 [mic5237, is similar to the Ana-PCC7120 orrA gene whose product is involved in osmoregulation [actM and pfnM, two genes that encode eukaryotic-like proteins, actin and profilin (an actin cognate binding partner), respectively, have been discovered in the Mic-PCC7806 genome. As shown by Guljamow et al. [Microcystis cells that inhabit the Braakman water reservoir (The Netherlands), which was cut off from the sea in the 20th century, and from which the Mic-PCC7806 strain was originally isolated.During the overwintering benthic phase of their life cycle, cal data . All the-PCC6803 . Anothergulation . A genomM. aeruginosa morphotypes have been described [mvn; mic3128), which binds specifically to a sugar moiety present on the surface of Mic-PCC7806 cells, and a binding partner has been identified in the lipoplysaccharide fraction [Microcystis morphotypes. Another protein, MrpC (microcystin-related protein C), has been shown to be a potential target of an O-glycosyltransferase of the SPINDLY family [In situ, this protein accumulates at the cell surface, and is involved in cellular interactions. Microcystins may therefore have an impact on the aggregation of Microcystis cells, which is very important for the competitive advantage of these organisms over other phytoplankton species. Mvn and MrpC are predominantly encoded in toxic strains [[mic0129) and a Ser/Thr phosphatase of the PPP family (mic4622) are found within two clusters that may be involved in cell wall synthesis. Mic-PCC7806 also has two genes that encode Wzc-like protein Tyr kinases (mic2086 and mic1089) and three genes coding for Wzb-like protein Tyr phosphatases . In E. coli, the function of these systems is known to be related to the synthesis of the cell wall and polysaccharides [gvpV and gvpW, are novel [mic1271 and mic1270 genes are highly similar to the genes coding for a light-regulated two-component system in Syn-PCC6803. This system, which consists of a cyanobacterial phytochrome (Cph1) and its response regulator (Rcp1), has been proposed to play a role in the control of processes required for the adaptation from light to dark conditions and vice-versa [Microcystis colonies in the water column are controlled by this phytochrome and by the circadian clock mechanism would be worth being tested.Although several different escribed , little fraction . A functY family . In situstrains [ and E. Dcharides . These kcharides . The Micre novel . The micce-versa . Moreovece-versa are presMicrocystis, oxidative stress was shown to induce programmed cell death (PCD) [mic1068 and mic5406 are expressed, and a cross-reaction with human caspase-3 polyclonal antisera was observed indicating that the proteins are synthesized (data not shown). Alignment of the regions containing the conserved caspase domains of Mic1068, Mic5406, MAE24870 and a yeast metacaspase shows that the Histidine-Cysteine catalytic diad of the key functionnal regions of the capases is conserved , proteins characterized by the presence of both His and Ser/Thr kinase domains [2-fixing cells in filamentous cyanobacteria [[M. aeruginosa, but it would be interesting to test whether these HstK-like protein kinases are involved in iron homeostasis and/or in the control of programmed cell death in response to oxidative stress. It has been proposed that the methionine recycling pathway may contribute to preventing oxidative stress in Bacillus subtilis [mtnW (rbcLIV), encodes a 2,3-diketo-5-methylthiopentyl-1-phosphate enolase that has been identified in all the Microcystis strains tested including Mic-NIES843 [Microcystis, Lyngbya and Cyanothece.In natural populations of th (PCD) . Accordir-IMS101 . Mic-PCC domains . Some ofacteria [ and C-C subtilis ,51. Inte-NIES843 ,52, but Cyanobacteria are known as prolific producers of natural products, in particular of the nonribosomal peptide and polyketide classes ,53. HowemcyA-J gene cluster encoding NRPS, PKS and tailoring enzymes [mcn cluster) could be assigned based on the amino acid specificities of the substrate-activating domains of a second NRPS gene cluster that was congruent with the amino acid moieties contained in the cyanopeptolin structure [mcn genes of Mic-PCC7806 display some similarity to the anabaenopeptilide genes of Anabaena strain 90 [Microcystis wesenbergii [Planktothrix agardhii Cya 126 [The strain Mic-PCC7806 is known to produce two isoforms of microcystin . The cor enzymes ,57 couldtructure while tcells/ml ). Microch phases , but to m et al. for SalmMicrocystis and Crocosphaera genera are required to clarify the molecular basis of their genome plasticity, at both the intergeneric and intraspecies levels. This will also provide a deeper understanding of the evolutionary significance of this mode of adaptation to the environment. The ongoing sequencing of such genomes should make it possible to reach this goal in the near future. More generally, large cyanobacterial genomes constitute excellent model systems for studying genome dynamics and the mechanism(s) by which some gene clusters may escape rearrangement and retain the same physical organization in several different lineages.More genome sequences of members of the Acaryochloris marina MBIC11017 (embl: CP000828)Ama-MBIC11017: Anabaena/Nostoc sp. PCC 7120 (embl: BA000019)Ana-PCC7120: Anabaena variabilis ATCC 29413 (embl: CP000117)Ava-ATCC29413: Cyanobium sp. PCC 7001 (gb: 1106012173546)Cbi-PCC7001: Cyanothece sp. ATCC 51142 (embl: CP000806)Cth-ATCC51142: Cyanothece sp. CCY0110 (gb: 1101676644636\u20131101676644658)Cth-CCY0110: Crocosphaera watsonii WH8501 (embl: AADV02000100)Cwa-WH8501: Cyanobacteria Yellowstone JA-3-3Ab (embl: CP000239)Syn-JA33Ab: Cyanobacteria Yellowstone JA-2-3B'a (embl: CP000240)Syn-JA23B'a: Gloeobacter violaceus PCC 7421 (embl: BA000045)Gvi-PCC7421: Lyngbya aestuari PCC 8106 (gb: 1099428180563\u20131099428180584)Lae-PCC8106: Microcoleus chthonoplastes PCC 7420 (gb:1103659003780\u20131103659003836)Mch-PCC7420: Microcystis aeruginosa NIES-843 (embl: AP009552)Mic-NIES843: Microcystis aeruginosa PCC 7806 (embl: AM778843\u2013AM778958)Mic-PCC7806: Nodularia spumigena CCY9414 (gb:1099428179735\u20131099428179797)Nsp-CCY9414: Nostoc punctiforme PCC 73102 (kindly provided by J. C. Meeks) [Npu-PCC73102: . Meeks) Prochlorococcus marinus SS120 (embl: AE017126)Pro-SS120: Prochlorococcus marinus AS9601 (embl: CP000551)Pro-AS9601: Prochlorococcus marinus MED4 (embl: BX548174)Pro-MED4: Prochlorococcus marinus MIT9211 (embl: AALP01000001)Pro-MIT9211: Prochlorococcus marinus MIT9215 (embl: CP000825)Pro-MIT9215: Prochlorococcus marinus MIT9301 (embl: CP000576)Pro-MIT9301: Prochlorococcus marinus MIT9303 (embl: CP000554)Pro-MIT9303: Prochlorococcus marinus MIT9312 (embl: CP000111)Pro-MIT9312: Prochlorococcus marinus MIT9313 (embl: BX572095)Pro-MIT9313: Prochlorococcus marinus MIT9515 (embl: CP000552)Pro-MIT9515: Prochlorococcus marinus NATL1A (embl: CP000553)Pro-NATL1A: Prochlorococcus marinus NATL2A (embl: CP000095)Pro-NATL2A: Synechococcus sp. BL107 (gb: 1099739244347)Syn-BL107: Synechococcus sp. CC9311 (embl: CP000435)Syn-CC9311: Synechococcus sp. CC9605 (embl: CP000110)Syn-CC9605: Synechococcus sp. CC9902 (embl: CP000097)Syn-CC9902: Synechococcus elongatus PCC 6301 (embl: AP008231)Syn-PCC6301: Synechococcus sp. PCC 7002 (embl: CP000951)Syn-PCC7002: Synechococcus sp. PCC 7335 (gb: 1103496006889\u20131103496006899)Syn-PCC7335: Synechococcus elongatus PCC 7942 (embl: CP000100)Syn-PCC7942: Synechococcus sp. RCC307 (embl: CT978603)Syn-RCC307: Synechococcus sp. RS9916 (gb: 1100013018508)Syn-RS9916: Synechococcus sp. RS9917 (gb: 1099465004208)Syn-RS9917: Synechococcus sp. WH5701 (gb: 1099465003749\u20131099465003864)Syn-WH5701: Synechococcus sp. WH7803 (embl: CT971583)Syn-WH7803: Synechococcus sp. WH7805 (gb: 1099646010155\u20131099646010157)Syn-WH7805: Synechococcus sp. WH8102 (gb: BX548020)Syn-WH8102: Synechocystis sp. PCC 6803 (embl: BA000022)Syn-PCC6803: Thermosynechococcus elongatus BP-1 (embl: BA000039)Tel-BP1: Trichodesmium erythreum IMS101 (embl: CP000393)Ter-IMS101: Mic-PCC7806/Cwa-WH8501Mic-PCC7806/Syn-PCC6803Cwa-WH8501/Syn-PCC6803Lae-PCC8106/Ter-IMS101Npu-PCC73102/Ava-ATCC29413Npu-PCC73102/Ana-PCC7120Npu-PCC73102/Nsp-CCY9414Nsp-CCY9414/Ana-PCC7120Ava-ATCC29413/Nsp-CCY9414Ana-PCC7120/Ava-ATCC29413Shigella dysenteriae, serovar 1, strain Sd97/Sd197 (CP000034_GR)Acidovorax avenae subsp. citrulli AAC00-1 (NC_008752)Agrobacterium tumefaciens str. C58 (NC_003062)Bacillus subtilis subsp. subtilis str. 168 (NC_000964)Bordetella parapertussis 12822 (NC_002928)Escherichia coli APEC O1 (NC_008563)Enterobacter sp. 638 (NC_009436)Janthinobacterium sp. Marseille (NC_009659)Klebsiella pneumoniae subsp. pneumoniae MGH 78578 (CP000647)Listeria monocytogenes EGD-e (NC_003210)Methylococcus capsulatus str. Bath (NC_002977)Ochrobactrum anthropi ATCC 49188 chromosome 1 (NC_009667)Polaromonas naphthalenivorans CJ2 (NC_008781)Pseudomonas aeruginosa PA7 (NC_009656)Pseudomonas fluorescens PfO-1 (NC_007492)Rhizobium etli CFN 42 (NC_007761)Rhizobium leguminosarum bv. viciae 3841 (NC_008380)Rhodobacter sphaeroides ATCC 17025 (NC_009428)Rhodoferax ferrireducens T118 (NC_007908)Shewanella loihica PV-4 (NC_009092)Shewanella oneidensis MR-1 (NC_004347)Shewanella sp. W3-18-1 (NC_008750)Shigella boydii Sb227 (NC_007613)Silicibacter sp. TM1040 (NC_008044)Yersinia enterocolitica subsp. enterocolitica 8081 (NC_008800)Yersinia pestis CO92 (NC_003143)Photorhabdus luminescens subsp. laumondii TTO1 (NC_005126)Microcystis aeruginosa PCC 7806 was gro, France . The genPlasmid DNA purification was performed using the Montage Plasmid Miniprep96 Kit or the TempliPhi DNA sequencing template amplification kit . BAC Miniprep96 Kit was used for BAC templates. Sequencing reactions were done, from both ends of DNA inserts, using ABI PRISM BigDye Terminator cycle sequencing ready reactions kit and run on a 3700 Genetic Analyzer . The trace file was used with the Phred-Phrap-Consed package to perform the assembly . SequencA dataset containing a concatenation of the 16S and 23S sequences was aligned by Muscle , and theTen orthologs located on either side of one pair of putatively orthologous CDS (linked by BDBH) were analyzed. For each pair of orthologous genes located in the proximity of the tested gene and of its ortholog, the synteny score was incremented by 1. Using this method of calculation, two totally syntenic genomes will have a score of 20 attributed to each of their orthologs, whereas two-non syntenic genomes will have a score of 0.Putative restriction enzymes were identified by Blast searching of known type I and II restriction enzymes against the Mic-PCC7806 genome. Because DNA methylases are more reliably identified by Blast than restriction enzymes, we also identified all methylases, and examined the surrounding genes for potential restriction enzymes.in silico horizontal gene transfer simulations, during which random genes from different genomes were introduced artificially into the genome sequences under study. The optimal threshold (0.1%) was defined for all the genomes of the group as the value at which the model had the highest detection of the in silico introduced genes (true positives), and the lowest detection of core genes .A first-order Markov model was built based on the dinucleotide composition of the core genes of a group of 8 selected cyanobacterial genomes Table , identifWe defined an initial cluster of at least 4 neighboring atypical genes which was allowed to grow (in both directions) searching for other nearby atypical genes, until regions containing 4 or more non-atypical genes appeared. By this process, a reduced number of less-atypical genes and of normal genes could be included in a larger CAG.CDS: coding sequence; HSP: high scoring segment pair; BDBH: bidirectional best hit; rDNA: ribosomal DNA; CAG: cluster of atypical gene; BV: bootstrap value. NRPS: nonribosomal peptide synthetase; PKS: polyketide synthase; N50: contig size such that all the larger contigs contain 50% of the bases of the assembly.LF carried out the bioinformatics studies. PQ and A\u2013MC carried out the molecular genetic studies. AB and A\u2013MC constructed the DNA libraries. CB, AL, PQ and A\u2013MC carried out the sequence of the genome. LF, PQ, A\u2013MC, NTM, ED, J\u2013FH, J\u2013CK, YZ, and NZ annotated the genome. HCPM, SB and NTM analyzed the metabolic pathways. DC carried out the CAG analyses. AT, PQ and LF carried out the enzyme and 6-mer analyses. SG and LF performed the phylogenetic analyses. CCZ, ET and AL analyzed the sensor and regulatory systems. NTM, LF and PQ designed the research. NTM coordinated the study. C\u2013CZ, SG, DC, HCPM and AT drafted the manuscript. LF, NTM, J\u2013FH, PQ and ED wrote the manuscript.All authors read and approved the final manuscript.Representation of the location of the genes of Mic-PCC7806-contig328 on the genome of Mic-NIES843.Click here for fileSimilarity of orthologous genes between Mic-PCC7806, Cwa-WH8501 and Syn-PCC6803.Click here for fileDistribution of genome lengths for several cyanobacterial genomes.Click here for fileSchematic representation of the phosphate transport gene cluster.Click here for fileDistribution of the intergenic distances in cyanobacterial and other bacterial genomes.Click here for filePutative restriction endonucleases in the genome of Mic-PCC7806.Click here for filePutative methylases and methyltransferases in the genome of Mic-PCC7806.Click here for fileIdentity of the 1% 6-mers that were least common in the genome of Mic-PCC7806.Click here for filednd gene products in cyanobacteria.Occurrence of the putative Click here for fileFermentation pathway adapted from Moezelaar and Stal .Click here for fileGenes of the circadian clock system.Click here for fileSaccharomyces cerevisiae.Alignment of the amino acid sequences of the putative caspases from Mic-PCC7806, Mic-NIES843 and a metacaspase from Click here for fileGenes of the methionine salvage pathway.Click here for file"} +{"text": "Escherichia coli by colicin M entering cells from the outside. Highly active colicin M preparations were inactive against fkpA mutant cells; 104-fold dilutions killed fkpA+ cells. Three previously isolated spontaneous mutants tolerant to colicin M carried a stop codon or an IS1 insertion in the peptidyl-prolyl-cis-trans-isomerase (PPIase) domain (C-domain) of FkpA, which resulted in deletion of the domain. A randomly generated mutant carried a G148D mutation in the C-domain. A temperature-sensitive mutant tolerant to colicin M carried a Y25N mutation in the FkpA N-domain. Mutants transformed with wild-type fkpA were colicin M-sensitive. Isolated FkpA-His reduced colicin M-His cleavage by proteinase K and renatured denatured colicin M-His in vitro; renaturation was prevented by the PPIase inhibitor FK506. In both assays, periplasmic SurA-His had no effect. No other tested periplasmic chaperone could activate colicin M. Among the tested colicins, only colicin M required FkpA for activity. Colicin M bound to cells via FhuA was inactivated by trypsin; unbound colicin M retained activity. We propose that colicin M unfolds during import across the outer membrane, FkpA specifically assists in folding colicin M into an active toxin in the periplasm and PPIase is essential for colicin M activity. Colicin M is a suitable tool for the isolation of FkpA mutants used to elucidate the functions of the FkpA N- and C-domains.Chaperones facilitate correct folding of newly synthesized proteins. We show here that the periplasmic FkpA chaperone is required for killing Escherichia coli that kill sensitive E. coli cells (E. coli cells carrying a ColBM plasmid. This colicin lyses sensitive cells by interfering with murein (peptidoglycan) biosynthesis (N-acetyl-muramyl pentapeptide-N-acetyl-glucosamine (lipid II), is transferred across the cytoplasmic membrane and incorporated into murein. Undecaprenyl pyrophosphate is released and converted to the monophosphate, which re-enters the reaction cycle. Colicin M inhibits this undecaprenylphosphate (lipid) carrier regeneration step of murein synthesis . These mutants are insensitive to colicin M, but the mutations map close to the streptomycin-resistance gene rpsL and not among the genes required for uptake activity .One mutant with a deleted periplasmic chaperone gene, t strain . Complemolicin M . StrainsfkpA mutant to the other FhuA ligands was as high as the sensitivity of the FkpA+ parent strain BW25113 insensitive to colicin M but fully sensitive to albomycin and phages T1 and T5 . The truncated FkpA derivatives comprise the entire FkpA N-domain ferrichrome was determined essentially as previously described (55Fe3+] (specific activity 81.5 kBq in 0.048 \u03bcg) and 5 \u03bcM deferri-ferrichrome. Samples were withdrawn after 5, 10, 15 and 25 min and filtered; the filters were dried, and the radioactivity was determined in a liquid scintillation counter.Transport of ["} +{"text": "A traditional single-stage reverse transcription-polymerase chain reaction (RT-PCR) procedure is effective in determining West Nile (WN) virus in avian tissue and infected cell cultures. However, the procedure lacks the sensitivity to detect WN virus in equine tissue. We describe an RT-nested PCR (RT-nPCR) procedure that identifies the North American strain of WN virus directly in equine and avian tissues."} +{"text": "Multidrug resistance-associated protein (MRP) and the canalicular multispecific organic anion transporter (cMOAT) are organic anion pumps that have been linked to cytotoxic drug resistance. We previously reported the isolation of three human MRP/cMOAT-related transporters, MOAT-B (MRP4), MOAT-C (MRP5) and MOAT-D (MRP3). In the present study we describe the fourth MRP/cMOAT-related transporter. We analysed ARA, a human cDNA reported to encode a 453 residue MRP-related transporter, and found that it represents a fused transcript composed of MRP sequences and partial sequences of a novel transporter. The complete coding sequence of this novel transporter, which we designated MOAT-E, was isolated. MOAT-E encodes a 1503 residue transporter that is most closely related to MRP (45%), MOAT-D (44%) and cMOAT (39%), both in terms of amino acid identity and sharing a common topology in which \u223c 17 transmembrane spanning helices are distributed within three membrane spanning domains. RNA blot analysis indicated that MOAT-E expression is restricted to kidney and liver. These observations suggest that MOAT-E may function as an organic anion transporter involved in cellular detoxification and possibly in the hepatobiliary and renal excretion of xenobiotics and/or endogenous metabolites. Isolation of MOAT-E helps to define the MRP/cMOAT subfamily of transporters. \u00a9 1999 Cancer Research Campaign"} +{"text": "When used as single agents at their maximum tolerated dose, all three novel compounds produced a significant growth retardation of BCNU-resistant murine colon and human breast xenografts. This in vivo anti-tumour effect was potentiated by BG, but was accompanied by severe myelotoxicity as judged by spleen colony forming assays. However, while tumour resistance to BCNU was overcome using BG, this was at the expense of enhanced bone marrow, gut and liver toxicity. Therefore, although this ATase-depletion approach resulted in improved anti-tumour activity for all three 5-FU:CNU molecular combinations, the potentiated toxicities in already dose-limiting tissues indicate that these types of agents offer no therapeutic advantage over BCNU when they are used together with BG. \u00a9 1999 Cancer Research CampaignPrevious studies have demonstrated that novel molecular combinations of 5-fluorouracil (5FU) and 2-chloroethyl-1-nitrosourea (CNU) have good preclinical activity and may exert less myelotoxicity than the clinically used nitrosoureas such as 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). This study examined the effect of O"} +{"text": "In the subclass of lesions with diameters below 10 mm the sensitivities were 72% (n = 13), 78% (n = 14) and 100% (n = 18) respectively. In 35 regional lymph nodes classified as benign lesions, FNAC was always negative while FNA-PCR produced one positive result. Neither of these methods did produce false positive results in 15 control lymph nodes of non-melanoma patients. We conclude, that FNA-PCR might have superior sensitivity as compared to FNAC or ultrasound B-scan, particularly in melanoma lesions with diameters below 10 mm. \u00a9 1999 Cancer Research CampaignPhysical examination and ultrasound B-scan screening are important follow-up procedures in melanoma patients with regional disease. However, they do not allow definite diagnosis of suspicious lesions. Fine-needle aspiration cytology (FNAC) enhances the diagnostic accuracy in such patients but, unfortunately, reaches its technical limits, particularly when very small or necrotic lesions are examined. We therefore tested whether tyrosinase reverse transcription polymerase chain reaction (RT-PCR) of fine-needle aspirates (FNA-PCR) could help to increase diagnostic sensitivity. With clinical follow-up in 69 melanoma patients 81 regional lymph nodes were detected by ultrasound B-scan examination, nine of whom appeared to be palpable. Technically, FNAC was successful in all 81 lymph nodes, while FNA-PCR failed to obtain RNA at detectable levels in two lymph nodes of two patients. Of 79 lesions which have been completely evaluated by B-scan, FNAC and FNA-PCR, 44 proved to be melanoma metastases by histopathology, while the remaining 35 lesions were finally classified as non-specific lymph nodes. Of the 44 melanoma metastases 80% ("} +{"text": "The effect of cetirizine on plasma membrane fluidity andheterogeneity of human eosinophils, neutrophils, platelets andlymphocytes was investigated using a fluorescence technique.Membrane fluidity and heterogeneity were studied by measuring thesteady-state fluorescence anisotropy and fluorescence decay of 1-(4-trimethylammonium-phenyl)-6-phenyl-1, 3, 5-hexatriene (TMA-DPH)incorporated in the membrane. The results demonstrate thatcetirizine (1 \u03bcg/ml) induced a significant increase in theHpid order in the exterior part of the membrane and a decrease inmembrane heterogeneity in eosinophils, neutrophils and platelets.Moreover, cetirizine blocked the PAF induced changes in membranefluidity in these cells. Cetirizine did not influence significantlythe plasma membrane of lymphocytes. These data may partially explainthe effect ofcetirizine on inflammatory cell activities."} +{"text": "COUP-TFII-null mice die during the early embryonic development due to angiogenesis and cardiovascular defects. To circumvent the early embryonic lethality and investigate the physiological function of COUP-TFII, we knocked out COUP-TFII gene in a time-specific manner by using a tamoxifen inducible Cre recombinase. The ablation of COUP-TFII during pre-pubertal stages of male development results in infertility, hypogonadism and spermatogenetic arrest. Homozygous adult male mutants are defective in testosterone synthesis, and administration of testosterone could largely rescue the mutant defects. Notably, the rescued results also provide the evidence that the major function of adult Leydig cell is to synthesize testosterone. Further phenotypic analysis reveals that Leydig cell differentiation is arrested at the progenitor cell stage in the testes of null mice. The failure of testosterone to resumption of Leydig cell maturation in the null mice indicates that COUP-TFII itself is essential for this process. In addition, we identify that COUP-TFII plays roles in progenitor Leydig cell formation and early testis organogenesis, as demonstrated by the ablation of COUP-TFII at E18.5. On the other hand, when COUP-TFII is deleted in the adult stage after Leydig cells are well differentiated, there are no obvious defects in reproduction and Leydig cell function. Taken together, these results indicate that COUP-TFII plays a major role in differentiation, but not the maintenance of Leydig cells.Chicken Ovalbumin Upstream Promoter-Transcription Factor II , is an orphan nuclear receptor of the steroid/thyroid hormone receptor superfamily. Male fertility is controlled by complex interactions among the hypothalamus, pituitary gland, and testis COUP-TFII null mutants die before E10.5 due to angiogenesis and cardiovascular defects COUP-TFII in the limbs, stomach, diaphragm, uterus and endothelial cells reveal that COUP-TFII plays a pivotal role in cell growth, differentiation, organogenesis and lineage determination COUP-TFII heterozygous female mice develop significantly reduced fecundity, and the expression level of steroid biosynthetic enzymes such as P450Scc, 3\u03b2\u2212HSD, and steroidogenic acute regulatory protein (StAR), were significantly reduced Chicken Ovalbumin Upstream Promoter-Transcription Factor II (COUP-TFII) is a member of the nuclear receptor superfamily COUP-TFII, a tamoxifen inducible TMCAGG-Cre-ER mouse line COUP-TFII gene. The promoter driving Cre-ERTM expression is a chimeric promoter of the cytomegalovirus immediate-early enhancer and chicken \u03b2-actin promoter/enhancer (CAGG). Here we report that the ablation of COUP-TFII at the pre-pubertal stage resulted in infertility, hypogonadism and arrest of spermatogenesis due to a defective testosterone synthesis in the null mice. We further demonstrated that COUP-TFII itself is indispensable for Leydig cell maturation, specifically from progenitor Leydig cells maturation into adult Leydig cells. Interestingly, we identified that COUP-TFII played roles in progenitor Leydig cell formation and early testis organogenesis, as demonstrated by the ablation of COUP-TFII at E18.5. In contrast, when COUP-TFII was deleted after Leydig cell differentiation was complete, no discernible phenotype was detected, suggesting that COUP-TFII is not important for the maintenance of Leydig cell function and male reproduction in the adult.To circumvent the early embryonic lethality and investigate the postnatal function of TM (+/\u2212) COUP-TFIIflox/floxCre-ER mice were generated by crossing TMCAGG Cre-ER mice flox/floxCOUP-TFII mice. To induce the deletion of COUP-TFII gene, one group of P14 TM (+/\u2212) COUP-TFIIflox/floxCre-ER animals were injected intraperitoneally with tamoxifen, while another group received only the corn oil carrier to serve as controls. A third group of flox/floxCOUP-TFII animals received IP tamoxifen to ascertain the effect of tamoxifen independent of the activation of the Cre recombinase , suggesting possible defects in reproduction. To test this hypothesis, tamoxifen-treated TM (+/\u2212) COUP-TFIIflox/floxCre-ER male mice and control male mice, including flox/floxCOUP-TFII and TM (+/\u2212) COUP-TFIIflox/+Cre-ER mice treated with tamoxifen, and TM (+/\u2212) COUP-TFIIflox/floxCre-ER treated with oil, were mated with wild-type females and the breeding capacity of each groups were monitored for 3 months. As shown in ombinase . After CCOUP-TFII is efficiently ablated. However, in the absence of tamoxifen, \u201cleakiness\u201d of Cre-ER was detected in several organs such as the adrenal glands and epididymis. In contrast, the leakage was not detected in the testes, ovary, seminal vesicles or the anterior prostate. These observations were further confirmed by immunohistochemistry and qRT-PCR analysis of COUP-TFII protein and COUP-TFII mRNA, respectively (data not shown).To verify the efficiency of Cre-mediated recombination and see whether there is any sporadic leakage independent of tamoxifen, major organs were isolated after the injection of tamoxifen or oil. As shown in COUP-TFII null mice displayed a hypogonadal phenotype (flox/floxCOUP-TFII mice and oil-treated TM (+/\u2212) COUP-TFIIflox/floxCre-ER mice revealed that tamoxifen alone slightly reduced the weight of seminal vesicles and anterior prostate (TM (+/\u2212) COUP-TFIIflox/floxCre-ER animal and hsp70-2 gene are transcribed before the first meiotic division, and are expressed during the premeiosis phase transition protein 1 (TP1), transition protein 2 (TP2), protamine 1 (Prm 1), and protamine 2 (Prm 2), are expressed only during the postmeiotic phase (spermatids) COUP-TFII null mice.As a further step toward understanding the molecular basis of the spermatogenesis defects illustrated in TM (+/\u2212) COUP-TFIIflox/floxCre-ER animals was confirmed by qRT-PCR (COUP-TFII was expressed in Leydig cells (arrowhead) and pertubular myoid cells (arrow) in adult . Further qRT-PCR . Given t qRT-PCR . These rCOUP-TFII null mice display Leydig cell hypoplasia, which would account for the testosterone production defect in the mutant mice.To ascertain the cause of testosterone deficiency in the mutants, we examined the histology of Leydig cells. As shown in flox/floxCOUP-TFII and TM (+/\u2212) COUP-TFIIflox/floxCre-ER were supplemented with testosterone (as an implant), at the same time as tamoxifen injection at P14. As shown in Since the pivotal role of androgens in spermatogenesis and male fertility is well established, we asked whether testosterone deficiency was the major reason for the defects in the mutant mice. Furthermore, we observed that testosterone treatment stimulated the growth of the seminiferous tubules, which contained all stages of spermatogenic cells, including mature sperm in the enlarged lumen ; arrow. flox/floxCOUP-TFII and TM (+/\u2212) COUP-TFIIflox/floxCre-ER received tamoxifen treatment to induce the deletion of COUP-TFII gene at two months of age, when Leydig cells were fully developed. After two months of COUP-TFII deletion, the histology of testis and epididymis revealed that spermatogenesis was normal in the mutants . In the rescued animals, the serum testosterone level was already increased two- to three -fold compared with the control mice. Notably, COUP-TFII is highly expressed in the stromal cells of other reproductive organs such as the epididymis, seminal vesicles and prostate at an early stage. It is possible that COUP-TFII deletion affected one or all three above organs contributing to infertility. For example, we still observed disorganized epithelial cells in the epididymides of rescued animals. Epididymal dysfunction may have affected sperm motility and male fertility. It is interesting that COUP-TFII not only impacts testosterone biosynthesis, but may also play important roles in other reproductive functions, which ultimately affects male fertility. Given that testosterone alone cannot rescue the infertility of mutant mice, we believe that COUP-TFII might be a potential drug target to treat male infertility.Cre-ER line has toxicity in hematopoiesis and the increase of apoptosis in many tissues during embryonic development, and the toxicity also depends on the injection dose of tamoxifen TM (+/\u2212) COUP-TFIIflox/+Cre-ER animal, excluding the possibility that the phenotype we observed in COUP-TFII null mice was due to Cre-ER toxicity. Not only will this study provide a new insight into the process of Leydig cell differentiation, but it also will further our understanding of the dysregulation of steroid hormone synthesis and male reproduction.In a recent study, it was reported that tamoxifen inducible flox/floCOUP-TFIIx mice and TM miceCAGG-Cre-ER (Jackson Lab) has been previously described COUP-TFII deletion after birth, mice were intraperitoneally injected with tamoxifen at 1 mg per 50 g of body weight for 5 consecutive days starting at P14 or P60. For the deletion in embryonic stages, embryos received tamoxifen through blood circulation from the mothers. Pregnant mothers (E18.5) were intraperitoneally injected with tamoxifen at 2 mg per animal.Generation of flox/floxCOUP-TF (Tamoxifen), TM (+/\u2212) COUP-TFIIflox/floxCre-ER (Oil), TM (+/\u2212) COUP-TFIIflox/+Cre-ER (Tamoxifen) and TM (+/\u2212) COUP-TFIIflox/floxCre-ER (Tamoxifen) mice by mating one male with two females for 3 months. Female mice were checked for vaginal plugs each morning, and litter sizes were recorded on delivery during two successive matings.We investigated reproductive capacities of 3, 1.2 mM KH2PO4, 1.2 mM MgSO4 and 1.3 mM CaCl2). The tissues were incubated at 37\u00b0C for 5 min to allow sperm to disperse, as described previously The caudal epididymis were removed and minced in 0.1 ml of motile buffer /PBS, dehydrated through graded ethanol, and processed for paraffin embedding. Primary antibodies used in this study are as follows: mouse monoclonal anti-COUP-TFII (Perseus Proteomics), Goat polyclonal anti\u20133\u03b2-HSD, Goat polyclonal anti-CYP19, rabbit polyclonal anti-GATA-1 (Santa Cruz Biotechnology), rabbit polyclonal anti-P450Scc (Chemicon), rabbit polyclonal anti-EST (Biovision). Biotinylated antibodies were used as secondary antibodies, followed by horseradish peroxidase\u2013conjugated streptavidin , and signals were developed with 3, 3\u2032-diaminobenzidine (DAB) substrate kit (Vector Laboratories) or tyramide signal amplification (TSA) kit (Molecular Probes). Hematoxylin was used for counterstaining in immunohistochemistry.Isolated tissues were homogenized and lysed with 1\u00d7RIPA buffer . Protein concentrations were determined by the BCA protein assay system . 20 \u00b5g of protein was loaded and separated on 4\u201315% polyacrylamide gels, then electroblotted onto nitrocellulose membranes. The membranes were blocked and then incubated overnight with primary antibody at 4\u00b0C, followed by incubation with HRP-conjugated second antibody (DAKO). Signals were visualized with ECL plus Western Blotting Detection System .P450Scc (Mm00490735_m1); StAR (Mm00441558_m1); 3\u03b2-HSD (Mm00476184_g1); COUP-TFII (Mm00772789_m1), TP1 (Mm00437165_g1); TP2 (Mm00726979_s1); Hsp70-2 (Mm00434069_s1); Prm1 (Mm01342731_g1); Prm2 (Mm03048199_m1); RLF (Mm01340353_m1); EST (Rn00820646_g1); Acro (Mm00496484_g1) and Eukaryotic 18S rRNA (4319413E).Total RNA was extracted by Trizol methods according to the manufacture protocol (Invitrogen) and reverse transcribed using TaqMan Reverse Transcription Reagents . Gene expression assay was performed using the ABI PRISM 7700 Sequence Detector System (Applied Biosystems). TaqMan Universal Master Mix reagents and inventoried primer/probe mixture (Applied Biosytems) were used for the reaction. Standard curves were generated by serial dilution of a preparation of total RNA, and all mRNA quantities were normalized against 18S RNA using ABI rRNA control reagents. The primers/probes used in this study as follows: Sixty-day time-release pellets containing 7.5 mg testosterone were subcutaneously implanted into null animals at P14 together with tamoxifen injection. The pellets represent a matrix-driven delivery system that effectively and continuously releases testosterone. Null mice for controls were implanted with control pellets. After 45 days of testosterone treatment, the animals were approximately 8 weeks older. Mice were sacrificed to recover the reproduction organs and blood.Total testosterone level was measured with ELISA kits . The serum LH and FSH levels were measured with radioimmunoassay by the core laboratory of University of Virginia Center for Research in Reproduction Ligand Assay and Analysis Core."} +{"text": "Both MLI-DCIS and MLI-INV were related to oestrogen receptor (ER) , grade of invasive tumour and classification of intraductal components . In theunivariate disease-free survival analysis, both MLI-DCIS and MLI-INV were found to be significant . However, in node-negative cases, only MLI-DCIS was significant (P = 0.0416). Multivariate analysis revealed that MLI-DCIS was significant not only in all cases, but also in node-negative cases , whereas MLI-INV was not. These findings indicate that MIB1-determined proliferative activity of intraductal components is a significant prognostic determinant of invasive ductal breast carcinoma in which intraductal components predominate. \u00a9 1999 Cancer Research CampaignThe prognostic significance of the proliferative activities in intraductal components and invasive foci was investigated using 157 cases of invasive ductal breast carcinoma in which intraductal components predominated. Proliferative activity was expressed as the number of MIB1-positive nuclei per 1000 cancer cells in the most active areas of intraductal components (MLI-DCIS) or invasive foci (MLI-INV). MLI-DCIS correlated closely with MLI-INV ("} +{"text": "HfGeTe4 is isostructural with stoichiometric ZrGeTe4 and the Hf site in this compound is also fully occupied. The crystal structure of HfGeTe4 adopts a two-dimensional layered structure, each layer being composed of two unique one-dimensional chains of face-sharing Hf-centered bicapped trigonal prisms and corner-sharing Ge-centered tetra\u00adhedra. These layers stack on top of each other to complete the three-dimensional structure with undulating van der Waals gaps.The title hafnium germanium telluride, HfGeTe DOI: 10.1107/S1600536808011380/gw2040Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Transgenic mice expressing the c-myc proto-oncogene under the control of the CD2-dominant control region show stochastic development of mainly clonal thymic lymphoma with long latency, indicating that cooperative events are needed for the development of the fully malignant phenotype. Previous studies have suggested that T-cell receptor-associated signals can contribute to tumour development. We have therefore used this transgenic model of T-cell transformation to determine whether antigen-specific responses could constitute an epigenetic event in lymphomagenesis. The T-cell receptor (TcR) repertoires of lymphoma clones were analysed with a panel of monoclonal antibodies (Abs) recognizing TcR Vbeta chains. The Vbeta repertoire of tumour clones arising in these mice was non-random with overrepresentation of Vbeta8.2 TcR species. The majority of Vbeta8.2+ clones were of a mature CD3+ CD8 single-positive (SP) phenotype. The biased TcR usage, together with a mature cell phenotype is consistent with the hypothesis that TcR-mediated signals cooperate with activated myc during T-cell transformation."} +{"text": "CEA immune complexes and free CEA were determined to 363 patients with histologically confirmed adenocarcinoma of the gastrointestinal tract before surgery and in a post-operative follow-up. Circulating CEA immune complexes (CEA-IC) could be detected preoperatively in 89 patients. Incidence of CEA-IC increased with increasing tumour extension; 72/89 patients with CEA-IC showed already metastatic disease progression, 40/89 had nonresectable tumours. Patients with preoperative CEA-IC had a poorer prognosis than patients without CEA-IC but with high levels of free CEA, or CEA-negative patients. The appearance of CEA-IC with consecutive increases in the postoperative follow-up indicated disease recurrence. In 32/55 relapse cases, circulating CEA-IC were detected postoperatively, all 32 cases developing metastatic spread of disease."} +{"text": "To compare the effectiveness of cromolyn sodium (CS)(10 mg) and nedocromil sodium (NS) (4 mg) administeredby a metered dose inhaler (MDI) with a spacerdevice in preventing exercise-induced asthma (EIA), eightasthmatic children with EIA were studied in arandomized double-blind, cross-over, placebo-controlledstudy, CS and NS provided significant, comparable protectionfrom EIA and both were better than placebo. Weconclude that CS and NS administered by a pressurizedaerosol with a spacer device provide equal protectionagainst EIA in children."} +{"text": "The immunogenicity of a soluble fraction containing Gross-virus-associated cell-surface antigen (GCSAa) obtained from (C58NT)D lymphoma cells either by detergent (NP40) solubilization or by 3M KCl extraction, was studied in syngeneic W/Fu rats. Rats immunized by 2 s.c. injections of soluble antigen or soluble antigen mixed with empty liposomes and emulsified in complete Freund's adjuvant (CFA) failed to produce significant levels of cytotoxic antibodies to GCSAa. On the other hand, rats similarly immunized by negatively charged liposomes containing NP40-solubilized GCSAa, and emulsified in CFA, developed high and persistent levels of cytotoxic antibodies, and their response could even mimic that induced by viable (C58NT)D cells. A similar response could also be obtained in rats immunized with liposome-associated NP40-solubilized GCSAa, but without CFA. Rats immunized by comparable amounts of liposome-assocated 3M KCl-extracted GCSAa developed only low levels of cytotoxic antibodies, and their response was of shorter duration. These results strongly suggest that inclusion into liposomes of a solubilized proteic tumour-associated cell-surface antigen can provide an immunogen as potent as viable tumour cells in inducing an antibody response, and that the solubilization method may be critical."} +{"text": "Transforming growth factor-alpha -mediated autocrine regulation in human non-small-cell lung cancer (NSCLC) cells NCI-H226 and its brain metastatic variant H226Br were compared. An enhanced TGF-alpha-induced dose-dependent mitogenic responsiveness in H226Br cells was observed. Neutralising antibody that binds TGF-alpha inhibits H226Br cell growth more effectively than NCI-H226 cell growth. Binding assay with 125I-labelled epidermal growth factor (EGF) revealed that H226Br has two types of EGF receptors (EGFRs), whereas the parental cell line, NCI-H226, has only one. H226Br cells contain twice as many EGFRs as H226 cells, as proved by Scatchard analysis and immune kinase assay. Northern analysis indicated that there is more EGFR transcript in H226Br than in NCI-H226, indicating a transcriptional EGFR gene elevation during metastasis progression. The level of accumulated immunoactive TGF-alpha is lower in the conditioned medium of H226Br than in that of NCI-H226. demonstrating down-regulation of TGF-alpha transcript. The accumulated data suggest an elevated and sensitive autocrine modulation by TGF-alpha and EGFR in immortalising the brain metastatic variant cells that were derived from a human NSCLC squamous cell line."} +{"text": "Experiments were undertaken with DMBA-induced mammary tumours of the rat to determine the anti-tumour properties of a new and potent luteinizing hormone releasing hormone (LH-RH) agonist, [D-Ser(But) 6Azgly10]-LH-RH (ICI 118630). Tumours were classified according to their oestrogen-receptor (ER) content. Twice daily i.m. injections of either 5 micrograms or 0.5 micrograms ICI 118630 in saline were as effective as ovariectomy or tamoxifen therapy in causing the regression of ER+ DMBA-induced mammary tumours. ER- mammary tumours showed a more equivocal overall response to ICI 118630, some tumours progressing, others regressing. About one-third of the ER+ tumours disappeared in the 20-day treatment period. Those tumours which did regrow after the cessation of treatment proved to be hormone-dependent. In addition to the inhibitory effects of the LH-RH agonist on pre-existing tumours, ICI 118630 also reduced the total number of new tumours formed during and after treatment."} +{"text": "A cytotoxicity assay was used to study the action of bacillus Calmette-Guerin (BCG) and cytokines on four human bladder cancer cell lines. Monocytes and lymphocytes from peripheral blood were incubated with or without BCG or cytokines for 24 h, after which [3H]thymidine-labelled target cells were added and the 72 h percentage specific release determined. BCG had a direct cytotoxic effect against tumour cells and significantly enhanced monocyte/macrophage and enhanced lymphocyte cytotoxicity against one cell line (UCRU-BL-17). Supernatants (SNs) from BCG-activated monocytes/macrophages and lymphocytes increased the percentage specific release of [3H]thymidine from UCRU-BL-17 cells. Interferon alpha and interleukin 2 (IL-2) were cytotoxic towards UCRU-BL-17. No synergy occurred between BCG and cytokines at the concentrations tested. The results suggest that BCG is superior to IFN-alpha, interferon gamma (IFN-gamma) and IL-2 in enhancing cell-mediated cytotoxicity."} +{"text": "Multiple sclerosis (MS) is an autoimmune disease where T-cells activated against myelin antigens are involved in myelin destruction. Yet, healthy subjects also harbor T-cells responsive to myelin antigens, suggesting that MS patient-derived autoimmune T-cells might bear functional differences from T-cells derived from healthy individuals. We addressed this issue by analyzing gene expression patterns of myelin oligodendrocytic glycoprotein (MOG) responsive T-cell lines generated from MS patients and healthy subjects. We identified 150 transcripts that were differentially expressed between MS patients and healthy controls. The most informative 43 genes exhibited >1.5-fold change in expression level. Eighteen genes were up-regulated including BCL2, lifeguard, IGFBP3 and VEGF. Twenty five genes were down-regulated, including apoptotic activators like TNF and heat shock protein genes. This gene expression pattern was unique to MOG specific T-cell lines and was not expressed in T-cell lines reactive to tetanus toxin (TTX). Our results indicate that activation in MS that promotes T-cell survival and expansion, has its own state and that the unique gene expression pattern that characterize autoreactive T-cells in MS represent a constellation of factors in which the chronicity, timing and accumulation of damage make the difference between health and disease."} +{"text": "Staphylococcus aureus (MRSA) strains different from those of an endemic healthcare-associated clone was conducted over 13 years in Geneva, Switzerland. We demonstrated strain diversity, including clones rarely found in Europe. Local epidemiology of community-associated MRSA is diverse and is evolving by importation and transmission of new strains.Molecular characterization of methicillin-resistant Staphylococcus aureus (CA-MRSA) is responsible for severe infections related to carriage of exotoxins such as the Panton-Valentine leukocidin (PVL), toxic shock syndrome toxin 1 (TSST-1), or exfoliatin A (Community-associated methicillin-resistant We selected 2 collections of strains with 151 nonduplicated MRSA isolates identified in patients or carriers treated at our institution. The first collection was from a retrospective review of laboratory records and included non\u2013multidrug-resistant (gentamicin- and ciprofloxacin-susceptible) strains collected during 1993\u20132002 that had a phenotype different from the endemic healthcare-associated MRSA (HA-MRSA) strain in Geneva. The prevalent HA-MRSA clone in Geneva is sequence type (ST) 228-MRSA-I (CC5), which shows resistance to gentamicin, ciprofloxacin, clindamycin, and erythromycin. HA-MRSA strain ST8-MRSA-IV has been sporadically introduced from France. This strain has the same phenotype as ST228-MRSA-I (CC5) except for its susceptibility to gentamicin (The second collection was isolates selected from patients prospectively identified as colonized or infected with CA-MRSA by the CA-MRSA surveillance program during 2003\u20132005 (mec (SCCmec) elements, accessory gene regulator group, and the PVL gene . Multiple-locus variable-number tandem repeat analysis, which consisted of a multiplex PCR with 10 primer pairs, and multilocus sequence typing were performed as reported were obtained from the retrospective specimen collection. Fifty-nine isolates were obtained from clinical specimens and 33 from screening swabs. Among these isolates, 46 were obtained from skin and soft tissue samples and 13 from other body sites. A total of 59 isolates were obtained from the prospective CA-MRSA surveillance system from 59 patients .Strains were rarely resistant to clindamycin (5%), gentamicin (8% only in isolates recovered after 2002), or rifampicin (<1%). Susceptibility to cotrimoxazole was 89% during the first period and 100% during the second period. PVL-positive isolates remained multidrug susceptible throughout the study period, and were distinct from our endemic nosocomial strain. A, shows the incidence of isolates fulfilling our entry criteria and the proportion of strains producing PVL or harboring SCCmec IV or V. An increase in non\u2013multidrug-resistant MRSA was observed during 1994\u20131997 , and a second peak was observed during 2002\u20132005 . Molecular characterization of the 151 strains showed that 124 (82%) harbored either SCCmec IV or V. A total of 92 isolates (61%) harbored at least 1 toxin gene, most frequently PVL (n = 60), followed by TSST-1 (n = 22) and exfoliatin A (n = 11). An isolate (ST149-MRSA-IV) from a Libyan patient harbored the PVL and TSST-1 genes . After 2002, these strains were less frequent and the proportion of other clonotypes increased.From 1994 through 1999, we identified 14 PVL-positive MRSA isolates. The The Several epidemiologically linked cases were identified in the second period ( B) showed a wide diversity of patterns. Most isolates (n = 33) were obtained from the first stain collection and showed many different genetic backgrounds (n = 21).Strains lacking toxins , panel Bmec IV, PVL positive); the PVL gene is disseminated in many genetic backgrounds; strains showed diversity of genomic content; several epidemiologic clusters were identified; and many cases were linked to migration and travel.We studied 2 collections of non-multiresistant MRSA strains identified over a 13-year period at our institution. Our analysis showed that sporadic PVL-positive CA-MRSA has been isolated in Geneva since 1994; the largest cluster corresponded to ST 80 . Second, retrospective case ascertainment does not distinguish invasive from colonizing strains in all patients. Finally, we cannot exclude detection bias caused by our active MRSA screening policy with a strain highly related to USA400; H isolate showing molecular content of the ST59 Pacific clone. Scale bar (lower left) shows relative distance between strains. B). Trees obtained by using MLVA-based genotyping for strains devoid of clinically important toxins. Year of isolation, SCCmec type, toxin content, MLST, and agr types are also indicated for each strain (year/SCCmec/ST/agr). A familial cluster of ST1-MRSA-V composed of a mother and her 2 children; B control strain MW2 (USA400); C control strain ST228-MRSA-I representing the common nosocomial strain in our area. Scale bar (lower left) shows relative distance between strains.Clustering trees. A) Trees obtained by using multiple-locus variable-number tandem repeat analysis (MLVA)\u2013based genotyping for strains of Staphylococcus aureus harboring clinically important toxins, Geneva, Switzerland, 1993\u20132005. Year of isolation, staphylococcal cassette chromosome mec (SCCmec) type, toxin content, multiple locus sequence type, and accessory gene regulator (agr) types are also indicated for each strain (year/SCCmec/ST/agr). Major clusters appear in gray. ND, not determined; NT, nontypeable. *Clonal strains isolated from the familial cluster of ST80-MRSA-IV harboring the Panton-Valentine leukocidin (PVL) gene. **First ST80-MRSA-IV harboring the PVL gene isolated in 1994. ***Aypical ST149 strain clustering with other ST149 isolates, showing 2 toxins."} +{"text": "SiHa cells exposed to paclitaxel underwent apoptosis, which was strongly inhibited by EGF. This inhibition of apoptosis by EGF was not altered by pharmacological blockade of phosphatidylinositol 3\u2032-OH kinase (PI-3K) with the PI-3K specific inhibitor LY294002 or blockade of the mitogen-activated protein kinase (MAPK) kinase (MEK) with the MEK specific inhibitor PD98059, or by transfection of the cells with PI-3K or MEK dominant-negative expression vectors. EGF did not stimulate PI-3K/Akt, MEK/MAPK, or p38 MAPK activity in SiHa cells but did transiently activate the c-Jun NH2-terminal kinase (JNK). Co-exposure of SiHa cells to SB202190 at concentrations that inhibit JNK abolished the protective effect of EGF on SiHa cells against paclitaxel-induced apoptosis. Our findings indicate that the JNK signaling pathway plays an important role in EGF-mediated protection from paclitaxel-induced apoptosis in SiHa cells. \u00a9 2001 Cancer Research Campaign"} +{"text": "The transfer of tumour-specific cytotoxicity against a murine fibrosar-coma has been demonstrated in vitro using xenogeneic RNA extracted from tumour-cell-immune animals. Poly(A)-tailed messenger RNA from immunogenic RNA was isolated by passage through an oligo(dT)-cellulose column, and evaluated to determine whether the same tumour-specific cytotoxicity could be transferred. Aliquots of normal C3H mouse lymphocytes were treated with poly(A)-containing immune RNA, whole-cell immune RNA lacking poly(A) and total cellular immune RNA. Treated cells were tested in vitro using an adaptation of the Takasugi and Klein microcytotoxicity assay. Percent cytotoxicity was calculted using cells treated with fractions of normal RNA as control. An increase in tumour cytotoxicity was found with poly(A)-containing immune RNA. The optimum dose of poly(A)-tailed immune RNA was estimated as 6.5 microgram of RNA per 4 x 10(6) lymphocytes. Populations of lymphocytes were separated using glass and nylon wool. T- and B-enriched populations were treated with various RNA components. The adherent cell population showed no significant cytotoxicity, whilst treatment of the nonadherent population with poly(A)-tailed immune RNA produced high levels of cytotoxicity."} +{"text": "Confocal microscopy was used to visualize the intracellular uptake of the fluorescent methotrexate analogue, fluorescein-MTX (F-MTX), in human leukaemic cell lines and leukaemic blasts. Cytosolic labelling of wild-type K562 human erythroleukaemia cells was detected during 3-60 min incubations with F-MTX (1 microM) and was completely inhibited by co-exposure to either methotrexate or the thymidylate synthase inhibitor, ZD1694. There was no significant intracellular F-MTX accumulation over this period in a K562 subline (K500E) with a documented defect (approximately 10% of wild type) in membrane transport by the reduced folate carrier (RFC). F-MTX uptake was re-established in K500E cells transfected with a cDNA to human RFC, establishing a role for RFC in the cellular uptake of this compound. High levels of intracellular labelling were detected in all cell lines after prolonged (24 h) F-MTX incubations, however F-MTX accumulation at this time was not inhibited by ZD1694. F-MTX uptake by RFC was also detected in leukaemic blasts from children with acute lymphoblastic leukaemia and could be blocked with ZD1694. In leukaemic blasts with a documented defect in MTX uptake, F-MTX accumulation was abolished in almost all the cells. These results display the power of confocal microscopy for directly visualizing RFC-mediated anti-folate uptake. Over short intervals, F-MTX uptake is mediated by RFC, however, RFC-independent processes predominate during long drug exposures. Direct assay by confocal microscopy may be better suited than other indirect methods (i.e. flow cytometry) for detecting low levels of RFC transport in leukaemic blasts from patients undergoing chemotherapy with methotrexate."} +{"text": "Epidermal growth factor (EGF) receptor-overexpression is characteristic of many human tumours of epithelial origin and has been correlated with unfavourable patient prognosis. Its involvement in the malignant process, its elevated expression in tumours and its accessibility on the tumour cell surface make the EGF receptor a potential target for directed tumour therapy. We have previously characterized a recombinant antibody - Pseudomonas exotoxin A fusion protein, scFv(225)-ETA, which displayes antitumoral activity towards EGF receptor-overexpressing tumour cells but is less potent in tumour cell killing than TGF-alpha-ETA, a recombinant toxin using the natural EGF receptor ligand transforming growth factor alpha as a targeting domain. Here, we describe the construction and functional characterization in vitro of a novel single-chain antibody-toxin, scFv(14E1)-ETA, based on the independently isolated EGF receptor-specific monoclonal antibody 14E1. ScFv(14E1)-ETA binds to an EGF receptor epitope that is very similar or identical to that of scFv(225)-ETA with nine times higher affinity than the latter and displays more than tenfold higher cytotoxic activity on EGF receptor-overexpressing tumour cells. ScFv(14E1)-ETA cell killing activity was very similar to that of TGF-alpha-ETA on receptor-overexpressing cells but, in contrast to the latter, scFv(14E1)-ETA was much more selective and did not display significant cytotoxic activity on cells expressing moderate EGF receptor levels."} +{"text": "Since Zileuton, also an inhibitor of 5-LOX, attenuates asthma but with an undesirable sideeffect, we investigated whether dietary GLA would suppress biosynthesis of PMN-LTB4 isolated fromasthma patients and attenuate asthma. Twenty-four mild-moderate asthma patients (16\u201375 years) wererandomized to receive either 2.0 g daily GLA (borage oil) or corn oil (placebo) for 12 months. Blooddrawn at 3 months intervals was used to prepare sera for fatty acid analysis, PMNs for determiningphospholipid fatty acids and for LTB4 generation. Patients were monitored by daily asthma scores,pulmonary function, and exhaled NO. Ingestion of daily GLA (i) increased DGLA (GLA metabolite) inPMN-phospholipids; (ii) increased generation of PMN-15-HETrE (5-LOX metabolite of DGLA).Increased PMN-DGLA/15-HETrE paralleled the decreased PMN generation of proinflammatory LTB4.However, the suppression of PMN-LTB4 did not reveal statistically significant suppression of theasthma scores evaluated. Nonetheless, the study demonstrated dietary fatty acid modulation ofendogenous inflammatory mediators without side effects and thus warrant further explorations into theroles of GLA at higher doses, leukotrienes and asthma.Dietary gammalinolenic acid (GLA), a potent inhibitor of 5-lipoxygenase (5-LOX) and suppressor ofleukotriene B"} +{"text": "Culturing heterologous tumour-specific cytotoxic T-cells with purified pancreatic tumour cell-mucin rendered them unresponsive to their target cells. Furthermore, purified mucin did not produce a mucin-specific response in mucinous pancreatic tumour patients' primary T-cells even in the presence of antigen-presenting cells. Our study finds no evidence for MHC-unrestricted recognition of mucin by pancreatic cancer patients' T-cells. \u00a9 2000 Cancer Research CampaignCytotoxic T-cells generated against heterologous, mucinous pancreatic tumour cells were shown to recognize mucin in a major histocombatibility complex (MHC)-unrestricted fashion. In contrast, the present study demonstrates a typical allogeneic response of heterologous cytotoxic T-cells established against mucin-expressing pancreatic tumour cells. Heterologous cytotoxic T cells lysed targets that were used as stimulators and other targets that shared human leucocyte antigen (HLA) with the stimulator. These cytotoxic T-cells lysed mucin-expressing stimulator cells but not autologous tumour cells in spite of expressing mucin on their surface. Likewise, tumour-infiltrating CD4"} +{"text": "In order to elucidate the EBV gene expression patterns in vivo, we examined eight patients with cutaneous EBV-related NK/T-cell lymphomas, including six patients with a NK-cell phenotype and two patients with a T-cell phenotype. The implication of EBV in the skin lesions was determined by the presence of EBV-DNA, EBV-encoded nuclear RNA (EBER) and a clonality of EBV-DNA fragments containing the terminal repeats. Transcripts of EBV-encoded genes were screened by reverse transcription- polymerase chain reaction (RT-PCR), and confirmed by Southern blot hybridization. The expression of EBV-related antigens was examined by immunostaining using paraffin-embedded tissue sections and cell pellets of EBV-positive cell lines. Our study demonstrated that all samples from the patients contained EBV nuclear antigen (EBNA)-1 mRNA which was transcribed using the Q\u2008promoter, whereas both the Q promoter and another upstream promoter (Cp/Wp) were used in EBV-positive cell lines, B95.8, Raji and Jiyoye. Latent membrane protein-1 (LMP-1) mRNA was detected in seven of eight patients and all cell lines, whereas EBNA-2 transcripts were found only in the cell lines. Immunostaining showed no LMP-1, EBNA-2 or ZEBRA antigens in the paraffin-embedded tissue sections, although they were positive in the cell line cells. Latent BHRF1 transcripts encoding bcl-2 homologue and BCRF1 transcripts encoding viral interleukin (vIL)-10 were detected in one and two of eight patients, respectively. A patient with NK-cell lymphoma expressing both transcripts died of rapid progression of the illness. Our results indicate that the restricted expression of the latency-associated EBV genes and the production of vIL-10 and bcl-2 homologue may favour tumour growth, evading the host immune surveillance. \u00a9 2001 Cancer Research Campaign"} +{"text": "ATP-dependent nucleosome-remodeling enzymes and covalent modifiers of chromatinset the functional state of chromatin. However, how these enzymatic activitiesare coordinated in the nucleus is largely unknown. We found that theevolutionary conserved nucleosome-remodeling ATPase ISWI and the poly-ADP-ribosepolymerase PARP genetically interact. We present evidence showing that ISWI istarget of poly-ADP-ribosylation. Poly-ADP-ribosylation counteracts ISWI functionin vitro and in vivo. Our work suggests that ISWI is a physiological target ofPARP and that poly-ADP-ribosylation can be a new, important post-translationalmodification regulating the activity of ATP-dependent nucleosome remodelers. The ISWI protein is a highly conserved nucleosome remodeler that plays essentialroles in regulating chromosome structure, DNA replication, and gene expression.The variety of functions associated with ISWI activity are probably connected tothe ability of other cellular factors to regulate its ATP-dependentnucleosome-remodeling activity. We identified one factor\u2014the poly-ADP-ribosepolymerase, PARP\u2014that can counteract ISWI function. PARP is an abundant nuclearprotein that catalyzes the transfer of ADP-ribose units to specific proteinsinvolved in DNA repair, transcription, and chromatin structure. Our worksuggests that the activity of an ATP-dependent remodeler can be modulated bypoly-ADP-ribosylation in order to regulate chromatin function in vivo. Enzymes that mediate nucleosome remodeling and poly-ADP-ribosylation playessential roles in the eukaryotic cell. A new study suggests a mechanism toexplain how two nuclear enzymes can coordinate their activities to regulatechromatin structure and function. Eukaryotic chromatin is packaged in a highly organized hierarchy of structuralbuilding blocks, all composed of the basic repeating unit of the nucleosome.ATP-dependent nucleosome-remodeling activities as well as covalent modifications ofchromatin components underlie the dynamic nature of chromatin structure and function,2. AlthoDrosophila, loss of ISWI function causes global transcriptiondefects and leads to dramatic alterations in higher-order chromatin structure,including the apparent decondensation of both mitotic and interphase chromosomesATP-3000 mmol-1 . Either 1 \u03bcgof activated DNA (Trevigen) or 100 ng of in vitro assembled chromatin are compared, by Immuno-dot, with serial dilutions of total PAR\u2014free andcovalently attached\u2014produced in standard ATPase/PARylation dual assayreactions in the presence of DNA [DNA] or UV-treated chromatin [CHR] . The amount of PAR produced by PARP, in the presenceof DNA or UV-treated chromatin was estimated by quantification of Westernblot with aPAR antibody.(B) ATP hydrolysis of recombinant ISWI incubated with activating DNA in thepresence of up to 100 pmol poly-ADP-ribose .(C) ATP hydrolysis of recombinant ISWI incubated with UV-treated chromatin inthe presence of up to 50 pmol of poly-ADP-ribose . Inconclusion, a 5-fold excess of free PAR over the amount produced in astandard ATPase/PARylation reaction does not inhibit ISWI DNA-dependent orchromatin-stimulated ATPase activity.(344 KB TIF)Click here for additional data file.Figure S5Schematic representation of the temporal order in which we added ISWI,polynucleosomes [Poly], mononucleosomes [Mono], PARP, activating DNA, and3-aminobenzamide [3-AB] in the mobility shift assays presented (A) in (141 KB TIF)Click here for additional data file.Figure S6hsp70 loci at 87A and 87C (red), was carried out onpolytene chromosomes prepared from C03256Parp third instar larvae maintained under (A) non\u2013heat-shock or (B)under heat-shock conditions. The data are presented as a merge chromosomeimage. Chromosomes were also stained with DAPI to visualize DNA (blue). Thecytological hsp70 loci 87A and 87C, are indicated byarrows. The C03256Parp mutant fails to respond to heat-shock stimuli since no puffing andno hyper-PARylation at 87A and 87C loci is observed after heat-shock. Theresidual PARP activity detected by the aPAR antibody is comparable to theone presented in Immuno-FISH using an aPAR antibody (green) and a DNA probe for thehsp70 loci 87A and 87C (red) are also compared, as a\u201csplit\u201d chromosome image, (C) before or (D) after heat shock. Arrowheadindicates ISWI binding at the 87A hsp70 locus. (-) nonheat-shock and (hs) heat-shock conditions. ISWI remains bound to the 87Alocus before and after heat-shock in the C03256Parp mutant, suggesting that ISWI binding at 87A locus is directlyregulated by the activity of PARP.Anti-ISWI immunostaining (green) and in situ hybridization for the(381 KB TIF)Click here for additional data file."} +{"text": "Non-alcoholic fatty liver disease (NAFLD), which is characterized by hepatic steatosis, can be reversed by early treatment. Several case reports have indicated that the administration of recombinant growth hormone (GH) could improve fatty liver in GH-deficient patients. Here, we investigated whether chronic exogenous GH levels could improve hepatic steatosis induced by a high-fat diet in rats, and explored the underlying mechanisms.High-fat diet-fed rats developed abdominal obesity, fatty liver and insulin resistance. Chronic exogenous GH improved fatty liver, by reversing dyslipidaemia, fat accumulation and insulin resistance. Exogenous GH also reduced serum tumour necrosis factor-alpha levels, and ameliorated hepatic lipid peroxidation and oxidative stress. Hepatic fat deposition was also reduced by exogenous GH levels, as was the expression of adipocyte-derived adipokines , which might improve lipid metabolism and hepatic steatosis. Exogenous GH seems to improve fatty liver by reducing fat weight, improving insulin sensitivity and correcting oxidative stress, which may be achieved through phosphorylation or dephosphorylation of a group of signal transducers and activators of hepatic signal transduction pathways.Chronic exogenous GH has positive effects on fatty liver and may be a potential clinical application in the prevention or reversal of fatty liver. However, chronic secretion of exogenous GH, even at a low level, may increase serum glucose and insulin levels in rats fed a standard diet, and thus increase the risk of insulin resistance. Non-alcoholic fatty liver disease (NAFLD) is a metabolic disorder characterized by fatty infiltration of the liver in the absence of alcohol consumption. It is the most common cause of chronic liver disease and represents a spectrum of liver diseases, which include simple fatty liver, steatohepatitis, and cirrhosis . NAFLD iHepatic steatosis is a hallmark of NAFLD and is caused by the accumulation of lipids, particularly triglycerides, in the liver. Hepatic steatosis, usually considered as an early stage of NAFLD, is generally benign, relatively non-aggressive and reversible. The symptoms are not obvious and the disease is often overlooked. However, because hepatic steatosis can progress to fibrosis (in 20-40% of patients), cirrhosis (in 30% of patients) or hepatocellular carcinoma , early pGrowth hormone (GH) has a pronounced lipolytic effect, particularly in abdominal fat . PreviouAdipocyte-derived adipokines such as adiponectin, leptin and resistin, are essential regulators of inflammation and the progression of fibrosis in various chronic liver diseases, and may be used in the treatment of NAFLD -16. SpecNM_000515) coding sequence (cds) was transferred in vivo by recombinant adeno-associated viral vectors pseudotyped with viral capsids from serotype 1 (rAAV2/1).Here, we investigated whether early GH administration has the preventive effects on hepatic steatosis (an early stage of NAFLD) in rats, and explored the underlying mechanisms. We also discuss limitations of GH administration for hepatic steatosis. Viral vectors can induce longer-lasting effects than administration of recombinant protein and avoid the inconvenience of repetitive subcutaneous injections. Therefore, we used GH gene delivery technology rather than injection of recombinant GH. The GH1 gene . Meanwhile, chronic exogenous GH improved the body composition and decreased visceral fat (VF) weight and VF percentage (VF%) in the GH group compared with the CH group . Hepatomegaly, which is common in NAFLD, is determined by liver wet weight (LWW) and the HI. LWW and HI were significantly increased in the CH group than in the CS group . Chronic exogenous GH levels prevented hepatomegaly, because LWW and HI decreased in the GH group compared with the CH group (p < 0.001), triglyceride (TG) (p < 0.001) and low-density lipoprotein cholesterol (LDL-C) (p < 0.001) and decreased the serum level of high-density lipoprotein cholesterol (HDL-C) in the CH group compared with the CS group. Chronic exogenous GH markedly reduced the serum TC (p < 0.01), TG (p < 0.001) and LDL-C levels (p < 0.01) and increased, albeit not significantly, the HDL-C levels (p > 0.05) in the GH group compared with the CH group, indicating that exogenous GH levels reversed the dyslipidaemia induced by the high-fat diet Table . SimilarPhotomicrographs of hepatic specimens stained with H&E are shown in Figure Serum TNF-alpha levels were remarkably elevated in the CH group compared with the CS group (p < 0.001). Although exogenous GH levels did not affect the serum TNF-alpha level in the GS group, it did partly prevent the high-fat diet-induced increase in serum TNF-alpha level in the GH group, as the serum TNF-alpha level was significantly lower in the GH group than in the CH (p < 0.001) Table . The hepBacterial translocation from mesenteric lymph nodes (MLNs) is considered one of the main events in the pathogenesis of spontaneous bacterial peritonitis and other infections in cirrhosis . TNF-alpThe full-length leptin receptor isoform, ob-rb, contains intracellular motifs required for the activation of the JAK/STAT signal transduction pathway, and is considered to be the functional receptor . The effThe high-fat diet induced marked changes in the expression of a group of signal transducers and activators in the CH group compared with the CS group, which were reversed by exogenous GH. Figure IR plays a crucial role in NAFLD , which ide novo lipogenesis and improve lipid profiles by decreasing TG and TC concentrations, and increasing HDL-C concentrations ) and total body weight (TBW) were measured by an electronic balance that was calibrated every day and an electronic digital calliper . Body mass index (BMI) was calculated using the formula BMI = TBW/(BL)g for 15 min at 4\u00b0C, and stored at -80\u00b0C until used for biochemical and hormone assays.Blood was collected via the aorta and allowed to clot at room temperature for 60 min to form serum. The serum samples were then centrifuged at 3000 \u00d7 The freshly dissected rat livers were immediately fixed in 4% paraformaldehyde, dehydrated, embedded in paraffin, and sectioned. Formalin-fixed, paraffin-embedded sections were cut (5 \u03bcm thick) and mounted on glass slides. The sections were deparaffinized in xylene, stained with haematoxylin and eosin (H&E) using standard techniques, and examined by an investigator blind to the treatment group. Biopsies were classified into four grades based on fat accumulation using the classification method devised by Brunt et al , where gSerum human GH , TNF-alpha , IGF-1 and insulin (ADL) was measured using enzyme-linked immunosorbent assay (ELISA) kits. All assay kits included quality controls. Each sample was assayed in duplicate. The serum levels of glucose, alanine aminotransferase (ALT), aspartate aminotransferase (AST), TG, TC, and HDL-C were measured using standard methods. LDL-C level was calculated using Friedwald's formula . IR was g for 10 min at 4\u00b0C. The supernatant was used for analysis. MDA was quantified using the thiobarbituric acid reaction as described by Ohkawa [To measure the level of hepatic MDA, 25 mg of liver tissue was added to 250 \u03bcl of radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitors. This mixture was sonicated for 15 s at 40 V over ice and centrifuged at 1600 \u00d7 y Ohkawa ,53, and Samples of MLNs and portal and peripheral blood were collected under sterile conditions before the rats were killed. The MLNs were homogenized in physiological (0.9%) saline, and 0.1 ml aliquots of the homogenate were cultured in MacConkey agar , Columbia sheep blood (Oxoid), and Esculin-Bile-Azide agar , and incubated at 37\u00b0C for 48 h. Bacterial translocation was defined as a positive culture of MLNs. Systemic infections were defined as a positive culture of any of the other biological samples, as previously described .\u00ae RNA clean-up kit . The mRNA analysis was carried out by quantitative real-time RT-PCR using LightCycler technology (Roche Diagnostics) for continuous fluorescence detection. The primers for rat leptin receptor isoform b (ob-rb) [Total RNA was extracted from the livers of each group of rats and isolated and purified with TRIzol reagent and NucleoSpin (ob-rb) ,56, adip (ob-rb) and resi (ob-rb) were use (ob-rb) ,55-57. A (ob-rb) was usedGoat polyclonal IgG antibodies against phospho-signal transducer and activator of Transcription 3 (p-STAT3) (Tyr705), total STAT3 (STAT3), phospho-signal transducer and activator of Transcription 5 (p-STAT5) (Tyr694/Tyr699), total STAT5 (STAT5), phospho-Janus kinase 2 (p-JAK2) (Tyr1007/1008) and total JAK2 (JAK2) were purchased from Santa Cruz Biotechnology . Rabbit polyclonal IgG antibodies against phospho-adenosine monophosphate-activated protein kinase-alpha (Thr172), total AMPK-alpha , phospho-extracellular signal-regulated kinase 1/2 (p-ERK1/2) (Thr202/Tyr204), total ERK1/2 (ERK1/2), phospho-c-Jun NH2-terminal kinase (p-JNK) (Thr183/Tyr185), total JNK (JNK), phospho-p38 MAPK (p-P38 MAPK) (Thr180/Tyr182) and total P38 MAPK (P38 MAPK) were obtained from Cell Signaling Technology . Rabbit polyclonal IgG antibodies against phospho-peroxisome proliferator-activated receptor -alpha (p-Ser21) and total PPAR-alpha were purchased from GenScript USA Incorporated .Soluble protein was extracted from rat livers using a protein extraction reagent and protein concentration was measured using a bicinchoninic acid assay kit . The extracted proteins were resolved by 6-8% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by electrophoretic transfer to nitrocellulose membranes. The membranes were then incubated in 1.0% non-fat dried milk in 50 mM Tris (pH 8.0) followed by incubation overnight with the primary antibodies at 300-500-fold dilution. The bound primary antibody was detected using biotinylated rabbit anti-goat or rabbit anti-rabbit antibody and visualized using 3', 3'-diaminobenzidine tetrahydrochloride. Detection was carried out using an electrochemiluminescence kit . Image-Pro Plus software version 6.0 was used to determine the mean optical density.Data are mean \u00b1 standard deviation. Statistical analyses were done using SPSS software version 13.0 . A bifactorial ANOVA (followed by post hoc protected least square difference method) were used for statistical comparison. The two fixed factors were treatment and diet . Weighted kappa values were calculated to determine inter-observer agreement in pathological evaluation. Values of p < 0.05 were considered significant.AMPK-alpha: monophosphate-activated protein kinase-alpha; ERK1/2: extracellular signal-regulated kinase 1/2; GH: growth hormone; GHD: GH deficiency; IGF-1: Insulin-like growth factor 1; IR: insulin resistance; JAK2: Janus kinase 2; JNK: c-Jun NH2-terminal kinase; MDA: malondialdehyde; NAFLD: non-alcoholic fatty liver disease; p38 MAPK: p38 mitogen-activated protein kinase; PPAR-alpha: peroxisome proliferator-activated receptor-alpha; rAAV: recombinant adeno-associated virus; rAAV2/1: recombinant adeno-associated viral vectors pseudotyped with viral capsids from serotype 1; STAT3: signal transducer and activator of transcription 3; STAT5: signal transducer and activator of transcription 5; TNF-alpha: tumour necrosis factor-alpha.The authors declare that they have no competing interests.Guarantor of integrity of entire study YQ and YPT; study concepts and design: YQ and YPT; data acquisition/analysis/interpretation: YQ and YPT, statistical analysis: YQ; obtained funding: YQ and YPT; manuscript drafting or revision for important intellectual content, literature research, manuscript editing, and manuscript final version approval: YQ, and YPT."} +{"text": "Insulin-like growth factor-1 (IGF-I) signalling is important for cancer initiation and progression. Given the emerging evidence for the role of the stroma in these processes, we aimed to characterize the effects of IGF-I on cancer cells and stromal cells separately.ex vivo culture model and measured gene expression changes after IGF-I stimulation with cDNA microarrays. In vitro data were correlated with in vivo findings by comparing the results with published expression datasets on human cancer biopsies.We used an P = 0.029 - Norway/Stanford and P = 7.96e-09 - NKI dataset). Furthermore, based on an IGF-I induced gene expression signature derived from primary lung fibroblasts, a separation of prognostically different lung cancers was possible (P = 0.007 - Bhattacharjee and P = 0.008 - Garber dataset).Upon stimulation with IGF-I, breast cancer cells and stromal fibroblasts show some common and other distinct response patterns. Among the up-regulated genes in the stromal fibroblasts we observed a significant enrichment in proliferation associated genes. The expression of the IGF-I induced genes was coherent and it provided a basis for the segregation of the patients into two groups. Patients with tumours with highly expressed IGF-I induced genes had a significantly lower survival rate than patients whose tumours showed lower levels of IGF-I induced gene expression (Expression patterns of genes induced by IGF-I in primary breast and lung fibroblasts accurately predict outcomes in breast and lung cancer patients. Furthermore, these IGF-I induced gene signatures derived from stromal fibroblasts might be promising predictors for the response to IGF-I targeted therapies.http://www.biomedcentral.com/1741-7015/8/2See the related commentary by Werner and Bruchim: In parallel, the overall survival rate was significantly lower for patients with up-regulation of the breast fibroblast derived IGF-I signature . The same coordinated behaviour and segregation of tumours could also be observed in a set of advanced breast cancers from Norway/Stanford signature) and down-regulated in primary breast fibroblasts upon IGF-I stimulation.Click here for fileFigure S4. Graphical visualization of the output from GO::Termfinder for biological process ontology. GOgraph layout that includes the significant GO nodes up-regulated in primary breast fibroblasts, derived from 186 clones compared to a background of 8918 clones. The colour of the nodes is an indication of their Bonferroni corrected P-value .Click here for fileFigure S6. Relationship of expression level of breast fibroblast derived insulin-like growth factor-1 (IGF-I) signature with distant metastasis free and overall survival applying continuous scoring. A. Continuous score based on average expression level of the signature in Netherlands Cancer Institute (NKI) patients. Colours correspond to score below (yellow) or above (blue) the median (red line). Overall (B) and metastasis free survival (C) analysis using a continuous score resulting from breast fibroblast derived IGF-I signature in early stage breast cancer patients from the NKI.Click here for fileTable S3. The detailed list of correlation values of breast fibroblast derived insulin-like growth factor-1 (IGF-I) signature to the previously published signatures and fibroblast derived IGF-I signature.Click here for fileFigure S7. Relationship of expression level of breast fibroblast derived insulin-like growth factor-1 (IGF-I) signature with overall survival and disease specific survival applying continuous scoring. A. Continuous score based on average expression level of the signature in Bhattacharjee dataset patients. Colours correspond to score below (yellow) or above (blue) the median (red line). Overall (B) and disease specific survival (C) analysis using a continuous score resulting from breast fibroblast derived IGF-I signature in Bhattacharjee dataset patients.Click here for fileFigure S5. Correlation of the fibroblast derived insulin-like growth factor-1 (IGF-I) signature and the breast fibroblast IGF-I induced signature centroids in the Netherlands Cancer Institute dataset. Pearson correlations for the signature and the P value are shown in the lower right part of the plot.Click here for file"} +{"text": "Recently we have reported membrane androgen receptors-induced apoptotic regression of prostate cancer cells regulated by Rho/ROCK/actin signaling. In the present study we explored the specificity of these receptors and we analyzed downstream effectors controlling survival and apoptosis in hormone refractory DU145-prostate cancer cells stimulated with membrane androgen receptor-selective agonists.Using membrane impermeable conjugates of serum albumin covalently linked to testosterone, we show here down-regulation of the activity of pro-survival gene products, namely PI-3K/Akt and NF-\u03baB, in DU145 cells. Testosterone-albumin conjugates further induced FasL expression. A FasL blocking peptide abrogated membrane androgen receptors-dependent apoptosis. In addition, testosterone-albumin conjugates increased caspase-3 and Bad protein activity. The actin cytoskeleton drug cytochalasin B and the ROCK inhibitor Y-27632 inhibited FasL induction and caspase-3 activation, indicating that the newly identified Rho/Rock/actin signaling may regulate the downstream pro-apoptotic effectors in DU145 cells. Finally, other steroids or steroid-albumin conjugates did not interfere with these receptors indicating testosterone specificity.Collectively, our results provide novel mechanistic insights pointing to specific pro-apoptotic molecules controlling membrane androgen receptors-induced apoptotic regression of prostate cancer cells and corroborate previously published observations on the potential use of membrane androgen receptor-agonists as novel anti-tumor agents in prostate cancer. Furt in vivo ,18. FinaIn the present study we have analyzed the specificity of mAR and the activity of downstream gene products playing a prominent role in survival and apoptosis in DU145 prostate cancer cells. Our results show that testosterone-BSA suppresses PI-3K activity, inhibits Akt function and finally inactivates the pro-survival transcription factor NF-\u03baB. We further report mAR-dependent suppression in the phosphorylation/inactivation of the pro-apoptotic Bad protein, stimulation of FasL expression and induction of caspase-3 activity. Taken together, our results provide new mechanistic insight into specific mAR-dependent apoptosis of prostate cancer cells.The DU145 human prostate cancer cell line was obtained from the American Type Culture Collection and was studied between passages 60 and 70. DU145 cells fail to respond to androgen treatment owing to the expression of non-functional iAR , or to c-5 M. The steroid-albumin conjugates-stock solutions were incubated for 30 min at room temperature with 0.3% charcoal and 0.03% dextran, centrifuged at 3000 \u00d7 g and passed through a 0.45 \u03bcm filter to remove any potential contamination with free steroid. Testosterone-BSA, estradiol-BSA, dexamethasone and DHT solutions were used at a final concentration of 10-7 M throughout all studies. If not otherwise stated all treatments and incubations with steroids including apoptosis assays were performed in serum-containing medium.Before each experiment testosterone-3-(O-carboxymethyl) oxime-BSA, referred to as testosterone-BSA, dihydrotestosterone (DHT), estradiol-BSA and dexamethasone (Sigma), were dissolved in serum-free culture medium at a final concentration of 10The Triton X-100 soluble G-actin containing and insoluble F-actin containing fractions of cells exposed to testosterone-BSA and DHT were prepared as previously described . An incr-7 M cytochalasin B , or 10 \u03bcM Y-27632 , and stimulated with 10-7 M testosterone-BSA for the time periods indicated in the figure legends.DU145 cells treated or not (control cells) with testosterone-BSA were washed twice with ice-cold phosphate-buffered saline and suspended in cold lysis buffer containing 1% Nonidet P-40, 20 mM Tris (pH 7.4) and 137 mM NaCl, supplemented with protease and phosphatase inhibitors. Cleared lysates were pre-adsorbed with protein A-Sepharose beads (Amersham) for 1 h at 4\u00b0C. Equal amounts of the supernatants were subjected to immunoprecipitation using an anti-phosphotyrosine (PY20) antibody (Santa Cruz Biotechnology) and protein A-Sepharose beads. For immunoblot analysis the immunoprecipitates and equal amounts of total protein extracts were suspended in Laemmli's sample buffer and separated by SDS-PAGE. For Fas ligand expression studies cells were pretreated or not with 10Proteins were transferred onto nitrocellulose membranes and blotted with rabbit polyclonal anti-PI-3K p85 (Upstate) (1:1000 dilution), rabbit polyclonal anti-phospho-Akt Ser473, anti-phospho-Akt Thr308 or anti-Akt (1:500 dilution), rabbit polyclonal anti-Fas-L (1:200 dilution), phospho- and total Bad antibodies . Secondary antibodies used were horseradish peroxidase-conjugated anti-mouse IgG (Chemicon), and horseradish peroxidase-conjugated anti-rabbit IgG . Then, the membranes were exposed to Kodak X-Omat AR films. A PC-based Image Analysis program was used to quantify the intensity of each band .-7 M cytochalasin B. After co-incubation the active form of NF-\u03baB contained in the nuclear extract binds to its consensus sequence. Thereafter, the extract/probe/buffer mixture was directly transferred to the streptavidin-coated plate. The biotinylated double stranded oligonucleotide bound by active NF-\u03baB protein was immobilized, and any inactive unbound material was washed away. The bound NF-\u03baB transcription factor subunits, p50/p65, were detected with specific primary antibodies. An HRP-conjugated secondary antibody was then used for detection and quantification in a spectrophotometric plate reader. By loading the same amount of total protein in each sample, we ensured the exact normalization for all cases.A non-radioactive NF-\u03baB p50/p65 Transcription Factor Assay was used to detect specific transcription factor DNA binding activity in nuclear extracts . A double stranded biotinylated oligonucleotide containing the consensus sequence for NF-\u03baB binding (5'-GGGACTTTCC-3'), was mixed with cellular (nuclear) extract pre-treated or not with 10APOPercentage apoptosis assay . In the presence or absence of 10-7 M flutamide (Sigma), cells were stimulated or not with 10-7 M of the following steroids in serum-supplemented medium: testosterone-BSA (Testo-BSA), dihydrotestosterone (DHT), estradiol-BSA (E2-BSA) and dexamethasone (DEXA) or 10-7 M BSA for 24 hours. Untreated cells cultured in serum free medium were used as positive control for the apoptotic response.DU145 cells were cultured in 96-well plates for the -7 M testosterone-BSA in serum-supplemented medium for the time periods indicated in the figure legends. Untreated cells cultured in serum-free medium were used as a positive control for the apoptotic response. At the end of the respective treatment cells were harvested in PBS and stained with the Annexin V-FITC Apoptosis Detection kit I according to the manufacturer's instructions. They were analyzed within 1 h by flow cytometry using a FACSArray Apparatus (BD Biosciences) and CellQuest (BD Biosciences) and ModFit LT software.DU145 cells were cultured in 60 mm plates for FACS analysis and determination of Fas expression levels. After pre-treatment with a monoclonal Ab to Fas, , cells were stimulated or not with 10-7 M cytochalasin B, or 10 \u03bcM Y-27632 and then stimulated with 10-7 M testosterone-BSA for the time periods indicated in the figure legends, using the Clontech ApoAlert\u00ae Caspase Colorimetric Assay kit according to the manufacturers' instructions. Caspase-3 activity was determined by incubating lysates with a caspase-3 substrate (the peptide DEVD conjugated to the chromophor p-nitroaniline) for 2 h at 37\u00b0C. The absorbance of each sample was measured at 405 nm by using a 96-well colorimetric plate reader.The activity of caspase-3 was measured in whole cell lysates pre-treated or not with either 10As previously reported, DU145 cells are iAR-negative and express functional mAR ,17,19. A+ LNCaP cells, testosterone-BSA-stimulated iAR- DU145 cells fail to rapidly activate FAK/PI-3K signaling [-7 M testosterone-BSA for various time periods and evaluated the tyrosine phosphorylation activity of the p85 regulatory subunit in IP-Western assays . Work fHaving established a clear role for testosterone-BSA in suppressing the activity of PI-3K/Akt and NF-\u03baB pro-survival pathways in DU145 cells, we sought to characterize the mechanism of apoptotic induction by testosterone-BSA. Since the CD95/FasL death pathway was recently shown to be involved in testosterone-BSA-induced apoptosis in LNCaP cells , we furtTo assess the participation of caspases as executors in mAR-dependent cell death, we measured caspase-3 activity in testosterone-BSA-treated DU145 cells. As presented in Fig Previous studies in prostate cancer cell lines have established a clear role for membrane androgen receptors in the induction of apoptotic responses via actin cytoskeleton reorganization . FurtherUsing pharmacological inhibitors, dominant negative alleles and various functional assays, we were able to identify in previous studies a series of key denominators of mAR function in prostate cancer cell lines ,17. SpecIn the present work we characterized the specificity of mAR by using a series of BSA-conjugated and free steroid hormones, providing clear evidence for testosterone specificity. We further explored the mechanism of cell death triggered by mAR-stimulation by analyzing the expression and activity of several gene products and pathways involved in the regulation of survival and apoptosis of DU145 prostate cancer cells. Using testosterone-BSA as a specific mAR ligand, we show here that mAR activation results in almost complete down-regulation of the activity of PI-3K, Akt and NF-\u03baB in DU145 cells. Concurrently, testosterone-BSA induces FasL expression, activates Bad and up-regulates the activity of caspase-3, indicating that mAR-stimulation affects prominent pro-apoptotic regulators ,40. Impo+ prostate cancer cells independently of the functional status of the intracellular androgen receptor. Interestingly, mAR is selectively over-expressed in biopsy samples from aggressive, high-Gleason prostate tumors in comparison to samples from benign prostate hyperplasia patients or healthy subjects [Although we cannot rule out that the observed changes in the expression and activity of all analyzed proteins are the consequence rather than the cause of mAR-dependent apoptosis, the data clearly underscore the key role of mAR-activating ligands in the selective elimination of DU145 cells. Moreover, these receptors are specific for testosterone and testosterone-albumin conjugates, since other steroid hormones-conjugated or not-failed to exhibit any pro-apoptotic activity. Notably, these cells typically represent an aggressive pre-clinical hormone-refractory cell line model used to assess the anti-tumor ability of chemotherapeutic drugs, as they (I) are devoid of functional intracellular androgen receptors (iARs) and (II) fail to respond to androgen treatment . Based osubjects .Future experiments will focus on the identification of additional signaling targets downstream of mAR and the characterization of functional synergies of mAR-dependent signals with other pathways activated in prostate cancer. Characterization of the functional interplay between membrane and intracellular androgen receptors may contribute to the understanding of the apparent discrepancy in the actions of androgens inducing both proliferation and death within a given cell. Our present findings elucidating at least parts of the mAR-induced molecular pro-apoptotic machinery in DU145 cells provide novel insights in membrane GPCR mediated non-genomic androgen actions.The authors declare that they have no competing interests.NP and IC carried out the analysis of all signaling molecules, apoptotic responses and actin cytoskeleton dynamics. VA and GK carried out the kinetics of pro-survival gene products and FACS analysis. MF participated in the design of the study and performed the statistical analysis. KA participated in the design of the study and drafted the manuscript. FL and AG participated in the coordination of the study and evaluation of the results. CS conceived of the study and participated in the design and coordination."} +{"text": "A recently identified genetic polymorphism located in the 5' region of the HLA-C gene is associated with individual variations in HIV-1 viral load and with differences in HLA-C expression levels. HLA-C has the potential to restrict HIV-1 by presenting epitopes to cytotoxic T cells but it is also a potent inhibitor of NK cells. In addition, HLA-C molecules incorporated within the HIV-1 envelope have been shown to bind to the envelope glycoprotein gp120 and enhance viral infectivity. We investigated this last property in cell fusion assays where the expression of HLA-C was silenced by small interfering RNA sequences. Syncytia formation was analyzed by co-cultivating cell lines expressing HIV-1 gp120/gp41 from different laboratory and primary isolates with target cells expressing different HIV-1 co-receptors. Virus infectivity was analyzed using pseudoviruses. Molecular complexes generated during cell fusion (fusion complexes) were purified and analyzed for their HLA-C content.HLA-C positive cells co-expressing HIV-1 gp120/gp41 fused more rapidly and produced larger syncytia than HLA-C negative cells. Transient transfection of gp120/gp41 from different primary isolates in HLA-C positive cells resulted in a significant cell fusion increase. Fusion efficiency was reduced in HLA-C silenced cells compared to non-silenced cells when co-cultivated with different target cell lines expressing HIV-1 co-receptors. Similarly, pseudoviruses produced from HLA-C silenced cells were significantly less infectious. HLA-C was co-purified with gp120 from cells before and after fusion and was associated with the fusion complex.Virionic HLA-C molecules associate to Env and increase the infectivity of both R5 and X4 viruses. Genetic polymorphisms associated to variations in HLA-C expression levels may therefore influence the individual viral set point not only by means of a regulation of the virus-specific immune response but also via a direct effect on the virus replicative capacity. These findings have implications for the understanding of the HIV-1 entry mechanism and of the role of Env conformational modifications induced by virion-associated host proteins. The siRNAs targeted different regions of the HLA-C mRNA.The HLA-C mRNA , LAI [GenBank: AF004394]; ADA [GenBank: AY426119]; 92UG024 [GenBank: U43386]; 93MW965 [GenBank: U08455]: 91US005 [GenBank: U27434]) were aligned and compared using CLC Sequence Viewer 4.6.2, developed by CLC bio A/S for Apple Mac OSX.HIV-1 Env sequences (NDK [GenBank: The authors declare that they have no competing interests.env sequence from the J500 primary isolate. AGS participated in the design and coordination of the study and drafted the manuscript. AB participated to study design, data analysis and gave a significant contribution in drafting and revising the manuscript. DZ produced Env-coding plasmids and stably transfected cell lines, did fusion complexes preparation and analysis, conceived the study and carried out its design, and, as corresponding author, carried out the drafting of the manuscript. All authors read and approved the final manuscript.AM carried out siRNA silencing, RT-PCR, cell transfections, ELISA, Western blot and FACS analysis, cellular fusions and pseudovirus infections experiments. PR carried out sequencing, pseudovirus preparation and titration and fusion complexes preparation and analysis. MB isolated and cloned the HLA-C insensitive"} +{"text": "We studied whether fibronectin (FN) enhances the activity of autologous tumour-reactive cytotoxic T lymphocytes (CTLs) generated from cancer patients. The proliferation of CTLs stimulated by immobilised anti-CD3 monoclonal antibody and interleukin 2 (IL-2) was enhanced three or four times by immobilised FN. whereas soluble FN did not alter the DNA synthesis of CTLs. Moreover, the cytotoxic activity of CTLs was augmented by FN stimulation against autologous tumour cells . The major cell surface phenotype of CTLs with FN was CD3+, CD4+ and CD25+ in 6 weeks' culture. Cytotoxicity against autologous tumour cells was inhibited by anti-HLA class I monoclonal antibody (MAb). The autologous tumour-killing activity of CTLs was suppressed by the elimination of CD4+ cells. Moreover, the cytokine production of CTLs was augmented by FN stimulation. Especially, the production of IL-2, interferon gamma (IFN-gamma), and granulocyte macrophage colony-stimulating factor (GM-CSF) was significantly augmented by FN stimulation (P<0.05). Thus, CTLs generated by FN might have both killer and helper functions, since they could lyse autologous tumour cells and secrete various cytokines, including IL-2."} +{"text": "Long-circulating liposomes coated with polyethylene glycol (PEG), which show reduced uptake by the reticuloendothelial system (RES) and enhanced accumulation in tumours, were used for conjugation to monoclonal antibodies (MAbs) as a drug-targeting device. A MAb (N-12A5) directed against erbB-2 oncoprotein, a functional surface antigen, was used. Amplification and overexpression of the erbB-2 gene product, being unique to malignancy, confer onto this antibody-mediated therapy high tumour specificity. In vitro binding of [3H]cholesteryl ether ([3H]Chol ether) labelled anti-erbB-2 conjugated liposomes to N-87 cells (erbB-2-positive human gastric carcinoma) was compared with the binding of non-targeted liposomes and indicated a 16-fold increase in binding for the targeted liposomes. No difference in binding to OV1063 cells (erbB-2-negative human ovary carcinoma) was observed. These results indicate highly selective binding of antibody-targeted liposomes to erbB-2-overexpressing cells. Despite increased cell binding, doxorubicin (DOX) loaded in anti-erbB-2-conjugated liposomes did not cause increased in vitro cytotoxicity against N-87 cells, suggesting lack of liposome internalisation. In vivo, the critical factor needed to decrease the non-specific RES uptake and prolong the circulation time of antibody-conjugated liposomes is a low protein to phospholipid ratio ( < 60 micrograms mumol-1). Using these optimised liposome preparations loaded with DOX and by monitoring the drug levels and the [3H]Chol ether label, biodistribution studies in nude mice bearing subcutaneous implants of N-87 tumours were carried out. No significant differences in liver and spleen uptake between antibody-conjugated and plain liposomes were observed. Nevertheless, there was no enhancement of tumour liposome levels over plain liposomes. Both liposome preparations considerably enhanced DOX concentration in the tumour compared with free drug administration. Therapeutic experiments with N-87 tumour-bearing nude mice indicated that anti-tumour activity of targeted and non-targeted liposomes was similar, although both preparations had an increased therapeutic efficacy compared with the free drug. These studies suggest that efficacy is dependent on drug delivery to the tumour and that the rate-limiting factor of liposome accumulation in tumours is the liposome extravasation process, irrespective of liposome affinity or targeting to tumour cells."} +{"text": "To understand the mechanisms and identify novel approaches to overcoming retinoic acid (RA) resistance in acute promyelocytic leukaemia (APL), we established the first human RA-resistant APL model in severe combined immunodeficiency (SCID) mice. UF-1 cells, an RA-resistant APL cell line established in our laboratory, were transplanted into human granulocyte-macrophage colony-stimulating factor (GM-CSF)-producing SCID (hGMTg SCID) mice and inoculated cells formed subcutaneous tumours in all hGMTg SCID mice, but not in the non-transgenic control SCID mice. Single-cell suspensions (UF-1/GMTg SCID cells) were similar in morphological, immunological, cytogenetic and molecular genetic features to parental UF-1 cells. All-trans RA did not change the morphological features of cells or their expression of CD11b. RA did not alter the growth curve of cells as determined by MTT assay, suggesting that UF-1/GMTg SCID cells are resistant to RA. These results demonstrate that this is the first RA-resistant APL animal model that may be useful for investigating the biology of this myeloid leukaemia in vivo, as well as for evaluating novel therapeutic approaches including patients with RA-resistant APL."} +{"text": "Somatic hypermutation (SHM) and class switch recombination (CSR) take place in B cells of the germinal center (GC) and are associated with DNA double-strand breaks (DNA-DSBs). Transcription favors the generation of DNA-DSBs in the V-regions and switch regions of Ig genes. Both SHM and CSR are controlled by the Activation Induced Cytidine Deaminase (AID), an enzyme exclusively expressed in B cells of the GC. Because AID is capable of deaminating deoxy-cytidine (dC) to deoxy-uracil (dU), it might directly induce nicks (single strand DNA breaks) and also DNA-DSBs via a U-DNA glycosylase mediated base excision repair pathway ('DNA-substrate model'). Alternatively, AID could function like its closest homologue Apobec-1 as a catalytic subunit of a RNA editing holoenzyme ('RNA-substrate model'). To determine whether AID lies upstream or downstream of the DNA lesions found in hypermutating Ig genes, we have analysed the V\u03bb locus of AID proficient and AID deficient GC B cells for the presence of DNA-DSBs. Although rearranged V\u03bb genes are preferred targets of SHM we find that AID-proficient and -deficient V\u03bb1/2-expressing GC B cells display a similar frequency, distribution and sequence preference of DNA-DSBs in rearranged and germline V\u03bb genes, favoring the idea that AID acts downstream of the DNA lesions to mediate error prone processing."} +{"text": "Mast cell amines, platelet-activating factor (PAF), thromboxanes and leukotrienes have been shown to be released during nitric oxide-synthase inhibition in the rat intestine. Mast cells in rat isolated omentum (OMCs) or isolated from the rat peritoneal cavity (PMCs) have been used here to investigate the relationship(s) between these agents. N-nitro-L-arginine methyl ester caused some degranulation of OMCs, but no enhancement of histamine release from PMCs. PAF (5 \u03bcM) and U46619 (1 \u03bcM) degranulated OMCs and enhanced histamine release from PMCs. Pre-treatment of the omentum with BN52021 (10 \u03bcM) inhibited degranulation of OMCs in response to L-NAME, PAF or U46619. Pretreatment with 1-benzylimidazole (5 or 50 \u03bcM) inhibited the effect of L-NAME but not that of PAF. Indomethacin (1 \u03bcM) or sodium nitroprusside (10 \u03bcM) also inhibited the effects of L-NAME, but nordihydroguaiaretic acid (30 \u03bcM) did not. In PMCs BN52021 inhibited PAF-induced, but not U46619-induced, release of histamine. These results suggest that inhibition of nitric oxidesynthase in the omentum by L-NAME allows thromboxanes to release PAF, which in turn degranulates and releases histamine from OMCs."} +{"text": "Serum GH levels increased andserum IGF-I levels decreased in the 30d DM group. Another group (30d DM + I)was given SC insulin, and its serum IGF-I levelsremained decreased. Liver GH receptor (GHR) andGH binding protein (GHBP) mRNA levels, as wellas liver membrane GH binding assays were deeplydecreased in the 30d DM group in comparison tocontrols. GHR message and binding capacity remaineddecreased in the 30d DM + I group. RenalGHR mRNA was decreased at 21d DM but not at14d DM, whereas GHBP mRNA remained unchangedthroughout the experiment. In conclusion,increased serum GH levels are documented in NODdiabetic mice, similarly to the changes described inhumans. The decrease in GHR levels and decreasedserum IGF-I in spite of increased circulating GHsuggest a state of GH resistance.We investigated the changes in GH-IGF-I axis innon-obese diabetic (NOD)-mice, a model of insulin dependentdiabetes mellitus. Diabetic female NODmice and their age- and sex-matched controls weresacrificed at 4, 14, 21 and 30 days ("} +{"text": "This paper discusses how enhanced FDC-B-cell interaction within SIV-infected germinal centers may result in a reduced ability to select high-affinity B cells and alter the dynamics of antibodyproducing- cell and memory-cell generation resulting in the observed hyperactivity.Human immunodeficiency virus (HIV) infections have been characterized by both polyclonal Bcell activation and enhanced responsiveness to B-cell growth factors on one hand and the loss of specific antibody (Ab) responses and refractoriness to the normal signals for B-cell activation on the other. Histopathological studies of lymph node from HIV- and simian immunodeficiency virus (SIV)-infected individuals have indicated initial follicular hyperplasia and the appearance of large irregular germinal centers that undergo progressive involution concomitant with follicular dendritic-cell (FDC) disruption. During this process, follicular dendritic-cell -enrichedlymph-node-cell cultures exhibit increased ability to induce cluster formation (\u201c"} +{"text": "High-fructose feeding (60 g/100 gdiet) to normal rats resulted in a significant increase inthe concentrations of cholesterol, triglycerides (TGs), freefatty acids (FFAs), and phospholipids in plasma, liver, kidney,and skeletal muscle. Reduced activities of lipoproteinlipase (LPL) and lecithin cholesterol acyl transferase(LCAT) and increased activity of the lipogenic enzymehydroxymethylglutaryl\u2013coenzyme A (HMG-CoA) reductasewere observed in plasma and liver. High-densitylipoprotein cholesterol (HDL-C) was significantly loweredand very low-density lipoprotein cholesterol (VLDL-C) andlow-density lipoprotein cholesterol (LDL-C) were significantlyelevated. Treatment with LA reduced the effects of fructose. The ratsshowed near-normal levels of lipid components on plasmaand tissues. Activities of key enzymes of lipid metabolismwere also restored to normal values. Cholesterol distributionin the plasma lipoproteins was normalized, resulting ina favorable lipid profile. This study demonstrates that LAcan alter lipid metabolism in fructose-fed insulin-resistantrats and may have implications in the treatment of insulinresistance.This study investigated the effect of administration of"} +{"text": "Using a regrowth-delay assay, we investigated structure/activity relationships for the enhancement by electron-affinic agents of the anti-tumour effect of the nitrosourea CCNU against the KHT sarcoma in C3H mice. A series of neutral 2-nitroimidazoles similar in electron affinity but varying in octanol/water partition coefficient (PC) over 4 orders of magnitude were examined at a fixed dose of 2.5 mmol/kg. A parabolic (quadratic) dependence of activity on log PC was observed. Analogues more hydrophilic than misonidazole (MISO) were inactive as were those with very high PCs (greater than 20). Those with PC 0.43--20 were usually more active than MISO, some considerably so. The fairly lipophilic 5-nitroimidazoles nimorazole and metronidazole (METRO) had similar activity to MISO, despite their reduced electron affinity. Two basic 2-nitroimidazoles more efficient as radiosensitizers in vitro likewise showed activity comparable to MISO. We also investigated several agents more electron-affinic than MISO, including some non-nitro compounds. Most were inactive at maximum tolerated doses, but nitrofurazone showed reasonable activity. Sensitizer dose-response curves were obtained for MISO, METRO and two of the most effective agents, benznidazole (Ro 07-1051) and Ro 07-1902. The two latter agents were both considerably more active than MISO at low doses (0.1--0.9 mmol/kg). These studies indicate that the structural features of electron-affinic agents responsible for the enhancement of KHT tumour response to CCNU, are quite different from those affecting radiosensitization, lipophilicity being particularly important. The microsomal enzyme-inhibitor SKF 525A increased the anti-tumour effect of CCNU, suggesting inhibition of CCNU metabolism as one possible mechanism contributing to chemosensitization by lipophilic electron-affinic agents in mice."} +{"text": "This article reviews recent basic and clinical studies of ginseng, particularly the anti-cancer effects and the potential chemopreventive actions by activating the transcriptional factor, nuclear factor (erythroid-derived 2)-like 2 (Nrf2 or NFE2L2)-mediated anti-oxidative stress or anti-inflammatory pathways. Nrf2 is a novel target for cancer prevention as it regulates the antioxidant responsive element (ARE), a critical regulatory element in the promoter region of genes encoding cellular phase II detoxifying and anti-oxidative stress enzymes. The studies on the chemopreventive effects of ginseng or its components/products showed that Nrf2 could also be a target for ginseng's actions. A number of papers also demonstrated the anti-inflammatory effects of ginseng. Targeting Nrf2 pathway is a novel approach to the investigation of ginseng's cancer chemopreventive actions, including some oxidative stress and inflammatory conditions responsible for the initiation, promotion and progression of carcinogenesis. Ginseng protects the cardiovascular system, stimulates the central nervous system and possvia the Nrf2 signalling pathway and the potential molecular mechanism of ginseng's anti-cancer effects.Nuclear factor (erythroid-derived 2)-like 2 (Nrf2 or NFE2L2) is a key regulator of the antioxidant responsive element (ARE)-mediated gene expression and therefore a potential anti-cancer target for chemopreventive compounds , includiAngelica sinensis (Danggui) pyrene; NF-kB: Nuclear factor-kappa-B; Gpx2: Glutathione peroxidase 2; COX-2: Cyclooxygenase-2; iNOS: Inducible nitric oxide synthase; \u03b3-GCL: \u03b3-glutamylcystein ligase; MAPKs: Mitogen-activated protein kinases; bZip: Basic leucine zipper partners; GSP: Ginseng polysaccharides; KPS: Karnofsky Performance Status Scale; NPC: Nasopharyngeal carcinoma; RT: Radiotherapy; NK: Natural killer; LAK: Lymphocyte activated killer; NQO1: NADPH: quinone oxidoreductase 1; AKR: Aldo-keto reductases.Nrf2 (NFE2L2): Nuclear factor (erythroid-derived 2)-like 2; ARE: Antioxidant responsive element; CNKI: Chinese National Knowledge Infrastructure; ROS: Reactive oxygen species; RNS: Reactive nitrogen species; GSH: Glutathione; GST: Glutathione The authors declare that they have no competing interests.CLLS planned this review. CLLS and QW performed the literature searches and drafted the manuscript. ANTK supervised the review process and revised the manuscript. All authors read and approved the final version of the manuscript.Clinical studies of ginseng Chinese medicine products as adjuvant therapy to cancer treatments.Click here for filePreclinical studies on ginseng and its extracts showing molecular activities on Nrf2 activation for potential chemopreventive use.Click here for file"} +{"text": "The results suggested that copper aspirinate inhibited platelet-neutrophil adhesionand resulted in a more potent antithrombotic activity.Antithrombotic effect of the copper-aspirin complex (dimeric copper(II) bis(o-acetoxybenzoate)was evaluated in the model of venous thrombosis; its effects on platelet-neutrophil adhesion wereinvestigated by use of rosette assay. The results showed that the intragastrically administered copper-aspirincomplex (5, 7, and 10 mg kg"} +{"text": "Great efforts have been made to develop novel and efficacious therapeutics against pancreatic cancer to improve the treatment outcomes. Tumor-necrosis factor-related apoptosis-inducing ligand (TRAIL) is such a therapeutic cytokine with selective killing effect toward malignant cells. However, some human pancreatic cancers are intrinsically resistant to TRAIL-mediated apoptosis or therapy. In this study, we have shown that the histone deacetylase inhibitor LBH589 can synergize with TRAIL to augment apoptosis even in TRAIL-resistant cells. LBH589 decreased c-FLIP levels in every tested cell line and survivin levels in some of the tested cell lines. Enforced expression of ectopic c-FLIP, but not survivin, abolished the cooperative induction of apoptosis by the combination of LBH589 and TRAIL, indicating that c-FLIP downregulation plays a critical role in LBH589 sensitization of pancreatic cancer cells to TRAIL. Moreover, LBH589 decreased c-FLIP stability and the presence of the proteasome inhibitor MG132 prevented c-FLIP from reduction by LBH589. Correspondingly, we detected increased levels of ubiqutinated c-FLIP in LBH589-treated cells. These data thus indicate that LBH589 promotes ubiqutin/proteasome-mediated degradation of c-FLIP, leading to downregulation of c-FLIP. Collectively, LBH589 induces c-FLIP degradation and accordingly sensitizes pancreatic cancer cells to TRAIL-induced apoptosis, highlighting a novel therapeutic regimen against pancreatic cancer. Pancreatic cancer is one of the most difficult cancers to treat although it accounts for only 3% of all cancers. Despite multiple clinical trials with new chemotherapeutic agents, over the past 25 years the 5-year survival rate of 5%, and median survival of 6 months has largely remained unchanged. The median survival is about 6 months Apoptosis is an essential part of mechanisms that maintain normal tissue homeostasis The death ligand TRAIL has recently emerged as potential cancer therapeutic agent because it preferentially induces apoptosis in transformed or malignant cells L and FLIPS are subject to regulation by ubiquitin/proteasome-mediated degradation Cellular FLICE-inhibitory protein (c-FLIP), which inhibits caspase-8 activation by preventing recruitment of caspase-8 to DISC, is the primary inhibitor of TRAIL/death receptor-induced apoptosis LBH589 (panobinostat) is a pan-histone deacetylase (HDAC) inhibitor with promising anticancer activity LBH589 was provided by Novartis . The soluble recombinant human TRAIL was purchased from PeproTech, Inc. . The proteasome inhibitor MG132 and the protein synthesis inhibitor cyclohexemide (CHX) were purchased from Sigma Chemical Co. . Rabbit polyclonal anti-DR5 antibody was purchased from ProSci Inc . Mouse monoclonal anti-DR4 antibody (B-N28) was purchased from Diaclone . Mouse monoclonal anti-caspase-3 antibody was purchased from Imgenex . Rabbit polyclonal anti-XIAP, anti-caspase-8, anti-Mcl-1, and anti-PARP antibodies and mouse monoclonal anti-survivin antibody were purchased from Cell Signaling Technology, Inc. . Mouse anti Bcl-2 antibody was purchased from Santa Cruz Biotechnology, Inc . Rabbit anti-GAPDH polyclonal antibody and mouse anti-Bax monoclonal antibody were purchased from Trevigen . Mouse monoclonal anti-\u03b2-actin antibody was purchased from Sigma Chemical Co.L and survivin, respectively, as described previously 2 and 95% air.Human pancreatic cancer cell lines used in this study were purchased from the American Type Culture Collection . For establishing pancreatic cancer cell lines that stably express ectopic c-FLIP or survivin, Panc-1 cells were infected with lentiviruses harboring lentiviral expression vectors of FLIPt tests by use of Graphpad InStat 3 software . Results were considered to be statistically significant at P<0.05.Cells were seeded in 96-well cell culture plates and treated the next day with the agents indicated. The viable cell numbers were determined using the sulforhodamine B (SRB) assay, as previously described Apoptosis was evaluated by annexin V staining using annexin V-PE apoptosis detection kit purchased from BD Biosciences following the manufacturer's instructions. We also detected caspase activation by Western blotting (as described below) as an additional indicator of apoptosis.Whole-cell protein lysates were prepared and analyzed by Western blotting as described previously L-5 cells, which stably express FLIPL, were transfected with HA-ubiquitin plasmid using the FuGENE 6 transfection reagent following the manufacturer's instruction. After 24 h, the cells were treated with LBH589 or MG132 plus LBH589 for 4 h and then were lysed for immunoprecipitation of Flag-FLIPL using Flag M2 monoclonal antibody as previously described L with Western blotting using anti-HA antibody .Panc-1/FLIPWe first determined the sensitivities of pancreatic cancer cell lines used in this study to TRAIL. As presented in L) than Bxpc-3 cells. Treatment of these cell lines with LBH589 decreased the levels of c-FLIP in all of the three cell lines in a concentration-dependent manner abolished LBH589's ability to enhance TRAIL-induced apoptosis (DR5 induction and c-FLIP downregulation are important mechanisms underlying drug-mediated augmentation or sensitization of TRAIL-induced apoptosis poptosis and 5. Cc-FLIP is known to be regulated by ubiquitin/proteasome-mediated degradation The maximal plasma concentrations of LBH589 in human cancer patients range from 200 nM to 1300 nM depending on doses tested"} +{"text": "Mesothelial activation with interleukin (IL)-1b or tumour necrosis factor (TNF)-a produced an up-regulation of mRNA for HB-EGF and VEGF, but not bFGF expression. IL-6 failed to stimulate growth factor expression, whereas IL-2 produced a marked suppression in HB-EGF and bFGF, but not VEGF expression. Mesothelial cells were shown to predominantly express mRNA for the intermediate affinity (bgc) IL-2 receptor. Cytokine-induced growth factor up-regulation was confirmed at the protein level using Western blotting of mesothelial cell lysates for HB-EGF and culture supernatant enzyme-linked immunosorbent assay for VEGF. The production of these growth factors by human mesothelial cells may play a significant role in post-operative peritoneal tumour recurrence. Their common heparin-binding property offers a potential therapeutic target for manipulating the growth factor environment of the human peritoneum. \u00a9 2000 Cancer Research CampaignCurative surgery for gastrointestinal malignancy is commonly thwarted by local tumour recurrence. The heparin-binding growth factors, basic fibroblast growth factor (bFGF), heparin-binding epidermal growth factor-like growth factor (HB-EGF) and vascular epidermal growth factor (VEGF) are all implicated in the metastatic process, but whether or not these essential growth factors are produced by the activated peritoneum is unknown. This study reveals that peritoneal mesothelial cells constitutively express mRNA for bFGF, HB-EGF and two VEGF spliced variants, VEGF"} +{"text": "Inorganic arsenic is a ubiquitous environmental carcinogen affecting millions of people worldwide. Evolving theory predicts that normal stem cells (NSCs) are transformed into cancer stem cells (CSCs) that then drive oncogenesis. In humans, arsenic is carcinogenic in the urogenital system (UGS), including the bladder and potentially the prostate, whereas in mice arsenic induces multiorgan UGS cancers, indicating that UGS NSCs may represent targets for carcinogenic initiation. However, proof of emergence of CSCs induced by arsenic in a stem cell population is not available.in vitro and determined the acquired cancer phenotype.We continuously exposed the human prostate epithelial stem/progenitor cell line WPE-stem to an environmentally relevant level of arsenic (5 \u03bcM) p63, ABCG2, BMI-1, SHH, OCT-4, NOTCH-1) during arsenite exposure was subsequently reversed as the tumor suppressor gene PTEN was progressively suppressed and the CSC-like phenotype acquired.WPE-stem cells rapidly acquired a malignant CSC-like phenotype by 18 weeks of exposure, becoming highly invasive, losing contact inhibition, and hypersecreting matrix metalloproteinase-9. When hetero-transplanted, these cells (designated As-CSC) formed highly pleomorphic, aggressive tumors with immature epithelial- and mesenchymal-like cells, suggesting a highly pluripotent cell of origin. Consistent with tumor-derived CSCs, As-CSCs formed abundant free-floating spheres enriched in CSC-like cells, as confirmed by molecular analysis and the fact that only these floating cells formed xenograft tumors. An early loss of NSC self-renewal gene expression (Arsenite transforms prostate epithelial stem/progenitor cells into CSC-like cells, indicating that it can produce CSCs from a model NSC population. This dein vitro . Althougin vitro . Typicalin vitro , rather in vitro, inorganic arsenic causes malignant transformation of various cells, including human prostate cells ]. We used arsenic at a concentration that approximates levels in drinking water in areas where arsenicosis is common (Leukemia SCs (LSCs) can be forced to arise from hematopoietic SCs (HSCs) after molecular manipulations of leukemogenesis genes . Howevers common ; and celRWPE-1 is a human papillomavirus (HPV)-18\u2013immortalized, nontumorigenic, prostate epithelial cell line derived from normal adult human prostate . WPE-stein vitro biomarkers of carcinogenic transformation every 3 weeks. Secreted matrix metalloproteinase-9 (MMP-9) activity was examined using conditioned medium as previously described in T-75 flasks; cells were then grown for 10 days and fed every 48 hr. Total spheres were then counted.Free-floating spheres of viable cells in culture are a characteristic of SCs and CSCs . To examFree-floating spheres were collected from flasks and dissociated into single-cell suspensions by pipette trituration then passage through a 40-\u03bcm cell strainer . Cells were suspended in 250 \u03bcL Matrigel (BD Biosciences) in 48-well plates and placed in incubators for 24 hr. Matrigel was then covered with 300 \u03bcL growth medium, which was changed every 48 hr. Images were taken 2 weeks later.p63 [tumor protein p63 (TP63)], BMI-1, ABCG2, SHH (sonic hedgehog), OCT-4 [POU class 5 homeobox 1 (POU5F1)], NOTCH-1 , K5 [keratin 5 (KRT5)], K18, and PTEN (phosphatase and tensin homolog). For gene and primer information, see Supplemental Material, Table 2 (doi:10.1289/ehp.0901059.S1). For Western blot analysis, protein extracts were collected using either NE-PER or M-PER extraction reagents , separated by SDS-PAGE, transferred to polyvinyl difluoride membranes, and probed with anti-\u0394Np63 or anti-cytokeratin-18 (Sigma) followed by horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz Biotechnology). Membranes were stripped and reprobed with anti-\u03b2-actin antibody followed by peroxidase-conjugated anti-mouse (Santa Cruz Biotechnology). We used the ImageJ analysis program and purified with RNeasy Mini Kit columns according to the manufacturer\u2019s protocol, and transcribed with MuLV (Moloney murine leukemia virus) reverse transcriptase and oligo-dT primers. Primers were designed with ABI Primer Express software . We used SYBR Green Master Mix for real-time PCR (polymerase chain reaction) analysis. Cycle times were normalized with \u03b2-actin and glyceraldehyde-3-phosphate from the same sample and normalized to passage-matched controls. Genes examined included We conducted immunocytochemistry for p63 protein as described previously . Chambernu; Charles River Laboratory, Wilmington, MA). Mice were housed at the NCI-Frederick animal facility , and animal care was provided in accordance with the Public Health Service policy on the care and use of animals were collected and injected under the renal capsules of mice. In the second study, arsenite-treated floating cells were first separated from adherent cells and 1 \u00d7 106 cells from each subgroup was injected subcutaneously into separate groups of mice. Animals were observed for tumor formation over a 6-month period.Once biomarkers of carcinogenic transformation suggested that arsenic-induced carcinogenic conversion had occurred, arsenic-treated and control cells were injected into male nude mice . We used an unpaired Student\u2019s MMP-9, an enzyme that when secreted digests extracellular matrix to aid in invasion and metastasis typical of cancer cells , includiTo establish malignant transformation, we inoculated cells under the renal capsules of mice. Arsenite-treated cells (As-CSCs) rapidly developed into highly pleomorphic tumors, with regional invasion and distant metastases that often dictated euthanasia in as little as 2\u20133 weeks. Tumors were highly undifferentiated, highly malignant, and composed of immature epithelial- and mesenchymal-like cells . Strong A common characteristic of SCs in culture is the formation of floating \u201cspheres\u201d of viable cells . Malignap63 during arsenic-induced malignant transformation, with an initial suppression followed by reactivation. p63 is essential for SC self-renewal and proliferation within the prostate and other tissues gene into hematopoietic committed progenitor cells (NSCs and CSCs share fundamental properties, including self-renewal capacity, which is typically dysregulated in oncogenesis . In this tissues . BMI-1 i tissues . In seve tissues . ABCG2 p tissues . OCT-4 iogenesis . The Notogenesis . In the ogenesis . Additioogenesis . The remor cells . A groupor cells . Likewisor cells . Thus, dor cells and cleaor cells , the prePTEN expression coincided with the reactivation of self-renewal genes and acquisition of malignant phenotype. Poor prostatic PTEN expression increases SC-like cells and causes cancer initiation (PTEN enhances self-renewal capacity of SCs without dramatically altering pluripotency (PTEN expression maintains HSCs in a quiescent state, and its deletion leads to HSC depletion and leukemia formation enriched for LSCs (PTEN provides a plausible mechanism through which As-CSC cells reactivate self-renewal, although dysregulated, and yet maintain and potentially distort multipotential differentiation capacity.During arsenite-induced transformation and formation of As-CSCs, the dramatic decrease in itiation and in pitiation . Furtheripotency . PTEN exfor LSCs . The losin vivo. The truly stunning capacity to rapidly produce highly pleomorphic tumors in xenograft studies and the fact that only cells from the arsenite-treated spheres formed tumors, together with strong evidence that CSCs reside in similar spheres derived from advanced cancers (Our data support the hypothesis that invokes emergence of CSCs from NSCs as a primal initiating event in oncogenesis , at leas cancers , support cancers .in utero life-stage susceptibility to chemical carcinogenesis (Overall, this study expands our understanding of the carcinogenic potential of arsenic, a common environmental contaminant , by indiogenesis . Therefo"} +{"text": "Sir,Elective nodal irradiation (ENI) was used in conventional radiotherapy (RT) target volume for limited-stage small-cell lung cancer (LS-SCLC) in contrast to involved-field RT only in some recent studies . In cont"} +{"text": "N-methyl-N-nitrosourea (MNU) elicited antibody production (detected by membrane immunofluorescence test in vitro) when injected into a highly inbred strain of rats from which the liver cells were originally isolated. In contrast, the control cells which were untreated did not evoke humoral antibodies. Antisera raised against the MNU-treated cells reacted not only with the immunizing cells, but also with 3'-methyl-4-dimethyl aminoazobenzene and 3-methylcholanthrene-treated cells. However, this antiserum failed to react with cells treated with either aflatoxin or N-acetoxy-2-acetyl-aminofluorene. Embryonic antigens were found to be absent from the normal adult liver cell line. Preliminary results indicated that none could be detected on carcinogen-treated cells either.Normal rat liver cell lines treated with the chemical carcinogen"} +{"text": "The RNA-binding protein tristetraprolin (TTP) regulates expression of many cancer-associated and proinflammatory factors through binding AU-rich elements (ARE) in the 3'-untranslated region (3'UTR) and facilitating rapid mRNA decay. Here we report on the ability of TTP to act in an anti-proliferative capacity in HPV18-positive HeLa cells by inducing senescence. HeLa cells maintain a dormant p53 pathway and elevated telomerase activity resulting from HPV-mediated transformation, whereas TTP expression counteracted this effect by stabilizing p53 protein and inhibiting hTERT expression. Presence of TTP did not alter E6 and E7 viral mRNA levels indicating that these are not TTP targets. It was found that TTP promoted rapid mRNA decay of the cellular ubiquitin ligase E6-associated protein (E6-AP). RNA-binding studies demonstrated TTP and E6-AP mRNA interaction and deletion of the E6-AP mRNA ARE-containing 3'UTR imparts resistance to TTP-mediated downregulation. Similar results were obtained with high-risk HPV16-positive cells that employ the E6-AP pathway to control p53 and hTERT levels. Furthermore, loss of TTP expression was consistently observed in cervical cancer tissue compared to normal tissue. These findings demonstrate the ability of TTP to act as a tumor suppressor by inhibiting the E6-AP pathway and indicate TTP loss to be a critical event during HPV-mediated carcinogenesis. E6 and E7. The HPV E7 protein neutralizes the retinoblastoma (Rb) tumor suppressor pathway by sequestering Rb from E2F and promoting its destabilization [hTERT) [hTERT gene expression [Cervical cancer is the second most common cancer among women worldwide . A neceslization ,4, while [hTERT) ,6. The [hTERT) . E6-AP [hTERT) and itspression . cis-acting adenylate- and uridylate (AU)-rich elements (ARE) in the mRNA 3' untranslated region (3'UTR). A primary function of the ARE is to target mRNAs for rapid decay through interaction with trans-acting RNA-binding proteins that have high affinity for AREs. Among the best characterized ARE-binding proteins involved in promoting ARE-mediated mRNA decay is tristetraprolin . TTP is a member of a small family of tandem Cys3His zinc finger proteins originally identified as an inducible immediate-early response gene [Messenger RNA turnover is a tightly regulated process that plays a central role in controlling mammalian gene expression. The significance of this is evident in disease and tumorigenesis where loss of post-transcriptional gene regulation accounts for the aberrant overexpression of a variety of genes encoding growth factors, inflammatory cytokines and proto-oncogenes ,12. A manse gene . Initialnse gene -16. The nse gene ,17-21. Tnse gene ,23. In this study, we examined the role of TTP in HPV-mediated cervical carcinogenesis. Expression of TTP in HPV18-positive HeLa cells dramatically inhibited cell growth by inducing cellular senescence through a mechanism involving p53 protein stabilization and inhibition of telomerase expression. It was found that TTP induced cellular senescence through rapid decay of E6-AP ubiquitin ligase mRNA that was mediated through the ARE-containing 3'UTR of E6-AP. Furthermore, we demonstrate that TTP expression is lost in cervical cancer compared to normal tissue, implying a tumor suppressor function for TTP in cervical tissue. These novel findings not only add another attribute to the already established anti-inflammatory role of this ARE-binding protein but also bring new insights into the mechanism of HPV-mediated cervical carcinogenesis. Based on its ability to control expression of ARE-containing mRNAs associated with various aspects of cellular transformation and tumorigenesis, TTP can serve in a tumor suppressor capacity. To test this in HPV-transformed cervical carcinoma cells, a tetracycline (Tet)-regulated TTP expression system in HeLa cells was developed. HeLa Tet-Off cells were stably transfected with a Flag epitope-tagged TTP cDNA in a Tet-regulated expression vector such that cells grown in the absence of doxycycline (Dox) allow for the expression of TTP Figure . To determine the consequence of TTP expression, we evaluated the ability of TTP to attenuate HeLa cell growth and proliferation. As shown in Figure HPV oncogenicity is mediated through the interaction between HPV E6 protein and the tumor suppressor p53 with E6 promoting accelerated ubiquitin-mediated degradation of p53 ,10. BaseActivation of p53 promotes its accumulation in the nucleus and transcription of p53-responsive promoters ,29. To Elevated telomerase activity is associated with approximately 85% of human cancers . In In high-risk HPV-transformed cells, the cellular ubiquitin ligase E6-associated protein (E6-AP) plays a central role in mediating the oncogenic functions of E6. E6-AP couples with E6 to target p53 for proteasomal degradation . This coHPV16 and HPV18 high-risk types are most frequently associated with cervical carcinomas . To detecis-acting AU-rich RNA elements (AREs) present in the 3'UTR of target transcripts [Rapid mRNA decay mediated by TTP occurs through nscripts ,34. Withnscripts ) suggesttC values were used to detect the presence of a specific mRNA , and protein expression was assayed in the presence or absence of TTP. We found no TTP-dependent changes in the amount of E6-AP protein expressed when the 3'UTR was absent Figure and exprP < 0.001). These results are consistent with recent findings demonstrating elevated TTP mRNA levels to be present in normal cervix tissue [Based on its ability to target E6-AP mRNA for rapid decay, these results suggested that the presence of TTP would be inhibitory to HPV-mediated tumorigenesis and loss of TTP would be observed in cervical cancer. To test this, TTP expression was evaluated by immunohistochemistry using human tissue arrays containing cervical tissue sections from both normal and squamous cell carcinoma Figure . In norm tissue and suggtrans-acting RNA-binding regulatory factors. Normal cellular growth is associated with rapid decay of ARE-containing mRNAs and targeted mRNA decay is an essential way of controlling their pathogenic overexpression. However, a number of observations have implicated loss of ARE-mediated post-transcriptional regulation in the neoplastic transformation of cells . Based uThrough their ability to selectively bind and control expression of many cancer-associated transcripts , ARE RNAThrough its ability to promote rapid mRNA decay, the tumor suppressor ability of TTP should reflect the ARE-containing mRNAs needed for enhanced tumor cell growth and survival. Previous findings have indicated that TTP overexpression can promote apoptosis in various cells lines . The resIn HeLa cells, repression of viral E6 and E7 oncogene expression can trigger endogenous senescence pathways -50. AlthActively growing HeLa cells maintain a dormant p53 pathway and elevated telomerase activities . The hTERT gene expression, which is the catalytic component of telomerase. Although the mechanism of E6-dependent activation of hTERT is not entirely defined in HPV-transformed cells, current observations indicate the involvement of E6-AP in targeting a regulator of hTERT expression [hTERT promoter activation. Replicative senescence in somatic cells is in part attributed to gradual loss of telomeres, while high telomerase activity is observed in a majority of cancer cells . Anotherpression ,33,57. pression , suggestCentral to the deregulation of these factors in cervical cancer is E6-AP and the results presented here are readily explained with E6-AP being a novel target of TTP-mediated post-transcriptional regulation Figure . Within Recent findings have demonstrated that loss of TTP expression is observed in a variety of tumor types ,36,44,60ZFP36) is located on 19q13.1 and does not appear to be a target of genomic loss or rearrangement in cervical cancer [The mechanisms underlying the loss of TTP expression in cervical cancer cells and tumors are largely undefined. The TTP gene , SiHa (HPV16+) and CaSki (HPV16+) cells were obtained from ATCC; HeLa Tet-Off cells were purchased from BD Clontech. Cells were maintained in DMEM supplemented with 10% fetal bovine serum ; HeLa Tet-Off cell media was supplemented with 100 \u03bcg/ml G418 (Cellgro). The Tet-responsive pTRE2hyg/TTP-Flag vector was created by cloning an N-terminal Flag epitope-tagged TTP cDNA from pcDNA3-Flag-TTP into pTRE2hyg (Clontech). HeLa Tet-Off cells were stably transfected with pTRE2hyg/TTP-Flag using Lipofectamine Plus (Invitrogen) according to the vendor's protocol. Stably transfected cells were selected in normal growth medium containing 100 \u03bcg/ml G418, 200 \u03bcg/ml hygromycin B (Invitrogen), and 2 \u03bcg/ml doxycycline (Dox) (Clontech) for 2-3 weeks. Individual clones were isolated by limiting dilution in 96-well plates. Positive HeLa-Tet-Off/TTP-Flag clones were screened by growing cells in the presence or absence of Dox (2 \u03bcg/ml) to induce expression of TTP-Flag, respectively; the absence of Dox allows for TTP-Flag expression. For stable cell maintenance the hygromycin B concentration was reduced to 100 \u03bcg/ml. Unless otherwise indicated, HeLa Tet-Off/TTP-Flag cells were grown in the absence of Dox for 48 hr to induce TTP-Flag expression. HeLa Tet-Off/TTP-Flag cells were transiently transfected with p53-responsive promoter luciferase reporter vector pp53-Luc or control vector pTA-Luc (Clontech) along with control pRL-TK renilla vector (Promega) using Lipofectamine Plus. The E6-AP coding region or 3'UTR were PCR amplified from HeLa cDNA as described . E6-AP TTP-Flag expressing adenovirus was created by cloning TTP-Flag cDNA into the shuttle vector Dual-CCM-CMV-EGFP (Vector Biolabs) that contains dual CMV promoters to drive expression of TTP-Flag and GFP. Construction of TTP-expressing adenoviral vector (AdGFP/TTP) and production of viral stocks were conducted by Vector Biolabs. Control GFP-expressing adenovirus (AdGFP) was purchased from Vector Biolabs. HeLa, SiHa and CaSki cells were infected with AdGFP or AdGFP/TTP using a MOI of 100 in serum-free DMEM for 2 hr after which FBS was added to a final concentration of 10%. 48 hr after infection, cells were harvested in SDS-PAGE lysis buffer for western blot analysis. Immunoblot analysis. Cells were lysed in SDS-PAGE lysis buffer and protein content was determined using a BCA protein assay with BSA as standard (Pierce Biotechnology). Where indicated, nuclear lysates were prepared by resuspending cells in lysis buffer containing 0.5 mM PMSF and protease inhibitor cocktail (Sigma) and incubated on ice for 10 min. Cells were centrifuged at 13000 rpm for 10 min and the nuclear pellet was washed 3 times with lysis buffer. Nuclei were lysed in RIPA buffer . Lysates (50 \u03bcg) were separated by 10% SDS-PAGE, transferred to PVDF membranes (Bio-Rad), and probed with antibodies against Flag epitope , TTP , p53 , hTERT , and E6-AP (BD Biosciences) at dilutions specified by the vendor. Blots were stripped and then probed with antibodies against \u03b2-actin or nucleoporin (BD Biosciences). Detection and quantitation of blots were performed as described [ escribed . mRNA analysis. Total RNA was extracted from cells using Trizol reagent (Invitrogen). Northern blotting was performed as previously described [32-labeled DNA probes synthesized for TTP, E6-AP and actin (Promega). cDNA synthesis and RT-PCR analysis of mRNA was accomplished as described [ escribed and probescribed . The Cell growth and senescence. Cell growth was assayed using the MTT-based cell growth determination kit (Sigma) as previously described [4 HeLa Tet-Off/TTP-Flag cells were grown in 35mm diameter dishes in the presence or absence of 2 \u03bcg/ml Dox. Twelve days later, the cells were stained at pH 6.0 with X-Gal to visualize senescence associated-\u03b2-galactosidase activity. escribed . For Fluorescence microscopy. HeLa Tet-Off/TTP-Flag cells were plated on coverslips in a 24-well plate and grown in the presence or absence of 2 \u03bcg/ml Dox. After 48 hrs, the cells were fixed in 4% paraformaldehyde for 10 min and permeabilized with 0.1% Triton X-100 in PBS for 5 min. The cells were blocked with 10% normal goat serum and 3% BSA diluted in PBST (PBS + 0.1% Tween-20) for 1 hr. Cells were incubated for 1 hr at RT with anti-p53 antibody diluted in blocking buffer. After washing, the cells were incubated with fluorescein-conjugated goat anti-mouse secondary antibody for 1 hr at RT. DAPI (Invitrogen) was used for nuclear counter-staining. Coverslips were mounted on glass slides and visualized using an Axiovert 200 inverted microscope (Zeiss). Cell morphology was examined by staining fixed and permeabilized cells with DAPI and rhodamine phalloidin (Invitrogen) according to the vendor's instructions. Telomerase activity. Telomerase activity was determined in HeLa Tet-Off/TTP-Flag lysates 48 hr after TTP induction using the PCR-based TRAP assay as previously described [ escribed . PCR escribed . Immunoprecipitations. HeLa Tet-Off/TTP-Flag (1.25 x 105 cells) were grown in 100 mm diameter dishes in the presence or absence of 2 \u03bcg/ml Dox for 48 hr. Cells were lysed in polysome lysis buffer containing 100 U/ml RNase inhibitor (Ambion) and protease inhibitor cocktail (Sigma). Cytoplasmic extracts were separated from nuclei by centrifugation at 20,000g for 30 min. 700 \u03bcl of IP buffer was added to 500 \u03bcg of lysate and immunoprecipitation of TTP-bound RNA was accomplished by incubating lysates with equal amounts (30 \u03bcg) of anti-Flag mAb or mouse IgG pre-coated to protein A/G PLUS agarose (Santa Cruz Biotechnology) overnight at 4\u00b0C. Immunoprecipitates were collected by brief centrifugation and washed 4 times with IP buffer. Total RNA was isolated using 1 ml Trizol per IP reaction and then used for cDNA synthesis [t to represent the abundance of E6-AP or GAPDH mRNA in each IP sample. ynthesis . Real-tiImmunohistochemical analysis. Immunohistochemical analysis of TTP expression was performed using cervical cancer tissue array CXC96101 (Pantomics) that contained 12 cases of normal and inflammatory tissues of cervix and 36 cases of cervical cancer graded by histology. TTP immunostaining was performed using TTP polyclonal antibody at 8 \u03bcg/ml (1:400). Standard staining protocol was performed and stained tissue sections were evaluated for intensity of staining as described [ escribed using twStatistical analysis. The data are expressed as the mean +/- SD. Student's t-test was used to determine significant differences. P-values less than 0.05 were considered significant."} +{"text": "The purpose of this study was to use an immunization protocol with Moloney sarcoma virus (MSV-M) as active immunogen against exogenous and endogenous leukaemia. The s.c. route was chosen since it offered advantages over the i.m. route: the primary sarcomas were smaller, the regression faster, there were fewer recurrences and there was good persistent immunity. Strong protection was obtained against primary leukaemias induced by Friend leukaemia virus (FLV), Moloney leukaemia virus (MLV), Rauscher leukaemia virus (RLV), Precerutti-Law leukaemia virus (PLLV/T2), and H179A leukaemia virus. It was not possible to protect against leukaemia induced by Gross leukaemia virus (GLV). With transplantable leukaemias the results varied: partial protection was observed against H110 leukaemia and R14 leukaemia (induced by X-irradiation) whilst no protection was obtained against P277 leukaemia (induced by Moloney leukaemia virus). As for spontaneous leukaemias, immunized BALB/c mice showed an increased incidence over the controls, while in F1 (Swiss x AKR) mice the incidence was similar but the latent period was shorter. Furthermore, in long-term observations the MSV-M-immunized mice showed an increased mortaltiy, which could be related to (1) new phenotypic mixtures between MSV-M and leukaemia viruses; (2) reactivation of MSV-M sarcoma-genesis with age, and (3) genotype susceptibility to MSV-M."} +{"text": "In a study involving 50 breast cancer tumours, we compared two oestrogen receptor (ER) detection methods developed by us--a microplate immunoenzymometric assay (EIA96) and an immunohistochemistry kit (HistoCIS-ER)--with the radioligand assay (RLA), the Abbott immunoenzymometric assay ER-EIA and the reverse transcriptase polymerase chain reaction technique (RT-PCR). Among the three ER protein cytosolic assays , the two EIAs showed the best agreement . At the calculated optimal cut-off values (8 and 14 fmol mg(-1) protein for EIA96 and RLA respectively), EIA96 was more sensitive than RLA , but slightly less specific . The Cox logistical regression model applied to EIA96, RLA and RT-PCR showed that EIA96 discriminated the best between ER-EIA+ and ER-EIA- samples. The RT-PCR technique and HistoCIS-ER both had a positivity-negativity concordance of 86% with EIA96."} +{"text": "Thereis currently little information as to the mechanism by which thesechannels are modulated. The effect of PAF on quasi steady-stateinwardly rectifying K+ currents (presumably of theIK1 type) of auricular, atrial and ventricular cardiomyocytes fromguinea-pig were studied. Applying the patch-clamp technique in thewhole-cell configuration, PAF (10 nM) reduced the K+currents in all three cell types. The inhibitory effect of PAFoccurred within seconds and was reversible upon wash-out. It wasalmost completely abolished by the PAF receptor antagonist BN 50730.Intracellular infusion of atrial cells with guanine5\u2032-(\u03b2-thio)diphosphate (GDPS) or pretreatment of cellswith pertussis toxin abolished the PAF dependent reduction of thecurrents. Neither extracellularly applied isoproterenol norintracellularly applied adenosine 3\u2032,5\u2032-cyclicmonophosphate (cyclic AMP) attenuated the PAF effect. Inmulticellular preparations of auricles, PAF (10 nM) inducedarrhythmias. The arrhythmogenic activity was also reduced by BN50730. The data indicate that activated PAF receptors inhibitinwardly rectifying K+ currents via a pertussistoxin sensitive G-protein without involvement of a cyclicAMP-dependent step. Since IK1 is a major component instabilizing the resting membrane potential, the observed inhibitionof this type of channel could play an important role in PAFdependent arrhythmogenesis in guinea-pig heart.Platelet-activating factor (PAF) inhibits single inwardly rectifyingK"} +{"text": "P = 0.002). 53 patients displaying stage Ib\u2013IIb cervical cancer underwent radical hysterectomy and pelvic lymphadenectomy. VEGF-C expression was significantly higher in tumours exhibiting deep stromal invasion, pelvic lymph node metastasis and lymph-vascular space involvement . Multivariate analysis revealed VEGF-C mRNA expression to be the sole independent factor influencing pelvic lymph node metastasis. Subjects demonstrating VEGF-C mRNA expression displayed significantly poorer prognoses than those lacking VEGF-C mRNA expression (P = 0.049). These findings provide evidence supporting the involvement of VEGF-C expression in the promotion of lymph node metastasis in cervical cancer. Furthermore, examination of VEGF-C expression in biopsy specimens may be beneficial in the prediction of pelvic lymph node metastasis. \u00a9 2001 Cancer Research Campaign http://www.bjcancer.comVascular endothelial growth factor-C (VEGF-C) has been implicated in lymphangiogenesis, the process of new lymphatics formation. The present study investigated VEGF-C mRNA expression in invasive cervical cancer tissue. Additionally, the association of VEGF-C mRNA with clinicopathological features was examined. VEGF-C mRNA expression was assessed by reverse transcription-polymerase chain reaction using \u03b2-action as an internal control. 75 patients presenting with invasive cervical cancer were included in the trial. VEGF-C mRNA expression was markedly higher in tumours in which pelvic lymph node metastasis was diagnosed by magnetic resonance (MR) imaging ("} +{"text": "Serum levels of cell-free interleukin-2 receptors were elevated above normal in mice bearing the IL-2R positive T-cell lymphoma Eb or its highly metastatic variant ESb. Although ESb cells expressed less IL-2R molecules than Eb cells on their cell surface, serum receptor levels were raised more quickly in ESb than in Eb tumour bearing animals. Elevated IL-2R serum levels were a sensitive tumour marker in animals bearing the aggressive variant ESb but not in animals bearing the low metastatic line Eb. Peritoneal ascites tumour-bearing animals had higher serum IL-2R levels than corresponding animals with subcutaneously growing tumours. Thus, serum IL-2R levels in tumour-bearing animals were dependent on the tumour line and influenced by the site and mode of tumour growth."} +{"text": "The preantral-early antral follicle transition is the penultimate stage of follicular development in terms of gonadotropin dependence and follicle destiny (growth versus atresia). Follicular growth during this period is tightly regulated by oocyte-granulosa-theca cell interactions. Formation of the theca cell layer is a key event that occurs during this transitional stage. Granulosal factor(s) stimulates the recruitment of theca cells from cortical stromal cells, while oocyte-derived growth differentiation factor-9 (GDF-9) is involved in the differentiation of theca cells during this early stage of follicular development. The preantral to early antral transition is most susceptible to follicular atresia. GDF-9 promotes follicular survival and growth during transition from preantral stage to early antral stage by suppressing granulosa cell apoptosis and follicular atresia. GDF-9 also enhances preantral follicle growth by up-regulating theca cell androgen production. Thecal factor(s) promotes granulosa cell proliferation and suppress granulosa cell apoptosis. Understanding the intraovarian mechanisms in the regulation of follicular growth and atresia during this stage may be of clinical significance in the selection of the best quality germ cells for assisted reproduction. In addition, since certain ovarian dysfunctions, such as polycystic ovarian syndrome and gonadotropin poor-responsiveness, are consequences of dysregulated follicle growth at this transitional stage, understanding the molecular and cellular mechanisms in the control of follicular development during the preantral-early antral transition may provide important insight into the pathophysiology and rational treatment of these conditions. The ovarian follicle, consisting of an oocyte surrounded by granulosa and theca cells, represents the basic functional unit of the ovary. Follicular growth can be classified into three phases according to their developmental stage and gonadotropin dependence -3 Fig. : 1) fol fol1,7,8The transition of the follicle from the preantral to early antral stage is the \"penultimate\" stage of development in terms of gonadotropin dependence and follicle destiny (growth versus atresia) Fig. 1)1). FolliThe role of theca cells in follicular function has received less attention compared with intensive investigation into the role of granulosa cells . Neverthi.e. insulin-like growth factor-I (IGF-I) and kit ligand (KL), increased gene expression for androgenic factors and androgen production in rat theca-interstitial cells [The origin of theca cells has been a long-standing research interest and whether the cortical or medullary stromal cells are thecal stem cells remains an unanswered question. Our recent studies with a bovine co-culture model ,23 indical cells . Using tal cells . Theca cal cells , suggestOocyte-somatic cell interaction plays a critical role in folliculogenesis, including activation of resting follicles, early growth, and terminal differentiation -31. Growe.g. caspase-8 and -9) and effector caspases (e.g. caspase-3). Although apoptosis can occur at all stages of follicular development, the early antral follicles are most susceptible to atreatogenic signals [In mammals, a single or small number of germ cell(s) will ovulate during an ovarian cycle, whereas most follicles undergo atresia by follicle cell apoptosis ,3,15, a signals ,41. In c signals . Accordiin vitro [in vitro, while the addition of recombinant GDF-9 enhances basal and FSH-induced follicular growth in rat [Deletion of GDF-9 in the oocyte results in decreased granulosa cell proliferation, abnormal oocyte growth, and failure of follicles to develop past the primary stage , demonstin vitro . We haveh in rat . There mh in rat . Although in rat , whetherIn vitro studies have shown that androgens stimulate preantral follicle growth and granulosa cell mitosis in mice [Ovarian androgens are produced by theca cells, and act via receptors (AR) localized to granulosa cells, stromal cells, and oocytes . Inactiv in mice , the tra in mice , and fol in mice .in vitro [We have recently shown that oocyte-derived GDF-9 enhances rat preantral follicle growth, and augments androgen production and CYP17A1 mRNA expression in the preantral follicles, whereas down-regulation of GDF-9 suppressed these responses . The spein vitro . The nonin vitro , indicatEvidence indicates that steroidal and nonsteroidal factors produced by granulosa and theca cells influence proliferation and differentiation of both cell types on opposite sides of a basement membrane during folliculogenesis ,15,55-58Our previous studies suggest that theca cell-derived soluble growth factors promote granulosa cell proliferation and suppThe preantral to early antral transition is the penultimate stage of follicular development in terms of gonadotropin dependence and follicle destiny (growth versus atresia). Follicular growth during the preantral-early antral transition is tightly regulated by intra-ovarian oocyte-granulosa-theca cell interactions Fig. . FormatiGDF-9: growth differentiation factor-9; PCOS: polycystic ovarian syndrome; LHR: LH receptor; IGF-I: insulin-like growth factor-I; KL: kit ligand; TGF-\u03b2: transforming growth factor-\u03b2; CYP17A1: 17\u03b1-hydroxylase/17,20 lyase; caspases: cysteine aspartate-specific proteases; BMP-15: Bone morphogenetic protein-15; ALK-5: activin-like receptor kinase-5; BMPRII: BMP receptor type II; AR: androgen receptor; DHT: 5\u03b1-dihydrotestosterone; EGF: epidermal growth factor; KGF: keratinocyte growth factor: HGF: hepatocyte growth factor; GC: granulosa cell; TC: theca cell; SC: stromal cell; FSHR: FSH receptor.The authors declare that they have no competing interests.MO and KT participated in drafting the full manuscript and creating figures. BKT and FK participated in substantial contribution to conception and revising it critically for important intellectual content. All authors read and approved the final manuscript."} +{"text": "MSI-H and K-ras mutation in Egyptians under age 40 were unusual , and schistosomiasis was associated with MSI and K-ras mutation. Cluster analysis identified 2 groups: predominantly young men with poorly differentiated mucinous and signet-ring cell colorectal carcinoma lacking K-ras mutation; older patients who had well- or moderately differentiated adenocarcinoma often with MSI-H, K-ras mutation and schistosomiasis. Our findings show that the molecular pathology of colorectal cancer in older as well as younger Egyptians has unique differences from Western patients, and schistosomiasis influences the molecular pathogenesis of some tumours. \u00a9 2001 Cancer Research Campaignhttp://www.bjcancer.comColorectal carcinoma is uncommon in Egypt, but a high proportion of cases occurs before age 40 years and in the rectum. We compared the molecular pathology of 59 representative Egyptian patients aged 10\u201372 to Western patients with sporadic, young-onset, or hereditary non-polyposis colorectal cancer syndrome (HNPCC)-associated carcinoma and found significant differences. Most Egyptian cancers were rectal (51%) and poorly differentiated (58%). High levels of microsatellite instability (MSI-H) were frequent (37%) and attributable in some cases (36%) to methylation of the promoter of the hMLH1 mismatch repair gene, but no MSI-H cancer had loss of hMSH2 mismatch repair gene product of the type seen with germline hMSH2 mutation in HNPCC. K-ras mutation was uncommon (11%). In subset analyses, high frequencies of MSI-H in rectal carcinomas (36%) and"} +{"text": "Functional homologous and non-homologous clusters of MIR genes that co-regulate target mRNA transcripts have been identified in plants MIRNA genes, in contrast to animal miRNAs, which are frequently clustered.MicroRNAs (miRNAs) are endogenous single-stranded small RNAs that regulate the expression of specific mRNAs involved in diverse biological processes. In plants, miRNAs are generally encoded as a single species in independent transcriptional units, referred to as Arabidopsis) and characterized miRNA clusters containing two to eight miRNA species. These clusters usually encode miRNAs of the same family and certain share a common evolutionary origin across monocot and dicot lineages. In addition, we identified miRNA clusters harboring miRNAs with unrelated sequences that are usually not evolutionarily conserved. Strikingly, non-homologous miRNAs from the same cluster were predicted to target transcripts encoding related proteins. At least four Arabidopsis non-homologous clusters were expressed as single transcriptional units. Overexpression of one of these polycistronic precursors, producing Ath-miR859 and Ath-miR774, led to the DCL1-dependent accumulation of both miRNAs and down-regulation of their different mRNA targets encoding F-box proteins.We performed a comparative genomic analysis in three model plants (rice, poplar and MIRNA precursors as a new molecular tool for plant biologists to simultaneously control the expression of different genes.In addition to polycistronic precursors carrying related miRNAs, plants also contain precursors allowing coordinated expression of non-homologous miRNAs to co-regulate functionally related target transcripts. This mechanism paves the way for using polycistronic MIRNA precursors that are able to fold-back into a stable secondary structure (stem loop or hairpin). miRNAs act in many developmental processes as well as environmental and pathogenic responses carbodiimide hydrochloride)-mediated cross-linking step was then performed as described [32P end-labeled oligonucleotides (20 pmoles) complementary to miRNAs, and at the same time with an end-labelled oligonucleotide U6 RNA probe as loading control.Tissues were frozen in liquid nitrogen, ground to a fine powder with a mortar and pestle, and then homogenized in TRI-Reagentescribed . Blots wArabidopsis non-homologous pri-MIRNAs were submitted to GenBank . For Ath-MIR842-846 loci, no amplification was obtained despite testing eight different primer combinations, even on genomic DNA (data not shown). A control without reverse transcriptase was systematically included.Total RNAs were extracted using the total RNA Isolation kit . cDNA was synthesized by reverse transcription of 1.5 \u03bcg of total RNAs using the SuperScript II Reverse Transcriptase and (T)16 A/G/C oligonucleotides. Primer pairs used for RT-PCR are listed in Additional data file 5. Specificity of amplification was checked by cloning and sequencing of PCR amplicons, and ESTs corresponding to \u00ae realplex real-time PCR system using FastStart Universal SYBR Green Master Mix (Rox) from Roche Applied Science . Technical triplicates were done for each datapoint, and two independent biological replicates (per condition and/or transgenic line) were assayed. Normalization was done with averaged reference genes TAIR:At1g13320, TAIR:At4g26410, and TAIR:At5g15710 [Real-time RT-PCR was performed on an Eppendorf Mastercyclert5g15710 , which wt5g15710 .MIRNA Ath-MIR859-774 was amplified by RT-PCR from seedling cDNA and cloned into pCR8\u00ae/GW/TOPO\u00ae TA Cloning\u00ae vector . The construct was then transferred to the destination vector pEarlyGate103 [A. thaliana plants by floral dipping [\u00ae solution successively at 12, 14, and 16 days after germination. Basta-resistant plantlets were then tested for transgene expression by real time RT-PCR as described above. Since amplification across the successive hairpin regions of the Ath-MIR859-774 pri-MIRNA was not efficient and quantitative enough for real time RT-PCR analyses, a GFP mRNA present 3' of the pEarlyGate103 vector cloning site, for which efficient and specific primers were available , was used as a 3' tag to analyze transgene expression.Firstly, pri- dipping . TransgeArabidopsis Small RNA Project; Ath: Arabidopsis thaliana; DCL1: Dicer-like enzyme 1; EST: expressed sequence tag; JR/MBP: Jacalin repeat/Myrosinase binding protein; miRNA: microRNA; Osa: Oryza sativa; pre-MIRNA: miRNA precursor; pri-MIRNA: miRNA primary transcript; Ptc: Populus trichocarpa; RGL: Repressor of gibberellic acid requiring (GA1)-LIKE; RISC: RNA-induced silencing complex; TAIR: The Arabidopsis Information Resource.ASRP: FM carried out the molecular genetic studies. AB conceived the study and carried out the bioinformatic analyses. MC drafted the manuscript and participated in its coordination. FF conceived the study, designed experiments and wrote the manuscript. All authors read and approved the final manuscript.Arabidopsis, rice and poplar genomes (Additional data file Arabidopsis, rice and poplar genomes (Additional data file Arabidopsis non-homologous polycistronic miRNA clusters (Additional data file The following additional data are available with the online version of this paper: a table listing the conserved and non-conserved miRNAs in Arabidopsis, rice and poplar genomes.Conserved and non-conserved miRNAs in Click here for fileArabidopsis, rice and poplar genomes.Clustered miRNAs in Click here for fileMIR395a-g and osa-MIR395h-l,y putative polycistronic homologous miRNA clusters. Figure S2: secondary structures of poplar and rice putative polycistronic non-homologous miRNA clusters. Figure S3: secondary structures of four Arabidopsis non-homologous polycistronic miRNA clusters: Ath-MIR397b-857, ath-MIR842-846, ath-MIR850-863, and ath-MIR851-771. Figure S4: small RNA hits in Arabidopsis polycistronic non-homologous miRNA clusters based on the 'Genome View' browser in the ASRP database.Figure S1: secondary structures of the rice osa-Click here for fileArabidopsis non-homologous polycistronic miRNA clusters.Targets predicted for Click here for filePrimers used in this study.Click here for file"} +{"text": "Creatine Kinases (CK) catalyze the reversible transfer of high-energy phosphate groups between ATP and phosphocreatine, thereby playing a storage and distribution role in cellular energetics. Brain-type CK (CK-B) deficiency is coupled to loss of function in neural cell circuits, altered bone-remodeling by osteoclasts and complement-mediated phagocytotic activity of macrophages, processes sharing dependency on actomyosin dynamics.Here, we provide evidence for direct coupling between CK-B and actomyosin activities in cortical microdomains of astrocytes and fibroblasts during spreading and migration. CK-B transiently accumulates in membrane ruffles and ablation of CK-B activity affects spreading and migration performance. Complementation experiments in CK-B-deficient fibroblasts, using new strategies to force protein relocalization from cytosol to cortical sites at membranes, confirmed the contribution of compartmentalized CK-B to cell morphogenetic dynamics.on-site availability of ATP generated by CK-B.Our results provide evidence that local cytoskeletal dynamics during cell motility is coupled to Distinctly different processes like protrusion and retraction of narrow surface projections by astrocytes, development of invadopodia by malignant cancer cells, pathogen binding and formation of phagocytic cups by macrophages, and the generation of protrusive structures at leading edges of migrating or extending cells share that they are driven by extensive local remodeling of the actin cytoskeleton For the dynamic polymerization and filament-bundling of the actin cytoskeleton and for the control of movement and force generation by associated non-muscle myosin ATPases appropriate spatiotemporal regulation of ATP supply is needed The intensively fluctuating rate of ATP turnover in mammalian cells with a high energy demand requires a robust system for supply, storage and distribution of energy in the form of high-energy phosphate compounds 2+-ATPases preferential access to ATP for calcium sequestration As shown by experiments in muscle tissue of AK-knockout mouse models, AK activity has an important role in the control of energetic economy Here, we provide evidence for a direct mechanistic connection between CK-B mediated phosphotransfer activity and local actin remodeling during cell migration and morphological changes. We show that the subcellular distribution of CK-B partially overlaps with that of dynamic actin in protrusion-active cortical areas of astrocytes and fibroblasts, but not with static F-actin of stress fibers in these cells. CK-B activity in astrocytes and fibroblasts directly facilitates actomyosin-driven motility, as demonstrated by cell spreading and migration assays. Additionally, by using deliberate positional swapping of CK-B from a cytosolic to membrane-bound location, we show that spatial confinement of CK-B activity controls local actin dynamics and therewith determines morphology and migration behavior.in vivo context- are cells with highly dynamic surface extensions. We applied Western blot analysis to confirm that CK-B protein expression is also high in cultured primary astrocytes, in vitro. As anticipated, astrocytes derived from wild type (WT) mice gave a prominent signal, while control astrocytes derived from CK-B deficient mice Within the brain, CK-B is expressed at high levels in astrocytes To study the possible coupling between CK-B accumulation and local actin dynamics in more detail, we analyzed astrocyte spreading in presence and absence of cytochalasin D, an inhibitor of actin polymerization. We took cell spreading as this is a process that is partially analogous to cell migration and dependent on actomyosin dynamics To study the functional contribution of CK-B to astrocyte spreading, we performed cell spreading assays using WT and CK-B deficient (\u2212/\u2212) astrocytes. As shown in Since spreading and migration events have analogous protrusive processes in common, we wondered whether astrocyte migration was also affected by absence or presence of CK-B. Using the barrier migration assay Cell shape changes require highly coordinated activities of actin machinery that is localized (close) to the plasma membrane Activity of the ATP-generating enzyme AK1, which is expressed in MEF cells, is also linked to control of cell migration Migration analysis domain. Upon addition of Rapalog , inducible translocation of a significant fraction of cytosolic distributed CK-B and CK-BC283S to membranes occurred, as verified by immunofluorescent staining CK-B to membranes, distances increased from 344\u00b115 to 434\u00b119 \u00b5m over the period measured . Addition of Rapalog did not alter locomotion of CK-BC283S expressing cells as archetypal motile cells for study of the localized role of CK-B in cell dynamics. Our data on recruitment of CK-B from the cytosolic pool and it's transient co-accumulation in areas with cortical F-actin but not with the less dynamic F-actin in stress fibers in both cell types support the idea that CK-B activity is generally most needed at sites where the actin cytoskeleton is highly dynamic. This idea was already proposed for the accumulation of CK-B in nascent phagosomes as well The direct functional link between CK-B activity and cell shape changes was clarified by our cell spreading and migration assays, in which ablation of CK-B slowed down these processes in both astrocytes and MEFs. Overall, findings between astrocytes and MEFs were comparable, only CK-B effects on spreading were not reproduced in MEFs. Cell spreading is a complex process consisting of substrate adherence and passive-descent events, later followed by active probing of the cellular environment C283S in the positional swapping/tagging experiments demonstrated that the contribution of CK-B to enhanced cell migration capacity did not depend merely on its structural interference, but that its catalytic ATP-generating activity is required.Importantly, the experiments with pharmacological inhibition with cyclocreatine or substitution of native CK-B by the catalytically dead CK-BWe propose that cells use CK-B to balance ATP/ADP ratios and buffer temporary demands for ATP in specialized structures with highly dynamic actin, such as lamellipodia and filopodia, to sustain optimal migration rates. The early observation that cyclocreatine was able to inhibit motility in tumor cells One most conspicuous finding in our studies was that manipulation of CK-B location had an effect on both migratory capacity and lamellipodial morphology in MEFs. Interestingly, targeting CK-B to membranes changed the lamellipodial dimensions. Formation of lamellipodia is largely dependent on branched actin polymerization at the cell's periphery, which requires the availability of free ATP-loaded actin monomers By using the Rapalog heterodimerizer system, we were able to manipulate the localization of CK-B without altering the total cellular levels of CK-B, thus excluding effects of variation in activity levels or in genetic or metabolic background between cells in our pool. As rapamycin is reported to inhibit F-actin reorganization Besides CK-B, also other enzymes have been reported to act as local distributors of ATP at actin-rich structures. For example, translocation of adenylate kinase (AK1) to focal contacts sites in MEFs induced migratory capacity of these cells In summary, data presented here strongly suggest that locally generated ATP is an important regulator for actin-based cytoskeletal dynamics involved in cell extension and motility and that CK-B is a controlling enzyme in the compartmentalization of ATP availability. This model also provides a plausible explanation for our earlier findings on synaptic function and development of neural cell networks in CK-B knockout mice Primary cultures of astrocytes were established from brains of embryonic wild type (C57BL/6\u00d7129Ola) or CK-B deficient mice C283S sequence in vector pLZRS-IRES-Zeo. Constructs and targeting principle are depicted in cDNA encoding CK-B, myristoylation tagged CK-B (MYR-CK-B) and their YFP controls were cloned into the EcoR1/Xho1 sites of the retroviral transfection vector pLZRS-IRES-Zeo 2HPO4, 2.8 mM KH2PO4, 0.05% Triton X-100, 0.3 mM DTT) containing protease inhibitor cocktail (Roche). After incubation on ice (20 min) the cell lysates were centrifuged for 10 min at 13000 rpm and supernatants were collected. MEFs were lysed directly in sample buffer or in NP-40 Buffer and protein content was determined using the Bradford method. Samples were resolved by 10% SDS-PAGE and blotted onto nitrocellulose membranes. Subsequently, the membranes were blocked with PBS containing 5% non-fat milk and blots were probed with anti-CK-B Astrocyte cell lysates were prepared in lysisbuffer . Subsequently, cells were permeabilized with 0.1% Triton X-100 or 0.1% saponin and incubated for 20 min in PBS containing 1% bovine serum albumin (BSA). Incubation with primary and secondary antibodies was done for 1 h and in between incubations the cells were washes thrice with PBS. Primary antibodies used were polyclonal anti-GFAP , monoclonal anti-CK-B and polyclonal anti-CK-B (1\u22362000). Secondary goat-anti-mouse and goat-anti-rabbit conjugated to Alexa 488 or Alexa 568 . F-actin was visualized by phalloidin conjugated to Alexa 660 or TexasRed (Molecular Probes). Samples were dehydrated in 70% and 100% ethanol, air dried and mounted onto microscope slides using mowiol. Samples were analyzed on a Biorad MRC1024 laser scanning confocal microscope using an oil immersion 60\u00d7 objective or on an Axiovert 35M fluorescence microscope (Carl Zeiss) using 63\u00d7 and 100\u00d7 oil immersion objectives.Cells were grown on glass cover slips, washed twice in PBS and fixed with 2% paraformaldehyde in PHEM buffer and divided by the number of cells to calculate the area per cell. Five random fields were analyzed in each of three independent experiments. For every experiment the ratio between knockout and wild type was calculated (wildtype set at 100%).Glass cover slips were coated with laminin (40 \u00b5g/ml) or fibronectin (50 \u00b5g/ml) for 2 hours at 37\u00b0C. Cells were collected by centrifugation, washed in DMEM/1% BSA (fatty acid free) and incubated in DMEM/1% BSA for 20 minutes at 37\u00b0C before seeding onto coated cover slips. After 30 minutes spreading on coated cover slips, cells were washed once with PBS and fixed in 2% paraformaldehyde. Cells were permeabilized and stained for CK-B and TexasRed conjugated phalloidin (Molecular Probes). For every cover slip 5\u20139 random fields were imaged with a Biorad Confocal Microscope MRC1024 using a 10\u00d7 objective. The total surface occupied by cells was determined by ImageJ software were sonificated and coated by incubation in carbonate buffer (pH 9.6) with fibronectin (100 \u00b5g/ml) for 2 hours. The beads were washed twice in PBS and resuspended in serum free DMEM containing cytochalasin D or DMSO as control before seeding onto cultured astrocytes. After incubation with FN-beads for 30 min at 37\u00b0C/5% COProliferation rates and apoptosis of cyclocreatine-treated and control cells were determined using the WST-1 proliferation assay (Roche) and the Apo-ONE Homogeneous Caspase 3/7 Assay (Promega) according to manufacturers' protocols. MEFs were seeded at 5000 and 10000 cells per well for proliferation and apoptosis assays respectively. Proliferation was measured for time points op to 72 h. Apoptosis was measured in semi-confluent MEFs treated with cyclocreatine for 24 h, staurosporin was used as positive control.2, 1% NP-40, 1 mM PMSF, 1\u00d7 protease inhibitor cocktail). Activity was normalized to CK-B content by performing quantitative western blotting in the same samples.CK activity was determined by enzyme coupled reactions using the Liquid NAC activated UV test according to manufactures' protocol. Cell lysates were prepared in NP-40 lysis buffer used for further analysis. Rapalog-treated and untreated cells were analyzed for subcellular positioning of CK-B or CK-BC283S and cell migration was monitored directly after Rapalog addition.Translocation of CK-B from the cytosolic pool to cellular membranes within the same cell was done using the dimerization strategy based on the heterodimeration of FKBP and FRB protein domains by Rapalog Data are presented as mean\u00b1SEM of at least three independent experiments. Groups were compared with Student's t-test for single values, one-sample t-test for relative values and with Ratio t-test for ratio-values and considered significant different when p<0.05.Figure S1Cyclocreatine does not alter proliferation and apoptosis. A) Proliferation of MEF-CK-B (left) and MEF-MYR-CK-B (right) cells with (red) and without (black) cCr (5 mM) treatment. Relative proliferation rates, taking non-treated cells as control, are shown of three independent experiments. (B) Apoptosis of MEF-CK-B and MEF-MYR-CK-B cells cultured without (open bars) and with (red bars) cCr (5 mM). Non-treated cells were set as 100% to compare cCr effects.(3.90 MB TIF)Click here for additional data file.Figure S2Morphology measurements of migrating MEFs. In the top panel, the analysis of migration morphology is illustrated. The number of lamellipodia per cell, the lamellipodium dimensions and tail length was measured. Lower panels show high magnification images of migration fronts of complemented MEFs and correspond to (2.72 MB TIF)Click here for additional data file.Figure S3MEF-MYR-CK-B cells spread out faster than MEF-CK-B cells. Quantification of MEF spreading on FN for 30 min, showing that expression of MYR-CK-B facilitates cell spreading. * p<0.05.(0.68 MB TIF)Click here for additional data file.Figure S4Rapalog-induced membrane localization of CK-B and CK-BC238S in MEFs. (A) MEF-BAK\u2212/\u2212 cells stably expressing MYR-FKBP were retrovirally transduced with FRB-CK-B (upper panels) or CK-BC283S (lower panels) and stained for CK-B. Rapalog treatment resulted in translocation of (a fraction of) CK-B and CK-BC283S to cellular membranes. Bar, 10 \u00b5m (B) High magnification images of migration fronts are shown, corresponding to (6.71 MB TIF)Click here for additional data file.Video S1CK-B mediates astrocyte migration. WT (upper panels) and CK-B\u2212/\u2212 (lower panels) astrocytes migrating along laminin for 48 h. Migration assays were performed without (left panels) and with (right panels) cyclocreatine treatment. Both CK-B knockout and inhibition strongly decreased migratory behavior of astrocytes. (9.22 MB MOV)Click here for additional data file.Video S2CK-B expression and localization affects MEF migration. YFP/MYR-YFP complemented (upper panels) and CK-B/MYR-CK-B complemented MEFs migration along FN for 24 h. Migration assays of non-targeted (left panels) and membrane-targeted (right panels) cells showed that CK-B induced migration and additionally that MEF-MYR-CK-B were the most motile. (4.76 MB MOV)Click here for additional data file.Video S3Cyclocreatine selectively inhibits CK-B-induced migration. MEF-YFP, MEF-CK-B, MEF-MYR-CK-B and MEK-AK1 cells were followed during migration along FN. In contrast to YFP and AK1 complemented MEFs, MEF-CK-B and MEF-MYR-CK-B migration was inhibited by cCr and a strong morphological effect was observed. (7.47 MB MOV)Click here for additional data file.Video S4Repositioning of CK-B to cellular membranes induces migration. MEF-MYR-FKBP cells transfected with FRB-CK-B (upper panels) or FRB-CK-BC283s (lower panels) were allowed to migrate along FN without (left panels) and with Rapalog (right panels). Rapalog-mediated targeting of CK-B to membranes, but not CK-BC283s membrane targeting, stimulated migration. (4.78 MB MOV)Click here for additional data file."} +{"text": "The protein components of mature skeletal muscle have largely been characterized, but the mechanics and sequence of their assembly during normal development remain an active field of study. Chaperone proteins specific to sarcomeric myosins have been shown to be necessary in zebrafish and invertebrates for proper muscle assembly and function.Xenopus tropicalis mutation dicky ticker results in disrupted skeletal muscle myofibrillogenesis, paralysis, and lack of heartbeat, and maps to a missense mutation in the muscle-specific chaperone unc45b. Unc45b is known to be required for folding the head domains of myosin heavy chains, and mutant embryos fail to incorporate muscle myosin into sarcomeres. Mutants also show delayed polymerization of \u03b1-actinin-rich Z-bodies into the Z-disks that flank the myosin-containing A-band.The dicky ticker phenotype confirms that a requirement for myosin-specific chaperones is conserved in tetrapod sarcomerogenesis, and also suggests a novel role for myosin chaperone function in Z-body maturation.The The sarcomere is the fundamental unit of the muscle myofibril, and mediates ATP-dependent contraction and relaxation. While the components of sarcomeres are well understood, the mechanics and sequence of their assembly remain under debate. A key early process in myofibrillogenesis is the polymerization of \u03b1-actinin-rich Z-bodies into the Z-discs that flank the myosin-containing A-band. In a mature vertebrate sarcomere, Z-discs are held in register with the central M-band by titin filaments tightly associated with filamentous actin ,2.uncoordinated nematode mutation .2O and 5 ng injected into both blastomeres at the two-cell stage.A translation-blocking AMO (ATG-MO 5'-CCTTTAGCTGCACTGGGTCTTCCAT-3') and a splice-blocking AMO spanning exon-intron boundary 6 (splice-MO 5'-AGCACACATTATTCTTACCCAGTAC-3') were obtained from GeneTools LLC . GeneTools standard control AMO was used for control injections. All were diluted in nuclease free Hunc45b (1444 - 2661 bp) cloned into pCRII-TOPO, linearised with HincII and transcribed with Sp6.WISH was performed according to Harland et al. . A 181 b\u00ae fluorophore conjugated secondary antibodies . F-actin was stained with Alexa\u00ae 543 phalloidin . Western blots were performed with anti-MyHC F59 [Embryos were staged according to Nieuwkoop & Faber (1967), then fixed in 4% paraformaldehyde. Antibodies used were as follows: MyHC (MHC) A4.1025 and TitiMyHC F59 , obtainein situ hybridization.AMO: antisense morpholino oligonucleotide; Arg: Arginine; Cys: Cysteine; MyHC: myosin heavy chain; SNP: Single Nucleotide Polymorphism; SSLP: Simple Sequence Length Polymorphism; WISH: whole mount TG & LZ conceived the project, designed the experiments and wrote the paper. TG performed the experiments. Both authors read and approved the final manuscript.dit embryos lack heartbeat. Beating heart at stage 40 in wild type (top) is not seen in dit (bottom).Click here for filedit tadpoles are paralyzed. Wild type tadpoles at stage 40 are motile (top); dit tadpoles (bottom) are paralyzed.Click here for filedit tailsMuscle structure is disrupted in . (A) Birefringence of polarized light in stage 43 wild type tail. (B) Birefringence is greatly reduced in dit tails of the same stage. (C) Phalloidin staining of stage 43 wild type tail muscle showing orderly myofibril structure. (D) Phalloidin staining in dit embryo tails shows disorganized myofibrils.Click here for filedit embryos with MyHC F59 antibodyWestern blot analysis of wild type and . Presence of MyHC as detected by the F59 antibody is reduced in dit compared to wild type embryos at both stages 40 and 43.Click here for file"} +{"text": "However, molecular events mediating PKC-dependent disruption of epithelial cell-cell contact remain poorly understood. In the present study we investigate mechanisms by which PKC activation induces disassembly of tight junctions (TJs) and adherens junctions (AJs) in a model pancreatic epithelium.Disruption of epithelial cell-cell adhesions represents an early and important stage in tumor metastasis. This process can be modeled Exposure of HPAF-II human pancreatic adenocarcinoma cell monolayers to either OI-V or 12-O-tetradecanoylphorbol-13-acetate caused rapid disruption and internalization of AJs and TJs. Activity of classical PKC isoenzymes was responsible for the loss of cell-cell contacts which was accompanied by cell rounding, phosphorylation and relocalization of the F-actin motor nonmuscle myosin (NM) II. The OI-V-induced disruption of AJs and TJs was prevented by either pharmacological inhibition of NM II with blebbistatin or by siRNA-mediated downregulation of NM IIA. Furthermore, AJ/TJ disassembly was attenuated by inhibition of Rho-associated kinase (ROCK) II, but was insensitive to blockage of MLCK, calmodulin, ERK1/2, caspases and RhoA GTPase.Our data suggest that stimulation of PKC disrupts epithelial apical junctions via ROCK-II dependent activation of NM II, which increases contractility of perijunctional actin filaments. This mechanism is likely to be important for cancer cell dissociation and tumor metastasis. Progression and dissemination of epithelial tumors is accompanied by a loss of morphological features of epithelial cells and acquisition of mesenchymal cell phenotype known as epithelial to mesenchymal transition (EMT) ,2. WeakeDisruption of TJs and AJs occurs at the early stage of EMT and has two major functional consequences in tumor cells. One is the increase in cell proliferation, and another is enhanced cell motility ,4. The fin vitro by exposing epithelial cells to growth factors or chemical tumor promoters [Disruption of epithelial junctions during EMT is commonly modeled romoters ,14. Amonromoters ,16. Thesromoters ,18. BecaA large body of evidence indicates that scattering/invasiveness of epithelial cells induced by PKC-targeting tumor promoters involves disassembly of intercellular junctions. Indeed, 12-O-tetradecanoylphorbol-13-acetate (TPA) was shown to disrupt AJs in Madin-Darby canine kidney (MDCK) cells -21, mousPKC is a powerful regulator of the actin cytoskeleton in a variety of cells , and phoThe aim of this study was to investigate the role of NM II in the disassembly of epithelial apical junctions caused by PKC-targeting tumor promoters, which mimic the disruption of epithelial cell-cell adhesions during EMT and tumor metastasis. We rationalized that appropriate model cell line for this study should fulfill the following criteria: 1) to be a human tumor cell line; 2) to have well-developed TJs and AJs; 3) to readily disassemble their junctions after exposure to PKC-activating tumor-promoters. However, MDCK and LLC-PK1 cell lines, which are widely used to study phorbol ester-induced junctional disruption, do not fulfill the first criteria since they are neither human, nor cancer cells. On the other hand, well characterized human colonic carcinoma cell lines, such as T84, Caco-2, and HT-29 do not respond to PKC activation by junctional disassembly -48. To o2) transepithelial electrical resistance (TEER) developed confluent cell monolayers with high and AJ (E-cadherin and \u03b2-catenin) proteins from the areas of cell-cell contacts and their accumulation in cytosolic dot-like structures Figure , arrows.Next we sought to elucidate which intracellular signal initiates OI-V-dependent junctional disassembly. While teleocidins and phorbol esters primarily signal through activation of PKC, several other target proteins, such as protein kinase D, chimaerins, and Munc 13 have been recently identified . TherefoTo address these questions, we first investigated which PKC isoforms are expressed in HPAF-II cells. RT-PCR and immunoblotting analyses revealed the presence of multiple isoforms including three classical and four novel isoenzymes on either one (Ser 19) or two (Thr18/Ser19) residues ,63. By u2 (98%) and from 1046 \u00b1 20 to 47 \u00b1 3 Ohm \u00d7 cm2 (95%) in vehicle- and blebbistatin-treated cell monolayers respectively. Together these data suggest that NM II plays an important role in the disruption of AJ and TJ structure caused by PKC activation in pancreatic epithelium.To obtain definitive evidence for the role of NM II in OI-V-induced junctional disassembly we inactivated NM II by using either the pharmacological inhibitor, blebbistatin or siRNASince PKC does not directly phosphorylate RMLC at Ser19 and Thr18 to stimulate actomyosin contractility ,66 theseTo elucidate whether ROCK II plays a unique role in PKC-dependent disruption of epithelial junctions, we next tested the involvement of alternative signaling pathways previously implicated in either NM II activation, or PKC-dependent disassembly of cell-cell adhesions ,42,43,69Clostridium botulinum C3 exoenzyme [Since ROCK represents the major downstream effector for Rho small GTPase ,71, we ixoenzyme and the in vitro model to study disruption of epithelial cell-cell adhesions during tumor progression/metastasis.Loss of epithelial cell-cell adhesions upon exposure to PKC-activating carcinogens is commonly used to model scattering of epithelial cells during tumor metastasis ,22,25. IExposure of HPAF-II cell monolayers to OI-V or TPA induced rapid opening of the paracellular barrier Figure and disaA key finding of this study is a critical role of NM II in disassembly of AJs and TJs upon PKC activation in HPAF-II epithelial cells. It should be noted that the involvement of actomyosin contractility in PKC-dependent disruption of cell-cell adhesions has been addressed in previous publication, which yielded conflicting results. Thus, the increased F-actin tension/contraction has been implicated in phorbol-ester-induced disruption of endothelial junctions in one , but notin vitro data, we hypothesize that stimulation of actomyosin contractility can also be involved in the loss of epithelial cell-cell contacts during metastatic scattering of tumor cells in vivo.Our study provides the first direct evidence implicating NM II activity in the disruption of epithelial apical junctions by PKC-activating tumor promoters. Such activation of NM II is likely to serve as a trigger for junctional disassembly by either breaking adhesive contacts formed by transmembrane junctional proteins, or by destabilizing perijunctional F-actin bundles. This may activate endocytosis of AJ/TJ proteins, thus leading to complete disintegration of apical junctions ,25. AlthAlthough PKC was shown to directly phosphorylate RMLC at Ser1/2 residues ,66, suchInhibition of NM II and ROCK while substantially attenuating OI-V-induced disintegration of AJ/TJ structure either did not affect, or only modestly attenuated the decrease in TEER Figures &7. ThisOne intriguing findings of this study is that the signaling cascade, which is triggered by PKC activation and mediated by ROCK-II does not involve RhoA GTPase Figure . This imRecent studies have unraveled a complexity of mechanisms regulating ROCK activity which also involves Rho-independent activatory modes. For example, a Rho-independent cleavage of ROCK, that renders this enzyme constitutively active, was reported by several groups -91. PartThis study dissected critical intracellular events which are involved in disassembly of epithelial apical junctions stimulated by the tumor promoter, octylindolactam V. These events involve activation of classical PKC isoforms, which bypasses RhoA and signals directly through ROCK-II to activate NM II. Activation of NM II stimulates reorganization/contractility of perijunctional actomyosin ring, which drives the disassembly of epithelial AJs and TJs. Similar mechanisms may contribute to the disruption of cell-cell adhesions during tumor progression and metastasis.The following primary polyclonal (pAb) and monoclonal (mAb) antibodies were used to detect junctional, cytoskeletal and signaling proteins: anti-occludin, ZO-1, E-cadherin, and \u03b2-catenin mAbs ; anti-NM IIA pAb ; anti-PKC\u03b1 mAb, anti-PKC \u03b2I, anti-ROCK-I (H-85), anti-ROCK-II (C-20 and H-85), and anti-RMLC pAbs ; anti-PKC\u03b4 pAb anti-mono, and di-phosphorylated RMLC pAbs ; anti-actin pAb . Alexa-488 or Alexa-568 dye conjugated donkey anti-rabbit and goat anti-mouse secondary antibodies and Alexa-labeled phalloidin were obtained from Molecular Probes ; horseradish peroxidase-conjugated goat anti-rabbit and anti-mouse secondary antibodies were obtained from Jackson Immunoresearch Laboratories . (-)-7-Octylindolactam V was obtained from Biomol International ; TPA, S(-)-Blebbistatin, W-7, ML-7 and rottlerin were purchased from Sigma; G\u00f6 6976, Y-27632, H-1152, and U0126 were purchased from EMD Biosciences ; GF-109203X was obtained from Axxora LLC ; a cell permeable Rho inhibitor was obtain from Cytoskeleton Inc. . All other reagents were of the highest analytical grade and obtained from Sigma.HPAF-II human pancreatic epithelial cells were grown in RPMI medium supplemented with 10% fetal bovine serum, 10 mM HEPES, 1 mM sodium pyruvate, 2 mM L-glutamine, 100 IU/ml penicillin and 100 \u03bcg/ml streptomycin, pH 7.4. For immunofluorescence labeling experiments epithelial cells were grown for 6\u201310 days on either collagen-coated permeable polycarbonate Transwell filters with 0.4 \u03bcm pore size or on collagen-coated coverslips. For biochemical experiments cells were cultured on either Transwell filters, or 6-well plastic plates. Junctional disassembly in HPAF-II cell monolayers was induced by incubation with either OI-V or TPA for indicated periods of time. For pharmacological inhibition experiments, cells were preincubated with inhibitors for 30 min followed by exposure to OI-V in the presence of inhibitors. Appropriate vehicle (DMSO) was added to all control samples.Cell monolayers were fixed/permeabilized in 100% methanol or 100% ethanol (-20\u00b0C for 20 min) and double-immunolabeled according to previously described protocols ,35,40. FCells were homogenized in a RIPA lysis buffer , containing a protease inhibitor cocktail and phosphatase inhibitor cocktails 1 and 2 . Lysates were cleared by centrifugation , diluted with 2\u00d7 SDS sample buffer and boiled. SDS-polyacrylamide gel electrophoresis and immunoblotting were conducted by standard protocols with equal amount of total protein (10 or 20 \u03bcg) per lane. Results shown are representative immunoblots of three independent experiments. Protein expression was quantified by densitometric analysis of at least three Western blot images each representing independent experiment, using Scion Image and UN-SCAN-IT digitizing software .2, 10 mM Hepes, pH 7.4) containing protease and phosphatase inhibitor cocktails and nitrogen cavitated . Nuclei were removed by centrifugation . Membranes were pelleted by ultracentrifugation of the post nuclear supernatants at 100,000 \u00d7 g for 60 min. Supernatants (cytosolic fraction) were collected and the pellets were resuspended in an equivalent volume of Hanks balanced salt solution containing 1% n-octylglucoside by sonication on ice. Equal volumes of the cytosolic and membrane fractions were subjected to immunoblotting analysis as described above.Cytosolic and membrane fractions of control or OI-V treated HPAF-II cells were prepared as described previously . BrieflyTotal RNA was isolated from confluent HPAF-II cells using Trizol LS extraction reagent according to manufacturer's protocol followed by DNase I digestion . RT-PCR reaction was performed using a SuperScript III One-Step RT-PCR System (Invitrogen). The primer sequences of different PKC isoforms are presented in the Additional File 5 plaque-forming units/ml for 36 h before OI-V treatment.siRNA-mediated knock-down was carried out using isoform-specific human NM IIA siRNA SmartPool , p160ROCK (ROCK-I) duplex 2 and ROCK-II duplex 1 respectively. Cyclophilin B siRNA SmartPool (Dharmacon) was used as a control. HPAF-II cells were transfected using the DharmaFect 1 transfection reagent (Dharmacon) in Opti-MEM I medium (Invitrogen) according to manufacturer's protocol with a final siRNA concentration of 50 nM. Cells were used in experiments 72\u201380 h post-transfection. Adenoviruses expressing EGFP-tagged dominant negative N19RhoA mutant, as well as control EGFP virus were provided by Dr. James Bamburg (Colorado State University) and produced as described previously . Cells wEffect of PKC activators on transepithelial electrical resistance was measured using an EVOMX voltohmmeter . The resistance of cell-free collagen-coated filters was subtracted from each experimental point.Confluent HPAF-II monolayers were incubated for 5 h with either vehicle, octylindolactam-V (1 \u03bcM), or camptothecin (2 \u03bcg/mL). Live cells were labeled with Poly Caspase FLICA detection Kit (Axxora LLC) according to the manufacturer protocol, fixed in 3.7% paraformaldehyde and examined by confocal microscopy as described above.Numerical values from individual experiments were pooled and expressed as mean \u00b1 standard error of the mean (S.E.) throughout. The results were compared by a post-hoc Bonferroni test following analysis of variance (ANOVA) with a statistical significance assumed at p < 0.05.AJs: adherens junctions; EMT: epithelial to mesenchymal transition; OI-V: octylindolactam V; NM II: nonmuscle myosin II; PKC: protein kinase C; ROCK: Rho-dependent kinase; TEER: transepithelial electrical resistance; TJs: tight junctions; TPA: 12-O-tetradecanoylphorbol-13-acetate; ZO-1: 'zonula occludens'-1.AII conceived of the study, carried out siRNA-mediated gene knockdown, inhibitory analysis, immunofluorescence labeling and drafted the manuscript. SNS participated in confocal microscopy data acquisition, permeability measurements and siRNA knockdowns. MB carried out Western blot analysis. CAP and AN participated in design and coordination of the study and helped to draft the manuscript.Detection of different PKC isoforms in HPAF-II cells. The data provided represent the RT-PCR analysis of different PKC isoform expression in confluent HPAF-II cell monolayers. (A) Agarose electrophoresis of PCR amplicons shows expression of two classical (PKCs \u03b1 and \u03b3) and four novel PKC isoforms. (B) Primer sequences that were used to detect expression of different PKC isoenzymes.Click here for fileOI-V induces membrane translocation of different PKC isoforms. The data presented show distribution of different PKC isoenzymes between cytosolic and plasma membrane fractions in control and OI-V-treated epithelial cells. Note that 1 h OI-V treatment rapidly increased the amount of both classical (\u03b1 and \u03b2I) and novel (\u03b4) PKC isoenzymes associated with cell membranes.Click here for fileNM IIA-specific siRNAs effectively downregulate expression of this protein. The data show the effectiveness of siRNA-mediated depletion of NM IIA. HPAF-II cells were transfected with either a control (cyclophilin B), or NM IIA-specific siRNA SmartPools and analyzed for NM IIA expression 84 h post-transfection.Click here for fileEffects of Rho inhibition on myosin phosphorylation and basal F-actin filaments in OI-V-treated and control epithelial cells. The presented data demonstrate different effects of Rho inhibition on actomyosin cytoskeleton in HPAF-II cells. (A) HPAF-II cells were treated for 3 h with either OI-V alone (1 \u03bcM), or in a combination with cell-permeable Rho inhibitor, C3 toxin (2 \u03bcg/ml). Immunoblotting analysis shows that Rho inhibitor fails to prevent OI-V-dependent increase in the amount of mono- and di-phosphorylated RMLC. (B) Control HPAF-II monolayers were treated for 3 h with either vehicle or C3 toxin with subsequent fixation and fluorescence labeling of F-actin. Note that Rho inhibitor causes disappearance of basal actin filaments in HPAF-II cell monolayers which indicates the efficiency of this inhibitor in our experimental conditions. Bar, 20 \u03bcm.Click here for fileOI-V-induced junctional disassembly is independent of apoptosis. These experiments probed the role of caspase activation in OI-V-induced junctional disassembly (A) Confluent HPAF-II cell monolayers were incubated for 5 h with either vehicle, or OI-V. Cell monolayers exposed for 5 h to a classical pro-apoptotic agent, camptothecin (2 \u03bcg/mL), were used as a positive control. Cell monolayers were fixed and probed with Poly Caspase FLICA detection kit. Note, that camptothecin-treated cells show a significant increase in the number of FLICA-positive caspase-activated cells (green), whereas OI-V does not induce such a caspase activation. (B) HPAF-II cell monolayers were treated for 3 h with either vehicle or 1 \u03bcM OI-V with or without pretreatment with a potent pan-caspase inhibitor zVAD-fmk (50 \u03bcM). Junctional integrity was evaluated by immunolabeling for E-cadherin. Note that caspase inhibition has no effect on AJ disassembly induced by OI-V. Bar, 20 \u03bcm.Click here for file"} +{"text": "Protonation and oxidationof metal-free GSH and its analogues were also studied in detail. The monoprotonated form HL2- of GSH isthe one most susceptible to oxidation, due to a salt bridge between S- and NH3+ groups, which activates thethiol.Glutathione, \u03a5-Glu-Cys-Gly, is one of the most abundant small molecules in biosphere. Its main form isthe reduced monomer (GSH), serving to detoxicate xenobiotics and heavy metals, reduce protein thiols,maintain cellular membranes and deactivate free radicals. Its oxidized dimer (GSSG) controls metal contentof metallothionein. The results presented provided a quantitative and structural description of Zn(II)-glutathione complexes, including a novel ternary Zn(II)-GSH-His complex. A solution structure for thiscomplex was obtained using 2D-NMR. The Complexes studied may contribute to both zinc and glutathionephysiology. In the case of Ni(ll) complexes an interesting dependence of coordination modes on the ratios ofreactants was found. At high GSH excess a Ni(GSH)2 complex is formed, with Ni(ll) bonded through S andN and/or O donor atoms. This complex may exist as a high- or low-spin species. Another goal of the studiespresented was to describe the catalytic properties of Ni(II) ions towards GSH oxidation, which appeared to bean important step in nickel carcinogenesis. The pH dependence of oxidation rates allowed to determine theNi(GSH)"} +{"text": "Thirty-eight patients with metastatic melanoma were investigated for lymphocyte function immediately prior to chemo-immunotherapy. The pre-treatment immune tests were compared with normal control values and with response to therapy. The \"non-responder\" group (but not \"responder\") had significantly reduced values for lymphocyte, null-cell and E-rosette-cell counts compared with controls. Lymphocytoxicity ( using a Chang target cell) showed the same pattern, with depression of direct and K-cell cytotoxic capacity in non-responders compared with controls. Eight patients were studied sequentially whilst on treatment, and demonstrated considerable change in lymphocytotoxicity, an untreated \"control\" patient showed little variation. \"Recall\"-antigen skin testing showed no statistically significant difference between the patient groups. The data indicate that \"non-T-cell activity\" may be associated with response to chemo-immunotherapy."} +{"text": "In June 2000, vancomycin-intermediate Staphylococcus aureus (VISA) was isolated from a 27-year-old home health-care patient following a complicated cholecystectomy. Two VISA strains were identified with identical MICs to all antimicrobials tested except oxacillin and with closely related pulsed-field gel electrophoresis types. The patient was treated successfully with antimicrobial therapy, biliary drainage, and reconstruction. Standard precautions in the home health setting appear successful in preventing transmission."} +{"text": "The BH3-only protein Bid is an important component of death receptor-mediated caspase activation. Bid is cleaved by caspase-8 or -10 into t-Bid, which translocates to mitochondria and triggers the release of caspase-activating factors. Bid has also been reported to be cleaved by other proteases.bid lead to a pronounced resistance to Fas/CD95- and TRAIL-induced caspase activation and apoptosis, and significantly increased clonogenic survival. While Bid-deficient cells were equally sensitive to ER stress-induced apoptosis, they showed moderate, but significantly reduced levels of apoptosis, as well as increased clonogenic survival in response to the genotoxic drugs Etoposide, Oxaliplatin, and Doxorubicin. Similar effects were observed using the Bid inhibitor BI6C9. Interestingly, Bid-deficient cells were dramatically protected from apoptosis when subtoxic concentrations of ER stressors, Etoposide or Oxaliplatin were combined with subtoxic TRAIL concentrations.To test the hypothesis that Bid is a central mediator of stress-induced apoptosis, we investigated the effects of a small molecule Bid inhibitor on stress-induced apoptosis, and generated HeLa cells deficient for Bid. Stable knockdown of Our data demonstrate that Bid is central for death receptor-induced cell death and participates in anti-cancer drug-induced apoptosis in human cervical cancer HeLa cells. They also show that the synergistic effects of TRAIL in combination with either ER stressors or genotoxic anti-cancer drugs are nearly exclusively mediated via an increased activation of Bid-induced apoptosis signalling. The members of the Bcl-2 family of proteins are key players in cellular life and death decisions during apoptosis bid is a p53 target gene Bid is a member of the Bcl-2-homology domain (BH)-3-only family of pro-apoptotic proteins that initiate apoptotic cell death bid-deficient mice presented with a relatively mild phenotype, but showed a prominent resistance to Fas-dependent apoptosis of hepatocytes bid-deficient murine cells could not identify such a role for Bid Interestingly, bid was performed as described in bid . Dose-response analyses demonstrated that the knockdown of bid potently reduced DEVD cleavage activity in response to both stimuli compared to a control clone transfected with a scrambled sequence , demonstrating that these cells did not acquire a general resistance against apoptosis activation .bid knockdown in a different approach, HeLa cells were treated with the synthetic Bid inhibitor BI6C9 To confirm the results of the N-linked glycosylation of nascent proteins, leading to an accumulation of hypoglycosylated proteins in the ER lumen, resulting in the activation of ER stress responses and, if persisting, to ER stress-induced apoptosis. Treatment of HeLa Bid kd cells and control HeLa cells with Tunicamycin resulted in a similar induction in protein levels of the ER resident molecular chaperones Grp78 and Grp94, which are known ER stress response target genes , Oxaliplatin (Ox), and Doxorubicin. Detection of effector caspase activation by activity assays indicated that the knockdown of ic drugs . A more ic drugs and clonic drugs indicateor BI6C9 .We next investigated the role of Bid in the potential synergism between genotoxic drugs and TRAIL. Oxaliplatin and Etoposide both upregulated DR5 receptor expression in a time-dependent manner, while DR4 levels remained constant . Similarpuma has been shown to be a pivotal regulator of apoptosis induced by DNA-damaging anticancer drugs in many types of cancer cells and in particular to mediate Oxaliplatin-induced apoptosis puma gene expression in HeLa control cells and HeLa Bid kd cells. puma mRNA levels were potently induced by Oxaliplatin as evaluated by qPCR . Treatment with puma siRNA significantly reduced levels of apoptosis in HeLa control cells exposed to Oxaliplatin can be generated by the activity of caspase-8 and -10 Activation of caspase-8 has also been suggested to play a significant role during ER stress-induced apoptosis DR5 expression but also bid expression Despite the considerable interest in TRAIL or agonistic TRAIL-receptor antibodies as anticancer agents, accumulating evidence suggests that indeed mono-therapies with these agents may have limited success, but that synergistic treatments with other apoptosis inducers may be a good rationale to efficiently activate apoptosis in many types of cancers puma has been suggested to largely mediate the pro-apoptotic activity of p53 puma was potently activated by Oxaliplatin in HeLa control and Bid kd cells, and transient RNA interference against puma was able to significantly reduce apoptosis in both cell lines. Despite the prominent role of PUMA in mediating genototoxic drug-induced apoptosis, several lines of evidence support the notion that Bid also partially contributes to apoptosis induced by DNA-damage protecting cells from genotoxic stress by promoting cell cycle arrest, thus facilitating DNA repair and potentially cellular survival bid gene deletion was explored in non-transformed murine cells In cancer cells treated with genotoxic drugs only, the p53 target gene A further level of complexity in regard to the role of Bid in the response to genotoxic stress lies in the fact that Bid can be activated through several distinct mechanisms after DNA damage. In addition to its transcriptional induction, Bid has been shown to be proteolytically activated by the initiator caspase, caspase-2, and generation of t-Bid has been suggested to be essential for apoptosis induced by caspase-2 Collectively, our data demonstrate that Bid is an indispensable component of death receptor-induced cell death, but also participates in DNA damage-induced apoptosis of human cervical cancer HeLa cells. Importantly, our data also show that the synergistic effects of the death ligand TRAIL in combination with either ER stressors or DNA damaging anti-cancer drugs are nearly exclusively mediated via an increased activation of Bid-induced apoptosis signaling.Human recombinant TRAIL was purchased from Leinco Technologies . Caspase substrate N-acteyl-Asp-Glu-Val-Asp-7-amino-4-methyl-coumarin (Ac-DEVD-AMC) and the pan-caspase inhibitor Z-Val-Ala-Asp(O-methyl)-fluoromethylketone (zVAD-fmk) were obtained from Bachem . All other chemicals came in analytical grade purity from Alexis or Sigma-Aldrich .2 containing atmosphere at 37\u00b0C. Cells were kept in logarithmic growth phase by routinely passaging them twice a week and were plated 24 h prior to treatments.HeLa cells were grown in RPMI 1640 medium supplemented with 10% (v/v) heat-inactivated fetal calf serum, 2 mM glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin (Sigma-Aldrich) in a humidified 5% COhttp://www.dharmacon.com/sidesign/): Bid-1 sense (5\u2032-AAGCTGTTCTGACAACAGC-3\u2032), Bid-2 sense (5\u2032-AAGGAGAAGACCATGCTGG-3\u2032) and Bid-3 sense (5\u2032-AAGAATAGAGGCAGATTCT-3\u2032). Bid-specific or control shRNA duplexes were ligated into the pSilencer 2.1-U6 hygro vector via their BamH I and Hind III sites. To generate stable knock down cell lines HeLa cells were transfected with the different shRNA constructs using Metafectene according to the manufacturer's instructions. 24 h post transfection the cells were diluted, transferred to 96-well plates and stable clones were selected using hygromycin B (160 \u00b5g/ml).The following shRNA sequences specific for human Bid mRNA were designed using the Dharmacon siRNA design tool puma mRNA were designed utilizing the RNA workbench (www.rnaworkbench.com). The annealed puma (5\u2032-GAUGGCCCAGCCUGUAAGAUACUdTdT-3\u2032) and control siRNA (5\u2032-UUCUCCGAACGUGUCACGUdTdT-3\u2032) duplexes were purchased from Sigma Proligo . 24 h after seeding into six-well plates, cells were transfected with 100 nM of the siRNA duplex using Metafectene Pro as per manufacturer's instructions. 24 h post transfection cells were treated as indicated.siRNA duplexes targeting Preparation of cell lysates and western blotting was performed as previously described The measurement Annexin-V-FITC-staining of apoptotic cells was performed on a Cyflow ML16 flow cytometer as described previously http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) and came from Sigma-Genosys . Sense and antisense primers were: \u03b2-actin: TCACCCACACTGTGCCCATCTA and CAGCGGAACCGCTCATTGCCAA; puma: CCATCTCAGGAAAGGCTGTT and ACGTTTGGCTCATTTGCTCT.Total RNA was extracted using the RNeasy Mini kit and 2 \u00b5g per sample were reverse-transcribed into first strand cDNA using random hexamer primers (50 pmol) and MMLV Reverse Transcriptase . Quantitative real-time PCR was conducted using the QuantiTech SYBR Green PCR kit (Qiagen) and the LightCycler as described previously Preparation of cell lysates to determine their caspase-3-like protease-activity was performed as previously described Cells were treated as indicated before transferring 1,000 cells to 60 mm dishes and culturing them for two weeks to allow for colony formation. Then the medium was removed, colonies were fixed and stained with a solution containing ethanol (50%) and methylene-blue (0.25%) for 45 min and the number of colonies per plate was counted.Data are given as means\u00b1SD or SEM. For statistical comparison, t-test or one-way ANOVA followed by Tukey test were employed using SPSS software . P-values smaller than 0.05 were considered to be statistically significant."} +{"text": "In vitro cytotoxicity of Kupffer cells and tumour infiltrating lymphocytes (TILs) on RCN-9 cells was evaluated using [3H]-release assay. RCN-9 cells were resistant to cytotoxicity mediated by cirrhotic Kupffer cells, but were sensitized to TIL-mediated killing after treatment with cirrhotic KCM. The specific killing induced by TILs was FasR-mediated, as it was inhibited by ZB4, an antagonistic anti-FasR antibody. In agreement, cirrhotic KCM increased recombinant Fas ligand-induced apoptosis of RCN-9 cells, and up-regulated FasR expression on RCN-9 cells as evaluated by RT-PCR and flow cytometry. These findings suggest that Kupffer cells in cirrhotic livers sensitize metastatic colon cancer cells to FasR-mediated apoptosis by up-regulating the receptors, which thus prepare them to be eliminated by infiltrating lymphocytes. \u00a9 2001 Cancer Research Campaign http://www.bjcancer.comMetastasis of colorectal carcinomas rarely occurs in cirrhotic livers. Our study investigated the influence of activated Kupffer cells from cirrhotic rat livers on hepatic colonization and FasR-mediated apoptosis of colon cancer cells. A rat colon cancer cell line, RCN-9, was used to inoculate rat livers. Treatment with conditioned media of Kupffer cells isolated from CCl"} +{"text": "The relative importance of different proteinases, and their inhibition, in the breakdown of human endothelial basement membrane (BM) by MDA-MB-231 and MCF7ADR human breast cancer cell lines has been studied using 35S-labelled BM-coated 96-well culture plates. Basement membrane degradation (BMD) was independent of cell proliferation above the seeding density. Inhibitors of aspartic (pepstatin and PD 134678-0073) and cysteine proteinases (E64) had little effect on BMD under normal culture conditions, suggesting that cathepsins D, B and L have only a minor role. In contrast, inhibitors of urokinase-type plasminogen activator (uPA) and/or plasminogen activation to plasmin all reduced BMD by MDA-MB-231 cells by approximately 30-40%, but only in the presence of serum or plasminogen. BB94, an inhibitor of matrix metalloproteinases (MMPs), also reduced BMD by about 30% under these conditions but was similarly effective in serum-free medium. Combinations of BB94 with any of the uPA/plasminogen activation inhibitors in serum-containing medium had additive effects, while BB94 with pepstatin and E64 under serum-free conditions reduced BMD to 16% of control. Serum-containing conditioned medium exhibited appreciable BMD, largely due to aprotinin-inhibitable activity. Although small reductions in cell proliferation were seen with some inhibitors, the combination of BB94 with E64 or E64d reduced the cell population by about 60% under serum-containing conditions. These in vitro observations suggest that combinations of proteinase inhibitors, particularly of uPA/plasminogen activation and MMPs, may merit clinical evaluation as potential antimetastatic therapy for breast cancer."} +{"text": "Gene-transfected tumour cells were used to cure mice bearing lung metastases by the parental, non-transduced mammary adenocarcinoma (TSA-pc). Repeated subcutaneous (s.c.) administrations of mitomycin C (MitC)-treated interferon gamma (IFN-gamma) transfectants induced a 90% inhibition in the number of lung metastases. Therapeutic effect required an intact T-cell response, as shown by the lack of efficacy in nude mice. Autocrine stimulation by IFN-gamma induces specific modifications in the phenotype of transfectants that acquire a high metastatic ability and show a high expression of IFN-responsive genes; these two features were exploited to design two experimental protocols to obtain an improvement of the therapeutic effect. The increased metastatic ability of IFN-gamma transfectants was used to deliver IFN-gamma selectively to the lungs of mice bearing TSA-pc pulmonary metastases. A significant therapeutic effect was obtained when TSA-pc experimental metastases were treated by repeated intravenous (i.v.) injections of MitC IFN-gamma transfectants. Since i.v. administrations of IFN-gamma transfectants did not induce immune memory, the therapeutical effect appeared to depend on the inflammatory-like response activated by local IFN release. To exploit the autocrine stimulation of IFN-sensitive genes an IFN-gamma transfectant clone was subjected to a second transfection with an allogeneic class I MHC gene (H-2K(b) or H-2D(h)). IFN-gamma plus MHC double transfectants maintained IFN-gamma release, showed a very high expression of the MHC gene products, stimulated both macrophages and T cells, and were less tumorigenic in immunocompetent mice than the parent IFN-gamma clone. Therapeutic efficacy of double transfectant IFN-gamma plus H-2D(b) cells against TSA-pc was superior to single transfectants, showing that the reaction elicited by genetically engineered cells can be selectively tuned to increase therapeutic efficacy."} +{"text": "Macrophage inflammatory protein 1alpha inhibits haemopoietic stem cell proliferation. This property has been exploited in a murine chemotherapy model and has been shown to ameliorate cytotoxic-induced myelosuppression after S-phase-specific cytotoxic therapy. We have now shown that BB-10010, a stable mutant of MIP-1alpha, (a) is more effective when administered as a continuous infusion than when bolus injected and (b), when administered via a 7-day infusion during and after cyclophosphamide treatment, results in an earlier recovery of leucocyte numbers. This effect was accompanied by progenitor cell mobilization into the peripheral blood and included primitive cells with marrow-repopulating ability (MRA). Maximal mobilization and recovery of leucocytes occurred when MIP-1alpha was combined with granulocyte colony-stimulating factor (G-CSF) therapy. The findings suggest that MIP1-alpha used alone or in combination with G-CSF may allow delivery of a greater chemotherapy dose intensity as a consequence of both accelerated leucocyte recovery and maintenance of high-quality mobilized progenitor cells for harvesting and peripheral blood stem cell transplantation."} +{"text": "Oxidative stress and inflammation contributed to the propagation of acute liver injury (ALI). The present study was undertaken to determine whether D-galactosamine induces ALI via the mitochondrial apoptosis- and proinflammatory cytokine-signaling pathways, and possible mechanism(s) by which green tea (GT) extract modulates the apoptotic and proinflammatory signaling in rat. D-GalN induced hepatic hypoxia/hypoperfusion and triggered reactive oxygen species (ROS) production from affected hepatocytes, infiltrated leukocytes, and activated Kupffer cells. D-GalN evoked cytosolic Bax and mitochondrial cytochrome C translocation and activated proinflammatory nuclear factor-kappa B (NF-\u03baB) and activator protein-1 (AP-1) translocation, contributing to the increase of intercellular adhesion molecule-1 expression, terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL)-positive hepatocytes, multiple plasma cytokines and chemokines release, and alanine aminotransferase (ALT) activity. An altered biliary secretion profile of several acute phase proteins directly indicates oxidative stress affecting intracellular trafficking in the hepatocyte. GT pretreatment attenuated ROS production, mitochondrial apoptosis- and proinflammatory cytokine-signaling pathway, plasma ALT and cytokines levels, biliary acute phase proteins secretion and hepatic pathology by the enhancement of anti-apoptotic mechanisms. In conclusion, D-GalN induced ALI via hypoxia/hypoperfusion-enhanced mitochondrial apoptosis- and proinflammatory cytokine-signaling pathway, contributing to oxidative stress and inflammation in the liver. GT can counteract the D-GalN-induced ALI via the attenuation of apoptotic and proinflammatory signaling by the upregulation of anti-apoptotic mechanism. WCC, HSL, and HMC provided important comments and excellent techniques in the paper. All authors read and approved the final manuscript."} +{"text": "II compounds as well as the Schiff bases with 2-furaldehyde (dienOO), 2-thiophenecarboxaldehyde (dienSS) and pyrrole-2-carboxaldehyde (dienNN) of dien and that of dpta with 2-thiophenecarboxaldehyde (dptaSS), were prepared and characterised. They were tested against Bacillus substilis, Bacillus cereus, Staphylococcus aureus, Escherichia coli, Proteus vulgaris and Xanthomonas campestris as antibacterial reagents, the highest activity being exhibited by Cu(dptaSS)(NO3)2 complex, which acts as antibiotic. In the antiproliferative tests the best resultswere obtained with Cu(dptaSS)2+ and Cu(dienSS)2+. Electronic structure calculations gave for dptaSS anddienSS the higher negative charges on the N atoms. The counter-ions play an importantrole by modulating the reagent's selectivity versus the bacteria [Gram(+) or Gram(-)], but they have no effecton the antiproliferative activity.Ethylenediamine (en), putrescine (pu), diethylenetriamine (dien), dipropylenetriamine (dpta),spermidine (spmd) and their Cu"} +{"text": "Even though many studies suggest that proteoglycans with their structurally determinative polysaccharide chains, the glycosaminoglycans (GAGs), are important mediators of cellular interactions, little is known about expression and possible functions of these macromolecules expressed by tumour cells during the transition from low to highly metastatic behaviour. Therefore, we investigated the cellular expression and secretion of GAGs in a syngeneic tumour system of DBA/2 mice consisting of a methylcholanthrene-induced low metastatic T lymphoma (Eb), its highly metastatic spontaneous variant (ESb), and a low metastatic derivative of ESb (ESb-MP), selected by its adherent growth properties. The [35S]-sulphate-labelled GAGs were isolated from in vitro cultivated cells and further characterized by separation on Sepharose CL 6B, on Mono-Q ion exchange chromatography, and alkali- and enzymatic digestion. In contrast to Eb-cells which produce chondroitin/dermatan sulphate (CS/DS) and heparan sulphate (HS) ESb- and ESb-MP-cells only express and secrete CS/DS. For ESb cells the CS portions consisted of 42% chondroitin-4-sulphate (CS-4) and 58% chondroitin-6-sulphate (CS-6), for ESb-MP cells of 23% CS-4 and 77% CS-6, for Eb cells of 16% CS-4 and 84% CS-6. The cell surface GAGs of the adherent variant ESb-MP contained a significantly higher portion of DS (65%) compared to ESb cells (25%). GAGs of all tumour cell lines studied had a mol. wt ranging from 35-40 kD compared to GAG molecular weight standards. Ion exchange chromatography indicated that differences in charge density between GAGs of these cell lines were minimal. These findings suggest that the different biological behaviour of the cell lines cannot be attributed to altered size and charge density of their GAG chains. However, highly metastatic ESb-cells secreted significantly more GAG than low metastatic Eb- and ESb-MP-cells. The possible consequences of the enhanced secretion of CS/DS by ESb-cells are discussed in terms of the postulated role of CS/DS in cellular adhesion, growth regulation and interactions with the immune system."} +{"text": "Thedifferences between these isozymes in susceptibility to inhibition by these metal complexes isdiscussed in relationship to the characteristic features of their active sites, and is rationalized interms useful for developing isozyme-specific CA inhibitors.Coordination compounds of 5-chloroacetamido-1,3,4-thiadiazole-2-sulfonamide (Hcaz)with V(IV), Cr(lll), Fe(ll), Co(ll), Ni(ll) and Cu(ll) have been prepared and characterized by standardprocedures . Some of these compounds showed very good"} +{"text": "Type and extent of endocrinological alterations were studied in long-term disease-free survivors after cisplatin-based chemotherapy for testicular cancer. A total of 63 patients with a median age of 30 (19-53) years, and median follow-up of 42 (16-128) months were included. Elevated serum follicle-stimulating hormone (FSH) levels were found in 63% of patients, 24% showed pathologically elevated luteinising hormone (LH) levels with normal and 10% with subnormal testosterone levels. The degree of gonadotropin elevation was highly significantly correlated with the cumulative platinum (P) dose. Patients treated with platinum-vinblastine-bleomycin regimens showed higher gonadotropin levels than those treated with platinum-etoposide-bleomycin. The adrenal androgen dehydroepiandrosterone (DHEA), pathologically elevated in 68% of patients, was significantly correlated with the cumulative doses of chemotherapy (ctx) used and to the gonadotropin levels. Treatment variables, such as type and dose of cytotoxic agents used, as well as degree of gonadotropin elevation were further correlated with changes in oestron, testosterone and 17 alpha-OH-progesterone levels. Cholesterol levels were elevated in 32% of patients and significant interactions between the steroid hormone levels and cardiovascular risk factors could be shown."} +{"text": "The current approaches to the quality control of herbal medicines are either compound-oriented or pattern-oriented, the former targeting specific components with some known chemical properties and the latter targeting all detectable components. The marker approach uses specific chemical compounds with known molecular structures, while the multi-compound approach uses both chemical compounds with known structures and those with partial chemical information e.g. retention times, mass spectra and ultraviolet spectra. Apart from chromatographic techniques, new techniques such as oscillating and electrochemistry fingerprints have been developed for quality control. Chemometric resolution methods are widely used for component deconvolution and data comparison. Pattern recognition techniques are used for authentication of herbal medicines. Cortex Cinnamomi (Rougui) revealed over 90 volatile components in a gas chromatography-mass spectroscopy (GC-MS) experiment, only 60 of which were chemically identified [High chemical complexity of herbal medicines makes quality control through chemical analysis difficult. For example, a common Chinese medicinal herb entified . Little entified . Mok andentified . For a rentified -6. Insteentified ,8. Metabentified .16H18O9) is recommended for the identification of Flos Lonicerae (Jinyinhua) (content \u2265 1.5%) and Flos Chrysanthemi (Juhua) (content \u2265 0.2%) [Fufang Danshen Diwan is a formulated herbal medicine that consists of three herbs but there is only one chemical marker salvianic acid A (C9H10O5) recommended for this particular medicine in the Chinese Pharmacopoeia (2005 edition) [Different ingredients within a herbal medicine may have synergistic effects. These active ingredients must be identified and quantified for better understanding of the action mechanisms of herbal medicines. For the moment, the conventional marker approach is not successful as only a few chemical markers are available for herbal medicines in the American Herbal Pharmacopeia and Chinese Pharmacopoeia. For example, in the Chinese Pharmacopoeia (2005 edition) only one chemical marker chlorogenic acid (C \u2265 0.2%) . These tedition) .The pattern-oriented approach such as fingerprinting is more useful than compound-oriented approach in most cases. Chemical fingerprints are characteristic for herbal medicines, and are therefore useful in the quality control of herbal medicines -15. The et al. used near infrared spectroscopy (NIR) to quantitatively determine the content of berberine and total alkaloid in Cortex Phellodendri (Guanhuangbo) [Qingfu Guanjie Shu (capsule) using four marker compounds, namely sinomenine, paeoniflorin, paeonol and curcumin. [et al. used liquid chromatography-tandem mass spectrometers (LC-MS/MS), solid phase extraction, and the marker glycyrrhetic acid to simultaneously validate Radix Glycyrrhizae (Gancao) and quantify the target compound in the samples [According to the Chinese Pharmacopoeia (2005 edition), identification and quantification of chemical markers are crucial to the quality control of herbal medicines. A total of 525 quantitative assays of chemical markers were documented in the Chinese Pharmacopoeia (2005 edition) for assessment of herbal medicinal materials, plant lipids, herbal extracts and formulations . Chemisthuangbo) . The conurcumin. . Lin et samples . Quantit samples -34. Gas samples -39.Compared with the marker approach, the multi-compound approach uses multiple compounds with known chemical properties and does not necessarily require chemical markers. Chemometric deconvolution and resolution are major methods in this approach. In the Chinese Pharmacopoeia (2005 edition) , multiplChemometric resolution methods (CRM) were used extensively in the past decades to 'purify' chromatographic peak profiles of complex mixture systems such as herbal medicines . The quaet al. identified 38 volatile chemical components of Radix Paeoniae Rubra (Chishao), which accounted for 95.21% of all detectable components [Radix Rehmanniae Preparata (Shudihuang) were separated, of which 59 were identified using standard spectra in the database of the National Institute of Standards and Technology (NIST). The 26 identified methyl esters accounted for about 94.29% of the total number of components [Rhizoma et Radix Notopterygii (Qianghuo) were identified out of the 98 separated chemicals. Qi et al. resolved the overlapping chromatographic peaks in Resina Draconis (Xuejie) using HPLC-DAD. Therefore, using chemometric methods with hyphenated instruments was powerful in the analysis of herbal medicines [et al. used PCA and generalized rank annihilation factor analysis (GRAFA) to process HPLC-DAD data sets obtained from Radix Salviae Miltiorrhizae (Danshen) and Radix Notoginseng (Sanqi) [et al. simultaneously analyzed seven major saponins of Danshen Diwan with HPLC-DAD and electron spray ionization-mass spectrometry (ESI-MS) [Previously, both iterative and non-iterative resolution methods were used to study the volatile and non-volatile components in herbal medicines -46. Manymponents . In anotmponents . Most ofmponents ,46. Withedicines . Zeng et (Sanqi) . Ye et a(ESI-MS) .Chemometric methods including spectral correlative chromatography (SCC), multi-component spectral correlative chromatography (MSCC), AMWFA were proposed for comparing three-dimensional (3D) or two-dimensional (2D) chromatographic profiles and integrating presence or absence information ,57-59. Set al. combined 3D fingerprints from HPLC-DAD instruments with principal component analysis (PCA) to monitor Qingkailing (a proprietary Chinese medicine injection formulation) produced by various manufacturers [Radix Salviae Miltiorrhizae (Danshen) collected from various localities [Caulophyllum robustum (Leiyemudan). Similarity index and the cluster analysis method were used to analyze the quality of ten batches of samples of Caulophyllum robustum [et al. developed multiple chromatographic fingerprinting including two HPLC fingerprints for the quality assessment of Danshen Diwan [The pattern-oriented approach analyzes fingerprints obtained from one-, two- or higher dimensional chromatographic and/or spectral instruments. Single pattern approach focuses on one type of pattern for the quality control of herbal medicines. Significant progress was made in 2D chromatographic profiles obtained from HPLC, CE, GC as well as 3D ones from hyphenated instruments ,19,67-70acturers . High-spcalities -74. Binarobustum . Fan et en Diwan . Thin laen Diwan ,77. Fouren Diwan ,28,78,79en Diwan , electroen Diwan ,82, X-raen Diwan and highen Diwan .et al. developed a software package Computer Aided Similarity Evaluation (CASE) for data processing under Matlab [et al. developed a series of chemometric methods to extract information from fingerprints [et al. used target peak alignment (TPA) to correct the retention time shifts and multiplicative scattering correction (MSC) for response correction [It is important to extract useful information from chromatographic/spectral fingerprints which contain chemical information of all detectable components of herbal medicines. Chau r Matlab . Gong eterprints ,86,87. Xrrection . In addirrection . Comparirrection -94. Inforrection -93. PCA,rrection . Data derrection -53. Furtrrection ,99.et al. found 32 potential bioactive components of Radix Angelicae Sinensis (Danggui) in rabbit plasma and over ten new compounds [Herba Houttuyniae (Yuxingcao) injection, Lu et al. evaluated its anti-inflammatory effects and established the correlation between the chemical components of the injection and the bioactivities of the active ingredients [et al. devised a method which combined metabolic profiling and liquid chromatography-diode array detection-mass spectroscopy (LC/DAD-MS) with chemometrics to study Danggui Buxue Tang, a Chinese medicine formulation [et al. discovered major bioactive components of Aquilegia oxysepala (Jianeloudoucai) [The multi-pattern approach assesses the quality of herbal medicines according to multiple patterns. For example, both chromatographic fingerprints and biological activity profiles are used for the quality control of herbal medicines. This approach targets the discovery of bioactive ingredients, assessment of medical effects, correlation between chemical fingerprints and pharmacological indices, and quality control -105. Somompounds . Using aredients ,102. Wanmulation . Aided budoucai) . EvidentThe compound-oriented and pattern-oriented approaches to the quality control of herbal medicines have been significantly improved in terms of analytical instruments, biological screening methods and chemometrics. Among all the advanced techniques, multi-pattern approach will have a great potential for further development.2D-IR: two-dimensional correlation infrared spectroscopy; AMWFA: alternative moving window factor analysis; CE: capillary electrophoresis; CRM: chemometric resolution methods; CZE: capillary zone electrophoresis; DAD: diode array detection; ESI-MS: electron spray ionization-mass spectrometry; EWOP: evolving window orthogonal projection; FSMWEFA: fixed-size moving window evolving factor analysis; FT-IR: Fourier transform infrared spectroscopy; GC: gas chromatography; GC-MS: gas chromatography-mass spectroscopy; GRAFA: generalized rank annihilation factor analysis; HELP: heuristic evolving latent projection; HPLC: High-performance liquid chromatography; HPLC-CEAD: high-performance liquid chromatography-coulometric electrode array detector; HPLC-DAD: high-performance liquid chromatography-diode array detection; HPLC-ESI-MSn: high-performance liquid chromatography electrospray ionization tandem mass spectrometry; HSCCC: high-speed counter-current chromatography; IOP: iterative orthogonal projection resolution; LC-DAD-APCI-MS: liquid chromatography-diode array detection-atmospheric pressure chemical ionization-mass spectroscopy; LC/DAD-MS: liquid chromatography-diode array detection-mass spectroscopy; LC-MS: liquid chromatography-mass spectroscopy; LC-MS/MS: liquid chromatography-tandem mass spectrometers; MS: mass spectroscopy, MSCC: multi-component spectral correlative chromatography; NIR: near infrared spectroscopy, OP: orthogonal projection technique; OPA: orthogonal projection approach, PCA: principle component analysis; PLS: partial least squares; SCC: spectral correlative chromatography; SFA: subwindow factor analysis, TLC: thin layer chromatography; TLC-UV: thin layer chromatography-ultraviolet spectrophotometry; XRD: X-ray diffraction.The authors declare that they have no competing interests.ZZ and FC conceived the classification of approaches to quality control of herbal medicines. ZZ drafted the manuscript. FC supervised the project and revised the manuscript. HC and CYC provided references and helped revise the manuscript. TL and SW advised on the manuscript. DM, COC and YL were responsible for some studies in this work. All authors read and approved the final manuscript."} +{"text": "We have investigated the regression phenomenon which occurs when EBV-infected peripheral blood mononuclear cells from seropositive individuals are cultured for one month at high cell concentration and have confirmed that regression is mediated by E+ lymphocytes. When helper/inducer (Leu 3a+) and suppressor/cytotoxic (Leu 2a+) cells are separated by fluorescence-activated cell sorting from fresh peripheral blood and co-cultured with EBV-infected autologous E- mononuclear cells, regression only regularly occurs in cultures receiving suppressor/cytotoxic lymphocytes. Titration experiments show that suppressor/cytotoxic lymphocytes are more active in the regression assay that unfractionated E+ cells. When Ia+ E+ and Ia- E+ cells are separated one week after initiation of co-cultures of E+ cells and EBV-infected E- cells, both Ia+ E+ and Ia- E+ cells are active in the regression assay although regression occurs earlier in cultures receiving Ia+ E+ cells. Experiments in which NK cells are isolated using the monoclonal antibodies H25 and H366 show that NK cells do not influence the regression phenomenon in normal individuals."} +{"text": "Inhibition of nitric oxide synthase increases microvascular permeability in rat small intestinal villi. To determine the mechanism(s) whereby this occurs we have perfused the vasculature of rat isolated small intestines with a gelatin-containing physiological salt solution. Inclusion of N-nitro-L-argintne methyl ester or indomethacin (1 \u03bcM) in the perfusate increased leakage of injected colloidal carbon into microvessel walls. Pre-treatment with sodium nitroprusside (10 \u03bcM) significantly reduced the effects of both L-NAME and indomethacin, whereas carbacyclin (1 \u03bcM) only reduced the effects of indomethacin. PD151242 (1 \u03bcM) showed some antagonism towards the effects of L-NAME, but nordihydroguaiaretic acid (3 \u03bcM) was inactive. Pre-tment with cyproheptadine (10 \u03bcM) reduced the effects of both L-NAME and indomethacin, and also significantly reduced background (control) colloidal carbon leakage. Small intestines from polymixin B-treated rats showed significantly reduced colloidal carbon leakage in response to L-NAME. This suggests that the leakage-enhancing effects of both L-NAME and indomethacin in this preparation may be mediated by mast cell-derived amines."} +{"text": "MAP (mitogen-activated protein) kinase plays a crucial role in cell proliferation anddifferentiation. Its impact on secretory events is lesswell established. The interplay of protein kinase C(PKC), PI3-kinase nd cellular tyrosine kinase withMAP kinase activity using inhibitors and compoundssuch as glucose, phorbol 12-myristate 13-acetate(PMA) and agonists of G-protein coupled receptorslike gastrin releasing peptide (GRP), oxytocin (OT)and glucose-dependent insulinotropic peptide (GIP)was investigated in INS-1 cells, an insulin secretingcell line. MAP kinase activity was determined byusing a peptide derived from the EGF receptor as aMAP kinase substrate and ["} +{"text": "Four Ge(IV)-octabutoxy-phthalocyanines (GePcs) bearing two alkyl-type axial ligands were assayed for their pharmacokinetic properties and phototherapeutic efficiency in Balb/c mice bearing an intramuscularly transplanted MS-2 fibrosarcoma. The GePcs were i.v. injected at a dose of 0.35 mumol kg-1 body weight after incorporation into either Cremophor emulsions or small unilamellar liposomes of dipalmitoyl-phosphatidylcholine (DPPC). Both the nature of the delivery system and the chemical structure of the phthalocyanine were found to affect the behaviour of the GePcs in vivo. Thus, Cremophor-administered GePcs invariably yielded a more prolonged serum retention and a larger association with low-density lipoproteins (LDLs) as compared with the corresponding liposome-delivered phthalocyanines. This led to a greater efficiency and selectivity of tumour targeting. These effects were more pronounced for those GePcs having relatively long alkyl chains (hexyl to decyl) in the axial ligands. Maximal tumour accumulation (0.67 nmol per g of tissue) was found for Ge-Pc(hexyl)2 at 24 h after injection. Consistently, the Ge-Pc(hexyl)2, administered via Cremophor, showed the highest phototherapeutic activity towards MS-2 fibrosarcoma."} +{"text": "Esters of succinic acid are potent insulin secretagogues,and have been proposed as novel antidiabeticagents for type 2 diabetes. This studyexamines the effects of acute and chronic exposureto succinic acid monomethyl ester (SAM) on insulinsecretion, glucose metabolism and pancreatic betacell function using the BRIN-BD11 cell line. SAMstimulated insulin release in a dose-dependentmanner at both non-stimulatory (1.1mM) and stimulatory(16.7mM) glucose. The depolarizing actionsof arginine also stimulated a significant increasein SAM-induced insulin release but 2-ketoisocaproicacid (KIC) inhibited SAM induced insulinsecretion indicating a possible competition betweenthe preferential oxidative metabolism of these twoagents. Prolonged (18hour) exposure to SAM revealeddecreases in the insulin-secretory responsesto glucose, KIC, glyceraldehyde and alanine.Furthermore, SAM diminished the effects of nonmetabolizedsecretagogues arginine and 3-isobutyl-1-methylxanthine (IBMX). While the ability ofBRIN-BD11 cells to oxidise glucose was unaffectedby SAM culture, glucose utilization was substantiallyreduced. Collectively, these data suggest thatwhile SAM may enhance the secretory potential ofnon-metabolized secretagogues, it may also serve asa preferential metabolic fuel in preference to otherimportant physiological nutrients and compromisepancreatic beta cell function following prolongedexposure."} +{"text": "We have used a pulldown assay for enrichment of the CpG methylated fraction of cellular DNA combined with microarray platforms, followed by extensive validation through conventional bisulfite-based analysis. Here, we demonstrate strikingly similar patterns of DNA methylation in non-transformed B[a]PDE-treated cells vs control using high-throughput microarray-based DNA methylation profiling confirmed by conventional bisulfite-based DNA methylation analysis. The absence of aberrant DNA methylation in our model system within a timeframe that precedes cellular transformation suggests that following carcinogen exposure, other as yet unknown factors (secondary to carcinogen treatment) may help initiate global loss of DNA methylation and region-specific gain of DNA methylation, which can, in turn, contribute to lung cancer development. Unveiling the initiating events that cause aberrant DNA methylation in lung cancer has tremendous public health relevance, as it can help define future strategies for early detection and prevention of this highly lethal disease.Global loss of DNA methylation and locus/gene-specific gain of DNA methylation are two distinct hallmarks of carcinogenesis. Aberrant DNA methylation is implicated in smoking-related lung cancer. In this study, we have comprehensively investigated the modulation of DNA methylation consequent to chronic exposure to a prototype smoke-derived carcinogen, benzo[ Etiologically, tobacco smoking continues to represent the single most important risk factor for lung cancer development Lung cancer is the chief cause of cancer-related mortalities, worldwide hypomethylation) and a locus/gene-specific gain of DNA methylation (hypermethylation) are two distinct hallmarks of carcinogenesis e.g., 80\u201390% of CpGs in the human genome are methylated e.g., \u223c70% of all human promoters encompass CpG islands per seEpigenetic mechanisms of carcinogenesis manifest as heritable changes in gene expression without involving alterations in the underlying DNA sequence e.g., dietary, and medicinal sources a]pyrene (B[a]P) is a prototype PAH, which requires metabolic activation to its ultimate carcinogenic form, B[a]P diol epoxide (B[a]PDE), to exert its biological effects in vivoa]P and/or B[a]PDE, as model tobacco-smoke carcinogens, to investigate the modulation of DNA methylation in vitroa]PDE resulted in impairment of DNA methyltransferase (DNMT) activities, manifested as inhibition of catalyzing reaction between the methyl donor S-adenosylmethionine (SAM) and the substrate DNA a]P caused a reduction in the 5-methylcytosine content of cellular DNA, albeit only in the latter cell line DNMT1 or the de novo DNMT3a or DNMT3b, there were increased levels of DNMT1 protein and promoter hypermethylation of several genes of the panel of 30 genes analyzed in the latter study DNMTs, either at the expression or activity level Polycyclic aromatic hydrocarbons (PAH) are a prominent class of carcinogenic compounds present in tobacco smoke, as well as in numerous other sources, including occupational, environmental, methylated-CpG island recovery assay (MIRA), in combination with microarray platforms a]PDE in vitro. For verification purposes, we have scrutinized the data obtained by our MIRA-assisted microarray analysis using the conventional combined bisulfite-restriction analysis (COBRA) We have recently developed a versatile DNA methylation detection method, the a]PDE. To fairly mimic a real life situation, we treated the cells repeatedly with biologically effective doses of B[a]PDE on a daily basis with 3-day-intervals in between the treatments. Of significance, we ensured that the administered doses of B[a]PDE did not severely affect the proliferative capacity of the cells because the maintenance of DNA methylation pattern is dependent upon DNA replication during cell division a]PDE with cellular DNA in carcinogen-treated normal human fibroblasts. In all cases, proliferatively-competent cell cultures treated with B[a]PDE did reach nearly full confluency, and required multiple rounds of passaging during the course of treatment.Using a well-defined validated cell culture model system and under strictly controlled experimental conditions, we have investigated the modulation of DNA methylation consequent to chronic exposure to the smoke-derived activated carcinogen, B[a]PDE-treated normal human fibroblasts, applying the MIRA-assisted microarray approach. As illustrated in a]PDE-treated DNA vs MIRA-enriched DMSO-treated DNA, (II) MIRA-enriched B[a]PDE-treated DNA vs Input non-enriched B[a]PDE-treated DNA, and (III) MIRA-enriched DMSO-treated DNA vs Input non-enriched DMSO-treated DNA. No PCR amplification was performed on the MIRA-enriched fractions before hybridization to the arrays. Applying very stringent bioinformatics criteria, we made comparative analysis between DNA methylation patterns found in various genomic regions in B[a]PDE-treated cells vs control. Overall, we observed strikingly similar patterns of DNA methylation in B[a]PDE-treated cells vs control. The remarkable resemblance of DNA methylation status between B[a]PDE-treated cells and control is shown at different representative genomic regions in a]PDE-treated cells vs control were deemed non-significant after statistical analysis. On average, the most pronounced fold-difference in the extent of DNA methylation between B[a]PDE-treated cells and control, as indicated by peaks, for example in a]PDE-treated cells vs control. For comparison, we have previously established the profile of DNA methylation in smokers' lung tumors vs adjacent non-tumorous tissues, as determined by parallel analysis vs normal lung) in the extent of DNA methylation reached more than 10 for several hundred hypermethylated targets, and more than 3 for several thousand hypomethylated targets a]PDE-treated cells and control (data not shown).Using NimbelGen tiling array , we have established the status of DNA methylation in chromosomes 7 and 8 in B[a]PDE-DNA adducts. The latter was to rule out the possibility that B[a]PDE-DNA adduction at methylated CpGs may adversely affect the formation of MBD2b/MBD3L1 complex at these dinucleotides, thus, impeding the MIRA pulldown procedure. As shown in a]PDE-DNA adducts.Furthermore, we performed an electromobility shift assay a]PDE-treated cells vs control, and established their methylation status, individually. In agreement with our MIRA-assisted microarray data, both the COBRA a]PDE-treated cells and control for all the analyzed targets. As shown in a]PDE-treated cells vs control.We validated the data obtained by MIRA-assisted microarray analysis using the conventional COBRA assay a]PDE-treated cells vs control. We adapted a published procedure in vitro .Because lung cancer is derived from the epithelial compartment of the lung, we also extended our DNA methylation analysis to normal human bronchial epithelial cells exposed repeatedly to BPDE a]PDE in vitro.Aberrant DNA methylation is the most-extensively studied epigenetic mechanism of carcinogenesis a]PDE, while allowing for the potential epigenetic effects to occur in proliferatively-competent cells. Using our high-throughput MIRA-assisted microarray analysis a]PDE-treated cells vs control. Methodologically, the MIRA enrichment procedure takes advantage of the property of the MBD2b/MBD3L1 complex to specifically bind methylated-CpGs a]PDE-DNA adduction at methylated CpGs may adversely affect the formation of MBD2b/MBD3L1 complex at these dinucleotides. Thus, we verified that MIRA-based analysis is appropriate for studying DNA methylation in B[a]PDE-treated cells herein.We set up a treatment protocol that resembled - as much as technically possible \u2013 a real life situation, in which normal human cells were exposed chronically to biologically effective doses of B[a]PDE-treated cells and control using conventional analysis of the representative targets identified by the high throughput MIRA-based analysis.Our MIRA-assisted microarray approach is a genome-scale interrogation assay for detecting aberrant DNA methylation, including global hypomethylation and locus/gene specific hypermethylation e.g., mutations in crucial genes that can directly or indirectly influence key pathways involved in DNA methylation. It can be envisaged that carcinogen-induced epigenetic or genetic alterations, which can affect the DNA methylation network, e.g., by up- or down-regulating the expression or activities of DNMTs or potential demethylase(s), or alternatively their upstream or downstream regulatory genes, may initiate global DNA hypomethylation and/or region-specific DNA hypermethylation, which can, in turn, give rise to lung tumorigenesis.Our study is unique in that we have comprehensively investigated the modulation of DNA methylation consequent to exposure to a smoke-derived carcinogen, in genomic regions of significance in lung cancer, in \u2018normal\u2019 human cells challenged with relevant doses of carcinogen. Previous studies have implicated a relationship between aberrant DNA methylation and smoking-related lung cancer e.g., naked DNA treatment with high concentrations of B[a]PDE e.g., DNMTs activities or expression in vitro modification of genomic DNA with extreme doses of B[a]PDE resulted in 12 adducts per 103 nucleotides. Such adduct levels of B[a]PDE are physiologically not attainable, e.g., leukocytes DNA from average smokers contains \u223c3 B[a]PDE-DNA adducts per 108 nucleotides comparability\u201d to normal human cells a]PDE-treated cells has proved unsuccessful a]PDE-DNA adducts with restriction enzyme digestion and/or PCR-amplification steps involved therein Previous studies by others have investigated indirectly and/or non-comprehensively the modulation of DNA methylation consequent to exposure to smoke-related carcinogens Currently, high throughput next-generation sequencing projects are analyzing large numbers of human lung tumors. These projects are poised to identify unique pathways that are adversely affected in human lung cancer. To infer causality, however, the aberration of these pathways does need to be experimentally recapitulated. For example, it is likely that next-generation sequencing of human lung tumors will elucidate genetic or epigenetic alterations that are specifically associated with exposure to tobacco smoke carcinogens. The relevance of such findings should be verified in validated experimental model systems under well-defined and controlled exposure conditions. As the upcoming data from the sequencing of smokers' lung-cancer genomes and epigenomes will become available, validated model systems should help delineate various aspects of the pathogenesis of this disease. Of importance, genetic or epigenetic mechanisms affecting specific pathways should be investigated so that their role as a driving force behind each individual pathway can be clearly established.a]PDE-treatment of normal human cells in the present study is a reasonable recapitulation of chronic exposure to smoke-derived carcinogens, albeit much shorter than what typical smokers' lung cells experience in vivo. Here, the resistance of normal human cells to undergo transformation in vitro prevented us from examining the possibility that aberrant DNA methylation may occur as a rare stochastic event in individual cells, which might then be selected for through a growth advantage a]PDE, are the culprit epimutagens that may cause aberrant DNA methylation in lung carcinogenesis.Lastly, we acknowledge that BPDE In conclusion, we have demonstrated that PLoS ONE Guidelines for Authors\u201d, all the authors of this manuscript confirm that, an ethics statement is not required for this work.Having read the \u201ca]PDE (1 \u00b5M) or control solvent [dimethylsulfoxide (DMSO)] were added to the media, and incubation was performed at 37\u00b0C for 20 minutes in the dark. Immediately after treatment, the cells were washed with PBS, fed with complete growth medium (DMEM plus 10% FBS), and cultivated for 3 days, after which an ensuing round of chemical treatment was carried out, as described above. When reaching approximately 90% confluency, all cultures underwent passaging (1 to 3 split) either 24- or 48 hours post chemical treatment. Three days after the 10th round of B[a]PDE treatment, the cells were harvested by trypsinization, and subjected to genomic DNA isolation using the DNeasy purification kit . The above-specified treatment protocol was based on our preliminary tests in which we established that normal human fibroblasts well-tolerate multiple rounds of treatment with 1 \u00b5M B[a]PDE, while having 83\u201389% survival rate and preserving their proliferation capacity by replicating once every 32\u201336 hours. All experiments were conducted in triplicate.The normal human fibroblast cells used in the present study are described in References a]PDE treatment in normal human fibroblasts, we used a standard immuno-dot-blot assay a]PDE-DNA adducts a]PDE-treated cells vs control was dot-blotted onto a nitrocellulose membrane using the Bio-Dot Microfiltration Apparatus . The membrane was laid over an absorbent paper pre-soaked with 0.4 N NaOH for 20 minutes at room temperature. Subsequently, the membrane was blocked by incubating in phosphate buffered saline plus 0.2% Tween 20 (PBS-T) containing 5% non-fat milk (NFM) at 4\u00b0C overnight. After multiple washes with PBS-T, the membrane was incubated with BP1-Ab antibody for 2 hours at room temperature. The membrane was washed thoroughly with PBS-T and further incubated with an anti-rabbit horseradish peroxidase conjugated immunoglobulin for 1 hour at room temperature . To reveal peroxidase activity, the membrane was stained with the Enhanced Chemiluminescence Detection System according to the manufacturer's instructions. The stained membrane was exposed to x-ray film, and the relative intensity of luminescence was determined using the Bio-Rad Imaging Equipment applying Quantity One image analyzer (Bio-Rad Laboratories).To verify the efficiency of B[a]PDE-treated normal human fibroblasts. We used our recently published protocol with some modifications a]PDE-treated cells vs control (30 \u00b5g each) was fragmented by sonication in a Branson Sonifier for five pulses of five seconds each, and one-minute interval among pulses. The average size of the fragments, determined by electrophoresis on 1.5% agarose gel, was between 500 to 800 bp. Purified GST-tagged MBD2b and His-tagged MBD3L1 proteins (60 \u00b5g each) were pre-incubated with a solution containing 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM EDTA, 1 mM DTT, 3 mM MgCl2, 0.1% Triton-X100, 5% glycerol, 25 mg/ml BSA, and sonicated JM110 dcm-) for 20 minutes at 4\u00b0C on a rocking platform. The fragmented DNA was then added to the pre-incubated mix, and binding of the MBD2b/MBD3L1 complex to methylated CpGs was achieved after an overnight incubation, as described above. The resultant was mixed with pre-washed MagneGST glutathione particles , and purified by magnetic capturing according to the manufacturer's instructions. The enriched MBD2b/MBD3L-bound methylated CpG fraction was further processed using the QIAquick PCR purification kit (Qiagen) to elute the methylated CpG fraction therein.To catalogue DNA methylation profile in chromosomal regions of significance in lung cancer, we performed MIRA-assisted microarray analysis a]PDE-treated cells vs respective DMSO-treated control or input DNA (non-enriched control) were labeled with Cy5-dCTP and Cy3-dCTP , respectively, using a BioPrime Array CGH Genomic Labeling kit from BPDE treated samples were identified by combining data from all three array designs, including (I) MIRA-enriched B[a]PDE-treated DNA vs MIRA-enriched DMSO-treated DNA, (II) MIRA-enriched B[a]PDE-treated DNA vs Input non-enriched B[a]PDE-treated DNA, and (III) MIRA-enriched DMSO-treated DNA vs Input non-enriched DMSO-treated DNA. First, methylation peaks in B[a]PDE treated samples were identified as described above using data on the array comparing MIRA-enriched B[a]PDE-treated DNA and Input non-enriched B[a]PDE-treated DNA and served as the potential candidates of hyper-methylated regions, which are regions only methylated in B[a]PDE-treated samples but not in DMSO-treated samples. The hyper-methylated regions were selected if they satisfied both of the following criteria: 1) the difference between the average log2 ratios of probes within these regions in B[a]PDE treated sample (MIRA-enriched B[a]PDE-treated DNA vs Input non-enriched B[a]PDE-treated DNA) and the average log2 ratios of probes in untreated sample (MIRA-enriched DMSO-treated DNA vs Input non-enriched DMSO-treated DNA) is more than 1 (2-fold); 2) the average log2 ratios of probes on array comparing MIRA-enriched B[a]PDE-treated DNA and MIRA-enriched DMSO-treated DNA were above 1 (2-fold higher comparing MIRA-enriched B[a]PDE-treated DNA vs MIRA-enriched DMSO-treated DNA). Similar analysis approach was used to identify hypo-methylated regions, except that the methylated regions in untreated sample were used as the starting point to look for difference and signal in MIRA-enriched B[a]PDE-treated DNA is more than 2-fold lower than that in MIRA-enriched DMSO-treated DNA.Hyper- and hypo-methylated regions in B[a]PDE-treated human fibroblasts. Briefly, total genomic DNA (1 \u00b5g) from B[a]PDE-treated cells vs control was subjected to sodium bisulfite treatment using the Qiagen EpiTect kit according to the manufacturer's instructions (Qiagen). The purified bisulfite-treated DNA was then analyzed by standard COBRA assay in vitro with M. SssI CpG methyltransferase , and served as positive control. For genomic sequencing, the PCR products obtained after bisulfite conversion of genomic DNA were cloned into the TOPO-TA cloning vector (Invitrogen Inc.) according to the manufacturer's instructions. Randomly selected clones from B[a]PDE-treated DNA vs control were sequenced using an ABI-3730 DNA Sequencer .To verify the data obtained by MIRA-assisted microarray analysis, we used both the COBRA Figure S1a]PDE is shown by chemical structures bound to the DNA fragments. Methylated and unmethylated CpGs are indicated as black and white lollipops, respectively.A schematic representation of MIRA-assisted microarray approach. Modification of DNA with B[(0.12 MB PDF)Click here for additional data file.Figure S2a]PDE-DNA adducts by immuno-dot-blot assay. Normal human fibroblasts were chronically treated in vitro with increasing concentrations of B[a]PDE vs control solvent (DMSO). Immediately after the end of last treatment, the cells were harvested and genomic DNA was subjected to immuno-dot-blot assay, as described in the text.Quantification of B[(0.29 MB PDF)Click here for additional data file.Figure S3a]PDE-treated cells and control by MIRA-assisted microarray analysis. Genomic DNA of normal human fibroblasts chronically treated with B[a]PDE vs control solvent (DMSO) was subjected to MIRA-assisted microarray analysis, as described in the text. Representative methylation array profiles from different chromosomal regions are shown with corresponding genomic coordinates (indicated on the top). MIRA-T/MIRA-UT'\u200a=\u200aMIRA-enriched B[a]PDE-treated DNA vs MIRA-enriched DMSO-treated DNA, 'MIRA-T/Input'\u200a=\u200aMIRA-enriched B[a]PDE-treated DNA vs Input non-enriched B[a]PDE-treated DNA, and 'MIRA-UT/Input'\u200a=\u200aMIRA-enriched DMSO-treated DNA vs Input non-enriched DMSO-treated DNA.Comparison of DNA methylation profiles between B[(0.06 MB PDF)Click here for additional data file.Figure S4a]PDE-DNA adducts determined by gel mobility shift assay. A 55-mer oligonucleotide, containing 1-10 symmetrically methylated CpG dinucleotides, was treated with increasing concentrations of B[a]PDE, and subsequently subjected to electromobility gel shift assay, as described earlier . Invariable formation of the MBD2b/MBD3L1 complex in the presence and absence of B[a]PDE-DNA adducts is indicated by an arrow. MBD2-Ab\u200a=\u200aNegative control, co-incubated with polyclonal antibody raised specifically against MBD2b protein. Representative result from the oligonucleotide with 10 methylated CpGs is shown.Affinity of the MBD2b/MBD3L1 complex for methylated CpGs in the presence and absence of B[(0.23 MB PDF)Click here for additional data file.Figure S5Conceptual framework for the methylation detection assay in repetitive DNA elements. The assay is an adaptation of a published procedure , which involves primer amplification of the consensus sequences from the repetitive DNA elements followed by appropriate restriction digestion or direct sequencing . Adopted from Ref. .(0.03 MB PDF)Click here for additional data file."} +{"text": "Since the oncogenes c- myc and TGF\u03b1 are frequently overexpressed in human lung bronchiolo-alveolar adenocarcinomas, these mouse lines are useful as models for human lung bronchiolo-alveolar adenocarcinomas. The average life expectancies of hemizygous and homozygous c- myc transgenics were 14.25 months and 9.2 months, respectively, suggesting that a dosage effect of c- myc caused an accelerated bronchiolo-alveolar adenocarcinoma formation. First analyses of double transgenics, hemizygous for both c- myc and IgEGF, show that these mice develop bronchiolo-alveolar adenocarcinomas at the average age of 9 months, indicating that these oncogenes cooperate during the lung cancer formation. Our results demonstrate that c- myc and EGF are directly involved and cooperate with one another during formation of bronchiolo-alveolar adenocarcinomas in the lung. \u00a9 2001 Cancer Research Campaign http://www.bjcancer.comTransgenic mouse models were established to study tumorigenesis of bronchiolo-alveolar adenocarcinomas derived from alveolar type II pneumocytes (AT-II cells). Transgenic lines expressing the murine oncogene c-"} +{"text": "We have studied the effects of hypoxia on aminolaevulinic acid (ALA)-induced protoporphyrin IX (PpIX) synthesis in EMT6 monolayer cultures characterized by different cell densities and proliferation rates. Specifically, after ALA incubation under hypoxic or normoxic conditions, we detected spectrofluorometrically the PpIX content of the following populations: (a) low-density exponentially growing cells; (b) high-density fed-plateau cells; and (c) high-density unfed-plateau cells. These populations were selected either for the purpose of comparison with other in vitro studies or as representatives of tumour regions adjacent to (high-density fed-plateau cells) and further away from (high-density unfed-plateau cells) capillaries. The amount of PpIX per cell produced by each one of these populations was higher after normoxic ALA incubation. The magnitude of the effect of hypoxia on PpIX synthesis was dependent on cell density and proliferation rate. A 42-fold decrease in PpIX fluorescence was observed for the high-density unfed-plateau cells. PpIX production by the low-density exponential cells was affected the least by ALA incubation under hypoxic conditions (1.4-fold decrease), whereas the effect on the high-density fed-plateau population was intermediate (20-fold decrease). \u00a9 1999 Cancer Research Campaign"} +{"text": "It is therefore necessary to design robust scalable methods to enable efficient expansion of bona fide CSCs in vitro. Here, we evaluated the applicability of computer-controlled bioreactors combined with 3D aggregate culture and microcarrier technology, widely used in stem cell bioprocessing, for the expansion and enrichment of CSCs isolated from different types of solid tumors\u2014colorectal cancer (CRC) and non-small-cell lung cancer (NSCLC) from two patients. Results show that these culture strategies improved cell expansion and CSC enrichment. Both patient-derived CSC lines were able to grow on microcarriers, the best results being achieved for PPlus 102-L, Pro-F 102-L, Fact 102-L, and CGEN 102-L beads . As for 3D aggregate culture strategy, the cell proliferation profile was donor dependent. NSCLC cells were the only cells able to form aggregates and proliferate, and the flat-bottom bioreactor vessel equipped with a trapezoid-shaped paddle impeller was the most efficient configuration for cell growth (21-fold increase in cell concentration achieved in 8 days). Serum-free medium promotes CSC enrichment in both 3D aggregate and microcarrier cultures. The protocols developed herein for CSC expansion have the potential to be transferred to clinical and industrial settings, providing key insights to guide bioprocess design towards the production of enriched CSC cultures in higher quantity and improved quality.Cancer stem cells (CSCs) have recently raised great interest as a promising biological system for designing effective cancer therapies. The scarcity of CSCs Cancer stem cells (CSCs) represent a promising target for effective anticancer therapies , 2 as thIt has been reported that CSCs exist within almost every solid tumor , 6 at a In this work, computer-controlled stirred tank bioreactors combined with 3D cell aggregate cultures as well as microcarrier technology were applied for the first time to expand and enrich CSCs from two different patient-derived cell lines\u2014non-small-cell lung cancer (NSCLC) and colorectal cancer (CRC). The findings reported herein provide novel knowledge to guide cell bioprocess design towards the production of CSC in higher quantity and improved quality, which are key requisites for their application in drug discovery and in the development of new cancer therapeutics.CSC lines were established in Merck Biopharma, ImmunoOncology, following a proprietary protocol. Tumor cells were derived from lung and colorectal cancer patients and purchased from Indivumed . Classification of the tumors was large-cell carcinoma, NOS, and colorectal carcinoma. CSC lines (CRC and NSCLC) were routinely propagated in collagen I-coated T-flasks as described in supplemental online data.6 cell/mL and cultured during 8 days in two different bioreactor configurations\u2014round-bottom bioreactor vessel equipped with a pitched 4-bladde impeller (BR-R/P4b) and flat-bottom bioreactor vessel equipped with a trapezoid-shaped paddle impeller (BR-F/T) . Two culture medium formulations (serum-containing medium (SCM) and serum-free medium (SFM)) and four cell aggregate dissociation protocols were tested .CSC lines were inoculated as single cells in computer-controlled stirred tank bioreactors at a concentration of 0.25\u2009\u00d7\u2009104 cell/cm2) in ultra-low-attachment plates and cultured for 6 days at 37\u00b0C in a humidified atmosphere of 5% CO2 using SCM or SFM. Eight commercially available microcarriers were tested: Cytodex1\u2122, Cytodex3\u2122, PPlus 102-L, Pro-F 102-L, Fact 102-L, CGEN 102-L, Cytopore2\u2122, and CultiSpher\u00ae-S as described in the supplemental online data.CSC lines were inoculated as single cells with empty microcarriers (data not shown). For their expansion, different bioreactor configurations and aggregate dissociation protocols were evaluated. In addition, microcarrier-based cultures were investigated; eight different microcarriers and non-small-cell lung cancer (NSCLC) from two patients, when routinely cultured in static adherent culture systems, show percentage of ALDHem cells , 11, sup\u03bcm) [+ cells (>70%) , contrasting to SCM cultures where no increase was attained (NSCLC cells were able to grow as aggregates in computer-controlled stirred tank bioreactors independently of the medium used (SCM or SFM) Figures . Importa\u03bcm) . Notewors (>70%) . Culture/T; SCM) .+ subpopulations (74-83%) were obtained in less culture time (6 days) when compared to aggregate culture in bioreactors, the exception being Cytopore2\u2122 beads . In addinoculum) .+ subpopulations in relation to the inoculum were observed for cultures using SFM (6 cells/mL) tested was observed for PPlus 102-L, Fact 102-L, CGEN 102, and Pro-F 102-L beads . The culsing SFM . The twosing SFM , Table 1This work describes, for the first time, the successful application of computer-controlled stirred tank bioreactors combined with 3D aggregate cultures as well as microcarrier technology to expand and enrich human CSCs. Despite the fact that there is no universal culture strategy capable of embracing different types/patient-derived CSCs, the protocols developed herein for CSC expansion can be easily screened prior to their transfer to clinical and industrial settings. This study also provides key insights to guide bioprocess design towards scalable production of patient-derived CSCs with improved quality. This will potentiate their application in drug discovery and for the development of new cancer therapeutics."} +{"text": "Although three-way repeated measures analysis of variance did not reveal significant interactions for any assessment of morphology, significant simple (muscle x time) effects were observed for CSA (p = 0.002) and FL (p = 0.016). Specifically, average CSA changes favored the VL over the RF , while significant decreases in average FL were noted for the RF but not the VL . No other significant differences were observed. The findings of this study demonstrate the occurrence of non-homogenous adaptations in RF and VL muscle size and architecture following 8 weeks of high-intensity resistance training in resistance-trained men. However, training does not appear to influence region-specific adaptations in either muscle.Resistance training may differentially affect morphological adaptations along the length of uni-articular and bi-articular muscles. The purpose of this study was to compare changes in muscle morphology along the length of the rectus femoris (RF) and vastus lateralis (VL) in response to resistance training. Following a 2-wk preparatory phase, 15 resistance-trained men completed pre-training (PRE) assessments of muscle thickness (MT), pennation angle (PA), cross-sectional area (CSA), and echo-intensity in the RF and VL at 30, 50, and 70% of each muscle\u2019s length; fascicle length (FL) was estimated from respective measurements of MT and PA within each muscle and region. Participants then began a high intensity, low volume lower-body resistance training program, and repeated all PRE-assessments after 8 weeks (2 d \u2219 wk"} +{"text": "Anti-Dense Fine Speckled 70 (DFS70) antibodies are a common finding in clinical laboratory referrals. High prevalence of DFS70 autoantibodies in healthy population and usual negative association with Antinuclear Antibody (ANA)-associated autoimmune rheumatic diseases (AARD) were reported. The aim of this study was to evaluate the prevalence of DFS70 autoantibodies and their association with other autoantibodies in the context of a routine ANA referral cohort. Consecutive sera submitted for ANA screening were analyzed for anti-DFS70 antibodies by indirect immunofluorescence (IIF) then confirmed by immunoblotting. Anti-DFS70 positive samples were also assayed for a large spectrum of other circulating autoantibodies. The prevalence of anti-DFS70 antibodies was 1.7% in the whole population and 4.6% in the ANA-positive samples. Comparison between DFS70 IIF and immunoblotting showed an excellent correlation between the two methods. The prevalence of anti-DFS70 positive was significantly higher in females than in males . Of note, no concomitant autoantibodies were found in the DFS70-positive male group compared with DFS70-positive females group that showed other serum autoantibodies in the 51% of cases. Anti-DFS70 reactivity in male population may represent an useful biomarker predicting the absence of other autoantibodies. On the contrary, the serological profile of DFS70-positive females required further investigations in order to define the presence of concomitant disease-marker autoantibodies. The presence of ANAs is a serological hallmark of systemic autoimmune rheumatic diseases, but their presence in sera from healthy people was also reported2. Anti-Dense Fine Speckled 70 (DFS70) antibodies, also known as lens epithelium-derived growth factor (LEDGF), were recently identified as associated to a specific ANA IIF pattern characterized by irregularly distributed, fine-granular fluorescence of the nuclei in the interphase and of the metaphase chromatin6. Current knowledge of the DFS70/LEDGF autoantigen-autoantibody system identifies the antigen as a transcription co-activator able to upregulate some stress protective and inflammatory genes. This function could contribute to the cellular survival under environmental stress factors, both in health and in disease context8. The alteration of DFS70 function or structure may trigger disease pathogenesis and autoantibody elicitation. The autoantibodies are preferentially of the IgG class and target a conserved region in DFS70 C-terminal domain. The prevalence of anti-DFS70 antibodies was analyzed in different cohorts with value ranged from 0.8% to 16.6%22. These variations may be related to differences in the patient selection criteria and methodological aspects as different HEp-2 substrates, to inter-reader variability in pattern assignment and variation in screening dilution. Furthermore, in some studies, no confirmatory analyte-specific immunoassays were employed. Also, the agreements between DFS70 IIF suspicion and confirmation by specific assays varied widely among studies. The discrepancies were related to different antigen exposure and selection (full length LEDGF or selected antigenic region), analytical sensitivity/specificity and manufacturer\u2019s cut-off of the various confirmatory assays23. However, use of DFS70-specific immunoassays was recently suggested by a study showing a low accuracy in assessing the DFS70 IIF pattern by experienced technologists24.Antinuclear antibodies (ANAs) detection by indirect immunofluorescence (IIF) represents a sensitive routine method thus recommended as the screening test of choice by a study group of the American College of Rheumatology28. It was reported that none of anti-DFS70 positive subjects showed symptoms suggestive of an AARD after clinical follow-up of 4 years18.Anti-DFS70 antibodies were initially described in patients with interstitial cystitis and later in heterogeneous chronic inflammatory conditions, tumours and even in apparently healthy individuals, but their clinical impact is still unknown31. Understanding the serological and clinical profile of anti-DFS70 positive subjects thus could avoid inappropriate referral to care specialists and follow-up tests for healthy people. Accordingly, the aim of this study was to define the prevalence of anti-DFS70 antibodies in a routine diagnostic laboratory setting and the associated serum autoantibodies to support the clinical use of these markers.Some previous studies suggested that the isolated anti-DFS70 reactivity could be taken as biomarker to exclude AARD (Antinuclear Antibody (ANA)-associated autoimmune rheumatic diseases) from ANA-positive healthy individualsCollected samples consisted of serum specimens sent to the Immunopathology Laboratory of the San Carlo Hospital of Potenza by outpatients or inpatients for ANA analysis with IIF. Paediatric subjects were excluded. Confirmed IIF DFS70 positive adult patients were asked to participate in the present study. Recruited patients have given their informed consent. Collection of patient samples and laboratory procedures were carried out according to Common Regional Ethical Committee of Basilicata (CEUR) (Authorization Number: 705/2017).ANA were detected by commercial ANA HEp-2000 Indirect Immunofluorescent assays . ANA Kits were used according to the appropriate manufacturer\u2019s instructions. The screening dilution was 1:160. Automated instrument for IIF preparation and anti-human IgG specific fluorescein-labelled (FITC) conjugate were used. Subsequently, slides were assessed with a fluorescence microscope. For each well, 4 microscopic fields with 20x objective with Image Navigator were captured. This instrument is based on a conventional fluorescence microscope with a motorized stage, high-intensity LED light source, digital camera, computer and proprietary software. Manufacturer\u2019 Fluorescence Index (F.I.) cut-off value for HEp-2000 is equal to 30. Positive/negative interpretation based on a F.I. was evaluated during focusing and confirmed by expert-readers. Positive samples were also tested using the conventional serial dilution method to assign end-point titer. The titer was defined as the reciprocal of the highest dilution of serum that still shows immunofluorescent staining.The serum samples positive for DFS70-like pattern in IIF were further processed for detection of human IgG autoantibodies against DFS70 specificity (truncated sequence of the DFS70 antigen (residues 349\u2013435)) by Immunoblotting assay. In addition, confirmed anti-DFS70 positive samples, were also evaluated for the antibodies against Sm, U1-RNP, Sm/RNP, SSA/Ro60kD, SSB, Scl-70, PM-Scl 100, Ku, CENP-A/B, PCNA, Mi-2 antigens using immunoblotting kit by automatic Blu Diver Instrument (BDI) , Italy). During the automated test procedure, the BDI sequentially incubates the strips in the wells of ready-to use reagent cartridges. Human antibodies bind the corresponding specific antigen(s) on the membrane and enzyme activity leads to development of purple dots on the membrane pads. The intensity of the coloration is directly proportional to the amount of antibody present in the sample. Semi-quantitative interpretation was done by Dr Dot Software (Alifax) and scanning system using Arbitrary Unit (AU).Crithidia luciliae IIF Test , Italy); IgG class of anti-mitochondrial (AMA), anti-smooth muscle (ASMA), anti-liver/kidney/microsome type 1 (LKM), and anti-parietal cells (PCA) by triple IIF test on rat liver, stomach and kidney substrates (Alphadia); anti-neutrophil cytoplasmic myeloperoxidase (MPO) and proteinase 3 (PR3) through enzyme immunoassay performed on automatic Immunomat Serion ELISA analyzer as recommended by the manufacturer; anti-Saccharomyces Cerevisiae IgG/IgA (ASCAs) and anti-cardiolipin/\u03b22-GPI complex and \u03b22-GPI isolated protein IgG (aCL) using immunodot kit, a previously described diagnostic assay platform.Anti-DFS70 positive samples were tested for the presence of other serum autoantibodies: anti-extractable nuclear antigen (ENA) , anti-thyroid peroxidase (TPO), anti-thyroglobulin (TGAb) and anti-tissue transglutaminase (tTg)-IgA by a fully automated chemiluminescence analyzer LIAISON XL , Italy) using a \u2018Flash\u2019 chemiluminescence technology (CLIA) with paramagnetic microparticle solid phase; anti-dsDNA by the 2); categorical variables were compared using Fisher\u2019 Test. A p value\u2009<\u20090.05 was considered statistically significant.Statistical analyses were performed using the Graph Pad Prism statistical package . Statistical significance was calculated using the non-parametric Mann-Whitney-U test and correlations were analyzed by the Spearman\u2019s rank correlation test and R-squared analyses .Selected cohort included 3175 sera from consecutive adult subjects submitted for routine ANA testing. Only 45.8% (55/120) of the presumptive DFS70 pattern on the ANA-IIF test were confirmed by immunoblotting, therefore sera negative by dot blot method were included in ANA positive group. Anti-DFS70 antibodies versus inpatients and Rheumatology also confirmed the relationship between IIF titers and immunoblotting AU . More than half of the patients presenting DFS70 monopattern showed high titers (\u22651:640) Fig.\u00a0. Regress AU Fig.\u00a0.Figure 3Collected DFS70-positive serum samples were assayed for a large spectrum of other circulating autoantibodies. By comparing DFS70-positive males with females, it emerged that isolated anti-DFS70 reactivity was found only in the male group. In contrast, in the 51% of DFS70-positive females, concomitant serum autoantibodies were found. Most common detected autoantibodies were anti-TPO (16.0%), anti-TG (11.0%), anti-tTg-IgA (9.0%) and ANCA . Only two subjects had both anti-TPO and anti-TG antibodies. ASCAs were detected only in one DFS70-positive female, also positive for anti-TPO, anti-TG and anti-tTg-IgA; anti-cardiolipin in two other and anti-ENA (anti-Mi-2 antibody) in one which also showed anti-TPO antibody positivity. No anti-DFS70 positive female, carried concomitant anti-dsDNA, anti-PCA, AMA, ASMA and anti-LKM was found and remaining suffered from various diseases. In the latter group 3 had spondyloarthritis (SpA), 1 morphea, 1 undifferentiated connective disease (UCTD), 3 rheumatoid arthritis (RA), 1 alopecia areata, 1 Crohn\u2019s disease, 4 celiac disease (CD), 1 autoimmune thyroiditis (AT), 1 RA-UCTD overlap syndrome, 1 CD plus AT, and 1 SpA plus AT.22. In details, a rate of positivity ranged from 0 to 5% was found in blood donors, healthy children and in routine ANA screening cohort. Higher frequencies were reported in ANA-positive populations. In addition, some studies examined serum samples only by IIF without specific confirmatory assays, resulting in higher rate of positivity. It has been showed that not all samples displaying DFS70 IIF pattern were then confirmed by specific CLIA or ELISA methods33. We analyzed 3175 consecutive unselected samples screened for ANA during the routine work-up by IIF at screening dilution of 1:160, then confirmed by immunoblotting of sera displaying the anti-DFS70 reactivity on HEp-2 substrate in a large cohort of samples screened for ANA in clinical laboratory. However, because our study was performed with ANA-requested specimens from outpatients and inpatients, the overall frequency of anti-DFS70 antibodies in general population is unknown. Gender differences with greater prevalence of female (4:1) was observed in anti-DFS70 negative/ANA positive group and within the anti-DFS70 positive samples. This is an important finding since the increased immune reactivity in females predisposes them to developing AARD. A number of factors may underlie this striking gender difference and further studies are needed to better understand these findings.Previous studies using ANA-IIF test on HEp-2 substrates provide the detection of high-titer autoantibodies with typical DFS70 staining pattern in 1% to 16% of the studied cohorts. These heterogeneous results were obtained in different patient cohorts, including blood donors, healthy individuals, routine ANA cohorts, selected ANA positive healthy individuals and routine ANA positive subjectsRegarding referring sources of anti-DFS70 positive samples, a comparative analysis highlighted that greater prevalence of anti-DFS70 positive subjects referred from the outpatients, with value of 2.1% in 64.3% of the DFS70 IIF-positive sera, with a titer greater than 1:2560 found only in one sample. Titer distribution revealed no statistically differences when compared with other ANA positivity (p\u2009>\u20090.05) or full-length DFS70 antigenic sequenceion Fig.\u00a0.et al., found a high prevalence of disease-related autoantibodies in anti-DFS70 positive patients, but of note, their cohort included only 22 anti-DFS70 positive females. Noteworthy, our data evidenced isolated reactivity of anti-DFS70 autoantibodies in male group, and high prevalence of disease-marker autoantibodies in females (51%). In particular, most common detected autoantibodies were anti-TPO, anti-TG, anti-tTg-IgA and ANCA (Fig.\u00a0We also analyzed the simultaneous presence of a broad spectrum of other circulating disease-markers autoantibodies. Muro females 1%. In paMajor strengths of our study are the use of anti-DFS70 confirmatory assay to avoid false positive results and the gender analysis in appraisal of the anti-DFS70 antibodies prevalence. The prevalence of anti-DFS70 was previously assessed in different cohorts mostly smaller than ours, therefore, we believe that our anti-DFS70 positive cohort should be considered suitable for a preliminary evaluation although a future larger confirmatory study is needed. In contrast, the scarce number of anti-DFS70 positive males could be considered inadequate to draw any firm conclusions. Therefore, based on low male prevalence, it is mandatory to screen larger cohort by future multicentre studies.According to previous observations performed on routine ANA cohorts, our prevalence of anti-DFS70 positivity was 1.7% of adult screened population. Serological profile of anti-DFS70 positive females required further clinical and laboratory investigations in order to define the presence of associated disease-marker autoantibodies and their clinical significance. In contrast, isolated anti-DFS70 specificity in male population suggests that the DFS70 could be considered reliable screening indicator for absence of other circulating autoantibodies resulting in considerable cost-saving potential."} +{"text": "Pre-school children spend an average of two-hours daily using screens. We examined associations between screen-time on pre-school behavior using data from the Canadian Healthy Infant Longitudinal Development (CHILD) study.CHILD participant parents completed the Child Behavior Checklist (CBCL) at five-years of age. Parents reported their child\u2019s total screen-time including gaming and mobile devices. Screen-time was categorized using the recommended threshold of two-hours/day for five-years or one-hour/day for three-years. Multiple linear regression examined associations between screen-time and externalizing behavior (e.g. inattention and aggression). Multiple logistic regression identified characteristics of children at risk for clinically significant externalizing problems (CBCL T-score\u226565).n = 24). Children with more than 2-hours of screen-time/day had a 7\u00b77-fold increased risk of meeting criteria for ADHD . There was no significant association between screen-time and aggressive behaviors (p>0.05).Screen-time was available for over 95% of children with CBCL data. Mean screen-time was 1\u00b74 hours/day at five-years and 1\u00b75 hours/day at three-years. Compared to children with less than 30-minutes/day screen-time, those watching more than two-hours/day (13\u00b77%) had a 2\u00b72-point increase in externalizing T-score ; a five-fold increased odd for reporting clinically significant externalizing problems ; and were 5\u00b79 times more likely to report clinically significant inattention problems . Children with a DSM-5 ADHD T-score above the 65 clinical cut-off were considered to have significant ADHD type symptoms (Increased screen-time in pre-school is associated with worse inattention problems. Childhood screen-time has increased over the years\u20134. IncreThere has been a significant increase in screen options in recent years, from device choices to streaming content, with rising concern that screen-time may have negative consequences for mental health. StudiesWe analyzed data from the population-based Canadian Healthy Infant Longitudinal Development (CHILD) birth cohort study to determine associations between screen-time and behavioral outcomes at age 5 years. Prolonged screen-time may displace time spent in other activities such as active play; important to promoting development in young children, 22. Thewww.childstudy.ca). CHILD is a naturalistic observational study initially designed to examine gene-environment interactions on the development of asthma and atopy[This study involved a population-based sample of 3,455 children recruited in four Canadian from the CHILD study or standardized questionnaires . A detailed overview of the covariates included in this analysis is provided in the Screen-time (hh:mm) was assessed at ages three and five-years. Parents reported their child\u2019s total screen-time/day, which included watching TV/DVD\u2019s, using a computer, tablet, mobile phone, or playing video games. Screen-time was grouped into three categories based on the recommended Canadian 24-hour Movement Guidelines for children 5\u201313 years(6): 1) less than 30-minutes/day; 2) between 30-minutes and two-hours daily; or 3) more than 2-hours. The upper threshold for total screen-time at age three-years was adjusted to one-hour per day based on the Canadian 24-hour Movement Guidelines for Young Children. The Caninternalizing problems , externalizing problems and total problems [The Child Behavior Checklist (CBCL) preschool version was compproblems), 29. Theproblems). Higher problems).Children with reported developmental or genetic disorders, such as autism, diagnosed by their healthcare professional, (n = 71) were removed from all analyses. Characteristics of those families who provided CBCL data were compared to those without CBCL data using chi-squared analysis for categorical variables and t-test for continuous variables.t-test for dichotomous predictors and linear regression for continuous variables, were used to identify associations between screen-time categories (primary exposure variable), sleep duration, sleep disordered breathing (SDB), physical activity, child and family characteristics and CBCL assessed externalizing (primary outcome), internalizing, and total behavior problems (secondary outcomes) at five-years.Univariate analyses, p\u22640\u00b705). The final model was determined based on the Akaike Information Criterion (AIC) where the lowest values indicated the best model fit. Missing values for all covariates were replaced with the mean for continuous for variables and the reference for categorical variables. A dummy variable was included in the analysis in order to account for the mean replacement for continuous variables. A sensitivity analysis was conducted to explore associations of movement behaviors known to interact and influence one another in accordance with the 24- Canadian 24-hour Movement Guidelines[p\u2019s\u22640\u00b705). We completed a sensitivity analysis (multiple logistic regression) to examine the association between screen-time and clinically significant behavior problems using a cut-off CBCL T-score of \u226565. Statistical analysis was completed using STATA, version 14.Multiple linear regression (entry method) was used to assess the association between screen-time and behavior problem scores while adjusting for child gender and factors significant in univariate analysis children had CBCL data at five-years. Those with CBCL data had a higher family income and were more likely to be Caucasian see . Parentsn = 28), while 2\u00b75% of children (n = 61) exhibited clinically significant internalizing behavior problems. Less than 1% of children (n = 18) had both clinically significant internalizing and externalizing behavior problems.Clinically significant externalizing behavior problems (T-score \u226565) was observed among 1\u00b72% of children (SD = 9\u00b79) than girls . Boys were more likely to be classified as having clinically significant (T-score \u226565) externalizing behavior problems than girls . There were no differences by gender observed for internalizing behavior problems.Boys had a higher CBCL externalizing T-score of children were exposed to more than 2-hours of screen-time/day while 83% of children met the Canadian recommended[Screen-time data was available for 96% 2322/2427) of participants whose parents had completed the CBCL questionnaire. At five-years, over 13% are presented in p\u22640\u00b7001; model 1, p\u22640\u00b7001). There were no significant interactions between screen-time and gender, physical activity, school enrolled, sleep duration, SDB, or parenting stress . The association between screen-time at five-years and increased externalizing behavioral morbidity remained significant when controlling for daily reported hours of screen-time at three-years (p\u22640\u00b705).Children exposed to more than 2-hours of screen-time had a 2\u00b72-point increase in externalizing behavior problem T-score : Parents of children exposed to more than two-hours of screen-time were 5-times more likely to report clinically relevant externalizing behavior problems . More than 2-hours of screen-time/day was significantly associated with an ADHD score above the clinical-cut-off of 65 after adjusting for physical activity, parent reported SDB symptoms, SES, breastfeeding, parenting stress, and maternal depression which limited our ability to determine directionality. As such, it is possible parents may respond to children who exhibit externalizing behavior difficulties by offering more screen-time or using increased opportunity for screen-time as a self-soothing strategy. Although we identified prior studies in school-aged children, 36, 37 7 which lOur results suggest that physicians and educators promote limiting young children\u2019s screen-time exposure in line with recommended guidelines, 41, 42.We provide results from one of the largest birth cohort studies to examine screen-time exposure and behavioral morbidity in pre-school children. Screen-time above the two-hours threshold at 5-years was associated with an increased risk of clinically relevant externalizing morbidity and specifically inattention problems. The association between screen-time and behavioral morbidity was greater than any other risk factor including sleep, parenting stress, and socio-economic factors. Our findings indicate that pre-school may be a critical period for supporting parents and families on education about limiting screen-time and supporting physical activity.S1 File(DOCX)Click here for additional data file.S2 File(DOCX)Click here for additional data file.S3 File(DOCX)Click here for additional data file.S1 TableaAnalyzed by One-way ANOVA *p\u22640.05 based on Tukey post hoc test.Note: SD = standard deviation; SES: socioeconomic status; SDB = sleep disordered breathing (DOCX)Click here for additional data file.S2 TableCaption: CI = confidence interval; SDB = sleep disordered breathing.(DOCX)Click here for additional data file.S3 Tablea Analyzed by One-way ANOVA *p\u22640.05 based on Tukey post hoc test.Note: SD = standard deviation; SES: socioeconomic status; SDB = sleep disordered breathing (DOCX)Click here for additional data file.S4 TableCaption: PSI-SF = Parenting Stress Index-Self Report, higher score presents increased levels of parenting stress; P-CDI = Parent-Child Dysfunction Index, higher scores reflect increased perceived difficulties; CES-D = Centre for Epidemiological Studies\u2014Depression, higher scores represent increased maternal symptoms of depression.(DOCX)Click here for additional data file.S5 TableNote: SD = standard deviation; SES: socioeconomic status; SDB = sleep disordered breathing a Analyzed by One-way ANOVA *p\u22640.05 based on Tukey post hoc test.(DOCX)Click here for additional data file.S6 TableCaption: PSI-SF = Parenting Stress Index-Self Report, higher score presents increased levels of parenting stress; P-CDI = Parent-Child Dysfunction Index, higher scores reflect increased perceived difficulties; CES-D = Centre for Epidemiological Studies\u2013Depression, higher scores represent increased maternal symptoms of depression.(DOCX)Click here for additional data file.S7 TableNote: SD = standard deviation; SES: socioeconomic status; SDB = sleep disordered breathing a Analyzed by One-way ANOVA *p\u22640.05 based on Tukey post hoc test.(DOCX)Click here for additional data file.S8 TableCaption: PSI-SF = Parenting Stress Index-Self Report, higher score presents increased levels of parenting stress; P-CDI = Parent-Child Dysfunction Index, higher scores reflect increased perceived difficulties; CES-D = Centre for Epidemiological Studies\u2014Depression, higher scores represent increased maternal symptoms of depression.(DOCX)Click here for additional data file.S9 TableCaption: SDB = Sleep Disordered Breathing, based on 6 items; PCD-I = Parent-Child Dysfunction Index, higher scores represents; PSI-SF = Parenting Stress Index-Self Report, higher score presents increased levels of parenting stress; CES-D = Centre for Epidemiological Studies\u2014Depression, higher scores represent increased maternal symptoms of depression.(DOCX)Click here for additional data file."} +{"text": "Immune checkpoint inhibitor (ICI) activates host\u2019s anti-tumor immune response by blocking negative regulatory immune signals. A series of clinical trials showed that ICI could effectively induce tumor regression in a subset of advanced cancer patients. In clinical practice, a main concerning for choosing ICI is the low response rate. Even though multiple predictive biomarkers such as PD-L1 expression, mismatch-repair deficiency, and status of tumor infiltrating lymphocytes have been adopted for patient selection, frequent resistance to ICI monotherapy has not been completely resolved. However, some recent studies indicated that ICI resistance could be alleviated by combination therapy with anti-angiogenesis treatment. Actually, anti-angiogenesis therapy not only prunes blood vessel which is essential to cancer growth and metastasis, but also reprograms the tumor immune microenvironment. Preclinical studies demonstrated that the efficacy of combination therapy of ICI and anti-angiogenesis was superior to monotherapy. In mice model, combination therapy could effectively increase the ratio of anti-tumor/pro-tumor immune cell and decrease the expression of multiple immune checkpoints more than PD-1. Based on exciting results from preclinical studies, many clinical trials were deployed to investigate the synergistic effect of the combination therapy and acquired promising outcome. This review summarized the latest understanding of ICI combined anti-angiogenesis therapy and highlighted the advances of relevant clinical trials. Immune checkpoint molecules mainly includes programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte antigen-4 (CTLA-4) . HoweverCompared with immune checkpoint inhibitor (ICI), anti-angiogenesis therapy attracted intensive attention earlier. Angiogenesis, mainly indicating the generation of new vessels from pre-existing ones, occurs in many physiological processes . In the The initial aim of anti-angiogenesis therapy is to reduce blood supply and starve tumor cell of oxygen and nutrients . However+ T cells infiltrate; (II) excluded infiltration type, where abnormal angiogenesis and immunosuppressive reactive stroma prevent the infiltration of T cell; (III) immune ignorance type, where tumor mutation burden and the expression of antigen presentation machinery marker are low .\u201370.68\u201370+/TIM-3+/LAG-3+ T cell) significantly increased in mice resistant to anti-PD-L1 [Meder et al. conducted a preclinical study by genetically engineered small-cell lung cancer (SCLC) mouse . All SCLti-PD-L1 . Howeverti-PD-L1 . To confti-PD-L1 . After sti-PD-L1 .+ T cell [In line with the finding of Meder and colleagues, Voron et al. observed that anti-VEGF could selectively inhibit the expression of immune checkpoint molecules on intratumoral CD8+ T cell . Voron e+ T cell . Therefo+ T cell .+/CD69+ CD8+ T cell increased [+ T cells accompanied the high expression of PD-L1 on tumor cell because of IFN-\u03b3-mediated negative feedback regulatory mechanism [Apart from VEGF signaling pathway, angiopoietin-2 (ANGPT2)/Tie 2 is another pro-angiogenic pathway which relates with resistance to anti-VEGF treatment \u201377. Schmncreased . In the echanism . Combinaechanism . The resechanism .+ CD8+ and IFN-\u03b3+ CD4+ T cell increased by twofold in pancreatic neuroendocrine tumor and mammary carcinoma. However, IFN-\u03b3+ CD8+ T cell modestly increased in just 50% of glioblastomas [Allen et al. investigated the efficacy of combination therapy of anti-PD-L1 (anti-PD-L1 mAb: B20S) and anti-VEGFR2 (anti-VEGFR2 mAb: DC101) in mice bearing pancreatic neuroendocrine tumor, mammary carcinoma, and glioblastoma . Combinalastomas . As the lastomas . Apart flastomas . Immunohlastomas \u201382. Similastomas . LT\u03b2R silastomas . Activatlastomas .As discussed above, the interaction between immunity and angiogenesis renders tumor immune escape and treatment resistance. Based on the encouraging results of preclinical studies, many clinical studies have been conducted to investigate the synergistic effect of ICI plus anti-angiogenesis therapy in patients Table\u00a0. SchmidtNCT00790010 is a phase I clinical trial to explore the effect of ipilimumab (anti-CTLA-4) plus bevacizumab (anti-VEGF) in metastatic melanoma patients . All 46 + T cells [Inspired by the significantly synergistic effect of anti-CTLA-4 plus anti-VEGF therapy, Wallin et al. conducted the clinical study (NCT 01633970) to explore the efficacy of anti-PD-L1 combined with anti-VEGF . NCT0163 T cells . Dynamic T cells . The eme T cells .In 2018, the results of the phase 3 study IMpower150 (NCT02366143) were reported. This study was aimed to evaluate the effect of combination therapy consisting of atezolizumab, bevacizumab, and chemotherapy in treatment-na\u00efve metastatic non-squamous non\u2013small-cell lung cancer patients . Among tIn most clinical studies by far, combination strategies consist of ICI and anti-angiogenesis mAb bevacizumab. In 2018 Choueiri et al. firstly reported the efficacy of avelumab plus anti-angiogenesis TKI axitinib therapy in treatment-na\u00efve advanced clear-cell renal-cell carcinoma . JAVELIN Renal 100 (NCT02493751) is a phase 1b study aiming to evaluate safety, pharmacokinetics, and pharmacodynamics of avelumab (anti-PD-L1) plus axitinib (VEGFR TKI) therapy . For a tLater, Xu et al. reported the results of another phase 1 clinical study (NCT02942329) which aimed to investigate the efficacy of SHR-1210 (anti-PD-1 antibody) plus apatinib (VEGFR2 TKI) in refractory hepatocellular cancer (HCC), gastric cancer (GC), and esophagogastric junction cancer (EGJC) patients . 15 patiFor ICI therapy, an important factor contributes to treatment discontinuation is the severe adverse event. Most adverse events are related with hyperactive immune response, showing T cell mediated auto-immune like inflammation . DisturbA series of preclinical and clinical studies indicated the mutually enhanced effect of anti-angiogenesis and ICI therapy. On the one hand, anti-angiogenesis blocks the negative immune signals by increasing ratio of anti\u2212/pro-tumor immune cell and decreasing multiple immune checkpoints expression. On the other hand, ICI therapy could restore immune-supportive microenvironment and promote vessel normalization. Besides, because of enhanced drug delivery benefiting from vessel normalization, smaller dose of ICI could be applied which reduces the risk of adverse event. A main problem needing to resolve is how to optimize the dose and schedule of anti-angiogenesis in the combination therapy. Extending window of vessel normalization and avoiding excessive vessel pruning would facilitate the maximized survival benefit. We believe ICI plus anti-angiogenesis would be a promising strategy to overcome treatment resistance and improve patients\u2019 prognosis."} +{"text": "We report two complete proviral genome sequences of human T-cell leukemia virus type 1 (HTLV-1) isolated from the peripheral blood specimens of acute type adult T-cell leukemia (ATL) patients in Oita Prefecture, Japan. Retroviridae, genus Deltaretrovirus, and an etiological agent for adult T-cell leukemia (ATL) (Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus belonging to the family ia (ATL) . Currentia (ATL) . HTLV-1 ia (ATL) . The expia (ATL) or epigeia (ATL) of the pia (ATL) .Here, we report the complete HTLV-1 provirus genome sequence of OATL9, a novel HTLV-1/EBV-infected B-cell line isolated from the acute type ATL patient . The stuIt remains to be elucidated how Tax and HBZ contribute to the viral tumorigenesis or how the integration site of the virus affects its gene expression property. We are conducting molecular biological analysis on this cell line to elucidate its mechanistic properties for hematologic immortalization ability.LC183873 and LC378575 .The genome sequences of these HTLV-1 proviral genomes have been deposited in GenBank under accession numbers"} +{"text": "BRAF-like and RAS-like thyroid cancers with up-regulation of genes involved in oxidative phosphorylation and cell adhesions, respectively. The third metagene signature representing up-regulation of immune-related genes further segregated BRAF-like and RAS-like PTCs into their respective subgroups of immunoreactive (IR) and immunodeficient (ID), respectively. BRAF-IR PTCs showed enrichment of tumor infiltrating immune cells, tall cell variant PTC, and shorter recurrence-free survival compared to BRAF-ID PTCs. RAS-IR and RAS-ID PTC subtypes included majority of normal thyroid tissues and follicular variant PTC, respectively. Immunopathological features of PTC subtypes such as immune cell fraction, repertoire of T cell receptors, cytolytic activity, and expression level of immune checkpoints such as and PD-L1 and CTLA-4 were consistently observed in two different cohorts. Taken together, an immune-related metagene signature can classify PTCs into four molecular subtypes, featuring the distinct histologic type, genetic and transcriptional alterations, and potential clinical significance.Papillary thyroid carcinoma (PTC) represents a heterogeneous disease with diverse clinical outcomes highlighting a need to identify robust biomarkers with clinical relevance. We applied non-negative matrix factorization-based deconvolution to publicly available gene expression profiles of thyroid cancers in the Cancer Genome Atlas (TCGA) consortium. Among three metagene signatures identified, two signatures were enriched in canonical Most patients with PTC have excellent prognosis after surgery. They are more likely to die from other diseases. However, recurrence and death can occur more than 30 years after initial diagnosis of PTC [The incidence of thyroid cancer has been rapidly increasing worldwide, especially in Korea (15 times of increase) over the past few decades . In the PTC is a heterogeneous disease characterized by more than 10 histologic variants with disparate molecular phenotypes and clinical behaviors . MicroscBRAF-like and RAS-like PTCs significantly differ in their genomic, transcriptomic, epigenomic, and proteomic profiles [BRAF-like PTCs have classical papillary morphology and high levels of mitogen-activated protein kinase (MAPK) pathway signaling whereas RAS-like PTCs show a follicular growth pattern and low levels of MAPK pathway signaling [BRAF-like PTCs are more clinically and molecularly heterogeneous than RAS-like tumors. In the initial analysis of the TCGA dataset, data on first recurrence were unavailable. The risk of recurrence was evaluated using the American Thyroid Association (ATA) risk stratification system [BRAF V600E are related to immune function pathways [BRAF V600E mutation have lower levels of immune/inflammation function gene expression and lymphocyte infiltration than BRAF-wild type PTCs. Another study using RNA sequencing data of TCGA has found that increased immune cell enrichment scores in PTCs are associated with low thyroid differentiation score and BRAF V600E mutation while the expression of immunosuppressive markers is higher in BRAF V600E positive PTCs [BRAF V600E and TERT promoter mutations is associated with aggressive clinical behavior and poor clinical outcome in PTC patients [Recent advances in next-generation sequencing based cancer genomic research have explored mutational and transcriptional landscape of PTCs. The Cancer Genome Atlas (TCGA) study of PTC has demonstrated that profiles . BRAF-liignaling . BRAF-lin system and metan system . A studypathways . PTCs wiive PTCs . These fpatients ,11,12. HA number of gene expression-based algorithms have been proposed for latent feature selection or deconvolution assuming that bulk-level sequencing of primary tumors represents an admixture of heterogeneous cell populations. As a technique of blind source separation , non-negBRAF-like and RAS-like PTCs. These identified PTC clusters were compared with previously proposed multiomics-based PTC clusters. They were also evaluated for immunologic features including immune cell abundance estimated by CIBERSORT algorithm. Of note, we evaluated the clinical utility of PTC clusters by correlative analyses with clinicopathological features including recurrence-free survival. Our observed findings were largely consistent across independent cohorts of our PTC expression profiles and a public one [In the present study, we obtained a large-scale gene expression profile of PTC including tumor-adjacent normal thyroid tissue from TCGA consortium . We applblic one .To identify key metagene signatures that could explain heterogeneous PTC gene expression profiles in reduced dimensions, we performed NMF clustering of 568 RNAseq-based gene expression profiles (501 PTCs with 8 matched metastatic and 59 matched tumor-adjacent non-tumors) available in TCGA consortium [BRAF-RAS classes annotated according to the presence of driver mutations of BRAF and RAS genes, metagene signatures 1 and 3 were mostly enriched in RAS-like and BRAF-like PTC classes, respectively, as shown in RAS-signature\u2019 and \u2018BRAF-signature\u2019, respectively. RAS-like and BRAF-like PTCs have been previously proposed as two molecular subtypes of PTC including a majority of histologic classes of predominantly follicular growth pattern and papillary growth pattern , respectively [RAS-like PTCs, as shown in RAS-like and BRAF-like PTCs into their respective two subgroups. In the case of RAS-like subtypes, metagene signature 2 was enriched in NMF cluster 1 that included all normal thyroid tissues while NMF cluster 2 was relatively enriched with follicular variant of PTC (FVPTC), as shown in BRAF-like PTCs: NMF cluster 3 PTCs were more enriched with TCVPTC than NMF cluster 4.ectively . In addiBRAF-like PTCs with enrichment of metagene signature 3/BRAF-signature. Those belonging to \u2018classical 2\u2019 cluster showed an enrichment of metagene signature 2. Although the presence of multiple PTC clusters has been previously proposed, multiomics data-driven PTC clusters have not been properly evaluated and a functional interpretation of metagene signature 2 is largely unknown.The clustering of PTC based on multiomics data such as mRNA, miRNA, and DNA promoter methylation has proposed the presence of multiple PTC clusters . Thus, wRAS-like and BRAF-like thyroid cancers can activate the metabolic pathway and cell adhesion molecule/extracellular matrix receptor interaction pathways, respectively [CCL21 and CCL19 encoding C-C motif chemokine ligands 21 and 19 precursors, respectively, as shown in RAS-IR, RAS-ID, BRAF-IR, and BRAF-ID, respectively.To functionally interpret metagene signatures, we performed pre-ranked gene set enrichment analysis (GSEA) . High-raectively , consistr = 0.82, 0.73, 0.66, and 0.60, respectively), as shown in BRAF-IR while they were suppressed in NMF cluster 2/RAS-ID and 4/BRAF-ID, as shown in r = \u22120.71) is likely due to the relationship between leukocyte fraction and tumor purity [We further evaluated other genomic-pathologic features associated with immune cell abundance such as tumor purity , total mr purity .p < 0.05; ANOVA). Various immune cell subtypes including B cells, T cells, macrophage M1, dendritic cells, and mast cells showed differential enrichments. The majority of them were enriched in NMF clusters one and three, consistent with the enrichment of immune-signature (metagene signature 2). These findings support that levels of metagene signature 2/immune-signature are associated with immune activity or the abundance of tumor infiltrating immune cells. The representative histologic images from each cluster are shown in We further explored which immune cell subsets were differently enriched across four NMF clusters by using the CIBERSORT algorithm . Figure BRAF-like molecular phenotype, and high risk of recurrence. In subgroup analysis between NMF clusters three and four, cluster three patients had significantly higher rate of TCVPTC, extrathyroid extension, thyroiditis, and advanced tumor stage.Baseline characteristics of NMF clustering subgroups of patients at initial surgery are shown in p = 0.032), extrathyroidal extension (p = 0.008), pT3-4 stage (p = 0.007), lymph node metastasis (p = 0.003), high burden of nonsynonymous mutations (p = 0.020), and NMF cluster 3 (p = 0.008). In multivariate analysis, as shown in p = 0.001), high burden of nonsynonymous mutations (p = 0.021), and NMF cluster 3 (p = 0.010) remained significant factors associated with the status of disease recurrence.In univariate logistic regression analysis, as shown in RAS-like/BRAF-like clustering, mRNA clusters (clusters 1\u20135), microRNA clustering (clusters 1\u20136), or DNA methylation clustering . In multivariate Cox regression analysis, lymph node metastasis, high burden of nonsynonymous mutations, NMF cluster three had a negative influence on recurrence free survival, as shown in In Kaplan\u2013Meier analyses of PTC recurrence-free probability, as shown in RAS-, BRAF- and immune-signatures) to yield four NMF clusters with a similar enrichment pattern of metagene signatures and the association with immune-histological presentations, as shown in RAS-IR and 3/BRAF-IR included the majority of normal thyroid tissue and TCVPTC, respectively. The level of immune-related features such as CYT, immune scores from ESTIMATE algorithm, TCR richness, and the expression level of CTLA-4 were highly elevated in NMF3/BRAF-IR PTCs. Correlation coefficients were also high with immune signature, as shown in p < 0.05; ANOVA) were illustrated, demonstrating that T cells and macrophages were highly infiltrated in tumors belonging to IR clusters (NMF3 and NMF1) compared to those belonging to ID clusters (NMF2 and NMF4), as shown in r > 0.7) with the level of signature 2 in SNU cohort. These findings suggest that our three metagene signature-based PTC classification is consistent across a number of PTC expression profiles. In addition, the relationship between NMF clusters, histologic type, and ATA recurrence risk were investigated across three cohorts, as shown in To evaluate three metagene signature-based classification of PTC in independent gene expression datasets, we performed RNAseq for 27 thyroid tumors and 14 tumor-adjacent normal thyroid tissues. A total of 41 thyroid expression profiles (CMC cohort) were subjected to metagene projection with three metagene signatures (RAS-like and BRAF-like tumors was further divided into NMF1 (RAS-IR), NMF2 (RAS-ID), NMF3 (BRAF-IR), and NMF4 (BRAF-ID). Normal thyroid tissue was enriched in NMF1. NMF3 showed BRAF-like molecular features and enrichment of tumor infiltrating immune cells. The level of immune-signature was suppressed in NMF clusters two and four. NMF3 was an independent prognostic marker for disease recurrence in TCGA cohort. We confirmed that immunopathological features and NMF classification of PTC developed from TCGA cohort were consistently replicated in SNU and CMC cohorts. Interestingly, NMF2 was enriched with miFTC and NIFTP in SNU and CMC cohorts, respectively. We identified clinically relevant metagene signatures that could classify PTCs into four groups and predict disease recurrence after initial treatment. The binomial classification of PTCs into BRAF V600E and RAS activating mutations [BRAF and RAS mutations in PTCs and their mutually exclusivity have led to the discovery of two molecular subtypes of PTC\u2013BRAF-like and RAS-like PTCs [BRAF-like and RAS-like PTCs had similar recurrence-free survival, as shown in Robust molecular classification of thyroid tumors with clinical implication is challenging. MAPK pathway is activated in approximately 70% of PTCs mainly by utations . The preike PTCs ,25. AlthBRAF- and RAS-like PTC subtypes. We noted that the remaining metagene signature was enriched with immune-related genes, suggesting that expression-level immune activity could be an additional feature in PTC categorization. Among the four PTC clusters, BRAF-IR/NMF3 cluster demarcated a subgroup of BRAF-like PTC with unfavorable prognosis, as shown in BRAF-ID PTCs, as shown in RAS-, BRAF- and immune-metagene signatures that can be projected onto microarray- or RNAseq-based PTC expression profiles are provided, as shown in NMF-based deconvolution technique has been proven to be useful in the decomposition of multiple cellular composition given that a bulk-level tumor transcriptome is a heterogeneous cellular admixture of tumor cells and tumor-infiltrating non-tumor cells such as immune and stromal cells . When apBRAF-IR compared to that in BRAF-ID PTCs, as shown in BRAF-IR PTCs may be candidates for PD1, PD-L1, or CTLA-4 blockade therapy. Along with unfavorable clinical outcomes of BRAF-IR PTCs, their potential eligibility to immune checkpoint blockade treatment should be investigated further.The recent success of immune checkpoint blockade treatment in various types of solid tumors ,27 with BRAF V600E mutation, low thyroid differentiation score, high expression of immunosuppressive markers , and shorter recurrence-free survival [BRAF mutation status, or expression of PD-L1 between the two groups. In the present study, expression levels of CTLA-4 and PD-L1 were up-regulated in BRAF-IR group with tumor infiltrating immune cells. The BRAF-IR group had the highest recurrence rate.Some studies have reported gene expression-based immunoprofiling of PTC using TCGA data. Na and Choi have employed gene expression-based PTC differentiation and immune scores . They obsurvival . Kuo et survival have divsurvival . HoweverBRAF-like and RAS-like PTCs instead of specific immune cell types. Across three cohorts examined, T lymphocytes and myeloid cells such as macrophages and dendritic cells were commonly overrepresented in IR PTCs. In the case of T cells, CD8+ lymphocyte infiltration within tumor cells has been generally considered as an unfavorable prognostic feature across cancers including thyroid cancers [CD8 mRNA level was associated with lymph node metastasis (p = 0.008) and BRAF-like group . Concomitant up-regulation of CYT scores in BRAF-IR PTCs suggest that these tumors are enriched with cytolytic CD8+ T cells. Up-regulation of CTLA-4 and PD-L1 is suggestive of the exhaustion of infiltrated T cells and may explain unfavorable clinical outcomes of this PTC subtype. Adverse effects of regulatory T cells (Tregs) in antitumor immune response should also be considered since Tregs are enriched in BRAF-/RAS-IR PTCs [BRAF-/RAS-IR PTCs. Tumor-associated macrophages (TAM) have been previously associated with clinical outcomes of PTC [Our analysis on the abundance of individual immune cell subsets showed overall enrichment of immune cells in cancers . Cunha e cancers . When we-IR PTCs . Myeloids of PTC . Furthers of PTC .n = 509) and tumor-adjacent normal thyroid tissues (n = 59) were obtained from TCGA consortium [https://gdac.broadinstitute.org). Among these 568 expression profiles, 501 were from primary PTC tumor tissues, 8 were matched metastatic tumors, and 59 were expression profiles of adjacent normal thyroid tissue. Clinicopathological information of these patients was obtained from TCGA data portal and the literature [We used two publicly available gene expression datasets. A total of 568 expression profiles for PTCs (http://www.ebi.ac.uk/data/view/PRJEB11591). We used FastQC and Trimmomatic [In addition to the TCGA cohort, we obtained gene expression data for 180 thyroid tumors including 25 follicular adenomas, 30 FTCs, 48 FVPTCs, 77 PTCs, and matched 81 normal thyroid tissues from a public resource . RNAseq mmomatic for qualmmomatic . Gene-lemmomatic . We callmmomatic .n = 9), TCVPTC (n = 7), invasive EFVPTC (n = 3), and NIFTP (n = 8). The enrollment of patients and the overall experimental process were approved by the Institutional Review Board of Seoul St. Mary\u2019s Hospital, the Catholic University of Korea (KC16SISI0709). Histological examination and tumor purity check were done by a board-certified pathologist. Tissue RNAs were extracted and converted into cDNAs. Sequencing library was prepared according to the manufacturer\u2019s instructions. Sequencing reads were generated using Illumina HiSeq2500 . Sequencing reads of FASTQ files were aligned and processed into gene-level expression profiles as described for SNU cohort. We describe the obtained 41 gene expression profiles as a Catholic Medical Center (CMC) cohort. The sequencing information of RNAseq is available in We performed RNAseq on an Illumina platform for 27 thyroid tumors and 14 matched normal specimens. The tumor set was composed of classical PTC (https://cran.r-project.org/package=NMF) was used to deconvolute log-transformed thyroid expression profiles of TCGA consortium. To determine the number of metagene signatures, we measured cophenetic correlation in a range of signature numbers (2 to 10 metagene signatures). The goal of NMF is to identify latent features in gene expression profiles by decomposing the original matrix into basis matrix or metagenes and metagene expression profiles [NMF implemented in R packages . Antigen retrieval was performed with high-pH EnVision FLEX Target Retrieval Solution (Agilent Technologies) for 30 min at 97 \u00b0C. Tissues sections were incubated with polyclonal rabbit anti-human CD3 antibody for 20 min at room temperature, followed by visualization with EnVision FLEX visualization system (EnVision/HRP for 20 min and chromogen substrate for 5 min). The specimens were then counterstained with Hematoxylin for 3 min.p values of less than 0.05 were considered to indicate statistically significant differences.Analysis of variance (ANOVA) was performed to compare means of gene expression values among groups. Relationships between clinicopathologic features and gene expression profiles were analyzed using parametric (chi-square test) and non-parametric (Fisher\u2019s exact) assessments where appropriate. Univariate binomial logistic regression analysis of variables was performed to determine whether clinicopathologic variables and molecular clustering were significantly associated with tumor recurrence. Disease recurrence free survival curves were plotted using the Kaplan\u2013Meier method. Statistical differences between survival curves were calculated using the log-rank test. For multivariate survival analysis of variables affecting disease-free survival, the Cox proportional-hazard model was used. All statistical values were calculated using Prism and statistical software program SPSS . BRAF-like PTCs. This new classification provides novel insights into our understanding of immune response in PTCs and clinical application of molecular classification for the treatment and management of this tumor.The immune-related metagene signature identified four clinically distinct subgroups of PTCs in the present study. The risk of recurrence of PTC after initial treatment was the highest in immune reactive"} +{"text": "This article comments on:et al. 2018. Three UDP-xylose transporters (UXTs) participate in xylan biosynthesis by conveying cytosolic UDP-xylose into the Golgi lumen in Arabidopsis. Journal of Experimental Botany 69, 1125\u20131134..Zhao X, Liu N, Shang N, A raft of recent studies, including a new paper by Plant science is approaching the vision of cell walls constructed \u2018fit for purpose\u2019 . More reMost nucleotide sugars are made from UDP-D-glucose (UDP-Glc) in the cytosol . This cein planta, of carbon bodies ending up as cell wall-associated xylose: glucuronoxylan (GX), xyloglucan (XG), xylogalacturonan (XGA) and complex N-linked glycan (CGL). Green arrows indicate the fate of carbon bodies ending up as arabinofuranose bound to cell wall polymers: rhamnogalacturonan I (RG I) and RG II, arabinogalactan protein (AGP) and extensin (EXT). The red arrow indicates the flux from UDP-Glc to UDP-GlcA that is shared between arabinose and xylose bodies and mediated by UDP-Glc dehydrogenase (UGD). Thin grey arrows indicate potential metabolic routes that are apparently not used in planta. The enzymes involved are UDP-Glc dehydrogenase (UGD), UDP-Xyl synthase (UXS), UDP-xylose 4-epimerase (UXE) bispecific UDP-Glc/UDP-Xyl 4-epimerase (UGE), arabinokinase (AK), UDP-sugar pyrophosphorylase (USP), and reversibly glycosylated polypeptide (RGP). Nucleotide sugar transporters are UDP-Xyl transporter (UXT), UDP-uronic acid transporter (UUAT), a hypothetical UDP-Arap export facilitator (grey), and UDP-arabinofuranose transporter (UAfT). The symbols for \u2018xylosyltransferase\u2019 (XT) and \u2018arabinosyltransferase\u2019 (AT) represent different glycosyltransferases generically. Note that pathways leading to UDP-galactose, UDP-L-rhamnose, and UDP-galacturonic acid or any GDP-sugars are not shown.Brown arrows indicate the fate, UXS3, -5 and -6 genes encoding cytosolic UXS and the UXS1, -2 and -4 loci encoding the Golgi-localized isoforms. Because UDP-Xyl is exclusively required inside the Golgi, the three cytosolic UXS isoforms appear redundant. More crucially, to give the cytosolic pool of UDP-Xyl access to the site of carbohydrate biosynthesis, a Golgi-localized UDP-Xyl transporter is needed. Indeed, three UDP-Xyl transporters (UXT1\u20133) were ingeniously identified using a biochemical assay and the uxt1 mutant showed a defect in glucuronoxylan (GX) structure and abundance. This phenotype suggested that polysaccharide biosynthesis somehow depended on cytosolic UDP-Xyl despite the presence of functional UXS in the Golgi. However, there was only a relatively minor reduction of total cell wall xylose in uxt1 and no carbohydrate other than GX was affected in the mutant. Moreover, the uxt2 uxt3 double mutant was phenotypically normal but not the hexagonal pyranose (Arap) form of L-arabinose is found in most cell wall polymers. However, a mutase is required for efficient conversion of the product of UXE UDP-Arap into UDP-Araf. This mutase is a protein complex encoded by the REVERSIBLY GLYCOSYLATED POLYPEPTIDE1, -2 and -5 loci and is partially bound to the cytosolic face of the Golgi. Knockdown of RGP1 and RGP2 resulted in massive growth defects and a near-complete loss of cell wall arabinose (p from the plentiful supply of UDP-Xyl by cytosolic UXS. This potential option, however, is contradicted by the strong arabinose deficiency in cell walls in the uxe1 mutant (uge1 uge3 double mutant (UDP-Araed L-Ara . And yet1 mutant and the"} +{"text": "Reversible deactivation radical polymerizations (RDRPs) have proven to be the convenient tools for the preparation of polymeric architectures and nanostructured materials. When biodegradability is conferred to these materials, many biomedical applications can be envisioned. In this review, we discuss the synthesis and applications of biodegradable polymeric architectures using different RDRPs. These biodegradable polymeric structures can be designed as well-defined star-shaped, cross-linked or hyperbranched via smartly designing the chain transfer agents and/or post-polymerization modifications. These polymers can also be exploited to fabricate micelles, vesicles and capsules via either self-assembly or cross-linking methodologies. Nanogels and hydrogels can also be prepared via RDRPs and their applications in biomedical science are also discussed. In addition to the synthetic polymers, varied natural precursors such as cellulose and biomolecules can also be employed to prepare biodegradable polymeric architectures. Biodegradable polymers refer to a category of polymers that can be cleaved into small polymer fragments in vivo. The biodegradability endows these polymers with many special applications particularly in drug delivery, tissue regeneration and biotherapeutics ,2,3. MetPolymeric architectures are very versatile. Based on the composition, they can be homopolymers, or block, statistical, gradient and graft copolymers. Based on the structure, they can be designed as linear, multi-armed, comb-like, networks, and hyperbranched polymers. They can also be tailored with single, multi-, homo-, hetero- or multi-functionalities. These broad polymeric architectures can be fabricated into various complicated particles via either self-assembly or designed interactions, such as micelles, vesicles, capsules, hydrogels and nanogels . BecauseIn addition to ionic and coordination ring-opening polymerization ,12, free\u22121 will exhibit significantly increased circulation time in the body since the glomerular filtration in the kidney has a molecular weight cut-off of about 50,000 g\u00b7mol\u22121 ..N-vinylc\u03b5-caprolactone) (PCL) and nondegradable polystyrene (PSt) and poly(methyl methacrylate) (PMMA) macro-initiators, which were subsequently cross-linked to generate core cross-linked star (CCS) polymers. By using the non-degradable divinylbenzene (DVB) and ethylene glycol dimethacrylate (EGDMA) as well as the degradable and 2,2-bis(\u03b5-caprolactone-4-yl)propane (BCP) monomers to cross-link the different macro-initiators, a range of CCS polymers were synthesized where either the arm or the core domain can be selectively degraded. Hydrolysis of PCL/PMMA/EGDMA miktoarm CCS polymer resulted in CCS polymer with a reduced number of arms, whereas PSt/BOD core-degradable CCS polymer yielded the original linear PSt arms upon hydrolysis.In another study reported by Qiao and Wiltshire, the synthesis of selectively degradable core cross-linked star polymers using ATRP and ROP was presented . In thei\u03b5-caprolactone)-b-poly(ethylene glycol) methacrylates (PEGMAs) using ATRP and ROP. These multi-armed star architectures exhibited unimolecular behavior and the capability of encapsulation of hydrophobic molecules, therefore they are potential candidates as hydrophobic anticancer drug carriers. Likewise, thermosensitive four armed triblock copolymers comprised of poly(\u03b5-caprolactone), poly(olego(ethylene oxide) methacrylate) and poly(di(ethylene oxide)methyl ether methacrylate) segments were also synthesized by ATRP and ROP joint methods using a four armed initiator. These four armed polymeric structures were found to be able to self-assemble into spherical micelles which undergo reversible sol-gel transitions between room temperature (22 \u00b0C) and human body temperature (37 \u00b0C) (PPS-b-PDMA-b-PNIPAAM) that forms physically cross-linked hydrogels when transitioned from mechanisms for reactive oxygen species (ROS) triggered degradation and drug release. At ambient temperature, PPS-b-PDMA-b-PNIPAAM assembled into 66 \u00b1 32 nm micelles comprising a hydrophobic PPS core and PNIPAAM on the outer corona. The PPS-b-PDMA-b-PNIPAAM micelles were preloaded with the model drug Nile red and the resulting hydrogels demonstrated ROS-dependent drug release. The hydrogels were cyto-compatible in vitro and demonstrated to have utility for cell encapsulation and delivery. These hydrogels also possessed inherent cell-protective properties and reduced ROS-mediated cellular death in vitro. Subcutaneously injected PPS-b-PDMA-b-PNIPAAM polymer solutions formed stable hydrogels that sustained local release of the model drug Nile red for 14 days in vivo. These collective data demonstrate the potential use of PPS-b-PDMA-b-PNIPAAM as an injectable, cyto-protective hydrogel that overcomes conventional PNIPAAM hydrogel limitations such as syneresis, lack of degradability, lack of inherent drug loading and environmentally responsive release mechanisms by a combination of ATRP and Michael-type addition reaction using biodegradable nanogel precursors, 2-hydroxyethyl p(OEO300MA-o-PHEMA) . RAFT ago-PHEMA) . It is wo-PHEMA) . A combichanisms 84]..co-methaNanogels have drawn enormous attention due to their applications as targeted drug delivery scaffolds in biomedical science. Matyjaszewski\u2019s group is pioneering the fabrication of hyperbranched polymeric architectures, particles, hydrogels and nanogels using ATRP strategies . They reRecent advances in drug carrier design in the field of photodynamic therapy (PDT) have stimulated the development of numerous sophisticated drug delivery carriers. Kim and coworkers designed a novel biodegradable and biocompatible nanogels used as PDT carriers. The nanogels were synthesized through ATRP method using inverse miniemulsion and their biodegradability was determined in the presence of glutathione. The model photosensitizer (PS) was encapsulated in the biodegradable nanogels by simple mixing and sonication. The cellular uptake and the cytotoxicity of the nanogels before and after laser irradiation were determined. The results showed that the Ce6-loaded nanogels did not influence the cellular viability of the cells before light irradiation. Under light exposure, the Ce6-nanogel complex revealed strong photoactivity. These nanogels may enhance therapeutic efficacy of PSs without any complex chemical modifications with PSs 89]..89].The stability of encapsulation in self-assembled system is usually limited by the requisite concentration for self-assembly formation. Once the encapsulation is achieved, the lack of targeting molecules on the drug carriers will compromise the efficiency for targeted delivery. To tackle this issue, Thayumanavan and coworkers successfully fabricated surface-functionalized polymer nanogels with facile hydrophobic guest encapsulation capabilities . These b\u22121 ..co-oligob-poly(St)-b-poly(PEG-A) by RAFT polymerization using a new bifunctional RAFT agent, S,S-bis trithiocarbonate (BDPET) (PCL-b-PMPC), which was synthesized using the combined methods of ROP, end-group modification and ATRP. These vesicles can be stabilized by sol-gel chemistry within the vesicle membrane ..\u03b5-caprolN-vinyl pyrrolidone) (PVPON) capsules with engineered biodegradable properties via LbL process mediated by hydrogen bonding interaction. Due to the introduction of intra-disulfide linkages among the capsules they underwent destruction within 4 h in the presence of 5 mM glutathione. The cross-linked multilayers endowed the capsule with low-fouling properties to a range of proteins, including fibrinogen, lysozyme, immunoglobulin G, and bovine serum albumin ..co-poly(co-3-hydroxyhexanoate) (P(HB-co-HHx)) ) a 117]. . co-3-hyer on it .b-poly(l-lactic acid), from the cellulose surface. The biodegradable poly(l-lactic acid) block further facilitates the biodegradability of the so-prepared architecture to afford biodegradable enzyme\u2013polymer conjugates. Bio-cleavage of the polymer chains from the GOx surface obviously recovered the enzymatic activity [si-RNA is considered an effective targeting molecule, its biodegradable polymer conjugates could be good candidates for potential bio-therapeutics [Davis and coworkers delivered elegant research on the preparation of biodegradable conjugates. Free thiol tethered biomolecule, e.g., bovine serum albumin (BSA), has been successfully modified with several polymers to afford biodegradable homo- or hetero-bioconjugates under ambient condition using room temperature initiation via RAFT polymerization d 20,135,137,138.activity . A latesactivity . These sapeutics . In addiapeutics ,142 and apeutics .This review has discussed the synthesis and applications of biodegradable polymeric architectures using different RDRPs. These biodegradable polymeric structures can be designed as well-defined star-shaped, cross-linked or hyperbranched, through which more complicated nanoparticles such as micelles, vesicles and capsules can be fabricated via either self-assembly or cross-linking methodologies. Nanogels and hydrogels can also be prepared via RDRPs. Their applications in biomedical science are also discussed. Biodegradable polymeric architectures can be prepared with both synthetic and natural precursors.As discussed in this review, RDRPs have proven to be convenient tools for the synthesis of the versatile biodegradable polymeric architectures to meet varied applications. Driven by the practical application and commercialization, the design of more complicated polymeric architectures with controllable biodegradability will be expected. However, it is worth noting that a fast biodegradable process in vivo is not desired in some situations. Therefore, designing and fabricating the polymeric architectures with controllable and slow biodegradability would be a critical issue in this field. To achieve this, many other different polymerization techniques are required besides RDRPs."} +{"text": "A 40-year-old HIV-positive female presented with an erythematous macular eruption involving the malar and periorbital area, the forehead and neck of six weeks of duration (A). She had initiated antiretroviral treatment with co-formulated tenofovir/lamivudine/efavirenz QD plus isoniazid preventive therapy (IPT) 18 weeks before with CD4 counts of 496 cells/\u03bcl (25%). Drug-induced subacute cutaneous lupus erythematous (DI-SCLE) secondary to isoniazid was clinically diagnosed and isoniazid was stopped. Antinuclear antibody (ANA), anti-SSA/Ro, anti-SSB/La, anti-Sm, anti-RNP and antihistone antibodies were negative. Complete blood count, eGFR and liver transaminases were normal. Three months after stopping isoniazid, the skin lesions resolved completely, supporting the diagnosis of isoniazid-induced SCLE (B). DI-SCLE with negative ANA has been described, but there are no reports in HIV-infected patients. With the current expanded provision of IPT to people living with HIV, it is important to be aware of this rare side effect of isoniazid despite the negativity of antibody assays."} +{"text": "The increased performance of microCLIP in CLIP-Seq-guided detection of miRNA interactions, uncovers previously elusive regulatory events and miRNA-controlled pathways.Argonaute crosslinking and immunoprecipitation (CLIP) experiments are the most widely used high-throughput methodologies for miRNA targetome characterization. The analysis of Photoactivatable Ribonucleoside-Enhanced (PAR) CLIP methodology focuses on sequence clusters containing T-to-C conversions. Here, we demonstrate for the first time that the non-T-to-C clusters, frequently observed in PAR-CLIP experiments, exhibit functional miRNA-binding events and strong RNA accessibility. This discovery is based on the analysis of an extensive compendium of bona fide miRNA-binding events, and is further supported by numerous miRNA perturbation experiments and structural sequencing data. The incorporation of these previously neglected clusters yields an average of 14% increase in miRNA-target interactions per PAR-CLIP library. Our findings are integrated in microCLIP ( AGO-PAR-CLIP is widely used for high-throughput miRNA target characterization. Here, the authors show that the previously neglected non-T-to-C clusters denote functional miRNA binding events, and develop microCLIP, a super learning framework that accurately detects miRNA interactions. They are small single stranded RNA molecules that are loaded into Argonaute (AGO) to induce target cleavage, degradation, or translational suppression enabled the high-throughput mapping of RNA-binding protein interactions. microRNAs (miRNAs) are central post-transcriptional gene expression regulators, actively researched for their role in most physiological and pathological conditions, as well as for their potential as biomarkers and/or therapeutic targetsion Fig.\u00a0. Photoac4 implementation employs a biophysical model, while PARma5 provides canonical miRNA seed family interactions by processing significantly overrepresented kmers. microMUMMIE6 is another state-of-the-art approach based on a six-state hidden Markov model for characterizing the background, the AGO-bound clusters and their flanking regions. Its core algorithm solely processes T-to-C enriched clusters determined by PARalyzer7 and recognizes miRNA-binding sites with (im)perfect seed complementarity. These approaches cannot be readily used on sequencing data, since they require extra pre-processing steps and the creation of non-standard file types.During the past few years, computational methods devoted to AGO-PAR-CLIP data analysis have been elaborated by employing different mathematical models and feature sets. MIRZA8 and depend strongly upon the induced T-to-C conversions creating a comprehensive collection of PAR-CLIP experiments, (b) implementing an extensive compendium of bona fide functional miRNA-binding events from highly specific techniques, and (c) analyzing 123 high-throughput miRNA expression perturbation datasets. This unprecedented list of in-house analyzed experiments enabled us to assess the impact of every algorithmic choice on the accuracy of the provided results.The most remarkable finding was that clusters depleted on T-to-C conversions, which are always filtered out in such analyses, can aid in the identification of functional miRNA-binding events Fig.\u00a0. Importa9. By using multiple combinations of classifiers, super learning outperforms a single prediction model.microCLIP integrates our findings and provides a robust pipeline for the analysis of all AGO-enriched regions Fig.\u00a0. It enco11. To this end, we paid specific attention to incorporate data from different cell types and experiments in our study.Gene expression regulation has been shown to be highly context-specific at both transcriptional and post-transcriptional levels. In a fashion similar to how transcription factor binding is controlled not only by the site sequence but also by numerous epigenomic mechanisms, microRNA binding and function is dictated by context that can differ significantly among cell types/conditions16, was incorporated in the algorithm\u2019s development and evaluation process T-to-C clusters and demarcate structural imprints of AGO-bound regions, we examined sequencing data from Parallel Analysis of RNA Structure (PARS) experiments for the first time in such a setting. All enriched regions exhibited strong structural accessibility in the miRNA seed site. non-T-to-C sites were also functionally investigated against 17 gene expression profiling datasets following up/downregulation of individual miRNAs. They proved to harbor functional miRNA-binding events and their incorporation in the analysis revealed an average increase of 14% in identified miRNA-target interactions per PAR-CLIP library.17.We subsequently processed an independent AGO-PAR-CLIP dataset in MCF7 cells to evaluate the impact of these neglected sites in downstream analyses. Their inclusion revealed critical pathway components under miRNA regulation that were previously undetected. Pathway members under miRNA control that remained uncovered with conventional pipelines, were validated in miRNA\u2013mRNA expression profiles retrieved from 271 ductal breast cancer samples indexed in The Cancer Genome Atlas (TCGA)microCLIP is the first algorithm for AGO-PAR-CLIP data providing more than 80% true positive miRNA-target predictions on a broad test set. Our approach detects 1.6-fold more validated miRNA-target sites when juxtaposed against state-of-the-art implementations, ushering in a new era of miRNA-target annotation. Use of microCLIP can unveil uncharted parts of the miRNA interactome in different physiological/pathological conditions.We extracted positive/negative miRNA-target pairs from direct low-yield techniques and miRNA perturbation high-throughput experiments to distinguish AGO-CLIP functional clusters (Methods). Our downstream evaluations included miRNA-binding sites residing on AGO-enriched regions derived from 26 AGO-PAR-CLIP libraries were derived from direct experimental techniques, while the rest originated from the analyzed miRNA high-throughput perturbation datasets (64 microarray and 12 RNA-Seq experiments) in our reference collection . We show for the first time, that features related to miRNA targeted sites on non-T-to-C clusters also significantly differentiate from relevant estimates corresponding to negative miRNA-target instances . These findings are consistent with our hypothesis that previously discarded non-T-to-C targeted regions display common characteristics with T-to-C sites regarding properties that are considered decisive for miRNA function.Importantly, we observed that ~28% of the positive MREs, including 1131 chimeric and reporter assay-verified interactions, were exclusively resolved by non-T-to-C AGO-enriched clusters Fig.\u00a0. Consequ10, we analyzed 10 AGO-PAR-CLIP libraries on virus-infected B-cell lines . Both site families exhibited exclusive binding events for a specific context as well as constitutive sites, common across experiments . In this approach, AGO-binding efficiency is revealed by RSS signatures observed on mRNA transcripts, since increased structural accessibility is expected in functional conformations. To this end, we investigated whether functional miRNA-target pairs residing on non-T-to-C clusters harbored similar structural properties. We calculated PARS sequencing profiles around AGO-PAR-CLIP-derived miRNA-binding sites in 4 EBV transformed lymphoblastoid cell lines2. The analysis of the respective RNase S1 or V1 nuclease signals/intensities at single base resolution enabled the assessment of miRNA site accessibilities in both T-to-C and non-T-to-C clusters. These measurements were juxtaposed against negative MREs comprising miRNAs expressed in the examined lymphoblastoid cell types. The per base averaged PARS scores indicate that strong structural accessibility occurs in the 3\u2032 end of miRNA-target sites and specifically on 2\u20134\u2009nt positions of the miRNA seed region. These results were identified on interactions residing on (non-)T-to-C clusters and significantly differ from respective base scores along negative MREs located on AGO-enriched peaks . The outcome of our analysis is consistent with previous observations19 and demonstrates that the highest accessibility segregating functional from non-functional binding sites resides towards the initiation of the direct miRNA seed pairing.In order to demarcate RNA Secondary Structures (RSS) of AGO-bound regions compared to a set of negative miRNA sites on mRNA transcripts, we estimated respective PARS scores as introduced by Wan et al.We incorporated all the aforementioned observations in an extensive in silico framework. microCLIP is based on ensemble super learning and provides a complete pipeline for experimentally supported miRNA targetome annotation, initiating from aligned (.sam/.bam) PAR-CLIP-sequencing reads. This algorithm, contrary to existing leading implementations, operates on every AGO-enriched cluster, utilizing the previously neglected non-T-to-C clusters. We designed a collection of 131 descriptors associated with CLIP-Seq attributes and miRNA/MRE hybrid derived characteristics , in order to explore the extent of miRNA-target pairs that remain uncovered using standard AGO-PAR-CLIP computational approaches. Processed CLIP-Seq libraries were accompanied by RNA-Seq and small RNA-Seq (sRNA-Seq) data to determine the set of expressed transcripts and miRNAs per cell type. By screening every AGO-enriched region, microCLIP reveals a significant portion of targeted genes distinguished only from CLIP clusters presenting no conversion sites. An average 11\u2009\u00b1\u20096.4% increase of detected targets was observed across the analyzed experiments. Figure\u00a020; Supplementary Table\u00a021, Farazi et al.3, Whisnant et al.22). Response of targeted mRNAs to miRNA deregulation was evaluated independently per tested cell type. In the conducted comparisons, we measured target fold changes in three distinct groups: (i) mRNAs presenting at least one predicted MRE on T-to-C clusters, (ii) mRNAs participating in interactions resolved only by non-T-to-C clusters, (iii) transcripts lacking sites for the examined miRNAs. The distributions of gene expression fold changes in these subgroups are presented in Fig.\u00a0P values T-to-C: 5.1\u2009\u00d7\u200910\u2212138\u201311\u2009\u00d7\u200910\u22123, P values non-T-to-C: 8.5\u2009\u00d7\u200910\u221229\u201337\u2009\u00d7\u200910\u22123, two-tailed Wilcoxon rank-sum test, 51\u2009<\u2009nT-to-C\u2009<\u20091569, 11\u2009<\u2009nnon-T-to-C\u2009<\u2009344, 2677\u2009<\u2009nno-site\u2009<\u200912,330). Regardless of the perturbation type, T-to-C clusters were observed to relate to more responsive targets at equal numbers of predicted sites : 2.7\u2009\u00d7\u200910\u221211\u20133.9\u2009\u00d7\u200910\u22122, two-tailed Wilcoxon rank-sum test, 11\u2009<\u2009nT-to-C/non-T-to-C\u2009<\u2009344). Still, our analysis outcomes confirm our initial assumption that there are functionally important non-T-to-C targets.To investigate the functional importance of miRNA sites residing on AGO-enriched regions presenting insufficient T-to-C substitutions, we utilized 17 public high-throughput gene expression profiling datasets following transfection or knockdown of specific miRNAs .To demonstrate the ability of detected non-T-to-C interactions to statistically empower downstream analyses, a functional enrichment investigation on KEGG pathways was conducted in highly scored miRNA-target pairs from an independent AGO-PAR-CLIP dataset in MCF7 cells , and one combining T-to-C and non-T-to-C targets (n\u2009=\u2009491). A total of 391 genes were common between the two. Pathway analysis of T-to-C targets resulted in 63 significantly enriched terms , while the combined set yielded 67 enriched terms T-to-C\u2009<\u200958). An average of 2.4 more targets per pathway was observed when non-T-to-C interactions were included.8921 and 846 unique interactions retrieved from T-to-C and non-T-to-C peaks, respectively, were utilized to form two gene sets: one containing unique T-to-C targets T-to-C\u2009=\u20093.5\u2009\u00d7\u200910\u22127, one-sided Fisher\u2019s exact test, Benjamini\u2013Hochberg adjustment, n(non-)T-to-C\u2009=\u200934), among targets derived only from non-T-to-C peaks T-to-C\u2009=\u20095.42\u2009\u00d7\u200910\u22127, one-sided Fisher\u2019s exact test, Benjamini\u2013Hochberg adjustment, n(non-)T-to-C\u2009=\u200923) revealed miRNAs targeting TGIF2, TFDP-1, and EP300, orchestrators of c-Myc and p15 transcription with involvement in cell cycle progression T-to-C\u2009=\u20091.26\u2009\u00d7\u200910\u22126, one-sided Fisher\u2019s exact test, Benjamini\u2013Hochberg adjustment, n(non-)T-to-C\u2009=\u200930), established as anti-proliferative and apoptotic marker in breast cancer cells28, was revealed to be regulated by non-T-to-C peaks .To further validate pathway-related interactions from (non-)T-to-C clusters, we investigated miRNA-target expression associations in 271 breast cancer patient samples indexed in TCGAThe MCF7 dataset case-study exhibited non-T-to-C peaks yielding breast cancer-related interactions that would be lost following standard analysis scenarios. Independent processing of miRNA/mRNA expression profiles from TCGA ductal breast cancer samples clearly distinguishes non-T-to-C miRNA-target pairs from randomly selected pairs lacking target sites for highly expressed miRNAs, validating the previous observations. We conclude that non-T-to-C targets, lost under conventional PAR-CLIP analysis practices, are functionally relevant and should be discerned from experimental noise.4, microMUMMIE6, and PARma5. In the evaluation process, AGO-CLIP-guided algorithm performance was also contrasted with Targetscan v730, a state-of-the-art computational approach that defines de novo miRNA-target pairs. Targetscan is a strong seed-based approach and is among the most extensively used target prediction algorithms with higher discriminative power compared to simple seed matching. The performance evaluation was initially accomplished against unified sets of four microarray and two RNA-Seq public datasets in which miRNAs were individually transfected into HEK293 cells . The retrieved MREs were juxtaposed with deregulated targets identified in the gene expression profiling experiments. To determine the ability of each method to identify the most strongly downregulated targeted genes, detected interactions were ranked according to their provided scores. The median fold changes (log2) of the top predicted targets for the different algorithms were subsequently estimated and accordingly compared by applying stepwise thresholds of total predictions. The performance of implementations was additionally evaluated against median fold-change values of randomly selected genes. In the examined miRNA perturbation experiments, microCLIP-detected targets revealed the strongest repression, compared to all the assessed approaches for the considered miRNAs. microCLIP exerted significant differences in expression changes compared to transcripts lacking any predicted binding site : 3.2\u2009\u00d7\u200910\u221271\u20131.3\u2009\u00d7\u200910\u22126, one-sided Kolmogorov\u2013Smirnov test, 6764\u2009<\u2009nno-site\u2009<\u200913,122). Compared to the other CLIP-guided implementations, microCLIP displayed the greatest site effectiveness in most cases : 3.1\u2009\u00d7\u200910\u221213\u20130.031, one-sided Kolmogorov\u2013Smirnov test, 70\u2009<\u2009n\u2009<\u2009321; Fig.\u00a0P values (g): 0.0005\u20130.1, one-sided Kolmogorov\u2013Smirnov test, n\u2009=\u2009192). In this evaluation, Targetscan achieved similar site efficacy as microCLIP in Fig.\u00a0P values (a-g): 0.002\u20130.5, one-sided Kolmogorov\u2013Smirnov test, 70\u2009<\u2009n\u2009<\u2009321).The performance of microCLIP, MIRZA, microMUMMIE, PARma, and Targetscan v7 was also tested using 3 HEK293 and 4 HeLa expression profiling datasets following miRNA perturbation . The utilized validation set is composed of 1674 chimeric and reporter assay-verified interactions from 125 miRNAs .Following thorough examination of properties underlying miRNA targeting efficiency, we observed that positive miRNA-binding sites identified on both T-to-C and non-T-to-C clusters significantly diverge from respective descriptors derived from negative MREs Fig.\u00a0. On the 10, the evaluation in Fig.\u00a0In order to further examine the functional significance of non-T-to-C interactions, we utilized an extensive collection of miRNA perturbation experiments. microCLIP-predicted MREs supported from clusters containing or lacking T-to-C mutations exhibited significantly increased repressive effectiveness compared to transcripts without miRNA-binding sites Fig.\u00a0. In accoStructural accessibility of miRNA-binding sites designates true targets and is considered of paramount importance in most prediction algorithms. RNA structural signatures of AGO-bound regions were characterized with PARS sequencing experimental data and assessed for the first time in (non-)T-to-C enriched regions. microCLIP-detected MREs residing on both non-T-to-C and T-to-C clusters, shared strong and common accessibility patterns towards the nucleotides initiating the direct miRNA seed pairing Fig.\u00a0. AdditioAn independent pathway enrichment analysis directly showed that miRNA regulation of important signal transduction members may remain elusive using conventional analyses. Enrichment analysis with T-to-C as well as with (non-)T-to-C targets in MCF7 AGO-PAR-CLIP data revealed numerous cancer-related terms to be among the most significantly enriched terms. non-T-to-C targets increased the number of miRNA-controlled genes per pathway, offering more candidate genes for examination, as well as achieving lower statistical significance levels 13 and CLEAR-CLIP 14 experiments, as well as additional miRNA-target chimeric fragments derived from a previous meta-analysis of published AGO-CLIP datasets12. In order to quantify miRNA-induced mRNA expression changes and to identify functional binding sites, 101 miRNA perturbation experiments were analyzed , were incorporated in our pipeline to identify non-specific AGO-bound transcripts and deduce more than 24,000 negative miRNA-binding sites. A compendium of 96 AGO-CLIP-Seq experiments was derived from DIANA-TarBase and used to further select background-derived MREs displaying no overlap with AGO-enriched regions , depending on the type of regulation, was applied to determine negative and positive interaction subsets. For GSE8501 experiment conducted in Rosetta\u2013Merck microarrays, error-weighted log10 intensity ratios were retrieved and transformed to log2-scale.A total of 44 microarray studies of distinct experimental conditions and RNA-Seq libraries treated with specific miRNA overexpression, 12 experimental conditions in total in HeLa cells following the individual overexpression of five human miRNAs or knockdown of let-7b (Supplementary Table\u00a039 and DDBJ40 repositories (Supplementary Table\u00a041 to infer expressed miRNAs and transcripts. (s)RNA-Seq datasets were derived from the ENCODE repository and from a series of studies . Adapter sequences were retrieved from the original publication or GEO/SRA entries, when available. Contaminants were detected utilizing in-house\u00a0developed algorithms and the Kraken suite42. Pre-processing was performed utilizing Cutadapt43. PAR-CLIP reads were aligned against human reference genome (GRCh37/hg19) with GMAP/GSNAP44 spliced aligner, appropriately parameterized to identify known and novel splice junctions. microRNA expression was quantified using miRDeep245. Ensembl v7546 and miRBase v1847 were used as annotation for genes and microRNAs, respectively. Top expressed miRNAs and AGO-PAR-CLIP data in each cell type, were jointly utilized as input to microCLIP in silico framework for miRNA-target identification. Specifications on the processed 36 datasets are provided in Supplementary Tables\u00a0Pre-processing and alignment of PAR-CLIP datasets was realized as described by Vlachos et al.For the analyses presented in Figs.\u00a018 . The identification of single or double stranded regions, across the human transcriptome, was derived from deeply sequenced RNA fragments generated from RNase S1 or V1 nuclease treatment of GM12878 cells, respectively.PARS sequencing data on total RNA isolated from lymphoblastoid cells were obtained from Wan et al. studyRaw reads of 51nt length, accordingly pre-processed for quality control and contaminant removal, were aligned against human reference genome (GRCh37/hg19) with GSNAP spliced aligner. This analysis resulted in ~130\u2009M uniquely mapped PE-sequenced fragments per sample. In order to derive structural signals in RNase S1 or V1 nuclease experiments at single base resolution, we calculated single(S1) and double(V1) stranded raw reads initiating on each nucleotide. The number of PARS tags per sample starting at each base were normalized by sequencing library depth. These base intensities were subsequently combined into the formula described by Wan et al. to compute PARS scores.2. miRNA-mRNA interactions were identified in both T-to-C and non-T-to-C PAR-CLIP clusters, corresponding to transcripts with\u2009>\u20091 TPM expression in GM12878 cells. For expressed miRNAs in respective EFD3-AGO2, LCL-BAC, LCL-BAC-D1, and LCL-BAC-D3 EBV infected lymphoblastoid cells, we included collapsed miRNA-binding sites residing within the PAR-CLIP clusters. For the performed comparisons, we incorporated negative MREs extracted from different high-throughput miRNA perturbation experiments . MREs utilized for the assessment of RSS signatures on AGO-bound clusters and the derivation of (non-)functional conformations of miRNA-target base pairings, were localized on coding and 3\u2032UTR regions. The examined sites had to present S1 and V1 signals in at least half of their occupied bases.RSS were defined by estimated PARS scores in the vicinity of PAR-CLIP-derived miRNA-binding sites in four lymphoblastoid cell lines from the study of Skalsky et al.sRNA-Seq and RNA-Seq datasets were retrieved from ENCODE consortium .Feature set description: A set of 131 descriptors and biases of codon usage were added. Entropy, enthalpy, free energy, and melting temperature (Tm) thermodynamic properties were calculated for MRE sequences in R.Moreover, single base and dinucleotide contents for miRNA-binding and respective flanking regions, complexity features for the MRE and proximal upstream/downstream sequences were introduced to microCLIP model. BLAST\u2019s DUST score50, positions and nucleotide composition of (un)paired nucleotides. Distinct features have been established to model (mis)matches, bulges, loops, and wobble pairs for miRNA-MRE hybrid structure and sub-domains encountered in the duplex. The distinct domains for miRNA sequences, as defined by microCLIP, are: (i) seed region (2\u20138 positions), (ii) central region (9\u201312 positions), (iii) 3\u2032 supplementary/compensatory region (13\u201316 positions), (iv) tail region (17-3\u2032 miRNA end). Similar regions were designated on the MREs based on the miRNA-binding anchors upon duplex formation.miRNA-target hybrids were associated with different descriptors such as the binding type, duplex structure energy calculated with the Vienna package51 in bigwig format and were utilized to deduce respective evolutionary rates. Conservation signals were computed as mean intensities of the phastCons base-wise scores on miRNA targeted regions, as well as their flanking regions. The conservation of the 5\u2032 MRE binding nucleotides was independently modeled. microCLIP integrates additional features corresponding to the location of the MRE within the AGO-enriched cluster and binding length ratios of miRNA and/or target regions.Our approach incorporates conservation of the MRE and upflank/downflank-MRE regions. phastCons pre-computed scores from genome-wide multiple alignments were downloaded from the UCSC repositoryThe applied super learning scheme benefits from the incorporation of the complete array of features, maximizing their contribution through their parallel use in different classification models in every node. The impact of weaker features and classifiers in optimal super learner design and behavior is shown in Supplementary Fig.\u00a0Description of the algorithm: microCLIP operates on AGO-PAR-CLIP-sequencing reads, requiring a SAM/BAM alignment file and a list of miRNAs as minimum input. It initially seeks for AGO-enriched regions and resolves coverage and observed transitions. A sensitive pipeline is adopted to scan read clusters for putative targeted sites including a wide range of binding types. The algorithm supports an extended set of (non-)canonical matches including 6mer to 9mer, offset 6mer, 3\u2032supplementary and compensatory sites as well as (im)perfect centered bindings. microCLIP extracts features for each candidate MRE and subsequently scores sites through a super learning scheme.The adopted framework incorporates two distinct levels of classification. The first layer comprises a group of 9 different nodes (base classifiers), which are aggregated in the meta-classifier of the second layer. The learning procedure is decentralized through the distinct nodes and relevant base classifiers that specialize in different subsets of features Fig.\u00a0. \u201cRegionEight of the nine base nodes adopt a super learning scheme that assembles the output of seven individual Random Forest (RF), Generalized Linear Model (GLM), Gradient Boosting Model (GBM), Deep Learning (DL) classifiers . The \u201cRegion features\u201d is analyzed by an RF classification scheme. The retrieved scores from each node are aggregated in a final GBM meta-classifier.52 R package. The RF, GBM, GLM learning models were developed, parameterized, and tuned with the caret53 and H2O52 R packages.Model training: The DL models developed for the microCLIP framework adopt a feed-forward multi-layer architecture. The input layers match the respective feature space and values are subsequently propagated within three hidden layers. We utilized a rectifier activation function to retrieve weighted combinations of the inputs transmitted to interconnected neuron units. Dropout regularization was added to achieve model optimization and avoid overfitting. A cross entropy cost-function was selected to adapt weights during the learning process by minimizing the loss. Bernoulli distribution function was used along with cross entropy (log-loss) to model the response variables. The output layer at the end of the network applies a Softmax activation function so that each neuron (predicted class) results in a probabilistic interpretation. The DL network depth, width, and topology, as well as activation functions and learning parameters were modeled with a tuning-in grid search algorithm using H2OBase classifiers were trained against a collection of 8693 positive and 21,789 negative miRNA interactions . mRNA and miRNA pre-computed expression values were RPM and RPKM units, respectively. In downstream analyses, miRNAs/mRNAs with zero expression value in at least 70% of the samples were filtered out. miRNAs presenting\u2009>\u200910 RPM in\u2009>\u200910% samples were included, based on miRBase criteria for defining a high confidence set47. The mRNA set was specified by applying a threshold of more than 1 RPKM in at least 10% of the processed samples. Zero mRNA expression values were replaced by the lowest non-zero RPKM value per sample. This pipeline resulted in a set of 13,346 mRNAs and 322 expressed miRNAs. Pearson correlation coefficient was computed for each miRNA-target pair across samples.271 TCGA ductal breast cancer RNA-Seq and sRNA-Seq samples were obtained from Firehose (3) with microCLIP in silico framework. The 100 most highly expressed miRNAs and their targets in 3\u2032 UTR regions were retained. Gene set enrichment analysis of AGO-PAR-CLIP-detected miRNA targets was performed for KEGG pathways54 using the R package limma35. Enrichment P values were corrected for multiple comparisons using Benjamini\u2013Hochberg false discovery rate and a 0.01 p-value threshold was applied. R package Pathview55 was used to visualize targeted pathway members in KEGG pathways.miRNA-target pairs were retrieved from the analysis of MCF7 PAR-CLIP library . The proposed settings for each implementation were retrieved from the relevant publications.In order to form a complete list of interactions for MIRZAhttp://www.clipz.unibas.ch). MIRZA input data were 51nt AGO-bound sequences centered on T-to-C sites and mature miRNA sequences of 21nt length as reported in the model\u2019s restrictions. The \u201ctarget frequency\u201d score was utilized to evaluate the quality of MIRZA-detected sites.The MIRZA biophysical model was executed in the \u201cnoupdate\u201d mode. The algorithm provides an optional parameterization to introduce miRNA expression profiles. We realized two different runs of MIRZA, with and without cell type-specific miRNA expression values that were extracted from the CLIPZ web server . Derived files, comprising annotated AGO clusters with positions of T-to-C transitions, constituted the input of the microMUMMIE core algorithm. Predictions with signal-to-noise ratio equal to 9.95 were retained, while posterior probabilities were utilized for the evaluation of microMUMMIE\u2019s performance.microMUMMIE algorithm was tested in both Viterbi and posterior decoding modes. Following microMUMMIE\u2019s constraints, we utilized PARalyzer v1.5PARma was applied on AGO-PAR-CLIP aligned data that were prepared following the algorithm\u2019s described format. The required input files contained genomic locations of aligned CLIP reads along with positions of observed conversion sites. PARma predictions are coupled with Cscore and MAscore scores for the cluster and miRNA seed family activity, respectively. The latter score was utilized for PARma-detected miRNA-target sites evaluation.http://www.targetscan.org/cgi-bin/targetscan/data_download.vert72.cgi). Targetscan v7 algorithm was additionally executed following the proposed settings in order to cover a greater transcript collection, as well as the whole spectrum of Targetscan-detected interactions including 6mer sites. Gene annotation files were retrieved from the Targetscan v7.2 official download page, and the miRNA seed sequence file that is a pre-requisite for the execution of the model was provided by Targetscan developers. The local Targetscan run complements the precompiled data with miRNA-target interactions on transcripts presenting the longest 3\u2032UTR, in cases they are not deposited on the online repository.Precompiled (non)conserved miRNA site context++\u2009scores for representative transcripts were downloaded from the Targetscan v7.2 site (2 fold change) following miRNA transfection and/or knockdown have been filtered out. Subsequent measurements were realized at the gene level. miRNA-gene interactions retrieved from each implementation were sorted according to their prediction scores. Each miRNA-target pair was characterized by the highest scored miRNA-binding site overlapping coding or 3\u2032UTR exons, since utilized algorithms provided MRE-oriented prediction scores. In cases of multiple transcript-gene associations, the transcript with the longest 3\u2032UTR was selected. Median expression log2 fold changes were estimated in consistence with the number of top predicted targets. Aggregated expression changes of genes were calculated by applying stepwise score thresholds. Paired comparisons required tested programs to have targets at every computational cutoff. Lower mean log2 fold changes correspond to stronger downregulation of the detected targets upon miRNA transfection. The statistical differences in the mean log2 fold-change values obtained by each implementation were assessed using two-tailed Wilcoxon signed-rank test. Identified targets by each algorithm were also juxtaposed against averaged log fold changes of 1000 randomly selected genes (without replacement). The mean log2 fold-change values of the randomly selected genes in different stepwise thresholds were taken and the median curve derived from these values was calculated. Genes with zero fold-change indication were filtered out from the random selection process.The comparison between microCLIP and existing implementations was performed using six gene expression profiling datasets following individual transfection of highly expressed miRNAs into HEK293 cells (GEO accessions GSE60426, GSE52531, GSE21901, GSE14537, GSE35621, Supplementary Table\u00a0P values\u2009<\u20090.05 were considered as statistically significant.Enrichment analyses were performed using one-sided Fisher\u2019s exact test. Correlations between quantitative parameters were assessed by calculating Pearson\u2019s correlation coefficient. Comparisons between two or more groups were conducted using Wilcoxon\u2019s rank-sum test and Kruskal-Wallis\u2019 test, respectively. In the latter, Wilcoxon\u2019s rank-sum test was performed as a post hoc test in order to assess between-group differences. The one-sided Kolmogorov\u2013Smirnov test was used to test for greater functional efficacy. In cases of multiple hypothesis testing, Benjamini\u2013Hochberg\u2019s false discovery rate was applied to control family-wise error rate. Supplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4"} +{"text": "Staphylococcus aureus (CA-MRSA) sequence type (ST) 398 type V (5C2&5) isolate in Australia in 2017. This CA-MRSA ST398 variant was highly virulent, similar to other related CA-MRSAs of ST398. This strain should be monitored to prevent more widespread dissemination.Using whole-genome sequencing, we identified a community-associated methicillin-resistant This program involves 38 hospitals across Australia continuously providing antimicrobial MIC data and demographic data on episodes of Staphylococcus aureus sepsis. Specimens are referred to a central reference laboratory where whole-genome sequencing is performed for all methicillin-resistant S. aureus (MRSA) isolates.The Australian Group on Antimicrobial Resistance 398 type V (5C2&5) isolate, typically referred to as livestock-associated MRSA (LA-MRSA), which has been isolated in many parts of the world including Australia (S. aureus by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using the Bruker MALDI Biotyper (https://www.bruker.com/) and confirmed this finding by DNA microarray using the S. aureus Genotyping Kit 2.0 . We performed susceptibility testing by using the VITEK 2 automated microbiology system and performed whole-genome sequencing of the isolate using the Illumina NextSeq 550 Sequencing System (https://www.illumina.com/). We performed DNA extraction on an overnight subculture using the Invitrogen MagMAX DNA Multi-Sample Kit . We prepared a library of the extracted DNA using the Illumina Nextera XT Library Preparation Kit and sequenced libraries having 150-bp paired-end chemistries. We identified single-nucleotide polymorphisms and performed core genome alignments using Snippy version 3.2 (https://github.com/tseemann/snippy). We constructed a phylogenetic tree using the resulting single-nucleotide polymorphisms in MEGA version 7.0 (https://www.megasoftware.net/) with the maximum parsimony algorithm. We identified antimicrobial resistance and virulence genes, multilocus sequence type, staphylococcal cassette chromosome mec (SCCmec) type, and spa type using available pipelines (https://cge.cbs.dtu.dk/services/) and confirmed the virulence and antimicrobial resistance gene profile by DNA microarray. We performed a phylogenetic comparison of S23009-2017 with 22 previously published MRSA ST398 whole-genome sequences and rooted the tree with an outgroup of single-locus Panton-Valentine leukocidin (PVL)\u2013positive variants.We identified the patient\u2019s isolate (S23009-2017) as spa type t011 and SCCmec element type V (5C2&5) are frequently identified in LA-MRSA ST398 isolates isolate. Phylogenetic analysis showed S23009-2007 had a much closer relationship to the PVL-negative MRSA ST398 isolates from China reported by He et al. than to LA-MRSA ST398 isolates from Australia (7). Themec, only 1 isolate harbored the type V (5C2&5) SCCmec element found in S23009-2017. Like S23009-2017, CA-MRSA ST398 isolates from China harbor sak, chp, and scn genes and lack the tetM resistance gene.He et al. reported several cases of severe and fatal infections with community-associated MRSA (CA-MRSA) ST398 and proposed that the strain arose from human-adapted predecessors and not from a livestock-adapted strain. Although all of the China isolates harbored a type V SCCMRSA ST398 has not been previously reported to cause serious disease in Australia. Although LA-MRSA ST398 is frequently identified in pig herds in Australia, the isolate from this patient was not LA-MRSA but CA-MRSA and presumably originated in China. Because of immigration irregularities, we were not able to investigate whether the patient had visited China shortly before his illness or if his house companions were from China. Unlike LA-MRSA ST398, CA-MRSA ST398 has been shown to be highly virulent and has become the predominant CA-MRSA circulating in Shanghai, China. Thus, continued monitoring of this strain\u2019s epidemiology and preventing its widespread transmission are essential."} +{"text": "C. elegans, which lives in reduced oxygen environments, engages in developmentally timed sleep (DTS) during larval stage transitions and engages in stress-induced sleep (SIS) during recovery from damaging conditions. Although DTS and SIS use distinct mechanisms to coordinate multiple sleep-associated behaviors, we show that movement quiescence in these sleep states is similarly integrated with the competing drive to avoid oxygen. Furthermore, by manipulating oxygen to deprive animals of SIS, we observe sleep rebound in a wild C. elegans isolate, indicating that sleep debt accrues during oxygen-induced SIS deprivation. Our work suggests that multiple sleep states adopt a common, highly plastic effector of movement quiescence that is suppressed by aversive stimuli and responsive to homeostatic sleep pressure, providing a limited window of opportunity for escape.Sleep is beneficial yet antagonistic to critical functions such as foraging and escape, and we aim to understand how these competing drives are functionally integrated. \u2022NPR-1 promotes locomotor quiescence during stress-induced sleep (SIS)\u2022npr-1(lf) is partly dependent on PDF secretionArousal from SIS in \u2022Oxygen levels dynamically influence SIS\u2022Sleep rebound is observed after SIS deprivation Physiology; Genetics; Behavioral Neuroscience Drosophila males during copulation suppresses daytime sleep in females, allowing increased foraging and egg-laying or lethargus mutants as well as wild isolates with low NPR-1 activity engage in DTS only under conditions that minimize arousing cues and other npr-1(lf) mutants engage in SIS when aggregating but not when solitary. The quiescence defect is specific to locomotion, with feeding quiescence intact, and is observed during both SIS and EGF-induced sleep. We find the locomotor arousal of npr-1 mutants during SIS to depend partly on the wake-promoting Pigment-Dispersing Factor PDF-1. We show that SIS in npr-1(lf) animals is rapidly responsive to changes in ambient oxygen and that the SIS defect observed under normoxic conditions is suppressed by mutations that interfere with oxygen sensation. Last, we use the wake-promoting effect of oxygen to deprive animals of SIS and observe sleep rebound in a wild (low NPR-1 activity) C. elegans isolate. Our results reveal that oxygen avoidance can dynamically override SIS and that sleep debt accrues during oxygen-induced SIS deprivation.In a genetic screen for SIS-defective mutants, we have uncovered C. elegans, we performed a non-clonal EMS screen for mutants that fail to engage in SIS following ingestion of the pore-forming toxin Cry5B, which triggers a robust bout of sleep that is dependent on the ALA neuron receptors mutant harbors a G-to-A transition at nucleotide 533 of the NPR-1 coding sequence, resulting in a Cys178Tyr substitution. Cys178 lies in the extracellular region between predicted transmembrane domains 4 and 5 mutants are severely impaired for both locomotor and feeding quiescence, whereas npr-1(lf) mutants are specifically defective in locomotor quiescence during SIS occurs during the period of cuticle restructuring that precedes each larval molt (npr-1(lf) during DTS has been associated with heightened sensitivity to touch (npr-1(lf) during SIS. We first examined the contribution of touch sensitivity.val molt . Interesval molt . Locomotto touch and to oto touch . We wishnpr-1(lf) animals from DTS has been found to be dependent on secretion of PDF-1, an arousal peptide acting via receptors in peripheral mechanosensory neurons and body muscles, possibly increasing the sensitivity of these tissues to stimulation (npr-1(lf) during SIS, we examined npr-1;pdf-1 double mutant animals. We found that pdf-1(lf) reduces but does not eliminate arousal from SIS in npr-1(lf) (npr-1(lf) animals did not increase under the same conditions A and 3B,pr-1(lf) and animpr-1(lf) C and thepr-1(lf) D, indicanditions D. We intnpr-1(lf) from SIS. Animals with reduced NPR-1 function are hypersensitive to oxygen (npr-1(lf) animals are competent to engage in DTS animals under controlled reduced-oxygen conditions (10% oxygen) and found that this potently suppressed locomotor arousal (npr-1(lf) animals become completely immobile within 15\u00a0s of oxygen reduction, an effect that is reversed equally rapidly upon exposure to normoxic conditions. A fraction of wild-type animals are aroused by the transition between oxygen concentrations, but this effect is very transient.Second, we examined SIS behavior of arousal B. This enpr-1(lf) mutants. Oxygen avoidance contributes to aggregation behavior in npr-1(lf), which is dependent on heightened activity of the RMG interneuron (2-sensing soluble guanylate cyclase encoded by gcy-35 abolish oxygen avoidance behaviors of npr-1(lf) channel subunit required for sensory transduction in the RMG circuit (npr-1(ad609) mutants , as do m circuit . We foun mutants C and 4D.C. elegans, including the Hawaiian strain CB4856, carry a lower-activity variant of NPR-1 (215F) than the laboratory strain N2 (NPR-1 215V) containing the NPR-1(215V) variant from N2 introgressed into the Hawaii genetic background . We wish-1 215V) A\u20135C. To ckground . We founC.\u00a0elegans DTS (npr-1(lf) and in the Hawaiian isolate CB4856 harboring a low-activity NPR-1 variant (215F). Following exposure to UV light, the exposed population was separated to two plates and placed in a low-oxygen chamber to allow animals to engage in SIS. After 55\u00a0min, one plate was removed to 21% oxygen for sleep deprivation and the control plate was returned to low-oxygen conditions. After 10\u00a0min the sleep-deprived animals were returned to the chamber and low-oxygen conditions were re-established. During each transition the control group experienced a maximum of 2\u00a0min of sleep deprivation. In both npr-1(lf) and the Hawaiian strain, we found an increase in locomotor quiescence among sleep-deprived animals relative to controls (npr-1(lf) animals ultimately becoming quiescent. This increase in quiescence may be influenced by, but is not likely attributable to, changes in bordering and aggregation behavior throughout these assays animals, which we have isolated in a genetic screen for SIS-defective mutants.Natural isolates of exposure . In thisSimilar to SIS, locomotor quiescence during DTS is rapidly reversed by hyperoxia avoidance in a manner that depends on NPR-1 . This ranpr-1(lf) animals, which are active with respect to both head movement and locomotion (but not feeding), it may be that arousal cues act solely on the locomotor circuit.During SIS, the coordinated suppression of multiple active behaviors is mediated by the ALA interneuron, which promotes quiescence via the collective action of neuropeptides with overlapping but distinct effects on the sub-behaviors of sleep, including feeding quiescence, head movement quiescence, and locomotion . InteresDrosophila males, courtship-associated sleep loss is not associated with homeostatic sleep rebound, indicating that sexual arousal can counteract sleep pressure (C. elegans SIS is not associated with the reduced viability normally associated with sleep loss under well-fed conditions (C. elegans SIS is subject to homeostatic regulation and that sleep pressure continues to accumulate in the hyperoxia-aroused state, an effect also apparent during DTS (C. elegans serve critical functions. Although the mechanism remains unclear, SIS appears to enhance cellular repair, as sleepless ALA-impaired mutant animals show reduced survival after noxious heat exposure (Drosophila promotes survival following heat exposure or infectious challenges (C. elegans and vertebrates.Sleep homeostasis, which manifests as increased sleep amount or depth following sleep deprivation, indicates that sleep serves a function that cannot be bypassed . Howeverpressure . Similarnditions . The datring DTS . These dexposure . This fuallenges , and sleallenges . Interesallenges , indicatAlthough the requirement for oxygen-sensing soluble guanylate cyclases and OSM-9 TRPV channel activity suggest involvement of the RMG sensory circuit, this study does not include cell-specific rescue experiments. Further analysis is required to identify the NPR-1 site of action in the modulation of SIS by hyperoxia.All methods can be found in the accompanying"} +{"text": "A 52-year-old woman presented with a complaint of neck swelling. The patient showed signs of hyperparathyroidism: hypercalcemia, and hypophosphatemia. Tc-99m MIBI dual-phase parathyroid scintigraphy and SPECT revealed increased activity in a regular-bordered, \u201cdoughnut\u201d-shaped mass on the left side of the thyroid gland with a central hypoactive area. The cervical ultrasound identified a mixed echoic thyroid nodule with a central large cystic portion, and no parathyroid gland abnormality. Total thyroidectomy and parathyroid exploration was performed. Pathological evaluation of the resected thyroid specimen reported a giant intra-thyroidal hemorrhagic parathyroid adenoma."} +{"text": "Physical illness confers risk for late-life suicide, yet few studies report whether risks differ with older age and among Veterans. We examined age-stratified associations between physical illness and suicide attempt among Veterans 65+ years from a larger retrospective case-control study that utilized secondary data from the Veterans Affairs Corporate Data Warehouse and Suicide Prevention Applications Network. Controls were matched by age, sex, and mental health treatment utilization. Risk estimates for 15 conditions and a combined comorbidity score were stratified by young-old (65-74), middle-old (75-84), and oldest-old (85+), adjusting for age and sex within strata. Neurodegenerative disorder or dementia diagnosis within 180 days conferred the highest risks across young-, middle-, and oldest-old. Cerebrovascular disorder was associated with higher risk among the oldest-old versus young-old (ORs=6.1 vs 2.2). Findings differ from reported risks for suicide death. Illness may be experienced differently across later-life."} +{"text": "In this data article, we provide field emission scanning electron microscopy (FE-SEM) and energy dispersive X-ray spectroscopy (EDS) images of wet-spun polyurethane (PU)-silver nanoparticles (AgNPs)/graphene nanoplatelets (GNPs) composite fibers according to the content of AgNPs and GNPs. In addition, microstructural changes of PU-AgNPs/GNPs composite fibers due to heat treatment at various temperatures are provided. The data collected in this article is directly related to our research article \u201cStretchable and Electrically Conductive Polyurethane- Silver/Graphene composite fibers prepared by wet-spinning process\u201d [1]. In addition, surface and cross-sectional EDS images of PU-AgNPs/GNPs composite fibers were provided. As shown in 2The PU-AgNPs/GNPs composite fibers were prepared by wet-spinning method"} +{"text": "Older adults with mild cognitive impairment (MCI) are at an elevated risk of falls. We conducted a pilot longitudinal observational study to examine the natural course of brain intrinsic functional connectivity (FC) and cognitive function changes in association to falling history in older adults with MCI. 15 MCI participants included 10 non-fallers and 5 fallers from Metro Vancouver, BC, Canada. At study entry and 1-year follow-up, participants completed brain scanning session of structural MRI and resting state (RS) functional MRI, the Montreal Cognitive Assessment (MoCA), and the Mini Mental State Examination (MMSE). Results indicated an interaction between time (baseline vs. follow-up) and falls history on RS-FC in individuals with MCI (p<0.001). At 1-year follow-up, MCI non-fallers showed increased FC between frontal, parietal and occipital cortex (from baseline R=0.141 to follow-up R=0.321) and lack of decline on cognitive measures. Meanwhile, MCI fallers showed weakening of FC between those brain regions (from baseline R=0.314 to follow-up R=0.201) with simultaneous cognitive deterioration. Significant relationships between FC strength and cognitive status existed only at follow-up , suggesting that the triggered functional compensatory brain mechanisms in MCI non-fallers are not successfully executed in MCI fallers. Together, our pilot data suggest that older adults with MCI who fall show more advanced brain functional degradation with adjacent cognitive decline as compared to MCI individuals who do not fall."} +{"text": "Background: This meta-analysis aimed to explore if immunotherapy or chemotherapy alone or in combination is a better first line treatment strategy for advanced non-small cell lung cancer (NSCLC) patients.Methods: Electronic databases including Google Scholar, PMC, PubMed, EMBASE, Scopus and the major conference proceedings were searched for relevant randomized controlled trials (RCTs) comparing outcomes of immune-checkpoint inhibitor combined with chemotherapy or immune-checkpoint inhibitor alone over chemotherapy alone in patients with advanced NSCLC without previous treatment. Study heterogeneity was assessed using the I2 test.Results: A total of 14 RCTs including 8,081 treatment na\u00efve advanced NSCLC patients were enrolled in this study. Our results showed that in comparison to chemotherapy alone, introducing immunotherapy into first-line chemotherapy has significant benefit in tumor response , progression-free survival (PFS) , and overall survival (OS) but with an increased risk of grade3 - 5 toxicity . The pooled results of comparison of immune therapy alone with chemotherapy alone in selected patients with positive expression of Programmed Death-ligament (PD-L1) or with a high tumor mutational burden, demonstrated similar tumor response , 3 - 5 grade toxicity and long-term outcomes, including OS and PFS .Conclusions: Our meta-analysis showed the superiority of combination therapy over monotherapy with chemotherapeutic agents in terms of tumor response, and long-term survival, but with an increased the 3 - 5 grade toxicity. And immune-checkpoint inhibitors alone showed similar tumor response, toxicity and long-term outcomes compared to platinum-based chemotherapy in selected patients. EGFR), anaplastic lymphoma kinase gene (ALK), and orphan receptor tyrosine kinase (ROS) Lung cancer is one of the most lethal diseases and has become the leading cause of cancer related deaths Drugs interrupting immune checkpoints, such as anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), anti-programmed cell death protein 1 (PD-1), anti-programmed cell death-ligand 1 (PD-L1), and others, can enhance anti-tumor immunity and mediate durable cancer regressions Over time several studies exploring the safety and efficacy of immunotherapy as the first-line treatment strategy for advanced NSCLC have been published st January, 2010 and 1st June, 2019. The following medical subject heading (MeSH) terms were used: (1) \"non-small cell lung cancer or NSCLC\"; (2) \"nivolumab or pembrolizumab or atezolizumab or ipilimumab or durvalumab\"; (3) \"PD-1 or PD-L1 or CTLA-4 or immune checkpoint inhibitor\"; (4) \"Randomized Controlled Trial or RCT\". All potentially eligible and relevant clinical studies were manually retrieved and examined. Studies that met the following criteria were included in this meta-analysis: (a) RCTs; (b) studies comparing the combination of immune therapy and chemotherapy with chemotherapy alone in the treatment of advanced treatment-naive NSCLC patients; and (c) studies comparing immune therapy alone with chemotherapy alone in the treatment of advanced treatment-naive NSCLC patients. Non-randomized controlled trials, or studies unrelated to the first-line immune therapy, were excluded. Ultimately, 14 RCTs were included for quantitative analysis were searched by two authors (Chen and Zhou) independently for RCTs published between 1The following information from the eligible studies were extracted by two authors (Tang and Chen) independently: year of publication, number of included patients, treatment regimen, and clinical outcomes. Clinical outcome measures included tumor response, long-term survival [progression-free survival (PFS) and overall survival (OS)], and the toxicity (3-5 grade toxicity and toxicity leading to discontinuation of treatment). Tumor response was stratified as objective responders who obtained a complete or partial response and as non-responders who experienced a stable or progressive disease according to the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 Two authors (Chen and Zhou) independently assessed the methodological quality of the eligible studies according to the Cochrane Collaboration guidelines v5.1.0 2 statistics, where an I2 value > 50% was defined as substantial heterogeneity according to the Cochrane Collaboration guidelines v5.1.0 2 was < 50%, the fixed-effects model was used to assess outcomes; otherwise, the random-effects model were preferred. Sensitivity analysis using both fixed and random-effect models for the same data was conducted to confirm the robustness and reliability of the results.All statistical analyses were performed using Stata12.0 software . Risk ratios (RRs) and hazard ratios (HRs) with 95% confidence intervals (CIs) were calculated for dichotomous data. Heterogeneity among these included studies was evaluated using IThe database search retrieved 5220 records. After deleting duplicate results, a total of 3410 abstracts were screened for eligibility, and 42 clinical trials were considered potentially eligible for inclusion based on titles and abstract review. After retrieving and further analyzing the full-text of these studies, another 28 studies were excluded; the remaining 14 studies 2 = 66.8%) 2 = 65.7%) 2 = 94.2%) 2 = 70.5%) Fig. A 27-30. 2 = 72.6%) . The median PFS of patients receiving nivolumab monotherapy (9.7 months) has been reported to be much longer than those receiving chemotherapy alone (5.8 months) Comparison of monotherapy arms of immune-checkpoint inhibitors and chemotherapy shows that selected patients with positive PD-L1 tumors Numerous limitations of these trials may hinder the fairness of this meta-analysis. Since only 14 RCTs were included in this meta-analysis, the results were underpowered. Furthermore, the HRs and corresponding 95% CIs were mainly extracted from the original studies without access to individualized data, which might have contributed to reporting bias. High heterogeneity was observed among the included studies, which may have decreased the strength of our meta-analysis. The high heterogeneity may be explained by the following: First, different immune checkpoint inhibitor regimens were used in different studies. Second, inclusion criteria differed among the included studies. Third, different treatment strategies had been used among studies.The results of this meta-analysis demonstrated the superiority of combined immunotherapy with chemotherapy over chemotherapy alone in terms of tumor response and long-term survival as a first-line treatment strategy for advanced NSCLC patients but with higher rate of 3 - 5 grade toxicity. And immune-checkpoint inhibitors alone showed similar tumor response, toxicity and long-term outcomes compared to platinum-based chemotherapy in selected patients."} +{"text": "Critical Care the effectiveness of Gram stain results to reduce the use of broad-spectrum antibiotics [The rapid pandemic spread of multidrug-resistant (MDR) pathogens and the paucity of new, effective antibiotics are placing patients\u2019 safety at risk worldwide . The Woribiotics . Here, wWe conducted a prospective observational study from July 2016 to June 2017. Patients diagnosed as having ventilator-associated pneumonia (VAP), defined by a modified clinical pulmonary infection score \u2265 5, were enrolled and treated according to a Gram stain-guided antibiotic choice algorithm years were enrolled during the study period. Clinical risk factors for MDR pathogens were present in 13 (68.4%) of the VAP patients. Pathogens isolated from endotracheal aspirates are presented in Additional\u00a0file\u00a09\u201381 yearClinicalTrials.gov NCT03506113, registered on 29 March 2018) [Our Gram stain-guided antibiotic choice algorithm was shown not only to safely guide appropriate initial antibiotic therapy but also to properly cure VAP. On the basis of the promising results of this study, we are conducting a multicentre, randomised, non-inferiority trial (GRam stain-guided Antibiotics ChoicE for Ventilator-Associated Pneumonia (GRACE-VAP)) to compare our Gram stain-guided treatment with guidelines-based treatment for patients with VAP Additional file 2:Table S2. Pathogens associated with ventilator-associated pneumonia. MRSA: methicillin-resistant Staphylococcus aureus. (DOCX 22 kb)Additional file 3:Table S3. Clinical response in the present study and previous studies. VAP: ventilator-associated pneumonia; HAP: hospital-acquired pneumonia. (DOCX 22 kb)"} +{"text": "Zaire ebolavirus (EBOV) within the eye. As part of a large research project aimed at defining the human RPE cell response to being infected with EBOV, this work focused on the microRNAs (miRNAs) associated with the infection.Survivors of Ebola virus disease (EVD) are at risk of developing blinding intraocular inflammation\u2014or uveitis\u2014which is associated with retinal pigment epithelial (RPE) scarring and persistence of live Using RNA-sequencing, we detected 13 highly induced and 2 highly repressed human miRNAs in human ARPE-19 RPE cells infected with EBOV, including hsa-miR-1307-5p, hsa-miR-29b-3p and hsa-miR-33a-5p (up-regulated), and hsa-miR-3074-3p and hsa-miR-27b-5p (down-regulated). EBOV-miR-1-5p was also found in infected RPE cells. Through computational identification of putative miRNA targets, we predicted a broad range of regulatory activities, including effects on innate and adaptive immune responses, cellular metabolism, cell cycle progression, apoptosis and autophagy. The most highly-connected molecule in the miR-target network was leucine-rich repeat kinase 2, which is involved in neuroinflammation and lysosomal processing. Our findings should stimulate new studies on the impact of miRNA changes in EBOV-infected RPE cells to further understanding of intraocular viral persistence and the pathogenesis of uveitis in EVD survivors. Zaire ebolavirus (EBOV) has been isolated from intraocular fluid after resolution of the viremia withComputational predictions of the targets of differentially expressed human miRNAs were performed using public databases with different algorithms: Diana microT CDS (v.5.0) , 15, filGene ontology and path7 reads per replicate . This trignaling . Our priignaling . MicroRNignaling .After entering the host cell, EBOV is transported via endolysosomal trafficking . InfecteAs applies to all viruses, EBOV infection involves hijacking translational machinery of the host cell to produce viral particles. Gene ontology enrichment analysis for molecular function showed strong activation of DNA-binding and transcription pathways in EBOV-infected human RPE cells. Known to bind double-stranded RNA, multiple genes in the 2\u2032-5\u2032-oligoadenylate synthetase pathway were upregulated in our gene ontology enrichment analyses, indicating a miRNA-facilitated host response to EBOV. Activation of kinases was prominent; pathways involving kinases\u2014such as protein kinase R\u2014are directly involved in the response to EBOV, binding to double-stranded RNA, activating cellular targets and inhibiting the host translational machinery .Virus-infected cells release exosomes containing host- and virus-derived miRNAs that modulate gene expression in uninfected neighbouring cells . Human RRNA viruses do not typically encode miRNAs . HoweverOur work provides new information about the potential post-transcriptional regulation of the human RPE cell response to infection with EBOV. Review of the biological targets of the 15 highly-induced or repressed miRNAs indicates a broad range of potential regulatory activities, including effects on immune responses, cellular metabolism, cell cycle progression, apoptosis and autophagy in the host cells. MicroRNA expression varies by tissue, including within the eye , 48; henWe studied EBOV infection in the ARPE-19 human RPE cell line, in place of cells isolated from human eyes. This cell line is a well-characterized, robust model for studying human retinal pigment epithelium , and thuMicroRNA expression was evaluated at one time-point post-infection. The time-point was selected in our previously published study , to be mHuman RPE cells were studied in isolation, while the intraocular environment includes multiple cell populations. However, this approach allowed us to focus specifically on gene expression in a key intraocular target cell for EBOV.Ebolavirus species may induce different miRNA expression. Cells were not co-infected with other pathogens: co-infection influences EVD outcome, which might in part reflect altered miRNA expression, as demonstrated in other infections [Cells were infected with virulent EBOV, which caused recent EVD epidemics; less virulent fections , 50.Additional file 1. Trimming statistics for RNA sequencing data generated for small RNA expressed in human RPE cells at 24\u00a0h following infection with EBOV or mock-infection.Additional file 2. Lists of total and differentially expressed host miRNAs expressed in human RPE cells at 24\u00a0h following infection with EBOV or mock-infection.Additional file 3. Graphic showing multidimensional scaling of small RNA expressed in human RPE cell at 24\u00a0h post-infection with EBOV.Additional file 4. List of EBOV miRNAs expressed in human RPE cells at 24\u00a0h following infection with EBOV or mock-infection.Additional file 5. List of predicted miRNA targets from Diana microT, miRDB and TargetScan databases.Additional file 6. List of validated miRNA targets from miRecords and miRTarBase databases.Additional file 7. List of differentially-expressed genes in human RPE cells at 24\u00a0h following infection with EBOV or mock-infection.Additional file 8. List of enriched gene ontology categories and KEGG pathways for biological targets of differentially expressed miRNAs in EBOV- versus mock-infected human RPE cells.Additional file 9. Results of network construction based around differentially expressed miRNAs in EBOV- versus mock-infected human RPE cells.Additional file 10. Enlargement of Fig."} +{"text": "S) from the Bartha-K61 (as backbone) and AH02LA strain (as template for gD and gC genes) through bacterial artificial chromosome (BAC) technology using homologous recombination. The growth kinetics of PRV B-gD&gCS was compared with Bartha-K61. Its safety was evaluated in 28-day-old piglets. Protection efficacy was tested in piglets by lethal challenge with AH02LA at 7\u2009days post vaccination, including body temperature, clinical symptoms, virus shedding, mortality rate, and lung lesions.Since 2011, pseudorabies caused by a variant PRV has re-emerged in many Chinese Bartha-K61-vaccinated pig farms. An efficacious vaccine is necessary to control this disease. We described the construction of a gD&gC-substituted pseudorabies virus (PRV B-gD&gCS clone were successfully generated. The growth kinetics of PRV B-gD&gCS strain on ST (Swine testicular) cells was similar to that of the Bartha-K61 strain. No piglets inoculated intramuscularly with PRV B-gD&gCS strain exhibited any clinical symptoms or virus shedding. After AH02LA challenge, all piglets in PRV B-gD&gCS and Bartha-K61 groups (n\u2009=\u20095 each) survived without exhibiting any clinical symptoms and high body temperature. More importantly, PRV B-gD&gCS strain completely prevented virus shedding in 2 piglets and reduced virus shedding post challenge in the other 3 piglets as compared with Bartha-K61 group.The results showed that a BAC clone of Bartha-K61 and a B-gD&gCS strain is a promising vaccine candidate for the effective control of current severe epidemic pseudorabies in China.Our results suggest that PRV B-gD&gC Pseudorabies is a highly contagious and economically significant porcine infectious disease in many countries \u20134. The pThe PRV genome is a 143-kb-long linear double-stranded DNA encoding for approximately 70 genes, including 11 different glycoproteins , 13. gB,In this study, we generated a gD&gC-susbtituted PRV mutant from the Bartha-K61 (as backbone) and AH02LA strain (as template for gD and gC genes) through bacterial artificial chromosome (BAC) technology using homologous recombination, and evaluated its growth kinetics as well as safety and immunogenicity for piglets. Our results showed that the gD&gC-substituted pseudorabies virus vaccine strain could not only provide complete clinical protection but also effectively reduce and may even prevent virus shedding against lethal AH02LA challenge, indicating that it might be an efficient vaccine to control this emergence disease in China.B-H2B DNA was transfected into GS1783 competent cells, generating the infectious clone for further gene manipulations through the En Passant method at 48\u2009h after co-transfection of Bartha-K61 DNA and the BAC transfer vector plasmid pHA2-Puc19-H1S-mini-F) Fig. . RFLP anS, co-transfection of DNA of pB-gDS-mini-F and H1-H2-gCA-T, non-fluorescent plaques were observed under UV light (488\u2009nm) challenge. More importantly, it greatly reduces and may even prevents virus shedding in piglets challenged with virulent AH02LA strain.For immunogenicity, after AH02LA challenge, all piglets in challenge control group showed typical clinical syndromes including prolonged high fever, sneezing, coughing, dyspnea and spirits atrophy. No clinical symptom was observed in any piglets in PRV B-gD&gCS) from the Bartha-K61 (as backbone) and AH02LA strain (as template for gC and gD genes). The growth kinetics of PRV B-gD&gCS were similar to that of Bartha-K61. Piglets experiments showed that PRV B-gD&gCS is safe for piglets. Furthermore, it provides complete clinical protection, and greatly reduces and may even prevent virus shedding against a pseudorabies variant (AH02LA) challenge.Since 2011, pseudorabies caused by a variant PRV has re-emerged in Bartha-K61-vaccinated pig farms in China . PreviouEn Passant protocol. PRV B-gD&gCS was obtained by homologous recombination between pB-gDA-mini-F and H1-H2-gCA-T. Compared to the parental Bartha-K61, PRV B-gD&gCS showed similar growth properties on ST cells.Mutants of herpesvirus were generated by the BAC mutagenesis protocol to study the mechanism of the viruses and construct vectored vaccines . In the S groups showed no clinical pseudorabies symptoms post inoculation. After AH02LA challenge, clinical symptom was observed in none of the piglets in PRV B-gD&gCS group. This result indicated that PRV B-gD&gCS was safe and provided effective protection against virulent PRV variant. Vaccine has played an important role to eradicate PRV, and it is crucial to reduce or prevent virus shedding post infection with virulent strain. Virus shedding is one of the important parameters to evaluate the efficacy of PRV vaccine [S group did not excrete PRV post immunization. Two piglets in piglets of PRV B-gD&gCS group did not shed virus post challenge, and the other three pigs recorded lower titers of shed virus compared with pigs in the Bartha group.Variant PRV infections cause high mortality and clinical symptoms including fever, and respiratory and neurologic symptoms in piglets . In the vaccine . The effS group had reduced virus shedding, but did not completely stop virus shedding post challenge.Mutations of glycoprotein genes may directly lead to the impaired protection of Bartha-K61 vaccination against PRV variant. In a previous study, the insertion or deletion mutations was found in gB, gC, gD, gE, gI and gN genes of AH02LA strain compared with Bartha . gB, gC S. PRV B-gD&gCS is safe for piglets and provides complete clinical protection against a pseudorabies variant (AH02LA) challenge. More importantly, it effectively reduces and may even prevent virus shedding in piglets challenged with virulent AH02LA strain. Future studies involving neutralizing antibody and cell-mediated immunity analysis are necessary to elucidate the mechanism underlining the virus shedding reduction in piglets vaccinated with PRV B-gD&gCS.We constructed an infectious BAC clone of Bartha-K61 and a gD&gC-substituted pseudorabies virus PRV B-gD&gC2 in a humidified incubator.PRV stain AH02LA was isolated and identified in our lab (CGMCC No. 10891) . The PRVB) and right homologous arm (H2B) of gC were amplified from the Bartha-K61. Both fragments H1B and H2B were cloned into pUC19 using restriction enzyme sites in the primers and gD (AH02LA) were amplified from the AH02LA by PCR and cloned into T Vector pMD19 (Takara), and the resulting constructs were named H1-H2-gCA-T and gDA-T, respectively. The plasmid gDA-KAN-T containing gDA and a kanamycin resistance gene inserted at the Sac I restriction site was constructed by cutting and ligating for further En Passant recombination . Growth kinetics curve was performed in triplicate.Multi-step growth kinetics of virus was tested on ST cells with a multiplicity of infection (MOI) of 0.01 as described previously . The culE.coli cells DH5\u03b1 (Takara), Electrocompetent MegaX DH10B T1R Electrocomp cells (Invitrogen) and GS1783 (provided by professor Nikolaus Osterrieder) were used for bacterial manipulations. Electroporation was carried to transform viral or BAC DNA into E.coli cells as described earlier [Commercial chemical competent earlier , 33, 34.A -T, a pair of specific primers , plaque purification was carried out to obtain the homogeneous viruses, named B-mini-F. DNA of B-mini-F was transferred into E.coli DH10B competent cells (Invitrogen) by electroporation. Restriction fragment length polymorphism (RFLP) with Hind III and Sph I were determined to select positive clone of pB-mini-F, which was then electroporated into E.coli GS1783 for mutagenesis of Bartha-K61 genome [A Bartha-K61 infectious clone was generated following the general technique of BAC . Briefly1 genome .A-KAN was insert into gD area of pB-mini-F through the En Passant [A-KAN, which contains 40-bp homologous sequences of Bartha-K61. PCR product was digested with Dpn I and was electroporated into GS1783 with pB-mini-F to achieve the first recombination at the gD sites. Through the second recombination, the target recombinant pB-gDS-mini-F clone was selected with the kanamycin resistance gene deletion , ST cells were transfected with pB-gDS-mini-F DNA and H1-H2-gCA (generated as described above). One to two days after transfection, non-fluorescent plaques (488\u2009nm) were selected and purified to obtain a homogeneous population. The substituted gD and gC genes were confirmed using PCR and sequencing.To replace the gD of Bartha-K61, gD Passant . Brieflyn\u2009=\u20095 each) based on equal body weights, and housed in separated facilities. Piglets were inoculated intramuscularly with 2\u2009mL of PRV B-gD&gCS , Bartha-K61 , PBS (group C) or PBS (group D). One week after inoculation, groups A, B and C were challenged intranasally with 2 LD50 PRV AH02LA to assess protection. All piglets were monitored daily for body temperature, clinical signs, and virus shedding from1 day post inoculation (dpi) to 14\u2009day post challenge (dpc). During the experimental period, the temperature of the pig house was maintained at 28\u2009\u00b1\u20092\u2009\u00b0C. All piglets were fed twice daily (at 8:00 and 17:00) and had free access to water via a low-pressure nipple drinker. Upon completion of the experiments, all animals were euthanized by intravenous injection of pentobarbital sodium (100\u2009mg/kg).This animal study was approved by the Institutional Animal Care and Use Committee at the Jiangsu Academy of Agriculture Sciences (authorization number SYXK (Su) 2015\u20130019) and Zhengzhuquan Pig Breeding Farm, and performed in strict accordance with the guidelines provided by the Institutional Biosafety Committee. Twenty 28\u2009days piglets free of PRV, porcine reproductive and respiratory syndrome virus, porcine parvovirus, porcine circovirus 2 and classical swine fever virus were obtained from Zhengzhuquan Pig Breeding Farm, Nanjing, China. All piglets were randomly assigned to four groups were used to detect gE or gB antibody in the serum according to the manufacturer\u2019s instructions.Nasal swab samples of all piglets were collected and weighed daily from 0 dpi to 14 dpc. After shaking and freeze-thaw cycles (\u2212\u200970\u2009\u00b0C and 37\u2009\u00b0C), samples were centrifuged and ten-fold serial dilutions of supernatants were used to infect ST cells to determine the viral titers."} +{"text": "To evaluate association of patients\u2019 clinicopathological data with expression of nicotinamide nucleotidetranshydrogenase (NNT) and naturally occurring antisense RNA of the same gene locus (NNT-AS1) in breast cancersamples. In the current case-control study, mean expressions of NNT and NNT-AS1 were assessedin 108 breast tissue samples including 54 invasive ductal carcinoma samples and 54 adjacent non-cancerous tissues(ANCTs) by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). NNT expression was not significantly different between tumor tissues and ANCTs. However, NNT-AS1expression was significantly down-regulated in tumor tissues compared to ANCTs .NNT-AS1 expression was significantly higher in estrogen receptor (ER) negative samples, in comparison with ERpositives (P=0.01). No considerable difference was found in the gene expressions between other subcategories ofpatients. Considerable correlations were detected between expression levels of these two genetic loci in both tumortissues and ANCTs.In the current study, for the first time we simultaneously assessed expression of NNT and NNT-AS1in breast cancer tissues. This study highlights association of ER status with dysregulation of NNT-AS1 in breastcancer tissues. Future researches are necessary to explore the function of this long non-coding RNA (lncRNA) in thepathogenesis of breast cancer. Breast cancer, as the most frequent type of femalecancer, has prompted several investigators to finddiagnostic or prognostic biomarkers among long noncodingRNAs (lncRNAs) . This siNicotinamide nucleotide transhydrogenase (NNT)and the related NAT in the pathogenesis of somehuman cancer types. NNT has an indispensible rolein the homeostasis of NADH and NADPH participates in proliferation, migration, invasionand metastasis of colorectal cancer (CRC) cells (NNT-AS1 expressionhas enhanced cell proliferation and inhibited cyclearrest as well as apoptosis through modulation of miR-363/CDK6 axis (Recent studies have highlighted contribution ofnd NADPH . Thesignd NADPH . NNT silpathways . The relC) cells . InhepaDK6 axis .NNT-AS1 in breast cancer tissues compared toANCTs in association with patients\u2019 clinicopathologicaldata, to find whether their transcript levels are alteredin breast cancer in parallel, or they can be used asbiomarkers of breast cancer.NNT loss has led to accumulation of acetylatedcatabolic substances of polyamines and a subsequentdiminution of spermine and spermidine. Polyaminecatabolism would mutually be elicited by oxidativestress and over-produce hydrogen peroxide, resultingin a malicious cycle that accelerates reactive oxygenspecies (ROS) production , 9. In sFor the current case-control study, a total of 108breast tissue samples -including 54 invasive ductalcarcinoma samples and 54 ANCTs- were excisedduring surgery from patients hospitalized in Farmaniehand Sina Hospitals during January2017-January 2018. Patients with definite diagnosisof invasive ductal carcinoma were included in thestudy. Those with other types of breast cancer andthose received chemo-/radio-therapy before surgerywere excluded from the study. All patients signed thewritten informed consent forms. The study protocolwas permitted by the Ethical Committee of ShahidBeheshti University of Medical Sciences (IR.REC.SBMU.1397.764), Iran. Clinical and demographicaldata of patients were collected through evaluation ofmedical reports and interviews with patients.Total RNA was extracted from tumor tissues and ANCTsusing the TRIzol\u2122 reagent based onthe company guidelines. In brief, we homogenized 75 mgof tissues in 1 ml TRIzol\u2122 reagent, followed by RNAprecipitation using isopropanol and washing it in 75%ethanol . After aHPRT1-F: 5\u02ca-AGCCTAAGATGAGAGTTC-3\u02ca,R: 5\u02ca-CACAGAACTAGAACATTGATA-3\u02ca,FAM-CATCTGGAGTCCTATTGACATCGC-TAMRA;NNT1-F: 5\u02ca-AGCCACCTTCTGTGTTACTTGC-3\u02ca,R: 5\u02ca-TAGCCCAGAGCTGCCATGAC-3\u02ca,FAM-TCAACCGTCAGGCTGCCACTGCTG-TAMRANNT-AS1-F: 5\u02ca-CTTCCACTCTCGGGGACAGG-3\u02ca,R: 5\u02ca-GCACCAGGTTTGATTGACAAGG-3\u02ca,FAM-TTGTCTCTGCCTCGGCCTGCGG-TAMRA.PCR efficiency and threshold cycle (Ct) values wereobtained to quantify relative expression of each geneticlocus in tumor tissues and ANCTs.SPSS software version 18.0 wasused for statistical analysis. Ct values obtained fromqRT-PCR experiments were adjusted based on the PCRefficiency values. Association of patients\u2019 data withrelative expression of the gene or lncRNA (down-/up-regulation in the tumor samples vs. ANCTs) wasevaluated using Chi-square test. Relative expression ofeach genetic loci in each tumor sample was calculatedusing EfficiencyCt reference/ EfficiencyCt target formula.Data is presented as mean \u00b1 SD. The difference inthese values between individual groups of patients wasevaluated using Tukey\u2019s honest significance test. Thepairwise correlation between relative transcriptionlevels of the genetic loci in each set of samples(tumor tissues and ANCTs) was calculated using theregression model. For all statistical analyses, P<0.05was regarded as significant. The receiver operatingcharacteristic (ROC) curve was plotted to assess thepower of genetic loci expression levels for diagnosisof disease status in breast samples. The Youden index(j) was applied to get the highest difference betweensensitivity (true-positive rate) and 1-specificity .Demographic and clinical information of the studyparticipants are reported in Table 1.NNT expression level was not significantly differentbetween tumor tissues and ANCTs. However, NNT-AS1expression was significantly down-regulated in tumortissues compared to ANCTs . Figure 1 shows relative expression of NNT andNNT-AS1 in each set of samples, as described by -\u0394Ctvalues (Ct reference-Ct target).Based on the transcriptions in each tumor samplecompared to related ANCT (<1 or >1), we categorizedpatients to up-/down-regulated groups. Then, weevaluated associations between such values andpatients\u2019 clinicopathological data. No association wasfound between relative expression of genetic loci intumor tissue vs. ANCT and any of tumor characteristics.Ct reference/EfficiencyCt target formula and compared these valuesbetween tumor subgroups. We detected significantlyhigher expression of NNT-AS1 in ER negative samplescompared to ER positive cases (P=0.01). No remarkabledifference was found between transcription levels ofthe other patient subcategories (We also calculated relative expression of eachgenetic loci in tumor samples using Efficiencytegories .NNT were correlated with the expression of NNTAS1in both ANCT and tumor samples (Correlations between transcript level of NNT andits naturally occurring antisense was assessed inboth tumor and ANCT samples. Transcription levelsof NNT-AS1 expression in prediction ofdisease status in breast samples was evaluated using ROCcurve (The power of OCcurve . AssessmNNT and NNT-AS1 in breast cancertissues in comparison with ANCTs and found downregulationof NNT-AS1 in tumor tissues, in spite of detectingsimilar level of NNT expression in tumor tissues and ANCTs.NNT-AS1 has previously been shown to exert oncogeniceffects in CRC, HCC, osteosarcoma and cervical cancer(NNT-AS1 silencing enhancescell migration and invasion, while it suppresses apoptosis(NNT-AS1 in thecurrent study is consistent with the previously reporteddysregulation of this molecule in ovarian cancer. ThislncRNA is transcribed in the opposite direction of NNTgene and it has no intersection with latter gene nucleotidesequence (NNT-AS1 in breast tumor tissues comparedto ANCTs in correlation with patients\u2019 survival and HER2,but not ER, status. In vitro experiments showed that NNT-AS1contributes to breast cancer pathogenesis via alteringmiR-142-3p/ZEB1 axis. The inconsistency between ourresults and their results might be due to a possible differencein the mean age of the study participants or an ethnic-basedmodulator of transcription. They have shown that ZEB1 ispositively regulated by NNT-AS1. In our previous study,on the same cohort of patients, we failed to demonstrateany significant change in ZEB1 expression between tumortissues and ANCTs (NNT-AS1might participate in the pathogenesis of breast cancerthrough other mechanisms, including regulation of NNTexpression. Significant correlation between NNT and NNT-AS1expressions, especially in non-tumor tissues, implies thepresence of a feed-forward loop between these two geneticloci which should be assessed in future studies.In the current study, for the first time we simultaneouslyassessed expression of l cancer, 11, 12.poptosis. So, thesequence . Most resequence demonstrnd ANCTs . So, we NNT-AS1 in tumortissues compared to ANCTs, we demonstrated higherexpression of it in ER negative tumor samples, comparedto ER positive samples, likely suggesting the importanceof this lncRNA in pathogenesis of ER negative breastcancers. Future studies are needed to evaluate expressionof NNT-AS1 in larger cohorts of patients with regards tohormone receptor status.In spite of totally down-regulation of NNT-AS1 expressionin prediction of the disease status in breast samples.Although transcription level of this genetic locus is notindividually a sensitive marker for prediction of breastcancer, it might increase specificity of other putativepanels of gene expression.Finally, we evaluated the power of NNT-AS1 inbreast cancer tissues compared to ANCTs in association withER status. Future studies are necessary to explore function ofthis lncRNA in the pathogenesis of breast cancerThe current study shows down-regulation of"} +{"text": "The purpose of this study is to compare the spatial concordance of 18F-DCFBC or 18F-DCFPyL) and 18F-NaF PET/CT. In pelvic and spinal lesions detected by both radiotracers, regions-of-interest (ROIs) derived by various thresholds of uptake intensity were compared for spatial colocalization. Overlap volume was correlated with uptake characteristics and disease status.Prostate cancer patients with known bone metastases underwent PSMA-targeted PET/CT (The study included 149 lesions in 19 patients. Qualitatively, lesions exhibited a heterogeneous range of spatial concordance between PSMA and NaF uptake from completely matched to completely discordant. Quantitatively, overlap volume decreased as a function of tracer intensity. and disease status, where lesions from patients with castration-sensitive disease showed higher spatial concordance while lesions from patients with castration-resistant disease demonstrated more frequent spatial discordance.As metastatic prostate cancer progresses from castration-sensitive to castration-resistant, greater discordance is observed between NaF PET and PSMA PET uptake. This may indicate a possible phenotypic shift to tumor growth that is more independent of bone remodeling via osteoblastic formation. A single, static whole body 18F-NaF PET/CT scan was performed 60 minutes after IV bolus (median injected dose 3.5 mCi [3.4-3.6 mCi]) of radiotracer. All imaging was performed on a Philips Gemini TF system . Low-dose CT transmission scans were obtained for attenuation correction and localization. Emission PET images were obtained at 2 minutes/bed position with 22 slices in bed overlap. The PET images were reconstructed using the Gemini TF ) followed by a static whole-body PET/CT performed at 120 minutes post-injection. The 18F-NaF imaging procedure was identical for both patient PSMA scan groups (median injected dose 3.46 mCi [3.1-5mCi]). Imaging was performed on a GE Discovery MI DR system . Low-dose CT transmission scans were obtained for attenuation correction and localization. Emission PET images were obtained at 3 minutes/bed position with 22 slices in bed overlap. PET images were reconstructed using Q. Clear method, a Bayesian penalized-likelihood TOF reconstruction algorithm with voxel size 2.73 \u00d7 2.73 \u00d7 3.27 mm3.Patients imaged between 2017-2018 underwent eviously . Patient18F-DCFBC, a first generation agent, suffered from relatively high retention in the blood resulting in slower clearance compared to 18F-DCFPyL [18F-DCFPyL and 18F-DCFBC agents are chemically related and bind with high affinity to the same PSMA epitope on prostate cancer cells, they are referred to collectively as 18F-PSMA scans in this analysis.F-DCFPyL . Howevermax, as described in Figure Standardized Uptake Values (SUVs) were calculated as the ratio of measured activity to injected dose per body weight (kilogram). Image review and analysis was performed using commercial software . Lesions determined to be highly suspicious for metastatic disease by consensus of 3 nuclear medicine physicians were considered for analysis. Of highly suspicious bone lesions detected both by NaF PET/CT and PSMA-targeting PET/CT, only those within the pelvis and spine were included in spatial analysis to reduce artifacts introduced from breathing motion (ribs) or patient-positioning motion . Regions-of-interest (ROI) for each tracer were obtained by semi-automated gradient-based method . ROIs were further segmented into areas of highest tracer uptake by 60%, 70% and 80% thresholds of lesion-specific SUVmax and NaF SUVmax. To avoid potential bias from partial-volume errors, the distance between center-of-mass and average pairwise distance from 80%-SUVmax PSMA ROIs and 80%-SUVmax NaF ROIs were also calculated.A two-step image registration procedure was completed using low dose CTs. First global skeletal alignment was achieved using rigid alignment. Next, lesion-based alignment was further achieved using local box-based optimization fit to bony regions containing each lesion . After registration, spatial concordance of NaF and PSMA ROIs was evaluated by assessing the degree of overlap volume in the area of increased radiotracer uptake, defined as the ratio of overlapping volume to minimum lesion volume. This calculation was repeated for NaF and PSMA overlap with CT. Each voxel was assigned to one of seven possible concordance categories: PSMA exclusive, NaF exclusive, CT exclusive, PSMA and NaF only, PSMA and CT only, NaF and CT only, or all matching . Distance between regions of highest tracer uptake was measured by, absolute distance from PSMA SUVVoxel-based accuracy of lesion-specific registration was evaluated using the Intraclass Correlation Coefficient (ICC), estimated from a mixed effect model of Hounsfield Units (HU) measurements with nested random effects for patient, lesions, skeletal region and PSMA tracers (DCFBC or DCFPyL) and fixed effect voxel-based concordance category. The significance of fixed effects was determined by the likelihood ratio test. Differences in lesion-based overlap volume as a function of ROI segmentation method was evaluated using paired Wilcoxon signed-rank test using Rosner\u2013Glynne\u2013Lee method to account for intra-patient correlation . Associa"} +{"text": "Fear-of-falling (FOF) can be adaptive or maladaptive depending on one\u2019s appraisal of knowledge and beliefs, but few have elucidated this cognitive process in older adults surrounding falls. We aim to identify risk factors for high FOF amongst community-dwelling older adults (OA) and middle-aged adults (MA) in Singapore. This was a cross-sectional survey of a nationally-representative sample of OA (\u226560 years) and MA (40-59 years) identified by stratified random sampling. Primary outcome was high FOF measured by a single-item . Independent variables were history-of-falls, quality-of-life, fall-related cognitive appraisal and knowledge indicators . MA were also asked if they\u2019re caregivers. Multiple logistic regressions identified risk factors for high FOF separately by age-groups, adjusting for socio-demographics and comorbidities. The final analysis included 549 OA (70.6\u00b16.88 years) and 309 MA (49.7\u00b15.89 years). No differences in high FOF was found among OA and MA , but there were more falls among OA . Higher knowledge of fall risk factors and self-reported balance problems were significant risk factors for high FOF among OA only, while a history-of-falls and being a caregiver were significant among MA only. Perceived importance to restrict activities was associated with high FOF in both age-groups. Although findings suggest differences in the mechanism of high FOF between OA and MA, both age-groups have maladaptive appraisal tendencies related to restrict activities to prevent falls."} +{"text": "Eisenia andrei (Ea) and striped E. fetida (Ef) lumbricid earthworms are hermaphrodites capable of self-fertilization, cross-fertilization, and asymmetrical hybridization. The latter was detected by genotyping of F1 and F2 progeny of the controlled Ea+Ef pairs by species-specific sequences of maternal mitochondrial COI genes and maternal/paternal nuclear S28 rRNA genes. Among F1offspring there were self-fertilized Ea (aAA), Ef (fFF), and cross-fertilized fertile Ea-derived hybrids (aAF); the latter mated with Ea and gave new generation of Ea and hybrids, while mated with Ef gave Ea, Ef, Ea-derived hybrids and sterile Ef-derived hybrids (fFA). Coelomic fluid of Ea exhibits unique fluorescence spectra called here the M-fluorescence considered as a molecular biomarker of this species. Since similar fluorescence was detected also in some Ef , the aim of present investigations was to identify the M-positive earthworms among families genotyped previously. It was assumed that factor/s responsible for metabolic pathways leading to production of undefined yet M-fluorophore might be encoded/controlled by alleles of hypothetical nuclear gene of Eisenia sp. segregating independently from species-specific S28 rRNA nuclear genes, where \u2018MM\u2019 or \u2018Mm\u2019 alleles determine M-positivity while \u2018mm\u2019 alleles determine M-negative phenotypes. Spectra of M-fluorescence were detected in all 10 Ea (aAAMM) and 19 Ea-derived hybrids (aAFMm), three of four Ef-derived hybrids (fFAMm) and one \u2018atypical\u2019 Ef (fFFMm) among 13 Ef earthworms. Among progeny of \u2018atypical\u2019 M-positive Ef (fFFMm) reappeared \u2018typical\u2019 M-negative Ef (fFFmm), confirming such hypothesis. Alternatively, the M-fluorescence might be dependent on unknown gene products of vertically-transmitted Ea-specific symbiotic bacteria sexually transferred to the Ef partner. Hypotheses of intrinsic and external origin of M-fluorescence might complement each other. The presence/absence of M-fluorophore does not correspond with body pigmentation patterns; Ef-characteristic banding appeared in posterior parts of hybrids body. In conclusion, Ea/Ef hybridization may serve for further studies on bi-directional gene flow.Uniformly pigmented Eisenia sp. are valuable models in various scientific disciplines like biochemistry, ecotoxicology, and biomedicine [Eisenia andrei (Ea) and striped E. fetida (Ef), originally described as pigmentation morphs of the one species spelled as Eisenia foetida, then as its two subspecies, and later on as two independent species with reproductive barrier [Lumbricid earthworms from medicine \u20135 where barrier forming barrier \u20139.E. andrei, hypothetically derived from 4-methylumbelliferyl \u03b2-D-glucoronide [Body pigmentation is often not conclusive, thus during our earlier studies of Ea/Ef delivered from France we have used various methods for proper distinction of specimens of these two species ; among ocoronide , called coronide ; 12; 13,coronide ; 12. Juscoronide capable coronide .Hybridization was detected by genotyping of F1 and F2 progeny of the controlled Ea+Ef pairs by species-specific sequences of both haploid mitochondrial COI genes of maternal origin ; 17 (\u2018a\u2019E. andrei could be transferred to some E. fetida earthworms?Since earthworm genotyping was performed on DNA extracted from amputated (and then regenerating ) tail tiEisenia sp. itself, or might be derived from vertically transmitted E. andrei-specific symbiotic bacteria that can \u2018infect\u2019 partners of copulation. The results of tracking the M-positive earthworms within their families from previous investigations were consistent with hypothesis of the intrinsic origin of fluorophore; the dominant M-allele might be transmitted from M-positive Ea (aAAMM) to fertile Ea-derived M-positive hybrids (aAFMm) and then to \u2018atypical\u2019 M-positive Ef (fFFmM) earthworm and sterile Ef-derived hybrids (fFAmM). Such intrinsic pathway was also consistent with reappearance of M-negative Ef (fFFmm) earthworms in long-lasting cultures of atypical M-positive Ef (fFFMm). However, hypothetical participation of microbiome-derived factors in production of M-fluorophore cannot be neglected. The presence/absence of M-fluorophore does not correspond with body pigmentation pattern.Hypothetically, the M-fluorescence might be dependent either on the metabolic pathway/s of Eisenia andrei (Ea) and Eisenia fetida (Ef) from laboratory colonies at the Lille University (France) were reared for generations in the Institute of Zoology and Biomedical Research of the Jagiellonian University, Krakow, Poland.Adult composting earthworms The main analyses of coelomic fluid were performed on 46 out of 158 descendants of laboratory-paired M-positive Ea and M-negative Ef specimens genotyped previously . In shorProof-of-concept investigations were performed on coelomic fluid of specimens from long-lasting cultures of M-positive Ea (EaMp), \u2018typical\u2019 M-negative Ef (EfMn), and \u2018atypical\u2019 M-positive Ef (EfMp). The EfMp individuals were identified in 2013 during our previous studies .Pigmentation patterns were photographically documented with the DSL camera (Sony SLT-A58).For main experiments, 46 genetically identified aAA, fFF, aAF, or fFA specimens from previous study of similSpectrofluorimetric analysis of the M-fluorophore in non-invasively retrieved coelomic fluid was performed by slightly modified method described previously ; 12; 13.E. andrei ova, i.e. 10 aAA and19 aAF hybrids, were M-positive. Thirteen specimens from E. fetida ova were M-negative, among them 12 fFF earthworm and one fFA hybrid; only one fFF specimen and three fFA hybrids were M-positive (The M-positive (Mp) earthworms exhibited distinct spectra of fluorescence with a peak of absorbance at 314\u2013320 nm (\u03bb = 380) and a peak of emission at 370\u2013380 nm (\u03bb = 320), while the M-negative (Mn) earthworms were devoid of such fluorescence in Triton-lysates of coelomic fluid Inset in . As visipositive .Genealogy of M-positive and M-negative descendants of Ea+Ef pairs has been shown on Eisenia sp. while two recessive \u2018mm\u2019 alleles determine M-negative phenotype. Thus genotypes of phenotypically M-positive earthworms are either of MM or Mm, while genotypes of M-negative specimens are always \u2018mm\u2019.Hypothetically the factor/s responsible for M-fluorescence might be encoded/controlled by the nuclear dominant \u2018M\u2019 allele of some unknown gene/s of Hypothetically, M/m alleles segregate independently from the nuclear A/F sequences of 28S rRNA gene. Therefore the genotype of M-positive Ea specimens may be either aAAMM or aAAMm, while the genotype of M-negative Ef specimens may be only fFFmm. Inter-specific hybrids might be either aAFMm/aAFmM or fFAMm/fFAmM, with the first written allele of each gene being of maternal origin, while Mm/mM have the same phenotypic effects.As illustrated on Theoretically, the M-positive aAFMm hybrids might produce four types of oocytes, aAM, aAm, aFM, and aFm, the two latter genotypes less probable due to mitochondrial-nuclear (aF) incompatibility \u201322, and The aAFMm hybrids gave a progeny in pairs with Ea or Ef specimens illustrated on Only M-positive offspring appeared in the aAFMm+aAAMM pairs, that was consistent with data on The offspring of aAFMm+fFFmm pairs from the hybrid\u2019s ova (aAM and aAm), excluding those with mito-nuclear-incompatibility (aFM and aFm), might give both M-positive (aAFMm) and M-negative (aAFmm) hybrids , but theEisenia sp. [Cinnamonum zeylanicum) [E. andrei was identified as the compound SP-8203, consisting two quinazoline-2,4-diones joined by an N-acetylspermine linker but its fluorescence spectra have not been analyzed in this paper [E. andrei and M-positive Ea-derived hybrids than M-negative E. fetida and rare infertile Ef-ova derived hybrids. Further studies on the selected M-positive E. fetida might be fruitful in testing such supposition.Characteristic fluorescence spectra of coelomic fluid of (MUGlcU) , called lanicum) . Biologilanicum) , anti-fulanicum) , anti-tulanicum) , anti-colanicum) , and antlanicum) activitiis paper . Compounis paper ; 34. TheVerminephrobacter localized in their excetory nephridia [E. andrei [E. andrei, responsible for metabolic pathways leading to production of M-fluorophore. It is also possible that both earthworm-derived and bacteria-derived factors must cooperate to give the final fluorescent product, that is either accumulated breakdown product being significant only as molecular biomarker, or may have unrecognized yet crucial biological significance.Speculations on hypothetical gene with the dominant M-allele are consistent with assumption of the intrinsic origin of M-fluorophore, being entirely dependent on the earthworm own metabolic pathways. Keeping in mind the peculiar copulatory behavior of lumbricid earthworms , hypotheephridia ; 36. Theephridia ; 38. Rec. andrei . Some prE. andrei, as its amount came back rapidly to the initial level after experimental expulsion of coelomic fluid [E.andrei/E.fetida complex, but other techniques shall be used to show conclusively its presence in various earthworm cell types lining coelomic cavity and/or other sources.On the basis of our previous results we may assume that coelomocytes are not the main cellular source of M-fluorophore in ic fluid , 19. ThiE. andrei and E. fetida. If M-fluorophore is genetically controlled by hypothetical gene of Eisenia sp. with the dominant M allele, then such allele may be inherited by Ea-derived hybrid from M-positive Ea parent, and then transferred during mating with M-negative Ef earthworm into some E. fetida and some Ef-derived hybrids. Even one \u2018atypical\u2019 M-positive Ef might propagate this allele by crossing with \u2018typical\u2019 M-negative Ef. Vice versa, in cultures of M-positive Ef earthworms might reappear \u2018typical\u2019 M-negative specimens. However, hypothesis of the microbial origin of F-fluorescence derived from E. andrei specific bacterial symbionts cannot be neglected. Moreover, both the intrinsic and external factors might cooperate to produce the M-fluorophore. The existence of Ea and Ef hybridization make these common species easily maintained in laboratory the attractive models for studies on interspecies gene flow, inter-specific transmission of bacterial symbionts, and hypothetical effects of external factors on these phenomena.Asymmetrical hybridization between Ea and Ef resulted in bi-directional gene flow resulting in two phenomena recognized in our laboratory. First, Ef-like body pigmentation pattern appeared in posterior body segments of hybrids, both Ef- and Ea-derived; second, Ea-specific M-fluorophore was transferred to majority of hybrids and some Ef earthworms. The chemical nature and biological significance of this fluorophore is still an open question, but its fluorescence spectra are reliable markers for tracking the gene flow between S1 Figa) Scheme of aAFMm parental cell, gametes and zygotes; b) Punnett square. Shadowed parts of part \u2018a\u2019 and crossed out parts of part \u2018b\u2019 indicate mitochondrial-nuclear conflicts. Framed genotypes were absent among investigated earthworms. Assumption is that M-fluorescence might be encoded/controlled by the nuclear gene with the dominant \u2018M\u2019 allele and the recessive \u2018m\u2019 allele segregating independently from the nuclear A/F sequences of 28s rRNA gene. The \u2018MM\u2019 and \u2018Mm/mM\u2019 determines the M-positive (Mp) phenotype (in orange) while \u2018mm\u2019 genotype determines the M-negative (Mn) phenotype (in blue). Punnett square is adapted to pairs of hermaphroditic earthworms able to self-fertilization; ova in yellow, spermatozoa in green. In each pair the first allele is that of maternal origin. Framed genotypes were apparently absent among investigated earthworms. Ova and resulted offspring with mito-nuclear incompatibility are crossed out.(TIF)Click here for additional data file."} +{"text": "We hypothesize that impaired signaling via the bile salt receptorFXR underlies the development of IFALD. The aim of this study was toinvestigate whether activation of FXR improves liver homeostasis duringchronic loss of bile in rats.Intestinal failure-associated liver disease (IFALD) is a clinical challenge.The pathophysiol-ogy is multifactorial and remains poorly understood.Disturbed recirculation of bile salts, To study consequences of chronic loss of bile, rats underwent externalbiliary drainage (EBD) or sham surgery for seven days, and the prophylacticpotential of the FXR agonist INT-747 was assessed.Fgf15was undetectable after EBD, and this was accompanied by an anticipatedincrease in hepatic Cyp7a1 expression, indicating increasedbile salt synthesis. Treatment with INT-747 improved serum biochemistry,reduced loss of bile fluid in drained rats and prevented development ofdrainage-associated histological liver injury.EBD for 7 days resulted in liver test abnormalities and histological liverdamage. Expression of the intestinal FXR target gene EBD results in extensive hepatobiliary injury and cholestasis. These datasuggest that FXR activation might be a novel therapy in preventing liverdysfunction in patients with intestinal failure.This study demonstrates that chronic loss of bile causes liver injury inrats. Abro-gated recycling of bile salts impairing of enterohepatic bilesalt/FXR signaling underlies these pathological changes, as administrationof FXR agonist INT747 prevents biliary drainage-induced liver damage.Phar-macological activation of FXR might be a therapeutic strategy to treatdisorders accompanied by a per-turbed enterohepatic circulation such asintestinal failure-associated liver disease. Thecliviz. fistula) fluid into the distal small intestine improvedliver injury, despite continued parenteral nutrition [It has been postulated that loss of enteric fluid from pancre-atobiliary andintestinal secretions, contributes to the devel-opment of IFALD -5. Thusutrition . In partutrition . CollectBile salts act as endogenous activating ligands of nuclear and plasma membranereceptors expressed in numerous tissues, but in particular in the small intestineand the liver . The farCyp7a1. Studies in several an-imalmodels with an obstructed enterohepatic circulation showed that disruption of theFXR-Fgf15 axis was associated with development of (cholestatic) liver injury [Previous studies established the role of the gut in regulating bile salt synthesis,12. In tAn abrogated entero-hepatic cycle is expected to result in impaired delivery of bilesalt ligands to bile salt receptors, in particular FXR that are essential forintestinal and hepatic function. Thus, we hypothesize that loss of bile fluid leadsto diminished activation of FXR, dysregulated bile salt homeostasis and compromisedhepatic and intestinal integrity, events that could underlie the development ofIFALD. The aim of the study was to investigate the effect of FXR agonism on prevention of entero-he-paticdysfunction in a rat model of IFALD due to continuous loss of bile.2.2.1Male Sprague Dawley rats (Charles River) weighing 300-350 grams, were housedunder controlled environmental con-ditions in separate cages at the animalhousing facility of Maastricht University. Animals had free access to regularchow and water throughout the experiment. The study was approved by the AnimalCare Committee of Maastricht Uni-versity (DEC 2009-170).After an acclimatization period of one week, external bili-ary drainage (EBD) wasperformed essentially as described by Kuipers et al. In briefIn a pilot experiment, rats underwent continuous EBD for three or seven days toinvestigate the severity of liver injury. Although inflammation was alreadyapparent after three days, other signs of histological injury and abnormalbiochemistry (cholestasis and hepatocellular damage) developed after 7 days ofcontinuous EBD (data not shown). This duration was cho-sen for the interventionstudy. Thus, rats underwent EBD for 7 days or were sham-operated (n = 8 pergroup). Immediately after surgery, animals received a daily intraperitoneal doseof the FXR agonist INT-747 or vehicle alone (corn oil with 5% DMSO). The final dose ofINT-747 was administered 24 hrs before sacrifice. Bile production in thedrainage groups was determined daily. Unimpeded bile flow was maintainedthroughout the experiment in all animals in the drainage groups.All animals were weighed daily, and none of the animals experienced significantweight changes during the course of the experiment (data not shown). Two rats inthe vehicletreated EBD group died as a result of biliary peritonitis, one rat inthe agonist-treated EBD group died as a result of dehydra-tion, and two rats inthe vehicle-sham group died because of abdominal wall dehiscence (n = 1) orunknown cause (n = 1).At the end of the experiments, rats were anesthetized with isoflurane andsacrificed through aortic puncture between 8: 00 and 12: 00 AM. Blood wastransferred to EDTA tubes and plasma was prepared by centrifugation. Terminalileum and liver were harvested and portions were snap-frozen or pro-cessed forembedding in paraffin. Plasma and tissue specimens were stored at -80\u00b0Cuntil analysis.2.2Liver damage was assessed by analysis of plasma levels of alanineaminotransferase (ALT), aspartate aminotransferase (AST), GGT, ALP, and totalbilirubin . Systemic inflammationwas evaluated by measuring plasma IL-6 levels using ELISA . Plasma levels of the enterocyte damage marker ILBP weredetermined by ELISA . Serum l2.3After deparaffinization and rehydration, H&E stained liver sections (4\u03bcm thickness) were scored individually on a 0 to 4 scale for inflammationby a blinded pathologist (MJG). Score 0 indicating no inflammation; score 1indicating mini-mal periportal inflammation; score 2 indicating mildinflam-mation ; score 3 indicating moderate periportal andsinusoidal inflammation and score 4 indicating severe peri-portal and sinusoidalinflammation. Fibrosis was scored on Sirius Red stained sections with score 0indicating no fibrosis; score 1 indicating mild periportal fibrosis; score 2indicating moderate periportal fibrosis with minimal sprouting; score 3indicating severe periportal fibrosis with moderate sprouting and score 4indicating bridging fibrosis. Bile duct proliferation was examined bymorphometric analysis of pan-cytokeratin stained (Dako) liver sections. Inbrief, cytokeratin-positive cell area (>5 \u03bcm2) and total cell area(H&E stained) were deter-mined in 10 random fields of each section by supervised analysis of auto-mated imageprocessing (Leica QWin v3 software). Only tan-gentially cut bile ductules wereanalyzed.2.4For immunoblot analysis, liver tissue was homogenized in lysis buffer . 20 \u00b5gsolubilized liver protein was separated by reducing SDS-PAGE and transferred toPVDF membrane. Following blocking of unoccupied binding sites with PBScontaining 5% non-fat dry milk powder, mem-branes were probed with rabbitanti-rat Cyp7a1 and rabbit anti-mouse \u03b2-actin (Sigma) antibodies. Secondary detectionconsisted of horseradish peroxidase-labelled goat anti- rabbit IgG antibody and immunocomplexes were visualizedusing enhanced chem-iluminescence (Thermo Scientific). Three independent liverhomogenates were analyzed per experimental group.2.5Hprt and Rplp0) was used as normalizationfactor, and values are graphically presented relative to median expression insham-operated controls.Total RNA was extracted from liver or ileal tissue using TRI reagent (Sigma). 750ng DNAse-treated RNA was con-verted to cDNA . qPCR reactions were conducted in a volume of 20\u00b5l containing cDNA equivalent to 10 ng total RNA, 1x Absolute qPCR SYBRGreen Fluorescein Mix and 150 nM of gene-specificprimers (Supplementary Table 1), and wereperformed in duplicate. Gene expression levels were determined with iQ5 software(Bio-Rad) using a \u0394\u0394Ct relative quantification model. Thegeometric mean of the expression levels of two reference genes and SPSS 22.0 .For histological analysis, multiple fields per section were scored and averagedper animal. Histological scores were tested for significance with theFisher\u2019s exact test. Effects of EBD or agonist treatment on serumbiochemistry, mRNA ex-pression, morphometric parameters, intestinalpermeability, enterocyte damage and systemic inflammation were evaluated withthe Mann-Whitney U test for unpaired samples. A Bon-ferroni correction formultiple testing was applied where ap-propriate. Differences in daily bileproduction in the drainage groups were tested with repeated measures ANOVA. Forvisu-al purposes, data in graphs are presented as means +/- standard error ofmean. 3.3.1P = 0.02, P= 0.065) towards elevated circulating IL-6 was noted in drained animalsreceiving vehicle or even com-pletely prevented by INT-747 . To furts of EBD . Despites of EBD .3.3P =0.005) in drained an-imals without affecting circulating LBP fluid back into theintestinal tract was shown to normalize the fistula output in patients with ahigh-output proximal ECF Toinves3.5Cyp7a1 mRNA , butFXR levels were similar in INT-747 treated groups of animals and theirrespective vehi-cle-treated controls (P = 0.44) and this was accompanied by increased7a1 mRNA and prot7a1 mRNA expressi7a1 mRNA , indicat7a1 mRNA .Hepatic7a1 mRNA . Insham7a1 mRNA . INT-74ani-mals.Nonetheani-mals. Despite andShp . A trend = 0.44) .4.Prolonged loss of bile fluid in patients with intestinal failure is associated withthe development of liver disease in the con-text of intestinal failure . The maji.e. ductular reaction) thatresults in expansion of the biliary net-work [e.g. toxic bile salts,endotoxins and nutri-tional deficits like essential fatty acid deficiency) indrained animals. Indeed, hepatic Il-6 expression, a target of theNF-\u03baB pathway [kB pathway, which is central to hepaticin-flammation [Prolonged EBD resulted in damage to the liver (ALT and AST elevations) and thebiliary compartment (ALP and GGT elevations). Hepatic inflammation may contribute toimpaired canalicular transport (hyperbilirubinemia) and development and/or worseningof cholestatic liver injury -27. Hepanet-work , 29. Thinet-work . The app pathway , was eleammation . Nonetheammation . Lack ofammation . Byactiammation .Il-6 expression. Furthermore, inflammatory signaling in theliver may interfere with proper function of tight junctions between hepatocytecouplets or bile duct epithelial cells [Mrp3. Dis-turbed feedback regulation of bile saltsynthesis on the other hand, results in enhanced production of bile salts assupported by elevated Cyp7a1 protein in drained animals. However, cir-culating C4levels did not reflect the elevated Cyp7a1 protein. Although, INT-747 treatment hadno significant effects on Cyp7a1 mRNA/protein expression and levelsof bile salts in the circulation and the liver, reduced bile flow suggests that FXRagonism prevents deregulated bile salt synthesis follow-ing prolonged EBD.Diminished bile salt synthesis in drained animals receiving INT-747 may result inreduced availability of substrates for Bsep and decreased biliary bile saltsecretion. The latter constitutes the main driving force for bile formation.What could be the mechanism of EBD-induced liver dam-age? Failed delivery ofactivating ligands re-sults in inadequate function of intestinalFXR during EBD. This potential mechanism has two functional consequences. Firstly,gut barrier integrity will become compromised as in-ferred from increased intestinalpermeability , and secal cells .Similaral cells -39. Amonal cells , an obliCyp7a1 is coun-teracted by INT-747,at least at the transcriptional level. Re-duced biliary output in drained animalsreceiving INT-747 can be interpreted as lowered bile salt synthesis and reducedavailability to the canalicular transporters responsible for bili-ary secretion ofthese osmolytically active molecules. En-hanced bile salt synthesis in drainedanimals may result in det-rimental levels of toxic intermediates, which have beenimpli-cated in liver injury in patients with genetic bile salt synthesis defects[The inflamed liver may be particularly sensitive to toxic ef-fects of excessive bilesalts. Nonetheless, 7 days of biliary di-version did not affect hepatic bile saltcontent, nor did FXR agonism result in significant lowering of hepatic bile salts indrained animals. FXR controls the composition of the bile salt pool and regulatestheir conjugation, and accordingly influ-ences toxic potential of bile salt species, 42. Dra defects. Toxic eAdditional protection may be conferred by preservation of intestinal integrity, thus,limiting first pass exposure of the liver to dietary and microbial insults. Impairedgut barrier function following EBD is evident from elevation of circulat-ing LBPlevels, which is already apparent after three days (da-ta not shown) . Thus,translocation of microbial products ap-pears an early event in hepatic injuryfollowing EBD. However, INT-747 did not reduce serum LPB levels. Likewise, there-ported anti-inflammatory effects of FXR agonism were not apparent from ouranalysis of hepatic inflammatory genes. Thus, the molecular pathways underlying thehepatoprotective action of INT-747 in drained animals remain elusive.The pathogenesis of liver disease in patients with intestinal failure has remainedlargely unknown, with studies mainly focusing on the effect of parenteral nutrition-46. The"} +{"text": "Gankyrin (GK) is an oncoprotein which regulates inflammatory responses and its inhibition is considered as a possible anti-inflammatory therapy for inflammatory bowel disease (IBD).Villin-Cre;Gankyrinf/f) and (ii) the distal intestine and colon .In this study, we investigated the role of GK in epithelial cells using mice with intestinal epithelial cell-specific GK deletion in (i) the entire small intestine and colon (Helicobacter japonicum and Bilophila were found to be over-represented in colitis induced Villin-Cre;Gankyrinf/f mice when compared to Gankyrinf/f control mice under the same condition.Unexpectedly, GK-deficiency in the upper small bowel augmented inflammatory activity compared with control mice when colitis was induced with dextran sodium sulfate. Biochemical analyses have revealed GK-deficiency to have caused reduction in the expression of antimicrobial peptides, \u03b1-Defensin-5 and -6, in the upper small bowel. Examination of human samples have further confirmed that the reduction of GK expression in the small bowel is associated with colonic involvement in human Crohn\u2019s disease. Through the sequencing of bacterial 16S rRNA gene amplicons, bacteria potentially deleterious to intestinal homeostasis such as These results highlight the distinct site dependence of the pro- and anti-inflammatory functions of GK and provide important insights into the pathogenesis of IBD. Inflammatory bowel disease (IBD) is characterized by a persistent inflammation in the colon and/or small intestine. Crohn\u2019s disease (CD) may affect any segment of the digestive tract, while ulcerative colitis is restricted to the colon. IBD arises due to disruption of immune tolerance to the gut microbiota that leads to chronic intestinal inflammation and mucosal damage in genetically predisposed hosts , 2. The In IBD, long-standing inflammation is a major risk factor for colorectal cancer, also called as colitis-associated cancer (CAC) . Indeed,Villin-Cre;Gankyrinf/f) and (ii) distal intestine and colon-specific GK-deficient mice . These mice were challenged by dextran sodium sulfate (DSS) which is a colitis-inducing agent. Surprisingly, Villin-Cre;Gankyrinf/f mice were more susceptible to DSS-induced colitis and showed an alteration of gut microbiota in comparison with Gankyrinf/f control mice. In contrast, Cdx2-Cre;Gankyrinf/f showed a similar level of inflammatory response as Gankyrinf/f control mice. This study suggests that GK in the upper small bowel is involved in the pathogenesis of colitis through affecting gut microbiota.To examine whether inhibition of GK might prevent colitis, we have employed 2 kinds of epithelial cell-specific GK-deficient mice, i.e. (i) small intestine and colon-specific GK-deficient mice (Gankyrinflox/flox [Villin-Cre and Cdx2-Cre mice (obtained from Jackson laboratory) were used to produce tissue-specific conditional GK knockout mice, designated as Villin-Cre;Gankyrinf/f and Cdx2-Cre;Gankyrinf/f mice, respectively. All mice were maintained in a specific-pathogen-free environment and housed under a 12-h dark-light cycle (light from 7:00 to 19:00). They were given free access to standard diet and water and were not fasted before the experiments. Sex- and age-matched Cdx2-Cre;Gankyrinf/f, Villin-Cre;Gankyrinf/f [Gankyrinf/f (control) mice (8\u201316\u2009weeks old) were administered with 2.5% (w/v) dextran sodium sulfate in drinking water for 7\u2009days. Inflammatory cell infiltration score was assessed using a method described in a previous study [lox/flox , 10, Vilkyrinf/f , and GanAll animal procedures were performed according to approved protocols and in accordance with the recommendations for the proper care and use of laboratory animals. The animal study protocol was approved by the Medical Ethics Committee of Kindai University Faculty of Medicine and Institutional Animal Care .n\u2009=\u200920) were obtained by endoscopy from patients with CD. Informed consent of all the patients was obtained. The clinical study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki and was approved by the institutional review board of Kindai University Faculty of Medicine.The upper small intestine tissues . Microarray analysis was performed by Hokkaido System Science Co., Ltd. .Real-time qPCR and immunohistochemistry were performed as previously described in the respective references , 11, 12.Frozen samples of caecum and rectum were thawed and homogenized through the usage of Zirconia/Silica Beads (BioSpec Products) and MagNALyzer (Roche Diagnostics). Next, DNA was extracted from the homogenized samples by the usage of QIAamp DNA Mini Kit according to the manufacturer\u2019s instructions . The variable V3\u2013V4 16S rRNA gene regions of the extracted DNA samples were amplified by PCR with 16S Amplicon PCR Forward primer 5\u2032-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -MID-GT-CCTACGGGNGGCWGCAG-3\u2032 and 16S Amplicon PCR Reverse primer 5\u2032-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-MID-GT-GACTACHVGGGTATCTAATCC-3\u2032. The preparation of sequencing libraries was conducted according to the protocol described in \u201816S Metagenomic Sequencing Library Preparation: Preparing 16S Ribosomal RNA Gene Amplicons for the Illumina MiSeq System\u2019 protocol with theAmplicon sequences were processed with the following procedures modified from our previous paper . Low-quaThe representative 16S rRNA sequences of Greengenes database were uset-test implemented in QIIME\u2019s compare_alpha_diversity.py with 10,000 Monte Carlo permutations. The P-values were adjusted for multiple testing using Holm\u2019s method.Rarefaction curves were generated by randomly sampling reads from each sample 10 times and calculating the average number of OTUs across different sampling depths with intervals of 5000 reads using QIIME\u2019s parallel_multiple_rarefactions.py. OTU richness and Shannon diversity index values wBeta diversity analysis was performed using unweighted and weighted UniFrac distanceP-value was repeated 10 times. We used the average of the P-values. The P-values were adjusted for multiple testing using Holm\u2019s method.The significance of the compositional difference between two groups of microbiotas was tested using Adonis implemenPrior to testing the significance of differential abundance of OTUs, OTUs supported by less than a total of 100 reads from samples in the pair of groups under comparison were omitted from analysis. Furthermore, OTUs observed in less than 25% of the total number of samples under comparison were also removed. These steps were done using QIIME\u2019s filter_otus_from_otu_table.py. The statistical significance of the differential abundance of OTUs between sample groups were tested using the DESeq2\u2019s negative binomial Wald test implemenIn some specific cases, we inspected detailed taxonomy of OTUs using homology searches. Homology searches of the representative sequences of OTUs against the NCBI\u2019s nucleotide collection (nr/nt) database were conducted using the online megablast tool , 31 witht-test with P value <\u20090.05 as the significance cut-off, unless otherwise stated.Statistical tests were performed using two-tailed Student\u2019s Gankyrinf/f mice [Villin-Cre and (ii) Cdx2-Cre transgenic mice to generate mice that lack GK levels in their intestinal epithelial cells of (i) the whole small intestine and the colon and (ii) the distal small intestine and the colon , respectively than in Gankyrinf/f control mice (n\u2009=\u20095) in small intestine sections by quantitative PCR with reverse transcription revealed significant decreases of defensin expression in upper small bowel in Villin-Cre;Gankyrinf/f mice, suggesting that GK is at least partially responsible for the transcription of defensins in this part of intestinal tract as well as control small intestine sections using whole-genome gene expression arrays. Data analysis revealed changes in the expression of several genes related to the regulation of stem cells. We confirmed the decrease in expression of R-spondin1 and Wnt5B by quantitative RT-PCR of the GK-deficient small intestine samples relative to control samples suffer from colonic involvement, while the disease is restricted to the small intestine in the remaining CD patients. Therefore, we examined whether GK in the upper small bowel contributes to colonic involvement in CD. Tissue samples of the upper small bowel from CD patients with colonic involvement showed a significantly lower level of GK expression than that in the samples from CD patients without colonic involvement for caecum and rectum samples, though sample size was small.To investigate the variation of bacterial composition among the samples, a principal coordinate analysis was conducted based on the unweighted UniFrac distances Fig. b. In thetum Fig. b. The coots Fig. b. The twVillin-Cre;Gankyrinf/f mice belonged to Bilophila , Helicobacteraceae , Bacteroidales S24\u20137 , Clostridiales , and Erysipelotrichaceae . BLAST searches for the three OTUs belonging to Helicobacteraceae revealed that the representative sequence of OTU-206538 was aligned with the 16S rRNA gene of Helicobacter japonicum (NR_149210.1 and EF373968.1) with 100% sequence identify. The other two OTUs were also aligned with the 16S rRNA gene sequences of Helicobacter species with 100% sequence identity . OTU-206538 (Helicobacter japonicum) showed a notably high relative abundance (average 18.8%) in the rectum samples of Villin-Cre;Gankyrinf/f mice , CW040 F16 , and Clostridiales .To better understand the effect of GK deficiency on the bacterial composition of intestinal microbiota, samples were compared to identify OTUs showing differential abundance. First comparisons were conducted between colitis-induced e) Figs. and 5. OVillin-Cre;Gankyrinf/f and colitis non-induced control mice. This comparison revealed 91 OTUs in caecum and 86 OTUs in rectum with differential abundance between the two mice groups , Bacteroidales , Lactobacillus reuteri , Turicibacter , Clostridiales , and Allobaculum . OTUs that were under-represented in caecum and/or rectum samples of Villin-Cre;Gankyrinf/f mice belonged to Bacteroidales , Mucispirillum schaedleri , Clostridiales , Alphaproteobacteria RF32 , and Desulfovibrionaceae .Next, we compared microbiotas of colitis non-induced Villin-Cre;Gankyrinf/f mice to DSS-induced colitis is associated with intestinal microbiota, we performed a co-housing experiment between Villin-Cre;Gankyrinf/f and control mice. Villin-Cre;Gankyrinf/f and control mice were co-housed for 21\u2009days before being challenged with DSS for 7\u2009days. In this case, elevated inflammatory cell infiltration into the colon was not observed in Villin-Cre;Gankyrinf/f . Comparison of the relative abundances of OTUs revealed 10 OTUs with differential abundance between the Villin-Cre;Gankyrinf/f and control mice . OTUs under-represented in Villin-Cre;Gankyrinf/f mice belonged to Helicobacteraceae (4 OTUs), Clostridiales (2 OTUs), and Enterobacteriaceae (1 OTU). The representative sequences of the four Helicobacteraceae OTUs were compared against the NCBI\u2019s nr/nt. None of them could be assigned to Helicobacter japonicum.Microbiota analysis was also conducted by adding the 16S amplicon data from co-housed mice complete small intestine and colon for Villin-Cre;Gankyrinf/f mice and (ii) distal small intestine and colon for Cdx2-Cre;Gankyrinf/f mice. Unexpectedly, the development of colitis that was induced by DSS treatment was significantly augmented in the colon of Villin-Cre;Gankyrinf/f mice as compared to that of control mice. In contrast, colitis that was induced by DSS treatment was comparable between Cdx2-Cre;Gankyrinf/f and control mice. Therefore, similar to nuclear factor-\u03baB (NF-\u03baB) signaling [Previously, we found that GK upregulates the expression of TNF-\u03b1 and IL-17 in lamina propria and Kupffer cells, which was accompanied by the enhanced MAP kinase level and expansion of cancer stem cells, subsequently leading to inflammation-associated carcinogenesis , 10. Regignaling \u201339, GK rVillin-Cre;Gankyrinf/f and colitis-induced control mice revealed 39 OTUs in caecum and 25 OTUs in rectum with differential abundance between the two groups of samples. Of these, two OTUs over-represented in colitis-induced Villin-Cre;Gankyrinf/f mice appeared to be related to the enhanced state of inflammation: Helicobacter japonicum (1 OTU) and Bilophila spp. (1 OTU). Helicobacter japonicum is a recently isolated species shown to cause augmented inflammation in IL-10 deficient mice through the upregulation of the inflammatory cytokines, inducible nitric oxide synthase (iNOS), IFN-\u03b3, IL-17A, and TNF-\u03b1 [Villin-Cre;Gankyrinf/f mice, which showed augmented inflammation. In addition, two OTUs belonging to Helicobacter spp. were over-represented in colitis-induced Villin-Cre;Gankyrinf/f mice. Enterohepatic Helicobacter species (EHS) are known to colonize the intestine and are also associated with IBD [H. pylori known to be associated with the attenuation of IBD [Helicobacter OTUs may have contributed to the augmented inflammation in colitis-induced Villin-Cre;Gankyrinf/f mice. Bilophila includes B. wadsworthia, a sulfate reducing bacterium which produces H2S [2S has been experimentally proven to promote intestinal inflammation when administered in amounts exceeding the detoxifying capacity of colonocytes in rats [Comparisons of gut microbiota between colitis-induced nd TNF-\u03b1 . Intereswith IBD . In factwith IBD . In addin of IBD was not uces H2S . H2S has in rats , 45. OveVillin-Cre;Gankyrinf/f mice and colitis non-induced control mice, numerous OTUs (91 OTUs in caecum and 86 OTUs in rectum) were differentially abundant. It is tempting to speculate that these differences is due to the reduced expression of defensin in the upper small bowel of Villin-Cre;Gankyrinf/f mice. It is notable that none of the differentially abundant OTUs between the two groups belonged to Helicobacter japonicum nor Bilophila spp. Therefore, the distinct composition of intestinal microbiome in colitis-induced Villin-Cre;Gankyrinf/f mice cannot be explained solely by the decreased defensin production. It appears that a shift in bacterial community was further induced upon colitis induction. Such an alteration of community composition may be a result of intricate dynamic interactions among intestinal inflammation, microbiome and a decreased level of defensin.Between colitis non-induced Villin-Cre;Gankyrinf/f and control mice. However, these differentially abundant OTUs provide some insights into the lowered inflammation for Villin-Cre;Gankyrinf/f mice. There was no differentially abundant OTU related to Helicobacter japonicum or Bilophila spp. This reinforces our idea that over-representation of the OTUs related to Helicobacter japonicum and Bilophila spp. were involved in the elevated intestinal inflammation in the non-cohoused colitis-induced Villin-Cre;Gankyrinf/f mice.In the mice co-housing experiment, there were several OTUs with significant differential abundances. Therefore, contrary to our expectation, co-housing did not lead to similar intestinal microbiomes between Epithelial antimicrobial peptides such as defensins act as important components within a complex gut barrier defense system. Deficiency of \u03b1-defensin in Paneth cells is known to cause significant changes in the composition of gut microbiota, suggesting an essential interaction between the gut microbiota and the host . Our resThe enhanced activation of GK induced by chronic inflammation promotes inflammatory response and inflammation-associated carcinogenesis , 10. HowAdditional file 1: Figure S1. Representative images of immunohistochemical detection of Gankyrin (GK) are shown in Gankyrinf/f (GKf/f), Cdx2-Cre;Gankyrinf/f and Villin-Cre;Gankyrinf/f mice. Scale bar, 100\u2009\u03bcmAdditional file 2: Figure S2. Principal coordinate analysis of weighted UniFrac distance between all samples based on bacterial operational taxonomic units (OTUs). The color codes for the samples are, green: colitis non-induced Gankyrinf/f control mice, orange: colitis non-induced Villin-Cre;Gankyrinf/f mice, blue: colitis-induced Gankyrinf/f control mice, red: colitis-induced Villin-Cre;Gankyrinf/f mice.Additional file 3: Figure S3. Bacterial OTUs under-represented in caecum samples of colitis non-induced Villin-Cre;Gankyrinf/f mice in comparison with caecum samples of colitis non-induced Gankyrinf/f control mice (FDR\u2009<\u20090.05). Each dot represents the relative abundance of OTUs within each sample.Additional file 4: Figure S4. Bacterial OTUs over-represented in caecum samples of colitis non-induced Villin-Cre;Gankyrinf/f mice in comparison with caecum samples of colitis non-induced Gankyrinf/f control mice (FDR\u2009<\u20090.05). Each dot represents the relative abundance of OTUs within each sample.Additional file 5: Figure S5. Bacterial OTUs under-represented in rectum samples of colitis non-induced Villin-Cre;Gankyrinf/f mice in comparison with rectum samples of colitis non-induced Gankyrinf/f control mice (FDR\u2009<\u20090.05). Each dot represents the relative abundance of OTUs within each sample.Additional file 6: Figure S6. Bacterial OTUs over-represented in rectum samples of colitis non-induced Villin-Cre;Gankyrinf/f mice in comparison with rectum samples of colitis non-induced Gankyrinf/f control mice (FDR\u2009<\u20090.05). Each dot represents the relative abundance of OTUs within each sample.Additional file 7: Figure S7. Principal coordinate analysis of (A) unweighted UniFrac distance and (B) weighted UniFrac distance between all samples, including samples from cohouse mice, based on bacterial operational taxonomic units (OTUs). The color codes for the samples are, light blue: co-housed colitis-induced Gankyrinf/f control mice, pink: co-housed colitis-induced Villin-Cre;Gankyrinf/f mice, green: colitis non-induced Gankyrinf/f control mice, orange: colitis non-induced Villin-Cre;Gankyrinf/f mice, blue: colitis-induced Gankyrinf/f control mice, red: colitis-induced Villin-Cre;Gankyrinf/f mice.Additional file 8: Figure S8. Bacterial OTUs showing differential abundance in comparison between the rectum samples of co-housed colitis-induced Villin-Cre;Gankyrinf/f mice vs. co-housed colitis-induced Gankyrinf/f control mice (FDR\u2009<\u20090.05). Each dot represents the relative abundance of OTUs within each sample.Additional file 9: Table S1. The corrected P-values and effect sizes reported by the Adonis test when performed on weighted unifrac distances between sample groupsAdditional file 10: Table S2. Number of sequence reads analyzed during the 16S rRNA amplicon analysis conducted to understand the intestinal microbiota in the co-housed mice groupsAdditional file 11: Table S3. Richness and Shannon diversity estimates of intestinal microbiota of co-housed Gankyrinf/f control mice and co-housed colitis induced Villin-Cre;Gankyrinf/f mice"} +{"text": "Chronic pain (CP) is a common, morbid, and costly disorder in older adults. Guidelines encourage clinicians to employ non-pharmacologic therapies for its management, but current psychological interventions have modest treatment benefits and their effects are largely unknown in older cognitively impaired adults. We developed PATH-Pain, an emotion regulation therapy focused on reducing negative emotions and augmenting positive emotions. PATH-Pain is appropriate for use by older adults with CP, negative emotions, and a wide range of cognitive functioning. Treatment consists of 8 weekly individual sessions followed by 4 monthly booster sessions. One hundred older adults (ages 60+) with CP (\u2265 3 months) and at least mild-to-moderate levels of negative emotions (per the Positive and Negative Affect Schedule) were randomized to receive PATH-Pain versus Usual Care (UC). Cognitive screening revealed that 44 participants were cognitively intact score \u226526), while 56 evidenced mild-to-moderate cognitive impairment (MoCA=16-25). Participants completed follow-ups at 5 (n=89) and 10 weeks (n=84), while 24-week assessments are ongoing. Examination of the treatment \u00d7 time interaction in a repeated-measures mixed model indicate the presence of treatment effects. PATH-Pain (vs. UC) participants experienced significant reductions in pain intensity (p<0.044) and pain-related disability (p<0.003). Reductions in pain-related disability score were more pronounced among cognitively impaired individuals. The PATH-Pain group also demonstrated significant reductions in emotional suppression (p<0.019) and depression (p<0.009) scores. These results suggest that PATH-Pain is an effective treatment for the management of pain in cognitively intact and cognitively impaired older adults."} +{"text": "There is an increasing awareness of the importance of a diet rich in fruits and vegetables for human health. Cancer stem cells (CSCs) are characterized as a subpopulation of cancer cells with aberrant regulation of self-renewal, proliferation or apoptosis leading to cancer progression, invasiveness, metastasis formation, and therapy resistance. Anticancer effects of phytochemicals are also directed to target CSCs. Here we provide a comprehensive review of dietary phytochemicals targeting CSCs. Moreover, we evaluate and summarize studies dealing with effects of dietary phytochemicals on CSCs of various malignancies in preclinical and clinical research. Dietary phytochemicals have a significant impact on CSCs which may be applied in cancer prevention and treatment. However, anticancer effects of plant derived compounds have not yet been fully investigated in clinical research. Despite progress in anticancer therapy, cancer is a major health problem and one of the leading causes of morbidity worldwide ,2. CanceThe review focuses on the anticancer effectiveness of dietary phytochemicals, either isolated or as mixtures via targeting CSCs. Firstly, it discusses the basics of CSCs and signaling pathways modulating their stem-like properties. The core of the review is the summary of preclinical and clinical studies evaluating whether dietary phytochemicals target CSCs in various malignancies. Plant-derived dietary compounds which are effective agents against CSCs in preclinical in vitro and in vivo research should be further evaluated in clinical research. We emphasize the need to include dietary phytochemicals in the current clinical research.Data were recovered from the biomedical literature published in the English-language literature by use of \u201ccancer stem cells\u201d and \u201cplant-based functional foods\u201c or \u201cphytochemicals\u201d or \u201cfruit\u201c or \u201cvegetables\u201d or \u201cherbs\u201d as either a keyword or medical subject heading (MeSH) term in searches of the PubMed bibliographic database. We emphasize the most recent scientific papers from the years 2013\u20132019. About 40 studies were selected with the database accessed between December 2018 and February 2019.CSCs are multipotent cells exhibiting stem-like properties and possessing the capability of the initiation of tumor growth, invasiveness, and dissemination to distant organs ,11. CSCsCSCs have been recognized in various types of tumors and it is now possible to identify and isolate them using a distinctive profile of surface or intracellular markers (e.g. ALDH) . Table 1Cluster of differentiation 44 (CD44) promotes increase in growth factor beta (TGF-\u03b2) leading to Epithelial\u2013mesenchymal transition (EMT). Binding of hyaluronic acid (HA) with CD44 activates Protein kinase C (PKC) which then phosphorylates transcription factor Nanog resulting in upregulation of ATP binding cassette subfamily B member 1 (ABCB1) contributing to multidrug resistance (MDR)). CD44 serves as a coreceptor for growth factors and stimulate CSCs self-renewal. Tumour necrosis factor alfa (TNF-\u03b1) upregulates CD44 through Janus kinase (JNK), thus inducing migration, invasion, metastasis or EMT. Cluster of differentiation 24 (CD24) stimulates metastasis formation via interaction with P-selectin and cancer progression and trigger EMT via activation of Notch1signaling. Cluster of differentiation 133 (CD133) is involved in tumour cell proliferation, metastasis, tumorigenesis or therapy resistance\u2014via activation of the phosphatidylinositol-3-kinase (PI3K)/Akt. Hypoxia (H) in stem cells and the tumour microenvironment promote CD133 expansion via upregulation of hypoxia-inducible factor 1-alpha (HIF-1\u03b1). Overexpression of CD133 is associated with tumour progression through epidermal growth factor receptor (EGFR)-dependent Akt activation. The role of cluster of differentiation 90 (CD90) in cancer depends on the cancer type and signaling mechanism. For example, cancer stem-like activity is elevated through up-activation of Notch pathway. Increased expression of cluster of differentiation13 (CD13) reduces reactive oxygen species (ROS) promoting CSCs survival via EMT. Leucine-rich repeat-containing G-protein-coupled 5 (LGR5) promotes proliferation of cancer cells via activation of Wnt/\u03b2-catenin pathway. Epithelial cell adhesion molecule (EpCAM) cleavages with its intracellular domain (EpICD) and provide key signals for achieving CSCs properties by modulation of Wnt pathway or LIF/STAT3. Activity of aldehyde dehydrogenase (ALDH) may protect CSCs against cell death caused by ROS. ALDHs metabolizes retinoic acid (RA) thus regulating stem-like properties of CSCs. Aberrantly regulated signaling pathways and cross-talks between them may ultimately influence their target genes such as c-Myc, cyclinD1, Survivin, Nanog, Oct-4, Sox2, etc.EMT is the reversible change occurring usually during embryogenesis in which epithelial cells acquire mesenchymal phenotypes ; moreoveStrictly regulated signaling pathways control the activity of stem cells . HoweverThe evolutionary conserved Notch signaling pathway possesses an important role in the balance of differentiation, cell cycle progression , survivaWnt signaling pathway is involved in embryonic development and homeostasis of tissues. Evolutionary conserved and highly complex Wnt signaling is considered to encompass two pathways which are not exclusive and cross-talk may occur between them. \u03b2-catenin-independent pathway with calcium as the major mediator regulates asymmetrical division of cells, cell polarity and migration ,45. On tThe Hedgehog (HH) signaling pathway plays a crucial role in the embryonic development, especially the development of skin, hair follicles and sebaceous glands and also in adult brain development . MoreoveThe phosphatidylinositol-3-kinase (PI3K)/Akt and the mammalian target of rapamycin (mTOR) is a signaling pathway playing crucial role in metabolism, proliferation, angiogenesis, differentiation, and survival of cells . It is aThe Janus kinase (JAK) and signal transducer and activator of transcription (STAT) signaling pathway possess and important role in cytokines and growth factor signaling affecting cell growth, proliferation and immune response. Aberrant regulation of the JAK/STAT pathway is associated and implied with maintenance of germ-line stem cell populations in various cancers . MoreoveAbberant regulation of Notch signaling may be modulated via abnormal expression of Notch ligands including Delta-like (DLL1/3/4) and JAGGED (JAG1/2), Notch receptors (Notch1\u20134) or Notch target genes (Hes1). Wnt/\u03b2-catenin pathway may contribute to cancer-like phenotype of cells via abnormal expression of Secreted frizzled-related proteins (SRFP-1), Wnt inhibitory factor (WIF), Dickkopf-related protein (DKK), Axis inhibition protein 2 (AXIN2) and increased levels of Wnt signaling proteins including Lymphoid enhancer-binding factor 1 (LEF-1) or T-cell factor 4 (TCF-4) binding to which \u03b2-catenin influence expression of target genes (eg. Cyclin D or c-Myc). Deregulation of Hedgehog (HH) pathway may be influenced via aberrant expression of HH ligands , receptors PATCHED (PTCH), transmembrane proteins SMOOTHENED (SMO) or transcription factors Zinc finger proteins (GLI1-3). Abnormal modulation of PI3K/Akt/mTOR may be based on dysregulated Protein kinase B (Akt) or negative regulator of the Phosphatase and tensin homolog (PTEN). JAK/STAT signaling may be deregulated via abnormal expression of Signal transducer and activator of transcription 3 (STAT3).Apoptosis is regulated by an extrinsic or intrinsic pathway. Dysregulated apoptosis is a hallmark of cancer, and failure in signaling either of extrinsic or intrinsic pathways occurs also in CSCs ,55. DefiIn conclusion, multiple mechanisms are involved in regulation of the stem-cells related processes of self-renewal, differentiation or apoptosis, many of them are deregulated in CSCs. Self-renewal, differentiation and maintenance of other stem-like properties may be mediated via modulation of signaling pathways including Notch, Wnt/\u03b2-catenin, Hedgehog, PI3K/Akt, JAK/STAT and others. cFLIPS, FLICE-like inhibitory proteins; IAPs, inhibitors of apoptosis proteins; Bcl-2, Bcl-2 family proteins; ABC, ATP-binding efflux multidrug resistance transporters; HIF-1, hypoxia-inducible factor 1; IL-4, interleukin-4; NF-\u03baB, nuclear factor-\u03baB are suggested to modulate CSCs resistance to death and cancer therapies.st lacking, a possible association between phytochemicals and their potential anti-CSCs effects was indicated by several studies. Jaiswal et al. [The importance of natural compounds in cancer treatment or prevention is supported by abundant evidence from cancer research . Naturall et al. and Ryu l et al. suggestel et al. . HoweverEpigallocatechin-3-gallate (EGCG) is the main constituents of green tea and its protective effects are associated with various human malignancies whereas the combination of EGCG and anticancer therapy is more effective for inhibiting CSCs . InteresResveratrol is a protective ingredient widely spread in the traditional Mediterranean diet which is considered to lower the risk of cancer. Resveratrol and its analogue pterostilbene target CSCs via multiple signaling pathways . Resveran-octyl genistein (DFOG) is a novel synthetic genistein analogue. DFOG inhibited stem-like properties and reverse EMT phenotype in gastric cancer stem-like cells in vitro [Genistein is a soy isoflavone functioning as a natural NF-\u03baB inhibitor . Genistein vitro .Curcuma longa. Curcumin targets CSCs through acting on the signaling pathways including Wnt, HH or Notch [Curcumin is a dietary polyphenol derived from or Notch ,77. Impoor Notch . Anotheror Notch . Curcumior Notch . Curcumior Notch .Isothiocyanates (ITCs) suppress cellular proliferation, EMT and self-renewal of CSCs via inhibition of oncogenic signaling pathways such as NF-\u03baB, STAT3 or other pathways which are found to be upregulated in various cancers . PhenethDiallyl trisulfide (DATS) is a garlic derived organosulfur suggested to possess anticancer properties. DATS reduced tumorsphere formation, decreased CSCs markers expression, inhibited proliferation and induced apoptosis via inhibition of Wnt/\u03b2-catenin pathway and its target genes in colorectal cancer cell line . SimilarCapsosiphon fluvescens glycoprotein downregulated the Wnt-1 signaling pathway in human gastric cell line, and therefore inhibited gastric cancer cell migration [Origanum vulgare L. in diet in the lower dose (0.1%) suppressed expression of CD24 by 34% and by 57% in the higher dose (1%). Moreover, the level of expression of EpCAM was decreased by 14% and 10% respectively. Furthermore, dietary administration of Syzygium aromaticum L. in high dose (1%) showed decrease in expression of CD24 and CD44 and increase in expression of ALDH1. These effects on CSCs were associated with significant chemopreventive activity in both studies [Punica granatum L.) is a fruit rich in nutrients and bioactive phytochemicals [Trianthema portulacastrum L. is an exotic plant exhibiting various pharmacological properties including antibacterial, antifungal, anti-inflammatory or antioxidant effects. T. portulacastrum extract (TPE) was found to prevent DMBA-induced breast carcinogenesis by anti-inflammatory mechanism mediated via modulation of NF-\u03baB and Nrf signaling pathways [Geissospermum vellosii also known as Pao Pereira, inhibited pancreatic CSCs via modulation of Wnt/\u03b2-catenin in vitro and in vivo [Rauwolfia vomitoria in vivo and in vitro also via modulation of Wnt/\u03b2-catenin signaling pathway [Myrica rubra) leaf proanthocyanidins (BLPs) containing epigallocatechin-3-O-gallate (EGCG) as their terminal and major extension units exhibited inhibitory effects on chemotherapy-resistant OVCAR-3 spheroid cells via modulation of cell viability and sphere and colony formation. Furthermore, BLPs also inhibited self-renewal abilities of CSCs via targeting Wnt/\u03b2-catenin signaling pathway [Hormophysa triquerta (HT-EA), Spatoglossum asperum (SA-EA) or Padina tetrastromatica (PT-EA) were explored utilizing pancreatic cancer (PC) stem cells grown ex vivo and mouse model of residual-PC. Results of the study demonstrated the ability of these extracts to target signaling pathways playing critical role in the regulation of EMT, pluripotency and maintenance of CSCs after first-line therapy [Gynura divaricata (GDE) was found to target liver CSCs in a moderate to weak level and to sensitize Huh7 cell to cisplatin therapy by regulation of Wnt/\u03b2-catenin pathway and target genes [Anti-cancer research demonstrated benefits of phytochemicals combinations over isolated phytochemicals . Plant-digration . Anticar studies ,92. Pomehemicals . A pomeghemicals . Furtherhemicals , and thipathways . Moreove in vivo . Similar pathway . Signifi pathway . The ant therapy . Water eet genes .Moreover, the efficacy of resveratrol (RSV) in combination with grape seed extract (GSE) was investigating in isolated human colon CSCs in vitro and in an azoxymethane-induced mouse model of colon carcinogenesis in vivo. RSV-GSE suppressed Wnt/\u03b2-catenin and induced mitochondrial-mediated apoptosis of CSCs . A summaClinicalTrials.gov Identifier: NCT02423811). Moreover, pancreatic ductal adenocarcinoma (PDA) stem cells were target of a pilot study initiated in 2013 with the aim to find whether the application of freeze-dried broccoli sprouts lead cancer inhibition in patients with advanced PDA . However, no results of these clinical trials are available at this time.Anti-CSCs potential of dietary phytochemicals (isolated or mixtures) was investigated in several previously mentioned preclinical studies ,99,100. S. aromaticum, C. fluvescens, O. vulgare, Chinese bayberry leaf proanthocyanidins (BLPs) and extracts of pomegranate, Trianthema portulacastrum, Gynura divaricata, Hormophysa triquerta (HT-EA), Spatoglossum asperum (SA-EA), Padina tetrastromatica (PT-EA) and resveratrol in combination with grape seed extracts (GSE) demonstrated anticancer properties via targeting CSCs-mediated pathways and thus modulating CSCs proliferation, invasiveness, migration, self-renewal, EMT and sensitivity to therapeutic approaches in preclinical research. The data evaluating effects of dietary phytochemicals in clinical research were insufficient. Ellagitannins-containing pomegranate extract (PE) and purified soy extract (G-2535) may modulate CSCs signaling at least partially. Resveratrol formulation and resveratrol-containing freeze-dried grape powder RSV/GP did not exhibit any prosperous effects in inhibition of CSCs pathways in cancer tissue. Clinical trials evaluating anticancer effects of broccoli sprouts and fursultiamine were initiated in 2013 and 2015, however no results were reported for these studies.Isolated dietary phytochemicals including diallyl trisulfide, pterostilbene, sulforaphane, resveratrol, curcumin, genistein, epigallocatechin-3-gallate (EGCG), phenethyl isothiocyanate (PEITC) and plant functional foods including There is great evidence suggesting that aberrant regulation of CSCs signaling pathways may lead to deregulation of self-renewal, apoptosis, proliferation, and importantly resistance to anti-cancer therapy. Considering the cancer research, phytochemicals (isolated or mixtures) are suggested to possess antioxidant, antiproliferative, and anticancer properties and also to have the ability to target aberrantly regulated signaling of CSCs. Importantly, the use of plant derived compounds is associated with no or very little adverse events. Phytochemicals are thought to modulate various signaling pathways of CSCs. Cross talk between these pathways influence self-renewal, differentiation, EMT, therapy resistance and other pro-cancer mechanism associated with stem-like cells. Here we summarized the current state of the anticancer effectiveness of different plant-derived dietary phytochemicals in preclinical and clinical research. In vitro and in vivo preclinical studies indicated significant anticancer effects of dietary phytochemicals mediated by CSCs targeting via modulation of signaling pathways, including Wnt, Notch, Hedgehog, or other, as well as via regulation of mechanisms involved in the processes of apoptosis or drug resistance. Based on the comparative preclinical oncology studies, functional foods are suggested to exhibit better anti-cancer activities (including the anti-CSCs properties) when compared to isolated phytochemicals. Importantly, each of the preclinical studies included in our review is specific in its aims and uses specifically designed methods. However, individual processes in the cell and therefore processes of carcinogenesis are complex and interconnected. Nevertheless, it would be beneficial to find out if there are associations or discrepancies between studies dealing with the same type of cancer, cell line, model, phytochemical or specific pathway responsible for anti-CSCs effects of particular substance. After all, more specific and comparative studies are needed for such analysis. Despite numerous preclinical studies, clinical research in this area is significantly lagging behind and only a few trials could be identified. On the contrary, we have encountered a large number of clinical studies focused on how are CSCs influenced by synthetic drugs; however, evidence of plant-derived foods or other dietary supplements as anti-CSCs agents is lacking. In conclusion, we emphasize the significant anti-cancer effects of dietary phytochemicals on CSCs in a wide range of cancer types via influencing multiple signaling mechanisms, and thus demonstrating the urgent need for their in-depth investigation in clinical research."} +{"text": "Disrupted microRNA biosynthesis and aberrant regulation contribute to the activation of mesenchymal programs in the kidney. miR-29 regulates the interaction between dipeptidyl peptidase-4 (DPP-4) and integrin \u03b21 and the associated active transforming growth factor \u03b2 (TGF\u03b2) and pro-EndMT signaling; however, miR-let-7 targets transforming growth factor \u03b2 receptors (TGF\u03b2Rs) to inhibit TGF\u03b2 signaling. N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) is an endogenous anti-fibrotic peptide, which is associated with fibroblast growth factor receptor 1 (FGFR1) phosphorylation and subsequently responsible for the production of miR-let-7. miR-29 and miR-let-7 family clusters participate in crosstalk mechanisms, which are crucial for endothelial cell homeostasis. The physiological level of AcSDKP is vital for the activation of anti-fibrotic mechanisms including restoration of anti-fibrotic microRNA crosstalk and suppression of profibrotic signaling by mitigating DPP-4-associated mesenchymal activation in the epithelial cells, endothelial cells, and M2 phenotype macrophages. The present review highlights recent advancements in the understanding of both the role of microRNAs in the development of kidney disease and their potential as novel therapeutic targets for fibrotic disease states.microRNAs (miRNAs) are small, non-coding nucleotides that regulate diverse biological processes. Altered microRNA biosynthesis or regulation contributes to pathological processes including kidney fibrosis. Kidney fibrosis is characterized by deposition of excess extracellular matrix (ECM), which is caused by infiltration of immune cells, inflammatory cells, altered chemokines, and cytokines as well as activation and accumulation of fibroblasts in the kidney. These activated fibroblasts can arise from epithelial cells It resu (EndMT) . The ava (EndMT) . Current (EndMT) ; hence, EMT involves a series of events through which epithelial cells lose their epithelial characteristics and acquire properties of typical mesenchymal cells . FigureTie2-Cre and LoxP-enhanced green fluorescent protein (EGFP) transgenic mice, the authors confirmed a large population of interstitial \u03b1SMA-positive cells of endothelial origin in the fibrotic kidneys of STZ-induced diabetic mice are well known for their regulatory role in diseases like diabetes, cancer, and fibrosis . They arvia targeting smad interacting protein 1 (SIP1), protein-tyrosine phosphatase (PTEN), and y-box binding protein 1 (Ybx1) played critical roles in collagen expression has been shown to decrease TGF\u03b21-induced expression of mesenchymal markers, i.e., fibronectin, N-cadherin, thrombospondin, and the notch ligand jagged-1 in HK-2 cells through a mechanism by inducing of miR-let-7c \u2013mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) pathway of new chemical molecules . miRNA-bSome miRNAs display down-regulated status in kidney disease, suggesting protective roles. Anti-fibrotic mechanisms of miRNAs could be dependent on signaling molecules in TGF\u03b2 pathways or independent from TGF\u03b2 pathways, i.e., targeting signaling molecules of ECM-secreting pathways. miRNA-based therapeutics are superior to those of conventional drug approaches because they are able to target complex pathogenic gene networks. Further benefits include sustained outcomes, expansion of drug-ridden targets to virtually any miRNA, rapid drug development, and limited potential for drug interactions . Using emicroRNAs can be used as biomarkers and therapeutic targets for kidney diseases . The chaAltered metabolic states can alter the expression level of pro-fibrotic and anti-fibrotic microRNAs. SS wrote the manuscript, made the figures, and provided intellectual output. AH helped in editing. KK and JG provided intellectual output in the manuscript.JG is supported by the National Heart, Lung and Blood Institute Grant R01-HL-131952.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "G-quadruplexes have gained prominence over the past two decades for their role in gene regulation, control of anti-tumour activity and ageing. The physiological relevance and significance of these non-canonical structures in the context of cancer has been reviewed several times. Putative roles of G-quadruplexes in cancer prognosis and pathogenesis have spurred the search for small molecule ligands that are capable of binding and modulating the effect of such structures. On a related theme, small molecule fluorescent probes have emerged that are capable of selective recognition of G-quadruplex structures. These have opened up the possibility of direct visualization and tracking of such structures. In this review we outline recent developments on G-quadruplex specific small molecule fluorescent probes for visualizing G-quadruplexes. The molecules represent a variety of structural scaffolds, mechanism of quadruplex-recognition and fluorescence signal transduction. Quadruplex selectivity and in vivo imaging potential of these molecules places them uniquely as quadruplex-theranostic agents in the predominantly cancer therapeutic context of quadruplex-selective ligands. Cancer biology is inherently complex owing to the varied cellular alterations and mutations juxtaposed with failures of biological safety mechanisms. Molecular complexity of tumors adds to complexity of the disease, which is evident from the fact that no universal cancer biomarker is yet to be documented . Identif+ concentrations such as 100 mM thereby attenuating transcription. Among other changes in aggressive tumors, over-expression of K+ membrane ion channels leads to depletion in intracellular K+ concentration to nearly 60 mM . It. It47]. In a novel strategy towards the development of G-quadruplex targeting ligands, the quadruplex structural motif has been sought as a template for the assembly of synthetic quartets . Signal Interaction of N-TASQ with suitable quadruplexes disrupts the electron transfer from guanines thereby leading to significant fluorescence enhancement. Multi-photon microscopy has been used to visualize N-TASQ in human breast cancer (MCF7), human osteosarcoma (U2OS) and murine melanoma B16F10, . StaininPyridodicarboxamide derivatives have been successful G4 specific ligands with their applications as potential anti-cancer and anti-inflammatory drugs. However, its unexplored fluorescent probe based application was brought to light by Yang and fellow researchers by synthesizing and studying its derivative, tetramethoxybis(4-aminobenzylidene) acetone, GD3 51]. Th. Th51]. A few other probes have been reported in addition to the above prominent G-quadruplex visualizing agents. Notably, the triangulenium derivatives ADOTA-M and DAOTA-M2 exhibit Over the past decade, massive headway has been made towards the development of G4-selective ligands that are capable of stabilizing the secondary structures. The quest for quadruplex-specific ligands has navigated the challenge of distinguishing quadruplexes from canonical DNA in the early days, to the challenge of distinguishing quadruplex topologies from one another at present time. Not surprisingly, fluorescent ligands capable of identifying quadruplex structures in vivo have faced similar challenges. Several scaffolds such as carbazoles and pyridinium derivatives have emerged as promising building blocks for construction of quadruplex-selective staining agents. G4-selective fluorescence probes most commonly rely on an end-stacking mechanism of recognition. Recurring motifs in these probes include heterocyclic moieties such as benzothiazole, presence of charged centers and extended conjugation. Considering the much smaller set of G4-selective fluorescent ligands, it is conceivable that such agents may fulfill a dual theranostic role enabling wider validation of G4-targets in disease contexts . CurrentBMVC3,6-bis(1-methyl-4-vinylpyridinium)carbazolediiodideTOThiazole OrangeIMTN-Isopropyl-2--6-methylbenzothiazoleN-TASQNaptho-Template Assisted Synthetic G-QuartetGD3Tetra-methoxy bis(4-aminobenzylidene) acetoneDAOTA-M2Morpholino containing bis-substituted trianguleniumADOTA-MMorpholino containing mono-substituted trianguleniumBPBC9-methyl-3,6-bis[5-(4-methylpiperazin-1-yl)-1Hbenzo[d]imidazol-2-yl]-9H-carbazole ATPDsGuanidino anthrathiophenediones"} +{"text": "Ambystoma mavortium) from 2013 and 2014 in Alberta, Canada. The genome lengths ranged from 106,258 to 106,915 bp and contained 108 open reading frames encoding predicted proteins larger than 50 amino acids.Complete genome sequences of six Ambystoma tigrinum viruses (ATV) were determined directly from tail clips of western tiger salamanders ( Ambystoma mavortium) from 2013 and 2014 in Alberta, Canada. The genome lengths ranged from 106,258 to 106,915 bp and contained 108 open reading frames encoding predicted proteins larger than 50 amino acids.Complete genome sequences of six Ambystoma tigrinum viruses (ATV) were determined directly from tail clips of western tiger salamanders ( Ranavirus and family Iridoviridae affect a wide range of amphibians, reptiles, and fish (Ambystoma tigrinum virus (ATV), a virus with a double-stranded DNA (dsDNA) genome of \u223c106 kb was 495 , which resulted in only a partial genome using SPAdes in metagenomics mode (metaSPAdes). Therefore, the sample was resequenced using probe capture enrichment and manu.4 and mMK580536]) and the small neurofilament triplet H1-like protein (NTHP) (corresponding to nucleotide position 83798 to 84790 of genome 2014-ATV-322 [MK580536]) , with thMK580531 and SAMN11127530 (2013-ATV-04); MK580532 and SAMN11127531 (2013-ATV-12); MK580533 and SAMN11127532 (2013-LL1); MK580534 and SAMN11127533 (2013-LL2); MK580535 and SAMN11127534 (2013-LL3); and MK580536 and SAMN11127535 (2014-ATV-322). The versions described in this paper for MK580531, MK580532, MK580533, and MK580535 are the second versions; all other versions described are the first versions.Nucleotide sequences, including annotations and raw sequencing reads, were deposited in GenBank and the Sequence Read Archive (SRA), National Center for Biotechnology Information, with the following GenBank and SRA accession numbers, respectively:"} +{"text": "Evidence-based data demonstrated good diagnostic performance of PET with different tracers in detecting brain tumors, in particular, radiolabelled amino acid tracers showed the highest diagnostic performance values. All the PET tracers evaluated had significant prognostic value in patients with glioma. Conclusions: Evidence-based data showed a good diagnostic performance for some PET tracers in specific indications and significant prognostic value in brain tumors.Background: Several meta-analyses reporting data on the diagnostic performance or prognostic value of positron emission tomography (PET) with different tracers in detecting brain tumors have been published so far. This review article was written to summarize the evidence-based data in these settings. Methods: We have performed a comprehensive literature search of meta-analyses published in the Cochrane library and PubMed/Medline databases (from inception through July 2019) about the diagnostic performance or prognostic value of PET with different tracers in patients with brain tumors. Results: We have summarized the results of 24 retrieved meta-analyses on the use of PET or PET/computed tomography (CT) with different tracers in brain tumors. The tracers included were: fluorine-18 fluorodeoxyglucose ( Positron emission tomography (PET) is a nuclear medicine imaging technique that, using different radiotracers evaluating different metabolic patterns, is able to detect in advance pathophysiological changes in oncological patients, including those with brain tumors. These functional changes usually occur before the development of morphological changes detected by conventional radiological imaging techniques such as computed tomography (CT) and magnetic resonance imaging (MRI) . MRI is 18F-FDG), carbon-11 methionine (11C-methionine), fluorine-18 fluoroethyltyrosine (18F-FET), fluorine-18 fluorodihydroxyphenylalanine (18F-FDOPA), fluorine-18 fluorothymidine (18F-FLT), and radiolabelled choline (11C-choline or 18F-choline).Different PET radiotracers have been used to evaluate brain tumors, including fluorine-18 fluorodeoxyglucose and subsequently phosphorylated through the activity of intracellular hexokinase. 18F-FDG allows the detection of neoplastic cells due to their frequently increased glucose metabolism methyl-L-tryptophan [Furthermore, there are no meta-analyses about the diagnostic performance of PET with other emerging tracers for evaluation of brain tumors, such as radiolabelled prostate-specific membrane antigen (PSMA) or fluorF-FACBC) , or withyptophan .Hybrid PET/MRI tomographs are now available for clinical use, and the role of PET/MRI using different PET tracers in brain tumors is promising, but more evidence-based data are needed in this setting .Large multicenter prospective studies and, in particular, more cost-effectiveness analyses comparing different PET tracers and different neuroimaging modalities in patients with brain tumors are warranted. To this end, some cost-effectiveness analyses on the use of amino acid PET or PET/CT in brain tumors are already available, demonstrating that the combination of amino acid PET and MRI may be cost-effective for target selection in patients with suspicious glioma, for the surgical plan in patients with high-grade glioma, and for the evaluation of patients with suspicious recurrent high-grade glioma or brain metastasis ,40,41,42"} +{"text": "Imaging with X - ray and ultrasound of KUB region demonstrated completely encrusted double - J ureteral catheter with giant stone formation (45 mm) at distal bladder end resembling a \u201chockey - stick\u201d for the last 12 months. He also presented history of several episodes of urinary tract infections (UTI) in the last 2 years. There was a non - documented history of undergoing left open pyelolithotomy for left renal calculus 5 - years ago. Apart from a scar present in left flank region, the rest of the general physical examination was unremarkable. Routine blood / urine examination revealed normal haemoglobin, blood counts, kidney function tests, random blood sugar levels and presence of 20 - 25 pus cells / hpf. Urine culture examination revealed presence of E coli (> 10- stick\u201d . After p- stick\u201d .Double - J ureteric catheters are an indispensable part of urology. Close follow-up of these patients is mandatory to prevent long - term complications arising from prolonged in situ ureteral catheter such as worsening of hydronephrosis, infections, encrustations, stent migration and renal damage . Combine"} +{"text": "Authors Ting-Yu Chen and Chi-Wen Kao are incorrectly noted as equal contributors to this work. The full, correct byline is as follows:Ting-Yu Chen, Chi-Wen Kao*, Shu-Meng Cheng\u2021, Yue-Cune Chang\u2021.\u2021 These authors contributed equally to this work* Corresponding author"} +{"text": "OBJECTIVES/SPECIFIC AIMS: The goal of this study was to investigate whether RF-RDN attenuates renal fibrosis and inflammation in SHR with established hypertension. METHODS/STUDY POPULATION: Twenty-two-week-old SHR received bilateral RF-RDN or Sham-RDN (Biosense Webster Stockert 70 generator and RF-probe). Four weeks later, SHR were sacrificed and paraffin sections of kidneys were stained for fibrosis by Masson\u2019s trichrome staining. Kidney tissue were homogenized for measurement of cytokines levels by ELISA. RESULTS/ANTICIPATED RESULTS: The results showed that Sham-RDN treated SHR had extensive fibrosis as demonstrated by moderate thickening of Bowman\u2019s capsule, collagen deposition in glomerulus, extensive tubulointerstitial fibrosis, and segmental glomerulosclerosis. In contrast, RF-RDN significantly reduced each of these pathological components of fibrosis in kidney cortex and medulla as compared with Sham-RDN treated kidneys. In other studies, RF-RDN decreased B cells, CD4+ T cells, and CD8+ T cells in the kidney of SHR as measured by flow cytometry. Meanwhile, kidney tissue levels of IL-17, INF-\u03b3, MIP-3a, TNF-\u03b1, and TGF-\u03b2 were decreased as compared with respective levels in Sham-RDN. DISCUSSION/SIGNIFICANCE OF IMPACT: Together, these findings demonstrate that removal of the influence of heightened renal sympathetic activity by RF-RDN decreases kidney inflammatory markers and attenuates renal fibrosis in hypertensive SHR."} +{"text": "Caring for a person with dementia (PWD) requires commitment, flexibility, and resilience - the ability to endure and recover from stressors that arise during the caring process. However, it is unknown what behaviors can indicate resilience in care partners (CPs) of PWDs. We examined 46 peer-reviewed articles (1990 to 2018) that included measures or definitions of resilience in CPs of PWDs. Our goal is to identify resilience-related behaviors and create a behavior-based model/framework for CPs of PWDs. Three major themes emerged: (1) Problem-response behaviors ; (2) self-growth behaviors ; (3) help-related behaviors (help-seeking and help-receiving). These findings informed the development of a behavior-based Care Partner Resilience (CPR) measure. Future steps in this research include evaluating to what extent behaviors in the CPR framework are associated with CPs\u2019 self-assessed resilience and can predict CPs\u2019 resilience following specific caregiving-related stressors."} +{"text": "Tumor metastasis is a hallmark of cancer, with distant metastasis frequently developing in lung cancer, even at initial diagnosis, resulting in poor prognosis and high mortality. However, available biomarkers cannot reliably predict cancer spreading sites. The metastatic cascade involves highly complicated processes including invasion, migration, angiogenesis, and epithelial-to-mesenchymal transition that are tightly controlled by various genetic expression modalities along with interaction between cancer cells and the extracellular matrix. In particular, microRNAs (miRNAs), a group of small non-coding RNAs, can influence the transcriptional and post-transcriptional processes, with dysregulation of miRNA expression contributing to the regulation of cancer metastasis. Nevertheless, although miRNA-targeted therapy is widely studied in vitro and in vivo, this strategy currently affords limited feasibility and a few miRNA-targeted therapies for lung cancer have entered into clinical trials to date. Advances in understanding the molecular mechanism of metastasis will thus provide additional potential targets for lung cancer treatment. This review discusses the current research related to the role of miRNAs in lung cancer invasion and metastasis, with a particular focus on the different metastatic lesions and potential miRNA-targeted treatments for lung cancer with the expectation that further exploration of miRNA-targeted therapy may establish a new spectrum of lung cancer treatments. Lung cancer constitutes the leading cause of cancer death worldwide , with moNumerous studies regarding tumor invasion and migration depict the interaction between tumor cells and adjacent tissues or microenvironments, reporting different mechanisms and various signal pathways related to tumor spreading. Specifically, the critical role of the epithelial-to-mesenchymal transition (EMT) in cancer invasion, migration, and metastasis provides a clue to prevent cancer spread and identify possible therapeutic targets .MicroRNAs (miRNAs), a group of small non-protein-coding RNAs (20\u201325 nucleotides), suppress gene expression primarily through direct interaction with the 3\u2032-untranslated region (3\u2032UTR) of corresponding target messenger RNAs (mRNAs) . Target Distant spreading of the primary tumor represents the major cause of cancer-related deaths in non-small cell lung cancer (NSCLC), especially metastasis to the brain ,12. MetaIn epithelial-to-mesenchymal transition (EMT), uncontrolled epithelial cells first reduce dependence on their normal tissue microenvironment and proliferate . PolarizNumerous genetic and epigenetic alternations are associated with type 3 EMT, which is driven by intrinsic oncogenic activation , human epidermal growth factor receptor 2 (Her2), hepatocyte growth factor receptor (MET), and epidermal growth factor receptor (EGFR)) ,24,25,26Accumulating evidence suggests that miRNAs comprise key regulators to control EMT signaling pathways and TFs. Because some miRNAs directly target EMT-TF, miRNAs and EMT-TF form tightly interconnected negative feedback loops that regulate the expression of TF, epithelial cell plasticity, and cell invasion/migration. Therefore, alterations in miRNAs expression have impacts on EMT program and metastasis cascade in cancer . AlthougThe Snail gene family encodes three TFs: SNAI1 , SNAI2 (SLUG), and SNAI3 (SMUC). Their activation down-regulates epithelial gene expression including E-cadherin, and up-regulates that of mesenchymal genes; e.g., N-cadherin, \u03b2-catenin, and fibronectin . VariousThe p53/miR-34 axis regulates Snail expression in different cancer cell lines including lung cancer. p53 knockdown induces SNAIL protein expression by down-regulating miR-34 expression . MiR-126MiR-137 promotes lung cancer invasion and metastasis by suppressing transcription factor AP-2 gamma (TFAP2C). Patients harboring lung adenocarcinoma with low-level Slug and miR-137 albeit high-level TFAP2C expression exhibit significantly longer overall survival (OS) . MiR-124MiR-1 also directly targets Slug to regulate EMT. MiR-1 overexpression in A549 lung cancer cells causes significant morphological change from mesenchymal to epithelial phenotype with increased E-cadherin, attenuating invasion and migration . VasculaZEB1/ZEB2, well-characterized EMT TFs, mediate cell plasticity, metastasis, and treatment resistance in different cancers ,108,109.The widely studied miR-200 family is crucial in cancer initiation and metastasis . Their pMiR-205 (miR-205-5p) also suppresses EMT by targeting the ZEB1 and ZEB2 3\u2032UTR . PatientMiR-455, a tumor suppressor, is significantly down-regulated in NSCLC tumor tissue samples and cell lines. MiR-455 (miR-455-3p) up-regulation inhibits cell proliferation, invasion, and migration by directly targeting ZEB1 . RestoriZEB2 also constitutes a direct target of miRNAs. Ectopic ZEB2 rescues the suppressed cell migration and invasion mediated by miR-132, which is significantly down-regulated in NSCLC cell lines and clinical NSCLC tissue samples. . MiR-138Twist, an EMT TF, is a target of miR-98, as their expression levels inversely correlates in clinical NSCLC tissue specimens. MiR-98 up-regulation suppresses cell invasion and migration by impeding Twist-induced EMT . BioinfoNumerous EMT-associated signaling modulators are also regulated by miRNAs . TGF-\u03b2, NSCLC cells exhibit lower miR-148b expression than that in normal bronchial epithelial cells. A miR-148 mimic increased epithelial-associated E-cadherin and decreased mesenchymal-associated N-cadherin and vimentin expression. MiR-148b regulates ROCK1, a downstream TGF-\u03b2 signaling factor, to inhibit cell proliferation and EMT, and increase sensitivity to radio-chemotherapy in NSCLC . MiR-155MiR-136 (miR-136-5p) inhibits lung cancer cell metastasis and EMT by directly targeting Smad2 and Smad3 . Among aIn NSCLC and hepatocellular carcinoma, MET oncogene activated miR-221 and miR-222 by activating the c-JUN TF. These miRNAs suppress phosphatase and tensin homolog (PTEN) and tissue inhibitor of metalloproteinases 3 (TIMP3), and promote cellular invasion and migration by activating the Akt murine thymoma viral oncogene homolog (AKT) pathway and metallopeptidase . SomaticTransmembrane serine protease 4, a membrane-anchored protease, mediates cell invasion and migration in a variety of cancers including lung cancer. This protein suppresses miR-205 (miR-205-5p) expression to promote EMT. In vivo, miR-205-5p expression inhibits cell growth, migration, and metastasis formation. MiR-205-5p directly targets integrin \u03b15, a pro-invasive protein in NSCLC. Down-regulated integrin \u03b15 expression in lung cancer cells completely abrogates cell migration, decreases the fibronectin adherence capacity, and reduces tumor growth in vivo .Polycomb repressive complex 2 subunit (SUZ12) is involved in NSCLC tumor progression by promoting cell proliferation and metastasis . MiR-489MiRNAs exhibit contradictory effects on EMT because their targets are cell-context dependent. MiR-590 (miR-590-3p) promotes A549 lung adenocarcinoma cell migration and invasion by targeting olfactomedin 4 (OLFM4), inhibiting tumor cell adhesion . ConversIn summary, the EMT plays a crucial role in tumor invasion and metastasis, and it is also complex, multifunctional, and tightly regulated developmental program. Accumulating evidence suggests that microRNAs tightly regulate EMT in lung cancer cells. MicroRNAs act as pro- or anti-EMT through different targets and signal pathways, which regulates lung cancer invasion and metastasis.In addition to predicting patient survival and tumor relapse, patients with NSCLC with and without metastasis exhibit different miRNA profiles ,144. NumBone metastasis occurs in approximately 15 to 30 percent of patients with lung cancer , represeA high-throughput sequencing study to explore the candidate bone metastasis-related miRNAs in lung adenocarcinoma generated two small RNA (corresponding to 18\u201330 nucleotides) libraries from the blood of patients with lung adenocarcinoma with and without bone metastasis. Expression profiling revealed 7 down-regulated and 21 up-regulated miRNAs in lung adenocarcinoma with bone metastasis. Bioinformatics analysis identified putative associated signaling pathways including MAPK, Wnt, and nuclear factor kappa light chain enhancer of activated B cells (NF-\u03baB), along with pathways involving cytoskeletal proteins, angiogenesis factors, and MMP .Moreover, 18 patients with NSCLC and vertebral column metastasis exhibited higher miR-21 expression levels than that in 20 patients with bone tuberculosis . MiR-21 n = 10) from lung cancer with that of primary lung cancers (n = 24) identified and validated a candidate viral miRNA, Hsv2-miR-H9-5p, encoded by herpes simplex virus type 2 latency-associated transcript [Some viruses regulate their own and/or host gene expression via aberrant miRNA expression ,154. Micanscript . Hsv2-mianscript . SOCS2 eanscript .MiR-139-5p serum levels from patients with lung adenocarcinoma and osteolytic bone metastasis are lower than those in patients with other organ metastasis. MiR-139-5p expression in mesenchymal stem cells (MSCs) significantly increases during osteogenic differentiation. Notch homolog 1, translocation-associated (Drosophila) (Notch1), a direct miR-139-5p target, exhibits significant down-regulation during MSC osteogenesis . Tumor tBrain metastasis affects approximately 25% of patients with NSCLC during their lifetime . HoweverMiRNA microarray-based comparison of expression profiles in five primary lung adenocarcinoma tumors versus three brain metastatic lung adenocarcinoma samples reveals obvious miR-145 down-regulation in brain metastatic samples, albeit no relationship between miR-145 and lymph node metastasis . Among mMiR-21 is a target of STAT3 ,180. In n = 7) and without (n = 8) brain metastasis. MiR-328 overexpression in A549 cells significantly promotes cell migration concomitant with protein kinase C alpha (PRKCA) up-regulation [MiR-4317 is significantly down-regulated in tumor tissues compared with that in paired normal tissues, whereas patients with early stages and non-lymph node metastasis exhibit higher miR-4317 levels. MiR-4317 up-regulation significantly suppresses cell proliferation, colony formation, invasion, and migration. It also hampers NSCLC cell cycling by directly targeting fibroblast growth factor 9 (FGF9) and cyclin D2 (CCND2). In mouse xenograft model, miR-4317 suppresses tumor growth and brain and lung metastasis . MiRNA mgulation .Overexpression of mir-423-5p, selected using microarray analysis of brain metastasis-related miRNAs and validated by quantitative PCR, promotes NSCLC cell colony formation, cell motility, migration, and invasion by direct targeting metastasis suppressor 1 (MTSS1). In clinical samples, lung adenocarcinoma tissues without brain metastasis exhibit positive staining for MTSS1 expression . MicroarRecently, increasing evidence revealed that exosomes play important roles in the tumor microenvironment and the mechanism of malignant tumor metastasis. Exosomes, consist of a phospholipid bilayer, which is composed mainly of proteins, lipids, carbohydrates, and nucleic acids ,182. ExoAstrocytes oppose brain metastasis via exosome-delivered miR-142-3p, which directly binds to the suppressing transient receptor potential ankyrin-1 (TRPA1) 3\u2032UTR. TRPA1 also directly targets the FGF receptor 2 C-terminal proline-rich motif, thereby constitutively activating the receptor and increasing lung adenocarcinoma progression and metastasis . TransfeLymphatic metastasis comprises an important mechanism in tumor spreading in addition to metastasis via blood vessels. The primary epithelial cancer cells enter into the lymphatic drainage system and spread to local or distal lymph nodes after penetrating the basement membrane . For patThe role of miR-200c in lung cancers is controversial. MiR-200c inhibits NSCLC cells invasion and migration, and expression of the miR-200c targets USP25 in NSCLC correlates with clinical stage and lymphatic node metastasis . Lower mMiR-125a-3p/5p is down-regulated in NSCLC tissues compared with adjacent normal lung tissues. However, the relationship with metastasis differs between the two mature miRNAs, which are derived from the 3\u2032 and 5\u2032 ends of pre-miR-125a. Patients with low miR-125a-3p and high miR-125a-5p expression exhibit increased lymph node metastasis . A similMiR-130 also plays a controversial role in NSCLC. MiR-130 is significantly down-regulated in NSCLC tumor tissues and cell lines. High miR-130 expression inversely correlates with lymph node metastasis and late stages. MiR-130 up-regulation significantly suppresses NSCLC cell growth and enhances cell apoptosis by directly targeting PTEN . MiR-130Serum miRNA levels also serve as biomarkers of NSCLC metastasis or prognosis ,213. HigMiRNAs are detected in sputum and plasma . BetweenMeta-analysis from the TCGA database demonstrated that lower miR-133a-3p correlates with negative lymph node metastasis and might act as a tumor suppressor . In lungGene promoter methylation generally results in down-regulation of gene expression. Aberrant miR-200c promoter methylation obviously negatively correlates with miR-200c expression and is associated with lymph node metastasis and poor clinical outcome . HistoneMiRNAs are also influenced by other non-coding RNAs, such as long noncoding RNA (lncRNA), which are over 200 nucleotides in length and exert their effects in the form of RNA. The lncRNA LINC00978 promotes cell proliferation and invasion in NSCLC by inhibiting miR-6754-5p . HOXD-ASIncreasing evidence demonstrates that miRNAs play pivotal roles in lung cancer invasion and metastasis. Studies using miRNA profiling to predict prognosis and clinical treatment response indicate that miRNA expression profiles can predict patient cancer relapse and clinical outcome in NSCLC . The EurFor predicting disease prognosis, gain- and loss-of-function studies of miRNAs have provided a rationale and innovative insight toward precision medicine by targeting miRNA to prevent tumor progression or spreading of cancer cells, because miRNAs can stably modulate gene networks . PossiblA critical challenge of targeted miRNA therapy is how to introduce the synthetic oligonucleotide or miRNA mimic into the cancer cells. Viral and non-viral vectors comprise commonly used vectors for miRNA delivery . HoweverClinicalTrials.gov identifiers: NCT01829971, NCT02862145) were terminated or withdrawn because the suitability of associated serious immune-related adverse events for clinical application was questioned. Several pre-clinical and clinical trials of miRNA as targeting therapy for lung cancer are ongoing . Let-7 sMesomiR 1 (NCT02369198), a first-in-man, phase I clinical trial, enrolled patients with NSCLC and malignant pleural mesothelioma to assess the safety and activity of TargomiRs as the second and third line of treatment . TargomiArgonaute-2 (AGO2) mediates post-transcriptional gene silencing, as an essential component of the RNA-induced silencing complex (RISC). After miRNA assembles into RISC, the activation complex silences and degrades the target mRNA transcripts . HoweverThere are several advantages of miRNA-based therapeutic in lung cancer over other treatment strategies, such as targeting growth factor receptors or enzymatic proteins. MiRNA-based therapies have emerged as promising therapeutic tools for cancer management due to highly specific in tissues and tumors. In addition, the advantage of using miRNA approaches is based on the ability to concurrently target multiple effectors of pathways involved in cell differentiation, proliferation, and survival. Therefore, miRNAs therapies have extremely efficiencies in regulating distinct biological cell processes relevant to malignant cell homeostasis ,373. TheHowever, there are still some problems with miRNA therapies. Therapeutic miRNAs is difficult to cross through cell membranes resulting in poor cellular uptake of oligonucleotides because of the size and negative charge of miRNAs. In addition, delivering a therapeutic miRNA to the associated target tissues also challenges. MiRNAs are relatively unstable and reduce their half-life substantially in the blood circulation due to subject to rapid degradation by RNases . It is aAlthough miRNA replacement therapy remains challenging with numerous problems needing to be resolved, several clinical trials with miRNA mimics have already been initiated. By developing more specific carriers and expression models, regulation of miRNA function will likely become more specific and effective for cancers. Cancer therapy through miRNA regulation may thus engender a new era for cancer patients in the near future.Lung cancer remains a major cause of cancer-associated deaths globally. The aggressive behavior of lung cancer involved in invasion and migration caused disease rapid progression despite standard treatment. MiRNAs regulate multiple genes and different signal pathways. Increasing studies suggested that miRNAs reveal discrete expression patterns in lung cancers. Dysregulation of miRNA expression regulates EMT and cancer metastasis by targeting various genes. Different miRNA expression in tumor tissues or sera is associated with different metastatic sites. These miRNA profiles also correlate with prognosis and clinical treatment response in lung cancers and could be potential targets of lung cancer treatment. More research on miRNA targeted therapies is necessary to increase the target specificity and potency and decrease the off-target effects and toxicity. Exploring miRNA-targeted therapy may establish a new spectrum of lung cancer treatments."} +{"text": "Drosophila telomere repeat HeT-A in oogenesis and early development with disrupted telomeric repeat silencing. In wild type ovaries, HeT-A expression is downregulated by the Piwi-interacting RNAs (piRNAs). By repressing piRNA pathway, we show that overexpressed HeT-A transcripts interact with their product, RNA-binding protein Gag-HeT-A, forming ribonucleoprotein particles (RNPs) during oogenesis and early embryonic development. Moreover, during early stages of oogenesis, in the nuclei of dividing cystoblasts, HeT-A RNP form spherical structures, which supposedly represent the retrotransposition complexes participating in telomere elongation. During the later stages of oogenesis, abundant HeT-A RNP are detected in the cytoplasm and nuclei of the nurse cells, as well as in the cytoplasm of the oocyte. Further on, we demonstrate that HeT-A products co-localize with the transporter protein Egalitarian (Egl) both in wild type ovaries and upon piRNA loss. This finding suggests a role of Egl in the transportation of the HeT-A RNP to the oocyte using a dynein motor. Following germline piRNA depletion, abundant maternal HeT-A RNP interacts with Egl resulting in ectopic accumulation of Egl close to the centrosomes during the syncytial stage of embryogenesis. Given the essential role of Egl in the proper localization of numerous patterning mRNAs, we suggest that its abnormal localization likely leads to impaired embryonic axis specification typical for piRNA pathway mutants.The study of the telomeric complex in oogenesis and early development is important for understanding the mechanisms which maintain genome integrity. Telomeric transcripts are the key components of the telomeric complex and are essential for regulation of telomere function. We study the biogenesis of transcripts generated by the major Telomeres are DNA-protein complexes that protect the ends of linear eukaryotic chromosomes. In most species telomeric repeats are synthesized by telomerase consisting of reverse transcriptase and an RNA template. Telomere-associated proteins form the telomere protection shelterin complex . RecentlDrosophila we conducted a systematic study of the biogenesis and function of telomeric transcripts and their associated proteins in the female germline and in early development. Telomerase was not found in the Drosophila genome [Drosophila telomeres is that they are composed of LINE (long interspersed nuclear element) retrotransposons; namely, HeT-A, TART and TAHRE, among which HeT-A is the most abundant [Drosophila and species encoding telomerase, the basic mechanisms of telomere maintenance are similar [Drosophila telomeric protein complex is structurally different from human shelterin, but is functionally analogous and protects the chromosome ends from degradation and fusion [Drosophila were described a decade earlier than human TERRA [Drosophila telomeric transcriptome in the germline consists of both long retrotransposon transcripts and small RNAs [HeT-A and TART produce multiple sense and antisense transcripts, the latter containing multiple introns [HeT-A and TART retrotransposons drive the bidirectional transcription [HeT-A sense transcripts may be considered as functional analogues of both the telomerase RNA template and TERRA RNA. Clearly, the expression of telomeric elements must be strictly controlled to ensure regulation of telomere length. In Drosophila ovaries, such control is mediated by a distinct mechanism of RNA interference, the piRNA (PIWI interacting RNA) pathway. In the germline, antisense transcripts of telomeric retrotransposons serve as piRNA precursors, regulating the abundance of sense coding transcripts [HeT-A expression in the germline leading to severe developmental defects [Using a genome . A uniquabundant . Despite similar . Drosophd fusion , 10. Traan TERRA , 12. Theall RNAs , 14. HeT introns . Unusualcription , 17. HeTnscripts , 14. In nscripts . piRNA p defects \u201321.Drosophila telomere biology is whether the level of HeT-A RNA could play a role in cellular response to the state of telomeres. To address this question, it is critical to examine the HeT-A RNA and HeT-A Gag biogenesis in normal development and with telomere dysfunctions.Telomeres have certain features of heterochromatin, therefore, their transcriptional activity in a normal cell is repressed. Increased level of telomere transcription in response to telomere damage may be a part of telomere signaling affecting various cellular processes . An impoHeT-A expression, abundant long telomeric RNAs accumulate in the germline, then transported to the oocyte and form aggregates at the mitotic spindles during the syncytial stage of embryogenesis [HeT-A RNA and Gag protein in the germline and early development upon piRNA pathway disruption.Previously, we have shown that the mechanisms of chromosome end protection are closely related to the silencing of telomeric repeats: upon depletion of the piRNA pathway components and other factors suppressing ogenesis . This phogenesis , 21. HowHeT-A and TART were detected at different stages of germline development in wild type Drosophila strains as well as upon depletion of telomere silencing components [Drosophila telomeric RNP in the germline and in early development is still poorly understood since most studies are focused on either HeT-A and TART RNA or their protein products separately.RNA-binding proteins are involved in RNA metabolism at different stages of its life cycle, from transcription to degradation. Transcripts and proteins encoded by mponents , 23, 24.Gaiano strain which is characterized by increased HeT-A and TART copy numbers [HeT-A Gag protein is barely detected in germ cells [HeT-A piRNAs in Gaiano ovaries [Drosophila telomeric RNP were recently obtained from studies of somatic tissues. HeT-A spherical particles consisting of HeT-A Gag and HeT-A RNA were discovered in proliferating neuroblasts where they associate with telomeres undergoing DNA replication and are considered a prerequisite for somatic HeT-A transposition [Due to robust piRNA-mediated silencing in the germline, telomeric retrotransposon products are present at very low levels in wild type ovaries. Even in the numbers , HeT-A Grm cells , which c ovaries . Valuablposition .HeT-A transcripts interact with the RNA-binding protein Gag-HeT-A, which they encode, and that they form RNP particles during most of their life cycle in oogenesis and early embryogenesis. In germarium, HeT-A RNP form spherical structures in the nuclei of dividing cystoblasts and seem to be the intermediates of telomere elongation in the germline. It has been shown that interaction of HeT-A RNPs with the main carrier protein Egalitarian (Egl) [HeT-A RNP in early development. Retention of Egl at HeT-A RNP causes its ectopic localization at syncytial stage of embryogenesis which likely impairs embryonic development in the piRNA pathway mutants.Here, we show that upon piRNA loss overexpressed an (Egl) is requiHeT-A element encoding Gag protein tagged with HA (hemagglutinin) and FLAG epitopes was cloned into pUASp-attB as described previously [HeT-A 3`UTR. The construct pUAST-attB-HeTA-HA-FLAG-mut_ms2 was created on the basis of pUAST-attB-HeTA-HA-FLAG-ms2 by cloning of the mutated HeT-A hairpin (Germline Knockdown) flies were F1 progeny of the genetic cross of a strain bearing a construct with short hairpin (sh) RNA and a driver strain #25751 . The transgenic strain expressing fused protein Egl-GFP was kindly provided by S. Bullock [Gaiano III (GIII) is a strain carrying a third chromosome with Tel locus derived from natural Gaiano strain characterized by extremely long telomeres [Full-length eviously . To gene hairpin . Constru Bullock . The Gai2, 0.5% NP-40, Complete mini protease inhibitor cocktail (Roche), 20 mM NaF, 0.2 mM NaVO4, 1 U/\u03bcl RiboLock RNase Inhibitor (ThermoScientific)). For RIP with anti-HA beads, lysis buffer was supplemented with 1% Triton X-100, 0.1% SDS and 10% glycerol. The extracts were cleared by centrifugation at 16,000 g at 4\u00b0 C for 10 min and diluted with 9 volumes of NT2 buffer . The lysates were incubated with anti-HA magnetic beads (Pierce) or with antibody-coated (5 \u03bcg of the primary antibodies per sample) Dynabeads Protein A (Invitrogen) for 2 h at 4\u00b0C on a rotator. After 3 washes in NT2 buffer, 1/10 of beads were saved for Western blot analysis. RNA was isolated from the remaining beads using Trizol reagent (Life Technologies). Reverse transcription was performed with a random hexanucleotide primer and SuperScriptIII reverse transcriptase (Life Technologies) according to the manufacturer\u2019s instructions. qPCR was performed on a LightCycler96 (Roche). Primers are listed in RIP was performed according to with modHeT-A riboprobe containing a fragment of the ORF (nucleotides 4330\u20134690 of GenBank sequence DMU06920) was used. The specificity of this probe was previously confirmed by Northern blotting and in situ hybridization with polytene chromosomes of salivary glands [RNA FISH combined with immunostaining was carried out according to the previously described procedure with mody glands , 26. To 2, 0.1% Triton X-100, Complete Mini protease inhibitor cocktail (Roche), 0,2 mM NaVO4, 20 mM NaF, RiboLock 1U/\u03bcl (ThermoScientific)). The extracts were cleared by centrifugation at 16,000 g at 4\u00b0C for 10 min. Supernatants were incubated with anti-HA magnetic beads for 30 min at room temperature on a rotator. After 3 washes in IP buffer the bound proteins were eluted from the beads by boiling in 100 \u03bcl of Laemmli protein loading buffer for 5 min. Beads were pelleted and the supernatant was saved for Western blot analysis. Samples were resolved on 8% SDS-PAGE gel and transferred onto Immobilon-P PVDF membrane (Millipore). Blots were developed using the Immun-Star AP detection system (Bio-Rad Laboratories), in accordance with the manufacturer\u2019s recommendations.To prepare embryonic extracts, 0-2-hour old embryos were dechorionated, frozen and stored at -80\u00b0C. Ovaries (400 pairs) or embryos (the volume of dechorionized embryos ~70 \u03bcl) were homogenized in a Dounce homogenizer in 9 volumes of cold IP Buffer and is referred to as cis-preference [Drosophila telomeric protein Gag, encoded by HeT-A, interacts with HeT-A RNA forming RNP in the germline. Recombinant engineering of epitope tags and inducible tissue-specific expression of transgene containing full-length HeT-A have made detection and purification of HeT-A Gag complexes routine [HeT-A is strongly repressed in the germline [HeT-A Gag is not detectable by Western blotting in the ovaries of GIII and transgenic strains expressing HeT-A-HA in contrast to somatic tissues combined with immunostaining. In the germarium region 2A, HeT-A transcripts co-localize with Gag-positive particles forming spherical RNPs of 1\u20132 microns in size we took advantage of another transgenic strain containing a HeT-A copy, tagged by the MS2 bacteriophage hairpins, and encoding for Gag-HA. Combined MS2 RNA FISH and HA-immunostaining shows colocalization of transgenic RNA and protein in the nuclei and cytoplasm of germ cells , oskar (osk) and gurken (grk) mRNAs are associated with Egl-BicD complex in wild type ovaries is presumably involved in the HeT-A RNA localization, and therefore, generated \u0430 transgenic strain, encoding HeT-A transcripts with impaired hairpin structure by Western blotting. Extracts were prepared from the GIII strain (first lane) and transgenic strains expressing UAS-HeT-A-HA in the germline on a wild type background (second lane) or upon spnE_GLKD (third lane). Antibodies are indicated to the left. In ovaries, HeT-A Gag is detected only upon piRNA pathway disruption (spnE_GLKD). HeT-A RNA FISH (green) combined with endogenous HeT-A Gag (red) immunostaining on ovaries of GIII strain. HeT-A RNPs are not reveled in the germ cells but detected in somatic cells of germarium in accordance to previously published observation (23). Intensive HeT-A staining observed in the nurse cell nuclei appear to be correspond to the transcribed telomeres (arrows) (C).(A) Detection of (TIF)Click here for additional data file.S2 Fig(A)HeT-A RNA (green) and HeT-A Gag-HA (red) form HeT-A RNPs (arrows) in the cytoplasm of nurse cells in the ovaries of nosGal4; UAS-HeT-A-HA; UAS-spnE_sh flies. Egg chamber at stage 7 of oogenesis is shown. (B) Colocalization of HeT-A RNA (green), HeT-A Gag-HA (red) and telomeric protein HipHop (magenta) is indicated by arrows. Nurse cell nucleus of a stage 6 is shown. (C) HeT-A RNA FISH (green) combined with endogenous HeT-A Gag (red) immunostaining on ovaries of non-transgenic spnE_GLKD strain. A fragment of a stage 7 egg chamber is shown. (D) HeT-A RNA FISH (green) combined with HeT-A Gag (red) immunostaining was performed on ovaries of yw wild type strain. An egg chamber at stage 7 of oogenesis is shown. DNA is stained with DAPI (blue).(TIF)Click here for additional data file.S3 FigHeT-A Gag-HA (green) (A) or Egl (green) (B) is shown. Stage 6 egg chambers of transgenic strains expressing UAS-HeT-A-Gag-HA in the germline in wild type background (bottom panels) or upon spnE_GLKD (top panels). Arrows indicate nuage. Egl colocalizes with Vasa in nuage, while HeT-A Gag-HA staining is more diffuse and only partially overlapped with Vasa. Magnification is 63x.Immunostaining of a nuage component Vasa (red) and (TIF)Click here for additional data file.S4 Figrp49 was used for normalization. Western blot analysis of co-immunoprecipitated proteins is shown to the right. The antibodies used for Western blotting are indicated to the right. The antibodies used for co-IP are indicated above the IP lanes. Lane designation: input , pellet (insoluble fraction), IP (precipitates). Anti-Egl immunoprecipitates both Egl and BicD proteins but not HeT-A Gag which is enriched in insoluble fraction. (B) RT-qPCR analysis of RNA precipitated with anti-GFP relative to negative control from ovary lysates of w; tub-Egl-GFP flies. Western blot analysis of co-immunoprecipitated proteins is shown to the right; the indications are as in (A). Anti-GFP immunoprecipitates both Egl-GFP and Egl as well as BicD indicating that Egl-BicD is an oligomeric complex. For RIP panels, the error bars represent SEM of 2 biological replicas.(A) RT-qPCR analysis of RNA precipitated with anti-Egl relative to negative control from ovary lysates of nosGal4; UAS-HeT-A-HA; UAS-spnE_sh flies. (TIF)Click here for additional data file.S5 FigHeT-A RNA (green), HeT-A Gag-HA (magenta) and Egl (red) form granules (arrows) in the cytoplasm of nurse cells in ovaries of nosGal4; UAS-HeT-A-HA; UAS-spnE_sh flies. Egg chamber at stage 7 of oogenesis is shown. (B) Egl (red) and endogenous HeT-A Gag (green) form granules (arrows) in ovaries of spnE_GLKD not carrying UAS-HeT-A-HA transgene. DNA is stained with DAPI (blue). (C) Co-IP of HeT-A Gag. Western blot analysis of proteins immunoprecipitated with anti-Gag HeT-A from ovaries of spnE_GLKD flies. Anti-Gag HeT-A immunoprecipitates Egl protein. The antibodies used for Western blotting are indicated to the right and the antibodies used for co-IP are indicated above the IP lanes.(A) (TIF)Click here for additional data file.S6 FigHeT-A localization signal 1) is revealed in the 3\u2019UTR of HeT-A copies . Folding of HLS1 is shown. (B) Sequence of mutated HeT-A hairpin is shown. (C) Colocalization of MS2 RNA (green), HeT-A Gag-HA (magenta) and Egl (red) in the cytoplasm of nurse cells (arrowheads) and in the oocyte (arrows) in ovaries of nosGal4; UAS-HeT-A-HA-ms2_mut flies upon spnE_GLKD. Two egg chambers at different stages of oogenesis are shown. (D) MS2 RNA FISH (green) combined with immunostaining of gamma-tubulin (red) was performed on 0-2-hour old embryos of nosGal4; UAS-HeT-A-HA-ms2_mut flies upon spnE_GLKD. DNA is stained with DAPI (blue). Syncytial metaphase is shown. (E) HeT-A Gag-HA (red) and Egl (green) immunostaining were performed on 0-2-hour old embryos of nosGal4; UAS-HeT-A-HA-ms2_mut flies upon spnE_GLKD. DNA is stained with DAPI (blue).(A) A putative HLS1 ((TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file."} +{"text": "A conventional region of interest analysis was also conducted to quantify BP changes within the sensorimotor network using the automated anatomical labeling atlas. DISCUSSION/SIGNIFICANCE OF IMPACT: CD patients have reduced GABA-A receptor binding compared with healthy controls, with the greatest reduction seen within the sensorimotor region of the thalamus. Furthermore, reductions in GABA-A binding in brain regions associated with coupling sensory and motor information predict motor severity. These findings support that reduced GABAergic signaling within sensorimotor integration regions is a key mechanism underlying dystonic symptoms in CD and could help inform the development of better, more targeted treatment options.OBJECTIVES/SPECIFIC AIMS: Determine whether GABA-A receptor binding is abnormal and linked to dystonia symptoms in cervical dystonia (CD). METHODS/STUDY POPULATION: There is increasing evidence that a key pathophysiological mechanism in adult-onset focal dystonia is a reduction in inhibitory control over the sensorimotor network. Results from a recent 11C-flumazenil PET imaging study suggest that abnormal inhibitory signaling in genetic and sporadic forms of dystonia may be due to reduced GABA-A binding. It remains unknown whether CD, the most common form of adult-onset focal dystonia, is associated with abnormal GABA-A binding. The goal of this research is to determine if GABA-A receptor binding is abnormal and linked to dystonia symptoms in CD. RESULTS/ANTICIPATED RESULTS: We investigated whole brain GABA-A binding in 15 CD patients and 15 healthy controls using 60-minute dynamic 11C-flumazenil PET scans. GABA-A receptor binding potential (BP) was estimated using a simplified reference tissue model. A 2-sample"} +{"text": "The sarcomeric troponin-tropomyosin complex is a critical mediator of excitation-contraction coupling, sarcomeric stability and force generation. We previously reported that induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from patients with a dilated cardiomyopathy (DCM) mutation, troponin T (TnT)-R173W, display sarcomere protein misalignment and impaired contractility. Yet it is not known how TnT mutation causes dysfunction of sarcomere microdomains and how these events contribute to misalignment of sarcomeric proteins in presence of DCM TnT-R173W. Using a human iPSC-CM model combined with CRISPR/Cas9-engineered isogenic controls, we uncovered that TnT-R173W destabilizes molecular interactions of troponin with tropomyosin, and limits binding of PKA to local sarcomere microdomains. This attenuates troponin phosphorylation and dysregulates local sarcomeric microdomains in DCM iPSC-CMs. Disrupted microdomain signaling impairs MYH7-mediated, AMPK-dependent sarcomere-cytoskeleton filament interactions and plasma membrane attachment. Small molecule-based activation of AMPK can restore TnT microdomain interactions, and partially recovers sarcomere protein misalignment as well as impaired contractility in DCM TnT-R173W iPSC-CMs. Our findings suggest a novel therapeutic direction targeting sarcomere- cytoskeleton interactions to induce sarcomere re-organization and contractile recovery in DCM. This highly sensitive mechanisms is fine- tuned by post-translational modifications, such as PKA-mediated phosphorylation of TnI. Mutations in the Tn-Tm complex lead to severe disease, such as dilated cardiomyopathy (DCM). DCM is featured by left ventricular dilatation, contractile dysfunction, and arrhythmias1 and represents a frequent cause of heart failure. More than 25% of DCM cases are caused by inherited mutations, particularly in sarcomeric proteins2. Recently, human iPSC-derived cardiomyocytes (iPSC-CMs) have been utilized for human genetic disease modeling6 and drug testing7. Here, we analyze a sarcomeric mutation in cardiac troponin T (TnT), TnT-R173W. This mutation is located within one of the two tropomyosin binding regions of TnT, the T1 domain8 and was the first DCM mutation reported in a human patient-specific iPSC-derived cardiomyocyte model9. This report demonstrated DCM patient-specific iPSC-CMs to display molecular disease-specific phenotypes such as abnormal sarcomeric structure, dysregulated Ca2+ signaling, and impaired contractility9. More recently, disturbed beta-adrenergic signaling due to epigenetic modulation has been attributed as a source for PDE2A and PDE3A upregulation, as well as dysregulated Ca2+ handling10. These and other studies14 show that sarcomeric microdomain organization plays an important role in maintenance of cardiomyocyte structure and function. Likewise, sarcomeric microdomains are critical for cytoskeleton filament integrity and mediate attachment with the plasma membrane15. Previously, cytoskeletal interactions with caveolin-enriched plasma membrane domains have been implied in sarcomere attachment as well as in signal transduction during heart failure16. Novel proteins contributing to these processes have been recently characterized, such as AMP-activated protein kinase (AMPK)18. The regulation of metabolism by AMPK in various cell types is well recognized, as well as the ability of AMPK to sense ATP levels20. Of note, AMPK has been shown to also act as a cytoskeleton remodeling protein23. Despite this progress, the contribution of cardiomyopathy mutations, such as DCM TnT-R173W, to dysfunction of local sarcomeric microdomains and their cytoskeleton/plasma membrane interactions is not yet clear.Sarcomeres are the basic contractile unit of cardiac cells, whose particularly specialized function depends on a highly organized structure. The troponin-tropomyosin (Tn-Tm) complex at sarcomeric thin filaments is a critical component for excitation-contraction coupling. Stability and anchoring of the Tn-Tm complex on sarcomeres is provided by binding of the troponin T (TnT) subunit to Tm and the troponin I subunit (TnI) on actin myofilaments. Tropomyosin (TPM) together with TnI regulates actin/myosin binding and ATPase function in presence of micromolar, cytocolic CaHere, we uncover novel mechanistic features underlying disorganization of sarcomeric protein- and microdomain array, as well as reduced contractility in DCM patient-specific iPSC- CMs. We generated CRISPR/Cas9 gene edited troponin T knock-out (TnT-KO) iPSC-CMs as an isogenic control for specificity. We report disturbed molecular interactions of troponin and tropomyosin in patient-specific iPSC-CMs carrying the DCM-TnT-R173W mutation, compared to gene edited TnT-KO iPSC-CMs and healthy controls (TnT-WT). Importantly, the DCM mutation TnT-R173W destabilizes TnT interactions with PKA, resulting in diminished troponin I (TnI) phosphorylation. This contributes to impaired force generation as well as reduced contractility, which we validated in a 3D engineered heart muscle (EHM) model. A Foerster Resonance Electron Transfer (FRET)-based molecular sensor and interrogation of sarcomeric PDE activity revealed dysregulation of local TnT microdomains in presence of DCM-TnT-R173W, which results in impaired interactions with cytoskeleton filaments as well as reduced plasma membrane attachment. We identified AMPK to assist in cytoskeleton filament interactions with both sarcomere- and plasma membrane junctions via myosin heavy chain 7 (MYH7) in DCM iPSC-CMs. We showed that AMPK activation can in part overcome destabilized microdomain interactions, as well as sarcomere protein misalignment and impaired contractility in presence of the TnT-R173W mutation. Our studies present new information regarding disturbed troponin complex interactions in patient-specific iPSC-CMs carrying an inherited DCM mutation. We contribute to novel understanding of local signal regulation in sarcomeric microdomains as well as sarcomere interactions with other cytoskeleton filament proteins and plasma membrane compartments. These findings may be exploited in the future for therapeutic manipulation of molecular disease mechanisms.9 as well as healthy control (WT) iPSCs. We generated an isogenic TnT knock-out iPSC line as a negative control, using site-specific CRISPR- Cas9 gene editing to induce a frameshift mutation at exon 2 -tagged TnT-WT and TnT-R173W. TnT-WT-DYK, TnT-R173W-DYK or DYK as a negative control were expressed in HEK 293\u2009T cells, immobilized on flag-decorated beads . Co-immunoprecipitation experiments indicated reduced binding capacity of TnT-R173W towards Tm, compared to iPSC-CMs TnT Fig.\u00a0. Moreove31. We therefore tested phosphorylation of TnI-Ser 23/24 in DCM patient-specific TnT-R173W iPSC- CMs, compared to healthy control iPSC-CMs and TnT-KO iPSC-CMs. In DCM iPSC-CMs, substantially reduced phosphorylation of TnI was detected -based readout for detection of locus-specific cAMP levels at the TnT complex. A targeted cAMP FRET-biosensor, CUTie, which has the PKA cyclic nucleoside binding domain fused to TnI (TnI-CUTie)CMs Fig.\u00a0 while inCMs Fig.\u00a0. InvestiCMs Fig.\u00a0. This ma10 we found increased phosphodiesterase PDE3A in TnT-R173W iPSC-CMs, compared to healthy controls . We therefore assessed interactions of sarcomere microdomains with cytoskeleton filament proteins and the PM in more detail. Of note, we found impaired integrity of sarcomere-cytoskeleton filament junctions in DCM TnT-R173W iPSC-CMs versus healthy controls 32. As myosin heavy chain 7 (MYH7) could also interact with troponin34, we speculated that the presence of the DCM mutation TnT-R173W may impair TnT interaction with MYH7. Destabilization of the TnT-MYH7 interaction could in turn affect AMPK-mediated regulation of cytoskeleton filaments. To probe integrity of MYH7 binding to TnT in DCM and control iPSC-CMs, we used immunoprecipitation from iPSC-CM cell lysates is an established modulator of cytoskeleton filament interactions, which is known to regulate cardiomyocyte metabolismtes Fig.\u00a0. In prestes Fig.\u00a0, confirmcle Fig.\u00a0. Interescle Fig.\u00a0. AMPK prcle Fig.\u00a0.Figure 5We next probed if increased AMPK activation in DCM iPSC-CMs could contribute to sarcomere microdomain organization as well as cytoskeleton-plasma membrane attachment in human iPSC-CMs. Interestingly, quantitative immunohistochemistry studies showed significantly reduced co-localization of MYH7 and AMPK in DCM iPSC-CMs compared to healthy controls Fig.\u00a0. Followi36 in WT iPSC-CMs have been studied previously43. To our best knowledge, this is the first time a cell-based assay with iPSC-CMs is used to examine TnT binding in presence of the TnT-R173W mutation. We found in this experimental system significantly reduced binding of TnT-R173W to tropomyosin compared to WT. In addition, we found lower PKA binding at sarcomeric microdomains in presence of TnT-R173W, resulting in diminished TnI phosphorylation. Moreover, presence of the TnT-R173W mutation disturbed sarcomere microdomain-cytoskeleton filament interactions via MYH7 and AMPK, contributing to disrupted sarcomere protein alignment and impaired contractility.Here, we utilized human patient-specific iPSC-CMs to characterize sarcomere microdomain interactions in presence of the DCM TnT-R173W mutation. We discovered binding of TnT- R173W to Tm to be reduced, which limits troponin anchoring on sarcomere filaments and destabilizes sarcomere protein alignment. A previous study which used recombinant pyrene-labeled tropomyosin did not detect any differences in the binding affinity of recombinant TnT-R173W compared to WT2+]. Reduced interaction with TnT in presence of DCM-TnT-R173W may affect correct relocation of Tm following Ca2+ binding to TnC as well as complete freeing of myosin- binding sites on actin, thereby limiting the initiation of contraction. We confirmed impaired force generation and contractility in presence of the DCM mutation using automated high-speed imaging-based analysis of contractility as well as a 3D engineered heart muscle (EHM) model.Firstly, biochemical assessment revealed reduced binding of mutated TnT-R173W with Tm, which stabilizes the troponin complex on sarcomeric myofilaments. This may directly lead to the disrupted sarcomere protein alignment and regularity observed in DCM iPSC-CMs, compared to healthy controls. Moreover, reduced force generation may be an important consequence of limited TnT-Tm interaction since Tm occupies the myosin-binding site on actin filaments under low intracellular [Ca45. Previously, TnT was identified as an A-kinase anchoring protein (AKAP) which brings PKA at the thin filaments into close proximity with its sarcomeric substrates, including TnI30. Generally, beta-adrenergic stimulation leads to a cell- wide increase of cAMP, which is important for local regulation of cAMP-PKA responses14. Using a sarcomeric TnI-localized FRET biosensor, we determined dysregulation of local sarcomeric cAMP/PKA pools in presence of the TnT-R173W mutation. DCM iPSC-CMs attempt an upregulation of local sarcomeric cAMP, likely to compensate for reduced PKA binding in presence of the TnT-R173W mutation, which limits TnI phosphorylation at local sarcomere microdomains, thereby diminishing contractility. On the other hand, we found a substantial upregulation of sarcomeric PDE activity in DCM TnT-R173W iPSC-CMs, in line with a recent report proposing upregulation of PDE isoform expression as a basis for deficient beta-adrenergic activation in DCM iPSC-CMs10. Thus, the observed slight increase in sarcomeric cAMP levels in DCM TnT-R173W iPSC-CMs may present a compensatory reaction and long- term-adaptation of cardiomyocyte signaling. Importantly, sarcomeric cAMP alterations could not counterbalance reduced PKA binding to TnT in presence of TnT-R173W, which may contribute to the impaired contractility observed in DCM TnT-R173W iPSC-CMs.Secondly, we discovered that the DCM mutation TnT-R173W contributes to dysregulation of local sarcomeric microdomains by limiting PKA binding and resulting in decreased PKA-mediated TnI phosphorylation at Ser-23/24. PKA-mediated phosphorylation is a critical regulatory switch for modulation of cardiac contraction46, we observe that AMPK activation improves contractility in cardiomyocytes. In our study, we utilize a recently developed, direct AMPK activator, A-769662, which is not expected to exert side-effects on e.g. metabolism-related enzymes48.Together, these findings indicate a highly sensitive balance of sarcomere microdomain regulation, which is disrupted in presence of DCM TnT-R173W. Consequently, as our results show, sarcomere-cytoskeleton filament attachment is mediated via MYH7 and AMPK and is impaired in DCM iPSC-CMs. Binding of cytoskeleton attachment proteins, such as MYH7 and filamin-C, with TnT was significantly diminished in presence of the TnT-R173W mutation. Also, sarcomere attachment with plasma membrane junctions appears to be impaired in DCM iPSC-CMs compared to healthy controls. Particularly, TnT binding to MYH7 implies a critical link of sarcomere protein alignment and organization to cytoskeleton filament stability and function. Our data suggest that disturbed binding of TnT-R173W to MYH7 directly destabilizes MYH7-AMPK interactions and AMPK-mediated modulation of cytoskeleton integrity. AMPK, a protein which assists cytoskeleton remodeling, was found to regulate sarcomere-cytoskeleton filament interactions in iPSC-CMs. Small molecule-based AMPK activation could ameliorate disruption of sarcomere-cytoskeleton protein interaction sites in DCM TnT-R173W iPSC-CMs. Importantly, activation of AMPK leads to improved contractility and sarcomeric protein alignment in DCM iPSC-CMs. Our findings suggest that in DCM TnT-R173W iPSC-CMs, disturbed interactions of TnT and resulting reduced sarcomere protein alignment and interactions require a substantial activation of AMPK to overcome the consequences of the TnT-R173W mutation. In line with a previous publicationFiguratively, these signaling events resemble a \u201cmolecular seesaw\u201d, in which the DCM mutation causes disturbed binding of TnT-R173W to MYH7, which in turn directly translates into impaired MYH7-AMPK interactions and AMPK-mediated modulation of cytoskeleton integrity. Conversely, activation of AMPK causes a recovery shift in the opposite \u201cseesaw\u201d direction, by restoring cytoskeleton-sarcomere interactions and thus partially recovering sarcomere protein alignment as well as contractility. Overall, our study reveals impaired sarcomere microdomain regulation in DCM iPSC-CMs such as disrupted binding of mutated TnT-R173W with tropomyosin as well as PKA, affecting sarcomeric protein alignment and local sarcomeric cAMP/PKA regulation. These molecular functions are modulated by AMPK and contribute to reduced sarcomere protein regularity and defective force generation observed in DCM iPSC-CMs. Moreover, our findings provide novel insight into the tightly regulated interplay of sarcomeric microdomains with cytoskeleton- as well as plasma membrane junctions in iPSC-CMs from DCM patients. Further studies of these molecular functions may assist the development of novel therapeutic strategies exploiting these regulatory mechanisms to combat cardiac disease.49. Subsequently, iPSCs were differentiated into beating cardiomyocytes with a small molecule\u2013based monolayer method described previously25. In short, a GSK inhibitor, Chir (Selleckchem) was applied for 48\u2009h, followed by addition of the canonical Wnt-signaling inhibitor, IWR2 (Selleckchem). Beating iPSC-derived cardiomyocytes (iPSC-CMs) were observed from day 7\u201310 onwards. Following differentiation, human iPSC-CMs were cultured in RPMI medium plus B-27 Supplement (Life Technologies). Human iPSC-CMs expressed typical cardiac markers such as cardiac troponin T (TNNT2), sarcomeric a-actinin (SAA), and myosin light chain 2a (MYL2a). Following 25 days of cardiac differentiation, beating iPSC-CM monolayers were dissociated using Accutase and plated in the required assay format. All protocols required for this study were approved by the Goettingen University Ethical Board. Informed consent was obtained from all participants and all research was performed in accordance with relevant guidelines and regulations.Human iPSCs were grown to 80\u201390% confluence using matrigel-coating and chemically defined E8 medium53. Briefly, ATG knock-out of troponin T was performed by oligonucleotide annealing the sgRNA sequences , subcloning into the pSpCas9(BB)-2A-Puro plasmid and subsequent iPSC transfection and single clone isolation. pcDNA 3.1 plasmids carrying the full-length TNNT2-C-terminal-DYK sequence were purchased from GenScript. The TNNT2-R173W was generated by PCR-based site-directed mutagenesis as described before54.CRISPR/Cas9-mediated gene editing was performed as described before54 with protease and phosphatase inhibitors. Eluted protein solutions as well as lysate input were subjected to immunoblot. For visualization of phosphorylated TnI (p-TnI), a phospho-TnI- Ser23/24 antibody was used. In addition, phos-tag acrylamide was used56. Non-phosphorylated and phosphorylated cTnI species were separated in one- dimensional SDS-PAGE with containing phos-tag acrylamide and transferred to Western blots.TnT-specific antibody (Thermo Fisher Scientific) was immobilized on IgG1 Protein G Sepharose beads according to the manufacturer\u2019s instructions. Subsequently, iPSC-CMs lysates were prepared in immunoprecipitation binding and wash buffer as described before6) were first gently mixed on ice with collagen type I and serum-free EHM medium and casted into custom- made molds according to a previously published protocol57. Following 5 days condensation in casting molds, EHMs were transferred onto mechanical stretchers for functional maturation for an additional 12\u201314 days. EHM media was changed every other day. Contractile forces generated by EHMs were measured in organ baths in Tyrode\u2019s solution containing 1.8\u2009mmol/L calcium under 1.5\u2009Hz field stimulation.Human ESC-CMs .An expanded Methods section is available in the online Data Supplement.Supplementary informationSupplementary movie 1Supplementary movie 2Supplementary movie 3"} +{"text": "Buttiauxella sp. strain A111, isolated on the basis of bioconversion activity of the plant growth-regulating compound 2-azahypoxanthine to 2-aza-8-oxohypoxanthine. The genome contains 4,388 protein-coding sequences, including several genes possibly involved in the metabolism of the plant growth-regulating compound.We report here the draft genome sequence of Buttiauxella sp. strain A111, isolated on the basis of bioconversion activity of the plant growth-regulating compound 2-azahypoxanthine to 2-aza-8-oxohypoxanthine. The genome contains 4,388 protein-coding sequences, including several genes possibly involved in the metabolism of the plant growth-regulating compound.We report here the draft genome sequence of Burkholderia contaminans CH-1 (Buttiauxella sp. bacterium (designated strain A111) from forest soil in Hokkaido, Japan, by screening based on the bioconversion activity from AHX to AOH. The genus Buttiauxella is a member of the family Enterobacteriaceae, which contains an endophytic bacterium, Buttiauxella sp. strain SaSR13, that improves the growth of a plant of the genus Crassulaceae (The efficient production method of 2-aza-8-oxohypoxanthine (AOH), a strong plant growth stimulator , is limiButtiauxella sp. strain A111 was extracted using a DNeasy blood and tissue kit and fragmented using a Covaris acoustic solubilizer. A library constructed using a TruSeq DNA PCR-free library preparation kit was sequenced using the Illumina MiSeq platform to generate 301-bp paired-end reads. The raw reads were cleaned up as described previously , average nucleotide identity (ANI) analysis (Buttiauxella ferragutiae ATCC 51602 genome (GenBank accession number LXEQ00000000). This ANI value was significantly lower than the species threshold of 95% (Buttiauxella sp. strain A111 was a novel species belonging to the genus Buttiauxella. The proteomes of Buttiauxella sp. strain A111 and B. contaminans CH-1 (GenBank accession numbers AP018357 to AP018360) and xanthine dehydrogenase accessory protein XdhC (BSPA111_08010) were encoded adjacent to the upstream area of the latter gene cluster in the Buttiauxella sp. strain A111 genome to compao 02430) , and theo 02430) . Unlike 1 genome and have1 genome , respectButtiauxella sp. strain A111 have been deposited in the DDBJ Sequence Read Archive under the accession number DRA008321. The draft genome sequence has been deposited in DDBJ/ENA/GenBank under the accession number BJFN00000000.The raw reads of"} +{"text": "Cultured human cells are pivotal models to study human gene functions, but introducing complete loss of function in diploid or aneuploid cells has been a challenge. The recently developed CRISPR/Cas9-mediated homology-independent knock-in approach permits targeted insertion of large DNA at high efficiency, providing a tool for insertional disruption of a selected gene. Pioneer studies have showed promising results, but the current methodology is still suboptimal and functional outcomes have not been well examined. Taking advantage of the promoterless fluorescence reporter systems established in our previous study, here, we further investigated potentials of this new insertional gene disruption approach and examined its functional outcomes.ULK1 gene at all four alleles, lacking intact FAT10 in all three alleles, or devoid of intact CtIP at both alleles. We have confirmed the depletion of ULK1 and FAT10 transcripts as well as corresponding proteins in the obtained cell clones. Moreover, consistent with previous reports, we observed impaired mitophagy in ULK1\u2212/\u2212 cells and attenuated cytokine-induced cell death in FAT10\u2212/\u2212 clones. However, our analysis showed that single-cell clones carrying complete disruption of CtIP gene at both alleles preserved in-frame aberrant CtIP transcripts and produced proteins. Strikingly, the CtIP-disrupted clones raised through another two distinct targeting strategies also produced varied but in-frame aberrant CtIP transcripts. Sequencing analysis suggested that diverse DNA processing and alternative RNA splicing were involved in generating these in-frame aberrant CtIP transcripts, and some infrequent events were biasedly enriched among the CtIP-disrupted cell clones.Exemplified by using hyperploid LO2 cells, we demonstrated that simultaneous knock-in of dual fluorescence reporters through CRISPR/Cas9-induced homology-independent DNA repair permitted one-step generation of cells carrying complete disruption of target genes at multiple alleles. Through knocking-in at coding exons, we generated stable single-cell clones carrying complete disruption of Multiallelic gene disruption could be readily introduced through CRISPR/Cas9-induced homology-independent knock-in of dual fluorescence reporters followed by direct tracing and cell isolation. Robust cellular mechanisms exist to spare essential genes from loss-of-function modifications, by generating partially functional transcripts through diverse DNA and RNA processing mechanisms.The online version of this article (10.1186/s12915-018-0616-2) contains supplementary material, which is available to authorized users. Recent breakthroughs in engineered nucleases have marked a new era for genome editing. Three main technologies, Zinc-finger nucleases (ZFNs) , transcrHuman cell lines maintained under culture conditions are pivotal models for direct analysis of human gene functions. Since most cultured cells possess diploid or hyperploid genomes, meaning that a single gene is often presented as two or more copies in the genome, knockout or targeted disruption to introduce complete loss of function of a selected gene has been technically challenging in these cells. HDR-based strategies require cloning of homology arms specific to each target locus, and it remains tedious to modify multiple alleles to abolish target gene function completely. Whereas, NHEJ-mediated mutagenesis or deletions provide no means for enrichment and isolation of target cells; hence, non-selective clonal expansion and thorough screening analysis, which are often labor-intensive, are required to identify target cells harboring desired genome modifications.GAPDH at 3\u2032-UTR using promoterless fluorescence reporters, we directly compared frequencies of knock-in mediated by CRISPR-induced NHEJ and HDR repair mechanisms -2,5 diphenyl tetrazolium bromide (MTT) assay as previously described . BrieflyAdditional file 1:Figure S1. Cytogenetic analysis of the human cell line LO2. Figure S2. NHEJ-based knock-in of ires-GFP reporter at coding exons allows tracing of the integration at target sites. Figure S3. Genome PCR of single-cell clones raised from targeting ULK1 and FAT10 genes. Figure S4.CtIP-disruption clones raised from targeted knock-in of ires donors. Figure S5. CtIP-disruption clones raised from simultaneous knock-in of dual pgk-GFP/Td donors at CtIP exon-7. Figure S6. Simultaneous knock-in of dual 5\u2032GFP/Tddonor at CtIP 5\u2032-UTR. Table S1. BAC clones and probes used for FISH analysis. Table S2. DNA sequences bound by sgRNAs. Table S3. Primers used for genome PCR and RT-PCR. (PDF 1413 kb)Additional file 2:Individual data values for Fig."} +{"text": "QIIME 2 microbiome bioinformatics platform that facilitates access, reproducibility, and interpretation of supervised learning (SL) methods for a broad audience of non-bioinformatics specialists.q2-sample-classifier is a plugin for the Microbiome studies often aim to predict outcomes or differentiate samples based on their microbial compositions, tasks that can be efficiently performed by SL methods . The goaq2-sample-classifier, a QIIME 2 plugin to support SL tools for pattern recognition in microbiome data. This plugin provides several SL methods, automatic parameter tuning, feature selection, and various learning algorithms. The visualizations generated provide portable, shareable reports, publication-ready figures, and integrated decentralized data provenance. Additionally, integration as a QIIME 2 plugin streamlines data handling and supports the use of multiple user interfaces, including a prototype graphical user interface (q2studio), facilitating its use for non-expert users. The plugin is freely available under the BSD-3-Clause license at https://github.com/qiime2/q2-sample-classifier.We describe The q2-sample-classifier plugin is written in Python 3.5 and employs pandas and numpThe standard workflow for classification and regression in q2-feature-classifier is shown in RFECV to select the features that maximize predictive accuracy. Hyperparameter tuning is automatically performed using a cross-validated randomized parameter grid search via scikit-learn\u2019s RandomizedSearchCV to find hyperparameter permutations (within a sensible range) that maximize accuracy.The user can enable automatic feature selection and hyperparameter tuning, and can select the number of cross-validations to perform for each (default = 5). Feature selection is performed using cross-validated recursive feature elimination via scikit-learn\u2019s The following scikit-learn SL estim"} +{"text": "However, recent data show that the molecular mechanisms underlying CD8+ T-cell failure in chronic HBV infection depend on the targeted antigen and thus different strategies to improve the HBV-specific CD8+ T-cell response are required. Here, we review the current knowledge about the heterogeneity of impaired HBV-specific T-cell populations and the potential consequences for T cell-based immunotherapeutic approaches in HBV cure.Chronic hepatitis B virus (HBV) infection is a major global health burden affecting around 257 million people worldwide. The consequences of chronic HBV infection include progressive liver damage, liver cirrhosis, and hepatocellular carcinoma. Although current direct antiviral therapies successfully lead to suppression of viral replication and deceleration of liver cirrhosis progression, these treatments are rarely curative and patients often require a life-long therapy. Based on the ability of the immune system to control HBV infection in at least a subset of patients, immunotherapeutic approaches are promising treatment options to achieve HBV cure. In particular, T cell-based therapies are of special interest since CD8 In fact, in this study, a prolonged viremia was observed until the reappearance of HBV-specific CD8+ T cells in the blood and liver represents one of the major causes of morbidity and mortality worldwide. Despite the availability of a prophylactic vaccine for over 30 years, the number of infections remains dramatically high. Approximately, 257 million people globally suffer from chronic HBV infection . Currentnd liver . In cont+ T-cell response. Although the HBV-specific CD8+ T-cell response is initially induced, several phenotypic and functional deficiencies can be observed under the conditions of antigen persistence. These so-called exhausted CD8+ T cells represent a unique immune cell differentiation stage that was first described in mice chronically infected with the lymphocytic choriomeningitis virus (LCMV). In chronic HBV, early studies analyzing HBV-specific CD8+ T cells during persistent HBV infection also showed an compromised functionality of HBV-specific CD8+ T cells including reduced production of the antiviral cytokine interferon \u03b3 (IFN\u03b3) and the immunomodulatory cytokine interleukin 2 (IL-2), as well as the loss of cytotoxicity and impaired proliferative capacities (+ T cells displayed a high expression of multiple inhibitory receptors like programmed cell death protein 1 (PD1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), lymphocyte-activation gene 3 (LAG3), T-cell immunoglobulin mucin domain-3 (TIM3), CD244/2B4, or CD160. The co-expression of these markers contributes to T-cell dysfunction by excessive T-cell suppression has been shown to be increased in chronically HBV-infected patients and to drive the decline of HBV-specific CD8+ effector T cells plays a central role , 45 and us (HIV) have alsory-like . In HBV,patients . Recentlet level . Based onfection .+ T cells is also existent on the level of the targeted antigens. Indeed, immunological characterization of transgenic mice has already shown a hierarchy of dominant and subdominant HBV antigens with various frequencies and antiviral activity (+ T cells targeting different HLA-A*02:01 restricted epitopes located in the core (core18\u221227: FLPSDFFPSV), envelope (env183\u2212191: FLLTRILTI), and polymerase (pol455\u2212463: GLSRYVARL) proteins indicating an antigen-related phenomena. In line with this observation, another recent study also highlighted different exhaustion profiles of HBV-specific CD8+ T cells targeting different HLA-A*11:01 restricted epitopes within the core and polymerase antigens (core169\u2212179: STLPETAVVRR and pol387\u2212396: LVVDFSQFSR) . Furthernfection . Still, + T cells specific for distinct HBV antigens are still unclear . Hence, + T-cell responses by blocking the interactions of inhibitory receptors with their ligands . In vitro studies have shown an at least partial restoration of HBV-specific CD8+ T cells including an enhanced proliferative potential and increased cytokine production upon checkpoint pathway blockade (+ T-cell subpopulation might endow with a better antiviral activity. The current findings suggest that core18-specific CD8+ T cells have a less impaired functionality than pol455-specific CD8+ T cells reflected by their mildly exhausted phenotype. Thus, this finding indicates that core18-specific CD8+ T cells may show an improved responsiveness to PD1 pathway blockade. In contrast, pol455-specific CD8+ T cells which exhibit a terminally exhausted phenotype may only poorly respond to PD1 pathway blockade as already described in the chronic LCMV mouse model (455-specific CD8+ T-cell response might be an efficient approach to achieve HBV cure. It is known that the pol455-specific CD8+ T-cell response is particularly impaired in chronic HBV infection compared to acutely resolved infection, whereas core18-specific CD8+ T cells are similar in their expansion capacity in both HBV infection settings (+ T cells highlight the difficulty of boosting HBV-specific CD8+ T cells. Interestingly, a marked recovery of antiviral capacity was achieved by treating core18-specific CD8+ T cells with mitochondrion-targeted antioxidant compounds in vitro (455-specific CD8+ T-cell responses since the combination of compounds targeting both, the impaired metabolism together with checkpoint blockade, provides a promising option in HBV treatment. In light of the recent finding that TOX represents a master regulator of T-cell exhaustion (+ T-cell dysfunction. Hence, to boost the defective pol455-specific CD8+ T-cell response, combining the PD1 pathway blockade with epigenetic modifications might be necessary. Early studies on the manipulation of epigenetic pathways have shown promising results to overcome T-cell exhaustion in chronic viral infections. Indeed, treatment of exhausted LCMV-specific CD8+ T cells with either histone deacetylate inhibitors (de novo DNA methylation (+ T cells from functional exhaustion. Thus, attempts to manipulate the epigenetic signature could have important clinical implications. However, such studies investigating therapeutic manipulations of epigenetic changes have not been performed yet in chronic viral hepatitis and probably face safety issues due to the broadly acting characteristics of currently available reagents. As a consequence of the terminally exhausted phenotype of pol455-specific CD8+ T cells and the complexity in restoring pol455-specific CD8+ T-cell immunity, another immunotherapeutic strategy is based on adoptive transfer of engineered T cells. Recently, it was demonstrated by Kah et al. that engineered T cells that transiently express HBV-specific T-cell receptors exert a great antiviral effect in HBV-infected humanized chimeric mice (183-specific CD8+ T cells might be caused by the induction of T-cell tolerance which results in reduced T-cell expansion and elevated T-cell apoptosis (183-specific CD8+ T cells in chronic HBV patients, the usage of adoptive transfer is also implicated. However, the induction of novel env183-specific CD8+ T-cell responses by therapeutic vaccines should additionally be considered. So far, different therapeutic vaccines are already used in several clinical trials with limited effect on chronic HBV infection (+ T cells suggest that HBV cure can probably not be achieved by only one single approach. Therefore, a combination of different immunotherapeutic interventions might be necessary to induce viral elimination or at least T cell-based control in chronic HBV infection.Since the discovery of T-cell exhaustion, researchers are investigating the potential for functional restoration of exhausted T-cell populations. Such approaches aim to restore an endogenous dysfunctional antiviral immune response. Indeed, great efforts have been made to boost HBV-specific CD8blockade , 11\u201314. blockade . Howeverblockade . The abose model , 35, 36.settings . Moreovein vitro . Furtherhaustion \u201342, it ihibitors or the bhylation rescued ric mice . The extpoptosis . Thus, fnfection \u201367. In s+ T cells specific for different HBV proteins harbor distinct phenotypical and more importantly functional features in chronically HBV-infected patients. This knowledge allows specializing future immunotherapeutic approaches to target the specific T-cell subpopulation and its molecular pathway that is suitable for the desired kind of immunomodulation. However, the current state of immunotherapeutic treatments suggests that the task of controlling chronic HBV infection is quite difficult. Further characterization of the recently discovered HBV-specific CD8+ T-cell subpopulations are needed to uncover new molecular pathways that could be additionally targeted by immunotherapeutic interventions.Exhausted T cells consist of functionally diverse T-cell subpopulations that co-exist during chronic HBV infection. These findings revealed that HBV-specific CD8All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Streptococcus mutans that jointly control bacteriocin gene expression, natural competence development, as well as a cell death pathway, yet they do not function via any of the currently recognized signal transduction paradigms. These systems, which we refer to as LytTR Regulatory Systems (LRS), minimally consist of two proteins, a transcription regulator from the LytTR Family and a transmembrane protein inhibitor of this transcription regulator. Here, we provide evidence suggesting that LRS are a unique uncharacterized class of prokaryotic sensory system. LRS exist in a basal inactive state. However, when LRS membrane inhibitor proteins are inactivated, an autoregulatory positive feedback loop is triggered due to LRS regulator protein interactions with direct repeat sequences located just upstream of the -35 sequences of LRS operon promoters. Uncharacterized LRS operons are widely encoded by a vast array of Gram positive and Gram negative bacteria as well as some archaea. These operons also contain unique direct repeat sequences immediately upstream of their operon promoters indicating that positive feedback autoregulation is a globally conserved feature of LRS. Despite the surprisingly widespread occurrence of LRS operons, the only characterized examples are those of S. mutans. Therefore, the current study provides a useful roadmap to investigate LRS function in the numerous other LRS-encoding organisms.The most commonly studied prokaryotic sensory signal transduction systems include the one-component systems, phosphosignaling systems, extracytoplasmic function (ECF) sigma factor systems, and the various types of second messenger systems. Recently, we described the regulatory role of two separate sensory systems in Streptococcus mutans. We employ these LRS as models to first define the key features of LRS and then demonstrate how some of these characteristics are likely universally conserved among the plethora of uncharacterized LRS in other organisms. Based upon these data, we further describe how these sensory systems are likely to function in diverse species and illustrate how to identify and investigate the function of novel LRS.The ability to sense stimuli triggered by the extracellular environment is a fundamental requirement of all cellular life. For prokaryotes, there are a variety of recognized classes of sensory systems that are used to detect and respond to environmental stimuli. In the current study, we provide the first evidence for the existence of a potentially new class of prokaryotic sensory system, which we refer to as LytTR Regulatory Systems (LRS). Here, we show that LRS are broadly distributed among prokaryotes and are distinct from the other commonly studied sensory systems like two-component signal transduction systems and ECF sigma factor systems. Presently, there are only two characterized examples of LRS, both from The capacity of bacteria to sense and respond to stimuli triggered by the extracellular environment is fundamental for survival, particularly in highly dynamic and/or competitive niches. Prokaryotes currently have several recognized classes of sensory signal transduction systems that are used specifically for this purpose. The most diverse class consists of the one-component systems, which contain single protein fusions of a signal-sensing input domain and a transcription regulatory output domain . The vasStreptococcus mutans, which we previously named HdrRM and BrsRM. Both systems share a variety of features and appear to be distinct from the aforementioned signal transduction system paradigms. Homologs of these two S. mutans systems, which we broadly refer to as LytTR Regulatory Systems (LRS), can be found in various bacteria, particularly within the Firmicutes phylum , SMU_1854/\u03941855 (hdrR\u0394M) LRS [hdrRM159-LF/hdrR(RenG)-R and (erm)hdrR-RF/hdrRM159-RR-2], SMU_2080/2081 (brsRM) LRS [brsM-LF/brsM(RenG)-R and (erm)brsM-RF/brsM-RR], SMU_2080/\u03942081 (brsR\u0394M) LRS [brsM-LF/brsR(RenG)-R and (erm)brsR-RF/brsM-RR]. All PCR amplicons were purified and mixed in equal molar concentrations and then subjected to a 4-fragment OE-PCR reaction using the respective upstream forward/downstream reverse primer pairs. The assembled PCR amplicons were transformed into S. mutans strain UA140 and selected on agar plates supplemented with erythromycin to obtain the following strains: 294-295-RenG, 294-RenG, 1070c-1069c-RenG, 1070c-RenG, hdrRM-RenG, hdrR-RenG, brsRM-RenG, and brsR-RenG. The wild-type SMU_433/434 and SMU_433/\u0394434 LRS luciferase reporter constructs were PCR amplified from strains 01-luc and 01-luc-434. The resulting PCR amplicons were then transformed into S. mutans strain UA140 and selected on agar plates supplemented with spectinomycin to obtain the strains 433-434-RenG and 433-RenG.The dhRenGSm using th pJY4164 using thS. mutans UA140, we first deleted SMU_433/434 using our previously described markerless mutagenesis protocol [p-chlorophenylalanine (4-CP) to remove the IFDC2 cassette and obtain the markerless deletion mutant. This strain was then used as a recipient for the sequential deletion of SMU_1070c/1069c, SMU_294/295, hdrRM, and brsRM using the same approach to obtain the final 5 LRS deletion strain ifdLRS.To create markerless in-frame deletions of all 5 LRS in protocol . Two fraprotocol using thGenomic DNA from strains 294-295-RenG, 1070c-1069c-RenG, hdrRM-RenG, brsRM-RenG, and 433-434-RenG were transformed into strain ifdLRS and selected on THYE plates contains erythromycin or spectinomycin to obtain the single LRS luciferase reporter strains ifdLRS/294-295-RenG, ifdLRS/1070c-69c-RenG, ifdLRS/hdrRM-RenG, ifdLRS/brsRM-RenG, and ifdLRS/433-434-RenG.hdrM and brsM in each of the single LRS reporter strains. The SMU_434 and SMU_1069c mutations were PCR amplified from d-smu434/UA140 and d-smu1069/UA140 and then transformed into the single LRS reporter strains.To examine potential cross-regulation between different LRS, ORFs encoding LRS membrane proteins were replaced by a kanamycin resistance cassette using the single LRS luciferase reporter strains as recipients. Briefly, upstream and downstream homologous fragments of SMU_295 were amplified using the primer pairs SMU294-LF/(kan)smu295-LR and (kan)smu295-RF/SMU295-RR as well as UA140 genomic DNA as a template. The kanamycin resistance gene was amplified using the primer pair kan-F/kan-R and plasmid pWVTKs as the tS. mutans firefly luciferase reporter strains used in hdrRM ORFs with that of luciferase, we first created an allelic replacement of the hdrRM ORFs with the counterselectable IFDC2 cassette [hdrRM operon were amplified with the primer pairs hdrRupF/hdrRupR-ldh and hdrMdnF-erm/hdrMdnR, respectively. The IFDC2 cassette was amplified using the primer pair ldhF/ermR. The three fragments were mixed and used as template for OE-PCR with the primer pair hdrRupF/hdrMdnR. The resulting OE-PCR product was transformed into UA140 and selected on medium containing erythromycin to obtain strain RMIFDC2. Next, a DNA fragment containing the hdrR upstream region and firefly luciferase ORF was amplified with the primer pair hdrRupF/lucR-1856 and strain LZ89-luc [hdrM downstream region was amplified with the primer pair 1856F-luc/hdrMDnR. The two fragments were mixed and assembled with OE-PCR using the primer pair hdrRupF/hdrMdnR. The OE-PCR amplicon was transformed into strain RMIFDC2 and selected on medium containing p-chlorophenylalanine (4-CP) to obtain strain RpLuc. To create strains Rp+1luc and Rp-10mluc, the upstream and downstream regions of the hdrRM operon were amplified from strain UA140 with the primer pairs hdrRupF/(luc)hdrRp-R or hdrRupF/(luc)hdrRp-10-R and (lucR)hdrMdn-F/hdrMDn-R, respectively. The luciferase ORF was amplified from strain RpLuc with the primer pair lucF/lucR. The three fragments were mixed and used as template for OE-PCR with the primer pair hdrRupF/hdrMdnR. OE-PCR products were transformed into RMIFDC2 and selected on medium containing 4-CP to obtain the strains Rp+1luc and Rp-10mluc. Strains Rp+1luc and Rp-10mluc were both transformed with the plasmid pHdrRoe [hdrR ectopic overexpression plasmid pJYROE, a fragment containing the hdrR ORF fused to the ldh promoter was first amplified from pHdrRoe using the primer pair ldhF-bamHI/hdrRR-hindIII. The resulting PCR amplicon was digested with BamHI and HindIII and then ligated to pJY4164 to obtain the suicide vector pJYROE. To create the hdrM ectopic overexpression plasmid pMOE, an ldh promoter-hdrM transcription fusion was assembled by first PCR amplifying the ldh promoter and hdrM ORF using the primer pairs ldhF-BamHI/ldhR-SpeI and hdrMF-SpeI/hdrMR-EcoRI as well as UA140 gDNA as a template. The resulting amplicons were then digested with BamHI/SpeI and SpeI/EcoRI and subsequently ligated to the BamHI/EcoRI restriction sites of the E. coli-Streptococcus shuttle vector pDL278 [The cassette . Using ULZ89-luc as a tem pHdrRoe to creatr pDL278 to creathdrRM ORFs, a DNA fragment containing the hdrR upstream region and IFDC2 were PCR amplified from strain RMIFDC2 with the primer pair hdrRupF/ermR-lucf. Using the genomic DNA of RpLuc as a template, the luciferase ORF was amplified with the primer pair lucF-erm/lucmR. The two amplicons were assembled using OE-PCR and the primer pair hdrRupF/lucmR. The resulting overlapping PCR products were transformed into RpLuc strain and selected on medium containing erythromycin to obtain the strain RMlucIFDC2. Next, two fragments encompassing the hdrRM locus were amplified from strain UA140 with the primer pair hdrRupF/MterR-luc, while the luciferase ORF was amplified from strain RpLuc with the primer pair lucF-Mter/lucmR. The PCR amplicons were mixed and assembled by OE-PCR using the primer pair hdrRupF/lucmR. The resulting OE-PCR amplicon was transformed into strain RMlucIFDC2 and selected on plates supplemented with 4-CP to obtain the strain hdrRMluc. To mutate hdrM in strain hdrRMluc, three fragments were amplified from this strain using the primer pairs hdrRupF/(spec)smu1853R, (spec)smu1853-hdrR-LF2/hdrM(TAA)R, and hdrM(TAA)F/lucmR. The spectinomycin resistance cassette aad9 was amplified from the E. coli-Streptococcus shuttle vector pDL278 [hdrRM promoter in strain dhdrMluc, two fragments were amplified from this strain using the primer pair hdrRupF/(repeat-m)hdrR-LR and (repeat-m)hdrR-RF/lucmR. The two PCR amplicons were mixed with hybridized EMSA-hdrRpm F/R primers and assembled using OE-PCR and the primers hdrRupF/lucmR. The resulting OE-PCR amplicon was transformed into strain hdrRMluc to obtain the strain dhdrMdDRluc.To insert the luciferase ORF downstream of the r pDL278 using thgusA transcription fusions to the brsRM operon, a brsRM upstream homologous fragment was amplified from strain UA140 or ifdLRS using the primer pair brsRM-LF/(gusA)brsRM-LR, while the brsRM downstream homologous fragment was amplified from strain UA140 using the primer pair (gusA)brsRM-RF/brsRM-RR. The gusA ORF was amplified from plasmid pZX7 [To create markerless mid pZX7 using thhimar transposon onto both ends of the PCR amplicon. The resulting amplicon was then ligated to the pGEM\u00ae-T vector (Promega) to obtain pT-MGL-erm. In vitro transposon mutagenesis was performed by combining MarC9 transposase, genomic DNA from strain ifdLRS, and plasmid pT-MGL-erm and then incubating at 30\u00b0C for 1 h. Transposon junctions were subsequently repaired and then the transposition reaction was transformed into strain ifdLRS/brsRM-gusA. Transposon mutants were selected on THYE plates containing erythromycin and 5-bromo-4-chloro-3-indolyl-\u03b2-D-glucuronic acid . After 5 days of incubation, blue colonies were selected. Transposon insertion sites were mapped according to the published protocol [\u00ae-T vector, transformed into E.coli DH5\u03b1, and then the resulting plasmid inserts were sequenced. PCR was used to confirm the expected locations of transposon insertions sites in each of the mutant strains. Genomic DNA from confirmed transposon mutants was also transformed into strain ifdLRS/brsRMp-gusA (\u0394brsRM) to compare its reporter activity with the corresponding transposon mutants obtained in the ifdLRS/brsRM-gusA (brsRM+) background.The ifdLRS/brsRM-gusA reporter strain transposon library was generated by a previously described transposon mutagenesis protocol . Brieflyprotocol , except hdrR ORF was amplified from strain UA140 using the primer pair hdrRF-NdeI/HdrRR-Hind. The amplicon was then digested with NdeI/HindIII and ligated to the expression vector pET29b to create the plasmid pEcROE.The E. coli BL21(DE3) pLysS expression system. Cultures were grown to OD600 0.6 at 37\u00b0C with aeration before adding 0.1 mM IPTG and culturing for an additional 12 hr. at 20\u00b0C. Cells were harvested by centrifugation , washed twice with binding buffer and then resuspended in 20 ml of the same buffer. Next, the cells were chilled on ice, lysed by sonication, centrifuged to recover supernatants , and then HdrR-His6 was purified using Ni-NTA agarose chromatography (Novagen). Proteins were eluted with 4 ml elution buffer and concentrated by ultrafiltration . Purified proteins were stored in 10% glycerol at -80\u00b0C.Recombinant HdrR was purified using pET29b and the 2, 50 mM NaCl and 1 mM EDTA, with the forward primer 5\u2032-end labeled with digoxigenin-11-ddUTP (Roche). The oligonucleotide pair EMSA-hdrRp-F/EMSA-hdrRp-R served as the wild-type probe, while the oligonucleotide pair EMSA-hdrRpm-F/EMSA-hdrRpm-R served as the direct repeat mutant probe. 1 ng of DNA probe was incubated individually with various concentrations of HdrR-His6 at 25\u00b0C for 20 min in a 20 \u03bcl reaction volume. After incubation, the reaction mixtures were separated by electrophoresis and electro-transferred to nylon membranes. Images were detected using chemiluminescence and X-ray films. For competition experiments, 50- and 200-fold excess of unlabeled probes (EMSAs were performed similarly as previously described . Brieflyd probes were add600) values. Luciferase activity was measured with a GloMax Discover 96-well luminometer (Promega).Assays of firefly and green renilla luciferase activity were performed using a previously described methodology with midTo identify homologs of LRS membrane proteins, we searched the NCBI non-redundant nucleotide collection (nr/nt) and whole-genome shotgun (wgs) databases using tBLASTn . These putative LRS membrane proteins (except for SMU_295 homologs) were then refined contingent on containing either DUF3021 or DUF2154 domains, as determined by NCBI RPS-tBLASTn . Qualifying LRS membrane protein results were further filtered based upon the presence of adjacent upstream LytTR Family transcription regulator homologs identified using tBLASTn .2 for 4 days. To assay the impact of purines on the transposon mutants of ifdLRS/brsRM-gusA, adenine and/or guanine was added to the CDM at a final concentration of 0.15 mM and/or 0.132 mM, respectively. Plates were incubated at 37\u00b0C with 5% CO2 for 2.5 days.To assess the effect of purines on the BrsRM LRS, overnight cultures of ifdLRS/brsRM-gusA and isogenic transposon mutants were harvested by centrifugation, washed thrice with an equal volume of 0.9% NaCl, and spotted on adenine/guanine-replete or adenine/guanine drop-out chemically defined medium (CDM) agar plates . Differet-tests with Welch\u2019s correction. Statistical significance was assessed using a cutoff value of P < 0.05.All statistical analyses were performed using GraphPad Prism software to calculate significance via two-tailed Student\u2019s S1 FighdrRM operon was replaced by a luciferase ORF, which was fused to the operon transcriptional start site (+1). For strain ROE, hdrR was ectopically expressed from a constitutive promoter on a multicopy plasmid. The orange bars correspond to the strains listed in hdrRM ORFs. The green bars correspond to the strains listed in hdrRM ORFs replaced by that of luciferase. For strain RMOE, the hdrR ORF was ectopically expressed in a single copy on the chromosome using a constitutive promoter, while the hdrM ORF was ectopically expressed from a constitutive promoter on a multicopy plasmid. Luciferase data are expressed as means \u00b1 s.d. (indicated by error bars) derived from four biological replicates.The specific activities of the reporter strains described in (TIF)Click here for additional data file.S2 FigS. mutans LytTR Family response regulators ComE and LytR along with the well characterized response regulators VicR and CiaR. Residues marked with an asterisk indicate conserved residues. The residues shown in red font represent the conserved aspartate residues that are the sites of phosphorylation from cognate sensor kinases. B) Clustal Omega was used to align the five S. mutans LRS regulators. Residues marked with an asterisk indicate conserved residues.A) Clustal Omega was used to align the (TIF)Click here for additional data file.S3 FigS. mutans LRS membrane protein as well as putative LRS membrane proteins from other species. For A-E, the predicted protein topology of each S. mutans LRS membrane protein was compared to its corresponding weakest similarity protein shown in Pseudobutyrivibrio ruminis . B) Comparison of SMU_433 with OEOE_0725 from Oenococcus oeni . C) Comparison of SMU_1069c with BUB90_RS22585 from Anaerosporobacter mobilis . D) Comparison of SMU_1855 (HdrM) with ERS095036_10318 from Chlamydia trachomatis . Residues shown in red represent a putative cleavable signal sequence. E) Comparison of SMU_2081 (BrsM) with TALC_RS05575 from the Thermoplasmatales archaeon BRNA1 . F) Predicted topology of SACOL_RS12400 from Staphylococcus aureus. Residues shown in red represent a putative cleavable signal sequence. G) Predicted topology of Btheta7330_RS19920 from Bacteroides thetaiotaomicron.Protter , 37 was (TIF)Click here for additional data file.S4 FigbrsRM-gusA reporter strain. Open reading frames are drawn to scale. Note: two identical, but independent tilS transposon insertion mutants were isolated.Red arrows mark the locations of transposon insertions resulting in activation of the (TIF)Click here for additional data file.S1 Table*Em\u2014erythromycin; Sp\u2014spectinomycin; Km\u2014kanamycin; Cm\u2014chloramphenicol; 4-CP\u2014p-chlorophenylalanine.(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file."} +{"text": "Advances in gene-transfer system and in-depth understanding of immune mechanism have made the immunotherapy a powerful tool for fighting against cancers. Recent studies demonstrated a therapeutic potential of T cells with chimeric antigen receptor (CAR) targeting CD19 in refractory hematopoietic malignancies. At the same time, however, hence the CD19 targeting results in normal cell destruction such as B cell aplasia, a novel marker that specifically expressed in malignant B cells should be applied. In this study, we developed anti-malignancy variant receptor (MVR) mAb that exclusively bound to malignant B cells but not to normal B cells, and demonstrated that autologous T cells expressing CAR construct with anti-MVR scFv (MVR-CAR T cells) efficiently suppressed the outgrowth of malignant B cells in lymphoid organs.+ B cells in vitro. Furthermore, when the MVR-CAR T cells were adoptively transferred into immune-deficient RAG2-/-\u03b3c-/- mice into which LCLs were subcutaneously injected 3 weeks previously, they efficiently suppressed the outgrowth of metastasized LCLs in secondary lymphoid organs in vivo.Malignant B cell-specific monoclonal antibody was isolated from the Balb/c mice immunized with Burkitt's lymphoma cell line, L3055. The antibody specifically recognized the established B lymphoma cell lines and malignant B cells derived from acute lymphoblastic leukemia, chronic lymphocytic leukemia, and diffuse large B cell lymphoma patients. Q-TOF analysis revealed that anti-MVR mAb recognized one of the CD74 variants that distinctively expressed in malignant B cells. We used anti-MVR mAb to generate CAR T cells for the rapid and efficient production of autologous T cells targeting malignant B cells. MVR-CAR T cells were generated by stimulating T cells with anti-CD2, CD3, CD28 Ab-coated beads and transducing MVR-CAR construct using lentiviral vector system. Autologous MVR-CAR T cells efficiently induced cytotoxicity against EBV-transformed LCLs but not against the normal CD19in vitro and in vivo. Considering the unique specificity on malignant B cells, anti-MVR mAb can be a therapeutic of B cell malignancies without normal B cell destruction.We developed anti-MVR mAb - a novel malignant B cell-specific antibody. Anti-MVR mAb recognized one of CD74 variants that exclusively expressed on malignant B cells. MVR-CAR T cells successfully induced LCL-specific cytotoxicity"} +{"text": "Endogenous Retroviruses (ERVs) constitute approximately 8% of every human genome and are relics of ancestral infections that affected the germ line cells. The ERV-W group contributed to primate physiology by providing an envelope protein (Syncytin-1) that has been adopted for placenta development in hominoids. Expression of Human ERV-W (HERV-W) sequences is investigated for a pathological role in various human diseases.Callithrix jacchus) and squirrel monkey (Saimiri boliviensis). We identified in both species proviral sequences, annotated as ERV1\u20131 in respective genome assemblies, sharing high sequence similarities with Catarrhini ERV-W. A total of 130 relatively intact proviruses from the genomes of marmoset and squirrel monkey were characterized regarding their structural and evolutionarily relationships with Catarrhini ERV-W elements. Platyrrhini ERV-W sequences share several structural features with Catarrhini ERV-W elements and are closely related phylogenetically with the latter as well as with other ERV-W-related gammaretrovirus-like ERVs. The ERV-W group colonized Platyrrhini primates of both Callitrichidae and Atelidae lineages, with provirus formations having occurred mostly between 25 and 15 mya. Two LTR subgroups were associated with monophyletic proviral bodies. A pre-gag region appears to be a sequence feature common to the ERV-W group: it harbors a putative intron sequence that is missing in some ERV-W loci, holding a putative ORF as well. The presence of a long pre-gag portion was confirmed among all gammaretroviral ERV analyzed, suggesting a role in the latter biology. It is noteworthy that, contrary to Catarrhini ERV-W, there was no evidence of L1-mediated mobilization for Platyrrhini ERV-W sequences.We previously characterized ERV-W group genomic sequences in human and non-human Catarrhini species. We now investigated ERV-W-like sequences in the parvorder Platyrrhini, especially regarding two species with complete genome assemblies, namely marmoset (Our data establish that ERV-W is not exclusive to Catarrhini primates but colonized both parvorders of Simiiformes, providing further insight into the evolution of ERV-W and the colonization of primate genomes. Such inAmong the various HERVs and squirrel monkey (Saimiri boliviensis) harboring reasonably intact LTRs and internal portions were selected for subsequent analysis ERV-W elements [pre-gag of Catarrhini ERV-W sequences harbored a putative ORF starting in the pre-gag portion and extending into the gag and pro genes [pre-gag, ranging from nt 992\u20131925 and nt 991\u20131949 of marmoset and squirrel monkey consensus sequences, respectively .Besides the common proviral genes, both consensus sequences showed an atypical elements , 15 , 15. Retnsensus) predicte2Cx4Hx4C amino acid sequence at nt 3219\u20133260 and nt 3243\u20133284, respectively; ii) a second modified Gag NC zinc finger characterized by loss of one of the variable residues (Cx2Cx3Hx4C) h HERV-H and HERVgag, pol and env genes by neighbor joining (NJ) analysis. As already observed for Catarrhini ERV-W sequences [Platyrrhini ERV-W-like elements retrieved from marmoset and squirrel monkey genome sequences display high nucleotide similarities with internal portions of Catarrhini ERV-W, yet are more diverged for LTR sequences . As prevequences , phylogeIn order to characterize sequence differences of above defined Platyrrhini ERV-W LTR subgroups, we generated a general LTR consensus was estimated by two different approaches based on a molecular clock, one based on LTR-LTR sequence divergence and another one based on sequence divergence to a gag gene consensus specific for each subgroup, as detailed in materials and methods. Hence, the ERV-W proviruses that were not included in any subgroup based on phylogenetic analyses were evaluated employing only LTR-LTR sequence divergence, due to the low reliability of a consensus built from a heterogeneous ensemble of sequences. With no well-established nucleotide substitution rate (SR) for Platyrrhini available, we estimated ages based on the human neutral SR (0.45% substitutions/nucleotide/million year), which has been previously used to estimate ages of ERVs in primates [t-test (p\u2009=\u20090.000018). With overlapping time periods of integrations in mind, we searched for homologous ERV-W-like loci shared between marmoset and squirrel monkey genome sequences. We identified at least 19 orthologous ERV-W-like insertions (data not shown), confirming that a proportion of ERV-W-like loci has been acquired before the evolutionary separation of the two Platyrrhini lineages that is thought to have occurred between 20 and 18 mya [The time of integration of marmoset and squirrel monkey ERV-W proviruses whose LTRs clustered in the above-mentioned subgroups and Atelidae (Ateles belzebuth), but not in Pitheciidae species (as well as in Tarsiiformes) in the NCBI nucleotide collection (nr/nt) for Platyrrhini species other than marmoset and squirrel monkey, specifically nucleotide sequences derived from families Atelidae, Cebidae and Pitheciidae, using the RepBase CalJac reference sequence as a query (data not shown). Preliminary evidence of ERV-W-like sequences was found for Platyrrhini species belonging to Cebidae (es) Fig. , panel BConsiderable sequence identity between ERV-W sequences in Catarrhini primates and sequences identified in marmoset and squirrel monkey strongly suggested closer evolutionary relationships between those ERVs. Phylogenetic analysis of Gag, Pol and Env putative proteins (puteins) obtained by RetroTector analysis , 37 of rpre-gag region located between PBS and gag gene. A portion of that pre-gag region was also found in marmoset and squirrel monkey ERV-W proviruses [pre-gag region in Catarrhini and Platyrrhini. Notably, more pronounced sequence similarities were limited to approximately 400\u2009nt at the 5\u2032 end when compared to HERV-W pre-gag . More specifically, the particular pre-gag portion displayed 82% sequence identity with a central portion of HERVIP10F ORF2 (nt 2786\u20134249 in the RepBase HERVIP10F reference sequence) pol gene region of an HERVIP10-like ERV group present in Platyrrhini. It is reasonable to speculate that the latter portion was acquired through a recombination event that occurred after the separation from Catarrhini. However, we note that an ERV-W locus on the chimpanzee Y chromosome, nt 21,951,590-21,956,101 (assembly Feb. 2011 - CSAC 2.1.4/panTro4), harbors a pre-gag sequence that has further 350 shared nucleotides in addition to the above 400, and lacks the downstream AG-rich repeat and the HERVIP10-like portion, thus being more similar to Platyrrhini ERV-W pre-gag sequence than to the one normally found in Catarrhini. In addition, the LTRs of that element (annotated as LTR12F) showed relatively high nucleotide similarity with Platyrrhini ERV-W LTRs. Comparative genomic analysis localized the sequence orthologous to this locus in human chromosome Yq11.221, nt 14,340,494-14,345,004 (assembly GRCh38/hg38), likewise annotated as LTR12F-HERV17-LTR12F. That human locus and other elements with similar structure were previously included in a sequence dataset of Catarrhini ERV-W elements showing low score identity to HERV17 [In order to gain further insight into the origin of the remaining approximately 1.5\u2009kb of the Catarrhini ERV-W rch tool Fig. 4)pre-gag ro HERV17 , being mpre-gag region entirely . Because of the fact that all the (H)ERV-W loci lacking the pre-gag portion are actually processed pseudogenes we hypothesized that the pre-gag portion has been removed occasionally through the splicing of proviral transcripts originating from one or several source elements. Thus the pre-gag region may represent an intron sequence. Accordingly, the pre-gag region being an intron is supported by remarkable sequence similarities with splice donor (SD) and splice acceptor (SA) sites , branch site , and SA stretches of sequence similarity with other repetitive elements (such as LINEs and MIR), yet subsequent RepeatMasker analysis did not corroborate the unexplained sequence portions as being derived from such repetitive elements (data not shown).Taken together, our analysis of the Catarrhini pre-gag portion was previously reported for HERV-H gammaretroviruses [gag gene and includes an ORF positioned like the N terminus of murine leukemia virus (MLV) \u201cglyco-Gag,\u201d potentially encoding a proline and serine-rich domain remotely similar to MLV pp12 [gag gene, and this element regulates central steps of viral replication, including splicing and - in some instances \u2013 ribosome occupancy [Besides the HERV-W group , the preoviruses . ParticuMLV pp12 . More inccupancy .pre-gag region could be a common feature of all gammaretroviral HERVs, possibly suggesting a functional role of pre-gag also in the ancestral exogenous viruses. Proviral consensus sequences generated during characterization of the ERV-W group in the human genome [pre-gag portion shared between Catarrhini and Platyrrhini ERV-W sequences showed partial nucleotide identity also in HERV9 and HERV30, possibly due to their closer sequence relationships with the ERV-W group. Of note, all the gammaretroviral HERV sequences taken into account showed an additional, intergenic portion between 5\u2019LTR and gag gene, similarly to the ones already reported for HERV-H [pre-gag region varied from 423 to about 2000 nucleotides in length, with an average value of 1021 bases. In contrast, the portion between 5\u2019LTR and gag gene in the reference sequences of members of spumaretroviruses (including HERV-S) and betaretroviruses (including HERV-K HML1 to 10) as well as exogenous members of the HERV-devoid retroviral genera alpha- and deltaretroviruses was overall remarkably shorter, being only 147 nucleotides in average of the HERV-9 and HERV-30 pre-gag reference sequences (data not shown).Hence, we asked whether such a n genome and marmn genome referencr HERV-H and HERVr HERV-H Fig. 6)pre-gag rBLAT searches in marmoset and squirrel monkey Platyrrhini genome assemblies with the HERV-W group RepBase reference sequence (LTR17-HERV17-LTR17) as a query identified ERV sequences not previously considered in the ERV-W context. Respective sequences were already annotated as \u201cERV1\u20131_CJa-I\u201d for the internal portion and \u201cERV1\u20131_CJa-LTR\u201d for LTR sequences by Repeatmasker/RepBase, yet those sequences and the corresponding ERV group were not characterized in more detail so far, to the best of our knowledge.Given that there is currently no taxonomical support and no correlation with other ERV1\u20131 groups annotated in RepBase for other vertebrates, and because of the high sequence identity with Catarrhini ERV-W elements and their close phylogenetic relationship at the amino acid level; we propose that the here characterized ERV sequences are members of the ERV-W group that colonized Platyrrhini species.Arg, as also observed for the HERV-W group [We have retrieved a total of 130 reasonably intact ERV loci with LTRs and flanking sequences from marmoset and squirrel monkey genome sequences and characterized these elements in terms of structure, phylogeny and estimated time of integration. Platyrrhini ERV-W sequences showed typical gammaretroviral structural features that they have in common with features already characterized in Catarrhini ERV-W sequences . In part-W group . Even if-W group . Accordi-W group .pre-gag region that was previously reported to be present in almost all Catarrhini ERV-W sequences examined [pre-gag sequences now revealed high sequence similarities along the first 400 nucleotides, while Catarrhini ERV-W pre-gag, but not Platyrrhini ERV-W pre-gag, harbors a portion highly similar in sequence to a region within HERVIP10 pol. Of note, some ERV-W loci previously characterized in Catarrhini species\u2019 Y chromosome [pre-gag portion more similar to Platyrrhini pre-gag. It is conceivable that recombination events occurred early after the evolutionary split of the two parvorders, and more ancestral ERV-W sequences could likely be present in Y chromosome due to the fact that much of it does not recombine, except for intrachromosomal/inverted repeat-mediated recombination. Such low recombination rate has been already involved in the Y chromosome delayed loss of Alu transposons as compared with the autosomes, in which genomic redistributions of retroelements is greatly facilitated [gag has been reported to be an unique genetic feature of exogenous gammaretroviruses, providing splicing signals and promoting ribosome synthesis of viral proteins independently of the 5\u2032 cap structure through an internal ribosome entry site (IRES) [gag has been found in all the gammaretroviral HERV groups analyzed, being instead absent in the other genera. This stable feature shared by ancient and existing gammaretroviruses further corroborates an important role in their replication cycle. Accordingly, MLV, feline leukemia virus, and koala retrovirus all harbor additional ORFs that are translated in the 5\u2032 leader and encode a glycosylated form of Gag, enhancing the infectivity of the viruses [pre-gag includes an ORF positioned like the N terminus of MLV gag, possibly encoding for a MLV pp12-like protein [pre-gag portion in both Catarrhini and Platyrrhini species, yet located in different subregions within pre-gag and thus showing a different nucleotide sequence. Identification of a small subset of Catarrhini ERV-W processed pseudogenes lacking the pre-gag region and presence of putative splicing donor and acceptor sites at the pre-gag 5\u2032 and 3\u2032 ends, respectively, suggests an alternative splicing strategy for the ancestral retroviral sequences. Overall, the fact that the ERV-W pre-gag harbors a putative ORF, presenting also splicing signals that occasionally led to the removal of such portion in ERV-W-derived processed pseudogenes, could indicate a similar function originally crucial for viral replication, and possibly removed by intronic splicing after endogenization due to the loss of replication competence in favor of a more compact (and hence transposable) genetic structure. Such strategy was already observed regarding the frequent loss of the env gene, a trait that together with retrotransposition led ERVs to became genomic superspreaders [pre-gag region and splicing within that region in ERV-W and other gammaretroviral ERVs.In addition, Platyrrhini ERV-W loci are characterized by a examined , 15. Fure (IRES) . A simile (IRES) as well e (IRES) . We exte viruses . Similar protein . Our anapreaders . FurtherPlatyrrhini ERV-W sequences were furthermore different from Catarrhini ERV-W in that there was no evidence of ERV-W loci being processed pseudogenes, that is ERV-W loci having been generated by LINE-1-mediated retrotransposition, which accounted indeed for approximately two-thirds of HERV-W loci in the human genome , 19, 24.pre-gag region.Phylogenetic analysis of marmoset and squirrel monkey ERV-W LTRs revealed at least 2 LTR subgroups, named A and B, that support the evolution of different LTRs associated with monophyletic proviral bodies, as already reported for Catarrhini ERV-W proviruses , 15. In The time period of integration of Platyrrhini ERV-W sequences into host genomes was estimated to have taken place between 25 and 15 mya, with the earlier provirus formations being associated with LTRs of subgroup A followed by the major wave of provirus formations with LTRs of subgroup B. The time period of genome colonization was furthermore supported by presence of orthologous ERV-W-like loci shared between marmoset and squirrel monkey genomes as well as related ERV-W elements in other Platyrrhini species belonging to Cebidae and Atelidae lineages.pre-gag region and an intron located within pre-gag appears as a common feature of the ERV-W group, and the biological relevance of this proviral region deserves further investigation especially with regard to the biology of ancestral gammaretroviruses.Besides Catarrhini species, Platyrrhini primates belonging to both Cebidae and Atelidae families were colonized by ERV-W as well, approximately between 25 and 15 mya. Such colonization has been sustained by at least two different ERV-W subgroups, which can be distinguished by alternative LTR types that were furthermore different in sequence from Catarrhini ERV-W LTRs, indicating that various ERV-W versions have colonized respective primate lineages. The Callithrix jacchus, assembly March 2009 - WUGSC 3.2/calJac3) and squirrel monkey . Sequences identified by BLAT searches have been annotated in the UCSC Genome Browser by RepeatMasker/RepBase [ERV-W like elements analyzed in this study were retrieved as previously described . Briefly/RepBase as ERV1\u2013/RepBase . We alsoBesides the above marmoset and squirrel monkey reference genome assemblies, presence of ERV-W like elements was also assessed in other Platyrrhini species belonging to Cebidae, Atelidae and Pitheciidae lineages by Blast searches of nucleotide collection (nt) database of the National Center for Biotechnogy Information (NCBI), using discontiguous megablast and a sequence comprised of ERV1\u20131 CJa-LTR\u2013CJa-I\u2013CJa-LTR as query.Nucleotide sequences were pairwisely and multiply aligned using Geneious bioinformatics software, version 8.1.4 applyingCompiled ERV-W-like sequences were multiply aligned and compared to an LTR17-HERV17-LTR17 proviral reference, obtained from RepBase Update . All thePhylogenetic analyses were performed from manually optimized sequence alignments using MEGA Software, version 6 . Phylogegag gene and a consensus generated for each subgroup (only for sequences that were included in subgroup A and B based on LTR phylogeny). The obtained D values were employed following previous methodologies [The time of integration of each ERV sequence was estimated through different approaches, all based on the percentage of divergent nucleotides (D) as calculated by MEGA software (version 6) . D was edologies to estimn years) .T values obtained from 5\u2032 and 3\u2019LTR D calculations were divided by a factor of 2, considering that each LTR evolved independently in the genome (T\u2009=\u2009D/SR/2). The resulting age of each sequence was expressed as the average of T obtained from the different approaches, excluding values with a standard deviation >\u200920%.http://retrotector.neuro.uu.se/) [https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) [pol ORF sequence translation and comparison with Pol protein sequences of other gammaretrovirus-like HERVs, as reconstructed from the most intact insertions present in human genome assembly GRCh37/hg19 by RetroTector software [Putative Reverse Transcriptase - Ribonuclease H (RT-RH) amino acid sequences of retrieved ERV-W proviruses and the other gammaretroviral ERV groups were inferred as follows. RT-RH portions in the HERV-W sequences and in the Platyrrhini ERV-W-like elements were identified in the respective proviral consensus sequences , 15 usin.uu.se/) ; ii) NCBpsb.cgi) ; and iiisoftware .Additional file 1: Figure S1. Graphical representation of the phylogeny of primates. Primate species addressed in the present study are indicated, including chimpanzee (Pan troglodytes), gorilla (Gorilla gorilla gorilla), orangutan (Pongo pygmaeus abelii), gibbon (Nomascus Leucogenys), various old world monkeys (OWM), marmoset , and squirrel monkey (Saimiri boliviensis). Numbers near nodes represent evolutionary divergence times of lineages (in millions of years ago) as estimated previously [eviously , 17.Additional file 2: Figure S2. Phylogenetic analysis of Marmoset and Squirrel Monkey ERV-W LTR sequences. Nucleotide sequences of Platyrrhini proviral 5\u2032 and 3\u2032 LTRs of marmoset and squirrel monkey ERV-W elements were multiply aligned and analyzed using the Neighbor-joining method and the Kimura-2-parameter model the applying pairwise deletion option. Phylogeny was tested using the Bootstrap method with 1000 replicates. The length of branches indicates the number of substitutions per site. LTR subgroups (see the main paper text) are indicated by squared brackets.Additional file 3: Figure S3. Phylogenetic analysis of the RT-RH region. Platyrrhini and Catarrhini ERV-W RT-RH amino acid sequences were inferred and translated bioinformatically from respective proviral consensus sequences, as detailed in materials and methods. RT-RH sequences of other gammaretroviral-like HERVs derive from amino acid consensus sequences reconstructed previously by RetroTector software [software . RT-RH aAdditional file 4: Figure S4. Splice signals in the pre-gag region. Panel A: 5\u2032 and 3\u2032 ends of HERV-W pre-gag regions display striking similarities with sequences of splice donor (SD) and splice acceptor (SA) sites. A multiple sequence alignment of HERV-W loci lacking the pre-gag region and HERV-W consensus sequences harboring the pre-gag region is shown. Note that only relevant 5\u2032 and 3\u2032 parts of the pre-gag region are depicted. Sequence logos depicting sequence conservation of SD and SA sites are shown. Note the similarities with sequences included in the multiple sequence alignment, supporting the idea that the pre-gag 5\u2032 and 3\u2032 ends represent intron ends. Panel B: further comparison of conserved splice signal sequences with HERV-W sequences identified in [pre-gag region, having the particular nucleotide.ified in . SD\u2009=\u2009spAdditional file 5. Fasta alignment of ERV-W proviral sequences retrieved from marmoset and squirrel monkey genome assemblies. A total of 59 and 71 reasonably complete ERV-W proviruses, i.e. having intact LTRs and internal portions, were retrieved from marmoset and squirrel monkey genome assemblies, respectively, and aligned with respect to the corresponding proviral consensus sequences [equences ."} +{"text": "This aptamer was selected for its ability to bind to suspensions of crude murine myelin in vitro. Our initial studies in vitro and in vivo involved a 40-nucleotide derivative (LJM-3064) of the original 100-nucleotide aptamer. LJM-3064 retained robust myelin-binding properties. Structural characterization of LJM-3064 revealed that the guanosine-rich 5\u2032 half of the sequence forms different G-quadruplex-type structures that are variably stable in the presence of physiologically relevant ions. We hypothesized that this structured domain is sufficient for myelin binding. In this study, we confirm that a 20-nucleotide DNA, corresponding to the 5\u2032 half of LJM-3064, retains myelin-binding properties. We then optimize this minimal myelin-binding aptamer via systematic evolution of ligands by exponential enrichment after sparse rerandomization. We report a sequence variant (LJM-5708) of the 20-nucleotide myelin-binding aptamer with enhanced myelin-binding properties and the ability to bind cultured human oligodendroglioma cells in vitro, providing the first evidence of cross-species reactivity of this myelin-binding aptamer. As our formulation of DNA aptamers for in vivo remyelination therapy involves conjugation to streptavidin, we verified that the myelin-binding properties of LJM-5708 were retained in conjugates to avidin, streptavidin, and neutravidin. DNA aptamer LJM-5708 is a lead for further preclinical development of remyelinating aptamer technologies.We previously reported the Multiple sclerosis (MS) is a central nervous system (CNS) demyelinating disease with increasing prevalence in the Western world, affecting \u223c0.1% of the global population . Most MSDisease-altering therapy for progressive forms of MS has been less effective, however, with only modest clinical benefit observed for ocrelizumab (anti-CD20) , azathioThe relative failure of immunosuppressive approaches to treat progressive MS suggests the need for other approaches to mitigate disease progression. Although many alternative therapeutic approaches have been proposed, regenerative therapies have emerged as one of the most promising avenues for future research. Example approaches include stem cell transplant to generate new oligodendrocytes , regulatInspired by reports of remyelination induced by natural human antimyelin immunoglobulin M (IgM) antibodies, we previously reported the application of systematic evolution of ligands by exponential enrichment (SELEX) using crude murine myelin as a selection target to yield a myelin-binding DNA aptamer (LJM-3064) capable of promoting remyelination in the Theiler's Murine Encephalomyelitis Virus model of MS . In addiWhen compared to IgM antibody-based approaches to stimulating CNS remyelination, DNA aptamer formulations are \u223c10-fold smaller, more stable, and easier to synthesize thus offering many practical advantages . Aptamerin vivo.In the present work, we apply a rational approach to truncate the 40-nucleotide aptamer LJM-3064 to a 20-nucleotide minimal myelin-binding sequence, and perform an unbiased optimization of this minimal sequence using optimization SELEX. We report the emergence of an optimized sequence (LJM-5708) with enhanced myelin-binding properties relative to the parental aptamer, while also conserving the ability to bind to human oligodendroglioma (HOG) cells in culture. We further show that LJM-5708 retains the G-quadruplex properties of the parental aptamer, with formation of a parallel-stranded G-quadruplex structure stabilized by potassium cations. In addition, we show conserved myelin-binding properties of streptavidin, neutravidin, and avidin conjugates of LJM-5708 and assess the role each protein core may play in myelin binding. These studies lay the framework for future testing of optimized remyelinating aptamer formulations 3TCG2CG3TG4TG3) was synthesized by Integrated DNA Technologies with 15% nucleotide randomization at each position, flanked by 5\u2032 (AGAC2AGAC2AGCTGATAC2AGTCGTG) and 3\u2032 (TACGC2A2GC2AC2TGCTC2TC2TGA) regions used for PCR amplification using forward primer 5\u2032-FAM-AGAC2AGAC2AGCTGATAC2AGTCGTG-3\u2032 and reverse primer (5\u2032-A20-spacer18-spacer18-TCAG2AG2AGCAG2TG2CT2G2CGTA-3\u2032). Optimization SELEX was then performed according to a previously described protocol with slight modification and subsequent phenol/chloroform extraction. This method was repeated over five rounds. Beginning with the third round, nonspecific competitor DNA was introduced to increase stringency of selection conditions. After five rounds of selection, recovered aptamers were PCR amplified, ligated into the pGEM-Teasy cloning vector, cloned into DH5\u03b1 cells, colonies grown, plasmids isolated, and aptamer sequences determined by Sanger sequencing.The pelleted material was then suspended in binding buffer and combined with aptamer in 100\u2009\u03bcL total volume. This mixture was incubated at 37\u00b0C for 30\u2009min with rotary mixing. Myelin was again pelleted by centrifugation at 4,700 in vitro was performed as previously described .in vitro myelin-binding assay and in vivo remyelination studies typically utilize streptavidin-conjugated biotinylated aptamer formulations, we further characterized the folded structures of 3\u2032-biotinylated LJM-5706 and LJM-5708 when conjugated to streptavidin showed strong convergent evolution identifying three nucleotides that differ from LJM-3064. Such strong convergence indicates that the optimized 20-nucleotide molecule (LJM-5708) is an improved myelin-binding aptamer within the constraints of the SELEX design .Indeed, we demonstrate that optimized antimyelin aptamer LJM-5708 has enhanced myelin-binding properties relative to the parental aptamer. LJM-5708 retains the G-quadruplex secondary structure of the parent molecule, which appears to be essential for strong myelin-binding activity. G-quadruplex-forming aptamers are not uncommon results of SELEX experiments using DNA libraries , and manImportantly, we also confirm that antimyelin aptamers LJM-3064 and LJM-5708 both have the ability to bind to cultured HOG cells. This is important because it is the first direct evidence of aptamer binding to an intact myelin-rich cell surface. The result also demonstrates that antimyelin aptamers selected against mouse myelin recognize HOG cells. These confocal microscopy and flow cytometry results are the first confirmation of cross-species molecular recognition of myelin by remyelinating DNA aptamers.Mouse-derived myelin suspensions, used in the myelin-binding assay, and human myelin sheaths, as studied in HOG cells, are of relatively similar composition. The classical human myelin proteins 2\u2032 3\u2032-cyclic-nucleotide 3\u2032-phosphodiesterase (CNP), myelin-associated glycoprotein (MAG), myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), oligodendrocyte specific protein (OSP), and myelin proteolipid protein (PLP) make up more than 90% of the rodent myelin proteome, although the remaining fraction does contain a range of proteins unique to rodent myelin . A totalOur study of protein conjugates indicates that bacterial streptavidin complexes bind myelin with the greatest specificity. Negative control aptamer complexes assembled with egg white avidin or neutravidin display higher background binding than with streptavidin. Although streptavidin, neutravidin, and avidin all bind biotin with extreme affinity, their structural differences likely are responsible for varied myelin specificity of conjugated DNA aptamers.in vivo. Such interactions may be relevant to the known remyelination activity of antimyelin aptamer streptavidin conjugates [About 10% of the total mass of avidin is contributed by charged carbohydrate modifications, yielding an isoelectric point (pI) of 10\u201310.5 . This menjugates .in vitro selection. The G-quadruplex secondary structure is retained in optimized DNA aptamer LJM-5708, although one of three base changes alters the central G-tetrad. Myelin-binding assays with biotinylated aptamers conjugated to three different core proteins suggest that streptavidin enhances binding specificity. Finally, a HOG cell-binding assay shows that streptavidin conjugates of optimized antimyelin DNA 20-nucleotide aptamer LJM-5708 bind oligodendrocytes comparably to the parent 40-nucleotide sequence. This evidence of cross-species specificity for myelin is a key component in the nomination of LJM-5708 as a lead molecule for further investigation. Optimized 20-nucleotide DNA aptamer LJM-5708 is thus a strong candidate for further preclinical testing, including pharmacokinetic analysis and remyelination assay in vivo [Our results indicate that the 5\u2032 20 nucleotides of previously identified DNA 40-nucleotide remyelinating aptamer LJM-3064 preserve a parallel G-quadruplex structure associated with myelin binding activity. This isolated 20-nucleotide aptamer shows strong myelin-binding activity that was increased significantly by further in vivo ,20."} +{"text": "PET/CT showed increased 18F-FDG uptakes in multifocal cutaneous lesions in both lower extremities. The patient was diagnosed with SCC via skin biopsy from the left lateral lower thigh. Ten months later, PET/CT showed increased FDG uptakes in the primary tumor area as well as the left inguinal and left supraclavicular lymph node regions. 18F-FDG PET/CT seems to be useful for re-staging and planning appropriate therapeutic strategy in DEB-patients with SCC.Dystrophic epidermolysis bullosa (DEB) is a rare, inherited skin fragility disorder characterized by blister formation in the sublamina densa. DEB is associated with aggressive squamous cell carcinoma (SCC) that has increased risk of metastases and poor prognosis. A 41-year-old woman with DEB underwent"} +{"text": "Dinoflagellates have some of the largest genome sizes, but lack architectural nucleosomes. Their liquid crystalline chromosomes (LCCs) are the only non-architectural protein-mediated chromosome packaging systems, having high degrees of DNA superhelicity, liquid crystalline condensation and high levels of chromosomal divalent cations. Recent observations on the reversible decompaction\u2013recompaction of higher-order structures implicated that LCCs are composed of superhelical modules (SPMs) comprising highly supercoiled DNA. Orientated polarizing light photomicrography suggested the presence of three compartments with different packaging DNA density in LCCs. Recent and previous biophysical data suggest that LCCs are composed of: (a) the highly birefringent inner core compartment (i) with a high-density columnar-hexagonal mesophase (CH-m); (b) the lower-density core surface compartment (ii.1) consisting of a spiraling chromonema; (c) the birefringent-negative periphery compartment (ii.2) comprising peripheral chromosomal loops. C(ii.1) and C(ii.2) are in dynamic equilibrium, and can merge into a single compartment during dinomitosis, regulated through multiphasic reversible soft-matter phase transitions. Dinoflagellate liquid crystalline chromosomes (LCCs) are strongly birefringent, when observed under polarizing light microscopy (live cells without fixation) ,2, sugge2+ concentrations [2+ could mediate the condensation of supercoiled DNA, but on its own would not incur reversibility in the soft-matter phase transition [Cation-chelation (1-6mM EDTA) induced orchestrated concentration-dependent decompaction of LCCs, and the associated loss of higher order could be partially repacked with the reintroduction of divalent cations . This fitrations . Mg2+ coansition , which wansition . Liquid ansition ,22,23,24ansition , observeTransmission electron microscopy (TEM) and DNA fluorescent microscopy of chelator-treated LCCs (5-6mM EDTA), revealed a decompacted screw thread-like appearance surrounding an extended central core . This deUnder physiological concentration ranges of DNA, the cholesteric mesophase (Cm) is the common liquid crystalline mesophase encountered, followed by columnar-hexagonal phase (C-Hm) with higher density than . A chole2+ was a stronger liquid crystalline cations then Mg2+ [2+ stimulated transcription in other eukaryotic cells, inhibited transcription in dinokaryon [Gene encoding region was the only fraction accessible for restriction enzyme digestion . The birhen Mg2+ ; Mn2+ stnokaryon . PCL couBased on the above interpretation of data, the Compartmental Superhelical Liquid Crystalline Model (CSLCM) is proposed:(a) LCCs are composed of modular units termed superhelical plectonemic modules (SPMs), which are formed by highly supercoiled DNA;(b) LCCs compose of an inner core C(i), a core surface C(ii.1) in dynamical equilibrium with C(ii.2) comprising of transcriptionally-active periphery chromosomal loops (PCL).The chromonema is composed of coil-spiralling SPMs in tandem . The des2+ concentrations, indicating that a proportion of higher divalent cation was attributed to counterions, which would not be disrupted by ethanol.LCC decompaction and remodeling were induced with a sample preparation of ultrastructural studies of dinokaryons and attributed to an insufficiency of DNA-protein chemical cross-links associated with a very low protein-DNA ratio . The decGenomes and associated molecular processes have to be optimally crafted for the physical realization of the genome carrier. There was a reduced diversity of specific transcription factors and DNA-binding proteins, but increase in predicted RNA-binding proteins, consistent with post-transcriptional regulation of gene expression (reviewed in ); genes A special dinoflagellate RNA leader sequence (CSL) was identified ,43. The cwater) with high concentrations of divalent counterions, which commonly led to reduce DNA rigidity [A substantial amount of dinoflagellate genomic thymine (60\u201370%) was replaced by 5-hydroxymethyluracil (5Hmu) ,51,52, wrigidity , would frigidity ,57; extrrigidity , consistLCCs had a high frequency of methylcytosine (~1%) , which wUp to 200 metres of DNA can be packaged into a dinokaryon; LCCs embody superhelical-liquid crystalline condensation for compacting some of the largest eukaryotic genomes. This partnership relies on multiphasic phase transitions in the crowded dinokaryon to coordinate multiple levels of chromosome structure. The selective placement of special bases, and high concentrations of selective cations, substantiate higher degrees of DNA superhelicity and anisotropy, likely trending with SPM modularity to reciprocate phase-transitional reversibility and dynamicity. CHLCM embraces modular SPMs, SPM coil-spiraling, special-base(s) placement, high concentrations of counterions, and PGK to optimize soft-matter phase transitions for three special compartments with different chromosomal functions."} +{"text": "Inhibition of pregnancy-associated plasma protein-A (PAPP-A), an upstream activator of the insulin-like growth factor (IGF) pathway, is known to augment sensitivity to platinum-based chemotherapy. This study further tests the efficacy of PAPP-A inhibition with a monoclonal antibody inhibitor (mAb-PA) in ovarian cancer (OC) platinum-resistant patient-derived xenograft (PDX) models.PAPP-A expression was quantitated in platinum-resistant PDX models by ELISA. A subset with High (n = 5) and Low (n = 2) expression were revived in female SCID/beige mice for studies with either saline, carboplatin/paclitaxel (CP) + mAb-PA, or CP + IgG2a. The primary endpoint was tumor area by ultrasound on day 28 relative to baseline. Conversion to platinum-sensitive was defined by average tumor regression below baseline. Statistical analyses included linear mixed effects modeling and Kaplan Meier curves. Response to therapy was correlated with changes in the ratio of phosphorylated/total AKT and ERK 1/2 using Wes analysis.The addition of mAb-PA to CP induced tumor regression below baseline in one High PAPP-A PDX model; another three models exhibited notable growth inhibition relative to CP + IgG2a. None of the Low PAPP-A PDX models regressed below baseline. The PDX model with the greatest magnitude of tumor regression from baseline after combination therapy was maintained on single agent mAb-PA or IgG2a, but no benefit was observed. Decreased phosphorylation of ERK1/2 correlated with conversion to platinum-sensitive.The addition of mAb-PA to CP overcame platinum-resistance in one of five High PAPP-A PDX models; three other models demonstrated improved platinum-response. This supports further clinical development of this novel therapeutic. Front line treatment of ovarian cancer (OC) is a combination of surgery and platinum-based combination chemotherapy. RecurreThe insulin-like growth factor (IGF) system is a signaling pathway that plays an important role in tumorigenesis and is a potential therapeutic target 4]. Acti. Acti4].An alternative approach to IGF targeting involves modulation of IGF binding proteins (IGFBPs). IGFBPs downregulate IGF-1R activity by decreasing the extracellular availability of unbound ligand. In contA barrier to the further study of the IGF-1 pathway and PAPP-A inhibition in OC has been the paucity of clinically-relevant models. OC cell lines are prone to genetic drift with genotypic and phenotypic divergence from the original tumor occurring over time . HoweverPreliminary studies have demonstrated that targeting PAPP-A may overcome platinum resistance . A high-The objective of this study was to confirm the therapeutic activity of mAb-PA and test the hypothesis that the combination of CP and mAb-PA will improve the efficacy of CP in a panel of OC PDX models that express PAPP-A. Positive results may help identify a predictive biomarker for eventual translation to clinical trials in patients with platinum-resistant OC.The development and characterization of the mAb-PA antibody and its effectiveness in inhibiting PDX tumor growth by inhibiting IGFBP-4 proteolysis have been reported previously. It is a3 per mouse) were injected intraperitoneally into at least 3 female SCID-bg mice , in accordance with the Mayo Clinic Institutional Animal Care and Use Committee (IACUC). Upon engraftment, tumor was collected and cryopreserved.After approval by the Mayo Clinic Institutional Review Board (IRB), fresh tissue from consenting patients with ovarian, primary peritoneal or fallopian tube cancer was obtained at the time of primary debulking surgery and immediately processed in the research laboratory. A unique identifier [patient heterotransplant (PH) number] was issued in accordance with the Health Insurance Portability and Accountability Act. Prior to injection, primary tumor specimens were minced and mixed with McCoy\u2019s media containing 10 mg/kg of rituximab to prevent human lymphoproliferation. Minced PDX tumor samples were snap frozen in liquid nitrogen and then pulverized using the Cellcrusher . Protein lysates were prepared in M-PER extraction buffer containing a protease inhibitor cocktail and NP40 cell lysis buffer. PAPP-A ELISA kits were generously provided by Ansh Labs , and were used to quantitatively measure human PAPP-A protein expression. This assay does not recognize murine PAPP-A. Tumors Platinum resistance was defined by a tumor fold change above baseline after four weeks of treatment with IP Carboplatin /Paclitaxel at 50 and 15 mg/kg, respectively, on days 1, 8, 15, and 22 as described, 27.scid Lystbg-J; Envigo, Indianapolis, IN, USA) were injected intraperitoneally with primary platinum-resistant OC patient tissue prepared as previously described and positive staining (red) for mAb-PA intratumor penetration [B]. A similar pattern was observed with PH271 [C and D], which did regress below baseline when treated with CP + mAb-PA. Tumors treated with CP + IgG2a had similar immunofluorescent staining patterns to panels [B] and [D] (not shown). DAPI was used to stain nuclei (blue).(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file."} +{"text": "Post-operative infections occur frequently following major surgery. The magnitude of the post-operative immune response is associated with an increased risk of post-operative infections, although the mechanisms driving post-operative immune-dysfunction and the potential reversibility of this response with immune stimulants are not well understood. This study aims to describe the immediate immune response to major surgery and establish links to both post-operative infection and functional aspects of immune dysregulation. We also investigate the potential of clinically available immune stimulants to reverse features of post-operative immune-dysfunction.Patients over 45 years old undergoing elective gastro-intestinal surgery with planned post-operative surgical ICU admission were recruited. The expression of selected genes was determined pre-operatively and at 2, 24 and 48 hours post-operatively using qRT-PCR. Circulating levels of Interleukin-10 protein were determined by ELISA. Peri-operative cell surface monocyte HLA-DR (mHLA-DR) expression was determined using flow cytometry. Gene expression and mHLA-DR levels were determined in healthy monocytes cultured in peri-operative serum with and without neutralising antibodies and immune stimulants.P<0.0001), peaking within 2 hours of the procedure. Higher post-operative Interleukin-10 mRNA (P = 0.007) and protein (P = 0.001) levels were associated with an increased risk of infection. Cell surface mHLA-DR expression fell post-operatively (P<0.0001). Reduced production, rather than intracellular sequestration, accounted for the post-operative decline in cell surface mHLA-DR expression. Interleukin-10 antibody prevented the decrease in mHLA-DR expression observed when post-operative serum was added to healthy monocytes. GM-CSF and IFN-\u03b3 prevented the decline in mHLA-DR production through distinct pathways.119 patients were recruited; 44 developed a post-operative infection. Interleukin-10 mRNA and protein increased 4-fold post-operatively (Monocyte dysfunction and features of immune suppression occur frequently after major surgery. Greater post-operative Interleukin-10 production is associated with later infection. Interleukin-10 is an important mediator of post-operative reductions in mHLA-DR expression, while clinically available immune stimulants can restore mHLA-DR levels. The most prevalent adverse event complicating major surgery is infection . More thSerial measurements of post-operative cytokine levels have shown an association between the magnitude of the immediate post-operative immune response and susceptibility to post-operative infections , 7, 8. DIn this study, we use a selected panel of transcriptomic, cytokine and functional biomarkers to describe the immediate immune response in patients admitted to the surgical intensive care unit (ICU) following major surgery and establish links both to post-operative infection and functional aspects of immune dysregulation. We also investigate the potential of clinically available immune stimulants, Interferon Gamma (IFN-\u03b3) and Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF), to reverse features of post-operative immune-dysfunction.This study was conducted at The Royal London Hospital, London, UK between June 2012 and November 2014. The study was approved by the North Wales Research Ethics Committee (Reference 10/WNo3/25).Consecutive patients undergoing elective surgery involving the gastrointestinal tract, requiring a general anaesthetic and at least an overnight stay in the surgical ICU were eligible for inclusion. Written informed consent was obtained. Exclusion criteria were: consent refusal, emergency surgery and surgery involving access to the thoracic cavity. All patients received peri-operative prophylactic antibiotics.Patients were examined daily until discharge from hospital by the clinical team for the presence of infection. Infections were confirmed by a member of the study team (HDTT & MJO\u2019D). The definition of infection was based on CDC criteria . As per \u00ae (whole blood) RNA tubes , citrated plasma and serum samples were collected immediately before induction of anaesthesia, at two hours , 24 and 48 hours following surgery.PAXGeneTo provide a representative overview of the peri-operative immune response we selected seven genes encoding key cytokines and transcription factors:Tumour Necrosis Factor-alpha (TNF\u03b1)\u2013common end-product of many innate and adaptive immune pathways.Interleukin-10 (IL-10)\u2013anti-inflammatory cytokine produced by many T cell subtypes and some macrophages.h1) response was analysed using FlowJo software 10.0.7 on a MAC\u00ae workstation. Quantibrite \u00ae PE calibration beads were run regularly to calibrate the flow cytometer. This also allowed the quantification of mHLA-DR expression as low, low-medium, medium-high or high, thus allowing calculation of HLA-DR antibodies per monocyte (Ab/C) describes an inverse relationship and [+ve] a direct relationship.Data in each cell describe the coefficient of determination or r(DOCX)Click here for additional data file.S1 Raw data(XLSX)Click here for additional data file."} +{"text": "OBJECTIVES/SPECIFIC AIMS: Understand the immunomodulatory effects of anti-thymocyte globulin (ATG) and granulocyte colony stimulating factor (G-CSF) on type 1 diabetes patients using samples and in the preclinical model, the nonobese diabetic mouse. METHODS/STUDY POPULATION: Flow cytometry analysis of phase 1 peripheral blood samples treatment of nonobese diabetic mouse with ATG and GCSF and flow cytometry analysis of immune organs . RESULTS/ANTICIPATED RESULTS: Changes in both innate and adaptive immune cell subsets including plasmacytoid dendritic cells, naive, memory, effector CD4+ and CD8+ T-cells, and CD4+ T-regulatory cells and CD8+ T-regulatory cells DISCUSSION/SIGNIFICANCE OF IMPACT: Understanding of immune cell targets for immunotherapy in new-onset type 1 diabetes patients."} +{"text": "This article has been corrected: The \u2018p\u2019 value in the second-last sentence of the Abstract section has been changed. The corrected text of the Abstract is shown below. The authors declare that these corrections do not change the results or conclusions of this paper.ABSTRACTin situ (pre-invasive lesion) or minimally invasive adenocarcinoma in stage IA lung cancer patients with solitary ground-glass opacity nodules. This retrospective study enrolled 58 consecutive stage IA pulmonary adenocarcinoma patients with solitary ground-glass opacity nodules. The characteristics and measurements of the ground-glass opacity nodules as pure ground-glass opacity nodules and mixed ground-glass opacity nodules in the pre-invasive or minimally invasive adenocarcinoma and invasive adenocarcinoma groups on Positron Emission Tomography/Computed Tomography and high-resolution CT were compared and analyzed. Ground-glass opacity nodules in the pre-invasive or minimally invasive adenocarcinoma group preferentially manifested as pure ground-glass opacity nodule (p < 0.01) compared to the invasive adenocarcinoma group. While cystic appearance was more common in the invasive adenocarcinoma group (p < 0.05). Significant differences were found in the diameter of the ground-glass opacity nodule itself and its solid component, and consolidation/tumor ratio between the two groups. The sensitivity in predicting invasive adenocarcinoma was higher with a combined consolidation/tumor ratio > 0.38 and SUVTo investigate the performance of combined 18F-FDG Positron Emission Tomography/Computed Tomography with high-resolution CT for differentiating invasive adenocarcinoma from adenocarcinoma max> 1.46 in mixed ground-glass opacity nodule when compared to those of SUVmaxp < 0.05). For a mixed ground-glass opacity nodule combined consolidation/tumor ratio > 0.38 and SUV> 0.95 alone or consolidation/tumor ratio> 0.39 alone (both max> 1.46 appears to better predict invasive adenocarcinoma in stage IA lung cancer patients with solitary ground-glass opacity nodules.https://doi.org/10.18632/oncotarget.15577Original article: Oncotarget. 2017; 8:23312-23321."} +{"text": "Invisibility and ageism are ever-present challenges to developing a work-force commensurate with the expanding older population . Intergenerational and experiential endeavors can counter stereotypes while highlighting gerontology career opportunities. Focusing workforce development efforts on the oft-overlooked population of high school students has numerous advantages, including: (1) identifying aging-related career paths prior to college; (2) reaching students eligible to become CNAs/PCAs; (3) uncovering potential benefits agencies may offer to student-employees and (4) countering aging stereotypes. This presentation reports on a multi-faceted undertaking which includes collaborating with local service agencies on a regional aging-workforce initiative, bringing high-school students to a college campus for CIAW events, participating in local/regional career events focused on high school students, and developing/implementing experientially-rich learning opportunities such as a college-credit bearing introductory course on aging to be taught at the high school."} +{"text": "A subclass of C fibre sensory neurons found in hairy skin are activated by gentle touch et al. find that gentle stroking of the skin at a frequency that stimulates C-fibre sensory neurons relieves pain in infants.Gursul Noxious-evoked brain activity in infants is similar to that observed when adults experience pain In Study 1, CT-optimal touch significantly reduced noxious-evoked brain activity following the first acute experimental noxious stimulus . CT-optimal stimulation significantly reduced the magnitude of noxious-evoked brain activity compared with age-matched controls who were not touched prior to lancing (p = 0.045, two-sided t-test; Consistent with Study 1, the magnitude of the reflex withdrawal was not significantly different between groups (p = 1, Mann-Whitney U-test; We also examined infants\u2019 behavioural responses in Study 2 by recording the duration of pain-related facial expressions see . A similMicroneurography has facilitated identification of CT fibres in adults, but the safe use of this invasive technique has not been established in infants"} +{"text": "Scientific Reports 10.1038/s41598-019-51868-5, published online 28 October 2019Correction to: The original version of this Article contained a typographical error in the Abstract.\u201cPatients with Snyder-Robinson Syndrome (SRS) exhibit deficient Spermidine Synthase (SMS) gene expression\u201dnow reads:\u201cPatients with Snyder-Robinson Syndrome (SRS) exhibit deficient Spermine Synthase (SMS) gene expression\u201dThis has now been corrected in the PDF and HTML versions of the Article."} +{"text": "A self-portrait is a common representation of an artist with different techniques by that artist and emerged since the 15th century. Albrecht D\u00fcrer was one of the first artists who performed various self-portraits during his life. The fascinating aspect from a gerontological point of view is that artists show themselves throughout the aging process as well as sometimes with manifest signs of diseases. The poster show self-portraits over the life course from Rembrandt van Rijn (1606-69), Vincent van Gogh (1853-90), Ferdinand Hodler (1853-1918), Lovis Corinth (1858-1925), Helene Schjerfbeck (1862-1946), Edvard Munch (1863-1944), K\u00e4the Kollwitz (1867-1945), Pablo Picasso (1881-1973), and Max Beckmann (1884-1950)."} +{"text": "OBJECTIVES/SPECIFIC AIMS: MicroRNAs are small, non-coding RNAs that control gene expression by inhibiting protein translation. Preclinical studies in rodent stroke models suggest that changes in microRNA expression contribute to neural repair mechanisms. To our knowledge, no one has previously assessed microRNA changes during the recovery phase of human stroke. Our goal was to determine whether patients with significant upper limb recovery following stroke have alteration of neural repair-related microRNA expression when compared to those with poor recovery. METHODS/STUDY POPULATION: Plasma was collected at 19 days post-stroke from 27 participants with mild-moderate upper extremity impairment enrolled in the Critical Periods After Stroke Study. MicroRNA expression was assessed using TaqMan microRNA assays (Thermo Fisher Scientific). Good recovery was defined as \u22656 point change in the Action Research Arm Test (ARAT) score from baseline to 6 months. Bioinformatics analysis compared the plasma microRNA expression profiles of participants with good Versus poor recovery. Candidate biomarkers were identified after correcting for multiple comparisons using a false discovery rate <0.05. RESULTS/ANTICIPATED RESULTS: Eleven microRNAs had significantly altered expression in the good (n=22) Versus poor (n=5) recovery groups, with 2 showing increased expression\u2014miR-371-3p and miR-520g, and 9 showing decreased expression\u2014miR-449b, miR-519b, miR-581, miR-616, miR-892b, miR-941, miR-1179, miR-1292, and miR1296. Three of these could be implicated in neural repair mechanisms. Elevated miR-371-3p levels increase the likelihood that pluripotent stem cells will differentiate into neural progenitors. MiR-892b decreases levels of amyloid precursor protein, which has been implicated as a regulator of synapse formation. Finally miR-941, the only known human-specific microRNA, downregulates the CSP\u03b1 protein which is involved in neurotransmitter release. DISCUSSION/SIGNIFICANCE OF IMPACT: This preliminary study suggests that circulating microRNAs in the plasma may help serve as biomarkers of neural repair and aid in understanding human neural repair mechanisms. If validated in larger studies with appropriate controls, these markers could aid in timing rehabilitation therapy or designing recovery-based therapeutics."} +{"text": "A 74-year-old man recently diagnosed with high-risk prostate cancer with high serum prostate specific antigen was referred to nuclear medicine for a technetium-99m-methylene diphosphonate (Tc-99m MDP) bone scan. On delayed three-hour anterior planar image, an unexpected round focus of intense uptake was found overlying the right orbit. Single-photon emission computed tomography/computed tomography localized the uptake to an ocular prosthesis. The hydroxyapatite composition of the ocular implant can be recognized by its bone-like density and its intense accumulation of Tc-99m MDP. Review of the patient\u2019s history revealed remote right eye evisceration secondary to a complication of cataract surgery, consistent with the findings."} +{"text": "EVs from the conditioned media, as well as from pregnant and non-pregnant sera, were enriched for exosomes. The RNA composition of these murine trophoblast-derived and pregnancy-associated exosome-enriched-EVs (ExoE-EVs) was determined using RNA-sequencing analysis and expression levels confirmed by qRT-PCR. Differentially abundant miRNAs were detected in syncytial differentiated ExoE-EVs, particularly from the X chromosome cluster . These were confirmed to be increased in pregnant mouse sera ExoE-EVs by qRT-PCR analysis. Interestingly, fifteen miRNAs were only present within the pregnancy-derived ExoE-EVs compared to non-pregnant controls. Mmu-miR-292-3p and mmu-miR-183-5p were noted to be some of the most abundant miRNAs in syncytial ExoE-EVs and were also present at higher levels in pregnant versus non-pregnant sera ExoE-EVs. The bioinformatics tool, MultiMir, was employed to query publicly available databases of predicted miRNA-target interactions. This analysis reveals that the X-chromosome miRNAs are predicted to target ubiquitin-mediated proteolysis and intracellular signaling pathways. Knowing the cargo of placental and pregnancy-specific ExoE-EVs as well as the predicted biological targets informs studies using murine models to examine not only maternal-fetal immune interactions but also the physiologic consequences of placental-maternal communication.The role of extracellular vesicles (EVs), specifically exosomes, in intercellular communication likely plays a key role in placental orchestration of pregnancy and maternal immune sensing of the fetus. While murine models are powerful tools to study pregnancy and maternal-fetal immune interactions, in contrast to human placental exosomes, the content of murine placental and pregnancy exosomes remains largely understudied. Using a recently developed Appreciation of intercellular communication has shifted dramatically since the identification and demonstrated role of extracellular vesicles (EVs). EVs are released from cells, carrying protein and nucleic acids that then impact function(s) of distant cells and tissues. The human placenta, a major regulator of maternal physiology during pregnancy, is a prime example of this type of cellular communication. The outer syncytiotrophoblast layer of the chorionic villi of human placentas is bathed in maternal blood and releases a wide range of EVs including exosomes, microvesicles, apoptotic bodies and large cellular fragments including syncytial nuclear aggregates . PregnanExosomes contain distinct repertoires of proteins, lipids, and RNAs originating from the host cell. Placental EVs carry a wide range of proteins with known or predicted functional effects on target cells including the pro-apoptotic protein members of the tumor necrosis factor (TNF) family, FAS ligand and TNF-related apoptosis inducing ligand (TRAIL) \u20136. HumanExosomes can transfer RNA to target cells where messenger RNA (mRNA) can be translated into protein and microRNAs (miRNAs) can alter recipient cell gene expression . The abuin vitro-derived murine trophoblastic exosomes and pregnancy-associated exosomes. A recently described in vitro model of mouse trophoblast syncytial differentiation from undifferentiated trophoblast stem cells (TSC) was used to generate both stem cell and syncytial derived trophoblast (SynT) EVs [Mice are often used to model normal and pathologic pregnancy, including studies investigating the biological mechanisms responsible for maternal-fetal tolerance in normal pregnancy and systemic alterations in disease. Determining the RNA cargo of placental exosomes is an important first step in understanding their role in pregnancy. We postulate RNAs that are enriched in or unique to pregnancy sera exosomes, and also present in trophoblast-derived exosomes, will reflect placenta-derived RNAs. Therefore, this study sought to characterize the exosomal RNA composition of both ynT) EVs . To gain2, 21%) result in fused, multinuclear syncytial-like cultures (SynT) [Murine TSCs derived from C57BL/6 mice were maintained on CellStart-CTS coated plastic plates in RPMI-1640 with L-glutamine (Hyclone SH30027) supplemented with 1% penicillin/streptomycin (Hyclone SV30010), 1% sodium pyruvate (Gibco 11360), 85.8 \u03bcM \u03b2-mercaptoethanol (Bio-Rad 161\u20130710), 1 \u03bcg/ml heparin (Sigma H3149), 25 ng/ml fibroblast growth factor 4 (FGF4) (Sigma F8424) to maintain stemness. TSCs cultured on plastic in the absence of FGF4 or heparin at normal oxygen tension (Os (SynT) . Cell diC57BL/6.J (B6) mice (JAX stock #000664) were purchased or bred and housed in a pathogen-free animal care facility at National Jewish Health (NJH) in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments at NJH. Mice were given ad lib access to food and water. All cages contained bedding and were on 12 hour light-day cycles. At 6\u20138 weeks of age, B6 mice were pairbred overnight. The presence of a vaginal plug marked gestational day (GD) 0.5. Pregnant female mice were sacrificed on GD 14.5. Age-matched, non-pregnant females (NP) were sacrificed in conjunction with each pregnant female. At time of sacrifice , blood was collected via cardiac puncture. Serum was prepared from the whole blood using venous blood collection serum separator tubes (BD Vacutainer #367986) per manufacturer\u2019s instructions. Serum was stored at -80\u00b0C.et al [Conditioned media (CM) was collected from independent TSC (n = 3\u20135) and SynT (n = 3\u20135) cultures. Cellular debris was removed by sterile filtering as described in the alternate filtration protocol by Th\u00e9ry et al . ExoE-EVExoE-EVs (10uL) were deposited on Formvar carbon coated, glow-discharged grids using a Denton Desk III TSC Sputter Coater . After 20 minutes of adsorption, grids were fixed with 4% PFA in NaCac for 10 minutes. Grids were then quenched with 50 mM of Glycine in PBS for five minutes then washed with PBS x 3 in 50 uL for 5 minutes each wash. The grids were incubated in blocking media containing 1% BSA in PBS for 20 minutes. After washing with PBS as indicated above, EVs were exposed to either anti-CD63 or anti-Tsg101 for 45 minutes followed by incubation with Protein A\u2013 10nm Gold Conjugate for 40 minutes at room temperature . Grids were fixed in 1% Glutaraldehyde and 2% PFA in NaCac. The grids were then stained with 4% uranylacetate in 0.15M Oxalic Acid, pH 7, embedded with 2% methylcellulose in 4% uranylacetate, and imaged in a JEOL, JEM1400 is a 120kV Transmission Electron Microscope with a LaB6 emitter and imaged using a Gatan Orius 10.7 megapixel CCD camera. Images were taken at either 25,000X or 40,000X.The concentration and size distribution profiles of ExoE-EV were evaluated using a NanoSight NS300 instrument following manufacturer\u2019s instructions. Videos were recorded at camera level 14. The following post acquisition settings were selected: minimum detection threshold of 3, automatic blur, and automatic minimum expected particle size. Samples were diluted 1:10\u20131:1000 in PBS and loaded using a syringe pump. For each sample, three 30-second videos were recorded and analyzed in the batch-processing mode using Nano Tracking Analysis (NTA) software.Whole cell lysates (WCL) and ExoE-EV lysates (EXO) from TSC and SynT cultures were prepared in lysis buffer containing: 25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, and 0.1% sodium dodecyl sulfate. Both WCL (n = 3) and EXO lysates (n = 3) were sonicated and protein concentration was determined by DC protein assay (Bio-Rad 5000112). 20 \u03bcg of each lysate were loaded into a 10% Bio-Rad Criterion gel for sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Protein was transferred to polyvinylidene difluoride membrane and blocked in 5% blotting-grade milk (Bio-Rad 1706404) in Tris-buffered saline containing 0.05% Tween 20 (TBS-T). Membranes were incubated with primary anti-CD63 antibody or Tsg101 in blocking buffer overnight at 4\u00b0C. Membranes were washed 3 times in TBS-T and probed with species appropriate secondary antibody . Following a final wash in TBS, immunoreactivity was visualized using Western-Lightning chemiluminescent substrate (Perkin-Elmer NEL103E001) per manufacturer\u2019s instructions and exposed to Ultra Blue X-Ray film (Light Labs X-3001).Sera and ExoE-EVs from pregnant and non-pregnant mice were prepared in 20uL M-PER Mammalian Proteins Extraction Reagent (Thermo Scientific # 78503). Samples were incubated at room temperature for 5 minutes to allow for complete lysis. The lysate was centrifuged at 14,000x g for 10 minutes to remove cell debris. The protein concentration was determined using NanoDrop A280 . Thirty micrograms of sera and exosome sera lysates were loaded onto a NuPAGE 10% Bis-Tris Protein Gel, 1.5 mm, 10-well . The gel ran for 1 hour at 200V with 1XNuPAGE MOPS SDS running buffer . The proteins from the gel were then transferred onto a polyvinylidene difluoride membrane using chilled 1X NuPage Transfer buffer . The membrane was then blocked with 5% blotting-grade nonfat dry milk in Tris-buffered saline containing 0.05% Tween-20 (TBS-T) at room temperature for 1 hour with shaking. The membranes were incubated with either primary antibody CD63 Tsg101 (C-2) or GRP94 . Membranes were washed 4 times in TBS-T (5 minutes per wash with shaking) then incubated with the appropriate secondary antibody: goat anti-rabbit HRP or anti-mouse IgG HRP . Membranes were washed with TBS-T as mentioned above and immunoreactivity was visualized using SuperSignal West Femto and imaged in an Odyssey FC imager controlled by Image studio software .RNA was purified from ExoE-EV using SeraMir RNA kits (System Biosciences) as previously detailed , 14. RNAAccession number to be provided once manuscript accepted.)The ExoE-EV RNA-Seq data analysis was performed by Maverix Biomics custom exosome RNA-Seq analysis pipeline. Data quality check of the input sequence was first performed using FastQC, an open-source quality control (QC) tool for high-throughput sequence data . Bowtie2RNA was purified from the ExoE-EV using SeraMir RNA kits (System Biosciences), as described above from independent cultures of TSC (n = 5) and SynT (n = 5) cells. Briefly, 100 ng of purified RNA from each sample was poly-adenylated, and cDNA was synthesized according to the QuantimiR protocol (System Biosciences). NP (n = 8) and GD 14.5 (n = 8) RNA was isolated as described above. Purified RNA from each serum sample was reverse-transcribed into cDNA. Relative miRNA abundance was determined with Maxima SYBR Green detection using Universal reverse primer and a forward primer designed for miRNAs of interest and an HPredicted miRNA-target interactions were determined by utilizing the multiMiR R package to simultaneously query eight publicly available databases: DIANA-microT-CDS, EIMMo, MicroCosm, miRanda, miRDB, PicTar, PITA, and TargetScan , 32. MulTo characterize the EVs isolated using ExoQuick, samples were examined by nano tracking analysis (NTA), electron microscopy (EM), and immunoblot analysis . ExoQuicRNA-Seq was performed to generate a preliminary, unbiased description of the total RNA composition of trophoblast and pregnancy ExoE-EVs. This schema allowed for the identification of all RNAs contained within each group, as well as comparison of the differential abundance of RNAs between groups (TSC vs. SynT and NP vs. GD 14.5).2 selected for convenience of numbers and a natural breakpoint in the data) in GD 14.5 compared to NP mice are shown in Quality, trimming, and read mapping of sequenced RNAs are summarized and presented in in vitro- and in vivo-derived ExoE-EVs revealed a subset of miRNAs that are likely to be associated with trophoblasts, pregnancy, or both. RNA sequencing presented in this study identified mmu-miR-322-3p as significantly more abundant in SynT compared to TSC ExoE-EVs , and mmu-miR-322-5p exhibited a trend in increased abundance with syncytial differentiation (log2 fold change = 2.17). To further quantify and validate the abundance of mature mmu-mir-322 products in our trophoblast- and pregnancy-derived samples, qRT-PCR was performed on a larger sample size of independent samples. Using this method, the relative abundance of mmu-miR-322-3p increased in SynT differentiation in vitro cell culture and in vivo during pregnancy . Signifi, n = 8) . These r2 fold change = 2.03; In addition to the X-chromosome derived mmu-miR-322-3p and mmu-miR-322-5p, abundance of mir-322/424 cluster member, mmu-miR-450a-5p, was found to be significantly increased in SynT compared to TSC and exhibited an increase in pregnancy samples for predicted targets. For each miRNA, hundreds to thousands of unique mRNA targets were identified by the eight databases. In order to reduce this to a meaningful list with more confidence in the miRNA-target prediction, targets with three or more of the eight databases predicting the miRNA-target interaction were considered to be of higher confidence. The high-confidence miRNA-target interactions are presented in the supplemental material . In totaAll unique targets with at least one high-confidence interaction predicted for mmu-miR-322-3p, mmu-miR-322-5p, mmu-miR-503-5p, or mmu-miR-542-3p from MultiMiR analysis were imported into the DAVID Bioinformatics Resource 6.7 for KEGG pathway analysis. Of the 855 unique Ensembl IDs imported, 846 matched DAVID IDs and were included in the subsequent KEGG analysis. All KEGG pathways significantly targeted (p<0.05) by predicted miRNA-target interactions are summarized in These results highlight important signaling pathways and protein family members likely to be targeted by X-chromosome cluster miRNAs upon target cell acquisition of circulating trophoblast-derived exosomes.Placenta-derived EVs, specifically exosomes, have been implicated in maternal-fetal communication where interactions occur with maternal cells including decidual cells, circulating immune cells, and endothelial cells , 39, 40.We identified five mature miRNAs from the mir-mmu-322 X chromosome cluster that were significantly increased in ExoE-EVs with trophoblast syncytial differentiation, and fifteen miRNAs that were unique to pregnancy ExoE-EVs compared to NP controls. To assess the potential targets of these miRNAs, we utilized the bioinformatics tool MultiMiR to simultaneously query eight publicly available miRNA target databases. X-chromosome miRNAs mmu-miR-322-3p, mmu-miR-322-5p, mmu-miR-503-5p, and mmu-miR-542-3p were predicted to target intracellular signaling pathways and ubiquitin-mediated proteolysis, and specifically several cullin family proteins. Pathways such as MAPK signaling and focal adhesion are critical to the functions of many cells, including trophoblasts, endothelial cells and immune cells, all potential cellular targets of circulating exosomes , 42. TroExosome-mediated targeting of the ubiquitin-mediated proteolysis pathway may alter protein localization and/or stability in target cells. Cullins carry crucial roles in the post-translational modification of proteins involving ubiquitin . We idenIn T cells, independent of hypoxia, Cul2 and Cul3 regulate interleukin-2 production (IL-2). Specifically, loss of Cul2 or Cul3 resulted in an increase in IL-2 production upon T cell receptor stimulation suggesting that repression of these cullins would impact intracellular signaling cascades to allow for increased cytokine release , 47. Prein vitro syncytialization, and targets fibroblast growth factor receptor 1 (FGFR1) [in vitro model. Together with previous data, these findings are consistent with the proposed role for hsa-miR-424/mmu-miR-322 in regulating trophoblast differentiation toward a syncytial fate [As expected we identified the expression of the placental miRNA mmu-miR-322-5p in our trophoblast-derived ExoE-EVs. The human ortholog of mmu-miR-322-5p, hsa-miR-424-5p, is known to be expressed in human trophoblasts, regulated by hypoxia, increased with (FGFR1) , 50. Thiial fate . In addiial fate . BDNF haial fate , 54. TheThe abundance of mmu-miR-292-3p and mmu-miR-183-5p increased with pregnancy, and were detected in trophoblast ExoE-EVs independent of differentiation state. This is somewhat surprising given that the mmu-miR-290 cluster of miRNAs was originally identified as being expressed exclusively by mouse embryonic stem cells (ESC) . ConsistWe were able to derive an enriched population of exosomes from the EVs from small blood volumes as well as obtain good quality RNA-seq data. Given that the EV material is not 100% exosomes, some of the identified RNA species may originate from other EVs or protein complexes contained within the plasma. Regardless of the exact origin, the fact that the miRNAs are increased or only present in pregnant plasma still highlights the role they may play in pregnancy. The RNA-seq data was limited in terms of the number and magnitude of expression difference that could be detected given the small sample size (n = 3). Utilizing the larger sample numbers for the qRT-PCR confirmation studies was valuable to validate not only the significant findings noted by RNA-seq but additional miRNAs that were not significant by RNA-seq. In the future, purifying trophoblast-derived exosomes from the pregnancy plasma would provide additional clarity to the origin of identified miRNAs.in vitro-derived murine trophoblastic and pregnancy-associated exosome-enriched EVs. Importantly, this study identifies specific miRNAs that have significant roles in pregnancy physiology and potentially maternal-fetal tolerance and warrant further mechanistic studies. Determining the cargo of placental and pregnancy-specific ExoE-EVs and their predicted biological targets is important to advance our understanding of how these small membrane packages mediate cell-cell communication in pregnancy, as well as where these interactions may go awry in pregnancy-specific diseases.Murine models are routinely used for the study of pregnancy and maternal-fetal tolerance, yet an important communication modality, placenta-derived EVs and specifically the miRNA cargo, has remained largely understudied. This study provides the first in-depth characterization of the RNA composition of Further investigation is necessary to determine the net biological effect of the multiple miRNAs contained within ExoE-EVs, on the vast number of each miRNA\u2019s predicted targets. Additionally, confirmation of miRNA cargo in a pure exosome population would be ideal for validating our findings in the exosome-enriched EVs. For trophoblast-derived miRNAs, it will be important to consider both the local effects on reproductive and immune cells, as well as the systemic implications for maternal immune and endothelial cells.S1 FigPanel A shows bright field light microscopy images (20x) of IV1 cells in culture (CellStart with FGF4 and heparin) and then 7 days in the absence of FGF4 and heparin that leads to differentiation and cell fusion into SynT. Panel B Immunoblot of nuclear enriched fractions (NE-PER Nuclear and Cytoplasmic Extract Reagent from ThermoFisher) from undifferentiated IV1 TSC (U) and differentiated syncytiotrophoblasts (D) showing downregulation of stem cell markers CDX2 (abcam 76541), EOMES (abcam ab23345), and ELF5 (abcam ab104410), and alpha tubulin serves as loading control.(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S5 Fig(TIF)Click here for additional data file.S6 FigC57BL/6.J (B6) mice were cared for in a pathogen-free animal care facility at National Jewish Health in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines.(PDF)Click here for additional data file.S1 Table(TIF)Click here for additional data file.S2 TableQuality, trimming, and read mapping of sequenced RNAs.(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(TIF)Click here for additional data file."} +{"text": "Heterotopic ossification (HO), either acquired (aHO) or hereditary, such as fibrodysplasia ossificans progressiva (FOP), is a serious condition without effective treatment. Understanding of the core process of injury-induced HO is still severely limited.Double-pulse thymidine analog labeling was used to explore the distinctive domains evolved in injury-induced lesions in an animal model of HO (Nse-BMP4). Histological studies were performed to see whether a similar zonal pattern is also consistently found in biopsies from patients with aHO and FOP. In vivo clonal analysis with Rainbow mice, genetic loss-of-function studies with diphtheria toxin A (DTA)-mediated depletion and lineage tracing with Zsgreen reporter mice were used to obtain further evidence that Tie2-cre-, Gli1-creERT-, and Glast-creERT-labeled cells contribute to HO as niche-dwelling progenitor/stem cells. Immunohistochemistry was used to test whether vasculature, neurites, macrophages, and mast cells are closely associated with the proposed niche and thus are possible candidate niche supportive cells. Similar methods also were employed to further understand the signaling pathways that regulate the niche and the resultant HO.We found that distinctive domains evolved in injury-induced lesions, including, from outside-in, a mesenchymal stem cell (MSC) niche, a transient domain and an inner differentiated core in an animal model of HO (Nse-BMP4). A similar zonal structure was found in patients with aHO and FOP. In vivo clonal analysis with Rainbow mice and genetic loss-of-function studies with DTA provided evidence that Tie2-cre-, Gli1-creERT-, and Glast-creERT-labeled cells contribute to HO as niche-dwelling progenitor/stem cells; consistently, vasculature, neurites, macrophages, and mast cells are closely associated with the proposed niche and thus are possible candidate niche supportive cells. Further mechanistic study found that BMP and hedgehog (Hh) signaling co-regulate the niche and the resultant HO.Available data provide evidence of a potential core mechanism in which multiple disease-specific cellular and extracellular molecular elements form a unique local microenvironment, i.e., an injury-induced stem cell niche, which regulates the proliferation and osteogenic differentiation of mesenchymal stem cells (MSCs). The implication for HO is that therapeutic approaches must consider several different disease specific factors as parts of a functional unit, instead of treating one factor at a time.The online version of this article (10.1186/s13287-018-1107-7) contains supplementary material, which is available to authorized users. Heterotopic ossification (HO), acquired or hereditary, is the formation of true bone in extraskeletal soft tissue \u20133. AcquiBased on long-term observations, we hypothesized that multiple disease-specific cellular and extracellular molecular elements form a unique and relatively stable local microenvironment, i.e., an injury-induced mesenchymal stem cell niche (MSC niche), which regulates the proliferation and subsequent osteogenic differentiation of mesenchymal stem cells (MSCs) and the downstream HO process. This study was designed to directly test this hypothesis. One commonly accepted approach for identifying and localizing stem cells in the niche is through the label-retaining cell (LRC) technique, which normally depends on administration of a marker that is incorporated into the DNA of proliferating cells and retained by stem cells for long periods of time after the labeling period because of the slow-cycling, quiescent nature of stem cells. To maximize the temporal-spatial resolution of the LRCs, we used a dual-pulse CldU and IdU labeling protocol at diffeWe also took advantage of the high spatial-temporal resolution afforded by Rainbow reporter mice to provide a way for in vivo clonal analyses of stem cells that contribute to HO. The R26R-Confetti allele in these mice acts as a stochastic multicolor Cre recombinase reporter of multiple fluorescent proteins from a single genomic locus . PreviouNumerous prior studies have implicated various signaling pathways, such as bone morphogenetic protein (BMP) BMP, wnt/\u03b2-catenin, fibroblast growth factor (FGF), hedgehog (Hh) signaling, and probably other conserved pathways that participate in the HO process . The BMPOverall, this proof-of-concept study provides evidence of a potential core mechanism in which multiple disease-specific cellular and extracellular molecular elements form a unique local microenvironment, i.e., a MSC niche, where BMP and Hh signaling co-regulate this process. The insights gained from this study have broad implications not only for prevention and treatment of HO, but also for potential translational applications of MSCs in a variety of injury or regenerative-related disorders.tm3(cre/ERT2)Alj/J (Gli1-creERT) \u00a0model,\u00a0\u00a0 are warranted. (2) Although the LRC approach is widely used to label and identify potential stem cell populations in many different tissues, it has a series of limitations [There are also several caveats regarding our findings: (1) Since the central hypothesis of this study aims to provide a potential common core mechanism for HO, testing this concept in multiple different models will be necessary to validate the extent to which it is generalizable. Even though we have tested this idea in injury-induced, BMP-dependent animal model (Nse-BMP4) and in human aHO and FOP samples, more detailed studies in different models, such as ACVR1itations . (3) Dueitations , have noAvailable data supported the central hypothesis, i.e., the formation of a relatively stable injury-induced local microenvironment (niche) might be a crucial core process, and this niche regulates the proliferation and osteogenic differentiation of mesenchymal stem cells (MSCs). The important implication\u00a0 for HO treatment is that therapeutic approaches must consider several different disease-specific factors as parts of the functional unit, instead of treating one factor at a time. The insights gained from this study have broad implications not only for the prevention and treatment of HO, but also for the potential translational applications of MSCs in a variety of injury or regenerative-related disorders.Additional file 1:Table S1. Summary of clinical data of HO patients. (DOCX 15 kb)Additional file 2:Table S2. Summary of the histomorphometric analysis of 8 aHO samples from 8 patients. (DOCX 75 kb)Additional file 3:Figure S1. Representative X-ray/CT images of 8 patients with aHO. The number in each panel is the patient\u2019s coded serial number after deidentification of the images. (JPG 603 kb)Additional file 4:Figure S2. Representative H&E images of 8 patients with aHO. The number in each panel is the patient\u2019s coded serial number after deidentification of the tissues. (JPG 2131 kb)Additional file 5:Figure S3. Validation of the dual-pulse CldU and IdU labeling procedure. To validate this procedure, we used two positive controls (A&B), and two negative controls (C&D). As expected, we observed plentiful specific staining, including CldU+/IdU\u2212 (red arrows), CldU+/IdU+ (yellow arrows) and CldU\u2212/IdU+ (green arrows) cells in intestinal mucosa (A) & hair follicles (B) but not in muscle (C&D). E) Experimental paradigm for dual-pulse labeling procedure. A-D are on the same scale, Bar\u2009=\u200950\u2009\u03bcm. (TIF 9688 kb)Additional file 6:Figure S4. LRC cells co-labeled with typical MSC markers. In the early stages of the lesion (A&C), and proposed niche (B&D), many CldU+/IdU\u2212 (quiescent stem cells) co-labeled with Stro1(A&B) and S100A4 (C&D). A-D are on the same scale, Bar\u2009=\u200950\u2009\u03bcm. (TIF 9632 kb)Additional file 7:Figure S5. The distribution of Cre-labeled cells outside of the target regions. A&B) the distribution of Gli1-creERT-labeled cells in Nse-BMP4;Gli1-creERT;R26R-Confetti mice outside of the target regions, i.e., A) in normal skeletal bone (growth plate of femur), and B) in differentiated core of chondrocyte of HO, away from the newly formed zonal region. C&D) the distribution of Glast-creERT labeled cells in Nse-BMP4;Glast-creERT;R26R-Confetti mice outside of the target regions, i.e., C) in the cerebellum, consistent with the known expression pattern in Bergmann glia, and D) in the skeletal muscle interstitium. E&F) the distribution of Tie2-cre labeled cells in Nse-BMP4;Tie2;R26R-Confetti and Nse-BMP4;Tie2-cre;Zsgreen mice outside of the target regions, i.e., E) The pattern of labeled cells in the adult brain of Nse-BMP4;Tie2-cre;Zsgreen, consistent with the known vascular expression pattern. F) The pattern of labeled cells in the early lesion of Nse-BMP4;Tie2-cre;R26R-Confetti. Note that the morphology of some labeled cells is consistent with the known vascular pattern. A-F are on the same scale, Bar\u2009=\u200950\u2009\u03bcm. (TIF 11999 kb)Additional file 8:Table S3. Summary of the histomorphometric analysis of Nse-BMP4;Glast-creERT;ROSA26-eGFP-DTA mice with or without TAM treatment. (DOCX 72 kb)Additional file 9:Figure S6. Conditional depletion of Glast-creERT+ cells resulted in less severe yet \u201ctypical\u201d HO. A&B) Gross image of HO harvested from TAM treated (A) and control (B) Nse-BMP4;Glast-creERT;ROSA26-eGFP-DTA mice after injury. Note that the gross morphology of HO in both groups was similar but the HO in the TAM treated group was smaller. Also note that a significant portion of harvest HO was not mature (without red bone marrow), which argued that quantification the immature HO with micro-CT could be misleading. C-H) Typical H&E images from treated and control groups both demonstrate typical features of fibro-proliferative (C&D), chondrocyte (E&F) and mature HO (G&H), though subtle differences do exist between the two groups. C-H are on the same scale, Bar\u2009=\u200950\u2009\u03bcm. (TIF 18128 kb)Additional file 10:Figure S7. Gli1-creERT-mediated DTA expression inhibited injury-induced HO. A&B) Typical x-ray images of control (A) and TAM treated (B) Nse-BMP4;Gli-creERT;ROSA26-eGFP-DTA mice after injury. C) HO incidence in control and TAM treated group. D) Quantification of wet weight of HO in the control and TAM treated groups. Note that depletion of Gli1-creERT-labeled cells partially inhibited but did not completely block HO. E) Typical fluorescence images from TAM treated (E) and control (F) Nse-BMP4;Gli1-creERT;ROSA26-eGFP-DTA mice. Note that in the TAM treated group (E), GFP- (recombined) cells were rarely found. G&H) H&E staining of sections from TAM treated (G) and control (H) Nse-BMP4;Gli1-creERT;ROSA26-eGFP-DTA mice. Note that both fluorescence images and H&E staining suggest that the proposed MSC domain (within dashed lines) was thinner in the TAM treated group. E-H are on the same scale, Bar\u2009=\u200950\u2009\u03bcm. (TIF 15685 kb)Additional file 11:Figure S8. Evidence of depletion of Gli1 in the target cells. The depletion of Gli1 in the target cells was confirmed by staining the tissue sections of Nse-BMP4;Gli1-creERT\u2212/\u2212;Zsgreen mice , and the tissues of Nse-BMP4;Gli1-creERT+/\u2212;Zsgreen mice with Gli1 antibody. Note that there is no specific staining of Gli1 (red) in the lesional tissues from Nse-BMP4;Gli1-creERT\u2212/\u2212;Zsgreen mice, while the specific staining of Gli1 (red) was observed in the Zsgreen+ cells in the proposed MSC niche in lesional tissues from Nse-BMP4;Gli1-creERT+/\u2212;Zsgreen mice. (JPG 822 kb)Additional file 12:Figure S9. Characterization of candidate niche supportive ECM molecules. A) Col4 was mainly involved in forming microtubular structures in the proposed MSC niche. B) Col6 was more ubiquitously upregulated in the proposed MSC niche. C) Interestingly, Col6 was closely associated with Tenascin C (TEN) only in early lesions. D) TEN was diffusedly upregulated in the early stages, but in the later stages, TEN was enriched mostly in mature domains. E&F) Similar to TEN, Chondroitin sulfate proteoglycan (CSPG) was also more or less evenly upregulated in early lesions but it became more defined as the proposed MSC niche formed. In contrast, laminin (LAM) was mainly involved in forming micro-tubular structures in the proposed MSC niche. A-F are on the same scale, Bar\u2009=\u200950\u2009\u03bcm. (TIF 9818 kb)Additional file 13:Figure S10. Working model of injury-induced MSC niche. Injury-induced local microenvironment (MSC niche) is composed of niche-dwelling progenitor/stem cells and niche supportive cells . The formation of the MSC niche likely initiates the pathological osteogenic cascade, under the co-regulation of BMP and Hh signaling through feedback and non-cell autonomous mechanisms. (TIF 1825 kb)"} +{"text": "OBJECTIVES/SPECIFIC AIMS: We hypothesized that CXCL12, as a biased dimer variant or secreted at dimer-dominant concentrations, would influence PDAC growth and progression. METHODS/STUDY POPULATION: PDAC cells were genetically manipulated to express dimer-promoting levels of CXCL12. These cells were studied in vitro or orthotopically implanted into the mouse pancreas for in vivo studies. As a second approach, recombinant wild-type or engineered CXCL12 monomer or dimer proteins were applied to cells in culture or administered intra-peritoneal to study the effects on tumor growth. RESULTS/ANTICIPATED RESULTS: Mice engrafted with CXCL12-expressing cells had a better survival rate, delayed tumor growth and smaller tumors. Tumors from these mice had significantly less proliferation, measured by Ki-67 staining. In vitro analysis of CXCL12-expressing cells showed decreased viability and growth rates. Percent of cells in the cell cycle G2 phase was also decreased, suggesting cell cycle progression blockade. Viability of human PDAC cells dose-dependently declined upon wild-type CXCL12 treatment, with the non-motile dimer-dominant dose (1000 nM) exhibiting maximal effect. Treatment in an allogeneic mouse model of PDAC with locked-dimer CXCL12, but not wild-type, reduced tumor burden. DISCUSSION/SIGNIFICANCE OF IMPACT: Our results support the notion that biased CXCL12 signaling may be therapeutically exploited to limit pancreatic cancer progression."} +{"text": "OBJECTIVES/SPECIFIC AIMS: MicroRNAs (miRNA) affect transcription of a number of genes involved in the development and progression of diabetic nephropathy (DN), and have become attractive therapeutic targets and biomarkers. Elevated renal gluconeogenesis, fibrosis, and albuminuria are early markers of incipient DN. Recent studies report that renal miRNA-451 may protect against DN and reduce renal gluconeogenesis in rodent models. MiRNA-451 is thought to act by targeting select factors resulting from disrupted insulin and growth factor signaling and the mechanistic-target of rapamycin (mTOR) in early DN. This study aimed to elucidate the role of miRNA-451 in the development and progression of DN. METHODS/STUDY POPULATION: To further elucidate the role of miRNA-451 in DN, we placed male insulin-resistant, TALLYHO/Jng mice on a high-fat diet . The mice were divided into 2 treatment groups and received 8 consecutive weekly intraperitoneal injections of locked nucleic acid (LNA) miR-451-inhibitor or LNA-scrambled compound . Mice were euthanized after 12 weeks (4 weeks sans injections) and kidneys, liver, pancreas and abdominal adipose tissue were harvested for analysis. RESULTS/ANTICIPATED RESULTS: Renal homogenate expression of miRNA-451 was drastically reduced in inhibitor-treated mice (~6-fold) in comparison with scramble-treated mice. Western blotting of cortex homogenates for indicators of fibrosis and targets of miRNA-451 revealed a significant reduction in collagen IV in inhibitor-treated mice. In addition, metalloproteinase type 9 , YWHAZ (a scaffolding protein and known target of miR-451), mTOR, and fructose bisphosphatase were significantly increased in this group in comparison to scramble-treated mice. However, no differences were found in protein levels for glucose-6-phosphatase (G-6-Pase) or phosphoenolpyruvate (PEPCK), 2 additional gluconeogenic rate-limiting genes. MiRNA-451 antagonist did not significantly affect final body weight or blood glucose; however, mean blood sodium concentrations were slightly, but significantly higher (2%) in the LNA-inhibitor treated group (when compared with the scramble-treated group). No differences in blood potassium or chloride were found. Anion gap was 90% higher in the LNA-inhibitor treated group when compared with scramble-treated mice. No differences in urinary albumin to creatinine ratio were found between the two treatment groups. However, Masson Trichrome scoring revealed a 59% increase in fibrosis in inhibitor-treated mice. DISCUSSION/SIGNIFICANCE OF IMPACT: Collectively, these findings support a potentially protective role of miRNA-451 in attenuating signaling via mTOR that may alter both renal gluconeogenic potential (contributing to the diabetic phenotype) and activation and progression of renal fibrosis. Therapies to enhance miRNA-451 signaling may be beneficial to reduce renal pathology associated with DN."} +{"text": "Siglecg deficiency attenuated TLR4-triggered pro-inflammatory cytokine production and increased anti-inflammatory cytokine [interleukin-10 [IL-10]] production in vivo and in vitro at both acute and immunosuppressive phases. Siglecg deficiency also protected mice from lipopolysaccharide (LPS)-induced sepsis with less inflammation in the lung and less tissue destruction in the spleen. Siglec-G inhibited proto-oncogene tyrosine-protein kinase Src (Src) activation via recruiting and activating tyrosine phosphatase Src homology region 2 domain-containing phosphatase-1 (SHP1) through immunoreceptor tyrosine-based inhibitory motif (ITIM) domain. Src could inhibit TLR4-induced inflammatory cytokines and promote anti-inflammatory cytokine IL-10. Mechanical investigation showed that Src could interact with and phosphorylate STAT3. Src could also promote HIF1\u03b1 degradation through activating GSK3\u03b2. Our study reveals that Siglec-G orchestrates TLR-induced inflammation, which outlines that blocking Siglec-G or activating Src may be a promising strategy for both acute and chronic inflammatory diseases.Hyper-inflammation during acute phase and sequential hypo-inflammation during immunosuppressive phase in macrophages/monocytes lead to multiorgan failure syndrome and immune collapse of sepsis, in which toll-like receptor (TLR)-triggered inflammatory responses play a major role. Here, we reported that Sepsis is a systemic inflammatory syndrome induced by infection, caused by the lack of normal immune homeostatic functions and excessive production of pro-inflammatory cytokines, which leads to multiorgan failure and immune collapse \u20137. Toll-Siglecs are divided into two groups on the basis of their molecular structure. The first group includes Siglec-1, Siglec-2, Siglec-4, and Siglec-15, which are structurally conserved between rodents, humans, and other vertebrates \u201319. The Siglecg deficiency protected mice from over-activation of acute inflammatory responses and death in TLR-triggered sepsis by attenuating TLR-triggered pro-inflammatory cytokine production and increasing anti-inflammatory cytokine IL-10 production. We further demonstrated that Siglec-G decreased Src activation through SHP1. Our results showed that Siglec-G-induced Src signaling could be a promising drug target to regulate immune homeostasis of pro-inflammation and anti-inflammation.In this study, we observed that Siglecg-deficient (Siglecg\u2212/\u2212) and control (Siglecg+/\u2212) mice with a non-lethal dose of LPS for indicated time, a sepsis model we previously reported (Siglecg\u2212/\u2212 mice produced less interleukin-6 (IL-6) and tumor necrosis factor-\u03b1 (TNF-\u03b1) than did Siglecg+/\u2212 mice productions, whereas it increased the anti-inflammatory cytokine IL-10 production compared with that in the control mice. We observed more severe infiltration of inflammatory cells in the lungs of Siglecg+/\u2212 mice than that in the lungs of Siglecg\u2212/\u2212 mice at both acute and immunosuppressive phases stimulation . To invereported . In resp+/\u2212 mice . Howevere phases . Accordisurvived , which iin vivo. To further confirm the function of Siglec-G in vitro, LPS-induced cytokine production in mouse peritoneal macrophages was tested using ELISA. In response to LPS administration, Siglecg\u2212/\u2212 peritoneal macrophages produced less pro-inflammatory cytokines (IL-6 and TNF-\u03b1) but more IL-10 than did Siglecg+/\u2212 (iglecg+/+ (Siglecg\u2212/\u2212) macrophages was increased in Siglecg\u2212/\u2212 macrophages than in Siglecg+/\u2212 macrophages production , which is more suitable to illustrate the function of Siglec-G. Siglecs are also endocytic receptors that constitutively cycle between the cell surface and intracellular endosome ligand-bearing cargo into the cell, in which the molecular basis for sialic acid specificity remains to be revealed . Siglecg+/\u2212 and Siglecg\u2212/\u2212 mice were derived from the Siglecg+/\u2212 heterozygote littermates. All animal experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, with the approval of the Scientific Investigation Board of Second Military Medical University, Shanghai.Mice homozygous for Siglec-G-deficient mice on a C57BL/6J background were generated as described previously and bredLPS O111:B4) used was described previously 1:B4 used. PP1 wasThe recombinant vectors encoding Src (NM_009271), SHP1 NM_011202.3), HIF1\u03b1 (NM_001313919), STAT3 (NM_213659), and Siglec-G (NM_172900.3) were constructed by PCR-based amplification from RAW264.7 cDNA and then subcloned into the pcDNA3.1 eukaryotic expression vector as described previously 202.3, HI. The CA-RAW 264.7 and HEK293T cell lines were obtained from the American Type Culture Collection. Six-week-old mice were injected with 3% (w/v) Merck thioglycollate medium into the peritoneal cavity of each mouse. Three days after the injection, the peritoneal macrophages were collected by flushing the peritoneal cavity with Roswell Park Memorial Institute (RPMI) 1640 and cultured for 1 h and washed with DMEM to clear the non-adherent cells; then the macrophages were further cultured in endotoxin-free DMEM with 10% fetal bovine serum (FBS) (Invitrogen) as previously reported , 25. OveQuantitative real-time RT-PCR analysis was performed by LightCycler and SYBR RT-PCR kit as described previously , 25. DatCells were lysed with radioimmunoprecipitation assay (RIPA) buffer or M-PER Protein Extraction Reagent supplemented with protease inhibitor cocktail. Protein concentrations of the extracts were measured with bicinchoninic acid (BCA) assay (Pierce). The immunoprecipitation and immunoblot assays were performed as described previously , 25.t test. Statistical significance was determined as P-values of <0.05 or 0.01.Results are given as means plus or minus standard deviation (SD). Comparisons between two groups were performed using Student's Publicly available datasets were analyzed in this study. This data can be found here: GSE39620. Other raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.The animal study was reviewed and approved by Scientific Investigation Board of Second Military Medical University, Shanghai.YLu, HY, and CH designed and supervised the study. WL, YLi, KQ, BD, and TL performed the experiments. WL, HY, CH, and YLu analyzed the data and wrote the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Induced pluripotency in cancer cells by ectopic expression of pluripotency-regulating factors may be used for disease modeling of cancers. MicroRNAs (miRNAs) are negative regulators of gene expression that play important role in reprogramming somatic cells. However, studies on the miRNA expression profile and the expression patterns of the mesenchymal-epithelial transition (MET)/epithelial-mesenchymal transition (EMT) genes in induced pluripotent cancer (iPC) cells are lacking.iPC clones were generated from two colorectal cancer (CRC) cell lines by retroviral transduction of the Yamanaka factors. The iPC clones obtained were characterized by morphology, expression of pluripotency markers and the ability to undergo in vitro tri-lineage differentiation. Genome-wide miRNA profiles of the iPC cells were obtained by microarray analysis and bioinformatics interrogation. Gene expression was done by real-time RT-PCR and immuno-staining; MET/EMT protein levels were determined by western blot analysis.The CRC-iPC cells showed embryonic stem cell-like features and tri-lineage differentiation abilities. The spontaneously-differentiated post-iPC cells obtained were highly similar to the parental CRC cells. However, down-regulated pluripotency gene expression and failure to form teratoma indicated that the CRC-iPC cells had only attained partial pluripotency. The CRC-iPC cells shared similarities in the genome-wide miRNA expression profiles of both cancer and pluripotent embryonic stem cells. One hundred and two differentially-expressed miRNAs were identified in the CRC-iPC cells, which were predicted by bioinformatics analysis be closely involved in regulating cellular pluripotency and the expression of the MET/EMT genes, possibly via the phosphatidylinositol-3 kinases-protein kinase B (PI3K-Akt) and transforming growth factor beta (TGF-\u03b2) signaling pathways. Irregular and inconsistent expression patterns of the EMT vimentin and Snai1 and MET E-cadherin and occludin proteins were observed in the four CRC-iPC clones analyzed, which suggested an epithelial/mesenchymal hybrid phenotype in the partially reprogrammed CRC cells. MET/EMT gene expression was also generally reversed on re-differentiation, also suggesting epigenetic regulation.Our data support the elite model for cancer cell-reprogramming in which only a selected subset of cancer may be fully reprogrammed; partial cancer cell reprogramming may also elicit an epithelial-mesenchymal mixed phenotype, and highlight opportunities and challenges in cancer cell-reprogramming.The online version of this article (10.1186/s12929-018-0461-1) contains supplementary material, which is available to authorized users. Induced pluripotent cancer (iPC) cells are iPSCs of cancer origin, which also exhibit the distinctive iPSC characteristics of self-renewal and trilineage differentiation . Wh. Wh. Theion Fig.\u00a0. The obsOSKM transduction, it was important to distinguish between ectopic and endogenous OSKM expression. For this purpose, RT-PCR was conducted using primers specific for the transgenes using total RNA isolated from iPC cells at passage 25 or later. In each case, two independent iHCT-15 clones C1 and C5, and iSK-CO-1 clones C1 and C2 were analyzed. The results showed that expression of the transgenes was extinguished with one notable exception , implying that the maintenance of pluripotency and self-renewal properties of the reprogrammed colorectal cancer cells may not be entirely dependent on the expression levels of the pluripotency genes.Endogenous expression levels of the pluripotency genes were quantified by real-time RT-PCR Fig. . Post-iPgramming , 21. SucPrevious reports have shown that reprogramming alters the miRNA expression profile in induced pluripotent stem cells (iPSCs) derived from both somatic and cancer cells , 24. In c-MYC and Lin28 [Hierarchical clustering analysis of the miRNA profiles showed that the differentially-expressed miRNAs could be grouped into three major clusters based on their expression patterns (FC)\u2009\u2265\u20092.0 or\u2009\u2264\u2009\u2212\u20092.0 and p-value <\u20090.05, a total of 102 statistically significant differentially-expressed miRNAs were thus identified, fifty of which were down-regulated and fifty-two miRNAs were up-regulated pathways were mapped, which showed that six of the top 10 KEGG pathways were related to cellular signaling and Snail1 (SNAI1) and two typical MET genes, E-cadherin (CDH1) and occludin (OCLN) Table\u00a0. For valsis Fig. . The presis Fig. .Table 2DSNAI1, and both the MET CDH1 and OCLN genes hybrid phenotype , 43. TheSOX2 and c-MYC have previously proposed a pluripotency hierarchy model in which the cancer cell-derived iPCs are thought to be induced to a pre-iPSC state, just a level above the multipotent mesenchymal stem cells . The preIn this work, Yamanaka factor-reprogrammed colorectal cancer-induced pluripotent cancer cells show ESC-like features and trilineage differentiation. Down-regulated expression of pluripotency genes and dissimilar miRNA expression profiles to that of pluripotent embryonic stem cells indicate incomplete reprogramming. Reprogramming activates miRNAs that target the TGF-\u03b2 signaling pathway to modulate expression of EMT/MET genes; however, dysregulated expression of EMT and MET proteins suggests an epithelial-mesenchymal hybrid phenotype, consistent with partial CRC reprogramming. Our data further highlight challenges in obtaining fully reprogrammed cancer cells likely due to the accumulated mutations and epigenetic modifications in the cancer cells. Nonetheless, the reprogrammed CRC-iPC cells offer an opportunity for the development of disease models to further elucidate the molecular mechanism in the tumorigenesis of colorectal cancer.Additional file 1:Table S1. Primer sequences of genes and miRNAs analyzed in RT-PCR or qRT-PCR. (PDF 99 kb)Additional file 2:Table S2. Regulated expression of miRNAs known to modulate cellular reprogramming. (PDF 38 kb)Additional file 3:Table S3. Full list of 102 differentially expressed miRNAs in 4 iPCs vs 2 CRCs. (PDF 220 kb)"} +{"text": "Newcastle disease virus (NDV) was detected by reverse transcriptase PCR (RT-PCR) from total RNA isolated from a chicken spleen of a backyard flock in Jordan. The complete coding genome sequence of NDV/chicken/Jordan/J11-spleen/2018 was obtained with MiSeq (Illumina) sequencing. Newcastle disease virus (NDV) was detected by reverse transcriptase PCR (RT-PCR) from total RNA isolated from a chicken spleen of a backyard flock in Jordan. The complete coding genome sequence of NDV/chicken/Jordan/J11-spleen/2018 was obtained with MiSeq (Illumina) sequencing. Phylogenetic analysis of the concatenated coding sequences classified the virus as class II subgenotype VIIi. Paramyxoviridae, genus Avulavirus (Newcastle disease virus (NDV), family ulavirus , causes ulavirus . A 4-monKY076035) (RNA extraction from fresh spleen tissue was performed with TRIzol (Ambion-Thermo Fisher Scientific) followed by purification with a Direct-zol kit (Zymo Research). RNA was quantified with spectrophotometry and Qubit (Invitrogen) fluorimetry. RNA was reverse transcribed, and DNA libraries for next-generation sequencing (NGS) were prepared using the KAPA stranded RNA-Seq library preparation kit according to the manufacturer\u2019s instructions. The size distribution and concentration of DNA in the prepared libraries were checked on a Bioanalyzer 2100 and a Qubit instrument using a high-sensitivity (HS) DNA kit and a Qubit double-stranded DNA (dsDNA) HS assay kit , respectively . Paired-Y076035) . This isMH614933.The complete genome sequence of Newcastle disease virus genotype VIIi has been deposited in GenBank under accession number"} +{"text": "Conformationally defective cystic fibrosis transmembrane conductance regulator (CFTR) including rescued \u0394F508-CFTR is rapidly eliminated from the plasma membrane (PM) even in the presence of a CFTR corrector and potentiator, limiting the therapeutic effort of the combination therapy. CFTR elimination from the PM is determined by the conformation-dependent ubiquitination as a part of the peripheral quality control (PQC) mechanism. Recently, the molecular machineries responsible for the CFTR PQC mechanism which includes molecular chaperones and ubiquitination enzymes have been revealed. This review summarizes the molecular mechanism of the CFTR PQC and discusses the possibility that the peripheral ubiquitination mechanism becomes a novel drug target to develop the CFTR stabilizer as a novel class of CFTR modulator. Figure 1).Cystic fibrosis (CF) is one of the most lethal autosomal-recessive diseases caused by mutation in CFTR . CFTR muN-glycosylated at the endoplasmic reticulum (ER) during translation and folded by the aid of chaperones such as calnexin (CNX), HSP70 and HSP90 (Nascent wild-type (WT) CFTR is nd HSP90 . Properlnd HSP90 . Properlnd HSP90 .N-glycosylation, especially the core-glycosylation, determines the CFTR PM stability likely by affecting the CFTR conformational stability and recycled back to the PM. Conformationally defective CFTR produced by genetic mutations and/or environmental stresses selectively undergoes ubiquitination at the PM by PQC machineries. The ubiquitinated CFTR is rapidly internalized and delivered to lysosome for degradation . Internatability . Proteintability . RPL12 Ktability and thertability . Thus, cPseudomonas aeruginosa (PA) is one of the most common bacteria found in CF respiratory tissue and responsible for lung injury in CF (The CFTR loss of function induces airway surface liquid (ASL) dysregulation which impairs clearance of infected bacteria and/or fungi, and increases the concentration of other soluble signal mediators such as cytokines, chemokines and growth factors. ry in CF . PA destry in CF . PA secrry in CF . PA alsory in CF . TGF-\u03b21 ry in CF althoughMore than 10 ppb of arsenic induces the WT-CFTR ubiquitination and lysosomal degradation via c-Cbl in CF bronchial epithelial (CFBE) cells . Importa2+ dependently. This observation suggesting that CS induces PM CFTR destabilization by stimulating internalization and aggregation in addition to suppressing CFTR functionality cells . CS promionality .Endocytosis is the critical step of elimination of PM CFTR as a part of PQC and is regulated by several molecules. WT-CFTR is internalized slowly by CME while misfolded r\u0394F508-CFTR endocytosis is accelerated . KD of C+/H+ exchanger regulatory factor (NHERF1) PDZ domain. NHERF1 tethers CFTR with Ezrin and works as a scaffold protein that supports CFTR efficient channel activation and apical PM localization binding motif at C-terminus and binds with Nalization . NHERF1 lization . An exchlization . EPAC1 alization .The CFTR-associated ligand (CAL) negatively regulates \u0394F508-CFTR PM abundance through its PDZ domain . CAL inhFilamin-A (FLN-A) is a membrane tethered actin adaptor protein and interacts with CFTR N-terminus region. S13F mutation of CFTR compromises FLN-A binding and consequently destabilizes the PM CFTR . FLN-A bThe CFTR PM stability is regulated by phosphorylation. CFTR is predominantly phosphorylated at the R domain and also at nucleotide binding domain 1 (NBD1) and C-terminus residues by protein kinase A (PKA), protein kinase C (PKC), casein kinase II (CK2) and AMP-activated protein kinase (AMPK) for the channel function . CK2 is Molecular chaperones selectively interact with and stabilize unfolded or partially folded protein to acquire a functionally active conformation. Nascent CFTR interacts with a panel of chaperones and co-chaperones including HSC70, HSP70, HSP90, and CNX at the ER . Even atUbiquitination determines CFTR elimination not only at the ER, but also from the PM. Ubiquitination is mediated by a sequential action of E1, E2, and E3 enzymes and this modification could be removed by DUB. Specifically, E3 Ub ligase has been proposed to determine the substrate specificity. CHIP is the first identified E3 ligase responsible for the CFTR PQC . ConsistE3 ligase c-Cbl may play a role in the CFTR peripheral QC, but its contribution could be modest since its KD slightly increases r\u0394F508-CFTR PM stability in CFBE cells . c-Cbl a+ Channel (ENaC) (Nedd4-2 is a member of homologous to the E6-AP carboxyl terminus (HECT) E3 which may regulate the CFTR PM expression. Nedd4-2 KD reduces \u0394F508-CFTR ubiquitination at the ER, and increases the PM expression and function in CF pancreatic adenocarcinoma cell 1 (CFPAC1) and IB3-1 cells . Nedd4-2l (ENaC) .A number of DUBs regulate the CFTR turnover. USP10, a DUB localized at early endosomes, interacts with WT-CFTR and reduces the CFTR poly-ubiquination in CFBE cells. The USP10-mediated deubiquitination enhances the endocytic recycling of WT-CFTR . The rolRecently, we have discovered RING finger and FYVE like domain containing E3 Ub protein ligase (RFFL) as a novel component of the CFTR PQC machineries by a comprehensive siRNA screen in CFBE cells . RFFL sePharmacological chaperones affect the CFTR PM stability by direct stabilization. CFTR corrector VX-809 is the first food and drug administration (FDA) approved CFTR corrector in combination with VX-770/ivacaftor (known as Orkambi). VX-809 selectively improves the processing of misfolded CFTR by stabilizing NBD1-membrane spanning domain (MSD) interface but not other misfolded proteins such as human ether-\u00e0-go-go-related gene (hERG) mutants . VX-809 The first FDA approved CFTR potentiator VX-770 improves the gating defect of some CFTR mutants. However, chronic VX-770 treatment destabilizes the PM r\u0394F508-CFTR in CFBE and \u0394F508 homozygous CF patient HBE (CF-HBE) cells . ImportaProteostasis regulating drugs that affect array of proteins regulating CFTR folding and QC also affect the CFTR PM stability. Histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) alters expression of a subset of CF-interacting gene products and sustains PM expression of \u0394F508-CFTR in CFBE cells . Tissue S-nitrosylation by S-nitrosoglutathione (GSNO) induces HOP degradation and increases \u0394F508-CFTR PM expression is an ER associated E3 Ub ligase that regulates early stage CFTR proteostasis at the ER . A RNF5 1. In contrast, RFFL could bind and regulate a limited number of substrates because of its nature of direct binding to the CFTR through the disordered regions . The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "The devices obtained from 2a\u2013b exhibit competitive power conversion efficiencies (PCEs) and improved open-circuit voltage (ocV) values (>1.0 V) in comparison to a reference cell based on phenyl-C61-butyric-acid methyl-ester (PC61BM). These results are rationalized in terms of a) the higher passivation ability of the open-cage fullerenes with respect to the other fullerenes, and b) a good overlap between the highest occupied molecular orbital/lowest unoccupied molecular orbital (HOMO/LUMO) levels of 2a\u2013b and the conduction band of the perovskite.The synthesis, characterization, and incorporation of open-cage [60]fullerene derivatives as electron-transporting materials (ETMs) in perovskite solar cells (PSCs) with an inverted planar (p-i-n) structure is reported. Following optical and electrochemical characterization of the open-cage fullerenes Perovskite solar cells (PSCs) are an emerging class of photovoltaic devices, which promise to rival the performance of state-of-the-art cells, with current record power conversion efficiencies (PCEs) recently reaching 24.2% A major ocV) values are difficult to achieve. Successful strategies to overcome this limitation rely on the incorporation of dopant materials fullerene in one step using our Rh(I)-catalyzed cycloaddition protocol were determined by cyclic voltammetry (CV) in ortho-dichlorobenzene (o-DCB) values were estimated from the ultraviolet (UV) and CV measurements fullerene derivatives"} +{"text": "Human T-cell leukemia virus type-1 (HTLV-1) causes adult T-cell leukemia/lymphoma (ATL), HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), and other inflammatory diseases. There is no disease-specific difference in viral strains, and it is unclear how HTLV-1 causes such different diseases manifesting as lymphoproliferation or inflammation. Although some progress has been made in therapies for these diseases, the prognosis for ATL is still dismal and HAM/TSP remains an intractable disease. So far, two regulatory proteins of HTLV-1, Tax and HBZ, have been well studied and shown to have pleiotropic functions implicated in viral pathogenesis. Tax in particular can strongly activate NF\u03baB, which is constitutively activated in HTLV-1-infected cells and considered to contribute to both oncogenesis and inflammation. However, the expression level of Tax is very low in vivo, leading to confusion in understanding its role in viral pathogenesis. A series of studies using IL-2-dependent HTLV-1-infected cells indicated that IL-10, an anti-inflammatory/immune suppressive cytokine, could induce a proliferative phenotype in HTLV-1-infected cells. In addition, type I interferon (IFN) suppresses HTLV-1 expression in a reversible manner. These findings suggest involvement of host innate immunity in the switch between lymphoproliferative and inflammatory diseases as well as the regulation of HTLV-1 expression. Innate immune responses also affect another important host determinant, Tax-specific cytotoxic T lymphocytes (CTLs), which are impaired in ATL patients, while activated in HAM/TSP patients. Activation of Tax-specific CTLs in ATL patients after hematopoietic stem cell transplantation indicates Tax expression and its fluctuation in vivo. A recently developed anti-ATL therapeutic vaccine, consisting of Tax peptide-pulsed dendritic cells, induced Tax-specific CTL responses in ATL patients and exhibited favorable clinical outcomes, unless Tax-defective ATL clones emerged. These findings support the significance of Tax in HTLV-1 pathogenesis, at least in part, and encourage Tax-targeted immunotherapy in ATL. Host innate and acquired immune responses induce host microenvironments that modify HTLV-1-encoded pathogenesis and establish a complicated network for development of diseases in HTLV-1 infection. Both host and viral factors should be taken into consideration in development of therapeutic and prophylactic strategies in HTLV-1 infection. H. H20]. HSuch spontaneous induction of HTLV-1 expression in culture of primary PBMCs is observed in asymptomatic HTLV-1 carriers, HAM/TSP patients and about half of ATL patients. In the remaining ATL cases, the ATL cells do not express Tax even after culture because of genetic alterations and epigenetic silencing of HTLV-1 gene expression .The mechanism of the on/off switch of HTLV-1 Tax expression in vitro and in vivo is not well understood. Requirement of TORC family proteins in long terminal repeat (LTR)-driven Tax expression and decrNegative regulation of HTLV-1 expression may involve type-I IFN responses. Although HTLV-1-transformed cells such as MT-2 cells are known to be resistant to type I IFN , IFN\u03b1 suFurthermore, when IL-2-dependent HTLV-1-infected cells are co-cultured with stromal cells, viral expression becomes undetectable, yet recovers again when the infected cells are re-isolated and cultured alone , 88. An Constitutive NF\u03baB activation in Tax-negative ATL cells is associated with increased NF-\u03baB-inducing kinase (NIK) . DownregAnother candidate that may mediate activation of NF\u03baB and ISG could be pattern recognition receptors (PRRs) that recognize various pathogen-associated molecular patterns (PAMPs) and potentially mediate NF\u03baB and IRF3 downstream . In de nCombination therapy with azidothymidine and IFN\u03b1 (AZT/IFN\u03b1) has been used for ATL patients , 98 and HTLV-1-specific T-cell responses are observed in most asymptomatic HTLV-1 carriers and HAM/TSP patients, but impaired in ATL patients. Tax is the major target antigen for T-cells , 25. EnvDespite Tax expression being undetectable by serological methods in vivo, evidence of Tax antigen presentation in ATL patients was obtained in ATL patients following hematopoietic stem cell transplantation (HSCT). Because frequent relapse is one of the reasons for poor prognosis in ATL patients, HSCT following chemotherapy is recommended in Japan and has been shown to achieve long-term survival in one-third of recipients , 115. InAnti-tumor effects of CD8+ Tax-specific CTLs have been shown in an animal model, in which HTLV-1-infected lymphomas in nude rats were eradicated by adoptive transfer of syngeneic Tax-specific CTLs that had been induced by vaccines using Tax-coding DNA or Tax pThe first anti-ATL therapeutic vaccine was designed to activate CD8+ Tax-specific CTLs by using Tax peptides as antigen and autologous DCs as adjuvant Fig.\u00a0b 28, 11. The TaxIn the initial pilot study, three ATL patients who were in stable conditions after other therapies except for HSCT were subcutaneously injected with Tax peptide-pulsed autologous DCs three times at fortnightly intervals. All three patients showed clear proliferative responses of Tax-specific CTLs after vaccination without severe adverse effects . ClinicaThese favorable clinical outcomes of the Tax-DC vaccine indicate the significance of Tax-specific CTLs in maintenance of remission, although they may become ineffective when Tax-negative ATL clones emerge.In ATL patients, half of the cases retain the ability to express Tax in ATL cells, while the other half lose this ability . TherefoIn ATL patients whose ATL cells lack Tax expression, Tax-specific CTLs cannot directly attack ATL cells. However, these CTLs may still control subdominant HTLV-1-infected cell clones that retain the ability to express Tax, because there are multiple HTLV-1-infected cell clones and the dominant ATL clones sometimes differ among tissues and change during the disease course , 126.Since it takes several weeks to induce immune responses, the Tax-targeted vaccine cannot be the first line therapy for aggressive types of ATL. In the clinical studies, Tax-DC vaccines were administered in ATL patients as a maintenance therapy after chemotherapy. Indolent types of ATL such as smoldering and chronic ATL may be more responsive to the vaccine, as ATL cells likely retain the ability to express Tax more frequently compared with aggressive ATL. Although HAM/TSP patients may also retain the ability to express Tax, HAM/TSP patients usually have active Tax-specific CTLs and less likely benefit from additional vaccines.Once its safety and efficacy are confirmed, Tax-targeted vaccines might potentially be applied for prophylaxis of ATL development in the future. A small proportion of ACs exhibit insufficient Tax-specific CTL response and elevated proviral load , both ofThe complexity of disease mechanisms in HTLV-1 infection results from the host immune responses in concert with HTLV-1-encoded genes (Fig.\u00a0The extremely low but not silent Tax expression in vivo can be partly explained by type I IFN and ISG, which suppress viral expression mainly at a post-transcriptional level, presumably maintaining HTLV-1 expression at low equilibrium levels in various tissues in vivo. The presence of an IFN signature in HTLV-1-infected cells implies continuous innate immune stimulations going on in these cells, which might also contribute to pathogenesis in HTLV-1 infection.While Tax protein is undetectable by serological means in vivo, Tax-specific CTLs still seem to recognize HTLV-1-infected cells to some extent. The results of a clinical study of a Tax-targeted therapeutic vaccine in ATL patients indicated a greater impact of Tax-specific CTLs on immune surveillance of HTLV-1 infected cells than expected, again suggesting the presence of Tax expression in vivo. Although further investigation is required, this opens up a new door to early therapy and prophylaxis against ATL."} +{"text": "Correction to: Biol Sex Differhttps://doi.org/10.1186/s13293-019-0221-2bold font. The errors do not impact the conclusions of the paper. We, the authors, apologize for any inconvenience this may have caused.Morphometrical analysis by stereology, the sentenceUnder the heading \u201cThese parameters were examined in gonadally intact males (Sham-orchiectomized), orchiectomized males, proestrous females (Shan-ovarictomized), or ovariectomized females\u201d should read as follows:sham-orchiectomized), orchiectomized males, proestrous females (sham-ovarictomized), or ovariectomized females.\u201d\u201cThese parameters were examined in gonadally intact males , orchiectomized males (ORX), Sham-ovariectomized proestrous females (proestrous), and ovariectomized females (OVX) were analyzed by two-way ANOVA\u201d should read:sham-orchiectomized males (intact), orchiectomized males (ORX), sham-ovariectomized proestrous females (proestrous), and ovariectomized females (OVX) were analyzed by two-way ANOVA.\u201d\u201cGroups of"} +{"text": "Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is involved in the pathophysiology of atherosclerosis and acute coronary syndromes (ACS). Circulating soluble LOX-1 (sLOX-1) has been linked to the risk of coronary artery disease (CAD). Our aim was to test if baseline serum sLOX-1 was associated with major adverse cardiovascular events (MACE) in patients with stable CAD.This multicentre pilot study enrolled 833 stable CAD patients. All patients were followed for two years. Serum sLOX-1 concentrations were detected by enzyme-linked immunosorbent assay (ELISA). The association between sLOX-1 concentrations and MACE was assessed by logistic regression, Kaplan-Meier survival curves and Cox proportional hazards analyses. Logistic regression analysis was employed to assess the predictors of complex lesion.Multivariate logistic regression analysis revealed that sLOX-1 concentration was an independent predictor of MACE . Kaplan-Meier cumulative survival curves showed that the incidence of MACE in patients with a high sLOX-1 concentration was significantly higher than in patients with an intermediate or low sLOX-1 concentration (P < 0.001). Soluble LOX-1 concentrations were independently correlated with coronary complex lesions .Baseline sLOX-1 concentrations were correlated with 2-year MACE in stable CAD patients. Furthermore, patients with high serum sLOX-1 concentrations had higher cumulative incidence of MACE compared to those with low serum sLOX-1 concentrations. Coronary artery disease (CAD) remains the leading cause of mortality and disability worldwide is a class E scavenger receptor that primarily binds oxidized low-density lipoprotein (ox-LDL) were enrolled. These patients did not participate in any other studies.The flowchart of study protocol is shown in Traditional cardiovascular risk factors including hypertension, DM and smoking status were included in the statistical analyses to appreciate sLOX-1 uniqueness. Hypertension was defined as systolic blood pressure (SBP) \u2265 140 mmHg or diastolic blood pressure (DBP) \u2265 90 mmHg, or recently taking medication for hypertension. Diabetes mellitus was defined as fasting blood glucose (FBG) value \u2265 7.0 mmol/L or non-fasting blood glucose \u2265 11.1 mmol/L and/or any hypoglycaemic medication taken at admission. Smoking status was defined as more than one cigarette a day and lasting at least half a year.At study entry, a baseline survey including age, gender, body mass index (BMI), blood pressure, cardiovascular risk factors and cardiovascular medication was performed. Cardiac functional status was classified according to the New York Heart Association (NYHA) functional classification.After recruitment, all patients were followed up for two years. During the 2-year follow-up period, patients were given standard medications including anti-platelet agents, statins, angiotensinogen converting enzyme inhibitors (ACEI)/angiotensin receptor blockers (ARB) and beta blocker according to up-to-date guidelines. The primary end-point of this study was the composite of MACE, which were identified as all-cause death, nonfatal AMI and readmission for Braunwald\u2019s class IIIb UA requiring treatment. Hospital readmissions and deaths were identified by electronic patient records and through direct contact with the patients or relatives.All patients have provided written informed consent. The study protocol was approved by the institutional ethics committee.Dimensional echocardiogram of the patients was measured using GE Vivid 3 cardiovascular ultrasound equipment .per minute (rpm) for 15 minutes. The serum samples were stored at - 80 \u00b0C until analysis. Routine laboratory parameters including FBG, triglycerides (TG), total cholesterol (CHOL), low density lipoprotein cholesterol (LDL), high density lipoprotein cholesterol (HDL), creatinine (CREA), urea and uric acid (UA) were measured using standard laboratory techniques by automatic biochemical analyser . N-terminal pro-brain natriuretic peptide (NT-proBNP) concentrations were determined on Cobas 6000 analyser series . High-sensitivity C-reactive protein (hs-CRP) concentrations were determined on Immage 800 Immunochemistry System . Homocysteine (Hcys) concentrations were determined on Abbott i2000SR analyser . Circulating sLOX-1 concentrations was measured using an enzyme-linked immunosorbent assay (ELISA) kit with an intra-assay and inter-assay CV < 5% according to a published experiment (Venous blood samples were drawn in a fasting state under standardized conditions (blood samples were collected from basilic vein into plain biochemistry tubes). After clotting, blood samples were centrifuged at 3000 rounds All patients underwent CAG, which was performed in the catheterization room according to standard protocols. Angiograms were analysed by two experienced interventional cardiologists blinded to the study protocol. Coronary lesion morphology was grouped into simple or complex lesion according to the Ambrose classification or intravascular ultrasound (IVUS) are needed to further evaluate the relationship between sLOX-1 and vulnerable plaques.In conclusion, baseline sLOX-1 concentrations were correlated with 2-year MACE in stable CAD patients. If future studies prove the causality of the observation, sLOX-1 might emerge as a promising biomarker for risk stratification and a target for secondary prevention in these patients."} +{"text": "Klebsiella pneumoniae strain was isolated in Finland. CAZ-AVI resistance was observed 34 days after CAZ-AVI treatment in a trauma patient transferred from a hospital in Greece who had been colonised with blaKPC-2-producing K. pneumoniae ST39, and later developed a bloodstream infection. The CAZ-AVI-resistant strain contained a novel 15 amino acid insertion in the KPC-2 protein causing structural changes proximal to the KPC-2 active site.In December 2018, a ceftazidime-avibactam (CAZ-AVI)-resistant KPC-2-producing Klebsiella pneumoniae carbapenemases (KPC). According to the rapid risk assessment published by the European Centre for Disease Prevention and Control (ECDC) in June 2018, the emergence of CAZ-AVI resistance in Europe is a very rare phenomenon but an important cross-border threat that should be monitored carefully [K. pneumoniae strain and describe a mutation associated with the resistance.Ceftazidime-avibactam (CAZ-AVI) is a promising novel \u03b2-lactam-\u03b2-lactamase inhibitor combination with activity against multidrug-resistant (MDR) Enterobacteriaceae for which there are limited treatment options, e.g. those producing Following a traffic accident, a patient without any known underlying diseases was hospitalised in Greece. The patient was first admitted to a local hospital before being transferred to a tertiary hospital 3 days later; 27 days after admittance the patient was moved to a university hospital in Finland. There was no information on carriage of carbapenemase-producing Enterobacteriaceae (CPE) in the medical notes received from the Greek hospital. The screening specimens for MDR bacteria were obtained at admission to the Finnish hospital according to the national guidelines [K. pneumoniae sequence type (ST)39 (isolate 1), which was susceptible to CAZ-AVI and resistant to carbapenems and several other antimicrobials (K. pneumoniae ST39 (isolate 2), susceptible to CAZ-AVI and resistant to carbapenems. The treatment with tigecyclin and CAZ-AVI was administered for 2 weeks. After 2 days without antimicrobials, the patient developed fever again and treatment with CAZ-AVI and fosfomycin was initiated and continued for 19 days. Ten days after treatment ended, the patient developed fever once more. Blood cultures were found to be positive for KPC-2-producing K. pneumoniae ST39 (isolate 3), resistant to both CAZ-AVI and carbapenems but sensitive to sulfamethoxazole-trimethoprim. Colistin and sulfamethoxazole-trimethoprim were administered for 12 days. The patient subsequently recovered from the infection.The screening specimen from sacrum decubitus was positive for KPC-2-producing crobials . The patK. pneumoniae ST39 isolates were characterised by whole genome sequencing method and subsequent data by core-genome multilocus sequence typing (cgMLST) as previously described [TEM-1, blaSHV-11 and blaKPC-2) and the third (isolate 3) had two such genes (blaSHV-11 and a blaKPC-2 variant). The third isolate had lower minimal inhibition concentrations (MIC) of meropenem and ertapenem than those of the first two isolates. The blaKPC-2 genes from the first two isolates were identical. The blaKPC-2 gene from the third isolate had an insertion of 45 nucleotide corresponding to 15 amino acid insertion after position 259 of the KPC-2 protein (GenBank accession number MK823188).All three KPC-2-producing escribed . The firK. pneumoniae with blaKPC gene isolated in Finland and the second isolated in Europe.After the ECDC published its rapid risk assessment of the emergence of CAZ-AVI resistance, the National Institute for Health and Welfare (THL) informed clinical microbiology laboratories in Finland and asked them to notify THL of any CAZ-AVI resistant isolates. To our knowledge, this is the first CAZ-AVI-resistant KPC-2-producing K. pneumoniae ST39 and later developed a blood stream infection. The strain isolated after CAZ-AVI treatment had a mutated blaKPC-2 gene encoding KPC-2 protein with 15 amino acid insertion; the observed mutation in blaKPC-2 gene has not been described previously. Comparative protein modelling [CAZ-AVI resistance was observed after 34 days of CAZ-AVI treatment in a patient who was colonised by blaodelling based anKPC genes have been associated with the development of CAZ-AVI resistance after CAZ-AVI treatment in a few occasions. The first report from the United States (US) identified K. pneumoniae ST258 isolates with three mutations in KPC-3 after 10\u201319 days of CAZ-AVI treatment [K. pneumoniae ST1519 strain from Italy where CAZ-AVI resistance arose after 17 days of CAZ-AVI treatment [K. pneumoniae ST258 isolate after 12 days of CAZ-AVI treatment [We hypothesise that the resulting structural change weakens avibactam's inhibitory effect by disrupting its ability to bind at the active site, thereby causing resistance. Mutations in blareatment .K. pneumonia with CAZ-AVI can select KPC-variants that cause CAZ-AVI resistance and simultaneously preserve carbapenem resistance. Therefore, careful monitoring of resistance development by bacterial cultures and subsequent susceptibility testing is important.In conclusion, treating infections caused by KPC-producing"} +{"text": "Chimeric antigen receptor T (CAR-T) cell therapy is an emerging and effective cancer immunotherapy. Especially in hematological malignancies, CAR-T cells have achieved exciting results. Two Anti-CD19 CAR-T therapies have been approved for the treatment of CD19-positive leukemia or lymphoma. However, the application of CAR-T cells is obviously hampered by the adverse effects, such as cytokines release syndrome and on-target off-tumor toxicity. In some clinical trials, patients quitted the treatment of CAR-T cells due to life-threatening toxicity. Seeking to alleviate these toxicities or prevent the occurrence, researchers have developed a number of safety strategies of CAR-T cells, including suicide genes, synthetic Notch receptor, on-switch CAR, combinatorial target-antigen recognition, bispecific T cell engager and inhibitory CAR. This review summarized the preclinical studies and clinical trials of the safety strategies of CAR-T cells and their respective strengths and weaknesses. Many studies have proven that immunity plays an essential role in the development of cancers , 2. TherThe cytokine release syndrome (CRS) is the most common toxicity of CAR-T cells due to the excessive cytokine release . When CABecause there are few available tumor-specific antigens (TSAs), the targets that recognized by CAR-T cells are always tumor-associated antigens (TAAs), which are weakly expressed in normal tissues. Therefore, the on-target off-tumor toxicity is an unavoidable side effect. CAR-T cells have achieved promising results in hematological tumors. However, since the target antigens are also expressed on some normal blood cells, on-target off-tumor toxicity such as B-cell aplasia become a major obstacle for application of CAR-T cells in hematological tumors . In soliAllogeneic hematopoietic stem cell transplantation is an important treatment strategy for hematological malignancies. However, the success of allo-HSCT may be hindered by graft-versus-host disease (GVHD). Donor-derived CAR-T cells may increase the risk of GVHD occurrence. Ghosh et al. demonstrated that CAR-T cells with cumulative TCR and CAR signaling could reduce the risk of GVHD . Most anHerpes simplex virus thymidine kinase (HSV-tk) is the best characterized suicide gene and wide+ hematopoietic stem cells also express CD33, it is essential to completely eliminate the CD33 CAR-T cells prior to stem cell infusion to reduce the potential risk of engraftment failure. The conditional administration of CID resulted in the elimination of only 76.4% anti-CD33 CAR-T cells [+ hematopoietic stem cell [The inducible safety switch caspase 9 (iCasp9) suicide gene contains a modified human caspase 9 fused to the human FK506 binding protein (FKBP). Conditional administration of a chemical inducer of dimerization (CID) AP1903) forms dimerization and activates the downstream caspase molecules, resulting in apoptosis of cells expressing the fusion protein could be efficiently and specifically eliminated with clinically approved monoclonal antibody rituximab or cetuximab through the complement dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) , 69 Figc. In par+PSCA+ but not PSMA+PSCA\u2212 target cells. However, when T cells transduced efficient activation CAR (high affinity) and a CCR recirculate, they could kill the PSMA+PSCA\u2212 target cells without requiring further costimulation, resulting in an \u201con-target off-tumor\u201d adverse effect on normal tissues [+ tumor cells, and produced modest IL-2 in response to co-stimulation with a second antigen when compared to a control CAR-T cells expressing CD28 and CD3\u03b6 endodomain [Dividing the traditional CAR into two complementary parts may be a promising approach to enhance safety. T cells were transduced a CAR with CD3\u03b6 recognizing one antigen and a chimeric costimulatory receptor (CCR) with CD28 and/or 4-1BB binding another antigen receptors . Wild-tyImmune inhibitory receptors, such as PD-1, CTLA-4, play a crucial role in the regulation of immune response, especially in attenuating or terminating T cells response . It is aT cells were engineered to express a CAR that binds a fluorescein isothiocyanate (FITC) molecule, termed \u201cuniversal\u201d anti-FITC\u2013directed CAR-T cell. These CAR-T cells do not directly recognize antigen on target cells, but they are recruited to effector cells through a bispecific small molecule Fig. h. TherefOn-switch CAR consists of an extracellular specific antigen-binding domain (scFv) with costimulatory domains and a key downstream signaling element: the ITAMs from the T cell receptor CD3\u03b6 subunit Fig. 1ii1i. RecoIn addition to the above superior safety strategies, other methods may also reduce the side effects of CAR-T cells. Regional delivery or intratumoral injection of CAR-T cells could be considered as a measure of reducing \u201coff-tumor on-target\u201d toxicity and enhancing antitumor efficacy \u201398. A laAdoptive cellular therapy with the engineered CAR-T cells is a promising approach for cancer treatment. And encouraging results have been achieved in hematological malignancies. But the toxicity of CAR-T cells has been a challenge and impedes the further development. Nowadays, numerous safety measures to overcome these adverse effects of CAR-T cells have been investigated in preclinical and clinical trials. Traditional suicide genes can quickly and effectively mitigate toxicity, but they also irreversibly eliminate therapeutic CAR-T cells which are expensive labor-intensive. Endogenous switches including synNotch, iCAR and CCR can cause intracellular regulation in a self-switch manner when CAR-T cells recognize responding antigens, which significantly reduce on-target off-tumor effects. However, these methods cannot control the timing and intensity of CAR-T cells activity. Bispecific T cell engager and on-switch CAR could achieve it by exogenously administrating small molecules. Of course, the exogenous small molecules should be safe and bio-inert, as well as have good pharmacokinetic properties. Although these strategies have potential shortcomings and need improvement or optimized new methods, we believe that the next generation CAR-T cells with safety switch could exhibit superior safety and efficacy, as well as bring more hopes to patients with malignant tumors."} +{"text": "Single-cell RNA-seq technologies require library preparation prior to sequencing. Here, we present the first report to compare the cheaper BGISEQ-500 platform to the Illumina HiSeq platform for scRNA-seq. We generate a resource of 468 single cells and 1297 matched single cDNA samples, performing SMARTer and Smart-seq2 protocols on two cell lines with RNA spike-ins. We sequence these libraries on both platforms using single- and paired-end reads. The platforms have comparable sensitivity and accuracy in terms of quantification of gene expression, and low technical variability. Our study provides a standardized scRNA-seq resource to benchmark new scRNA-seq library preparation protocols and sequencing platforms.The online version of this article (10.1186/s13059-019-1676-5) contains supplementary material, which is available to authorized users. Single-cell RNA-seq (scRNA-seq) has become the established approach to dissect cellular heterogeneity, unravel cell states, and identify subpopulation structures across different cell types \u20134. The dThe most widely used Illumina platform uses a stepwise sequencing by polymerase approach. The libraries are made by fragmentation of bulk or single-cell cDNA, followed by the addition of custom adaptors. The template is flooded across a patterned flow cell to bind with immobilized primers and washed to remove unbound ends. The bound but free template ends further interact with nearby primers, forming bridge structures. The second strand is synthesized by PCR using the same primers, followed by washing and re-formation of bridges. This bridge amplification typically generates more than a million copies of each template within the tight physical cluster on the flow cell (reviewed in ). A simpThe BGISEQ-500 is an alternative short-read sequencing platform, developed by BGI (Beijing Genomics Institute). The BGISEQ-500 works use combinatorial probe-anchor synthesis (cPAS) that combines DNA-Nanoball (DNBs) arrays with stepwise sequencing using DNA polymerase on a flow cell on BGISEQ-500 is 40\u201360% of Illumina HiSeq4000 platform, without accounting for physical sequencer cost on single cells using two different protocols and across Illumina and BGISEQ-500 sequencing platform.We perform two different scRNA-seq methods (SMARTer and Smart-seq2) on mouse embryonic stem cells (mESCs) and human K562 cells , 13. We R) between estimated spike-in expression from sequencing and the a priori known input spike-in concentration (ground truth) in log space, for each individual cell for batch-matched mESCs and K562s, which is a large data resource for comparison of new protocols and benchmarking computational methods.We performed two scRNA-seq protocols (SMARTer and Smart-seq2) in parallel on 288 single-mESCs using both ERCCs and SIRVs spike-ins on Fluidigm C1-system , 13. TheWe used the same \u201cmatched\u201d single-cell cDNA from 288 cells, across SMARTer and Smart-seq2 protocols, and generated 576 single-cell libraries for both Illumina HiSeq2500 and BGISEQ-500 platforms Fig.\u00a0a [5]. HeFor each single cell, we converted the aligned reads to normalized transcript per million (TPM) units and re-computed sensitivity and accuracy. This allows us to compare the platforms at different sequencing depths and also estimate where saturation of sequencing occurs. Both sensitivity and accuracy were highly similar across single cells between scRNA-seq protocols and sequencing platforms, upon downsampling , with the sensitivity reaching saturation around ~\u20092.8 million reads between platforms, reaching saturation at ~\u20092.5 million reads or for spike-ins alone. We accounted for sequencing depth by downsampling to 1 million reads per single cell, as this has shown to be enough for single-cell analysis . We perfR\u2009=\u20090.14~0.52; Additional\u00a0file\u00a0R\u2009=\u20090.52~0.70).We re-performed PCA using both sets of spike-ins (without genes) on matched single cells to re-validate the subpopulations and to control for any technical bias arising from endogenous genes. As expected, the matched cells in resulting PCA were uniform with similar distances and low PC1 (~\u20097%) and PC2 (~\u20095\u20136%) variation between platforms .We also compared the single-cell sensitivities for matched cells both before and after downsampling. The rationale is that without downsampling, single-cell correlations would be poorer due to sequencing depth variation and skewed towards the more deeply sequenced BGISEQ-500. As expected, the correlations were poorer before downsampling and can capture underlying biological subpopulations at single-cell level. The BGISEQ-500 offers a cost-effective alternative to Illumina platform with similar yields.Next, we repeated our benchmarking comparison using plate-based Smart-seq2 protocol on a smaller subset of 82 mESCs and 98 K562s using ERCC spike-ins only. We chose plate-based Smart-seq2 as its most widely used full-length protocol and avoid cell capture biases in the C1-platform. We used the matched single-cell cDNA from mESCs and K562s to generate 600 BGISEQ-500 sequencing libraries in both single- and paired-end configurations and 121 HiSeq 4000 paired-end sequencing libraries for both cell types, indicating similar performance metrics across sequencing platforms , two scRNA-seq protocols , two technologies , and matched 1297 libraries across Illumina and BGISEQ-500 sequencing platform , and reproducibility for the first critical step have been recently evaluated and benchmarked using synthetic RNA spike-in molecules \u20137. HowevHere we explore an alternative BGISEQ-500 short-read sequencing platform for scRNA-seq that uses combinatorial probe-anchor synthesis (cPAS). Unlike Illumina, BGISEQ-500 platform performs template enrichment using rolling circle amplification on DNA-Nanoballs combined with stepwise sequencing for template amplification (AdditioOur study is the first to utilize BGISEQ-500 platform for scRNA-seq. Our comprehensive benchmarking of performance metrics utilizes two scRNA-seq protocols (SMARTer and Smart-seq2), multiple spike-ins , two different cell lines , and two technologies across Illumina HiSeq and BGISEQ-500 platform. Utilizing 468 single K562 and mESCs and matched 1297 single-cell libraries, we observe BGISEQ-500 to be highly comparable in sensitivity, accuracy, and reproducibility to Illumina platform, while being considerably more cost-effective.From our mESC scRNA-seq dataset, we could distinguish technical artifacts (sequencing depth) from biological variation (subpopulations) across both sequencing platforms. We observe differential alternative splicing events between K562s and mESCs across both sequencing platforms. We observe some RNA degradation in few single-cell libraries, which we believe is due to transport of samples. Our data using mESCs and K562s across two scRNA-seq protocols supports the notion that minimal variability is introduced during library preparation and sequencing for both Illumina and BGISEQ-500 platforms. In combination with our previous framework [We observe minimal variability in cDNA processing across different library preparation and sequencing platforms. In the current study, we did not perform scRNA-seq protocols with Unique Molecular Identifiers (UMIs) that account for PCR amplification biases. Given that UMIs primarily address biases during the RNA-to-cDNA stage (and to cDNA amplification), this would have minimal or no impact on our assessment of sequencing platforms. The scRNA-seq UMI-based protocols could easily be extended to be compatible with sequencing on BGISEQ-500 platform. Our large resource for benchmarking scRNA-seq data suggests that the BGISEQ-500 platform is suitable for plate-based (microwell or nanowell), droplet, and microfluidics technologies.In addition to benchmarking, we provide a large comprehensive multi-cell type, protocol, and platform scRNA-seq dataset spanning 468 cells and 1297 libraries in both single- and paired-end configuration to the community. Given the large research initiatives profiling transcriptomes of single cells in mouse , 17 and Additional file 1:Supplementary figures. (PDF 1140 kb)Additional file 2:Supplementary methods. (PDF 608 kb)Additional file 3:Table S1. The sequencing throughput and costs for single- and paired-end reads across both Illumina and BGISEQ-500 platform. (XLSX 9 kb)Additional file 4:Table S2. Single-cell library statistics computed from raw paired-end sequencing reads for mESCs using SMARTer and Smart-seq2 protocols and sequenced across HiSeq2500 and BGISEQ-500 platforms. (CSV 50 kb)Additional file 5:Table S3. Single-cell library statistics computed from randomly downsampled 1 million paired-end sequencing reads for mESCs performed using SMARTer and Smart-seq2 protocols and sequenced across HiSeq2500 and BGISEQ-500 platforms. (CSV 48 kb)Additional file 6:Table S4. Single-cell library statistics computed from raw paired-end sequencing reads for mESCs and K562 using plate-based Smart-seq2 protocol and sequenced across HiSeq4000 and BGISEQ-500 platforms. (CSV 87 kb)Additional file 7:Table S5. Single-cell library statistics computed from randomly downsampled 1 million paired-end sequencing reads for mESCs and K562 using plate-based Smart-seq2 protocol and sequenced across HiSeq4000 and BGISEQ-500 platforms. (CSV 82 kb)Additional file 8:Table S6. Single-cell library statistics computed from raw single-end sequencing reads for mESCs and K562 using plate-based Smart-seq2 protocol and sequenced across HiSeq4000 and BGISEQ-500 platforms. (CSV 75 kb)Additional file 9:Table S7. Metadata for all single-cell libraries profiled in this manuscript. Each cell is labeled with a sample id (accession number), protocol, place of experiment, read type, and sequencing platform."} +{"text": "T-cell prolymphocytic leukemia (T-PLL) is a mature T-cell leukemia typically presenting at stages of exponentially rising lymphocyte counts in peripheral blood, accompanied by splenomegaly and bone marrow involvement , 2. T-PLSchrader et al [The past years have witnessed a significantly improved molecular understanding of T-PLL, which culminated in a newly proposed disease model by er et al . This coer et al \u20138, this Schrader et al, we describe that in virtually all T-PLL the genomic landscape is dominated by lesions activating the TCL1 oncogene and by those compromising the DNA repair master regulator ATM [TCL1up/ATMdef genotype mainly affects key signaling branches and functions of DNA damage response (DDR) pathways, i.e. perturbations of ATM\u2019s safeguarding tasks by TCL1 resulting in cell-death evasion. TCL1up/ATMdef jointly confer a functional signature of cellular inefficiency in alleviation of high burdens of reactive oxygen species, in maintaining telomere and genome integrity, and in activating protective p53 programs. Further alterations include oncogenes like MYC, epigenetic modifiers like EZH2, KMTs and HDACs as well as elements of micro-RNA processing (e.g. AGO2). We also extrapolate that overt-stage autonomous proliferation including clonal escape from niche-defined homeostatic control relies on independence from milieu input, as potentially conveyed by the highly prevalent activating JAK/STAT mutations or by other modes of net-activated JAK/STAT signaling [In ator ATM . This coignaling .via the ATM/CHK2 axis. Explained by their hypomorphic ATM, T-PLL cells uniformly failed to generate an adequate DSB-induced p53 response [TP53 and its immediate regulators are infrequent in T-PLL [via de-/acetylation (through HATs/HDACs) regulate central steps of the DDR by (i) direct histone modulation and by (ii) modifying \u2018non-histone\u2019 proteins like p53 or ATM. Consequently, we showed the efficacy of targeting such (dys)regulated acetylation via (H)DAC inhibitors (HDACi\u2019s) [ex vivo drug profiling studies in primary T-PLL cells [Schrader et al [A pro-apoptotic response to most kinds of DNA damage is relayed through activation of p53 LL cells \u20138. In th(i) it\u00b4s function as a transcription factor that stimulates the expression of pro-apoptotic Bcl-2 family genes and through (ii) direct transcription-independent effects at the mitochondrial membrane (Figure BCL2-family genes [L to be highly active in T-PLL cells as well [Apoptosis induction downstream of p53 is mediated through e Figure . Overall as well -8. Venet as well .Taking together, we are witnessing the exciting transition of an advanced understanding of the key molecular lesions of T-PLL towards their clinical exploitation. Within the past 2 years highly promising substance categories that specifically address the vulnerabilities of T-PLL have emerged Figure . Namely,"} +{"text": "Particulate matter (PM) air pollution is a global environmental health problem contributing to more severe lung inflammation and injury. However, the molecular and cellular mechanisms of PM-induced exacerbation of lung barrier dysfunction and injury are not well understood. In the current study, we tested a hypothesis that PM exacerbates vascular barrier dysfunction via ROS-induced generation of truncated oxidized phospholipids (Tr-OxPLs). Treatment of human pulmonary endothelial cells with PM caused endothelial cell barrier disruption in a dose-dependent fashion. Biochemical analysis showed destabilization of cell junctions by PM via tyrosine phosphorylation and internalization of VE-cadherin. These events were accompanied by PM-induced generation of Tr-OxPLs, detected by mass spectrometry analysis. Furthermore, purified Tr-OxPLs: POVPC, PGPC and lyso-PC alone, caused a rapid increase in endothelial permeability and augmented pulmonary endothelial barrier dysfunction induced by submaximal doses of PM. In support of a role of TR-OxPLs-dependent mechanism in mediation of PM effects, ectopic expression of intracellular type 2 platelet-activating factor acetylhydrolase (PAFAH2), which specifically hydrolyzes Tr-OxPLs, significantly attenuated PM-induced endothelial hyperpermeability. In summary, this study uncovered a novel mechanism of PM-induced sustained dysfunction of pulmonary endothelial cell barrier which is driven by PM-induced generation of truncated products of phospholipid oxidation causing destabilization of cell junctions. Particulate matter (PM) is the most common air pollutant with a serious global health threat contributing to millions of premature deaths annually worldwide. The role of PM air pollution in the development or exacerbation of various heart and lung diseases is becoming increasingly recognized \u20133. AmongThe endothelial barrier formed by the cell-cell junction complexes controls the passage of fluids, solutes and circulating cells across the vascular wall. Compromised EC barrier integrity leads to the excessive leakage and development of life-threatening conditions such as pulmonary edema and sepsis . Adherensn-glycero-3-phosphorylcholine (PAPC) represents the major plasma membrane phospholipid and its oxidation generates a heterogeneous mixture of oxygenated full-length products such as 1-palmitoyl-2--sn-glycero-3-phsphocholine , 1-palmitoyl-2--sn-glycero-3-phsphocholine ; and truncated products of PAPC oxidation including 1-palmitoyl-2--sn-glycero-phosphocholine (POVPC), 1-palmitoyl-2-glutaroyl-sn-glycero-phosphocholine (PGPC) and lysophosphatidyl choline (lyso-PC). Importantly, these truncated oxidized phospholipids (Tr-OxPLs) have been shown to induce endothelial barrier disruption [Phospholipids provide the structural basis for the formation of cell membranes. In turn, phospholipid oxidation generates bioactive lipid mediators that play an important role in cellular signaling , 31. Thesruption .In this study, we tested the hypothesis that PM challenge triggers the production of bioactive Tr-OxPLs by pulmonary EC, which cause AJ breakdown and endothelial barrier dysfunction. We also evaluated potential molecular approaches to reduce the levels of Tr-OxPLs and mitigate barrier disruptive effects of PM on pulmonary vascular endothelium.Human pulmonary artery endothelial cells and endothelial growth media were obtained from Lonza . Cells were used at passages 5\u20138 and all cell stimulations were carried out in medium containing 2% fetal bovine serum unless otherwise specified. Texas Red-conjugated phalloidin and Alexa Fluor 488-labeled secondary antibodies were purchased form Molecular Probes . Antibodies to phospho-VE-cadherin (pTyr-658 and pTyr-731) were obtained from Invitrogen and VE-cadherin antibody was from Santa Cruz Biotechnology . p120-Catenin antibody was from BD biosciences and HRP-linked anti-mouse and anti-rabbit IgG were obtained from Cell Signaling . N-acetyl cysteine and amifostine were obtained from Sigma . For PM, we used an urban PM 1649b collected from ambient air in Washington, DC and characterized by National Institute of Standards and Technology . Purified POVPC, PGPC and lyso-PC were obtained from Avanti Polar Lipids .Human pulmonary endothelial cells were grown in 96-well plate and 5-(and-6)-carboxy-2\u2032, 7\u2032-dichlorodihydrofluorescein diacetate was added to final concentration of 10 \u03bcM. Cells were incubated in serum-free medium for 30 min at 37\u00b0C, protected from light, then stimulated with PM. ROS measurement was performed using the Image LIVE Green Reactive Oxygen Species Detection Kit from Molecular probes according to the manufacturer\u2019s instructions.The cellular barrier properties were analyzed by measurements of transendothelial electrical resistance (TER) across confluent human pulmonary endothelial monolayers using an electrical cell-substrate impedance sensing system as previously described . EndotheThis assay was performed as we previously described . BrieflyAfter agonist stimulation, cells were and lysed with cold TBS-NP40 lysis buffer supplemented with protease and phosphatase inhibitor cocktails . Cytosolic (soluble) and membrane/cytoskeletal (particulate) fractions were isolated as described previously . ProteinFollowing agonist stimulation, endothelial cells were fixed in 3.7% formaldehyde solution in PBS for 10 min at 4\u00b0C, washed with PBS, permeabilized with 0.1% Triton X-100 in PBS for 30 min at room temperature and blocked with 2% BSA in PBS for 30 min. Incubation with VE-cadherin antibody was performed in blocking solution (2% BSA in PBS) for 1 hr at room temperature followed by staining with Alexa 488-conjugated secondary antibody. Actin filaments were stained with Texas Red-conjugated phalloidin diluted in the blocking solution. After immunostaining, the slides were analyzed using an inverted microscope Nikon Eclipse TE300 connected to SPOT RT monochrome digital camera and image processor . The images were processed with Adobe Photoshop 7.0 . For each experimental condition at least 10 microscopic fields in each independent experiment were analyzed.Human lung EC were transfected with PAFAH2 DNA plasmid harboring Myc-FLAG-tags using Lipofectamine 2000 reagent as recommended by the manufacturer. Then, cells were treated with vehicle or PM after 24 hr of transfections and assayed for permeability measurements or other biochemical analysis.All animal care and treatment procedures were approved by the University of Maryland and University of Chicago Institutional Animal Care and Use Committees. 8-10-week old male and female C57Bl/6j mice were purchased from Jackson Laboratories . Animals were handled according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. PM was administered as suspension in saline (100 \u03bcg/mouse). Animals were anesthetized with a 0.03 ml intraperitoneal injection of ketamine (12 mg/kg). Proper anesthesia was assessed by paw and tail pinching, and additional anesthetic was administered as necessary. Animals were sacrificed at 3 hrs or 24 hrs after PM administration by exsanguination under ketamine anesthesia. After perfusion with Hank\u2019s balanced salt buffer supplemented with 0.5 mM phenyl-methyl-sulfonyl-fluoride (PMSF) and 0.1 mM Pefa-block, lungs were snap frozen in liquid nitrogen and used for MS analysis of Tr-OxPL content.The levels of Tr-OxPLs in endothelial cells and mouse lungs was determined by mass spectrometric analysis as recently described by our group . BrieflyResults are expressed as means \u00b1 SD of three to five independent experiments. Stimulated samples were compared with controls by unpaired Student\u2019s t-test. For multiple-group comparisons, one-way analysis of variance (ANOVA) followed by the post hoc Fisher\u2019s test were used. P<0.05 was considered statistically significant.In situ biotinylation assay of cell surface proteins showed a marked decrease of biotinylated VE-cadherin in PM stimulated cells suggesting VE-cadherin internalization using ECIS array and evaluation of endothelial cell monolayer permeability for FITC-avidin as described in Methods. Stimulation of pulmonary endothelial cell monolayers with PM caused a rapid and sustained dose-dependent decrease in TER in the dose range of 10\u201350 \u03bcg/ml of PM reflecting endothelial cell barrier dysfunction . Accordilization . PM alsolization . The decSince earlier studies have suggested the generation of ROS as a key mechanism of PM-induced pathologies , 29, we is a unique enzyme which may inactivate pro-inflammatory TR-OxPLs [Since our results strongly indicate that production of Tr-OxPLs is the major mechanism that mediates PM-induced endothelial cell dysfunction, we tested whether inhibition of Tr-OxPLs restores endothelial function. Hydrolysis and deactivation of truncated products of phospholipid activation via cleavage of truncated/oxidized fatty acid moiety present at TR-OxPLs . To evalPM air pollution has emerged as a severe environmental health concern with over 4 million premature deaths annually worldwide from various cardiopulmonary disorders . PM is kin vivo oxidation of circulating lipoproteins in patients [In agreement with previous report of the role of ROS in PM-induced EC dysfunction , our datpatients . WR-1065patients and mitipatients , 47. Thu658 and Tyr731 is known to prevent its binding to partners, p120- and \u03b2-catenin, leading to weakened AJ [in vitro as well as in vivo that cause PM-induced endothelial dysfunction.Furthermore, we also show that PM-induced disruption of AJ complex caused by the loss of major AJ proteins VE-cadherin and p120-catenin mediates endothelial cell barrier disruption. Phosphorylation of VE-cadherin at Tyrkened AJ , 49. Thekened AJ . Our datkened AJ , 29. Mosin vitro and in vivo lung injuries [Lipid peroxidation products such as 4-hydroxy-2-nonenal and 8-isoprostane have been found in increased concentrations in patients with ARDS and other lung injuries and correlated with severity of disease \u201352. Howeinjuries , 53\u201356. injuries , 58. Andinjuries .Our results strongly suggest that PM-induced rapid endothelial barrier disruption occurs through the production of various Tr-OxPLs including PGPC, POVPC and a number of structural variants of lyso-PC, since each of these Tr-OxPLs species identified by mass spectrometry analysis of PM-challenged lungs and pulmonary endothelial cells caused a dose-dependent increase in endothelial permeability. Furthermore, the robust potentiating effects of POVPC in PM-induced endothelial barrier disruption strongly suggest that generation of Tr-OxPLS is essential for PM pathologic effects on lung function. These potentiating effects of POVPC were also observed with respect to PM-induced VE-cadherin phosphorylation and internalization, where minimal effects of low dose POVPC were augmented by PM co-treatment. This phenomenon may be of clinical significance. It is conceivable that as normal healthy endothelium may not become affected by low PM exposure. However, during various pathological conditions (i.e. septic inflammation) associated with elevated Tr-OxPLs, the pulmonary endothelium becomes more vulnerable to even low doses of PM. This \"two-hit\" model could also be valid in other aspects of lung dysfunction. For example, in healthy individuals, epithelial barrier traps PM preventing their penetration to the lung parenchyma and translocation into the circulation. But lung epithelial barrier compromise caused by infection, toxins, mechanical injury, etc., may be further exacerbated by PM leading to PM interactions with lung endothelium and propagation of ALI-associated vascular permeability and inflammation.A definitive role of Tr-OxPLs mediated VE-cadherin phosphorylation and internalization as a mechanism of PM-induced endothelial barrier dysfunction was further supported by experiment with ectopic expression of PAFAH2, a Tr-OxPLs-specific acetyl hydrolase, which markedly attenuated PM-induced VE-cadherin phosphorylation and restored VE-cadherin presence in plasma membrane. Consistently, PAFAH2 overexpression also significantly attenuated PM-induced endothelial permeability. This finding not only confirms the vital role of Tr-OxPLs generation in exacerbation of PM-induced endothelial cell dysfunction, but also presents a promising therapeutic strategy for targeted elimination of deleterious Tr-OxPLs without global inhibition of redox-dependent processes, since ROS signaling also plays an essential role in lung physiology, innate immunity and lung recovery. In conclusion, our study demonstrates a novel pathologic mechanism of PM-induced endothelial dysfunction via production of bioactive Tr-OxPL products. These findings also highlight PAFAH2-mediated inhibition of Tr-OxPLs production as a potential therapeutic approach to alleviate PM-induced complications of lung injury and other acute cardiovascular inflammatory disorders."} +{"text": "Microvesicles are the body's most powerful intercellular communication system and cancer-initiating cell microvesicles (CIC-TEX) reprogram Non-CIC towards fortified malignancy. Claudin7, a CIC-biomarker in gastrointestinal tumors, is recovered in TEX. Recent evidence suggesting individual cells delivering distinct microvesicles became of particular interest for claudin7, which is part of tight junctions (TJ) and glycolipid-enriched membrane domains (GEM), GEM-located claudin7 is palmitoylated. This offered the unique possibility of exploring the contribution of a CIC marker and its origin from distinct membrane domains on CIC-TEX biogenesis and activities. Proteome and miRNA analysis of wild-type, claudin7-knockdown and a rescue with claudin7 harboring a mutated palmitoylation site (mP) of a rat pancreatic and a human colon cancer line uncovered significant, only partly overlapping contributions of palmitoylated and non-palmitoylated claudin7 to TEX composition. Palmitoylated claudin7 facilitates GEM-integrated plasma membrane and associated signaling molecule recruitment; non-palmitoylated claudin7 supports recruitment of trafficking components, proteins engaged in fatty acid metabolism and TJ proteins into TEX. Claudin7mP also assists TEX recovery of selected miRNA. Thus, distinctly located claudin7 affects CIC-TEX composition and TJ-derived cld7 might play a unique role in equipping CIC-TEX with transporters and lipid metabolism-regulating molecules, awareness of distinct TEX populations being crucial facing therapeutic translation. Cancer-initiating cells (CIC) are the major obstacle in curative treatment after tumor spread from the primary location Claudins are four-pass membrane proteins. They are essential for the formation of tight junctions (TJ) Exosomes are a subpopulation of small 40-130nm microvesicles. Many cells and abundantly tumor cells deliver exosomes We here asked whether palmitoylated and possibly TJ-derived, non-palmitoylated cld7 contribute distinctly to the TEX composition and ILV loading. We approached the question using the rat PaCa ASML and the human CoCa SW948 lines as CIC-TEX donors. A cld7-knockdown (kd) in these lines results in a loss of CIC features CIC-TEX promote metastasis by altering host cells and non-CIC Cld7 is detected in the cell membrane at two different locations, in TJ and GEM Western blot (WB) and flow-cytometry of ASML cells and TEX confirmed the efficacy of the cld7kd and the partial rescue in cld7mP cells and TEX, rescued cld7mP being non-palmitoylated Proteins displaying reduced expression in ASML-cld7kd and -cld7mP cells and TEX cover a wide range of functions. In ASML-cld7kd and -cld7mP cells, mostly metabolism- and trafficking/transport-engaged proteins are reduced. In cld7mP cells, a reduction in trafficking/transport is dominating Fig. C. In TEXIn concern about the impact of CIC-TEX on cld7kd cells, proteins selectively enriched in ASML-wt TEX are particularly important. ASML-wt TEX showed loss or reduced recovery of 887 proteins compared to wt cells; 87 proteins were enriched in wt TEX compared to wt cells and 137 proteins compared to cld7kd cells Table A. STRINGTo confirm the validity of cld7 contributing to TEX biogenesis and a suggested impact of wt TEX on cld7kd cells the proteome analysis was repeated with wt and cld7kd SW948 cells and TEX Table A-S2D. SiBriefly, signal transducing and transporter molecules are highly enriched in TEX compared to cells. However, differences due to palmitoylation-competent versus palmitoylation-deficient cld7 are weaker in TEX than cells.To pursue the question of a cld7-selective contribution to TEX loading, ASML-wt and -cld7mP cells and TEX were precipitated with anti-cld7 comparing the pattern of coimmunoprecipitating molecules. According to the mild lysis condition, the precipitates include loosely attached molecules. Precipitates were lysed, separated by SDS gel and analyzed by mass spectrometry (nanoLC-ESI-MS/MS on an LTQ orbitrap).First to note, more proteins coimmunoprecipitate with cld7 in ASML-wt TEX 213) than cells (110). This likely depends on the TEX membrane lipid composition that supports palmitoylated or myristoylated protein attachment Fig. A. In ASM than celImportantly, STRING network analysis uncovered striking differences in the function of proteins coimmunoprecipitating with wt versus cld7mP in cells and TEX. Proteins overrepresented in coimmunoprecipitates in ASML-wt cell lysates were mostly engaged in repair Fig. A, while Thus, in cells the majority of cld7-coimmunoprecipitating molecules are associated with palmitoylated, GEM-located cld7, whereas in TEX significantly more proteins are associated with non-palmitoylated cld7. These proteins are frequently not plasma membrane-integrated, indicating TEX recruitment of proteins that associate with cytoplasmic, non-palmitoylated cld7. The exclusive recovery of TJ components in cld7mP cell and TEX precipitates and the strong enrichment of proteasome components in cld7mP coimmunoprecipitating proteins in TEX confirm the distinct palmitoylated versus non-palmitoylated cld7 recruitment and indicate a considerable contribution of recycling cld7 to the TEX armament.Briefly, cld7 contributes to shaping TEX in ASML and SW948. Distinct to cells, where mostly GEM-located, palmitoylated cld7 accounts for differences in the protein profile, in TEX cytoplasmic, non-palmitoylated cld7 is decisive.As the rules for recruiting miRNA into ILV differ from that of proteins, we finally analyzed, whether cld7 affects miRNA recruitment. Differences in the miRNA profile between wt cells and TEX confirmed selective miRNA recruitment into TEX http://www.microrna.org, http://www.targetscan.org). IPA-based STRING pathway analysis revealed that from these 16 miRNA 10 targeted mRNA engaged in cancer relevant pathways, mostly in molecular mechanism in cancer, cell cycle and TJ regulation. IPA was used defining, which of the predicted targets are discovered at a higher level in cld7kd- and cld7mP-TEX. Only for 6 miRNA high in wt-TEX, predicted targets were found at a higher level in cld7kd- and/or cld7mP-TEX. Only 5 from 11 predicted targets were relevant for cancer, being engaged in molecular mechanisms of cancer of 4 of 11 miRNA higher in wt-TEX showed reduced expression in wt-TEX cld7. As elaborated for ASML TEX, recruitment of miR-200c-3p was palmitoylated cld7-dependent, recruitment of an additional 3 miRNA was cld7-dependent irrespective of palmitoylation. In concern about a contribution of cld7 to miRNA recruitment into TEX, a significant number of miRNA is distinctly recovered in wt- versus cld7kd- or cld7mP-TEX without corresponding changes in cells. We interpret these findings that cld7/cld7mP contributes to MVB transport, but is not decisive for ILV-loading. Steps/molecular complexes of MVB transport that are supported by palmitoylated or non-palmitoylated cld7 require further elaboration.Irrespective of miRNA recruitment, there was no evidence that TEX miRNA affected TEX mRNA/protein. This accounted for predicted target mRNA/protein of miRNA high in TEX. In addition, from 70 proteins higher in cld7kd- and/or cld7mP- than wt-TEX, only 5 were predicted targets of 4 miRNA lower in cld7kd- than wt-TEX. Instead, from 49 proteins with lower expression in wt- than cld7kd-cells, 17 are predicted targets of miRNA higher in wt-TEX than cld7kd-cells, the predicted targets mostly being engaged in central signaling activities. The finding is in line with the suggested impact of TEX miRNA on target cell mRNA In brief, mostly non-palmitoylated cld7 contributes to the transport of miRNA-loaded MVB into TEX. The target proteins of these miRNA are not reduced in TEX, indicating that miRNA recruitment was independent of mRNA/protein recruitment and that TEX miRNA does not affect mRNA within TEX. Instead, predicted mRNA targets of abundant TEX miRNA were reduced in wt- compared to cld7kd-cells, which suggests TEX miRNA supporting \u201ctumor competence\u201d of cld7kd-cells Tumor lines: The human CoCa lines SW948 and SW948-cld7kd Antibodies are listed in Table CIC enrichment and TEX preparation: SW948-CIC are enriched by spheroid growth 4/10mM NaF/protease inhibitor mix , mild lysis conditions were used for avoiding the destruction of loosely attached protein complexes. Lysates were centrifuged , mixed with antibody and incubated with ProteinG-Sepharose (1h). Washed complexes were dissolved in Laemmli buffer. For WB, lysed cells and TEX were dissolved in Laemmli buffer and resolved on 10%-12% SDS-PAGE. After protein transfer, blocking, blotting with antibodies, blots were developed with enhanced chemiluminescence reagent.Immunoprecipitation (IP) and WB: Cells and TEX were lysed in HEPES buffer/1%Lubrol/1mM PMSF/1mM NaVOProtein elution, tryptic digestion, mass spectrometry and database searches: Cell and TEX lysates and dissolved immunoprecipitates were separated by 1D SDS gel electrophoresis. After staining with Coomassie lanes were cut into ten slices. After tryptic digestion, peptides were analyzed by nanoLC-ESI-MS/MS and subjected to quantification .miRNA: TEX were pretreated with RNase. Following the supplier's suggestion , the miRNeasy Minikit was used to extract cell and TEX miRNA.http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi? acc=GSE119031, GSE119032, -GSE 11903, rat miRNA: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi? acc=GSE120185). Mean values of normalized data were compared. Differential recovery was defined by \u22652-fold changes in signal strength.Microarray miRNA analysis: miRNA analysis of cells and TEX was performed at the Core facility of EMBL, Heidelberg, using the Agilent microRNA microarray evaluating quadruplicates of three independent preparations. Data are deposited at GEO (human miRNA: http://pantherdb.org), KEGG (http://www.kegg.jp), Reactome (https://reactome.org), andSTRING (http://string-db.org) databases were used for pathway analysis. IPA was used for miRNA analysis and for correlating miRNA with protein expression after predicted targets were selected by miRNA databases .Proteome and miRNA analysis: PANTHER : Real-time polymerase chain reaction (PCR) was performed using a standard TaqMan PCR kit protocol on an Applied Biosystems 7900HT Sequence Detection System (Applied Biosystems). Primers are listed in Table Flow-cytometry of cells followed routine procedures. TEX (10-15\u00b5g) were incubated with 1\u00b5l aldehyde-sulfate latex beads (LB) (4\u00b5m) (Invitrogen) in PBS/1% BSA . After centrifugation, free binding sites were blocked . TEX-coated LB were washed (PBS/1% BSA). Staining of 1\u00b5l TEX-coated LB followed the protocol for cell staining. For intracellular staining, cells/TEX were fixed and permeabilized. Samples were analyzed in a FACSCalibur using the CellQuest program.t test, analysis of variance. If not indicated otherwise, p-values <0.05 were considered significant.Statistics: IBM SPSS software was used for statistical evaluation. In vitro assays were run in triplicates and repeated at least three times. P-values are derived from two-tailed Student's The CIC marker cld7 is abundantly recovered in PaCIC- and CoCIC-TEX, which transfer CIC messages into host cells and non-CIC. As TEX-cld7 can be derived from GEM or TJ, defining the contribution to TEX biogenesis became important. Palmitoylated cld7 assists the recruitment of GEM-associated proteins including membrane-attached cytoskeletal components and signaling molecules. Non-palmitoylated, likely TJ-derived cld7 most prominently recruits transporters and assists miRNA-loaded vesicle transport. Thus, TEM- and TJ-derived cld7 distinctly, but additively affect TEX composition. Awareness of distinct TEX populations delivered by a single cell only recently received attention, but essentially needs to be taken into account optimizing therapeutic strategies. The information on cld7-dependent TEX assembly provides a solid ground elaborating at the molecular level optimized strategies to interfere with TEX-cld7 metastasis-promoting activities.Supplementary figures and tables.Click here for additional data file."} +{"text": "Ischemic heart disease (IHD) is the major cause of death in patients with cardiovascular disease. Cardiac remodeling is a common pathological change following myocardial infarction (MI), and cardiomyocyte apoptosis plays a key role in this change. Transcription factor recombination signal-binding protein-J (RBP-J)-mediated Notch signaling pathway has been implicated in several inherited cardiovascular diseases, including aortic valve diseases, but whether the RBP-J-mediated Notch signaling pathway plays a role in cardiomyocyte apoptosis after MI is unclear.fl/fl mice and Myh6-Cre/Esr1 transgenic mice to delete RBP-J in vivo and to partly inhibit the canonical Notch signaling pathway. MI was induced in mice by permanent ligation of the left anterior descending coronary artery followed by the knockout of RBP-J. Cardiac function and morphology were assessed by echocardiography and histological analysis 4 weeks after infarction. In addition, the expression and regulation of apoptosis-related molecules were examined by real time PCR and western blot. We crossed RBP-J RBP-J knockout decreased the survival rate and deteriorated post-MI remodeling and function in mice, and this effect was associated with increased cardiomyocyte apoptosis. The potential mechanisms might be related to the downregulated expression of bcl-2, upregulated expression of bax, and cleaved-caspase 3 to exacerbate cardiomyocyte apoptosis. These findings show that the RBP-J-mediated Notch signaling pathway in cardiomyocytes limits ventricular remodeling and improves cardiac function after MI. The RBP-J-mediated Notch signaling pathway has a protective role in cardiomyocyte apoptosis following cardiac injury. With the population growth and aging, the global burden of ischemic heart disease (IHD) is high , 2. How A number of signaling pathways are involved in the regulation of cardiomyocyte apoptosis after MI. Notch signaling is an evolutionarily conserved pathway that governs cell fate specification. Activation of the Notch signaling pathway exhibits an improved hemodynamic function and reduced myocardial fibrosis after MI \u20139. And tfl/fl mice and Myh6-Cre/Esr1 transgenic mice were kindly granted by Professor Li Hongliang . loxP sites were introduced on both sides of the RBP-J exons encoding its DNA-binding domain in RBP-Jfl/fl mice. To generate Myh6-RBP-Jfl/wt mice, the RBP-Jfl/fl mice were crossed with the Myh6-Cre/Esr1 transgenic mice with the use of the Cre-loxP system in which transgenic Cre expression is driven by the cardiac-specific mouse \u03b1-myosin heavy chain (Myh6) promoter [fl/wt mice were injected with 20\u2009mg/kg Tamoxifen , which was prepared in ethanol and diluted with corn oil; this mixture was administered by intraperitoneal injection once per day for 5 consecutive days at the age of 6-8 weeks [RBP-Jpromoter , 19. To fl/wt mice (n = 30) and RBP-Jfl/fl mice (n = 30) underwent MI surgery by permanent coronary ligation (n = 15 per group) or sham operation (n = 15 per group) 7-10 days after the intraperitoneal injection of Tamoxifen [Myh6-RBP-Jamoxifen , 22. In amoxifen .Echocardiography was performed with a MS400 probe on a Visual Sonics Vevo2100 small animal ultrasound scanner to assess cardiac function at day 0 and day 28. Mice were anesthetized (1.5% isoflurane and oxygen) and put in a supine position. Both two-dimensional and M-mode images were recorded. The left ventricular systolic dimension (LVDs), left ventricular diastolic dimension (LVDd), and septum and posterior wall thicknesses were measured. The left ventricular systolic volume (LV Vol s), left ventricular diastolic volume (LV Vol d), left ventricular ejection fraction (LVEF), and fractional shortening (FS) were computed from these measurements .\u03bcm thickness. Hematoxylin and eosin (H&E) staining, Masson's trichrome staining, and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining were performed according to standard methods. Using H&E-stained sections, the cross-sectional area of cardiomyocytes was measured at the border zone. For Masson's trichrome-stained sections, photomicrographs were taken and collagen-positive areas were measured using ImageJ. TUNEL staining was performed to detect apoptosis. The nuclei of apoptotic cardiomyocytes were stained dark brown. Finally, the sections were counterstained with methyl green and then coverslipped. The sections were observed by light microscopy, and the apoptosis ratio was measured using ImageJ.Hearts were harvested 28 days after operation. Hearts from the mice were isolated and fixed in 4% para-formaldehyde overnight. The hearts were then embedded in paraffin and sectioned at 3\u2009\u00b5g) were separated by 10% SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane. After blocking for 1\u2009h at room temperature, the membranes were probed with rabbit antibodies against RBP-J (1:500), bax (1:500), bcl-2 (1:500) , cleaved-caspase 3 (1:1000) , and \u03b2-actin mouse monoclonal antibody (1:1000) overnight at 4\u00b0C, followed by incubation with secondary horseradish peroxidase-conjugated anti-rabbit IgG antibody (1:1000) or horseradish peroxidase-conjugated anti-mouse IgG antibody (1:1000) for 1\u2009h at room temperature. Proteins were detected by exposing the blots to X-ray film (Roche Applied Science).Left ventricular tissues were ground in liquid nitrogen. Nuclear protein and cytoplasmic protein extracts were made using a Nuclear Protein and Cytoplasmic Protein Extraction kit according to the manufacturer's instructions. The protein concentrations were determined with a BCA Protein Assay Kit . Samples containing equal amounts of protein according to the manufacturer's instructions. The concentration of RNA was measured using a NanoDrop ND-1000 spectrophotometer . One microgram of total RNA was reverse transcribed with Oligo (dT) primer using M-MLV reverse transcriptase (Invitrogen). Real time PCR was performed using SYBR Premix EX Taq (Takara) and the ABI PRISM 7500 real time PCR system. The PCR conditions involved a denaturation step (95\u00b0C for 10\u2009sec), and amplification and quantification were repeated 40 times . Sequences of the primers used in real time PCR were RBP-J (NM009035), forward: GAATGTACTTGTGCCTTTCTCAAGAAAG; reverse: CTGTAAGTTCAAGGATTGCTACGTCCCC; Hes1 (NM008235), forward: AGAGGCGAAGGGCAAG; reverse: AGGTGCTTCACAGTCATTT; Hey1 (NM010423), forward: GCATACGGCAGGAGGGAAA; reverse: CTGGGAAGCGTAGTTGTTGAGAT; Notch1 (NM008714), forward: TGCCAGGACCGTGACAACTC; reverse: CACAGGCACATTCGTAGCCATC; Bax (NM007527), forward: TCCACCAAGAAGCTGAGCGAG; reverse: GTCCAGCCCATGATGGTTCT; Bcl-2 (NM009741), forward: TTCTTTGAGTTCGGTGGGGTC; reverse: TGCATATTTGTTTGGGGCAGG; \u03b2-actin (M12481), forward: CATCCGTAAAGACCTCTATGCCAAC; reverse: ATGGAGCCACCGATCCACA. The relative target mRNA levels were determined using the 2\u2212\u0394\u0394Ct method.Real time PCR was performed to quantify and validate the specific target genes hairy and enhancer of split 1 (Hes1), the hes-related family bHLH transcription factor with YRPW motif 1 (Hey1), bax, and bcl-2 mRNA expression. P < 0.05 was considered statistically significant.The statistical analysis was performed with the SPSS 19.0 program. The results were expressed as the means\u00b1SD. The comparisons between two groups were undertaken using an unpaired Student's t-test. The multiple comparisons were first analyzed by one-way ANOVA followed by Student-Newman-Keuls test to determine significance between groups. fl/wt mice as the experiment group and RBP-Jfl/fl mice as the control. Mice were injected with Tamoxifen for 5 days to induce Cre-mediated recombination and RBP-J knockout. The real time PCR and western blot analysis of the expression level of the RBP-J mRNA and protein in Myh6-RBP-Jfl/wt mice and RBP-Jfl/fl mice showed strong expression of the RBP-J mRNA and protein in the hearts of the RBP-Jfl/fl mice .fl/wt-MI mice and in RBP-Jfl/fl-MI mice, whereas LVEF and FS were decreased compared with Myh6-RBP-Jfl/wt-sham mice and RBP-Jfl/fl-sham mice. These results suggest that RBP-J knockout induces the deterioration of cardiac function following MI.To determine whether RBP-J knockout induces the deterioration of cardiac function following MI, echocardiography was performed at day 0 and day 28. We assessed LV Vol s, LV Vol d, LVEF, and FS in vivo as depicted in fl/wt-MI mice and RBP-Jfl/fl-MI mice, the surviving myocardial cells were found in the border zones and arranged irregularly. The cross-sectional area of cardiomyocytes at the border zone was measured and demonstrated that Myh6-RBP-Jfl/wt mice with MI had an increased cross-sectional area of the border zone compared with RBP-Jfl/fl-MI mice (fl/wt mice with MI had significantly increased fibrosis and collagen deposition compared with RBP-Jfl/fl-MI mice (After 4 weeks, as shown by H&E staining, in both Myh6-RBP-J-MI mice . The ana-MI mice .fl/wt mice compared with RBP-Jfl/fl mice after MI, the number of apoptotic cells was significantly increased (fl/wt mice compared with the RBP-Jfl/fl mice with MI, whereas bcl-2 expression was significantly decreased in Myh6-RBP-Jfl/wt mice compared with RBP-Jfl/fl mice following MI. The protein expression of cleaved-caspase 3 and bax was increased in Myh6-RBP-Jfl/wt mice compared with the RBP-Jfl/fl mice with MI, whereas bcl-2 protein expression had the same tendency as bcl-2 mRNA expression in Myh6-RBP-Jfl/wt mice compared with RBP-Jfl/fl mice following MI. These results indicated that in spite of activation of Notch 1 receptor, blockade of RBP-J-mediated Notch signaling pathway could deteriorate cardiomyocyte apoptosis induced by myocardial ischemia injury.TUNEL staining was undertaken to analyze whether RBP-J knockout increased cardiomyocyte apoptosis. The results showed that in the border zone of ischemic heart tissue of Myh6-RBP-Jncreased . We detencreased and the ncreased to confiThis study produced that genetic blockade of RBP-J-mediated Notch signaling pathway receded the protective effect brought by activation of Notch 1 receptor. And this result was related to increased cardiomyocyte apoptosis in the RBP-J deficient mice after MI. The potential mechanism is involved in the modulation of an increased expression of bax and a decreased expression of bcl-2 after blockade of RBP-J-mediated Notch signaling pathway. These results indicate that endogenous RBP-J-mediated Notch signaling is critical for ischemia-induced myocardial injury, and this signaling pathway may serve as a therapeutic target.Notch signaling is highly related to cardioprotective effects after myocardial injury. Previous studies found that Notch 1 was activated in myocardial injury . And thiApoptosis is uncommon in a normal heart but is a frequent process in cardiac remodeling, particularly in IHD \u201328. And With increasing incidence in recent years, the mitochondria are vulnerable to damage in response to ischemia/hypoxia, leading to excessive oxidative stress and apoptosis in cardiomyocytes , 31. BclIn conclusion, we used genetic knockout of RBP-J-mediated Notch signaling pathway to demonstrate the roles of RBP-J-mediated Notch signaling in ischemia-induced myocardial injury. RBP-J-mediated Notch signaling also protects against ischemia-induced myocardial injury through the modulation of the expression of bax and bcl-2, which activates caspase 3 expression and leads to cardiomyocyte apoptosis. These findings suggest new therapeutic targets to limit ischemia-induced myocardial injury. However, the detailed underlying mechanism of the regulation of bcl-2 family members by the canonical Notch signaling requires further investigation in future studies."} +{"text": "Listeria monocytogenes strains isolated from meat and milk products in Switzerland. All of these strains carry premature stop codons or amino acid deletions in inlA.Here, we report the whole-genome sequences of six Listeria monocytogenes strains isolated from meat and milk products in Switzerland. All of these strains carry premature stop codons or amino acid deletions in inlA.Here, we report the whole-genome sequences of six Listeria monocytogenes is a Gram-positive foodborne pathogen and the causative agent of listeriosis, which poses a significant public health risk because of its high case fatality rate, ranging from 15 to 30 deaths per 100 cases . Quality filtering and trimming of adapters were performed using Trimmomatic with default settings (Listeria pathogenicity island 1 (LIPI-1)-associated genes were present and are flanked by the following conserved housekeeping genes: prs, a phosphoribosyl synthetase gene, and ldh, a lactate dehydrogenase gene, as described by V\u00e1zquez-Boland et al. verifiedd et al. . None ofce genes , were prinlA gene, which codes for a protein that interacts with host cell receptors. The stop codons occurred at different nucleotide positions . The other three strains all carried the same three amino acid deletions in inlA at nucleotide position 2221.Three of the strains carried premature stop codons (PMSC) in the QEMB00000000 (N11-1848), QEMA00000000 (N12-0460), QELZ00000000 (N13-0703), QELY00000000 (N13-0836), QELX00000000 (N13-1184), and QELW00000000 (N14-0261). The raw reads were deposited in the Sequence Read Archive (SRA) under accession no. SRS3471743 (N11-1848), SRS3471742 (N12-0460), SRS3471740 (N13-0703), SRS3471738 (N13-0836), SRS3471739 (N13-1184), and SRS3471737 (N14-0261).All sequences have been published in GenBank under accession no."} +{"text": "Kaposi\u2019s sarcoma (KS) herpesvirus (KSHV) causes KS, an angiogenic AIDS-associated spindle-cell neoplasm, by activating host oncogenic signaling cascades through autocrine and paracrine mechanisms. Tyrosine kinase receptor (RTK) proteomic arrays, identified PDGF receptor-alpha (PDGFRA) as the predominantly-activated RTK in KSHV-induced mouse KS-tumors. We show that: 1) KSHV lytic replication and the vGPCR can activate PDGFRA through upregulation of its ligands PDGFA/B, which increase c-myc, VEGF and KSHV gene expression in infected cells 2) KSHV infected spindle cells of most AIDS-KS lesions display robust phospho-PDGFRA staining 3) blocking PDGFRA-signaling with N-acetyl-cysteine, RTK-inhibitors Imatinib and Sunitinib, or dominant-negative PDGFRA inhibits tumorigenesis 4) PDGFRA D842V activating-mutation confers resistance to Imatinib in mouse-KS tumorigenesis. Our data show that KSHV usurps sarcomagenic PDGFRA signaling to drive KS. This and the fact that PDGFRA drives non-viral sarcomas highlights the importance for KSHV-induced ligand-mediated activation of PDGFRA in KS sarcomagenesis and shows that this oncogenic axis could be successfully blocked to impede KS tumor growth. Signaling mimicry is a key mechanism whereby oncoviruses can usurp host-regulatory pathways leading to acquisition of tissue-specific cancer hallmarks. A critical question in the KS field is the identification of this host pathways activated by KSHV that could provide novel insights on KSHV-pathobiology, elucidating new druggable pathways. Here we show that KSHV lytic replication as well as the KSHV-oncogene vGPCR activates PDGFRA signaling through upregulation of its ligands PDGFA/B, and that blocking of PDGFRA signaling is anti-tumorigenic. This indicates that approaches that fully and stably inhibit PDGFR-signaling could lead to successful treatments for KS, validating this receptor-ligand signaling-axis as a therapeutic target. Kaposi\u2019s sarcoma herpesvirus (KSHV) is the etiological agent of Kaposi\u2019s sarcoma (KS) \u20134. KS isin vivo, pointing to the upregulation of various receptor tyrosine kinase signaling axes ..in vitroSince vGPCR is a potent inducer of KS growth factors, we used a Tet-inducible vGPCR construct to mimic the vGPCR-overexpression status of tumor forming mECK36 that display a 160 fold up-regulation of vGPCR mRNA levels. We found that vGPCR upregulates PDGF in mECK36 via a Rac1-NOX-ROS oxidative stress axis . In contThe involvement of the Rac1-NOX-ROS pathway in vGPCR upregulation of PDGFB and in PDGFRA downstream signaling supports the role of oxidative stress in KSHV oncogenesis , 50. ThiIn accordance with the promising results of the Imatinib AIDS-KS trials , 34, phaOur results point to a mechanism for PDGFRA activation in mECK36 tumors mediated by KSHV genes. We found that mECK36 explanted from tumors that have lost KSHV, induced tumorigenesis with very strong PDGFRA activation and low levels of PDGF-AA expression and secretion . We founWe resorted to genetic PDGFRA-signaling blockade using a tyrosine kinase truncated dominant negative mutant as a specific approach to suppress PDGFR signaling , 45. TheThe clinical relevance of our findings to KS is underscored by the analysis of AIDS-KS tumor biopsies that showed co-distribution of phosphorylated PDGFRA to areas infected with KSHV. Analysis of an AIDS-KS TMA showed strong phospho-PDGFRA staining in areas of KSHV-infected LANA+ve spindle cells in ninety percent of the biopsies , indicatThe finding that a mutation occurring in KSHV-negative mouse-KS, which is known to confer Imatinib resistance , 42, sugA major breakthrough in targeted therapies for sarcomas is the recent FDA approval of an anti-PDGFRA therapeutic antibody to treat soft tissue sarcomas ; which, In summary, our results identify a KSHV-dependent-ligand mediated PDGFR activation pathway that drives KS tumorigenesis. We found that this mechanism operates in human KS and in our mouse model can be targeted therapeutically with NAC, Imatinib/Gleevec, Sunitinib or with dominant-negative genetic intervention to block tumorigenesis. We found that host mutations in this pathway could confer resistance to PDGFR inhibition and should be explored for improving treatment decision options. Taken together, our results identify and validate the PDGFR activation axis as a key vulnerability and therapeutic target in KS .mECK36 cells were obtained and cultured as previously described . HEK293TR&D Systems' Mouse Phospho-Receptor Tyrosine Kinase (RTK) Array Kit was used to detect levels of phosphorylation of 39 RTKs in mECK36 tumors.RayBiotech C-Series Mouse Growth Factor Antibody Array Kit , was used to detect 30 Mouse Growth Factors in KSHV+ve mECK36 and KSHV-ve mECK36 tumors.Soluble Platelet-Derived Growth Factor AA (PDGF-AA) and Platelet-derived growth factor subunit BB (PDGF-BB) were determined using American Research Products ELISA kit .Tetracycline-inducible vGPCR construct (TET-vGPCR) was cloned into the TRIPZ vector (Open Biosystems) using AgeI and ClaI restriction sites at the 5\u2019 and 3\u2019 ends, respectively. Tetracycline-inducible control vector (TET-RFP) was purchased from Open Biosystems. Expression constructs: vGPCR was cloned into pcDNA3 vector using EcoRI and XhoI restriction sites; and Rac1QL (constitutively active) and Rac1N17 (dominant negative) were cloned into pcDNA3 using KpnI and XhoI restriction sites. PDGFB-Luc construct was cloned from a 450 bp fragment of the human PDGFB promoter and cloned into pGL2 vector using KpnI and HindIII restriction sites as previously described .2 incubator at 37\u00b0C (dark conditions), in the presence of growth factors and inhibitors if required. Cells were then washed 3 times with HBSS and images in the red channel were taken using a Zeiss ApoTome Axiovert 200M microscope.DHE staining was done as previously . BrieflyHEK293T cells were transfected using Lipofectamine 2000 (Invitrogen). The renilla luciferase plasmid pRL-TK was used as control for transfection efficiency. Transfected cells were assessed for luciferase activity using the Dual-Luciferase Reporter Assay System .\u2013\u0394\u0394CT.RNA was isolated with RNeasy Plus Kit with on-column DNase treatment. 500 ng of RNA was transcribed into cDNA using Reverse Transcription System according to manufacturer's instructions. RT-qPCR was performed using an ABI Prism 7000 Sequence Detection System (Applied Biosystems) with SybrGreen PCR Master Mix (Quanta Biosciences). The following primer sets were used: GAPDH ; LANA ; c-Myc ; VEGFA . In every run, melting curve analysis was performed to verify specificity of products as well as water and\u2013RT controls. Data were analyzed using the \u0394\u0394CT method as previously described . Target Serum-starved mECK36 cells were stimulated with PDGF-BB (40 ng/mL) for 10 min. Rac1 inhibitor EHT1864 (50 \u03bcM) was added to culture media 10 min before PDGF stimulation. Rac1 pull-down assays were performed using the Rac1 Activation Assay Kit following manufacturer\u2019s instructions. Cell lysates were pre-cleared with GST-agarose beads for 20 min at 4\u00b0C. After removal of GST-beads, lysates were incubated at 4\u00b0C for 60 min with p21-activated kinase 1-binding domain (PBD)-agarose beads, then washed four times with MLB buffer and resuspended in 2x Laemmli buffer and resolved in a 12% SDS-PAGE gel. GST-PBD bound active Rac1 (Rac1-GTP) was detected by immunoblotting using a specific antibody against Rac1. Total Rac1 was detected by immunoblotting in samples from corresponding cell lysates.Immunostaining was performed as previously described . BrieflyProtein concentrations in cell and tumor lysates were quantified using the DC Protein Assay . 20 \u03bcg of proteins were mixed with Laemmli buffer, boiled for 5 min, resolved by SDS-PAGE and transferred to PVDF membranes . Membranes were blocked with 5% nonfat milk/PBS for 1 hr and incubated with primary antibodies . After 3 TBS/Tween washes, membranes were incubated with HRP-labeled secondary antibodies for 1 hr at room temperature. Protein bands were developed using ECL Plus Detection Reagents and quantified by densitometry with QuantityOne software . To analyze multiple proteins on the same membrane, membranes were washed with Restore PLUS Western Blot Stripping Buffer according to manufacturer\u2019s protocol.5 cells) as previously described [2\u00d7 0.52][All mice were housed under pathogen-free conditions. Tumor studies were done in 4- to 6- week-old nude mice obtained from the National Cancer Institute. Tumors were generated by subcutaneous injection of mECK36 cells tissue microarray. Immunohistochemistry of clinical tissue microarrays was performed using a standard protocol of the Immunohistochemistry Laboratory of the Department of Pathology at the University of Miami. Antibody staining of p-PDGFRA from R&D Systems was diluted to 1:30 and LANA from Abcam was diluted 1:40. The expression of p-PDGFRA and LANA were classified into three levels depending on the signal strength.Statistical significance of the data was determined using two-tailed Student\u2019s t-test. A p-value lower than 0.05 was considered significant. Statistical analysis was performed using Unistat Statistical Package for Microsoft Excel. All values were expressed as means \u00b1 standard deviation.The animal experiments have been performed under UM IACUC approval number 13\u2013093. The University of Miami has an Animal Welfare Assurance on file with the Office of Laboratory Animal Welfare (OLAW), National Institutes of Health. Additionally, UM is registered with USDA APHIS. The Council on Accreditation of the Association for Assessment and Accreditation of Laboratory Animal Care has continued the University of Miami\u2019s full accreditation.S1 Fig(A-B) Proliferation of serum-starved mECK36 cells stimulated with PDGF-BB (40 ng/mL) in the presence of increasing concentrations of Imatinib (A) and Sunitinib (B) for 24 hs. (C-D) VEGF secretion of serum-starved mECK36 cells stimulated with PDGF-BB (40 ng/mL) in the presence of increasing concentrations of Imatinib (C) and Sunitinib (D) was determined by ELISA. (E-F) Phosphorylated and total PDGFR levels of serum-starved mECK36 cells stimulated with PDGF-BB (40 ng/mL) in the presence of increasing concentrations of Imatinib (E) and Sunitinib (F) were determined by immunoblotting.(TIF)Click here for additional data file.S1 TableInhibitory data is expressed as IC50 in nM concentration.(TIF)Click here for additional data file."} +{"text": "There exists a general recognition of the fact that mitochondrial remodelling as a result of aerobic glycolysis ensures human somatic cells to revert to a more primitive-form exhibiting stem-like phenotype. The present study is an attempt to demonstrate that miR-2909 RNomics within human peripheral blood mononuclear cells (PBMCs) has the inherent capacity to re-program these cells to exhibit mitochondrial remodelling paralleled by aerobic glycolysis together with intracellular lipid inclusions. Such re-programmed PBMCs also expressed genes having ability to sustain their de-differentiation state and survival.Glut-1), hexokinase (HK), hypoxia inducia factor-1 (HIF-1\u03b1), c-Myc, p53,mechanistic target of rapamycin complex (mTORC1), polycombcomplex protein (Bmi-1), Notch,Nanog,Tie-2, Oct-4,CD59, p53, CD34, B-cell lymphoma-2 (Bcl2),sterol regulatory element-binding protein2 (SREBP2), peroxisome proliferator-activated receptor gamma (PPAR\u03b3) nuclear respiratory factor 1 (NRF1), mitochondrial transcription factor A (Tfam), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1\u03b1) within miR-2909 expression vector transfected human PBMCs as well as PBMCs transfected with control vector containing scrambled sequence after 48h post-transfection using RT-qPCR and cellular ultrastructural features induced by miR-2909 ectopic expression were observed using transmission electron microscopy and morphometric analysis of an electron micrograph.Human PBMCs were programed to ectopically express miR-2909. Expression levels of genes including glucose transporter-1 , quantification of ATP levels, GSSG/GSH ratio, mitochondrial cytochrome Based upon these results we propose that AATF gene-encoded miR-2909 may act as an epigenetic switch for cellular aerobic-glycolysis to ensure de-differentiation. HIF-1\u03b1) and c-Myc but also promotes lipogenesis through the upregulation of sterol regulatory element-binding protein2 (SREBP-2) gene [Although the underlying epigenetic mechanism that governs the reprogramming of somatic cells into a stem-like phenotype is far from clear several recent findings have revealed that mitochondrial maturation and remodelling drives these somatic cells into either a differentiated or dedifferentiated state . Upon re-2) gene ,7. The t-2) gene as well -2) gene ,10. A ne-2) gene . Various-2) gene ,13. Howe-2) gene . Further5 cells per well in 6 well culture plate and were maintained in RPMI-1640 culture medium containing 10% FCS, at 37\u00b0C in 5% CO2 atmosphere.The present study included 30 healthy volunteers who had come to donate blood in Blood Transfusion Department of our Institute hospital. Approximately 10 ml of blood was withdrawn from the subjects after their informed consent on prescribed format approved by our Institutional Ethics Committee, Postgraduate Institute of Medical Education and Research (PGIMER), Chandigarh. The Institutional Ethics Committee evaluated and provided ethics clearance/approval for the submitted project and the design of this study in accordance with the principles outlined in the declaration of Helsinki . The incGenomic DNA was extracted from the mononuclear cells by standard phenol-chloroform procedure . For conUCP2), the present study was directed to explore whether or not the miR-2909 RNomics has the ability to regulate genes that play crucial role in cellular energy metabolism , dedifferentiation , apoptosis (Bcl2) and intracellular lipid synthesis and storage , transcriptional regulators of mitochondrial OXPHOS genes .The primers\u2019 sequences used in this study are listed in Keeping in view our recent findings regardinc oxidase activity and secreted lactate concentrations in human PBMCs transfected with either miR-2909 expression vector or control vector after 48h post-transfection using their respective kits. Cells were lysed using cell lysis buffer supplied by Biovision. ATP levels were quantified using ATP kit according to the manufacturer's instructions. ATP values were normalized to protein concentrations quantified with the Bradford assay protocol [c oxidase activity was measured using the cytochrome c oxidase assay kit supplied by Biovision using automatic plate reader .Bradford assay was used to quantify the protein concentration. Secreted lactate concentrations in cell culture media of control and miR-2909 transfected PBMCs were determined by using L-Lactate Assay Kit . The fluorescent product was analyzed with an excitation wavelength of 530\u2013540 nm and an emission wavelength of 585\u2013595 nm using automatic plate reader . Lactate values were normalized to protein concentrations quantified with the Bradford assay protocol [To scrutinize the role of miR-2909 on mitochondrial function, various parameters include quantification of ATP levels, mitochondrial cytochrome protocol . Mitochoprotocol .N- to glutathione reduced (GSH), activity of antioxidant enzymes; superoxide dismutase (SOD), reactive oxygen species (ROS) coupled withnitric oxide (NO) production were measured in human PBMCs that were ectopically overexpressing miR-2909 as well as human PBMCs transfected with null vector containing scrambled sequence. GSSG to GSH ratio was determined using GSH/GSSG Ratio Detection Assay Kit II; Fluorometric\u2014Green (Abcam). GSH (reduced) and total GSH (GSH + GSSG) was measured directly with their standards provided by the kit. GSSG was then calculated by subtracting the GSH amount from the total GSH + GSSG amount, thus providing the final GSH:GSSG ratio. Fluorescence was monitored at Ex/Em = 490/520 nm with Infinite M200 Microplate Reader (Tecan). ROS was detected using the dye DCFH-DA according to the procedure described by Wu and Yotnda, 2011 . InhibitTo assess the role of miR-2909 in the formation of intracellular lipid droplets, levels of esterified cholesterol and triglycerides were estimated. Control and miR-2909 transfected PBMCs were lysed using cell lysis buffer supplied by Biovision. The levels of cholesteryl esters were determined by subtracting the value of free cholesterol from the total (cholesterol plus cholesteryl esters) by using Total Cholesterol Assay Kit (Cell Biolabs). The levels of triglyceride in control and miR-2909 transfected PBMCs were estimated using the commercial kit provided by Kee Diagnostics. Total protein was estimation by BCA assay.Cellular ultrastructural features were observed using transmission electron microscopy. After 48 h post-transfection, human PBMCs transfected with either miR-2909 expression vector or control vector were fixed in 3% glutaraldehyde in sorenson\u2019s phosphate buffer, pH 7.2 for 4\u20136 hr followed by secondary fixation in 1% osmium tetraoxide in Millonig\u2019s phosphate buffer pH 7.2. Cells were processed and embedded in Taab-812 embedding medium. Ultrathin sections of 60nm thickness were cut on Leica EMUC-6 ultra-microtome and taken on nickel grids. Ultrathin sections were stained with uranyl acetate and lead citrate and examined at 80kV accelerating voltage at JEOL Transmission Electron Microscope; JEM-1400 Plus equipped with XR81M-B Camera . Digital electron micrographs of control and miR-2909 transfected PBMCs were acquired using AMT Image Capture Engine V602 software using Image J software equipped with XR81M-B Camera. The volume density (Vv) of mitochondria was expressed as percent volume: Vv = (Pm/Pc)*100 (%), where Pm is the pixel size occupied by each mitochondrial structure and Pc is the total cell mass in pixel size occupied by the cell ,23.p < 0.01 and p<0.05.Statistical analyses were performed by SPSS window version 19. Data was expressed as mean \u00b1 SD of all the experiments done independently. Unpaired Student\u2019s T test was used for comparison between two groups. The difference was considered statistically significant when UCP2) gene which favours aerobic glycolysis as the energy driving force for such cells. The present study revealed that the miR-2909 ectopic expression within human PBMCs , hexokinase (HK) that have intrinsic ability to favour aerobic glycolysis. This phenomenon induced by miR-2909was paralleled by downregulation of p53 c oxidase activity. Such a study revealed that ectopic miR-2909 expression within human PBMCs reduced their cytochrome c oxidase activity by about 4-fold ratio as well as lipid inclusion bodies , the prep53 and mitochondrial uncoupling protein (UCP2) assumes importance because UCP2 initiates aerobic glycolysis by shunting pyruvate out of mitochondria [p53 expression restricts aerobic glycolysis and favours aerobic respiration [p53 gene expression as well as influences p53 protein stability through the induction of genes coding for c-Myc and Bmi-1 [AATF, c-Myc, UCP2 and Bmi-1 as well as downregulation of p53 gene [AATF gene and its encoded miR-2909 ensures regulation of UCP2 in a cyclic fashion [c oxidase (COX) activity recognized widely as the rate-limiting step in cellular aerobic-respiration [mTORC1 gene ensures enhanced cellular lipogenesis (via its positive regulation of SREBP2) as well as increased formation of intracellular lipid droplets rich in esterified cholesterol and triglycerides via positive regulation of PPAR\u03b3 gene as wellene Figs . This phogenesis ,34. All S1 Table(DOCX)Click here for additional data file.S1 Fig(A) Decrease in OD at 550nm was observed in human PBMCs transfected with null vector containing scrambled sequence in contrast to no change in OD in human PBMCs transfected with miR-2909 expression vector. The decrease in OD was recorded over a period of 45 min using automatic plate reader. The rate of the reaction was calculated in the linear range by subtracting the initial OD reading from the final OD, t1 and t2 represents linear rate of reaction. (B) Flow-cytometric detection of reactive oxygen species (ROS) generation in human PBMCs transfected with null vector containing scrambled sequence compared with corresponding control cells transfected with miR-2909 expression vector.(DOCX)Click here for additional data file.S2 FigqRT-PCR analysis for relative expression of miR-2909 in human PBMCs transfected with null vector containing scrambled sequence, human PBMCs transfected with miR-2909 expression vector and human PBMCs co-transfected with miR-2909 expression vector and antagomiR-2909 expression vector.(DOCX)Click here for additional data file.S3 Fig(A) Representative TEM images indicating N/C ratio in human PBMCs transfected with null vector containing scrambled sequence, (B) human PBMCs transfected with miR-2909 expression vector and (C) human PBMCs co-transfected with miR-2909 expression vector and antagomiR-2909 expression vector. N/C ratio was calculated using Image J software.(DOCX)Click here for additional data file.S4 Fig(A-B) Globular shaped mitochondria (M) coupled with cristae-poor morphology in human PBMCs transfected with miR-2909 expression vector in contrast to normal elongated mitochondrial morphology with well aligned lamellar cristae in human PBMCs transfected with control null vector containing scrambled sequence. (C) However no appreciable change in mitochondrial morphology was observed in healthy PBMCs co-transfected with miR-2909 expression vector and antagomiR-2909 vector. (D-E) Increased formation of intracellular lipid inclusion bodies (LI) and myelin figures (MF) in human PBMCs transfected with miR-2909 expression vector compared with human PBMCs transfected with control null vector containing scrambled sequence. (F) However on co-transfection of human PBMCs with E2F-miR-2909 expression vector and antagomiR-2909 vector around 60% of cells showed depletion of intracellular lipid inclusion bodies. Scale 800nm; Final Magnification 14110X. 50cells/case was studied under TEM for the observation of apoptotic changes in human PBMCs transfected with above-mentioned expression vectors after 48h incubation period. We observed that few cells under TEM were indicating early apoptotic changes and formation of phagolysosome (PL) in human PBMCs transfected with control null vector containing scrambled sequence and human PBMCs co-transfected withmiR-2909 expression vector and antagomiR-2909 expression vector (H) however no apoptotic changes were observed in healthy PBMCs transfected with miR-2909 expression vector.Representative images of electron microscopy (TEM) of human PBMCs transfected with null vector containing scrambled sequence, human PBMCs transfected with miR-2909 expression vector and human PBMCs co-transfected with miR-2909 expression vector and antagomiR-2909 expression vector. (DOCX)Click here for additional data file."} +{"text": "The purpose of this study was to conduct an 8-year follow-up of the National Elder Mistreatment Study (NEMS) and specify risk ratios for negative outcomes of elder abuse, including DSM-5 defined depression, generalized anxiety disorder (GAD), post-traumatic stress disorder (PTSD), and poor self-reported health. Methodology: Attempts were made to re-contact, via Computer Assisted Telephone Interview, all 752 NEMS participants who reported mistreatment since age 60 at Wave I, as well as a randomly selected sample of non-mistreated NEMS participants Results: 183 NEMS Wave I elder abuse victims and 591 non-victims provided data. In bivariate analyses, elder mistreatment 8 years earlier increased risk of negative outcomes by 200-700%. However, multivariate analyses revealed that Current (Wave II) social support was highly protective against most negative outcomes (excepting PTSD), and even appeared to nullify effects of mistreatment on GAD and poor self-reported health. Conclusions: Outcomes of elder mistreatment have not been studied prospectively in a national sample. The NEMS 8-year follow-up findings indicate a strong relationship between elder mistreatment at Wave I and negative emotional and physical health 8 years later. Fortunately, current (Wave II) social support appears to be both consistently and powerfully protective against most negative outcomes."} +{"text": "MicroRNA-21 (miR-21), probably one of the most studied miRNAs to date, is found pleiotropic in various biological events. Its emerging role in pulmonary remodeling has attracted extensive attention. This review summarizes the genomic information of its primary transcript and various transcriptional regulations on its promoter. In addition, the role of miR-21 in pulmonary remodeling related signaling such as transforming growth factor \u03b2 (TGF-\u03b2), bone morphogenetic protein (BMP), epidermal growth factor receptor (EGFR) and Notch signaling is discussed. Various validated miR-21 target genes participate in controlling of the overactive cell accumulation, smooth muscle contraction, inflammatory stress (trigger for lung epithelium damage), extracellular matrix deposition and hypoxia-induced disorders. Moreover, we focus on its particular implication in events including inflammatory stress-driven epithelium damage, epithelial-to-mesenchymal transition (EMT), transdifferentiation of fibroblasts into myofibroblasts, hypoxia stimuli and ROS response, as well as some other pulmonary remodeling related events such as overactive fibroblast (myofibroblast) accumulation, extracellular matrix deposition, and angiogenesis. Here, we summarize the strong potential of miR-21 in pulmonary remodeling and provide novel clues for further research in this area. MicroRNA-21 (miR-21) is a pleiotropic miRNA with its frequent appearance in researches concerning the promotion of cell proliferation, inflammation, angiogenesis, and immune destruction. Its significance in molecular function has been focused on the strong implication in human cancers , head anIt was found that miR-21 is also strongly implicated in pulmonary inflammation in our previous study . As the Plenty of evidence indicated that miR-21 might play an important role in pathological remodeling. Our review aimed to summarize the regulation potential of miR-21 in pathological pulmonary remodeling and provide novel clues for further research.According to the recently released information on miRBase , the sinNuclear factor-kappa B (NF-\u03baB) p65 subunit direct binds to pri-miR-21 promoter, which is identified by chromatin immunoprecipitation (ChIP) analysis and confGfi1\u2212/\u2212 mice display overexpression of miR-21 [miR-21 transcription is found to be repressed by NFI and C/EBP\u03b1. NFIB protein usually binds to the miR-21 promoter as a negative regulator in HL-60 human promyelocytic cells. During miR-21 independent suppression of NFIB, NFIB is swept off from the promoter, and as a miR-21 direct target protein, NFIB expression is also inhibited at translational level . BCL-6 if miR-21 .1 arrest and more sensitivity to cisplatin in MCF-7 breast cancer cells [miRNAs undergo a classical cropping and dicing process before they become mature and functional molecules . Firstlyer cells . TGF-\u03b2 aer cells .As we mentioned above, there are multiple players involved during the transcription and biogenesis of miR-21 . Both signaling pathways are crucial for lung vasculature remodeling . TGF-\u03b2 aEGFR gene is associated with airway hyper-reactivity (AHR) in patients . EGFR acNotch signaling pathway plays a crucial role in angiogenesis and vascular remodeling. mRNA expressions of Notch 1 receptor and downstream factors are induced with a peak at 1\u20132\u00a0weeks in pulmonary vascular remodeling of rats with pulmonary hepertension . Its sigThese observations indicated that transcriptional regulation on miR-21 could be an important mechanism to understand the role of TGF, BMP, EGFR and Notch signaling during pulmonary remodeling.Till now, mounting putative miR-21 targets have been validated based on experimental evidence. Among them, several targets also play their role in multiple events of pulmonary remodeling -phosphatase that inhibits phosphoinositide-3-kinase (PI3K) pathway, blocks AKT signaling activation and hence acts as a tumor suppressor. PTEN is initially identified as a miR-21 target in Mz-ChA-1 human cholangiocarcinoma cells [Phosphatase and tensin homolog (PTEN), a phosphatidylinositol-3,4,5-trisphosphate 3 (PIPma cells . Besidesma cells .Sprouty homolog (spry) suppresses proliferation through extracellular response kinase (ERK)-mitogen activated protein kinase (MAPK) signaling. Spry 1 and 2 are expressed both in epithelial and peripheral mesenchymal cells of the lung during embryonic development . NegativAs we mentioned, among multiple validated miR-21 targets, the PTEN and SPRY were considered as crucial players in regulating hyperproliferation during different pathogenesis. Their involvement might help us understand the role of miR-21 during overactive fibroblast accumulation.Tropomyosin 1 (TPM1), a rod-like helical protein that dimerizes and binds to actin, controls smooth muscle contraction and relaxation, and thus has a major role in the regulation of cell shape and function . It is iLung epithelium damage induced by inflammatory stress is a major trigger for pulmonary remodeling. As we know, during this process, the skewed Th1/Th2 balance is entirely responsible. Interleukin-12 (IL-12) is a heterodimer composed of the 35kD subunit named IL-12p35 (IL-12A) and another 40kD subunit. IL-12 is required for T-cell-independent IFN-\u03b3 induction. Hence it is considered as a molecule germane to Th1 cell polarization. Recently, IL-12p35 is identified as a direct miR-21 target, and its expression reduction is found in asthmatic mice lungs .Programmed cell death 4 (PDCD4) promotes apoptosis and positively regulates E-Cadherin and tissue inhibitor of metalloproteinase 2 (TIMP2) . Three SIt was possible that the dysregulated miR-21 could repress its target IL-12p35 and PDCD4 hence contribute to an imbalanced Th1/Th2 and M1/M2 polarization and the inflammatory stress driven epithelium damage, which eventually leads to the pulmonary remodeling.Matrix metalloproteinase (MMPs) related tissue damage is crucial in extracellular matrix deposition. Reversion-inducing-cysteine-rich protein with Kazal motifs (RECK) participates in tumor metastasis and angiogenesis by modulating matrix metalloproteases (MMPs) including MMP 2, 9, 14, and is validated as a target of miR-21 in gastric cancer cells . RECK isPeroxisome proliferator-activated receptor-\u03b1 (PPAR-\u03b1), a nuclear receptor acts as a translational factor which is involved in cell proliferation, cell differentiation and inflammation responses, is identified as a miR-21 target, and may participate in hypoxia-induced PASMC proliferation . More inPreviously, miR-21 was found to strongly implicated in fibrosis of various tissues including lung . In this+ T cells produce more IFN-\u03b3 and less IL-4. The evidence demonstrates that miR-21 might act as a major regulator of Th1 vs. Th2 responses [Alveolar epithelium plays a vital role in maintaining lung homeostasis. However, inflammatory stress and physical injury may cause epithelium damage and hence trigger airway remodeling . The skeesponses .1 induced primary parenchymal lung fibroblasts based on microarray data and further validated using RT-qPCR [In tissue remodeling, fibroblast is very likely to derive from epithelial or endothelial cells via epithelial-to-mesenchymal transition (EMT) or endothelial-to-mesenchymal transition (EndMT). During lung remodeling, the contribution of alveolar epithelial cells (AECs) to effective myofibroblast through EMT process has also been well acknowledged . TGF-\u03b2 s RT-qPCR . For nowThe transdifferentiation of fibroblasts into myofibroblasts (MFs) plays an essential part in tissue remodeling. Pathological remodeling can be induced by TGF-\u03b2 cytokine or triggered by injury. In vascular remodeling, miR-21 is found to be overexpressed in MFs with a 4.8-fold change against adventitial fibroblasts (AFs) expression. Overexpression of miR-21 by using pre-miR-21 treatment increases the proliferation and decreases apoptosis of AFs and MFs. Furthermore, miR-21 inhibition can reverse the vascular remodeling induced by balloon injury . In card1-induced fibroblast-to-myofibroblast transdifferentiation in cancer stroma by targeting PDCD4. miR-21 is upregulated in TGF-\u03b2 and cancer medium activated fibroblasts. Gain or loss of function assay further demonstrates the effect of miR-21 on TGF-\u03b21-induced MF transdifferentiation [1 enhances miR-21 in primary pulmonary fibroblasts, and more interestingly, this elevated miR-21 expression can amplify a circuit which eventually leads to TGF-\u03b21 signaling activation by negatively targeting an inhibitory SMAD protein, the SMAD7 [miR-21 also participates in TGF-\u03b2ntiation . Up-reguntiation . Immunohhe SMAD7 .Here, mounting evidence seems to suggest that up-regulation of miR-21 could lead to the transdifferentiation of fibroblasts into myofibroblasts, which is the cellular signature for the EMT process during pulmonary remodeling.2), and miR-21 can induce proliferation in normoxia condition. Reduction of miR-21 leads to a significant decrease in hypoxia-induced cell proliferation [One of the most critical lung functions is to maintain adequate oxygen. Chronic hypoxia is an important trigger for pulmonary vascular remodeling. Hypoxia favors ROS and NOS production causing pulmonary oxidative stress . Hypoxiaferation . Moreoveferation .2O2) induces apoptosis and cell death, as well as the up-regulation of miR-21. Pre-miR-21 plays a protective role in VSMC apoptosis by targeting PDCD4 [2O2 mediated injury [In vascular smooth muscle cells (VSMC), hydrogen peroxide (Hng PDCD4 . miR-21 d injury . In humad injury . Nitric d injury .Such evidence indicates that dysregulated miR-21 could actively participate in hypoxia stimuli and ROS response during pulmonary remodeling.In other pulmonary remodeling related events such as overactive fibroblast (myofibroblast) accumulation, extracellular matrix deposition, angiogenesis and tissue regeneration, there is also plenty of observation support that miR-21 might also play a crucial part.In bleomycin-treated lungs , increased miR-21 expression is primarily localized in the sheets of the lung parenchymal with fibroblast/MF accumulation . miR-21 Transcriptional regulation on miR-21 could be an essential mechanism to understand the role of TGF, BMP, EGFR and Notch signaling during pulmonary remodeling. Several miR-21 targets play their role in multiple events of pulmonary remodeling, e.g., the overactive cell accumulation, smooth muscle contraction, inflammatory stress (trigger for lung epithelium damage), extracellular matrix deposition and hypoxia-induced disorders. Dysregulated miR-21 expression profile was found in many aspects of pulmonary remodeling including inflammatory stress-driven epithelium damage, EMT transition, transdifferentiation of fibroblasts into myofibroblast, hypoxia stimuli and ROS response, as well as some other pulmonary remodeling related events.In summary, miR-21 might play a crucial role in pulmonary remodeling, which makes it a promising candidate miRNA for further study. We tried to follow many clues for its role in pulmonary remodeling. However, the direct proof is still in need. Hence, our work calls for the experimental investigation into above-mentioned aspects and hope such novel clues could be quite useful for further research in this area."} +{"text": "DNA methylation (DNAm) based biomarkers collectively known as \u201cepigenetic clock\u201d yielding accurate measure of age in any tissue across the entire life course have associated with a large host of age-related conditions. To study their implications in metabolic syndromes, we analyzed computed tomography (CT) derived measures of adiposity with 1) three widely-used clocks: Horvath, Hannum, and Levine (PhenoAge), and a newly developed clock DNAmGrimAge, and 2) seven DNAm based plasma proteins, the components comprising the DNAmGrimAge. Age-adjusted DNAmGrimAge and several age-adjusted DNAm plasma proteins including plasminogen activation inhibitor 1 (PAI-1) stand our followed by adrenomedullin and leptin. Fatty liver and excess visceral adipose tissue fat are the most significant CT-based measures of DNAmPAI-1 and DNAmGrimAge. Furthermore, age-adjusted DNAmGrimAge and age-adjusted DNAmPAI-1 continued showing the most significant relationship with lifestyle factors including healthy diet and educational attainment in expected directions."} +{"text": "Rationale: The sustained and severe endoplasmic reticulum (ER) stress in cancer cells may contribute to apoptotic cell death, thus representing a potential target for cancer therapy. Brigatinib is an anaplastic lymphoma kinase (ALK) inhibitor approved for the treatment of ALK-positive non-small-cell lung cancer (NSCLC). However, it remains unclear if brigatinib has alternative model of action to exert antitumor effect in ALK-negative cancers.Methods: ALK-positive NSCLC cells and various human ALK-negative cancer cells, especially human colorectal cancer (CRC) cells were used to examine the tumor suppression effect of brigatinib alone or in combination with autophagy inhibitors in vitro and in vivo. A variety of biochemical assays were conducted to elucidate the underlying mechanisms of brigatinib in CRC cells.Results: Here, we show the significant anti-cancer effect of brigatinib in CRC through induction of apoptosis by sustained ER stress. Mechanistically, brigatinib induces ER stress via promoting the interaction between ubiquitin-specific peptidase 5 (USP5), a deubiquitinase, and oxysterol-binding protein-related protein 8 (ORP8), leading to ORP8 deubiquitination, accumulation and growth inhibition. Furthermore, we find that brigatinib-mediated ER stress simultaneously induces autophagic response via ER-phagy that acts as a protective mechanism to relieve excessive ER stress. As such, combination of brigatinib with autophagy inhibitors significantly enhances the anti-CRC effect of brigatinib both in vitro and in vivo, supporting the repurposing of brigatinib in CRC, independently of ALK.Conclusion: The unearthed new molecular action of brigatinib suggests that therapeutic modulation of ER stress and autophagy might represent a valid strategy to treat CRC and perhaps other ALK-negative cancers. Endoplasmic reticulum (ER) stress and unfolded protein response (UPR) have attracted much attention as therapeutic targets for cancer treatment in recent years Macroautophagy is responsible for the degradation and recycling of macromolecules and organelles to maintain cellular homeostasis and can be activated in stress conditions, including ER stress Repurposing existing drugs offers a cost-effective and time-saving approach for drug discovery in cancer therapy. Drug repurposing is based on two concepts: on-target repurposing (finding new therapeutic indications based on relevant mechanisms) and off-target repurposing (identifying new targets for known compounds) in vitro and in vivo. Meanwhile, ER-phagy is stimulated to relieve ER stress in brigatinib-treated CRC cells. Collectively, our results provide novel insights into the molecular basis for the anti-cancer effect of brigatinib and demonstrate the potential of brigatinib for the treatment of ALK-negative cancer.In this study, we found an ALK-independent anti-cancer mechanism of brigatinib in colorectal cancer (CRC). Brigatinib stimulates ER stress by USP5-mediated ORP8 stabilization to promote apoptotic cell death of CRC cells both 2. Human CRC cell lines , human colon mucosal epithelial cell line NCM460, human NSCLC cell line A549, human HCC cell line Hep3B and human prostate cancer cell line Du145 were purchased from ATCC . NSCLC cell lines H3122 and H2228 were kindly provided by Prof. Yong Peng . Du145, H3122 and H2228 cell lines were cultured in RPMI Medium 1640, other cell lines were cultured in high glucose Dulbecco's Modified Eagle Medium and were supplemented with 10% FBS (Biowest), 100 U/mL penicillin, 100 U/mL streptomycin (Hyclone).Cell culture was performed at 37\u00b0C in a humidified atmosphere containing 5% COAll commercial antibodies used in this study were obtained from the following suppliers: BiP (sc-376768), CHOP (sc-56107), PERK (sc-377400), p-PERK (sc-32577), IRE1\u03b1 (sc-390960), ORP8 (sc-134409), USP5 (sc-390943), JNK (sc-7345), p-JNK (sc-81502), horseradish peroxidase-conjugated anti-rabbit secondary antibody (sc-2004) and horseradish peroxidase-conjugated anti-mouse secondary antibody (sc-2005) were obtained from Santa Cruz Biotechnology; p-IRE1\u03b1 (ab48187) and HA (ab9110) were obtained from Abcam; LC3 (NB100-2220) was obtained from Novus; FAM134B (PA5-64943), goat anti-mouse Alexa Fluor 488 (A28175), goat anti-rabbit Alexa Fluor 488 (A27034), goat anti-mouse Alexa Fluor 594 (A21044) and goat anti-rabbit Alexa Fluor 594 (A32740) were obtained from Invitrogen. PARP (9532), Cleaved PARP (5625), Caspase-3 (9662), Cleaved caspase-3 (9664), ATG5 (12994S), ATG7 (8558S), Beclin1 (3738), ATG14 (96752) and Bcl-2 (15071) were obtained from Cell Signaling Technology.The following commercial chemical reagents were obtained from the following suppliers: brigatinib (HY-12857), Z-VAD(OMe)-FMK (HY-16658), 3-Methyladenine (HY-19312), bafilomycin A1 (HY-100558), 5-FU (HY-90006) and Cycloheximide (HY-12320) were obtained from MedChemExpress; 4-PBA (1716-12-7) and chloroquine diphosphate salt (C6628) were obtained from MilliporeSigma. All chemicals were handled in accordance with the supplier's recommendations.The growth of brigatinib-treated cells was determined using the MTT assay. Cells were plated in 96-well plates at 5,000 cells per well and subjected to different treatments. The detailed procedure has been previously described Lactate dehydrogenase release was used to detect cytotoxicity following different treatments, using a lactate dehydrogenase (LDH) test kit . Studies were performed according to the supplier's instruction.The DeadEndTM Fluorometric TUNEL system (Promega) was used to detect apoptotic cells according to the manufacturer's instructions. The apoptotic and non-apoptotic signals were recorded using a fluorescent microscope and the percentage of cells with DNA nick end-labelling evaluated.The apoptotic ratio was determined with a FITC-Annexin V Apoptosis Detection kit . In brief, cells were harvested and washed once with PBS, and then resuspended in PI/Annexin-V solution for apoptosis analysis. The detailed procedures were performed according to the corresponding manufacturer's instructions. At least 10,000 live cells were analyzed on a FACSCalibur flow cytometer (Becton Dickinson). Data were analyzed by using FlowJo software.Cells were rinsed in precooled PBS and lysed in RIPA lysis buffer . Equal amounts of soluble proteins (15-30 \u03bcg) were used for immunoblotting analysis. For immunoprecipitations, cells were lysed with IP lysis buffer . The whole-cell lysates were subjected to immunoprecipitation overnight at 4\u00b0C with 1 \u03bcg of the indicated antibodies, and the immunoprecipitated protein was pulled down with Protein A agarose beads for 4 h. Proteins were visualized with Immobilon Western HRP Substrate . The images were obtained using a ChemiScope 6000 Touch .3 cells/well). After being fixed with 4% paraformaldehyde (Sigma) for 30 minutes, cells were permeabilized with 0.4% Triton X-100 followed by blocking with 5% fetal bovine serum after being washed 3 times with PBS. The treated cells were then incubated with the indicated primary antibodies and Alexa Flour secondary antibodies. Lastly, cells were visualized using a Zeiss LSM 510 confocal microscope. For ER labelling, live cells were previously stained with 100 nM ER-Tracker Blue for 30 minutes at 37\u2103.Cells were cultured on glass coverslips overnight and treated with different agents in 24-well plates and were transfected with Lipofectamine 3000 reagent (Thermo Fisher Scientific) for 48 h according to the manufacturer's protocol. The sequences of siRNA involved in this study were as follows: siThe human FAM134B coding region with C-terminal HA tag was cloned using PCR and was ligated into the pcDNA3.1(+) vector. The origin PCR primers for wild type FAM134B sequence were as follows: Sense primer: 5'-CCCGGATCCATGGCGAGCCCG-3'; Anti-sense primer: 5'-CCGCTCGAGTTAATGGCCTCCCAG-3'. The PCR primers for LIR-motif mutant type FAM134B sequence were as follows: Sense primer: 5'-ACTGAAGAAGGTGCTGCCGCTGCAGCAGCTGACCAGTCAGAG-3'; Anti-sense primer: 5'-GCTGCTGCAGCGGCAGCACCTTCTTCAGTGTCTGTGTCCTC-3'.7 cells/mouse) were suspended in PBS and injected subcutaneously into mice. When the tumor volume reached ~100 mm3, the mice were randomized into vehicle and treatment groups. The mice were treated once daily by oral gavage . Tumor volumes were measured each day and evaluated according to the following formula: tumor volume (mm3) = (length\u00d7width2)/2. When significant differences between each group were obtained, the mice were euthanized and tumor tissues isolated and fixed immediately in 10% formalin. All animal experiments were approved by the Institutional Animal Care and Treatment Committee of Sichuan University.Female nude mice at 6 weeks of age were purchased from HFK Bioscience . For the subcutaneous xenograft model, DLD-1 cells . One-way ANOVA or Student's t test was used to analyze statistical differences. A value of To examine whether brigatinib exhibits a growth inhibition effect in ALK-negative cancer cells, ALK-positive NSCLC cell line (H3122 and H2228) and various ALK-negative cancer cells lines were treated with brigatinib. As shown in Figure in vitro.To ascertain the anti-cancer effect of brigatinib against CRC cell lines, cell growth was assessed following brigatinib treatment in a variety of CRC cell lines and a human colon mucosal epithelial cell line, NCM460. As expected, all tested CRC cells were sensitive to brigatinib at 2 \u03bcM for 24 hours, whereas NCM460 cells demonstrated higher tolerance to brigatinib in ALK-negative cancer cell lines Figure A, but noNext, we set out to investigate the mechanism underlying brigatinib-induced ER stress. Phospholipids, the main component of biological membranes, play a crucial role in the functional and structural aspects of ER ORP8 significantly countered brigatinib-induced up-regulation of ER stress markers and activation of caspase time-course analysis showed that brigatinib reduced the rate of ORP8 degradation Figure E-F. We tATG5, ATG7 or BECN1 silencing prominently decreased LC3B lipidation in brigatinib-treated cells than autophagosomes resulted in a further increase in LC3B lipidation Figure H. Moreov) Figure C-D. TakeORP8 or combined with 4-PBA. As shown in Figure ORP8 or 4-PBA obviously prevented brigatinib-induced up-regulation of autophagy-related proteins. In contrast, an early autophagy inhibitor had no obvious effect on the expression of classic ER signaling protein markers in brigatinib-treated cells , the first-line chemotherapeutic drug for CRC treatment, by performing the Chou-Talalay method. The combination index (CI) was calculated to evaluate the synergism (CI < 1) or antagonism (CI > 1) for each drug combination. Our results showed that 1 \u03bcM brigatinib combined with 25 \u03bcM 5-FU exhibited remunerative anti-cancer effect in CRC cells as evidenced by reduced cell growth and colony formation. In addition, the CI value of brigatinib combined with 5-FU treatment also showed the sufficient synergy Figure A-C. TakeBrigatinib is an ALK inhibitor used to treat ALK-positive NSCLC. In this study, we demonstrate its anti-cancer effect and the underlying mechanisms in CRC which is ALK-negative. We show that brigatinib triggers apoptosis in CRC via the induction of ORP8/USP5-mediated ER stress. In addition, we show that brigatinib activates ER stress-induced UPR to provoke both apoptosis and protective autophagy/ER-phagy Figure . CombinaDrug repurposing, which has been proposed as a strategy for developing new therapies, offers a cost-effective and time-saving approach for developing promising drug for cancer therapies in vitro and in vivo. This combinatorial treatment showed a comparable growth inhibition efficacy to that in ALK-positive NSCLC cells. In addition, blockage of ER-phagy by FAM134B knockdown also aggravated cell death induced by brigatinib treatment, suggesting that combinatorial treatment of brigatinib with ER-phagy inhibitor might benefit ALK-negative cancer patients. Moreover, targeting ER-phagy is more specific than targeting autophagy, which implicates notable advantage through avoiding potential side effects induced by autophagy inhibition.ER-phagy, a selective autophagy process targeting the ER, requires the activation of UPR and the core autophagy machinery Our study uncovered a novel mechanism of the USP5/ORP8 axis in the regulation of ER stress induced by brigatinib in CRC cells. USP5, a deubiquitinase, cleaves both linear and branched multi-ubiquitin polymers to influence proteasomal protein degradation in vitro and in vivo, and excessive ER stress might be a pivotal molecular event for tumor suppression in brigatinib-treated CRC cells. Interestingly, autophagy/ER-phagy played a protective role in response to ER stress and ameliorated the efficacy of brigatinib, whereas inhibition of autophagy/ER-phagy potentiated its anti-cancer effect. These results provided novel insights into the mechanism underlying brigatinib-induced CRC suppression, implying that brigatinib could act as a potential therapeutic agent in CRC and highlighting the possibility of autophagy/ER-phagy inhibition in optimizing cancer therapeutics.Taken together, our results indicate that brigatinib could be a promising anti-cancer drug for CRC therapy both Supplementary figures.Click here for additional data file."} +{"text": "Drosophila melanogaster accessory glands secrete seminal fluid components that enhance fecundity. In humans, the prostate, stimulated by environmentally regulated endocrine and local androgens, grows throughout adult life. We previously showed that in fly accessory glands, secondary cells (SCs) and their nuclei also grow in adults, a process enhanced by mating and controlled by bone morphogenetic protein (BMP) signalling. Here, we demonstrate that BMP-mediated SC growth is dependent on the receptor for the developmental steroid ecdysone, whose concentration is reported to reflect sociosexual experience in adults. BMP signalling appears to regulate ecdysone receptor (EcR) levels via one or more mechanisms involving the EcR\u2019s N terminus or the RNA sequence that encodes it. Nuclear growth in virgin males is dependent on ecdysone, some of which is synthesised in SCs. However, mating induces additional BMP-mediated nuclear growth via a cell type\u2013specific form of hormone-independent EcR signalling, which drives genome endoreplication in a subset of adult SCs. Switching to hormone-independent endoreplication after mating allows growth and secretion to be hyperactivated independently of ecdysone levels in SCs, permitting more rapid replenishment of the accessory gland luminal contents. Our data suggest mechanistic parallels between this physiological, behaviour-induced signalling switch and altered pathological signalling associated with prostate cancer progression.Male reproductive glands like the mammalian prostate and the paired Hormone receptor-dependent growth of secondary cells in the accessory glands of male fruit flies switches to a hormone-independent mechanism after mating, reminiscent of changes seen in advanced prostate cancer in humans. Drosophila melanogaster, perform related functions and can also substantially alter female behaviour after mating, increasing egg laying, promoting sperm storage, and reducing female receptivity to subsequent mating attempts ), does not affect EcR levels. (C-J) Expression of EcR-B1 (C), -B2 (E), -A (G), and -C (I) in main cells leads to accumulation of EcR in these cells, in contrast to SCs. Coexpression with TkvACT does not appear to alter either the levels or subcellular localisation of EcR . Scale bars, 100 \u03bcm. Acp, AG protein; AG, accessory gland; BMP, bone morphogenetic protein; EcR, ecdysone receptor; GFP, green fluorescent protein; SC, secondary cell; Tkv, Thick veins.Images show the AG epithelium dissected from 6-day-old virgin males expressing nuclear GFP and other transgenes in main cells under Acp26Aa-GAL4 control and stained with a pan-EcR antibody. Note that GFP is also observed in the main cell cytoplasm when expressed at high levels in these cells. Nuclei are stained with DAPI (blue). Merge does not include DAPI channel for increased clarity. Expression of Tkv(TIF)Click here for additional data file.S5 FigtsF/O control. Panels show a single z-plane and therefore do not include all SCs in each gland; also, not all migrated SCs express GFP at sufficiently high levels to be detected at this magnification. In 16-day-old virgin males, an average of 11 \u00b1 3 SCs expressing TkvACT (B) and 4 \u00b1 1 SCs expressing EcR-RNAi (C) migrate to the proximal end of the accessory gland (marked with white arrows), whereas no cells migrate from the distal tip (red arrows) in controls (A). Overexpression of EcR-B1 (D) or EcR-C (F) has no effect on SC migration. However, coexpression of EcR-B1 with TkvACT (E) strongly suppresses cell migration, as does coexpression of TkvACT with EcR-C (G). (H) Data analysed using the Kruskal-Wallis test with Dunn\u2019s multiple-comparisons test. ****p < 0.0001, n = 15. Underlying data for this figure can be found in esg, escargot; esgtsF/O, the yeast transcription factor GAL4 expressed under the control of the promoter of the gene esg in a temperature-dependent fashion; GFP, green fluorescent protein; RNAi, RNA interference; SC, secondary cell; Tkv, Thick veins.(A-G) Confocal images of whole accessory glands expressing nuclear GFP and other transgenes under esg(TIF)Click here for additional data file.S1 Data(XLSX)Click here for additional data file."} +{"text": "Here, we present a case of plasmablastic lymphoma involving the uterus in a 54-year-old human immunodeficiency virus-negative female patient. The torso positron emission tomography/CT scan revealed intense A 54-year-old female with a history of acute myeloid leukaemia, successfully treated with chemotherapy and haplo-related allo-peripheral blood stem cell transplantation, remained in remission for 4 years. A large pelvic mass was incidentally picked up on a routine follow-up CT scan of the abdomen and pelvis. The patient remained asymptomatic.\u22121, normal range 0\u201335), lactate dehydrogenase and immunoglobulin M . Serological examination was all negative for HBsAg, human immunodeficiency virus (HIV), hepatitis C virus, HBsAb, HBeAb and HBcAb. Immunofixation electrophoresis examinations of serum and urine were both negative.Laboratory findings showed an increased serum level of CA125 (89.2 U ml18F-fludeoxyglucose (18F-FDG) positron emission tomography (PET)/CT scan showed a huge intense hypermetabolic lesion at the uterus involving the uterine cervix and right pelvic side wall. The maximum standardized uptake value (SUVmax) of the most metabolically active portion was 37.1. 18F-FDG PET/CT scan revealed multifocal malignant hypermetabolic bone involvement in multiple bones , and malignant hypermetabolic lymph nodes in the neck , thoracic , presacral and intrapelvic cavity were observed. In addition, diffuse hypermetabolism in the intraperitoneal space was seen, suggesting peritoneal seeding and radiological (huge pelvic mass) findings. Histological findings of the retroperitoneal mass demonstrated diffuse sheets of medium to large atypical lymphoid cells . The tumfindings . This im18F-FDG PET/CT scan for treatment response assessment. Her follow-up 18F-FDG PET/CT showed complete remission for the previously noted hypermetabolic malignant lesions of thoracoabdominal lymph nodes and peritoneal seeding.After four cycles of EPOCH chemotherapy, the patient underwent follow-up 1Plasmablastic lymphoma (PBL) is a very rare tumour occurring predominantly in the oral cavity in immunocompromised or HIV-positive groups.2PBL should be differentiated from other malignant tumours such as gastrointestinal stromal tumours, poorly differentiated carcinomas, multiple myeloma, diffuse large B-cell lymphoma, anaplastic large cell lymphoma, Burkitt\u2019s lymphoma and haematological malignancies such as leukaemia.18F-FDG PET/CT scan have been reported in the literature.7 To the best of our knowledge, this is the first case to report PBL of uterus using 18F-FDG PET/CT scan.A few cases of PBL using 18F-FDG PET/CT scan was useful in evaluating the malignant potential of the uterine tumour and detection of distant metastasis for operability. Although rare, uterine PBL should be considered one of the differential diagnosis of uterine lesions detected by 18F-FDG PET/CT scan.A 18F-FDG PET/CT scan can prove the high glucose metabolism of PBL, which is a very rare and aggressive type of lymphoma.18F-FDG PET/CT scan is useful in identifying the treatment response of lymphoma including PBL.Written informed consent was obtained from the patient(s) for publication of this case report, including accompanying images."} +{"text": "Carcinogenesis occurs in elastin-rich tissues and leads to local inflammation and elastolytic proteinase release. This contributes to bioactive matrix fragment (Matrikine) accumulation like elastin degradation products (EDP) stimulating tumour cell invasive and metastatic properties. We previously demonstrate that EDPs exert protumoural activities through Hsp90 secretion to stabilised extracellular proteinases.EDP influence on cancer cell blebbing and extracellular vesicle shedding were examined with a videomicroscope coupled with confocal Yokogawa spinning disk, by transmission electron microscopy, scanning electron microscopy and confocal microscopy. The ribosomal protein SA (RPSA) elastin receptor was identified after affinity chromatography by western blotting and cell immunolocalisation. mRNA expression was studied using real-time PCR. SiRNA were used to confirm the essential role of RPSA.We demonstrate that extracellular matrix degradation products like EDPs induce tumour amoeboid phenotype with cell membrane blebbing and shedding of extracellular vesicle containing Hsp90 and proteinases in the extracellular space. EDPs influence intracellular calcium influx and cytoskeleton reorganisation. Among matrikines, VGVAPG and AGVPGLGVG peptides reproduced EDP effects through RPSA binding.Our data suggests that matrikines induce cancer cell blebbing and extracellular vesicle release through RPSA binding, favouring dissemination, cell-to-cell communication and growth of cancer cells in metastatic sites. Meaningfully, the partial proteolytic degradation of elastic fibres by several elastases causes formation of EDPs (a category of matrikines3), revealing cryptic sites that might potentially interact with cell receptors. EDPs have been blamed for their contribution to several pathologies like tumour progression and metastasis.6 EDPs promote the invasive and metastatic properties of tumour cells. Among EDPs, VGVAPG and AGVPGLGVG peptides were reported to stimulate cell invasion.7 They increase MMP-2, MT1-MMP, uPA and Hsp90 secretion by cancer cells. Hsp90 was shown to interact with many extracellular proteins involved in cancer progression and metastasis and to be of critical importance.8 Extracellular Hsp90 is associated with increased tumour invasiveness. Hsp90 increases MMP-2 stability and enzymatic activity.9 Hostile environmental conditions such as serum starvation, hypoxia and oxidative stress promote Hsp90 extracellular location through extracellular vesicles (EVs),10 like exosomes and ectosomes.Local invasion and metastasis of most malignant tumours require secretion of proteinases that degrade local extracellular matrix (ECM). This process causes accumulation of ECM degradation products. These products then interact with cell receptors and regulate tumour invasion and metastatic dissemination.11 (ii) \u03b1v\u03b23 integrin;12 (iii) \u03b1v\u03b25 integrin;13 (iv) galectin-3; and (v) ribosomal protein SA (RPSA).14 Expressed at all stages, RPSA was shown to exert many physiological roles. The RPSA was originally identified as a 37-kDa/67-kDa binding protein for laminin but is also a membrane receptor for growth factors, prion and pathogens.16 It contributes to the crossing of the blood\u2013brain barrier by neurotropic viruses and bacteria. Contributing to cell receptor, RPSA also regulates ribosome biogenesis, cytoskeletal organisation, and nuclear functions.17 It governs critical cellular processes including growth, survival, migration, protein synthesis, development, and differentiation. RPSA has been associated with neurodegenerative diseases and developmental abnormalities and is a biomarker of metastasis.23Five cell surface receptors mediate EDP effects: (i) the elastin receptor complex (ERC) i concentration was derived from the ratio of the fluorescence intensities for each of the excitation wavelengths (F350/F380), and from the Grynkiewicz equation. The cells were continuously perfused with saline solution and EDPs were added as 50\u2009\u00b5g/mL via a whole chamber perfusion system. The flow rate of the whole cell chamber perfusion system was set to 1\u2009mL\u2009min\u22121 and the chamber volume was 500\u2009mL.The cytosolic calcium concentration was measured using FURA-2-loaded HT-1080 cells. The glass coverslip was mounted in a chamber on a Zeiss microscope equipped for fluorescence . Acquisitions were performed using a Cool SNAP HQ camera and Metafluor software (version 7.1.7.0) was used for acquisition and analysis. The i. Nifedipine did not inhibit EDP-dependent calcium flux, excluding a L-channel type role39 , phosphorylated LIMK1/2, phosphorylated ERM (P-ERM) and cofilin presented a normal localisation in all cell body Fig. . During ing Fig. . Consisting Fig. , showinging Fig. ; video 75 As Hsp90 was reported to localise in all blebs and extracellular vesicles, we decided to study its expression in different cell lines can overcome this difficulty.To date, only two cell receptor have been described to mediate membrane blebbing: neurokinin-1 receptor and P2X7 receptor.60 Taken together, our results suggest that EDP-induced blebbing involve the RhoA/ROCK pathway, the calpain activation, the phosphorylation of ERM proteins leading to the actin cytoskeletal reorganisation and bleb formation.After EDP/RPSA interaction, membrane bleb formation involves well already define signalling pathways like RhoA/ROCK/MLC cascade. Our results demonstrate that Y27632, a ROCK inhibitor, and blebbistatin, a myosin II inhibitor, inhibit the EDP-induced release of Hsp90, MMPs and uPA, showing the involvement of the RhoA/ROCK/MLC pathway. EDPs trigger a calcium influx that activates calpain. Calpain was reported to induce cytoskeletal protein degradation and actomyosin reorganisation. The breakdown of the actomyosin network associated with the hydrostatic pressure causes the membrane bleb formation. ROCK was previously reported to phosphorylate ERM that participates in actin cytoskeleton reorganisation and bleb formation.38 We demonstrate a correlation between EDP-dependent bleb formation, shed extracellular vesicles and Hsp90 secretion. Highly invasive cancer cells undergo membrane bleb formation and a high Hsp90 release under EDP influence. Extracellular Hsp90 exacerbate cancer cell invasion as it participates in many proteinase activation and prevents their inactivation.61 Hsp90 secretion depends on yet unknown regulating mechanisms. We show in this paper that Hsp90 is strongly linked to bleb formation and extracellular vesicle shedding. The presence of Hsp90 in the shed extracellular vesicles could explain the high level of Hsp90 in plasma of cancer patients.Extracellular vesicle were reported to contain a large amount of Hsp90 protein.62 Blebs and shed extracellular vesicles presented common markers, like high amount of RPSA, phospho-ERM, Hsp90, \u03b1V integrin subunit and some proteinases. Tumour extracellular vesicles have been linked to Hsp90 secretion and tumour invasion via release of proteinases.63 Extracellular vesicles provide a large membrane surface for the activation of membrane-associated proteinases involved in extracellular matrix degradation and tissue invasion.36 The tumour cell release vesicles that exhibit membrane phosphatidylserine redistribution, one of the major events required of extracellular vesicle formation.Besides the amoeboid migration movement, membrane blebbing allows the extracellular vesicle shedding in the cellular microenvironment.Taken together, our results demonstrate that matrikines induce cancer cell blebbing and extracellular vesicle release through RPSA binding, favouring dissemination, cell-to-cell communication and growth of cancer cells in metastatic sites. This process involves normal blebbing signalling pathway: calcium influx, RhoA/ROCK/MLC, calpain activation and ERM phosphorylation. Hsp90 and proteinase expression and release are increased after EDP treatment and is correlated with blebbing and Matrigel invasion.Video G - Spinning disk microscopy of GFP-Hsp90 HT-1080 cell in presence of EGCG and in absence of EDPsVideo H - Spinning disk microscopy of GFP-Hsp90 HT-1080 cell in presence of EGCG and EDPsS Fig 1 - EDPs stimulate cell membrane blebbing in 3D collagen matrix. GFP-Hsp90 transfected HT-1080 cells were seeded in a 3D collagen matrix in the presence of EDPsS Fig 2 - 2D Time-lapse snapshots of blebbing HT-1080 cells in presence of EDPs at t0min and at t75minS Fig 3 - Cell proliferation and cell survival testsS Fig 4 - 2D Time-lapse snapshots of cell-to-cell communication between a blebbing cell and a mesenchymal cell in presence of EDPsS Fig 5 - Extracellular vesicles were prepared from cell-conditioned medium by centrifugation and ultracentrifugation after 24h of incubationS Fig 6 - Signalling pathway immunostaining quantifications and localizations using the ImageJ softwareS Fig 7 - Blebbistatin and Y27632 inhibit EDP-stimulated blebbing, Hsp90 and proteinase secretionsVideo 1. Spinning disk microscopy of a mesenchymal GFP-Hsp90 HT-1080 cell in absence of EDPVideo 2. Spinning disk microscopy of a blebbing GFP-Hsp90 HT-1080 cell in presence of EDPsVideo 3. Live videomicroscopy of the reversible blebbing in presence of EDPsVideo 4 - Spinning disk microscopy of cell-to-cell communication via shed extracellular vesicles in presence of EDPsVideo 5 - Spinning disk microscopy of mesenchymal mCherry-MLC HT-1080 cells in absence of EDPVideo 6 - Spinning disk microscopy of blebbing mCherry-MLC HT-1080 cells in presence of EDPsVideo 7 - Spinning disk microscopy of blebbing GFP-Hsp90 HT-1080 cells and shed microsicles in presence of EDPsS Table 1. Immunostaining quantification and localization in HT-1080 cells using ImageJ pluginS Table 2. Blebbing quantification in HT-1080 cells in presence of different elastin receptor inhibitorsS Fig 8 - Identification of the RPSA protein as the VGVAPG receptor by affinity chromatographyS Fig 9 - EGCG inhibits EDP-stimulated blebbingVideo A - Spinning disk microscopy of a mesenchymal GFP-Hsp90 HT-1080 cell in absence of EDPVideo B - Spinning disk microscopy of blebbing GFP-Hsp90 HT-1080 cell in presence of EDPsVideo C - Live videomicroscopy of blebbing HT-1080 cells in presence of EDPsVideo D - Live videomicroscopy of cell-to-cell communication via shed microvesicle in presence of EDPsVideo E - Spinning disk microscopy of GFP-Hsp90 HT-1080 cell in absence of EDPsVideo F - Spinning disk microscopy of GFP-Hsp90 HT-1080 cell in presence of EDPs"} +{"text": "V600E mutation is the most prevalent driver mutation of sporadic papillary thyroid cancers (PTC). It was previously shown that prenatal or postnatal expression of BRAFV600E under elevated TSH levels induced thyroid cancers in several genetically engineered mouse models. In contrast, we found that postnatal expression of BRAFV600E under physiologic TSH levels failed to develop thyroid cancers in conditional transgenic Tg(LNL-BrafV600E) mice injected in the thyroid with adenovirus expressing Cre under control of the thyroglobulin promoter (Ad-TgP-Cre). In this study, we first demonstrated that BrafCA/+ mice carrying a Cre-activated allele of BrafV600E exhibited higher transformation efficiency than Tg(LNL-BrafV600E) mice when crossed with TPO-Cre mice. As a result, most BrafCA/+ mice injected with Ad-TgP-Cre developed thyroid cancers in 1 year. Histologic examination showed follicular or cribriform-like structures with positive TG and PAX staining and no colloid formation. Some tumors also had papillary structure component with lower TG expression. Concomitant PTEN haploinsufficiency in injected BrafCA/+;Ptenf/+ mice induced tumors predominantly exhibiting papillary structures and occasionally undifferentiated solid patterns with normal to low PAX expression and low to absent TG expression. Typical nuclear features of human PTC and extrathyroidal invasion were observed primarily in the latter mice. The percentages of pERK-, Ki67- and TUNEL-positive cells were all higher in the latter. In conclusion, we established novel thyroid cancer mouse models in which postnatal expression of BRAFV600E alone under physiologic TSH levels induces PTC. Simultaneous PTEN haploinsufficiency tends to promote tumor growth and de-differentiation.The BRAF RAS/BRAF genes or chromosomal rearrangements such as RET/PTC translocations [BRAF gene, the T1799A transverse point mutation results in a mutant BRAF, BRAFV600E, which exhibits constitutive serine/threonine kinase activity.Sporadic thyroid cancers usually develop via abnormal activation of the RAS-RAF-MEK-ERK signaling pathway , primarily as a result of point mutations in the ocations . In the V600E in the thyroid glands was first demonstrated in vivo in Tg-BrafV600E transgenic mice expressing BRAFV600E under control of thyroid-specific thyroglobulin (Tg) promoter; these mice developed thyroid cancers very early in life [V600E was expressed in all thyroid cells from the fetal period, suggesting that this is a model of hereditary rather than sporadic thyroid cancers; (ii) serum TSH levels were elevated by BRAFV600E-mediated suppression of thyroid function, which by itself can induce thyroid goiters and sometimes tumors; and (iii) BRAFV600E expression was controlled by the Tg promotor rather than the original Braf promoter [LSL-BrafV600E;TPO-Cre mice expressed BRAFV600E in all the thyroid cells from the fetal period, with ~8- to 80-fold increases in TSH, although TSH was expressed at physiologic levels under the control of the chromosomal promoter [BrafCA;Thyro::CreER mice were generated to control expression of BRAFV600E by tamoxifen in the postnatal period, but untreated mice displayed increased thyroid volumes 1 month after birth, presumably due to aberrant nuclear localization of CreERT2 in the absence of tamoxifen [BrafCA mice carried a Cre-activated allele of BrafV600E [LSL-BrafV600E mice mentioned above [Tg-rtTA/tetO-BrafV600E mice expressed BRAFV600E in all the thyroid cells, with >100-fold increases in TSH, although expression began after birth (after administration of doxycycline) [BrafCA;TPOCreER mice were reported to develop thyroid cancers after birth (after administration of tamoxifen), although TSH increased slightly (<10-fold) [The carcinogenicity of BRAF in life . Howeverpromoter . These lpromoter . BrafCA;amoxifen . In thated above . Leakinecycline) . FinallyTg(LNL-BrafV600E) mice. Upon injection of adenovirus expressing Cre under control of the Tg promoter (Ad-TgP-Cre) into their left thyroid lobes at age of ~4 weeks, these mice expressed BRAFV600E in a fraction of the thyroid cells. As such, serum TSH remained within physiologic range, and mice did not develop thyroid cancer [V600E alone in a small number of thyroid cells under normal TSH levels is insufficient for thyroid cancer development. However, this model also had a drawback; a comparison of data from the previous reports [Tg(LNL-BrafV600E);TPO-Cre mice than LSL-BrafV600E;TPO-Cre mice, as serum TSH levels increased in the latter not the former.To establish an ideal mouse model of sporadic thyroid cancer, we previously generated d cancer . From th reports , 4 suggeBrafCA;TPO-Cre mice compared with Tg(LNL-BrafV600E);TPO-Cre mice in our laboratory and then used BrafCA mice rather than Tg(LNL-BrafV600E) mice to re-evaluate the carcinogenesis of BRAFV600E in the context of our experimental setting with Ad-TgP-Cre. Here, we show that postnatal BRAFV600E expression alone under physiologic TSH levels is sufficient for thyroid cancer development. In addition, we also studied the effect of concomitant PTEN haploinsufficiency on BRAFV600E-induced thyroid cancers and show that the simultaneous reduction of PTEN expression tends to promote tumor growth and de-differentiation. Our results also demonstrate development of thyroid hyperplasia/adenoma in Pten\u0394/+ mice (but not Ptenf/+ mice) injected with Ad-TgP-Cre, suggesting that the timing of PTEN reduction is critical for tumorigenicity of PTEN in the thyroid.In the present study, therefore, we first confirmed the higher transformation efficiency of Cre-mediated DNA recombination in BrafV600E mice (Tg(LNL-BrafV600E)#213MM) and TPO-Cre mice were previously described [BrafCA (B6.129P2(Cg)-Braftm1Mmcm/J, stock# 017837) mice [Pten\u0394/+ mice were obtained from National Cancer Institute at Frederick, MD, USA) [TPO-Cre, which were FVB/NCr.Conditional transgenic escribed . BrafCA All mice were kept in a specific pathogen-free facility. Animal care and all experimental procedures were performed in accordance with the Guideline for Animal Experimentation of Nagasaki University with approval of the Institutional Animal Care and Use Committee (permission number: 1309021089). All surgeries were performed under isoflurane anesthesia, and every effort was made to minimize suffering.Ad-TgP-Cre was used in this study, as described previously .9 adenovirus particles/mouse were injected. The number of mice in each group was shown in Brafthyr-V600E and 6 Brafthyr-V600E;Ptenthyr-\u0394/+ mice).Surgery and injection of adenovirus into the left lobe of the thyroid of ~4-week-old mice were performed as described previously . A totalV600E , rabbit monoclonal anti-Ki-67 , rabbit monoclonal anti-thyroglobulin or rabbit monoclonal anti-phospho-p44/42 MAPK (ERK1/2) . It should be noted here that the protein recognized by anti-PAX8 mentioned above is called \"PAX\" throughout the paper, because, although the immunogen for this antibody was a part of human PAX8 (212 amino acids), its specificity to PAX8 has not been confirmed. The primary antibody was followed by incubation with secondary antibody: swine anti-rabbit IgG/HRP or rabbit anti-mouse IgG/HGRP . Color was developed with 3, 3\u2019-diaminobenzidine substrate. Slides were analyzed using an All-in-One BZ-9000 Fluorescence Microscope . A total of 1,500 cells were evaluated to determine the percentage of Ki67-positive cells.Tissues were fixed in 10% neutral-buffered formalin and then embedded in paraffin. Sections (4-\u03bcm-thick) were prepared and stained with hematoxylin eosin (H & E) or immunostained with primary antibody: rabbit polyclonal anti-surfactant protein A , rabbit monoclonal anti-PTEN , rabbit polyclonal anti-PAX8 , mouse monoclonal anti-BRAFTerminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) was performed with the Apop-tag\u2122 Fluorescein Direct in situ apoptosis detection kit . Slides were embedded with VECTASHIELD mounting medium containing DAPI and analyzed using an All-in-One BZ-9000 Fluorescence Microscope (Keyence). A total of 1,500 cells were evaluated in each sample to determine the percentage of TUNEL-positive cells.Serum TSH was measured using a specific mouse TSH RIA with mouse TSH/LH reference (AFP9090D), mouse TSH antiserum (AFP98991) and rat TSH antigen (NIDDK-rTSH-I-9) as described previously , 14. Thet-test. A p-value of less than 0.05 was considered statistically significant.All data were analyzed for significant differences using the Student\u2019s Tg(LNL-BrafV600E)#213MM (a high expressor);TPO-Cre mice exhibited a slightly (but not significantly) enlarged thyroid with focal neoplastic lesions and normal TSH levels, whereas BrafCA/+;TPO-Cre mice exhibited a greatly enlarged thyroid with diffuse neoplastic lesions and elevated TSH levels #213MM;TPO-Cre mice, and controls exhibited serum TSH levels of 43.1 \u00b1 56.6, 0.9 \u00b1 0.2 and 1.0 \u00b1 0.2 ng/ml, respectively, and thyroid weights of 122.0 \u00b1 63.6, 8.0 \u00b1 4.3 and 6.7 \u00b1 1.8 mg, respectively. The lower transformation efficiency in Tg(LNL-BrafV600E)#213MM as compared with BrafCA mice may explain our previous failure of tumor induction in Tg(LNL-BrafV600E)#213MM mice with intrathyroidal injection of Ad-TgP-Cre in our previous study [BrafCA rather than Tg(LNL-BrafV600E)#213MM mice to re-evaluate the carcinogenesis of BRAFV600E with our thyroid cancer model with Ad-TgP-Cre. We also examined the carcinogenesis of PTEN haploinsufficiency using Ptenf/+ mice and BrafCA/+;Ptenf/+ mice, as reduced PTEN expression alone and in combination with BRAFV600E reportedly plays a significant role in the carcinogenesis of various organs [In previous research reported by us and otheH levels , at agess organs \u201317.BrafCA/+, BrafCA/+;Ptenf/+ and Ptenf/+ mice . Because it was totally unknown whether thyroid tumors developed and if so when, we decided to observe the mice either until some symptoms appeared or for 26 and 52 weeks. Because no symptom developed, the mice were sacrificed at 2 time points, as originally scheduled. The thyroid lobe was macroscopically normal in all mice at 26 weeks (data not shown), but at 52 weeks, the left lobe was enlarged in Brafthyr-V600E mice (8/9) and Brafthyr-V600E;Ptenthyr-\u0394/+ mice (9/9), but not Ptenthyr-\u0394/+ mice (0/7) and also each lobe of the controls. The left lobe tended to be heavier in Brafthyr-V600E;Ptenthyr-\u0394/+ mice compared with Brafthyr-V600E mice, but the difference was not statistically significant (Ad-TgP-Cre was injected into the left thyroid lobe of 4-week-old ce (0/7) , Fig 2. Brafthyr-V600E and Brafthyr-V600E;Ptenthyr-\u0394/+ mice typical nuclear features of human PTC such as intranuclear cytoplasmic inclusions and nuclear grooves and/or (ii) invasion of the extrathyroidal tissues surrounding the thyroid glands were readily diagnosed as cancers. Some tumors in vs. 5.6 \u00b1 4.6) were compensated by higher cell death rates as determined by TUNEL staining (1.1 \u00b1 0.9 vs. 0.4 \u00b1 0.4) in Brafthyr-V600E;Ptenthyr-\u0394/+ mice as compared with Brafthyr-V600E mice [V600E-induced lung adenomas [Macroscopic lung nodules were observed in 2 of 9 nodules excludedadenomas . A spontadenomas also staPtenthyr-\u0394/+ mice, most Pten\u0394/+ mice developed thyroid hyperplasia/adenoma by the age of 6 to 8 months . As BRAFV600E is frequently found in sporadic thyroid cancers in euthyroid subjects, BRAFV600E should be expressed in a small fraction of thyroid cells after birth under physiologic TSH levels. In this regard, our experimental design\u2014that is, intrathyroidal injection of Ad-TgP-Cre into one side of the thyroid lobes of genetically engineered mice harboring the loxP sequences\u2014is likely ideal. The feasibility of adenovirus-mediated Cre gene transfer to temporally and spatially control Cre expression has been well demonstrated [did develop in Ad-TgP-Cre-injected BrafCA mice, indicating that postnatal expression of BRAFV600E alone under physiologic TSH levels is sufficient for thyroid cancer development. Similar preliminary results were reported by McFadden et al .Tg(LNL-BrafV600E) mice [Tg(LNL-BrafV600E) vs. B6 in BrafCA) and/or different promoters (CAG promoter vs. the endogenous Braf promoter) could have affected our previous results. Different recombination frequencies of distinct alleles have been reported [V600E-expressing normal, differentiated thyroid cells into malignant cells is extremely low.Our previous failure with 0E) mice appearedreported . PresumaBrafCA;TPOCreER mouse model with tamoxifen reported by McFadden et al. may also be ideal, although the TSH levels increased slightly (<10 fold) [The lightly < fold [10Tg-BrafV600E;Tshr-/-mice [LSL-BrafV600E;TPO-Cre;Tshr-/- mice [LSL-BrafV600E;TPO-Cre mice (with high TSH levels) into nude or syngeneic immuno-competent mice leads to regression and senescence [Significant increases in TSH levels (up to 500 fold) have been noted in other models , 9, 10. -/-mice and LSL--/- mice , both ofnescence .Regarding the question as to how many mutations are required for full development of differentiated thyroid cancer, recent studies using human samples show that number of non-synonymous mutations in exomes is ~0.4/Mb \u201331, and V600E was first discovered in malignant melanoma, but later also found to be present in benign nevi, which seldom progress to melanoma unless additional mutations occur [V600E alone cannot induce melanoma, but it can in combination with PTEN loss or activating PI3KCA mutations [BRAFns occur . In accoutations , 34. Conutations , 35. Of utations , 31.V600E and reduced PTEN expression tended to induce larger and more undifferentiated thyroid cancers in our study, and these data were similar to those in LSL-BrafV600E;Ptenf/f;TPO-Cre mice in which PTC rapidly progressed to poorly differentiated thyroid cancers as compared with LSL-BrafV600E;TPO-Cre mice [Thyro::CreER;BrafCA/+;Pik3calat-1047R/+ mice, which developed anaplastic cancers as compared with Thyro::CreER;BrafCA/+ mice [Pten gene are not common [The combination of BRAFCre mice and alsoA/+ mice . Althougt common , reducedt common .PTEN gene induces thyroid multinodular goiter and adenoma [et al. using PtenL/L;TPO-Cre mice [Pten\u0394/+ mice used in our study, the majority of mice in the 129Sv genetic background developed well-circumscribed follicular adenomas and nodular hyperplasia, often characterized by increased cellularity and mitotic figures at 8 to 10 months of age. However, no thyroid tumors were observed in Ptenf/+ mice injected with Ad-TgP-Cre in our study. These data clearly indicate that the tumorigenic potential of reduced PTEN expression differs between the prenatal and postnatal periods.Tumorigenesis associated with PTEN loss by itself is well known in human Cowden syndrome, in which a germline loss-of-function mutation in the adenoma . ExperimCre mice . Thus, sV600E was expressed aberrantly from the Tg promoter, even when the volume of adenovirus injected was low (1 \u03bcl) and highly thyroid specific Tg promoter was used. Thus, one of the limitations of our study is the leakiness of locally injected adenovirus as well as leakiness of the Tg promoter. Our model is therefore not suitable for study of metastasis. Only 2 reports of lung metastasis have been reported, one by Rusinek et al. using transgenic Tg-2HA-BrafV600E mice [TPOCreER;BrafCA/+;p53LSL-R270H/+ mice [We interpret our data on lung tumors as showing that adenovirus injected into the thyroid lobes leaked, disseminated systemically, and reached the lung, where BRAF00E mice and the H/+ mice . AnotherV600E alone under physiologic TSH levels is sufficient for development of thyroid cancer and that simultaneous reduced expression of PTEN tends to promote tumor growth and de-differentiation. It will be of interest in the future to compare the differences/similarities of thyroid cancers associated with postnatal vs. prenatal expression of BRAFV600E. Our data also indicate that the effects of BRAFV600E expression and reduced PTEN expression differ between the prenatal vs. postnatal periods. Thus, unlike BRAFV600E, the tumorigenic potential of PTEN depends on a prenatal reduction in expression.In conclusion, using our mouse model with Ad-TgP-Cre, we show that postnatal expression of BRAF"} +{"text": "Optimizing Dementia Care in Veterans with Dementia is a randomized, controlled, pilot study examining outcomes for Veterans and their caregivers at 6- and 12-months for two telephone-based interventions: a) Benjamin Rose Institute\u2019s (BRI) Care Consultation (CC), and b) CC + Counseling (CC+C). Counseling modules are integrated into the existing BRI CC framework using guided mindfulness-based skill-building exercises on various content domains . Sixty-four caregivers and 47 Veterans have been randomized in this ongoing pilot study. Caregivers are 91% female, 32% Black/African American, and 72% spouses. Preliminary implementation and 6-month outcome data is discussed using within-group paired samples t-tests for the 32 dyads randomized to CC+C. Lessons learned include strategies for adapting mindfulness-based approaches over the telephone to enhance access for Veterans and caregivers across geographic regions."} +{"text": "Therefore, the tissue-agnostic cancer drugs represent current treatment options against NTRK fusion-positive or MSI-H/dMMR MUOs. Other pipelines under development include: entrectinib for the therapy of pediatric and adult patients with recurrent or refractory extracranial solid tumors harboring NTRK, c-Ros oncogene 1 (ROS1) or anaplastic lymphoma kinase (ALK) gene fusions; BLU-667 and Loxo-292 for solid neoplasms with alterations in the \u201crearranged during transfection\u201d (RET) gene; and Loxo-195 for NTRK fusion-positive solid cancers. [Metastasis of unknown origin (MUO) means that a cancer is detected when it is already in metastasis, but without any evidence of the primary tumor, even after a full clinico-radiological workup, histological examination, immunohistochemical investigations and tissue-of-origin testing. The occucancers. \u201311 Prelicancers. All this"} +{"text": "Subsequent large-scale validation and genotyping of these SNPs discovered 619 desi accessions-derived (DAD) SNPs, 531 kabuli accessions-derived (KAD) SNPs, 884 multiple accessions-derived (MAD) SNPs and 1083 two accessions (desi ICC 4958 and kabuli CDC Frontier)-derived (TAD) SNPs that were mapped on eight chromosomes. These informative SNPs were annotated in coding/non-coding regulatory sequence components of genes. The MAD-SNPs were efficient to detect high intra-specific polymorphic potential and wide natural allelic diversity level including high-resolution admixed-population genetic structure and precise phylogenetic relationship among 291 desi and kabuli accessions. This signifies their effectiveness in introgression breeding and varietal improvement studies targeting useful agronomic traits of chickpea. Six trait-associated genes with SNPs including quantitative trait nucleotides (QTNs) in combination explained 27.5% phenotypic variation for seed yield per plant (SYP). A pentatricopeptide repeat (PPR) gene with a synonymous-coding SNP/QTN significantly associated with SYP trait was found most-promising in chickpea. The essential information delineated can be of immense utility in genomics-assisted breeding applications to develop high-yielding chickpea cultivars.We discovered 2150 Cicer arietinum) is a self-pollinated, diploid (2n\u2009=\u200916) and economically important legume food crop rich in human dietary proteins1. The chickpea is primarily represented by its desi and kabuli cultivars exhibiting a distinct differentiation in both agro-morphological and architecture traits, which genomes have been sequenced recently5. Substantial enhancement of its yield and productivity by developing high-yielding a/biotic stress tolerant and climate-ready cultivars is essential at present to sustain the global food security. To accomplish these objectives, many significant efforts involving classical genetics as well as genomics-assisted breeding strategies like quantitative trait loci (QTL) mapping, fine-mapping/map-based cloning and association analysis have been made to decipher the inheritance pattern and quantitative dissection of complex yield and stress tolerance traits for genetic improvement of chickpea30. However, these are constrained by narrow genetic base and low intra-specific marker polymorphism among desi and kabuli accessions due to combined strong impact of four major domestication bottlenecks in chickpea as compared to other food crop plants35. To overcome the aforesaid limitations, development and high-throughput genotyping of large-scale sequence-based informative markers like single nucleotide polymorphism (SNPs) exhibiting a higher potential of polymorphism among cultivated desi and kabuli accessions is a prerequisite for their use in genomics-assisted breeding applications and genetic improvement of chickpea.Chickpea (desi and kabuli) and wild accessions are found efficient in fast discovery of numerous SNPs at a genome-wide scale in chickpea44. The large-scale validation and high-throughput genotyping of these SNPs mined from limited number of sequenced accessions using diverse array-based genotyping assays commonly exhibit less potential of inter-/intra-specific polymorphism and natural allelic diversity among cultivated and wild accessions to be deployed efficiently for genetic enhancement of chickpea27. However, degree of these polymorphic and diversity potential can be enriched by using the genotyping information of SNPs derived from whole genome resequencing data that are generated from a large number of desi, kabuli and wild accessions of chickpea. Essentially, this approach requires huge cost, time and resources for developing and genotyping of SNPs at a whole genome/gene level in chickpea. In this regard, NGS-based low sequencing-depth genome coverage sequencing strategies like genotyping-by-sequencing (GBS) assay seems quite promising for rapid discovery and genotyping of genome-wide SNPs simultaneously among numerous cultivated (desi and kabuli) and wild accessions in order to drive high-throughput genetic analysis in chickpea with sub-optimal expense of cost, labour and resources27. However, these low genome coverage sequencing assays often generate erroneous and non-uniform SNP genotyping data across accessions, thereby further large-scale revalidation and genotyping of GBS-derived SNPs is needed currently prior to their use in genetic improvement of chickpea. In this perspective, high-throughput validation and genotyping of numerous GBS-derived high-quality SNPs discovered by assembling all the sequence reads generated from large number of accessions belonging to each of the desi and kabuli cultivars individually (cultivar-wise) can be useful for their deployment in genomics-assisted crop improvement of chickpea. In view of aforementioned prospects, efforts were made in the current study for large-scale validation and high-throughput genotyping of informative SNPs, discovered by desi and kabuli cultivar-wise individual assembling of the sequence reads, generated from 92 accessions using GBS assay at a genome-wide scale in chickpea -led genome and transcriptome sequencing of multiple cultivated -, kabuli accessions-derived (KAD)- and multiple accessions-derived (MAD)-SNPs which were further compared with two accessions (ICC 4958 and CDC Frontier) derived (TAD) SNPs for evaluating the potential of these developed markers in large-scale genetic analysis in chickpea sequence depth of coverage. This exertion overall produced 3.3 to 5.7 million of high-quality sequence reads per accession covering on an average 5.4-fold (X) sequence depth of coverage. More than 85% of de-multiplexed high-quality sequence reads generated from accessions were mapped separately (desi and kabuli cultivar-wise) to unique physical locations on desi reference genome4. The desi and kabuli cultivar-wise reference genome-based GBS analysis altogether detected 2150 (DAD-SNPs) and 2199 (KAD-SNPs) high-quality SNPs from the accessions which were further validated through PCR amplicons-based sequencing assay. For large-scale validation and high-throughput genotyping, a representative set of high-quality 672 DAD-, 576 KAD- and 1152 TAD-SNPs differentiating 39 desi, 53 kabuli and 2 (ICC 4958 and CDC Frontier) accessions, respectively, were selected based on their uniform physical distribution on eight chromosomes. These physically mapped SNPs were genotyped using the genomic DNA of 291 desi and kabuli chickpea accessions through a 34-plex high-throughput matrix assisted laser desorption ionization-time of flight (MALDI-TOF) Sequenom Mass Array genotyping assay. The optimized multiplex assay successfully validated 619 DAD-, 531 KAD- and 1083 TAD-SNPs based on their homozygous and heterozygous allele discrimination as evident from the call cluster plots of low and high mass homo- and hetero-zygote yields with a 92\u201394% genotyping success rate followed by biological process and cellular component . The transcription factor (TF) genes with MAD- and TAD-SNPs belonging to TF families like basic helix-loop-helix (bHLH), basic leucine zipper (bZIP) and APETALA2 ethylene-responsive element binding proteins (AP2-EREBP) were found predominant.The detailed structural annotation of 884 MAD- and 1083 TAD-SNPs exhibited the occurrence of 664 (75.1%) and 837 (77.3%) SNPs in 436 and 461 chickpea genes, respectively Fig.\u00a0\u2013S4. The desi and kabuli chickpea accessions than that of 102 kabuli accessions . Notably, a higher potential of MAD-SNPs followed by DAD-SNPs and a lower potential of TAD-SNPs for detecting polymorphism among accessions belonging to desi and kabuli cultivars was observed and kabuli (0.58) chickpea and kabuli (0.31) chickpea.The molecular diversity and population genetic structure among 291 kabuli and 29 desi accessions while POP II comprised of 129 desi and 60 kabuli accessions. The K value outcome with the best replicate was ascertained by estimating the average LnP(D) (log-likelihood) (exhibiting the greatest apparent inflection) and the second order statistics of \u0394K . All these SNPs detected a higher polymorphic potential in POP II (mean PIC: 0.31\u20130.43) than that of POP I. A significant population divergence based on pair-wise FST (P\u2009<\u20090.001) was estimated as 0.69 between POP I and POP II. Highest FST-based population differentiation of 0.30 was observed within POP II. All 291 desi and kabuli chickpea accessions clearly belonged to a structured population in which 69% of their inferred ancestry was derived from one model-based population and the remaining 31% had an admixed ancestry value of 2 as POP and POP II Fig.\u00a0. The POPdesi and kabuli chickpea accessions (association panel). The use of these SNPs classified these desi and kabuli accessions into aforesaid two distinct population groups (POP I and POP II) was observed among accessions belonging to the association panel based on multi-environments field data. A normal frequency distribution of quantitative SYP trait among accessions of an association panel was evident and replicated multi-location/years seed yield per plant (SYP) field phenotyping data of 291 \u22126 among desi and kabuli accessions belonging to an association panel. The combined SYP trait phenotypic variation explained by all significant six gene-based SNP loci was 27.5%. Notably, one synonymous SNP derived from a gene encoding pentatricopeptide repeat (PPR)-containing protein exhibited strong association (24.2% PVE and 1.2\u2009\u00d7\u200910\u22128 P) with SYP trait in chickpea cut-off \u22640.05 using the genotyping data of 1967 SNPs identified six genomic loci exhibiting significant association with SYP trait at a P\u2009\u2264\u200910\u22126 Fig.\u00a0. Interesome Fig.\u00a0. A maximome Fig.\u00a0. The SYPdesi and kabuli accessions. To overcome these, large-scale discovery and genotyping of informative sequence-based markers like SNPs differentiating the maximum number of desi and kabuli accessions together and exhibiting high intra-specific potential among cultivated accessions using a user-friendly, rapid and cost-effective genotyping assay is essential at a genome-wide scale45. This will essentially accelerate the generation of genotyping information of numerous genome-wide SNP markers differentiating large-scale accessions for high-resolution association mapping and efficient dissection of complex quantitative traits in order to drive genetic enhancement of chickpea. In this perspective, validation and genotyping of numerous non-erroneous and robust SNPs that are discovered by desi and kabuli cultivar-wise individual assembling of low-sequencing depth-coverage sequence reads generated from numerous germplasm accessions using GBS assay, can be an efficient alternative genome-wide strategy in chickpea. Adopting this strategy, the current study has developed diverse kinds of genome-wide 1967 SNPs including 884 DAD-, KAD-, MAD-SNPs and 1083 TAD-SNPs to evaluate their potential for discriminating cultivated desi and kabuli accessions and exhibiting association with SYP traits in chickpea. The structurally and functionally annotated informative natural SNP allelic variants discovered by us can be utilized for rapid identification of functionally relevant molecular tags associated with multiple agronomic traits in chickpea. These developed SNPs will further serve as a useful genomic resource for cultivar-wise selection of informative SNPs differentiating desi and kabuli accessions at a genome-wide scale to expedite high-throughput genetic analysis in chickpea.Genomics-assisted crop improvement of chickpea is usually hindered due to its narrow genetic base and low intra-specific polymorphism among cultivated desi and kabuli chickpea germplasm accessions was much higher as compared to that reported in earlier studies with SNP markers48. Nevertheless, the nucleotide diversity measured by DAD-, KAD- and MAD-SNPs among 189 desi and 102 kabuli accessions was almost comparable to that documented using the genome-wide resequencing- and GBS-derived SNP markers45. The level of molecular diversity detected especially by MAD-SNPs among 291 cultivated desi and kabuli chickpea accessions is much higher as compared to that documented earlier with SNP markers48. Distinct differentiation and clustering of 291 accessions into two major population groups are in accordance with their cultivar-specific origin, pedigree relationships and parentage that well-corresponded with previous studies of Upadhyaya et al.49 and Kujur et al.45.The intra-specific polymorphic potential especially detected by SNPs among 291 cultivated desi and kabuli accessions. The continuous crop genetic improvement breeding efforts with an aim to develop high-yielding stress tolerant desi and kabuli cultivars may influence their domestication pattern and genetic structure immensely therefore, detection of numerous admixture traces among these cultivated 291 desi and kabuli chickpea accessions is expected. The determination of high-resolution population genetic structure among accessions is vital to assess the accuracy (non-spurious) of robust SNPs associated with a particular agronomic trait in crop plants. In this context, high-resolution admixed population genetic structure can essentially be effective in detecting non-spurious genomic loci associated significantly with target traits in chickpea.The clustering of accessions into a specific population group was strongly influenced by geographical origin as well as adaptive environment rather than their known parentage and pedigree. The observed admixed population genetic structure inferred the occurrence of multiple domestication events along with strong evolutionary bottleneck among desi and kabuli chickpea accessions can be deployed in marker-aided introgression breeding for varietal improvement targeting multiple traits of agronomic importance in chickpea.While trying to evaluate the potential of the developed markers in explaining molecular diversity and population genetic structure in chickpea it was observed that large-scale genome-wide MAD-SNPs discovered by especially comparing the low-sequencing depth-coverage sequence-reads of multiple accessions exhibited a higher potential of intra-specific polymorphism as well as a wider natural allelic diversity than that of two accessions (TAD-SNPs). Therefore, these informative MAD-SNPs with the requisite potential to establish precise phylogenetic relationships and determine admixed population genetic structure among 291 desi and kabuli chickpea accessions at a genome-wide scale to evaluate their potential for marker-trait association and identify potential genomic loci and natural allelic variants governing complex seed yield quantitative trait in chickpea. The high-resolution association study altogether detected six genomic SNP loci including QTNs significantly associated with SYP traits. None of these six major SYP trait-associated QTNs identified has been documented by earlier association and QTL mapping studies in chickpea. This infers the novelty of the detected gene-based natural SNP allelic variants in governing seed yield trait in chickpea. Essentially, the five protein-coding genes with synonymous and non-synonymous SNP loci associated with SYP trait delineated by high-resolution association mapping have functional relevance for quantitative dissection of complex seed yield traits in chickpea. These SYP trait-associated SNPs will be of immense use in establishing marker-trait linkages and identification of natural allelic variants in the genes regulating SYP trait, thereby can accelerate the marker-assisted selection to develop desirable cultivars with high seed yield.The current investigation developed numerous informative DAD-, KAD-, MAD- and TAD-SNPs differentiating 291 precisely field-phenotyped 56. Essentially, the role of delineated most-promising SYP trait-associated PPR genes with synonymous SNPs in regulating fertility restoration, heterosis breeding and hybrid seed production is well-documented in crop plants including legumes57. Modulation of their gene expression can thereby enhance the overall seed yield. Henceforth, functionally relevant molecular signatures including candidate genes, transcription factors and natural SNP alleles delineated in the present study can essentially be deployed for genomics-assisted crop improvement to develop high seed-yielding cultivars of chickpea.The delineated genes encoding response regulator, arginine decarboxylase, Mediator transcription factor and PPR protein with synonymous and non-synonymous coding and intronic SNPs associated with SYP trait in chickpea are known to control gene expression dynamics and transcriptional regulatory pathways underlying growth, development and yield traits in multiple crop plantsC. arietinum desi (189 accessions) and kabuli (102) cultivars were selected from available chickpea reference core/mini-core germplasm collections49 as per Kujur et al.45 belonging to 45 Table\u00a0. Of thesea Table\u00a0. The gendesi and kabuli chickpea accessions using GBS assay were acquired from NCBI SRA database (Accession ID SRX845396) and de-multiplexed into individual sequences by using their unique barcodes in accordance with Kujur et al.45. The high-quality sequence reads of 92 accessions after filtering with NGS QC Toolkit58, were grouped into desi (39 accessions) and kabuli (53) cultivar-wise separately and further mapped individually on desi chickpea genome sequences4 using BWA59. The SNPs were mined by desi and kabuli cultivar-wise comparison of the mapped sequences among accessions using Samtools (http://www.htslib.org) and subsequently high-quality SNPs supported by at least 10 sequence reads were screened following Srivastava et al.30. A set of chromosome-wise uniformly mapped high-quality SNPs exhibiting differentiation only between two desi (ICC 4958) and kabuli (CDC Frontier) cultivars (which draft genomes are sequenced) were screened for further analysis. The structural and functional annotation of SNPs in diverse coding (synonymous and non-synonymous substitutions) and non-coding sequence components of genes and genomes (chromosomes/pseudomolecules and unanchored scaffolds) were performed as per the desi (CGAP v2.04) genome annotation.The raw FASTQ sequence reads generated by sequencing of 92 desi accessions (39)-derived (DAD) SNPs, kabuli accessions (53)-derived (KAD) SNPs, multiple accessions (92 desi and kabuli)-derived (MAD) SNPs and two accessions (ICC 4958 and CDC Frontier)-derived (TAD) SNPs were genotyped in the genomic DNA of 291 chickpea accessions using Sequenom MALDI-TOF MassARRAY assay (http://www.sequenom.com). The SNP loci along with their flanking 100-bp sequences were curated and a 34-plex MassARRAY multiplex iPLEX assay including adapter-ligated pre-amplification and unextended/nucleotide extension PCR primers targeting each SNP were designed by MassARRAY Assay Design software v3.1 following the criteria of Saxena et al.61. The multiplex PCR amplification, incubation, primer extension, resin clean-up and mass spectrometry-based SNP genotyping were carried out as per the manufacturer\u2019s instructions of Sequenom iPLEX Gold amplification kit. The SNP genotyping data detected in iPLEX spectrochip bio-arrays were analysed by a MassARRAY Typer 3.4. The genotyping information of SNPs based on allele-specific differences in mass between extension products was visualized and documented among 291 chickpea accessions following Saxena et al.61.For genotyping of SNPs, desi and kabuli chickpea accessions. The amplicons were sequenced and high-quality sequences were aligned and compared among accessions to detect SNPs following Kujur et al.8 and Saxena et al.60.For validation of high-quality SNPs, primer-pairs were designed targeting 100-bp either side flanking sequences of 192 representative SNP loci and PCR amplified using the genomic DNA of 48 selected 62 to estimate the per cent polymorphism and PIC (polymorphism information content) among 291 desi and kabuli chickpea accessions. The SNP genotyping information was further analysed on a 100-kb non-overlapping sliding window approach of TASSEL v5.063 (http://www.maizegenetics.net) to measure one of the major nucleotide diversity parameter-\u03b8\u03c0 among chickpea accessions as per Kujur et al.45 and Bajaj et al.18.The genotyping data of DAD-, KAD-, MAD- and TAD-SNPs were analysed with PowerMarker v3.51desi and kabuli chickpea accessions. The cluster analysis among accessions was carried out based on Nei\u2019s genetic distance64 using the neighbour-joining (NJ) method (with 1000 bootstrap replicates) of PowerMarker, and unrooted phylogenetic tree was constructed employing MEGA v7.065.The genotyping data of SNPs were used to assess the molecular diversity and for establishing phylogenetic relationships among 291 66 as per Kujur et al.45. The optimum population number (K) was determined following the ad hoc66 and delta K67 methods. Using the optimum K, the population structure model representing better relationships among chickpea accessions was determined. Diverse population genetic parameters including genetic divergence (FST) and degree of admixture among different population groups were estimated.To determine the population structure among 291 chickpea accessions, the genotyping data of SNPs were analysed in a model-based program STRUCTURE v2.3.4desi (189) and kabuli (102) germplasm accessions were planted in the experimental field with 35\u2009\u00d7\u200910\u2009cm row-to-row and plant-to-plant spacing, respectively, following standard agronomic practices. These accessions were grown in the field as per \u2018alpha-lattice\u2019 design with two replications during crop season for two consecutive years at two diverse geographical locations of India. Four selected desi (ICCV 10 and ICCV 93954) and kabuli (ICC 12968 and Annigeri) chickpea accessions sown after every 10 individual plants were served as reference in the field experimental design to ascertain the homogeneity of association panel across two environments. Seed yield per plant (SYP) was estimated by measuring the average weight (g) of fully matured dried seeds (at 10% moisture content) harvested from 10\u201315 representative plants of each accession. The genetic inheritance pattern of SYP trait based on diverse statistical parameters including coefficient of variation (CV), broad-sense heritability (H2) and frequency distribution were estimated in an association panel as per Bajaj et al.18 and Das et al.23.For large-scale phenotyping, an association panel consisting of 291 diverse desi and kabuli accessions belonging to an association panel of chickpea. To perform association analysis, the compressed mixed linear model (CMLM) and population parameters previously determined (P3D) interfaces of GAPIT were utilized as per Thudi et al.14, Kujur et al.10 and Kumar et al.65. To determine the accuracy of SNP marker-trait association, we performed the quantile-quantile (Q-Q) plot-based false discovery rate (FDR cut-off \u22640.05) corrections for multiple comparisons between observed/expected \u2212log10(P)-values and adjusted P-value threshold of significance in accordance with Benjamini and Hochberg68. The SNP loci associated with SYP trait at a lowest FDR adjusted P-value (threshold P\u2009<\u20091\u2009\u00d7\u200910\u22126) and highest R2 were identified to be highly significant in chickpea. Following FDR-controlling method of model with the SNPs and adjusted P-value, R2 representing the magnitude of SNP marker-SYP trait association was estimated. The trait association outcomes were revalidated through a fast multi-locus random-SNP-effect EMMA (FASTmrEMMA) model of GWAS as per Wen et al.69. The QTNs with LOD >3.0 and P-value\u2009<\u20090.005 were identified to be significantly associated with studied trait.For efficient trait association mapping, we employed the environment-wise SYP phenotyping data measured across two diverse geographical locations and experimental years from each of the selected accession for estimating the mean SYP trait value in an individual accession. The SNP genotyping data was correlated with multi- environments replicated field phenotyping data of SYP trait as well as population structure (Q), kinship (K) matrix and principal component analysis (PCA) (P) information of 291 Table S1Table S2Table S3Table S4Table S5Table S6"} +{"text": "KrasG12D and KRAS-mutant human cancer cells took up markedly higher levels of tetramethylrhodamine (TMR)-dextran than the corresponding wild-type cells. siRNA-mediated inhibition of TFEB did not influence extracellular TMR-dextran uptake, but significantly attenuated lysosomal degradation of extracellular protein. Bovine serum albumin (BSA) treatment restored p-S6K levels and cell proliferation suppressed by leucine deprivation, and these effects were blocked by siTFEB. Collectively, our results show that TFEB plays a role in macropinocytosis-mediated KRAS-mutant cell growth under nutrient deprivation by promoting lysosomal degradation of extracellular proteins.Macropinocytosis is a regulated form of endocytosis that mediates the nonselective uptake of nutrients to support growth under nutrient-deprived conditions. KRAS-mutant cancer cells upregulate macropinocytosis to import extracellular proteins, which subsequently undergo proteolytic degradation in the lysosome. Although transcription factor EB (TFEB) is a master regulator of lysosomal biogenesis and function, its role in the degradation of extracellular protein from macropinocytosis in KRAS-mutant cells has not previously been elucidated. In this study, we investigated the role of TFEB in the recovery of macropinocytosis-mediated mTORC1 activity and cell growth under nutrient depletion. Mouse embryonic fibroblasts (MEFs) expressing Macropinocytosis is a fluid-phase endocytic process whereby extracellular fluid and its content are internalized into cells through large uncoated vacuoles called . OncogenTranscription factor EB (TFEB), a member of the MiTF/TFE family, is a master regulator of multiple cellular processes, including lysosomal biogenesis and autophagy . TFEB reKrasG12D and KrasWT mouse embryonic fibroblasts (MEFs) were obtained from Cre recombinase-induced SV40 large T-immortalized Lox-Stop-Lox-K-RasG12D MEF [G12D MEF . MEFs anCell were treated with 1 mg/mL TMR-dextran 70 kDa in leucine-free medium for 3 h, or with 0.5 mg/mL DQ-BSA (Invitrogen) in leucine-free medium for 6 h. Cells were washed three times with ice-cold phosphate-buffered saline (PBS), and then fixed with 3.7% formaldehyde. After fixation, cells were mounted in mounting solution containing DAPI . For imaging of DQ-BSA fluorescence and lysosome, cells were treated with 0.5 mg/mL DQ-BSA (Invitrogen) for 3 h and 50 nM Lysotracker Red (Invitrogen) for 1 h prior to analysis in leucine-free medium. Nuclei were stained using NucBlueTM Live ReadyProbesTM Reagent (Invitrogen). Images were analyzed using the \u2018Analyze Particle\u2019 tool in Image J (a Java-based image processing program).KRAS, and siTFEB using Lipofectamine RNAiMAX reagent (Invitrogen).For gene silencing, cells were transfected with scrambled siRNA, si4P2O7, 100 mM NaF, 2 mM Na3VO4, 1% NP-40, and protease and phosphatase inhibitors. Proteins from cellular lysates were resolved on a 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred to a polyvinylidene difluoride (PVDF) membrane. Membranes were blocked with 5% skimmed milk and incubated overnight at 4 \u00b0C with primary antibodies at the indicated dilutions: antibodies against p70 S6 kinase, phospho-p70 S6 Kinase (Thr389), and TFEB (1:1000) were obtained from Cell Signaling Technology , and antibody against \u03b2-actin (1:5000) was obtained from Santa Cruz Biotechnology . After incubation with primary antibodies, membranes were washed three times with Tris-buffered saline containing 0.1% Tween 20 (TBST), and then incubated with horseradish peroxidase (HPR)-conjugated rabbit secondary antibody . HRP was detected using the WEST-QueenTM Western Blot Detection Kit .Cells were lysed in lysis buffer containing 20 mM Tris-HCl (pH 7.4), 5 mM EDTA (pH 8.0), 10 mM NaTFEB for 24 h and then maintained in leucine-free medium with or without 3% BSA and EIPA (Sigma) for 72 h. Cell proliferation was measured using a CCK-8 assay .Cells were transfected with scrambled siRNA or sit-test. p < 0.05 was considered statistically significant.All values are presented as means \u00b1 SEM. Statistical analysis was performed using an unpaired KrasG12D MEFs and KRAS-mutant human pancreatic (MIA PaCa-2) and lung (A549) cancer cells with the corresponding KRAS wild-type cells . Macropinosomes were detected using tetramethylrhodamine-labeled high-molecular-mass dextran (TMR-dextran), an established marker of macropinocytosis, on the basis of cells\u2019 ability to internalize extracellular fluid and its contents [G12D MEFs and KRAS-mutant human cancer cells than in the respective wild-type controls -amiloride (EIPA) D,E.TFEB did not affect macropinocytotic uptake, as measured by TMR-dextran incorporation under leucine-depleted conditions . However, this recovery of mTORC1 activity was prevented when TFEB was silenced, indicating that TFEB restored mTORC1 activity in response to BSA stimulation through macropinocytosis. By contrast, treatment of KrasWT MEFs with BSA did not influence mTORC1 activity in the leucine deprivation condition; neither was affected by silencing of TFEB. Consistent with the changes in mTORC1 activity, treatment with BSA exhibited recovery of cell proliferation, which was also suppressed by leucine starvation in KrasG12D MEFs, MIA PaCa-2 and A549 cells, but not in KrasWT MEFs (TFEB and EIPA reduced BSA-treated KrasWT MEF proliferation in the leucine-starved condition, their effect on proliferation in these cells was minimal compared with their effect on proliferation in KRAS-mutant cells. Collectively, these data demonstrated that TFEB contributes to sustained cell proliferation by promoting lysosomal catabolism of extracellular albumin taken up by macropinocytosis.Because mTORC1 activity is suppressed under amino acid starvation, we asked whether TFEB-mediated lysosomal degradation of extracellular protein could restore suppressed mTORC1 activity in leucine-depleted cells. As shown in sWT MEFs C. In addsWT MEFs C. MacropsWT MEFs C. AlthouIn this study, we demonstrated that TFEB plays a pivotal role in acquisition of nutrients required for growth in KRAS-mutant cancer cells under nutrient-depleted conditions. Our results showed that TFEB does not affect macropinocytic uptake, but is responsible for lysosomal catabolism of internalized protein and maintenance of intracellular amino acid availability in KRAS-mutant cancer cells. Therefore, inhibition of TFEB could prevent metabolic adaptation of cancer cells to energy starvation, and thus represents a promising strategy for the treatment of cancers harboring KRAS mutations. KRASWT cells, and can therefore sustain rapid proliferation in the absence of nutrients.Cancer cells can maintain metabolic homeostasis to sustain growth and survival under nutrient limitation by activating stress responses ,17. SeveIn addition to oncogenic signaling, several factors are necessary for coordinately regulating macropinocytosis . AlthougIn summary, this study shows that TFEB is an important metabolic regulator in macropinocytosis-mediated KRAS-mutant cell growth under nutrient-deprived conditions. TFEB initiates anabolic pathways and cellular adaptation to metabolic stress by promoting lysosomal degradation of extracellular proteins. TFEB-mediated lysosomal catabolism of macropinocytic proteins confers a growth advantage on KRAS-mutant cells. Future studies should explore how the function of TFEB could be therapeutically targeted."} +{"text": "Scientific Reports 10.1038/s41598-017-10130-6, published online 29 August 2017Correction to: This Article contains an error in the Introduction where,\u201cActive projects include KiteGen (drag) and Makani (lift), both are under active development and pilot studies.\u201dshould read:\u201cActive projects include KiteGen (ground-based generator system) and Makani (on-board generator system), both are under active development and pilot studies.\u201d"} +{"text": "OBJECTIVES/SPECIFIC AIMS: Allogeneic hematopoietic stem cell transplantation is a curative procedure for hematological malignancies. Chronic graft Versus host disease (cGVHD) is a lethal complication that often develops after allo-HCT. Fli-1 is an aberrantly expressed protein in cancers including erythroleukemia and melanoma, while being implicated in pathogenesis of systemic lupus in mice and humans, a disease with marked similarity to cGVHD. METHODS/STUDY POPULATION: cGVHD was induced using hematopoietic cells from conditional knock-out mice deficient for the fli-1 gene specifically on T cells and progression of cGVHD in murine allo-HCT recipients was monitored using a clinical scoring system, and changes in activation status of hematopoietic cell populations were quantified using flow cytometry. RESULTS/ANTICIPATED RESULTS: Recipients transplanted with fli-1 deficient T cells exhibited reduced cGVHD clinical scores compared with littermate wild-type controls. Donor-grafts containing fli-1 deficient T cells were associated with restrained T-cell responses including reduced Interferon-y cytokine production, PD-1 expression, and differentiation into follicular helper T cells. fli-1 T-cell deficient donor-grafts also improved donor B-cell reconstitution and reduced plasma cells in allo-HCT recipients relative to littermate wild-type control donor-graft recipients. DISCUSSION/SIGNIFICANCE OF IMPACT: Thus, inhibiting Fli-1 represents a promising therapeutic strategy for the goal of preventing cGVHD after allo-HCT while also directly targeting cancers which aberrantly express Fli-1."} +{"text": "Over 1 in 5 HIV-positive women reported discrimination in the healthcare system based on HIV status (21.3%). Report of discrimination based on drug/alcohol use was higher among HIV-negative participants . Among women with longitudinal sustained viremia, report of discrimination based on race ethnicity and sexual orientation were higher than within the nonviremic and intermittent trajectory groups. DISCUSSION/SIGNIFICANCE OF IMPACT: Physician trust did not associate with increased longitudinal viral suppression among HIV-positive women in Washington, DC. Lack of physician trust among high-risk HIV-negative women could have implications for uptake of prevention methods. Reports of discrimination vary between HIV-positive and HIV-negative women in the Washington, DC area. The findings of healthcare system distrust among HIV-negative women has implications outside the realm of HIV, as this lack of trust may impact risk for other disease states among similar populations of women.OBJECTIVES/SPECIFIC AIMS: Discrimination within the healthcare system and physician distrust have been associated with adverse clinical outcomes for people living with HIV; however, many studies do not link these variables to biological data. We hypothesize that perceived healthcare discrimination and physician distrust associates with higher longitudinal viremia among HIV-positive women. METHODS/STUDY POPULATION: A 2006 cross-sectional survey assessed healthcare-based discrimination and physician trust in 92 HIV-positive and 46 high-risk HIV-negative women from the Washington DC Women\u2019s Interagency HIV Study (DC-WIHS). In addition, we identified HIV viral load trajectories and demographics from the HIV-positive women who contributed\u22654 semi-annual visits from 1994 to 2015. Viral suppression was defined by assay detection limits (<80 to <20 copies/mL). Group-based probability trajectory analyses grouped women based on longitudinal viral load patterns, and identified 3 groups: sustained viremia (n=32) with low-viral suppression over time, intermittent viremia (n=27) with varying suppression over time, and non-viremia (n=33) with high-longitudinal viral suppression. Ordinal logistic regression models assessed trajectory group and discrimination variables, controlling for demographics, using stepwise selection with significance level of \u03b1=0.05. RESULTS/ANTICIPATED RESULTS: Most women were African American (60%), insured at the time of visit (89%) and nonsmokers (56%). While physician trust did not differ by HIV viral trajectory group, trust was lower among HIV-negative women compared with HIV-positive women ("} +{"text": "We forward a methodological approach, using model-based cluster analyses, and ambulatory assessments of cognition (2 indicators from each task), to derive subgroups of interest for tailored clinical follow-up in a longitudinal framework. Community dwelling adults were asked to complete 14 consecutive days of ecological momentary assessments (EMAs) using smartphones, including measures of cognitive performance, and self-reported physical and mental health outcomes . A stable four-cluster solution emerged, labelled as: (1) a high-risk cognitive group ; (2) subjective risk group ; (3) normative aging ; (4) super-cognitive agers . In conclusion, these findings highlight the potential of a cluster-based approach for risk classification, uncovering different profiles of poor performance that may represent different etiologies."} +{"text": "C. elegans as a regulator of developmental timing [Over the past twenty years, mounting evidence showed that microRNA (miR) plays indispensable roles in various biological processes including aging process, immune cell responses and metabolic reprogramming through posttranscriptional gene targeting . MiR-encl timing . The Letl timing . An intrl timing . Based oGlucose and glutamine are two principle nutrients in B cell activation, proliferation, and differentiation. Regulation of glucose and glutamine metabolism plays a key role in B cell activation . Our stuWe reported that comprehensive repression of all let-7 members by overexpressing Lin28a in B cells contributes to both T cell-independent (TI) and T cell-dependent (TD) antigen-induced antibody production . Lin28a Future studies using profiling technologies such as RNA immunoprecipitation sequencing and liquid chromatography-tandem mass spectrometry would provide a global picture of miR-mediated gene and metabolic regulations in anti-CD40-activated Lin28a iTg B cells.Although we showed that the let-7adf cluster is essential to TI responses in splenic B cells by tracing and analyzing both glucose and glutamine metabolism , many quOur study focused on the physiological role of let-7adf cluster in B cell activation in young adult mice , but it"} +{"text": "Arterial stiffness and blood pressure (BP) are contributors to cognitive decline and dementia. Lower global cerebral blood flow (CBF) is one of the earliest manifestations of biological alterations linked to cognitive decline, nevertheless the best cardiovascular predictor of CBF in gray-matter (CBF-GM) remains to be identified. Our objective is to determine the best predictor of CBF-GM levels amongst cardiovascular parameters. Eigthy-four healthy participants between 60-80 years-old were evaluated. The measured parameters for arterial stiffness were the carotid-femoral pulse wave velocity (cf-PWV) and the augmentation index (Aix), measured by applanation tonometry (SphygmoCor). Mean systolic BP (SBP) was monitored over 24-hours and analyzed following Hypertension-Canada\u2019s guidelines(2018). The coefficient of variation for 24-hours SBP was calculated by dividing the standard deviation by the mean. Resting CBF-GM was quantified from pseudocontinuous arterial-spin-labeling using Neurolens 2.0(pcASL), and acquired on a 3T scanner (MAGNETOM Prisma-Fit). We created multiple linear regression models for each independent variable using age, sex, schooling and body mass index as covariates. Multiple linear regression models demonstrated that two independent variables could predict CBF-GM levels: a)PWV (p=0.010) and b)Aix (p=0.006). In this cohort, we demonstrated that while PWV and Aix are both predictors of CBF-GM levels, it is Aix which has the highest predictive value and could be a useful tool to understand the interplay between lower CBF-GM and arterial stiffness. These results also indicate that Aix may be a good therapeutic target to preserve brain health and cognition."} +{"text": "MYC amplification), which includes 50\u201360% of patients and has a 5-year event-free survival of 75\u201385%. Within standard-risk medulloblastoma, patients in the WNT subgroup are established as having a favourable prognosis; however, outcome prediction for the remaining majority of patients is imprecise. We sought to identify novel prognostic biomarkers to enable improved risk-adapted therapies.Most children with medulloblastoma fall within the standard-risk clinical disease group defined by absence of high-risk features . We assessed the clinical behaviour of the molecularly defined WNT and SHH subgroups, and identified novel independent prognostic markers and models for standard-risk patients with non-WNT/non-SHH disease. Because of the scarcity and low quality of available genomic material, we used a mass spectrometry-minimal methylation classifier assay (MS-MIMIC) to assess methylation subgroup and a molecular inversion probe array to detect genome-wide copy number aberrations. Prognostic biomarkers and models identified were validated in an independent, demographically matched cohort (n=70) of medulloblastoma patients with non-WNT/non-SHH standard-risk disease treated with conventional therapies associated treatment centres between 1990 and 2014. These samples were analysed by Illumina 450k DNA methylation microarray. HIT-SIOP PNET 4 is registered with TP53mut) or chromosome 17p loss. A novel whole chromosomal aberration signature associated with increased ploidy and multiple non-random whole chromosomal aberrations was identified in 38 (42%) of the 91 samples from patients with non-WNT/non-SHH medulloblastoma in the HIT-SIOP PNET 4 cohort. Biomarkers associated with this whole chromosomal aberration signature predicted favourable prognosis. Patients with non-WNT/non-SHH medulloblastoma could be reclassified by these markers as having favourable-risk or high-risk disease. In patients in the HIT-SIOP PNET4 cohort with non-WNT/non-SHH medulloblastoma, with a median follow-up of 6\u00b77 years (IQR 5\u00b78\u20138\u00b72), 5-year event-free survival was 100% in the favourable-risk group and 68% in the high-risk group. In the validation cohort, with a median follow-up of 5\u00b76 years (IQR 3\u00b71\u20138\u00b71), 5-year event-free survival was 94\u00b77% (95% CI 85\u00b72\u2013100) in the favourable-risk group and 58\u00b76% (95% CI 45\u00b71\u201376\u00b71) in the high-risk group . Our comprehensive molecular investigation identified subgroup-specific risk models which allowed 69 (51%) of 134 accessible patients from the standard-risk medulloblastoma HIT-SIOP PNET 4 cohort to be assigned to a favourable-risk group.We analysed methylation subgroup, genome-wide copy number aberrations, and mutational features in 136 assessable tumour samples from the HIT-SIOP PNET 4 cohort, representing 40% of the 338 patients in the trial cohort. This cohort of 136 samples consisted of 28 (21%) classified as WNT, 17 (13%) as SHH, and 91 (67%) as non-WNT/non-SHH . Favourable outcomes for WNT tumours were confirmed in patients younger than 16 years, and all relapse events in SHH (four [24%] of 17) occurred in patients with TP53 mutation (TP53wild-type tumours should be considered for therapy de-escalation in future biomarker-driven, risk-adapted clinical trials. The remaining subgroups of patients with high-risk medulloblastoma might benefit from more intensive therapies.We define a whole chromosomal signature that allows the assignment of non-WNT/non-SHH medulloblastoma patients normally classified as standard-risk into favourable-risk and high-risk categories. In addition to patients younger than 16 years with WNT tumours, patients with non-WNT/non-SHH tumours with our defined whole chromosomal aberration signature and patients with SHH-Cancer Research UK, Swedish Childhood Cancer Foundation, French Ministry of Health/French National Cancer Institute, and the German Children's Cancer Foundation. TP53wild-type, SHH-TP53mut, and non-WNT/non-SHH).Medulloblastoma, the most common malignant childhood brain tumour, is now recognised as an umbrella term for different molecular pathological disease entities. These entities differ in their progenitor cells, characteristic mutations, biological profiles, and clinical behaviour. Currently, WHO classification of CNS tumours recognises four distinct genetically defined entities . Standard-risk, non-infant disease represents the largest clinical treatment group of patients. The ongoing pan-European SIOP PNET 5 MB clinical trial defines standard-risk, non-infant disease as the absence of high-risk clinical features such as metastatic disease or subtotal resection, molecular features (MYC or MYCN amplification or TP53 mutation in SHH medulloblastoma), and histological characteristics (large-cell/anaplastic disease). These definitions were established based on previous disease-wide studies. The SIOP PNET 5 MB trial is investigating reduced-intensity therapies for patients classified as standard-risk with expected good prognosis , aimed at maintaining overall survival while minimising late toxicities. However, biomarkers that stratify risk within remaining standard-risk patients with non-WNT medulloblastoma have not been identified. Moreover, novel non-WNT/non-SHH medulloblastoma epigenetic subtypes have been recognised; however, these subtypes remain to be validated and implemented clinically. Our own reviews of the literature formed the foundation for the present study; we did not carry out any formal literature searches before the study start date .International consensus and the 2016 WHO classification recognise the following distinct clinico-molecular disease entities in medulloblastoma: WNT, SHH-Added value of this studyTo our knowledge, HIT-SIOP PNET 4 is the only completed pan-European clinical trial in patients with standard-risk medulloblastoma. However, to date, systematically collected biological material remaining from this trial was not amenable to contemporary molecular analysis. Application of novel methods to enable assessment of this cohort, and investigation of an independent demographically matched standard-risk medulloblastoma validation cohort, allowed derivation and validation of biomarker-driven, risk-stratification models on the basis of the molecular pathology of standard-risk medulloblastoma, including a novel whole chromosomal cytogenetic aberration signature within standard-risk non-WNT/non-SHH medulloblastoma. These newly described whole chromosomal cytogenetic aberration signatures allowed reallocation of more than 50% of HIT-SIOP PNET 4 patients with standard-risk medulloblastoma into a favourable-risk group, while the remaining patients were classified as high risk. Therefore, findings from this study resolve current patients with standard-risk medulloblastoma into biomarker-defined distinct favourable-risk and high-risk groups, and represent a substantial step in our ability to risk stratify and clinically manage medulloblastoma.Implications of all the available evidenceThe results of this study redefine the concepts of risk stratification in standard-risk medulloblastoma, providing insight into its molecular subtypes, their underpinning biology, and clinical application. Stratification of standard-risk medulloblastoma by use of the biomarkers and validated schemes we describe could allow assignment of 150\u2013200 patients per year in Europe into a favourable-risk group, and such patients could benefit from reduction of treatment intensity. Patients not classified as favourable-risk should be considered high-risk and might benefit from treatment intensification. The molecular risk groups and biomarker schemes presented in this study are amenable to routine diagnostic assessment and provide a foundation for future clinical trials and research investigations.MYC or MYCN amplification, large-cell/anaplastic histology, metastatic disease, or subtotal resection, define high-risk disease .NCT02066220) and SJMB12 (NCT01878617) trials, which aim to improve outcomes through reduced-intensity therapies for favourable-risk patients and randomised assessment of adapted therapies in the remaining patients.Discovery and validation of clinically meaningful medulloblastoma features in previous clinical trial cohorts have driven advances in the clinical management of the disease. Children younger than 16 years of age at diagnosis with WNT-activated medulloblastomas have consistently achieved favourable outcomes ,TP53mut.TP53wild-type medulloblastoma13Standard-risk medulloblastoma represents the predominant clinical treatment group (around 60% of patients) and is defined by the absence of clinical, molecular, and histopathological high-risk features. This group encompasses tumours of all variants except high-risk SHH-To our knowledge, HIT-SIOP PNET 4Formalin-fixed, paraffin-embedded (FFPE) tumour material for biological studies was prospectively collected, which enabled confirmation of favourable outcomes in patients with WNT medulloblastoma (defined by immunohistochemistry [IHC]) and identification of chromosome 17 imbalances on a diploid background as a marker of poor prognosis.In this Article, we report comprehensive molecular and pathological characterisation of the HIT-SIOP PNET 4 cohort using novel technologiesNCT01351870).2 intravenously) and lomustine (75 mg/m2) on day 1, and vincristine (1\u00b75 mg/m2 intravenously) on days 1, 8, and 15, were given with a 6 week interval between each cycle.In this retrospective analysis, we assessed remaining tumour samples from patients from the HIT-SIOP PNET 4 trial or tumour sections, originally intended for FISH and IHC,We validated and extended our findings in a second independent, demographically matched, retrospective cohort of patients with non-WNT/non-SHH standard-risk medulloblastoma (n=70) collected at UK Children's Cancer and Leukaemia Group and European Society for Paediatric Oncology (SIOPE) associated treatment centres between 1990 and 2014. Patients in this cohort received equivalent therapies of 70 patients]).Written informed consent for tumour collection for biological studies was obtained from patients or their parents. Tumour investigations were done with approval from Newcastle and North Tyneside Research Ethics Committee (study reference 07/Q0905/71)\u2014all tumour material was collected in accordance with this approval.Because only material of mostly low quantity and quality was available, the HIT-SIOP PNET 4 samples were unsuitable for subgroup assessment using conventional approaches in samples with CTNNB1wild-type WNT medulloblastoma. We used a molecular inversion probe array to identify aberrant changes in genomic copy number in samples from the HIT-SIOP PNET 4 trial.We assessed amplification of Event-free survival was defined as the time from surgery to first event (progression or relapse), or date of last follow-up. Patients whose follow-up time exceeded 10 years were right-censored at 10 years. Clinical follow-up data were collected according to the HIT-SIOP PNET 4 trial protocol.Using hierarchical clustering, we clustered samples classified as non-WNT/non-SHH medulloblastoma subtype by their recurrent whole chromosomal aberration , cytogenetic markers , and cumulative numbers of total whole chromosomal aberrations (gains vs losses). We verified the proportionality assumption for Cox modelling using scaled Schoenfeld residuals. We derived pragmatic assignments of patient risk by combining whole chromosomal aberrations that were significantly different in univariate testing to define risk groups and assessed their predictive value by calculating total area under the curve (AUC), sensitivity, and specificity at 5 years since diagnosis . Group3 and Group4 had very similar event-free survival curves (With a median follow-up of 6\u00b77 years (IQR 5\u00b76\u20138\u00b74) in the HIT-SIOP PNET 4 cohort, 5-year event-free survival was equivalent between patients who received standard radiotherapy and those who received hyperfractionated radiotherapy of 28 WNT tumours and we identified APC frameshift deletions (E1309fs \u0394AAAAG and Q1062fs \u0394ACAAA). Outcomes within the WNT subgroup were age-dependent. We observed a 5-year event-free survival of 100% in patients younger than 16 years at diagnosis, and all WNT relapses (three [11%] of 28 WNT tumours) occurred in patients aged 16\u201320 years and TP53 mutations were associated with each other . We did not observe MYCN amplifications in tumours classified as SHH medulloblastoma, including TP53mut tumours. A previously reported SHH disease risk model (of chromosome 14 loss and GLI2 amplification)Tumours classified as SHH in the HIT-SIOP PNET 4 cohort (17 [13%] of 136 patients) also had few whole chromosomal aberrations . Chromosvs 1\u00b782 [SD 2\u00b756] for WNT and 1\u00b776 [SD 2\u00b700] for SHH; MYCN amplifications, OTX2, CCND2, and 18q12 [TPTE2] gains or amplifications, SNCAIP duplications, and 13q11\u201312 [SETBP1] loss; MYCN amplification, i17q alterations, and subtotal resection)The 91 (67%) non-WNT/non-SHH tumours in the HIT-SIOP PNET 4 cohort of 136 were characterised by a higher incidence of whole chromosomal aberrations in the high-risk group molecularly characterised patients with medulloblastoma from the HIT-SIOP PNET 4 cohort with a favourable prognosis .A pooled analysis applied the validated whole chromosomal aberration signature-based risk-stratification model to the merged non-WNT/non-SHH medulloblastomas from the HIT-SIOP PNET 4, and validation cohorts (n=161) and classified 58 (36%) non-WNT/non-SHH medulloblastomas as favourable-risk and 103 (64%) as high-risk; 5-year event-free survival was 98\u00b73% (95% CI 94\u00b79\u2013100) in the favourable-risk group Implementation of enabling technologies (MS-MIMIC and molecular inversion probe assay) allowed us to systematically assess the molecular pathology of the standard-risk medulloblastoma clinical group within the HIT-SIOP PNET 4 patient cohort. To our knowledge, no equivalent multicentre, prospective investigations of standard-risk medulloblastoma have been reported. Although wider, retrospective medulloblastoma datasets are available, these typically lack the full clinical and molecular annotation necessary to define the standard-risk medulloblastoma group. The standard-risk medulloblastoma group displayed distinct demographics versus disease-wide cohorts.TP53 mutations (SHH-TP53wild-type) or chromosome 17p loss similarly had a favourable prognosis. These data validate independent previous findingsSMO inhibitors).The favourable prognosis of patients with WNT medulloblastoma was confirmed in patients from the HIT-SIOP PNET 4 cohort who were younger than 16 years at diagnosis. However, patients older than 16 years did not share this good prognosis, consistent with previous reports.Development of biomarker-driven treatment strategies for the large remaining group of patients with non-WNT/non-SHH disease represents the largest ongoing challenge for standard-risk medulloblastoma. In the absence of high-risk features,Group4-LowRisk,Group4-HighRiskIn this whole chromosomal aberration group, using multivariable event-free survival analysis and risk modelling, we deduced a whole chromosomal aberration signature , which best defined patients with non-WNT/non-SHH medulloblastoma with favourable prognosis. We validated these findings in an independent demographically matched standard-risk medulloblastoma cohort, and they were reproducible when Group4 patients were considered in isolation. This whole chromosomal aberration signature was detected within a number of novel methylation subgroups within non-WNT/non-SHH medulloblastoma, and associated with the low-risk MBBiologically and clinically significant whole chromosomal phenotypes are a notable feature of childhood malignancies other than medulloblastoma. Characteristic patterns of non-random whole chromosomal aberrations in neuroblastoma This common involvement of whole chromosomal aberration signatures provides strong impetus to understand the underlying molecular pathomechanisms, including errors in mitotic control, chromosome segregation, and function of the spindle apparatus. Although beyond the scope of this study, investigation of associated biology and the involvement of specific chromosomes , is essential to improve understanding and therapeutic targeting. Potential opportunities include agents that target the spindle apparatus or mitotic control. For instance, vincristine (a component of medulloblastoma treatment regimens) directly targets the spindle apparatus, and the excellent whole chromosomal aberration signature-associated outcomes might be explained by high sensitivity to such treatments. Indeed, the association between HeH acute leukaemia and chemosensitivity associated with increased DNA content has been long established.This study has some limitations. The developed risk stratification scheme applies only to non-infant, standard-risk medulloblastoma treated with standard multimodal therapies. Children younger than 4 years, patients treated with chemotherapy only, and high-risk patients require independent assessment and development of appropriate risk stratification schemes. However, our biomarker-driven risk stratification schemes for standard-risk medulloblastoma are readily testable in routine molecular diagnostic practice and, following their validation in independent clinically controlled and biomarker-defined cohorts, could form the basis of international clinical trials aimed at improving outcomes.In summary, our molecular pathological characterisation of the HIT-SIOP PNET 4 cohort identified and independently validated a whole chromosomal aberration signature-defined subgroup of non-WNT/non-SHH medulloblastomas associated with good prognosis. Combination of these newly defined subtypes with the favourable-risk WNT and SHH medulloblastomas validated in our study redistributed around 50% of patients with standard-risk medulloblastoma into a favourable-risk group, who could benefit from reduced-intensity therapies aimed at maintaining overall survival while reducing treatment-associated toxicities and late effects. Patients not classified into this favourable-risk group had a 5-year event-free survival of around 60% and should be considered high risk. In the HIT-SIOP PNET 4 cohort, this model compared favourably with published and currently accepted risk stratification schemes (eg, Shih and colleagues"} +{"text": "Terpenoids comprise tens of thousands of small molecule natural products that are widely distributed across all domains of life. Plants produce by far the largest array of terpenoids with various roles in development and chemical ecology. Driven by selective pressure to adapt to their specific ecological niche, individual species form only a fraction of the myriad plant terpenoids, typically representing unique metabolite blends. Terpene synthase (TPS) enzymes are the gatekeepers in generating terpenoid diversity by catalyzing complex carbocation-driven cyclization, rearrangement, and elimination reactions that enable the transformation of a few acyclic prenyl diphosphate substrates into a vast chemical library of hydrocarbon and, for a few enzymes, oxygenated terpene scaffolds. The seven currently defined clades (a-h) forming the plant TPS family evolved from ancestral triterpene synthase- and prenyl transferase\u2013type enzymes through repeated events of gene duplication and subsequent loss, gain, or fusion of protein domains and further functional diversification. Lineage-specific expansion of these TPS clades led to variable family sizes that may range from a single TPS gene to families of more than 100 members that may further function as part of modular metabolic networks to maximize the number of possible products. Accompanying gene family expansion, the TPS family shows a profound functional plasticity, where minor active site alterations can dramatically impact product outcome, thus enabling the emergence of new functions with minimal investment in evolving new enzymes. This article reviews current knowledge on the functional diversity and molecular evolution of the plant TPS family that underlies the chemical diversity of bioactive terpenoids across the plant kingdom. Among the wealth of small molecule natural products, terpenoids form an especially diversified and evolutionary ancient superfamily, which likely emerged alongside the formation of primitive membranes at the very origins of cellular life . UbiquitThe biological and economic relevance of terpenoids has fostered long-standing efforts in understanding the metabolic enzymes that generate terpenoid chemical diversity. Following common metabolic patterns of scaffold-forming and tailoring reactions in specialized metabolism , terpeno5 isoprenoid precursor isopentenyl diphosphate (IPP) and its double-bond isomer dimethylallyl diphosphate (DMAPP) (C-methyl-D-erythritol-4-phosphate (MEP) pathway and carrot (Daucus carota) (Gossypium hirsutum) showed contribution of the MVA pathway to C10\u2013C40 terpenoid biosynthesis . Unlike pathway . Similarynthesis .via the MVA and MEP pathways is not solely routed through IPP and DMAPP but can involve a pool of the respective isopentenyl and dimethylallyl monophosphates, IP, and DMAP and sesquiterpenes produced (25\u201331%). Conversely, overexpression of AtIPK in transgenic Nicotiana tabacum led to a 3-fold and 2-fold increase in sesquiterpenes and monoterpenes, respectively. Further efforts to understand the formation of IP/DMAP in plants identified a role of Nudix hydrolases, a superfamily of two-domain hydrolases/peptidases broadly found in bacteria, animals, and plants , monoterpenes (148\u2013503%), and sterols (\u223c50%) whereas overexpression of these enzymes in N. tabacum resulted in decreased production of monoterpenes (\u223c50%) and sesquiterpenes (57\u201388%). Although understanding the broader relevance of IP kinase and Nudix hydrolase genes in plant terpenoid metabolism requires further studies, these collective findings highlight the potential of these pathway reactions to possibly function as additional regulatory mechanisms for balancing the IP/DMAP and IPP/DMAPP pools in the biosynthesis of terpenoids and other isoprenoids , C15 , and C20 intermediates as precursors in mono-, sesqui-, and di-terpenoid metabolisms, respectively products catalyze the sequential condensation of isoprenoid units ecursors . As alteectively . Similarhosphate . Recent species . Arabidoproducts .cis- or trans-C\u2013C double-bond configuration of their product and SARM (DDx2D), which are critical for substrate binding, whereas cis-PTs lack these motifs, and substrate binding is controlled by Asp and Glu residues broadly distributed within the active site (30) PTs more commonly produce cis-prenyl diphosphate compounds, most prominently represented by cis-PTs of rubber biosynthesis (10\u201325) prenyl diphosphates occur in the trans configuration. However, a number of cis-PTs have been identified in certain species that produce intermediates featuring cis- and trans-configured double bonds. For example, short-chain cis-PTs were identified in tomato (Solanum sp.) to form (+)-\u03b1-santalene, (+)-endo-\u03b2-bergamotene, and (\u2212)-endo-\u03b1-bergamotene (trans-FPP synthases (Solanum lycopersicum) identified neryl-diphosphate synthase 1 (NDPS1) catalyzing the formation of a cis-neryl diphosphate (NPP) as a precursor for a range of monoterpenoids in addition to the canonical trans-substrate GPP (cis-PT (CPT2) that produces the cisoid C20 GGPP variant Z,Z,Z-nerylneryl diphosphate (NNPP) . In the gamotene . Notablyynthases . Furtherrate GPP . More ree (NNPP) . NNPP coe (NNPP) . Combinaert NNPP . Togethe5n prenyl diphosphate substrates. At the core of TPS product specificity is the intramolecular rearrangement of highly reactive carbocation intermediates. Although with far greater variation, these reactions are mechanistically analogous to those observed in PT enzymes synthase from Streptomyces platensis empirically demonstrated this typical \u03b1-barrel \u03b2\u03b3-domain structure featuring a Dx4E motif closely related to the DxDD signature motif critical for the activity of plant class II diTPSs and some phytopathogens such as species of Xanthomonas and Erwinia (ent-CPP and ent-kaurene synthases are core enzymes in the formation of bioactive gibberellin (GA) phytohormones, and all so far characterized bacterial ent-CPP and ent-kaurene synthases function as part of GA-biosynthetic operons, albeit with some end product variation ranging from GA precursors to bioactive GA4 as compared to 12\u201314 introns in classical plant TPSs such as Laurencia pacifica,Porphyridium purpureum, and Erythrolobus australicus . A family of MTPSL was first discovered by Li et al. in the ant TPSs . The 48 ant TPSs . Secondlant TPSs . Lastly,y origin . Despitendorffii . Followilineages -kaurene-producing bifunctional diTPSs underwent neo-functionalization toward the biosynthesis of diterpenoids with specialized functions in a number of species. The moss Hypnum plumaeforme contains a bifunctional diTPS that forms syn-pimara-7,15-diene, a diterpenoid also present in rice as precursor of anti-microbial and allelopathic momilactones , namely, normal (9S10S) CPP or (+)-CPP as an intermediate (SmMDS) and labda-7,13E-dien-15-ol (SmCPS/KSL1) via labda-15-en-8-ol diphosphate (LPP) (Ginkgo biloba (Abies) (Picea) (Pinus) , namely, normal (9S10S) CPP or (+)-CPP, as an intermediate en route to the their individual diterpene products. In addition, some species feature diTPSs that have undergone additional neo-functionalization. For example, balsam fir contains a cis-abienol synthase (AbCAS) that catalyzes conversion of GGPP into cis-abienol via the C-8 hydroxylated CPP intermediate LPP also observed in S. moellendorffii . Althougte (LPP) . In contte (LPP) . Bifuncto biloba , species (Abies) , spruce (Picea) , and pin (Pinus) . Notablyndorffii .Ascomycota and some Basidiomycota species where they function in pathogenic or plant growth\u2013promoting pathways (ent-CPP/ent-kaurene synthase activity was first identified as a part of a GA-biosynthetic gene cluster in Gibberella fujikuroi (genus Fusarium), the causal agent of to bakanae disease in rice (Oryza sativa) . Beyond ma betae or the eamygdali . Recent I diTPSs . This hyse genes . Interesse genes , such asn toxins .ent-CPP synthase) activity in the \u03b2\u03b1-domain and the other copy acting as a monofunctional class I diTPS (ent-kaurene synthase) with a functional \u03b1-domain -CPP, first identified as an intermediate of gymnosperm class II/I diTPSs (Lamiaceae species (Poaceous crops such as maize (Zea mays) and wheat (Triticum aestivum) appear to be of narrower taxonomic distribution with current examples limited to some Poaceous grasses -CPP, and syn-CPP are the most commonly occurring labdane diterpene precursors, variable series of 1,2-methyl and/or hydride migrations prior to carbocation neutralization can result in other isomeric structures , and CelastraceaeTripterygium wilfordii . Class I grasses . While eructures . Exampleilfordii ; 7,13-CP robusta ; and mos robusta . In addirmediate . Here, ormediate . But alsuenching .ent-kaurene synthases within the TPS-e/f clade and lodgepole pine (Pinus contorta) that derived from bifunctional progenitors and are capable of utilizing the (+)-CPP intermediate produced by bifunctional class II/I enzymes to form a set of pimarane-type labdanes (Thuja plicata) , and ultimately, sesqui-TPSs and mono-TPSs have arisen . Biochemvia hydroxylation in a manner analogous to class II diTPSs that produce C-8 or C-9 hydroxylated prenyl diphosphate products KSL5 and maize TPS1 spanning mono-, sesqui-, and di-terpenoid products and 113 calyptus , enablinTaxus spp.) that produce the taxane scaffold in the biosynthesis of the chemotherapeutic agent Taxol (Pseudolarix amabilis) that forms an unusual 5\u20137-ring scaffold en route to the bioactive anti-cancer compound pseudolaric acid B -4,8,12-trimethyltrideca-1,3,7,11-tetraene (TMTT) with anti-herbivory activity are \u03b3\u03b2\u03b1-domain diTPSs that uniquely catalyze the ionization of the GGPP diphosphate ester bond without subsequent cyclization to occur Figure 5activity . Intriguqui-TPSs . Althougqui-TPSs . Conversrbiaceae , cembrat species , Arabidosynthase , and mosvulgaris (Taxus brevifolia taxadiene synthase (class I diTPS) (A. grandis abietadiene synthase (class II/I diTPS) , Taxus bI diTPS) , A. granI diTPS) , and a fI diTPS) . The conA. grandis demonstrated that class II catalysis uses a general aid-base mechanism to bring about the cyclo-isomerization of GGPP, whereby the acid function is provided by the middle aspartate of the conserved DxDD motif -CPP to 8\u03b1-hydroxy-CPP -CPP synthases is represented by a hydrogen-bonded histidine-tyrosine pair -CPP synthase as well as other Lamiaceae class II diTPSs showed a dramatic impact on product outcome. Likewise, alanine substitution of a corresponding phenylalanine residue in a 8,13-CPP synthase from switchgrass (PvCPS3) resulted in both positional isomers and hydroxylated forms of the native 8,13-CPP product showed that substitution of the corresponding phenylalanine residue and a proximal tryptophan in MvCPS1 also redirected product outcome to yield a halimadienyl diphosphate scaffold residue in the rice hosphate and polar (P. trichocarpa) showed that mutagenesis of the corresponding methionine residues in these enzymes had the reciprocal effect by redirecting product specificity toward 16\u03b1-hydroxy-ent-kaurane instead of ent-kaurene illustrated that reciprocal exchange of a largely conserved SIAL/SISL motif located at this hinge region resulted in the complete interconversion of the respective abietadiene and isopimaradiene synthase activities 3-carene synthase, and sabinene synthase associated with tree resistance against white pine weevil has been reported (Ricinus communis) that catalyze epoxidation and oxidation reactions converting macrocyclic casbene and neocembrene scaffolds in Euphorbiaceae species -dependent dioxygenases (2-ODDs) , for exain planta and under various environmental conditions. To this end, gene editing and transformation techniques applicable to a broader range of model and non-model species that produce species-specific blends of bioactive terpenoids will be critical , by the DOE Joint Genome Institute Community Science Program (grant# CSP2568 to PZ), and by the DOE Early Career Research Program (grant# DE-SC0019178 to PZ).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Stair climbing assesses neuromuscular components of movement, including muscle power (force*velocity) which may decline earlier in aging vs. strength. We hypothesized age and age-related factor associations to stair climb total time (sec), ascend lap time degradation (lap 1 minus 3), power (W/kg body weight) and power degradation (lap 1 minus 3). Adjusting for demographic, lifestyle and age-related comorbidity factors using multivariate linear regression, older age independently related to slower total time and lower power. Non-white ethnicity had slower total time , higher ascend time degradation (Hispanic), and lower power vs. Whites. Higher 36-Item Short Form Health Survey (SF-36) and Modified Baecke physical activity scores indicated better performance: lower total time, higher power (SF-36 only), and less degradation in ascend time and power. Stair climb time and power in early old age may capture initial functional loss targets for interventions to prevent late-life disability."} +{"text": "Abnormally upregulated cholesterol and lipid metabolism, observed commonly in multiple cancer types, contributes to cancer development and progression through the activation of oncogenic growth signaling pathways. Although accumulating evidence has shown the preventive and therapeutic benefits of cholesterol-lowering drugs for cancer management, the development of cholesterol-lowering drugs is needed for treatment of cancer as well as metabolism-related chronic diseases. Ursolic acid (UA), a natural pentacyclic terpenoid, suppresses cancer growth and metastasis, but the precise underlying molecular mechanism for its anti-cancer effects is poorly understood. Here, using sterol regulatory element (SRE)-luciferase assay-based screening on a library of 502 natural compounds, this study found that UA activates sterol regulatory element-binding protein 2 (SREBP2). The expression of cholesterol biosynthesis-related genes and enzymes increased in UA-treated hepatocellular carcinoma (HCC) cells. The UA increased cell cycle arrest and apoptotic death in HCC cells and reduced the activation of oncogenic growth signaling factors, all of which was significantly reversed by cholesterol supplementation. As cholesterol supplementation successfully reversed UA-induced attenuation of growth in HCC cells, it indicated that UA suppresses HCC cells growth through its cholesterol-lowering effect. Overall, these results suggested that UA is a promising cholesterol-lowering nutraceutical for the prevention and treatment of patients with HCC and cholesterol-related chronic diseases. Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer, which is closely associated with chronic liver diseases, particularly viral hepatitis and metabolic steatohepatitis, and is the third most common cause of cancer-related deaths worldwide ,2,3.Ocimum sanctum L.), thyme (Thymus vulgaris L.), lavender (Lavandula augustifolia), catnip (Nepeta sibthorpii), peppermint leaves (Mentha piperita L.), or fruit peel [Ursolic acid (UA) is a pentacyclic terpenoid and a secondary plant metabolite, usually found in the holy basil /protein kinase B (AKT) and epidermal growth factor receptor (EGFR)/mitogen-activated protein kinase (MAPK) pathways, and oncogenic transcription factors, such as nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB), signal transducer and activator of transcription 3 (STAT3), and hypoxia-inducible factor-1\u03b1 (HIF-1\u03b1), in several types of cancer . HoweverMammalian cells synthesize cholesterol through a series of 21 enzymatic steps, including the mevalonate (MVA) pathway, generating various metabolites that are required for maintenance of physiological and developmental processes . EnricheAlthough the main source of cholesterol is diet, intracellular cholesterol levels are carefully regulated and balanced by sterol regulatory element-binding protein 2 (SREBP2)-mediated transcriptional programming . When inStatins, inhibitors of HMG-CoA reductase, which is the rate-limiting enzyme in cholesterol biosynthesis, are widely used as cholesterol-lowering drugs ,27. EmerA growing body of preclinical, epidemiological, and clinical evidence has shown the various side effects of the most commonly used cholesterol-lowering nutraceuticals . HoweverSterol regulatory element-binding protein 2 (SREBP2) undergoes maturation and activation upon cholesterol depletion, subsequently triggering the enhancement of intracellular cholesterol biosynthesis, for maintaining cholesterol homeostasis . ParadoxTo investigate whether UA activates SREBP2, the expression of cholesterol biosynthesis-related genes was analyzed in the absence or presence of UA. The increased expression was observed for genes related to cholesterol biosynthesis , fatty acid synthesis (SREBP1a and SREBP1c), and cholesterol uptake (LDL-R) in UA-treated SK-HEP-1 cells, and this UA-induced gene expression was similar to that observed with simvastatin treatment A. Given Cholesterol, a component of lipid rafts, promotes cell growth signaling factors, such as PI3K/AKT and EGFR/MAPK, by mediating the trafficking of oncogenic growth factor receptors . In addiAs UA dramatically suppresses the oncogenic growth signaling, the growth-suppressive effect of UA in HCC cells was further tested. Here, this study found that UA, at a concentration of 10\u201320 \u03bcM, strongly inhibits cell viability in SK-HEP-1 A, Huh7 B, and HeThe insufficient cholesterol biosynthesis and supplementation increases the incidence of cell cycle arrest and apoptotic cell death ,61. In aTo understand whether UA suppresses HCC cell growth through a cholesterol-lowering effect, the alteration in cell growth by UA was further measured in the absence or presence of cholesterol. Initially, a strong suppression of cell viability by UA upon FBS deprivation, was observed in SK-HEP-1, Hep3B, and Huh7 cells. This indicates that FBS, containing high levels of cholesterol in the form of lipoproteins may modulate the anti-cancer effects of UA A. ImportA library of 502 natural compounds was obtained from Enzo Biochem . Ursolic acid (U6753), water-soluble cholesterol (C4951) and simvastatin (S6196) were purchased from Sigma Aldrich and Santa Cruz Biotechnology . The antibodies recognizing HMGCS1 (CST-36877), HMGCR (ab174830), FDPS (ab189874), SREBP2 (ab30682), AKT (CST-4691), phospho-AKT (CST-4060), MEK (CST-9122), phospho-MEK (CST-9154), ERK1/2 (sc-94), phospho-ERK1/2 (CST-4370), phospho-GSK3\u03b2 (CST-5558) and \u03b2-actin (sc-47778) were purchased from Cell Signaling Technology , Santa Cruz Biotechnology, and Abcam .Hepatocellular carcinoma , lung adenocarcinoma (A549 and NCI-H1666), malignant melanoma (A375 and A2058), breast cancer (MCF7 and MDA-MB-231) and cervical cancer (HeLa) cells were obtained from the Korean Cell Line Bank and American Type Culture Collection and cultured in Dulbecco\u2019s modified Eagle\u2019s medium (DMEM) and Minimum Essential Medium Eagle alpha medium (MEM-\u03b1) supplemented with 10% fetal bovine serum (FBS) and antibiotics. Lipoprotein depleted FBS (d \u2264 1.25 g/mL), which produced and qualified by ultracentrifugation and agarose gel electrophoresis, respectively, was used for cholesterol deprivation. Cell viability was measured as previously described [For western blotting, total protein samples were extracted by using a protein extraction buffer , 10 mM NaF, 0.1 mM EDTA, and a protease inhibitor cocktail) as previously described [The mRNA encoding cholesterol biosynthesis-related enzymes expression was measured by quantitative real-time polymerase chain reaction (PCR) performed as previously described . Total RThe cell cycle analysis was performed as previously described . The cul5 cells/well) in the absence or presence of UA were collected into fresh tubes, and the cells were then washed with cold PBS and centrifuged at 2000 rpm for 2 min at room temperature. The cell pellets were mixed and reacted with 100 \u03bcL of Muse\u2122 Annexin V and Dead Cell kit reagents for 20 min. The apoptotic cells population was measured by using Mini Flow Cytometry Muse\u2122 Cell Analyzer .Annexin-V staining to measure apoptotic cell death was performed as previously described . The culLuciferase assay was performed as previously described . HEK293TIntracellular total cholesterol levels were measured by using the Amplex Red cholesterol assay kit in accordance with the manufacturer\u2019s protocols as previously described . Brieflyt-test and one-way ANOVA with Tukey post hoc test. A p value of < 0.05 was considered statistically significant.The data are represented as the mean \u00b1 standard deviation (SD). All statistical analyses were performed using two-tailed Student\u2019s Despite the accumulating evidence that UA exerts an anti-cancer effect in multiple types of cancer, the precise underlying molecular mechanism is not clear. The major finding of this study is that UA exerts its anti-cancer effect through a cholesterol-lowering effect in hepatocellular carcinoma (HCC) cells. Taken together, these results indicate that UA, as a promising cholesterol-lowering drug, may be useful for the development of drugs and/or functional foods for the prevention and treatment of HCC as well as dysregulated cholesterol metabolism-related chronic diseases."} +{"text": "Diffusion Tensor Imaging (DTI) can evaluate microstructural tissue damage in the optic radiation (OR) of patients with clinically isolated syndrome (CIS), early relapsing-remitting multiple sclerosis and neuromyelitis optica spectrum disorders (NMOSD). Different post-processing techniques, e.g. tract-based spatial statistics (TBSS) and probabilistic tractography, exist to quantify this damage.To evaluate the capacity of TBSS-based atlas region-of-interest (ROI) combination with 1) posterior thalamic radiation ROIs from the Johns Hopkins University atlas (JHU-TBSS), 2) Juelich Probabilistic ROIs (JUEL-TBSS) and tractography methods using 3) ConTrack (CON-PROB) and 4) constrained spherical deconvolution tractography (CSD-PROB) to detect OR damage in patients with a) NMOSD with prior ON (NMOSD-ON), b) CIS and early RRMS patients with ON (CIS/RRMS-ON) and c) CIS and early RRMS patients without prior ON (CIS/RRMS-NON) against healthy controls (HCs).Twenty-three NMOSD-ON, 18 CIS/RRMS-ON, 21 CIS/RRMS-NON, and 26 HCs underwent 3\u202fT MRI. DTI data analysis was carried out using JUEL-TBSS, JHU-TBSS, CON-PROB and CSD-PROB. Optical coherence tomography (OCT) and visual acuity testing was performed in the majority of patients and HCs.2 for CSD-PROB and JHU-TBSS , compared to CON-PROB and JUEL-TBSS . Differences between CIS/RRMS-NON and HC were only observable in CSD-PROB . No significant differences between CIS/RRMS-ON and HC were detected by any of the methods.Absolute OR fractional anisotropy (FA) values differed between all methods but showed good correlation and agreement in Bland-Altman analysis. OR FA values between NMOSD and HC differed throughout the methodologies . ROC-analysis and effect size estimation revealed higher AUCs and RAll DTI post-processing techniques facilitated the detection of OR damage in patient groups with severe microstructural OR degradation. The comparison of distinct disease groups by use of different methods may lead to different - either false-positive or false-negative - results. Since different DTI post-processing approaches seem to provide complementary information on OR damage, application of distinct methods may depend on the relevant research question. \u2022We describe a fully-automated optic radiation probabilistic tractography approach (CSD-PROB) that is feasible in healthy controls and patients with neuroimmunological diseases.\u2022We demonstrate CSD-PROB to yield superior sensitivity and specificity in differentiating NMOSD from healthy controls compared to TBSS-based optic radiation ROI methods and ConTrack-based probabilistic tractography.\u2022CSD-PROB shows significant differences between healthy controls and patients with clinically isolated syndrome and early multiple sclerosis without prior optic neuritis.\u2022The comparison of different subject groups by use of different DWI post-processing methods may lead to either false-positive or false-negative results, which may be of particular significance for future optic radiation DTI comparison in studies and meta-analyses. DiffusiA multitude of DW-MRI post-processing techniques have been used in recent studies to investigate OR damage in neuroinflammatory disorders . TBSS isPrevious investigations using CON-PROB found OR DTI metrics to be altered in long-standing MS patients compared to healthy controls with correlations between OR FA and OR T2 lesion volume . A studyValidation studies of sensitivity, specificity and technical advantages and disadvantages of different DTI post-processing methods are thus highly required. Unfortunately, there is no \u201cgold-standard\u201d for non-invasive DTI-based OR tract-probing , making The purpose of our study was to compare distinct TBSS-based and probabilistic tractography-based approaches in the delineation of OR and the detection of OR damage. We therefore investigated OR damage with different severity levels and compared a) NMOSD patients with prior ON with suspected severe OR damage, b) clinically isolated syndrome (CIS) and early relapsing-remitting multiple sclerosis (RRMS) patients with ON and suspected moderate OR damage and c) CIS and early RRMS patients without prior ON and potential OR damage against healthy controls (HCs). We evaluated inter-method agreement of FA values and compared the capacity of all methods to detect OR FA differences in all patient cohorts compared to HCs.22.1Sixty-two patients were retrospectively analyzed from our research database. This included CIS and early RRMS with ON (CIS/RRMS-ON), CIS and early RRMS without ON (CIS/RRMS-NON), NMOSD with ON (NMOSD-ON) as well as 26 HCs see . All patClinicalTrials.gov Identifier: NCT01371071; EA1/182/10). CIS/RRMS-ON patients were investigated following a first-time ON attack after 4.61\u202f\u00b1\u202f5.51\u202fmonths on average (range: 1\u201324\u202fmonths) and showed no other neurological symptoms than ON-related visual dysfunction. All CIS/RRMS-ON patients presented with unilateral optic neuritis as their first clinical symptom. At the time of MRI examination, 3 of these patients fulfilled the 2010 revised McDonald criteria for MS , a volumetric high-resolution T1 weighted magnetization prepared rapid acquisition gradient echo (MPRAGE) sequence as well as a volumetric high-resolution fluid-attenuated inversion recovery sequence (3D FLAIR) . 3D FLAIR images of all patients were checked and verified for total lesion volume and OR-specific lesion volume by three expert raters under the supervision of a board-certified radiologist. Whole-brain segmentation and quantification of lesions of FLAIR images were performed using ITK-SNAP (www.itksnap.org) using a single-shot echo planar imaging DTI sequence .2.32.3.1DTI data analysis was carried out using TBSS with tooFirst, eddy-current and motion correction were run in FSL, then FA images were created by fitting a tensor model to the raw diffusion data using a least-squares algorithm in FDT, and then brain-extracted using BET . FA dataTBSS skeleton masks were overlaid with two different atlas masks: (A) OR ROIs derived from the Juelich 1\u202fmm probabilistic atlas optic radiation ROI thresholded to exclude the lowest 10% (JUEL-TBSS) and (B) Johns Hopkins University 1\u202fmm white matter tractography probabilistic atlas' posterior thalamic radiation ROI (JHU-TBSS) .2.3.2http://vistalab.stanford.edu/software). Probabilistic fiber tracking was performed using the Contrack algorithm (CON-PROB) , designeON-PROB) . MeasureON-PROB) to combi2.3.3http://www.mrtrix.org/) (We applied a combination of previously published OR tractography based on high order fiber orientation distributions estimated with CSD (CSD-PROB) and weigix.org/) . First, ix.org/) .Maps of the fiber orientation distributions (FODs) were calculated using CSD with a maximum harmonic order of 6 . OR reconstruction pipeline was modified after Mart\u00ednez-Heras et al. and Lim et al. with non2.4Optical coherence tomography (OCT) investigations were performed in all CIS/RRMS-NON patients, in 17 out of 18 CIS/RRMS-ON patients, in 22 out of 23 NMOSD-ON patients and in 21 out of 26 HC using a Heidelberg Engineering Spectralis spectral domain OCT with automatic real-time (ART) function for image averaging. The peripapillary retinal nerve fiber layer (pRNFL) was measured with activated eye tracker using 3.4-mm ring scans around the optic nerve head . Segmentation of global RNFL was performed semiautomatically using software provided by the OCT manufacturer . Visual acuity tests were performed by either using ETDRS charts or the Traditional Snellen Eye Chart in all CIS/RRMS-NON, in 17 out of 18 CIS/RRMS-ON patients, in 21 out of 23 NMOSD-ON patients and in 21 out of 26 HC. Visual testing outcomes were converted in decimals.2.5ROCt and ggplot. For comparison and correlation of absolute FA values between methods we used separate FA values of left and right OR and conducted a two-way repeated measures ANOVA to account for the effect of 1) method choice and 2) OR side on FA values within each patient group and an intraclass correlation coefficient (ICC) analysis. Agreement of FA values between methods was evaluated by Pearson's correlation coefficient analysis and Bland-Altman plots .For statistical analysis we used Graphpad Prism 6.0 software and R version 3.1.2 with packages psych, geepack, irr, ICC, lme4, 2 effect size measures estimation. A receiver operating characteristic (ROC) analysis was used to assess sensitivity and specificity of methods to discriminate each patient group from healthy controls corrected for age. Comparison of tract profiles was conduected using two-way ANOVA comparing FA values of patient groups in every node against HC group. Correction for multiple comparison was performed using Bonferroni correction. Correlations between OR FA values and OR T2 lesion volume, RNFL and visual acuity were performed using linear model analysis. For all statistical analyses, a p-value of <0.05 was regarded as significant. Data are presented as mean\u202f\u00b1\u202fSD, except for tract profling results that are displayed in mean\u202f\u00b1\u202fstandard error of the mean (SEM).Exploratory comparisons of patient groups regarding T2 lesion volume, RNFL and visual acuity of worse eye were conducted using one-way ANOVA. For group comparisons and correlation analyses with clinical data, we combined FA measures of left and right optic radiation and calculated the simple mean of both values in JHU-TBSS, JUEL-TBSS and CON-PROB. Since OR volumes differed between right and left side in CSD-PROB, we used weighted mean of both values for CSD-PROB based group comparison and correlation analyses. Comparisons of patient groups regarding FA values were assessed using linear model analyses to account for FA values with subsequent R33.13.1.1All four methods successfully generated visually appropriate OR tracts, with the exception of one subject in the CIS/RRMS-ON group using the CSD-PROB method.3.1.2Coefficient of variation in HC group was lowest in JUEL-TBSS (3.99%) and highest in CON-PROB (13.54%) with comparable coefficients of variation in JHU-TBSS (5.88%) and CSD-PROB (7.21%).3.1.3Absolute FA value distribution of the different methods for ORs of both sides within each subject group are shown in 3.1.4r ranging from 0.2730 (JUEL-TBSS vs. CON-PROB) to 0.8714 were observed between values of both TBSS-based approaches . CSD-PROB revealed significant FA differences between CIS/RRMS-ON patients and HCs, that were not observable when other methods were applied.Comparison of patient groups against healthy controls regarding T2 lesion volume, OCT RNFL, visual acuity and optic radiation FA values are shown in 3.2.2AUC values to discriminate HCs from NMOSD-ON were highest in CSD-PROB (AUC\u202f=\u202f0.812), while slightly lower in CON-PROB (AUC\u202f=\u202f0.742), JHU-TBSS (AUC\u202f=\u202f0.756) and JUEL-TBSS . Both, TBSS and probabilistic tractography methods detected microstructural damage in NMOSD-ON patients compared to HCs.4.1CSD-PROB failed to generate OR tracts in one CIS/RRMS-ON patient, while successfully generating tracts in all other subjects. All other methods successfully identified the ORs in all subjects. It has been reported, that extensive white matter lesions in neurological disorders, such as stroke or multiple sclerosis, may lead to erroneous termination of the tracking algorithm or may cause a deviation of the bundles at the level of the lesions . A previ4.2In our study, CON-PROB showed highest coefficient of variation of FA in HC, while JUEL-TBSS showed lowest coefficient of variation. Supposing that a homogeneous and normally distributed cohort was investigated, low coefficients of variation may suggest a correlate of good method quality. High coefficients of variation in HCs in CON-PROB, possibly caused by the mainly manual approach, might impair the validity of the method. However, high coefficients may on the other hand indicate higher method sensitivity. A recently published study compared a) individual CON-PROB with b) healthy control-based CON-PROB template OR reconstructions and c) Juelich histological atlas-based OR ROI approach in 35 healthy controls and 70 MS patients . DespiteBy contrast, the distribution of absolute OR FA values significantly differed in our study between nearly all methods in all patient groups and showed poor absolute agreement in the ICC analysis, except for JHU-TBSS and CSD-PROB. We conclude that differences between absolute OR FA values may impede comparisons of previous and future DTI study results investigating microstructural OR damage. The application of the exact same method is therefore necessary to allow for any statements on possible differences between OR FA values within a specific cohort of patients. These findings may be of particular significance in any case of OR DTI comparison, regardless of within-study analyses or comparisons of OR DTI results between studies, for example in meta-analyses. Comparisons of absolute OR DTI values that did not use the same post-processing approach are not valid and must therefore be avoided.4.3OR FA values of all methods showed significant correlations suggesting underlying associations of FA values and actual OR specific microstructural damage regardless of method choice. Subgroup analyses of Pearson correlation coefficient analyses revealed best correlations of OR FA values in CIS/RRMS-ON and CIS/RRMS-NON. These findings are in line with a recent study reporting on good agreement between CON-PROB, template-based OR reconstruction and a Juelich OR ROI-based approach in HC and MS measured by Pearson correlation coefficents and Bland-Altman analysis .By contrast, only limited correlations of OR FA values were seen in our study in the non-damage group (HC) and patients with suspected extensive OR damage (NMOSD-ON).In a recent study, CSD-PROB was investigated in ten HCs and five MS patients to compare tractography results with histological masks. It showed a good sensitivity ranging from 65% to 81% and a specificity up to 100% . AnotherThe presence of systematic bias and proportional errors in the comparison of DTI TBSS-based and tractography based methods may lead to false positive or false negative results when different patient groups are compared by different methods. While one method might produce significant differences in group comparison due to underestimation of low FA values, another method may yield non-significant results due to relative overestimation of low FA values. These findings might be a causative factor of today's equivocal findings of previ4.4Group comparison showed FA differences between NMOSD-ON and HCs throughout all TBSS and probabilistic tractography based methods. Best effect size and AUC values to distinguish both groups were observed for CSD-PROB. JHU-TBSS, JUEL-TBSS and CON-PROB showed slightly lower AUC values and effect size. Our study results are in line with previous investigations using DTI reporting on microstructural degradation with significant FA reduction within the OR . A previAlthough clinical history of our NMOSD patients with prior unilateral or bilateral ON, long disease duration and pronounced visual impairment and OCT RNFL thinning suggests the presence of attack-related optic radiation FA decrease in our NMOSD cohort, we did not find any direct associations between OCT RNFL or visual acuity and optic radation FA, irrespective of the method. However, our data mirror the clinical experience as well as findings from conventional imaging studies showing that neurological disability and tissue damage in the visual pathway are on average more pronounced in NMOSD as compared to MS/CIS, as it can be seen in the relatively frequent bilateral manifestation of optic neuritis in our NMOSD-cohort compared to CIS/RRMS-ON .4.5No difference of OR FA between CIS/RRMS-ON and HC was seen in any of the methods used. CSD-PROB showed differences between HC and CIS/RRMS-NON. In CIS and early stages of MS, OR microstructural damage is most likely caused by 1) trans-synaptic neurodegeneration after ON and 2) iDamage in the OR of ON patients due to inflammatory T2 lesions has been investigated previously. Raz et al. reported on reduced OR FA by making use of CON-PROB in patients with clinically isolated ON compared to healthy controls. In this study, reduced OR FA was associated with OR specific T2 lesion volume suggesting FA differences to be explained by intrabundle lesions . In our 4.6Tract-profiling group differences between HC and NMOSD were seen in higher proportion of nodes in CON-PROB compared to CSD-PROB, indicating CON-PROB tract-profiling to yield higher sensitivity for the detection of microstructural OR damage in NMOSD compared to CSD-PROB tract profiling. Using CON-PROB, tract-profiling enabled the distinction between OR fibers affected by T2 lesions and non-lesional OR fibers. Radial diffusivity, mean diffusivity and FA changes were detected along the entire OR, while axial diffusivity changes were confined to the posterior half of the OR. This discrepancy implied distinct pathophysiologic processes to be detectable by DTI tract profiling .In our study, tract profiling showed middle and posterior parts of the OR to be more affected than anterior OR sections in NMOSD compared to HC. These findings may suggest distinct regions of the OR to exhibit more pronounced damage by trans-synaptic neurodegeneration or distinct OR T2 lesional damage affecting only specific regions of the OR due to Wallerian degeneration . However4.7TBSS can be implemented fully-automated requiring no manual intervention. In TBSS, the average FA may be affected by surrounding structures due to partial voluming . Probabi4.8Given the multitude of methods that exist for tractography and the comparison of DWI measures, our study naturally fails to comprehensively include all alternative methods for comparison. Our study is limited by the small sample size of the respective subpopulations mitigating validity of our cross-method comparison.5To our knowledge, this is the first study to compare multiple probabilistic tractography and TBSS-based approaches to quantify microstructural OR damage in patients with neuroinflammatory visual pathway damage. We proved TBSS-based and probabilistic tractography based DWI processing techniques to be feasible in detecting microstructural damage within the OR. Absolute FA values differed between the methods, preventing comparisons of OR FA analyses of previous and future studies with different post-processing approaches. Correlation and agreement of all methods' FA values were best in patients with suggested mild to moderate OR FA damage (CIS/RRMS patients), indicating methods to be exchangeable \u2013 at least to a certain extent \u2013 in the analysis of CIS/RRMS patients but not if healthy controls or patients with suspected severe damage (NMOSD-ON) are investigated. Due to systematic bias and proportional errors of FA between methods, the comparison of subject groups by use of different methods leads to different results. Although the pattern of differences between the patient cohorts was similar in our study, CSD-PROB showed significant FA differences between HC and CIS/RRMS-NON patients. Although these CSD-PROB derived differences between the groups could result from the above-mentioned systematic bias, we suggest CSD-PROB to be more sensitive to early neuroinflammatory damage, partially associated with lesions. All methods were successful in differentiating NMOSD-ON patients from HCs. Given that CSD-PROB showed highest AUC and effect size followed by JHU-TBSS, JUEL-TBSS and CON-PROB, CSD-PROB approach might be the method of choice to further investigate differential diagnostic aspects between HC and NMOSD. Tract-profiling differences between HC and NMOSD were more pronounced in CON-PROB, which might be the method of choice for tract profiling assessments. In our study, TBSS-based approaches showed better correlations with OR specific lesions, which could favor them as the method of choice for future studies to investigate the relationship between T2 lesions and DTI. Given the lack of a \u201cgold-standard\u201d for non-invasive DW-MRI OR delineation , future Supplementary materialImage 1The following are the supplementary data related to this article."} +{"text": "Atg) genes are positively regulated by the homeodomain transcription factor Cut, and that basal autophagy functions as a downstream effector pathway for Cut-mediated dendritic terminal branching in Drosophila multidendritic (md) sensory neurons. Further, loss of function analyses implicate Atg genes in promoting cell type-specific dendritic arborization and terminal branching, while gain of function studies suggest that excessive autophagy leads to dramatic reductions in dendritic complexity. We demonstrate that the Atg1 initiator kinase interacts with the dual leucine zipper kinase (DLK) pathway by negatively regulating the E3 ubiquitin ligase Highwire and positively regulating the MAPKKK Wallenda. Finally, autophagic induction partially rescues dendritic atrophy defects observed in a model of polyglutamine toxicity. Collectively, these studies implicate transcriptional control of basal autophagy in directing dendritic terminal branching and demonstrate the importance of homeostatic control of autophagic levels for dendritic arbor complexity under native or cellular stress conditions.Dendrites function as the primary sites for synaptic input and integration with impairments in dendritic arborization being associated with dysfunctional neuronal circuitry. Post-mitotic neurons require high levels of basal autophagy to clear cytotoxic materials and autophagic dysfunction under native or cellular stress conditions has been linked to neuronal cell death as well as axo-dendritic degeneration. However, relatively little is known regarding the developmental role of basal autophagy in directing aspects of dendritic arborization or the mechanisms by which the autophagic machinery may be transcriptionally regulated to promote dendritic diversification. We demonstrate that autophagy-related ( Neurons exhibit a vast array of morphological architectures due in part to their distinct and elaborate patterns of dendritic arborization. Dendritic arbor diversity across neuronal subtypes plays a pivotal role in regulating synaptic and sensory integration, functional connectivity, electrotonic properties and neuronal computation . TherefoC. elegans, D. melanogaster and mammals [Atg genes [In addition to these cellular processes, recent studies demonstrate that the autophagy pathway is one mechanism involved in maintaining neuronal morphology that is evolutionarily conserved across species including mammals . Macroau mammals ,5. Autop mammals \u20138. Durin mammals ,10. The tg genes ,11. Thestg genes .Atg genes and autophagic function has been shown to lead to the accumulation of ubiquitin-positive and other abnormal protein aggregates known to contribute to a variety of neurodegenerative disease states including Parkinson\u2019s and Huntington\u2019s [Post-mitotic neurons are known to require high levels of basal autophagy for cellular homeostasis in terms of clearing misfolded proteins and damaged organelles . Autophaington\u2019s ,17\u201319.Atg genes and the developmental role of basal autophagy in promoting dendritic arbor diversity both remain largely unknown. Furthermore, while significant evidence has emerged that complex transcriptional regulatory programs function to generate the array of neuronal dendritic architectures, much remains to be discovered regarding the downstream cellular and molecular mechanisms through which these transcriptional codes are implemented to drive dendritic diversification [Drosophila has proven a powerful model for investigating autophagy due to the evolutionary conservation of the core machinery involved in the autophagic process [Drosophila multidendritic (md) sensory neurons have served as a robust system for characterizing dendrite morphogenesis [Despite the importance of the autophagy pathway in neuronal function, the transcriptional mechanisms controlling cell type-specific expression of fication ,20. Dros process ,11. Moreogenesis . These sogenesis ,22.Drosophila md sensory neuron subtypes. We demonstrate that the homeodomain transcription factor Cut positively regulates the expression of Atg genes linked to autophagic induction, Atg protein cycling, and vesicle completion and that basal autophagy functions as a downstream effector of Cut-mediated dendritic terminal branching. Genetic analyses reveal that insufficient or excessive autophagic activity leads to defects in dendritic arborization and higher order complexity indicative of a homeostatic role for autophagy in directing cell type-specific features contributing to dendritic diversification. Genetic interaction studies identify a regulatory relationship between autophagy and the DLK pathway. Finally, we demonstrate that under conditions of cellular stress, autophagic induction can partially rescue dendritic atrophy phenotypes exhibited in a model of polyglutamine toxicity, providing a link between upregulation of autophagy and mitigation of dendritic neurodegenerative-like defects.Here we functionally connect transcriptional regulation to autophagy in directing cell type-specific dendritic arborization in Drosophila stocks were maintained at 25\u00b0C on standard molasses-cornmeal agar. The following strains were obtained from Bloomington Drosophila Stock Center: UAS-RNAi lines ; GAL4477,UAS-mCD8::GFP; UAS-Atg16A; UAS-Atg16B; UAS-GFP-hiwA; UAS-MJD-78Q; UAS-eGFP-Atg5; UAS-Atg8a.GFP; UAS-wndK188A. Additional strains from other sources included the pan-md reporter strain GAL421-7,UAS-mCD8::GFP [GAL4221,UAS-mCD8::GFP [GAL419-12,UAS-hCD4::tdGFP [; nompC-GAL4,UAS-mCD8::GFP [ppk-GAL4,UAS-mCD8::GFP, ppk-GAL80 [GAL4477,UAS-mCD8::GFP; ppk1.9-GAL4,UAS-mCD8::GFP [ppk::hCD4::tdTomato (donated by Dr. Yuh-Nung Jan) [; UAS-cut; UAS-Atg1K38Q [UAS-Hiw\u0394RING and UAS-wnd.C [UAS-RNAi (IR) lines were used for each Atg gene to control for any potential off-target effects and a representative IR line for each Atg gene is depicted in the figures. Detailed genotypes for each figure are reported in HMS00924 ; Atg1JF04::tdGFP ; nompC-Gpk-GAL80 ; class ICD8::GFP ; and ppkung Jan) ; UAS-cutichigan) ,34. A miGAL42-21,UAS-mCD8::GFP (C-I) [ppk-GAL4,UAS-mCD8::GFP, ppk-GAL80 (C-III) [ppk1.9-GAL4,UAS-mCD8::GFP (C-IV) [UAS-mCD8::GFP under the control of GAL42-21 in the presence or absence of UAS-cut [D. melanogaster genome oligo microarrays (4x44K) and statistical analyses of microarray data were performed as previously described [Class-specific isolation and purification of md neurons was performed as previously described ,35,36. F (C-III) , and ppkP (C-IV) age-matc UAS-cut ,35\u201337. M UAS-cut ; GSE6935escribed . Raw micDissection and immunofluorescent labeling of third instar larval filets was performed as previously described . PrimaryFlyboys (freely available upon request). For total dendritic length measurements, images were processed and skeletonized in ImageJ [Live neuronal imaging was performed as previously described ,30. We fn ImageJ . Quantitn ImageJ . Branch cut-IR expressing neurons was done in quadruplicates as previously described [UAS-mCD8::GFP expressing C-IV md neurons were isolated using superparamagnetic beads that were coupled to biotinylated anti-CD8a antibody (eBioscience). RNA was then isolated from these cells using the miRCURY RNA Isolation Kit (Exiqon) and qRT-PCR was performed using the following pre-validated QuantiTect Primer Assays: Atg1 (QT00963536), Atg2 (QT00956963), Atg5 (QT00499723), Atg8a (QT00919695) and Atg18 (QT00960337). Expression data was normalized to RpL32 (QT00980007).qRT-PCR analysis of WT and escribed . BrieflyStatistical analyses of neuromorphometric data and data plotting were performed using GraphPad Prism 7. Error bars reported in the study represent SEM. Statistical analyses were performed using either two-tailed unpaired t-test with Welch\u2019s correction or one-way ANOVA using Dunnett\u2019s multiple comparisons test when data sets were normally distributed as determined by the Shapiro-Wilk normality test. When data was not normally distributed, appropriate non-parametric tests were used (see figure legends for specific tests used in each case). Significance scores indicated on graphs are . Detailed information on statistical analyses for each figure is reported in The homeodomain transcription factor Cut has been shown to promote dendritic diversification in morphologically complex C-III and C-IV md neurons via regulation of a variety of cellular processes including the secretory pathway and the cytoskeleton ,23,36,43Atg genes. Relative to wild-type (WT) control C-I md neurons, Cut misexpressing C-I neurons exhibited significantly increased expression of the following Atg genes -associated 13 domain containing protein involved in Atg protein cycling between peripheral sites and the phagophore assembly site (PAS); Atg5, encoding an E3-like ubiquitin ligase component involved in autophagosome vesicle completion via lipidation of Atg8; Atg8a, encoding a ubiquitin-like protein involved in autophagosome vesicle completion; and Atg18, encoding a WD40 repeat domain phosphoinositide-interacting protein involved in protein cycling and autophagosome formation [Bioinformatic analyses of Cut-mediated differential gene expression identified a variety of intriguing cellular pathways including coordinated upregulation of numerous tg genes : Atg1, eogenesis ; Atg2, eormation .To validate the putative regulatory relationship between Cut and Atg proteins independent of the neurogenomic analyses, we performed immunofluorescence analyses to determine whether Cut can directly increase Atg protein expression. For these analyses we examined the expression of Atg8a based upon characterized and available antibodies. Relative to WT control C-I md neurons, we observed a significant increase in Atg8a expression levels in C-I neurons ectopically expressing Cut (p<0.0001) .Atg gene expression in C-III and C-IV md neurons displayed any relationship to Cut protein expression levels in these neurons. Based upon previously published WT C-I, C-III, and C-IV md neuron transcriptome expression profiling data [Atg genes identified above were largely correlated with differential Cut protein expression levels. With the exception of Atg5, we observed the highest levels of Atg gene expression in C-III neurons followed by C-IV neurons, which corresponds with previously reported Cut differential expression levels .cut (UAS-cut-IR) using a pan-md GAL4 driver and labeled third instar larval filets with anti-Atg8a. We focused our analyses on C-I, C-III, and C-IV md neurons based upon differential Cut protein expression levels and our neurogenomic analyses. In WT control neurons, Atg8a protein expression appeared in punctate cytosolic vesicular structures representing presumptive autophagosomes throughout the md neuron cell bodies (cut knockdown in the C-III (p<0.0001) and C-IV (p = 0.0093) md neurons compared to WT controls, supporting a positive regulatory relationship between Cut and Atg8a (cut knockdown in the C-I md neurons (p = 0.1203), which was predicted based upon the lack of detectable Cut protein expression in C-I neurons [To complement the mRNA expression analyses, we performed RNAi-mediated knockdown of l bodies . Quantitnd Atg8a . In cont neurons causes a dramatic increase in dendritic branching complexity characterized by extensive de novo formation of dendritic terminal filopodia-like branches emanating from the primary arbors displayed suppression of Cut-mediated de novo dendritic arborization, which was particularly notable with respect to Cut-induced short terminal dendritic filopodial branching , Parkinson\u2019s (PD) and Huntington\u2019s (HD) diseases . These findings suggest that homeostatic regulation of autophagy is important for maintaining neuritic architecture and neuronal survival. To investigate how excessive autophagic induction may impact md neuron dendritic development, we conducted gain-of-function phenotypic analyses of the Atg1 initiator kinase in C-III and C-IV neurons. Relative to controls, neurons resultin neurons , total d neurons , and num neurons in both neurons . This ob neurons , total dc length , and numc length that werAtg genes appears to largely suppress the formation of Cut-induced dendritic growth and terminal filopodial branching in which the Hiw E3 ubiquitin ligase RING domain is mutated generating a loss-of-function effect revealed defects in dendritic arbor growth and branching decrease in the levels of GFP-tagged Hiw in neurons co-expressing UAS-Atg1 relative to controls, thereby suggesting that Atg1 overexpression negatively regulates Hiw expression in C-IV neurons increase in levels of GFP-tagged Hiw pathway. Previous studies demonstrated that the autophagy pathway regulates synaptic and axonal growth via degradation of the E3 ubiquitin ligase Highwire (Hiw) in CNS neurons , and tharanching reminiscransgene . In contransgene in a manransgene . We therpression . Consistpression , howeverpression , suggestransgene in C-IV neurons . To furtgged Hiw . Based ugged Hiw . Quantitgged Hiw . These rUAS-MJD-78Q) that retains a long polyQ repeat leads to severe defects in dendritic growth and higher order terminal branching presumptively linked to polyQ toxicity ectopic expression of Cut in C-I md neurons reveals upregulated mRNA expression of Atg genes involved in autophagic induction, Atg protein cycling, and autophagosome vesicle completion; (2) Cut ectopic expression upregulates Atg8a protein expression, whereas Cut-specific knockdown in C-III and C-IV md neurons, which normally express Cut, leads to downregulation of Atg8a protein expression as well as downregulation of Atg1, Atg2, Atg5, Atg8a, and Atg18 mRNA expression levels; (3) genetic suppressor analyses involving Atg gene knockdown in C-I md neurons ectopically expressing Cut support a role for basal autophagy as a downstream effector of Cut-mediated dendritic growth and terminal branching; and (4) overexpression of Atg genes in a cut loss-of-function background can partially rescue defects in C-III md neuron dendritic growth and terminal branching. While these analyses are indicative of a positive regulatory relationship between Cut and components of the autophagic machinery, whether Cut directly or indirectly regulates these Atg genes remains to be determined. Our work demonstrates, however, that Cut is not absolutely required for Atg protein expression or autophagosome formation as Atg8a labeling of autophagosomes is observed in C-I md neurons that do not normally express detectable levels of Cut, suggesting that other transcriptional regulators likely also play important roles in directing Atg gene expression. The observations that disruption of individual Atg genes only partially suppresses Cut overexpression effects on C-I md neuron dendritic growth and filopodial-like terminal branching and that overexpression of Atg1 alone is insufficient to fully rescue cut loss-of-function dendritic defects in C-III md neurons can be explained in light of recent studies demonstrating that Cut functions via a variety of cellular processes in regulating dendritic development [Our results suggest that homeostatic regulation of the basal autophagy pathway is important in mediating aspects of cell type-specific dendritic arborization including terminal branching in ntegrity . Our wor levels; genetic elopment and funcelopment ,43,57,58elopment ,59, cellelopment , and ribelopment as downsAtg gene function leads to reductions in growth and terminal dendritic branching. In the case of C-III md neurons, this branching defect appears to manifest as a reduction in terminal branches, whereas in C-IV md neurons, we observed variable defects upon Atg gene knockdown leading to reductions in dendritic growth and terminal branching. Interestingly, C-IV expression of the kinase dead Atg1K38Q transgene led to the most dramatic phenotypic effect with notable reductions in terminal arbor branching. Thus, insufficient autophagy appears to have major effects on cell type-specific dendritic terminal branching in C-III and C-IV neurons, whereas lower order dendritic branches do not appear to be affected. In contrast to lower order branches, studies have demonstrated that higher order terminal branches of md neurons are highly dynamic with respect to extension and retraction [Insufficient or excessive levels of autophagic activity can lead to neuritic degeneration, highlighting the importance of homeostatic regulation of autophagy . With retraction \u201362. WhilA growing body of evidence indicates that excessive autophagy can also contribute to neurodegeneration and dystrophy of axo-dendritic processes, though the mechanism of action remains incompletely understood. In the case of neurodegenerative diseases such as AD, PD, and HD, degenerating neurites observed in brain tissue samples display increased levels of autophagy-related vesicular compartments and in LDrosophila CNS, autophagy has been demonstrated to positively regulate the development of the larval neuromuscular junction (NMJ) [hiw E3 ubiquitin ligase mutants and this overgrowth phenotype can be suppressed by the MAPKKK protein Wnd. Autophagy interacts with the DLK pathway by negatively regulating Hiw expression in order to promote NMJ growth [How might the autophagy pathway mechanistically interact with other cell signaling pathways to regulate cell type-specific dendritic morphogenesis? A pair of intriguing studies have suggested that interaction with the DLK pathway may be one mechanism by which autophagy can exert control over this process ,45. In ton (NMJ) . IncreasJ growth . InteresAtg5 [Atg12 and p62 [Drosophila eye degeneration models. This suggests that autophagy may play a neuroprotective role by clearing pathogenic aggregates. Using a Drosophila model of SCA3 polyQ toxicity, we found that autophagic induction via Atg1 overexpression resulted in a partial rescue of neurodegenerative-like dendritic arbor atrophy defects. These findings are consistent with recent evidence demonstrating that upregulation of autophagy via overexpression of Beclin-1 (Atg6) enhances SCA3 aggregate clearance [Conditions of cellular stress can trigger an acute upregulation of basal autophagy as a mechanism to clear toxic protein aggregates or damaged organelles that may otherwise contribute to neurodegenerative states. Recent studies demonstrate that knockdown of autophagy via Atg5 as well and p62 led to alearance , and sugIn summary, these studies provide mechanistic insights into transcriptional regulation of basal autophagy and highlight the importance of homeostatic control of basal autophagy function in promoting cell type-specific dendritic arborization during normal development and under conditions of cellular stress.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file."} +{"text": "Furthermore, clinical outcomes correlated with DC migration to vaccine-site draining lymph nodes measured by Indium-111 labeling of RNA-loaded DCs and SPECT/CT imaging. Although these studies demonstrated that tracking DC migration may be an important clinical biomarker for response to DC vaccination, the complexity and regulatory requirements associated with nuclear labelling to track DC migration limits widespread application of this technique. We have therefore developed RNA-loaded magnetic nanoparticles (RNA-NPs) to enhance DC migration to LNs and track that migration with a widely available imaging modality . METHODS/STUDY POPULATION: Cationic liposomes were loaded with iron oxide nanoparticles with or without cholesterol. The resulting nanoparticles were complexed with RNA and used to transfect DCs ex vivo. RNA-NP-loaded DsRed+ DCs were then injected intradermally into mice and tracked noninvasively with T2-weighted 11T MRI before excision and quantification with flow cytometry. RESULTS/ANTICIPATED RESULTS: In vitro experiments demonstrate that iron oxide loading does not reduce RNA-NP-mediated transfection of DCs. Additionally, replacement of cationic lipids with cholesterol increased RNA-NP transfection of the DC2.4 cell line and enhanced the T cell stimulatory capacity of treated bone marrow-derived dendritic cells (BMDCs). Compared to electroporation, RNA-NPs enhanced DC migration to lymph nodes and reduced T2 MRI intensity in DC-bearing lymph nodes. DISCUSSION/SIGNIFICANCE OF IMPACT: This data suggests that iron oxide-loaded RNA-NPs enable noninvasive cell tracking with MRI and enhance DC migration to lymph nodes. We have further shown that inclusion of cholesterol in RNA-NPs augments the stimulatory capacity of transfected DCs. Future work will consider effects of RNA-NPs on antitumor immune responses and the utility of MRI-detected DC migration as a biomarker of vaccine efficacy.OBJECTIVES/SPECIFIC AIMS: Despite aggressive chemotherapy, surgical resection, and radiation therapy, glioblastoma remains almost universally fatal. In a pilot, randomized, and blinded clinical trial, we recently demonstrated that administration of RNA-loaded DC vaccines was associated with significantly improved progression-free and overall survival in patients with glioblastoma (Mitchell"} diff --git a/PMC_clustering_475.jsonl b/PMC_clustering_475.jsonl new file mode 100644 index 0000000000000000000000000000000000000000..39021577ecd7d9eb3d5bc59b039dcb0e1bd4d276 --- /dev/null +++ b/PMC_clustering_475.jsonl @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:7dfbf6bfe068afb3cba70a4d0f186a898b36b2a7aa24949b0457a1352cf2735a +size 27527034 diff --git a/PMC_clustering_476.jsonl b/PMC_clustering_476.jsonl new file mode 100644 index 0000000000000000000000000000000000000000..731c211881ca4235082cc9843671d435db93ddee --- /dev/null +++ b/PMC_clustering_476.jsonl @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:0e0a8f6ed2e1ca271e4862d8698abf3bd9d98d1d5992aa911c63f4c6c1a6505b +size 36844358 diff --git a/PMC_clustering_477.jsonl b/PMC_clustering_477.jsonl new file mode 100644 index 0000000000000000000000000000000000000000..6bf09c0ccfaaae55719b4152a52d9b7d0318ce9a --- /dev/null +++ b/PMC_clustering_477.jsonl @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:92c5f0c294a14fa9966164d7a0d8320b6e5d88c222b9253b8c830cd5032461f6 +size 22630362 diff --git a/PMC_clustering_478.jsonl b/PMC_clustering_478.jsonl new file mode 100644 index 0000000000000000000000000000000000000000..b83017608248bf5fed31950eb59af136dc37af3e --- /dev/null +++ b/PMC_clustering_478.jsonl @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:42effe33d2d371fa34e10eb3058ce27001d7b97ca4fa70f62f9fe343e03ab351 +size 64483141 diff --git a/PMC_clustering_479.jsonl b/PMC_clustering_479.jsonl new file mode 100644 index 0000000000000000000000000000000000000000..4fa29c2bcd9d2306b0731710a5995b834f2c8985 --- /dev/null +++ b/PMC_clustering_479.jsonl @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:578ca0d4030338d4442b63e9719be62412e00ddaa449251d5bf5d4400d20a806 +size 59271816 diff --git a/PMC_clustering_480.jsonl b/PMC_clustering_480.jsonl new file mode 100644 index 0000000000000000000000000000000000000000..e502df958cb0dfd6d332988e8cc49f0330c9f6e7 --- /dev/null +++ b/PMC_clustering_480.jsonl @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:27a231458f617e4c858a615c9210c6c1eee857cb29044c0c30af696a15c72491 +size 45200276 diff --git a/PMC_clustering_481.jsonl b/PMC_clustering_481.jsonl new file mode 100644 index 0000000000000000000000000000000000000000..ceab2d0d54c357fcc89acad419d01dac5c0351c9 --- /dev/null +++ b/PMC_clustering_481.jsonl @@ -0,0 +1,878 @@ +{"text": "Nature Communications 10.1038/s41467-020-16633-7, published online 5 June 2020.Correction to: In the original version of this Article, Author Kylie M. Quinn, was incorrectly assigned a present address.This error has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Correction to: BMC Psycholhttps://doi.org/10.1186/s40359-019-0369-xFollowing publication of the original article , the autPlease find the corrected name in the author list of this article.The error has now been corrected in the original article.The authors apologize for any inconvenience caused."} +{"text": "Scientific Reports 10.1038/s41598-019-49975-4, published online 25 September 2019Correction to: This Article contains errors in Table 4. In the HTML and PDF versions of this Article, the positions of the mutation in the gene are inaccurate. The correct Table"} +{"text": "Scientific Reportshttps://doi.org/10.1038/s41598-020-73191-0, published online 30 September 2020Correction to: The original version of this Article contained a typographical error in the spelling of the author Matthias E. Meunier, which was incorrectly given as Matthias Meunier. This has now been corrected in the PDF and HTML versions of the Article, and in the accompanying Supplementary Information file."} +{"text": "Nature Communications 10.1038/s41467-020-18369-w, published online 2 October 2020.Correction to: The original version of this Article omitted the following from the Acknowledgements:Research of T.H. and P.G. in the Baumeister lab was supported by the Deutsche Forschungsgemeinschaft DFG .This has now been corrected both the PDF and HTML versions of the Article."} +{"text": "The mcr-1 gene was detected in one colistin-resistant E. coli isolated from a diarrheic dog. The isolate exhibited additional resistance to multiple antimicrobials, including fluoroquinolones and third-generation cephalosporins. The mcr-1 carrying isolate belonged to ST160. The pulsed-field gel electrophoresis pattern of our strain differed from those ST160 E. coli strains previously identified from chickens in Korea. The mcr-1 gene was identified in the IncI2 plasmid. It was also transferred to E. coli J53 recipient strain, with a conjugation efficiency of 2.8 \u00d7 10\u22124. Average nucleotide identity analysis demonstrated that the mcr-1-carrying plasmid in this study was closely related to those from patients in Korea. To the best of our knowledge, this is the first report of mcr-1 carrying E. coli from a companion animal in South Korea. Our findings support One Health approach is necessary to prevent the dissemination of this high-risk gene.We studied the presence of the mobile colistin resistance gene The mcrosporins . While, mcr-1-carrying plasmid pK19EC149 from this study was closely related to those from patients in Korea (GenBank accession no. KY657476 and KY657478) had a similar size and highly conserved backbone (>96%) to other IncI2 plasmids detected in E. coli strains from humans (KY657476 and KY657478) and chickens in Korea . Additioin Korea . The finmcr-1 carrying E. coli was mainly identified in food animals in the country [mcr-1 gene. Nonetheless, we isolated the mcr-1 carrying E. coli from a dog in the urban areas of the Seoul metropolitan. This implies that the dog had minimal or no contact with food animals. Considering the close and frequent contact between humans and companion animals, our results might suggest that mcr-1-carrying E. coli could be transferred between dogs and humans. Consistent with this study, the transmission of mcr-carrying E. coli between humans and companion animals, especially dogs, was reported in China [E. coli harboring the mcr-1 gene has been frequently identified from environmental samples [mcr-1-carrying E. coli isolated from the environment and those from humans and dogs. Therefore, the mcr-1-carrying E. coli in this study might also originate from the natural environment.Colistin is not commonly used to treat companion animals in Korea; however, it is widely used in the livestock industry. Additionally, country ,14. Thusin China ,10. Addi samples ,34,35. G samples showed amcr-1 carrying E. coli, adding another layer of intricacy to the rapidly evolving epidemiology of plasmid-mediated colistin resistance in the community. Thus, a \u201cOne Health\u201d based strategy and a detailed knowledge of antimicrobial usage in humans and companion animals are needed to reduce the dissemination of colistin-resistant E. coli.In conclusion, dogs can serve as a reservoir of"} +{"text": "Nature Communications 10.1038/s41467-020-18764-3, published online 2 October 2020.Correction to: The original version of this Article contained an error in the spelling of the author Trine H. Mogensen, which was incorrectly given as Trine Mogensen. This has now been corrected in both the PDF and HTML versions of the Article."} +{"text": "Communications Biology 10.1038/s42003-020-01501-3, published online 15 December 2020.Correction to: A CAG CAA : QQQQ\u201d was incorrectly bolded and underlined, changing the scientific interpretation of the text. The error has been corrected in the PDF and HTML versions of the Article.In the original published version of the Article, the string of glutamines (QQQQ) in the Results section text \u201ce.g., CAG CA"} +{"text": "Nature Communications 10.1038/s41467-019-13265-4, published online 11 December 2019.Correction to: The original version of this Article omitted the following from the Acknowledgements:This project has received funding from the H2020 MSCA-RISE-HALT under grant agreement No. 823937.This has now been corrected in both the PDF and HTML versions of the Article."} +{"text": "Communications Biology 10.1038/s42003-020-01357-7, published online 30 October 2020.Correction to: In the original published version of the article, the middle initial for author Paul W. Franks was incorrectly omitted. The error has been corrected in the PDF and HTML versions of the article."} +{"text": "Communications Biology 10.1038/s42003-020-0977-2, published online 21 May 2020.Correction to: In the original published version of the article, an error was introduced into Fig. 1a during the typesetting process. The error has been corrected in the PDF and HTML versions of the paper."} +{"text": "The authors wish to make the following erratum to this paper .The Funding is incorrect and must be replaced by the following Funding:Funding: This work was co-funded by the European Union from the European Regional Development Fund under the Smart Growth Operational Programme 2014\u20132020, Sub-Measure 4.1.4 Application Projects under grant no. POIR.04.01.04-00-0101/16.The authors would like to apologize for any inconvenience caused to the readers by these changes."} +{"text": "Scientific Reports 10.1038/s41598-020-64977-3, published online 26 May 2020Correction to: The original version of this Article contained a typographical error in the spelling of the author M. Ka\u017amierczak, which was incorrectly given as M. Kazmierczak. This has now been corrected in the PDF and HTML versions of the Article, and in the accompanying Supplementary Information file."} +{"text": "Nature Communications 10.1038/s41467-020-16857-7, published online 19 June 2020.Correction to: The original version of this article contained an error in the author affiliations.Serghei Mangul was incorrectly associated with Quantitative and Computational Biology, University of Southern California, Los Angeles, CA 90089, USA.This has now been corrected in both the PDF and HTML versions of the article."} +{"text": "Nature Communications 10.1038/s41467-019-12481-2, published online 2 October 2019.Correction to: 12. The unit should be 1017. This has been corrected in the PDF and HTML versions of the Article.In the original version of this article, the unit in the right y-axis of Fig. 4c was mislabelled as 10"} +{"text": "Nature Communications 10.1038/s41467-019-13550-2, published online 5 December 2019.Correction to: The original version of this Article contained an error in Fig.\u00a05. In panel a, the multiple sequence alignment of ASH2L Linker-IDR region was not displayed.This has now been corrected in both the PDF and HTML versions of the Article."} +{"text": "Various cardiac arrhythmias, e.g. atrial fibrillation and ventricular tachycardia, can be treated by electrophysiological (EP) interventions . ApplyinAll experiments were performed on a clinical whole-body 1.5 T MR scanner equipped with an in-room display and an additional MR-EP-workstation (MR-EP-WS) including a standard EP-recorder . This workstation, located next to the scanner, combines and displays incoming real-time 2D and 3D images and real-time tracking positions from the MR scanner as well as real-time EP-data from the EP-recorder.A 7F diagnostic EP catheter Fig. with twoConventional diagnostic EP catheters and MR-EP catheters were compared under X-ray. Bipolar intracardiac electrograms (IEGM) were acquired with both catheters at corresponding locations .Temperature recordings during a typical real-time bFFE sequence were performed for the MR-EP and the conventional EP catheter. The catheters were equipped with fiber optic temperature probes and were inserted into the RA.The RA and RV were mapped using the MR-EP system and catheters. 3D bFFE and 3D CE-MRA datasets were acquired prior to catheterization of the animals. All MR and EP data can be combined and displayed on the MR-EP-WS for guidance, including a surface model of the cardiac vessels, reformatted slices at the catheter position either manually angulated or using the real-time MR imaging geometry.IEGMs acquired with the MR-EP catheter were equivalent in quality to those acquired with the conventional EP catheter Fig. .Figure 2The MR-EP catheter's maximal temperature increase after 10 min of RF transmission at 4 W/kg was 0.7 K Fig. almost cIn contrast, an increase of up to 7.5 K in only 80 s was observed at the tip of the conventional catheter Fig. .The MR-EP-WS enabled a fast mapping, e.g. 40 points in RV in 20 min. The in-bore IEGM recordings were comparable to those under X-ray (Fig. Furthermore, atrial and ventricular pacing was achieved via the MR-EP catheters. Successful stimulation was confirmed by a second MR-EP catheter and was also clearly visible in the surface ECG.Recording of intracardiac electrograms is feasible with the MR-EP catheter. EP data quality is equivalent to conventional EP catheters. The combined use of highly resistive wires and a transformer-based transmission line for active tip tracking effectively suppresses RF-heating even during high SAR MRI.The prototype setup of the MR-EP system provided excellent guidance and an efficient workflow for diagnostic MR-EP interventions."} +{"text": "Scientific Reportshttps://doi.org/10.1038/s41598-020-75538-z, published online 29 October 2020Correction to: The Supplementary Information file that accompanies this Article contains errors, where the numbering of tables and references is incorrect. Additionally, the reference list in the Supplementary Information file is omitted.The corrected Supplementary Information file is linked to this correction notice.Supplementary Information."} +{"text": "Scientific Reports 10.1038/s41598-020-58171-8, published online 03 February 2020Correction to: The original version of this Article contained an error in the email address given for the author Robert J. Griffitt. The correct email address is given below:joe.griffitt@usm.eduAdditionally, this Article contained a truncated version of Figure\u00a03.These errors have been fixed in the HTML and PDF of the original Article."} +{"text": "Scientific Reports 10.1038/s41598-019-42364-x, published online 24 June 2019Correction to: In the original version of this Article, Tao Fan was omitted as a corresponding author. Correspondence and request for materials should also be addressed to fant@ccmu.edu.cn. This error has now been corrected in the HTML and PDF versions of the Article."} +{"text": "Nature Communications 10.1038/s41467-020-15124-z, published online 11 March 2020.Correction to: The original version of this article contained an error in the spelling of the author Michael V. Airola, which was incorrectly given as Michael V. V. Airola. This has now been corrected in the HTML version of the article. The PDF was correct at the time of publication."} +{"text": "Nature Communications 10.1038/s41467-020-19366-9, published online 5 January 2021.Correction to: The original version of this Article contained an error in Fig. 2, in which panels a and b were inadvertently swapped.This has now been corrected in the PDF and HTML versions of the Article."} +{"text": "Nature Communications 10.1038/s41467-019-08750-9, published online 12 March 2019.Correction to: This article contained an error in the ordinate axis in Fig.\u00a02d, where the units were reported as nmol/L. The unit should have been reported as pg/ml. This has now been corrected in both the PDF and HTML versions of the Article."} +{"text": "Correction to: BMC Musculoskelet Disord 20, 157 (2019)https://doi.org/10.1186/s12891-019-2548-6i)The authors conducted both studies using data from the same patients. The authors did not reference the related Chinese language article . This ciii)t-test was used in Chinese language article [The main differences between the two publications were as followed: First, the two publications did not use the same design. The English language article was desi article .Following publication of this article , the autBoth the original article and the"} +{"text": "Nature Communications 10.1038/s41467-020-16281-x, published online 26 May 2020.Correction to: The original version of this Article contained an incorrect hyperlink in reference 17. This has now been corrected in the PDF and HTML versions of the Article."} +{"text": "Correction to: BMC Cancer (2013) 13:21https://doi.org/10.1186/1471-2407-13-21Following publication of the original article , the autThe correct name of the cell line should be Hep3B, instead of HepB3. The correct figure is displayed below. The results and conclusions described therein are not affected by these corrections. The authors sincerely apologize for the error."} +{"text": "Nature Communications 10.1038/s41467-020-16861-x, published online 19 June 2020.Correction to: The original version of this Article contained an error in the labelling of the cross-section in Fig. 2g and the vertical axis in Fig. 2b.This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Nature Communications 10.1038/s41467-020-16184-x, published online 19 May 2020.Correction to: The current Figs. 1 and 2 have incorrect numbers shown on the purple bars. The source data was also incorrect in the original version of the Article.This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Scientific Reportshttps://doi.org/10.1038/s41598-019-46236-2, published online 05 July 2019.Correction to: In the original version of this Article, Dr Kazuki Matsuura was incorrectly listed as the corresponding author. The correct corresponding author for this Article is Dr Dai Miyazaki.Correspondence and request for materials should be addressed to miyazaki-ttr@umin.ac.jp. This error has now been corrected in the HTML and PDF version of the Article."} +{"text": "Scientific Reports 10.1038/s41598-019-49141-w, published online 05 September 2019Correction to: The original version of this Article contained missing angle brackets in equation\u00a011. This has now been corrected in the PDF and HTML versions of the Article."} +{"text": "Nature Communications10.1038/s41467-020-17186-5, published online 8 July 2020.Correction to: The original version of this Article contained an error in Fig. 1. On the x axis of Fig. 1e the labels for mesenchymal and astrocytic cells were swapped. This error has now been corrected in the Pdf and HTML versions of the Article."} +{"text": "Communications Biology 10.1038/s42003-020-0935-z, published online 4 May 2020.Correction to: In the original published version of the article, co-author Katuhiko Shirahige was incorrectly indicated as a jointly supervising author instead of Ki-Hyeon Seong. The error has been corrected in the HTML and PDF versions of the article."} +{"text": "Nature Communications 10.1038/s41467-020-18130-3, published online 08 September 2020.Correction to: The original version of this manuscript did not include a link to the RNA Extraction Procotol scripts that are publically available at Zenodo. The scripts can be found at the following URL: 10.5281/zenodo.4021454In addition, the original version of this Article omitted a declaration from the Competing Interests statement, which should have included the following: \u201cM. Priestman and M. Ciechonska are co-founders of Salient Labs. All other authors declare no competing interests.\u201dThis has now been corrected in both the PDF and HTML versions of the Article."} +{"text": "Communications Biology 10.1038/s42003-020-0908-2, published online 8 May 2020.Correction to: In the original published version of the article, the link to the Figshare dataset referred to in the Data availability statement and the Supplementary Information was missing. The link is 10.6084/m9.figshare.11987448.v1. The errors have been corrected in the HTML and PDF versions of the article and in the \u2018Description of Additional Supplementary Files\u2019 PDF."} +{"text": "Nature Medicine 10.1038/s41591-019-0654-5, published online 2 December 2019.Correction to: In the version of this article initially published, the sixth author\u2019s surname (Jerby-Amon) was incorrect. The correct surname is \u2018Jerby-Arnon\u2019. The error has been corrected in the HTML and PDF versions of the article."} +{"text": "Scientific Reports 10.1038/s41598-019-39755-5, published online 01 March 2019Correction to: This original version of this Article contained errors in Table 1. Due to a production error, the version of Table 1 originally published with this Article contained typesetting information. This has now been corrected in the PDF and HTML versions of the paper."} +{"text": "Nature Communications 10.1038/s41467-019-13720-2, published online 7 January 2020.Correction to: The original version of this Article omitted the following from the Acknowledgements:R.M.W. was supported by National Science Foundation award EAR-1918126 and by the US Department of Energy award DESC0019759.This has now been corrected in both the PDF and HTML versions of the Article."} +{"text": "Nature Communications 10.1038/s41467-019-09374-9, published online 27 March 2019.Correction to: The original version of the Supplementary Information associated with this Article did not include Supplementary Figure 13. The HTML has been updated to include a corrected version of the Supplementary Information."} +{"text": "Nature Communications 10.1038/s41467-019-14151-9, published online 24 January 2020.Correction to: The original version of this Article contained an error in the author affiliations.Cindy Lin was incorrectly associated with \u2018Program in Molecular and Cellular Oncogenesis, The Wistar Institute, Philadelphia, PA 19104 USA\u2019.This has now been corrected in both the PDF and HTML versions of the Article."} +{"text": "Nature Communications 10.1038/s41467-019-12888-x, published online 21 October 2019.Correction to: The original version of this Article mistakenly included the following sentence in the Acknowledgement section:P.P.-R. receives support from a program by the Deputacion de Coru\u00f1a (BINV-CS/2019). This has now been corrected in both the PDF and HTML versions of the Article."} +{"text": "Nature Communications 10.1038/s41467-020-18820-y, published online 6 October 2020.Correction to: The original version of this Article omitted Hao Tian and Huajun Tian as equally contributing authors.This has now been corrected in both the PDF and HTML versions of the Article."} +{"text": "Scientific Reportshttps://doi.org/10.1038/s41598-020-74011-1, published online 07 October 2020Correction to: In the original version of this Article, Ziqiang Wu, Huan Yao and Huan Xu were omitted as equally contributing authors. This has now been corrected in the PDF and HTML versions of the Article, and in the accompanying Supplementary Information files."} +{"text": "Nature Communications 10.1038/s41467-020-19639-3, published online 24 November 2020.Correction to: The original version of this Article contained an error in the spelling of the author Hong X. Do, which was incorrectly given as X. Do Hong. This has now been corrected in both the PDF and HTML versions of the Article."} +{"text": "Nature Communications 10.1038/s41467-020-14914-9, published online 27 February 2020Correction to: The original version of this Article contained an error in the author affiliations.The affiliation of Weilin Xu with \u2018University of Science and Technology of China, 230026 Anhui, P.R. China\u2019 was inadvertently omitted.This has now been corrected in both the PDF and HTML versions of the Article."} +{"text": "Retraction to: Cell Death and Disease10.1038/cddis.2017.223 published online 25 May 2017The Editors-in-Chief have retracted this article because the PTEN-shRNA panel in Fig. 6d appears to be the same as the Anti3142+PTEN-shRNA panel in Fig. 6h. The Editors-in-Chief therefore no longer have confidence in the integrity of the data in Fig. 6. All of the authors disagree with this retraction."} +{"text": "Nature Communications 10.1038/s41467-020-19650-8, published online 17 November 2020.Correction to: In this Article, there was an error in the spelling of the name of the first author of reference 32. This author should have been cited as Nahmad, A. D. not as Nahmed, A. D.This error has been corrected in the HTML and pdf versions of the Article."} +{"text": "Scientific Reports10.1038/s41598-020-66103-9, published online 03 June 2020Correction to: The original version of this Article contained a typographical error in the spelling of the author Deborah N. Huntzinger, which was incorrectly given as Deborah N. Huntinzger. This has now been corrected in the PDF and HTML versions of the Article."} +{"text": "Correction to: BMC Microbiol 20, 295 (2020)https://doi.org/10.1186/s12866-020-01981-7After publication of the original article , the autThe correct Fig. 5A is presented below.-The update of this figure does not change the results, discussion, and conclusion of the original article. We apologize for the inconvenience caused."} +{"text": "Scientific Reportshttps://doi.org/10.1038/s41598-020-58969-6, published online 13 February 2020.Correction to: The original version of this Article contained an error in the title of the paper, where \u2018Full-length title:\u2019 was incorrectly included. This has now been corrected in the PDF and HTML versions of the Article and in the Supplementary Information File."} +{"text": "Communications Biology 10.1038/s42003-020-01227-2, published online 08 September 2020.Correction to: In the originally published version of the Article, an incorrect file was uploaded as Supplementary Movie 1. The error has been corrected in the HTML version of the Article."} +{"text": "Nature Communications 10.1038/s41467-020-20556-8, published online 12 January 2021.Correction to: P < 0.001) was mistakenly positioned over the blue bars , whereas it should have been positioned over the closed red and closed blue bars . The data were indicated correctly in the main text. This error has now been corrected in the PDF and HTML versions of the Article.The originally published version of this Article contained an error in Figure 5. In panel 5g, a statistical significance line (indicating"} +{"text": "Nature Communications 10.1038/s41467-020-16256-y, published online 4 May 2020.Correction to: The competing interests section of the original article contained an error. In the sentence \u201cA patent application has been filed on 12 March 2020 on monoclonal antibodies targeting SARS-CoV-2 (United Kingdom patent application no. 2003632.3\u201d, the number 2003632 was hyperlinked in error to an irrelevant page. The link has been removed both from the PDF and the HTML version of the article."} +{"text": "Nature Communications 10.1038/s41467-020-15141-y, published online 16 March 2020.Correction to: The original version of this Article did not acknowledge Renhao Dong as a corresponding author. This has now been corrected in both the PDF and HTML versions of the Article."} +{"text": "Nature Communications 10.1038/s41467-020-16520-1, published online 8 June 2020.Correction to: The original version of this Article contains an error in Fig.\u00a03 in which panel B was inadvertently duplicated from panel A. This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Nature Communications 10.1038/s41467-020-19593-0, published online 20 November 2020.Correction to: This Article contained an error in Fig. 8. In Fig. 8h multiple white and blue bars denoting CEBPB were present in the histogram.This has now been corrected in the PDF and HTML versions of the Article."} +{"text": "Nature Communications 10.1038/s41467-020-18093-5, published online 01 September 2020.Correction to: The original version of the Supplementary Information associated with this Article contained errors in Supplementary Fig. 9a and on page 7. In both cases, chemical formula of the liquid crystalline medium was drawn incorrectly. The HTML has been updated to include a corrected version of the Supplementary Information; the original incorrect versions of these Figs. can be found as Supplementary Information associated with this Correction.Supplementary Information"} +{"text": "Nature Communications 10.1038/s41467-020-14582-9, published online 21 February 2020, and 10.1038/s41467-018-05653-z, published online 28 September 2018.Correction to: A correction was issued for the original version of this Article which contained an error in the spelling of the author Ana Os\u00f3rio Oliveira, which was incorrectly written as Anna Osorio Oliviera. This has now been corrected in both the PDF and HTML versions of the Article and of the Correction."} +{"text": "Communications Biology 10.1038/s42003-020-0878-4, published online 27 April 2020.Correction to: In the original published version of the article references 56, 57, and 58 contained errors. These have been corrected in the HTML and PDF versions of the article."} +{"text": "Significance [Sci. Rep. 3 (2013) 2930], and analyzed its critical behaviors based on the theoretical derivation of critical number of communities and the phase diagram in community-partition transition. It was revealed that Significance exhibits far higher resolution than the traditional Modularity when the intra- and inter-link densities of communities are obviously different. Following the critical analysis, we developed a multi-resolution version of Significance for identifying communities in the multi-scale networks. Experimental tests in several typical networks have been performed and confirmed that the generalized Significance can be competent for the multi-scale communities detection. Moreover, it can effectively relax the first- and second-type resolution limits. Finally, we displayed an important potential application of the multi-scale Significance in computational biology: disease-gene identification, showing that extracting information from the perspective of multi-scale module mining is helpful for disease gene prediction.Community detection in complex networks is an important issue in network science. Several statistical measures have been proposed and widely applied to detecting the communities in various complex networks. However, due to the lack of flexibility resolution, some of them have to encounter the resolution limit and thus are not compatible with multi-scale structures of complex networks. In this paper, we investigated a statistical measure of interest for community detection, Complex systems, including the artificial and natural ones in the real world, can be properly described as complex networks that consist of vertices and links. Typical examples contain the social, biological, and computer information networks. Currently, it has been recognized that networked description of complex systems is a kind of useful approach to study the structures of and dynamical processes on these systems. Although networked structures are abstracted out from different complex systems, they exhibit many common topological properties , 2, suchBayesian inference ). It seems that Significance tends to favor the detection of small-scale structures, potentially returning partitions with more communities (i.e. groups) than other methods such as those based on Modularity Maximization. It is convenient, then, to use one of these alternative methods to judge the benefits of the Significance as compared to that of Modularity. Otherwise, the better performance could just be the outcome of chance.2. Significance seems particularly insensitive to the resolution parameter. In some Figures gamma runs over 14 orders of magnitude. This may become a problem with networks presenting several levels or scales of organization. See for instance [E4], where benchmark networks with more than 2 levels of hierarchical organization are introduced.3. The description of the community-loop networks seems inadequate. Readers may find difficult to reproduce the results if they are not able to appropriately generate such networks. Please improve and clarify the description. In particular, an extra figure illustrating a few example of community-loop networks could be of help.4. It seems there is definition of $n_s$ around Eq.~1. Please add a sentence defining $n_s$.5. There are many grammatical and orthographic errors, and a few typos (e.g. lever instead of level). Please check and correct them. Use a check speller.6. Please consider sharing your code, if any.Extra references[E1] Vinh, N. X.; Epps, J.; Bailey, J. (2009). \"Information theoretic measures for clusterings comparison\". Proceedings of the 26th Annual International Conference on Machine Learning - ICML '09. p. 1. doi:10.1145/1553374.1553511. ISBN 9781605585161.[E2] Meila, M. (2007). \"Comparing clusterings\u2014an information based distance\". Journal of Multivariate Analysis. 98 (5): 873\u2013895. doi:10.1016/j.jmva.2006.11.013.https://arxiv.org/abs/1907.12581[E3] M. E. J. Newman, George T. Cantwell, Jean-Gabriel Young, Improved mutual information measure for classification and community detection (2019), [E4] Z. Yang, J. I. Perotti, C. J. Tessone, Hierarchical benchmark graphs for testing community detection algorithms, Phys. Rev. E 96, 052311 (2017)**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article digital diagnostic tool, 6 Dec 2019Dear Dr. Claudio J. Tessone and Reviewers, Thank you for your comments concerning our manuscript entitled \u201cSignificance-based multi-scale method for network community detection and its application in disease-gene prediction\u201d (ID: PONE-D-19-25876). Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our researches. We have studied Journal requirements and reviewer\u2019s comments carefully and have made revisions which hope meet with approval. Significant changes in the text have been marked in blue for easy check purpose. The main corrections in the paper and the responds to your comments are as flowing. Journal Requirements:1. When submitting your revision, we need you to address these additional requirements.http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdfPlease ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at Response: We have carefully checked the manuscript and ensure that it meets PLOS ONE\u2019s style requirements.Response to the reviewers\u2019 comments:Reviewer #1: General Comment: In the manuscript \"Multi-scale community detection in complex networks by Significance\", the authors have investigated a statistical measure in community detection, i.e. \"Significance\". They have compared the resolution of significance against modularity and surprise. After that, the authors have developed a multi-resolution significance and examined the performance of this measure.The research question of this paper is well explained and is relevant. However, I would like to make some suggestions to the paper.Response: Thanks. We are very glad that you affirm the value of our research. We are responding positively to your comments listed as follows.Comment (1-1): (1) In \"2.1 - Critical analysis of Significance\", it would be great if the authors can conduct more analysis. Please see Fig. 2 ~ 4 in \"Xiang, J., Li, H. J., Bu, Z., Wang, Z., Bao, M. H., Tang, L., & Li, J. M. (2018). Critical analysis of (Quasi-) Surprise for community detection in complex networks. Scientific reports, 8(1), 14459\" for details.Response: Thanks for reviewer\u2019s useful suggestion. This reference provided a good example for critical analysis. In this submitted manuscript, for the integrity of this work, we first discussed the critical characteristics of Significance in community detection, because it is based on the critical analysis of Significance that our multi-scale method was proposed. In order to help readers better understand this work, we further supplemented the theoretical derivation process of the critical parameter in the phase transition, and provided the theoretical proof that there is no \u201cpotential well\u201d effect in the significance compared with the surprise. In order to enrich the manuscript, we further applied our multi-scale method to a hot issue in computational biology: disease-gene identification. The results showed that extracting information from the perspective of multi-scale module mining is helpful for disease gene prediction, and its combination with other methods can effectively improve the overall performance of prediction methods . This provides important insights for our next research. In the future work, we will further study the applications of the multi-scale method in computational biology. Thanks again for the reviewer's useful suggestion Comment (1-2): (2) In Fig. 3, how large is the network? Does network size play a role here?Response: Thanks for reviewer\u2019s useful suggestion. Indeed, the network-size effect should be considered in detail. Therefore, we comprehensively computed three metrics of these methods for the different network sizes. In Fig. 4, the results for three metrics have been added. In the LFR networks, all metrics indicate that Significance gets the somewhat better performance than Surprise, and significantly overcomes Modularity. Also, six subfigures have been added to demonstrate the network-size effects in text). Interestingly, with the increase of network size, NMI, AMI and ARI for both Significance and Surprise gradually increase, while decrease for Modularity, indicating that Significance and Surprise have better performance for the large networks than Modularity.Comment (1-3): (3) In Fig. 4 and 5, the authors have compared the NMI of significance and modularity in community-loop networks and LFR networks as a function of resolution parameter. However, the x-axis of these figures have different scales. This makes it difficult to compare the results. Please fix it.Authors\u2019 Response: Thanks you for pointing out this question. For ease to compare, in the revised manuscript, we adjusted the scales in these Figures. In addition, the main purpose of Fig.4 and 5 is to demonstrate the region of resolution parameter where the predefined communities can be exactly identified. It was found: (1) when gamma<1, Significance has successfully identified all predefined communities, while for Modularity, in order to exactly detect all predefined communities, it is required that the resolution parameter must be larger than 1. (2) In order to exactly indentify all predefined communities, the region of resolution parameter for Significance is wider than that for Modularity, indicating that our method can find out the predefined community structure more easily than the multi-resolution Modularity. Comment (1-4): (4) Similar to (3), the scales of x-axis are different in Fig. 6 - 8. For panel (c) & (d) in these figures, could the authors increase the range of x-axis to 10^1?Authors\u2019 Response: As has been demonstrated in Fig.4 and 5, Significance and Modularity show the different regions of resolution parameter where the predefined network partition can be exactly detected. Thus, in order to demonstrate those promising network partitions and the tolerance to the second-type resolution limit, the scales of resolution parameters are set to be different for different methods. Because Significance has a higher resolution, in the region of gamma<1, it has successfully detected the predefined community structure. Therefore, the results for gamma>1 did not continue to be shown. Comment (1-5): (5) What is the computational complexity of multi-resolution significance?Authors\u2019 Response: This is an important problem. In general, the multi-resolution Significance divided the networks into communities at each resolution by Louvain process. Louvain process is a widely used and efficient algorithm, but its exact computational complexity is not known1. Most of its computational effort is spent on the optimization at the first level, taking a time O(nkmf) if we control the maximal iteration times, where n is the number of nodes, km is the mean degree of nodes, and f is the number of operations of calculating S-value each time (on average the number of communities that each node connects to is less than the number of neighbors of the vertex). https://perso.uclouvain.be/vincent.blondel/research/louvain.html1Comment (1-6): Overall, I like the idea of this paper and I hope my comments help in the development of the paper.Authors\u2019 Response: Thanks you for your valuable comments. All of them are very helpful for improving our manuscript. We appreciate for your warm work earnestly, and hope that these corrections will meet with your approval.-------------------------------------------------------------------------------------------------------Reviewer #2: The work by K. Hu et al. focuses on the problem of community detection in complex networks. In particular, it comparatively studies the \"Significance\" of Traag et al. against the more traditional \"Modularity\" of Girvan and Newman, for the detection of communities within networks with multi-scale structures. Moreover, the present work introduces and studies a multi-resolution variation of the \"Significance\", essentially encompassing the novelty of the present contribution.In my opinion the work presents interesting results, so I recommend it for publication after the following issues are appropriately addressed.Response: Thanks. We are very glad that you affirm the value of our research. We are responding positively to your questions pointed out in the referee report.Comment (2-1): 1. The NMI may result significantly non-zero when two random partitions with large numbers of groups are compared, because random coincidences become likely in this case. Similarly, it may result in artificially large values, even when two non-random partitions are compared if these have a large number of groups. To counter balance for such bias, several metrics alternative to the NMI were introduced (see [E1-E3]). It seems that Significance tends to favor the detection of small-scale structures, potentially returning partitions with more communities (i.e. groups) than other methods such as those based on Modularity Maximization. It is convenient, then, to use one of these alternative methods to judge the benefits of the Significance as compared to that of Modularity. Otherwise, the better performance could just be the outcome of chance.Authors\u2019 Response: Thanks for the reviewer\u2019s useful suggestion. For comparison, we added the results of the adjusted mutual information (AMI) and adjusted Rand index (ARI). We find that the results for both ARI and AMI are similar to those of NMI, which indicate the better performance of Significance . Also, in Fig 4, we present the results of network-size effect to demonstrate the performances of these methods.Comment (2-2): 2. Significance seems particularly insensitive to the resolution parameter. In some Figures gamma runs over 14 orders of magnitude. This may become a problem with networks presenting several levels or scales of organization. See for instance [E4], where benchmark networks with more than 2 levels of hierarchical organization are introduced.Authors\u2019 Response: In reference E4, Yang et al. proposed a good hierarchical benchmark graph (named as RB-LFR network) for testing various community detection algorithms. In our manuscript, we employed the RB-LFR networks with three levels to test both Modularity and Significance. The results have been shown in Fig 10. For two typical mixing parameters of seed LFR benchmark, three different ground truths: seed-replica-replica, replica-replica-seed and flat, are well identified. In order to obtain a richer hierarchical community structure, we also extended the RB-LFR network by setting different probabilities of randomly removing connections between the seed communities and the replicas for the different hierarchies. In these extended RB-LFR benchmarks with three levels, three different community structures corresponding to three different hierarchies can be well defined for each of mixing parameters. For instance, when the mixing parameter is small enough and the probabilities p1 and p2 of removing connections are small , the communities for every LFR (including seed LFR and its replicas) can been well defined on the first level (or upper level), and two levels of community structures , corresponding to the second and the third hierarchy, can be then defined. When the mixing parameter is large and the probabilities p1 and p2 of removing connections are large enough , the first level is the same as the case for small mixing parameter, and the second and third levels are refereed to two kinds of flats. In view of the explicit hierarchical community structures of the extended RB-LFR benchmark, we also test both Significance and Modularity in these benchmark networks. The results show that the multi-resolution Significance and Modularity can well identify the predefined community structures at every level .Thanks you for your valuable suggestion.Comment (2-3): 3. The description of the community-loop networks seems inadequate. Readers may find difficult to reproduce the results if they are not able to appropriately generate such networks. Please improve and clarify the description. In particular, an extra figure illustrating a few example of community-loop networks could be of help.Authors\u2019 Response: We added a figure illustrating an example of community-loop networks. Thanks for your useful suggestion.Comment (2-4): 4. It seems there is definition of $n_s$ around Eq.~1. Please add a sentence defining $n_s$.Authors\u2019 Response: Thanks you for pointing out this question. We added a sentence defining $n_s$.Comment (2-5): 5. There are many grammatical and orthographic errors, and a few typos (e.g. lever instead of level). Please check and correct them. Use a check speller.Authors\u2019 Response: We revised and checked the manuscript carefully. Thanks you for these useful suggestions.Comment (2-6): 6. Please consider sharing your code, if any.yu.sunny@126.com). Authors\u2019 Response: The readers could request the code of the paper by the corresponding author . \"Information theoretic measures for clusterings comparison\". Proceedings of the 26th Annual International Conference on Machine Learning - ICML '09. p. 1. doi:10.1145/1553374.1553511. ISBN 9781605585161.[E2] Meila, M. (2007). \"Comparing clusterings\u2014an information based distance\". Journal of Multivariate Analysis. 98 (5): 873\u2013895. doi:10.1016/j.jmva.2006.11.013.https://arxiv.org/abs/1907.12581[E3] M. E. J. Newman, George T. Cantwell, Jean-Gabriel Young, Improved mutual information measure for classification and community detection (2019), [E4] Z. Yang, J. I. Perotti, C. J. Tessone, Hierarchical benchmark graphs for testing community detection algorithms, Phys. Rev. E 96, 052311 (2017)We tried our best to revise our manuscript according to the comments. Also, several minor problems have been corrected. Four figures and some references (including E1-E4) have been added, and thus the figures and references are renumbered. Fig 4 has been recalculated and re-plotted.Once again, we would like to express our great appreciation to you for helpful comments on our manuscript.With our thanks and best regards.Yours sincerely,Yun-Xia Yuyu.sunny@126.comE-mail: AttachmentResponse to Reviewers.pdfSubmitted filename: Click here for additional data file. 17 Dec 2019Significance-based multi-scale method for network community detection and its application in disease-gene predictionPONE-D-19-25876R1Dear Dr. Yu,We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.https://www.editorialmanager.com/pone/, click the \"Update My Information\" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact With kind regards,Claudio J. Tessone, PD Ph.D.Academic EditorPLOS ONEAdditional Editor Comments :Dear Dr Yun-Xia YuWe are happy to confirm that your manuscript entitled \"Significance-based multi-scale method for network community detection and its application in disease-gene prediction\" has been accepted for Publication in PLOS ONE. This decision follows from your careful reply to the reviewer's comments.Reviewers' comments: 4 Mar 2020PONE-D-19-25876R1 Significance-based multi-scale method for network community detection and its application in disease-gene prediction Dear Dr. Yu:I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. For any other questions or concerns, please email Thank you for submitting your work to PLOS ONE.With kind regards,PLOS ONE Editorial Office Staffon behalf ofDr. Claudio J. Tessone Academic EditorPLOS ONE"} +{"text": "Scientific Reportshttps://doi.org/10.1038/s41598-020-79675-3, published online 11 January 2021Correction to: This Article contained an error. Camille No\u00fbs, listed as the tenth author on the paper, is fictitious and therefore does not fulfil the requirements for authorship. The name is now removed from the author list. Additionally, the following sentence was added in the Acknowledgements:https://www.cogitamus.fr/camilleen.html), and is hereby acknowledged.\"\"Camille No\u00fbs, a fictitious author, embodies the ideals of the collegial construction of the standards of science through developing the methodological framework, the state-of-the-art, and by ensuring post-publication follow-up ("} +{"text": "Nature Communications 10.1038/s41467-020-16844-y, published online 10 June 2020.Correction to: The original version of this Article contained errors in the Acknowledgements:F.Z. is supported by ARO under Grant No. W911NF-18-1-0416 and NSF under Grant No. DMR-1921581 (DMREF). This was incorrectly given as F.Z. is supported by the University of Texas at Dallas research enhancement funds.Funding from the NSF MRSEC program (DMR-1719875) was incorrectly given as Funding from the NSF MRSEC program (DMR-1120296).This has now been corrected in both the PDF and HTML versions of the Article."} +{"text": "Kidney transplantation (KT) is the treatment of choice for end-stage chronic kidney disease (CKD) and is well known to improve the clinical outcome of patients. However, the impact of KT on comorbid psychological symptoms, particularly depression and anxiety, is less clear, and recipients of living-donor (LD) organs may have a different psychological outcome from recipients of dead-donor (DD) organs.In total, 152 patients were included and analyzed using a cross-sectional design. Of these patients, 25 were pre-KT, 13 were post-KT with a LD transplant and 114 were post-KT with a DD transplant. The patients were tested for a variety of psychometric outcomes using the Hospital Anxiety and Depression Scale, the 12-Item Short Form Health Survey , the Resilience Scale, the Coping Self-Questionnaire and the Social Support Questionnaire.The mean age of the patients was 51.25\u2009years and 40 per cent of the patients were female. As expected, the post-KT patients had significantly better scores on the physical component of the Short Form Health Survey than the pre-KT patients, and there were no significant differences between the two post-KT groups. There were no significant differences among the groups in any of the other psychometric outcome parameters tested, including anxiety, depression and the mental component of health-related quality of life.KT and the origin of the donor organ do not appear to have a significant impact on the psychological well-being of transplant patients with CKD. Although the diagnosis and early treatment of psychological symptoms, such as depression and anxiety, remain important for these patients, decisions regarding KT, including the mode of transplantation, should not be fundamentally influenced by concerns about psychological impairments at the population level.CKD is a serious condition involving profound impairment of the physical and psychological well-being of patients. KT is considered the treatment of choice for most of these patients. KT has notable advantages over dialysis with regard to the long-term physical functioning of the renal and cardiovascular system and increases the life expectancy of patients. However, the data on the improvement of psychological impairments after KT are less conclusive. CKD= chronic kidney disease;DD= dead donor;FKV= Freiburger Fragebogen zur Krankheitsverarbeitung;F-SozU= Social Support Questionnaire;HADS= Hospital Anxiety and Depression Scale;KT= kidney transplantation;LD= living donor;PROs= patient-reported outcomes; andRS= resilience scale.Chronic kidney disease (CKD) is a serious condition involving profound impairment of the physical and psychological well-being of patients. Kidney transplantation (KT) is considered the treatment of choice for most of these patients. KT has notable advantages over dialysis with regard to the long-term physical functioning of the renal and cardiovascular system and increases the life expectancy of patients , we compared patients waiting for transplantation with kidney-transplanted patients regarding a variety of psychometric parameters and found no significant differences in depression, anxiety, resilience and health-related quality of life among the groups (PI-KT study) ;patients after KT with a LD organ (post-KT-LD); andpatients after KT with a DD organ (post-KT-DD).The clinical patient groups eligible to participate were as follows:The study was evaluated and approved by the Ethics Committee of the University of Erlangen-Nuremberg. All patients received detailed information regarding the study design and the procedures and signed a written informed consent form before inclusion. Data acquisition for individual patients was performed on a single date, typically during a routine KT-protocol visit and, generally, took approximately 45\u2009min for each proband, which is comparable to the duration of a normal psychiatric consultation.The general somatic and nephrological comorbidity burdens of all probands were also collected.Hospital Anxiety and Depression Scale (HADS-D/A) (The ADS-D/A) is a valResilience Scale (RS) is a 25-Coping Self-Questionnaire (The German) comprise12-Item Short-Form Health Survey . The descriptive data analysis was performed for all demographic data, somatic data and patient-reported outcomes (PROs). The descriptive data are shown as the median [interquartile range] unless otherwise indicated. The data from the psychometric questionnaires were inspected for normality of distribution using both numerical and graphical methods (Shapiro\u2013Wilk test and Q-Q plots). Because the data from some questionnaires were not normally distributed, we used nonparametric statistics. Potential differences among the pre-KT, KT-LD and KT-DD patient groups were tested using the Kruskal\u2013Wallis test for interval data, and differences in frequency distributions were tested using the N = 118), multiple assessments of CKD patients over time, and patients who had experienced a failed KT and were waiting a new transplant (N = 8). Accordingly, our final data set included 152 patients. In total, 59.9 per cent of the patients were males and 40.1 per cent were females. The mean \u00b1 standard deviation age was 51.25\u2009\u00b1\u200915.12\u2009years. Among these patients, 25 were pre-KT, 13 were post-KT-LD and 114 were post-KT-DD.For this analysis, we considered a total of 342 CKD patient visits, excluding evaluations with missing data on the type of donor organ (p = 0.014) and duration of dialysis (p = 0.001). Post hoc testing showed that the post-KT-DD patients were older than the pre-KT patients (p = 0.004) (pre-KT = 43.3[27.0] and post-KT-DD = 52.8[22.6]). No significant age differences were identified between post-KT-LD patients and those in the other two groups. All three subgroups differed from one another in the duration of dialysis (prior to KT) with the shortest period of dialysis in the pre-KT groups (10.00[35.0]) followed by the post-KT-DD (54.00[79.0]) and post-KT-LD groups (117.0[160.0]). All of the aforementioned significant post hoc tests remained significant after Bonferroni\u2013Holm adjustment.Demographic characteristics, including age, gender, relationship status, educational status and duration of dialysis, are shown in p = 0.007), whereas only the post hoc test for pre-KT (53.44[6.1]) vs post-KT-DD (44.69[7.1]) patients remained significant (p = 0.002) after adjustment, suggesting reduced physical well-being among post-KT-DD patients compared to that among the pre-KT subgroup. No significant difference was found in the SF-12 physical component scale between the post-KT-LD and post-KT-DD groups. Additionally, a cluster analysis showed no significant differences in the nephrological disease that led to KT but was carefully interpreted with regard to the sample sizes.The mean scores obtained on the various psychometric questionnaires are shown in p-values of the comparisons of each questionnaire are shown in Furthermore, no significant differences were found among the three subgroups of patients in any of the other PROs. The corresponding \u03c72-test .Regarding the symptoms of depression and anxiety, the mean HADS total scores and the anxiety and depression subscale scores were similar among the groups, ranging from 4 to 6. The rate of patients scoring above the cut-off value for clinically relevant depression/anxiety on the HADS depression and anxiety scales was not related to patient subgroup according to the The results of the present study are consistent with our recent finding that psychiatric symptoms, including depression, anxiety and health-related quality of life, did not significantly change after KT compared to those of the pre-KT state .The study received intramural funding from the Department of Psychiatry of the Friedrich-Alexander-University Erlangen-Nuremberg (Head of Department: Prof Dr Johannes Kornhuber) and within the funding program NoneConsent for publication was obtained from all probands of this study.All data are presented in the manuscript. There are no additional data.H.H.O.M., J.M.M. and J.K. designed the study. H.H.O.M., T.S. and M.S.W. participated in data acquisition. H.H.O.M. and C.L. wrote the manuscript . C.L. and M.E. provided expert opinions on the statistical procedures and analyzed the data. M.S.W. and K.U.E. provided expert opinions on the nephrology and transplantation procedures. J.K. and A.P. advised regarding the psychometric measurements of anxiety and affective disorders. H.H.O.M., T.S., J.K., K.U.E. and J.M.M. provided expert advice on the diagnoses of psychiatric disorders in somatically ill probands. J.M.M. was the senior author and critically revised the versions of the manuscript. All authors participated in writing and revising the final manuscript.All authors declare that they have no competing interests."} +{"text": "Nature Communications 10.1038/s41467-020-15787-8, published online 23 April 2020.Correction to: The original version of this Article contained an error in Fig. 5 that was inadvertently introduced during production. The black line depicting the confidence interval was shifted in Fig. 5e, first row, right panel. This error has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Communications Biology 10.1038/s42003-020-01128-4, published online 28 July 2020.Correction to: In the original published version of the article, an equal contributions statement for authors Saurabh Verma and Sudhanva S. Kashyap was inadvertently omitted. The error has been corrected in the HTML and PDF versions of the article."} +{"text": "Correction to: BMC Med Genethttps://doi.org/10.1186/s12881-020-0950-4Following publication of the original article , the autThe spelling has since been corrected in the original article and is included in this correction.The authors apologize for any inconvenience caused."} +{"text": "Scientific Reports 10.1038/s41598-019-38941-9, published online 21 February 2019Correction to: In the original version of this Article, the ORCID ID for J. P. Allain was omitted.This error has now been corrected in the HTML and PDF versions of the Article."} +{"text": "Nature Communications 10.1038/s41467-020-18399-4, published online 15 September 2020.Correction to: The original version of this Article omitted the following from the Acknowledgements:L.N.S. was supported by the Training Grant in Oncogenic Signals and Chromosome Biology T32 CA108459.This has now been corrected in both the PDF and HTML versions of the Article."} +{"text": "Correction to: Surg Case Rep 6, 164 (2020)https://doi.org/10.1186/s40792-020-00897-8Acknowledgement.Following publication of the original article , the autThe content currently reads:We would like to thank Edanz Company for the English language editing.The content should be changed to:M.E would like to thank the Ministry of Higher Education and Scientific Research, Cultural Affairs and Missions Sector, Egypt for the educational scholarship."} +{"text": "Cell Death DiscoveryCorrection to: 10.1038/s41420-020-00329-4 published online 28 September 2020In the original version of this article, Figs. 1\u20136 and their legends were mismatched. This has now been corrected in the PDF and HTML versions of the article."} +{"text": "Nature Communications 10.1038/s41467-019-12489-8, published online 4 October 2019.Correction to: The original version of this article omitted the following from the Acknowledgements:H.X. is a CPRIT Scholar in Cancer Research.This has now been corrected in both the PDF and HTML versions of the article."} +{"text": "Scientific Data 10.1038/s41597-020-0380-3, published online 10 February 2020Correction to: During the typesetting process, multiple errors were introduced into the numbering of the references in this Data Descriptor. These errors have been corrected in the HTML and PDF versions."} +{"text": "Nature Communications 10.1038/s41467-018-05970-3, published online 24 August 2018.Correction to: The original version of this article contained an error in the Methods section, which incorrectly read \u2018nuclear nonproliferation treaty\u2019. The correct version removes this phrase. This has been corrected in both the PDF and HTML versions of the article."} +{"text": "Correction to: World J Emerg Surg (2020) 15:2https://doi.org/10.1186/s13017-019-0281-yThe original article containeAs such, the original article has since been corrected to reflect the correct authorship.Furthermore, this error was mistakenly introduced by the production team handling this article and, as such was not the fault of the authors."} +{"text": "Scientific Data 10.1038/s41597-020-0445-3, published online 14 April 2020Correction to: In the original version of this Data Descriptor, the link to the metadata files produced during the journal\u2019s curation process was incorrect. The correct link is 10.6084/m9.figshare.11967924This has now been updated in the HTML version of the manuscript."} +{"text": "Communications Biology 10.1038/s42003-020-1001-6, published online 1 June 2020.Correction to: In the original published version of the Supplementary Information file, Supplementary Figure 10 was inadvertently omitted, although the figure caption was present. This has been corrected in the Supplementary Information file online."} +{"text": "Correction to: BMC Pulm Med (2020) 20:48https://doi.org/10.1186/s12890-020-1081-6Following publication of the original article , the autPlease note that the correct spelling is \u2018Eyob Alemayehu Gebreyohannes\u2019.The spelling has since been corrected in the original article and, furthermore, is provided in the author list of this correction.The authors apologize for any inconvenience caused."} +{"text": "Nature Communications 10.1038/s41467-020-18617-z, published online 24 September 2020.Correction to: The original version of this Article contained an error in the spelling of the author Ahmed I. Younes, which was incorrectly given as Ahmed Younes. This has now been corrected in both the PDF and HTML versions of the Article."} +{"text": "Nature Communications 10.1038/s41467-020-14816-w, published online 26 February 2020.Correction to: The original version of this Article contained an error in the spelling of the authors Huan Tran and Thomas R. Pauly, which were incorrectly given as Tran D. Huan and Thomas J. Pauly. This has now been corrected in both the PDF and HTML versions of the Article."} +{"text": "Light: Science & ApplicationsCorrection to: 10.1038/s41377-021-00466-0 published online 25 January 2021We would like to correct the SEM image of Fig. 3a on page 5.The SEM image of Fig. 3a in the original article should be corrected to the Fig. We would like to apologize for any inconvenience this may have caused."} +{"text": "Nature Communications 10.1038/s41467-020-15326-5, published online 30 March 2020.Correction to: The original version of this Article contained an error in the Methods section, which incorrectly read \u2018A step-by-step protocol describing the differentiation protocol can be found at Nature Protocol Exchange (ref. 36)\u2019. The correct version states \u2018Protocol Exchange\u2019 in place of \u2018Nature Protocol Exchange\u2019. This has been corrected in both the PDF and HTML versions of the Article.Nat. Protoc.\u2019 and incorrect DOI. The correct form of ref. 36 is:The original version of this Article contained an error in ref. 36, which was incorrectly given with the wrong journal name as: \u2018Protoc. Exch. 10.21203/rs.3.pex-635/v1 (2020).Plaza Reyes, A. et al. Xeno-free, chemically defined and scalable protocol to produce hPSC-derived RPE monolayer. This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Nature Communications 10.1038/s41467-020-18494-6, published online 5 October 2020.Correction to: The original version of this Article contained errors in Figs. 3b, c and 6h in which the edges connecting the network nodes have been missing. This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Nat. Commun. 10.1038/s41467-020-19813-7, published online 8 December 2020.Correction to: The original version of this Article contained an error in the author contributions, which incorrectly omitted the following: \u2018J.W. led the experiments to study the genetic mechanism underlying the emergence of the non-Dun coat colors of donkeys, including performing RNA-seq of croup skin samples of Dun and non-Dun donkeys, performing the immunohistochemistry and immunofluorescence assays against donkey TBX3 protein and bioinformatics analysis of the 1\u2009bp deletion downstream the TBX3 gene. J.W. also contributed to the plotting of Figures\u2019.The original version of this Article also contained an error in the author affiliations. The affiliation of Antonia Noce with Leibniz-Institute for Farm Animal Biology (FBN), Dummerstorf 18196, Germany, was inadvertently omitted.These have now been corrected in both the PDF and HTML versions of the Article."} +{"text": "Nature Communications 10.1038/s41467-019-13244-9, published online 21 November 2019.Correction to: The original version of this Article contained an error in the spelling of the author Wilma D.J. van de Berg, which was incorrectly given as Wilma D.J. van den Berg. This has now been corrected in both the PDF and HTML versions of the Article."} +{"text": "Scientific Reportshttps://doi.org/10.1038/s41598-020-63811-0, published online 27 April 2020Correction to: The original version of this Article contained an error in the title of the paper, where the word \u201cResearch Article\u201d was incorrectly added. This has now been corrected in the PDF and HTML versions of the Article."} +{"text": "Cell Death & DiseaseCorrection to: 10.1038/s41419-020-02767-5, published online date 23 July 2020In the original version of this article, Fig. 6 appeared incorrectly in the PDF. It showed panels d and e of Fig. 5 instead of the intended Fig. 6, which was omitted. This has now been corrected in the PDF version of the Article."} +{"text": "Scientific Reports 10.1038/s41598-020-73180-3, published online 01 October 2020Correction to: In the original version of this Article, the affiliations given for Francesco Cavallin were incomplete.The correct affiliations are listed below:Solagna, ItalyFrancesco Cavallin is an independent statistician.This error has now been corrected in the PDF and HTML versions of the Article."} +{"text": "Scientific Reportshttps://doi.org/10.1038/s41598-020-64275-y, published online 06 May 2020Correction to: A supplementary file containing Tables S1 and S2 was omitted from the original version of this Article. This has been corrected in the HTML version of the Article; the PDF version was correct at time of publication."} +{"text": "Nature Communications 10.1038/s41467-019-13707-z, published online 18 December 2019.Correction to: Equation (1) in the original version of this Article contained an error. This has now been corrected in the PDF and HTML versions of the Article."} +{"text": "Nature Biotechnology 10.1038/s41587-020-0508-1, published online 11 May 2020.Correction to: In the version of this article initially published online, a plot from a different dataset was substituted for the left panel of Fig."} +{"text": "Communications Biology 10.1038/s42003-020-01132-8, published online 3 August 2020.Correction to: In the original published version of the article, co-author Seoyeon Lee\u2019s name was incorrectly spelled as Seoyoen Lee. The error has been corrected in the HTML and PDF versions of the article."} +{"text": "Nature Communications 10.1038/s41467-020-15328-3, published online 23 March 2020.Correction to: The original version of this Article contained an error in Fig. 2b, in which error bars were missing. This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Scientific Reports 10.1038/s41598-020-60434-3, published online 26 February 2020Correction to: In the original version of this Article, Chaoyun Wang was omitted as a corresponding author. Correspondence and request for materials should be addressed to ibfcwcy@139.com. This error has now been corrected in the HTML and PDF versions of the Article."} +{"text": "Nature Communications 10.1038/s41467-020-15632-y, published online 14 April 2020.Correction to: The original version of this Article contained an error in the spelling of the author Maria C. Puertas, which was incorrectly given as Mari Carmen Puertas. This has now been corrected in both the PDF and HTML versions of the Article."} +{"text": "Nature Communications 10.1038/s41467-019-14069-2, published online 31 January 2020.Correction to: The original version of the Peer Review File associated with this Article was updated after publication to redact two figures in the interest of confidentiality.Peer Review File"} +{"text": "Nature Communications 10.1038/s41467-020-20781-1, published online 21 January 2021.Correction to: The original version of this Article contained an error in Fig. 2 that inverted the order of the zones. This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Nature Communications 10.1038/s41467-020-15903-8, published online 30 April 2020.Correction to: GFP-UVSSA vs GFP-UVSSA+UV). This has been corrected in both the PDF and HTML versions of the Article.The original version of this Article contained an error in the hyperlink to an interactive volcano plot for Figure 5 panel b ("} +{"text": "International Journal of ObesityCorrection to: 10.1038/s41366-020-00735-9The original version of this article unfortunately contained a mistake in the ESM. The original article has been corrected.Supplementary Information"} +{"text": "Correction to: Scand J Trauma Resusc Emerg Med 28, 112 (2020)https://doi.org/10.1186/s13049-020-00801-1Following the publication of the original article , the autThe new corresponding author Manuel F. Struck is shown in the authorship of this Correction as well as in that of the original article."} +{"text": "Correction to: BMC Genethttps://doi.org/10.1186/s12863-020-0824-yFollowing publication of the original article , the autThe author list has now been corrected in the original article.Please also find the corrected author list in this correction article.The publisher apologizes for this processing error."} +{"text": "This article has been corrected: Due to an entry error found in one of the triplicate data for the 48-h time-point of stability in human serum in vitro, the half-life for drug release has been corrected to 18.82 h from the stated 23.98 h in 22496-22512. https://doi.org/10.18632/oncotarget.4318Original article: Oncotarget. 2015; 6:22496\u201322512."} +{"text": "Correction to: BMC Genomics (2020) 21:331https://doi.org/10.1186/s12864-020-6748-0Following the publication of the original article , it was The correct Fig. The publisher apologizes to the authors and readers for the inconvenience."} +{"text": "RMD Open 2021;7:e001324. doi: 10.1136/rmdopen-2020-001324Boegel S, Castle JC, Schwarting A. Current status of use of high throughput nucleotide sequencing in rheumatology. The article has been corrected since it was published online. The funding statement has been updated as follows.We wish to acknowledge the RARENET EU-Interreg for supporting this study. Furthermore, SB wishes to acknowledge and thank the Mainz Research School of Translational Biomedicine (TransMed) for support."} +{"text": "Nature Communications 10.1038/s41467-020-17136-1, published online 06 July 2020.Correction to: The original version of this Article contained an error in the author affiliations.The affiliation of Kerry Hilligan with \u2018Immune Cell Biology Programme, Malaghan Institute of Medical Research, Wellington 6012, New Zealand\u2019 was inadvertently omitted.This has now been corrected in both the PDF and HTML versions of the Article."} +{"text": "APVV grant is APVV-17-0642. The corrected statement appears below.There is an error in the Funding statement. The correct number for the first The work was supported by research grants APVV-17-0642, APVV-18-0515, VEGA 2/0181/17 VEGA 2/0135/18, and VEGA 2/0167/17.The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated."} +{"text": "Nature Communications 10.1038/s41467-020-20091-6, published online 4 December 2020.Correction to: In the original version of this Article, contributions from Jennifer R. Baker and Cecilia C. Russell from the University of Newcastle for synthesizing batches of Dynole 34-2 were not acknowledged. This error has now been corrected in both the PDF and HTML versions of the Article."} +{"text": "Scientific Reportshttps://doi.org/10.1038/s41598-020-74761-y, published online 20 October 2020Correction to: In the original version of this Article, Jianbo Dong and Betty Huang were omitted as equally contributing authors. This has now been corrected in the PDF and HTML versions of the paper."} +{"text": "Scientific Data 10.1038/s41597-021-00865-3, published online 05 March 2021Correction to: In the corrected version of this Data Descriptor, an error was introduced in the spelling of H. Peter Soyer which was incorrectly given as H. Peter Seter. This has now been corrected in both the PDF and HTML versions of the Data Descriptor."} +{"text": "Scientific Reports 10.1038/s41598-020-78516-7, published online 09 December 2020Correction to: In the original version of this Article, Jianchang Wang was incorrectly listed as the corresponding author. The correct corresponding author for this Article is Zhanguo Jin. Correspondence and request for materials should be addressed to ccjzg@qq.com. This error has now been corrected in the HTML and PDF versions of the Article."} +{"text": "Nature Communications 10.1038/s41467-020-18870-2, published online 9 October 2020.Correction to: Y. enterocolitica, Y. enterocolitica and K. aerogenes superimposed. In the correct version, panel 3a shows the structure of the Y. enterocolitica urease only. This has been corrected in both the PDF and HTML versions of the Article.The original version of this Article contained an error in Fig. 3, in which panel 3a showed the structures of the ureases from"} +{"text": "Scientific Reports 10.1038/s41598-020-62831-0, published online 23 April 2020Correction to: In the original version of this Article, Rachana Singh was incorrectly listed as the corresponding author. The correct corresponding author for this Article is Sheo Mohan Prasad. Correspondence and request for materials should be addressed to profsmprasad@gmail.com. This error has now been corrected in the HTML and PDF of the article."} +{"text": "Cell Death and DiseaseCorrection to: 10.1038/s41419-018-1264-8published online 10 January 2019In the original published version of this article, Fig. This has been corrected in both the PDF and HTML versions."} +{"text": "Nature Communications 10.1038/s41467-020-18800-2, published online 2 October 2020.Correction to: The original version of the Supplementary information associated with this Article omitted the Supplementary References. The HTML has been updated to include a corrected version of the Supplementary information."} +{"text": "Scientific Reports 10.1038/s41598-020-68875-6, published online 20 July 2020Correction to:In the original version of this Article, F. Rossella was incorrectly listed as a corresponding author. The correct corresponding author for this Article is C.S. Pomelli. Correspondence and request for materials should be addressed to christian.pomelli@unipi.it.This error has now been corrected in the HTML and PDF versions of the Article."} +{"text": "Nature Communications 10.1038/s41467-020-15961-y, published online 1 May 2020.Correction to: a was duplicated in panel b. The correct version of Fig. 3 is:The original version of this article contained an error in Fig. 3, in which panel which replaces the previous incorrect version:This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Correction to: J Exp Clin Cancer Res 36, 115 (2017)https://doi.org/10.1186/s13046-017-0585-2Following publication of the original article , the autIn Fig. In Fig. In Fig. The corrected figures are given below. The corrections do not have any effect on the final conclusions of the paper."} +{"text": "Nature Communications 10.1038/s41467-018-05151-2, published online 20 July 2018.Correction to: The original version of this Article omitted from the author list the third author Alexander Kazakov who is from the \u2018Edmond and Lily Safra Center for Brain Sciences, The Hebrew University of Jerusalem, Israel\u2019. The following was added to the Author contributions: \u2018A.K. established the freely-behaving worm tracking system\u2019. This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Communications Biology 10.1038/s42003-020-01332-2, published online 23 October 2020.Correction to: In the original published version of the article, the labels to Fig. 1e and Fig. 1f were corrupted. The errors have been corrected in the HTML and PDF versions of the article."} +{"text": "Obesity and diabetes mellitus are known to lead to the development of metabolic syndrome and non-alcoholic fatty liver disease (NAFLD). The mechanisms of programmed cell death are actively involved in maintaining cellular homeostasis along development of NAFLD. Proteins of the BCL-2 family are key regulators of physiological and pathological apoptosis. Homozygous males of BKS.Cg-Dock7mLeprdb/+/+/J mice (db/db mice) are characterized by progressive obesity and the development of type 2 diabetes mellitus (DM2) with severe hyperglycemia at 4\u20138 weeks and organ lesions at 8\u201310 weeks of age. The aim of this research was to study the expression of molecular cell regulators of apoptosis in liver cells of db/db mice males at different stages of obesity and diabetes development (at the age of 10 and 18 weeks). Immunohistochemical analysis (using the indirect avidin-biotin peroxidase method) and morphometric evaluation of the expression of the antiapoptotic protein Bcl-2 and the proapoptotic protein Bad in liver cells of studied animals at different stages of obesity and DM2 were carried out. An excess of the value of the Bcl-2 protein staining area over the Bad protein staining area was revealed in the liver of 10-week-old animals. The Bcl-2/Bad expression area ratio in 10-week-old animals was twice as high as in 18-week-old animals, which indicates the presence of conditions for blocking apoptosis in the liver of younger db/ db mice. At the 18th week of life, db/db mice displayed an almost threefold increase in the expression area of the Bad protein against the background of an unchanged expression of the Bcl-2 protein. The decrease in the Bcl-2/Bad staining area ratio in 18-week-old animals was due to the increase in the Bad expression area, which indicates the absence of antiapoptotic cell protection and creates conditions for activation of the mitochondrial pathway of apoptosis in the liver of male db/db mice with pronounced signs of obesity and DM2. Mechanisms of programmed cell death are actively involvedin maintaining cell homeostasis in the development of nonalcoholicfatty liver disease (NAFLD) . Obesity and related metabolic disorders, includinglipid accumulation in the liver and inflammation, play animportant role in liver carcinogenesis. Recent data indicatethat obesity and diabetes lead to the development of metabolicsyndrome and NAFLD, which can progress in patients withthis disease to non-alcoholic steatohepatitis, which includesthe risk of cirrhosis and hepatocellular carcinoma . Proteins of the BCL-2 family are key regulatorsof physiological and pathological apoptosis. According tothe modern model of apoptosis regulation, the ratio of theapoptosis regulator proteins Bcl-2, Bad and Bax determinesthe sensitivity of cells to the effects of apoptotic factors andis a \u201cmolecular switch\u201d that determines whether tissue growthor atrophy will occur . Molecular features ofthe development of the mitochondrial pathway of apoptosisin the liver of male db/db mice in postnatal ontogenesis atdifferent development stages of obesity and type 2 diabetesmellitus (DM2) have not yet been studied.The aim of this research \u2013 to study the expression of apoptosismolecular cell regulators from the BCL-2 family proteins:the antiapoptotic protein Bcl-2 and the proapoptoticprotein Bad in liver cells of male db/db mice at differentstages of obesity and DM2 development (at the age of 10 and18 weeks).The experiments were carried out in the SPF Vivarium of theInstitute of Cytology and Genetics, SB RAS, on homozygousmales of BKS.Cg-Dock7m+/+Lepr db/J mice (db/db mice). Homozygousindividuals of this strain have a defect of the leptinreceptor (spontaneous mutation Lepr db) and are characterizedby polyphagia, progressive obesity from 3\u20134 weeks of life,severe hyperglycemia from 4\u20138 weeks of life, the developmentof organ lesions after 8\u201310 weeks. Animals were stored in aroom with a regular light cycle (14 h light/10 h darkness), aconstant room temperature of 24 \u00b1 2 \u00b0C and a relative humidityof 45 \u00b1 10 %. The mice were kept on a standard food and water ad libitum.Studies were conducted on mice aged 10 (n = 7) and 18(n = 7) weeks, which is comparable to 10 and 18 years ofman age, respectively . Animals weresacrificed by cranio-cervical dislocation and liver sampleswere taken for light-optical and immunehistochemical studies.All experiments were performed in compliance with theprinciples of humanity and carried out in accordance with the \u201cRules for the Use of Experimental Animals\u201d and the European Unity Directive (86/609/EEC). Thestudy was approved by the local ethics committee (ProtocolNo. 128 of 15 March 2017).The liver pieces were fixed in 10 % buffered formalin for 48 h, dehydrated in a series of alcoholsof increasing concentration and enclosed in histomix (BioVitrum).The organ slices 3 \u03bcm in thickness were obtainedusing a LEICA RM2155 microtome (Germany) Liver preparationswere stained with Mayer hematoxylin and eosin forlight-optical examination.An immunohistochemical study of the Bcl-2 and Badprotein expression was performed on liver paraffin sectionsusing the indirect avidin-biotin-peroxidase method using theVectaStain Universal Elite ABC Kit . At the last stage, immunohistochemicalstaining was carried out in a chromogenic substratecontaining diaminobenzidine . Some sectionswere stained with Mayer hematoxilin, washed with distilledwater and, after dehydration, mounted under the cover glass.To quantify the expression of Bcl-2 and Bad in the mouseliver, a computer-assisted morphometric analysis of digitalphotographs obtained using a LEICA DM 2500 microscopewith a LEICA DFC425C video camera (Germany) at \u00d7400magnification was performed. Using the Image J softwareprogram, the average area of the staining zones on Bcl-2 andBad was determined on digital images. The ratio of Bcl-2expression area to Bad expression area was calculated.Statistical processing of research results was carried outusing Statistica 6.1 . Toanalyze the data obeying the normal distribution (the averagestaining area of Bcl-2 and Bad proteins), the arithmetic meanand standard error of the arithmetic mean were calculated; thesignificance of differences between the studied groups wasestablished using Student\u2019s t-test. The significance of datadifferences other than the normal distribution (the ratio ofBcl-2 expression area to Bad expression area) was determinedusing the nonparametric Mann\u2013Whitney test. Differencesbetween the values compared were considered statisticallysignificant at p < 0.05.In the liver of the male mice studied at the age of 10 weeks,stagnations in the interlobular veins, dilatation of lymphaticvessels and bile ducts were detected. Signs of protein dystrophyand lipid accumulations, mainly of small droplet nature, were found in some hepatocytes and in groups of parenchymalcells located mainly in the intermediate zones of the hepaticlobules.Immunohistochemically, a weak B\u0430d-positive signal wasidentified in individual hepatocytes and in the heterogeneouspopulation of sinusoidal cells of liver blood capillaries involved in the formation of the blood-lymph barrierin the liver, including endotheliocytes, Kupffer cells, Itocells and Pit cells . At the same time,pronounced immunohistochemical staining was also observedin liver cells for the antiapoptotic protein Bcl-2. In the hepaticlobules, the marker studied was accumulated mainly in theendothelial cells of the lining of blood sinusoidal capillariesand in single hepatocytes .Quantitative evaluation of the expression of the antiapoptoticprotein Bcl-2 and the proapoptotic protein Bad showedan excess of the immunohistochemical staining area for theBcl-2 protein over the value of this parameter for the Badprotein in the liver of db/db mice males aged 10 weeks .In the liver of male db/db mice at the age of 18 weeks, signsof nonalcoholic fatty liver disease (NAFLD) developmentwere more pronounced than in animals aged 10 weeks. Diffuseaccumulation of medium-sized and large lipid dropletswas found in parenchymal cells of all hepatic lobule zones.It developed against the background of disturbances in microcirculation,intraorgan bile transport, a significant dilatation ofblood and lymph vessels in the triad system and central veins.The study of the expression of apoptosis molecular-cellregulators of BCL-2 family proteins in the liver of male db/ dbmice at the age of 18 weeks revealed a pronounced immunohistochemicalstaining for the proapoptotic protein Bad ofendothelial cells of blood sinusoidal capillaries. A strong Badpositive signal was detected in hepatocytes located mainly inperiportal zones and around central veins as well asin the ductal epithelium of triad bile ducts. At the same time,weak immunohistochemical staining for the antiapoptoticprotein Bcl-2 was detected in cells of the blood-lymph barrierin the liver and in single hepatocytes of \u0435\u0440\u0443 animals studiedat the age of 18 weeks .Morphometric analysis of the liver of 18-week-old animalsshowed an increase in the Bad protein expression area, comparedto 10-week-old mice. At the same time, the stainingBcl-2 protein area did not change in comparison with theanimals at the age of 10 weeks .Evaluation of the ratio of Bcl-2/Bad expression areasrevealed a significant decrease in this index in 18-week-olddb/ db mice compared to 10-week-old animals , due toan increase, mainly, in the Bad expression area in the animalsaged 18 weeks. Data obtained indicate the absence of antiapoptoticcell protection of organ cells, which creates conditionsfor activation of the mitochondrial pathway of apoptosis inliver cells of the db/db mice at the age of 18 weeks.It is known that the development of programmed cell death isinfluenced by posttranslational modifications of BCL-2 familyproteins. One of the ways to regulate the activity of apoptosisinducingproteins is the phosphorylation/dephosphorylationprocess, which affects their ability to form heterodimers withother members of the BCL-2 family proteins. According tocurrent data, the induction of the antiapoptotic protein Bcl-2expression causes the closure of mitochondrial membranechannels and prevents the release of the protease AIF (apoptosisinducing factor) and cytochrome C, thereby protectingthe cell from apoptosis. At the same time, Bcl-2 blocks lipidperoxidation reactions in cell membranes, protecting cellsfrom damage by free radicals and thus preventing the developmentof apoptosis . We had previously found that db/db mice were already obese by10 weeks of age and had severe hyperglycemia with plasmaglucose levels of 506 mg/dL (28.1 mmol/L) and higher. Therewere no significant differences in glucose, triglyceride, totalcholesterol, ALT, or GGT levels in the db/db mice aged 10 and18 weeks . At the same time, as wasfound in this study, the expression area of the antiapoptoticprotein Bcl-2 exceeded the value of the imunohistochemicalstaining area of the proapoptotic protein Bad in the liver of the10-week-old animals. Results obtained indicate the presenceof antiapoptotic protection of liver cells at this stage of theNAFLD development.We have previously identified ultrastructural disordersof the energy and protein-synthesis apparatus in liver cells,carbohydrate and fat metabolism disturbances in the liverof 18-week-old male db/db mice with DM2, which leads tothe development of protein and fat dystrophy in hepatocytes.Disorders of blood circulation and lymph flow in the db/ dbmouse liver lead to a disruption in the morphological organizationof the blood-lymph barrier in the liver, and causea decrease in LYVE-1 receptor expression on endothelialsinusoid cell membranes. Such morphological rearrangementscontribute to the development of tissue hypoxia, oxidativestress and mitochondria damage, which are the inducers ofcell death . Underthese conditions, the mitochondrial pathway of cell apoptosisis launched using the BCL-2 protein family. When the outermembrane of mitochondria is disturbed, a thermolabile factoris also released from the intermembrane space, catalyzingreactions with O2 and leading to the development of oxidativestress. In this case, reactive oxygen species (ROS) areformed that destroy mitochondria and are powerful inducersof apoptosis .The development of microvesicular steatosis is also consideredto be a consequence of severe mitochondrial dysfunction. The same mitochondrialdisorders are thought to be a common cause of small-bubblesteatosis and apoptosis development in obese mice .In this study, we identified the greatest changes in the endothelialcells of the liver blood sinusoidal capillaries. We arejust beginning to understand the complexity of the endothelialcell functions. It is now proven that these cells control liverregeneration as \u201ca spatiotemporal rheostat\u201d. Dynamicallyregulating the angiopoietin-2 expression, they coordinate theirown regeneration and proliferation of hepatocytes, support therestoration of connective tissue, and control the maturationand resting state of blood vessels . The endotheliumacts as the first line of defense against invasion bypathogenic microorganisms, and also regulates vascular toneand permeability. Since damaged endotheliocytes can separatefrom their basement membrane and circulate freely in theblood, the possibility of detecting endothelial apoptosis in vivowas discussed. The degree of development of vascular injuriesdirectly correlates with organ trauma in critically ill patients. The pronounced immunohistochemicalstaining revealed by us in the liver of the 18-week-old maledb/db mice for the proapoptotic protein Bad of endothelialcells of the blood sinusoid capillaries with a low level of the antiapoptotic protein Bcl-2 expression in them indicates thedevelopment of a mitochondrial pathway of apoptosis in thecells of the blood-lymph barrier in the liver under NAFLD.Since apoptosis is triggered through the inactivation ofBcl-2 upon its binding to the Bad protein, the increase in theproapoptotic Bad staining area established by us indicatesthe absence of antiapoptotic protection and the apoptosisdevelopment along the mitochondrial pathway in liver cells.This is also confirmed by a decrease in the Bcl-2/Bad liverexpression area ratio in the male db/db mice at the 18th weekof life.Immunohistochemical analysis and morphometric evaluationof the expression of apoptosis molecular-cell regulatorsof BCL-2 family proteins \u2013 the antiapoptotic protein Bcl-2and the proapoptotic protein Bad \u2013 in the liver cells of maledb/ db mice were carried out at different stages of obesity andtype 2 diabetes mellitus development. An excess of the valueof the Bcl-2 protein staining area over the Bad protein stainingarea was revealed in the liver of 10-week-old animals.The Bcl-2/ Bad expression area ratio was twice as high in the10-week-old animals as in the 18-week-old animals, indicatingthe presence of conditions for blocking apoptosis in the liverof younger mice. At the 18th week of life, mice displayed analmost threefold increase in the expression area of the Badprotein against the background of an unchanged expressionof the Bcl-2 protein. The decrease in the ratio of Bcl-2/Badstaining areas in the 18-week-old animals was due to the increasein the Bad expression area. The obtained results indicatethe absence of antiapoptotic cell protection and the creationof conditions for activation of the mitochondrial pathway ofapoptosis in the liver of the male db/db mice with pronouncedsigns of obesity and DM2.The authors declare no conflict of interest.Begriche K., Massart J., Robin M.A., Borgne-Sanchez A., FromentyB. 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DOI 10.1111/jgh.12212.Shimizu M., Yasuda Y., Sakai H., Kubota M., Terakura D., Baba A.,Ohno T., Kochi T., Tsurumi H., Tanaka T., Moriwaki H. Pitavastatinsuppresses diethylnitrosamine-induced liver preneoplasms in maleC57BL/KsJ-db/db obese mice. BMC Cancer. 2011;11:281. DOI10.1186/1471-2407-11-281.Sun D., Li S., Wu H., Zhang M., Zhang X., Wei L., Qin X., Gao E.Oncostatin M (OSM) protects against cardiac ischaemia/reperfusioninjury in diabetic mice by regulating apoptosis, mitochondrial biogenesisand insulin sensitivity. J. Cell. Mol. Med. 2015;19(6):1296-1307. DOI 10.1111/jcmm.12501.Trak-Smayra V., Paradis V., Massart J., Nasser S., Jebara V., FromentyB. Pathology of the liver in obese and diabetic ob/ob and db/dbmice fed a standard or high-calorie diet. Int. J. Exp. Pathol. 2011;92(6):413-421. DOI 10.1111/j.1365-2613.2011.00793.x."} +{"text": "Nature Microbiology 10.1038/s41564-020-00817-4, published online 4 January 2021.Correction to: In the version of this Brief Communication originally published, in Fig. 1e, the cytometry data were mistakenly omitted from all three graphs. This error has now been corrected and the updated figure is available online."} +{"text": "The correct version of the figure appears below. Additionally, the funding information for this Article was not acknowledged. The Acknowledgements should have included:This work was supported by the Centre National de la Recherche Scientifique (CNRS), a grant from the Agence Nationale de la Recherche (ANR) to T.L. (ANR-14-CE11-0006-01) and by support from the Association pour la Recherche sur le Cancer (ARC) (grant 7801 and SFI20121205586) to T.L., E.H. and F.L. were supported by grants from the Minist\u00e8re de la Recherche et de l'Enseignement Sup\u00e9rieur and by a 4th year of PhD fellowship from the ARC.Figure 4"} +{"text": "Nature Communications 10.1038/s41467-020-16429-9, published online 21 May 2020.Correction to: The original version of this Article contained an error in the spelling of the author Suman R. Das, which was incorrectly given as Suman Das. This has now been corrected in both the PDF and HTML versions of the Article."} +{"text": "Allotraeus asiaticus Schwarzer and Callidiellum villosulum Fairmaire are repeatedly intercepted in wood and wood products all over the world. As two common stem borers of Cunninghamia lanceolata (Lambert) Hooker, to further understanding of the differences in their living habits, behaviors and the mechanism of insect-host chemical communication, we observed the external morphology, number and distribution of antennal sensilla of A. asiaticus and C. villosulum with scanning electron microscopy (SEM), respectively. The results showed that 1st-5th subsegments of the flagellum are spined endoapically in A. asiaticus which is different from the previous report (1st-3rd of the flagellomere). Meanwhile, there were five subsegments on the flagellum of C. villosulum that were clearly specialized as serrated shapes on the 4th-8th flagellomeres. Four types (ten subtypes) of sensilla were both found on the antennae of these two fir longhorn beetles, named B\u00f6hm bristle (Bb), sensilla trichodea (ST I and II), sensilla basiconica , sensilla chaetica . There is one additional kind of morphological type of sensilla found on the antennae of C. villosulum compared to A. asiaticus which was related to their habit of laying eggs only on dry and injured fir branches, named sensilla campaniformia (SCa). These differences may vary according to their own biological habits. For research purposes, the observed difference in the sensillum distribution and function between the two fir longhorn beetles will greatly facilitate the design of better semiochemical control methods of these insect pests. Cunninghamia lanceolata (Lambert) Hooker, Chinese fir, is one of the most commonly planted species in cultivated fast-growing timber forests of East and Southeast Asia, such as China, Japan, Laos, Vietnam, and neighboring countries, is popularly used for home and gardens due to its soft but durable wood that is easily workable [Allotraeus asiaticus Schwarzer and Callidiellum villosulum Fairmaire are two reported pest species of China fir (C. lanceolata) [A. asiaticus [C. villosulum is only found in East Asia, but is occasionally intercepted in wood and wood products in US, Malta and Japan [workable . Therefoceolata) ,3. Indigsiaticus . On the nd Japan ,5,6. To As phytophagous insects, Cerambycidae are economically important pests of street trees and forests , with moInsect antennae have sensory receptors called sensilla. Several morphological structures are adapted to the primary function of insect antennae, such as omnidirectional movements and sensing adequate area. Antennae shaking during walking, the antennal sensilla can facilitate the insect to recognize different stimuli in the environment and are specialized for taste, olfaction, hygroreception, thermoreception or mechanoreception . BecauseA. asiaticus and C. villosulum and between the sexes of each species via scanning electron microscopy (SEM) techniques. To our knowledge, there have been no studies on the description of the antennal sensilla of these two species using SEM techniques. The two species of beetle both belong to family Cerambycidae, and the main flight season is from March to May [The purpose of this study was to compare antennal morphology and sensilla ultrastructure between h to May ,22. ThusA. asiaticus and C. villosulum . The antennae were stored in a 75% alcohol solution until examination. All animal experiments were approved by the Institutional Animal Care and Use Committee of Guangxi University and animal care and use protocol are based upon the National Institutes of Health (NIH), USA.The adults of llosulum were capThe antennae were cleaned three times by distilled water in an ultrasonic bath JP-010T at 250 W for 360 seconds each, and were then fixed separately in 2.5% glutaraldehyde at 4\u00b0C for 12 h. The antennae were dehydrated through an ascending ethanol series of 75%, 80%, 85%, 90%, 95% and 100% at 10 min intervals. The prepared antennae were stored in a cleaned and dried glass petri dishes container that was air-dried for 12 h. After drying, the specimens were mounted on a holder using double-sided sticky tape and sputter coated with gold-palladium. Samples were put into a holder with double-sided adhesive tape . Subsequently, the prepared samples were scanned by the electron microscope at an accelerating voltage of 5\u201310 kV. Images were named respectively and stored on a computer.A. asiaticus and C. villosulum were also drawn by Adobe Photoshop Version CS6. The quantity and distribution of each type of sensillum was analyzed between the antennae of both sexes of the two beetles. One-way ANOVA was applied to determine possible differences in antennal sensilla between the sexes of each species and the qualities of spine between different flagellomeres of the same sex of A. asiaticus, by using SPSS statistical software package version 25.0 . In all, individual samples per type were subjected to a quantitative analysis in both side of antenna. A t-test or one-way ANOVA was applied to determine possible differentiation of the number, length, and width of antennal sensilla, and values were reported as mean \u00b1 SE (standard error). The significance level was set at 0.05. All graphs were made by SPSS 25.0 and GraphPad prism 6.01 .Identification and classification of the sensilla types and the terminology used in this work was based on studies of Schneider and ZachA. asiaticus and C. villosulum in the shape of the flagellum. The nine flagellomeres of A. asiaticus are all columnar , pedicel (Pd) and flagellum (nine flagellomere). There is a great difference between nar Figs , but theic prism . Besidesth sexes . In C. vxes Figs . And thead. Figs and 3B.A. asiaticus, there are no significant difference between males and females in the length of antennae (p = 0.00) and 3rd , 4th , 7th , 9th subsegments of the flagellum (In antennae . In termlagellum .C. villosulum (11.25 \u00b1 0.27 mm) were distinct significantly longer than female (p = 0.00), pedicel and 3rd of flagellum (p = 0.04) and 9th subsegments of the flagella.However, the antennae of male = 0.00) . Meanwhilagellum . On the A. asiaticus were greater in antennal length than C. villosulum both in female and male shown wider than female C. villosulum in 4th flagella , 7th , 8th , and 9th subsegment of the flagella . In termflagella . But it flagella .A. asiaticus and C. villosulum: B\u00f6hm bristle (Bb), sensilla trichodea , sensilla basiconica , sensilla chaetica . One more morphological type of sensilla was found on the antennae of C. villosulum compared to A. asiaticus, which named sensilla campaniformia (SCa). There was no clear sexual dimorphism in the species and distribution of the antennal sensilla between different sexes of two fir longhorn beetles, respectively. However, there was significant difference in the number of the antennal sensilla between different sexes and species were widely distributed just on the ventral sides all over the antennae except the last segements of flagella . Moreovellosulum .C. villosulum and male (7th-9th subsegments of the flagella) on the ventral sides of C. villosulum. Over all, the length of this sensilla type in males was longer than females in both A. asiaticus and C. villosulum (Sensilla chaetica type II (SCh II) were the largest number of sensilum which looked slightly curved. It had a tight sockets, grooved wall and gradually tapers into a slender tip . This tyllosulum . SCh II llosulum .A. asiaticus and C. villosulum , but there was a big difference between the venter and dorsal sides of its number both in two beetles (A. asiaticus and C. villosulum (Sensilla chaetica type III (SCh III) were visible at the junction of each segment of the flagellomeres . This ki beetles ; Fig 5. 3.28 \u03bcm) . Meanwhillosulum .C. villosulum in both sexes and female (3rd-5th subsegments of the flagella) A. asiaticus than A. asiaticus were distributed from Sc to 6th flagella in both dorsal and ventral side of th sexes ; Fig 5 wth sexes . Meanwhisiaticus ; Fig 5. A. asiaticusin both sexes and width (3.46 \u00b1 0.11 \u03bcm) were observed on the base of the scape and pedicel, between the Head-Scape and Scape-Pedicel, respectively. However, no Bb were found on the ventral side of pedicel in th sexes ; Fig 7. th sexes . In fema0.11 \u03bcm) . The numectively .A. asiaticus and C. villosulum. It had hair-like protruding receptors with grooves and perforated surfaces. The sensillum could been further classified into two subtypes based on their morphological and ultramicro-structural characteristics: ST I, II. The number of ST varies in the dorsal and ventral side of the antennae of different segments of both sexes of A. asiaticus and C. villosulum, respectively and C. villosulum (Sc-3rd flagella) (C. villosulum (417.80 \u00b1 19.44 \u03bcm) than in both sexes of two beetles (C. villosulum were the shortest (321.32 \u00b1 15.72 \u03bcm) (Sensilla trichodea type I (ST I) was present only on the dorsal side of the antennae of lagella) ; Fig 7. lagella) . ST I wa beetles . In cont5.72 \u03bcm) .A. asiaticus and C. villosulum. Where as, it shown differences between A. asiaticus (Sc-9th flagellomere) and C. villosulum (Sc-3rd flagellomere) on the ventral side. In total number, male A. asiaticus exhibited large quantities in 1st-3rd subsegments of the flagella while females had high abundance distributed in the 7th-9th subsegments of the flagella were visible at the junction of flagellomeres which waflagella . On the C. villosulum which were distributed from 1st-9th subsegments of the flagella on the dorsal side were more pronounced than males (Sensilla campaniformia (SCa) were only found on the antennae of sal side . Howeversal side . It is asal side . This se0.12 \u03bcm) .A. asiaticus, sensilla basiconic scattered over 1st-9th subsegments of the flagella on the dorsal side while it distributed in 6th-9th subsegments on ventral side and female (7th-9th flagella). On the other hand, it could be characterized as a cone-shaped prominence with a pedestal shape or a conically uplifted base. In the center of the base, there are small cone-shaped receptors with different shapes, which has chemical sensory functions such as smell perception and taste perception. The basiconic sensilla could be further classified into three types based on their surface micro-morphology: Sensilla SB I, II and III.Sensilla basiconic were interspersed on the flagellomeres . The disral side . MeanwhiC. villosulum was longer than A. asiaticus both without sexual differences were cone-shaped and straight with a slightly pointed tip and a smferences . In term overall .A. asiaticus (11.47 \u00b1 0.38 \u03bcm) was longer than female . However, there was no sexual dimorphism in C. villosulum were significantly different compared to male on the 9th flagellomere (Sensilla basiconic type II (SB II) has no basic sockets and gradually tapers into the blunt tip with a smooth surface and only distributed on the flagellum . The len0.27 \u03bcm) . Female ellomere .C. villosulum than A. asiaticus both without sexual differences had a smooth surface and ridgy socket with a slender tip in llosulum . Howeversiaticus . In addiferences . In termo female . But in th sides .A. asiaticus and C. villosulum. From this study, we found several significant differences between the two fir longhorn beetles in the antennal shape. The five flagellomeres of C. villosulum were specialized as serrated shapes in 4th-8th subsegments of the flagella with abundant olfactory sensillum. Those characters can greatly increase the effective sensing area of the antennae, enhancing the inductive force [A. asiaticus in 1st-6th subsegments of the flagella were pentagonal , ST , SB . Although they have the same subtypes of olfactory sensilla, there are some differences in quantity, length and width between the sensillum with the results of this study, will provide valuable insights into possible functions of each sensillum. These results provide the necessary background information for future studies on the chemical ecology of these economically important longhorn beetle species. The observed difference in the sensilla distribution and function will greatly facilitate the design of better semiochemical control methods, for example more effective lures for survey and detection, for these pest insects.This study aims to identify and characterize the external morphology and distribution of antennal sensillum types of C. villosulum are sexual dimorphism, but A. asiaticus is opposite. Coincidentally, the conclusions in our past field experiment can reasonably prove this result. Moreover, there\u2019s a big difference between A. asiaticus and C. villosulum on antennae in terms of interspecies. First of all, the morphological characteristics of their antennae are quite different which related to their specific and unknown living habits and behaviors. This needs further study. Secondly, common temperature and humidity sensing sensilla were spread all over flagellomeres in C. villosulum which was related to the habit of laying eggs only on dry and injured fir branches. Last, the number of sensillum in the same types varied greatly among species. The result can well explain that the two longicorn beetles responded to different pheromones in our field experiment. These results merit further study with a particular focus on the chemosensory characteristics, which will greatly facilitate the design of better semiochemical control methods for these two fir longhorn beetles.In this research, we found that the antennae of S1 File(XLSX)Click here for additional data file. 26 Aug 2020PONE-D-20-25310First description and comparison of the morphological and ultramicro characteristics of the antennal sensilla of two fir longhorn beetlesPLOS ONEDear Dr. Lu,Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE\u2019s publication criteria as it currently stands. 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The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. \"We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:\"This work was also supported financially by degree construction - funds for doctoral study abroad of Guangxi University in 2019.\"https://www.youtube.com/watch?v=_xcclfuvtxQ5. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to \u2018Update my Information\u2019 (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1:\u00a0The study proposed by Done et. al is based on antennal morphology analyses of A. asiaticus and C. villosulum using scanning electron microscopy based approach. The study described in this manuscript was conducted in China and it reports the first comprehensive list of antennal sensilla of two fir longhorn beetles, important pests in this region of the world. The overall objective of the study was accomplished as stated. The new information reported in the study builds upon what is already known about the antennal sensilla of other insects. The comparative analyses also validate some of the previously published information on morphological and ultramicro of antennal sensilla in other insect pests. Information from this study, can provide a better understanding of the molecular basis of olfaction and other chemical cues that regulate behavior in A. asiaticus and C. villosulum. This in turn, can provide a basis for developing alternative and better methods of controlling this pest.This study is the first of its type on A. asiaticus and C. villosulum and it provides useful information on the comparative morphological and ultramicro analyses on the economically important insect pests. I believe it will contribute to the body of knowledge that is available on A. asiaticus and C. villosulum. In this regard I will recommend the manuscript for acceptance for publication subject to the comments addressed here.1. As a whole\uff0cthis article is tedious and repeated, such as the abstract, which should be compressed to more describe morphological and ultramicro characteristics.2. There are many logical problems in this paper, such as \u201cthe study on the comparison between\u2026\u201d (lines 316-319), \u201cduring field trials, C. villosulum shown\u2026\u201d (lines 368-371) and \u201cthis is related to the identification of\u2026\u201d (lines 378-383), move logically from one idea to the nextlogically from one idea to the next and don't skip steps, revise it again.3. Overall, the authors paid attention to details in writing of the manuscript.Reviewer #2:\u00a0In this study, the authors compared the antennal morphology and sensilla ultrastructure between A. asiaticus and C. villosulum and between the sexes of each species via scanning electron microscopy (SEM) techniques. The findings of the current study can contribute to a better understanding of the differences in their living habits and behaviors. Meanwhile, this descriptive work will also provide the theoretical basis for future work on pheromone identification and development of prevention and control techniques for these two pests. Overall, this study is interesting, the methods used are standard, the manuscript is well written scientifically and the data is sufficient to support its main conclusion. However, I have a few comments for improvement in my view.Some suggestions:Lines 15-17: Please rewrite this sentence more clearly.Lines 21-22: I think no need of this sentence.Lines 26-29: The given reference focus Chinese firs only in Fujian Province, China. Please provide references for their distribution in other countries or revised the statement which focus only China.Lines 31: Replace indigenous by Indigenous..Lines 43-45: Please provide references for these sentences.Lines 108: Please use full scientific name at the start of a sentence. Check whole MS and correct it.Lines 110-114: The use of three adversative conjunction \u2018Interestingly\u2019, \u2018Meanwhile\u2019 and \u2018Nevertheless\u2019 led to a logic miss. Please consider to rewrite these three sentences. Also, please check whole MS regarding this kind of mistakes.Lines 131-132: P should be italic. Correct this in whole MS.Lines 142-143: A. asiaticus and C. villosulum, should be italic.Lines 158: Remove \u201ca large\u201d from sentence.Lines 159: Replace sex by \u201csexes\u201d.Lines 177; 297-299; 378-379: The contractions in English should be avoided in scientific article. Please remove \"What's more\" and revise the sentence accordingly.Lines 302: Replace significant by \u201csignificantly\u201dLines 314-315: Please add references.Lines 368-369: Replace \u201cOne past study\u201d by \u201cPrevious study\u201d. Also, please provide references.Lines 401-404: Please remove \u201cGenerally speaking\u201d and revise the sentence accordingly.Lines 412-430: The conclusion section is too large, please reduce the text and conclude concisely. 13 Sep 2020Dear Academic Editor,We really appreciate your earnest and careful review of our manuscript ID PONE-D-20-25310 entitled \u201cFirst description and comparison of the morphological and ultramicro characteristics of the antennal sensilla of two fir longhorn beetles\u201d. We also thank you very much for giving us an opportunity to consider again our revised paper.Constructive comments from the reviewer was appreciated and carefully considered with a complete revision.The response to manuscript revision instructions and reviewer\u2019s comments point by point as listed below. Revised contents are showed by red fonts in the revised version.Sincerely,Zishu Dong30 August, 2020-------------------------------------------------------------------------------------------------------Response to Manuscript revision instruction:Response: Thank you for your suggestion. In the revised version, revised contents is mainly shown in the following aspects:(1)Revised contents are showed by red fonts in the revised version.(2)We have checked carefully that our manuscript meets PLOS ONE's style requirements.(3)We\u2019ve integrated title page and main body together to make sure that the title page within our main document.(4)Sorry for we forgot to provide a statement in our methods section that the works are feasibly. And we have added a statement in the methods section of manuscript and in the \u201cEthics Statement\u201d field of the submission form. (5)For the acknowledgments section, we have revised according to your suggestion which avoided the appearance of any funding-related text in this manuscript. And we make sure that the funding information doesn\u2019t appear in the Acknowledgments section or other areas of our manuscript. Besides, we have provided funding information that is currently declared in our Funding Statement. Moreover, we include the updated Funding Statement in your cover letter. Our final Funding Statement is \u201cThis research was funded by National Natural Science Foundation of China (31660626). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript\u201d. We have deleted the study abroad fund program of Guangxi University in 2019. The cover letter also clearly states that the project only supported by National Natural Science Foundation of China (31660626). What's more, we also make a selection mark in the selection box of Current Funding Sources List of funding imformation.(6)Corresponding author have completed the authorization process of ORCID iD.-------------------------------------------------------------------------------------------------------Response to reviewer 1:Comments 1: \u201cAs a whole\uff0cthis article is tedious and repeated, such as the abstract, which should be compressed to more describe morphological and ultramicro characteristics\u201d.Response: Thanks for your suggestion. In the revised version, we rephrased our abstract and conclusions according to your comments.Comments 2: \u201cThere are many logical problems in this paper, such as \u201cthe study on the comparison between\u2026\u201d (lines 316-319), \u201cduring field trials, C. villosulum shown\u2026\u201d (lines 368-371) and \u201cthis is related to the identification of\u2026\u201d (lines 378-383), move logically from one idea to the nextlogically from one idea to the next and don't skip steps, revise it again\u201d.Response: Thank you for your meaningful suggestions. After careful verification and cautious thinking, we revised the sentences respectively :(1)At present, the study of antennae ultrastructure of longicorn beetles is focused on the comparison between male and female adults of each species. However, the interspecific comparison of antennae ultrastructure of different longicorn beetles with the same host plants is very rare . (2)The results of field trials show that C. villosulum shown specific attraction to the blend of 3-hydroxyhexan-2-one and the pyrrole, while A. asiaticus was only specifically attracted to the pyrrole as a single component . (3)This result suggests that these two sensilla very likely involved in the identification of sex pheromone components . Comments 3: \u201cOverall, the authors paid attention to details in writing of the manuscript\u201d.Response: Thanks for your recognition of our work. What\u2019s more, we are also particularly grateful to you for your careful review and appropriate evaluation of our article.-------------------------------------------------------------------------------------------------------Response to reviewer 2:Comments 1: \u201cLines 15-17: Please rewrite this sentence more clearly\u201d.Response: According to your suggestion. We rephrased the sentence into \u201c Four types (ten subtypes) of sensilla were both found on the antennae of these two fir longhorn beetles, named B\u00f6hm bristle (Bb), sensilla trichodea (ST I and II), sensilla basiconica , sensilla chaetica \u201d .Comments 2: \u201cLines 21-22: I think no need of this sentence\u201d.Response: We have deleted this sentence according to your comments .Comments 3: \u201cLines 26-29: The given reference focus Chinese firs only in Fujian Province, China. Please provide references for their distribution in other countries or revised the statement which focus only China\u201d.Response: Thank you for your suggestion. We have replaced a new reference .https://www.conifers.org/cu/Cunninghamia.phpEarle CJ. Cunninghamia. The Gymnosperm Database. 2020 May 20. Available from: Comments 4: \u201cLines 31: Replace indigenous by Indigenous\u201d.Response: We have revised it according to your suggestion .Comments 5: \u201cLines 43-45: Please provide references for these sentences\u201d.Response: Sorry for my inappropriate expression. This is our own statement without reference. So, we change the sentence into \u201c To avoid its proliferation in other regions of the world, it is necessary to systematically study the two beetles in order to plan preventive measures such as developing detection tools\u201d . Comments 6: \u201cLines 108: Please use full scientific name at the start of a sentence. Check whole MS and correct it\u201d.Response: Thank you for your reminder. We have checked whole MS, and just found the problem in this sentence. We changed this sentence into \u2018There is a great difference between A. asiaticus and C. villosulum in the shape of the flagellum\u2019 .Comments 7: \u201cLines 110-114: The use of three adversative conjunction \u2018Interestingly\u2019, \u2018Meanwhile\u2019 and \u2018Nevertheless\u2019 led to a logic miss. Please consider to rewrite these three sentences. Also, please check whole MS regarding this kind of mistakes\u201d.Response: This is a meanful suggestion. We have rephrased this sentence as your comments . After checked whole MS, we also rephrased some sentence with similar problem .Comments 8: \u201cLines 131-132: P should be italic. Correct this in whole MS\u201d.Response: We examined the whole MS carefully, and all p have been kept italic according to your comments .Comments 9: \u201cLines 142-143: A. asiaticus and C. villosulum, should be italic\u201d.Response: We examined the whole MS carefully, and all latin names have been kept italic according to your comments .Comments 10: \u201cLines 158: Remove \u2018a large\u2019 from sentence\u201d.Response: We have removed \u2018a large\u2019 from sentence according to your comments .Comments 11: \u201cLines 159: Replace sex by \u2018sexes\u2019 \u201d.Response: We have replaced \u2018sex\u2019 by \u2018sexes\u2019 according to your comments .Comments 12: \u201cLines 177; 297-299; 378-379: The contractions in English should be avoided in scientific article. Please remove \u2018What's more\u2019 and revise the sentence accordingly\u201d.Response: Thank you for your suggestion. We have rephrased this sentence according to your comments . And we also have removed \u2018What's more\u2019 and revise the sentence accordingly .Comments 13: \u201cLines 302: Replace significant by \u2018significantly\u2019 \u201d.Response: We have replaced \u2018significant\u2019 by \u2018significantly\u2019 according to your comments .Comments 14: \u201cLines 314-315: Please add references\u201d.Response: Thanks for your suggestion. We have added reference for this sentence .Comments 15: \u201cLines 368-369: Replace \u2018One past study\u2019 by \u2018Previous study\u2019. Also, please provide references\u201d.Response: Thanks for your suggestion. We have rephrased this sentence according to your comments Comments 16: \u201cLines 401-404: Please remove \u2018Generally speaking\u2019 and revise the sentence accordingly\u201d.Response: Thanks for your suggestion. We have rephrased this sentence according to your comments .Comments 17: \u201cLines 412-430: The conclusion section is too large, please reduce the text and conclude concisely\u201d.Response: We rephrased our conclusion according to your comments .AttachmentResponse to Reviewers.docxSubmitted filename: Click here for additional data file. 9 Oct 2020First description and comparison of the morphological and ultramicro characteristics of the antennal sensilla of two fir longhorn beetlesPONE-D-20-25310R1Dear Dr. Lu,We\u2019re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you\u2019ll receive an e-mail detailing the required amendments. When these have been addressed, you\u2019ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they\u2019ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact Kind regards,Yulin GaoAcademic EditorPLOS ONE 19 Oct 2020PONE-D-20-25310R1 First description and comparison of the morphological and ultramicro characteristics of the antennal sensilla of two fir longhorn beetles Dear Dr. Lu:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. If we can help with anything else, please email us at Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staffon behalf ofDr. Yulin Gao Academic EditorPLOS ONE"} +{"text": "Nature Communications 10.1038/s41467-021-21184-6, published online 11 February 2021.Correction to: 9.a had one linker misplaced and one missing. This has been corrected in both the PDF and HTML versions of the Article.The original version of this Article contained an error in Fig. 3a, in which the topological scheme representing the protein SBP1"} +{"text": "Scientific Reportshttps://doi.org/10.1038/s41598-020-66656-9, published online 17 June 2020Correction to: The title in the original version of this Article contained a typographical error of \u2018unresponsive\u2019. The error has now been corrected in the PDF and HTML versions of the Article."} +{"text": "Scientific Reportshttps://doi.org/10.1038/s41598-020-65634-5, published online 01 June 2020Correction to: A supplementary file containing Supplementary Video S1 was omitted from the original version of this Article. This has been corrected in the HTML version of the Article; the PDF version was correct at time of publication."} +{"text": "Scientific Reports 10.1038/s41598-018-28164-9, published online 14 September 2018Correction to: The original version of this Article contained errors in the figures. Figure\u00a01 was truncated so that the right edge was missing. Additionally, in Figure\u00a02, the graphs for UCM-MSCs and UCB-MSCs were presented as UCB-MSCs and UCM-MSCs respectively.These errors have now been fixed in the HTML and PDF versions of the Article."} +{"text": "Scientific Reports 10.1038/s41598-019-56237-w, published online 27 December 2019Correction to: In the original version of this Article, Bj\u00f6rn Hegner was incorrectly listed as the corresponding author. The correct corresponding author for this Article is Duska Dragun. Correspondence and request for materials should be addressed to duska.dragun@charite.de. This error has now been corrected in the HTML and PDF versions of the Article."} +{"text": "Scientific Reports 10.1038/s41598-019-53277-0, published online 14 November 2019Correction to: In the original version of this Article, Seung Woo Park was incorrectly listed as the corresponding author. The correct corresponding author for this Article is Sung-Ji Park. Correspondence and requests for materials should be addressed to tyche.park@gmail.com. This error has now been corrected in the HTML and PDF versions of the Article."} +{"text": "The Pharmacogenomics JournalCorrection to: 10.1038/s41397-019-0140-yThe original version of this article did not include Juan C. Celed\u00f3n, Erick Forno and Glorisa Canino in the list of authors and incorrectly cited Gerard H. Koppelman, Maria Pino-Yanes and Steve Turner as authors. This has now been corrected in both the PDF and HTML versions of the article."} +{"text": "Scientific Reports 10.1038/s41598-020-64572-6, published online 18 May 2020Correction to: The original version of this Article contained a typographical error in the spelling of the author M.E. Navarro Sanchez, which was incorrectly given as Navarro Sanchez.This has now been corrected in the PDF and HTML versions of the Article."} +{"text": "Communications Biology 10.1038/s42003-021-01648-7, published online 29 January 2021.Correction to: which replaces the previous incorrect version. The error has been corrected in the HTML and PDF versions of the Article.The original version of the Article contained an error in Fig.\u00a05d, in which a white rectangle was introduced, obscuring part of the data in the row marked 588. The correct version of Fig.\u00a05 is:"} +{"text": "Correction to: Journal of Exposure Science & Environmental Epidemiology10.1038/s41370-019-0172-zIn the original Article, Eric D. McCollum\u2019s name was incompletely stated (as Eric McCollum). This has been corrected in the HTML, PDF and XML versions of this article."} +{"text": "Nature Communications 10.1038/s41467-020-19702-z, published online 17 November 2020.Correction to: The original version of this Article contained an error in the spelling of the author Lisa C. Zaba, which was incorrectly given as Lisa Zaba. This has now been corrected in both the PDF and HTML versions of the Article."} +{"text": "Correction to: BMC Bioinformatics 21, 315 (2020)https://doi.org/10.1186/s12859-020-03613-3Following publication of the original article , it was The details of the supplement in which this article ought to have been published are given below:About this supplementhttps://bmcbioinformatics.biomedcentral.com/articles/supplements/volume-21-supplement-5.This article has been published as part of BMC Bioinformatics, Volume 21 Supplement 5, 2020: Proceedings of the 13th International Workshop on Data and Text Mining in Biomedical Informatics (DTMBIO 2019). The full contents of the supplement are available at The publisher apologises for any inconvenience caused."} +{"text": "Nature Methods 10.1038/s41592-019-0658-6, published online 28 November 2019.Correction to: In the version of this article initially published, the institution name was missing from author Dmytro Panchenko\u2019s affiliation. The full affiliation is Kharkiv National University of Radioelectronics, Kharkiv, Ukraine. The error has been corrected in the PDF and HTML versions of the article."} +{"text": "Correction to: Public Health Rev 41, 10 (2020)https://doi.org/10.1186/s40985-020-00126-5In the original publication of this article a large The full table is published in this correction article. The original publication has been updated.The publisher apologizes to the authors & readers for the inconvenience."} +{"text": "Nature Communications 10.1038/s41467-018-07701-0, published online 10 December 2018.Correction to: The original version of this Article contained an error in Fig. 1, in which the labels for Fig. 1b and Fig. 1c were incorrectly swapped. This has been corrected in the PDF and HTML versions of the Article."} +{"text": "Nature Communications 10.1038/s41467-019-13259-2, published online 25 November 2019.Correction to: The original version of this article omitted the following from the Acknowledgements:R.G. was supported by a Scholar in Clinical Research award from The Leukemia & Lymphoma Society.This has now been corrected in both the PDF and HTML versions of the article."} +{"text": "Nature Communications 10.1038/s41467-020-16000-6, published online 24 April 2020.Correction to: The original version of this Article contained an error in Fig. 2d, in which all the numbers appeared as bold instead of either normal or bold type setting to indicate statistical significance of FDR < 0.05. This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Nature Communications 10.1038/s41467-020-14298-w, published online 21 January 2020.Correction to: y-axis. This has been corrected in both the PDF and HTML versions of the article.The original version of this article contained an error in Fig.\u00a02a, in which additional tick marks were added to the"} +{"text": "Nature Communications 10.1038/s41467-020-20121-3, published online 17 December 2020.Correction to: The original version of this Article omitted the following from the Acknowledgements: Veterans Administration Grant BX003893.This has now been corrected in both the PDF and HTML versions of the Article."} +{"text": "Nature Communications 10.1038/s41467-020-14390-1, published online 24 January 2020Correction to: The original version of this Article contained an error in Fig. 7, in which panels s, t and u were described as being treated with Ha-pl-BG. They were treated with Ha-TMP. This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Nature Communications 10.1038/s41467-020-15434-2, published online 27 March 2020.Correction to: The original version of this article contained an error in Fig. 3d, in which the plot border and the x- and y-tick marks of Fig. 3d were missing.This error has now been corrected in both the PDF and the HTML versions of the Article."} +{"text": "Communications Biology 10.1038/s42003-020-01287-4, published online 9 October 2020.Correction to: In the original version of the published Article, Fig. 2 was mistakenly replaced by another copy of Fig. 3.This has now been corrected in the PDF and HTML versions of the Article."} +{"text": "Scientific Reports 10.1038/s41598-020-66487-8, published online 17 June 2020Correction to: The original version of this Article contained a typographical error in the spelling of the author Eric J. Marrotte, which was incorrectly given as Eric J. Marotte. This has now been corrected in the PDF and HTML versions of the Article."} +{"text": "Neuropsychopharmacology 10.1038/s41386-019-0381-0, published online 6 April 2019.Correction to: Shortly after publication, the Authors noticed that two of the equations in the Methods section contained errors. The original Article contained a typographic error in Model-free Q-learning models 1 and 3. The correct equations are as follows:Model-free Q-learning: model 1Model-free Q-learning: model 3The correct equations were used for the data analyses performed in the original Article. The analyses, results and conclusions of the paper were not affected by the typographic error. This has now been corrected in both the PDF and HTML versions of the Article."} +{"text": "Communications Biology 10.1038/s42003-021-01743-9, published online 12 February 2021.Correction to: n\u2009=\u200910; the correct sample sizes are n\u2009=\u200922 for each label, respectively: Late-Severe, Early-Severe, Late-Mild, and Early-Mild. The correct version of Fig.\u00a01 is which replaces the previous incorrect version. The error has been corrected in the HTML and PDF versions of the Article.The original version of the Article contained an error in Fig.\u00a01b, in which the sample sizes for the following labels were incorrectly given as"} +{"text": "Nature Communications 10.1038/s41467-020-16497-x, published online 2 June 2020.Correction to: The original version of the Supplementary Information associated with this Article contained an error in Supplementary Fig. 4b, in which both panels of Supplementary Fig. 4a were duplicated in error in Supplementary Fig. 4b. The correct version of Supplementary Fig. 4b is given with this paper, which replaces the previous incorrect version.The HTML has been updated to include a corrected version of the Supplementary Information."} +{"text": "Sci. Rep. 9, 1\u201314. doi: 10.1038/s41598-019-54316-6 as Leong et al., 2019. It should be Ieong et al., 2019 in all occurrences in the text.In the original article, the name of one of the authors was incorrectly spelled in the reference for Ieong, H. F. H., Gao, F., and Yuan, Z. (2019). Machine learning: assessing neurovascular signals in the prefrontal cortex with non-invasive bimodal electro-optical neuroimaging in opiate addiction. The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated."} +{"text": "Correction to: BMC Genomics (2020) 21:833https://doi.org/10.1186/s12864-020-07243-0Following publication of the original article , it was Furthermore, the layout of the online versions of Tables 1 and 2 have been updated to improve the presentation of the genotype comparisons.The correct Fig."} +{"text": "Communications Biology 10.1038/s42003-020-01136-4, published online 6 August 2020.Correction to: In the original published version of the Article, contributing author Adrienne C. Greene of the University of California, Berkeley was incorrectly listed as Adrianne C. Green of the National Cancer Centre Singapore. The error has been corrected in the HTML and PDF versions of the Article."} +{"text": "Correction to: BMC Public Health 20, 1036 (2020)https://doi.org/10.1186/s12889-020-09164-9In the publication of the original article , the autThe collaboration group has been updated above. The original article has beenThe publisher apologizes for the inconvenience caused to the authors and readers."} +{"text": "Communications Biology 10.1038/s42003-019-0443-1, published online 20 May 2019.Correction to: In the original published version of this article the grant number attributed to the Italian Society for Celiac Disease was incorrectly given as Project No. 053_FC_2013. The correct grant number is Grant 005_FC_2016, awarded to Monia Porpora. The error has been corrected in the HTML and PDF versions of the article."} +{"text": "Scientific Reports 10.1038/s41598-020-62391-3, published online 27 March 2020Correction to: In the original version of this Article, Michael V. Holmes and Robin G. Walters were omitted as jointly supervising this work. This has now been corrected in the PDF and HTML versions of the paper."} +{"text": "Correction to: BMC Pulmonary Medhttps://doi.org/10.1186/s12890-020-1053-xFollowing publication of the original article , the autThe content of Table\u00a0Furthermore, in the (non-PDF) version of Table These errors have now been corrected in the original article.Please also find the corrected tables below for reference.The publisher apologizes for this technical error."} +{"text": "Nature Communications 10.1038/s41467-019-13797-9, published online 10 January 2020.Correction to: The original version of this Article contained an error in the Peer Review file, which incorrectly omitted the last page. This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Nature Communications 10.1038/s41467-020-16289-3, published online 15 May 2020.Correction to: The original version of the Supplementary Information associated with this Article contained errors in Supplementary Figs. 8 and 10. In Supplementary Fig. 8, the axes and the grid lines were missing from all six panels. In panel d of Supplementary Fig. 10, the figure element linking the sequences to the gene structure diagram was missing. The HTML has been updated to include a corrected version of the Supplementary Information."} +{"text": "Scientific Reports 10.1038/s41598-020-72241-x, published online 16 September 2020.Correction to: In the original version of this Article, Hirohiko Niioka was omitted as a corresponding author. Correspondence and request for materials should also be addressed to niioka@ids.osaka-u.ac.jp. This error has now been corrected in the HTML and PDF versions of the Article."} +{"text": "Scientific Reports 10.1038/s41598-023-38978-x, published online 14 August 2023Correction to: The original version of this Article contained an error in the spelling of the author Paulina Ratajczyk, which was incorrectly given as Paulina Ratajczak.The original Article has been corrected."} +{"text": "Scientific Data 10.1038/s41597-022-01899-x, published online 3 January 2023.Correction to: An author of the paper was omitted in the original version . This has been corrected in the pdf and HTML versions of the paper, and the associated metadata."} +{"text": "Correction to: Naunyn-Schmiedeberg\u2019s Archives of Pharmacology10.1007/s00210-022-02351-yIn the published version, the name of the last author contained an error.Hayat H.M. El-Nour.The correct name of the last author should be The original article has been corrected."} +{"text": "Scientific Reports 10.1038/s41598-023-38019-7, published online 04 July 2023Correction to: In the original version of this Article, author Arash Tirandaz was omitted as a corresponding author. Correspondence and requests for materials should also be addressed to a.tirandaz@basu.ac.ir.The original Article has been corrected."} +{"text": "Nature Communications 10.1038/s41467-023-37937-4, published online 08 June 2023Correction to: The original version of this Article contained an error in Fig. 1a, in which part of the \u2018LUCAS Vegetation cover\u2019 legend was omitted. This has now been corrected in the PDF and HTML versions of the Article."} +{"text": "Retraction: Parasites & Vectors (2017) 10:303 10.1186/s13071-017-2239-9The Editor-in-Chief has retracted this article. After publication, concerns were raised regarding western blot similarities in Figs. 5B and 6C . Additional checks by the Publisher have found a number of unexpected breaks in the western blot backgrounds in Figs. 3C\u2013F and 5B.The Editor-in-Chief therefore no longer has confidence in the presented data.Sujata Kumari agrees to this retraction. Sudha Verma does not agree to this retraction. None of the other authors have responded to any correspondence from the editor or publisher about this retraction."} +{"text": "Scientific Reports 10.1038/s41598-023-41765-3, published online 02 September 2023Correction to: The original version of this Article contained an error in the spelling of the author Tadeusz A. Wysocki, which was incorrectly given as Tadeusz A. Wysoki.The original Article has been corrected."} +{"text": "Scientific Data 10.1038/s41597-023-02119-w, published online 15 April 2023Correction to: The original manuscript contained errors in the reference list order, meaning some reference numbers pointed to the wrong citation. The affected references numbers are 28\u201332, 36, and 52-53.This has been corrected in the HTML and PDF versions of the manuscript. No references were added or removed, just re-ordered vs their original reference numbers."} +{"text": "Nature Nanotechnology 10.1038/s41565-022-01310-1. Published online 26 January 2023.Correction to: In the version of this article originally published, a conversion error led to parts of the structures in Figure 1a not appearing. The figure has been corrected in the HTML and PDF versions of the article."} +{"text": "Correction: Journal of Nanobiotechnology (2022) 20: 426 10.1186/s12951-022-01634-zFollowing publication of the original article , the autIn addition, in the Live/Dead cell staining experiments, the culture time of cells and hydrogels should be revised to \"3\u00a0days\".The author apologizes for any inconvenience caused.The original article has been"} +{"text": "Correction to: Breast Cancer Research, Treatment 10.1007/s10549-023-06870-xIn the original publication of the article, the reference citations in the text have been incorrectly processed. The original article has been corrected."} +{"text": "Diazoxide post-conditioning activates the HIF-1/HRE pathway to induce myocardial protection in hypoxic/reoxygenated cardiomyocytes by Chen X-Y, Wang J-Q, Cheng S-J, Wang Y, Deng M-Y, Yu T, Wang H-Y and Zhou W-J (2021). Front. Cardiovasc. Med. 8:711465. doi: 10.3389/fcvm.2021.711465A Corrigendum on Incorrect FundingIn the published article, there was an error in the Funding statement. \u201cThis work was supported by Master's Research Foundation of the Affiliated Hospital of Zunyi Medical College [Grant No. (2016)49] and the Science and Technology Fund Projects of Guizhou Provincial Health department (Grant No. gzwjkj2019-1-162).\u201dThe correct Funding statement appears below.FUNDINGThis work was supported by Master's Research Foundation of the Affiliated Hospital of Zunyi Medical College [Grant No. (2016)49] and the Science and Technology Fund Projects of Guizhou Provincial Health department (Grant No. gzwjkj2019-1-163).The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."} +{"text": "Correction: BMC Bioinformatics (2023) 24:39https://doi.org/10.1186/s12859-023-05160-zFollowing publication of the original article , it was The details of the supplement in which this article ought to have been published are given below:About this supplementhttps://bmcbioinformatics.biomedcentral.com/articles/supplements/volume23-supplement-9.This article has been published as part of BMC Bioinformatics Volume 23 Supplement 9, 2022: Proceedings of the 15th International Conference on Data and Text Mining in Biomedical Informatics (DTMBIO 2021). The full contents of the supplement are available online at The publisher apologizes for any inconvenience caused."} +{"text": "Nature Communications 10.1038/s41467-023-35912-7, published online 20 January 2023Correction to: The original version of this Article contained an error in Fig. 6e, in which Fig. 6e was mistakenly given as a duplicate of Fig. 6d. This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Correction to: Angiogenesis 10.1007/s10456-023-09885-6In the original publication of the article, the author wishes to update the funding section. The updated funding information is provided below.The original article has been corrected."} +{"text": "Correction to: GeroScience10.1007/s11357-023-00725-5Unfortunately, the authors for this published article noticed an error in the article title.The word verses in the article title should be corrected to versus. The corrected article title is shown above."} +{"text": "Cell Death & Disease 10.1038/s41419-020-2250-5, published online 22 January 2020Retraction to: Two images in Fig. 4h appear highly similar to those in Fig. 1f of [The images in Fig. 7b appear highly similar to those in Fig. 8a of with difThe images in Fig. 8a appear highly similar to those in Figs. 4j and 5h of .The Editors-in-Chief have retracted this article. After publication, concerns were raised regarding image similarities between this article and previous papers from different author groups, specifically:Fig. 4d SKBR-3-TR CQ+ GAPDH appears highly similar to Fig. 5d SKBR-3-TR Control GAPDH.Fig. 4d BT474-TR CQ- and CQ+ GAPDH appear highly similar to Fig. 5d BT474-TR Control and miR-567 GAPDH, respectively.Some of the blots in Figs. 7d and 8c appear to have irregularities in the backgrounds.The GSE104076 microarray dataset information [Further checks by the Publisher identified a number of further concerns:The Editors-in-Chief therefore no longer have confidence in the presented data and the conclusions of the article.None of the authors have responded to any correspondence from the editor or publisher about this retraction."} +{"text": "Retraction Note: Journal of Cardiothoracic Surgery (2020) 15:309https://doi.org/10.1186/s13019-020-01355-0The Editor-in-Chief has retracted this article due to the lack of adherence to the journal's ethical standards and editorial policy. After publication, concerns were raised regarding the trial registration and ethics approval of this study. The authors have provided the ethics approval, which was dated after the patient recruitment start date.All authors agree to this retraction."} +{"text": "Nature Cell Biology 10.1038/s41556-022-00984-y. Published online 13 September 2022.Correction to: In the version of this article initially published, author Andy L. Cox was mistakenly omitted from the author list. The error has been corrected in the HTML and PDF versions of the article."} +{"text": "Cell Death and Disease 10.1038/s41419-020-03266-3, published online 12 December 2020Retraction to: The Editors-in-Chief have retracted this article at the authors\u2019 request. After publication, concerns were raised regarding image similarities between this article and a number of previously published articles, specifically:Several images in Fig. 6D and E appear highly similar to those in Fig. 6C and D in , Fig. 6DFig. 5E input appears highly similar to Fig. 4E input in .The authors have stated that the images in Fig. 6 were misused and could not be reproduced, which undermined the reliability of the conclusions. The Editors-in-Chief therefore no longer have confidence in the presented data and the conclusions of the article.All authors agree to this retraction."} +{"text": "Correction to: Journal of Clinical Immunology10.1007/s10875-023-01582-9Due to typesetting mistake, there were overlapping values in the first histogram of Figure\u00a02b, which were not there in the proof reading of this figure.The original version has been corrected."} +{"text": "Retraction note: Nanoscale Res Lett (2021) 16:28https://doi.org/10.1186/s11671-021-03477-3The Editors in Chief have retracted this article. After publication, concerns were raised about the appearance of Western blots and the data in Table 1 that indicates that miR-124-3p may have been the target of the assays instead of miR-124-5p. The authors were unable to provide uncropped gels and blots and stated that some blots have been modified for \"aesthetics \" reasons. The Editors, therefore, have lost confidence in the integrity of the article's findings. Jian Hu agrees with this retraction. Chunling Tang did not respond to correspondence from the Editor about this retraction."} +{"text": "Retraction: Biol Res (2017) 50:28 https://doi.org/10.1186/s40659-017-0134-7The Editor in Chief has retracted this article because of significant text overlap with a previous work by the same authors . FurtherTherefore, the Editor has lost confidence in the data presented in this article.Author Md. Abdullah Al Mamun does not agree to this retraction. Authors Mohammad Jakir Hosen, Amina Khatun, M. Masihul Alam and Md. Abdul Alim Al-Bari have not responded to correspondence from the Editor or Publisher about this retraction."} +{"text": "Scientific Reports 10.1038/s41598-023-32731-0, published online 04 April 2023Correction to: In the original version of this Article, Hyun\u2011Ju Kim, Junghwa Seo and Minji Bang were incorrectly listed as corresponding authors. The correct corresponding author for this Article is Sang-Hyuk Lee. Correspondence and request for materials should be addressed to drshlee@cha.ac.kr.The original Article has been corrected."} +{"text": "Oncogenesis 10.1038/s41389-020-0230-3, published online 12 May 2020Retraction to: The Editor-in-Chief has retracted this article. Concerns were raising regarding Figure 2H. This figure appears to overlap with Figure 3G in an article by different authors that was simultaneously under consideration in another journal .The authors have provided partial raw data to address this concern. However, they have stated that some of the original data are no longer available. The Editor-in-Chief therefore no longer has confidence in the presented data.All authors agree to this retraction."} +{"text": "Nature Communications 10.1038/s41467-023-39872-w, published online 18 July 2023Correction to: The original version of this Article contained an error in Fig. 6, in which the brain maps in panels b and d were inadvertently swapped. This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Neuropsychopharmacology 10.1038/s41386-022-01520-0, published online 16 December 2022Correction to: In the original article, the 4th image of Fig. 2A was inadvertently an incorrect version and has now been replaced. This error does not affect the conclusions of the article. The original article has been corrected."} +{"text": "Correction to: Current Diabetes Reports10.1007/s11892-023-01520-4The original version of this article contained a mistake in the name of the 6th author. It should be Dianna J Magliano rather than Diana J Magliano.The original article has been corrected."} +{"text": "Nature Cell Biology 10.1038/s41556-022-01037-0. Published online 19 December 2022.Correction to: In the version of this article initially published, an incorrect file for Source Data Fig. 2 was uploaded. The file has now been replaced and the error has been corrected in the HTML and PDF versions of the article."} +{"text": "Nature Communications 10.1038/s41467-022-34902-5, published online 15 December 2022Correction to: The original version of this Article contained an error in Fig. 2, in which the bottom captions for the heatmap in Fig. 2b were inadvertently shifted downward into Fig. 2c. This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Nature Microbiology 10.1038/s41564-023-01326-w. Published online 9 February 2023.Correction to: In the version of this article initially published, the author list did not display first names, as are now provided in the HTML and PDF versions of the article."} +{"text": "Nature Communications 10.1038/s41467-023-36994-z, published online 15 March 2023Correction to: In the original version of this article, Fig. 2 was inadvertently duplicated from Fig. 5. The correct version of Fig. 2 is:which replaces the previous incorrect version.This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "It has been reported an inflammatory state in schizophrenia, with altered levels of some cytokines . Recent publications have shown the importance of IL- 33, a member of the IL-1 cytokine family which acts as an alarmin . The role of this cytokine as a biomarker has been investigated in schizophrenia . However, results are controversial. Some studies have not found significant associations between IL-33 and chronic schizophrenia , while other papers have reported increased levels . In all these studies, levels of IL-33 were measured in a single daily measure, so that it has not been studied if IL-33 has changes during hospitalization.To study the serum level of IL-33 at 12:00 and 00:00 hours in schizophrenia patients at admission and before hospital discharge.Fifteen inpatients with diagnosis of paranoid schizophrenia according to ICD-10 criteria were studied. Patients were hospitalized at the University Hospital of the Canary Islands psychiatric ward because of an acute relapse. A total of four blood samples were taken from each patient: at 12:00 and 00:00 hours the day after admission and at 12:00 and 00:00 hours the day before discharge. Serum IL-33 levels were measured by ELISA techniques. Daytime and nighttime IL-33 serum levels at admission and discharge were compared using a non-parametric Wilcoxon signed-rank test.In table 1 the results of the comparison of IL-33 at admission and discharge are presented. There is a significant reduction of IL-33 levels at 00:00 h. at discharge in comparison with the IL-33 levels at 00:00 h. at admission (p=0.028). No other statistically significant differences were observed.The decrease of serum IL-33 at 00:00 at discharge compared to the 00:00 IL-33 serum level at admission points to the utility of this biomarker as a surrogate of brain inflammation.None Declared"} +{"text": "Correction to: Journal of Clinical Immunologyhttps://doi.org/10.1007/s10875-023-01459-xDue to typesetting mistake, the institutional authors of the French COVID cohort study group, were mistakenly listed in the author group which resulted to redundancy. Also, Yves L\u00e9vy should be the last author from the list as shown above.The original version has been corrected."} +{"text": "Nature Methods 10.1038/s41592-022-01748-0. Published online 16 January 2022.Correction to: In the version of this article originally published, Shuoguo Li and Ziyan Wang were missing descriptors as equally contributing authors. The error has been corrected in the HTML and PDF versions of the article."} +{"text": "Scientific Reports 10.1038/s41598-023-35342-x, published online 22 May 2023Correction to: In the original version of this Article, Michael Lintner was incorrectly listed as a corresponding author. The correct corresponding author for this Article is Petra Heinz. Correspondence and request for materials should be addressed to petra.heinz@univie.ac.at.The original Article has been corrected."} +{"text": "Dis. Model. Mech. (2012) 5, 785-795 (doi:10.1242/dmm.009449).There was an error in The name of the author Hung-Chi Tu was spelled incorrectly (Hung-Chi Du). The correct name appears above, and the author contributions have been updated.The authors apologise for this error and any inconvenience it may have caused. Both the online full-text and PDF versions of the article have been updated."} +{"text": "Correction to: Statistical Methods & Applications 10.1007/s10260-023-00690-5In the published version of the paper, two figures have been uploaded from a previous version. In view of that, the captions of Figs.\u00a0We report below Figs."} +{"text": "Scientific Reports 10.1038/s41598-022-19167-8, published online 29 August 2022Correction to: In the original version of this Article, the Supplementary Information file was omitted from the Supplementary Information section.The Supplementary Information file now accompanies the original Article."} +{"text": "Correction: Journal of Health, Population and Nutrition (2023) 42:74https://doi.org/10.1186/s41043-023-00423-0Following publication of the original article , Figs. 3The updated version aligns with ethical guidelines and policies and includes sufficient reference to previous articles. The authors sincerely apologize for the errors. The errors do not affect the conclusion of the article.Figures"} +{"text": "Nature Communications 10.1038/s41467-023-40220-1, published online 9 August 2023Correction to: The original version of this Article contained an error in Fig. 4, in which several datapoints did not display. This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Scientific Reports 10.1038/s41598-023-46816-3, published online 17 November 2023Correction to: The original version of this Article contained an error in the name of Lianjing Liang, which was incorrectly given as Lianjin Liang.The original Article has been corrected."} +{"text": "Nature Communications 10.1038/s41467-023-42655-y, published online 18 November 2023Correction to: The original version of this Article contained an error in Fig. 3, in which the insets of Fig. 3a and 3b were not correctly visualized. The correct version of Fig. 3 is:which replaces the previous incorrect version:This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Correction: Surgical Endoscopyhttps://doi.org/10.1007/s00464-023-10081-2The original online version of this article was revised to correct the presentation of the name of author Neda D. Jooya.The original article has been corrected."} +{"text": "Correction to: Current Treatment Options in Oncology10.1007/s11864-023-01126-8The original version of this article, unfortunately, contained mistakes. The 2nd author name in the author group was incorrectly written as Camille Le Brooy. The correct spelling should be Camille La Brooy.The original article has been corrected."} +{"text": "Correction to: Archives of Virology (2023) 168:182 10.1007/s00705-023-05793-8Legends for Fig. 1 and Fig. 2 were incorrectly published in the original version, in which these had been interchanged. This is corrected now (Figs. Errors in Table The original article has been corrected."} +{"text": "Nature Communications 10.1038/s41467-023-39248-0, published online 19 June 2023Correction to: The original version of this Article contained an error in Fig. 1, in which panel d was inadvertently a duplicate of panel a. This has now been corrected both in the html and pdf version."} +{"text": "Correction to: Functional & Integrative Genomics 2023https://doi.org/10.1007/s10142-023-01079-zThe online version of the article contains an error during publication. In Funding, the first project number, SQ2022YFD1600330, is the number automatically generated at the time of the project application, not the final project number. The correct project number is 2022YFD1602203.The original article has been corrected."} +{"text": "Longitude variation of the microRNA-497/FGF-23 axis during treatment and its linkage with neoadjuvant/adjuvant trastuzumab-induced cardiotoxicity in HER2-positive breast cancer patients By Liu H, Hu X, Wang L, Du T, Feng J, Li M, Liu L and Liu X (2022) Front. Surg. 9:862617. doi: 10.3389/fsurg.2022.862617A Retraction of the Original Research Article The journal and Chief Editors retract the 11 May 2022 article cited above.This article is retracted by Frontiers. The publisher has discovered that the authors provided false information for the peer-review process. As the scientific integrity of the article cannot be guaranteed, and adhering to the recommendations of the Committee on Publication Ethics (COPE), the publisher therefore retracts the article.The authors have not responded to this retraction.This retraction was approved by the Chief Editors of Frontiers in Surgery and the Chief Executive Editor of Frontiers."} +{"text": "Nature Medicine 10.1038/s41591-023-02469-3. Published online 24 July 2023.Correction to: NCT04215471, which has now been inserted in the HTML and PDF versions of the article.In the version of this article initially published, the Abstract was missing the ClinicalTrials.gov identifier,"} +{"text": "Correction to: Journal of Materials Science: Materials in Medicine10.1007/s10856-023-06711-9The original version of this article unfortunately contained mistakes.1. The second author Zahra Behroozi had two affiliations listed. Her affiliation is only the affiliation listed as follows: Physiology Research Center, Institute of Neuropharmacology, Kerman University of Medical Science, Kerman, Iran.2. The full name of the third author is Ali Motamednezhad.3. Atousa Janzadeh\u2019s academic e-mail address is janzadeh.at@iums.ac.irThe original article has been corrected."} +{"text": "Early decrease of blood myeloid-derived suppressor cells during checkpoint inhibition is a favorable biomarker in metastatic melanoma. Journal for ImmunoTherapy of Cancer 2023;11:e006802. doi: 10.1136/jitc-2023-006802Gai\u00dfler A, Bochem J, Spreuer J, The licence was updated to CC-BY in September 2023. The funding statement has been updated to include the following \"we acknowledge support from the Open Access Publishing Fund of the University of T\u00fcbingen\"."} +{"text": "Correction to: Surgical Endoscopy 10.1007/s00464-022-09803-9The original online version of this article was revised to correct an error in the Discussion section, related to the presentation of data regarding the mesh-to-defect (M/D) ratio in hernia repairs. The correct M/D ratio is 20 (13 to 43).The original article has been corrected."} +{"text": "Communications Biology 10.1038/s42003-023-04569-9, published online 20 February 2023.Correction to: d of figure 9 was used. In the new version the correct panel for Figure 9 d is used.The original version of this Article contained an error in which an incorrect panel"} +{"text": "Nature Chemical Biology 10.1038/s41589-022-01247-5. Published online 13 February 2023.Correction to: In the version of this article initially published, Nicholas Barnes was not properly acknowledged as a co-author. He has now been included in the current author list, and his contributions are specified in the author contributions statement. The error has been corrected in the HTML and PDF versions of the article."} +{"text": "Correction to: Parasitology Research10.1007/s00436-022-07776-1The authors regret that there are some errors in their article, including the mis-identification of the fish host species.The original article has been corrected."} +{"text": "Scientific Reports 10.1038/s41598-023-42838-z, published online 30 October 2023Correction to: The original version of this Article contained an error in the spelling of the author Yosra A. Helmy which was incorrectly given as Yosra A. Hemly.The original Article has been corrected."} +{"text": "Nature Cell Biology 10.1038/s41556-023-01150-8. Published online 15 May 2023.Correction to: x-axis DLK1 and GCG gene labels, respectively, in the wrong order. The error does not affect the results or conclusions in the study. The figure is now updated in the HTML and PDF versions of the article.In the version of this article initially published, the Fig. 1c heatmap for verifying the identified cell types with key gene markers presented the"} +{"text": "Scientific Reports 10.1038/s41598-023-33047-9, published online 12 April 2023Correction to: The Acknowledgements section in the original version of this Article was incomplete. It now reads:This study was supported by the Ministry of Health, Czech Republic, project No. NU20-03-00360. We are grateful to Dr. Petr Krupa and Dr. Michael Bartos from Department of Neurosurgery, Faculty Hospital in Hradec Kralove, for providing GBM source samples.The original Article has been corrected."} +{"text": "Depression has been recognized as a common comorbidity in patient with epilepsy and is associated with low quality of life. Regular screening for depression may aid in early detection and enhance quality of life.To validate the Thai version of the Neurological Disorders Depression Inventory for Epilepsy (NDDI-E).The English version of NDDI-E was translated into Thai. Patients with epilepsy were enrolled at the outpatient neurology clinic from May 2019 to September 2019. Demographic data and clinical characteristics were collected. Participants underwent a psychiatric structured interview using the Mini-International Neuropsychiatric Interview (M.I.N.I.) as a gold standard for the diagnosis of major depressive disorder. Then, participants completed the NDDI-E. The internal consistency was measured by Cronbach\u2019s alpha coefficient. The validity of the Thai version of the NDDI-E was assessed using the receiver operating characteristic (ROC) curve analysis. Youden\u2019s index was used to determine the optimal cut-off score of the Thai version of the NDDI-E.A total of 115 patients with epilepsy completed the evaluation. Twenty-three patients (20%) had major depressive disorder according to M.I.N.I. criteria. The Cronbach\u2019s alpha coefficient of the Thai version of the NDDI-E was 0.826. The area under the ROC curve was 0.995. A cut-off score greater than 17 provided a sensitivity of 95.65%, a specificity of 97.83%, a positive predictive value of 91.67% ,and a negative predictive value of 98.90%.The Thai version of the NDDI-E is a valid screening tool for major depressive disorder in patients with epilepsy.None Declared"} +{"text": "Nature Communications 10.1038/s41467-023-42093-w, published online 10 October 2023Correction to: In the original version of this Article, there were errors in Fig. 2g and h. In both cases, the asterisks denoting statistical significance were inadvertently misaligned with the brackets indicating the categories being compared. These errors have now been corrected in both the PDF and HTML versions of the Article."} +{"text": "Publisher Correction: Genome Biol 24, 70 (2023)https://doi.org/10.1186/s13059-023-02893-1Following publication of the original article , the autwww.humancellatlas.org/publications.Further to this, the authors would like to add the following statement to the Acknowledgement section: This publication is part of the Human Cell Atlas\u2014The correct Fig."} +{"text": "Correction: BMC Primary Care 23:256 (2022)10.1186/s12875-022-01861-1Following publication of the original article , the autDr. Laura Nimmon is requesting removal of authorship. Based on the ICMJE criteria for author contributions Dr. Nimmon does meet the requirements for authorship. All authors have agreed to this change.The authorship of this article and the original article has been corrected."} +{"text": "Translational Psychiatry 10.1038/s41398-019-0662-8 published online 28 November 2019Correction to: The original version of this article contained an error. In the original file of Fig. 4g, the loading controls were accidentally mirror-image changed. The loading controls of Fig. 4g should be the same as the loading controls of Fig. 3c, because in the western blotting experiment, the two proteins came from the same piece of separation glue and shared the same loading controls. The authors apologize for the error. The original article has been corrected."} +{"text": "Nature Communications 10.1038/s41467-022-33399-2, published online 29 September 2022Correction to: The original version of this Article contained an error in Fig. 5, in which the labels in panels a and b were interchanged.This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Nature Communications 10.1038/s41467-023-36798-1, published online 02 March 2023Correction to: The original version of this Article contained an error in Fig. 2a, in which the graph in panel i (for peptide ALHAP) was duplicated in panel v. This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Translational Psychiatry 10.1038/s41398-023-02549-5, published online 18 July 2023Correction to: The Funding information section was missing from this article and should have read: The present work was supported by the Research Fund of Istanbul University. Project No. TYL-2019-33725.The original article has been corrected."} +{"text": "Nature Microbiology 10.1038/s41564-023-01353-7. Published online 6 April 2023.Correction to: In the version of this article initially published, the author list did not display first names. The list has been revised in the HTML and PDF versions of the article."} +{"text": "Environmental Management 10.1007/s00267-022-01723-7Correction to: In this article, the designations a, b and c were missing on the individual panels in Figure 4. The Figure 4 has now been corrected.The original article has been corrected."} +{"text": "Scientific Reportshttps://doi.org/10.1038/s41598-023-37048-6, published online 29 June 2023Correction to: The original version of this Article contained an error in the spelling of the author Jason S. Link which was incorrectly given as Jason M. Link.The original Article and accompanying Supplementary Information file have been corrected."} +{"text": "Cell Death & Differentiation 10.1038/s41418-023-01126-z, published online 24 February 2023Correction to: The original version of this article contained an error in the author list. During the revision process, Dr. Paul M. Nguyen contributed to the design and assisted with the experiment presented in Supplemental Figure 4g/h. Paul\u2019s name was omitted from the manuscript in error when it was re-submitted. The original article has been corrected."} +{"text": "Correction: Cell Regen 12, 13 (2023)https://doi.org/10.1186/s13619-023-00157-8Following publication of the original article (Ye et al. The original version was:The authors declare no competing interests.The updated version is:Yufang Shi is a member of the Editorial Board for Cell Regeneration. He was not involved in the journal\u2019s review of, or decisions related to, this manuscript.The original article (Ye et al."} +{"text": "Communications Biology 10.1038/s42003-023-05325-9, published online 22 September 2023.Correction to: The original version of this Article contained an error in Fig. 1d, in which the UMAP graph was blank. The error has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Correction: Hereditas (2021) 158:1010.1186/s41065-021-00171-3Following the publication of the original article , the comDr. Yongyong Shi is a Chief Scientist for Dynegene and a Co-Chief Editor for Hereditas. All the other authors declare that they have no competing interests."} +{"text": "Correction to: Journal of Neurology 10.1007/s00415-023-11844-6The original version of this article unfortunately contained a mistake. The acknowledgements section was incorrect.The correct acknowledgement section should read asMassimiliano Filosto, Francesca Trojsi, Letizia Mazzini. Fabiola De Marchi, Raffaele Dubbioso and Valentina Virginia Iuzzolino are members of the European Reference Network Euro-NMD. All authors are member of the MND Italian Study Group.The original article has been corrected."} +{"text": "Nature Immunology 10.1038/s41590-023-01588-w, published online 10 August 2023.Correction to: In the version of the article originally published, the middle panel in Fig. 3b was mistakenly a duplicate of the upper panel. The figure has now been corrected in the HTML and PDF versions of the article."} +{"text": "Environmental Health (2023) 22:4310.1186/s12940-023-00987-8https://iris.who.int/handle/10665/347877.Following the publication of the original article, the author reported that the link in reference 216 leads to the main WHO website. The link should be updated to The original article has been updated."} +{"text": "Cancer Cell International (2020) 20:20210.1186/s12935-020-01287-8.The Editors-in-Chief have retracted this article. Concerns were raised regarding Fig.\u00a03g, which appears to overlap with Fig.\u00a02h in an article by different authors that was simultaneously under consideration with another journal .The Editors-in-Chief therefore no longer have confidence in the results and conclusions of this article. The authors have not responded to correspondence regarding this retraction."} +{"text": "Scientific Reportshttps://doi.org/10.1038/s41598-023-35416-w, published online 27 May 2023Correction to: The original version of this Article contained an error in the spelling of the author Samuel Weinstein which was incorrectly given as Samuel Weinsein.The original Article has been corrected."} +{"text": "Correction to: Journal of Mathematical Biology (2023) 86:39https://doi.org/10.1007/s00285-023-01870-3In the original publication of the article, the article title was published incorrectly. The correct title is provided in this Correction.The original article has been corrected."} +{"text": "Nature Communications 10.1038/s41467-018-05275-5, published online 09 August 2018Correction to: The original article contained an error made during the assembly of Fig. 2b. In Fig. 2b, the same BRAF IHC picture from the BRAF mutated sample was selected by mistake for the two panels shown.The corrected version of Fig. 2b is shown below.The figure has been corrected in the PDF and HTML versions of the article.A pdf file containing the raw data for the same panels is appended below.Supplementary Raw Data"} +{"text": "Nature Communications 10.1038/s41467-022-35480-2, published online 13 December 2022Correction to: The published version of the article contained an error in Figure 2, where the incorrect panel d had been included. This has been corrected in the HTML and PDF versions."} +{"text": "Cell Death Discovery 10.1038/s41420-023-01438-6, published online 29 April 2023Correction to: The original version of this article contained a mistake. Due to an error in production Hyun-Soo Cho was omitted to be named as a corresponding author. We apologize for the mistake. The original article has been corrected."} +{"text": "Correction: Cell Commun Signal 20, 137 (2022)https://doi.org/10.1186/s12964-022-00923-2Following publication of the original article , the autThis project was supported by the Polish National Agency for Academic Exchange.Further to this, the correspondence email address for this article has been updated to kmmarycz@ucdavis.edu.The original article has been"} +{"text": "South African Journal of Communication Disorders, 69(2), a898. https://doi.org/10.4102/sajcd.v69i2.898, an author name was incorrectly spelt as Monroe. The correct spelling is Moroe.In the published article, Khoza-Shangase, K., Monroe, N., & Sebothoma, B. (2022). Conducting clinical research in the era of the COVID-19 pandemic: Challenges and lessons for Speech-Language Pathology and Audiology research. The publisher apologises for this error. The correction does not change the study\u2019s findings of significance or overall interpretation of the study\u2019s results or the scientific conclusions of the article in any way."} +{"text": "Correction to: Ambio 10.1007/s13280-023-01906-4In the original publication of the article, the three right panel images of Fig. The original article has been corrected."} +{"text": "Correction to: Stem Cell Research & Therapy 10.1186/s13287-021-02678-yIn the original article, the authors identified two editing errors: Methods section, concretely in the Study population subsection, a typing error was made, since it states that twenty-seven women were included in the study (14 with GDM and 14 with NGT), but indeed there are twenty-eight women.1. In the 2. In Table"} +{"text": "Correction to: Clinical Oral Investigations10.1007/s00784-022-04814-1In the published paper, the supplementary file is containing an additional page, which was not intended to make public. Unfortunately, it was made online during the publication process.The original article has been corrected."} +{"text": "Open Biol.9, 190095 (Published online 4 September 2019). (https://doi.org/10.1098/rsob.190095)The Editor-in-Chief in agreement with the Publisher has retracted this article. Following an investigation, flaws and inconsistencies in the sequence analyses and western blots were found (figs. 4 and 5). The authors have not been able to supply the raw data as requested, thus, the editors consider the conclusions of this article to be invalid. The authors have not responded to correspondence about this retraction.https://royalsocietypublishing.org/doi/10.1098/rsob.200165.The image integrity standards and policies for the journal can be found here: Prof. Jon Pines FRSOpen Biology.Editor-in-Chief,"} +{"text": "Scientific Reports 10.1038/s41598-023-34354-x, published online 02 May 2023Correction to: The original version of this Article contained an error in the name of the author Haralambos Mouratidis, which was incorrectly given as Haris Mouratidis.The original Article has been corrected."} +{"text": "Scientific Reportshttps://doi.org/10.1038/s41598-023-40570-2, published online 16 August 2023Correction to: The original version of this Article contained an error in the name of Donghwan Lee, which was incorrectly given as Dongwhan Lee.The original Article has been corrected."} +{"text": "Correction to: BMC Pulmonary Medicine (2023) 23:9310.1186/s12890-023-02374-yFollowing publication of the original article, the authors flagged an error in Fig. 1: the figure had been erroneously replaced by a duplicate of Fig. 7. The article has since been updated to correct the figure. The authors thank you for reading and apologize for any inconvenience caused."} +{"text": "Scientific reports 10.1038/s41598-023-32440-8, published online 01 April 2023Correction to: The original version of this Article contained an error in the spelling of the author Ioanna Minopoulou, which was incorrectly given as Ioanna Minnopoulou.The original Article has been corrected."} +{"text": "Scientific Reportshttps://doi.org/10.1038/s41598-023-31157-y, published online 17 March 2023Correction to: The original version of this Article contained an error in the spelling of the author Nicola Taccardi, which was incorrectly given as Nicola Tacardi.The original Article has been corrected."} +{"text": "ALOX12 gene polymorphism and bone mineral density (BMD) has also been verified,ALOX12 variation, contributing to the peak BMD and the development of osteoporosis. Another multidomain enzyme involved in DNA methylation, cystathionine beta-synthase (CBS), regulates the conversion of homocysteine into glutathione. Mutations in CBS lead to the production of more sulfur end-products from the methylation cycle. The newborn CBS-knockout (KO) mice who received treatment with recombinant poly ethylene glycol human truncated CBS (PEG-CBS) were rescued from osteoporosis-like symptoms.CBS was found to be down-regulated in femur tissues, leading to a lower BMD.As a bone-confined chronic degenerative disorder, the progression of osteoporosis is characterized by abnormal crosstalk between osteoblasts and osteoclasts, leading to an imbalance of bone remodeling in adults.ALOX12 (No. 2021-YS-093). For age- and sex-matched rats, animals were fed and maintained under specific pathogen-free conditions following the criteria of the National Institutes of Health Guide for the Care and Use of Laboratory Animals with the approval of the Ethics Committees of Shanghai Sixth People's Hospital Affiliated to Shanghai Jiao Tong University (No. 2022\u20130271).S.G. wrote the main manuscript and acquired the funding; Y.W. contributed to the methodology of experiments needed and data analysis; W.C. and P.H. did the investigation and data curation; W.C. reviewed the writing; H.W. did the validation; Y.W. and B.Z. made the supervision and reviewed and edited the manuscript.The authors declare no conflict of interests.10.13039/501100004921Shanghai Jiao Tong University (No. YG2019QNA23).This work is supported by grants from the Interdisciplinary Program of"} +{"text": "Retraction Note: Journal of Orthopaedic Surgery and Research (2021) 16:290 https://doi.org/10.1186/s13018-021-02423-4The Editor-in-Chief has retracted this article. After publication, concerns were raised regarding potential reuse of the data reported in the article. Specifically:The article appears to include a subset of data from without The control group data and results appear to be highly similar to those published in the authors' other articles \u20134.The Hyaluronan group T0 values in Table 4 appear to match those for the Control group in \u20134.The authors have been contacted to address these concerns. The corresponding author stated that the data are unique to each article. However, based on the extent of data similarities among these articles, the Editor-in-Chief no longer has confidence in the presented data.J. C. Alves and C. Lavrador do not agree to this retraction. A. Santos, P. Jorge and L. Miguel Carreira have not responded to any correspondence from the editor or publisher about this retraction."} +{"text": "Correction to: Internal and Emergency Medicine 10.1007/s11739-023-03254-3In this article the names of a few collaborators and some data in Table 5 were missing. It has been corrected.The original article has been corrected."} +{"text": "Translational Psychiatry 10.1038/s41398-021-01216-x, published online 01 February 2021Correction to: The original version of this article contained a figure error. The authors state the following: In the original file of Fig. 4A, the illustration of CA2/3 region of CUS/running group was misplaced. The authors apologize for the error."} +{"text": "Correction to: BMC Health Services Research(2023) 23:875.10.1186/s12913-023-09928-0.Following publication of the original article , the autThis error is corrected in the affiliations list of this Correction article and the original article has been"} +{"text": "Nature Communications 10.1038/s41467-022-35555-0, published online 19 December 2022Correction to: The original version of this Article contained an error in the first sentence of the Author Contributions Section, both in the html and in the PDF version. The sentence incorrectly read:\u201cC.Z and J.X. conceived the concept.\u201dWhile the correct form should be:\u201cJ. C. and J. X. conceived the concept.\u201dThis has been corrected in both versions of the Article."} +{"text": "Communications Biology 10.1038/s42003-022-04067-4, published online 16 October 2022.Correction to: The original version of the Article contained an error in Figure 1a showing the structure of the compound CIMSS.The original article has been corrected."} +{"text": "Correction: Thrombosis J 21, 73 (2023)10.1186/s12959-023-00518-yFollowing publication of the original article , it was The author group has been updated above and the original article has been corrected.The publisher would like to apologize for any inconvenience this may have caused."} +{"text": "Correction to: Diabetologiahttps://doi.org/10.1007/s00125-022-05729-yIde panel was a duplication of the image used for the control panel. The authors assert that this mistake had no impact on the data analysis, interpretation or conclusions drawn. Figure Unfortunately, in the BrdU staining of alpha-TC1.9 cells shown in Fig."} +{"text": "BMC Public Health (2023) 23:30810.1186/s12889-023-15176-yIn the original publication of this article there was an error in an author name. The correct and incorrect information is listed below. The original article has been updated. The publisher apologizes for the inconvenience caused to the authors.IncorrectAndrea KozakCorrectAndrea T Kozak"} +{"text": "Correction to: Journal of Cancer Education10.1007/s13187-023-02268-xThe original version of this article unfortunately contained a mistake.Table 3, p.8 of the article, the header REMINDER LETTER should be replaced with EDUCATIONAL VIDEO.The original article has been corrected."} +{"text": "Correction: Surgical Endoscopyhttps://doi.org/10.1007/s00464-023-10278-5The original online version of this article was revised to correct the information for affiliation 10. The correct information is: Department of Surgery and Division of Foregut Surgery, IRCCS Policlinico San Donato, University of Milan, Milan, Italy. The article was also updated to reflect that author Luigi Bonavina has this affiliation.The original article has been corrected."} +{"text": "Correction to:BMC Gastroenterol23, 339 (2023).10.1186/s12876-023-02975-1.Following publication of the original article , it was The correct authorship is given in this Correction article and the original article has been"} +{"text": "Scientific Reports 10.1038/s41598-023-37606-y, published online 26 June 2023Correction to: The original version of this Article contained an error in one of the author names. Peder L. Myhre was incorrectly given as Peder Myhre.The original Article has been corrected."} +{"text": "Correction to: Neurosurgical Reviewhttps://doi.org/10.1007/s10143-023-02109-xThe authors regret that the author names that appears in the original article were incorrect. The first and last names were swapped.The original article has been corrected."} +{"text": "Nature Chemical Biology 10.1038/s41589-022-01162-9. Published online 20 October 2022.Correction to: In the version of this article originally published, the Acknowledgements omitted thanks to the Swiss National Fund for grant number 200020_178763. The error has been corrected in the HTML and PDF versions of the article."} +{"text": "Scientific Reports 10.1038/s41598-023-31441-x, published online 16 March 2023Correction to: In the original version of this Article, the author name P. Blasiak was inadvertently duplicated in the author list.The original Article has been corrected."} +{"text": "Nature Genetics 10.1038/s41588-023-01472-1, published online 24 August 2023.Correction to: In the version of the article initially published, grants to L.M. from the Associazione Italiana per la Ricerca sul Cancro (AIRC) and Cancer Research UK, FC AECC and AIRC under the International Accelerator Award Program (C355/A26819 and 22796) were missing from the Acknowledgments section. The grant information has been inserted in the HTML and PDF versions of the article."} +{"text": "Communications Medicine 10.1038/s43856-023-00331-8, published online 22 July 2023.Correction to: y-axis labels of Figures 3 and 4 of this article. For both figures, commas were positioned or added incorrectly in the numbers on the y-axis. These errors have now been corrected in both the PDF and HTML versions of this article.There were errors in the"} +{"text": "Correction: Cell Biosci (2019) 9:34 https://doi.org/10.1186/s13578-019-0296-9The position of DAPI panels in Fig.\u00a04g was reversed. The authors would like to provide a revised Fig. 4g with reorganized DAPI panels.The Emerin blot of Fig.\u00a02c was the unpublished data from the same Co-IP experiment published in Wang et al. . The autIn the original version of the article, the authors wish to make the following corrections:The correct version of figures is given in this correction."} +{"text": "Correction to: AIDS and Behavior https://doi.org/10.1007/s10461-023-04052-wDue to a mistake on the Publisher\u2019s end, the original version of this article contained errors in Table 3. Table The original article has been corrected."} +{"text": "Scientific Reports 10.1038/s41598-023-29997-9, published online 24 February 2023Correction to: In the original version of this Article, author G. Ha was omitted as a corresponding author. Correspondence and requests for materials should also be addressed to gwanghui.ha@gmail.com.The original Article has been corrected."} +{"text": "Eye 10.1038/s41433-022-02097-0, published online 25 May 2022Correction to: The original version of this article unfortunately contained an error in an author name. The correction is to the first name of Hrvoje Bogunovic, currently spelt \u2018Hvroje\u2019. The authors apologize for the error. The original article has been corrected."} +{"text": "Scientific Reportshttps://doi.org/10.1038/s41598-023-43618-5, published online 28 September 2023Correction to: In the original version of this Article, the Supplementary Information file which contains a high-resolution version of all the Figures was omitted.The Supplementary Information file now accompanies the original Article."} +{"text": "Nature 10.1038/s41586-022-05640-x Published online 25 January 2023Correction to: In the version of this article initially published, the affiliation listed for Weiqing Zeng was incorrect ; it should have read International Flavors & Fragrances, Wilmington, DE, USA. The affiliation has been corrected in the HTML and PDF versions of the article."} +{"text": "Scientific Reports 10.1038/s41598-023-40652-1, published online 19 August 2023Correction to: The original version of this Article contained an error in the spelling of the author Young H. Kim which was incorrectly given as Young Kim.The original Article has been corrected."} +{"text": "Scientific Reports 10.1038/s41598-023-41711-3, published online 02 September 2023Correction to: In the original version of this Article Hezerul Abdul Karim was omitted as a corresponding author. Correspondence and requests for materials should also be addressed to hezerul@mmu.edu.my.The original Article has been corrected."} +{"text": "Nature Biotechnology 10.1038/s41587-022-01520-x. Published online 2 January 2023.Correction to: In the version of this article initially published, Cristina Leal Rodr\u00edguez was omitted from the author list. The error has been corrected in the HTML and PDF versions of the article."} +{"text": "Bone Research 10.1038/s41413-023-00261-0, published online 19 April 2023Correction to: 1 it was reported that the name of author Scott Stewart has been mistakenly typed as Scott Stuart.Following publication of this article,The correct name is Scott Stewart.1 was updated.The original article"} +{"text": "Communications Biology 10.1038/s42003-023-05308-w, published online 09 September 2023.Correction to: In this article, Fig. 6d contained an error related to the position of the (\u2212) and (+) symbols on top of the Western blots; the figure should have appeared as shown below. The original article has been corrected."} +{"text": "Nature Communications 10.1038/s41467-023-40575-5, published online 15 August 2023Correction to: The original version of this Article contained an error in the email address provided in the Discussion section which was incorrectly given as fast_del@novartis.com. The correct email address is fast.del@novartis.com. This has now been corrected in both the PDF and HTML versions of the Article."} +{"text": "Scientific Reports 10.1038/s41598-023-31610-y, published online 16 March 2023Correction to: The original version of this Article contained an error in the name of author Akimichi Morita, which was incorrectly given as Morita Akimichi.The original Article has been corrected."} +{"text": "Scientific Reportshttps://doi.org/10.1038/s41598-023-40973-1, published online 22 August 2023Correction to: The original version of this Article contained an error in the name of INSIST investigator Simon F. de Meyer, which was incorrectly given as Simon de Meyer.The original Article has been corrected."} +{"text": "Correction to: Infectious Agents and Cancer (2023) 18:7.10.1186/s13027-023-00485-z.Following publication of the original article , the autThis error is corrected in the author list of this Correction article and the original article has been"} +{"text": "Correction to: Journal of Applied Genetics (2023)https://doi.org/10.1007/s13353-023-00749-9Originally, the article was published with error. In the Abstract, the second line should be \"in the twentieth century\" instead of \"in the twenty-first century\".The original article has been corrected."} +{"text": "Scientific Reportshttps://doi.org/10.1038/s41598-022-26744-4, published online 10 January 2023Correction to: The original version of this Article contained an error in the spelling of the author Alexander S. Vedenkin which was incorrectly given as Alexander A. Vedenkin.The original Article has been corrected."} +{"text": "Correction: Eating and Weight Disorders: Studies on Anorexia, Bulimia and Obesity (2022) 27:2595\u20132604https://doi.org/10.1007/s40519-022-01398-3Given name is Kamolthip and family name is Ruckwongpatr.Given name is Chirawat and family name is Paratthakonkun.In this article, the given and family names of the following two authors were incorrect. The correct names are given below.The original article has been"} +{"text": "Correction to: Social Psychiatry and Psychiatric Epidemiology 10.1007/s0017-03-0484-2In Table 4 of this article, the data in the row 6th headed Natural sciences were incorrected. It has been corrected. The correct Table The original article has been corrected."} +{"text": "In the above-mentioned article an author's name was corrected. This was corrected in the online version on 10.08.2023."} +{"text": "Nature Medicine 10.1038/s41591-023-02566-3, published online 9 October 2023.Correction to: In the version of this article initially published, the first name of Jaclyn B. Fahey appeared as Jacyln and has now been corrected in the HTML and PDF versions of the article."} +{"text": "Scientific Reportshttps://doi.org/10.1038/s41598-023-36047-x, published online 01 June 2023Correction to: The original version of this Article contained an error in the spelling of the author Luai A. Ahmed, which was incorrectly given as Luai Ahmed.The original Article has been corrected."} +{"text": "Correction to: Surgical Endoscopy 10.1007/s00464-023-10081-2The original online version of this article was revised to correct the presentation of one coauthor's name. The coauthor's correct name is Matthew W. Lee.The original article has been corrected."} +{"text": "This article has been corrected to remove a protocol that was mistakenly included and referred to as a clinical trial:\u00a0Ornowska M, Wong H, Ouyang Y, et al.: https://trialsjournal.biomedcentral.com/articles/10.1186/s13063-022-06671-5 Control of Line Complications with KiteLock (CLiCK) in the critical care unit: study protocol for a multi-center, cluster-randomized, double-blinded, crossover trial investigating the effect of a novel locking fluid on central line complications in the critical care population. Trials. 2022, 23: https://trialsjournal.biomedcentral.com/articles/10.1186/s13063-022-06671-5 10.1186/s13063-022-06671-5The following changes have been made as a result of this removal:The references and citations to the protocol, Ornowska et al., are removed from the tables, the text, and the reference section.All the citation numbers are adjusted accordingly.The PRISMA flow chart shows the updated number of finalized articles included.The quality appraisal table (Table 2) and the summary of characteristics table (Table 3) have been edited accordingly.The removal of the above protocol did not affect\u00a0the overall results or the conclusion of the systematic review. The authors regret that this error was not identified prior to submission and publication of this article."} +{"text": "Nature Aging 10.1038/s43587-023-00462-6, published online 10 August 2023.Correction to: In the version of Supplementary Data initially published with this article, data were missing from Table S1.13 , which now appear in an updated Supplementary Data file in the online version of the article."} +{"text": "PLoS Comput. Biol. 8(3): e1002385. doi: 10.1371/journal.pcbi.1002385. Our investigation, conducted in accordance with Frontiers' policies, confirmed the similarity, and so the article has been retracted.Our office received a complaint claiming that the cited article is unacceptably similar to the article: Shimazaki H, Amari S-i, Brown EN, Gr\u00fcn S (2012) State-Space Analysis of Time-Varying Higher-Order Spike Correlation for Multiple Neural Spike Train Data. This retraction was approved by the Chief Editors of Frontiers in Computational Neuroscience and the Chief Executive Editor of Frontiers. The authors did not agree to this retraction."} +{"text": "Translational Psychiatry 10.1038/s41398-023-02361-1, published online 11 March 2023Correction to: The original version of this article contained errors in the authorship and the affiliations. The correct authorship and affiliations can be found above. The original article has been corrected."} +{"text": "Scientific Reports 10.1038/s41598-023-31121-w, published online 11 March 2023Correction to: In the original version of this Article, Mar\u00eda Cecilia De Rossi was omitted as a corresponding author. Correspondence and requests for materials should also be addressed to mcecilia.dr@gmail.com.The original Article has been corrected."} +{"text": "Correction: J Exp Clin Cancer Res 39, 235 (2020)10.1186/s13046-020-01739-zFollowing publication of the original article , incorreThe correction does not affect the overall result or conclusion of the article. The original article has been corrected.Below is the link to the electronic supplementary material.Table S1. Primers and oligonucleotides sequences used in this study."} +{"text": "Scientific Reports 10.1038/s41598-023-45181-5, published online 20 October 2023Correction to: The original version of this Article contained an error in the name of Anass Hafnaoui, which was incorrectly given as Anass Hafnoaui.The original Article has been corrected."} +{"text": "The word \"appropriate\" is misspelled in the article title. The correct title is: The Pathogen- and Incidence-Based DALY Approach: An Appropriate Methodology for Estimating the Burden of Infectious Diseases.The correct citation is: Mangen M-JJ, Plass D, Havelaar AH, Gibbons CL, Cassini A, et al. (2013) The Pathogen- and Incidence-Based DALY Approach: An Appropriate Methodology for Estimating the Burden of Infectious Diseases. PLoS ONE 8(11): e79740. doi:10.1371/journal.pone.0079740In the Affiliation, the line \"Membership of the Mouse Genome Sequencing Consortium is provided in the Acknowledgments\" should read \"Other members of the BCoDE consortium are listed in the Acknowledgments.\""} +{"text": "One of the author\u2019s institutions found that the data were not reproducible from the described methods, but an investigation by the author\u2019s other institution did not find the data or their interpretation suspicious. Given the conflicting conclusions of these investigations, the Editors advise the readers to interpret the data with due caution. We apologize to all affected parties.After publication of this article (Fernandez et al., BMC Genomics 2011, BMC Genomics 2011, 12:604. doi: http://dx.doi.org/10.1186/1471-2164-12-604. URL http://www.biomedcentral.com/1471-2164/12/604.Ariel Fernandez, Yun-Huei Tzeng and Sze-Bi Hsu. Subfunctionalization reduces the fitness cost of gene duplication in humans by buffering dosage imbalances."} +{"text": "Two errors were made in this article. First, a reference was omitted from the second sentence of the fourth paragraph in the Introduction section. The sentence should read: SidD lacks any obvious sequence homology with the AT-N or other known proteins, although fold recognition analysis of the N-terminal portion of SidD predicted limited resemblance with members of the metal-dependent protein phosphatase (PPM) family (Rigden 2011).The reference is: Rigden DJ. (2011) Identification and modelling of a PPM protein phosphatase fold in the Legionella pneumophila deAMPylase SidD. FEBS Lett. 585:2749-54.doi: 10.1016/j.febslet.2011.08.006.Second, there were some omissions from the acknowledgements section, which should instead read as follows:We thank K. B. Decker, Y. Lin, and A.H. Gaspar for comments on the manuscript, and A. Vidaurrazaga for assistance with biochemical experiments. This study made use of the European Synchrotron Radiation Facility under the Block Allocation Group (BAG) MX1420, the Diamond Light Source under the rapid access mode MX7512 and BAG MX8302, the beamline PROXIMA1 at the SOLEIL synchrotron , the X-ray crystallography platform and proteomics platform (member of CIBERehd and ProteoRed-ISCIII) at CIC bioGUNE , and the SGIker analytical facility at UPV/EHU . We thank all the staff from these facilities for technical and human support. Access to synchrotron facilities was part funded by the the BioStruct-X project ."} +{"text": "The authors are grateful to Dr. Bernard Henrissat , Aix-Marseille Universit\u00e9, France) for his comments on this article. Due to his comments, the authors wish to make the following corrections:1. In the legend of Figure 2, this note should be included: \"GH5a* represents cellulose-degrading glycoside hydrolases in the GH family 5 while GH5b* represents hemicellulose-degrading glycoside hydrolases in the GH family 5.\"2. For Table S2, CE10 should be removed because this family no longer belongs to CAZymes according to the new version of Carbohydrate-active enzymes database (www.cazy.org).http://cricket.ornl.gov/cgi-bin/cat.cgi?tab=Home), using the Carbohydrate Active Enzymes (CAZy) database (http://www.cazy.org)\" 3. The description of the annotation method of CAZymes, \"The search and functional annotation for carbohydrate-active modules in V. volvacea were performed by the software CAZYmes Analysis Toolkit (CAT) (should be replaced with this description:http://cricket.ornl.gov/cgi-bin/cat.cgi?tab=Home). In order to confirm the correct of annotation, we annotated the CAZymes of V. volvacea with two methods: 1. Pfam-based annotation ; 2. Sequence-based annotation . Then the non-redundant CAZyme genes of V. volvacea were created by merging the two methods, removing the genes only annotated by one of the methods.\"\"The search and functional annotation for carbohydrate-active modules in V. volvacea were performed by the software CAZYmes Analysis Toolkit (CAT) (Recently (2013.5.6), we re-annotated the CAZymes of V. volvacea. We found 3 new CAZymes in GH128 family and 14 new CAZymes in GT2 family .A new Table S2 is available here: Click here for additional data file."} +{"text": "The Publication Date given in the PDF version and expressed in the Citation and Footer in the PDF version of the article is incorrect. The correct publication date is September 25, 2013. The correct Citation is: Chu H-Y, Liao Y-L, Tsai Y-J, Chu Y-C, Wu S-Y, et al. (2013) Use of Extraocular Muscle Flaps in the Correction of Orbital Implant Exposure. PLoS ONE 8(9): e72223. doi:10.1371/journal.pone.0072223. The Footer in the bottom right corner of each page of the PDF should read: 2013 | Volume 8 | Issue 9 | e72223."} +{"text": "The journal has been informed by the authors' institution that, contrary to the statement in this article , the MacThe pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2474/12/200/prepub"} +{"text": "The Editor listed in the published manuscript is incorrect. The correct Editor is Dr. Heidar-Ali Tajmir-Riahi. His affiliation is the University of Quebec at Trois-Rivieres, Canada."} +{"text": "The journal has been informed by the authors' institution that, contrary to the statement in this article , the MacThe pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2474/12/158/prepub"} +{"text": "There is an error in the funding statement.The correct statement is: The excavations and scientific research associated with Gorham's Cave have been funded by the Government of Gibraltar and, additionally between 2002\u201304, by the European Community - Program Interreg IIIB: 2002-02-4, 1-U-048 -. R. Blasco and J. Rosell are part of the following projects: 2009 SGR 188 of Generalitat de Catalunya and CGL2009-12703-C03-02 of the Spanish Goverment. A. S\u00e1nchez Marco belongs to the CGL2011-28681 project of the Spanish Government, and J.S. Carri\u00f3n to CGL-2009-06988/BOS-FEDER. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."} +{"text": "There is an error in the Funding section. http://www.dfg.de/en/index.jsp) to A.B. and the University of Wuerzburg in the funding programme Open Access Publishing. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The correct funding information is as follows: This work was supported by grants BU951/3-1, BU951/3-2 and BU951/4-1 by the German Research Foundation (DFG) ("} +{"text": "The following information was omitted from the Funding section:This work was funded by the grant of Polish Ministry of Science and Higher Education N N303 550439 and by intramural grant of University of Warsaw 501/86- 100036. The research for this paper was in part supported by the EU through the European Social Fund, contract number UDA-POKL.04.01.01-00-072/09-00. PR obtained short term fellowship in JFC lab from EMBO no. ASTF 450.00-2010."} +{"text": "There was an error in the Funding statement. The correct version of the statement is available below.This study was financially supported by the Balassi Institute - Hungarian Scholarship Board Office and Institute for Ethnic and National Minority Studies of the Hungarian Academy of Sciences. Bakker Brothers, Bejo Zaden B.V., Daehnfeldt A/S and Syngenta Seeds Inc. are acknowledged for providing seeds for this study. Author BVN was supported by FAPESP (09/54292-7). The publication of this study was supported by the grant TAMOP 4.2.2/B-10/1-2010-0023. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."} +{"text": "The authors would like to add the following to the funding information: \"The Danish National Research Foundation, the Danish Cancer Society, the European Commission (projects Biomedreg-CZ.1.05/2.1.00/01.0030 and Infla-Care), and the Lundbeck Foundation (R13-A1287)."} +{"text": "The journal has been informed by the authors' institution that, contrary to the statement in this article , the MacThe pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2474/12/159/prepub"} +{"text": "The structures of new compounds were elucidated by extensive spectroscopic methods. The known compounds were identified by comparing their spectroscopic data with those reported in the literature. Five new 5,6-Supplementary material is available for this article at 10.1007/s13659-013-0003-1 and is accessible for authorized users. Supplementary material, approximately 4.09 MB."} +{"text": "The seventh author\u2019s name is spelled incorrectly. The correct name is: Saeed A. Al-Harthi.The affiliation for the seventh author is incorrect and the correct affiliation is not indicated. Saeed A. Al-Harthi is not affiliated with #3. The correct affiliation is: Department of Parasitology, Faculty of Medicine, Umm AL-Qura University, Makkah, Kingdom of Saudia Arabia."} +{"text": "To explore the association of lipid ratios and triglyceride (TG) with insulin resistance (IR) in a Chinese population. We also provide the clinical utility of lipid ratios to identify men and women with IR.This cross-sectional study included 614 men and 1055 women without diabetes. Insulin resistance was defined by homeostatic model assessment of IR > 2.69. Lipid ratios included the TG/ high density lipoprotein cholesterol (HDL-C), the total cholesterol (TC)/HDL-C and the low density lipoprotein cholesterol (LDL-C)/HDL \u2013C. Logistic regression models and accurate estimates of the area under the receiver operating characteristic (AUROC) curves were obtained.In normal-weight men, none of lipid ratios nor TG was associated with IR. In overweight/obese men, normal-weight women and overweight/obese women, the TG/HDL-C, the TC/HDL-C and TG were significantly associated with IR, and the associations were independent of waist circumference. All of the AUROCs for the TG/HDL-C and TG were > 0.7. The AUROCs for TC/HDL-C ratio were 0.69\u20130.77. The optimal cut-offs for TG/HDL-C were 1.51 in men and 0.84 in women. The optimal cut-offs for TG were 1.78 mmol/L in men and 1.49 mmol/L in women, respectively. In men, the optimal cut-off for LDL-C/HDL-C is 3.80. In women, the optimal cut-off for LDL-C/HDL-C is 3.82.The TG/HDL-C, the TC/HDL-C and TG are associated with IR in overweight/obese men, normal-weight and overweight/obese women. The LDL-C/HDL-C is only associated with IR in normal-weight women. The TG/HDL-C and TG might be used as surrogate markers for assessing IR. Insulin resistance (IR) is a major risk of type 2 diabetes and cardiovascular diseases . The golApart from the TG/HDL-C ratio, other lipid ratios or TG might also be used to predict IR. One study indicated that the low density lipoprotein cholesterol (LDL-C)/HDL-C ratio might be the best reliable marker of IR in non-obese Japanese adults . TG has The aim of the present study is to explore the associations of lipid ratios and TG with IR in a Chinese population. We also provide the clinical utility of lipid ratios to identify men and women with IR.All of data were drawn from a population-based, cross-sectional survey in Southern China. We have described the epidemiology study elsewhere . The stuThis study has been approved by the ethics committee of the Third Affiliated Hospital of Southern Medical University. Written informed consents were obtained. All of the work has been carried out in accordance with the Declaration of Helsinki.We used HOMA-IR to assess IR. Homeostatic model assessment of insulin resistance was calculated as the following formula: fasting plasma glucose (mmlo/L) \u00d7 fasting insulin (IU/L)/22.5 . Using bFasting serum lipids were determined using blood specimens after an overnight fasting for at least 10 hours. Using calorimetric methods with the Roche assay (Roche cobas6000), serum total cholesterol (TC), serum TG, and serum HDL- C were determined . Low denInformation on age, sex, education attainment, personal and family health history and lifestyle habits was obtained by questionnaires . AccordiAnthropometric measurements including height, weight, and waist circumference were measured by the World Health Organization recommended protocols . Waist cAll statistical analyses were performed using Stata (version 11). One previous study indicated that the association of the TG/HDL-C ratio with IR might be sex-dependant . Body fa2 in Chinese population [Insulin resistance exists not only in obese individuals but also in individuals with normal weight . The mecpulation . ClinicaTo explore whether lipid ratios and TG are associated with IR in Chinese individuals, logistic regression models were used and odds ratios (ORs) and 95% confidence interval (CI) were calculated. Lipid ratios and TG were used as an independent variable, respectively. Insulin resistance was defined as HOMA-IR > 2.69 and insulin sensitivity was defined if HOMA-IR < or = 2.69 . In the We used HOMA-IR as a continuous variable to examine the correlations of HOMA-IR with the lipid ratios and TG. The models were adjusted for age, educational attainment, current smoking, current alcohol use, physical inactivity, waist circumference, systolic blood pressure and diastolic blood pressure. All variables with a skewed distribution including HOMA-IR, TG, the TG/HDL-C ratio, the TC/HDL-C ratio and the LDL-C/HDL-C ratio were logarithmically transformed in the regression models.We also run the full models to explore the associations of TG/HDL-C ratio with IR in the whole population. Next, sex*TG/HDL-C ratio and BMI category* TG/HDL-C ratio were added into the adjusted models, respectively. The p-values for the interaction terms of sex* TG/HDL-C ratio and BMI category* TG/HDL-C ratio in the full models of IR were explored.Accurate estimates of the area under the receiver operating characteristic (AUROC) curve analysis was conducted by using the TG/HDL-C ratio, the TC/HDL-C ratio, the LDL-C/HDL-C ratio, and TG as continuous variables in the logistic regression models. Accurate estimates of AUROC were obtained. Insulin resistance is mostly caused by abdominal obesity and waist circumference is used as an alternative method to assess IR , 26. So Youden\u2019s index was calculated as (specificity + sensitivity \u22121) and used to select the optimal cut-offs for each lipid ratio and TG.Characteristics of male and female subjects in different BMI categories were presented in The association of the TG/HDL-C ratio and IR was first explored in the whole population. Next, sex*TG/HDL-C ratio and BMI category*TG/HDL-C ratio were added into the adjusted models, respectively. The interaction term of sex*TG/HDL-C ratio is 0.1 in the full model of insulin resistance and that of BMI category*TG/HDL-C ratio is <0.001.In normal-weight men, none of lipid ratios nor serum TG was associated with IR in the adjusted models. In overweight/obese men, the TG/HDL-C ratio, the TC/HDL-C ratio and TG were significant associated with IR, and the associations were independent of waist circumference and other potential confounders. The OR was 1.80 for one unit increase in the TG/HDL-C ratio, 1.67 for one unit increase in the TG/HDL-C ratio, or 1.36 for one mmol/L increase in TG, respectively.In normal-weight women, all of lipid ratios and TG were significantly associated with IR and the associations were independent of other confounders. Every unit increase in the TG/HDL-C ratio was associated with OR 3.17 . Every unit increase in the TC/HDL-C ratio was associated with OR 1.67 . Every unit increase in the LDL-C/HDL-C ratio was associated with OR 1.55 . One mmol/L increase in TG was associated with OR 2.62 .In overweight/obese women, the TG/HDL-C ratio, the TC/HDL-C ratio, and TG were associated with IR. The OR for the TG/HDL-C ratio (every unit increase) was 2.85 . The OR for the TC/HDL-C ratio (every unit increase) was 2.01 . The OR for TG (each mmol/l increase) was 2.02 , respectively.When HOMA-IR was used as a continuous variable in the adjusted regression models, TG, the TG/HDL-C ratio and the TC/HDL-C ratio were significantly association with HOMA-IR in both the normal-weight or overweight/obese men and women (P < 0.05). The Pearson correlation coefficient between TG and HOMA-IR was 0.32\u20130.37. The Pearson correlation coefficient between the TG/HDL-C ratio and HOMA-IR was 0.33\u20130.41. The Pearson correlation coefficient between the TC/HDL-C ratio and HOMA-IR was 0.24\u20130.33. The LDL-C/HDL-C ratio was only associated with HOMA-IR in normal-weight women (The Pearson correlation coefficient was 0.07\u20130.22).In logistic regression models, waist circumference was associated with IR in normal-weight men (not shown in the table). The AUROC for waist circumference was 0.71 (95% CI 0.61\u20130.81) . In overIn normal-weight women, waist circumference, the TG/HDL-C ratio, the TC/HDL-C ratio, the LDL-C/HDL-C ratio and TG were suitable predictors for IR. Among four variables, the TG/HDL-C ratio and TG were better predictors for IR than waist circumference and the TC/HDL-C ratio. The TG/HDL-C ratio and TG had significantly higher AUROCs than waist circumference (P < 0.05) . In overBMI was also a suitable predictor for IR in men and normal-weight women (the AUROC > 0.7), but it was not better than waist circumference, TG and the TG/HDL-C ratio .The optimal cut-offs for lipid ratios and TG are listed in 2 in men and 23.44 kg/m2 in women).The cut-offs for waist circumference were 87 cm in men and 81 cm in women. Men and women had different cut-offs for BMI could provide a predictive value . The TC 2 in men and women in Chinese population [2 in men and 23.44 kg/m2 in women).In the present study, it is shown that the optimal cut-off for the TG/HDL-C ratio is 1.51 for men and this result is similar to the previous study . Based opulation . HoweverIn the present study, we did not use the gold standard method, the hyperinsulinemiceuglycemic clamp, to detect IR. This is a major limitation of the present study. Several alternative methods can be used to detect IR. These methods included HOMA-IR, fasting insulin, Quantitative insulin sensitivity check index (QUICKI) , glucoseIn the present study, we found that in men with normal weight, none of the lipid ratios nor TG was a reliable marker of IR. Because the sample size is relatively small, the associations of lipid ratios and IG with IR need to be further explored. Waist circumference can be used to predict IR in normal-weight men, and the AUROC for waist circumference was 0.86. In our previous study, it was found that waist circumference is associated with IR and metabolic syndrome in normal-weight subjects . But in Other limitations of the present study include two additional points. This was only a cross-sectional study not a longitudinal study. The sample might be bias because only 37% of subjects were men. However, we have analyzed the data in men and women, respectively. The main advantages included using three lipid ratios including the TG/HDL-C ratio, the TC/HDL-C ratio and the LDL-C/HDL-C ratio and the sample was relatively large compared with previous studies , 10, 11.The TG/HDL-C ratio, the TC/HDL-C ratio and TG are associated with IR in overweight/obese men and both normal-weight and overweight/obese women. All of the AUROCs for the TG/HDL-C, and TG were > 0.7. The AUROCs for TC/HDL-C ratio were 0.69\u20130.77. The LDL-C/HDL-C ratio should not be used as a reliable predictor for IR in both men and overweight women. In normal-weight men, none of the lipid ratios nor TG was associated with IR, and waist circumference is significantly associated with IR in normal-weight men. The TG/HDL-C ratio and TG might be recommended as surrogate markers of IR in overweight/obese men, normal-weight and overweight/obese women."} +{"text": "The structures of the new compounds were elucidated on the basis of spectroscopic and chemical methods.Two new apotirucallane triterpenoids, namely chisiamols G (Supplementary material is available for this article at 10.1007/s13659-012-0065-5 and is accessible for authorized users. Supplementary material, approximately 3.74 MB."} +{"text": "Cationic antimicrobial peptides are major components of innate immunity and help control the initial steps of the infectious process. They are expressed not only by immunocytes, but also by epithelial cells. They share an amphipathic secondary structure with a polar cationic site, which explains their tropism for prokaryote membranes and their hydrophobic site contributing to the destructuration of these membranes. LL-37 is the only cationic antimicrobial peptide derived from human cathelicidin. LL-37 can also cross the plasma membrane of eukaryotic cells, probably through special domains of this membrane called lipid rafts. This transfer could be beneficial in the context of vaccination: the activation of intracellular toll-like receptors by a complex formed between CpG oligonucleotides and LL-37 could conceivably play a major role in the building of a cellular immunity involving NK cells. Epithelia are constantly exposed to potential pathogens. The digestive tract, the upper respiratory airways, the genital tract and the skin are, among others, body surfaces interacting with the surroundings and very often a door to aggression by a pathogen. The response of adaptative immunity to this aggression is highly efficient and very often successful in fighting an infection, but adaptation is slow and linked to delay. In a first step, cells producing membrane-anchored antibodies with reasonable affinity to the antigen have to be selected. In a second step, these antibodies are secreted in the extracellular medium. The tremendous dilution of these proteins decreases their concentration to extremely low values, which jeopardizes their interaction with the antigen. The dilution must be compensated by an increase of the affinity of the protein for the antigen. This affinity maturation is the consequence of a very high mutational rate of the gene, a high proliferation rate of the cells and a very strong selective pressure. This process is considered a paradigm for evolution, but at a timescale of a few days rather than a few million years . A similDefensins are small (approximately 4 kDa), cysteine-rich, cationic peptides which have been identified in plants, insects, and a large variety of higher-level mammals . They haN-terminus, followed by a propeptide sequence, and the mature defensin peptide at the C-terminus [N-terminal sequence of about 40 amino acids. This prosequence is anionic which probably accounts for the binding of the mature defensin to the propeptide. \u03b1-Prodefensin acts as a chaperone and contributes to the structure of mature defensin. It also blocks the activity of the mature defensin preventing damages to the producing cells [N-terminal extension of the proform has a very short amino acid sequence; the proform has bactericidal activity [The genes for \u03b1- and \u03b2-defensins are located on chromosome 8. They form a cluster within a < 1 Mb region at the locus p22-23 [ng cells . In man,ng cells , kallikrng cells or metalng cells . The \u03b1-dng cells . The \u03b2-dactivity and evenactivity . The matactivity . They aractivity . Both \u03b1-activity . et al. [cathepsin Linhibitor. The human cathelicidin has 18 kDa (hCAP-18) and is a major protein in specific granules of neutrophils [The first antimicrobial peptide of this group was isolated from pig . The teret al. to descrtrophils . It is atrophils and in ttrophils ,38. Sevetrophils ,40,41. Ptrophils . The preC-terminal domain of the protein. Zaiou et al. [C-terminal domain of cathelicidin is very variable among species. In man, the only peptide derived from hCAP has 37 amino acids and two leucines at its N-terminal, hence the acronym LL-37 [Considering the conservation of this proregion during evolution, it might play an important biological function with respect to the maturation of the antimicrobial peptide which is the u et al. reportedym LL-37 . The humym LL-37 , a locusym LL-37 . This miym LL-37 ,48. The ym LL-37 ,50, thenym LL-37 . The relym LL-37 or elastym LL-37 . In vagiym LL-37 . In humaym LL-37 . These fym LL-37 . These nym LL-37 . It neutym LL-37 and it iym LL-37 . It alsoym LL-37 . Salmonella typhimurium survive better in macrophages from mice which do not express the cathelicidin related antimicrobial peptide (CRAMP), the murine analog of LL-37, than from wild-type (WT) mice [Staphylococcus aureus [et al. [Pseudomonas aeruginosa. They also showed that the transfer of the LL-37/hCAP18 gene restored bacterial (Pseudomonas aeruginosa and Staphylococcus aureus) killing in a human cystic fibrosis bronchial xenograft model [Patients with atopic dermatitis suffer not only from chronic cutaneous inflammation but are also affected by recurrent infections provoked by bacteria, viruses or fungi . The skiWT) mice . These ms aureus or to mes aureus and to is aureus . Convers [et al. demonstrft model . Cathelift model . It is aft model .At the structural level, efforts were made to understand how LL-37 and related peptides interact with the membrane and what type of perturbation they may cause. To have access to the molecular level, simplified systems were used, comprising phospholipid bilayers made of a limited series of commonly found phospholipids. Since cathelicidins have a positive balance of cationic amino acid side-chains, the focus was put on the possible difference between zwitterionic and acidic phospholipid-containing membranes.versus eukaryotic membranes was partly addressed at the lipid level, using lipids present in both types of organisms, but which are not fully exposed to the outer membrane leaflet, such as the acidic phospholipid phosphatidylserine (PS), phosphatidylglycerol (PG) and the non-bilayer forming unsaturated phosphatidylethanolamine (PE), the latter being abundant in prokaryotes. Whereas it was initially demonstrated that similar leakage occured in zwitterionic palmitoyl-oleoyl-phosphatidyl-choline (POPC) vesicles as well as in charged palmitoyl-oleoyl-phosphatidyl serine/palmitoyl-oleoyl-phosphatidylcholine (POPS/POPC) vesicles when considering K+ permeabilization [p-xylene-bis-pyridinium bromide (ANTS/DPX) with a slight preference for the PC:SM:CHOL mixture at low peptide concentration, especially with the orang-utan orthologue of the human LL-37 [The interpretation of initial circular dichroic studies on the peptide diluted in pure water led to conclude that LL-37 adopted a random conformation in aqueous solution. It became rapidly obvious that the peptide could structure itself either in the presence of the salts found in physiological fluids or at hilization , furtherlization . In anotan LL-37 .et al. [et al. [In accordance with typical antimicrobial peptide behavior, cholesterol diminished LL-37 induced leakage in the study of Zhang et al. , whereas [et al. . From th [et al. . Clearly [et al. or membr [et al. . Many st [et al. . Demonst [et al. .et al. [N-terminal is still able to aggregate. In contrast, deletion of the 14 C-terminal residues greatly reduces the aggregation of LL-23 [C-terminal fragment (IG-25) retains the toxic activity on bacteria and eukaryotic cells [C-terminal tripeptide from FK-16 yields FK-13, a peptide nearly devoid of any activity on bacteria and eukaryotic cells. KR-12 has three arginines and two lysines. It covers the cationic rich region of LL-37. This peptide is the smallest fragment of LL-37 that is still active against Gram-negative bacteria [LL-37 is a peptide with 35% hydrophobic residues. It has a high content of basic residues and at neutral pH it has a positive charge (+6). In spite of their very diverse primary structures, antimicrobial peptides seem to share some common structural characteristics . They all form amphipathic secondary structures with a cationic and a hydrophobic face ,83. Thiset al. have suget al. . KR-20, of LL-23 . KE-18 aof LL-23 ,93 and iof LL-23 . When LLic cells . The sambacteria . A longebacteria . This dibacteria . The lonper se, and may resemble classical cell-penetrating peptides in both structure and mode of action. These peptides, with the 16-residue penetratin as their prototypical representative, have been the subject of many studies over the past decade [versus lysine [As discussed in the previous section, the mode of action of LL-37 requires the peptide to adopt an amphipathic helix conformation. However, shortened versions like KR-12, FK-16, and especially KR-20, are not amphipathic t decade and are t decade . Despitet decade with pept decade . For pept decade . Molecult decade ; bindingt decade , a secont decade . Surprist decade , althougt decade . Adsorptt decade . Howevert decade . Investit decade and a get decade . The rolt decade ,101,108 t decade ,109 and t decade . The fret decade , making t decade . The fort decade . The keyt decade ,111,112.t decade , solid-st decade . Althougs lysine . The strs lysine . Complexs lysine ,115, allStreptococcus pyogenes secrete a protein which binds to and neutralizes LL-37 [LL-37 interacts with the membrane of bacteria mostly by electrostatic interactions. The outer membrane of many Gram-negative bacteria contains LPS with lipid A, an anionic lipid forming the outer leaflet of the membrane. The transfer of aminoarabinose to lipides LL-37 . Inhibites LL-37 .Staphylococcus aureus on intravenous catheters [et al. [Pseudomonas aeruginosain vitro. It also affected preexisting biofilms. Instillation of LL-37 also inhibited the formation of a biofilm by Pseudomonas aeruginosa in a model of sinusitis developed in rabbits [et al. [Staphylococcus epidermidis in wells of microtitre plates and the formation of a biofilm by these bacteria. Amer et al. [Francisella.Bacteria spend most of their life not in planktonic conditions but within a biofilm. In a first step the bacteria adhere to a support. Planktonic and adherent bacteria have a different transcriptome . The celatheters . Overhag [et al. reported rabbits . More re [et al. reportedr et al. used a set al. [et al. [11 receptor of glial cells, was activated by LL-37. This interaction provoked the expression of various cytokines and the activation of phosphorylation pathways. The activation of mast cells by LL-37 might also be mediated by a GPCR. Indeed, the peptide was chemotactic for these cells and stimulated their degranulation [et al. [7 receptors. It was not secondary to the release of ATP since apyrase, an enzyme which catabolizes extracellular ATP, had no effect on the response to LL-37. These results suggested that the P2X7 receptor was a target for LL-37. This receptor is an ionotropic purinergic receptor with two transmembrane domains [4 receptors [et al. [7 receptors with their ability to form an alpha-helix and to aggregate. LL-37 also provokes cell proliferation and wound healing [The original description of cationic peptides as antimicrobial agents put the emphasis on their deleterious effects on bacteria. The specificity of cationic antimicrobial peptides for bacterial membranes was explained by the anionic properties of bacterial surfaces . The higet al. reported [et al. observednulation and theinulation . These rnulation . Elssner [et al. reported domains . After aeceptors . More re [et al. correlat healing . It can healing ,155,156. healing . This ac7 receptors by modifying the physico- chemical state of the membrane rather than by directly interacting with the receptor. Such a model has been proposed by Zughaier et al. [et al. [2 and by inhibiting phospholipase D [et al. [7 receptors by binding to the intracellular C-terminal domain of the receptor [7 has a very long C-terminal extension through which this receptor interacts with intracellular proteins [et al. reported that the peptide could cross the plasma membrane [These multiple effects of LL-37 and the diversity of plasma membrane receptors which might be affected by the peptide raised some doubts on a direct interaction between these receptors and the peptide ,158. Forr et al. and by D [et al. to expla [et al. . This delipase D . Accordilipase D , these e [et al. , who sugreceptor . Indeed,proteins . In 2004membrane . The pepmembrane . Such a membrane . AccordiN-terminal glutamate (RQIKIWFQNRRMKWKK) (peptide 43-58) [Shigella dysenteriae (RFRQIQRGFR) or in the A chain of ricin from Ricinus communis (RTRIRYNRR) [et al. [et al. [et al. [LL-37 belongs to the cell-penetrating peptide family ,169. Thee 43-58) , and thee 43-58) , the firRIRYNRR) . In soluRIRYNRR) ,175. In RIRYNRR) ,177,178.RIRYNRR) . These pRIRYNRR) . They miRIRYNRR) ,182, or RIRYNRR) . The intRIRYNRR) . The intRIRYNRR) , but thiRIRYNRR) and the RIRYNRR) . This isRIRYNRR) . These pRIRYNRR) ,190. Con [et al. showed t [et al. confirme [et al. reported [et al. . Indeed, [et al. which ac [et al. and NK c [et al. . Activat [et al. . They al [et al. . The act [et al. . The inj [et al. . Tumours [et al. . These p [et al. ,204. The [et al. reportedLL-37 was originally described as an antimicrobial peptide. The development of analogs of the peptide with increased resistance to proteases and less cytotoxicity on eukaryotic cells, or the synthesis of molecules mimicking LL-37 is a promising strategy to eradicate multiresistant bacteria . Low, no"} +{"text": "The major adverse consequences of obesity are associated with the development of insulin resistance (IR) and adiposopathy. The Homeostasis Model Assessment-Adiponectin (HOMA-AD) was proposed as a modified version of the HOMA-IR, which incorporates adiponectin in the denominator of the index.Evaluate the performance of the HOMA-AD compared with the HOMA-IR as a surrogate marker of IR in women, and to establish the cutoff value of the HOMA-AD.2 and non-diabetic. The IR was assessed by the indexes HOMA-IR and HOMA-AD and by the hyperglycemic clamp (n=49). Metabolic syndrome was defined using the IDF criteria.The BRAMS is a cross-sectional multicenter survey. The data from 1.062 subjects met the desired criteria: 18-65 yrs. old, BMI: 18.5-49.9 Kg/mFor the IR assessed by the clamp, the HOMA-AD demonstrated a stronger coefficient of correlation (r=-0.64) compared with the HOMA-IR (r=-0.56); p<0.0001. In the ROC analysis, compared with the HOMA-IR, the HOMA-AD showed higher values of the AUC for the identification of IR based on the clamp test (AUC: 0.844 vs. AUC: 0.804) and on the metabolic syndrome (AUC: 0.703 vs. AUC: 0.689), respectively; p<0.001 for all. However, the pairwise comparison did not suggest superiority for the HOMA-AD in the diagnostic of IR (p>0.05). The optimal cutoff identified for the HOMA-AD for the diagnosis of IR was 0.51.HOMA-AD was demonstrated to be a useful surrogate marker for detecting IR among adult women and presented a similar performance as the HOMA-IR."} +{"text": "There is an error in the first sentence of the Funding section. The correct sentence is: The work was supported by the National Science Centre under the project DEC-2013/09/B/ST6/03117 and European Social Fund project UDA-POKL.04.01.01-00-106/09.The correct, complete Funding section should read: The work was supported by the National Science Centre under the project DEC-2013/09/B/ST6/03117 and European Social Fund project UDA-POKL.04.01.01-00-106/09. The work was performed using the infrastructure supported by POIG.02.03.01-24-099/13 grant: \u201cGeCONiI---Upper Silesian Center for Computational Science and Engineering\u201d. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."} +{"text": "The UK College of Emergency Medicine recommends that level 1 ultrasound competency is a basic standard for EM doctors and is now mandatory for career progression. Focused Assessment with Sonography in Trauma to include the detection of pleural fluid and pneumothorax (the Extended-FAST scan) forms part of this competency. We compare the diagnostic accuracy of E-FAST with the \u201cgold standard\u201d of CT or operative intervention. Trauma team leaders were asked to evaluate point-of-care ultrasound in their decision-making and patient management.Royal London Hospital. The Major Trauma Centre for North East London and base for London's Air Ambulance. Approximately 2500 adult trauma cases seen per year.Prospective observational study, comprising convenience sample of adult major trauma presenting to Royal London between October 2012-March 2013 leading to activation of a \u201ctrauma call\u201d.To assess the diagnostic accuracy of E-FAST in the detection of haemorrhage (free fluid) and pneumothorax in major trauma.To assess the impact of E-FAST on trauma team leader's decision-making process in major trauma care.Free fluid or pneumothorax formally reported on CT or found at time of surgical intervention117 patients initially recruited, 45 allowed comparison to reference standard. Sensitivity, Specificity, Positive and Negative Predictive Values for E-FAST were 68.4% (43.5-87.4), 96.3% (81-99.4), 92.9% (66.1-98.8) and 81.3% (63.6-92.8) respectively. 58% of team leaders stated that ultrasound guided their decision-making.E-FAST has limited sensitivity but high specificity when used in isolation. It influenced trauma team leader's decision-making 58% of the time, despite reported low sensitivity. The major role of ultrasound is the rapid triage of unstable patients and localization of major haemorrhage to help guide immediate life-saving intervention in this subgroup of patients. May reduce CT load in selected patients but further research needed."} +{"text": "The access control for the article was CC-BY-NC-ND, and has since been changed to CC-BY. The copyright statement now reads as follows:An error appeared in 1 August 2013 issue of the journal [Nicol MP, Spiers K, Workman L, et al. Xpert MTB/RIF Testing of Stool Samples for the Diagnosis of Pulmonary Tuberculosis in Children. \u00a9 The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.The authors regret this error."} +{"text": "Dis. Model. Mech. 8, 1323-1337.There was an error published in PSD-95 gene by RT-PCR. Therefore, the data shown in Fig.\u00a07I do not represent the expression levels of the PSD-95 mRNA. This error has null impact on the main conclusions of the study.An erroneous TaqMan assay was used to evaluate the expression of the The authors apologise to the readers for this error."} +{"text": "In the Funding section, the following grant number from the Grant Agency of the Czech Republic is missing: P303-11-2378. The correct, complete funding information is as follows: This study was supported by the grants GA CR 13-02154S, P303-11-2378 and P304/12/G069 from the Grant Agency of the Czech Republic, by the project BIOCEV CZ.1.05/1.1.00/02.0109 from the European Regional Development Fund and by the Operational Programme Education for Competitiveness CZ.1.07/2.3.00/30.0045 from the European Social Fund and the state budget of the Czech Republic. The authors state that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."} +{"text": "In the original version of the manuscript there waThe correct versions of http://www.mdpi.com/1422-0067/15/6/9789/s1.The corrected version of the paper can be accessed at"} +{"text": "CDC is assisting ministries of health and working with other organizations to end the ongoing epidemic of Ebola virus disease (Ebola) in West Africa . The updAccording to the latest World Health Organization update on January 28, 2015 , a totalEight cases and six deaths were previously reported from Mali ,5. No nehttp://www.cdc.gov/vhf/ebola/outbreaks/2014-west-africa/index.html. The most up-to-date infection control and clinical guidelines for the Ebola epidemic in West Africa are available at http://www.cdc.gov/vhf/ebola/hcp/index.html.The latest updates on the ongoing Ebola epidemic in West Africa, including case counts, are available at"} +{"text": "The CE-MARC study was the largest prospective evaluation of the diagnostic accuracy of cardiovascular magnetic resonance (CMR) in coronary artery disease (CAD), demonstrating its superiority over single-photon emission computed tomography . The triAll patients from the CE-MARC population were studied. Visual CMR analyses were from the original, blinded read. Pre-specified sub-analysis of the four individual core components of the CMR protocol was performed in isolation, as a paired component (perfusion and LGE) and as a triplet and compared to the full multi-parametric protocol.Both CMR and X-ray angiography were available in 676 patients. The sensitivity of the combined CMR protocol was 86.5%, specificity 83.4%, PPV 77.2% and NPV 90.5%. The diagnostic accuracy of the individual components and paired and triplet combinations compared to the full multi-parametric protocol are presented in Table A combined multi-parametric CMR protocol has higher sensitivity, PPV and NPV that the individual components however the specificity of the individual components of perfusion, ventricular function and late gadolinium enhancement (LGE) all performed significantly better than the multi-parametric protocol.This study was funded by the British Heart Foundation (RG/05/004). J.P.G and S.P. received research grant from Philips Healthcare. S.P. is funded by a British Heart Foundation fellowship (FS/10/62/28409)."} +{"text": "Scientific Reports6: Article number: 2254610.1038/srep22546; published online: 03042016; updated: 04202016.The Acknowledgements section in this Article is incomplete.\u201cThis work has been supported by the Russian Fund for Basic Research within the project 16-52-00112. The calculations of multipole moments and fields has been supported by the Russian Science Foundation Grant No. 16-12-10287.\u201dshould read:\u201cThis work has been supported by the Russian Fund for Basic Research within the project 16-52-00112. A.S.S. acknowledges the support of the President of Russian Federation in the frame of Scholarship SP-4248.2016.1. The calculations of multipole moments and fields has been supported by the Russian Science Foundation Grant No. 16-12-10287.\u201d"} +{"text": "Temporomandibular joint (TMJ)-related pain also includes the craniomandibular and cervicocranial areas due to the topographic-functional closeness.To determine the relationship between types of headaches and cervical spine (CS) disorders in a patients with osteoarthritis (OA) of TMJ with one-year-follow-up.65 patients were consecutively treated for signs and symptoms of OA of TMJ. A definitive diagnosis of OA was confirmed by magnetic resonance imaging. The patients were examined by a dentist, a neurologist, and a physiatrist-rheumatologist. Pain intensity in TMJ was shown on the visual-analogue scale (VAS 0-10). They were treated by an occlusal splint and physical therapy with one-year-follow-up.The applied treatment modalities achieved a significant reduction of pain (p<0.001) in the TMJ at first examination and one-year-follow-up . 46.2% of patients did not have a CS diagnosis and 53.9% of patients did not have headaches. 16.9% of them had migraines, 23.1% had CS-related headache and 6.1% of patients had tension-types headaches. Cervical syndrome was found in 10.8% of patients. 26.1% had cervicobrachial syndrome, 7.7% had cervicocephalic syndrome and 9.2% of patients had both. The SC syndrome was significant regarding the patients\u2019 age , whereas there were no differences for headaches.The relationship of SC disturbances with the higher age of patients was determined in patients with OA of TMJ. The existence of comorbidity with headaches does not affect treatment success of TMJ.No conflict of interest."} +{"text": "There is an error in the beginning of the second paragraph of the \u201cSystematic description of identified woods\u201d sub-section of the Observations and Results. The correct sentence is: Artocarpus sp. cf. A. lacucha Buch-Ham., Moraceae, Figure S1, 1-5 in File S1; Figured Specimen \u2013 BSIP Museum No. 40081.There is an error in reference 91. The correct reference is: Rajendran CP, Rajagopalan G, Narayanaswamy (1989) Quaternary geology of Kerala: evidence from radiocarbon dates. Journal of Geological Society of India 33: 218 - 222.There is an error in the legend for"} +{"text": "Flavobacterium psychrophilum strain KTEN-1510, with genotype A/G-C. This strain was isolated in October 2015 from the gills of an ayu in the upper Kagami River in central Kochi Prefecture on Shikoku Island, Japan.In this paper, we describe the draft genome sequence of Plecoglossus altivelis altivelis) is a popular angling fish in Japan. A large number of juvenile ayu reared in hatcheries or collected from Lake Biwa, local rivers, and marine coasts are released annually in fishing areas to enhance ayu stocks. Flavobacterium psychrophilum, a member of the family Flavobacteriaceae .Ayu . In this paper, we describe the draft genome sequence of F. psychrophilum strain KTEN-1510, isolated in October 2015 from the gills of an ayu caught in the upper reaches of the dam using the Japanese fishing method known as Tomozuri.The Kagami River is located in the central Kochi Prefecture on Shikoku Island in Japan. The river is 31\u00a0km long, with a drainage basin area of 170\u00a0kmde novo Assembler version 2.9 software (Roche). The assembly of strain KTEN-1510 consisted of 182 contigs (>500\u00a0bp) totaling 2,705,210\u00a0bp, with a G+C content of 32.5%. We annotated the sequence using the Microbial Genome Annotation Pipeline (MiGAP) version 2.19 assay of the peptidyl-prolyl cis-trans-isomerase C (PPIC) gene. Type A was detected only in isolates from ayu. Fujiwara-Nagata et al. (gyrA) gene. Type G-C showed strong pathogenicity to ayu, whereas the other three were not or only weakly pathogenic. Both PCR-RFLP- and SNP-based genotyping identified strain KTEN-1510 as type A/G-C (data not shown). The present whole-genome analysis of the virulent strain of BCWD increases our understanding of the mechanisms underlying the pathogenicity of F. psychrophilum.The genomic DNA of KTEN-1510 was extracted, purified, and sequenced according to our previously used method . Genome BCNG00000000. The version described in this paper is the first version, BCNG01000000.The draft genome sequence of strain KTEN-1510 has been deposited in GenBank under accession no."} +{"text": "The abbreviation for Herion self-administration (HSA) is incorrectly written as HAS in two locations in the Materials and Methods section. The subheading for section 2.9.1 should read: Acquisition of HSA. The subheading for section 2.9.2 should read: Dose-reduction and reinstatement of HSA. The publisher apologizes for this error."} +{"text": "There is an error in the first name of the co-author, Dr. Xian-Tao Liu. The correct name for this co-author is: Dr. Xiang-Tao Liu."} +{"text": "After publication of the original article it was bhttp://www.usefil.eu), as well as the LLM Care (www.llmcare.gr) self-funded initiative that emerged as the business exploitation of the Long Lasting Memories (LLM Project) (www.longlastingmemories.eu) originally funded by the ICT-CIP-PSP Programme.This work was supported in part by the European Union\u2019s Seventh Framework Programme (Project USEFIL, GA 288532;"} +{"text": "S. aureus. The Introduction should state that ClfA was the first fibrinogen \u03b3-chain-binding protein isolated in S. aureus.There is an error in the published article. Paragraph eight of the Introduction incorrectly states that ClfA was the first fibronectin-binding protein isolated in"} +{"text": "Nature Communications 10.1038/s41467-022-31506-x, published online 08 July 2022Correction to: The original version of this Article contained an error in Fig. 1 and Fig. 3. In the original version of Figs. 1 and 3, the sugars were drawn with the L form instead of the D form. The sugars in this work were obtained from the naturally occurring D enantiomers.The correct version of Fig. 1 is:which replaces the previous incorrect version:The correct version of Fig. 3 is:which replaces the previous incorrect version:This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Nature Communications 10.1038/s41467-022-34884-4, published online 25 November 2022Correction to: The original version of this Article omitted an acknowledgement to the source of Supplementary Fig.\u00a0ACS Energy Lett. 6, 2369\u20132377 (2021).The original version of this Article contained wrong reference for Ref. 33. The correct Ref. 33 is: Mavrikis, S. et al. Effective hydrogen peroxide production from electrochemical water oxidation. These have been corrected in the PDF and HTML versions of the Article.Updated Supplementary Information"} +{"text": "Nature Communications 10.1038/s41467-020-17878-y, published online 13 August 2020Correction to: The original version of this Article contained an error in Fig. 6d, in which an incorrect western blot was included. This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Scientific Reports 10.1038/s41598-022-14426-0, published online 17 June 2022Correction to: In the original version of this Article, reference 9 was erroneously cited twice. As a result, the subsequent references were incorrectly numbered. The reference list was correct at the time of publication.The original Article has been corrected."} +{"text": "Correction to: BMC Nursing (2022) 21:262. 10.1186/s12912-022-01037-3.Following publication of the original article [1], the authors identified an error in the author names. The given names and family names of three of the authors were erroneously transposed.The incorrect author names are: Lu Dongyan, Xu Mengqi and Hu Sanlian.The correct author names are: Dongyan Lu, Mengqi Xu and Sanlian Hu.The author group has been updated above and the original article has been"} +{"text": "Cell Death Discov 10.1038/s41420-021-00776-7, published online 9 December 2021Correction to: The original version of this article unfortunately contained errors in Figures 2 and 4. The authors apologize for the mistake. The corrected figures can be found below. The original article has been corrected."} +{"text": "Experimental & Molecular Medicine 10.1038/s12276-018-0164-4, published online 29 October 2018Correction to: After online publication of this article, the authors noticed an error in the Results section.In the original article, there was a mistake in Fig. 6A as published. The original Fig. 6A was provided to examine the infection efficiency of the recombinant lentivirus , in which duplicated images were found among the Lenti-NC, Lenti-over/miR-29b-3p and Lenti-inhibit/miR-29b-3p groups. The authors have provided new versions of Fig. 6A. The corrections will not affect the results and scientific conclusions of the article.The authors apologize for any inconvenience caused.The original article has been corrected."} +{"text": "Communications Chemistry 10.1038/s42004-020-0285-2, published online 26 March 2020.Correction to: The original version of this Article incorrectly designated Kun Wang as a corresponding author and listed their e-mail address in error. This has now been corrected in the PDF and HTML versions of the Article."} +{"text": "Nature Communications 10.1038/s41467-019-11591-1, published online 14 August 2019.Correction to: The original version of this article contained errors in Tables 1 and 2, which don\u2019t affect the conclusions. In Table 1, several numbers were incorrectly given in columns corresponding to the samples #548327, #721214 and #809653. The previous version of Table 1 was:Original Table 1. Overview of mutation discovery and detection in eWGS and scRNA-seq data.The correct version appears as:Revised Table 1. Overview of mutation discovery and detection in eWGS and scRNA-seq data.In Table 2, several numbers were incorrectly given in columns corresponding to the samples #548327, #721214, and #809653. The previous version of Table 2 was:Original Table 2. Frequency of cells containing multiple mutations in each case.The correct version appears as:Revised Table 2. Frequency of cells containing multiple mutations in each case.These errors have been corrected in the PDF and HTML versions of the article."} +{"text": "Eye 10.1038/s41433-021-01746-0, published online 13 August 2021Correction to: Unfortunately, a comment from the author to our vendor was published in the part Data collection.We apologize for this mistake.The original article has been corrected."} +{"text": "Cell Death & Disease 10.1038/s41419-022-04588-0, published online 08 February 2022Correction to: The original version of this article unfortunately contained two errors. For figure 5D an incorrect image was used during the figure preparation. The corrected figure can be found below. In addition, the product number of the antibody to myocardin (MYOCD) should be SAB4200539. The authors apologize for the errors. The original article has been corrected."} +{"text": "Nature Communications 10.1038/s41467-017-02811-7, published online 2 February 2018.Correction to: Since the publication of this work, Eli M Carrami has changed their name from Mohammad Karaminejadranjbar. This has now been amended in the HTML and PDF versions of the article."} +{"text": "Scientific Reports 10.1038/s41598-022-18814-4, published online 05 September 2022Correction to: The original version of this Article contained an error in the spelling of the author Hamimatunnisa Johar, which was incorrectly given as Hamimatunissa Johar.The original Article has been corrected."} +{"text": "Nature Communications 10.1038/s41467-022-28836-1, published online 3 March 2022.Correction to: The original version of this Article contained an error in Fig. 5, in which the legend incorrectly states that the magenta/black shading indicates downward/upward wave propagation This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Nature Communications 10.1038/s41467-021-26788-6, published online 11 November 2021.Correction to: The original version of this Article incorrectly acknowledged Eytan Ruppin as the sole corresponding author instead of Aniruddha J. Deshpande and Eytan Ruppin as co-corresponding authors. This has now been corrected in both the PDF and HTML versions of the Article."} +{"text": "Nature Communications 10.1038/s41467-022-28225-8, published online 04 February 2022Correction to: The original version of this Article contained an error in Fig. 1c. The image shown in the bottom right panel was inadvertently duplicated from the top right panel. This error was introduced during the revision process. This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Communications Biology 10.1038/s42003-022-04270-3, published online 26 November 2022.Correction to: The original version of this Article contained a formatting error in Fig. 1C, in which the example images for the CNN classification are not displayed properly. The correct version of Fig. 1C is:which replaces the previous incorrect version:This has now been corrected in both the PDF and HTML versions of the Article."} +{"text": "Correction to: Clinical Rheumatologyhttps://doi.org/10.1007/s10067-022-06047-9In the original version of the above article, The figures and legends used were incorrect. The correct figures are presented as follows Figs., and 4:The original article has been corrected."} +{"text": "Correction to: Health Justice 9, 1 (2021)https://doi.org/10.1186/s40352-020-00127-1Following the publication of the original article . Statutory rape: a guide to state laws and reporting requirements. Office of the Assistant Secretary for Planning and Evaluation The original article (Cook et al.,"} +{"text": "Scientific Reports 10.1038/s41598-022-12040-8, published online 18 May 2022Correction to: In the original version of this Article, Mohammadreza Samaei was omitted as a corresponding author. Correspondence and requests for materials should also be addressed to mrsamaei@sums.ac.ir."} +{"text": "Nature Communications 10.1038/s41467-022-32987-6, published online 21 October 2022Correction to: The original version of this Article contained an error in Fig. 5, in which the schematic arrows were incorrectly numbered. This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Scientific Reportshttps://doi.org/10.1038/s41598-018-31688-9, published online 06 September 2018Correction to: This article contains an error in the Acknowledgements section.This work was supported by the Fonds National de la Recherche Luxembourg .should read:This work was supported by the Fonds National de la Recherche Luxembourg ."} +{"text": "Signal Transduction and Targeted Therapy 10.1038/s41392-021-00765-3, published online 22 October 2021Correction to: 1. The correct data are provided as follows. The key findings of the article are not affected by these corrections. The original article has been corrected.Scale bar in Fig. During the preparation of Fig. During the preparation of Supplementary Fig. In the process of collating the raw data, the authors noticed several inadvertent mistakes occurred in Fig. Supplementary MaterialsRevised Supplementary figure 1"} +{"text": "Curr Issues Crim Just. (2005) 17:284\u201390. doi: 10.1080/10345329.2005.12036355\u201d was included but not referenced. It has been removed from the reference list.In the original article, the reference for \u201cGrewcocForensic Sci Int: Genet Suppl Ser. (2017) 6:e43-e45. doi: 10.1016/j.fsigss.2017.09.009\u201d was included but not referenced. It has been removed from the reference list.In the original article, the reference for \u201cWard J.Psychol Med. (2019) 49:2764\u201371. doi: 10.1017/S0033291718003793\u201d was included but not referenced. It has been removed from the reference list.In the original article, the reference for \u201cIsuru AAus J For Sci. (2018) 50:708\u201322. doi: 10.1080/00450618.2018.1466535\u201d was not cited in the article. The citation has now been inserted in Section Introduction, Paragraph one:In the original article, reference , \u201cWard J\u201cThe expectation is that some of these unknown remains will be linked to known missing persons, who in some cases have been absent for decades .\u201dIdentifying Victims Using DNA: A Guide for Families. Washington, DC: U.S. Department of Justice (2005). Available online at: https://www.ojp.gov/ncjrs/virtual-library/abstracts/identifying-victims-using-dna-guide-families-guia-para-las-familias\u201d was not cited in the article. The citation has now been inserted in Section Introduction, paragraph two:In the original article, reference , \u201cPresidhttps://www.missingpersons.gov.au/support/national-dna-program-unidentified-and-missing-persons) for families to aid their understanding of the use of DNA for identifying human remains.\u201d\u201cAdditionally, there are recently published international guidelines for police and forensic investigators regarding the use of DNA for humanitarian and mass disaster operations \u20139, and pJ For Sci. (2020) 65:791\u20139. doi: 10.1111/1556-4029.14284\u201d was not cited in the article. The citation has now been inserted in Introduction, Paragraph six:In the original article, reference , \u201cGin K,\u201cUnlike DNA identification of disaster victims, which are typically identified rapidly due to the high profile and public nature of the event, community expectations and provision of adequate resources, DNA identification may take an extended period of time for routine missing persons cases .\u201dThe references have been renumbered as a result of other reference updates.The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The updated reference list appears below. The original article has been updated.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."} +{"text": "Nature Biotechnology 10.1038/s41587-022-01278-2, published online 25 April 2022.Correction to: In the version of this article initially published, there were errors in the sequences shown in Supplementary Fig. 8, which has been replaced in the online version of the article."} +{"text": "Nature Human Behaviour 10.1038/s41562-022-01394-8, published online 11 July 2022.Correction to: https://github.com/deepmind/physical_concepts),\u201d originally listed placeholder text instead of the dataset link. The error has been corrected in the HTML and PDF versions of the article.In the version of this article initially published, in the sixth paragraph of the main text, the text now reading \u201cIn the present work, we introduce a much richer video corpus, the Physical Concepts dataset ("} +{"text": "Research of COVID-19-Pandemic mental health impact focus on three groups: the general population, (2) so called vulnerable groups and (3) individuals suffering COVID-19 including Long-COVID syndromes.We investigate whether individuals with a history of depression in the past, react to the COVID-19 pandemic with increased depressive symptoms.Longitudinal Data stem from the NAKO-Baseline-Assessment and the subsequent NAKO-COVID-Assessment (5-11/2020). The sample for analysis comprises 115.519 individuals. History of psychiatric disorder was operationalized as lifetime self-report for physician-diagnosed depression. Depressive symptoms were measured with the PHQ 9.Mean age of the sample at baseline was 49.95 (SD 12.53). It comprised 51.70 women; 14 % of the individuals had a history of physician-diagnosed depression. Considering a PHQ-Score with cut-off 10 as a clinical relevant depression, 3.65 % of the individuals without history of depression and 24.19 % of those with a history of depression were depressed at baseline. The NAKO-COVID-Assessment revealed 6.53 % depressed individuals without any history of depression and a similar rate of 23.29 % in those with history of depression.In contrast to that what we expected, individuals with a history of a physician-diagnosed depression, did not react with increasing depressiveness during the first phase of the pandemic in Germany. Several reasons could be discussed. Whether there medium and long-term impact remains open.No significant relationships."} +{"text": "Correction to: European Radiology10.1007/s00330-022-09025-6The original version of this article, published on 04 October 2022, unfortunately contained a mistake. The first names of all authors were given as initials and have now been written out in full. The original article has been corrected."} +{"text": "Nature Communications 10.1038/s41467-022-31326-z, published online 01 July 2022Correction to: Voc\u201d data (the symbols) in Fig. 2a and 2b. The correct version of Fig. 2 is:The original version of this Article contained an error in Fig. 2, in which three data sets were shifted along the x-axis by one data point, leading to an incorrect representation of the \u201cExp. suns-which replaces the previous incorrect version:This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Nature Microbiology10.1038/s41564-022-01091-2, published online 25 April 2022.Correction to: In the version of this article initially published, a production error resulted in partially duplicated circles representing the various isolates in the phylogenetic analysis of Fig. 1c.The figure has been corrected in the HTML and PDF versions of the article."} +{"text": "Translational Psychiatry 10.1038/s41398-022-01973-3, published online 18 May 2022Correction to: The original version of this article contained an error in an author name. The correct full author name is Vinicius Elias de Moura Oliveira. Thus the name in the manuscript should be Vin\u00edcius E. de M. Oliveira. The original article has been corrected."} +{"text": "Correction: BMC Genomics 22, 920 (2021)https://doi.org/10.1186/s12864-022-08609-2Following publication of the original article , it was The incorrect author name is: Yuri Iwasaki.The correct author name is: Yuki Iwasaki.Furthermore, it was reported that the Given and Family names of Trieu-Duc Vu were transposed in the original publication.The author group has been updated above and the original article has been"} +{"text": "Translational Psychiatry 10.1038/s41398-021-01545-x, published online 13 August 2021Correction to: The original version of this article unfortunately contained a mistake. Recently, the authors found that there is an error in the equation 2.6. The original are has been corrected."} +{"text": "European Journal of Clinical Nutrition 10.1038/s41430-022-01068-8 published online 09 February 2022Correction to: The original version of the article contained some errors. Tables 1, 2, and 3 have been updated. Furthermore, the affiliation of the Past Steering Committee member James H. Ware was corrected. The original article has been corrected."} +{"text": "Bioscience Reports at the request of the Editor-in-Chief and the Editorial Board following receipt of a notification from a reader, alerting the Editorial Board to multiple similarities between the figures in this manuscript and those in other articles. These include similarities between:The Figure 4C AMC-HN-8/GAPDH bands with the Fig.6A A549/GAPDH bands from Wang et al. 2019 (doi: 10.1042/BSR20182433) and the Fig.3F GAPDH bands from Chen et al. 2019 (doi: 10.1042/BSR20191050),The Figure 2C miR-625 mimics panel with Fig.6E from Xiao et al. 2020 (doi: 10.18632/aging.103762) and Fig.1D from Hu et al. 2019 (doi: 10.1002/2211-5463.12693)The Figure 2C mimics control/Tu-177 panel with a panel from Fig.1D from Hu et al. 2019 (doi: 10.1002/2211-5463.12693)The Figure 3B mimics control/Tu-177 panel and the 5C miR-625+NC/Tu-177 panel with panels from Fig.4B from Cheng et al. 2019 (doi: 10.1042/BSR20180906)This article is being retracted from The authors have been contacted with regards to the retraction and have not responded to the Journal's queries or the concerns raised. Given the extent of the issues raised, the Editorial Board stand by the decision to retract the article."} +{"text": "Nature Communications 10.1038/s41467-021-24680-x, published online 20 July 2021.Correction to: Fig. 4 was missing from the PDF version of this article; the figure should have appeared as shown below. The original article PDF has been corrected. The HTML version was unaffected."} +{"text": "Cell Death and Disease 10.1038/s41419-020-2272-z, published online 05 February 2020Correction to: The original version of this article contained an error. The authors put the wrong picture (upper right) in Fig. 4B. The authors apologize for the error. The corrected Figure can be found below."} +{"text": "Scientific Reports 10.1038/s41598-021-89015-8, published online 06 May 2021Correction to: The original version of this Article contained an error in Table 1, where the formula given for the MCC score was incorrect. The correct and incorrect values appear below.Incorrect:Correct:The original Article has been corrected."} +{"text": "Communications Chemistry 10.1038/s42004-020-0283-4, published online 25 March 2020.Correction to: The original version of this Article incorrectly designated Yoonmook Kang as a corresponding author and listed their e-mail address in error. This has now been corrected in the PDF and HTML versions of the Article."} +{"text": "Correction to: Trials 22, 550 (2021)https://doi.org/10.1186/s13063-021-05461-9measuredsolutions.com.au).Following the publication of the original article [1], we were notified that the Flu-iiQ\u2122 table included in the supplementary material of original publication needs to be deleted. The authors were not at liberty to publish the table in full as a number of the items listed are confidential and commercially sensitive. For full details of the Flu-iiQ\u2122 questionnaire please contact Measured Solutions P/L, PO Box 5127, Alphington VIC 3079, Australia (The original article has been corrected."} +{"text": "British Journal of Cancer 10.1038/s41416-021-01488-6, published online 22 July 2021Correction to: The original version of this article unfortunately contained an error. The labels were swapped in Figure 3a. The WT should be purple and the FGFR altered should be red. The original article has been corrected."} +{"text": "Scientific Reports 10.1038/s41598-022-12463-3, published online 24 May 2022Correction to: In the original version of this Article, the transcript abundance table was omitted from the Supplementary Information section.The Supplementary Information file now accompanies the original Article.Supplementary Table 1."} +{"text": "Retraction Note: BMC Public Health 20, 1657 (2020)https://doi.org/10.1186/s12889-020-09765-4The Editor has retracted this article. After publication, concerns were raised regarding the data analysis and conclusions in the paper. The authors have provided raw data, and post-publication review found inconsistencies in methodology and major misinterpretation of the primary result. None of the authors agree to this retraction."} +{"text": "Scientific Reports 10.1038/s41598-022-17922-5, published online 16 August 2022Correction to: In the original version of this Article, Amin Mekacher was incorrectly listed as the corresponding author. The correct corresponding author for this Article is Andrea Baronchelli. Correspondence and request for materials should be addressed to abaronchelli@turing.ac.uk.The original Article has been corrected."} +{"text": "Nature Biotechnology 10.1038/s41587-022-01393-0, published 18 July 2022.Correction to: In the version of this article initially published, several sequences in Supplementary Table 1 were listed incorrectly, which have now been replaced in the online version of the article."} +{"text": "Correction to: World Journal of Surgeryhttps://doi.org/10.1007/s00268-022-06871-9In the original online version of this article, A. Al Samaraee's name in Ref. 7 was misspelled.The original article was corrected."} +{"text": "Correction: The Cerebellumhttps://doi.org/10.1007/s12311-021-01337-5The original version of this article unfortunately contained an error.The Methods section in the original version of the article, \u201cfive-point scale\" should read \u201cseven-point scale\u201d.This typographical mistake has been amended in the corrected version of the article."} +{"text": "Communications Biology 10.1038/s42003-022-04232-9, published online 19 November 2022.Correction to: In this Article Sun Hee Do should have been denoted as a corresponding author.This has now been corrected in the PDF and HTML versions of the Article."} +{"text": "Nature Communications 10.1038/s41467-022-29925-x, published online 25 April 2022.Correction to: The original version of the Supplementary Information associated with this Article included an incorrect Supplementary Data 1 file, which was an incorrect version of the Source Data. The original Source Data file is correct. The HTML has been updated to remove Supplementary Data 1."} +{"text": "Scientific Reports 10.1038/s41598-023-27462-1, published online 16 January 2023Correction to: In the original version of this Article, Piotr Winkielman was omitted as a corresponding author. Correspondence and requests for materials should also be addressed to pwinkielman@ucsd.edu.The original Article has been corrected."} +{"text": "Nature Human Behaviour 10.1038/s41562-022-01383-x, published online 4 July 2022.Correction to: In the version of this article initially published, author Jan Balaguer\u2019s name was presented as Balaguer Jan. The error has now been corrected in the HTML and PDF versions of the article."} +{"text": "Nature Communications 10.1038/s41467-022-33595-0, published online 03 October 2022Correction to: The original version of this Article contained an error in the Acknowledgements, in which a funding agent was omitted. The correct version contains \u2018L. Wu and Y. Zhu were supported by the U.S. Department of Energy, Office of Basic Energy Science, Division of Materials Science and Engineering, under Contract No. DE-SC0012704.\u2019 This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Mod Pathol 10.1038/s41379-021-00983-8, published online 3 December 2021Correction to: The original version of this article unfortunately contained a mistake in the coloring of Table 3. We apologize for the error. The correct table can be found below. The original article has been corrected."} +{"text": "Retraction Note: J Exp Clin Cancer Res 40, 299 (2021)https://doi.org/10.1186/s13046-021-02090-7The Editor in Chief has retracted this article. After publication, concerns were raised about an apparent partial overlap between the leftmost SNU-449 migration panel in Fig. 2C and the top LNCAROD+2-DG panel in Fig. 4G. The authors were unable to provide a satisfactory set of raw data on request. Additionally, the issues regarding the provenance of cell lines were not completely resolved in the published version of the article. The editor, therefore, has lost confidence in the integrity of the findings of this article. The authors have not explicitly stated that they agree to this retraction."} +{"text": "Nature Communications 10.1038/s41467-023-35878-6, published online 13 January 2023Correction to: The original version of this Article contained an error in Fig. 3, in which the panel 3b was incorrectly displayed. This correct version of Fig. 3b is:which replaces the previous incorrect version:This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Retraction Note:J Exp Clin Cancer Res39, 186 (2020)https://doi.org/10.1186/s13046-020-01697-6Fig. 4C appears to contain image overlap between different treatment groups and cell lines.Fig. 4F appears to contain overlapping areas with two articles on unrelated circRNAs that were under consideration within a similar time frame , 2.Figs. 6G and 7G appear to contain image overlap between different treatment groups and cell lines.The Editor-in-Chief has retracted this article at the authors' request. After publication, concerns were raised regarding the published images. Specifically:The authors have stated that the microscopy images in Fig. 4F were provided by a third party.Due to the number of concerns with the images, the Editor-in-Chief and the authors no longer have confidence in the presented data.All authors agree to this retraction."} +{"text": "Communications Biology 10.1038/s42003-022-03228-9, published online 31 March 2022.Correction to: The original version of this Article contained an error in Fig. 2f, in which the incorrect axis label and values were included for the second graph . The correct version of Fig. 2f is:which replaces the previous incorrect version:This has now been corrected in both the PDF and HTML versions of the Article."} +{"text": "Nature Cancer 10.1038/s43018-022-00431-9, published online 5 September 2022.Correction to: In the version of this article initially published, the affiliation listed for Jer-Tsong Hsieh was incorrect. The correct affiliation has now been inserted in the HTML and PDF versions of the article."} +{"text": "Scientific Reportshttps://doi.org/10.1038/s41598-022-08349-z, published online 15 March 2022Correction to: In the original version of this Article, Annika Reuser was incorrectly listed as a corresponding author. The correct corresponding author for this Article is Martin Bahls. Correspondence and request for materials should be addressed to martin.bahls@uni-greifswald.de.The original Article has been corrected."} +{"text": "Nature 10.1038/s41586-021-04069-y Published online 14 October 2021Correction to: In the version of this article initially published, the name of author Erik Volz appeared as \u201cErik M. Volz\u201d in The COVID-19 Genomics UK (COG-UK) Consortium contributions listings. Further, the affiliation listed for COG-UK Consortium member Adam A. Witney was shown incorrectly and has been now amended to the Institute for Infection and Immunity, St George\u2019s Hospital of London, London, UK. The changes have been made to the HTML and PDF versions of the article."} +{"text": "Cell Death and Disease 10.1038/s41419-022-04741-9, published online 22 April 2022Correction to: The original version of this article unfortunately contained a mistake. Due to a typesetting error the graphical abstract was omitted. We apologize for the error. The graphical abstract can be found below. The original article has been updated."} +{"text": "Cell Death and Disease 10.1038/s41419-021-04442-9, published online 17 December 2021Correction to: The original version of this article unfortunately contained a mistake. For the author Gautam Mehta one affiliation was omitted. The omitted affiliation is: The Roger Williams Institute of Hepatology, Foundation for Liver Research, London, UK. The original article has been corrected."} +{"text": "Staphylococcus warneri was detected in the CSF. The patient had an infected, ulcerated, and hyperpigmented wound in the left nose-cheek region, which was said to have been present for 2-3 years . More pr-3 years . The his"} +{"text": "We hereby retract this article due to the method for administering the monoclonal anti-TLR4 antibody (ATAB) described in the original paper being inaccurate, as this may influence the readers' understanding of the effect of the ATAB. This inaccuracy was brought to our attention by a Letter to the Editor (https://doi.org/10.1128/spectrum.01863-22). We have responded to the concerns regarding the original paper in our reply to the letter (https://doi.org/10.1128/spectrum.02377-22).Volume 10, no. 3, e00647-22, 2022, We apologize for any inconvenience caused to the readers."} +{"text": "Nature Communications 10.1038/s41467-022-34514-z, published online 05 November 2022Correction to: The original version of this Article omitted listing Dr. Deqiang Sun as a corresponding author. This has now been corrected in both the PDF and HTML versions of the Article."} +{"text": "Pediatric Research 10.1038/s41390-021-01602-7, published online 19 June 2021Correction to: In the original article, the order of authors was wrong and a footnote was missing. The original article has been corrected."} +{"text": "Nature Genetics 10.1038/s41588-021-00972-2, published online 2 December 2021.Correction to: In the version of this article initially published, \u201cEuropean Research Council grant no. 853546\u201d was missing from the Acknowledgements section. The error has been corrected in the HTML and PDF versions of the article."} +{"text": "Translational Psychiatry 10.1038/s41398-022-01919-9, published online 11 April 2022Correction to: The original version of this article unfortunately contained a mistake in an author name. The correct name is Kelli Lehto. The authors apologize for the error. The original article has been corrected."} +{"text": "Molecular Psychiatry 10.1038/s41380-022-01567-x, published online 28 April 2022Correction to: The wrong Supplementary file was originally published with this article; it has now been replaced with the correct file, and referenced appropriately in the main text.The original article has been corrected."} +{"text": "Nature Communications 10.1038/s41467-022-30600-4, published online 23 May 2022.Correction to: The original version of this Article incorrectly included the 6th author Cyriel M. A. Pennartz as a corresponding author. The correct version lists only 7th author Umberto Olcese as corresponding author. In addition, the original version of the Article omitted to include a statement to indicate that Cyriel M. A. Pennartz and Umberto Olcese jointly supervised the work. This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Correction: Mol Cell Pediatr 9, 14 (2022)https://doi.org/10.1186/s40348-022-00146-yFollowing publication of the original article , authorsInfant formulas with synthetic oligosaccharides and respective marketing practices: Position Statement of the German Society for Child and Adolescent Medicine e.V. (DGKJ), Commission for Nutrition.The correction does not have any effect on the results or conclusions of the paper. The original article has been corrected."} +{"text": "Nature Communications 10.1038/s41467-019-14275-y, published online 21 February 2020.Correction to: Since the publication of this work, Eli M Carrami has changed their name from Mohammad Karaminejadranjbar. This has now been amended in the HTML and PDF versions of the article."} +{"text": "Scientific Reportshttps://doi.org/10.1038/s41598-022-21779-z, published online 03 November 2022Correction to: The original version of this Article contained an error in the spelling of the author Carolina Inostroza which was incorrectly given as Carolina Inotroza.The original Article has been corrected."} +{"text": "Cell Death and Disease 10.1038/s41419-018-0426-z, published online 10 May 2018Correction to: The original version of this article unfortunately contained an error in figure 3. The authors apologize for the mistake. The corrected figure can be found below."} +{"text": "Nature Biomedical Engineering 10.1038/s41551-022-00911-4, published online 28 July 2022.Correction to: In the version of this article initially published, individual data points in Fig. 5f\u2013k were omitted, and have now been restored in the HTML and PDF versions of the paper."} +{"text": "Nature Communications 10.1038/s41467-022-30965-6, published online 08 June 2022Correction to: In the Acknowledgements section of this article the grant number relating to U.S. National Science Foundation was incorrectly given as 2016136 and should have been 2137642. The original article has been corrected."} +{"text": "Nature Communications 10.1038/s41467-021-24999-5, published online 19 August 2021.Correction to: The original version of this article contained errors in equal contribution attributions that were inadvertently introduced during typesetting. These errors have now been corrected in both the PDF and HTML versions of the article."} +{"text": "Nature Communications 10.1038/s41467-022-31055-3, published online 08 July 2022Correction to: a and d was corrupted. This has been corrected in the HTML version of the Article. The PDF version was correct from the time of publication.The original HTML version of this Article contained an error in Fig. 5, in which text in panels"} +{"text": "Cell Death Discovery 10.1038/s41420-022-01138-7, published online 20 September 2022Correction to: The original version of this article contained a mistake in Figure 2D. The authors apologize for the error. The correct figure can be found below. The original article has been corrected."} +{"text": "Nature Communications 10.1038/s41467-022-29379-1, published online 12 April 2022.Correction to: The original version of this Article contained an error in Fig. 1, in which Atlantic was spelt incorrectly. This has been corrected in both the PDF and HTML versions of the article."} +{"text": "Oncogene 10.1038/s41388-022-02314-w, published online 18 March 2022Correction to: Following the publication of this article, the authors noted an error in Fig. 6C. The merged (bottom left) panel for the human ILC organoid KCL320, did not display the correct z-plane. The correct merged image has now been provided. The authors confirm this does not affect the conclusions of the study in any way.The original article has been corrected."} +{"text": "Correction: Journal of Cardiothoracic Surgery (2022) 17:224 https://doi.org/10.1186/s13019-022-01958-9.Following publication of the original article , the autfromResults: A total of 9 RCTs were included in this meta-analysis, enrolling a total of 2031 patients. Colchicine significantly reduces the incidence of POAF . Subgroup analyses indicated that the protective effect of colchicine on POAF was slightly stronger in the long-duration group than in the short-duration group .Conclusion: Colchicine is effective in preventing the occurrence of POAF. The efficacy of colchicine can be slightly increased over treatment duration, with no obvious adverse reactions.to.Results: A total of 9 RCTs were included in this meta-analysis, enrolling a total of 2031 patients. Colchicine significantly reduces the incidence of POAF . Subgroup analyses indicated that the protective effect of colchicine on POAF was almost the same (P\u2009=\u20090.71) in the long-duration group and the short-duration group .Conclusion: Colchicine is effective in preventing the occurrence of POAF. The efficacy of colchicine cannot be increased over treatment duration, with no obvious adverse reactions.The original article has been corrected."} +{"text": "Nature Communications 10.1038/s41467-021-22197-x, published online 25 March 2021.Correction to: In Figure 2, panels (a) and (b) were inadvertently swapped. The correct version of this figure appears below. This has been corrected in the HTML and PDF version of this article."} +{"text": "Scientific Reportshttps://doi.org/10.1038/s41598-022-24607-6, published online 21 November 2022Correction to: The original version of this Article contained an error in the name of the author Brett A. Halperin which was incorrectly given as Brett Halperin.The original Article has been corrected."} +{"text": "Nature Communications 10.1038/s41467-022-30922-3, published online 09 June 2022Correction to: Since the publication of this work, Lena Langenohl has changed her name from Lena Frommeyer. This has now been amended in the HTML and PDF versions of the article."} +{"text": "Nature Communications 10.1038/s41467-023-36322-5, published online 11 February 2023Correction to: The original version of the published article omitted acknowledgement to the U.S. DOE. The following statement is now added in the Acknowledgement section in both HTML and the PDF versions of the article:\u201cF.A-P. would like to acknowledge support from the U.S. DOE, Office of Science, Office of Basic Energy Sciences, Chemical Sciences, Geosciences, and Biosciences Division, Catalysis Science Program to the SUNCAT Center for Interface Science and Catalysis.\u201d"} +{"text": "Correction to: Neurochemical Research.10.1007/s11064-022-03786-8.In the original version of the article, unfortunately the typesetting team was missed to incorporate the article note \u201cKai Gao and Congying Wu contributed equally\u201d. This has been corrected by publishing this correction article. The original version has been updated."} +{"text": "Nature 10.1038/s41586-022-04950-4Published online 6 July 2022Correction to: C. roseus and S. nux-vomica),\u201d where Supplementary Fig. 7 was originally cited. The error has been corrected in the HTML and PDF versions of the article.In the version of this article initially published, in the fourth paragraph of the main text, there was a typographical error in the first sentence, now reading in part, \u201c\u2026(see Supplementary Fig. 6 for the phylogenetic relationship of"} +{"text": "Biol. Open (2016) 5, 62-71 (doi:10.1242/bio.015776).There was an error published in In Fig.\u00a04, the Autumn-control image in panel B was erroneously a duplication of the Autumn-HU image in panel D.The corrected figure is shown below.The authors apologise to readers for this error, which does not impact the results or conclusions of this paper."} +{"text": "Nature Communications 10.1038/s41467-021-21146-y, published online 08 February 2021Correction to: The original version of this Article contained an error in Fig. 4b, in which patient 4 was incorrectly labelled as Patient 3. The correct version of Fig. 4b is:which replaces the previous incorrect version:This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Scientific Reports 10.1038/s41598-023-28574-4, published online 27 January 2023Correction to: In the original version of this Article, Miguel Nakamura was omitted as a corresponding author. Correspondence and requests for materials should also be addressed to nakamura@cimat.mx.The original Article has been corrected."} +{"text": "Scientific Reportshttps://doi.org/10.1038/s41598-022-12735-y, published online 23 May 2022Correction to: The original version of this Article contained an error in the spelling of the author Razieh Mansoori which was incorrectly given as Razieh Mansouri.The original Article has been corrected."} +{"text": "The article has been updated accordingly. Authors of the above-mentioned article have informed that Abubaker Elamin and PanagiotisTsoutsanis are the co-first authors. The original article was published on September 26,2022 in issue 8.3. DOI:"} +{"text": "Retraction Note: BMC Oral Health (2019) 19:290 10.1186/s12903-019-0980-5The Editor has retracted this article. After publication concerns were raised that the data presented are owned by the National Congenital Malformation Registries network of Italy [Emilia-Romagna Registry of Birth Defects (IMER) and Registro Toscano Difetti Congeniti (RTDC)]. The authors have not been able to demonstrate ownership of the data in this article. G. Galluccio and A. Impellizzeri do not agree to this retraction. I. Giannantoni, A. Polimeni and E. Barbato have not responded to correspondence from the Editor about this retraction."} +{"text": "Nature Microbiology 10.1038/s41564-022-01059-2, published online 14 February 2022.Correction to: In the version of this article initially published, in the first sentence of the last paragraph of the \u201cGeneration of AlgoCIS mutants\u201d section in the Methods now reading \u201cThe in-frame deletions of the AlgoCIS genes were generated utilizing our pCHIP3 suicide plasmid ,\u201d Extended Data Fig. 5e was originally cited in error. Further, ref. 49 has been updated with complete publication information, as below. The errors have been corrected in the HTML and PDF versions of the article.Nat. Microbiol. 10.1038/s41564-021-01055-y (2022).49. Weiss, G. L. et al. Structure of a thylakoid-anchored contractile injection system in multicellular cyanobacteria."} +{"text": "Scientific Reportshttps://doi.org/10.1038/s41598-022-14454-w, published online 16 June 2022Correction to: The original version of this Article contained an error, where an erroneous figure was uploaded instead of a flowchart of the recruited samples in this study.The original Figure The original Article has been corrected."} +{"text": "Cell Death and Disease 10.1038/s41419-017-0091-7, published online 19 January 2018Correction to: The original version of this article contained errors in Figures 6a and 7a. The correct figures can be found below. The authors apologize for the error."} +{"text": "To the Editor: We read with interest the observations by Manning et al. from P. falciparum\u2013exposed persons in a high-transmission setting in western Kenya for the presence of SARS-CoV-2 neutralizing antibodies (nAbs) using the GenScript SARS-CoV2 sVNT assay (https://www.genscript.com). Monthly P. falciparum real-time PCR results were collected in a previous study (P. falciparum at least 1 time in 2019, suggesting that most persons included in this screening had been recently exposed to malaria parasites.One hypothesis for the unexpectedly moderate burden of SARS-CoV-2 in malaria-endemic countries in Africa is that exposure to P. falciparum. Although nAbs are subject to decay after infection (P. falciparum infections elicit functional humoral responses against COVID-19.Consistent with findings in Manning et al. ("} +{"text": "Scientific Reports 10.1038/s41598-022-22101-7, published online 14 October 2022Correction to: The original version of this Article contained an error in the spelling of the author Chunguang Liang, which was incorrectly given as Liang Chunguang.The original Article has been corrected."} +{"text": "Communications Biology 10.1038/s42003-022-04141-x, published online 16 November 2022.Correction to: In the original version of the Article, an incorrect additional description of panel b in Figure 1 was included. The following sentence has now been removed:b Lignocellulose is a complex and recalcitrant polymer built up from cellulose, xylan (hemicellulose), and lignin. Its degradation requires the synergistic action of various different enzymes."} +{"text": "Retraction Note: Cellular Oncology (2020) 43:1017\u2013103310.1007/s13402-020-00553-1The Editors-in-chief have retracted this article because after publication concerns have been raised regarding a number of figures, specifically:Figure 3a: the 24h panels for m-NC and i-NC appear to be identical.Figure 4f: the AGS and BGC-823 panels for miR-4490/USP22 appear to partially overlap.The Editors-in-Chief therefore consider the data reported in this the article to be unreliable.Jide Wang does not agree to this retraction. The editor was not able to obtain a current email address for Tianming Chen. None of the other authors have responded to any correspondence regarding this retraction."} +{"text": "Cell Death & DifferentiationCorrection to: 10.1038/s41418-019-0483-6.The original version of this article unfortunately contained a mistake. Following publication of this article, the authors noticed that there was an error in the image used to compile the Fig. 6E shV image. An adjacent serial image section from Fig. 6D shV image was inadvertently used for Fig. 6E shV image during image compilation. The corrected images are provided below. The authors apologise for this error."} +{"text": "Correction to: Space Science Reviewshttps://doi.org/10.1007/s00484-022-02238-wDue to figure processing errors introduced during typesetting via the publisher, the article was originally published with Figure 1 missing and Figure 2 appearing twice .This correction stands to support the update to the original article. The original article has been corrected."} +{"text": "Scientific Reports 10.1038/s41598-022-25110-8, published online 28 November 2022Correction to: In the original version of this Article, Moslem Abdipour was omitted as a corresponding author. Correspondence and requests for materials should also be addressed to abdipur.m@gmail.com.The original Article has been corrected."} +{"text": "Correction to: Archives of Public Health 80, 109 (2022)https://doi.org/10.1186/s13690-022-00856-9Following publication of the original article , due to Marjan Meurisse and Adrien Lajot contributed equally to this work.The correct information has been provided in this Correction and the original article has been"} +{"text": "Nature Communications 10.1038/s41467-022-32639-9, published online 05 September 2022Correction to: The original version of this Article contained errors in Fig. 1 and Fig. 2. In the original version of Fig. 1, several labels in figure panels were illegible. The correct version of Fig. 1 is:which replaces the previous incorrect version:In the original version of Fig. 2, several labels in figure panels were illegible. The correct version of Fig. 2 is:which replaces the previous incorrect version:This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Scientific Reports 10.1038/s41598-022-21999-3, published online 15 November 2022Correction to: The original version of this Article contained an error in the name of the author A. I. Sharshir which was incorrectly given as Ahmed Ibrahim Sharshir.The original Article has been corrected."} +{"text": "Cell Death and Disease 10.1038/s41419-021-04103-x, published online 03 September 2021Retraction to: The authors have retracted this article. After publication the authors found that the staining done in Figure 5D had not been done with the antibody SOCS3 as stated in the article. The authors have, therefore, lost confidence in their results. All authors agree to this retraction."} +{"text": "Nature Communications 10.1038/s41467-022-34321-6, published online 04 November 2022Correction to: The original version of this Article contained errors in the y-axis of Fig. 3a and 3b, in which Fig. 3a had an incorrect title in the y-axis, and fig. 3b had incorrect numbers in the y-axis. This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Cell Death & Differentiation 10.1038/s41418-019-0351-4, published online 24 May 2019Correction to: The original version of this article unfortunately contained an error in figure 5B. The corrected figure can be found below. This error does not affect the results, discussion, or conclusions. The authors sincerely apologize to the editors and readers for any inconvenience or confusion."} +{"text": "Laboratory Investigation 10.1038/s41374-022-00819-2, published online 08 August 2022Correction to: The original version of this article unfortunately contained mistakes in Figs. 4 and 6. The authors found that the Tunnel staining of Fig. 4 was the same as Fig. 6. The correct figures can be found below. The original article has been corrected."} +{"text": "Scientific Reports 10.1038/s41598-022-18849-7, published online 17 September 2022Correction to: The original version of this Article contained an error in the spelling of the author Kholood Alkamis which was incorrectly given as Kh. M. Alkhamis.The original Article has been corrected."} +{"text": "Correction to: Nature Communications 10.1038/s41467-022-33223-x, published online 26 September 2022Peer-to-Peer Netw. Appl.15, 1577\u20131594 (2022). This has been corrected in the PDF and HTML versions of the Article.The original version of this Article contained an error in Ref. 39, in which the title of the cited work was missing. The complete citation is: Bereketli, A. Interference-free source deployment for coverage in underwater acoustic backscatter networks."} +{"text": "Cell Death and Disease (2021) 12:1041 10.1038/s41419-021-04337-9, published online 01 November 2021Correction to: The original version of this article contained a mistake. During the eproofing procedure, the authors found that the entire 74 references of the 2 tables were not included. All these references should be additionally included in the review. All references can be found below. We apologize for this error Tables and 2.Ta"} +{"text": "QuantiFERON-TB Gold Plus (QFT-Plus) is an important test that has emerged in recent years for detecting TB infection. We conducted a review to compare the sensitivity, specificity and positive rate of QFT-Plus with that of QuantiFERON-TB Gold In-Tube (QFT-GIT), T-cell spot of tuberculosis assay (T-SPOT.TB) and Tuberculin test (TST).Mycobacterium tuberculosis Infections\u201d and \u201cQuantiFERON-TB-Plus\u201d as search phrases. We estimated the sensitivity from studies of patients with active tuberculosis, specificity from studies of populations with very low risk of TB exposure, and positive rate from studies of high-risk populations. The methodological quality of the eligible studies was assessed, and a random-effects model meta-analysis was used to determine the risk difference (RD). We assessed the pooled rate by using a random-effects model. This study was registered in PROSPERO (CRD 42021267432).PubMed and Embase were searched, without language restrictions, from 1 January 2015 to 31 March 2022 using \u201cOf 3996 studies, 83 were eligible for full-text screening and 41 were included in the meta-analysis. In patients with active TB, the sensitivity of QFT-Plus was compared to that of QFT-GIT and T-SPOT.TB, respectively, and no statistically differences were found. In populations with a very low risk of TB exposure, the specificity of QFT-Plus was compared with that of QFT-GTI and T-SPOT.TB, respectively, and no statistically differences were found. Two studies were eligible to compare the specificity of the QFT-Plus test with that of the TST test, and the pooled RD was 0.12 (95% CI 0.02 to 0.22). In high-risk populations, 18 studies were eligible to compare the positive rate of the QFT-Plus test with that of the QFT-GIT test, and the pooled RD was 0.02 (95% CI 0.01 to 0.03). The positive rate of QFT-Plus was compared with that of T-SPOT.TB and TST groups, and no statistically differences were found.The diagnostic performance of QFT-Plus was similar to that of QFT-GIT and T-SPOT.TB, but was slightly more specific than TST.The online version contains supplementary material available at 10.1186/s12879-023-08008-2. Mycobacterium tuberculosis. Approximately one-quarter of the world\u2019s population is currently infected with Mycobacterium tuberculosis, most of whom also have latent TB infections and divided as follows: 1\u201330 per 100,000 persons; 31\u2013100 per 100,000 persons; 100\u2013200 per 100,000 persons and 200-per 100,000 persons. In populations with a very low risk of TB exposure, we used TB burden of the areas and number of participants for subgroup analysis to compare QFT-Plus with QFT-GIT. In high-risk populations, we used age of the participants, TB burden of the areas, number of participants and population for subgroup analysis, and when multiple TST cut-off results were reported in the same study, the TST-5 (cut-off 5\u00a0mm) results were retained to calculate the pooled RD value.In patients with active TB, we used the age of the participants (children were defined as those aged <\u200918\u00a0years), TB burden of the areas and number of participants for subgroup analysis to compare QFT-Plus with QFT-GIT and used participants for subgroup analysis to compare QFT-Plus with T-SPOT.TB. TB burden of the areas considered was determined using data from the WHO website [I2 statistic was used to assess the heterogeneity of the included studies, with I2\u2009>\u200950% indicating significant heterogeneity. We assessed publication bias using \u201cPeters\u201d test. All p-value were two-sided. A p-value of less than 0.05 was considered to be significant [Sensitivity, specificity and positive rate were pooled using a general linear random-effects mixed model . The I2 nificant , 19. Thenificant .We identified 3966 studies; 83 were selected for full-text review and 42 articles were excluded . Among the specificity tested in populations with a very low risk of TB exposure, QFT-Plus showed hardly any difference in specificity compared to QFT-GIT and T-SPOT.TB. However, compared to TST, QFT-Plus shows significant advantages, and we speculate that it is possible that prior BCG vaccination or non-TST test \u201361. AmonWe did not find evidence that QFT-Plus has better sensitivity and specificity than QFT-GIT. In a previous analysis, it was shown that QFT-Plus has a higher sensitivity than QFT-GIT . In contIn the study, the authors asked whether QFT-Plus use might be more advantageous than QFT-GIT in an immunosuppressed population because QFT-Plus has two TB antigen tubes that stimulate IFN-\u03b3 production by CD8+ T cells and CD4+ T cells. Therefore, we conducted a subgroup analysis of high-risk populations. Results of this analysis showed that both tests performed similarly in the immunosuppressed population, with no significant advantage of QFT-PLUS use revealed. However, we can see that QFT-Plus, with its two TB antigen tubes, may have a genuine advantage in detecting positive rate in high-risk groups. It is possible that the small amount of data we included may have biased the results obtained; therefore, it is recommended that subsequent researchers continue to focus on this issue and include more data to obtain more reliable results.There are also some limitations to our study. First, the sensitivity and specificity of our findings are underestimated because TB exposure may still be present in a population with very low risk of TB exposure and patients with active TB have partially compromised basic immunity, resulting in a reduced power to detect TB. Second, the absence of HIV infection in high-risk populations and the rare inclusion of children may have influenced our assessment of the positivity rate in high-risk populations.The use of QFT-Plus in clinical situations can be convenient and affordable. However, with respect to convenience, the procedures for both tests are similar. With respect to affordability, the cost difference between using the QFT-Plus and QFT-GIT tests is not significant. However, in our study, we found a slight advantage of QFT-Plus over QFT-GIT in positive rate, which may not be sufficient to use QFT-Plus as a recommended method for detecting positivity rate in high-risk populations; therefore, more data need to be included in subsequent studies.The detection performance of QFT-Plus is not significantly improved compared with QFT-GIT and T-SPOT.TB, and the findings of this systematic\u00a0review should encourage people to choose methods that are more convenient and economical for TB testing.Additional file 1: Table S1. PRISMA checklist. Table S2. Search strategy. Table S2. Search strategy. Table S3. Inclusion and partial exclusion for patients with active TB. Table S4. Inclusion and partial exclusion for populations with very low risk of TB exposure. Table S5. Inclusion and partial exclusion for high-risk populations. Table S6. Populations considered high-risk. Table S7. Details of excluded criteria. Table S8. QUADAS-2 adapted quality assessment criteria for patients with active TB. Table S9. Quality score of 12 studies for patients with active TB. Table S10. QUADAS-2 adapted quality assessment criteria for populations with very low risk of TB exposure. Table S11. Quality score of 7 studies for populations with very low risk of TB exposure. Table S12. QUADAS-2 adapted quality assessment criteria for high-risk groups. Table S13. Quality score of 31 studies for high-risk groups. Table S14. Reasons for exclusion of 42 studies that were read in full-text review. Table S15. Characteristics of the 12 studies included in the sensitivity analysis. Table S16. Characteristics of the 7 studies included in the specificity analysis. Table S17. Characteristics of the 31 studies included in the positive rates. Table S18. Linearregression test of funnel plot asymmetry results of QFT-PLUS compared to QFT-GIT, T-SPOT.TB and TST in three populations. Figure S1. Forest plot of studies estimating the sensitivity of QFT-Plus (A) and QFT-GIT (B) in patients with active tuberculosis. Figure S2. Forest plot of studies estimating the sensitivity of QFT-Plus (A) and T-SPOT.TB (B) in patients with active tuberculosis. Figure S3. Forest plot of studies estimating the specificity of QFT-Plus (A) and QFT-GIT (B) in populations with very low risk of TB exposure. Figure S4. Forest plot of studies estimating the specificity of QFT-Plus (A) and T-SPOT.TB (B) in populations with very low risk of TB exposure. Figure S5. Forest plot of studies estimating the specificity of QFT-Plus (A) and TST (B) in populations with very low risk of TB exposure. Figure S6. Forest plot of studies estimating the positive rate Plus (A) and QFT-GIT (B) in high-risk populations. Figure S7. Forest plot of studies estimating the positive rate of QFT-Plus (A) and T-SPOT.TB (B) in high-risk populations. Figure S8. Forest plot of studies estimating the positive rate of QFT-Plus (A) and TST (B) in high-risk populations. Figure S9. Forest plot of studies estimating the sensitivity in patients with active tuberculosis for age of the participants (A), TB burden of the areas (B) and number of participants (C) subgroup analysis of QFT-PLUS compared with QFT-GIT. Figure S10. Forest plot of studies estimating the sensitivity in patients with active tuberculosis for number of participants subgroup analysis of QFT-PLUS compared with T-SPOT.TB. Figure S11. Forest plot of studies estimating the Specificity in populations with very low risk of TB exposure for TB burden of the areas (A) and number of participants (B) subgroup analysis of QFT-PLUS compared with QFT-GIT. Figure S12. Forest plot of studies estimating the positive rate in high-risk populations for age of the participants (A), TB burden of the areas (B), number of participants (C) and population (D)subgroup analysis of QFT-PLUS compared with QFT-GIT. Figure S13. Forest plot of studies estimating the positive rate in high-risk populations for age of the participants (A), TB burden of the areas (B), number of participants (C) and population (D)subgroup analysis of QFT-PLUS compared with T-SPOT.TB. Figure S14. Forest plot of studies estimating the positive rate in high-risk populations for age of the participants (A), TB burden of the areas (B), number of participants (C) and population (D)subgroup analysis of QFT-PLUS compared with TST. Figure S15. Sensitivity analysis of QFT-PLUS compared to QFT-GIT (A) and T-SPOT.TB (B) in patients with active TB. Figure S16. Sensitivity analysis of QFT-PLUS compared to QFT-GIT (A), T-SPOT.TB (B) and TST(C) in populations with very low risk of TB exposure. Figure S17. Sensitivity analysis of QFT-PLUS compared to QFT-GIT (A), T-SPOT.TB (B) and TST(C) in high-risk populations. Figure S18. Funnel plot of QFT-PLUS compared to QFT-GIT in patients with active TB. Figure S19. Funnel plot of QFT-PLUS compared to QFT-GIT (A) and TST (B) in high-risk populations."} +{"text": "Communications Chemistry 10.1038/s42004-019-0249-6, published online 3 January 2020.Correction to: The original version of this Article contained an error in reference 2. The title in reference 2 was incorrectly written as \u2018The Staudinger ligation-a.pngt to chemical biology\u2019. This error has been corrected in the HTML and PDF versions of the Article."} +{"text": "Nature Communications 10.1038/s41467-022-30627-7, published online 30 May 2022.Correction to: The original version of this Article contained an error in Figure 5, which read DFGZ instead of DGFZ. This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Correction to: European Radiology10.1007/s00330-022-09024-7The original version of this article, published on 04 October 2022, unfortunately contained a mistake. The first names of all authors were given as initials and have now been written out in full. The original article has been corrected."} +{"text": "Nature Communications 10.1038/s41467-022-32551-2, published online 22 August 2022Correction to: The original version of this article contained an error in the Abstract, which incorrectly omitted from the end the following: \u2018Trial Registration: ClinicalTrials.gov Identifier: NCT04518410\u2019. This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Nature Metabolism 10.1038/s42255-022-00677-8. Published online 21 November 2022.Correction to: In the version of this article originally published, the surname of co-author Thomas Topilko was misspelled as Tolpiko. The error has been corrected in the HTML and PDF versions of the article."} +{"text": "Nature Communications 10.1038/s41467-022-35385-0, published online 03 January 2023Correction to: The original version of this Article incorrectly made the peer review reports for this paper available. In the correct HTML version, the link to the peer review file has been removed."} +{"text": "Correction to: European Journal of Nuclear Medicine and Molecular Imaginghttps://doi.org/10.1007/s00259-022-05991-7The authors regret that their names in the original article are incorrect as the first and last names were interchanged. It is now corrected in this erratum article.https://doi.org/10.1007/s00259-022-05991-7.The original article can be found at"} +{"text": "Scientific Reports 10.1038/s41598-022-18992-1, published online 26 August 2022Correction to: In the original version of this Article, Sheng-Che Chou was incorrectly listed as the corresponding author. The correct corresponding author for this Article is Sheng-Hong Tseng. Correspondence and request for materials should be addressed to shenghongtseng@gmail.com.The original Article has been corrected."} +{"text": "Experimental & Molecular Medicine 10.1038/s12276-021-00716-6, published online 21 December 2021Correction to: After online publication of this article, the authors noticed an error in Figure 1i.The correct image of this plot should have shown as below.The corrected data were printed below. These corrected results do not alter the conclusions of this article. The authors apologize for any inconvenience caused.The original article has been corrected."} +{"text": "Correction to: Journal of Child & Adolescent Trauma 10.1007/s40653-022-00486-xThe original online version of this article was revised. The surname of the second author was misspelled. Correct info below. Also, Figures 1 and 2 are difficult to read in black and white, textured graphs can be found below.Catriona O\u2019TooleMira Dobutowitsch"} +{"text": "Nature 10.1038/s41586-022-04666-5 Published online 4 May 2022Correction to: In the version of this article initially published, Extended Data Fig. 5 was a duplicate of Extended Data Fig. 6. The correct image is now in place in the HTML and PDF versions of the article."} +{"text": "Scientific Reports 10.1038/s41598-022-08632-z, published online 23 March 2022Correction to: In the original version of this Article, Conrad Strydom was omitted as a corresponding author. Correspondence and requests for materials should also be addressed to conradstryd@gmail.com.The original Article has been corrected."} +{"text": "Protozoan parasites are known to attach specific and diverse group of proteins to their plasma membrane via a GPI anchor. In malaria parasites, GPI-anchored proteins (GPI-APs) have been shown to play an important role in host\u2013pathogen interactions and a key function in host cell invasion and immune evasion. Because of their immunogenic properties, some of these proteins have been considered as malaria vaccine candidates. However, identification of all possible GPI-APs encoded by these parasites remains challenging due to their sequence diversity and limitations of the tools used for their characterization.Plasmodium falciparum strain 3D7 and Plasmodium vivax strain Sal-1, a heuristic method was defined to select the most sensitive and specific FT-GPI software parameters.The FT-GPI software was developed to detect GPI-APs based on the presence of a hydrophobic helix at both ends of the premature peptide. FT-GPI was implemented in C\u2009++and applied to study the GPI-proteome of 46 isolates of the order Haemosporida. Using the GPI proteome of P. falciparum and P. vivax, including the identification of novel GPI-APs. Orthology- and synteny-based analyses showed that 19 of the 37 GPI-APs found in the order Haemosporida are conserved among Plasmodium species. Our analyses suggest that gene duplication and deletion events may have contributed significantly to the evolution of the GPI proteome, and its composition correlates with speciation.FT-GPI enabled revision of the GPI-proteome of FT-GPI-based prediction is a useful tool for mining GPI-APs and gaining further insights into their evolution and sequence diversity. This resource may also help identify new protein candidates for the development of vaccines for malaria and other parasitic diseases.The online version contains supplementary material available at 10.1186/s12936-022-04430-0. Plasmodium, remains one of the deadliest infectious diseases affecting humans. In 2019, the World Health Organization (WHO) recorded 227 million cases, and this figure rose to 241 million in 2020 due to major disruptions to health infrastructures during the COVID-19 epidemic . Human-infecting parasites are in bold. PlasmoDB and other source of information were used to establish the host and geographic origin of the different isolates. P. faciparum 3D7 is a reference laboratory strain and first described GPI-proteome in apicomplexa [17]. Cloned: genome sequence was obtained from long-term, established laboratory strains that often pass through a purification step. All P. falciparum isolates are of clinical origin. Clinical: non-P. falciparum isolates that were recovered from malaria patientsAdditional file 4: Table S4. Description of proteins from the GPI-AP reference sets. A. P. falciparum reference set from Gilson, et al. [17]. Protein size, number of TM and signal 4.0 prediction were recovered from PlasmoDB. SignalP 5.0 and PredGPI were performed online . Essential genes were described by a Mutation Index Score (MIS) and Fitness score (MFS) from transposon saturation mutagenesis experiments [77]. Sequences were downloaded from PlasmoDB release 51. B. P. vivax reference set from Carlton et al [38]. The list of GPI-APs was given in Suppl. Table 8 of the P. vivax genome paper. Sequence analysis was performed as in (A). No information was available concerning gene essentialness in P. vivax. Negative predictions are in red. * Some proteins had no gene names and were arbitrarily labelled from gpi1 to gpi4. P. falciparum Pf_gpi1 P. vivax ortholog was absent from Carlton et al list [38]. P. falciparum P36 ortholog was not in Gilson et al list [17]. The gpi3 protein was labelled as P32 in some references.Additional file 5: Table S5. TMPred analysis of GPI-AP reference proteins. TMPred provided three parameters for each predicted TM: the first and last coordinates as well as a score. Data are presented for the first and last TM, called N-term and C-term TM respectively. The middle of the table provided information for all TM predicted in between. The distance to end parameter of the last TM was computed as the difference between the protein length and last coordinate. Proteins in bold indicate those which were positively detected by TMpred for the presence of two TM at both ends of the protein, by PredGPI for being a GPI-AP and by SignalP for the presence of a signal peptide. Missing TM and outliers with extreme TM values are in red. A. P. falciparum GPI-APs reference set. Genes with no P. vivax orthologs were in italic. B. P. vivax GPI-APs reference set. Genes with no P. falciparum orthologs are in italic.Additional file 6: Table S6. FT-GPI parameter set efficacy. The Positive and Negative prediction of each combination of FT-GPI parameters was evaluated based on the detection of the 30 reference GPI-APs of P. falciparum and P. vivax in combination with cytosolic, membrane or among the full proteome. Cytosolic proteins had no TM and signal peptide according to PlasmoDB data (Suppl. Table 4). Membrane proteins presented more than 3 TM according to the same source of information. The formula used to compute the parameters of the test are given with the title of each subsection of the Table. AUC was computed using the binary output of FT-GPI (1 for positive detection and 0 if not).Additional file 7: Table S7. GPI-APs detection using different FT-GPI settings in 46 Haemosporida isolates and comparison with orthologs of the P. falciparum 3D7 reference set from Gilson et al [17]. The 31 combination of FT-GPI parameters are described in Suppl. Table 2. Orthologs were obtained from OrthoMCL analysis at PlasmoDB. Efficacy of the FT-GPI set of parameters was computed using the formula described in Suppl. Table 8.Additional file 8: Table S8. Composition of the GPI-proteome of 46 Haemosporida isolates according to FT-GPI PLA001 parameter set. Proteomes were analysed using FT-GPI software. The OrthoMCL annotation (cog_id) and synteny available at PlasmoDB were used to assess the presence of genes in the different isolates. Our analysis was based on proteins presenting more that 210 amino acids. Colour coding was used to determine OrthoMCL group encoding orthologs. The table refers to the number of paralogs found in each genome. Lines merging information from the different orthoMCL groups are marked as Summary in column G. The left part of the table displays the number of orthologs present in PlasmoDB, the FT-GPI detection with the 31 combinations of FT-GPI parameters and the specific detection of PLA001 and PLA030. The GPI-AP provided gene names of the 33 GPI-AP candidates validated in the present study. Four were new GPI-AP compared with P. falciparum 3D7 and P. vivax Sal-1 GPI-proteome . SRA detection as GPI-AP was species specific. The sum of validated GPI-AP is given at the top of the table as well as the not-validated putative GPI-AP (from line 121 to 174). Some orthologs with size below 210 aa were introduced in the analysis and are in italics. P. falciparum 3D7 and P. vivax Sal-1 columns are in bold. The right part of the table provides information about mean, median and standard deviation of the size of protein sequences in recorded per line. * indicates the duplicated OrthoMCL IDs encompassing genes from different loci.Additional file 9: Table S9. Composition of the GPI-proteome of 46 Haemosporida isolates according to FT-GPI PLA030 parameter set. Proteomes were analysed using FT-GPI software. OrthoMCL annotation (cog_id) and synteny available at PlasmoDB were used to assess the presence of genes in the different isolates. Analysis was based on protein presenting more than 210 amino acids. Colour coding was used to determine OrthoMCL group encoding orthologs. The table refers to the number of paralogs found in each genome. Lines merging information from the different orthoMCL groups are marked as summary in column G. The left part of the table displays the number of orthologs present in PlasmoDB, FT-GPI detection with the 31 combinations of FT-GPI parameters and specific detection of PLA001 and PLA030. GPI-AP provided gene names of the 33 GPI-AP candidates validated in the present study. Four were new GPI-AP compared with P. falciparum 3D7 and P. vivax Sal-1 GPI-proteome . SRA detection as GPI-AP was species specific. The sum of validated GPI-AP is given at the top of the table as well as the sum not-validated putative GPI-AP (from line 121 to 177). Some orthologs with size below 210 aa were introduced in the analysis and are in italics. P. falciparum 3D7 and P. vivax Sal-1 columns are in bold. The right part of the table provides information about mean, median and standard deviation of the size of protein sequences in recorded per line. * indicates the duplicated OrthoMCL IDs encompassing genes from different loci.Additional file 10: Fig. S1. Size distribution of GPI-AP present in UNIPROT database. The 29,901 proteins recovered using a query (keyword:KW-0336) were distributed among 2,136 organisms. A. Distribution of the log10 of GPI-AP protein sizes. The graph was generated with ggplot. The colour gradients distinguish organisms within each vertical bar. B. Distribution density based on the hypothesis that the histogram in (A) is a mixture of two normal distributions. Functions were obtained using normalmixEM function from the mixtools library in R. Similar results were obtained using the Mclust function (not shown). Fig. S2. Detection of orthologs of the P. falciparum 3D7 reference set from Gilson, et al. [17] using FT-GPI with varying parameter combinations in 46 Haemosporida isolates. These 31 FT-GPI parameter sets are described in Suppl. Table S2. The horizontal bars depict the number of GPI-AP orthologs detected by each setting of FT-GPI in each isolate. Bars colours are arbitrary. Fig. S3. Evolution of the GPI-Proteome among Haemosprida using PLA001 FT-GPI parameters set. Gene encoding proteins with size over 210 aa were selected for this analysis. A. Heatmap representing the distribution of genes among species. The presence of orthologs and paralogs was established using OrthoMCL annotation. Only orthology groups presenting orthologs in more than four species were included in the present analysis. Presence of paralogs were detected for some genes and represented by the red colour scale. A GPI-AP was absent (white) either because it was not detected by PLA001 or the gene was not present in the genome. Some genes were represented by more than one OrthoMCL group. The discrepancy between synteny and orthology groups was due to rapid sequence evolution and shared homologies. Complete species name is given in given in B and suppl Table 3. Laverania-Pg differentiated P. gaboni and close species from the P. falciparum/P. reichenowi group of parasites [68]. B. Evolution of the GPI-proteome is related to speciation. The genes were the same as in A. The number of paralogs was set to 1 to compute Jaccard distance. The Ward-2 method was used to build the tree."} +{"text": "Nature Communications 10.1038/s41467-022-30819-1, published online 07 June 2022.Correction to: The original version of this Article contained an error in Fig. 5, in which the reference number used in the Fig. 5c did not match with that used in the legend. This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Correction to: Cell Stress and Chaperoneshttps://doi.org/10.1007/s12192-022-01311-yIn the original version of this article, the first author's given and family names were transposed and should have read Lukas Salvermoser.The original article has been corrected."} +{"text": "Cell Death Discovery 10.1038/s41420-022-01011-7, published online 27 April 2022Correction to: The original version of this article unfortunately contained mistakes in figures 1 and 5. The authors apologize for the errors. The correct figures can be found below. The original article has been corrected."} +{"text": "Cell Death and Disease 10.1038/s41419-022-05082-3, published online 20 July 2022Correction to: The original version of this article unfortunately contained an error in Fig. 6F. The authors apologize for the error. The correct figure can be found below. The original article has been corrected."} +{"text": "Correction to: Journal of Cardiovascular Translational Researchhttps://doi.org/10.1007/s12265-022-10288-zDue to an error during production, the graphical abstract for this article was missing from the article as originally published.The original article has been corrected."} +{"text": "British Journal of Cancer 10.1038/s41416-022-02107-8, published online 04 January 2023Correction to: The original version of this article contained an error in two author names. The correct names are Steffan N Ho and Janette Rawlinson. The authors apologize for the errors. The original article has been corrected."} +{"text": "Scientific Reportshttps://doi.org/10.1038/s41598-022-21592-8, published online 09 December 2022Correction to: The original version of this Article contained an error in the name of the author Friedemann Paul, which was incorrectly given as Paul Friedemann.The original Article has been corrected."} +{"text": "Scientific Reportshttps://doi.org/10.1038/s41598-022-26579-z, published online 02 January 2023Correction to: Tables 1 and 2 in the original version of this Article presented redundant for the research information. To increase their readability, data in both Tables was summarized.The original Article has been corrected."} +{"text": "Bioscience Reports at the request of the authors, who found miscalculations in their raw data and are therefore concerned about the results and conclusions. It also follows receipt of a notification from a reader, alerting the Editorial Office to similarities between the Figure 3A panels of this paper and those from other articles. These other papers do not share any common authors, and include Figure 2B from Shi et al. 2018 (doi: 10.1186/s11658-018-0094-0), Figure 3A from Wang et al. 2020 (doi: 10.1242/bio.049478), and Figure 3A from Qui et al. 2018 (doi: 10.3892/ijo.2018.4483). The authors wish to retract the article. The Editor-in-Chief and Editorial Board agree with the retraction.This article is being retracted from"} +{"text": "Scientific Reports, 10.1038/s41598-022-18371-w, published on 22 August 2022Correction to:The original version of this Article contained an error in the spelling of one of the author names. Lorna M. Hatch was incorrectly given as Lorna A. Hatch.The original Article has been corrected."} +{"text": "Nature 10.1038/s41586-022-04755-5 Published online 8 June 2022Correction to: In the version of this article initially published, the name of author P. Wang appeared incorrectly (as P. Wan); the name has been corrected in the HTML and PDF versions of the article."} +{"text": "Nature Immunology 10.1038/s41590-021-01121-x, published online 20 January 2022.Correction to: https://github.com/angelolab/publications/tree/master/2022-McCaffrey_etal_HumanTB.In the version of the article originally published, the link provided in the Code availability section was invalid and has now been replaced in the HTML and PDF versions of the article with the following:"} +{"text": "Open Biol.\u200910, 20170247. (Published online 1 January 2017) (https://doi.org/10.1098/rsob.160247)Following an investigation, we have found that in figures 4, 5 and 6 of the manuscript, there appears to be multiple duplications, re-positioning and alterations to the western blot images. In further review of the images and the raw data supplied by the authors, we are unable to verify the conclusions of the paper or validity of the data.https://royalsocietypublishing.org/doi/10.1098/rsob.200165.The image integrity standards and policies for the journal can be found here: Open BiologyProf. Jon Pines FRS Editor-in-Chief,\u2009Open Biology Editorial Team"} +{"text": "Correction: Int J Behav Nutr Phys Act 19, 43 (2022)https://doi.org/10.1186/s12966-022-01269-1Following publication of the original article , the autbold typeface.The updated text is given below and the changes have been highlighted in The original article has beenBelow is a table of corrections which have been implemented in the original article."} +{"text": "Nature Human Behaviour 10.1038/s41562-022-01470-z. Published online 24 November 2022.Correction to: In the version of this article initially published, the Acknowledgements information for Aleksandra Kanjuo Mr\u010dela did not include thanks for support from the Slovenian Research Agency (ARRS) under grant no. P5-0193. The error has been corrected in the HTML and PDF versions of the article."} +{"text": "Cell Death and Disease 10.1038/s41419-018-0758-8, published online 18 June 2018Correction to: The original version of this article contained errors in figures 2 and 3. The author apologize for the errors. The corrected figures can be found below."} +{"text": "Correction to: Neurochemical Research 10.1007/s11064-022-03756-0In the original online version of this article unfortunately contains errors, the authors would like to issue the following corrections:1. Professor Hao Yang (yanghao.71_99@yahoo.com) should be incorporated as co-corresponding author.2. The resolution of the Fig.\u00a0Those changes do not affect the results of the study. We apologize to readers for those errors. The original version of this article is updated."} +{"text": "Cell Discovery (2022) 8:85Correction to: 10.1038/s41421-022-00442-x published online 06 September 2022In this article, the authors have found an error in Data Availability section, the iProX ID is incorrect. The correct one is IPX0001444000. We are sorry for the inconvenience."} +{"text": "Correction to: European Radiology10.1007/s00330-022-08840-1The original version of this article, published on 18 May 2022, unfortunately contained some mistakes. The affiliations were incorrectly rearranged during the typesetting stage. They are now corrected in this paper.The original article has been corrected."} +{"text": "Cell Death and Disease 10.1038/s41419-021-03662-3, published online 08 April 2021Correction to: The original version of this article unfortunately contained an error. For Figure 2I, the authors accidentally misplaced the image derived from Immunohistochemistry assays of anti-CC3 in Vec group during the last typesetting process (CDDIS-20-4660R), as inadvertent duplication the image of the WT+ shp63 group. The correct figure can be found below. The authors apologize for the error. The original article has been corrected."} +{"text": "Nature 10.1038/s41586-022-04402-z Published online 9 February 2022Correction to: In the version of this article initially published, the first sentence of the Author contributions section was truncated. The section has been amended to begin \u201cV.B.K. performed protein purification, atomic model building and structural interpretation.\u201d The error has been corrected in the HTML and PDF versions of the article."} +{"text": "Retraction Note: Cancer Cell Int (2019) 19:156https://doi.org/10.1186/s12935-019-0879-xThe Editors-in-Chief have retracted this article at the corresponding author's request. After publication, the authors found that the data could not be replicated. This was caused by mislabelling of patient blood samples and the use of contaminated cell lines in this study.The authors therefore no longer have confidence in the presented results.All authors agree to this retraction."} +{"text": "Correction: Mol Cancer 16, 171 (2017)https://doi.org/10.1186/s12943-017-0737-1Fig. Fig. Fig. Fig. Following publication of the original article , the autThe authors provided the Journal with the original data files. The corrected figure is provided here. The corrections do not have any effect on the results or conclusions of the paper."} +{"text": "Journal of Perinatology 10.1038/s41372-022-01422-5, published online 11 June 2022Correction to: In the original version, there was an error in the inscription of Fig. 2. The arrow next to \u2018Anatomical dead space\u2019 should be UP and not DOWN. The figure should have appeared as shown below.The original article has been corrected."} +{"text": "Cryptography deals with designing practical mathematical algorithms having the two primitive elements of confusion and diffusion. The security of encrypted data is highly dependent on these two primitive elements and a key. S-box is the nonlinear component present in a symmetric encryption algorithm that provides confusion. A cryptographically strong bijective S-box structure in cryptosystem ensures near-optimal resistance against cryptanalytic attacks. It provides uncertainty and nonlinearity that ensures high confidentiality and security against cryptanalysis attacks. The nonlinearity of an S-box is highly dependent on the dispersal of input data using an S-box. Cryptographic performance criteria of chaos-based S-boxes are worse than algebraic S-box design methods, especially differential probability. This article reports a novel approach to design an 8 \u00d7 8 S-box using chaos and randomization using dispersion property to S-box cryptographic properties, especially differential probability. The randomization using dispersion property is introduced within the design loop to achieve low differential uniformity possibly. Two steps are involved in generating the proposed S-box. In the first step, a piecewise linear chaotic map (PWLCM) is utilized to generate initial S-box positions. Generally, the dispersion property is a post-processing technique that measures maximum nonlinearity in a given random sequence. However, in the second step, the concept is carefully reverse engineered, and the dispersion property is used within the design loop for systematic dispersal of input substituting sequence. The proposed controlled randomization changes the probability distribution statistics of S-box\u2019s differentials. The proposed methodology systematically substitutes the S-box positions that cause output differences to recur for a given input difference. The proposed S-box is analyzed using well-established and well-known statistical cryptographic criteria of nonlinearity, strict avalanche criteria (SAC), bit independence criteria (BIC), differential probability, and linear probability. Further, the S-box\u2019s boomerang connectivity table (BCT) is generated to analyze its strength against boomerang attack. Boomerang is a relatively new attacking framework for cryptosystem. The proposed S-box is compared with the state-of-the-art latest related publications. Results show that the proposed S-box achieves an upper bound of cryptographic properties, especially differential probability. This work hypothesizes that highly dispersive hamming distances at output difference, generated a systematic S-box. The mixing property of chaos generated trajectories utilized for decimal mapping. To test the randomness of generated chaotic trajectories, a cryptographically secure pseudo-random sequence was generated using a chaotic map that was tested using the National Institute of Standards and Technology (NIST) NIST-800-22 test suit. Cryptography aids individual users and corporate organizations in protecting their digital data and information. With the prevalence of cryptography , digitalData confidentially in cryptography is related to the encryption of digital data. Modern block ciphers, including DES and variAn S-box is a bijective mapping analysis , an expaanalysis . It was Differential cryptanalysis is a beneficial attack on block ciphers, also known as a chosen-plaintext attack. To mount this attack, a cryptanalyst first chooses input differential attacker . The folattacker .Definition 1: Let x, \u2206y) with input difference The DP is considered a stochastic variable and can only take limited values of either 0 or multiple of Rijndael S-box was a two-step algebraic design based on AES\u2019s GF(256) inverse and affine transformation. It was based on the NIST criteria, inspired by the linear and differential attack . The intn) are stored in an integer table. Secondly, a 2D Baker map permutes the integer table to obtain the final S-box. Later on, many researchers showed their potential and improved chaos-based S-boxes utilizing 1-dimensional chaotic maps is the initial value and p \u2208 is the control factor. Any arbitrary chosen initial condition can be used. It is well established that the randomness of the RNG numbers directly affects encryption applications\u2019 security. Hence they have crucial importance. Therefore, a successful bitstream selected as a key for encryption possesses a property that should have a uniform probability distribution of 1\u2032s and 0\u2032s. It means that the number of 1\u2032s and 0\u2032s in the bitstream should be equal or nearly equal. A PWLCM generates floating-point numbers in the given range of . We used the default parameters for all tests to test our proposed RNG using the NIST test. The value of \u03b1 was chosen equal to 0.01, which means that for a test to be successful, the P-value obtained must be greater than or equal to 0.01. The random bit stream obtained from the proposed RNG using PWLCM passed all NIST tests presented in where, Generally, the dispersion property is a post-processing technique used to measure the randomness in a sequence. The proposed novel methodology uses dispersion property within the design loop for systematic S-box design. The dispersion property can be defined as:Definition 2:The dispersion measures the irregularity in output spreadfor a given input spreadFor a given substitution \u03c0, the list of dispersion pairs of \u03c0 is defined aswhere The utilization of dispersion property within the design loop under proposed design conditions requires an understanding of the computation of DM. The dispersion matrix is filled with the spread pair , strict 1. Bijective2. NonlinearityThe bijection test evaluates the uniqueness of the output of an S-box. If an S-box fulfills the bijection criterion, its output values are unique and non-repeating in the interval The nonlinearity (NL) test measures the smallest Hamming distance of the reference function from all the affine functions . It reprwhere The maximum possible nonlinearity value in 2n\u22122n2\u22121 . Hence, 3. Strict avalanche criterionStrict Avalanche Criterion (SAC) measures4. Bit independence criteriaThe output bit independence criterion (BIC) is a crucial property for any cryptographic system and was introduced by where, 5. Linear approximation probabilityThe linear approximation probability (LP) measures the maximum imbalance between input and output bits . Mathemawhere 6. Differential approximation probabilityThe differential approximation probability (DP) exhibits the differential uniformity of an S-box , which i7. Correlation analysis: sensitivity among S-boxesTo study the randomness among S-boxes, the correlation coefficient is measured. It determines the similarities among S-boxes with a slight change in the initial condition. The correlation coefficient, \u03c1, is measured as:n = size of the S-box. The initial condition is changed to the 4th decimal digit for the analysis, and 500 S-boxes are generated. x-axis shows the number of inputs, the y-axis shows the number of S-boxes, and the z-axis shows the values of correlation coefficients. The upper and lower bound of the achieved correlation coefficient ranges from \u22120.2139 to 0.2667. It is quite evident from the where 8. Boomerang connectivity table (BCT)The boomerang attack, proposed by where and DDT . The numThe entries in the first row and first column of BCT are all 256. For a better illustration of the internal structure of BCT, 9. Feistel counterpart of BCT (FBCT)A detailed analysis is provided in Another related extension of BCT for ciphers following Feistel construction was proposed by (1) Symmetry: for all (2) Fixed value :\u2003(a) First row: for all \u2003(b) First column: for all \u2003(c) Diagonal: for all (3) Multiplicity: for all (4) Equalities: for all The FBCT was given mentclasspt{minimaThe suitability of the proposed S-box is evaluated as an application in image encryption. Image encryption, measures the strength and robustness of the proposed S-box, is performed using majority logic criteria (MLC) . It is ptitution . This imIt can be observed from The coefficient value of approximately 0 indicates no or weak correlation between images. The proposed S-box\u2019s correlation parameter value is 0.0398, close to 0, and comparable with AES S-box. The proposed S-box enhances the spread and dispersion among input and output pixels. Thus, it results in a weak correlation among pixels values. Further, the proposed S-box enhances the modern encryption properties of confusion and diffusion. The contrast parameter value of the proposed S-box is 9.9895. The constant image entails a contrast value of 0. A high value of contrast indicates randomness in the image. Due to systematic nonlinear mapping using the proposed S-box, Objects in the plain image are dispersed completely. Therefore, we achieved a high value of contrast in encrypted that indicates strong encryption. The homogeneity parameter measures the closeness of the distributed pixels of GLCM to its diagonals. The achieved homogeneity results using the proposed S-box and AES S-box are comparable and show strong encryption. Using majority logic, the image substitution analysis entails the proposed S-box results comparable to the state-of-the-art results in The visual demonstration of plain image substituWe proposed a systematic S-box to achieve near-optimal cryptographic properties of bijective, nonlinearity, SAC, BIC, DP, and LP. The generated S-box is given in This article introduced a relatively new cryptanalysis known as a boomerang attack. The BCT table is generated to individually analyze the differential characteristics of cryptosystem system components. BCT provides more versatility than DDT and finds the nonzero pairs P-values were well under the accepted range . The NIST suite\u2019s frequency and block frequency test also validates the balance properties. We also employed our S-box in image encryption algorithm and performed various statistical tests to investigate the proposed S-box\u2019s performance and suitability in image encryption applications. The proposed S-box shows excellent statistical entropy, energy, correlation, contrast, homogeneity.Further, the proposed nonlinear mapping, real to decimal, generated cryptographically secure PRN were tested using the NIST approved test suite. All of the tests in the NIST suite were passed, and A novel method to generate a near-optimal S-box is proposed. A chaotic multilevel map is employed for initial chaotic trajectories. The given PWLCM generates cryptographically secure PRN, vetted through the NIST test. Under given design conditions, the dispersion matrix is systematically employed within the proposed design loop. The proposed design criteria efficiently substitute weak S-box positions for a robust S-box structure and near-optimal results. The proposed S-box also exhibits high dispersion in design which is critical to achieving the notion of confusion. The proposed S-boxes were evaluated based on expanded S-box design criteria. The proposed S-boxes were comparable to recently published state-of-the-art S-box designs in the field. Our results demonstrate that the proposed S-box has excellent cryptographic properties. The nonlinearity value is in the range of 100 to 108 and achieves the differential uniformity of 8. A systematic and robust methodology of chaos-based S-box is required to achieve the DP in the range of 4 to 10. The strength of our proposed S-box was also tested against new boomerang cryptanalysis. Therefore, the BCT and FBCT table of the proposed S-box was generated to find the maximum BCT/FBCT differential probability. The proposed S-box had a maximum BCT and FBCT differential probability of 0.0625 and 0.0468, respectively. The BCT/FBCT analysis provides a new insight to design and analyze the S-box for cryptosystem. Our proposed S-box shows an upper-bound value of LP of 0.1028. It is evident from the results presented in this paper that our S-box achieves an upper bound of cryptographic properties. To validate the suitability of the proposed S-box as a confusion component in image encryption algorithms, a substitution-based statistical test of entropy, energy, correlation, contrast, and homogeneity was performed to achieve the values of 7.358, 0.016, and 0.033, 10.11, 0.406, respectively. Our S-box show excellent performance against these tests and is suitable for image encryption applications.In the future, this work can be extended to design key-based S-boxes. The S-boxes are based on chaotic parameters, where the S-boxes are dynamically generated in each round of encryption to obtain a more secure cryptosystem. Further, the differential uniformity of BCT/FBCT of chaos-based S-boxes will be analyzed to study the resistance against boomerang attack. Furthermore, the applications of the proposed S-boxes in image encryption and watermarking can be investigated.10.7717/peerj-cs.940/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj-cs.940/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj-cs.940/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj-cs.940/supp-4Supplemental Information 4Click here for additional data file.10.7717/peerj-cs.940/supp-5Supplemental Information 5Click here for additional data file.10.7717/peerj-cs.940/supp-6Supplemental Information 6Click here for additional data file.10.7717/peerj-cs.940/supp-7Supplemental Information 7Click here for additional data file."} +{"text": "Nature Communications 10.1038/s41467-022-30905-4, published online 8 June 2022.Correction to: The original version of this Article contained an error in the Acknowledgement Section, which incorrectly read\u2018The authors thank N. Perez and M. Pankhurst for a sample permit\u2019.The correct version removes this sentence. This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Scientific Reports 10.1038/s41598-022-10807-7, published online 25 April 2022Correction to: The original version of this Article contained an error in the spelling of the author Min A Yoon which was incorrectly given as Min A. Yoon.The original Article has been corrected."} +{"text": "Correction to: BMC Pregnancy Childbirth 22, 3 (2022)https://doi.org/10.1186/s12884-021-04307-1P Value of 0.906 for preterm and 0.122 for low birthweight. This has been corrected in table 1 and the corrections do not change the results of the data or the conclusions.Following publication of the original article , the aut"} +{"text": "Translational Psychiatry 10.1038/s41398-021-01562-w, published online 17 September 2021Correction to: The original version of this article unfortunately contained a mistake. The correction is to swap the labels for figure 3, representing Lo and Hi Probability in panels G, H, and K. The corrected figure can be found below. The original article has been corrected."} +{"text": "Cell Death and Disease 10.1038/s41419-022-04790-0, published online 09 April 2022Correction to: The original version of this article unfortunately contained an error in Fig. 5. The authors found that the Fig. 5B and D (the second panel) in the online version are different from those in the version they submitted. We apologize for the error. The correct figure can be found below. The original article has been corrected."} +{"text": "Mucosal Immunol 10.1038/s41385-021-00470-y, published online 30 Nov 2021.Correction to: The original version of this article unfortunately contained a mistake. Due to a typesetting error Box 1 was omitted. We apologize for the error. Box 1 can be found below. The original article has been corrected."} +{"text": "Nature Communications 10.1038/s41467-022-28290-z, published online 04 February 2022.Correction to: The original version of this Article contained an error in the numbering of the reference list, with references 132\u2013134 being 154\u2013156 and references 135\u2013156 being 132-153. This has been corrected in the PDF and HTML versions of the Article."} +{"text": "Cell Death Discovery 10.1038/s41420-021-00720-9, published online 28 October 2021Correction to: The original version of this article contained an error in an author name. The name of the corresponding author Dr. Kent should be \u201cCraig K. Kent\u201d, currently Craig is listed as the middle name. We apologize for the error. The original article has been corrected."} +{"text": "Scientific Reportshttps://doi.org/10.1038/s41598-021-01808-z, published online 24 November 2021Correction to: The original version of this Article contained an error in the spelling of the author Bijan Fallah which was incorrectly given as Bijan H. Fallah. The original Article and accompanying Supplementary Information file have been corrected."} +{"text": "Nature 10.1038/s41586-022-04470-1 Published online 2 March 2022Correction to: In the version of this article initially published, Extended Data Fig. 4 was an inadvertent duplicate of Extended Data Fig. 2. The image has been replaced in the HTML and PDF versions of the article"} +{"text": "Correction to: J Exp Clin Cancer Res 37, 76 (2018)https://doi.org/10.1186/s13046-018-0739-xFollowing publication of the original article , the autThe corrected figure is provided here. The correction does not have any effect on the results or conclusions of the paper. The original article has been corrected."} +{"text": "Correction to: J Exp Clin Cancer Res 39, 82 (2020)https://doi.org/10.1186/s13046-020-01581-3In Fig. Following publication of the original article , the autThe corrected figure is given here. The corrections do not have any effect on the final conclusions of the paper. The original article has been corrected."} +{"text": "Nature Communications 10.1038/s41467-021-24343-x; published online 02 July 2021.Correction to: The original version of this Article contained an error in the author affiliations.Luca G. Campana was incorrectly associated with Medical Oncology, The Christie NHS Foundation Trust, Manchester, UK.This has now been corrected in both the PDF and HTML versions of the Article."} +{"text": "The Editor-in-Chief expresses his gratitude to the following individuals, who reviewed manuscripts for Food Safety Vol. 7to Vol. 9."} +{"text": "Nature Communications 10.1038/s41467-021-22581-7, published online 19 April 2021.Correction to: The original version of this Article contained an error in the caption of Fig. 2, where the sentence \u2018Yield-gap fractions close to zero show low-yielding croplands with high yield gaps.\u2019 should have been deleted. This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Nature Structural & Molecular Biology 10.1038/s41594-021-00590-w, published online 27 May 2021.Correction to: In the version of this article initially published, the surname of author Mayra L. Ruiz Tejada Segura was misspelled as Ruiz Tejeda Segura. The error has been corrected in the online version of the article."} +{"text": "Scientific Reports 10.1038/s41598-021-89351-9, published online 11 May 2021.Correction to: The original version of this Article contained an error in the spelling of the author H. R. Abd El-Mageed which was incorrectly given as H. R. Abdel El-Mageed. The original Article and accompanying Supplementary Information file have been corrected."} +{"text": "Correction to: Confl Health 15, 38 (2021)https://doi.org/10.1186/s13031-021-00371-8The findings and conclusions in this article are those of the authors and do not necessarily represent any official position or policy of the U.S. National Institutes of Health, the U.S. Department of Health and Human Services, or any other institutions with which authors are affiliated.The original publication of this article was missThe original article has been updated."} +{"text": "Scientific Reports 10.1038/s41598-021-86181-7, published online 23 March 2021Correction to: The original version of this Article contained an error in the spelling of the author A. Laref which was incorrectly given as A. Lareef. The original Article has been corrected."} +{"text": "Correction to: BMC Cancer 21, 682 (2021)https://doi.org/10.1186/s12885-021-08274-wFollowing publication of the original article , it was The publisher apologises to the authors and readers for the inconvenience caused by the error."} +{"text": "Nature Communications 10.1038/s41467-021-22244-7, published online 06 April 2021.Correction to: The original version of the Peer Review File associated with this Article was updated shortly after publication to redact reviewer names. This has been corrected in the PDF.Peer Review File"} +{"text": "To the Editor,https://kmplot.com/analysis/) has been used to clarify the prognostic significance of cofilin isoforms in PDAC [Proteomics is a technique for analyzing protein expression at the cellular level. Proteomic technology that combines two-dimensional gel electrophoresis 2-DE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) has a high throughput and accuracy . This te-DE and l in PDAC . Using t"} +{"text": "Scientific Reports 10.1038/s41598-020-70093-z, published online 06 August 2020Correction to: The original version of this Article contained an error in the spelling of the author Gerald C. Gooden which was incorrectly given as Gerald C. Gooden II.The original Article has been corrected."} +{"text": "Communications Biology 10.1038/s42003-021-02982-6, published online 21 January 2022.Correction to: The original version of this Article contained an error in Fig. 4d, in which a representative microscopy image for TRAP-6 stimulated platelets in Li-Heparin was erroneously used for the K2-EDTA panel. The correct version of Fig. 4 is:which replaces the previous incorrect version.The link to the raw data has been updated in the Data Availability statement to: \u201810.5281/zenodo.4461273.The error has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Scientific Reports 10.1038/s41598-022-05877-6, published online 07 February 2022Correction to: In the original version of this Article, Sandra Steiger was incorrectly indicated as a corresponding author. The correct corresponding author for this Article is Jacqueline Sahm. Correspondence and request for materials should be addressed to Jacqueline.Sahm@uni-bayreuth.de.The original Article has been corrected."} +{"text": "Light: Science & ApplicationsCorrection to: 10.1038/s41377-021-00662-y, published online 28 October 20213) after treatment was incorrectly positioned on the Control sample (PAT-treated MAPbI3). In addition, the color scales were drawn in the wrong orientation.In the version of this article initially published, there were errors in Fig. 3a. The PL mapping image of Target sample (Au@PAT-modified MAPbISo we would like to correct Fig. 3 in the initial published version to the following figure:We would like to apologize for any inconvenience this may have caused.The original article has been updated."} +{"text": "Communications Biology 10.1038/s42003-021-02944-y, published online 17 January 2022.Correction to: The original version of this Article contained an error in Fig. 1f, in which the labels for pSTAT3, STAT3 and Histone H3 were omitted. The correct version of Fig. 1f is:which replaces the previous incorrect version:The error/errors have been corrected in both the PDF and HTML versions of the Article."} +{"text": "Scientific Reports 10.1038/s41598-021-97090-0, published online 06 September 2021.Correction to: In the original version of this Article, Lin Zhu was omitted as a corresponding author. Correspondence and requests for materials should also be addressed to zhulinkin@foxmail.com.The original Article has been corrected."} +{"text": "Correction to: J Exp Clin Cancer Res 40, 153 (2021)https://doi.org/10.1186/s13046-021-01959-xFollowing publication of the original article , the autThe corrected figure is given here. The correction does not have any effect on the results or conclusions of the paper. The original article has been corrected."} +{"text": "Scientific Reports 10.1038/s41598-020-80182-8, published online 13 January 2021Correction to: The original version of this Article did not include the link to the code repository. This is now included in the Code Availability section which reads:https://github.com/nonstop1962/pytorch-dental_age_estimation.The code underlying this study is available at: The Article has been corrected."} +{"text": "Scientific Reports 10.1038/s41598-021-94720-5, published online 27 July 2021Correction to: The Acknowledgments section in the original version of this Article was incomplete.\"The part of nanowire growth was supported by RFBR 19-02-00981, a theoretical part was supported by RSF 20-69-47013. O.V.S. acknowledges the support of the scholarship of the President of the Russian Federation for the study of electronic transport of the samples. D.R. acknowledges COST Action CA16218\u2014Nanoscale Coherent Hybrid Devices for Superconducting Quantum Technologies.\"now reads:\"The part of nanowire growth was supported by RFBR 19-02-00981, a theoretical part was supported by RSF 20-69-47013. O.V.S. acknowledges the support of the scholarship of the President of the Russian Federation for the study of electronic transport of the samples. D.R. acknowledges COST Action CA16218\u2014Nanoscale Coherent Hybrid Devices for Superconducting Quantum Technologies. The work is partially supported by the Twinning project SPINTECH under Grant Agreement Number 810144.\"The original Article has been corrected."} +{"text": "Nature Communications 10.1038/s41467-021-23369-5, published online 25 May 2021.Correction to: The original version of this Article did not acknowledge Prof. Jun Fan as a corresponding author. This has now been corrected in both the PDF and HTML versions of the Article."} +{"text": "Cell Death Discovery 10.1038/s41420-020-00367-y, published online 02 December 2020Correction to: The original version of this article unfortunately contained a mistake in figure 5. The correct figure can be found below. The authors apologize for the error. The original article has been corrected."} +{"text": "Scientific Reports 10.1038/s41598-021-87170-6, published online 08 April 2021Correction to: In the original version of this Article, Lei Shi was omitted as a corresponding author. Correspondence and requests for materials should also be addressed to 964673743@qq.com.The original Article has been corrected."} +{"text": "Correction to: J Exp Clin Cancer Res 36, 172 (2017)https://doi.org/10.1186/s13046-017-0635-9Figure Fig. SFollowing publication of the original article , minor eThe corrected figures, produced using the original data, are given here. The correction does not have any effect on the final conclusions of the paper.Additional file 4. The wound healing and chamber invasive assay revealed that breast cancer cell migration and invasion were inhibited by EA in a time- and dose-dependent manner."} +{"text": "Nature Communications 10.1038/s41467-021-23351-1, published online 24 May 2021.Correction to: The original version of this Article contained an error in the spelling of the author L. David Sibley, which was incorrectly given as David L. Sibley. This has now been corrected in both the PDF and HTML versions of the Article."} +{"text": "Publisher Correction to: BMC Biol 19, 99 (2021)https://doi.org/10.1186/s12915-021-01043-yFollowing publication of the original article , it was The publisher apologises to the authors and readers for the inconvenience caused by the error."} +{"text": "Correction to: J Exp Clin Cancer Res 40, 52 (2021)https://doi.org/10.1186/s13046-021-01857-2st row, 3rd column)Fig. Following publication of the original article , the autThe authors provided the Journal with the original data files. The corrected figure is provided here. The correction does not have any effect on the results or conclusions of the paper. The original article has been corrected."} +{"text": "Nature Communications 10.1038/s41467-021-25031-6, published online 4 August 2021.Correction to: The original version of this Article omitted an equal contribution statement for Tae Jin Jang and Won Seok Choi. This has now been corrected in both the PDF and HTML versions of the Article."} +{"text": "Nature Communications 10.1038/s41467-021-21939-1, published online 23 March 2021Correction to: The original version of this Article contained an error in Figs. 3 and 8.In the original version of Fig. 3, the references reported in panels a and c are not correct.The correct version of Fig. 3 is:which replaces the previous incorrect version:In the original version of Fig. 8, the reference reported in panel b is not correct.The correct version of Fig. 8 is:which replaces the previous incorrect version:This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Correction to: Scientific Reports, 10.1038/s41598-021-02728-8, Published online 02 December 2021The Supplementary Information published with this Article contained an error, where the layout for Table S1 was incorrect.This error has now been corrected in the Supplementary Information file that accompanies the original Article."} +{"text": "Scientific Data 10.1038/s41597-021-00860-8, published online 02 March 2021Correction to: The original version of this Data Descriptor omitted the following author from the Author List: Ying Taur. The URL in reference 35 has also been corrected to 10.6084/m9.figshare.c.5271128. These errors have now been corrected in both the PDF and HTML versions of the Article."} +{"text": "Correction to: BioPsychoSocial Med 15, 22 (2021).https://doi.org/10.1186/s13030-021-00223-0Following publication of the original article , the autIn the old Fig. In the old Table\u00a0The correct figure and table have been included in this correction, and the original article has been corrected."} +{"text": "Scientific Reportshttps://doi.org/10.1038/s41598-021-92469-5, published online 21 June 2021Correction to: The original version of this Article contained an error in the spelling of the author Robert A. McCleery which was incorrectly given as Robert A. McCeery. The original Article has been corrected."} +{"text": "Scientific Reports 10.1038/s41598-021-96855-x, published online 30 August 2021Correction to: In the original version of this Article, Sang Yong Song was omitted as a corresponding author. Correspondence and requests for materials should also be addressed to yodasong@gmail.com.The original Article and accompanying Supplementary Information file have been corrected."} +{"text": "Nature Communications 10.1038/s41467-020-17892-0, published online 13 August 2020.Correction to: The original version of this Article contained an error in the Acknowledgements, which incorrectly listed the NIH grant \u2018AI142759\u2019. This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Communications Biology 10.1038/s42003-020-01335-z, published online 26 October 2020.Correction to: A. The units were given as mA. The correct units are mM. The error has been corrected in the HTML and PDF versions of the Article.In the original version of the Article, Figure 3 panels b\u2013d displayed incorrect units for F-actin Concentration, C"} +{"text": "Scientific Reports 10.1038/s41598-022-05798-4, published online 25 February 2022Correction to: The original version of this Article contained an error in the spelling of the author Grace M. Hwang, which was incorrectly given as Grace Hwang.The original Article has been corrected."} +{"text": "Nature Neuroscience 10.1038/s41593-020-0685-8, published online 24 August 2020.Correction to: In the version of this article initially published, multiple errors appeared in the author and affiliations lists. The errors have been corrected in the PDF and HTML versions of this article."} +{"text": "Nature Communications 10.1038/s41467-021-22255-4, published online 6 April 2021.Correction to: The original version of this Article contained several typographic errors. These have been corrected in both the PDF and HTML versions of the Article."} +{"text": "BMJ Open Resp Res 2021;8:e000857.Wilcox CR, Islam N, Dambha-Miller H. Association between influenza vaccination and hospitalisation or all-cause mortality in people with COVID-19: a retrospective cohort study. The authors want to alert readers to the following correction made to the published version.Christopher Wilcox and Nazrul Islam are the joint first authors."} +{"text": "Nature Communications 10.1038/s41467-021-23690-z, published online 8 June 2021.Correction to: The original version of this Article contained an error in Fig. 1c, in which two single-headed arrows were inadvertently illustrated as double-sided arrows. This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Nature Communications 10.1038/s41467-021-21372-4, published online 19 February 2021.Correction to: The original version of this Article contained an error in the Code availability statement, which omitted a link to the in-house Fiji macro. The Code availability statement should read:https://github.com/timjedwar/Vangl2-cell-morpho.112. The in-house Fiji macro developed to identify and extract the surface of 3D structures is available from https://github.com/DaleMoulding/Fiji-Macros and related resources are available from the corresponding author on reasonable request or previously published14.\u201d\u201cCellpose processing is available on GitHub This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Correction to: BMC Psychiatry 21, 627 2021https://doi.org/10.1186/s12888-021-03615-2Following the publication of the original article , the autThe publisher apologizes to the authors and readers for the inconvenience.The original article has been"} +{"text": "Nature Communications 10.1038/s41467-021-22799-5, published online 25 May 2021.Correction to: The original version of this Article contained an error in Fig. 2, in which a legend has been missing. This has been corrected in both the PDF and HTML versions of the Article.The original version of the Peer Review File associated with this Article was updated shortly after publication to redact private notes from the authors.Peer Review File"} +{"text": "Scientific Reportshttps://doi.org/10.1038/s41598-022-07306-0, published online 03 March 2022Correction to: The original version of this Article contained an error in the spelling of the author Ruben R. G. Soares, which was incorrectly given as Ruben G. Soares.This error has now been corrected in the original Article."} +{"text": "Communications Biology 10.1038/s42003-021-02155-5, published online 9 June 2021.Correction to: The original version of this Article contained an error in the spelling of the author Han Zuilhof, which was incorrectly given as Han T. Zuilhof. This has now been corrected in both the PDF and HTML versions of the Article."} +{"text": "Correction to: BMC Genomics 22, 809 (2021)https://doi.org/10.1186/s12864-021-08132-wFollowing publication of the original article , it was In Fig. In Fig. In Fig. The correct figures are presented in this Correction and the original article has been"} +{"text": "Scientific Reports 10.1038/s41598-021-93861-x, published online 15 July 2021Correction to: The original version of this Article contained an error in Figure 6, as the method of VitC administration was incorrectly described as a subcutaneous injection, rather than intraperitoneal.The original Figure The original Article has been corrected."} +{"text": "Cell Death & Disease 10.1038/s41419-021-04410-3, published online 27 November 2021Correction to: The original version of this article unfortunately contained a mistake. There was an error in figure 6b. The authors apologize for the error. The corrected figure can be found below. The original article has been corrected."} +{"text": "Scientific Reportshttps://doi.org/10.1038/s41598-021-88954-6, published online 03 June 2021Correction to: The original version of this Article contained an error.The Data Availability section was omitted. It has now been included and states:Data AvailabilityS. obliqnus WT sample and two S. obliqnus mutant SO120G samples are available at the National Center for Biotechnology Information SRA database under the accession PRJNA805302. The RNA-seq reads of the other reported samples are not available due to file corruption during storage.RNA-seq reads for one The original Article has been corrected."} +{"text": "Nature Ecology & Evolution 10.1038/s41559-019-0853-y, published online 15 April 2019.Correction to: The version of this article originally published was not open access, but should have been open access. The error has been corrected, and the paper is now open access with a CC-BY license."} +{"text": "The Department of Defense Grant award that helped support this work was W81XWH-16-1-0025. Therefore, he needed to change financial support and sponsorship in his publication in our journal.According to the evidence provided by the corresponding author, this work was supported by the National Cancer Institute Grant (R01CA200232-05) and the Department of Defense Grant (W81XWH-16-1-0025) both to Maki CG, and all of the other authors of this paper agreed with the change mentioned above. Therefore, we publish this erratum to announce this change.Cancer Drug Resist 2021;4:85-95. http://dx.doi.org/10.20517/cdr.2020.85Cite this article: Shim D, Duan L, Maki CG. P53-regulated autophagy and its impact on drug resistance and cell fate."} +{"text": "Scientific Reportshttps://doi.org/10.1038/s41598-021-88240-5, published online 26 April 2021Correction to: The original version of this Article contained an error in the spelling of the author Anne Philippe, which was incorrectly given as Anne Phillippe. The original Article and accompanying Supplementary Information file have been corrected."} +{"text": "Communications Biology 10.1038/s42003-021-02863-y, published online 25 November 2021.Correction to: hsp-12.8 as one of the differentially-expressed stress response genes. The correct gene symbol is hsp-12.6. The error has been corrected in the HTML and PDF versions of the Article.In the original published version of the Article, the abstract incorrectly listed"} +{"text": "Cell Death Discovery7:137; 10.1038/s41420-021-00496-y; published online 10 June 2021Correction to: The original version of this article unfortunately contained a mistake in the funding section. This research was funded by a Ph.D. studentship from the Royal Thai Government. Additional funding was provided by Cyclacel Ltd. This study was supported by the Glasgow Experimental Cancer Medicine Center, which is funded by Cancer Research UK [C58789/A251741] and the Chief Scientist\u2019s Office, Scotland. Cell-sorting facilities were funded by the Kay Kendall Leukemia Fund (KKL501) and the Howat Foundation. The authors apologize for the mistake. The original article has been corrected."} +{"text": "Scientific Reports 10.1038/s41598-020-69540-8, published online 28 July 2020Correction to: The original version of this Article contained an error in the spelling of the author Anastasia I. Shpichka which was incorrectly given as Anastasia A. Shpichka.The original Article and accompanying Supplementary Information file have been corrected."} +{"text": "Nature Communications 10.1038/s41467-021-22233-w, published online 30 March 2021.Correction to: The original version of this Article contained an error in Fig. 3c, in which the yellow dots were offset with respect to the background STM image. This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Nature Communications 10.1038/s41467-021-22845-2, published online 7 May 2021.Correction to: The original version of this Article omitted the following from the Acknowledgements:Y.Q. was supported by the National Research Foundation (NRF) of Singapore, NRF-NRFF12-2020-0006.This has now been corrected in both the PDF and HTML versions of the Article."} +{"text": "Nature Communications 10.1038/s41467-021-23168-y, published online 08 June 2021.Correction to: The original version of this Article contained an error in Box 2, in which the Seychelles Conservation and Climate Adaptation Trust funding was written as . The correct value is USD 700,000. This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Correction to: BMC Public Health 22, 257 (2022)https://doi.org/10.1186/s12889-022-12604-3During the publication process of the original article several citation errors were introduced. The correct and incorrect information is shown below. The original article has been updated. The publisher apologizes to the authors & readers for the inconvenience caused."} +{"text": "Nature Communications 10.1038/s41467-021-22605-2, published online 23 April 2021.Correction to: The original version of this Article contained an error in Fig. 4. In Fig. 4b the asterisks denoting significance were incorrectly shown as red instead of black. This error has been corrected in the PDF and HTML versions of the Article."} +{"text": "Correction to: Global Health 17, 15 (2021)https://doi.org/10.1186/s12992-021-00663-xFollowing publication of the original article , it was The affiliation details have been updated in the original article and the correct details can be seen in this correction.The authors apologize for any inconvenience caused."} +{"text": "Nature Communications 10.1038/s41467-021-25213-2, published online 11 August 2021.Correction to: The original version of this Article incorrectly acknowledged Quirijn de Mast as a corresponding author instead of Ramnik J. Xavier. This has now been corrected in both the PDF and HTML versions of the Article."} +{"text": "Scientific Reports 10.1038/s41598-021-96221-x, published online 06 September 2021Correction to: In the original version of this Article, ShiQi Wang was omitted as a corresponding author. Correspondence and requests for materials should also be addressed to wangshiqi@ivpp.ac.cn.The original Article has been corrected."} +{"text": "Scientific Reports 10.1038/s41598-021-91711-4, published online 11 June 2021Correction to: In the original version of this Article, Kumarasamy Thangaraj was omitted as a corresponding author. Correspondence and request for materials should also be addressed to thangs@ccmb.res.in. In addition, the email address of the co-corresponding author Gyaneshwer Chaubey was incorrectly given as thangs@ccmb.res.in. Correspondence and request for materials should also be addressed to gyaneshwer.chaubey@bhu.ac.in.The original Article has been corrected."} +{"text": "Nature 10.1038/s41586-020-2287-8Published online 27 May 2020Correction to: In this Article, author Marquis P. Vawter was missing from the Genome Aggregation Database Consortium list. They are associated with the affiliation: \u2018Department of Psychiatry & Human Behavior, University of California Irvine, Irvine, CA, USA\u2019, and contributed to the generation of the primary data incorporated into the gnomAD resource. In addition, the following statement should have been included in the Acknowledgements section: \u2018L.C.F. was supported by the Swiss National Science Foundation (Advanced Postdoc.Mobility 177853).\u2019 The original Article has been corrected online."} +{"text": "Nature Communications 10.1038/s41467-021-22687-y, published online 17 May 2021.Correction to: In the original version of this Article, Figures 9 and 10 were erroneously swapped. This has now been corrected in the PDF and HTML versions of the Article."} +{"text": "Cell Death and Disease 10.1038/s41419-021-04244-z, published online 13 October 2021Correction to: The original version of this article unfortunately contained a mistake. There was an error in Fig. 7. The authors apologize for the mistake. The original article has been corrected."} +{"text": "Light: Science & ApplicationsCorrection to: 10.1038/s41377-021-00537-2 published online 12 May 20211, it is noticed the Fig. 2 contained an error: three of the curves were cut. The correct Fig. 2 has been provided in this Correction.Following publication of this articleThe original article has also been updated."} +{"text": "Scientific Reports 10.1038/s41598-021-02272-5, published online 24 November 2021Correction to: The original version of this Article contained an error in the spelling of the author Kasia M. Bieszczad, which was incorrectly given as Kasia Bieszczad.The original Article has been corrected."} +{"text": "Correction to: Journal of Bioenergetics and Biomembranes.10.1007/s10863-021-09882-8The original version of this article unfortunately a mistake. Table 1 should not be included in the proofs.The original article has been corrected."} +{"text": "Correction to: Mol Cancer 18, 167 (2019)https://doi.org/10.1186/s12943-019-1097-9Following publication of the original article , the autThe corrected figure is given here. The correction does not have any effect on the results or conclusions of the paper.The original article has been updated."} +{"text": "Nature Communications 10.1038/s41467-021-21570-0, published online 26 February 2021.Correction to: The original version of this Article omitted Zhilun Zhao and Chen Chen as equally contributing authors. This has now been corrected in both the PDF and HTML versions of the Article."} +{"text": "The ISME Journal 10.1038/s41396-021-01045-2, published online 1 July 2021Correction to: Following the publication of this article, the authors noted an error in Table 1. In the first column it should be DNA, not RNA.The original article has been corrected."} +{"text": "Nature Communications 10.1038/s41467-018-03476-6, published online 13 March 2018.Correction to: The original version of this article contained an error in the author list. The author, Frederick F. Lang, was incorrectly listed as Frederick Lang. This error has been corrected in the HTML and PDF versions of the article."} +{"text": "Nature Communications 10.1038/s41467-019-11390-8, published online 06 August 2019.Correction to: This Article contains an error in Fig. 4, in which panel 4b was inadvertently copied from panel 4a. The correct version of Fig. 4 is:The error has not been corrected in the PDF or HTML versions of the Article."} +{"text": "Laboratory Investigation 10.1038/s41374-021-00653-y, published online 26 August 2021Correction to: The original version of this article unfortunately contained a mistake. The Tumor Profiler Consortium was published as Supplementary Information and was therefore not indexed by PubMed. The error has now been corrected."} +{"text": "Communications Biology 10.1038/s42003-021-02354-0, published online 5 July 2021.Correction to: In the original version of the manuscript the authors had included an incorrect image in Fig. 3a due to a copy and paste error. This has been corrected in the HTML and PDF versions of the article."} +{"text": "Scientific Reports 10.1038/s41598-022-05870-z, published online 09 February 2022Correction to: The original version of this Article contained an error in the spelling of the author Taosheng Liu which was incorrectly given as Taosheng S. Liu.The original Article has been corrected."} +{"text": "In the original article, an author name was incorrectly spelled as Raphael N. Vuille-dit-Bile. The correct spelling is Raphael N. Vuille-dit-Bille.The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated."} +{"text": "Correction to: J Exp Clin Cancer Res 40, 308 (2021)https://doi.org/10.1186/s13046-021-02103-5In Fig. In Fig. Following publication of the original article , the autThe corrected figures are given here.In addition, the Acknowledgements section has been corrected; the updated text is as follows:AcknowledgementsThe authors thank Dr Guoqing Ji for critical technical support.The corrections do not have any effect on the final conclusions of the paper. The original article has been corrected."} +{"text": "Scientific Reportshttps://doi.org/10.1038/s41598-021-93595-w, published online 09 July 2021Correction to: The original version of this Article contained an error in the spelling of the author Albert Sneppen which was incorrectly given as Albert Snepppen.The original Article has been corrected."} +{"text": "Scientific Reports 10.1038/s41598-021-98559-8, published online 30 September 2021Correction to: In the original version of this Article, Masaki Satoh was incorrectly listed as an author of the original Article, and has subsequently been removed.The original Article has been corrected."} +{"text": "We read with interest the article by Li et al. on the association between the use of ACE inhibitors (ACE-Is) and angiotensin receptor blockers (ARBs) and in-hospital mortality among patients with COVID-19 of 0.196 in the African American population and an OR of 0.687 (95% CI 0.427\u20131.106) in the non\u2013African American population. Accordingly, the interaction term was not significant (95% CI 0.185\u20131.292; The potential association between ACE-I/ARB use and COVID-19 in-hospital mortality is of great interest to the medical community. Further, the ability to provide reliable subgroup analyses is vital in clinical decision-making . Interac"} +{"text": "Scientific Reports 10.1038/s41598-021-96121-0, published online 20 August 2021Correction to: The original version of this Article contained an error in the spelling of the author Bhonde R. which was incorrectly given as Bhonde Ramesh R. The original Article and accompanying Supplementary Information file have been corrected."} +{"text": "Nature Communications 10.1038/s41467-021-21151-1, published online 17 February 2021.Correction to: x-axis were incorrectly shifted to the right.The original version of this article contained an error in Fig. 1, in which for the two left-hand plots depicted in Fig. 1c, the time labels (and associated dotted lines) on the The correct version of Fig. 1 is:which replaces the previous incorrect version:This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Correction to: Genes Nutr. 16, 15 (2021)https://doi.org/10.1186/s12263-021-00696-2Following publication of the original article , it was The original article has since been updated and the corrected author list can be found in this correction.The publisher apologizes for this processing error."} +{"text": "Cell Death Discovery 10.1038/s41420-021-00562-5, published online 08 July 2021Correction to: The original version of this article unfortunately contained a mistake. After publication of this article, the authors noticed an accidental duplication of Fig. 5E: the microscopy imagines of transwell for migration in the Syncytin-1+JTP-74057 and Syncytin-1+GDC-0994 groups. All text referring to the figure, including the legend, are correct and this does not impact the findings of the study. The corrected figure is supplied below. The authors apologize for the mistake. The original article has been corrected."} +{"text": "Correction to: BMC Public Health (2020) 20:1571.http://orcid.org/10.1186/s12889-020-09681-7It was highlighted that in the original article , in the To collect data an electronic self-designed questionnaire based on the EPPM was used in order to measure the risk perception related to the COVID-19.The results revealed significant differences in efficacy, defensive responses, and perceived threat among different population groups particularly among those aged 60 and over."} +{"text": "There is an error in the seventh sentence of the abstract. The correct sentence is: From 2005\u20132009 to 2015\u20132019, the median monthly product price over all distinct indications of all products decreased by 7.56% , whereas it increased by 73.7% for monoclonal antibodies.https://orcid.org/0000-0003-2621-5512).The ORCID iD is missing for the second author. Author Christoph Jakob Ackermann\u2019s ORCID iD is: 0000-0003-2621-5512 (The publisher apologizes for the errors."} +{"text": "Scientific Reports 10.1038/s41598-020-73930-3, published online 13 October 2020Correction to: In the original version of this Article, Shintaro Azuma was incorrectly listed as the corresponding author. The correct corresponding author for this Article is Haruki Uojima. Correspondence and request for materials should be addressed to kiruha@kitasato-u.ac.jp.This error has now been corrected in the HTML and PDF versions of the Article."} +{"text": "Nature Communications 10.1038/s41467-020-20789-7, published online 20 January 2021.Correction to: The original version of this Article contained an error in the Acknowledgements, which incorrectly listed the NIH grant \u2018AI142759\u2019. This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Open Biol.10, 190173. (Published 29 April 2020). (doi:10.1098/rsob.190173)The Editor-in-Chief in agreement with the Publisher has retracted this article. Following an investigation, several flaws and inconsistencies in the flow-cytometry cell-cycle analyses and western blots were found. The authors have not been able to supply the raw data as requested, thus, the editors consider the conclusions of this article to be invalid. The authors have not responded to correspondence about this retraction.https://royalsocietypublishing.org/doi/10.1098/rsob.200165.The image integrity standards and policies for the journal can be found here: Open Biology.Jonathon Pines FRS, Editor-in-Chief,"} +{"text": "Scientific Reportshttps://doi.org/10.1038/s41598-020-77429-9, published online 23 November 2020.Correction to: Hidekuni Inadera was omitted as a member of the consortium The Japan Environment, Children\u2019s Study (JECS) Group in the original version of this Article.The affiliation for Hidekuni Inadera is listed below:University of Toyama, Toyama, Japan.This has now been corrected in the HTML and PDF versions of this Article."} +{"text": "Correction to: J Exp Clin Cancer Res 38, 183 (2019)https://doi.org/10.1186/s13046-019-1177-0In Fig. st row, 3rd column)In Fig. Following publication of the original article , the autThe authors provided the Journal with the original data. The corrected figures are given here. The corrections do not have any effect on the final conclusions of the paper. The original article has been corrected."} +{"text": "Scientific Reportshttps://doi.org/10.1038/s41598-022-07914-w, published online 07 March 2022Correction to: The original version of this Article contained an error in the spelling of the author Michael R. Ehrenstein which was incorrectly given as Michael E. Ehrenstein. The original Article has been corrected."} +{"text": "Cell Death and Disease 10.1038/s41419-020-02930-y, published online 16 September 2020Correction to: The original version of this article unfortunately contained a mistake in figure 2d. The correct figure can be found below. The authors apologize for the mistake. The original article has been corrected."} +{"text": "Nature Communications 10.1038/s41467-021-22265-2, published online 6 April 2021.Correction to: The original version of this Article contained an error in Fig. 2, in which the plot shown in panel c was inadvertently duplicated in panel d. The correct version of Fig. 2 is:which replaces the previous incorrect version:"} +{"text": "In the original article, we neglected to include the funder Marga und Walter Boll-Stiftung, grant no.: 210-04.00-16 to Stephan Bender. The corrected funding statement is shown below.This research was funded by the Deutsche Forschungsgemeinschaft \u2013 Project-ID 431549029 \u2013 SFB 1451 and Marga und Walter Boll-Stiftung, grant no.: 210-04.00-16.The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated."} +{"text": "The Plant Cell, koab051, https://doi.org/10.1093/plcell/koab051During the conversion of black-and-white figures into color figures, the authors misplaced a sub-figure of LFY expression in Figure 4, panel F. A corrected version of the figure panel appears below and the original article has been updated. The authors apologize for the error."} +{"text": "Nature Medicine 10.1038/s41591-021-01453-z, published online 4 August 2021.Correction to: In the version of this article initially published, Andrew Fry was incorrectly listed as an equally contributing author. The footnote should read \u2018These authors contributed equally: Lia Bally, Roman Hovorka\u2019. The error has been corrected in the HTML and PDF versions of the article."} +{"text": "Scientific Reportshttps://doi.org/10.1038/s41598-020-62952-6, published online 08 April 2020Correction to: The original version of this Article contained an error in the spelling of the author Bevan M. Lewellen which was incorrectly given as Beven M. Lewellen. The original Article and accompanying Supplementary Information file have been corrected."} +{"text": "Nature Communications 10.1038/s41467-019-12042-7, published online 04 September 2019.Correction to: The original version of this Article contained an error in Fig. 7b, in which the x-axis was incorrectly labelled the same as Fig. 7a. This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Scientific Reportshttps://doi.org/10.1038/s41598-021-92542-z, published online 22 June 2021Correction to: The original version of this Article contained an error in the spelling of the author Marco Laiolo which was incorrectly given as Laiolo Marco. The original Article and accompanying Supplementary Information files have been corrected."} +{"text": "Correction to: Annals of Hematology10.1007/s00277-021-04448-5In the above-mentioned article, the affiliation of one author Magdalena Olszewska-Szopa is not correct. It is presented as 8 . It should be 9 .The original article has been corrected."} +{"text": "Scientific Reports, 10.1038/s41598-021-88945-7, published online 11 May 2021Correction to: The original version of this Article contained an error in the spelling of the author T. Ian Simpson which was incorrectly given as Thomas I. Simpson. The original Article has been corrected."} +{"text": "Nature Communications 10.1038/s41467-021-21625-2, published online 5 March 2021.Correction to: The original version of this Article omitted DMR-1420570 from the Acknowledgements.This has now been corrected in both the PDF and HTML versions of the Article."} +{"text": "Cell Death and Disease 10.1038/s41419-021-04101-z, published online 27 August 2021Correction to: The original version of this article unfortunately contained a mistake. An image of the STING KO mice (without DSS) was mistakenly used for the representative colon H&E images for wild type mice (without DSS) during the compilation of Fig. 5E. The authors apologize for the error. The correct Fig. 5E can be found below."} +{"text": "Nature Communications 10.1038/s41467-021-24743-z, published online 21 July 2021.Correction to: The original version of this Article contained an error in the spelling of the author William T. Pu, which was incorrectly given as William William Pu. This has now been corrected in both the PDF and HTML versions of the Article."} +{"text": "Correction to: BMC Nephrol 22, 39 (2021)https://doi.org/10.1186/s12882-021-02235-yAcknowledgements section by giving credit to someone who played a key role in the initial design of the study as follows:Following publication of the original article , the autWe would like to thank dr. G.A. de Wit, associate professor Health Technology Assessment at the University Medical Center Utrecht, for her work on the design of DIALOGICA."} +{"text": "Nature Communications 10.1038/ncomms9494, published online 25 September 2015.Correction to: This Article contains an error in Figure 2. In Fig. 2c the AMD3100 image was inadvertently duplicated from the anti-CXCR4-Ab image. The incorrect images are shown below.The correct images are shown below.This error has not been corrected in the PDF or HTML versions of the Article."} +{"text": "Translational Psychiatry (2019) 9:190; 10.1038/s41398-019-0515-5; published online 5 August 2019.Correction to: The original version of this article unfortunately contained a mistake. After the publication of the article the authors noticed that names of two authors have not been spelled fully: M. Subramaniapillai and R. S. McIntyer, which should appear as Mehala Subramanieapillai and Roger S. McIntyre. The original article has been corrected."} +{"text": "Correction to: J Exp Clin Cancer Res 34, 56 (2015)https://doi.org/10.1186/s13046-015-0139-4nd row, 2nd column); the image has been replaced with the correct imageFig. Following publication of the original article , the autThe corrected figure is given here. The correction does not have any effect on the final conclusions of the paper."} +{"text": "Scientific Reports 10.1038/s41598-020-65874-5, published online 03 June 2020Correction to: The original version of this Article contained an error in the spelling of the author Sarit Naishlos, which was incorrectly given as Sarit Naishols.The original Article has been corrected."} +{"text": "Nature Communications 10.1038/s41467-021-21161-z, published online 18 February 2021.Correction to: 36\u2019. The correct version states \u2018once we became aware of the structure fitting reported by Yamane et al.36, first deposited as a preprint\u2019 in place of the above.The original version of this Article contained an error in the 14th sentence of the third paragraph of the \u2018Refinement of ice XIX from neutron powder data\u2019 section, which incorrectly read \u2018once we became aware of the structure fitting reported by Yamane et al. on Research Squarehttps://www.researchsquare.com/article/rs-64673/v1 (2020)\u2019. The correct form of ref. 36 is \u2018Yamane, R. et al. Experimental evidence for the existence of a second partially-ordered phase of ice VI. Nat. Commun. 12, 1129 (2021)\u2019.The original version of this Article also contained an error in ref. 36, which incorrectly cited a preprint of the work: \u2018Yamane, R. et al. Experimental evidence for the existence of a second partially-ordered phase of ice VI. Nat. Commun. This has been corrected in the PDF and HTML versions of the Article."} +{"text": "Nature Communications 10.1038/s41467-021-23178-w, published online 18 May 2021.Correction to: The original version of this Article contained an error in Fig. 1, in which the species names are incorrectly displayed. The correct version of Fig. 1 is:The error has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Nature Communications 10.1038/s41467-021-25284-1, published online 17 August 2021.Correction to: The original version of this Article contained an error in the author affiliations.Stephen H. Hughes was incorrectly associated with \u2018Chromatin Structure & Mobile DNA Laboratory, The Francis Crick Institute, London, UK\u2019.This has now been corrected in both the PDF and HTML versions of the Article."} +{"text": "Communications Biology 10.1038/s42003-021-01876-x, published online 19 March 2021.Correction to: In the original version of the Article, the Acknowledgments listed the award numbers UG3DE027695 and UH3 DE002212. Those award numbers should have been UH3 DE027695 and UG DE027695, respectively.This has now been corrected in the PDF and HTML versions of the Article."} +{"text": "Nature Communications 10.1038/s41467-021-24717-1, published online 22 July 2021.Correction to: The original version of this Article contained an error in Figure 3d. The label \u2018ChRmine-eYFP\u2019 was incorrectly shown in orange font instead of green font. This error has been corrected in the HTML and PDF versions of the Article."} +{"text": "Scientific Reportshttps://doi.org/10.1038/s41598-018-30403-y, published online 10 August 2018Correction to: The Acknowledgements section in the original version of this Article is incomplete.\u201cB.G. is supported by the National Science Foundation Graduate Research Fellowship under Grant No. DGE-1256259. This work was supported by startup funds from UW Madison. We also thank Middleton Spectral Vision for access to their hyperspectral imaging systems.\u201dshould read:\u201cB.G. is supported by the National Science Foundation Graduate Research Fellowship under Grant No. DGE-1256259. This work was partially supported by startup funds from UW Madison, and partially by the AFOSR (FA9550-18-1-0146). We also thank Middleton Spectral Vision for access to their hyperspectral imaging systems.\""} +{"text": "Nature Communications 10.1038/s41467-020-20059-6, published online 10 December 2020.Correction to: The original version of this Article contained an error in Fig. 4a. The labels of the t-SNE plot in Fig. 4a incorrectly corresponded to those of Fig. 4c.This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Correction to:Thrombosis J20, 3 (2022).https://doi.org/10.1186/s12959-021-00359-7Following publication of the original article , it was The corrected figures and table are included in this Correction article and the original article has been"} +{"text": "Scientific Reportshttps://doi.org/10.1038/s41598-021-99102-5, published online 04 October 2021.Correction to: The original version of this Article contained an error in the spelling of the author Jihane Hajj which was incorrectly given as Hajj Jihane.The original Article has been corrected."} +{"text": "Communications Biology 10.1038/s42003-020-01230-7, published online 11 September 2020.Correction to: The original version of the Article contained errors in the Algorithm formulas in the Methods section. The formulahttps://github.com/WangX-Lab/DEPTH).\u201dIn addition, the sentence \u201cThe DEPTH algorithm (R package) is available are available at Github is available at This has now been corrected in the PDF and HTML versions of the Article."} +{"text": "Scientific Reports 10.1038/s41598-021-99143-w, published online 05 October 2021Correction to: The original version of this Article contained an error in the spelling of the author Frank W. Sellke which was incorrectly given as Frank W. Selke.The original Article has been corrected."} +{"text": "Scientific Reports 10.1038/s41598-021-03333-5, published online 14 December 2021Correction to: In the original version of this Article, Doh Young Lee was omitted as a corresponding author. Correspondence and requests for materials should also be addressed to gedo0212@naver.com."} +{"text": "Nature Communications 10.1038/s41467-020-18669-1, published online 25 September 2020.Correction to: The original version of this Article omitted Yilin Liu as an equally contributing author. This has now been corrected in both the PDF and HTML versions of the Article."} +{"text": "Correction to: J Exp Clin Cancer Res 37, 25 (2018)https://doi.org/10.1186/s13046-018-0697-3Figure\u00a0Following publication of the original article , the autThe authors provided the Journal with the original data files. The corrected figure is provided here. The correction does not have any effect on the results or conclusions of the paper. The original article has been corrected."} +{"text": "Scientific Reports 10.1038/s41598-021-95406-8, published online 10 August 2021Correction to: The Supplementary Information\u00a0file published with the original version of this Article was incomplete. It now also contains Tables A-I, and DNA sequences for eight CRISPR-PLUS proteins. The original Supplementary Information file is provided below.This error has now been corrected in the Supplementary Information file that accompanies the original Article.Supplementary Information."} +{"text": "Nature Communications 10.1038/s41467-022-28613-0, published online 17 February 2022.Correction to: In the Acknowledgments section, Dr. Hong Zhang was omitted.The funding from National Key Research and Development Project of China (2021YFA1300200) was omitted.The original article has been corrected."} +{"text": "Nature Communications 10.1038/s41467-021-23221-w, published online 3 June 2021.Correction to: The original version of this Article contained errors in the text , which incorrectly cited reference 7 in several places. This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Scientific Reports 10.1038/s41598-021-92014-4, published online 16 June 2021Correction to: The original version of this Article contained an error in the spelling of the author Ana Carolina de J. Paniza which was incorrectly given as Ana C. de J. Paviza.The original Article has been corrected."} +{"text": "Scientific Reports 10.1038/s41598-021-88570-4, published online 27 April 2021Correction to: The original version of this Article contained an error in the spelling of the author Venkateshbabu Nagendrababu, which was incorrectly given as Venkatesh Nagendrababu. The original Article has been corrected."} +{"text": "Nature Communications 10.1038/s41467-021-25569-5, published online 06 September 2021.Correction to: The original version of this Article contained an error in Fig.\u00a07, in which P atoms in the catalyst structures were labelled as PH not P. This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Nature Communications 10.1038/s41467-022-28729-3, published online 09 March 2022.Correction to: The original version of this Article contained an error in Fig. 4, in which the label \u201cc\u201d was missing. This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "International Journal of ObesityCorrection to: 10.1038/s41366-019-0459-0The original version of this article unfortunately contained a mistake. There was a typographical error in Table 2. The 7th row of the table should read \u201cChange in MVPA (min/day/year)\u201d. The authors apologize for the error. The original article has been corrected."} +{"text": "Scientific Reports 10.1038/s41598-021-97220-8, published online 16 September 2021Correction to: The original version of this Article contained an error in the spelling of the author J. Marc Simard which was incorrectly given as Marc J. Simard. The original Article has been corrected."} +{"text": "Nature Communications 10.1038/s41467-022-28194-y, published online 31 January 2022Correction to: Diopatra tube mimic was missing. This has been corrected in both the PDF and HTML versions of the Article.The original version of this Article contained an error in Fig. 1, in which the illustration depicting a"} +{"text": "Correction to: Environ Health Prev Med 26, 45 (2021)https://doi.org/10.1186/s12199-021-00968-8Following the publication of the original article the authIt was written as below.2000 were included in the study.The older adults aged 64 years who underwent medical check-up between 1996 and However, the correct writing is as follows.2005 were included in the study.The older adults aged 64 years who underwent medical check-up between 1996 and The original article has been"} +{"text": "Nature Communications 10.1038/s41467-020-14570-z, published online 06 February 2020.Correction to: The original version of this Article contained an error in Fig. 1, in which the labels were inadvertently omitted from the pie chart. This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Nature Communications 10.1038/s41467-021-22720-0, published online 30 April 2021.Correction to: In this Article there was an error in the Author contribution statement. The final sentence of the statement \u2018The last two authors (D.-C.L. and B.P.B.) are co-senior authors who jointly supervised the work, and they have the right to list their names last in their CV.\u2019 was omitted.This has been corrected in the pdf and HTML versions of the Article."} +{"text": "Cell Death and Disease 10.1038/s41419-022-04572-8, published online 4 February 2022Correction to: The original version of this article unfortunately needed to be updated. A related file was included as supplementary and the author would like it removed. The original article has been corrected."} +{"text": "Communications Biology 10.1038/s42003-020-01518-8, published online 18 December 2020.Correction to: The original version of the Article was missing the following text from the Acknowledgements: \u201cFigure cartoons created with BioRender.com\u201d. The error has been corrected in the HTML and PDF versions of the Article."} +{"text": "Scientific Reports 10.1038/s41598-020-80954-2, published online 15 January 2021Correction to: In the original version of this Article, Llu\u00eds Armengol was omitted as a corresponding author. Correspondence and request for materials should also be addressed to luis.armengol@qgenomics.com. This error has now been corrected in the HTML and PDF versions of the Article."} +{"text": "A Correction to this paper has been published: https://doi.org/10.1038/s41467-021-23162-4 Nature Communications 10.1038/s41467-020-20496-3, published online 17 February 2021.Correction to: The original version of this Article contained an error in the spelling of the author Heiko Becher, which was incorrectly given as Heko Becher. This has now been corrected in both the PDF and HTML versions of the Article."} +{"text": "Nature Communications 10.1038/s41467-021-27056-3, published online 18 November 2021.Correction to: The original version of this Article contained an error in Fig. 1d. In Fig. 1d the labelling of the EVs and cell lysate in the bottom panel was inadvertently switched. This error has been corrected in the PDF and HTML versions of the Article."} +{"text": "Correction to: BMC Psychiatry 14, 319 (2014)https://doi.org/10.1186/s12888-014-0319-3bold typeface.Following publication of the original article , the autThe sentences currently read:In format A (GST-A), two-year GST consists of 124 groups sessions with a duration of 90 minutes.In addition, in GST-A a total of up to 18 individual sessions can be used at the patients discretion or in times of crisis.In total, patients in this condition receive 74 group sessions and 62 individual sessions.The sentences should read:118 groups sessions with a duration of 90 minutes.In format A (GST-A), two-year GST consists of 17 individual sessions can be used at the patients discretion or in times of crisis.In addition, in GST-A a total of up to 63 group sessions and 61 individual sessions.In total, patients in this condition receive The original article has been"} +{"text": "Cell Death and Disease 10.1038/s41419-021-04365-5, published online 17 January 2022Correction to: The original version of this article unfortunately contained a mistake in figures 1 and 2. The authors apologize for the mistake. The original article has been corrected."} +{"text": "Nature Communications 10.1038/s41467-021-22948-w, published online 11 May 2021.Correction to: The original version of this article inadvertently acknowledge Xiangbin Cai as a co-corresponding author instead of co-first author.This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Scientific Reports 10.1038/srep12477, published online 24 July 2015Retraction of: Fig. 4a: partial overlap between the panels for 35S-GFP and 35S-SAUR76-GFP.Fig. 9a: duplication of Coomassie blue bands for the SAUR78-FLAG time-course and seedling treatment.The authors have stated they are unable to locate the original data. The Editors therefore no longer have confidence in the reliability of the data reported in the Article.The Editors have retracted this Article. After publication, concerns were raised regarding a number of figure panels, specifically:None of the authors agree to this retraction."} +{"text": "Scientific Reportshttps://doi.org/10.1038/s41598-021-00764-y, published online 27 October 2021Correction to: The original version of this Article contained an error in the spelling of the author Ayman S. Soliman which was incorrectly given as Ayman Samaan. The original Article has been corrected."} +{"text": "Correction to: Mol Cancer 17, 157 (2018)https://doi.org/10.1186/s12943-018-0906-xFollowing publication of the original article , a minorFig. The corrected figure is given here. The correction does not have any effect on the results or conclusions of the paper."} +{"text": "Communications Biology 10.1038/s42003-021-02404-7, published online 15 July 2021.Correction to: Figure 4 was missing from the PDF of this article. The original PDF has been corrected. The HTML version of the Article was correct at the time of publication."} +{"text": "Correction to: BMC Plant Biol 21, 472 (2021)https://doi.org/10.1186/s12870-021-03246-5Following publication of the original article , due to The correction does not have any effect on the results or conclusions of the paper. The original article has been corrected."} +{"text": "Nature Communications 10.1038/s41467-022-27973-x, published online 19 January 2022.Correction to: The original version of this Article contained an error in Fig. 1, Fig. 3, Fig. 4 in which the individual data points were inadvertently omitted. The correct version of Fig. 1 is:which replaces the previous incorrect version.The correct version of Fig. 3 is:which replaces the previous incorrect version.The correct version of Fig. 4 is:which replaces the previous incorrect version.This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Communications Biology 10.1038/s42003-021-01819-6, published online 10 March 2021.Correction to: The original version of this Article contained an error in the spelling of the author Sanford M. Simon, which was incorrectly given as Simon M. Sandford.This has now been corrected in both the PDF and HTML versions of the Article."} +{"text": "Nature Communications 10.1038/s41467-021-20903-3, published online 27 January 2021.Correction to: In the original version of the published article, the Acknowledgements section was missing a statement to acknowledge funding from an Alpha-1 Foundation Research grant. The correct Acknowledgements paragraph is:"} +{"text": "Nature Communications 10.1038/s41467-021-26595-z, published online 5 November 2021.Correction to: The original version of this Article contained an error in Fig. 4b, in which a variable (IMAGE 3.0) for one scenario for the year 2050 was incorrectly reported/placed. This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Correction to: Mol Neurobiol10.1007/s12035-020-02276-8The original version of this article unfortunately contained some mistakes. The current Electronic Supplementary file is not correct and should be updated.The original paper has been corrected."} +{"text": "Scientific Reports 10.1038/s41598-021-89758-4, published online 20 May 2021Correction to: In the original version of this Article Saabah B. Mahbub and Long T. Nguyen were omitted as equally contributing authors.Additionally, Sonia Saad and Ewa M. Goldys were omitted as jointly supervised authors.This error has now been corrected in the PDF version of the Article; the HTML version was correct from the time of publication."} +{"text": "Scientific Reports 10.1038/s41598-019-39568-6, published online 26 February 2019Correction to: In the original version of this Article, Fernando F\u00e9lix Bravo-Almonacid was omitted as a corresponding author. Correspondence and request for materials should also be addressed to fbravo@dna.uba.ar.This error has now been corrected in the HTML and PDF versions of the Article."} +{"text": "Scientific Reports 10.1038/s41598-022-05389-3, published online 24 January 2022Correction to: The original version of this Article contained an error in the author list. Rita G. Hazboun was incorrectly listed twice. The second time the author\u2019s name was listed in error.The original Article has been corrected."} +{"text": "Scientific Reports 10.1038/s41598-022-06908-y, published online 21 February 2022Correction to: In the original version of this Article, Meiling Liu and Junfeng Chen were omitted as a corresponding author. Correspondence and requests for materials should also be addressed to meilingliu@163.com and drchenjf@126.com.The original Article has been corrected."} +{"text": "Scientific Reports 10.1038/s41598-021-97303-6, published online 08 September 2021Correction to: The original version of this Article contained errors in the spelling of the authors Peter A. Lynn and Scott R. Sponheim which were give as Peter Lynn and Scott Sponheim respectively.The original Article has been corrected."} +{"text": "Nature Communications 10.1038/s41467-021-25279-y, published online 17 August 2021.Correction to: The original version of this Article contained an error in Fig. 3C furthest right panel, in which two white dots were inadvertently introduced. This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Communications Biology 10.1038/s42003-021-02432-3, published online 23 July 2021Correction to: which replaces the previous incorrect version .In the original PDF version of this Article, the image for Fig. 2 was inadvertently replaced with the image from Fig. 3. The online version of the Article was not affected. The correct version of Fig. 2 is This error has now been corrected in the PDF version of the Article."} +{"text": "Correction to: Implementation Sci 15, 41 (2020)https://doi.org/10.1186/s13012-020-0971-6Following the publication of the original article , it was The publisher apologizes to the authors and readers for the inconvenience."} +{"text": "The publisher has been informed of an error that occurred on pages 113-120 which the study type in the title must be changed to \u201cnon-randomized clinicaltrial\u201d. The publisher wishes to apologize for this error. The online version of the articlehas been updated on April 16, 2021 and can be found at http://journals.ssu.ac.ir/ijrmnew/article-1-1422-en.html (doi.org/10.18502/ijrm.v18i2.6423)."} +{"text": "Nature Communications 10.1038/s41467-020-18265-3, published online 9 September 2020.Correction to: The original version of this Article contained an error in Fig. 3, in which the incorrect images were used in Fig. 3a for the ratio of 1:2. This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Nature Communications 10.1038/s41467-021-25786-y, published online 7 October 2021.Correction to: This Article contained an error in the author list. The original author list omitted Silvia Turchetto as an equally contributing first author. This has been corrected in the author list and author contribution statement. The PDF and HTML versions of the Article have been updated."} +{"text": "Cell Death & Disease 10.1038/s41419-021-04440-x, published online 10 December 2021Correction to: The original version of this article unfortunately contained a mistake. The authors found that two images in Fig. 3F and Fig. 4D were the same. The authors apologize for the error. The original article has been corrected."} +{"text": "Nature Communications 10.1038/s41467-021-23169-x, published online 14 May 2021.Correction to: The original version of this Article contained an error in Fig. 1, in which several of the silhouettes did not appear in panel j. The original version of Fig. 1 isand the corrected version isThis has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Scientific Reports 10.1038/s41598-019-56025-6, published online 18 December 2019Correction to: In the original version of this Article, Fu-Zong Wu was incorrectly listed as a corresponding author. The correct corresponding author for this Article is only Yun-Pei Lin. Correspondence and request for materials should be addressed to fc760203@gmail.com.This error has now been corrected in the HTML and PDF versions of the Article and in the accompanying Supplementary Information file.Supplementary Tables."} +{"text": "Correction to: European Radiology.10.1007/s00330-020-07579-xThe original version of this article, published on 14 January 2021, unfortunately contained a mistake. The following correction has therefore been made in the original: The affiliations 5 and 6 were presented incorrectly. The corrected affiliations are given below. The original article has been corrected."} +{"text": "Scientific Reports 10.1038/s41598-021-04732-4, published online 18 January 2022Correction to: In the original version of this Article, Zhong Yang was omitted as a corresponding author. Correspondence and requests for materials should also be addressed to yz@jit.edu.cn.The original Article has been corrected."} +{"text": "Correction to: Thrombosis J 19, 37 (2021)https://doi.org/10.1186/s12959-021-00292-9Following publication of the original article , it was In the Abstract it was stated in the Results sub-section that a total of 31 cases were treated with anticoagulation, but the correct total is 33.In Fig. The original article has been updated."} +{"text": "Cell Death and DiseaseCorrection to: 10.1038/s41419-021-03493-2 published online 24 February 2021The original version of this article unfortunately contained an error in Figure 4. The H&E stained picture in the HOPE group in Figure 4 was wrong. The authors apologize for the error. The correct Figure 4 can be found below. The original article has been corrected."} +{"text": "Nature Microbiology 10.1038/s41564-021-01020-9, published online 30 December.Correction to: In the version of this article initially published, the images for Figs. 2 and 3 were in the wrong order, although figure callouts, titles and captions were correct. The images have now been reordered in the online version the article."} +{"text": "Scientific Reports 10.1038/s41598-021-92895-5, published online 30 June 2021Correction to: G. Mena and L. Matas were omitted from the Surveillance of Hospitalized Cases of Severe Influenza in Catalonia Working Group in the original version of this Article.The original Article has been corrected."} +{"text": "Nature Human Behaviour 10.1038/s41562-019-0772-6, published online 2 December 2019.Correction to: In the version of this article initially published, the surname of author Marcus Munaf\u00f2 was misspelled. The error has been corrected in the HTML and PDF versions of the article."} +{"text": "Communications Biology 10.1038/s42003-021-02218-7, published online 7 July 2021.Correction to: In the original published version of the Article, Fig. 1 contained errors affecting several of the text labels. The original and corrected versions of the figure are shown below. The errors have been corrected in the HTML and PDF versions of the Article.Original published version:Corrected version:"} +{"text": "Nature Communications 10.1038/s41467-021-25808-9, published online 14 September 2021.Correction to: The original version of this Article contained an error in Fig. 2, in which outdated Cr isotopic data has been reported. This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Correction to:10.1038/s41398-021-01375-x published online 5 May 2021The original version of this article unfortunately contained a mistake. There was an error in the reference order in the discussion section. The original article has been corrected accordingly."} +{"text": "Correction to: Molecular Neurobiology10.1007/s12035-021-02296-yThe original version of this article unfortunately contained some mistakes.There are changes in the authorship order and the list of authors. Dr. Momchil Ninov is added as the fifth author."} +{"text": "Translational Psychiatry 10.1038/s41398-021-01705-z, published online 10 November 2021Correction to: The original version of this article unfortunately contained a spelling mistake in the name of Anthony A. Grace. The authors apologize for the error. The original article has been corrected."} +{"text": "Nature Communications 10.1038/s41467-021-24169-7, published online 23 June 2021.Correction to: The original version of this Article contained an error in the spelling of the author Estefan\u00eda Sanabria-Reinoso, which was incorrectly given as Estefan\u00eda Sanbria-Reinoso. This has now been corrected in both the PDF and HTML versions of the Article."} +{"text": "Scientific Reports 10.1038/s41598-021-97444-8, published online 09 September 2021.Correction to: In the original version of this Article, the image on the right in Table Additionally, legends for Table The original Table The original Article has been corrected."} +{"text": "Nature Communications 10.1038/s41467-022-28245-4, published online 01 February 2022.Correction to: The Supplementary Software was inadvertently omitted from the original version of the published article. This has now been corrected in the HTML version of the Article.Supplementary Software"} +{"text": "Nature Communications 10.1038/s41467-021-24771-9, published online 26 July 2021.Correction to: In the original version of this Article, Fig. 2 was inadvertently omitted in the PDF version of the Article. This has now been corrected in the PDF version of the Article."} +{"text": "Cell Death and Disease 10.1038/s41419-021-04103-x, published online 03 September 2021Correction to: The original version of this article unfortunately contained an error in figure 5d. The authors apologize for the error. The amended figure can be found below. The original article has been corrected."} +{"text": "Communications Biology 10.1038/s42003-021-02488-1, published online 13 August 2021.Correction to: The original version of this Article contained an error in the author affiliations.The affiliation of Xiaofei Sun and S. K. Dey with \u201cDivision of Reproductive Sciences, Cincinnati Children\u2019s Hospital Medical Center, Cincinnati, OH 45229, USA\u201d was inadvertently omitted.This has now been corrected in both the PDF and HTML versions of the Article."} +{"text": "Scientific Reports 10.1038/s41598-020-58114-3, published online 31 January 2020Correction to: The original version of this Article contained an error in the spelling of the author Patricia J. Goedecke which was incorrectly given as Patricia J. Goeske.The original Article has been corrected."} +{"text": "The ISME Journal 10.1038/s41396-021-01161-z, published online 03 December 2021Correction to: Following the publication of this article it was noted that there were a number of typographical errors. These have now been corrected.The conclusions of the article remain unchanged.The original article has been corrected."} +{"text": "Excessive use of the emergency department (ED) is associated with increased costs and workload in the ED, patients' inconvenience and disruption of the continuity of care. The study's goal was to describe trends in ED utilization among Bedouins living in southern Israel. A retrospective cross-sectional study was conducted in primary care clinics in southern Israel. Patients included Bedouin and Jewish patients insured by Clalit Health Services. Data was retrieved from a central database. The number of visits to the ED and age-adjusted rates of ED visits during 2000-2003 were determined in the Bedouin vs. Jewish population. All visits that ended in hospitalization were excluded. Data was stratified according to patients' residence (semi-nomadic vs. urban Bedouins) and referral origin. Age-adjusted rates of ED visits decreased from 42.9/1000 patients/month in 2000 to 38.3/1000 patients/month in 2003. There were more ED visits in the Bedouin as compared to Jewish population (38.3/1000 vs. 21.8/1000 patients/month). The decrease in ED utilization was more prominent among adult semi-nomadic Bedouins (from 60.8/1000 to 40.3/1000 patients/month). The proportion of referrals by the family physician to ED significantly decreased , while the proportion of selfreferrals and referrals from physicians other than the family physician increased. A decrease in ED utilization by the Bedouin population during the last years was demonstrated. Utilization of ED services is still increased as compared to the non-Bedouin population. Interventions to control excessive use of ED services in the Bedouin population are currently underway."} +{"text": "Correction to: Nature Communications 10.1038/s41467-018-03225-9, published online 28 February 2018.In the originally published version of this Article, images in Fig. 5n were inadvertently replaced with duplicates of images in Fig. 5o during the production process. This has now been corrected in both the PDF and HTML versions of the Article."} +{"text": "Scientific Reports 10.1038/s41598-017-01451-7, published online 03 May 2017Correction to: The original Supplementary Information file published with this Article was incorrect. This version included errors in the Greenland temperatures, and the ice core depth information was omitted. The correct Supplementary Information file now accompanies the Article."} +{"text": "There are errors in the Funding section. The correct funding information is as follows: The Research was supported by two grants to AD from MHRD-CEMA F.NO-5-3/2015-TS VII and BUILDER program BT/PR12153/INF/22/200/2014. JJ acknowledges the financial support provided through the UGC-BSR fellowship sponsored by the University Grants Commission, New Delhi, India. This research was supported in part by two grants to MK from the U.S. National Institutes of Health, National Institutes of Allergy and Infectious Diseases: R21AI105472 and UO1053877.There are errors in the Acknowledgments section. The correct Acknowledgments information is as follows: The authors greatly acknowledge Dr. Michael Kron for providing the construct. We also thank Dr. Suvro Chatterjee of Vascular Biology Lab, AU-KBC Research Centre, Anna University MIT Campus, Chennai for help with angiogenesis assays."} +{"text": "Correction to: Cell Death and Disease (2018) 9:359. 10.1038/s41419-018-0394-3; Article published online: 02 Mar 2018.The name of the one of the authors was misspelt. The author\u2019s surname is Rodriguez, not Rodriquez as originally published. This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Scientific Reports 10.1038/s41598-017-00907-0, published online 11 April 2017Correction to: In the original version of this Article, Ting Yang was omitted as a corresponding author. Correspondence and request for materials should also be addressed to tyang@ippcaas.cn. This error has now been corrected in the HTML and PDF versions of the Article."} +{"text": "Nature Communications10.1038/s41467-017-01691-1, pubilshed online 17 November 2017Correction to: The original version of this article contained an error in the spelling of the author Christian A.M. Wilson, which was incorrectly given as Christian M.A. Wilson. This has now been corrected in both the PDF and HTML versions of the article."} +{"text": "BMC Genetics continues the series of BioMed Central special post-conference issues presenting materials from the conferences on bioinformatics and systems biology BGRS\\SB (Bioinformatics of Genome Regulation and Structure\\Systems Biology) annually held in Novosibirsk. Here we present the papers discussed at and Belyaev Conference-2017 \u201cBelyaev Readings - 2017\u201d (BR-2017). The Year 2017 marks the 100-th anniversary since birth of Full Member of the USSR Academy of Sciences, Professor Dmitry K. Belyaev (1917\u20131985), an outstanding scientist, evolutionist and geneticist. In view of this memorable date, the Institute of Cytology and Genetics of the Siberian Branch of the Russian Academy of Sciences (ICG SB RAS) held international Belyaev Conference on Genetics and Evolution .This thematic issue of BMC Genetics, BMC Genomics and BMC Evolutionary Biology covered the proceeding of BGRS\\SB-2016 conference and SBB-2015 School in Novosibirsk [https://bmcgenomics.biomedcentral.com/articles/supplements/volume-15-supplement-12). First BMC Genetics special issue paper in the area of genetics was presented at the BGRS\\SB-2014 conference series in Novosibirsk [Previously published special issues of http://vavilov.elpub.ru/jour/issue/view/32/showToc). In particular, the article by Prof. V. K. Shumny [The memorial Belyaev conference-2017 continued a tradition of BGRS\\SB series on bioinformatics and systems biology. In 2017 \u201cVavilov Journal of Selection and Breeding\u201d published a series of memoirs publications about Prof. Belyaev (. Shumny , 9: N. K. Shumny and P. D. Shumny .BMC Genetics.Genetics-related works discussed at Belyaev conference-2017 are collated in present issue of The work by Triska and co-authors (, this isThe paper by Ranajit Das and Priyanka Upadhyai (, this isNikolay S. Yudin and colleagues (, this isNETO2 gene upregulation along with deregulation of eight epithelial-mesenchymal transition-related genes in colorectal cancer.The paper by Fedorova et al. (, this isCNTN6 gene, which encodes the contactin-6 protein, is responsible for severe neurodevelopmental impairments, often in combination with facial dysmorphias. Mice carrying megabase-scale deletions, duplications, and inversions involving the full-sized Cntn6 gene were thoroughly characterized.Alexei N. Korablev and colleagues (, this isT. monococcum, T. durum, T. compactum, and T. spelta, and showed that DEP1 does not directly participate in the control of the spike architecture.Valeriya Vavilova and co-authors (, this isBMC Evolutionary Biology, BMC Plant Biology, BMC Genomics, BMC Medical Genomics and BMC Neuroscience. The Proceedings of the conference are available at http://conf.bionet.nsc.ru/belyaev100/en/ and http://conf.bionet.nsc.ru/belyaev100/wp-content/uploads/sites/14/2017/01/BELYAEV_conf_2_08_2017.pdf.Follow-on series of related works in the areas of classical and medical genomics, genetics, and plant biology discussed at \u201cBelyaev conference \u2013 2017\u201d and other related meetings in Novosibirsk and Moscow are published in Special Issues of The readers are welcome to visit Novosibirsk at the time of next XI-the BGRS\\SB-2018 conference on August 20-28th in 2018."} +{"text": "Nature Communications 10.1038/s41467-017-02802-8, published online 24 January 2018Correction to: The original version of this Article contained errors in Fig. 3g and Fig. 4d. In Fig. 3g, the grey shaded area was rendered incorrectly. The correct version of Fig. 3g is:which replaces the previous incorrect version:In Figure 4d, the black data points and error bars were omitted. The correct version of Fig. 4d is:which replaces the previous incorrect version:These errors have now been corrected in both the PDF and HTML versions of the Article."} +{"text": "Erratum to: npj Primary Care Respiratory Medicine (2017); doi:10.1038/s41533-017-0039-5; Published 09 June 2017The original version of this article contained an error in the spelling of the author I. G. Tsiligianni, which was incorrectly given as K. W. I. G. Tsiligianni. This has now been corrected in both the PDF and HTML versions of the article."} +{"text": "Scientific Reports 10.1038/s41598-017-06873-x, published online 27 July 2017Correction to: The Acknowledgments section of this Article is incomplete.\u201cThis work was partially supported by DTRA Award No.HDTRA1-09-1-0049, by the Army Research Laboratory under Cooperative Agreement Number W911NF-09-2-0053 (the ARL Network Science Collaborative Technology Alliance). The views and conclusions contained in this document are those of the authors and should not be interpreted as representing the official policies either expressed or implied of the U.S. Army Research Laboratory or the U.S. Government\u201d.should read:\u201cThis work was partially supported by DTRA Award No. HDTRA1-09-1-0049, by the Army Research Laboratory under Cooperative Agreement Number W911NF-09-2-0053 (the ARL Network Science Collaborative Technology Alliance), and by the Army Research Office under grant W911NF-16-1-0524. The views and conclusions contained in this document are those of the authors and should not be interpreted as representing the official policies either expressed or implied of the U.S. Army Research Laboratory or the U.S. Government\u201d."} +{"text": "Scientific Reports 10.1038/s41598-018-25438-0, published online 4 May 2018Correction to: The original version of this Article contained a typographical error in the spelling of the author G. Diane Shelton, which was incorrectly given as Diane G. Shelton. This has now been corrected in the PDF and HTML versions of the Article and Supplementary Information file."} +{"text": "Scientific Reports 10.1038/s41598-017-02767-0, published online 31 May 2017Correction to: The original version of this Article contained a typographical error in the spelling of the author Clement C. Tham, which was incorrectly given as Clement C. C. Tham. This has now been corrected in the PDF and HTML versions of the Article, and in the accompanying Supplementary Information file."} +{"text": "The authors acknowledge that there are overlaps between the text of this article and the following previously published work, which was not cited:10.1186/1471-2229-10-14Hu R, Guang Q, Yingzhen K, Dejing K, Gao Q and Zhou G (2010) Comprehensive Analysis of NAC Domain Transcription Factor Gene Family in Populus trichocarpa. BMC Plant Biology. DOI: The authors apologize for the overlap in text.PLOS ONE Editors have also identified overlaps between the text of this article and a number of previously published works. Text was duplicated verbatim or with minor modifications from the sources listed below:The 10.1007/s11033-010-0650-9 (cited as Reference 11 in the published article)Genome wide identification of Dof transcription factor gene family in sorghum and its comparative phylogenetic analysis with rice and Arabidopsis. Molecular Biology Reports. DOI: 10.1093/pcp/pch055 (cited as Reference 14 in the published article)Dof Domain Proteins: Plant-Specific Transcription Factors Associated with Diverse Phenomena Unique to Plants. Plant & Cell Physiology. DOI: 10.1111/jipb.12043 (cited as Reference 57 in the published article)Genome-wide Analysis of Plant-specific Dof Transcription Factor Family in Tomato. Journal of Integrative Plant Biology. DOI: 10.1016/j.gene.2012.02.035 (uncited in the published article)Identification of cis-regulatory elements specific for different types of reactive oxygen species in Arabidopsis thaliana. Gene. DOI: 10.1186/1471-2229-12-202 (cited as Reference 54 in the published article)The family of DOF transcription factors in Brachypodium distachyon: phylogenetic comparison with rice and barley DOFs and expression profiling. BMC Plant Biology. DOI: PLOS ONE Editors retract this article.In light of the concerns identified, the authors and the"} +{"text": "Scientific Reports 10.1038/s41598-018-21932-7, published online 22 February 2018Correction to: The original version of this Article contained a typographical error in the spelling of the author Marcos J. Ara\u00fazo-Bravo, which was incorrectly given as Marcos-Arauzo Bravo. This has now been corrected in the PDF and HTML versions of the Article, and in the accompanying Supplementary Information file."} +{"text": "Scientific Reports 10.1038/s41598-018-24392-1, published online 12 April 2018Correction to: In the Supplementary Information file originally published with this Article, an equation was omitted. This error has been corrected in the Supplementary Information that now accompanies the Article."} +{"text": "Nature Communications 10.1038/s41467-017-02032-y, published online 05 December 2017Correction to: In the original version of this Article, the text labels on the bottom-right panel in Fig. 1c were inadvertently displaced during the production process. The correct version is:Fig. 1which replaces the previous incorrect version :Fig. 2This has now been corrected in both the PDF and HTML versions of the Article."} +{"text": "Nature Communications 10.1038/s41467-018-03921-6, published online 23 April 2018Correction to: The original version of this Article contained an error in Fig. 3. Panel b was inadvertently duplicated and the correct panel c was originally omitted. This error has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Nature Communications 10.1038/s41467-017-01096-0, published online 10 November 2017.Correction to: The original version of the Supplementary Information associated with this Article inadvertently omitted Supplementary Table 1. The HTML has now been updated to include a corrected version of the Supplementary Information."} +{"text": "Scientific Reports 10.1038/s41598-017-04621-9, published online 27 June 2017Correction to: Due to a typesetting error, Figure\u00a01 in the original version of this Article was incorrect. This error has now been corrected in the PDF and HTML versions of the Article."} +{"text": "Scientific Reports 10.1038/s41598-017-18466-9, published online 09 January 2018Correction to: In the original version of this Article, Seok Ho Kang was incorrectly listed as the corresponding author. The correct corresponding author for this Article is Sung-Hoo Hong. Correspondence and request for materials should be addressed to toomey@catholic.ac.kr. This error has now been corrected in the HTML and PDF versions of the Article."} +{"text": "Scientific Reports7:6121; doi:10.1038/s41598-017-05015-7; Article published online 21 July 2017The original version of this Article contained a typographical error in the spelling of the author T. Kapitaniak, which was incorrectly given as T. Kapitaniakenglish. This has now been corrected in the PDF and HTML versions of the Article."} +{"text": "Scientific Reports 10.1038/s41598-018-19716-0, published online 22 January 2018.Correction to: Marc Dussauze, Bruno Bousquet and Evelyne Fargin were omitted from the author list in the original version of this Article. This has been corrected in the PDF and HTML versions of the Article."} +{"text": "Nature Communications8:1127 doi:10.1038/s41467-017-01278-w; Article published online: 24 October 2017The original version of this Article contained an error in the spelling of the author Steven M. Coyne, which was incorrectly given as Stephen M. Coyne. This has now been corrected in both the PDF and HTML versions of the Article."} +{"text": "There are errors in the Funding section. The correct funding information is as follows: This research was supported in part by the Ministry of Science and Technology, R.O.C. under grant MOST 104-2221-E-007-061-MY3. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."} +{"text": "This article was originally published online on 22 December 2017 without proper credit in the caption to Fig. 15. The online version of the article was corrected on 28 December 2017. The corrected figure caption appears below:13(9), 4524\u20134534 (2017). Copyright 2017 American Chemical Society.] (d) X-ray structure of inactive rhodopsin (PDB code 1U19). The black lines indicate the location of the cell membrane. The yellow circle shows the location of the G-protein interaction site. (e) Alignment of the active and inactive conformation of rhodopsin, viewed from the intracellular side. Corresponding PDB codes are listed in the same colour as the colour of the protein structure. The main structural changes due to activation are indicated by yellow arrows. (f) X-ray structure of inactive rhodopsin (PDB code 1U19) in which specific regions that are especially affected by retinal's cis-trans isomerization are highlighted. The retinal moiety is shown in orange.FIG. 15. Rhodopsin's activation mechanism. (a) 11-cis to all-trans photoisomerisation and later deprotonation of covalently bound retinal in rhodopsin due to exposure to light. (b) View from the extracellular region of the retinal moiety in the active site. (c) View of the retinal moiety, turned 90\u00b0 compared to image (b), with the Lys296 part located in front. [(b) and (c) Reprinted with permission from S. C. Van Keulen, A. Solano, and U. Rothlisberger, J. Chem. Theory Comput."} +{"text": "Scientific Reports 10.1038/s41598-017-15792-w, published online 13 November 2017Correction to: The Acknowledgements section in this Article is incomplete.\u201cThe authors are indebted to Prof. Nicholas A. Kotov at the Michigan University for fruitful discussions and recommendations in the preparation of the manuscript. This work was supported by the Russian Government, Ministry of Education and by the RFBR, Research Project Nos.14-03-31046 and 14-03-00502. The support from the Swedish Research Council (Vetenskapsr\u00e5det), Grant No. 2014-3938, is gratefully acknowledged.\u201dshould read:\u201cThe authors are indebted to Prof. Nicholas A. Kotov at the Michigan University for fruitful discussions and recommendations in the preparation of the manuscript. This work was supported by the Russian Government, Ministry of Education and by the RFBR, Research Project Nos.14-03-31046 and 14-03-00502. The support from the Swedish Research Council (Vetenskapsr\u00e5det), Grant No. 2014-3938, is gratefully acknowledged. Vladimir V. Vinogradov acknowledges the Ministry of Education and Science of Russian Federation (Project 4.8955.2017/8.9).\u201d"} +{"text": "Nature Communications(2017) 10.1038/s41467-017-02420-4, published online 04 January 2018Correction to: The original version of this Article contained an error in the spelling of the author Benjamin H. Williams, which was incorrectly given as Benjamin H. Willams. This has now been corrected in both the PDF and HTML versions of the Article."} +{"text": "Nature Communications 10.1038/s41467-017-01119-w, published online 19 October 2017Correction to: The original version of this Article contained an error in the spelling of the author Amos B. Smith, III, which was incorrectly given as Amos B. SmithIII. This has now been corrected in both the PDF and HTML versions of the Article."} +{"text": "Nature Communications 10.1038/s41467-017-00284-2, published online 04 Aug 2017Correction to: The original version of this Article contained an error in the spelling of the author Joseph S. Manser, which was incorrectly given as Joseph M. Manser. This has now been corrected in both the PDF and HTML versions of the Article."} +{"text": "Nature Communications 10.1038/s41467-017-02332-3, published online on 22 December 2017.Correction to: The originally published version of this Article contained an error in the spelling of the author Nathaniel W. Oswald, which was incorrectly given as Nathaniel W. Olswald. This has now been corrected in\u00a0both the PDF and HTML versions of the Article."} +{"text": "Nature Communications 10.1038/s41467-018-04221-9, published online 09 May 2018.Correction to:\u00a0The original version of this article contained an error in the spelling of Richard Thomson-Luque, which was incorrectly given as Richard Thomson Luque. This error has now been corrected in both the PDF and HTML versions of the Article."} +{"text": "Nature Communications 10.1038/s41467-018-03121-2, published online: 08 March 2018.Correction to: The original version of this article contained an error in the email address of the corresponding author Jun Qin. The correct email is jqin1965@126.com. The error has been corrected in the HTML and PDF versions of the article."} +{"text": "Scientific Reports 10.1038/s41598-017-06982-7, published online 27 July 2017Correction to: The original version of this Article contained errors in the spelling of the author Sanford L. Boye, which was incorrectly given as L. Boye Sanford. This has now been corrected in both the PDF and HTML versions of the Article."} +{"text": "Correction to: npj Precision Oncology1:29; 10.1038/s41698-017-0036-8, Published online: 07 September 2017In the original version of this Article the Supplementary Information was missing. This file has now been added to the HTML version of this Article."} +{"text": "Scientific Reports 10.1038/s41598-017-08164-x, published online 10 August 2017Correction to: The original version of this Article incorrectly included Zhi Zhang as a corresponding author. For correspondence and requests for materials, please contact Rihuan Cong (congrh@mail.hzau.edu.cn). This error has now been corrected in the PDF and HTML versions of this Article."} +{"text": "There is an error in affiliation 3 for author Stephan Naji. Affiliation 3 should be: New York University-CIRHUS, New York City, NY, United States of America.The second affiliation for author Stephan Naji is not indicated. Stephan Naji is also affiliated with: Univ. C\u00f4te d\u2019Azur, CNRS, UMR 7264-CEPAM, France.The following information is missing from the Acknowledgments section: The authors would also like to thank the Agence National de la Recherche (project CemeNTAA ANR-14-CE31-0011) for organizational help."} +{"text": "Scientific Reports 10.1038/s41598-017-05453-3, published online 14 July 2017Correction to: In the original version of this Article, the Author information section was incomplete. Alain Baulard was omitted as having jointly supervised this work. This has now been corrected in the HTML and PDF versions of this Article."} +{"text": "Scientific Reports 10.1038/s41598-017-14970-0, published online 13 November 2017Correction to: The original version of this Article contained a typographical error in the spelling of the author William E. Carson, which was incorrectly given as William E. Carson 3rd. This has now been corrected in the PDF and HTML versions of the Article, and in the accompanying supplementary material."} +{"text": "In the Methods section, the Nomenclatural Acts sub-section is missing. The correct sub-section is:http://zoobank.org/\u201d. The LSID for this publication is: urn:lsid:zoobank.org:pub: 092F06B7-B0F4-40F2-BF6A-4077A590766D. The electronic edition of this work was published in a journal with an ISSN, and has been archived and is available from the following digital repositories: PubMed Central, LOCKSS.The electronic edition of this article conforms to the requirements of the amended International Code of Zoological Nomenclature, and hence the new names contained herein are available under that Code from the electronic edition of this article. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix \u201cBuergeria otai sp. nov. is missing. The correct LSID is: urn:lsid:zoobank.org:act:BAF9221D-D0BE-4441-AC2B-46BFF9057E7D.In the Species description sub-section of the Discussion section, the LSID for"} +{"text": "Nature Communications8:272 doi:10.1038/s41467-017-00309-w; Article published online 16 August 2017H. pylori SS1 strain for this work and participated in the design of H. pylori infection studies, was inadvertently omitted from the author list. This has now been corrected in both the PDF and HTML versions of the Article.Marygorret Obonyo, who provided the"} +{"text": "Scientific Reports 10.1038/s41598-017-12509-x, published online 25 September 2017Correction to: The original version of this Article contained a typographical error in the spelling of the author J. Torres-Torronteras, which was incorrectly given as J. Torres-Torrenteras. This has now been corrected in the PDF and HTML versions of the Article and in the accompanying supplementary material."} +{"text": "Scientific Reports6:454; doi:10.1038/s41598-017-00581-2; Article published online 28 March 2017In the original version of this Article, Pi-I. D. Lin, and not Ming-Tsang Wu, was listed as the corresponding author. Correspondence and request for materials should be addressed to Prof. Ming-Tsang Wu at e_encourage@yahoo.com. This has now been corrected in the HTML and PDF versions of the Article."} +{"text": "Scientific Reports 10.1038/s41598-017-14302-2, published online 27 October 2017Correction to: The original version of this Article contained a typographical error in the spelling of the author Carlo A.J.M. Gaillard, which was incorrectly given as Carlo AJM Gaillard. This has now been corrected in the PDF and HTML versions of the Article."} +{"text": "Nature Communications 10.1038/s41467-017-01936-z, published online 12 December 2017Correction to: In the original version of this Article, Fig. 3 contained several errors in the chemical structures. The correct version is:Fig. 1which replaces the previous incorrect version :Fig. 2This has now been corrected in both the PDF and HTML versions of the Article."} +{"text": "Nature Communications8:742 doi:10.1038/s41467-017-00788-x; Article published online: 29 Sep 2017In the PDF version of this article, Eq. 5 is missing all elements after the equals sign. The correct version of Eq. 5 is given below. The HTML version of the paper was correct from the time of publication."} +{"text": "West Nile virus Epidemic\u201d by Thomas Briese et al., errors occurred in the text and figure legend. On page 529, right column, line 25, and in the figure legend on page 530, the host species for ISR-00PigC is pigeon. Additionally, in the figure legend, the GenBank accession no. for ISR-00PigC is AF380671, and the GenBank accession no. for WNV-ROM96(0334)-1996 is AF205879.In \u201cPhylogenetic Analysis of a Human Isolate from the 2000 Israel http://www.cdc.gov/ncidod/EID/vol8no5/01-0324.htm has been corrected.The online article at We regret any confusion these errors may have caused."} +{"text": "Purpose.To look at a method for treating soft tissue sarcoma of the retroperitoneal area. Patient. We report the case of a 38-year-old woman with a well-differentiated liposarcoma. Results. Complete excision was achieved resulting in only minor morbidity and complete local control."} +{"text": "S. mutans biofilms.A biofilm is a complex community of microorganisms that develop on surfaces in diverse environments. The thickness of the biofilm plays a crucial role in the physiology of the immobilized bacteria. The most cariogenic bacteria, mutans streptococci, are common inhabitants of a dental biofilm community. In this study, DNA-microarray analysis was used to identify differentially expressed genes associated with the thickness of S. mutans genes showed differential expression in both 400- vs. 100-microns and 200- vs. 100-microns biofilms. All of these genes were upregulated.Comparative transcriptome analyses indicated that expression of 29 genes was differentially altered in 400- vs. 100-microns depth and 39 genes in 200- vs. 100-microns biofilms. Only 10 As sucrose is a predominant factor in oral biofilm development, its influence was evaluated on selected genes expression in the various depths of biofilms. The presence of sucrose did not noticeably change the regulation of these genes in 400- vs. 100-microns and/or 200- vs. 100-microns biofilms tested by real-time RT-PCR.luxS- mutant strain. The expression of those genes was not radically changed in the mutant strain compared to wild-type bacteria in planktonic condition. Only slight downregulation was recorded in SMU.2146c, SMU.574, SMU.609, and SMU.987 genes expression in luxS- bacteria in biofilm vs. planktonic environments.Furthermore, we analyzed the expression profile of selected biofilm thickness associated genes in the S. mutans. Expression of these genes is apparently not regulated directly by luxS and is not necessarily influenced by the presence of sucrose in the growth media.These findings reveal genes associated with the thickness of biofilms of S. mutans is considered to be the most important etiological agent in dental caries. It forms biofilms on tooth surfaces and causes dissolution of enamel by acid end-products resulting from carbohydrate metabolism /2), the average expression level for the gene across all arrays and channels) of more than 8.5, leaving out genes with an average intensity in both channels less than 256. The microarray data were deposited in the GEO public repositories with accession number GSE12496.The arrays consisted of 1948 70-mer oligonucleotides representing 1960 open reading frames (ORF) from escribed . In brieescribed , availabn of 0.3 . To idenn of 0.3 . Accordin of 0.3 . B can tS. mutans (Acc. No. X58303) as an internal standard. Each assay was performed with at least two independent RNA samples in duplicate.Quantitative SYBR green PCR assays employing an ABI-Prism 7300 Light Cycler System were performed as described previously . The corAHL: acyl homoserine lactone; AI-2: autoinducer-2; BHI: brain heart infusion; CDFF: constant-depth film fermenter; CLSM: confocal laser scanning microscope; EPS: extracellular polysaccharides; QS: quorum sensing; PTFE: polytetrafluoroethylene; RT-PCR: reverse transcription polymerase chain reaction; TIGR: The Institute for Genomic Research.MS planed and carried out the experiments, performed the array and real time RT-PCR analyses and wrote the original manuscript. AT assisted in biofilms generation, RNA extraction, RT-PCR and CLSM experiments. MK performed the probe labeling, hybridization and array data normalization for DNA-microarrays. MF helped in setting up and performing the CDFF experiments to generate different depths of biofilm. DS conceived the study and oversaw its execution; he also revised the manuscript critically for important intellectual content. MS and DS integrated all of the data throughout the study and crafted the final manuscript. All authors read and approved the manuscript."} +{"text": "In 2001, a 71-year old male was admitted to our hospital with unstable angina. The angiography revealed 2-vessel disease with a 90% stenosis of the proximal LAD. A bare-metal stent was implanted. Four years later the angiography showed a 80% instent-stenosis in the bare-metal stent but no progress at the other coronary arteries. A DES was implanted. Again, four years later, the patient presented with non-ST-elevation myocardial infarction. Angiography showed a 90% instent-restenosis, again without any progession of coronary artery disease in the other vessels. Again a DES implanted. Therefore the processes involved in the late instent-stenosis were not influenced by the antiproliferative agent sirolimus The angiography revealed 2-vessel disease with a 90% stenosis of the proximal LAD was admitted to our hospital with unstable angina. Blood pressure at admission was 160/80 mmHg, LDL cholesterol was 127 mg/dl and no diabetes exist. The electrocardiogram showed persistent atrial fibrillation with negative T-waves in leads VD Figure . A bare-D Figure . AspirinD Figure . This tiD Figure . AspirinD Figure . Again, D Figure .This case is unusual, because a late instent-stenosis was observed twice without any difference in the time interval of the clinical occurrence of the restenosis between bare-metal and drug-eluting stent -3. UsualWritten informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.FT collected the data and wrote the case report. RZ reviewed the report and gave suggestions. UZ reviewed the report and gave suggestions.All authors have read and approved the final manuscript."} +{"text": "Scientific Reports 10.1038/s41598-019-42438-w, published online 11 April 2019Correction to: In the original version of this Article, Chuan Yang was incorrectly listed as the corresponding author. The correct corresponding author for this Article is Jiang Yu. Correspondence and request for materials should be addressed to yujianggz@163.com. This error has now been corrected in the HTML and PDF versions of the Article."} +{"text": "Scientific Reports 10.1038/s41598-017-15240-9, published online 06 November 2017Correction to: In the original version of this Article, Fei Liu was incorrectly listed as one of the corresponding authors. The correct corresponding author for this Article is Weijun Kang. Correspondence and request for materials should be addressed to: kangwj@hebmu.edu.cn. This error has now been corrected in the HTML and PDF versions of the Article."} +{"text": "Nature Communications; 10.1038/s41467-019-08541-2, published online 06 February 2019Correction to: y axis. This error has been corrected in both the PDF and HTML versions of the Article.The original version of this Article contained an error in Fig.\u00a07. In panel b, the survival curves were shifted relative to the"} +{"text": "Scientific Reports 10.1038/s41598-018-35595-x, published online 11 March 2019Correction to: In the original version of this Article, Rajesh Arora was omitted as a corresponding author. Correspondence and request for materials should also be addressed to rajesharoratejas@gmail.com. This error has now been corrected in the HTML and PDF versions of the Article."} +{"text": "Scientific Reports 10.1038/s41598-018-26458-6, published online 23 May 2018Correction to: The Acknowledgements section in this Article is incorrect.\u201cThis study has been supported in part by the Russian Science Foundation (Grant No. 16-13-10295) and by the grant from Sechenov First Moscow State Medical University, Russia, including preparation of the ternary DMG-PVP-CT compositions, analysis of their photocatalytic activity and temperature dependencies, acquisition of spectra, and in part by the Russian Foundation for Basic Research , including measurements of the sizes of porphyrin aggregates and polymers by dynamic light scattering and laser elastic scattering. The authors wish to thank Dr. T.G.Rudenko and Dr. A.B.Shekhter for the animal studies.\u201dshould read:\u201cThis study has been supported by the Russian Science Foundation (Grant No. 16-13-10295) including preparation of the ternary DMG-PVP-CT compositions, analysis of their photocatalytic activity and temperature dependencies, acquisition of spectra and in part by the Russian Foundation for Basic Research (Grant No. 17-02-00294), including measurements of the sizes of porphyrin aggregates and polymers by dynamic light scattering and laser elastic scattering. The authors wish to thank Dr. T.G.Rudenko and Dr. A.B.Shekhter for the animal studies.\u201d"} +{"text": "Nature Communications 10.1038/s41467-019-12831-0, published online 29 October 2019.Correction to: The original version of this Article contained an error in the spelling of the author Haralampos N. Miras, which was incorrectly given as Harry N. Miras. This has now been corrected in both the PDF and HTML versions of the Article."} +{"text": "Scientific Reports 10.1038/s41598-018-25557-8, published online 09 May 2018Correction to: In the original version of this Article, the author Menno van Lookeren Campagne was incorrectly indexed.Additionally, in the original version of this Article, Figure 8g contains an incorrect key.These errors have now been corrected in the HTML and PDF versions of this Article."} +{"text": "The Editor-in-Chief is retracting this article . After p"} +{"text": "Nature Communications 10.1038/s41467-018-02899-5, published online 30 January 2018.Correction to: The original version of this Article contained an error in the author affiliations. The affiliation of Soonbeom Seo with \u2018Los Alamos National Laboratory, Los Alamos, NM 87545, USA\u2019 was inadvertently omitted. Additionally the original version omitted the following from the Acknowledgements: \u2018S.S. acknowledges a Director's Postdoctoral Fellowship through the Los Alamos Laboratory Directed Research and Development program.\u2019 This has now been corrected in both the PDF and HTML versions of the Article."} +{"text": "Correction to: BMC Med Educhttps://doi.org/10.1186/s12909-019-1569-zFollowing publication of the original article , the autIncorrect heading for Table\u00a03 in the original article:Radiographic examination of BH (changes in bone height surrounding the implant).Correct heading for Table\u00a03:Overview of the sub-categories, categories, sub-themes and main themes which emerged in the study.The original article has been corrected."} +{"text": "Scientific Reports 10.1038/s41598-019-39291-2, published online 22 February 2019Correction to: The original version of this Article contained an error in the spelling of the author P. Garcia-Alfonso, which was incorrectly given as P. Garcia. This error has now been corrected in the PDF and HTML versions of this Article."} +{"text": "Scientific Reports 10.1038/s41598-019-48078-4, published online 12 August 2019Correction to: In the original version of this Article, the author Paolo De Coppi was incorrectly indexed.Additionally, Paolo De Coppi and Anna L. David were omitted as equally contributing senior authors.These errors have now been corrected in the PDF and HTML versions of the Article."} +{"text": "This article has been corrected: Due to errors in image selection, two small pictures were partially overlapped in Figure 3A - the mimics-NC are the same as the inhibitor-NC in the migration group of 786-O. The corrected Figure 3 is shown below. The authors declare that these corrections do not change the results or conclusions of this paper.13201-13215. https://doi.org/10.18632/oncotarget.3915Original article: Oncotarget. 2015; 6:13201\u201313215."} +{"text": "Communications Biology 10.1038/s42003-019-0698-6, published online 03 December 2019.Correction to: In the original published version of the article, funding information for co-author James C. Paulson was inadvertently omitted from the acknowledgements. The error has been corrected in the HTML and PDF versions."} +{"text": "Scientific Reports 10.1038/s41598-017-18076-5, published online 20 December 2017Correction to: The original version of this Article contained an incorrect email address for Jie Liu, which was incorrectly listed as jieliu28@fudan.edu.cn. This error has now been corrected in the HTML and PDF versions of the Article."} +{"text": "Nature Communications; 10.1038/s41467-018-07805-7; published online: 18 December 2018Correction to: Nat. Biotechnol. 21, 1069\u20131074 (2003)\u2019. This has been added as reference 42. The following has been added after the third sentence of the fifth paragraph of the Discussion: \u2018Integration could also allow more sophisticated information processing, for example as shown by the classic deoxyribozyme-based automaton that plays tic-tac-toe42, to direct structural reconfiguration (Supplementary Discussion)\u2019. This has been corrected in the PDF and HTML versions of the Article.The original version of this Article omitted a reference to previous work in \u2018Stojanovic, M. N. & Stefanovic, D. A deoxyribozyme-based molecular automaton."} +{"text": "Nature Communications 10.1038/s41467-018-06444-2; published online 1 October 2018Correction to: The original HTML version of this Article omitted to list Harold Y. Hwang as a corresponding author and incorrectly listed Adrian G. Swartz as a corresponding author. This has been corrected in the HTML version of the Article. The PDF version was correct from the time of publication."} +{"text": "Scientific Reports 10.1038/s41598-018-29428-0, published online 30 July 2018Correction to: The original version of this Article contained a typographical error in the spelling of the author T. Vo-Dinh, which was incorrectly given as T. Vo Dinh. This has now been corrected in the PDF and HTML versions of the Article."} +{"text": "Correction to: Blood Cancer Journal 9:510.1038/s41408-018-0169-1 published online 15 January 2019After publication of the original article, the authors realised there was an incorrect image in Fig. S5B. The correct image is reproduced here. The Supplementary File of the original article has also been replaced with this error corrected. This error does not affect the conclusions drawn in the article."} +{"text": "Scientific Reports 10.1038/s41598-017-01372-5, published online 28 April 2017Correction to: In the original version of this Article, the author Anne S. De Groot was incorrectly indexed.In addition, the publication date was omitted from the original PDF version of this Article.These errors have now been corrected in the HTML and PDF versions of the Article."} +{"text": "Nature Communications 10.1038/s41467-018-08034-8, published online 25 January 2019.Correction to: The original version of this Article contained an error in Fig.\u00a05. In the panel c, the right bar chart was inadvertently replaced with a duplicate of the left bar chart. This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Z.J. Zhang, M.L. Zheng, Y. Nie and Z.Q. NiuDepartment of Anesthesiology, the Cangzhou Central Hospital, Cangzhou, Hebei, ChinaRetraction for: Braz J Med Biol Res | doi: 10.1590/1414-431X20176825 | PMID: 29267506Click here to view Specialist message]The Brazilian Journal of Medical and Biological Research was contacted by one specialist questioning the validity of this study. [The authors were contacted on May 7 and 22, 2018 to answer the questions one-by-one. As of June 29, 2018, we did not receive an answer from the authors.10.1590/1414-431X20176825> PMID: 29267506.The Editors decided to Retract the article: \u201cComparison of Arndt-endobronchial blocker plus laryngeal mask airway with left-sided double-lumen endobronchial tube in one-lung ventilation in thoracic surgery in the morbidly obese\u201d that was published in volume 51 no. 2 (2018) in the Brazilian Journal of Medical and Biological Research 0.05) with RHL took anticholinergic drugs. These data suggest differential use of anticholinergic drugs is not likely to be responsible for differences in brain volumes observed in the current study.In addition, given the adverse cognitive impacts of psychotropic drug use in the elderly, particularly those with anticholinergic profiles, we examined whether use of anticholinergic drugs differed between RHL and NHL groups. Using updated Beers list criteria for anticholinergic drugs , we obseIn the current study, 257 brain regions were measured in 229 cognitively normal subjects, 308 MCI subjects and 188 AD subjects, and these subjects were further divided into those with self-report of hearing loss (RHL) or with no self-report of hearing loss (NHL). RHL was not associated with differences in brain volumes or surface areas in cognitively normal control or MCI subjects. In contrast, bilateral cerebellar volumes and brainstem volume were diminished in AD subjects with RHL compared to the NHL group. Two of these six brain regions (left and right cerebellar white matter) had effect sizes greater than 1.0, indicating a strong effect of reported hearing loss . A statiThis study employed subjective reports of HL, rather than objectively measured HL, such as can be measured with a pure-tone audiogram or auditory brainstem response. Use of subjective measures was required because hearing was not objectively measured in this dataset. Previous work has shown that subjective hearing loss shows an approximately 65\u201377% concordance rate with objectively measured hearing loss . Thus, iSimilar to previous work , when suThe presence of AD-related pathology in the brainstem and cerebellum suggests that these brain regions may be vulnerable in the setting of an additional insult, such as peripheral deafferentation. Both brain regions receive substantial input from the peripheral auditory system. For example, the eighth cranial nerve provides dense input to the cochlear nucleus which is situated at the pontomedullary junction. The cochlear nucleus then sends projections throughout the brainstem to the superior olive and nuclei of the lateral lemniscus . The cerThus, we speculate that loss of afferent acoustic input, in the setting of pre-existing AD pathology, induces downstream atrophy of synaptic targets in the brainstem and cerebellum. This atrophy is likely not seen in the cognitive-normal and MCI subjects because of the lower levels of AD pathology present in these populations. It is notable that brain regions associated with higher acoustic processing, such as regions of the superior temporal gyrus, which houses the auditory cortex, did not show this relationship, as has been seen in a previous study . Note thn = 42), and too small to parse effects of other important factors such as ApoE4 status. Thus, future work, particularly large-scale studies where both hearing levels and MRI volumes are quantitatively measured, will be needed to examine the relationship between hearing impairment and brain volume changes more closely.Thus, in the current study, we observed that in the context of AD, RHL was associated with lowered volumes of the brainstem and cerebellum, as well as faster rates of declines in these regions. These data suggest that pathological changes that occur in the brainstem and cerebellum in AD increase the vulnerability of these regions to auditory deafferentation-related pathology. Given the growing literature suggesting that ARHL may predispose to AD, it is possible that acoustic deafferentation may trigger brainstem pathology. Given early pathological changes that occur in the brainstem in AD , it is phttp://adni.loni.usc.edu/.Publicly available datasets were analyzed in this study. This data can be found here: The study was conducted across multiple clinical sites and was approved by the Institutional Review Boards of all of the participating institutions. Informed written consent was obtained from all participants at each site. The following individual ethics boards approved the study: Albany Medical College Institutional Review Board, Boston University Medical Campus Institutional Review Board (BU IRB), Butler Hospital Institutional Review Board, Cleveland Clinic Institutional Review Board, Columbia University Institutional Review Board, Dartmouth-Hitchcock Medical Center Committee for the Protection of Human Subjects, Duke University Health System Institutional Review Board, Emory University Institutional Review Board Georgetown University Institutional Review Board, Human Investigation Committee Yale University School of Medicine, Human Subjects Committee, University of Kansas Medical Center, Indiana University Institutional Review Board, Research Compliance Administration, Institutional Review Board of Baylor College of Medicine, Institutional Review Board of the Mount Sinai School of Medicine, Johns Hopkins University School of Medicine Institutional Review Boards, Lifespan\u2014Rhode Island Hospital Institutional Review Board, Mayo Clinic Institutional Review Board, Nathan Kline Institute Rockland Psychiatric Center Institutional Review Board (NKI RPC IRB), New York University Langone Medical Center School of Medicine, Institutional Review Board Human Research Program, Northwestern University Institutional Review Board Office, Office of the Washington University School of Medicine IRB (OWUMC IRB), Oregon Health and Science University Institutional Review Board, Partners Human Research Committee, Research Ethics Board Jewish General Hospital, Research Ethics Board Sunnybrook Health Sciences Centre, Roper St. Francis Institutional Review Board, Rush University Medical Center Institutional Review Board, Stanford University, Administrative Panel on Human Subjects in Medical Research, The Ohio State University Institutional Review Board, The University of Texas Southwestern Medical Center Institutional Review Board, UCLA Office of the Human Research Protection Program Institutional Review Board, UCSD Human Research Protections Program, University Hospitals Case Medical Center Institutional Review Board, University of Alabama at Birmingham Institutional Review Board, University of British Columbia, Clinical Research Ethics Board (CREB), University of California Davis Office of Research IRB Administration, University of California Irvine Office Of Research Institutional Review Board (IRB), University of California San Francisco Committee on Human Research (CHR), University of Iowa Institutional Review Board, University of Kentucky Office of Research Integrity, University of Michigan Medical School Institutional Review Board (IRBMED), University of Pennsylvania Institutional Review Board, University of Pittsburgh Institutional Review Board, University of Rochester Research Subjects Review Board (RSRB), University of South Florida Division of Research Integrity & Compliance, University of Southern California Health Science Campus Institutional Review Board, University of Western Ontario Research Ethics Board for Health Sciences Research Involving Human Subjects (HSREB), University of Wisconsin Health Sciences Institutional Review Board, Wake Forest University Institutional Review Board, Weill Cornell Medical College Institutional Review Board, Western Institutional Review Board and Western University Health Sciences Research Ethics Board. The patients/participants provided their written informed consent to participate in this study.DL and VD conceived of the project. DL, SK, and VD wrote the manuscript. VD did data analysis. All authors contributed to the article and approved the submitted version.VD was an employee of Eisai Pharmaceuticals. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."} +{"text": "In the article by Chen et\u00a0al.,\u00a01 State Key Laboratory of Bioreactor Engineering, School of Biotechnology, East China University of Science and Technology, Shanghai, China2 Shanghai Engineering Research Center of Molecular Therapeutics and New Drug Development, School of Chemistry and Molecular Engineering, East China Normal University, Shanghai, China3 NYU\u2010ECNU Center for Computational Chemistry at NYU Shanghai, Shanghai, China"} +{"text": "The second affiliation for co-Author Michal Marczyk was inadvertently omitted. The following affiliation has been added: Department of Data Science and Engineering, Silesian University of Technology, Gliwice, Poland. The HTML and PDF versions of the Article have been corrected."} +{"text": "There is an error in the Funding statement. The correct number for the Natural Science Foundation of Fujian Province of China is No. 2018J01134.The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.Incorrect Affiliation.In the published article, there was an error in affiliation 1. Instead of \u201cFujian Provincial Key Laboratory of Neurodegenerative Disease and Aging Research, Institute of Neuroscience, Department of Pathology, Basic Medicine, Medical College, Xiamen University, Xiamen, China\u201d, it should be \u201cCancer Research Center, Department of Basic Medical Sciences, Fujian Provincial Key Laboratory of Neurodegenerative, Disease and Aging Research, School of Medicine, Xiamen University, Xiamen, China\u201d.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."} +{"text": "There is an error in affiliation 2 for authors Mark P. Shephard and Narisong Huhe. The correct affiliation 2 is: School of Government and Public Policy, University of Strathclyde, Glasgow, United Kingdom."} +{"text": "In the published article, there were errors in affiliations 1 and 4. For affiliation 1, instead of \u201cSchool of Management, Shandong University, Jinan, China\u201d, it should be \u201cSchool of Economics, Shandong University, Jinan, China.\u201d For affiliation 4, instead of \u201cInstitute of Science and Technology for Development of Shandong, Qilu University of Technology, Shandong Academy of Science, Jinan, China\u201d, it should be \u201cInstitute of Science and Technology for Development of Shandong, Qilu University of Technology (Shandong Academy of Sciences), Jinan, China.\u201dThe authors apologize for these errors and state that they do not change the scientific conclusions of the article in any way.The original article has been updated.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."} +{"text": "The Irving S. Wright Award of Distinction Lecture will feature an address by the 2021 recipient Malene Hansen, PhD of the Buck Institue for Research on Aging. The Vincent Cristofalo Rising Star Award in Aging Research lecture will feature an address by the 2021 recipient, Morgan Levine, PhD, of Yale University. The Terrie Fox Wetle Award lecture will feature an address by the 2020 recipient, Kali Thomas, PhD, FGSA of Brown University and an address by the 2021 recpient, Andrea Gilmore-Bykovskyi of the University of Wisconsin, Madison. These awards are given by the American Federation for Aging Research, Inc."} +{"text": "There is an error in affiliation 2 for author Rahim Ahmadvand. The correct affiliation 2 is: Department of Vegetables Research, Seed and Plant Improvement Institute, Agricultural Research, Education & Extension Organization, Karaj, Iran."} +{"text": "Mahabir Gupta was born in India in 1942. He received his Bachelor and Master's degrees in Pharmacy from the University of Rajasthan and Banaras Hindu University, respectively, and earned his Ph.D. at Washington State University, Pullman, WA, USA.He joined the College of Pharmacy of the University of Panama in 1972, dedicating his highly productive professional life to pharmacognosy research in Panama and several other countries, especially Latin America. Over the years, in additional to his professorial position at the University of Panama, he served as Scientific Advisor to the Vice-President for Research and Graduate Studies, Director of International Technical Cooperation, Head of the Department of Medicinal Chemistry & Pharmacognosy, Advisor to the President of University of Panama International Technical Cooperation, and Dean, College of Pharmacy.In addition to the above, Dr. Gupta was an International Coordinator of the Natural Products Drug Discovery Program of the Ibero-American Program of Science and Technology for Development (CYTED) during 1995\u20132005, and Coordinator, Area of Health, CYTED 2011\u20132014, for 19 countries of Latin America, Spain and Portugal, with a network of over 1500 scientists. He was the Executive Director of the INTERCIENCIA Association for the promotion of science in the Americas.At the time of his death, he was Emeritus Professor of Pharmacognosy and Founding Director of the Centre for Pharmacognostic Research on Panamanian Flora at the University of Panama, Panama City, Panama, and Manager, Health Area, Iberoamerican Program of Science and Technology for Development CYTED, Madrid.In recognition of his outstanding performance and training of researchers, Dr. Gupta received many awards and honours. Examples include the National Research Achievement Award, National Secretary of Science Technology, and Innovation Panama; 2010 Science Award, Panamanian Association for the Advancement of Science; Distinguished Scientist \u2212 2008 in the National Investigators System National Secretariat for Science Technology & Innovation of Panama; Interciencia Award 2008 for Health Sciences; Honorary Professor National University of San Marcos, Per\u00fa; Vice Chairman, Plant Sciences Group, International Organisation of Chemical Sciences for Development (IOCD), since 2000; Member Dietary Supplements Botanical Expert Committee, U. S. Pharmacopoeia, USA 2005\u20132010; Member Medicinal Plants Expert Group, International Centre for Science and High Technology (ICS/UNIDO), Trieste, Italy; Recipient of International Scientific Cooperation Award from the American Association for the Advancement of Science; Member Board of Directors of International Foundation for Science; Member Board of Directors of the International Council of Medicinal and Aromatic Plants; Member of the International Union for Conservation of Nature and Natural Resources/Species Survival Commission, Ethnobotany Specialist Group; Member, ICSU Regional Committee for Latin America and the Caribbean; Academy of Sciences of Latin America, Caracas, Venezuela; Royal Academy of Pharmacy, Madrid, Spain; Panamanian Academy of Medicine and Surgery; Panamanian Association for the Advancement of Science \u2013 Founding Member and Permanent Secretary (Represents in IANAS because of lack of a science academy in Panama).Honoris causa) from the Universidad Nacional Aut\u00f3noma de Amazonia Peruana, Iquitos, Per\u00fa in 2005, and the University of Vale do Itaja\u00ed (UNIVALI Itaja\u00ed-Brazil) in 2014.Further, Dr. Gupta was elected Twas Fellow, TWAS World Academy of Sciences for the Advancement of Science in Developing Countries, and he received the maximum honour of"} +{"text": "This article has been corrected: The authors requested to modify the affiliation. The authors declare that these correction does not change the results or conclusions of this work.The corrected affiliation is posted below.Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China"} +{"text": "The correct affiliation is: Traditional Medicine and Hydrotherapy Research Center, Ardabil University of Medical Sciences, Ardabil, Iran.This articleThe publishers apologise for the omission."} +{"text": "There is an error in affiliation 1 for authors Guoqiang Du and Liangtao Bu. The correct affiliation 1 is: College of Civil Engineering, Hunan University, ChangSha, Hunan Province, China."} +{"text": "Therefore, the affiliations for this paper should have been written as follows:1*, JINGJING MENG2* and YU ZHANG3TING LIU1Nuclear Medicine and 2Thyroid and Breast Surgery, The Affiliated Wuhan Central Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430014; 3Department of Surgery II, Shanghai Municipal Hospital of Traditional Chinese Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai 200071, P.R. ChinaDepartments of Contributed equally*Furthermore, the authors have also realized that they overlooked acknowledging a source that contributed towards the paper's funding in the Funding section of the declarations. Accordingly, the following information should have been included in the paper regarding the funding received:"} +{"text": "In this article, the affiliation \u2018Department of Ophthalmology & Vision Science, UC Davis, School of Medicine, Davis CA 95618, USA\u2019 for Author Min Zhao was missing.The affiliation has been updated above and the original article has been"} +{"text": "The original version of this article was published with two of the author affiliations listed in the incorrect order and subsequently three authors were listed with institutions incorrectly assigned to them.The transposed institutions were \u2018National Reference Center for Influenza, Robert Koch Institute, Berlin, Germany State Institute of Public Health\u2019 and \u2018Bavarian Health and Food Safety Authority, Oberschlei\u00dfheim, Germany\u2019.The affected authors are listed with their correct affiliations belowBarbara Biere: National Reference Center for Influenza, Robert Koch Institute, Berlin, GermanyBernhard Liebl: State Institute of Public Health, Bavarian Health and Food Safety Authority, Oberschlei\u00dfheim, Germany; Ludwig Maximilians-Universit\u00e4t, Munich, GermanyAndreas Sing: Department of Public Health Microbiology, Bavarian Health and Food Safety Authority, Oberschlei\u00dfheim, Germany; State Institute of Public Health, Bavarian Health and Food Safety Authority, Oberschlei\u00dfheim, Germany; Ludwig Maximilians-Universit\u00e4t, Munich, GermanyThe original version of the article has now been corrected."} +{"text": "The lecture will feature an address by the 2020 Pollack Award recipient, Karl Pillmer, PhD, FGSA of Cornell University. The 2021 Pollack Award recipient is Namkee G. Choi, PhD, FGSA, of the University of Texas at Austin. The Maxwell A. Pollack Award for Contributions to Healthy Aging Award recognizes instances of practice informed by research and analysis, research that has directly improved policy or practice, and distinction in bridging the worlds of research and practice."} +{"text": "In the published article, there was an error in the affiliation. Instead of \u201cDivision of Life Sciences and Medicine, Department of Obstetrics and Gynecology, The First Affiliated Hospital of USTC, University of Science and Technology of China, Hefei, China\u201d it should be \u201cDepartment of Obstetrics and Gynecology, The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, China\u201d.The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated."} +{"text": "An affiliation was missed for Thomas G. Schulze. The missed affiliation was for the Department of Psychiatry and Behavioral Sciences, SUNY Upstate Medical University, Syracuse, NY, USA. The Publisher apologises for this omission."} +{"text": "In the publication of the original article (Hill et al. Asia Pacific College of Business and Law, Charles Darwin University, AustraliaCollege of Indigenous Futures, Education & Arts, Charles Darwin University, AustraliaSchool of Humanities, Arts and Social Sciences, University of New England, Armidale, AustraliaThe correct affiliations of the authors are:The original paper has been updated."} +{"text": "There is an error in affiliation 2 for authors Zawiyah Mohammad Yusof and Hazura Mohamed. The correct affiliation 2 is: Faculty of Information Science and Technology, Universiti Kebangsaan Malaysia, Selangor, Malaysia."} +{"text": "This article refers incorrectly to the affiliation of author Evelien Brouwers. The correct affiliation for this author is Tranzo, Scientific Center for Care and Wellfare, Tilburg School of Social and Behavioral Sciences, Tilburg University."} +{"text": "In the original article there were errors in the Affiliation section. Affiliations 3 and 4 need to be in reversed order. Affiliation 3 should be \"Aston Institute of Photonic Technologies, Aston University, Birmingham, United Kingdom\". Affiliation 4 should be \"Department of Bioengineering and COMSET, Clemson University, Clemson, SC, United States\". Affiliation 1 is also corrected to \"Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province, College of Physics and Optoelectronics Engineering, Shenzhen University, Shenzhen, China\".The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated."} +{"text": "This article has been corrected: The authors requested to update affiliation 3 which was not completed. This correction does not change the content of the publication. The correct affiliation is provided below:3Department of Neurosurgery, Shandong Provincial Hospital, Cheeloo College of Medicine, Shandong University, Jinan, Shandong, China"} +{"text": "Instead of \u201cDepartment of Oncology, The First Affiliated Hospital, Guangxi University of Chinese Medicine, Chengdu, China\u201d, it should be \u201cDepartment of Oncology, The First Affiliated Hospital, Guangxi University of Chinese Medicine, Nanning, China\u201d.In the published article, there was an error in The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated."} +{"text": "A second affiliation is missing for the first author. Ruiyi Dong is also affiliated with Department of Hand Surgery, The Third Affiliated Hospital of Hebei Medical University, Shijiazhuang, Hebei, 050051, China."} +{"text": "In the published article, there was an error in affiliation 1. Instead of \u201cAnhui Clinical and Preclinical Key Laboratory of Respiratory Disease, Department of Respiratory Disease, The First Affiliated Hospital of Bengbu Medical College, Bengbu, China\u201d, it should be \u201cSchool of Laboratory Medicine, Hangzhou Medical College, Hangzhou, China\u201d.The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated."} +{"text": "The affiliations of Xiao\u2010Jie Huang are incorrect and should have been changed to Center for Infectious Diseases, Beijing YouAn Hospital, Capital Medical University, Beijing, Chinahongshang100@hotmail.com)The main corresponding author for this article should be Hong Shang. Correspondence address: NHC Key Laboratory of AIDS Immunology , National Clinical Research Center for Laboratory Medicine, The First Affiliated Hospital of China Medical University, Shenyang, China (In the article , there w"} +{"text": "In the originally published version of this manuscript, the affiliation was incorrect for Ramadhan T Othman. The correct affiliation is College of Medicine, University of Duhok, Kurdistan, Iraq. This error has been corrected online."} +{"text": "Following publication of the original article , it was Il-Seok Oh was incorrectly assigned affiliation 1. The correct affiliation is: Department of Computer Science and Engineering, Jeonbuk National University, Jeonju, Korea.The original article has been"} +{"text": "Kunio Aoki, Professor Emeritus, Nagoya University School of Medicine, and Dr. Haruo Sugano, the former Director of the Cancer Institute, Tokyo -- in collaboration with 36 active epidemiologists at 24 different institutions.1-3Approximately 127,500 healthy inhabitants from 45 areas throughout Japan who responded to the study questionnaire between 1988 and 1990, were enrolled into the study. Among this group, a total of 110,792 individuals aged 40-79 years were followed up for a period of at least 10 years. Descriptions for the initial stage of the JACC Study have been published elsewhere.Sixteen years have now passed since the JACC Study was first established with the support of Monbusho . ThroughThe aims of the present supplement are: to present a summary of the data derived from the JACC Study; to clarify the details of the project; and to publish validity studies for each variable investigated, including major lifestyle factors such as smoking, drinking, dietary habits, physical exercise and serum chemistry.The authors anticipate that this supplement will be a valuable resource for better understanding of the JACC Study.The present investigators involved, with the co-authorship of this paper, in the JACC Study and their affiliations are as follows: Dr. Akiko Tamakoshi (present chairperson of the study group), Nagoya University Graduate School of Medicine; Dr. Mitsuru Mori, Sapporo Medical University School of Medicine; Dr. Yutaka Motohashi, Akita University School of Medicine; Dr. Ichiro Tsuji, Tohoku University Graduate School of Medicine; Dr. Yosikazu Nakamura, Jichi Medical School; Dr. Hiroyasu Iso, Institute of Community Medicine, University of Tsukuba; Dr. Haruo Mikami, Chiba Cancer Center; Dr. Yutaka Inaba, Juntendo University School of Medicine; Dr. Yoshiharu Hoshiyama, University of Human Arts and Sciences; Dr. Hiroshi Suzuki, Niigata University School of Medicine; Dr. Hiroyuki Shimizu, Gifu University School of Medicine; Dr. Hideaki Toyoshima, Nagoya University Graduate School of Medicine; Dr. Kenji Wakai, Aichi Cancer Center Research Institute; Dr. Shinkan Tokudome, Nagoya City University Graduate School of Medical Sciences; Dr. Yoshinori Ito, Fujita Health University School of Health Sciences; Dr. Shuji Hashimoto, Fujita Health University School of Medicine; Dr. Shogo Kikuchi, Aichi Medical University School of Medicine; Dr. Akio Koizumi, Graduate School of Medicine and Faculty of Medicine, Kyoto University; Dr. Takashi Kawamura, Kyoto University Center for Student Health; Dr. Yoshiyuki Watanabe, Kyoto Prefectural University of Medicine Graduate School of Medical Science; Dr. Tsuneharu Miki, Graduate School of Medical Science, Kyoto Prefectural University of Medicine; Dr. Chigusa Date, Faculty of Human Environmental Sciences, Mukogawa Women\u2019s University ; Dr. Kiyomi Sakata, Wakayama Medical University; Dr. Takayuki Nose, Tottori University Faculty of Medicine; Dr. Norihiko Hayakawa, Research Institute for Radiation Biology and Medicine, Hiroshima University; Dr. Takesumi Yoshimura, Fukuoka Institute of Health and Environmental Sciences; Dr. Akira Shibata, Kurume University School of Medicine; Dr. Naoyuki Okamoto, Kanagawa Cancer Center; Dr. Hideo Shio, Moriyama Municipal Hospital; Dr. Yoshiyuki Ohno, Asahi Rosai Hospital; Dr. Tomoyuki Kitagawa, Cancer Institute of the Japanese Foundation for Cancer Research; Dr. Toshio Kuroki, Gifu University; and Dr. Kazuo Tajima, Aichi Cancer Center Research Institute."} +{"text": "An additional affiliation is missing for the first author. Peng Lu is also affiliated with School of Life Sciences, Jiangsu Normal University, Xuzhou, China."} +{"text": "The affiliation for the third author is incorrect. Akira Niwa is not affiliated with #2 but with #1: Department of Clinical Application, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan."} +{"text": "Instead of \u201cDivision of Life Sciences and Medicine, Department of Pharmacy, Anhui Provincial Cancer Hospital, The First Affiliated Hospital of USTC, University of Science and Technology of China, Hefei, China\u201d, it should be \u201cDepartment of Pharmacy, Anhui Provincial Cancer Hospital, The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, China\u201d.In the published article, there was an error in affiliation 3. Instead of \u201cDivision of Life Sciences and Medicine, Department of Pharmacy, Anhui Provincial Hospital, The First Affiliated Hospital of USTC, University of Science and Technology of China, Hefei, China\u201d, it should be \u201cDepartment of Pharmacy, Anhui Provincial Hospital, The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, China\u201d.In the published article, there was an error in affiliation The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated."} +{"text": "Affiliation 1.In the published article, there was an error in Instead of \u201cNational Clinical Research Center for Metabolic Diseases, Key Laboratory of Diabetes Immunology , Ministry of Education, Metabolic Syndrome Research Center, and Department of Metabolism and Endocrinology, The Second Xiangya Hospital of Central South University, Changsha, China\u201d, it should be split into two separate affiliations \u201cNational Clinical Research Center for Metabolic Diseases, and Department of Metabolism and Endocrinology, The Second Xiangya Hospital of Central South University, Changsha, China\u201d and \u201cKey Laboratory of Diabetes Immunology, Ministry of Education, and Metabolic Syndrome Research Center, The Second Xiangya Hospital of Central South University, Changsha, China\u201d.The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."} +{"text": "There is an error in affiliation 3 for author Kai Hu. The correct affiliation 3 is: Department of Dermatological, Wuhan Fourth Hospital; Puai Hospital, Tongji Medical College, Huazhong University of Science and Technology."} +{"text": "Real-world evidence and comparison among commonly seen chronic obstructive pulmonary disease (COPD) phenotypes, i.e., asthma\u2013COPD overlap (ACO), bronchiectasis\u2013COPD overlap (BCO), and their coexistence (ABCO) have not been fully depicted, especially in Chinese patients.Data were retrieved from an ongoing nationwide registry in hospitalized patients due to acute exacerbation of COPD in China (ACURE).2 (IQR: 19.47\u201323.97) and 21.79 kg/m2 (IQR: 19.49\u201324.22), respectively. The COPD phenotype had more male gender (79.90%) and smokers (71.12%) with a longer history of smoking . The ACO and ABCO phenotypes suffered more prior allergic episodes with a proportion of 18.05 and 19.05%, respectively. The ACO phenotype exhibited a higher level of eosinophil and better lung reversibility. Moreover, the four phenotypes showed no significant difference neither in all-cause mortality, intensive care unit admission, length of hospital stay, and COPD Assessment Test score change during the index hospitalization, and nor in the day 30 outcomes, i.e., all-cause mortality, recurrence of exacerbation, all-cause, and exacerbation-related readmission.Of the eligible 4,813 patients with COPD, 338 (7.02%), 492 (10.22%), and 63 (1.31%) were identified as ACO, BCO, and ABCO phenotypes, respectively. Relatively, the ABCO phenotype had a younger age with a median of 62.99 years [interquartile range (IQR): 55.93\u201369.48] and the COPD phenotype had an older age with a median of 70.15 years (IQR: 64.37\u201376.82). The BCO and COPD phenotypes were similar in body mass index with a median of 21.79 kg/mThe ACO, BCO, ABCO, and COPD phenotypes exhibited distinct clinical features but had no varied short-term prognoses. Further validation in a larger sample is warranted. Patients with chronic obstructive pulmonary disease (COPD), comorbid with asthma or bronchiectasis as well as their coexistence (ABCO), are commonly seen phenotypes, which have been broadly discussed whether they were distinct disease entities, but there is no concluded consensus yet . The conThe prevalence of the above-mentioned phenotypes of COPD varied across studies. Alshabanat A et al. reported a pooled prevalence of ACO phenotype among patients with COPD was 27% and 28% in population- and hospital-based studies, respectively. Hosseini et al. found thBoth asthma and bronchiectasis could incur an exacerbation of the chronic pulmonary disease (AECOPD). The ACO and BCO phenotypes present common and distinct clinical characteristics and prognoses. In general, the ACO phenotype exhibits a higher eosinophil level and better bronchodilator reversibility, and the BCO phenotype presents more neutrophil and nonreversible characteristics , 24. AddAlthough numerous studies have been conducted, findings were still controversial . The notClinicalTrials.gov registry number: NCT02657525). The ACURE study was started on September 1, 2017 and planned to recruit 7,600 hospitalized patients with AECOPD who were admitted to 161 participating medical centers across China with a maximum of 3-year follow-up. The protocol of the ACURE registry and baseline characteristics of the study population have been published (Data was retrieved from an acute exacerbation of COPD inpatient registry (ACURE), which was initiated in China to investigate the demographic characteristics, clinical features, diagnoses and treatments, and prognoses among hospitalized patients with COPD who were suffering an acute exacerbation episode and other local participating centers. All the participating patients have provided written informed consent.The ACURE participants underwent screenings at index hospital admission to confirm their eligibilities for enrollments. Subjects would be enrolled if they fulfilled the following eligibility criteria: 1) aged 18 years or older; 2) confirmed or suspected to be hospitalized due to AECOPD; 3) not participating in other clinical trial or intervention studies; and 4) agreeing to sign the informed consent. As of February 25, 2020, 4,813 eligible patients met the inclusion and exclusion criteria.In the ACURE registry, patient management was at the discretion of clinical physicians. Well-designed and sophisticated questionnaires were administrated to enrolled participants at baseline, i.e., during the index hospitalization, and at planned follow-up visits, i.e., at day 30 (\u00b12 days), month 6 (\u00b112 days), month 12 (\u00b112 days), month 24 (\u00b112 days), and month 36 (\u00b112 days), respectively after the index hospital discharge by trained investigators. The questionnaires consisted of contents on basic and demographic information, inclusion and exclusion criteria, current diagnoses (including symptoms and signs), objective examinations , history and management of the disease , predisposing factors and prevention of the exacerbation, pharmacological and nonpharmacological treatments in the hospital, cost, outcomes at hospital discharge, and management and outcomes during the follow-ups. Data of any exacerbations that did not occur in the scheduled visits were also collected.All data were uploaded to an online electronic data capture system. Data quality was regularly monitored by a concerted project and data management team. For instance, missing values, outliers, and illogical information will be sent to the local participating centers for timely amendment.In current analyses, spirometric COPD was diagnosed as the presence of a post-bronchodilator forced expiratory volume in 1 s (FEV1) divided by the forced vital capacity (FVC) with a value of less than 0.70, which indicated a persistent airflow limitation according to the Global Initiative for Chronic Obstructive Lung Disease (GOLD) 2021 report . Asthma Data on demographic and clinical characteristics, laboratory, lung function, and image tests, short- and long-term prognoses were comprehensively collected. Lung function test was performed when the situation of the patient was relatively stable after admission. If multiple lung function tests were conducted, the latest result was chosen before discharge. Bronchiectasis was diagnosed once any of the CT scan result was confirmed during hospitalization. Classification of the severity of airflow limitation was categorized into four grades based on the 2021 GOLD report: GOLD 1 , GOLD 2 , GOLD 3 , and GOLD 4 .In this manuscript, short-term clinical outcomes referred to the length of index hospital stay, recurrence of exacerbation, and exacerbation-related hospital readmission within 30 days after the index hospital discharge. Definitions of clinical outcomes are explicated in the t-test or Wilcoxon rank-sum test or Kruskal\u2013Wallis test as appropriate for continuous variables, and using Pearson's Chi-square test or Fisher's exact test as appropriate for categorical ones.Mean and standard deviation (SD) were calculated for normally distributed continuous variables, otherwise median and interquartile range (IQR) were presented for abnormally distributed ones. Frequencies and percentages were calculated for categorical variables. Characteristics of patients within phenotypes were compared with the utilization of Student's p < 0.10), and factors that previously had been reported to be associated with prognoses of COPD were further adjusted in the multivariable statistical models . Multivariable Cox proportional hazards regression model was adapted to investigate the associations with day 30 outcomes . Variables that showed significant associations in univariable analyses and R Project (version 4.0.5).Statistical significance was defined as achieving a two-sided Of the overall 4,813 eligible patients with AECOPD, 63 (1.31%) patients were comorbid both with asthma and bronchiectasis (ABCO), 338 (7.02%) and 492 (10.22%) patients were identified as ACO and BCO phenotypes, respectively, and 3,920 (81.45%) patients did not coexist with asthma or bronchiectasis .2 (IQR: 19.47\u201323.97) and 21.79 kg/m2 (IQR: 19.49\u201324.22), respectively. In addition, patients without asthma or bronchiectasis were more of the male gender (79.90%) and smokers (71.12%) with a longer history of smoking . The ACO and ABCO phenotype patients suffered more prior allergic episodes with a proportion of 18.05 and 19.05%, respectively. The ACO phenotype patients exhibited higher level of eosinophil and better lung reversibility. The four phenotype patients showed no significant difference in all-cause mortality, intensive care unit (ICU)/respiratory intensive care unit (RICU) admission, length of stay, and COPD assessment test (CAT) score change in the index hospitalization, and neither in the day 30 outcomes, i.e., all-cause mortality, recurrence of exacerbation, and all-cause and exacerbation-related hospital readmission , levels of peripheral biomarkers , and lung function. Differences between ACO and BCO phenotypes were similar to previous research , 24, 27,via international multicenter collaboration between countries and ethnicities.The prevalences of ACO and BCO phenotypes in our study were lower than previous reports \u201322, 36, Furthermore, the entity of ACO phenotype has been debated for years, and the diagnosis criteria are developing. Cosio BG et al. used a composite criteria to define ACO, which included positive bronchodilator response, medical history of asthma, and higher levels of blood eosinophil and IgE . Hansen Current analyses were based on the largest ongoing multicenter registry on hospitalized patients with AECOPD in China. Characteristics and short-term prognoses of common but distinct phenotypes of COPD, i.e., ACO, BCO, ABCO phenotypes and those patients without the two diseases, were fully described. The findings of current analyses could provide the real-world evidence, as well as hints for disease management and further research. Meanwhile, several limitations of current analyses should be stated. First, some estimations of the associations lacked precision, i.e., a broad confidence interval might be due to the limited number of patients or events. Second, some potential impact factors and outcomes were not considered to avoid amplification of multiple testing and the type I error. Third, data on other common phenotypes such as chronic bronchitis and emphysema were not included. Additionally, some factors that would have influences on outcomes of interests were not collected, such as secondhand smoking, biomass fuel exposure, outdoor air pollution, etc, and alternative clinical outcomes including composite ones should be considered. With the ongoing recruitment and follow-up of the ACURE registry, a larger number of patients and long-term prognosis analyses in the future are possible.Current findings revealed that ACO, BCO, their overlaps (ABCO), and those patients without the two comorbidities had distinct clinical features, but did not differ in short-term prognoses. Further replication and validation in a larger and independent sample are warranted.The raw data supporting the conclusions of this article will be made available from the corresponding authors on reasonable request, without undue reservation.The study involving human participants was reviewed and approved by the Institutional Review Board of the China-Japan Friendship Hospital . The participants provided their written informed consent to participate in this study.We deeply appreciated continuous supports and contributions from the following 161 hospitals and the local investigators: The First Affiliated Hospital of Hunan University of Medicine, Hunan Province; The First People's Hospital of Huaihua City, Hunan Province; Affiliated Hospital of Inner Mongolia University for the Nationalities, Inner Mongolia Autonomous Region; The People's Hospital of the Xishuangbanna Dai Nationality Autonomous Prefecture, Yunnan Province; Tongji Hospital, Tongji Medical College, Huazhong University of Science & Technology, Hubei Province; Zhangjiagang First People's Hospital, Jiangsu Province; The First Affiliated Hospital of Zhengzhou University, Henan Province; First Hospital of Shanxi Medical University, Shanxi Province; Central Hospital Affiliated to ShenYang Medical College, Liaoning Province; Liuzhou Workers Hospital, Guangxi Zhuang Autonomous Region; LongHua Hospital Shanghai University of Traditional Chinese Medicine, Shanghai City; The First Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangdong Province; People's Hospital of Ji'an County, Jiangxi Province; Gongzhuling Central Hospital, Jilin Province; Chongqing Sixth People's Hospital, Chongqing City; Shanghai Putuo District Central Hospital , Shanghai City; The Second Affiliated Hospital of Tianjin University of Traditional Chinese Medicine, Tianjin City; Shanxi Fenyang Hospital, Shanxi Province; Taihe Hospital , Hubei Province; Ulanqab Second Hospital, Inner Mongolia Autonomous Region; Nanjing Pukou District Central Hospital , Jiangsu Province; Benxi Central Hospital, Liaoning Province; The Third People's Hospital of Jingdezhen, Jiangxi Province; Chongqing Jiulongpo District First People's Hospital, Chongqing City; Second Hospital of Shanxi Medical University, Shanxi Province; Ulanqab Central Hospital, Inner Mongolia Autonomous Region; Mianyang Central Hospital, Sichuan Province; Suining Central Hospital, Sichuan Province; The People's Hospital of Qitaihe, Heilongjiang Province; Affiliated Hospital of Zunyi Medical University, Guizhou Province; Inner Mongolia Baogang Hospital, Inner Mongolia Autonomous Region; Qiqihar Traditional Chinese Medicine Hospital, Heilongjiang Province; The Second Xiangya Hospital of Central South University, Hunan Province; Chengdu Qingbaijiang District People's Hospital, Sichuan Province; Minority Hospital of Guangxi Zhuang Autonomous Region , Guangxi Zhuang Autonomous Region; Meihekou Central Hospital, Jilin Province; The People's Hospital of Yi County, Liaoning Province; The Central Hospital of Yongzhou, Hunan Province; Fujian Provincial Hospital, Fujian Province; The First Affiliated Hospital of Guiyang College of Traditional Chinese Medicine, Guizhou Province; The People's Hospital of Wanzhou District, Chongqing City; Renhe Hospital, Baoshan District, Shanghai City; The People's Hospital of Nanchuan, Chongqing City; Miyun District Hospital, Beijing City; The First People's Hospital of Chuzhou, Anhui Province; Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai City; The Second Affiliated Hospital of Guangxi Medical University, Guangxi Zhuang Autonomous Region; Chengdu Second People's Hospital, Sichuan Province; 903 Hospital, Sichuan Province; The People's Hospital of Gaozhou, Guangdong Province; The Fourth Affiliated Hospital of Anhui Medical University, Anhui Province; Henan Provincial People's Hospital, Henan Province; The Second Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Shandong Province; The Affiliated Hospital of Southwest Medical University, Sichuan Province; The Second People's Hospital of Guiyang, Guizhou Province; The Second Affiliated Hospital of Guilin Medical University, Guangxi Zhuang Autonomous Region; Inner Mongolia Xing'an League People's Hospital, Inner Mongolia Autonomous Region; Affiliated Hospital of Jiangxi University of Traditional Chinese Medicine, Jiangxi Province; The First Hospital of Kunming, Yunnan Province; The People's Hospital of Langfang City, Hebei Province; Shanghai Traditional Chinese Medicine-Integrated Hospital, Shanghai City; The First Hospital of Changsha, Hunan Province; The Fifth People's Hospital of Datong, Shanxi Province; Yichang Central People's Hospital, Hubei Province; Hebei Provincial Hospital of Traditional Chinese Medicine, Hebei Province; Fuling Central Hospital, Chongqing City; Jiangxi Pingxiang People's Hospital, Jiangxi Province; Shanxi Bethune Hospital , Shanxi Province; Ths People's Hospital of Maoming, Guangdong Province; People's Hospital of Anshun City, Guizhou Province; China Resources WISCO General Hospital, Hubei Province; Qinghai Provincial People's Hospital, Qinghai Province; Shanghai Dongfang Hospital , Shanghai City; Xinjiang Uygur Autonomous Region People's Hospital, Xinjiang Uygur Autonomous Region; The People's Hospital of Cangnan County, Zhejiang Province; The Second Hospital of Harbin City, Heilongjiang Province; The Fourth Affiliated Hospital of Harbin Medical University, Heilongjiang Province; Shanghai Fifth People's Hospital, Fudan University, Shanghai City; Fujian Provincial People's Hospital, Fujian Province; Liuzhou City Liutie Central Hospital, Guangxi Zhuang Autonomous Region; XinSteel Center Hospital, Jiangxi Province; Liaocheng Hospital of Traditional Chinese Medicine, Shandong Province; Cangzhou People's Hospital, Hebei Province; The First Hospital of Lanzhou University, Gansu Province; Xinzhou People's Hospital, Shanxi Province; Affiliated Hospital of Guangdong Medical University, Guangdong Province; The Sixth Hospital of Beijing, Beijing City; Panjin Liaoyou Gem Flower Hospital , Liaoning Province; The People's Hospital of Nanping City, Fujian Province; The First People's Hospital of Qinzhou, Guangxi Zhuang Autonomous Region; The First Hospital of Tianjin, Tianjin City; Yichun People's Hospital, Jiangxi Province; The Central Hospital of Ningcheng County, Inner Mongolia Autonomous Region; The First Affiliated Hospital of Bengbu Medical College, Anhui Province; The Second People's Hospital of Lianyungang, Jiangsu Province; Gansu Gem Flower Hospital, Gansu Province; The People's Hospital of Lhasa, Tibet Autonomous Region; Anhui No. 2 Provincial People's Hospital, Anhui Province; Qingdao Municipal Hospital, Shandong Province; Maoming Hospital of Traditional Chinese Medicine, Guangdong Province; The People's Hospital of Pingliang, Gansu Province; Guizhou Provincial People's Hospital, Guizhou Province; Zhangjiajie City People's Hospital, Hunan Province; The Affiliated Hospital of Inner Mongolia Medical University, Inner Mongolia Autonomous Region; First Hospital of Qinhuangdao, Hebei Province; Chifeng Municipal Hospital, Inner Mongolia Autonomous Region; Jiading District Central Hospital, Shanghai City; The First Affiliated Hospital of Anhui University of Traditional Chinese Medicine, Anhui Province; The Affiliated Hospital of Shandong University of Traditional Chinese Medicine , Shandong Province; Yanbian No. 2 People's Hospital, Jinlin Province; The Third People's Hospital of Yichang City, Hubei Province; The Second Affiliated Hospital of Liaoning University of Traditional Chinese Medicine, Liaoning Province; Shenzhen Traditional Chinese Medicine Hospital, Guangdong Province; The First People's Hospital of Jiangxia District, Wuhan City, Hubei Province; The First People's Hospital of Xining, Qinghai Province; Xiamen Haicang Hospital, Fujian Province; Tibet Autonomous Region People's Hospital, Tibet Autonomous Region; The Third Hospital of Xiamen, Fujian Province; Nanjing Jiangning District Hospital of Traditional Chinese Medicine, Jiangsu Province; Sichuan Academy of Medical Sciences, Sichuan Provincial People's Hospital , Sichuan Province; Wuzhou Gongren Hospital, Guangxi Zhuang Autonomous Region; Wang Jing Hospital of China Academy of Chinese Medical Sciences, Beijing City; Shanghai Seventh People's Hospital, Shanghai City; The Fifth People's Hospital of Chongqing City, Chongqing City; The People's Hospital of Dongying City, Shandong Province; The Affiliated Hospital of Hangzhou Normal University, Zhejiang Province; The First Affiliated Hospital of Shantou University Medical College, Guangdong Province; Xinyu Hospital of Traditional Chinese Medicine, Jiangxi Province; Xishan Coal Electricity Group Gujiao Mining Area General Hospital, Shanxi Province; Ruikang Hospital Affiliated to Guangxi Universtiy of Chinese Medicine, Guangxi Zhuang Autonomous Region; The Third Affiliated Hospital of Guangzhou Medical University, Guangdong Province; Yu Tian Xian Zhong Yi Yuan, Hebei Province; People's Hospital of Changshou Chongqing, Chongqing City; Beijing Tiantan Hospital, Capital Medical University, Beijing City; General Hospital of Heilongjiang Provincial Agricultural Reclamation Bureau, Heilongjiang Province; The People's Hospital of Xishui County, Hubei Province; The First Hospital of Hunan University of Chinese Medicine, Hunan Province; The First People's Hospital of Huainan City, Anhui Province; The Second Affiliated Hospital of Nanjing Medical University, Jiangsu Province; Shanxi Provincial People's Hospital, Shanxi Province; West China Hospital of Sichuan University, Sichuan Province; Zhongshan Hospital Xiamen University, Fujian Province; Zheng Zhou Shi Zhong Yi Yuan, Henan Province; Xiangya Ping Mine Cooperative Hospital, Jiangxi Province; Northen Jiangsu People's Hospital, Jiangsu Province; Huzhou Hospital of Traditional Chinese Medicine affiliated to Zhejiang University of Traditional Chinese Medicine, Zhejiang Province; Guangdong Second Provincial General Hospital , Guangdong Province; Huainan Xinhua Hospital, Anhui Province; Third People's Hospital of Jiujiang City, Jiangxi Province; Panzhihua Hospital of Integrated Traditional Chinese and Western Medicine, Sichuan Province; The Central Hospital of Xuhui District, Shanghai City; Yueyang Integrated Traditional Chinese and Western Medicine Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai City; The Second Affiliated Hospital of Xiamen Medical College, Fujian Province; Hegang People's Hospital, Heilongjiang Province; Jiaozuo People's Hospital, Henan Province; Traditional Chinese Medicine Hospital of Kunshan, Jiangsu Province; The First People's Hospital of Nanning, Guangxi Zhuang Autonomous Region; Shandong Provincial Qianfoshan Hospital, Shandong Province; Beijing Hospital of Traditional Chinese Medicine, Capital Medical University, Beijing City; Tangshan People's Hospital, Hebei Province; Chongqing Traditional Chinese Medicine Hospital, Chongqing City.JL conceptualized this study, did all statistical analyses, interpreted the data, wrote the first draft, and critically revised the manuscript. CL, KH, and SW took part in manuscript revision, project, and data management. TY and CW supervised the work, had full access to all of the data in the study, and took responsibility for the integrity of the work as a whole, from inception to the published article. All authors have read and approved the final manuscript to be published.This work was supported by the Chinese Academy of Medical Science (CAMS) Innovation Fund for Medical Sciences (CIFMS) (nos. 2021-I2M-1-049 and 2020-I2M-2-008) and the Chinese Academy of Medical Science (CAMS) Basic Scientific Research Business Fee Fund of Central Level Public Welfare Scientific Research Institutes (no. 2019TX320005). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."} +{"text": "Instead of \u201c1Department of Geriatrics of Huashan Hospital, National Clinical Research Center for Aging and Medicine, Fudan University, Shanghai, China\u201d and \u201c4Department of Translational Neuroscience, Jing\u2019an District Centre Hospital of Shanghai and State Key Laboratory of Medical Neurobiology and MOE Frontiers Center for Brain Science, Institutes of Brain Science, Fudan University, Shanghai, China,\u201d it should be 1Department of Geriatrics of Huashan Hospital, National Clinical Research Center for Aging and Medicine, Department of Translational Neuroscience, Jing\u2019an District Centre Hospital of Shanghai, State Key Laboratory of Medical Neurobiology and MOE Frontiers Center for Brain Science, Institutes of Brain Science, Fudan University, Shanghai, China.\u201dIn the published article, there was an error in affiliation The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated."} +{"text": "Instead of 1School of Clinical Medicine, Cheeloo College of Medicine, Shandong University, Jinan, China, it should be 1School of Medicine, Cheeloo College of Medicine, Shandong University, Jinan, China.In the published article, there was an error in affiliation 3. Instead of 3Department of Radiation Oncology, Shandong Cancer Hospital and Institute, Shandong Cancer Hospital Affiliated to Shandong University, Shandong First Medical University and Shandong Academy of Medical Sciences, Jinan China, it should be 3Shandong Cancer Hospital, Cheeloo College of Medicine, Shandong University, Jinan, China.There was another error in The authors apologize for these errors and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."} +{"text": "Originally published affiliation: Department of Oncology, The First Affiliated Hospital of Anhui Medical University, Hefei 230022, ChinaCorrected affiliation: Department of Thoracic Surgery, The First Affiliated Hospital of Anhui Medical University, Hefei 230022, ChinaFollowing the publication of the original article , we wereThe original article has been corrected."} +{"text": "Following publication of the original article , the aut2 Center for Neural Science and WCI Center for Functional Connectomics, Korea Institute of Science and Technology (KIST), Seoul 136-791, South Korea.4 KU-KIST Graduate School of Converging Science and Technology, Korea University, Seoul 136-701, South Korea.The author list has been updated in this Correction article and the original article has been"} +{"text": "Instead of \u201c**Department of Infectious Disease, State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, National Clinical Research Center for Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China,**\u201d it should be \u201c**State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, National Clinical Research Center for Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, China.**\u201dIn the published article, there was an error in affiliation **Division of Hepatobiliary and Pancreatic Surgery, Department of Surgery, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, China.**\u201d Aforementioned corrections have been made and consequently, all affiliation numbers and order have been revised.Also, authors \u201cZheyue Shu\u201d and \u201cMin Zhang\u201d were missing an affiliation: \u201cThe authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated."} +{"text": "In the originally published version of this paper, the affiliation of author, Yoshifumi Mizuguchi, was incorrect. The paper has been corrected to include the correct affiliation, Department of Cardiovascular Medicine, Hokkaido University Hospital."} +{"text": "There is an error in affiliation 5 for author Pascale Salameh. The correct affiliation 5 is: University of Nicosia Medical School, Nicosia, Cyprus."} +{"text": "In \u201cDevelopment and Validation of a Scale to Measure Intimate Partner Violence Among Transgender and Gender Diverse Populations: Protocol for a Linear Three-Phase Study (Project Empower)\u201d :e23819), corrections have been made to the authorship list and to the text of the paper to clarify the methods of the protocol.Author Erin E Bonar has been added to the authorship list of the revised paper. The revised list of authors and affiliations is as follows:1*, MA, MSc, PhD; Kieran Todd2, MPH; Kristi E Gamarel3, PhD; Erin E Bonar2,4,5, PhD; Sarah Peitzmeier1*, PhDRob Stephenson1Center for Sexuality and Health Disparities and The School of Nursing, University of Michigan, Ann Arbor, MI, United States2Center for Sexuality and Health Disparities, University of Michigan, Ann Arbor, MI, United States3Center for Sexuality and Health Disparities and The School of Public Health, University of Michigan, Ann Arbor, MI, United States4Addiction Center, Department of Psychiatry, University of Michigan, Ann Arbor, MI, United States5The Injury Prevention Center, University of Michigan, Ann Arbor, MI, United States*these authors contributed equallyTo clarify the age of the participant group, mentions throughout the paper of participants as \u201caged over 15 years\u201d have been changed to \u201caged 15 and above.\u201dThe section \u201cProtection of Human Subjects\u201d has been edited to clarify the risk management plan for participants.References 40 and 45 in the original manuscript have been removed. 3 new references have been added to the reference list as References 41-43 and are cited in the \"Health Outcomes\" section. References have been renumbered accordingly. The full revised reference list appears below .In the \u201cHealth Outcomes\u201d section, the measure of suicidal ideation has been removed. The measure of substance abuse has been changed to the following:Substance use measures will assess the use and frequency of use of alcohol and other drugs in the past 3 months and are based on prior work -44.The correction will appear in the online version of the paper on the JMIR Publications website on May 12, 2021, together with the publication of this correction notice. Because this was made after submission to PubMed, PubMed Central, and other full-text repositories, the corrected article has also been resubmitted to those repositories."} +{"text": "There is an error in affiliation 2 for the second author Armando Santoro. The correct affiliation 2 is: Humanitas Clinical and Research Center\u2013IRCCS, Humanitas Cancer Center, Milan, Italy. An additional affiliation is also missing for the second author. Armando Santoro is also affiliated with Humanitas University, Department of Biomedical Sciences, Milan, Italy."} +{"text": "The correct affiliation is Center for Mental Health in Old Age, Landeskrankenhaus, Department of Psychiatry and Psychotherapy, University Medical Center, Mainz, Germany. This article has been corrected.1In the Original Investigation titled \u201cBupropion for the Treatment of Apathy in Alzheimer Disease: A Randomized Clinical Trial,\u201d"} +{"text": "The correct affiliation should be \u2018Division of Family Medicine, Department of Family Medicine, Faculty of Health Sciences, University of The Witwatersrand, Johannesburg, South Africa\u2019 instead of \u2018Division of Family Medicine, Department of Family Medicine, Faculty of Sciences, University of The Witwatersrand, Johannesburg, South Africa\u2019.In the version of this article initially published, Phukuta NSJ, Omole OB. Prevalence and risk factors associated with postnatal depression in a South African primary care facility. Afr J Prm Health Care Fam Med. 2020;12(1), a2538. This correction does not alter the study\u2019s findings of significance or overall interpretation of the study\u2019s results. The publisher apologises for any inconvenience caused."} +{"text": "In the published article, there was an error in affiliations 1 and 2. For affiliation 1, instead of \u201cVascular Cognitive Impairment and Neurodegeneration Program, Oklahoma Center for Geroscience and Healthy Brain Aging, Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, United States,\u201d it should be \u201cDepartment of Physiology, Semmelweis University, Budapest, Hungary.\u201d For affiliation 2, instead of \u201cDepartment of Biochemistry and Molecular Biology, Oklahoma Center for Geroscience and Healthy Brain Aging, University of Oklahoma Health Sciences Center, Oklahoma City, OK, United States,\u201d it should be \u201cVascular Cognitive Impairment and Neurodegeneration Program, Oklahoma Center for Geroscience and Healthy Brain Aging, Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, United States.\u201dThe authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated."} +{"text": "In the published article, there was an error in affiliations 1 and 3. For affiliation 1, instead of \u201cDepartment of Radiology, School of Clinical Medicine, Shanghai Tenth People's Hospital, Nanjing Medical University, Shanghai, China,\u201d it should be \u201cDepartment of Radiology, Shanghai Tenth People's Hospital, School of Clinical Medicine of Nanjing Medical University, Shanghai, China.\u201d For affiliation 3, instead of \u201cDepartment of Colorectal and Anal Surgery, School of Clinical Medicine, Shanghai Tenth People's Hospital, Nanjing Medical University, Shanghai, China,\u201d it should be \u201cDepartment of Colorectal and Anal Surgery, Shanghai Tenth People's Hospital, School of Clinical Medicine of Nanjing Medical University, Shanghai, China.\u201dThe authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."} +{"text": "Following the publication of the original article , errors boldtypeface.The changes have been highlighted in Affiliation:Center for Biotechnology Khalifa University, KhalifaUniversity of Science and Technology, Abu Dhabi, United Arab Emirates.Department of Electrical Engineering and Computer Science, Funding note:The study is funded by Khalifa University Collaborative Internal Research AwardCIRA-2019-076.The original article hasbeen"} +{"text": "Instead of \u201cDivision of Gastroenterology, Tongji Medical College, Union Hospital, Huazhong University of Science and Technology, Wuhan, China,\u201d it should be \u201cDivision of Gastroenterology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.\u201dIn the published article, there was an error in affiliation **2**. Instead of \u201cDepartment of Integrated Chinese and Western Medicine, Tongji Medical College, Union Hospital, Huazhong University of Science and Technology, Wuhan, China,\u201d it should be \u201cDepartment of Integrated Chinese and Western Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.\u201dIn the published article, there was an error in affiliation The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."} +{"text": "The affiliation for the fourth author is incorrect. Saswata Dutt is not affiliated with #5 but with #4:Department of Prevention, Care and Treatment, Institute of Human Virology, Abuja, Nigeria"} +{"text": "Youming Zhang\" and the authors \"Yunli Shen, Qizheng Lu,\" should be Affiliation 2 \"Department of Cardiology, Shanghai East Hospital, School of Medicine, Tongji University, Shanghai 200092, People\u2019s Republic of China.\" And the affiliation of the author \"Zichun Cai\" should be Affiliation 3 \"Department of Cardiology, Shanghai East Hospital of Clinical Medical College, Nanjing Medical University, Nanjing 211166, People\u2019s Republic of China.\"Following publication of the original article , the autThe original article has been revised."} +{"text": "Nature Communications 10.1038/s41467-021-26124-y, published online 5 October 2021.Correction to: In this article the affiliation details for all authors were incorrectly given as \u2018Division of Nanomaterials & Chemistry, Hefei National Laboratory for Physical Sciences at the Microscale, Hefei, China\u2019 but should have been \u2018Division of Nanomaterials & Chemistry, Hefei National Laboratory for Physical Sciences at the Microscale, University of Science and Technology of China, Hefei 230026, China\u2019. The original article has been corrected."} +{"text": "In \u201cElectronic Video Consent to Power Precision Health Research: A Pilot Cohort Study\u201d :e29123) the authors noted six errors.The title of the originally published article appeared as follows:Electronic Video Consent to Power Precision Research: A Pilot Cohort StudyIn the corrected version, the title has been revised to:Electronic Video Consent to Power Precision Health Research: A Pilot Cohort StudyIn the original version, author Liliana Johansen's name was displayed incorrectly as follows:Lilliana JohansenIn the revised version, it has been corrected as follows:Liliana JohansenIn the originally published paper, Affiliation 1 appeared as follows:UCLA Center for SMART Health, Clinical Translational Science Institute, David Geffen School of Medicine at UCLA, Los Angeles, CA, United StatesIn the revised version, it has been revised to:UCLA Center for SMART Health, Clinical and Translational Science Institute, David Geffen School of Medicine at UCLA, Los Angeles, CA, United StatesIn the originally published paper, Affiliation 2 appeared as follows:David Geffen UCLA School of Medicine, Los Angeles, CA, United StatesIn the revised version, it has been revised to:David Geffen School of Medicine at UCLA, Los Angeles, CA, United StatesIn the originally published paper, Affiliation 4 appeared as follows:Office of Clinical Research, Clinical and Translational Science Institute, David Geffen School of Medicine at UCLA, Los Angeles, CA, United StatesIn the revised version, it has been revised to:Embedded Clinical Research and Innovation Unit, CTSI Office of Clinical Research, Los Angeles, CA, United StatesIn the originally published paper, the following email address of the corresponding address was provided:arashnaeim@gmail.comIn the revised version, it has been changed to:anaeim@mednet.ucla.eduThe corrections will appear in the online version of the paper on the JMIR Publications website on October 21, 2021, together with the publication of this correction notice. Because this was made after submission to PubMed, PubMed Central, and other full-text repositories, the corrected article has also been resubmitted to those repositories."} +{"text": "There is an error in affiliation 17 for author Massimo Volpe. The correct affiliation should be two separate affiliations. The correct affiliation 17 is: Department of Clinical and Molecular Medicine, School of Medicine and Psychology, Sapienza University of Rome, Rome, Italy.th author is not indicated. Massimo Volpe is affiliated with: IRCCS Neuromed, Sapienza University Sant\u2019Andrea Hospital, Pozzilli (Isernia), Italy.An affiliation for the 19"} +{"text": "In \u201cConstructing High-Fidelity Phenotype Knowledge Graphs for Infectious Diseases With a Fine-Grained Semantic Information Model: Development and Usability Study\u201d :e26892) the authors noted three errors.1. In the originally published manuscript, affiliation 1, 2, and 3 were incorrectly mentioned as follows:\u00a01Suzhou Institute of Systems Medicine, Suzhou, China2Jiangsu Institute of Clinical Immunology, Jiangsu Key Laboratory of Clinical Immunology, The First Affiliated Hospital of Soochow University, Suzhou, China3Center of Systems Medicine, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, ChinaThese have been corrected to the following:\u00a01Center of Systems Medicine, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China2Suzhou Institute of Systems Medicine, Suzhou, China3Jiangsu Institute of Clinical Immunology, Jiangsu Key Laboratory of Clinical Immunology, The First Affiliated Hospital of Soochow University, Suzhou, China\"Center of Systems Medicine, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China\" was originally listed only for author Taijiao Jiang. This affiliation has now been added to authors Lizong Deng, Luming Chen, Tao Yang, and Shicheng Li in addition to author Taijiao Jiang.2. The affiliation 3. The address of the corresponding author was originally published as follows:Chongwen Road 100Suzhou Industrial ParkSuzhou, 215000ChinaThis has been corrected to the following:#5 Dong Dan San TiaoDongcheng DistrictBeijing, 100005ChinaThe correction will appear in the online version of the paper on the JMIR Publications website on July 13, 2021, together with the publication of this correction notice. Because this was made after submission to PubMed, PubMed Central, and other full-text repositories, the corrected article has also been resubmitted to those repositories."} +{"text": "The authors wish to make a correction to the published version of the paper .In the original publication, the funder \u201cOffice of Education Research, National Institute of Education, Nanyang Technological University, Singapore, grant number PG 01/21 SFB, to Stephen F. Burns\u201d was not included. The funder \u201cNTU Research Student Scholarship, National Institute of Education, Nanyang Technological University, Singapore, to Zhi Sen Tan\u201d was also not included.The authors apologize for any inconvenience caused and state that the scientific conclusions are unaffected. The original publication has also been updated.The paper should be as follows:Funding: This research was funded by a Planning Grant from the Office of Education Research, National Institute of Education (NIE), Nanyang Technological University, Singapore, grant number PG 01/21 SFB. The first author (Z.S.T.) was supported by an NTU Research Student Scholarship at the National Institute of Education, Nanyang Technological University, Singapore."} +{"text": "There is an error in affiliation 1 for authors Cinzia Cruder and Marco Barbero. The correct affiliation 1 is: Rehabilitation Research Laboratory 2rLab, Department of Business Economics, Health and Social Care, University of Applied Sciences and Arts of Southern Switzerland, Manno, Switzerland.There is also an error in affiliation 4 for author Emiliano Soldini. The correct affiliation 4 is: Research Methodology Competence Centre, Department of Business, Health and Social Care, University of Applied Sciences and Arts of Southern Switzerland (SUPSI), Manno, Switzerland.The publisher apologizes for the error."} +{"text": "Affiliation 2 for author Dinggang Shen is incorrect. The correct affiliation 2 is: Department of Research and Development, Shanghai United Imaging Intelligence Co., Ltd., Shanghai, China."} +{"text": "The following affiliation should be added as his second affiliation: Division of Family Medicine, School of Public Health and Family Medicine, University of Cape Town, Cape Town, South Africa.In the version of the article initially published, David N, Mash R. Community-based screening and testing for Coronavirus in Cape Town, South Africa: Short report. Afr J Prm Health Care Fam Med. 2020;12(1), a2499. This correction does not alter the study\u2019s findings of significance or overall interpretation of the study\u2019s results. The authors apologise for any inconvenience caused."} +{"text": "In \u201cComparison of Intercom and Megaphone Hashtags Using Four Years of Tweets From the Top 44 Schools of Nursing: Thematic Analysis\u201d :e25114 doi: 10.2196/25114), the author noted one error.In the originally published paper, the Acknowledgments section contained the following line about the source of funds for the 2016-2018 Twitter data:The September 29, 2016, to February 22, 2018, Twitter data for this project were purchased with funds provided by the George Washington University School of Nursing\u2019s Center for Health Policy and Media Engagement.To increase clarity regarding the amount and original sources of funding provided for the purchase of data, this has been corrected to:The September 29, 2016, to February 22, 2018, Twitter data for this project were purchased by the George Washington University School of Nursing\u2019s Center for Health Policy and Media Engagement for $1000 with funds received from the Gordon and Betty Moore Foundation, Robert Wood Johnson Foundation, Beatrice Renfield Foundation, Sigma Theta Tau International, American Association of Critical-Care Nurses, Donald and Barbara Jonas Foundation, National League for Nursing, OnCourse Learning, American Association of Colleges of Nursing, American Organization of Nurse Executives, and Wolters Kluwer Health. No funding was provided for this study beyond the $1000 used for the purchase of data.The correction will appear in the online version of the paper on the JMIR website on April 29, 2021, together with the publication of this correction notice. Because this was made after submission to PubMed, PubMed Central, and other full-text repositories, the corrected article has also been resubmitted to those repositories."} +{"text": "On January 6\u20137, 2022, computer-based testing (CBT) was adopted for the Korean Medical Licensing Examination (KMLE) Fig. 1)Fig. 1). On March 21, 2003, presenters first proposed CBT and computerized adaptive testing (CAT) for the KMLE at a seminar held by the Institute of Medical Education of Hallym University at the Chuncheon Sejong Hotel. At that time, the staff members of the Korea Health Personnel Licensing Examination Institute (KHPLEI) attended the seminar. Thus, 19 years have passed since CBT and CAT were suggested for licensing examinations in Korea.In November 2011, the Standing Committee of the KMLE recommended introducing computerized testing to the KMLE, including CBT and CAT . I also CBT has already been a very common testing platform at most medical schools. For example, at Hallym University, CBT was introduced in 1999 for clinical course testing. In my parasitology class, CBT was already introduced in 1993. My students have had no difficulties in taking CBT. A new task after adopting CBT is to guarantee item quality, including that of audio-visual materials. CBT items should be more clinically oriented than paper-and-pencil materials to test examinees\u2019 clinical competency. Furthermore, the number of items to measure the examinee\u2019s latent traits should be re-considered. A correct distribution of core clinical content is required, and the balance between reliability and core clinical presentations should be guaranteed to ensure desirable item numbers. The standard setting for CBT, including the modified Angoff, modified Ebel, and Hofstee methods, should be implemented soon, instead of using a cut score to ensure a success rate of 60% ,8. It isTwo invited reviews were published last year: \u201cSample size determination and power analysis using the G*Power software\u201d by Kang and \u201cEduAlthough Dr. Kang is an anesthesiologist, he studied statistics after graduating from Korea National Open University with a major in Statistics. I invited him to serve as a statistical editor in 2021. I asked him to submit a review on determining the study size for authors and reviewers of the journal. When I read manuscripts submitted to the journal, the most challenging task is to check the appropriateness of study size estimation, especially since many manuscripts do not describe it. If estimation before the study was not possible, it is recommended to conduct a post-hoc analysis. Dr. Kang stated that \u201cthe null and alternative hypothesis, effect size, power, alpha, type I error, and type II error should be described when calculating the sample size or power\u201d . I hope The year 2021 was the year of the metaverse . AlthougJournal of Educational Evaluation for Health Professions (JEEHP): VR endotracheal intubation training [I did a PubMed search with the keyword \u201cmetaverse,\u201d and only 11 articles were found on January 8, 2022. However, this does not mean that the number of articles on metaverse is small. Before that, articles on the 4 types of the metaverse have been published frequently. These publications soared in 2021 and the training ; digitaltraining ; a simultraining , and VR training .According to the usage trends of the term \u201cmetaverse,\u201d a review article was invited as an abridged English translation of the issue report by the Korea Education and Research Information Service . Dr. BokIn 2021, JEEHP received its first Journal Citation Indicator (JCI) value, 0.51, in June 2021. This score means that the citation impact of articles from 2017 to 2019 was about half of the average citation impact of Web of Science Core Collection journals. JEEHP\u2019s JCI ranking in the scientific education category was 9th out of 35 Emerging Sources Citation Index (ESCI) journals (74.3%) and 47th out of 78 SCIE (Science Citation Index Expanded) and ESCI journals (39.8%) [https://www.scopus.com/sourceid/21100467423. This value is higher than that in 2020 (1.7)Bibliometric statistics for the author\u2019s country and total cites are presented in I should be cautious when selecting manuscripts for review. The number of rejected or withdrawn manuscripts after review was 16 in 2020 and 10 in 2021. The number of accepted manuscripts after review was 26 out of 43 (70.3%) in 2021. In 2022, the editorial office will do its best to select acceptable manuscripts for review to save reviewers\u2019 time.I am deeply indebted to the reviewers who voluntarily devoted themselves to reviewing manuscripts. Their role is essential for maintaining the journal quality. I regret that I sent them some manuscripts that were finally rejected. Below are the reviewers\u2019 names and affiliations by country.Australia: Boaz Shulruf, University of New South Wales; Elio Stefan Arruzza, University of South AustraliaChile: Castillo Ni\u00f1o Manuel, University of ChileIndonesia: Armyanti Ita, Tanjungpura University; Romiko, Muhammadiyah University of PalembangIndia: Upreet Dhaliwal, University of Delhi; Kiran Goswami, All India Institute of Medical Sciences; T. S. Gugapriya, All India Institute of Medical Science, Manjiri Phansalkar, Pondicherry Institute of Medical SciencesIsrael: Colin Block, The Hebrew University of JerusalemItaly: Colaceci Sofia, Saint Camillus International University of Health SciencesKorea: A Ra Cho, The Catholic University of Korea; Yera Hur, Hallym University; Junyong In, Dongguk University; Hyun Kang, Chung-Ang University; Jae Hyun Kim, Dankook University; Mi Kyoung Yim, Korea Health Personnel Licensing Examination Institute; Sun Kim, The Catholic University of Korea; Young Ju Kim, Ewha Womans University; Dong Kyu Lee, Dongguk University; Keumho Lee, Korea Institute for Research in the Behavioral Sciences; Younjae Oh, Hallym University; Janghee Park, Soonchunhyang University; Jeong Yun Park, University of Ulsan; Jungchul Park, Dankook University; Jungkyu Park, Kyungpook National University; Kyung Hye Park, Yonsei University; Song Yi Park, Dong-A University; Won Kyun Park, Keimyung University; Dong Gi Seo, Hallym University; Ji-Hyun Seo, Gyeongsang National University; Aeree Son, Samyook University; Sanghee Yeo, Kyungpook National University; Hyun Bae Yoon, Seoul National University; Dong-Mi Yoo, The Catholic University of KoreaMalaysia: Fata Nahas Abdulrahman, International Islamic University; Jamshid Shazia, Sultan Zainal Abidin UniversityMorocco: Hassouni Kenza, Mohammed VI University of Health Sciences; El Hajji Mohamed, Ibn Zohr UniversityPakistan: Rano Mal Piryani, Liaquat University of Medical and Health SciencesPortugal: Jos\u00e9 Miguel Padilha, Oporto Nursing SchoolSouth Africa: Richard Cooke, University of the WitwatersrandThailand: Poonpong Boonbrahm, Walailak University; Marisa Krairiksh, Khon Kaen University; Manoch Namfu, Rajamangala University of Technology LannaUnited Kingdom: Oliver Kemp, Gloucestershire NHS Foundation Trust; Marios Nicolaides, Queen Mary University of London; Cesar A. Orsini, Norwich Medical School, University of East Anglia; Emily Suckling, University Hospitals Bristol and Weston NHS Foundation TrustUnited States of America: Chad Cook, Duke University; Robert Cunningham, Maryville University; Mariana D\u2019amico, Nova Southeastern University; Jessica Fino, Midwestern State University; Sandra Groth, Midwestern State University; Junguk Hur, University of North Dakota; Myunghee Jun, University of Wisconsin-Green Bay; Hyekyung Kim, University of Wisconsin-Green Bay; Seock-Ho Kim, The University of Georgia; Amteshwar Singh, Johns Hopkins University; Jessyca Wagner, Midwestern State UniversityVietnam: Bach Xuan Tran, Ha Noi Medical UniversityTom Huh, a graduate student in the Division of Life Sciences, College of Life Sciences and Biotechnology, Korea University, Seoul, Korea, voluntarily made audio recordings of some abstracts.In the screening process of a submitted manuscript, the fitness of the manuscript in terms of both aims and scope and style and format will be meticulously checked before dispatching it for review. Reviewers or editors cannot determine originality; this determination is dependent on readers\u2019 experience. Therefore, the review has been focused on scientific integrity. A description according to reporting guidelines will be more strictly required. It should be demonstrated that the study design follows specific reporting guidelines. JEEHP is a non-commercial scholarly journal, so the number of published articles is not critical\u2014but scientific integrity is essential. The editorial team and I will do our best to maintain these policies in 2022 to save reviewers\u2019 time."} +{"text": "One of the assigned affiliations for the fifth author is incorrect. Dara Rahmati is not affiliated with #1, but with #5: Department of Computer Science and Engineering, Shahid Beheshti University, Tehran, Iran."} +{"text": "Following publication of the original article , it was The incorrect version was:Norwegian Institute of Public Health, Institute of Health and Society, University of Oslo, Oslo, Norway.The correct version is:Institute of Health and Society, University of Oslo, Oslo, Norway.The original article has been updated."} +{"text": "The authors wish to correct the following in this paper .1\u00a0Graduate Institute of Life Sciences, National Defense Medical Center, Taipei 114, Taiwan3\u00a0School of Public Health, National Defense Medical Center, Taipei 114, TaiwanThe fourth author, Ching-Huang Lai, should be affiliated with:The authors would like to apologize for any inconvenience caused to the readers by this change."} +{"text": "In the originally published article, affiliations 1 and 3 were presented incorrectly.Affiliation 1 was presented as \u201cDepartment of Cancer Epidemiology and Prevention, Henan Engineering Research Center of Cancer Prevention and Control, Henan International Joint Laboratory of Cancer Prevention, Henan Cancer Hospital, The Affiliated Cancer Hospital of Zhengzhou University, Zhengzhou, China\u201d; it should be \u201cDepartment of Cancer Epidemiology and Prevention, Henan Engineering Research Center of Cancer Prevention and Control, Henan International Joint Laboratory of Cancer Prevention, The Affiliated Cancer Hospital of Zhengzhou University, Henan Cancer Hospital, Zhengzhou, China\u201d.Affiliation 3 was presented as \u201cDepartment of Radiology, Henan Cancer Hospital, The Affiliated Cancer Hospital of Zhengzhou University, Zhengzhou, China\u201d; it should be \u201cDepartment of Radiology, The Affiliated Cancer Hospital of Zhengzhou University, Henan Cancer Hospital, Zhengzhou, China\u201d.The authors apologize for these errors and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."} +{"text": "In the published article, there was an error in affiliation 1. Instead of \u201cDepartment of Metabolism and Endocrinology, National Clinical Research Center for Metabolic Diseases, Metabolic Syndrome Research Center, The Second Xiangya Hospital of Central South University, Changsha, China\u201d, it should be \u201cNational Clinical Research Center for Metabolic Diseases, Metabolic Syndrome Research Center, and Department of Metabolism and Endocrinology, The Second Xiangya Hospital of Central South University, Changsha, China\u201d.The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated."} +{"text": "Instead of \u201c1 National Clinical Research Center for Mental Disorders, Changsha, China, 2 Department of Psychiatry, The Second Xiangya Hospital of Central South University, Changsha, China 3 Shanghai Mental Health Center, Shanghai Jiao Tong University School of Medicine, Shanghai, China, 4 Department of Psychiatry, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, China, 5 Department of Psychiatry, Hunan Brain Hospital , Changsha, China,\u201d it should be \u201c1 Department of Psychiatry, and National Clinical Research Center for Mental Disorders, The Second Xiangya Hospital of Central South University, Changsha, China, 2 Shanghai Mental Health Center, Shanghai Jiao Tong University School of Medicine, Shanghai, China, 3 Department of Psychiatry, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, China, 4 Department of Psychiatry, Hunan Brain Hospital , Changsha, China.\u201dIn the published article, there was an error in affiliation The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."} +{"text": "The correct affiliation is \u2018Department of Food, Nutrition, Dietetics and Health, Kansas State University, Manhattan, KS, USA\u2019.This has now been corrected in both the PDF and HTML versions of the Article."} +{"text": "An additional affiliation is missing for the sixth author. Danielle Daniels-Felix is also affiliated with School of Public Health, University of the Witwatersrand, Johannesburg, South Africa."} +{"text": "There is an error in affiliation 1 for authors Tengyang Wang and Guanghua Liu. The correct affiliation 1 is: Department of pediatrics, Fujian Provincial Maternity and Children\u2019s Hospital, Affiliated Hospital of Fujian Medical University, Fuzhou, Fujian, China"} +{"text": "Nature Communications 10.1038/s41467-021-23501-5, published online 21 June 2021Correction to: The original version of this Article contained an error in the author\u2019s affiliations.The affiliation of Zhimin Lu with the Department of Thoracic Surgery, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China, was inadvertently omitted.This has now been corrected in both the PDF and HTML versions of the Article."} +{"text": "We continue the practice of transparency of potential competing interests for us as editors of the Journal of Global Health, following the International Committee of Medical Journal Editors (ICMJE) Recommendations for the Conduct, Reporting, Editing, and Publication of Scholarly work in Medical Journals . As we eWe declare below the following conflicts of interest, in alphabetical order. We will regularly publish updated declarations when there is a change in editors\u2019 activities and relationships.Prof. Campbell is employed by the University of Edinburgh, where he holds a position as Professor in the Usher Institute, College of Medicine and Veterinary Medicine. He is also co-Director of the WHO Collaborating Centre in Population Health Research and Training and co-Director of the NIHR Global Respiratory Health Unit. He currently receives research funding from the European Commission (Innovative Medicines Initiative), WHO, UK NIHR and the Baszucki Brain Research Fund. He has received consultancy payments, paid via the University of Edinburgh, from WHO, the Bill and Melinda Gates Foundation, the UK Funding Councils [Research Excellence Framework] and Sanofi in the past 10 years.Prof. Campbell holds a position as the co-Editor in Chief. He occasionally reimburses expenses related to Journal\u2019s work, including travel and consumables. Prof. Campbell is a member of the International Society of Global Health (ISoGH).Numerous technical advisor appointments to WHO in the past 10 years.Prof. Maru\u0161i\u0107 is fully employed by the University of Split School of Medicine, where she holds a tenured position as Professor and Chair of the Department of Research in Biomedicine and Health. She receives funding support for research from the Croatian Science Foundation and the European Commission under Horizon research framewrok. She serves as an external expert for the European Commission.Prof. Maru\u0161i\u0107 holds a position as the Editor in Chief. She occasionally reimburses expenses related to Journal\u2019s work, including travel and consumables. Prof. Maru\u0161i\u0107 is a member of the International Society of Global Health (ISoGH).Honorary Professor, College of Medicine and Veterinary Medicine, University of Edinburgh, Edinburgh, Scotland, UK;Visiting Professor, School of Medicine, University of Sao Paulo, Sao Paulo, Brazil:Steering Group, EQUATOR Network;Co-Chair, Cochrane Scientific Committee;Research Coordinator, Cochrane Croatia;Council member, Committee on Publication Ethics (COPE);Advisory board or editorial board member for a number of medical journals.Prof. Rudan is employed by the University of Edinburgh, where he holds a position as Professor and Co-Director of the Centre for Global Health Research. He is also co-Director of the WHO Collaborating Centre in Population Health Research and Training.Prof. Rudan holds a position as the co-Editor in Chief. He occasionally reimburses expenses related to Journal\u2019s work, including travel and consumables. Prof. Rudan is a member of the International Society of Global Health (ISoGH), and serves as its President.Prof. Rudan has numerous technical advisor appointments to WHO, UNICEF and the World Bank."} +{"text": "In the published article, there was an error in affiliation 1. Instead of \u201cDepartment of Respiratory Medicine, Diagnosis and Treatment Center of Respiratory Disease, The Second Xiangtan Hospital of Central South University, Changsha, China,\u201d it should be \u201cDepartment of Respiratory Medicine, Diagnosis and Treatment Center of Respiratory Disease, The Second XiangYa Hospital of Central South University, Changsha, China.\u201dThe authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated."} +{"text": "Trond Nordfj\u00e6rn. In addition to the affiliation Department of Psychology, Norwegian University of Science and Technology (NTNU), 7491 Trondheim, Norway should be Department of Research and Development, Clinic of Substance Use and Addiction Medicine, St. Olavs University Hospital, 7006 Trondheim, Norway. The authors apologize for any inconvenience caused and state that the scientific conclusions are unaffected. The original article has been updated.In the published article , there w"} +{"text": "The authors regret that the affiliations for Professor F. Puglisi are incorrect. They should be:CRO Aviano National Cancer Institute IRCCS, Aviano, Italy and Department of Medicine (DAME), University of Udine, Udine, ItalyThe authors would like to apologise for any inconvenience caused."} +{"text": "In the published article, there was an error in affiliations 1,2,3,4, and 5. Instead of \u201c1 Mental Health Institute of the Second Xiangya Hospital, Central South University, Changsha, China, 2 China National Clinical Research Center on Mental Disorders, Changsha, China, 3 China National Technology Institute on Mental Disorders, Central South University, Changsha, China, 4 Hunan Key Laboratory of Psychiatry and Mental Health, Chinese Academy of Sciences, Changsha, China, 5 Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China,\u201d it should be \u201c1 National Clinical Research Center for Mental Disorders, and Department of Psychiatry, The Second Xiangya Hospital of Central South University; National Technology Institute on Mental Disorders; Hunan Key Laboratory of Psychiatry and Mental Health, Changsha, China, 2 Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.\u201dThe authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated."} +{"text": "The lecture will feature an address by the 2020 Pollack Award recipient, Karl Pillmer, PhD, FGSA of Cornell University. The 2021 Pollack Award recipient is Namkee G. Choi, PhD, FGSA, of the University of Texas at Austin. The Maxwell A. Pollack Award for Contributions to Healthy Aging Award recognizes instances of practice informed by research and analysis, research that has directly improved policy or practice, and distinction in bridging the worlds of research and practice."} +{"text": "The first author\u2019s affiliation was linked to the first and second affiliations in the published version, which is incorrect as she is only affiliated to \u2018The First Affiliated Hospital of Guanzhou Medical University, Guanzhou, China\u2019. The correct presentation is listed below.In an article1 | Zhaoyang Zeng2 | Yuao Deng3 | Weifeng Feng4 | Lun Huang1 | Huiling Liu1 | Jiazhi Lin1 | Chen Zhang5 | Yue Fan6 | Longyang Liu7Yingxia Ning1Department of Gynaecology and Obstetrics, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China2Department of Gynecology and Obstetrics, The Integrated Hospital of Traditional Chinese Medicine, Southern Medical University, Guangzhou, China3Department of Gynaecology and Obstetrics, Shenzhen People's Hospital, Shenzhen, China4The First Affiliated Hospital of Jinan University, Guangzhou, China5Department of Clinical Pharmacy, Guangzhou First People\u2019s Hospital, Guangzhou, China6Cancer Research Institute, Southern Medical University, Guangzhou, China7Cancer Center, Integrated Hospital of Traditional Chinese Medicine, Southern Medical University, Guangzhou, ChinaThe authors would like to insert additional text in the Acknowledgments section and it should read:ACKNOWLEDGEMENTSWe gratefully thank all of the authors for this study. This study was conducted both in \u201cthe First Affiliated Hospital of Guangzhou Medical University\u201d and \u201cIntegrated hospital of Traditional Chinese Medicine, Southern Medical University\u201d. Thanks for the help of Integrated Hospital of Traditional Chinese Medicine, Southern Medical University.The authors apologize for the errors."} +{"text": "Kunio Aoki, Professor Emeritus, Nagoya University School of Medicine, and Dr. Haruo Sugano, the former Director of the Cancer Institute, Tokyo, with 36 active epidemiologists throughout Japan.2-5Cancer prevention has been one of the major issues on health in Japan because 30% of total deaths are accounted for cancer.2-4Approximately 127,500 healthy inhabitants from 45 areas throughout Japan were enrolled into the study. Among this group, a total of 110,792 individuals aged 40-79 years, were followed up for a period of at least 10 years. Details of this cohort study was described elsewhere.5 besides original papers published from the JACC Study.Also, descriptive data for the JACC Study and validity studies were shown in the Journal of Epidemiology Supplement 1 in 2005,In 1996, 10 years after the establishment of this cohort study, Professor Yoshiyuki Ohno, Nagoya University School of Medicine, succeeded Professor Kunio Aoki as chairman of the JACC study group. Under the leadership of Prof. Ohno, the JACC study was greatly supported by Dr. Tomoyuki Kitagawa, Chairman of Grant-in-Aid for Scientific Research on Priority Area \u2018Cancer\u2019, for serum analysis in 1998-99. Since 2000, granting system of the study has been changed. Now this study has been supported by the Committee of Scientific Research on Priority Areas of Cancer under the Chairman of the Committee, Dr. Kazuo Tajima, and conducted under the leadership of Dr. Akiko Tamakoshi. The granting system of the study is going to be changed again in 2005. Taking this opportunity, in the present supplement, authors intend to review present status of cancer sites in Japan based on the data obtained from the JACC Study.We hope that this supplement 2 of 2005 also help you to obtain latest descriptive data for each specific cancer site with the supplement 1 of the Journal of Epidemiology.The present investigators involved, with the co-authorship of this paper, in the JACC Study and their affiliations are as follows: Dr. Akiko Tamakoshi (present chairman of the study group), Nagoya University Graduate School of Medicine; Dr. Mitsuru Mori, Sapporo Medical University School of Medicine; Dr. Yutaka Motohashi, Akita University School of Medicine; Dr. Ichiro Tsuji, Tohoku University Graduate School of Medicine; Dr. Yosikazu Nakamura, Jichi Medical School; Dr. Hiroyasu Iso, Institute of Community Medicine, University of Tsukuba; Dr. Haruo Mikami, Chiba Cancer Center; Dr. Yutaka Inaba, Juntendo University School of Medicine; Dr. Yoshiharu Hoshiyama, University of Human Arts and Sciences; Dr. Hiroshi Suzuki, Niigata University School of Medicine; Dr. Hiroyuki Shimizu, Gifu University School of Medicine; Dr. Hideaki Toyoshima, Nagoya University Graduate School of Medicine; Dr. Kenji Wakai, Aichi Cancer Center Research Institute; Dr. Shinkan Tokudome, Nagoya City University Graduate School of Medical Sciences; Dr. Yoshinori Ito, Fujita Health University School of Health Sciences; Dr. Shuji Hashimoto, Fujita Health University School of Medicine; Dr. Shogo Kikuchi, Aichi Medical University School of Medicine; Dr. Akio Koizumi, Graduate School of Medicine and Faculty of Medicine, Kyoto University; Dr. Takashi Kawamura, Kyoto University Center for Student Health; Dr. Yoshiyuki Watanabe, Kyoto Prefectural University of Medicine Graduate School of Medical Science; Dr. Tsuneharu Miki, Graduate School of Medical Science, Kyoto Prefectural University of Medicine; Dr. Chigusa Date, Faculty of Human Environmental Sciences, Mukogawa Women\u2019s University ; Dr. Kiyomi Sakata, Wakayama Medical University; Dr. Takayuki Nose, Tottori University Faculty of Medicine; Dr. Norihiko Hayakawa, Research Institute for Radiation Biology and Medicine, Hiroshima University; Dr. Takesumi Yoshimura, Fukuoka Institute of Health and Environmental Sciences; Dr. Akira Shibata, Kurume University School of Medicine; Dr. Naoyuki Okamoto, Kanagawa Cancer Center; Dr. Hideo Shio, Moriyama Municipal Hospital; Dr. Yoshiyuki Ohno, Asahi Rosai Hospital; Dr. Tomoyuki Kitagawa, Cancer Institute of the Japanese Foundation for Cancer Research; Dr. Toshio Kuroki, Gifu University; and Dr. Kazuo Tajima, Aichi Cancer Center Research Institute."} +{"text": "BJS Open, 2021, DOI: 10.1093/bjsopen/zraa071The following funder information was omitted from this article upon first publication but has now been added:This work was funded by the UK National Institute for Health Research (Grant RfPB PB-PG-0816-20005) and Allergan PLC. Neither funder was involved in the planning, methodology, analysis or write-up of the research. National Institute of Health Research, Room 132, Richmond House, 79 Whitehall, London, SW1A 2NS. Allergan Plc, Clonshaugh Business and Technology Park, Coolock, Dublin, D17 E400, Ireland.S.M. was supported by NIHR Birmingham Biomedical Research Centre at the University Hospitals Birmingham NHS Foundation Trust and the University of Birmingham during this work. S.H. is supported by the NIHR University College London Hospitals Biomedical Research Centre.This report presents independent research supported by the National Institute for Health Research (NIHR). The views and opinions expressed by authors in this publication are those of the authors and do not necessarily reflect those of the NHS, the NIHR, or the Department of Health."} +{"text": "In the published article, there was an error in affiliation 1. Instead of \u201cTongji Medical College, Wuhan Union Hospital, Huazhong University of Science and Technology, Wuhan, China,\u201d it should be \u201cDepartment of Neurology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.\u201dAdditionally, there was an error in affiliation 2. Instead of \u201cTongji Medical College, Huazhong University of Science and Technology, Wuhan, China,\u201d it should be \u201cDepartment of Pharmacology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.\u201dLastly, the affiliation superscript was missing for author Gang Li. This author should be affiliated to affiliation 1\u2014\u201cDepartment of Neurology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.\u201dThe authors apologize for these errors and state that they do not change the scientific conclusions of the article in any way. The original article has been updated."} +{"text": "The institution that the second author, Ursula Neumann, is affiliated with has updated their name. The correct affiliation is: Fraunhofer IIS, Fraunhofer Institute for Integrated Circuits IIS, Division Supply Chain Services, Group Data Science."} +{"text": "Instead of \u201c2Department of Clinical Medicine, Faculty of Dental Medicine and Health Osijek, Zagreb, Croatia; 3PhD Program Translational Research in Biomedicine\u2014TRIBE, School of Medicine, University of Split, Zagreb, Croatia.,\u201d it should be \u201c2Department of Clinical Medicine, Faculty of Dental Medicine and Health Osijek, Osijek, Croatia; 3PhD Program Translational Research in Biomedicine\u2014TRIBE, School of Medicine, University of Split, Split, Croatia.\u201dIn the published article, there was an error in The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated."} +{"text": "Instead of \u201cDivision of Life Science and Medicine, Department of Medical Oncology, The First Affiliated Hospital of USTC, University of Science and Technology of China, Hefei, China,\u201d it should be \u201cDepartment of Medical Oncology, The First Affiliated Hospital of USTC, Division of Life Science and Medicine, University of Science and Technology of China, Hefei, China.\u201dIn the published article, there was an error in affiliation The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated."} +{"text": "The affiliation for the eleventh author is incorrect. Sung Bae Lee is not affiliated with #7 but with #6: Department of Brain & Cognitive Sciences, Daegu Gyeongbuk Institute of Science and Technology, Daegu, Republic of Korea."} +{"text": "The Editor was Burkhard Rost, Columbia University, United States of America."} +{"text": "PLoS Biology, volume 3, issue 4, DOI: 10.1371/journal.pbio.0030127In The figure credits for Figures 1 and 2 are missing from the PDF versions of the article. For Figure 1, images are courtesy of Langdon Quetin and Robin Ross, researchers at the Marine Science Institute, University of California, Santa Barbara, and funded by the Office of Polar Programs, National Science Foundation. For Figure 2, images are courtesy of Donna Fraser."} +{"text": "In the Funding section, the grant numbers from the National Science Foundation of China are listed incorrectly. The correct grant numbers are: 41273122, 41472038, 51509093."} +{"text": "There are errors in the author affiliations. The affiliations should appear as shown here:7, Elena Iakhiaeva7Terry Smith7 University of Texas Health Science Center, Tyler, Texas, United States of America"} +{"text": "The following information is missing from the Funding section: This study was supported by research grants from the Natural Sciences and Engineering Research Council of Canada (NSERC) to E.M.B. and K.A.H., Conservation Funds from the Alberta Conservation Association to E.M.B., an operating grant from Environment Canada to K.A.H., a NSERC Postgraduate Scholarship, Queen Elizabeth II Graduate Scholarship (University of Alberta), grants from the Canadian Circumpolar Institute and the Northern Scientific Program (University of Alberta), and a Dissertation Fellowship (University of Alberta) to S.H. Funding in support of the Boreal MAPS in the Oil Sands Program was provided by Syncrude Canada Ltd., Hammerstone Corporation, Canadian Natural Resources Limited., Cenovus Energy, ConocoPhillips Canada, Devon Energy, Husky Oil Operations Ltd., Imperial Oil Ltd., Suncor Energy, and TOTAL E&P Canada.The following information is missing from the Competing Interests section: The authors received funding in support of the Boreal MAPS in the Oil Sands Program from Syncrude Canada Ltd., Hammerstone Corporation, Canadian Natural Resources Limited, Cenovus Energy, ConocoPhillips Canada, Devon Energy, Husky Oil Operations Ltd., Imperial Oil Ltd., Suncor Energy, and TOTAL E&P Canada, all commercial companies, for this study."} +{"text": "Scientific Reports7: Article number: 4358510.1038/srep43585; published online: 03082017; updated: 04102017In this Article, an affiliation has been omitted for Jilai Ding. The correct affiliations are listed below:Center for Nanophase Materials Science, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA.School of Materials Science and Engineering, Georgia Institute of Technology, Atlanta, GA 30332, USA.In addition, the Acknowledgements section in this Article is incomplete.\u201cResearch supported by Oak Ridge National Laboratory\u2019s (ORNL) Center for Nanophase Materials Sciences (CNMS), which is a U.S. Department of Energy (DOE), Office of Science User Facility , by the Division of Materials Sciences and Engineering, Office of Basic Energy Sciences, DOE (ARL) and by ORNL\u2019s Laboratory Directed Research and Development Program, which is managed by UT-Battelle LLC for the U.S. DOE (SJ)\u201d.should read:\u201cResearch supported by Oak Ridge National Laboratory\u2019s (ORNL) Center for Nanophase Materials Sciences (CNMS), which is a U.S. Department of Energy (DOE), Office of Science User Facility , by the Division of Materials Sciences and Engineering, Office of Basic Energy Sciences, DOE (ARL), by the ORNL GO! Fellowship program (JD) and by ORNL\u2019s Laboratory Directed Research and Development Program, which is managed by UT-Battelle LLC for the U.S. DOE (SJ)\u201d."} +{"text": "There is an error in affiliation 1 for author Ting Li. Affiliation 1 should be: School of Clinical Medicine, Southeast University, Nanjing, China. Additionally, Xianghua Huang and Zhihong Liu should also be affiliated with School of Clinical Medicine, Southeast University, Nanjing, China.The following information is missing from the Funding section: This work was supported by grants from the Jiangsu Provincial Medical Youth Talent (QNRC2016895), and Key Research and Development Plan Project of Jiangsu Province\u2014Social Development Projects (BE2017721). The publisher apologizes for this error."} +{"text": "There is an error in the fifth author\u2019s affiliation. The correct affiliation is: Department of Pediatrics, School of Medicine, College of Medicine, Taipei Medical University, Taipei City, Taiwan."} +{"text": "In the report \u201cCDC Grand Rounds: National Amyotrophic Lateral Sclerosis (ALS) Registry Impact, Challenges, and Future Directions,\u201d on page 1382, the list of author affiliations should have read as follows: 1Environmental Health and Surveillance Branch, Division of Toxicology and Human Health Sciences, Agency for Toxic Substances and Disease Registry, CDC; 2Cynthia Shaw Crispen Chair, ALS Research, Department of Neurology, Lexington, University of Kentucky; 3person living with ALS; 4Office of the Associate Director for Science, CDC; 5Office of the Associate Director for Communication, CDC."} +{"text": "The affiliation for author Hansaim Lim should read \u201cThe Ph.D. Program in Biochemistry, The Graduate Center, The City University of New York, New York, New York, United States\u201d and the affiliation for author Di He should read \u201cThe Ph.D. Program in Computer Science, The Graduate Center, The City University of New York, New York, New York, United States\u201d."} +{"text": "There was a mistake in the affiliation of the authors. Instead of \u201cState University, Saint Petersburg, Russia\u201d it should be \u201cSaint Petersburg State University, Saint Petersburg, Russia\u201d:1Laboratory of Neuromorphology, Pavlov Institute of Physiology RAS, Saint Petersburg, Russia2Laboratory of Neuroprosthetics, Institute of Translational Biomedicine, Saint Petersburg State University, Saint Petersburg, Russia3Laboratory of Motor Physiology, Pavlov Institute of Physiology RAS, Saint Petersburg, Russia4Laboratory of Neurophysiology and Experimental Neurorehabilitation, Children's Surgery and Orthopedic Clinic, Department of Non-pulmonary Tuberculosis, Research Institute of Phthysiopulmonology, Saint Petersburg, RussiaThis error does not change the scientific conclusions of the article in any way.The original article has been updated.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "One of the affiliations for the 4th author is not indicated. Aman Aggarwal is affiliated with National Centre for Biological Sciences, Tata Institute of Fundamental Research, GKVK Campus, Bangalore, India, and also with Manipal University, Manipal, India."} +{"text": "There is an error in the affiliation for all authors. The affiliation should be: Department of Radiology, Seoul National University Hospital, Seoul National University College of Medicine, Seoul, Korea."} +{"text": "Dr. Debbie van den Berg should be included in the author byline instead of the Acknowledgments. She should be listed as the fifteenth author, and her affiliation is: Neural Stem Cell Biology Laboratory, The Francis Crick Institute, London, United Kingdom. Her current address should be: Department of Cell Biology, Erasmus Medical Center, Rotterdam, The Netherlands. The contribution of this author is as follows: Investigation."} +{"text": "Scientific Reports7: Article number: 46220; 10.1038/srep46220 published online: 04122017; updated: 12222017.The original version of this Article contained an error in Affiliation 1 which was incorrectly listed as \u2018Department of Obstetrics and Gynaecology, Nagasaki University Graduate School of Medicine, Nagasaki, Japan\u2019. The correct affiliation is listed below:Department of Obstetrics and Gynaecology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan.These errors have now been corrected in the PDF and HTML versions of the Article, and in the accompanying Supplementary Information file."} +{"text": "Dr. Hongdong Chen is affiliated with both the Department of Endocrinology, Guang\u2019anmen Hospital of China, Academy of Chinese Medical Sciences and the Department of Endocrinology, Beijing Hepingli Hospital. Dr. Jing Guo is affiliated with the Department of Endocrinology, Guang\u2019anmen Hospital of China, Academy of Chinese Medical Sciences. Dr. Zhongchen He is affiliated with the Department of Endocrinology, Beijing Hepingli Hospital. Dr. Xiaolin Tong is affiliated with the Department of Endocrinology, Guang\u2019anmen Hospital of China, Academy of Chinese Medical Sciences.In the article, \u201cRetrospective analysis of the overt proteinuria diabetic kidney disease in the treatmentof modified Shenzhuo formula for 2 years\u201d,"} +{"text": "The affiliation for the last author is incorrect. Takanori Shibata is not affiliated with #6 but with #2 Showa University, School of Medicine, Division of Nephrology, Department of Medicine, Tokyo, Japan. The publisher apologizes for the error."} +{"text": "An affiliation for the last author is missing. Vincent C. Emery is also affiliated with Department of Microbial and Cellular Sciences, University of Surrey, Guildford, Surrey, United Kingdom."} +{"text": "The affiliation of the third author is incorrect. Mohamad Alaa Terkawi is not affiliated with #1 but with #2 National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, Japan.The affiliation of the fourteenth author is incorrect. Yoshifumi Nishikawa is not affiliated with #3 but with #2 National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, Japan.The affiliation of the fifteenth author is incorrect. Xuenan Xuan is not affiliated with #3 but with #2 National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, Japan."} +{"text": "Scientific Reports 10.1038/s41598-017-10450-7, published online 29 August 2017Correction to: The original version of this Article omitted an additional affiliation for the authors Mu Qiao, Honglin Liu and Shensheng Han. The correct affiliations for these authors are listed below:Key Laboratory for Quantum Optics and Center for Cold Atom Physics, Shanghai Institute of Optics and Fine Mechanics, Chinese Academy of Sciences, Shanghai, 201800.University of Chinese Academy of Sciences, Beijing, 100049, China.This has now been corrected in the HTML and PDF versions of this Article."} +{"text": "The affiliation for the fourth author is incorrect. Ermias Kebreab is not affiliated with #3 but with #4 The Department of Animal Sciences, University of California, Davis, Davis, California, United States of America."} +{"text": "Scientific Reports 10.1038/s41598-018-21451-5, published online 14 February 2018Correction to: The original version of this Article listed incorrect affiliations for Shuo Wang and Les Copeland. The correct affiliations are listed below:Shuo Wang: Research center of Food Science and Human Health, School of Medicine, Nankai University, Tianjin, 300071, China.Les Copeland: The University of Sydney, Sydney Institute of Agriculture, School of Life and Environmental Sciences, Sydney, NSW, 2006, Australia.In addition, the footnote of the PDF version of this article contained a typographical error in the spelling of the author Shuo Wang, which was incorrectly given as Shou Wang.These errors have now been corrected in the HTML and PDF versions of this Article."} +{"text": "Scientific Reports7: Article number: 4213310.1038/srep42133; published online: 02102017; updated: 06302017In the original version of this Article, there were errors in the affiliation which was incorrectly listed as \u2018Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, People\u2019s Republic of China\u2019. The correct affiliation is listed below:National Engineering Research Center of Biological Feed, Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, People\u2019s Republic of China.These errors have now been corrected in the PDF and HTML versions of the Article."} +{"text": "Zhao's affiliations were originally given incorrectly. The correct affiliations are as follows:School of Medicine and Life Sciences, University of Jinan-Shandong Academy of Medical Sciences, Jinan, China andDepartment of Radiation Oncology, Shandong Cancer Hospital and Institute, Shandong Academy of Medical Sciences, Jinan, China.In the article \u201cThe malignant transformation of retrorectal cystic hamartomas with blood irregular antibodies positive: A case report\u201d,All other affiliations appeared correctly in the original article."} +{"text": "Jun-Pyo MyongThe current affiliation,\u2019 Occupational Safety and Health Research Institute, Korea Occupational Safety and Health Agency\u2019 is incorrect.The corrected affiliation should be:Department of Occupational and Environmental Medicine, Seoul St. Mary\u2019s Hospital, College of Medicine, Catholic University of Korea, Seoul, Republic of KoreaAfter publication of the original article the auth"} +{"text": "There is an affiliation missing for the first author. Qian Zhang should also be affiliated with: University of Chinese Academy of Sciences, Beijing, China."} +{"text": "Jun-Pyo MyongThe current affiliation,\u2019 Occupational Safety and Health Research Institute, Korea Occupational Safety and Health Agency\u2019 is incorrect.The corrected affiliation should be:Department of Occupational and Environmental Medicine, Seoul St. Mary\u2019s Hospital, College of Medicine, Catholic University of Korea, Seoul, Republic of KoreaJong Heon ParkThe current affiliation,\u2019 Occupational Safety and Health Research Institute, Korea Occupational Safety and Health Agency\u2019 is incorrect.The corrected affiliation should be:Big Data Steering Department, National Health Insurance Service, Wonju, Republic of KoreaAfter publication of the original article the auth"} +{"text": "The second affiliation for the tenth author is not indicated. Jian-Ming Li is also affiliated with: Pathology Center and Department of Pathology, Soochow University, Suzhou, China."} +{"text": "Scientific Reports6: Article number: 2214710.1038/srep22147; published online: 02262016; updated: 11142016The original version of this Article contained errors in the affiliations.\u2018Institute of Basic Medical Sciences, National Cheng Kung University, Tainan, Taiwan\u2019was incomplete, and now reads:\u2018Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan, Taiwan\u2019This error has now been corrected in the HTML and PDF versions of this Article."} +{"text": "There is an error in affiliation 2 for authors Richard Tobin and Claire Grover. Affiliation 2 should be: Language Technology Group, The University of Edinburgh, Edinburgh, Scotland, United Kingdom. The publisher apologizes for the error."} +{"text": "The secondary affiliation for the fifth author is not indicated. Tiago Mestre is also affiliated with the Division of Neurology, Department of Medicine, The Ottawa Hospital Research Institute, University of Ottawa Brain and Mind Institute, Canada."} +{"text": "The affiliation for the fifth author is incorrect. Siaw-Chuing Loo is not affiliated with #3 but with #4 Centre of Building, Construction & Tropical Architecture, Faculty of Built Environment, University of Malaya, Kuala Lumpur, Malaysia. The publisher apologizes for the error."} +{"text": "The affiliation for the third author is incorrect. Yueh-Lun Lee is affiliated with: Department of Microbiology and Immunology, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan."} +{"text": "Scientific Reports6: Article number: 35205; 10.1038/srep35205 published online: 10172016; updated: 01192017.The original version of this Article contained a typographical error in Affiliation 1. The affiliation:Institute of Neurological Disease, Department of Anesthesiology and Translational Neuroscience Center, the state key laboratory of Biotherapy, West China Hospital, Sichuan University, Kunming 650500, China.now readsInstitute of Neurological Disease, Department of Anesthesiology and Translational Neuroscience Center, the state key laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu 610041, China.In addition the Acknowledgements section in this Article is incomplete.\u201cWe thank Dr. Zhi-cheng Xiao for discussion and critical comments, Dr. Visar for close editing of the manuscript.\u201dnow reads\u201cWe thank Dr. Zhi-cheng Xiao for discussion and critical comments, Dr. Visar for close editing of the manuscript. This research was supported by a Grant of National Science Foundation of China, No. 81271358, and a Grant of National Science Foundation of China, No. 81271358, and a Grant of National Science Foundation of China, No. 81471268. This study was also supported by the Program for IRTSTYN, together with the program Innovative Research Team in Science and Technology in Yunnan province\u201d.These errors have been corrected in the HTML and PDF versions of this Article."} +{"text": "After publication of the original article , the autIn the original version of the manuscript, the affiliation is listed as affiliation 4 from the Author details list:Psycho-oncology Co-operative Research Group (PoCoG), School of Psychology, Level 6, Chris O\u2019Brien Lifehouse (C39Z), The University of Sydney, Sydney, NSW 2006, Australia.However this is incorrect and the correct affiliation should be affiliation 1 from the Author details list:Clinical Research Unit for Anxiety and Depression (CRUfAD), UNSW School of Psychiatry at St Vincent\u2019s Hospital, Level 4, O\u2019Brien Centre, St Vincent\u2019s Hospital, 394 Victoria Street, Sydney, NSW 2010, Australia.This has been updated in the affiliations used for this Correction."} +{"text": "Zhao's affiliations were originally given incorrectly. The correct affiliations are as follows:School of Medicine and Life Sciences, University of Jinan-Shandong Academy of Medical Sciences, Jinan, China andDepartment of Radiation Oncology, Shandong Cancer Hospital and Institute, Shandong Academy of Medical Sciences, Jinan, China.In the article \u201cUrachal adenocarcinoma that metastasized to breast was misinterpreted as primary breast mucinous carcinoma: A rare case report and literature review\u201d,All other affiliations appeared correctly in the original article."} +{"text": "There is an error in the first author\u2019s affiliation. The correct affiliation is: Department of Pediatrics, School of Medicine, College of Medicine, Taipei Medical University, Taipei City, Taiwan."} +{"text": "The affiliation for the eleventh and twelfth authors is incorrect. Chen-Yong Lin and Michael D. Johnson are not affiliated with #10, but with #8, Lombardi Comprehensive Cancer Center, Department of Oncology Georgetown University Washington DC, United States of America. There is also an error in affiliation #6 for author Shun-Cheng Chang. Affiliation #6 should be Department of Surgery, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan."} +{"text": "There is an error in affiliation 3 for the author Mehdi Mojadam. Affiliation 3 should be: Public Health Department, School of Health, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran."} +{"text": "The affiliation for the fifth author is incorrect. Michel Kliot is affiliated with: Department of Neurosurgery, Stanford University School of Medicine, Stanford, California, United States of America."} +{"text": "There is an error in the current address for author Halil Bisgin. The current address should be: Department of Computer Science, Engineering and Physics, University of Michigan-Flint, Flint, Michigan, United States of America."} +{"text": "There is an error in the authors\u2019 affiliation. The affiliation should be: Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China."} +{"text": "Nature Communications8: Article number: 15870; DOI: 10.1038/ncomms15870 (2017); Published online 06192017; Updated 03302018In the original version of this Article, the affiliation details for Yan Huang and Li Zhang were incorrectly given as \u2018Department of Medical Oncology, Sun Yat-Sen University Cancer Center, Guangzhou 510060, China\u2019, and should have been \u2018State Key Laboratory of Oncology in South China, Department of Medical Oncology, Sun Yat-Sen University Cancer Center, Guangzhou 510060, China\u2019. This has now been corrected in both the PDF and HTML versions of the Article."} +{"text": "The affiliation for the seventh author is incorrect. Ihssan Masad is affiliated with: Department of Biomedical Systems and Informatics Engineering, Hijjawi Faculty for Engineering Technology, Yarmouk University, Irbid 21163, Jordan."} +{"text": "Dr. Yong Mao is affiliated with the Department of Epidemiology and Health Statistics, School of Public Health, Kunming Medical University, Kunming. Dr. Yang Xu is affiliated with the Department of Anesthesiology, Xiangyang No. 1 People's Hospital, Hubei University of Medicine, Xiangyang. Dr. Leihong Lu is affiliated with the Department of Dermatology, Linyi People's Hospital, Linyi, China.In the article \u201cThe nonlinear association between apolipoprotein B to apolipoprotein A1 ratio and type 2 diabetes\u201d,"} +{"text": "One affiliation for the second author is not indicated. Kostas Marias is also affiliated with: Department of Informatics Engineering, Technological Educational Institute of Crete, Heraklion, Greece."} +{"text": "An affiliation for the 3rd author is not indicated. Ibrahim Mohamed is also affiliated with: Soil and Water Department, Faculty of Agriculture, Benha University, Egypt."} +{"text": "The second affiliation for the third author is not indicated. Dingquan Zou is also affiliated with:Department of Anesthesiology, Second Xiangya Hospital, Central South University, Changsha, Huna, China."} +{"text": "The affiliations for the first four authors are incorrect. Nargis Bibi is not affiliated with #1 but with #2, Department of Computer Science, Fatima Jinnah Women University, Rawalpindi, Pakistan. Shabieh Farwa, Nazeer Muhammad, and Adnan Jahngir are not affiliated with #2 but with #1, Department of Mathematics, COMSATS Institute of Information Technology, Wah Cantt., Pakistan."} +{"text": "There is an error in affiliation 6 for author Ofer Amram. Affiliation 6 should be: Department of Nutrition and Exercise Physiology, Elson S. Floyd College of Medicine, Washington State University, USA."} +{"text": "Dear Editor,Costusspeciosus, Smilax menispermoidea, Trigonella foenum, species of Paris, Aletris, Trigonella, and Trillium, and many species of Dioscorea .Dioscorea tokoro Makino in 1935 . The bial., 1952) suggestal., 1952).In the pharmaceutical industry, diosgenin is the principal precursor compound in the manufacture of several synthetic steroidal drugs . DiosgeThis work was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries(IPET) through Advanced Production Technology Development Program, funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA) (116115-03-1-CG000). This research was supported by the Bio & Medical Technology Development Program of the National Research Foundation (NRF) funded by the Ministry of Science, ICT & Future Planning (2016M3A9A5919548).The authors declare no conflict of interest."} +{"text": "The affiliation for the last author is incorrect. Yun Kyu Oh is not affiliated with #5 but with #2 Department of Surgery, Seoul National University Boramae Medical Center, Seoul, Korea."} +{"text": "An affiliation for the 6th author is not indicated. Trude Helen Flo is affiliated with: Centre for Molecular Medicine Norway, Nordic EMBL Partnership, University of Oslo and Oslo University Hospital, Oslo, Norway."} +{"text": "After publication of the original article it was nDepartment of Breast and Thyroid Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Number 1277, Jiefang Road, Wuhan, Hubei Province, China\u201d.The address affiliation address listed currently reads \u201c\u201cDepartment of Breast and Thyroid Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China\u201d.At present the correct address is This error had since been acknowledged and corrected in this correction article. The authors also wish to apologize for any inconvenience caused."} +{"text": "Proceedings of the National Academy of Sciences of the United States of America.\u201dIn Volume 94, Issue 9, September 2016, page 639, the third paragraph should read: \u201cGlobal consumption of antimicrobials in food animals was estimated at 63 151 tons in 2010, of which the largest share, 23%, was in China, 13% in the United States of America, 9% in Brazil and 3% in India, according to Thomas Van Boeckel and colleagues\u2019 2015 study in"} +{"text": "The affiliation for the eighth author is incorrect. Mariana Beltran is not affiliated with #7 but with #8 Queen\u2019s Medical Research Institute, University of Edinburgh, Edinburgh, United Kingdom. The publisher apologizes for the error."} +{"text": "The authors world like to amend the following affiliation for Xiao Zhao and Runqiu Huang of paper :1 State Key Laboratory of Geohazard Prevention & Geoenvironment Protection, Chengdu University of Technology, Chengdu 610019, Sichuan, ChinaThe authors apologize for any inconvenience caused to the readers."} +{"text": "There is an error in affiliation 1 for authors Iyad Aqra, Tutut Herawan, and Norjihan Abdul Ghani. Affiliation 1 should be: Department of Information Systems, Faculty of Computer Science and Information Technology, University of Malaya, Kuala Lumpur, Malaysia.The following information is missing from the Funding section: This research is supported by University of Malaya Postgraduate Research Grant (PPP) no. vote PG106-2015B, and it is supported by Faculty of Computer Science & Information Technology University of Malaya."} +{"text": "There are errors in the Funding section. The correct funding information is as follows: This work was supported by the Biotechnology and Biological Sciences Research Council; and the Natural Environment Research Council [Grant number NE/M009106/1], by a Soils Training and Research Studentships (STARS) grant to JB-H. STARS is a consortium consisting of Bangor University, British Geological Survey, Centre for Ecology and Hydrology, Cranfield University, James Hutton Institute, Lancaster University, Rothamsted Research and the University of Nottingham. The James Hutton Institute receives funding from the Scottish Government."} +{"text": "The affiliation for the last author is incorrect. Kyung Seok Park is not affiliated with #1 but with #2 Department of Neurology, Seoul National University, College of Medicine, Seoul, Korea and #4 Department of Neurology, Seoul National University Bundang Hospital, Seongnam, Korea."} +{"text": "The following information is missing from the Funding section: The National Health and Medical Research Council and Department of Medicine, Faculty of Medicine, Dentistry and Health Sciences funded the researchers who worked on this analysis. The Australian Bureau of Statistics provided population based data used in comparison analysis and web-based data was deidentified and provided by Fleur Streets at Sisu Wellness. Funding for the Healthy Ageing Project (HAP) has been provided by the National Health and Medical Research Council , Ramaciotti Foundation, Australian Healthy Ageing Organisation, the Brain Foundation, the Alzheimer's Association (NIA320312), Australian Menopausal Society, Bayer Healthcare, Shepherd Foundation, Scobie and Claire Mackinnon Foundation, Collier Trust Fund, J.O. & J.R. Wicking Trust, Mason Foundation and the Alzheimer's Association of Australia."} +{"text": "The affiliation for the third author is incorrect. Maryam Marzban is not affiliated with #3 but with the Department of Public Health, School of Public Health, Bushehr University of Medical Science, Bushehr, Iran."} +{"text": "Scientific Reports 10.1038/s41598-017-06203-1 published online 19 July 2017Correction to: The original version of this Article contained an error in Affiliation 2, which was incorrectly given as \u2018University of the Chinese Academy of Sciences, Beijing, 100049, China\u2019. The correct affiliation is listed below:University of Chinese Academy of Sciences, Beijing, 100049, China.This error has now been corrected in the HTML and PDF versions of the Article."} +{"text": "The affiliation for the last author is incorrect. Zhiyun Jia is not affiliated with #2 but with #1 Department of Nuclear Medicine, West China Hospital, Sichuan University, Chengdu, Sichuan, People\u2019s Republic of China."} +{"text": "The second affiliation for author Pablo Rovira Kaltwasser is not indicated. Pablo Rovira Kaltwasser is also affiliated with: Dept. of Economic and Business, University of Leuven, Leuven, Belgium."} +{"text": "The affiliation for the first author is incorrect. Yueh-Lun Lee is affiliated with: Department of Microbiology and Immunology, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan."} +{"text": "The affiliation of the ninth author is incorrect. Dinant GeertJan is not affiliated with #4 but with #2 Maastricht University (Netherlands), Department of Family Medicine, Faculty of Health, Medicine and Life Sciences, Maastricht, The Netherlands."} +{"text": "Reason for Corrigendum:We forgot to add the State Key Laboratory for Biology of Plant Diseases and Insect Pests before the Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China.Correction:1State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China. 2College of Plant Protection, Southwest University, Chongqing, China.The affiliations of authors should be: The authors apologize for this error.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Incorrect Affiliation of first author:1 Beijing Anding Hospital of Capital Medical University, Beijing, China2 Department of Psychiatry, The Second Hospital of Shanxi Medical University, Taiyuan, Chinas for Feng Tian. As well as having Affiliation 1, they should also have Beijing Anding Hospital of Capital Medical University, Beijing, China. Meanwhile, Beijing Anding Hospital of Capital Medical University as the first affiliation is vital. Department of Psychiatry, The Second Hospital of Shanxi Medical University, Taiyuan, China must be the second affiliation.In the published article, there was an error regarding the affiliationThe authors apologize for this error and state that this does not change the scientific conclusions of the article in any way.The original article has been updated.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Dr. Larry D. Spears is not included in the author byline. He should be listed as the thirteenth author and affiliated with the Division of Endocrinology, Metabolism & Lipid Research, Washington University, 660 South Euclid Avenue, St. Louis, MO, 63110, United States of America. The contributions of this author are as follows: methodology and investigation.Dr. Clay F. Semenkovich is not included in the author byline. He should be listed as the fourteenth author and affiliated with the Division of Endocrinology, Metabolism & Lipid Research, Washington University, 660 South Euclid Avenue, St. Louis, MO, 63110, United States of America. The contributions of this author are as follows: conceptualization and supervision."} +{"text": "The affiliation for the eighth author is incorrect. Pei Liu is not affiliated with #2 but with #1 Department of Epidemiology and Biostatistics, School of Public Health, Southeast University, Nanjing, China."} +{"text": "The affiliation for the seventh author is incorrect. Yueh-Lun Lee Lee is affiliated with: Department of Microbiology and Immunology, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan."} +{"text": "There is an error in affiliation 3 for author Amishi Jha. Affiliation 3 should be: University of Miami, Coral Gables, Florida, United States of America. The publisher apologizes for the error."} +{"text": "An affiliation for the 11th author is not indicated. Jahson Alemu is also affiliated with Institute of Marine Affairs, Chaguaramas, Trinidad, Trinidad and Tobago."} +{"text": "There is an error in affiliation 1 for authors Hao Li and Kebin Zhang. Affiliation 1 should be:School of Soil and Water Conservation, Beijing Forestry University, Beijing, China."} +{"text": "The affiliation for the second author is incorrect. Chien-Liang Liu is not affiliated with #3 but with #2 Department of Emergency Medicine, China Medical University Hospital, Taichung, Taiwan."} +{"text": "The affiliation for the first author is incomplete. Qiong Gao is also affiliated with #2 Institute for Tropical Ecosystem Studies, University of Puerto Rico\u2014Rio Piedras, San Juan, Puerto Rico, United States of America."} +{"text": "There is an error in the Correction published on August 15, 2017. There is an error in affiliation 1. The publisher apologizes for the error. The correct text is:The second affiliation for the first author is missing. Yulong Duan is affiliated with #1 Key Laboratory of Extreme Environmental Microbial Resources and Engineering, Gansu Province, Northwest Institute of Eco-Environment and Resources, Chinese Academy of Sciences, Lanzhou, P.R.China and #5 University of Chinese Academy of Sciences, Beijing, P.R.China."} +{"text": "The affiliation for the thirteenth author is incorrect. Susan K. Dutcher is not affiliated with #6, and only with #7 Department of Genetics, Washington University School of Medicine, St. Louis, Missouri, United States of America. The publisher apologizes for the error."} +{"text": "The affiliation for the second author is incorrect. Rituraj Batth is not affiliated with #2 but with #1 Faculty of Life Sciences and Biotechnology, Plant Molecular Biology Laboratory, South Asian University, Akbar Bhawan, Chanakyapuri, New Delhi 110021, India. The publisher apologizes for the error."} +{"text": "The affiliation for the eighth author is incorrect. Alberto Bartelli is not affiliated with #3 but with #1 The CRUK Gene Function Laboratory, The Institute of Cancer Research, London, SW3 6JB, United Kingdom and #2 Breakthrough Breast Cancer Research Centre, The Institute of Cancer Research, London, SW3 6JB, United Kingdom."} +{"text": "There is an error in the authors\u2019 affiliation. The correct affiliation is: Department of Anesthesiology and Critical Care, Medical Center\u2013University of Freiburg, Faculty of Medicine, University of Freiburg, Germany."} +{"text": "Scientific Reports5: Article number: 1498510.1038/srep14985; published online: 10082015; updated: 11142016The original version of this Article contained errors in the affiliations.\u2018Department of Cell Biology and Anatomy, National Cheng Kung University, North District, Tainan City, Taiwan\u2019was incomplete, and now reads:\u2018Department of Cell Biology and Anatomy, College of Medicine, National Cheng Kung University, Tainan, Taiwan, ROC\u2019This error has now been corrected in the HTML and PDF versions of this Article."} +{"text": "Dr. Ruth Rodriguez should be marked as an equal contributor alongside Dr. Putcha and Dr. Yu.Dr. Ruth Rodriguez\u2019s correct affiliation is \u2018Department of Pathology, Icahn School of Medicine at Mount Sinai , New York, NY 10029-6574, USA\u2019 and not \u2018Department of Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA\u2019 as the original article states.After the publication of this article the auth"} +{"text": "Scientific Reports7: Article number: 4058710.1038/srep40587; published online: 01102017; updated: 02092017In the original version of this Article, Hongfu Zhang was incorrectly affiliated with \u2018Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100094, China\u2019.The correct affiliation is listed below:State Key Laboratory of Animal Nutrition, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100094, China.This error has now been corrected in the PDF and HTML versions of the Article."} +{"text": "The Sino-Austrian High-Tech Acupuncture Research Network was founded in 2005 and has been growing ever since. The network comprises many partners from China and is highly involved in research and education activities. This report introduces the network\u2019s activities in the year 2017. The High-Tech Acupuncture Network was founded in 2005 by Professor DDr. Gerhard Litscher from the Medical University of Graz Research Center Graz) and comprises many partners from China see .In 2017, the Sino-Austrian High-Tech Acupuncture Network has grown very fast. In the following, milestones from the year 2017 are listed chronologically and at the end of the report recent literature from the network is cited ,13,14,158 January 2017: World Health Organization (WHO): Professor Gerhard Litscher has been invited by the WHO in Geneva to be an official participant in the following two working groups:WHO Benchmark for Practice in AcupunctureWHO Benchmark for Practice in Tuina16 February 2017: Acupuncture Student Lecture, Professor Liang Fengxia, Graz, Austria.Professor Liang Fengxia, Associate Dean, Acupuncture and Moxibustion Orthopaedic College and Deputy Director, Acupuncture and Moxibustion Institute at the Hubei University of Chinese Medicine, performed a 3-hour student lecture at the Medical University of Graz , coordinator Dr. Andrea Pribyl).12 March 2017: Eurasia Pacific Uninet (EPU). Joint project with the Hubei University of Chinese Medicine, Wuhan, China and the Medical University of Graz, Graz, Austria.2 with a total construction of about 470,000 m2. The university has 15 departments, 17 specialties for the bachelor\u2019s degree and 9 specialties for professional training, 19 specialties for the master\u2019s degree and 12 specialties for the doctor degree, and more than 60 bases for clinical practice, including 6 affiliated hospitals, and 21 State-Level, Province-Level, or College-Level laboratories for teaching. At present, the university has about 15,000 students.Established in 1958, the Hubei University of Chinese Medicine has three secondary schools, four affiliated hospitals, four State-Level (highest level) Medical Research Centers, and 10 Research Institutions. The university occupies 0.65 kmAcupuncture is one of the key disciplines of research of this renowned university. The Medical University of Graz (Professor Gerhard Litscher) works in close cooperation with the Hubei University of Chinese Medicine (with Professor Wang Hua and Professor Liang Fengxia) on the topic of high-tech acupuncture research. Within the few last years, several joint SCI/PubMed-listed publications have been published. Professor Litscher from the Medical University of Graz is Visiting Professor at the Hubei University of Chinese Medicine and at the Hubei Provincial Collaborative Innovation Center of Preventive Treatment by Acupuncture and Moxibustion (Director: Professor Wang Hua). The cooperation is also supported by the Austrian Ministry of Science, Research, and Economy and by Eurasia Pacific Uninet.21 May 2017: Editor\u2019s Meet in Beijing: Medicines, Beijing, China (G. Litscher: Editor-in-chief of Medicines).21 May 2017: World Federation of Chinese Medicine Societies, Beijing, China. Professor G. Litscher: Executive Council Member (December 2016\u2013December 2020). Topic: Heart Rate Variability.22 May 2017: Sino-Austrian Project Meeting 2017. Sino-Austrian TCM Research on Lifestyle Related Diseases, Beijing, China.23 May 2017: Tianjin University of Traditional Chinese Medicine, Acupuncture Seminar, Tianjin, China.24 May 2017: Beijing Tongren Hospital affiliated to Capital Medical University (CMU), Beijing, China, Project Discussion: Department of Anesthesiology.24 May 2017: China Academy of Chinese Medical Sciences (CACMS), Beijing, China.25 May 2017: Peking University Health Science Center and Eurasia Pacific Uninet (EPU) PhD Interviews: Professors. D. Rausch and G. Litscher, Beijing, China.26 May 2017: Capital Medical University (CMU): Beijing Hospital of TCM, Beijing, China.9\u201310 June 1017: 12th International ISLA Congress, Beverungen, Germany.9 June 2017: Meeting with Representatives from WALT (World Association of Laser Therapy), Lauenf\u00f6rde, Germany.24\u201325 June 2017: Interdisciplinary Acupuncture Symposium V, Athens, Greece: Science focuses on Pain.26 June 2017: China Academy of Chinese Medical Sciences at the Medical University of Graz, Graz, Austria.Established in 1955, China Academy of Chinese Medical Sciences (CACMS) is China\u2019s largest comprehensive research institute combining scientific research, medical treatment, and teaching that is directly under the State Administration of TCM . It boasts various disciplines, advanced equipment, and great research strength and has under it 17 research institutes, six medical institutions, one graduate school, two branch schools, two pharmaceutical companies, and publishing houses of ancient books on Chinese medical science. Besides, it is a founder of 18 kinds of academic journals on Chinese medical science.What is especially worth mentioning is the achievement in artemisinin research, which has provided a powerful weapon for humans against malaria, saved hundreds of thousands of lives, and made tremendous contributions to human health. Thus, CACMS was awarded a Lasker Medical Research Award and in 2015 the Nobel Prize in Medicine.Acupuncture is one of the key disciplines of research of this renowned university. The Medical University of Graz works in close cooperation with China Academy of Chinese Medical Sciences on the topic of high-tech acupuncture research. Within the last 12 years, more than 65 joint SCI/PubMed-listed publications have been published with CACMS together. Professor Litscher from the Medical University of Graz is Visiting Professor at the Institute of Acupuncture and Moxibustion at CACMS. This appointment was renewed last year during the last visit of CACMS to the Medical University of Graz with President Professor Zhang Boli.2 July 2017: 10th Anniversary of the Traditional Chinese Medicine (TCM) Research Center Graz, Austria, Europe.From acupuncture to the many hundreds of different medical herbs: Traditional Chinese Medicine (TCM) is booming. In addition, it is effective: TCM has been practised successfully for more than 4000 years, and the Western demand for something to complement classical Western medicine has been increasing for years. Graz is going to play a central role in TCM research: the \u201cTCM Research Center Graz\u201d was founded in early March 2007 by Karl-Franzens-University Graz and the Medical University Graz, and subsequently became a competence center that is unique worldwide.Dealing with acupuncture and Chinese medical herbs has a long tradition in Graz: Professor Rudolf Bauer, Head of the Institute of Pharmaceutical Sciences at Karl-Franzens-University, has been researching the active pharmaceutical ingredients and quality of Chinese medicinal herbs now for 26 years. Professor Gerhard Litscher, Head of the Research Unit of Biomedical Engineering in Anesthesia and Intensive Care Medicine at the Medical University of Graz, has dedicated himself to the research of acupuncture using the latest high-tech methods for 20 years. For both, scientific work is the basis for the modernization of TCM: \u201cTCM is a kind of medicine that can be evaluated scientifically\u201d, says Rudolf Bauer, \u201cit possesses comprehensible diagnostic methods and uses precise and controllable therapies\u201d. So, all research work is done based on scientific methods. Gerhard Litscher: \u201cWe are interested in basic research and those aspects of TCM that have not been given much attention so far; for example, the quantification of new acupuncture techniques, such as the painless laser needle acupuncture and electro acupuncture. Possible effects of acupuncture in combination with other methods are also subject to scientific research\u201d.A scientific report summarizes some of the research activities within the last 10 years and the chairmen of the center would like to use the opportunity to thank everyone for their generous support of the TCM Research Center Graz.8 August 2017: Lecture about High-Tech Acupuncture at Eu Yan Sang Intern. Ltd.\u2014Singapore, Republic of Singapore.10\u201312 August 2017: 9th International Symposium on Auriculotherapy, Singapore, Republic of Singapore. Report published in Medicines [11\u201315 September 2017: Acupuncture Congress: Germany, Timmendorfer Strand, Germany.Gerhard Litscher .23 September 2017: Project Meeting: China Academy of Chinese Medical Sciences, Beijing, China.25\u201327 September 2017: 4th Annual World Congress (BIT) of High-Tech Acupuncture and Integrative Medicine (HTA&IM): 2017, and 2nd Annual World Congress (BIT) of Modern Chinese Medicine: 2017, Xi\u2019an, China.Professor Gerhard Litscher, Chairman of the two World Congresses, Sheraton Hotel, Xi\u2019an, China, 400 participants at 4 congresses (opening ceremony), 110 participants at HTA&IM (57 speakers from 12 countries). Report published in Medicines [27 September 2017: Capital Medical University, Beijing Hospital of TCM, China.27 September 2017: People\u2019s Medical Publishing House, Beijing, China.28 September 2017: Editor\u2019s Meet in Beijing: Medicines (Pubmed-listed since September 2017), Beijing, China.28 September 2017: Beijing Tongren Hospital affiliated to Capital Medical University: Project Meeting Swissotel, Beijing, China, Project Discussion: Department of Anesthesiology and Department of Acupuncture and Moxibustion.17 October 2017: Project Cooperation: People\u2019s Liberation Army General Hospital, Beijing University of Chinese Medicine, and Medical University of Graz, Graz, Austria, Project Discussion.9\u201311 November 2017: 1st World Congress (BIT) of Biomedical Engineering: 2017, Xi\u2019an, China. Hilton, Xi\u2019an, China, 380 participants, Report published in Medicines [edicines .30 November 2017: Beijing Hospital of TCM affiliated to Capital Medical University (CMU), Beijing, China. Project Meeting (EPU-Project 05/2017).1 December 2017: Editorial Board Meeting in Beijing: Medicines (Pubmed-listed since September 2017), Beijing, China.2\u20134 December 2017: The 9th General Assembly of the World Federation of Acupuncture: Moxibustion Societies (WFAS) and WFAS Congress, Beijing, China.4 December 2017: The 5th Editorial Board of the World Journal of Acupuncture: Moxibustion. Beijing, China.5 December 2017: Cooperation between the Medical University of Graz (TCM Research Center) and Hubei University of Chinese Medicine, Wuhan, China.6 December 2017: Huazhong University of Science and Technology, Tongji Medical College, School of Nursing, Wuhan, China."} +{"text": "There is an error in the affiliation for Yongmin Li, Jingwen Li, He Liu, Yanlong Liu, and Binbin Cui. The affiliation should be: Department of Colorectal Surgery, Harbin Medical University Cancer Hospital, Harbin, China.There are omissions in the Funding section. The correct funding information is as follows:This work was supported by the National Natural Science Foundation of China , the Science&Technology Bureau of Harbin (No. 2014RFQGJ and 2015RAXYJ063) and Research Fund for the Doctoral Program of Higher Education , Heilongjiang postdoctoral fund (No. LBH-Z12155) and Harbin Municipal Science and Technology Committee of Harbin outstanding academic leaders plan (No. 2015RAXYJ063). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."} +{"text": "Correction to:Cell Discovery (2017) 3, 17006; doi:10.1038/celldisc.2017.6; published online 21 March 2017In the initial published version of this article, a mistake was made in the affiliation 1, where \u2018Sun Yat-sen University\u2019 should be added after the School of Life Sciences. The corrected affiliation 1 is displayed below. This addition does not affect the results, figures and conclusion of the paper.1Key Laboratory of Gene Engineering of the Ministry of Education, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, China;"} +{"text": "They should have appeared as:In the article, \u201cLow serum levels of uric acid and albumin in patients with GuillainBarre syndrome\u201d,a Department of Neurology, The First Affiliated Hospital of Wenzhou Medical University, Wenzhoud Department of Endocrinology, The First Affiliated Hospital of Wenzhou Medical University, Wenzhoug Department of Infection and Liver Diseases, Liver Research Center, The First Affiliated Hospital of Wenzhou Medical University, Wenzhouh Department of Cardiovascular Medicine, The Heart Center, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou"} +{"text": "One of the affiliations for the sixth and seventh authors is not indicated. Ki-Woong Kim and In-Young Yoon are both affiliated with: Department of Psychiatry, Seoul National University College of Medicine, Seoul, South Korea."} +{"text": "There is an error in affiliation 6 for author Ranganatha Sitaram. Affiliation 6 should be: Institute for Biological and Medical Engineering, and Department of Psychiatry and Division of Neuroscience, Schools of Engineering, Biology & Medicine, Pontificia Universidad Cat\u00f3lica de Chile.Ranganatha Sitaram is not affiliated with #1, #2, or #3 but only with #6: Institute for Biological and Medical Engineering, and Department of Psychiatry and Division of Neuroscience, Schools of Engineering, Biology & Medicine, Pontificia Universidad Cat\u00f3lica de Chile."} +{"text": "K. Rahul Reddy and Tushar Mokashi are not included in the author byline.K. Rahul Reddy should be the seventh author and affiliated with Healthcare Financing Division, National Health Systems Resource Centre, Ministry of Health and Family Welfare, Government of India. The contributions of the author are as follows: conceived and designed the experimentsTushar Mokashi should be the eighth author and affiliated with Healthcare Financing Division, National Health Systems Resource Centre, Ministry of Health and Family Welfare, Government of India. The contributions of the author are as follows: conceived and designed the experimentsThe following information is missing from the Methodology section: The methodology was adopted from measurement of district level health financing indicators for Universal Health Coverage tool kit designed by National Health System Resource Center, New Delhi . The reference is: Sundararaman T, Vidyanathan G, Vaishnavi SD, Reddy KR, Mokashi T, Sharma J et al. Measuring Progress towards Universal Health Coverage\u2014An Approach in the Indian Context. Econ Polit Wkly. 2014 Nov 22; XLIX(47):60\u201365Some information is omitted from the Acknowledgements section. The Acknowledgements should read: We are grateful to National Rural Health Mission Punjab for providing financial support for conducting this survey, and National Health System Resource Center, New Delhi for supporting us with research design, sampling, providing the questionnaire and training of investigators."} +{"text": "Scientific Reports6: Article number: 2409610.1038/srep24096; published online: 06142016; updated: 11022016.In this article, the Acknowledgements section is incomplete:\u2018The authors appreciate the help of Professor Arokia Nathan and Dr. Sungsik Lee in University of Cambridge and Professor Mutsumi Kimura in Department of Electronics and Informatics, Ryukoku University. This work was supported by Research Exchanges with India and China scheme, Royal Academy of Engineering, UK, National Natural Science Foundation of China (Grant No. 61106090), the Spring Project in Ningbo Institute of Material Technology and Engineering, the Ningbo Natural Science Foundation of China , and Youth Innovation Promotion Association, Chinese Academy of Sciences. Professor Mutsumi Kimura from Department of Electronics and Informatics, Ryukoku University is appreciated for his explanation of CV measurement and calculation for DOS\u2019.Should read:\u2018The authors appreciate the help of Professor Mutsumi Kimura in Department of Electronics and Informatics, Ryukoku University. This work was supported by the National Natural Science Foundation of China , Ningbo Municipal Natural Science Foundation (No. 2014A610011), the Ningbo Natural Science Foundation of China , the State Key Basic Research Program of China (2013CB922300), Youth Innovation Promotion Association, Chinese Academy of Sciences and Royal Academy of Engineering, UK. The author appreciates the K.C. Wong Magna Fund in Ningbo University, China\u2019."} +{"text": "Part of the affiliation for the authors Stuart Aitken and Colin A. Semple is missing. The corrected affiliation is as follows:1 MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, United Kingdom"} +{"text": "Affiliation 2 for author Chulyong Park is incomplete. Affiliation 2 should be: Department of Occupational and Environmental Medicine, Samsung Changwon Hospital, Sungkyunkwan University School of Medicine, Changwon, Korea."} +{"text": "The affiliation for the tenth author is incorrect. Kainne Dokubo is not affiliated with #2 but with #3 Division of Global HIV/AIDS, Center for Global Health, U.S. Centers for Disease Control & Prevention, Atlanta, Georgia, United States of America."} +{"text": "There are errors in the Funding section. The correct funding information is as follows: This work was funded by the National Nature Science Foundation of China , State Key Laboratory of Cryospheric Science (SKLCS-ZZ-2017), the Academy of Finland (decision number 268170).1 Laboratory of Green Chemistry, Lappeenranta University of Technology, Mikkeli, Finland and 3 Key Laboratory of Tibetan Environment Changes and Land Surface Processes, Institute of Tibetan Plateau Research, Chinese Academy of Sciences, Beijing, China. Chaoliu Li is affiliated with both 3 Key Laboratory of Tibetan Environment Changes and Land Surface Processes, Institute of Tibetan Plateau Research, Chinese Academy of Sciences, Beijing, China and 5 CAS Center for Excellence in Tibetan Plateau Earth Sciences, Chinese Academy of Sciences, Beijing, China.Additional affiliations for the first and third authors are missing. Bin Qu is affiliated with both"} +{"text": "The author affiliation of Rosario Rizzuto mentioned in the final version is:2 Venetian Institute of Molecular Medicine, Padova, Italy.This is incorrect and should instead be:3 Department of Biomedical Sciences, University of Padova, Padova, Italy.We apologize for the error."} +{"text": "After publication of the original article , it cameFirstly, the affiliation of Meta Roestenberg was originally given as 1\u2014Department of Medical Microbiology and Radboud Center for Infectious Diseases, Radboud University Medical Center. In fact Meta Roestenberg is affiliated to 2\u2014Department of Infectious Diseases, Leiden University Medical Center.Secondly, the name of a department and two individuals should have been added to the Acknowledgements section. Eric Brienen, Lisette van Lieshout and the Department of Parasitology of the LUMC should have been included. The Acknowledgements section therefore read as follows:\u2018We would like to thank Geert-Jan van Gemert, Marga van de Vegte-Bolmer, Rianne Siebelink-Stoter, Wouter Graumans and Theo Arens for their support of the controlled human malaria infection studies at the Radboud University Medical Center. We thank all the clinical staff who performed the controlled human malaria infection trials, Matthew McCall, An-Emmie Nieman, Else Bijker, Guido Bastiaens, Maurits van Meer, Linda Wammes, Andre van der Ven, Quirijn de Mast, Perry van Genderen, Jaap van Hellemond, Jorien Wiersma, Eric Brienen, Lisette van Lieshout and the entire staff of the Clinical Research Center Nijmegen and the Departments of Medical Microbiology and Parasitology of the LUMC. We thank Anja Scholzen for her advice and input in the data analysis.\u2019"} +{"text": "Scientific Reports6: Article number: 2833810.1038/srep28338; published online: 06202016; updated: 05092017In the original version of this Article, Wenhui Yang was incorrectly listed as being affiliated with \u2018Key Laboratory of Risk Assessment and Control for Environment and Food Safety, Tianjin Institute of Health and Environmental Medicine, Tianjin, 300050, China\u2019. The correct affiliation is listed below:State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, 100071, China.Enterobacter aerogenes phage. The correct In addition, Table 1 previously contained the results of the host range of an incorrect These errors have now been corrected in the HTML and PDF versions of the Article."} +{"text": "Scientific Reports 10.1038/s41598-018-25091-7, published online 27 April 2018Correction to: In the original version of this Article, there were errors in Affiliation 2 which was incorrectly listed as \u2018Graduate School of Life Science, National Defense Medical Center, Taipei, 114, Taiwan\u2019. The correct affiliation is listed below:\u2018Graduate Institute of Life Science, National Defense Medical Center, Taipei 114, Taiwan.\u2019These errors have now been corrected in the PDF and HTML versions of the Article, and in the accompanying Supplementary Information file."} +{"text": "There is an affiliation missing for the fifth author. Marwa Eltoweissy is also affiliated with: Department of Zoology, Faculty of Science, Alexandria University, Alexandria, Egypt."} +{"text": "Scientific Reports 10.1038/s41598-017-19060-9, published online 17 January 2018Correction to: In the original version of this Article, there were errors in Affiliation 2 which was incorrectly listed as \u2018Institute of Infectious Disease and Molecular Medicine (IDM), Division of Immunology, University of Cape Town, Cape Town, South Africa.\u2019 The correct affiliation is listed below:Institute of Infectious Disease and Molecular Medicine (IDM), Division of Immunology, University of Cape Town and South African Medical Research Council (SAMRC), Cape Town, South Africa.In addition, the author Natalie Eva Nieuwenhuizen was incorrectly indexed.These errors have now been corrected in the PDF and HTML versions of the Article."} +{"text": "The equal contributions of the last six authors are displayed incorrectly. The equal contributions for these authors should be stated as follows: \u201cEB, BMP, SS, JMS, VG and CMvD contributed equally to this work.\u201d The publisher apologizes for the error.rd author have been omitted. Joshua M. Shulman is also affiliated with: Department of Neurology, Baylor College of Medicine, Houston, Texas, United States of America, and Department of Neuroscience, Baylor College of Medicine, Houston, Texas, United States of America.Two affiliations for the 53"} +{"text": "The affiliation for the eighth author is incorrect. Anna Weston is not affiliated with #3 but with #10 Electronics and Computer Science, University of Southampton, Southampton, Hampshire, UK."} +{"text": "The correct affiliation is: Department of Microbiology, Faculty of Science, Prince of Songkla University, Hat Yai, Thailand.The affiliation of the 14"} +{"text": "The affiliation for the first author is incomplete. Nahla Saeed AL-Wajeeh is affiliated with the following: Department of Biomedical Science, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia; Department of Molecular Medicine, AL-Gumhouri Teaching Hospital, Ministry of Public Health & Population, Sana'a, Yemen; Ministry of Higher Education & Scientific Research, Yemen.The following information is missing from the funding section: This study was supported grants from the University of Malaya ."} +{"text": "Feng's affiliation was originally given incorrectly. The correct affiliation is as follows:In the article \u201cRisk of anxiety and depressive disorders in patients with myocardial infarction: A nationwide population-based cohort study\u201d,Graduate Institute of Medical Sciences and School of Nursing, National Defense Medical Center, Taipei City, Taiwan, Republic of China"} +{"text": "The wrong Academic Editor name and affiliation was included on the original published article. The publisher apologizes for the error. The correct Academic Editor name and affiliation is: Karunesh Ganguly, University of California, San Francisco, UNITED STATES."} +{"text": "The affiliation for the third and seventh author is incorrect. Mohamed Abdou and Ola Zyaan are not affiliated with the Department of Entomology, University of Maryland, College Park, Maryland, United States of America.The correct affiliation is the Department of Entomology, Faculty of Science, Ain Shams University, Cairo, Egypt."} +{"text": "There is an error in affiliation 3 for author Soichiro Miura. Affiliation 3 should be: Department of Internal Medicine, National Defense Medical College, Tokorozawa, Saitama, Japan."} +{"text": "Harrison C. Spencer, Jr., MD, MPH, DTM&H, CPH, died on Wednesday, August 10, 2016, in Washington, DC. He is survived by his wife of 39 years, Christine M. Spencer, and two sons, Harrison (Trey) Spencer III, and Peter M. Spencer, daughter-in-law Katherine Spencer, and granddaughters Gigi and Maggie. He is also survived by two brothers and their wives, Fred and Jackie Spencer and Timothy and Ann Spencer. He was predeceased by his parents, Harrison C. Spencer, Sr., MD, and Dorothy (Stokes) Spencer and by a sister, Harriet Spencer Hoxton.At the time of his death, Dr. Spencer was president and chief executive officer of the Association of Schools and Programs of Public Health (ASPPH). He joined the Association in 2000 when it was known as the Association of Schools of Public Health (ASPH). He led the ASPH's transformation to ASPPH in 2013.Dr. Spencer was born at the Johns Hopkins Hospital in Baltimore on September 22, 1944. He lived in Baltimore until moving at the age of 6 to Abingdon, VA. After receiving his BS degree from Haverford College in 1965, Dr. Spencer received his MD degree from the Johns Hopkins University School of Medicine in 1969. Following an internship in medicine at Vanderbilt University Hospital in Nashville, he completed residencies in medicine and preventive medicine as a member of the U.S. Public Health Service in San Francisco and at the University of California, Berkeley, CA. While at Berkeley, he received an MPH degree from the School of Public Health. In 1972, he received a Diploma in Tropical Medicine and Hygiene from the London School of Hygiene and Tropical Medicine. After serving at the Centers for Disease Control and Prevention (CDC) in Atlanta, he returned to San Francisco to complete a residency in internal medicine at University of California, San Francisco. He subsequently became board certified in Internal Medicine and Preventive Medicine.Dr. Spencer was affiliated with the U.S. Public Health Service and the CDC from 1970 to 1991. His service included appointments as Epidemic Intelligence Service Officer, CDC Atlanta, GA (1972\u20131974); Medical Officer, Central America Research Station, CDC, San Salvador, El Salvador (1975\u20131977); Medical Officer, Bureau of Tropical Diseases, CDC, Atlanta (1977\u20131979); Senior Physician and Malaria Coordinator, Clinical Research Center, Kenya Medical Research Institute, and Senior Lecturer, Department of Community Medicine, University of Nairobi Medical School (1979\u20131984); Senior Medical Officer, World Health Organization, Geneva, Switzerland (1984\u20131987); and Chief, Parasitic Diseases Branch, Division of Parasitic Diseases, CDC, Atlanta (1987\u20131991).From 1991 to 1995, Dr. Spencer served as dean of the Tulane University School of Public Health and Tropical Medicine in New Orleans. He was a member of the American Society of Tropical Medicine and Hygiene, and served as a councilor from 1993 to 1995. He subsequently served as dean of the London School of Hygiene and Tropical Medicine, University of London from 1996 to 2000.At the time of his death, Dr. Spencer was an adjunct professor in the Department of International Health at the Johns Hopkins Bloomberg School of Public Health and in the Department of Epidemiology at the Milken Institute School of Public Health at George Washington University. Earlier in his career, he held senior academic appointments at the London School of Hygiene and Tropical Medicine, Tulane University School of Public Health and Tropical Medicine, Tulane University Medical School, and the Morehouse School of Medicine.Dr. Spencer was the author of dozens of scientific papers and book chapters published in leading peer-reviewed scientific journals and textbooks.A member of the National Academy of Medicine (formerly the Institute of Medicine), Dr. Spencer was also a founding fellow of the UK Academy of Medical Sciences, an honorary fellow of the UK Faculty of Public Health Medicine, a fellow of the American College of Physicians, a fellow of the American College of Preventive Medicine, and a fellow of the Royal Society of Tropical Medicine and Hygiene. He received the U.S. Public Health Service Commendation Medal in 1984 and 1991 and the Outstanding Service Medal in 1989.Throughout his long and distinguished career, Dr. Spencer served on dozens of committees and boards. At the time of his death, he was chair of the Interprofessional Education Collaborative and a member of the National Academy of Medicine's Leadership Consortium for a Value and Science-Driven Health System. He earlier had served as the chairman of the World Health Organization's Task Force on Malaria.In addition to his immediate family, Dr. Spencer leaves countless public health professionals, academic public health leaders, and association colleagues inspired by his intellect, passion, leadership, and compelling ethical values."} +{"text": "The second affiliation for the fourth author was incorrectly omitted. Eun Jin Jang is affiliated with both National Evidence-Based Healthcare Collaborating Agency, Seoul, Republic of Korea, and Department of Information Statistics, College of Natural Science, Andong National University, Andong, Republic of Korea."} +{"text": "The affiliation for the 13th author Alan D. Attie was incorrect. The correct affiliation is: Department of Biochemistry, University of Wisconsin, Madison, Wisconsin, United States of America"} +{"text": "Drs. Antonio Graziano and Antonio Giordano were not included in the author byline. Antonio Graziano should be listed as the sixth author and is affiliated with Dipartimento di Scienze Odontostomatologiche, Ortodontiche e Chirurgiche, Secondo Ateneo di Napoli, Napoli, Italy. His contributions were as follows: Performed the experiments.Antonio Giordano should be listed as the 11th author and is affiliated with Sbarro Institute for Cancer Research and Molecular Medicine, Center of Biotechnology, College of Science and Technology, Temple University, Philadelphia, Pennsylvania, United States of America, and Department of Human Pathology and Oncology, University of Siena, Siena, Italy. His contributions were as follows: Conceived and designed the experiments, contributed reagents/materials/analysis tools."} +{"text": "We examined the relation between coffee drinking and hepatocellular carcinoma (HCC) mortality in the Japan Collaborative Cohort Study for Evaluation of Cancer Risk (JACC Study). In total, 110\u2009688 cohort members aged 40\u201379 years were grouped by coffee intake into three categories: one or more cups per day, less than one cup per day and non-coffee drinkers. Cox proportional hazards model by SAS was used to obtain hazard ratio of HCC mortality for each coffee consumption categories. The hazard ratios were adjusted for age, gender, educational status, history of diabetes and liver diseases, smoking habits and alcohol. The hazard ratio of death due to HCC for drinkers of one and more cups of coffee per day, compared with non-coffee drinkers, was 0.50 , and the ratio for drinkers of less than one cup per day was 0.83 . Our data confirmed an inverse association between coffee consumption and HCC mortality. Hepatocellular carcinoma (HCC) has a high incidence in Africa and Asia where Hepatitis B virus (HBV) or Hepatitis C virus (HCV) infection are major risk factors . Heavy aCoffee drinking has been inversely related to the risk of liver cirrhosis in several studies aged 40\u201379 years from JACC Study, the design of which has been previously described . Subjects with HCC at baseline or died from HCC within 2 years after registration in the study were excluded from the analysis. Subjects coded C22.9 were also excluded from the analysis. The total number of subjects was 110\u2009688 . The subjects who died of HCC during the observation periods were 287 male and 114 female subjects.After obtaining the informed consent to participate the study, subjects were interviewed or completed questionnaire. A self-administered questionnaires for the survey included past and family history, health condition and lifestyle habits such as smoking, alcohol and non-alcohol beverages, diet, physical exercise, occupation and others. On the questionnaire, habitual coffee consumption was queried by the question\u2019 Do you drink coffee? \u2018The response sets was: \u2018almost everyday\u2019; \u20183\u20134 cups per week\u2019; \u20181\u20132 cups per week\u2019; \u20181\u20132cups per month\u2019; \u2018scarcely any\u2019. Those who answered \u2018almost everyday\u2019 were asked to report the number of cups of coffee per day. Study participants were grouped into three groups as follows: one or more cups per day, less than one cup per day and 0 cup per day (non coffee drinkers). Drinkers of less than one cup per day included those of \u20183\u20134 cups per week\u2019, \u20181\u20132 cups per week\u2019 and \u20181\u20132cups per month\u2019.Each data set was transformed into the format of the JACC Study standard questionnaire, submitted to central office. Integrated data were tested in distribution and logical accuracy by working group for data clean up. More detailed process was described elsewhere was used for the statistical analysis. To examine the association between the potential confounding factors and coffee consumption, we calculated the age-adjusted proportions and mean values for each factor at each coffee level. Cox proportional hazards model by SAS PHREG with strata statement (difference of collaborating institutes) was used to obtain hazard ratio (HR) of HCC mortality for each coffee consumption categories. Multiple logistic regression analysis was used to analyse the trends in adjusted means and proportion. This study was approved by the Ethics Committee of the Kurume University School of Medicine.No consistent association emerged between coffee consumption and the risk of HCC. Recently, In Japan, approximately 80% of HCC cases are associated with HCV . The mulThe present investigators involved, with the co-authorship of this paper, in the JACC Study and their affiliations are as follows: Dr Akiko Tamakoshi (present chairman of the study group), Nagoya University Graduate School of Medicine; Dr Mitsuru Mori, Sapporo Medical University School of Medicine; Dr Yutaka Motohashi, Akita University School of Medicine; Dr Ichiro Tsuji, Tohoku University Graduate School of Medicine; Dr Yosikazu Nakamura, Jichi Medical School; Dr Hiroyasu Iso, Institute of Community Medicine, University of Tsukuba; Dr Haruo Mikami, Chiba Cancer Center; Dr Yutaka Inaba, Juntendo University School of Medicine; Dr Yoshiharu Hoshiyama, University of Human Arts and Sciences; Dr Hiroshi Suzuki, Niigata University School of Medicine; Dr Hiroyuki Shimizu, Gifu University School of Medicine; Dr Hideaki Toyoshima, Nagoya University Graduate School of Medicine; Dr Kenji Wakai, Aichi Cancer Center Research Institute; Dr Shinkan Tokudome, Nagoya City University Graduate School of Medical Sciences; Dr Yoshinori Ito, Fujita Health University School of Health Sciences; Dr Shuji Hashimoto, Fujita Health University School of Medicine; Dr Shogo Kikuchi, Aichi Medical University School of Medicine; Dr Akio Koizumi, Graduate School of Medicine and Faculty of Medicine, Kyoto University; Dr Takashi Kawamura, Kyoto University Center for Student Health; Dr Yoshiyuki Watanabe, Kyoto Prefectural University of Medicine Graduate School of Medical Science; Dr Tsuneharu Miki, Graduate School of Medical Science, Kyoto Prefectural University of Medicine; Dr Chigusa Date, Faculty of Human Environmental Sciences, Mukogawa Women's University; Dr Kiyomi Sakata, Wakayama Medical University; Dr Takayuki Nose, Tottori University Faculty of Medicine; Dr Norihiko Hayakawa, Research Institute for Radiation Biology and Medicine, Hiroshima University; Dr Takesumi Yoshimura, Fukuoka Institute of Health and Environmental Sciences; Dr Akira Shibata, Kurume University School of Medicine; Dr Naoyuki Okamoto, Kanagawa Cancer Center; Dr Hideo Shio, Moriyama Municipal Hospital; Dr Yoshiyuki Ohno, Asahi Rosai Hospital; Dr Tomoyuki Kitagawa, Cancer Institute of the Japanese Foundation for Cancer Research; Dr Toshio Kuroki, Gifu University; and Dr Kazuo Tajima, Aichi Cancer Center Research Institute."} +{"text": "There was an error in affiliation assignment for author Sylvia Knapp. This author should also be affiliated with #5, Department of Medicine 1, Division of Infectious Diseases and Tropical Medicine, Medical University Vienna, Vienna, Austria."} +{"text": "Lellean JeBailey should also be affiliated with: GeneGo Inc., St. Joseph, Michigan, United States of America. The Competing Interests statement should also be amended to read: LJ is an employee of GeneGo Inc."} +{"text": "There is an error in the authors' affiliation. The Affiliation should be: NASA Ames Research Center, Moffett Field, California, United States of America"} +{"text": "The affiliation for the fourth author was incorrect. Marina Tiemi Shio is not affiliated with #3 but with #1 The Research Institute of the McGill University Health Centre, Centre for the Study of Host Resistance, Departments of Medicine, Microbiology and Immunology, McGill University, Montr\u00e9al, Canada."} +{"text": "The sixth author's affiliation was incorrect. Surya P. Manandhar is affiliated with #3: University of Georgia, Department of Biochemistry and Molecular Biology, Athens, Georgia, United States of America."} +{"text": "The authors wish to note the work of an additional author, Naomi Sengamalay, whose name was not published previously. Dr. Sengamalay is the 7th author on this paper, and was affiliated with affiliation number 3: The J. Craig Venter Institute, Rockville, Maryland, United States of America.This author has a current address of: The Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, Maryland, United States of America.The contributions of this author are as follows: Performed the experiments."} +{"text": "Author affiliation 1 is incorrect. The correct affiliation is: Key Laboratory of Systems Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China"} +{"text": "The affiliation for 65th author Johanna Kuusisto and 115th author Markku Laakso was incorrect. The correct affiliation is: Department of Medicine, University of Kuopio and Kuopio University Hospital, Kuopio, Finland."} +{"text": "An affiliation for the fourth author was not indicated. Affiliation #1 for Luonan Chen should be: Key Laboratory of Systems Biology, SIBS-Novo Nordisk Translational Research Centre for PreDiabetes, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China. Affiliations #3 and #4 are his second and third affiliation, respectively."} +{"text": "An author was omitted from the published paper. Emma Hall should be listed as second author, affiliated with: MRC Human Genetics Unit and Institute of Genetics and Molecular Medicine, Western General Hospital, Edinburgh, United Kingdom. Ms. Hall's contribution to the paper was: contributed and analysed data."} +{"text": "Richard Franka was not included in the author byline. He should be listed as the ninth author, and his affiliation is 7: WHO Collaborating Centre for Reference and Research on Rabies, Rabies Section, Division of Viral and Rickettsial Diseases, Viral and Rickettsial Zoonoses Branch, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America. The contributions of this author are as follows: Performed the experiments, analyzed the data, contributed reagents/materials/analysis tools."} +{"text": "Dr. Prakash Srinivasan was not included in the author byline. He should be listed as the ninth author and affiliated with Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland, United States of America. The contributions of this author are as follows: Developed pbCAP380 antibody."} +{"text": "The affiliation for the third author Alberto Inga was incorrect. Dr. Inga is only affiliated with institution number 3, not number 1. The correct, complete affiliation is: Unit of Molecular Mutagenesis and DNA Repair, National Institute for Cancer Research, IST, Genoa, Italy"} +{"text": "There is an error in the first affiliation. It should appear:Institute for Human Evolution and the Bernard Price Institute for Palaeontology, School of GeoSciences, University of the Witwatersrand, Johannesburg, South Africa."} +{"text": "The affiliation of the first author was incorrect. Kenji Nishide should be affiliated not with #2 but with #3 Department of Biology, Graduate School of Science, Kobe University, Kobe, 657-8601, Japan."} +{"text": "The affiliation for the sixth author is incorrect. Jacopo Guccione is not affiliated with #4 but with #3: Department of Veterinary Medicine and Animal Productions, University of Napoli Federico II, Napoli, Italy."} +{"text": "The affiliation for the sixth author is incorrect. Michael Frachetti is not affiliated with #2 but with #3 Anthropology Department, Washington University in St. Louis, St. Louis, Missouri, United States of America"} +{"text": "There is an error in affiliation 1 for author Seungbum Koo. The correct affiliation 1 is: Department of Mechanical Engineering, Korea Advanced Institute of Science and Technology (KAIST), Daejon, Korea."} +{"text": "In \u201cA Virtual Counseling Application Using Artificial Intelligence for Communication Skills Training in Nursing Education: Development Study\u201d by Shorey et al :e14658), the affiliation of authors Shefaly Shorey and Emily Ang has been corrected from \u201cDepartment of Mechanical Engineering, National University of Singapore\u201d to \u201cAlice Lee Centre for Nursing Studies, National University of Singapore\u201d.The contact information for Shefaly Shorey has also changed from \u201cDepartment of Mechanical Engineering, National University of Singapore, Singapore, Singapore\u201d to \u201cAlice Lee Centre for Nursing Studies, National University of Singapore, Clinical Research Centre 10 Medical Drive, Singapore, 117597, Singapore\u201d.The correction will appear in the online version of the paper on the JMIR website on November 26, 2019, together with the publication of this correction notice. Because this was made after submission to PubMed, PubMed Central, and other full-text repositories, the corrected article has also been resubmitted to those repositories."} +{"text": "An additional affiliation for the first author is not indicated. Min Hyung Kim is also affiliated with: Department of Internal Medicine Yonsei University College of Medicine, Seoul, Korea."} +{"text": "There is an error in affiliation 1 for author Zhujia Yin. The correct affiliation 1 is: School of Economics and Management, Changsha University of Science & Technology, Changsha, China."} +{"text": "The sixth International Conference on Intelligent Biology and Medicine (ICIBM) took place in Los Angeles, California, USA on June 10\u201312, 2018. This conference featured eleven regular scientific sessions, four tutorials, one poster session, four keynote talks, and four eminent scholar talks. The scientific program covered a wide range of topics from bench to bedside, including 3D Genome Organization, reconstruction of large scale evolution of genomes and gene functions, artificial intelligence in biological and biomedical fields, and precision medicine. Both method development and application in genomic research continued to be a main component in the conference, including studies on genetic variants, regulation of transcription, genetic-epigenetic interaction at both single cell and tissue level and artificial intelligence. Here, we write a summary of the conference and also briefly introduce the four high quality papers selected to be published in BMC Genomics that cover novel methodology development or innovative data analysis. The 2018 International Conference on Intelligent Biology and Medicine (ICIBM 2018) was held from June 10th to 12th, 2018 in Los Angeles, California, USA. This is the sixth ICIBM conference and it became the first official conference of The International Association for Intelligent Biology and Medicine (IAIBM). Since its inception in 2012, ICIBM conference series aim to: 1) foster interdisciplinary and multidisciplinary research in bioinformatics, systems biology, intelligent computing/artificial intelligence, bioengineering, and data sciences, and 2) offer an educational program for trainees and young investigators in multiple scientific disciplines to learn or exchange the new methods/tools/discoveries in these areas and to build a professional network among both the established and junior investigators.We have expanded our ICIBM conference each year since 2012. The summary of the previous conferences is reported in previous introduction article . The ICIThe ICIBM 2018 scientific program includes eleven regular scientific sessions, four tutorials, one poster session, four keynote talks, and four eminent scholar talks. It covered a variety of the topics in bioinformatics, systems biology, machine learning, data sciences, and biomedical informatics.All of Us Research Program, which will make millions of individuals available with dense molecular and phenotypic data. (2) Paul D. Thomas, Ph.D., an Associate Professor in the Preventive Medicine Department, and who heads the Bioinformatics Division at the University of Southern California Keck School of Medicine. Dr. Thomas is the leader of the Gene Ontology project, which is among the world\u2019s largest bioinformatics projects. In Dr. Thomas presentation entitled \u201cReconstructing the large-scale evolution of genomes and gene functions\u201d, he gave an overview of the reconstruction methods and the related findings, introduced his work that reconstructed the evolutionary history of over 1 million genes in 15,000 gene families covering all domains of life, and also his work inferring human gene function from experiments in \u201cmodel organisms\u201d such as the fruit fly and yeast.Four distinguished scientists were invited to deliver the keynote speeches to the ICIBM attendees. These speakers were: (1) Joshua C. Denny, MD, MS, a Professor of Biomedical Informatics and Medicine, Director of the Center for Precision Medicine and Vice President of Personalized Medicine at Vanderbilt University Medical Center. He is a Fellow of the American College of Medical Informatics and a member of the National Academy of Medicine. He presented \u201cHuge cohorts, genomics, and clinical data to personalize medicine\u201d. In his talk, Dr. Denny demonstrated how electronic health records (EHRs) and genomic data can be combined for phenome-wide association studies, leading to personalized medicine. He also introduced the era of huge international cohorts such as the UK Biobank, Million Veteran Program, and the newly started In the third keynote speech, Dr. Alexander Hoffmann, a Professor of Microbiology, Immunology, and Molecular Genetics, and Director of the Institute for Quantitative and Computational Biosciences (QCB) at UCLA, demonstrated \u201cLearning how to predict immune responses\u201d. He discussed how molecular network dynamics and molecular noise affect immune cell function, and some of the modeling strategies that allow for prediction and insight. The fourth keynote speaker was Dr. Jason Moore, who holds the Edward Rose Professor of Informatics and Director of the Penn Institute for Biomedical Informatics. He also serves as Senior Associate Dean for Informatics and Chief of the Division of Informatics in the Department of Biostatistics, Epidemiology, and Informatics. Dr. Moore is a fellow of the American Association for the Advancement of Science (AAAS), the American College of Medical Informatics (ACMI), the American Statistical Association (ASA), and a Kavli fellow of the National Academy of Sciences. In his presentation, entitled \u201cAccessible artificial intelligence for data science\u201d, Dr. Moore introduced the history of artificial intelligence (AI) and then demonstrated the PennAI, an accessible, open-source, and user-friendly AI system at the University of Pennsylvania. PennAI brings AI and automated machine learning technology to everyone who wants to incorporate this technology into their big data analytics agenda.ICIBM 2018 also featured four eminent scholar talks. These talks were delivered by four renowned researchers in their specific fields. (1) Dr. Xinghua Lu, a professor in the Department of Biomedical Informatics, University of Pittsburgh, presented \u201cFrom big data to bedside (BD2B): Precision oncology in an era of artificial intelligence\u201d. (2) Ting Wang, Ph.D., an Associate Professor of Genetics, Computer Science and Engineering, and Biostatistics, and the Director of Computational and Systems Biology Program at Washington University School of Medicine, talked about \u201cExploring the dark matter in genomics data\u201d. Dr. Xinshu (Grace) Xiao, a Professor and Vice Chair of the Department of Integrative Biology and Physiology at the University of California, Los Angeles, gave the presentation entitled \u201cDeciphering the function of single-nucleotide variants in the RNA\u201d. Finally, Dr. Charles Wang, the founding Director of the Center for Genomics and a professor in the School of Medicine, Loma Linda University (LLU), gave the lecture focusing on \u201cVegetarian diet-modulated epigenetic reprogramming and longevity\u201d.As before, ICIBM offered tutorials to help trainees or other investigators learn the frontier informatics technologies. ICIBM 2018 provided four tutorials: Deep architectures are not necessary for anomaly classification of matrix-formed omics data , Single-cell sequencing analysis , Workshop on 3D genome organization , and WashU Epigenome Browser . All the tutorial lecturers are the experts in the related techniques or tools, or the developers of the tools.\u25e6 Session I: NGS & Tools I\u25e6 Session II: Systems Biology I\u25e6 Session III: Bioinformatics\u25e6 Session IV: NGS & Tools II\u25e6 Session V: Systems Biology II\u25e6 Session VI: Medical Informatics\u25e6 Session VII: Cancer Genomics I\u25e6 Session VIII: Systems Biology III\u25e6 Session IX: Computational Drug Discovery\u25e6 Session X: International PI Talk\u25e6 Session XI: Cancer Genomics IIICIBM 2018 included eleven concurrent scientific sessions and one poster session. Speakers in the regular sessions were chosen from those top ranked manuscripts after peer review. The topics in these session covered bioinformatics, genomics, systems biology, intelligent computing, data sciences, computational drug discovery, and biomedical informatics. To promote international collaboration, we first time organized an International PI talk session. Several principal investigators (PIs) from oversea presented their exciting projects in this session followed by extensive discussion. In addition, two best papers were selected and honored in the conference. The eleven session are:Since 2010, ICIBM meetings have been covering the latest developments in the entire pipeline of genomics research from low level data processing, to modeling, prediction and visualization, as well as to application to the real data sets. The tasks have grown from single feature to network analysis, and the scale of studies have grown from single data set to joint analysis of large scale cohorts or integration of data from multiple types and sources. The ICIBM 2018 reflects the trend to harvesting signal from increasing amount of data and keeping the development of computational methods to meet the needs of emerging biotechnology that presents new data types. Below, we summarize the contribution of ten papers included in this supplement issue.The paper by Du et al. exploredRNA-sequencing (RNA-seq) has now become a routine technique in genomic studies and its application has expanded beyond quantifying transcription activity. Mohammad et al. introducEmerging technologies that bring in new data types have become a constant phenomenon in the genomics era. The paper by Liu et al. presentsIn addition to technology advancement, innovative analysis using existing datasets continue to bring discoveries. Pei et al. investigWe would like to express our sincere gratitude to the members of the Steering, Program, Publication, Workshop/Tutorial, Publicity, Award, Trainee and Local Organization Committees, as well as to all the reviewers, volunteers and invited speakers, who spent their valuable time and effort on making ICIBM 2018 a success. The conference accomplishments are the results of support and hard work of all these people.The International Association for Intelligent Biology and Medicine (IAIBM), The University of Texas Health Science Center at Houston , National Science Foundation, UTHealth Center for Precision Health, and UTHealth Data Science and Informatics Core for Cancer Research.General Chair Zhongming Zhao .Steering Committee Yidong Chen , Kun Huang (Indiana University), Tony Hu (Drexel University), Lang Li (The Ohio State University), Yi Xing , Jiajie Zhang , Wenjin J. Zheng .Program Committee Chair: Kai Wang , Co-Chair: Degui Zhi . Members: Genevera Allen (Rice University), Jianlin Cheng (University of Missouri Columbia), Luca Giancardo , Assaf Gottlieb , Mike Guo (University of New Mexico), Yan Guo (University of New Mexico), Zhi Han (Indiana University School of Medicine), Matthew Hayes (Xavier University of Louisiana), Kun Huang (The Ohio State University), Weichun Huang , Yang Huang (Kaiser Permanente), Yufei Huang (University of Texas at San Antonio), Zhi-Linag Ji , Peilin Jia , Victor Jin (University of Texas at San Antonio), Yufang Jin (University of Texas at San Antonio), Jun Kong (Emory University), Dmitry Korkin (Worcester Polytechnic Institute), Rui Kuang (University of Minnesota Twin Cities), K.B. Kulasekera (University of Louisville), Qiwei Li (Rice University), Fuhai Li (The Ohio State University), Tao Li , Zhaohui Li , Aimin Li , Ping Liang , Liao Li (University of Delaware), Honghuang Lin (Boston University), Nan Liu , Yin Liu , Xiaoming Liu , Yunlong Liu (Indiana University), Zhandong Liu (Baylor College of Medicine), Zhiyong Lu , Mirjana Maletic (Baylor College of Medicine), Patricio A Manque , Huaiyu Mi , Nitish Mishra , Qiangxing Mo (Baylor College of Medicine), Tabrez Mohammad , Hatice Ozer (Ohio State University), Ranadip Pal (Texas Tech University), Jiang Qian (Johns Hopkins University), Guimin Qin (Xidian University), Thomas Rindflesch , Jianhua Ruan (The University of Texas at San Antonio), Bairong Shen , Xiaofeng Song , Fengzhu Sun , Wing-Kin Sung , Manabu Torii (Kaiser Permanente), Ying-Wooi Wan (Baylor College of Medicine), Jun Wan (Indiana University), Yufeng Wang (University of Texas at San Antonio), Junbai Wang , Qingguo Wang , Jiaying Wang , Chaochun Wei , Xiwei Wu , Yonghui Wu (University of Florida), Zhijin Wu (Brown University), Junfeng Xia , Lu Xie , Lei Xie (City University of New York), Hua Xu , Jianhua Xuan (Virginia Tech), Yu Xue , Zhenqing Ye , Sungroh Yoon , Feng Yue (Penn State University), Habil Zare (Texas State University), Rui Zhang (University of Minnesota), Shaojie Zhang , Han Zhang , Bing Zhang (Baylor College of Medicine), Zhongming Zhao , Min Zhao , Jim W. Zheng , Xianghong Zhou , Yunyun Zhou (University of Mississippi).Publication Committee Chair: Zhijin Wu (Brown University), Co-Chair: Jianhua Ruan (The University of Texas at San Antonio).Workshop/Tutorial Committee Chair: Feng Yue (Pennsylvania State University), Co-Chair: Yan Guo (University of New Mexico).Publicity Committee Chair: Lana Garmire (University of Hawaii), Co-Chair: Sun Kim , Co-Chair: Yu Xue .Award Committee Chair: Fuhai Li (Ohio State University), Co-Chair: Lei Xie (City University of New York).Trainee Committee Chair: Abolfazl Doostparast (Columbia University), Co-Chair: Qian Liu .Local Organization Committee Chair: Jessica Li , Co-Chair: Matteo Pellegrini , Co-Chair: Xiaoming Liu ."} +{"text": "The affiliation for the third author is incorrect. Heesoo Pyo is not affiliated with #3 but with #2 Molecular Recognition Research Center, Korea Institute of Science and Technology, Seoul, Republic of Korea."} +{"text": "There is an error in affiliation 5 for author Mehdi Hedayati. Affiliation 5 should be: Cellular and Molecular Endocrine Research Center, Research Institute for Endocrine Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran ."} +{"text": "There is an error in affiliation 1 for authors Qinquizi Yi and Naohiro Hohashi. Affiliation 1 should be: Division of Family Health Care Nursing, Graduate School of Health Sciences, Kobe University, Japan."} +{"text": "There is an error in affiliation 3 for author Ana Maria Trindade Gr\u00e9gio Hardy. The correct affiliation 3 is: Department of Physiology and Pharmacology, College of Medicine and Life Sciences, University of Toledo, Ohio, United States of America."} +{"text": "The creation of the Medical Library Center of New York (MLCNY) was a significant contribution to the history of health sciences librarianship as a model for cooperative, democratic, and practical solutions to the issues of storage and resource sharing. The MLCNY\u2019s founding director, Erich Meyerhoff, was a key figure in the successful start-up and ongoing operations of the center, which operated from 1960\u20132003 and served the greater New York area and beyond. This essay traces the evolution of the center including the creation of the Union Catalog of Medical Periodicals and the demise of the center occasioned by changes in scholarly publishing, technology, and constituent needs. New York City and its environs have a large number of major health sciences libraries, all located within a 40-mile radius: 7 medical schools, several major research institutes, and an extensive medical society library. In a 1967 article, McCormack estimated that in New York City, there were 7 medical schools; 156 hospitals ; 18,600 physicians ; and 10% of all internships. McCormack also estimated that 12.8% of all residencies in the United States were located in New York City some of his own social beliefs\u201d . In the Patricia E. Gallagher, AHIP, FMLA,patriciaegallagher@gmail.com, Librarian, National Information Center on Health Services Research and Health Care Technology, National Library of Medicine, Bethesda, MD"} +{"text": "The Vincent Cristofalo Rising Star Award in Aging Research lecture will feature an address by the 2018 recipient, Nathan K. LeBrasseur, PT, PhD, of the Robert and Arlene Kogod Center on Aging, titled \u201cBiomarkers of Senescent Cell Burden.\u201d The Irving S. Wright Award of Distinction Lecture will feature an address by the 2018 recipient Pinchas Cohen, MD, of the USC Leonard Davis School of Gerontology, titled \u201cMitochondrial System Biology as a Window Into Diseases of Aging.\u201d These awards are given by the American Federation for Aging Research, Inc."} +{"text": "There is an error in affiliation 3 for authors Faruq Mohammad and Hamad A. Al-lohedan. Affiliation 3 should be: Surfactant Research Chair, Department of Chemistry, College of Science, King Saud University, Riyadh, Kingdom of Saudi Arabia. The publisher apologizes for the error."} +{"text": "The affiliation for the eleventh author is incorrect. Lea Barfod is not affiliated with #6 but with #5 The Jenner Institute, University of Oxford, Oxford, United Kingdom."} +{"text": "In the published version of this article, author Guan Feng failed to include \u201cKey Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, China,\u201d as his first organization in affiliation section.The original text and the modified text are shown below:The original version:Ganglioside GM1 promotes contact inhibition of growth by regulating the localization of epidermal growth factor receptor from glycosphingolipid\u2010enriched microdomain to caveolae1, Guan Feng2,*Zhuo Dinghao1Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, China.2Provincial Key Laboratory of Biotechnology, Joint International Research Laboratory of Glycobiology and Medicinal Chemistry, College of Life Science, Northwest University, Xi'an, China.The modified version:Ganglioside GM1 promotes contact inhibition of growth by regulating the localization of epidermal growth factor receptor from glycosphingolipid\u2010enriched microdomain to caveolae1, Guan Feng1,2,*Zhuo Dinghao1Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, China.2Provincial Key Laboratory of Biotechnology, Joint International Research Laboratory of Glycobiology and Medicinal Chemistry, College of Life Science, Northwest University, Xi'an, China.The author would like to sincerely apologize for the inconvenience."} +{"text": "There is an error in affiliation 3 for author Haeyoung Lee. The correct affiliation 3 is: Red Cross College of Nursing, Chung-Ang University, Seoul, Republic of Korea."} +{"text": "One of the affiliations for the first author is not indicated. Xiaojian Ye is affiliated with: Department of Surgery, Shangrao First People's Hospital, Shangrao Jiangxi Province, China, and Department of General Surgery, The First Affiliated Hospital of Nanchang University, Yongwai Zhengjie, Nanchang, Jiangxi Province, China."} +{"text": "An additional affiliation is missing for the fourth author. Lin Ma is also affiliated with State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China."} +{"text": "The affiliation for the twelfth author is incorrect. Hongjie Yu is not affiliated with #4 but with #1 and #5:Division of Infectious Diseases, Key Laboratory of Surveillance and Early\u2013warning on Infectious Disease, Chinese Center for Disease Control and Prevention, Beijing, China.School of Public Health, Fudan University, Key Laboratory of Public Health Safety, Ministry of Education, Shanghai, China. The publisher apologize for the error."} +{"text": "The affiliations for the second and third authors are incorrect. Simon Dixon and Mark Sandler are not affiliated with #1 but with #2: School of Electronic Engineering and Computer Science, Queen Mary University of London, London, England."} +{"text": "The authors wish to make the following corrections to their paper : The order of the author Zheng-Jun Xie\u2019s affiliations was incorrect in our published paper in Sensors . Therefo1\u2003State Key Laboratory of Dairy Biotechnology, Shanghai Engineering Research Center of Dairy Biotechnology, Dairy Research Institute, Bright Dairy & Food Co., Ltd., Shanghai 200436, China to 1\u2003School of Food Science and Technology, Jiangnan University, Wuxi 214122, China The manuscript will be updated and the original will remain online on the article webpage.The authors would like to apologize for any inconvenience caused."} +{"text": "The affiliation for the second author is incorrect. The correct affiliation for Li Zeng is Department of Clinical Laboratory, The First Affiliated Hospital of Zhengzhou University, Zhengzhou City, China."} +{"text": "Scientific Reports 10.1038/s41598-017-02853-3, published online 06 June 2017Correction to: In the original version of this Article, there were errors in Affiliations 1 and 2 which were incorrectly listed \u2018The Key Laboratory of Aquatic Biodiversity and Conservation of Chinese Academy of Science, Institute of Hydrobiology, Chinese Academy of Science, Wuhan, Hubei, 430072, China\u2019 and \u2018Institute of Hydrobiology, University of Chinese Academy of Science, Beijing, 10001, China\u2019 respectively. The correct affiliations are listed below;Affiliation 1:The Key Laboratory of Aquatic Biodiversity and Conservation of Chinese Academy of Sciences, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei, 430072, ChinaAffiliation 2:University of Chinese Academy of Sciences, Beijing, 100049, ChinaThese errors have now been corrected in the PDF and HTML versions of the Article and in the accompanying Supplementary Information file."} +{"text": "In the article titled \u201cBilateral Proximal Tibia Stress Fractures through Persistent Physes\u201d , there wThe view(s) expressed herein are those of the author(s) and do not reflect the official policy or position of Brooke Army Medical Center, the U.S. Army Medical Department, the U.S. Army Office of the Surgeon General, the Department of the Army, the Department of the Air Force, or the Department of Defense or the U.S. Government."} +{"text": "Scientific Reports 10.1038/s41598-017-06386-7, published online 20 July 2017Correction to: The original version of this Article omitted an affiliation for Jung Hee Kim. The correct affiliations for Jung Hee Kim are listed below:Department of Internal Medicine, Seoul National University College of Medicine, Seoul, Republic of KoreaDepartment of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergence Science and Technology, Seoul National University, Seoul, Republic of Korea.This has now been corrected in the HTML and PDF versions of this Article."} +{"text": "The affiliation for the fifth author is incorrect; the correct affiliation is not indicated. Ernst Spaan is not affiliated with #3, but with: Radboud Institute for Health Science, Department for Health Evidence, Radboud University Medical Centre, Nijmegen, The Netherlands"} +{"text": "The second affiliation for the eleventh author is incorrect. Kazuhiko Nakamura is not affiliated with #4 but with #3 Department of Neuropsychiatry, Graduate School of Medicine, Hirosaki University, Hirosaki, Aomori, Japan."} +{"text": "Dr. Paul Munson should be included in the author byline. He should be listed as the seventh author, and his affiliation is 6: the Department of Microbiology, University of Washington, Seattle, Washington, United States of America. The contributions of the author are as follows: Data curation, formal analysis, methodology, and validation."} +{"text": "The affiliation of the fifth author is incorrect. Lucianne Cople Maia is not affiliated with #2 but with #3 Department of Pediatric Dentistry and Orthodontics, School of Dentistry, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil."} +{"text": "Dan Hu and Xiandong Lin. The correct affiliation is provided below. The authors declare that this correction does not change the results or conclusions of this paper. The authors sincerely apologize for this error.2Department of Pathology, Fujian Cancer Hospital and Fujian Medical University Cancer Hospital, Fuzhou, Fujian, China"} +{"text": "An additional affiliation is missing for the seventh author. Mariarosa Maiorana is also affiliated with Clinical and Experimental Medicine PhD Program, University of Modena and Reggio Emilia, Modena, Italy."} +{"text": "GBD 2017 Risk Factor Collaborators. Global, regional, and national comparative risk assessment of 84 behavioural, environmental and occupational, and metabolic risks or clusters of risks for 195 countries and territories, 1990\u20132017: a systematic analysis for the Global Burden of Disease Study 2017. Lancet 392: 1923\u201394\u20142018; The funding of this paper has been updated to \u201cBill & Melinda Gates Foundation and Bloomberg Philanthropies\u201d and the first sentence of the acknowledgments has been changed to \u201cResearch reported in this publication was supported by the Bill & Melinda Gates Foundation, Bloomberg Philanthropies, the University of Melbourne, Public Health England, the Norwegian Institute of Public Health, St Jude Children's Research Hospital, the National Institute on Ageing of the National Institutes of Health , and the National Institute of Mental Health of NIH (award R01MH110163)\u201d. These corrections have been made to the online version as of Jan 10, 2019."} +{"text": "The second affiliation for the fifth author is not indicated. Ehsan Oskoueian is affiliated with: Mashhad Branch, Agricultural Biotechnology Research Institute of Iran (ABRII), Agricultural Research, Education, and Extension Organization (AREEO), Mashhad, Iran."} +{"text": "There is an error in affiliation 2 for author Erica Hayes. Affiliation 2 should be: North Carolina State University Libraries, Raleigh, North Carolina, United States of America."} +{"text": "Myeong Soo Lee's affiliation should appear as Clinical Medicine Division, Korea Institute of Oriental Medicine, Daejeon, Republic of Korea.In the article, \u201cEunkyosan for treatment of the common cold: A protocol for the systematic review of controlled trials\u201d,"} +{"text": "Dr. Yinglong Chen is not included in the author byline. He should be listed as the seventh author and affiliated with State Key Laboratory of Soil Erosion and Dryland Farming on the Loess Plateau, Northwest A&F University, Chinese Academy of Sciences, Yangling, Shaanxi 712100, China, and the UWA Institute of Agriculture, & School of Agriculture and Environment, The University of Western Australia, LB 5005, Perth 6001, Australia. The contributions of this author are as follows: Contributed resources, supervision, and validation.There are errors in the Funding section. The correct funding information is as follows: This work was supported by National Natural Science Foundation of China (31471946) and Chinese Academy of Sciences ."} +{"text": "There is an error in affiliation 2 for author Theresa M. Rossouw. The correct affiliation 2 is: Department of Immunology, University of Pretoria, Pretoria, South Africa.The affiliation for the fourth author is incorrectly indicated as their current address. Martin Nieuwoudt is affiliated with: Institute for Biomedical Engineering (IBE), Mechanical and Mechatronic Engineering,Stellenbosch University, Western Cape, South Africa."} +{"text": "In the above article, there were errors in authors\u2019 affiliations. The correct affiliations are as shown in bold below.1Cancer Control Center, Osaka International Cancer Institute, Osaka, Japan2Department of Biostatistics, School of Public Health, the University of Tokyo, Tokyo, Japan3Department of Environmental Health, National Institute of Public Health, Saitama, Japan4School of Health Sciences, University of Occupational and Environmental Health, Fukuoka, Japan5Health Promotion Research Center, Institute of Community Medicine, Japan Association for Development of Community Medicine, Tokyo, Japan6Division of Epidemiology, Department of Health Informatics & Public Health, Tohoku University School of Public Health, Graduate School of Medicine, Miyagi, Japan"} +{"text": "In the original publication of the article, a mistake was introduced in affiliation of Dr. Michael Nelson; the correct affiliation is:Michael NelsonPublic Health Nutrition Research Ltd, London, UK"} +{"text": "One of the affiliations for the second author is not indicated. Gholamreza Abdi is affiliated with the Department of Life Sciences and Systems Biology, Innovation Centre, University of Turin, Turin, Italy and the Department of Biotechnology, Persian Gulf Research Institute, Persian Gulf University, Bushehr, Iran."} +{"text": "The affiliation for the third author is incorrect. Linlin Zhang is not affiliated with #2 but with #1 Department of Acupuncture and Cerebropathy, Second Affiliated Hospital of Tianjin University of Traditional Chinese Medicine, Tianjin, China."} +{"text": "Journal of Global Health [Journal, it is reviewed independently from the editor, who has no access to or influence on the review process and editorial decision-making. This is always indicated in the Competing Interests declaration at the end of the published article.We continue the practice of transparency of potential competing interests for us as editors of the l Health , followil Health . As we eWe declare below the following conflicts of interest for the current year.Prof. Maru\u0161i\u0107 is fully employed by the University of Split School of Medicine, where she holds a tenured position as Professor and Chair of the Department of Research in Biomedicine and Health. She receives funding support for research from the Croatian Science Foundation and the European Commission under the Horizon 2020 Programme. She serves as an external expert for the European Commission and Council of Europe. She is also a Visiting Professor at the NTU Lee Kong Chian School of Medicine, Singapore.Prof. Maru\u0161i\u0107 holds a position as the Editor in Chief. She occasionally reimburses small expenses related to Journal\u2019s work, including travel and consumables.Honorary Professor, College of Medicine and Veterinary Medicine, University of Edinburgh, Edinburgh, Scotland, UK;Visiting Professor, School of Medicine, University of Sao Paulo, Sao Paulo, Brazil:Past President, European Association of Science Editors;Steering Group, EQUATOR Network;Co-Chair, Cochrane Scientific Committee;Research Coordinator, Cochrane Croatia;Advisory Board Member, the Balkan Medical Journal.Prof. Rudan is employed by the University of Edinburgh, where he holds a position as Professor and Co-Director of the Centre for Global Health Research. He is also co-Director of the WHO Collaborating Centre in Population Health Research and Training. He currently receives research funding support from the Gates Foundation.Prof. Rudan holds a position as the co-Editor in Chief. He occasionally reimburses small expenses related to Journal\u2019s work, including travel and consumables.Prof. Rudan has numerous technical advisor appointments to WHO, UNICEF and the World Bank.Prof. Campbell is employed by the University of Edinburgh, where he holds a position as Professor, Co-Director of the Centre for Global Health Research and acting Dean of Molecular, Genetic and Population Health Sciences in the College of Medicine and Veterinary Medicine. He is also co-Director of the WHO Collaborating Centre in Population Health Research and Training and co-Director of the NIHR Global Respiratory Health Unit. He currently receives research funding support from the European Commission (Innovative Medicines Initiative), WHO, UK NIHR, Cancer Research UK, UK MRC, Sanofi (for work on specific GI and respiratory infections) and the Gates Foundation.Prof. Campbell holds a position as the co-Editor in Chief. He occasionally reimburses small expenses related to Journal\u2019s work, including travel and consumables.Member, MRC Africa Research Excellence Fund (AREF), College of Experts, since 2016;Member, WHO PIP burden of disease advisory group, since 2015;Member of international Scientific Advisory Group, INDEPTH network, since 2014;Member of Expert Advisory Group for \u201cThe Making of Genomic Medicine Wellcome Trust Programme; University of Edinburgh), since 2013;Member, Public Health, Health Services Research and Primary Care Research Excellence Framework (REF), 2018-2021;Numerous technical advisor appointments to WHO in the past 5 years."} +{"text": "The affiliation for the second author is incorrect. Michele Maltz is not affiliated with #2 but with #1: Yale School of Public Health, Department of Epidemiology of Microbial Diseases, New Haven, Connecticut, United States of America.Additionally, the current address for Michele Maltz is: Southern Connecticut State University, New Haven, Connecticut, United States of America."} +{"text": "There is an error in affiliation 3 for author Koichi Nakayama. The correct affiliation 3 is: Department of Regenerative Medicine and Biomedical Engineering, Faculty of Medicine, Saga University, Saga, Japan."} +{"text": "In the Acknowledgments, there is an error in affiliation for Abdulrahman O. Musaiger of the Global Burden of Diseases Nutrition and Chronic Diseases Expert Group (NutriCoDE) authorship group. The correct affiliation is: Arab Center for Nutrition, University of Bahrain, Bahrain."} +{"text": "I have the pleasure to announce that Greg Seymour, Emeritus Professor, The University of Queensland, Australia, and John Taylor, Senior Lecturer in Molecular Immunology, School of Dental Sciences, Newcastle University, UK are new Associate Editors in the Journal of Oral Microbiology. Their major field will be immunology.I am also happy to tell that Professors Ann Progulske-Fox, College of Dentistry, University of Florida, and Bruce J Paster, Forsyth Institute/Harvard School of Dental Medicine will still be involved as Associate Editors in the journal in the field of microbiology.Professor Philip Marsh has retired from his post at Public Health England and has therefore decided to step down from his role as Associate Editor in Journal of Oral Microbiology. I will miss him deeply as co-worker and thank him for his generous work for the journal over the years.Oslo, June 1, 2018Ingar OlsenEditor-in-Chief"} +{"text": "There is an error in affiliation 4 and 5 for authors Shengjie Lai and Hongjie Yu. Affiliation 4 should be: WorldPop, Department of Geography and Environment, University of Southampton, Southampton, UK. Affiliation 5 should be: School of Public Health, Fudan University, Key Laboratory of Public Health Safety, Ministry of Education, Shanghai, China. The publisher apologizes for the error[s]."} +{"text": "In the published article, there was an error in affiliation 1. Instead of \u201cDepartment of Human Parasitology, School of Basic Medical Science, Shiyan, China,\u201d it should be \u201cDepartment of Human Parasitology, School of Basic Medical Science, Hubei University of Medicine, Shiyan, China.\u201dThe authors apologize for this error and state that this does not change the scientific conclusions of the article in any way."} +{"text": "The affiliation for the fifth author is incorrect. Sang Joon Lee is not affiliated with #1 and #3 but with #1 School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology (POSTECH), Pohang, Gyeongbuk, Korea, and #4 Department of Mechanical Engineering, Pohang University of Science and Technology (POSTECH), Pohang, Gyeongbuk, Korea."} +{"text": "Dr Huillard et al. suggested that we report on mental health outcomes in breast cancer survivors with and without history of mental disorders. Twenty-two of the 60 studies excluded participants with history of mental disorders. Of the 38 studies that did not mention psychiatric history in their exclusion criteria, three accounted for it either through matching or adjustment in multivariable analyses; only one study explored the role of psychiatric history (it showed no correlation between psychiatric history and symptoms of posttraumatic stress).Dr Huillard et al. noted that in a previous study, an increased risk of mental disorders was only observed among cancer patients who had a history of mental disorder . As noteAffiliations of authors: Department of Non-Communicable Disease Epidemiology, Faculty of Epidemiology and Population Health, London School of Hygiene & Tropical Medicine, London, UK ; Clinical Practice Research Datalink (CPRD), Medicines and Healthcare products Regulatory Agency, London, UK (RW); Department of Emergency Medicine, Inselspital, Bern University Hospital, University of Bern, Bern, Switzerland (MM); Institute of Health Economics and Clinical Epidemiology, University Hospital of Cologne, Cologne, Germany (MM); Department of Medicine, The Royal Marsden NHS Foundation Trust, London and Surrey, UK (SS)."} +{"text": "It has been highlighted that the original article containsThis was incorrectly captured as Shaheed Beheshti University of Medical Sciences. This correction article shows the correct and incorrect affiliation:Shaheed Beheshti University of Medical Sciences, Tehran, IranIncorrect affiliation:Shahid Beheshti University of Medical Sciences, Tehran, IranCorrect affiliation:"} +{"text": "The affiliation and current address for the first author are swapped. Affiliation 1 should be: School of Health Sciences, Macao Polytechnic Institute, Macau Special Administration Region, China. The current address of Ying Lau should be: Department of Alice Lee Centre for Nursing Studies, Yong Loo Lin School of Medicine, National University of Singapore, Singapore. The publisher apologizes for the errors."} +{"text": "The affiliation for the seventh author is incorrect. Ziaul Islam is not affiliated with #5 but with #3: Health System and Population Studies Division, International Centre for Diarrheal Disease Research, Bangladesh , Dhaka, Bangladesh."} +{"text": "The symposium is a follow-up of the Latsis Symposium 2017 held at ETHZ, which raised major interest nationally and internationally and was highly successful. The 2019 symposium will gather leaders from different disciplines working on epigenetic inheritance, and cover scientific aspects from behavior to metabolism in humans and various animal models. It will feature keynote lectures from leaders in the field, and short talks from young researchers, and provide a platform for discussion and debate about the current state of research, new findings and discoveries, the challenges of the discipline and the perspectives for biology, medical research and the society.Main Themes:Epidemiological evidence and animal modelsReprogrammingTransmission mechanismsConceptual challenges and computational aspects of epigenetic mappingImpact on society and evolutionConfirmed Speakers:Patrick Allard; University of California, Los Angeles, USRomain Barres; University of Copenhagen, DKQi Chen; University of Nevada, Reno, USVictor Corces; Emory University, Atlanta, USLucia Daxinger; Leiden University Medical Center, NLJill Escher; Escher Fund for Autism, USLarry Feig; Tufts University, Boston, USAnne Ferguson-Smith; University of Cambridge, GBRandy Jirtle; North Carolina State University, Raleigh, USAlexander Meissner; Max-Plank Institute for Molecular Genetics, Berlin, DERuth M\u00fcller; Munich Center for Technology in Society, DEAntoine Peters; Friedrich Miescher Institute for Biomedical Research, Basel, CHAndrew Pospisilik, Max-Planck Institute of Immunobiology and Epigenetics, Freiburg, DEMark Robinson; University of Zurich, CHJulia Schr\u00f6der; Imperial College London, GBUpasna Sharma; University of California, Santa Cruz, USMichael Skinner; Washington State University, Pullman, USCorrado Spadafora; Italian National Research Council, Rome, ITAzim Surani; University of Cambridge, GBhttps://www.epigenetic-inheritance-zurich.ethz.ch/registration/, registration is open until 31 July 2019. Please feel free to share the information in your community and invite your co-workers, students and colleagues to participate.There will also be poster sessions during the meeting and a workshop \u2018Meet the experts: Questions and Answers\u2019 on the last day for students and postdocs (separate registration). You can register here: Look forward to seeing you at the symposium next August."} +{"text": "An affiliation for the first author is not indicated. Maria Simak is also affiliated with: Institute of Bioinformatics and Systems Biology, National Chiao Tung University, Hsinchu, Taiwan."} +{"text": "There is an error in affiliation 3 for author Maryam Tohidi. The correct affiliation 3 is: Prevention of Metabolic Disorders Research Center, Research Institute for Endocrine Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran."} +{"text": "In the published article, there was an error in affiliation one. Instead of \u201cYunnan Academy of Tobacco Agricultural Science, Kunming, China\u201d, it should be \u201cKey Laboratory of Microbial Resources Collection and Preservation, Ministry of Agriculture, Institute of Agricultural Resources and Regional Planning, Chinese Academy of Agricultural Sciences, Beijing, China.\u201d In addition, there was an error regarding the affiliation for Zhenyuan Xia. As well as having affiliation one, they should also have \u201cKey Laboratory of Microbial Resources Collection and Preservation, Ministry of Agriculture, Institute of Agricultural Resources and Regional Planning, Chinese Academy of Agricultural Sciences, Beijing, China.\u201dThe authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated."} +{"text": "The corresponding author information should appear as The Second Affiliated Hospital of Tianjin University of Traditional Chinese Medicine, NO. 69, Zengchan Avenue, Hebei District, Tianjin City 300250 China (luomingchimed@foxmail.com).In the article, \u201cClinical utility of miniprobe endoscopic ultrasonography for prediction of invasion depth of early gastric cancer: A meta-analysis of diagnostic test from PRISMA guideline\u201d,Mingchi Luo's affiliation should be The Second Affiliated Hospital of Tianjin University of Traditional Chinese Medicine, Tianjin City 300250 China."} +{"text": "One of the affiliations for the ninth author is incorrect. Pierre Zalloua is not affiliated with #3 but with #2: School of Medicine, Lebanese American University, Beirut, Lebanon."} +{"text": "An additional affiliation for the first author is not indicated. Dr. Huaguo Zheng is also affiliated with: University of Chinese Academy of Sciences, Beijing, China"} +{"text": "The affiliation for the seventh author is improperly abbreviated. The correct affiliation for Abdulaziz Bin Saeed is: Department of Family and Community Medicine, College of Medicine, King Saud University, Riyadh, Saudi Arabia."} +{"text": "The affiliation for the first author is incorrect. Xiaojian Ye is not affiliated with #1 but with #2 Department of General Surgery, The First Affiliated Hospital of Nanchang University, Yongwai Zhengjie, Nanchang, Jiangxi Province, China."} +{"text": "PLOS Biology to clarify that the affiliation \u2018Alaska Department of Fish and Game\u2019 listed for co-authors Sterling D. Miller and John W. Schoen are for their former affiliations with that agency, prior to retirement 20 years ago, and that the viewpoints expressed in the article do not reflect the position of the Alaska Department of Fish and Game.The Alaska Department of Fish and Game has asked Similarly, co-author Sanford P. Rabinowitch is retired from the United States National Park Service and his co-authorship does not reflect the position of that agency.The author byline and affiliations should read as follows:1, Sterling D. Miller2,\u00a4a, John W. Schoen2,\u00a4b, Sanford P. Rabinowitch3,\u00a4bWilliam J. Ripple1 Global Trophic Cascades Program, Department of Forest Ecosystems and Society, Oregon State University, Corvallis, Oregon, United States of America, 2 Alaska Department of Fish and Game (ret.), Anchorage, Alaska, United States of America, 3 United States National Park Service (ret.), Anchorage, Alaska, United States of America\u00a4a Current address: Missoula, Montana, United States of America\u00a4b Current address: Anchorage, Alaska, United States of America"} +{"text": "There is an error in the Funding section. The complete, correct funding information is: This research is funded by the National Natural Foundation of China 71661018, 71663034, CMB16-261, Project of Science and Technology Department 20161BBA10031."} +{"text": "There is an error in affiliation 4 for authors Hye Yun Park and Man Pyo Chung. The correct affiliation 4 is: Division of Pulmonary and Critical Care Medicine, Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea."} +{"text": "Due to a production error, affiliation 2 and 3 were inverted and therefore attributed to the wrong authors.The correct affiliation for Christopher Michael Jones is: Department of Psychology, The Ohio State University, Columbus, OH, United States. The correct affiliation for Roy Luria is: Sagol School of Neuroscience and the School of Psychological Science, Tel Aviv University, Tel Aviv, Israel.The publisher apologizes for this mistake. The original article has been updated."} +{"text": "The affiliation for the fifth author is incomplete. Istv\u00e1n Ulbert is also affiliated with #2: Institute of Cognitive Neuroscience and Psychology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary"} +{"text": "Dr. Melissa Suh is not included in the author byline. She should be listed as the twelfth author and her affiliation is 1: Department of Surgery, University of Nebraska Medical Center, Omaha, Nebraska, United States of America. The contributions of this author are as follows: Data Curation, Methodology, Writing\u2013Original Draft Preparation, and Writing\u2013Review & Editing."} +{"text": "The second affiliation for the second author is not indicated. Jisang Han is also affiliated with: Department of Ophthalmology, Kangbuk Samsung Hospital, Sungkyunkwan University School of Medicine, Seoul, South Korea."} +{"text": "Correction to: Genetics in Medicine, 10.1038/s41436-018-0015-7, published online 12 June 2018Hao Win, Hui Ma and Sajjad Hussain were incorrectly affiliated to \u2018Department of Radiation Oncology, The Houston Methodist Research Institute, Houston, TX 77030 USA\u2019. These authors should only have been affiliated to \u2018Hefei National Laboratory for Physical Sciences at Microscale, The First Affiliated Hospital of USTC, USTC-SJH Joint Center for Human Reproduction and Genetics, The CAS Key Laboratory of Innate Immunity and Chronic Diseases, School of Life Sciences, CAS Center for Excellence in Molecular Cell Science, Collaborative Innovation Center of Genetics and Development, University of Science and Technology of China, Hefei 230027, China\u2019. They were also not noted as contributing equally to the paper. Both these errors have now been corrected in the PDF and HTML versions of the paper."} +{"text": "The affiliation for the second author is incorrect. Kijung Yoon is not affiliated with #2 but with: Independent researcher, Ridgewood, New Jersey, United States."} +{"text": "The authors wish to correct the affiliation of co-author Guangyao Si, due to name changes of which he was unaware during his leave of absence. The correct affiliation is 3. School of Minerals and Energy Resources Engineering, University of New South Wales, Sydney, NSW 2052, Australia and 4. State Key Laboratory of Coal Resources and Safe Mining, China University of Mining and Technology, Xuzhou 221116, China. The authors would like to apologize for any inconvenience caused to the readers by these changes."} +{"text": "An additional affiliation is missing for the third author. Mohamed Eltohamy is also affiliated with Department of Glass Research, National Research Centre, Dokki, Cairo, Egypt."} +{"text": "The stressful experience of infertility is associated with a wide range of psychological damage (1), so infertility affects people's mental health and all aspects of an individual's life (2). Since women in the family are considered to be the main pillars of the community and they are also more vulnerable to illnesses, therefore consideration of their health is also very important (3). The objective of the current letter is investigating the mental health status of infertile women and its related factors as predictors of mental health in infertile women.This is a descriptive study conducted on 100 infertile women referred to the infertility treatment centers in Mazandaran province, North of Iran. The General Health Questionnaire was provided to the infertile women. The questionnaire is a self-reporting questionnaire that is used clinically to track those who are prepared for mental illness (4).Based on the findings, Total Scale of General Health Questionnaire was 33.18 Therefore, based on our findings and the level of women's mental health, there is a need for a psychologist or midwifery counselor in the infertility treatment centers to improve the mental health of women. In addition, since the mental disease may also affect the outcome of the treatment, attention to the mental health of infertile women is really importance.Zeinab Hamzehgardeshi1Sexual and Reproductive Health Research Center, Mazandaran University of Medical Sciences, Sari, Iran.2Department of Reproductive Health and Midwifery, School of Nursing and Midwifery, Mazandaran University of Medical Sciences, Sari, Iran.3Traditional and Complementary Medicine Research Center, Mazandaran University of Medical Sciences, Sari, Iran.4Student Research Committee, School of Nursing and Midwifery, Shahid Beheshti University of Medical Sciences, Tehran, Iran.5Department of Psychiatry, Sexual and reproductive health research center, Psychiatry and Behavioral Sciences Research Center, Addiction Institute, School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran.6Health Science Research Center, Addiction Institute, Mazandaran University of Medical Sciences, Sari, Iran.7IVF Ward, Mazandaran University of Medical Sciences, Sari, Iran.8Department of Reproductive Health and Midwifery, Tehran Nursing and Midwifery Faculty, Tehran university of medical science, Tehran, Iran.9Department of Medical Physics, Mazandaran Medical University, Mazandaran, Iran.10Hazrat_e Maryam Fertility Center, Sari, Iran."} +{"text": "Scientific Reports 10.1038/srep30386, published online 09 August 2016Correction to: This Article contains errors in the Affiliations.Hyun Woo Shin is incorrectly affiliated with \u2018School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology, Pohang, Republic of Korea\u2019. The correct affiliation is listed below:Department of Mechanical Engineering, Pohang University of Science and Technology, Pohang, Republic of KoreaAdditionally, this Article omits an affiliation for Jaesung Park. The correct affiliations for Jaesung Park are listed below:Department of Mechanical Engineering, Pohang University of Science and Technology, Pohang, Republic of KoreaSchool of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology, Pohang, Republic of Korea"} +{"text": "There is an error in affiliation 3 for author Salah Elsayed. The correct affiliation 3 is: Evaluation of Natural Resources Department, Environmental Studies and Research Institute, University of Sadat City, Menoufia, Egypt."} +{"text": "There is an error in the affiliation for author Wei Li. The affiliation should be:Center of Interventional Oncology and Liver Diseases, Beijing You\u2019an Hospital, Capital Medical University, Beijing, China."} +{"text": "Correction to: Cell Death and Disease10.1038/s41419-018-0567-0 published online 14 May 2019After publication of this article, it was realized that for the authors Xiaoying Luo, Dan Wang, Xintao Zhu and Xu Li the Nanfang Hospital at Southern Medical University was accidentally omitted from their affiliation designation. The correct full affiliation for the State Key Laboratory of Organ Failure Research is:State Key Laboratory of Organ Failure Research, Guangdong Provincial Key Laboratory of Viral Hepatitis Research, Department of Infectious Diseases, Nanfang Hospital, Southern Medical University, Guangzhou, China.The authors apologize for this omission."} +{"text": "An additional affiliation for the author is not indicated. Alemnesh Mirkuzie is also affiliated with Center for International Health, University of Bergen, Bergen, Norway."} +{"text": "In the article titled \u201cHealth Status and the Demand for Healthcare among the Elderly in the Rural Quoc-Oai District of Hanoi in Vietnam\u201d , additio\u201cThis research was financially supported by the JW LEE Center for Global Medicine of Seoul National University College of Medicine, Seoul, Republic of Korea. This project is part of a collaborative project by Hanoi University of Public Health, Hanoi Medical University, University of Medicine and Pharmacy of Ho Chi Minh City, and the JW LEE Center for Global Medicine of Seoul National University College of Medicine, Seoul, South Korea.\u201d"} +{"text": "Nature Communications 10.1038/s41467-018-07929-w; published online 10 January 2019.Correction to: The original version of this Article contained an error in the author affiliations.Affiliation 2 incorrectly read \u2018Department of Neurology of the Second Affiliated Hospital, Department of Neurobiology, Key Laboratory of Medical Neurobiology of the Ministry of Health of China, Key Laboratory of Neurobiology, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310007, China\u2019 and affiliation 3 incorrectly read \u2018Qiushi Academy for Advanced Studies, Zhejiang University, Hangzhou, Zhejiang 310058, China.\u2019This has now been corrected in both the PDF and HTML versions of the Article."} +{"text": "Therefore, the corrected list of authors and affliliations for this paper are shown below.Subsequently to the publication of this article, the authors have realized that the name and the affliation of the eighth author were presented incorrectly. The name was spelt as \u2018Fatemh Nasiri\u2019, whereas the correct spelling should have been \u2018Fatemeh Nasiri-1, ARAM TIRGAR2, MAHMOD HAJIAHMADI3, AKRAM HOSSEINI4, BEHZAD HEIDARI5, FATEMEH GHAFFARI6, ABBAS EBADI7, FATEMEH NASIRI-AMIRI8 and MOJGAN FIROUZBAKHT1MARYAM NIKPOUR1Student Research Committee; 2Social Determinants of Health Research Center; 3Department of Biostatistics, Non Communicable Pediatric Disease Research Center; 4Clinical Research Development Center, Shahid Beheshti Hospital; 5Mobility Impairment Research Center; 6Nursing Care Research Center, Health Research Institute, Babol University of Medical Sciences, Babol 47745.47176; 7Behavioral Sciences Research Center, Life Style Institute, Faculty of Nursing, Baqiyatallah University of Medical Sciences, Tehran 14359.16471; 8Infertility and Health Reproductive Research Center, Health Research Institute, Babol University of Medical Sciences, Babol 47745.47176, Iran.The authors regret that these errors were not recognized and corrected prior to the publication of the above article, and apologize for any inconvenience caused."} +{"text": "There is an error in affiliation 6 for the author Lavinia Fabeni. The correct affiliation 6 is: Department of Experimental Medicine, University of Rome \"Tor Vergata\", Rome, 00133, Italy."} +{"text": "Nature Communications 10.1038/s41467-019-12056-1, published online 12 September 2019.Correction to: The original version of this Article contained an error in the author affiliations. The fifth affiliation incorrectly read \u2018Beijing Advanced Innovation Center for Materials Genome Engineering, Institute for Advanced Materials and Technology, University of Science and Technology, Beijing 100083, China.\u2019 The correct version reads \u2018Beijing Advanced Innovation Center for Materials Genome Engineering, Institute for Advanced Materials and Technology, University of Science and Technology Beijing, Beijing 100083, China.\u2019 This has now been corrected in both the PDF and HTML versions of the Article."} +{"text": "Peter D. Kwong and Xuejun Chen are not included in the author byline. Xuejun Chen should be listed as the fifth author and Peter D. Kwong should be listed as the fifteenth author. Both are affiliated with Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA. The contributions of these authors are as follows: Investigation, Resources."} +{"text": "In the original article \"Patency of Individual and Sequential Coronary Artery Bypass in Patients with Ischemic Heart Disease: A Meta-analysis\", published in the Brazilian Journal of Cardiovascular Surgery 34.4, pages 420 to 427, in the article that the first unit of Zeshu Li belongs to Department of Thoracic and Cardiovascular Surgery, Shandong Provincial PKUcare Luzhong Hospital, Zibo, Shandong, People\u2019s Republic of China and the second unit is Department of Cardiac Surgery, Shandong Provincial Qianfoshan Hospital, Shandong University, Jinan, Shandong, People\u2019s Republic of China. It is the correct that the author of Zeshu Li first belongs to Department of Cardiac Surgery, Shandong Provincial Qianfoshan Hospital, Shandong University, Jinan, Shandong, People\u2019s Republic of China and the second unit it's the Department of Thoracic and Cardiovascular Surgery, Shandong Provincial PKUcare Luzhong Hospital, Zibo, Shandong, People\u2019s Republic of China."} +{"text": "The authors wish to make the following corrections to their paper :The author Yizhang Liu\u2019s affiliations were incorrect in our published paper in Sensors . Therefo1 Department of Food and Environmental Engineering, Vocational and Technical College, Chuzhou 239001, China\u201c, College of Chemistry and Materials Science, Anhui Normal University, Wuhu 241000, China)\u201d to 1 Department of Food and Environmental Engineering, Chuzhou Vocational and Technical College, Chuzhou 239001, China\u201c, College of Chemistry and Materials Science, Anhui Normal University, Wuhu 241000, China)\u201d. The manuscript will be updated and the original will remain online on the article webpage.The authors would like to apologize for any inconvenience caused."} +{"text": "Nature Communications\u00a010.1038/s41467-018-04980-5; published online: 03 July 2018Correction to: The original version of this Article incorrectly gave the second address in the list of affiliations as \u201cState Key Laboratory of Palaeobiology and Stratigraphy & Center for Excellence in Life and Paleoenvironment, Nanjing Institute of Geology and Palaeontology, Chinese Academy of Sciences, 210008 Nanjing, China\u201d, instead of the correct \u2018State Key Laboratory for Mineral Deposits Research, School of Earth Sciences and Engineering, Nanjing University, Nanjing 210023, China\u201d. This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "An affiliation is missing for the first author. In addition to the current affiliation information, Dionysios C. Watson is affiliated with: Department of Medicine, University of Patras, Greece."} +{"text": "Xin Wang is affiliated with both #2 and #3, Shenzhen Research Institute, City University of Hong Kong, Shenzhen, China.An affiliation for the 6The following information is missing from the Funding section: The authors acknowledge the support received from Shenzhen Research Institute, City University of Hong Kong."} +{"text": "There is an error in affiliation 4 for author Sukma Putra. The correct affiliation 4 is: International Business Management Program, Management Department, BINUS Business School Undergraduate Program, Bina Nusantara University, Jakarta, Indonesia."} +{"text": "In addition to the noted affiliation, Ruma Chandran is affiliated with: School of Life Sciences, Manipal Academy of Higher Education, Manipal, Karnataka, India."} +{"text": "The affiliation for the second author is incorrect. Riccardo Poli is not affiliated with #2 but with #1: Brain Computer Interfaces and Neural Engineering Laboratory, School of Computer Science and Electronic Engineering, University of Essex, Colchester, United Kingdom."} +{"text": "One of the affiliations for the first author is missing. Rattiporn Kosuwin is also affiliated with: Inter-Department Program of Biomedical Sciences, Faculty of Graduate School, Chulalongkorn University, Bangkok, Thailand."} +{"text": "The affiliation for the fifth author is incorrect. David W. Cadotte is affiliated with University of Calgary, Calgary, Alberta, Canada."} diff --git a/PMC_clustering_485.jsonl b/PMC_clustering_485.jsonl new file mode 100644 index 0000000000000000000000000000000000000000..b91c59914ad96d45e88a75f4466fe82c93af5785 --- /dev/null +++ b/PMC_clustering_485.jsonl @@ -0,0 +1,286 @@ +{"text": "This article has been corrected: The Acknowledgements section has been updated as follows:These studies were supported by funds from IRG-16-124-37 from the America Cancer Society and University of Arizona Career Development Award to EMRR and by NIH award R01CA177585 to L.H.H.1758-1776. https://doi.org/10.18632/oncotarget.27589Original article: Oncotarget. 2020; 11:1758\u20131776."} +{"text": "Palate herpes simplex virus infection. The Pan African Medical Journal. 2020;35: 123. doi: 10.11604/pamj.2020.35.123.18748. The error affected only the abstract of the PMC version of the article. The PDF and versions of the article on the journal website were not affected.This corrigendum modifies the abstract of the article: An error during the XML generation of the article \u201cPalate herpes simplex virus infection\u201d resulted"} +{"text": "In the article titled \u201cA New Device Improves Signs and Symptoms of TMD\u201d , we woulThe title has been revised to \u201cA Device Improves Signs and Symptoms of TMD.\u201d Ruggero Cattaneo and Dino Cappar\u00e8 have been removed from the author list, and I.A.P.N.O.R. has been added. Furthermore, the methods have been revised to accurately reflect the protocol for the clinical implementation of E.Li.Ba., aka ELIBA, as published by I.A.P.N.O.R. in 2010 .The device is the Elevatore Linguale Balercia , developed by the late Prof. Luigi Balercia, the founder of I.A.P.N.O.R., and described in 1998 and 1999We would also like to clarify that the study is part of the BENEFIT trial, which includes an additional arm on mandibular physiotherapy that is not yet published."} +{"text": "Scientific Reports 10.1038/s41598-020-64119-9, published online 28 April 2020Correction to: The Acknowledgements section in this Article is incomplete.\u201cA.M., M.M. and B.S. gratefully acknowledge Dr Michael Poppelreiter for facilitating access to geophysical data for research purposes and for acquiring permission to publish the same.\u201dshould read:\u201cA.M., M.M. and B.S. gratefully acknowledge Dr Michael Poppelreiter for facilitating access to geophysical data for research purposes and for acquiring permission to publish the same. The authors thank Petroliam Nasional Berhad (PETRONAS), Malaysia, for access to the data and Rasidah Husain and Kamal Embong for supporting the project.\u201d."} +{"text": "Scientific Reports 10.1038/s41598-018-38161-7, published online 14 February 2019Correction to: The Acknowledgements section in this Article is incomplete.\u201cThe authors wish to thank A. Shekhter for useful discussions. B.J.R. acknowledges funding from LANL LDRD 20160616ECR \u2018New States of Matter in Weyl Semimetals\u2019, from the DOE-BES \u2018Science of 100 Tesla\u2019 program, and from the National Science Foundation under Grant No. 1752784. Work at Los Alamos National Laboratory was supported by the LDRD Program. T.M. is funded by Deutsche Forschungsgemeinschaft through GRK 1621, SFB 1143, and the Emmy-Noether program ME 4844/1. P.J.M. is supported by the Max-Planck-Society and the European Research Council (ERC) under the European Union\u2019s Horizon 2020 research and innovation programme (grant agreement No. 715730).\u201dshould read:\u201cThe authors wish to thank A. Shekhter for useful discussions. B.J.R. acknowledges funding from LANL LDRD 20160616ECR \u2018New States of Matter in Weyl Semimetals\u2019, from the DOE-BES \u2018Science of 100 Tesla\u2019 program, and from the National Science Foundation under Grant No. 1752784. Work at Los Alamos National Laboratory was supported by the LDRD Program. T.M. is funded by Deutsche Forschungsgemeinschaft through GRK 1621, SFB 1143, and the Emmy-Noether program ME 4844/1. P.J.M. is supported by the Max-Planck-Society and the European Research Council (ERC) under the European Union\u2019s Horizon 2020 research and innovation programme (grant agreement No. 715730). A portion of this work was performed at the National High Magnetic Field Laboratory, which is supported by the National Science Foundation Cooperative Agreement No. DMR-1157490, the State of Florida and the United States Department of Energy.\u201d"} +{"text": "Erdheim Chester disease (ECD) is very rare in pediatrics with no standard treatment guidelines. Here we present the case of a pediatric ECD patient who was cured with a Langerhan cell histiocytosis (LCH) directed chemotherapy protocol.The aim of the report was to publish this rare presentation of ECD in pediatrics and highlight the complete response obtained to treatment.The details of the patient were extracted by a retrospective review of her clinical records.An 11 years old girl presented with fever and bone pain. On investigating she had multiple lytic bony lesions scattered throughout her skeleton. A biopsy from one of the bone lesions confirmed the diagnosis to be ECD. ECD is very rare in pediatrics and this case adds to the existing list of 11 previously reported ones. Also, worth mention is the fact that the child presented with isolated skeletal involvement in form of multiple osteolytic lesions. The child was started on the LCH\u2010III protocol on which she achieved a cure.Lytic bone lesions in a child may be present in ECD. A subset of ECD may have good response to LCH like chemotherapy. The name of the disease comes from Jacob Erdheim and William Chester, who initially described it in 1939.2An 11\u2009years old girl presented to our hospital with a history of intermittent fever for 5 months. The fever was high grade and was relieved temporarily by antipyretics. The fever used to occur irregularly with intermittent periods of defeverescence. She had been treated with multiple courses of antibiotics without relief. Her chest x ray (CXR) had revealed lytic lesions in her ribs after which she had been referred to our centre.Upon presentation to our hospital she had been afebrile, but had bone pain at multiple sites. There were no other complains. On examination, she had tenderness on deep palpation of the long bones. The systemic examination was otherwise unremarkable. Her sexual maturity rating was appropriate for age.Her complete blood count, liver function tests and renal function tests were normal. The urine investigations were unremarkable. The CXR showed a normal lung parenchyma and lytic lesions in the ribs, clavicle, sternum and scapulae. The Mantoux test was negative. A skeletal survey by X\u2010ray and computed tomography (CT) scan revealed lytic lesions in multiple bones Figure . The posA CT guided biopsy was done from one of the FDG avid bone lesions that revealed a foamy histiocytic infiltrate with pale eosinophilic abundant cytoplasm in a background of fibroblastic proliferation, and lymphocytic infiltrate. The histiocytic cells were CD1a and S\u2010100 negative but showed positivity for CD68 Figure . A diagnThere is no standard treatment for ECD. Previous pediatric patients have been treated with various agents like interferon\u2010\u03b1, vinca alkaloids, steroids, anthracyclines and radiotherapy. After taking into consideration the feasibility and financial status of the family the child was given a trial on the LCH III protocolThe child achieved partial remission after the first phase of treatment consisting of prednisolone and weekly vinblastine . Subsequently a second cycle was administered following which a complete response was obtained and the child was then put on a 12\u2009months continuation treatment consisting of daily oral 6\u2010mercaptopurine and three weekly vinblastine and prednisolone. The PET scan post completion of treatment showed complete remission of the disease signalling pathway, most commonly involving the BRAF gene.The child responded to the conventional chemotherapy known to be effective in LCH. The patient is now 12\u2009months post treatment completion and continues to be in good health. Previous pediatric patients have been treated on either steroids or interferon\u2010The authors declare no potential conflict of interest.Conceptualization, A.K.G.; Data curation, A.K.G., A.W.M., S.T.A., A.M., P.N., R.K., R.S.; Investigation, A.K.G., A.W.M., J.P.M., S.T.A., A.M., P.N., R.K., R.S.; Resources, A.M., P.N., R.K., R.S.; Writing \u2010 Original Draft, A.K.G., A.W.M.; Writing \u2010 Review & Editing, A.K.G., A.W.M., J.P.M., S.T.A., A.M.,P.N., R.K., R.S.; Visualization, A.K.G., J.P.M., R.S.; Supervision, A.K.G., R.S.All authors had full access to the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. The informed and written consent to publish this case report was obtained from the patient's father and the publication was done in accordance to the institute ethics committee guidelines."} +{"text": "The Author contributions have been updated to the following: R.A., N.G., H.R., and O.E. should be considered as co-first authors.The HTML and PDF versions of the Article have been corrected."} +{"text": "The authors wish to make the following corrections to this paper :Funding: The authors acknowledge support from the Center for Nanophase Materials Sciences, which is a DOE Office of the Science User Facility. The authors (O.D. and S.J.) acknowledge support by the U.S. Department of Energy, Office of Science, Basic Energy Sciences, Materials Science and Engineering Division. C.Z. and M.G.S. acknowledge support from the U.S. Department of Energy (DOE) under grant no. DE-SC0002136. D.A.G. acknowledges the support by NSF CBET-1603780.The funding section needs to be corrected. The updated information is included below:This addendum does not cause any changes to the results or conclusions in the original published paper.The authors would like to apologize for any inconvenience caused to the readers by these changes."} +{"text": "TARDBP) nuclear-to-cytoplasmic mislocalization is a fatal neurodegenerative disease characterized by the progressive loss of upper and lower motor neurons. Although its precise aetiopathogenesis remains unclear, cellular hallmarks of the disease include the deregulation of RNA metabolism and the mislocalization of RNA binding proteins (RBPs) from the nucleus to the cytoplasm . We receNEAT1_2, enhanced paraspeckle formation has been observed in both sporadic , the UK Medical Research Council (FC010110), and the Wellcome Trust (FC010110). R.P. holds an MRC/MND Association Lady Edith Wolfson Senior Clinical Fellowship [MR/S006591/1] and is supported by the National Institute for Health Research University College London Hospitals Biomedical Research Centre. N.M.L. is supported by a Wellcome Trust Senior Investigator Award [103760/Z/14/Z], an MRC eMedLab Medical Bioinformatics Infrastructure Award to N.M.L. (MR/L016311/1). N.M.L. is a Winton Group Leader in recognition of the Winton Charitable Foundation\u2019s support towards the establishment of the Francis Crick Institute. R.L. is supported by the Idiap Research Institute.The authors report no competing interests."} +{"text": "M. chirigota and MN816441 for M. gaditana. Please see the complete, correct There are a number of errors in"} +{"text": "Nature Communications 10.1038/s41467-020-16549-2, published online 4 June 2020.Correction to: The original version of this Article\u2019s Acknowledgement section incompletely read:This project has received financial support from the European Union\u2019s Horizon 2020 research and innovation programme under the Marie Sk\u0142odowska Curie Grant Agreement No. 675585 SyMBioSys and under Grant Agreement No. 686070.Instead of:We would like to thank Dr. G. Fengos, Dr. J.P. Vieira and Dr. D.R. Weilandt for constructive discussions and feedback; and Dr. K. Butler for valuable comments proofreading this paper. This project has received financial support from the European Union\u2019s Horizon 2020 research and innovation programme under the Marie Sk\u0142odowska Curie Grant Agreement No. 675585 SyMBioSys, and under Grant Agreement No. 686070, and from the RTD project MicroScapeX (grant 2013/158) funded by SystemsX.ch, the Swiss Initiative for Systems Biology evaluated by the Swiss National Science Foundation.This has now been corrected in both the PDF and HTML versions of the Article."} +{"text": "The original version of this article unfortunately contained a mistake. In the author list, the first and last names of two authors, S. Matthijs Boekholdt and R. Nils Planken, were tagged incorrectly. Therefore, author names are abbreviated wrongly in Springerlink. The first and last names should be as follows:First name: S. MatthijsLast name: BoekholdtFirst name: R. NilsLast name: Planken"} +{"text": "Nature Communications 10.1038/s41467-020-18446-0, published online 15 September 2020.Correction to: The original version of this Article contained an error in the Competing interest statement. It was incorrectly stated that D.S.V. sits on the board of Life Biosciences and Iduna. The correct declaration is D.A.S sits on the board of both companies. This has now been corrected in both the PDF and HTML versions of the Article."} +{"text": "Smoking has been associated with worse colorectal cancer patient survival and may potentially suppress the immune response in the tumor microenvironment. We hypothesized that the prognostic association of smoking behavior at colorectal cancer diagnosis might differ by lymphocytic reaction patterns in cancer tissue.KRAS, BRAF, and PIK3CA mutations.Using 1474 colon and rectal cancer patients within 2 large prospective cohort studies , we characterized 4 patterns of histopathologic lymphocytic reaction, including tumor-infiltrating lymphocytes (TILs), intratumoral periglandular reaction, peritumoral lymphocytic reaction, and Crohn\u2019s-like lymphoid reaction. Using covariate data of 4420 incident colorectal cancer patients in total, an inverse probability weighted multivariable Cox proportional hazards regression model was conducted to adjust for selection bias due to tissue availability and potential confounders, including tumor differentiation, disease stage, microsatellite instability status, CpG island methylator phenotype, long interspersed nucleotide element-1 methylation, and Pinteraction = .009). No statistically significant interactions were observed in the other patterns of lymphocytic reaction.The prognostic association of smoking status at diagnosis differed by TIL status. Compared with never smokers, the multivariable-adjusted colorectal cancer\u2013specific mortality hazard ratio for current smokers was 1.50 in tumors with negative or low TIL and 0.43 in tumors with intermediate or high TIL (2-sided The association of smoking status at diagnosis with colorectal cancer mortality may be stronger for carcinomas with negative or low TIL, suggesting a potential interplay of smoking and lymphocytic reaction in the colorectal cancer microenvironment. Cigarette smoking is an established risk factor for incidence of colon and rectal cancer . CurrentA variety of endogenous and exogenous factors may exert effects on the host immune response to colorectal cancer . Tumor-iTo test our hypothesis, we used 2 large US nationwide prospective cohort studies with covariate data of 4420 colorectal cancer patients and a molecular pathological epidemiology database of 1474 patients. This comprehensive dataset enabled us to examine the prognostic association of smoking status at diagnosis according to lymphocytic reaction in the colorectal cancer tissue.We collected data from 2 prospective cohort studies in the United States: the Nurses\u2019 Health Study and the Health Professionals Follow-up Study . In bothWe included 1474 patients with available data on smoking exposure at diagnosis and immune profiles including lymphocytic reaction in colorectal cancer tissue. We included both colon and rectal carcinomas based on the colorectal continuum model . PatientInformed consent was obtained from all participants in this analysis. The study procedures and protocols were approved by the institutional review boards at the Harvard T.H. Chan School of Public Health, Brigham and Women\u2019s Hospital , and those of participating registries as required.Data on smoking status were collected in the 2 cohorts as reported previously . CurrentFour components of lymphocytic reaction to tumors, including TIL, intratumoral periglandular reaction, peritumoral lymphocytic reaction, and Crohn\u2019s-like lymphoid reaction, were histopathologically evaluated as previously reported . BrieflyCACNA1G, CDKN2A, CRABP1, IGF2, MLH1, NEUROG1, RUNX3, and SOCS1) , BRAF (codon 600), and PIK3CA (exons 9 and 20) ; MSI-high was defined as the presence of instability in at least 30% of the markers (d SOCS1) was deted SOCS1) . Methyla and 20) .P values were 2-sided. Our primary hypothesis test was an assessment of a statistical interaction between smoking status at diagnosis and lymphocytic reaction in tumor tissue (binary classification of negative/low and intermediate/high) in a multivariable Cox proportional hazards regression model using the Wald test on the cross-product. We used the 2-sided \u03b1-level of .005 . All of .005 . The haz of .005 .Primary outcome endpoint of this study was colorectal cancer\u2013specific mortality and the secondary endpoint was overall mortality. For colorectal cancer\u2013specific survival analyses, deaths of other causes and patients with missing data on cause of death were censored. Survival time was defined as the period from diagnosis of colorectal cancer to death or the end of follow-up, whichever came first.In all survival analyses, we used covariate data of 4420 incident colorectal cancer patients and the IPW method ,20. The KRAS, BRAF, and PIK3CA mutations. A backward elimination with a threshold of P = .05 was performed to select variables for the final models. The proportionality of hazards assumption was assessed using a time-varying covariate, which is an interaction term of survival time and smoking status at diagnosis. The proportionality of hazards assumption was generally satisfied for cancer-specific survival (P > .28). All statistical tests were 2-sided.IPW-adjusted, multivariable Cox proportional hazards regression models were used to adjust for potential confounders and initially included the following: sex , age at diagnosis, year of diagnosis, family history of colorectal cancer, body mass index at diagnosis, alcohol consumption at diagnosis; physical activity at diagnosis; processed meat intake at diagnosis; total fiber intake at diagnosis; tumor location; tumor differentiation; disease stage; MSI status; CIMP; long-interspersed nucleotide element-1 methylation level; and We included 1474 colorectal cancer patients with available data on smoking status at diagnosis and lymphocytic reaction among 4420 incident colorectal cancer patients in the 2 prospective cohort studies . The frePinteraction = .009; In our primary hypothesis testing, we evaluated the association of smoking status at diagnosis with colorectal cancer\u2013specific survival according to tumor lymphocytic reaction. We found a trend of a statistical interaction between smoking status at diagnosis and TIL in relation to colorectal cancer\u2013specific survival in the IPW-adjusted Cox model was approximately 10 to 20\u2009months , colorecThe major strength of this study was the use of a molecular pathological epidemiology databaseIn conclusion, the association of smoking behavior at diagnosis with colorectal cancer survival appears to differ by the host immune system, and tumors with negative or low lymphocytic reaction resulted in poor survival among current smokers compared with never smokers. Our findings emphasize the link between smoking and tumor immunity, both of which may interactively influence colorectal cancer progression.This work was supported by US National Institutes of Health (NIH) grants ; by Cancer Research UK\u2019s Grand Challenge Award ; by Nodal Award (2016-20) from the Dana-Farber Harvard Cancer Center (to S.O.); by Stand Up to Cancer Colorectal Cancer Dream Team Translational Research Grant (SU2C-AACR-DT22-17 to C.S.F. and M.G.), administered by the American Association for Cancer Research, a scientific partner of SU2C; and by grants from the Project P Fund, The Friends of the Dana-Farber Cancer Institute, Bennett Family Fund, and the Entertainment Industry Foundation through National Colorectal Cancer Research Alliance and SU2C. K.F. and K.H. were supported by fellowship grants from the Uehara Memorial Foundation. K.F. was supported by fellowship grants from the Grant of The Clinical Research Promotion Foundation (2018). Y.C. and L.L. were supported by a scholarship grant from Chinese Scholarship Council. K.H. was supported by fellowship grants from the Mitsukoshi Health and Welfare Foundation. T.U. and K.A. were supported by a grant from Overseas Research Fellowship from Japan Society for the Promotion of Science. A.T.C. is a Stuart and Suzanne Steele MGH Research Scholar. M.G. is supported by an ASCO Conquer Cancer Foundation Career Development Award.Role of the funders: The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Disclaimer: The content is solely the responsibility of the authors and does not necessarily represent the official views of NIH.Disclosures: A.T.C. previously served as a consultant for Bayer Healthcare and Pfizer Inc. This study was not funded by Bayer Healthcare or Pfizer Inc. J.A.M has received institutional research funding from Boston Biomedical, has served as an advisor/consultant to Ignyta and COTA Healthcare, and served on a grant review panel for the National Comprehensive Cancer Network funded by Taiho Pharmaceutical. C.S.F. previously served as a consultant for Agios, Bain Capital, Bayer, Celgene, Dicerna, Five Prime Therapeutics, Gilead Sciences, Eli Lilly, Entrinsic Health, Genentech, KEW, Merck, Merrimack Pharmaceuticals, Pfizer Inc, Sanofi, Taiho, and Unum Therapeutics; C.S.F. also serves as a Director for CytomX Therapeutics and owns unexercised stock options for CytomX and Entrinsic Health. R.N. is currently employed by Pfizer Inc; she contributed to this study before she became an employee of Pfizer Inc. M.G. receives research funding from Bristol-Myers Squibb and Merck. This study was not funded by any of these commercial entities. No other conflicts of interest exist. The other authors declare that they have no conflicts of interest.Author contributions: All authors contributed to review and revision. M.G., J.A.N., and S.O.: developed the main concept and designed the study. R.N., A.T.C., C.S.F., M.G., and S.O.: wrote grant applications. K.F., Y.C., K.H., T.U., J.K., T.H., L.L., J.B., D.A.D, M.S., A.T.C., E.L.G., J.A.M., C.S.F., R.N., J.K.L., J.A.N., and S.O.: were responsible for collection of tumor tissue, and acquisition of epidemiologic, clinical and tumor tissue data, including histopathological, immunohistochemical, and immunofluorescent characteristics. K.F., Y.C., T.U., J.K., K.H., C.S.F., R.N., X.Z., K.W., and S.O.: performed data analysis and interpretation. K.F., Y.C., K.H., T.U., J.K., and S.O.: drafted the manuscript. M.Z., K.A., J.P.V., M.C.L., S.G., S.S., N.A., T.S.T., M.S., R.N., M.G., J.A.N., X.Z., K.W., and S.O.: contributed to editing and critical revision for important intellectual contents.pkaa040_Supplementary_DataClick here for additional data file."} +{"text": "Ecology and Evolution. 2020; 10: 10965\u201310973. https://doi.org/10.1002/ece3.6353Pierson, K. Building a richer understanding of diversity through causally consistent evenness measures. wileyonlinelibrary.com), has been retracted by agreement between the author, the journal's Editors in Chief and John Wiley & Sons Ltd. The retraction has been agreed due to the paper substantially overstating the magnitude of the problem it addresses. This has caused a change in the fundamental message of the paper.The above article, published online on 26 May 2020 in Wiley Online Library ("} +{"text": "Scientific Reports 10.1038/s41598-020-68338-y, published on 20 July 2020Correction to: The Acknowledgements section in this Article is incomplete.\u201cWe thank S. Perry, S. Hrdy, and reviewers for comments and suggestions. K.R. was supported by a National Science Foundation Graduate Research Fellowship. S.G. was supported by the National Institute for Mathematical and Biological Synthesis through National Science Foundation awards EF-0832858 and DBI-1300426, with additional support from The University of Tennessee.\u201dshould read:\u201cWe thank S. Perry, S. Hrdy, and reviewers for comments and suggestions. K.R. was supported by a National Science Foundation Graduate Research Fellowship. S.G. was supported by the National Institute for Mathematical and Biological Synthesis through National Science Foundation awards EF-0832858 and DBI-1300426, with additional support from The University of Tennessee. Funding for open access to this research was provided by University of Tennessee\u2019s Open Publishing Support Fund.\u201d"} +{"text": "Nature Communications 10.1038/s41467-020-18985-6, published online 14 October 2020.Correction to: The original version of this Article\u2019s Acknowledgment section incorrectly read:This work was supported by funding from Cohen Veteran Biosciences, NIMH , and the Frazier Institute at McLean Hospital. C.C. was supported by the 2019 Seed Grant (through NIMH P50-MH115874). N.P.D. was supported by a NARSAD Young Investigator grant and an appointed KL2 award from Harvard Catalyst/The Harvard Clinical and Translational Science Center (NCATS KL2TR002542 and UL1TR002541).The correct version reads:This work was supported by funding from Cohen Veterans Bioscience, NIMH , and the Frazier Institute at McLean Hospital. C.C. was supported by the 2019 SPARED Center Seed Grant (through NIMH P50-MH115874). G.M. was supported by a NARSAD Young Investigator grant. N.P.D. was supported by a NARSAD Young Investigator grant and an appointed KL2 award from Harvard Catalyst/The Harvard Clinical and Translational Science Center (NCATS KL2TR002542 and UL1TR002541).This has now been corrected in both the PDF and HTML versions of the Article."} +{"text": "Nature Communications 10.1038/ncomms5430, published online 18 July 2014.Correction to: The original version of this Article omitted the following from the Acknowledgements:L.C.L. and M.M.P. were supported by CIRM (RN2-00931).This has not been corrected in the PDF and HTML versions of the Article."} +{"text": "Nature Communications 10.1038/s41467-020-19441-1, published online 04 November 2020.Correction to: The original version of this Article contained an error in the Acknowledgements, which incorrectly read \u2018This work was supported by the Fujian Agriculture and Forestry University (FAFU) Construction Project for Technological Innovation and Service System of Tea Industry Chain (K1520005A02) and other funds from FAFU to X.Y., Z.Y. and R.L.\u2019 The correct version states \u2018Y.Y.\u2019 in place of \u2018Z.Y.\u2019. This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "We present a mean-field formalism able to predict the collective dynamics of large networks of conductance-based interacting spiking neurons. We apply this formalism to several neuronal models, from the simplest Adaptive Exponential Integrate-and-Fire model to the more complex Hodgkin\u2013Huxley and Morris\u2013Lecar models. We show that the resulting mean-field models are capable of predicting the correct spontaneous activity of both excitatory and inhibitory neurons in asynchronous irregular regimes, typical of cortical dynamics. Moreover, it is possible to quantitatively predict the population response to external stimuli in the form of external spike trains. This mean-field formalism therefore provides a paradigm to bridge the scale between population dynamics and the microscopic complexity of the individual cells physiology.NEW & NOTEWORTHY Population models are a powerful mathematical tool to study the dynamics of neuronal networks and to simulate the brain at macroscopic scales. We present a mean-field model capable of quantitatively predicting the temporal dynamics of a network of complex spiking neuronal models, from Integrate-and-Fire to Hodgkin\u2013Huxley, thus linking population models to neurons electrophysiology. This opens a perspective on generating biologically realistic mean-field models from electrophysiological recordings. Brain dynamics can be investigated at different scales, from the microscopic cellular scale, describing the voltage dynamics of neurons and synapses , to the In their pioneering work , Wilson Moreover, a large effort has been made to derive population descriptions from the specificity of the network model under consideration. This bottom-up approach permits to obtain a dimensionally reduced mean-field description of the network population dynamics in different regimes , 1998. ODuring awake states, cortical dynamics generally show asynchronous spiking activity, where individual neurons are characterized by an irregular firing pattern . In thisIn this article we present a general approach to determine the transfer function for complex models, from the Adaptive Exponential Integrate-and-Fire (AdEx) to the Hodgkin\u2013Huxley (HH) and the Morris\u2013Lecar (ML) models. As a result, we obtain mean-field equations for the population dynamics in Asynchronous Irregular regimes as observed in cortical regions for highly detailed models, creating a bridge between electrophysiology at the microscopic scale and the details of the famous transfer function first used by Wilson and Cowan as a sigmoid.Finally, we test not only the ability of our mean-field models to describe spontaneous activity of the considered neuronal populations, but also their predictive power for network response to external stimuli. We show that, provided the stimuli are fairly slow, the mean-field model gives good quantitative predictions.We describe here the neuronal and network models used in this study. We also introduce mean-field equations describing population dynamics and the template to estimate the transfer function that we apply to all the neuronal models under consideration.N = 104 cells, among which 80% are regular-spiking (RS) excitatory (E) and 20% are fast-spiking (FS) inhibitory (I) neurons. The connections between pairs of neurons are set randomly with a fixed probability (P = 0.05). Unless otherwise stated, the same network and synaptic constants are used for all the neuronal models . The dynamics of each node k followsk, synI, is modeled aseQ (iQ) is the excitatory (inhibitory) quantal conductance. The variable \u03c4 = 5 ms is the decay timescale of excitatory and inhibitory synapses and \u0398 is the Heaviside step function. The summation runs over the overall presynaptic spiking times spt(n). For both Hodgkin\u2013Huxley and Adaptive Exponential Integrate-and-Fire models we set eQ\u2009=\u20091.5 nS and iQ\u2009=\u20095 nS, while for the Morris\u2013Lecar model eQ\u2009=\u20094 nS and iQ\u2009=\u200910 nS. On top of inputs coming from other neurons in the network, each excitatory and inhibitory neuron receives an external drive in the form of a Poissonian excitatory spike train at a constant firing rate drivev = 4 Hz, if not stated otherwise.We consider a random directed network of We describe here the neuronal models used in the rest of the paper, starting from the Integrate-and-Fire up to the Morris\u2013Lecar and Hodgkin\u2013Huxley models.i is described by the following 2D . A stepMI current in the case of Hodgkin\u2013Huxley model. The possibility to include a conductance based adaptation to this formalism, e.g., by considering the slow dynamics of MI current, is a stimulating perspective for future works and will permit to obtain mean-field models for realistic neuronal models beyond the asynchronous irregular regime.We also reported that, in the framework of the Asynchronous regimes here considered, corrections to first order mean field due to second order terms see . NeverthMoreover, beyond the input-output transfer function used here, a more complex transfer function has been used to take into account other features of neuron response, e.g., response in frequency . The addAnother possible extension is to apply the same formalism to complex neuronal models that include dendrites. A first attempt has been made in this direction by consiFinally, the positive results obtained here for complex models, by showing the generality of our approach, motivate the future step of the application of this technique directly to experimental data. To this end, neurons must be recorded intracellularly in the absence of network activity , and many combinations of excitatory and inhibitory inputs must be injected as conductances (using the dynamic-clamp technique). The first attempt of this sort was realized on the layer 5 neurons from mouse primary visual cortex , where tResearch supported by the Centre National de la Recherche Scientifique and the European Union (Human Brain Project H2020-720270 and H2020-785907). M. Jedynak acknowledges support from the European Research Council under the European Union\u2019s Seventh Framework Programme (FP/2007-2013)/ERC Grant Agreement No. 616268 F-TRACT and the European Union\u2019s Horizon 2020 Framework Programme for Research and Innovation under Specific Grant Agreement No. 785907 (Human Brain Project SGA2).No conflicts of interest, financial or otherwise, are declared by the authors.A.D. and M.d.V. conceived and designed research; M.C., O.C., L.D.P., D.D., C.H., M.J., E.K.E., P.M., S.S., and M.d.V. performed experiments; M.d.V. analyzed data; Y.Z., A.D., and M.d.V. interpreted the results of experiments; M.C., O.C., L.D.P., D.D., C.H., E.K.E., P.M., S.S., and M.d.V. prepared figures; M.d.V. drafted manuscript; M.C., O.C., L.D.P., D.D., C.H., M.J., E.K.E., P.M., S.S., C.C., Y.Z., A.D., and M.d.V. edited and revised manuscript; M.C., O.C., L.D.P., D.D., C.H., M.J., E.K.E., P.M., S.S., C.C., Y.Z., A.D., and M.d.V. approved final version of manuscript."} +{"text": "Scientific Reports 10.1038/s41598-019-49908-1, published online 19 September 2019Correction to: Fatmah Alkindi was omitted from the author list in the original version of this Article. This has been corrected in the PDF and HTML versions of the Article.The Author Contributions section now reads:\"M. Rezeq conceived the idea and supervised the experimental work and paper writing. Y. Abbas and A. Rezk performed the sample preparation, electrical and physical characterization along with the manuscript drafting. F. Alkindi performed data analysis and characterization. A. Nayfeh and I. Saadat contributed to data analysis and manuscript editing. All authors reviewed the manuscript.\""} +{"text": "Trachemys scripta troostii was sequenced and was characterized, which comprised 37 genes and a non-coding control region. Phylogenetic analysis based on the full mt genome indicated that T. s. troostii was more closely related to T. scripta from Canada than to T. s. elegans from China or T. s. scripta fom China. This is the first complete mt genome from T. s. troostii, which provides data for further study of phylogeny in Emydidae.The complete mitochondrial (mt) genome of Trachemys scripta troostii) is native in Cumberland and Tennessee Rivers, from south-eastern Virginia and Kentucky to north-eastern Alabama in the United States DNA fragments , Korea. We extracted the total genomic DNA from the tail using the DNeasy Blood & Tissue kit according to the manufacturer\u2019s protocol and the extracted DNA sample was deposited at the Museum of Wildlife, located in Research Center of Ecomimeitcs, Chonnam National University, Korea . We constructed the complete mt genome by primer walking method with the primer pairs (Supplementary Table 1). The complete mt genome was sequenced using Applied Biosystems 3730XL DNA Analyzer . We used Needleman-Wunsch algorithm on NCBI to align the sequences, checked quality control from chromatogram data provided by Bionics which offers DNA sequencing services. Each read was aligned and annotated by comparing T. scripta mt genome (Accession No.\u00a0FJ392294), T. s. elegans mt genome (Accession No. KM216748), and T. s. scripta mt genome (Accession No. KM216749) in GenBank.The T. s. troostii was 16,810\u2009bp in length deposited in GenBank (Accession No. MW122292), and contained 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, 2 ribosomal RNA (rRNA) genes (12\u2009s and 16\u2009s), and a putative non-coding control region (NCR). Twelve PCGs, 14 tRNAs, 2 rRNAs were predicted to be located in the heavy strand, whereas 1 protein-coding gene (NADH dehydrogenase subunit 6) and 8 tRNAs were encoded in the light strand. The nucleotide composition of the T. s. troostii from Korea mt genome was same with that of T. scripta from Canada , and similar with that of T. s. elegans from China , and T. s. scripta from China\u00a0. The\u00a0sequence comparison with T. s. troostii from Korea and T. scripta from Canada indicated a 99.97% sequence identity. But the sequence identity was 99.68% between T. s. troostii from Korea and T. s. elegans from China, and was 99.41% between T. s. troostii from Korea and T. s. scripta from China.The complete mt genome of T. s. troostii from Korea, the full mt genome sequences of 12 turtle species were extracted from Genbank. Based on other studies (Krenz et\u00a0al. Pelomedusa subrufa as an outgroup which belongs to the suborder \u2018Pleurodira.\u2019 The sequences were aligned using MUSCLE algorithm (Edgar T. scripta subspecies is clustered in a monophyletic group. T. s. troostii from Korea is more closely related to T. scripta from Canada than to T. s. elegans from China or T. s. scripta from China (T. s. troostii.In order to investigate the phylogenetic position of hm Edgar , and thehm Edgar . It showom China . These d"} +{"text": "The tenth author's name is spelled incorrectly. The correct name is: G. M. K. Mehaisen."} +{"text": "Nature Communications10.1038/s41467-020-15210-2, published online 20 March 2020.Correction to: The original version of this Article omitted some funding sources in the Acknowledgements. The correct version of the Acknowledgments is:\u201cThis work was financially supported by the European Research Council Consolidator Grant NanoSurfs (no. 615233), the Advanced Grant (no. 694097), the Horizon 2020 research and innovation program 2D ink (no. 664878), and the National Science Foundation of China (no. 11974403 and Sino-German Project no. 51761135130). W.A. acknowledges funding by the DFG via a Heisenberg professorship. M.R., R.B., and X.F. thank the German Research Foundation (DFG) within the Cluster of Excellence \u201cCenter for Advancing Electronics Dresden (cfaed)\u201d and EnhanceNano (No. 391979941). A.P.P. and A. Rubio thank the Cluster of Excellence \u201cAdvanced Imaging of Matter (AIM)\u201d and Grupos Consolidados (IT1249-19). M.G. acknowledges funding by the H2020-MSCA-IF\u22122014 program under GA no. 658070 (2DNano).\u201dWhich replaces the previous incorrect version:\u201cThis work was financially supported by the European Research Council Consolidator Grant NanoSurfs (no. 615233), the Horizon 2020 research and innovation program 2D ink (no. 664878) and the National Science Foundation of China (no. 11974403 and Sino-German Project no. 51761135130). W.A. acknowledges funding by the DFG via a Heisenberg professorship. M.R., R.B., and X.F. thank the German Research Foundation (DFG) within the Cluster of Excellence \u201cCenter for Advancing Electronics Dresden (cfaed)\u201d and EnhanceNano (No. 391979941). M.G. acknowledges funding by the H2020-MSCA-IF\u22122014 program under GA no. 658070 (2DNano).\u201dThis has now been corrected in both the PDF and HTML versions of the Article."} +{"text": "The authors regret that an incorrect reference was cited in the original version of their paper. The reference and citation of Kishimoto, 1989 have been replaced with Sehgal et al., 1989. The revised sentence and complete reference appear below. All versions of the article have been corrected.\u201cThe molecule was first designated IL-6 in 1988 at a conference entitled \u2018Regulation of the Acute Phase and Immune Responses: A New Cytokine\u2019 .\u201dAnn. N Y Acad. Sci. 557:1\u2013583.Sehgal, P.B., G. Grieninger, and G. Tosato, editors. 1989. Regulation of the Acute Phase and Immune Responses: Interleukin-6."} +{"text": "The original version of this article unfortunately contained a mistake. The joint first authorship was not mentioned in the paper.The article note should be:Wouter J. C. van Ballegoij, Sander C. Kuijpers and Irene C. Huffnagel are contributed equally to this work."} +{"text": "Scientific Reports 10.1038/s41598-020-61615-w, published online 19 March 2020Correction to: The Acknowledgements section in this Article contains errors.\u201cThe authors acknowledge the Office for Geology and Building Materials Testing (D. Costantini), the Regional Warning Centre (O. Formaggioni) and the Hydrographic Office of the Autonomous Province of Bolzano/Bozen - South Tyrol for providing the event catalogues, the meteorological data and useful comments on the manuscript. We acknowledge F. Pacini and H. Gaumont (Terradue) for developing and maintaining the GEP as well as P. Blanco (Tre-Altamira) in FASTVEL algorithm use. We thank M. Melillo (CNR IRPI) for helping in the reconstruction of rainfall events. We acknowledge S. Natali (Sistema) for enabling climate data access on ADAM platform and A. Jacob (Eurac Research) for the collection and interpretation of updated snow cover data. R.S.\u2019s work was supported by an ESA Climate Office research fellowship. S.L.G. was granted by a Regione Puglia - Civil Protection Department research fellow (project id: B82F16003840006). C.K. was supported by the Stiftung S\u00fcdtiroler Sparkasse/Fondazione Cassa di Risparmio di Bolzano PhD funding program on future-relevant topics for South Tyrol (grant number: 2017.0160).\u201dshould read:\u201cThe authors acknowledge the Office for Geology and Building Materials Testing (D. Costantini), the Regional Warning Centre (O. Formaggioni) and the Hydrographic Office of the Autonomous Province of Bolzano/Bozen - South Tyrol for providing the event catalogues, the meteorological data and useful comments on the manuscript. We acknowledge F. Pacini and H. Caumont (Terradue) for developing and maintaining the GEP as well as P. Blanco (Tre-Altamira) in FASTVEL algorithm use. We thank M. Melillo (CNR IRPI) for helping in the reconstruction of rainfall events. We acknowledge S. Natali (Sistema) for enabling climate data access on ADAM platform and A. Jacob (Eurac Research) for the collection and interpretation of updated snow cover data. R.S.\u2019s work was supported by an ESA Climate Office research fellowship. S.L.G. was granted by a Regione Puglia - Civil Protection Department research fellow (project id: B82F16003840006). C.K. was supported by the Stiftung S\u00fcdtiroler Sparkasse/Fondazione Cassa di Risparmio di Bolzano PhD funding program on future-relevant topics for South Tyrol (grant number: 2017.0160).\u201d"} +{"text": "Scientific Reportshttps://doi.org/10.1038/s41598-023-31008-w, published online 08 March 2023Correction to: The Funding section in the original version of this Article was omitted. The Funding section now reads:\u201cThis study and support for M.B. and D.F. were funded by the New Zealand Strategic Science Investment Fund: Antarctic Science Platform Contract ANTA1801. A.F.-V. was supported by the Deep South National Science Challenge. The mooring G data were collected in the framework of MORSea projects supported by the Italian National Program for Antarctic Research\u2010PNRA, which provided financial and logistic support. P.C. was funded by the PNRA, Grant PNRA18_00256. The PNRA is funded by the Italian Minister of the University\u201d.The original Article has been corrected."} +{"text": "Basal cisternostomy for severe TBI: Surgical technique and cadaveric dissection By Giammattei L, Starnoni D, Messerer M and Daniel RT. (2022) Front. Surg. 9:915818. doi: 10.3389/fsurg.2022.915818An Erratum on An omission to the funding section of the original article was made in error. The following sentence has been added: \u201cOpen access funding provided by University of Lausanne\u201d.The original version of this article has been updated."} +{"text": "Scientific Reportshttps://doi.org/10.1038/s41598-023-36604-4, published online 12 June 2023Correction to: Andrei Bieger was omitted from the author list in the original version of this Article.The Author Contributions section now reads:\u201cAll authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by [M.M.], [A.L.B.] and [R.M.C]. R codes were developed and implemented by [A.B.]. The first draft of the manuscript was written by [M.M.] and [R.M.C]; and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u201dThe original Article and accompanying Supplementary Information file have been corrected."} +{"text": "Neuroblastic tumors of the adrenal gland in elderly patients: A case report and review of the literature By Deslarzes P, Djafarrian R, Matter M, La Rosa S, Gengler C, Beck-Popovic M and Zingg T. (2022) Front. Pediatr. 10:869518. doi: 10.3389/fped.2022.869518An Erratum on An omission to the funding section of the original article was made in error. The following sentence has been added: \u201cOpen access funding was provided by the University of Lausanne\u201d.The original version of this article has been updated."} +{"text": "Future opportunities for the athlete biological passport By Krumm B, Botr\u00e8 F, Saugy JJ and Faiss R. (2022) Front. Sports Act. Living 4:986875. doi: 10.3389/fspor.2022.986875An Erratum on An omission to the funding section of the original article was made in error. The following sentence has been added: \u201cOpen access funding was provided by the University of Lausanne.\u201dThe original version of this article has been updated."} +{"text": "The stapatients . The ovepatients B. Analyspatients .Figure\u00a01in vitro and in vivo.To study the role of ZYX in GC cells, we measured the expression of ZYX in normal gastric mucosal epithelial cells (GES-1), a panel of GC cell lines , and a primary GC cell line XN0422 by real-time PCR and western blotting. Both mRNA and protein levels of ZYX were increased in GC cell lines compared to normal cells . We consTo explore potential signaling pathways involved in the ZYX-regulated mobility of GC cells, we analyzed GC databases from TCGA_STAD, GSE35809, and GSE2685 via Gene Set Enrichment Analysis (GSEA).To identify potential ZYX-related kinases in GC cells, we examined kinases activated by ZYX using a human phospho-kinase antibody microarray. The results showed that the phosphorylation levels of eight kinases in XN0422-ZYX cells were higher than those in XN0422-Ctrl cells, among which the elevated level of p-WNK1 was the most significant F. To verWe further evaluated the relationship between ZYX and SNAI1 using TCGA_STAD and GSE35809 databases. mRNA levels of ZYX and SNAI1 were significantly and positively correlated with each other . GSEA shFinally, we examined the effect of WNK1 deletion on the ZYX-induced invasion of GC cells. Knockdown of WNK1 remarkabAltogether, our studies revealed that GC patients with high ZYX had a worse prognosis. ZYX might regulate EMT in GC through the WNK1/SNAI1 axis to promote the invasion and metastasis of GC cells, which was reversed by pharmaceutical inhibition of WNK1. Therefore, ZYX/WNK1 could be potential therapeutic targets for the treatment of GC.All patients in this study signed an informed consent form, and the subject was approved by the Ethics Committee of Southwest Hospital of Army Military Medical University. All animal experiments were approved by the Institutional Animal Care and Use Committee of the Southwest Hospital in accordance with the Guide for the Care and Use of Laboratory Animals.Designing research studies: Y.W., Y.H.C., and X.W.C.; Conducting experiments: J.Y., D.F.X., X.M.W., M.M.H., and X.L.G.; Acquiring data: Y.Q., X.X.Y., Z.X.Y., and Y.R.; Analyzing data: Y.W., X.W.B., Y.H.C., and X.W.C.; Writing the manuscript: Y.W., Y.H.C., and X.W.C.The authors declare that they have no conflict of interests.This work was supported by the Chongqing Academician Program (No. cstc2019yszx-jcyjX0008 to Y.W.) and The Subject of Health Commission of Hubei Province, China (No. WJ2021M222 to X.-M.W.)."} +{"text": "Hydromorphone prescription for pain in children\u2014What place in clinical practice? By Rodieux F, Ivanyuk A, Besson M, Desmeules J and Samer CF. (2022) Front. Pediatr. 10:842454. doi: 10.3389/fped.2022.842454An Erratum on An omission to the funding section of the original article was made in error. The following sentence has been added: \u201cOpen access funding was provided by the University of Geneva\u201d.The original version of this article has been updated."} +{"text": "Cell Prolif. 2019;52:e12591. https://doi.org/10.1111/cpr.12591Shen Q\u2010Y, Yu S, Zhang Y, et al. Characterization of porcine extraembryonic endoderm cells. Figures 1A and 1B are duplicates. The corrected image for Figure 1 is below:The error does not affect the overall results and conclusions of the study but may lead to confusion regarding the specific cell images.The online version of this article was corrected.We apologize for this error."} +{"text": "Park H\u2010B, Lee J, Hong Y, et al. Risk factors based vessel\u2010specific prediction for stages of coronary artery disease using Bayesian quantile regression machine learning method: results from the PARADIGM registry. Clin Cardiol. 2023;46:320\u2010327. doi:10.1002/clc.23964The first name of the third author, Dr. Hong, was misspelled as Yongtaek in the published version. The correct spelling is Youngtaek.We apologize for this error."} +{"text": "Long term NIV in an infant with Hallermann-Streiff syndrome: A case report and overview of respiratory morbidity By Guerin S, Blanchon S, de Halleux Q, Bayon V and Ferry T. (2022) Front. Pediatr. 10:1039964. doi: 10.3389/fped.2022.1039964An Erratum on An omission to the funding section of the original article was made in error. The following sentence has been added: \u201cOpen access funding was provided by University of Lausanne\u201d.The original version of this article has been updated."} +{"text": "Bacillus cereus food poisoningMulti-organ failure caused by lasagnas: A case report of By Thery M, Cousin VL, Tissieres P, Enault M and Morin L. (2022) Front. Pediatr. 10:978250. doi: 10.3389/fped.2022.978250An Erratum on An omission to the funding section of the original article was made in error. The following sentence has been added: \u201cOpen access funding was provided by the University Of Geneva\u201d.The original version of this article has been updated."} +{"text": "Correction: Biology of Sex Differences (2021) 12:17 10.1186/s13293-021-00360-9Following publication of the original article , the aut18ZXDBSY00120 to J.N.L) and National Key R&D Program of China .This work was partially supported by Grants from Natural Science Foundation of Tianjin (19JCZDJC36100 to C.J.L and"} +{"text": "Nature Communications 10.1038/s41467-022-31432-y, published online 04 July 2022Correction to: The original version of this Article contained an error in Figure 5c, and Supplementary Figures\u00a0The original version of this Article contained an error in the Author Contribution section, which incorrectly read \u2018E.R., D.C., K.K., G.B.-.A. and F.C. contributed to the interpretation of the results and the final form of the manuscript.\u2019 The correct version states \u2018E.R., D.C., K.K., G.B.-.A. and F.L. contributed to the interpretation of the results and the final form of the manuscript.\u2019 This has been corrected in both the PDF and HTML versions of the Article.Updated Supplementary Information"} +{"text": "Scientific Reports 10.1038/s41598-022-15782-7, published online 13 July 2022Correction to: The original version of this Article contained an error in the Competing Interests statement.\u201cThe authors declare no competing interests.\u201dnow reads:\u201cThis study was a research cooperation between the University of Bonn and Bayer AG. Bayer AG sponsored parts of the study. M.R., A.D., U.C., K.T., O.G., C.S., M.W., S.M., M.S., M.T.M., and S.G. were employees of Bayer AG during this research and supported the manuscript as indicated in the contribution section. S.S.H. was employed by the University of Bonn during his contributions to this research and is now an employee of BASF Vegetable Seeds. Any statements, findings or conclusions made in this publication are those of the authors and are not influenced by the sponsor or employer.\u201dThe original article has been corrected."} +{"text": "Nature Communications 10.1038/s41467-022-31483-1, published online 28 June 2022Correction to: The original version of this Article omitted from the author list the following authors: the 2nd author Solip Park, who is from the \u2018Centro Nacional de Investigaciones Oncol\u00f3gicas (CNIO), Madrid, Spain\u2019; the 3rd author Jose Espinosa-Carrasco, who is from the \u2018Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology (BIST), Barcelona, Spain\u2019; and the 4th author Daniel Ortiz-Mart\u00ednez, who is from the \u2018Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology (BIST), Barcelona, Spain\u2019. The corrected version of the Acknowledgements also removes the following from the original version: \u2018We thank Solip Park for method consultations, comments, and discussions.\u2019Additionally, the Author Contributions statement has been corrected as follows: \u2018M.V.P. collected and curated the data, implemented software, performed all analyses, visualized the data, and interpreted the results. S.P. supervised the processing of common germline variants for the PCA analysis and the definition of rare germline variants. J.E.C. performed data retrieval and bioinformatics preprocessing of the germline data of a part of the genomic data analyzed . D.O.M. performed data retrieval and bioinformatics preprocessing of a part of the genomic data analyzed (ICGC WGS data sets ESAD-UK and MELA-AU). F.S. and B.L. conceptualized the analysis, devised the methodology, interpreted results and supervised the study. M.V.P., B.L. and F.S. drafted the manuscript jointly.\u2019This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "The LAUsanne STAPHylococcus aureus ENdocarditis (LAUSTAPHEN) score: A prediction score to estimate initial risk for infective endocarditis in patients with S. aureus bacteremia By Papadimitriou-Olivgeris M, Monney P, Mueller L, Senn L and Guery B. (2022) Front. Cardiovasc. Med. 9:961579. doi: 10.3389/fcvm.2022.961579An Erratum on An omission to the funding section of the original article was made in error. The following sentence has been added: \u201cOpen access funding was provided by the University of Lausanne\u201d.The original version of this article has been updated."} +{"text": "Impact of central venous pressure during the first 24\u2005h and its time-course on the lactate levels and clinical outcomes of patients who underwent coronary artery bypass grafting By Zhao Y, Zhang H, Wang X and Liu D. (2023) Front. Cardiovasc. Med. 10:1036285. doi: 10.3389/fcvm.2023.1036285A Corrigendum on In the published article, there was an error in the order of the Funding statement. The original text \u201cThanks to the Beijing Municipal Science & Technology Commission for the financial support (no. Z201100005520038). Also to National High Level Hospital Clinical Research Funding (2022-PUMCH-B-026)\u201d has been corrected. The new version appears below."} +{"text": "A new dynamic word learning task to diagnose language disorder in French-speaking monolingual and bilingual children By Matrat M, Delage H and Kehoe M. (2023) Front. Rehabilit. Sci. 3:1095023. doi: 10.3389/fresc.2022.1095023An Erratum on An omission to the funding section of the original article was made in error. The following sentence has been added: \u201cOpen access funding was provided by the University of Geneva\u201d.The original version of this article has been updated."} +{"text": "Laparoscopic adrenalectomy has the advantage of reduced blood loss, early convalescence, and shorter hospital stays. Retroperitoneal laparoscopic approach was first demonstrated by Mercan et al. in 1995 and is easier for adrenal pathologies. Adrenal-preserving surgeries may prevent adrenal insufficiency from developing over long time. There are many studies in the literature which have compared the appropriate approach and the extent of resection for the functional adrenal tumor. But it remains unclear which is the optimal option. Here we present a video of laparoscopic partial adrenalectomy by a retroperitoneal approach in a patient with primary hyperaldosteronism.This procedure was performed by laparoscopic retroperitoneal approach for a small right adrenal lesion, which was an aldosterone-secreting benign tumor diagnosed preoperatively. Initially after creating a retroperitoneal space with the help of a balloon, the procedure was carried out with 2 working ports apart from the camera port. Intraoperatively, a well-circumscribed 2 cm adrenal lesion was identified. A partial adrenalectomy was performed.Total operating time was 1 hour 40 minutes. Estimated blood loss was 100 mL. There was no intraoperative or postoperative complication. In the postoperative period, the patient got relieved of the symptoms, and her antihypertensive drug requirements were reduced from 4 to a single drug. The patient was discharged on the third postoperative day.In summary, retroperitoneal laparoscopic partial adrenalectomy is an easy, fast, and organ-preserving approach that can be performed for unilateral hormone-secreting tumors.Video see link: https://doi.org/10.5152/tud.2023.23102This study was approved by Ethics Committee of Gujarat University of Transplantation Sciences (GUTS) University).Verbal informed consent was obtained from the patient who agreed to take part in the study.Externally peer-reviewed.Concept \u2013 S.R., K.M.; Design \u2013 S.R., K.M.; Supervision \u2013 S.R., K.P.; Resources \u2013 S.P., K.P.; Materials \u2013 K.M., K.P.; Data Collection and/or Processing \u2013 K.M., S.P.; Analysis and/or Interpretation \u2013 S.R., K.M.; Literature Search \u2013 K.M., S.P.; Writing \u2013 K.M., S.P.; Critical Review \u2013 S.R., K.M.The authors have no conflict of interest to declare.The authors declared that this study has received no financial support."} +{"text": "Prevalence of active trachoma infection and associated factors post\u2010war resettled population in raya kobo districts, North East Ethiopia: A community\u2010based cross\u2010sectional study in 2022. F. Kebede, M. Jamal. Health Science Reports. 2023. 10.1002/hsr2.1486.]Retraction: [wileyonlinelibrary.com), has been retracted by agreement between the journal's Editor\u2010in\u2010Chief, Dr Charles Young, and John Wiley & Sons, Inc. The retraction has been agreed due to major overlap with a previously published article from the same group of authors.The above article, published online on 06 August 2023 in Wiley Online Library ("} +{"text": "Scientific Reportshttps://doi.org/10.1038/s41598-022-23964-6, published online 18 November 2022Correction to: The original version of this Article contained errors.Salah Al-Ofi was incorrectly listed as an author of the original Article, and has subsequently been removed.The Author Contribution section now reads:\u201cS.A. and M.M. contributed to conception and planning of the study. S.A. and M.M. organized and supervised the work. S.A. performed the experiments. S.A. performed data analysis and representation. S.A. wrote the first draft of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.\u201dAdditionally, the Acknowledgement section:\u201cThe authors of this paper gratefully acknowledge the support provided by the department of Petroleum Engineering at King Fahd University of Petroleum & Minerals (KFUPM).\u201dnow reads:\u201cThe authors express their sincere gratitude to Schlumberger Dhahran Center for Carbonate Research (SDCR) for providing the necessary facilities and equipment to conduct the laboratory experiments featured in this study. This work was carried out in collaboration with the Department of Petroleum Engineering at King Fahd University of Petroleum & Minerals (KFUPM). The authors would like to acknowledge Mr. Salah Al-Ofi for his help and support.\u201dThe original Article has been corrected."} +{"text": "Mutation of residue 313 in the viral nucleoprotein from F/L to Y/V facilitates IAVs to escape from BTN3A3 restriction on virus replication. It is worrisome that regional migration of migratory birds, intensified livestock production, and global population mobility accelerate the cross\u2010species transmission of IAVs from other host species to humans.The spillover of avian IAVs from avian to human has caused several global pandemics, seriously affecting global economic development and threatening the health of humans and animals.H.F. designed the research. H.F., L.T., and M.L. read the papers and analyzed the data. L.T. and H.F. wrote and revised the manuscript. All authors have read and approved the final manuscript.The authors declare they have no conflicts of interest.2Not applicable."} +{"text": "Palliative care provision for people living with heart failure: The Geneva model By Hentsch L, Sobanski PZ, Escher M, Pautex S and Meyer P. (2022) Front. Cardiovasc. Med. 9:933977. doi: 10.3389/fcvm.2022.933977An Erratum on An omission to the funding section of the original article was made in error. The following sentence has been added: \u201cOpen access funding was provided by the University Of Geneva\u201d.The original version of this article has been updated."} +{"text": "The Xylariaceae and its relatives rank as one of the best-known members of the Ascomycota. They are now well recognized for their diversity, global distribution, ecological activities and their outstanding novel metabolites with wide ranging bioactivity. Professor Jack Rogers in his presidential address to the Mycological Society of America entitled \u2018The Xylariaceae: systematics, biological and evolutionary aspects\u2019 Rogers triggereHypoxylon\u2019 . In attempts to make their identification more manageable, the genus was arranged in subgroups based on size of the stromata the X. phyllocharis group and (3) the X. heloidea group based on stromatal shape and/or the conspicuousness of perithecial mounds on the stromatal surface Grev., which is its closest relative at a bootstrap value of 74 followed closely by C. fangjingshanensis Q.R. Li., J.C. Kang & K.D. Hyde, R. necatrix (Berl. ex Prill.) and R. aqulia (Fr.) De Not. Fr. from Iran and Europe comparing morphological, chemotaxonomic, and phylogenetic evidence and described the new species of H. eurasiaticum Pourmoghaddam, Krisal-Greihuber & Khodapand., H. pseudofuscum Pourmoghaddam, Khodap. In the UK H. fuscum usually grows on Corylus forming hemispherical to pulvinate stromata on bark or frequently effused-pulvinate stromata on decorticated wood. Collections made on Alnus or Betula deserve further examination since H. pseudofuscum appears to be strongly associated with Alnus, Quercus and Salix with H. eurasiaticum mainly associated with Quercus, thus, highlighting the importance of the host identity J.Kickx on Fagus, H. cercidicola (Berk. & M.A. Curtis ex Peck) Y.-M. Ju & J.D. Rogers and H. intermedium (Schwein: Fr.) Y.-M. Ju & J.D. Rogers on Fraxinus and several others as detailed in the monograph (Ju and Rogers Xylaria longipes Nitschke mainly found on Acer and Jackrogersella cohaerens (Pers.) L.Wendt, Kuhnert, & M. Stadler on Fagus There are also examples where the species are seen to be faithful to their host tree species. Dematophora buxi (Fabre) C. lambert, K. Wittstein & M. Stadler has been reported as occurring on Buxus sempervirens and is distributed in Southern France and Great Britain L\u00e6ss\u00f8e & Spooner ex Fr. West. and N. uda (Pers.) Gray. Interestingly both species are associated only with Quercus J.Kickx, Podosordaria Ellis & Holw., Poronia Willd. and Wawelia Namys. They are united by ascospores possessing dark walls, sticky sheaths, and multinucleate condition resembling those of their sordariaceous relatives S C. Jong & E.E. Davis has now been shown to have affinities with the coprophilous Xylariaceae, based on morphological, molecular, and chemical data Fr. on elephant dung (Deepa Latha and Manimohan P. elephanti was not recorded and Podosordaria leporina (Ellis & Everh.) Dennis was the only member of the Xylariaceae reported. Richardson Ces. & De Not. on fallen Fraxinus, its usual host in the UK, has been observed to develop over several years to maturity in water stressed environmental conditions. In Europe D. caldariorum Henn. is only found on burnt and weathered gorse, Ulex europaeus, which is another water stressed environment Rehm growing on a log pile or on fallen trunks or large branches in clearings in the forest where they are exposed to the sun and therefore experience periodic drying Whalley, E.B.G. Jones, K.D. Hyde & L\u00e6ss\u00f8e from Florida is now recognized as a common and widespread mangrove species S.M. Francis especially in crowded, humid conditions and not R. herpotrichoides Hepting & R.W. Davidson as was frequently cited as the culprit. Rosellinia herpotrichoides is noted to occur on Tsuga branches in more open situations rather than in overcrowded and humid environments and is restricted to the USA Fr. causes a serious root decay in macadamia in Hawaii P.M.D. Martin has long been recognized as causing a serious root rot in rubber trees (Vargese K. deusta (Hoffm: Fr.) P.M.D. Martin causes rot at the bases of a variety of tree trunks J.D. Rogers & Y.M. Ju or previous infections by wilt causing fungi. In quaking aspen, the gall forming insect Saperda inornata provides infection wounds for E. mammata J.D. Rogers & Y.M. Ju is, however, an important cause of canker in quaking aspen (Populus tremuloides Michx) responsible for a major loss of young trees in the forests of the Lake States of the USA Kuntze in Southern Europe (Vannini and Scarascia Mugnozza B. nummularia (Bull: Fr.) Kuntze) with beech in Sicily Pouzar causes a serious drought related disease on oaks (Thompson Camillea punctulata (Berk. & Rav.) L\u00e6ss\u00f8e, J.D. Rogers and Whalley infects oak in south eastern U.S.A. where it is linked with trees which are water stressed following prior infection with the oak wilt fungus Ceratocystis fagacearum (Bretz) J. Hunt (Davis C. tinctor (Berk.) L\u00e6ss\u00f8e, J.D. Rogers and Whalley is associated with canker of American sycamore (McAlpine1961) and plane (Hepting Hypoxylon rubiginosum (Pers.) Fr. and catalpa which was reported from the campus of the University of Georgia USA for bait by beating the trunks of the trees with clubs to dislodge them, thus causing localized injury leading to the development of cancer caused by Hypoxylon in Chang Mai Province at the beginning of the rainy season endophytic Xylariaceae proved to be the most frequent Pouzar, H. haematostroma (Mont) Fr.. and X. cubensis (Mont.) Fr. were frequent isolates with Daldinia being detected early in the rainy season and Xylaria species occurred later. The overwhelming number of publications on endophytes makes it impossible to do them justice here. This major interest in endophytes was certainly stimulated by the discovery of many novel compounds and the promising leads in new drug discovery in Thailand involved sampling in 3 geographical sites to allow comparisons between their endophytic assemblages and to evaluate these data in relation to differences in plant diversity, density and the local environment. Overall, members\u2019 species of Xylaria and Daldinia were most frequently isolated but representatives of Nemania and Hypoxylon were also recorded . Xylaria is the only major genus awaiting a World monograph. In certain genera distribution is tropical or subtropical whilst others occur in temperate countries. There are also those found mainly in either the North or South hemispheres. Camillea has for many years been considered generally restricted to the Amazon region. Now that there is a modern monograph, several species placed in Hypoxylon in secondary forest where the mangrove is in decline Y.M. Ju, J.D. Rogers & H.M. Hsieh was originally described from Argentina, but is also known from Australia, Indonesia, and New Zealand, probably associated with Nothofagus species L.E. Petrini which both exhibit adaptations to life in cooler situations. They occur at high altitudes and the ascospores of E. sinensis can only be germinated at low temperature Fr.PK108, X. cf. cubensis SWUF08-86, X. allantoidea (Berk.) Fr. SWUF76, and X. sp. SWUF08-37) all of which exhibited a degree of toxicity /against human cancer cell lines Y.-M. Ju, J.D. Rogers & H.M. Hsieh SWUF09-030 and bergamotene, guaiane, phthalide derivatives from Biscogniauxia whalleyi N.Wangsawat, C. Phosri and N. Suwanassai SWUF13-085 which showed both cytotoxic and anti-inflammatory activities (Pimjuk et al. Annulohypoxylon spougei Suwannasai, M.P. Martin, Phosri & Whalley showed significant effects against both radish and ruzi grass radicle elongation, which were comparable to the commercial herbicide, glyphosate (Pimjuk et al. Xylaria species in Thailand have indicated both promising antioxidant, antimicrobial and anticancer activities (Orachaipun et al. (Early studies on metabolites of the Xylariaceae reported that n et al. ; Orachain et al. .The publication \u2018The Xylariaceae: systematic, biological and evolutionary aspects Rogers \u2019 initiat"} +{"text": "Dear Editor,ex vivo therapy (Electroporation (EP) is a non-viral method for introducing macromolecules such as DNA, protein, and messenger RNA (mRNA) into mammalian cells for both basic research and therapy . Due to therapy . EP appl therapy . Since m therapy . TherefoThe CRISPR\u2013Cas9 genome editing system has been widely used due to its simplicity and high efficiency . The CRIWe performed a high-throughput screening of 10 different EP buffers paired with up to 45 EP programs in four different cell lines . By compCell density can greatly affect EP outcomes by modulating the electric current . The LonTo test whether it is compatible in our EP system, BSA or DMSO at the indicated dose was added to the EP buffer. Compared with the control, adding BSA or DMSO did not further improve the transfection .TRAC gene encoding T cell receptor (TCR). Since TCR is expressed on Jurkat cells, TCR expression detected by fluorescence-activated cell sorting (FACS) was used to assay the editing efficiency and knock in a GFP expression cassette into the RAB11A locus in Jurkat cells , the National Natural Science Foundation of China (31972936 to Y.Z.), Medical Science Advancement Program of Wuhan University (TFJC2018005), the Fundamental Research Funds for the Central Universities (to Y.Z.), and the startup funding from Wuhan University (to Y.Z.). Y.Z., H.Y., C.-P.Z., and J.A. have filed a patent application on buffer composition through Wuhan University . Y.Z. conceived, designed, and managed the project with support from K.-Q.Z.; C.-P.Z. designed, performed experiments, and analyzed data; H.-Y.Q. and Y.-M.Z. helped with experiments; C.-X.Z. and Y.-Z.Z. drew the heatmap; C.-P.Z. and Y.Z. wrote the paper with input from H.Y.]mjad018_Supplemental_FileClick here for additional data file."} +{"text": "The first sentence of these should read: \u201cThis material is based upon work supported by the National Science Foundation under Grant No. MCB1817764 to P.J.G.\u201d instead of: \u201cThis work was supported by the National Science Foundation (NSF-MCB1817764) to P.J.G.\u201d.This error has been corrected in the article."} +{"text": "Exceptional performance in competitive ski mountaineering: an inertial sensor case study By Kayser B and Mariani B. (2022) Front. Sports Act. Living 4:854614. doi: 10.3389/fspor.2022.854614An Erratum on An omission to the funding section of the original article was made in error. The following sentence has been added: \u201cOpen access funding was provided by the University of Lausanne\u201d.The original version of this article has been updated."} +{"text": "Assessment of human factors after advanced life support courses comparing simulated team and real team assessment: A randomized controlled cohort trial By Nabecker S, Huwendiek S, Seidl C, Hana A, Theiler L and Greif R (2022) Front. Cardiovasc. Med. 9:840114. doi: 10.3389/fcvm.2022.840114An Erratum on An omission to the funding section of the original article was made in error. The following sentence has been added: \u201cOpen access funding was provided by the University of Bern\u201d.The original version of this article has been updated."} +{"text": "Journal of Experimental Psychology: General. Advance online publication. November 30, 2020. https://doi.org/10.1037/xge0000991), formatting for UK Research Councils funding was omitted. The author note and copyright line now reflect the standard acknowledgment of and formatting for the funding received for this article.In the article \u201cUncertainty and Predictiveness Modulate Attention in Human Predictive Learning\u201d by Chang-Mao Chao, Anthony McGregor, and David J. Sanderson (All versions of this article have been corrected."} +{"text": "Surgery is the primary treatment that can offer potential cure for gastric cancer, but is associated with significant risks. Identifying optimal surgical approaches should be based on comparing outcomes from well designed trials. Currently, trials report different outcomes, making synthesis of evidence difficult. To address this, the aim of this study was to develop a core outcome set (COS)\u2014a standardized group of outcomes important to key international stakeholders\u2014that should be reported by future trials in this field.Stage 1 of the study involved identifying potentially important outcomes from previous trials and a series of patient interviews. Stage 2 involved patients and healthcare professionals prioritizing outcomes using a multilanguage international Delphi survey that informed an international consensus meeting at which the COS was finalized.Some 498 outcomes were identified from previously reported trials and patient interviews, and rationalized into 56 items presented in the Delphi survey. A total of 952 patients, surgeons, and nurses enrolled in round 1 of the survey, and 662 (70 per cent) completed round 2. Following the consensus meeting, eight outcomes were included in the COS: disease-free survival, disease-specific survival, surgery-related death, recurrence, completeness of tumour removal, overall quality of life, nutritional effects, and \u2018serious\u2019 adverse events.A COS for surgical trials in gastric cancer has been developed with international patients and healthcare professionals. This is a minimum set of outcomes that is recommended to be used in all future trials in this field to improve trial design and synthesis of evidence. Outcomes in surgical trials for gastric cancer are reported heterogeneously and do not sufficiently reflect the priorities of key stakeholders, including patients. This contributes to outcome reporting bias and research waste. To address these challenges, a core outcome set\u2014a minimum group of critically important outcomes to be reported in future trials\u2014 has been developed through an international consensus process involving patients and healthcare professionals. Gastric cancer is a significant global health burden which is associated with poor survivalHomogeneity in the selection and reporting of key outcomes between studies is necessary if useful synthesis of evidence is to be achieved. However, outcome reporting in surgical trials for gastric cancer is heterogeneous and not based on methodologically robust standardsTo address this challenge, the GASTROS study was undertaken to develop a core outcome set (COS) for surgical trials in gastric cancerhttps://www.comet-initiative.org) is an initiative that aims to promote COS development,These challenges in outcome reporting are not limited to the field of gastric cancer and affect virtually all clinical areas. COMET Fig. 1. This publication describes stages 1 and 2.The scope, objectives, and methodological approaches of this study have been described previously in detailThe GASTROS study aimed to consider the views of key stakeholders during the process, namely patients, surgeons, and oncology nurses. The guiding principle was to promote the patient voice as they are the beneficiaries of trials in this field and have all-important lived experience. Surgeons provide a clinical perspective and the experience of treating large volumes of patients. Oncology nurses were invited to participate given their central roles as caregivers, patient advocates, and core members of the clinical team. Participation in the study was open to all interested stakeholders who fulfilled the following criteria: surgeons who had completed their training and routinely treat gastric cancer, oncology nurses with a recognized proportion of their role involved in the care and follow-up of patients with gastric cancer, and patients who have undergone surgical resection for gastric cancer with the intention of cure. Patients and healthcare professionals were identified through local, regional, and national clinical and research networks. Support from patient groups, charities, and professional societies was also key.Fig. 1) identified outcomes that may be important to stakeholders and stage 2 subsequently prioritized outcomes for inclusion in the final COS (what to measure). In addition, the GASTROS study aimed to collate the corresponding outcome measurement instruments used in surgical trials and to determine the variability in measurement time points, for use in future outcomes research (to determine how and when to measure) in gastric cancer.Stage 1 was initiated through discussion within the Study Management Group (SMG) to merge closely related items and map them against a taxonomy developed for COSsThe rationalization process in a two-round online Delphi survey. Participants were given the opportunity to add outcomes they considered were missing, for consideration by participants in round 2. Suggested additional outcomes were considered by the SMG and reviewed independently by an external methodologist with experience of cancer-related surgical COSs. The scores of each stakeholder group were collated and summarized separately to ensure an equal voice among stakeholder groups. Participants who had completed 50 per cent of the first survey were included in the round 1 analysis and invited to participate in round 2. They were then presented with group scores (presented as score distribution charts) for each stakeholder group from round 1, and given the opportunity to reflect on the opinions of others before deciding whether to change their scores for each outcome in round 2. Those who changed scores between rounds were able to provide a reason for thisAny outcome scored as critically important (7\u20139) by more than 70 per cent and not important (1\u20133) by less than 15 per cent in all three stakeholder groups was categorized for inclusion. Any outcome scored as critically important (7\u20139) by less than 50 per cent in all three stakeholder groups was categorized for exclusion. These criteria were adapted from established COS methodologySurvey participants were invited to attend a consensus meeting in Manchester (UK) during March 2020. The aim of the meeting was to review the results of the Delphi survey and consider the outcomes for which no consensus was reached before finalizing the COS. Participants could take part by attending the meeting venue in person, or through an online platform. The meeting was undertaken in English and chaired by a clinical academic from the SMG with experience in COS development, and with no clinical expertise in the management of gastric cancer.Following discussion, stakeholders were asked to score outcomes using the same criteria as was set out in the Delphi survey. Similarly, scores from each stakeholder group were considered separately to mitigate for imbalance in the numbers of each participant type. Turning Point software was used to support voting at the venue and online simultaneously. Participants were also asked to complete an online voting form to mitigate against software malfunction. Outcomes reaching the original consensus criteria for inclusion in the final COS were to be added to those included from the Delphi survey.t test to examine for statistically significant differences at the 0.05 level. Furthermore, the characteristics of stakeholders participating in both rounds were compared with those who only completed round 1. A descriptive analysis was undertaken, and the c2 test applied to examine for statistically significant differences at the 0.05 level.To assess the impact of attrition bias between survey rounds, mean scores of participants completing both rounds of the Delphi survey were compared against those of participants completing round 1 alone. Mean scores of those who took part in the Delphi survey but did not attend the consensus meeting were compared against the mean scores of those who attended to assess the degree to which consensus meeting participants were representative of those who participated in the survey. Both analyses were undertaken using a A guiding principle of the GASTROS study was that patients\u2019 voices should be represented at each stage of the project. Patient representatives were integral with membership in the SAG and support from international charities. The dissemination of results from this study will be supported by a network of international charities and patient support groups. The patient representatives opted to participate in the patient-focused study report of results rather than the scientific dissemination.http://www.comet-initiative.org/studies/details/764?result=true) before commencement. The study protocol has been described previouslyThe GASTROS study was registered in the COMET database and governance approvals by Central Manchester University Hospital National Health Service (NHS) Foundation Trust. The Delphi survey was given ethical approval by the North West\u2014Greater Manchester East Research Ethics Committee (18/NW/0347) and governance approvals by Manchester University Hospitals NHS Foundation Trust. Both the patient interviews and Delphi survey were adopted on to the National Institute for Health Research (NIHR) Clinical Research Network Portfolio (IDs 33312 and 38318). Ethical approval for international participants was sought and obtained locally by IWG collaborators.Fig. 2. The 498 outcomes identified from the systematic review, patient-reported outcome measures, and patient interviews were rationalized by the SMG into 58 items, which were presented to the SAG. The SAG merged chyle leak, nutritional complications, respiratory function, surgical-site infection, and time to ambulation into other existing outcomes. Bleeding, anaesthetic complications, and destination on discharge were expanded or added as separate outcomes, which meant that a total of 56 items were presented to participants in the Delphi survey (Table S3).The results for each stage of the study are summarized in Table 1 summarizes the characteristics of those included in the analyses. Table S4 details the results of voting in both rounds. One additional outcome (duration of stay in an intensive care ward) suggested by participants in round 1 was presented in round 2 along with the original 56 outcomes for rescoring . Although other outcomes were suggested by participants in round 1, these were deemed by the SMG and an external reviewer as either direct duplication of outcomes already included or not sufficiently unique that they warranted being presented separately (Table S5). Scores from 662 participants in round 2 were included in the final analysis representing an attrition rate of 30 per cent. Some 557 participants (84 per cent) changed the score of least one answer from round 1, with 191 (29 per cent) participants changing a score to cross a boundary . A detailed analysis exploring the reasons for changing scores has been reported previouslyA total of 1021 patients, surgeons, and oncology nurses registered for the Delphi survey, of whom 952 from 55 countries across six continents fulfilled the criteria for inclusion in the round 1 analysis. Consensus was reached to include 13 outcomes: disease-free survival, disease-specific survival, surgery-related death, recurrence of cancer, completeness of tumour removal, overall quality of life, nutritional effects, all-cause complications, intraoperative complications, anaesthetic complications, anastomotic complications, multiple organ failure, and bleeding.Thirteen outcomes were categorized for exclusion, and no consensus was reached for 31 outcomes, which were subsequently discussed at the consensus meeting. An analysis exploring the relationship between participant characteristics and their impact on how outcomes were scored is under reviewP\u2009=\u20090.76).There was no statistically significant difference between the mean scores of participants completing both survey rounds and those completing round 1 only (mean(s.d.) difference 0.17(0.1), largest difference 0.4; P < 0.001).Forty-three Delphi survey participants attended the consensus meeting in person (18) or using the online platform (25). Fourteen countries from four continents were represented. A full breakdown of the regional origin of participants is described in In preparation for the consensus meeting, the SMG reviewed and discussed the Delphi results. Of the 13 outcomes that reached consensus to be included, six related to perioperative complications. The SMG took the view that, as the outcome all-cause complications was voted for inclusion, by extension, all complications would need to be measured and reported by researchers as a minimum. However, 14 complication-type outcomes from the list of 57 did not reach consensus for inclusion and a further two outcomes reached consensus for exclusion from the COS. The SMG decided to present this seemingly contradictory position at the consensus meeting for further discussion and voting on a desired final position.After an interactive debate, participants were asked to vote for one of five propositions: all complications to be reported individually as a minimum; all \u2018serious\u2019 (without defining the term serious) complications to be reported as a minimum; outcomes meeting the criteria as core as set out by the GASTROS study to be included; unsure; and other options. The result of this live vote was presented to participants, who were given an opportunity for further discussion ahead of a final vote. The result of the second vote is shown in Table 2. Participants agreed that future work on complications, definitions, and when outcomes should be reported should involve both patients and healthcare professionals.Non-complication-type outcomes for which there was no consensus to include or exclude in the Delphi survey were then discussed. Results from the subsequent voting are presented in The original study protocol described a three-round Delphi survey. Based on emerging evidence at that timeThe GASTROS study has developed the first COS for use in surgical trials for gastric cancer. This represents a significant step towards addressing the current challenges related to outcome reporting, synthesis of evidence, and research waste in this field. Outcomes in the set were identified as critically important through an inclusive international consensus process involving patients and healthcare professionals. Further work is required to develop the COS, in particular, finalizing definitions, seeking consensus on how complications should be included, and identifying appropriate outcome measurement instruments. In its present form, the COS will guide trialists to which outcome domains should be reported.A COS can only achieve its stated aims if it is used by researchers. From the outset of the study, the SMG set out a clear strategy to ensure buy-in by researchers and professional bodies. This resulted in broad international support, and the development of a network that enabled the recruitment of over 1000 participants to the Delphi survey. These participants were well balanced in terms of regional origins and personal or professional experience of gastric cancer. A strength of the study is that the methodology used is based on consensus guidelines and has been transparently reported in detail at each stageA COS is a minimum set of critically important outcomes. It does not limit trialists in their reporting of other outcomes of interest. Furthermore, it should be noted that some grant-awarding bodies can make their own recommendations regarding which outcomes should be reported as a minimum. An example would be the recommendation to report overall survival, which was not prioritized through the present consensus process,The study was unable to achieve agreement with respect to which complications should be included in the COS. The consensus meeting could not decide whether all complications or only \u2018serious\u2019 complications should be reported as a minimum. However, the overwhelming majority voted for one of these options, which will be the focus of future work in this area. Other surgical cancer-related COSs have differed in their recommendations for the reporting of complications. Some have included only a small number of \u2018serious\u2019 or \u2018core\u2019 complication-related outcomesThe term \u2018serious\u2019 was purposefully not defined at the consensus meeting so as not to remove focus from the discussions. Others have already attempted to define this; the Gastrectomy Complications Consensus Group (GCCG) is a collaboration of European surgeons who have prioritized a list of 27 clearly defined complications which should be reported as a minimum in research, audits, and registriesDefining outcomes is an area that deserves further consideration. The present approach was to use plain-language descriptions to define outcomes presented in the Delphi survey and consensus meetings. These were developed with the support of the SAG and an independent patient group. This was necessary to ensure that patients were engaged throughout the study and made translations easier. It is acknowledged that these may not be adequately detailed for use in trials, and more work is required with researchers and patients to address this for the outcomes included in the COS. Substantial work has already been undertaken by the StEP-COMPAC (Standardized Endpoints in Perioperative Medicine\u2014Core Outcome Measures in Perioperative and Anaesthetic Care) group to identify available definitions for outcomes from several systematic reviews,Identifying which outcomes to measure is the first step in standardizing outcome reporting in this field. Although many outcomes in the COS are event-type outcomes , some are composite outcomes which require the use of an instrument to measure . There is currently no standardized approach to measuring these outcomesThere are limitations to the present study that require discussion. It could be argued that, given the multimodal nature of treatment for gastric cancer, the COS would be more relevant if it incorporated all therapies including chemotherapy, radiotherapy, and endotherapy. However, at the time that GASTROS was conceived, there were 24 ongoing surgical trials planning to recruit 11 000 patients for whom non-surgical-related outcomes would not be applicable or relevant. The timing of support from Japanese and Korean collaborators meant that their participation was through the English-language Delphi survey which likely influenced the recruitment of patients from these countries. Nonetheless, an exploratory analysis of the Delphi survey results suggested that patients from Eastern countries did not prioritize outcomes differently from their Western counterpartsAnother consideration relates to the type of stakeholder groups recruited to the study. It was agreed to limit participation to patients, nurses, and surgeons as this represented a balance of a broad spectrum of opinion but ensured that the study\u2019s coordination and data analysis were manageable. It should be acknowledged that other groups, such as caregivers, allied health professionals, regulators, policymakers, and grant awarding bodies, will also provide valuable opinion. Inclusion of these groups will be considered in future stages of the GASTROS study and when the COS is reviewed.The consensus meeting was held in English, limiting participants to English speakers only. Although there was a broad spectrum of international representation from the surgeon group, this was not mirrored by the patient or nurse stakeholders who were primarily UK-based. That said, no further outcomes were added following discussions, supporting the validity of the Delphi process, which recruited widely in terms of regional origin and other demographic characteristics across all three stakeholder groups.GASTROS International Working Group Collaborators: S. Li; Y. L. He; Z. Xu; Y. Xue; H. Liang; G. Li; E. Zhao; P. Neumann; L. O'Neill; E. Guinan; D. Zanotti; G. de Manzoni; E. R. C. Hagens; M. I. van Berge Henegouwen; P. Lages; S. Onofre; R. M. Restrepo Nu\u00f1ez; G. Salcedo Caba\u00f1as; M. Posada Gonzalez; C. Marin Campos; B. Candas; B. Emre Baki; M. Selim Bodur; R. Yildirim; A. Burak Cekic; J. Brown; K. Hayes. Participants in international Delphi survey: I. Daher; R. H. Gianchandani Moorjani; A. Adetoyese Adeyeye; A. Sulaiman Olayide; A. Mitsuo Leon-Takahashi; A. Pueyo Rabanal; A. Peri; A. Boddy; A. Novotny; A. Charalabopoulos; A. Alemdar; A. Souadka; A. M. Rodrigues Gomes; A. L\u00e1zaro; A. Maciel Da Silva; A. do Ros\u00e1rio da Concei\u00e7\u00e3o Silva e Santos; A. Guidi; A. J. Silva Bernardes; A. Quinn; A. Isik; A. A Slipek; B. Canda\u015f Altinba\u015f; B. Johnson Alegbeleye; B. Wool Eom; B. Frittoli; B. Lonsdale; B. Rogers; B. J. Ammori; B. Rau; B. Molteni; B. E. Byrne; B. A. Villac\u00eds-Bermeo; B. E. Villac\u00eds Gallardo; B. K\u00f6se; C. J. Sampedro Nogueira; C. Loureiro; C. M. Oliveira de Sousa; C. G. Collins; C. Nonso Ekwunife; C. Chukwunwendu Osuagwu; C. L.-Y. Wong; C. Winkler; D. Reim; D. W. Kj\u00e6r; D. Cooper; D. Horner; D. Irvine; D. J. Bowrey; D. J. Chuter; D. Elliot; D. McGhee; D. Toth; D. \u00d6fner; D. K. Manatakis; D. R. Silveira Martins; E. J. T. Belt; E. Cattaneo; E. Samadov; E. Colak; E. Treppiedi; E. Guglielmi; E. Redondo-Villahoz; E. Ciferri; E. Tiemens-de Graaf; E. Cocozza; E. Pape; E. S. Drozdov; F. Enrico; F. Rashid; F. Marco; F. Rosa; F. Mingol Navarro; F. Simionato Perrotta; F. S.-Y. Chan; F. D. Saavedra Tomasich; F. R. Takeda; F. Farrell; F. Olanike Wuraola; G. Rosero; G. Bevilacqua; G. Baronio; G. Mura; G. de Manzoni; G. D'Eugenio; G. Ortega-Perez; G. Tilt; G. Sutcliffe; G. Mureddu; G. Guerra Jacob; G. H. Daneri; H. Olufemi Gbenga; H. Okabe; I. Kingsford Smith; I. Olawale Lateef; I. Garosio; \u0130. Hatipo\u011flu; I. Gockel; I. Negoi; I. S.-H. Min; I. M. M. Mesquita; I. Diez del Val; J. H. F. Leemhuis; J. A. Gossage; J. Weindelmayer; J. R. Izbicki; J. McKenzie Manson; J. Kelly; J. H. M. B. Stoot; J. W. Haveman; J. D. Brown; J. Sultan; J. Hassall; J. van Sandick; J. H. Saunders; J. K. Clarke; J. Heisterkamp; J. I. Vargas R; J. M. Couselo Villanueva; J. Ingmire; J. McEwen; J. Galindo \u00c1lvarez; J. Turner; J. Peng; K. Roberts; K. G. Brandon; K. Mitchell; K. McCarthy; K. Akhtar; K. N. Mikhailovich; L. Corbelli; L. Medeiros Milhomem; L. Solaini; L. Fengyuan; L. Xinchun; L. Timmermans; L. Porritt; L. Taglietti; L. Bonavina; L. F. Pinheiro; M. de los Angeles Mayo Ossorio; M. Schiavo; M. Marchesiello; M. das Dores Vieira Leite; M. DeMois; M. Posada Gonzalez; M. T. Di Felice; M. I. van Berge Henegouwen; M. D. de Sousa; M. Takahashi; M. Forshaw; M. Berselli; M. Paro; M. A. Usta; M.-H. Yan; M. Pinchin; M. CapriolI; M. Rubbini; M. Cowen; M. A. Herrera Servin; M.-Z. Li; M. Sasako; M. Shukri Jahit; M. Ngonyoku Muhinga; M. A. Tareen; M. F. Ahmad; M. S. Bodur; M. Kaban; N. Farooq; N. Coburn; N. Cooper; N. S. Blencowe; N. Loria; N. de Vries; N. Adami Andreollo; N. K\u00f6ksal; N. Zanini; N. Kreuser; N. Okkabaz; O. Damiana; O. Afuwape; O. Kayode Fasiku; O. Comensoli; O. F. Koroye; P. Capener; P. Morgagni; P. M. Pernadas Lages; P. M. Wilkerson; P. Turner; P. Dutton; P. Hayes; P. Vorwald; P. Singh; Q. Gan; R. Kottayasamy Seenivasagam; R. Ayloor Seshadri; R. Guevara Castro; R. Douglas; R. M. Koshy; R. Y\u0131ld\u0131r\u0131m; R. J. E. Skipworth; R. A. Gould; R. C. Wetherill; R. Shaw; R. A. Burley; R. Palatucci; R. Racalbuto; R. M. Correia Casaca; S. M. Lagarde; S. Gana; S. Marietti; S. Qureshi; S. Morales-Conde; S. Molfino; S. G. Barreto; S. Turkyilmaz; S. Turan-Trabzon; S. Frisch; S. Castoldi; S. Belloni; S. Flisi; S. Galloway; S. R. Maria; S. Royston; T. Boyle; T. \u00d6. Sezer; V. Mengardo; V. Concepci\u00f3n Mart\u00edn; V. Lee Wills; V. Owen-Holt; V. Casagrande; W. Al-Khyatt; W. Jansen; W. Wang; W. Eshuis; W. P. Polkowski; X. Huang; X. Wang; X.-Z. Chen; Y. Gonzalez Dominguez; Y. Wang; Y. K. S. Viswanath; Y.-L. He; Z. Demir; Z. Na.www.igca.info), Association of Upper Gastrointestinal Surgeons of Great Britain and Ireland (www.augis.org), Brazilian Gastric Cancer Association (www.abcg.org.br), Canadian Gastric Cancer Association (www.gastriccancer.ca), Chinese Gastric Cancer Association, Dutch Upper Gastrointestinal Cancer Group (www.ducg.nl), GASTRODATA group (www.gastrodata.org), Italian Research Group for Gastric Cancer (www.gircg.it), Korean Gastric Cancer Association (www.kgca-i.or.kr), Oesophago-Gastric Surgery Section of the Asociaci\u00f3n Espa\u00f1ola de Cirujanos\u2014Spain (www.aecirujanos.es), Upper Gastrointestinal International Robotic Association (www.ugira.org), United Kingdom Oncology Nursing Society (www.ukons.org.uk), European Oncology Nursing Society (www.cancernurse.eu), Oesophageal Patients Association\u2014United Kingdom (www.opa.org.uk), My Gut Feeling\u2014Canada (www.mygutfeeling.ca), No Stomach for Cancer\u2014USA (www.nostomachforcancer.org), Vivere Senza Stomaco\u2014Italy, Gastro/Oesophageal Support and Help Cancer Group (Bristol)\u2014UK, and Greater Manchester Oesophago-Gastric Patient Support Group.The authors highlight the role undertaken by A. Metryka, Senior Clinical Trials Coordinator, who facilitated the running of this study; and thank the following associations and groups for their support in facilitating recruitment to the GASTROS study Delphi survey: International Gastric Cancer Association . J.M.B. is partially funded by the NIHR Bristol Biomedical Research Centre and the Medical Research Council (MRC) ConDUCT-II Hub for Trials Methodology Research. P.R.W. was funded by the MRC North West Hub for Trials Methodology Research (MR/K025635/01).This paper presents independent research funded by the NIHR. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health.P.R.W. and I.A.B. are joint senior authors. The data sets analysed for the present study are available from the corresponding author on reasonable request.Disclosure. The authors declare no conflict of interest.znab192_Supplementary_DataClick here for additional data file."} +{"text": "The affiliation for the third author is incorrect. Lin Ling is not affiliated with #1 but only with #2: Inspection and Quarantine Technology Communication Department, Shanghai Customs College, 201204, Shanghai, P.R. China.flyrui0318@163.com.Lin Ling\u2019s email address is also incorrect. The correct email address is:"} +{"text": "Dear EditorJournal Advances in Nutrition in 2023 . N.T. is the Director of the Nutrition Coalition, U.S., a non-profit group that has a policy not to accept any industry funding."} +{"text": "Quality of life and sexual health after perineal reconstruction in Fournier gangrene using pedicled anterolateral thigh flaps By Rossi SA, de Schoulepnikoff C, Guillier D, Raffoul W and di Summa PG. (2022) Front. Surg. 9:994936. doi: 10.3389/fsurg.2022.994936An Erratum on An omission to the funding section of the original article was made in error. The following sentence has been added: \u201cOpen access funding was provided by the University of Lausanne\u201d.The original version of this article has been updated."} +{"text": "Andrew W. Liebau has been changed to Rowyn C. Liebau."} +{"text": "The Pan African Medical Journal. 2022;43: 74. doi: 10.11604/pamj.2022.43.74.35639. The error affected only the author\u2019s name and an orthographic error in the abstract of the article. The name of the author was written Dale Banhart instead of Dale Barnhart in the article \u201cGlycemic control among patients with type 2 diabetes in a low resource setting in Rwanda: a prospective cohort study. The Pan African Medical Journal. 2022;43: 74\u201d . This er"} +{"text": "In the published version of this article, there was an omission in the Funding Information.The BBSRC funder grant reference number, BB/R009724/1, should have been included.Previously, the Funding Information appeared as below:This work was funded by a Wellcome Trust funded Investigator award to R.C.M. (grant reference number: 212258/Z/18/Z), and a grant from the BBSRC. D.A. is funded by a PhD studentship awarded by the Saudi Arabian Cultural Bureau.The correct Funding Information should have appeared as:This work was funded by a Wellcome Trust funded Investigator award to R.C.M. (grant reference number: 212258/Z/18/Z), and a grant from the BBSRC (grant reference number: BB/R009724/1) . D.A. is funded by a PhD studentship awarded by the Saudi Arabian Cultural Bureau.The author apologizes for any inconvenience caused."} +{"text": "Comparison of treatment outcomes of stable and unstable developmental dysplasia of the hip with the T\u00fcbingen splint By Chaibi E, Saugy C-A, Samara E, Zambelli P-Y and Merckaert SR. (2022) Front. Pediatr. 10:976367. doi: 10.3389/fped.2022.976367An Erratum on An omission to the funding section of the original article was made in error. The following sentence has been added: \u201cOpen access funding was provided by the University of Lausanne\u201d.The original version of this article has been updated."} +{"text": "Scientific Reports 10.1038/s41598-020-76169-0, published online 05 November 2020Correction to: The original version of this Article contained errors.Firstly, Eleni Spanidi and Konstantinos Gardikis were omitted from the author list in the original version of this Article.The Author Contributions section now reads:\u201cS.L. conceived, planned, and oversaw the experiments. A.B. carried out the experiments on the RT-qPCR analysis. A.H. and J.R. initiated the callus cell line and carried out experiments on the antioxidant assay and on HPLC analysis. S.L. analyzed and integrated the datasets and drafted the manuscript. K.G. conceived the project. K.G. revised the manuscript. E.S. collected the material and carried out experiments on antioxidant assays. All authors critically read and contributed to improving the MS.\u201dSecondly, the Acknowledgements section of this Article: \u201cThis study was made possible thanks to financial support provided by APIVITA SA.\u201d has been removed.Paeonia officinalis var. mascula\u201d (and the corresponding abbreviation \u201cPOCE\u201d) is now\u00a0used throughout the article. Additionally, sentences implying potential application of the plant extract in cosmetics have been removed.Lastly, two synonymous names have been used in the paper to describe the studied species. This has been corrected for consistency, and \u201cThe original Article and accompanying Supplementary Information file have been corrected."} +{"text": "Karunakarnta Goswami, born on 2 June 1947, was as active member of I.S.A. We regret to inform the sad demise of Dr. Goswami who passed away on 21Dr. Goswami was the Vice-President of Guwahati City Branch of I.S.A. at the time of his death. He did M.D. (Anaesthesiology) in 1986 from Guwahati Medical College and after that was engaged in freelance practice and established himself as a renowned anaesthesiologist of Guwahati. He took special interest in pain management and was managing his own acupuncture clinic. Dr. Goswami was very regular in attending national conference of I.S.A. He left behind his wife Dr. Iva Goswami, a prominent Gynaecologist, a son and daughter. Guwahati City Branch of I.S.A. deeply mourns the untimely demise of Dr. Goswami."} +{"text": "There is an error in the third sentence of the funding section. The sentence should read: \"J.W.S. is grateful to the School of Dentistry, University of Liverpool, for financial support for D.P.B.\""} +{"text": "There was information omitted from the Funding statement. The correct version of the statement is available below.This work was supported by National Institutes of Health grants 1R01GM088344-01 and P50GM068763. Edoardo M. Airoldi was supported by NSF CAREER IIS-1149662, NIH R01 GM096193, and a Alfred P. Sloan Research Fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."} +{"text": "Author My N. Helms should not have been given Equal Contribution. The two Equally Contributing first authors are Charles A. Downs and David Q. Trac."} +{"text": "The Funding section was missing information. The complete Funding statement reads: \"This work was supported by the CARDIA contracts (N01-HC-48047 \u2013 N01-HC-48050 and N01-HC-95095), by grants P30 AG028748, 5R01 AG26105-3 to A.K., R01-AG034588 and R01-AG026364 to M.R.I., and P30 AG017265 to T.E.S. from the National Institute on Aging of the US National Institutes of Health, and VA Greater Los Angeles Healthcare System Geriatric Research, Education and Clinical Center; and VA Advanced Geriatrics Fellowship to S.I. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\"The legend for Figure 1 was incorrect. The correct legend for Figure 1 reads: The predicted probabilities were computed from multiple logistic regression models while all other covariates were held constant . Panel A. Model-predicted probabilities of one-time and repeated CRP elevation. Panel B. Model-predicted probabilities of repeated CRP elevation among those with one or more CRP elevations above 10mg/L."} +{"text": "The following article was found to be a redundant publication and is retracted herewith with this notice.Safaei AA, Maleknejad S. Clinical and laboratory findings and therapeutic responses in children with nephrotic syndrome. Indian J Nephrol 2010;20:68-71.Editor, Indian Journal of Nephrology"} +{"text": "The following information was missing from the Funding section: B.L.M. was supported by the Childhood Infections Research Program (ChIRP) training program (1T32AI095202-01)."} +{"text": "There was an error in the sixth author's name. Karel G.M. Moons is correct."} +{"text": "The name of the seventh author is incorrect. The correct name is: Matthew B.A. McDermott."} +{"text": "The following grant was missing from the Funding section: T.A. was a predoctoral appointee of a National Cancer Institute training grant (T32CA009140)."} +{"text": "The name of the sixth author was spelled incorrectly. The correct name is: Hasabelrasol F.A. Elhaj."} +{"text": "Salary support for A.P.P. Zhang was provided by training grant 5T32 AI007244 from the National Institute of Allergy and Infectious Disease (NIAID)."} +{"text": "There was a name missing from the Acknowledgments. The following is the correct version:We thank A. Miyazawa, A. J. Driessen and D. Oliver for providing the strains and the plasmids. We thank R.Henderson for providing the programs TILTDIFF and TILTDIFFMULTI. We also thank the comments of H.W. Wang."} +{"text": "The equal contributions designation was erroneously omitted from the published manuscript. Authors Eoin J. O\u2019Gorman and David W. E. Hone contributed equally to this work."} +{"text": "The authors wish to add the following funding sources to their financial disclosure information: RO1 EY017653 and The Edward N. Della L. Thome Memorial Foundation, Bank of America N.A., Trustee."} +{"text": "Several grants are missing from the funding information. The complete funding information is:\"This work was supported by grants from the US National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS) NS061152 (Z.Y.), NS060809 (Z.Y.), RNS055683A (Z.Y.), Michael J. Fox Foundation (Z.Y.), the US National Institutes of Health/National Center for Research Resources (NIH/NCRR) RR00862 (B.T.C.), RR022220 (B.T.C.), and 2P20 RR020171 (Q.J.W.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\""} +{"text": "A grant to the first and third authors was incorrectly omitted from the Funding Statement. The following sentence should be read following the first sentence in the Funding Statement: \"Z.X. and P.R. are also supported by Robert A. Welch Foundation (F-1691).\""} +{"text": "There was a formatting error in the title. The correct title is: PEG Precipitation Coupled with Chromatography is a New and Sufficient Method for the Purification of Botulinum Neurotoxin Type B. The correct citation is: Zhao Y, Kang L, Gao S, Gao X, Xin W, et al. (2012) PEG Precipitation Coupled with Chromatography is a New and Sufficient Method for the Purification of Botulinum Neurotoxin Type B. PLoS ONE 7(6): e39670. doi:10.1371/journal.pone.0039670"} +{"text": "The authors' names were incorrectly listed as Kroeger N., Belldegrun A. S., and Pantuck A. J.; the correct format is shown above."} +{"text": "The following authors should have been designated as contributing equally to the article, and thus sharing first authorship: Judith Spijkerman and Sabine M.P.J. Prevaes."} +{"text": "The name of the sixth author was given incorrectly. The correct name is: Luiz R. G. Bechara. The correct abbreviation in Citations is: LRGB."} +{"text": "Multiple grants and a funding organization were incorrectly omitted from the Funding Statement. The Funding Statement should read: \"This work was funded by grants from the National Institutes of Health DK078851, DK090262 (C.N.L.), DK091976 (D.L.M.) and HD007513 (K.S.). Additional support from Endocrine Fellows Foundation and Pediatric Endocrine Society for K.S.. The funders had no role in study design, data collection, decision to publish, or preparation of the manuscript.\""} +{"text": "The following information was incorrectly omitted from the Funding section: \"J.T. was in part supported by US National Institutes of Health grant R03AR061565.\""} +{"text": "A funder is incorrectly omitted from the Funding statement. The correct Funding statement is as follows:This work was supported by a Polish-German cooperation grant (S007/P-N/2007/01); National Science Center grant N N401 324739; EMBO Short-Term Fellowship to E. Z. (ASTF 211.00.2007); GA No 264173 (Bio-Imagine) and statutory funds for the Nencki Institute. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."} +{"text": "Molecular Vision v18:280-289), the following corrections were made:In this article by Lisa J. Roberts and colleagues (A correction was made to Table 4, page 286. The numbering of the nucleotide change was corrected from \"c.1885C>T\" to \"c.1804C>T\"."} +{"text": "The name of the second author is incorrect. The correct name is: Mazhar I. Khan. The correct abbreviation for this author's name in in the Author Contributions statement is: MIK. The name of the third author is also incorrect. The correct name is: Ion M\u0103ndoiu. The correct abbreviation in the Author Contributions statement for this author's name is: IM. The correct Citation is: Huang Y, Khan MI, M\u0103ndoiu I (2013) Neuraminidase Subtyping of Avian Influenza Viruses with PrimerHunter-Designed Primers and Quadruplicate Primer Pools. PLoS ONE 8(11): e81842. doi:10.1371/journal.pone.0081842."} +{"text": "Clonorchis sinensis is a carcinogenic human liver fluke that is widespread in Asian countries. Increasing infection rates of this neglected tropical disease are leading to negative economic and public health consequences in affected regions. Experimental and epidemiological studies have shown a strong association between the incidence of cholangiocarcinoma and the infection rate of C. sinensis. To aid research into this organism, we have sequenced its genome.de novo sequencing with computational techniques to provide new information about the biology of this liver fluke. The assembled genome has a total size of 516 Mb with a scaffold N50 length of 42 kb. Approximately 16,000 reliable protein-coding gene models were predicted. Genes for the complete pathways for glycolysis, the Krebs cycle and fatty acid metabolism were found, but key genes involved in fatty acid biosynthesis are missing from the genome, reflecting the parasitic lifestyle of a liver fluke that receives lipids from the bile of its host. We also identified pathogenic molecules that may contribute to liver fluke-induced hepatobiliary diseases. Large proteins such as multifunctional secreted proteases and tegumental proteins were identified as potential targets for the development of drugs and vaccines.We combined C. sinensis and adds to our knowledge on the biology of the parasite. The draft genome will serve as a platform to develop new strategies for parasite control.This study provides valuable genomic information about the human liver fluke Clonorchis sinensis, the oriental liver fluke, is an important food-borne parasite that causes human clonorchiasis in most Asian countries, including China, Japan, Korea, and Vietnam . The genome is available at the NCBI [NCBI: 72781], and the sequences are also available, from GenBank [GenBank: BADR00000000.1].All of the genome shotgun and transcriptome data are available in the NCBI Sequence Read Archive [SRA: 029284 and 035384]. The assembled genome and gene models are available at . The genbp: base pair; CCA: cholangiocarcinoma; DMRT1: mab-3 related transcription factor 1; ES: excretory-secretory; EST: expressed sequence tag; FASN: fatty acid synthase; KEGG: Kyoto Encyclopedia of Genes and Genomes; miRNA: microRNA; NCBI: National Center for Biotechnology Information; ORF: open reading frame; PKA: cyclic AMP-dependent protein kinase; SOX6: sex determining region Y-box 6.The authors declare that they have no competing interests.XBY designed the study. XYW and WJC prepared the DNA samples, interpreted the data and wrote the paper. YH prepared the DNA samples and generated figures and figure legends. JFS prepared the DNA samples. FL prepared the DNA samples, analyzed the genome data and interpreted the data. HLL and LG analyzed the genome data, interpreted the data and generated figures and figure legends. JTM, XLL, CHD, CHZ, XRL, XCH and JX wrote the paper. CL, YXF and LSH generated figures and figure legends. All authors read and approved the final manuscript.C. sinensis. Genome assembly and genome features of Figure S1: 17-mer depth distribution of the sequencing reads. Figure S2: features of the assembled C. sinensis genome. Figure S3: distribution of heterozygosity in C. sinensis. Figure S4: protein domain analysis of C. sinensis, S. mansoni, and S. japonicum. Table S1: main features of C. sinensis genome sequencing data. Table S2: numbers of reads mapped to the assembled C. sinensis genome. Table S3: genome validation by PCR products. Table S4: genome validation by Sanger ESTs. Table S5: repeat composition of C. sinensis genome. Table S6: summary of predicted protein-coding genes by different methods. Table S7: statistics of the reliable gene set with homology, or functional annotation or putative full-length ORF support [C. sinensis genome. support . Table SClick here for fileC. sinensis miRNA precursorsSummary of .Click here for fileC. sinensis versus S. japonicum and C. sinensis versus S. mansoni, respectivelyDetailed information on putative syntenic blocks of .Click here for fileC. sinensis. Important metabolism pathways of Figure S5: the glycolytic pathway of C. sinensis. All the key enzymes required for glycolysis were identified, indicating that the glycolytic pathway of C. sinensis is intact. EC numbers marked in red indicate the presence of the genes in the genome of C. sinensis. Figure S6: the Krebs cycle of C. sinensis. The Krebs cycle of C. sinensis is intact, reflected by related key enzymes present in C. sinensis genome, demonstrating that the liver fluke can generate energy from aerobic or anaerobic metabolism. EC numbers marked in red indicate the presence of the genes in the genome of C. sinensis. Figure S7: the fatty acid metabolism pathway of C. sinensis. C. sinensis can metabolize fatty acids as all required enzymes in the fatty acid metabolism pathway have been discovered. EC numbers marked in red indicate the presence of the genes in the genome of C. sinensis. Figure S8: the fatty acid biosynthesis pathway of C. sinensis. Only three enzymes in the fatty acid biosynthesis pathway were identified, indicating that C. sinensis cannot synthesize endogenous fatty acids. EC numbers marked in red indicate the presence of the genes in the genome of C. sinensis.Click here for fileC. sinensis, S. japonicum and S. mansoniComprehensive analysis of genes involved in fatty acid biosynthesis in .Click here for fileC. sinenisisKey molecules of . Table S12: glycolysis molecules of C. sinensis. Table S13: protease molecules of C. sinensis. Table S14: kinase molecules of C. sinensis. Table S15: phosphatase molecules of C. sinensis. Table S16: tegument molecules of C. sinensis. Table S17: ES molecules of C. sinensis. Table S18: host binding molecules of C. sinensis. Table S19: sex determination molecules of C. sinensis. Table S20: CCA-related molecules of C. sinensis.Click here for fileSources of gene sets used for comparative analysis.Click here for file"} +{"text": "The published funding statement was incorrect. The correct funding is:This work was supported by the NIH (R01-HL70748 to W.R.M.), Ruth Kirschstein National Research Service Award (GM07185 and T32 HL69766 to B.V.H), the Cardiovascular Development Funds and Alberta Heritage Foundation for Medical Research Fellowship (CDF and AHFMR to A.N), the BMBF (0316059), Fraunhofer-Gesellschaft Internal programs (Attract 692263) as well as the Ministry of Science, Research and the Arts of Baden-W\u00fcrttemberg (33-729.55-3/214) (to K.S.-L.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."} +{"text": "The following information was missing from the Funding section: This work was also supported by the National Institute of Health grant 1R03TW008739-01 (E.K. and D.H.T), and National Science Center grant N N301 788 440 (E.K.)."} +{"text": "The last sentence of the Abstract is incorrect. Please see the corrected sentence here:T. polonicum, T. petropavlovskyi, T. carthlicum, T. turgidum, and T. compactum indicates an age of 0.78 million years.\"\"The dating of divergence among The last sentence of the Discussion is incorrect. Please see the corrected sentence here:T. petropavlovskyi has an origin starting with a natural cross between T. aestivum and T. polonicum.\"\"It is most likely that"} +{"text": "Dis. Model. Mech.6, 571\u2013579.There was an error published in The Funding section should read: This study was supported by the European Commission (InfraCoMP \u2013 grant no. 284501 \u2013 to INFRAFRONTIER) and by the UK Medical Research Council.The authors apologise for this mistake."} +{"text": "There was an author name missing from the Acknowledgments. The following is the correct version:We thank A. Miyazawa, A. J. Driessen and D. Oliver for providing the strains and the plasmids. We thank R.Henderson for providing the programs TILTDIFF and TILTDIFFMULTI. We also thank the comments of H.W. Wang."} +{"text": "There were errors in the Funding statement and Author Contributions.The correct version of the Funding is: This work was supported by the NOFAR grant of the Israel Ministry of Industry and Trade. V.L. acknowledges support from the Lady Davis Fellowship Foundation. B.A. acknowledges support of the Israel Science Foundation (Grant 1167/08) and of the Israel Cancer Association. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.The correct version of the Author Contributions are: Conceived and designed the experiments: VY TM MG BA. Performed the experiments: VY TM. Analyzed the data: VY TM MG. Contributed reagents/materials/analysis tools: VY VL MG BA. Wrote the paper: VY TM VL MG BA DD. Conceived and supervised the project: MG BA DD."} +{"text": "Scientific Reports5: Article number: 11344;10.1038/srep11344 published online: 06122015; updated: 03292016The Acknowledgements section in this Article is incomplete.\u201cWe thank Ben Cooper for helpful discussions, Ritva Syrj\u00e4nen for pointing out useful references on the seasonality of colonization, Marc Lipsitch for advice in parametrizing the effects of previous exposures in the model and Dan Weinberger on sharing the point estimates on the Navajo colonization study, visualized in Fig. 1. E.N. and J.C. were funded by ERC grant no. 239784 and Academy of Finland grant no. 251170. P.T. was funded by the Wellcome Trust of Great Britain (Grant No. 083735/Z/07/Z).\u201dshould read:\u201cWe thank Ben Cooper for helpful discussions, Ritva Syrj\u00e4nen for pointing out useful references on the seasonality of colonization, Marc Lipsitch for advice in parametrizing the effects of previous exposures in the model and Dan Weinberger on sharing the point estimates on the Navajo colonization study, visualized in Fig. 1. E.N. and J.C. were funded by ERC grant no. 239784 and Academy of Finland grant no. 251170. P.T. was funded by the Wellcome Trust of Great Britain (Grant No. 083735/Z/07/Z). This research was supported by the National Institute for Health Research Biomedical Research Centre at Great Ormond Street Hospital for Children NHS Foundation Trust and University College London.\u201d"} +{"text": "Scientific Reports6: Article number: 20480; 10.1038/srep20480 published online: 02092016; updated: 03042016.The Acknowledgements section in this Article is incomplete.\u201cS.J.H. and E.A.L. acknowledge funding from the Engineering and Physical Sciences Research Council (EPSRC) UK Grants EP/G035954/1 and EP/J021172/1 and from the Defence Threat Reduction Agency Grant HDTRA1-12-1-0013. E.A.L. would like to thank the North West Nanoscience Doctoral Training Centre (NOWNano DTC) for supporting his studentship. The authors wish to acknowledge the support from H.M. Government (UK) for the provision of the funds for the FEI Titan G2 80-200 S/TEM associated with research capability of the Nuclear Advanced Manufacturing Research Centre.\u201dshould read:\u201cS.J.H. and E.A.L. acknowledge funding from the Engineering and Physical Sciences Research Council (EPSRC) UK Grants EP/G035954/1 and EP/J021172/1 and from the Defence Threat Reduction Agency Grant HDTRA1-12-1-0013. E.A.L. would like to thank the North West Nanoscience Doctoral Training Centre (NOWNano DTC) for supporting his studentship. The authors wish to acknowledge the support from H.M. Government (UK) for the provision of the funds for the FEI Titan G2 80-200 S/TEM associated with research capability of the Nuclear Advanced Manufacturing Research Centre. Open access for this article was funded by King\u2019s College London.\u201d"} +{"text": "Regarding the paper titled: \u201cEstimating family planning coverage from contraceptive prevalence using national household surveys\u201d by Aluisio J. D. Barros, Ties Boerma, Ahmad R. Hosseinpoor, Mar\u00eda C. Restrepo-M\u00e9ndez, Kerry L. M. Wong, Cesar G. VictorGlobal Health Action 9 November 2015. Citation: Glob Health Action 2015, 8: 29735 - http://dx.doi.org/10.3402/gha.v8.29735Published in The conflict of interest and funding section is incomplete.This section currently reads:Competing interests and funding: The authors have not received any funding or benefits from industry or elsewhere to conduct this study.The section should read:Competing interests and funding: The activities of the International Center for Equity in Health (ICEH) and the authors Aluisio J D Barros and Cesar G Victora are supported by a Investigator Award granted by the Wellcome Trust, UK."} +{"text": "Eric.Jackson@genmills.com. The publisher apologizes for this error.The email for corresponding author Eric W. Jackson is incorrect. The correct email is"} +{"text": "Scientific Reports5: Article number: 1446510.1038/srep14465; published online: 09252015; updated: 12142015The Acknowledgements section in this Article is incomplete.\u201cThis work was supported by MOST project 2011CB808804 and 2014AA02200, NSFC project 31370282 and 41030210 to Q.W. and by Tsinghua University Initiative Scientific Research Program 2011Z02296 and 2012Z08128 to J.D.\u201dshould read:\u201cThis work was supported by MOST project 2011CB808804 and 2014AA02200, NSFC project 31370282 and 41030210 to Q.W. and by Tsinghua University Initiative Scientific Research Program 2011Z02296 and 2012Z08128 to J.D., and partially by MOST project 2012AA02A701."} +{"text": "Scientific Reports6: Article number: 2246310.1038/srep22463; published online: 03012016; updated: 05312016Toshinori Matsushima was omitted from the author list in the original version of this Article. This has been corrected in the PDF and HTML versions of the Article.The Acknowledgements section now reads:This work was supported by the Exploratory Research for Advanced Technology (ERATO) and the Kumamoto Collaborations on Organic Electronics under the Regional Innovation Strategy Support Program sponsored by the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan. We would like to acknowledge Mr. Ko Inada for his assistance with calculation of molecular energy levels. We also thank Dr. William J. Potscavage Jr. for his assistance with the preparation of this manuscript.The Author Contributions section now reads:D.T. performed the OLED and lifetime experiments. T.M. performed the TSC measurements and analysis. D.T. and C.A. wrote the paper with input from T.M. C.A. supervised the project."} +{"text": "The second affiliation for author David M. Guidot is not indicated. Dr. Guidot is also affiliated with: Atlanta VA Medical Center, Decatur, GA 30033"} +{"text": "The fifth author\u2019s first and last names were inadvertently switched. The first name appears as the last name and the last name appears as the first name. The correct name is: Tadepally Lakshmikanth. The correct citation is: La Rocca R, Tallerico R, Talib Hassan A, Das G, Lakshmikanth T, Matteucci M, et al. (2014) Mechanical Stress Downregulates MHC Class I Expression on Human Cancer Cell Membrane. PLoS ONE 9(12): e111758. doi:10.1371/journal.pone.0111758"} +{"text": "Northern and tropical peatlands represent a globally significant carbon reserve accumulated over thousands of years of waterlogged conditions. It is unclear whether moderate drying predicted for northern peatlands will stimulate burning and carbon losses as has occurred in their smaller tropical counterparts where the carbon legacy has been destabilized due to severe drainage and deep peat fires. Capitalizing on a unique long-term experiment, we quantify the post-wildfire recovery of a northern peatland subjected to decadal drainage. We show that the moderate drop in water table position predicted for most northern regions triggers a shift in vegetation composition previously observed within only severely disturbed tropical peatlands. The combined impact of moderate drainage followed by wildfire converted the low productivity, moss-dominated peatland to a non-carbon accumulating shrub-grass ecosystem. This new ecosystem is likely to experience a low intensity, high frequency wildfire regime, which will further deplete the legacy of stored peat carbon. Sphagnum mosses that carpet most peatland surfaces lead to an array of negative feedbacksSphagnum species that dominate high latitude peatlands resist all but the most intense wildfires by retaining high near-surface moisture contentsWildfire has been an important disturbance within the boreal region of North America throughout the HoloceneMajor hydrological disruption in tropical peatlands has resulted in catastrophic wildfires and the loss of peatland ecosystems. In 1997, wildfires in drained Indonesian peatlands led to the release of 0.95\u20132.57 Gt of carbon into the atmosphere7To investigate the fate of high latitude carbon stocks following drainage and wildfire, we capitalized on a unique long-term experiment. A treed fen in Alberta, Canada was partially drained for silviculture in 1983. The water table position was immediately lowered by about 0.25\u2005m, comparable to projections of future water table reductions (ranging from 0.05 to 0.6\u2005m)In the absence of drainage, the peatland followed a typical pattern of post-fire recovery6Sphagnum moss cover at the drained site and a 77% reduction in total moss cover in vegetation between drained and undrained plots ten years post-fire. We observed the absence of ss cover . This wass cover . This siSphagnum recolonisationSphagnum stress . The study site, with a maximum peat depth of 4\u2005m, is characteristic of this broad class of fen system that dominates Canada's Boreal Plains. The response of the site to disturbance is thus suggestive of the general response of these peatland ecosystems. In 1983 a 50-ha portion of the poor fen was drained through the construction of 0.9\u2005m deep ditches spaced 40\u2005m apart. The drained and undrained portions of the fen subsequently burned in 2001 as part of a 105,000\u2005ha wildfire (LWF-063)Sampling transects established prior to the wildfire in the drained and undrained portions of the fenThe loss of water required to lower the water table from the surface to a depth of 0.4\u2005m within drained and undrained areas prior to fire were determined from specific yield measurements conducted by 2 quadrats randomly distributed within the vicinity of each well transect. Sampling occurred during July 2010 while herbaceous and sedge species were flowering to ease species identification. Species presences within each quadrat was determined and percentage ground cover was also estimated for each quadrat. Species similarity between the drained and undrained portion of the peatland was determined using S\u00f8renson's quotient of similarity (Q/S): j the number of species common to both samples, a is the total number recorded in the first sample, b is the number recorded in the second sample.Understory and surface plant species were identified within eight 0.5\u2005mLight transmissivity and leaf area index (LAI) were calculated using gap light photography. Hemispherical photographs were captured adjacent to the drained and undrained transects (n = 40) and within the center of the 16 species identification plots using a Nikon D60 camera and a Sunex 185\u00b0 Super Fisheye lens. Photographs were processed using GLA v.2 softwareN.K., M.R.T. and J.M.W. wrote the manuscript and devised the conceptual understanding. J.H.S., J.M.W., M.R.T., D.K.T. and B.W.B. devised the field research, J.H.S., D.K.T. and C.A.M. undertook the field research and J.H.S., N.K. and J.M.W. carried out the data analysis. M.D.F. and B.M.W. provided specific expertise and knowledge throughout the project. J.H.S., D.K.T., B.W.B., M.D.F., B.M.W. commented on the manuscript through its development. Funding was provided by M.R.T., B.M.W. and J.M.W.Table S1"} +{"text": "There are errors in the Funding section. The correct funding information is as follows: This work was supported by NIH grants AI089935 and AI108651 (L.-F.L). L.-F.L. is a Kimmel Scholar and a Hellman Fellow. H.-M.L is an Irvington Fellow of the Cancer Research Institute. The conditional IFN\u03b3R2 mutant mouse generation was supported by DFG (SFB 621 to W.M.) and MUGEN LSHG-CT-2005-005203. The initial functional characterization of IFN\u03b3R2 mutant mice was supported by the BBSRC PhD program at the University of Manchester. The aforementioned funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."} +{"text": "The following information is missing from the Funding section of the published article: E.J.L. was funded by NIH grant GM033048-26S1. The publisher apologizes for the error."} +{"text": "The Authors correct list is: Narese D, Bracale U. M., Vitale G., Porcellini M., Midiri M., Bracale G. This paper was published on Pubmed listing the Authors by their first names rather than by their surnames."} +{"text": "Nutrition & Metabolism and contributing their cherished work to this journal.We and the Editorial Board sincerely acknowledge and thank all the reviewers for their active participation and contribution during 2015 (Volume 12). We greatly appreciate their dedication and behind the scenes contribution. It is largely due to their support and expertise that we have been able to publish high standard manuscripts. We would also like to thank authors for choosing A. SchlegelUnited States of AmericaA. A. TinkovRussian FederationAbdalla El-mowafyEgyptAhmed BakillahUnited States of AmericaAlan DucatmanUnited States of AmericaAlan HayesAustraliaAltan OnatTurkeyAmit TyagiIndiaAmy BentleyUnited States of AmericaAnna LitwicUnited KingdomAnssi ManninenFinlandAntonio CamargoSpainArpita BasuUnited States of AmericaB. ScolnickUnited States of AmericaB. A. PatelUnited KingdomBalachandar NedumaranUnited States of AmericaBettina PfleidererGermanyBill LandsUnited States of AmericaBrian BennettUnited States of AmericaBruce AmesUnited States of AmericaBruno G. OertelGermanyC. A. CooperUnited States of AmericaCarrie DurwardUnited States of AmericaChen ZhengUnited States of AmericaChiara FerrarioItalyChris McEntyreNew ZealandChristine MetzUnited States of AmericaChristos DerdemezisGreeceC-Y. Oliver ChenUnited States of AmericaD. ChartoumpekisUnited States of AmericaDamien BelobrajdicAustraliaDaniel KonradSwitzerlandDarius BatuleviciusLithuaniaD. C. DamascenoBrazilD. D. LeonidasGreeceDdo N. MarreiroBrazilD. L. WilliamsonUnited States of AmericaD. M. CameraAustraliaDonald LaymanUnited States of AmericaDong ChengUnited States of AmericaDonghui ZhangUnited States of AmericaDouglas KalmanUnited States of AmericaE. James SquiresCanadaE. SaadEgyptE. ZambranoMexicoEI Magalh\u00e3esBrazilEirik DegerudNorwayElizabeth BeierleUnited States of AmericaE. M. PereiraBrazilEverson NunesBrazilFabio LiraBrazilFabrice TranchidaFranceFareeba SheedfarNetherlandsFasika TedlaUnited States of AmericaFederico SalomoneItalyFei FangUnited States of AmericaFelipe Vadillo-OrtegaMexicoForrest NielsenUnited States of AmericaFotios IliadisGreeceG. JamarBrazilG. \u0141ysiakPolandGary LopaschukCanadaGm Dallinga-ThieNetherlandsGustavo PimentelBrazilH. KajiJapanH. MirmiranpourIslamic Republic of IranH. YegerCanadaHans DegensUnited KingdomHossam ElnoamanyEgyptJ. FarinhaBrazilJ. LiangChinaJ. PalmfeldtDenmarkJ. PepeItalyJahangir IqbalUnited States of AmericaJamie BaumUnited States of AmericaJan PalmbladSwedenJavier MontalvoMexicoJ. H. LeeUnited States of AmericaJ. J. DiNicolantonioUnited States of AmericaJ. J. WorthingtonUnited KingdomJ. L. BurkheadUnited States of AmericaJ. L. Hern\u00e1ndezSpainJohanna AproSwedenJonathan Steven AlexanderUnited States of AmericaJong RhoCanadaJoost WillebrordsBelgiumJ\u00f8rgen JensenNorwayJose AntonioUnited States of AmericaJose Donato Jr.United States of AmericaJoshua MeidenbauerUnited States of AmericaJ. R. TrevithickCanadaJt Jr NickelsUnited States of AmericaJuei-Tang ChengTaiwanJung-Su ChangTaiwanJ. Y. ChangTaiwanK. JarukamjornThailandK. RoyAustraliaKatherine CrewUnited States of AmericaKatherine VerasBrazilKe LiUnited States of AmericaKevin MakiUnited States of AmericaKezhi DaiUnited States of AmericaKezhi DaiUnited States of AmericaKiatlyn LiuUnited States of AmericaKM WeinbergeUnited KingdomKun-Ruey ShiehTaiwanL. DuplombFranceLarry RudelUnited States of AmericaLatha DeviUnited States of AmericaL. D. KongChinaLei HaoUnited States of AmericaLeng Huat FooMalaysiaLeonardina CiccarelliItalyLharbi DridiCanadaLianfeng WuUnited States of AmericaLing YangUnited States of AmericaLiye ZhouUnited States of AmericaL. R. BrunhamCanadaLuis Salazar-OlivoMexicoLydie CombaretFranceM. AlemanySpainM. B\u00f6hmAustraliaM. DixitIndiaM. FexSwedenM. FisbergBrazilM. HoriuchiJapanM. YamatoJapanMahmood HussainUnited States of AmericaMarco MancaNetherlandsMaria Victoria Garcia-MediavillaSpainMark HermanUnited States of AmericaMartha BeluryUnited States of AmericaMartha Rodriguez-MoranMexicoMartin BurtscherAustriaMartin Joyce-BradyUnited States of AmericaMary HonorsUnited States of AmericaMasanori YoshizumiJapanMatthew CannonUnited States of AmericaMatthew PickloUnited States of AmericaM. C. Gomes-MarcondesBrazilMeghan WalshUnited States of AmericaMelissa BentonUnited States of AmericaMengwei ZangUnited States of AmericaM. H. SharawyEgyptM'hamed BentourkiaCanadaMin YangChinaMingming GaoUnited States of AmericaM. J. KeenanUnited States of AmericaM. J. MorrisAustraliaM. J. RowlingUnited States of AmericaMohammed Al-GayyarEgyptMostafa WalyOmanM. S. FawzyEgyptN. Keleku-LukweteJapanNeha GargUnited States of AmericaNicholas DavidsonUnited States of AmericaN. J. KellowAustraliaO. D. Rangel-HuertaSpainP. BhattacharyyaPuerto RicoP. LiuChinaPalanikumar ManoharanUnited States of AmericaP. C. LisboaBrazilP. D. Portales-P\u00e9rezMexicoPhilip CalderUnited KingdomPhilippe CostetUnited States of AmericaPietro RagonesiItalyP. K. DudejaUnited States of AmericaP. O. PradaBrazilQiang FuChinaQiang LiUnited States of AmericaR. FujiwaraJapanR. VijIndiaR. A. G\u00f3mez-D\u00edazMexicoRai Ajit SrivastavaUnited States of AmericaRandolph MatthewsUnited States of AmericaRebecca MacPhersonCanadaRen XuUnited States of AmericaRobert HarrisUnited States of AmericaRosa LucianoItalyRosana PaganoBrazilRyuichi KawamotoJapanS. ChungUnited States of AmericaS. EghbalsaiedIslamic Republic of IranS. KlausGermanyS. ReddiIndiaS. Sanchez-EnriquezSpainS. A. KumarAustraliaSaihan BorghjidUnited States of AmericaSalman AzharUnited States of AmericaSamy McFarlaneUnited States of AmericaSandra PetersCanadaSara AuharekBrazilSelma LiberatoAustraliaS. H. HyunDemocratic People\u2019s Repubilc of KoreaShanshan PeiUnited States of AmericaShi SunUnited States of AmericaSilvia SavastanoItalySrinidi MohanUnited States of AmericaStanley HazenUnited States of AmericaStephane WalrandFranceStephanie ChungUnited States of AmericaStreamson ChuaUnited States of AmericaSunil PanchalAustraliaSusan ArthurUnited States of AmericaSushma Reddy GundalaUnited States of AmericaT. LuoChinaT. A. WihastutiIndonesiaTakeshi HaseJapanTam\u00e1s DecsiHungaryTatjana StojakovicAustriaThad RosenbergerUnited States of AmericaThirupathi MuthusamyUnited States of AmericaTia RainsUnited States of AmericaT. J. HuppertUnited States of AmericaT. K. LeeAustraliaT. L. MarinUnited States of AmericaToshiaki TamakiJapanTracy AnthonyUnited States of AmericaTyler BarkerUnited States of AmericaUwe TietgeNetherlandsV. VelardeChileVasilios AthyrosGreeceVathsala MohanNew ZealandVenu VelagapudiUnited States of AmericaVera MazurakCanadaVidya VelagapudiFinlandVikas KumarIndiaW. WangChinaW. ZhangUnited States of AmericaWayne GrodyUnited States of AmericaWei SongUnited States of AmericaW. M. XuChinaWolfram DoehnerGermanyX. PrieurFranceXi LiChinaXian-Cheng JiangUnited States of AmericaXiaoyue PanUnited States of AmericaXin BiUnited States of AmericaXinyin JiangUnited States of AmericaXueying ChenChinaXueying ChenUnited States of AmericaY. KawanoJapanYang ZhihongUnited States of AmericaYiyi MaUnited States of AmericaYong HuUnited States of AmericaYong ZhuUnited States of AmericaY. T. KuoJapanYvonne FierzSwitzerlandZ. NingChinaZehra Ilke AkyildizTurkeyZengying WuUnited States of America"} +{"text": "AbstractAs part of ongoing exploration of the mayflies of hill streams of the southern Western Ghats of India, we establish a new record of mayfly.Potamanthelluscaenoides Ulmer 1939 is newly recorded based on larval collection from the upstream of Silent Valley National Park of the southern Western Ghats. Brief ecological notes are appended. Ephemeroptera is a biogeographically significant archaic order of aquatic insects abounding in several enigmatic families in the pantropical region, especially in the Oriental Realm. Neoephemeridae is a small group of mayflies presently confined to Holartic and Oriental regions. PotamanthellusNeoephemeraOchernovaLeucorhoenanthusNeoephemera. This is not accepted by Neoephemeridae have unique operculate gills on the second abdominal segment that are fused medially. The larvae of Potamanthellus are distinguished from those of Neoephemera and Ochernova by their densely setate mouthparts, by their lack of well developed lateral expansions of the pronotum and mesonotum, and by their possession of rows of long setae on the caudal filaments .Ulmer 1939Type status:Other material. Occurrence: individualCount: 7; sex: male & female; lifeStage: Larva; Taxon: kingdom: Animalia; phylum: Arthropoda; class: Insecta; order: Ephemeroptera; family: Neoephemeridae; genus: Potamanthellus; specificEpithet: caenoides; taxonRank: species; taxonomicStatus: accepted; Location: country: INDIA; stateProvince: Kerala; municipality: Silent Valley National Park; locality: Poochipara; verbatimElevation: 935 m; verbatimLatitude: 11\u00b006\u201949.5\u201d N; verbatimLongitude: 76\u00b025\u201952.4\u201d E; Identification: identifiedBy: C. Selvakumar & K. G. Sivaramakrishnan; Event: samplingProtocol: Hand picking; year: 2013; month: April; day: 18; habitat: CascadePotamanthelluscaenoides is distinguished from other species of Potamanthellus by the following combination of characters in larvae: (i) a distinct diagonal ridge on operculate gills posteromedian tubercle on abdominal terga 1\u20132 and 6\u20138 distinct dills Fig. ; (iii) dlls Fig. ; (iv) rells Fig. and (v) lls Fig. . P.caenIndonesia , fallen leaves and detritus. The water temperature in April ranges 18-23\u00b0C. Larvae were collected by Kick samples and hand picking.The larvae of P.amabilis (P.caenoides (P.chinensis (P.edmundsi (P.ganges (P.shaowuensis (P.unicutibius (P.ganges is known from India from the tributary of Ganges (P.caenoides (Presently this genus consists of seven species viz. amabilis , P.caenaenoides , P.chinhinensis , P.edmuedmundsi , P.gang.ganges , P.shaoowuensis and P.ucutibius . Howeverf Ganges . P.caenaenoides is new r"} +{"text": "Sir,et al.1 that fast genotypic methods will play an increasingly prominent role in drug susceptibility testing for the Mycobacterium tuberculosis complex (MTBC).3 We would, however, like to point out that the embB (Rv3795) Glu378Ala polymorphism, which is detected by probe 3 of their newly developed low-density DNA array, is not a marker for ethambutol resistance.7 Instead, Ala represents the ancestral amino acid at this codon .We agree with Moure 10In light of these data, the results of probe 3 would be predicted to lead to systematic false-positive reports, which calls into question the validity of this probe. This underlines that the entire MTBC diversity has to be considered when designing and validating genotypic drug susceptibility testing assays.Wellcome Trust and the Health Innovation Challenge Fund (HICF-T5-342 and WT098600 to S. J. P.), Public Health England (to S. J. P.), the Medical Research Council (to J. M. B.) and the Wellcome Trust Sanger Institute (WT098051 to J. P. and J. M. B). C. U. K. is a Junior Research Fellow at Wolfson College, Cambridge. I. C. is supported by a Ram\u00f3n y Cajal fellowship from the Spanish Government (RYC-2012-10627).This work was supported by a grant from the Department of Health, J. P. has received funding for travel and accommodation from Pacific Biosciences Inc. and Illumina Inc. S. J. P. is a consultant for Pfizer Inc. and has received funding for travel and accommodation from Illumina Inc. All other authors: none to declare.This publication presents independent research supported by the Health Innovation Challenge Fund (HICF-T5-342 and WT098600), a parallel funding partnership between the Department of Health and Wellcome Trust. The views expressed in this publication are those of the authors and not necessarily those of the Department of Health or Wellcome Trust."} +{"text": "Scientific Reports5: Article number: 1215310.1038/srep12153; published online: 07162015; updated: 02082016.Ping Wang and Michael J. Sadowsky were omitted from the author list in the original version of this Article. This has been corrected in the PDF and HTML versions of the Article.The Author Contributions section now reads:\u201cRV and TC designed and conducted the experiments. RV developed the equations, performed data reduction, prepared the figures and wrote the manuscript. JC performed the statistical analyses. PW, MS and FB-S developed the qPCR methods. FB-S conducted the qPCR analyses. TC, JC and FB-S revised and wrote specific sections of the manuscript.\u201d"} +{"text": "The Excel file is absent from the published S5 FileA. chinensis cv. 'Hongyang'. Table B. List of novel transcripts, alternative splicing and genes extended identified through analysis of the 'Hongyang' transcriptome. Table C. Verification of RNA-seq results by qPCR in A. chinensis cv. 'Hongyang'. Table D. Differentially expressed genes between two neighboring stages of fruit development in A. chinensis cv. \u2018Hongyang\u2019. Table E. List of genes involved in ABA, CK, AUX and GA3 biosynthesis and signal transduction in developing fruit of A. chinensis cv. 'Hongyang'. Table F. List of genes involved in biosynthesis and metabolism of sugar and starch in developing fruit of A. chinensis cv. 'Hongyang'. Table G. List of genes involved in L-ascorbic acid biosynthesis and metabolism in developing fruit of A. chinensis cv. 'Hongyang'. Table H. List of genes involved in flavonoid biosynthesis and regulation in developing fruit of A. chinensis cv. 'Hongyang'.Table A. The expression profiles of all expressed genes in developing fruit of (XLS)Click here for additional data file."} +{"text": "Scientific Reports5: Article number: 15673; 10.1038/srep15673 published online 10292015; updated: 03102016The Acknowledgements section is incomplete.\u201cC.R.P. acknowledges financial support for the development of the X-ray imaging modality by the U.S. Department of Energy under Grant DE-FG02-08ER15937. J.R.W. acknowledges financial support by the National Institutes of Health under Grant CA123544. The authors thank Virginia Hovanesian for her assistance with immunofluorescence studies.\u201dshould read:\u201cC.R.P. acknowledges financial support for the development of the X-ray imaging modality by the U.S. Department of Energy under Grant DE-FG02-08ER15937. J.R.W. acknowledges financial support by the National Institutes of Health under Grant CA123544. The authors thank Virginia Hovanesian for her assistance with immunofluorescence studies. The authors also thank Vivian Ortiz for her help with data collection and figure preparation, and for valuable discussions on these topics.\u201d"} +{"text": "The last two authors, Philip M. Hansbro and Paul S. Foster, should be noted as contributing equally to this work."} +{"text": "Following publication of this article we notedThe authors contributions section should be updated as follows:A.Al-T. designed this study. Wedad Saleem performed data analysis and drafted the manuscript. N. Al-A. provided assistance with data collection and manuscript revision. S.D. collected samples and clinical data, participated in the design of the study and performed the statistical analysis. G.W. was involved in the concept and design of the study and he analysed the data. All authors read and approved the final manuscript."} +{"text": "Nucl. Acids Res. 43 (W1): W527\u2013W534. doi:10.1093/nar/gkv344The authors wish to make the following corrections to their article:In Table The results and conclusion of the article are not affected and remain valid. The authors apologise to the readers for the inconvenience caused."} +{"text": "There is an error in the co-author, Dr. Damien Griffin\u2019s, first name. The correct name for this co-author is Dr. Damian Griffin."} +{"text": "The original version of this article unfortunGeorge P. Tiley* and J. Gordon BurleighThe original article was upda"} +{"text": "Nature Communications6 Article number: 748110.1038/ncomms8481 (2015); Published 06262015; Updated 02052016The financial support for the work described in this Article was not fully acknowledged. The Acknowledgements should have included the following:This work was supported in part by a grant to H.J.G., B.H. and N.T. (European Union's Seventh Framework Program (FP/2007/2013)/European Research Council (ERC) Grant Agreement 322820)."} +{"text": "Ming-Chia Lin's affiliation was not reported in full. Dr. Lin is affiliated with the E-Da Hospital. The articles have since been corrected online.In the articles \u201cAssociation of Head and Neck Cancers in Chronic Osteomyelitis: A National Retrospective Cohort Study\u201d,"} +{"text": "The funding source \"E-rare project Transposmart\" was missing from the Funding section. Please see the corrected Funding statement here:DFG grant SPP 1230 (A.E.), the E-rare project Transposmart (A.E. and Z.I.), and EU Framework Programme 7 (Persistent Transgenesis)(A.E.). W.Z. was supported by a fellowship from the Chinese Scholarship Council (CSC) in cooperation with Northwestern A&F University, Yangling, China. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."} +{"text": "The publisher apologizes for the error.The last author\u2019s name is spelled incorrectly. The correct name is: Jane R. Taylor. The correct citation is: Harb R, Taylor JR (2015) The Fragrant Power of Collective Fear. PLoS ONE 10(5): e0123908. doi:"} +{"text": "The Competing Interests section is incorrect. The Competing Interests section should read: Joshua Field, M.D., M.S., Joel Linden, Ph.D. and David Nathan, M.D. each have consulting agreements with NKT Therapeutics, Inc. Joel Linden, Ph.D. is an inventor on a patent issued to the University of Virginia, which claims the use of adenosine A2A agonists for the treatment of sickle cell disease; he owns shares in Adenosine Therapeutics LLC, which licensed this patent. This does not alter our adherence to all the PLOS ONE policies on sharing data and materials."} +{"text": "Enas A. A.Abdallah's name was inadvertently omitted, and Dr. Adnan A. Alsulaimani'sname was incorrectly included. Dr. Mostafa A. Salama's name was also incorrectlylisted as Mostafa Abdelazim. The article has since been corrected online.In the article \u201cOutcomes of Early Ligation of Patent Ductus Arteriosus in Preterms,Multicenter Experience\u201d,"} +{"text": "There is a missing grant number in the Financial Disclosure (FD) statement of this article. Please refer to the correct FD statement below:This work was supported by The Scientific and Technological Research Council of Turkey (TUBITAK) 1001 Grant number: 112T272 and Sabanci University. D.G. and A.K are recipients the Turkish Academy of Sciences (TUBA) GEBIP Award. D.G. is a recipient of the EMBO Strategical Development and Installation Grant (EMBO-SDIG) and A.K. is a recipient of the TUBITAK Incentive Award. G.K. and K.A.T. are recipients of Yousef Jameel and TUBITAK-BIDEB PhD Scholarships, respectively. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."} +{"text": "Scientific Reports5: Article number: 1188510.1038/srep11885; published online: 07082015; updated: 09242015.This Article contains a typographical error in the grant number in the Acknowledgements section.\u201cThis study was supported by the Grant Agency of the Czech Republic (P305/10/2141) to Z.K. by the Ministry of Education, Youth and Sports of the Czech Republic (LM2011022) to P.B. and by IMG institutional support RVO68378050\u201d.should read:\u201cThis study was supported by the Grant Agency of the Czech Republic (P305/10/2141) to Z.K. by the Ministry of Education, Youth and Sports of the Czech Republic (LO1220) to P.B. and by IMG institutional support RVO68378050\u201d."} +{"text": "Antognazza C.M., Andreou D., Zaccara S. & Britton J.R.Ecology and Evolution 2016; 6(5): 1280\u2013129210.1002/ece3.1906doi: The date of the first fish introduction for River Great Ouse should have been in the 1890s, instead of 1990s. The below table has the correct number. This change has no impact in the overall conclusions of the paper. The authors regret this error."} +{"text": "Correction for the Acknowledgement section, the correct text should be:We are grateful to Drs. C. Bittencourt, M. Prato, L. Ballerini, and M. Nesl\u00e1dek for discussions. We are also grateful to Drs. Henry Markram, S\u00e9bastien Lasserre, and the Blue Brain Project team at the EPFL for contributing the graphical abstract. Financial support from the European Commission , from the Flanders Research Foundation (contract no. G088812N), and from the Belgian Science Policy Office (BELSPO) is kindly acknowledged."} +{"text": "Key messageImmunosuppressants repair nailfold capillary abnormalities in patients with early-stageSSc.Dear Editor, We read the paper by Matsuda et al. [a et al. with greAs the authors pointed out, few reports exist on the improvements in nailfold capillaryabnormalities after immunosuppressive treatment in patients with SSc. Among them, CYC improvedthe nailfold capillary abnormalities of SSc when using iloprost in five of eight patients. Autologet al. [We reported that in five patients with SSc treated with CYC i.v. therapy, findings ofenlarged capillaries, giant capillaries and haemorrhage among the nailfold capillaryabnormalities improved after 6\u2009months .Additioet al. . She waset al. [et al. [et al. [In the recent study by Matsuda et al. , tacroli [et al. . We cons [et al. were mil [et al. , 6, 7. Wet al. [In summary, we argue that early-stage nailfold capillary abnormalities can be improved withimmunosuppressive agents alone. As pointed out by Matsuda et al. , furtherFunding: This work was supported in part by Japan Society for the promotionof science KAKENHI (grant numbers 19K18499 to S.M. and 19K07940 to S.H.), MitsubishiFoundation to S.M., Takeda Science Foundation to S.M., Mochida Memorial Foundation for Medicaland Pharmaceutical Research to S.M., Japanese Respiratory Foundation Grant to S.M. and JapanRheumatism Foundation to S.M.Disclosure statement: The authors have declared no conflicts of interest.Patient consent: Written consent was obtained from the patient regarding thepublication of this case. This study was approved by the clinical ethics committee ofHiroshima University Hospital .The data underlying this article will be shared on reasonable request to the correspondingauthor."} +{"text": "The authors wish to update their Acknowledgements to recognise the European Synchrotron Radiation Facility. The full and correct Acknowledgements can be found below:\u201cThe authors acknowledge the Research Foundation Flanders through project fundings and through a PhD scholarship to G. R. (grant 11C6922N), as well as iBOF-21-085 PERSIST. T. A. and S. V. A. acknowledge funding from the University of Antwerp Research fund (BOF). J. H. acknowledges the Flemish government through long-term structural funding Methusalem and the MPI as MPI fellow. M. R. acknowledges funding by the KU Leuven Research Fund (C14/19/079). S. B. and S. V. A. acknowledge funding from the European Research Council under the European Union's Horizon 2020 Research and Innovation Program (ERC Consolidator Grants No. 815128 \u2013 REALNANO and No. 770887 \u2013 PICOMETRICS). We acknowledge the European Synchrotron Radiation Facility for the provision of synchrotron radiation facilities, and we would like to thank Dr D. Chernyshov for assistance in using beamline BM01\u201d.This new text supersedes the Acknowledgements originally provided with the article.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "Keratinocyte cancers are the commonest cancers in white populations, with rates increasing worldwide.George W. M. Millington: Writing \u2013 original draft (lead); Writing \u2013 review & editing (lead).George W. M. Millington is the Editor in Chief of Skin Health and Disease."} +{"text": "J. van der Fels-Klerx was missing. The panel legend for Figs. 1 and 2 inadvertently swapped. The original article has been corrected."} +{"text": "In the article titled \u201cEffectiveness of Transcutaneous Electrical Nerve Stimulation with Taping for Stroke Rehabilitation\u201d , the aut\u201cAll authors have read and approved the final version of the manuscript. T.-s.I and J.-h.J are co-first authors. Conceptualization was performed by K.-s.J. and T.-s.I.; writing was performed by T.-s.I.; writing (review and editing) was performed by K.-s.J. and H.-y.C.; methodology was performed by H.-y.C.; formal analysis was performed by K.-s.J.; investigation was performed by J.-h.J.; measurement visualization was performed by J.-h.J.\u201d"} +{"text": "The Funding statement is incomplete. The correct Funding statement is:The overall project was supported by a grant from the German Research Foundation (DFG) to S.H.R. (RI 2488/3 1). Project parts conducted within the laboratory of H.W. in Switzerland were additionally supported by a grant from the Swiss National Science Foundation to H.W. (SNF Grant No. 310030\u2013179254). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."} +{"text": "Ovarian clear cell carcinoma (OCCC) is a relatively uncommon epithelial ovarian malignancy with unique clinical, histopathologic and genetic characteristics. Patients with advanced OCCC have poor outcomes and are resistant to standard chemotherapy. Targeted therapy offers a novel approach for treating OCCC. We report the case of a 45-year-old female patient with advanced OCCC who experienced relapse after standard treatment. Further, a frameshift mutation in the homologous recombination repair-related gene RAD50 (RAD50-p.I371Ffs*8) was identified by genetic testing. Next, the patient had received targeted combination therapy with poly (ADP-ribose) polymerase (PARP) inhibitor pamiparib and bevacizumab, achieving partial remission. Patient\u2019s symptoms improved significantly compared to before. To date, the patient has been followed up for more than half a year with favorable survival and high quality of life. The case report suggested that parmiparib-targeted therapy is a viable treatment option for advanced OCCC patients with RAD50 mutation. Epithelial ovarian cancer (EOC) has the highest mortality rate among gynecologic malignancies . There aA 45-year-old female patient who presented with worsening epigastric pain was diagnosed with OCCC on 1 November 2020 . Tumor mutation burden (TMB) was 0.45 Muts/Mb. The HLA-typing analysis identified a heterozygous mutation in RAD50, indicating better clinical outcomes. Then, on 25 August 2021 the patient was received intraperitoneal perfusion chemotherapy . After one cycle of treatment, the ascites was significantly reduced , a rare mutation in OCCC, resulted in significant relief of clinical symptoms in patients after targeted therapy with the combination of pamiparib and bevacizumab. Overall, genetic testing-based approaches and combined with targeted therapy is a feasible treatment option for advanced or recurrent OCCC.The authors thank all colleagues in the Department of Oncology, 900TH Hospital of Joint Logistics Support Force, Fujian Province for their support in reporting this case.The authors received no financial support for the research, authorship, and/or publication of this article.Conception and design: X.H., X.He., D.L., X.C. and Xi.C. Administrative support: D.L., X.C. and Xi.C. Provision of study materials or patients: X.H., D.L., X.C. and Xi.C. Collection and assembly of data: X.H., X.He. Data analysis and interpretation: X.H., X.He. Manuscript writing: all authors; supervision, X.H., D.L., X.C. and Xi.C. All authors have read and agreed to the published version of the manuscript.The study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of 900TH Hospital of Joint Logistics Support Force. Written informed consent was obtained from the patient for publication of this case report and accompanying images.There are no conflicts of interest."} +{"text": "British and Irish Orthoptic Journal, 18(1), pp. 93\u2013100. DOI: http://doi.org/10.22599/bioj.271.This article details a correction to: O\u2019Connor, A., King, C., Milling, A. and Tidbury, L., 2022. Using a Computerised Staircase and Incremental Optotype Sizes to Improve Visual Acuity Assessment Accuracy. The original article was erroThe affected sentences are corrected below.The first sentence should read:The study was approved by the Committee on Research Ethics at the University of Liverpool and informed consent was obtained prior to testing.The first sentence should read:Participants were recruited into the study from the student population at the University of Liverpool."} +{"text": "Dear Editor,Morella rubra Lour., 2n\u2009=\u20092x\u2009=\u200916) is an evergreen fruit tree native to southern China and the only domesticated species in family Myricaceae in Zhejiang province [Compared with the previous study of Liu . 2015) , our stu , our stprovince , which gFST\u2009=\u20090.45), in which a significant reduction of genetic diversity was also found. This suggests that improvement events play a more crucial role in the development of selected traits of extant cultivars. The first improvement, which gave rise to the common ancestor of all cultivars, took place 1890 (480\u20132800) years ago, approximately corresponding to the Western Han Dynasty or even earlier. The fruits of Chinese bayberry were discovered in the array of funeral objects in \u2018Mawangdui\u2019 tombs, the most famous Han Dynasty tombs . The first documentary about the cultivation of Chinese bayberry was also made in the Western Han Dynasty by Chen (1996)[Paeonia suffruticosa, Prunus mume, Prunus persica, Prunus salicina, and M. rubra [et al., 2004). The two most popular cultivars, BQ and DK, were derived from the same ancestor and developed from a third independent improvement ~100 (60\u2013540) years ago, approximately in the Qing Dynasty. A recent study of the genomic DNA footprint of 14 DK individuals suggests that they were all propagated from one mother tree ~180\u00a0years old that lives in Huangyan, Zhejiang province [et al. (2013) [On the contrary, the major genetic differentiation was observed between two elite cultivars DK and BQ (n 1996). The sec96. The . (2013) reportedM. rubra and provided valuable information for future research and breeding activities based on genetic diversity.On summary our study has for the first time clarified the domestication history of This work was supported by the National Natural Science Foundation of China and the Zhejiang Provincial Natural Science Foundation (grant LY19C030007). We sincerely thank Yonghua Zhang and Luxian Liu for their great help with collecting plant materials.P.L., C.F., J.L., and J.C. conceived the study; L.L., N.C., and P.L. contributed to the sampling; L.L. and N.C. performed the experiments; J.L., J.C., and P.L. analyzed the data. The manuscript was written by J.L. and J.C. and revised by K.M.C., P.L., C.F., and X.L.https://github.com/JUNKELII/RAD/blob/main/supplementary.docx.The raw resequencing data have been deposited in the Sequence Read Archive of the National Center for Biotechnology Information (NCBI) with BioProject accession number PRJNA606201. The detailed supplementary material and sampling information are available at"} +{"text": "Dear Editor,Helicobacter pylori infection status\u201d by Adachi and his colleagues with great interest.H. pylori infectionH. pylori eradication.H. pylori infection, and antral nodularity is also improved by H. pylori eradication.We have read the article \u201cendoscopic findings of cardiac lymphoid hyperplasia and The authors declare no conflict of interest.None"} +{"text": "We thank Zhang and colleagues for their readership and letter.The statistical analyses of our manuscript were based on a five\u2010tiered biopsy and cytology diagnostic categorization. Two cut\u2010offs for positive/negative test result were explored, considering malignant diagnoses (B5/C5) only as positive or including the category of suspicious for malignancy (B4/C4) as positive. All subsequent analyses were based on two\u2010by\u2010two tables of pleural biopsies and pleural effusion cytology specimens. Sensitivity (Sn), specificity (Sp), positive predictive value (PPV), negative predictive value (NPV), accuracy, and risk of malignancy (ROM) between biopsy and cytology, and subgroup stratified by disease types were compared.Our study addressed the difference in diagnostic performance of biopsy versus cytology, as such, only paired specimens were included. Statistics were selected for comparing two\u2010by\u2010two tables, we regarded the most direct metrics, namely Sn, Sp, PPV, NPV, accuracy and ROM sufficient for the purpose. It is possible to extrapolate the DOR and LR from the raw data. However, as the majority of biopsies and cytology specimens received in a hospital laboratory are unpaired, the samples included in our study were discontinuous, thus not representative for assessment of overall diagnostic performance. Guidelines and cohorts have established references for these metrics.On the same note, AUC may be more suited for determining thresholds for continuous variables.The role of effusion cytology is irreplaceable in the investigation of pleural diseases, but not without certain limitations. We highly appreciate the opportunity Zhang and colleagues offered in further deliberation on the issue.Ivan K. Poon: Writing \u2013 original draft . Ronald C. K. Chan: Validation . Joseph S. H. Choi: Writing \u2013 review and editing . Joanna K. M. Ng: Validation . Katsie T. Tang: Validation . Yolanda Y. H. Wong: Validation . Ka Pang Chan: Validation . Wing Ho Yip: Validation . Gary M. Tse: Writing \u2013 review and editing . Joshua J. X. Li: Writing \u2013 original draft .The authors declare that there is no conflict of interest regarding the publication of this paper."} +{"text": "Risk factors for tuberculous or nontuberculous spondylitis after percutaneous vertebroplasty or kyphoplasty in patients with osteoporotic vertebral compression fracture: A case-control study by Zheng B-W, Liu F-S, Zheng B-Y, Niu HQ, Li J, Lv G-H, Zou1 M-X and Xu Z. (2022) Front. Surg. 9: 962425. doi: 10.3389/fsurg.2022.962425A corrigendum on Incorrect FundingIn the published article, there was an error in the Funding statement. [This work was supported by the National Natural Science Foundation of China (81871821 to JL and 82002364 to MXZ) and Project for Clinical Research of Hunan Provincial Health Commission ]. The correct Funding statement appears below.FundingThe authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated."} +{"text": "Nature Communications 10.1038/s41467-021-21643-0, published online 04 March 2021Correction to: In the Acknowledgements section of this article the grant number relating to NSF DMR given for authors X.D. and G.K. was incorrectly given as 173307 and should have been 1733071. The original article has been corrected."} +{"text": "S. aureus-induced mammary gland tissues by H & E staining as published. We used the wrong picture of the XD69 group in In the original publication , there w"} +{"text": "Urogymnus polylepis is a threatened species that is vulnerable to riverine and coastal marine pressures. Despite its threatened status, the range of U.\u00a0polylepis is still being determined. In this study, photographic evidence of U.\u00a0polylepis in Myanmar was provided through market surveys (2017\u20132018) and social media . Urogymnus polylepis is exposed to fisheries and habitat degradation pressures in Myanmar; therefore, conservation management is likely needed to ensure populations persist into the future.The giant freshwater whipray In the tropics, riverine environments have degraded through a range of human\u2010induced activities, such as the construction of water retention structures fisheries . Vidthayanon et al.\u00a0 between 2017 and 2018. These surveys aimed to obtain baseline biological information on shark and ray species in Rakhine State . The authors followed the survey protocol outlined in the Wildlife Conservation Society (WCS) Field Manual for Shark and Ray Fisher, Trader & Market Based Surveys in Rakhine, Myanmar on 9 June 2021, where users were invited to share any photographs of large stingrays caught locally within riverine environments. An additional five records were received from Facebook users, and consent for use of their images in publication was received. These records were dated between 2016 and 2021 from fishing communities along the Mayu and Kalatan Rivers in Rakhine and Chin States inland fisheries of inland fisheries in Myanmar, limiting the amount of catch that is sold in markets and thus more easily observable. Apart from fishing, riverine environments in Myanmar have been significantly degraded by land repurposing activities and potentially from mining pollutants siamensis in the late 19th century and continue to have broad applications to conservation of elasmobranchs globally.M.I.G. and M.M. were involved in conception of this study; A.B., T.H., A.M., K.M.M., T.R., M.K.S. and M.M. helped in data generation; M.I.G., T.H., A.M., T.M., K.M.M., T.R., M.K.S., W.T.W., K.Z.Y. and M.M. assisted with data analysis; M.I.G and M.M. were involved in manuscript preparation; all other authors contributed to editing the manuscript."} +{"text": "It was the first new journal from the British Association of Dermatologists in over 100 years.G. W. M. Millington is the Editor in Chief of Skin Health and Disease.G. W. M. Millington: Writing \u2013 original draft."} +{"text": "Nature Communications 10.1038/s41467-022-28520-4, published online 16 February 2022.Correction to: th author Srinivasrao Ganipisetti, who is from the University of Louisville. Consequently, Srinivasrao Ganipisetti\u2019s initials were added to the Competing Interests section, which now reads as follows: \u2018Seoul National University and AUTOTAC Bio, Inc. have filed patent applications based on the results of this study. The remaining authors declare no competing interests.\u2018 Additionally, Srinivasrao Ganipisetti\u2019s initials were added to the Author Contributions, and the corrected sentence reads as follows: \u2018ATLs binding the p62-ZZ domain were synthesized and modeled by S.G., K.Y.K., J.E.N., and H.T.K.\u2019 This has been corrected in both the PDF and HTML versions of the Article.The original version of this Article omitted from the author list the 8"} +{"text": "Molecular Genetics & Genomic Medicine, 8(8), e1349. https://doi.org/10.1002/mgg3.1349Huang, Y. F., Lu, L., Shen, H. L., & Lu, X. X. (2020). LncRNA SNHG4 promotes osteosarcoma proliferation and migration by sponging miR\u2010377\u20103p. www.wileyonlinelibrary.com), has been retracted by agreement between the Editor in Chief Prof. Suzanne Hart and John Wiley & Sons. The retraction has been agreed due to concerns raised about image manipulation. The journal team has investigated and determined that the image manipulation undermines the reliability of the data presented and the article's conclusions.The above article published online on 14 June 2020 in Wiley Online Library ("} +{"text": "The Role of the Family in Deceased Organ Procurement. A Guide for Clinicians and Policy Makers. Alphen aan den Rijn, Netherlands: Transplantation (2019)\u201d 103(5):e112\u201318. doi: 10.1097/TP.0000000000002622.\u201dThe full reference is \u201cDelgado J, Molina-Perez A, Shaw D, Rodriguez-Arias D. The Role of the Family in Deceased Organ Procurement: A Guide for Clinicians and Policymakers. The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated."} +{"text": "Analysis on the MRI and BAEP results of neonatal brain with different levels of bilirubin by Lu Z, Ding S, Wang F and Lv H. (2022) Front. Pediatr. 9: 719370. doi: 10.3389/fped.2021.719370A corrigendum on In the published article, author Zhongxing Lu was missing an affiliation: \u201cChildren\u2019s Hospital of Soochow University, Suzhou, China\u201d. The author affiliation list has now been updated.The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated."} +{"text": "The authors have requested that the following changes be made to their paper .The original authors wish to add Dr. Sung Hyun Chung and Dr. Mi Kyung Pyo as a coauthor to this paper. The author contributions were: S.H.C. and M.K.P. contributed to funding acquisition and production of GS-KG9 and GS-E3D, respectively. The updated \u201cAuthor Contributions\u201d and \u201cFunding\u201d are provided below.We would like to apologize for any inconvenience caused to the authors and readers by this mistake. The published version will be updated on the article webpage, with a reference to this notice."} +{"text": "The affiliation for the fourth author is incorrect. Kramat Hussain is not affiliated with #2 but with #3: College of Management and Economics, Tianjin University, P.R. China."} +{"text": "Scientific Reports 10.1038/s41598-021-83240-x, published online 25 March 2021Correction to: Nesrine Baatalah was omitted from the author list in the original version of this Article.The Author Contributions section now reads:\u201cS.B.: conducted and acquired cellular biology experiments, analyzed and interpreted the data. A.E.: conducted and acquired molecular docking and dynamics simulations experiments, analyzed and interpreted the data. G.C: conducted and acquired chemistry experiments, analyzed and interpreted the data. I.P.: conducted and acquired cellular biology and electrophysiology experiments, analyzed and interpreted the data. B.C.: conducted and acquired cellular biology experiments, analyzed and interpreted the data. F.B.: conducted and acquired chemistry experiments, analyzed and interpreted the data. B.H.: conducted and acquired molecular docking and dynamics simulations experiments, analyzed and interpreted the data. N.S.: conducted and acquired patch clamp experiments, analyzed and interpreted the data. N.S. conducted and acquired Western Blott experiments, analyzed and interpreted the data. D.T.: conducted and acquired Western Blott experiments, analyzed and interpreted the data. A.H.: conducted and acquired and electrophysiology experiments, analyzed and interpreted the data. C.M.: conducted and acquired Western Blott experiments, analyzed and interpreted the data. M.F.D.C.: conducted and acquired cellular biology and electrophysiology experiments, analyzed and interpreted the data. AP: performed chemistry experiments.\u00a0A.L.: conducted and acquired and electrophysiology experiments, analyzed and interpreted the data. A.H.i: participated to the design and interpretation of the experiments. J.P.M.: participated to the design of molecular docking experiments and interpretation of the experiments. G.P.: participated to the design of chemistry experiments and interpretation of the experiments. A.E.: conceived the study, analyzed and interpreted the data and participated to the writing of the manuscript. I.C.: designed the molecular docking and dynamics simulation analyzed and interpreted the data and participated to the writing of the manuscript. C.G.P.: designed the chemistry experiments analyzed and interpreted the data and participated to the writing of the manuscript. I.S.G.: conceived the study, analyzed and interpreted the data and wrote the manuscript\u201d.The original Article and accompanying Supplementary information file has been corrected."} +{"text": "Sci., 2019, 10, 2653\u20132662, DOI: 10.1039/C8SC03426E.Correction for \u2018A universal method for sensitive and cell-free detection of CRISPR-associated nucleases\u2019 by Kurt J. Cox In the original article, incorrect grant information from the Department of Energy was provided. The corrected Acknowledgements section is provided below, with the correct grant number:This work was supported by the Burroughs Wellcome Fund (Career Award at the Scientific Interface to A. C.), DARPA (Brdi N66001-17-2-4055 to A. C.), NIH (1R21AI126239-01 to A. C.), Army Research Office award W911NF1610586 (to A. C.), and by the Department of Energy through grant DE-SC0010595 to E. F., which supported the salary of H. K. K. S. This work is dedicated to Professor Ronald T. Raines on the occasion of his 60th birthday.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "Scientific Reportshttps://doi.org/10.1038/s41598-021-97596-7, published online 09 September 2021Correction to: Dajie Zhang was omitted from the author list in the original version of this Article.The Author Contributions section now reads:\u201cJ.S.K. led the electrochemical characterization of the catalysts. N.Q.L. performed the theoretical calculations. D.R.S. assisted with PFAS analysis. J.K.J. contributed to data interpretation. D.Z. contributed to the synthesis of catalysts. Z.X. was responsible for conceiving and planning of the project. All authors contributed to drafting of the manuscript.\u201dThe Acknowledgements section now reads:\u201cThe authors acknowledge the support from the Independent Research and Development (IRAD) Fund from the Research and Exploratory Development Mission Area of the Johns Hopkins University Applied Physics Laboratory. The authors also thank Dr. Tim Montalbano for the assistance with SEM. The XANES work used the Beamline for Materials Measurement (6-BM) of the National Synchrotron Light Source II, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Brookhaven National Laboratory under Contract No. DE-SC0012704.\u201dThe original Article has been corrected."} +{"text": "Nature Communications 10.1038/s41467-021-23659-y, published online 07 June 2021.Correction to: The original version of this Article contained an error in the author affiliations. Affiliation 1 incorrectly read \u201cDepartment of Chemistry, The University of Hong Kong, Hong Kong S.A.R., People\u2019s Republic of China\u201d. This has now been corrected to \u201cDepartment of Chemistry, State Key Laboratory of Synthetic Chemistry, CAS-HKU Joint Laboratory of Metallomics on Health and Environment, The University of Hong Kong, Hong Kong S.A.R., People\u2019s Republic of China\u201d. in both the PDF and HTML versions of the Article."} +{"text": "We also detect transcripts associated with oxidation of iron and sulfur, and with reduction of arsenate, selenate and nitrate. Given limited input of electron acceptors from outside the system, these results suggest that the microbial communities use an unexpectedly diverse variety of electron acceptors. Products of water radiolysis and their interactions with sediment continuously provide diverse electron acceptors and hydrogen. Cryptic microbial utilization of these oxidized substrates and H2 may be an important mechanism for multi\u2010million\u2010year survival under the extreme energy limitation in subseafloor sediment.Microbial gene expression in anoxic subseafloor sediment was recently explored in the Baltic Sea and the Peru Margin. Our analysis of these data reveals diverse transcripts encoding proteins associated with neutralization of reactive oxygen species, including catalase, which may provide an Among tet al.,\u00a0et al.,\u00a0Roseobacter spp. to Mn(IV). Interestingly, fungal peroxidases, including manganese and heme peroxidases, are also thought to result in extracellular manganese oxidation . Constitutive expression of functional genes from early in the sediment history might conceivably occur if microbes persist from that time without reproduction might explain PETs for ROS and diverse electron acceptors in marine sediment older than a few million years support for microbial communities buried in deep anoxic sediment.et al.,\u00a0et al.,\u00a0et al.,\u00a0Information regarding collection, handling and processing of samples for the original transcriptomic studies can be found in the following publications: Peru Margin \u2010 Orsi SRA Toolkit v.2.8.2 . Site metadata, sequence trimming, assembly and read map details are provided in the Metatranscriptomic data from Peru Margin and Baltic Sea marine sediment were downloaded from the Sequence Read Archive (SRA) using the LithoGenie and RosGenie. These software packages and the HMM libraries employed by each tool are public: https://github.com/Arkadiy-Garber/LithoGenie and https://github.com/Arkadiy-Garber/RosGenie. Software details, including marker gene selection, HMM calibrations and contaminant screening are provided in the To identify transcripts relevant to cryptic metabolisms and ROS neutralization, we developed two novel bioinformatics tools: G.A.R. was funded by an NSF C\u2010DEBI post\u2010doctoral fellowship. S.D. and G.A.R. were funded by the U.S. National Science Foundation through grants NSF\u2010OCE\u20101130735 and NSF\u2010OCE\u20100939564. S.M.M. was funded by the Joint Institute for the Study of the Atmosphere and Ocean (JISAO) under the NOAA Cooperative Agreement NA15OAR4320063.G.A.R., S.D. and A.I.G. conceived the study; A.I.G. and G.A.R. performed bioinformatics analyses; A.I.G., G.A.R., S.M.M., W.O. and S.D. interpreted data and wrote the manuscript.Appendix S1. Supporting information.Click here for additional data file."} +{"text": "Rheumatology 2020;59:2563\u2013257110.1093/rheumatology/kez671doi:In the above article, image C and D in Figure 2 was originally a duplicate of image A and B. This has been corrected online and in print. The author apologises for the error."} +{"text": "Cerebral Blood Flow and Oxygen Consumption in Man. This concept suggested a relatively broad mean arterial pressure range (~60\u2013150\u00a0mmHg) wherein cerebral blood flow remains constant. However, the assumption that this wide cerebral autoregulation plateau could be applied on a within\u2010individual basis is incorrect and greatly variable between individuals. Indeed, each data point on the autoregulatory curve originated from independent samples of participants and patients and represented interindividual relationships between cerebral blood flow and mean arterial pressure. Nonetheless, this influential concept remains commonly cited and illustrated in various high\u2010impact publications and medical textbooks, and is frequently taught in medical and science education without appropriate nuances and caveats. Herein, we provide the rationale and additional experimental data supporting the notion we need to lose this dogmatic view of cerebral autoregulation.In 1959, Niels Lassen illustrated the cerebral autoregulation curve in the classic review article entitled In 1959, Niels Lassen illustrated the cerebral autoregulation curve in the classic review article entitled Cerebral Blood Flow and Oxygen Consumption in Man. This concept suggested a relatively broad mean arterial pressure range wherein cerebral blood flow remains constant. Herein, we provide the rationale and additional experimental data supporting the notion we need to lose this dogmatic view of cerebral autoregulation. However, some of these methods to manipulate mean arterial pressure have the ability to influence CBF, independently from cerebral autoregulation, via direct effects on tone or indirect effects on arterial carbon dioxide tension or cerebral metabolism. In addition, the effects that some anesthetic agents have on CBF (i.e., isoflurane and sevoflurane can increase CBF while propofol can reduce CBF (Slupe & Kirsch, 8Considering the compelling evidence currently available, it is now time for the medical community to move away from the commonly touted view of cerebral autoregulation presented in \u201cCerebral Blood Flow and Oxygen Consumption in Man\u201d Figure . As suchThe authors declare no conflict of interest.P.B. contributed to the original idea of the review article; L.L. analyzed the data; P.B., L.L., and P.N.A. interpreted the results; L.L. prepared Figure 4; P.B. drafted the article; L.L., J.D.S., M.M.T., H.G.C., R.L.H., S.J.E.L, A.Y.D., E.J.C., and P.N.A. edited and revised the manuscript; all authors approved the final version of manuscript."} +{"text": "The eighth author\u2019s name is spelled incorrectly. The correct name is: James R. Walters. There is also an error in affiliation 3 for author James R. Walters. The correct affiliation 3 is: Department of Ecology and Evolutionary Biology, University of Kansas, KS, United States of America."} +{"text": "Scientific Reportshttps://doi.org/10.1038/s41598-021-87339-z, published online 13 April 2021Correction to: The original version of this Article contained an error in the author list. Xin Lou was incorrectly listed as an author of the original Article, and has subsequently been removed.The Acknowledgments section now reads:The authors would like to thank Professor Lou Xin for her technical guidance.The Author Contributions section now reads:W.W. collected data and wrote the paper. T.W. and Z.S. performed data analysis. L.M., and G.W. conceived the research, provided guidance, discussed the data, revised and improved the paper.The Funding section \u201cThis study was funded by the National Natural Science Foundation of China (Contract Grant Numbers: 81671126 to X.L.).\u201d has been removed.The original Article and accompanying Supplementary Information file have been corrected."} +{"text": "In vivo modulation of ubiquitin chains by N-methylated non-proteinogenic cyclic peptides\u2019 by Joseph M. Rogers et al., RSC Chem. Biol., 2021, 2, 513\u2013522, DOI: 10.1039/D0CB00179A.Correction for \u2018 The author regrets that the funding information was incorrectly shown in the acknowledgements section of the original manuscript.This study was also funded by the National Institutes of Health under grant GM065334 to D.F. and A.B.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "The authors declare no competing interests.A.L.\u2010P., C.L.\u2010C., M.G.\u2010S., M.A., R.A., J.P., and B.P.: molecular analysis; A.A., A.A.\u2010C., and F.J.R.: patients\u2019 recruitment, clinical evaluation, and clinical score calculation; I.M.\u2010A. and P.G.\u2010P.: bioinformatics studies and variants interpretation; A.L.\u2010P., J.P., and B.P.: manuscript writing, collection, and assembly of data; F.J.R., J.P., and B.P.: manuscript editing and approval of the manuscript. All authors have read and agreed to the submitted version of the manuscript.The employed procedure was reviewed and approved by the Ethics Committee of Clinical Research from the Government of Arag\u00f3n . All human subjects participating in the research signed the informed consent. An additional informed consent was collected and signed for the publication of subjects\u2019 photographs.Dear Editor,ANKRD11 and NIPBL were identified.The diagnosis success rates for developmental disorders have greatly improved in the last years mainly due to the widespread use of DNA next\u2010generation sequencing. Nevertheless, several studies have stressed the importance of a critical reconsideration of genetic results and a further implementation of protocols for variant\u2010level reevaluation and case\u2010level reanalysis ]. The variant, that induces changes in the surface charge of the protein , body weight , and head circumference were all normal. She was referred to our hospital at the age of 3\u00a0years because of facial dysmorphism, gastroesophageal reflux, and motor developmental delay. After a comprehensive physical evaluation by our clinical geneticist, she was clinically diagnosed as CdLS with a clinical score of 10, mainly due to the synophrys (HP:0000664), thick eyebrows (HP:0000574), concave nasal ridge (HP:0011120), downturned corners of mouth (HP:0002714), global developmental delay (HP:0001263), small hands (HP:0200055) and feet (HP:0001773), short fifth finger (HP:0009237), and hirsutism (HP:0001007)] , triangular face (HP:0000325), or bulbous nasal tip (HP:0000414). An additional clinical analysis was carried out with Face2Gene. At 3\u00a0years old, KBGS and CdLS were the first and second syndromes suggested, respectively, whereas at 6\u00a0years old, CdLS diagnosis did not appear between the top\u20105 diagnosis provided by Face2Gene Figure . A molec] Figure . This va] Figure . The varANKRD11 variants (Parenti et al., ANKRD11 in gene panels used for molecular testing of individuals presenting with clinical characteristics of CdLS.Considerable efforts have been made to standardize the interpretation of genetic variants in the laboratory (Latorre\u2010Pellicer et al.,"} +{"text": "The original version of this article unfortunately contained a mistake. The given name and family name of authors were swapped.The correct given name and family name should be:Vanessa S. FearCatherine A. ForbesSamuel A. NeeveScott A. FisherJonathan CheeJason WaithmanShao Kang MaRichard LakeAnna K. NowakJenette CreaneyMatthew D. BrownChristobel SaundersBruce W. S. RobinsonThe original article has been corrected."} +{"text": "Phil. Trans. R. Soc. B376, 20200297. (Published 4 October 2021). (doi:10.1098/rstb.2020.0297)Funding information for one author was omitted from the funding statement.S.L. is part of the relationship programme supported by The Medical Research Council and Scotland's Chief Scientist Office (grant no. MC_UU_00022/3) and with CSO funding of the Relationships programme (grant no. SPHSU18).This has been updated on the publisher's website."} +{"text": "Dear Editor,Atherosclerosis, as the leading cause of coronary artery disease is one of the major contributors of death globally.We reanalyzed scRNA\u2010seq data of COVID\u201019 patient blood samples and found SARS\u2010CoV\u20102 infection may affect CD36 and MSR1 expressions which mediate lipid uptake of macrophages contributing to atherosclerosis progression , and a grant from the Department of Science and Technology, Zhejiang Province (Grant no. LGF19H020011), a Grant from the Department of Science and Technology, Zhejiang Province (Grant no. Z16H020001) and the Natural Science Foundation of China (Grant no. 81873484), People's Republic of China.Z.L.R., Z.J., and W.S. conceived the research and participated in the study design. C.M., H.J., Y.X., and H.X.T. assisted in collecting the data. Z.W.T., W.Z., and C.W.J. analyzed the data and drafted the manuscript. All authors read and approved the final manuscript.Supporting InformationClick here for additional data file."} +{"text": "The ninth author\u2019s name is incorrect. The correct name is: Daniela E. Kirwan.The ORCID iD is missing for the ninth author. Author Daniela E. Kirwan\u2019s ORCID iD is: 0000-0003-3989-0795.The affiliation for the ninth author is incorrect. The correct affiliation is not indicated. Daniela E. Kirwan is not affiliated with #1 but with: Institute for Infection & Immunity, St George\u2019s University of London, London, United Kingdom.The following information is missing from the Funding statement: DEK is supported by Medical Research Council, UK Fellowship MR/P019978/2."} +{"text": "Dear Editor,3AR axis. Fatty acid transport 2 (Fatp2) may serve as an important factor to mediate the browning\u2010inducing effects of celastrol. This study may provide a basis for exploring a new strategy for the treatment of obesity.Despite advances in therapy for obesity, effective therapeutic strategies for weight loss are still needed.Celastrol is a leptin sensitizer.3\u2010adrenergic receptor (AR) abrogated the weight\u2010reducing effects of celastrol without affecting food intake diminished the expression of WAT browning\u2010associated genes , the National Key Research and Development Program of China , the Beijing Municipal Natural Science Foundation , the Peking University Research Foundation , and the Peking University People's Hospital Research and Development Funds .B.W. and X.Y. performed the experiment, analyzed the data, made the figures, and wrote the paper. M.Z., Z.S., Z.H., C.Z., B.G. and J.L. participated in experiments. L.Q. and W.Z. edited the manuscript. R.Z. conceived the study, designed experiments, and wrote and edited the paper. All authors reviewed and approved the manuscript for submission.All\u00a0data\u00a0associated with this study are present in the paper or Supplementary Materials.The authors declare no conflict of interest.Supporting informationClick here for additional data file.FigureS1Click here for additional data file.FigureS2Click here for additional data file.FigureS3Click here for additional data file.FigureS4Click here for additional data file.FigureS5Click here for additional data file.FigureS6Click here for additional data file."} +{"text": "Nature Communications 10.1038/s41467-021-24788-0, published online 22 July 2021.Correction to: The original version of this Article contained an error in the Acknowledgments, which omitted the grant numbers relating to the Swiss National Science Foundation and the European Research Council given for P.L.H. The original version read \u2018This work was funded with grants from Swiss National Science Foundation, European Research Council, and Fondation Aclon to P.L.H\u2019. The correct version replaces this sentence with \u2018This work was funded with grants from the Swiss National Science Foundation (#310030_192496), the Fondation Aclon and the European Research Council to P.L.H\u2019. This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Journal of Experimental Psychology: General. Advance online publication. July 15, 2021. http://dx.doi.org/10.1037/xge0001071), acknowledgment of and formatting for Economic and Social Research Council funding was omitted. The author note and copyright line now reflect the standard acknowledgment of and formatting for the funding received for this article.In the article \u201cPrior Experience With Unlabeled Actions Promotes 3-Year-Old Children\u2019s Verb Learning\u201d by Suzanne Aussems, Katherine H. Mumford, and Sotaro Kita (All versions of this article have been corrected."} +{"text": "There are two tiers of programs in the Project P.A.T.H.S. . In the Tier 1 Program, teaching units based on different positive youth development constructs are covered. Pre- and post-test data utilizing the Chinese Positive Youth Development Scale (CPYDS) and post-test subjective outcome evaluation data were collected from 546 students who participated in the 20h Tier 1 Program of the P.A.T.H.S. Project. Results showed that high proportions of the respondents had positive perceptions of the program and the instructors, with 85.3% of the respondents regarding the program as helpful to them. Positive changes in the program participants in many measures of positive youth development were also observed. Although there were some increases in problem behavior in some areas, adolescent problem behavior was generally stable. The present study provides preliminary support for the effectiveness of the Tier 1 Program of the Project P.A.T.H.S. in Hong Kong."} +{"text": "The authors have requested the following corrections to their paper .1,*\u201d has been changed to \u201cSo Young Kim 1\u201d. So Young Kim is not the corresponding author.\u201cSo Young Kim The email address of So Young Kim has been added to the Affiliation 1: Department of Clinical Nutrition, Research Institute & Hospital, National Cancer Center, Goyang 10408, Korea; drewpia@ncc.re.kr (S.Y.K.); gawie@ncc.re.kr (G.-A.W.); yacho@ncc.re.kr (Y.-A.C.); saltstars@ncc.re.kr (H.-h.K.); rka2007@ncc.re.kr (K.-A.R.); mkyoo52@ncc.re.kr (M.-K.Y.)The Correspondence section has been changed to \u201cCorrespondence: hjjoung@snu.ac.kr; Tel.: +82-2-880-2716\u201d.The authors apologize for any inconvenience caused to the readers by the change. The change does not affect the scientific results. The manuscript will be updated and the original will remain online on the article webpage."} +{"text": "Scientific Reports6 Article number: 3057810.1038/srep30578; published online: 08012016; updated on: 02222017The Acknowledgements section in this Article is incomplete.E. coli strains; Knut Drescher for P. aeruginosa PA14 strains\u201d.\u201cThis work was supported by NSF Grant No. DMR-1120901 (Penn MRSEC) and PIRE-1545884. T.H.R.N. was supported by the Postdoctoral Fellowship for Academic Diversity Program (University of Pennsylvania) and the Consortium for Ocean Leadership, Inc. (Prime award #SA15-19). H.K. was supported by NIH/NIDCR grants R01DE018023 and R01DE025220. L.H. and H.J. were supported by Natural Science Foundation of China (NSFC) grant #11372093 and China Scholarship Council (CSC) Grant #201306120117. We also thank Dacheng Ren (Syracuse University) for providing the should read:E. coli strains; Knut Drescher for P. aeruginosa PA14 strains. Data are publicly available through the Gulf of Mexico Research Initiative Information & Data Cooperative (GRIIDC) at https://data.gulfresearchinitiative.org \u201d.\u201cThis research was made possible in part by a grant from The Gulf of Mexico Research Initiative, NSF Grant No. DMR-1120901 (Penn MRSEC), and PIRE-1545884. T.H.R. N. was supported by the Postdoctoral Fellowship for Academic Diversity Program (University of Pennsylvania). H.K. was supported by NIH/NIDCR grants R01DE018023 and R01DE025220. L.H. and H.J. were supported by Natural Science Foundation of China (NSFC) grant #11372093 and China Scholarship Council (CSC) Grant #201306120117. We also thank Dacheng Ren (Syracuse University) for providing the"} +{"text": "Scientific Reports6: Article number: 32609; 10.1038/srep32609 published online: 09092016; updated: 10102016.In this Article additional disclosures should have been included in the Competing Financial Interests statement. The corrected statement is included below. The Authors apologize for these omissions.The Competing financial interests statement in this Article should read:\u201cS.P. and V.P. are employed by the Chopra Center. D.C. is a co-founder and a co-owner of the Chopra Center. The Chopra Center offers the Perfect Health program commercially; however, none of the study subjects were charged for the participation in the program.\u201d"} +{"text": "Scientific Reports7:1454; doi:10.1038/s41598-017-01626-2; Article published online 03 May 2017Funding sources and the scientific contributions of B Shillito, J Ravaux and G Hamel were not acknowledged in this Article. The authors apologise for these accidental omissions. The Acknowledgements section,\u201cWe thank the captain and crew of the RV Atalante, the DSV Nautile group (IFREMER), along with N. Le Bris (UMR8222) and F. Lallier (UMR7144), chief scientists of the MESCAL cruises. We thank A.S. Leport (UMR7144) for her technical assistance and B. Shillito (BOREA) and S. Hourdez (UMR7144) for the use of BALIST and DESEARES, respectively. We also acknowledge I. Probert for the English proofreading. This work was funded by the Universit\u00e9 de Lille (BQR), the CNRS, the GDR ECCHIS and the R\u00e9gion Nord Pas de Calais-FRB (VERMER project)\u201d.should read:\u201cWe thank the captain and crew of the RV Atalante, the DSV Nautile group (IFREMER), along with N. Le Bris (UMR8222) and F. Lallier (UMR7144), chief scientists of the MESCAL cruises. We thank A.S. Leport (UMR7144) for her technical assistance and B. Shillito (BOREA) and S. Hourdez (UMR7144) for the use of BALIST and DESEARES, respectively. We also acknowledge I. Probert for the English proofreading. This work was funded by the Universit\u00e9 de Lille (BQR), the CNRS, the GDR ECCHIS and the R\u00e9gion Nord Pas de Calais-FRB (VERMER project). We thank B Shillito, J Ravaux (UMR 7208 BOREA) and G Hamel (UMR 7590 IMPMC),\u00a0for conducting and performing the BALIST pressure experiments, and for providing access to the corresponding samples. The isobaric sampling and transfer equipments used in this study were funded by the EXOCET/D project , the BALIST project , and by the University Pierre et Marie Curie (BQR)\u201d.In addition, the Author Contributions Statement is incorrect.\u201cC.P. generated and analyzed the sequence dataset. D.J. designed and contributed to the genetic analysis and performed the sampling with A.T. A.T. conducted and performed the experiments under pressure and the RT-qPCR. F.M. and C.P. participated in writing the paper. A.T. and D.J. supervised the work and wrote the paper\u201d.should read:\u201cC.P. generated and analyzed the sequence dataset. D.J. designed and contributed to the genetic analysis and performed the sampling with A.T. F.M. and C.P. participated in writing the paper. A.T. and D.J. supervised the work and wrote the paper\u201d."} +{"text": "Nature Communications7 Article number: 1231610.1038/ncomms12316 (2016); Published 08242016; Updated 09282016.The financial support for this Article was not fully acknowledged. The Acknowledgements should have included the following: Y.M.C. gratefully acknowledges the financial support from International Science and Technology Cooperation Program supported by National Natural Science Foundation of China (No. 11674263)."} +{"text": "Nature Communications8: Article number: 15112; DOI: 10.1038/ncomms15112 (2017); Published 04252017; Updated 05232017A patent based on the work reported in this Article was inadvertently omitted from the Competing interests section of this article. The Competing interests statement should read:E.M., A.F. and M.D. are co-authors on a patent entitled \u2018Extracorporeal life support system and methods of use thereof' (Patent no. WO2014145494 A1). The remaining authors declare no competing financial interests."} +{"text": "There are errors in the Author Contributions. The correct contributions are: Conceptualization: MS PM. Data curation: NA MS. Funding acquisition: PM JW. Investigation: MS MR. Methodology: MS PM. Project administration: PM JW. Resources: PM. Software: MS NA. Supervision: PM JW. Visualization: MS. Writing\u2013original draft: MS. Writing\u2013review & editing: MS PM MR JW.The following information is missing from the Funding section: We acknowledge support for the Article Processing Charge by the German Research Foundation and the Open Access Publication Fund of the Technische Universit\u00e4t Ilmenau.The publisher apologizes for the errors."} +{"text": "Scientific Reports7:1412; doi:10.1038/s41598-017-01456-2; Article published online 03 May 2017The original PDF version of this Article contained an error in the order of corresponding authors:\u201cCorrespondence and requests for materials should be addressed to D.W. (email: cruise00@126.com) or H.-P.L. (email: liw2013@126.com)\u201dnow reads:\u201cCorrespondence and requests for materials should be addressed to H.-P.L. (email: liw2013@126.com) or D.W. (email: cruise00@126.com)\u201d.This has been corrected in the PDF version of this Article; the HTML version was correct at the time of publication."} +{"text": "In this presentation we illustrate the effects of combining eye movement desensitization and reprocessing (E.M.D.R) therapy and theory of structural dissociation of the personality (T.S.D.P) on dissociative and post-traumatic stress disorder (P.T.S.D) symptoms. We first briefly describe both theories and conclude why combining them in the treatment of severely traumatized adolescents with PTSD may be beneficial.E.M.D.R therapy is an empirically valid treatment for P.T.S.D, based on numerous randomized controlled trials and several meta-analyses personification or a sense of ownership and knowing \u2018this is what happened to me\u2019 and knowing feelings and thoughts about it and (2) presentification being grounded in the present while able to integrate the past and the possibilities of the future. Knowing that this has happened in the past and the present and future is no longer dictated by the traumatic past (van der Hart, Nijenhuis & Steele, Young severely traumatized adolescents with PTSD who have been early victims of emotional, physical and sexual abuse within an interpersonal relationship and have exhibited dissociative symptoms, have been treated in the University Child and Adolescent Psychiatry Department, Athens, Greece by applying E.M.D.R therapy as a treatment intervention and the theory of T.S.D.P as a theoretical and conceptual tool for understanding the presenting dissociative symptoms. The theory of T.S.D.P was utilized to conceptualize the cases in terms of dissociative and P.T.S.D symptoms as they were measured at baseline, during therapy and at the end of therapy to assess change. We observed that their P.T.S.D and dissociative symptoms would decrease when they felt safe and were able to trust their own bodily reactions and emotions. Following a prolonged stabilization phase the adolescents were more integrated and able to reprocess their traumatic memories and put together their own life story.In conclusion, by applying E.M.D.R therapy while being informed by T.S.D.P we may better understand and support severely traumatized adolescents who have been victims of early interpersonal abuse. While working with them can be very challenging, we saw promising results while applying E.M.D.R therapy guided by the concepts of T.S.D.P. There is promise in combining E.M.D.R therapy and T.S.D.P and future research should focus on its effects in terms of understanding dissociation of the personality following interpersonal trauma."} +{"text": "Nature Communications5: Article number: 4562; DOI: 10.1038/ncomms4562(2014); Published: 04152014; Updated: 10142016.Nature Communications is publishing an editorial expression of concern on the manuscript \u2018Experimental orthotopic transplantation of a tissue-engineered oesophagus in rats\u2019 from Sj\u00f6qvist et al. to alert our readership to concerns regarding the integrity of the study. An investigation related to this research has been conducted by the Expert Group for Misconduct in Research at the Swedish Central Ethical Review Board on behalf of Karolinska Institutet. A statement on behalf of the Expert Group summarizing the results of this investigation ( http://www.epn.se/media/2374/o-1-2016-statement-expert-group-for-misconduct-in-research-160906-eng.pdf) raises concerns regarding the extent to which the data presented in this Article accurately report and are fully representative of the results of the experiments that were carried out. Concerns have been raised regarding the in vitro characterization of the oesophageal scaffold, in vivo imaging of the transplanted oesophageal scaffold and the degree to which its transplantation into animals was successful. We are currently following our established process to investigate these issues further.P.M., M.A.B., H.K., P.D., B.J. and D.A.T. agree, whereas S.S., P.J. and M.L.L. disagree with this notice. The other authors could either not be reached or did not provide a clear response."} +{"text": "Primula undulifolia G. Hao, C.M. Hu & Y. Xu, sp. nov. [urn:lsid:ipni.org:names: 60472693\u20132] .The appropriate GUID numbers are missing from the \u201cTaxonomic treatment\u201d subsection of the Results. The publisher apologizes for the error. The correct sentence should read as: There is an error in both Fig 2 and Fig 3. The scale bar is incorrect. Please see the corrected figures here."} +{"text": "In the publication of this article , there iThe error: \u2018This work has been founded by IRE Internal Projects to G. P. and E. V.\u2019Should instead read: \u2018This work has been founded by IRE Internal Projects to G. P. and E. V. and Lazio Region BTO project to E. V.\u2019This has now been included in this erratum."} +{"text": "Sir,in silico approach could predict with 81.3% accuracy whether a patient would have good or bad outcome.Recently, in silico\u2019, which We would like to highlight that these results effectively replicate findings from a study we published in mid-2016 and the regions of brain tissue resected. This allows predictions of the model to be validated. Ultimately the reported predictive capacity of both approaches is broadly similar in terms of sensitivity (91% versus 87.5%) and specificity (80% versus 75%) . Howeverin silico approaches to predict postsurgical outcome. A particularly important result is that predictions derived from interictal, rather than ictal data were found to be promising, which could be beneficial for patients undergoing presurgical monitoring, as seizures may not need to be observed. Such approaches offer exciting new possibilities to develop surgical and other treatment strategies for people with medically intractable epilepsies.In summary, the replication of our earlier findings by M.G., M.P.R. and J.R.T. gratefully acknowledge the financial support of the EPSRC via grant EP/N014391/1. They further acknowledge funding from Epilepsy Research UK via grant number A1007 and the Medical Research Council via grant MR/K013998/1. The contribution of M.G. and J.R.T. was generously supported by a Wellcome Trust Institutional Strategic Support Award (WT105618MA). M.P.R. is supported by the National Institute for Health Research (NIHR) Biomedical Research Centre at the South London and Maudsley NHS Foundation Trust. C.R. and A.E. were supported by the Swiss National Science Foundation (grant SPUM 140332). K.S. is grateful for support from the Swiss National Science Foundation (grants 122010 and 155950)."} +{"text": "Carinina ochracea . The correct citation is: von D\u00f6hren J (2016) Development of the Nervous System of Carinina ochracea . PLoS ONE 11(10): e0165649. doi:10.1371/journal.pone.0165649. The publisher apologizes for the error.The word \u201cPalaeonemertea\u201d is misspelled in the article title. The correct title is: Development of the Nervous System of"} +{"text": "Scientific Reports6: Article number: 2029010.1038/srep20290; published online: 02052016; updated: 09022016Vinodkumar Etacheri was omitted from the author list in the original version of this Article. This has now been corrected in the PDF and HTML versions of the Article, as well as the Supplementary Information file that now accompanies the Article.The Acknowledgements section now reads:The authors thank Purdue University and its School of Chemical Engineering for their generous start-up funding. Electron microscopy studies at Birck nanotechnology center were funded by Kirk exploratory research grant and were conducted by Arthur D. Dysart (SEM). XPS measurement was conducted by Dr. Dmitry Zemlyanov at Brick nanotechnology center. We also thank Thermo Scientific for DXR Raman microscope. J. Tang is indebted to Hoosier Heavy Hybrid Center of Excellence (H3CoE) fellowship (funded by US Department of Energy) for its financial support.The Author Contributions section now reads:J.T. conceived and conducted the experiment; V.E. collected the TEM images; J.T. and V.G.P. analyzed the data; J.T. wrote the manuscript and V.G.P. revised the manuscript."} +{"text": "There are errors in the Funding section. The correct funding information is as follows: This study was supported by University of Florida Departmental funds (UF project #00127916(C.S.) and #00045513(M.L.B)), and European Brain Research Institute funds (PAINCAGE FP7 #603191 (I.A.)). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.The publisher apologizes for the error."} +{"text": "Scientific Reports 10.1038/s41598-017-05055-z, published online 07 July 2017Correction to: The Acknowledgements section in this Article is incomplete.\u201cWork at Argonne was supported by the US Department of Energy, Office of Science, Basic Energy Sciences, Materials Sciences and Engineering Division. Use of the Center for Nanoscale Materials was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under contract no. DE-AC02-06CH11357. Y.S. acknowledges the NSF (Grant #1002410) for supporting the guest graduate studentship at Argonne National Laboratory. R. A. acknowledges the NSF (Grant #1002410) for graduate fellowship at Univ. Puerto-Rico. B.K. acknowledges the Brain Korea program for the guest graduate studentship at Argonne National Laboratory.\u201dshould read:\u201cC.P. and S.H., were supported by the US Department of Energy, Office of Science, Basic Energy Sciences, Materials Sciences and Engineering Division. Use of the Center for Nanoscale Materials was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under contract no. DE-AC02-06CH11357. Y.S. acknowledges the NSF (Grant #1002410) for supporting a guest graduate studentship at Argonne National Laboratory. R. A. acknowledges the NSF (Grant #1002410) for a graduate fellowship at Univ. Puerto-Rico. B. K. acknowledges the Brain Korea program for supporting a guest graduate studentship at Argonne National Laboratory.\u201d"} +{"text": "Women's knowledge and attitudes surrounding abortion in Zambia: a crosssectional survey across three provinces. BMJ Open 2016;6:e010076. This paper has been resupplied with the CC BY license.Cresswell JA, Schroeder R, Dennis M,"} +{"text": "The following information is missing from the Funding section: Daniel J. C. Kronauer (DJCK) was supported by The Carl & Marian Rettenmeyer Ant-Guest Endowment Award.In the Author Contributions section, Daniel J. C. Kronauer (DJCK) should be listed as one of the persons who wrote the paper."} +{"text": "Nature Communications7: Article number: 12621; DOI: 10.1038/ncomms12621 (2016); Published: 09012016; Updated: 11222016The financial support for this Article was not fully acknowledged. The Acknowledgements should have included the following:M.B.H. and P.I. are grateful for computational resources provided by the North-German Supercomputing Alliance and the ZEDAT cluster Soroban of the Freie Universit\u00e4t Berlin."} +{"text": "The ability to perform laboratory testing near the patient and with smaller blood volumes would benefit patients and physicians alike. We describe our design of a miniaturized clinical laboratory system with three components: a hardware platform that performs preanalytical and analytical processing steps using miniaturized sample manipulation and detection modules, an assay\u2010configurable cartridge that provides consumable materials and assay reagents, and a server that communicates bidirectionally with the miniLab to manage assay\u2010specific protocols and analyze, store, and report results . The miniLab can detect analytes in blood using multiple methods, including molecular diagnostics, immunoassays, clinical chemistry, and hematology. Analytical performance results show that our qualitative Zika virus assay has a limit of detection of 55 genomic copies/ml. For our anti\u2010herpes simplex virus type 2 immunoglobulin G, lipid panel, and lymphocyte subset panel assays, the miniLab has low imprecision, and method comparison results agree well with those from the United States Food and Drug Administration\u2010cleared devices. With its small footprint and versatility, the miniLab has the potential to provide testing of a range of analytes in decentralized locations. The system includes a hardware platform that performs the tests , an assay\u2010configurable cartridge containing all necessary consumable assay materials, and a server that communicates bidirectionally with the miniLab to manage assay\u2010specific instructions and results . We demonstrate the functionality of the system with analytical performance metrics across a variety of analyte classes, as represented by a Zika virus22.1The disposable assay\u2010configurable cartridge, which contains the blood sample and all reagents and consumable vessels required to conduct one or more analytical tests, inserts into the miniLab hardware platform that performs immunoassays, general chemistry, nucleic acid, and cellular characterization assays direction and has independent piston actuation. The larger pipette is also capable of actuating a magnetic rod that allows for magnetic bead handling operations. Additionally, the robot manipulates the contents of the inserted cartridge, including the transfer of vessels, engagement of tips, puncturing of sealed vessels, removing plugs from vessels, and holding vessels in place during interrogation and readout in the detector modules. The pipette module is mounted on the gantry module. The gantry module translates the pipette module horizontally (X\u2010Y plane) relative to the rest of the miniLab with a positional resolution of 2 \u00b5m with simultaneous control of motion. The gantry module also orients and couples the pipette module to the base plate of the device, to which all other modules are mounted, and provides a means for the pipette module to access all other modules in the miniLab.The miniLab's material\u2010handling robot consists of a pipette module and a gantry module to enable pick\u2010and\u2010place and liquid transfer functions during the execution of assay\u2010specific protocols. The pipette module consists of three small pipettes and one large pipette that aspirate, dispense, and mix fluids. All four pipettes are piston\u2010driven air displacement pipettes. Each pipette can be independently positioned in the vertical for processing specimens with a relative centrifugal field of up to 3,000The thermal cycling and isothermal fluorescence detection system includes a thermocycler and a photodetector. These can be used to amplify and qualitatively detect the products of nucleic acid amplification (NAA) reactions. The thermocycler uses a thermoelectric heater with forced air cooling of the heat sink. The isothermal fluorescence detection module is composed of a compact grid of 64 independently controlled excitation/emission channels. A variable\u2010gain detection system is optimized for fluorescence measurements with light emitting diode (LED) excitation (600\u2013630 nm) and a photodetector to measure epifluorescence (670\u2013800 nm). A thermal control system of resistive heaters, heat pipes, and fans maintain the isothermal set point (34\u201395\u00b0C) throughout the real\u2010time photodetection of amplification.The general\u2010purpose photodetector module contains optics and a high\u2010sensitivity optical detector to measure fluorescence and chemiluminescence. For fluorescence measurements, the LED light source excites the sample at 420\u2013460 nm with a radiant flux of 1 pW to 1 nW. The high\u2010sensitivity optical detector detects fluorescence emission at 570\u2013600 nm. For chemiluminescence measurements, the module detects light emitted with a radiant flux of 500 fW to 2 nW, and the high\u2010sensitivity optical detector detects the luminescence emission between 400 and 600 nm.The spectrophotometer module is a crossed Czerny\u2010Turner spectrograph featuring a broadband pulsed\u2010Xenon lamp, allowing for simultaneous quantification of sample absorbance levels from 300 to 800 nm, a minimum spectral resolution of 10 nm, and better than 2.5 nm spectral accuracy.X\u2010Y plane) and an independent Z\u2010axis for auto\u2010focusing (with 1\u2010\u03bcm precision). An apochromatic objective lens magnifies objects onto a high\u2010sensitivity image sensor. The microscope uses three laser diode light sources to excite samples, and the image sensor detects multiple spectral channels through selectable optical filters. Additionally, the module incorporates a ring light for dark\u2010field microscopy, allowing for the visualization of elements with differential light scattering properties.The microscope module detects cells and other components in samples by epifluorescence and dark\u2010field microscopy with a minimum lateral resolution of 1.5 \u00b5m. The microscope includes a precision stage for scanning the sample are designed and preloaded to contain all consumable materials required for single or multiple assay(s) on one sample .2.2.5The miniLab's core capability for small volume processing with the necessary flexibility to accommodate various assay types is underpinned by accurate, precise, and reliable liquid handling by the material\u2010handling robot, as well as small volume consumable components that are compatible with the on\u2010board detection systems. In selecting raw materials to manufacture the consumable components of the cartridge, we balanced the requirements of small volume handling, including surface, optical, and physical properties. Furthermore, we devised and implemented methods to mitigate evaporation of small liquid volumes, such as capping aqueous reagents with oil or wax.Z\u2010axis motion. In order to achieve the required liquid handling performance and reliability, we developed the hardware and control algorithm solutions described below.Due to the wide range of analyte classes and matrices the miniLab was designed to be able to test, the material\u2010handling robot needed to be capable of aspirating, dispensing, and mixing diverse liquids with varying rheological properties . This dictated that the pipettes on the material\u2010handling robot have a wide dynamic range of speed, precise control, and simultaneous pump and Z\u2010axis motion and the gantry module motion in the X\u2010Y plane.We used custom\u2010engineered canted coil spring shaft seals and centerless ground piston shafts in the pipettes. We designed a plastic bearing to constrain the piston motion. We optimized the gear ratio between the piston lead screw and the motor to achieve a sufficiently high number of encoder positions per unit of piston travel. We designed custom motor control algorithms and tuned the corresponding gains to achieve good steady state error, position overshoot, speed, and trajectory tracking performance for aspirating and dispensing fluids. In order to make the pipette robust, we used a profile rail linear guide for smooth motion and added a breakout printed circuit board assembly to the motor/encoder and vent valve so that all moving conductors would be combined in a high\u2010flex, long\u2010life cable. We also had custom metal gears designed so we could weld the gears to the motor and lead screw shafts. Lastly, the custom motor control algorithms used to control pump axis motion were also used to control pipette 2.3To demonstrate the analytical performance of the miniLab, we performed analytical sensitivity, precision, and method comparison studies across four disparate assays that represent each of the major analyte classes and use each of the miniLab's detector modules. The functionality of the thermocycler and isothermal fluorescence detector was assessed with a Zika virus nucleic acid test; the photodetector was assessed with an anti\u2010HSV\u20102 IgG immunoassay; the spectrophotometer was assessed with a lipid panel general chemistry assay; and the microscope was assessed with a lymphocyte subset panel hematology assay. Details of each assay methodology and workflow can be found in the Supporting Information.2.3.1We developed a qualitative nucleic acid test to detect Zika virus RNA in blood samples on the miniLab platform. We evaluated the analytical sensitivity of the assay by examining the limit of detection (LoD) using Zika virus spiked into whole blood with concentrations ranging from 0 to 3,520 genomic copies/ml. A minimum of six replicates were measured for each concentration tested, followed by 20 replicates at the putative limit of detection. The lowest Zika virus RNA concentration in whole blood at which a minimum of 95% of results were positive (55 genomic copies/ml) was confirmed as the assay LoD Table . The ana2.3.2We measured repeatability (within\u2010day and within\u2010miniLab), between\u2010day, between\u2010miniLab, and reproducibility (across miniLab and day) with standard deviations or percent coefficients of variation (CV) across three miniLabs over 5 days with five replicates per day at two medically relevant measurand concentrations for the anti\u2010HSV\u20102 IgG, the lipid panel, and the lymphocyte subset panel assays and to assess each of the detector modules built into the miniLab. These tests also demonstrated the coordination and functionality of the miniLab's sample handling components , which together replicate many of the steps typically done by laboratory personnel in central laboratories. Most pre\u2010analytical and all analytical steps were self\u2010contained and automated within the miniLab. Our analytical sensitivity results for the Zika virus NAA assay showed that the miniLab was able to detect Zika virus at 55 copies/ml of whole blood. While this is in the same range as other tests for Zika virus RNA,Precision studies for anti\u2010HSV\u20102 IgG, lipid panel, and lymphocyte subset assays showed low imprecision for all three assays/panels and were within industry standards or similar to comparator methods. Unlike the performance characteristics of many clinical laboratory analyzers, most of the variability was captured in the repeatability component with minimal day\u2010to\u2010day or device\u2010to\u2010device variability across the three miniLab devices. Thus, the miniLabs operate consistently across devices and days.When we compared the miniLab assays to FDA\u2010cleared methods, our results showed that the anti\u2010HSV\u20102 IgG assay had high sensitivity and the lipid panel and lymphocyte subset panel had excellent correlation with their respective comparator methods, with most measurands having minimal bias. Two lipid panel analytes (HDL\u2010cholesterol and LDL\u2010cholesterol) had biases relative to the comparator method that exceed the recommended limits Table .35, 36 DOne benefit of the miniaturized clinical laboratory system is its ability to conduct newly developed tests. The miniLab hardware can accept any new assay\u2010configurable cartridge and download the protocol to manipulate the contents of the cartridge from the virtual analyzer. As new tests become available for a given miniLab configuration, the device itself does not require any modifications. Another advantage is that the system is designed to be able to multiplex tests that comprise frequent ordering patterns, including those from different analyte classes, on each sample. The number of tests the system may ultimately be able to multiplex on a single sample depends on the sample volume and type required for each test, number of reagents and space available on the cartridge, and overlap of requirements between test types. We envision all assays functioning with blood specimens either in the absence of anticoagulants (serum) or the presence of a limited set of common assay\u2010compatible anticoagulants . Limiting the number of anticoagulants may increase the potential to multiplex tests across analyte classes and could reduce the number of separate specimens required for larger test order sets. In addition, the system is compatible with other specimen types such as urine. Finally, if desired, laboratory personnel may remotely oversee and interpret control and test results through the virtual analyzer, thus allowing for a similar review as is typically available in a clinical laboratory setting.The design of the system presents some limitations. As with certain point of care analyzers, the miniLab can test only one cartridge at a time, which limits the throughput of a single device. We plan to shorten the assay run times to increase the miniLab's throughput and utility. The data presented here show only one assay or panel per cartridge, though the system is designed to be capable of multiplexing disparate assays or panels together using a single cartridge and associated protocol. Finally, the current version of the platform cannot contain all the modules in the same chassis. Future versions of the platform will contain the full functionality of all modules within a single device.4The miniLab has a unique potential as a clinical laboratory testing platform, and the system is designed to fill several unmet opportunities in the field of diagnostic testing. The system requires smaller specimen volumes than many other analyzers. This makes frequent testing more feasible for populations for whom required blood volume may limit laboratory testing . The miniLab's compact size, coupled with the virtual analyzer's connectivity and the versatile assay\u2010configurable cartridges, has the potential to provide high\u2010quality testing of diverse analytes in decentralized laboratory and other near\u2010patient locations, which could expand access to clinical services and expedite diagnoses and therapies. By augmenting access to clinical laboratory testing options, the miniaturized clinical laboratory system has the potential to complement the arsenal of technologies available to the clinical laboratory community.5Extended methods and Supporting Information Figures, Tables, and Video are available online.Low [lymphocyte count]; for the microscope). All miniLab assays were conducted by loading up to 160 \u00b5l of sample into an assay\u2010configurable cartridge and inserting it into the miniLab. Basic assay specifications (see Supporting Information Table 1) and extended details of each assay methodology are provided in the Supporting Information.To demonstrate the analytical capabilities of each detector module, we performed the following illustrative assays on the miniLab using automated protocols: Zika virus NAA test , anti\u2010HSV\u20102 IgG (for the photodetector), lipid panel , and lymphocyte subset panel . For real\u2010time monitoring of the isothermal amplification, the inflection cut\u2010off time was taken as the time at which 92.6% sensitivity (positive sample detection rate) and 95.0% specificity (rate of correctly identified negative samples) were achieved via receiver operating characteristic analysis. The LoD study design followed the March 9, 2016, FDA draft interactive emergency use authorization review template for molecular assays (Zika virus\u2010specific). The preliminary LoD concentration was determined by testing contrived venous whole blood samples of decreasing Zika virus concentrations in replicates of six or more until the assay no longer detected the virus at the tested concentration. The LoD was confirmed by identifying the lowest concentration exhibiting at least a 95% Zika virus detection rate after testing an additional 20 replicates.For the anti\u2010HSV\u20102 IgG, lipid panel, and lymphocyte subset panel tests, we measured precision by testing two sample pools at low and high measurand concentrations over 5 days, with five replicates each day on each of three miniLabs, following the multidevice precision guidelines in the Clinical Laboratory Standards Institute (CLSI) EP05\u2010A3 .We performed method comparison studies for the anti\u2010HSV\u20102 IgG, lipid panel, and lymphocyte subset panel assays by testing a minimum of 100 samples on the miniLab and an FDA\u2010cleared comparator or reference method.We tested fresh and archived plasma clinical specimens in duplicate on the miniLab and the comparator method for the lipid panel method comparison study . We tested fresh venous whole blood and contrived samples in singlicate on the miniLab and in duplicate on the comparator method, Becton Dickinson (BD) Biosciences Multitest 6\u2010Color TBNK , for the lymphocyte subset method comparison study .We purchased all anti\u2010HSV\u20102 IgG specimens (202 samples) and some lipid panel specimens (21 samples) used for method comparison studies as de\u2010identified clinical specimens from commercial vendors (see Supporting Information). We collected blood specimens for all other studies from 224 healthy adult donors by venipuncture using BD Vacutainers . Veritas Institutional Review Board, Inc. approved the study protocols. All subjects gave informed consent, and consent was waived for commercially purchased specimens. Theranos manufactured all miniLabs, reagents, cartridges, and consumable materials for each assay in Newark, CA.5.1Prior to analysis, invalid miniLab results were determined by applying the following exclusion criteria: tests which failed to collect all assay data, flagged integrity checks , and traceable human error. Results from comparator methods that exceeded the analytical measuring range as reported in the package inserts were also excluded, as well as any incomplete data sets. In some cases of excluded tests where sample volume and time permitted, samples were retested on a new cartridge.Statistical analysis was performed with R statistical software . The precision analysis was performed with two\u2010way nested analysis of variance with random effects (\u201cday\u201d nested within \u201cdevice\u201d) to determine the components of the variance for each assay. We used Grubbs' test at the 99% level to exclude up to one outlier per miniLab per measurand and per sample pool, as recommended in the CLSI EP05\u2010A3 guideline.The sensitivity, specificity, and their CIs were calculated as described in CLSI EP12\u2010A.r) for each measurand.Quantitative method comparison of the first replicate (lipid panel) or singlicate (lymphocyte subset) result from the miniLab and mean of duplicate results from the comparator methodsFor bias calculations and plots, mean of duplicate (lipid panel) or singlicate (lymphocyte subset panel) miniLab results were compared to the mean of duplicate results for the comparator method for each study subject:Median absolute bias or median proportional bias was calculated and the approximate 95% CI was obtained by an application of the binomial distribution.5.2https://osf.io/ur7kw/The data and analysis scripts presented in this paper are available at All authors of this paper, as either current or former Theranos employees, may have received or may in the future receive equity compensation from the Company. S.G.A., K.E., E.A.H., T.M.K., A.N., M.B.N., J.O., C.H.P., P.J.P., J.R., C.S., S.S., B.S.\u2010P., and D.L.Y. are inventors on patents or patent applications related to the material presented here, all of which are assigned to Theranos.S.G.A., P.B., S. Chow, K.D.H., E.A.H., J.O., C.H.P., J.R., N.V.S., C.S., L.T., T.C.W., P.Z. designed and/or optimized functional components of the miniLab device. S.G.A., K.D.H., E.A.H., J.O., C.H.P., J.R., C.S., B.S.\u2010P., A.T. designed and/or optimized consumable materials contained in the miniLab cartridge. S. Chandrasekaran, E.G., X.G., K.J., R.S. wrote software for the miniLab and/or virtual analyzer. S. Chandrasekaran, X.G., A.J., K.J., J.O. wrote software to generate miniLab protocols. K.Q., T.C.W. designed and implemented the embedded control software for custom electronics. S.G.A., S. Chow, X.G., K.D.H., J.O., J.R., J.F.S., A.T., T.C.W., P.Z. optimized hardware integration and control processes for miniLab. D.P.B., X.G., K.D.H., K.J., A.N., J.O., C.H.P., J.F.S., A.T., D.W., A.Y. wrote and optimized protocol instructions for the miniLab. S.G.A., D.P.B., Y.C., U.D., K.E., S.F.G., K.D.H., E.A.H., R.H., L.H., A.N., M.B.N., J.O., C.H.P., A.R.R., J.F.S., N.V.S., C.S., S.S., L.T., A.T., D.W. designed and/or optimized the assay methods. T.M.K. specified requirements for anti\u2010HSV\u20102 IgG assay calibration. Y.C., U.D., K.E., K.D.H., R.H., L.H., Y.L.L., M.B.N., J.O., C.H.P., P.L.R., A.R.R., J.F.S., N.V.S., C.S., B.S.\u2010P., L.T., A.T., D.W. developed and/or optimized assay reagents. D.P.B., Y.C., S. Chow, K.E., S.F.G., X.G., K.D.H., R.H., L.H., K.J., A.N., M.B.N., J.O., P.J.P., A.R.R., J.F.S., N.V.S., C.S., S.S., B.S.\u2010P., D.L.Y., designed experiments and/or research plans. J.A.B., D.P.B., Y.C., K.E., S.F.G., K.D.H., R.H., L.H., A.N.K., L.S.L., Y.L.L., A.N., J.O., A.R.R., J.F.S., C.S., L.T., A.T., D.W. performed experiments. J.A.B., D.P.B., Y.C., K.E., E.G., S.F.G., X.G., K.D.H., L.H., A.J., K.J., A.N.K., M.B.N., J.O., A.R.R., J.F.S., N.V.S., C.S., B.S.\u2010P., D.W. analyzed and visualized data. S.G.A., J.A.B., D.P.B., Y.C., S. Chow, K.E., S.F.G., X.G., R.H., K.J., A.N.K., Y.L.L., M.B.N., J.O., A.R.R., J.F.S., N.V.S., C.S., S.S., B.S.\u2010P., A.T., D.W., D.L.Y. discussed and interpreted results. C.H.P., P.J.P., N.V.S., C.S., S.S., D.L.Y. supervised projects. E.A.H., C.H.P., D.L.Y. conceived the overall system design. K.E., M.B.N., C.R.R. wrote the manuscript. All authors critically reviewed and/or revised the manuscript. All authors have read and approved the final version of the manuscript.Additional Supporting Information may be found online in the supporting information tab for this article.Supporting Information VideoClick here for additional data file.Supporting InformationClick here for additional data file."} +{"text": "Synapse loss is a key feature of dementia, but it is unclear whether synaptic dysfunction precedes degenerative phases of the disease. Here, we show that even before any decrease in synapse density, there is abnormal turnover of cortical axonal boutons and dendritic spines in a mouse model of tauopathy-associated dementia. Strikingly, tauopathy drives a mismatch in synapse turnover; postsynaptic spines turn over more rapidly, whereas presynaptic boutons are stabilized. This imbalance between pre- and post-synaptic stability coincides with reduced synaptically driven neuronal activity in pre-degenerative stages of the disease. \u2022Density of cortical axonal boutons and dendritic spines is reduced early in tauopathy\u2022Abnormalities in synaptic stability and size exist before decreases in synapse density\u2022Turnover of dendritic spines is elevated, whereas presynaptic boutons are stabilized\u2022Neuronal activity is reduced at stages associated with mismatched synaptic turnover Using in\u00a0vivo two-photon imaging in the rTg4510 tauopathy mouse model, Jackson et\u00a0al. find that synapse stability is altered during the pre-degenerative stages of tauopathy. Mismatched abnormalities in pre- and post-synaptic turnover coincide with disrupted neuronal activity. MAPT) gene that causes Frontotemporal Dementia with Parkinsonism-17 is expressed in excitatory neurons of the forebrain, recapitulates many characteristics of neurodegenerative disease of GFP-expressing pyramidal neurons in the somatosensory cortex of rTg4510 mice and littermate controls. The same regions of interest containing several sections of neurite were imaged weekly . To span several months at weekly intervals, we studied three groups of animals at different ages ranging from early to intermediate to advanced stages, when clear neurodegeneration has taken root in the cortex .As a potential indicator of synaptic degeneration, we compared the relative density of spines A and TBsThe relative density of axonal boutons mirrored the age-dependent decreases in dendritic spine density D. No difOngoing addition and removal of a small but significant fraction of synapses is thought to underlie the continual tuning of neuronal function to ongoing cognitive demands or in response to injury or disease . PerturbTurnover of dendritic spines was significantly higher in both age groups in rTg4510 dendrites than in WT dendrites A. NotablComparing spine and TB dynamics within individual animals, we observed that turnover of pre- and post-synaptic elements was relatively balanced in WT animals . For spines present at 21\u00a0weeks of age, there was no difference between rTg4510 and WT in month-long survival fraction Act 1986 and subject to internal ethical review. Further experimental detail can be found in J.S.J., J.W., M.C.A., A.R., M.L.H., M.J.O., and J.I. designed the study; J.S.J. and J.W. carried out the in\u00a0vivo experiments; J.S.J., J.W., J.D.J., and M.C.A. analyzed in\u00a0vivo data; and Z.A. and M.W. carried out the histology experiments and data analysis. M.C.A., J.T.I., A.D.R., and M.J.O. jointly supervised the project. All authors contributed to the manuscript preparation."} +{"text": "Scientific Reports6: Article number: 34592; 10.1038/srep34592 published online: 10042016; updated: 10202017.The Acknowledgements section in this Article is incomplete.\u201cThis work was supported by NIH grants HL109015 (to J.I.S. and Z.X.), HL071556 (to J.I.S.) and HL105649 as well as HL55601 and HL34300 (to N.G.A.); the Brickstreet Foundation (to J.I.S. and N.G.A.); F32DK104615 (to C.A.D.); AHA 14SDG18650010, The Boone Foundation and an Early Career Development Award from the Central Society for Clinical and Translational Research (to D.J.K.) and the Huntington Foundation Inc.\u201dshould read:\u201cThis work was supported by NIH grants HL109015 (to J.I.S. and Z.X.), DK-106666 (to J.L.), HL071556 (to J.I.S.) and HL105649 as well as HL55601 and HL34300 (to N.G.A.); the Brickstreet Foundation (to J.I.S. and N.G.A.); F32DK104615 (to C.A.D.); AHA 14SDG18650010, The Boone Foundation and an Early Career Development Award from the Central Society for Clinical and Translational Research (to D.J.K.) and the Huntington Foundation Inc.\u201d"} +{"text": "Nature Communications 10.1038/s41467-017-00985-8, published online 13 October 2017Correction to: In the original version of this Article, financial support and contributions in manuscript preparation were not fully acknowledged. The PDF and HTML versions of the Article have now been corrected to include the following:\u2018M.P. and P.O. would like to thank Prof. Roderick Y.H. Lim for advice during manuscript preparation and for providing the laboratory and microscopy infrastructure.\u2026 [We also thank] the NanoteraProject, awarded to the PATLiSciII Consortium (M.P and P.O)\u2026\u2019"} +{"text": "Following publication of the original article it was b\u201cN.T. (006/P&C/CORE/2013/OXFSTATS), and C.F. (006/PSS/CORE/2016/OXFORD) receive funding from the British Heart Foundation (BHF). CV is grateful for Clarendon and Nuffield Department of Population Health funding.\u201d"} +{"text": "Understanding the factors that affect water quality and the ecological services provided by freshwater ecosystems is an urgent global environmental issue. Predicting how water quality will respond to global changes not only requires water quality data, but also information about the ecological context of individual water bodies across broad spatial extents. Because lake water quality is usually sampled in limited geographic regions, often for limited time periods, assessing the environmental controls of water quality requires compilation of many data sets across broad regions and across time into an integrated database. LAGOS-NE accomplishes this goal for lakes in the northeastern-most 17 US states.LAGOS-NE contains data for 51\u2009101 lakes and reservoirs larger than 4 ha in 17 lake-rich US states. The database includes 3 data modules for: lake location and physical characteristics for all lakes; ecological context for all lakes; and in situ measurements of lake water quality for a subset of the lakes from the past 3 decades for approximately 2600\u201312\u2009000 lakes depending on the variable. The database contains approximately 150\u2009000 measures of total phosphorus, 200\u2009000 measures of chlorophyll, and 900\u2009000 measures of Secchi depth. The water quality data were compiled from 87 lake water quality data sets from federal, state, tribal, and non-profit agencies, university researchers, and citizen scientists. This database is one of the largest and most comprehensive databases of its type because it includes both in situ measurements and ecological context data. Because ecological context can be used to study a variety of other questions about lakes, streams, and wetlands, this database can also be used as the foundation for other studies of freshwaters at broad spatial and ecological scales. A major concern for water quality in freshwaters globally is cultural eutrophication, or excess nutrient inputs from human activities that lead to increased plant and algal growth. In many parts of the world, runoff from land, or nonpoint-source pollution, has replaced discharges of sewage, or point-source pollution, as the primary driver of lake and reservoir eutrophication . In lakeIn practice, measures of water quality are often collected from a relatively small number of lakes within individual regions. In the United States, large investments have been made in water quality monitoring by federal, state, local, and tribal governments; and many, but not all, of the data sets have been placed in government data repositories such as the USGS National Water Information System (NWIS) and the USEPA Storage and Retrieval (STORET) database. Unfortunately, these data repositories do not currently allow us to study lake water quality at broad scales. Despite the large number of water quality records in these systems, a recent analysis of stream nutrient data obtained from NWIS, STORET, and more than 400 other organizations determined that more than half of the data records lacked the most critical metadata necessary to make the data usable , and we 2).We created a database called LAGOS-NE, the \u201clake multi-scaled geospatial and temporal database\u201d for thousands of inland lakes in 17 of the most lake-rich states in the upper Midwest and Northeastern United States to facilitate further development of our basic understanding of lake water quality at broad scales using water quality data on thousands of lakes collected over the last several decades obtained from existing national-scale GIS (geographic information system) data sets and measured in multiple zones around all lakes. This module also contains some temporal data for climate, land use/cover, and atmospheric deposition variables. LAGOS-NELIMNO v1.087.1 includes in situ measurements of lake water quality for a subset of the above lakes. These 87 data sets of lake water quality were obtained from a combination of sources including government, tribal agencies, university researchers, citizen scientists, and non-profit agencies. Samples were taken during any season of the year from the most recent decades, mostly from the late 1980s to 2012.LAGOS-NE comprises 3 data modules that, although integrated in the same database, were derived using different data sources and data integration methods, and thus must be version-controlled separately. LAGOS-NEThe largest challenge in building LAGOS-NE was the heterogeneity of the data set formats, variable conventions and units, and metadata, none of which were standardized. Many steps of data integration required manual input from experts in diverse fields and close collaboration among specialists in ecoinformatics, database design, freshwater ecology, and geography; all combined, the effort took 6 years and involved \u223c15 individuals, spread across numerous institutions.We designed the database using principles of open science so future users could ask new research questions by using the existing database or adding new data modules to the database. To ensure that users could do this, we documented the major steps of data set integration and carefully integrated metadata directly into the database itself, we emphasized data provenance, and we used a database versioning system. In this data paper, we make the following research products available: (i) data tables with the data that make up LAGOS-NE and an R package for accessing the data and integrating the tables; (ii) for each of the 87 water quality data sets, we provide the ecological metadata language (EML) metadata files that we authored after receiving the data, the data files that we processed to import into LAGOS-NE and the R-script that we wrote to process the data; and (iii) GIS coverages of the underlying freshwater geographic features that are linked to the data tables for GIS processing by researchers.We selected an area of the United States known to have large numbers of lakes, well-developed lake water quality sampling programs, and that spans diverse geographic conditions and thus gradients of ecological context Table . Our stuAlthough the majority of the data that we provide are for lakes \u22654 ha (see below for reasons for using this threshold), we do include some data on lakes \u22651 ha and <4 ha if data were available. Although there may be water quality data for some lakes in this smaller size range, ecological context variables are not available for these lakes.LAGOS-NE includes some data on all lakes in a study area , which we call the \u201ccensus\u201d population of lakes. The census population of lakes is a critical feature of LAGOS-NE because it allows us to characterize the ecological context of every lake in our study population and to identify whether the lakes for which we have water quality data are biased in any way. LAGOS-NE includes 3 main categories of variables: (i) variables that describe the physical characteristics and location of lakes themselves; (ii) variables that describe in situ water quality; and (iii) variables that describe a lake's ecological context at multiple scales and across multiple dimensions based on the principles of landscape limnology , 18\u201320. There was a number of constraints for each of the categories of data that had to be considered. For creating the census population of lakes , we relied on a single source of data . For theWe organized these 3 categories of data into database \u201cmodules\u201d that had similar data types and sources so that we could develop procedures and set standards for each module Fig. . The modThe design of LAGOS-NE and the workflow for its construction have been described previously in detail . In partLOCUS module includes data on the physical location, some features, and unique identifiers for all lakes in the study area \u22651 ha, which means this data file has information on 141\u2009378 lakes. Note that, because we detected errors in the digitization of lakes between 1 and 4 ha, we have chosen to define our census population of lakes as only those \u22654 ha, but we still make data available for lakes smaller than 4 ha when available in this and the LAGOS-NELIMNO data module. However, we recommend caution in analyses, interpretation, and inference for lakes <4 ha in this database that depend on NHD\u2019s spatial representation and detection of water bodies. The data in this module include lake unique identifiers, perimeter, area, latitude and longitude , GNIS name, and the zone IDs that the lake is located within . The GIS data sets that we also make available provide the lake polygon features associated with this module, as well as coverages for lake watersheds, streams, wetlands, spatial classifications, and glaciation history.The LAGOS-NEWe defined lakes previously in Soranno et\u00a0al. as folloGEO for further details.We calculated lake watersheds as \u201cinter-lake watersheds\u201d (IWS), defined as the area of land draining directly into the lake as well as the area that drains into upstream-connected streams and lakes <10 ha Fig. . We defiFor some of our analyses, we delineated boundaries in other ways than political boundaries that were more ecologically relevant, which resulted in the inclusion of some lakes outside of the exact 17-state border. This fact allowed us to include more in situ data collected by state and citizen sampling programs that do not always follow strict state borders and may include lakes that are outside of state lines. Although most of these border lakes have hydrologic and topographic calculations or water quality data, some measures of ecological context may be missing. For example, for lakes in Canada, we were not able to estimate any data that relied on national data sets that stopped at the Canadian border; one exception is the NHD, which extends into Canada to retain hydrologic boundaries.Detailed information on data sources are found in Additional file 5 in Soranno et\u00a0al. . BrieflyAll methods to create this module are described in Soranno et\u00a0al. . The mosThe full description of error analysis for this module is described in Soranno et\u00a0al. . However2 rectangles from each state then compared the number of lakes occurring in the NHD GIS coverage with the number of lakes in the best available aerial imagery from a range of sources to calculate the percentage of lakes missing from the NHD. The average percentage of lakes missing from the NHD was 58% for the \u22651 ha 4-state test and 13% for the \u22654 ha 7-state test. Because an average of 87% of lakes \u22654 ha that are present in high-resolution aerial imagery are also present in the NHD, we chose this surface area as our cut-off and accepted this error rate.We selected 4 states in which to evaluate error rates of water body identification for lakes \u22651 ha and 7 states in which to evaluate error rates for lakes \u22654 ha. We randomly selected three 100-kmFigure LIMNO module includes in situ measurements of lake water quality. We included variables that are most commonly measured by state agencies and researchers for studying eutrophication , detection limits (if available), and standardized censor codes from our quality control procedures . Finally, we include documentation about each source program that is linked to each data value.The LAGOS-NEC]) Fig. . For eacC]) Fig. , includiLIMNO by contacting individuals at each of the 17 state and 5 tribal agencies. These contacts helped us to identify the state agency\u2013collected data set required by the Clean Water Act that was most likely to be in the public domain. In this way, we were able to acquire at least 1 data set from each of the 17 states. Because state and tribal agencies vary in sampling approach and intensity (see below for details), we sought to supplement these data sets with other known sources of water quality data, including university researchers, federal agencies, and non-profit groups, to integrate into the LAGOS-NELIMNO module. The full list of data sources acquired is in Soranno et\u00a0al. ).Creation of LAGOS-NE required a strong focus on team science, and in particular the roles of and incentives for early-career researchers in such efforts. This type of research cannot be conducted in a single-investigator mode, but requires a highly collaborative and effective team-based model e.g., \u201352). We . We 52])The decision of how to disseminate the database documentation needs to be considered early in the project. For example, database documentation papers are rare, especially in ecology, but are very important. The documentation and procedural approaches for developing this large, integrated, and heterogeneous database had to be disseminated through publication prior to making the database available and prioOne challenge is to prioritize research areas and to identify the types of data sets that may benefit from a similar type of integration. State, federal, tribal, and citizen science water quality data sets were an excellent source of quality data for integration and conducting broad-scale research on aquatic systems. There are likely other such data sources that would benefit from being integrated as we have done here. We recommend the following strategies to make the best use of future data integration efforts.Project name: LAGOS-NEhttps://github.com/cont-limno/LAGOSProject home page: Operating system(s): e.g., platform independentProgramming language: ROther requirements: R packages required (with associated versions): dplyr (\u22650.7.0), rappdirs (\u22650.3.1), lazyeval (\u22650.2), purrr (\u22650.2.2.2), magrittr (\u22651.5), sf, curl (\u22652.7.0), stringr (\u22651.2.0)License: GPLLAGOS-NE-LOCUS v1.01 ;LAGOS-NE-LIMNO v1.087.1 ;LAGOS-NE-GEO v1.05 ;LAGOS-NE-GIS v1.0 ;GigaScience repository, GigaDB [Snapshots of the R package in the LAGOS GitHub page are also available in the , GigaDB .The data sets supporting the results of this article are available in the Ecological Data Initiative repository, including the following specific components:Soranno_etal_2017_Additional file 1_8SEP17_final.docxSoranno_etal_2017_Additional file 2_qaqc-limno_v2.docxCHAG: Climate, Hydrology, Atmospheric deposition of nitrogen and sulfur, and surficial Geology; CONN: connectivity and abundance ; CSI: cross-scale interactions; DOC: dissolved organic carbon; EML: ecological metadata language; GIS: Geographic Information System; HUC: Hydrologic Unit Code; IQR: interquartile range; IWS: interlake watershed; LAGOS-NE: LAke multi-scaled GeOSpatial and temporal database for the 17 Northeastern and Midwest US states; LULC: land use land cover; MAV: maximum allowable value; NHD: National Hydrography Dataset; SRP: soluble reactive phosphorus; TDN: total dissolved nitrogen; TN: total nitrogen; TP: total phosphorus; US EPA: United States Environmental Protection Agency; USGS: United States Geological Survey; WBD: Watershed Boundary Dataset.The authors declare that they have no competing interests.The creation of LAGOS-NE was supported by the National Science Foundation (NSF) MacroSystems Biology Program in the Emerging Frontiers Division of the Biological Sciences Directorate and the United States Department of Agriculture National Institute of Food and Agriculture, Hatch project 176820 to P.A.S. K.E.W. thanks the STRIVE Programme (2011-W-FS-7) from the Environmental Protection Agency, Ireland. S.M.C. thanks the NSF Division of Biological Infrastructure (1401954).The water quality data that are incorporated into LAGOS-NE were originally funded by the following sources: State of Maine; Michigan Agricultural Experiment Station; Fisheries Division, Michigan Department of Natural Resources; New York State Division of Water Quality; Wisconsin Department of Natural Resources; University of Wisconsin-Madison; State/Trust; Michigan State University Agriculture Experimental Station Disciplinary Research Grant Program; US EPA; US EPA Section 106/319 Grants; Tribal General Fund; US Army Corps of Engineers Federal Lakes Operation and Maintenance Funds; Aquatic Plant Management Society; Aquatic Ecosystem Restoration Foundation; Michigan State University; Michigan State University Department of Fisheries and Wildlife; EPA Star Fellowship to K.S.C. (U-915342\u201301-0); Andrew W. Mellon Foundation; Federal Aid in Sport Fish Restoration Program administered jointly by the US Fish and Wildlife Service and the Ohio Department of Natural Resources, Division of Wildlife; Iowa Department of Natural Resources (Contract #ESD04HALFasch110155); Minnesota Pollution Control Agency; NSF-Division of Environmental Biology; Ohio Department of Natural Resources Division of Wildlife; University of Rhode Island Watershed Watch; NSF Kellogg Biological Station Long Term Ecological Research (LTER) Program, DEB 1027253; NSF North Temperate Lakes LTER Program, DEB 1440297; Lac du Flambeau Band and Bureau of Indian Affairs; Indiana Department of Environmental Management; Missouri Department of Natural Resources; Clean Water Act Section 16; Michigan Department of Environmental Quality; Massachusetts Water Supply Protection Trust; US EPA Clean Air Markets Division (LTM Network); US EPA Office of Research and Development; New York City Department of Environmental Protection (NYSDEP); City of New York; USGS Water Availability and Use Science Program (WAUSP); US Geological Survey; New York State Energy Research and Development Authority; National Institute of Food and Agriculture, US Department of Agriculture, Hatch Grant 1003732; the New York State Department of Environmental Conservation; Lake Sunapee Protective Association; National Oceanic and Atmospheric Administration; Gull Lake Quality Organization; Clean Michigan Initiative; NSF grant DEB-1455461.LIMNO were conducted by N.R.L.; the quality control of LAGOS-NEGIS was led by C.E.S. and S.M.C. and conducted by C.E.S., S.M.C., C.E.F., N.K.S., and K.E.W. The quality control of LAGOS-NELOCUS was conducted by E.G.B. Many authors who were part of the database integration team wrote the technical documentation; J.F.L. served as editor of these technical documents. Tables and figures were prepared by S.M.C., K.B.S.K., J.F.L., N.R.L., A.C.P., N.K.S., and P.A.S. and edited by many of the contributing authors. S.K.O. and J.J.S. wrote the LAGOS-NE R package. N.J.S. prepared the GIS data and their corresponding metadata. P.A.S. coordinated the writing of the manuscript, and major parts of the manuscript were written by P.A.S., K.S.C., S.M.C., J.F.L., N.R.L., S.K.O., J.J.S., E.H.S., P.N.T., T.W., and S.Y. After the lead author, authors are listed alphabetically.Data for the database were contributed by L.C.B., M.B., K.E.B., M.G.B., M.T.B., S.R.C., J.W.C., K.S.C., M.C., J.D.C., J.A.D., J.D., C.T.F., C.S.F., M.J.G., L.T.G., J.D.F., S.K.H., P.C.H., E.H., C.H., J.R.J., K.J.H., L.L.J., W.W.J., J.R.J., C.M.K., S.A.K., B.L., J.A.L., Y.L., N.R.L., J.A.L., L.J.M., W.H.M., K.E.B.M., B.P.N., S.J.N., M.L.P., D.C.P., A.I.P., D.M.P., P.O.R., D.O.R., K.M.R., L.G.R., O.S., N.J.S., P.A.S., N.R.S., E.H.S., J.L.S., J.M.T., T.P.T., M.V., G.W., K.C.W., K.E.W., J.D.W., and M.K.W. The idea to create the database was conceived by P.A.S. and K.S.C. P.A.S. coordinated the different activities across team members to build LAGOS-NE. The database was designed by E.G.B., P.N.T., C.G., and P.A.S. and created and managed by E.G.B. The following authored metadata for the individual water quality data sets using information provided by the data providers: M.T.B., C.K.B., K.S.C., S.M.C., C.E.F., C.T.F., E.N.H., N.R.L., S.K.O., N.K.S., P.A.S., E.H.S., and K.E.W. C.E.F. prepared the integrated LAGOS-NE metadata and developed the protocols for authoring the EML metadata, and C.E.F. and C.K.B. created EML metadata for the 87 water quality data sets. S.K.O. wrote the final variables\u2019 definitions for the integrated metadata. C.G. helped to prepare the needed metadata and documentation for loading the data in the data repository. Code for importing the data sets into the database was written by E.G.B., S.T.C., N.R.L., and S.Y. N.J.S. and S.B.S. performed geospatial analyses and created the LAGOS-GIS Toolbox. The conceptual foundation for measuring freshwater connectivity was led by C.E.F. S.B.S. developed the methods to delineate lake watersheds. The quality control methods development and analysis on LAGOS-NEGIGA-D-17-00112_Original-Submission.pdfClick here for additional data file.GIGA-D-17-00112_Revision-1.pdfClick here for additional data file.GIGA-D-17-00112_Revision-2.pdfClick here for additional data file.Response-to-Reviewer-Comments_Original-Submission.pdfClick here for additional data file.Response-to-Reviewer-Comments_Revision-1.pdfClick here for additional data file.Reviewer-1-Report-.pdfClick here for additional data file.Reviewer-1_Original-Submission-(Attachment).pdfClick here for additional data file.Reviewer-2-Report-.pdfClick here for additional data file.Reviewer-3-Report-.pdfClick here for additional data file.Additional FileClick here for additional data file.Additional FileClick here for additional data file."} +{"text": "There is an error in the Acknowledgements section: Prof. Yuji Tachibana should be Dr. Hiroshi Tachibana. The correct Acknowledgements section should read: We thank Dr. Seiki Kobayashi of the Keio University School of Medicine, and Dr. Hiroshi Tachibana of the Tokai University School of Medicine, for providing E. histolytica clinical strains and nucleotide samples of clinical samples. We thank Dr. Nicholas E. Sherman, W.M. Keck Biomedical Mass Spectrometry Laboratory, University of Virginia for assistance with mass spectrometric analysis. We thank Ms. Kumiko Shibata and Mihoko Imada for technical assistance."} +{"text": "Following the publication of this article it was bReference 12 currently reads: Chiumento A. A haven of greenspace. 2012.The completed reference should read: Chiumento A. A haven of greenspace. YoungMinds Magazine 2012;118:32\u201334."} +{"text": "There is an error in affiliation 5 for the author Chih-Chung Shiao. Affiliation 5 should read: Saint Mary\u2019s Junior College of Medicine, Nursing and Management. Yilan, Taiwan (R.O.C)."} +{"text": "In the original article, the reference for Shaw et al. (2015) was incorrectly written as:Proceedings of the 15th Australasian International Conference on Speech Science and Technology (Christchurch), 3\u20135.Shaw, J. A., Catherine, T. B., Karen, E. M., Gerard, D., Bronwen, G. E., Paul, F., et al. (2015). \u201cEffects of short-term exposure to unfamiliar regional accents: Australians' categorization of London and Yorkshire English consonants,\u201d in It should be:Proceedings of the 15th Australasian International Conference on Speech Science and Technology (Christchurch), 3\u20135.Shaw, J. A., Best, C. B., Mulak, K. E., Docherty, G. J., Evans, B. G., Foulkes, P., et al. (2015). \u201cEffects of short-term exposure to unfamiliar regional accents: Australians' categorization of London and Yorkshire English consonants,\u201d in The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way.The original article has been updated.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Nature Communications8: Article number: 14967; DOI: 10.1038/ncomms14967 (2017); Published 04032012; Updated 05172017The financial support for this Article was not fully acknowledged. The Acknowledgements should have included the following:[***Human Frontiers Science Program (HFSP) funds the labs of A.I.D. and P.C. ***]."} +{"text": "Scientific Reports6: Article number: 2321610.1038/srep23216; published online: 03152016; updated: 05022017The Acknowledgements section in this Article is incomplete.\u201cWe thank Dr. Robert Friesel for critical reading of the manuscript. We thank MMCRI histology core Armie Mangoba, Katrina Abramo and Dr. Volkhard Lindner for creating breast cancer tissue array (TMA) and histochemistry analysis. We thank MMC BioBank Robert Ackroyd, Dr. Ivette F. Emery and Dr. Anne Breggia for coordinating studies on clinical specimens. This study was supported by the Maine Cancer Foundation Accelerate Grant and a Maine Medical Center Research Program Grant to X.Y\u201d.Should read:\u201cWe thank Dr. Robert Friesel for critical reading of the manuscript. We thank MMCRI histology core Armie Mangoba, Katrina Abramo and Dr. Volkhard Lindner for creating breast cancer tissue array (TMA) and histochemistry analysis. We thank MMC BioBank Robert Ackroyd, Dr. Ivette F. Emery and Dr. Anne Breggia for coordinating studies on clinical specimens. This study was supported by the Maine Cancer Foundation Accelerate Grant and a Maine Medical Center Research Program Grant to X.Y. This work was supported by a grant from the Maine Cancer Foundation and NIH grants P30 GM1033465 to D. Wojchowski, PI, and P30 GM103392 (Histopathology and cell imaging cores) to R. Friesel, PI, and generous support from the Maine Medical Center\u201d."} +{"text": "Scientific Reports 10.1038/s41598-017-16922-0, published online 01 December 2017Correction to: This Article contains errors in the Author Contribution Statement.\u201cChan P. and Wu T. designed the experiments and revised the manuscript; Mi T., Mei S. and Gao L. carried out data collection; Mi T. carried out data analysis and drafted the manuscript; Liang P. and Li K. carried out functional fMRI scan\u201d.should read:\u201cChan P., Wu T. and Li K. designed the experiments and revised the manuscript; Mi T., Mei S. and Gao L. carried out data collection; Mi T. carried out data analysis and drafted the manuscript; Liang P. carried out functional MRI scan and data analysis\u201d."} +{"text": "The middle initial for two authors, Jesse Smith and Kevin Kuntz, are incorrect. Their names should read Jesse J. Smith and Kevin W. Kuntz respectively."} +{"text": "The Worldwide PDB recently launched a deposition, biocuration, and validation tool: OneDep. At various stages of OneDep data processing, validation reports for three-dimensional structures of biological macromolecules are produced. These reports are based on recommendations of expert task forces representing crystallography, nuclear magnetic resonance, and cryoelectron microscopy communities. The reports provide useful metrics with which depositors can evaluate the quality of the experimental data, the structural model, and the fit between them. The validation module is also available as a stand-alone web server and as a programmatically accessible web service. A growing number of journals require the official wwPDB validation reports (produced at biocuration) to accompany manuscripts describing macromolecular structures. Upon public release of the structure, the validation report becomes part of the public PDB archive. Geometric quality scores for proteins in the PDB archive have improved over the past decade. \u2022Validation reports are available for X-ray, NMR, and EM structures in the PDB\u2022Preliminary reports obtained at deposition, stand-alone servers, and programmatically\u2022Official reports from biocuration should be submitted with manuscripts\u2022Quality metrics for protein structures have improved over the past decade Gore et\u00a0al. describe the community-recommended validation reports, produced by wwPDB at deposition and biocuration of PDB submissions, and integrated into the archive of publicly released PDB entries. The authors also show that the quality of protein structures has improved over the last decade. Clearly, raw experimental data, before application of any transformations which may lead to loss of information, and devoid of interpretation, would lend the ultimate support of the final model and allow an independent verification of the results, leading to novel validation tools. Efforts to archive such raw data are under way through established archives for X-ray diffraction images , X-ray fhttps://deposit.wwpdb.org) and the\u00a0stand-alone wwPDB validation server .To incorporate state-of-the-art validation tools into the wwPDB biocuration pipeline, and to provide useful validation metrics to depositors and other PDB users, the wwPDB convened Validation Task Forces (VTFs) for crystallography and NMR R and Rfree shows key information about the entry, such as the experimental technique employed to determine the structure, a proxy measure of information content of the analyzed data , and a number of percentile scores (\u201csliders\u201d), comparing the validated structure to the entire PDB archive (Rfree factor) and by the fraction of residues that locally do not fit the electron density well , specifies the type of the report , shows basic administrative information about the uploaded data or the PDB entry, lists the software packages and versions that were used to produce the report, and provides a URL to access help text at archive . Table 1e, RSRZ) . These c angles) . IdeallyThe percentile ranks are followed by a graphical summary of chain quality . Each stThe section on overall quality is followed by one on entry composition, which describes each unique molecule present in the entry. For NMR entries, a separate section on ensemble composition is also included. As most NMR structures are deposited as ensembles of conformers, this section reports on what parts of the entry are deemed to be well-defined or ill-defined and alsoThe section on residue quality highlights residues that exhibit at least one kind of issue, i.e., color coded yellow, orange, or\u00a0red, as described above . While uR and Rfree values are presented as provided by the depositor and as recalculated by the wwPDB from structure-factor amplitudes and the model. The Rfree value measures how well the atomic model predicts the structure factors for a small subset of the reflections that were not included in the refinement protocol . MolProbity also performs protein-backbone and side-chain torsion-angle analysis (Ramachandran plot and rotameric state) and RNA-backbone and ribose-pucker analysis. For X-ray crystal structures of proteins, cases where 180\u00b0 flips of histidine rings and glutamine or asparagine side chains improve the hydrogen-bonding network without detriment to the electron density fit are also reported. The MAXIT software is also used to identify and report cis-peptides and stereochemistry issues, such as chirality errors and polymer linkage artifacts.The section on model validation provides further details for\u00a0each criterion covering polypeptides, ribonucleic acids, small\u00a0molecules, and non-standard polymer residues. The bond lengths and bond angles of amino acid and nucleotide residues are checked by MolProbity's Dangle module against The geometry of all non-standard or modified residues of a polymer, small-molecule ligands, and carbohydrate molecules is analyzed with the Mogul software . For eaccalc map) and one calculated based on model and experimental data . The fit is analyzed on a per-residue basis for proteins and polynucleotides, and reported as the real-space R value (RSR) are reported. LLDF for a ligand or non-standard residue is calculated as follows: all standard amino acid or nucleotide residues within 5.0\u00a0\u00c5 distance of any atom of the ligand or\u00a0non-standard residue are identified by the CCP4 NCONT program, taking crystallographic symmetry into account . LLDF values greater than 2 are highlighted in the reports . The wwPDB partners and the crystallography community are evaluating this and other metrics to reliably assess the fit to electron density for bound ligands, following the recommendations of the wwPDB/CCDC/D3R Ligand Validation Workshop is analyzed by the procedure developed for the Uppsala Electron Density Server . Electroue (RSR) . These Reld RSRZ . Residue account . The meaWorkshop .RCI) for each residue, which estimates how likely the residue is to be disordered. In a bar-graph representation of RCI for each polypeptide chain, each residue considered to be ill-defined from the analysis of the NMR ensemble of conformers . Therefore, each chemical-shift list is treated independently. For each list, a table summarizing any parsing and mapping issues between the chemical shifts and the model coordinates helps depositors detect and correct data entry errors. For entries containing proteins, the PANAV package is invokThe OneDep validation module is used at various points during PDB data deposition and biocuration . When daThe same validation module is available from the wwPDB stand-alone validation web server and from an application programming interface (API) designed for use by structure determination, refinement, and visualization software . The priThe wwPDB validation pipeline orchestrates execution of each community-recommended validation tool , extracthttps://wwpdb.org/validation/schema/wwpdb_validation_v002.xsd). Summary reports are composed using TeX formatting instructions and rendered in PDF format for delivery.The modules access data and validation tools through a collection of APIs shared by all of the wwPDB OneDep system components. These core APIs provide uniform access to the diverse set of pipeline dependencies, including both locally developed and community-supported tools and libraries. As the pipeline executes each module, it\u00a0records names and versions of each validation tool together with the completion status for the tool. Pipeline results are recorded in data files and summarized in formatted reports. The data file organization is documented in the XSD format schema files . Installation and user documentation for the Python API and CLI\u00a0are provided at https://wwpdb.org/validation/onedep-validation-web-service-interface.The web-service API is supported by both a client-side Python implementation and a Unix command-line interface (CLI). Execution of the wwPDB validation pipeline using the API involves multiple steps performed in the context of a validation session. Within a session, the API provides methods to upload data files, queue validation pipeline requests, check completion status, and recover result files. The API steps are summarized in https://www.rabbitmq.com/) message broker and the supporting AMQP (https://www.amqp.org/) Python client library.Future resource requirements of the web-service API are anticipated to be significantly greater than those of the web user interfaces. As a result, a different workflow system has been developed to support the web-service deployment. This system uses a message broker to route requests from the web-service API to a distributed collection of task queues. Queued validation task requests are handled by a set of back-end services. The volume of back-end services can be adjusted quickly in response to changes in workload. Our current implementation uses the RabbitMQ , percentile ranks of structures also change. To account for such changes, wwPDB validation reports are regenerated annually for the entire\u00a0public archive, with recalculated statistics underlying the percentile ranks based on the state of the PDB archive on December 31 of the preceding calendar year. Following internal review, the updated reports replace the older versions in the public wwPDB FTP areas. The most recent update took place on March 15, 2017. Older reports continue to be accessible via yearly snapshots of the wwPDB FTP area.For most entries, changes in the percentile ranks are modest year-on-year. However, with improved tools for structure determination and more awareness of the importance of validation, it is hoped that erroneous features will become increasingly rare in newly deposited structures. As a result, the percentile ranks for older structures are expected to slowly decline, reflecting an increase in overall quality of structures in\u00a0the PDB archive.Official wwPDB validation reports provide an assessment of\u00a0structure quality using widely accepted and community-recommended standards and criteria. To help deliver the best possible quality in the PDB archive, the wwPDB partners strongly encourage journal editors and referees to request these reports from authors as part of the manuscript submission and review process. To achieve this goal, wwPDB partners have formally approached the journals responsible for publishing most structures to request them to implement mandatory submission of official wwPDB validation reports together with manuscripts describing the structures. , the Nature Publishing Group journals, FEBS Journal, the Journal of Immunology, and Angewandte Chemie International Edition in English as part of their manuscript submission process. Submission of official wwPDB validation reports is further recommended by Cell, Molecular Cell, and Cell Chemical Biology. The interaction between wwPDB and journals is an ongoing effort. More journals have expressed interest recently, and we expect that additional publishers will commence requiring wwPDB validation reports as part of their manuscript review process.At the time of writing, submission of official wwPDB validation reports is required by ng Group , eLife, https://wwpdb.org/validation/validation-reports. These materials include explanatory notes for each kind of validation report , frequently asked questions, and instructions for use of the web-service API.To assist the structural biology and wider scientific community in interpreting the valuable information contained in wwPDB validation reports, the OneDep team has made available an extensive set of documentation materials at R value of their models.\u201d Thus, another contributing factor to the improving statistics may be the fact that deposition of experimental data has become mandatory since then. This important development enabled better validation of structures, calculation of electron density maps for all crystal structures , and re, in EDS ).Figure\u00a0Ramachandran analysis is perhaps the best-known and most widely used geometric quality metric for experimentally determined models. For NMR entries, the analysis of validation metrics reveals fewer trends. There has been no perceptible change in the\u00a0fraction of Ramachandran outliers, residue side chains modeled in non-rotameric conformations, or clashscores since 2006, and the observed distributions of these metrics are considerably wider than seen for X-ray crystal structures D\u20133F. NevThe OneDep validation module will continue to be developed and improved as the wwPDB partnership receives further recommendations from the expert VTFs for X-ray, NMR, and 3DEM, the OneDep system is refined, and feedback is received from PDB depositors and users alike.Analyses of wwPDB biocuration efficiency have sugThe wwPDB validation reports for NMR structures do not yet include analysis of NMR restraints. To achieve this goal, the wwPDB in partnership with Leicester University has convened a working group for standardization of restraint representation . The resThe wwPDB validation reports for 3DEM structures currently include only assessment of geometric parameters for the map-derived atomic coordinates. In the near future, we will add basic information about the experimental map and map-model fit, integrating some of the features from the EM map visual analysis software . Recent Major contributors to this project are S.G., E.S.G., P.M.S.H., A.G., J.D.W., Z.F., and H.Y. The X-ray validation pipeline was implemented by S.G., Z.F., H.Y., O.S.S., and J.D.W. The NMR validation pipeline was implemented by P.M.S.H., A.G., Z.F., O.S.S., and S.M. The 3DEM validation pipeline was implemented by E.S.G. and O.S.S. The validation pipeline was integrated in the OneDep deposition and biocuration system by J.D.W., T.J.O., E.P., Z.F., E.S.G., P.M.S.H., L.M., O.S.S., and J.M.B. The stand-alone validation web server was implemented by E.S.G., E.P., T.J.O., P.M.S.H., and J.D.W. The validation web-service API was implemented by J.D.W. Annual report recalculations were performed by S.G. and O.S.S. Testing of integrated systems and feedback on the report content were provided by S.S., J.Y.Y., J.M.B., G.S., A.M., C.S., E.P., B.P.H., M.R.S., C.L.L., A.P., A.G., Y.I., N.K., K.B., E.L.U., and R.Y. Project management was provided by A.G., A.P., M.Q., J.D.W., and J.Y.Y. Overall project direction was provided by J.L.M., H.N., H.M.B., S.K.B., S.V., and G.J.K. The manuscript was written by A.G., J.Y.Y., J.D.W., C.L.L., J.M.B., O.S.S., and A.P."} +{"text": "Scientific Reports 10.1038/s41598-017-16926-w, published online 05 December 2017Correction to: The Acknowledgements section in this Article is incomplete.\u201cThe financial support of the National Science Foundation to R.A.M. (grants 1310350 and 1429329), is gratefully acknowledged. Thanks are extended to Dr. Zheng Wei for assistance with X-ray crystal structure determination and Prof. Kathy Dunn at SUNY Polytechnic Institute for assistance with TEM imaging.\u201dshould read:\u201cThe financial support of the National Science Foundation to R.A.M. , is gratefully acknowledged. Thanks are extended to Dr. Zheng Wei for assistance with X-ray crystal structure determination and Prof. Kathy Dunn at SUNY Polytechnic Institute for assistance with TEM imaging.\u201d"} +{"text": "The following information is missing from the Funding section: A.D. was supported by an American Cancer Society (ACS) New faculty Career Development Grant, and by a \u201cWalter B. Frommeyer Jr. Fellowship in investigative medicine.\""} +{"text": "Megastigmuspistaciae Roques & Copeland sp. n.On p. 81 the header for the new species reads Megastigmusozoroae Roques & Copeland sp. n. The name was misspelled when the PDF file was generated.The CORRECT species name is"} +{"text": "Scientific Reports6: Article number: 2887310.1038/srep28873; published online: 07012016; updated: 04202017The Acknowledgements section in this Article is incomplete.Tg zebrafish, respectively. We thank Z. Wang for conceiving of the name N-CISSOR. This work was supported by MEXT KAKENHI Grant Number 22122007 (to A. Sehara-Fujisawa) and 15H02370 (to K. Kawakami); JSPS KAKENHI Grant Numbers 26650077 (to A. Sehara-Fujisawa) and 14J05637 (to A. Kamezaki); the National BioResource Project (to K. Kawakami); the Cooperative Research Program of Institute for Frontier Medical Sciences of Kyoto University (to K. Kawakami); and the Platform for Dynamic Approaches to Living System from the Ministry of Education, Culture, Sports, Science and Technology, Japan (to A. Sehara-Fujisawa and K. Aoki)\u201d.\u201cWe are grateful to Drs K. Shimamura, J. Hatakeyama, M. Suzuki, and K. Tsumagari for helpful discussions. We also thank Drs Y. Kanaho and S. Higashijima for providing the murine neuroblastoma cell line N1E-115 and the should read:HuC:Gal4-VP16) zebrafish, respectively. We thank Z. Wang for conceiving of the name N-CISSOR. This work was supported by MEXT KAKENHI Grant-in-Aid for Scientific Research on Innovative Areas 22122007 and 15H05938 (to A. Sehara-Fujisawa) and 15H02370 (to K. Kawakami); JSPS KAKENHI Grant Numbers 26650077 and (B) 16H04793 (to A. Sehara-Fujisawa) and 14J05637 (to A. Kamezaki); the National BioResource Project (to K. Kawakami); the Cooperative Research Program of Institute for Frontier Medical Sciences of Kyoto University (to K. Kawakami); and the Platform for Dynamic Approaches to Living System from the Ministry of Education, Culture, Sports, Science and Technology, Japan (to A. Sehara-Fujisawa and K. Aoki)\u201d.\u201cWe are grateful to Drs K. Shimamura, J. Hatakeyama, M. Suzuki, and K. Tsumagari for helpful discussions. We also thank Drs Y. Kanaho and S. Higashijima for providing the murine neuroblastoma cell line N1E-115 and the Tg ("} +{"text": "Scientific Reports6: Article number: 36132; 10.1038/srep36132 published online: 10312016; updated: 06262017.This Article contains errors. The Acknowledgements section is incomplete.\u201cWe thank the Department of Biotechnology (DBT), Government of India for research support to the Institute of Bioinformatics (IOB), Bangalore. We thank the \u201cInfosys Foundation\u201d for research support to IOB. AC was supported by Department of Science and Technology (DST) grants (SERC/LS-439/2011 and SR/SO/HS/0208/2013). IOB is supported by DBT Program Support on Neuroproteomics and infrastructure for proteomic data analysis (BT/01/COE/08/05). AR, GS and SC are recipients of Senior Research Fellowship from Council of Scientific and Industrial Research (CSIR), Government of India. RR is a recipient of research associateship from DBT. We thank Dr. S. K. Shankar and Dr. Anita Mahadevan of National Institute of Mental Health and Neurological Sciences (NIMHANS), for providing access to the microscopy imaging facility. We thank Dr. V. Ravi for providing access to advanced FACS facility in NIMHANS. We thank Ms. Neha Deshpande for technical inputs with fluorescence imaging\u201d.should read:\u201cWe thank the Department of Biotechnology (DBT), Government of India for research support to the Institute of Bioinformatics (IOB), Bangalore. We thank the \u201cInfosys Foundation\u201d for research support to IOB. AC was supported by Department of Science and Technology (DST) grants (SERC/LS-439/2011 and SR/SO/HS/0208/2013) and FAMRI-funded 072017_YCSA. IOB is supported by DBT Program Support on Neuroproteomics and infrastructure for proteomic data analysis (BT/01/COE/08/05). AR, GS and SC are recipients of Senior Research Fellowship from Council of Scientific and Industrial Research (CSIR), Government of India. RR is a recipient of research associateship from DBT. We thank Dr. S. K. Shankar and Dr. Anita Mahadevan of National Institute of Mental Health and Neurological Sciences (NIMHANS), for providing access to the microscopy imaging facility. We thank Dr. V. Ravi for providing access to advanced FACS facility in NIMHANS. We thank Ms. Neha Deshpande for technical inputs with fluorescence imaging\u201d.Additionally in Figure 4b, the label \u2018Harmine (15\u2009mg/kg)\u2019 is incorrectly given as \u2018Harmine (30\u2009mg/kg)\u2019. The correct Figure 4 appears below as"} +{"text": "Nature Communications8: Article number: 13937; DOI: 10.1038/ncomms13937 (2017); Published 01182017; Updated 03212017The Author Mi Yan was incorrectly omitted from the list of corresponding Authors in the PDF of this Article, and the author Tianyu Ma was incorrectly listed as a corresponding author; the HTML version of the paper was correct from the time of publication. The correct information for correspondence is: \u2018Correspondence and requests for materials should be addressed to X.R. ren.xiaobing@nims.go.jp) or to M.Y. mse_yanmi@zju.edu.cn).'"} +{"text": "Erratum to: Neth Heart J (2016) DOI: 10.1007/s12471-016-0874-yIn the version of the article originally published online, there was an error in the acknowledgement section. The authors wrote: \u2018and Dr. S.H.K.\u00a0The (Bethesda Diabetes Research Center)\u2019. The line should have read: \u2018and Dr. S.H.K.\u00a0The (Treant Zorggroep \u2013 locatie Bethesda)\u2019."} +{"text": "Scientific Reports6: Article number: 38224; 10.1038/srep38224 published online: 12022016; updated: 02012017.The Acknowledgements section in this Article is incomplete.\u201cThis work was supported by the Wessex Medical Trust, the Alzheimer\u2019s Society and the Henry Smith\u2019s foundation. Thanks to Prof. St. Johnstone for providing the dtau antibody and Dr. Peter Davies for providing the PHF-1 antibody\u201d.should read:\u201cThis work was supported by the Wessex Medical Trust, the Alzheimer\u2019s Society and the Henry Smith\u2019s foundation. Thanks to Prof. St. Johnstone for providing the dtau antibody and Dr. Peter Davies for providing the PHF-1 antibody. Thanks to the ARUK South Coast Network for contributing to publication costs\u201d."} +{"text": "Reference 49 contains two individual references. The correct reference 49 is: Beverton RJH, Holt SJ. On the dynamics of exploited fish populations. London: Her Majesty\u2019s Stationary Office. 1957. The correct reference 50 is: Hilborn R, Walters CJ. Quantitative fisheries stock assessment and management. Chapman-Hall: New York. 1992. All subsequent references should be shifted up by one.Reference 79 is incorrectly included in the article. All of the in-text citations appear correctly throughout the paper."} +{"text": "The Pan African Medical Journal. 2017;25:142. doi:10.11604/pamj.2016.25.142.7193.This erratum corrects The error was not present in both the PDF and HTML versions of the article. The author\u2019s name should be abbreviated as Nansseu JR.The name of the author of this article ["} +{"text": "Scientific Reports5: Article number: 1599310.1038/srep15993; published online: 01062016; updated: 11112016Joong Sun Kim and Wol Soon Jo did not make contributions that warrant authorship of this paper and have been removed from the author list.The Acknowledgements section now reads:This work was supported by the National Research Foundation of Korea (DIRAMS) grant funded by the Korea government (MSIP) (50597-2014).We thank Min Young Kim, and Min Su Ju for administrative support and J.S. Kim and W.S. Jo for the data analysis and scientific comments.The Author Contributions statement now reads:C.G.L. and J.Y.H. designed and wrote the manuscript. J.Y.H., Y.K.H., G.-Y.P. and S.D.K. performed the experiments. All authors reviewed the manuscript.These errors have now been corrected in the PDF and HTML versions of the Article, as well as the previous Corrigendum that accompanies the Article."} +{"text": "In the original article, we neglected to acknowledge the Gulf of Mexico Research Initiative (231612-00), the Prince Albert II Foundation (Project 1265) and the National Science Foundation (1536776) for supporting DB.Lutibacter profundi sp. nov., isolated from a deep-sea hydrothermal system on the Arctic Mid-Ocean Ridge and emended description of the genus Lutibacter. Int. J. Syst. Evol. Microbiol. 66, 2671\u20132677. doi: 10.1099/ijsem.0.001105\u201d as \u201cBauer, S. L. M.\u201d. It should be \u201cLe Moine Bauer, S.\u201d. The authors apologize for these errors and state that this does not change the scientific conclusions of the article in any way.Also, the name of one of the authors was incorrectly spelled in the reference for \u201cBauer, S. L. M., Roalkvam, I., Steen, I. H., and Dahle, H. (2016). The original article has been updated.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Scientific Reports 10.1038/s41598-017-16283-8, published online 22 November 2017Correction to: Clark Zahn and Dennis Friedrich were omitted from the author list in the original version of this Article. This has been corrected in the PDF and HTML versions of the Article, and the Supplementary Information file that accompanies the Article.The Acknowledgements section now reads:\u201cThe authors would like to thank Fanxing Xi for SEM analysis and the ATM deposition, Moritz K\u00f6lbach for SEM/ EDX analysis, Paul Plate for XPS, and Sean Berglund for his help with the PEC measurements. The research association Optotransmitter-Umweltschutz-Technologie e.V. is acknowledged for the preparation of the TiN:O back contact layers. F.B. acknowledges the Ministry of Education and Science of the Republic of Kazakhstan for a research fellowship under the program no. 0115\u0420\u041a03029 \u201cNU-Berkeley strategic initiative in warm-dense matter, advanced materials and energy sources for 2014\u20132018\u201d and grants from Tomsk (Russia) Polytechnic University Competitiveness Enhancement Program and National Nanotechnology Laboratory of open type of Al-Farabi Kazakh National University, Almaty (Kazakhstan), 0263/PSF.\u201cThe Author Contributions section now reads:\u201cK.E. and F.B. conceived the study. F.B. and K.H. performed the deposition experiments. F.B. did the XRD and PEC measurements of the films. C.Z. and D.F. measured and analyzed the TRMC data. F.B. and K.E. analyzed the data. F.B. drafted the manuscript and K.E. finished the manuscript using the contributions from the other authors.\u201d"} +{"text": "Scientific Reports6: Article number: 3721710.1038/srep37217; published online: 11152016; updated: 05092017The authors wish to acknowledge other contributors and other sources of funding.The Author Contributions statement should read:G.A. and S.B. contributed to the design of the experiments. G.A. contributed to the development of the microscope. G.A. and S.B. collected the data. S.B. contributed to the development of cell protocols and sample preparation. G.A. and S.B. wrote the manuscript.The Acknowledgements should read:The authors would like to thank Prof Peter T\u00f6r\u00f6k and Dr Carl Paterson for their advice, guidance on and contributions to the design of the microscope; Prof T\u00f6r\u00f6k and Dr Darryl Overby for their advice and guidance on the experimental design; and Dr Overby for his advice and guidance on the development of cell protocols and sample preparation. Mr Martin Kehoe and Mr Simon Johnson are acknowledged for their valuable help in designing and manufacturing parts of the optomechanical setup. The authors wish to thank Margherita Rossi for her contribution to the design of the figures. The authors acknowledge funding from the EPSRC Doctoral Training Account (G.A.) and a PhD studentship from the Department of Bioengineering, Imperial College London (S.B.), as well as funding from a National Institutes of Health grant EY019696 (Dr Overby) and EPSRC Pathways to Impact Grant (Prof. T\u00f6r\u00f6k)."} +{"text": "Scientific Reports 10.1038/s41598-017-05763-6, published online 18 July 2017Correction to: The Acknowledgements section in this Article is incomplete:\u201cWe thank the Atlantic and Gulf Rapid Reef Assessment Program, especially K. Marks, J. Lang, and P. Kramer for providing data. Thanks also to S. J. van Woesik for editorial comments. This paper is Contribution No. 148 from the Institute for Research on Global Climate Change at the Florida Institute of Technology.\u201dshould read:\u201cWe thank the Atlantic and Gulf Rapid Reef Assessment Program, especially K. Marks, J. Lang, and P. Kramer for providing data. Thanks also to S. J. van Woesik for editorial comments. This paper is Contribution No. 148 from the Institute for Research on Global Climate Change at the Florida Institute of Technology. CJR was supported by a National Science Foundation (NSF) OCE grant 1535007 to Richard B. Aronson. RvW acknowledges funding from the National Science Foundation (NSF) OCE grant 1657633.\u201d"} +{"text": "Nature Communications 10.1038/s41467-018-04074-2, published online 26 April 2018Correction to: The original version of the Article was missing an acknowledgement of a funding source. The authors acknowledge that A. Safaie and K. Davis were supported by National Science Foundation Award No. 1436254 and G. Pawlak was supported by Award No. 1436522. This omission has now been corrected in the PDF and HTML versions of the Article."} +{"text": "Scientific Reports6: Article number: srep3848810.1038/srep38488; published online: 11052016; updated: 04062017The Acknowledgments section of this Article is incomplete, where:\u201cR.S. and M.K.S. acknowledge the Ramalingaswami fellowship and grant from the Department of Biotechnology, Government of India\u201d.should read:\u201cR.S. and M.K.S. acknowledge the Ramalingaswami fellowship and grant from the Department of Biotechnology, Government of India. Authors also acknowledge the financial support through PURSE grant from Department of Science & Technology, Government of India\u201d."} +{"text": "There are errors in the Funding section. The correct funding information is as follows: The present study was supported by the Hong Kong Government Research Grant Council grant, Collaborative Research Fund CRFHKU6/CRF/11G and Hong Kong University 764812M to B.K.C.C. & International Scientific Partnership Program ISPP at King Saud University through ISPP# 008."} +{"text": "Indian Journal of Anaesthesia Mar-Apr 2010; Vol. 54; Issue 2Article Type: ObituaryPage 156; Column: Bottom of the page; Title Heading and First lineDr. Chinnappa S. MetgudShould be read asDr. C H MetgudThe error is regretted.Editor, Indian Journal of Anaesthesia"} +{"text": "An investigation was made into the effect of Corynebacterium parvum therapy on cerebral tumours in mice. I.v. C. parvum caused a slight but significant increase in the survival of BALB/c mice injected intracerebrally (i.c.) with not more than 50 Meth A cells. C. parvum was most effective if given on the same day or 5 days after tumour. If this interval was increased there was no effect. Multiple i.v. injections were no more effective than a single dose. I.v. C. parvum had no influence on the survival of C57BL mice injected i.c. with Lewis tumour cells, and had little effect on the induction of i.c. or s.c. tumours by methylcholanthrene. It was concluded that C. parvum therapy was of little use in the treatment of cerebral tumour in mice. The clinical implications of these findings are discussed."} +{"text": "Acta Cryst. (2007), E63, o3999.Corrigendum to Acta Cryst. (2007), E63, o3999] is corrected.The name of the first author in the paper by Chen & Cao [ Acta Cryst. (2007), E63, o3999], the name of the first author is incorrect. The correct name is given above.In the paper by Chen & Cao ["} +{"text": "The funding information for this paper is missing. It should read:This work was supported by the Israel Science Foundation (O.M.), the Binational Science Foundation (O.M.), the AICR (O.M), the ICRF (O.M.), the European Commission (LSHC-CT-2002-518178 and MRTN-CT-2005 to O.M.), the Israel Ministry of Health (A.P.), the Cooperation Program in Cancer Research of the Deutsches Krebsforschungszentrum (DKFZ), and Israeli's Ministry of Science and Technology (MOST) (A.P.)."} +{"text": "The surname of co-author Barend J. M. de Wet was misspelled in the originally published article."} +{"text": "Gallstone dissolution may be possible only in selected patients. Patients with calcified or large gallstonesare not suitable for dissolution. Citrate is normally present in bile and an oral citrate load can increasebiliary citrate.\t\tin vitro. Patients with calcified or large gallstones were treated with a combination of C.D.C.A. and citrate. Partial decalcification was achieved in seven out of twenty patients withcalcified stones (35%) and complete decalcification in four patients (20%). One of the patients withlarge stones had complete dissolution.A combination of chenodeoxycholic acid (C.D.C.A.) and citrate has been shown to dissolve calcifiedcholesterol gallstones \t\tFive patients who were suitable for C.D.C.A. treatment but did not respond were also treated withC.D.C.A. and citrate. One of the patients in this latter group had complete dissolution. Oral citrate candecalcify some calcified gallstones."} +{"text": "The ninth author's name was spelled incorrectly. The correct name is: Saffron A. G. Willis-Owen."} +{"text": "A funding source was omitted from this article. Additional funding for this study was provided by a grant from the Pediatric Low Grade Astrocytoma Foundation. The corrected funding statement of this article should read: This work was supported by the National Cancer Institute (R21CA126674) to L.A.G., (K08CA134931) to A.J.B., and the Dana-Farber Cancer Institute. A.J.B. was supported by the Harvard Clinical Investigator Training Program. Funding for analysis of gastrointestinal tumors was supplied the American Society of Clinical Oncology, Friends of the Dana-Farber Cancer Institute and the H.T. Berry Foundation. Additional funding was provided by a grant from the Pediatric Low Grade Astrocytoma Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."} +{"text": "Dr. Ashok Jain joined G.R. Medical College, Gwalior in 1973 and completed his MBBS., and D.A. in 1981. During his life time, he started his career as Anaesthesiologist in Cancer Hospital and Research Center, Gwalior. Later on he left the job and became first Full Time anaesthesiologist in private sector of Gwalior. He was one of active member of I.S.A. and having dynamic personality.He was also first anaesthesiologist who started Pain \u2013 Clinic at Gwalior. Since last two year, he was suffering from undiagnosed disease of spine.He survived by his wife and two sons, elder one is an engineer and working in U.S.A., while younger is doing B.E. from Indore.May his soul rest in peaceGwaliorDate: 15-05-2010"} +{"text": "P. falciparum and P. vivax lactate dehydrogenase were chosen, synthesized and coupled to rabbit albumin carrier. Anti-peptide antibodies were raised in chickens against the peptides and against each of the recombinant proteins and affinity purified. An antibody against a common epitope detected recombinant P. falciparum, P. vivax and P. yoelii and native P. falciparum and P. vivax protein lactate dehydrogenase. Antibodies against species specific lactate dehydrogenase epitopes differentiated between P. falciparum and P. vivax lactate dehydrogenase. The study supports an anti-peptide antibody approach for the design of malaria diagnostic reagents.Lactate dehydrogenase is one of the antigens targeted in lateral flow immunochromatographic rapid diagnostic tests for the diagnosis of malaria. Unique diagnostic peptide targets based on the respective amino-acid sequences to differentiate between"} +{"text": "After publication of this manuscript , we becaWe regret any inconvenience that these omissions might have caused and thank Prof. J.P.W. Young for bringing this matter to our attention."} +{"text": "A competing interest statement was erroneously omitted for the 2nd and 4th authors, Sreedhar R. Nallapareddy and Barbara E. Murray. The Competing Interests section should read: Drs. Barbara E. Murray and Sreedhar R. Nallapareddy are co-inventors of useful improvements in COLLAGEN-BINDING PROTEINS FROM ENTEROCOCCAL BACTERIA, a patent for which the rights have been assigned to The Texas A&M University System and the Board of Regents of the University of Texas System ."} +{"text": "The name of the 84th author contained an error. Mark I. Greene should be Mark H. Greene."} +{"text": "The 11th and 12th authors' names were misspelled. The correct names read: Andrew P. Waters and Chris J. Janse"} +{"text": "Scientific Reports6; Article number: 2937810.1038/srep29378; published online: 07112016; updated: 06282018The Acknowledgements section in this Article is incomplete.\u201cWe like to thank to K. Schoknecht, S. Bernhardt, and A. Kolchmeier for excellent technical assistance. This work was supported by grants of the Deutsche Forschungsgemeinschaft to K.B. and V.N. and of the Friedrich Schiller University to V.N.\u201dshould read:\u201cWe like to thank to K. Schoknecht, S. Bernhardt, and A. Kolchmeier for excellent technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft and by the Friedrich Schiller University to V.N.\u201d"} +{"text": "The following information is missing from the Funding statement: M.J.B. and A.H. received funding from the Intramural Research Program of the NIH; National Institute of Environmental Health Sciences (ZIC ES103326 to M.J.B).The Data Availability statement for this paper is incorrect. The correct statement is: The cryo-EM data of CH235 UCA bound to Man5-enriched CH505.N279K.G458Y.SOSIP.664 have been deposited to EMDB database with the accession code EMD-20735, and the fitted coordinates have been deposited to the RCSB database with accession number 6UDA."} +{"text": "Nature Communications 10.1038/s41467-018-03134-x, published online 19 March 2018Correction to: The original version of this Article omitted the author N. Penny Holliday from the National Oceanography Centre, European Way, Southampton SO14 3ZH, UK.Consequently, the following was originally omitted from the Acknowledgements: \u2018N.P.H. and the JR302 cruise were funded through the UK Natural Environment Research Council programmes UK OSNAP (NE/K010875/1), RAGNARRoCC (NE/K002511/1) and the Extended Ellett Line \u2019. The corrected version of the Acknowledgements also removes the following from the original version: \u2018The Zonally Accumulated Heat Transport in observation was calculated by N.P.H. from the National Ocean Center, United Kingdom\u2019.Additionally, the following was originally omitted from the Author Contributions: \u2018N.P.H. calculated the observed Zonally Accumulated Heat Transport along the OSNAP east section\u2019.This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Scientific Report 10.1038/s41598-019-50048-9, published online 26 September 2019Correction to: The Acknowledgements section in this Article is incomplete.TM, Dr. Ourania M. Andrisani\u2019s lab and Dr. Emily C. Dykhuizen\u2019s lab for their qPCR machines.\u201d\u201cL.M. is grateful to the partial support by the Purdue Polytechnic Institute Dean\u2019s award GRA \u2013 FY16. Publication of this article was funded by Purdue University Libraries Open Access Publishing Fund. We thank Victoria Hedrick of the Purdue Proteomics Facility for support in LC-MS/MS data collection and for critical reading of the manuscript. All the mass spec experiments were performed at the Purdue Proteomics Facility, Bindley Bioscience Center. The Q Exactive Orbitrap HF and UltiMate 3000 HPLC system used for LC-MS/MS analysis were purchased from funding provided by the Purdue Office of the Executive Vice President for Research and Partnership. We thank Alexis A. Musleh for help in maintaining the cell cultures and assistance in experiments and Vishak Raman for useful discussions. The authors are grateful to Dr. Richard J. Kuhn\u2019s lab for the use of spectrophotometer, Dr. Chun-Ju Chang\u2019s lab for the Nanodropshould read:TM, Dr. Ourania M. Andrisani\u2019s lab and Dr. Emily C. Dykhuizen\u2019s lab for their qPCR machines. The authors are grateful to Prof. Morris Levy for his excellent edits.\u201d\u201cL.M. is grateful to the partial support by the Purdue Polytechnic Institute Dean\u2019s award GRA \u2013 FY16. Publication of this article was funded by Purdue University Libraries Open Access Publishing Fund. We thank Victoria Hedrick of the Purdue Proteomics Facility for support in LC-MS/MS data collection and for critical reading of the manuscript. All the mass spec experiments were performed at the Purdue Proteomics Facility, Bindley Bioscience Center. The Q Exactive Orbitrap HF and UltiMate 3000 HPLC system used for LC-MS/MS analysis were purchased from funding provided by the Purdue Office of the Executive Vice President for Research and Partnership. We thank Alexis A. Musleh for help in maintaining the cell cultures and assistance in experiments and Vishak Raman for useful discussions. The authors are grateful to Dr. Richard J. Kuhn\u2019s lab for the use of spectrophotometer, Dr. Chun-Ju Chang\u2019s lab for the Nanodrop"} +{"text": "Communications Biology 10.1038/s42003-019-0587-z, published online 17 September 2019.Correction to: The original version of this Article contained errors in the author affiliations. The affiliations of Adriana Coricello with Dipartimento di Scienze della Salute and Net4Science academic spinoff at Universit\u00e0 \u201cMagna Gr\u00e6cia\u201d di Catanzaro were inadvertently omitted. This has now been corrected in both the PDF and HTML versions of the article.The original version of this Article also omitted the following from the Acknowledgements:This work was supported by Cardiff University , the National Institutes of Health [R01 GM111695 to Y.T.], the National Science Foundation [MCB-1157688 to Y.T.], and the UK Biotechnology and Biological Sciences Research Council (BBSRC) (BB/I003703/1 to D.W.R. and BB/P018017/1 to N.G.J.R.). Funding was also provided by the University of Florida Undergraduate Scholar\u2019s Program (A.H.B.) and a Short-Term Scientific Mission grant from COST Action CA15135 (A.C.).This has now been corrected in both the PDF and HTML versions of the article."} +{"text": "Grant number GRDC 9176106 to S.L.N and GRDC 9176093 to G.M.R. The publisher apologizes for the error.There are errors in the Funding section. The correct funding information is as follows: This research was funded by the Grain Research and Development Corporation ("} +{"text": "In the Author Contributions, James F. A. Traniello (JFAT) should be listed as a contributor to Conceptualization and Project Administration."} +{"text": "Ulrich Gembruch's degree should appear as Prof. Dr. Med. and Dr. Guido Kukuk's degree should appear as PD. Dr. Med.In the article, \u201cEnteritis as initial manifestation of systemic lupus erythematosus in early pregnancy: A case report\u201d,"} +{"text": "Lateolabrax maculatus) is a valuable commercial fish that is widely cultured in China. While analyses using molecular markers and population genetics have been conducted, genomic resources are lacking.The spotted sea bass (Here, we report a chromosome-scale assembly of the spotted sea bass genome by high-depth genome sequencing, assembly, and annotation. The genome scale was 0.67 Gb with contig and scaffold N50 length of 31 Kb and 1,040 Kb, respectively. Hi-C scaffolding of the genome resulted in 24 pseudochromosomes containing 77.68% of the total assembled sequences. A total of 132.38 Mb repeat sequences were detected, accounting for 20.73% of the assembled genome. A total of 22,\u2009015 protein-coding genes were predicted, of which 96.52% were homologous to proteins in various databases. In addition, we constructed a phylogenetic tree using 1,586 single-copy gene families and identified 125 unique gene families in the spotted sea bass genome.We assembled a spotted sea bass genome that will be a valuable genomic resource to understanding the biology of the spotted sea bass and will also lead to the development of molecular breeding techniques to generate spotted sea bass with better economic traits. Lateolabrax maculatus) belongs to the family Moronidae (Perciformes) and has characteristic clear black dots on the lateral side of its body taxonomy ID 315\u00a0492) that was obtained from Haiyang Yellow Sea Fisheries Co. . Genomic DNA was isolated and processed according to DNA extraction protocol BUSCO: Benchmarking Universal Single-Copy Orthologs; GWAS: genome-wide association study; Mya: million years ago; NCBI: National Center for Biotechnology Information; SNP: single-nucleotide polymorphism; TE: transposable element.The authors declare that they have no competing interests.This work was supported by AoShan Talents Program Supported by Qingdao National Laboratory for Marine Science and Technology (2017ASTCP-OS15) to S.C., Technological Innovation Project financially supported by Qingdao National Laboratory for Marine Science and Technology (2015ASKJ02\u201303) to S.C., and Taishan Scholar Climbing Project of Shandong to S.C. and Taishan Scholar Project of Shandong for Young Scientists to C.S., and AoShan Talents Program Supported by Qingdao National Laboratory for Marine Science and Technology (2017ASTCP-ES06) and International Scientific Partnership Program ISPP at King Saud University for funding this research work through ISPP No.0050.S.C., C.S., and X.L. designed the project. C.L., Q.L., Y.Z., W.X., Q.Z., and C.S. analyzed the data. N.W., Y.Q., X.L., X.C., and S.L. prepared the samples and conducted the experiments. C.S., C.L., S.M., X.L., and S.C. wrote and revised the manuscript."} +{"text": "Scientific Reports 10.1038/s41598-019-51605-y, published online 24 October 2019Correction to: In the original version of this Article, Yibo Wang and Rocio K. Finol-Urdaneta were omitted as equally contributing authors.Furthermore, the Acknowledgments section in this Article was incomplete.https://prism.ucalgary.ca/handle/11023/3077. All of the computations were performed on the West-Grid/ Compute Canada facilities, and the University of Calgary TNK and GlaDos clusters acquired with direct support by the Canada Foundation for Innovation and NSERC RTI awards.\u201d\u201cThis work was supported by grants from the Natural Sciences and Engineering Research Council (Canada) to S.Y.N. (NSERC RGPIN-315019) and the Alberta Innovates Technical Futures Strategic Chair in (Bio)Molecular Simulations. V.A.N. was financially supported by Postdoctoral Fellowships (AIHS and CIHR) during 2015\u20132018, and by LANL Director\u2019s Fellowship during 2018\u20132021 (V.A.N.). The classification number for this publication issued by LANL is LA-UR-19-29893. The experiments and simulations reported here also featured in the doctoral thesis of Y.W. now reads:https://prism.ucalgary.ca/handle/11023/3077. All of the computations were performed on the West-Grid/ Compute Canada facilities, and the University of Calgary TNK and GlaDos clusters acquired with direct support by the Canada Foundation for Innovation and NSERC RTI awards. Preliminary experiments by Ahmed Al-Sabi suggested the interesting difference in permeation of ammonium and hydrazinium through wild-type NaChBac, and its mutant, E191D.\u201d\u201cThis work was supported by grants from the Natural Sciences and Engineering Research Council (Canada) to S.Y.N. (NSERC RGPIN-315019), and to R.J.F. (NSERC RGPIN-418658) and the Alberta Innovates Technical Futures Strategic Chair in (Bio)Molecular Simulations to S.Y.N. V.A.N. was financially supported by Postdoctoral Fellowships (AIHS and CIHR) during 2015\u20132018, and by LANL Director\u2019s Fellowship during 2018\u20132021 (V.A.N.). The classification number for this publication issued by LANL is LA-UR-19-29893. The experiments and simulations reported here also featured in the doctoral thesis of Y.W. This has now been corrected in the HTML and PDF versions of the Article."} +{"text": "The authors wish to make the following change to their paper (Che Idris et al. 2018 [Conflicts of Interest: C.A.C.I. is currently working for the Malaysian Palm Oil Board, K.S. is currently from the Malaysian Palm Oil Council which previously served the Malaysian Palm Oil Board, and A.F.A.R. did his research internship at the Malaysian Palm Oil Board."} +{"text": "Milagros C. Rosal's name was missing the middle initial and has an MS as well as a PhD. Dr. Rosal's affiliation should appear as Department of Population and Quantitative Health Sciences, University of Massachusetts Medical School, Worcester, MA. Dr. Julie Donohue's affiliation should appear as University of Pittsburgh Graduate School of Public Health.In the article, \u201cOptimism may moderate screening mammogram frequency in Medicare: A longitudinal study\u201d,"} +{"text": "However, the underlying regulatory network of cell fate commitment during early neural differentiation remains elusive.Investigating cell fate decision and subpopulation specification in the context of the neural lineage is fundamental to understanding neurogenesis and neurodegenerative diseases. The differentiation process of neural-tube-like rosettes cis-regulatory elements for transcription factors known to have a key role in neural differentiation as well as for those that we suggest are also involved. Further, communication network analysis demonstrated that cellular interactions most frequently occurred in the embryoid body stage and that each cell subpopulation possessed a distinctive spectrum of ligands and receptors associated with neural differentiation that could reflect the identity of each subpopulation.In this study, we investigated the genome-wide transcriptome profile of single cells from six consecutive reprogramming and neural differentiation time points and identified cellular subpopulations present at each differentiation stage. Based on the inferred reconstructed trajectory and the characteristics of subpopulations contributing the most toward commitment to the central nervous system lineage at each stage during differentiation, we identified putative novel transcription factors in regulating neural differentiation. In addition, we dissected the dynamics of chromatin accessibility at the neural differentiation stages and revealed active Our study provides a comprehensive and integrative study of the transcriptomics and epigenetics of human early neural differentiation, which paves the way for a deeper understanding of the regulatory mechanisms driving the differentiation of the neural lineage. Moreover, the limited accessibility of human abortive fetuses at such an early stage precludes a thorough investigation of human early neural development.The nervous system contains complex molecular circuitry in developmental processes. In humans, there is a paucity of data describing early neural development and the corresponding cellular heterogeneity at various stages. To our knowledge, neural tube formation and closure are crucial for embryonic central nervous system (CNS) development and the process of neurulation. Previous studies have reported that neural tube closure is strongly controlled by both genetic and epigenetic factors and is sensitive to environmental influences . Perturbin vitro model for tracing early cell lineages and studying the cell fate specification of human neural differentiation 884) funded by Development and Reform Commission of Shenzhen Municipality; and Shenzhen Key Laboratory of Neurogenomics (CXB201108250094A) funded by Science, Technology and Innovation Commission of Shenzhen Municipality. D.C. is supported by China Postdoctoral Science Foundation (grant 2017M622795).Z.S., Z.G., and X.X. conceived and designed the project. Z.S., D.C., Q.W., Shengpeng W., and Q.D. conducted the majority of experiments and data analysis. Shengpeng W., L.W., X.D., Shiyou W., and J.Z. performed computational analyses and prepared figures. C.L. participated in validation experiments and assisted with figure preparation for revision. D.Z., X.C., and F.C. contributed to sample collection. H.Y., X.X., Z.G., and Z.S. supervised the entire study. X.L. contributed to the design of the revision and jointly supervised the validation work. Z.S. wrote the manuscript with input from D.C., Q.W., L.L., J.L.F., Z.G., and X.X. D.C., L.L., J.L.F., S.Z., F.C., Z.G., and X.X. contributed to the discussion and revision of the manuscript. All authors read and approved the final manuscript.Authors_Response_To_Reviewer_Comments_Revision_1.pdfClick here for additional data file.GIGA-D-18-00097_Original_Submission.pdfClick here for additional data file.GIGA-D-18-00097_Revision_1.pdfClick here for additional data file.GIGA-D-18-00097_Revision_2.pdfClick here for additional data file.Response_To_Reviewer_Comments_.pdfClick here for additional data file.Reviewer_1_Report_ -- Cedric Bardy4/23/2018 ReviewedClick here for additional data file.Reviewer_1_Report_Revision_1 -- Cedric Bardy7/29/2018 ReviewedClick here for additional data file.Reviewer_2_Report_ -- Jerome Mertens4/27/2018 ReviewedClick here for additional data file.Reviewer_2_Report_Revision_1 -- Cedric Bardy10/8/2018 ReviewedClick here for additional data file.Supplemental FilesClick here for additional data file."} +{"text": "G3: Genes|Genomes|Genetics 8: 2603-2615) entitled \u201cA Dual sgRNA Approach for Functional Genomics in Arabidopsis thaliana\u201d on page 2613 in the first sentence of the Acknowledgments section, incomplete funding information was provided related to support from Research Foundation Flanders. The sentence has been corrected as follows:In the article by L. Pauwels, R. De Clercq, J. Goossens, S. I\u00f1igo, C. Williams, M. Ron, A. Britt, and A. Goossens (This work was supported by the Research Foundation Flanders through the projects 1507013N, G005212N, G005312N and G005915N and a postdoctoral fellowship to L.P.; the Belgian Science Policy Organization for a postdoctoral fellowship to S.I. and the Agency for Innovation by Science and Technology in Flanders for a predoctoral fellowship to J.G.; the National Science Foundation for M.R.\u2019s contribution to this work by grant # 1636397."} +{"text": "M\u00f8ller Foundation for the Advancement of Medical Science (https://www.apmollerfonde.dk/ansoegning/laegefonden.aspx); The Danish Bio and Genome Bank (http://www.regioner.dk/rbgben).The Funding statement is incorrect. The publisher apologizes for the error. The correct Funding statement is as follows: The Danish Blood Donor Study was funded by: The Danish Council for Independent Research - Medical Sciences , Health Research Fund of Central Denmark Region (https://www.rm.dk/sundhed/faginfo/forskning/region-midtjyllands-sundhedsvidenskabelige-forskningsfond/), Aase & Ejnar Danielsens Fond (https://danielsensfond.dk), The H\u00f8jmoseg\u00e5rd Grant, and A.P. M\u00f8ller Foundation for the Advancement of Medical Science. None of the funders had any influence on study design, data collection and analysis, decision to publish, or preparation of the manuscript.The first author was funded by Aarhus University ("} +{"text": "Nature Communications 10.1038/s41467-019-10244-7, published online 24 May 2019.Correction to: The original version of this Article omitted the following from the Acknowledgements:This research was funded by a NERC BETR grant (NE/P013678/1 to D.P. and J.V.), and the European Union\u2019s Horizon 2020 research and innovation programme under the Marie Sk\u0142odowska-Curie grant agreement (764840 to D.P. and M.G.).This has now been corrected in both the PDF and HTML versions of the Article."} +{"text": "Scientific Reports 10.1038/s41598-018-34172-6, published online 02 November 2018Correction to: The Author Contributions section in this Article is incorrect.\u201cM.G. did all necessary experimental work , wrote main manuscript text and prepared all figures. S.D. did experimental work for Fig. 2. P.A.A. wrote the original proposal which was funded, and then served as the basis of this research project, supervised the work and modified the manuscript for important intellectual content.\u201dshould read:\u201cM.G. did all necessary experimental work , wrote main manuscript text and prepared all figures. S.D. was an undergraduate researcher who was predominantly in charge of work shown in Figure 2, and was involved from the beginning of this project including all repetitions of the work performed by M.G. P.A.A. wrote the original proposal which was funded, and then served as the basis of this research project, supervised the work and modified the manuscript for important intellectual content.\u201d"} +{"text": "Nature Communications 10.1038/s41467-018-06929-0; published online 26 October 2018Correction to: The original version of this Article omitted from the author list the 7th author Bridget Young, who is from the \u2018Department of Pediatrics, Section of Nutrition, University of Colorado Anschutz Medical Campus, Aurora 80045 CO, USA\u2019 and \u2018Present address: Department of Pediatrics; Allergy and Immunology, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA\u2019, and the 8th author Nancy F.\u00a0Kriebs, who is from the \u2018Department of Pediatrics, Section of Nutrition, University of Colorado Anschutz Medical Campus, Aurora 80045 CO, USA\u2019.Consequently, \u2018L.A.B., and N.F.K.\u2019 was added to the fourth sentence of the Acknowledgements: \u2018This study was supported by the American Diabetes Association/Glaxo Smith Kline Targeted Research Award , [\u2026]\u2019. Plus the following was added to the end of the Acknowledgements: \u2018B.Y. and N.F.K. were supported by NIH/National Institute of Diabetes and Digestive and Kidney Diseases T32 DK007658 and NIH/Child Health and Development grant F32-0978068 (B.Y.).\u2019In addition, the following was added to the Author contributions: \u2018B.Y. and N.F.K. helped to design the clinical study and participated in the sample collection during the infant visits.\u2019This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Nature Communications; 10.1038/s41467-018-05853-7; published online 12 September 2018Correction to: The original version of this Article omitted a declaration from the Competing interests statement, which should have included the following: \u2018A patent has been applied for by Emory University with F.E.L., I.S., and D.C.N. as named inventors. The patent application number is PCT/US2016/036650\u2019. This has now been corrected in both the PDF and HTML versions of the Article."} +{"text": "Scientific Reports 10.1038/s41598-017-14910-y, published online 03 November 2017Correction to: The original version of this Article contained a typographical error in the Abstract.\u2018In Experiment 2, 64 participants performed the SRRL task in both a low-stress VRE and a high-stress VRE .\u2019now reads:\u2018In Experiment 2, 66 participants performed the SRRL task in both a low-stress VRE and a high-stress VRE .\u2019In addition, the original version of this Article contained a typographical error in the Author Contributions section.\u2018Z.C. and Y.M. conducted the experiment and wrote the manuscript; Z.C. created all the virtual reality environments and undertook the coding of VR experiments and the data analysis. Z.C. also took the figures and supplementary videos . Y.M. and L.Z. interpreted the results and revised the manuscript.\u2019now reads:\u2018Z.C. and Y.W. conducted the experiment and wrote the manuscript; Z.C. created all the virtual reality environments and undertook the coding of VR experiments and the data analysis. Z.C. also took the figures and supplementary videos . Y.W. and L.Z. interpreted the results and revised the manuscript.\u2019"} +{"text": "Scientific Reports 10.1038/s41598-017-16558-0, published online 27 November 2017Correction to: The Acknowledgements section in this Article is incomplete.\u201cY.S. is supported by the Israel Foundation grant numbers 711631, the ERC (StG-335377), the MRA and by the Knell Family and the Hamburger Family. This work was supported by ISF and BSF to RS. R.S. is an incumbent of the Yale S. Lewine and Ella Miller Lewine Professional Chair for Cancer Research. R.A. is supported by the Clore Foundation\u201d.should read:\u201cY.S. is supported by the Israel Foundation grant numbers 711631, the ERC (StG-335377), The Minerva Foundation Grant, the MRA and by the Knell Family and the Hamburger Family. This work was supported by ISF and BSF to RS. R.S. is an incumbent of the Yale S. Lewine and Ella Miller Lewine Professional Chair for Cancer Research. R.A. is supported by the Clore Foundation\u201d."} +{"text": "Sci., 2016, 7, 5893\u20135899.Correction for \u2018Face and edge directed self-assembly of Pd An incorrect grant number was quoted for this paper and the corrected Acknowledgement section is as shown below:P. S. M. thanks the DST (New Delhi) for the research grant [DST/SJF/CSA-01/2011-2012(G)]. P. H. is grateful to Mr Sayantan Chatterjee for the rheology experiments and also thankful to Mr Shaunak Chakraborty for fruitful discussion on the single crystal structure analysis.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "Scientific Reports 10.1038/srep46062, published online 10 April 2017Correction to: The Acknowledgements section in this Article is incomplete.\u201cSudesh, P. Kumar and P. Neha acknowledge UGC-D. S. Kothari fellowship, JNU (New Delhi) and UGC-BSR, respectively for financial support. Authors are thankful to AIRF (JNU) for access to the PPMS, EDAX and TEM facilities. We thank Prof A. K. Rastogi and Dr. Chandra Shekhar for useful discussions. Low\u2013temperature high magnetic field at JNU is supported under the FIST program of Department of Science and Technology, Government of India. T. Das\u2019s work is supported by SERB young scientist Startup research grant. We thank Dr. Dinabandhu Das for advice on single crystal diffraction data analysis.\u201dshould read:\u201cSudesh, P. Kumar and P. Neha acknowledge UGC-D. S. Kothari fellowship, JNU (New Delhi) and UGC-BSR, respectively for financial support. Authors are thankful to AIRF (JNU) for access to the PPMS, EDAX and TEM facilities. We thank Prof A. K. Rastogi and Dr. Chandra Shekhar for useful discussions. Low\u2013temperature high magnetic field at JNU is supported under the FIST program of Department of Science and Technology, Government of India. We also acknowledge financial support received from DST-PURSE grant (Government of India) to fund the open access charges for this manuscript. T. Das\u2019s work is supported by SERB young scientist Startup research grant. We thank Dr. Dinabandhu Das for advice on single crystal diffraction data analysis.\u201d"} +{"text": "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.There is information missing from the Funding section. The complete, correct Funding information is as follows: H.T. and C.M. were funded by JSPS KAKENHI Grant Numbers JP16K20066 and JP17K08726 (There is information missing from the Acknowledgements. The complete, correct Acknowledgments is as follows: We are grateful to Ms. Sachiko Yasui for technical assistance. This work was partly supported by JSPS KAKENHI Grant Numbers JP16K20066 to H.T. and JP17K08726 to C.M."} +{"text": "The second sentence in the Introduction should have cited reference 14 instead of 2. The correct sentence should read: Granger [14] followed this notion of causality, and applied it to the analysis of economic time series data.14. Granger CWJ. Investigating causal relations by econometric models and cross-spectral methods. Econ.1969; 37: 424\u2013438.The fifth sentence in the Introduction should have cited reference 15 instead of 3. The correct sentence should read: Geweke [15] expanded on Granger\u2019s idea to define a measure for model comparison based on using the quasi-likelihood function.15. Geweke J. Measurement of linear dependence and feedback between multiple time series. J. Amer. Stat. Assoc.; 1982: 77:304\u2013313.The seventh sentence in the Introduction should have cited reference 4 instead of 5. The correct sentence should read: Here, we define feedback as being present when given bivariate time series, each of them is mutually causal to the other [4].4. Akaike H. On the use of a linear model for the identification of feedback systems, Annal. Inst. Stat. Math. 1968; 20: 425\u2013439.A reference is omitted from the penultimate sentence of the first paragraph in the Introduction. The correct sentence should read: The methodology is referred to as the Akaike\u2019s total causality approach [5].5. Ozaki T. Time Series Modeling of Neuroscience Data. Chapman & Hall/CRC. 2012.The final sentence beneath the \u201cmultivariate auto-regressive model and model selection\u201d subheading in the Methods should have cited reference 17 instead of 2. The correct sentence should read: The AR order is identified by statistical model selection approaches, such as, the Akaike Information Criterion (AIC) [17], defined by17. Akaike H. A new look at statistical model identification, IEEE Tran. Automat. Contrl. 1974; AC-19: 716\u2013723.The reference in eighth sentence beneath the \u201cStudy 1\u201d subheading of the Simulation study section is incorrect. The correct sentence should read: To avoid multiple testing problem, False Discovery Rate (FDR) analysis was applied to the p-values.The correct citation is: Benjamini, Y. and Hochberge, Y. 1995 Controlling the false discovery rate: a practical and powerful approach to multiple testing, J.R.Statistics, Soc.B, 57:289\u2013300.Beneath the \u201cData\u201d subheading of the Causal analyses of Barents Sea capelin population dynamics section, reference 1 is mistakenly cited in the first half of the eight sentence. The correct sentence should read: The data are taken from the database of the WGIBAR , and for particularly for capelin, the survey procedure and biomass calculations can be found in Gj\u00f8s\u00e6ter et al. [24].The correct citation is: ICES. 2016 Final report of the working group on the integrated assessment of the Barents Sea (WGIBAR). ICES CM, 2016/SSGIEA:04:123 p.The 23. Hjermann D\u00d8, Stenseth NC, and Ottersen G, Indirect climate forcing of the Barents Sea capelin: a cohort effect. Mar. Ecol. Prog. Ser. 2004; 273: 229\u2013238.Beneath the \u201cThe influence from temperature as an abiotic factor subheading\u201d in the Results and Discussion, reference 1 is incorrectly omitted from the fourteenth sentence. The correct sentence should read: Since MAR model includes full coefficients, the model that the abiotic factor is treated as an exogeneous variable seen in could capture more significant relationship between capelin and herring.3. Francis T., Wolkovich E.M., Scheuerell M.D., Katz S.L., Holmes E.E., and Hampton S.E. Shifting regimes and changing interactions in the Lake Washington, U.S.A., plankton community from 1962\u20131994. PLOS ONE, 2014; 9; 2110363."} +{"text": "Scientific Reports 10.1038/s41598-019-41643-x, published online 26 March 2019Correction to: The Acknowledgements section in this Article is incomplete.\u201cThe research was partially supported by EDUFI Fellowships and the Academy of Finland . IM also acknowledges partial support from the MEPhI Academic Excellence Project (Contract No. 02.a03.21.0005) and National Research Tomsk State University Academic D.I. Mendeleev Fund Program\u201d.should read:\u201cAuthors acknowledge the initial involvement of Dr. M. Kinnunen , Prof. A.V. Priezzhev and Dr. A. Karmenyan at the early stage of the optical tweezers development and providing nanodiamonds (A.K.). The research was partially supported by EDUFI Fellowships and the Academy of Finland . IM also acknowledges partial support from the MEPhI Academic Excellence Project (Contract No. 02.a03.21.0005) and National Research Tomsk State University Academic D.I. Mendeleev Fund Program\u201d."} +{"text": "Nature Communications 10.1038/s41467-019-12020-z, published online 4 October 2019.Correction to: The original version of this Article has been corrected to include an extended Competing interests declaration: \u2018KF and GB are co-developers of the River Styles Framework. River Styles foundation research has been supported through competitive grant schemes and university grants. Consultancy-based River Styles short courses taught by KF and GB are administered by Macquarie University. River Styles contract research is administered by Macquarie University and University of Auckland. River Styles as a trade mark expires in May 2020. TD declares no conflict of interest.\u2019 This replaced the original Competing interests declaration: \u2018K.F. and G.B. are co-developers of the River Styles Framework. T.D. declares no competing interests.\u2019 This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "Scientific Data 10.1038/sdata.2018.249, published online 20 November 2018Correction to: J. H. Burns was omitted in error from the author list of the original version of this Data Descriptor. This omission has now been corrected in both the HTML and PDF versions."} +{"text": "Suad A. Telity.Following publication of the original article , the aut"} +{"text": "Scientific Reports6; Article number: 2652510.1038/srep26525; published online: 06102016; updated 06272018The Acknowledgements section in this Article is incomplete.\u201cThis work was supported by NIH EY019101 to M.Z. This study was supported in part by an Unrestricted Grant from Research to Prevent Blindness, Inc., and an NEI core grant. Y.S. is supported by a fellowship from the China Scholarship Council. F.F. is supported by Funda\u00e7\u00e3o para a Ci\u00eancia e Tecnologia (FCT) grant SFRH/BD/87256/2012. M.F.N. is supported by NIH HL098200 and HL121059. We thank Dr. James Jester (UC Irvine) for the generous gift of hTCEpi cells. We are grateful to Bradley Shibata and Dr. Paul Fitzgerald, Dept. of Cell Biology and Human Anatomy, UC Davis, for help with cornea histology , and Madeline Nieves, Dept. of Pharmacology, UC Davis, for technical support. We also thank Dr. Vijay Raghunathan for help with cell culture and imaging. We are also grateful to Dr. Rivkah Isseroff and Michelle So for helpful discussions and initial tissue samples\u201d.should read:\u201cThis work was supported by NIH EY019101 to M.Z. This study was supported in part by Air Force Office of Scientific Research under award number FA9550-16-1-0052, R21EB015737, and an Unrestricted Grant from Research to Prevent Blindness, Inc.. Y.S. is supported by a fellowship from the China Scholarship Council. F.F. is supported by Funda\u00e7\u00e3o para a Ci\u00eancia e Tecnologia (FCT) grant SFRH/BD/87256/2012. M.F.N. is supported by NIH HL098200 and HL121059. We thank Dr. James Jester (UC Irvine) for the generous gift of hTCEpi cells. We are grateful to Bradley Shibata and Dr. Paul Fitzgerald, Dept. of Cell Biology and Human Anatomy, UC Davis, for help with cornea histology , and Madeline Nieves, Dept. of Pharmacology, UC Davis, for technical support. We also thank Dr. Vijay Raghunathan for help with cell culture and imaging. We are also grateful to Dr. Rivkah Isseroff and Michelle So for helpful discussions and initial tissue samples\u201d."} +{"text": "Proc. R. Soc. B286, 20182539. (Published Online 23 January 2019) (doi:10.1098/rspb.2018.2539)Corrected funding statementT.B. was supported by two Monash University postgraduate scholarships and by CSIRO Data61. Model development and fieldwork were partially supported by Australian Research Council grants no. DP140103946 to M.B. and no. DP110101413 to B.M. Laboratory work was supported by Universities Australia-DAAD project 57219808 \u2018Division of Labour and Collective Homeostasis in Dynamic Environments\u2019 and by DFG Centre of Excellence 2117 \u2018Centre for the Advanced Study of Collective Behaviour\u2019 (ID: 422037984)."} +{"text": "Nature Communications; doi:10.1038/s41467-018-05515-8; published online: 07 Aug 2018Correction to: Philos. Trans. R. Soc. Lond. B \u2019 in the eighth sentence of the last paragraph of the Introduction, and the last sentence of the first paragraph of the \u2018Study population\u2019 section of the Methods. As this paper does not exist, this reference has been removed and all following citations have been renumbered as appropriate. This has been corrected in both the PDF and HTML versions of the Article.The original version of this Article incorrectly cited as ref. 45 the paper \u2018Lynch, E. C., Lahdenper\u00e4, M., Mar, K. U. & Lummaa, V. The evolutionary significance of maternal kinship in a long-lived mammal."} +{"text": "Scientific Reports 10.1038/s41598-018-36608-5, published online 31 January 2019Correction to: Laura Buttitta was omitted from the author list in the original version of this Article.The Acknowledgements section now reads:\u201cThis research was supported by the National Science Foundation (NSF CAREER Award DMR-0845592 to K.K.), the National Cancer Institute , the Department of Defense , the Prostate Cancer Foundation , Grants-in-Aid for Challenging Research from the Japan Society for the Promotion of Science (JSPS), and Department of Biologic and Materials Sciences, School of Dentistry, University of Michigan. R.T. receives support as the Major McKinley Ash Collegiate Professor. This work was also supported by JSPS Postdoctoral Fellowships for Research Abroad (No. 26-774 to H.T.). We also thank Dr. Robertson Davenport at University of Michigan Hospital for providing RBCs. We thank Dr. Yuji Mishina and Dr. Maiko Omi for help on cell imaging and Dr. Dan Sun for assistance with generating cell lines.\u201dThe Author Contributions section now reads:\u201cH.T. synthesized and characterized the copolymers, evaluated the cytotoxicity of copolymers to proliferating cancer cells and normal cells, performed LDH leakage assay and cell imaging. K. Yumoto characterized the primary normal cells and dormant cancer cells, evaluated the cytotoxicity of copolymer to normal cells and dormant cancer cells, and prepared cell samples for SEM. K. Yasuhara performed the lipid vesicle assays. E.T.N. synthesized and characterized the copolymers. Y.K. supervised the assays using primary normal cells. L.B. devised and supervised the development of cell lines to monitor dormancy. R.S.T. devised the project and supervised the assays using dormant cancer cells. K.K. devised the project, helped with data analysis, and prepared the manuscript. All authors assisted with editing the manuscript.\u201dThese errors have now been corrected in the PDF and HTML versions of the Article, and in the accompanying Supplementary Information file."} +{"text": "Correction to:British Journal of Cancer (2019) 121, 474\u2013482; 10.1038/s41416-019-0540-4, published online 7 August 2019.Since the publication of this paper, the authors noticed that the funding information for E.D. was not included. The correct funding information is now shown in the Funding section below.Funding: Funding for this study was provided by: Cancer Research UK (C6199/A10417 and C399/A2291), the European Union Seventh Framework Programme (FP7/207- 2013) grant 258236 collaborative project SYSCOL, European Research Council project EVOCAN, the Oxford Cancer Centre, the Oxford NIHR Comprehensive Biomedical Research Centre (BRC), and core funding to the Wellcome Trust Centre for Human Genetics from the Wellcome Trust (090532/Z/09/Z). M.G. is funded by a Wellcome Trust Clinical Training Fellowship. E.D. is supported by the S:CORT consortium which is funded by a grant from the Medical Research Council and Cancer Research UK. D.N.C. is funded by a Health Foundation/Academy of Medical Sciences Clinician Scientist Fellowship. A.S. is funded by a Norwegian Cancer Society Scientist Fellowship (no. 6824048-2016). R.A.L. is supported by the Research Council of Norway (no. 250993) and the Norwegian Cancer Society (no. 182759-2016). M.N. and D.O. were supported by Cancer Research UK and the National Institute for Health Research through the UCL Experimental Cancer Medicine Centre (to D.O.) and UCL Hospitals Biomedical Research Centre (to M.N.). The views expressed are those of the authors and not necessarily those of the NHS, the NIHR, the Department of Health or the Wellcome Trust. The costs of open access publishing were funded by The Wellcome Trust (090532/Z/09/Z). The study funders had no role in the design, analysis, interpretation, or manuscript preparation for this biomarker study. D.N.C. had full access to all study data and had final responsibility for submission of this report."} +{"text": "Nature Communications 10.1038/s41467-018-05435-7; published online 27 July 2018Correction to: The original PDF version of the Article contained an error in the last sentence of the author affiliation information, which incorrectly read \u2018Correspondence and requests for materials should be addressed to T.$.R. or to Y.F. (e-mail: yejun@oist.jp)\u2019. The correct version states \u2018T.F.R.\u2019 in place of \u201cT.$.R.\u2019. This has been corrected in the PDF version of the Article. The HTML version was correct from the time of publication."} +{"text": "It has been brought to our attention that the affiliation of Dr. Jerzy Pieczykolan at the time when he was responsible for the work described in the paper was not We would like to change the Dr. Jerzy Pieczykolan\u2019 affiliation on Page 1 of paper [Preclinical Development Department, R&D Celon Pharma Inc., 05-092 Lomianki/Kielpin, Poland; jerzy.pieczykolan@celonpharma.com.to the correct version, as follows:Drug Discovery Department, Adamed Group, Czosnow 05-152, Poland; jerzy.pieczykolan@adamed.com.pl.And add the following affiliation as the current address of Dr. Jerzy Pieczykolan:Preclinical Development Department, R&D Celon Pharma Inc., 05-092 Lomianki/Kielpin, Poland; jerzy.pieczykolan@celonpharma.com.This correction does not cause any changes to results and conclusions in the original published paper."} +{"text": "Scientific Reports 10.1038/s41598-018-21254-8, published online 12 February 2018Correction to: The Acknowledgements section in this Article is incomplete.\u201cThis research was supported by the NIH , with additional funding from Research to Prevent Blindness (Baylor College of Medicine), the Retina Research Foundation (S.M.W. and B.J.F.) and Hong Kong Polytechnic University grant # G-UA7J (D.Y.T.). We thank Dr. Yong Park for critically reading the manuscript.\u201dshould read:\u201cThis research was supported by the NIH , with additional funding from Research to Prevent Blindness (Baylor College of Medicine), the Retina Research Foundation (S.M.W. and B.J.F.) and Hong Kong Polytechnic University grant # G-UA7J (D.Y.T.). We thank Dr. Yong Park for critically reading the manuscript.\u201d"} +{"text": "Scientific Reports 10.1038/s41598-018-37557-9, published online 04 February 2019Correction to: This Article contains an error in the Acknowledgements section.Diap1-lacZ fly strain. We acknowledge Bloomington Stock Center, Drosophila Genetic Resource Center and Developmental Studies Hybridoma Bank for fly strains and antibodies. This research was supported by National Research Foundation R1A1A301573 and N01170193, and National Research Council of Science and Technology Grant DRC-14-2-KRISS\u201d.\u201cWe are grateful to K.-W. Choi, S.-T. Hong and colleagues in our lab for discussion and comments on manuscript. We also thank to D.-G. Cho for irradiating flies, and O.-K. Lee and B.-S. Kim for technical assistance and fly maintenance. We thank S. J. Yoo for Diap1 antibody and J.-K. Chung for should read:Diap1-lacZ fly strain. We acknowledge Bloomington Stock Center, Drosophila Genetic Resource Center and Developmental Studies Hybridoma Bank for fly strains and antibodies. This research was supported by National Research Foundation R1A1A301573 and 2017R1A2B4009254, and National Research Council of Science and Technology Grant DRC-14-2-KRISS\u201d.\u201cWe are grateful to K.-W. Choi, S.-T. Hong and colleagues in our lab for discussion and comments on manuscript. We also thank to D.-G. Cho for irradiating flies, and O.-K. Lee and B.-S. Kim for technical assistance and fly maintenance. We thank S. J. Yoo for Diap1 antibody and J.-K. Chung for"} +{"text": "Sci., 2015, 6, 2575\u20132583.Correction for \u2018Pattern-based detection of anion pollutants in water with DNA polyfluorophores\u2019 by Hyukin Kwon On page 2581, an NIH grant was inadvertently omitted in the Acknowledgements. The Acknowledgements should read as follows:We thank Eni S.p.A. and the U.S. National Institutes of Health (GM106067) for support.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."} +{"text": "Two references are omitted from the from the second sentence of the third paragraph in the Introduction section.The sentence should read: Indeed, the faunal and botanical assemblages from early sixth millennium BC sites in southeast Europe have been shown to vary in the proportions of food plant and animal taxa [7\u201315] .The references are:Kreuz A, Marinova E, Sch\u00e4fer E, Wiethold J. A comparison of early Neolithic crop and weed assemblages from the Linearbandkeramik and the Bulgarian Neolithic cultures: differences and similarities. Vegetation History and Archaeobotany. 2005;20:333\u201348.10.1007/s00334-017-0643-x.Kreuz A, Marinova E. Archaeobotanical evidence of crop growing and diet within the areas of the Karanovo and the Linear Pottery Cultures: a quantitative and qualitative approach. Veget Hist Archaeobot. 2017; 26:639\u201357. doi:"} +{"text": "The original version of this article unfortunately contained a mistake in coauthor's given name. The abbreviated given name \"M.D.\" for coauthor M.D.P. Elfferich's name has been inadvertently deleted during the production process.The original article has been corrected."} +{"text": "Following publication of the original article , the aut2Incorrect: Peter M. Mollica2Correct: Peter A. MollicaThe publishers apologise for this error. The original article has been"} +{"text": "Nature Communications 10.1038/s41467-019-08593-4, published online 08 February 2019.Correction to: The original version of this Article omitted from the Author Contributions statement that \u2018R.S. and J.G.R. contributed equally to this work.\u2019 This has been corrected in both the PDF and HTML versions of the Article."} +{"text": "U.S. President George W. Bush's nod to scientific evidence for human industrialization's major role in the onset of global climate change grabs both top story positions in Nature and Science this week."} +{"text": "J.B. Cui, Q.Q. Chen, T.T. Liu, S.J. LiNeurosurgery Intensive Care Unit, Weifang People\u2019s Hospital, Weifang, ChinaRetraction for: Braz J Med Biol Res | doi: http://10.1590/1414-431X20176830 | PMID: 29791584The Brazilian Journal of Medical and Biological Research was contacted by one specialist questioning the validity of this study.http://dx.doi.org/10.1590/1414-431X20176830> PMID: 29791584 | PMCID: PMC5972009The Editors decided to retract the article: \u201cRisk factors for early-onset ventilator-associated pneumonia in aneurysmal subarachnoid hemorrhage patients\u201d that was published in volume 51 no. 7 (2018) in the Brazilian Journal of Medical and Biological Research